Synthesis and antitumor activity evaluation of quinazoline derivatives bearing piperazine-1-carbodithioate moiety at C4-position

Article abstract of DOI:10.1016/j.bmcl.2016.08.060

A series of quinazoline derivatives bearing piperazine-1-carbodithioate moiety at the C4-position were synthesized using piperidine and 1-bromo-3-chloropropane as starting materials via eight steps. Final compounds 8a–q and 9a–i were evaluated for their antiproliferative activity against human lung cancer A549, breast adenocarcinoma MCF-7, and colorectal cancer HCT-116 cell lines. The results showed that fourteen of twenty-six final compounds inhibited the proliferation of three cancer cell lines with IC₅₀values less than 10?μM. When treated with a representative compound 8n, HCT-116 cells were arrested at G0/G1 phase of the cell cycle. This provided a clue to further investigation of the mechanism of action.

Full text of DOI:10.1016/j.bmcl.2016.08.060

Accepted Manuscript
Synthesis and antitumor activity evaluation of quinazoline derivatives bearing
piperazine-1-carbodithioate moiety at C4-position
Ying Zhang, Chao-Rui Yang, Xue Tang, Sheng-Li Cao, Ting-Ting Ren, Man
Gao, Ji Liao, Xingzhi Xu
PII:
S0960-894X(16)30893-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.060
BMCL 24186
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
13 February 2016
6 August 2016
19 August 2016
Please cite this article as: Zhang, Y., Yang, C-R., Tang, X., Cao, S-L., Ren, T-T., Gao, M., Liao, J., Xu, X., Synthesis
and antitumor activity evaluation of quinazoline derivatives bearing piperazine-1-carbodithioate moiety at C4-
position, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.08.060
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Synthesis and antitumor activity evaluation of quinazoline derivatives bearing
piperazine-1-carbodithioate moiety at C4-position
Ying Zhanga, Chao-Rui Yanga, Xue Tanga, Sheng-Li Caoa,*, Ting-Ting Rena, Man Gaoa, Ji Liaob,c,
Xingzhi Xub,c*
a Department of Chemistry, Capital Normal University, Beijing 100048, China
b School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, China
cBeijing Key Laboratory of DNA Damage Response, College of Life Science, Capital Normal
University, Beijing 100048, China
∗Corresponding authors:
Sheng-Li Cao, Department of Chemistry, Capital Normal University, Beijing 100048, China; Tel/Fax:
+86-10-68902974; E-mail: slcao@cnu.edu.cn
Xingzhi Xu, School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, China; Tel/Fax:
+86-755-86671981; E-mail: xingzhi.xu@szu.edu.cn
ABSTRACT
A series of quinazoline derivatives bearing piperazine-1-carbodithioate moiety at the C4-position
were synthesized using piperidine and 1-bromo-3-chloropropane as starting materials via eight steps.
Final compounds 8a−q and 9a−i were evaluated for their antiproliferative activity against human lung
cancer A549, breast adenocarcinoma MCF-7, and colorectal cancer HCT-116 cell lines. The results
showed that fourteen of twenty-six final compounds inhibited the proliferation of three cancer cell lines
with IC50 values less than 10 µM. When treated with a representative compound 8n, HCT-116 cells
were arrested at G0/G1 phase of the cell cycle. This provided a clue to further investigation of the
mechanism of action.
Key words: Quinazoline, piperazine-1-carbodithioate, synthesis, antiproliferative activity, cell cycle.
1

Piperazine is a common heterocycle present in a number of well established and commercially
available drugs including antitumor agents. Diversity of structural modifications on the piperazine ring
has made piperazine derivatives indispensable anchors for the development of novel therapeutic
agents.1, 2 In the last decade, piperazine-1-carbodithioate and piperazine-1-carbothioyl derivatives have
attracted considerable attention due to their potent biological effects, especially antitumor activity. For
example,
Li
and
co-workers
synthesized
4-methyl-piperazine-1-carbodithioc
acid
3-cyano-3,3-diphenylpropyl ester hydrochloride (TM208, Fig. 1), which significantly inhibited the
growth of xenografted hepatocellular carcinomas and gastric carcinomas in nude mice with low
toxicity.3, 4 Recently, Liu's group synthesized a series of 1,2,3-triazole-dithiocarbamate hybrids, and
found that compound I (Fig. 1) exhibited the most specific and robust inhibition of lysine specific
demethylase 1 (LSD1) and cytotoxicity against LSD1 overexpressing gastric cancer cell lines
6
MGC-803 and HGC-27.5,
Several antitumor agents bearing piperazine-1-carbodithioate or
piperazine-1-carbothioyl moiety have been developed by other researchers.7-11
In our previous work, 4-substituted-piperazine-1-carbodithioate moiety had been incorporated into
the C6-position of 2-methyl-4-oxoquinazoline or 2,4-diaminoquinazoline to synthesize target
compounds with the general formulas II or III (Fig. 1).12, 13 Among them, compounds IIIa−c exhibited
cytotoxicity with IC50 values in the range of 1.58−2.27, 1.84−3.27 and 1.47−4.68 µM, respectively,
against A549, MCF-7, HeLa, HT29 and HCT-116 cells, inducing a G2/M phase arrest in HCT-116
cells.13 Besides, we had also synthesized a series of 4-substituted-piperazine-1-carbothiohydrazide
derivatives of indolin-2-one (IV, Fig. 1), and most of them exhibited antiproliferative activity against
A549, MCF-7 and HCT-116 cell lines. Notably, it was found that removal of the piperazine moiety
between the hydrazinecarbothioyl moiety and phenyl ring resulted in a decrease or loss in
antiproliferative activity.14
On the other hand, 4-substituted-quinazolines have been identified as an important class of
compounds for the development of antitumor agents, of which several 4-anilinoquinazoline kinase
inhibitors have been approved for the treatment of cancer, as exemplified by Gefitinib and Erlotinib.15,
16 Moreover, several 4-piperazinylquinazoline derivatives were reported to exhibit attractive antitumor
efficacy. For instance, Tandutinib (CT53518, Fig. 1) has entered phase II clinical trials for the treatment
of myeloid leukemia and advanced myelodysplasia.17, 18 Taking inspiration from reported antitumor
activities of 4-substituted-quinazoline derivatives and encouraged by our promising results of novel
2

compounds containing the piperazine-1-carbodithioate moiety, we attempted to introduce this moiety
into the C4-position of quinazoline core to synthesize compounds 8a−q (Scheme 1 and Table 1). The
target compounds could be considered as Tandutinib analogs designed by the isosteric replacement of
formamide moiety in Tandutinib with carbodithioate, which would be tested for their cytotoxicity and
examined the effect on cell cycle progression.
Fig. 1. Structures of TM208, Tandutinib, and compounds I−IV.
The synthetic pathway of final compounds 8a−q is outlined in Scheme 1, in which the
preparation methods of intermediates 4−6 are similar to those in the synthesis of Tandutinib utilized
by Knesl et al.18 Briefly, reaction of piperidine with 1-bromo-3-chloropropane in tetrahydrofuran
furnished 1-(3-chloropropyl)piperidine (1), which underwent etherification with methyl
4-hydroxy-3-methoxybenzoate using potassium carbonate as a base in N,N-dimethylformamide (DMF)
to give methyl 3-methoxy-4-(3-(piperidin-1-yl)propoxy)benzoate (2). Nitration of intermediate 2 with
65% nitric acid in acetic acid, followed by the reduction with SnCl2·2H2O in concentrated hydrochloric
acid at room temperature generated methyl 2-amino-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzoate
(4). Cyclized product, 6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4(3H)-one (5), was obtained
by heating 4, triethyl orthoformate and ammonium acetate in methanol for 9 h. Treatment of 5 with a
mixture of phosphorus pentachloride and phosphorus oxychloride furnished 4-chloroquinazoline
intermediate 6, which was used in the next step without purificaton. Refluxing 6 with excess piperazine
in isopropanol for 3 h gave 4-(piperazin-1-yl)quinazoline 7, which reacted with carbon disulfide and
the appropriate benzyl bromide, substituted-benzyl bromide, cyclohexyl bromide, or
chloromethylpyridine hydrochloride in the presence of potassium phosphate to afford target
3

compounds 8a−q.
Scheme 1. Synthetic pathway of final compounds 8a−q. Reagents and conditions: (a)
1-Bromo-3-chloropropane, K2CO3, THF, 45 °C, 6 h; (b) Methyl 4-hydroxy-3-methoxybenzoate, K2CO3,
DMF, 75 °C, 6 h; (c) 65% HNO3, AcOH, 45 °C, 6 h; (d) SnCl2·2H2O, concentrated HCl, rt, overnight;
(e) HC(OEt)3, AcONH4, MeOH, reflux, 9 h; (f) PCl5, POCl3, reflux, 15 h; (g) Piperazine, isopropanol,
reflux, 3 h; (h) RCH2Br/RCH2Cl, CS2, K3PO4, DMF, rt, 2 h.
The
MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium, inner salt] cell proliferation assay was used to evaluate the antiproliferative activity of
final compounds against three human cancer cell lines, namely, A549 (lung cancer), MCF-7 (breast
adenocarcinoma), and HCT-116 (colorectal cancer). The inhibition of cell proliferation was determined
72 h after cells were exposed to the tested compounds at a concentration of 50 µM. The compounds
with 50% or more inhibition compared with vehicle-treated cells were considered active. Inhibition of
cell proliferation by these active compounds at various concentrations were further measured, and their
IC50 (the concentration that causes 50% of cell proliferation inhibition) values were determined and
summarized in Table 1. 5-Fluorouracil (5-FU), Sunitinib, and Tandutinib were used as positive
controls.
4

Table 1.
Antiproliferative activity of compounds 8a−q against A549, MCF-7 and HCT-116 cell lines.
IC50a, µM
Compound
R
A549
MCF-7
HCT-116
C6H5
8a
15.67 0.29
4.51 0.44
4.25 0.13
4.58 0.15
4.31 0.26
4.71 0.22
4.25 0.20
5.44 0.37
4.90 0.37
4.58 0.40
> 50
28.01 0.91
3.73 0.43
4.16 0.24
3.86 0.09
3.93 0.66
3.94 0.24
4.20 0.22
4.67 0.77
3.07 0.65
4.14 0.54
> 50
28.83 0.62
5.46 0.49
4.92 0.77
8.01 0.42
4.52 0.30
8.04 0.22
4.50 0.36
4.64 0.21
5.96 0.10
4.45 0.39
> 50
4-CH3C6H4
4-CH3OC6H4
3-CH3OC6H4
4-BrC6H4
8b
8c
8d
8e
4-ClC6H4
8f
4-FC6H4
8g
8h
2,4-F2C6H3
3,4-F2C6H3
2,3,4,5,6-F5C6
4-HOOCC6H4
4-CNC6H4
4-NO2C6H4
Cyclohexyl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
8i
8j
8k
8l
9.52 0.53
5.36 0.45
4.06 0.22
28.78 1.31
19.46 1.86
> 50
10.07 0.11
6.89 0.15
3.99 0.53
20.68 0.73
22.20 2.41
> 50
8.67 0.54
5.65 0.21
4.55 0.28
20.62 0.09
22.21 1.43
> 50
8m
8n
8o
8p
8q
5-FU
Sunitinib
Tandutinib
3.52 0.46
2.44 0.22
14.75 0.87
32.18 1.13
6.29 0.22
14.70 0.95
5.53 0.90
4.71 0.32
18.13 1.06
a IC50: The concentration that causes 50% of cell proliferation inhibition. Data are expressed as mean
SD from triplicate determination from three independent experiments.
In view of the structural feature of 4-substituted-quinazoline-based antitumor agents,19 especially
for Tandutinib, a methoxy and a (3-(piperidin-1-yl)propoxy group were arrayed on the 6- and
7-position of the quinazoline ring, respectively, while the influence of R group in the
piperazine-1-carbodithioate moiety on antiproliferative activity would be mainly investigated in this
study. As shown in Table 1, compound 8a (R = C6H5), which can be regarded as the parent compound,
exhibited moderate antiproliferative activity with IC50 values in the range of 15.67−28.83 µM against
5

three cancer cell lines. Introduction of a methyl or a methoxy group into the 4-position of the phenyl
ring of 8a (viz., 8b, R = 4-CH3C6H4, and 8c, R = 4-CH3OC6H4) led to an obvious increase in
antiproliferative activity against three cancer cell lines.
Similar to the compounds bearing electron-donating groups (8b−d), compounds bearing a
moderate electron-withdrawing halogen substituent (namely, 8e−g, R = 4-BrC6H4, 4-ClC6H4, 4-FC6H4)
were more active than the parent compound 8a as well. Interestingly, further introduction of a fluoro
substituent into the 2- or 3-position, or four fluoro substituents into the 2, 3, 5, and 6-position of
compound 8g, resulted in compounds 8h−j (R = 2,4-F2C6H3, 3,4-F2C6H3, or 2,3,4,5,6-F5C6) which
possessed the similar activity to compound 8g. As for the compounds bearing strong
electron-withdrawing groups, however, compound 8k (R = 4-HOOCC6H4) was inactive, while 8l (R =
4-CNC6H4) and 8m (R = 4-NO2C6H4) were less active than compounds 8b−j. Although compounds 8l
and 8m were more cytotoxic against the cancer cell lines tested than the parent compound 8a, a strong
electron-withdrawing group on the phenyl ring was in general unfavorable to the antiproliferative
activity, in comparison with the electron-donating groups or even moderate electron-withdrawing
groups.
Replacement of the phenyl ring in the parent compound 8a with a cyclohexyl group resulted in
compound 8n, which exhibited significant cytotoxicity with IC50 values of 4.06, 3.99 and 4.55 µM
against A549, MCF-7 and HCT-116 cell lines, respectively. Substitution of the phenyl ring by a
pyridinyl group resulted in compounds 8o−q (R = pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl).
Compounds 8o and 8q exhibited comparable activity against A549, MCF-7 and HCT-116 cell lines to
the parent compound 8a, whereas compound 8q was inactive at 50 µM concentration. These results
indicated that a cycloalkyl group is more favorable than an electron-deficient heteroaryl group to the
antiproliferative activity. Interestingly, compound 8n exhibited comparable activities with the positive
controls 5-FU and Sunitinib against A549 and HCT-116 cells, and stronger activity than 5-FU and
Sunitinib against MCF-7 cells. Moreover, it was more potent than Tandutinib against all three cancer
cell lines tested (4.06, 3.99, 4.55 versus 14.75, 14.70, 18.13 µM, respectively).
Given that compound 8n showed decent antiproliferative potency, we replaced the cyclohexyl
group in 8n with alkyl or aliphatic heterocyclyl groups to synthesize compounds 9a−g for further
investigation of the structure-activity relationship (see Supplementary data). As shown in Table 2,
replacement of the cyclohexyl group with an open-chain alkyl group (R1 = methyl, ethyl, propyl, or
6

isopropyl) led to a decrease in antiproliferative activity. With reduction of the bulk of alkyl group, the
antiproliferative activity decreased obviously. Similarly, compounds containing an aliphatic
heterocyclyl group, viz., 9e (R1 = tetrahydrofuran-2-yl), 9f (R1 = 1,3-dioxolan-2-yl), 9g (R1
=
tetrahydropyran-4-yl) exhibited weaker activity in comparison with 8n. However, compound 9g
containing a six-membered heterocycle was more active than those containing a five-membered
heterocycle (9e, 9f). These results indicated that the bulk of alkyl or aliphatic heterocyclyl groups plays
an important role in exerting the antiproliferative effect.
From the first series of compounds (8a−q), it suggests that aryl groups bearing an
electron-donating substituent, but not aryl groups bearing an electron-withdrawing substituent and
electron-deficient pyridinyl groups, are favorable to the antiproliferative activity. We thus synthesized
two compounds containing an electron-rich heterocycle, namely, 9h (R1 = furan-2-yl) and 9i (R1 =
thiophen-3-yl). As shown in Table 2, these two compounds exhibited higher antiproliferative activity
than the compounds containing pyridinyl groups (8o−q), which was comparable with the compounds
containing aryl groups with an electron-donating substituent, e.g. 8b−d.
Table 2.
Antiproliferative activity of compounds 9a−i against A549, MCF-7 and HCT-116 cell lines.
IC50a, µM
Compound
R1
A549
MCF-7
HCT-116
CH3
33.35 1.17
18.36 0.80
8.07 0.11
11.27 0.93
22.80 1.00
24.62 0.44
19.14 0.68
6.58 0.43
4.84 0.24
27.52 0.77
16.62 0.32
10.54 0.94
8.37 0.21
22.71 0.51
29.55 1.17
18.35 0.46
7.29 0.31
5.17 0.14
24.35 1.52
10.35 0.48
8.67 0.65
10.44 1.18
19.22 0.80
23.92 0.64
15.08 0.40
7.30 0.60
5.64 0.22
9a
9b
9c
9d
9e
9f
CH3CH2
CH3CH2CH2
(CH3)2CH
Tetrahydrofuran-2-ylCH2
1,3-Dioxolan-2-ylCH2
Tetrahydropyran-4-ylCH2
Furan-2-ylCH2
Thiophen-3-ylCH2
9g
9h
9i
a Data are expressed as mean SD from triplicate determination from three independent experiments.
7

To investigate the preliminary mechanism of action, compound 8n was employed to examine the
effect on cell cycle distribution in HCT-116 cells. HCT-116 cells were treated with compound 8n (1 ×
IC50, 1.25 × IC50, or 1.5 × IC50), 5-FU (IC50), or Sunitinib (0.1 µM) for 24 h. The cells were then
harvested, fixed in 70% ice-cold ethanol, stained with propidium iodide (PI), and analyzed for DNA
content by flow cytometry. Three independent experiments were performed, and the results from one
representative experiment were shown in Fig. 2, and the percentages of the cells in G0/G1, S, and
G2/M phases were calculated and presented in Table S1 (see Supplementary data).
Fig. 2. Effect of compound 8n on cell cycle progression. HCT-116 cells were treated with 8n (1 ×, 1.25
×, and 1.5 × IC50), 5-FU, or Sunitinib for 24 h. And then, cells were harvested, fixed, stained with PI
and analyzed with a flow cytometer. (A) DMSO; (B) 5-FU; (C) Sunitinib (0.1 µM); (D) 8n (1 × IC50);
(E) 8n (1.25 × IC50); (F) 8n (1.5 × IC50); (G) Histogram representing effects of DMSO, 5-FU, Sunitinib,
and 8n on cell cycle progression.
5-FU is an inhibitor of thymidylate synthase (TS), blocking DNA synthesis in a variety of cancer
8

cells to arrest cell cycle in S-phase, while Sunitinib is an inhibitor of multiple tyrosine kinase receptors
inducing G1 cell cycle arrest.20 Our results indicated an increase of G0/G1 and a decrease of S and
G2/M phase after 8n treatment at its IC50 concentration for 24 h in HCT-116 cells. Moreover, a
dose-dependent accumulation of G0/G1cells was observed with the concentrations increased to 1.5
times of the IC50 value of 8n. Additionally, a dose-dependent increase in the sub-G1 phase was also
observed after treatment with 8n for 24 h, which was similar to the behavior of Sunitinib (Fig. 2, C and
F). These properties are quite different from those of 2,4-diaminoquinazoline derivatives connecting
piperazine-1-carbodithioate moiety at its C6-position (IIIa−c, Fig. 1),13 which induced a G2/M phase
arrest in HCT-116 cells, without an obvious sub-G1 peak in the cell cycle profile.
In conclusion, we synthesized
a
series of quinazoline derivatives bearing
piperazine-1-carbodithioate moiety at the C4-position and tested for their antiproliferative activity
against A549, MCF-7 and HCT-116 cell lines. Preliminary structure-activity relationship analysis
revealed that an electron-donating or a moderate electron-withdrawing group on the phenyl ring is
more favorable than a strong electron-withdrawing group to the antiproliferative activity. Replacement
of the phenyl ring in the parent compound 8a by a cyclohexyl (8n) or an electron-rich heterocyclyl
group (9h, 9i), but not an open-chain alkyl or an electron-deficient pyridinyl group, resulted in an
obvious enhancement in activity. Similar to the multiple tyrosine kinase receptor inhibitor, Sunitinib,
compound 8n induced a dose-dependent arrest of HCT-116 cells at G0/G1 phase after treatment for 24
h. Therefore, our newly synthesized compounds might exert an effect on cell proliferation through the
mode similar to Sunitinib, and the mechanism of action is under investigation in our laboratory.
Acknowledgments
This work was supported by the 973 project (Grant No. 2013CB911002 and 2015CB910601),
National Natural Science Foundation of China (Grant No. 20972099, 31530016, and 31461143012),
and Beijing Municipal Commission of Education (Grant No. KZ201210028035).
Supplementary data
Supplementary data associated with this article can be found, in the online version, at...
9

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Figure captions
Fig. 1. Structures of TM208, Tandutinib, and compounds I−IV.
Fig. 2. Effect of compound 8n on cell cycle progression. HCT-116 cells were treated with 8n (1 ×, 1.25
×, and 1.5 × IC50), 5-FU, or Sunitinib for 24 h. And then, cells were harvested, fixed, stained with PI
and analyzed with a flow cytometer. (A) DMSO; (B) 5-FU; (C) Sunitinib (0.1 µM); (D) 8n (1 × IC50);
(E) 8n (1.25 × IC50); (F) 8n (1.5 × IC50); (G) Histogram representing effects of DMSO, 5-FU, Sunitinib,
and 8n on cell cycle progression.
Scheme 1. Synthetic pathway of final compounds 8a−q. Reagents and conditions: (a)
1-Bromo-3-chloropropane, K2CO3, THF, 45 °C, 6 h; (b) Methyl 4-hydroxy-3-methoxybenzoate, K2CO3,
DMF, 75 °C, 6 h; (c) 65% HNO3, AcOH, 45 °C, 6 h; (d) SnCl2·2H2O, concentrated HCl, rt, overnight;
(e) HC(OEt)3, AcONH4, MeOH, reflux, 9 h; (f) PCl5, POCl3, reflux, 15 h; (g) Piperazine, isopropanol,
reflux, 3 h; (h) RCH2Br/RCH2Cl, CS2, K3PO4, DMF, rt, 2 h.
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Graphical Abstract
Synthesis and antitumor activity evaluation of quinazoline derivatives bearing
piperazine-1-carbodithioate moiety at C4-position
Ying Zhanga, Chao-Rui Yang a, Xue Tanga, Sheng-Li Caoa,*, Ting-Ting Rena, Man Gaoa, Ji Liaob,c,
Xingzhi Xub,c,
*
4-Quinazolinylpiperazine-1-carbodithioates 8a−q and 9a−i were synthesized. Most of them exhibited
promising cytotoxicity, and 8n induced a dose-dependent G0/G1 phase arrest in HCT-116 cells.
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