Total synthesis and anti-tubulin activity of epi-C3 analogues of cryptophycin-24

Article abstract of DOI:10.1021/jm030555f

Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR breast cancer cell lines and in an in vitro tubulin assembly assay. The results demonstrate that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity and the m-Cl substituent present on the C10 side chain did not induce any large change in activity.

Full text of DOI:10.1021/jm030555f

J . Med. Chem. 2004, 47, 3697-3699
3697
Tota l Syn th esis a n d An ti-Tu bu lin Activity of Ep i-C3 An a logu es of
Cr yp top h ycin -24
Suzanne B. Buck,† J acquelyn K. Huff,‡ Richard H. Himes,‡ and Gunda I. Georg*,†
Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Drive, Lawrence, Kansas 66045-7582, and
Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, Kansas 66045-7534
Received November 4, 2003
Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes
were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR
breast cancer cell lines and in an in vitro tubulin assembly assay. The results demonstrate
that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity
and the m-Cl substituent present on the C10 side chain did not induce any large change in
activity.
In tr od u ction
The cryptophycins,1 natural products isolated from
the blue-green algae Nostoc sp. ATCC 53789 and Nostoc
sp. GSV 224, possess antimitotic activity. Cryptophy-
cin-1 (1, Figure 1)2 exhibits potent cytotoxicity in breast
cancer cell lines with IC50 values in the low picomolar
range and maintains its potency in resistant cancer cell
lines that overexpress P-glycoprotein.3 Cryptophycin-
24 (arenastatin A, 2)4 differs from cryptophycin-1 (1)
F igu r e 1. Structures of selected cryptophycins.
in that it lacks both the chloro substituent on the C10
side chain and the methyl group at C6. Cryptophycin-
24 (2) is also cytotoxic at picomolar concentrations.4
The cryptophycins have been shown to inhibit tubulin
polymerization in vitro,5-8 induce microtubule depolym-
erization in cells,3 potently decrease microtubule dy-
namics,9,10 and interact with tubulin to form ring
structures.11,12 The cryptophycins have also been found
to activate the caspase (ICE/CED3) protein cascade13
and induce the phosphorylation of the Bcl-2 family of
proteins at picomolar concentrations.14 Studies have
revealed that the cryptophycins interact at or near the
Vinca binding domain,5-7,15 as well as at the rhizoxin/
maytansine binding site.16
A recent review reported that all of the stereocenters
of cryptophycin-1 (1) were required for optimal biological
activity,17 although to our knowledge, no synthesis of a
C3 epimer of any cryptophycin has been reported. We
are now detailing the synthesis and evaluation of C3-
epi-cryptophycin-24 (5, Figure 2), C3-epi-m-chloroben-
zyl-cryptophycin-24 (6), and the corresponding styrenes
3 and 4. These were tested in vitro for their ability to
(1) inhibit microtubule polymerization in a tubulin
assembly assay and (2) induce cytotoxicity in the MCF-7
and the MCF-7/ADR breast cancer cell lines.
F igu r e 2. Structures of novel analogues.
halves” 13 and 14. The northern half 15 was obtained
through asymmetric synthesis,18 while southern halves
13 and 14 were formed by coupling easily accessed
building blocks. The synthesis of the southern halves
13 and 14 began with the conversion of D-leucic acid
(7)19 to the corresponding benzyl ester (Scheme 1). The
leucic acid benzyl ester was subsequently esterified with
N-Boc-protected â-alanine to afford 8. Intermediate 8
was deprotected using trifluoroacetic acid and then
treated with DIEA, DCC, HOBT, and acid 9 or 10 to
afford the protected southern half 11 or 12. The benzyl
esters were cleaved using palladium(II) hydroxide and
hydrogen gas to reveal acids 13 and 14. Acids 13 and
14 were activated using the Yamaguchi reagent, 2,4,6-
trichlorobenzoyl chloride, and reacted with building
Ch em istr y
Analogues 5 and 6 were prepared by epoxidation of
styrenes 3 and 4, which were constructed from two key
fragments: the “northern half” 15 and the “southern
* To whom correspondence should be addressed. Phone: 785-864-
4498. Fax: 785-864-5836. E-mail: georg@ku.edu.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biosciences.
10.1021/jm030555f CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/02/2004

3698 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Brief Articles
Sch em e 1a
range with IC50 values of 13.7 and 10.6 µM, respectively,
in the same range as cryptophycin-24 (2, IC50 ) 15.8
µM). Cryptophycin-1 (1) was the most potent inhibitor
of tubulin assembly, with an IC50 of 3.4 µM. Epoxide
analogues 5 and 6 were active with IC50 values of 0.088
and 0.28 nM, respectively, in the MCF-7 breast cancer
cell line compared to cryptophycin-1 (1, IC50 ) 0.009 nM)
and cryptophycin-24 (2, IC50 ) 0.13 nM). With an IC50
of 0.92 nM, 6 was approximately one-third as active in
the MCF-7/ADR MDR cell line as in the MCF-7 cell line.
Compound 5 was 3% as active in the resistant cell line
as in the MCF-7 cell line, with an IC50 of 2.4 nM. The
activities of cryptophycin-1 (1) and cryptophycin-24 (2)
were reduced by 50% in the MDR MCF-7/ADR cell line
compared to the MCF-7 cell line. In vitro testing of
styrenes 3 and 4 revealed that the compounds had IC50
values greater than 25 nM in both the MCF-7 and the
MCF-7/ADR cell lines.
Con clu sion s
The results demonstrate that the C3 stereocenter is
not required to be the S configuration for cryptophycin-
24 (2) to induce cytotoxicity in the MCF-7 cell lines. Both
epoxide analogues 5 and 6, which possess the R config-
uration at C3, had activities comparable to that of
cryptophycin-24 (2) in the MCF-7 cytotoxicity and
tubulin assays. Overall, the general trend in activity of
the epimeric C3 analogues tested was consistent with
reports of other analogues in vitro; the styrene ana-
logues were inactive in the tubulin assembly assay and
had much reduced activity in the cytotoxicity assays,
while the epoxide analogues possessed good activi-
ties.17,21 The presence of the m-Cl substituent on the
C10 side chain of epoxide 6 provided a slight increase
in activity in the MCF-7/ADR cell line, but no general
effect was noted in the MCF-7 cell line. Inversion of the
C3 center did not reduce the ability of the cryptophycins
to evade the P-glycoprotein drug efflux pump, which is
overexpressed in the MCF-7/ADR breast cancer cell line.
a
(a) BnBr, Bu4NI, K2CO3, 76%; (b) N-Boc-â-alanine, DCC,
DMAP, 90%; (c) TFA; (d) DIEA, DCC, HOBT, 9 or 10, 48-68%,
two steps; (e) Pd(OH)2, H2, 95-98%; (f) DIEA, DMAP, 2,4,6-
trichlorobenzoyl chloride, 82-85%; (g) TFA; (h) HBTU, DIEA, 60-
74%, two steps; (i) DMD, 48-84%.
Ta ble 1. Biological Results for in Vitro Tubulin Assembly
Assay and Cytotoxicity Studies of C3-Epi Analogues
tubulin
assay
IC50 (µM)
MCF-7
IC50 (nM)
MCF-7/ADR resistance
IC50 (nM)
compd
factora
Exp er im en ta l Section . Syn th eses
1
2
3
4
5
6
3.4 ( 0.80
0.009 ( 0.003 0.018 ( 0.007
2.0
1.9
P r ep a r a tion of In ter m ed ia tes 3-6, 8, 11-14, 16, a n d
17. These compounds were synthesized in a manner similar
to that described in our earlier work22 except that D-leucic
acid19 was substituted for L-leucic acid in the synthesis.
15.8 ( 0.14 0.13 ( 0.06
0.25 ( 0.20
>25
>100
>100
13.7
>25
>25
0.088
0.28
>25
2.4
27.3
3.3
P r ep a r a tion of Ep oxid es 5 a n d 6. To a solution of styrene
3 (27.7 mg, 0.047 mmol) in acetone (2.0 mL) was added a
solution of DMD20 in acetone (2.0 mL), and the mixture was
stirred at room temperature for 5 h. After the mixture was
concentrated, the residue was purified using column chroma-
tography on silica gel (40% EtOAc/hexanes). The diastereo-
meric mixture of epoxides was separated using HPLC (Vydac
C18, internal diameter of 8 mm, eluent (isocratic), MeOH/H2O
65:35, flow rate of 3 mL/min). The total yield of a mixture of
5 and R-5 (â/R, 2:1), a white solid, was 13.6 mg, 48%. The
synthesis and isolation of 6 was carried out in the same fashion
and provided 22.8 mg (84%) of a mixture of 6 and R-6 (â/R,
2:1) as a white solid.
10.6
0.92
a
The resistance factor is defined as the IC50 of the resistance
cell line divided by the IC50 of the sensitive cell line.
block 15 to form esters 16 and 17.18 The N-Boc group
and the tert-butyl ester were simultaneously cleaved
from intermediates 16 and 17 using trifluoroacetic acid.
The macrocycles were closed using HBTU (O-benzot-
riazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate) to form styrenes 3 and 4.18 Epoxidation
using dimethyldioxirane (DMD)20 provided mixtures of
the R and â epoxides of 5 and 6. The epoxides were
separated using HPLC, and the â epoxides were tested
in vitro.
Ack n ow led gm en t. The authors thank Dmitry Ya-
kovlev of the Biological Research Services Laboratory
at the University of Kansas for conducting the HPLC
separation of the R and â epoxides. S. B. Buck also
thanks the Department of Defense for a predoctoral
fellowship (Grant DAMD17-98-1-8200) and the NIH for
a predoctoral training grant (Grant NIH-GM-07775).
Biologica l Testin g
In the tubulin assembly assay,5 styrene analogues 3
and 4 were inactive below 100 µM (Table 1). Epoxide
analogues 5 and 6 were active in the low micromolar

Brief Articles
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