The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4

Article abstract of DOI:10.1016/S0960-894X(01)00436-X

A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently described tubulin binding benzo[b]thiophenes 3-5. The most active thiophene compounds identified in this study were 11, 14, and 18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3-5. A structure-activity relationship of these compounds is considered.

Full text of DOI:10.1016/S0960-894X(01)00436-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2341–2343
The Synthesis and Tubulin Binding Activity of Thiophene-Based
Analogues of Combretastatin A-4
Bernard L. Flynn,a,* Guy P. Flynn,a Ernest Hamelb and M. Katherine Jungc
aDepartment of Chemistry, The Faculties, Australian National University, Canberra, ACT 0200, Australia
bScreening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA
cScience Applications International Corporation-Frederick, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, MD 21702, USA
Received 21 March 2001; accepted 18 June 2001
Abstract—A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl
groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and
iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently
described tubulin binding benzo[b]thiophenes 3–5. The most active thiophene compounds identified in this study were 11, 14, and
18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3–5. A structure–
activity relationship of these compounds is considered. # 2001 Elsevier Science Ltd. All rights reserved.
Compounds that bind to tubulin and prevent its poly-
merization into microtubules are effective anti-mitotic
agents.1 The naturally occurring cis-stilbene com-
bretastatin A-4 (1) is particularly effective in this regard
(Fig. 1).2 It displays exceptional cytotoxicity towards a
variety of cancer cell lines.2
Using a novel approach to benzo[b]thiophenes, we pre-
pared 3 and a number of analogues.5 Two of these
analogues, compounds 4 and 5, exhibited greater activ-
ity than 3 (Table 1).5 These compounds inhibited both
the rate and extent of tubulin assembly but again were
less active than 1.
Tubulin binding agents have also proven to be effective
in targeting the tumor vasculature system.3 The water
soluble prodrug form of 1, combretastatin A-4 disodium
phosphate (2), is currently undergoing clinical trials as a
tumor vascular targeting agent.
Recently, Pinney and co-workers described a new tubu-
lin binding agent 3, containing a benzo[b]thiophene
core.4 It was much less active than 1 and only reduced
the rate but not the extent of tubulin assembly (Table 1).4
It was postulated that this is due to the poor solubility
of 3. Pinney and co-workers also reported an X-ray
crystal structure of 3, which revealed a pseudo-p-stack-
ing arrangement of the D and C rings.4a,c This suggested
the possibility that the D and C rings in 3 may corre-
spond to the two phenyl rings in the cis-stilbene 1.
*Corresponding author. Fax: +61-2-6125-0760; e-mail: flynn@rsc.
anv.edu.au
Figure 1. Tubulin binders.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00436-X

2342
B. L. Flynn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2341–2343
In order to further investigate the structural elements
within 3–5 that are necessary for activity, we prepared
the simple (non-benzofused) thiophene analogues 11,
14, 18, and 19. The synthetic approach used was similar
to the palladium-mediated coupling/iodocyclization
approach we developed for gaining access to
benzo[b]thiophenes 3–5.5,6
DDQ oxidation to give 16. Lithiation of 16 and reaction
with benzaldehyde 17, followed by in situ methanolysis
of the acetate, gave the diol 18 in high yield. Oxidation
of 18 to ketone 19 using DDQ also proceeded smoothly.
Compounds 18 and 19 were obtained in a 63 and 62%
overall yield, respectively, from 3-butynol (Scheme 2).
Compounds 11, 14, 18, and 19 were first evaluated for
inhibition of tubulin assembly (Table 1). Those that
displayed an inhibitory effect were also examined for an
inhibitory effect on the binding of [3H]colchicine to
tubulin and for cytotoxicity against MCF-7 human
breast carcinoma cells (Table 1).
The synthesis of thiophenes 11 and 14 began with 3-
butynol, which was easily converted to the benzyl 3-
butynyl sulfide 6 (Scheme 1). Sonogashira coupling of 6
with aryliodide 7 afforded 8 in high yield. Treatment of
8 with iodine resulted in a rapid and efficient 5-endo-dig-
iodocyclization to give 9.6 Cross-coupling of vinyliodide
9 with arylzinc 10 and in situ hydrolysis of the acetate
group produced 11. Aromatization of 9 with DDQ and
acetate hydrolysis afforded 12. Treatment of 12 with 3
equivalents of t-BuLi, lithiated the phenol and the C-3
postion of the thiophene ring.7 Reaction of this dilithio
species with 3,4,5-trimethoxybenzoyl chloride 13 affor-
ded 14 upon protic workup. All reactions proceeded in
good yield, giving 11 and 14 in a 71 and 51% overall
yield, respectively, from 3-butynol.
Compound 19 did not inhibit tubulin assembly at con-
centrations as high as 40 mM and was not further
examined. Compounds 11, 14, and 18 all inhibited
tubulin assembly. Compound 14 showed greater
potency than combretastatin A-4 1 in this regard. In the
competitive binding studies all compounds were less
active than 1 at inhibiting the binding of [3H]colchicine
to tubulin. They were also much less cytotoxic towards
MCF-7 human carcinoma cells as compared to
combretastatin A-4. Interestingly, both 11 and 14 were
significantly more potent inhibitors of [3H]colchicine
binding to tubulin than the benzo[b]thipohene com-
pounds 3–5.
Preparation of 18 and 19 began with Sonogashira
coupling of 6 and 15 followed by iodocyclization and
In terms of the structure–activity relationships (SARs),
the activity associated with compound 14 supports the
notion that the activity of 3 and 4 results from the cor-
respondence of their C and D rings, to the two phenyl
rings in 1. However, our previous studies have shown
that the activity of 3 and 4 appears also to be highly
dependent upon the presence of a 6-methoxy substituted
A ring and a one-carbon linker between the B and D
rings since 20 to 22 are inactive (Fig. 2).5 Interestingly,
these two features suggest a correspondence between the
Scheme 1. Reagents and conditions: (i) KOH, TosCl, CH2Cl2; (ii)
NaH, BnSH, THF, 18 ꢀC; (iii) 7, Pd(PPh3)2Cl2 2.0 mol%, CuI 4.0
mol%, DMF/Et3N 3:1, 18 ꢀC; (iv) I2, CH2Cl2; (v) 10 (from 3,4,5-tri-
methoxyiodobenzene, 2equiv t-BuLi, 1 equiv ZnCl2), Pd(PPh3)2Cl2
5.0 mol%, THF, 18 ꢀC 4 h followed by MeOH, K2CO3; (vi) DDQ,
CH2Cl2 (vii) MeOH, K2CO3; (viii) 3 equiv t-BuLi, À78 ꢀC then 13.
Scheme 2. Reagents and conditions: (i) 15, Pd(PPh3)2Cl2 2.0 mol%,
CuI 4.0 mol%, DMF/Et3N 3:1, 18 ꢀC; (ii) I2, CH2Cl2; (iii) DDQ,
CH2Cl2; (iv) n-BuLi, À78 ꢀC then 17 followed by MeOH, K2CO3.

B. L. Flynn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2341–2343
2343
Table 1. Effects of thiophenes and benzo[b]thiophenes on tubulin polymerization, colchicine binding and growth of MCF-7 human breast carci-
noma cells8
Compound
Inhibition of tubulin polymerizationa
Inhibition of colchicine binding (% inhibition)b
Inhibition of cell growth
IC50 (nM)
IC50 (mM)
5 mM inhibitor
50 mM inhibitor
1
3
4
5c
11
14
18
19
2.1Æ0.1c
>40*d,e
3.4Æ0.22
6.1Æ0.8
3.6Æ1.0
1.0Æ0.1
8.8Æ0.9
>40
98Æ3
—
—
2
—
73
390
—
11Æ4
e
6840Æ10
520Æ400
1
Æ10
f
5
—
64Æ288
67Æ10
26Æ3
—
Æ100
300Æ400
500Æ300
—
74
—
aThe tubulin concentration was 10 mM. Inhibition of extent of assembly was the parameter measured.
bThe tubulin concentration was 1.0 mM and the [3H]colchicine concentration was 5.0 mM.
cData from ref 5.
dThe asterisk indicates that the rate but not the extent of assembly was reduced by compound concentrations as high as 40 mM.
eData from ref 4a.
fCompound 5 was not tested against the MCF-7 cell line but was tested against the Burkitt lymphoma CA46 cell line (IC50>1000 nM); see ref 5.
Schmidt, J. M.; Hogan, F. J. Med. Chem. 1995, 38, 1666. (b)
Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry
1989, 28, 6984. (c) Pettit, G. R.; Cragg, G. M.; Singh, S. B. J.
Nat. Prod. 1987, 60, 1374. (d) Pettit, G. R.; Singh, S. B.;
Cragg, G. M. J. Org. Chem. 1985, 50, 3404. (e) Pettit, G. R.;
Cragg, G. M.; Herald, D. L.; Schmidt, J. M.; Lohavanijaya, P.
Can. J. Chem. 1982, 60, 1374.
3. (a) Chaplin, D. J.; Pettit, G. R.; Parkins, C. S.; Hill, S. A.
Br. J. Cancer 1996, 74, S86. (b) Dark, G. G.; Hill, S. A.; Prise,
V. E.; Tozer, G. M.; Pettit, G. R.; Chaplin, D. J. Cancer Res.
1997, 57, 1829. (c) Tozer, G. M.; Prise, V. E.; Wilson, J.;
Locke, R. J.; Vojnovic, B.; Stratford, M. R. L.; Dennis, M. F.;
Chaplin, D. J. Cancer Res. 1999, 59, 1626. (d) Iyer, S.; Chap-
lin, D. J.; Rosenthal, D. S.; Boulares, A. H.; Li, Lu-Y.; Smul-
son, M. E. Cancer Res. 1998, 58, 4510. (e) Grosios, K.;
Holwell, S. E.; McGown, A. T.; Pettit, G. R.; Bibby, M. C. Br.
J. Cancer 1999, 81, 1318. (f) Pettit, G. R.; Rhodes, M. R. Anti-
Cancer Drug Des. 1998, 13, 183. (g) Pettit, G. R.; Rhodes,
Figure 2.
M. R.; Herald, D. L.; Chaplin, D. J.; Stratford, M. R. L.;
Hamel, E.; Pettit, R. K.; Chapuis, J.-C.; Oliva, D. Anti-Cancer
Drug Des. 1998, 13, 981.
4. (a) Pinney, K. G.; Bounds, A. D.; Dingeman, K. M.;
Mocharla, V. P.; Pettit, G. R.; Bai, R.; Hamel, E. Bioorg.
Med. Chem. Lett. 1999, 9, 1081. (b) Pinney, K. G.; Pettit, G.
R.; Mocharla, V. P.; Del Pilar Majia, M.; Shirali, A. PCT Int.
Appl. WO 9839323, 1998. Chem. Abstr. 1998, 129, 245037. (c)
Mullica, D. F.; Pinney, K. G.; Mocharla, V. P.; Dingeman,
K. M.; Bounds, A. D.; Sappenfield, E. L. J. Chem. Cryst.
1998, 28, 289.
A and D rings in 3 and 4 to the two phenyl rings in 1. A
more definitive understanding of the relationship
between the A, C, and D rings in 3 and 4 and the phenyl
rings in 1 is being pursued through the synthesis of
additional analogues.
5. Flynn, B. L.; Verdier-Pinard, P.; Hamel, E. Org. Lett. 2001,
3, 651.
Acknowledgements
6. (a) For some other examples of iodocyclization involving
alkynyl benzyl sulfides, see: Ren, X.-F.; Turos, E. Tetrahedron
Lett. 1993, 34, 1575. (b) Ren, X.-F.; Turos, E.; Lake, C. H.;
Churchill, M. R. J. Org. Chem. 1995, 60, 6468. (c) Ren, X.-F.;
Konaklieva, M. I.; Shi, H.; Dickey, S.; Lim, D. V.; Gonzalez,
J.; Turos, E. J. Org. Chem. 1998, 63, 8898.
The authors thank the Australian Research Council for
financial support including an Australian Research Fel-
lowship to BLF. This work was supported in part by NCI,
National Institutes of Health Contract N01-CO-56000.
7. We found that attempted metalation of the 3-iodo-4,5-
dihydrothiophenes results in ring opening to give lithium sul-
fides. This ring opening has also been observed by Ren, X.-F.
et al.; see ref 6c.
References and Notes
8. For experimental procedures, see: Verdier-Pinard, P.; Lai,
J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu,
M.; White, J. D.; Falck, J. R.; Gerwick, W. H.; Day, B. W.;
Hamel, E. Mol. Pharmacol. 1998, 53, 62 .
1. Hamel, E. Med. Res. Rev. 1996, 16, 207.
2. (b) Sackett, D. L. Pharmacol. Ther. 1993, 59, 163. (a) Pettit,
G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.;