1 The Epothilones Brian Lucas October 16, 2003 2 Epothiwhat? Epoxide, thiazole, ketone = epothilone Epothilone A, R = H Epothilone B, R = CH 3

1

The Epothilones

OH

OOHO

O

S

N

O R

Brian Lucas

October 16, 2003

2

Epothiwhat?

OH

OOHO

O

S

N

O R

Epoxide, thiazole, ketone = epothilone

Epothilone A, R = H Epothilone B, R = CH3

3

Overview

The epothilones are extremely cytotoxic

agents with a similar mode of action to Taxol® Epo B is more active than Taxol (in vitro) Comparatively simple in structure

OH

OOHO

O

S

N

O

NH

O

OH

O

O

OH

O OH

OHO

O

AcO

AcO

Epothilone B Taxol

4

Overview

The epothilones are active against Taxol resistant cancer cell lines The epothilone scaffold is easier to derivatize than Taxol The epothilones are more water soluble than Taxol

OH

OOHO

O

S

N

O

NH

O

OH

O

O

OH

O OH

OHO

O

AcO

AcO

Epothilone B Taxol

5

Outline

Discovery and Background Mode of Action Biosynthesis Initial Synthetic Efforts Selected Total Syntheses Structure Activity Relationships (SAR) In vivo Studies and Phase I/II Clinical

Results

6

Discovery

First isolated from the common soil

bacteria Sorangium cellulosum as early

as 1987 by Höfle and Reichenbach (GBF) Exhibited a narrow antifungal spectrum

(against Mucor hiemalis only) Found to be too toxic for use as an

antifungal

Höfle, G. Bedorf, N.; Gerth, K.; Reichenbach, H. (GBF), DE-B 4138042, 1993, [Chem. Abstr. 1993, 120, 52841]Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed. 1998, 37, 2014.

7

Cytotoxicity

Bollag (Merck) discovered taxol-like cytotoxicity Epo A and B were competitive inhibitors

of taxol binding IC50 values comparable (Epo A) or better (Epo B) than Taxol Active against Pgp MDR (“Taxol refractory”) cells

Bollag, D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Leisch, J.; Goetz, M.; Lazarides, E.; Woods, C.M.

Cancer Res. 1995, 55, 2325.

8

Comparison of IC50 Values [nM]

Altmann, K.H. Mini-Reviews in Medicinal Chemistry 2003, 3, 149.

Altmann, K.H.; Wartmann, M.; O’Reilly, T. Biochim. Biophys. Acta 2000, 1470, M79

The epothilones retain activity in taxol-resistant

cancer cell lines

9

Mode of Action

Like Taxol, the epothilones are microtubule stabilizers Both bind to β tubulin

Competitive binding suggests same

binding site Epo B efficacy against tubulin-mutated

cell lines suggests different interactions

Bollag, D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Leisch, J.; Goetz, M.; Lazarides, E.; Woods, C.M.

Cancer Res. 1995, 55, 2325.

10

Epothilone B / Taxol Mode I

NH

O

OH

O

OH

O OH

OHO

O

AcO

AcO

OHO

OHO

O

O

SN

O

X

X

6.95

6.93

13 16 1 2

1 13

12

75

Giannakakou, P.; Gussio, R.; Nogales, E. Downing, K.H.; Zaharevitz, D.; Bollubuck, B.; Poy, G.; Sackett, D.; Nicolaou, K.C.

Fojo, T. Proc. Natl. Acad. Sci. USA, 2000, 97, 2904

Carbon: Taxol, Epothilone B

Nitrogen, Oxygen

11

Epothilone B / Taxol Mode II

NH

O

OH

O

HO

OOH

OHBzO

OAc

O

OHO

HO

OOO

OX

X

6.95

6.93N

S

O

CH3

13

161

1

3

75

12

13

Giannakakou, P.; Gussio, R.; Nogales, E. Downing, K.H.; Zaharevitz, D.; Bollubuck, B.; Poy, G.; Sackett, D.; Nicolaou, K.C.

Fojo, T. Proc. Natl. Acad. Sci. USA, 2000, 97, 2904

Carbon: Taxol, Epothilone B

Nitrogen, Oxygen

12

Formation of Microtubules

Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed. 1998, 37, 2014.

13

Stabilized Microtubules BlockMitosis

Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed. 1998, 37, 2014.

Epothilone Biosynthesis

15

Biosynthesis

Produced by the myxobacterium Sorangium cellulosum So Ce90 ~20 mg/L 2:1 Epo A : Epo B

The So Ce90 genome sequenced: Nine modules of polyketide synthase

(PKS) One nonribosomal peptide synthetase

(NRPS) Cytochrome P450 monooxygenase

(epoxidation)Molnar, I.; Schupp, T.; Ono, M.; Zirkle, R.E.; Milnamow, M.; Nowak-Thompson, B.; Engel, N.; Toupet, C.; Stratmann, A.;

Cyr, D.D.; Gorlach, J.; Mayo, J.M.; Hu, A.; Goff, S.; Schmid, J. Ligon, J.M. Chem. Biol. 2000, 7, 97.Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Kholsa, C.; Julien, B. Science 2000, 287, 640

16

Epothilone Gene Cluster

Walsh, C.T.; O’Connor, S.E.; Schneider, T.L. J. Ind. Microbiol. Biotechnol. 2003, 30, 448.

SO

HS NHO

B:

SO

SN

OH

B:

H

SO

SN

[O]

17

Post Assembly Line Epoxidation

OH

OOHO

O

S

N

Epothilone D

P450 Monooxygenase

OH

OOHO

O

S

N

O

Epothilone B

OH

OOHO

O

S

NOH

OOHO

O

S

N

O

Epothilone C Epothilone A

OH

OO

OH

S

N

SEnz

TE domain

HOHSEnz

OH

OO

OH

S

N

O

TE domain

Cyclorelease

Boddy, C.N.; Scheider, T.L.; Hotta, K.; Walsh, C.T.; Khosla, C. J. Am. Chem. Soc. 2003, 125, 3428Crystal Structure of Epo D and Epo B bound Cytochrome P450EpoK : Nagano, S.; Huiyang, L.; Shimizu, H.; Nishida,

Ogura, H.; Ortiz de Montellan, P.R.; Poulos, T.L. JBC Papers in Press, 2003.

18

Cloning and Heterologous Expression

The epothilone gene cluster has been expressed in Streptomyces coelicolor CH999. Doubling time of 2h vs. 16h

Expression in Myxococcus xanthus 1:10 Epo A: Epo B “Large Scale”

Reengineering of the biosynthetic pathway can lead to novel epothilones

Arslanian, R.L.; Parker, C.D.; Wang, P.K.; McIntire, J.R.; Lau, J.; Starks, C.; Licari, P.J. J. Nat. Prod. 2002, 65, 570.

O’Connor, S.E.; Walsh, C.T.; Liu, F. Angew. Chem. Int. Ed. 2003, 42, 3917.

Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Kholsa, C.; Julien, B. Science, 2000, 287, 640

Initial Synthetic Efforts

Höfle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H. Angew. Chem. Int. Ed. 1996, 35, 1567.

20

The Race is On

EpoA Oct. 17, 1996 EpoA Nov. 25, 1996 EpoA Dec 28, 1996

21

Retrosyntheses

OH

OOHO

O

S

NOH

OOHO

O

S

N

O

Epothilone C

Epothilone A

RCM (Nicolaou, Schinzer, Danishefsky)

Macrolactonization(Danishefsky, Nicolaou) Macroaldolization

(Danishefsky)

All chose a late-stage epoxidation Key issues: E / Z isomers, stereochemistry,

epoxidation selectivity

22

Danishefsky’s Macroaldolization

Balog, A.; Meng, D.; Kamenecka, T.; Bertinato, P.; Su, D.-S., Sorensen, E.J.; Danishefsky, S.J. Angew. Chem. Int. Ed. 1996, 35, 2801

OTBS

OTPS

(MeO)2HC

O

O

S

N

I 9-BBN, then

PdCl2(dppf)2,CsCO3, Ph3As

71%

+O

S

NOTBS

OTPS

OTBS

OTPSOHO

O

S

N

O OMe

MeO

1. pTSOH, dioxane, H2O

2. KHMDS, -78°C 0°C Quench 51%

OH

OOHO

O

S

N

Epothilone C

-50°C, 45%

6 : 13S:3R

OH

OOHO

O

S

N

O

Epothilone A

20:1O O

CH3H3C

4 steps73% yield

23

Nicolaou’s Macrolactonization

Nicolaou, K.C.; Sarabia, F.; Ninkovic, S.; Yang, Z. Angew. Chem. Int. Ed. 1997, 36, 525.Nicolaou, K.C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M.R.V.; Yang, Z. J.Am. Chem. Soc. 1997,

119, 7974.

OH

OTBSO

OTBS

S

N

OTBS

S

N

OTBS

9:1 Z : E1. CSA2. SO3•Py3. LDA,

OTBSO

HOOC

O

HO

1. TBS-OTf2. K2CO3 / MeOH3. TBAF 79% (3 steps)

31% 6R, 7S

30% 6S, 7R

+

6

7

OTBS

OOTBSO

O

S

N

1. TFA 92%

O O

CF3

2.

75%

OH

OOHO

O

S

N

O5:1

(Wittig)

Epothilone A

O

Cl

Cl

Cl Cl

4.

NEt3, DMAP 90%

24

Schinzer’s RCM

Schinzer, D.; Limberg, A.; Bauer, A.; Bohm, O.M.; Cordes, M. Angew. Chem. Int. Ed. 1997, 36, 523.

OH

OOHO

O

S

N

O

O

HO

OTBS O OTBS

Aldolsingle isomer70%

OH

S

N

DCC, DMAP

80%

(resolution)

36

7

OTBS

OOTBSO

O

S

N

RuPh

DCM, RT, 12 hr

OTBS

OOTBSO

O

S

N

Z : E1 : 1

94%

1. HF, MeCN, Et2O 65%

2. DMDO, DCM, -35°C 48%

5:1

Cl

ClPCy3

PCy3

25

Nicolaou’s RCM

Yang, Z.; He, Y.; Vourlumis, D.; Vallberg, H.; Nicolaou, K.C. Angew. Chem. Int. Ed. 1997, 36, 166.Nicolaou, K.C.; He., Y.; Vourloumis, D.; Vallberg, H.; Roschanger, F.; Sarabia, F.; Ninkovic, S.; Yang, Z.; Trujillo, J.I. J. Am.

Chem. Soc. 1997, 119, 7960.

OH

OOHO

O

S

N

O

O

HO

OTBS O OH

Aldol (2:1)(6R, 7S : 6S, 7R) OH

S

N

DCC, DMAP

80%

36

7

OH

OOTBSO

O

S

N

DCM, RT, 12 hr

OH

OOTBSO

O

S

N

Z : E1.4 : 1 85%

1. TFA 98%

2. mCPBA, PhH, 0°C 55%

3:1

Brown Reagent74%

20% epoxide

RuPh

Cl

ClPCy3

PCy3

26

Danishefsky’s RCM

OTPSO OTBS

O

S

N

OTi / BinolSn Allylation95% ee

KHMDS, 65%

OTBS

O

O

S

N

OH OTPS

1:1 Dess-Martin, NaBH4

( OH to OH

Recycle:

Benzene

OTBS

OTPSOHO

O

S

NEpothilone A

86%

Z : E1 : 3

RuPh

Cl

ClPCy3

PCy3

Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc. 1997, 119, 10073Meng, D.; Su, D.-S.; Balog, A.; Bertinato, P.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. J. Am.

Chem. Soc. 1997, 119, 2733.

27

Substituent Effects on RCM

Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc. 1997, 119, 10073Meng, D.; Su, D.-S.; Balog, A.; Bertinato, P.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. J. Am.

Chem. Soc. 1997, 119, 2733.

OR

O

O

S

N

Y X

Benzene

OR

O

O

S

N

Y X

RuPh

Cl

ClPCy3

PCy3

28

Overall # of Steps / Yields

29

Summary of Initial Synthesesof Epothilone A Remarkably short period of time from

Höfle’s crystal structure to first syntheses Numerous synthetic challenges were

identified: Z:E selectivity about C12-C13

RCM problematic Epoxidation yields / selectivity moderate Stereochemical outcome of aldol reactions

highly dependent on substrate

Selected Total Syntheses

31

More Epothilone Syntheses

Epo B Danishefsky (1997) Subsequent syntheses by Nicolaou,

Schinzer, Grieco, Mulzer, White, Sinha- Lerner, Panek, Shibasaki, Carreira, Ley,

Taylor OH

OOHO

O

S

N

O

Epothilone B

32

Mulzer’s “Early Epoxide” Route

Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000, 39, 581.

S

N

O

CO2Me

OTBSO

OEt

OPMB

12 steps,67% overallyield

1. DIBAL-H, -80°C 93%

2. n-BuLi 0°-RT 92%

PO

EtO

EtOO

NS

O O

S

N

O

OTBS O

X*

L-Selectride-78°C to -60°C,

then HMPA, MeI-78°C to RT. 78%

S

N

O

OTBS O

X*

1. TBAF, RT2. TES-Cl, Et3N 85% (2 step)

3. DIBAL-H -95° to -80°C 93%

S

N

O

OTES O

98:2

33

Mulzer’s “Early Epoxide” Route


S

N

O

OTES O

OH

OOHO

O

S

N

O

Epothilone B

LDA, -78°C, 92%

TBSO OS

N

O

OTES

OH

TBSOO

95:5 ds

1. TrocCl, py 94%2. OsO4, NMO3. NaIO44. HF•py

5. NaClO2, NaH2PO42,2 dimethyl-2-butene63% over 4 steps

S

N

O

OH

OTroc

TBSOO

O

HO

1. 2,4,6 trichlorobenzoylchloride, Et3N 65%

2. Zn, NH4Cl, 80°C3. HF•py, py, 30°C 7 days62% for 2 steps

34

Epoxide Stability

Reductive DIBAL-H (neutral) L-Selectride (ionic) Zn (metallic)

Oxidative OsO4 / NaIO4

NaOCl2

Basic TBAF DMAP LDA Enolates

Electrophilic Acyl Chloride


35

Ley’s Resin Route to Epo C

OH

OOHO

O

S

N

Epothilone C

Wittig

aldol

Yamaguchi

Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed. 2003, 42, 2521.

36

Ley - Fragment A


HO

TBS-Cl

DMAP

CH2Cl2, RT, 96%

TBSO

O

2. O3

PPh2

-78°C to RTCH2Cl2, 93%

1.

PhTsN BH

O

O

OTMS

OMe

NOH

OH

CH2Cl2 -93°C to -78°C92%

TBSO

OH O

OMe

>92% ee

1. TBS-OTf

NEt2

100%CH2Cl2, 0°C to RT

2. TMSCH2Li

CO2H

100%

TBSO

TBSO OLDA, MeI

CO2HTBSO

TBSO O

Fragment A-78°C to -15°CTHF 94%

37

Ley – Fragment B


Br OH

O

SO3H

100%

1.

2. NaI, 2-butanone,75°C, "SiO2 filter"96%

I OTHP

CuI,

MgBr

CO2H

THF, -10°C to 0°C

NNH2

NH2

97%

OTHP

1. MeOH,

SO3H, RT, 97%

2.

N•HCrO3Cl

CH2Cl2, RT, 80%

O

Fragment B

38

Ley – Fragment C


CO2H, 98%

O

OH

O

1. TBS-Cl

DMAP

CH2Cl2, RT, 97%

2. MeLi, THF -78°C O

OTBS

OH

TBS-Cl

DMAP

CH2Cl2, RT, 98%

O

OTBS

OTBS

S

N Cl•HCl

NEt3 NaCO31.

MeOH, RT, 98%

2. P(OEt)3160°C, 84%

S

N P

O

OEt

OEt

nBuLi, THF/ Hex-78°C

+

CO2H, quant.

O

H

S

N

OTBS

OTBS

CSA, 1:1CH2Cl2:MeOH

NEt3 NaCO3

quant.

S

N

OTBS

OH

I2, PPh2

Imidazole, MeCN

NEt2

S

N

OTBS

I 73%

PPh2

PhMe, 90°C

S

N

OTBS

PPh2I

Fragment C

D

D

39

Ley - Convergence


O

TBSO

TBSO OLDA, THF, -78°C to -40°Cthen AcOH, then

NH

NH2

100%, 13.5:1

+A

BOOTBS

TBSO

OH

1. TBS-OTf, CH2Cl2,RT NEt2

99%

2. O3, DCM, -78°C

PPh2 100%OOTBS

TBSO

OTBS

S

N

OTBS

PPh2I

Fragment C

1. NaHMDS,-78°C, then C93%

2. CSA,

NEt3 NaCO3

OOTBS

OTBS

O

S

N

OH

TBSO

99%1. TPAP, NMOCH2Cl2, 93%

2.

NMe3 ClO2-

RT 99%

3. TBAF, RT, 95%

O

HO2C

OTBS

OTBS

S

NOH

1.

O

Cl

Cl

Cl Cl

DMAP

SO3H,

then, NH3 / MeOH81% (2 steps)

Epo C

COLUMN

40

Ley’s Resin Route

29 total steps 17 step longest linear sequence from

commercially available materials 1 column Most complex natural product built by

these techniques

41

Danishefsky’s “Scalable” Synthesis

Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett. 2000, 2, 1633.

Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc. 2001,

123, 5249.

OH

OOHO

O

S

N

O

OH

OOHO

O

S

N

Epothilone B Epothilone D

O

OO

OTBS

S

N

I

O

OtBu

Suzuki

Yamaguchi

C

Aldol

Noyori

A

B

36

7

1213

815

42

Subunit A

OTBS

S

N

I1213

15

O

I-9-BBN,

NaOH 65%

O

I

TMSI, HMDS

OTMS

I

OsO4 1%

AD-Mix MeSO2NH2

55% two steps

O

I

HO TES-Cl

Imid. 85%

O

I

TESO

O

Cl ClS

NH2

1. Acetone

2. ZnCl2, MeOH, 60%

+

N

S

Cl

HOPPh3Cs2CO3

Bu4NI,CH2Cl297%

N

S

P

O

PhPh

n-BuLi98%

A



123, 5249.

43

Subunit B



123, 5249.

B

HO

SAE

98%,82% ee

HOO

NaCNBH3

BF3•Et2O

52%

HO

OH

NaIO4

81%

O

44

Subunit C / BC coupling



123, 5249.

C

tBuO

OO

NaH, nBuLi

O

Cl

71%OO O

tBuO TMSCHN2

iPr2NEt 74% OO OCH3

O

B

LDA

60%

OO OCH3

OH1. Troc-Cl, py

2. pTsOH

OO O

OTroc

BC

tBuO

tBuOtBuO

~6:1 ds83%, 2 steps

45

Final Convergence

OO O

OtBu

OTroc

BC

OTES

S

N

I

A

1. 9-BBN

CsCO3,

Pd(dppf)2Cl2 Ph3As

OO

OTroc

OH

S

N

tBuCO2

OOH

OTroc

OH

S

N

tBuCO2

[RuCl2((R)-BINAP)][NEt3] HClH2 (1200 psi)(88%, 95:5 dr)

2. HCl/MeOH 85% (2 steps)

1. TES-OTf

2. HCl / MeOH 77% 2 steps

3. Yamaguchi 78%

OTroc

OOTESO

O

S

N



123, 5249.

46

Completion of Epo D

23 – 25 total steps 16 (13) step longest linear sequence 6.2% (17%) overall yield



123, 5249.

OTroc

OOTESO

O

S

N

1.SmI2, NiI2 95%

2. HF•py 98%

OH

OOHO

O

S

N

Epothilone D

Epothilone B in vivo

48

Epothilone B in vivo (MSK)

Found to be toxic in non-tumor-bearing nude mouse models 0.6 mg/kg/day x 4 given i.p. resulted in 8/8

deaths (days 5-7)

MX-1

xeno

graft

Chou, T.C.; Zhang, X.-G.; Balog, A.; Shu, D.-S.; Meng, D.; Savin, K.A.; Bertino, J.R.; Danishefsky, S.J. Proc. Nat. Acad. Sci. USA 1998 95, 9642.

49

Epothilone B in vivo (Novartis)

Significant tumor regression in Pgp MDR

cells (HCT-15, KB-8511) relatively narrow therapeutic window

t½≈ 40 min in mouse plasma stability in human plasma is much greater

Altmann, K.H., Wartmann, KM.; O’Reilly, T. Biochem. Biophys. Acta. 2000, 1470, M79

Structure-Activity

Relationships

OH

OOHO

O

S

N

O

Epothilone B

1 2 34

5 678

910111213

14151617

A

B

C

D

By 1998, >300 epothilone analogues had been made None were more active in vitro than Epo B

52

Region A SAR

Ring size important (14-18 membered rings evaluated)

6S, 7R stereochemistry crucial 8,8 dimethyl or 8-desmethyl not tolerated 9,10 unsaturation leads to

increased activityOH

678

10

( )n

OH

OOHO

O

S

N

O

References 2,4,5,6, 36-44

53

Region B SAR

C12 substituent important (Me, Et, Pr, Hex, CF3, CN tolerated)

Epoxide not essential replaceable with episulfide, alkene,

cyclopropane, aziridine C12-C13 geometry not important

However, C12-C13 hydrogenation leads to complete loss of activity

C15 stereochemistry importantO

O12

13

1415

OH

OOHO

O

S

N

O


54

Region C SAR

Thiazole, oxazole, pyridyl tolerated Nitrogen location essential

C16-C17 unsaturation important Must be E

C16 methyl group can be removed C26 hydroxyl, primary amine

tolerated

(small groups only)

OH

OOHO

O

S

N

O

S

N1617

1819

26

20


55

Region D SAR

C3 stereochemistry crucial C2-C3 E olefin tolerated

C5 ketone important C4 gem-dimethyl can be replaced

with cyclopropyl

OOHO

O 12 3

45

OH

OOHO

O

S

N

O


Current Generation

Epothilone Analogues

57

Nicolaou’s Latest

OH

OOHO

O

S

NH3CS

Nicolaou, K.C.; Sasmal, P.K.; Rassias, G.; Reddy, M.K.; Altmann, K.H.; Wartmann, M.; O’Brien, A.; Giannakakou, P. Angew.

Chem. Int. Ed. 2003, 42, 3515.

IC50 [nM]

58

(E)-9,10-dehydroEpoB

~ 3 fold more potent than EpoB in vitro Significant in vivo growth inhibition @

0.4 mg/kg

OH

OOHO

O

S

N

O

Yosimura, F.; Rivkin, A.; Gabarda, A.E.; Chou, T.C.; Dong, H.; Sukenik, G.; Morel, F.F.; Talor, R.E.; Danishefsky, S.J.

Angew. Chem. Int. Ed. 2003, 42, 2518.

59

(E)-9,10-dehydroEpoB IC50 [nM]

Yosimura, F.; Rivkin, A.; Gabarda, A.E.; Chou, T.C.; Dong, H.; Sukenik, G.; Morel, F.F.; Talor, R.E.; Danishefsky, S.J.

Angew. Chem. Int. Ed. 2003, 42, 2518.

OH

OOHO

O

S

N

O

60

Epo D (dEpoB, KOS 862)

Advanced early on by MSK group as

as alternative to Epo B ~10 fold less active in vitro than Epo B Potentially less toxic / broader

therapeutic index Presently in Phase II OH

OOHO

O

S

N

Epothilone D

61

Epo D (dEpoB, KOS 862)

Epo D causes significant regression in MX-1

xenograft mice

Curative in 5/5 mice at 30 mg/kg QD2d x 6

Chou, T.C.; Zhang, X.-G.; Harris, C.F., Kuduk, S.D.; Balog, A. Savin, K.A.; Bertino, J.R.; Danishefsky, S.J. Proc. Natl. Acad. Sci. USA 1998 95, 15798.

Chou, T.C.; Zhang, X.-G.; Balog, A.; Shu, D.-S.; Meng, D.; Savin, K.A.; Bertino, J.R.; Danishefsky, S.J. Proc. Natl. Acad. Sci. USA 1998 95, 9642.

62

BMS-247550

OH

OOHO

O

S

N

O

10% Pd(PPh3)4

NaN3, THF-H2O20 min

OH

OOH

O

O

S

N

O

Pd

-N3

OH

OOHO

HO

S

N

O

N3

1. PMe3

2. EDCI-HOBT

OH

OOHO

HN

S

N

O

15-Aza-Epothilone BBMS-247550

25% overall yield"one-pot"

Borzilleri, R.M.; Zheng, X.; Schmidt, R.J.; Johnson, J.A.; Kim, S.-H.; DiMarco, J.D.; Fairchild, C.R.; Gougoutas, J.Z.;

Lee, F.Y.F.; Long, B.H.; Vite, G.D. J. Am. Chem. Soc. 2000, 122, 8890.

63

BMS-247550

IC50 values in [nM]

BMS-247550 is considerably more stable to

esterases in mouse plasma The amide linkage makes BMS-247550 a substrate

for the P-glycoprotein efflux pump Curative in > 50% of HCT-116 mice Significant oral bioavailability in mouse models

Lee, F.Y.F.; Borzilleri, R. Fairchild, C.R.; Kim, S.-H.; Long, B.H.; Reventos-Suarez, C.; Vite, G.D.; Rose, W.C., Kramer, R.A.

Clin. Cancer Res. 2001, 7, 1429.

Lin, N.; Brakora, K.; Seiden, M. Curr. Opin. Invest. Drugs. 2003, 4, 746.

64

BMS-310705

Hofle, G.; Glaser, N.l Miffe, M.; Hecht, H.J.; Sasse, F.; Reichenbach, H. Angew. Chem. Int. Ed. 1999, 38, 1971.

OH

OOHO

O

S

N

O

Epothilone B

mCPBA

55%

OH

OOHO

O

S

N

O

O MeOH, NH3

OH

OOHO

O

S

N

O

HO

85%

O

F3C O

O

CF3

Epothilone F

1. DPPA, DBU, THF 94%

2. PMe3, THF-H2O 91%

OH

OOHO

O

S

N

O

H2N

BMS-310705

65

BMS-310705

Stable to esterases (t½ = 8.1 hr in PLE

vs Epo B t½ = 1.2 hr)

Similar activity in vivo to BMS-247550 Improved water solubility (1 mg/mL) Currently in Phase I trials

OH

OOHO

O

S

N

O

H2N

BMS-310705

Hofle, G.; Glaser, N.; Miffe, M.; Hecht, H.J.; Sasse, F.; Reichenbach, H. Angew. Chem. Int. Ed. 1999, 38, 1971.

Epothilones in the Clinic

67

Epothilones in the Clinic

OH

OOHO

O

S

N

O

Epothilone B (EPO906)

OH

OOHO

O

S

N

Epothilone D (KOS 862)

OH

OOHO

HN

S

N

O

15-Aza-Epothilone BBMS-247550

OH

OOHO

O

S

N

O

H2N

BMS-310705

Novartis (BMS / NCI)

Kosan Biosciences / MSK

68

BMS-247550

Phase II study of 49 women with metastatic breast cancer Prior taxane based therapy 12% partial response 39% stable disease

Phase II study of 77 patients with

non small cell lung cancer 13.1% objective response

Lin, N.; Brakora, K.; Seiden, M. Curr. Opin. Invest. Drugs. 2003, 4, 746.Thomas, E.; Taberno, J.; Fornier, P.; Fumoleau,P.; Lluch, A.; Viens, P.; Vahdat, P. Proc. Am. Soc. Clin. Onc. 2003, 22, 8.

Bristol-Meyers-Squibb Press Release, June 2, 2003.

69

BMS-247550 Phase II study of 61 women with metastatic

breast cancer Prior anthracycline based therapy 44% partial response 34% stable disease

Phase II study of 12 men with progressive metastatic prostate cancer Combination therapy w/ estramustine 50% PSA decline in 92% of patients 1 complete response, 3 partial, 5 SD

Smaletz, O.; Galsky, M.; Scher, H.I.; DeLaCruz, A.; Slovin, S.F.; Morris, M.J.; Solit, D.B.; Davar, U.; Schwartz, L.; Kelly, W.K.Annals of Oncology, 2003, 14, 1518.

Borzilleri, R.M.; Vite, G.D. Drugs of the Future 2002, 27, 1149

70

Summary

The epothilones are a class of microtubule stabilizing compounds Similar to Taxol Complementary to Taxol

Extensive multidisciplinary research has greatly advanced this class of compounds towards becoming a viable cancer treatment

71

Acknowledgements

Andy Hawk, Keunho Kim, Amy Lee, Eric Voight, John Campbell, Bill Lambert, Val Keller, Chris Marvin, Greg Hanson, Laura Luther

Susie Przybylinski, Jack Sadowsky, Jason Pontrello, Laura Wysocki, Andrew Dilger

Dr. Ilia Guzei

Documents

1 The Epothilones Brian Lucas October 16, 2003 2 Epothiwhat? Epoxide, thiazole, ketone = epothilone Epothilone A, R = H Epothilone B, R = CH 3