-1-

The Chemistry and Biology of Discodermolide

OO

OH

HO

OH

OH O

NH2

O

Yueheng JiangNovember 20, 2003

-2-

OO

OH

HO

OH

OH O

NH2

OOutline

Discovery and Biological Activity

Total Syntheses

Structure-Activity Relationships (SAR)

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DiscoveryIsolated by Gunasekera and co-workers in 1990 from the Caribbean deep-sea sponge (Discodermiadissoluta).0.002% w/w isolation yield (7 mg/434 g of sponge).Found initially to have immunosuppressive and antifungal activities.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.

-4-

Microtubule-stabilizing agents

O

NH2

O OH OH

HO

O

HO

O

AcO

O

O OH

HOO

O

PhOH

NH

O

R

H

H

O

OH

AcOH

BzO

O

OOHO

HO

O

N

S

RH

H

O

OMeO

O

OAc

OH

OHCO2Me O

OH O O

OOH

O

discodermolideR = Ph Taxol (BMS)R = OtBu Taxotere (Aventis)

R = H epothilone AR = Me epothilone B sarcodictyin Aeleutherobin laulimalide

ON

NMe

OON

NMe

O

-5-

Cytotoxicity

Cytotoxic over a variety of cell lines (IC50 3-80 nM)More potent than TaxolCompetitively inhibits the binding of Taxol to tubulinActive 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.

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Mechanism of actionPromote tubulin polymerization in vitroStabilize microtubules against depolymerizationInterfere with Taxol-binding to microtubulesInduce microtubule bundles in cells

Consequences:

Interfere with proper formation of mitotic spindle

Cause arrest of cell cycle

Cell death by apoptosis

-7-

Dynamic Equilibria of Tubulin-Microtubules

Taxol or Discodermolide

Taxol or

Discodermolide

Taxol or Discodermolide

Heterodimer Formation Initiation Polymerzation/Elongation

α-tubulin~50 kD

β-tubulin~50 kD

KD = 10-5

(α.β)

Nucleationcenter

StabilizedNucleation

centers

5 nm

(+) End (more grown)

(-) End (less grown)

Microtubule

Stabilizedmicrotubules

Map's

Mg2+ ; G

TP

Ca2+ ; 0

o C

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

-8-

Microtubule Bundling

Influence of discodermolide on a transformed mouse fibroblast.

Discodermolide induces microtubule bundling (tubulinappears 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.

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Unique Activities

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

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.

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Potential Candidate for Cancer Chemotherapy

Novel structureGreater or comparable efficacy to TaxolPoor substrate for P-glycoprotein (Pgp).Synergistic effect in combination with TaxolGreater 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.

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Taxol (semi-synthesis)Epothilone (fermentation)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)

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Selected Total Synthesis of (+)-Discodermolide

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

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

PatersonNovel approaches

NovartisMeet supply needs for clinical studies by total synthesis

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Structure and conformation of (+) discodermolide

OO

OH

HO

OH

OH O

NH2

O1 13

57

17 19

21

24

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

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

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General Features

O

OH

OH O

NH2

OO

OH

HO

1

724

13* * *

* * ** **

OH* * *

repeating anti, syn-stereotriaddivided into three fragmentsof roughly equal complexity

HOCO2Me

Roche ester

17

Introduction of two Z-alkenes and terminal Z-diene

Fragment Coupling

-15-

Strategy by Scheiber

O

OH

OH O

NH2

OO

OH

HO

O

OTBS

H

O

PhS

OTBS

I

OPiv

O OPMB

TBSO OH

TBSO OH TBSO OH

OHCO2Me

1 5 13

24

Nozaki-Kishi coupling enolate alkylation

71

815

16

24

Still-GennariHWE olefination

Negishi coupling

+ +

Roush crotylation Roush crotylation

Roche ester

(+)-discodermolide 1

AB C

23 2

158

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Strategy by Smith2nd Generation

O

OTBS

OTBS

O

OTBS TBSO

I

O

OH

OH O

NH2

OO

OH

HO

H

O

PMBOI

O O

PMP

OH

NO

PMBO

O

1

9

13

1924

Wittig olefination Negishi coupling

14 15

Yamamotoolefination

19

9

81

Zhao-Wittigolefination

Evans aldolreaction

OHCO2Me

Roche ester

A BC

Evans aldol

+

(+)-discodermolide 1

Common Precursor 2

15

+

-17-

Strategy by Paterson 1st generation

O

OH

OH O

NH2

OO

OH

HO

1

724

13

boron aldol

TBSOO O

HMeO2CTBSO O

O OOBz

PMBO

OArO

PMBOO

BnO

HOCO2Me

+ +

boron aldol

6

lithium aldol

16

9 1617

24

boron aldol

Claisen [3,3]

Nozaki-Hiyama/Peterson elimination

PMB boron aldolA B

C

(+)-discodermolide 1

Roche ester

2 3 4

9

EtO2C OH

ethyl-(S)-lactate

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Synthesis of Stereotriadby Schreiber

B O

O CO2iPr

CO2iPr

B O

O CO2iPr

CO2iPr

TBSO OH TBSO OH

(R,R)-(E)-crotylboronate (S,S)-(Z)-crotylboronateTBSO O

H

2 3

Roche-ester

Roush crotylation

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.

-19-

Synthesis of stereotriadby Smith

CO2MeHO

PMBO CCl3, PPTS

NH

1

2 LAH3 Swern Oxidation

PMBO H

O

N O

Ph

OOn-Bu2BOTf, Et3N

52-55%, 4 steps

N O

O OOH

PMBO

Ph

AlMe3, THF, 98%

N

OOH

PMBOOMeNH(OMe).HCl

Common Precursor 2

3

4

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.

-20-

Synthesis of stereotriadby Paterson

O

BnO

cHex2BClEt3N, Et2O

H

O O

BnO

OB

LL

+ -

LiBH4

OH

BnO

OH2

6 stepsSubunit A

O

PMBO

cHex2BCl, Et3Nmethacrolein

95%, >30:1 dr OPMBO OH

Me4NBH(OAc)3

94%, > 30:1 dr OHPMBO OH3 7 8

6 stepsSubunit B

OOBz

cHex2BCl, Me2NEt> 97% ds

TBSO

OPMBO

TBSOCHO

4 10

OBz6 steps

Subunit C

5 6

86%> 30:1 dr

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.

-21-

Synthesis of trisubstituted (Z)-alkeneSchreiber

TBSO OTBS

KHMDS,(CF3CH2O)2POCHMeCO2Me

TBSO OTBS

CO2Me

Sch-5 Sch-8

O

(Still-Gennari HWE olefination)93%, Z:E >20:1

14

H

OTBSO

PMBO

(Zhao-Wittig olefination)46%, 3 steps, Z:E = 8-17:1 TBSO

PMBO

I

9

Subunit B

1 Ph3PEtI, n-BuLi, 0oC2 0.1 M I2 in THF, -78oC3 NaHMDS, -23oC

4 Add 6, -33oC13 14

Sm-5

Smith

Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405.Chen, J.; Wang, T.; Zhao, K. Tetrahedron Lett. 1994, 35, 2827.

-22-

Synthesis of trisubstituted (Z)-alkenePaterson

OPMBO O

PhSe

1 NaIO4, NaHCO3 MeOH, H2O, rt

2 DBU, xylene, reflux 82%

OO

MePMBO

Me

++

O

MePMBO

MeO 3 steps, 81%

OTBS

PMBO

OAcO

O

PMBO

O

169

9

11 13

9

15

Subunit B

[3, 3]

14

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

-23-

Synthesis of terminal (Z)-dieneSchreiber

TBSO OPMB

I Pd(PPh3)4H2C CHZnBr

80% TBSO OPMB

Sch-6 Sch-7

NaHMDSPh3P+CH2I I-

TBSO OH(Stork-Zhao Wittig olefination)

O75%

OPMBO

OTBS

TrOTBS

O Ph2P Ti(OiPr)4Lithen MeI

76%, 2 steps Z:E = 12:1 OPMBO

OTBS

TrOTBS

Yamamoto Olefination

21 24

9

Sm-8

Sch-4

Sm-7

Smith

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

-24-

Synthesis of terminal (Z)-diene

Paterson

TBSO H

OPMBO

TBSO

OHPMBO

SiMe3

LnCr SiMe3

KH, 98%

Z:E = > 20:1 TBSO

OPMB

11 12

13

Nozaki-Hiyamaallylation

Peterson elimination

24

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.

-25-

Fragment couplingSchreiber

0.011% NiCl2/CrCl2, 65%2:1 dr at C7

O

OTBS

PhS

OTBS

HO OPiv

5 steps

O

OTBS

PhS

OTBS

TBSO Br

LDA (1.1 eq), THF, 0oC65%

O

OTBS

O OPMBPhS

OTBS

TBSO

O

OTBS

H

O

PhS

O OPMB

OTBS

I

OPiv8

15

71

1

15

16 2415

1

2415

7

9

1110

Subunit B

A

(Nozaki-Kishi coupling)16

C

O

OTBS

OPhS

OTBS

TBSO

LiN(SiMe2Ph)2 (5 eq)MeI, 47%

3:1 dr at C16

16

24

12

19

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.

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Fragment couplingSmith

O OTBSO

I

PMP

1 ZnCl2 (1.15 eq.)2 t-BuLi (3.45 eq.), -78oC to rt

TBSO

PMBOI

3 Pd(PPh3)4 (0.05 eq.), 66% O OO

PMPOTBSTBS

8 steps15

1414

19

subunit Csubunit B

6PMBO

OPMBO

OTBS

-IPh3+P

TBS

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

OO

OTBS

OTBS

O

H

(1.05 eq.)

69%Z:E = 24:1

O

OTBS

O OPMBO

OTBS

TBSO

TBS

89

924

10

11

A

8

-27-

Fragment couplingPaterson

PMBO

O O

OTBS

PMBO

OTBS

OH OPMB

HO1 LiTMP, LiBr2 LiAlH4, 62%> 30:1 dr

16

Subunit C

1716

Subunit B16

H

OPMBO

1724

OTBS

O OTBS

NH2

OOTBS

O OTBS

NH2

OOMeO2C

OTBS

O

H

(+)-Ipc2BCl

87%, 5:1 at C77 7

2

24

17

HO

MeO2CTBSO O

61

Subunit A

9 steps

18

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.

-28-

ImprovementSmith

OPMBO

OTBSTBS

9 OPMBO

OTBS

-IPh3+P

TBS9

1 PPh3, I22 PPh3, i-Pr2NEt 12.8 kbar, 6d 82%, 2 steps

12.8 kbar = 12600 atm = 186,000 psi !!

H

OTBSMe

Me

I

HR

HH

OTBSMe

MeH

HH

I -R

+

R

TBSO

R

TBSO

+

OH

OPMBO

OMOMTBS

9 OPMBO

OMOM

-IPh3+P

TBS9

1 PPh3, I22 PPh3, i-Pr2NEt, 100oC 70%, 2 steps

ambient pressureOH

11 11

1111

2nd Generation: ultrahigh pressureUndesired intramolecular cyclization

3rd Generation: improvementReduction of the steric bulk at C11

Smith, A.B. III; Kaufman, M. D.; Beauchamp, T. J.; LaMarche, M. J.; Arimoto, H. Org. Lett. 2003, 5, 4405.

-29-

Paterson 1st generationProblem

OTBS

O OTBS

NH2

OOTBS

O OTBS

NH2

OOMeO2C

OTBS

O

H

(+)-Ipc2BCl

87%, 5:1 at C7

H HO

H Me

RL

Nu

++

7 7

2

24

17

18a: R1 = OH, R2 = H18b: R1 = H, R2 = OH (desired)

Hreagent 18a : 18b yield

c-Hex2BCl 7 : 1 67%(+)-Ipc2BCl 1 : 5 87%

R1R2

MeO2CTBSO O

61

710

10

18

-30-

Paterson 2nd Generation

100% substrate controlO

OH

OH O

NH2

OO

OH

HO

TBSO

O OPMB

H

MeO2CTBSO O

O

H

PMBO

OArO

PMBO

OH

OH O

NH2

O

O

OH

OHPMBO

boron aldol

61

lithium aldol

56 11

1624

24

9

14

1524

16

boron aldol

Still-GennariHWE olefination

1

alkylationA

B

C

common precursor 19

56

+

+

5

reduced overall steps from 42 to 35

-31-

Paterson 2nd Generation

Improvement

OTBS

O O

O

NH2

OTBS

O

B

L

L

+

++

-O

HH

R

cHex2BCl, Et3N

64%, 20:1 dr at C5HO

OTBS

O O

NH2

O

OH

O

TBSMeO2C

RL

616

10

10

19

20

5

8 24

Subunit A

MeO2CTBSO O

H51

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

-32-

Strategy by Novartis

O

OH

OH O

NH2

OO

OH

HO

boron aldol (Paterson)

O O

16

9

14

18

6N

OO

TBS OH

OH O

NH2

O

Mg2+-chelated aldol (novel)

9

14

18MeO2C+

OH

O

9

13

MeO2C

24

18

O

H 19

Zhao-Wittig Olefination(Smith)

Negishi coupling(Smith)

Nozaki-Hiyama/Peterson elimination(Paterson)

OH

PMBO

Roush Crotylation(Schreiber)

O

PMBO H

O

PMBO H

+

CO2MeHO

CO2MeHO

commercially available

Francavilla, C.; Chen, W.; Kinder, F. R. Jr. Org. Lett., 2003, 5, 1233.

-33-

Next Generation ???

10.3%

6%

N.A.21Novartis

23Paterson

2.2%29Marshall

1.4%25Myles

24Smith

4.3%24Schreiber

YieldStepsLongest Linear Sequences

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

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

A549 MG63Discodermolide 1 3.6 6

(R = Me)2 (R = H; 16-demethyl) n.d. 10

Acetylated analogues:3a (3-OAc) 3.83b (7-OAc) 0.83c (3,7-OAc) 0.83d (3,11-OAc) 1643e (3,17-OAc) 524

OO

OH

HO

OH

R

OH O

NH2

O

3

711

1617

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.

-35-

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

OO

R

OH

OH O

NH2

O

3

7OO

OH

HO

OH

OH O

NH2

O14

A5494a (14-cis, demethyl) 7.84b (14-trans, demethyl) 485

A5494c (3-dehydro, R = OH) 1.84d (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.

-36-

Summary

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

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.

-37-

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 KuangLei Chen, Dr. Gopinadhan Anilkumar