4-Chlorocolchicine derivatives bearing a thiourea side chain at the C-7 position as potent anticancer agents

Article abstract of DOI:10.1039/c3md00287j

A series of 4-substituted colchicine derivatives were synthesized and evaluated with an eye toward developing new anticancer agents. As a result, 4-chlorocolchicine derivatives bearing a thioureide side chain at the C-7 position were found to exhibit significant cytotoxicities to three human cancer cell lines (A549, HT-29, and HCT116). In particular, compound 26 having an ethylthioureide group at the C-7 had high antitumor activity in vivo and a broad effective dosage range. Furthermore, compound 58, which has a (5-methylpyrazol-3-yl)thioureide group at the C-7 side chain, exhibited strong cytotoxicity and desirable metabolic stability in vitro.

Full text of DOI:10.1039/c3md00287j

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4-Chlorocolchicine derivatives bearing a thiourea
side chain at the C-7 position as potent anticancer
agents†
Cite this: Med. Chem. Commun., 2014,
5, 452
Hiroyuki Nishiyama,ac Masahiro Ono,a Takuya Sugimoto,a Toshio Sasai,a
Naoyuki Asakawa,a Satoshi Ueno,a Yoshitaka Tominaga,b Takashi Yaegashi,a
Masato Nagaoka,a Takeshi Matsuzaki,a Noriyuki Kogure,c Mariko Kitajimac
c
*
and Hiromitsu Takayama
A series of 4-substituted colchicine derivatives were synthesized and evaluated with an eye toward
developing new anticancer agents. As a result, 4-chlorocolchicine derivatives bearing a thioureide side
chain at the C-7 position were found to exhibit significant cytotoxicities to three human cancer cell lines
(A549, HT-29, and HCT116). In particular, compound 26 having an ethylthioureide group at the C-7 had
high antitumor activity in vivo and a broad effective dosage range. Furthermore, compound 58, which
has a (5-methylpyrazol-3-yl)thioureide group at the C-7 side chain, exhibited strong cytotoxicity and
desirable metabolic stability in vitro.
Received 30th September 2013
Accepted 20th December 2013
DOI: 10.1039/c3md00287j
www.rsc.org/medchemcomm
Results and discussion
Introduction
We initially synthesized 4-(mono-, di-, and tri-)uoromethyl
derivatives 5, 6, and 7 (Scheme 1), because, it was reported that
the biological activity of a chloro-containing compound was
improved when the chlorine group was substituted with a
uorinated methyl group.22–24 First, colchicine (1) was converted
into 4-formylcolchicine (8) by reacting with Cl2CHOMe in the
presence of SnCl4. The reduction of 8 with NaBH4 gave
4-hydroxymethyl derivative 9 and subsequent uorination with
DAST gave 4-uoromethyl derivative 5. In turn, compound 8 was
converted into 4-diuoromethylcolchicine (6) via 4-(1,3-dithian-
2-yl)colchicine (10). 4-(Triuoromethyl)colchicine (7) could be
synthesized in good yield by treatment of 4-iodocolchicine (11)
with FSO2CF2COOMe and CuI in a sealed tube.25,26
Colchicine (1), the major alkaloid isolated from Colchicum
autumnale, is a well-known antimitotic agent that acts by
binding to tubulin.1 A number of research groups have
poured much effort into the development of potential anti-
cancer agents derived from colchicine.2–15 However, the
inherent adverse effects of alkaloids have hampered the
development of colchicine-derived anticancer drugs.16–19
We reported that 4-chlorocolchicine (2) exhibited potent
activities in vitro and in vivo.20 Furthermore, we found that
derivatives 3 and 4, which possess an a-hydroxyalkanamide
side chain at the C-7 position, had signicant antitumor
activities in vivo with broad effective dosage ranges.21
Therefore, we looked into the potency of C-4 substituted
colchicines in greater detail. In this paper, we report the
synthesis of 4-substituted colchicines having newly designed
side chains at the C-7 position and their antitumor activities
(Fig. 1).
The cytotoxicities of derivatives 5–7 were evaluated in three
human cancer cell lines (A549, HT-29, and HCT116) (Table 1).
aYakult Central Institute for Microbiological Research, 1798 Yaho, Kunitachi-shi,
Tokyo 186-8650, Japan
bPharmaceutical Division, Yakult Honsha Co., Ltd., 7-16-21 Ginza, Chuo-ku, Tokyo
104-0061, Japan
cGraduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-
ku, Chiba-shi, 260-8675, Japan. E-mail: htakayam@p.chiba-u.ac.jp; Fax: +81-43-226-
2914; Tel: +81-43-226-2914
† Electronic supplementary information (ESI) available: Synthetic procedures and
characterization data of all compounds, as well as the methodology used in
cytotoxicity evaluation, antitumor experiments, metabolic stability experiments,
and docking simulations. See DOI: 10.1039/c3md00287j
Fig. 1 Structures of colchicine (1), 4-chlorocolchicine (2), and their
analogues 3 and 4 with an a-hydroxyalkylamide function at the C-7
position.
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Scheme 1 Reagents and conditions: (a) Cl2CHOMe, SnCl4, CH2Cl2, rt, overnight; (b) NaBH4, MeOH, rt, 3 h; (c) DAST, CH2Cl2, 0 ꢀC, 2 h; (d) 1,3-
propanedithiol, I2, CHCl3, rt, overnight; (e) NIS, HF$Py, CH2Cl2, ꢁ78 ꢀC to rt, then NaBH4, MeOH, rt, 0.5 h; (f) NIS, AcOH, 70 ꢀC, 7 h and (g)
FSO2CF2COOMe, CuI, NMP, in a sealed tube, 120 ꢀC, overnight.
Table 1 Cytotoxicities and S log P values of 4-substituted colchicine
derivatives
IC50 value (nM)
Compound
no.
Metabolic stability
A549
HT-29 HCT116 S log Pa (mL minꢁ1 mgꢁ1
)
1
2
5
6
7
54.4
9.9
8793
55.4
55.2
8.2
7.9
2615
48.1
35.0
10.6
8.1
4753
45.8
37.9
2.59
3.24
3.32
3.62
3.92
0.001
0.028
NTb
NT
NT
Scheme 2 Reagents and conditions: (a) R0SO2Cl, Et3N, CH2Cl2, 0 ꢀC, 2
h; (b) R0N]C]O, CH2Cl2 or MeOH–H2O, 0 ꢀC; and (c) R20 NCOCl,
Et3N, CH2Cl2, reflux.
a
b
S log P: the index of hydrophobicity was estimated by MOE. Not
tested.
Fluorinated derivatives 6 and 7 exhibited moderate activities isocyanates. Similarly, ureides (21–23) were synthesized by
but were disappointingly inferior to 4-chlorocolchicine (2). 4- acylation using the corresponding carbamoyl chlorides.
Fluoromethyl derivative
activity.
5
showed markedly diminished
Table 2 enumerates the cytotoxicities of sulfonamides
(13–17) and ureides (18–23). Sulfonamides (13–17) and urea-
Consequently, we shied our attention again to the 4-chloro type compounds 21–23 showed modest IC50 values regardless of
derivatives having a newly designed side chain, such as sulfon- the length and type of the group (cyclic or acyclic), whereas the
amide, urea, or thiourea, at the C-7 position. The synthesis of metabolic stability in vitro of compound 14 was comparable to
compounds 13–23 is shown in Scheme 2. Sulfonamides (13–17) the favorable stability of colchicine (1) (see Tables 1 & 2). The
were obtained by treatment of 4-chlorodeacetylcolchicine (12) cytotoxicities of urea compounds 18 and 19 were low. On the
with the corresponding sulfonyl chlorides. Compounds 18–20 other hand, compounds 17 and 20 possessing an aromatic ring
were prepared from 12 by employing the corresponding exhibited slightly improved cytotoxicities.
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Table 2 Cytotoxicities, S log P values, and metabolic stabilities of 4-
chlorocolchicine derivatives
all cyclic thioureides (35–38) possessed good potencies. On the
other hand, N-(hydroxyethyl)thioureides (30 and 34) resulted in
diminished activities in spite of the fact that 4-chlorocolchicine
derivatives bearing a hydroxyalkanamide side chain at the C-7
position showed high antitumor activities and favorable meta-
bolic stabilities.21
IC50 value (nM)
Compound
no.
Metabolic stability
A549 HT-29 HCT116 S log Pa (mL minꢁ1 mgꢁ1
)
13
14
15
16
17
18
19
20
21
22
23
45.4
48.1
44.9
40.8
36.8
247
289
48.1
192
48.3
26.6
38.6
42.6
42.2
36.4
8.7
81.8
181
35.2
53.2
43.9
42.6
36.9
39.9
40.7
33.2
25.8
134
189
36.0
2.65
3.04
3.43
3.68
4.08
3.42
3.81
4.93
3.37
4.15
4.30
NTb
0.005
NT
NT
NT
NT
NT
NT
NT
Docking simulations in silico were carried out to estimate
why the thiourea-type derivatives showed higher activities than
the urea-type derivatives. The thioureide–NH group of derivative
26 interacts with Thr179 of the tubulin a-subunit through a
hydrogen bond. In addition, the C-9 carbonyl group in
compound 26 and Lys254 of the tubulin b-subunit are held
together by a hydrogen bond (Fig. 2b). On the other hand, urea
derivative 18 has no such favorable interaction with tubulin
(Fig. 2a). The reason is inferred as follows. It is known that the
hydrogen-bond donating ability of a thioureide–NH group is
generally higher than that of the corresponding ureide–NH
group, because the pKa value of a thioureide–NH group is lower
than that of the homologous ureide–NH group.27 Therefore, the
interaction of Thr179 of tubulin with the thioureide group of
compound 26 presumably determines the orientation of
compound 26 in the binding pocket, and compound 26 is better
for accepting hydrogen-bonding with Lys254 of tubulin than
compound 18.
Table 4 presents the cytotoxicities of phenylthioureides (39–
47). Phenyl compound 39, and meta- and para-methoxyphenyl
compounds 41 and 42 showed good potencies but ortho-
methoxyphenyl derivative 40 exhibited weaker activity than 41
and 42. A similar tendency was observed in the cytotoxicity of
cyanophenyl derivatives (ortho: 43, meta: 44, and para: 45). para-
(Dimethylamino)phenyl derivative 47 showed stronger activity
than meta-substituted compound 46.
Next, we selected four compounds 26, 32, 38, and 47 with
potent cytotoxicities in vitro to examine antitumor activity in
vivo using HCT116 transplanted nude mice. The results are
summarized in Table 5. All of the tested compounds in vivo were
found to possess signicant antitumor activities at half of the
maximum dose (1/2MD) and no signs of toxicity, such as weight
loss and mortality, were noted in the mice.28 Thioureide-type
4-chlorocolchicines (26, 32, 38, and 47) exhibited broad effective
dosage ranges, similar to our previously reported amide-type
derivatives.21 It should be stressed that compound 26 showed
high potency (44.4% IR) at even 1/4MD.
50.4
44.3
42.4
0.021
0.043
a
Estimated by MOE. b Not tested.
As a matter of course, thiourea-type derivatives were
synthesized next (Scheme 3). Derivatives 24–63 were prepared
from 4-chlorodeacetylcolchicine (12) by condensation with the
corresponding isothiocyanates or substitution of the homolo-
gous imidazole-1-carbothioamides. In another experiment,
deacetyl compound 12 was converted into isothiocyanate (64)
with thiophosgene, and this was followed by the addition of
amines to afford 4-chlorocolchicines having various thiourea
side chains.
Ethylthioureide (26) exhibited more potent cytotoxicity than
ethylureide (18) (see Tables 2 & 3). The results prompted us to
investigate the antitumor potency of thiourea-type derivatives in
detail (Table 3). N-Monosubstituted thiourea-type derivatives
24–28 exhibited comparable activities to 4-chlorocolchicine (2).
However, n-hexylthioureide (29) showed decreased activity.
Similarly, in N,N-disubstituted derivatives 31–33, compounds
31 and 32 showed potent antitumor activities, whereas the
activity of N,N-dipropylthioureide (33) was decreased. Moreover,
Aromatic thioureide (47) showed signicant antitumor activity
in in vivo experiments, in contrast to the corresponding aromatic
amide-type derivative that had no activity in vivo.21 However, the
metabolic stability of 47 was lower than that of colchicine (1) (see
Tables 1 & 4). Therefore, heteroaromatic thioureides (48–63)
having lower S log P values than compound 47 were synthesized
and evaluated in vitro with a view to improving metabolic stabil-
ities (Scheme 3 and Table 6). Six-membered heteroaromatic-type
compounds 48–53 exhibited moderate cytotoxicities compared to
compound 47. On the other hand, 2-thiazolylthioureide (54)
demonstrated potent activity. All of the ve-membered derivatives
55–63, except compound 61, showed strong activities. Notably,
3-pyrazolylthiourea (56), 5-methyl-3-pyrazolylthiourea (58), 3-tri-
azolylthiourea (59), and 5-methyl-3-triazolylthiourea (62) exhibited
Scheme 3 Reagents and conditions: (a) RN]C]S, CH2Cl2 or MeOH–
H2O, 0 ꢀC, 1.5 h; (b) N-R-1H-imidazole-1-carbothioamide; (c) thio-
phosgene, Et3N, CH2Cl2, 0 ꢀC, 2 h; and (d) RNH2 or R2NH, CH2Cl2 or
MeOH–H2O, rt, overnight.
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Table 3 Cytotoxicities, S log P values, and metabolic stabilities of 4-chlorocolchicine derivatives
Compound
No.
IC50 value (nM)
A549
Metabolic stability
R1
R2
HT-29
HCT116
S log Pa
(mL minꢁ1 mgꢁ1
)
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
H
H
H
Me
H
Me
45.0
10.9
37.9
9.9
7.4
212
6.1
2.8
8.6
1.6
1.0
176
52.7
8.8
9.0
6.3
7.9
4.3
1.8
171
89.7
8.4
2.94
3.20
3.59
3.98
4.37
5.15
2.56
3.54
4.32
5.10
2.90
3.68
4.07
4.46
3.31
NTb
NT
0.124
NT
NT
NT
NT
NT
0.541
NT
NT
Et
nPr
nBu
nC6H13
CH2CH2OH
Me
251
10.7
10.1
40.3
44.9
33.3
12.2
9.6
Et
Et
6.1
6.9
nPr
nPr
38.2
30.4
5.8
7.5
6.1
34.4
32.6
8.4
7.2
6.9
Me
CH2CH2OH
–CH2CH2CH2–
–CH2CH2CH2CH2–
–CH2CH2CH2CH2CH2–
–CH2CH2OCH2CH2–
NT
NT
NT
0.061
44.9
7.2
9.1
a
Estimated by MOE. b Not tested.
Table 5 Antitumor activities of 4-chlorocolchicine derivatives
Dose
Total dosea
IR
(%)
Compound no.
(mg kgꢁ1 dayꢁ1
)
(mg kgꢁ1
)
Mortality
26
32
38
47
20
60
120
75
150
75
150
30
60
44.4
62.6
29.5
54.6
31.3
52.7
32.6
52.5
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
40b
25
50b
25
50b
10
20b
a
b
Dosing schedule on days 1, 5, and 9. Half of the maximum dose.
Fig. 2 Docking modes of compounds 18 (a), 26 (b), and 58 (c) at the
colchicine binding site of tubulin (green: hydrophobic regions and
pink: polar regions).
potent activities of sub-nanomole order against two or all three of
the human cancer cell lines examined. Of particular interest was
that the metabolic stability of compound 58 was comparable to
that of colchicine (1).
Table 7 shows the data for inhibition of tubulin polymeri-
zation by 1, 26, and 58. As expected, 58 showed the most potent
antitubulin activity among the three assay samples, and
compounds 26 and 58 were revealed to have same mode of
action as 1. The antitubulin activities of three compounds
almost paralleled those of the cytotoxic activities. On the other
hand, big differences between the IC50 values of cytotoxicities
and of anti-tubulin activity on compound 58 were observed.
Almost the same result has been reported in the study of tubulin
polymerization inhibitors.29 Possibly, this result is due to the
difference in experimental conditions, i.e., the cell-based assay
(cytotoxicity) or the cell-free assay (tubulin assembly) and/or due
to the cytotoxic effect of the compound through some addi-
tional target besides tubulin.
Table
derivatives
4 Cytotoxicities and S log P values of 4-chlorocolchicine
Compounda
No. R1
IC50 value (nM)
A549 HT-29 HCT116 S log Pb (mL minꢁ1 mgꢁ1
Metabolic stability
)
39 Ph
24.4
77.6 15.0
23.3
8.0
0.4
1.2 5.09
29.4 5.10
1.4 5.10
1.4 5.10
NTc
NT
NT
40 Ph-2-OMe
41 Ph-3-OMe
42 Ph-4-OMe
43 Ph-2-CN
44 Ph-3-CN
45 Ph-4-CN
46 Ph-3-NMe2
47 Ph-4-NMe2
0.5
0.3
NT
3795
126
67.8
38.4
1.8
866
2204
4.96
25.3 4.96
25.0 4.96
24.2 5.16
1.1 5.16
NT
NT
NT
NT
6.7
6.4
4.4
0.3
In silico simulation was conducted to uncover the factors
responsible for the quite strong antitubulin activity in vitro of
compound 58. Compound 58 interacted not only with Thr179
0.152
a
b
R2 ¼ H. Estimated by MOE. c Not tested.
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Table 6 Cytotoxicities, S log P values, and metabolic stabilities of 4-chlorocolchicine derivatives
Compounda
No.
IC50 value (nM)
A549
Metabolic stability
R1
HT-29
34.6
HCT116
61.7
S log Pb
(mL minꢁ1 mgꢁ1
)
48
49
50
170
186
247
4.48
NTc
31.6
23.5
6.3
41.7
34.1
13.2
4.48
3.90
3.88
NT
NT
NT
51
52
53
39.6
941
154
177
3.88
3.88
NT
NT
37.4
19.4
33.0
54
55
56
57
10.1
7.6
7.8
5.8
1.1
0.4
0.3
0.7
6.5
2.4
0.9
1.3
4.55
4.85
3.81
4.18
NT
NT
0.188
0.037
58
2.0
0.3
0.3
4.12
0.004
59
60
0.5
0.2
6.0
0.3
8.8
3.21
3.58
0.088
NT
34.8
61
62
40.3
1.7
18.0
0.3
33.9
0.5
3.58
3.52
NT
0.223
63
42.5
5.6
7.3
4.39
NT
a
b
R2 ¼ H. Estimated by MOE. c Not tested.
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Table 7 Inhibition of tubulin assembly
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Compound no.
IC50 value (mM)
1
26
58
8.12
2.36
1.11
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and Lys254 of tubulin through the formation of hydrogen bonds
with the C-7 thioureide side chain and the C-9 carbonyl group,
respectively, but also Asn258 of the tubulin b-subunit through
CH–p interaction with the terminal pyrazole ring at the C-7 side
chain (Fig. 2c). It is speculated that the CH–p interaction with
the pyrazole ring in compound 58 confers potent activity rela-
tive to compound 26.
Conclusions
4-Chlorocolchicine derivative 26, which has an ethylthioureide
side chain at the C-7 position, was found to have high antitumor
activity in vivo and a broad effective dosage range. Furthermore, we
developed a new derivative, 5-methylpyrazol-3-ylthioureide (58),
which exhibited the strongest cytotoxicity to HCT116 cell line in
our series of colchicine studies and possessed desirable metabolic
stability in vitro. Further evaluation of compounds 26 and 58 is in
progress to examine their potential as anticancer agents.
Acknowledgements
The authors are grateful to Dr Seigo Sawada, Dr Ryuta Yama-
zaki, and Akinobu Kurita for useful and helpful discussion. We
also thank Tsuneo Matsumoto, Fukiko Nishisaka, Yukiko
Nishiyama, and Miyuki Uchida for excellent technical assis-
tance. Part of this work was supported by a Grant-in-Aid for
Scientic Research from the Japan Society for the Promotion of
Science.
21 H. Nishiyama, M. Ono, T. Sugimoto, T. Sasai, N. Asakawa,
T. Yaegashi, M. Nagaoka, T. Matsuzaki, N. Kogure,
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28 The maximum dose (MD) was dened as the maximum
single dose that induced less than 20% weight loss when
the derivative was intravenously administered to a mouse
on days 1 and 5.
29 C.-Y. Nien, Y.-C. Chen, C.-C. Kuo, H.-P. Hsieh,
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458 | Med. Chem. Commun., 2014, 5, 452–458
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