Design, synthesis and biological evaluation of novel histone deacetylase1/2 (HDAC1/2) and cyclin-dependent Kinase2 (CDK2) dual inhibitors against malignant cancer

Article abstract of DOI:10.1016/j.ejmech.2020.112322

In the current study, we have designed and synthesized a series of novel histone deacetylase1/2 (HDAC1/2) and cyclin-dependent kinase2 (CDK2) dual inhibitors by integrating purine-based pharmacophore into the recognition cap group of CS055. The representative compound 14d with excellent antiproliferative activities towards five solid cancer cells, showed potent inhibitory activities against HDAC1, HDAC2 and CDK2 with IC₅₀ values of 70.7 nM, 23.1 nM and 0.80 μM, respectively. Besides, compound 14d could effectively block the cell cycle in the G2/M phase and induce apoptosis, which might be related to increasing intracellular ROS levels. Importantly, compound 14d exhibited desirable pharmacokinetic (PK) properties with the intraperitoneal bioavailability of 50.8percent in ICR mice, and potent in vivo antitumor activity in the HCT116 xenograft model. Therefore, compound 14d could be considered as a promising lead compound for the development of multitargeting anticancer agents.

Full text of DOI:10.1016/j.ejmech.2020.112322

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Design, synthesis and biological evaluation of novel histone Deacetylase1/2
(HDAC1/2) and cyclin-dependent Kinase2 (CDK2) dual inhibitors against malignant
cancer
Fan Yun, Chunhui Cheng, Sadeeq Ullah, Qipeng Yuan
PII:
S0223-5234(20)30291-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112322
EJMECH 112322
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To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2019
Revised Date: 7 April 2020
Accepted Date: 7 April 2020
Please cite this article as: F. Yun, C. Cheng, S. Ullah, Q. Yuan, Design, synthesis and biological
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Design, Synthesis and Biological Evaluation of Novel
Histone Deacetylase1/2 (HDAC1/2) and Cyclin-dependent
Kinase2 (CDK2) Dual Inhibitors against Malignant Cancer
Fan Yun†, Chunhui Cheng†, Sadeeq Ullah, Qipeng Yuan*
Key Laboratory of Biomedical Materials of Natural Macromolecules (Beijing University of Chemical Technology),
Ministry of Education. College of Life Science and Technology, Beijing University of Chemical Technology, 15
Beisanhuan East Road, Beijing 100029, China.
† These two authors contributed equally to this work.
*E-mail addresses of the corresponding authors: yuanqp@mail.buct.edu.cn

Graphic abstract

Design, Synthesis and Biological Evaluation of Novel
Histone Deacetylase1/2 (HDAC1/2) and Cyclin-dependent
Kinase2 (CDK2) Dual Inhibitors against Malignant Cancer
Fan Yun†, Chunhui Cheng†, Sadeeq Ullah, Qipeng Yuan*
Key Laboratory of Biomedical Materials of Natural Macromolecules (Beijing University of Chemical Technology),
Ministry of Education. College of Life Science and Technology, Beijing University of Chemical Technology, 15
Beisanhuan East Road, Beijing 100029, China.
† These two authors contributed equally to this work.
*E-mail addresses of the corresponding authors: yuanqp@mail.buct.edu.cn
ABSTRACT
In the current study, we have designed and synthesized a series of novel histone
deacetylase1/2 (HDAC1/2) and cyclin-dependent kinase2 (CDK2) dual inhibitors by
integrating purine-based pharmacophore into the recognition cap group of CS055.
The representative compound 14d with excellent antiproliferative activities towards
five solid cancer cells, showed potent inhibitory activities against HDAC1, HDAC2
and CDK2 with IC50 values of 70.7 nM, 23.1 nM and 0.80 μM, respectively. Besides,
compound 14d could effectively block the cell cycle in the G2/M phase and induce
apoptosis, which might be related to increasing intracellular ROS levels. Importantly,
compound 14d exhibited desirable pharmacokinetic (PK) properties with the
intraperitoneal bioavailability of 50.8 % in ICR mice, and potent in vivo antitumor
activity in the HCT116 xenograft model. Therefore, compound 14d could be
considered as a promising lead compound for the development of multitargeting
anticancer agents.
Key words: Histone deacetylase1/2, Cyclin-dependent kinase2, Enzyme inhibitory
activity, Pharmacokinetic properties, In vivo antitumor activity
1. Introduction
Single target drugs often lead to functional compensation of other pathways in
the cancer networks, accompanied by inevitable resistance and side effects.
Therefore, single-target drugs fail to achieve the desired therapeutic efficacy in
complex tumor disease[1-5]. The combination drug treatment can overcome the
limitations, and be widely applied for clinical work. However, this combination
treatment causes the risks including drug-drug interactions, unforeseen
pharmacokinetic (PK) properties and weak patient compliance[6, 7]. On the contrary,

a single molecule acting on multiple targets can simultaneously modulate different
pathways of the tumor to acquire favorable therapeutic efficacy and reduce adverse
effects[8-12]. Consequently, developing multitarget drugs provided an effective
strategy for cancer therapy.
Epigenetic regulation plays an essential role in the occurrence, development and
metastasis of cancer and other human diseases[13]. Histone deacetylases (HDACs)
and histone acetyltransferases (HATs) are a pair of epigenetic antagonistic enzymes
that modulate the acetylation status of lysine residues in histones. Overexpressed
HDACs enhance the electrostatic interaction between histone and DNA, leading to
chromatin pyknosis and transcriptional termination, and finally cause the
tumorigenesis [14-16]. Thus, HDAC inhibitors (HDACIs) have been considered as
powerful antineoplastic agents for cancer therapy[17]. Up to date, four HDAC
inhibitors have been approved by the U.S. FDA, namely, vorinostat (SAHA, Figure
1), romidepsin (FK228, Figure 1), belinostat (PXD-101 Figure 1) and panobinostat
(LBH-589 Figure 1). Chidamide (CS055, Figure 1) has been launched on the market
of China in 2014[18-22]. However, most of the marketed HDAC inhibitors are in
clinical application for hematological malignancies, but show weak inhibition toward
solid tumors. Many researchers have focused on the discovery of HDAC-based
muititargeting agents.
The precise cell division cycle is one of the most fundamental requirements for
all living cells[23]. The cell cycle commonly progresses through four sequential
phases: G1, S, G2 and M phases. Cyclin-dependent kinases (CDKs) that belong to a
serine-threonine protein kinases family, could form heterodimeric structures with
relevant cyclins to control the cell cycle. The interaction between CDKs and cyclins
are closely associated with the correct timing and order of each phase[24]. CDK1,
CDK2, CDK3, CDK4 and CDK6 directly regulate the cell cycle, and CDK7, CDK8,
CDK9 and CDK12 are involved in governing transcription[25-29]. CDKs that
aberrantly expressed in various durable diseases and malignancies have emerged as a
key target for cancer treatment [30-33]. Up to now, two CDK inhibitors, palbociclib
(Pfizer Figure 1) and ribociclib (Novartis Figure 1), have been launched on the
market for the treatment of breast cancer and HER2-negative advanced breast cancer
respectively [34-36].
Recent studies demonstrated that vorinostat (SAHA) in combination with
flavopiridol, a pan-CDK inhibitor, was utilized for the treatment of neuroblastoma
and melanoma[37]. Yang’s group further confirmed that a single molecule targeting
HDAC1 and CDK4/9 was achieved for the treatment of cancer[38]. Based on this
evidence, we conceived that designing a dual-action inhibitor using the
pharmacophore of CDK inhibitor in combination with the HDAC targeting drug is a
compelling strategy for oncology disease treatment. Roscovitine (CYC202), a
purine-based CDK2 inhibitor, showed diverse effects on cell proliferation, cell cycle
progression and apoptosis induction towards different cancer cells[39-41]. CYC202
has been in the clinical trial.[42, 43]. The chemical scaffold of HDAC inhibitors
contains three parts: (1) a surface recognition cap group, which interacts with the
surface of the active site pocket; (2) a zinc binding group (ZBG) such as hydroxamic

acid and aminobenzamide, which is the core pharmacophoric unit of HDAC inhibitors;
(3) a linker to connect the cap group and ZBG [44, 45]. We conceived that the
purine-based pharmacophore of CYC202 was merged with benzamide moiety to
obtain a single inhibitor targeting CDK2 and HDAC (Figure 2). Herein, we designed
and synthesized a series of dual CDK2/HDAC inhibitors, and then evaluated for their
antitumor activity.
Figure 1. The structures of approved HDAC and CDK inhibitors.
Figure 2. Design of HDAC-CDK2 dual inhibitors. (A) The binding modes of HDAC
and CDK2 inhibitors. (B) The design strategy of HDAC-CDK2 dual inhibitors.
2. Results and discussion
2.1. Chemistry

The synthetic procedures of final compounds were illustrated in Schemes 1-3.
Products 8a-8g and 11a-11f were prepared according to Scheme 1. Starting from 1
and various relative amines 2, compounds of type 3 was synthesized in microwave
irradiation. Then nucleophilic substitution of compounds of type 3 with various alkyl
bromides provided compounds of type 4, which was condensed with ethanolamine to
give compounds of type 5. The treatment of compounds of type 5 with methyl
4-(bromomethyl)benzoate and methyl (Z)-3-(4-(bromomethyl)phenyl)acrylate
afforded compounds of type 6 and compounds of type 9, respectively. Hydrolysis of
compounds of type 6 and compounds of type 9 gave the acetic compounds of type 7
and 10, followed by condensation with o-phenylenediamine in the presence of BOP
and triethylamine to yield the final products 8a-8g and 11a-11f.
Products 14a-14i and 17a-17h were synthesized according to the synthetic routs
in Scheme 2. Commercially available relative amines 2 were condensed with starting
material 1 to give intermediates of type 3. 14a-14i and 17a-17h were obtained by
intermediates of type 3 with three steps, which was similar to the synthesis of
compounds of type 8 and 11. Similarly, the treatment of 1 with 18 in the presence of
triethylamine and butanone afforded intermediate 19, which was further reacted with
various alkyl bromides to obtain compounds of type 20. As outlined in Scheme 3,
final products 22a-22f were prepared by a similar protocol with compounds 8a-8g in
Scheme 1.

Scheme 1. Reagents and conditions: (a) relative amines (compound 2), microwave,
butanone, 70 °C; (b) K2CO3, KI, butanone, reflux; (c) microwave, 170 °C; (d) Cs2CO3,
acetonitrile, reflux; (e) 1, NaOH, MeOH, reflux; 2, HCl, 0 °C; (f)
benzene-1,2-diamine, BOP, Et3N/DMF, room temperature, overnight;
Scheme 2. Reagents and conditions: (a) oil bath, butanone, 90 °C; (b) K2CO3, DMF,

room temperature, overnight; (c) 1, NaOH, 1,4-dioxane, reflux, 2, HCl, 0 °C; (d)
benzene-1,2-diamine, BOP, Et3N/DMF, room temperature, overnight.
Scheme 3. Reagents and conditions: (a) triethylamine, butanone, 90 °C; (b) K2CO3,
DMF, 40-70 °C; (c) 1, NaOH, 1,4-dioxane, reflux; 2, HCl, 0 °C; (d)
benzene-1,2-diamine, BOP, Et3N/DMF, room temperature, overnight.
2.2. Biological evaluation
2.2.1. In vitro antiproliferation and enzyme inhibition assays
Table 1
In vitro inhibition of HDAC1 and CDK2 and cellular antiproliferative activity of
compounds 8a-8g, 11a-11f, 14a-14i, 17a-17h and 22a-22g;
HCT116
HepG2
H460
HDAC1
CDK2
(inhib%) a
(inhib%) a
(inhib%) a
(inhib%) a (inhib%) a
Compds
2 µM 8 µM 2 µM 8 µM 2 µM 8 µM
58.86 71.87 30.57 63.09 56.89 62.04
72.89 74.88 46.60 70.67 27.70 51.27
10 µM
84.92
82.92
43.57
49.15
36.82
44.87
10.52
24.63
56.32
25.38
30.29
10 µM
74.66
72.51
38.42
50.24
39.20
79.40
23.25
34.52
47.73
24.39
24.03
8a
8b
8c
8d
8e
8f
8g
11a
11b
11c
11d
6.85
7.12
18.82
63.44
6.58
1.48
50.23 15.02 34.27
60.53 28.94 43.22
29.16 40.59 32.59 63.19 36.10 49.16
75.15 76.93 37.51 66.88 41.16 56.13
9.41
4.76
6.58
9.04
25.82
4.85
36.22 33.48 41.59
10.85 20.45 30.42 25.66 51.11
51.88 27.88 59.34 23.14 34.22
9.19
9.67
2.93
14.75 37.05 46.07
18.75 52.03 59.70
10.49 17.34

2.53
18.51 27.87
41.70
1.46
6.34
40.77 41.01 53.14
49.86 53.01 61.06
17.68
21.81
61.32
43.90
84.03
88.65
76.04
75.33
93.24
80.50
37.43
23.61
41.80
22.51
14.83
4.87
25.69
40.32
28.24
90.90
89.72
94.71
9.56
31.81
20.04
83.04
54.28
90.68
79.71
96.18
90.21
94.89
76.44
18.53
38.20
43.75
10.71
4.87
11e
11f
56.19 78.36 29.85 66.68 10.33 66.97
69.49 73.74 11.51 41.22 18.14 30.76
71.42 83.52 49.57 74.80 42.66 77.75
76.30 81.50 76.16 81.98 67.34 82.49
68.31 78.35
70.94 78.60
14a
14b
14c
14d
14e
14f
14g
14h
14i
17a
17b
17c
17d
17e
17f
17g
17h
22a
22b
22c
22d
22e
22f
7.55
1.22
62.83 44.72 61.17
65.34 11.84 40.92
77.68 80.70 56.92 71.31
71.58 72.77 60.37 74.99 13.45 55.05
1.60 46.59 1.88 31.21 47.70 62.44
12.94 15.81 35.75 49.32 7.11 29.40
15.03 33.57 42.95 31.27 51.27
5.05
46.96
0.27
2.85
7.75
4.24
17.40 11.51 37.67
30.91 16.40 41.93
26.87
4.62
2.05
8.24
1.24
2.10
8.09
49.06
28.67
10.77
37.92
66.68
24.92
8.73
32.19
7.58
17.82 55.39 18.99 40.85
2.80
3.34
75.61 80.60 54.72 74.86 42.39 57.41
73.20 76.04 63.77 74.41 54.15 80.49
74.59 75.14 44.99 65.01 47.30 67.69
30.61
43.05
19.32
87.84
73.17
76.44
6.32
5.23
18.69
16.22 37.64
3.83 42.65
4.89
40.89
6.80
19.60 19.66 23.67
28.48 29.62 12.36 14.10 31.01 39.19
7.70
6.39
11.42
27.61
29.53
96.39
12.27
11.33
21.17
11.45
11.92
5.01
8.52
8.36
5.32
43.77 46.39
34.24 43.70 51.16
22g
CS055
b
65.68 73.41
40.60
1.73
7.73
38.24
15.56
-
b
Roscovitine 23.50 29.10 12.01 22.11
-
81.32
a The values represent the means of three separate experiments with SD less than 10 %.
b Not tested.
Table 2
Antitumor activities (IC50, µM) of representative compounds against 5 different
cancer cell lines a.
IC50 (µM)
HepG2
8.66±1.27
4.09±0.88
6.02±1.33
9.80±2.01
Compds
H460
15.00±2.80 6.82±1.01
8.78±1.09 4.48±0.53
18.06±9.07 8.70±1.97
6.58±1.21 6.08±0.92
A375
HCT116
2.12±0.42
1.19±0.37
0.96±0.29
Hela
6.78±0.37
3.29±0.32
8.70±2.05
8a
8b
8f
14a
14b
14c
14d
14e
2.34±0.64 11.32±1.79
18.39±6.66 18.54±5.56 30.05±7.32
1.25±0.42
1.07±0.28
0.58±0.18
1.06±0.36
>20
5.74±0.97
1.59±0.44
2.80±0.35
0.47±0.14
4.18±0.94
0.86±0.28
7.72±1.25
2.92±0.25
1.05±0.23
6.77±0.71
11.16±2.06 5.83±0.72

7.40±0.68
6.09±1.29
6.16±1.59
5.78±0.53
3.41±0.50
4.94±0.44
4.24±0.80
2.68±0.38
1.60±0.23
2.79±0.42
2.17±0.30
2.88±0.27
5.18±0.53
2.82±0.72
2.01±0.67
3.43±0.51
3.32±0.72
2.99±0.56
0.95±0.32
0.45±0.19
0.31±0.16
0.85±0.22
0.80±0.32
0.72±0.26
4.50±0.32
2.03±0.32
1.17±0.13
3.02±0.29
2.13±0.13
3.03±0.36
14f
14g
14h
22a
22b
22c
CS055
13.34±2.10 6.72±0.63 11.67±1.62
1.49±0.56 10.97±2.18
6.76±0.54 11.21±1.28
Roscovitine 11.90±2.21 11.79±0.88 8.53±0.87
The values represent the means of three separate experiments with SD less than
a
10 %.
Figure 3. Cell morphology of five cancer cell lines was treated with 14d at the
indicated concentration or DMSO (0.1 %) for 48 h. Detection by microscope (10×).
The results of in vitro antiproliferation and enzyme inhibition assays were listed
in Table 1 with CS055 and roscovitine as the reference drugs. The target compounds
were examined at the concentrations of 8 µM and 2 µM against HCT116 cell line
(human colorectal cancer), HepG2 cell line (human liver cancer) and H460 cell line
(human lung cancer). We also tested the inhibitory activities of compounds at 10µM
towards HDAC1 and CDK2. The introduction of different substituents at C-2, C-6
and N-9 positions of 2,6-dichloro-9H-purine ring possessed considerable effects on
antiproliferative activity. Compounds 11a-11f and 17a-17h containing
a
phenylacrylamide group from series I and II decreased the potency relative to 8a-8g
and 14a-14i with a benzamide group against three cancer cells. When the cyclopentyl
group was at the R2 position, compound 8f with a phenylamino group at the R1
position was more active than compounds 8e and 8g. Among compounds 14a-14i
(series II), compounds 14a and 14c-14h revealed potent antiproliferative activity,
however, the bulk substituted groups, such as (4-(pyridin)-2-yl) phenyl (14b) and

3-(trifluoromethoxy)phenyl (14i) at R1 position caused an obvious reduction of
antiproliferative activity. Compounds 22a-22c revealed higher cell growth inhibition
compared with other compounds from series III. Moreover, the antiproliferative
activities towards three cell lines were consistent with HDAC1 and CDK2 inhibitory
activities. For example, compounds 22a-22c with better cell growth inhibition
exhibited more potent inhibitory activity against HDAC1 and CDK2 compared to
compounds 22d-22g.
As a consequence, the IC50 values of compounds that possessed superior or
comparable inhibitory activity relative to positive controls, were tested toward five
solid tumor cell lines: HCT116 (colon cancer), HepG2 (liver cancer), H460 (lung
cancer), Hela (cervical cancer), A375 (malignant melanoma cancer) by the CCK-8
assay. The results were presented in Table 2. Compound 8b exhibited higher
inhibitory activity, with IC50 values ranging from 1.19 to 8.78 µM against five cell
lines compared with other compounds from series I. Among compounds 22a-22c
from series III, 22b showed the most potent active, particularly for HCT116 cell line
(IC50=0.80 µM). Among the tested compounds 14a-14h (series II), compound 14b
showed strong activity against HCT116 cells with IC50 of 1.25 µM but had a poor
inhibitory effect on the other cancer cells. Compound 14c with a phenylamino group
at the R1 position was more active than compound 14e with a benzylamino group.
Compound 14h, in which the 4-methylpiperazine group was at the R1 position, had
better antiproliferative activities compared to other heterocyclic derivatives 14f and
14g. Compound 14d with a p-methylphenyl group at the R1 position showed excellent
antiproliferative activities against H460, A375, HepG2, HCT116 and Hela cells with
IC50 values of 1.59, 0.47, 0.86, 0.58 and 1.05 µM, respectively. These data indicated
that compound 14d was the greatest active among all the compounds from series I, II
and III, and was considered for further evaluations. In the following study, 14d was
examined for the cell morphology assay towards all cell lines. The results listed in
Figure 3 indicated that 14d could alter cancer cell morphology at low concentrations.
2.2.2. In vitro enzyme inhibitory activity assay
Table 3
Inhibitory activities of target compound 14d against CDK2 and HDAC1/2.
Compds
IC50 of HDAC1 IC50 of HDAC2 IC50 of CDK2
(nM) a
(nM) a
(μM) a
70.7±5.54
260±20.0
23.1±1.05
66.5±3.44
0.80±0.06
14d
CS055
Roscovitine
b
-
b
b
-
-
0.11±0.08
a Values are the means of at least two separate determinations and expressed as
mean ±SD.
b Not tested.
The representative compound 14d was investigated for HDAC1, HDAC2 and
CDK2 inhibitory activities with CS055 and roscovitine as reference drugs. As
depicted in Table 3, 14d displayed remarkable inhibitory activities against HDAC1

and HDAC2, which was above 3-fold and 2-fold more potent than CS055. Moreover,
compound 14d could effectively inhibit CDK2 with IC50 of 0.80 μM, showing a
similar submicromolar level of CDK2 inhibition to roscovitine (IC50=0.11 μM). From
these results, we can determine that compound 14d could possess HDAC1/2 and
CDK2 inhibitory activity.
2.2.3. Molecular docking
Figure 4. Compound 14d docked into CDK2 (PDB code: 1PYE), HDAC1 (PDB code:
4BKX) and HDAC2 (PDB code: 4LXZ). Compound 14d was represented by grey
carbon in the stick. (A) Predicted binding mode of 14d with CDK2. (B) Proposed
binding mode of compound 14d in the active site of HDAC1. (C) Predicted binding
mode of 14d in the active site of HDAC2. (D) 14d interacted with the active site of
CDK2. (E) Molecular surface of HDAC1 binding pocket with 14d. (F) 14d interacted
with the active site of HDAC2.
To demonstrate the potential binding modes of highly active compound 14d with
CDK2, HDAC1 and HDAC2, computational molecular docking assays were
accomplished. The crystal structures of HDAC1, HDAC2 and CDK2 were obtained
from the RCSB PDB (Protein Data Bank) website. As shown in Figure 4A and 4D,
14d could be closely integrated with CDK2 in the ATP binding site. The
9H-purin-6-amine moiety of 14d formed two potential hydrogen bonds with amino
acids (LEU83 and PHE82), which were critical for binding to CDK2. Besides, the
benzene-1,2-diamine moiety of 14d could interact with CXL13 and LYS33 of CDK2
to form another two hydrogen bonds. The binding modes of compound 14d with
HDAC1 were shown in Figure 4B and 4E. As expected, compound 14d were buried
into the narrow active-site tunnel, and the 2-aminobenzamide group chelated with
zinc ion, which was important for HDAC1 inhibitory effects of compound. The zinc
binding group of 14d was involved in the H-bonding network with HIS140, HIS141,
ASP176, GLY149 and ZN600 at the HDAC1 active site. Similarly, compound 14d
projected into the active-site tunnel of HDAC2 and chelated with zinc ion (Figure

4F). It was observed that 14d formed five hydrogen bonds with ASP181, HIS145,
HIS146, GLY154, TYR308 (Figure 4C). It might be the reason why compound 14d
had better inhibitory activity for HDAC2 than that for HDAC1.The computational
docking results might provide an effective explanation for the binding affinity of 14d
for HDAC1/2 and CDK2.
2.2.4. Migration assay
Figure 5. Inhibition of 14d on migration was determined by the scratch test. A375
and H460 cells were treated with 14d, CS055, roscovitine and blank (DMSO) for 48 h.
At least two independent experiments were conducted.
To confirm the inhibition of 14d to cell scratch, A375 cells and H460 cells were
seeded into 12-well plates, and then were treated with 14d, CS055 and roscovitine for
48 h, respectively. The results of the wound healing test were demonstrated in Figure
5. As expected, 14d significantly inhibited the migration of H460 and A375 cells
within 48 h compared to the positive drugs CS055 and roscovitine.
2.2.5. Cell cycle analysis
Figure 6. The effects of compound 14d on the cell cycle assay. (A) A375 cell line. (B)
HCT116 cell line. (C) H460 cell line. (D) Hela cell line.
In the following study, compound 14d was assessed for its effect on the cell
cycle towards A375 cells, HCT116 cells, H460 cells and Hela cells. Cells were
treated with 14d, CS055 and roscovitine at concentrations of 2, 1 and 0.5 μM for 24 h

respectively, then stained with propidium iodide (PI), and finally analyzed by flow
cytometry. As presented in Figure 6, compared to the blank (DMSO), 14d induced a
loss of G0/G1 phase cells and an increase of G2/M phase cells in four cancer cells.
14d led to an apparent accumulation of A375, HCT116 and Hela cells in G2/M phase
at low concentration of 0.5 μM (A375, the percentage from 13.70 (0.1 % DMSO) to
57.03 %; HCT116, from 27.46 to 76.99 %; Hela, from 7.89 % to 51.85 %. See
Supporting Information). In general, compound 14d was able to significantly block
the cell cycle and showed a similar blocking pattern to the reference drugs.
2.2.6. Cell apoptosis analysis
Figure 7. Effects of 14d, CS055 and roscovitine on the induction of cell apoptosis in
four cancer cell lines. Cells were exposed to compound 14d, CS055 and roscovitine
for 48 h, and were analyzed by flow cytometry with FITC-Annexin V/PI staining.
These data represent two separate experiments.
To evaluate the efficacy of compound 14d to cause cell death through apoptosis,
we performed cell apoptosis assay against A375 cell line, HCT116 cell line, H460 cell
line and Hela cell line. Cells were treated with compound 14d, CS055 and roscovitine
as positive drugs for 48 h. The flow cytometry analysis was shown in Figure 7. 14d
could promote cancer cell apoptosis in a dose-dependent manner. Compared with the
blank group, 14d was more effective in apoptosis induction towards four cancer cells.
Besides, the apoptosis rates of A375, HCT116, H460 and Hela cells by 14d were
91.99 %, 89.60 %, 59.10 % and 22.36 % respectively, which were higher than that of
CS055 and roscovitine at the concentration of 2 μM. 14d were more active in the
apoptosis induction of A375 and HCT116 cells with the apoptosis rates above 70 %.
Overall, 14d could lead to cell death by promoting apoptosis.

2.2.7. Immunofluorescence assay
Figure 8. A375 cells were treated with 14d at 1 μM or DMSO for 12 h.
Immunofluorescence images of (A) CDK2, DAPI and (B) AcH3K9, DAPI staining
and merge in untreated or treated cells.
Since the computational molecular docking assays illuminated strong affinities
between 14d with HDAC and CDK2, we evaluated the effect of 14d on the inhibition
of CDK2 and the deacetylation of histone H3K9. A375 cells were selected as a
representative model because A375 cells had good cell adherence property and
compound 14d exhibited a favorable antiproliferative effect on them. As shown in
Figure 8, A375 cells were treated with 14d at the concentration of 1 μM for 12 h.
Compared with the blank (DMSO), 14d significantly inhibited CDK2 (Figure 8A)
and increased the acetylation level of histone H3 (Figure 8B). The
immunofluorometric analysis illustrated that compound 14d could inhibit CDK2 and
HDAC activity, causing cancer cell death.
2.2.8. Intracellular ROS assay
Figure 9. Effects of 14d (1 μM) and blank (DMSO) on the ROS generation assay
in the A375 cell line.

Reactive oxygen species (ROS) play an important role in regulating cell
proliferation, angiogenesis and other cell processes. It seems necessary to classify that
ROS levels in cancer cells are slightly higher than those in normal cells due to the
relatively exuberant metabolism of cancer cells. However, the high levels of ROS
could irreversibly damage DNA, ultimately leading to cancer cell death [46-48]. A375
cells were treated with 14d at the concentration of 1 μM for 24 h. We applied a
ROS-sensitive fluorescent dye (DCFH-DA) to detect intracellular ROS levels. As
shown in Figure 9, after treatment with 14d, cells presented a bright green
fluorescence signal compared with the blank, indicating that 14d strongly increased
ROS levels in A375 cells. Accordingly, 14d might cause cancer cell death by
improving intracellular ROS levels.
2.2.9. In vivo pharmacokinetic (PK) assay
Table 4
Preliminary pharmacokinetic (PK) profile of 14d in ICR mice (N=9, male).
Dose
(mg/kg)
4
adminis
tration
IV
T
(h)
1.48
2.84
Tmax
(h)
-
Cmax
AUC0-t
(ng h/mL)
2850
MRT0-t
(h)
0.563
4.54
CL
(mL/ (min kg))
23.3
F
½
(ng/mL)
(%)
a
a
a
-
-
a
20
IP
2
1360
7240
-
50.8
a Not tested.
Encouraged by its excellent in vitro antiproliferative activity, the
pharmacokinetic (PK) profile of compound 14d was evaluated in ICR mice. 14d was
administered intravenously (IV) at 4 mg/kg and intraperitoneally (IP) at 20 mg/kg. As
presented in Table 4, after intravenous administration, the half-life (T1/2) was 1.48 h
and AUC0-t was 2850 ng h/mL. The clearance (CL) was found to be 23.3 mL/(min kg).
When administrated intraperitoneally, the half-life was increased to 2.84 h, the
bioavailability (F) was 50.8 %, and AUC0-t reached 2850 ng h/mL. These data
indicated that compound 14d exhibited desirable pharmacokinetic (PK) properties.
2.2.10. In vivo antitumor activity

Figure 10. Antitumor efficacy of compound 14d in the HCT116 xenograft model. (A)
Body weight and (B) tumor volume measurements after 21 days of 14d. (C)
Comparison of the final tumor weights in each group after the 21 days treatment
period of 14d. (D) Image of dissected HCT116 tumor tissues.
The in vivo antitumor effect of compound 14d was evaluated in the HCT116
nude mice xenograft model. When the tumor volumes reached 100-300 mm3, BALB/c
nude mice were treated by intraperitoneal injection (IP) with 14d at 25, 50 and 100
mg/kg once daily (QD) for 21 days. As presented in Figure 10A, there was no
significant change in the body weight of all mice. 14d could significantly inhibit the
tumor growth compared to the blank. The tumor growth inhibitions (TGI) were 28 %,
40 % and 44 % at doses of 25, 50 and 100 mg/kg, respectively (Figure 10B-D). These
results demonstrated that compound 14d showed potent in vivo antitumor activity in
the HCT116 xenograft model.

3. Conclusion
In summary, novel HDAC1/2 and CDK2 dual inhibitors were designed,
synthesized and evaluated for their antitumor activity. Among the derivatives,
compound 14d showed excellent antiproliferative activity towards H460 cells, A375
cells, HepG2 cells, HCT116 cells and Hela cells with IC50 values of 1.59, 0.47, 0.86,
0.58 and 1.05 µM, respectively. Compound 14d also exhibited potent inhibitory
activity against HDAC1, 2 and CDK2 with IC50 values of 70.7 nM, 23.1 nM and 0.80
μM, respectively. Furthermore, the flow cytometry assay showed that 14d arrested the
cancer cell cycle in the G2/M phase, and significantly induced apoptosis. The
immunofluorometric analysis indicated that 14d cause an obvious inhibition of CDK2
and an increase of the acetylation of H3. In addition, compound 14d significantly
inhibited the migration of A375 and H460 cells. The in vivo antitumor activity assay
showed that compound 14d possessed desirable pharmacokinetic (PK) properties in
ICR mice, and potent in vivo antitumor activity in the HCT116 xenograft model
(TGI=44 %). The current study provided a new strategy for the development of
multitargeting antitumor drugs.
4. Experimental Section
4.1. Chemistry
Unless otherwise specified, all starting materials were obtained commercially
and were used without further purification.1 H NMR and 13 C NMR spectra were
determined in MeOD-d4 or DMSO-d6 at 400 MHz. High-resolution mass spectra
(HRMS) were measured by an Agilent 6540 UHD Q-TOF mass spectrometer.
Microwave reactions were performed by a Preekem NOVA-2S reactor. Thin layer
chromatography (TLC) analysis was performed on precoated plates with silica gel
F254 (Qingdao Haiyang Chemical Co. Ltd). The purity of each final compound was
≥90%. It was tested by high-performance liquid chromatography (HPLC) on a
Shimadzu LC-20AT system (column, C18, 5 mm, 4.6 mm×250 mm; UV wavelength,
254 nm; temperature, 35 °C; mobile phase, gradient elution of methanol/ H2O (0.1 %
H3PO4); flow rate, 1.0mL/min). The flow rate of solvent A with the gradient started at
40% of methanol and increased to 90 % over 25 min for 14c-14h, 14i, 17g, 17h and
22a-22d. The 5 % of methanol was run at a flat rate of 1.0 mL/min over 10 min for
the rest of compounds. Analyses indicated by the symbols of the elements or
functions were within 0.4 % of the theoretical values.
4.1.1 General method for the synthesis of 8a-8g and 11a-11f;

Step-1: 15 mL n-BuOH, 2,6-dichloro-9H-purine 1 (1.0 equiv.) and relative amines 2
(3.0 equiv.) were added in a microwave reaction tube. The vessel was heated to 70 °C
for 1 h. The reaction was cooled to room temperature, the suspension was filtered and
washed with methanol to afford the compound 3 as a yellow solid.
Step-2: To a 100 mL, one-necked, round-bottomed flask, sequentially charged with
compound 3 (1.0 equiv.), 1-bromobutane (1.2 equiv.), K2CO3 (2.0 equiv.), KI (2.0
equiv.) and 30 mL of 2-Butanone. The reaction was heated at reflux for 3-6 h. When
TLC analysis revealed the completion of the reaction, the mixture was cooled to room
temperature and filtered. Then the solvent was removed under reduced pressure. The
white solid crude compound 4 was taken to the next step directly without further
purification.
Step-3: In a microwave tube, compound 4 was suspended in opportune
2-aminoethan-1-ol. The sealed tube was heated in the microwave at 170 °C for 10
min, the solution was poured into water with vigorous stirring overnight. The
resulting solid was filtered off to provide compound 5 as a white solid.
Step-4: To a 100 mL, one-necked, round-bottomed flask, sequentially charged with
compound
5
(1.0 equiv.), methyl 4-(bromomethyl)benzoate or methyl
(Z)-3-(4-(bromomethyl)phenyl)acrylate (1.2 equiv.), Cs2CO3 (2.0 equiv.) and 30 mL
of acetonitrile. The reaction was heated at reflux for 1-2 h. After TLC analysis
showed the completion of the reaction, the mixture was cooled to room temperature,
and filtered through Celite. The solution was concentrated in vacuo to get compound
6 or compound 9. Without further purification, the compound 6 or compound 9 was
dissolved in 1,4-dioxane (15 mL). An aqueous sodium hydroxide solution (2 mol/L,
10 equiv.) was added. The reaction was heated at 80 °C for 1 h. When TLC analysis
indicated the completion of the reaction. the mixture was cooled to room temperature
and acidified to with 2N HCl to pH<7. The obtained solid was filtered to afford
compound 7 or compound 10.
Step-5: A solution of the compound 7 or compound 10 (1 equiv.), DMF (10 mL),
BOP (1.2 equiv.) and Triethylamine (4.0 equiv.) was stirred for 10 min at room
temperature. Benzene-1,2-diamine (1.2 equiv.) was added into the reaction. The
reaction was stirred overnight at room temperature. The reaction was monitored by
TLC. After completion of the reaction, the reaction was poured into 500 mL water
with vigorous stirring for 30 min, the obtained solid was filtered to afford compound
8 or compound 11. The final product was purified by silica gel chromatography.
4.1.1.1
N-(2-aminophenyl)-4-((2-((9-butyl-6-((3-chlorophenyl)amino)-9H-purin-2-yl)amino)
ethoxy)methyl)benzamide (8a)
Light yellow solid; Yield 63.7 %. HPLC Rt = 2.748 min. H NMR (400MHz,
1
MeOD-d4): δ=7.88(2H, d, J=7.7Hz), 7.61(1H, s), 7.47-7.45(2H, m), 7.31-7.17(5H, m),
7.07(1H, t, J=7.1Hz), 6.90-6.88(1H, d, J=7.4Hz), 6.79(1H, t, J=8.1Hz), 5.66(2H, s),
4.08(2H, t, J=7.1Hz), 3.63(2H, s), 3.43(2H, s), 1.82(2H, m), 1.35(2H, m), 0.97(3H,
J=7.1Hz); 13C NMR (100MHz, MeOD-d4): δ=167.3, 159.1, 154.1, 153.6, 145.4,
142.8, 142.3, 138.1, 133.7, 133.0, 129.6, 127.6, 127.1, 127.0, 126.2, 125.8, 125.2,

123.9, 118.3, 117.3, 114.1, 61.2, 53.9, 43.8, 42.6, 31.6, 19.4, 12.5ppm. HRMS (ESI):
Calculated for C31H33ClN8O2 m/z (M+H)+: 585.2494; found: 585.2498.
4.1.1.2
N-(2-aminophenyl)-4-((2-((9-butyl-6-(phenylamino)-9H-purin-2-yl)amino)ethoxy)me
thyl)benzamide (8b)
1
Light yellow solid; Yield 83.2 %; HPLC Rt = 2.740 min. H NMR (400MHz,
MeOD-d4): δ=7.88-7.86(2H, m), 7.57(2H, d, J=8.2Hz), 7.46-7.44(4H, m),
7.32-7.23(4H, m), 7.16(1H, m), 7.09-7.05(1H, m), 6.90-6.75(2H, m), 5.65(2H, s),
4.06(2H, t, J=7.1Hz), 3.61(2H, m), 3.41(2H, t, J=7.1Hz), 3.32(2H, m), 1.85-1.77(2H,
m), 1.38-1.30(2H, m), 0.97(3H, t, J=7.5Hz); 13C NMR (100MHz, MeOD-d4): δ=167.3,
159.1, 154.4, 153.3, 144.1, 143.1, 142.3, 137.7, 132.8, 128.5, 127.7, 127.4, 127.1,
126.2, 126.0, 123.9, 118.3, 117.3, 114.0, 61.3, 54.3, 43.8, 42.6, 31.6, 19.4, 12.5ppm;
HRMS (ESI): Calculated for C31H34N8O2 m/z (M+H)+: 551.2884; found: 551.2882.
4.1.1.3
N-(2-aminophenyl)-4-((2-((9-isopropyl-6-(phenylamino)-9H-purin-2-yl)amino)ethox
y)methyl)benzamide (8c)
Light yellow solid; Yield 65.2 %; HPLC Rt = 2.723 min. H NMR (400MHz,
1
MeOD-d4): δ=7.88-7.86(2H, d, J=8.4Hz), 7.65(1H, s), 7.46-7.44(2H, d, J=7.7Hz),
7.34-7.28(2H, m), 7.23-7.16(4H, m), 7.09-7.05(1H, m), 6.91-6.88(1H, m),
6.78-6.74(1H, m), 5.66(2H, s), 4.68 (1H, m), 3.62(2H, t, J=5.1Hz), 3.41(2H, t,
J=5.0Hz), 1.55-1.53(6H, d, J=6.7Hz); 13C NMR (100MHz, MeOD-d4): δ=167.4,
159.0, 154.4, 152.9, 144.2, 143.2, 142.3, 135.2, 132.8, 128.5, 127.7, 127.4, 127.1,
127.0, 126.2, 125.9, 123.9, 118.3, 117.3, 114.4, 61.3, 54.3, 46.3, 43.7, 21.1ppm;
HRMS (ESI): Calculated for C30H32N8O2 m/z (M+H)+: 537.2727; found: 537.2715.
4.1.1.4
N-(2-aminophenyl)-4-((2-((6-((3-chlorophenyl)amino)-9-isopropyl-9H-purin-2-yl)am
ino)ethoxy)methyl)benzamide (8d)
Light yellow solid; Yield 33.2 %; HPLC Rt = 2.727 min. H NMR (400MHz,
1
MeOD-d4): δ=7.90-7.88(2H, d, J=8.1Hz), 7.69(1H, s), 7.47-7.45(2H, d, J=8.0Hz),
7.30-7.05(6H, m), 6.90-6.88(1H, d, J=7.5Hz), 6.76(1H, t, J=7.4Hz), 5.66(2H, s),
13
4.69(1H, m), 3.64(2H, m), 3.44(2H, m), 3.32(2H, s), 1.56-1.54(6H, d, J=6.8Hz); C
NMR (100MHz, MeOD-d4): δ=167.3, 159.0, 154.1, 153.2, 145.5, 142.8, 142.3, 135.6,
133.7, 133.0, 129.6, 127.6, 127.6, 127.1, 126.9, 126.2, 125.8, 125.1, 123.9, 118.3,
117.3, 114.5, 61.2, 53.9, 46.4, 43.8, 21.1ppm; HRMS (ESI): Calculated for
C30H31ClN8O2 m/z (M+H)+: 571.2337; found: 571.2328.
4.1.1.5
N-(2-aminophenyl)-4-((2-((6-((3-chlorophenyl)amino)-9-cyclopentyl-9H-purin-2-yl)a
mino)ethoxy)methyl)benzamide (8e)
Light yellow solid; Yield 62.7 %; HPLC Rt = 2.758 min. H NMR (400MHz,
1
MeOD-d4): δ=7.90-7.87(2H, d, J=8.4Hz), 7.65(1H, s), 7.47-7.45(2H, d, J=7.6Hz),

7.30-7.25(2H, m), 7.21-7.14(3H, m), 7.09-7.05(1H, m), 6.90-6.88(1H, m), 6.76(1H, t,
J=7.3Hz), 5.66(2H, s), 4.77(1H, m), 3.64(2H, m), 3.43(2H, m), 3.32(2H, s),
2.24-2.18(2H, m), 2.05-1.92(4H, m), 1.80-1.75(2H, m); 13C NMR (100MHz,
MeOD-d4): δ=167.3, 159.0, 154.1, 153.6, 145.5, 142.8, 142.3, 136.2, 133.7, 133.0,
129.6, 127.6, 127.6, 127.1, 126.9, 126.2, 125.7, 125.1, 123.9, 118.3, 117.3, 114.5,
61.2, 55.5, 53.9, 43.8, 31.8, 23.6ppm; HRMS (ESI): Calculated for C32H33ClN8O2 m/z
(M+H)+: 597.2494; found: 597.2493.
4.1.1.6
N-(2-aminophenyl)-4-((2-((9-cyclopentyl-6-(phenylamino)-9H-purin-2-yl)amino)etho
xy)methyl)benzamide (8f)
Light yellow solid; Yield 44.3 %; HPLC Rt = 2.760 min. H NMR (400MHz,
1
MeOD-d4): δ=7.88-7.86(2H, d, J=8.2Hz), 7.61(1H, s), 7.46-7.44(2H, d, J=8.1Hz),
7.31(2H, m), 7.22-7.16(4H, m), 7.07(1H, m), 6.90-6.88(1H, m), 6.76(1H, t, J=7.1Hz),
5.65(2H, s), 4.77(1H, m), 3.62(2H, t, J=5.6Hz), 3.41(2H, t, J=5.6Hz), 2.24-2.17(2H,
m), 2.03-1.91(4H, m), 1.79-1.75(2H, m); 13C NMR (100MHz, MeOD-d4): δ=167.3,
159.0, 154.4, 153.3, 144.2, 143.2, 142.3, 135.7, 132.8, 128.5, 127.7, 127.4, 127.1,
127.0, 126.2, 125.9, 123.9, 118.2, 117.3, 114.4, 61.3, 55.5, 54.3, 43.8, 31.8, 23.6ppm;
HRMS (ESI): Calculated for C32H34N8O2 m/z (M+H)+: 563.2884; found: 563.2894.
4.1.1.7
4-((2-((6-([1,1'-biphenyl]-4-ylamino)-9-cyclopentyl-9H-purin-2-yl)amino)ethoxy)met
hyl)-N-(2-aminophenyl)benzamide (8g)
Light yellow solid; Yield 52.9 %; HPLC Rt = 2.744 min. H NMR (400MHz,
1
MeOD-d4): δ=7.95-7.86(2H, m), 7.63-7.55(5H, m), 7.49-7.39(4H, m), 7.33-7.26(3H,
m), 7.17-7.15(1H, d, J=7.3Hz), 7.06(1H, t, J=7.2Hz), 6.90-6.87(1H, d, J=7.2Hz),
6.75(1H, t, J=7.9Hz), 5.67(2H, m), 4.79-4.75(1H, m), 3.65-3.60(2H, m), 3.44(2H, t,
J=5.3Hz), 2.20-2.17(2H, m), 2.02-1.92(4H, m), 1.79-1.74(2H, m); 13C NMR
(100MHz, MeOD-d4): δ=167.4, 159.0, 154.4, 153.4, 143.5, 143.2, 142.3, 140.4, 138.8,
135.9, 132.9, 128.4, 127.7, 127.5, 127.1, 127.0, 126.9, 126.8, 126.5, 126.2, 126.1,
123.9, 119.8, 118.2, 117.3, 114.5, 61.3, 55.7, 55.5, 54.1, 43.8, 31.8, 23.6ppm; HRMS
(ESI): Calculated for C38H38N8O2 m/z (M+H)+: 639.3197; found: 639.3190.
4.1.1.8
(E)-N-(2-aminophenyl)-3-(4-((2-((9-butyl-6-((3-chlorophenyl)amino)-9H-purin-2-yl)
amino)ethoxy)methyl)phenyl)acrylamide (11a)
Light yellow solid; Yield 78.5 %; HPLC Rt = 2.754 min. H NMR (400MHz,
1
MeOD-d4): δ=7.65-7.52(5H, m), 7.37-7.35(2H, d, J=8.0Hz), 7.29-7.19(4H, m),
7.15-7.13(1H, d, J=8.2Hz), 7.05(1H, m), 6.9-6.73(4H, m), 5.61(2H, s), 4.08(2H, t,
J=7.3Hz), 3.63 (2H, m), 3.43(2H, m), 3.33(2H, m), 1.82(2H, m), 1.35(2H, m),
13
0.97(3H, t, J=7.3Hz); C NMR (100MHz, MeOD-d4): δ=165.6, 159.1, 154.1, 153.5,
145.5, 141.7, 141.0, 140.7, 138.0, 133.8, 133.7, 129.6, 128.1, 127.9, 127.9, 127.8,
127.7, 127.1, 126.8, 126.5, 125.8, 125.3, 123.8, 120.1, 118.1, 117.2, 114.1, 61.2, 54.0,

43.8, 42.6, 31.6, 19.5, 12.5ppm; HRMS (ESI): Calculated for C33H35ClN8O2 m/z
(M+H)+: 611.2651; found: 611.2655.
4.1.1.9
(E)-N-(2-aminophenyl)-3-(4-((2-((9-butyl-6-(phenylamino)-9H-purin-2-yl)amino)eth
oxy)methyl)phenyl)acrylamide (11b)
Light yellow solid; Yield 73.7 %; HPLC Rt = 2.753 min. H NMR (400MHz,
1
MeOD-d4): δ=7.63-7.56(2H, m), 7.48(2H, d, J=8.2Hz), 7.34-7.29(4H, m),
7.23-7.17(4H, m), 7.04(1H, m), 6.88-6.86(1H, m), 6.80-6.72(2H, m), 5.58(2H, s),
4.05 (2H, t, J=7.1Hz), 3.61(2H, m), 3.40(2H, t, J=7.1Hz), 3.32(2H, m), 1.18(2H, m),
13
1.33(2H, m), 0.98(3H, t, J=7.5Hz); C NMR (100MHz, MeOD-d4): δ=165.6, 159.1,
154.5, 153.3, 144.2, 141.7, 141.1, 137.7, 133.6, 128.5, 128.2, 127.6, 127.2, 126.8,
126.0, 125.3, 123.8, 119.9, 118.1, 117.2, 114.0, 61.3, 54.3, 43.8, 42.6, 31.6, 19.5,
12.5ppm; HRMS (ESI): Calculated for C33H36N8O2 m/z (M+H)+: 577.3040; found:
577.3050.
4.1.1.10
(E)-N-(2-aminophenyl)-3-(4-((2-((6-((3-chlorophenyl)amino)-9-cyclopentyl-9H-purin
-2-yl)amino)ethoxy)methyl)phenyl)acrylamide (11c)
Light yellow solid; Yield 57.1 %; HPLC Rt = 2.749 min. H NMR (400MHz,
1
MeOD-d4): δ=7.64-7.60(2H, m), 7.51-7.49(2H, m), 7.35-7.27(2H, m), 7.19-7.13(4H,
m), 7.11-7.06(1H, m), 7.06-7.02(1H, m), 6.88-6.86(1H, m), 6.81-6.72(2H, m),
5.59(2H, s), 4.76(1H, m), 3.64(2H, m), 3.43(2H, m), 3.32(2H, m), 2.22-2.17(2H, m),
2.02-1.91(4H, m), 1.78(2H, m); 13C NMR (100MHz, MeOD-d4): δ=169.5, 162.9,
158.0, 157.5, 149.5, 145.6, 145.0, 144.6, 140.0, 137.6, 133.6, 132.0, 131.6, 130.9,
130.8, 129.7, 129.2, 129.1, 127.7, 124.0, 122.1, 121.2, 118.4, 65.2, 59.5, 57.9, 47.7,
35.7, 27.5ppm; HRMS (ESI): Calculated for C34H35ClN8O2 m/z (M+H)+: 623.2651;
found: 623.2653.
4.1.1.11
(E)-N-(2-aminophenyl)-3-(4-((2-((9-cyclopentyl-6-(phenylamino)-9H-purin-2-yl)ami
no)ethoxy)methyl)phenyl)acrylamide (11d)
Light yellow solid; Yield 54.2 %; HPLC Rt = 2.745 min. H NMR (400MHz,
1
MeOD-d4): δ=7.64-7.60(2H, m), 7.50-7.48(2H, d, J=8.1Hz), 7.35-7.29(4H, m),
7.22-7.17(4H, m), 7.06-7.02(1H, m), 6.89-6.86(1H, m), 6.81-6.72(2H, m), 5.59(2H, s),
4.80-4.72(1H, m), 3.62(2H, m), 3.41(2H, t, J=5.4Hz), 2.21-2.18(2H, m),
2.01-1.91(4H, m), 1.78-1.75(2H, m); 13C NMR (100MHz, MeOD-d4): δ=165.6, 159.0,
154.5, 153.3, 144.3, 141.7, 141.1, 135.7, 133.6, 128.5, 128.2, 127.6, 127.1, 126.8,
125.9, 125.3, 123.8, 119.9, 118.1, 117.2, 114.4, 61.3, 55.5, 54.3, 43.8, 31.8, 23.6ppm;
HRMS (ESI): Calculated for C34H36N8O2 m/z (M+H)+: 589.3040; found: 589.3050.
4.1.1.12
(E)-N-(2-aminophenyl)-3-(4-((2-((9-isopropyl-6-(phenylamino)-9H-purin-2-yl)amino
)ethoxy)methyl)phenyl)acrylamide (11e)

1
Light yellow solid; Yield 32.8 %; HPLC Rt = 2.749 min. H NMR (400MHz,
MeOD-d4): δ=7.65-7.60(2H, m), 7.50-7.48(2H, m), 7.35-7.29(4H, m), 7.23-7.18(4H,
m), 7.06-7.02(1H, t, J=7.3Hz), 6.88-6.86(1H, m), 6.81-6.72(2H, m), 5.59(2H, s),
4.71-4.64(1H, m), 3.62(2H, m), 3.41(2H, t, J=5.4Hz), 1.54-1.53(6H, d, J=6.7Hz); 13C
NMR (100MHz, MeOD-d4): δ=165.6, 159.0, 154.5, 152.9, 144.3, 141.7, 141.1, 135.1,
133.6, 129.2, 128.5, 128.2, 127.6, 127.1, 126.8, 125.9, 125.4, 123.8, 119.9, 118.1,
117.2, 114.3, 61.3, 54.3, 46.3, 43.8, 21.1ppm; HRMS (ESI): Calculated for
C32H34N8O2 m/z (M+H)+: 563.2884; found: 563.2882.
4.1.1.13
N-(2-aminophenyl)-4-((2-((9-isopropyl-6-(phenylamino)-9H-purin-2-yl)amino)ethox
y)methyl)benzamide (11f)
Light yellow solid; Yield 23.5 %; HPLC Rt = 2.740 min. H NMR (400MHz,
1
MeOD-d4): δ=7.68-7.47(4H, m), 7.34-7.17(6H, m), 7.12-7.10(1H, d, J=8.0Hz),
7.03(1H, t, J=7.5Hz), 6.87-6.71(3H, m), 5.58(2H, s), 4.70-4.64(1H, m), 3.66-3.62(2H,
m), 3.44-3.41(2H, t, J=5.4Hz), 1.54-1.52(6H, d, J=7.0Hz); 13C NMR (100MHz,
MeOD-d4): δ=165.6, 159.0, 154.1, 153.1, 145.5, 141.7, 141.0, 140.7, 135.5, 133.7,
129.6, 128.0, 127.7, 127.0, 126.8, 125.8, 125.3, 125.2, 123.8, 120.1, 118.1, 117.2,
114.5, 61.2, 53.9, 46.4, 43.8, 21.1ppm; HRMS (ESI): Calculated for C32H33ClN8O2
m/z (M+H)+: 597.2494; found: 597.2474.
4.1.2 General method for the synthesis of 14a-14i and 17a-17h;
Step-1: 15 mL n-BuOH, 2,6-dichloro-9H-purine 1 (1.0 equiv.) and relative amines 2
(3.0 equiv.) were added in a microwave reaction tube. The vessel was heated to 70 °C
for 1 h. The reaction was cooled to room temperature, the suspension was filtered and
washed with methanol to afford the compound 3 as a yellow solid.
Step-2: To a 100 mL round-bottomed flask, sequentially charged with compound 3 (1
equiv.),
methyl
(E)-3-(4-(bromomethyl)
phenyl)
acrylate
or
methyl
(Z)-3-(4-(bromomethyl)phenyl)acrylate (1.2 equiv.), K2CO3 (2.0 equiv.) and 30 mL
DMF. The reaction was heated to 45 °C for 2 h. After TLC analysis indicated the
completion of the reaction, the mixture was poured into water with vigorous stirring
for 30 min, the obtained solid was filtered to afford compound 12 or compound 15
without further purification.
Step-3: The compound 12 or compound 15 was dissolved in 1,4-dioxane (15 mL). An
aqueous sodium hydroxide solution (2 mol/L, 10 equiv.) was added. The reaction was
heated at 80 °C for 2 h. TLC analysis indicated the complete of the reaction. The
reaction mixture was cooled to room temperature and acidified with 2N HCl to pH<7.
The obtained solid was filtered to afford compound 13 or compound 16.
Step-4: A solution of the compound 13 or compound 16 (1.0 equiv.), DMF (10 mL),
BOP (1.2 equiv.) and triethylamine (4.0 equiv.) was stirred for 10 min at room
temperature. Benzene-1,2-diamine (1.2 equiv.) was added into the reaction. The
reaction was stirred overnight at room temperature. After TLC analysis indicated the
completion of the reaction, the mixture was poured into water with vigorous stirring

for 30 min, the obtained solid was filtered to afford the product 14 or product 17. The
final product was purified by silica gel chromatography.
4.1.2.1
N-(2-aminophenyl)-4-((2-chloro-6-((3-chlorophenyl)amino)-9H-purin-9-yl)methyl)be
nzamide (14a)
1
Light yellow solid; Yield 12.7 %; HPLC Rt = 2.575 min; H NMR (400MHz,
DMSO-d6): δ=10.52(1H, s), 9.62(1H, s), 8.49(1H, s), 8.03(1H, s), 7.95-7.93(1H, d,
J=8.2Hz), 7.80(1H, d, J=7.8Hz), 7.42-7.32(3H, m), 7.13-7.11(2H, m), 6.94(1H, t,
J=7.6Hz), 6.85(1H, s), 6.76-6.73(1H, d, J=8.3Hz), 6.62-6.54(1H, m), 5.50(2H, s),
4.86(2H, m); 13C NMR (100MHz, DMSO-d6): δ=165.4, 152.8, 152.6, 151.9, 151.4,
143.6, 143.3, 140.9, 140.1, 139.7, 134.7, 133.3, 130.6, 128.7, 128.5, 127.7, 127.1,
127.0, 125.4, 123.6, 123.5, 120.9, 119.8, 119.5, 116.7, 116.5, 30.9ppm; HRMS (ESI):
Calculated for C25H19Cl2N7O m/z (M+H)+: 504.1107; found: 504.1101.
4.1.2.2
N-(2-aminophenyl)-4-((2-chloro-6-((4-(pyridin-2-yl)phenyl)amino)-9H-purin-9-yl)me
thyl)benzamide (14b)
Light yellow solid; Yield 20.4 %; HPLC Rt = 2.545 min. H NMR (400MHz,
1
DMSO-d6): δ=10.55(1H, s), 9.69(1H, s), 8.65-8.64(1H, d, J=4.4Hz), 8.51(1H, s),
8.12(2H, d, J=8.8Hz), 7.95(5H, m), 7.85(1H, t, J=7.4Hz), 7.44-7.42(2H, d, J=8.2Hz),
7.35-7.29(1H, m), 7.17-7.15(1H, d, J=7.5Hz), 6.96(1H, d, J=7.5Hz), 6.78-6.76(1H, d,
13
J=7.1Hz), 5.52(2H, s), 4.91(2H, s); C NMR (100MHz, DMSO-d6): δ=165.4, 156.1,
153.0, 152.7, 151.4, 149.9, 143.6, 143.1, 140.2, 137.6, 134.7, 134.1, 128.8, 127.7,
127.2, 127.0, 123.6, 122.6, 121.5, 120.2, 119.5, 116.6, 116.5, 46.7ppm; HRMS (ESI):
Calculated for C30H23ClN8O m/z (M+H)+: 547.1762; found: 547.1756.
4.1.2.3
N-(2-aminophenyl)-4-((2-chloro-6-(phenylamino)-9H-purin-9-yl)methyl)benzamide
(14c)
1
White solid; Yield 19.6 %; HPLC Rt = 15.951 min. H NMR (400MHz, DMSO-d6):
δ=10.34(1H, s), 9.63(1H, s), 8.64(1H, m), 7.69-7.82(3H, m), 7.42-7.33(5H, m),
7.15-7.07(2H, m), 6.95(1H, t, J=7.6Hz), 6.77-6.75(1H, d, J=7.8Hz), 6.57(1H, t,
13
J=7.6Hz), 5.49(2H, s), 4.90(2H, s); C NMR (100MHz, DMSO-d6): δ=165.4, 153.0,
152.9, 151.2, 143.6, 142.9, 140.2, 139.2, 134.7, 129.4, 129.2, 129.0, 128.7, 128.4,
127.9, 127.7, 127.1, 127.0, 124.0, 123.6, 121.8, 119.3, 116.7, 116.5, 46.7ppm; HRMS
(ESI): Calculated for C25H20ClN7O m/z (M+ H)+: 470.1497; found: 470.1494.
4.1.2.4
N-(2-aminophenyl)-4-((2-chloro-6-(p-tolylamino)-9H-purin-9-yl)methyl)benzamide
(14d)
1
Gray solid; Yield 53.3 %; HPLC Rt = 17.735 min. H NMR (400MHz, DMSO-d6):
δ=10.24(1H, s), 9.62(1H, s), 8.44(1H, s), 7.96-7.94(2H, d, J=7.9Hz), 7.69-7.67(2H, d,
J=8.5Hz), 7.41-7.39(2H, d, J=8.0Hz), 7.16-7.14(3H, m), 6.95(1H, t, J=7.2Hz),

6.77-6.75(1H, d, J=7.5Hz), 6.58(1H, t, J=7.4Hz), 5.49(2H, s), 4.91(2H, s), 2.28(3H, s);
13C NMR (100MHz, DMSO-d6): δ=165.4, 153.1, 153.0, 151.1, 143.5, 142.7, 140.2,
136.6, 134.7, 133.1, 129.4, 128.7, 127.7, 127.1, 127.0, 123.6, 122.0, 119.2, 116.7,
+
116.5, 46.7, 21.0ppm; HRMS (ESI): Calculated for C26H22ClN7O m/z (M+ H) :
484.1653; found: 484.1651.
4.1.2.5
N-(2-aminophenyl)-4-((6-(benzylamino)-2-chloro-9H-purin-9-yl)methyl)benzamide
(14e)
1
Light yellow solid; Yield 66.4 %; HPLC Rt = 15.466 min. H NMR (400MHz,
DMSO-d6): δ=9.61(1H, s), 8.87(1H, s), 8.33(1H, s), 7.95-7.89(2H, m), 7.40-7.22(7H,
m), 7.16-7.14(2H, d, J=7.2Hz), 6.96(1H, t, J=7.3Hz), 6.78-6.75(1H, d, J=7.8Hz),
6.58(1H, t, J=7.1Hz), 5.44(2H, s), 4.89(2H, s), 4.66(2H, s); 13C NMR (100MHz,
DMSO-d6): δ=165.4, 155.4, 153.7, 150.4, 143.6, 142.0, 140.3, 139.7, 134.7, 128.8,
128.7, 127.8, 127.7, 127.3, 127.1, 127.0, 123.6, 118.7, 116.6, 116.5, 46.6, 43.6ppm;
HRMS (ESI): Calculated for C26H22ClN7O m/z (M+ H) +: 484.1653; found: 484.1653.
4.1.2.6
N-(2-aminophenyl)-4-((2-chloro-6-(4-methylpiperidin-1-yl)-9H-purin-9-yl)methyl)be
nzamide (14f)
1
Light yellow solid; Yield 24.1 %; HPLC Rt = 18.323 min. H NMR (400MHz,
DMSO-d6): δ=9.63(1H, s), 8.32(1H, s), 7.96-7.94(2H, d, J=7.7Hz), 7.40-7.38(2H, d,
J=8.4Hz), 7.17-7.15(1H, d, J=7.7Hz), 6.96(1H, t, J=7.3Hz), 6.79-6.77 (1H, d,
J=7.5Hz), 6.59(1H, t, J=7.4Hz), 5.44(2H, s), 4.91(2H, s), 1.75-1.72(4H, d, J=11.8Hz),
1.13-1.10(4H, m), 0.91-0.90(4H, m); 13C NMR (100MHz, DMSO-d6): δ=165.4, 153.7,
153.3, 152.1, 143.6, 140.7, 140.3, 134.6, 128.7, 127.7, 127.1, 127.0, 123.6, 118.4,
116.7, 116.5, 46.5, 34.2, 30.8, 22.1ppm; HRMS (ESI): Calculated for C25H26ClN7O
m/z (M+H)+: 476.1966; found: 476.1968.
4.1.2.7
N-(2-aminophenyl)-4-((2-chloro-6-morpholino-9H-purin-9-yl)methyl)benzamide
(14g)
1
Light yellow solid; Yield 26.3 %; HPLC Rt = 2.595 min. H NMR (400MHz,
DMSO-d6): δ=9.63(1H, s), 8.37(1H, s), 7.96-7.94(2H, m), 7.39-7.37(2H, d, J=8.2Hz),
7.16-7.14(1H, d, J=7.4Hz), 6.96(1H, m), 6.78-6.76(1H, d, J=7.8Hz), 6.59(1H, t,
J=7.3Hz), 5.46(2H, s), 4.91(2H, s), 3.72(4H, s); 13C NMR (100MHz, DMSO-d6):
δ=165.4, 153.9, 153.2, 152.2, 143.6, 141.2, 140.2, 134.6, 128.7, 127.6, 127.1, 127.0,
123.6, 118.5, 116.7, 116.5, 66.5, 46.6ppm; HRMS (ESI): Calculated for
C23H22ClN7O2 m/z (M+ H) +: 464.1603; found: 464.1605.
4.1.2.8
N-(2-aminophenyl)-4-((2-chloro-6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)methyl)be
nzamide (14h)

1
Light yellow solid; Yield 21.5 %; HPLC Rt = 3.486 min. H NMR (400MHz,
DMSO-d6): δ=9.60(1H, s), 8.33(1H, s), 7.93-7.86(2H, m), 7.37-7.35(2H, d, J=8.2Hz),
7.14-7.12(1H, d, J=7.4Hz), 6.94(1H, t, J=7.2Hz), 6.76-6.73(1H, d, J=6.6Hz), 6.56(1H,
13
t, J=7.1Hz), 5.43(2H, s), 4.86(2H, s), 2.42(4H, s), 2.20(3H, s); C NMR (100MHz,
DMSO-d6): δ=165.4, 153.8, 153.2, 152.2, 143.6, 141.1, 140.2, 134.6, 128.7, 127.6,
127.1, 127.0, 123.6, 118.5, 116.7, 116.5, 54.8, 46.6, 46.1ppm; HRMS (ESI):
Calculated for C24H25ClN8O m/z (M+ H) +: 477.1919; found: 477.1919.
4.1.2.9
N-(2-aminophenyl)-4-((2-chloro-6-((3-(trifluoromethoxy)phenyl)amino)-9H-purin-9-
yl)methyl)benzamide (14i)
Light yellow solid; Yield 19.4 %; HPLC Rt = 22. 854 min. H NMR (400MHz,
1
DMSO-d6): δ=10.67(1H, s), 9.65-9.58(1H, m), 8.53(1H, s), 8.08-7.89(3H, m),
7.51-6.94(8H, m), 6.79-6.76(2H, m), 6.59(1H, m), 5.53-5.47(2H, m), 4.90(2H, s); 13C
NMR (100MHz, DMSO-d6): δ=165.4, 155.8, 154.7, 152.7, 152.6, 151.5, 148.9, 143.6,
143.3, 141.1, 140.1, 134.7, 128.8, 128.4, 127.8, 127.8, 127.2, 127.0, 123.6, 119.9,
119.5, 119.3, 116.7, 116.5, 115.6, 113.4, 46.8ppm; HRMS (ESI): Calculated for
C26H19ClF3N7O2 m/z (M+ H) +: 554.1320; found: 554.1318.
4.1.2.10
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-((3-chlorophenyl)amino)-9H-purin-9-yl)me
thyl)phenyl)acrylamide (17a)
White solid; Yield 42.7 %; HPLC Rt = 2.583 min. H NMR (400MHz, MeOD-d4):
1
δ=10.45(1H, s), 9.45(1H, s), 8.39(1H, s), 7.95(1H, s), 7.75-7.73(1H, d, J=8.1Hz),
7.53-7.41(3H, m), 7.30-7.26(4H, m), 7.04(1H, d, J=7.3Hz), 6.81(2H, m), 6.65(1H, d,
J=7.8Hz), 6.46(1H, t, J=7.5Hz), 5.36(2H, s), 2.40(2H, m); 13C NMR (100MHz,
MeOD-d4): δ=163.9, 152.8, 152.6, 151.5, 143.2, 142.1, 140.9, 139.3, 138.2, 135.0,
133.3, 130.6, 128.5, 126.2, 125.2, 124.0, 123.4, 123.2, 120.9, 119.8, 119.4, 116.7,
116.4, 46.7ppm; HRMS (ESI): Calculated for C27H21Cl2N7O m/z (M+H)+: 530.1264;
found: 530.1255.
4.1.2.11
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-(p-tolylamino)-9H-purin-9-yl)methyl)phen
yl)acrylamide (17b)
White solid; Yield 20.8 %; HPLC Rt = 2.587 min. 1H NMR (400MHz, DMSO-d6):
δ=10.24(1H, s), 9.40(1H, s), 8.42(1H, s), 7.69-7.50(5H, m), 7.33(3H, t, J=9.0Hz),
7.16-7.15(2H, d, J=7.3Hz), 6.92-6.86(2H, m), 6.75-6.73(1H, d, J=7.8Hz), 6.56(1H, t,
J=7.4Hz), 5.43(2H, s), 5.01(2H, s), 2.28(3H, s); 13C NMR (100MHz, DMSO-d6):
δ=163.9, 153.1, 153.0, 151.1, 142.7, 142.1, 139.4, 138.4, 136.6, 135.0, 133.1, 129.4,
128.5, 128.5, 126.3, 125.2, 123.9, 123.1, 122.0, 119.1, 116.8, 116.5, 46.6, 21.0ppm;
HRMS (ESI): Calculated for C28H24ClN7O m/z (M+H)+: 510.1810; found: 510.1802.

4.1.2.12
(E)-N-(2-aminophenyl)-3-(4-((6-(benzylamino)-2-chloro-9H-purin-9-yl)methyl)phen
yl)acrylamide (17c)
White solid; Yield 27.4 %; HPLC Rt = 2.530 min. 1H NMR (400MHz, DMSO-d6):
δ=9.39(1H, s), 8.87(1H, s), 8.29(1H, s), 7.60-7.49(3H, m), 7.34-7.27(7H, m),
7.23-7.19(1H, m), 6.92-6.84(2H, m), 6.74-6.72(1H, d, J=8.1Hz), 6.56(1H, t, J=7.5Hz),
5.37(2H, s), 4.93(2H, s), 4.64(2H, s); 13C NMR (100MHz, DMSO-d6): δ=163.9, 155.4,
153.8, 150.4, 142.1, 142.0, 139.7, 139.4, 138.5, 134.9, 128.8, 128.5, 127.8, 127.3,
126.3, 125.2, 123.9, 123.0, 118.6, 116.7, 116.5, 46.5, 43.6ppm; HRMS (ESI):
Calculated for C28H24ClN7O m/z (M+H)+: 510.1810; found: 510.1792.
4.1.2.13
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-(phenylamino)-9H-purin-9-yl)methyl)phen
yl)acrylamide (17d)
1
Light yellow solid; Yield 54.6 %; HPLC Rt = 2.568 min. H NMR (400MHz,
DMSO-d6): δ=10.33(1H, s), 9.39(1H, s), 8.43(1H, s), 7.83-7.81(2H, d, J=7.9Hz),
7.61-7.50(3H, m), 7.36-7.29(5H, m), 7.10-7.04(1H, m), 6.92-6.85(2H, m),
13
6.74-6.72(1H, d, J=8.6Hz), 6.55(1H, t, J=7.9Hz), 5.43-5.39(2H, m), 4.9(2H, s); C
NMR (100MHz, DMSO-d6): δ=163.9, 153.0, 152.9, 151.2, 142.8, 142.1, 139.4, 139.2,
138.4, 135.0, 129.4, 129.0, 128.5, 128.5, 126.3, 125.2, 124.0, 123.1, 121.8, 119.3,
116.8, 116.5, 46.7ppm; HRMS (ESI): Calculated for C27H22ClN7O m/z (M+H)+:
496.1653; found: 496.1648.
4.1.2.14
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-(4-methylpiperidin-1-yl)-9H-purin-9-yl)met
hyl)phenyl)acrylamide (17e)
Light yellow solid; Yield 32.7 %; HPLC Rt = 2.728 min. H NMR (400MHz,
1
DMSO-d6): δ=9.42(1H, s), 8.29(1H, s), 7.68-7.51(3H, m), 7.35-7.32(3H, m),
6.93-6.87(2H, m), 6.77-6.75(1H, d, J=8.0Hz), 6.57(1H, t, J=7.7Hz), 5.38(2H, s),
4.96(2H, s), 1.73-1.70(4H, d, J=12.1Hz), 1.20-1.05(4H, m), 0.89-0.88(4H, d,
J=5.6Hz); 13C NMR (100MHz, DMSO-d6): δ=163.9, 153.7, 153.3, 152.1, 142.1,
140.6, 139.5, 138.5, 134.9, 128.4, 126.3, 125.2, 123.9, 123.0, 118.4, 116.7, 116.5,
46.5, 34.2, 30.8, 22.0ppm; HRMS (ESI): Calculated for C27H28ClN7O m/z (M+H)+:
502.2123; found: 502.2101.
4.1.2.15
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-morpholino-9H-purin-9-yl)methyl)phenyl)a
crylamide (17f)
1
Light yellow solid; Yield 31.2 %; HPLC Rt = 2.598 min. H NMR (400MHz,
DMSO-d6): δ=9.41(1H, s), 8.35(1H, s), 7.61-7.50(3H, m), 7.34-7.29(3H, m),
6.93-6.86(2H, m), 6.76-6.74(1H, d, J=8.1Hz), 6.57(1H, t, J=7.2Hz), 5.40(2H, s),
4.96(2H, s), 3.72(4H, s); 13C NMR (100MHz, DMSO-d6): δ=163.9, 153.9, 153.2,
152.2, 142.1, 141.2, 139.4, 138.4, 134.9, 128.5, 128.4, 126.3, 125.2, 123.9, 123.0,

118.5, 116.7, 116.5, 66.5, 46.5ppm; HRMS (ESI): Calculated for C25H24ClN7O2 m/z
(M+H)+: 490.1759; found: 490.1754.
4.1.2.16
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-((3-(trifluoromethoxy)phenyl)amino)-9H-p
urin-9-yl)methyl)phenyl)acrylamide (17g)
Light yellow solid; Yield 36.2 %; HPLC Rt = 20. 873 min. H NMR (400MHz,
1
DMSO-d6): δ=10.66(1H, s), 9.42(1H, s), 8.51(1H, s), 8.08(1H, s), 7.91-7.88(1H, m),
7.63-7.46(4H, m), 7.38-7.33(3H, m), 7.07-7.05(1H, d, J=8.2Hz), 6.93-6.84(2H, m),
13
6.77-6.74(1H, d, J=7.9Hz), 6.57(1H, t, J=7.1Hz), 5.47-5.42(2H, m), 4.97(2H, s); C
NMR (100MHz, DMSO-d6): δ=163.9, 152.8, 152.6, 151.5, 148.9, 143.3, 142.1, 141.1,
139.4, 138.3, 135.0, 130.6, 128.8, 128.5, 128.2, 126.3, 125.2, 123.9, 123.1, 119.9,
119.5, 119.2, 116.7, 116.5, 115.6, 113.4, 46.7ppm; HRMS (ESI): Calculated for
C28H21ClF3N7O2 m/z (M+H)+: 580.1476; found: 580.1477.
4.1.2.17
(E)-N-(2-aminophenyl)-3-(4-((2-chloro-6-(cyclohexylamino)-9H-purin-9-yl)methyl)p
henyl)acrylamide (17h)
Light yellow solid; Yield 22.4 %; HPLC Rt = 18.848 min. H NMR (400MHz,
1
DMSO-d6): δ=9.40(1H, s), 8.26(1H, m), 8.12(1H, m), 7.68-7.42(3H, m),
7.32-7.29(2H, m), 7.08-7.04(1H, d, J=10.7Hz), 6.91-6.85(1H, m), 6.75-6.73(1H, d,
J=9.1Hz), 6.58-6.54(1H, t, J=7.3Hz), 5.37-5.35(2H, d, J=9.3Hz), 4.94(1H, s),
3.49(1H, s), 1.91-1.58(5H, m), 1.41-1.03(5H, m); 13C NMR (100MHz, DMSO-d6): δ=
164.9, 163.8, 155.2, 154.7, 153.9, 150.2, 142.1, 141.5, 140.8, 139.4, 138.6, 138.3,
135.3, 134.9, 128.8, 128.5, 128.4, 128.3, 126.3, 125.2, 123.9, 123.0, 119.6, 118.4,
116.7, 116.5, 49.4, 46.5, 41.9, 32.5, 25.4, 15.7, 13.7ppm; HRMS (ESI): Calculated for
C27H28ClN7O m/z (M+H)+: 502.2123; found: 502.2121.
4.1.3 General method for the synthesis of 22a-22g;
Step-1: 30 mL n-BuOH, 2,6-dichloro-9H-purine (1.0 equiv.) and methyl
4-(aminomethyl)benzoate 18 (1.2 equiv.) were added in a 100 mL round-bottomed
flask. The reaction was heated to 90 °C for 2 h. The reaction was cooled to room
temperature. The suspension was filtered to afford the compound 19 as a white solid.
Step-2: To a 100 mL round-bottomed flask, sequentially charged with compound 19
(1 equiv), bromocyclopentane (2.0 equiv.), K2CO3 (2.0 equiv.) and 30 mL DMF. The
reaction was heated to 90 °C for 2 h. After TLC analysis indicated the completion of
the reaction, the mixture was poured into water with vigorous stirring for 30 min, the
obtained solid was filtered to afford compound 20 without further purification.
Step-3: The compound 20 was dissolved in 1,4-dioxane (15 mL). An aqueous sodium
hydroxide solution (2 mol/L, 10 equiv.) was added. The reaction was heated at 80°C
for 2 h. When TLC analysis indicated the completion of the reaction, the mixture was
cooled to room temperature and acidified with 2N HCl to pH<7. The obtained solid
was filtered to afford compound 21.

Step-4: A solution of the compound 21 (1.0 equiv.), DMF (10 mL), BOP (1.2 equiv.)
and triethylamine (4.0 equiv.) was stirred for 10 min at room temperature.
Benzene-1,2-diamine (1.2 equiv.) was added into the mixture. The reaction was
stirred overnight at room temperature. After TLC analysis indicated completion of the
reaction, the mixture was poured into water with vigorous stirring for 30 min. The
obtained solid was filtered to afford product 22. The final product was purified by
silica gel chromatography.
4.1.3.1
N1-(4-(((2-chloro-9-cyclopentyl-9H-purin-6-yl)amino)methyl)phenyl)benzene-1,2-di
amine. (22a)
White solid; Yield 23.2 %; HPLC Rt = 15.080 min. 1H NMR (400MHz, MeOD-d4):
δ=8.12(1H, s), 7.97(2H, d, J=8.2Hz), 7.56-7.49(2H, m), 7.20-7.18(1H, d, J=7.1Hz),
7.08(1H, t, J=7.7Hz), 6.92-6.90(1H, d, J=8.0Hz), 6.77(1H, t, J=7.7Hz), 4.85-4.83(2H,
m), 2.30-2.22(2H, m), 2.05-1.94(4H, m), 1.83-1.78(2H, m); 13C NMR (100MHz,
MeOD-d4): δ=167.3, 153.9, 150.0, 143.1, 142.3, 139.4, 137.8, 133.0, 127.7, 127.4,
127.1, 126.2, 123.9, 118.3, 117.3, 56.2, 43.4, 32.0, 23.5ppm; HRMS (ESI): Calculated
for C24H24ClN7O m/z (M+H)+: 462.1810; found: 462.1813.
4.1.3.2
N-(2-aminophenyl)-4-(((2-chloro-9-isopropyl-9H-purin-6-yl)amino)methyl)benzamid
e. (22b)
White solid; Yield 19.7 %; HPLC Rt = 12.230 min. 1H NMR (400MHz, DMSO-d6):
δ=9.79(1H, s), 8.90(1H, s), 8.31(1H, s), 7.95-7.93(2H, d, J=7.5Hz), 7.48-7.46(2H, d,
J=7.9Hz), 7.24-7.22(1H, d, J=7.7Hz), 7.05(1H, t, J=7.1Hz), 6.93(1H, d, J=8.0Hz),
13
6.78(1H, t, J=7.5Hz), 5.24(1H, s), 4.72-4.64(4H, m), 1.51-1.49(6H, d, J=6.6Hz); C
NMR (100MHz, DMSO-d6): δ=165.8, 155.4, 153.2, 149.9, 143.5, 140.0, 133.4, 128.4,
127.5, 127.2, 127.0, 125.6, 119.4, 119.0, 118.3, 47.3, 43.5, 22.6ppm; HRMS (ESI):
Calculated for C22H22ClN7O m/z (M+H)+: 436.1653; found: 436.1656.
4.1.3.3
N-(2-aminophenyl)-4-(((9-(sec-butyl)-2-chloro-9H-purin-6-yl)amino)methyl)benzami
de. (22c)
1
White solid; Yield 20.9 %; HPLC Rt = 13.740 min. H NMR (400MHz, DMSO-d6):
δ=9.59(1H, s), 8.88(1H, s), 8.28(1H, s), 7.93-7.87(2H, m), 7.46-7.44(2H, d, J=8.1Hz),
7.16-7.14(1H, d, J=7.5Hz), 6.95(1H, t, J=7.6Hz), 6.78-6.75(1H, d, J=7.9Hz), 6.58(1H,
t, J=7.5Hz), 4.92(2H, s), 4.71-4.69(2H, d, J=5.6Hz), 4.48-4.39(1H, m), 1.95-1.79(2H,
m), 1.49-1.47(3H, d, J=7.0Hz), 0.73(3H, d, J=7.5Hz); 13C NMR (100MHz,
DMSO-d6): δ=165.6, 155.4, 153.2, 150.2, 143.4, 140.5, 133.7, 128.3, 127.5, 127.1,
126.9, 123.9, 118.9, 116.8, 116.6, 53.0, 43.5, 29.1, 20.7, 11.0ppm; HRMS (ESI):
Calculated for C23H24ClN7O m/z (M+H)+: 450.1810; found: 450.1794.