Discovery of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as novel FAK inhibitors with antitumor and anti-angiogenesis activities

Article abstract of DOI:10.1016/j.ejmech.2019.05.048

A series of 2,4-diarylaminopyrimidine derivatives containing dithiocarbamate moiety were designed by molecular hybridization strategy and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most of these compounds exhibit significant a

Full text of DOI:10.1016/j.ejmech.2019.05.048

Accepted Manuscript
Discovery of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as
novel FAK inhibitors with antitumor and anti-angiogenesis activities
Yue Su, Ridong Li, Xianling Ning, Zhiqiang Lin, Xuyang Zhao, Juntuo Zhou, Jia Liu,
Yan Jin, Yuxin Yin
PII:
S0223-5234(19)30458-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.048
EJMECH 11358
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2018
Revised Date: 17 May 2019
Accepted Date: 17 May 2019
Please cite this article as: Y. Su, R. Li, X. Ning, Z. Lin, X. Zhao, J. Zhou, J. Liu, Y. Jin, Y. Yin, Discovery
of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as novel FAK inhibitors with
antitumor and anti-angiogenesis activities, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.048.
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Graphical abstract

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Discovery of 2,4-diarylaminopyrimidine derivatives bearing
dithiocarbamate moiety as novel FAK inhibitors with antitumor
and anti-angiogenesis activities
a
a
Yue Su a, c, 1, Ridong Li a, 1, Xianling Ning , Zhiqiang Lin , Xuyang Zhao a, Juntuo
Zhou a, Jia Liu a, Yan Jin a, Yuxin Yin a, b, c,*
aInstitute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center,
Beijing 100191, PR China
bPeking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing 100191, PR China
cDepartment of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing
100191, PR China
Abstract
A series of 2,4-diarylaminopyrimidine derivatives containing dithiocarbamate
moiety were designed by molecular hybridization strategy and synthesized for
screening as inhibitors of focal adhesion kinase (FAK). Most of these compounds
exhibit significant antiproliferative activities on human cancer cell lines expressing
high levels of FAK at nanomolar concentrations. The compound 14z was identified as
the most potent FAK inhibitor among these candidates. 14z has excellent
anti-proliferative effect with IC50 values from 0.001 µM to 0.06 µM on HCT116, PC-3,
U87-MG and MCF-7 cell lines and relatively less cytotoxicity to a nonmalignant cell
line MCF-10A compared with MCF-7 cells (SI value > 10). 14z also exhibits
significant FAK inhibitory activity (IC50 = 0.07 nM). In addition, compound 14z
causes cell cycle arrest at G2/M and prompted apoptosis in both HCT116 and MCF-7
cells in a dose-dependent manner. Further studies show that compound 14z inhibits
migration of MCF-7 and has anti-angiogenesis effect on HUVEC cells.
Keywords:
FAK inhibitors; 2,4-Diarylaminopyrimidine; Dithiocarbamate; Anti-tumor activity;
Anti-angiogenesis
*Corresponding author.
E-mail address: yinyuxin@hsc.pku.edu.cn.
1These authors contribute equally to this work.
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1. Introduction
Cancer is still one of the major causes of the death worldwide. However the
traditional chemotherapy is facing the obstacles that lacking of selectivity towards
tumor cells and causing drug resistance [1]. Thus a lot of efforts have been put into
designing small molecular compounds with specific targets to enhance the selectivity
and efficacy in recent years [2-4].
Focal adhesion kinase (FAK) is one of these targets. FAK is a non-receptor tyrosine
kinase and plays a vital role in regulating cancer cell survival, proliferation, migration,
and angiogenesis. It is overexpressed in a number of human cancers, including
gliomas, breast, colon, prostate carcinomas and others [5-7]. Inhibition of FAK has
exhibited efficacy in limiting tumor progression and metastasis. Therefore this kinase
is emerging as a promising cancer therapeutic target.
In recent years, several FAK inhibitors have been developed and some of them
have entered clinical trials (Fig.1) [8]. PF-573,228 is one of the first FAK inhibitors
and was highly specific for FAK (IC50 = 4 nM) compared with other related enzymes.
However it only inhibits cell migration rather than inhibits cell proliferation or
induces apoptosis in tumor cells [9]. Another FAK inhibitor TAE226 (IC50 = 5.5 nM)
exhibits antitumor activity in vitro and in vivo against several types of malignancies
including glioma [10], breast cancer [11], ovarian cancer [12] and gastrointestinal
stroma tumor [13]. But it has never entered clinical trials for its severe side effect in
affecting glucose metabolism and glucose blood levels [14]. CEP-37440 and
PF-562,271 have currently completed the phase study. CEP-37440 (IC50 = 2 nM)
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displayed favorable in vitro ADME properties [15]. PF-562, 271 （IC50 = 1.5 nM）not
only inhibits the proliferation of cancer cells but also exerts a novel mechanism in
altering the tumor microenvironment [16]. In the phase Ⅰ study of PF-562,271,
although several patients have experienced prolonged disease stabilization of 6
months or longer, no objective response has been reported. Defactinib (VS-6063) is a
second-generation inhibitor of FAK (IC50 = 0.6 nM). In the phase I dose-escalation
study in patients with advanced solid malignancies, some adverse events have been
reported, including nausea, diarrhea, hyperbilirubinemia and so on. Defactinib
prevents tumor invasion and dissemination rather than reducing tumor volume [17].
Recently, the phase II clinical trials for Defactinib have been completed in patients
with KRAS mutant non-small cell lung cancer. In summary of these currently
available FAK inhibitors, their antitumor activity is not enough when used alone and
needed to be combined with other therapies. Up till now, there is no FAK inhibitor
coming into the market. So there is an urgent need to develop novel FAK inhibitors
with higher antitumor activity to exert more potent antitumor effect in patients.
Among the potent FAK inhibitors aforementioned, a panel of compounds possesses
2,4-diarylaminopyrimidine scaffold as a pivotal pharmacophore. Generally, the
2,4-diarylaminopyrimidine core may functionally interact with FAK at Cys502,
Asp564 or other sites within the ATP binding pocket [8]. The researches of other FAK
inhibitors with 1,2,4-triazine or imidazo[1,2 ‑ a][1,3,5]triazinesas scaffold are
underway [18, 19].
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Fig.1. Structures of several known FAK inhibitors.
Molecular hybridization strategy is a common concept to design new drugs based
on the combination of different bioactive moieties to produce a new hybrid with
improved affinity and efficacy [20]. Recently, dithiocarbamates have received
considerable attention due to their potent anticancer activities [21-26]. In this
reasearch, molecular hybridization technique based on the incorporation of
2,4-diarylaminopyrimidine and dithiocarbamate into one molecular platform has the
potential to improve the anti-FAK and antiproliferative efficacy.
Herein, a series of C-2 dithiocarbamate-substituted 2,4-diarylaminopyrimidine
derivatives (Fig. 2) were synthesized to improve inhibitory potency toward FAK
kinase and antiproliferative activity of cancer cells. To the best of our knowledge,
there have been no reports regarding 2,4-diarylaminopyrimidine-dithiocarbamate
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hybrids so far. These novel compounds showed significant inhibitory potency against
FAK enzyme and antitumor activities in HCT116, PC-3, U87-MG, and MCF-7 cell
lines. SAR of these compounds was discussed in this manuscript. The most effective
compound 14z was also evaluated for its effects on cell cycle arrest, apoptosis, cell
migration and angiogenesis.
Fig.2. Designed strategy of the dithiocarbamate-substituted 2,4-diarylaminopyrimidine derivatives
2. Results and discussion
The synthesis of dithiocarbamate-substituted diphenylaminopyrimidine derivatives
(14a-14u) was depicted in Scheme 1. 2-nitrobenzoyl chloride was reacted with
methanamine in the presence of sodium bicarbonate to produce
2-nitro-(N-methyl)benzamide (6). 2-amino-N-methylbenzamide (7) was obtained via
the reduction of compound 6 by using the Fe-NH4Cl reduction reagent. Compound 8
was produced by the same way as compound 6 started from 4-nitrobenzoyl chloride.
Compound 9 was produced by the same way as compound 7. The C-4 chlorine atom
of 2,4,5-trichloropyrimidine was first region selectively replaced by compound 7 to
obtain 10a. Then compound 9 was coupled with the C-2 of 2,5-dichloropyrimidine
from compound 10a and produced intermediate 11a. The compound 11a was stirred
in the solution of trifluoroacetate and dichloromethane (1:1) to get rid of Boc group.
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Then the product was concentrated and reacted with carbon disulfide in DMF with
triethylamine as base as previously reported [27].Without further purification, a
variety of halogen or alkene substituted side chains that were commercially available
or prepared by literature [28] were reacted with the product mentioned above
respectively, followed by chromatographic purification to generate the target
compounds 14a-14u.
O2N
H2N
Boc
O2N
Boc
a
N
N
b
Cl
N
N
O
O
O
9
8
Scheme 1. Synthesis of dithiocarbamate-substituted diphenylaminopyrimidine derivatives (14a-14u). Reagents
and conditions:(a) Methanamine hydrochloride or tert-butyl piperazine-1-carboxylate, NaHCO3, acetonitrile,
reflux, 4 h; (b) Fe-NH4Cl, EtOH-H2O, reflux, 2 h; (c) Compound 7; DIPEA, i-PrOH, reflux 6 h (d) Compound 9,
Pd(OAc)2, Xantphos, K3PO4, DMF, Ar, reflux, 22 h; (e) i: TFA, DCM, rt., 2 h; ii: CS2, Et3N, DMF, rt., 1 h; iii:
RBr/RCl/Methyl acrylate, DMF, 50 , overnight.
In order to test our design rationale, we took 14a which bears a propanenitrile
moiety at R as our new lead to test the inhibitory activity against FAK. The result
revealed that 14a is a potent FAK inhibitor (inhibition rate = 50% at 1µM). Then
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according to bioisosterism principles, the cyano group in 14a was replaced with
alkenyl, methoxy, ester, methanesulfonyl, hydroxyl, amines and five or six-membered
aliphatic heterocycles, respectively (14b-14p). As a comparison, in order to
investigate the influence of aromatic group on the inhibitory activity, we used benzyl
group (14q), phenylethyl group (14r) and pyridinemethyl (14s-14u) to replace
propanenitrile moiety. Compounds 14a-14u were all evaluated as FAK inhibitors by
ADP-Glo kinase assay [29] at the concentration of 1 µM. TAE226 and Defactinib were
used as positive control. As shown in Table 1, most of the novel derivatives exhibited
potent anti-FAK activity at 1 µM, especially compounds 14i and 14j. The replacement
of cyano group with ester (14d-14f), hydroxyl (14h, 14i), amine (14j, 14k, 14n) or
aliphatic heterocycle containing oxygen atom (14o, 14p) can increase the inhibitory
activity against FAK. However, when the propanenitrile moiety of 14a was replaced
by phenylethyl group (14r) or pyridinemethyl (14s-14u), the inhibitory activity
decreased.
To test the anticancer activity of synthesized compounds, we evaluated
antiproliferative activities of compounds 14a-14u against four FAK over-expressed
human tumor cell lines derived from human colon cancer (HCT116), prostate cancer
(PC-3), glioma (U87-MG), and breast cancer (MCF-7) by MTS assay. TAE226 and
Defactinib were also used as positive control. As shown in Table 1, most compounds
exhibited potent anti-cancer activities against selected cancer cell lines with IC50 less
than 1µM. Among all of the compounds 14c, 14h-14m exhibited more significant
efficacy against all of four cancer cell lines. Among these cancer cell lines, HCT116
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cell line was the most sensitive to these compounds with most IC50 values in a
nanomolar range. Compared with TAE226 and Defactinib, 14a significantly increased
the activity against HCT116 cells (IC50 = 0.03 µM) and PC-3 cells (IC50 = 0.17 µM).
Replacing the propanenitrile moiety of 14a with allyl (14b), or ethyl acetate (14e)
resulted in decrease in potency against HCT116, PC-3 and U87-MG cell lines.
However, the replacements of propanenitrile by ethyl pyruvate (14f, IC50 = 0.52 µM),
2-(1,3-dioxolan-2-yl)ethyl
(14o,
IC50
=
0.21
µM)
or
(tetrahydro-2H-pyran-4-yl)methyl (14p, IC50 = 0.54 µM) significantly enhanced the
inhibitory against MCF-7 cells. Replacements of cyano group of 14a by methoxy
(14c), methanesulfonyl (14g), hydroxyl (14h, 14i) or amine (14j-14m) resulted in a
significant improvement of activity against U87-MG and MCF-7 cells and
maintaining the inhibitory against HCT116 and PC3 cells except 14j which increased
the inhibitory against HCT116 and PC3 greatly. However, when the cyano group of
14a was replaced by morpholine (14n), the potency against HCT116 cells decreased
approximately 117-fold. Introducing the benzyl group (14q) or phenylethyl group
(14r) at the R position led to significant or slight reduction of activities against all of
the tested cancer cell lines. However, when pyridinemethyl (14s-14u) was introduced
at the R position, the corresponding compounds (14s-14u) displayed 9~18-fold
increases in inhibitory potency against MCF-7 cells compared with compound 14a.
The position of nitrogen atom in the pyridine ring has little effect on activity.
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Table 1.
FAK inhibitory activities and antiproliferative activities of target compounds 14a-14u against four human tumor cell
lines.
Antiproliferative activity (IC50 / µM)
FAK inhibition%
Compd.
R
HCT116
PC-3
U87-MG
MCF-7
(1 µM)
50
45
69
73
0.03±0.01
0.11±0.01
0.04±0.01
0.26±0.05
0.17±0.03
1.02±0.47
0.46±0.02
1.99±0.26
1.73±0.59
>20
7.17±3.33
1.38±0.73
0.30±0.08
2.29±0.68
14a
14b
14c
14d
0.61±0.09
1.31±0.07
55
57
20
4.69±0.81
0.06±0.02
0.03±0.01
3.53±0.91
0.47±0.03
0.52±0.14
7.54±0.22
1.16±0.04
0.92±0.43
9.93±0.10
0.52±0.05
0.40±0.07
14e
14f
14g
53
71
0.02±0.01
0.03±0.01
0.24±0.02
0.20±0.04
0.71±0.21
0.87±0.41
0.11±0.09
0.11±0.10
14h
14i
73
55
44
50
62
63
62
52
0.004±0.00
0.01±0.00
0.01±0.00
0.01±0.00
3.52±0.30
0.05±0.00
0.09±0.04
0.05±0.02
0.09±0.01
0.22±0.06
0.38±0.02
0.26±0.01
0.23±0.03
0.69±0.37
0.31±0.06
17.00±5.15
0.20±0.03
0.20±0.02
0.28±0.05
0.62±0.28
0.47±0.13
2.74±1.13
>20
0.08±0.02
0.16±0.01
0.27±0.04
0.22±0.02
0.33±0.05
0.21±0.01
0.54±0.17
>20
14j
14k
14l
14m
14n
14o
14p
14q
3.36±1.91
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39
3.78±1.22
0.06±0.03
0.06±0.05
0.03±0.02
>20
>20
11.69±4.43
0.77±0.22
0.39±0.18
0.39±0.18
14r
14s
14t
14u
0
0.36±0.15
0.40±0.03
0.45±0.05
1.70±0.42
2.53±0.12
1.53±0.60
41
23
TAE226
----
----
65
89
0.46±0.02
2.65±0.63
1.21±0.12
>20
1.58±1.40
0.45±0.05
8.97±1.47
Defactinib
10.27±1.04
In view of the above results, we found that different structural units at R have great
impact on enzymatic and cellular inhibitory activity. Compound 14j exhibits the
highest inhibitory activities against FAK and four tested tumor cell lines among all of
target compounds modified at R. Therefore, compound 14j was chosen as a new
starting point for further optimization.
In order to evaluate the influence of different substituents on 5-position of the
pyrimidine ring, we kept N,N-dimethylaminoethyl as R and modified the 5-position
substitution of pyrimidine. Thus, analogs 14v-14z with chlorine replaced by different
substitutions at X were synthesized. The synthetic routes were depicted in Scheme 2
and Scheme 3. When the substitute at the 5-position of pyrimidine was H, F, CH3,
OCH3, the C-4 chlorine atom of different 5-substituted-2,4-dichloropyrimidines was
first region selectively replaced by compound 7 to obtain 10b-10e respectively. Then
compound 9 was coupled with the C-2 of different 5-substituted-2-chloropyrimidines
from compounds 10b-10e and produced intermediates 11b-11e accordingly. As for the
substitute at the 5-position of pyrimidine was trifluoromethyl (Scheme 3), compound
9
was
first
installed
selectively
to
2-position
of
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2,4-dichloro-5-trifluoromethylpyrimidine to generate intermediate 12 mediated by
Lewis acid [30]. Then compound 12 was subjected to coupling with compound 7 to
produce intermediate 13. The compounds 11b-11e and 13were stirred in the solution
of trifluoroacetate and dichloromethane (1:1) to get rid of Boc group as
aforementioned. Then the products were concentrated and reacted with carbon
disulfide in DMF with triethylamine as base. Without further purification,
2-bromo-N,N-dimethylethan-1-amine was reacted with the products mentioned above
respectively followed by chromatographic purification to generate the target
compounds 14v-14z.
Scheme 2. Synthesis of compounds 14v-14z. Reagents and conditions: (a) Compound 7; X = H, F: DIPEA,
i-PrOH, reflux 6 h; X = CH3, OCH3: NaH, DMF, 0 →rt.,24 h; (b) Compound 9, Pd(OAc)2, Xantphos, K3PO4,
DMF, Ar, reflux, 22 h; (c) i: TFA, DCM, rt.,
2 h; ii: CS2, Et3N, DMF, rt., 1 h; iii:
2-bromo-N,N-dimethylethan-1-amine hydrobromide, DMF, 50 , overnight.
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Scheme 3. Synthesis of intermediate 13. Reagents and conditions:(a) i: 2,4-dichloro-5(trifluoromethyl)pyrimidine,
ZnCl2 (1.0 M solution in ether), DCE-tBuOH (1:1), 0 , 1 h; ii: Et3N, compound 9, 0 , 1.5 h; (b) Compound 7,
DIPEA, 1-butanol, reflux 24 h.
The newly synthesized compounds 14v-14z were then evaluated against FAK and
the same four human cancer cell lines. As shown in Table 2, anti-FAK activity of
TAE226 was 6.21 nM and Defactinib was 0.77 nM, which were consistent with
previously reported [14, 17]. Replacement of chlorine atom with fluorine atom (14w)
caused slight decrease in FAK inhibition. When the chlorine group was removed (14v)
or replaced with methyl group (14x) and methoxy group (14y), the kinase inhibitory
activity decreased significantly. However, introducing a strong electron-withdrawing
group such as trifluoromethyl group (14z) increased FAK inhibitory activity
dramatically with an IC50 of 0.07nM. Compared with 14j, the inhibitory effects of 14v
against the four tested cancer cell lines decreased dramatically. Compounds 14w, 14x
and 14y resulted in 4~40-fold decreases in potency. Nevertheless, 14z increased the
potency against HCT116, PC-3, U87-MG and MCF-7 cell lines by 3~4-fold. These
results suggest that trifluoromethyl group may form an important interaction with the
target protein. SAR analysis result of FAK inhibitory activity of these tested
compounds was in agreement to that of their antitumor activities, indicating that the
potent antitumor activities of these target compounds were likely related to their FAK
inhibitory activities.
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To evaluate the safety of the target compounds 14j and 14v-14z, they were also
tested in human normal mammary epithelial cell MCF-10A (Table 2). The selectivity
index (SI) values were calculated to determine the toxicities of these compounds on
normal cells compared with breast cancer cell line MCF-7. Compounds 14x and 14z
showed considerable safety (SI value >10).
Table 2.
FAK inhibitory activities and antiproliferative activities of target compounds 14j, 14v-14z against human tumor cell
lines and normal cell line.
FAK
inhibition
( IC50/nM)
8.57
Antiproliferative activity (IC50/µM)
SI
valuea
Compd.
X
HCT116
PC-3
U87-MG
MCF-7
MCF-10A
Cl
H
0.004±0.00 0.09±0.02 0.36±0.15
0.44±0.05 3.01±0.43 2.80±1.05
0.03±0.02 0.36±0.10 0.69±0.25
0.01±0.01 0.27±0.02 0.28±0.03
0.16±0.05 0.92±0.11 1.61±0.36
0.001±0.00 0.03±0.00 0.06±0.01
0.29±0.04 1.68±0.34 1.66±0.87
0.09±0.02
2.01±0.14
0.16±0.03
0.08±0.01
0.36±0.02
0.02±0.01
0.56±0.10
0.72±0.01
6.19±0.42
1.75±0.02
1.08±0.62
2.43±0.04
0.28±0.05
8.76±0.40
8.00
3.08
14j
14v
316.30
32.70
F
5.09
14w
Me
OMe
CF3
----
----
175.40
506.70
0.07
13.25
6.75
14x
14y
14.00
15.64
1.89
14z
6.21
TAE226
0.77
3.02±0.40
>20
10.21±1.02 9.76±1.53 18.42±1.21
Defactinib
aSI = IC50 for nonmalignant cell line / IC50 for MCF-7 cell line.
According to the results above, compound 14z was chosen to further investigate the
mechanism of the antiproliferative effect of these compounds. After exposure of
MCF-7 to compound 14z with different concentrations for 24 h, the cell cycle was
analyzed using flow cytometry. As shown in Fig. 3, treatment of compound 14z
increased the fraction of cells arrested at G2/M phase. Compared with control (without
14z), when MCF-7 cells were treated with increasing concentrations (0.005 µM, 0.01
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µM and 0.05 µM) of compound 14z, the percentage of cells in the G2/M phase
increased from 11.48% to 86.08%, whereas cells in S and G0/G1 phase decreased
concomitantly. Compound 14z was also able to induce significant cell cycle arrest in
HCT-116 in a dose-dependent manner (Fig. S1, Supporting Information).
Control
0.005 µM
G0/G1:62.45%
G2/M:11.48%
G0/G1:41.93%
G2/M:36.67%
S:26.07%
S:21.4%
0.01 µM
0.05 µM
G0/G1:9.52%
G2/M:81.73%
S:8.75%
G0/G1:6.72%
G2/M:86.08%
S:7.20%
Fig.3. Compound 14z induced cell cycle arrest in G2/M phase of MCF-7 cells in a dose-dependent manner. MCF-7
cells were treated with 0.005 µM, 0.01 µM and 0.05 µM compound 14z for 24 h. Cells were fixed, stained
with 7-AAD, and were assessed by flow cytometry Cells untreated were used for control.
.
Next, in order to examine whether compound 14z induces apoptosis in MCF-7 and
HCT116 cell lines. Cells were treated with compound 14z with increasing
concentrations for 24 h and then examined for expression of both annexin V and
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7-AAD by flow cytometry. As shown in Fig. 4, compound 14z induced significant
apoptosis in a dose-dependent manner compared with control. The result was
consistent with pro-apoptosis effect of compound 14z in HCT116 cell line (Fig. S2,
Supporting Information). These results suggested that compound 14z may display
antitumor activity by inducing both cell cycle arrest and cell apoptosis.
0.005 µM
Control
Q1:0.16%
Q2:7.63%
Q3:2.04%
Q4:90.2%
Q1:0.16%
Q2:16.5%
Q3:9.11%
Q4:74.2%
0.01 µM
0.05 µM
Q1:0.47%
Q2:21.4%
Q3:6.95%
Q4:71.2%
Q1:0.56%
Q2:25.7%
Q3:13.7%
Q4:60.1%
Fig.4. Compound 14z induced apoptosis of MCF-7 cells in a dose-dependent manner. MCF-7 cells were treated
with 0.005µM, 0.01µM and 0.05µM compound 14z for 24 h. Cells were incubated with annexin V-FITC and
7-AAD and analyzed using flow cytometry.
Metastasis is a major cause of mortality in patients with cancer [31]. Therefore, the
potency of compound 14z to inhibit tumor cell migration was also evaluated using
Transwell migration assay as reported [32]. As shown in Fig.5, when MCF-7 cells
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was exposed to compound 14z with concentrations of 0.05 µM or 0.1 µM for 24 h,
the number of the cells that penetrate through the filters was significantly decreased
compared with control. This observation indicates that compound 14z possesses the
potency to inhibit the migration of tumor cells.
Control
0.005µM
0.1µM
0.05µM
Fig.5. Effects of compound 14z on the migrations of MCF-7. Cells seeded onto the upper chamber in serum-free
medium were exposed to compound 14z for 24 h.
Angiogenesis also plays an important role in development of tumor [33]. HUVEC
(human umbilical vein endothelial cells) is a kind of cell line that has been widely
used to imitate the neovascularization of tumor in vitro [34-36]. In order to investigate
whether compound 14z could interrupt the angiogenesis of tumor, HUVEC cells tube
formation assay was carried out. After incubation for 6 h, the untreated endothelial
cells formed tube-like networks (Fig.6). At the concentration of 0.005 µM, 0.05 µM
and 0.1 µM, compound 14z significantly inhibited the tube formation by reducing the
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tube-like structure in a dose-dependent manner.
Control
0.005 µM
0.1 µM
0.05 µM
Fig.6. Anti-angiogenesis potency of compound 14z by inhibiting the tube formation in HUVECs. The cells were
incubated with or without compound 14z in the Matrigel coated 96-wells plate for 6 h and then the images were
taken.
3. Conclusion
A new series of 2,4-diarylaminopyrimidine derivatives containing dithiocarbamate
moiety were synthesized and evaluated for their in vitro FAK enzyme and antitumor
inhibitory activities. Most of the compounds showed obvious activity promotion
compared with Defactinib while some of them exhibited better bioactivities than
TAE226. Particularly, the most effective compound 14z displayed superior
antiproliferative effect with IC50 values from 0.001 to 0.06 µM against four FAK over
expressed human tumor cell lines, which were more potent 10~100-fold than TAE226
and more potent 100~1000-fold than Defactinib.
Accordingly, compound 14z inhibited FAK enzyme activity with an IC50 of 0.07
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nM. In addition, compound 14z showed good safety for the selectivity between
human normal cells and MCF-7 tumor cells with SI value > 10. Compound 14z was
thus identified as the most potent antitumor agent and was used for studies of its
antitumor mechanism in this paper. In summary, compound 14z exerted antitumor
effect by arresting cell cycle in G2/M phase and inducing apoptosis in both MCF-7
and HCT116 cell lines. In addition to antiproliferative activities, it also showed the
potency of inhibiting the migration of tumor cells and disrupting the angiogenesis
within nanomolar concentrations. Therefore, we propose that compound 14z is a
promising candidate for further preclinical studies.
4. Experimental Procedures
4.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and were used
without further purification. The positive control moleculars TAE226 and Defactinib
were purchased from TargetMol. Melting points were determined on X4 microscope
1
and are uncorrected. H NMR and 13C NMR spectra were recorded on Bruker
AVANCEⅢ 400 MHz and 100 MHz spectrometer respectively. High resolution mass
spectrum (HRMS) was recorded on a Thermo Scientific Orbitrap Elite MS. Thin Layer
Chromatography (TLC) was performed on TLC Silica gel 60 F254 aluminium sheets
(Merck, Darmstadt, Germany) to check the purity of the compounds.
4.1.1. Procedure for the synthesis of N-methyl-2-nitrobenzamide (6)
A mixture of 2-nitrobenzoyl chloride (3.9 g, 30 mmol), methylamine hydrochloride
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(3 g, 45 mmol) and sodium bicarbonate (12.6 g, 150 mmol) in acetonitrile (30 mL)
was heated under reflux for 4 h. The reaction was cooled to room temperature. The
mixture was concentrated under vacuum and extracted with ethyl acetate (20 mL × 3).
The organic phase was washed with 1N hydrochloric acid (15 mL × 3) and water (15
mL × 2), dried over anhydrous Na2SO4 and concentrated under vacuum to afford
compound 6 as white solid (4.55 g, yield: 84%). Mp: 86-87 °C.1H NMR (400 MHz,
DMSO-d6) δ 8.59 (d, J = 2.5 Hz, 1H), 8.03 – 8.01 (m, 1H), 7.77 (td, J = 7.6, 0.8Hz,
1H), 7.68 (td, J = 8.4, 1.6 Hz, 1H), 7.60 (dd, J = 7.6, 1.2 Hz, 1H), 2.76 (d, J = 4.4 Hz,
3H).13C NMR (100 MHz, DMSO-d6) δ165.78, 147.16, 133.50, 132.52, 130.65,
128.98, 124.03, 26.10.
4.1.2. Procedure for the synthesis of 2-amino-N-methylbenzamide (7)
To a solution of compound 6 (4.80 g, 27 mmol) in H2O (8 mL) and EtOH (24 mL)
was added Fe (6.7 g, 120 mmol) and NH4Cl (2.25 g, 42 mmol). The mixture was
heated under reflux for 2 h. The solid was filtered off and the liquid was concentrated
under vacuum to afford the crude product. The crude product was purified by column
chromatography to provide compound 7 as white solid (3.73 g, yield: 92%). Mp:
66-67 °C.1H NMR (400 MHz, CDCl3) δ 7.30 – 7.27 (m, 1H), 7.20 – 7.15 (m, 1H),
6.66 (dd, J = 8.0, 0.8 Hz, 1H), 6.62 – 6.58 (m, 1H), 6.30 (s, 1H), 5.51 (s, 2H), 2.92 (d,
J = 4.8 Hz, 3H).13C NMR (100 MHz, CDCl3) δ169.99, 148.41, 132.03, 127.10, 117.14,
116.49, 116.18, 26.37.
4.1.3. Procedure for the synthesis of tert-butyl 4-(4-nitrobenzoyl)piperazine-1-
carboxylate (8)
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A mixture of 4-nitrobenzoyl chloride (3.7 g, 20 mmol), tert-butyl
piperazine-1-carboxylate (5.6 g, 30 mmol) and sodium bicarbonate (8.4 g, 100 mmol)
in acetonitrile (30 mL) was heated under reflux for 4 h. The reaction was cooled to
room temperature. The mixture was concentrated under vacuum and extracted with
ethyl acetate (20 mL × 3). The organic phase was washed with 1N hydrochloric acid
(15 mL × 3) and water (15 mL × 2), dried over anhydrous Na2SO4 and concentrated
under vacuum to afford compound 8 as white solid (6.0 g, yield: 90%). Mp:
133-134 °C.1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4
13
Hz, 2H), 3.64 – 3.28 (m, 8H), 1.42 (s, 9H). C NMR (100 MHz, DMSO-d6) δ167.25,
153.76, 147.80, 142.02, 128.31, 123.71, 79.20, 43.11, 42.81, 27.95.
4.1.4. The procedure for the synthesis of tert-butyl 4-(4-aminobenzoyl)piperazine-1-
carboxylate (9)
To a solution of compound 8 (6.0 g, 20 mmol) in H2O (8 mL) and EtOH (24 mL)
was added Fe (4.5 g, 80 mmol) and NH4Cl (1.5 g, 28 mmol). The mixture was heated
under reflux for 2 h. The solid was filtered off and the liquid was concentrated under
vacuum to afford the crude product. The crude product was purified by column
chromatography to provide compound 9 as yellow solid (5.7 g, yield: 93%). Mp:
1
155-156 °C. H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4
13
Hz, 2H), 3.94 (s, 2H), 3.59 (s, 4H), 3.44 (s, 4H), 1.47 (s, 9H). C NMR (100 MHz,
CDCl3) δ171.02, 154.54, 148.36, 129.26, 124.44, 114.10, 80.15, 44.01, 43.30, 28.29.
4.1.5. Methods for the synthesis of compounds 10a-10e
For the 5-subsititution was H, F or Cl, the procedure was as follows: A mixture of
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compound 7 (0.79 g, 5.25 mmol), 5-substituted-2,4-dichloropyrimidine (5 mmol) and
DIPEA (1.05 mL, 6 mmol) in i-PrOH was heated under reflux for 6 h. Water (100mL)
was added and the solid was filtered off, washed with water anddried to obtain
compounds 10a-10c. For the 5-subsititution was methyl or methoxy, the procedure
was as follows: To a mixture of compound 7 (1.26 g, 8.4 mmol)
and5-substituted-2,4-dichloropyrimidine (8 mmol) was added NaH (60%, 0.38 g, 9.6
mmol) in DMF (6 mL) under ice bath. Then the mixture was stirred for 24 h at room
temperature. Water was added dropwise to stop the reaction. The reaction mixture was
extracted with dichloromethane (20 mL × 3), and the combined organic layer was
washed with brine (15 mL × 2), dried over anhydrous Na2SO4 and concentrated under
vacuum to afford the crude product. The residue was purified by column
chromatography to obtain compound 10d and 10e.
4.1.5.3. 2-((3,6-dichloropyridin-2-yl)amino)benzamide (10a)
Yield 80%. MP: 222-223 °C.1H NMR (400 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.88
(d, J = 4.4 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.46 (s, 1H), 7.82 (dd, J = 7.6, 0.8 Hz,
13
1H), 7.62 – 7.58 (m, 1H), 7.24 – 7.21 (m, 1H), 2.84 (d, J = 4.8 Hz, 3H). C NMR
(100 MHz, DMSO-d6) δ 168.70, 156.63, 156.12, 155.25, 138.33, 131.84, 128.11,
123.06, 120.92, 120.77, 114.93, 26.36.
4.1.5.1. 2-((6-chloropyridin-2-yl)amino)benzamide (10b)
Yield 65%. Mp: 167-168°C.1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.65
(d, J = 4.4 Hz, 1H), 8.20 (d, J = 5.6 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.69 (dd, J = 8.0,
1.2 Hz, 1H), 7.56 – 7.52 (m, 1H), 7.22– 7.18 (m, 1H), 6.87 (d, J = 5.6 Hz, 1H), 2.79
21

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(d, J = 4.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ168.43, 161.13, 159.28, 157.58,
137.70, 131.31, 128.27, 124.19, 123.23, 122.10, 106.52, 26.21.
4.1.5.2. 2-((6-chloro-3-fluoropyridin-2-yl)amino)benzamide (10c)
Yield 82%.Mp: 219-220 °C.1H NMR (400 MHz, DMSO-d6) δ 12.27 (d, J = 2.0 Hz,
1H), 8.86 (d, J = 4.0 Hz, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.39 (d, J = 3.2 Hz, 1H), 7.82
(dd, J = 8.0, 1.2 Hz, 1H), 7.62 – 7.58 (m, 1H), 7.23 – 7.19 (m, 1H), 2.82 (d, J = 4.8
Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 168.79, 152.72 (d, J = 3.6 Hz), 150.37 (d,
J = 10.8 Hz), 146.92, 144.36, 141.35 (d, J = 20.8 Hz), 138.41, 132.03, 128.13, 122.85,
120.49 (d, J = 15.8 Hz), 26.27.
4.1.5.4. 2-((6-chloro-3-methylpyridin-2-yl)amino)benzamide (10d)
Yield 40%. MP: 232-233 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.83
(d, J = 4.0 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H), 8.14 (s, 1H), 7.80 (dd, J = 8.0, 1.2 Hz,
1H), 7.60 – 7.55 (m, 1H), 7.18 – 7.14 (m, 1H), 2.83 (d, J = 4.4 Hz, 3H), 2.18 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ169.07, 159.46, 156.63, 156.19, 139.50, 131.89,
128.05, 122.09, 120.45, 120.17, 115.40, 26.31, 12.98.
4.1.5.5. 2-((6-chloro-3-methoxypyridin-2-yl)amino)benzamide (10e)
Yield 41%. MP: 220-221 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.77
(d, J = 4.0 Hz, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.02 (s, 1H), 7.78 (d, J = 7.2 Hz, 1H),
7.58 (t, J = 7.2 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H), 3.99 (s, 3H), 2.83 (d, J = 4.4 Hz, 3H).
13C NMR (100 MHz, DMSO-d6) δ168.71, 151.66, 149.16, 140.23, 138.84, 135.80,
131.78, 128.10, 122.11, 120.61, 120.14, 56.82, 26.29.
4.1.6. The general procedure for the synthesis of compounds 11a-11e
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The mixture of compound 10a-10e (3.24 mmol), compound 9 (1.04g, 3.4 mmol),
Pd(OAc)2 (0.11g, 0.5 mmol), Xantphos (0.28g, 0.5 mmol), and K3PO4（1.38g, 6.48
mmol）in DMF (8 mL) was heated 22 h at 125°C under argonfollowed by cooling to
room temperature.The mixture was extracted with dichloromethane (20 mL × 3), the
combined organic phase was washed with water (15 mL × 2), dried over anhydrous
Na2SO4 and concentrated to afford the crude product. The crude product was purified
by column chromatography to obtain compounds 11a-11e.
4.1.6.3. Tert-butyl 4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin
-2-yl)amino)benzoyl)piperazine-1-carboxylate (11a)
Yield 62%. MP: 227-228 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 9.73
(s, 1H), 8.77 (dd, J = 17.2, 4.4 Hz, 2H), 8.28 (s, 1H), 7.78 (d, J = 8.8 Hz, 3H), 7.56 –
7.52 (m, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 3.50 – 3.36 (m, 8H),
2.83 (d, J = 4.4 Hz, 3H), 1.42 (s, 9H).13C NMR (100 MHz, DMSO-d6) δ169.32,
168.87, 157.39, 155.07, 154.53, 153.83, 141.78, 139.15, 131.45, 127.99, 122.13,
121.58, 121.01, 118.47, 105.80, 79.16, 43.56, 42.90, 28.02, 26.31.
4.1.6.1. Tert-butyl4-(4-((4-((2-(methylcarbamoyl)phenyl)amino) pyrimidin-2-yl)amino)
benzoyl)piperazine-1-carboxylate (11b)
Yield 60%. MP: 206-207 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.55
(s, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 5.6 Hz, 1H),
7.85 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.2 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.37 (d, J =
8.4 Hz, 2H), 7.12 (t, J = 7.2 Hz, 1H), 6.37 (d, J = 6.0 Hz, 1H), 3.51 (s, 3H), 3.41
(s,5H). 2.82 (d, J = 4.4 Hz, 3H), 1.42 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
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δ169.47, 168.97, 159.89, 159.28, 156.68, 153.86, 142.34, 139.58, 131.33, 128.11,
128.03, 127.54, 121.90, 121.62, 118.15, 99.85, 79.17, 43.73, 42.98, 28.03, 26.24.
4.1.6.2. Tert-butyl 4-(4-((5-fluoro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin
-2-yl)amino)benzoyl)piperazine-1-carboxylate (11c)
1
Yield 62%. MP: 216-217 °C. H NMR (400 MHz, DMSO-d6) δ 11.81 (d, J = 2.0
Hz, 1H), 9.62 (s, 1H), 8.82 (t, J = 4.8 Hz, 2H), 8.23 (d, J = 3.2 Hz, 1H), 7.82 – 7.79
(m, 3H), 7.57 – 7.53 (m, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.18 – 7.14 (m, 1H), 3.50 –
3.38 (m, 8H), 2.83 (d, J = 4.4 Hz, 3H), 1.42 (s, 9H).13C NMR (100 MHz, DMSO-d6)
δ 169.41, 169.06, 155.09 (d, J = 3.0 Hz),153.84, 149.07 (d, J = 10.0 Hz), 142.40 (d, J
= 25.0 Hz), 140.51 – 140.06 (m), 139.47, 131.80, 128.06, 127.63, 121.82, 120.78,
120.78, 117.87, 79.17, 43.81, 42.82, 28.02, 26.30.
4.1.6.4. Tert-butyl 4-(4-((5-methyl-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin
-2-yl)amino)benzoyl)piperazine-1-carboxylate (11d)
Yield 58%. MP: 232-233 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 9.41
(s, 1H), 8.87 (d, J = 8.4 Hz, 1H), 8.76 (d, J = 4.4 Hz, 1H), 8.02 (s, 1H), 7.82 (d, J =
8.4 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.8 Hz,
2H), 7.10 (t, J = 7.2 Hz, 1H), 3.50 – 3.36 (m, 8H), 2.83 (d, J = 4.4 Hz, 3H), 2.09 (s,
3H), 1.42 (s, 9H).13C NMR (100 MHz, DMSO-d6) δ169.49, 169.32, 158.47, 157.85,
155.53, 153.84, 142.65, 140.50, 131.58, 128.07, 127.09, 121.10, 119.95, 117.75,
107.34, 79.16, 43.58, 42.93, 30.68, 28.03, 26.33.
4.1.6.5. tert-butyl 4-(4-((5-methoxy-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin
-2-yl)amino)benzoyl)piperazine-1-carboxylate (11e)
24

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1
Yield 59%. MP: 220-221 °C. H NMR (400 MHz, DMSO-d6) δ 11.43 (s, 1H),
9.15z (s, 1H), 8.93 (d, J = 8.4 Hz, 1H), 8.70 (d, J = 4.4 Hz, 1H), 7.95 (s, 1H), 7.84 (d,
J = 8.4 Hz, 2H), 7.76 (d, J = 6.8 Hz, 1H), 7.55 – 7.51 (m, 1H), 7.36 (d, J = 8.4 Hz,
2H), 7.11 (t, J = 7.2 Hz, 1H), 3.92 (s, 2H), 3.52 – 3.39 (m, 8H), 2.84 (d, J = 4.4 Hz,
13
3H), 1.43 (s, 9H). C NMR (100 MHz, DMSO-d6) δ169.56, 168.93, 153.84, 153.04,
150.95, 143.01, 139.74, 136.13, 135.50, 131.49, 128.13, 126.66, 121.19, 120.51,
117.12, 79.14, 56.89, 43.55, 42.94, 28.01, 26.29.
4.1.7. The procedure for the synthesis of tert-butyl4-(4-((4-chloro-5-(trifluoromethyl)
pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylate (12)
To a solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (0.46 mL,3.4 mmol) in
DCE (12 mL) and tBuOH (12 mL) was added 4 mL ZnCl2 solution (1M in diethyl
ether). The mixture was stirred under ice bath for 1 h. Then the mixture was added
compound 21(1.04 g, 3.4 mmol in 3mL DCE: tBuOH = 1:1) and stirred for another
1.5 h under ice bath. Then the mixture was added Et3N (0.52 mL, 3.7 mmol) dropwise.
The mixture was concentrated under vacuum and extracted with dichloromethane (20
mL × 3). The organic phase was washed with brine (15 mL × 3) and dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by
column chromatography to get compound 12 as white solid (0.76 g, 46%). Mp:
1
182-183 °C. H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.85 (s, 1H), 7.82 (d, J
= 8.0 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 3.40 (s, 8H), 1.42 (s, 9H).13C NMR (100
MHz, DMSO-d6) δ 168.96, 160.37, 158.08 (d, J = 5.0 Hz),157.73, 153.81, 139.80,
130.26, 128.07, 119.55, 112.32 – 111.32 (m), 79.14, 43.49, 42.90, 27.99.
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4.1.8. Procedure for the synthesis oftert-butyl 4-(4-((4-((2-(methylcarbamoyl)phenyl)
amino)pyrimidin-2-yl)amino)benzoyl)piperazine-1-carboxylate (13)
The mixture of compound 7 (0.24 g, 1.58 mmol), compound 12 (0.73 g, 1.5 mmol)
and DIPEA (0.31 mL, 1.8 mmol) in 1-Butanol was heated under reflux for 24 h. The
mixture was concentrated under vacuum and extracted with dichloromethane (20
ml×3). The organic phase was washed with brine (15 mL × 3), dried over anhydrous
Na2SO4 and concentrated under vacuum. The crude product was purified by column
chromatography to get compound 13 as yellow solid (0.51 g, 55%). Mp: 203-204 °C.
1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 10.07 (s, 1H), 8.76 (d, J = 3.6 Hz,
1H), 8.50 (s, 2H), 7.75 (d, J = 6.0 Hz, 3H), 7.53 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.0
Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 3.49 – 3.36 (m, 8H), 2.80 (d, J = 3.6 Hz, 3H), 1.42
(s, 9H).13C NMR (100 MHz, DMSO-d6) δ 169.16, 168.73, 160.59, 156.24, 155.94,
153.83, 140.89, 138.44, 131.12, 129.08, 127.85, 125.83, 123.23 (d, J = 17.4
Hz),122.82, 122.53 – 122.41 (m), 119.43, 79.16, 28.01, 26.21.
4.1.9. General procedure for the synthesis of compounds 14a-14z
To the suspension of compound 11a-11e or 13 (1.5 mmol) in DCM (3 mL) was
added TFA (3 mL). The mixture was stirred at room temperature for 2 h and was
concentrated under vacuum. The product was then dissolved in DMF (6 mL) and
added Et3N (1 mL) without further purification. The reaction mixture was stirred for 1
h and was added CS2 (0.14 mL, 2.25 mmol) to continuously stir for 30 min. A series
of different of halogen or alkene substituted compounds (1.5 mmol) were added
respectively and the mixture was stirred at 50 °C overnight. The mixture was cooled
26

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to room temperature and extracted with dichloromethane (20 mL × 3). The organic
phase was washed with brine (15 mL × 3), dried over anhydrous Na2SO4 and
concentrated under vacuum. The residue was purified by column chromatography to
provide the target products.
4.1.9.1.
2-cyanoethyl
4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)benzoyl)piperazine-1-carbodithioate(14a)
Yield 76%. Mp: 287-288 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 9.76
(s, 1H), 8.77 (dd, J = 18.4, 4.0 Hz, 2H), 8.28 (s, 1H), 7.79 (t, J = 7.6 Hz, 3H), 7.55 (t,
J = 7.6 Hz, 1H), 7.42 (d, J = 8.4 Hz,2H), 7.18 (t, J = 7.2 Hz, 1H), 4.33 (s, 2H), 4.04 (s,
2H), 3.70 (s, 4H), 3.57 (t, J = 6.8 Hz, 2H), 2.97 (t, J = 6.8 Hz, 2H), 2.83 (d, J = 4.4Hz,
3H).13C NMR (100 MHz, DMSO-d6)δ193.92, 169.38, 168.88, 157.37, 155.08, 154.54,
142.01, 139.14, 131.48, 128.21, 128.04, 127.66, 122.17, 121.61, 121.04, 119.19,
118.40, 105.87, 50.84, 49.46, 31.41, 26.34, 17.28.HRMS m/z: calcd. for
C27H28ClN8O2S2 [M+H]+: 595.1465; found: 595.1439.
4.1.9.2.allyl 4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)
amino)benzoyl)piperazine-1-carbodithioate (14b)
Yield 84%. Mp: 247-248 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 9.75
(s, 1H), 8.78 (t, J = 8.8 Hz, 2H), 8.28 (s, 1H), 7.82 (dd, J = 11.2, 8.8 Hz, 3H), 7.55 (t,
J = 7.6 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 5.96 – 5.85 (m, 1H),
5.34 (d, J = 16.8 Hz, 1H), 5.16 (d, J = 10.0 Hz, 1H), 4.34 – 3.70 (m, 6H), 3.70 (s, 4H),
13
2.85 (d, J = 4.4 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ195.05, 169.37, 168.86,
157.34, 155.05, 154.45, 142.00, 139.16, 131.40, 128.15, 127.98, 127.63, 122.06,
27

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121.57, 120.99, 118.72, 118.39, 105.88, 54.87, 50.53, 49.24, 26.30. HRMS m/z: calcd.
for C27H29ClN7O2S2 [M+H]+:582.1513;found: 582.1506.
4.1.9.3.2-methoxyethyl 4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)
pyrimidin -2-yl)amino)benzoyl)piperazine-1-carbodithioate (14c)
Yield 82%. Mp: 215-216 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.76
(s, 1H), 8.80 – 8.73 (m, 2H), 8.28 (s, 1H), 7.82 – 7.77 (m, 3H), 7.55 (t, J = 7.2 Hz,
1H), 7.42 (d, J = 8.8 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 4.33 (s, 2H), 4.06 – 4.03 (m,
2H), 3.68 (s, 4H), 3.58 – 3.55 (m, 2H), 3.50 (t, J = 6.0 Hz, 2H), 3.27 (s, 3H), 2.83 (d,
J = 4.4 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ195.54, 169.40, 168.89, 157.36,
155.07, 154.49, 142.04, 139.19, 131.45, 128.20, 128.02, 127.64, 122.10, 121.60,
120.99, 118.40, 105.92, 69.86, 57.89, 50.70, 49.18, 35.82, 26.33. HRMS m/z: calcd. for
C27H31ClN7O3S2 [M+H]+: 600.1618 ; found: 600.1602.
4.1.9.4. Methyl 3-((4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin
-2-yl)amino)benzoyl)piperazine-1-carbonothioyl)thio)propanoate(14d)
Yield 83%.Mp: 180-181 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.74
(s, 1H), 8.78 – 8.72 (m,2H), 8.28 (s, 1H), 7.80 – 7.76 (m, 3H), 7.57 – 7.52 (m, 1H),
7.40 (d, J = 8.4 Hz, 2H), 7.19 – 7.15 (m, 1H), 4.31 (s, 2H), 4.00 (s, 2H), 3.65 (d, J =
17.2 Hz, 7H), 3.48 (t, J = 7.2 Hz, 2H), 2.82 (d, J = 4.4 Hz, 3H), 2.78 (t, J = 6.8 Hz,
2H).13C NMR (100 MHz, DMSO-d6) δ 195.86, 169.36, 168.88, 157.37, 155.08,
154.55, 142.00, 139.15, 131.48, 128.20, 128.04, 127.70, 122.15, 121.60, 121.01,
118.38, 105.87, 66.76, 50.48, 49.48, 34.19, 32.02, 26.33.HRMS m/z: calcd. for
C28H31ClN7O4S2 [M+H]+: 628.1568; found:628.1561.
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