-1-
The Chemistry and Biology of Discodermolide
OO
OH
HO
OH
OH O
NH2
O
Yueheng JiangNovember 20, 2003
-2-
Discovery and Biological Activity
Total Syntheses
Structure-Activity Relationships (SAR)
Outline OO
OH
HO
OH
OH O
NH2
O
-3-
Discovery Isolated by Gunasekera and co-workers in 1990
from the Caribbean deep-sea sponge (Discodermia dissoluta).
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
Ph
OH
NH
O
R
H
H
O
OH
AcOH
BzO
O
OOHO
HO
O
N
S
RH
H
O
OMe
O
O
OAc
OH
OHCO2Me O
OH O O
O
OHO
discodermolideR = Ph Taxol (BMS)R = OtBu Taxotere (Aventis)
R = H epothilone AR = Me epothilone B
sarcodictyin Aeleutherobin laulimalide
O
NNMe
OO
NNMe
O
-5-
Cytotoxicity
Cytotoxic over a variety of cell lines (IC50 3-80 nM)
More potent than Taxol Competitively inhibits the binding of Taxol to tubulin 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.
-6-
Mechanism of action Promote tubulin polymerization in vitro Stabilize microtubules against depolymerization Interfere with Taxol-binding to microtubules Induce 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
'sM
g2+ ; G
TPCa2+
;
0oC
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 (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.
-9-
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 structure Greater or comparable efficacy to Taxol Poor substrate for P-glycoprotein (Pgp). Synergistic effect in combination with Taxol 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.
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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)
Taxol (semi-synthesis) Epothilone (fermentation) Discodermolide ( ???)
-12-
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
X-ray structure of discodermolide;hydrogen atoms omitted for clarity
OO
OH
HO
OH
OH O
NH2
O1 13
57
17 19
21
24
Paterson, I.; Florence, G. J. Eur. J. Org. Chem., 2003, 2193.
-14-
General Features
O
OH
OH O
NH2
OO
OH
HO
1
724
13* * *
* * *
* **
OH
* * *
repeating anti, syn-stereotriad
divided 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
OH
CO2Me
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
-16-
Strategy by Smith2nd Generation
O
OTBS
OTBS
O
OTBS TBSO
I
O
OH
OH O
NH2
OO
OH
HO
H
O
PMBO
I
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
OH
CO2Me
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
HMeO2C
TBSO O
O O
OBz
PMBO
OArO
PMBO
O
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
-18-
Synthesis of Stereotriadby Schreiber
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.
B O
OCO2iPr
CO2iPr
B O
OCO2iPr
CO2iPr
TBSO OH TBSO OH
(R,R)-(E)-crotylboronate (S,S)-(Z)-crotylboronate
TBSO O
H
2 3
Roche-ester
Roush crotylation
Subunit A & C Subunit B
-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
PMBOO
MeNH(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.
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Synthesis of stereotriadby Paterson
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.
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
O
OBzcHex2BCl, Me2NEt> 97% ds
TBSO
OPMBO
TBSOCHO
4 10
OBz6 steps
Subunit C
5 6
86%> 30:1 dr
-21-
Synthesis of trisubstituted (Z)-alkene
TBSO OTBS
KHMDS, (CF3CH2O)2POCHMeCO2Me
TBSO OTBS
CO2Me
Sch-5 Sch-8
O
(Still-Gennari HWE olefination)
93%, Z:E >20:114
H
OTBSO
PMBO
(Zhao-Wittig olefination)
46%, 3 steps, Z:E = 8-17:1TBSO
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
Schreiber
Smith
Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405. Chen, J.; Wang, T.; Zhao, K. Tetrahedron Lett. 1994, 35, 2827.
-22-
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
Synthesis of trisubstituted (Z)-alkene
Paterson
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)-diene
Schreiber
TBSO OPMB
IPd(PPh3)4
H2C CHZnBr
80% TBSO OPMB
Sch-6 Sch-7
NaHMDSPh3P+CH2I I-
TBSO OH(Stork-Zhao Wittig olefination)
O
75%
OPMBO
OTBS
TrOTBS
O Ph2P Ti(OiPr)4Lithen MeI
76%, 2 steps Z:E = 12:1 OPMBO
OTBS
TrOTBS
Yamamoto Olefination
2124
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
1112
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
7
1
1
15
16 2415
1
24
15
7
9
11
10
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.
-26-
Fragment couplingSmith
O OTBSO
I
PMP
1 ZnCl2 (1.15 eq.)2 t-BuLi (3.45 eq.), -78oC to rt
TBSO
PMBO
I3 Pd(PPh3)4 (0.05 eq.), 66% O OO
PMPOTBSTBS
8 steps
15
1414
19
subunit C
subunit B6
PMBO
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
9
24
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 B
16
H
OPMBO
17
24
OTBS
O O
TBSNH2
OOTBS
O O
TBSNH2
OOMeO2C
OTBS
O
H
(+)-Ipc2BCl
87%, 5:1 at C77 7
2
24
17
HO
MeO2C
TBSO 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 generation
Problem
OTBS
O O
TBSNH2
OOTBS
O O
TBSNH2
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)
H
reagent 18a : 18b yield
c-Hex2BCl 7 : 1 67%
(+)-Ipc2BCl 1 : 5 87%
R1R2
MeO2C
TBSO O
61
710
10
18
-30-
Paterson 2nd Generation
100% substrate controlO
OH
OH O
NH2
OO
OH
HO
TBSO
O OPMB
H
MeO2C
TBSO O
O
H
PMBO
OArO
PMBO
OH
OH O
NH2
O
O
OH
OHPMBO
boron aldol
6
1
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-
OTBS
O O
O
NH2
O
TBS
O
B
L
L
+
++
-O
HH
R
cHex2BCl, Et3N
64%, 20:1 dr at C5
HO
OTBS
O O
NH2
O
OH
O
TBSMeO2C
RL
6
16
10
10
19
20
5
824
Subunit A
MeO2C
TBSO O
H51
Paterson 2nd Generation
Improvement
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
O
O
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 ???
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 …
-34-
OO
OH
HO
OH
R
OH O
NH2
O
3
711
1617
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
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-
OO
R
OH
OH O
NH2
O
3
7
SAR SummaryAntiproliferative Potencies (IC50, nM) Against A549 Cells of Discodermolide (1), and Analogues 4a-4d.
Martello, L. A.; LaMarche, M. J.; He, L.; Beauchamp, T. J.; Smith, A. B. III; Horwitz, S. B. Chem. Biol. 2001, 8, 843.
OO
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
-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