Novel sulfonate analogues of combretastatin A-4: Potent antimitotic agents

Article abstract of DOI:10.1016/S0960-894X(01)00098-1

Sulfonate analogues of combretastatin A-4 have been prepared. These compounds compete with colchicine and combretastatin A-4 for the colchicine binding site on tubulin and are potent inhibitors of tubulin polymerization and cell proliferation. Importantly, these compounds also inhibit the proliferation of P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.

Full text of DOI:10.1016/S0960-894X(01)00098-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 871–874
Novel Sulfonate Analogues of Combretastatin A-4:
Potent Antimitotic Agents
Stephen L. Gwaltney, II,* Hovis M. Imade, Kenneth J. Barr, Qun Li, Laura Gehrke,
R. Bruce Credo, Robert B. Warner, Jang Yun Lee, Peter Kovar, Jieyi Wang,
Michael A. Nukkala, Nicolette A. Zielinski, David Frost, Shi-Chung Ng
and Hing L. Sham
Cancer Research, D47B, Abbott Laboratories, Abbott Park, IL 60064-6101, USA
Received 6 September 2000; accepted 22 January 2001
Abstract—Sulfonate analogues of combretastatin A-4 have been prepared. These compounds compete with colchicine and com-
bretastatin A-4 for the colchicine binding site on tubulin and are potent inhibitors of tubulin polymerization and cell proliferation.
Importantly, these compounds also inhibit the proliferation of P-glycoprotein positive (+) cancer cells, which are resistant to many
other antitumor agents. # 2001 Elsevier Science Ltd. All rights reserved.
Combretastatin A-4 (CA-4, Fig. 1) is a naturally occur-
ring stilbene that inhibits tubulin polymerization by
binding to tubulin at the colchicine binding site.1 This
phenomenon leads to potent antitumor activity against
a wide variety of cell lines, including multidrug resistant
lines.2 However, the limited water solubility and poor
oral bioavailability of this compound have limited its in
vivo efficacy,3 and efforts to develop more efficacious
analogues and prodrugs are ongoing.4
Scheme 1. Synthesis of sulfonate analogues of combretastatin A-4.
Figure 1. Combretastatin A-4.
As part of our program to identify novel antimitotic
agents, we have synthesized a series of sulfonate analo-
gues of CA-4 as shown in Scheme 1. 3,4,5-Trimethoxy-
phenylsulfonyl chloride5 was treated with phenols
under standard conditions6 to provide sulfonates A.
Alternatively, the reversed sulfonates B could be pre-
pared by treatment of 3,4,5-trimethoxyphenol with aryl
sulfonyl halides.
Most of the phenols and sulfonyl chlorides used were
commercially available. The others were synthesized by
literature methods7 or as shown in Scheme 2.
The structures of the diarylsulfonates synthesized are
shown in Scheme 3. The yields for the sulfonylation
reactions are shown in parentheses. In some cases, fur-
ther modification of the initial sulfonate was performed
to provide others. Compound 12 was prepared by
treating 4-methoxymetanilyl fluoride with 3,4,5-tri-
methoxyphenol and triethylamine in methylene chloride.
*Corresponding author. Tel.: +1-847-935-6604; fax: +1-847-938-
5034; e-mail: stephen.gwaltney@abbott.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00098-1

872
S. L. Gwaltney, II et al. / Bioorg. Med. Chem. Lett. 11 (2001) 871–874
Figure 2. Olefins 16 and 17.
The compounds above were tested for their ability to
inhibit the proliferation of two cancer cell lines (Table
1).8 NCI-H460 is a human lung carcinoma that does not
express P-glycoprotein, while HCT-15 is a human colon
carcinoma that expresses a high level of this protein.
HCT-15 therefore shows multidrug resistance. This fact
can be appreciated by examining the inhibition data for
a P-glycoprotein substrate such as paclitaxel against the
two cell lines.
Many of the sulfonate analogues show excellent anti-
proliferative activity. In fact, the potency of compounds
2 and 12 rivals that of CA-4 itself. Importantly, the
potent sulfonates in Table 1 maintain their activity
against the multidrug resistant HCT-15 cell line. The
orientation of the sulfonate group relative to the two
aryl rings makes little difference in the antiproliferative
activity of the compounds, as demonstrated by com-
pound pairs 3/10, 4/9, and 7/11. That the sulfonate is an
effective replacement for the cis olefin of CA-4 can be
appreciated by comparing the activities of compounds
Scheme 2. Synthesis of phenols and sulfonyl chlorides. Reagents: (a)
NaH, MeI, 55%; (b) H2, Pd–C, 67–91%; (c) NaCNBH3, AcOH, 38%;
(d) ClSO3H, 27%; (e) Me2SO4, NaOH, 77%; (f) NaNO2; SO2, CuCl2,
41%.
To evaluate the properties of the sulfonates prepared
above, they were compared to several Z-olefins. Com-
bretastatin A-4 was prepared as in the literature.1 Using
the same method, we prepared olefins 16 and 17 (Fig. 2)
which share substituents with sulfonates 4, 7, 9, and 11.
Scheme 3. Sulfonate analogues of combretastatin A-4. Structures, sulfonylation yields, and synthetic modifications. Reagents: (a) H2, Pd–C, 36%;
(b) (CH2O)n, NaCNBH3, 49%; (c) MeI, 135 ꢀC, 45%.

S. L. Gwaltney, II et al. / Bioorg. Med. Chem. Lett. 11 (2001) 871–874
873
Table 1. Inhibition of cellular proliferation (IC50) for sulfonate
analogues of CA-4, olefins 16 and 17, CA-4, and paclitaxel
Table 3. Percent inhibition of colchicine (5 mM) binding to tubulin
(10 mM) for compounds 2, 9, and CA-4 at 5 mM and 50 mM
Compounds
IC50 (nM), HCT-15a
IC50 (nM), NCI-H460b
Compounds
% Inhibition
1
2
3
4
35 (Æ1.8)
3.3 (Æ2.8)
71 (Æ0.85)
36 (Æ9.9)
>10,000
47 (Æ14)
3.1 (Æ1.6)
620 (Æ71)
67 (Æ4.0)
>10,000
5 mM
50 mM
2
63
43
53
95
80
93
9
CA-4
5
6
7
8
9
10
11
12
13
14
15
16
700 (Æ81)
740 (Æ13)
17 (Æ20)
29 (Æ22)
580 (Æ53)
56 (Æ22)
550 (Æ55)
140 (Æ160)
540 (Æ120)
41 (Æ12)
their ability to compete with colchicine for its binding
site on tubulin10 (Table 3).
250 (Æ2.8)
30 (Æ8.8)
4.1 (Æ0.064)
440 (Æ200)
670 (Æ270)
>10,000
2.7 (Æ1.5)
480 (Æ250)
900 (Æ510)
>10,000
The data shown above demonstrate that these sulfonate
analogues of CA-4 inhibit the polymerization of tubulin
and compete effectively for the colchicine binding site
on tubulin. Of the sulfonates in Tables 2 and 3, com-
pound 2 proved to be the best antimitotic agent, inhi-
biting tubulin polymerization with an IC50 similar to
that of CA-4 and competing more effectively than CA-4
for the colchicine binding site.
8.7 (Æ1.2)
74 (Æ3.1)
1.7 (Æ1.1)
450 (Æ190)
60 (Æ4.5)
140 (Æ140)
3 (Æ0.66)
15 (Æ6.0)
17
CA-4
Paclitaxel
aMDR(+), multidrug resistant.
bMDR( À), not multidrug resistant.
The pharmacokinetic behavior of several of the more
active analogues shown above was examined. These
compounds are characterized by low aqueous solubility,
short iv half-lives, and low oral bioavailablity (F). This
problem can be ameliorated in some cases through the
preparation of prodrugs. For example, in rats, com-
pound 12 has an iv half-life of 0.56 h, and F=0%;
however, prodrug 18 (Fig. 3) provided improved plasma
levels of 12 after a 10 mg/kg oral dose (t1/2=1.82 h,
4/9 with that of 16, and comparing the activities of
compounds 7/11 with that of 17.
Our efforts to replace the 3-hydroxy-4-methoxyphenyl
ring of CA-4 with other groups in our sulfonates led to
several findings. The 5-substituted indole nucleus (as in
compound 1), and especially N-methyl indole (as in
compound 2), are good replacements for the 3-hydroxy-
4-methoxyphenyl ring of CA-4. Conversion of the
indole to an indoline as in 13 leads to significant loss of
potency. It has been previously established that the 3-
amino-4-methoxyphenyl moiety is also a good replace-
ment for the 3-hydroxy-4-methoxyphenyl ring of CA-
4.4d,j This is supported by the potency reported here for
12. Pyridones have been incorporated into CA-4 analo-
gues and these compounds have shown good anti-
tumor activity.4g With this in mind, compounds 8
and 15 were designed to improve water solubility;
however, these compounds suffered substantial losses in
potency.
.
AUC(0À6 h)=0.8 mcg h/mL, Fꢁ100%).
Figure 3. Prodrug 18.
Selected compounds that showed significant anti-
proliferative effects were also tested for their ability to
inhibit the polymerization of tubulin9 (Table 2) and
Compound 18 was tested in vivo against M5076, a
murine cell line. Mice were dosed once daily on days 1–5
of the trial with either 200, 150, or 50 mg/kg. Tumor
growth was measured and compared to tumor growth
in untreated animals. Compound 18 did not show
appreciable efficacy in this model.
Table 2. Inhibition of tubulin polymerization (IC50) for sulfonate
analogues of CA-4, olefins 16 and 17, CA-4, and paclitaxel
In conclusion, we have synthesized sulfonate analo-
gues of combretastatin A-4 and have found many of
these compounds to be potent in vitro inhibitors of
the proliferation of two cancer cell lines. These com-
pounds inhibit the polymerization of tubulin and
compete effectively for the colchicine binding site on
tubulin. Examination of the SARin this series of
compounds reveals that the sulfonate replacement of
the cis olefin and various replacements for the 3-
hydroxy-4-methoxyphenyl ring of CA-4 are well
tolerated.
Compounds
IC50 (mM)
1
2
9
10
11
12
6.6 (Æ1.3)
1.4 (Æ0.15)
8.2 (Æ2.5)
>100
16 (Æ1.3)
6.7 (Æ3.7)
1.3 (Æ0.89)
1.7 (Æ0.042)
1.2 (Æ0.76)
1.3 (Æ0.50)
16
17
CA-4
Paclitaxel

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S. L. Gwaltney, II et al. / Bioorg. Med. Chem. Lett. 11 (2001) 871–874
K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.;
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