Opportunity, challenge and Scope of Natural Products in Medicinal Chemistry

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry Editor

Vinod K. Tiwari Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Co-editor

Bhuwan B. Mishra Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Research Signpost 2011; Rights Reserved Research Signpost T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Vinod K. Tiwari Co-editor Bhuwan B. Mishra Managing Editor S.G. Pandalai Publication Manager A. Gayathri Research Signpost and the Editors assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-308-0448-4

Preface Natural products extracted from tissues of terrestrial plants, marine organisms or microorganism fermentation broths are the evolutionary shaped molecules with a profound impact on human health. Nature's biosynthetic engine produces innumerate metabolites with distinct biological properties that make them valuable as health products or as structural templates for drug discovery. In the early 1900s, before the ‘‘Synthetic Era’’, 80% of all medicines were obtained from roots, barks and leaves with a belief that for every ill there exists a cure in the plants of field and forest. However, with the advent of robotics, bioinformatics, high throughput screening, molecular biology-biotechnology, combinatorial chemistry, in silico (molecular modelling) and other methodologies, the pharmaceutical industry largely moved away from plant derived natural products as a source for leads and prospective drug candidates. There are also several misconceptions that constrained the utilization of plant products for discovery and development of pharmaceuticals. Among some of practical aspects while trying to explain the difficulties associated with natural product research are: low yield, one-sampleone-source problem; high structural complexity and occurrence of multiple stereoisomer; lacking of follow-up studies, since most efforts (e.g. in academic environments) are not the part of focused drug development programs. Pharmaceutical discovery is a numbers game in which thousands of chemicals must be evaluated to find a hit. The interesting chemicals identified as natural products are derived from the phenomenon of biodiversity in which the interaction of organisms among each other and their environment formulate the evolution of diverse complex natural entities in the organisms that enhance their survival by protecting them against a wide variety of microorganisms, arthropods and vertebrates and maintain competitiveness in the ecosystem. Importantly, nature has been “doing” combinational chemistry for eons and supplying almost unimaginable chemical diversity, which yields stereochemically complex structures with diverse functional groups and molecules ideal for interacting specifically with biological targets. As Aristotle said, ‘‘Nature does nothing without purpose or uselessly”, the world of plants, and indeed all natural sources, represents a virtually untapped pool of novel drugs awaiting imaginative and progressive organizations. This book covers almost all natural product drug discoveries that have been made in past few decades. The book editorial (Chapter 1) sumarises the natural products, semi-synthetic natural products and natural product derived compounds that have been registered, undergoing registration or in clinical

development, while chapters 2-12 are focused on natural product drug discoveries by disease area i.e. infectious (bacterial, fungal and parasitic etc.) diseases and Oncology. The chapter 13 is focused on mutasynthesis that couples the power of chemical synthesis with molecular biology to generate derivatives of medicinally important natural products while the last chapter of the book highlights significance of carbohydrate containing natural products in medicinal chemistry. I am thankful to all the authors and reviewers who helped me in compiling this book. Lastly, I want to draw the attention of readers about the increasing loss of much of the world’s forests, particularly in the tropics, where the potentially remarkable properties of plant constituents not yet discovered are threatened. Several plant species are on the brink of extinction and in need of urgent conservation measures, otherwise, many future drugs and other useful plant products would remain undiscovered and the often surprising chemical structures produced by the genetic diversity of plants might not be envisioned by future chemists. Vinod K. Tiwari

Editorial Advisory Board Prof. A. D. Kinghorn, USA Prof. A. W. Lipkowski, Poland Prof. Atta-ur-Rahman, Pakistan Prof. B. Pirotte, Belgium Prof. D. L. Boger, USA Prof. D. S. Bhakuni, India Prof. D. Strack, Germany Prof. De-Yun Wang, Singapore Prof. G. A. Cordell, USA Prof. G. Appendino, Itly Prof. G. Bringmann, Germany Prof. G. H. Veeneman, Netherland Prof. G. P. Bolwell, UK Prof. G. S. Singh, Botswana Prof. G. Vo-Thanh, France Prof. Ganesh Pandey, India Prof. Guisen Zhao, China Prof. H. Ila, India Prof. J. A. R Rodrigues, Brazil Prof. J. C. Stockert, Spain Prof. J. D. Connolly, UK Prof. J. S. Yadav, India Prof. Jamie Simpson, Australia Prof. K. C. Nicolau, USA Prof. K. D. Janda, USA Prof. M. Garson, Austria Prof. M. I. Choudhary, Pakistan Prof. M. J. Chan-Bacab, Mexico Prof. M. P. Kaushik, India Prof. M. Salzet, France

Prof. M. Shibasaki, Japan Prof. N. Tagmatarchis, Greece Prof. Norbert Haider, Austria Prof. P. G. Wang, USA Prof. P. S. Portoghese, USA Prof. Prabhat Arya, Canada Prof. Pradeep Kumar, India Dr. Prabhu P Mohapatra, USA Prof. R. A. Lewis, Switzerland Prof. R. M. Singh, India Prof. R. P. Tripathi, India Prof. R. R. Schmidt, Germany Prof. S. Neidle, UK Prof. R. Robins, France Prof. S. Chandrashekhar, India Prof. S. Komatsu, Japan Prof. Seokjoon Lee, South Korea Prof. Shang-Cheng Hung, Taiwan Prof. Thomas Kurz, Germany Prof. V. K. Singh, India Prof. W. Boland, Germany Prof. Xi Chen, USA Prof. Y. Asakawa, Japan Prof. Y. B. Tripathi, India Prof. Y. H. Wong, Hong Kong Prof. Y. Hashimoto, Japan Prof. Y. Hu, China Prof. Y. Yamamoto, Japan Prof. Yogendra Singh, India Prof. Nity anand, India

Contents

Chapter 1 Natural products in drug discovery: Clinical evaluations and investigations Bhuwan B. Mishra and Vinod K. Tiwari

1

Chapter 2 Natural products in discovery of potential and safer antibacterial agents Girija S. Singh and Surendra N. Pandeya

63

Chapter 3 Anti-tubercular activity of natural products: Recent developments L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov

103

Chapter 4 Scope of natural products in fighting against leishmaniasis B. B. Mishra, R. R. Kale, V. Prasad, V. K. Tiwari and R. K. Singh

121

Chapter 5 Naturally occurring antihyperglycemic and antidyslipidemic agents T. Narender, T. Khaliq and G. Madhur

155

Chapter 6 Bio-flavonoids with promising anti-diabetic potentials: A critical survey Goutam Brahmachari

187

Chapter 7 Marine natural alkaloids as anticancer agents Deepak Kumar and Diwan S. Rawat

213

Chapter 8 Microtubule binding natural substances in cancer chemotherapy Ram C. Mishra

269

Chapter 9 Natural products: Anti-fungal agents derived from plants Tasleem Arif, T. K. Mandal and Rajesh Dabur

283

Chapter 10 Sesquiterpene lactones: Structural diversity and their biological activities Devdutt Chaturvedi

313

Chapter 11 A review on natural products with mosquitosidal potentials Navneet Kishore, Bhuwan B. Mishra, Vinod K. Tiwari and Vyasji Tripathi Chapter 12 Soybean constituents and their functional benefits Ajay K. Dixit, J. I. X. Antony, Navin K. Sharma and Rakesh K. Tiwari

335

367

Chapter 13 Mutasynthesis of medicinally important natural products through manipulation of gene governing starter unit Deepak Sharma, Syed Khalid Yousuf and Debaraj Mukherjee

385

Chapter 14 Carbohydrate-containing natural products in medicinal chemistry Hongzhi Cao, Joel Hwang and Xi Chen

411

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 1-62 ISBN: 978-81-308-0448-4

1. Natural products in drug discovery: Clinical evaluations and investigations Bhuwan B. Mishra and Vinod K. Tiwari Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Abstract. Natural products (NPs) have provided the source for the majority of FDA-approved agents and continue to be one of the major sources of inspiration for future drug discovery. The R&D thrust in the pharmaceutical sector today is focused on development of new drugs, innovative/indigenous processes for known drugs, development of NP-based drugs through investigation of leads obtained from the traditional systems of medicine as well as other resources. Present review describes natural products (NPs), semi-synthetic NPs and NP-derived compounds that have been registered, undergoing registration or in clinical development since 1998 till June 2010 by disease area i.e. infectious (bacterial, fungal, parasitic and viral), immunological, cardiovascular, neurological, inflammatory and related diseases and Oncology. This review also highlights the recently launched natural product-derived drugs, new natural product templates and late-stage development candidates.

1. Introduction The interesting chemicals identified as NPs are derived from the phenomenon of biodiversity in which the interactions among organisms and their environment formulate the diverse complex chemical entities within the Correspondence/Reprint request: Dr. Vinod K. Tiwari, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. E-mail: [email protected]

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Bhuwan B. Mishra & Vinod K. Tiwari

organisms that enhance their survival and competitiveness. Today, R&D thrust in the pharmaceutical sector is focused on development of new drugs, innovative/indigenous processes for known drugs and development of plant-based drugs through investigation of leads obtained from the traditional systems of medicine as well other resources [1,2]. History of medicine dates back practically to the existence of human civilization and use of NPs by human have been traced from ancient records such as the use of Artemisia annua in China, opium poppy (active principle morphine) in Egypt, snakeroot plant (active principle reserpine) in India, willow tree (salicin) & foxglove (active principle digitalis - a mixture of compounds such as digitoxin, digitonin, digitalin) in England and ipecacuanha root (active principle emetine), coca bush (active principle cocaine) and cinchona bark (active principle quinine) in Mesoamerica. The current accepted modern medicine or allopathy has gradually developed over the years by scientific and observational efforts of scientists. However, the basis of its development remains rooted in traditional medicine and therapies. Plants have always been a rich source of NP leads e.g. morphine, cocaine, digitalis, quinine, tubocurarine, nicotine, muscarine, paclitaxel (TaxolTM) and artemisinin. The success of penicillin encouraged the discovery of new antibiotics from microorganisms. Mining of the bacterial genome and identification of crucial targets followed by study of new bacterial or fungal strains have resulted in discovery of significant antibacterial agents such as the cephalosporins, tetracyclines, aminoglycosides, rifamycins and chloramphenicol. Since past five decades, marine sources e.g. coral, sponges, fish and marine microorganisms have attracted scientists from different disciplines leding to the discovery of several marine NPs with promising biological activity such as curacin A, eleutherobin, discodermolide, bryostatins, dolostatins, and cephalostatins. Venoms and toxins (peptides and non-peptides) occurring in snakes, spiders, scorpions, insects, and other microorganisms are also significant in drug discovery due to their specific interactions with macromolecular targets in the body, and have been proved crucial while studying receptors, ion channels, and enzymes. Toxins like α-bungarotoxin (from cobras), tetrodotoxin (from puffer fish) and teprotide (from Brazilian viper) etc. are in clinical trials for drug development. Similarly, the neurotoxins obtained from Clostridium botulinum (responsible for botulism, a serious food poisoning), has been found significant to prevent muscle spasm. The review summarizes the 3 groups of compounds classified as NPs, semi-synthetic NPs and NP-derived compounds that have been registred, undergoing registration or in clinical development since 1998 to June 2010 by disease area i.e. infectious (bacterial, fungal, parasitic and viral),

Natural products in drug discovery

3

immunological, cardiovascular, neurological, inflammatory and related diseases and oncology. The compounds which have biological activities and are derived from natural sources, e.g., plants, animals and microorganisms have been grouped as NPs. The compounds that are derived from a NP template using semi-synthesis have been grouped in semi-synthetic NPs while the compounds that were synthetically derived or in some cases inspired from a NP template have been classified as NP-derived compounds [3-5]. The review also presents an update of previous reviews published in relevance to present context [6-10]. Table 1. NP-deived drugs launched during 1998-2004; lead compounds and therapeutic area. Year 1998 1998 1999 1999 1999 2000 2001 2001 2001 2001 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2004

Trade name orlistat (Xenical®) cefoselis (Wincef®) dalfopristin and quinupristin (70 :30 mixture) (Synercid®) valrubicin (Valstar®) colforsin daropate (Adele, Adehl®) arteether (Artemotil®) ertapenem (InvanzTM) caspofungin (Cancidas®) telithromycin (Ketek®) 55 pimecrolimus (Elidel®) galantamine (Reminyl®) micafungin (Funguard®) amrubicin hydrochloride (Calsed®) biapenem (Omegacin®) nitisinone (Orfadin®) miglustat (Zavesca®)

Lead compound lipstatin cephalosporin streptogramin B 44 & streptogramin A 45

Disease area Antiobesity Antibacterial Antibacterial

doxorubicin 164 forskolin

Oncology Cardiotonic

artemisinin 65 thienamycin 5 pneumocandin B erythromycin 51 ascomycin galantamine FR901379 doxorubicin 164

Antimalarial Antibacterial Antifungal Antibacterial Atopic dermatitis Alzheimer’s disease Antifungal Oncology

thienamycin 5 leptospermone 1-deoxynojirimycin

mycophenolate sodium (Myfortic®) rosuvastatin (Crestor®) pitavastatin (Livalo®) daptomycin (CubicinTM) everolimus (CerticanTM) 24

mycophenolic acid

Antibacterial Antityrosinaemia Type 1 Gaucher disease Immunosuppression

mevastatin mevastatin daptomycin sirolimus 10

Dyslipidemia Dyslipidemia Antibacterial Immunosuppression

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Bhuwan B. Mishra & Vinod K. Tiwari

2. Drug approval processes The Investigational New Drug (IND) application is submitted to the FDA or EMA before commencement of clinical trials. Once clinical trials are successfully completed, the applicant files New Drug Application (NDA) in the US or Marketing Authorization Application (MAA) in Europe seeking drug’s approval for marketing, to which the agency replys in the form of “approval letter”, “nonapproval letter” or “approvable letter”. An ‘‘approval letter’’ allows the applicant to begin marketing of product, while a ‘‘non-approval letter’’ rejects the application. An ‘‘approvable letter’’ informs the applicants that the agency have completed their scientific review and determined that the application can be approved pending resolution of minor deficiencies identified in the letter or during an inspection of the manufacturing facilities. Table 2. NP-deived drugs launched during 2005-2010; lead compounds, and therapeutic area. Year 2005

Lead compound Dronabinol 1/cannabidol 2 Fumagillin 3 Thienamycin 5

Disease area Pain

2005 2005

Trade name Dronabinol 1/Cannabidol 2 (Sativex®) Fumagillin 3 (Flisint®) Doripenem 4 (Finibax®/DoribaxTM)

2005 2005 2005

Tigecycline 6 (Tygacil®) Ziconotide 8 (Prialt®) Zotarolimus 9 (EndeavorTM stent)

Tetracycline 7 Ziconotide 8 Sirolimus 10

2006

Anidulafungin 11 (EraxisTM/EcaltaTM) Exenatide 13 (Byetta®) Lisdexamfetamine 14 (VyvanseTM) Retapamulin 16 (AltabaxTM/AltargoTM) Temsirolimus 18 (ToriselTM) Trabectedin 19 (YondelisTM) Ixabepilone 20 (IxempraTM) Methylnaltrexone 22 (Relistor®) Everolimus 24 (Afinitor®) Telavancin 25 (VibativTM) Romidepsin 27 (Istodax®) Capsaicin 28 (Qutenza®) Monobactam aztreonam 29 (CaystonTM)

Echinocandin B 12

Antibacterial Pain Cardiovascul ar surgery Antifungal

Exenatide-4 13 Amphetamine 15 Pleuromutilin 17

Diabetes ADHD Antibacterial

Sirolimus 10 Trabectedin 19 Epothilone B 21 Naltrexone 23 Sirolimus 10 Vancomycin 26 Romidepsin 27 Capsaicin 28 Aztreonam 29

Oncology Oncology Oncology Pain Oncology Antibacterial Oncology Pain Antibacterial

2006 2007 2007 2007 2007 2007 2008 2009 2009 2009 2009 2010

Antiparasitic Antibacterial

Natural products in drug discovery

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3. NP based drugs approved during 1998-2004 A total of 21 NP and NP-derived drugs were launched in the United States, Europe or Japan during 1998-2004 that can be classified as 3 NPs, 10 semi-synthetic NPs and 8 NP-derived drugs (Table 1).

3.1. NP based drugs approved during 2005-2010 A total of 19 NP based drugs were approved for marketing worldwide in between the year 2005 to April 2010, among which 7 being classified as NPs, 10 semi-synthetic NPs and 2 NP-derived drugs (Table 2). VeregenTM (Polyphenon® E ointment), a defined mixture of catechins obtained from green tea, is the first ever herbal medicine to receive FDA approval in 2006. VeregenTM was developed by MediGene AG and launched in the US by Bradley Pharmaceuticals in December 2007 for topical use against genital warts. In March 2010, Solvay launched Veregen® (10 % ointment) in Germany. Sativex®, a mixture of dronabinol 1 and cannabidol 2 obtained from the cannabis plant, is the world's first pharmaceutical prescription medicine that was launched in Canada (April 2005) and was later approved by Health Canada (August 2007) as adjunctive analgesic for severe pain in advanced cancer patients [11]. Sativex® has been recommended by FDA to enter directly in Phase III trials and as of November 2009, GW Pharmaceuticals have completed the recruitment for Phase II/III trial against cancer pain. In March 2010, GW Pharmaceuticals provided an update on the progress of regulatory submission for Sativex® oromucosal spray for the treatment of the symptoms of spasticity due to multiple sclerosis. Fumagillin (Flisint®, Sanofi-Aventis) 3, an endothelial cell proliferation inhibitor isolated from Aspergillus fumigatus [12], was approved in France in September 2005 for the treatment of intestinal microsporidiosis. Fumagillin 3 can also block the blood vessel formation through binding to methionine aminopeptidase II and is under clinical investigtions as an angiogenesis inhibitor for the treatment of cancer. Among carbapenem-type β-lactams, doripenem (Finibax®, DoribaxTM) 4 is an ultra-broad spectrum injectable antibiotic launched in Japan (2005) by Shionogi & Co. while ertapenem, a NP derived compound based on structure of thienamycin 5 is being marketed by Merck as InvanzTM. In October 2007, Johnson & Johnson (J&J) obtained formal FDA approval for use of 4 in intraabdominal and urinary tract infections. Tigecycline (Tygacil®) 6, a glycylcycline antibiotic structurally similar to teracycline 7, was approved by FDA in June 2005 against intra-abdominal

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Bhuwan B. Mishra & Vinod K. Tiwari

and complicated skin and skin structure infections (SSSIs). Tigecycline 6 was developed by Francis Tally and contains a centralised four-ring carbocyclic skeleton substituted at the D-9 position confering broad spectrum activity. Tigecycline 6 inhibits protein translation by connecting with 30S ribosome and hinders amino-acyl tRNA molecules coming to A site ribosomal subunit [13]. As of May 2006, the 6 has been approved in Europe and later a supplementary NDA for community-acquired pneumonia (CAP) was submitted to the FDA in October 2007. Ziconotide (Prialt®) 8, a synthetic-conotoxin and calcium channel blocker, isolated from Conus magus [14], causes pain relief by inhibiting pro-nociceptive neurochemical releases in the brain and spinal cord [15]. In December 2004, the FDA approved 8 when delivered as infusions into the cerebrospinal fluid using intrathecal pump system. In 2005, Elan launched 8 in US and Europe while rights for marketing 8 in Europe were obtained by Eisai in March 2006. CH3

CH3

OH

OH

H2C H3C O

H3C

CH3 HO

CH3

CH3

O

CH3

2

1 CH3

HO

H

H CH3

H

CH3

H3C

O

S N

OCH3

H N

O O

O

HO O

N H

NH2 S O

O

CO2H

3 HO H

4 H3C

CH3

H

CH3

H3C

N

NH2

CH3 N

H

H OH

H3C

O

S N

H N

H3C

O

N H

H3C

O

HO

CH3

5

O

HO OH

CONH2 O

6 H3C

HO

OH

CH3 N

CH3

H

OH

H2N-CKGKGAKCSRLMYDCCTGSCRSGKC-CONH2 CONH2

OH OH

O

OH

7

O

8

Natural products in drug discovery

7

Zotarolimus 9, a derivative of sirolimus 10, is an active principle of EndeavorTM stent that is being used as anti-proliferative agent by Medtronic [16,17]. In July 2005 EndeavorTM was approved by European comission for the sale while FDA approved it in February 2008 for the treatment of coronary artery disease. Anidulafungin 11 (EraxisTM in US, EcaltaTM in Europe), a semi-synthetic derivative of echinocandin B 12, was originally developed by Eli Lilly against invasive and oesophageal candidiasis and candidemia. Anidulafungin 11 was later licensed to Vicuron Pharmaceuticals, which was purchased by Pfizer in June 2005. Pfizer gained FDA approval for EraxisTM in the US (February 2006) and EcaltaTM in Europe (July 2007). Exenatide 13 (Byetta®), a 39 amino acid peptide isolated from the oral secretions of Heloderma suspectum (Gila monster) [18], mimics the antidiabetic or glucose-lowering properties of incretins. In April 2005, Eli Lilly obtained FDA approval for 13 while EMEA in November 2006 approved it to Amylin Pharmaceuticals for use in type 2 diabetes mellitus [19]. Amylin Pharmaceuticals, Eli Lilly and Alkermes submitted a NDA in May 2009 for subcutaneous dosing of 13 once weekly that was accepted in July 2009 by the FDA. Attention-Deficit Hyperactivity Disorder (ADHD), a neurodevelopmental disorder in which dopaminergic and noradrenergic neurotransmission are supposed to be dysregulated, is primarily characterized by the co-existence of attentional problems and hyperactivity. Despite abuse potentials methylphenidate and amphetamines were used for Attention-Deficit Hyperactivity Disorder (ADHD) management since many years. 14 Lisdexamfetamine (VyvanseTM, NRP104) consisting of dextroamphetamine coupled with L-lysine was designed by New River Pharmaceuticals produces effects similar to placebo on intravenous administration, however on oral administration it converts into D-amphetamine 15 in the gastrointestinal (GI) tract [20]. In February 2007, FDA approved 14 to treat ADHD. Pleuromutilin 16, a fungal metabolite inhibiting protein synthesis in bacteria [21], is the lead compound of retapamulin (SB-275833) 17 developed by GlaxoSmithKline. In June 2007, EMEA approved an ointment containing 1% retapamulin 17 called AltabaxTM in the US and AltargoTM in Europe for topical treatment of impetigo caused by Staphylococcus aureus or Streptococcus pyogenes. Temsirolimus (Torisel®, CCI-779) 18, a derivative of 10 and mTOR inhibitor [22] developed by Wyeth in various Phase III trials was approved in May 2007 by the FDA and November 2007 by the EMEA for the treatment of renal cell carcinoma (RCC) [23].

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Bhuwan B. Mishra & Vinod K. Tiwari

Trabectedin (Yondelis®, ecteinascidin-743, ET-743) 19, an alkaloid obtained from Ecteinascidia turbinate [24], is a DNA minor groove binder that inhibits cell proliferation by disrupting the cell cycle. Trabectedin 19 is sold by Zeltia and J&J against advanced soft tissue sarcoma (STS). In September 2007, the EMEA has approved 19 for use against ovarian cancer and STS. In November 2009, Yondelis® received its second marketing authorisation from the European Commission against relapsed platinumsensitive ovarian cancer when administered in combination with DOXIL®/ Caelyx®. R O CH3

CH3 O

OH

O CH3

O CH3

O N

CH3

O CH3 CH3

O

N

CH3

9R=

O

N N

N

O HO

10 R =

O H3C

OH

CH3

HO

O

OH

O HO

11 R =

R

NH NH

H3C

O

N HN

O

H3C

O

CH3 H3C

N

H N

HO

O

O

NH

HO

OH

O OH O

OH

12 R = H3C

HO

Natural products in drug discovery

9

13 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS NH2 H N

NH2 NH2

CH3

CH3

O

15

14 O

OH

CH2

CH3

O

CH3 OH

OH O

CH3

O R

CH3

H O CH3 O

H3C H3C O

CH3 CH3

O N

S

O

O

16 R = OH 17 R =

OH

CH3

O CH3

CH3

O CH3

NCH3 O O O CH3 H3C

HO H3C

HO

OCH3

O

CH3

O

18

H

H3C S

N

CH3

O

N

CH3

S

O O

OH O

O

H3CO

NH

H3C

OH H3C CH3 H3C

N X

CH3 O

HO

19

CH3

OH

O

20 X = NH 21 X = O

Ixabepilone (IxempraTM, BMS-247550) 20, a semi-synthetic derivative of epothilone B 21 produced by Sorangium cellulosum [25], was developed by Bristol-Myers Squibb (BMS) as an anticancer drug that binds to β-tubulin and suppresses the dynamics of microtubule. In October 2007, BMS gained FDA approval for 20 as a monotherapy and in combination with Xeloda® for the treatment of breast cancer, resisting standard therapy [26]. Methylnaltrexone (MOA-728, Relistor® by Wyeth) 22, a derivative of naltrexone 23 that blocks peripheral opioid receptors activated by opioids and

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Bhuwan B. Mishra & Vinod K. Tiwari

thus is significant in management of alcohol and opioid dependence [27]. Wyeth and Progenics in May 2007 filed an NDA for subcutaneous doses of 22 against opioid induced constipation (OIC) and other pain indications that was approved in April 2008 by Health Canada and the FDA. As of May 2009 an oral formulation of 22 is under Phase II trials against OIC in chronic pain. HO

O

CH3 O

H3C

O N HO

O

H

O CH3

O

HO

O

OH

HO CH3

O

22

N

CH3

O CH3

CH3

O CH3

O

O HO

O

N Br CH3

H

O

HO O H3C

CH3

24

23

Everolimus (LuveniqTM or LX211) 24, an mTOR inhibiting derivative of 10 is marketed as immunosuppressant by Novartis under ZortressTM (USA) and CerticanTM (Europe and other countries) in transplantation medicine, and Afinitor® for use in advanced renal cell carcinoma (RCC). CerticanTM was approved in 2004 as immunosuppressant while in March 2009 the FDA has approved 24 against advanced RCC after failure of Sutent® (sunitinib) or Nexavar® (sorafenib). OH R1 HN H3C

O CH3

OH O

OH

O

O

OH

Cl

O

O OH

HO Cl O

O

O

O

H N

H N

H N N H

N H

N H

HN

O

CH3 CH3

O O

HO

CH3

NH2 O OH

H N

OH

CH3

R1 = HO

R2

25 R2 =

N H

26 R1 = H, R2 = H

PO3H2

Natural products in drug discovery

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Telavancin (VibativTM, TD-6424) 25, a semisynthetic derivative of vancomycin 26 that inhibits bacterial growth through binding to D-Ala-D-Ala [28], was developed by Theravance in partnership with Astellas for use against Gram-positive cSSSIs and MRSA that was approved in September 2009 by the FDA. Theravance has also submitted telavancin 25 to the FDA in a second indication against nosocomial pneumonia or hospital aquired pneumonia (HAP). In November 2009, the FDA released a complete response letter to Theravance for telavancin 25 NDA against nosocomial pneumonia. Romidepsin (depsipeptide, FK228, FR901228, Istodax®) 27 extracted from the bacteria Chromobacterium violaceum, is a histone deacetylase (HDAC) inhibitor [29] developed by Gloucester Pharmaceuticals under National Cancer Institute (NCI) sponsorship for treatment of cutaneous and peripheral T-cell lymphoma (TCL). In November 2009, the FDA approved 27 to use in the treatment of selective cutaneous TCL patients previously treted with minimum of one prior systemic therapy. In January 2010, Celgene completed the acquisition of Gloucester Pharmaceuticals. Capsaicin (Qutenza®) 28, isolated from chili peppers of genus Capsicum [30], produces burning sensation on contact to tissues though binding to vanilloid receptor subtype 1 (VR 1) [31]. In November 2009, the FDA approved Qutenza® (a transdermal 8% patch of 28) to use in treatment of neuropathic pain combined with postherpetic neuralgia. In April 2010, NeurogesX launched Qutenza® in US. Aztreonam lysine (CaystonTM) 29 is an inhaled lysine salt formulation [32] that was evaluated by Gilead in various Phase III trials against cystic fibrosis (CF) patients infected with the Gramnegative bacteria Pseudomonas aeruginosa. In February 2010, the FDA approved 29 against CF patients. O H3CO

O

CH3

H N

H3C

NH

CH3

S O

S O O

H3C

CH3 CO2H O

N

NH

H N

N O

CH3

H2N H3C

27

CH3

28

HO

O HN

CH3

N H

CH3

S

O

N

O

29

O S

O

OH

12

Bhuwan B. Mishra & Vinod K. Tiwari

4. Infectious diseases 4.1. Antibacterial NP-derived drugs have played their crucial role in anti-infective drug discovery and the majorities of antibacterial drugs currently in clinical use are NPs or were designed using NP templates. Despite having complex structure the development of a NP to an antibacterial drug entirely depends on its ability to penetrate bacterial cell membranes. The success of penicillin encouraged the discovery of other compounds from natural sources against bacterial infections and as a result nearly all novel classes of antibiotics belong to NP sourced scaffolds [33]. Ceftobiprole medocaril (BAL-5788) 30, a cephalosporin antibiotic with excellent activity against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, Pseudomonas aeruginosa, and Enterococci [34], was filed for regulatory approval in the US and Europe in July 2007 by Basilea Pharmaceutica and J&J affiliated Cilag GmbH International to use in the treatment of cSSSIs. In November 2008, the approval of 30 was declined by the FDA with recommendation of two new studies to access safety and efficacy in treatment of cSSSIs. Additionly, various Phase III trials are underway for HAP/CAP. Ceftaroline acetate (PPI0903, TAK-599) 31, discovered by Takeda and licensed to Cerexa, shows efficacy against the penicillin-resistant S. pneumoniae and is under Phase II development by Forest Laboratories to use in the treatment of cSSSIs and CAP [35]. Tebipenem pivoxil (ME-1211, L-084) 32, an oral carbapenem antibiotic is under Phase III clinical development by Meiji Seika in Japan for treatment of otolaryngological/respiratory infections. Tomopenem (CS-023, RO4908463, R1558) 33 [36], by Daiichi Sankyo for treatment of common nosocomial infections and PZ601 (SM-216601, Protez) 34 [37], against MRSA and Pseudomonas aeruginosa, are currently in Phase II trials. ME1036 (CP5609) 35, a DHP-1-stable parenteral carbapenem having excellent in vitro activity against multidrug-resistant (MDR) staphylococci and Enterococcus faecalis was licensed by Cerexa and Forest Laboratories from Meiji Seika Kaisha. ME1036 35 is currently under Phase I evaluation. Likewise, sulopenem (CP-70429) 36, is being evaluated by Pfizer in various Phase I trials [38]. Faropenem daloxate (SUN-208, BAY-56-6824) 37 is a penem-type β-lactam licensed to Replidyne by Daiichi Suntory Pharma for marketing in conjunction with Forest Pharmaceuticals [39]. In December 2005, Replidyne submitted an NDA to the FDA for use of 37 in the treatment of bacterial

Natural products in drug discovery

13

sinusitis (BS), chronic bronchitis (CB), CAP and uncomplicated (SSSIs). In response to Replidyne’s NDA, the FDA in March 2007 agreed for Phase III placebo-controlled trials of 37, one each in BS and CB along with two non-inferiority CAP trials. However, these additional trials have certainly delayed the launch of drug. OH

N H2N

H

H N

N S N

O

O

S

N

N

N O HO

CH3

H3C

O

O

O

S

N O O

O

O

O

OH H H

CH3

H3C H3C CH3

30

N

O

O

O

S N

32

CH3 CH3 N OH HO

P

S

O

N

OH H H

O H

H N

N

H N

N

O

OH H H

H3C

S

S

S

N

N

H N

N H

N H CH3

S

O O HN

36 S

O

Cl H N

O

H N

NH

O

HO

O OH OH

O

HO

OH OH

O H N

O

38 R = 39 R = OH

O

O

Cl HO

OH O

O

37

N H

O

O

H

N O

N H

O

R

HH

H3C

OH CO2H

O

O

HO

O

CH3

O

OH

H3C

S

CH3

N

O

NH2

35

O

O

O

HO

O

H3C

OH

N

O

O

HO

N

S

NH2 O NH

N CH3

HH

O N

33

OH

CH3

H3C

O

O

H

NH

34 OH H H

O

S

O

S

N

O

HO

CH3

H3C

CH3

N

O

N HO

S

N

N

O

31

CH3

CH3 N

CH3

N H

CH3

14

Bhuwan B. Mishra & Vinod K. Tiwari

Dalbavancin (Zeven®, BI-397) 38, a semi-synthetic derivative of the teicoplanin analogue A40926 39, was discovered by Biosearch Italia and being developed by Pfizer for the treatment of cSSSIs [40]. In February 2005, Vicuron Pharamaceutical (now a part of Pfizer) filed an NDA for 38 to use in the treatment of patients suffering from cSSSIs. In response, the FDA released an approval letter in December 2007, however, as of September 2008 Pfizer have withdrawn all the marketing applications of 38 for running another Phase III trial. Oritavancin (NuvocidTM, LY-333328) 40, a chloroeremomycin 41 derivative inhibiting cell-wall synthesis, was discovered and developed by Eli Lilly and acquired by InterMune in 2001 and later tansferred to Targanta Therapeutics in 2005. In February 2008, Targanta submitted an NDA for 40 to the FDA that was not approved due to insufficient data. Aditionally, a MAA was submitted by Targanta for 40 to EMEA that was accepted for review in June 2008. TD-1792 42, a vancomycin-cephalosporin heterodimer successfully evaluated by Theravance in Phase II trials against cSSSIs including MRSA, has been designed to target 2 key targets in bacterial cell wall synthesis. In July 2007, Theravance disclosed to meet primary and secondary endpoints of non-inferiority trial compared to vancomycin 26. Ramoplanin factor A2 (known as ‘‘ramoplanin’’) 43, the major component of the lipopeptide antibiotic drugs obtained from Actinoplanes ATCC 33076 [41], inhibits cell wall synthesis in bacteria by forming U-shaped structures that are able to bind and capture Lipid II (C35-MurNAcpeptide-GlcNAc), a specific intermediate in membrane formation [42]. Oscient Pharmaceuticals hold the North American right and are evaluating orally active doses of 43 in Phase II trials against Clostridium difficile associated GI tract infections [43]. NXL-103 (XRP2868), an orally available mixture (70:30) of flopristin (RPR132552A, streptogramin A-type) 44 and linopristin (RPR202698, streptogramin B-type) 45 that inhibit bacterial protein synthesis through the synergistic binding to different sites on the peptidyltransferase domain of the 50S ribosomal subunit [44], was discovered by Sanofi-Aventis [45]. Novexel in October 2008 announced for positive Phase II trials of NXL-103 against CAP and cSSSIs including MRSA. Friulimicin B 46, a lipopeptide antibiotic produced by Actinoplanes friuliensis HAG 010964 [46], exerts activity through complex formation with bactoprenol-phosphate, resulting in inhibition of peptidoglycan and teichoic acid biosynthesis in bacteria [47], is under Phase I clinical development (July 2007) by MerLion Pharmaceuticals. Structure of friulimicin B 46 was confirmed after the crystal structure of amphomycin tsushimycin (A-1437 B) 47, an aspartic acid analogue of 46 was published in late 2005.

Natural products in drug discovery

15 R HN HO H3C

O CH3

H2N HO H3C

OH O

O

O

O CH3

Cl

O

O

Cl H N

O

OH OH

OH

O O

O

N H

HN

O H N

N H

O

HO

H N

N H

O

O

CH3

NH2

O

CH3 CH3

OH OH

HO

40 R =

Cl

41 R = H OH H3C

NH2 O CH3 O

OH OH

O

O

OH

Cl

O

O OH

HO

Cl

O O

H N

N H

HN

O

O

H N

N H

O

H N

N H

O

O

CH3

NH2 NH

N

42

H N

N H2N S

OH OH

HO

O

H

S

N

O

Cl

CH3 CH3

O

N

O CO2H

OH

O

OH H2N

OH

H3C

O H N

H3C

O

HN

OH

H N

N H

O

O H N

N H O

O

O

O

N H NH

N H O

NH2 O O

H2N

O

OH

O

H3C O

HN H N

OH

O H N

N H

O

O H N

N H

O

O

NH2

NH

N H

CH3

OH OH

O O O OH

OH

OH

43

CH3

OH

CH3 CH3

O

HO HO HO

O

H3C

Cl

16

Bhuwan B. Mishra & Vinod K. Tiwari CH3

H3C N O

N

N O F

CH3

O

CH3

N

N

N O

O O

O

CH3

H N

HO

N

H3C

CH3

O

O

H N

N H N

NH

OH

N H

N

45 COOH

O

O

CH3 O O

O CH3

44

O

HN

O

HN

O

R

O

O

H3C

O H3C

NH H3C N O

O

O

N H COOH

HN

HOOC CH3 O N H H3C

H N

HN

O O

O NH

NH2

46 R = NH2 47 R = OH H3C

CH3

Moli1901 (duramycin, 2262U90) 48, obtained from Streptomyces cinnamoneum [48], inhances the chloride transport and increases fluid secretion in vitro, thus finds significance for the treatment of CF [49]. Moli1901 48 is currently under clinical development by AOP Orphan in colaboration with Lantibio in Europe. In March 2007, Lantibio announced the positive results of Phase II trial of aerosolized 48 in adolescents and adults suffering from CF. An ophthalmic solution of 48 for treatment of dry eye syndrome is also under Phase II trials by Lantibio. Omiganan 49, originally purified from neutrophils of bovine, is an indolicidin 50 derivative that can interact with the bacterial cytoplasmic membrane and has been found significant against antibiotic-resistant and sensitive bacterial infections [50]. Omiganan 49 was developed by MIGENIX and later licensed to Cadence Pharmaceuticals and Cutanea Life Sciences for catheter-related infections (coded OmigardTM, CPI-226, MBI-226) and dermatological diseases (coded as CLS001, MX-594AN), respectively. Cadence Pharmaceuticals are currently evaluating a gel-based formulation of 49 in Phase III trials while another phase III trials for treatment of rosacea, a chronic inflammatory skin disorder are underway.

Natural products in drug discovery

17 NH

H-Ala-Lys-Gln-Ala-Ala-Ala-Phe-Gly-Pro-Phe-Abu-Phe-Val-Ala-HOAsp-Gly-Asn-Abu-LysOH S S S

48 ILRWPWWPWRRK-NH2

49

ILPWKWPWWPWRR-NH2

50

Erythromycin 51, macrolide antibiotic produced by actinomycetes, exerts antibacterial activity through inhibition of protein synthesis by binding to peptidyltransferase site of 50S subunit [51]. Among other derivatives, cethromycin 52, EP-420 53 and BAL-19403 54 are currently in clinical development. Cethromycin (ABT-773) 52 was discovered by Abbott Laboratories and later acquired by Advanced Life Sciences to use in the treatment of CAP and anthrax [52]. Advanced Life Sciences in October 2008 submitted a NDA to use 52 in the treatment of mild-to-moderate CAP which was accepted by FDA in December 2008. The cethromycin 52 (RestanzaTM) has demostrated clinically and statistically significant survival rate in placebo-controlled non-human primate studies with anthrax, plague and tularemia. In September 2009, the FDA has given orphan drug designation to 52 for the treatment of plague and tularemia. Likewise, EP-420 (EP-013420) 53, a bridged bicyclic derivative of 51 is currently under Phase II clinical development by Enanta and Shionogi for treatment of CAP [53]. BAL19403 54, a macrolide antibiotic significant against clinical isolates of Propionibacterium acnes with mutations in the 2057 to 2059 region of 23S rRNA conferring resistance to 51, is under clinical development by Basilea for the treatment of acne [54]. Telithromycin (Ketek®) 55 is the first approved ketolide developed by Sanofi-Aventis that received approval from the European Commission (July 2001) and the FDA (in 2004) for treatment of respiratory infections. Telithromycin 55 displays bactericidal activity by blocking the progression of the growing polypeptide chain through binding with the 50S subunit of ribosome. Tiacumicin B (OPT-80, PAR-101) 56, a macrolactone isolated by Abbott [55] from actinomyces, inhibits RNA synthesis and is under phase III clinical development by Optimer Pharmaceuticals for the treatment of Clostridium difficile-associated diarrhea (CDAD) [56]. PTK-0796 (MK-2764) 57 is an aminomethylcycline inhibiting protein synthesis in bacteria, was discovered and evaluated by Paratek in Phase II trials for the treatment of common hospital infections. PTK-0796 57 was in-licensed

18

Bhuwan B. Mishra & Vinod K. Tiwari O

N

H3C

CH3

H3C H3C

OH OH

HO

CH3 HO O

H3C O O

O H3C N O

CH3

O

O

H3C N

CH3 HO O

CH3 H3C O

H3C

CH3 OH

CH3

CH3 O

CH3

OCH3

O

H3C H N

O

O

O

CH3 CH3

O

CH3

CH3

51

52 N

N

N

N

N

HO

CH3

O

H3C

H3C H3C

H3C

CH3 HO O

O

CH3 OCH3 H3C N CH3 CH3 HO CH3 O O

O

O

H3C O

O

H C S 3

CH3 N

O

N

N

O

H

O N

O

CH3 H3C O

H3C

CH3 CH3

O

OCH3

O CH3

O

O

CH3

54

53 H3C

N

H3C

HO O

O H3C

CH3

N H CH3 H3C O

O H3C O

O O

OCH3

O

H3C H3C

CH3 OHO

O

N CH3 CH3

OH

H3C OH O

N N

CH3 OH CH3

H3C

H

CH3 H3C

CH3 OCH3 O

OH O

O O

OH Cl

O H3C

OH

CH3

O

OH CH3 Cl

CH3

55

56

by Novartis form Paratek for collaborative Phase III clinical development. In October 2009, Novartis gained exclusive marketing rights of 57 to use in the treatment of MRSA, MDR Streptococcus pneumoniae and vancomycinresistant enterococci. Eritoran (E5564) 58, a second-generation lipid A antagonist [57] designed by Eisai from Rs-DPLA 59 isolated from Rhodopseudomonas sphaeroides [58], inhibits endotoxin response through antagonism of the Toll-like receptor 4 (TLR4) [59,60].

Natural products in drug discovery

19

CBR-2092 60, a hybrid antibiotic inhibiting RNA and DNA synthesis is being developed by Cumbre Pharmaceuticals for treatment of gram-positive cocci infections. CBR-2092 60 is supposed to exert antimicrobial activity through combined effects on RNA polymerase, DNA topoisomerase IV and DNA gyrase. Currently, CBR-2092 60 is in Phase IIa trial by Cumbre for treatment of infections caused by gram-positive cocci [61]. H3C CH3

H3C

N

CH3

H3C H

H

N

CH3 OH

H N

H3C

OH

CONH2

OH OH O

O

57 OCH3 H2O3PO

OH

O O

HO HN

O

O H2O3PO

O O

O

HN OPO3H2 O O

HO

O

O

H3CO

CH3

O HN

HO

O

O

O

HO O

CH3

CH3

O O HN OPO3H2 O O

CH3 H3C H3C

H3C CH3

CH3

58 H3C

59 O

CH3 CH3 CH3

O H3CO

OH CH3 OH

OH O OH

H3C

CH3 NH

O

O OH

O CH3

O

N

F

N N CH3

N

CO2H

N CH3

60

4.2. Antifungal Invasive fungal infections – infections of the bloodstream and organs within the body (e.g. meningitis, pneumonia, peritonitis) – are important causes of morbidity and mortality in liver, pancreas, heart, kidney and lung (i.e. solid organ) transplant recipients [62]. Fungi are eukaryotes and, despite

20

Bhuwan B. Mishra & Vinod K. Tiwari

the presence of a cell wall, fungi are more similar to mammalian cells on a cellular level than to bacteria, making the treatment of mycotic infections difficult [63]. Only 2 NP-derived compounds, aminocandin 61 and SPK-843 62 are undergoing clinical evaluation. Due to lack of biological target, 1,3-βD-glucan synthesis in human, echinocandin derivatives have been considered significant against refractory aspergillosis and invasive infections by Candida species [64]. Among other semi-synthetic echinocandins, caspofungin (launch 2001, Cancidas®, Merck), micafungin (launch 2002, Mycamine®/Funguard®, Astellas) and anidulafungin 11 (launch 2006, EraxisTM/EcaltaTM, Pfizer) have been approved. Deoxymulundocandin 63, isolated from Aspergillus sydowii [65], is the lead compound of aminocandin (NXL-201, IP960, HMR-3270) 61 and exhibit excellent activity against Candida albicans and C. tropicalis by destabilizing the fungal cell membrane. SPK-843 62, a semi-synthetic derivative of patrician-A 64, is under clinical development by Dutch company APARTS BV [66] that has acquired world wide rights for the development of 62.

4.3. Antiparasitic The use of medicinal plants against parasitic diseases has been traced to ancient times i.e. bark of Cinchona calisaya and Strychnos pseudoquina, root and leaves of Deianira erubescens, bark of Remijia ferruginea [67]. Artemisinin (Artemotil®) 65, obtained from traditional Chinese medicine Artemisia annua, was approved in the year 2000 for the treatment of chloroquine-resistant Plasmodium falciparum malaria and cerebral malaria. The World Health Organization has strongly discouraged the use of 65 as a monotherapy since malarial parasites are developing resistance to the drug. However, combination therapies that include 65 are the preferred treatment for malaria and are both effective and well tolerated in patients. Artemotil® is currently used only as a second line drug in severe cases of malaria and is also increasingly being used against vivax malaria. As of May 2009, arterolane (RBx11160, OZ-277) 66, a trioxolane modelled on artemisinin 65 pharmacophore, is under Phase III clinical development for the treatment of malaria by Ranbaxy in combination with piperaquine [68]. Paromomycin 67 (HumatinTM, King Pharmaceuticals), an orphan drug extracted from Streptomyces krestomuceticus [69], was approved in September 2006 by Drug-Controller General of India for the treatment of patients suffering from visceral leishmaniasis (VL). Paromomycin 67 was developed by the Institute for OneWorld Health [70] and is an off-patent antibiotic marketed in the US to treat intestinal parasites.

Natural products in drug discovery

H3C

O

HO

NH2

H N HO

21 OH O

O HO

O

NH

NH N H

N

NH H3C

HN

O

O

N

O OH

HN

O

OH

O HO O

CH3

O

NH

HO

N

H N

O

NH O

CH3 N

H N

OH

H3C OH

O

OH

H3C

O

OH H3C HO

HO

61

H3C

63

H N OH

OH HO O

O

H3C

O

OH

OH

OH

OH

OH

O

R1

O

O

O

H N

N

62 R1 =

CH3 R2 =

NH

OH

CH3 N

CH3 OH

O

O

H3C

R2

CH3

CH3 OH

64 R1 = OH, R2 = H H H3C

O O

O O

H

O

O

HO HO

CH3

O

NH O

NH2 O

HO

CH3 CH3 NH2

O H2N HO

NH2

O OH

O OH

CH3 O

H2N

O

NH2 OH

65

66

67

4.4. Antiviral Virus is a small infectious agent that can replicate only inside the living cells of organisms bringing most common (i.e. cold, influenza, chickenpox and cold sores) to greatest human health risk (i.e. ebola, AIDS, avian influenza and SARS). Researches over last 25 years have resulted in the identification of many natural product templates significant to antiviral drug discovery, however fewer are in clinical investigation.

22

Bhuwan B. Mishra & Vinod K. Tiwari

Betulinic acid 68, a topoisomerase I inhibitor isolated from bark of Betula pubescens [71], is currently in Phase I clinical development. Bevirimat (PA-457) 69, obtained from Syzygium claviflorum, was evaluated by Panacos in Phase IIb trial for development as combination therapy with other standard antiviral drugs. Bevirimat 69 inhibits the final step of the HIV Gag protein processing and thus blocks HIV maturation [72]. In January 2009, Myriad Genetics announced for the acquisition of all rights from Panacos for 69. Ribavarin 70, a NP-derived compound structurally similar to pyrazomycin and showdomycin, was marketed as ‘Rebetol’ until 2005 by Schering Plough with Valeant Pharmaceuticals in the US. Valeant Pharmaceuticals are developing taribavirin (Viramidine®, ribamidine) 71, a liver-targeting prodrug of ribavirin 70 [73], is in various Phase II/III trials for the treatment of chronic hepatitis C virus (HCV). In 2006, 71 failed to meet the non-inferiority efficacy endpoints in Phase III trials by Valeant. In 2007, Valeant initiated another Phase IIb trial for 71 with higher doses and reported the final results in June 2009 against HCV. MBI-3253 (celgosivir, 6-O-butanoylcastanospermine) 72, a glucosidase inhibitor and semi-synthetic derivative of indolizine alkaloid castanospermine 73 isolated from Castanospermum australe seeds [74], is an investigational antiviral drug under clinical development by MIGENIX. As of January 2009, MIGENIX has completed Phase II clinical studies of 72 as a ‘‘triple combination’’ (with peginterferon α-2b and ribavirin 70) and a ‘‘double combination’’ (with peginterferon α-2b) in HCV patients. After discontinuation of exclusive option agreement with United Therapeutics Corporation (UTC) in April 2009, MIGENIX are seeking other strategic options for further development of 72. Cyclosporin 74, a cyclophilin inhibitor obtained from Beauveria nivea, exerts significant antiviral activity. However, due to calcineurin-related and immunosuppressive side effects development of 74 as antiviral drug is not possible [75]. NIM 811 (SDZ NIM 811, cyclosporin 29, MeIle4-cyclosporin) 75, discovered by Sandoz (now Novartis) with 1700 times less immunosuppressive activity than cyclosporin 74 [76], was evaluated in Phase I trial for anti-HIV and HCV activity. Likewise, debio-025 (UNIL025, MeAla3EtVal4-cyclosporin) 76, a cyclophilin inhibitor with 7000 times less immunosuppressive activity than 74, is being evaluated by Debiopharm in various phase IIb trials for the treatment of HCV [77,78]. In February 2010, Novartis in-licensed the exclusive rights to develop and market 76, as potential first-in-class antiviral agent except in Japan. 4-Methylumbelliferone (Heparvit®) 77 is a naturally occurring coumarin that is in Phase II development by MTmedical Institute of Health and BioMonde for the treatment of HBV and HCV. 1,5-DCQA (1,5-di-O-

Natural products in drug discovery

23

caffeoylquinic acid) 78, a HIV-1 integrase inhibitor extracted from Inula Britannic, is under human clinical trials by Chinese Academy of Military Medical Sciences for treatment of HIV/AIDS and hepatitis B [79]. WAP-8294A2 (JA-002) 79, produced by the Gram-negative Lysobacter species exerts antibacterial activity by interacting selectivly to membrane phospholipids and causes sever damage to bacterial membrane [80]. The aRigen Pharmaceuticals are evaluating injectible, gel and cream of 79 in various Phase I/II trials to treat MRSA and acne. In August 2009, New Energy and Industrial Technology Development Organization (NEDO), Japan has decided for funding two-thirds R&D costs to aRigen Pharmaceuticals until February 2011 for development of 79 as first-line anti-MRSA product candidate. CH2 H3C

R H

N CO2H

CH3 CH3 H H RO H3C

O

CO2H

CH3 HO CH3 O

H3C

H3C

70 R = O

72 R =

71 R = NH

73 R = H

CH3

N

N

O H3C O N CH3

CH3

N

CH3

N

CH3 O H H N

CH3 O CH3 CH3 O H N N H CH3 O

76

CH3

74 R =

N R N CH3 O O CH3CH3 O CH3 H3C CH3 H3C O CH3 O CH3 H N N N N O N H H O H3C CH3 O CH3 H3C N

HO CH3 CH3 O

O CH3

O

CH3 O H H N

CH3

H3C

OH

CH3

CH3

O CH3

OH

H

OHOH

69 R =

N

N HO

H CH3

OR

N

N

CH3

68 R = H OH C CH 3 3

H3C

NH2

HO

CH3 CH3 CH3

75 R =

CH3

HO CH3 CH3

CH3 N

N

O CH3 CH3

O

HO

CH3

CH3CH3 O H3C CH3 CH3 N

O

77

N H

O

HO O HO

HO2C

O

O

HO O

78

OH OH

24

Bhuwan B. Mishra & Vinod K. Tiwari NH2

HO H N O H N HN

O O

NH2

H3C

O H N

NH2

N

H3C

N H

O

O

O

O

H N H

O O

N H H3C OH H3C

O

O CH3 HO

CH3

H N

CH3

N O

O

O N H O

CH3 O

NH OH

NH2

79

5. Neurological diseases Historically, the alkaloids like morphine 80 isolated from Papaver somniferum and physostigmine 81 extracted from Physostigma venenosum, were used to treat sever pain and diseases of central nervous system (CNS). (+)-huperzine A 82, a sesquiterpene alkaloid and acetylcholinesterase (AChE) inhibitor extracted from Huperzia serrata [81], is being evaluated by Chinese scientists against Alzheimer’s disease. The National Institute on Aging (NIA) is evaluating orally administered formulation of 82 in Phase II trials against Alzheimer's disease [82]. Morphine-6-glucuronide (M6G) 83, produced by metabolism of artemisone (BAY 44-9585) 84 (obtained through semi-synthesis from artemisinin 63) in human body, was evaluated successfully by CeNeS Pharmaceuticals as significant analgesic in Phase III trials in Europe. PAION in June 2008 acquired CeNeS Pharmaceuticals and later in November 2008 disclosed for completion of two Phase III trials. A spicamycin derivative KRN-5500 85, obtained from Streptomyces alanosinicus [83], was evaluated by DARA BioSciences in Phase I trials against neuropathic pain. DARA BioSciences are currently running Phase IIa trials of 85 given intravenously (IV) to cancer patients suffering from neuropathic pain [84]. Debio 9902 (ZT-1) 86, synthesized by Shanghai Institute of Material Medica, is a prodrug of 82 licensed to Debiopharm. Debiopharm in June 2007 announced the positive results of a Phase IIa trial of 86 against mild Alzheimer’s disease. As of October 2008, Debiopharm have started tablet

Natural products in drug discovery

25

formulation bridging study of 86 as Investigational New Drug (IND) to treat Alzheimer’s patients. Lobeline 87, a VMAT2 ligand [85] reducing the methamphetamine induced dopamine release, is a significant tobacco smoking cessation agent occurring in Hippobroma longiflora [86]. Lobeline 87 is being evaluated by Yaupon Therapeutics and NIH as a dopamine modulating agent under Phase II trials against ADHD and methamphetamine addiction. Anabaseine 88, isolated from marine worms of the phylum Rhynchocoela [87], stimulates the neuronal nicotinic receptors thus has been considered significant in the treatment of Alzheimer’s disease as Alzheimer’s brain loses many of its nicotinic receptors by the time of death [88]. The 3(2,4-dimethoxybenzylidene)-anabaseine (DMXBA; also called GTS-21) 89, a synthetic derivative of 88, was evaluated against Alzheimer’s disease in a sponsored research by Taiho Pharmaceutical to Kem’s University of Florida. HO

O H

N

H

CH3

H3C

H3C

H N

O N O

CH3

N H

RO

CH3

80 R = H

81 HO

83 R =

O

OH OH CO2H CH3

H3C

H N

H3C

H H

O

N O O

S

O

O O

O

H3C H2N

CH3

82

84

H3C

N H

85

OH

HO H N

O

O

N

O HO

N

N H OH

NH N

26

Bhuwan B. Mishra & Vinod K. Tiwari CH3 O

OCH3

OH

H N H3C

CH3

O N

OCH3

87

HO N H3CO

Cl

86

N

N

88

N

89

The University of Florida licensed 89 to Osprey Pharmaceuticals whose assets were purchased by CoMentis (previously Athenagen) in April 2006. CoMentis are currently assessing 89 in various Phase I/II trials for safety assessment and cognitive improvement in ADHD patients. Tetrodotoxin (TectinTM, Wex Pharmaceuticals) 90, extracted from the puffer fish [89], blocks the action potentials in nerves through binding to sodium channels in cell membrane [90]. Wex are evaluating 90 in colaboratin with Chinese medical institute against cancer pain and management of opiate withdrawal symptoms in Phase III and I trials, respectively. Also a Phase IIa trial of 90 against neuropathic pain caused by cancer chemotherapy is underway by Wex Pharmaceuticals. Capsaicin 28 and related compounds (called as capsaicinoids) are produced by chili peppers as irritants against certain herbivores and fungi. Among capsaicinoids, Xen-2174 91, obtained from venom of Conus marmoreus targeting norepinephrine transporter (NET) was discovered by researchers at the University of Queensland. Xenome are associated with Phase II development of 91 against acute post-operative and chronic pain in cancer patients resistant to morphine and hydromorphone. Anesiva are evaluating capsaicin 28 (coded 4975, ALGRX 4975, AdleaTM) in various clinical trials against pain indications such as severe post-surgical pain, posttraumatic neuropathic pain and musculoskeletal diseases [91]. Anesiva in December 2008, disclosed to meet primary end point in a phase III trial of 28 against acute pain following orthopedic surgery. Winston Laboratories are associated with Phase III trials of civamide (cis-capsaicin, zucapsaicin, WL-1001) to treat episodic cluster headache and knee osteoarthritis. Winston in October 2008 filed a NDS to Canada for Civanex® (civamide 0.075%) to use against osteoarthritis pain. In February 2009, an orphan drug designation to Civanex® was given by FDA with NON release to Winston Pharmaceuticals in October 2009.

Natural products in drug discovery

27

Phlorizin 92, a flavonoid that belongs to the group of dihydrochalcones obtained from bark of pear (Pyrus communis), apple, cherry and other fruit trees (family-Rosaceae), is a sodium glucose co-transporters (SGLTs) inhibitor that lowers glucose plasma level and improves insulin resistance [92] but has poor intestinal absorption and become inactive by lactasephlorizin hydrolase. Dapagliflozin (BMS-512148) 93, a 92 derivative that selectivly inhibits SGLT2, is under clinical development by Bristol-Myers Squibb (BMS) in collaboration with AstraZeneca for the treatment of 2 diabetes. In October 2009, the BMS announced the positive results of Phase III placebo controlled trial of 93. O HO H2N

O N HN H HO

OH

O

NGVCCGYKLCHOC OH

90

OH

91

Resveratrol 94, a triphenolic stilbene occurring in many plants is significant against clinical indications such as cancer, ischemic injuries and cardiovascular disease [93]. Resveratrol 94 is an agonist of Saccharomyces cerevisiae silent information regulator (Sir2) protein, a class III histone deactylase whose presence causes extention of lifespan in S. cerevisiae, Caenorhabditis elegans and Drosophila melanogaster [94]. Italian scientists in 2006 observed 56% increase in median life span of Nothobranchius furzeri [95], a fish when supplemeted with 94. SRT-501, a formulation of 94 by Sirtris Pharmaceuticals, acts by increasing mitochondrial activity and is under clinical investigations against diabetes and obesity. Sirtris has announced the positive results of Phase IIa trial in which oral doses of 1.25 or 2.5 grams of SRT501 was found safe at twice daily dosing for 28 days in type 2 diabetes. A similar Phase IIa cancer trial with SRT501 is under way. Cannabinoids are a group of secondary metabolites responsible for pharmacological properties of Cannabis sativa (cannabis plant) [96]. CP 7075 (IP 751, ajulemic acid, CT-3) 95, a synthetic cannabinoid, suppressing IL-1β and matrix metalloproteinases (MMPs) through a peroxisome proliferator-activated receptor (PPAR) γ-mediated mechanism [97], was investigated by Indevus Pharmaceuticals in pre-clinical studies. In October 2007, the drug was licensed by Cervelo Pharmaceuticals for Phase I trials in neuropathic pain.

28

Bhuwan B. Mishra & Vinod K. Tiwari HO HO

HO

OH

HO

O

HO

O

HO O

OH

OH

OH

Cl

O OH

O

CH3

93

92 CO2H

OH

OH HO

H3C OH

94

H3C

CH3 H3C CH3

95

6. Cardiovascular and metabolic diseases Natural products have played an important role in development of drugs against cardiovascular and metabolic diseases. Simvastatin (Zocor®, Merck), a lipid-lowering statin obtained from fermentation product of Aspergillus terreus, inhibits 5-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase. Orlistat (Xenical®), a lipstatin derivative isolated from Streptomyces toxytricini [98], inhibits pancreatic lipases and used for the treatment of obesity. Captopril, ramipril and quinapril are the examples of some antihypertensive angiotensin-converting enzyme (ACE) inhibitors derived from the snake venom. An endopeptidase (NEP) inhibitor, ilepatril (AVE-7688) 96, is being developed by Sanofi-Aventis in various Phase IIb/III trials to treat hypertension while phase II trial for diabetic nephropathy. (+)-1-Deoxygalactonojirimycin 97 and (+)-galactonojirimycin 98 obtained from Streptomyces species [99] display strong inhibitory activity toward several β-galactosidases. Miglustat (Zavesca®) was used earlier to treat Type 1 Gaucher disease (GD1) by Actelion. Migalastat (AmigalTM, AT1001, 1-deoxygalactonojirimycin, 1-deoxygalactostatin) 97, a semisynthetic derivative of 98, stabilizes protein structures and restores correct folding through binding with them. (+)-1-Deoxygalactonojirimycin 97 is an orphan designated drug by European Commission in May 2006 to use in treatment of Fabry disease. As of January 2010, the 97 is being evaluated under Phase III trials by Amicus Therapeutics in collaboration with Shire

Natural products in drug discovery

29

Pharmaceuticals against Fabry disease. Isofagomine (PliceraTM, AT2101) 99, an aza-sugar that mimics the carbocation transition state used by glycosidases [100], is under clinical development by Amicus Pharmaceuticals to treat Gaucher’s disease [101]. Amicus in October 2009 announced the positive results of a Phase II trial for two dose regimens consisting of 225 mg of 99 given three days on/four days off and seven days on/seven days off. Ruboxistaurin (LY333531) 100, inhibiting protein kinase C (PKC), is being developed by Eli Lilly to use in the treatment of microvascular complications in diabetes mellitus [102]. In February 2006, Lilly submitted a NDA for use of 100 in diabetic peripheral retinopathy. In August 2006, the FDA essued an “approvable” letter to Lilly while suggesting another Phase III trial for additional efficacy. SCH 530348 (TRA) 101, a PAR-1 antagonist [103] similar to himbacine 102 obtained from Galbulimima baccata, is in Phase III clinical trials by Schering-Plough for the treatment of cardiovascular diseases such as atherosclerosis, ischemia, myocardial infarction and stroke. H N CO2H

O NH

H3C

H N

HO

O

H N

R

HO

OH

HO

OH

OH

OH SAc

H3C

97 R = H 98 R = OH

96

H N

O

O

O

H

H

H 3C

N

O

H N

O

N

99

O O

H

H

CH3

H

H

H

H

O H3C

H3C

N CH3

O N

H3C CH3 F

100

101

102

N

30

Bhuwan B. Mishra & Vinod K. Tiwari

Genaera Corporation are associated with clinical development of trodusquemine (MSI-1436) 103 and squalamine 104 extracted from Squalus acanthias [104]. MSI-1436 103 is a protein tyrosine phosphatase 1B inhibitor [105] that is being evaluated by Genaera in a second Phase I trial using an ascending single dose in overweight type 2 diabetics under the obesity IND. Ouabain (g-strophanthin) 105, a cardiac glycoside occurring in ripe seeds of Strophanthus gratus and bark of Acokanthera ouabaio, involves binding to and inhibition of the plasma membrane Na+/K+-ATPase attainable in vitro or with intravenous dosage [106]. Likewise, digoxin 106, isolated from Digitalis lanata (foxglove plant) [107], also exists in the human adrenal gland and is significant in atrial fibrillation and atrial flutter. Rostafuroxin (PST 2238) 107, an ouabain antagonist, is under Phase II development by Sigma-Tau to use in the treatment of chronic arterial hypertension. H3C

OSO3H

CH3 H CH3

H

H

H N R

N H

N H

CH3

H3C H OH

H

103 R = N H

104 R = H

NH2 O

O

HO

CH3

HO OH

O

H CH3

HO

H

H3C

OH

OH

CH3

105

O

O

OH O HO

O O

H

H

106

O

CH3

OH CH3

H3C

H3C H3C OH O

O

CH3 HO O

HO

CH3 O

N O

OCH3

O CH3

H

CH3

H3C

O

H3C O

H OH

OH

O

O

OH

OH

CH3

O

O

CH3 OH

CH3

NH2

CH3 HO

OH H3C

N

CH3

107

H

H

H

H3C

H3C

H3C

OH

O

OH

H

108

109

Natural products in drug discovery

31

Mitemcinal (GM-611) 108, an agonist of motilin that lacks the antibiotic properties of 51 and increases the amplitude & frequency of antral contractions and initiates gastric contractions, was discovered by Chugai Pharma. Phase I trials of 108 in Japan has been completed by Chugai while Phase II trials in US are still running against diabetic reflux oesophagitis and idiopathic gastroparesis [108]. Chugai are also conducting Phase II trials of 108 against irritable bowel syndrome (IBS). Pyridoxamine (PyridorinTM) 109, consisting of a pyridine ring bearing hydroxyl, methyl, aminomethyl, and hydroxymethyl substituents, is a vitamin B6 analogue [109] that was evaluated by BioStratum in two phase II trials demonstrating retardation of diabetic nephropathy. In October 2006, BioStratum licensed 109 to NephroGenex, which has initiated a new Phase IIb trial in patients with type 2 diabetes. Taisho Pharmaceutical is evaluating 109 (coded as K-163) in Phase II trials against diabetic nephropathy. In January 2009, the FDA ruled for regulation of 109 as a pharmaceutical drug and awarded a fast track drug designation.

7. Immunological, inflammatory and related diseases Autoimmune and inflammatory disease condition arises through aberrant reactions of the human adaptive or innate immune systems. Aspirin, discovered in the late 1890s, is still a significant analgesic and antiinflammatory drug. Salbutamol, a β2-adrenergic receptor agonist, is marketed by GlaxoSmithKlinen to treat asthma and chronic obstructive pulmonary disease. Cyclosporin 74 (1983), tacrolimus (1993), sirolimus 10 (1999), mycophenolate sodium (2003) and mycophenolate mofetil (1995) are among some important immunosuppressive drugs sourced from natural products. Everolimus (LuveniqTM or LX211) 110, a derivative of 10 inhibiting mTOR, is marketed by Novartis as immunosuppressant Certican® in organ transplantation. Voclosporin (ISA-247, R1524, LX211) 111, a derivative of 74 inhibiting calcineurin [110], is under Phase IIb trial to prevent kidney graft rejection and Phase III trial against psoriasis. Voclosporin 111 was licensed by Lux Biosciences from Isotechnika for ophthalmic indications. As of March 2009, Lux Biosciences have completed Phase III trials of 111 oral capsules against uveitis. In February 2010, Lux Biosciences filed a NDA to the FDA and MAA to the EMA for 111 under LuveniqTM against non-infectious uveitis, which were accepted by respective agencies in March 2010. Eupatilin 112, a flavone isolated from Korean traditional medicine Artemisia argyi possess efficacy against chronic diarrhea [111]. DA-6034 113, a synthetic 112 derivative, is being developed by Dong-A Pharmaceuticals in Phase I and II trials against dry eye and gastritis, respectively.

32

Bhuwan B. Mishra & Vinod K. Tiwari

H3C

O CH3 O

O

OH

O CH3

O HO

CH3

O N

CH3

O CH3

CH3

O CH3 O

O HO O H3C

CH3

110 H3C

CH2 CH3

HO

CH3

O

CH3 H H N

N

H3C

N O

CH3

O

CH3 CH3

CH3 O

CH3

O H N

CH3

CH3

H3C CH3

N H CH3

O

N

N CH3

CH3

N

CH3 H3C

CH3 N

N

O

H3C

O

N H

O

O

O CH3 CH3

111

OCH3 HO

OCH3

O O

O OCH3

O

HO

OCH3

H3CO OH

O

112

OCH3 O

113

8. Oncological diseases 8.1. Small-molecule anticancer agents 8.1.1. Plant-derived compounds Camptothecin 114, a topoisomerase I inhibitor isolated from Camptotheca acuminata [112], exhibits significant anticancer activity. Among other camptothecin class of drugs, belotecan (Camptobell, CDK-602) was developed and launched in 2004 by Chung Kun Dong in Korea [113]. BNP-1350 (Karenitecin®) 115 is an investigational drug under clinical

Natural products in drug discovery

33

development by BioNumerik Pharmaceuticals for cancer chemotherapy. As of February 2008, BioNumerik are running the Phase III trial of 115 against ovarian cancer [114]. Diflomotecan (BN80915) 116, a 115 analogue [115], is being developed by Ipsen under Phase II trials to treat advance metastatic cancers. Gimatecan (ST-1481) 117, an oral topoisomerase I inhibitor, is currently in Phase II development by Novartis against solid tumors [116]. Elomotecan (BN-80927, LBQ707, R-1559) 118, inhibiting topoisomerase I and II, is a promising Phase I pipeline by Ipsen in oncology (e.g. colon, breast and prostate cancer) [117]. DRF 1042 105 is a 114 derivative evaluated by Dr. Reddy’s Laboratories in Phase I trials to use in the treatment of various cancers [118]. Dr. Reddy’s Laboratories in September 2006 collaborated with ClinTec International for joint Phase II/III development of 119. In September 2006, Sonus Pharmaceuticals initiated a Phase I study of SN2310 120, a prodrug of SN-38 121 to address cancer is presently ongoing. In May 2008, Sonus murged with OncoGenex Technologies and the new company, OncoGenex Pharmaceuticals has included 120 as a strong oncology pipeline. R F

O

O N

N F

N

114 R = H

N O

O

H3C HO

H3C

Si(CH3)3

117 R =

N

O

HO

O

115 R =

116

C(CH3)3

O

H3C OH N

O O

H3C

N

O N

Cl

N

N O H3C

CH3

CH3

CH3

HO

O

HO

118

O

H3C

O

119 CH3 H3C

CH3 O

O

H3C

O CH3

O

CH3

O

O N

120

N O H3C HO

O

34

Bhuwan B. Mishra & Vinod K. Tiwari

Combretastatin A-4 phosphate (ZybrestatTM, CA4P) 121, a prodrug of combretastatin A-4 122 obtained from South African Bush Willow Combretum caffrum [119], is a reversible tubulin depolymerizing agent that causes tumour-associated endothelial cells to change from a flat to a round shape, thus by plugging the blood vessels deprives the tumour from oxygen and nutrients. Oxigene are evaluating 121 as a vascular disrupting agent (VDA) [120] and as on September 2008, Phase III trial against anaplastic thyroid cancer (ATC) is underway. Oxigene in November 2009 disclosed the positive results of a Phase II trial of 121 in non-small cell lung cancer (NSCLC). Ombrabulin (AVE8062) 123, another 122 derivative licensed to Sanofi-Aventis from Ajinomoto, is under Phase III trials in advanced STS patients. Combretastatin A-1 diphosphate (OXi4503) 124, a pro-drug of combretastatin A-1 125 that is capable of binding to proteins and nucleic acids [121], is under various Phase I trials by OXGENE to use in the treatment of advanced-stage solid tumors. Noscapine (CB3304, noscapine) 126, a benzylisoquinoline alkaloid occurring in the plants of family Papaveraceae, is a microtubule targeting antitussive currently in Phase I/II trials by Cougar Biotechnology for the treatment of multiple myeloma [122]. Vinblastine (Alkaban-AQ®, Velban®) 127, a microtubule inhibitor isolated from Catharanthus roseus [123], has been found significant when given intravenously to patients suffering from Hodgkin's disease, nonHodgkin's lymphoma, Kaposi's sarcoma, choriocarcinoma, TCL, breast, testicular, lung, neck and head cancers. Vinflunine (Javlor®) 128, a fluorinated vinca alkaloid [124] discovered by Laboratoires Pierre Fabre, was submitted for registration with the EMEA in June 2008, after positive Phase III trial for metastatic treatment of bladder cancer. In June 2009, Pierre Fabre received a positive opinion with recommendation for marketing authorization of 128 in the metastatic treatment of bladder cancer. Paclitaxel (TaxolTM, AbraxaneTM) 129, isolated from Taxus brevifolia [125], is a mitotic inhibitor that stabilizes microtubules and interferes with the normal breakdown of microtubules during cell division. Bristol-Myers Squibb (BMS) are associated with commercial development of 129. Cabazitaxel (XRP6258) 130 and larotaxel (XRP9881) 131 have been designed by Sanofi-Aventis as poor substrates for membrane-associated P-glycoprotein (P-gp), overexpressed in taxane resisting cells [126] and are in Phase III trials against pancreatic and hormone-refractory prostate cancers [127]. Luitpold Pharmaceuticals are developing DHA-paclitaxel (Taxoprexin®) 132, a fatty acid conjugate of 129, in Phase III trials against metastatic melanoma [128]. Spectrum are associated with Phase I/II development of intravenous/oral ortataxel (IDN-5109, BAY-59-8862) 133, a third generation taxane with toxicity/tolerance profile similar to 129. As of

Natural products in drug discovery

35

June 2009, the 133 is under Phase II trials in taxane-refractory solid tumors [129]. Milataxel (MAC-321, TL-00139) 134, a poor substrate for P-gp, is under Phase II clinical development by Wyeth Pharmaceuticals to use in the treatment of colorectal neoplasms [130]. Tesetaxel (DJ-927) 135, an orally administered semisynthetic taxane, was evlaulated by Genta in various Phase I/II trials against advanced gastric and breast cancer [131]. The Phase II clinical trials of 135 are running for the treatment of patients with advanced melanoma having a normal serum lactate dehydrogenase (LDH) and have progressed after one chemotherapy regimen. Other taxanes that are in Phase II clinical development, i.e. TPI-287 136 by Tapestry Pharmaceuticals and BMS-188797 137 by Bristol-Myers Squibb, to treat patients suffering from pancreatic and advanced malignancies, respectively are currently in Phase II clinical development [132]. R

OCH3 H3CO

NH2

H N O OCH3

OH

OCH3 H3CO

OCH3

OCH3 OCH3

121 R = OPO3Na2 122 R = OH

123

O

OR

H

OR

N

O OCH3 H3CO

CH3 O

H3C

O

OCH3

O

OCH3 H3C

124 R = PO3Na2 125 R = H

O

O

126

OH CH3

N

H

H

N OAc H CH3 CO2CH3

N OAc H CH3 CO2CH3

128 CH3 CH3

O H3C H3C

O

O

O CH3OH

O

CH3 NH

H3CO H3C

O

CH3

O HO

O CH3 CH3 O

CH3

CH3

OH

O

H

CH3

OH O O

129

CH3

OH

H3CO

127

O

N

N H H3CO2C

CH3

OH

H3CO

F CH3

N

N H H3CO2C

NH

F

H

N

O

CH3

O

O

HO

H

O O

O

O O

H3C

O H3C

130

36

Bhuwan B. Mishra & Vinod K. Tiwari

Acronycine 138, an alkaloid isolated from Acronychia baueri, exhibits activity against various solid tumors such as S-180 and AKR sarcomas, X-5563 myeloma, S-115 carcinoma and S-91 melanoma. S23906-1 139, a benzoacronycine derivative inhibiting DNA synthesis and S-phase cell cycle arrest, is currently in Phase I trials for the treatment of solid tumors [133].

CH3 CH3 O O

O

O

CH3 NH

H3C H3C

O

O

O

O

NH

CH3

O

CH3 HO

O

H3C

CH3

O

O

O O

O

O

H3C

132 CH3 CH3

O H3C

O

O O CH3 OH

H3C CH3 CH3

O

NH

O

O

O

O

H3C

CH3

OH

O

HO

O O

O OH H3C O CH3 O CH3

O O

O

CH3

H

O

133

HO

H3C

O OH

H

6

NH

CH3

O

O

131

O

O CH3 OH

CH3

O O

H3C

CH3

O

O

H

O

CH3 CH3

H3C H3C O

OH

O

O

O

O

O

O H3C

H3C

H

O

134

Homoharringtonine (omacetaxine mepesuccinate, Ceflatonin®) 140, a myelosuppressive alkaloid isolated from Cephalotuxus fortuneii, inhibits Mcl-1 protein synthesis and induces apoptosis [134]. The European Commission in October 2004 granted orphan designation to Stragen France SAS for 140 against acute myeloid leukemia (AML) that was later transferred to ChemGenex Europe SAS in January 2009. The FDA in January 2009 designated 140 as orphan drug against myelodysplastic syndromes (MDS). In September 2009, a NDA for 140 under Omapro™ (omacetaxine mepesuccinate) was submitted by ChemGenex to the FDA for use in the treatment of chronic myelogeneous leukemia (CML) patients having T315I

Natural products in drug discovery

37

mutation or failed in imatinib therapy. ChemGenex are also running Phase II trial of 140 for treatment of refractory or relapseed AML patients failed to intensive chemotherapy. CH3 CH3 O

CH3

CH3 O

O

NH

CH3 CH3

CH3 N

O

CH3 CH3 CH3

N O

CH3

H3C

O

H3C

O

O NH

O O CH3 O

H3C

O

CH3 CH3

O

H

OH

CH2

O

H

OH

F

HO

O

H3C

O

O

CH3

HO

O

O

O O

O

NH

O H3C

136

O

H3C H3C

O

O

H3C

135

O

O

O CH3 OH

O

OCH3

CH3 O

CH3

H

OH HO

N O

O

O H3CO

137

O

O

O

CH3

CH3 R

CH3 R

138 R = H 139 R = OAc

3′-O-methyl-nordihydroguaiaretic acid (NDGA) 141, a lignan isolated from Larrea divaricatta exhibits significant anticancer activity by retardation of tumor cell proliferation through inhibition of insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu receptor. Terameprocol 142, a synthetic 141 derivative that induces apoptosis in cancer cells through inactivation of maturation promoting factor, was licensed by Erimos from The Johns Hopkins University. Terameprocol 142 is currently in various Phase I/II trials by Erimos against solid tumors, glioma and leukemia [135]. Epipodophyllotoxin (F11782) 143, a non-intercalating dual inhibitor of both topoisomerases I and II, was originally isolated from root of Podophyllum peltatum [136]. Tafluposide 144, a 143 derivative, is being developed by Pierre Fabre under Phase I/II trials for various tumor types [137]. Ingenol 145, isolated from the sap of Euphorbia peplus, is under clinical development by Peplin Biotech for topical treatment of basal cell carcinomas and squamous cell carcinomas [138]. Afer merger of Peplin with LEO Pharma in November 2009, ingenol mebutate (PEP005) 146, a 145 derivative that

38

Bhuwan B. Mishra & Vinod K. Tiwari

activates PKC, is currently in Phase III trials against actinic keratosis (AK). In December 2009, LEO Pharma disclosed the positive results of 146 in two Phase III trials against AK lesions on head (including the face and scalp) while announced to meet the primary endpoint in February 2010 with disappearance of AK lesions in non-head locations. Daidzein 147, an isoflavone occurring in Pueraria Mirifica, soybeans and soy products, exhibits clinical indication against tumors [139]. Phenoxodiol 148 is a synthetic 147 derivative that was licensed by Marshall Edwards from Novogen for development as combination therapy against ovarian cancer and as mono-therapeutic agent for the treatment of prostate and cervical cancers, resistant to standard chemotherapy [140]. Phenoxodiol 148 is supposed to inhibit sphingosine-1-phosphate and is under Phase III development by Marshall Edwards to restore chemosensitivity in patients with ovarin cancer resisting platinum drugs. A phase II trial of 148 against castrate and noncastrate prostate cancer is also uderway. Triphendiol (NV-196), an orally-delivered chemosensitizing derivative of 148 that was licensed to Marshall Edwards by Novogen, is under Phase I trials for use in combination therapy against cholangiocarcinoma, advanced prostate cancer and melanoma. An orphan drug status was granted to 148 by the FDA for cholangiocarcinoma, prostate cancer and stage IIb-IV malignant melanoma. In January 2009, FDA granted IND approval to 148. Genistein 149, a soy-derived antineoplastic phytoestrogen, inhibits protein-tyrosine kinase and induces cell differentiation, is under Phase I/II trials by Astellas, Bausch & Lomb for treatment of tumors. Genistein 149 is also supposed to inhibit topoisomerase-II, resulting in DNA fragmentation and apoptosis. (-)-Gossypol (AT-101) 150, a pan-Bcl-2 inhibitor isolated from the cottonseed plant of genus Gossypium [141], is under Phase I/II clinical development by Ascenta Therapeutics to address prostate, brain and lung cancers. In October 2009 Ascenta announced the results of Phase I trial for two combination regeims containing 150 for the treatment of malignant brain tumor. OH H3C

CH3 H3C

OH

CH3 O

O

OH O

O

H

OCH3 H3C

O

O O

O

H3CO

OR OR

O H

OR

H3CO

OCH3 OCH3

N

O

141 R = H 140 142 R = CH3

143

Natural products in drug discovery

39

ASA 404 (vadimezan, AS1404 and DMXAA) 151, a tumor-VDA and derivative of flavone-8-acetic acid 152, was discovered at Auckland Cancer Society Research and later in-licensed by Antisoma. Novartis AG in April 2007 signed an agreement with Antisoma for worldwide rights and co-selling of 151 in the US. As of April 2008, the 151 is currently in Phase III clinical trials by Novartis as a second line treatment for NSCLC. The β-Lapachone (ARQ-501) 153, isolated from Tabebuia avellanedae, induces expression of cyclin dependent kinase inhibitor 1A (CDKN1A or p21) and exerts anti-tumor effect by sustained increase of the pro-apoptotic protein E2F-1 [142]. The β-Lapachone 153 is currently in Phase II trials by ArQule as a combination therapy against pancreatic and ovarian cancer. Alvocidib (Flavopiridol, HMR 1275) 154, a CDK inhibitor and synthetic derivative of rohitukine 155 isolated from Dysoxylum binectariferum [143], is being developed by Sanofi-Aventis in collaboration with NCI. As on May 2009, the 154 is under late Phase III clinical development by Sanofi-Aventis against NSCLC while Phase IIb trial for the treatment of chronic lymphocytic leukemia (CLL). F

CH3

F

CH3 F

O

H3C

F F

F

H3C

O

O

F

F

O

F

O

H3C

O

O

RO HO

HO HO

F

O O

O

O

145 R = H

O

CH3

O

146 R =

O

CH3

O H

O

OH R

H3CO

OCH3 OH

O P

144

O

O

OH

HO

O

147

Curcumin 156, isolated from Curcuma longa roots, can interfere with the p53 tumor suppressor pathway and is under various Phase I/II trials world wide while a Phase III trial for the treatment of metastatic colon cancer (MCC) is underway [144]. RTA 402 (CDDO-Me, Bardoxolone methyl) 157,

40

Bhuwan B. Mishra & Vinod K. Tiwari

an IkB alpha kinase activation inhibitor and synthetic derivative of oleanolic acid 158 [145], is being evaluated by Reata Pharmaceuticals under Phase I/II trials against prostate cancer and Phase II trials for the treatment of type 2 diabetes with chronic kidney disease (CKD). RTA 402 157 is an orphan drug by the FDA against prostate cancer. In January 2010, Kyowa Hakko Kirin gained exclusive rights from Reata Pharmaceuticals to develop and commercialize 157 in Japan and other selected Asian regions to treat type 2 diabetes with CKD. O OH OH

O

OH HO

OH

O

OH HO

HO HO

O

HO

H3C

O

CH3 H3C

CH3

148

OH

CH3

149

150 OH

O

O

O

O

H3C

Cl O HO

O OH

O

O

CH3 CH3

HO2C

O N

HO2C

151

H3C

152

CH3

CH3

154

153

CH3 O

O HO

O N

HO OH

155

CH3

H3CO

O OCH3

HO

OH

156

Betulinic acid (ALS-357) 68, a topoisomerase I inhibitor isolated from Betula pubescens [146], is an orphan drug (by the FDA) in Phase I trial by Advanced Life Sciences for the treatment of malignant melanoma. Silybin 159, a flavonolignan isolated from Silybum marianum, is the active constituent of IdB 1060 (silybin-phosphatidylcholine complex, Siliphos®). Silybin 159 is currently in Phase II trials by American college of gastroenterology for chemoprevention of cancer [147].

Natural products in drug discovery H3C

41

CH3

H3C

CH3 OH

O

OCH3

O

H

OH CH3

CH3

N

CO2CH3 CH 3

H

CH3 O H3C

H HO

H CH3

CH3

H3C

H

O

CO2H O

HO

CH3

OH

H CH3

OH

157

O

159

158

8.1.2. Microorganism-derived compounds 8.1.2.1. Actinomycetes Pladienolide D 160, obtained from fermentation broth of Streptomyces platensis Mer-11107, exerts significant antiproliferative activities against variety of cancer cell lines. E7107 161, a synthetic 160 derivative that binds with spliceosome-associated protein 130 (SAP130) and inhibits the splicing of pre-mRNA, is in various Phase I trials by Eisai against solid tumors [148]. Chartreusin (U-7257) 162 isolated from Streptomyces chartreuses and elsamicin A (BMY-28090, elsamitrucin) 163 isolated from actinomycete strain J907-21, are the antibiotics that inhibit RNA synthesis and result in single-strand scission of DNA [149]. Elsamicin A 163 is also a topoisomerase I/II inhibitor being developed by Spectrum Pharmaceuticals in Phase II trials to use in the treatment of advanced solid tumors. OR OH CH3 OH H3C

H3C

O

O O

CH3

H3C

OH

160 R = Ac O 161 R =

N

N

OH

CH3

Doxorubicin 164, an anthracycline antibiotic capable of intercalating with DNA, was isolated from bacteria occurring in soil samples taken from Castel del Monte, an Italian castle. Doxorubicin 164 is an orphan drug by the FDA against acute lymphocytic leukemia (ALL) and AML. Valrubicin (Valstar®), a semi-synthetic 164 derivative was approved in 1999 for the treatment of bladder cancer but was withdrawn in 2002 due to some manufacturing issues and has been relaunched in September 2009. L-annamycin 165, a topoisomerase II inhibitor that was developed at the MD Anderson Cancer Center, is currently in Phase I/IIa trials by Callisto

42

Bhuwan B. Mishra & Vinod K. Tiwari

Pharmaceuticals for the treatment of younger and adults with refractory or relapsed ALL or AML. Berubicin (RTA744, WP744) 166, a DNA intercalator capable of crossing the BBB, hence is significant for the treatment of primary brain tumor. Reata Pharmaceuticals are associated with Phase II development of 166 against malignant gliomas. Likewise, sabarubicin (MEN-10755) 167 [150], a topoisomerase II inhibitor and disaccharide analogue of 164, is currently in Phase II clinical trials by Menarini Pharmaceuticals against solid tumors [151]. Nemorubicin (MMDX, PNU-152243A) 168, a 3′-deamino-3′[2-(S)-methoxy-4morpholinyl] derivative of 164, is a topoisomerase I inhibitor exhibiting activity against selected tumors resistant to current treatment. Nerviano Medical Sciences are evaluating 168 in Phase I/II trials. Distamycin A 169, a DNA minor grove binder (MGB) and lead compound of brostallicin (PNU-166196) 170, was originally developed by Nerviano [152]. Nerviano had transferred the exclusive world right of 170 to Systems Medicine Inc (SMI) which was taken over by the Cell Therapeutics. Currently, the 170 is in phase II trials by Cell Therapeutics as monotherapy against metastatic or advanced stage STS. H3C

H3C O OH HO

OH

O

O

OH

HO

CH3

O CH3

O

HO

H3C

OH

H3C

H3C

OH

O

OH

O

O

CH3 O

O NH2

R

OH

164 R = H 166 R =

163 O

O

H3C

O

162

O

O

O

O

H2N

O OH

OH OH

O

CH3 O

O

O

O O

CH3 O

OH

O

O

OH

O O

OH

O

OH

O OH

OH

OH

OH

OH O

OH

O O

O

OH

H3C HO

O

HO O

O HO

CH3

H3C

O

OH

O

H3C O

H3C

CH3 O

OH N

O OH

165

O

H3C

O

NH2

167

168

Natural products in drug discovery

43

Geldanamycin 171 is an antineoplastic benzoquinone ansamycin antibiotic and was discovered from broth and mycelium of Streptomyces species [153]. Tanespimycin (17-AAG, KOS-953, NSC-330507) 172, a comparatively less toxic antibiotic derived from 171, can bind to HSP90 and interrupts the MAPK pathway. As on November 2009, Kosan have completed a Phase II/III trial of 172 in combination with Velcade® against relapsed-refractory multiple myeloma. Alvespimycin (17-DMAG, KOS1022, NSC-707545) 173, a second generation HSP90 inhibitor [154] is in clinical development by Kosan to use in the treatment of solid tumors. As on January 2008, 173 is in Phase I trials in combination with trastuzumab & paclitaxel (Taxol®) against solid tumors, Phase II monotherapic trials against HER2-positive metastatic breast cancer and Phase I trials for the treatment of solid tumors. Retaspimycin (IPI-504, 17-AAG hdroquinone salt) 174, a HSP90 inhibitor, is being developed by Infinity Pharmaceuticals in Phase I/II clinical trials to address certain cancers. Currently, the Infinity are evaluating 174 in a Phase II trial against NSCLC while enrolling patients for another Phase II trial in combination with Herceptin® against breast cancer. H NH O H N Br

N

H2C

CH3

H N

O N

NH

CH3

O

H N

NH2

N

169

N CH3

H N

O O

CH3

NH

H N

O N CH3

H N

O

NH

N CH3

170

H N

O

N H

N CH3

NH2

O

Deforolimus (AP23573, MK-8669) 175, is an mTOR inhibitor codeveloped by Merck and ARIAD Pharmaceuticals to address several tumor types including sarcoma. The name of 175 was changed to ‘ridaforolimus’ by ARIAD in May 2009 and as on December 2009, the enrollment for a Phase III study in patients with metastatic STS and bone sarcomas has been completed by ARIAD. Besides, ARIAD are also running several Phase I/II trials of 175 as a single agent and in combination therapies. Salinosporamide A (NPI-0052) 176, a proteasome inhibitor produced by a marine bacterium

44

Bhuwan B. Mishra & Vinod K. Tiwari

Salinispora tropica [155], exerts activity by modifying the threonine residues of the 20S proteasome. Nereus are associated with Phase I clinical development of 176 to use in the treatment of solid tumors and lymphomas. As on April 2008, Nereus Pharmaceuticals are enrolling patients for a Phase Ib trial of 176 in combination with vorinostat (Zolinza®, Merck & Co.) against selected solid tumor malignancies. O H

R

H

OH

N

O

O CH3

N H O H3C H3C

O

171 R = OCH3 172 R =

CH3 OH

Cl

O CH3

173 R = NH2

H3C

O

O

N H N H

CH3

H2C CH2

OH

CH3

H3C

N

H3C

CH3

CH3 OH O H3C

O CH3

O O

174

NH2

Staurosporine 177, isolated from bacterium Streptomyces staurosporeus [156], a precursor of protein kinase inhibitors like enzastaurin (LY317615) 178 and midostaurin (PKC-412, CGP 41251, 4’-N-Benzoyl-staurosporine) 179, has significant anticancer potenticals. Enzastaurin (LY317615) 178 is a serine/threonine kinase inhibitor [157] that was evaluated in Phase II trials by Eli Lilly to use in the treatment of NSCLC patients. As of April 2010, the 178 is under Phase III trials for the treatment of diffuse large B-cell lymphoma. Midostaurin (PKC-412) 179 inhibits protein kinases including FLT3 [158] and is in Phase II trials by Novartis to treat AML patients carrying FLT3 mutations. K252a 180, an alkaloid isolated from Nocardiopisis species, is the lead compound of lestaurtinib (CEP-701, KT-5555) 181 that inhibits FLT3 and tyrosine phosphorylation of Trk A. As of 2008, the 181 is in Phase II trials against myeloproliferative disorders and Phase III trials for the treatment of AML. Likewise, KRX-0601 (UCN-01, KW-2401) 182, inhibiting a broad spectrum of kinases including CDKs, is being developed by Keryx (Kyowa Hakko) in Phase II clinical trials under sponsorship of NCI against melanoma, TCL and SCLC. Diazepinomicin (ECO-4601, TLN-4601) 183, a dibenzodiazepine alkaloid isolated from the culture of a marine actinomycete of the genus Micromonospora [159], can bind to peripheral benzodiazepine receptor (PBR) and inhibits the Ras/MAP kinase signaling pathway involved in cellular proliferation and migration [160]. ECO-4601 183 was found safe and well-tolerated in Phase I/II trials conducted by the NCI and Thallion. ECO-4601 183 can cross the BBB and as on September 2008, Thallion are enrolling patients for Phase II trial of 183 as a second line treatment for GBM.

Natural products in drug discovery

45

CH3 OH

CH3 O

O

OH

O OH

O

HO P O

O

O CH3

CH3

O CH3

O N

CH3

HN

O

Cl

CH3

CH3

O CH3

O H N

O

176

O

O HO O H3C

CH3 H N

O

175

N

N CH3

N N

O

N CH3 N

OCH3 NHCH3

178

177

H N

O

H N

O

N

N

O

CH3

N

N

O

OCH3

CH3

N

OH

CH3

R

O

180 R = CO2CH3

179

181 R = CH2OH

H N

O

OH O

CH3

CH3

N N

O

N

HO

CH3

HN

CH3 OCH3

HO

OH

HN CH3

182

CH3

183

46

Bhuwan B. Mishra & Vinod K. Tiwari

8.1.2.2. Eubacteria Prodigiosin (Streptorubin B) 184, a Bcl-2 inhibitor and lead compound of obatoclax (GX15-070) 185, is produced by strains of the bacterium Serratia marcescens [161]. Gemin X are developing intravenous infusion of 185 in multiple Phase I/II trials as a monotherapy in hematological and solid tumors while in combination with carboplatin & etoposide to treat SCLC and with bortezomib (Velcade®) against mantle cell lymphoma (MCL). In March 2009, Gemin X launched a Phase II study of 185 as first-line treatment for SCLC while disclosed the results of a Phase Ib trial in May 2009 against extensive-stage SCLC. 8.1.2.3. Myxobacteria Patupilone (epothilone B, EPO-906) 21, produced by the myxobacterium Sorangium cellulosum, is a microtubule-stabilizing agent currently in Phase III trials by Novartis against ovarian cancer [162]. Sagopilone (ZK-EPO, ZK-219477) 186, a synthetic 21 derivative, can retain activity in MDR cancer cells overexpressing the P-gp [163]. As of February 2010, Schering AG is evaluating 186 in Phase II trials for the treatment of lung, ovarian and prostate cancers. Epothilone D (desoxyepothilone B) 187, a natural polyketide inhibits the disassembly of microtubules by binding to tubulin. 9,10-Didehydroepothilone D (KOS-1584) 188 [164], a 187 derivative, being evaluated by Kosan Pharmaceuticals to use in the treatment of multiple solid tumors. In Phase I dose escalation trials by Kosan, 188 has demonstrated efficacy and tolerability against patients with ovarian cancer and NSCLC. As of February 2007, Kosan were planning to initiate Phase II clinical development of 188 against multiple solid tumors in collaboration with Roche. OCH3 H C 3 N H

OCH3

N N H

N

HN HN H3C

184

185

CH3

Natural products in drug discovery

47

8.1.2.4. Fungi NPI-2350 (halimide, phenylahistin) 189 is a tubulin-depolymerizing agent isolated from a marine fungi Aspergillus ustus [165]. Nereus are developing plinabulin (NPI-2358) 190, a synthetic 189 analog in various clinical trials for the treatment of NSCLC [166]. In November 2009, Nereus announced the positive results of a Phase II trial in NSCLC patients. Irofulven (MGI-114, HMAF) 191 is a DNA synthesis inhibitor and analog of illudin S 192, a sesquiterpene toxin found in mushrooms of the genus Omphalotus [167]. Eisai (MGI Pharma) are currently evaluatig 191 in various Phase II/III trials in patients with advanced-stage prostate cancer and GI solid tumors. 8.1.3. Marine-derived compounds Plitidepsin (Aplidin®) 193, extracted from Aplidium albicans [168], is being evaluated by PharmaMar in Phase II trials to use in the treatment of hematological and solid tumors. Plitidepsin 193 inhibits the vascular endothelial growth factor (VEGF) and is currently in Phase II trials by PharmaMar as a first-line monotherapy treatment and in combination with dacarbazine for advanced unresectable melanoma [169]. Halichondrin B 194, isolated from Halichondria okadai sponge [170], was identified as a significant anticancer agent by NCI. Eribulin mesylate (E7389, ER-086526, NSC-707389) 195, a 194 analog, is being developed by Eisai against advanced breast cancer patients. Eribulin 195 is a microtubule dynamics inhibitor and in March 2010, Eisai has submitted regulatory applications to agencies in Japan, US and EU for approval of 195 to use in the treatment of locally advanced or metastatic breast cancer. Hemiasterlin 196, derived from marine sponges [171], is capable of inhibiting tubulin assembly and disrupts normal microtubule dynamics by depolymerizing the microtubules. E7974 197, a synthetic analogue of 196, can bind to α- and β-tubulin and is under Phase I clinical development by Eisai against a variety of human tumor xenografts. Psammaplin A 198, an inhibitor of key several enzymes that control gene expression, DNA replication and angiogenesis, was originally isolated from the marine sponge Psammaplinaplysilla. Panobinostat (LBH-589) 199, a synthetic 198 analog and pan-deacetylase inhibitor that induces death of tumor cell lines but not the normal cells, is in Phase Ib/II clinical trials by Novartis to use as monotherapy and in combination with chemotherapy and/or targeted therapy against Hodgkins lymphoma, malignant melanoma, AML/MDS and other hematological malignancies [172]. Currently, Novartis are enrolling patients for Phase III trial in relapsed malignant melanoma.

48

Bhuwan B. Mishra & Vinod K. Tiwari

O

S

CH3

CH3

H3C

S

O

N

H3C

O OH

OH

N

CH3

H3C CH3 H3C

O

H3C

H3C

CH3

CH2 O

OH

O

O

OH

186

S H3C

CH3

187

CH3 O

O

O

N

OH NH H3C

H3C

N NH

CH3

HN

CH2

CH3 O

O

H3C

OH

CH3

CH3

189

188

O OH

CH3 NH

CH3

OH

N NH

HN O

CH3

HO CH3 H3C CH3

H3C

CH3 OH HO H3C

O

O

192

191

190

OCH3 H3C

CH3

O

N

O

H3C

NH

O

CH3 O

O

O O

H3C H3C

O

N

O

N

OH NH

H3C

N

N H

O CH3

CH3

H3C H3C

CH3

193

O

O O

CH3

Natural products in drug discovery

49

CH3 H

H

H

H

O

O

O

O

O

O

O

H

HO H

H O

O

O H

CH3 H

H

H

O H

H

CH2 O

O

H

CH3

O

CH3

O

OH O

HO

194 H3C

O

H

H2C

H O H

HO

O

O

O O

H NH2

H3C

CH3

H3C O

CH3 CH3 CH3

H

CH2 O O CH3

O

N

CO2H

N H O

O H3C

HN

N

CH3

CH3 CH3

H3C

O

H2C

195

196 OH

H3C

CH3 H C 3 O N

N H

CH3 CH3 CH3 N O H3C

Br

O

CO2H CH3 CH3

HO

N H

N

S

N

H N

S

Br O

198

197

H3C H3C HO

OAc O

O N H

H N

O

OH

O

O HO

O OCH3

HN

HO O

OCH3

CH3 O O

H3C

200

OH H3C

H3C

CH3

199

OH

OH

O

50

Bhuwan B. Mishra & Vinod K. Tiwari

Bryostatin 1 200, a macrolide lactone isolated from Bugula neritina collected from the Gulf of California and Mexico, inhibits PKC [173] and was granted with orphan drug status by the FDA (2001) and a similar designation by the EU (2002) for use as combination therapy with TaxolTM against esophageal cancer. In 2001, Arizona State University licensed 200 to GPC Biotech, which are associated with current Phase I/II trials under the guidance of the NCI. Jorumycin 201, isolated from Jorunna funebris produces cytotoxic effects through binding to DNA [174] and is the lead compound of Zalypsis® (PM00104/50) 202 being developed by PharmaMar in Phase I trials for the treatment of solid tumors or lymphoma. As on November 2009, the 202 is in Phase II trials for treating cervical and endometrial cancer patients previously treated with standard chemotherapy. Dolastatin 15 203, is an antimitotic agent structurally related to dolastatin 10 205, a five-subunit peptide obtained from Dolabella auricularia [175]. Tasidotin (synthadotin, ILX-651) 204, an analog 203, induces G2/M phase cell cycle arrest by inhibiting tubulin polymerization was evaluated by Genzyme in Phase I/II trials against solid tumors. In May 2009, Genzyme signed an agreement with Ergomed for the co-development of 204 as an antineoplastic agent. Soblidotin (YHI-501, TZT-1027, auristatin PE) 206, a derivative of 205, inhibits tubulin polymerization and is under Phase II trials by Yakult Honsha for treatment of solid tumors. Kahalalide F 207, obtained from the Hawaiian sea slug Elysia rufescens, can alter lysosomal membrane function [176] and is in Phase II trials since October 2008 for the treatment of severe psoriasis. In June 2009, the 207 was licensed by Medimetriks Pharmaceuticals from PharmaMar for uses outside of oncology and neurology. PM02734 (Irvalec®) 208, another 207 derivative, is in Phase II trials against solid tumors by PharmaMar. As on February 2010, PharmaMar are recruiting patients for Phase I trial of 207 in combination with erlotinib against advanced malignant solid tumors.

8.2. NP-antibody anticancer conjugates Anticancer agents conjugated with various supports (antibodies, polymers, liposomes and nanoparticles etc.) have been extensively explored during the last few decades [177]. Zinostatin stimalamer (ZSS), conjugated with a molecule of neocarzinostatin (NCS) chromoprotein and two molecules of polystyrene-co-maleic acid [178], was launched by Yamanouchi (now Astellas) in Japan against hepatocellular carcinoma. Gemtuzumab ozogamicin (Mylotarg®) 209 linked to calicheamicin 210 (obtained from Micromonospora echinospora), was co-developed by Wyeth and

Natural products in drug discovery

51

OCH3 O H

H3C

N

H3CO O

N

N

CH3

O

OH OAc

NH

N H

N O

CH3

CF3

O

N

CH3

OH

O

202

CH3H3C

O

N

O

O

O

OCH3

N

O H3C

CH3O

203 H3C H3C

CH3H3C

CH3H3C

N H

N CH3

CH3 N

N

O

O

CH3

H N

N

O

O

CH3 CH3 CH3

O

204 H3C

CH3 H3C

CH3 H3C

CH3 CH3

H3C

N N H

N CH3

O

N CH3 OCH3 O

O

OCH3 O

H N

S

205 H3C

CH3 H3C

CH3 H3C

CH3 CH3

H3C

N N H

N CH3

O

N O

CH3

N

O

C CHH 33

N

CH3

H

H3C

O

CH3

O

201

H3C

HO

CH3 H3C

O

H3C

OCH3

O

CH3 OCH3 O

206

OCH3 O

H N

N

52

Bhuwan B. Mishra & Vinod K. Tiwari CH3 H2N

CH3

O

O

CH3

O N H CH3

N

N H O

H3C O H3C H3C

HN

H3C

HO

O

NH CH3 H3C H3C

NH

CH3

CH3

207 R =

NH O

CH3

O

CH3 O

O

O CH3 HN

NH H3C

NH

O

O H3C

H3C

NH CH3

H N O

208 R = O

CH3

O NH R

UCB Pharma. Likewise, inotuzumab ozogamicin (CMC-544), a calicheamicinantibody conjugated with CalichDMH and hydrazone linker attached to humanized IgG4 anti-CD22 [179], is being developed by Wyeth and UCB Pharma in Phase II/III trials against non-Hodgkin’s lymphoma in combination with rituximab, a chimeric human IgG1 antibody that targets another B-lymphoid lineage-specific molecule, CD20 [180]. Maytansine 211, isolated from plants of the genus Maytenus, is a microtubule inhibitor that failed to show significant activity at non-toxic concentrations in Phase I/II trials. ImmunoGen are associated with clinical development of IMGN-242 (HuC242-DM4) 212, a maytansinoid DM4 and huC242 conjugate, currently in Phase II trials for CanAg-expressing cancers. In June 2009, ImmunoGen discontinued the development of 212 and are seeking for out-licensing. ImmunoGen are also evaluating IMGN-901 (HuN901-DM1) 213, a maytansinoid DM1 and huN901 congugate targeting CD56 expressing tumors, is under Phase I trials against multiple myeloma while Phase II trial for the treatment of SCLC. The FDA in March 2010 awarded orphan drug designation to 213 for use against merkel cell carcinoma (MCC).

9. Conclusion Natural products have been the major sources of chemical diversity for starting materials while driving pharmaceutical discovery over the past century.

Natural products in drug discovery

53

H N O

hP67.6

O

O H3C CH3

H3C

N H

O H3C

S O

H3CO

H3C

O

OH

OCH3

O

H3C O

N H3CO

O

209

CH3

HO

S

O H3C

I

H3C O

H3C HO H3CO

O O

H3C

OCH3

O

N H

N H HO

OCH3

O

O O

HN H3CO

OH

210 CH3

H3C

O

Cl H3C H3CO

O

O O CH3

N CH3

O

HO N H CH3

OCH3

211

O

S S H3C

O

S OH

H

O

H3C

OH

OCH3

O

N H HO

OCH3

O

O

N H

S

S

H3C HO

O

N

I

O

HO

CH3

O

H

O OCH3

54

Bhuwan B. Mishra & Vinod K. Tiwari

CH3 H3C

N

O

O

H3C

O

CH3 S S

N H

huC 242

O O

Cl H3C H3CO

O CH3

N CH3

O

HO

CH3

OCH3

N H

O 3-4

212 CH3 H3C

N

O

O

CH3 S

O O

Cl H3C H3CO

H N

S

HuN901

O

O CH3

N CH3

O

HO N H CH3

O

OCH3 3-4

213

Today, NPs are finding increasing use as probes to interrogate biological systems as part of chemical genomics and related researches. The modification of natural products in an effort to alter their biochemical capacity is a common technique utilized by synthetic and medicinal chemists. There have been remarkable achievements in the field of ‘natural products drug discovery’ during last three decades and several compounds having profound biological activities have been searched out with the help of modern and sophisticated techniques. The quality of leads arising from NP discovery is better and often more bio-friendly, due to their co-evolution with the target sites in biological systems.

Natural products in drug discovery

55

The large number of NP-derived compounds in various stages of clinical development indicates that the use of NP templates is still a viable source of new drug candidates. In future, the ‘natural products drug discovery’ will be more holistic, personalized and involve wise use of ancient and modern therapeutic skills in a complementary manner so that maximum benefits can be accrued to the patients and the community.

Acknowledgement Authors are grateful to Prof. Dr. Richard R. Schmidt, Department of Chemistry, Universitat Konstanz, Germany for his useful discussions during the preparation of manuscript. Financial assistance from DST, New Delhi has been greatly acknowledged.

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18. 19.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 63-101 ISBN: 978-81-308-0448-4

2. Natural products in discovery of potential and safer antibacterial agents 1

Girija S. Singh1 and Surendra N. Pandeya2

Chemistry Department, University of Botswana, Gaborone, Botswana; 2Pharmaceutics Department Saroj Institute of Management & Technology, Lucknow-226001, India

Abstract. This article describes the significance of natural products in discovery of potential and safer antibacterial agents. The introductory paragraph is followed by various classes of compounds depending on the structural class. These include β-lactams, macrolides and ketolides, lincosamides, furanomycin, pyrrolidinediones, tetrahydropyrimidinones, biphenomycins, tuberactinomycin and capreomycin, glycopeptides, lysobactins, enopeptin depsipeptides, tetracyclines and aminoglycosides. A brief historical development of each class is described followed by its mechanism of action. Several semi-synthetic compounds are described. Synthetic methods are described in selected cases.

1. Introduction Today, infectious diseases are the second major cause of death worldwide and third leading cause of death in economically advanced countries [1]. Bacterial pathogens are responsible for several serious diseases (Table 1). Strains are getting resistant to antibiotics in clinical use and hence posing threat to mankind (Table 2). The ability of bacteria to deceive any kind of conventional therapy has become apparent and pathogens resistant to one or more antibiotics are emerging and spreading worldwide [2]. Unnecessary use of antibiotics has further fuelled this problem. Correspondence/Reprint request: Prof. Girija S. Singh, Chemistry Department, University of Botswana, Gaborone Botswana. E-mail: [email protected]

64

Girija S. Singh & Surendra N. Pandeya

Table 1. Key bacterial pathogens and related diseases.

Pathogens S. aureus S. pneumonia

Infectious Diseases Skin and wound infections, endocarditis Upper respiratory tract infection, pneumonia, sinusuitis, meningitis Pharyngitis, tonsillitis, skin and soft tissue infection S. pyogenes E. faecalis Endocarditis, urinary tract infection E. faecium Peritonitis, endocarditis, bacteremia E. coli Urinary tract infection, bacteremia, gastrointestinal infection Bacteremia, pneumonia K. neumoniae H. influenza Respiratory tract infection, sinusuitis, meningitis P. aeruginosa Bacteremia, burn infection M. tuberculosis Tuberculosis Table 2. Prevalence of resistance in hospital-acquired infections in USA (2004). Antibiotics Methicillin Vancomycin Cephalosporins (3rd generation)

Imipenem Quinolones (synthetic)

Pathogen S. aureus Enterococci Enterobacter sp. P. aeruginosa E. coli K. pneumoniae P. aeruginosa P. aeruginosa

Resistance (%) 59.5 28.5 31.1 31.9 5.8 20.6 21.4 29.5

The discovery of vancomycin resistant S. aureus (VRSA) and multiresistant S. aureus has generated worldwide concern. It has thus become evident that there is urgent need for novel antibacterial drugs with broader spectrum, lesser side effects, and without cross-resistance to antibiotics in use. More initiative is required to foster the responsible and appropriate use of antibiotic that is another issue. The traditional medicine system based on natural products continues to play an important role in treatment of many diseases especially the infectious diseases. According to the WHO estimation, approximately 80% of the world’s population relies mainly on traditional medicine for their primary health. Indian traditional medicine system relied on plants and their parts to treat various infectious diseases (Table 3). Hundreds of herbs are known to be used for various diseases including many infectious diseases. Acacia, garlic, turmeric, neem, ginger, clove, plum and pomegranate are only few to name.

Antibacterial natural products

65

Table 3. Some traditionally used herbs and their bioactivity. Plant Triphala i.e. three fruits viz. Haritaki (Terminalia chebula), Bibhitaka (Terminalia belerica) & Amalaki (Emblica officinalis) Turmeric (Curcuma longa)

Plant Parts 90 % ethanolic & aqueous extracts of Triphala

Bioactivity Significant activity on S. aureus, E. coli and P. areoginosa

Leaf extract

Fenugreek (Methika) (Trigonella faenum- graecum) Wildrue (Peganum harmal)

Seed extract

Antibacterial and antifungal activity Antibacterial and antifungal activity Antibacterial and antifungal activity Antibacterial and antifungal activity

Gokarna (Clitoria Ternatea) Sharapunkha (Tephrosia puropurea) Brahmi (Bacopa monnieri) Tulsi (Ocimum sanctum)

Aqueous seed extract Hexane & methanolic root extract Root extract Ethanolic extract of aerial parts Leaf extract

Antibacterial and antifungal activity Antihelminth activity Antibacterial and antifungal activity

Later on extracts from many of such plants and herbs have been screened by investigators in quest for potential and safer antibacterial agents [3-8]. Many comprehensive review articles have been published on the role of natural products in the discovery of antibacterial agents [9-12]. The plethora of literature in the area indicates an urgent need for a coordinated effort for meaningful research and discovery of novel antimicrobial agents. Most of the antibacterial agents in use today are either natural products or their semi-synthetic variations or improved subclasses. The success of natural products as guideposts to new drugs is most obvious in antibacterials (Table 4). Over 75% of new chemical entities submitted between 1984 and 2004 were based on natural product lead structures [13]. This article focuses on significance of selected natural products in discovery of new antibacterial agents with broader spectrum and least side effects. The sections are classified according to established structural characteristics. A brief historical development of each class is described followed by its mode of action. Selected recent examples of synthetic modification of natural antibiotics are discussed. More emphasis is given on β-lactam antibiotics as it consists of several subclasses such as penems, cephems etc. As mentioned in the preceding paragraph there are plethora of

66

Girija S. Singh & Surendra N. Pandeya

Table 4. Some potential antibiotics and their targets. Class

Representative example H N

β-Lactams

Target

S

O

Cell wall

N O

COOH

Penicillin G (1) OH

NMe2 OH

H

Polyketides

Protein biosynthesis

NH2 OH

OH OH O

O

O

Tetracycline (2) O2 N

NHCOCHCl2 OH

Phenylpropanoids

Protein biosynthesis

OH

Chloramphenicol (3) NH2

Aminoglycosides

O

HO

NH2 O HO

Protein biosynthesis

H2N NH2

O

OH OH

O

HO

NH2

Tobramycin (4) O

Macrolides

Protein biosynthesis

OH

HO OH

HO

O

O

NMe2

OMeO O

O

OH

O

Erythromycin A (5) OH NH

2

Glycopeptides

H3C

O CH3 O

HO O O NH

HO

N H

O

O

O

Cl H N

O

OH Cl OH OH

O

N H

O H N O

O NH2

O

OH OH HO

Cell wall

OH O

Vancomycin

N H

H N

CH3 CH3

CH3

Antibacterial natural products

67

Table 4. Continued Streptogramins

Me N

N

NMe2

O O HN O O N

O

O O

Protein biosynthesis

N O NH

O NH OH

Pristinamycin IA (7) OH O N O

NH

O

O O N O

Pristinamycin IIA (8)

literature available in the area and each class has been reviewed by several others from different angles it was considered pertinent to bring a concise account of the material for the convenience of readers.

2. β-Lactam antibiotics The group of antibiotics containing four-membered cyclic amides (azetidin-2-ones) is commonly known as β-lactam antibiotics. It is the first class of antibiotics to be used as a therapeutic treatment for bacterial infections (Figure 1). The first member penicillin was discovered by Fleming from the cultures of Penicillium notatum in 1928. Since then this group has maintained its charm among synthetic and medicinal chemists [14,15]. About half of the antibacterial drugs prescribed today belong to this class. Their broad antibacterial spectrum, clinical efficacy and excellent safety profile make them preeminent in pharmaceutical drug discovery. As a result of extensive research cephalosporins have reached their fourth generation (cefepime). The main approaches in design of the new cephem derivatives involve structural modifications at positions C-3 and C-7, and the development of cephem prodrugs. The compounds with a methoxy [16], carbamoyloxy [17] or heteroaryl ring such as tetrazole [18] or thiazole [19] in the C-3 side chain are known to have potent antibacterial activity.

68

Girija S. Singh & Surendra N. Pandeya OH H

H N

R O

N

N O

O

CO2H

OH H

O N

O

oxapenem

H N

R O

N CO2H

H N

R R

oxacephem

N

O

N

Cl CO2H

S

O

N O

H2N

N CO2-

Cefepime

H N

R N

O

OMe H N

S

S CO2H

CO2H sulbactam

carbapenem

O

O

NH

O

O

CO2H

NH

N

CO2H

N

O

oxapenam

NH2 OH H

O S

N

O CO2H

penem

penam

O

OH

O

S

S

O

carbacephem

S N

OAc

O CO2H cephem

H N

R O

O

N SO H 3

monobactam

Figure 1

Natural penicillin G, the first therapeutic antibiotic and lead structure of this class still had a few critical features such as narrow antibacterial spectrum, instability in acidic and alkaline environments, limited solubility and pronounced sensitivity to hydrolyze by bacterial penicillase enzymes that are needed to be improved. For about thirty years, penicillins (penams) and cephalosporins (cephems) remained the only examples of β-lactam antibiotics. Many related subgroups such as the monobactams (aztreonam), oxacephems (moxalactam), carbacephems (loracarbef), oxapenams (clavulanic acid), penems (faropenem), carbapenems (imipenem), and oxapenems (AM-112) were discovered during 1970s and 1980s either from microbes or by synthetic efforts. Numerous structural variations of these βlactam scaffolds provided derivatives with increased potency, low host toxicity, improved physicochemical and pharmacokinetic profiles. The β-lactam antibacterials act on bacteria by inhibiting the final step of the bacterial cell wall biosynthesis. Although several mechanisms might be operating in this inhibition, the most important is probably the inhibition of the terminal peptidoglycan cross-linking. Bacteria have a cytoplasmic membrane similar to that of eukaryotes. This membrane is surrounded by a periplasmic space, which is in turn enclosed by a peptidoglycan layer, and finally the outer membrane. The peptidoglycan layer is a cross-linked polymer that forms a net-like structure, which provides structural rigidity to

Antibacterial natural products

69

the organism, and allows it to survive in mediums to which it may be strongly hypertonic. As a bacterium grows, a series of covalent cross-links must be formed between adjoining peptidoglycan strands in the cell wall. These cross-links are stitched together by transpeptidase enzymes in the cell membrane through the replacement of a terminal D-alanine unit on one peptidoglycan strand with a glycine residue on a neighboring peptidoglycan strand. The initial cleavage of the D-alanine residue by transpeptidase occurs by a nucleophilic addition of an active site serine onto the amide functionality, as shown in Scheme 1. In a subsequent amidation step, the resulting enzyme-linked peptidoglycan is converted to the cross-linked material, which releases serine for further catalysis. Penicillins, cephalosporins and related β-lactam drugs possess an unusual ability to interrupt this crucial cross-linking process by an irreversible acylation of the hydroxy group of the catalytic serine unit within the enzyme active site resulting in the formation of a catalytically inactive stable enzyme-drug adduct (Scheme 2). The net result is decrease in the number of cross-linked residues within the cell wall making it weak and prone to rupture. Inhibition of the transpeptidase thus inhibits the bacterial growth. The sequences leading to cidal action are still not clearly understood. Nicks may be produced at the growth site of the cell wall. If these nicks are sufficiently severe, the protoplast may protrude into the medium and burst resulting in bacterial death. The major limitation to the potentials of β-lactam antibacterials is the ability of bacteria to produce a family of enzymes called β-lactamases. These enzymes hydrolyze the β-lactam ring which is required for antibacterial activity. There are different types of β -lactamases and their efficacy in

ngtp

H N

CO2-

O N H

Me

Me

ngtp

OH

H N

O O Me enzyme-serine

ngtp

H N

O

Me

N H

H N ptgn O

OH

enzyme serine

ptgn = Peptidoglycan

enzyme serine

Scheme 1 ROCHN

ROCHN

S N

O

CO2H OH

enzyme-serine

S

HN OO

CO2H enzyme-serine

Scheme 2

Bacterial-death

70

Girija S. Singh & Surendra N. Pandeya

hydrolyzing the ring varies widely. There are four distinct classes of βlactamases, of which class A enzymes are the most common. In order to counter the hydrolysis by β-lactamases, some antibiotics are administered in combination with a β-lactamase inhibitor drug. For example, amoxicillin is administered in combination with clavulanic acid, itself also a β-lactam (oxapenam). However, the discovery of new variants of β-lactamases, which are resistant to known β-lactamase inhibitors, has caused great concern worldwide. The major thrust areas in research on β-lactams have been the development of new stereoselective methodologies to construct the β-lactam ring, and structural modifications in compounds, especially carbapenems and cephems with known activity, to design and develop new molecules with i) a broad spectrum of activity, specially against resistant strains and, ii) least side effects. The succeeding paragraphs describe the synthesis of some new cephems. The biological activity is discussed in selected examples. A series of β-[(Z)-2-(2-aminothiazol-4-yl)-2-hydoxyiminoacetamido]-3[(E) and (Z)-2-substituted vinyl] cephalosporin derivatives 9,10 have been synthesized using palladium-catalyzed coupling reaction of a 3methanesulfonoxy-3-cephem and an E substituted vinyl stannane (Scheme 3) or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an aldehyde (Scheme 4) as key steps [20]. OH N H N

N O H2N

S

S N

R

O CO2H

9,10

R = H (Cefdinir), -CH2CH2OMe, -CH2CH2OTMS, -CH2CH2OH, CH2CH2OCONH2, CH2CH2OAc a O

b

c

O

d

O

e O N

N N

f

g

j

h

N

N

k

N

i

N

l

N

m

Antibacterial natural products OHCHN

71 OHCHN

n-Bu3Sn

S

S

R

a, b, c N

O

OMs CO2CHPh2

H2N

N

O

H2N

R CO2CHPh2

i)

R CO2CHPh2 c

H2N

COCl

iii)

S OAc N

H N

N

vi), vii)

S

O H2N

BocHN

R CO2CHPh2

OAc

N N

O

N

O

S N

S

ii)

N

S

R

O

S

CO2CHPh2

N

O

iv), v) CO2CHPh2 iv)

TFA H2N

N 1. iii) H2N

H N

N

S

S

O

N

O

OH N

COCl

N

S

OAc

H2N

2. v)

S

N

R

O

CO2CHPh2

CO2H

9a,c-e

Reagents and conditions: i) Pd(CH3CN)2Cl2, LiBr, DMF; ii) cHCl, MeOH; iii) BSA, CH2Cl2; iv) TFA, anisole, CH2Cl2; v) NaHCO3, NH4Cl, MeOH-H2O; vi) Boc2O, MSA, THF; vii) MeCOCl, Et3N, CH2Cl2

Scheme 3 BocHN O

N

BocHN

R-CHO a,b,f,m

S PPh3I

BocHN

S N

O

i)

CO2CHPh2

R CO2CHPh2

O

CO2CHPh2

OH N

a,e,m

a, e-m b

1. iv) or v) 2. vi) 3. vii)

c H N

N O H2N

S

R

N

j,m j,m

S

+

d

S N

O

ii) iii)

R

9j,m = E isomerCO2H 10a,c,d,f-l = Z isomer Reagents and conditions: i) a. 1N NaOH, aq. NaCl, CH2Cl2, b. separation; ii) cHCl, MeOH; iii) a. Cl3CONCO, CH2Cl2, b. SiO2, CHCl3, MeOH; iv) TFA, anisole, CH2Cl2; v) HCO2H, cHCl; vi) BSA or MSA, (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl chloride hydrochloride, CH2Cl2; vii) NaHCO3, NH4Cl, MeOH, H2O

Scheme 4

The research findings at Lilly in the nineteen seventies that 2,5-dichlorophenylthioacetamido at C-7 as a lipophilic side chain conferred excellent Gram-(+) activity to the cephem class have been exploited further [21]. Through a series of optimization at C-3 and C-7, four cephalosporins 11, 12, 13 and 14 possessing a 2,5-dichlorophenylthioacetamido group at C-7 and a polar thiopyridinium group at C-3 with potent in vitro and in vivo

72

Girija S. Singh & Surendra N. Pandeya

anti-MRSA activity have been reported [22]. The C-3 thiopyridinium ring was substituted with amino acid and pyruvic acid groups that were designed to provide aqueous solubility as required for IV formulation. These compounds have excellent in vitro activity against a variety of Gram-(+) bacteria including resistant strains such as penicillin-resistant S. pneumoniae, methicillin-resistant S. epidermitis and S. haemolyticus (Table 5). Furthermore, all of them were efficacious in a systemic murine model of infection with PD5os ranging from 4.8-9.6 mg/kg. The aqueous solubility of 13 and 14 was much more (23 and 40 mg/mL, respectively at pH 7) in comparison to 11 (2-3 mg/mL at pH 7 and at room temperature). H N

R O

S N

H N

R S

O

O CO2

S

N O

N

CO2H NH3

11, R = diClPh (double zwitterion) 12, R = diClpyr

S

O2C

N O

CO2

13, R = diClPh 14, R = diClpyr

Table 5. Antibacterial Activity of Cephalosporin Derivatives. Organism S. aureus/Hetero MR S. aureus/ + 50% calf serum S. aureus/Hetero MR S. aureus/Hetero MR S. aureus /Homo MR S. aureus /Hetero MR S. aureus /Homo MR S. aureus /Homo MR S. aureus /Homo MR S. aureus / MR, PPBP2a IC50 (μg/mL)

A No. A27218

11 0.5

12 1

13 0.5

14 1

M 32

IM 1

A27218

0.5

1

1

1

8

NT

A27217

0.5

0.5

0.5

1

64

1

A25795

1

2

1

1

128

8

A27223

2

2

2

2

128

32

A27223

4

4

16

2

64

NT

A27621

1

2

2

2

64

16

A27295

2

4

4

2

128

64

A27226

1

2

1

2

64

4

A27225

1

2

2

2

128

NT

28

NT

10

4.5

100

NT

MIC in μg/mL, MR = methicillin-resistant, P- = penicillin negative, M = methicillin, IM = imipenem, NT = not tested.

Antibacterial natural products

73

The in vitro and in vivo activity of 7-α-methoxy-cephems and 7-αmethoxy-oxacephems 15-18 and their demethoxy congeners 15a-18a on H. felis and H. pylori, human pathogens associated with type B gastritis, peptic ulcer disease and gastric cancer have been studied that showed the significance of 7-α-methoxy substituents in dealing with these bacteria [23]. The in vivo antibacterial activity was studied on a mouse helibacter infection model after oral administration, in which mice were infected with H. felis. All of the compounds except 18 and 18a exhibited very similar MICs for both H. felis (0.25-0.5 mg/L) and H. pylori (0.5-1.0 mg/L). Compounds 18 and 18a had lower MIC for H. felis (0.13 mg/L) than for H. pylori (1.0-2.0 mg/L). Even though the MICs of all four pairs of compounds were within 1to 2-fold dilution for H. felis and H. pylori, the 7-α-methoxy compounds were at least 4-fold more active at bacterial eradication than their demethoxy counterparts (Table 6). Intravenous administration of flomoxef resulted in extremely low eradication activity compared with oral administration. These results, together with the fact that flomoxef is not absorbed orally, indicated that the compound had direct access to the bacteria in the stomach after oral administration. H Y N

R1 O

X N

O 15-18

Compounds 15 Flomoxef

X O

15a Dimethoxyflomoxef O

R2 CO2H

Y OMe

R1

H

F F

16 1-thiaflomoxef

O

OMe

16a Dimethoxy-1thiaflomoxef 17 Cefmetazole 17a dimethoxycefmetazole 18 M-1

S

H

S

OMe

O

OMe

18a H-1

O

H

H

S

S

S

N

N N

HO

NC

S

R2

S

N

N N

N

N

74

Girija S. Singh & Surendra N. Pandeya

The activity of RWJ-54428, a new parenteral cephalosporin originally developed by the R. W. Johnson Pharmaceutical Research Institute, NJ, USA, against recent isolates of Gram-(+) bacteria, including staphylococci with decreased susceptibility to vancomycin 6 has been reported [24]. This compound has been shown to be active against a wide range of multiply resistant Gram-(+) pathogens, including oxacillin-resistant S. aureus (MRSA), E. faecalis (MIC90 = 0.5 mg/L), vancomycin-resistant E. faecalis (MIC90 = 0.25 mg/L), and penicillinresistant pneumococci and streptococci (MIC90 = 1 mg/L). The only group of organisms for which the MIC90 was >2 mg/L was ampicillin-resistant E. faecium. Reinert and coworkers have evaluated cefditoren against penicillin-susceptible strains of S. pneumoniae and penicillin-intermediate strains of S. pneumoniae isolated from patients with respiratory tract infections and suggested it as a promising agent for the treatment of infections caused by pneumococci with reduced penicillin susceptibility [25]. Gerber and coworkers have reported cefepime, considered as fourth generation cephalosporin, having an excellent CSF penetration with level ranging between 10 and 16 mg/L after two intravenous injections (100 mg/kg). The bactericidal activity of cefepime was superior to ceftriaxone and vancomycin in the treatment of rabbits with meningitis caused by an isolate highly resistant to penicillin [26]. Schito and coworkers have studied the activity of many cephalosporins against some common respiratory tract pathogens such as S. pneumoniae, H. influenzae and M. catarrhalis etc., isolated from the patients in Italy, Spain, and Austria [27]. Cefpodoxime has been reported as a suitable choice for use. Table 6. Clearance and Eradication Doses of compound. Compounds 15 Flomoxef 15a Demethoxyflomoxef 16 1-thia-flomoxef 16a Demethoxy-1-thiaflomoxef 17 Cefmetazole 17a Demethoxycefmetazole 18 M-1 18a H-1 Amoxicillin 15 Flomoxef (iv) a

50% Clearance dose (mg/kg/dose)a 1.00 4.00 0.97 3.84 1.00 5.79 0.47 3.59 1.92 >15.0

50% Eradication dose (mg/kg/dose)b 3.67 17.4 3.38 >60 3.67 58.8 Not tested Not tested 9.12 58.8

Compounds were administered orally twice a day for 1 day and mice were killed on the following day. b Compounds were administered orally twice a day for 5 days and mice were killed after 14 days.

Antibacterial natural products

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Some amides 19 and imines 20 containing 5-nitrofuryl and 3-methoxy-2nitrophenyl groups from 7-β-aminocephalosporanic acid and 7-βaminodesacetoxycephalosporanic acid have been synthesized and evaluated for antibacterial activity [28]. Many compounds, especially with 5-nitrofuryl moiety, exhibited an activity equal to or better than those of ampicillin or cephalexin against the majority of Gram-(+) organisms tested. None of the compounds showed appreciable activity against E. coli. ArOCHN O

S N

CH2OCOMe CO2Na

19

ArHC N O

S N

CH2OCOMe CO2Na

20

Ishikawa and coworkers are involved in the development of new cefozopran (CZOP) 21 derivatives for use against MRSA [29,30]. They observed that the CZOP with lipophilic alkoxyimino groups in the C-7 acyl moiety showed potent anti-MRSA activity. Cyclopentyloxyimino derivatives with amino-based substituent(s) in the C-3’ azole moiety had anti-MRSA activity comparable to vancomycin. In order to further increase the activity they have modified the C-3 linked spacers of cephem derivatives bearing a 1-methylimidazo-[1,2-b]-pyridazinium-6-yl group at the C-3’ position and a 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-cyclopentyloxyiminoacetyl group at the C-7 position [31]. They have found that the optimal spacers are (E)-2vinyl and (E)-2-thiovinyl groups. The anti-MRSA activity of the compounds 22a,b bearing these spacers were 16-32 times higher than CZOP. Taking these two spacers they have modified the alkoxyimino group in the C-7 acyl moiety and the 1-alkylimidazo-[1,2-b]-pyridazinium moiety at C-3’ and discovered compound 22c with anti-MRSA activity comparable to vancomycin both in vitro and in vivo, high affinity (IC50 = 2.7 mg/mL) for PBP2’ of MRSA and potent activity against Gram-(-) bacteria as well. Hakimelahi and coworkers are working on the concept of using antibacterial prodrugs. They have reported the synthesis, antibacterial and β-lactamase inhibitor activity of clavunate derivatives of amoxicillin. The compounds screened by them showed better antibacterial activity than amoxicillin and clavulanic acid combination augmentin [32]. Since the discovery of thienamycin (23a) from the fermentation broth of soil bacteria Streptomyces cattleya in 1976 several carbapenem derivatives have been synthesized and evaluated for their antibacterial activity. Thienamycin itself has excellent antibacterial activity against both Gram-(+) and Gram-(-) bacteria, and is resistant to β -lactamases. Numerous methods

76

Girija S. Singh & Surendra N. Pandeya

N N

OMe H N

H2N S N

O O

N

N

S

N

N

N

OMe H N

S

H2N

N

S N

O

N

O

CO2Na

21

CO222a

1

N N

OR H N O O

N

N Me

S

H2N S N

N

R2

N

N

S

N N

CO2-

22b, R1 = cyclopentyl; R2 = Me 65b 22c, R1 = CH2F; R2 = Me 65c

are described in literature for the total synthesis of thienamycin [33]. Currently, two 1-H carbapenems, imipenem (23b) and panipenem (23c), and one βmethyl carbapenem, meropenem (24) are available in the market for clinical use [34-36]. Although arbapenems have a broad antimicrobial spectrum and potent bactericidal activity [37] most of them have some limitations as well from the view point of clinical application. For example, imipenem is unstable to the renal dehydropeptidase-I (DHP-I) and has epileptic side effect. Meropenem has good stability to DHP-I due to steric hindrance of β-methyl group at C-1 and an excellent spectrum against Gram-(-)bacteria, but it is relatively less active against Gram-(+) bacteria than imipenem. The studies on carbapenems from a pharmaceutical point of view in the previous decade have been devoted mainly to the synthesis and evaluation of 1-β-methylcarbapenem derivatives as antibacterials. New methodologies for construction of the carbapenem skeleton are under investigation by Mori and

Me

OH H H

X R

N O

CO2H

X = 23a-c: H; 24: Me R = 23a: -SCH2CH2NH2 (Thienamycin) 23b: -SCH2CH2NHCH=NH (Imipenem) 23c:

N

S

Me (Panipenem) NH

Me2NOC 24 S

NH

(Meropenem)

Antibacterial natural products

77

Kozawa [38,39]. A new method involving palladium-catalyzed C-N bond forming reaction (Scheme 5) in azetidinone 25 leading to the synthesis of carbapenem 26 with a carboxylic group on C-3 of the five–membered ring has been reported by them [40]. 10 mol% Pd(OAc)2 15 mol% DP Ephos PhMe, 100 oC, base (2 equiv.)

OSi H H

N

NH O

X

OSi H H

O

CO2Et

CO2Et 26

25

Scheme 5

3. Macrolide and ketolide antibiotics Macrolide antibiotics belong to the subgroup of polyketide natural products and constitute an important therapeutic class. They act against community-acquired respiratory infections such as community-acquired pneumonia, acute bacterial exacerbations of chronic bronchitis, acute sinusitis and tonsillitis [41,42]. Macrolides account for 20% of all the antibiotics prescribed. The principal representative of macrolides erythromycin A 5 was first isolated from Streptomyces erythreus at the Lilly in 1952 [43]. Its absolute configuration was established by NMR spectroscopic studies and X-ray crystallographic data [44,45]. It helps against the major respiratory pathogens, is considered safe and is widely prescribed for children. However, it has a limited antibacterial spectrum and limited solubility in acidic medium. The second generation macrolide antibiotics, 27-29 have gradually replaced erythromycin A because of their higher potency, broader spectrum of activity, improved physicochemical and pharmacokinetic profiles, and attenuated side effects [46]. However, similar to erythromycin A, the second generation variants also have poor activity against macrolide resistant pathoges. The mode of action of macrolide antibiotics involves blocking bacterial protein biosynthesis by binding to the 23S ribosomal RNA of the 50S subunit and interfering with the elongation of nascent peptide chains during translation [47]. Located in domain V, near the peptyl transferase site, macrolide antibiotics obstruct the peptide exit tunnel without affecting peptidyl transferase activity. Ketolides [48] are derived from 14-membered macrolides by removal of L-cladinone under acidic conditions and selective oxidation of the resulting 3-hydroxy group to the corresponding carbonyl group. The first semisynthetic

78

Girija S. Singh & Surendra N. Pandeya O N

O

O

OMe

OMe

HO OH

HO

O

OH

HO

O

OH

NMe2

OMeO O

O

O

NMe2

OMeO O

O OH

O

HO

O

OH O Roxithromycin (28)

Clarithromycin (27) Me N

N OH

HO OH

HO

O O

N

O

N

O

O O

N

HO

NMe2 O

OMeO O O

OH

OH

Azithromycin (29)

O

O O

NMe2 O

Telithromycin (30)

ketolide RU-64004 (HMR 3004) was synthesized at Roussel Uclaf [49]. This ketolide was stable in acidic media, showed good intracellular penetration, and demonstrated potent activity against erythromycin A resistant and penicillin resistant streptococci and H. influenzae. Systematic SAR studies led to the discovery of several ketolide lead structures such as telithromycin, cethromycin, and EP-013420 with potent activity and improved pharmacokinetic profile. Telithromycin 30 was the first ketolide to be approved in Europe (2001), Japan (2003) and in the US (2004) for the once daily oral dose for treatment of respiratory tract infections. It was synthesized from clarithromycin in eight steps [50].

4. Lincosamides Lincomycin 31 and its semi-synthetic congener clindamycin 32 were introduced into clinical use as oral antibiotics in 1960 and 1969, respectively [51]. They exhibit a similar spectrum as macrolides, including activity against most gram-(+) organisms and the anaerobes, but not the Gram-(-) and enterococci [52]. Now a day they are not in much use due to their limited antibacterial spectrum, the emergence of resistance, and severe side effects of this class.

Antibacterial natural products

79

Clindamycin is a semi-synthetic derivative of the natural product lincomycin, which is produced by fermentation of Streptomyces lincolnensis [53]. This transformation involves selective transformation of only one secalcoholic group of the three present (Scheme 6).

Me N

HO O N H HO

O

SMe

NCS, PPh3 THF, Δ, 18h

OH

Me N

O

Cl N H HO

O

SMe OH

OH

OH

84% 32

31

Scheme 6

The mode of action of lincosamide involves binding to the ribosome and inhibiting bacterial protein synthesis. Specifically, macrolides, lincosamides, and streptogramin B type antibiotics bind to adjacent sites on the 50S ribosomal subunit. The complexes of bacterial ribosomes with these antibiotics have been studied by X-ray crystallography [54,55]. Several methods for semi-synthetic modification of lincomycin and clindamycin have been published in addition to the methodologies developed during the total synthesis of natural product [56,57]. Substitution of the 7-hydroxy group by a methyl group in conjunction with novel amides resulted into discovery of VIC-105404 33 and VIC-105555 34 (Scheme 7) [58,59]. The latter compound has rapidly been progressed into preclinical development. Hopefully these achievements will translate into clinical benefit.

5. Furanomycin L-(+)-Furanomycin 35 is a low molecular weight (157 g/M) antibacterial natural product. It is a α-amino acid isolated by Katagiri and coworkers in 1967 from the fermentation broth of Streptomyces threomyceticu L-803 (ATCC15795) [60]. It inhibits the growth of bacteria such as M. tuberculosis, E. coli, B. subtilis, and some Shigella- and Salmonella species in the μM range. Its initially assigned absolute configuration was later on revised to (+)(αS,2R,5S) by synthesis starting from D-glucose and by X-ray crystallographic study of the N-acetyl derivative [61-63]. Furanomycin 35 is accepted as a substrate by isoleucyl aminoacyl-t-RNA synthetase and its antibacterial activity results from a substitution for isoleucine during the bacterial protein translation [64]. Therefore, the antibacterial activity of furanomycin is antagonized by isoleucine. Furanomycin hampers the formation

80

Girija S. Singh & Surendra N. Pandeya Me N

HO O

TMSO O

N H HO

SMe

i), ii), iii)

O

BocHN

OH

TMSO

OH

SMe

iv), v)

OTMS OTMS

31

O

BocHN TMSO

SMe

H2N

vi), vii), viii)

BocHN

OTMS OTMS

O

HO

Boc N CO2H

SMe

OH

H3C(H2C)4 ix), x), xi)

xii), xiii) NH

Boc N

OH

HO

O

O

HN BocHN

O

HO

N

SMe

HN BocHN

H3C(H2C)4

OH OH

O

HO

33 VIC-10555

34 VIC-105404

SMe OH

OH

Reagents and conditions: i) N2H4, H2O; II) (Boc)2O, Et3N, MeOH; iii) BSTFA, Et3N, DMF; iv) DMSO, (COCl)2, Et3N, CH2Cl2, -70 - 40oC; (v) PPh3Me+Br-, t-BuOK; vi) Dowex H+, MeOH; vii) H2 (65psi), Pd/C; viii) TFA/H2O (9:1); ix) HBTU, Et3N; x) TFA/H2O (9:1); xi) oxirane, Et3N; xii) HBTU, Et3N; xiii) TFA/H2O (9:1)

Scheme 7

of isoleucine-tRNA in E. coli, whereas other aminoacyl-tRNA are not affected [65,66]. Aminoacyl-tRNA synthetases are essential in all living organisms and have attracted considerable interest as novel targets in bacterial protein synthesis [67-70].

O H2N

CO2H 35

O H2N

CO2H 36

H2N

CO2H 37

Antibacterial natural products

81

Several approaches have been developed towards the synthesis of Furanomycin 35. Many syntheses start from carbohydrates such as D-glucose [51], D-ribose [71], D-glucosamine [72], L-xylose [73], and D-mannitol [74]. Glycine [75], serine [76], furans [77], and dimethyl L-tartarate [78], have also been used as substrate for the synthesis of Furanomycin 35. Approaches involving amino acids are appealing as they involve lesser steps. A SAR was studied using many synthetic isomers and derivatives of natural products. Unfortunately, all of them showed either no activity or poor activity against a panel of selected Gram-(+) and Gram-(-) pathogens including S. aureus, and E. coli. Only L-(+)-dihydrofuranomycin 36 showed borderline MIC (32-64 μg/mL) against S. aureus. The chiral carbon analogue 37 exhibited weak antibacterial activity (4 μg/mL) against an efflux-pum-deficient E. coli. Furanomycin, thus, proved an insufficient lead and could not be a valuable compound as a starting point for an antibacterial drug discovery program.

6. Pyrrolidinedione antibacterials Komura and coworkers in 1987 isolated natural peptide antibiotic compound andrimid 38 from cultures of a symbiont of the brown planthopper Nilaparvata lugens [79]. Later on moiramide 39 was discovered in a marine isolate of Pseudomonas fluorescens obtained from a tunicate collected in Moira Sound at Prince of Wales Island, Alaska [80]. The structures of these metabolites contain four characteristic elements: a pyrrolidinedione head group, a valine derived β-ketoamide, a (S)-β-phenylalanine moiety, and an N-terminal polyunsaturated fatty acid. Various diastereoselective and asymmetric total syntheses of andrimid and moiramides are known that allow ready access to these antibiotics [81,82]. Pyrrolidinedione antibiotics act by inhibiting the first committed step in bacterial fatty acid biosynthesis, a reaction catalyzed by the carboxyltransferase subunit of the multimeric bacterial enzyme acetyl-CoA carboxylase [83]. For most living organisms, fatty acid biosynthesis is a vital metabolic process, but the pathway in bacteria and mammals are different. Acetyl-CoA carboxylase is essential for microbial growth and is broadly conserved amongst bacteria [84].

O O

O NH

n N H

N H

O

O

n = 2: Andrimid 38; n = 1 moiramide B 39

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Girija S. Singh & Surendra N. Pandeya

Using (S)-(-)-methylsuccinic acid 40 as a substrate, the synthesis of a pyrrolidinedione antibacterial 41 is shown in Scheme 8. Through this route, and also by solid-phase synthesis starting with polymer bound (S)-β-phenylalanine, a wide variety of pyrrolidinedione antibacterial are known in literature [85,86]. Broad structural variations were tolerated at the fatty acid side chain without adversely affecting the bioactivity. Inhibitory values (IC50) remained in the nM range for the E. coli and S. aureus acetyl-CoA carboxylase enzymes with polar and lipophilic side chains as well. CO2H

O i), ii)

HO2C

CO2H 40

86%

O

iii)

iv), v), vi)

NHBoc N OBn

N OBn 40%

O

BocHN

68%

O

O

O O NC

N H

O

O

O

vii)

NH H2N

O

CO2H

66%

O N H

NC

NH N H

O

O

41

Reagents and conditions: i) MeCOCl, 4h, 60 oC; ii) O-benzylhydroxylamine, CDI, CH2Cl2, 12h, rt; iii) 1. NBoc-(2S)-cyclopentyl glycine, CDI, THF; 2. LiHMDS, THF, 15 min, -65 oC; 3. conc. aq. NH4Cl,- 65 oC - rt; iv) H2, Pd/C (10%), EtOH, 1h, rt; v) 2'-bromoacetophenone, Et3N, cat DMAP, MeCN, 20h, rt; vi) 4N HCl in 1,4-dioxane, 2h, rt; vii) HATU, iPr2EtN, CH2Cl2, DMF, 10h, 0 oC - rt.

Scheme 8

Apparently, the side chain is not involved in key interactions with the enzyme and could be used for tuning the physicochemical profile. On the other hand, the nature of the side chain has a significant effect on antibacterial activity. A comparison of compounds 41a and 41b demonstrated that despite excellent target activity of 41a and the benefit of a polar substituent for other parameters, such as solubility, reasonable lipophilicity was required for penetration into bacterial cells and for good MIC values. Replacement of the (S)-β-phenylalanine by non-aromatic β-amino acids led to a loss in activity. On the other hand significant activity was observed by varying the lead’s β-ketoamide part, for example, by replacing (S)-valine with (2S)-cyclopentyl glycine, whereas aromatic amino acids in this position rendered the molecule inactive.

Antibacterial natural products

83

O O HO2C

O N H

O O

NH N H

O

O N H

O

41a

NH N H

O

O

41b

7. Tetrahydropyrimidinone antibiotics The titled class of antibiotics was first isolated by scientist from the Takeda Foundation in Japan from Flexibacter species found in soil samples of the Nachi mountain area of the Wakayama prefecture of Japan [87]. The structures of TAN-1057A-D 42-45 were disclosed in 1993 [88]. The epimeric tetrahydropyrimidinones TAN-1057A/B 42,43 were isolated from Flexibacter species, PK-74, whereas the epimeric dioxo diazepans TAN 1057C/D 44,45 resulted from Flexibacter species PK-176. Total synthesis endeavors and medicinal chemistry optimization focused on TAN-1057A/B 42,43 due to instability problem of TAN 1057C/D 44,45. The antibacterial activity of TAN1057A 42 was studied in detail. Its in vitro antibacterial activity against Gram-(+) organisms such as S. aureus and S. pneumoniae was mediocre under standard conditions (6.25-12.5 μg/mL). However, its in vivo activity was reported superior to vancomycin and imipenem in a murine S. aureus sepsis model. NH O NH H2N

N H

Me N 5 NH2 O

O

42 TAN-1057A (5S) 43 TAN-1057B (5R)

H2N NH O N H

NH2

N H

HN

N Me 2

NH H N HN 44 TAN-1057C (2R) O 45 TAN-1057D (2S) O

NH2

TAN-1057A/B acts by blocking bacterial protein biosynthesis [89]. The detailed studies found that it inhibited bacterial growth through binding to the 50S subunit of ribosomes [90]. The synthesis of TAN-1057A/B 42,43 was reported by Yuan and Williams which involved a rather linear approach starting from triple-protected β-homoarginine (Scheme 9) [91]. Meijere and coworkers published a more convergent synthesis shortly after [92]. The attractive antibacterial properties and the structure of natural antibiotics TAN-1057A/B attracted several synthetic research groups. Systematic SAR exploration required novel routes to β-lysine and β-homolysine derivatives. The synthetic pyrimidinones (n = 1) 46 and (n = 2) 47 readily accessible on large scale exhibited improved cytotoxicity and tolerability while retaining eminent potency of the natural compound.

84

Girija S. Singh & Surendra N. Pandeya

8. Biphenomycin An antibiotic with unusual biological properties, LL-AF283a was isolated by fermentation of S. filipinesis at the Lederle Laboratories in 1967 [93,94]. Later on the discovery of peptide antibiotic biphenomycin A 48 (WS-43708A) was reported by scientists from Fujisawa in 1984 [95-97]. In 1991, Border and coworkers found that the two antibiotics were identical [98]. Biphenomycins have unique structure with a cyclic tripeptide containing a biphenyl moiety in a 15-membered ring. The in vitro activity of biphenomycin A was almost limited to Cornbacterium xerosis. It could not affect the growth of other bacteria, such as S. aureus, E. coli, or S. pyogenes up to 200 mg/mL. However, it was NH Z

N H

OH

N Z

NH

NH

O

i), ii), iii)

Z

+ Me

H N

Z

51%

N H

Me N

N Z

NH O

79%

O

NHMe

N H CO2H

Z NPhth CO2tBu NH2 O

iv)

O

NH Z

N H

Me N

N Z

H N

SMe

NHBoc

, v) NHZ

52%

CO2H

NH O

N

Z NH Z

vi), vii) TAN-1057A/ TAN-1057B 42,43

Me N

N Z

N H

66%

NH Z

O O MeS

H N

NHBoc

N

O N H

NHZ

Reagents and conditions: i) BOPCl, 16h; ii) MeNH2, MeOH, 5min; iii) TFA/anisole 25:1, 0 oC to rt, 1h; iv) Boc2O, Et3N, H2O/dioxane (1:1), 16h; v) EDC, DMAP, CH2Cl2, 16h; vi) TFA/anisole 10:1, 15 min, evaporation, then Et3N, THF, 10 min; vii) PdCl2, H2, MeOH/CH2Cl2, 2:1, 99%.

Scheme 9 Me N 5

H2N

n

NH O

NH2 O

N H

O 46 n = 1, 47 n = 2

HO

OH H N

H2N O

O

R CO2H N H OH

NH2 48 R = OH, Biphenomycin A 49 R = H, Biphenomycin B

NH2

Antibacterial natural products

85

highly effective in vivo in a murine sepsis model. It protected mice from an otherwise lethal infection against S. aureus Smith (ED50 1.0 mg/kg) and was five times more effective than vancomycin on subcutaneous administration. The reason for this discrepancy between in vitro and in vivo activity is yet not clear. Although both the biphenomycins and vancomycin have a biphenyl group, there is no evidence of binding of to the cell-wall analogues of N-AcD-Ala-D-Ala. Instead, bacterial protein biosynthesis appeared to be the target of these antibacterials [99]. The biphenomycins represented an attractive starting point for an optimization program in medicinal chemistry and need for its synthesis was felt. The first total synthesis of Biphenomycin B 49 was reported by Schmidt and coworkers in 1991 [100]. Its sequence involved 1) synthesis of (S,S)isotyrosine, 2) formation of an ansa-tripeptide, 3) macrocyclization, and 4) removal of protecting groups (Scheme 10). However, besides the total synthesis of natural biphenomycins A and B, neither derivative nor close analogues had been prepared. A first series of simplified amide and ester derivatives, including derivatization at the peptide backbone have been reported recently [101,85]. The novel congeners 50 and 51 of biphenomycin B 49, showed improved in vitro activity (Table 7) [102]. Table 7. In vitro antibacterial activity of 50 and 51 against Gram-(+) pathogens, MIC (μg/mL). Compd No. 50 51

S. aureus 1.5 0.1

E. faecalis 1.0 3.0

B. catarrhalis 1.0 1.0

Thus, the route for the total synthesis of natural compounds and their congeners with improved in vitro efficacy has been established. A further insight into the molecular target of these compounds will definitely pave the way for further development.

HO

OH H N

H2N O

HO

OH

H

O

N H O OH

NH2

H N

H2N O

NH2 50

H

O

N H O OH NH2

51

OMe

86

Girija S. Singh & Surendra N. Pandeya

BnO

OBn i), ii)

O

CHO

BnO

OBn

O

94%

BocHN

BocHN

CO2Me

vii), viii), ix 88%

BocHN CO2TMSE

ZHN

CO2Bn

BocHN

CO2Bn

CO2H O

OBn

OBn

OHC

69%

O

O

BnO

iii), iv), v), vi) BnO

BnO

x), xi)

N Z

ZHN ETMSO

71%

OBn O BocHN O

N H O

CO2Bn

N Z xii), xiii) HO

OH H N

H2N O

O

H CO2H N H OH

81%

BnO xiv), xv)

ZHN

60% C6F5O

NH2 Biphenomycin B (49)

OBn O BocHN O

N H OH

CO2Bn

NHZ

Reagents and conditions: i) methyl N-tert-butoxycarbonyl(dimethoxyphosphoryl)glycinate, LiCl, DBU, MeCN, rt, 1h; ii) Et3N, C, EtOH/CHCl3, (1:1), rt, 2days; iii) LiOH, H2O, dioxane, rt, 12h; iv) [Rh(cod)dipamp)]BF4, H2, MeOH, rt, 72h; v) BnOH, DCC, DMAP, EtOAc, - 15 to 20oC, 12h; vi) PPTS, acetone, H2O, Δ, 6h; vii) Nbenzyloxycarbonyl(dimethoxuphosphoryl)glycine tromethyl-silyl ester, LiCl, DBU, MeCN, rt, 2h; viii) Et3N, C, EtOH/CHCl3, (1:1), rt, 2 days; ix) [Rh(cod)dipamp)]BF4, H2, MeOH, rt, 72h; x) HCl, dioxane, 20oC, 2h; xi) EDC, HOBt, CH2Cl2, 15-20oC, 14h; xii) AcOH/H2O (9:1), 50oC; xiii) Bu4NF, DMF, rt, 1h, C6F5OH, EDC, CH2Cl2, - 15 to 20oC, 14h; xiv) HCl, dioxane/CH2Cl2 (1:1) 0oC, evaporation, CHCl3, NaHCO3, 20oC, 5min; xv) trimethylsilyl trifluromethanesulfonate, thioanisole, TFA, rt, 30 min.

Scheme 10

9. Tuberactinomycins and capreomycins Tuberactinomycins 52-55 and capreomycins 56-59 are closely related cyclic homopentapeptides. The first member viomycin (Tuberactiinomycins B) 52 was discovered in 1951 [103] and marketed by Ciba and Pfizer as a tuberculostatic agent in the 1960s. The capreomycins were isolated from the fermentation of Streptomyces capreolus as a four-component mixture, with 56 and 57 as the major and 58 and 59 as the minor components [104]. Both subclasses showed good activity against Mycobacteria including multi-drug resistant strains but had only limited activity against other species [105].

Antibacterial natural products

87

R1 H2N

OH H N

O

H N

N H

NH2 O

NH O

OH

O

O H N

HN

H N

O

O NH R2

N H

NH2

NH

52 R1 = H, R2 = O, Tuberactinomycin B; 53 R1 = R2 = OH, Tuberactinomycin A (viomycin); 54 R1 = OH, R2 = H, Tuberactinomycin N; 55 R1 = H, R2 = H, Tuberactinomycin O

Tuberactinomycins exert their antibacterial activity as potent inhibitors of the translation step of prokaryotic protein biosynthesis by inhibiting both the initiation and elongation steps. A detailed report on the interaction of tuberactinomycin at the target level illustrated how these compounds interacted with RNA [106]. When tested against a panel of M. tuberculosis strains in vitro, capreomycins compared favorably with streptomycin, cycloserine, and kanamycin [107]. No cross resistance of tuberactinomycins with kanamycin, lividomycin, or paronomycin was observed [108]. The in vivo efficacy of these compounds was low after oral dosing, but good after subcutaneous administration in experimental murine tuberculosis models [109]. Although tuberactinomycins were not devoid of toxicological problems their toxicity profile after i.v. and preoral administration was quite favorable [110]. These biological features warranted further evaluation of this class for their clinical use as antitubercular agents. R1

O H2N

H N

N H NH O

O H N O NH

HN

N H O H N

R2

NH2 O

N NH H 56 R1 = OH, R2 = β-lysyl, Capreomycin IA; 57 R1 = H, R2 = β-lysyl, Capreomycin IB; 58 R1 = OH, R2 = H, Capreomycin IIA; 59 R1 = R2 = H, Capreomycin IIB

88

Girija S. Singh & Surendra N. Pandeya

Most of the derivatives prepared for biological testing have been obtained through fermentation and semisynthesis. The acetylation of the terminal amino group 60 or both amino groups of β-lysine (Scheme 11) led to a complete loss in activity [111]. Acylation with uncharged or acidic amino acids at the same position also produced inactive compounds whereas introduction of a basic amino acid 61 maintained the original MIC [112]. Similarly, blocking of the serine hydroxyl groups 62 left the activity unaltered [113]. Surprisingly, hydrolysis of the urea functionality produced 63 with comparable in vitro activity as observed for viomycin 52. Oxidation of 52 yielded the inactive bisamide 64 [114]. Finally, reductive opening of the capreomycidine ring of 52 forming 65 led to a complete loss in activity [115]. O P(OiPr)2 O H N

O H2N NH2

N H

NH2 O

O

61

i) N H

iv) OH H N

O

H N

H2N

O 62

ii), iii)

O

N H

NH2 O

NH

NH2 O O 60

O HN

H N O NH

vii)

HO

NH2 N H

HO 65

vi)

O

O

O P(OiPr)2

N H

OH O H N

NH2 O

NH

v)

52 HN

HN

OH O 64

63

Reagents and conditions: i) N-acetoxysuccinimide, Et3N, carbonate buffer, dioxane, 1h; ii) Z-D-Orn(Z)OSuc, Et3N, carbonate buffer, THF, 0oC, 12h; iii) H2, Pd, DMF; v) HOH; vi) KMnO4 ; vii) NaBH4

Scheme 11

Antibacterial natural products

89

Similar modifications of tuberactinomycine N 54, tuberactinomycine O 55, and capreomycins did not result in an improved activity against Micobacteria or an extension of the antibacterial spectrum. The 3,4-dichlorophenylamino analogue 66 of viomycine exhibited good MICs against the animal pathogens P. multocida (MIC = 0.39 μg/mL) but only mediocre activity against E. faecium and E. faecalis (MIC = 25 and 12.5 μg/mL, respectively). Further variation of the substituted ureido analogues of capreomycines IA/IB (mixture of 56 and 57) yielded novel compounds with activities against several multidrug resistant gram-(+) pathogens and gram-(-) E.coli [116]. Despite proven in vitro and in vivo efficacy of the novel analogues no further clinical development in this class has taken place.

H2N

OH H N

O

H N

N H

NH2 O

NH O

O HN

H N

OH O H N

O NH

Cl Cl

HO

N H

NH

66

10. Glycopeptide antibiotics Vancomycine 6, the first gylcopeptide introduced into clinical practice in 1959 was isolated from Streptomyces orientalis from soil samples by the Lilly in the mid 1950s [117]. Its structure was unequivocally established in the early 80s [118, 119]. Teicoplanin 67 is the only additional member of this class that is available for human use. Both drugs are unaltered natural antibiotics of the large dalbaheptide group that is produced by various actinomycetes. Their common structural element is a linear heptapeptide backbone (configuration R,R,S,R,R,S,S) in which some aromatic amino acids residues are cross linked (biphenyl and diphenyl ether motives) and build a rigid concave shape. Glycopeptides inhibit bacterial cell walls biosynthesis by recognizing and strongly binding to the L-Lys-D-Ala-D-Ala termini of peptidoglycan precursor strands at the external side of the membrane. In this way, transpeptidases are prevented from executing their cross linking job [120].

90

Girija S. Singh & Surendra N. Pandeya

Glycopeptides antibiotics are restricted to treating gram-(+) infections as they cannot penetrate the outer membrane of gram-(-) bacteria. With the rise of MRSA infections in hospitals, vancomycines became the antibiotic of last resort but, due to its frequent use, resistant gram-(+) pathogens, in particular vancomycine-resistant enterococci (VRE) has emerged and worryingly spread. By 2003, more than half of the clinical VRE isolates in the US had become resistant to glycopeptides. Three semi-synthetic second generation drugs oritavancin (Ly-333328) [121], dalbavancin (Bi397) [122], and telavancin (TD-6424) [123] have been advanced to clinical developments [124]. O OH O

N H

OH OH

O Cl

OH HO HO

O

O Cl

O O

NH O

O HN

O

O

H N

N H

O

O

H N

N H

O

NH2

N H

CO2H HO HO

O

O OH

OH OH O

OH OH

OH

67

11. Lysobactins The lysobactins are good examples of structurally exciting natural products that were isolated from urban soil organisms. Ketanosin B 68 was isolated from the fermentation broth of Lysobacter sp. SC-14076 (ATCC 53042) by scientists from Squibb [125,126]. So far its total synthesis has not

Antibacterial natural products

91

been published. Recently, katanosin A has been found as a minor metabolite of the Lysobacter sp. ATCC53042 starin [127]. Lysobactin and katanosin A are highly active against Gram-(+) bacteria such as staphylococci and enterococci. Their excellent in vitro antibacterial activity was maintained in vancomycin-resistant enterococci. Promising therapeutic in vivo efficacy has been demonstrated in a systemic murine S. aureus infection model (ED50 = 1.8 mg/kg, i. v., CFU = 105) [128]. The primary target of lysobactin antibiotics appears to be the bacterial biosynthesis. These compounds inhibit consumption of the cell-wall precursor [14C]GlcNAc, a very good indicator for interference with the peptidoglycan biosynthesis. The inhibition of peptidoglycan formation is most likely induced by the binding of lysobactin to lipid intermediates (not through binding to biosynthetic enzymes) that occurs as biosynthetic precursors downstream of the muramyl pentapeptide. The lysobactins are interesting antibacterial lead structures with promising in vitro activity and in vivo efficacy. So far, the knowledge about this class is based on these natural products and some semi-synthetic Edman derivatives. A preliminary SAR has been established for the amino acid position 1 within the liner segment. Further investigations need to be done for a real assessment of the potential of this class.

H2N O

HO

OH H N

H N

NH

O

O

O O

H2N

N H

NH

HN

N H

HO

NH

O

O

O

O

O O

O

NH

O

HO

N H NH2

HN NH 68

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Girija S. Singh & Surendra N. Pandeya

12. Enopeptin depsipeptide antibiotics The name of the family was derived from two depsipeptides enopeptin A 69 and depsipeptides enopeptin B 70 isolated in 1991, from a culture broth of Streptomyces sp. RK-1051, found in a soil sample collected in Tsuruoka city of Japan [129]. They consist of a 16-membered lactone ring made up of five (S)-amino acids and a lipophilic polyene side chain attached to the serine N-terminus [130]. About a decade ago the Eli Lilly published the isolation of a similar depsipeptide antibiotic A54556 71,72, a complex of eight depsipeptide factors A-H, which was produced by aerobic fermentation of Streptomyces hawaiiensis (NRRL 15010) [131]. Mode of action studies with B. subtilis demonstrated impaired bacterial cell division and induction of filamentation. It has been shown by using RG techniques that lead structures inhibited bacterial growth by binding to caseine lytic protease [132,133]. R O N O O

NH N

O

O

O N

O N H

HO O

HN

N H

O

O

69 R = Me, Enopeptin A 70 R = H, Enopeptin B 71 R = Me, A54556A 72 R = H, A54556B

O

The natural enopeptin depsipeptides antibiotics had promising in vitro activity against enterococci and streptococci but only moderate in vitro potency against staphylococci and were inactive against Gram-(-) bacteria. Both lead structures were not effective in vivo in standard lethal bacterial infection models in mice and their ADME profile was critical. Their chemical stability proved to be rather limited. Their solubility was insufficient for parenteral application and they were readily cleared from the body. The in vivo antibacterial activity of some natural and synthetic enopeptins is described in Table 8.

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93

O R O

O O

NH

N

N

O

O O

N H

F

N

O

O

O

F

O

N

HN

NH

C6H11

O

O

O

N H

N

N

HN

O

O 77

73-76

F

O O NH

O

F

N

N

O

F

O

F

O

O N

N

O N H

O O

HN

C4H9

RO

NH

O

N

O

O N

O N H

HN

C4H9 O

O 79, 80

78

Table 8. Antibacterial activity of some natural and synthetic enopeptins. No. 71 72 73 74 75 76 77 78 79 80

R Me H H 3-F 3,5-F2 3,4,5-F3 H COCH2NMe2

S. aureus 8 16 > 64 1 0.5 8 1 ≤ 0.125 0.25 0.5

S. pneumoniae 0.5 1 > 64 0.25 ≤ 0.125 2 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

E. faecium 1 2 > 64 ≤ 0.125 ≤ 0.125 2 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

E. faecalis 1 2 > 64 0.125 0.125 1 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

13. Tetracycline antibiotics Tetracyclins belong to the group of polyketides. They are old-known class of broad-spectrum antibiotics whose use has been reduced in recent times with the onset of bacterial resistance. They consist of an octahydrotetracene-2-carboxamide skeleton [134]. The first member of the group, chlorotetracycline 81 was discovered in 1940s from a golden-colored fungus-like, soil-dwelling bacterium called Streptomyces aureofaciens. Soon after oxytetracycline 82 was discovered from a similar soil bacterium called Streptomyce srimosus. The structure of oxytetracycline was determined by

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Woodword and coworkers [135], which led to its synthesis by Conover and coworkers. Doxycycline 83 is the most commonly known semi-synthetic drug of this class. In 2005, tigecycline [136] 84 belonging to the subclass of glycylcyclines was introduced to treat infections that were resistant to other antimicrobics including conventional tetracyclins [137]. Newer versions of tetracyclins are currently in trials. Tetracyclins inhibit the protein biosynthesis by inhibiting the binding of aminoacyl-tRNA to the mRNAribosome complex. Tetracyclins have also been found to inhibit matrix metalloproteinase. This mechanism does not contribute to their antibiotic activity, but has led to extensive research on chemically modified tetracyclins. Tetracyclins inhibit cell growth by inhibiting translation. It binds to the 16S part of the 30S ribosomal subunit and prevents the aminoacyl tRNA from binding to the A site of the ribosome. The binding is reversible in nature [138].

OH

O

O OH

OH OH

O

O

O OH

OH OH

H2N

H2N O N

HO

H

OH Cl

N

81

H

OH 82

OH O

OH O OH

O NH2

H

H OH

OH

83

N H N

H

N

O N H

OH O

OH O 84

OH OH O

NH2

OH

Antibacterial natural products

95

14. Aminoglycoside antibiotics Aminoglycoside antibiotics are among the oldest known class of antibiotics [139, 140]. Although much has been written on this class it would be worth mentioning the representatives of this class streptomycin 85, neomycin 86 and gentamycin 87 for completeness of the article. The well-known streptomycin was isolated by Waksman and coworkers in 1944 from cultures of Streptomyces griseus. It was the first effective drug for treatment of tuberculosis. Aminoglycosides are often administered into veins or muscle to treat serious bacterial infections. Some aminoglycosides are also used orally to treat intestinal infections or topically to treat eye infections. Among several modes of actions some are protein biosynthesis inhibitors and thus compromising the structure of the bacterial cell-wall. The mechanism of resistance to aminoglycoside antibiotics has been investigated [141]. Recent advances in structure, molecular mechanism, SAR, aminoglycoside mimetics have been reviewed by Silva and Carvalk [142]. The current efforts to develop new aminoglycoside derivatives with modification and reconstruction on each sugar ring and advances in SAR have also been reviewed by Zhou and coworkers [143]. The recent emergence of infections due to Gram-(-) bacterial strains with advanced patterns of antimicrobial resistance has prompted reevaluation of the use of aminoglycoside antibacterial agents [144]. This revived interest has brought back to light the debate on the two major issues related to these compounds, namely the spectrum of antimicrobial susceptibility and toxicity. Current evidences show that aminoglycosides retain activity against the majority of Gram-(-) clinical bacterial isolates in many parts of the world. OH

Me OHC O

HO O HO HO OH

OH

H2N

NH2

N O

O N H

OH

HO

HO

N

Me

O

HO

NH2 NH2

O

O

NH2

HO

H2N

O

O OH

OH

O

85

NH2

NH2 H2N OH Me HN Me

OH

H2N

O OH O H2N

O

HO OH 87

O NH2

Me NH2

86

NH2

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Girija S. Singh & Surendra N. Pandeya

However, the relatively frequent occurrence of nephrotoxicity and ototoxicity during aminoglycoside treatment makes physicians reluctant to use these drugs in everyday practice. Recent advances in the understanding of the effect of various dosage schedules of aminoglycosides on toxicity have provided a partial solution to this problem, although more research still needs to be done in order to overcome this problem entirely [145].

15. Concluding remarks Pathogenic bacteria are increasingly evading the standard treatment for antibacterial infections as resistance to multiple antibiotics is spreading worldwide. Resistant pathogens lead to higher expenditure on treatments due to extended stay in hospitals and expensive medicines. There is an urgent need for a sustainable supply of new, potential and safer antibacterial drugs having no cross-resistance to currently used antibiotics. Nature so far has been proved the treasure of potential remedies for diseases. It is more relevant for treatment of infectious diseases. Most of the antibacterials in use today could be discovered on the basis of information obtained from the study of natural products from microbes, marines and plants. Medicinal chemists do systematic studies and provide the tools for optimization of natural products to obtain drug molecules with improved pharmacokinetic, physicochemical and toxicological properties. The diligent selection of natural antibiotics lead structures for medicinal chemistry programs and guideposts for valid targets can reveal pathways for future therapies. Advance studies have been conducted on many classes in order to identify the drug target and mechanism of action. There is still a plenty of scope even within the known antibiotics. Many old classes have not been thoroughly investigated and only partial SAR information is available on their backbone structures. Macrolides are an excellent example that many old classes have not been completely explored. Undoubtedly, more could be done to fully exploit the weapons against bacteria. β-Lactams continue to be one of the most important classes in antibacterial research and development. Their efficacy and compatibility has allowed their broad therapeutic application. Further investigations on second generation lincosamides might be useful for successful treatment of infections by enterococci. Biphenomycins appear promising for the future studies. Further work is anticipated on lysobactins for a real assessment of this class.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 103-120 ISBN: 978-81-308-0448-4

3. Anti-tubercular activity of natural products: Recent developments L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov Department of Chemistry, Russian Acad Sci, Siberian Div, Vorozhtsov Inst Organ Chem Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia

Abstract. An increasing incidence of deaths due to tuberculosis and the known drawbacks of the current existing drugs including the emergence of multi drug-resistant strains have led to a renewed interest in the discovery of new anti-tubercular agents. The recent researches focused on natural products have shown a useful way to obtain a potentially rich source of drug candidates. This review covers the most active naturally occurring compounds with antitubercular properties at minimal inhibitory concentrations (MICs) of 5 mg/mL or less. The literature from January 2001 to 2009 is reviewed. The compounds are presented in order of chemical type, namely alkynes, heterocyclic compounds, phenols and quinones, peptides, alkaloids, terpenoids and steroids.

1. Introduction Tuberculosis is a chronic infectious disease, one of the major enemies of the humanity from times immemorial. Today it still remains one of the most serious medical and social problems. It is responsible for 3 million deaths per year and around 8 million cases of first-recorded disease. The advances in the chemotherapy of tuberculosis in the mid-20th century have recently given way to anxiety over the evolution of drug resistance based on the genetically Correspondence/Reprint request: Prof. L. N. Rogoza, Department of Chemistry, Russian Acad Sci, Siberian Div, Vorozhtsov Inst Organ Chem, Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia E-mail: [email protected]

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fixed mutations of M. tuberculosis. Moreover, nearly all drugs used for the treatment of tuberculosis and possessing different mechanisms of activity are able to cause adverse side effects on the human organism. Therefore, it is extremely important to search for new, low-toxic substances superior to the available drugs in their activity and efficiency. This primarily concerns the agents possessing activity against M. tuberculosis strains with multidrug resistance. Modern tuberculosis is generally associated with M. tuberculosis and M. bovis, mycobacteria that are pathogenic to the human organism. Because of slow growth and pathogenicity of M. tuberculosis H37Rv, many research groups used fast-growing and/or nonpathogenic mycobacteria including M. tuberculosis H37Ra, M. smegmatis, M. aurum, and others as organisms to be tested. The antimycobacterial activity was also investigated on M. avium and M. intracellulare, which cause bird tuberculosis and are associated with human diseases in advanced countries (AIDS patients and immunocompromised individuals), to find compounds with a wide range of activity. A special group of research works includes investigations on M. tuberculosis clinical isolates and strains possessing multidrug resistance. Multidrug-resistant tuberculosis (MDRTB) is strictly defined as M. tuberculosis strains showing resistance simultaneously against isoniazid and rifampicin [1, 2]. Tuberculosis with a different drug resistance (DDRTB) involves M. tuberculosis strains displaying mono- or polyresistance not including associated resistance against isoniazid and rifampicin [3]. The M. tuberculosis strains may be sensitive (inhibited by first series drugs such as isoniazid) or resistant (not inhibited by isoniazid). Since researchers use different analytical procedures and/or organisms under test, care should be taken in comparing the biological activities obtained by different authors. The review covers publications from 2001 to first half 2009; the selected structures have minimum inhibiting concentrations (MIC) of 5 μg/mL or less. Due to this limitation, the most effective compounds were analyzed within one review. For better insight into the "structure-property" relationship, we occasionally gave structures with higher MIC values. The review includes the introduction section, two chapters on synthetic and natural compounds with an antimycobacterial activity, and the conclusions section. To reveal possible "structure-activity" relationships, we grouped the data according to chemical structures.

2. Alkynes and heterocyclic compounds The metabolite of several strains of the endophytic fungus of the genus Phomopsis, 3-nitropropionic acid (1), actively inhibited growth of

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M. tuberculosis H37Ra (MIC 0.4 μg/mL). Although the high neurotoxicity of this compound was a hindrance to its use as a pharmaceutical, it could be used as a model for the synthesis of new inhibitors of isocitratelyase, an enzyme essential to the catabolism of fatty acids and virulence of M. tuberculosis [4]. Linoleic acid (2) that inhibits growth of M. phlei (MIC 2 μg/mL) is extracted from the plant Humulus lupulus. An example of polyacetylene compounds is 3S,8R stereoisomer (3) isolated from Anethum graveolens and having MIC 2-4 μg/mL when tested on a group of fast growing mycobacteria (M. fortuitum ATCC 6841, M smegmatis ATCC 14468, M. phlei ATCC 11758, M. aurum Pasteur Institute 104482, and M. abscessus ATCC 19977; for ethambutol, MIC 0.5-4 μg/mL) [5]. However, cytotoxicity of this class of polyacetylene compounds can lower the interest in their biological activity [6]. Compounds (4a) and (4b), the synthetic analogs of the natural antibiotic thiolactomycin, inhibit growth of M. tuberculosis with MIC 1-16 μg/mL, including drug-resistant strains [7]. OH HOOC

NO2

7

8

CO2H 3

(2)

(1)

C7H15 (3)

OH

S

O

O O

O

H

O

H

C8H17

OH

R (4a), R = O(CH2)10Br (4b), R = O(CH2)8SCH2COOMe

7

(5)

(6)

Investigation of the components of the plant Cinnamomum kotoense led to the isolation of a number of compounds, of which lincomolide В (5) with MIC 2.8 μg/mL had the highest antituberculosis activity [8]. Micromolide (6), which is a γ-lactone derivative of oleic acid, was isolated from the stem bark of Micromelum hirsutum and has MIC 1.5 μg/mL against M. tuberculosis (H37Rv). Further evaluation of activity on J774 mice cells infected with a more virulent strain of M. tuberculosis Erdman gave MIC 5.6 μg/mL [9]. 2-Substituted furans (7a,b) and (8a,b) isolated from the roots of Polyalthia evecta possess activity against M. tuberculosis (MIC 3.1 and 6.25 μg/mL, respectively) [10]. The synthesized natural compound pamamycin-607 (9) inhibits growth of M. bovis BCG, M. smegmatis and M. tuberculosis (MIC 0.54.7 μg/mL). It does not show cross resistance to isoniazid and rifampicin [11].

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(CH2)13 O

O

n OR

(7a) R = H (7b) R = Me

(8a) n = 5 R = C15H31 (8b) n = 3 R = (CH2)13OH

NMe2 O O

O O

O

O O

(9)

3. Phenols and quinones Phenylpropanoids (10) and (11), metabolites of Pimpinella sp., inhibit growth of a number of mycobacteria, including M. intracellulare, M. smegmatis, M. aurum, and M. phlei (MIC 1.25-10 μg/mL) [12]. The tricyclic diphenol ether engelhardion (12) is very active against M. tuberculosis H37Rv (MIC 0.2 μg/mL) [13]. (-)-4-Hydroxy-1-tetralone (13, MIC 4.0 μg/mL) & 3-methoxycarboxy-1,5-dihydroxyantraquinone (15, MIC 3.125 μg/mL) were isolated as an antituberculosis component of Engelhardia roxburghiana [13]. As is known, the level of the intra- and extracellular inhibition of M. tuberculosis by 7-methyljuglon (14) (MIC 0.5 μg/mL) extracted from the plant Е. natalenis, is comparable to that of streptomycin and ethambutol (MIC 1 and 2 μg/mL, respectively). Its derivatives, namely, 5-hydroxy-, 5-alkoxy-, and 5-acetoxy-8-substituted naphthoquinones, are less active (MIC 2.5- >20 μg/mL) and possess low antituberculosis selectivity, probably because of their nonspecific activity with various disulfide reductases found in mammal cells. Optimization of the specificity of these compounds for mycothiol disulfide reductase, which is one of the several biological targets for the antituberculosis activity of naphthoquinones of this structure, is required [14].

Anti-tubercular natural products

107 HO

OH O

O O

H

H O

O

O

O

O

(11)

(10) O

(12)

O

O

OH

OCH3

H OH

OH

(13)

O

OH

O

O (15)

(14)

Marine metabolites pseudopyronines A and B (16a,b, MIC 0.78-3.125 μg/mL) inhibit the growth M. tuberculosis H37Rv [15]. Pyrone (17, MIC 4 μg/mL) is a component of Piper sanctum that is active against M. tuberculosis H37Rv [16]. Ferulenol (18a) isolated from the Sardinian giant fennel Ferula communis is effective against M. smegmatis (MIC 0.5 μg/mL), as well as M. fortuitum, M. phlei and M. aurum (MIC 2 μg/mL). The analogs of this compound, (18b-d), were isolated from the same plant; compound (18b) with a benzyloxy group retained its activity against M. smegmatis and M. phlei, and, to a lesser extent, against M. fortuitum and M. aurum, while the activity of (18c) and (18d) with the hydroxy and acetoxy groups is considerably lower [17]. Ostruthin (19), the metabolite of Peucedanum ostruthin Koch, inhibit the growth M. aurum (МIC 3.4 μg/mL) [18]. OH

R

OMe

O

O

O (16a), R = C5H11 (16b), R = C7H15

O

O

O

(17)

OH R O

O (18a), R = H (18b), R = OBz (18c), R = OH (18d), R = OAc

HO

O (19)

O

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O

OH R

OH

O

OH

O O

O

O

O

O

O

O OH

HO

R2

O O (21)

R1 (20a) (20b) (20c) (20d) (20e) (20f)

(20g) R = O (20h) R = OH

R1 = OH, R2 = H R1 = OH, R2 = H, Δ saturated R1 = O, R2 = H R1 = O, R2 = H, Δ saturated R1 = O, R2 = OMe, Δ saturated R1 = O, R2 = OH, Δ saturated

HO

OH

OH

O

OH

OR

O

OAc

(22c)

RO

OR

O AcO

OAc

OH

O

O

O HO

OR

O

OAc

OH

O

OH

(22a) R = Ac (22b) R = H

Compounds (20a-h), isolated from the lichen fungus Microsphaeropsis sp., show different activities against M. tuberculosis H37Ra (MIC 25, 3.12, 3.12–6.25, 6.25, 12.5, 25, 1.56-3.12, 50 μg/mL, respectively), but are also characterized by cytotoxicity [19]. The dibenzofuran derivative, usnic acid (21), which is a secondary metabolite of lichen, inhibits growth of M. tuberculosis (MIC 2.5–5 μg/mL) [20]. One of the xanthone dimers isolated from the endophytic fungus of the genus Phomopsis, phomoxanthone A (22a), is very active against M. tuberculosis H37Ra (0.5 μg/mL), while its deacetylated derivative (22b) is inactive. Phomoxanthone B (22c) is less active (MIC 6.25 μg/mL). Both active compounds are cytotoxic [21]. The anthraquinone celastramycin B (23), isolated from the unknown species Streptomyces, is active against M. Vaccae (MIC 3.1 μg/mL) [22]. The anti-HIV agent (+)-calanolide A (24) was tested for the antituberculosis activity; a combination of the anti-HIV and antituberculosis activities in one agent is very attractive in view of the concurrence of these diseases. This compound, isolated from the tropical tree Calophyllum lanigerum, also has an antituberculosis activity against M. tuberculosis (MIC 3.13 μg/mL) and a number of drug resistant strains (MIC 8–16 μg/mL) [23].

Anti-tubercular natural products

OH

109

O

O

OH

Cl O O (23)

OH

O

O

O

OH (24)

4. Peptides Four cyclic peptides, namely, enniatins H (25a), I (25b), B (25c), and B4 (25d), which are the components of the pathogenic fungus Verticillium hemipterigenum, inhibit growth of M. tuberculosis H37Ra (MIC 3.12–6.25 μg/mL) [24]. Syringomycin E (26), isolated from Pseudomonas syringae pv. Syringae, is active against M. smegmatis (MIC 1.5 μg/mL) [25]. The metabolite of Nocardia sp. (ATCC 202099), namely, the thiazole peptide nocathiacin (27) shows activity against M. tuberculosis ATCC 35828, M. avium A26778, and M. avium A26640 (MIC ≤ 0.008, 0.06, and 0.25 μg/mL, respectively). Unfortunately, compounds from this class typically show poor pharmacokinetics and solubility (the latter problem can be solved by synthesizing analogs with higher solubility in water) [26].

5. Alkaloids Two compounds, namely, the known antibiotic pyrrolnitrin (28) and banegasine (29), isolated from the zoobacterium Aristabacter necator, act synergetically against M. smegmatis (MIC (29) >0.5 μg/mL, (28) 0.3 μg/mL, (28) + (29) 0.075 μg/mL) [27]. Their analog celastramycin A (30), which is a dichloropyrrole metabolite of the Streptomyces strain, has a broad spectrum of antimycobacterial activity (MIC 0.05-3.1 μg/mL against M. smegmatis, M. aurum, M. vaccae, and M. fortuitum) [22]. The bis-1-oxaquinolizidine alkaloid (−)-araguspongine С (32), isolated from the sea sponge Xestospongia exigua, inhibits growth of M. tuberculosis H37Rv (MIC 1.9 μg/mL) [28]. In the series of quinolone alkaloids (33a-d), isolated from the fruits of Evodia rutaecarpa, compounds with usaturated aliphatic chains at 2-position exhibited better antimycobacterial activity as compared with saturated chain compounds [18]. Agelasine E (33a) and agelasine D (33b) were previously isolated from the sea sponge Agelas nakamurai. While agelasine Е is inactive, its methoxy analogs (33c-g), having different terpenoid side chains,

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L. N. Rogoza et al. R1 O

O N

R6

O

R4

O

O

O

O O

O

3

R

O

N

S

HN

N

NH2

S

O

H N

NH2

O

N

S

O

N OH

N

O

O O

N

S

NH

N

O O

HO

(CH2)2NH2

(26):R = NHCOCH2CH(OH)(CH2)8CH3 OH

O

NH

N H

S H N

NH

H N O

H N

CH2OH

O

O

N H

HO

Me2N

NH

O

R1 = R2 = R3 = R5 = R6 = i-Pr; R4 = s-Bu; R1 = R2 = R3 = R6 = i-Pr; R4 = R5 = s-Bu; R1 = R2 = R3 = R4 = R5 = R6 = i-Pr; R1 = i-Bu; R2 = R3 = R4 = R5 = R6 = i-Pr

MeO

NH

H2N(H2C)2

R5

O (25a) (25b) (25c) (25d)

R2

N

N

O

CH(OH)CH2Cl R O CH(OH)CO2H

HN

O

O

H N

O N

O

OH

O (27)

Cl

Cl

OH

NH2

NO2

NH Cl

Cl

N H

N H (28)

OH

COOH

(30)

(29)

(31a) N

R

(31b) (31c)

O

(31d) HO H

O N

N O

H OH (32)

O

Cl

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demonstrate high activity against M. tuberculosis H37Rv (MIC 3.13, 1.56, and 3.13 μg/mL respectively). Possibly, the presence of an alkoxy group at the terminal nitrogen atom is a very important factor for the antimycobacterial activity of these compounds. However, there is only slight difference between the activities of agelasine D (33b) and its alkoxy derivatives (33f) and (33g) [29]. It is interesting that the simpler analog of the compounds, 9-methyladenine (33h), has MIC of 6.25 μg/mL [30]. The tetracyclic alkaloid cryptolepine (34a), isolated from Cryptolepis sanguinolenta, is active against a number of fast-growing mycobacteria, including M. aurum (MIC 2 μg/mL), M. phlei (MIC 4 μg/mL), and M. fortuitum (MIC 16 μg/mL) [31]. Metabolite of Allium neapolitanium (34b) R1O NH2

N

R N

N N

N

HN

N

NH2

R

N

N

N

N N

Me

Me

Me (33a) R = c (33b) R = d

(33c) R = a, R1 = Me (33d) R = b, R1 = Me (33e) R = c, R1 = Me (33f) R = d, R1 = Me (33g) R = d, R1 = t-Bu

(33h)

c

a n

b

d

Me N R N H

N

N

O

(34a)

N

(34b), R = H (34c), R = OH

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displayed enhanced activity against the M. smegmatis (mc22700), when compared with that for (34c) (MIC 2-8 μg/mL). Furthermore, the activity of (34b) was greater against M. smegmatis (mc2 2700) than M. smegmatis (ATCC 14468) (MIC 16 μg/mL for (34c) and 8 μg/mL for (34b) [32]. The metabolites of the Thailand pathogenic fungus Hirsutella nivea BCC 2594 hirsutellones A-D (35a-d) inhibit growth of M. tuberculosis H37Ra (MIC 0.78, 3.125, 0.78, 0.78 μg/mL, respectively). Compound (35d) exhibits moderate in vitro cytotoxicity, while other compounds are less cytotoxic [33]. Hirsutellone F (35e), which is a new dimer alkaloid isolated, together with known hirsutellones A, B, and C, from the seeds of the fungus Trichoderma sp. BCC 7579 shows a weaker antituberculosis activity against M. tuberculosis H37Ra (MIC 3.12 μg/mL) than hirsutellones А, В, and С [34]. The known alkaloid ecteinascidin 770 (36a) and the new one, ecteinascidin 786 (36b), both isolated from Ecteinascidia thurstoni, inhibit growth of M. tuberculosis H37Ra (MIC 0.1 and 1.6 μg/mL, respectively) [35]. Manzamine alkaloids isolated from sea sponges are promising from the viewpoint of their antituberculosis activity. Manzamines А (37a), Е (37c), and F (37d) and their hydroxyl derivatives 6-hydroxymanzamine Е (37e) and

H

O NH

O

O

H

H

H

H

NH

O

O

O

H

(35a) R = H (35b) R = Me

O

O

H

H

H

R

H

OH

OH

H

H

OH

O

H

H

H

H

H

O

H

NH N H

H

(35d) H

O

H

H

(35c)

H

O

O

O O

(35e)

H

O

H

O

H

NH O

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(+)-8-hydroxymanzamine А (37b) show activity against M. tuberculosis H37Rv (MIC 1.5, 3.8, 2.6, 0.4, and 0.9 μg/mL, respectively) [36]. Manadomanzamines A (38a) and B (38b) inhibit growth of M. tuberculosis H37Rv (MIC 1.9 and 1.5 μg/mL, respectively) [37].

OMe R

HO OAc

H

H

H

N N

R2

N H

H

N O O MeO

O

H

OH

N

H

R1

R1 N

O NH

HO (37a), R = R1 = H (37b), R = H, R1 = OH

(36a) R1 = CN, R2 = none (36b) R1 = CN, R2 = O

R N

N H

H

OH

N

H R1

22 O

N H N

N H H H OH

N HN

O (37c), R = R1 = H (37d), R = H, R1 = OH (37e), R = OH, R1 = H

(38a), 22βH (38b), 22αH

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6. Terpenes Compound (39), isolated from Indigofera longeracemosa, is active against M. tuberculosis (MIC 0.38 μg/mL) [39]. Diterpenes (40) and (41) from Calceolaria pinnifolia [40], the structurally related lecheronol A (42), isolated from Sapium haematospermum (MIC 4 μg/mL) [40], and metabolite of Melica volkensii 6-hydroxyculactone (43) [18] have the same value of MIC against M. tuberculosis H37Rv. Ugandensidial (44, from Warbugia ugandensis) inhibit growth of M. aurum and M. phlei at this value of MIC [18].

OMe O

O OAc CH2OCOCH2COOH

MeOOCH2COCOH2C

(40)

(39)

(41)

OH

O

OHC

OH CHO

OH

O H OCOCH3

O

O H

OH

(42)

(43)

(44)

The diterpenes diaportheines A (45a) and B (45b) were isolated from the fungus Diaporthe sp. Compound (45b) has antituberculosis activity against M. tuberculosis H37Ra (MIC 3.1 μg/mL) and cytotoxicity, while compound (45a) is much less active and cytotoxic (MIC 200 μg/mL) [41]. These data indicate that the presence of a carbonyl group is important for the antituberculosis activity. A metabolite of the African tree Combretum imberbe, traditionally used in folk medicine is imberbic acid (46), which shows activity against M. fortuitum (MIC 1.56 μg/mL) [42].

Anti-tubercular natural products

115

HOOC HO OH O OH

OH R2 R1

OH

HO Me

(46)

(45a) R1 = OH, R2 = H (45b) R1 + R2 = O

The chemical modifications of the parent structure of ursolic acid (at the C-3 position to cinnamate-based esters) resulted in an 4-fold increase in antimycobacterial activity ((47а), MIC 3.13 μg/mL for M. tuberculosis H37Ra, for ursolic acid MIC 12.5 μg/mL) [43].

COOH O CO R MeO

(47a) R = OAc (47b) R = OH

O R1

COOH

R2

MeO2C MeO2C

HO

(48)

(49a) R1 + R2 = O (49b) R1 = H, R2 = OH

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24 OH H H

H

HO

(51) saturated (52) unsaturated

(50a) 24R (50b) 24S

R2

O O

O O

R1O

HO

(54a) R1 = CO(CH2)16Me, R2 = Me (54b) R1 = H, R2 = Me (54c) R1 = H, R2 = Et

(53)

R

H H O

HO

H CHO

OH

(54d)

(55a) R = Me (55b) R = Et

Triterpene (48), isolated from Elateriospermum tapos, is active against M. tuberculosis H37Ra (MIC 3.13 μg/mL, for isoniazide and kanamicin sulfate MIC 0.05 & 1.25 μg/mL, respectively) [44]. Aegicerin (49a) and protoprimulagenin A (49b) were isolated from Aegiceras sp., Embelia Schimperi, and the Peruvian plant Clavija procera. Aegicerin (49a) was tested on 37 different strains of tuberculosis (MIC 1.6-3.1 μg/mL against one strain of H37Rv, 21 sensitive clinical strains, two clinical isolates resistant to isoniazid, and 13 MDR clinical strains). The inactivity of protoprimulagenin

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A (49b) (MIC 200 μg/mL) demonstrates that as in the case of (45a) and (45b), the presence of a carbonyl group is critical for the antituberculosis activity. For the first time, an oleane type triterpene shows uniformly high activity against a wide range of both sensitive and resistant strains. Regretfully, for many MDR strains, its excellent antituberculosis activity (for comparison, MIC is 4-32 μg/mL for isoniazid and 2-16 μg/mL for rifampicin) has not yet been effected [45].

7. Steroids Saringosterol, isolated from brown seaweeds Sargassum ringgoldianum and Lessonia nigrescens in the form of a 1:1 mixture of the 24R isomer (50a) and 24S isomer (50b), inhibits growth of M. tuberculosis H37Rv (MIC 0.25 μg/mL) and has low cytotoxicity. In pure form these isomers possess different levels of activity (MIC is 0.125 μg/mL for the 24R isomer and 1 μg/mL for the 24S isomer) [46]. Lipids that inhibit growth of M. tuberculosis H37Rv were isolated from an extract from Morinda citrifolia (Rubiaceae), traditionally used in folk medicine in the Philippines for the treatment of tuberculosis and respiratory diseases. The highest activity was found for a mixture of (51) and (52) (MIC IC50>0.39 μg/mL) in compared to 36, 37 and 38 (6.25>IC50>1.56 μg/mL) [26]. O

O CH3

CH3 CH3

CH3

H

H

H

CH3

CH3 H

H

H

H

OH

H2N

H2N 35

O CH3

CH3

O

36

CH3

H

H CH3 H

CH3 O O

H

H

H NH H3C

H OCH3

37

CH3

CH3

H

H

OH CH3 O O H H NH2 H OCH3

H

38

H OH

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133

6.6. Benzoquinolizidine alkaloids Klugine (39), cephaeline (40), isocephaeline (41) and emetine (42), demonstrating significant leishmanicidal activity against L. donovani have been isolated from Psychotria klugii (Rubiaceae). Among these metabolites, the compound 39 (IC50 of 0.40 μg/mL) and 41 (IC50 0.45 μg/mL) exhibit 5-fold more in vitro activity against L. donovani when compared to pentamidine and amphotericin-B, respectively. The alkaloid 42, exhibits activity against L. donovani with an IC50 value 0.03 μg/mL, however produces toxicity in treatment of cutaneous leishmaniasis caused by L. major [27]. H3CO

R1

N

N H3CO

H

H3CO

H

H

CH3

CH3 R2

H

H

H

OCH3

H OCH3

HN

HN

R

OH 41 R = OCH3 42 R= OH

39 R1 = OH; R2 = OH 40 R1 = OCH3; R2 = H

6.7. Diterpene alkaloids The alkaloids, 15,22-O-Diacetyl-19-oxo-dihydroatisine (43), azitine (44) and isoazitine (45), isolated from Aconitum, Delphinium and Consolida species, show significant leishmanicidal activities. The metabolite 45 exhibits strongest activity against promastigotes of L. infantum with IC50 values 44.6, 32.3 and 24.6 μM at 24, 48 and 72 h of culture, respectively. The compound 44 and 43 with IC50 values of 33.7 and 27.9 μM at 72 h of culture, respectively, exhibit activity against promastigotes of L. infantum [28]. CH2

CH2

CH2

OAc

N

H

OAc

N

H

OH

HN

OH

H

CH3 CH3 O

43

CH3

CH3 44

45

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6.8. Pyrrolidinium alkaloids The pyrrolidinium alkaloid (2S,4R)-2-carboxy-4-(E)-p-coumaroyloxy1,1-dimethylpyrrolidinium inner salt (46), isolated from Phlomis brunneogaleata (Lamiaceae), display activity with an IC50 of 9.1 μg/mL against axenic amastigotes of L. donovani [29].

6.9. Acridone alkaloids The rhodesiacridone (47) and gravacridonediol (48) isolated from Thamnosma rhodesica (Rutaceae), exhibit 69% and 46% inhibition at 10 μM concentration, respectively against promastigotes of L. major. The compounds also display activity against L. major amastigotes and cause over 90% and 50% inhibition at 10 and 1 μM concentration, respectively [26]. O

OH

OH O

OOC

N H3C

N CH3

O

O CH3 46

R 47 R = C(OH)(CH2OH)COCH3 48 R = C(OH)(CH3)CH2OH

6.10. β-Carboline alkaloids The harmaline (49), isolated from Peganum harmala (Nitrariaceae), exhibits amastigotespecific activity (IC50 of 1.16 μM). Harmine (50) isolated from same plant species reduces spleen parasite load by approximately 40, 60, 70 and 80% in free, liposomal, niosomal and nanoparticular forms, respectively in mice model. Canthin-6-one (51) and 5-methoxycanthin-6-one (52) occurring in plant species of Rutaceae and Simaroubaceae, demonstrate in vivo activity against L. amazonensis in BALB/c mice model. Nhydroxyannomontine (53) and annomontine (54) isolated from Annona foetida (Annonaceae), show efficient leishmanicidal potentials. The SAR studies suggest that the metabolite 54 (IC50 = 34.8 μM) displays 6 times more activity compared to 53 against L. braziliensis promastigotes. The compound 53 also exhibits activity against promastigotes of L. guyanensis while 54 remain inactive [25].

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N

N N

H3CO

N

N

N H

R N

O R

R H2N

49 R = H 50 R = CH3

51 R = H 52 R = OCH3

N

53 R = OH 54 R = H

6.11. Alkaloids from marine sources Many marine sponges e.g. Amphimedon viridis, Acanthostrongylophora species, Neopetrosia species, Plakortis angulospiculatus and Pachymatisma johnstonii serve as rich sources of alkaloids with significant antileishmanial potentials. Renieramycin A (55) isolated from Neopetrosia species, is a La/egfp (expressing enhanced green fluorescent protein) inhibitor that shows efficient antileishmanial activity against L. amazonensis with IC50 0.2 μg/mL. Araguspongin C (56), isolated from a marine sponge Haliclona exigua, displays leishmanicidal activity against promastigotes as well as amastigotes at 100 μg/mL concentrations [30]. N

OCH3

H CH3

O

O H

H

O

H

H3C

N

CH3

N

H3CO O

O

OH

O

CH3

H

H

O O

H N

CH3 55

56

Among the ciliatamides A-C (57-59) isolated from Aaptos ciliate, the peptide 57 and 58 at 10.0 μg/mL concentrations inhibit 50% growth L. major promastigotes [31]. The lipopeptides, almiramides A-C (60-62) isolated from cyanobacterium Lyngbya majuscule, exhibit significant in vitro antileishmanial activity against L. donovani. The SAR studies among these peptides suggest that 61 and 62 exhibit strong activity against L. donovani

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with EC50 values of 2.4 and 1.9 μM, respectively. The metabolites 61 and 62 also display weak cytotoxicity to mammalian Vero cells at 52.3 and 33.1 μM concentrations, respectively [32]. Dragonamide A (63), E (64) and herbamide B (65), isolated from same cyanobacterium strain, exhibit in vitro activity against L. donovani with EC50 values of 6.5, 5.1 and 5.9 μM, respectively [33]. Viridamide A (66) isolated from Oscillatoria nigro-viridis, shows activity against L. mexicana with EC50 of 1.5 μM [34]. Venturamides A (67) and B (68) obtained from cyanobacterium Oscillatoria species, exhibit activity against L. donovani with EC50 >19.0 μM. Valinomycin (69), a dodecadepsipeptide isolated from Streptomyces strains, exhibits activity against promastigotes of L. major with EC50 < 0.11 μM, but at the same time shows cytotoxicity to 293T kidney epithelial cells and J774.1 macrophages [35].

7. Quinones Primin (2-methoxy-6-pentylcyclohexa-2,5-diene-1,4-dione), occurring in Primula obconica and other species (Primulaceae), shows significant leishmanicidal activity against L. donovani with an IC50 of 0.711 μM. Diospyrin (70), a bis-naphthoquinone inhibiting topoisomerase I, isolated from the bark of Diospyros Montana (Ebenaceae), demonstrates antileishmanial activity against L. donovani promastigotes with an MIC of 1.0 μg/mL [36]. The hydroxylated derivative of 70 at 3 μM concentration eliminates 73.8% of amastigotes in infected macrophages [37]. Plumbagin (72), originally isolated from Plumbago zylenica, shows leishmanicidal activity against amastigotes of L. donovani (IC50 = 0.42 μg/mL) and L. amazonensis (IC50 = 1.1 μg/mL). At a concentration of 10 μg/mL, the

O

O

O

O H N

H N

HN

N O

R

HN

N O

CH3

60 R = 61 R = 62 R =

H3C CH3

R

CH3 59

57 R = (CH2)7CHCH2 58 R = (CH2)6CH3 O

CH2

CH3 N

O H3C

O N CH3 CH3

CH3 H N O H3C

CH3

O

N

N CH3 CH3

CH3

O

O NH2

Scope of natural products against leishmaniasis CH3

H3C O

CH3

H3C O

CH3

N O

CH3

O

CH3

H3C

CH3

CH3

CH3

H N

Cl3C NH2

N

N CH3

CH3

O

CH3

N

R

137

CH3

O N

S

63 R = 65 64 R = CH3

CH3 OCH3

O

HC

H N

N

H3C

N H3C

NH

N O

HN

H3C

N CH3 S

NH

HN

O

O

S O

H3C

CH3

O

O

O

H3C

68

O NH

N H

O

CH3 CH3

HN O

CH3

H3C

67

O

N

N

O

H3C

S

N H

O

N

CH3

OH

O

S

N H

O

CH3 H3C

H3C

COOCH3

O

66

CH3

O

N

O

O H3C

CH3

CH3

O

N

N H

O H3C

CH3

CH3 CH3

H3C O

CH3

H3C

O

CH3

CH3

CH3

CH3 CH3

O

O HN

H3C

H3C

NH CH3

CH3

O

CH3

O

O

O

O

O

CH3

N H CH3

O

H3C

CH3

69

compound 72 presents an amastigote survival index (SI) of 16.5% against L. amazonensis with the absence of toxic effects against the macrophages. The metabolite 72 also shows in vivo activity against L. amazonensis and L. venezuelensis at concentrations 2.5 and 5 mg/kg/day, respectively. The mechanism of the action of compounds 72 and 71 involves generation of oxygen free radicals from which the parasites remain unable to defend. The dimeric products 3,3-biplumbagin (73) and 8,8′-biplumbagin (74), isolated from the bark of Pera benensis (Euphorbiaceae), display significant

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antileishmanial activity. Among these, the metabolite 73 shows lower activity (IC90 = 50 μg/mL) compared to 72 and 75 (IC90 = 50 μg/mL) against L. braziliensis, L. amazonensis, and L. donovani promastigotes [38,39]. Lapachol (75), a prenylated hydroxynaphthoquinone isolated from Tecoma species (Bignoniaceae), displays activity with mechanism of action similar to 71 and 72 against L. donovani amastigotes in peritoneal mice macrophages. The metabolite 3,4-dihydronaphthalen-1(2H)-one (76), isolated from the bark of Ampelocera edentula (Ulmaceae), exhibits leishmanicidal activity (IC90 of 10 μg/mL) against L. braziliensis, L. amazonensis and L. donovani promastigotes. The metabolite 76 demonstrates strong in vivo activity on subcutaneous treatment in BALB/c mice infected with L. amazonensis or L. venezuelensis when compared to Glucantime® (25 mg/kg/day vs 56 mg SbV/kg/day). However, the use of tetralones is limited due to cytotoxic, carcinogenic and mutagenic properties in animals [40]. Jacaranone (77), a quinone isolated from the leaves of Jacaranda copaia (Bignoniaceae), exhibits a strong activity with an ED50 of 0.02 mM against L. amazonensis promastigotes but at the same concentration shows toxicity to peritoneal mice macrophages. The prenylated dihydroquinone hydropiperone (78), isolated from Peperomia galioides (Piperaceae), shows activity at a concentration of 25 μg/mL against promastigote forms of L. braziliensis, L. donovani and L. amazonensis. At 100 μg/mL concentration the metabolite 78 causes total lysis of the parasites [41]. OH

O

OCH3 OH OH

O

O

H3C

OCH3 OH

CH3

H3C O H3C

OH H3C

OH

O

OH 72

71

70 O

OH

O

O CH3 O

OH

OH O

H3C

CH3

CH3

O OH

O

O H3C

O

CH3

O OH 73

O 74

75

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The anthraquinone-2-carbaldehydes, 79 and 80, isolated from the roots of Morinda lucida (Rubiaceae), shows leishmanicidal potential selective to L. major promastigotes. SAR studies suggest that presence of an aldehyde group at C-2 and a phenolic hydroxy group at C-3 in both structures, are essential for their antiprotozoal activity [42]. O

O

O H3C

HO

CH2COCH3

OH 76

77 O

R1

CH3

CH3

CH3 CH3

OH CH3 O

OH

O

78 OH

CHO

OH O 79 R1 = OCH3 80 R1 = H

CH2OH O 81

The aloe-emodin (81) isolated from Stephania dinklagei (Menispermaceae), shows leishmanicidal activity at IC50 values of 185.1 and 90 μM against L. donovani promastigotes and amastigotes, respectively [43]. Vismione D isolated from Vismia orientalis (Clusiaceae) exhibits activity against axenic amastigotes of L. donovani with an IC50 value of 0.37 μg/mL but shows cytotoxicity when tested on human L6 cells (IC50 of 4.1 μg/mL) [29].

8. Terpenes 8.1. Iridoids Iridoids, a class of monoterpenoid glycosides often serve as intermediates in the biosynthesis of indole alkaloids are well known for significant leishmanicidal activity. The arbortristosides-A (82), B (83), C (84) and 6-βhydroxyloganin (85), isolated from Nyctanthes arbortristis (Oleaceae) exhibit in vitro activity against L. donovani amastigotes. The in vivo studies using intraperitoneal and oral treatment (10 and 100 mg/kg concentrations for 5 days) of hamsters infected with L. donovani, the metabolite 82 displays significant leishmanicidal activities [44]. Picroside I (86) and kutkoside (87),

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obtained from Picrorhiza kurroa, exhibits a high degree of protection against the infection of promastigotes of L. donovani in hamsters [45]. Picroliv, a standardized fraction of iridoid glycosides 86 and 87, increases the nonspecific immune response and induces a high degree of protection against the infection of promastigotes of L. donovani in hamsters. Picroliv is an adjuvant proposed to increase the efficacy of leishmanicidal drugs and has demonstrated excellent therapeutic index in Phase I and II clinical trials [46]. HO

H

OH H CO2CH3 O R1O

O O

R1O

H OR2 O O OH OH OH 86 R1 = Vanilloyl, R2 = H 87 R1 = H, R2 = Cinnamoyl

OH CH3H O O HO OH OH 82 R1 = p-Methoxycinnamoyl 83 R1= Caffeoyl 84 R1 = Coumaroyl 85 R1 = H

Amarogentin (88), a secoiridoid glycoside isolated from Swertia chirata (Gentiaceae), produces leishmaincidal effect at a concentration > 60 μM against L. donovani through inhibition of catalytic activity of topoisomerase I [47]. The metabolite 88 exerts inhibitory effect with a mechanism of action similar to Pentostam® i.e. by binding to the enzyme and preventing the formation of a binary complex with DNA. The evaluation of 88 in the form of liposomes and niosomes shows an enhanced leishmanicidal activity (without toxic effects) than those observed for free 88 when tested in hamsters [48]. O

O

OH

H

O

H CH2

O O

HOH2C

O O

C HO

OH

HO OH 88

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8.2. Monoterpenes Espintanol (89), isolated from the bark of Oxandra espintana (Annonaceae), shows antileishmanial activity against promastigotes of twelve Leishmania species. However, the metabolite 89 exhibits only a weak activity in vivo in mice infected with L. amazonensis. Grifolin (90) and piperogalin (91) obtained from Peperomia galoides, causes total lysis of L. braziliensis, L. donovani and L. amazonensis promastigotes at 100 μg/mL concentrations. At 10 μg/mL concentration, metabolite 91 causes more than 90% lysis of the promastigotes [49]. CH3

OH

CH3

CH3

CH3 OH

H3C

90

OH

CH3

CH3

CH3

OH OH

H3C H3CO H3C

CH3

OCH3 CH3 89

H3C

CH3 91

8.3. Sesquiterpenes A sesquiterpene lactone, dehydrozaluzanin C (92), isolated from the leaves of Munnozia maronii (Asteraceae), shows activity at concentrations between 2.5-10 μg/mL against promastigotes of eleven Leishmania species. The in vivo test using the metabolite 92 in BALB/c mice results in reduction of the lesions caused by L. amazonensis [50]. Sesquiterpene dilactone, 16,17-dihydrobrachycalyoxide (93), isolated from Vernonia brachycalyx (Asteraceae), exhibits activity (IC50 = 17 μg/mL) against L. major promastigote but also inhibits the proliferation of human lymphocytes [51]. Kudtriol (94), a sesquiterpene alcohol isolated from the aerial parts of Jasonia glutinosa (Asteraceae), shows toxic activity against promastigotes of L. donovani at 250 μg/mL concentration. SAR study with metabolite 94 indicates that the presence of a C-5 hydroxy group in the α-orientation is essential for the expression of the leishmanicidal activity [52]. The (+)-curcuphenol (95), isolated from sponge Myrmekioderma styx, exhibits in vitro anti-leishmanial activities against L. donovani with an EC50 of 11.0 μM [53].

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B. B. Mishra et al. H2C H

O

C2H5 HO H CH2

H O O

CH3

O

O H3C O 93 OH

CH3

CH3

OH H3C OH H3C

94

O O

92

OH CH2 H3C

OH

O O CH 3

H

O H2C

H2C

O

CH3

95

8.4. Diterpenes A phorbol diester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) 96, also known as phorbol 12-myristate 13-acetate (PMA), was originally identified from the croton plant, which at a concentration of 20 ng/mL displays ability to cause a variety of structural changes in the parasites of L. amazonensis by activation of protein kinase C, an important enzyme in the development of several cellular functions [54]. Among the other diterpenoids isolated from Euphorbiaceae species with leishmanicidal potentials are jatrogrossidione (97) and jatrophone (98). These metabolites possess toxic activity against the promastigote forms of L. braziliensis, L. amazonensis and L. chagasi. SAR studies with these metabolites revealed that 97 with IC100 value of 0.75 μg/mL displays activity higher than 98 (IC100 = 5 μg/mL), but remains inactive in vivo [55]. The 15-monomethyl ester of dehydropinifolic acid (99), obtained from the stem bark of Polyalthia macropoda (Annonaceae), and ribenol (100), an ent-manoyl oxide derivative isolated from Sideritis varoi (Lamiaceae), show in vitro activity against promastigotes of L. donovani [56]. Also the different derivatives of this metabolite, obtained through chemical or biological transformations, exhibit strong leishmanicidal activity. Additionally, 6-βhydroxyrosenonolactone (101), a diterpene isolated from the bark of Holarrhena floribunda (Apocynaceae), has a moderate and weak activity against promastigotes and amastigotes of L. donovani, respectively [57].

Scope of natural products against leishmaniasis

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H3C H3C(H2C)12OCO H3C OCOCH3 CH3

H

H3C

HO O HO

CH3 H

HO H

CH3 CH3

CH2 H3C

O

O

H O

H

H3C CH2OH

CH3

O

O

H2C

CO2CH3

CH3

CH3

97

96

98

H3C CH3 CH3 CH3

CH3 O

CH2

CH2 CH3

O

CH2 O

CH3

HO H H3C CO2H 99

H O

H3C CH3

H CH3 100

OH 101

8.5. Triterpenes The ursolic acid (102) and betulinaldehyde (103), obtained from the bark of Jacaranda copaia and the stem of Doliocarpus dentatus (Dilleniaceae), respectively show activity against the amastigotes of L. amazonensis. However, the metabolite 103 exhibits toxicity to peritoneal macrophages in mice while 102 displays limited activity in vivo. The triterpenes, (24Z)-3-oxotirucalla-7,24-dien-26-oic acid (104) and epi-oleanolic acid (105), isolated from the leaves of Celaenododendron mexicanum (Euphorbiaceae), display leishmanicidal activity against L. donovani with IC50 values of 13.7 and 18.8 μM, respectively. The quassinoids, simalikalactone D (106) and 15-β-heptylchaparrinone (107), obtained from species of Simaroubaceae family show activity against promastigotes of L. donovani but at the same time exhibit toxicity to macrophages [58]. Triterpene glycosides obtained from marine sources e.g. holothurins A (108), isolated from the sea cucumber Actinopyga lecanora, causes 73.2 ± 6.8% and 65.8 ± 6% inhibition of L. donovani promastigotes and amastigotes, respectively at 100 μg/mL concentration. The other isomer B (109) obtained from same source shows 82.5 ± 11.6% and 47.3 ± 6.5% inhibition against promastigotes of L. donovani at 100 and 50 μg/mL concentrations, respectively [59].

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CH2

CH3 H3C

H3C H

CH3 H

CH3

CO2H

CH3

H

CH3

CHO

CH3 CH3

HO

HO H CH3

H3C

H CH3

H3C 102

103 H

CH3

H

CO2H CH3

CH3

CH3 H

CH3

H3C

CH3

H3C

CO2H

CH3

CH3

CH3

O CH3

H3C

H CH3

CH3

105

104

OH OH HO OH CH3

HO OH CH3

CH3 O

(CH2)6CH3

OCOCH(CH3)C2H5

H

H

O

O H

H

O

O H

H

O

H

O

CH3 O

CH3

H

CH3 107

106

CH3 HO

CH3

O

O

O OH

CH3 CH3 O O

H3C

CH3

O NaO3SO HO H3C

O OH OH

OH HO O

HO OR 108 R1 = HO MeO 109 R2 = H

HO

OH

CH3

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9. Saponins The α-hederin (110), β-hederin (111) and hederagenin (112), obtained from the leaves of Hedera helix (Araliaceae), show lishmanicidal activity against L. infantum and L. tropica. Among these, the metabolite 112 also shows significant activity against the amastigote forms while both 110 and 111 exhibit strong anti-proliferative activity on human monocytes [60]. The saponins 110-112 appear to inhibit the growth of Leishmania promastigotes by acting on the membrane of the parasite with induction of a drop in membrane potential [61]. The hederecolchiside-A1 (113), isolated from Hedera colchica, shows strong activity against the promastigotes and amastigotes of L. infantum, but also displays a notable activity on human monocytes. The saponin, mimengoside-A (114), isolated from the leaves of Buddleja madagascariensis (Loganiaceae) [62], exhibits activity against promastigotes of L. infantum. Muzanzagenin (115), obtained from the roots of Asparagus africanus (Liliaceae), displays activity with an IC50 value 31 μg/mL against the L. major promastigotes. However, the metabolite 115 also inhibits the proliferation of human lymphocytes [63].

10. Phenolic derivatives 10.1. Chalcones The chalcone, (E)-1-[2,4-hydroxy-3-(3-methylbut-2-enyl)phenyl]-3-[4hydroxy-3-(3-methylbut-2-enyl)phenyl]-prop-2-en-1-one (116) shows toxicity to promastigotes of L. donovani, while 2′,6′-dihydroxy-4′methoxychalcone (117), isolated from inflorescences of Piper aduncum (Piperaceae), exhibits significant in vitro activity against promastigotes and amastigotes of L. amazonensis by affecting the ultrastructure of the parasite mitochondria without causing damage or inducing NO production in the macrophages [64,65]. The metabolite 117 with an IC50 value of 0.5 μg/mL shows strong antileishmanial activity against the promastigotes of L. amazonensis, while exhibit lower activity (IC50 = 24 μg/mL) against amastigote forms. Encapsulated formulation of 117 when administered at 1.0 μg/mL causes the reduction in the level of L. amazonensis infected macrophages by 53% [66]. Ultrastructural studies suggest that 117 produces selective toxicity to the intracellular amastigotes without affecting macrophage organelles even when exposed to 80 μg/mL concentration. The licochalcone-A (118), isolated from roots of the Chinese licorice plant Glycyrrhiza species (Fabaceae), shows in vitro

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H3C

CH3

CH3

CH3

CO2H

CH3 R1O CH3

R2H2C

110 R1 = Ara 2-1 Rha, R2 = OH 111 R1 = Ara 2-1 Rha, R2 = H 112 R1 = H, R2 = OH 113 R1 = Ara [Glc 4-1] 2 Rha, R2 = H Ara: α -L-arabinopyranose Glc: β -D-glucopyranose Rha:α -L-rhamnopyranose Fuc: β -D-fucopyranose H3C

CH3

O CH3

CH3 CH3

RO H3C OH 114 3-0-α -L-rhamnopyranosyl-(1-4)- β -D-glucopyranosyl(1-3)-[ β -D-glucopyranosyl-(1-2)]-β -D-fucopyranoside of 16-dehydroxysaikogenin G H

H

H

H OH CH3

CH3

CH3

O

H H

HO

H

H

O 115

O CH3

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OH CH3 H3CO

HO

OH

CH3

H3C CH3

OH

OH

O

O

117

116 CH2

HO

OH

H3C H3C

HO

O

OH

HO

O

O

OCH3 118

119

OH HO

R1O

OH

O

O

HO OH

OH

O

OH 120

OR2

O

O

121 R1 = H, R2 = H 122 R1 = H, R2 = OCH3 123 R1 = OCH3, R2 = OCH3

activity against L. major and L. donovani promastigotes. The intraperitoneal administration of 118 prevents the development of lesions in BALB/c mice infected with L. major [67,68]. The intraperitoneal and oral administration of 118 significantly reduces the parasite load in the spleen and liver of hamsters infected with L. donovani. The compound 118 appears to affect the parasite respiratory chain without damaging the organelles of macrophages or phagocytic function by altering the ultrastructure and function of mitochondria only. However, at lower concentrations 118 inhibits the proliferation of human lymphocytes. Subsituents that hinder free rotation in chalcones have been demonstrated to be inactive. The introduction of polar chemical moieties (like hydroxyl and glycosyl groups) led to a reduction of the antileishmanial activity. The modification at the α,β-double bond in chalcones results in marginal reduction of the leishmanicidal activity compared to parent compounds, thus this part is just a chemical spacer

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necessary only. The sulfuretin (2-[(3,4-dihydroxyphenyl)methylene]-6hydroxybenzofuran-3(2H)-one) (119), is an aurone, a group of metabolites related biosynthetically to the chalcones, exhibit activity with EC50 values of 0.09-0.11 μg/mL against promastigotes of Leishmania species. The metabolite 119 with an EC50 value of 1.24 μg/mL displays activity against L. donovani amastigotes, but remains non-toxic to bone marrow-derived macrophages [69].

10.2. Flavonoids The compound 5,7,4′-trihydroxyflavan (120) shows activity against the amastigotes of L. amazonensis [70], while the biflavonoids amentoflavone (121), podocarpusflavone A (122) and B (123), isolated from the leaves of Celanodendron mexicanum, exhibit weak activity against L. donovani promastigotes. The flavones fisetin (124) (isolated from Acacia greggii and A. berlandieri), 3-hydroxyflavone (125), luteolin (126) (isolated from Salvia tomentosa), and quercetin (127) (isolated from plants of family Alliaceae) exhibit potent antileishmanial activity against the intracellular forms of the L. donovani with IC50 values 0.6, 0.7, 0.8 and 1.0 μg/mL, respectively. Biochanin A (128), an O-methylated isoflavone occurring in legumes, shows activity against L. donovani with an IC50 value of 2.5 μg/mL [3]. O

HO

OH

OH

O

OH

HO

O

OH

OH

OH

O

O OH

124

125

O 126

OH HO

HO

O OH OH

O 127

O

OH OH

O

OCH3

128

10.3. Lignans The lignans (+)-medioresinol (129), (-)-lirioresinol B (130) and (+)nyasol (131), show activity against the amastigotes of L. amazonensis, whereas 131 also exhibits high selectivity in its activity against the promastigotes of L. major. Dyphillin, isolated from Haplophyllum bucharicum (Rutaceae), modulates phagocytosis of macrophages and selectively inhibits the amastigotes of L. infantum with an IC50 value 0.2 μg/mL [71].

Scope of natural products against leishmaniasis

149

R2 HO HO

O

H3CO H

H OCH3

O

H2C

OH OH

R1 129 R1 = R2 = OCH3 130 R1 = R2 = CH3

131

10.4. Coumarins The coumarin isomers 2-epicycloisobrachycoumarinone (132) and cycloisobrachycoumarinone (133), isolated from Vernonia brachycalyx (Asteraceae), display selective activity against promastigotes of L. major. R1 CH3

O

O

R2 CH3

O

HO

OH

R1

R2

O

CH3 O

132 R1 =CH3, R2 = H 133 R1 =H, R2 = CH3

CH3

O

OH

134 R1 = R2 = OCH3 135 R1 = H, R2 = OCH3 136 R1 = H, R2 = H

10.5. Curcumins The curcumins, curcumin (134), desmethoxycurcumin (135) and bis-desmethoxycurcumin (136), isolated from the rhizomes of Curcuma longa, show significant anti-leishmanial activity against promastigotes of L. major. However, these metabolites also inhibit the proliferation of human lymphocytes [72].

11. Other metabolites Acetogenins like senegalene (137), squamocine (138), asimicine (139) and molvizarine (140), isolated from the seeds of Annona senegalensis (Annonaceae), show activity against promastigotes of L. major and L. donovani at concentrations that vary between 25 and 100 μg/mL. However, these metabolites also show cytotoxicity greater than that of

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vinblastine against KB and VERO cell lines [73]. Other acetogenins such as rolliniastatin-1 (141), isolated from Rollinia emarginata (Annonaceae), annonacin A (142) and goniothalamicin (143), obtained from Annona glauca (Annonaceae), display promicing activity against the promastigote of L. braziliensis, L. donovani, L. amazonensis, however a clear SAR has not been established [74]. O O

OH

OH

OH O

H3C

(CH2)3CH3

7

7 OH O O

137 R1

OH O

H3C

R2

OH O

*

n

(CH2)5CH3

threo-trans-threo-trans-* 138 R1 = H, R2 = OH, n = 10, *= erythro 139 R1 = OH, R2 = H, n = 10, *= threo 140 R1 = OH, R2 = H, n = 8, *= erythro

O

O

OH

CH3

OH O

H3C

O

8

CH3

6 141 R2

H3C

m

*

O

OH

R1

O OH n

O CH3

OH

142 R1 = OH, R2 = H, n = 5, m = 8, *= erythro 143 R1 = H, R2 = OH, n = 3, m = 10, *= threo

Future prospectives Despite the advances in the parasitological and biochemical researches using various species of Leishmania, the treatment options available against leishmaniasis are far from satisfactory. In current situation, development of new drugs to combat leishmaniasis require increased input from the disciplines of chemistry, pharmacology, toxicology and pharmaceutics to complement the advances in molecular biology that have been made in past 21 years. Natural products are potential sources of new and selective agents for the treatment of important tropical diseases caused by protozoans and other parasites. The tremendous chemical diversity present in natural products and the promising leads that have already been demonstrated significant against parasitic diseases are needed to be addressed also against leishmania

Scope of natural products against leishmaniasis

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parasites. The development of antileishmanial natural products or their analogs in accordance to the considerations outlined above would have a dramatic positive impact on the treatment of leishmaniasis. A safe, non-toxic and cost-effective drug is urgently required to eliminate this problem from every corner of world. A safer, shorter & cheaper treatment, identification of the most cost effective surveillance system and control strategies, suitable vector control approach are among some important aspect for the control and complete eradication of this deadly disease.

Acknowledgement Financial assistance from DST, New Delhi is greatly acknowledged.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Renslo, A.R., McKerrow, J.H. Nat. Chem. Biol., 2006, 2, 701. Balana-Fouce, R., Reguera, R.M., Cubria, J.C., Ordonez, D. Gen. Pharmacol., 1998, 30, 435. Ioset, J.R., Curr. Org. Chem., 2008, 12, 643. Cruz, I., Nieto, J., Moreno, J., Canavate, C., Desjeux, P., Alvar, J. Indian J. Med. Res., 2006, 123, 357. Mathur, P., Samantaray, J.C., Vajpayee, M., Samanta, P. J. Med. Microbiol., 2006, 55, 919-922. Desjeux, P., Alvar, J. Ann. Trop. Med. Parasitol., 2003, 97, S3-15. Zijlstra, E.E., el-Hassan, A.M., Ismael, A. Am. J. Trop. Med. Hyg., 1995, 52, 299. Saraiva, E.M., Pinto-Da-Silva, L.H., Wanderley, J.L.M., Bonomo, A.C., Barcinski, M.A., Moreira, M.E.C. Exp. Parasitol., 2005, 110, 39. McConville, M.J., Souza, D., Saunders, E., Likic, V.A., Naderer, T. Trends Parasitol., 2007, 23, 368. Glew, R.H., Saha, A.K., Das, S., Remaley, A.T. Micro. Rev., 1988, 54, 412. Veeken, H., Ritmeijer, K., Seaman, J., Davidson, R. Trop. Med. Int. Health, 2000, 5, 312. Jha, T.K. Trans. R. Soc. Trop. Med. Hyg., 1983, 77, 167. Thakur, C.P., Singh, R.K., Hassan, S.M., Narain, R.K., Kumar, S.A. Trans. R. Soc. Trop. Med. Hyg., 1999, 93, 319. Sundar, S., Jha, T.K., Sindermann, H., Junge, K., Bachmann, P., Berman, J. Pediatr. Infect. Dis. J., 2003, 22, 434. Sundar, S., Jha, T.K., Thakur, C.P., Sinha, P.K., Bhattacharya, S.K. N. Engl. J. Med., 2007, 356, 2571. Wasunna, M.K., Rashid, J.R., Mbui, J., Kirigi, G., Kinoti, D., Lodenyo, H., Felton, J.M., Sabin, A.J., Horton, J. Am. J. Trop. Med. Hyg., 2005, 73, 871. Fournet, A., Gantier, J.C., Gautheret, A., Leysalles, L., Munos, M.H., Mayrargue, J., Moskowitz, H., Cave, A., Hocquemiller, R. J. Antimicrob. Chemother., 1994, 33, 537.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 155-185 ISBN: 978-81-308-0448-4

5. Naturally occurring antihyperglycemic and antidyslipidemic agents T. Narender, T. Khaliq and G. Madhur Medicinal and Process Chemistry Division, Central Drug Research Institute Lucknow-226 001, U.P., India

Abstract. Diabetes mellitus is an independent risk factor for the development of coronary artery diseases, myocardial infarction, hypertension, and dyslipidemia. Clinically diabetic patients are characterized by marked increase in blood glucose level followed by mild hyperlipidemia. Non-insulin dependent diabetes mellitus (NIDDM) accounts for approximately 80–90% of all cases and it is the fastest growing global threat to public health. If the current trend continues, it is likely to result in an estimated 215 million sufferers from NIDDM worldwide by the year 2010. When carbohydrates are in low supply or their breakdown is incomplete, fats become the preferred source of energy. Fatty acids are mobilized into the general circulation leading to secondary triglyceridemia in which total serum lipids in particular triglycerides as well as the levels of cholesterol and phospholipids increases.This rise is proportional to the severity of the diabetes. Uncontrolled diabetes is manifested by a very high rise in triglycerides and fatty acid levels. These conditions are responsible for one third of deaths in industrialized nations Plants have always been a rich source of drugs and many of the currently available drugs have been derived either from natural products or its templates. We here in present a precise description of naturally occurring compounds possessing potential antihyperglycemic action or antidyslipidemic action against specific drug targets. Correspondence/Reprint request: Dr. T. Narender, Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow-226 001, U.P., India. E-mail: [email protected]

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1. Diabetes mellitus Diabetes is a disease in which the body does not produce or properly use insulin. Insulin is a hormone that converts sugar, starch and other food into energy needed for daily life. The causes of diabetes are not known clearly, although both genetics and environmental factors such as obesity and lack of exercise appear to play roles. Diabetes mellitus and glucose intolerance are common in adolescent and adult patients with cystic fibrosis. Diabetes is invariably associated with pancreatic exocrine dysfunction (malabsorption). The prevalence in patients over 20 years of age may be as high as 53% [1]. The major types of diabetes include type-I and type-II diabetes. The former results from the body's failure to produce insulin, the hormone that "unlocks" the cells of the body, allowing glucose to enter and fuel them while the latter results from insulin resistance, a condition in which the body fails to properly use insulin combined with relative insulin deficiency. Type-II insulin-resistant diabetes mellitus accounts for 90-95% of all diabetes. This heterogeneous disorder afflicts an estimated 6% of the adult population in western society; its worldwide frequency is expected to continue to grow by 6% per annum, potentially reaching a total of 200-300 million cases in 2010 [2].

2. Drug targets At present, therapy for type-II diabetes relies mainly on several approaches intended to reduce the hyperglycemia itself. Table 1. Current therapeutic agents for type-II diabetes. Drug class

Molecular target

Site(s) of action

Insulins

Insulin receptor

Liver, muscle, fat Hypoglycemia, weight gain Pancreatic β-cell Hypoglycemia, weight gain

Sulphonylureas SU receptor/ K+ (e.g. ATP channel glibenclamide) plus nateglinide & repaglinide Biguanides Unknown Metformin Acarbose

α-glucosidase

Thiazolidinediones PPARγ Rosiglitazone, Pioglitazone

Liver (muscle) Intestine Fat, muscle, liver

Adverse events

Gastrointestinal disturbances, lactic acidosis Gastrointestinal disturbances Weight gain, anemia, oedema,

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3. Antihyperglycemic isolates from nature Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them. The ethnobotanical information reports that about 800 plants may possess anti-diabetic potential [3]. Several such herbs have depicted antidiabetic activity while assessed using currently available experimental techniques [4]. A wide array of plant derived active principles representing numerous chemical compounds has demonstrated activity consistent with their possible use in the treatment of non-insulin dependent diabetes mellitus (NIDDM) [5]. Amongst these are flavonoids, alkaloids, glycosides, polysaccharides, peptidoglycans, hypoglycans, guanidine, steroids, carbohydrates, glycopeptides, terpenoids and amino acids. Even the discovery of widely used hypoglycemic drug, metformin was developed on the basis of the natural products lead isolated from Galega officinalis [6]. Thus, plants are a potential source of anti-diabetic drugs. Herein, is presented a precise description of naturally occurring compounds possessing potential antihyperglycemic action.

3.1. Flavonoids The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain. Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognized as flower pigments in most angiosperm families (flowering plants). However, their occurrence is not restricted to flowers but include all parts of the plant. They show wide variety of activities including antihyperglycemic activity. Bio-flavonoids with promising anti-diabetic potential: A critical survey by Goutam Brahmachari will give comprehensive information on the flavonoids and their antihyperglycemic activity.

3.2. Triterpenoids and steroids There are at least 4000 known triterpenes, which are derived from mevalonic acid pathway. Triterpenes are precursors to steroids in both plants and animals. Steroids are hormonal substances in animals, but they are components of membranes in most organisms. Many triterpenes and sterols occur free, but others occur as glycosides or in special combined forms. Momordica charantia belongs to the family of Cucurbitaceae the fruits of the plant is also known as bitter melon or bitter guard. Cucurbitane class of

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triterpenoids isolated from M. charanta such as 5-β,19-epoxy-3-β,25dihydroxycucurbita-6,23-(E)-diene (1) and 3-β,7-β,25-trihydroxycucurbita5,23-(E)-dien-19-al (2) have blood hypoglycemic effects in the diabetesinduced male ddY mice strain at 400 mg/kg [7]. Hypoglycemic activity guided fractionation together with chemical analysis on the stem of Agarista mexicana led to the isolation of 12-ursene (3) and 23,24-dimethyl-24-ethyl-stigmast-25-ene (4) from the chloroform fraction. The isolated triterpenes showed hypoglycemic activity in normal and alloxan-diabetic CD1 mice at a dose of 50 mg/kg body weight. Comparison was made between the action of the triterpenes and a known hypoglycemic drug, tolbutamide (50 mg/kg). The 12-ursene (3) was found to be less potent than tolbutamide where as 23,24-dimethyl-24-ethyl-stigmast25-ene (4) was shown to be more effective than tolbutamide [8]. 20 17

20

OH

17

OHC

OH

H O

HO

3

6

OH

HO

3

6

2

1

3

4

From the roots of Salacia oblonga a friedelane-type triterpene, kotalagenin 16-acetate (5), maytenfolic acid (6), 3β,22α-Dihydroxyolean-12en-29-oic acid (7) and a unique thiosugar sulfonium sulfate named Salacinol (60) was isolated. They were screened for inhibitory activity on aldose reductase and were found to be responsible components for the inhibitory activity [9]. Bioassay-guided isolation work on Cabernet Sauvignon’s grape skin yielded antihyperglycemic active compounds which were identified as, oleanolic acid (8) and oleanolic aldehyde (9). These compounds were assayed for insulin production using an INS-1 cell assay. In a dose-response study,

Naturally occurring antihyperglycemic and antidyslipidemic agents

O

H

H

159

COOH

COOH

OH

OH

H OAc OH

O

HO

HO

6

5

7

oleanolic acid stimulated insulin production of INS-1 cells by 20.23, 87.97, 1.13 and 6.38 ng of insulin/ mg of protein at a dose of 6.25, 12.5, 25 and 50 μg/mL respectively. The activity was similar to the dose-dependent insulin production of INS-1 cells by glucose. Oleanolic aldehyde also showed a dose-dependent insulin production in the same assay [10]. Our activity guided fractional and isolation work on the plant Ficus racemosa yielded moderately active antihyperglycemic principle, α-amyrin acetate (10). Several ester derivatives of α-amyrin were prepared to study their structure activity relationship [11].

CHO

COOH

O HO

HO

O

8

9

10

Triterpenoid and steroidal glycosides referred to collectively as saponins are bioactive compounds present naturally in many plants and known to possess potent hypoglycemic activity [12]. Glucuronide saponin named betavulgaroside (11) was isolated from the roots and leaves of Beta vulgaris L. (sugar beet) exhibited hypoglycemic effect in rats [13].

H CO2H HO2C

H

O HO HO2C HO2CH2CO

O

O O

H

OH 11

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The root cortex of Aralia elata provided another triterpnoid glycoside, Elatoside E (12), which was shown to affect the elevation of plasma glucose level by oral sugar tolerance test in rats [14]. Hypoglycemic activity-guided fractionation on the rhizomes of Anemarrhena asphodeloides yielded steroidal glycosides, pseudoprotoimosaponin AIII, (13) and prototimosaponin AIII, (14). These compounds exhibited hypoglycemic effects in a dose-dependent manner in streptozotocin-diabetic mice but showed no effects on glucose uptake and insulin release, suggesting that the hypoglycemic mechanism may be due to inhibition of hepatic gluconeogenesis and/or glycogenolysis [15]. HO

HO O

R=

O HO

RO

OH

OH

COO

O O

OH

O OH

OH OH

O OH OH OH

12

HO OGlu

OGlu

O

Glu GalO 2

O

Glu 13

GalO 2 14

Charantin (15) a steroidal saponin, obtained from Momordica charantia is known to have an insulin-like activity [16]. Charantin stimulates the release of insulin and blocks the formation of glucose in the bloodstream. Similar steroidal saponin (16) was isolated from the fruiting bodies of Ganoderma applanatum, which exhibits Rat lens aldose reductase (RLAR) inhibiting activity. The same plant also produced few other class of compounds (65-67) with RLAR inhibiting property [17]. A steroidal saponin, chloragin (17) was isolated from the aerial part of Chlorophytum nimonii (Grah) Dalz. The saponin characterized as tigogenin3-O-α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 3)-β-Dxylopyranosyl-(1 →4)-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside showed potent antihyperglycemic activity in streptozotocin induced diabetic rats [18].

Naturally occurring antihyperglycemic and antidyslipidemic agents

OH OH OH

161

OH O OH OH

O OH

O O OH

O 16

15

O H

OH OH OH

O OH

O OH

O OH

O OH

O

OH O

OH

OH O OH

H

O OH

O

O

H

H

OH

17

Yoshikawa and co-workers isolated elatoside G (18), H (19) and I (20) from a garnish foodstuff "Taranome," the young shoot of Japanese Aralia elata were found to exhibit potent hypoglycemic activity in rats [19]. From Gynostemma pentaphyllum Makino (Cucurbitaceae) a gypenoside saponin, named phanoside (21,23-epoxy-3-β-20,21-trihydroxydammar-24ene-3-O-([α-D-rhamnopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-Dlyxopyranoside) (21), has been isolated. Phanoside is a dammarane-type saponin and found to stimulate insulin release from isolated rat pancreatic islets. Phanoside (40 and 80 mg/mL) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia, when given orally to rats [20]. Coagulin C (22), 17β-hydroxywithanolide K (23), withanolide F (24), coagulanolide (25) and coagulin L (26), isolated from the fruits of Withania somnifera, showed significant inhibition on postprandial rise in hyperglycemia post sucrose load in normoglycemic rats and in streptozotocin-induced diabetic rats. Coagulin L (26) showed significant fall in peripheral blood glucose profile and also improved the glucose tolerance of db/db mice [21]. Methanolic extract of the leaves of Boussingaultia baselloides yielded four nor-saponins and a saponin with hypoglycemic activity (27-31). Amongst these, boussingoside A1 (31) exhibited very strong hypoglycemic activity in rats [22].

3.3. Diterpenoids Diterpenoids are composed of four isoprene units and have the molecular formula C20H32, which are derived from geranylgeranylpyrophosphate pathway. Andrographolide (32), a diterpenoid lactone, obtained from

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COOH

COOH

OH

COOH O O OH

OH

COOH CH2OH

CH2OH

O

O

O CH2OH

O

OH OH

OH

OH

OH

OH

OH 19

18

O HO

OH

COOH OH COOH O O O

CH2OH O

O CH2OH OH OH

OH OH

OH

O

O

CH2OH

O

O

O

O

OH OH

OH

O

OH

OH OH

OH

HO

OH

OH

20

21

OH HO O

H O O

H

O

OH

H O O

OH

H

H

H O

O

O

OH

H

OH

H

23

22

24 OH

OH H O O

OH

H H

OH 25

OH

O

OH

H

H O O

O

H

OH HO HO

O

H

OH

O OH

OH

26

O

Naturally occurring antihyperglycemic and antidyslipidemic agents

163

H

H CO2R1 R3 HO HO

O HO HO

O R2

OH

CO2 H

HOOC O HO O

O O OH

OH

27- R1 = H, R2 = Me, R3 = CO2H 28- R1 =β-D-glucosyl, R2 = Me, R3 = CO2H 29- R1 =β-D-glucosyl, R2 = R3 = CH2OH 30- R1 = H, R2 = CH2OH, R3 =CO2H

31

Andrographis paniculata was found to possess significant hypoglycemic activity [23]. A modified diterpene, saudin (33) was isolated from the leaves of Cluytia richardiana (Euphorbiaceae) growing in Saudi Arabia. It is related to the labdane-type of diterpenes with a novel rearrangement of lactone groups, was found to possess hypoglycemic activity when tested in alloxan induced diabetic rats [24]. Bioassay-guided fractionation of the EtOH extract of Maprounea africana, on noninsulin-dependent diabetes mellitus db/db mouse model, resulted in the isolation of a daphnane-type diterpenoid, maprouneacin (34) which showed potent glucose-lowering properties by the oral route [25]. O O

HO

O O CH2

H

O O

HO

CH2OH 32

O H Me

O

O O H

O

O

O O

O

O 33

O

O

O

OH OH

OH 34

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3.4. Sesquiterpenoids Sesquiterpeniods consist of three isoprene units and have the molecular formula C15H24. A sesquiterpene lactone, lactucain C (35) and furofuran lignan, lactucaside (77), were isolated from Lactuca indica which showed in vivo antihyperglycemic activity profile Δ -22.74 ± 12.53% and Δ -17.95 ± 5.63% using STZ-diabetic rats at a dose of 1 μM/kg [26]. O 14

O

13'

1 3

H 11'

9

12' O 6'

5'

O

5

1'

H R

15

O

12

R

15'

13

O

14'

O

35: R =

16 O C

H2C

18

21

OH

O

3.5. Alkaloids An alkaloid is a naturally occurring nitrogenous organic molecule that has a pharmacological effect on humans and other animals. Berberine (36) is known to have potent hypoglycemic activity. It was obtained from the traditional medicinal plant Tinospora cordifolia [27]. The mode of its antihyperglycemic activity was investigated in the Caco-2 cell line. Berberine effectively inhibited the activity of disaccharidases in Caco-2 cells, decreased sucrase activity after pre-incubation with Caco-2 cells for 72 h but failed to produce any significant effect on gluconeogenesis and glucose consumption of Caco-2 cells, suggesting that the antihyperglycemic activity of berberine is at least partly due to its ability to inhibit α-glucosidase and decrease glucose transport through the intestinal epithelium [28]. Other alkaloids such as catharanthine (37), vindoline (38) and vindolinine (39) obtained from Catharanthus roseus also lower blood sugar level [29]. Arecoline (40), an alkaloid isolated from Areca catechu was investigated and reported to have hypoglycemic activity in an animal model of diabetes upon subcutaneous administration [30].

Naturally occurring antihyperglycemic and antidyslipidemic agents

165

Cryptolepine (41) is a rare example of a natural product whose synthesis was reported prior to its isolation from Cryptolepis sanguinolenta. Cryptolepine and its salts form lower blood glucose in rodent models of type II diabetes. To optimize this natural product lead, a series of substituted and hetero substituted cryptolepine analogs was synthesized [31]. Aegeline (42), an alkaloidal-amide from the leaves of Aegle marmelos, was isolated by our group and was found to have antihyperglycemic activity as depicted from the lowering of the blood glucose levels by 12.9% and 16.9% at 5 and 24 h, respectively, in sucrose challenged streptozotocin induced diabetic rats (STZ-S) model at the dose of 100 mg/kg body weight. The reasonable mapping of compound to a validated pharmacophoric hypothesis and 3D QSAR model with an estimated activity (283 nM) suggested that aegeline might be a β3-adregenic receptor (AR) agonist [32]. Hypoglycemic activity of trigonelline (43) and 4-hydroxyisoleucine (57) isolated from seeds of Trigonella foenum graecum viz was evaluated in alloxan induced diabetic mice. The combination of 4-hydroxyisoleucine and trigonelline [4-HIT, 40: 30, 120 mg/kg] was administered orally in alloxan induced diabetic mice. After 28 days treatment with 4-HIT, there was significant decrease in blood glucose level. 4-HIT increased the glucose threshold as compared to only alloxan treated group. Histology of pancreas showed formation of new islets near the vicinity of the pancreatic duct. Glyburide was used as a standard antidiabetic drug and its effect on pancreatic cell was also studied. The pancreatic β cells of glyburide treated mice did not show any islets in the vicinity of pancreatic duct. LD50 was found to be more than 5000 mg/kg. These results suggested that 4-HIT showed hypoglycemic effect in alloxan induced diabetic mice. The presence of the pancreatic islets in the vicinity of duct suggested that 4-HIT might act by regeneration of new islets [33]. The therapeutic potential of Galega officinalis for the management of diabetes was defined in the first half of the twentieth century. G. officinalis is a rich source of guanidine and related molecules, which account for its biological effects. The toxicity of guanidine precludes its use clinically, and experiments by Georges Tanret in the years immediately before the Great War identified a less toxic guanidine-like alkaloid, galegine (44) [34]. The synthetic biguanides such as metformin (45) and its analogues were synthesized on the basis galegine chemical structure.

3.6. β-Carbolines The β-carboline alkaloids Harmane (46), norharmane (47) and pinoline (48), were found to increase insulin secretion two to three-fold from isolated

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T. Narender et al. O N

O

N

+ N

OCOCH3

OCH3 N H

OCH3

COOC H3

36

COOCH3 OH

N H

H3CO

38

37 O

N

N O N

N COOCH3

N H

41

40

39

O OH

H N + N

O

MeO

NH

O_

42

H2N

NH

N H

N

44

43

NH

N H

NH2

45

human islets of langerhans. Harmane and norharmane obtained from Tribulus terrestris may account for the hypoglycemic property of the plant [35]. Harmane stimulates insulin secretion in a glucose-dependent manner. The results strongly substantiated the claim of β-carbolines as potent insulin secretagogues [36]. Harmine (49) is found in Syrian rue (Peganum harmala) and other plants. Recently Waki and co-workers through a small-molecule library screen has identified it as a proadipogenic that acts by inducing PPARγ expression. Obese (db/db) mice treated with harmine show a delay in the onset of diabetes, coincident with increased oxygen consumption and thermogenesis. A 2-fold increase in PPARγ levels was selectively seen in white adipose tissue, while there was a 50% decrease in PPARγ levels in the liver and no change in muscle, brown adipose tissue, or kidney. The effect of harmine on PPARγ expression in the brain and pancreas is currently unknown [37].

N

N

N

NH

H3CO

N H

N H

N H

46

47

48

N H

MeO

49

Naturally occurring antihyperglycemic and antidyslipidemic agents

167

3.7. Carbohydrates Two hypoglycemic principles, ganoderan B (50) and C (51), isolated from the fruit bodies of Ganoderma lucidum were shown to be peptidoglycans with mol wts of 7400 and 5800, respectively. Physicochemical and chemical studies demonstrated that the backbone and side chains of ganoderan B contain D-glucopyranosyl β-1→3 and β-1→6-linkages while those of ganoderan C contain D-glucopyranosyl β-1→3 and β-1→6-linkages and a D-galactopyranosyl α-1→6-linkage [38]. β -D-Glcp1

3)- β -D-Glcp-(1

6β -D-Glcp1

6 β -D-Glcp

1

1

6

6

3)-β -D-Glcp-(1

[ (1

50

3)-β -D-Glcp-(1 ]5 [

3)- β -D-Glcp

6)-α -D-Galp-(1

]1

51

3.8. Amino acids FR225659 (52) and four related compounds (53-56) are gluconeogenesis inhibitors that consisted of an acyl-group and three unusual amino acids. They were isolated from the culture broth of Helicomyces sp. and purified by absorptive resin and reverse-phase column chromatography. They were found to be potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents [39]. T. foenum-graecum (Leguminosae family) is an annual herbaceous plant commonly known as fenugreek and is widely distributed across Asia, Africa, and Europe. Fowden [40] was the first to isolate and identify the unusual amino acid, 4-hydroxyisoleucine (57). Christophe et al. [41] discovered that the major isomer 2S,3R,4S of 4-hydroxyisoleucine induces insulin secretion through a direct effect on pancreatic β cells in rats and humans. Recent studies by our group also confirm the antihyperglycemic activity [42]. The plant Blighia sapida belongs to sapindacae family, which is known for its poisonous properties. Two unusual amino acids such as hypoglycin A (58) and hypoglycin B (59) isolated from this plant possess antihyperglycemic activity [43].

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T. Narender et al. NH2 HO N

O

OH

HN

NH2 COOH

NH

Cl

57

NH O R1

N

R2

NH2

O

O

R2

R3

52

-OH

-CH3

53 54

-OH

-OH -OH -H

55

-OCH3

-OH

-CH3 -CH2CH3

-OCH3

-OH

-CH3

56

58

COOH

R1

-OH

COOH

R3

HN

NH2

HN

-CH2CH3 COOH

COOH

59

3.9. Miscellaneous Salacinol (60) has been isolated from an antidiabetic ayurvedic traditional medicine, Salacia reticulata, through bioassay-guided separation and was found to be most potent natural α-glucosidase inhibitor [44]. Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) (61), a sulphur compound isolated from garlic (Allium sativum) has resulted in pronounced hypoglycemia in mildly diabetic rabbits upon oral administration (0.25 mg/kg) [45]. S-allyl cysteine sulphoxide (62), a sulphur containing amino acid which is the precursor of allicin and garlic oil, has been found to show significant antidiabetic effects in alloxan diabetic rats at a dose of 200 mg/kg body weight [46]. Leporin B (63), a demethylated analog of leporin A (64) was isolated from a taxonomically unidentified fungal strain to discover compounds with the ability to increase expression levels of the enzyme hexokinase II [47].

O O HO HO

-O3SO S+

CH2OH

H H CH2OH OH 60

O

S S

S

H2N OH

N O

O 61

RO

62

63 R=Me 64 R=H

Naturally occurring antihyperglycemic and antidyslipidemic agents

169

Rat lens aldose reductase (RLAR) inhibitors (65-67 and 16) from the fruiting bodies of Ganoderma applanatum were isolated, protocatechualdehyde (67) was the most potent RLAR inhibitor (IC50 = 0.7 μg/mL) equivalent to that of the positive control TMG (IC50 = 0.6 μg/ml) [17]. O CHO OH

OMe HOOC

HO HO HO

n 65

O

NH O

n

HO

n

OH

n = 12-15

66

n = 7-9

OH OH 67

2-Arylbenzofuran, puerariafuran (68) was isolated from MeOH extract of the roots of Pueraria lobata as active constituent, using an in vitro bioassay based on the inhibition of advanced glycation end products (AGE). The compound (68) and coumestrol (69) exhibited a superior inhibitory activity against AGEs formation with IC50 values of 0.53 and 0.19 μM, respectively, compared to a well known positive control, aminoguanidine (IC50 value of 473 μM) [48]. Two compounds viz, kodaistatin A (70) and kodaistatin C (71) were isolated from cultures of Aspergillus terreus. The kodaistatins are effective inhibitors of the glucose-6-phosphate translocase component of the glucose6-phosphatase system (EC 3.1-3.9), an enzyme system which is important for the control of blood glucose levels. The IC50 was 80 nM for kodaistatin A and 130 nM for kodaistatin C [49]. H HO

O

O

O

OH HO

O H3CO

O

68

69 HO O

O

HO

O

HO OH HO O

O R OH

70 R = H 71 R = OH

OH

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T. Narender et al.

The glucose lowering effect of mangiferin (72), a xanthone glucoside, isolated from the leaves of Mangifera indica was studied in streptozotocininduced diabetic rats. Hypoglycemic activity of mangiferin (10 and 20 mg/kg, i.p. once daily for 28 days) at different time intervals in STZ induced diabetic rats and improvement in oral glucose tolerance in glucose-loaded normal rats upon chronic administration (10 and 20 mg/ kg, i. p.) for 28 days was observed [50]. O

HO O

HO HO

O

HO

OH

OH

O

O

OH

O OH

O

OH

72

O

OH

73

A xanthone, which is close analogue of mangiferin was isolated from the hexane fraction of the plant, Swertia chirayita, identified as 1,8-dihydroxy3,5-dimethoxyxanthone (swerchirin: 73). It has a very significant blood sugar lowering effect in fasted, fed, glucose loaded, and tolbutamide pretreated albino rat models. The ED50 for 40% blood sugar lowering in CF male albino rats (body weight 140-165 g) is 23.1 mg/kg/oral [51]. Various active components like (−)-epicatechin (74), the benzofuranone, marsupsin (75) and the stilbene, pterostilbene (76) isolated from the bark and heartwood of Pterocarpus marsupium were evaluated for their putative antihyperglycemic activity against streptozotocin-induced hyperglycemic rats and were found to possess blood sugar lowering activity. The phenolic constituents viz, marsupsin (75) and pterostilbene (76) significantly decreased the plasma glucose level of STZ-induced diabetic rats by -33% and -42% respectively. The antidiabetic activity of pterostilbene (-42%) was comparable to that of the reference compound, metformin (-48%) [52]. OH HO

HO

O

O OH

OH OH

OH OH

OH 74

OCH3 O 75

MeO

OMe 76

Naturally occurring antihyperglycemic and antidyslipidemic agents

171

A furofuran lignan, lactucaside (77) along lactucain (35) was isolated from Lactuca indica which showed in vivo antihyperglycemic activity profile Δ -17.95 ± 5.63% using STZ-diabetic rats at a dose of 1 μM/kg [26]. OCH3 OH O 9' 7

HO

4

1

8' 1'

R

O 4' HO

R = glucose

3' OCH3 77

HO OH

MeO 78

O

H 79

O

Ferulic acid (78) is polyphenolic compound found in many medicinal plants such as Curcuma longa. Ohnishi and co-workers from Japan demonstrated its antihyperglycemic activity in insulin dependent (IDD) and non-insulin dependent diabetes milletus models (NIDDM) [53]. Similar class of compound that is cinnamaldehyde (79) was isolated from Cinnamonum zeylanicum (cinnamon) exhibits potent antihyperglycemic activity in streptozotocin (STZ)-induced male diabetic wistar rats [54]. Both the compounds also possesses hypolipidemic properties [51,52].

4. Dyslipidemia Dyslipidemia is elevation of plasma cholesterol, triglycerides (TGs), or both, or a low high density lipoprotein level that contributes to the development of atherosclerosis. Causes may be primary (genetic) or secondary. Diagnosis is by measuring plasma levels of total cholesterol, TGs, and individual lipoproteins. When carbohydrates are in low supply or their breakdown is incomplete, fats become the preferred source of energy in diabetic patients. As a result, the fatty acids are mobilized into the general circulation leading to secondary triglyceridemia in which total serum lipids in particular triglycerides as well as the levels of cholesterol and phospholipids

172

T. Narender et al.

increase. This rise is proportional to the severity of the diabetes. Uncontrolled diabetes is manifested by a very high rise in triglycerides and fatty acid levels. An increase in plasma lipids, particularly cholesterol, is a common feature of atherosclerosis, a condition involving arterial damage, which may lead to ischemic heart disease, myocardial infarction, and cerebrovascular accidents. These conditions are responsible for one-third of deaths in industrialized nations [55].

5. Current therapeutics Current antidyslipidemia drugs include statins, fibrates, niacin, ezetimibe, and bile acid binding resins (Table-2). These drugs target one component of the lipid profile, with smaller additional effects on other parameters. For instance, statins and fibrates produce sizable reductions primarily in plasma LDL-C and TG, respectively. Meanwhile, niacin has the greatest HDL-C raising capacity. However, many high CHD risk patients fail to reach strict guideline target levels with currently Table 2. Currently available pharmaceuticals for dyslipidemia. Medication

Effects on lipid parameters ↓ ↓ LDL-C, ↓ TG Minimal effects on HDL-C (rosuvastatin can increase HDL-C levels)

Adverse effects

Fibrates (PPAR- α agonists)

↓ LDL-C, ↓ ↓ TG, ↑ HDL-C (mild)

Myalgias, Rhabdomyolysis Cholelithiasis, Elevations in serum creatinine

Ezetimibe (intestinal cholesterol absorption inhibitor) Niacin

↓ LDL-C, ↓ TG

Myalgias (very rare) Rhabdomyolysis (very rare)

Statins (HMG-CoA reductase inhibitors)

Myalgias, Myositis/rhabdomyolysis Transaminitis

↓ ↓ TG, ↑ ↑ HDL-C, ↓ Flushing/vasodilation Impair insulin sensitivity ↓ LDL-C, ↓ ↓ LP (a) Gout, gastric

Bile acid ↓ LDL-C resins (inhibitors of enterohepatic circulation)

↑ TG Bloating, constipation Interference with absorption of other, medications such as levothyroxine, warfarin, digoxin, statins

Naturally occurring antihyperglycemic and antidyslipidemic agents

173

available drugs. A small but clinically relevant proportion of patients experience adverse effects. Thus, additional pharmaceutical strategies are required to fill these gaps in efficacy and tolerability. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them.

5.1. Sterols and triterpenoids A number of studies, both in animal models and human clinical trials, have shown that guggulipid (80,81) isolated from the Resin of the gum of the guggul tree, Commiphora mukul, has beneficial effects on serum lipoprotein profiles [56]. A pregnane glycoside roylenine (82) was isolated from Marsdenia roylei. The glycoside (82) and its acetylated derivative showed singnificant antioxidant and antidyslipidemic activities [57]. H3C

OH

O

O

OH O

O

80

O

H3C

81

OH 82

OH

H3C AcO AcO

O

O O

A steroidal saponin, chloragin (17) [tigogenin-3-O-α-Lrhamnopyranosyl-(1 → 4)- -D-glucopyranosyl-(1 → 3)-β-D-xylopyranosyl(1 → 4 )-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside] was isolated from the aerial part of Chlorophytum nimonii (Grah) which showed potent antidyslipidemic activities in albino rats [18]. Coagulin L (26) isolated from Withania somnifera showed significant fall in peripheral blood glucose profile and also improved the glucose tolerance of db/db mice. It also showed antidyslipidemic activity in db/db mice that is comparable to median effective dose of fenofibrate i.e., 50 mg/kg body weight [21]. Sudhahar and co-workers reported hypercholesterolemia in lupeol (83) and linoleate ester of lupeol (84) [58]. We have also prepared several ester derivatives of lupeol and studied their structure activity relationship. Some of the derivative showed potent activity than the lupeol. Lupeol nicotenate (85) was found to be the most potent triglyceride lowering agent in addition to antihyperglycemic activity [59].

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T. Narender et al.

O

O O

O

HO N 84

83

85

Wiedendiol-A (86) and B (87), sesquiterpene-hydroquinones which inhibit cholesteryl ester transfer protein (CETP), have been isolated from the marine sponge Xestoepongia wiedenmayeri [60] HO

HO

HO

HO OCH3 CH3

OCH3 CH3

H

H 87

86

Statins are currently marketed drugs used to lower the plasma cholesterol levels in humans. Natural statins obtained from different genera and species of filamentous fungi. Lovastatin (88) is mainly produced by Aspergillus terreus strains and mevastatin (89) by Penicillium citrinum. Pravastatin (90) was obtained by the biotransformation of mevastatin by Streptomyces carbophilus and simvastatin (91) by a semi-synthetic process, involving the chemical modification of the lovastatin side chain. The hypocholesterolemic effect of statins lies in the reduction of the very low-density lipoproteins (VLDL) and LDL involved in the translocation of cholesterol, and in the increase in the high-density lipoproteins (HDL), with a subsequent reduction of the LDL- to HDL-cholesterol ratio, the best predictor of atherogenic risk [61]. HO O

O O

H

HO

O

O

HO

O

O O

O O

H

H

COOH OH

HO O

O O

H

HO 88

89

90

O

91

Naturally occurring antihyperglycemic and antidyslipidemic agents

175

Several synthetic statins such as atorvastatin (92), cerivastatin (93), pitavastatin (94) and rosuvastatin (95) were developed on the basis of structures of natural statins. HO

COOH OH

HO

F F

N

HO

COOH OH

COOH OH

O

N

N

N

O 92

COOH OH

F

F

HN

HO

S O

93

94

N N O 95

A diterpene, (96) which has close structural features of statins was isolated from the leaves of Polyalthia longifolia [62]. This compound showed significant antidyslipidemic activity in high diet (HFD) fed dyslipidemic hamsters at different doses. O

O

OH

96

5.2. Polyphenolic compounds Few naturally occurring flavanones and their glycosides such as hesperetin (97), hesperidin (98), naringenin (99), and naringin (100) have been reported as potential agents for improving the cholesterol metabolism in diet-induced hypercholesterolemic animals [63]. We also isolated three modified furano-flavonoids (101-103) and a rare flavonol glycoside (104) as an antidyslipidemic agents from the aerial parts of Indigofera tinctoria [64]. Flavonoid mixture (101 and 102) showed potent triglyceride lowering activity in high fat fed hamster model.

176

T. Narender et al. O O H

O

O H H

O

R2 RO

O

O

O

O 101

O

OH O

H O

R1

O H H

O

O 102 OR

O 97: R= H; R1=OH; R2=OCH3 98: R= β -D-Rutinoside; R1=OH; R2= OCH3 O 99: R = H; R1=H; R2=OH 100: R=Neohespiridoside; R1=H; R2=OH

RO

O O

O OH

O OH O

O 103

104: R= Rhamnose

Eriocitrin (105) (eriodictyol 7-O-β-rutinoside) is the main flavonoid in lemon fruit (Citrus). Eriocitrin was effective in lowering effect on serum and hepatic lipids in high-fat and high-cholesterol fed rats [65]. OH HO

OH OH

H3C

O

O

OH O

HO

O

O

OH OH

OH O

105

Pterosupin (106) and liquiritigenin (107) were isolated from the heartwood of Pterocarpus marsupium showed hypolipidemic activity in Triton model. Both the compounds lowered the serum cholesterol and LDL-cholesterol. Pterosupin also lowered the triglycerides [66]. OH HO

OH

HO

O

Glc OH O 106

OH

O 107

Naturally occurring antihyperglycemic and antidyslipidemic agents

177

Rutin (108) is flavonoid glycoside found in many plants and is also an important dietary constituent of food and plant-based beverages. Several studies demonstrated lipid lowering effect of rutin. Recently Amir and co-workers reported its anti-hyperchloesterolaemic effect (plasma cholesterol and LDL-C) in rat model [67]. Odbayar and co-workers from Japan studied the effect of quercetin (109) and its glycoside (rutin) and their studies indicated that quercetin better than the rutin in reduction of hepatic lipogenesis (hypolipidemic effect) [68]. OH OH HO

O

OH HO O

OH

O

OH OH OH O

H3C O HO HO

HO

O OH

OH

OH

O 109

108

Tso-Hsiao Chen and co-workers studied about 40 flavonoids for their HMG-Co-Enzyme reductase activity. Astilbin (110) was the only effective HMG-Co-Enzyme reducates inhibitor in their studies, which demonstrates its hypochelestereamic activity [69]. OH O

HO

OH

O

O HO

OH OH O

CH3 OH

110

Kurarinol (111) is a prenylated flavanone, which is known for its alpha glucosidase, beta amylase and diacylglyceral transferase activity. Kuraridinol (112) is a prenylated chalcone. Both were isolated from the Sophora flavescens showed significant hyperlipidemic and hypercholesterolemic effect. Kuraridinol was more potent than kurarinol in their studies [70].

178

T. Narender et al. OH

OH

OH

OH HO

HO

O

OH OH

OH O

O

O

O 112

111

Resveratrol (113), a naturally occurring stilbenoid commonly available in red wine act as a free-radical trap to halt the progression of LDL oxidation. It is very strong antioxidant and mild lipid lowering agent, which certain extent prevents the development of atherosclerosis [71]. Resveratrol derivatives such as, pterostelbene (76) and trimethylated resveratrol (114) and its analogue Piceatannol (115) have been studied for their PPAR alpha activity and in-vivo hyperlipidemic activity. Pterostelbene showed good PPAR alpha agonist activity and hypolipidemic activity than other compounds [72]. Polydatin (116) is glycoside of resveratrol isolated from Polygonum cuspidtum also has been reported for its lipid lowering effect in high fat diet fed hamster [73]. OH HO

HO

MeO

OH

OH OH

OMe 113

OH

OMe

114

115

Mangiferin (72) a xanthone glucoside, isolated from the leaves of Mangifera indica showed significant antihyperlipidemic activity at a dose of 10 and 20 mg/kg, i.p. Further, in streptozotocin-induced diabetic rats it showed antiatherogenic activities as evidenced by significant decrease in plasma total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C) levels coupled together with elevation of high density lipoprotein cholesterol (HDL-C) level and diminution of atherogenic index in diabetic rats [50].

Naturally occurring antihyperglycemic and antidyslipidemic agents

179

HO H OH

H OH H O HO H HO H

O H H OH

116

Bergenin (117) is commonly available in many plants of Euphorbiaceae, Saxifragaceae and Myrsinaceae. It is a C-glucoside of 4-O-methylgallic acid. Oral administration of bergenin isolated from the leaves of Flueggea microcarpa reduced the serum cholesterol, triglycerides, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL)-cholesterol levels were significantly [74]. OH OH

OH O MeO

OH O

HO O 117

5.3. Alkaloids Berberine (36), a natural plant alkaloid isolated from the root of Berberis oblonga. In vitro and in vivo studies have showed its effects on hyperglycemia and dyslipidemia [75]. Our activity guided fraction and isolation work o the leaves of A. marmelos led to isolate an alkaloidal-amide, Aegeline (42) and found to have antihyperglycemic activity as well as hypolipidemic activity [32]. Aegeline has strong triglyceride lowering activity in our studies and the activity was comparable with the marketed drug i.e. fenofirbrate. Hsu and coworkers showed that arecoline (40) inhibited adipogenesis as determined

180

T. Narender et al.

by oil droplet formation and adipogenic marker gene expression. There further studies indicated that arecoline induced lipolysis in an adenylyl cyclase-dependent manner [30].

5.4. Amino acid We isolated an unusual amino acid 4-hydroxyisoleucine (57) from the seeds of T. foenumgraecum, which significantly decreased the plasma triglyceride levels by 33% (P < 0.002), total cholesterol (TC) by 22% (P < 0.02), and free fatty acids by 14%, accompanied by an increase in HDL–C/ TC ratio by 39% in the dyslipidemic hamster model [11]. 4-Hydroxyisoleucine is also very good insulin releasing agent.

5.5. Miscellaneous C60-polyprenol (118) was isolated from the chloroform fraction of the ethanol extract of Coccinia grandis. It significantly decreased serum TG by 42%, total cholesterol (TC) 25% and glycerol (Gly) 12% and increased HDL-C/TC ratio by 26% in high fat diet (HFD)-fed dyslipidemic hamsters at the dose of 50 mg/kg body weight as compared to the standard drug fenofibrate at the dose of 108 mg/kg [76]. O S 7

OH H2N OH O

118

119

S-methyl cysteine sulfoxide –SMCS (119) isolated from Allium cepa was investigated for its lipid lowering action in SD rats. SMCS at a dose of 200 mg/kg body weight for 45 days enhanced the hyperlipidemic condition. Concentrations of cholesterol, triglyceride and phospholipids were significantly reduced with respect to control [77]. Itokawa and co-workers also reported the lipid lowering activity in S-methyl cysteine sulfoxide (SMCS) and S-allylcysteine sulfoxide (62) [78]. Ferulic acid (78) and cinnamaldehydes (79) which are commonly available in many medicinal plants have been reported for their lipid lowering activity as well as anthyperglycemic activity [51,52].

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6. Conclusion Type-II diabetes poses a lethal threat to mankind in the present health scenario. The more alarming situation has raised owing to the secondary complications such as atherosclerosis, (ischemic heart disease, myocardial infarction, and cerebrovascular accidents) associated with this silent killer. So, there is an urgent need for broad based drugs which can ameliorate this complex menace. Natural products have always been the inexhaustible source of new drugs from the time immemorial. Notwithstanding the significant headways in synthetic chemistry in the management of hyperglycemia and hyperlipidemia, chemical entities emanating from the natural source still hold promise in alleviating the blood glucose levels and lipids and its concurrent ailments. More has been done but much has remained unexplored in the drug discovery paradigm of natural products attributed with therapeutic virtues. Some targets have been identified for the active principles but unless, their mechanism of action is not determined and clinical studies not performed, their potential as antihyperglycemics and antidyslipidemics will remain unearthed. Moreover, the combination of plant based drugs and synthetic pharmaceuticals for correcting this metabolic error could pave way for costeffective therapies. The scope of plant drugs lies in the rectifying the problem of adverse side effects generated by synthetic drugs, cost-effectiveness and minimal side-effects. The resurgence of natural products in the drug discovery and development may hold the key in the proper utilization of biodiversity for the management of hyperglycemia and hyperlipidemia.

Acknowledgements The authors are grateful to the Director, CDRI, Lucknow for constant encouragement for the program on Indian medicinal plants, CSIR, New Delhi for financial support.

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 187-212 ISBN: 978-81-308-0448-4

6. Bio-flavonoids with promising antidiabetic potentials: A critical survey Goutam Brahmachari Department of Chemistry, Visva-Bharati University, Santiniketan-731 235 West Bengal, India

Abstract. Bio-flavonoids comprise a group of phenolic secondary plant metabolites that are widespread in nature. Major flavonoids that have well categorized structures and well defined structure function-relationships are: flavans, flavanones, flavones, flavonols, flavanols, flavanonols, cetechins, anthocyanidins and isoflavones. Bio-flavonoids are well-known for their multi-directional biological activities including anti-diabetic efficacy. Numerous studies have been carried out to explore their potential role in the treatment of diabetes. A good number of studies have already demonstrated the hypoglycemic effects of flavonoids using different experimental models and treatments - the drug candidates have been shown to exert such beneficial effects against the disease manifestation, either through their capacity to avoid glucose absorption or to improve glucose tolerance. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications; besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues, and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. As a result, bio-flavonoids are now-a-days regarded as promising and significantly attractive natural substances to enrich the current therapy options against diabetes. Correspondence/Reprint request: Dr. Goutam Brahmachari, Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India. E-mail: [email protected]

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The purpose of this resume is to represent promising anti-diabetic flavonoid candidates highlighting their absorption and metabolism along with their mode of action in regulating diabetic symptoms.

1. Introduction Diabetes mellitus is the most prevalent metabolic syndrome world-wide with an incidence varying between 1 to 8% [1,2]. The disease arises when insufficient insulin is produced, or when the available insulin does not function properly. Thus diabetes is characterized by hyperglycaemia (elevation in blood sugar levels) resulting in various short-term metabolic changes in lipid and protein metabolism and long-term irreversible vascular changes. The long-term manifestation of diabetes can result in the development of some complications, broadly classified as microvascular or macrovascular disease. Microvascular complications include neuropathy (nerve damage), nephropathy (renal disease) and vision disorders (retinopathy, glaucoma, cataract and corneal diseases), while macrovascular complications include heart disease, stroke and peripheral vascular disease, which can lead to ulcers, gangrene and amputation [3]. These complications are also found in non-diabetic population, but have a two to five-fold increase in diabetic subjects [4]. The last century has seen a rapid increase in the global prevalence of coronary artery disease (CAD) [5,6]. Current estimates from different countries in Europe and the United States have shown that diabetes and its complications account for 8-16% of the total health costs for society and this will increase dramatically unless major efforts are made to prevent the ongoing epidemic. There are two major categories of diabetes - insulin dependent diabetes mellitus (IDDM, Type 1 diabetes mellitus) and non-insulin dependent diabetes mellitus (NIDDM, Type-2 diabetes mellitus). Type 1 diabetes occurs due to almost 95% destructions of β-cells of islets of Langerhans in the endocrine pancreas caused by an autoimmune process, usually leading to absolute insulin deficiency, this type has an early onset, most often between the ages of 10 and 16 yrs. Insulin resistance in peripheral tissue and an insulin secretive defect of the β-cells characterizes Type-2 diabetes mellitus (NIDDM). It is the most common form of diabetes mellitus constituting above 90% of the diabetic population and highly associated with a family history of diabetes, older age, obesity and lack of exercise [3]. The global prevalence of diabetes is estimated to increase, from 4% in 1995 to 5.4% by the year 2025 [7]. The World Health Organization (WHO) has predicted that the major burden will occur in the developing countries, there will be a 42% increase from 51 to 72 million in the developed countries while 170% increase from 84 to

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228 million, in the developing countries [8]. Prevalence of the complications is greater among the lower socio-economic people due to lack of good control of glycaemia and hypertension and also due to behavioral factors. The direct and indirect costs involved in the treatment of the chronic disease especially when associated with the vascular complications are enormous. The overall global scenario urges to implement cost-effective and at the same time efficacious preventive measures against diabetes to reduce the high morbidity and mortality [4].

2. Currently available therapies Currently available therapies for diabetes include insulin and various oral anti-diabetic agents such as sulfonylureas, biguanides, α-glucosidase inhibitors, and glinides, which are used as monotherapy or in combination to achieve better glycemic regulation. Many of these oral anti-diabetic agents suffer from various adverse effects, thus, managing diabetes without any side effects is still a challenge to the workers [9], and hence the search for more effective and safer therapeutic agents in eradiating diabetic syndromes has continued to be an important area of investigation. Both fasting and postprandial impaired glucose tolerance are associated with an increased risk of developing Type-2 diabetes mellitus and therefore form an important target group for interventions aimed at preventing diabetes [10]. The pharmacological agents with the greatest effect on postprandial hyperglycemia include insulin lispro, amylin analogues, and α-glucosidase inhibitors. In hyperglycemia associated with diabetes, the use of aldose reductase inhibitors has been reported for the treatment of diabetic complications [11]. Aldose reductase as a key enzyme in the polyol pathway has been reported to catalyze the reduction of glucose to sorbitol. Sorbitol does not readily diffuse across cell membranes, and the intracellular accumulation of sorbitol has been implicated in the chronic complications of diabetes such as peripheral neuropathy, retinopathy, and cataracts [12]. A recent study reported that aldose reductase may also be involved with another signal transduction pathway in the pathogenesis of diabetic nephropathy [13].

3. Back to the plant kingdom The use of ethnobotanicals has long folkloric history for the treatment of blood sugar abnormalities. In the India, indigenous remedies have been used in the treatment of diabetes since the time of Charaka and Sushruta (6th century B.C.) [14]. Plants have always been exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly

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from them. The ethnobotanical information reports about 800 plants that may possess anti-diabetic potential [15]. Many of such plants have exhibited anti-diabetic activity when assessed using presently available experimental techniques [17-20]. It may be mentioned in this connection that the discovery of widely used hypoglycaemic drug, metformin came from the traditional approach of using Galega officinalis. In spite of all these, the indigenous system has not yet gained enough momentum in the scientific community. The reasons may be many including lack of belief among the practitioners of conventional medicine over alternative medicine, alternative form of medicine are not very well-defined and natural drug may vary tremendously in content, quality and safety. To cope with severe problems associated with using of synthetic anti-diabetic drugs, there is a need to look for more efficacious drugs with lesser side effects and also of low cost. It is the high time to turn our attention to the plant kingdom in search of natural drugs for diabetes following an integrated approach and using correct procedures. The hypoglycemic effect of several plants used as anti-diabetic remedies has already been confirmed, and the mechanisms of hypoglycemic activity of these plants are being studied; if even a single plant material stands the acidtest of efficacy comparable to commonly used synthetic oral drugs already marketed, it will herald the discovery of cheap and relatively nontoxic drug.

4. Purpose of the present review A number of review articles on the uses of various plants (different parts of plant materials, crude extracts, herbal formulations, etc.) as anti-diabetic agents have been published time to time [22-26]. Naturally occurring chemotypes of varying structural skeletons have also been reported to possess anti-diabetic properties [27,28], and the purpose of this resume is to represent promising anti-diabetic bio-flavonoids highlighting their absorption and metabolism along with mode of action in regulating diabetic symptoms.

5. Anti-diabetic bio-flavonoids of promise Bio-flavonoids comprise a group of phenolic secondary plant metabolites that are widespread in nature. Major flavonoids that have well categorized structures and well defined structure function-relationships are: flavans, flavanones, flavones, flavonols, flavanols, flavanonols, cetechins, anthocyanidins and isoflavones. Bio-flavonoids are well-known for their multi-directional biological activities including anti-diabetic efficacy [29-32]. Numerous studies have been carried out to explore their potential role in the treatment of diabetes [27,28,33]. A good number of studies have already demonstrated the

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hypoglycemic effects of flavonoids using different experimental models and treatments - the drug candidates have been shown to exert such beneficial effects against the disease manifestation, either through their capacity to avoid glucose absorption or to improve glucose tolerance. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications; besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues, and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. As a result, bio-flavonoids are now-a-days regarded as promising and significantly attractive natural substances to enrich the current therapy options against diabetes. This present section embodies the information on promising anti-diabetic efficacies of certain bio-flavonoids. Choi et al. [34] demonstrated that intraperitoneal administration of prunin (naringenin 7-O-β-D-glucoside) produces a significant hypoglycemic effect in diabetic rats. Anti-hyperglycemic effects have also been demonstrated for various flavonoids including chrysin and its derivatives, silymarin, isoquercetrin and rutin [35-37]. Long-term studies carried out with rutin orally administered to diabetic rats showed that it decreased the plasma glucose levels by up to 60% when compared to the control group. However, oral administration of rutin to normal rats did not show any significant effect on fasting plasma glucose levels [38]. Chronic treatment with hesperidin and naringin was found to lower the blood glucose level of db/db mice compared with the control group [39]. Myrciacitrins I, II, III, IV and V (1-5) isolated from the dried leaves of Myrcia multiflora DC. (family: Myrtaceae) were reported to possess significant rat lens aldose reductase inhibitory activity [40], the IC50 values for the flavonoids 1-5 were determined as 3.2 x 10−6, 1.5 x 10−5, 4.6 x 10−5, 7.9 x 10−7, 1.6 x 10−5 and 1.3 x 10−5 M, respectively [40,41]. Hence, myrciacitrin IV (4) exhibited the most potent activity, although it had less activity than epalrestat, a commercially available synthetic aldose reductase inhibitor (IC50 = 7.2 x 10−8 M) [40]. Kawabata et al. [42] isolated five 6-hydroxy-flavonoids (6-10) from the methanol extract of Origanum majorana L. (family: Lamiaceae) leaves and studied their α-glucosidase enzyme inhibitory activity, three of these flavonoids: 6-hydroxyapigenin (scutellarein) (6), 6-hydroxyapigenin-7-O-βD-glucopyranoside (7), 6-hydroxyluteolin-7-O-β-D-glucopyranoside (8) are previously known [43-47], and the other two feruloylglucosides namely, 6-hydroxyapigenin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (9) and 6hydroxyluteolin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (10) are novel compounds. All the isolates showed rat intestinal α-glucosidase inhibitory activity, at an equal concentration of 500 μM, the flavonoid candidates 6-10

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inhibited the enzyme activity by 81%, 44%, 55%, 25% and 26%, respectively. The respective IC50 values for 6-10 were determined as 12, >500, 300, >500 and >500 μM. Another flavonoid, 6-hydroxyluteolin (11) [48], was also found to exhibit potent α-glucosidase inhibitory activity (92% inhibition at a concentration of 500 μM) with an IC50 value of 10 μM [42]. The same group [49] also evaluated 5,6,7-trihydroxyflavone (baicalein, 12), the flanonoid constituent of Scutellaria baicalensis, as an important inhibitor against rat intestinal α-glucosidase (IC50 = 32 μM). The investigators also observed that apigenin (5,7,4′-trihydroxyflavone, 13) and luteolin (5,7,3′,4′-tetrahydroxyflavone, 14), both lacking the 6-hydroxyl substituent, showed negligible activity (12% and 22% inhibition at 500 μM, respectively) in the α-glucosidase inhibitory assay. From their study, the present investigators suggested that 5,6,7-trihydroxyflavone skeleton is crucial for high α-glucosidase inhibitory activity regardless of B-ring hydroxylation, in addition, glycosation of 7-hydroxyl substituent as well as acylation of the sugar reduces the enzyme inhibitory activity [49]. Haraguchi et al. [50] isolated C-glucosidic flavone derivative named as isoaffineyin (5,7,4,3′,5′-pentahydroxyflavone-6-C-glucoside, 15) from Manikara indica (family: Sapotaceae), the flavonoid candidate exerted promising inhibition against porcine lens aldose reductase activity with an IC50 value of 4.6 μM (epalrestat was used as positive control, IC50 = 0.87 μM).

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The genistein derivatives (16-19) isolated from an EtOAc-soluble partition of the MeOH extract of a branch of Tetracera scandens (family: Dilleniaceae) were evaluated to possess promising activities on Type-2 diabetes mellitus treatment since the test compounds significantly stimulated the uptake of glucose, adenosine monophosphate-activated kinase (AMPK), glucose transport protein-4 (GLUT4) and GLUT1 mRNA expressions and protein tyrosine phosphatase 1B (PTP1B) inhibition in L6 myotubes [51]. The IC50 values for isofavonoids 16-19 in inhibiting PTP1B activities were

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determined as 31.75 ± 0.27, 28.13 ± 0.19, 20.63 ± 0.17 and 37.52 ± 0.31 μM, respectively (ursolic acid was used as positive control with IC50 value of 5.13 ± 0.45μM). No muscle cell toxicity was reported with compounds 17-19, while compound 16 reduced muscle cell viability with IC50 value of 18.69 ± 0.19 μM. The investigators, thus, demonstrated that the isoflavonoids constituents (16-19) of T. scandens stimulate glucose-uptake in basal and insulin-stimulated L6 myotubes in a dose-dependent manner - AMPK activation, GLUT4 and GLUT1 expressions and PTP1B inhibition by these bioactive constituents appeared to be involved in the mechanism of the stimulation of basal and insulin-responsive glucose-uptake. Hence, compounds 16-19 may be possible candidates of a novel therapeutic strategy for Type-2 diabetes mellitus treatment, although further studies will be required to clarify the molecular mechanism of these bioactive constituents [51].

Isoorientin (20), isolated from the water and butanolic extracts of Cecropia obtusifolia (family: Ceropiaceae), exhibited potent hypoglycemic activity comparable to that of glibenclamide at a dose of 3 mg/kg body weight in diabetic rats [52]. Kim et al. [53] isolated a new flavonol glycoside, quercetin 3-O-α-L-arabinopyranosyl-(1Æ2)-β-D-glucopyranoside (21) along with the known flavonoid glycosides such as kaempferol 3-O-β-D-glucopyranoside

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(astragalin) (22a) and quercetin 3-O-β-D-glucopyranoside (isoquercetin) (22b) from the leaves of Eucommia ulmoides (family: Eucommiaceae), these flavonoid constituents were found to be glycation inhibitors having comparable activity to that of aminoguanidine, a known glycation inhibitor. The IC50 values for the test compounds 21, 22a and 22b were determined as: 2.95 x 10−7, 4.86 x 10−7, and 3.20 x 10−7 M, respectively (aminoguanidine was used as positive control, IC50 = 4.45 x 10−7 M) [53].

Tabopda et al. [54] reported that six unusual C-4′-prenylated flavonols, dorsilurins F-K (23-28), isolated from the roots of Dorstenia psilurus (family: Moraceae), were found to exhibit glycosidase enzyme inhibitory activity against α-glucosidase, β-glucosidase, and α-mannosidase. Compound 23, with three unmodified prenyl groups, showed the best α-glucosidase inhibitory activity (IC50 4.13 μM), while compound 28, with only one unmodified prenyl group, showed the least α-glucosidase inhibitory activity (IC50 43.95 μM). Thus, it was suggested that α-glucosidase inhibitory activity of the compounds increased with the number of unmodified prenylated groups present. These compounds (23-28) showed very weak enzyme inhibitory activities against β-glucosidase and α-mannosidase [54].

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Two dihydroflavonol glycosides such as engeletin (29) and astilbin (30), isolated from the leaves of Stelechocarpus cauliflorus (family: Annonaceae), exhibited inhibitory activity against a recombinant human aldose reductase, the inhibitory activity of 29 (IC50 = 1.16 μM) was found to be twice that of quercetin (positive control, IC50 = 2.48 μM), and 23 times greater than that of 30 (IC50 = 26.7 μM) [55].

Flavonoid glycosides (FG 1 and FG 2), isolated from Phyllanthus fracternus (family: Euphorbiaceae), at a dose of 100 mg/kg p.o. were found to be hypoglycaemic in alloxanised rats (20 and 25%) at 3 hrs, however, no

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blood sugar lowering was observed in normal rats [56]. A neoflavonoid, coutareagenin [5-hydroxy-7-methoxy-4-(3,4-dihdroxyphenyl)-2H-benzo-1pyran-2-one] isolated from the bark of Hintonia latiflora (family: Rubiaceae), exhibited promising anti-diabetic efficacy in streptozotocin-induced Wistar rats as well as in menopausal diabetic women [57,58]. Kaempferol-3,7-O-(α)-dirhamnopyranoside (kaempferitrin, 31), isolated from the n-butanol fraction of the leaves of Bauhinia forficata (family: Leguminosae), exhibited significant hypoglycemic effect in normal and alloxan-induced diabetic rats on oral administration. In normal rats, reduction in blood glucose level was noticed only with the higher dose of 31 (200 mg/kg) at 1 h after treatment, whenever such efficacy of the test compound in diabetic rats was evident at all doses administered (50, 100, and 200 mg/kg), and this profile was found to be maintained throughout the period studied for both higher doses. However, in glucose-fed hyperglycemic normal rats, kaempferitrin could not down-regulate blood glucose levels [59]. Kaempferol3-neohesperidoside, a glycosylated flavonoid structurally very similar to kaempferitrin, was also shown to demonstrate promising hypoglycemic effect in both oral and intraperitoneal treatments in diabetic rats, in addition, kaempferol-3-neohesperidoside-VO(IV) complex showed potent hypoglycemic efficacy throughout the post-treatment period studied when compared with zero time [60]. When complexed with vanadium, quercetin also demonstrated much promising insulin-enhancing activity in STZ-diabetic mice with no effect on the blood glucose level of normal mice, which is in agreement with the results for kaempferitrin and kaempferol-3-neohesperidoside- VO(IV) complexes [60,61]. Quercetin itself was evaluated to possess anti-diabetic effect by reducing the blood glucose level of diabetic rats in 8-10 days of treatment [62], in the same study by Vessal and his group, the test compound exerted no effect on the glucose tolerance curve either in normoglycemic or in STZ-diabetic rats [62]. These results support the views of Shetty et al. [63] for hypoglycemic effects of quercetin in diabetic rats. Three prenylated flavanones (33-35) isolated from stem barks of Erythrina abyssinica (family: Liguminosae) exhibited inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) in dose-dependent manner with IC50 values >60, 18.9±1.9 and 15.7±0.4 μM, respectively [64], hence, the flavanone (32) bearing a 2,2-dimethylpyran moiety on B ring is less potent than the other two (33 & 34) in the series. The investigators, thus, suggested that substitution of prenyl groups on flavonoids may be important for in vitro PTP1B inhibitory activity and cyclization between a hydroxy group and the prenyl group in B ring without prenyl or methoxy groups may reduce the activity [64]. One more isoprenyl flavonoid (35) isolated from the root barks of Erythrina mildbraedii were also found to exhibit inhibitory

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activity against PTP1B enzyme in dose-dependent manner with IC50 values 21.2±1.6 μM. The present investigators argued that substitution of isoprenyl groups on ring-B might be important for PTP1B inhibitory activity in vitro, and introduction of one more hydroxyl group to C-5 of ring-A or one of the isoprenyl groups in ring-B might be responsible for a loss of such activity [65]. Isorhamnetin 3-O-β-D-glucoside (36) isolated from the ethylacetate fraction of Salicornia herbacea (family: Chenopodiaceae) was evaluated to possess significant inhibitory activity against rat lens aldose reductase (RLAR) in vitro with an IC50 value of 1.4 mM, which is similar to that of tetramethylene glutaric acid (IC50 = 1.7 mM) [66]. The flavonol glycoside (36), when administered orally at 25 mg/kg in streptozotocin (STZ)-induced diabetic rats, caused not only a significant inhibition of serum glucose concentration but also sorbitol accumulation in the lenses, red blood cells (RBC), and sciatic nerves, thereby, advocating the test compound from S. herbacea as a leading compound for further study as a new drug for the prevention and/or treatment of diabetes and its complications [66]. Luteolin 6-C-(6′′-O-trans-caffeoylglucoside) (37) isolated from Phyllostachys nigra (family: Gramineae) showed inhibitory efficacy against advanced glycation end products (AGEs), hence, this compound could be offered as a leading compound for its further study towards development of new natural products drug for diabetic complications [67]. Jang et al. [68] reported two flavan-3-ol derivatives (38 and 39) from the roots of Actinidia arguta (family: Actinidiaceae) that were found to exhibit inhibitory activity in vitro on the formation of advanced glycation end products with IC50 values of 13.5 and 17.9 μg/mL, respectively. Few more advanced glycation end products (AGEs) inhibitors such as the dihydroflavonol glycosides (40 and 41) [55], isoflavone C-glucosides (42 and 43) [69] and the 2,3-dioxygenated flavanone erigeroflavanone (44) have also been reported [70]. The isoflavone C-glucosides (42 and 43) isolated from the roots of Pueraria iobata (family: Pueraria) showed more potent in vitro inhibitory activity against AGEs formation with IC50 values 8.7 and 24.9 μg/mL, respectively [69]. The present investigators [69] suggested that the compound (42) is worthy of consideration as a therapeutic agent for diabetic complications or related diseases. Yoo et al. isolated the 2,3dioxygenated flavanone, erigeroflavanone (44) from the flowers of Erigeron annuus (family: Asteraceae/Compositae), and evaluated its inhibitory activity against AGEs formation with an IC50 value 22.7 μM [70]. A flavone xylopyranoside, 4',5-dihyroxy-6,7-dimethoxyflavone-3-O-βD-xylopyranoside (45), isolated from the roots of Euphorbia leucophylla (family: Euphorbiaceae) by Satyanarayana et al., was found to reduce the

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blood glucose levels (BGLs) and increase the serum insulin levels in normal and diabetic rats [71]. One flavone [1′′(R)-5,4′,1′′-trihydroxy-6,7-(3′′,3′′dimethylchromano)flavone, 46] and one flavanone [(2S)-4′-O-methyl-6methyl-8-prenylnaringenin, 47) both isolated Eysenhardtia platycarpa (family: Leguminosae) were evaluated to possess promising anti-

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hyperglycemic activity by decreasing glucose level of streptozotocin (STZ)induced diabetic rats (31 mg/kg of body weight, P < 0.05) [72]. Matsuda et al. [12] examined a variety of flavonoids for their rat lens aldose reductase inhibitory activity to study structure-activity relationships. Among the flavone constituents, 3′,4′-dihydroxyflavone (48), 3′,4′,7-trihydroxyflavone (49), luteolin (50), and luteolin 7-O-β-D-glucopyranoside (51) were found to possess potent inhibitory activity with IC50 values of 0.37, 0.30, 0.45 and 0.99 μM, the flavonoid glycosides, quercitrin (52), guaijaverin (53) and desmanthin-1 (54) also showed the most potent activity against the enzyme with respective IC50 values of 0.18, 0.18 and 0.082 μM [12]. The activity of desmanthin-1 (54) was equivalent to that of a commercially available synthetic aldose reductase inhibitor, epalrestat (IC50 = 0.072 μM). From their detailed studies, Matsuda et al. suggested the following structural requirements of flavonoids for aldose reductase inhibitory activity - (i) the 5-hydroxyl moiety has no effect, (ii) the 3-hydroxyl and 7-Oglucosyl moieties reduce the activity, (iii) the 2-3 double bond enhances the activity, and (iv) the flavones and flavonols having the catechol type moiety at the B ring (the 3′,4′-dihydroxyl groups) exhibit stronger activities than those of pyrogallol-type moiety (the 3′,4′,5′-trihydroxyl groups) [12].

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6. Absorption and metabolism of flavonoids 6.1. Absorption of flavonoids As far as reports are available, the absorption of dietary flavonoids may be influenced by the matrix in which they are consumed, with enhanced excretion in urine of easily recognized mammalian conjugates observed when presented in foods with a higher fat content [73-78] - although certain reports are there in contrast to [79-83]. However, an important factor in the absorption efficiency of flavonoid glycosides in the intestine is the sugar moiety, as demonstrated for quercetin glycosides, its aglycone and rutin supplements in healthy ileostomy volunteers [84]. Flavonoid aglycones, being hydrophobic in nature, can be transported across membranes by passive diffusion, whereas in flavonoid glycosides the sugar moiety enhances the hydrophilicity of the flavonoid molecules as a whole, thereby, reducing the possibility of passive transport. Hence, it may be argued that flavonoids are absorbed by active transport [85]. A good number of studies in human and animals are in agreement with the fact that some dietary flavonoids such as flavanols [86], quercetin-3-glucoside and quercetin-4′-glucoside [87-89] can be absorbed in the small intestine — however, quercetin, quercetin-3galactoside, quercetin-3-rutinoside (rutin), naringenin-7-glucoside, genistein7-glucoside and cyanidine-3,5-diglucoside have been found not to be [89,90]. It has been suggested that before absorption flavonoids are cleaved by specific enzymes either in the lumen or inside the cells of the gut. Lactasephlorizin hydrolase (LPH) is anchored in the brush-border membrane in the small intestine and catalyzes extracellular hydrolysis of some glucosides [91,92]. Another enzyme, located intracellularly and with broad specificity, is the cytosolic β-glucosidase (CBG). It is found in abundance in the small intestine, liver and kidney of mammals and requires active transport of hydrophilic glucosides into the cells [93]. Concerning LPH activity, it has been shown that the enzyme cleaves some flavonol and isoflavone glycosides such as quercetin-4′-glucoside, quercetin-3-glucoside, quercetin-3,4′glucoside, 3′-methylquercetin-3-glucoside, genistein-7-glucoside, and

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daidzein-7-glucoside. However, quercetin-3-rhamnoglucoside and naringenin-7-rhamnoglucoside (naringin) are not substrates for this enzyme [91,93]. In addition, β-glucosidase activity is reported to act on flavonoid and isoflavone glycosides according to the position and the structure of the sugar moiety attached to the flavonoid aglycone [94]. Mechanism of absorption have still not been completely elucidated but is believed to involve inter alia interaction of certain glucosides with the active sugar transporter-1 (SGLT-1) and luminal lactase-phlorizin hydrolysate (LHP), passive diffusion of the more hydrophobic aglycones, or absorption of the glycoside and interaction with cytosolic β-glucosidase (CBG).

6.2. Metabolism of flavonoids After being absorbed in body, flavonoids undergo three main types of conjugations such as methylation, sulfation and glucuronidation [95-97]. The most important enzymes involved in flavonoids metabolism are catechol-Omethyltransferase (COMT, EC 2.1.1.6), phenol sulfotransferase (P-PST, SULT, EC 2.8.2.1) and UDP glucuronosyl transferase (UDPGT, UGT, EC 2.4.1.17). Catechol-O-methyltransferase methylates polyphenols and has the highest activity in the liver and kidneys [98]. Phenol sulfotransferases are cytosolic enzymes that transfer sulfate moieties to hydroxyl groups from substrates such as iodothyronines, phenols and hydroxyarylamines mainly in the liver [96,97,99]. UDP glucuronosyl transferase catalyzes the conjugation of polyphenols to glucuronic acid in endoplasmic reticulum in the intestine, liver and kidney. In humans, the liver has the greatest capacity for glucuronidation while in rats, the highest level of glucuronyl transferase activity was observed in the intestine [99-101]. Conjugation reactions with glucuronic acid and/or sulfate appear to be the most common type of metabolic pathways for the flavonoids first occurring in the gut barrier [85] and these conjugates then reach the liver, where they are further metabolized [81,99,102]. Otake et al. [103] showed that hepatic UDP-glucuronosyl transferase isoforms were the main factors responsible for galangin metabolism into two major glucuronides conjugated at the 7- and 3- positions by using human liver microsomes. Also, Vaidyanathan and Walle [104] demonstrated no glucuronidation of (−)-epicatechin by human liver and small intestinal microsomes. However, in rats, (−)-epicatechin was efficiently metabolized by liver microsomes with formation of two glucuronides. In the same study, the authors concluded that sulfation also occurred in both the liver and intestine in human and rats. Three (−)-epicatechin metabolites such as (−)-epicatechin-3′-Oglucuronide, 4′-O-methyl-(−)-epicatechin-3′-O-glucuronide, and 4′-Omethyl(−)-epicatechin-5 or 7-O-glucuronide have been isolated from human

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urine [105], whereas the exact fate of (+)-catechin is not known although there is evidence for the formation of (+)-catechin sulfates, sulfoglucuronides, and 4′-methylated conjugates in plasma and urine [76,106]. In contrast, (−)-epicatechin gallate and (−)-epigallocatechin gallate appear to be excreted in bile [79,86,107,108]. The (−)-epicatechin gallate is extensively methylated by human liver catechol O-methyl transferase at the 4′-position and to a lesser extent at the 3′-position [109,110], while (−)-epigallocatechin gallate is metabolized first to the 4′′-methyl ether and then to the 4′,4′′dimethyl ether [110]. Flavonoid glycosides that are not absorbed in the small intestine along with the conjugated metabolites that are excreted in bile can be metabolized by microflora when they reach the colon. Glycoside flavonoid-hydrolyzing enzymes have been identified in fecal flora cultures. Bokkenheuser et al. [111] recovered three enzyme-producing strains that, using β-glucosidases, α-rhamnosidases, and/or β-galactosidases, were capable of converting rutin to quercetin. Also, it was shown that at least some of the bacterial glycosidases are able to cleave glycosidic bonds and flavonoid-saccharide bonds in the gut [91]. Genistein-7-glucoside and daidzein-7-glucoside have not been found in human plasma [112] but the aglycones have been observed [113]. Human metabolism of isoflavone glycosides produces genistein and daidzein 7-glucuronides/7-sulfates and 4′,7-diconjugates (including diglucuronides and mixed conjugates), with monoglucuronides predominant [114,115]. The profile of metabolites has been demonstrated in studies with quercetin, rutin and naringin. The flavonoid metabolism produces aromatic acids such as phenylvaleric, phenylpropionic, phenylacetic and benzoic acids with easy absorption through the colonic barrier [116-118]. Flavonol glycosides and quercetin aglycone have not been convincingly demonstrated in plasma [119-121], although kaempferol aglycone has been detected [122]. The main kaempferol metabolite in human plasma is the 3-glucuronide [122]. The three major metabolites of quercetin are: quercetin-3-glucuronide, quercetin-3′-sulfate, and isorhamnetin-3-glucuronide. Apigenin glucuronides have been detected in urine after volunteers consumed parsley [123], luteolin aglycone administered to volunteers has been detected in plasma as a monoglucuronide accompanied by a trace of unconjugated luteonin [124,125]. Chrysin is transformed primarily to the 7-glucuronide with much smaller yields of the 7-sulfate [126]. Metabolites of flavonoids in general (and also microflora metabolites), aglycones, glycosides and conjugated metabolites which are not absorbed, may follow two pathways of excretion: via the biliary or the urinary route. Large conjugated metabolites are more likely to be eliminated in the bile whereas small conjugates such as monosulfates are preferentially excreted in

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urine [100]. When excreted in bile, the flavonoids are passed to the duodenum and metabolized by intestinal bacteria, which results in the production of fragmentation products and/or the hydrolysis of glucurono- or sulfoconjugates [127]. The resulting metabolites which are released may be reabsorbed and enter an enterohepatic cycle or being excreted in feces [128,129]. For each flavonoid, the beneficial effect will be dependent upon their absorption and availability in the body. Thus, these factors should be considered in any interpretation of the potential health effects of flavonoids.

7. Mode of action of flavonoids Very recently, Cazarolli et al. [130] reviewed on the mode of action of flavonoids including cellular and molecular mechanism. In their review, the authors thoroughly discussed about the various effects of the drug candidates in regulating diabetic syndromes. It has been demonstrated that flavonoid compounds act against diabetes mellitus either through their capacity to avoid glucose absorption or to improve glucose tolerance. In vitro studies have shown that a soybean extract containing the isoflavones genistein and daidzein inhibits glucose absorption into the intestinal brush border membrane vesicles of rabbits [131]. Naringenin was also found to reduce glucose uptake in the intestinal brush border membrane vesicles of diabetic rats to a level similar to that of normal rats [132]. The (−)-epicatechin gallate, myricetin, quercetin, apigenin, (−)-epigallocatechin gallate, and (−)epigallocatechin demonstrated a marked reduction in glucose absorption, when compared with the control, by competitive inhibition of sodiumdependent glucose transporter-1 [133]. The non-glycosylated flavonoids were shown to reduce glucose absorption under sodium-dependent conditions in vivo and in vitro in animal tissues [134,135]. Besides reducing glucose absorption, another possible mechanism followed by flavonoid compounds to control blood glucose levels is the inhibition of α-glucosidase activity in the intestine. Such inhibitory effects against α-glucosidase activity were observed when luteolin, kaempferol, chrysin and galangin were used both in vitro and in vivo to study the potential role in the absorption and metabolism of carbohydrates [136]. Kim et al. [137] also demonstrated the α-glucosidase inhibitory activity of flavonoids in a study, where it was shown that luteolin, amentoflavone, luteolin 7-O-glucoside and daidzein are the strongest inhibitors of the compounds tested. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications, besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues,

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and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. In an experimental study by Liu et al. [138], genistein was found to act directly on pancreatic β-cells, leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect. Interestingly, it has found that epigallocatechin 3-gallate mimics the effects of insulin on the gene expression reduction of phosphoenolpyruvate carboxykinase and G-6-Pase in the mouse liver [139], like insulin, the drug candidate enhances tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), mitogen-activated protein kinase, p70s6k, and PI3K activity, and reduces phosphoenolpyruvate carboxykinase gene expression mediated by PI3K [140]. Furthermore, epigallocatechin 3gallate upregulates glucokinase mRNA expression in the liver of db/db mice [141]. In another study, oral administration of rutin to diabetic rats resulted in a decrease in plasma glucose and increase in insulin levels, and restored the glycogen content and hexokinase activity. The activity of enzymes such as G-6-Pase and fructose-1,6-bisphosphatase significantly decreased in the liver and muscles of rutin-treated diabetic rats [142]. Kaempferol-3neohesperidoside has been shown to have the efficacy for prompt stimulating of glycogen synthesis in rat soleus muscle by approximately 2.38-fold, it has also been demonstrated that the phosphatidylinositol-3-kinase (PI3K) glycogen synthase kinase-3 (GSK-3) pathway and mitogen-activated protein kinase (MEK) - protein phosphatase-1 (PP-1) pathway are involved in the stimulatory kaempferol-3-neohesperidoside effect on the glycogen synthesis [143]. Very recently, Cazarolli et al. [144,145] have reported on the mechanism of action of the anti-diabetic effects of apigenin-6-C-β-L-fucopyranoside and apigenin-6-C-(2′′-O-α-L-rhamnopyranosyl)-β-L-fucopyranoside – the former drug candidate was evaluated to stimulate insulin secretion and potentiated glucose-induced insulin secretion in hyperglycemic rats, in addition, this flavonoid stimulated glycogen synthesis in rat soleus muscle through mechanisms well known to insulin signal transduction, thereby, establishing the dual effects of apigenin-6-C-β-L-fucopyranoside as an anti-hyperglycemic (insulin secretion) as well as an insulino-mimetic (glycogen synthesis) agent [144]. In another study, the same group of investigators has characterized apigenin-6-C-(2′′-O-α-L-rhamnopyranosyl)-β-L-fucopyranoside as both an insulin secretagoge and an insulin-mimetic agent [145].

8. Conclusions Diabetes mellitus has already emerged as an alarming disease worldwide affecting the public health much. Though presently available therapies

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against the disease reduce the sufferings to some extent, still it remains inadequate and at the same time is costly, and also associated with a lot of side effects. Hence, there is an urgent need for search of more efficacious drugs with no or minimum side effects. There has been a growing interest in anti-diabetic agents from natural products, particularly those derived from plants. Flavonoids are naturally occurring phenolic compounds with a broad range of biological activities and the beneficial effects of flavonoids have been studied in relation to diabetes mellitus, either through the inhibition of intestinal α-glucosidase enzyme or through their capacity to avoid glucose absorption and/or to improve glucose tolerance. A good number of bio-flavonoids reported over the past 15-20 years discussed in this review clearly demonstrate that these exogenous substances represent an unparalleled source of molecular diversity in relation to the drug discovery process in the treatment of Type-2 diabetes. Although there has been considerable scientific progress over the past few years in unraveling of the effect and mechanism of action of flavonoids, we still need to define the missing steps in the flavonoid-signaling network and elucidate the mechanism of cross-talk based on the complex mechanism of insulin action, in order to provide new insights into the potential role of flavonoids in diabetes treatment. Further study is required concerning safety (assessment of toxic effect) and human trial to develop potential anti-diabetic remedies of choice.

Acknowledgement The author greatly appreciates financial support under Major Research Grant from the University Grants Commission (UGC), New Delhi, India [Project No. F.34-357/2008(SR) dt 02.01.2009].

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 213-268 ISBN: 978-81-308-0448-4

7. Marine natural alkaloids as anticancer agents+ Deepak Kumar and Diwan S. Rawat Department of Chemistry, University of Delhi, New Delhi-110007, India

Abstract. Alkaloids are naturally occurring nitrogen containing biologically active heterocyclic compounds. Over the last few years, a large number of biologically important alkaloids with antiviral, antibacterial, anti-inflammatory, antimalarial, antioxidant and anticancer activities have been isolated from marine source. Present article summarizes the isolation and anticancer activity evaluation of natural marine alkaloids and their synthetic analogues that includes pyridoacridine, indole, pyrrole, pyridine, Isoquinoline, guanidine and steroidal alkaloids.

Introduction Since ancient times nature has been a source of medicines to cure many deadly diseases. Majority of drugs in use today are either natural products (NP), their derivatives (ND), natural products mimics (NPD) or semisynthetic derivatives (SSD) [1-4]. In natural sources, plants, animals and microorganisms have been the main source of biologically important molecules. Ocean has been considered as the main source of medicines and during the past two decades thousands of compounds and their metabolites with several different type of biological activity such as antimicrobial, anti-inflammatory, antimalarial, antioxidant, anti HIV and anticancer activity have been isolated from marine microorganisms [5-12]. But till date only few anticancer drugs such as citarabine, +

Dedicated to Dr. DS Bhakuni and Prof. Deepak Pental

Correspondence/Reprint request: Prof. Diwan S. Rawat, Department of Chemistry, University of Delhi New Delhi-110007, India. E-mail: [email protected]

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vidarabine etc have been commercially developed from marine compounds while several others are currently in different stages of clinical trials [13]. Over 18000 compounds have been isolated from marine source and approximately 150 compounds are cytotoxic against the different tumor cells [14,15]. Some of the prominent anticancer compounds which are in different stages of clinical trials include aplidine, ecteinascidin-734 (Yondelis), bryostatin-1, squalamine, dolastatin10, ILX651, and KRN7000 (α-galactosylceramide) [16]. The present article summarises the recent development in the area of marine alkaloids that includes pyridoacridine, indole, pyrrole, pyridine, isoquinoline, guanidine and steroidal alkaloids.

1. Pyridoacridine alkaloids Pyridoacridines are highly coloured marine natural products having polycyclic planar heteroaromatic 11H-pyrido[4,3,2,mn]acridine system (1) [17]. They are probably the largest class among marine alkaloids and are almost universally isolated from sponges, ascidians as well as from a mollusc and a coelenterate [18]. Pyridoacridine alkaloids show significant biological activity primarily cytotoxicity and certain specific biological properties viz. fungicidal and bactericidal properties, inhibition of topoisomerase II, anti HIV, intercalation of DNA property, Ca+2 releasing activity, production of reactive oxygen species [19-22]. These activities depends upon the substitution pattern of the basic structure of pyridoacridine, therefore many synthetic analogues have also been synthesized keeping the basic skeleton of pyridoacridine in mind. The synthesis of these analogues and their biological activity evaluation revealed that in most of the cases cytotoxicity of the analogues has improved compared to the parent molecule [23, 24]. During the last few years, numerous additional compounds of this family were isolated; most of them are polycyclic with different substituents such as shermilamine, kuanoniamine, neoamphimedine, arnoamines and styelsamines. It has been observed that almost all the pyridoacridines shows promising cytotoxicity against different type of tumors. Therefore a great interest was developed to modify the pyridoacridine moiety for developing a new generation of therapeutic agents. The first review article on marine pyridoacridines alkaloids was published by Molinski in 1993 [25] followed by Ding et al. in 1999 [26]. The cytotoxicity of the compounds of this family is a manifestation of their DNA binding properties, topoisomerase II inhibition and the production of reactive oxygen species. Pyridoacridines vary structurally by attachment of different side chains or fusion of different rings to ring C of the basic structure (1) and less often to the acridine nitrogen. Halogen substitution in pyridoacridines is quite rare; even if it is present, then it is always bromine at C2 in ring A. Oxidation states of the rings are variable and in some cases ring D is partially saturated. Additional rings are

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often attached to ring C. Pyridoacridines can be divided into tetracyclic, pentacyclic, hexacyclic, heptacyclic and octacyclic alkaloids. 2 1

3 A

11a 11 HN

4 B

10a

N

4a 5

10 C

D

8

7a N 7

9

1

6

N O

2

1.1. Tetracyclic alkaloids In 1988, Kobayashi et al. reported the isolation of three tetracyclic alkaloids, cystodytins A-C (3-5) from tunicate Cystodytes dellechiajei collected from Okinawa [27]. Then in 1991, the same group reported six other novel tetracyclic alkaloids of cystodytin family, cystodytins D-I (6-11) along with cystodytins A (3) and B (4) [28]. Thus cystodytins A-C (3-5) are the first pyridoacridine alkaloids isolated from a marine tunicate and therefore the first tetracyclic member of this class. The common heterocyclic nucleus of cystodytins A-C (3-5) is an iminoquinone substituted at C10 with a 2-amidoethyl side chain. The N-acyl groups are derived from β,β´-dimethylacrylic, tiglic and 3-hydroxy-3methylbutanoic acids, respectively. Cystodytins D-I (6-11) are chiral, levorotatory compounds. Cystodytins F-I (7-10) are substituted with an O-methyl ether or O-9-octadecenoate ester. The isomeric pairs of cystodytin β,β´-dimethylacrylate and tiglate amides could not be separated and were characterized as 7:2 mixtures. Hydration of cystodytin A (3) in presence of 6% aq. HC1 at 100 οC gives cystodytin C (5). When treated with diazomethane, it afforded monomethyl ether (12) with 23% yield. This transformation is unusual as it constitutes a formal reductive methylation. Cystodytin A (3) is readily reduced in the ionization stage of a mass spectrometer as observed for quinones. Cystodytin A (3), when hydrogenated over Adams catalyst in acetic acid yielded (13) by reduction of the side chain and disubstituted benzene ring, but the iminoquinone part remains intact. Compounds 3, 4 and 5 showed potent cytotoxicity against L-1210 with IC50 values of 0.22, 0.22 and 0.24 µg/mL, respectively. Cystodytins D-I (6-11) were also found to be cytotoxic against murine lymphoma L-1210 cells with IC50 values of 1.1 (6 and 7), 0.068 (8 and 9) and 0.080 (10 and 11) µg/mL and values of 1.4 (6 and 7), 0.078 (8 and 9) and 0.092 (10 and 11) µg/mL against human epidermoid carcinoma KB cells in vitro.

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Deepak Kumar & Diwan S. Rawat O

R2

H N

R1

H N

X=

N

HN

O O N

N Y=

O 3, R2 = X, R1 = H O 4, R2 = Y, R1 = H 5, R2 = Z, R1 = H 6, R2 = X, R1 = OH Z= 7, R2 = Y, R1 = OH 8, R2 = X, R1 = OMe 9, R2 = Y, R1 = OMe 10, R2 = X, R1 = OCO(CH2)7CH=CH(CH2)7CH3 11, R2 = Y, R1 = OCO(CH2)7CH=CH(CH2)7CH3

12 H N OH

OMe

N

O N 13

O

Cystodytin J (14) was isolated from a ascidian Cystodytes sp. [29]. Cystodytin J (14) showed cytotoxic activity against HCT and xrs-6 with IC50 values of 1.6 and 135.6 µM, respectively. It also inhibited the topoisomerase (TOPO) II-mediated decatenation with IC90 value of 8.4 µM. Recently, Appleton et al. reported isolation of cystodytins K (15), a new member of cystodytins, from the extract of an ascidian Lissoclinum notti collected near Leigh Harbour, Northland, New Zealand [30]. Structure of compound was determined by spectroscopic techniques, including 2D 1H-15N NMR experiments and was found to be 12-methoxy derivative of cystodytin J (14). Cystodytins K (15) exhibited cytotoxic activity against P-388 murine leukaemia cell line with IC50 value of 1.3 µM. Two bright crimson pigments, Varamine A (16) and B (17) were isolated from the Fijian ascidian Lissoclinum vareau [31]. Varamines A (16) and B (17) have parent tetracyclic aromatic ring system at the same oxidation level as the methylation product of cystodytin A (12). Varamines also contain a methyl thioether substituent at C9. Varamine A (16) was readily oxidised by aq. cerric ammonium nitrate to imonoquinone (18) with 90% yield. Varamine A (16) and B (17) exhibited cytotoxicity towards L-1210 murine leukemia cells with IC50 values of 0.03 and 0.05 µg/mL, respectively. In 1989, Ireland et al. isolated a new tetracyclic alkaloid, diplamine (19) from the tunicate Diplosoma sp. collected from the Fiji Island [32]. The structure was established by interpretation of spectral data and chemical analysis. Diplamine (19) was found to be cytotoxic towards L-1210 murine leukemia cells with IC50 value of 0.02 µg/mL. Recently, two novel alkaloids, isodiplamine (20) and lissoclinidine (21) along with known diplamine (19) were isolated from an ascidian Lissoclinum notti collected near Leigh Harbour, Northland, New Zealand. All the compounds (19-21) were tested for their cytotoxicity against murine leukaemia (P-388), human colon tumour (HCT-116) and non-malignant African Green Monkey kidney (BSC-1) cell lines. Diplamine (19) was found to be the

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most active compound among the three and it was observed that movement of the thiomethyl group from C-9 (diplamine) to C-5 (isodiplamine) decreases cytotoxicity against all the cell lines and the same pattern also observed, when the thiomethyl group is cyclised into a benzoxathiole ring (lissoclinidine). These results were also found to be consistent with the proposed mechanism of cytotoxicity of diplamine, which includes DNA intercalation, inhibition of topoisomerase II and other DNA processing enzymes and bioreductive activation. Lissoclinidine (21) was also evaluated against the NCI 60 cell line panel and demonstrated moderate activity and selectivity with panel average values of GI50 = 1.0 mM, TGI = 6.9 mM and LC50 = 29 mM.

H N

H N

N

O S

N

N O

O

14

15

HN

N

O

16, R = CH2CH3 17, R = CH3 O HN

N

R1

N

N

R2

R3 S

N

S O

O

O

N H CF3COO

21

20, R1 = H, R2 = SCH3, R3 = H 19, R1 = SCH3, R2 = H, R3 = H

18

N OMe

O H N

HN

N

O MeO

O

H N

R

In 1998, Copp et al. reported the isolation of four new tetracyclic pyridoacridine alkaloids, styelsamines A-D (22-25) from an extract of the ascidian Eusynstyela latericius [33]. The structures of all the compounds were determined on the basis of 1D and 2D NMR spectroscopy. Styelsamines A-D (22-25) exhibited mild cytotoxicity toward the human colon tumor cell line (HCT-116) with IC50 values of 33, 89, 2.6 and 1.6 µM, respectively. OH H

N

HO

R

N H

CF3COO

22, R =

NH3

23, R =

NHCOMe

24, R = 25, R =

CHO NH3

CF3COO

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Deepak Kumar & Diwan S. Rawat

1.2. Pentacyclic alkaloids Amphimedine (26) was the first example of pyridoacridine alkaloids to be fully characterized [34]. In 1983, Schmitz et al. isolated amphimedine as a sparingly soluble yellow pigment from Amphimedon sp. The structure of amphimedine was established on the basis of spectroscopic data analysis. High resolution mass spectral analysis established the molecular formula C19H11N3O2 (m/e = 313.08547, + 0.35 mass error) for amphimedine. In mass spectrum very few fragments were observed corresponding to loss of CH, CO, CHO and HCN. The UV spectra of compound (26) in absolute ethanol showed absorption at λmax 210 nm (19690), 233 nm (39393), 281 nm (9099), 341 nm (6060). Significant changes were observed upon addition of NaBH4 [λmax 235 nm (12879), 280 nm (9090)], indicating the presence of α,β-unsaturated ketone, which was further supported by the strong absorbtion at 1690 cm-1. Further presence of amide functionality was confirmed by IR and 13C NMR. No OH or NH absorptions were observed in the IR and due to low solubility of compound (26) in common organic solvents, NMR spectral data were obtained in trifluoroacetic acid-d4 and CDCl3 (2:1). The 2D NMR techniques (1H-1H correlation and 13C-13C INADEQUATE NMR) were also used to confirm the structure of amphimedine (26). In 1999, Ireland et al. reported the isolation of a new pyridoacridine, neoamphimedine (27) along with amphimedine (26) from Xestospongia sp. from the Philippines and Xestospongia cf. carbonaria from Micronesia [35]. He deduced the molecular formula for neoamphimidine as C19H11N3O2 by high-resolution fast atom bombardment (FAB) mass spectral analysis. Both amphimedine and neoamphimedine have the same molecular formula hence they are isomers. Recently, deoxyamphimedine (28) along with two known compounds (26 and 27) was isolated from two tropical Xestospongia sponges [36]. Amphimedine, neoamphimedine and deoxyamphimedine have the same skeleton, but they differ in biological activities and this is probably due to the the differences in their structures. Literature servey reaveled that amphimidine relatively inactive compared to neoamphimedine and deoxyamphimedine. Neoamphimedine inhibits topoisomerase II while amphimedine is relatively nontoxic at the same dose level [37] and deoxyamphimedine damages DNA independent of topoisomerase enzymes through the generation of reactive oxygen species [38].

N

N

N

O N

N O 26

N

N O

N

N

O

O

27

28

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219

Schmitz et al. reported the isolation of three new alkaloids 29-31 from two ascidians. The meridine (29) and a relatively stable tautomer of meridine i.e. 30 were isolated from Amphicarpa meridiana collected at Stenhouse bay, South Australia [39]. The structure of meridine (29) was determined by X-ray analysis while that of 31 was established by spectral analysis. The third alkaloid, 11hydroxyascididemin (31) was isolated from a Leptoclinides sp. from Truk Lagoon. All three alkaloids (29-31) were found to be cytotoxic. Recently, Menendez et al. synthesized a regioisomer of meridine named as 9 Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-one (32) from 5,8dimethoxy-6-nitro-4(1H)-quinolinone in eight steps with 23% overall yield [40]. Compound (32) was tested for cytotoxicity against different tumor cell lines and exhibited mild to strong cytotoxic activity against P-388, A-549, HT-29 and MEL-28 with IC50 values of 4.18, 0.03, 0.40 and 0.17, whereas IC50 values for meridine were 0.08, 0.08, 0.84 and 0.08, respectively. Compound 32 and the natural meridine (29) were also tested in vitro for Topoisomerase II inhibitory activity. Meridine showed mild activity (IC50 = 3 mM), whereas compound 32 was found to be inactive even at the highest concentration (33 mM). In 1988, a novel pentacyclic alkaloid, ascididemin (33) was isolated from brown colored tunicate Didemnum sp. collected at Kerama Islands, Okinawa [41]. The structure of compound was elucidated on the basis of spectroscopic data. Ascididemin (33) was found to be cytotoxic against L-1210 murine leukemia cells in vitro with IC50 value of 0.39 µg/mL. Delfourne et al. synthesized an isomer of ascididemin, named as 9H-quino[4,3,2-de][1,7]phenanthroline-9-one (34) starting from 1,4-dimethoxyacridine with an overall yield of 12% along with other derivatives (35-39) of compound 34 [42]. These compounds were tested in vitro at six different concentrations on 12 different human cancer cell lines such as glioblastomas, breast, colon, lung, prostate and bladder cancers. Almost all the compounds showed significant cytotoxic activity and compound 34 was found as much potent or slightly less potent as the natural ascididemin (33). Ascididemin (33) and the isomer (34) exhibited cytotoxicity against U-87MG (0.07, 0.8 µM), U-373MG (0.5, 0.8 µM), SW1088 (0.6, 3 µM), T-47D (0.6, 0.7 µM), MCF-7 (0.07, 0.9 µM), Lovo (0.9, 0.7 µM), HCT-15 (0.06, 0.4 µM), A-549 (0.2, 7 µM), A-427 (0.06, 0.08 µM), PC-3 (0.008, 0.09 µM), T-24 (0.8, 0.1 µM) and J-82 (0.3, 1 µM), respectively. A new pentacyclic alkaloid, cystodamine (40) was isolated from a mediterranean ascidian Cystodytes dellechiajei collected near the bay of Gabes, at Skhira, Tunisia [43]. The structure was determined by extensive 2D NMR data analysis and was found to contain a phenanthroline unit fused with 7 aminopyridine moiety. Cystodamine (40) showed cytotoxic activity against CEM human leukemic lymphoblasts with IC50 value of 1.0 µg/mL. Later, Delfourne et al. revised the structure of cystodamine (40) to 11-hydroxyascididemin (31) by comparison of the spectroscopic data with those of synthetic cystodamine, meridine and 11hydroxyascididemin [44]. 11-Hydroxyascididemin had been previously isolated by Schmitz et al. from the other marine source Amphicarpa meridian.

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In 1988, Scheuer et al. reported the isolation of a new pentacyclic alkaloid, shermilamine A (41) from purple colonical tunicate Trididemnum sp. [45]. After one year, shermilamine B (42) was reported by two groups simultaneously Scheuer [46] and Kashman [47]. In 1994, McDonald et al. isolated shermilamine C (43) from a Fijian ascidian Cystodytes sp. [48]. Shermilamine A (41) contains a pentacyclic pyridoacridine thiazinone system while shermilamine B (42) is a debromo analogues of shermilamine A (41). Two novel shermilamine alkaloids, shermilamine D (44) and E (45) were isolated from the Indian Ocean tunicate Cystodytes violatinctus collected at the Mayotte Lagoon, Comoros Islands, northwest of Madagascar [49]. Shermilamine D (44) exhibited cytotoxicity against P-388, A-549, HT-29 and MEL-28 cancer cell lines with IC50 values of 0.53, 0.27, 2.66 and 0.53 µM, respectively [50]. A new member of shermilamines, cycloshermilamine D (46) was isolated from the same marine tunicate Cystodytes violatinctus [51]. The structure of cycloshermilamine D (46) was established mainly on the basis of NMR spectroscopic data and was found to be closely related to shermilamine D (44) having hexacyclic structure. Kuanoniamines A-D (47-50) were isolated along with the known shermilamine B (42) from a tunicate and its prosobranch mollusc predator Chelynotus simperi [52]. The structures were established by extensive NMR analysis and correlations spectroscopy. Kuanoniamines C (49) and D (50) were also isolated from another tunicate of the genus cystodytes collected in Pohnpei [53]. Kuanoniamines B (48) and D (50) are homologues of kuanoniamine C (49) having isovaleramide and acetamide side chains, respectively. Kuanoniamine A (47) is structurally different from the other three alkaloids and lacks the 2-amidoethyl side chain and contains an iminoquinone moiety. In 1994,

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McDonald et al. isolated dehydrokuanoniamines B (51) from a Fijian ascidian Cystodytes sp. [54]. More recently, the N-deacyl derivative (52) was isolated from the sponge Oceanapia sp. collected at Truk Lagoon, Micronesia along with its two parent molecule (49) and (50) [55]. Kuanoniamines A-D (47-50) showed weak cytotoxicity. Kuanoniamine A (47) was found to be the most active compound of the group and inhibits the proliferation of KB (human pharyngeal cancer) cell lines in vitro with IC50 value of 1 µg/mL. Dehydrokuanoniamine B (51) and kuanoniamines D (50) were found to have comparative potentials in vitro against HCT (IC50 = 8.3 and 7.8 µM) and xrs-6 cells (IC50 = 80 and 88.9 µM). The N-deacyl derivative (52), kuanoniamine C (49) and D (50) were tested in vitro against two human cancer cell lines, HeLa cells and MONO MAC-6 cells. Kuanoniamine C (49), D (50) and N-deacyl derivative (52) exhibited IC50 values of 5.1, 1.4 and 1.2 µg/mL (HeLa) and values of 1.2, 0.8 and 2.0 µg/mL (MONO MAC-6).

O

H N

N O

S

N H

R1

H N

N

N

N

N

S

N

N

S

N

S

N H

O R2

41, R1 = Br, R2 = NHCOMe 42, R1 = H, R2 = NHCOMe 43, R1 = H, R2 = NHCOCH=C(Me)2 44, R1 = H, R2 = NMe2 45, R1 = H, R2 = N(O)Me2

H

NMe2 R

46

47

48, R = NHCOCH2CH(CH3)2 49, R = NHCOCH2CH3 50, R = NHCOCH3 51, R = NHCOCH=C(CH3)2 52, R = NH3

In 1988, Gunawardanda et al. isolated dercitin (53) from the deep water marine sponge Dercitus sp. collected from Bahamas [56]. The structure of dercitin was assigned on the basis of spectroscopic data. This structure (53) was subsequently revised to structure (54) by the interpretation of the magnitude of long range protoncarbon coupling constants. Dercitin (54) exhibited in vitro antitumor activity against P-388 (IC50 = 0.05 µg/mL) and human tumor cells (HCT 8, A-549, T47D) with IC50 value of 1.0 µg/mL. Dercitin (54) also showed in vivo activity against P-388 (T/C 170%, 5 mg/kg). One year later, the same group isolated three new pentacyclic pyridoacridine alkaloids, nordercitin (55), dercitamine (56) and dercitamide (57) from the extract of a red coloured sponge Stelletta sp. collected in Bahamas [57]. Later dercitamide (57) was found to be identical to kuanoniamine C (49). Compounds (5557) inhibited the proliferation of P-388 murine leukemia cells in vitro with IC50 values of 4.79, 26.7 and 12.0 µM, respectively. Two new pyridoacridine alkaloids, arnoamines A (58) and B (59) were isolated from the ascidian Cystodytes sp. collected in the vicinity of Arno Atoll, Republic of Marshall Islands [58]. They were supposed to be the first members of

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Deepak Kumar & Diwan S. Rawat

pentacyclic pyridoacridine alkaloids having a pyrrole ring fused with the pyridoacridine ring system. The structures of 58 and 59 were established on the basis of spectroscopic data, particularly those obtained from HMBC and NOE NMR experiments. The arnoamines A (58) and B (59) displayed a much unexpected chemical reactivity. The pyrrole ring hydrogens labelled as Ha and Hb showed duterium exchange, when NMR were recorded in CDCl3/TFA-d4. Arnoamine A (58) exhibited cytotoxicity against the MCF-7, A-549 and HT-29 cell lines with GI50 values of 0.3, 2.0 and 4.0 µg/mL, respectively, whereas Arnoamine B (59) showed GI50 values of 5.0, 2.0 and 3.0 µg/mL against the MCF-7, A-549 and HT-29 cell lines, respectively. The methanol extract of the ascidian Cystodytes dellechiaijei, collected in Brazil yielded two novel alkaloids, sebastianine A (60) and B (61) [59]. The structures of both the compounds were established by analysis of spectroscopic data. Sebastianine A (60) was found comprising of a pyridoacridine system fused with a pyrrole unit and sebastianine B (61) is having a pyridoacridine system fused with a pyrrolidine system condensed with R-hydroxyisovaleric acid. Sebastianine A (60) and B (61) showed cytotoxic activity against a panel of HCT-116 colon carcinoma cells. N

N

Ha

S

X

N

Hb Y

N

N

N H N R

NMe2

OR

55, R = N(CH3)2 56, R = NHCH3 57, R = NHCOCH2CH3

53, X = S, Y = N 54, X = N, Y = S

58, R = H 59, R = Me O

N

N

N H

N

N

O

O

H

O

N OH

N H

NH

H N

O

CF3

N

O

60

61

62, R = H 63, R = OH

Recently, Davis et al. isolated two new pyridoacridine alkaloids, ecionines A (62) and B (63) from Australian sponge Ecionemia geodides [60]. Both the compounds were found to contain an imine moiety, which is very rarely found in pyridoacridine class of compounds. Both the compounds were tested against a panel of human bladder cancer cell lines (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2) and the superficial bladder cancer cell line 5637. Compound (63) showed moderate cytotoxicity against all the cell lines, with IC50 values

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of 6.48 mM (TSU-Pr1), 6.49 mM (TSU-Pr1-B1), 3.55 mM (TSU-Pr1-B2) and 3.66 mM (5637), whereas Compound (64) showed cytotoxic effect on 5637 and TSU-Pr1-B2 cells at 10 mM, with cell growth inhibitions of 54% and 51% cells, respectively, but did not have any effect on TSU-Pr1-B1 cells at 10 mM.

1.3. Hexacyclic alkaloids The extracts of a deep violet sponge Dercitus sp. collected in the Bahamas yielded a hexacyclic alkaloid cyclodercitin (64). The sixth ring in cyclodercitin (64) is formally derived by cyclization of the 2-aminoethyl side chain to the acridine nitrogen, while the pyridine ring is substituted with an N-methyl group. Cyclodercitin (64) inhibited the proliferation of P-388 murine leukemia cells in vitro with IC50 value of 1.9 µM. Recently, stellettamine (65) was isolated from a deep water marine sponge Stelleta sp. [61]. The molecular formula, C20H14N4S was determined by high resolution FAB mass spectroscopy. The structure of the compound was established on the basis of 1H-13C correlation spectroscopy except the orientation of thiazole ring. Therefore complete structure of stellettamine (65) was determined by a single-crystal X-ray diffraction experiment.

1.4. Heptacyclic alkaloids Eilatin (66) is the only known heptacyclic pyridoacridine alkaloid of the marine origin [62]. Molecular formula of eilatin (66) was determined as C24H12N4 by high-resolution EIMS. 1H NMR spectrum showed only six aromatic protons that could agree with the common six protons of the benzodiazaphenanthroline system. The 13C NMR spectrum exhibited only 12 carbon lines (6 for monoprotonated carbons and 6 nonprotonated carbons). This suggests a symmetrical dimeric structure for eilatin (66). Various 2D NMR experiments such as 1H-13C correlations and a HETCOSY experiment were failed to deduce the structure and finally it was determined by a single-crystal X-ray analysis. Eilatin (66) was found to exhibit cytotoxic activity against HCT cell line with IC50 value of 5.3 µM.

N

N

S

N

N

N

N

N

S

N N NMe2

64

65

N

66

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Deepak Kumar & Diwan S. Rawat

1.5. Octacyclic alkaloids In 1991, Faulkner et al. isolated two novel optically active octacyclic alkaloids, eudistones A (67) and B (68) from the Seychelles tunicate Eudistoma sp. [63]. Eudistone A (67) was obtained as an amorphous yellow powder. The molecular formula C27H19N5O for eudistone A (67) was determined by high resolution mass spectroscopy, which implies 21 degrees of unsaturation. The 13 C NMR signal at 191.8 ppm and an IR band at 1660 cm-1 indicated the presence of an unsaturated ketone and the broad bands at 3360 and 3220 cm-1 attributed for primary or secondary amines. The complete structure of the compounds was determined on the basis of other correlations NMR techniques such as COSY, NOE, HMBC and HMQC. Eudistone B (68) was obtained as a white amorphous powder. The molecular formula C27H17N5O for eudistone B (68) was determined and has one more degree of unsaturation than that present in eudistone A (67). Therefore eudistone B (68) is a dehydrogenation product which was also supported by air oxidation of eudistone A (67) to eudistone B (68). When air is bubbled through a solution of eudistone A (67) in DMSO at 60oC for 48 hrs, the dihydropyridine ring of eudistone A (67) is aromatized to yield eudistone B (68). N OCH2CH3 O HN

O

N

HN

N

N N

N

N

HN

N N

NH

67

68

N H

N N

69

Recently, Demeunynck et al. synthesized an octacyclic analogue (69) of eilatin [64]. The compound (69) was tested against two cancer cell lines, HT-29 (human colon adenocarcinoma) and A-431 (human epithelial carcinoma). Unfortunately due to its low solubility in water, the compound could only be tested at low concentration (5 µM) and did not show any activity against HT-29 and 85% survival on A-431 cell lines.

2. Indole alkaloids Indole-containing alkaloids have frequently been isolated from diverse marine invertebrates including bryozoans, coelenterates, sponges, tunicates, algae, symbiotic bacteria and fungi [65-72]. Indole alkaloids show different type of biological activities such as cytotoxic, antitumor, antiviral, antimicrobial,

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antiparasitics, antiserotonin and anti-inflammatory activities [73]. Due to the interesting biological activities and unique structural features, the indole series have become an attractive research field for the development of new pharmacological lead compounds. In the past few years, some of the isolated natural organic compounds and their derivatives have been synthesized by chemists and evaluated for their biological activity to find new lead compounds against different infectious diseases [74-79].

2.1. Bisindole alkaloids In 1988, Kohmoto et al. isolated a bisindole alkaloid, dragmacidin (70) from a deep water marine sponge Dragmacidin sp. [80]. Dragmacidin was found to contain two indole groups joined by a piperazine ring system which had not been found before in marine natural products. The molecular formula of dragmacidin was deduced as C21H19Br3N4O from FAB HRMS data analysis. Several 2D NMR experiments such as COSY, HETCOR, COLOC and HETCOSY were performed in order to determine the structure of the compound. Dragmacidin (70), when treated with excess acetic anhydride and pyridine overnight at room temperature yielded the triacetate derivative (71). An ethanolic solution of dragmacidin (70) on treatment with 10% Pd/C at room temperature under 20 psi of hydrogen gives tridebromodragmacidin (72). Dragmacidin (70) exhibited in vitro cytotoxicity with IC50 values of 15 µg/mL against P-388 cell lines and 1-10 µg/mL against A-549 (human lung), HCT-8 (human colon) and MDAMB (human mammary) cancer cell lines. The pacific sponge Hexadella sp. collected from the coast of British Columbia yielded two other members of dragmacidin family, dragmacidon A (73) and dragmacidon B (74) along with a new alkaloid, topsentin C (75) [81]. The structures of the compounds 73-75 were proposed on the basis of spectroscopic analysis. Dragmacidon A (73) showed in vitro cytotoxicity in the L-1210 assay with ED50 value of 10 mg/mL, whereas topsentin C (75) and dragmacidon B (74) were found to be inactive. In 1995, Capon et al. reported the isolation of dragmacidin D (76) from a deep water marine sponge Spongosorites sp. collected from the southern Australian coast [82]. Dragmacidin D (76) was found to be active against human lung tumor cell lines and inhibited in vitro growth of the P-388 murine and A-549 with IC50 values of 1.4 and 4.5 μg/mL, respectively. Four new bisindole alkaloids, nortopsentins A-D (77-80) were isolated from the Caribbean deep sea sponge Spongosorites ruetzleri [83]. The structures of nortopsentins A-D (77-80) were established mainly on the basis of NMR spectroscopic data and were found to contain an imidazole ring between two indole units. Compounds (77-80) exhibited cytotoxic activity against P-388 cells with IC50 values of 7.6, 7.8, 1.7 and 0.9 µg/mL, respectively.

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The sponge Topsentia genitrix, collected from Banyuls (France) yielded two bisindole alkaloids, topsentin (81) and bromotopsentin (82). They were found to contain 2-acyl imidazole moiety inserted between two indole units with different substitution on benzene rings [84]. In 1995, Capon et al. reported the isolation of isobromotopsentin (83) from the deep water sponge Spongosorites sp. collected from the coast of southern Australia [85].

R1

R4 N

R3 R2

N H

N

Br

N R5

HN

Br N H

N H

70, R1 = OH, R2 = R3 = Br, R4 = Me, R5 = H 71, R1 = OAc, R2 = R3 = Br, R4 = Me, R5 = OAc 72, R1 = OH, R2 = R3 = H, R4 = Me, R5 = H 73, R1 = H, R2 = R3 = Br, R4 = Me, R5 = H 74, R1 = H, R2 = R3 = Br, R4 = Me, R5 = Me H N O

O

N

R3

75

OH

H N

R2

HN

R1

N HN

NH

Br N HN

HN

76

H2N

NH

77, R1 = R2 = Br 78, R1 = Br, R2 = H 79, R1 = H, R2 = Br 80, R1 = R2 = H

Topsentin (81) inhibited proliferation of cultured human and murine tumor cells. It exhibited in vitro activity against P-388 with IC50 value of 3 μg/mL, human tumor cell (HCT-8, A-549, T47D) with IC50 value of 20 μg/mL and in vivo activity against P-388 (T/C 137%, 150 mg/kg) and B16 melanoma (T/C 144%, 37.5mg/kg) [86]. Bromotopsentin (82) showed antiproliferative activity against human bronocopuemonary cancer cells (NSCLC-N6) with an IC50 = 12 μg/mL [87]. Deoxytopsentin (84) was isolated from the sponge Hexadella sp collected in Jervis Inlet, British Columbia [88]. In 1999, bromodeoxytopsentin (85) and isobromodeoxytopsentin (86) were isolated from sponge Spongosorites genitrix collected from Jaeju Island Korea by Shin et al. [89]. Structurally topsentin (81) and deoxytopsentin (84) are the same except the indole ring which is unsubstituted in case of deoxytopsentin (84). Deoxytopsentin (84) showed the antiproliferative activity against human bronocopulmanary cancer cells (NSCLC-N6) with an IC50 value of 6.3 μg/mL. It also displayed moderate activity against breast cancer and hepatoma (HepG2) with an IC50 of 10.7 and 3.3 μg/mL, respectively.

Marine natural alkaloids as anticancer agents O

N

N

NH

R1

N H

O

NH

R3 R1

N H

R2

227

R3 N H

N H

R2

84, R1 = R2 = R3 = H 85, R1 = Br, R2 = R3 = H 86, R1 = R2 = H, R3 = Br

81, R1 = R2 = H, R3 = OH 82, R1 = Br , R2 = H, R3 = OH 83, R1 = H , R2 = OH, R3 = Br

Recently, Kobayashi et al. isolated a new cytotoxic bis-indole alkaloid, hyrtinadine A (87) from Okinawan marine sponge Hyrtios sp. [90]. The structure elucidation was achieved on the basis of spectroscopic data. Hyrtinadine A (87) was supposed to be the first example of a bisindole alkaloid with a 2,5-disubstituted pyrimidine ring between two indole units. Hyrtinadine A (87) exhibited in vitro cytotoxicity against murine leukemia L-1210 and human epidermoid carcinoma KB cells with IC50 values of 1.0 and 3 µg/mL, respectively. Hyrtiosins A (88) and B (89) were also isolated together with known 5-hydroxyindole-3-aldehyde (90) from the Okinawan marine sponge Hyrtios erecta [91]. Compound (90) exhibited cytotoxic activity against human epidermoid carcinoma KB cells in vitro with IC50 value of 4.3 µg/mL, while hyrtiosins A (88) and B (89) were less cytotoxic than 5-hydroxyindole-3-aldehyde (90) and showed 21% and 16% inhibition, respectively, at 10 µg/mL against KB cells. HO

OH

O

HN

N

N H

NH

87

O

OH

OH HO

HO

N

O

88

O

H

HO N H 89

N H

N H 90

2.2. Indolocarbazoles Staurosporine (91) was first isolated from Streptomyces staurosporeus Awaya (AM-2282) [92,93] and subsequently from other actinomycetes e.g. Streptomyces actuosus [94] and Streptomyces species strain M-193 [95]. The structure and stereochemistry of the compound in its MeOH-H2O solvate form was deduced by X-ray crystallography. Staurosporine (91) exhibited in vitro activity against several different type of tumors such as human neuroblastoma cell line (NB-1), HeLa S3 cells, B16 melanoma cells and P-388 leukemia cells [96,97]. Cordell et al. evaluated the cytotoxicity of staurosporine (91) towards the murine P-388 lymphocytic leukemia and human carcinoma KB cell lines. Staurosporine (91) showed potent cytotoxic activity with ED50 value of 0.0024 µg/mL for the KB system and 100 ng/mL each. The percentage inhibition of RNA and DNA synthesis of compounds 91 and 94 were 93 and >98, 98 and >98, respectively. Compound (98) inhibited the proliferation of human cancer A-549, BEL-7402, HL-60 cells and mouse leukemia P-388 cells with the percentage inhibition of 82.6%, 57.3%, 76.1%, 62.2% in the SRB assay [101]. It also inhibited the proliferation of mouse cancer tsFT210 cells with the inhibition rates of 28.3% at 21 μM and 20.5% at 2.1 μM in the SRB assay. Analysis of structure activity relationship demonstrated that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety causes an increase in antiproliferative activity, while hydroxylation at 11th position resulted in a decrease in activity. All these data suggested that not only the presence or absence of hydroxyl group, but also the position of OH group is crucial to determine the antiproliferative properties of the various staurosporine analogues.

O

H N

H N

O

H N

O

R1 N O N H H H H

Me R4 R3

HH R2

91, R1 = H, R2 = CH3, R3 = OCH3, R4 = H 92, R1 = OH, R2 = CH3, R3 = OH, R4 = H 93, R1 = H, R2 = H, R3 = OCH3, R4 = OH 94, R1 = OH, R2 = CH3, R3 = OCH3, R4 = H 95, R1 = H, R2 = CH3, R3 = OH, R4 = H 96, R1 = H, R2 = H, R3 = OCH3, R4 = H

N H

N H

N

N O

O

97

N Me

O

O

98

CHO N OH OH O

O 99

H

Marine natural alkaloids as anticancer agents

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A novel carbazole alkaloid, coproverdine (99) was isolated from an unidentified ascidian Anchorina sp. collected from the north Island of New Zealand [102]. The structure of 99 was established on the basis of extensive spectroscopic data analysis. Coproverdine (99) was evaluated against a variety of murine and human tumor cell lines such as P-388, A-549, HT-29, MEL-28 and DU-145 exhibiting IC50 values of 1.6, 0.3, 0.3, 0.3 and 0.3 µM, respectively.

2.3. Ergoline alkaloids Makarieva et al. isolated pibocin A (100) from the far-eastern ascidian Eudistoma sp. [103]. Its structure and absolute stereochemistry were established on the basis of spectroscopic and X-ray data analysis and was supposed to represent the first example of marine ergoline alkaloids. Pibocin A (100) exhibited moderate cytotoxicity against mouse Ehrlich carcinoma cells with ED50 value of 12.5 µg/mL. Recently, pibocin B (101) was isolated from the colonial ascidian Eudistoma sp. [104]. Its structure was established as (8β)-2-bromo-N-Omethyl-6,8-dimethylergoline on the basis of NMR, FAB and MALDI TOF MS data and chemical means. Pibocin B (101) exhibited moderate cytotoxic activity against mouse Ehrlich carcinoma cell with an ED50 value of 25 µg/mL. H3C

N

CH3 H

H3C

N

CH3

H Br

Br N OCH3

N H

100

101

2.4. Peptidoindoles Styelin D, a 32-residue, C-terminally amidated peptide was isolated from the blood cells of the solitary ascidian Styela clava [105]. It was found to contain two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual amino acids including, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. Styelin D exhibited cytotoxicity against HCT-116 cells with IC50 value of 10.1 µg/mL, and human ME-180 cervical epithelial cells with ED50 value of 50 µg/ mL. Nakao et al. isolated kapakahine B (102) from the marine sponge Cribrochalina olemda collected at Pohnpei, Micronesia [106]. Kapakahine B (102) was found having a cyclic hexapeptide with an α-carboline ring system and showed moderate cytotoxicity against P-388 murine leukemia cells with an IC50 value of 5.0 µg/mL.

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O

O

O N

N

N H

O NH

O HN

HN

O

NH O

O NH2 NH

N H

O

O

NH N

N

HO

N H O OH

O H N O H NH

NH

NH

N O HN

O NH O

HO

O OH

O H

NH

N O H NH

O

102

103

104

Two isomeric cycloheptapeptides, phakellistatin 3 (103) and isophakellistatin 3 (104), were isolated from the Western Indian marine sponge Phakellia carteri [107]. They were supposed to represent the first examples of photo-Trp serving as a natural peptide unit. A significant difference in the activity was also observed with the photo-Trp indole ring juncture. Phakellistatin (trans-ring juncture) exhibited inhibition of P-388 (ED50 = 0.33 µg/mL) while isophakellistatin (cis-ring juncture) showed no significant effects.

2.5. β-Carbolines Eudistomin K (105) was isolated from the Caribbean ascidian Eudistoma olivaceum and found to exhibit antitumor activity against L-1210, A-549, HCT-8 and P-388 cell lines with IC50 of 0.01 µg/mL against P-388 cell line [108]. Recently Kobayashi et al. reported the isolation and structure elucidation of a new β-carboline alkaloid, eudistomidin G (106) from the Okinawan marine tunicate Eudistoma glaucus [109]. Eudistomidins G (106) exhibited significant cytotoxic activity against L-1210 murine leukemia cells with IC50 value of 4.8 µg/mL in vitro. Adesanya et al. reported the isolation of two novel brominated β-carbolines, eudistalbin A (107) and B (108) from the marine tunicate Eudistoma album along with the known compound eudistomin E (109) [110]. The cytotoxicity of these compounds was tested using the human nasopharyngeal carcinoma KB cell lines. Eudistomin E (109) exhibited 100% cytotoxicity at seven concentrations ranging from 10 to 0.005 µg/mL (ED50