Design, synthesis, and biological evaluation of novel alkylsulfanyl-1,2,4-triazoles as cis-restricted combretastatin A-4 analogues

Article abstract of DOI:10.1016/j.ejmech.2016.10.051

Thirty-two novel 3-alkylsulfanyl-1,2,4-triazole derivatives, designed as cis-restricted combretastatin A-4 analogues, were synthesized and evaluated for their antiproliferative activities. The results indicated that analogue 20 showed more potent antiproliferative activities against PC-3?cell lines than positive control CA-4. Particularly, the most promising compound 25 displayed 5-fold improvement compared to CA-4 in inhibiting HCT116?cell proliferation with IC₅₀values of 1.15?μM. Further flow-activated cell sorting analysis revealed that compound 20 displayed a significant effect on G₂/M cell-cycle arrest in a dose-dependent manner in PC-3?cells. From this study, analogues 20 and 25 were the most potent anti-cancer agents in this structural class, and were considered lead compounds for further development as anti-cancer drugs.

Full text of DOI:10.1016/j.ejmech.2016.10.051

Accepted Manuscript
Design, synthesis, and biological evaluation of novel alkylsulfanyl-1,2,4-triazoles as
cis-restricted combretastatin A-4 analogues
Yan-Hong Li, Bei Zhang, Hai-Kui Yang, Qiu Li, Peng-Cheng Diao, Wen-Wei You, Pei-
Liang Zhao
PII:
S0223-5234(16)30913-8
10.1016/j.ejmech.2016.10.051
EJMECH 9016
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2016
Revised Date: 25 September 2016
Accepted Date: 21 October 2016
Please cite this article as: Y.-H. Li, B. Zhang, H.-K. Yang, Q. Li, P.-C. Diao, W.-W. You, P.-L. Zhao,
Design, synthesis, and biological evaluation of novel alkylsulfanyl-1,2,4-triazoles as cis-restricted
combretastatin A-4 analogues, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.10.051.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Design,
Synthesis,
and
Biological
Evaluation
of
Novel
Alkylsulfanyl-1,2,4-triazoles as Cis-Restricted Combretastatin A-4
Analogues
Yan-Hong Li#, Bei Zhang#, Hai-Kui, Yang, Qiu Li, Peng-Cheng Diao, Wen-Wei You*, Pei-Liang
Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science,
Southern Medical University, Guangzhou 510515, P.R.China
N
N
N
Bridge
N
N
S
N
A
O
Bridge-
modifications
N
S-Linker
2
B
N
N
1,2,4-triazole
scaffold
OH
N
O
N
25
HCT116: IC50 = 1.15
1 (CA-4)
R
3: R = OCH3
4: R = N(CH3)2

ACCEPTED MANUSCRIPT
Design,
Synthesis,
and
Biological
Evaluation
of
Novel
Alkylsulfanyl-1,2,4-triazoles as Cis-Restricted Combretastatin A-4
Analogues
5
Yan-Hong Li#, Bei Zhang#, Hai-Kui, Yang, Qiu Li, Peng-Cheng Diao, Wen-Wei You*, Pei-Liang
Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science,
Southern Medical University, Guangzhou 510515, P.R.China
10
ABSTRACT: Thirty-two novel 3-alkylsulfanyl-1,2,4-triazole derivatives, designed as cis-restricted
combretastatin A-4 analogues, were synthesized and evaluated for their antiproliferative activities.
The results indicated that analogue 20 showed more potent antiproliferative activities against PC-3
cell lines than positive control CA-4. Particularly, the most promising compound 25 displayed
5-fold improvement compared to CA-4 in inhibiting HCT116 cell proliferation with IC50 values of
1.15 ꢀM. Further flow-activated cell sorting analysis revealed that compound 20 displayed a
significant effect on G2/M cell-cycle arrest in a dose-dependent manner in PC-3 cells. From this
study, analogues 20 and 25 were the most potent anti-cancer agents in this structural class, and were
considered lead compounds for further development as anti-cancer drugs.
15
20
Keywords: Alkylsulfanyl-1,2,4-triazoles; Synthesis; Antiproliferative activity
——————————
Corresponding author. Tel./fax: +86(0)20 61648196.
25
Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao), youww@smu.edu.cn (W.-W.
You)
# These authors contributed equally to this work.
1

ACCEPTED MANUSCRIPT
1. Introduction
30
35
40
45
50
Natural products are widely used as lead compounds for the discovery of new drugs with novel
structures and mechanisms, which is reflected by the fact that increasing commercial drugs are
either natural products, or their derivatives [1–5]. Combretastatin A-4 (CA-4, Figure 1) is among the
most well-known anticancer agents, originally isolated from the South African bush willow tree,
Combretum caffrum Kuntze (Combretaceae) [6,7], and its water-soluble prodrug (CA-4P) has now
shown promising results in human cancer clinical trials [8]. The discovery of CA-4 has led to a
diverse library of antitumor agents designed, due to its simple structure and great anticancer
potency [9–16].
The structure-activity relationship (SAR) studies indicated that the presence of a
3,4,5-trimethoxyphenyl ring-A and cis-double bond is essential for potent antiproliferative activity
[17]. As a result, a significant number of the conformationally restricted analogues, obtained by
incorporating the cis-olefin bridge into a heterocyclic ring system, have been investigated [18–25].
Among them, 1,2,4-triazole-containing analogues (2, 3, 4) have been reported to possess potent
antiproliferative activities comparable to CA-4 [26]. Besides, many biologically active compounds
and drugs comprise the S-linkers which were reported to improve important drug-like parameters:
decrease lipophilicity, increase water solubility, and serve as good hydrogen bond acceptors [27].
Thus, in view of the previous rationale, we developed an idea that introducing diverse alkylsulfanyl
as S-linkers into the C3-position of the analogues 3 and 4 might result in an interesting scaffold
structure with potent anticancer activities (Fig. 1). Herein, we described the synthesis and
antiproliferative activity of a series of 3-alkylsulfanyl-1,2,4-triazoles as cis-restricted CA-4
analogues 5~36. To our knowledge, in the thirty-two target analogues, only two compounds 5 and
18 have been reported [28], but their antiproliferative activity has not been tested so far.
<Insert figure 1 here>
55
2. Chemistry
The synthetic route of the 3-alkylsulfanyl-1,2,4-triazole derivatives (5~36) was outlined in
Scheme 1. 3,4,5-trimethoxybenzoic acid (A) was treated with concentrated sulfuric acid in
2

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anhydrous ethanol at reflux to yield ethyl 3,4,5-trimethoxybenzoate (B), which was then converted
to hydrazide (C) in almost quantitative yield, according to our previously reported approach [29].
The condensation of the hydrazide with the commercially available suitably substituted
isothiocyanates resulted in the formation of the intermediates (D), which then underwent
intramolecular ring closure to generate the corresponding substituted 4H-1,2,4-triazole-3-thiol ring
5–6 in the presence of sodium hydroxide as base in refluxing water, as reported in the literature [30].
Subseqently, analogues 7~36 were easily accessible by S-alkylation directly from intermediates 5~6
with various commercial halogen compounds using K2CO3 as base in anhydrous
N,N-dimethylformamide at room temperature. All of the synthesized target compounds were given
satisfactory analytical and spectroscopic data, which were in full accordance with their depicted
structures.
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65
70
<Insert scheme 1 here>
3. Pharmacology Results and Discussion
All synthesized analogues, 5~36, were tested for in vitro cytotoxic activity against a panel of
human tumor cell lines by MTT method [31]. Cell lines included HT-29 (human colon carcinoma
75
cells), HepG2 (human hepatoma cells), PC-3 (human prostate cancer cell lines), and HCT116
(human colon cancer cell lines). CA-4 was selected as a positive control and the results expressed as
IC50 (ꢀM) were summarized in Table 1. Here, the IC50 value represents the concentration of one
compound resulting in a 50% inhibition in cell growth after a 48 h incubation, and is the average of
three independent experiments.
80
<Insert table 1 here>
For the sake of convenience, 4-methoxy and N,N-dimethyl analogues were used to represent
compounds 5~20 and 21~36, respectively, throughout the text. Among the 4-methoxy analogues,
initially, the importance of substitutions on alkylsulfanyl group was simply explored. As indicted in
Table 1, the results displayed that the alkylsulfanyl moiety and its substitutions were critical for
3
85

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keeping antiproliferative effect and the antiproliferative activities were almost lost when the thiol
group was free or replaced by methylthio or ethylthio (5, 6 and 7). Secondly, the free thiol group
was subsequently converted to other alkylthios, including benzylthio, acetylthio, and acetamidethio
(8~11, 12~18 and 19~20) in order to investigate the influence on the antiproliferative activity. As
presented in Table 1, when changing the thiol group of the 3-position of triazole ring with
benzylthio (8~11), the cytotoxic activities against HepG2, PC-3 and HCT116 cells were
significantly increased by the chain elongation. Meanwhile, the introduction of electron
withdrawing groups such as fluoro atom on the benzyl group (11), also caused a slight enhancement
of the antiproliferative activity. These results suggest that electronic effect of substituents on benzyl
group plays a crucial role on antitumor activities. Besides, linker-length of alkylsulfanyl moiety has
also profound effects on the antiproliferative activities. Introduction of phenyl acetylthio substitutes
on the 3-position of triazole ring (12~14) leads to dramatical enhancement of antiproliferative
activities against HepG2 cell lines, but naphthyl, cyclopropyl and ethoxyl groups (16~18) result in
dramatical decrease of the activities. It is worth noting that compound 20, with N-4-chlorophenyl
acetamidethio substitute, showed more potent in vitro cytotoxic activities against PC-3 with IC50
values of 6.29 ꢀM, which represented 3-fold improvement in activity compared to CA-4. Moreover,
further flow-activated cell sorting analysis revealed that compound 20 displayed a significant effect
on G2/M cell-cycle arrest in a dose-dependent manner in PC-3 cells.
90
95
100
105
Within the series of N,N-dimethyl analogues (21~32), the effects of substituents on the
antiproliferative activities was strongly correlated with the 4-methoxy analogues. Meanwhile,
analogue 25 was an exception, which displayed 5-fold improvement compared to CA-4 in inhibiting
HCT116 cell proliferation with IC50 values of 1.15 ꢀM.
In order to investigate the mode of action of these compounds on cancer cells, one selected
analog 20 was examined for its influence on the cell cycle progression. In this study, PC-3 cells
were treated with compound 20 at 3, 6, 12 ꢀM concentrations for 48 h. As shown in Figure 2 and
Table 2, the G2/M peak significantly increased from 15.81% to 20.06% (3 ꢀM), 24.23% (6 ꢀM),
and 34.80% (12 ꢀM) after 12 h of treatment. These cell cycle analysis results revealed that the
compound arrested the cell cycle at G2/M phase in a dose-dependent manner, when compared to
untreated control cells.
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115
4

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<Insert figure 2 here>
<Insert table 2 here>
120
125
130
135
140
Based on the obtained IC50 values against the four cancer cell lines, represent fourteen active
analogues were evaluated for in vitro inhibitory effects on tubulin polymerization at 10 ꢀM
concentration and CA-4 was also used as the reference and the results expressed as as a percentage
were summarized in Table 2. Interestingly, to some extent, there is correlation with respect to
anti-tubulin activity and cytotoxicity. For example, compound 25 (most active analogues) also
displayed the most potent antitubulin activity with an inhibition percentage of 49% at 10ꢀM
concentration. Meanwhile, compound 20 also exhibited significant inhibition of tubulin
polymerization with a percentage of 31%.
4. Conclusion
In conclusion, by introducing diverse alkylsulfanyl as S-linkers, a series of novel
alkylsulfanyl-1,2,4-triazoles as cis-restricted CA-4 analogues exhibiting significantly
antiproliferative activities were successfully identified. Analogue 20 showed more potent
antiproliferative activities against PC-3 cell lines than positive control CA-4. Particularly, the most
promising compound 25 displayed 5-fold improvement compared to CA-4 in inhibiting HCT116
cell proliferation with IC50 values of 1.15 ꢀM. More interestingly, the analogue 25 also displayed
the most potent antitubulin activity with a percentage of 49% at 10ꢀM concentration. Moreover,
further flow-activated cell sorting analysis revealed that compound 20 displayed a significant effect
on G2/M cell-cycle arrest in a dose-dependent manner in PC-3 cells. Further research on the
mechanisms of these compounds and modification is underway.
5. Experimental protocols
5.1 Chemistry
Unless otherwise noted, all chemical and biological reagents were purchased from commercial
suppliers and used without further purification while solvents were dried in a routine way and
redistilled before use. 1H and 13C NMR spectra were recorded on a Mercury-Plus 400 spectrometer
145
5

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in CDCl3 or DMSO-d6 solution and chemical shifts were recorded in parts per million (ppm) with
TMS
as
the
internal
reference.
ESI-MS/MS spectra were
recorded
using an Agilent QTOF 6540 mass spectrometer. The melting points were taken on a Buchi B-545
melting point apparatus and are uncorrected. The yields were not optimized.
150
5.1.1 General procedure for the target compounds 5~6
To a 100 mL round-bottom flask, 3,4,5-trimethoxybenzoic acid (10.6 g, 50 mmol), 1 mL of
concentrated sulfuric acid and 30 mL of ethanol were added gradually. The resulting mixture was
stirred at reflux for 12 h and was then concentrated on a rotary evaporator. The crude product
obtained was poured into 50 ml of water and extracted with ethyl acetate. The combined ethyl
acetate extracts were washed with water, dried with anhydrous magnesium sulfate, filtered off by
suction and the solvent was evaporated to give crude ethyl 3,4,5-trimethoxybenzoate, which and
hydrazine hydrate (7.5 g, 150 mmol, 60%) in 50 mL of ethanol was heated under reflux for 6 h.
Excess ethanol was distilled out and the contents were allowed to cool. The solid product obtained
was filtered, washed thoroughly with water, and dried to give 3,4,5-trimethoxybenzohydrazide in
total yield of 83%, which was used for the next reactions without further purification. A solution of
3,4,5-trimethoxybenzohydrazide (3.39 g, 15 mmol) and appropriate isothiocyanatobenzenes (15
mmol) in ethanol (40 mL) was heated at reflux for 1 h, then cooled at room temperature. The
suspension was filtered, and the solid was washed with ethanol and dried to give the intermediate
hydrazinecarbothioamide as white solid. Then 1 N NaOH (30 mL) was added to this solid and the
mixture was heated at reflux for 1 h. The resulting solution was cooled at room temperature and
acidified to pH 5-6 with 1 M HCl. The precipitate was filtered, washed with water, and dried to
obtain the title compounds as white solid.
155
160
165
170
5.1.1.1 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol (5).Yield, 76%;
1
mp 245.8–247.3°C (Lit.28 252–253°C); H NMR (400 MHz, DMSO-d6) δ: 3.57 (s, 6H, OCH3-3'',
-5''), 3.65 (s, 3H, OCH3-4'), 3.81 (s, 3H, OCH3-4''), 6.62 (s, 2H, trimethoxyPh-H), 7.07 (d, J = 8.8
Hz, 2H, ArH), 7.30 (d, J = 8.8 Hz, 2H, ArH), 14.06 (br, 1H, SH). 13C NMR (100 MHz, DMSO-d6) δ:
55.9, 56.1, 60.5, 106.1, 115.0, 121.3, 127.8, 130.5, 139.3, 150.7, 153.0, 160.1, 169.3. HRMS (ESI)
m/z: calcd for C18H19N3O4S, [M+H]+ 374.11298, found 374.11702.
5.1.1.2 4-(4-methoxyphenyl)-3-(methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole(6).Yield,
6

ACCEPTED MANUSCRIPT
175
180
185
93%; mp 100.9–101.2°C; 1H NMR (400 MHz, CDCl3) δ: 2.73 (s, 3H, SCH3), 3.66 (s, 6H, OCH3-3'',
-5''), 3.84 (s, 3H, OCH3-4'), 3.88 (s, 3H, OCH3-4''), 6.70 (s, 2H, trimethoxyPh-H), 7.03 (d, J = 8.8
Hz, 2H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 14.8, 56.0, 56.1,
60.5, 105.7, 115.5, 122.3, 126.9, 129.6, 138.8, 153.0, 153.5, 154.6, 160.6. HRMS (ESI) m/z: calcd
for C19H21N3O4S, [M+H]+ 388.12863, found 388.13213.
5.1.2 General procedure for the target compounds 7~36
A mixture of 4-substituted-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiols 5 or 6 (1.0
mmol), various commercial halogen compounds (1.0 mmol) and potassium carbonate (K2CO3)
(0.15 g, 1.1 mmol) in 5.0 mL of anhydrous N,N-dimethylformamide was stirred at room
temperature for 1.0–2.0 h. After the reaction was complete according to the TLC detection, the
mixture was quenched with water, and then the mixture extracted with ethyl acetate. The organic
layer was separated, washed with water and brine, dried over anhydrous magnesium sulfate, filtered,
and then concentrated to give the crude product followed by recrystallation from ethanol to afford
the target compounds in yields of 62–94%.
5.1.2.1 3-(ethylthio)-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole (7). Yield,
78%; mp 79.3–80.5°C; 1H NMR (400 MHz, CDCl3) δ: 1.47 (t, J = 7.2 Hz, 3H, SCH2CH3), 3.35 (dd,
J1 = 7.2 Hz, J2 = 14.8 Hz, 2H, SCH2CH3), 3.69 (s, 6H, OCH3-3'', -5''), 3.86 (s, 3H, OCH3-4''), 3.90
(s, 3H, OCH3-4'), 6.85 (s, 2H, trimethoxyPh-H), 7.08 (d, J = 8.8 Hz, 2H, ArH), 7.26 (d, J = 8.8Hz,
2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 15.2, 26.8, 56.0, 56.1, 60.5, 105.7, 115.4, 122.3,
127.0, 129.7, 138.8, 152.5, 153.0, 154.5, 160.5. HRMS (ESI) m/z: calcd for C20H23N3O4S, [M+H]+
402.14428, found 402.15155.
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5.1.2.2 3-(benzylthio)-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole (8). Yield,
1
81%; mp 109.4–111.2°C; H NMR (400 MHz, DMSO-d6) δ: 3.57 (s, 6H, OCH3-3'', -5''), 3.65 (s,
3H, OCH3-4'), 3.80 (s, 3H, OCH3-4''), 4.41 (s, 2H, SCH2), 6.68 (s, 2H, trimethoxyPh-H), 7.07 (d, J
= 8.4 Hz, 2H, ArH), 7.37 –7.24 (m,7H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 36.5, 56.0, 60.5,
105.7, 115.4, 122.2, 126.8, 127.9, 128.9, 129.4, 129.6, 137.5, 138.8, 152.2, 153.0, 154.6, 160.5.
HRMS (ESI) m/z: calcd for C25H25N3O4S, [M+H]+ 464.15993, found 464.16431.
200
5.1.2.3 4-(4-methoxyphenyl)-3-((4-methylbenzyl)thio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole
(9). Yield, 82%; mp 97.7–98.4°C; 1H NMR (400 MHz, CDCl3) δ: 2.33 (s, 3H, Ph-CH3), 3.65 (s, 6H,
7

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OCH3-3'', -5''), 3.85 (s, 6H, OCH3-4'', -4',), 4.47 (s, 2H, SCH2), 6.70 (s, 2H, trimethoxyPh-H), 6.97
205
(d, J = 8.8 Hz, 2H, ArH), 7.09 (dd, J1= 8.4 Hz, J2 = 14.4 Hz, 4H, ArH), 7.27 (d, J = 7.6 Hz,2H,
13
ArH). C NMR (100 MHz, DMSO-d6) δ: 21.1, 36.3, 56.0, 56.1, 60.5, 105.7, 115.4, 122.2, 126.9,
129.3, 129.4, 129.6, 134.4, 137.1, 138.9, 152.2, 153.0, 154.6, 160.5. HRMS (ESI) m/z: calcd for
C26H27N3O4S, [M+H]+ 478.17558, found 478.17993.
5.1.2.4 3-((4-methoxybenzyl)thio)-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4
-triazole (10). Yield, 93%; mp120.6–121.8°C; 1H NMR (400 MHz, CDCl3) δ: 3.65 (s, 6H, OCH3-3'',
-5''), 3.80 (s, 3H, OCH3-4''), 3.85 (s, 6H, OCH3-4', PhCH2OCH3), 4.46 (s, 2H, SCH2), 6.70 (s, 2H,
trimethoxyPh-H), 6.83 (d, J = 8.4 Hz, 2H, ArH), 6.97 (d, J = 8.4 Hz, 2H, ArH), 7.08 (d, J = 8.4 Hz,
2H, ArH), 7.30 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 36.1, 55.5, 56.0, 56.1,
60.5, 105.7, 114.3, 115.4, 122.3, 126.9, 129.2, 129.6, 130.7, 138.9, 152.3, 153.0, 154.5, 159.1,
160.5. HRMS (ESI) m/z: calcd for C26H27N3O5S, [M+H]+ 494.17050, found 494.17495.
210
215
5.1.2.5 3-((4-fluorobenzyl)thio)-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole
(11). Yield, 79%; mp 116.1–117.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.57 (s, 6H, OCH3-3'', -5''),
3.65 (s, 3H, OCH3-4'), 3.80 (s, 3H, OCH3-4''), 4.39 (s, 2H, SCH2), 6.66 (s, 2H, trimethoxyPh-H),
7.16–7.06 (m, 4H, ArH), 7.27 (d, J= 8.8 Hz, 2H, ArH), 7.42 (dd, J1= 5.4 Hz, J2= 8.4 Hz, 2H, ArH).
13C NMR (100 MHz, DMSO-d6) δ: 35.5, 56.0, 60.5, 105.7, 115.4, 115.5, 115.7, 122.2, 126.8, 129.6,
131.4, 131.5, 133.9, 134.0, 138.9, 152.1, 153.0, 154.6, 160.5, 160.7, 163.1. HRMS (ESI) m/z: calcd
for C25H24FN3O4S, [M+H]+ 482.15051, found 482.15576.
220
5.1.2.6 2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1-phenyleth
1
anone (12). Yield, 85%; mp 125.6–126.3°C; H NMR (400 MHz, DMSO-d6) δ: 3.58 (s, 6H,
225
OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4'), 3.83 (s, 3H, OCH3-4''), 4.92 (s, 2H, SCH2), 6.66 (s, 2H,
trimethoxyPh-H), 7.13 (d, J = 8.8Hz, 2H, ArH), 7.39 (d, J = 8.8Hz, 2H, ArH), 7.58 (t, J = 7.6
13
Hz,2H, ArH), 7.70 (t, J = 7.4 Hz,1H, ArH), 8.03 (t, J = 4.0 Hz, 2H, ArH). C NMR (100 MHz,
DMSO-d6) δ: 56.1, 60.6, 105.8, 115.6, 122.1, 126.7, 128.8, 129.3, 129.6, 134.3, 135.7, 138.9, 152.2,
153.1, 154.7, 160.7, 193.6. HRMS (ESI) m/z: calcd for C26H25N3O5S, [M+H]+ 492.15485, found
492.16234.
230
5.1.2.7 1-(4-methoxyphenyl)-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-
8

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3-yl)thio)ethanone (13). Yield, 88%; mp146.6–147.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.58 (s,
6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4'), 3.82 (s, 3H, OCH3-4''), 3.87 (s, 3H, COPh-OCH3), 4.85
(s, 2H, SCH2), 6.66 (s, 2H, trimethoxyPh-H), 7.10 (dd, J1 = 9.0 Hz, J2 = 14.2 Hz, 4H, ArH), 7.39 (d,
J = 9.2 Hz,2H, ArH), 8.00 (d, J = 8.8 Hz,2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 56.1, 60.5,
105.7, 114.5, 115.5, 122.2, 126.8, 128.6, 129.6, 131.2, 138.9, 152.1, 153.0, 154.6, 160.6, 164.0,
191.9. HRMS (ESI) m/z: calcd for C27H27N3O6S, [M+H]+ 522.16541, found 522.17349.
235
5.1.2.8 1-(4-fluorophenyl)-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-
1
yl)thio)ethanone (14). Yield, 86%; mp 178.5–180.2°C; H NMR (400 MHz, DMSO-d6) δ: 3.58 (s,
240
6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4'), 3.82 (s, 3H, OCH3-4''), 4.89 (s, 2H, SCH2), 6.66 (s, 2H,
trimethoxyPh-H), 7.12 (d, J = 8.4 Hz, 2H, ArH), 7.40 (t, J = 9.4 Hz, 4H, ArH), 8.12 (t, J = 7.0 Hz,
13
2H, ArH). C NMR (100 MHz, DMSO-d6) δ: 56.1, 60.5, 105.7, 115.5, 116.2, 116.4, 122.2, 126.8,
129.6, 131.9, 132.0, 132.5, 138.9, 152.0, 153.0, 154.6, 160.6, 164.4, 167.0, 192.3. HRMS (ESI) m/z:
calcd for C26H24FN3O5S, [M+H]+ 510.14542, found 510.15315.
245
5.1.2.9 1-(3-hydroxy-4-methoxyphenyl)-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-
1,2,4-triazol-3-yl)thio)ethanone (15). Yield, 68%; mp176.6–177.5°C; 1H NMR (400 MHz,
DMSO-d6) δ: 3.58 (s, 6H, OCH3-3'', -5''), 3.66 (s, 3H, OCH3-4'), 3.83 (s, 3H, OCH3-4''), 3.88 (s, 3H,
OHPh-OCH3), 4.83 (s, 2H, SCH2), 6.67 (s, 2H, trimethoxyPh-H), 7.07 (d, J = 8.8 Hz, 1H, ArH),
7.13 (d, J = 8.4 Hz, 2H, ArH), 7.40 (d, J = 5.2 Hz, 3H, ArH), 7.56 (d, J = 8.0 Hz,1H, ArH),9.49 (s,
1H, OH). 13C NMR (100 MHz, DMSO-d6) δ: 56.0, 56.1, 56.2, 60.5, 105.7, 111.7, 115.0, 115.5,
122.1, 122.2, 126.8, 128.7, 129.6, 138.9, 146.9, 152.2, 153.0, 154.6, 160.6, 192.0. HRMS (ESI) m/z:
calcd for C27H27FN4O4S, [M+H]+ 538.1540, found 538.1231.
250
5.1.2.10 2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1-(naphth
1
alen-2-yl)ethanone (16). Yield, 67%; mp 165.4–166.8°C; H NMR (400 MHz, DMSO-d6) δ: 3.58
255
(d, J = 3.6 Hz, 6H, OCH3-3'', -5''), 3.65 (d, J = 3.6 Hz, 3H, OCH3-4'), 3.82 (d, J = 3.6 Hz,3H,
OCH3-4''), 4.96 (d, J = 3.6 Hz, 2H, SCH2), 6.65 (d, J = 3.6 Hz, 2H, trimethoxyPh-H), 7.10 (dd, J1 =
4.0 Hz, J2 = 8.4 Hz, 2H, ArH), 7.32 (dd, J1 = 3.8 Hz, J2 = 8.6 Hz, 2H, ArH), 7.65 (dd, J1 = 6.4 Hz,
J2 = 18.8 Hz, 3H, ArH), 8.04 (s, 1H, ArH), 8.25–8.19 (m, 2H, ArH), 8.42 (s, 1H, ArH).13C NMR
(100 MHz, DMSO-d6) δ: 42.6, 56.0, 56.1, 60.5, 105.7, 115.5, 122.1, 125.2, 125.7, 126.7, 127.0,
128.3, 128.9, 129.1, 129.5, 129.9, 133.4, 133.9, 134.2, 138.9, 152.2, 153.0, 154.6, 160.6, 197.1.
260
9

ACCEPTED MANUSCRIPT
HRMS (ESI) m/z: calcd for C30H27N3O5S, [M+H]+ 542.17050, found 542.17429.
5.1.2.11 1-cyclopropyl-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)
thio)ethanone (17). Yield, 66%; mp 138.5–140.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 0.94 (dd, J1
= 5.6 Hz, J2 = 23.6 Hz, 4H, cyclopropyl CH2), 2.28–2.25 (m, 1H, cyclopropyl CH), 3.58 (s, 6H,
OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4'), 3.82 (s, 3H, OCH3-4''), 4.39 (s, 2H, SCH2), 6.66 (s, 2H,
trimethoxyPh-H), 7.13 (d, J = 8.8 Hz, 2H, ArH), 7.38 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (100
MHz, DMSO-d6) δ: 11.4, 19.9, 42.6, 56.0, 56.1, 60.5, 105.7, 115.5, 122.2, 126.8, 129.6, 138.9,
152.1, 153.0, 154.6, 160.6, 203.9. HRMS (ESI) m/z: calcd for C23H25N3O5S, [M+H]+ 456.15485,
found 456.16171.
265
270
5.1.2.12 ethyl2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)
acetate (18). Yield, 79%; mp104.3–106.0°C (Lit.28 103°C); 1H NMR (400 MHz, DMSO-d6) δ: 1.20
(t, J= 7.0 Hz, 3H, CO2CH2CH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (d, J = 1.2 Hz, 3H, OCH3-4'),
3.82 (s, 3H, OCH3-4''), 4.08 (s, 2H, SCH2), 4.13 (dd, J1 = 7.0 Hz, J2 = 14.2 Hz, 2H, CO2CH2CH3),
6.67 (s, 2H, trimethoxyPh-H), 7.13 (d, J = 7.6 Hz, 2H, ArH), 7.38 (d, J = 8.0 Hz, 2H, ArH).13C
NMR (100 MHz, DMSO-d6) δ: 14.4, 34.3, 56.1, 60.5, 61.7, 105.8, 115.5, 122.1, 126.7, 129.6, 138.9,
151.8, 153.0, 154.7, 160.7, 168.5. HRMS (ESI) m/z: calcd for C22H25N3O6S [M+H]+: 460.14976,
found 460.15305.
275
5.1.2.13 N-(4-methoxyphenyl)-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
1
triazol-3-yl)thio)acetamide (19). Yield, 94%; mp 209.8–210.9°C; H NMR (400 MHz, CDCl3) δ:
280
3.66 (s, 6H, OCH3-3'', -5''), 3.79 (s, 3H, OCH3-4'), 3.86 (s, 3H, OCH3-4''), 3.89 (s, 3H,
NHPh-OCH3), 4.18 (s, 2H, SCH2), 6.72 (s, 2H, trimethoxyPh-H), 6.83 (s, 1H, ArH), 6.83 (s, 1H,
ArH), 6.86 (s, 1H, ArH), 7.04 (s, 2H, ArH), 7.06 (s, 1H, ArH), 7.25 (d, J = 3.2 Hz, 1H, ArH), 7.28
(s, 1H, ArH), 7.60 (s, 1H, ArH), 7.63 (s, 1H, ArH), 10.34 (br, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 37.2, 55.6, 56.0, 56.1, 60.5, 105.7, 114.3, 115.5, 121.1, 122.2, 126.8, 129.6, 132.4,
138.9, 152.3, 153.0, 154.6, 155.8, 160.6, 165.4. HRMS (ESI) m/z: calcd for C27H28N4O6S, [M+H]+
537.17631, found 537.18231.
285
5.1.2.14 N-(4-chlorophenyl)-2-((4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-
1
3-yl)thio)acetamide (20). Yield, 93%; mp 230.6–232.3°C; H NMR (400 MHz, DMSO-d6) δ: 3.58
10

ACCEPTED MANUSCRIPT
(s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4'), 3.82 (s, 3H, OCH3-4''), 4.18 (s, 2H, SCH2), 6.67 (s,
290
2H, trimethoxyPh-H), 7.11 (d, J = 8.8 Hz, 2H, ArH), 7.39 (dd, J1 = 3.8 Hz, J2 = 8.6 Hz, 4H, ArH),
13
7.61 (d, J = 8.8 Hz, 2H, ArH), 10.50 (br, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 37.2, 56.0,
56.1, 60.5, 105.7, 115.5, 121.1, 122.1, 126.7, 127.5, 129.1, 129.6, 138.2, 138.9, 152.2, 153.0, 154.7,
160.6, 166.1. HRMS (ESI) m/z: calcd for C26H25ClN4O5S, [M+H]+ 541.12677, found 541.13289.
5.1.2.15 4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol
1
295
(21). Yield, 79%; mp 224.5–225.8°C; H NMR (400 MHz, DMSO-d6) δ: 2.94 (s, 6H, 2×NCH3),
3.57 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 6.64 (s, 2H, trimethoxyPh-H), 6.80 (d, J = 8.8
Hz, 2H, ArH), 7.13 (d, J= 8.8 Hz, 2H, ArH), 14.00 (br,1H, SH). 13C NMR (100 MHz, DMSO-d6) δ:
40.6, 56.1, 60.5, 106.0, 112.6, 121.4, 123.3, 129.6, 139.2, 150.7, 151.7, 153.0, 169.5. HRMS (ESI)
m/z: calcd for C19H22N4O3S, [M+H]+ 387.14462, found 387.15019.
300
5.1.2.16 N,N-dimethyl-4-(3-(methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)ani line
1
(22). Yield, 81%; mp 184.6–187.5°C; H NMR (400 MHz, CDCl3) δ: 2.75 (s, 3H, SCH3), 3.05 (s,
6H, 2×NCH3), 3.69 (s, 6H, OCH3-3'', -5''), 3.86 (s, 3H, OCH3-4''), 6.79 (d, J= 9.2 Hz, 2H, ArH),
13
6.87 (s, 2H, trimethoxyPh-H), 7.13 (d, J= 9.2 Hz, 2H, ArH). C NMR (100 MHz, DMSO-d6) δ:
14.7, 56.1, 60.5, 105.6, 112.8, 122.1, 122.5, 128.7, 138.7, 151.4, 153.0, 153.9, 154.6. HRMS (ESI)
m/z: calcd for C20H24N4O3S, [M+H]+ 401.16027, found 401.16721.
305
5.1.2.17 4-(3-(ethylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)-N,N-dimethylaniline
1
(23). Yield, 70%; mp164.1–165.5°C; H NMR (400 MHz, DMSO-d6) δ: 1.33 (t, J = 7.2 Hz, 3H,
SCH2CH3), 2.96 (s, 6H, 2×NCH3), 3.13 (dd, J1= 7.2 Hz, J2= 14.8 Hz, 2H, SCH2CH3), 3.58 (s, 6H,
OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 6.71 (s, 2H, trimethoxyPh-H), 6.80 (d, J = 9.2 Hz, 2H, ArH),
13
310
7.18 (d, J = 8.8 Hz, 2H, ArH). C NMR (100 MHz, DMSO-d6) δ: 15.2, 26.6, 56.0, 60.5, 105.6,
112.8, 122.0, 122.4, 128.7, 138.7, 151.4, 153.0, 154.6. HRMS (ESI) m/z: calcd for C21H26N4O3S,
[M+H]+ 415.17592, found 415.18366.
5.1.2.18 4-(3-(benzylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)-N,N-dimethylaniline
1
(24). Yield, 67%; mp 168.1–170.1°C; H NMR (400 MHz, CDCl3) δ: 3.01 (s, 6H, 2×NCH3), 3.66
315
(s, 6H, OCH3-3'', -5''), 3.84 (s, 3H, OCH3-4''), 4.50 (s, 2H, SCH2), 6.70 (d, J = 8.8 Hz, 2H,
trimethoxyPh-H), 6.77 (s, 2H, ArH), 6.98 (d, J = 8.8 Hz, 2H, ArH), 7.26 (s, 1H, ArH), 7.31 (d, J =
11

ACCEPTED MANUSCRIPT
13
6.8 Hz, 2H, ArH), 7.39 (d, J = 6.8 Hz, 2H, ArH). C NMR (100 MHz, DMSO-d6) δ: 36.3, 56.1,
60.5, 105.6, 112.7, 122.1, 122.4, 127.8, 128.7, 128.8, 129.4, 137.6, 138.8, 151.4, 152.6, 153.0,
154.6. HRMS (ESI) m/z: calcd for C26H28N4O3S, [M+H]+ 477.19157, found 477.19620.
320
5.1.2.19 4-(3-((4-methoxybenzyl)thio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)-N,N-dime
1
thylaniline (25). Yield, 77%; mp 170.1–171.1°C; H NMR (400 MHz, CDCl3) δ: 3.01 (s, 6H,
2×NCH3), 3.66 (s, 6H, OCH3-3'', -5''), 3.80 (s, 3H, OCH3-4''), 3.84 (s,3H, CH2Ph-OCH3), 4.46 (s,
2H, SCH2), 6.71 (d, J = 8.4 Hz, 2H, ArH), 6.77 (s, 2H, trimethoxyPh-H), 6.83 (d, J = 8.0 Hz, 2H,
ArH), 7.00 (d, J= 8.8 Hz, 2H, ArH), 7.32 (d, J = 8.4 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6)
δ: 35.8, 55.5, 56.0, 60.5, 105.6, 112.7, 114.2, 122.1, 122.5, 128.7, 129.3, 130.7, 138.8, 151.3, 152.7,
153.0, 154.5, 159.0. HRMS (ESI) m/z: calcd for C27H30N4O4S, [M+H]+ 507.20213, found
507.20448.
325
5.1.2.20 N,N-dimethyl-4-(3-((4-methylbenzyl)thio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol
1
-4-yl)aniline (26). Yield, 68%; mp 177.6–179.1°C; H NMR (400 MHz, DMSO-d6) δ: 2.27 (s, 3H,
330
CH3), 2.94 (s, 6H, 2×NCH3), 3.57 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.35 (s, 2H, SCH2),
6.70 (s, 2H, trimethoxyPh-H), 6.78 (d, J= 9.2 Hz, 2H, ArH), 7.10 (t, J = 9.4 Hz, 4H, ArH), 7.25 (d,
J = 8.0 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 21.1, 36.0, 56.0,60.5, 105.5, 112.7, 121.9,
122.3, 128.7, 129.3, 129.4, 134.3, 137.1, 138.8, 151.4, 152.8, 153.0, 154.6. HRMS (ESI) m/z: calcd
for C27H30N4O3S, [M+H]+ 491.20722, found 491.21129.
335
5.1.2.21 4-(3-((4-fluorobenzyl)thio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)-N,N-dime
1
thylaniline (27). Yield, 72%; mp 170.1–172.6°C; H NMR (400 MHz, DMSO-d6) δ: 2.94 (s, 6H,
2×NCH3), 3.57 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.39 (s, 2H, SCH2), 6.70 (s, 2H,
trimethoxyPh-H), 6.78 (d, J = 8.8 Hz, 2H, ArH), 7.16–7.09 (m, 4H, ArH), 7.43 (dd, J1 = 5.6 Hz, J2
= 8.4 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 35.3, 56.0, 60.5, 105.6, 112.8, 115.5, 115.7,
122.0, 122.4, 128.7, 131.4, 131.5, 134.0, 134.1, 138.8, 151.4, 152.5, 153.0, 154.6, 160.6, 163.1.
HRMS (ESI) m/z: calcd for C26H27FN4O3S, [M+H]+ 495.18214, found 495.18709.
340
5.1.2.22 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1
1
-phenylethanone (28). Yield, 81%; mp160.8–162.8°C; H NMR (400 MHz, DMSO-d6) δ: 2.96 (s,
6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.91 (s, 2H, SCH2), 6.70 (s, 2H,
12

ACCEPTED MANUSCRIPT
345
trimethoxyPh-H), 6.83 (d, J = 8.8 Hz, 2H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.58 (t, J = 7.8
Hz,2H, ArH), 7.70 (t, J = 7.4 Hz,1H, ArH), 8.04 (d, J = 7.6Hz, 2H, ArH).13C NMR (100 MHz,
DMSO-d6) δ: 56.1, 60.5, 105.6, 112.8, 122.0, 122.4, 128.7, 128.8, 129.2, 134.1, 135.8, 138.8, 151.4,
152.5, 153.0, 154.7, 193.7. HRMS (ESI) m/z: calcd for C27H28N4O4S, [M+H]+ 505.18648, found
505.19214.
350
5.1.2.23 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1-
1
(4-methoxyphenyl)ethanone (29). Yield, 86%; mp 145.2–147.8°C; H NMR (400 MHz, DMSO-d6)
δ: 2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 3.87 (s, 3H,
COPh-OCH3), 4.84 (s, 2H, SCH2), 6.70 (s, 2H, trimethoxyPh-H), 6.82 (d, J = 9.2 Hz, 2H, ArH),
7.09 (d, J = 8.8 Hz,2H, ArH), 7.20 (d, J = 8.8 Hz,2H, ArH), 8.01 (d, J = 8.8 Hz, 2H, ArH).13C NMR
(100 MHz, DMSO-d6) δ: 561, 60.5, 105.6, 112.8, 114.5, 122.0, 122.4, 128.6, 128.7, 131.2, 138.8,
151.4, 152.5, 153.0, 154.6, 164.0, 192.0. HRMS (ESI) m/z: calcd for C28H30N4O5S, [M+H]+
535.19705, found 535.20162.
355
5.1.2.24 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1
-(4-fluorophenyl)ethanone (30). Yield, 64%; mp 172.6–174.1°C; 1H NMR (400 MHz, DMSO-d6) δ:
2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.88 (s, 2H, SCH2), 6.69
(s, 2H, trimethoxyPh-H), 6.82 (d, J = 8.8 Hz, 2H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.40 (t, J =
8.6 Hz,2H, ArH), 8.12 (t, J = 7.0 Hz,2H, ArH).13C NMR (100 MHz, DMSO-d6) δ: 56.0, 60.5, 105.6,
112.8, 116.2, 116.4, 121.8, 122.2, 128.7, 131.8, 131.9, 132.5, 138.8, 151.4, 152.5, 153.0, 154.7,
164.4, 167.0, 192.3. HRMS (ESI) m/z: calcd for C27H27FN4O4S, [M+H]+ 523.17706, found
523.18156.
360
365
5.1.2.25 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1-
1
(3-hydroxy-4-methoxyphenyl)ethanone (31). Yield, 66%; mp 173.6–174.7°C; H NMR (400 MHz,
DMSO-d6) δ: 2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 3.87 (s, 3H,
HOPh-OCH3), 4.81 (s, 2H, SCH2), 6.70 (s, 2H, trimethoxyPh-H), 6.83 (d, J = 8.4 Hz, 2H, ArH),
7.06 (d, J = 8.8 Hz, 1H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.39 (s, 1H, ArH),7.57 (d, J = 8.4 Hz,
1H, ArH), 9.50 (s, 1H, OH).13C NMR (100 MHz, DMSO-d6) δ: 56.1, 56.2, 60.5, 111.7, 112.8, 115.0,
122.0, 122.1, 122.4, 128.7, 138.8, 146.9, 151.4, 152.6, 153.0, 154.6, 192.1. HRMS (ESI) m/z: calcd
for C27H27FN4O4S, [M+H]+ 551.1920, found 551.1902.
370
13

ACCEPTED MANUSCRIPT
5.1.2.26 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-1-
(naphthalen-2-yl)ethanone (32). Yield, 62%; mp159.6–160.3°C; 1H NMR (400 MHz, DMSO-d6) δ:
2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.95 (s, 2H, SCH2), 6.69
(s, 2H, trimethoxyPh-H), 6.80 (d, J = 8.4 Hz, 2H, ArH), 7.14 (d, J = 8.4 Hz, 2H, ArH), 7.65 (dd, J1
= 7.0 Hz, J2 = 14.6 Hz, 3H, ArH), 8.04 (d, J = 7.2 Hz, 1H, ArH), 8.22 (dd, J1 = 7.6 Hz, J2 = 17.6 Hz,
2H, ArH), 8.42 (d, J = 8.4 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) δ: 42.4, 56.1, 60.5, 105.6,
112.8, 121.9, 122.3, 125.2, 125.7, 127.0, 128.3, 128.6, 128.9, 129.1, 129.7, 130.0, 133.4, 133.9,
134.3, 138.8, 151.4, 152.6, 153.0, 154.6, 197.2. HRMS (ESI) m/z: calcd for C31H30N4O4S, [M+H]+
555.20213, found 555.20602.
375
380
5.1.2.27 1-cyclopropyl-2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
1
triazol-3-yl)thio)ethanone (33). Yield, 64%; mp152.6–154.1°C; H NMR (400 MHz, DMSO-d6) δ:
385
0.94 (dd, J1 = 5.2 Hz, J2 = 22.4 Hz, 4H, 2×CH2), 2.27 (dd, J1 = 5.2 Hz, J2 = 22.4 Hz, 1H, CH), 2.96
(s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.38 (s, 2H, SCH2), 6.70 (s,
2H, trimethoxyPh-H), 6.82 (d, J = 8.8 Hz, 2H, ArH), 7.20 (d, J = 8.4 Hz, 2H, ArH). 13C NMR (100
MHz, DMSO-d6) δ: 11.4, 19.9, 42.4, 56.1, 60.5, 105.6, 112.8, 121.9, 122.4, 128.7, 138.8, 151.4,
152.4, 153.0, 154.6, 204.0. HRMS (ESI) m/z: calcd for C24H28N4O4S, [M+ H]+ 469.18648, found
469.19303.
390
5.1.2.28 ethyl2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)
1
thio)acetate (34). Yield, 79%; mp 124.5–125.4°C; H NMR (400 MHz, DMSO-d6) δ: 1.20 (t, J =
7.2 Hz, 3H, CO2CH2CH3), 2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H,
OCH3-4''), 4.07 (s, 2H, SCH2), 4.13 (dd, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H, CO2CH2CH3), 6.70 (s, 2H,
13
395
trimethoxyPh-H), 6.83 (d, J = 9.2 Hz, 2H, ArH), 7.20 (d, J = 9.2 Hz, 2H, ArH). C NMR (100
MHz, DMSO-d6) δ: 14.4, 34.1, 56.1,60.5, 61.7, 105.6, 112.8, 121.8, 122.3, 128.7, 138.8, 151.4,
152.2, 153.0, 154.7, 168.6. HRMS (ESI) m/z: calcd for C23H28N4O5S, [M+H]+ 473.18140, found
473.18882.
5.1.2.29 2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-N-
(4-methoxyphenyl)acetamide (35). Yield, 92%; mp 193.9–195.3°C; 1H NMR (400 MHz, DMSO-d6)
δ: 2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 3.72 (s, 3H,
400
14

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NHPh-OCH3), 4.14 (s, 2H, SCH2), 6.71 (s, 2H, trimethoxyPh-H), 6.81 (d, J = 9.2 Hz, 2H, ArH),
6.90 (d, J = 8.8 Hz, 2H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.49 (d, J = 8.8Hz, 2H, ArH), 10.22
(br, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 37.0, 55.6, 56.1, 60.5, 105.6, 112.8, 114.3, 121.0,
121.9, 122.4, 128.7, 132.4, 138.8, 151.4, 152.8, 153.0, 154.6, 155.8, 165.5. HRMS (ESI) m/z: calcd
for C28H31N5O5S, [M+H]+ 550.20794, found 550.21313.
405
5.1.2.30 N-(4-chlorophenyl)-2-((4-(4-(dimethylamino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
triazol-3-yl)thio)acetamide (36). Yield, 88%; mp 181.7–182.9°C; 1H NMR (400 MHz, DMSO-d6) δ:
2.96 (s, 6H, 2×NCH3), 3.58 (s, 6H, OCH3-3'', -5''), 3.65 (s, 3H, OCH3-4''), 4.17 (s, 2H, SCH2), 6.70
(s, 2H, trimethoxyPh-H), 6.81 (d, J = 8.8 Hz, 2H, ArH), 7.21 (d, J = 8.8 Hz, 2H, ArH), 7.38 (d, J =
8.8 Hz, 2H, ArH), 7.61 (d, J = 8.8 Hz, 2H, ArH), 10.50 (br, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 37.0, 56.1, 60.5, 105.6, 112.8, 121.1, 121.8, 122.2, 127.5, 128.7, 129.1, 138.1, 138.8,
151.4, 152.7, 153.0, 154.7, 166.2. HRMS (ESI) m/z: calcd for C27H28ClN5O4S, [M+H]+ 554.15841,
found 554.16218.
410
415
420
5.2 Pharmacology evaluation
5.2.1 Antitumor activity
The antitumor activities of compounds 5–36 were evaluated with HT-29, HepG2, PC-3, and
HCT116 cell lines by the standard MTT assay in vitro. The cancer cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
Cells were splitted at 70-80% confluence, about twice a week by trypsinization.
Exponentially growing cells were plated in 96-well plates (5000 cells/well) and incubated at
37 °C for 24 h for attachment. Test compounds were prepared by dissolving in dimethyl sulfoxide
(DMSO) at 20 mM and diluted with the medium into a series of concentrations. The culture
medium was then changed, and cells grew in medium with the test compounds. DMSO (0.1%) was
used as negative control. Cells were incubated at 37 °C for 48 h. Then the medium was replaced
with MTT solution (5 mg/mL, 100 ꢀL) followed by incubation for another 4 h. The medium was
then aspirated and formazan crystals were dissolved in DMSO (150 ꢀL) for about 10 min. The
absorbance at 570 nm (Abs) of the suspension was measured by an enzyme-linked immunosorbent
assay (ELISA) reader. The inhibition percentage was calculated using the following formula:
% inhibition = (Abscontrol－Abscompound)/Abscontrol×100%. The IC50 values of the test compounds and
15
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CA-4 were measured by treating cells with drugs of various concentrations, and analyzed by use of
the prism statistical package (GraphPad Software, San Diego, CA, U.S.A.).
5.2.2 Flow-activating cell sorting analysis (FACS)
435
440
445
The effect of compound 20 on cell cycle phase distribution of human prostate cancer PC-3 was
assessed using flow cytometry. When the cells grew to about 70% confluence in 60 mm dishs over
night, they were treated with compound 20 at given concentrations (3, 6, 12 ꢀM). After 48h, cells
were harvested by trypsinization, washed with PBS, and fixed in 75% ice cold (4 °C) ethanol
overnight. Then, they were washed with PBS, incubated with RNase (10 ꢀg/mL final concentration)
at 37 °C for 30 min, stained with propidium iodide (50 ꢀg/mL final concentration), and analyzed by
flow cytometry (Beckman Coulter).
5.2.3 In vitro tubulin polymerization assay
Pig brain microtubule protein was isolated by employing three cycles of temperature-dependent
assembly/disassembly according to method described by Shelanski, et al [32]. Homogeneous
tubulin was prepared from microtubule protein by phosphocellulose (P11) chromatography as has
been described previously [33]. The purified proteins were stored in aliquots at -70 °C.
Microtubule polymerization of tubulin protein, in solutions containing different concentrations
of compounds in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA), 1 mM GTP, and
5% glycerol, was monitored at 37 °C by using light scattering at 340 nm with a SPECTRA MAX
190 (MD) spectrophotometer. Plateau absorbance values were used for calculations. CA-4 was used
as standard inhibitor of tubulin polymerisation, while DMSO was used as negative control. The
percent inhibition values for selected compounds were compared to the value of CA-4 and
measured the same day under the same conditions.
450
455
Acknowledgments
This work was supported by National Natural Science Foundation of China (21372113 and
21102069), and the project of the Outstanding Young Teachers in Guangdong Province.
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585
Figure captions
Fig. 1. Structures of CA-4 and some 1,2,4-triazole-based CA-4 analogues.
590
595
Fig. 2. Effect of compound 20 on cell cycle and apoptosis in PC-3 cells. Flow cytometry analysis of PC-3 cells
treated with 20 for 48 h. (A) Control; (B) 20, 3 ꢀM; (C) 20, 6 ꢀM; (D) 20, 12 ꢀM.
600
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Table 1. Cytotoxic activities of compounds 5~36 against human tumor cells.
In vitro cytotoxicity IC50(ꢀM) a
Tubulin
Comp.
R1
R2
polymerization
(% inhibition) b
HT-29
>60
HepG2
57.50
>60
PC-3
>60
HCT116
>60
5
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
H
NT c
NT
NT
16
6
CH3
>60
>60
>60
7
C2H5
>60
>60
57.70
34.10
56.50
40.31
34.97
>60
>60
8
C6H5CH2
>60
>60
42.37
33.30
49.64
38.74
>60
9
4-CH3C6H4CH2
4-CH3OC6H4CH2
4-FC6H4CH2
>60
31.59
41.10
34.97
16.21
18.12
10.74
>60
15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
>60
29
50.70
>60
NT
NT
30
>60
>60
>60
42.50
>60
58.25
>60
>60
12
>60
NT
NT
36
53.40
>60
>60
>60
>60
>60
37.20
>60
>60
>60
>60
>60
NT
10
45.25
58.02
>60
>60
25.83
6.29
>60
>60
>60
31
H
CH3
>60
>60
>60
NT
NT
NT
NT
>60
>60
>60
>60
C2H5
>60
>60
46.18
55.82
>60
C6H5CH2
>60
>60
54.04
22

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25
26
27
28
29
30
31
32
33
34
35
36
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
4-CH3OC6H4CH2
4-CH3C6H4CH2
4-FC6H4CH2
>60
27.45
44.30
45.15
63.96
78.05
>60
>60
>60
>60
37.32
46.45
38.60
22.00
42.30
>100
18.06
45.92
>60
1.15
>60
49
21
26.40
18.90
18.77
>60
>60
NT
16
42.53
>60
38
31.08
>100
54.48
>60
NT
NT
26
>100
>60
>60
>60
>60
NT
NT
NT
17
28.80
51.37
>60
>60
>60
>60
56.07
>60
>60
>60
39.26
6.10
CA-4
1.96
5.29
22.03
80
a IC50 values are presented as mean values of three independent experiments done in quadruplicates.
Coefficients of variation were <10%. b Compounds were tested at a final concentration of 10ꢀM. C NT: not
tested.
620
625
Table 2. Effect of compound 20 on cell cycle distribution in PC-3 cells.
Concentration Sub-G1(%) G0/G1(%)
S(%)
31.7
G2/M(%)
15.81
0ꢀM
3ꢀM
6ꢀM
12ꢀM
0.29
0.43
0.62
0.37
51.58
47.98
46.05
37.76
30.76
31.71
27.08
20.06
24.23
34.80
630
23

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Figure 1. Structures of CA-4 and some 1,2,4-triazole-based CA-4 analogues.
635
A
B
640
C
D
Figure 2. Effect of compound 20 on cell cycle and apoptosis in PC-3 cells. Flow cytometry analysis of PC-3 cells
treated with 20 for 48 h. (A) Control; (B) 20, 3 ꢀM; (C) 20, 6 ꢀM; (D) 20, 12 ꢀM.
24

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645
Scheme 1. Synthesis of the target compounds 5
~36. Reagents and conditions: (a) Con. H2SO4, ethanol, reflux; (b)
NH2NH2 H2O, ethanol, reflux; (c) isothiocyanate, ethanol, reflux; (d) 1M NaOH, reflux; (e) K2CO3, RX, DMF,rt.
25

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Highlights
• 32 novel 1,2,4-triazole derivatives were designed and synthesized as cis-restricted
combretastatin A-4 analogues. • Antiproliferative activity of these compounds was
evaluated. • Analogues 20 and 25 exhibited much stronger antitumor activity than
CA-4. • Flow-activated cell sorting analysis suggested that compound 20 mainly
arrested PC-3 cells in G2/M stage.