Synthesis and biological evaluation of quinazolin-4(3 H)-one derivatives bearing dithiocarbamate side chain at C2-position as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2015.11.044

A series of quinazolin-4(3H)-one derivatives bearing dithiocarbamate side chain at the C2-position were synthesized and evaluated for their antiproliferative activities against A549, MCF-7, HeLa, HT29 and HCT-116 cell lines. Most of the synthesized compounds exhibited broad spectrum antitproliferative activity against five cell lines, of which 5c was the most potent against HT29 cell line with an IC₅₀ value of 5.53 μM, inducing a G2/M phase arrest in HT29 cells. Treatment of HT29 cells with 5c resulted in BubR1 phosphorylation and an increase of mitotic index in a time-dependent manner. Furthermore, 5c promoted tubulin polymerization in vitro. These results demonstrate that quinazolin-4(3H)-one derivatives bearing dithiocarbamate side chain at C2-position may be potentially novel antitumor agents targeting tubulin to activate the spindle assembly checkpoint.

Full text of DOI:10.1016/j.ejmech.2015.11.044

Accepted Manuscript
Synthesis and biological evaluation of quinazolin-4(3H)-one derivatives bearing
dithiocarbamate side chain at C2-position as potential antitumor agents
Pan-Pan Ding, Man Gao, Bei-Bei Mao, Sheng-Li Cao, Cui-Huan Liu, Chao-Rui Yang,
Zhong-Feng Li, Ji Liao, Hongchang Zhao, Zheng Li, Jing Li, Hailong Wang, Xingzhi
Xu
PII:
S0223-5234(15)30380-9
10.1016/j.ejmech.2015.11.044
EJMECH 8234
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 June 2015
Revised Date: 24 November 2015
Accepted Date: 25 November 2015
Please cite this article as: P.-P. Ding, M. Gao, B.-B. Mao, S.-L. Cao, C.-H. Liu, C.-R. Yang, Z.-F. Li, J.
Liao, H. Zhao, Z. Li, J. Li, H. Wang, X. Xu, Synthesis and biological evaluation of quinazolin-4(3H)-one
derivatives bearing dithiocarbamate side chain at C2-position as potential antitumor agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.044.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of quinazolin-4(3H)-one derivatives bearing
dithiocarbamate side chain at C2-position as potential antitumor agents
Pan-Pan Dinga, Man Gaoa, Bei-Bei Maoa, Sheng-Li Caoa*, Cui-Huan Liua, Chao-Rui Yanga,
Zhong-Feng Lia, Ji Liaob, Hongchang Zhaob, Zheng Lib, Jing Lib, Hailong Wangb, and Xingzhi Xub*
a Department of Chemistry, Capital Normal University, Beijing 100048, PR China
b Beijing Key Laboratory of DNA Damage Response, College of Life Science, Capital Normal University, Beijing 100048, PR
China
Compounds 5a−t were synthesized and evaluated as antitumor agents. Among them, 5c inhibited the
proliferation of HT29 cells by interfering with tubulin, leading to a cell cycle arrest at G2/M phase.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of quinazolin-4(3H)-one derivatives bearing
dithiocarbamate side chain at C2-position as potential antitumor agents
Pan-Pan Dinga, Man Gaoa, Bei-Bei Maoa, Sheng-Li Caoa*, Cui-Huan Liua, Chao-Rui Yanga,
Zhong-Feng Lia, Ji Liaob, Hongchang Zhaob, Zheng Lib, Jing Lib, Hailong Wangb, Xingzhi Xub*
a Department of Chemistry, Capital Normal University, Beijing 100048, PR China
b Beijing Key Laboratory of DNA Damage Response, College of Life Science, Capital Normal University, Beijing 100048, PR
China
Abbreviations: 5-FU, 5-Fluorouracil; ATR, attenuated total reflection; BubR1, Bub1-related protein
kinase; CPT, Camptothecin; 7,8-DHF, 7,8-dihydrofolate; DHFR, dihydrofolate reductase; DSB, DNA
double-strand break;
-H2AX, histone H2AX phosphorylated on serine 139; MTS,
γ
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;
NADPH, nicotinamide adenine dinucleotide phosphate; Noc, Nocodazole; TS, thymidylate synthase.
* Corresponding authors. Tel/fax: +86 10 68902974 (S. L. Cao), +86 10 68902240 (X. Xu).
E-mail address: slcao@cnu.edu.cn (S. L. Cao), xingzhi_xu@mail.cnu.edu.cn (X. Xu).
ABSTRACT: A series of quinazolin-4(3H)-one derivatives bearing dithiocarbamate side chain at the
C2-position were synthesized and evaluated for their antiproliferative activities against A549, MCF-7,
HeLa, HT29 and HCT-116 cell lines. Most of the synthesized compounds exhibited broad spectrum
antitproliferative activity against five cell lines, of which 5c was the most potent against HT29 cell line
with an IC50 value of 5.53 µM, inducing a G2/M phase arrest in HT29 cells. Treatment of HT29 cells
with 5c resulted in BubR1 phosphorylation and an increase of mitotic index in a time-dependent
manner. Furthermore, 5c promoted tubulin polymerization in vitro. These results demonstrate that
quinazolin-4(3H)-one derivatives bearing dithiocarbamate side chain at C2-position may be potentially
novel antitumor agents targeting tubulin to activate the spindle assembly checkpoint.
Keywords: Quinazolin-4(3H)-one; dithiocarbamate; synthesis; antiproliferative activity; G2/M arrest;
spindle assembly checkpoint.
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1. Introduction
Quinazoline derivatives bearing side chains at the C5 or C6-position of the core structure possess
promising antitumor property by targeting folate dependent enzymes including thymidylate synthase
(TS) and dihydrofolate reductase (DHFR). TS catalyzes the reductive methylation of
2′-deoxyuridine-5′-monophosphate (dUMP) to 2′-deoxythymidine-5′-monophosphate (dTMP). This
reaction requires 5,10-methylenetetrahydrofolate (5,10-CH2THF) as the donor of the methyl group,
which is oxidized to 7,8-dihydrofolate (7,8-DHF). DHFR catalyzes the conversion of 7,8-DHF to
5,6,7,8-tetrahydrofolate (5,6,7,8-THF) using nicotinamide adenine dinucleotide phosphate (NADPH) as
the reductant. Thus, TS and DHFR are crucial for the synthesis of dTMP in dividing cells, and TS
and/or DHFR inhibitors have found clinical utility as antitumor agents [1,2]. The quinazoline skeleton
is similar to the pteridine ring in folic acid molecule, and therefore its derivatives resembling the folic
acid structure are of importance for the discovery of antitumor antifolates interfering with folate
dependent enzymes.
The quinazoline derivatives containing L-glutamic acid moiety are generally referred to as
classical antifolates such as ZD1694 [3] and CB3717 [4], while the derivatives containing lipophilic
side chains are called non-classical antifolates such as AG337 [5] and Trimetrexate [6] (Fig. 1). Several
classical or non-classical antifolates have been approved for clinical use or entered clinical trials [1,2].
In our attempts to explore quinazoline-based antitumor agents, the dithiocarbamate moiety that has
been recognized as an efficient antitumor pharmacophore [7-9] was incorporated into the C6-position
of 2-methylquinazlin-4(3H)-one as a lipophilic side chain. Among the synthesized compounds, I and II
(Fig. 1) inhibited the proliferation of several human cancer cell lines with IC50 values at the level of
micromolar concentrations [10-12].
Recently, increasing evidences indicate that quinazlin-4(3H)-one derivatives carrying various
substituents at the C2-position can also exert inhibitory effects on tumor cells. Nevertheless, they may
act through the mechanism of action different from that of quinazoline-based antifolates. For example,
Ispinesib (Fig. 2), a kinesin spindle protein inhibitor, causes mitotic arrest and growth inhibition in
several human tumor cell lines and is currently tested in multiple phase II clinical trials [13].
Compound III (Fig. 2) inhibits the growth of human MCF-7 breast carcinoma cells and Burkitt
lymphoma CA46 cells with IC50 values of 0.34 and 1.0 ꢀM, respectively. At 3.4 ꢀM, it caused
disruption of the cellular microtubule system of the MCF-7 cells, while at 10 ꢀM it induced G2/M
arrest in Burkitt cells. These cellular effects are consistent with its mechanism of action resulting from
its inhibitory effect on tubulin assembly [14]. These facts and our previous work motivated us to
synthesize a new series of compounds 5a−t (Fig. 2) by incorporating the dithiocarbamate moiety into
the C2-position of quinazlin-4(3H)-one. The target compounds would be evaluated for their
antiproliferative activity against human cancer cell lines and investigated the possible mechanism of
action.
2. Chemistry
As outlined in Scheme 1, heating 2-aminobenzoic acid (1) in acetic anhydride under reflux yielded
2-methyl-4H-benzo[d][1,3]oxazin-4-one (2), which was directly used in the next step without
purification. Reaction of 2 with 25% aqueous ammonia in ethanol gave 2-methylquinazolin-4(3H)-one
(3). According to the reported method [15], bromination of 3 with N-bromosuccinimide (NBS)
generated 2-(bromomethyl)quinazolin-4(3H)-one (4), which reacted with carbon disulfide and various
arylmethylamines or heterocyclylmethylamines in the presence of potassium phosphate [16,17] to
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afford quinazolin-4(3H)-one derivatives bearing dithiocarbamate side chain at the C2-position viz.,
target compounds 5a−t.
3. Biology
The
MTS
inner
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
salt] cell proliferation assay was used to evaluate the antiproliferative activity of the synthesized
compounds against five human cancer cell lines including A549 (lung cancer), MCF-7 (breast
adenocarcinoma), HeLa (cervical carcinoma), HT29 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) was
used as a positive control.
Among the tested compounds, 5c exhibited significant antiproliferative activity against HT29 cells
with an IC50 value of 5.53 µM. To gain further insight into the mechanisms of action of this new and
active compound, we examined if treatment of HT29 cells with compound 5c would lead to cell cycle
arrest and DNA damage. HT29 cells were treated with compound 5c or 5-FU at a concentration
equivalent to its IC50 for 24 h. 5-FU is an inhibitor of TS, blocking DNA synthesis in a variety of
cancer cells. Cells were harvested for cell cycle analysis by flow cytometry. Three independent
experiments were performed, and the results were shown in Table 2. Meanwhile, indirect
immunofluorescence assay and immuno-blotting analysis using antibody specific for the DNA
double-strand break (DSB) marker γ-H2AX (histone H2AX phosphorylated on serine 139) were
performed to examine whether these compounds would induce DSBs in cultured cancer cells. The
results were demonstrated in Fig. 3. Mitotic index and BubR1 (Bub1-related protein kinase)
phosphorylation levels were determined to assess the activation of the G2/M checkpoint or the spindle
assembly checkpoint. The results were illustrated in Table 3 and Fig. 4. Furthermore, in vitro tubulin
polymerization assays were conducted to evaluate if compound 5c would interfere with tubulin
polymerization in vitro, and the results were shown in Fig. 5.
4. Results and discussion
4.1. Antiproliferative activity
As shown in Scheme 1 and Table 1, we first synthesized compound 5a (R = C6H5) to serve as a
parent compound, which exhibited moderate antiproliferative activity against five cancer cell lines with
IC50 values in the range of 10.70−31.33 µM. And then, compounds 5b−g bearing electron-donating
groups on the phenyl ring of the side chain were prepared and evaluated for their antiproliferative
activity. Introduction of a methyl group into the 4-position of the phenyl ring (5b, R = 4-CH3C6H4) led
to a decrease in activity against four tumour cell lines. However, compound 5c (R = 4-CH3OC6H4)
bearing a methoxyl group at the 4-position of the phenyl ring exhibited higher antiproliferative activity
against A549 (7.27 versus 23.72 µM), MCF-7 (10.86 versus 31.33 µM) and HT29 (5.53 versus 14.45
µM) cell lines, and slightly weaker activity against HeLa and HCT-116 cell lines than the parent
compound 5a. In comparison with compound 5c, 5d (R = 2-CH3OC6H4) was less active against each
cell line, suggesting that a methoxyl group at the 4-position of the phenyl ring is favorable for the
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antiproliferative activity. Compounds 5e (R = 2,4-diCH3OC6H3), 5f (R = 3,4,5-triCH3OC6H2) and 5g (R
= 3,4-methylenedioxyC6H3) were in general less active than 5c, indicating that one methoxyl group at
the 4-position of the phenyl ring is adequate for generating the antiproliferative activity.
As for compounds bearing electron-withdrawing groups on the phenyl ring, compound 5h (R =
4-BrC6H4) was less active than the parent compound 5a, whereas 5i (R = 4-ClC6H4) was inactive
against five tumor cell lines. In contrast with 5e versus 5c, 5j bearing two chlorine atoms at both the 2
and 4-position of the phenyl ring was more active against A549, HeLa, HT29 and HCT-116 cell lines
than 5i with one chlorine atom at the 4-position. Furthermore, compound 5k (R = 4-FC6H4) bearing a
fluorine atom at the 4-position of the phenyl ring was more potent than its 4-bromo or 4-chloro
counterparts 5h and 5i. However, compounds 5l (R = 2-FC6H4) and 5m (R = 2,4-diFC6H3) exhibited
lower or comparable activity to the 4-fluoro substituted compound 5k, which IC50 was similar to
compounds 5d, 5e and 5c. In addition, compound 5n bearing a stronger electron-withdrawing nitro
group possessed comparable activity to 5k, although both of them were less active than 5c against four
tumour cell lines. These results suggest that the electronic and steric characters of the substituent are
not the critical determinant of activity.
Replacement of the phenyl group in the parent compound 5a by thiophenyl, furanyl or pyridinyl
group resulted in compounds 5o−t. Compound 5o (R = thiophen-2-yl) exhibited comparable activity
against A549, HT29 and HCT-116 cell lines to compound 5a, but higher activity against MCF-7 or
lower activity against HeLa cell line than compound 5a. Compound 5p (R = furan-2-yl) was less active
against each cell line than 5a, while 5q (R = pyridin-2-yl) was inactive at the concentration of 50 µM.
Contrary to 5q, compounds 5r (R = pyridin-3-yl) and 5t (R = pyridin-4-yl) exhibited higher activity
against A549, MCF-7 and HCT-116 cell lines than 5a, or comparable activity against HeLa, and HT29
cell lines to 5a. In addition, the antiproliferative activity of 5s bearing an electron-withdrawing group
(trifluoromethyl) at the 6-position of the pyridine ring was close to its counterpart 5r. These results
indicate that the orientation of a heterocyclic substituent is crucial to the antiproliferative activity, while
the density of electron cloud on the heterocycles exerts little effect on the activity.
Among the compounds synthesized, 5t was the most active member with IC50 values of 6.13 and
16.67 µM against A549 and HCT-116 cell lines, respectively, whereas 5c was the most potent one
against HT29 cell line. Both compounds are worthy to be considered as leads for further studies. Since
5c was more potent than the clinically used anticancer drug 5-FU against HT29 cells (5.53 versus 24.50
µM), it would be used for further investigation on the mechanism of action.
4.2. Compound 5c induced G2/M arrest in HT29 cells
From the MTS assay results, we found that compound 5c inhibited the proliferation of HT29 cells
with an IC50 value of 5.53 µM. To reveal the molecular mechanism of 5c-induced cytotoxicity, we
determined the cell cycle profile of HT29 cells upon treatment with 5c. HT29 cells were treated with
compound 5c, 5-FU at the concentrations equivalent to their IC50, or Taxol (0.18 µM) for 24 h, and data
were shown in Table 2. In agreement with previous reports, HT29 cells treated with 5-FU or Taxol for
24 h were arrested in S-phase or G2/M phase, respectively; while treatment with compound 5c for 24 h
only induced a G2/M arrest in HT29 cells without an obvious sub-G1 peak in the cell cycle profile.
4.3. Compound 5c did not induce DNA damage in HT29 cells
Given that compound 5c induces the G2/M arrest in HT29 cells, one of its potential mechanisms is
that it induces DNA damage. To test this possibility, we treated HT29 cells with Camptothecin (CPT, 2
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µM) or compound 5c for 1 or 2 h. And then, indirect immunofluorescence assay and immuno-blotting
analysis using antibody specific for the DSB marker γ-H2AX were performed to examine whether
these compounds would induce DSBs in cultured cancer cells. The results were demonstrated in Fig. 3.
CPT is an inhibitor of topoisomerase I, which induces replication-dependent DNA damage. We found
that treatment with CPT, but not compound 5c, resulted in distinct γ-H2AX focus formation by
immunofluorescence staining analysis and an increase of γ-H2AX protein levels (Fig. 3). It has been
well established that γ-H2AX is a marker for DSBs [18-20]. These results indicated that compound 5c
might not be able to induce DNA damage.
4.4. Compound 5c activated the spindle assembly checkpoint
A G2/M arrest may result from DNA damage-induced G2/M checkpoint activation or tubulin
poison-induced spindle assembly checkpoint activation. Given that compound 5c failed to induce DNA
damage, we sought to determine if compound 5c would activate the spindle assembly checkpoint to
elicit the G2/M arrest. To this end, we determined the mitotic indexes of HT29 cells treated with 5-FU,
CPT, Noc or compound 5c for different periods of time. The mitotic index was determined by the
percentage of cells positive of immunostaining with the phospho-Histone H3 on serine 10 (H3S10)
antibody. The H3S10 phosphorylation starts at prophase and quickly disappears when cells move from
metaphase to anaphase. Both Noc and Taxol elicit their function in the spindle assembly checkpoint
activation mainly through binding to tubulin and arresting cells at metaphase. The former leads to
destabilization of tubulin polymerization, while Taxol treatment increases polymerization of tubulin
units. As shown in Table 3, 5-FU treatment reduced the mitotic index because it arrested cells at the S
phase, CPT treatment reduced the mitotic index as well since it activated the G2/M checkpoint to
prevent the mitotic entry, while Noc treatment led to a continuing increase of mitotic index between the
2 hour time point and the 16 hour time point. Compound 5c treatment resulted in a similar, but less
extent, continuing increase of mitotic indexes (Table 3). As presented in Fig. 4, Taxol treatment
increased phospho-BubR1 levels, while a less extent increase of BubR1 phosphorylation levels was
seen when cells were treated with compound 5c for the same period of time. BubR1 is a mitotic
regulatory kinase that ensures accurate segregation of chromosomes through its role in the mitotic
checkpoint and the establishment of proper microtubule-kinetochore attachments. BubR1
phosphorylation is an indicator of the spindle assembly checkpoint activation [21,22]. Taken together,
these results demonstrated that compound 5c activates the spindle assembly checkpoint.
4.5. Compound 5c promoted tubulin polymerization in vitro
Finally, in vitro tubulin polymerization assays were conducted to evaluate if compound 5c would
interfere with tubulin polymerization in vitro, and the results were shown in Fig. 5. The in vitro tubulin
polymerization assay confirmed that Taxol eliminated the nucleation phase and shortened the time for
tubulin polymerization in vitro to reach the plateau (i.e., Vmax) in comparison to control. Compound
5c reduced the nucleation phase in comparison to the control, while enhanced Vmax of the growth
phase of tubulin polymerization in vitro in comparison to Taxol. These data indicate that compound 5c
may target tubulin and promote tubulin polymerization in vitro. Further investigation to uncover the
biochemical and molecular mechnism how compund 5c binds to tubulin and promotes its
polymerization is warranted.
Introduction of new moieties into the C2-position of quinazoline has led to series of novel
anticancer compounds. For examples, Ispinesib, a kinesin spindle protein inhibitor, is in phase II
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clinical trials [13], while compound III exhibits its cytotoxic effect in cancer cells by inhibiting tubulin
polymerization[14]. Our newly synthesized quinazolin-4(3H)-one derivatives bearing dithiocarbamate
side chain at C2-position, compound 5c in particular, elicit cytotoxic effects in different cancer cells by
promoting tubulin polymerization and consequently activating the spindle assembly checkpoint.
Therefore, exploration of C2-derivatives of quinazoline may yield promising anticancer compounds
targeting G2/M phase of the cell cycle.
5. Conclusion
By transferring the dithiocarbamate side chain from the C6-position to the C2-position of
quinazolin-4(3H)-one, a new series of compounds 5a−t were designed and synthesized, and most of
them exhibited antiproliferative activities against A549, MCF-7, HeLa, HT29 and HCT-116 cell lines.
Among the active compounds, 5c significantly inhibited the proliferation of HT29 cells with an IC50
value of 5.53 µM and induced a G2/M arrest by promoting tubulin polymerization in vitro and
subsequently activating the spindle assembly checkpoint. These results have demonstrated that the
potential target of compound 5c is tubulin. How compound 5c binds to tubulin and promotes its
polymerization warrants further exploration.
6. Experimental
6.1. Chemistry
Melting points were determined on an XT5B microscopic melting point apparatus and are
uncorrected. 1H NMR spectra were recorded on a Varian VNMRS-600 spectrometer at 600 MHz using
tetramethylsilane (TMS) as internal standard (Abbreviations: Ph
=
phenyl, Quin
=
quinazolin-4(3H)-one). 13C NMR spectra were recorded on a Varian VNMRS-600 spectrometer at 150
MHz using tetramethylsilane (TMS) as internal standard. Infrared (IR) spectra were recorded on a
Bruker Tensor 27 spectrometer with an ATR (attenuated total reflection) accessory. High-resolution
electrospray ionization (HR-ESI) mass spectra were recorded on a Thermo Scientific LTQ Orbitrap
Discovery (Bremen, Germany) mass spectrometer. Column chromatography was carried out on silica
gel (200−300 mesh). High performance liquid chromatography (HPLC) analyses were performed on an
Agilent 1200 Series HPLC instrument with a G1314B VWD (variable-wavelength detector) detector,
and the results were presented in the Supplement materials. Arylmethylamines or
heterocyclylmethylamines were obtained commercially and used without further purification.
6.1.1. Preparation of 2-methylquinazolin-4(3H)-one (3)
A mixture of 2-aminobenzoic acid (1) (1.37 g, 10 mmol) and acetic anhydride (10 mL) was stirred
under reflux for 2 h. Most of excess acetic anhydride was removed by rotary evaporation and the
residue was cooled to room temperature. The separated solid was filtered and dried to give 1.47 g of
2-methyl-4H-benzo[d][1,3]oxazin-4-one (2) as a yellow solid, which was used directly in the next step
without purification.
A solution of 2 (1.47 g, 9.1 mmol) and 25% aqueous ammonia (37 mL) in ethanol (20 mL) was
stirred at room temperature for 48 h. After removing most of the solvent and cooling, the precipitate
was collected by filtration and dried in air, which was purified by recrystallization from ethanol to give
1.12 g (70%, overall yield of two steps) of 3 as a white solid, mp 233−236 °C (Lit. [23] mp
237−239 °C). 1H NMR (600 MHz, DMSO-d6)
δ: 2.61 (s, 2H, CH3), 7.65 (t, J = 7.8 Hz, 1H, Quin 6-H),
7.83 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.96 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.16 (d, J = 7.8 Hz, 1H, Quin
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5-H). 13C NMR (150MHz, DMSO-d6) δ: 21.11, 49.02, 120.69, 125.20, 126.37, 126.91, 135.18, 156.22,
161.61. IR (ATR, cm-1)
ν: 3409, 3036, 2872, 2785, 1657, 1608, 1464, 1385, 1319, 1138, 942, 873, 771,
691. ESI-HRMS m/z: calcd for C9H9N2O ([M+H]+): 161.0715; found: 161.0709.
6.1.2. Preparation of 2-(bromomethyl)quinazolin-4(3H)-one (4)
A stirred solution of 2-methylquinazolin-4(3H)-one (3) (6.40 g, 40 mmol), NBS (7.12 g, 40 mmol)
in anhydrous DMF (35 mL) was heated at 40 °C for 24 h. The formed solid was collected by filtration
and washed with ether and dry to give 7.37 g (77%) as a white solid, mp 227–230 °C. 1H NMR (600
MHz, DMSO-d6) δ: 4.41 (s, 2H, CH2Br), 7.55 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.67 (d, J = 7.8 Hz, 1H,
Quin 8-H), 7.84 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.12 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.57 (br s, 1H, NH).
13C NMR (150MHz, DMSO-d6) δ: 30.18, 121.54, 126.28, 127.66 (2C), 135.06, 148.76, 153.27, 161.94.
IR (ATR, cm-1)
ν: 3037, 2887, 2799, 1683, 1609, 1463, 1323, 1148, 897, 772, 674. ESI-HRMS m/z:
calcd for C9H8BrN2O ([M+H]+): 238.9820, 240.9800; found: 238.9813, 240.9790.
6.1.3. General procedure for the preparation of compounds 5a–t
A suspension of amine (1.5 mmol), carbon disulfide (0.6 mL, 10 mmol), and finely powdered
potassium phosphate (0.40 g, 1.9 mmol) in DMF (20 mL) was stirred at room temperature for 0.5 h.
After adding 2-(bromomethyl)quinazolin-4(3H)-one (4) (0.36 g, 1.5 mmol), stirring was continued for
1−3 h (for 5a-c, 5h-n, 1 h; for 5d-g, 2 h; for 5o-t, 3 h). The mixture was poured into water (150 mL)
and the resulting precipitate was collected by filtration, dried in air. The crude product was purified by
column chromatograph on silica gel using the solvent indicated below as eluent to give compounds
5a–t.
6.1.3.1. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl benzylcarbamodithioate (5a)
1
Yield 63%, yellowish solid, mp 172–174 °C (eluent: CH2Cl2). H NMR (600 MHz, DMSO-d6)
δ
:
4.45 (s, 2H, SCH2), 4.85 (d, J = 5.4 Hz, 2H, CH2NH), 7.29 (m, 1H, Ph 4’-H), 7.33 (m, 4H, Ph 2’-H,
3’-H, 5’-H, 6’-H), 7.42 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.49 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (t, J =
7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.70 (t, J = 5.4 Hz, 1H, CH2NH), 12.41 (s,
1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.91, 50.28, 121.38, 126.26, 127.06, 127.19, 127.77,
128.23 (2C), 128.86 (2C), 134.91, 137.38, 148.62, 153.89, 161.82, 195.52. IR (ATR, cm-1)
ν: 3179,
2920, 2874, 1665, 1604, 1549, 1401, 1319, 926, 903, 765, 695. ESI-MS m/z: 342 [M+H]+. ESI-HRMS
m/z: calcd for C17H16N3OS2 ([M+H]+): 342.0735; found: 342.0723.
6.1.3.2. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-methylbenzylcarbamodithioate (5b)
Yield 51%, off-white solid, mp 186–187 °C (eluent: CH2Cl2/CH3OH = 95:5). 1H NMR (600 MHz,
DMSO-d6) δ: 2.29 (s, 3H, CH3), 4.44 (s, 2H, SCH2), 4.80 (d, J = 5.4 Hz, 2H, CH2NH), 7.14 (d, J = 7.8
Hz, 2H, C6H4-H), 7.22 (d, J = 7.8 Hz, 2H, C6H4-H), 7.38 (d, J = 8.4 Hz, 1H, Quin 8-H), 7.49 (t, J = 7.8
Hz, 1H, Quin 6-H), 7.76 (t, J = 8.4 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.66 (t, J =
5.4 Hz, 1H, CH2NH), 12.40 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 21.15, 37.90, 50.12,
121.37, 126.24, 127.06, 127.17, 128.30 (2C), 129.39 (2C), 134.29, 134.87, 136.98, 148.58, 153.94,
161.81, 195.23. IR (ATR, cm-1)
ν: 3139, 2919, 2811, 1673, 1610, 1524, 1468, 1401, 1316, 1075, 921,
807, 764, 683. ESI-HRMS m/z: calcd for C18H18N3OS2 ([M+H]+): 356.0891; found: 356.0881.
6.1.3.3. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-methoxybenzylcarbamodithioate (5c)
Yield 61%, yellowish solid, mp 178–179 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 3.74 (s, 3H, OCH3), 4.43 (s, 2H, SCH2), 4.77 (d, J = 5.4 Hz, 2H, CH2NH), 6.89 (d, J =
8.4 Hz, 2H, C6H4 3’-H, 5’-H), 7.27 (d, J = 8.4 Hz, 2H, C6H4 2’-H, 6-H), 7.39 (d, J = 7.8 Hz, 1H, Quin
7

ACCEPTED MANUSCRIPT
8-H), 7.49 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.76 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.08 (d, J = 7.8 Hz, 1H,
Quin 5-H), 10.63 (t, J = 5.4 Hz, 1H, CH2NH), 12.40 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ:
37.88, 49.89, 55.54, 114.22 (2C), 121.37, 126.25, 127.07, 127.18, 129.23, 129.79 (2C), 134.87, 148.58,
153.97, 159.09, 161.81, 194.99. IR (ATR, cm-1)
ν: 3137, 2896, 1672, 1616, 1511, 1404, 1304, 1247,
1073, 920, 848, 765. ESI-HRMS m/z: calcd for C18H18N3O2S2 ([M+H]+): 372.0840; found: 372.0831.
6.1.3.4. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 2-methoxybenzylcarbamodithioate (5d)
Yield 49%, yellowish solid, mp 157–158 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 3.80 (s, 3H, OCH3), 4.42 (s, 2H, SCH2), 4.77 (d, J = 5.4 Hz, 2H, CH2NH), 6.90 (t, J = 7.8
Hz, 1H, C6H4 5’-H), 7.03 (d, J = 7.8 Hz, 1H, C6H4 3’-H), 7.21 (d, J = 7.8 Hz, 1H, C6H4 6’-H), 7.30 (t, J
= 7.8 Hz, 1H, C6H4 6’-H), 7.38 (d, J = 8.4 Hz, 1H, Quin 8-H), 7.50 (t, J = 8.4 Hz, 1H, Quin 6-H), 7.77
(t, J = 8.4 Hz, 1H, Quin 7-H), 8.09 (d, J = 8.4 Hz, 1H, Quin 5-H), 10.55 (t, J = 5.4 Hz, 1H, CH2NH),
12.41 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.94, 46.04, 55.76, 111.23, 120.57, 121.35,
124.54, 126.26, 127.07, 127.13, 129.13, 129.28, 134.91, 148.56, 154.05, 157.41, 161.80, 195.16. IR
(ATR, cm-1)
ν: 3173, 2922, 1672, 1610, 1462, 1321, 1243, 1079, 927, 749, 684. ESI-HRMS m/z:
calcd for C18H18N3O2S2 ([M+H]+): 372.0840; found: 372.0833.
6.1.3.5. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 2,4-dimethoxybenzylcarbamodithioate (5e)
Yield 51%, yellowish solid, mp 162–163 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 3.76 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.38 (s, 2H, SCH2), 4.67 (d, J = 5.4 Hz, 2H,
CH2NH), 6.48 (dd, J = 8.4, 2.4 Hz, 1H, C6H3 5’-H), 6.59 (d, J = 2.4 Hz,1H, C6H3 3’-H), 7.15 (d, J =
8.4 Hz, 1H, C6H3 6’-H), 7.33 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.50 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.76 (t,
J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d,J = 7.8 Hz, 1H, Quin 5-H), 10.64 (t, J = 5.4 Hz,1H, CH2NH), 12.40
(s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.94, 45.97, 55.70, 55.93, 98.86, 104.89, 116.65,
121.33, 126.25, 127.08, 127.10, 130.50, 134.87, 148.48, 154.18, 158.61, 160.81, 161.79, 194.56. IR
(ATR, cm-1)
ν: 3162, 2923, 1671, 1625, 1536, 1508, 1465, 1417, 1317, 1208, 1156, 1127, 1070, 1038,
912, 773. ESI-HRMS m/z: calcd for C19H20N3O3S2 ([M+H]+): 402.0946; found: 402.0933.
6.1.3.6. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 3,4,5-trimethoxybenzylcarbamodithioate (5f)
Yield 48%, yellowish solid, mp 168–170 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 3.64 (s, 3H, OCH3), 3.70 (s, 6H, 2OCH3), 4.44(s, 2H, SCH2), 4.76 (d, J = 4.8 Hz, 2H,
CH2NH), 6.68 (s, 2H, C6H2 2’-H, 6’-H), 7.34 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.49 (t, J = 7.8 Hz, 1H,
Quin 6-H), 7.74 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.64 (t, J = 4.8 Hz,
1H, CH2NH), 12.42 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.93, 50.87, 56.26 (2C), 60.43,
105.99 (2C), 121.34, 126.26, 127.05, 127.09, 132.71, 134.85, 137.30, 148.53, 153.30 (2C), 154.10,
161.78, 195.25. IR (ATR, cm-1)
ν: 3250, 2980, 2920, 1690, 1609, 1464, 1340, 1314, 1250, 1119, 1004,
927, 764, 686. ESI-HRMS m/z: calcd for C20H22N3O4S2 ([M+H]+): 432.1052; found: 432.1038.
6.1.3.7. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 3,4-methylenedioxybenzylcarbamodithioate (5g)
Yield 47%, yellowish solid, mp 176–177 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 4.43 (s, 2H, SCH2), 4.74 (d, J = 4.8 Hz, 2H, CH2NH), 6.0 (s, 2H, OCH2O), 6.82 (d, J =
7.8 Hz, 1H, C6H3 6’-H), 6.86 (d, J = 7.8 Hz, 1H, C6H3 5’-H), 6.90 (s, 1H, C6H3 2’-H), 7.42 (d, J = 7.8
Hz, 1H, Quin 8-H), 7.49 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.08 (d, J =
7.8 Hz, 1H, Quin 5-H), 10.62 (t, J = 4.8 Hz, 1H, CH2NH), 12.41 (s, 1H, NH). 13C NMR (150MHz,
DMSO-d6) δ: 37.87, 50.13, 101.40, 108.55, 108.90, 121.36, 121.82, 126.24, 127.06, 127.15, 131.02,
134.88, 146.97, 147.71, 148.58, 153.92, 161.80, 195.18. IR (ATR, cm-1)
ν: 3132, 3037, 2905, 2812,
1674, 1619, 1529, 1498, 1401, 1318, 1251, 1075, 1004, 923, 864, 812, 763, 686. ESI-HRMS m/z: calcd
for C18H16N3O3S2 ([M+H]+): 386.0633; found: 386.0622.
8

ACCEPTED MANUSCRIPT
6.1.3.8. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-bromobenzylcarbamodithioate (5h)
Yield 71%, yellowish solid, mp 192–193 °C (eluent: CH2Cl2/CH3OH = 95:5). 1H NMR (600 MHz,
DMSO-d6) δ: 4.43 (s, 2H, SCH2), 4.80 (d, J = 5.4 Hz, 2H, CH2NH), 7.26 (d, J = 8.4 Hz, 2H, C6H4 2’-H,
6’-H), 7.43 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.47 (t, J = 7.8 Hz, Quin 6-H), 7.50 (d, J = 8.4 Hz, 2H, C6H4
3’-H, 5’-H), 7.76 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.07 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.67 (t, J = 5.4,
1H, CH2NH), 12.40 (br s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.91, 49.46, 120.81, 121.38,
126.25, 127.06, 127.19, 130.37 (2C), 131.71 (2C), 134.88, 136.88, 148.62, 153.76, 161.80, 195.80. IR
(ATR, cm-1)
ν: 3140, 2924, 1673, 1613, 1525, 1468, 1398, 1315, 1076, 1008, 923, 845, 803, 766, 681.
ESI-HRMS m/z: calcd for C17H15BrN3OS2 ([M+H]+): 419.9840, 421.9819; found: 419.9833, 421.9801.
6.1.3.9. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-chlorobenzylcarbamodithioate (5i)
1
Yield 50%, pink solid, mp 188–190 °C (eluent: CH2Cl2/CH3OH = 95:5). H NMR (600 MHz,
DMSO-d6) δ: 4.45 (s, 2H, SCH2), 4.84 (d, J = 5.4 Hz, 2H, CH2NH), 7.33 (d, J = 8.4 Hz, 2H, C6H4 2’-H,
6’-H), 7.38 (d, J = 8.4 Hz, 2H, C6H4 3’-H, 5’-H), 7.45 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.50 (t, J = 7.8 Hz,
1H, Quin 6-H), 7.78 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.69 (t, J = 5.4
Hz, 1H, CH2NH), 12.41 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.93, 49.43, 121.39, 126.27,
127.07, 127.19, 128.80 (2C), 130.04 (2C), 132.34, 134.89, 136.46, 148.64, 153.78, 161.82, 195.81. IR
(ATR, cm-1)
ν: 3141, 2922, 1675, 1616, 1524, 1399, 1314, 1079, 923, 846, 804, 764, 730, 682.
ESI-HRMS m/z: calcd for C17H15ClN3OS2 ([M+H]+): 376.0345, 378.0316; found: 376.0338, 378.0300.
6.1.3.10. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 2,4-dichlorobenzylcarbamodithioate (5j)
1
Yield 57%, brown solid, mp 179–180 °C (eluent: CH2Cl2/CH3OH = 98:2). H NMR (600 MHz,
DMSO-d6) δ: 4.46 (s, 2H, SCH2), 4.86 (d, J = 4.8 Hz, 2H, CH2NH), 7.35 (d, J = 8.4 Hz, 1H, C6H3
6’-H), 7.39 (dd, J = 8.4, 1.8 Hz, 1H, C6H3 5’-H), 7.49 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.51 (t, J = 7.8 Hz,
1H, Quin 6-H), 7.63 (d, J = 1.8 Hz, 1H, C6H3 3’-H), 7.79 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.10 (d, J = 7.8
Hz, 1H, Quin 5-H), 10.66 (t, J = 4.8 Hz, 1H, CH2NH), 12.43 (s, 1H, NH). 13C NMR (150MHz,
DMSO-d6) δ: 37.99, 47.70, 121.39, 126.27, 127.07, 127.19, 127.74, 129.23, 131.16, 133.23, 133.67,
133.78, 134.90, 148.63, 153.69, 161.81, 196.32. IR (ATR, cm-1)
ν: 3151, 2924, 1676, 1617, 1535, 1466,
1404, 1318, 1079, 928, 812, 769, 684. ESI-HRMS m/z: calcd for C17H14Cl2N3OS2 ([M+H]+): 409.9955,
411.9926; found: 409.9944, 411.9911.
6.1.3.11. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-fluorobenzylcarbamodithioate (5k)
1
Yield 64.9%, white solid, mp 167–169 °C (eluent: CH2Cl2/CH3OH = 98:2). H NMR (600 MHz,
DMSO-d6) δ: 4.45 (s, 2H, SCH2), 4.83 (d, J = 5.4 Hz, 2H, CH2NH), 7.16 (t, J = 8.4 Hz, 2H, C6H4 3’-H,
5’-H), 7.37 (dd, J = 8.4, 5.4 Hz, 2H, C6H4 2’-H, 6’-H), 7.45 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.50 (t, J =
7.8 Hz, 1H, Quin 6-H), 7.78 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.09 (dd, J = 7.8, 1.2 Hz, 1H, Quin
5-H), 10.68 (t, J = 5.4 Hz, 1H, CH2NH), 12.41 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.92,
49.48, 115.61 (d, 2C, J = 21.0 Hz), 121.39, 126.27, 127.06, 127.18, 130.31 (d, 2C, J = 7.5 Hz), 133.60
(d, J = 3.0 Hz), 134.90, 148.63, 153.82, 161.82, 161.89 (d, J = 241.5 Hz), 195.60. IR (ATR, cm-1)
ν:
3144, 2920, 2870, 1671, 1608, 1507, 1400, 1318, 1222, 1077, 922, 854, 768, 661. ESI-HRMS m/z:
calcd for C17H15FN3OS2 ([M+H]+): 360.0641; found: 360.0631.
6.1.3.12. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 2-fluorobenzylcarbamodithioate (5l)
Yield 69%, yellowish solid, mp 159–160 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 4.45 (s, 2H, SCH2), 4.86 (d, J = 4.8 Hz, 2H, CH2NH), 7.15 (t, J = 7.2 Hz, 1H, C6H4 3’-H),
7.21 (t, J = 9.0 Hz, 1H, C6H4 5’-H), 7.36 (m, 2H, C6H4 4’-H, 6’-H), 7.46 (d, J = 7.8 Hz, 1H, Quin 8-H),
7.50 (t, J = 7.8 Hz, 1H, Quin 6-H ), 7.78 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.09 (dd, J = 7.8, 1.2 Hz,
1H, Quin 5-H), 10.66 (t, J = 4.8 Hz, 1H, CH2NH), 12.41 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6)
9

ACCEPTED MANUSCRIPT
δ: 37.97, 44.33 (d, J = 4.1 Hz), 115.73 (d, J = 21.2 Hz), 121.39, 124.01 (d, J = 14.7 Hz), 124.80 (d, J =
3.3 Hz), 126.27, 127.06, 127.19, 130.01 (d, J = 8.0 Hz), 130.55 (d, J = 4.1 Hz), 134.92, 148.65, 153.82,
160.65 (d, J = 243.9 Hz), 161.82, 195.91. IR (ATR, cm-1)
ν: 3182, 2982, 1679, 1607, 1377, 1313, 1222,
1067, 923, 888, 758. ESI-HRMS m/z: calcd for C17H15FN3OS2 ([M+H]+): 360.0641; found: 360.0630.
6.1.3.13. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 2,4-difluorobenzylcarbamodithioate (5m)
Yield 78%, yellowish solid, mp 162–163 °C (eluent: CH2Cl2/CH3OH = 95:5). 1H NMR (600 MHz,
DMSO-d6) δ: 4.45 (s, 2H, SCH2), 4.82 (d, J = 4.8 Hz, 2H, CH2NH),7.05 (td, J = 8.4, 1.8 Hz, 1H, C6H3
3’-H), 7.25 (td, J = 9.9, 1.8 Hz, 1H, C6H3 5’-H), 7.43 (m, 1H, C6H3 6’-H), 7.50 (m, 2H, Quin 8-H, 6-H),
7.79 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.63 (t, J = 4.8 Hz, 1H,
CH2NH), 12.41 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.96, 43.88, 104.31 (t, J = 25.6 Hz),
111.81 (dd, J = 21.2, 3.6 Hz), 120.44 (dd, J = 15.0, 3.6 Hz), 121.38, 126.28, 127.07, 127.13, 131.87 (dd,
J = 9.8, 5.7 Hz), 134.92, 148.60, 153.80, 160.72 (dd, J = 246.8, 12.6 Hz), 161.85, 162.20 (dd, J = 244.8,
12.0 Hz), 195.96. IR (ATR, cm-1)
ν: 3149, 2883, 2810, 1678, 1609, 1540, 1504, 1465, 1409, 1319,
1078, 922, 767, 686. ESI-HRMS m/z: calcd for C17H14F2N3OS2 ([M+H]+): 378.0546; found: 378.0539.
6.1.3.14. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl 4-nitrobenzylcarbamodithioate (5n)
Yield 41%, yellowish solid, mp 179–180 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 4.48 (s, 2H, SCH2), 4.99 (d, J = 5.4 Hz, 2H, CH2NH), 7.49 (m, 2H, Quin 8-H, 6-H), 7.52
(d, J = 8.4 Hz, 2H, C6H4 3’-H, 5’-H), 7.78 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin
5-H), 8.18 (d, J = 8.4 Hz, 2H, C6H4 2’-H, 6’-H), 10.79 (t, J = 5.4 Hz, 1H, CH2NH), 12.43 (s, 1H, NH).
13C NMR (150MHz, DMSO-d6) δ: 37.98, 49.35, 121.40, 123.96 (2C), 126.27, 127.06, 127.23, 128.95
(2C), 134.88, 145.48, 147.06, 148.67, 153.60, 161.82, 196.55. IR (ATR, cm-1)
ν: 3168, 2927, 2852,
1666, 1612, 1515, 1337, 1316, 1080, 925, 852, 762, 680. ESI-HRMS m/z: calcd for C17H15N4O3S2
([M+H]+): 387.0586; found: 387.0576.
6.1.3.15. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl thiophen-2-ylmethylcarbamodithioate (5o)
Yield 46%, yellowish solid, mp 176–178 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6)δ: 4.44 (s, 2H, SCH2), 5.02 (d, J = 3.0 Hz, 2H, CH2NH), 6.99 (t, J = 4.2 Hz, 1H,
thiophen-2-yl 4’-H), 7.10 (d, J = 2.4 Hz, 1H, thiophen-2-yl 3’-H), 7.44 (d, J = 4.8 Hz, 1H,
thiophen-2-yl 5’-H), 7.48 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.49 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (t, J =
7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 10.75 (br s, 1H, CH2NH), 12.41 (s, 1H,
NH). 13C NMR (150MHz, DMSO-d6) δ: 37.94, 45.01, 121.38, 126.25, 126.38, 127.06 (2C), 127.24,
127.57, 134.92, 139.44, 148.64, 153.79, 161.81, 195.39. IR (ATR, cm-1)
ν: 3164, 2976, 2928, 2896,
1678, 1609, 1467, 1382, 1318, 1256, 1076, 1062, 1003, 925, 889, 855, 770, 705, 684. ESI-HRMS m/z:
calcd for C15H14N3OS3 ([M+H]+): 348.0299; found: 348.0293.
6.1.3.16. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl (furan-2-yl)methylcarbamodithioate (5p)
Yield 48%, yellowish solid, mp 166–168 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) δ: 4.40 (s, 2H, SCH2), 4.80 (d, J = 5.4 Hz, 2H, NHCH2), 6.36 (d, J = 3.0 Hz, 1H, furan-2-yl
3’-H), 6.40 (m, 1H, furan-2-yl 4’-H), 7.47 (m, 2H, Quin 6-H, 8-H), 7.60 (m, 1H, furan-2-yl 5’-H), 7.76
(td, J = 7.8, 1.8 Hz, 1H, Quin 7-H), 8.06 (dd, J = 7.8, 1.8 Hz, 1H, Quin 5-H), 10.64 (t, J = 5.4 Hz, 1H,
CH2NH), 12.38 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.94, 43.53, 109.21, 111.03, 121.36,
126.25, 127.06, 127.23, 134.93, 143.18, 148.60, 150.05, 153.90, 161.79, 195.63. IR (ATR, cm-1)
ν:
3182, 2987, 1681, 1609, 1468, 1377, 1328, 1185, 1086, 1008, 935, 890, 769, 750, 686. ESI-HRMS m/z:
calcd for C15H14N3O2S2 ([M+H]+): 332.0527; found: 332.0519 .
6.1.3.17. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl pyridin-2-ylmethylcarbamodithioate (5q)
Yield 48%, yellowish solid, mp 147–149 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
10

ACCEPTED MANUSCRIPT
DMSO-d6)
δ
: 4.46 (s, 2H, SCH2), 4.93 (d, J = 3.0 Hz, 2H, CH2NH), 7.30 (m, 2H, pyridin-2-yl 3’-H,
5’-H),7.49 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.53 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.74 (d, J = 7.8 Hz, 1H,
pyridin-2-yl 4’-H), 7.79 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.52 (d, J =
4.2 Hz, 1H, pyridin-2-yl 6’-H), 10.80 (br s, 1H, CH2NH), 12.42 (s, 1H, NH). 13C NMR (150MHz,
DMSO-d6) δ: 37.99, 51.92, 121.37, 122.09, 122.93, 126.26, 127.05, 127.23, 134.82, 134.92, 137.20,
149.53, 153.82, 156.63, 161.83, 196.11. IR (ATR, cm-1)
ν: 3178, 2895, 1680, 1609, 1467, 1376, 1329,
1246, 1101, 936, 889, 770, 687. ESI-HRMS m/z: calcd for C16H15N4OS2 ([M+H]+): 343.0687; found:
343.0681.
6.1.3.18. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl pyridin-3-ylmethylcarbamodithioate (5r)
Yield 52%, yellowish solid, mp 180–182 °C (eluent: CH2Cl2/CH3OH = 98:2).1H NMR (600 MHz,
DMSO-d6) δ: 4.46 (s, 2H, SCH2), 4.87 (d, J = 5.4 Hz, 2H, CH2NH), 7.35 (m, 1H, pyridin-3-yl 5’-H),
7.48 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.50 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.72 (d, J = 7.8 Hz, 1H,
pyridin-3-yl 4’-H), 7.78 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.49 (d, J =
7.8 Hz, 1H, pyridin-3-yl 6’-H), 8.56 (s, 1H, pyridin-3-yl 2’-H), 10.71 (t, J = 5.4 Hz, 1H, CH2NH),
12.42 (s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.93, 47.82, 121.39, 123.96, 126.27, 127.06,
127.20, 133.08, 134.93, 136.00, 148.66, 148.95, 149.57, 153.73, 161.82, 195.99. IR (ATR, cm-1)
ν:
3150, 2966, 2930, 1704, 1609, 1541, 1468, 1406, 1321, 1082, 924, 770, 715, 687. ESI-HRMS m/z:
calcd for C16H15N4OS2 ([M+H]+): 343.0687; found: 343.0682.
6.1.3.19.
(4-Oxo-3,4-dihydroquinazolin-2-yl)methyl
(6-(trifluoromethyl)pyridin-3-yl)methylcarbamodithioate (5s)
Yield 50%, yellowish solid, mp 164–166 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6) : 4.48 (s, 2H, SCH2), 4.97 (s, 2H, CH2NH), 7.50 (m, 2H, Quin 8-H, 6-H), 7.77 (t, J = 7.8
δ
Hz, 1H, Quin 7-H), 7.87 (d, J = 7.8 Hz, 1H, pyridin-3-yl 5’-H), 7.97 (d, J = 7.8 Hz, 1H, pyridin-3-yl
4’-H), 8.09 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.73 (s, 1H, pyridin-3-yl 2’-H), 10.79 (s, 1H, CH2NH), 12.42
(s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.97, 47.32, 120.95 (q, J = 2.5 Hz), 121.41, 122.1 (q,
J = 271.5 Hz), 126.27, 127.06, 127.21, 134.88, 137.26, 137.77, 145.74 (q, J = 33.5 Hz), 148.66, 150.01,
153.59, 161.82, 196.57. IR (ATR, cm-1)
ν: 3144, 2929, 2875, 1679, 1614, 1327, 1176, 1134, 1085, 927,
855, 772. ESI-HRMS m/z: calcd for C17H14F3N4OS2 ([M+H]+): 411.0561; found: 411.0552.
6.1.3.20. (4-Oxo-3,4-dihydroquinazolin-2-yl)methyl pyridin-4-ylmethylcarbamodithioate (5t)
Yield 50%, yellowish solid, mp 148–150 °C (eluent: CH2Cl2/CH3OH = 98:2). 1H NMR (600 MHz,
DMSO-d6)δ: 4.48 (s, 2H, SCH2), 4.88 (d, J = 4.8 Hz, 2H, CH2NH), 7.27 (d, J = 4.8 Hz, 2H,
pyridin-4-yl 3’-H, 5’-H), 7.52 (m, 2H, Quin 8-H, 6-H), 7.80 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.10 (d, J =
7.8 Hz, 1H, Quin 5-H), 8.73 (d, J = 4.8 Hz, 2H, pyridin-4-yl 2’-H, 6’-H), 10.75 (s, 1H, CH2NH), 12.43
(s, 1H, NH). 13C NMR (150MHz, DMSO-d6) δ: 37.96, 48.88, 121.40, 122.74 (2C), 126.29, 127.08,
127.23, 134.96, 146.48, 148.70, 150.01 (2C), 153.64, 161.84, 196.66. IR (ATR, cm-1)
ν: 3386, 2876,
2792, 1654, 1609, 1577, 1469, 1377, 1328, 1100, 933, 896, 773, 692, 634. ESI-HRMS m/z: calcd for
C16H15N4OS2 ([M+H]+): 343.0687; found: 343.0681.
6.2. Cell Proliferation Assay
Cell proliferation assay was performed as described [24]. Cell viability was tested with CellTiter
96 AQueous One Solution Cell Proliferation Assay kit (Promega), based on the use of MTS. The data
were obtained from triplicate wells in three independent experiments.
6.3. Distribution of HT29 cells in cell cycle and mitotic indexes evaluation
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Cell cycle profiles were determined by propidium iodide staining and fluorescence-activated cell
sorter analysis. Mitotic index was determined essentially as described previously [25] using
phospho-Histone H3 (Ser-10) rabbit polyclonal antibodies (dilution 1:1000, Bethyl Laboratories, Inc).
6.4. Indirect immunofluorescence assay
Indirect immunofluorescence assay was performed as described before [26,27]. Mouse
monoclonal antibody against γ-H2AX (clone JBW301) was purchased from Millipore and used in a
dilution of 1:1500.
6.5. Immuno-blotting analysis
Immuno-blotting analysis was performed as described before [26,27]. Mouse monoclonal
antibody against β-actin was from Sigma, and rabbit polyclonal antibodies against BubR1, γ-H2AX,
Histone H3, and phospho-Histone H3 at Ser 10 (pH3S10) were purchased from Bethyl Laboratories,
Inc and used at a dilution of 1:5000, 1:1000, 1:5000, 1:2000, and 1:1000, respectively.
6.6. Tubulin polymeration assay
In vitro tubulin polymerization assays were performed essentially as described by the
manufacturer (Cytoskeleton, catalogue #BK006P). Polymerizations were monitored by an increase in
absorbance at 340 nm over a 60 minute period at 37 °C. OD at 340 nm was determined using a
SpectraMax M5 spectrometer (Molecular Devices).
Acknowledgements
This work was supported by the 973 project 2013CB911002, National Natural Science Foundation
of China (Project No. 20972099, 31461143012 and 31130017), and Beijing Municipal Commission of
Education (Project No. KZ201210028035).
References
[1] A. Gangjee, H.D. Jain, S. Kurup, Recent advances in classical and non-classical antifolates as
antitumour and antiopportunistic infection agents: Part I, Anti-Cancer Agents Med. Chem. 7 (2007)
524−542.
[2] A. Gangjee, H.D. Jain, S. Kurup, Recent advances in classical and non-classical antifolates as
antitumour and antiopportunistic infection agents: Part II, Anti-Cancer Agents Med. Chem. 8
(2008) 205−231.
[3] A.L. Jackman, F.T. Boyle, K.R. Harrap, Tomudex (ZD1694), from concept to care, a program in
rational drug discovery, Invest. New Drugs 14 (1996) 305–316.
[4] T.R. Jones, A.H. Calvert, A.L. Jackman, S.T. Brown, M. Jones, K.R. Harrap, A potent antitumor
quinazoline inhibitor of thymidylate synthase: synthesis, biological properties, and therapeutic
results in mice, Eur. J. Cancer 17 (1981) 11–19.
[5] S. Webber, C.A. Bartlett, T.J. Boritzki, J.A. Hilliard, E.F. Howland, A.L. Johnston, M. Kosa, S.A.
Margosiak, C.A. Morse, B.V. Shetty, AG337, a novel lipophilic thymidylate synthase inhibitor: In
vitro and in vivo preclinical studies, Cancer Chemother. Pharmacol. 37 (1996) 509–517.
[6] A. Gangjee, W. Li, J. Yang, R.L. Kisliuk, Design, synthesis, and biological evaluation of classical
and nonclassical 2-amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as dual
thymidylate synthase and dihydrofolate reductase inhibitors, J. Med. Chem. 51 (2008) 68–76.
12

ACCEPTED MANUSCRIPT
[7] P. Csomόs, I. Zupkό, B. Réthy, L. Fodor, G. Falkay, G. Bernáth, Isobrassinin and its analogues:
Novel types of antiproliferative agents, Bioorg. Med. Chem. Lett. 16 (2006) 6273–6276.
[8] X. Hou, Z. Ge, T. Wang, W. Guo, J. Cui, T. Cheng, C. Lai, R. Li, Dithiocarbamic acid esters as
anticancer
agent.
Part
1:
4-Substituted-piperazine-1-carbodithioic
acid
3-cyano-3,3-diphenyl-propyl esters, Bioorg. Med. Chem. Lett. 16 (2006) 4214–4219.
[9] M.A.-H. Zahran, T.A.-R.Salem, R.M. Samaka, H.S. Agwa, A.R. Awad, Design, synthesis and
antitumour evaluation of novel thalidomide dithiocarbamate and dithioate analogs against Ehrlich
ascites carcinoma-induced solid tumour in Swiss albino mice, Bioorg. Med. Chem. 16 (2008)
9708–9718.
[10] S.L. Cao, Y.P. Feng, Y.Y. Jiang, S.Y. Liu, G.Y. Ding, R.T. Li, Synthesis and in vitro antitumour
activity of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains, Bioorg. Med. Chem.
Lett. 15 (2005) 1915−1917.
[11] S.L. Cao, Y.P. Feng, X.L. Zheng, Y.Y. Jiang, M. Zhang, Y. Wang, M. Xu, Synthesis of
substituted-benzylamino-
and
heterocyclylmethylamino-carbodithioate
derivatives
of
4(3H)-quinazolinone and their cytotoxic activity, Arch. Pharm. 339 (2006) 250−254.
[12] S.L. Cao, Y. Wang, L. Zhu, J. Liao, Y.W. Guo, L.L. Chen, H.Q. Liu, X. Xu, Synthesis and
cytotoxic activity of N-((2-methyl-4(3H)-quinazolinon-6-yl)methyl)dithiocarbamates, Eur. J. Med.
Chem. 45 (2010) 3850−3857.
[13] L. Lad, L. Luo, J.D. Carson, K.W. Wood, J.J. Hartman, R.A. Copeland, R. Sakowicz, Mechanism
of inhibition of human KSP by ispinesib, Biochemistry 47 (2008) 3576–3585.
[14] D. Raffa, M.C. Edler, G. Daidone, B. Maggio, M. Merickech, S. Plescia, D. Schillaci, R. Bai, E.
Hamel, Synthesis, cytotoxicity, and inhibitory effects on tubulin polymerization of a new
3-heterocyclo substituted 2-styrylquinazolinones, Eur. J. Med. Chem. 39 (2004) 299–304.
[15] J. Bergman, A. Brynolf, Synthesis of chrysogine, a metabolite of penicillium chrysogenum and
some related 2-substituted 4-(3H)-quinazolinones, Tetrahedron 46 (1990) 1295−1310.
[16] R.T. Li, P.Y. Ding, M. Han M.S. Cai, A simple one-pot preparation of dithiocarbamates in the
,
presence of anhydrous potassium phosphate, Synth. Commun. 28 (1998) 295−300.
[17] R.T. Li, Z.M. Ge, T.M. Cheng, M.S. Cai, New synthetic method for dithiocarbamates, Chem. J.
Chin. Univ. 20 (1999) 1897−1902.
[18] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA double-stranded breaks
induce histone H2AX phosphorylation on serine 139, J. Biol. Chem. 273 (1998), 5858−5868.
[19] E.P. Rogakou, C. Boon, C. Redon, W.M. Bonner, Megabase chromatin domains involved in DNA
double-strand breaks in vivo, J. Cell. Biol. 146 (1999), 905−915.
[20] T.T. Paull, E.P. Rogakou, V. Yamazaki, C.U. Kirchgessner, M. Gellert, W.M. Bonner, A critical
role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage, Curr.
Biol. 10 (2000), 886−895.
[21] W.Q. Li, Z.D. Lan, H.Y. Wu, S.C. Wu, J. Meadows, J. Chen, V. Zhu, W. Dai, BUBR1
phosphorylation is regulated during mitotic checkpoint activation, Cell Growth Differ. 10 (1999),
769−775.
[22] G.K.T. Chan, S.A. Jablonski, V. Sudakin, J.C. Hittle, T.J. Yen, Human BUBR1 is a mitotic
checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC,
J. Cell. Biol. 146 (1999), 941−954.
[23] S.A. Rocco, J.E. Barbarini, R. Rittner, Synthese of some 4-anilinoquinazoline derivatives,
Synthesis (2004) 429−435.
13

ACCEPTED MANUSCRIPT
[24] S. L. Cao, Y. Han, C. Z. Yuan, Y. Wang, Z. K. Xiahou, J. Liao, R. T. Gao, B. B. Mao, B. L. Zhao,
Z.
F.
Li,
X.
Xu,
Synthesis
and
antiproliferative
activity
of
4-substituted-piperazine-1-carbodithioate derivatives of 2,4-diaminoquinazoline, Eur. J. Med.
Chem. 64 (2013) 401−409.
[25] X. Xu, J.H. Lee, D.F. Stern, Microcephalin is a DNA damage response protein involved in
regulation of CHK1 and BRCA1, J. Biol. Chem. 279 (2004) 34091–34094.
[26] X. Xu, D.F. Stern, NFBD1/MDC1 regulates ionizing radiation-induced focus formation of DNA
checkpoint signaling and repair factors, FASEB J. 17 (2003) 1842–1848.
[27] X. Xu, D.F. Stern, NFBD1/KIAA0170 is a chromatin-associated protein involved in DNA
damage signaling pathways, J. Biol. Chem. 278 (2003) 8795–8803.
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Figure captions
Fig. 1. Structures of ZD1694, CB3717, AG337, Trimetrexate and compounds I, II.
Fig. 2. Structures of Ispinesib, compound III and target compounds 5a−t.
Fig. 3. Compound 5c does not induce DNA damage. (A) Compound 5c failed to induce γ-H2AX foci
formation. HT29 cell were treated with Camptothecin (CPT), compound 5c or DMSO (dilutent for CPT
and 5c) for 1 or 2 h. Cells were fixed and immuno-stained with a mouse monoclonal antibody against
γ-H2AX, and DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar: 20 µM. (B)
Compound 5c failed to increase γ-H2AX protein levels. HT29 cells were treated as described in (A),
total lysates were harvested and immunoblotted with antibodies as indicated. Different sections from
the same membrane were blotted with different antibodies according to the molecular weight of
targeting antigens. The molecular weight of γ-H2AX and β-actin is 15 kDa and 42 kDa, respectively.
Fig. 4. Compound 5c induced BubR1 phosphorylation. After HT29 cells were treated with Taxol for 16
h or compound 5c for 2, 4, 8, and 16 h, total lysates were harvested for immuno-blotting with
antobsodies as indicated. Different sections from the same membrane were blotted with pH3S10,
BubR1, and β-actin antibodies according to the molecular weight of targeting antigens, while the
pH3S10 blot was striped and re-probed with an anti-Histone H3 antibody.
Fig. 5. Compound 5c promotes tubulin polymerization in vitro. In vitro tubulin polymerization assays
were performed using purified porcine neuronal tubulin according to the manufacturer’s instructions. I,
II, and III denote the nucleation phase, the growth phase, and the steady state equilibrium phase of
tubulin polymerization in vitro, respectively.
Scheme 1. Synthetic route to compounds 5a−t. Reagents and conditions: (a) Ac2O, reflux, 2 h. (b) 25%
NH3·H2O, EtOH, rt, 48 h. (c) NBS, DMF, 40 °C, 24 h. (d) RCH2NH2, CS2, K3PO4, DMF, rt, 1−3 h.
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Table 1. Antiproliferative activity of compounds 5a−t against five human cancer cell lines.
IC50a, µM
HeLa
Compound
R
A549
MCF-7
HT29
HCT-116
5a
5b
5c
5d
5e
5f
C6H5
23.72 ± 0.77
32.58 ± 0.75
7.27 ± 0.75
11.97 ± 1.40
10.47 ± 0.51
10.42 ± 1.50
44.58 ± 2.86
18.19 ± 1.68
> 50
31.33 ± 1.27
21.36 ± 1.48
10.86 ± 1.22
14.84 ± 0.83
13.48 ± 0.64
8.97 ± 0.57
21.66 ± 0.41
39.17 ± 0.79
> 50
10.70 ± 0.35
20.70 ± 0.89
12.19 ± 0.60
16.49 ± 0.56
15.99 ± 1.03
22.95 ± 0.79
20.28 ± 0.21
22.89 ± 0.50
> 50
14.45 ± 1.28
16.34 ± 0.45
5.53 ± 0.41
12.28 ± 3.06
10.08 ± 0.54
7.16 ± 0.96
19.27 ± 1.38
32.27 ± 2.49
> 50
18.12 ± 1.05
23.17 ± 1.46
22.72 ± 3.20
30.60 ± 1.80
25.47 ± 0.46
17.31 ± 0.98
34.25 ± 1.47
45.28 ± 4.82
> 50
4-CH3C6H4
4-CH3OC6H4
2-CH3OC6H4
2,4-diCH3OC6H3
3,4,5-triCH3OC6H2
3,4-MethylenedioxyC6H3
4-BrC6H4
5g
h
5i
4-ClC6H4
5j
2,4-diClC6H3
4-FC6H4
12.83 ± 0.38
10.71 ± 0.31
21.21 ± 1.57
30.58 ± 0.1
11.44 ± 1.50
25.67 ± 1.73
42.56 ± 1.95
> 50
> 50
28.97 ± 2.12
21.17 ± 0.76
13.15 ± 0.59
20.05 ± 0.58
28.39 ± 0.89
26.12 ± 0.70
31.20 ± 3.44
> 50
11.37 ± 0.75
18.57 ± 0.92
16.42 ± 0.60
15.65 ± 1.38
17.21 ± 2.37
16.05 ± 0.84
33.71 ± 2.61
> 50
29.73 ± 3.12
20.13 ± 0.13
20.3 ± 1.90
22.31 ± 0.79
16.38 ± 2.64
16.09 ± 2.20
24.62 ± 4.98
> 50
5k
5l
17.36 ± 0.33
20.49 ± 3.55
20.45 ± 1.30
19.28 ± 1.05
23.93 ± 1.33
> 50
2-FC6H4
5m
5n
5o
5p
5q
5r
5s
2,4-diFC6H3
4-NO2C6H4
Thiophen-2-yl
Furan-2-yl
Pyridin-2-yl
Pyridin-3-yl
6-CF3-pyridin-3-yl
Pyridin-4-yl
> 50
14.77 ± 1.63
12.87 ± 0.41
6.13 ± 1.04
3.52 ± 0.46
20.23 ± 0.64
19.25 ± 2.13
16.08 ± 0.96
32.18 ± 1.13
11.01 ± 2.13
28.43 ± 0.60
12.52 ± 2.48
43.71 ± 3.49
14.21 ± 1.40
16.48 ± 1.15
15.98 ± 1.47
24.50 ± 2.62
11.18 ± 0.29
16.66 ± 0.49
6.67 ± 0.85
5.53 ± 0.90
5t
5-FU
a IC50: The concentration that causes 50% of cell proliferation inhibition. Data are expressed as mean ± SD from triplicate
determination from three independent experiments.

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Table 2. Effects of compound 5c on HT29 cell distribution in cell cycle.
Compound
Cell cycle distribution (%)a
G0/G1
S
G2/M
DMSO
5-FU
Taxol
5c
50.25 ± 1.19
5.42 ± 1.07
1.7 ± 0.55
16.45 ± 2.15
33.05 ± 2.22
91.11 ± 0.81
17.6 ± 6.57
28.25 ± 3.82
16.7 ± 1.55
3.48 ± 0.25
80.71 ± 6.94
55.31 ± 2.96
a Data are expressed as mean ± SD from triplicate determination from three independent experiments.

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Table 3. Mitotic indexes of HT29 cells treated with compound 5c.
Compound
Mitotic indexa
2 h
4 h
8 h
16 h
DMSO
5-FU
CPT
2.47 ± 0.71
2.28 ± 0.97
1.55 ± 0.78
5.22 ± 1.66
4.66 ± 1.99
3.13 ± 1.01
2.09 ± 1.08
1.11 ± 0.67
11.4 ± 3.8
7.75 ± 4.86
2.81 ± 0.82
2.87 ± 0.82
0.23 ± 0.12
0.29 ± 0.10
0.33 ± 0.05
25.61 ± 3.67
10.15 ± 1.83
Noc
64.81 ± 3.54
37.85 ± 3.77
5c
a
Data are expressed as mean ± SD from triplicate determination from three independent experiments.
The mitotic index for untreated cells was 2.94 ± 0.59.

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Research highlights
•
•
•
•
Twenty dithiocarbamate derivatives of quinazolin-4(3H)-one were synthesized.
Antiproliferative activities of 5a−t against five cancer cell lines were evaluated.
Compound 5c induced a G2/M arrest in HT29 cells.
5c promoted tubulin polymerization and activated the spindle assembly checkpoint.