The Chemistry and Biology of Discodermolide

HO

O O OH O O

OH NH2 OH

Yueheng Jiang November 20, 2003

-1- HO Outline O O OH O O OH NH2 OH

¾ Discovery and Biological Activity

¾ Total Syntheses

¾ Structure-Activity Relationships (SAR)

-2- Discovery ƒ Isolated by Gunasekera and co-workers in 1990 from the Caribbean deep-sea sponge (Discodermia dissoluta). ƒ 0.002% w/w isolation yield (7 mg/434 g of sponge). ƒ Found initially to have immunosuppressive and antifungal activities. ƒ Further revealed to be a potent microtubule stabilizer.

Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1991, 56, 1346. Longley, R. E.; Caddigan, D.; Harmody, D.; Gunasekera, M.; Gunasekera, S. P.; Transplantation 1991, 52, 650. ter Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.; Gunasekera, S. P.; Rosenkranz, H. S.; Day, B. W. Biochemistry 1996, 35, 243. -3- Microtubule-stabilizing agents

AcO O O OH

R NH O O O OH OH Ph O H O NH2 OH HO AcO BzO HO R = Ph Taxol (BMS) HO R = OtBu Taxotere (Aventis) discodermolide O

O O

O O R NMe O H N S O NMe O H N OH HO N O O H O O OMe O H OH OO O OH O OAc OH CO2Me O O R = H epothilone A OH eleutherobin sarcodictyin A laulimalide R = Me epothilone B OH

-4- Cytotoxicity

ƒ Cytotoxic over a variety of cell lines (IC50 3-80 nM) ƒ More potent than Taxol ƒ Competitively inhibits the binding of Taxol to tubulin ƒ Active against multi-drug resistant (MDR) and Taxol- resistant (Pgp mediated MDR) cell lines

ter Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.; Gunasekera, S. P.; Rosenkranz, H. S.; Day, B. W. Biochemistry 1996, 35, 243. Kowalski, R. J.; Giannakakou, P.; Gunasekera, S. P.; Longley, R. E.; Day, B. W.; Hamel, E.; Mol. Pharmacol. 1997, 52, 613. -5- Mechanism of action

ƒ Promote tubulin polymerization in vitro ƒ Stabilize microtubules against depolymerization ƒ Interfere with Taxol-binding to microtubules ƒ Induce microtubule bundles in cells Consequences: Interfere with proper formation of mitotic spindle

Cause arrest of cell cycle

Cell death by apoptosis

-6- Dynamic Equilibria of Tubulin-Microtubules

Heterodimer Formation Initiation Polymerzation/Elongation

(+) End (more grown)

Nucleation center

's P p T 5 nm a G Microtubule M + ; 2 g M o C (-) End (less grown) ; 0 α-tubulin 2+ ~50 kD Ca

D is Ta c o x -5 de ol KD = 10 r o m r (α.β) ol id e Taxol or Discodermolide β-tubulin ~50 kD Stabilized Nucleation centers Stabilized microtubules Taxol or Discodermolide

Nicolaou, K. C.; Roshangar, F.; Vourlounis, D. Angew. Chem. Int. Ed., 1998, 37, 2014. -7- Microtubule Bundling

Influence of discodermolide on a transformed mouse fibroblast. Discodermolide induces microtubule bundling (tubulin appears green), which is clearly seen in the pseudopodia and near the nucleus (appears blue). As a result of this microtubule bundling, the cell is undergoing apoptosis and fragmentation of the nucleus can be seen.

Sasse, F. Current Biology, 2000, 10, R469.

-8- Unique Activities

ƒ Discodermolide could not substitute for Taxol in a Taxol-resistant cell line (A549-T12) that requires low concentrations of Taxol for normal growth.

ƒ Exhibits synergistic effects with Taxol in several cultured cell lines (not observed with Taxol/epothilones or Taxol/eleutherobin).

Martello, L. A.; McDaid, H. M.; Regl, D. L.; Yang, C. H.; Ment, D. T.; Pettus, R. R.; Kaufman, M. D.; Arimoto, H.; Danishefsky, S. J.; Smith, A. B. III; Horwitz, S. B. Clin. Cancer Res. 2000, 6, 1978. Giannakakou, P.; Fojo, T. Clin. Cancer Res. 2000, 6, 1613. -9- Potential Candidate for Cancer Chemotherapy

ƒ Novel structure ƒ Greater or comparable efficacy to Taxol ƒ Poor substrate for P-glycoprotein (Pgp). ƒ Synergistic effect in combination with Taxol ƒ Greater water solubility (100-fold > Taxol)

Entered Phase I clinical trials in 2002 as a chemotherapeutic agent for use against solid tumors.

Myles, D. C. Annual Reports in Med. Chem., 2002, 37, 125.

-10- ƒ Taxol (semi-synthesis) ƒ Epothilone (fermentation) ƒ Discodermolide ( ???)

Total Synthesis of Discodermolide ! (-)-Discodermolide (+)-Discodermolide

S. L. Schreiber (1993) S. L. Schreiber (1996) A. B. Smith (1995) J. A. Marshall (1998) D. C. Myles (1997) A. B. Smith (1999, 2003) I. Paterson (2000, 2002) Novartis (2003) D. C. Myles (2003)

-11- Selected Total Synthesis of (+)-Discodermolide

Schreiber 1st total synthesis of (+/-)-discodermolide established absolute configuration

Smith Delivered ~1 g of (+)-discodermolide (2nd generation)

Paterson Novel approaches

Novartis Meet supply needs for clinical studies by total synthesis

-12- Structure and conformation of (+) discodermolide

24 HO 17 19 7 21 O O 5 OH O O 1 13 OH NH2

OH

X-ray structure of discodermolide; hydrogen atoms omitted for clarity

Paterson, I.; Florence, G. J. Eur. J. Org. Chem., 2003, 2193.

-13- General Features

24 HO 7 17 * * * O O OH O O * * * 1 13 * * * OH NH2 OH

* * * OH repeating anti, syn-stereotriad divided into three fragments Introduction of two Z-alkenes of roughly equal complexity and terminal Z-diene

HO CO2Me Roche ester Fragment Coupling

-14- Strategy by Scheiber

Nozaki-Kishi coupling enolate alkylation 24 8 HO 15 O O OH O O (+)-discodermolide 1 1 5 13 OH NH2

OH I 8 24 PhS O 7 H 15 OPiv 1 16 O + +

OTBS OTBS Negishi coupling A O OPMB B Still-Gennari C HWE olefination

Roush crotylation Roush crotylation

TBSO OH TBSO OH 2 TBSO OH 3 2

Roche ester CO2Me OH -15- Strategy by Smith 2nd Generation Wittig olefination Negishi coupling 24 HO 15 19 9 Yamamoto O O OH O O olefination 1 13 OH NH2

OH (+)-discodermolide 1

O Evans aldol O O I 8 H 1 PMBO 14 I 15 19 + OTBS + 9 OTBS TBSO O O OTBS A B Zhao-Wittig C PMP olefination

PMBO N CO2Me O Common Precursor 2 OH OH O Roche ester Evans aldol reaction -16- Strategy by Paterson 1st generation lithium aldol

boron aldol 24 16 HO 7 9

O O OH O O (+)-discodermolide 1 1 13 OH NH2

OH Nozaki-Hiyama/ boron aldol boron aldol ArO O Peterson elimination

24 6 PMBO 9 16 ++H 17 MeO2C TBSO O C A B O O TBSO PMB boron aldol Claisen [3,3]

BnO PMBO OBz O O 2 3 O 4 HO CO2Me EtO2C OH Roche ester ethyl-(S)-lactate

-17- Synthesis of Stereotriad by Schreiber

CO2iPr CO2iPr Roche-ester O O CO2iPr CO2iPr B O B O H (R,R)-(E)-crotylboronate (S,S)-(Z)-crotylboronate TBSO OH TBSO O TBSO OH 2 Roush crotylation 3

Subunit A & C Subunit B

Hung, D. T.; Nerenberg, J. B.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 11054. Roush, W. R.; Palkowitz, A. D.; Ando, K. J. Am. Chem. Soc. 1990, 112, 6348.

-18- Synthesis of stereotriad by Smith

Ph

NH N O

1 PMBO CCl , PPTS O O HO 3 PMBO H n-Bu2BOTf, Et3N CO2Me 2 LAH 3 Swern Oxidation O 52-55%, 4 steps 3

Ph

O MeNH(OMe).HCl PMBO N PMBO N O

OH O O AlMe3, THF, 98% OH O

4 Common Precursor 2

Smith, A. B. III; Beauchamp, T. J.; LaMarche, M. J.; Kaufman, M. D.; Qiu, Y.; Arimoto, H.; Jones, D. R.; Kobayashi, K. J. Am. Chem. Soc. 2000, 122, 8654. Smith, A. B. III; Kaufman, M. D.; Beauchamp, T. J.; LaMarche, M. J.; Arimoto, H. Org. Lett. 1999, 1, 1823. Iversen, T.; Bundle, D. R. J. Chem. Soc., Chem. Commun. 1981, 1240. -19- Synthesis of stereotriad by Paterson

cHex2BCl Et3N, Et2O BnO LiBH BnO 4 BnO 6 steps Subunit A O + O - O 86% O B > 30:1 dr OH OH 2 H 5 L L 6

cHex2BCl, Et3N methacrolein Me4NBH(OAc)3 6 steps PMBO Subunit B 94%, > 30:1 dr 95%, >30:1 dr PMBO OH OH 3 O PMBO O OH 7 8

TBSO CHO TBSO 6 steps OBz OBz Subunit C O cHex2BCl, Me2NEt PMBO O 4 > 97% ds 10

Paterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P. Angew. Chem. Int. Ed. 2000, 39, 377. Paterson, I.; Arnott, E. A. Tetrohedron Lett. 1998, 39, 7185. Paterson, I.; Wallace, D. J.; Cowden, C. J. Synthesis 1998, 639.

-20- Synthesis of trisubstituted (Z)-alkene Schreiber

KHMDS, CO2Me O (CF3CH2O)2POCHMeCO2Me 14 TBSO OTBS 93%, Z:E >20:1 TBSO OTBS

Sch-5 (Still-Gennari HWE olefination) Sch-8

Smith

o 1 Ph3PEtI, n-BuLi, 0 C o 2 0.1 M I2 in THF, -78 C I 3 NaHMDS, -23oC PMBO 13 H PMBO 9 14 4 Add 6, -33oC TBSO TBSO O 46%, 3 steps, Z:E = 8-17:1 Sm-5 (Zhao-Wittig olefination) Subunit B

Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405. Chen, J.; Wang, T.; Zhao, K. Tetrahedron Lett. 1994, 35, 2827. -21- Synthesis of trisubstituted (Z)-alkene Paterson

+

9 1 NaIO4, NaHCO3 Me MeOH, H2O, rt O [3, 3] PMBO O O O 2 DBU, xylene, reflux Me 82% PMBO PhSe 9 14 AcO O PMBO 11 13 PMBO 9 16 Me O 3 steps, 81% O O

Me 15 PMBO O OTBS Subunit B

Burton, J. W.; Clark, J. S.; Derrer, S.; Stork, T. C.; Bendall, J. G.; Holmes, A. B. J. Am. Chem. Soc. 1997, 119, 7483.

-22- Synthesis of terminal (Z)-diene Schreiber

NaHMDS I + - Pd(PPh3)4 Ph3P CH2I I O H2CCHZnBr 75% TBSO OPMB TBSO OH 80% TBSO OPMB (Stork-Zhao Sch-4 Wittig olefination) Sch-6 Sch-7

Smith

i 24 21 O Ph2P Ti(O Pr)4Li then MeI TrO O OPMB TrO O OPMB TBS 76%, 2 steps Z:E = 12:1 OTBS 9 TBS OTBS Sm-7 Yamamoto Olefination Sm-8

1. Stock, G.; Zhao, K. Tetrahedron Lett. 1989, 30, 2173. Ikeda, Y.; Ukai, J.; Ikeda, N.; Yamamoto, H. Tetrahedron 1987, 43, 723.

-23- Synthesis of terminal (Z)-diene

Paterson

SiMe3

TBSO H LnCr SiMe3 TBSO

PMBO OH PMBO O Nozaki-Hiyama 11 allylation 12

KH, 98% 24 Z:E = > 20:1 TBSO

Peterson OPMB elimination 13

Takai, K.; Kuroda, T.; Nakatsukasa, S.; Oshima, K.; Nozaki, H. Tetrahedron Lett. 1985, 26, 5585. Aicher, T. D.; Kishi, Y. Tetrahedron Lett. 1987, 28, 3463. Ager, D. Org. React., 1990, 38, 1.

-24- Fragment coupling Schreiber I 8 PhS O 7 H HO OPiv 15 OPiv 1 15 O 7 A PhS O OTBS 1 5 steps OTBS OTBS 0.011% NiCl2/CrCl2, 65% Subunit B 2:1 dr at C7 OTBS 9 (Nozaki-Kishi coupling) 24 16 TBSO Br TBSO 15 15 16 24 PhS O O OPMB PhS O C 1 O OPMB OTBS OTBS 11 LDA (1.1 eq), THF, 0oC OTBS 10 65% OTBS 24 TBSO 19 16

LiN(SiMe2Ph)2 (5 eq) PhS O O MeI, 47% OTBS 3:1 dr at C16 12 OTBS

Takai, K.; Kuroda, T.; Nakatsukasa, S.; Oshima, K.; Nozaki, H. Tetrahedron Lett. 1985, 26, 5585. Aicher, T. D.; Kishi, Y. Tetrahedron Lett. 1987, 28, 3463.

-25- Fragment coupling Smith

1 ZnCl2 (1.15 eq.) I 15 2 t-BuLi (3.45 eq.), -78oC to rt 19 8 steps TBSO OO 3 Pd(PPh3)4 (0.05 eq.), 66% O OO I 14 TBS PMP PMBO 14 PMBO OTBS PMP subunit C 6 TBSO subunit B

NaHMDS (0.95 eq.), THF, -20oC

O - + IPh3 P O OPMB O O 8 H 9 TBS 69% OTBS OTBS Z:E = 24:1 10 OTBS A (1.05 eq.) 8 24 TBSO 9

O O OOPMB TBS OTBS 11 OTBS

-26- Fragment coupling Paterson

1 LiTMP, LiBr HO O O 2 LiAlH4, 62% > 30:1 dr PMBO 17 PMBO 9 steps 16 16 24 OH OPMB H 17 OTBS OTBS O OPMB 16 Subunit B Subunit C

24 HO (+)-Ipc2BCl O 7 7 87%, 5:1 at C7 O O O O MeO2C H O O O TBS 6 OTBS NH2 TBS 1 2 OTBS NH MeO C 2 2 OTBS 17 TBSO O 18 Subunit A

Paterson, I.; Goodman, J. M.; Lister, M. A.; Schumann, R. C.; McClure, C. K.; Norcross, R. D. Tetrahedron, 1990, 46, 4663. Mulzer, J.; Berger, M. J. Am Chem. Soc. 1999, 121, 8393. -27- Improvement

2nd Generation: ultrahigh pressure Smith Undesired intramolecular cyclization 1 PPh3, I2 2 PPh3, i-Pr2NEt 12.8 kbar, 6d 82%, 2 steps -IPh +P 11 O OPMB 9 11 O OPMB 12.8 kbar = 3 TBS 12600 atm = 186,000 psi !! 9 TBS OH OTBS OTBS

H R R I H H H H OTBS OTBS + Me R Me Me - + I Me TBSO TBSO R H H

3rd Generation: improvement Reduction of the steric bulk at C11

1 PPh3, I2 o 2 PPh3, i-Pr2NEt, 100 C 70%, 2 steps - + 9 11 O OPMB IPh3 P 11 O OPMB TBS ambient pressure 9 TBS OH OMOM OMOM

Smith, A.B. III; Kaufman, M. D.; Beauchamp, T. J.; LaMarche, M. J.; Arimoto, H. Org. Lett. 2003, 5, 4405. -28- Paterson 1st generation Problem

24 (+)-Ipc BCl R2 2 1 O 7 R 7 87%, 5:1 at C7 O 10 O O O H O O O MeO2C TBS 1 6 TBS OTBS NH2 OTBS NH2 MeO2C 2 17 TBSO O OTBS 18

18a: R1 = OH, R2 = H Nu 1 2 reagent 18a : 18b yield 18b: R = H, R = OH (desired) + H + H Me c-Hex2BCl 7 : 1 67% O 10 (+)-Ipc2BCl 1 : 5 87% 7 H H RL

-29- Paterson 2nd Generation boron aldol 24 HO 100% substrate control O O 6 OH O O 1 5 OH 1 NH2 lithium aldol OH 24 O 16

5 H + 11 OH O O MeO2C 6 TBSO O A OH NH2 alkylation ArO O 24 PMBO H 15 9 16 + 14 O OPMB

TBSO C Still-Gennari B HWE olefination

boron aldol reduced overall steps PMBO PMBO OH from 42 to 35 O OH common precursor 19 -30- Paterson 2nd Generation Improvement

O cHex2BCl, Et3N 16 6 O O O 10 1 5 H TBS MeO2C OTBS NH2 TBSO O 19 Subunit A R L + 10 24 8 L O H 64%, 20:1 dr at C5 H - HO OOO B MeO2C O L 5 TBS OTBS NH2 O R + OH 20

Paterson, I.; Delgado, O.; Florence, G. J.; Lyothier, I.; Scott, J. P.; Sereinig, N. S. Org. Lett. 2003, 5, 35.

-31- Strategy by Novartis

boron aldol (Paterson) 24 18 HO 9

O O 6 OH O O 1 14 2+ OH NH2 Mg -chelated aldol (novel)

OH

18 6 9 N MeO2C O + OH O O O O O 14 TBS OH NH2

Zhao-Wittig Olefination (Smith) Roush Crotylation (Schreiber) MeO C 9 18 2 + H 19 Nozaki-Hiyama/ PMBO O Peterson elimination 13 O (Paterson) OH OH Negishi coupling (Smith)

PMBO H PMBO H HO HO CO Me O CO2Me 2 O commercially available

Francavilla, C.; Chen, W.; Kinder, F. R. Jr. Org. Lett., 2003, 5, 1233. -32- Next Generation ???

Longest Linear Sequences Steps Yield Schreiber 24 4.3% Smith 24 6% Myles 25 1.4% Marshall 29 2.2% Paterson 23 10.3% Novartis 21 N.A.

Despite considerable synthetic efforts, there continues to be a pressing demand for a more practical and scaleable total synthesis …

-33- SAR Summary

Antiproliferative Potencies (IC50, nM) Against A549 or MG63 Cells of Discodermolide (1), and Analogues 2-3e.

A549 MG63 Discodermolide 1 3.6 6 R (R = Me) HO 17 7 16 2 (R = H; 16-demethyl) n.d. 10 O O 11 OH O O

3 OH NH2 Acetylated analogues: OH 3a (3-OAc) 3.8 3b (7-OAc) 0.8 3c (3,7-OAc) 0.8 3d (3,11-OAc) 164 3e (3,17-OAc) 524

N. Choy, Y. Shin, P. Q. Nguyen, D. P. Curran, R. Balanchandran, C. Madiraju, B. W. Day, J. Med. Chem., 2003, 46, 2846-2864. Nerenberg, J. B.; Hung, D. T.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 11054. Gunasekera, S. P.; Longley, R. E.; Isbrucker, R. A. J. Nat. Prod. 2001, 64, 171. -34- SAR Summary

Antiproliferative Potencies (IC50, nM) Against A549 Cells of Discodermolide (1), and Analogues 4a-4d.

HO R 7 O O 14 OH O O O O OH O O

OH NH2 OH NH2 OH 3

A549 A549 4a (14-cis, demethyl) 7.8 4c (3-dehydro, R = OH) 1.8 4b (14-trans, demethyl) 485 4d (3-dehydro, R = H) 11.4

Martello, L. A.; LaMarche, M. J.; He, L.; Beauchamp, T. J.; Smith, A. B. III; Horwitz, S. B. Chem. Biol. 2001, 8, 843. -35- Summary

ƒ Discodermolide, a marine natural product, shares the same microtubule-stabilizing mechanism as Taxol and has a promising anticancer profile.

ƒ However, the supply problem is still hampering further biological and SAR studies. To date, total synthesis is the only economical means of providing useful quantities of Discodermolide. Despite considerable synthetic efforts, there continues to be a demand for a more practical and scaleable total synthesis.

-36- Acknowledgements

Prof. Stephen F. Nelsen, Prof. Steven D. Burke

Brian Lucas, Andy Hawk

Dr. Jian Hong, Dr. Lei Jiang

Dr. Stuart Rosenblum, Dr. Michael Wong, Dr. Ron Kuang Lei Chen, Dr. Gopinadhan Anilkumar

-37-