Cryptophycin affinity labels: Synthesis and biological activity of a benzophenone analogue of cryptophycin-24

Article abstract of DOI:10.1016/S0960-894X(02)01023-5

An efficient synthesis of a C16 side chain benzophenone analogue of cryptophycin-24 using a crotylboration reaction and Heck coupling as key steps is described. In an in vitro tubulin assembly assay, the benzophenone analogue of the β isomer (IC₅₀

Full text of DOI:10.1016/S0960-894X(02)01023-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 757–760
Cryptophycin Affinity Labels: Synthesis and Biological Activity
of a Benzophenone Analogue of Cryptophycin-24
Ramdas Vidya,a MariJean Eggen,a Gunda I. Georga,* and Richard H. Himesb
aDepartment of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, USA
bDepartment of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
Received 8 May 2002; accepted 7 October 2002
Abstract—An efficient synthesis of a C16 side chain benzophenone analogue of cryptophycin-24 using a crotylboration reaction and
Heck coupling as key steps is described. In an in vitro tubulin assembly assay, the benzophenone analogue of the b isomer
(IC50=7.4 mM) is twice as active as cryptophycin-24 (IC50=15 mM).
# 2003 Elsevier Science Ltd. All rights reserved.
The cryptophycins, isolated from blue-green algae
(Nostoc sp.), are a potent tumor-selective class of tubu-
lin-binding antimitotic agents that show excellent activ-
ity against multi-drug resistant (MDR) cancer cell lines
and against mammary derived tumors.1,2 Cryptophycin-1
(1, Fig. 1) is the major cytotoxin in Nostoc sp.3,4 and
displays IC50 values in the pM range. Of special impor-
tance is the reduced susceptibility of the cryptophycins
to P-glycoprotein-mediated multiple drug resistance in
comparison to vinblastine, colchicine, and paclitaxel.5
In vivo studies (human tumor xenografts) with crypto-
phycin-1 demonstrated a remarkable reduction of
tumor burden.4 A structurally related compound cryp-
tophycin-24, (2, Fig. 1, also named arenastatin A), iso-
lated from the Okinawan marine sponge Dysidea
arenaria6 and later from Nostoc sp. strain GSV 224,7 is
also a potent inhibitor of tubulin polymerization.8 The
IC50 for arenastatin A cytotoxicity against KB cells was
5 pg/mL.6,9 A hydrolytically more stable synthetic ana-
logue, cryptophycin-52 (3), was selected for clinical
trials.10À12
It has been demonstrated that cryptophycin-1 inhibits
vinblastine binding to tubulin.13,15À17 Thus, cryptophy-
cin-1 belongs to a growing groupof compounds that
bind to the vinca binding domain on tubulin. However,
due to the structural differences between vinca alkaloids
and cryptophycins it may be that the binding domains
simply overlap.11,17 The possibility of covalent binding
of cryptophycin-1 to tubulin has also been studied and
the results demonstrate that a covalent addition of
cryptophycin to tubulin does not occur.14
Cryptophycins are one of the best recent lead in the
search for anticancer therapies. Although relatively little
is known about the interactions of cryptophycins with
tubulin, it is believed that the cryptophycins may inter-
act in a manner different from those of other tubulin-
binding antimitotic agents. For the development of
these promising compounds into useful chemothera-
peutic agents, detailed information about the binding
domain of the cryptophycins is essential. Therefore, we
planned to prepare a cryptophycin analogue bearing a
The interaction of cryptophycin-1 with tubulin and
microtubules in vitro showed that cryptophycin is an
effective inhibitor of tubulin polymerization at sub-
stoichiometric concentrations.13 Cryptophycin-1 causes
tubulin to aggregate and depolymerizes microtubules
into linear polymers as seen by electron microscopy.13,14
*Corresponding author. Tel.:+1-785-864-4498; fax:+1-785-864-5326;
e-mail: georg@ku.edu
Figure 1. Structures of cryptophycins.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01023-5

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R. Vidya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 757–760
photoaffinity label for tubulin labeling studies. Because
structural changes at the C16 side chain aromatic group
are tolerated without loss of activity,1,2 we targeted this
position for the placement of a photoreactive functionality
to study the cryptophycin binding site on tubulin.
98% yield. Deprotection of the p-methoxybenzyl ether
with DDQ followed by DMP-oxidation of the resulting
alcohol furnished the aldehyde 11 in 82% yield over two
steps. Wittig–Horner olefination of 11 provided the a,b-
unsaturated tert-butyl ester 12 in 95% yield. Heck
coupling37,38 on 12 with 4-bromobenzophenone in the
presence of palladium acetate and sodium bicarbonate
furnished the required ester 13 in 60% yield. Deprotection
of the silyl ether with tetrabutylammonium fluoride led
to the isolation of the desired octadienoate ester 6 in
71% yield.
Formal and total syntheses of the cryptophycins have
been published by several groups.1,18À28 Also, SAR
studies based on naturally occurring cryptophycins and
various synthetic analogues have been reported.2,29À32
Cryptophycin 1 (1) and the structurally less complex
arenastatin A (2, cryptophycin-24) are close structural
analogues (Fig. 1) and have very similar properties with
regard to tubulin binding.8 Therefore, we prepared an
affinity label of cryptophycin-24 for our labeling studies,
because it can be prepared in fewer synthetic steps than
the cryptophycin-1 analogues. The enzymatic/hydrolytic
instability33 of cryptophycin-24 should not pose a
problem during the in vitro tubulin investigations. The
retrosynthetic analysis of a benzophenone analogue of
cryptophycin-24 (4) reveals that the desepoxy analogue
5 can be derived from two main building blocks, the
octadienoate ester 6, and the peptide unit 7 (Scheme 1).
The second key synthon 7 was readily synthesized
starting from a N-Boc protected d-tyrosine deriva-
tive.39,27 Key synthons, octadienoate ester 6 and peptide
fragment 7 were subjected to the Yamaguchi coupling
reaction.40 The acid 7 was activated with 2,4,6-tri-
chlorobenzoyl chloride in the presence of Hunig’s base
and a catalytic amount of DMAP. Addition of the
alcohol 6 to the mixed anhydride afforded the inter-
mediate 14 in 85% yield (Scheme 3). Simultaneous
deprotection of the tert-butyl ester and the N-Boc with
trifluoroacetic acid produced the cyclization precursor
and HBTU activation provided the desired macrocycle
15 in 56% yield. Epoxidation of 15 with m-CPBA or
dimethyl dioxirane (DMD)41 furnished a diastereomeric
mixture of epoxides 4 in the ratios of a:b=2:1 and 1:2,
respectively (Scheme 3).42 The mixture was separated by
HPLC.
We and others have reported an efficient proto-
col26,27,34,35 for the synthesis of octadienoate ester 12
using a crotylboration approach to set both stereo-
centers in a single step.36 This method allows for the
convergent synthesis of analogues modified at the
phenyl group of the C16 side chain using Heck chem-
istry. The key steputilized the crotylboration of
8
The biological testing was carried out individually for
the a & b-isomers of 4. In the tubulin assembly assay,
the benzophenone analogue 4 (b) of cryptophycin-24
(IC50=7.4 mM) was half as active as cryptophycin-1
(IC50=3.7 mM) and twice as active as cryptophycin-24
(IC50=15 mM) (Table 1).32,43 In the cytotoxicity studies,
analogue 4 (b) had reduced activity against the MCF7
(Scheme 2) with crotyl diisopinocampheylborane (pre-
pared from (+)-B-methoxydiisopinocampheyl-borane)
to generate the desired stereochemistry at the two chiral
centers of 9 in 77% yield (91% ee). Silyl protection of
the secondary alcohol 9 with tert-butyldimethylsilyl
chloride and imidazole afforded the silyl ether 10 in
Scheme 1.
Scheme 2.

R. Vidya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 757–760
759
the tubulin assembly assay and is therefore a suitable
candidate for further studies to explore the tubulin
binding domain of cryptophycin. Our plans are to make
this derivative in a radioactive form for photolabeling
studies.
Acknowledgements
We thank the National Institutes of Health (NCI) for
financial support (CA 70369). The Department of the
Army is acknowledged for post-doctoral fellowships
from the Breast Cancer Research Program to M. E. and
R. V. This work was supported in part by the Kansas
Technology Enterprise Corporation through the Cen-
ters of Excellence Program.
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