Discovery and optimization of 3,4,5-trimethoxyphenyl substituted triazolylthioacetamides as potent tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.11.024

Based on our previous research, three series of new triazolylthioacetamides possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities and inhibition of tubulin polymerization. The most promising compounds 8b and 8j demonstrated more significant antiproliferative activities against MCF-7, HeLa, and HT-29 cell lines than our lead compound 6. Moreover, analogues 8f, 8j, and 8o manifested more potent antiproliferative activities against HeLa cell line with IC₅₀ values of 0.04, 0.05 and 0.16 μM, respectively, representing 100-, 82-, and 25-fold improvements of the activity compared to compound 6. Furthermore, the representative compound, 8j, was found to induce significant cell cycle arrest at the G₂/M phase in HeLa cell lines via a concentration-dependent manner. Meanwhile, compound 8b exhibited the most potent tubulin polymerization inhibitory activity with an IC₅₀ value of 5.9 μM, which was almost as active as that of CA-4 (IC₅₀ = 4.2 μM). Additionally, molecular docking analysis suggested that 8b formed stable interactions in the colchicine-binding site of tubulin.

Full text of DOI:10.1016/j.bmcl.2018.11.024

Accepted Manuscript
Discovery and optimization of 3,4,5-trimethoxyphenyl substituted triazolylth-
ioacetamides as potent tubulin polymerization inhibitors
Fang Yang, Cai-Ping He, Peng-Cheng Diao, Kwon Ho Hong, Jin-Jun Rao, Pei-
Liang Zhao
PII:
S0960-894X(18)30890-4
https://doi.org/10.1016/j.bmcl.2018.11.024
BMCL 26135
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 September 2018
5 November 2018
12 November 2018
Please cite this article as: Yang, F., He, C-P., Diao, P-C., Ho Hong, K., Rao, J-J., Zhao, P-L., Discovery and
optimization of 3,4,5-trimethoxyphenyl substituted triazolylthioacetamides as potent tubulin polymerization
inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.024
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Discovery
and optimization of
3,4,5-trimethoxyphenyl
substituted
triazolylthioacetamides as potent tubulin polymerization inhibitors
Fang Yanga,#, Cai-Ping Hea,#, Peng-Cheng, Diaoa, Kwon Ho Hongb, Jin-Jun Raoa,*, Pei-Liang
Zhaoa,*
aGuangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
bDepartment of Medicinal Chemistry and Institute for Therapeutics Discovery and
Development, College of Pharmacy, University of Minnesota, Minneapolis 55414, United States
ABSTRACT: Based on our previous research, three series of new triazolylthioacetamides
possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative
activities and inhibition of tubulin polymerization. The most promising compounds 8b and 8j
demonstrated more significant antiproliferative activities against MCF-7, HeLa, and HT-29 cell
lines than our lead compound 6. Moreover, analogues 8f, 8j, and 8o manifested more potent
antiproliferative activities against HeLa cell line with IC50 values of 0.04, 0.05 and 0.16 μM,
respectively, representing 100-, 82-, and 25-fold improvements of the activity compared to
compound 6. Furthermore, the representative compound, 8j, was found to induce significant cell
cycle arrest at the G2/M phase in HeLa cell lines via a concentration-dependent manner. Meanwhile,
compound 8b exhibited the most potent tubulin polymerization inhibitory activity with an IC50
value of 5.9 μM, which was almost as active as that of CA-4 (IC50 = 4.2 μM). Additionally,
molecular docking analysis suggested that 8b formed stable interactions in the colchicine-binding
site of tubulin.
Keywords: Triazolylthioacetamides; Synthesis; Tubulin polymerization; Antiproliferative activity.
——————————
 Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao), raojj@smu.edu.cn (J.-J. Rao)
# These authors contributed equally to this work.
1

Microtubules have been a highly attractive therapeutic target for the development of anticancer
agents due to their crucial roles in numerous cellular processes, such as maintenance of cell shape,
regulation of motility, transportation of vesicles and mitochondria, and cell division.1-3 Crucially,
microtubules form highly dynamic spindle fibers required for the segregation of duplicated
chromosomes during mitosis.4,5 Microtubule-targeting agents disrupting microtubule dynamics can
be divided into two classes based on their mode of action: inhibitors of tubulin polymerization and
blockers/inhibitors of microtubule disassembly.6,7 During the last few decades, many efforts have
been devoted to the identification of structurally diverse tubulin inhibitors including both natural
and synthetic molecules.8-13 Among them, epothilone, paclitaxel, and vindesine have already been
launched. However, they have certain limitations in their clinical utility due to
P-glycoprotein-mediated drug resistance, and difficulty in synthesis and isolation.14 Therefore,
development of small moleculars as potent tubulin inhibitors with better therapeutic properties for
clinical use are urgently needed.
Fig. 1. Structures of selected tubulin inhibitors, including CA-4, triazole-based compounds 2-6, and newly
designed compounds 7-9.
Over the past few years, one of the most active is the combretastatin A-4 (CA-4, Fig. 1), which
provides a simple structural template for the development of novel CA-4 analogues as potential
anticancer agents.15-18 To date, 1,2,4-triazole-based CA-4 analogues have attracted increasing
attentions and have been identified as potent tubulin polymerization inhibitors, such as the recently
reported 1H-indolyl-1,2,4-triazole 2, 5-amino-1,2,4-triazole derivative 3, and triazole-containing
analogue 4 possessing submicromolar antiproliferative activities.19-21 More recently, we described
2

two series of 3-alkylsulfanyl-1,2,4-triazole analogues, exemplified by compounds 5 and 6 (Fig. 1),
which exhibited remarkable antiproliferative activities against a panel of cancer cell lines.22,23 Initial
structure–activity relationship (SAR) studies indicated that both the 3,4,5-trimethoxyphenyl group
of the 5-position on the 1,2,4-triazole skeleton and the N-(substituted phenyl)thioacetamide group at
the 3-alkylsulfanyl moiety were essential for an optimal activity. Herein, we investigated the
biological importance of the substitution of 4-position and the N-substitution acetamide moiety of
3-position on the 1,2,4-triazole skeleton by introducing various groups to generate
3,4,5-trimethoxyphenyl substituted triazolylthioacetamides 7a-f, 8a-r, and 9a-e (Fig. 1). These
synthesized compounds were evaluated for their antiproliferative activities, and the most promising
derivatives were further evaluated for the inhibition of tubulin polymerization and the effects on the
cell-cycle. To our knowledge, of the twenty-nine target analogues, compound 8b was already
reported and its growth-inhibitory effect was characterized by us.23 Additionally, effect of these
compounds on tubulin-polymerization has not been reported so far.
The reaction sequence employed for the preparation of 29 triazolylthioacetamides was
depicted in Scheme 1. Briefly, the required diverse N-substituted 2-chloroacetamides were first
3

synthesized by acylation of substituted anilines (10) with chloroacetyl chloride (11).24
3,4,5-Trimethoxybenzohydrazide (14) was prepared from 3,4,5-trimethoxybenzoic acid via a
two-step operation empolying esterification and condensation with hydrazine. The
benzoylhydrazine was then treated with ammonium thiocyanate (NH4SCN) under acidic conditions
to give corresponding aroylthiourea, which was converted into the 4H-1,2,4-triazole-3-thiol (15) at
reflux in the presence of sodium hydroxide. Subsequently, the target triazolylthioacetamides 7a-f
were generated by a nucleophilic substitution reaction under alkaline conditions. Similarly, the
target compounds 8a-r were obtained by the reaction of N-substituted 2-chloroacetamides with
4-amino-1,2,4-triazole-3-thiol (16) which was prepared according to our reported procedure.23
Additionally, the key intermediate (16) was converted into the pyrrole-substituted 1,2,4-triazole (17)
by the reaction with 2,5-dimethoxytetrahydrofuran at reflux. Stirring compound 17 with different
2-chloroacetamides at room temperature afforded the desired compounds 9a-e in moderate to good
isolated yields ranging from 74% to 81%.
The structures of the newly synthesized 3,4,5-trimethoxyphenyl substituted
triazolylthioacetamides 7a-f, 8a-r, and 9a-e were characterized by 1H NMR, 13C NMR, and HRMS
spectroscopic techniques, and the results are shown in the Experimental section. In addition, the
representative compound 8p was further confirmed by single-crystal X-ray diffraction. As shown in
Fig. 2, the crystal structure showed that 8p had a C-shape, while the planes of the triazole and
3,4,5-trimethoxyphenyl ring were nearly parallel, with a dihedral angle of 4.83°.
Fig. 2. Molecular structure of compound 8p (CCDC 1847532).
4

The prepared triazolylthioacetamides were evaluated for their in vitro antiproliferative
activities against four human cancer cell lines, including MCF-7 (mammary adenocarcinoma cells),
HeLa (human cervical cancer cells), HCT116 (human colon carcinoma cells), and HT-29 (human
colon carcinoma cell line) through MTT screening assay. For comparison, a potent tubulin-binding
anticancer agent CA-4 and our previously reported compound 6 were employed as the positive
controls, and the results expressed as IC50 (μM) were summarized in Table 1. Here, the IC50 value
represents the concentration of a compound resulting in 50% inhibition of cell growth after 48 h
incubation with the compound, and is the average of three independent experiments.
Table 1 Cytotoxic activities of compounds 7~9 against human tumor cells.
In vitro cytotoxicity IC50(μM) a
Comp.
n
R
MCF-7
>100
>100
>100
>100
>100
>100
>100
HeLa
>100
HCT116
>100
HT-29
>100
>100
>100
>100
>100
>100
1.21±0.31
0.82±0.23
HEK-293 b
NT c
NT
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
9a
9b
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NCH3
O
4-CH3Ph
4-CH3OPh
4-CH3CH2Ph
4-CH3(CH2)2Ph
3-OH,4-CH3OPh
3-CH3OPh
3,4-(CH3)2Ph
4-CH3Ph
4-CH3OPh
>100
>100
33.89±1.20
>100
>100
>100
NT
NT
NT
NT
NT
NT
NT
NT
>100
>100
>100
>100
0.88±0.27
1.61±0.38
2.96±0.58
54.23±5.49
82.82±10.99
0.04±0.02
54.93±2.74
59.68±7.35
2.96±0.58
>100
3.45±0.47
61.10±6.69 15.16±2.16
26.83±3.96
>100
3,4-(OCH3)2Ph
2,4,6-(CH3)3Ph
2-F,4-CH3Ph
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.13±0.16
75.28±11.26
>100
4.87±1.47
0.84±0.41
>100
NT
61.09±6.12
NT
N
80.64±2.79
47.1±5.94
25.36±4.49
>100
>100
41.22±4.30
>100
NT
NT
>100
NT
NT
NT
NT
>100
NT
NT
3-CH3Ph
4-CH3CH2Ph
4-(CH3)2CH Ph
4- (CH3)3CPh
4-CH3(CH2)2Ph
4-N(CH3)2Ph
2-CH3OPh
3-OH,4-OCH3
4-CH3Ph
4-CH3Ph
4-CH3Ph
4-CH3OPh
0.05±0.017 71.36±6.24
13.73±1.22 74.77±1.63
49.64±12.59
>100
>100
18.67±0.57 41.81±3.32 50.75±8.11
23.82±0.36
0.16±0.05
34.76±3.4
21.32±0.37
>100
>100
82.85±2.11
>100
43.11±3.28
0.13±0.17
>100
>100
>100
2.94±0.02
>100
>100
>100
>100
>100
>100
>100
>100
>100
NT
NT
NT
NH
NH
>100
>100
26.58±3.22
>100
5

9c
9d
9e
NH
/
/
3,4-(CH3O)2Ph
4-CH3Ph
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NT
NT
NT
>100d
NT
4-ClPh
6
52.56±8.05
0.041±0.0013 0.007±0.001 6.10±0.14
4.02±0.31d 0.68±0.09 d
3.15±0.01
1.96±0.62
CA4
a
IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients of variation were
<10%. b Normal human embryonic kidney (HEK-293) cell lines. c NT: not tested.d Data taken from ref.[23].
It was interesting to note that almost all of the N-unsubstituted and N-pyrrole
triazolylthioacetamides 7a-f and 9a-e were inactive (IC50 >100 μM) against four tested cell lines,
while most N-amino substituted analogues 8a-r exhibited moderate to strong antiproliferative
activities against the tested cell lines. Particularly, analogues 8b and 8j displayed higher
antiproliferative activities against MCF-7, HeLa and HT-29 cell lines than our previously reported
compound 6. Moreover, compounds 8f, 8j and 8o showed 100-, 82-, and 25-fold improvement
compared to lead compound 6 in inhibiting HeLa cell proliferation with IC50 values of 0.04, 0.05,
and 0.16 μM, respectively. These results clearly demonstrate that N-amino analogues 8a-r manifest
better antitumor activities against tested cell lines than N-unsubstituted 7a-f and N-pyrrole
derivatives 9a-e. More interestingly, analogues 8f, 8j and 8o exhibited weak inhibitory effect on the
normal human embryonic kidney cells, HEK-293, suggesting that the compound may be selective
against cancer cells.
Within the series of N-amino analogues 8a-r, further analysis clearly revealed that different
antiproliferative activities were observed when various R groups were introduced. Our previous
SAR studies23 indicated that introduction of electron-donating groups on the phenyl group (R) could
improve antiproliferative activities, and the newly synthesized analogue 8b, with 4-methyl
substituent on the phenyl ring, displayed strong antiproliferative activity against four tested cell
lines. However, introduction of more electron-donating groups on the phenyl group results in
significant decrease of antiproliferative activities (8b vs 8d, 8e, 8k, 8l, 8m, 8n). It is worth noting
that replacement of 4-methyl with 4-ethyl group leads to obvious enhancement in antiproliferative
activities against MCF-7, HT-29 and HeLa cell lines (8b vs 8j). Most interestingly, compound 8q
with N-CH3 as a linker, and compound 8r bearing an oxygen atom as a linker almost did not show
any antiproliferative activities, indicating that the NH linker (X) might play a crucial role in
modulating the antitumor activity.
6

In order to investigate the mode of action of these compounds on cancer cells, one selected
analogue 8j was examined for the influence on the cell cycle progression. In this study, HeLa cells
were treated with compound 8j at given concentrations (0.25, 0.5 μM) for 48h. As shown in Fig. 3
and Table 2, the cell cycle analysis results revealed that the G2/M peak significantly increased from
12.91% to 27.30% (0.25 μM), and 54.77% (0.5 μM) after 48 h of treatment. These data suggest that
compound 8j induced a significant cell cycle arrest at the G2/M phase in a concentration-dependent
manner, compared to untreated cells.
A
B
C
Fig. 3. Effect of compound 8j on cell cycle and apoptosis in HeLa cells. Flow cytometry analysis of HeLa cells
stained with propidium iodide and treated with 8j for 48 h. (A) Control; (B) 8j, 0.25 μM; (C) 8j, 0.5 μM.
Table 2 Effect of compound 8j on cell cycle distribution in HeLa cells.
Concentration G0/G1(%)
S(%)
20.31
25.35
2.00
G2/M(%)
12.91
0μM
66.77
47.35
43.23
0.25μM
0.50μM
27.30
54.77
7

Cell cycle analysis results revealed a concentration dependent increase in HeLa cells arrested
at the G2/M phase, which is in accord with the typical propensity of tubulin polymerization
inhibitors. To evaluate whether the newly synthesized triazolylthioacetamides could potentially
interact with tubulin, ten representative analogues were selected for the evaluation of their in vitro
inhibitory activities against tubulin polymerization at 10 μM concentration and CA-4 was also used
as the reference. The results were summarized in Table 3. To some extent, there is correlation with
respect to antitubulin activity and antiproliferative activity of the tested compounds. For example,
two triazolylthioacetamides 8b and 8j were found to be potent anti-cancer agents against all the
tested cell lines, and also exhibited much higher than 35% inhibition of the tubulin polymerization
at a concentration of 10 μM. Notably, compound 8b displayed anti-tubulin activity with an IC50
value of 5.9 μM, which was almost as active as that of CA-4 (IC50 = 4.2 μM).
Table 3 Tubulin polymerization inhibitory activities of representative selected compounds
Tubulin polymerization
Comp.
X
R
% inhibition a
IC50(μM)
b
7c
8a
8b
8c
8i
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
4-CH3CH2Ph
3,4-(CH3)2Ph
4-CH3Ph
4-CH3OPh
3-CH3Ph
4-CH3CH2Ph
4-CH3(CH2)2Ph
2-CH3OPh
3-OH,4-OCH3
4-CH3Ph
11
18
55
24
25
38
19
25
21
17
80
-
-
5.9
-
-
-
-
-
-
-
8j
8m
8o
8p
9a
CA-4
4.22
a Compounds were tested at a final concentration of 10μM. b -: not tested.
The possible binding mode for the most active compound 8b with tubulin was investigated by
docking simulations, using Glide and the pose with the best emodel score (docking score -7.0, glide
emodel -71.1 kcal/mol) was used for MM-GBSA relative binding free energy calculation using
Prime.25,26 As shown in Fig. 4, docking studies suggest various hydrogen bonding and hydrophobic
interactions that appear to play a key role in binding. NH of the 4-methylphenylamine moiety of
compound 8b forms a hydrogen bonding interaction with Thr179 (3.0 Å) of α-subunit. Meanwhile,
the carbonyl of the thioacetamide group establishes hydrogen bonds with Asn258 (2.9 Å) of
8

β-subunit. In addition, the 3,4,5-trimethoxyphenyl triazole moiety of compound 8b occupies the
hydrophobic pocket formed by Tyr202, Val238, Cys241, Leu242, Ala250, Leu252, Leu255, Ala316,
Ala317 and Ile378.
A
B
Fig.4. Docking pose of compound 8b in tubulin with (A) and without (B) the surface of the pocket (PDP ID:
1SA0). Amino acid residues of tubulin beta chain are labeled in yellow and those in tubulin alpha in orange.
MM-GBSA ΔG is -114.0 kcal/mol.
In conclusion, based on our previously reported antiproliferative compound 6, three series of
novel triazolylthioacetamide derivatives bearing 3,4,5-trimethoxyphenyl were synthesized and
evaluated for their antiproliferative activities. The most promising compounds 8b and 8j displayed
higher antiproliferative activities against MCF-7, HeLa and HT-29 cell lines than lead compound 6.
Moreover, compounds 8f, 8j, and 8o showed more potent antiproliferative activities against HeLa
cell proliferation with IC50 values of 0.04, 0.05 and 0.16 μM, respectively, representing 100-, 82-,
and 25-fold improvement compared to lead compound 6. Additionally, in mechanistic studies, the
9

representative compound 8j was found to induce significant cell cycle arrest at the G2/M phase in
HeLa cell lines in a concentration-dependent manner. Notably, the compound 8b exhibited the most
potent antitubulin activity with an IC50 value of 5.9 μM, almost as active as that of CA-4 (IC50 = 4.2
μM). Furthermore, docking studies revealed that 8b, as
a
typical potent
tubulin polymerization inhibitor, formed stable interactions in the colchicine binding site in the
tubulin. These preliminary results encourage further investigation on triazolylthioacetamides aiming
to develop new potential tubulin polymerization inhibitors with anticancer activity.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China
(No. 21372113), and the Science and Technology Program of Guangzhou, China (No.
201707010198).
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmcl.2018.xx.xxx.
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12

Discovery
and optimization of
3,4,5-trimethoxyphenyl
substituted
triazolylthioacetamides as potent tubulin polymerization inhibitors
Fang Yanga,#, Cai-Ping Hea,#, Peng-Cheng, Diaoa, Kwon Ho Hongb, Jin-Jun Raoa,*, Pei-Liang
Zhaoa,*
aGuangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
bDepartment of Medicinal Chemistry and Institute for Therapeutics Discovery and
Development, College of Pharmacy, University of Minnesota, Minneapolis 55414, United States
13

Highlights
►
Based on our previous work, 29 novel triazolylthioacetamides possessing
3,4,5-trimethoxylphenyl groups were synthesized. ► 8f and 8j showed potent antiproliferative
activities against Hela with IC50 values of 0.04 and 0.05 μM, respectively. ► 8b exhibited potent
antitubulin activity with IC50 values of 5.9 μM, which was proximate to CA-4 (IC50 = 4.2 μM).
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