Synthesis and activity of Combretastatin A-4 analogues: 1,2,3-thiadiazoles as potent antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2006.11.060

A series of 4,5-disubstitute-1,2,3-thiadiazole compounds were designed and synthesized as potent anticancer agents, some of them exhibited excellent in vitro and in vivo inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2006.11.060

Bioorganic & Medicinal Chemistry Letters 17 (2007) 869–873
Synthesis and activity of Combretastatin A-4 analogues: 1,2,3-
thiadiazoles as potent antitumor agents
Maojiang Wu, Qiming Sun, Chunhao Yang,* Dongdong Chen, Jian Ding,*
Yi Chen, Liping Lin and Yuyuan Xie
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Shanghai 201203, PR China
Received 7 July 2006; revised 2 November 2006; accepted 22 November 2006
Available online 29 November 2006
Abstract—A series of 4,5-disubstitute-1,2,3-thiadiazole compounds were designed and synthesized as potent anticancer agents, some
of them exhibited excellent in vitro and in vivo inhibitory activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Malignant tumor is one of the most serious threats
against human health in the world, and the clinical prog-
nosis remains relatively poor. Therefore, it needs to
develop the new therapeutic strategies for the improve-
ment of drugs that are currently in use.
There has been more and more interest in the search for
antitumor compounds with high efficacy, low toxicity in
recent years. For example, Combretastatin A-4 (CA-4)
is one of the excellent substances for antitumor activity,
which was first isolated from the South African bush
willow tree Combretum caffrum. Because of its structural
simplicity and potent cytotoxicity, CA-4 is a very attrac-
tive lead compound, and many synthetic analogues of
CA-4 have been developed.1–3 The structure–activity
relationship demonstrated that the cis configuration of
double bond and 3,4,5-trimethoxyphenyl group are fun-
damental.4 And the restricted rotation of rings A and B
Figure 1. Combretastatin and its derivatives.
of CA-4 can be maintained by introducing suitable con-
formationally restricted heterocycles such as dioxolane,5
Synthetic methods for the preparation of target com-
thiazole,6 and imidazole (Fig. 1).7 According to the
SAR, we designed and synthesized a series of cis restricted
analogues with 1,2,3-thiadiazole instead of the
CA-4’s olefin group (4a–t).
pounds are summarized as follows.
Deoxybenzoins8 3a–i were prepared as shown in Scheme
1. 3,4,5-Trimethoxybenzaldehyde was converted to
phosphonate by Pudovik’s reaction with di-n-butyl-
amine in ether,9 which was protected by 3,4-dihydro-
2H-pyran (THP), and condensed with the appropriate
aromatic aldehyde in the presence of EtONa or t-BuOK
(in case R = NO2), followed by hydrolysis with diluted
acid to afford the deoxybenzoins 3a–i.
Keywords: Combretastatin A-4 analogue; 1,2,3-Thiadiazoles; Tubulin;
Antitumor.
*
Corresponding authors. Tel./fax: +86 21 50806770 (C.Y.); tel./fax:
+86 21 50806722 (J.D.); e-mail addresses: chyang@mail.shcnc.ac.cn;
jding@mail.shcnc.ac.cn
Compounds 3j–l were easily prepared by Friedel–Crafts
reaction of phenylacetic acid with appropriate aryl ether
Those authors contributed equally.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.060

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M. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 869–873
Scheme 3. Reagents and conditions: (a) TSH, EtOH, rt, 1 h; (b)
NaOH, EtOH, reflux, 18 h; (c) TBAF, THF, rt, 1 h.
Scheme 1. Reagents and conditions: (a) dimethyl phosphite, n-Bu2NH,
ether, rt, 3h; (b) THP, TsOH, benzene, 50 ꢁC, 0.5 h; (c) NaH/EtOH or
t-BuOK, benzene, rt, 18 h; (d) CH3OH, HCl aq, rt, 1 h; (e) Pd/C, H2,
rt, 2 h.
in polyphosphoric acid (PPA) at 85–90 ꢁC with mechan-
ical stirring for 2 h (Scheme 2).
And 3m–r were synthesized according to Scheme 3.
The trimethoxybenzaldehyde was converted to the cor-
responding tosylhydrazones and then reacted with
appropriate benzaldehydes to afford deoxybenzoins
3m–r.10
At last, as shown in Scheme 4, substituted deoxybenzo-
ins and TSH (toluenesulfonhydrazide) were condensed
in absolute ethanol, and the intermediates were used
directly for cyclization with SOCl2 in dry chloroform.11
The temperature of step b must be kept under 25 ꢁC.12
The final compounds 4a–t were characterized by 1H
NMR and MS.
Scheme 4. Reagents and conditions: (a) EtOH, reflux, 3 h; (b) SOCl2,
CHCl3, 0–5 ꢁC, 4 h; (c) AcOH, Zn, rt, 2 h.
Cytotoxicity assay. Series of the test compounds
including compounds 4a–t and CA-4 were evaluated
for their antiproliferation activities against three types
of human cancer cell lines, human myeloid leukemia
cells HL-60, human colon adenocarcinoma cells
HCT-116, and human microvascular endothelial cell
line (HMEC-1) (Table 1) cells.
As shown in Table 1, there was an interesting phenom-
enon that if the 3,4,5-trimethoxyphenyl is at 4 position
in 1,2,3-thiadiazole, many of the test compounds (4b–
e, 4i, and 4r) have good activities, if at 5 position, only
4s displays activity. This finding is thought to be benefi-
cial for further SAR studies of CA-4 analogues.
Effects on tubulin polymerization. To investigate whether
the antiproliferation effects of these compounds were
related to the interaction with the microtubule system,
six of the most active compounds (4b and 4d, 4e and
4i, 4r and 4s) and control compound CA-4 were evaluat-
Scheme 2. Reagents and conditions: (a) PPA, 90 ꢁC, 2 h.

M. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 869–873
Table 1. IC50 Values (nM SD) of compounds 4a–t and CA-4
871
To further confirm the effects of these compounds on
tubulin and compared with that of CA-4, we evaluated
their activities on the alteration of the microtubule net-
work by indirect immunostaining. As shown in the Fig-
ure 2, 4r, 4i, 4e, and 4b, like CA-4, disrupt the
cytoskeleton and alter the morphology of HMEC-1 cells
significantly.
Compound
Cell line
HCT116
HL-60
HMEC-1
4a
4b
4c
4d
4e
4f
>1000
>1000
>1000
14.5 6.3
50.2 29.5
60.9 30.8
13.4 1.1
ND
25.0 8.2
86.6 2.3
50.9 7.5
30.2 2.0
>1000
24.6 14.9
73.9 18.0
75.8 27.8
26.4 18.5
>1000
Cell cycle analysis. That compounds exhibiting activity
on tubulin binding should cause the alteration of cell cy-
cle parameters leading to a preferential G2/M arrest,13
we next investigated whether the compounds 4b and
4d influence the cell cycle using flow cytometry assay.
Treatment with 4b and 4d for 24 h at different
concentration, HMEC-1 cells revealed a dose-dependent
increase of cells in G2/M and a simultaneous decrease in
S and G1 cells compared to control cells. As indicated
by the data shown in Table 3, the effects of the com-
4g
4i
23.6 5.1
1.5 0.1
537 116
>1000
18.5 1.7
3.0 1.3
616 291
>1000
ND
3.9 2.8
>1000
>1000
4j
4k
4l
>1000
>1000
>1000
>1000
>1000
>1000
4m
4n
4o
4p
4r
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1.8 1.3
40.9 20.8
>1000
3.7 1.3
70.4 10.4
>1000
2.5 0.1
41.0 5.1
>1000
4s
Table 3. Effects of 4b, 4d, and CA-4 on mitosis in HMEC-1 cells
4t
CA-4
1.9 0.7
3.0 1.2
3.5 0.9
Concn (nM)
G1 (%)
G2/M (%)
S (%)
SD, standard deviation. All experiments were independently per-
formed at least three times. ND, not determined.
Control
CA-4
41
2
20
67
88
83
80
80
14
28
40
78
87
24
25
33
61
78
39
31
8
6.25
12.5
25
4
4
13
16
0
ed for the inhibition of the polymerization of purified
tubulin. The results indicate that tubulin is the intracel-
lular target of these six compounds because they were all
potent inhibitors of tubulin polymerization with activi-
ties quantitatively similar to those of CA-4 (Table 2)
(Fig. 2).
50
4
100
6.25
12.5
25
16
55
39
34
11
4
4b
4d
31
33
27
11
9
50
100
6.25
12.5
25
36
35
38
12
1
40
40
29
27
21
Table 2. Inhibitory effect of tubulin polymerization by compounds 4b,
4d, 4e, 4i, 4r, 4s, and CA-4
50
100
Compound
4b
4d
4e
4i
4r
4s
CA4
Cell cycle distribution was analyzed by the standard propidium iodide
procedure as described in the literature.13
IC50 (lM)
0.86
0.72
2.13
0.7
0.68
0.86
0.81
Figure 2. Effects of 4r, 4i, 4e, and 4b on the cytoskeleton and cell morphology of HMEC-1 cells. Cells cultured on slides were exposed to compounds
at 1 lM for 30 min, followed by localization fixed in 3% formaldehyde and permeabilizing with 0.1% Triton X-100, and then incubated with primary
antibodies and Alex488-conjugated second antibodies sequentially. Slides were mounted in Vectashield with 4,6-diamidino-2-phenylindole (DAPI) to
visualize nuclei. Fluorescent images were acquired with Leica Confocal Scanner.

872
M. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 869–873
Table 4. Effect of the compounds on the in vivo growth of mice S-180 sarcoma
Group
Dosage (mg/kg/day)
Mice (n)
Initial/end
Body weight (g)
Initial/end
Tumor weight (g)
SD
Inhibition rate (%)
x
NS
CA-4
4b
—
20/20
10/8
17.4/26.6
17.0/22.6
17.2/18.3
17.0/23.6
17.5/21.8
17.2/—
1.79 0.56
0.64 0.33
0.34 0.28
1.25 0.58
0.64 0.45
—
—
40 · 4
40 · 5
10 · 7
100 · 7
20 · 1
10 · 1
20 · 4
10 · 7
50 · 2
64.2a
81.0a
30.2
64.2a
—
10/10
10/10
10/9
4e
4r
10/0
10/0
17.3/—
—
—
4i
10/6
10/9
17.1/19.5
17.3/23.3
17.0/23.0
0.84 0.59
1.46 0.66
0.35 0.20
53.1a
18.4
80.4a
5-Fu
10/10
a p < 0.01 versus NS group
pounds on the cell cycle suggest that this class of mole-
cules arrest the cell cycle at G2/M phase selectively.
In conclusion, we have synthesized a series of 1,2,3-thia-
diazole derivatives as tubulin polymerization inhibitors.
The compounds were evaluated for their antiprolifera-
tion activities and some of them displayed potent anti-
tumor effect in vivo. The 3,4,5-trimethoxyphenyl at 5
position in 1,2,3-thiadiazole (4j–p, 4t) led to the loss
of antiproliferative activity except for 4s. Six of the
most cytotoxic compounds were evaluated for effects
on tubulin assembly and displayed as potent inhibition
as that of CA-4. Cell-cycle distribution analysis shows
that 4b acts on the G2/M phase of the cell cycle. In vivo
antitumor effect indicated that 4b and 4e exhibited
favorable activity and 4b is promising for further stud-
ies because of its excellent effect and relatively low
toxicity.
In vivo antitumor effect of the test compounds. According
to the in vitro cytotoxicities of the test compounds, we
chose four compounds 4i, 4b, 4e, 4r, and CA-4 to inves-
tigate their in vivo antitumor effect. Table 4 and Figure 3
present the results of the experimental therapeutic effica-
cy of those compounds on mice S180 sarcoma trans-
plant model. After daily i.p. administration with
different dosage for 5–7 days, animals were euthanized
and the tumors were excised and weighted. As shown
in Table 4, the test compounds displayed different
anti-cancer efficacy. The compounds shown significant
anti-cancer efficacy were marked with ‘ ’ (p < 0.01).
*
As shown in Table 4 and Figure 3, when mice were
treated with CA-4 at 40 mg/kg for 4 days, compound
4b at 40 mg/kg for 5 days, compound 4e at 100 mg/kg
for 7 days, and compound 4i at 20 mg/kg for 4 days,
the growth inhibition rate of sarcoma S180 reached
64.2%, 81.0%, 64.2%, and 53.1%, respectively. When
treated with compound 4r at 20 mg/kg or 10 mg/kg,
all mice died after 1 day because of toxicity. It seems
that the amino group evidently increases the toxicity
of the compound though it would improve the water
solubility. The same result was observed by Ohsumi’s
group.3
Acknowledgment
This work was financially supported in part by the
Shanghai Basic Research Project from the Shanghai Sci-
ence and Technology Commission (No03DZ19228) and
National Natural Science Foundation Grant 30572201.
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tumors were excised and are shown in the picture.

M. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 869–873
873
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