Organic Chemistry of Natural Products

菅裕明 (Hiroaki Suga, Bioorganic Chemistry Lab.) Robert Campbell（Biomolecular Chemistry Lab.）佐竹真幸 (Masayuki Satake, Natural Product Chemistry Lab.) 後藤佑樹 (Yuki Goto, Bioorganic Chemistry Lab.) 加藤敬行 (Takayuki Katoh, Bioorganic Chemistry Lab.) Tentative lecture schedule: date No. instructor subject 9/26 1 Goto overview of natural products 10/3 2 Goto terpenes 10/10 3 Goto terpenes 10/17 4 Katoh amino acids and peptides 10/24 5 Katoh amino acids and peptides 10/31 6 Satake polyketides 11/14 7 Satake polyketides 11/21 8 Satake polyketides 11/28 9 Satake chikimic acid pathway and its products 12/5 10 Satake chikimic acid pathway and its products 12/12 11 Campbell topic: protein engineering for diagnostics and therapeutics 12/19 12 Campbell topic: protein engineering for diagnostics and therapeutics 12/26 13 Suga topic: discovery and application of pseudo-natural peptides 1/9 14 Suga topic: quorum sensing controlled by auto inducers Natural products chemistry

instructors: 佐竹真幸 (Masayuki Satake, Natural Product Chemistry Lab.) 菅裕明 (Hiroaki Suga, Bioorganic Chemistry Lab.) 後藤佑樹 (Yuki Goto, Bioorganic Chemistry Lab.) Tentative lecture schedule: date No. instructor subject 9/29 1 Goto Overview: natural products 10/6 2 Goto Terpenoids 10/13 3 Goto Amino acids and peptides 10/20 4 Goto TOPIC: ribosomal synthesis of pseudo-natural peptides 10/27 5 “free study” 11/8 6 “free study” 11/10 7 Suga TOPIC: discovery and application of pseudo-natural peptides 11/17 8 Suga TOPIC: quorum sensing controlled by auto inducers 11/24 9 Satake TBA 12/1 10 Satake TBA 12/8 11 Satake TBA 12/15 12 Satake TBA 12/22 13 Satake TBA 1/5 14 Satake TBA

Grading: Final exam + attendance

Suggested reference textbooks: 医薬品天然物化学（南江堂）、海老塚豊監訳マクマリー生化学反応機構（東京化学同人）、長野哲雄監訳 Medicinal Natural Products: A Biosynthetic Approach, Paul M. Dewick The Organic Chemistry Of Biological Pathways, John E. McMurry & Tadhg P. Begley What is natural product? What is natural product? It is a chemical compound produced by a living organism—that is, found in nature

In a narrow sense, natural products often means secondary metabolites that have intriguing bioactivities. essential and universal What are secondary metabolites? (↔ primary metabolites) They are organic compounds generated in organisms (metabolites) that are not essential in the reproduction, development, or normal growth.

They often provide evolutionary advantages for the producing organisms. e.g.) "chemical weapon" agents against prey, predators, and competing organisms spider venom frog toxins anti-bacterials What is natural product? What is natural product? It is a chemical compound produced by a living organism—that is, found in nature

In a narrow sense, natural products often means secondary metabolites that have intriguing bioactivities. Humans use secondary metabolites discovered from nature as medicines, agents, and favorings. e.g.)

Paclitaxel Artemisinin Avermectin anti-cancer drug anti-malarial drug anti-parasitic worm drug isolated from a plant isolated from a plant produced by a bacterium (Taxus brevifolia) (Artemisia annua) (Streptomyces avermitilis)

Physiology/Medicine, 2015 Physiology/Medicine, 2015 What is natural products chemistry?

What is natural products chemistry? A feld of organic chemistry that deals with “natural products”.

It is related to various chemistry felds such as medicinal chemistry, synthetic chemistry, and agricultural chemistry.

Japanese scientists have historically shown large presence. Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Elucidation of the mode of action

Total synthesis

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Willow bark can be used for Morays sometimes cause A fungus kills microbials suppression of toothache food poisoning

Isolation of objective bioactive compounds from the organisms

Structural determination Elucidation of the mode of action Total synthesis

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms meat of morays culture medium of fungi willow bark (e.g. 850 morays, 4 tons!)

(0.3 mg of fnal compound) Purifying the natural samples by means of various separation methods, and hunting the fractions that shows the objective bioactivity

extraction liq.-liq. separation chromatography crystallization

Structural determination Elucidation of the mode of action Total synthesis

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Determination of the structure of the bioactive compound by means of elementary analysis, crystal structure analysis, and comprehensive spectrum analyses (e.g., MS, MS-MS, IR, NMR, etc.)

H N S OH

O N O O OH O OH benzylpenicillin salicylic acid ciguatoxin

Total synthesis Elucidation of the mode of action

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Total synthesis (de novo chemical synthesis of natural products) available reagents

HOOC O BnO O O I O tBuO C BnO 2 O OH BnO OPMB O O O SPh MP MOMO OBn O SPh

Science, 294, 5548 (2001)

Elucidation of the mode of action

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Total synthesis (de novo chemical synthesis of natural products)

Purpose/motivation: Confrmation of proposed structures

Mass supply of useful natural products

Derivatization of natural products

Artistic synthesis of sophisticated structures

Elucidation of the mode of action

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Total synthesis

Elucidation of the mode of action

transpeptidase

Penicillin

Derivatization of natural products aiming at development of novel drugs Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination Elucidation of the mode of action Total synthesis

Derivatization of natural products aiming at development of novel drugs

H N S O N O OH O O benzylpenicillin OH (penicillin G) O O O OH OH salicylic acid acetylsalicylic acid NH O 2 H N S H (aspirin) N S O N O O O N OH O O OH O Ampicillin methicillin Typical ﬂow in natural products chemistry

Discovery of organisms that show interesting bioactivities

Isolation of objective bioactive compounds from the organisms

Structural determination

Elucidation of the mode of action

Total synthesis

Derivatization of natural products aiming at development of novel drugs AnCommunications example to the of Editor drug development basedJ. Am. Chem. on SOC.. naturalVol. 107, No. 16, 1985products 4797

1985: Uemura et al. homogenization, extraction, liq.-liq. separation, chromatography Figure 1. Computer-generated perspective drawing of the final X-ray model of the p-bromophenacyl ester 2.

and Tsukitani resulted in the identification of okadaic acid2 as a cytotoxic constituent of this animal. However, our interestDiscovery in and identifcation of novel compounds 3162the same animal focused on the fact that spongeJ. extractsAm. Chem. exhibited SOC. 1992, 114,'OT: 3162-3164"/ 0 remarkable in vivo antitumor acti~ity.~Bioassay against B- 16 with potent cytotoxicity radical 5 which, after recombination with the flavin semiquinone, melanoma cells guided the isolation of halichondrins in low yield, features, （couplednamed with thishalichondrins fact, encouraged） synthetic efforts 600leads includingkg toof covalent seanorhalichondrin sponge modification A ( ofHalichondria(1):4 FAD amorphous (Scheme solidI, route okadai [(5 B). X lo")%Since) toward this class of natural product^.^,^ In this paper, we report theyield reactions fromfrom wet of carbonanimals]; Miura radicals, [aID Peninsula -47.8'particularly (MeOH, cyclopropylcarbinyl c 1.13). the first total synthesisOR of halichondrinPure BAppl. andOR norhalichondrin Chem. 58 (5): B, 701–710 and homoallylic species, with molecular oxygen are well docu- which has, we believe, potential to meet the demand. mented,14 the inactivation derailment may be envisioned as trapping the acyclic radical 5 with O2to form a transient peroxy radical 6 which, upon reduction by one-electron transfer from the active-site-bound flavin semiquinone, gives rise to a peroxy anion 7. Since the partition ratio of this inactivation is approximately R= COC6H4Br 3, reacting with oxygen instead of coupling with the flavin co- Figure 2. Exciton chirality of the 12,13-bis(p-bromobenzoate)system in enzyme is clearly a more facile process for the ring-opened radical 3. intermediate 5. As depicted in Scheme 11, this reroute is cul- minated by an intramolecular epoxidationls converting 7 via a 1,2-dioxolanylcarbiny1 anion 8 to the observed turnover product and 0.1 102 for the enantiomer. The computer-generated per- 9.16 The mechanistic insights derived from this study provide spective drawings8 of a molecule of 2 is shown in Figure 1, in- highly convincing evidence sustaining our early notion that including its absolute configuration. activation of GADnorhalichondrin by MCPA-CoA is likely Ato proceed through Application of the nonempiricalhalichondrin dibenzoate chirality B methodg a radical mechanism. These results may also be extrapolated to was also consistent with the X-ray crystallographic result. suggest that GAD is capable of mediating one-electron oxida- The SIMSisolation spectrum showed yield: the highest 35 mg mass peak at m/z Treatmentcytotoxicity of 1 with p-bromobenzoyl against B-16 chloride melanoma in pyridine at 80cells tion-reduction. 1127 "C yielded product 3 with the y-lactone [IR (CHC13) cm-'1; (M 1) corresponding to the final molecular formula CS9H82021. 1775 0.00005%+ of the original sample! 3 contains two p-bromobenzoylIC50 = 0.093 groups ng/mL judging (= from 80 the pM) IH NMR TheAcknowledgment. IR spectrum (KBr) We indicated thank Dr. the Vikram presence Roongta of hydroxyls for his (3450 as- cm-I), lactone larger than five-membered ring or ester (1740 cm-'), spectrum [C&, 6 6.9-7.8 (8 H, m), 5.89 (1 H, s, H13)]. This sistance with NMR measurements. This work was supported by reaction seems to proceed by reasonable3 : homohallchondrin C13-Cl2 acyl B migra- a andNational carboxylate Institutes (1580 ofMode cm-I).Health The grantof lHaction: (GM and 13C40541). NMRInhibition H.-w.L. spectra alsowere of cell division via targeting tubulin not so fruitful for structural elucidation because of their com- tion,1° followed by ordinary acylation of the secondary alcohol. thanks the National Institutes of Health for a Research Career The bis(p-bromobenzoate) 3 possessingJ. Biol. the 1,2-dibenzoateChem. 266 system(24): 15882–9 Developmentplexity. However, Award two (GM sets 00559). of exomethylene ('H NMR 6 4.81, 4.87,5.02, 5.06, 1 H each; I3C NMR 6 104.81,105.76, 153.17, showed a typical positive split CD; EtOH 241 (A6 -19.3) and 257 153.32),four carbon atoms bearing two oxygen atoms (13C NMR nmScheme (A€ 22.1)." I outlines This the observation synthesis confirmedof the right the half chirality of the of hali- the 6 (14) (a) Nakamura, E.; Inubushi,and T.; Aoki,four S.; secondaryMachii, D. methyl J. Am. Chem.groups C13~COC6H4Br/C12~Coc~H4Bras shown in Figure 2. The SOC.98.51,1991, 112.86,113, 8980.113.38, (b) Beckwith, 114.87), A. L. J. Tetrahedron 1981, 37, 3073. chondrin Bs. We planned to form the C-21-C-22 bondS via a ('H NMR 6 0.96,0.97, 1.06, 1.09,3 H each as a doublet; 13C Horner-Emmonsnovel 2,6,9-trioxatricyclo[3,3,2,03~7]decane reaction, followed by conjugatesystem reduction. in 1-3 wasWe (c) Porter, N. A. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic proven by the full analysis with homonuclear correlated spec- Press:NMR New 6 15.88,York, 1980; 17.47, Vol. 18.13, IV, p 18.42),261. (d)and Beckwith, then carbonyl A. L. J.; Ingold, group(s) K. were concerned with double-bond isomerization from the C- 19 U.(I3C In Rearrangements NMR 6 172.81) in Ground were recognized and Excited byStates; the NMRde Mayo, spectra P., Ed.; in exocyclictroscopy (COSY).12to the C- 19-C-20As expected, endocyclic this moietyposition was in thisnot labileprocess. in AcademicCD,OD. Press: New York, 1980. Thisbasic transformation media whereas was acid accomplished solution caused via obvious the preparation decomposition. of the (I 5) (a) Clark, C.; Hermans, P.; Meth-Cohn, 0.;Moore, C.; Taljaard, H. C.; vanNorhalichondrin Vuuren, G. J. Chem. A Soc.,(1) Chem.was treated Commun. with 1986,1378. p-bromophenacyl (b) Rontani, aldehydeNorhalichondrin from the primary A (1) alcoholconsists 36.7 of bya polyoxygenated Des-Martin oxidation: Cs3car- J.-F.bromide Tetrahedron and triethylamineLett. 1991,6551. in (c) DMF Dowd, at P.; 50 Ham, OC. S.After W. J. Am.separation Chem. Horner-Emmonsboxylic acid which reaction may act under in an carefullyimportant controlled role in the conditions, extension SOC.by preparative1991, 113, 9403. TLC, the desired crystalline pbromophenacyl ester andmechanism the conjugate of naturally reduction occurring of the long-straight resulting enone carbon by thechains Stryker such 2(16) [IR An (CHC13) alternate 1735, mechanism 1705, is 1590 also (weak)conceivable cm-'1 involving was recrystallized an intramo- reagent:as pa1yt0xin.l~ without Biosynthetic double-bond studies isomerization. on 1 as wellHydride as brevetoxin14 reduction lecular cyclization of 6 to produce a 1,2-dioxolanylcarbinyIradical (Feldman, may lead us to a general concept. Our studies on the biological K.from S.; Simpson, acetone-methanol, R. E. J. Am. Chem. furnishing SOC.1989, well-formed, 111, 4878) monoclinicand then a of the resulting saturated ketone yielded approximately a 1:l one-electroncrystals: reductionmp 173.5-175.0 to give the OC. corresponding Its structure anion has 8. been unambig- mixtureorigin of of 1 the and two its possible congeners diastereomers. are currently As under the stereochemistry way. uously determined as follows. The space roup is P21and lattice of diastereomeric alcohols could not be firmly established at this constants are a = 44.654 A, b = 9.264 1, c = 9.494 A, and p stage, both diastereomers were transformed separately into the = 93.72'. The structure was solved by the Monte-Carlo direct corresponding(8) ORTEP crystallographic mesylates and illustration used for program: the next Johnson, coupling C. K.reaction. Report methodSwith the aid of MULTAN 78 program system6 using 3080 However,ORNL-3794, it Oakis important Ridge National to noteLaboratory, that the Oak two Ridge, diastereomeric TN. unique reflections [IF,I I3o(F,,)] .' Full-matrix least-square alcohols(9) Harada, were readilyN.; Nakanishi, interconvertible K. J. Am. Chem. via the SOC. Mitsunobu1969,91, reaction.1°3989-3991. refinement with anisotropic temperature factors for the non-hy- (10) See, for example: "Molecular Rearrangements, Part Two"; de Mayo, Totaldrogen Synthesis atoms and ofanomalous Halichondrin dispersion B correctionsand Norhalichondrin have converged Ed.; Interscience Publishers: New York, 1964; pp 763-769, 1121-1 127. Bto a standard crystallographic residual of 0.1092 for the structure (1 1) The UV spectrum of 3 is as follows: A,,, (EtOH) 247 nm (e 38 000). ~~ (12) Nagayama, K.; An& K.; Wuthrich, K.; Ernst, R. R. J. Magn. Reson. (3) For the synthetic work from this laboratory, see: (a) Aicher, T. 1980, 40, 321-334. Assignments of each proton signal in 1 have been at-D.; Thomas D. Aicher, Keith R. Buszek, Francis G. ~Fang, Kishi, Y. Tetrahedron Lett. 1987,28, 3463-3466. (b) Aicher, T. D.; Buszek, tempted on the basis of the structure 2. Each carbon signal has been also Craig(4) J.Norhalichondrin Forsyth, Sun A Ho inhibited Jung, growth Yoshito of 8-16 Kishi,* melanoma cells in vitro K.assigned R.; Forsyth, by INEPT C. andJ.; Fang,2D ('H-"C F. G.; chemicalJung, S. shift H.; correlation)Kishi, Y.; Scola, experiments. P. M. Michaelat 5 ng/mL C. ICso.Matelich, Paul M. Scola, Denice M. Spero, and Tetrahedron Lett., in press. (c) Buszek, K. R.; Forsyth, C. J.; Fank, F. G.; (5) Furusaki, A. Acta Crystallogr., Sect. A 1969, A35, 22C-224. Jung,(13) S. (a) H.; Cha,Kishi, J. Y.; K.; Scola, Christ, P. W.M.; J.;Yoon, Finan, S. K.J. M.;Tetrahedron Fujioka, Lett.,H.; Kishi, in press. Y.; Suk(6) Kyoon Main, Yoon P.; Hull, S. E.; Lessinger, L.; Germain, G.;Declercq, J. P.; (d)Klein, Fang, L. F. L.; G.; KO, Kishi, S. S.; Y.; Leder, Matelich, J.; McWhorter,M. C.; Scola, W. P. M.W., Tetrahedron Jr.; Pfaff, K.-P.; Lett., Woolfson, M. M. MULTAN 78, a system of computer programs for the auto- inYonaga, press. M.; Uemura, D.; Hirata, Y. J. Am. Chem. SOC. 1982, 204, matic solution of crystal structure from X-ray diffraction data, University of 7369-7371.(4) For the(b) synthetic Moore, R.work E.; fromBartolini, other G.; laboratories, Barchi, J.; Bothner-By,see: (a) Kim, A. A,; Department of Chemistry, Harvard University Dadok, J.; Ford, J. J. Am. Chem. Soc. 1982, 104, 3776-3779. S.; York, York,12 England,Oxford 1978.Street, Cambridge, Massachusetts 021 38 Salomon, R. G. Tetrahedron Lett. 1989, 30, 6279-6282. (b) Cooper, A. J.; (7) An automated four-circle diffractometer Rigaku AFC-5R was used Salomon,(14) Lin, R. Y.-Y.;G. Tetrahedron Risk, M.; Ray,Lett. S.1990,31, M.; Engen, 3813-3816. D. V.; Clardy, (c) Burke, J.; Golik, S. D.;J.; throughout this work. Buchanan,James, J. C.; J. L.;Nakanishi, Rovin, J. K. D. J. Tetrahedron Am. Chem. Soc.Lett. 1981,1991, 103,32, 3961-3964.6773-6775. Received December 20, 1991 (5) The numbering adopted in this paper corresponds to that of halichondrins. Halichondrins are a class of polyether macrolides isolated or- (6) This substance was synthesized from 2-deoxy-~-arabinosediethyl iginally from the marine sponge Hulichondria okadui Kadota.'s2 thioacetal 4,s-acetonide (Wong, M. Y. H.; Gray, G. R. J. Am. Chem. SOC. 1978, 100, 3548) in 47% overall yield in 13 steps: (1) AcOH/H,O/room Halichondrins, especially halichondrin B and homohalichondrin temperature. (2) TBDPSiCl/imidazole. (3) I,/NaHCO,/H,O/acetone. (4) B, exhibit extraordinary in vitro and in vivo antitumor activity. Ac,O/pyridine/room temperature. (5) CH,=CHCH2TMS/BF3.0Et2/ However, the very limited supply of halichondrins from natural CHaCN/O OC. (6) (a) 9-BBN; (b) H20,. (7) MMTrCI/Et3N/CH2CI2. (8) sourm has prevented further evaluation of their potential clinical K,C03/MeOH. (9) Swern oxidation. (10) MeOH/PPTS. (1 1) Tebbe reagent (Tebbe, F. N.; Parshall, G. W.; Reddy, G. S. J. Am. Chem. Soc. 1978, application thus far. Their intriguing and challenging structural 100, 3611-3613. Cannizzo, L. F.; Grubbs, R. H. J. Org. Chem. 1985, 50, 2386-2397). (12) PvCl/pyridine. (13) TBAF. (7) Satisfactory spectroscopic data ('H and I3C NMR, HRMS, MS, IR, UV, [a]~)were obtained for all new compounds reported in this paper. (1) (a) Uemura, D.; Takahashi, K.; Yamamoto, T.; Katayama, C.; Tanaka, (8) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156. J.; Okumura, Y.; Hirata, Y. J. Am. Chem. SOC.1985,107,4796-4798. (b) (9) (a) Mahoney, W. S.; Brestensky, D. M.; Stryker, J. M. J. Am. Chem. Hirata, Y.; Uemura, D. Pure Appl. Chem. 1986, 58, 701-710. Soc. 1988,110,291-293. (b) Mahoney, W. S.;Stryker, J. M. J. Am. Chem. (2) A recent publication (Bai, R.; Paull, K. D.; Herald, C. L.; Malspeis, SOC.1989, 111, 8818-8823. (c) We are indebted to Professor Stryker for a L.;Pettit, G. R.; Hamel, E. J. Bioi. Chem. 1991, 266, 15882-15889) implies sample of this reagent. that halichondrin B and homohalichondrin B are isolated from Axinella (10) For a review on this reaction, see: Mitsunobu, 0. Synthesis 1981, sponges. 1-28. 0002-7863/92/1514-3162$03.00/0 0 1992 American Chemical Society 3162 J. Am. Chem. SOC.1992, 114, 3162-3164 Anradical example 5 which, after recombination of drug with the development flavin semiquinone, features, based coupled on with thisnatural fact, encouraged products synthetic efforts leads to covalent modification of FAD (Scheme I, route B). Since toward this class of natural product^.^,^ In this paper, we report the reactions of carbon radicals, particularly cyclopropylcarbinyl the first total synthesis of halichondrin B and norhalichondrin B, and homoallylic species, with molecular oxygen are well docu- which has, we believe, potential to meet the demand. mented,141992: the Kishiinactivation et derailmental. may be envisioned as trapping the acyclic radical 5 with O2to form a transient peroxy Totalradical 6 synthesiswhich, upon reduction of halichondrin by one-electron transfer B from the active-site-bound flavin semiquinone, gives rise to a peroxy anion 7.commercially Since the partition ratioover of 160this inactivation steps chemical is approximately reactions 3, reacting with oxygen instead of coupling with the flavin co- enzymeavailable is clearly a more facile process for the ring-opened radical intermediatereagents 5. As depicted in Scheme 11, this reroute is cul- minated by an intramolecular epoxidationls converting 7 via a 1,2-dioxolanylcarbiny1 anion 8 to the observed turnover product 9.16 The mechanistic insights derivedAdditional from this study provide Reading:halichondrin B highly convincing evidence sustaining our early notion that in- J. Am. Chem. Soc. 114 (8): 3162–3164. activation2004: of GAD Eisai by MCPA-CoA Co., Ltd. is likely to proceed through a radical mechanism. TheseHalichondrin results may also be extrapolated to B and E7389 suggest that GAD is capable of mediating one-electron oxidation-reduction.Simplifcation of halichondorins - development of E7389 Acknowledgment. We thank Dr. Vikram Roongta for his as- sistance with NMR measurements. This work was supported by 3 : homohallchondrin B a National Institutes of Health grant (GM 40541). H.-w.L. also thanks the National Institutes of Health for a Research Career Development Award (GM 00559). Scheme I outlines the synthesis of the right half of the hali- (14) (a) Nakamura, E.; Inubushi, T.; Aoki, S.;Machii, D. J. Am. Chem. SOC.1991, 113, 8980. (b) Beckwith, A. L. J. Tetrahedron 1981, 37, 3073. chondrin Bs. We planned to form the C-21-C-22 bondS via a (c) Porter, N. A. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic Horner-Emmons reaction, followed by conjugate reduction. We Press: New York, 1980; Vol. IV, p 261. (d) Beckwith, A. L. J.; Ingold, K. were concerned with double-bond isomerization from the C- 19 U. In Rearrangements in Ground and Excited States; de Mayo, P., Ed.; exocyclic to the C- 19-C-20 endocyclic position in this process. Academic Press: New York, 1980. This transformation was accomplished via the preparation of the (I 5) (a)• HalichondrinClark, C.; Hermans, P.; Meth-Cohn,B is a highly 0.;Moore, cC.;ytotoxic Taljaard, H. (antimitotic2/3 molecular) marine natural weight andproduct. chiral centers C.; van Vuuren, G. J. Chem. Soc., Chem. Commun. 1986,1378. (b) Rontani, aldehyde from the primary alcohol 36.7by Des-Martin oxidation: J.-F. Tetrahedron Total Lett. synthesis: 1991,6551. (c) Dowd, Kishi P.; Ham,, 1992 S. W. J. Am. Chem. Horner-Emmonsimproved reaction under in carefullyvivo stability controlled conditions, SOC.1991, 113, 9403. and the conjugate reduction of the resulting enone by the Stryker (16) An• alternateRecent mechanism diverted is also conceivable total synthesis involving an intramo- has ledreagent: to simplified without double-bond analog isomerization. E7389 with Hydride reduction lecular cyclization of 6 to produce a 1,2-dioxolanylcarbinyIradical (Feldman, FDA approval in 2010! K. S.; Simpson, similar R. E. J. a Am.ntimitotic Chem. SOC. 1989, activity. 111, 4878) Also, and then replacement a of the resulting of lactone saturated withketone k yieldedetone approximately a 1:l one-electron reduction to give the corresponding anion 8. mixture of the two possible diastereomers. As the stereochemistry has made E7389 more robust in vivo of diastereomeric alcohols could not be firmly established at this stage, both diastereomers were transformed separately into the •E7389 currently in phase I clinical trialscorresponding mesylates and used for the next coupling reaction. However, it is important to note that the two diastereomeric alcohols were readily interconvertible via the Mitsunobu reaction.1° Total Synthesis of Halichondrin B and Norhalichondrin B Aicher, T. D.; Buszek, K. R.; Fang, F. G.; Forsyth, C. J.; Jung, S. H.; Kishi, Y.; Matelich, M. C.; Scola, P. M.; Spero, D. M.; Yoon, S. K. J. Am. Chem. Soc.~~ 1992, 114, 3163. (3) For the synthetic work from this laboratory, see: (a) Aicher, T. D.; ThomasZhe nD.g, Aicher,W. J.; SKeithelets R.ky ,Buszek, B. M.; PFrancisalme, MG.. HFang,.; Ly don, P .J.; SKishi,inge rY., L Tetrahedron .A.; Cha sLett.e, C 1987,28,. E.; L e3463-3466.melin, C .(b) Aicher, T. D.; Buszek, Craig A J..; Forsyth,Shen, Y Sun. C.; HoDa vJung,is, H .Yoshito; Tremb Kishi,*lay, L. ; Towle, M. J.; SalvK.at oR.;, K Forsyth,. A.; W C.el sJ.;, B Fang,. F.; F.A aG.;lfs ,Jung, K. K S..; H.; Kishi, Y.; Scola, P. M. Michael Kis hC.i ,Matelich,Y.; Littlef iPauleld, BM.. A Scola,.; Yu, MDenice. J. B iM.oo rSpero,g. Me dand. C hem. LeTetrahedrontt. 2004, 1 Lett.,4, 5 5in5 press.1. (c) Buszek, K. R.; Forsyth, C. J.; Fank, F. G.; Jung, S. H.; Kishi, Y.; Scola, P. M.; Yoon, S. K. Tetrahedron Lett., in press. SukP Kyoonaters oYoonn, I.; Anderson, E. Science 2005, 310, 451. (d) Fang, F. G.; Kishi, Y.; Matelich, M. C.; Scola, P. M. Tetrahedron Lett., in press. Department of Chemistry, Harvard University (4) For the synthetic work from other laboratories, see: (a) Kim, S.; 12 Oxford Street, Cambridge, Massachusetts 021 38 Salomon, R. G. Tetrahedron Lett. 1989, 30, 6279-6282. (b) Cooper, A. J.; Salomon, R. G. Tetrahedron Lett. 1990,31, 3813-3816. (c) Burke, S. D.; Buchanan, J. L.; Rovin, J. D. Tetrahedron Lett. 1991, 32, 3961-3964. Received December 20, 1991 (5) The numbering adopted in this paper corresponds to that of halichondrins. Halichondrins are a class of polyether macrolides isolated or- (6) This substance was synthesized from 2-deoxy-~-arabinosediethyl iginally from the marine sponge Hulichondria okadui Kadota.'s2 thioacetal 4,s-acetonide (Wong, M. Y. H.; Gray, G. R. J. Am. Chem. SOC. 1978, 100, 3548) in 47% overall yield in 13 steps: (1) AcOH/H,O/room Halichondrins, especially halichondrin B and homohalichondrin temperature. (2) TBDPSiCl/imidazole. (3) I,/NaHCO,/H,O/acetone. (4) B, exhibit extraordinary in vitro and in vivo antitumor activity. Ac,O/pyridine/room temperature. (5) CH,=CHCH2TMS/BF3.0Et2/ However, the very limited supply of halichondrins from natural CHaCN/O OC. (6) (a) 9-BBN; (b) H20,. (7) MMTrCI/Et3N/CH2CI2. (8) sourm has prevented further evaluation of their potential clinical K,C03/MeOH. (9) Swern oxidation. (10) MeOH/PPTS. (1 1) Tebbe reagent (Tebbe, F. N.; Parshall, G. W.; Reddy, G. S. J. Am. Chem. Soc. 1978, application thus far. Their intriguing and challenging structural 100, 3611-3613. Cannizzo, L. F.; Grubbs, R. H. J. Org. Chem. 1985, 50, 2386-2397). (12) PvCl/pyridine. (13) TBAF. (7) Satisfactory spectroscopic data ('H and I3C NMR, HRMS, MS, IR, UV, [a]~)were obtained for all new compounds reported in this paper. (1) (a) Uemura, D.; Takahashi, K.; Yamamoto, T.; Katayama, C.; Tanaka, (8) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156. J.; Okumura, Y.; Hirata, Y. J. Am. Chem. SOC.1985,107,4796-4798. (b) (9) (a) Mahoney, W. S.; Brestensky, D. M.; Stryker, J. M. J. Am. Chem. Hirata, Y.; Uemura, D. Pure Appl. Chem. 1986, 58, 701-710. Soc. 1988,110,291-293. (b) Mahoney, W. S.;Stryker, J. M. J. Am. Chem. (2) A recent publication (Bai, R.; Paull, K. D.; Herald, C. L.; Malspeis, SOC.1989, 111, 8818-8823. (c) We are indebted to Professor Stryker for a L.;Pettit, G. R.; Hamel, E. J. Bioi. Chem. 1991, 266, 15882-15889) implies sample of this reagent. that halichondrin B and homohalichondrin B are isolated from Axinella (10) For a review on this reaction, see: Mitsunobu, 0. Synthesis 1981, sponges. 1-28. 0002-7863/92/1514-3162$03.00/0 0 1992 American Chemical Society Drug development using artifcial compounds

Drug development based on natural products chemistry Discover the “seeds” of objective drugs from nature, and brush them up to develop desirable drugs

Drug development using artifcial compounds de novo development of drug seeds using synthetic chemical approaches Drug development using artifcial compounds

High-throughput screening (HTS) of chemical libraries Chemical library HTS

collection of various compounds diversity = 105～106

Structure-activity relationship (SAR) study drug candidates “hit” compounds (lead compounds) (drug seeds) derivatization Drug development using artifcial compounds High-throughput screening (HTS) of chemical libraries REVIEWS REVIEWS

Chemical library Lead identification by fragment linking HTS Lead identification by fragment linking TABLES 3,4 show examples in which two fragments have TABLES 3,4 show examples in which two fragments have been identified that bind in separate binding sites that been identified that bind in separate binding sites that are close enough to each other to be chemically linked are close enough to each other to be chemically linked (FIG. 5).For this to be an efficient lead-identification (FIG. 5).For this to be an efficient lead-identification approach, one needs to both identify the initial frag- approach, one needs to both identify the initial fragments and also have a process that allows the appropriate ments and also have a process that allows the appropriate compoundslinking to be achieved in an efficient manner. initiallinking hitto be achieved in an efficient manner. The potency increase achievable from optimally The potency increase achievable from optimally after SARlinking studytwo fragments is often assumed to benefit from compoundlinking two fragments is often assumed to benefit from an approximate additivity effect, such that the free energy an approximate additivity effect, such that the free energy of binding of the joined molecule is approximately equal of binding of the joined molecule is approximately equal Figure 3 | Schematic representation of a low-quality Figure 3 | Schematic representation of a low-quality imageHTS hit. The high-throughput of the screening interaction (HTS) hit is large and of to the sum of the free energies of binding ofimageHTS the hit.frag-The high-throughput of the screening interaction (HTS) hit is large and of to the sum of the free energies of binding of the frag- makes surface contact with the receptor without forming ments (that is, two millimolar fragments whenmakes joined surface contact with the receptor without forming ments (that is, two millimolar fragments when joined high-thequality f interactionsnal drug in key pockets. candidates The affinity is spread together lead to a micromolar inhibitor)5.Suchhigh- additivitythequality initial interactions in hitkey pockets. compounds The affinity is spread together lead to a micromolar inhibitor)5.Such additivity throughout the entire molecule and, in the absence of requires that the contribution from the linker isthroughout negligible the entire molecule and, in the absence of requires that the contribution from the linker is negligible collectionstructural information, of the medicinal chemist does not know and that the loss in rigid-body entropy on bindingstructural of all information, the medicinal chemist does not know and that the loss in rigid-body entropy on binding of all which areas of the molecule to focus on during hit optimization. components to the enzyme is very small. Recently,which areas an of the molecule to focus on during hit optimization. components to the enzyme is very small. Recently, an variousExperience compounds shows that optimization of these kinds of hits is Experience shows that optimization of these kinds of hits is analysis of the experimental energetics associated with analysis of the experimental energetics associated with very difficult. This5 is in contrast6 to the schematic in FIG. 4, in very difficult. This is in contrast to the schematic in FIG. 4, in diversitywhich = fragment 10 1 ～is much10 smaller, makes high-quality contacts optimally linked fragments has suggested thatwhich the rigid- fragment 1 is much smaller, makes high-quality contacts optimally linked fragments has suggested that the rigid- with the receptor and has relatively weak affinity. It has been body entropy loss on protein binding constituteswith a barrier the receptor and has relatively weak affinity. It has been body entropy loss on protein binding constitutes a barrier shown that such fragments can often be built up into attractive of around three orders of magnitude to theshown binding that such fragments can often be built up into attractive of around three orders of magnitude to the binding leads with the aid of structural information (for example, TABLE 2) affinity, and that this barrier is essentially independentleads with of the aid of structural information (for example, TABLE 2) affinity, and that this barrier is essentially independent of molecular mass25.The analysis implies that there should molecular mass25.The analysis implies that there should Structure-activitybe a super-additivity relationship effect when two fragments are be a super-additivity effect when two fragments are Table 2, entry 7. NMR screening (that is, structure–activity linked in an optimal fashion. Such super-additivityTable 2, entry is 7. NMR screening (that is, structure–activity linked in an optimal fashion. Such super-additivity is relationships (SAR) by NMR7) was used to identify a (SAR)observed study for entries 2 and 7 in TABLE 3. relationships (SAR) by NMR7) was used to identify a observed for entries 2 and 7 in TABLE 3. series of triazine-containing compounds that bind in the series of triazine-containing compounds that bind in the“hit” compounds drug candidatesmM range to ErmAM methyl transferase24 (for example, Table 3, entry 1. SAR by NMR7 was used to mMidentify range a to ErmAM methyl transferase24 (for example, Table 3, entry 1. SAR by NMR7 was used to identify a

TABLE 2,entry 7, NMR Kd = 1 mM), an enzyme target for potent 49 nM inhibitor of the FK506-bindingTABLE protein 2,entry 7, NMR Kd = 1 mM), an enzyme target for (drugpotent 49 nM seeds) inhibitor of the FK506-binding protein (lead compounds)ameliorating antibiotic resistance. Optimization of this (FKBP) binding domain by linking twoameliorating weaker antibiotic resistance. Optimization of this (FKBP) binding domain by linking two weaker initial lead using parallel synthesis led to inhibitors in the inhibitors (2 µM and 100 µM). NMR screeninginitial of alead set using parallelderivatization synthesis led to inhibitors in the inhibitors (2 µM and 100 µM). NMR screening of a set

low µΜ range (for example, table entry Ki = 7.5 µΜ). of 1,000 fragments — including pipecolinic acidlow deriv-µΜ range (for example, table entry Ki = 7.5 µΜ). of 1,000 fragments — including pipecolinic acid deriv- NMR and X-ray structures show that these non-nucleo- atives, a class of compounds known to bind toNMR FKBP and — X-ray structures show that these non-nucleo- atives, a class of compounds known to bind to FKBP —

sidecompounds bind to the S-adenosylmethionine- identified the pipecolinic acid (Kd = 2 µM)sid andecompounds the bind to the S-adenosylmethionine- identified the pipecolinic acid (Kd = 2 µM) and the 15 13 15 13 binding site on the Erm protein and that there is scope for diphenyl amide (Kd = 100 µM). Use of N- bindingC-filtered site on the Erm protein and that there is scope for diphenyl amide (Kd = 100 µM). Use of N- C-filtered the incorporation of additional binding interactions. protein–ligand NUCLEAR OVERHAUSER EFFECT (NOE)the incorporation data of additional binding interactions. protein–ligand NUCLEAR OVERHAUSER EFFECT (NOE) data

Table 1 | Comparison of fragment-based approaches and high-throughput screening Table 1 | Comparison of fragment-based approaches and high-throughput screening Fragment-based approaches High-throughput screening (HTS) Fragment-based approaches High-throughput screening (HTS) Emphasis on efficiency Emphasis on potency Emphasis on efficiency Emphasis on potency Typically screen a few hundred–few thousand compounds Typically screen hundreds of thousands of compoundsTypically screen a few hundred–few thousand compounds Typically screen hundreds of thousands of compounds

Mr range ~150–300 Mr range ~250–600 Mr range ~150–300 Mr range ~250–600 Hit activity in the range mM–30 µMHit activity in the range ~30 µM–nM Hit activity in the range mM–30 µMHit activity in the range ~30 µM–nM Hits have clearly defined binding interactions; high Hits can contain functional groups that contributeHits poorly have clearly defined binding interactions; high Hits can contain functional groups that contribute poorly proportion of atoms directly involved in protein binding to protein binding or act primarily as scaffolding (asproportion shown of atoms directly involved in protein binding to protein binding or act primarily as scaffolding (as shown schematically in FIG. 3) schematically in FIG. 3) Biophysical screening techniques (NMR, X-ray) are direct In vitro bioassay-based screening. Can generate falseBiophysical screening techniques (NMR, X-ray) are direct In vitro bioassay-based screening. Can generate false measurements of binding interaction. Can screen against positives and high attrition in hit-validation stage measurements of binding interaction. Can screen against positives and high attrition in hit-validation stage ‘inactive’ forms of the target protein (for example, kinases) ‘inactive’ forms of the target protein (for example, kinases) Protein-structure-based information key in validating and Chemistry (re)synthesis resource usually required Protein-structure-basedto information key in validating and Chemistry (re)synthesis resource usually required to prioritizing chemistry hits validate and prioritize screening hits prioritizing chemistry hits validate and prioritize screening hits Hit-to-lead chemistry usually requires synthesis of only a High attrition of chemical series in hit-to-lead stage.Hit-to-lead chemistry usually requires synthesis of only a High attrition of chemical series in hit-to-lead stage. NUCLEAR OVERHAUSER EFFECTS few compounds designed to add additional, specific binding Usually requires NUCLEARseveral iterationsOVERHAUSER of EFFECTShigh-throughputfew compounds designed to add additional, specific binding Usually requires several iterations of high-throughput (NOEs). Changes in the interactions chemistry. Attrition(NOEs). rates Changes can be in improved the with interactions chemistry. Attrition rates can be improved with intensity of NMR signals, which knowledge of proteinintensity structure of NMR signals, which knowledge of protein structure are caused by through-space Design-intensive Resource-intensiveare caused by through-space Design-intensive Resource-intensive dipole–dipole coupling. Upper dipole–dipole coupling. Upper distance constraints obtained Requires expertise and knowledge in protein structure, HTS requires extensivedistance constraintsinfrastructure obtained for storing andRequires expertise and knowledge in protein structure, HTS requires extensive infrastructure for storing and from 1H–1H NOEs are used for protein–ligand-binding interactions and fragment design handling compoundfrom 1collections,H–1H NOEs are screening, used for automation,protein–ligand-binding interactions and fragment design handling compound collections, screening, automation, data processing and chemistry follow-up data processing and chemistry follow-up NMR structure determination NMR structure determination of biological macromolecules. NMR, nuclear magnetic resonance. of biological macromolecules. NMR, nuclear magnetic resonance.

Fragment-based drug discovery (FBDD) REVIEWS

small compounds monitoringcaspase-3,a cysteine the protease interaction34.Mass spectrometry was“extended”these library fragments were coupled to known reversible REVIEWS REVIEWS (fragments) by NMRused to and/oridentify the crystallography library members that had formedfragment a cysteine-binding library elements to generate potent reversible

disulphide bond to the tethered thiol.Lead identification Subsequently, by fragment linkingmolecules. DNA gyrase TABLES 3,4 show examples in which two fragments have caspase-3,a cysteine protease34.Mass spectrometrybeen was identified thatthese bind in library separate binding fragments sites that were coupled to known reversible Table 2 | Lead identification by fragment evolution* are close enough to each other to be chemically linked used to identify the library members that had formed(FIG. 5).F aor this tocysteine-binding be an efficient lead-identification elements to generate potent reversible approach, one needs to both identify the initial frag- Entry Target/methoddisulphide bond to Fragment the tethered thiol. Subsequently,Evolvedments and fragmentalso havemolecules. a process that allows the appropriate Lead 1 linking to be achieved in an efficient manner. 1 DNA gyrase / H The potencyH increase achievable from optimally H VS and SBD N linking twoN fragments is often assumed to benefit from N Table 2 | Lead identification by fragment evolution*N an approximateN additivity effect, such that the free energy N REVIEWS REVIEWSof binding of the joined molecule is approximatelyO equal O REVIEWS Figure 3 | Schematic representation of a low-quality O O Entry Target/method FragmentHTS hit. The high-throughput screeningEvolved (HTS) hit is large fragment and to the sum of the free energies of bindingLead of the frag- 2 4 ments (that is,S two millimolar fragments when joined O S Lead identification by fragment linking diversity = 10 ～1 10 makes surface contact with the receptor without forming 1 DNA gyrase / Hhigh-quality interactions in key pockets. The affinity is spreadH together lead to a micromolar inhibitor)5.Such additivity H TABLES 3,4 show examples in which two fragments have VS and SBD Nthroughout the entire molecule and, in the absence of N requires that the contribution from the linker is negligible N been identified that bind in separate binding sites that 34 structuralK information,d = 10 the mM medicinal (by chemist NMR) does not know MNEC = 8 µg per ml 34MNEC = 30 ng per ml are close enough to each other to be chemically linked caspase-3,a cysteine protease .Mass spectrometry was these library fragmentsN were coupled to known reversibleN and thatcas thepase-3 loss in rigid-body,a cysteine entropy on proteasebinding of all .Mass spectrometryN was these library fragments were coupled to known reversible which areasMNEC of the molecule >250 to focus µg on per during ml hit optimization. components to the enzyme is very small. Recently, an (FIG. 5).For this to be an efficient lead-identification Experience shows that optimization of these kinds of hits is O O analysisused of the experimentalto identify energetics the associatedlibrary with members that had formed a cysteine-bindingapproach, one elements needs to both to identify generate the initial potent frag- reversible used to identify the library members that had formed a cysteine-binding elementsvery difficult. This to is in generatecontrast to the schematic potent in FIG. reversible 4, in O O CO H 2 Thymidylate optimally linked fragments has suggested that the rigid- 2 ments and also have a process that allows the appropriate 19 which fragment 1 is much smaller, makes high-quality contactsS disulphide bond to the tetheredO thiol.S Subsequently, molecules. disulphide bond to the tethered thiol. Subsequently, molecules. with the receptor and has relatively weak affinity. It has been body entropy loss on protein binding constitutes a barrier linking to be achieved in an efficient manner. synthase / CO2H tethering shown that such fragments can often be built up into attractive of around threeredesigned orders of magnitude to the binding O NH H The potency increase achievable from optimally K = 10 mMleads with (by the aidNMR) Oof structural information MNEC (for example, = T ABLE8 µ 2)g peraffinity, ml and that this barrier is essentiallyMNEC independent = 30 of ng per ml linking two fragments is often assumed to benefit from and SBD d 25 O S CO H MNEC >250 gO perS T mlable 2 | Lead identificationmolecularO chemical mass by.T fragmenthe analysisNH implies libraryH thatevolution*REVIEWS there should 2 an approximate additivity effect, such that the free energy Table 2 | Lead identification by fragment evolution* µ be a super-additivity effect when two fragments are O of binding of the joined molecule is approximately equal O S CO H NFigure 3 | Schematic representation of a low-quality Table 2, entry 7. NMR screening (that is, structure–activity linked in an optimal fashion. Such super-additivity2 is to the sum of the free energies of binding of the frag- 2 drug candidatesThymidylate N Entry Target/method Fragment HTSEvolved hit. The high-throughput COfragment2H screening (HTS) hit is large and Lead Entry Target/method Fragment Evolved fragment Lead relationships (SAR) by NMR7) was used to identify a observed for entries 2 andO 7 in TABLE 3. makes surfaceO contact with the receptor without forming ments (that is, two millimolar fragments when joined 19 O NLead identification by fragment linking 5 synthase / series of triazine-containing compounds that bind in the1 high-quality interactions in key pockets. The affinity is spread together lead to a micromolar inhibitor) .Such additivity 1 1 DNA gyrase / TABLES 3,4 show examplesCO2H in which two fragments have 1 DNA gyrase / H (leadH compounds) mM range to ErmAMH methyl transferase24 (for example, Table 3, entryO 1. SAR by NMRH 7 was usedO to identify a NHthroughout theH Hentire molecule and, in the absence of requires that the contribution fromH the linker is negligible tethering O been identified that bind inN separate binding sites that structuralNH information,N the medicinal chemist does not know N N N TABLE 2,entry 7, NMRNOH K = 1 mM), anVS enzyme and target SBD for potent 49 nM inhibitor of the FK506-binding protein and that the loss in rigid-body entropy on binding of all VS and SBD and SBD d are close enough to each other toO be chemicallyS linked which areasCO of theH molecule to focus on during hit optimization. O S O NH H N 2 N components to the enzyme is very small.N Recently, an N N ameliorating antibioticN resistance. Optimization of this (FKBP)(FIG. 5) OH.F bindingor this to domain be an efficient by linking lead-identification two weaker Experience shows that optimization of these kinds of hits is O O analysis of the experimental energetics associated with O O initial lead using parallel synthesis ledO to inhibitorsS Oin the inhibitorsapproach, (2CO µ oneM2 Hand needs 100 to µ M).both NMR identify screeningN the initial of a setfrag- very difficult. This is in contrast to the schematicO in FIG. 4, in O O N low range (for example, table entry OK = 7.5 ). of 1,000 fragments — including pipecolinic acid deriv- which fragment CO1 is much2H smaller, makes high-quality contacts optimally linked fragments has suggested that the rigid- µΜ i µΜ ments and also have a process that allows the appropriate O O with the receptor and hasS relatively weak affinity. It has been body entropy loss on proteinO binding constitutesS a barrier S NMRO andIC OX-ray= structures 1.1 mMS show that theseN non-nucleo- IC ativlinking=es, 24 a class to be MIof achieved compounds in an known efficient to manner.bind to FKBP — C= 330 nM sidecompounds50 bind to the S-adenosylmethionine- 50identified µthe pipecolinic acid (K = 2 µM) and the 50shown that such fragments can often be built up into attractive of around three orders of magnitude to the binding O The potency increase achievabled from optimally leads with the aid of structural information (for example, TABLE 2) binding site on the Erm protein and that there is scope for diphenyl amide (K = 100 M). Use of 15N-13C-filteredNH affinity, and that this barrier is essentially independent of 2 linking two fragmentsK d = 10 isµ often mM assumed (by NMR) to benefit from MNEC = 8 g per ml MNEC = 30 ng per ml K = 10 mM (by NMR) MNEC = 8 g per3 ml p38 kinase MNEC/ =OH 30 ng per ml d N µ molecular mass25.The analysis implies that there should d µ the incorporation of additional binding interactions. protein–ligandan approximate NUCLEAR additivity OVERHAUSER effect, such EFFECT that(NOE) the free data energy MNEC >250 µg per ml be a super-additivity effect when two fragments are MNEC >250 µg per ml NMR OH of binding of theS joined molecule is approximately equal Figure 3 | Schematic representation of a low-quality Table 2, entry 7. NMRCH screening3 (that is, structure–activity linked in an optimal fashion. Such super-additivity is THTSable hit.1 | ComparisonThe high-throughput of fragment-basedscreening (HTS) hit is large approaches and to and the sumhigh-throughput of the free energies screening of bindingCO2H of the frag- 7 CO2H 2 CO2HThymidylate CO2Hrelationships (SAR) by NMR ) was used to identify a observed for entries 2 and 7 in TABLE 3. 2 Thymidylate makes surface contact with the receptor without forming19 ments (that is, two millimolar fragments when joined N 19 Fragment-based approaches High-throughputN screening (HTS) series of triazine-containing compounds that bind in the high-quality interactionsN in key pockets.synthaseCO The2 affinityH is spread/ 5 CO H synthase / IC = 1.1 mM IC = 24 MItogether lead to a micromolar inhibitor)C=.Such 330 additivity nM 24 2 7 CO2H 50 Emphasis on efficiency 50 µ Emphasis on potency 50 mM range to ErmAM methyl transferaseCO2H(for example, Table 3,O entry 1. SAR by NMRNHwas usedH to identify a tethering O throughout theNH entire moleculeH and, in tetheringthe absence of requires that the contributionO from the linker is negligible N O Tstructuralypically screen information, a few thehundred–few medicinal chemist thousand does compounds not know Typically screen hundreds of thousands of compounds TABLE 2,entry 7, NMR Kd = 1 mM), an enzyme target for potent 49 nM inhibitor of the FK506-binding protein 2 and SBD and that the loss in rigid-body entropy onN binding of all O S CO2H and SBD 3 p38 kinase / O S Mwhichrange areas ~150–300 of the moleculeCO 2toH focus on during hit optimization.F M range ~250–600O S amelioratingO antibiotic resistance.NH OptimizationH of this (FKBP) binding domain by linking two weaker O NH H r r components to the enzyme is very small. Recently, an O S Experience shows that optimization of these kinds of hits is initial lead using parallel synthesis led to inhibitors in the inhibitors (2 µM and 100 µM).O NMR screening of a set NMR Hit activity in theO range mM–30 µMHS itanalysis activity in of the the range experimental ~30 µM–nM energetics associated withF O S CO2H N O S CO2H N very difficult. This is in contrast to the schematic in FIG. 4, in N CHlow3 µΜ range (for example, table entry K = 7.5 µΜ). of 1,000 fragments — including pipecolinic acid deriv- Hitswhich have fragment clearly 1 defined is much binding smaller, interactions;makes high-quality high contacts Hitsoptimally can contain linked functionalCO fragmentsH groups has that suggested contribute that poorly the rigid- i N 2 NMR and X-ray structures showO that these non-nucleo- atives, a class of compoundsO known to bind to FKBP — O proportionOwith theK receptor of= atoms 1 and mM directly has relatively involved weak in proteinaffinity. It binding has been K to= proteinbody 200 entropy binding M loss or act on primarilyprotein bindingasO scaffolding constitutes (as shown a barrierK = 200 nM O d N d µ N i N N N shown that suchCO fragments2H can often be built up into attractive schematically in FIG. 3) sidecompounds bind to the S-adenosylmethionine- identified the pipecolinic acid (Kd = 2 µM) and the of around three orders of magnitude to the binding O 15 13 O 20 Biophysicalleads with the screening aid of structural techniques information (NMR, (for X-ray) example, are directTABLE 2) In vitro bioassay-based screening. Can generate false binding site on COthe Erm2H protein and that there is Nscope for diphenyl amideNH (Kd = 100 µM). Use of N- C-filtered 4 p38 kinase / NH affinity, and that this barrierOH is essentially independentN of OH measurements of binding interaction.N Can screen against positivesmolecular and highmass attrition25.The analysisin hit-validation implies stage that there should the incorporation of additional binding interactions. protein–ligand NUCLEAR OVERHAUSER EFFECT (NOE) data X-ray ‘inactive’ forms of the target protein (for example, kinases) F be a super-additivity effect when two fragments are OH OH Protein-structure-based information key in validating and Chemistry (re)synthesis resource usually required to and SBD Table 2, entry 7. NMR screening (that is, structure–activity linked in an optimal fashion. Such super-additivity is Table 1 | Comparison of fragment-based approaches and high-throughput screening prioritizing chemistry hits validate and prioritize screening hits F CO2H relationshipsCO2H (SAR) by NMR7) was used to identify a observed for entries 2 and 7 in TABLE 3. Hit-to-lead chemistry usually requires synthesis of only a High attrition of chemical series in hit-to-lead stage. Fragment-basedH Happroaches High-throughput screening (HTS) series of triazine-containing compounds that bind in the NUCLEAR OVERHAUSERK EFFECTSd = 1 mMfew compounds designed to add additional,Kd = 200 specific µ bindingM Usually requires severalIC50 iterations= 1.1 ofmM high-throughputKi = 200 nM ICEmphasis50 = N24on efficiency µMIN EmphasisC50 = on 330 potency nM IC50 = 1.1 mM IC50 = 24 µMIC50 = 330interactionsmM nM range to ErmAM methyl transferase24 (for example, chemistry.Table 3, Attrition entry 1. ratesSAR can by be NMR improved7 was withused to identify a N (NOEs). Changes in the Typically screen a few hundred–few thousand compounds Typically screen hundreds of thousands of compounds 20 intensity of NMR signals, which TABLE 2,entry 7, NMR K = 1 mM), an enzyme target2 for knowledgepotent 49 of nMprotein inhibitor structure of the FK506-binding protein N 2 4 p38 kinase / 3 d p38 kinase / N N are caused by through-space Mr range ~150–300O Mr range ~250–600 3 p38 kinase / N Design-intensiveamelioratingN antibiotic resistance. Optimization of this Resource-intensive(FKBP) binding domain by linking two weaker N X-ray dipole–dipole coupling. Upper initial lead using parallel synthesis NMRled to inhibitors in the inhibitors (2 µM and 100 µM). NMR screening of a set Hit activity in the rangeS mM–30 µMHit activity in the range ~30 µM–nM NMR S distance constraints obtained Requires expertise andN knowledge in protein structure, HTS requires extensive infrastructure for storing and H CH3 1 1 protein–ligand-bindingCH interactions and fragment design handling compound collections, screening, automation, CO H and SBD from H– H NOEs are used for low µΜ range3 (for example, table entry Ki = 7.5 µΜ). of 1,000 fragments — including pipecolinic acid deriv- Hits have clearly defined binding interactions; high2 Hits can contain functional groups that contribute poorly CO2H H data processing and chemistry follow-up proportion of atoms directly involved in protein binding to protein binding or act primarily as scaffolding (as shown NMR structure determination NMR and X-ray structures show that these non-nucleo- atives, a class of compounds known to bind to FKBP — N N N N CO H schematically in FIG. 3) N of biological macromolecules. NMR,sidec nuclearompounds magnetic bind resonance. to the S-adenosylmethionine- identified the pipecolinic acid (K = 2 µM)2 andH the H N CO2H d CO2H IC50 = 33 µMICO H 15 13 N CN50 =Biophysical 142 nM screening techniques (NMR, X-ray) are direct In vitro bioassay-based screening. Can generate false binding site on the Erm protein 2and that there is scope for diphenyl amide (Kd = 100 µM). Use of NN- C-filtered N N measurements of binding interaction. Can screen against positives and high attrition in hit-validation stage 5Urokinase21/ the incorporation of additional binding interactions. protein–ligand NUCLEAR OVERHAUSER EFFECT (NOE) data ‘inactive’F forms of the target protein (for example, kinases) F NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | AUGUSTN 2004 | 663 O bioassay Protein-structure-basedN information key in validating and ChemistryF (re)synthesis resource usually required to Table 1HN | Comparison of fragment-based approaches and high-throughput screening prioritizing chemistry hits validate and prioritize screening hits and SBD F N HN H O H Fragment-based approaches High-throughputK =screening 1 mM (HTS) KHit-to-lead= 200 chemistry µM usually requires synthesis of only a HighK attrition= 200 of chemicalnM series in hit-to-lead stage. K = 1 mM K = 200 M K = 200 nM d NUCLEAR OVERHAUSER EFFECTSHN fewd compounds designed to add additional, specificNH binding Usuallyi requires several iterations of high-throughput d d µ i Emphasis on efficiency Emphasis on potency interactions chemistry. Attrition rates can be improved with H2N 20 H N (NOEs). Changes in the HN N Typically screen a few hundred–few thousand compounds2 Typically screen hundreds of thousandsintensity of of NMR compounds signals, which knowledge of protein structure 20 IC50 = 33 µMI4 p38N kinase / C50 = 142 nM 4 p38 kinase / M range ~150–300 M range ~250–600 are causedN by through-space H2N Design-intensive Resource-intensive N 21 r X-ray r dipole–dipole coupling. Upper X-ray 5Urokinase / Requires expertise and knowledge in protein structure, HTS requires extensive infrastructure for storing and Hit activity in the range mM–30 µMHand SBD it activity in the range ~30 µM–nMdistance constraints obtained from 1H–1H NOEs are used for protein–ligand-binding interactions and fragment design handling compound collections, screening, automation, and SBD bioassay Hits haveKi = clearly not defined reported binding interactions; high Ki = Hits5.9 can µ containM functional groups that contribute poorly Ki = 6.3 nM data processing and chemistry follow-up HN proportion of atoms directly involvedHN in protein binding to protein binding or act primarilyNMR as scaffolding structure determination (as shown O H H and SBD NMR, nuclear magnetic resonance. H H schematically in FIG. 3) of biological macromolecules. N N 6FABP4 (REF. 23)/ CO2H HN CO2H NH NBiophysicalN screening techniques (NMR, X-ray) are direct In vitro bioassay-based screening. Can generate false N H2N N NMR measurements of binding interaction.H2N Can screen against positives and high attrition in hit-validation stage HN N ‘inactive’ forms of the target protein (for example, kinases) NATURE REVIEWS | DRUG DISCOVERY OVOLUME 3 | AUGUST 2004 | 663 and SBD N O H N N Protein-structure-based informationN key in validating and Chemistry (re)synthesis resource usually2 required to H prioritizing chemistry hits H validate and prioritize screeningN hits N H H K = not reportedHit-to-lead chemistry usually requiresK = synthesis 5.9 µ ofM only a High attrition of chemical series in hit-to-leadK = 6.3 stage. nM NUCLEAR OVERHAUSERi EFFECTS few compounds designed to add additional,i specific binding Usually requires several iterations of high-throughputi interactionsHO chemistry. Attrition rates can be improved with HO (NOEs). Changes in the IC50 = 33 µMIC50 = 142 nM IC = 33 µMI6FABP4 (REF. 23)/intensity of NMR signals,C which= 142 nMCO2H knowledge of protein structure CO2H 50 50 21 NMR are caused by through-space Design-intensiveIC = 5U590 µMIrokinase / Resource-intensive C= 10 µM 5Urokinase21/ dipole–dipole coupling. Upper 50 50 distance constraints obtained Requires expertise and knowledgebioassay in protein structure, HTS requires extensive infrastructure for storing and and SBD protein–ligand-binding interactions and fragment design handling compound collections, screening, automation, bioassay 7 Ermfrom methyl 1H–1H NOEs are used for HN HN O and SBD data processing and chemistry follow-up HN HN NMR structure determination24 O HN and SBD transferase / HN NH of biological macromolecules. NMR, nuclear magnetic resonance.NH2 NH HN CH3 NH H2N NMR N H2N HN H2N H N HO HN N HO HN N 2 H N NATURE REVIEWS | DRUG DISCOVERYS N N N VOLUME 3 | AUGUST 2004 | 663 2 HIC2N = 590 MIC= 10 M N 50 µ N N 50 µ K = not reported K = 5.9 µM NH2 K = 6.3 nM NH2 i i i Ki = not reported Ki 7= 5.9 µM Erm methyl Ki = 6.3 nM NH2 transferase24/ 6FABP4 (REF. 23)/ CO2H OH HN CO2H 6FABP4 (REF. 23)/ CO H NH2 CO H 2 CH 2 NMR NH NMR NMR 3 N Kd = 1 mM (by NMR)and SBD N Kd = 75 µM HNKi = 7.5 µM N and SBD S N *See also FIG. 4. FABP, fatty-acid-binding protein; MNEC, maximal non-effectiveN concentration;N NMR, nuclear magnetic resonance; SBD, structure-N based design; VS, virtual screening. N N NH2 NH2 NH2 HO HO HO HO IC = 590 MIOH C= 10 M NATURE REVIEWS | DRUG DISCOVERY 50 µ VOLUME 3 | AUGUST 2004 | 66550 µ IC50 = 590 µMIC50 = 10 µM Kd = 1 mM (by NMR)7 KErmd = 75 methyl µM Ki = 7.5 µM 24 7 Erm methyl *See also FIG. 4. FABP, fatty-acid-binding protein; MNEC, maximal non-effective concentration;transferase NMR,/ nuclear magnetic resonance; SBD, structure-based design; VS, virtual screening. HN 24 HN NH2 NH transferase / NMR CH3 NH2 NH N NMR CH3 N HN N N N S N HN N N N N S N N N N N N NH N NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | AUGUST 2004 | 665 2 N NH2 NH2 NH2 NH2 NH2 OH OH

Kd = 1 mM (by NMR) Kd = 75 µM Ki = 7.5 µM K = 1 mM (by NMR) K = 75 µM K = 7.5 µM d d i *See also FIG. 4. FABP, fatty-acid-binding protein; MNEC, maximal non-effective concentration; NMR, nuclear magnetic resonance; SBD, structure-based design; VS, virtual screening. *See also FIG. 4. FABP, fatty-acid-binding protein; MNEC, maximal non-effective concentration; NMR, nuclear magnetic resonance; SBD, structure-based design; VS, virtual screening.

Pros and cons of drug development approaches based on natural products

approach based on natural products approach using artifcial compounds

Strong bioactivity obtained Limitation in diversity of during the process of evolution available chemical libraries

Initial hit compounds are Optimizations of hit compounds often drug-ready molecules are generally required

Highly rely on the compounds Rational design strategies of produced in nature novel compounds are possible

Supply by chemical synthesis The drugs can be readily is sometimes challenging. supplied by chemically synthesis J. Med. Chem. 2008, 51, 2589–2599 2589

Natural Products as Leads to Potential Drugs: An Old Process or the New Hope for Drug Discovery?

David J. Newman† Natural Products Branch, DeVelopmental Therapeutics Program, DCTD, National Cancer InstitutesFrederick, P.O. Box B, Frederick, Maryland 21702

ReceiVed April 5, 2007

I. Introduction From approximately the early 1980s, the “influence of natural products” upon drug discovery in all therapeutic areas apparently has been on the wane because of the advent of combinatorial chemistry technology and the “associated expectation” that these techniques would be the future source of massive numbers of novel skeletons and drug leads/new chemical entities (NCEa) where the intellectual property aspects would be very simple. Large presence of natural products in drug development As a result, natural product work in the pharmaceutical industry, except for less than a handful of large pharmaceutical compa- nies, effectively ceased from the end of the 1980s. Figure 1. Source of small molecule drugs, 1981–2006: major What has now transpired (cf. evidence shown in Newman categories, N ) 983 (in percentages). Codes are as in ref 1. Major and Cragg, 20071 and Figures 1 and 2 below showing the categories are as follows: “N”, natural product; “ND”, derived from a natural product and usually a semisynthetic modification; “S”, totally continued influence of natural products as leads to or sources synthetic drug often found by random screening/modification of an of drugs over the past 26 years (1981–2006)) is that, to date, existing agent; “S*”, made by total synthesis, but the pharmacophore there has only been one de novo combinatorial NCE approved naturalis/was from products a natural product. natural products anywhere in the world by the U.S. Food and Drug Administra- mimics natural products tion (FDA) or its equivalent in other nations for any human derivatives disease, and that is the kinase inhibitor sorafenib (1, Chart 1), which was approved by the FDA in late 2005 for renal carcinoma. However, the techniques of combinatorial chemistry have 58% of the approved drugs revolutionized the deVelopment of active chemical leads where are natural products-related. currently, instead of medicinal chemists making derivatives from scratch, a procedure is used whereby syntheses are based on combinatorial processes so that modifications can be made in sources of small molecule drugs, 1981–2006 (N = 983) an iterative fashion. An example of such a process would be Figure 2. Sources of small molecule drugs, 1981–2006: all categories,J. Med. Chem., 51, 2589–2599 (2008) the methods underlying the ultimate synthesis of the antibiotic N ) 983 (in percentages). Codes are as in ref 1. Major categories are linezolid (Zyvox, 2) by the Pharmacia (now Pfizer) chemists as follows: “N”, natural product; “ND”, derived from a natural product starting from the base molecules developed in the late 1980s and usually a semisynthetic modification; “S”, totally synthetic drug by DuPont Pharmaceutical, who reported the underlying anti- often found by random screening/modification of an existing agent; biotic activity and mechanism of action of this novel class of “S*”, made by total synthesis, but the pharmacophore is/was from a molecules, the oxazolidinones.2–6 natural product. The subcategory is as follows: “NM”, natural product mimic.

† Contactinformation.Telephone:+301.846.5387.Facsimile:+301.846.6178. Although the early (late 1980s to late 1990s) combinatorial E-mail: [email protected]. The views expressed in this review are those of chemical literature is replete with examples of libraries contain- the author and are not necessarily the position of the U.S. Government. ing hundreds of thousands to millions of new compounds, as a Abbreviations: !-AST-IV, !-arylsulfotransferase-IV; BIOS, biology- oriented synthesis; Cdks, cyclin dependent kinases; DOS, diversity-oriented stated rather aptly by Lipinski in the early 2000s, if the early synthesis; E-FABP, epidermal fatty acid binding protein; EGFR, epidermal libraries had been disposed of, the productivity of pharmaceuti- growth factor receptor; FKBP-12, FK binding proteins; FXR agonists, cal drug discovery would have materially improved in the prior farnesoid X receptor agonist; GPCR, G-protein-coupled receptor; hERG, decade.7 However, in the late 1990s, synthetic chemists realized human ether-a-go-go-related-gene K+ channel; HIF-1R, hypoxia-inducible factor-1R;IGF1R,insulin-likegrowthfactor1receptor;LTA4H,leukotriene that the combinatorial libraries that had been synthesized up to A4 hydrolase/aminopeptidase; mEST, murine estrogen sulfotransferase; that time (with the exception of those based on intrinsically M6p-IGFR2, insulin-like growth factor II/mannose 6-phosphate receptor; bioactive compounds such as nucleosides, peptides, and to some NCE, new chemical entity; NMDA, N-methyl-D-aspartate; NodH sulfotransferase (gene product from Rhizobium NodH); PAPS, 3′-phosphoadenosine extent carbohydrates) lacked the “complexity” normally associ- 5′-phosphosulfate; PFT, protein fold topology; PI3K, phosphoinositol-3- ated with bioactive natural products, items such as multiple kinase; PKC, protein kinase C, exists in multiple isoforms; PSSC, protein chiral centers, heterocyclic substituents, and polycyclic structures. structure similarity clustering; SCONP, structural classiﬁcation of natural Although chemists had probably accepted that as a “basic products; SEA, similarity ensemble approach; Shp-2, Src homology 2 domain containing tyrosine phosphatase 2; TOR, target of rapamycin; rule”, natural products were different from synthetic compounds; VEGFR-2, vascular endothelial growth-factor receptor-2. the 1999 analysis by Henkel et al.8 was perhaps the ﬁrst of the

10.1021/jm0704090 This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society Published on Web 04/05/2008 Power of natural products –1 五大抗生物質五大抗生物質 Antibiotics Antibiotics are compounds that kill bacteria or suppress bacteria growth

ペニシリン Many natural products exhibit antibiotic activities. ストレプトマイシン五大抗生物質ペニシリンストレプトマイシン

クロラムフェニコールペニシリン penicillin ストレプトマイシンchloramphenicolクロラムフェニコール tetracyclineテトラサイクリンテトラサイクリン抗生物質には、語尾に「-mycin」（マイシン）がつくエリスロマイシンエリスロマイシン抗生物質には、語尾に「ものが多い -mycin」（マイシン）がつくものが多い O H クロラムフェニコール N S テトラサイクリン O O N O エリスロマイシン抗生物質には、語尾に「-mycin」（マイシン）がつく OH ものが多い O vancomycin methicillin Power of natural products –1

Development of antibiotics changed the world.

changes in causes of death http://www2.ttcn.ne.jp/honkawa/2080.html pneumonia

gastroenteritis cancers

tuberculosis heart diseases

cerebrovascular disease

benzylpenicillin release in 1942 emergence of MRSA in 1980s Power of natural products –2

Immunosuppressive drugs Immunosuppressive drugs are compounds that prevent the immune system.

They drastically increased success rate of organ transplantation. Number of transplantation in US kidney cyclosporin heart liver discovered by Sandoz (now Novartis) approved in 1983

tacrolimus discovered by Fujisawa (now Astellas) approved in 1994

http://www.medi-net.or.jp/tcnet/tc_1/1_2.html Power of natural products –3

Statins They reduce cholesterol in blood and are used for hyperlipidemia treatment.

This class of drugs resulted in many blockbusters.

Mevastatin Mevalotin natural product natural product derivative Daiichi-Sankyo/BMS 4,746 million $ sales in 2003 (約5000億円) Major categories of natural products

alkaloids (highly modifed amino acids) steroids (terpenes with a specific ring system)

cocaine caﬀeine cholesterol testosterone

terpenoids (oligomerized isoprenes) glycoside (highly modifed sugars)

menthol retinol salicin kanamycin Major categories of natural products polyphenol polyketide (oligomerized malonic acid)

catechin sesamin erythromycin tetracycline peptides (amino acid oligomers)

cyclosporin vancomycin