Synthesis of (1,3,4-thiadiazol-2-yl)-acrylamide derivatives as potential antitumor agents against acute leukemia cells

Article abstract of DOI:10.1016/j.bmcl.2020.127114

A lead compound with the (1,3,4-thiadiazol-2-yl)-acrylamide scaffold was discovered to have significant cytotoxicity on several tumor cell lines in an in-house cell-based screening. A total of 60 derivative compounds were then synthesized and tested in a CCK-8 cell viability assay. Some of them exhibited improved cytotoxic activities. The most potent compounds had IC₅₀ values of 1–5 μM on two acute leukemia tumor cell lines, i.e. RS4;11 and HL-60. Flow cytometry analysis of several active compounds and detection of caspase activation indicated that they induced caspase-dependent apoptosis. It was also encouraging to observe that these compounds did not have obvious cytotoxicity on normal cells, i.e. IC₅₀ > 50 μM on HEK-293T cells. Although the molecular targets of this class of compound are yet to be revealed, our current results suggest that this class of compound represents a new possibility for developing drug candidates against acute leukemia.

Full text of DOI:10.1016/j.bmcl.2020.127114

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of (1,3,4-thiadiazol-2-yl)-acrylamide derivatives as potential
antitumor agents against acute leukemia cells
⁎
⁎
⁎
Qing Lia,1, Ran Anb,1, Yaochun Xua, Mi Zhoua, Yan Lic, , Chun Guob, , Renxiao Wanga,c,d,
a State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
c Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, People’s Republic of China
d Shanxi Key Laboratory of Innovative Drugs for the Treatment of Serious Diseases Basing on Chronic Inflammation, College of Traditional Chinese Medicines, Shanxi
University of Chinese Medicine, Taiyuan, Shanxi 030619, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A lead compound with the (1,3,4-thiadiazol-2-yl)-acrylamide scaffold was discovered to have significant cyto-
toxicity on several tumor cell lines in an in-house cell-based screening. A total of 60 derivative compounds were
then synthesized and tested in a CCK-8 cell viability assay. Some of them exhibited improved cytotoxic activities.
The most potent compounds had IC50 values of 1–5 μM on two acute leukemia tumor cell lines, i.e. RS4;11 and
HL-60. Flow cytometry analysis of several active compounds and detection of caspase activation indicated that
they induced caspase-dependent apoptosis. It was also encouraging to observe that these compounds did not
have obvious cytotoxicity on normal cells, i.e. IC50 > 50 μM on HEK-293T cells. Although the molecular targets
of this class of compound are yet to be revealed, our current results suggest that this class of compound re-
presents a new possibility for developing drug candidates against acute leukemia.
(1,3,4-thiadiazol-2-yl)-acrylamide
Cytotoxicity
Cell apoptosis
Antitumor agents
Acute leukemia
With the advances in molecular biology and biochemistry, target-
based drug discovery, also known as ‘rational drug discovery’, is pre-
valent in the development of new drugs since this century.1,2 However,
many kinds of research indicate that all the major diseases are multi-
factorial and involve complex gene or target networks. This may ex-
plain the dilemma faced by the target-based drug discovery.3 Thus, the
traditional cell-based screening remains essential for discovering com-
pounds with desired potencies, as a complementary approach to target-
based screening.
cytotoxicity especially on two types of acute leukemia tumor cell lines,
RS4;11 and HL-60. The inhibition ratio at this concentration was over
50% on RS4;11 and HL-60 cells. The IC50 value on HL-60 cells was
12 μM. Current anti-tumor drugs on the market are mainly used for the
therapies of common cancers with high incidences, such as liver cancer,
lung cancer, and stomach cancer. While for some rare malignant tu-
mors, like cervical cancer, acute leukemia, etc., there are still relatively
few types of small-molecule drugs available, and the treatment is
usually limited.5,6 Therefore, compound 1 could be a good starting
point for developing effective candidate drug against acute leukemia.
In terms of chemical structure, compound 1 contains two distinct
moieties: a 1,3,4-thiadiazol moiety and a sulfonic acid phenolate
moiety. Our literature survey indicates that some known compounds
containing the 1,3,4-thiadiazol moiety are reported to have anti-tumor
effects (Fig. 2). For example, Kumar et al reported a class of 2-ar-
ylamino-5-aryl-1,3,4-thiadiazoles (compound 2), which showed cyto-
toxicity on several cancer cell lines, such as prostate, pancreatic and
breast tumor cells.7 Compound 4, i.e. N-((5-(substituted methylene
amino)-1,3,4-thiadiazol-2-yl)-methyl) benzamide, also had anti-tumor
In our efforts on the development of anti-tumor agents, we screen all
compounds available at the cellular level. These compounds are pur-
chased from a commercial resource or synthesized by ourselves. In our
cell-based screening protocol, each compound is tested at a single
concentration of 20 μM in a CCK-8 cell viability assay on five selected
tumor cell lines,4 including A549 (human alveolar epithelial cell), HeLa
(human cervical tumor cell), MBA-MD-231 (human breast tumor cell),
RS4;11 (human acute lymphoblastic leukemia tumor cell) and HL-60
(human protomyeloid leukemia tumor cell). For example, compound 1
(Fig. 1) was discovered in our screening, which exhibited significant
⁎ Corresponding authors at: Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, People’s Republic
of China (R. Wang).
E-mail addresses: li_yan@fudan.edu.cn (Y. Li), chunguo@syphu.edu.cn (C. Guo), wangrx@fudan.edu.cn (R. Wang).
1 Authors who have equivalent contributions to this work.
https://doi.org/10.1016/j.bmcl.2020.127114
Received 5 February 2020; Received in revised form 12 March 2020; Accepted 14 March 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:QingLi,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127114

Q. Li, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Chemical structure of compound 1 (left) and its cytotoxicity on five tumor cell lines (middle). Its IC50 value on HL-60 cells is 12 μM (right).
effect on several types of tumor cells.8 The inhibitory activity of 1,3,4-
thiadiazole derivatives on leukemia cells has also been reported, such as
compound 3 and 5.9,10 Therefore, we consider 1,3,4-thiadiazole moiety
as a valuable scaffold for developing new anti-tumor compounds. The
sulfonic acid group usually exists in the salt form in a drug molecule,
which leads to good solubility and other pharmacological properties.11
However, the downside is that the sulfonic acid group can easily form a
sulfonate with alkyl alcohol, such as methyl methanesulfonate, methyl
benzenesulfonate and etc. The sulfonate can directly or indirectly react
with DNA to transfer the alkyl group and cause damage12. Nevertheless,
compound 1 contains a phenol ester, which is in principle more stable
than alcohol esters and thus may avoid the potential genotoxicity. Thus,
we believe that it is possible to develop a new class of anti-tumor
compounds against acute leukemia based on the scaffold of compound
1.
In this work, a total of 60 derivatives of compound 1 were synthe-
sized with the methods outlined in Scheme 1 and 2. Using the condi-
tions described in literature,13,14 carboxylic acid and hy-
drazinecarbothioamide were mixed to produce the intermediate 5-
substituted-1,3,4-thiadiazol-2-amine (6) via a condensation reaction.
Then, under basic condition (NaOMe/MeOH), compound 6 was reacted
with ethyl cyanoacetate to obtain 2-cyano-N-(5-substituted-1,3,4-thia-
diazole-2-)acetamide (7). The aromatic aldehyde intermediate 8 was
Fig. 2. Examples of anti-tumor compounds which contain the 1,3,4-thiodiazole
prepared by hydroxybenzaldehyde and the corresponding substituted
scaffold.
benzenesulfonyl chloride in the mixture of DMAP/TEA/CH2Cl2.15 At
the last step, 7 and 8 were refluxed in the solution of NH4OAc/HOAc/
PhMe16 for 8 h to produce compounds 1, 13–27, and 30–72 with
overall yields of 20–80%.
To obtain the benzenesulfonamide derivatives, we adopted a mod-
ified synthetic route as illustrated in Scheme 2. Ethyl cyanoacetate was
first condensed with nitrobenzaldehyde to obtain 10, which was sub-
sequently reduced to its amine 11 with SnCl2/MeOH.17 11 was then
reacted with a sulfonyl chloride to produce the intermediate 12. Under
the catalysis of the alkaline reagent, 6 and 12 underwent transamina-
tion at basic condition (CH3ONa/CH3OH) to produce the final targets
28 and 29.
All of the synthesized compounds were then tested at a single
concentration of 20 μM on RS4;11 and HL-60 cells. The HEK293T cell
line was used as a model of normal cells in our work. The CCK-8 cell
viability assay was used to determine the inhibition ratio of the tested
compounds on tumor cell growth. The results obtained in this assay are
summarized in Table 1. One can see that when the sulfonate or sulfo-
namide group is at the ortho position on the phenyl ring, the corre-
sponding compounds with an electron donating group as R2 achieve
good inhibitory activities against two tumor cells. In contrast, electron
withdrawing groups as R2 reduce the inhibitory activities of the cor-
responding compounds, such as 14, 16, and 17. Their inhibition ratios
on two tumor cells are lower than 50%. A similar trend is observed
among the compounds where the sulfonate or sulfonamide group are at
the meta position on the phenyl ring.
Scheme 1. Synthetic route for obtaining compounds 1, 13–27, and 30–72 re-
The compounds with inhibition ratio over 50% in the initial
ported here. Reagents and conditions: (a) POCl3, 70 °C; (b) i. EtO2CCH2CN, ii.
screening were selected to be further tested at multiple concentrations
NaOMe/MeOH, reflux, 5 h; (c) i. NH4OAc, AcOH ii. tulene, reflux, 8 h.
to derive their concentrations at half inhibition (IC50). In this assay,
2

Q. Li, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 2. Synthetic route for obtaining compounds 28 and 29. Reagents and conditions: (a) i. AcOH/AcONH4, ii. PhMe, reflux, 5 h; (b) i. SnCl2·2H2O (5 equiv.), ii.
EtOH/HCl; (c) i. CH2Cl2, pyridine, rt, ii. RSO2Cl; (d) CH3ONa/CH3OH, reflux, 8 h.
Table 1
Chemical structures of compounds 1 and 13–72 and their cytotoxicity on three cell lines.
ID
R1
R2
Location of substituents on phenyl group
X
Inhibition ratioa (%) at 20 μM
RS4;11
HL-60
HEK293T
1
Me
4-Me-Ph
4-CF3-Ph
4-Cl-Ph
ortho
ortho
ortho
ortho
ortho
ortho
ortho
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
para
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
33
58
22
76
40
39
26
33
28
35
79
44
41
25
33
N.A.
22
40
13
70
78
70
68
79
80
73
78
58
66
68
68
69
78
55
17
67
23
48
59
20
71
63
73
67
73
62
71
30
N.A.
77
N.A.
25
100
51
25
20
25
19
98
18
23
15
12
16
35
57
46
40
33
42
53
20
27
52
N.A.
55
N.A.b
43
20
50
29
21
19
21
30
17
18
34
6
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
thiophene
thiophene
thiophene
thiophene
thiophene
2-indol
4-OMe-Ph
4-NO2-Ph
4-OCF3-Ph
4-OCF3-Ph
Me
Me
Me
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
4-NHAc-Ph
thiophene
4-F-Ph
Ph
4-OMe-Ph
2-Cl-Ph
2-OMe-Ph
thiophene
thiophene
thiophene
thiophene
thiophene
thiophene
4-OMe-Bn
4-OMe-Bn
4-OMe-Bn
4-OMe-Bn
4-OMe-Bn
4-OMe-Bn
1-methylnaphthyl
1-methylnaphthyl
1-methylnaphthyl
1-methylnaphthyl
1-methylnaphthyl
1-methylnaphthyl
Bn
4-OMe-Ph
Me
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
4-F-Ph
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
23
29
25
N.A.
16
15
15
4-F-Ph
4-Me-Ph
(continued on next page)
3

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Table 1 (continued)
ID
R1
R2
Location of substituents on phenyl group
X
Inhibition ratioa (%) at 20 μM
RS4;11
HL-60
HEK293T
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
ABT-199c
Bn
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
ortho
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
78
57
71
73
69
72
76
55
61
68
59
56
74
65
68
71
72
69
77
66
65
64
64
66
79
64
N.A.
72
73
95
100
20
12
26
22
N.A.
100
18
35
20
13
11
100
18
23
42
22
15
99
34
19
15
20
17
100
17
33
38
11
98
23
N.A.
10
25
6
Bn
Bn
Bn
Bn
4-F-Ph
4-F-Bn
4-F-Bn
4-F-Bn
4-F-Bn
4-F-Bn
4-F-Bn
3-F-Bn
3-F-Bn
3-F-Bn
3-F-Bn
3-F-Ph
3-F-Bn
4-Cl-Bn
4-Cl-Bn
4-Cl-Bn
4-Cl-Bn
4-Cl-Bn
4-Cl-Bn
4-Me-Bn
4-Me-Bn
4-Me-Bn
4-Me-Bn
4-Me-Bn
4-Me-Bn
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
16
20
8
6
6
4-F-Ph
N.A.
9
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
29
N.A.
N.A.
29
17
20
26
19
20
14
14
22
28
9
4-F-Ph
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
4-F-Ph
4-Me-Ph
4-OMe-Ph
4-NO2-Ph
2-NO2-Ph
4-Cl-Ph
6
8
4-F-Ph
11
N.A.
a
b
c
Cell growth inhibition ratio was measured after 48-hour treatment.
No significant inhibitory activity observed at the test concentration.
ABT-199 was tested at the concentration of 1 μM.
Table 2
summarized in Table 2. Here, compounds 32, 38, 44, 50, 56, 62 and 68
exhibit the most potent inhibitory activities on both RS4;11 and HL-60
cell lines (IC50 < 5 μM). Notably, all these compounds have 4-meth-
oxyphenyl as the R2 group. If this group is replaced by other groups,
such as 4-nitrophenyl and 4-chlorophenyl, their inhibitory activities
will decrease by 3–20 folds (Table S1 in Supporting Information). It is
also encouraging to observe that compounds 32, 38, 44, 50, 56, 62 and
68 are generally less toxic on HEK-293 T cells (IC50 > 50 μM), de-
monstrating the desired selectivity between tumor cells and normal
cells.
Cytotoxicity profiling of the most potent compounds.
Compd
Cytotoxicity (IC50, μM)a
RS4;11
> 20
HL-60
HEK-293T
1
11.47
17.46
5.23
11.06
5.78
8.75
7.94
8.37
3.12
10.90
11.42
4.70
3.17
4.05
3.88
4.36
4.08
0.92
1.60
> 20
12.31
> 20
14.38
> 50
> 20
> 20
> 20
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
13
15
18
20
25
26
29
32
33
35
38
44
50
56
62
68
10.54
4.75
> 20
> 20
> 20
> 20
> 20
0.84
3.67
4.02
1.36
1.06
1.21
1.21
1.25
1.01
2.01
0.12
2.45
1.46
0.85
0.83
0.74
2.38
0.92
0.61
0.05
In order to explore their mechanism in inducing apoptosis, we chose
to characterize mitochondrial potential reduction, transfer of phos-
phatidylserine (PS), and change of plasma membrane permeability
caused by these active compounds through flow cytometry analysis.
Compounds 32, 50 and 62 were selected for flow cytometry analysis
with Mitodamage staining, which could detect the above three events in
apoptosis. After the treatment by the selected compounds at 5 μM,
10 μM, 20 μM, and 40 μM for 48 h, RS4;11 cells were stained with 7-
AAD, annexin V, and MitoSense Red and then were analyzed. Here,
Annexin V and 7-AAD were used to detect the state of PS and the
plasma membrane permeability of cells, respectively. Combinations of
7-AAD and annexin V were used to distinguish cells in early apoptosis
(i.e. annexin V (+) and 7-ADD (−)) and late apoptosis (i.e. annexin V
(+) and 7-ADD (+)), corresponding to the bottom right and upper
right sections in Fig. 3. For the cells treated by DMSO, about 5% of them
were observed in early and late apoptosis. After the test compound was
added, the ratio of apoptotic cells increased with the compound con-
centration (see Fig. 3 and Fig. S1 in Supporting Information). When the
cells were treated by these compounds at 40 μM, the ratio of apoptotic
cells reached 78%, 68%, and 81% for compounds 32, 50 and 62,
0.05
0.25
0.20
0.06
0.07
0.04
0.11
0.02
0.09
0.42
0.29
0.09
0.18
0.62
0.69
0.43
0.53
a
Each compound was tested in triplicate; the data are presented as the
mean
SD.
venetoclax (ABT-199), which is a potent apoptosis inducer and ap-
proved by FDA for treating adults with chronic lymphocytic leukemia
(CLL) or small lymphocytic lymphoma (SLL), and treating adults with
newly-diagnosed acute myeloid leukemia (AML) in combination
therapies,18,19 was used as the positive control compound. The IC50
values of all active compounds (on one or both tumor cell lines) are
4

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Fig. 3. Results of flow cytometric analysis with Annexin V and 7-AAD on RS4;11 cells for three selected compounds. Cells were treated for 48 h. Cells with the
treatment of DMSO and ABT-199 were used as the negative and the positive controls respectively in this assay.
Compound 32
Compound 50
Compound 62
Fig. 4. Results of flow cytometric analysis with MitoSense Red on RS4;11 cells for three selected compounds. Cells were treated at 5 μM (orange), 10 μM (light green),
20 μM (dark green) and 40 μM (skin pink) for 48 h. Cells with the treatment of DMSO (red) and ABT-199 (blue) were used as the negative and the positive controls
respectively in this assay.
respectively. The MitoSense Red was used to detect the changes in
mitochondrial membrane potential. Uninduced cells with intact mi-
tochondrial membrane are associated with high Red2 fluorescence;
while cells with impaired mitochondrial membrane are associated with
low Red2 fluorescence. As one can see in Fig. 4, Red2 fluorescence
shifted significantly to the left when the concentration of the compound
under test reached 40 μM, indicating an obvious damage to
mitochondrial membrane. This is consistent with the results from 7-
AAD/annexin V staining, indicating that these compounds under test
are capable of inducing cell apoptosis.
Poly-ADP-ribose polymerase protein (PARP) is a major substrate of
activated caspases. Cleavage of PARP is a critical indicator of cell
apoptosis. In our work, the three compounds that exhibited significant
pro-apoptotic signals in flow cytometry analysis, i.e. 32, 50 and 62,
5

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Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
to 40 μM, the cleaved PARP, cleaved caspase-3 and cleaved caspase-9
were increased generally in a dose-dependent manner. This observation
again confirms the results of our flow cytometry analysis that these
compounds are able to induce the caspase-dependent apoptosis.
In conclusion, we have developed a class of compounds with the
(1,3,4-thiadiazol-2-yl)-acrylamide scaffold, which exhibited significant
cytotoxicity on two acute leukemia cells, i.e. RS4;11 and HL-60. The
most potent compounds have IC50 values lower than 5 μM. Flow cyto-
metry analysis and cleaved caspase detection indicate that these com-
pounds are capable of inducing caspase-dependent apoptosis in a dose-
dependent manner. Moreover, these compounds do not have obvious
cytotoxicity on normal cells, i.e. IC50 > 50 μM on HEK-293T cells.
Although the molecular targets of this class of compound are yet to be
revealed, our current results suggest that this class of compound re-
presents a new possibility for developing drug candidates against acute
leukemia.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors are grateful to the financial supports from the National
Natural Science Foundation of China (Grant No. 81430083, 81725022,
21472227, and 21661162003), the Ministry of Science and Technology
of China (National Key Research Program, Grant No.
2016YFA0502302), and Chinese Academy of Sciences (Strategic
Priority Research Program, Grant No. XDB20000000).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127114.
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