Complex target-oriented total synthesis in the drug discovery process: The discovery of a highly promising family of second generation epothilones

Article abstract of DOI:10.1021/ja029695p

The total synthesis of a family of (E)-9,10-dehydro derivatives of epothilone D (i.e., 12,13-desoxyepothilone B) is described. The route is particularly concise and amenable to production of new congeners. Furthermore, the chemistry described herein constitutes a major simplification in the total synthesis of EpoD, which is in human clinical trials. This new family of epothilones shows major advantages in terms of their potency and pharmacostability relative to the wild-type saturated analogues in the D series. From the perspective of compound availability through synthesis, potency, and pharmacokinetic properties, these compounds could well warrant advancement to clinical evaluation in humans. Copyright

Full text of DOI:10.1021/ja029695p

Published on Web 02/06/2003
Complex Target-Oriented Total Synthesis in the Drug Discovery Process:
The Discovery of a Highly Promising Family of Second Generation
Epothilones
Alexey Rivkin,† Fumihiko Yoshimura,† Ana E. Gabarda,† Ting-Chao Chou,‡ Huajin Dong,‡
William P. Tong,§ and Samuel J. Danishefsky*,†,|
Laboratory for Bioorganic Chemistry, Preclinical Pharmacology Core Facility, and Analytical Pharmacology Core
Facility, Sloan-Kettering Institute for Cancer Research, 1275 York AVenue, New York, New York 10021, and
Department of Chemistry, Columbia UniVersity, HaVemeyer Hall, New York, New York 10027
Received December 11, 2002 ; E-mail: s-danishefsky@ski.mskcc.org
The emergence of the epothilones as promising new anticancer
agents has led to a worldwide effort to synthesize new analogues,
and to establish their SAR with a view for identifying and
developing later generation agents for clinical evalution.1 Human
clinical trials seeking to assess issues of toxicity, optimal dosage,
and likely efficacy of several epothilones as drugs are well
underway.2 For instance, 12,13-desoxyepothilone B (1) (cf. inter
alia (dEpoB or EpoD)), initially developed in our laboratory via
Figure 1. Structures of epothilones.
total synthesis, is now undergoing human clinical trials following
highly favorable findings in various in vivo animal settings (Figure
1).3
as well as highly encouraging pharmacokinetic properties, rendering
them exciting prospects for advancement to become the next
generation of tubulin directed anticancer drugs.
It might have been thought that given the massive effort which
has already been expended on the epothilones, there is little to be
gained from still another such exploration in search of new
congeners. We show below that this need not be the case. In seeking
to deepen our grasp of the epothilone SAR estate, we initially
undertook the preparation of 12,13-desoxyepothilone analogues
containing a 12-trifluoromethyl group (cf. 2). Our menu of
epothilone synthesis strategies, then in existence, failed to encom-
pass this goal. The problem arose from a breakdown in the
feasibility of ring-forming olefin metathesis in a context where we
were trying to form a C10-C11 olefin with a CF3 group in place at
The synthesis of the RCM precursors commenced with the
preparation of acyl sector 11 (Scheme 1). Ketone 57c was subjected
to an aldol reaction with the readily available aldehyde 6.8 Upon
deprotonation and reaction of “lithio” 5 with 6, smooth condensa-
tion gave rise to aldol product 7, with acceptible diastereo-
selectivity (dr, 5.7:1). Protection of the C7 alcohol as a TBS silyl
ether followed by hydrolysis of the diisopropyl acetal group afforded
keto aldehyde 8, setting the stage for the second aldol reaction.
Following the previously practiced “titano” tert-butyl ester
method,7c with the new aldehyde 8 as the coupling partner, aldol
product 9 was obtained in high diastereoselectivity and high yield
(dr > 20:1). Protection of the C3 alcohol of 9 with a TES silyl
group was followed by deprotection of the benzyl ether. The
remaining straightforward steps to 11 are shown in Scheme 1.
Esterification of the resultant hydroxyketone 129 with the C1-
C9 acid fragment 11 provided the corresponding RCM cyclization
precursor 14 in 81% yield (Scheme 2). The ring-closing metathesis
C
12 (case 1).4 Earlier, we had demonstrated that viability for RCM
is restored when there is a carbon spacer between the CF3 group
and the RCM reaction center (case 2).5 Of course, this resulted in
formation of a 17-membered macrolide. We wondered whether we
could reach the 16-membered ring by moving the acyl side vinyl
group to C8 (see projected case 3).
To explore this new ring-forming strategy (vide infra), we also
targeted (E)-9,10-dehydro-dEpoB, that is, compound 3. At the time
we undertook this effort, it was thought that it was a known
compound, indeed with a disappointing early in vitro screening
profile.6 Hence efforts directed to 3 were viewed solely from the
perspective of it functioning as a synthetic intermediate to validate
the feasibility of RCM in the context of establishing a C9-C10
double bond. Of course, if all went smoothly, selective reduction
of the 9,10-double bond of 3 might constitute an even more efficient
route to 1 than is currently practiced7 (vide infra).
The key findings disclosed herein are as follows. (i) A highly
concise route to reach the synthesis targets has been accomplished;
(ii) contrary to our expectations, compound 3 had actually not been
prepared before; this family of epothilones is new; (iii) moreover,
early indications suggest that several new (E)-9,10-dehydro-dEpoB
congeners exhibit highly promising in vitro and in vivo potencies
† Laboratory for Bioorganic Chemistry.
‡ Preclinical Pharmacology Core Facility.
§ Analytical Pharmacology Core Facility.
| Columbia University.
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J. AM. CHEM. SOC. 2003, 125, 2899-2901
2899

C O M M U N I C A T I O N S
Scheme 1. Synthesis of Acyl Sector 11a
Table 1. In Vitro Cytotoxicities (IC50) with Tumor Cell Linesa
CCRF-CEM(C)
C/VBL100
C/Taxol
compound
(µM)
(µM)
(µM)
1 (dEpoB)
0.0036
0.0041
0.0009
0.0035
0.016
0.080
0.0042
0.0210
0.0046
0.018
0.0012
0.0057
2
3
4
a XTT assay following 72 h inhibition. CCRF-CEM is a human T-cell
acute lymphoblastic leukemia cell line. The CCRF-CEM/VBL100 cell line
is resistant to vinblastine, and CCRF-CEM/Taxol is resistant to taxol.
a Reagents and conditions: (a) LDA, THF, -90 °C (78% based on
aldehyde); (b) (i) TBSOTf, 2,6-lutidine, CH2Cl2, -40 to -20 °C, 97%, (ii)
p-TsOH‚H2O (cat.), THF-H2O (4:1), 64 °C, 98% (two steps, 95%); (c)
t-butyl acetate, LDA, CpTiCl(OR)2 (R ) 1,2:5,6-di-O-isopropylidene-R-
L-glucofuranos-3-O-yl), Et2O, -78 °C, 86%, (dr > 20:1); (d) (i) TESCl,
imidazole, DMF, 0 °C to room temperature, 98%, (ii) H2, Pd/C (10%),
EtOH, 83%, (iii) TPAP, NMO, CH2Cl2, 95%, (iv) MePPh3I, n-BuLi, THF,
-78 to -5 °C, 78% (four steps, 60%); (e) TESOTf, 2,6-lutidine, CH2Cl2,
0 °C to room temperature.
Scheme 2. Synthesis of Epothilone Analoguesa
Figure 2. Plasma stability of epothilones 1-4 murine plasma.
a Reagents and conditions: (a) EDCI, DMAP, CH2Cl2, 11, 0 °C to room
temperature (81%, starting from 10, two steps); (b) toluene, 110 °C, 20
min, 78%; (c) (i) KHMDS, 16, THF, -78 to -20 °C, 76%, (ii) HF-pyridine,
THF, 97% (two steps, 74%); (d) TrisNHNH2, Et3N, ClCH2CH2Cl, 50 °C,
91%.
Figure 3. Therapeutic effect of (E)-9,10-dehydro-dEpoB (3) in nude mice
bearing HCT-116 xenograft (iv infusion, Q2Dx7, n ) 3). Arrows indicate
drug administrations.
compound that possesses substantially improved cytotoxicity rela-
tive to that of dEpoB (1).
reaction10 of 14 was then carried out in toluene using the recently
described Grubbs catalyst.11 Happily, the reaction afforded exclu-
sively the trans isomer 15 in 78% yield. Installation of the thiazole
moiety via the protocol12 shown in Scheme 2 was followed by
deprotection of the two silyl ethers with HF-pyridine, thereby
leading to 3. The structure of 3 was rigorously corroborated by its
high yielding conversion to 1.9 We note in passing that the total
synthesis of 1 has been very substantially simplified relative to
previously practiced routes. Thus, the use of the readily available
6, obtained from the chiral pool, is certainly a large improvement
relative to reliance on (S)-2-methyl-4-pentenal, whose synthesis
requires intervening chiral auxiliaries. The new strategy was then
successfully applied to the total synthesis of the corresponding
trifluoro analogues 2 and 4.
With compound 3 of rigorously proven structure in hand, we
were surprised to find that its spectral properties were not congruent
with those previously reported for a compound presumed to be the
same entity. The actual structure of the compound previously
assigned as 3 has now been reevaluated and will be disclosed in
short order.13 However, it is clear in retrospect that 3 had not been
previously prepared and, in fact, the whole family of (E)-9,10-
dehydroepothilones reported here is a new genus.
The impressive cell growth inhibition exhibited by epothilones
2-4 across a range of various drug-resistant tumors prompted
determination of the blood plasma stability of these new (E)-9,10
congeners. For instance, the recently described (E)-10,11-dehydro-
dEpoB (of case 1 with a CH3 group at C12) exhibits very poor
plasma stability with respect to lactone opening. It is this plasma
instability which has stifled advancement of (E)-10,11-dehydro-
dEpoB. By contrast, on exposure of 2-4 to murine plasma, we
observed a much slower drug degradation as compared to dEpoB
(1) by a factor of 7. This stability constitutes a substantial advance
from a drug availability perspective relative to dEpoB (Figures 2
and 3).
The combination of the cytotoxicity and plasma stability data
encouraged us to synthesize substantial amounts of 3 to determine
its in vivo efficacy, in nude mice bearing human tumor xenografts.
The direct and high quality total synthesis described aboVe allowed
us to indulge these interests. Happily, epothilone 3 demonstrated a
markedly improved potency in inhibiting the growth of implanted
tumors, relative to dEpoB. The improved potency and plasma
stability allows very substantial reduction of drug dosing (an order
of magnitude) in the context of xenografts of 3.
In summary, delineated above is a powerful stereoselective total
synthesis of 3 and, following site-selective diimide reduction, dEpoB
(1) itself. The described herein strategy was then straightforwardly
applied to the preparation of the corresponding trifluoro analogues
2 and 4. The data reported above in conjunction with the findings
Examination of synthetic analogues (2-4), in cell culture settings,
revealed stronger inhibitory effects on various sensitive and MDR
tumor cell lines than are exhibited by our clinical entry dEpoB (1)
(Table 1). We note that 3 is the first 12,13-desoxyepothilone
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C O M M U N I C A T I O N S
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to be reported in due course point to the emergence of a most
promising new family of anticancer drugs appropriate for further
evaluation en route to the possible advancement to a human clinical
setting. Furthermore, the new synthesis strategy comprises a
significant practical improvement in the total synthesis of dEpoB.
Parenthetically, the study serves to underscore the potential
applicability of target directed total synthesis, even in a multistep
setting, in the quest for new substances of material clinical benefit.
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Acknowledgment. This research was supported by the National
Institutes of Health (grant numbers: S.J.D. (CA-28824), T.-C.C.
(CA-08748)). Postdoctoral fellowship support is gratefully ac-
knowledged by A.R. (NIH Cancer Pharmacology Training Grant,
T32-CA62948) and F.Y. (Uehara Memorial Foundation). The
authors wish to thank Dr. George Sukenick (NMR Core Facility,
Sloan-Kettering Institute, and CA-08748) for mass spectral and
NMR analysis.
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Note Added after ASAP: In the version of this manuscript
published on the Web 2/06/2003, the phrase “Thus, the use of the
readily available 11...” on the second page was incorrect and should
read “Thus, the use of the readily available 6...”. The final version
published 2/10/2003 and the print version are correct.
Supporting Information Available: Experimental procedures and
characterization data for all new compounds (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
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