Discovery of novel pyrimidine-based benzothiazole derivatives as potent cyclin-dependent kinase 2 inhibitors with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2019.06.055

To develop novel CDK2 inhibitors as anticancer agents, a series of novel pyrimidine-based benzothiazole derivatives were designed and synthesized. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against five cancer cell lines. Especially, the analogue 10s exhibited approximately potency with AZD5438 toward four cells including HeLa, HCT116, PC-3, and MDA-MB-231 with IC₅₀ values of 0.45, 0.70, 0.92, 1.80 μM, respectively. More interestingly, the most highly active compound 10s in this study also possessed promising CDK2/cyclin A2 inhibitory activities with IC₅₀ values of 15.4 nM, which was almost 3-fold potent than positive control AZD5438, and molecular docking studies revealed that the analogue bound efficiently with the CDK2 binding site. Further studies indicated that compound 10s could induce cell cycle arrest and apoptosis in a concentration-dependent manner. These observations suggest that pyrimidine-benzothiazole hybrids represent a new class of CDK2 inhibitors and well worth further investigation aiming to generate potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2019.06.055

Accepted Manuscript
Discovery of novel pyrimidine-based benzothiazole derivatives as potent cyclin-
dependent kinase 2 inhibitors with anticancer activity
Peng-Cheng Diao, Wei-Yuan Lin, Xie-Er Jian, Yan-Hong Li, Wen-Wei You, Pei-Liang
Zhao
PII:
S0223-5234(19)30582-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.055
EJMECH 11458
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 May 2019
Revised Date: 19 June 2019
Accepted Date: 19 June 2019
Please cite this article as: P.-C. Diao, W.-Y. Lin, X.-E. Jian, Y.-H. Li, W.-W. You, P.-L. Zhao, Discovery
of novel pyrimidine-based benzothiazole derivatives as potent cyclin-dependent kinase 2 inhibitors
with anticancer activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.055.
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ACCEPTED MANUSCRIPT
Discovery of novel pyrimidine-based benzothiazole derivatives as potent
cyclin-dependent kinase 2 inhibitors with anticancer activity
#
#
Peng-Cheng Diao , Wei-Yuan Lin , Xie-Er Jian, Yan-Hong Li, Wen-Wei You*, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China

ACCEPTED MANUSCRIPT
Discovery of novel pyrimidine-based benzothiazole derivatives as potent
cyclin-dependent kinase 2 inhibitors with anticancer activity
#
#
Peng-Cheng Diao , Wei-Yuan Lin , Xie-Er Jian, Yan-Hong Li, Wen-Wei You*, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
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ABSTRACT: To develop novel CDK2 inhibitors as anticancer agents, a series of novel
pyrimidine-based benzothiazole derivatives were designed and synthesized. Initial biological
evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro
against five cancer cell lines. Especially, the analogue 10s exhibited approximately potency with
AZD5438 toward four cells including HeLa, HCT116, PC-3, and MDA-MB-231 with IC values
1
5
0
of 0.45, 0.70, 0.92, 1.80 µM, respectively. More interestingly, the most highly active compound 10s
1
in this study also possessed promising CDK2/cyclin A2 inhibitory activities with IC values of
5
0
15.4 nM, which was almost 3-fold potent than positive control AZD5438, and molecular docking
studies revealed that the analogue bound efficiently with the CDK2 binding site. Further studies
indicated that compound 10s could induce cell cycle arrest and apoptosis in a
concentration-dependent manner. These observations suggest that pyrimidine-benzothiazole hybrids
represent a new class of CDK2 inhibitors and well worth further investigation aiming to generate
potential anticancer agents.
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Keywords: Pyrimidine; Benzothiazole; Antiproliferative activity; CDK2 inhibitor
2
5
—
—————————
∗
Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao), youww@smu.com (W.-W. You)
#
These authors contributed equally to this work.
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1. Introduction
The uncontrolled and sustained cell division has been identified as a hallmark feature of cancer
1-2], and since cyclin-dependent kinases (CDKs) are distinguished by their vital roles in regulating
[
cell divisions, they present one of the most attractive targets for cancer therapy [3-5]. As a member
of the CDK family, CDK2 has been considered to be fundamental to the regulation of the cell cycle
progression [6]. Furthermore, CDK2 was found to play a pivotal role in cell differentiation [7], and
apoptosis [8-9]. Even more interestingly, CDK2 is frequently over-expressed in human tumors
3
5
[
10-11], while most normal tissues have low expression of CDK2 [12]. For these reasons, CDK2
has been emerged as one of the most promising therapeutic target for the discovery of highly
efficient antitumor agents [13-15].
40
45
50
Fig. 1. The structures of representative CDK2 inhibitors in clinical evaluation.
In recent years, a large number of structurally diverse CDK2 inhibitors have been carried out
and several small molecules inhibitors, exampled with AZD5438, Dinaciclib, Milciclib, Roniciclib,
TG02 and SNS-032 have entered clinical trials (Fig. 1) [16-21] to treat numerous solid tumors and
hematopoietic malignances. It is worth mentioning that most CDK2 inhibitors in clinical evaluation
2
4
contain aminopyrimidine scaffold. In our previous efforts, a series of novel N , N -disubstituted
pyrimidine-2,4-diamines were designed and synthesized, and some of them emerged as potent
CDK2 inhibitors and anticancer agents with low toxicity [22-24]. On the other hand,
2
-aminobenzothiazole is a well-represented scaffold in medicinal chemistry and generally exists in
bioactive molecules, particularly in cancer agents, exampled by compounds Ⅰ , Ⅰ , Ⅰ and Ⅰ (Fig. 2)
25-28]. These observations provoked us to design a series of benzothiazole-based
[
diaminepyrimidine analogues to further optimize the structure with improved potency. In the
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present study, we propose to replace the 4-alkyl or arylamino moiety in our reported compounds
with 2-aminobenzothiazole to search for the novel CDK2 inhibitors as anticancer agents (Fig. 3).
We report herein our studies on the synthesis, biological evaluation and their CDK2-Cyclin A2
inhibitory activity of designed pyrimidine-benzothiazole hybrids. To the best of our knowledge,
there are no pyrimidine-based benzothiazole derivatives as CDK2 inhibitors reported and there has
not been a systematic investigation of the substitution effect so far.
5
5
6
0
Fig. 2. Various 2-aminobenzothiazole derivatives with reported anticancer activity.
Fig. 3. Design and optimization of pyrimidine-benzothiazole derivatives.
. Chemistry
2
6
5
As illustrated in Scheme 1, pyrimidine-benzothiazole hybrids 4a–j were synthesized by a two
step synthetic approach according to our previously reported methods [23]. The key intermediate
-
N-(2-chloro-5-methylpyrimidin-4-yl)benzo[d]thiazol-2-amine 3 was firstly synthesized from the
commercially available 2,4-dichloro-5-methylpyrimidine 1 as starting material by a nucleophilic
substitution reaction at C-4 position of pyrimidine ring with 2-aminobenzothiazole in the presence
of sodium hydroxide (NaOH) at the room temperature. In the second step, the C-2 chlorine was
substituted by various phenylamines under rigorous conditions (120°C) gave rise to the
corresponding 2,4-disubstituted pyrimidine derivatives 4a–j in moderate isolated yields ranging
from 50% to 76%.
7
0
The target compounds 10a–x were obtained following the procedures described in Scheme 2.
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7
5
According to the related literatures [29], the intermediates 2-aminobenzothiazoles 7 were prepared
through intramolecular cyclization of corresponding phenyl thioureas 6 in the presence of bromine,
which were synthesized by condensation ammonium thiocyanate with various substituted anilines 5.
Subsequently, the intermediates 7 were reacted with substituted 2,4-dichloropyrimidines 8 to afford
key intermediates
9
which were further functionalized at C-2 position with
8
0
4-(methylsulfonyl)aniline or 4-aminobenzenesulfonamide to successfully provide the desirable
target compounds 10a–x by heating at 120 °C in 2-methoxyethanol. The structures of the target
1
13
derivatives were characterized through H NMR, C NMR, and HRMS spectroscopic techniques,
and the spectral data were in full agreement with the expected structures (see Experimental Section
and Supporting Information).
N
N
a
N
N
b
N
N
+
NH2
R
S
N
N
N
S
Cl
N
1
Cl
Cl
N
N
S
H
H
H
2
4a-j
3
o
Scheme 1. Reagents and conditions: (a) DMF, NaOH, r.t.; (b) various aromatic amines, con. HCl, 2-methoxyethanol, 120 C.
8
5
3. Results and Discussion
9
0
3.1 In vitro antiproliferative activity
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to
3
investigate the in vitro antiproliferative activities of all synthesized pyrimidine-based benzothiazole
derivatives 4a–j and 10a–x against five human cancer cell lines (MDA-MB-231, MCF-7, HeLa,
HCT116, and PC-3). The results expressed as the half-maximal inhibitory concentration (IC ) were
5
0
9
5
summarized in Table 1 and 2, and AZD5438 (Fig. 1) was used as a control to make a comparison of
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the potency of the synthesized target compounds.
Initially, analogues 4a–j were evaluated for their antiproliferative activities in order to simply
investigate the effect of substitutions on the aminophenyl group, with AZD5438 as the positive
control. As depicted in Table 1, the results suggested that the different substitutions at the C-4
position of aminophenyl group had a significant influence on antiproliferative activity, which
indicated that the aminophenyl moiety contributed much to the potency. No obvious activity was
observed for compounds 4b, 4c and 4i with an electron-donating substitution at the R position.
While introducing an electron-withdrawing group, such as nitro, methylsulfonyl and sulfamoyl
significantly increased the antiproliferative activity (compare 4a, 4d, 4e with 4b, 4c, 4i). Among
them, analogue 4d with methylsulfonyl group was identified as the most highly active compound
against HCT116, HeLa, and MCF-7 cancer cell lines with IC values at 0.76, 0.46, and 6.81 µM,
100
105
110
5
0
respectively, which were similar to that of AZD5438. Meanwhile compounds 4e bearing sulfamoyl
group also displayed remarkable antiproliferative activity for three tested cell lines. Hence,
methylsulfonyl and sulfamoyl groups were selected as two preferred substitutions for further
optimization.
Table 1 Antiproliferative activities of compounds 4a–j against five human cancer cell lines
a
IC (µM)
5
0
Comp.
R
HCT116
6.33±0.70
>50
MDA-MB-231
22.49±1.17
>50
MCF-7
>50
HeLa
34.28±2.78
>50
PC-3
>50
4a
4b
4c
4-NO2
4-OCH
3-OCH
3
3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4d
4-(SO )CH
0.76±0.20
1.66±0.13
>50
27.75±2.40
>50
>50
18.74±2.48
0.47±0.14
14.76±1.53
16.75±2.11
>50
6.55±1.05
>50
0.46±0.05
2.80±0.17
>50
16.26±1.70
>50
2
3
4e
4-(SO )NH
2
2
4f
4-Cl
3-F
4-F
>50
>50
>50
>50
4
4
g
h
>50
>50
>50
4
4
i
j
4-CH(CH3)2
-(piperidin-1-yl)
>50
4.34±0.27
4
37.23±0.19
7.20±0.55
>50
AZD5438
10.67±0.69 0.73±0.12 0.36±0.09
a
50% inhibitory concentration and mean ± SD of three independent experiments performed in duplicate.
115
Next, our research was focused on the modification of benzothiazole and pyrimidine rings at
the right part of the scaffold. As shown in Table 2, we firstly investigated the effect of substitutions
of benzothiazole ring of the scaffold. The results demonstrated that most of compounds 10a–f,
5

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modified by introduction of a fluoro, chloro, methyl or methoxyl group in benzothiazole ring,
showed stronger antitumor activities than the corresponding analogues (4d, 4e) against MCF-7 and
PC-3. Unfortunately all these derivatives led to less active than corresponding compounds 4d, and
120
125
130
4e against HCT116 and HeLa cell lines. Later, our concern was performed to the different
2
substituents R at the C-5 position of pyrimidine ring. Generally, replacement of the methyl of
pyrimidine scaffold with fluoro atom (10g–o) resulted in the diminished potency against HCT116
2
and HeLa. To our delight, almost all of the derivatives (10p–x) with a hydrogen as R group
exhibited excellent antiproliferative activities in the single-digit micromolar range against
MDA-MB-231, HeLa, HCT116, and PC-3 cells. Noteworthy, analogues 10r, 10s, and 10u
exhibited much greater antitumor activities than AZD5438 in MDA-MB-231 and HeLa cells. More
interestingly, the most promising compound 10s displayed approximately potency with AZD5438
in HeLa, HCT116, PC-3, and MDA-MB-231 cells with IC values of 0.45, 0.70, 0.92, 1.80 µM,
5
0
respectively. These encouraging findings suggest that pyrimidine-benzothiazole hybrids may have
potential for clinical development as anticancer agents.
Table 2 Antiproliferative activities of compounds 10a–x against five human cancer cell lines
a
In vitro cytotoxicity IC (µM)
1
2
3
50
Comp.
R
R
R
HCT116
MDA-MB-231
>50
MCF-7
3.26±0.29
>50
HeLa
>50
PC-3
>50
6.98±0.45
1
0a
0b
0c
0d
0e
0f
F
F
CH3 CH3 24.00±1.15
CH3 NH2 2.01±0.49
1
7.58±0.12
3.46±0.13
4.18±0.27
3.73±0.28
3.28±0.40
36.74±4.39
3.17±0.63
4.52±0.56
>50
1
Cl
Cl
CH3 CH3 2.50±0.35
CH3 NH2 2.52±0.51
5.90±0.42
9.42±0.41
9.08±0.73
>50
4.32±0.49
8.43±0.84
>50
>50
6.88±0.35
>50
13.39±1.64
5.40±0.73
2.71±0.61
>50
1
1
CH3 CH3 NH2 3.87±0.67
OCH3 CH3 NH2 7.86±0.98
H
H
1
1
0g
0h
F
F
F
F
F
F
F
F
F
H
H
H
CH3 3.27±0.44
NH2 3.26±0.16
CH3
4.98±0.54
22.63±2.45
8.90±0.77
2.79±0.26
11.05±1.17
6.39±0.17
14.33±0.80
26.60±1.93
4.74±1.15
5.91±0.23
5.00±0.16
2.08±0.69
2.83±0.23
>50
1
1
0i
0j
F
>50
>50
>50
1
Cl
Cl
NH2 8.62±0.88
CH3 20.69±1.58
NH2 1.46±0.36
CH3 9.18±1.06
NH2 11.49±1.24
CH3 2.63±0.15
CH3 8.37±0.68
NH2 5.17±0.77
CH3 0.86±0.15
8.34±1.29
>50
8.77±0.69
>50
1.43±0.43
>50
4.96±0.77
>50
1
0k
0l
0m
1
1
CH3
CH3
OCH3
OCH3
H
24.54±1.80 1.89±0.16
>50
>50
17.20±2.97
12.98±0.85
1
1
1
1
1
0n
0o
0p
0q
>50
5.85±0.21 25.36±1.68
10.62±2.72 11.26±1.32
15.28±1.33 2.06±0.10
1.27±0.10
1.71±0.09
3.10±0.07
1.94±0.31
H
F
0r
>60
0.56±0.24
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1
1
0s
0t
F
Cl
CH3
CH3
OCH3
OCH3
H
H
H
H
H
H
NH2 0.70±0.03
1.80±0.22
>50
10.91±2.01 0.45±0.09
0.92±0.58
>50
1.64±0.51
>50
3.03±0.36
2.70±0.15
0.36±0.09
CH3 1.35±0.15
NH2 0.90±0.14
CH3 1.75±0.49
NH2 1.48±0.12
CH3 2.27±0.23
0.47±0.14
>50
5.74±0.62
>50
1.40±0.08
0.66±0.05
2.30±0.17
1
1
0u
0v
3.87±0.28
4.44±1.17
6.24±0.73
32.74±5.23
7.20±0.55
1
1
0w
0x
10.95±0.66 1.73±0.11
>50 4.13±0.38
10.67±0.69 0.73±0.12
AZD5438
a
5
0% inhibitory concentration and mean ± SD of three independent experiments performed in duplicate.
135
3.2. Inhibitory assay of CDK2 in vitro
In order to investigate the CDK2 enzyme inhibitory activity of the synthesized analogues,
thirteen of the most antiproliferative derivatives were chosed to further evaluate against
CDK2/cyclin A2 in vitro at a final concentration of 1 µM, and AZD5438 was also employed as a
positive control. As illustrated in Table 3, all compounds with potent antiproliferative activities also
displayed excellent inhibitory activities, which indicated that inhibition of CDK2 could be
responsible for the antiproliferative of these derivatives. Moreover, further evaluation indicated that
these analogues, manifesting significant inhibition activity (>90%), generally exhibited more potent
activities than the positive compound AZD5438 with IC values in the low nanomolar range.
140
5
0
145
Notably, the promising analogue 10s also demonstrated outstanding CDK2 inhibitory activity (IC50
15.4 nM), which was almost 3-fold potent than AZD5438 which exhibited IC value of 45 nM.
=
5
0
Table 3 CDK2 inhibitory activities of the most active compounds
CDK2/A2
1
2
3
Comp.
R
R
R
a
%
inhibition
IC (nM)
50
4
4
d
e
H
H
Cl
Cl
H
H
CH3
H
CH3
CH3
CH3
CH3
F
F
F
H
H
CH3
NH2
CH3
NH2
CH3
NH2
NH2
CH3
NH2
CH3
90
91
81
90
95
93
92
80
84
89
37.8±1.1
21.7±0.5
ND
ND
12.7±1.6
16.5±2.1
21.4±3.3
ND
b
10c
10d
10g
10h
10l
1
1
0p
0q
H
F
ND
ND
10r
H
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1
0s
F
CH3
CH3
H
H
H
NH2
NH2
CH3
94
82
77
15.4±4.4
ND
1
1
0u
0v
ND
c
AZD5438
45
a
b
c
[30]
150
Compounds were tested at a final concentration of 1µM. ND: not determined. Reported by the literature
3.3. Molecular studies
To explore the potential binding pose for this pyrimidine-benzothiazole hybrids, the most
highly active analogue 10s was selected to perform the molecular docking studies with CDK2
crystal structure (PDB: 6GUE). As given in Fig. 4, 10s was located deeply into the ATP-binding
site, where the analogue was found to be overlayed very well with AZD5438, the inhibitor of
co-crystal, and five potential hydrogen bond interactions with amino acids of CDK2 were also
observed. The oxygen as well as nitrogen atom of sulfamoyl moiety interact with the residue Asp86
through hydrogen bonding, which might explain that the derivatives with methylsulfonyl and
sulfamoyl groups are more potent than the analogues with other substitutions. Moreover, the residue
of Leu83 forms two hydrogen bonds with 2-amino moiety and nitrogen atom on pyrimidine ring.
Another important hydrogen bonding was also detected between sulphur atom on benzothiazole
ring and the residue Lys33. These binding model further supported the above biological assay data
and suggested that analogue 10s may be a potential CDK2 inhibitor.
1
55
160
1
65
70
Fig. 4. The predicted docked pose of compound 10s (magenta stick) overlayed with AZD5438 (cyan) in the
CDK2 binding site (PDB code: 6GUE). The main interacting residues were labeled, and the black dashed lines
were the potential H-bonds between Asp86 (1.8, 1.9 Å), Leu83 (1.9, 2.2 Å), Lys33 (3.1 Å).
1
3.4. Flow cytometry analysis
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In order to study the mechanism of action of the series of analogues against cancer cells, the
highly active compound 10s was selected to evaluate for the effects on the cell cycle progression by
flow cytometry. In present work, HCT116 cells were treated with 0.35, 0.70 and 1.40 µM
concentrations of analogue 10s for 24 h. As depicted in Fig. 5, compound 10s demonstrated 24.36%
(0.35 µM), 33.51% (0.70 µM), and 46.92% (1.40 µM) of cell accumulation in G2/M phase,
respectively, nevertheless 17.48% of G2/M phase for control (untreated cells) was detected. These
175
findings clearly demonstrated that derivative 10s induced a significant G /M cell cycle arrest,
2
compared with untreated cells.
0
3
2
3
.74%
0
3
1
4
.00%
6.61%
4.36%
9.03%
7.26%
7.48%
5.26%
180
A
B
6
3
3
3
.86%
39.30%
34.57%
46.92%
18.51%
5.61%
3.51%
0.88%
C
D
Fig.5. Effect of compound 10s on cell cycle in HCT116 cells. Flow cytometry analysis of HCT116 cells treated
with 10s for 24 h. (A) Control; (B) 10s, 0.35 µM; (C) 10s, 0.70 µM; (D) 10s, 1.40 µM.
185
3.5. Cell apoptosis study
To explore the preliminary molecular mechanisms of action, cell apoptosis analysis of HCT116
cells treated with the different concentrations of optimal derivative 10s (0.35, 0.70 and 1.4 µM) was
carried out using Annexin V-FITC and PI to stain cells and flow cytometry calculation. As
illustrated in Fig. 6, HCT116 cells treated with 0.35 µM of analogue 10s for 24 h displayed a
significant increase in the percentage of Annexin-V-positive cells, from 2.31% of vehicle control to
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19.17% in treated cells (10.1% and 9.07% of cells in early and late apoptotic cells , respectively).
After increasing the concentration of the compound to 0.70 and 1.4 µM, the percentages of
Annexin-V-positive cells substantial rose to 35.3%, and 48.2%, respectively. Hence, these
observations demonstrated that compound 10s induced obviously apoptosis in HCT116 cells in a
concentration-dependent manner.
195
2
00
05
Figure 6. Flow cytometry analyses of apoptosis induction in HCT116 cells after treated by different
concentrations of compound 10s (0.35, 0.70 and 1.4 µM) and no treatment (control) as a reference control for 24 h.
The lower left quadrant represents live cells, the lower right is for early apoptotic cells, upper right is for late
apoptotic cells, and the upper left represents cells necrosis.
2
4. Conclusion
In summary, a series of novel pyrimidine-based benzothiazole derivatives were designed,
synthesized and optimized for cancer therapy. These new derivatives were evaluated for their
antiproliferative activities against five cancer cell lines. Several of these compounds exhibited more
remarkable antitumor activity than AZD5438 against one or more cancer cell lines employed in this
study. Particularly, the most promising compound 10s displayed approximately potency with
AZD5438 toward four cells including HeLa, HCT116, PC-3, and MDA-MB-231 with IC values
210
5
0
1
0

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of 0.45, 0.70, 0.92, 1.80 µM, respectively. In addition, preliminary investigations of CDK2
inhibitory displayed that the analogue 10s also manifested outstanding CDK2 inhibitory activity
(
IC = 15.4 nM), which was almost 3-fold potent than AZD5438. Further studies demonstrated that
50
215
220
225
compound 10s could induce cell cycle arrest and apoptosis in a concentration-dependent manner.
These observations made in this work highlighted the potential of novel pyrimidine-benzothiazole
hybrids for further development as potent antitumor agents.
5
. Experimental protocols
5.1 Chemistry
All reagents and solvents obtained from commercially available sources were used without
1
13
further treatment. H NMR and C NMR spectra were acquired in DMSO-d or CDCl solution
6
3
with a Mercury-Plus 400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts (δ) were
given in parts per million with tetramethylsilane as internal reference and coupling constants were
expressed in hertz. High-resolution mass spectra (HRMS) measurements were carried out using
an Agilent QTOF 6540 mass spectrometer. Melting points (mp) were determined on a Buchi B-545
apparatus and are uncorrected.
5
5
.2 General procedure for the synthesis of analogues 4a–j and 10a–x.
.2 .1 General procedure for preparation of compounds 4a–j.
230
N-(2-chloro-5-methylpyrimidin-4-yl)benzo[d]thiazol-2-amine (3).
To a solution of 2,4-dichloro-5-methylpyrimidine 1 (0.24 g, 1.5 mmol), and
benzo[d]thiazol-2-amine 2 (0.41 g, 1.5 mmol) in anhydrous DMF (5 mL) was added NaOH (0.06
g, 1.5 mmol). The resulting mixture was stirred at room temperature for about 12 h. After
completion of the reaction as indicated by TLC, the mixture was treated with sodium bicarbonate
solution (15 mL) and extracted with EtOAc (3 × 20 mL). The obtained organic layer was then dried
over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The crude product was
purified by recrystallization from methanol to afford compound 3 as a pale yellow solid. Yield, 85%;
235
1
H NMR (400 MHz, DMSO-d ) δ: 2.26 (s, 3H, CH ), 7.29 (t, J = 4.2 Hz, 1H, ArH), 7.44 (t, J = 4.8
6
3
Hz, 1H, ArH), 7.67 (s, 1H, ArH), 7.99 (s, 1H, ArH), 8.27 (s, 1H, ArH), 11.73 (s, 1H, NH).
11

ACCEPTED MANUSCRIPT
240
General procedure for preparation of compounds 4a–j.
To a solution of N-(2-chloro-5-methylpyrimidin-4-yl)benzo[d]thiazol-2-amine 3 (0.28 g, 1.0
mmol), and various aromatic amines (1.0 mmol) in anhydrous 2-methoxyethanol (8 mL) was
added concentrated hydrochloric acid (0.1 mL). The resulting mixture was stirred at 120 °C for
about 20 h. After completion of the reaction as indicated by TLC, the mixture was treated with
sodium bicarbonate solution (30 mL) and extracted with EtOAc (3 × 30 mL). The obtained organic
layer was then dried over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The
crude product was purified by silica gel flash chromatography (ethyl acetate/petroleum ether as an
eluent) to afford the pure desired compounds 4a–j in yields of 50–76%.
245
4
2
5
.2.1.1 N -Benzothiazol-2-yl-5-methyl-N -(4-nitro-phenyl)-pyrimidine-2,4-diamine (4a). Yield, 53%;
1
2
50
55
mp 285.0–286.1°C; H NMR (400 MHz, DMSO-d ) δ: 2.24 (s, 3H, CH ), 7.29 (t, J = 7.6 Hz, 1H,
6
3
ArH), 7.44 (t, J = 7.6 Hz, 1H, ArH), 7.70 (d, J = 7.6 Hz, 1H, ArH), 7.89 (d, J = 7.6 Hz, 1H, ArH),
.10 (d, J = 9.2 Hz, 2H, ArH), 8.19 (dd, J = 1.6 Hz, J = 6.8 Hz, 3H, ArH), 9.93 (s, 1H, NH), 11.22
8
1
2
1
3
(
s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 157.02, 156.64, 147.83, 140.55, 126.45, 125.24,
6
+
1
23.35, 121.59, 118.32, 13.74. HRMS m/z (ESI) calcd for C H N O S [M+H] 379.0977, found
1
8
14
6
2
2
379.0968.
4
2
5
5
6
.2.1.2 N -Benzothiazol-2-yl-N -(4-methoxy-phenyl)-5-methyl-pyrimidine-2,4-diamine (4b). Yield,
1
9%; mp 207.6–209.2°C; H NMR (400 MHz, DMSO-d ) δ: 2.18 (s, 3H, CH ), 3.76 (s, 3H, CH O),
6
3
3
.91 (d, J = 8.8 Hz, 2H, ArH), 7.26 (t, J = 7.4 Hz, 1H, ArH), 7.41 (t, J = 7.4 Hz, 1H, ArH), 7.61 (d,
J = 8.8 Hz, 2H, ArH), 7.68 (s, 1H, ArH), 7.80 (d, J = 7.6 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 8.91 (s,
1
3
260
265
270
1H, NH), 10.95 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.62, 158.35, 157.59, 155.10,
6
1
33.93, 126.24, 123.10, 122.95, 121.27, 114.04, 55.62, 13.50. HRMS m/z (ESI) calcd for
+
C H N OS [M+H] 364.1232, found 364.1223.
19
17
5
4
2
5
.2.1.3 N -Benzothiazol-2-yl-N -(3-methoxy-phenyl)-5-methyl-pyrimidine-2,4-diamine (4c). Yield,
1
6
1%; mp 210.1–211.0°C; H NMR (400 MHz, DMSO-d ) δ: 2.21 (s, 3H, CH ), 3.75 (s, 3H, CH O),
6
3
3
6.56 (dd, J = 2.4 Hz, J = 8.0 Hz, 1H, ArH), 7.19 (t, J = 8.2 Hz, 1H, ArH), 7.28 (t, J = 7.6 Hz, 1H,
1
2
ArH), 7.42 (dd, J = 7.8 Hz, J = 15.4 Hz, 2H, ArH), 7.49 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.85 (d, J
1
2
1
3
=
7.6 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 9.10 (s, 1H, NH), 11.02 (s, 1H, NH). C NMR (100 MHz,
DMSO-d ) δ: 159.88, 157.73, 157.40, 142.14, 129.43, 126.32, 123.19, 121.39, 112.53, 107.18,
6
+
1
06.10, 55.30, 13.57. HRMS m/z (ESI) calcd for C H N O [M+H] 364.1232, found 364.1227.
19
17
5
S
4
2
5.2.1.4 N -Benzothiazol-2-yl-N -(4-methanesulfonyl-phenyl)-5-methyl-pyrimidine-2,4-diamine (4d).
1
Yield, 56%; mp 232.6–233.6°C; H NMR (400 MHz, DMSO-d ) δ: 2.24 (s, 3H, CH ), 3.17 (s, 3H,
6
3
CH ), 7.29 (t, J = 7.4 Hz, 1H, ArH), 7.44 (t, J = 7.6 Hz, 1H, ArH), 7.73 (s, 1H, ArH), 7.83 (d, J =
3
1
2

ACCEPTED MANUSCRIPT
8.8 Hz, 2H, ArH), 7.90 (s, 1H, ArH), 8.09 (d, J = 8.8 Hz, 2H, ArH), 8.19 (s, 1H, ArH), 9.69 (s, 1H,
1
3
NH), 11.14 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 157.16, 157.00, 145.86, 132.48, 128.25,
6
+
275
280
285
126.43, 123.34, 121.55, 118.87, 44.45, 13.67. HRMS m/z (ESI) calcd for C H N O S [M+H]
19
17
5
2 2
412.0902, found 412.0896.
5
.2.1.5 4-[4-(Benzothiazol-2-ylamino)-5-methyl-pyrimidin-2-ylamino]-benzenesulfonamide (4e).
1
Yield, 51%; mp 271.4–272.5°C; H NMR (400 MHz, DMSO-d ) δ: 2.23 (s, 3H, CH ), 7.18 (s, 2H,
6
3
NH ), 7.29 (t, J = 7.4 Hz, 1H, ArH), 7.44 (t, J = 7.4 Hz, 1H, ArH), 7.74 (d, J = 8.8 Hz, 3H, ArH),
2
7.89 (s, 1H, ArH), 7.99 (d, J = 8.8 Hz, 2H, ArH), 8.17 (s, 1H, ArH), 9.54 (s, 1H, NH), 11.11 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d ) δ: 157.20, 144.18, 136.44, 126.80, 126.40, 123.29, 121.51,
6
+
1
18.86, 13.65. HRMS (ESI) m/z: calcd for C H N O S [M+H] 413.0854, found 413.0847.
1
8
16
6
2 2
4
2
5
.2.1.6 N -Benzothiazol-2-yl-N -(4-chloro-phenyl)-5-methyl-pyrimidine-2,4-diamine(4f). Yield,
1
5
7%; mp 254.2–256.3°C; H NMR (400 MHz, DMSO-d ) δ: 2.21 (s, 3H, CH ), 7.28 (t, J = 7.2 Hz,
6
3
1H, ArH), 7.34 (d, J = 8.8 Hz, 2H, ArH), 7.43 (t, J = 7.4 Hz, 1H, ArH), 7.71 (s, 1H, ArH), 7.85 (d, J
1
3
=
8.8 Hz, 3H, ArH), 8.11 (s, 1H, ArH), 9.27 (s, 1H, NH), 11.05 (s, 1H, NH). C NMR (100 MHz,
DMSO-d ) δ: 157.51, 157.27, 140.04, 128.57, 126.33, 125.25, 123.21, 121.51, 121.43, 13.60.
6
+
HRMS (ESI) m/z: calcd for C H ClN S [M+H] 368.0737, found 368.0728.
1
8
14
5
4
2
5
.2.1.7 N -Benzothiazol-2-yl-N -(3-fluoro-phenyl)-5-methyl-pyrimidine-2,4-diamine (4g). Yield,
1
2
90
95
61%; mp 199.4–201.2°C; H NMR (400 MHz, DMSO-d ) δ: 2.22 (s, 3H, CH ), 6.73–6.78 (m, 1H,
6
3
ArH), 7.26–7.34 (m, 2H, ArH), 7.43 (t, J = 7.6 Hz, 1H, ArH), 7.57 (d, J = 8.0 Hz, 1H, ArH), 7.70 (s,
1
3
1
H, ArH), 7.84–7.88 (m, 2H, ArH), 8.14 (s, 1H, ArH), 9.35 (s, 1H, NH), 11.08 (s, 1H, NH). C
NMR (100 MHz, DMSO-d ) δ: 163.92, 161.54, 157.38, 143.01, 142.90, 130.20, 130.10, 126.37,
6
1
23.27, 121.44, 115.46, 107.89, 107.68, 106.46, 106.20, 99.93, 13.62. HRMS (ESI) m/z: calcd for
+
2
C H FN S [M+H] 352.1032, found 352.1021.
18
14
5
4
2
5
6
2
.2.1.8 N -Benzothiazol-2-yl-N -(4-fluoro-phenyl)-5-methyl-pyrimidine-2,4-diamine (4h). Yield,
1
3%; mp 195.5–198.6°C; H NMR (400 MHz, DMSO-d ) δ: 2.20 (s, 3H, CH ), 7.14 (t, J = 8.8 Hz,
6
3
H, ArH), 7.27 (t, J = 7.6 Hz, 1H, ArH), 7.42 (t, J = 7.6 Hz, 1H, ArH), 7.69 (d, J = 7.2 Hz, 1H,
ArH), 7.77 (dd, J = 5.2 Hz, J = 8.8 Hz, 2H, ArH), 7.84 (d, J = 7.6 Hz, 1H, ArH), 8.07 (s, 1H, ArH),
1
2
1
3
3
00
05
9.13 (s, 1H, NH), 11.03 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.54, 158.96, 157.87,
6
1
57.40, 156.59, 137.31, 126.31, 123.18, 122.25, 122.18, 121.38, 115.33, 115.11, 13.55. HRMS (ESI)
+
m/z: calcd for C H FN S [M+H] 352.1032, found 352.1021.
1
8
14
5
4
2
5
.2.1.9 N -Benzothiazol-2-yl-N -(4-isopropyl-phenyl)-5-methyl-pyrimidine-2,4-diamine (4i). Yield,
1
7
6%; mp 218.3–219.7°C; H NMR (400 MHz, DMSO-d ) δ: 1.21 (d, J = 7.2 Hz, 6H, 2 × CH ),
6
3
3
2.20 (s, 3H, CH ), 2.80–2.90 (m, 1H, CH), 7.16 (d, J = 8.4 Hz, 2H, ArH), 7.26 (t, J = 7.6 Hz, 1H,
3
ArH), 7.41 (t, J = 7.6 Hz, 1H, ArH), 7.67 (t, J = 10.0 Hz, 3H, ArH), 7.82 (d, J = 8.0 Hz, 1H, ArH),
1
3

ACCEPTED MANUSCRIPT
1
3
8
.05 (s, 1H, ArH), 9.01 (s, 1H, NH), 11.00 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.57,
6
158.04, 157.49, 142.11, 138.56, 126.41, 126.26, 123.11, 121.28, 120.85, 33.26, 24.51, 13.55.
+
HRMS (ESI) m/z: calcd for C H N S [M+H] 376.1596, found 376.1589.
2
1
21
5
4
2
3
10
15
5.2.1.10 N -Benzothiazol-2-yl-5-methyl-N -(4-piperidin-1-yl-phenyl)-pyrimidine-2,4-diamine (4j).
1
Yield, 50%; mp 243.8–245.8°C; H NMR (400 MHz, DMSO-d ) δ: 1.54 (d, J = 4.4 Hz, 2H, CH ),
6
2
1
.65 (d, J = 4.0 Hz, 4H, 2 × CH ), 2.18 (s, 3H, CH ), 3.07–3.09 (m, 4H, 2 × CH ), 6.91 (d, J = 8.8
2 3 2
Hz, 2H, ArH), 7.26 (t, J = 7.4 Hz, 1H, ArH), 7.41 (t, J = 7.6 Hz, 1H, ArH), 7.51 (d, J = 8.4 Hz, 2H,
ArH), 7.68 (s, 1H, ArH), 7.79 (d, J = 7.2 Hz, 1H, ArH), 8.02 (s, 1H, ArH), 8.81 (s, 1H, NH), 10.92
1
3
3
(s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 158.46, 157.64, 147.92, 132.57, 126.22, 123.05,
6
+
1
4
22.71, 121.25, 116.75, 50.91, 25.87, 24.32, 13.48. HRMS (ESI) m/z: calcd for C H N S [M+H]
23 24 6
17.1861, found 417.1856.
5.2 .2 General procedure for preparation of compounds 10a–x.
Substituted N-(2-chloro-pyrimidin-4-yl)benzo[d]thiazol-2-amine (9).
3
20
To a solution of substituted benzo[d]thiazol-2-amine 7 (1.5 mmol) which were prepared
according to the literature procedure [29], and 2,4-dichloro-5-substituted pyrimidine 8 (1.5 mmol)
in anhydrous DMF (5 mL) was added NaOH (0.06 g, 1.5 mmol). The resulting mixture was stirred
at room temperature for about 12 h. After completion of the reaction as indicated by TLC, the
mixture was treated with sodium bicarbonate solution (15 mL) and extracted with EtOAc (3 × 20
mL). The obtained organic layer was then dried over anhydrous magnesium sulfate, filtered, and
evaporated in vacuo. The crude product was purified by recrystallization from methanol to afford
325
the key intermediates 9 as pale yellow solids.
1
(
2-Chloro-5-methyl-pyrimidin-4-yl)-(6-fluoro-benzothiazol-2-yl)-amine (9a). Yield, 76%; H NMR
(
400 MHz, DMSO-d ) δ: 2.27 (s, 3H, CH ), 7.26–7.32 (m, 1H, ArH), 7.67 (s, 1H, ArH), 7.94 (dd,
6
3
330
J = 2.2 Hz, J = 8.6 Hz, 1H, ArH), 8.28 (s, 1H, ArH), 11.90 (s, 1H, NH).
1 2
1
(
6-Chloro-benzothiazol-2-yl)-(2-chloro-5-methyl-pyrimidin-4-yl)-amine (9b). Yield, 79%; H NMR
(
400 MHz, DMSO-d ) δ: 2.27 (s, 3H, CH ), 7.45 (dd, J = 1.8 Hz, J = 8.6 Hz, 1H, ArH), 7.65 (s,
6
3
1
2
1H, ArH), 8.14 (s, 1H, ArH), 8.29 (s, 1H, ArH), 11.99 (s, 1H, NH).
1
(
2-Chloro-5-methyl-pyrimidin-4-yl)-(6-methyl-benzothiazol-2-yl)-amine (9c). Yield, 72%; H NMR
335
(400 MHz, DMSO-d ) δ: 2.26 (s, 3H, CH ), 2.41 (s, 3H, CH ), 7.26 (d, J = 8.0 Hz, 1H, ArH), 7.56
6
3
3
(d, J = 8.4 Hz, 1H, ArH), 7.78 (s, 1H, ArH), 8.27 (s, 1H, ArH), 11.62 (s, 1H, NH).
1
(2-Chloro-5-methyl-pyrimidin-4-yl)-(6-methoxy-benzothiazol-2-yl)-amine (9d). Yield, 77%;
H
NMR (400 MHz, DMSO-d ) δ: 2.25 (s, 3H, CH ), 3.81 (s, 3H, CH O), 7.04 (dd, J = 2.4 Hz, J = 8.8
6
3
3
1
2
1
4

ACCEPTED MANUSCRIPT
Hz, 1H, ArH), 7.58 (d, J = 8.8 Hz, 1H, ArH), 7.62 (s, 1H, ArH), 8.24 (s, 1H, ArH), 11.62 (s, 1H,
340
NH).
1
Benzothiazol-2-yl-(2-chloro-5-fluoro-pyrimidin-4-yl)-amine (9e). Yield, 81%; H NMR (400 MHz,
DMSO-d ) δ: 7.32 (t, J = 7.4 Hz, 1H, ArH), 7.47 (t, J = 7.4 Hz, 1H, ArH), 7.52 (s, 1H, ArH), 7.99 (d,
6
J = 7.6 Hz, 1H, ArH), 8.48 (d, J = 2.4 Hz, 1H, ArH), 13.41 (s, 1H, NH).
1
(
2-Chloro-5-fluoro-pyrimidin-4-yl)-(6-fluoro-benzothiazol-2-yl)-amine (9f). Yield, 73%; H NMR
345
(400 MHz, DMSO-d ) δ: 7.26–7.31 (m, 1H, ArH), 7.58 (dd, J = 4.6 Hz, J = 8.6 Hz, 1H, ArH), 7.91
6 1 2
(
dd, J = 2.4 Hz, J = 8.8 Hz, 1H, ArH), 8.42 (d, J = 3.2 Hz, 1H, ArH), 13.19 (s, 1H, NH).
1 2
1
(
6-Chloro-benzothiazol-2-yl)-(2-chloro-5-fluoro-pyrimidin-4-yl)-amine (9g). Yield, 76%; H NMR
(
400 MHz, DMSO-d ) δ: 7.44 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H, ArH), 7.55 (d, J = 8.4 Hz, 1H, ArH),
6
1
2
8.10 (d, J = 2.0 Hz, 1H, ArH), 8.39 (d, J = 2.8 Hz, 1H, ArH), 13.06 (s, 1H, NH).
1
350
355
360
(2-Chloro-5-fluoro-pyrimidin-4-yl)-(6-methyl-benzothiazol-2-yl)-amine (9h). Yield, 71%; H NMR
400 MHz, DMSO-d ) δ: 2.40 (s, 3H, CH ),7.25 (d, J = 8.0 Hz, 1H, ArH), 7.41 (d, J = 8.0 Hz, 1H,
(
6
3
ArH), 7.75 (s, 1H, ArH), 8.42 (s, 1H, ArH),13.21 (s, 1H, NH).
1
(
2-Chloro-5-fluoro-pyrimidin-4-yl)-(6-methoxy-benzothiazol-2-yl)-amine (9i). Yield, 75%; H NMR
(
400 MHz, DMSO-d ) δ: 3.81 (s, 3H, CH O),7.07 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H, ArH), 7.47 (d, J
6
3
1
2
= 8.0 Hz, 1H, ArH), 7.64 (d, J = 2.4 Hz, 1H, ArH), 8.44 (d, J = 2.8 Hz, 1H, ArH), 13.17 (s, 1H,
NH).
1
Benzothiazol-2-yl-(2-chloro-pyrimidin-4-yl)-amine (9j). Yield, 63%; H NMR (400 MHz, DMSO-d )
6
δ: 7.17 (t, J = 7.6 Hz, 1H, ArH), 7.25 (t, J = 7.6 Hz, 1H, ArH), 7.53 (d, J = 7.6 Hz, 1H, ArH), 7.65
(
d, J = 8.4 Hz, 1H, ArH), 8.16 (d, J = 5.2 Hz, 1H, ArH), 8.90 (d, J = 5.2 Hz, 1H, ArH), 12.35 (s, 1H,
NH).
1
(
2-Chloro-pyrimidin-4-yl)-(6-fluoro-benzothiazol-2-yl)-amine (9k). Yield, 61%; H NMR (400 MHz,
DMSO-d ) δ: 7.17 (d, J = 6.0 Hz, 1H, ArH), 7.60 (s, 1H, ArH), 7.68 (d, J = 8.0 Hz, 1H, ArH), 8.12
6
(d, J = 2.4 Hz, 1H, ArH), 8.63 (d, J = 6.8 Hz, 1H, ArH),12.50 (s, 1H, NH).
1
(
6-Chloro-benzothiazol-2-yl)-(2-chloro-pyrimidin-4-yl)-amine (9l). Yield, 65%; H NMR (400 MHz,
365
DMSO-d ) δ: 7.17 (d, J = 5.6 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.72 (d, J = 8.8 Hz, 1H, ArH), 8.18
6
(d, J = 2.0 Hz, 1H, ArH), 8.45 (d, J = 6.0 Hz, 1H, ArH), 12.45 (s, 1H, NH).
1
(
2-Chloro-pyrimidin-4-yl)-(6-methyl-benzothiazol-2-yl)-amine (9m). Yield, 70%; H NMR (400
MHz, DMSO-d ) δ: 2.42 (s, 3H, CH ),7.19 (d, J = 5.2 Hz, 1H, ArH), 7.24–7.27 (m, 1H, ArH), 7.62
6
3
(d, J = 8.0 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 8.43 (d, J = 6.0 Hz, 1H, ArH), 12.28 (s, 1H, NH).
1
370
(2-Chloro-pyrimidin-4-yl)-(6-methoxy-benzothiazol-2-yl)-amine (9n). Yield, 62%; H NMR (400
1
5

ACCEPTED MANUSCRIPT
MHz, DMSO-d ) δ: 3.81 (s, 3H, CH O), 7.03 (dd, J = 2.2 Hz, J = 9.0 Hz, 1H, ArH),7.16–7.21 (m,
6
3
1
2
1
H, ArH), 7.57–7.64 (m, 2H, ArH),8.41 (dd, J = 1.4 Hz, J = 5.8 Hz, 1H, ArH), 12.22 (s, 1H, NH).
1 2
General procedure for preparation of compounds 10a–x.
To a solution of the above intermediates 9 (1.0 mmol), and 4-(methylsulfonyl)aniline or
4-aminobenzenesulfonamide (1.0 mmol) in anhydrous 2-methoxyethanol (8 mL) was added
concentrated HCl (0.1 mL). The resulting mixture was stirred at 120 °C for about 20 h. After
completion of the reaction as indicated by TLC, the mixture was treated with sodium bicarbonate
solution (30 mL) and extracted with EtOAc (3 × 30 mL). The obtained organic layer was then dried
over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The crude product was
purified by silica gel flash chromatography (acetone/petroleum ether as an eluent) to afford the pure
target compounds 10a–x in yields of 47–80%.
3
75
380
385
390
4
2
5
.2.2.1 N -(6-Fluoro-benzothiazol-2-yl)-N -(4-methanesulfonyl-phenyl)-5-methyl-pyrimidine-2,4-
1
diamine (10a). Yield, 47%; mp 253.3–254.1°C; H NMR (400 MHz, DMSO-d ) δ: 2.23 (s, 3H,
6
CH ), 3.17 (s, 3H, CH ), 7.29 (t, J = 7.8 Hz, 1H, ArH), 7.75 (t, J = 8.2 Hz, 2H, ArH), 7.82 (d, J =
3
3
8.8 Hz, 2H, ArH), 8.09 (d, J = 8.8 Hz, 2H, ArH), 8.18 (s, 1H, ArH), 9.72 (s, 1H, NH), 11.18 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d ) δ: 159.94, 157.56, 157.24, 156.95, 145.84, 132.52, 128.26,
6
+
118.89, 114.48, 114.24, 107.81, 44.46, 13.65. HRMS (ESI) m/z: calcd for C H FN O S [M+H]
19 16 5 2 2
430.0808, found 430.0799.
5
.2.2.2 4-(4-(6-fluorobenzo[d]thiazol-2-ylamino)-5-methylpyrimidin-2-ylamino)benzenesulfon
1
-amide (10b). Yield, 51%; mp 278.7–280.6°C; H NMR (400 MHz, DMSO-d ) δ: 2.22 (s, 3H, CH ),
6
3
7
8
1
.23 (s, 2H, NH ), 7.26–7.31 (m, 1H, ArH), 7.70–7.77 (m, 4H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH),
2
1
3
.15 (s, 1H, ArH), 9.58 (s, 1H, NH), 11.14 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.92,
6
57.54, 157.33, 157.15, 144.11, 136.54, 126.80, 118.93, 114.45, 114.21, 13.63. HRMS (ESI) m/z:
+
calcd for C H FN O S [M+H] 431.0760, found 431.0752.
1
8
15
6
2 2
4
2
3
95
00
5.2.2.3 N -(6-Chloro-benzothiazol-2-yl)-N -(4-methanesulfonyl-phenyl)-5-methyl-pyrimidine-2,4-
1
diamine (10c). Yield, 66%; mp 283.4–285.4°C; H NMR (400 MHz, DMSO-d ) δ: 2.24 (s, 3H,
6
CH ), 3.19 (s, 3H, CH ), 7.46 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H, ArH), 7.70 (s, 1H, ArH), 7.84 (d, J =
3
3
1
2
8
.8 Hz, 2H, ArH), 7.96 (s, 1H, ArH), 8.09 (d, J = 8.8 Hz, 2H, ArH), 8.20 (s, 1H, ArH), 9.73 (s, 1H,
1
3
NH), 11.26 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 157.39, 156.95, 145.79, 132.61, 128.29,
6
+
4
127.33, 126.76, 120.99, 118.91, 44.46, 13.65. HRMS (ESI) m/z: calcd for C H ClN O S [M+H]
19
16
5
2 2
446.0512, found 446.0503.
5.2.2.4 4-(4-(6-Chloro-benzothiazol-2-ylamino)-5-methylpyrimidin-2-ylamino)-benzenesulfonamide
1
(
10d). Yield, 57%; mp 298.8–299.2°C; H NMR (400 MHz, DMSO-d ) δ:2.22 (s, 3H, CH ), 7.20 (s,
6
3
1
6

ACCEPTED MANUSCRIPT
2
H, NH ), 7.45 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H, ArH), 7.69 (d, J = 8.4 Hz, 1H, ArH), 7.76 (d, J =
2
1
2
405
8.4 Hz, 2H, ArH), 7.96 (t, J = 8.4 Hz, 3H, ArH), 8.17 (s, 1H, ArH), 9.57 (s, 1H, NH), 11.22 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d ) δ: 157.49, 157.16, 144.10, 136.55, 127.29, 126.82, 126.74,
6
+
1
4
20.96, 118.95, 99.92, 13.61. HRMS (ESI) m/z: calcd for C H ClN O S [M+H] 447.0465, found
1
8
15
6
2 2
47.0458.
5
.2.2.5 4-(5-Methyl-4-(6-methyl-benzothiazol-2-ylamino)-pyrimidin-2-ylamino)-benzenesulfon-
1
4
10
15
amide (10e). Yield, 58%; mp 282.2–283.9°C; H NMR (400 MHz, DMSO-d ) δ: 2.22 (s, 3H, CH ),
6
3
2
.44 (s, 3H, CH ), 7.19 (s, 2H, NH ), 7.25 (d, J = 8.0 Hz, 1H, ArH), 7.61 (s, 1H, ArH), 7.67 (s, 1H,
3 2
ArH), 7.74 (d, J = 9.2 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 8.15 (s, 1H, ArH), 9.53 (s, 1H,
1
3
NH), 11.00 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 157.22, 144.21, 136.42, 132.69, 127.67,
6
+
1
26.80, 121.15, 118.85, 21.42, 13.68.HRMS (ESI) m/z: calcd for C H N O S [M+H] 427.1011,
1
9
18
6
2 2
4
found 427.1005.
5
.2.2.6 4-(4-(6-Methoxy-benzothiazol-2-ylamino)-5-methylpyrimidin-2-ylamino)-benzenesulfon-
1
amide (10f). Yield, 49%; mp 266.8–268.0°C; H NMR (400 MHz, DMSO-d ) δ: 2.22 (s, 3H, CH ),
6
3
3
.83 (s, 3H, CH O), 7.04 (dd, J = 2.8 Hz, J = 8.8 Hz, 1H, ArH), 7.20 (s, 2H, NH ), 7.41 (d, J = 2.4
3 1 2 2
Hz, 1H, ArH), 7.61 (d, J = 8.4 Hz, 1H, ArH), 7.75 (d, J = 8.8 Hz, 2H, ArH), 7.97 (d, J = 9.2 Hz, 2H,
1
3
4
20
25
ArH), 8.13 (s, 1H, ArH), 9.50 (s, 1H, NH), 10.97 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ:
6
1
57.25, 157.07, 156.13, 144.18, 136.50, 126.81, 119.01, 114.68, 112.83, 104.92, 56.04, 13.64.
+
HRMS (ESI) m/z: calcd for C H N O S [M+H] 443.0960, found 443.0955.
1
9
18
6
3 2
4
2
5
.2.2.7 N -Benzothiazol-2-yl-5-fluoro-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-diamine (10g).
1
Yield, 53%; mp 271.6–273.6°C; H NMR (400 MHz, DMSO-d ) δ: 3.18 (s, 3H, CH ), 7.30 (t, J =
6
3
4
7.4 Hz, 1H, ArH), 7.45 (t, J = 7.6 Hz, 1H, ArH), 7.64 (d, J = 8.0 Hz, 1H, ArH), 7.85 (t, J = 9.0 Hz,
3
1
1
H, ArH), 8.06 (d, J = 8.8 Hz, 2H, ArH), 8.35 (d, J = 2.8 Hz, 1H, ArH), 9.85 (s, 1H, NH), 12.48 (s,
1
3
H, NH). C NMR (100 MHz, DMSO-d ) δ: 154.51, 154.47, 145.64, 142.57, 142.37, 132.79,
6
28.28, 126.80, 123.62, 121.89, 118.84, 49.01, 44.43. HRMS (ESI) m/z: calcd for C H FN O S
2 2
1
8
14
5
+
[
M+H] 416.0651 found 416.0643.
4
30
35
5.2.2.8 4-[4-(Benzothiazol-2-ylamino)-5-fluoro-pyrimidin-2-ylamino]-benzenesulfonamide (10h).
1
Yield, 50%; mp > 300°C; H NMR (400 MHz, DMSO-d ) δ: 7.21 (s, 2H, NH ), 7.29 (t, J = 7.4 Hz,
6
2
1H, ArH), 7.44 (t, J = 7.2 Hz, 1H, ArH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.75 (d, J = 8.8 Hz, 2H,
ArH), 7.86 (d, J = 7.6 Hz, 1H, ArH), 7.96 (d, J = 8.8 Hz, 2H, ArH), 8.33 (d, J = 3.2 Hz, 1H, ArH),
1
3
9
.71 (s, 1H, NH) , 12.16 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 154.66, 148.48, 142.64,
6
4
142.44, 142.18, 130.06, 126.77, 125.48, 123.58, 121.79, 116.15, 113.47. HRMS (ESI) m/z: calcd
+
for C H FN O S [M+H] 417.0604, found 417.0596.
1
7
13
6
2 2
4
2
5
.2.2.9 5-Fluoro-N -(6-fluoro-benzothiazol-2-yl)-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-
1
7

ACCEPTED MANUSCRIPT
1
diamine (10i). Yield, 52%; mp 293.5–294.2°C; H NMR (400 MHz, DMSO-d ) δ: 3.18 (s, 3H,
6
CH ), 7.28–7.33 (m, 1H, ArH), 7.69 (dd, J = 4.2 Hz, J = 8.2 Hz, 1H, ArH), 7.77 (d, J = 8.0 Hz, 1H,
3
1
2
4
40
45
ArH), 7.84 (d, J = 8.8 Hz, 2H, ArH), 8.05 (d, J = 8.8 Hz, 2H, ArH), 8.37 (d, J = 2.4 Hz, 1H, ArH),
1
3
9
1
.89 (s, 1H, NH), 12.34 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 160.07, 157.69, 154.43,
6
45.55, 142.69, 142.49, 132.90, 128.30, 118.86, 114.75, 114.51, 108.40, 108.12, 44.41. HRMS (ESI)
+
m/z: calcd for C H F N O S [M+H] 434.0557, found 434.0550.
1
8
13
2
5
2 2
5
.2.2.10 4-(4-(6-Chloro-benzothiazol-2-ylamino)-5-fluoropyrimidin-2-ylamino)-benzenesulfon-
1
4
amide (10j). Yield, 56%; mp 299.5–300.0°C; H NMR (400 MHz, DMSO-d ) δ: 7.23 (s, 2H, NH ),
6
2
7.48 (d, J = 8.8 Hz, 1H, ArH), 7.69 (d, J = 8.4 Hz, 1H, ArH), 7.78 (d, J = 8.4 Hz, 2H, ArH), 7.95 (d,
1
3
J = 9.2 Hz, 3H, ArH), 8.38 (d, J = 2.0 Hz, 1H, ArH), 9.79 (s, 1H, NH), 12.34 (s, 1H, NH). C NMR
(
100 MHz, DMSO-d ) δ: 154.73, 143.84, 143.02, 142.82, 136.92, 127.65, 127.04, 126.86, 121.24,
6
+
1
19.02. HRMS (ESI) m/z: calcd for C H ClFN O S [M+H] 451.0214, found 451.0208.
1
7
12
6
2 2
4
2
4
50
55
5.2.2.11 N -(6-Chloro-benzothiazol-2-yl)-5-fluoro-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-
1
diamine (10k). Yield, 52%; mp 281.6–282.1°C; H NMR (400 MHz, DMSO-d ) δ: 3.18 (s, 3H,
6
CH ), 7.47 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H, ArH), 7.85 (d, J = 8.8 Hz, 2H, ArH),
3
7
.96 (s, 1H, ArH), 8.04 (d, J = 8.4 Hz, 2H, ArH), 8.38 (s, 1H, ArH), 9.92 (s, 1H, NH), 12.44 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d ) δ: 154.48, 145.53, 142.88, 142.68, 132.93, 128.31, 127.65,
6
+
4
127.04, 121.28, 118.88, 44.42. HRMS (ESI) m/z: calcd for C H ClFN O S [M+H] 450.0261,
1
8
13
5
2 2
found 450.0255.
5
.2.2.12 4-[5-Fluoro-4-(6-methyl-benzothiazol-2-ylamino)-pyrimidin-2-ylamino]-benzenesulfon-
1
amide (10l). Yield, 54%; mp 267.2–269.2°C; H NMR (400 MHz, DMSO-d ) δ: 2.43 (s, 3H, CH ),
6
3
7
.22 (s, 2H, NH ), 7.27 (dd, J = 0.8 Hz, J = 8.4 Hz, 1H, ArH), 7.54 (d, J = 8.4 Hz, 1H, ArH), 7.65
2 1 2
460
(s, 1H, ArH), 7.77 (d, J = 8.8 Hz, 2H, ArH), 7.96 (d, J = 8.8 Hz, 2H, ArH), 8.34 (d, J = 2.8 Hz, 1H,
1
3
ArH), 9.73 (s, 1H, NH), 12.32 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 154.76, 143.98,
6
142.57, 136.72, 133.08, 128.01, 126.83, 121.51, 118.83, 21.38. HRMS (ESI) m/z: calcd for
+
C H FN O S [M+H] 431.0760, found 431.0752.
18
15
6
2 2
2
4
5
.2.2.13 5-Fluoro-N -(4-methanesulfonyl-phenyl)-N -(6-methyl-benzothiazol-2-yl)-pyrimidine-2,4-
1
4
65
70
diamine (10m). Yield, 49%; mp244.4–246.6°C; H NMR (400 MHz, DMSO-d ) δ: 2.43 (s, 3H,
6
CH ), 3.18 (s, 3H, CH ), 7.31 (d, J = 8.4 Hz, 1H, ArH), 7.42 (d, J = 8.4 Hz, 1H, ArH), 7.67 (s, 1H,
3
3
ArH), 7.86 (d, J = 8.8 Hz, 2H, ArH), 8.03 (d, J = 8.8 Hz, 2H, ArH), 8.42 (d, J = 2.4 Hz, 1H, ArH),
1
3
9
.39 (s, 1H, NH), 9.97 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 160.10, 152.21, 145.62,
6
1
35.32, 132.85, 132.71, 129.23, 128.76, 128.38, 125.87, 123.12, 118.73, 113.09, 44.44, 21.13.
+
4
HRMS (ESI) m/z: calcd for C H FN O S [M+H] 430.0808, found 430.0799.
1
9
16
5
2 2
5.2.2.14 4-[5-Fluoro-4-(6-methoxy-benzothiazol-2-ylamino)-pyrimidin-2-ylamino]-benzenesulfon-
1
8

ACCEPTED MANUSCRIPT
1
amide (10n). Yield, 80%; mp 255.5–257.7°C; H NMR (400 MHz, DMSO-d ) δ: 3.83 (s, 3H, CH ),
6
3
7
.06 (dd, J = 2.8 Hz, J = 8.8 Hz, 1H, ArH), 7.22 (s, 2H, NH ), 7.41 (d, J = 2.8 Hz, 1H, ArH), 7.59
1 2 2
(d, J = 8.8 Hz, 1H, ArH), 7.77 (d, J = 8.8 Hz, 2H, ArH), 7.94 (d, J = 8.8 Hz, 2H, ArH), 8.33 (d, J =
1
3
4
75
80
3.2 Hz, 1H, ArH), 9.70 (s, 1H, NH), 12.18 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 156.32,
6
1
54.80, 154.77, 143.94, 142.53, 142.34, 136.83, 126.84, 119.05, 114.87, 105.33, 56.07. HRMS (ESI)
+
m/z: calcd for C H FN O S [M+H] 447.0709, found 447.0705.
1
8
15
6
3 2
2
4
5
.2.2.15 5-Fluoro-N -(4-methanesulfonyl-phenyl)-N -(6-methoxy-benzothiazol-2-yl)-pyrimidine-2,4-
1
Diamine (10o). Yield, 57%; mp 280.7–282.5°C; H NMR (400 MHz, DMSO-d ) δ: 3.18 (s, 3H,
6
4
CH ), 3.82 (s, 3H, CH ), 7.06 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H, ArH), 7.42 (d, J = 2.0 Hz, 1H, ArH),
3
3
1
2
7.58 (d, J = 8.8 Hz, 1H, ArH), 7.84 (d, J = 8.8 Hz, 2H, ArH), 8.04 (d, J = 8.8 Hz, 2H, ArH), 8.34 (d,
1
3
J = 2.8 Hz, 1H, ArH), 9.83 (s, 1H, NH), 12.23 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ:
6
156.33, 154.55, 145.63, 142.40, 142.20, 132.86, 128.29, 118.92, 114.87, 105.37, 56.06, 44.40.
+
HRMS (ESI) m/z: calcd for C H FN O S [M+H] 446.0757, found 446.0749.
1
9
16
5
3 2
4
2
4
85
90
5.2.2.16 N -Benzothiazol-2-yl-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-diamine (10p). Yield,
1
6
1
6%; mp 296.0–297.9°C; H NMR (400 MHz, DMSO-d ) δ: 3.19 (s, 3H, CH ), 6.75 (d, J = 4.4 Hz,
6
3
H, ArH), 7.28 (t, J = 7.2 Hz, 1H, ArH), 7.43 (t, J = 7.4 Hz, 1H, ArH), 7.72 (d, J = 8.0 Hz, 1H,
ArH), 7.88 (dd, J = 8.0 Hz, J = 17.2 Hz, 3H, ArH), 8.12 (d, J = 8.4 Hz, 2H, ArH), 8.33 (d, J = 5.2
1
2
1
3
Hz, 1H, ArH), 9.99 (s, 1H, NH) , 11.87 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 158.88,
6
4
158.72, 158.06, 157.77, 149.72, 145.53, 132.95, 132.65, 128.27, 126.42, 123.31, 121.53, 120.35,
+
1
19.29, 101.09, 44.41. HRMS (ESI) m/z: calcd for C H N O S [M+H] 398.0745, found
1
8
15
5
2 2
398.0741.
5.2.2.17 4-[4-(Benzothiazol-2-ylamino)-pyrimidin-2-ylamino]-benzenesulfonamide (10q). Yield,
1
6
9%; mp 264.2–267.6°C; H NMR (400 MHz, DMSO-d ) δ: 6.72 (d, J = 4.8 Hz, 1H, ArH), 7.24 (s,
6
4
95
00
2H, NH ), 7.29 (d, J = 7.6 Hz, 1H, ArH), 7.43 (t, J = 7.4 Hz, 1H, ArH), 7.72 (d, J = 8.0 Hz, 1H,
2
ArH), 7.78 (d, J = 8.4 Hz, 2H, ArH), 7.90 (d, J = 7.6 Hz, 1H, ArH), 8.01 (d, J = 8.8 Hz, 2H, ArH),
1
3
8
.32 (d, J = 5.6 Hz, 1H, ArH) , 9.77 (s, 1H, NH) , 11.84 (s, 1H, NH). C NMR (100 MHz,
DMSO-d ) δ: 158.88, 158.02, 157.84, 149.70, 143.82, 136.88, 132.65, 126.80, 126.42, 123.27,
6
+
1
21.51, 120.32, 119.32, 100.71. HRMS (ESI) m/z: calcd for C H N O S [M+H] 399.0698, found
17
14
6
2 2
5
399.0689.
4
2
5.2.2.18
N -(6-Fluoro-benzothiazol-2-yl)-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-diamine
1
(
10r). Yield, 63%; mp > 300°C; H NMR (400 MHz, DMSO-d ) δ: 3.19 (s, 3H, CH ), 6.73 (d, J =
6
3
5
.6 Hz, 1H, ArH), 7.25–7.30 (m, 1H, ArH), 7.72 (dd, J = 4.8 Hz, J = 8.8 Hz, 1H, ArH), 7.78 (dd,
1 2
J = 2.6 Hz, J = 8.6 Hz, 1H, ArH), 7.85 (d, J = 8.8 Hz, 2H, ArH), 8.10 (d, J = 8.8 Hz, 2H, ArH),
1
2
1
3
505
8.33 (d, J = 5.6 Hz, 1H, ArH), 9.88 (s, 1H, NH) , 11.86 (s, 1H, NH). C NMR (100 MHz,
DMSO-d ) δ: 159.91, 158.78, 158.70, 157.98, 157.81, 157.53, 146.46, 145.50, 134.69, 133.73,
6
1
9

ACCEPTED MANUSCRIPT
133.03, 128.27, 121.40, 119.31, 114.48, 114.24, 108.14, 107.87, 101.00, 44.41. HRMS (ESI) m/z:
+
calcd for C H FN O S [M+H] 416.0651, found 416.0644.
1
8
14
5
2 2
5.2.2.19 4-[4-(6-Fluoro-benzothiazol-2-ylamino)-pyrimidin-2-ylamino]-benzenesulfonamide (10s).
1
5
10
15
Yield, 68%; mp > 300°C; H NMR (400 MHz, DMSO-d ) δ: 6.70 (d, J = 5.2 Hz, 1H, ArH), 7.22 (s,
6
2
3
1
1
H, NH ), 7.25–7.30 (m, 1H, ArH), 7.72 (dd, J = 4.8 Hz, J = 8.8 Hz, 1H, ArH), 7.78 (d, J = 8.8 Hz,
2 1 2
H, ArH), 7.99 (d, J = 8.4 Hz, 2H, ArH), 8.31 (d, J = 5.6 Hz, 1H, ArH), 9.75 (s, 1H, NH), 11.83 (s,
1
3
H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.90, 158.88, 158.78, 157.89, 157.52, 146.45,
6
43.78, 136.98, 133.73, 126.80, 121.28, 119.44, 114.46, 114.23, 108.09, 107.83, 100.60. HRMS
+
5
(ESI) m/z: calcd for C H FN O S [M+H] 417.0604, found 417.0597.
1
7
13
6
2 2
4
2
5
.2.2.20 N -(6-Chloro-benzothiazol-2-yl)-N -(4-methanesulfonyl-phenyl)-pyrimidine-2,4-diamine
1
(
10t). Yield, 57%; mp > 300°C; H NMR (400 MHz, DMSO-d ) δ: 3.19 (s, 3H, CH ), 6.74 (d, J =
6
3
5
.6 Hz, 1H, ArH), 7.44 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H, ArH), 7.70 (d, J = 8.8 Hz, 1H, ArH), 7.86
1 2
(d, J = 8.8 Hz, 2H, ArH), 7.97 (d, J = 2.0 Hz, 1H, ArH), 8.09 (d, J = 8.8 Hz, 2H, ArH), 8.34 (d, J =
1
3
5
20
25
5.6 Hz, 1H, ArH) , 9.90 (s, 1H, NH) , 11.93 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 159.57,
6
1
1
58.71, 157.91, 148.63, 145.46, 134.33, 133.07, 128.28, 127.29, 126.76, 121.50, 121.05, 119.35,
+
01.11, 44.41. HRMS (ESI) m/z: calcd for C H ClN O S [M+H] 432.0356, found 432.0350.
1
8
14
5
2 2
5
.2.2.21 4-(4-(6-Methyl-benzothiazol-2-ylamino)-pyrimidin-2-ylamino)-benzenesulfonamide (10u).
1
Yield, 61%; mp >300°C; H NMR (400 MHz, DMSO-d ) δ: 2.42 (s, 3H, CH ), 6.71 (d, J = 5.2 Hz,
6
3
5
1H, ArH), 7.22 (s, 2H, NH ), 7.24 (s, 1H, ArH), 7.60 (d, J = 8.0 Hz, 1H, ArH), 7.67 (s, 1H, ArH),
2
7
1
1
.77 (d, J = 8.4 Hz, 2H, ArH), 8.00 (d, J = 8.4 Hz, 2H, ArH), 8.30 (d, J = 5.6 Hz, 1H, ArH), 9.73 (s,
1
3
H, NH) , 11.72 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 158.93, 158.06, 157.76, 143.85,
6
36.87, 132.67, 127.69, 126.79, 121.15, 119.99, 119.29, 100.65, 21.40. HRMS (ESI) m/z: calcd for
+
C H N O S [M+H] 413.0854, found 413.0850.
18
16
6
2
2
2
4
5
30
35
5.2.2.22 N -(4-Methanesulfonyl-phenyl)-N -(6-methyl-benzothiazol-2-yl)-pyrimidine-2,4-diamine
1
(
10v). Yield, 47%; mp 293.7–296.5°C; H NMR (400 MHz, DMSO-d ) δ: 2.43 (s, 3H, CH ), 3.19 (s,
6
3
3
H, CH ), 6.75 (d, J = 5.2 Hz, 1H, ArH), 7.23 (d, J = 8.4 Hz, 1H, ArH), 7.60 (d, J = 8.0 Hz, 1H,
3
ArH), 7.67 (s, 1H, ArH), 7.85 (d, J = 8.8 Hz, 2H, ArH), 8.11 (d, J = 8.8 Hz, 2H, ArH), 8.32 (d, J =
1
3
6
.0 Hz, 1H, ArH), 9.86 (s, 1H, NH) , 11.75 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 158.77,
6
5
158.06, 157.69, 147.71, 145.56, 132.94, 132.72, 128.25, 127.70, 121.17, 120.02, 119.26, 101.03,
+
4
4.43, 21.40. HRMS (ESI) m/z: calcd for C H N O S [M+H] 412.0902, found 412.0898.
1
9
17
5
2 2
5.2.2.23
4-[4-(6-Methoxy-benzothiazol-2-ylamino)-pyrimidin-2-ylamino]-benzenesulfonamide
1
(
10w). Yield, 48%; mp 298.0–298.3°C; H NMR (400 MHz, DMSO-d ) δ: 3.82 (s, 3H, CH O), 6.72
6
3
(
d, J = 5.2 Hz, 1H, ArH), 7.03 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H, ArH), 7.23 (s, 2H, NH ), 7.43 (d, J
1 2 2
540
= 2.4 Hz, 1H, ArH), 7.62 (d, J = 8.8 Hz, 1H, ArH), 7.78 (d, J = 8.8 Hz, 2H, ArH), 7.98 (d, J = 8.8
2
0

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1
3
Hz, 2H, ArH), 8.29 (d, J = 6.0 Hz, 1H, ArH) , 9.71 (s, 1H, NH) , 11.66 (s, 1H, NH). C NMR (100
MHz, DMSO-d ) δ: 158.95, 158.01, 157.68, 156.96, 156.10, 143.83, 136.95, 133.83, 126.80,
6
+
1
4
20.93, 119.46, 114.63, 105.07, 100.50, 56.05. HRMS (ESI) m/z: calcd for C H N O S [M+H]
18 16 6 3 2
29.0804, found 429.0797.
2
4
5
45
50
5.2.2.24 N -(4-Methanesulfonyl-phenyl)-N -(6-methoxy-benzothiazol-2-yl)-pyrimidine-2,4-diamine
1
(
10x). Yield, 52%; mp 277.7–280.0°C; H NMR (400 MHz, DMSO-d ) δ: 3.19 (s, 3H, CH ), 3.82
6
3
(
s, 3H, CH O), 6.75 (d, J = 4.8 Hz, 1H, ArH), 7.03 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H, ArH), 7.44 (d, J
3
1
2
=
1.6 Hz, 1H, ArH), 7.62 (d, J = 8.8 Hz, 1H, ArH), 7.85 (d, J = 8.8 Hz, 2H, ArH), 8.10 (d, J = 8.4
1
3
Hz, 2H, ArH), 8.31 (d, J = 5.6 Hz, 1H, ArH) , 9.83 (s, 1H, NH) , 11.68 (s, 1H, NH). C NMR (100
5
MHz, DMSO-d ) δ: 158.79, 158.06, 157.67, 156.97, 156.11, 145.57, 143.90, 133.83, 132.98, 128.26,
6
1
20.96, 119.34, 114.62, 105.09, 100.91, 56.04, 44.40. HRMS (ESI) m/z: calcd for C H N O S
19 17 5 3 2
+
[
M+H] 428.0851, found 428.0845.
5.3 Biological evaluation
555
560
565
570
5.3.1 Antiproliferative MTT assay
The antiproliferative activities of analogues 4a–j and 10a–x were evaluated by the usual MTT
assay with five human cancer cell lines (MDA-MB-231, MCF-7, HeLa, HCT116, and PC-3) in
vitro. MDA-MB-231, MCF-7, HeLa, and HCT116 cells were grown in RPMI-1640 medium
whereas PC-3 cell was cultured in DMEM/F12 in a humidified atmosphere containing 5% CO at
2
37 °C. DMEM/F12 and RPMI-1640 were supplemented with 10% fetal bovine serum (FBS). The
solid investigated compounds were dissolved in DMSO (dimethyl sulfoxide) at concentration of 60
mM, and then diluted with the medium into a range of concentrations. The cells were plated at a
3
density of 5×10 cells/well in a 96-well plate and incubated at 37 °C for attachment. Subsequently,
the medium was altered when the cells reached 70%-80% confluence, and the cells grew in new
medium with tested analogues. AZD5438 and DMSO (0.1%) were then added to the wells as
positive and negative controls, respectively. The cells with tested analogues were incubated at 37°C
for 48 h. After reatment, MTT solution (5 mg/mL) was added (10 µL/well), and plates were
incubated at 37°C for 4 h. Medium in the well was replaced by DMSO (150 µL), and slightly
shaken for 10 minutes. At 570 nm, the absorbance of suspension was determined by a microplate
reader (Infinite® 200 PRO NanoQuant – Switzerland Tecan). The formula: % inhibition =
(Abscontrol－Abscompound)/Abscontrol×100%, was used to calculate inhibition percentage. After the cells
2
1

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were treated by drugs of various concentrations, the IC values of tested compounds and AZD5438
5
0
were investigated using the prism statistical package (GraphPad Software, San Diego, CA, U.S.A.).
5.3.2 Flow-activating cell sorting analysis
575
The influence of analogue 10s on cell cycle of human colon cancer cell lines (HCT116) was
analyzed by flow cytometry. The cells were plated in 6-well plate and incubated overnight at 37 °C.
When the cells reached 70%-80% confluence, they were incubated in new medium with compound
10s at 0.35, 0.70 and 1.40 µM concentrations for 24 h. After treatment, cells from control and
treatment groups were digested, washed with phosphate buffer saline and fixed in 75% pre-cooled
ethanol at 4 °C overnight (over 18 h). Subsequently, they were washed with phosphate buffer saline
and stained with 50 µg/mL of propidium iodide supplemented with 50 µg/mL of RNase at 37 °C for
580
30 min, and the fluorescence intensity was then determined by flow cytometry.
5.3.3 Apoptosis analysis.
5
HCT116 cells were seeded in 6-well plates (3×10 cells/well), incubated in the presence or
585
absence of compound 10s at 0.35, 0.70 and 1.40 µM concentrations for 24 h. After incubation, cells
were harvested and incubated with 5 µL of Annexin-V/FITC in binding buffer (10 mM HEPES, 140
mM NaCl, and 2.5 mM CaCl at pH 7.4) at room temperature for 15 min. PI solution (10 µL) was
2
then added to the medium for another 10 min incubation. Almost 10000 events were collected for
each sample and analyzed with flow cytometry. The percentage of apoptotic cells was calculated
using FlowJo 7.6 analysis software.
590
5.3.4 In vitro CDK2 inhibitory assay
In vitro CDK2/CyclinA2 inhibitory assay was completed in the Huawei Pharmaceutical Com.
Ltd., China. Kinase assay was carried out as has been previously described [31]. All of the
enzymatic reactions were conducted at 30°C for 40 min. The 50 µL reaction mixture contains 40
595
mM Tris, pH 7.4, 10 mM MgCl , 0.1 mg/mL BSA, 1 mM DTT, 10 µM ATP, 0.2 µg/mL Kinase and
2
100 µM lipid substrate (0.1 mg/mL Histone H1, 10 µM ATP). 5µL of compound (in 10% DMSO)
was then added to the 50 µL reaction so that the final concentration of DMSO is 1% in all of
reactions. The assay was performed using Kinase-Glo Plus luminescence kinase assay kit, and the
kinase activity was determined by quantitating the amount of ATP remaining in solution following
a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP
600
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2

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present and is inversely correlated with the amount of kinase activity. Finally, the IC values were
5
0
calculated using nonlinear regression with normalized dose−response fit employing Prism
GraphPad sofeware.
5.3.5 Molecular modeling
605
610
615
Molecular docking studies were performed with the Surflex module in SYBYL 7.3 [32], and the
CDK2 complex with AZD5438 (PDB code: 6GUE) was chosed as our modeling system. The
selected molecule structure was initially prepared in Sybyl 7.3 and minimized using the Tripos
force field with a distance–dependent dielectric and powell gradient algorithm with a convergence
criterion of 0.001 kcal/molñÅ. In addition, the partial charges were calculated using
Gasteiger–Hückel method and all other parameters were given with their default values throughout
the docking procedure. The molecular interactions between ligand and receptor were visualized
with Pymol [33] software.
Acknowledgments
This present research was supported by the Natural Science Foundation of Guangdong
Province, China (Grant No. 2018B030311067), and the Science and Technology Program of
Guangzhou City, China (Grant No. 201707010198).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.ejmech.2019.xx.xxx.
620
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Highlights
•
A series of novel pyrimidine-based benzothiazole derivatives were synthesized. •
IC of analogue 10s were 0.45, 0.70, 0.92, 1.80 µM against HeLa, HCT116, PC-3,
5
0
and MDA-MB-231 cells, respectively. • 10s also possessed excellent CDK2/cyclin A
inhibitory activities with IC values of 15.4 nM, which was almost 3-fold potent than
5
0
AZD5438. • Further studies indicated compound 10s could induce cell cycle arrest
and apoptosis in a concentration-dependent manner.