The Epothilones

O R S N O O OH O Brian Lucas October 16, 2003 OH 1

Epothiwhat?

O R S N OH O O OH O Epo xide, thi azo l e, ket one = epothilone Epothilone A, R = H Epothilone B, R = CH 3 2

Overview

S N  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 O O O OH O Epothilone B OH O NH O OH O Taxol AcO O OH OH O H O AcO O 3

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 S Taxol O O O OH NH O AcO OH N O O OH O O OH O OH H O AcO O Epothilone B Taxol 4

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 5

Discovery

 First isolated from the common soil bacteria

Sorangium cellulosum

as early as 1987 by H öfle and Reichenbach (GBF)  Exhibited a narrow antifungal spectrum (against

Mucor hiemalis

only)  Found to be too toxic for use as an antifungal H öfle, 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.

6

Cytotoxicity

 Bollag (Merck) discovered taxol-like cytotoxicity  Epo A and B were competitive inhibitors of taxol binding  IC 50 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.

7

Comparison of IC

50

Values [nM]

8  The epothilones retain activity in taxol-resistant cancer cell lines 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

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.

9

Epothilone B / Taxol Mode I

AcO O OH NH O OH O O X 13 16 1 2 OH 6.95

H O AcO O O Carbon: Taxol , Epothilone B Nitrogen, Oxygen O 5 OH X 7 OH O 1 6.93

O 13 12 O S N 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 10

Epothilone B / Taxol Mode II

O NH OH O O 13 OAc O 16 1 HO BzO X H O 6.9

5 O OH O O 5 7 CH 3 OH 3 HO O X 1 O 6.9

3 13 O 12 N S Carbon: Taxol , Epothilone B Nitrogen, Oxygen 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 11

Formation of Microtubules

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

37

, 2014.

Stabilized Microtubules Block Mitosis

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

37

, 2014.

Epothilone Biosynthesis

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 15

Epothilone Gene Cluster

S B: O HS O NH S S O B: N H OH [O] S O S N Walsh, C.T.; O’Connor, S.E.; Schneider, T.L. J. Ind. Microbiol. Biotechnol. 2003 ,

30

, 448.

16

Post Assembly Line Epoxidation

S N Enz OH S O S N O O O OH O Epothilone C OH HO HSEnz TE domain OH S N S N OH O O TE domain O O O OH O Epothilone A OH O OH S N Cyclorelease O O OH O Epothilone D P450 Monooxygenase OH S N O O O OH O Epothilone B OH Boddy, C.N.; Scheider, T.L.; Hotta, K.; Walsh, C.T.; Khosla, C. J. Am. Chem. Soc. 2003 ,

125

, 3428 Crystal 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. 17

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 Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Kholsa, C.; Julien, B.

Science

,

2000

,

287

, 640 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.

18

Initial Synthetic Efforts

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

35

, 1567.

The Race is On

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

Retrosyntheses

S N O O O OH O Epothilone A OH RCM (Nicolaou, Schinzer, Danishefsky) S N O Macrolactonization (Danishefsky, Nicolaou) O OH O OH (Danishefsky) Epothilone C Macroaldolization  All chose a late-stage epoxidation  Key issues: E / Z isomers, stereochemistry, epoxidation selectivity 21

22

Danishefsky’s Macroaldolization

S N O O I

+

(MeO) 2 HC OTPS OTBS 9-BBN, then PdCl 2 (dppf) 2 , CsCO 3, Ph 3 As

71%

S N O O MeO OMe OTPS OTBS 1. pTSOH, dioxane, H 2 O 2. KHMDS, -78°C 0°C Quench

51%

S N O

20:1

 O O OH O Epothilone A OH O O H 3 C CH 3 -50°C,

45%

S N O O OH O Epothilone C OH

4 steps 73% yield

S N OTBS O O OH

6 : 1 3S:3R

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

35

, 2801

Nicolaou’s Macrolactonization

S (Wittig) 9:1 Z : E 1. CSA 2. SO 3 •Py 3. LDA, S N OTBS OTBS HOOC TBSO N O OTBS HO O TBSO 1. TBS-OTf 2. K 2 CO 3 / MeOH 3. TBAF

79% (3 steps)

4.

6 O Cl 7 OH

31%

6

R

, 7

S

+

30%

6

S

, 7

R

O Cl 5:1  Cl Cl NEt 3 , DMAP

90%

O S S OH 1. TFA

92%

N OTBS N O 2.

O O O O OH O Epothilone A CF 3

75%

O OTBS O 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.

23

Schinzer’s RCM

HO O 3 OTBS O 6 7 OTBS (resolution) Aldol single isomer 70% DCC, DMAP S N

80%

OH S N S N O

5:1

 O O OH O OH 1. HF, MeCN, Et 2 O

65%

S N 2. DMDO, DCM, -35°C

48%

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

36

, 523.

OTBS O O Z : E 1 : 1 OTBS O PCy 3 Ru Cl Ph Cl PCy 3 DCM, RT, 12 hr

94%

OTBS O O OTBS O 24

Nicolaou’s RCM

HO O 3 OTBS O 6 7 OH Brown Reagent

74%

DCC, DMAP Aldol (2:1) (6

R

, 7

S

: 6

S

, 7

R)

S N 20% epoxide O S 3:1  N O O OH O OH

80%

OH 1. TFA 98% 2.

m

CPBA, PhH, 0°C 55

%

S N S N OH O O Z : E 1.4 : 1 OTBS O PCy 3 Ru Cl Ph Cl PCy 3 DCM, RT, 12 hr

85%

O O OTBS O OH 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. 25

26

Danishefsky’s RCM

S S OTBS N O TPSO OTBS N O O KHMDS,

65%

Ti / Binol Sn Allylation 95% ee O O OH OTPS 1:1  Recycle: Dess-Martin, NaBH 4 (  OH to  OH  Z : E 1 : 3 PCy 3 Ru Cl Cl PCy 3 Benzene Ph

86%

S Epothilone A N OTBS O O OH OTPS Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc. 1997 ,

119

, 10073 Meng, 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.

S N

Substituent Effects on RCM

OR

Cl PCy 3 Ru Cl PCy 3 Ph S N O O Benzene O

Y

O

Y X X OR

27 Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc. 1997 ,

119

, 10073 Meng, 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.

Overall # of Steps / Yields

28

Summary of Initial Syntheses of Epothilone A

 Remarkably short period of time from H öfle’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 29

Selected Total Syntheses

More Epothilone Syntheses

 Epo B Danishefsky (1997)  Subsequent syntheses by Nicolaou, Schinzer, Grieco, Mulzer, White, Sinha Lerner, Panek, Shibasaki, Carreira, Ley, Taylor S O OH N O O OH O Epothilone B 31

Mulzer’s “Early Epoxide” Route

OPMB OEt O S N 12 steps,

67%

overall yield O OTBS S N O OTBS L-Selectride -78°C to -60°C, O X* then HMPA, MeI -78°C to RT.

78%

CO 2 Me 1. DIBAL-H, -80°C

93%

2. n-BuLi 0°-RT

92%

EtO EtO P O O N S O O O S N OTBS 98:2 O X* 1. TBAF, RT 2. TES-Cl, Et 3 N

85%

(2 step) 3. DIBAL-H -95° to -80°C

93%

S N O OTES Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000 ,

39

, 581.

O 32

Mulzer’s “Early Epoxide” Route

S N S N O OTES O TBSO O LDA, -78°C,

92%

O S N 1. TrocCl, py

94%

2. OsO 4, NMO 3. NaIO 4 4. HF •py OH OTES 95:5 ds O TBSO 5. NaClO 2 , NaH 2 PO 4 2,2 dimethyl-2-butene

63%

over 4 steps O O O OH O Epothilone B OH 1. 2,4,6 trichloro benzoylchloride, Et 3 N

65%

2. Zn, NH 4 Cl, 80°C 3. HF •py, py, 30°C 7 days

62%

for 2 steps S N O OH HO O TBSO O OTroc Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000 ,

39

, 581.

33

Epoxide Stability

 Reductive  DIBAL-H (neutral)   L-Selectride (ionic) Zn (metallic)  Oxidative   OsO 4 / NaIO 4 NaOCl 2  Basic  TBAF    DMAP LDA Enolates  Electrophilic  Acyl Chloride Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000 ,

39

, 581.

34

Ley’s Resin Route to Epo C

Wittig S N O Yamaguchi O OH O Epothilone C OH aldol Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed. 2003 ,

42

, 2521.

35

Ley - Fragment A

HO TBSO 1.

TBS-Cl DMAP CH 2 Cl 2 , RT,

96%

2. O 3 PPh 2 -78°C to RT CH 2 Cl 2 ,

93%

TBSO TBSO O O O Ph O TsN BH OTMS TBSO OMe OH N OH CH 2 Cl 2 -93°C to -78°C

92%

LDA, MeI TBSO O TBSO CO 2 H -78°C to -15°C THF

94%

Fragment A 1. TBS-OTf OH O NEt 2 CH 2 Cl 2 , 0°C to RT

100% >92% ee

OMe 2. TMSCH 2 Li CO 2 H

100%

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

42

, 2521.

36

Ley – Fragment B

Br 1.

SO 3 H OH O

100%

2. NaI, 2-butanone, 75°C, "SiO 2 filter"

96%

I OTHP CuI, MgBr THF, -10°C to 0°C

97%

CO 2 H N NH 2 NH 2 1. MeOH, SO 3 H, RT,

97%

2.

N •HCrO 3 Cl CH 2 Cl 2 , RT,

80%

O Fragment B Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed. 2003 ,

42

, 2521.

OTHP 37

Ley – Fragment C

O OH O 1. TBS-Cl DMAP CH 2 Cl 2 , RT,

97%

2. MeLi, THF -78°C CO 2 H,

98%

S N •HCl 1.

NEt 3 NaCO 3 MeOH, RT,

98%

Cl 2. P(OEt) 3 160°C,

84%

S N

Fragment C

OTBS O OH TBS-Cl DMAP CH 2 Cl 2 , RT,

98%

O OTBS OTBS + D S N OTBS PPh 2 I D O OEt P OEt PPh 2 PhMe, 90°C S N OTBS I nBuLi, THF/ Hex -78°C O H CO 2 H,

quant.

S N OTBS OTBS CSA, 1:1 CH 2 Cl 2 :MeOH

quant.

I 2 , PPh 2 Imidazole, MeCN NEt 3 NaCO 3 S N OTBS

73%

NEt 2 OH Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed. 2003 ,

42

, 2521.

38

Ley - Convergence

TBSO A TBSO + O B Epo C 1.

Cl O Cl O Cl Cl DMAP LDA, THF, -78°C to -40°C then AcOH, then N H

100%, 13.5:1

NH 2 TBSO SO 3 H, then, NH 3 / MeOH

81%

(2 steps)

COLUMN

S N OH HO 2 C OTBS O OTBS O OH 1. TBS-OTf, CH 2 Cl 2 , RT NEt 2

99%

2. O 3, DCM, -78°C PPh 2

100%

TBSO O OTBS O S N OTBS

93%

OTBS 1. NaHMDS, -78°C, then 2. CSA, C Fragment C NEt 3 NaCO 3 PPh 2 I

99%

1. TPAP, NMO CH 2 Cl 2 ,

93%

OTBS 2.

NMe 3 ClO 2 S N TBSO OTBS OH OTBS O RT

99%

3. TBAF, RT,

95%

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

42

, 2521.

39

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 40

Danishefsky’s “Scalable” Synthesis

O S N O O OH O Epothilone B OH S N OH O O OH O Epothilone D Suzuki Yamaguchi S N A 13 15 OTBS 12 I B 8 O 6 7 Aldol tBu O O 3 O C O Noyori 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.

41

Subunit A

I-9-BBN, O NaOH

65%

I O TMSI, HMDS I OTMS OsO 4 1% AD-Mix  MeSO 2 NH 2

55% two steps

HO I O TES-Cl TESO Imid.

85%

O I Cl O Cl + S NH 2 1. Acetone 2. ZnCl 2 , MeOH, 

60%

S N HOPPh 3 Cs 2 CO 3 Cl Bu 4 NI, CH 2 Cl 2

97%

S N O P Ph Ph n-BuLi

98%

S 13 12 I 15 N A OTBS 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.

42

Subunit B

HO SAE

98%, 82% ee

HO O NaCNBH 3 BF 3 •Et 2 O

52%

HO OH NaIO 4

81%

O B 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.

43

Subunit C / BC coupling

t

BuO O O NaH, nBuLi Cl O

71%

t

BuO O O O TMSCHN 2 iPr 2 NEt

74%

t

BuO O OCH 3 O C LDA O B

60%

t

BuO O O BC O OTroc 1. Troc-Cl, py 2. pTsOH

83%, 2 steps

t

BuO O OCH 3 O OH ~6:1 ds 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.

44

Final Convergence

S N I A OTES O tBu O O BC O OTroc S 1. 9-BBN N CsCO 3, Pd(dppf) 2 Cl 2 Ph 3 As 2. HCl/MeOH

85% (2 steps)

t

OH BuCO 2 [RuCl 2 ((R)-BINAP)][NEt 3 ] O O OTroc HCl H 2 (1200 psi)

(88%, 95:5 dr)

S S 1. TES-OTf N OTroc N OTroc O O OTES O 2. HCl / MeOH

77%

2 steps 3. Yamaguchi

78%

OH

t

BuCO 2 OH O 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.

45

S N

Completion of Epo D

O O OTES O OTroc 1.SmI

2, NiI 2

95%

2. HF •py

98%

S N O O OH O Epothilone D OH  23 – 25 total steps  16 (13) step longest linear sequence  6.2% (17%) overall yield 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.

46

Epothilone B in vivo

Epothilone B in vivo (MSK)

MX-1 xeno graft  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) 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.

48

Epothilone B in vivo (Novartis)

 Significant tumor regression in Pgp MDR cells (HCT-15, KB-8511)   t ½ ≈ 40 min in mouse plasma  relatively narrow therapeutic window stability in human plasma is much greater 49 Altmann, K.H., Wartmann, KM.; O’Reilly, T.

Biochem. Biophys. Acta

.

2000

,

1470

, M79

Structure-Activity Relationships

C S N B O 17 16 13 15 O 14 12 11 10 1 2 3 4 9 8 7 5 6 O OH O

Epothilone B

OH D A  By 1998, >300 epothilone analogues had been made  None were more active in vitro than Epo B

Region A SAR

S N O O O OH O OH  Ring size important (14-18 membered rings evaluated)  6

S

, 7

R

stereochemistry crucial  8,8 dimethyl or 8-desmethyl not tolerated  9,10 unsaturation leads to 10 increased activity ( ) n 8 OH 7 6 52 References 2,4,5,6, 36-44

Region B SAR

S N O O O OH O OH  C12 substituent important (Me, Et, Pr, Hex, CF 3 , 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 13 O 12 C15 stereochemistry important O 15 14 References 2,4,5,6, 36-44 53

Region C SAR

S N O O O OH O OH  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 20 S (small groups only) 26 N 19 18 17 16 References 2,4,5,6, 36-44 54

Region D SAR

S N  C3 stereochemistry crucial  C2-C3 E olefin tolerated  C5 ketone important  C4 gem-dimethyl can be replaced with cyclopropyl O 1 O 2 3 OH 4 5 O References 2,4,5,6, 36-44 O O O OH O OH 55

Current Generation Epothilone Analogues

IC 50 [nM]

Nicolaou’s Latest

H 3 CS S N O O OH O OH 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.

57

(

E

)-9,10-dehydroEpoB

O S N OH O O OH O  ~ 3 fold more potent than EpoB in vitro  Significant in vivo growth inhibition @ 0.4 mg/kg 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.

58

(

E

)-9,10-dehydroEpoB IC

50

[nM]

O S N OH O O OH O 59 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.

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 S N O O OH O Epothilone D OH 60

Epo D (dEpoB, KOS 862)

 Epo D causes significant regression in MX-1 xenograft mice 61  Curative in 5/5 mice at 30 mg/kg QD2d x 6 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.

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.

S N

BMS-247550

O O O OH O OH 10% Pd(PPh 3 ) 4 NaN 3, THF-H 2 O 20 min S N Pd N 3 O O O OH O OH O O S S N OH 1. PMe 3 N HN O OH O 15-Aza-Epothilone B BMS-247550 2. EDCI-HOBT 25% overall yield "one-pot" N 3 HO O OH O 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.

OH 62

BMS-247550

IC 50 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.

63

BMS-310705

S N HO S N O O O OH O Epothilone B OH

m

CPBA

55%

S N O O O O OH O O O OH F 3 C O CF MeOH, NH 3

85%

3 O O O OH Epothilone F O OH 1. DPPA, DBU, THF

94%

2. PMe 3 , THF-H 2 O

91%

H 2 N S N O O O OH O BMS-310705 OH Hofle, G.; Glaser, N.l Miffe, M.; Hecht, H.J.; Sasse, F.; Reichenbach, H. Angew. Chem. Int. Ed. 1999 ,

38

, 1971.

64

BMS-310705

 Stable to esterases (t ½ = 8.1 hr in PLE vs Epo B t ½ = 1.2 hr)  Similar activity in vivo to BMS-247550  Improved water solubility (1 mg/mL)  Currently in Phase I trials S O H 2 N N O O OH O BMS-310705 OH Hofle, G.; Glaser, N.; Miffe, M.; Hecht, H.J.; Sasse, F.; Reichenbach, H. Angew. Chem. Int. Ed. 1999 ,

38

, 1971.

65

Epothilones in the Clinic

Epothilones in the Clinic

S N S N O OH O O OH O Epothilone B (EPO906) Novartis OH H 2 N O O OH O Epothilone D (KOS 862) Kosan Biosciences / MSK S N S N O OH HN O OH O 15-Aza-Epothilone B BMS-247550 (BMS / NCI) O O O OH O BMS-310705 OH 67

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 Thomas, E.; Taberno, J.; Fornier, P.; Fumoleau,P.; Lluch, A.; Viens, P.; Vahdat, P. Proc. Am. Soc. Clin. Onc. 2003 ,

22

, 8.

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

4

, 746.

Bristol-Meyers-Squibb Press Release

, June 2, 2003.

68

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 69

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 70

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 71