Design, synthesis and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2018.05.024

Cyclin-dependent kinase 2 (CDK2) plays a key role in eukaryotic cell cycle progression which could facilitate the transition from G1 to S phase. The dysregulation of CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied kina

Full text of DOI:10.1016/j.bmc.2018.05.024

Accepted Manuscript
Design, synthesis and biological evaluation of pyrimidine derivatives as novel
CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells
Yiting Wang, Yanmei Chen, Xiaoling Cheng, Ke Zhang, Hangyu Wang, Bo
Liu, Jinhui Wang
PII:
S0968-0896(18)30171-8
https://doi.org/10.1016/j.bmc.2018.05.024
BMC 14366
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
26 January 2018
27 April 2018
16 May 2018
Please cite this article as: Wang, Y., Chen, Y., Cheng, X., Zhang, K., Wang, H., Liu, B., Wang, J., Design, synthesis
and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle
arrest in breast cancer cells, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.05.024
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Design, synthesis and biological evaluation of pyrimidine
derivatives as novel CDK2 inhibitors that induce
apoptosis and cell cycle arrest in breast cancer cells
Yiting Wang1#, Yanmei Chen1#, Xiaoling Cheng 1, Ke Zhang1, Hangyu Wang1*, Bo Liu1*,
Jinhui Wang1,2,3
*
1Key Laboratory of Xinjiang Phytomedicine Resource and Utilization, Ministry of
Education, College of Pharmacy, Shihezi University, Shihezi 832002, China
2School of Pharmacy, Xinjiang Medical University, Urumqi, 830054, China
3College of Pharmacy, Harbin Medical University, Harbin 150081, China
#These authors contributed equally to this work. *Correspondence should be addressed to
18909932852@189.cn (Hangyu Wang), profbl@126.com (Bo Liu) and profwjh@126.com
(Jinhui Wang).

Abstract
Cyclin-dependent Kinase 2 (CDK2) plays a key role in eukaryotic cell cycle
progression which could facilitate the transition from G1 to S phase. The dysregulation of
CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied
kinase targets in oncology. In this article, 24 benzamide derivatives were designed,
synthesized and investigated for the inhibition activity against CDK2. Our results revealed
that the compound 25 is a potent CDK2 inhibitor exhibiting a broad spectrum
anti-proliferative activity against several human breast cancer cells. Additionally,
compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in
MDA-MB-468 cells. These findings highlight a rationale for further development of CDK2
inhibitors to treat human breast cancer.
Key words: CDK2 inhibitor; cell cycle arrest; apoptosis; anti-proliferative activity
1. Introduction
Breast cancer is the most common form and the second most lethal cancer in women,
and the number of patients will increase in the next decade.1, 2 The integrative therapies
are extensively utilized in the clinic, including chemotherapy, radiotherapy, targeted drug
therapy, breast-conserving surgery and mastectomy.3 Breast cancer can be divided into
four different molecular subtypes according to the presence or absence of hormone
receptors (HR) and human epidermal growth factor receptor 2 (HER2) whether or not
overexpressed: luminal A (HR+/HER2-), luminal B (HR+/HER2-), HER2-enriched
(HR-/HER2+), and triple negative (HR-/HER2-).4 Among them, the proportion of triple
negative breast cancer (TNBC) in all is 12%~23%.5
Cyclin-dependent kinase (CDK) is a type of serine/threonine family protein kinase
that regulates mammalian cell cycles.6 The cell cycle regulation disorder is closely linked
to the abnormal expression of CDKs, which is prevalent in all kinds of cancers.7, 8 CDK2, a
member of the CDK family protein, is crucial for the transition of G1 to S phase in the cell
cycle.9 Additionally, increasing studies have shown that inhibition of CDK2 could induce

cancer cell apoptosis with no damage to normal cells.10 CDK2 has emerged as a highly
selective and low toxic cancer therapy target.
A great amount of effort has been made to develop new inhibitors of CDK2 in recent
years (Figure. 1). The ATP binding site of CDKs is located deep at the junction of the N
and C domains, including the pivotal catalytic residues with high sensitivity and kinase
hinge (residues Glu81, Phe82, and Leu83).11 Diaa et al designed and synthesized
derivatives of 2,4,5,6-tetrasubstituted pyrimidine and 4-aminopyrido[2,3-d]pyrimidine as a
CKD2 inhibitor, 8b, and 11a, and these compounds could inhibit CDK2 with an IC50 value
of
0.3µM
and
0.09µM.12,13
It
was
reported
that
compound
with
5-Nitrosopyrimidine-4-amine could be a potent and selective CDK2 inhibitor, such as
NU6027.14 Lees et al discovered a potent and selective CDK inhibitor (Dinaciclib), a
pyrazolo[1,5-a]pyrimidin-7-amine scaffold derivative, which inhibits CDK2, CDK5, CDK1, and
CDK9 activity in vitro with IC50 values of 1, 1, 3, and 4 nM respectively and is assayed in phase
III.15 Milciclib was identified as a potent, orally available cyclin-dependent kinase inhibitor with
IC50 value of 45 nM for CDK2. Milciclib showed good efficacy in the preclinical xenograft A2780
human ovarian carcinoma model and was well tolerated upon repeated daily treatments, which
is currently undergoing phase I and phase II clinical trials.16 NU6300, a covalent
ATP-competitive inhibitor, could bind irreversibly to CDK2 by modifying cysteine residues,
providing a novel rationale to develop the CDK2 inhibitor.17 In addition, Roscovitine, a
potent and selective inhibitor of the cyclin-dependent kinases CDC2, CDK2 and CDK5, inhibits
the proliferation of mammalian cell lines with IC50 value of 16 µM and induces L1210 cells
arrest in G1 and accumulate in G2.18 Another novel CDK2 inhibitor PHA-703887 was reported
as a cancer cell proliferation inhibitor harboring potent inhibitory activity over several
cancer cell lines including COLO-205, C-433, DU-145, A375, PC3, MCF-7 and so forth.19
Recent years, several CDK2 allosteric inhibitors have been discovered. K03861, an
allosteric inhibitor of CDK2, could bind in the absence of Cyclin.20 On the basis of
structure analysis, most of the inhibitors shared a similar amino-substituted heterocyclic
skeleton, which structurally played an essential role in the improvement of affinity on
CDK2. Despite the advance of CDK2 inhibitors, there are still some limitations to impede

their usage in the clinic, such as resistance drug, lack of specificity and efficiency. It is
considered as a promising opportunity to develop novel and special-small molecule
inhibitors targeting CDK2 with potent inhibitory activity in vivo and in vitro for breast cancer
therapy.
In this article, based on the aminopyrimidine scaffold, 24 benzamide derivatives were
designed, synthesized and investigated for the inhibition activity against CDK2. Our
results revealed that the compound 25 is a potent CDK2 inhibitor exhibiting a broad
spectrum anti-proliferative activity against human breast cancer cells. Additionally,
compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in
MDA-MB-468 cells.
Figure. 1 The structures of CDK2 inhibitors.
2. Results and discussion
2.1 Chemistry

The intermediates 3-6 were prepared by the reaction of commercially available
2,4-dichloropyrimidine (1a) or 2,4-dichloro-6-methylpyridine (1b) and substituted aniline
(2a-c) in ethanol (Scheme 1). The intermediates 3-6 reacted with tert-butyl
piperidine-4-ylcarbamate to give intermediates 7-10. Treatment of intermediates 7-10 with
trifluoroacetic acid afforded intermediates 11-14. The target compounds 15-38 were
obtained by reaction of intermediates 11-14 with different acyl chlorides in
dichloromethane.
Scheme 1 General synthesis of compounds 15-38. Reagents and conditions: (i) ethanol,

DIEA, 78℃, reflux, 8 h; (ii) isopropanol; DIEA, 85℃, reflux, overnight; (iii) trifluoroacetic
acid, r.t, 30min, (iv) dichloromethane, triethylamine, r.t, 8h.
2.2 SARs of compounds
To obtain potent candidate compounds based on the experimentally determined
inhibition against CDK2 (Table 1), we designed various substituents to explore the
structure diversity, yielding compounds 15-38. The in vitro cytotoxicity-test results in
different cell lines revealed that most of these compounds showed weak to moderate
cytotoxicity (Figure S1). We preferentially induced differently substituted benzoyl to the
4-amine of piperidine group to give compounds 15-20 when preserving the o-toluidine on
pyrimidine, revealing that compounds 18 and 19 have a comparable activity (IC50 ≈ 280
nM). Whereas other compounds (16, 17, 20) showed a weak inhibitory activity, suggesting
that alkyl substituents may be superior to halogens. Subsequently, 4-fluoroaniline was
incorporated into the pyrimidine scaffold to yield compounds 21-26. Most of these
compounds showed a significant improvement in potency against CDK2. Especially
compound 25 have 6 fold improvement compared with 18. We further confirmed that the
4-methoxy (R3) might have a favorable contribution to compound activity and the 4-fluoro
(R2) is more favorable than 2-methyl. To further explore the effect of R2 on the activity, we
expanded the R2 group ranging 4-methoxyl to 4-fluoro, and R1 was replaced with a methyl
group, yielding compounds 27-38. The results showed that the incorporation of
4-methoxyl at aniline had an adverse effect on the improvement of activity, and the
replacement of R1 with methyl group showed no obvious contribution to activity.
Collectively, compound 25 possessed the most potent activity against CDK2. In addition,
we tested the anti-proliferative activity of these compounds against several cancer cells,
including MDA-MB-231, MDA-MB-468, 786-O, HepG2, WM-266-4 and MCF-7 cells. The
results revealed that compound 25 showed the most potent anti-proliferative activity in
MDA-MB-468 cells. Compound 25 is a potent CDK2 inhibitor with a robust
anti-proliferative activity.
To further characterize the selectivity of compound 25 to other CDK family proteins,

we determined the binding affinity of compound 25 to CDK2, CDK4, CDK6 and CDK7 by
using ADP-GloTM Kinase assay kit. The results revealed that Compound 25 showed the
most potent selectivity against CDK2 with IC50 value of 54.3 nM, but there is no significant
difference from CDK6 (IC50 = 54.3 nM) and CDK7 (IC50 = 89.8 nM), and a weaker
inhibitory activity against CDK4 (IC50 = 278.2 nM) (Figure 2).
Table 1 The inhibition CDK2 activity of compounds 15-38
No.
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
R1
R2
R3
Inhibition of CDK2 (IC50, nM)a
458.5 7.2
398.7 8.0
500.7 6.6
287.5 7.5
285.8 7.5
566.7 8.1
151.1 7.8
240.9 6.5
206.2 7.1
386.5 6.6
45.8 6.4
H
2-methyl
2-methyl
2-methyl
2-methyl
2-methyl
2-methyl
4-fluoro
4-fluoro
H
2-fluoro
H
3-chloro
3-methyl
4-methoxyl
4-methyl
4-fluoro
H
H
H
H
H
4-fluoro
2-fluoro
H
4-fluoro
3-chloro
3-methyl
4-methoxyl
4-methyl
4-fluoro
H
H
4-fluoro
4-fluoro
H
4-fluoro
276.5 11.6
405.0 6.2
279.6 7.3
313.5 6.4
473.6 10.8
276.5 7.1
378.2 5.7
153.1 7.6
204.7 9.9
286.9 6.1
379.5 7.5
54.2 3.2
H
4-methoxyl
4-methoxy
4-methoxy
4-methoxy
4-methoxy
4-methoxy
4-fluoro
H
2-fluoro
H
3-chloro
3-methyl
4-methoxyl
4-methyl
4-fluoro
H
H
H
methyl
methyl
methyl
methyl
methyl
4-fluoro
2-fluoro
4-fluoro
3-chloro
3-methyl
4-methoxyl
4-fluoro
4-fluoro

38
methyl
4-fluoro
4-methyl
288.2 7.4
a. Performed at least for three independent biological replicates
Figure 2 The enzymatic potency of Compound 25 against CDK2, CDK4, CDK6, and
CDK7.
2.4 Molecular Docking and Molecular Dynamic stimulation
In order to elucidate the binding mode of compound 25 to CDK2, molecule docking
simulation was performed by using Discovery Studio 3.5 (Figure 3A). Compound 25
could form a hydrogen bond with Leu83 residue located in the kinase hinge of ATP binding
site. An additional hydrogen bond was observed between amide group of ligand and
Gln131 residue. A Pi-anion interaction was shaped through the aromatic ring of ligand and
Asp86 residue. Above mentioned interactions contributed to the binding affinity of
compound 25 and CDK2 (Figure 3B). To further explore the stability of the compound
25-CDK2 complex, 20 ns MD simulations were performed. The low root-mean-square
deviation (RMSD) fluctuations and low binding free energy indicated that the system was
stable and converged (Figure 3C and D).

Figure 3. Molecular docking and molecular dynamics simulations. (A) Chemical structure
of compound 25. (B) Molecular docking of compound 25 and CDK2. (C) Molecular
dynamics simulation of the compound 25-CDK2 complex. (D) The binding free energy of
complex.
2.5 Compound 25 induced cell cycle arrest in MDA-MB-468 cells
Many cells smoothly step into the next cell cycle by building up CDK2 activity from a
moderate level, while cells lack CDK2 activity will enter a transient state of quiescence.21
Deregulation of CDK2 occurs frequently in certain types of cancer and many tumorigenic
events ultimately drive proliferation by up-regulating CDK2 activity. Much of the control
over CDK2 involves the synthesis and availability of the cyclins, especially Cyclin E.22
Considering the close relationship between CDK2 and cell proliferation, we used the
specific small-interfering RNA for silencing CDK2 expression. We found CDK2 silencing
could decrease the proliferation of MDA-MB-468 cells (Figure S2) and this as well

confirmed the importance of CDK2 on MDA-MB-468 cell proliferation. Then we
determined the anti-proliferative effect of compound 25 on MDA-MB-468 cells. Compound
25 exhibited considerable anti-proliferative effect (IC50:14.28 µM at 24h, IC50: 8.96µM at
48h, IC50:5.74 µM at 72h) in a time- and concentration-dependent manner (Figure 4A）.
Next, we performed PI staining to detect the cell cycle after compound 25 treatment.
Compound 25 treated cells exhibited obvious cell cycle arrest with more cells remain in
G0 or G1 instead of entering into S phase (Figure 4B, C). Western Blot analysis
demonstrated compound 25 inhibited CDK2 activity with the obviously down-regulation of
the phosphorylation level of CDK2 and Cyclin E (Figure 4D). These results suggest that
compound 25 induced cell cycle arrest via CDK2 inhibition.
Figure 4. Compound 25 induced cell cycle arrest via CDK2 inhibition. (A) MDA-MB-468
cells were treated with different concentrations of compound 25 for 24 h, 48h, and 72 h.
MTT assay was used to detect the IC50 of compound 25. (B, C) MDA-MB-468 cells were
treated with 15µM compound 25 for 24 h, flow cytometry was used to measure the cell
cycle arrest. (D) MDA-MB-468 cells were treated with 15µM compound 25 for 12h, 24h,
36h and then the expressions of CDK2, p-CDK2, CyclinE, and p-CyclinE were detected by
western blot analysis.

2.6 Compound 25 induced apoptosis through CDK2 inhibition
Previous studies have been reported that deregulation of CDK2 could cause
Bim-mediated apoptosis.23 In our study, Hoechst 33258 staining, Annexin-V-PI staining
(Figure 5A, B) and western blot in Figure 5C all demonstrated that Compound 25 could
induce obviously apoptosis. Next, we used the specific small-interfering RNA for silencing
CDK2 expression. We found that Cyclin E and Bim were almost not affected in
CDK2-siRNA treated cells after Compound 25 treatment (Figure 5D). Interestingly, we
found CDK2 silencing could up-regulate the expression of Bim (Figure 5D) and induce
apoptosis in MDA-MB-468 cells (Figure S3), this was consistent with previous studies.23
These results suggest that Compound 25 induced cell cycle arrest and apoptosis were
dependent on CDK2 inhibition.
Figure 5. Compound 25 induced apoptosis through CDK2 inhibition. (A) MDA-MB-468

cells were treated with 15µM compound 25 for 6 h, 12h, and 24 h. Hoechst 33258 was used to
detect the DNA fragmentation. Scale bar: 20 µm. (B) Annexin V-PI were used to measure the
ratio of apoptosis after 15µM compound 25 treated for the indicated time. (C) MDA-MB-468
cells were treated with 15µM compound 25 for 12h, 24h, 36h and then the expressions of Bax,
Bim, Caspase3 and PARP were detected by western blot analysis.
3. Conclusions
CDK2 is a very important target for tumor therapy research. CDK2 and Cyclin E can
modulate the cell cycle from G1 to S period, inhibit CDK2 can lead to cell cycle block in
the G1 period, thus restricting the proliferation of tumor cells and causing tumor cells to
die, delaying the growth of tumor cells. In this study, we synthesis 24 structural analogs as
candidate compounds. Molecular docking and MD simulation reveal that compound 25-
CDK2 is a stable complex system. The MTT assay demonstrated that compound 25
caused a time-dependent triple-negative breast cancer cells proliferation inhibition, and
this anti-proliferative effect appears to be due to its ability to induce S-phase arrest and
apoptotic cell death. In addition, compound 25 induces apoptosis could be observed by
fluorescence microscopy assays, flow cytometry, and western blot analysis. These
findings will provide some valuable hints for the development of small molecule drugs for
TNBC therapy.
4. Experimental
4.1 Materials and measurements
1H-NMR spectra were recorded at 400 MHz (Bruker-ARX-400). Data were reported
as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet), coupling constants (Hz), integration. 13C-NMR data were collected at 100 MHz
with complete proton decoupling. Chemical shifts were reported in ppm from the
tetramethylsilane with the solvent resonance as internal standard. ESI-HRMS spectra
were recorded on a commercial apparatus and methanol was used to dissolve the sample.
All chemicals were obtained from commercial sources (Agilent 6890) and used without
further purification. Column chromatography was carried out on silica gel (300-400 mesh,

Qingdao Marine Chemical Ltd, Qingdao, China). Thin layer chromatography (TLC) was
performed on TLC silica gel 60 F254 plates. Melting points are uncorrected and were
determined by Shenguang melting point apparatus (SGW X-4).
4.2 Synthesis
4.2.1 General procedure synthesis of intermediates 3-6
After removing the solvent under reduced pressure to give the crude oil. The crude oil
was purified by silica gel flash chromatography (petroleum ether/ethyl acetate 5:1) as the
intermediates.
2-chloro-N-(o-tolyl)pyrimidin-4-amine (intermediate 3) white solid, yield 65%, m.p.
120.2-122.9℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 9.51 (1H, brs), 8.07 (1H, d, J = 5.9
Hz), 7.31(2H, m), 7.24 (1H, dd, J = 7.7, 6.9 Hz), 7.19 (1H, td, J = 7.3, 1.6 Hz), 6.48 (1H,
brs), 2.19 (3H, s), HER-ESI-MS m/z 220.0643 M+H+ (calcd. For C11H11ClN3, 220.0642).
2-chloro-N-(4-fluorophenyl)pyrimidin-4-amine (intermediate 4) white solid, yield 70%,
m.p. 167.9-170.6℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 10.02 (1H, brs), 8.15 (1H, d,
J = 5.9 Hz), 7.59 (2H, ddm, J = 8.6, 5.0 Hz), 7.21 (2H, ddd, J = 9.0, 8.6, 3.4 Hz), 6.71 (1H,
d, J = 5.9 Hz), HER-ESI-MS m/z 224.0385 M+H+ (calcd. For C10H8ClFN3, 224.0391)
2-chloro-N-(4-methoxyphenyl)pyrimidin-4-amine (intermediate 5) white solid, yield
70%, m.p. 173.2-176.5℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 9.83 (1H, brs), 8.08 (1H,
d, J = 6.0 Hz), 7.44 (2H, d, J = 7.5 Hz), 6.96 (2H, d, J = 7.5 Hz), 6.63 (1H, d, J = 6.0 Hz),
3.75 (3H, s), HER-ESI-MS m/z 236.0594 M+H+ (calcd. For C11H11ClN3O, 236.0591).
2-chloro-N-(4-fluorophenyl)-6-methylpyrimidin-4-amine (intermediate 6) white solid,
1
yield 70%, m.p. 144.7-146.7℃, H NMR (400 MHz, DMSO-d6), δ(ppm): 9.94 (1H, brs),
7.58 (2H, ddm, J = 9.0, 5.2 Hz), 7.21 (2H, ddd, J = 9.0, 8.5, 2.1 Hz), 6.56 (1H, s), 2.28 (3H,
s), HER-ESI-MS m/z 237.0474 M+H+ (calcd. For C11H10ClFN3, 237.0469).
4.2.2 General procedure synthesis of intermediates 7-10
To a solution of intermediates 3-6 (5mmol) respectively in n-butyl alcohol (100 ml),
DIEA (2 eq) was added at room temperature, N-(piperidin-4-yl)pivalamide (10 mmol) was
added and the resulting mixture was allowed to warm up to 120℃ and stirred for
additional 12 hours continually. After removing the solvent under reduced pressure to give
the crude oil. The crude oil was purified by silica gel flash chromatography
(dichloromethane/methanol 20:1) as the intermediates 7-10.
tert-butyl (1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)carbamate (intermediate 7)
1
white solid, yield 83%,m.p. 124.6-127.2℃, H NMR (400 MHz, DMSO-d6), δ(ppm): 8.44
(1H, brs), 7.85 (1H, d, J = 5.6 Hz), 7.45 (1H, d, J = 7.9 Hz), 7.23 (1H, d, J = 7.4 Hz), 7.17
(1H, t, J = 7.9 Hz), 7.05 (1H, t, J = 7.4 Hz), 5.85 (1H, d, J = 5.6 Hz), 5.75 (1H, s), 4.45 (2H,
m), 3.38 (1H, m), 2.84 (2H, m), 2.21 (3H, s), 1.70 (2H, m), 1.38 (9H, s), 1.25 (2H, m),

HER-ESI-MS m/z 384.2405 M+H+ (calcd. For C21H30N5O2, 384.2400).
tert-butyl
(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)carbamate
(intermediate 8) white solid, yield 79%, m.p. 140.1-142.2℃, 1H NMR (400 MHz, DMSO-d6),
δ(ppm): 1H NMR (400 MHz, DMSO-d6), δ(ppm): 9.22 (1H, brs), 7.90 (1H, d, J = 5.6 Hz),
7.60 (2H, ddm, J = 8.8, 4.1 Hz), 7.14 (2H, ddd, J = 9.0, 8.8, 2.3 Hz), 5.97 (1H, d, J = 5.6
Hz), 4.47 (2H, m), 3.51 (1H, m), 2.93 (2H, m), 1.75 (2H, m), 1.39 (9H, s), 1.29 (2H, m),
HER-ESI-MS m/z 388.2150 M+H+ (calcd. For C20H27FN5O2, 388.2149).
tert-butyl
(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)carbamate
(intermediate 9) white solid, yield 75%, m.p. 198.2-201.1℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.07 (1H, brs), 7.85 (1H, d, J = 5.4 Hz), 7.49 (2H, d, J = 8.6 Hz), 6.89
(2H, d, J = 8.6 Hz), 5.94 (1H, d, J = 5.4 Hz), 4.49 (2H, m), 3.73 (3H, s), 3.51(1H, m), 2.89
(1H, m), 1.74 (2H, m), 1.39 (9H, s), 1.28 (2H, m), HER-ESI-MS m/z 400.2346 M+H+ (calcd.
For C21H30N5O3, 400.2349).
tert-butyl(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)carbamat
1
e (intermediate 10) white solid, yield 69%, m.p. 128.4-129.6℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.08 (1H, brs), 7.58 (2H, ddm, J = 8.7, 4.7 Hz), 7.12 (2H, ddm, J = 9.1,
8.7 Hz), 5.84 (1H, s), 4.52 (2H, m), 3.48 (1H, s), 2.91 (2H, m), 2.12 (3H, s), 1.75 (2H, m),
1.39 (9H, s), 1.27 (2H, m), HER-ESI-MS m/z 402.2309 M+H+ (calcd. For C21H29FN5O2,
402.2305).
4.2.3 General procedure synthesis of intermediates 11-14
To a solution of intermediates 7-10 (5mmol) respectively in dichloromethane (25 ml),
trifluoroacetic acid (25 ml) was added at room temperature and the resulting mixture was
stirred for additional 30 minutes continually. After removing the solvent under reduced
pressure to give the mixture. the residue was concentrated and added 100 ml water, the
mixture was basified with aqueous NaOH (1 mol/L) till pH = 10 and extracted with ethyl
acetate (3 x 100 ml). The combined organic layers were washed with saturated aqueous
sodium bicarbonate and brine, and then dried over anhydrous sodium sulfate. After
removing the solvent under reduced pressure to give the crude oil. The crude oil was
purified by silica gel flash chromatography (dichloromethane/methanol 10:1) as the
intermediates 11-14.
2-(4-aminopiperidin-1-yl)-N-(o-tolyl)pyrimidin-4-amine (intermediate 11) yellow oil,
yield 72%, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 8.48 (1H, brs), 7.87 (1H, d, J = 5.7 Hz),
7.46 (1H, dd, J = 7.7, 1.3 Hz), 7.24 (1H, dd, J = 7.4, 1.3 Hz), 7.18 (1H, td, J = 7.7, 1.3 Hz),
7.06 (1H, td, J = 7.4, 1.3 Hz), 5.88 (1H, d, J = 5.7 Hz), 4.48 (2H, m), 2.97 (1H, m), 2.84 (2H,
m), 2.22 (3H, s), 1.77 (2H, m), 1.22 (2H, m), HER-ESI-MS m/z 284.1872 M+H+ (calcd. For
C16H22N5, 284.1875).
2-(4-aminopiperidin-1-yl)-N-(4-fluorophenyl)pyrimidin-4-amine (intermediate
12)
yellow oil, yield 77%, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 9.24 (1H, brs), 7.91 (1H, d, J

= 5.6 Hz), 7.62 (2H, ddm, J = 8.8, 4.9 Hz), 7.14 (2H, ddd, J = 9.3, 8.8, 3.7 Hz), 5.98 (1H, d,
J = 5.6 Hz), 4.46 (2H, m), 2.93 (2H, m), 2.84 (1H, m), 1.76 (2H, m), 1.18 (2H, m),
HER-ESI-MS m/z 288.1622 M+H+ (calcd. For C15H19FN5, 288.1624).
2-(4-aminopiperidin-1-yl)-N-(4-methoxyphenyl)pyrimidin-4-amine (intermediate 13)
1
white solid, yield 67%, m.p. 128.3-131.5℃, H NMR (400 MHz, DMSO-d6), δ(ppm):9.00
(1H, brs), 7.86 (1H, d, J = 5.7 Hz), 7.50 (2H, d, J = 8.9 Hz), 6.90 (2H, d, J = 8.9 Hz), 5.93
(1H, d, J = 5.7 Hz), 4.46 (2H, m), 3.73 (3H, s), 2.91 (1H, m), 2.79 (2H, m), 1.73 (2H, m),
1.15 (2H, m), HER-ESI-MS m/z 300.1828 M+H+ (calcd. For C16H22N5O, 300.1824).
2-(4-aminopiperidin-1-yl)-N-(4-fluorophenyl)-6-methylpyrimidin-4-amine (intermediate
14) yellow oil, yield 76%, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 9.12 (1H, brs), 7.60 (2H,
ddm, 8.8, 5.0 Hz), 7.12 (2H, ddd, 9.1, 8.8, 2.3 Hz), 5.86 (1H, s), 4.50 (2H, m), 2.92 (1H, m),
2.90 (2H, m), 2.13 (3H, s), 1.77 (2H, m), 1.20 (2H, m), HER-ESI-MS m/z 302.1785 M+H+
(calcd. For C16H21FN5, 302.1781).
4.2.4 General procedure synthesis of compounds 15-38
Method A. To a solution of intermediates 7-10 (100mg) respectively in
dichloromethane (25 ml), triethylamine (2 eq) was added at 0℃, 4-fluorobenzoyl (1.2 eq)
chloride was added and the mixture was stirred for additional 30 minutes continually at 0℃
then transferred to room temperature for additional 6 hours. Upon completion the reaction
mixture was filtered and the cake was washed twice by dichloromethane to give the
compounds 15, 21, 27, 33.
Method B. To a solution of intermediates 7-10 (100mg) respectively in
dichloromethane (25 ml), triethylamine (2 eq) was added at 0℃, 2-fluorobenzoyl chloride,
3-chlorobenzoyl chloride, 3-methylbenzoyl chloride, 4-methoxybenzoyl chloride,
4-methylbenzoyl chloride (1.2 eq) was added respectively and the mixture was stirred for
additional 30 minutes continually at 0℃ then transferred to room temperature for
additional 10 hours. After removing the solvent under reduced pressure to give the crude
oil. The crude oil was eluted with dichloromethane/methanol (60-85%) as the compounds
16-20, 22-26, 28-32, and 34-38.
4-fluoro-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide (compound 15),
white solid, yield 63%, m.p. 188.5-189.8℃,1H NMR (400 MHz, DMSO-d6), δ(ppm): 8.48
(1H, brs), 8.26 (1H, brd, J = 7.8 Hz), 7.91 (2H, dd, J = 7.8, 6.8 Hz) , 7.87 (1H, d, J = 5.7
Hz), 7.46 (1H, d, J = 7.7 Hz), 7.25 (2H, dd, J = 9.0, 7.8 Hz), 7.20 (1H, d, J = 7.6 Hz), 7.18
(1H, td, J = 7.7, 1.0 Hz), 7.06 (1H, td, J = 7.5, 1.0 Hz), 5.88 (1H, d, J = 5.6 Hz), 4.56 (2H,
m), 4.05 (1H, m), 2.90 (2H, m), 2.22 (3H, s), 1.80 (2H, m), 1.45 (2H, m), 13C NMR
(100MHz, DMSO-d6): δ (ppm): 164.4, 163.8 (247.4 Hz), 161.5, 161.1, 156.8, 138.0, 132.1,
131.1 (2.8 Hz), 130.4, 129.9 (10.3), 129.9 (10.3), 126.5, 124.9, 124.6, 115.1 (20.7), 115.1
(20.7), 94.9, 47.2, 42.6, 42.6, 31.1, 31.1, 18.0, HER-ESI-MS m/z 406.2040 M+H+ (calcd.
For C23H25FN5O, 406.2043).

2-fluoro-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide (compound 16),
white solid, yield 73%, m.p. 121.6-124.1℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm): 8.48
(1H, brs), 8.24 (1H, brd, J = 7.7 Hz), 7.88 (1H, d, J = 5.7 Hz), 7.53 (1H, t, J = 7.9 Hz ), 7.48
(1H, d, J = 6.8 Hz ), 7.26 (2H, m), 7.24 (1H, m) 7.15 (1H, t, J = 7.6 Hz), 7.07 (1H, m), 5.88
(1H, d, J = 5.7 Hz), 4.50 (2H, m), 4.03 (1H,m), 2.97 (2H, m), 2.22 (3H, s), 1.83 (2H, m),
1.42 (2H,m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.5, 164.9, 163.0, 161.9, 158.8
(248.9 Hz), 138.0, 132.6, 132.5 (8.6 Hz), 131.6, 131.5, 131.0, 130.4 (2.3 Hz), 126.5, 125.4,
125.2 (15.3 Hz), 125.0, 124.8 (3.3 Hz), 115.6 (23.0 Hz), 95.3, 47.6, 43.1, 31.6, 18.5,
HER-ESI-MS m/z 406.2043 M+H+ (calcd. For C23H24FN5O, 406.2043).
3-chloro-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound
17), white solid, yield 66%, m.p. 156.2-157.9℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm):
8.50 (1H, brs), 8.38 (1H, d, J = 8.1 Hz), 7.89 (1H, d, J = 5.7 Hz), 7.81 (1H, d, J = 7.8 Hz),
7.59 (1H, m), 7.50 (1H, m), 7.48 (1H, d, J = 7.4 Hz), 7.23 (1H, d, J = 7.4 Hz), 7.15 (1H, td,
J = 7.4, 1.0 Hz),7.07 (1H, td, J = 7.8, 1.3 Hz), 5.88 (1H, d, J = 5.7 Hz), 4.57 (2H, m), 4.04
(1H, m), 2.92 (2H, m), 2.23 (3H, s), 1.81 (2H,m), 1.46 (2H, m), 13C NMR (100MHz,
DMSO-d6): δ (ppm): 164.5, 161.9, 161.5, 156.8, 138.0, 137.1, 133.6, 132.5, 131.4, 130.9,
130.7, 127.5, 126.5, 126.5, 125.4, 125.0, 95.4, 47.7, 43.1, 43.1, 31.5, 31.5, 18.5,
HER-ESI-MS m/z 422.1744 M+H+ (calcd. For C23H25ClN5O, 422.1748).
3-methyl-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound
18), white solid, yield 67%, m.p. 127.5-129.6℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm):
8.49 (1H, brs), 8.20 (1H, d, J = 7.7 Hz), 7.89 (1H, d, J = 5.6 Hz), 7.66 (1H, brs), 7.60 (1H,
m), 7.47 (1H, d, J = 7.7 Hz), 7.33 (1H, m), 7.24 (1H, d, J = 7.4 Hz ), 7.15 (1H, td, J =
7.7,1.0 Hz), 7.07 (1H, td, J = 7.4,1.0 Hz), 5.88 (1H, d, J = 5.6 Hz), 4.57 (2H, m), 2.90 (2H,
m), 2.51 (1H, m), 2.35 (3H, s), 2.23 (3H, s), 1.80 (2H, m), 1.47 (2H, m), 13C NMR (100MHz,
DMSO-d6): δ (ppm): 166.1, 161.9, 161.5, 156.8, 138.0, 137.9, 135.1, 132.5, 132.0, 131.0,
128.5, 128.2, 126.5, 125.4, 125.0, 124.9, 95.3, 47.5, 43.1,43.1, 31.6, 31.6, 21.3, 18.5,
HER-ESI-MS m/z 402.2292 M+H+ (calcd. For C24H28N5O, 402.2294).
4-methoxy-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide (compound
1
19), white solid, yield 66%,m.p. 168.2-170.1℃, H NMR (400 MHz, DMSO-d6), δ(ppm):
8.48 (1H, brs), 7.88 (1H, d, J = 5.7 Hz), 7.83 (2H, d, J = 8.6 Hz), 7.46 (1H, d, J = 7.7 Hz),
7.24 (1H, d, J = 7.0 Hz), 7.19 (1H, t, J = 7.7 Hz), 7.06 (1H, d, J = 7.0 Hz), 6.97 (2H, d, J =
8.7 Hz), 5.87 (1H, d, J = 5.7 Hz), 4.57 (2H, m), 4.05 (1H, m), 3.80 (3H, s), 2.88 (2H, m),
2.22 (3H, s), 1.79 (2H, m), 1.45 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.4,
161.9, 161.9, 161.5, 156.8, 138.0, 132.5, 130.9, 129.5, 129.5, 127.3, 126.5, 125.4, 125.0,
113.8, 113.8, 95.3, 55.8, 47.5,43.1, 43.1, 31.7, 31.7, 18.5, HER-ESI-MS m/z 418.2241
M+H+ (calcd. For C24H28N5O2, 418.2243).
4-methyl-N-(1-(4-(o-tolylamino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound
20), white solid, yield 79%, m.p. 167.6-170.1℃, 1H NMR (400 MHz, DMSO-d6), δ(ppm):

8.48 (1H, brs), 7.88 (1H, d, J = 5.7 Hz), 7.74 (2H, d, J = 7.6 Hz), 7.46 (1H, dd, J = 7.7 Hz),
7.24 (1H, m), 7.22 (2H, d, J = 7.4 Hz), 7.18 (1H, td, J = 7.7, 1.0 Hz), 7.06 (1H, td, J = 7.4,
1.3 Hz), 5.87 (1H, d, J = 5.7 Hz), 4.56 (2H, m), 4.05 (1H, m), 2.89 (2H, m), 2.34 (3H, s),
2.22 (3H, s), 1.79 (2H, m), 1.46 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.8,
161.9, 161.5, 156.8, 141.3, 138.0, 132.5, 132.3, 130.9, 129.1, 129.1, 127.7, 127.7, 126.5,
125.4, 125.0, 95.3, 47.5, 43.1, 43.1, 31.7, 31.7, 21.4, 18.5, HER-ESI-MS m/z 402.2289
M+H+ (calcd. For C24H28N5O, 402.2294).
4-fluoro-N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound 21), white solid, yield 62%,m.p. 216.8-218.3℃, 1H NMR (400 MHz, DMSO-d6),
δ(ppm): 9.25 (1H, brs), 7.93 (1H, d, J = 5.7 Hz), 7.92 (2H, ddm, J = 8.9, 3.4 Hz), 7.62 (2H,
ddm, J = 9.1, 5.0 Hz), 7.27 (2H, ddm, J = 8.9, 1.9 Hz), 7.14 (2H, ddd, J = 9.1, 8.6, 2.1 Hz),
6.00 (1H, d, J = 5.8 Hz), 4.58 (2H, m), 4.08 (1H, m), 2.98 (2H, m), 1.85 (2H, m), 1.49 (2H,
m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 164.9, 164.5 (250.8 Hz), 161.5, 160.8, 157.6
(238.6 Hz), 156.7, 137.1 (2.9 Hz), 131.6 (2.9 Hz), 130.3 (9.3 Hz), 130.3 (9.3 Hz), 121.6
(7.3 Hz), 121.6 (7.3 Hz), 115.6 (21.7 Hz), 115.6 (21.7 Hz), 115.5 (21.7 Hz), 115.5 (21.7 Hz),
96.6, 47.6, 43.4, 43.4, 31.5, 31.5, HER-ESI-MS m/z 410.1787 M+H+ (calcd. For
C22H22F2N5O, 410.1792).
2-fluoro-N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound 22), white solid, yield 70%, m.p. 169.8-171.8℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.25 (1H, brs), 8.26 (1H, brd, J = 7.4 Hz), 7.94 (1H, brd, J = 5.5 Hz),
7.62 (2H, ddm, J = 9.0, 5.2 Hz), 7.56 (1H, ddd, J = 7.8, 7.4, 1.6 Hz), 7.50 (1H, ddd, J = 7.8,
5.8, 1.6 Hz), 7.26 (2H, ddm, J = 7.8, 2.6 Hz), 7.15 (2H, ddd, J = 9.0, 8.7, 2.0 Hz), 6.00 (1H,
d, J = 5.5 Hz), 4.50 (2H, m), 4.06 (1H, m), 3.05 (2H, m), 1.86 (2H, m), 1.46 (2H, m), 13C
NMR (100MHz, DMSO-d6): δ (ppm): 163.6, 161.5, 160.8, 159.4 (246.3 Hz), 156.7, 157.7
(238.5 Hz) 137.1 (2.4 Hz), 132.5 (8.0 Hz), 130.3 (3.2 Hz), 125.2 (15.1 Hz), 124.8 (4.0 Hz),
121.2 (7.6 Hz), 121.2 (7.6 Hz), 116.5 (22.3 Hz), 115.6 (22.3 Hz), 115.6 (22.3 Hz), 96.6,
47.4, 43.1, 43.1, 31.4, 31.4, HER-ESI-MS m/z 410.1786 M+H+ (calcd. For C22H22F2N5O,
410.1792).
3-chloro-N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound 23), white solid, yield 71%, m.p. 198.3-199.4℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.30 (1H, brs), 8.39 (1H, d, J = 7.8 Hz), 7.94 (1H, d, J = 5.7 Hz), 7.89
(1H, brs), 7.80 (1H, brd, J = 7.9 Hz), 7.61 (2H, ddm, J = 8.5, 4.9 Hz), 7.59(1H, m), 7.50
(1H, dd, J = 8.0, 7.6 Hz), 7.16 (2H, ddm, J = 9.0, 8.5 Hz), 6.02 (1H, brd, J = 5.8 Hz), 4.58
(2H, m), 4.09 (1H, m), 3.01 (2H, m), 1.86 (2H, m), 1.50 (2H, m), 13C NMR (100MHz,
DMSO-d6): δ (ppm): 164.6, 160.8, 157.9 (235.7 Hz), 155.7, 137.0, 136.9, 133.6, 131.4,
130.7, 127.5, 126.5, 121.8, 121.7 (8.2 Hz),121.7 (8.2 Hz), 115.7 (22.4 Hz), 115.7 (22.4 Hz),
96.8, 47.6, 43.5, 43.5, 31.4, 31.4, HER-ESI-MS m/z 426.1491 M+H+ (calcd. For
C22H22ClFN5O, 426.1497).

N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)-3-methylbenzamide
(compound 24), white solid, yield 64%,m.p. 181.4-184.1℃, 1H NMR (400 MHz, DMSO-d6),
δ(ppm): 9.30 (1H, brs), 7.94 (1H, d, J = 5.7 Hz), 7.81 (1H, d, J = 7.8 Hz), 7.64 (2H, m),
7.62 (2H, ddm, J = 8.5, 4.9 Hz), 7.31 (1H, d, J = 7.2 Hz), 7.16 (2H, dd, J = 8.9, 8.5 Hz),
6.02 (1H, d, J = 5.7 Hz), 4.58 (2H, m), 4.09 (1H, m), 3.01 (2H, m), 2.35 (3H, s), 1.86 (2H,
m), 1.50 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 166.1, 161.5, 160.8, 157.7
(236.9 Hz), 156.7, 137.9, 137.1 (2.3 Hz), 135.2, 132.0, 128.5, 128.2, 124.9, 121.6 (7.9 Hz),
121.6 (7.9 Hz), 115.7 (22.6 Hz), 115.7 (22.6 Hz), 96.6, 47.5, 43.4, 43.4, 31.6, 31.6, 21.4,
HER-ESI-MS m/z 406.2046 M+H+ (calcd. For C23H25FN5O, 406.2043).
N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)-4-methoxybenzamide
(compound 25), white solid, yield 60%, m.p. 187.0-188.9℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.26 (1H, brs), 8.09 (1H, brd, J = 8.0 Hz), 7.94 (1H, d, J = 5.3 Hz),
7.83 (2H, d, J = 8.6 Hz), 7.63 (2H, ddm, J = 9.0, 5.0 Hz), 7.15 (2H, dd, J = 9.0, 8.6 Hz),
6.97 (2H, d, J = 8.6 Hz), 6.01 (1H, d, J = 5.3 Hz), 4.59 (2H, m), 4.09 (1H, m), 3.80(3H, s),
2.97 (2H, m), 2.83 (2H, m), 1.50 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.4,
161.9, 161.5, 160.8, 157.8(238.6 Hz), 156.6, 137.1 (2.3 Hz), 129.5, 129.5, 127.3, 121.6
(8.2 Hz), 121.6 (8.2 Hz), 115.7 (23.5 Hz), 115.7 (23.5 Hz), 113.8, 113.8, 96.6, 55.8, 47.4,
43.5, 43.5, 31.6, 31.6, HER-ESI-MS m/z 422.1993 M+H+ (calcd. For C23H25FN5O2,
422.1992).
N-(1-(4-((4-fluorophenyl)amino)pyrimidin-2-yl)piperidin-4-yl)-4-methylbenzamide
(compound 26), white solid, yield 68%, m.p. 210.2-213.1℃, 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.25 (1H, brs), 8.20 (1H, d, J = 7.5 Hz), 7.94 (1H, d, J = 5.8 Hz), 7.65
(2H, m), 7.62 (2H, m), 7.32 (2H, brd, J = 8.0 Hz), 7.15 (2H, ddm, J = 9.2, 8.3 Hz), 6.01 (1H,
d, J = 5.8 Hz), 4.59 (2H, m), 4.08 (1H, m), 2.98 (2H, m), 2.34 (3H, s), 1.85(2H, m), 1.49
(2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.8, 161.5, 160.8, 157.7 (238.4 Hz),
156.7, 141.3, 137.1 (2.3 Hz), 132.3, 129.1, 129.1, 127.7, 127.7, 121.6 (8.3 Hz), 121.6 (8.3
Hz), 115.6 (25.3 Hz),115.6 (25.3 Hz), 96.6, 47.5, 43.5, 43.5, 31.6, 31.6, 21.4,
HER-ESI-MS m/z 406.2043 M+H+ (calcd. For C23H25FN5O, 406.2043)
4-fluoro-N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound 27), white solid, yield 76%, m.p. 210.7-212.6℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.03 (1H, brs), 7.91 (2H, ddm, J = 8.8, 5.6 Hz), 7.89(1H, d, J = 5.8 Hz),
7.50 (2H, d, J = 7.6 Hz), 7.28 (2H, ddm, J = 9.0, 8.8), 6.90 (2H, dd,J = 9.0, 2.3 Hz), 5.93
(1H, d, J = 5.8 Hz), 4.46 (2H, m), 3.73 (3H, s), 2.98 (2H, m), 1.85 (2H, m), 1.50 (2H, m),
1.15 (2H,m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 164.9, 164.3 (248.1 Hz), 161.6,
161.0, 156.3, 155.0, 133.7, 131.6 (2.7 Hz), 130.4, 130.3(8.1 Hz), 130.3 (8.1 Hz), 121.8,
115.5 (21.5 Hz), 115.5 (21.5 Hz), 114.4, 114.4, 96.3, 55.6, 47.6, 43.4, 43.4, 31.5, 31.5,
HER-ESI-MS m/z 422.1990 M+H+ (calcd. For C23H25FN5O2, 422.1992).
2-fluoro-N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide

(compound 28), white solid, yield 62%, m.p. 152.4-154.6℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.03 (1H, brs), 8.26 (1H, d, J = 7.6 Hz), 7.88 (1H, d, J = 5.5 Hz), 7.55
(1H, t, J = 7.6, 7.1 Hz), 7.49 (2H, d, J = 8.6 Hz), 7.27 (2H, ddm, J = 10.0, 7.6 Hz), 6.89 (2H,
d, J = 8.6 Hz), 5.95 (1H, d, J = 5.5 Hz), 4.53 (2H, m), 4.04 (1H, m), 3.73 (3H, s), 3.03 (2H,
m), 1.87 (2H, m), 1.44 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 163.7, 161.5,
161.0, 159.4 (246.7 Hz), 156.4, 155.0, 133.7, 132.5 (8.4 Hz), 130.3, 130.3, 125.2 (15.4
Hz), 124.8 (2.8 Hz), 121.8, 116.5 (22.3 Hz), 114.4, 114.4, 96.3, 55.6, 47.5, 43.0, 43.0, 31.4,
31.4, HER-ESI-MS m/z 422.1990 M+H+ (calcd. For C23H25FN5O2, 422.1992).
3-chloro-N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide,
(compound 29), white solid, yield 68%, m.p. 166.8-169.3℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.04 (1H, brs), 8.39 (1H, d, J = 7.8 Hz), 7.89 (1H, d, J = 5.7 Hz), 7.88
(1H, brs), 7.81(1H, dm, J = 7.9 Hz),7.59(1H, dm,J = 8.0 Hz),7.50(2H, d, J = 7.9 Hz), 6.90
(2H, d, J = 7.9 Hz), 5.96(1H, d, J = 5.7 Hz), 4.61 (2H, m), 4.07(1H, m),3.73 (3H, s), 3.03
(2H, m), 1.87 (2H, m), 1.54 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 166.6, 164.6,
161.6, 161.0, 156.3, 154.9, 137.1, 133.7, 133.6, 131.4, 130.7, 127.5, 126.5, 126.5, 114.4,
114.4, 96.3, 55.6, 47.7, 43.3, 43.3, 31.4, 31.4, HER-ESI-MS m/z 438.1698 M+H+ (calcd.
For C23H25ClN5O2, 438.1697).
N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)-3-methylbenzamide
(compound 30), white solid, yield 69%, m.p. 172.9-174.3℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.02 (1H, brs), 7.89 (1H, dd, J = 5.5 Hz), 7.66(1H, brs), 7.64(1H,
m),7.50(2H, d, J = 8.9 Hz), 7.32(1H, m), 7.31(1H, m), 6.90 (2H, d, J = 8.9 Hz), 5.96(1H, d,
J = 5.5 Hz), 4.60 (2H, m), 4.09(1H,m),3.73 (3H, s), 2.98 (2H,m), 2.34 (3H, s), 1.84 (2H, m),
1.50 (2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 166.1, 161.6, 161.0, 156.4, 155.0,
137.9, 135.2, 133.7, 132.0, 128.5, 128.2, 124.9, 124.9, 121.8, 114.4, 114.4, 96.3, 55.6,
47.5, 43.4, 43.4, 31.6, 31.6, 21.4, HER-ESI-MS m/z 418.2240 M+H+ (calcd. For
C24H28N5O2, 418.2243).
4-methoxy-N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)benzamide
(compound 31), white solid, yield 63%, m.p. 194.1-195.4℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.02 (1H, brs), 7.89 (1H, d, J = 5.7 Hz), 7.83 (2H, d, J = 8.8 Hz), 7.50
(2H, d, J = 9.0 Hz), 6.97 (2H, d, J = 8.8), 6.90 (2H, d, J = 9.0 Hz), 5.96 (1H, d, J = 5.7 Hz),
4.61 (2H, m), 4.09 (1H, m), 3.80 (3H, s), 3.73 (3H, s), 2.96 (2H, m),1.83 (2H, m), 1.49 (2H,
m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.4, 161.9, 161.6, 161.0, 156.4, 155.0,
133.7, 129.5, 129.5, 127.3, 127.4, 121.8, 114.4, 114.4, 113.8, 113.8, 96.3, 55.8, 55.6, 47.5,
43.4, 43.4, 31.7, 31.7, HER-ESI-MS m/z 434.2191 M+H+ (calcd. For C24H28N5O3,
434.2192).
N-(1-(4-((4-methoxyphenyl)amino)pyrimidin-2-yl)piperidin-4-yl)-4-methylbenzamide
(compound 32), white solid, yield 75%, m.p. 210.8-212.2℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.03 (1H, brs), 7.89 (1H, d, J = 5.7 Hz), 7.75 (2H, d, J = 8.2 Hz), 7.50

(2H, d, J = 9.0 Hz), 7.25 (2H, d, J = 8.2 Hz), 6.90 (2H, d, J = 9.0 Hz), 5.96 (1H, d, J = 5.7
Hz), 4.60 (2H, m), 4.08 (1H, m), 3.7 (3H, s), 2.97 (2H, m), 2.34 (3H, s), 1.84 (2H, m), 1.50
(2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 165.8, 161.6, 161.0, 156.4, 155.0, 141.3,
133.7, 132.3, 129.1, 129.1, 127.7, 127.7, 121.8, 121.8, 114.4, 114.4, 96.3, 55.6, 47.5, 43.4,
43.4, 31.6, 31.6, 21.4, HER-ESI-MS m/z 418.2241 M+H+ (calcd. For C24H28N5O2,
418.2243).
4-fluoro-N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)benza
1
mide (compound 33), white solid, yield 65%, m.p. 203.2-205.1℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.14 (1H, brs), 7.92 (2H, ddm, J = 8.6, 5.8 Hz), 7.61 (2H, dd, J = 8.9,
4.9 Hz), 7.30 (2H, ddd, J = 8.9, 8.6, 2.3 Hz), 7.13 (2H, ddm, J = 9.2, 2.8 Hz), 5.89 (1H, s),
4.62 (2H, m), 4.07 (1H, m), 2.96 (2H, m), 2.15 (3H, s), 1.86 (2H, m), 1.50 (2H, m), 13C
NMR (100MHz, DMSO-d6): δ (ppm): 165.6, 164.9, 164.3 (245.0 Hz), 161.5, 161.4, 158.6
(237.3 Hz), 137.3 (2.5 Hz), 132.5 (9.3 Hz), 131.6 (3.0 Hz), 130.4 (9.3 Hz), 130.4 (9.3 Hz),
121.5 (7.5 Hz), 121.5 (7.5 Hz), 116.1 (22.3 Hz), 115.6 (8.1 Hz), 115.5 (7.5 Hz), 94.8, 47.6,
43.4, 43.4, 31.6, 31.6, 24.4, HER-ESI-MS m/z 424.1949 M+H+ (calcd. For C23H24F2N5O,
424.1949).
2-fluoro-N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)benza
1
mide (compound 34), white solid, yield 69%, m.p. 151.0-152.4℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.12 (1H, brs), 7.62 (2H, m), 7.57 (1H, m), 7.51 (1H, ddd, J = 9.7, 7.6,
2.1 Hz), 7.27 (2H, ddd, J = 8.4, 7.6, 1.6 Hz), 7.14 (2H, ddd, J = 9.0, 8.8, 2.2 Hz), 5.88 (1H,
s), 4.55 (2H, m), 4.05 (1H, m), 3.03 (2H, m), 2.15 (3H, s), 1.88 (2H, m), 1.46 (2H, m), 13
C
NMR (100MHz, DMSO-d6): δ (ppm): 165.5, 163.6, 161.4, 161.3, 159.5 (248.0 Hz), 157.6
(237.6 Hz), 137.3 (2.3 Hz), 132.5 (8.5 Hz), 130.3 (3.1 Hz), 125.2 (15.5 Hz), 124.8 (3.1 Hz),
121.5 (11.7 Hz), 121.5 (11.7 Hz), 116.4 (21.6 Hz), 115.6 (22.4 Hz), 115.6 (22.4 Hz), 94.8,
47.5, 47.5, 43.0, 31.5, 31.5, 24.4, HER-ESI-MS m/z 424.1942 M+H+ (calcd. For
C23H24F2N5O, 424.1949).
3-chloro-N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)benza
1
mide (compound 35), white solid, yield 73%, m.p. 154.3-156.5℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.16 (1H, brs), 7.90 (1H, m), 7.82 (1H, dt, J = 7.8, 1.1 Hz), 7.61 (2H,
ddm, J = 8.9, 5.1 Hz), 7.58 (1H, m), 7.49 (1H, t, J = 7.8 Hz), 7.14 (2H, ddd, J = 8.9, 7.8, 2.3
Hz), 5.89 (1H, s), 4.62 (2H, m), 4.08 (1H,m), 2.97 (2H, m), 2.15 (3H, s), 1.86 (2H, m), 1.51
(2H, m), 13C NMR (100MHz, DMSO-d6): δ (ppm): 166.5, 165.5, 164.5, 161.5, 161.4, 157.6
(238.5 Hz), 137.2 (2.3 Hz), 133.6, 131.4, 130.7, 127.5, 126.5, 121.5 (7.9 Hz), 121.5 (7.9
Hz), 115.6 (21.5 Hz), 115.6 (21.5 Hz), 94.9, 47.8, 43.4, 43.4, 31.5, 31.5, 24.4,
HER-ESI-MS m/z 440.1648 M+H+ (calcd. For C23H24ClFN5O, 440.1653)
N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)-3-methylbenza
1
mide (compound 36), white solid, yield 77%, m.p. 164.3-167.4℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.12 (1H, brs), 7.67(1H, brs), 7.62 (2H, ddm, J = 8.8, 5.1 Hz), 7.60(1H,

m), 7.32 (2H, m), 7.13 (2H, ddd, J = 9.0, 8.8, 2.2 Hz), 5.88 (1H, s), 4.62 (2H, m), 4.08 (1H,
m), 2.96 (2H, m), 2.35 (3H, s), 2.15 (3H, s), 1.85 (2H, m), 1.53 (2H, m), 13C NMR (100MHz,
DMSO-d6): δ (ppm): 166.1, 165.6, 161.5, 161.4, 157.6(238.2 Hz), 137.9, 137.3 (2.2 Hz),
135.2, 132.0, 128.5, 128.2, 124.9, 121.5(8.9 Hz), 121.5 (8.9 Hz), 121.5, 115.6 (22.3 Hz),
115.6 (22.3 Hz), 94.8, 47.5, 43.4, 43.4, 31.6, 31.6, 21.4, HER-ESI-MS m/z 420.2196
M+H+ (calcd. For C24H27FN5O, 420.2200).
N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)-4-methoxyben
1
zamide (compound 37), white solid, yield 67%, m.p. 215.3-216.4℃, H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.12 (1H, brs), 7.83 (2H, d, J = 8.9 Hz), 7.61 (2H, ddm, 9.2, 4.9 Hz),
7.14 (2H, ddd, J = 9.2, 8.8, 2.3 Hz), 6.98 (2H, d, J = 8.9 Hz), 5.88 (1H, s), 4.62 (2H, m),
4.08 (1H, m), 3.80 (3H, s), 2.95 (2H, m), 2.15 (3H, s), 1.84 (2H, m), 1.49 (2H, m), 13C NMR
(100MHz, DMSO-d6): δ (ppm): 165.6, 165.5, 161.9, 161.5, 161.4, 157.6 (237.5 Hz), 137.3
(2.2 Hz), 129.5, 129.5, 127.3, 121.5 (7.6 Hz), 121.5 (7.6 Hz), 115.6 (22.1 Hz), 115.6 (22.1
Hz), 113.8, 113.8, 94.8, 55.8, 47.5, 43.5, 43.5, 31.7, 31.7, 24.4, HER-ESI-MS m/z
436.2141 M+H+ (calcd. For C24H27FN5O2, 436.2149).
N-(1-(4-((4-fluorophenyl)amino)-6-methylpyrimidin-2-yl)piperidin-4-yl)-4-methylbenzamide
(compound 38), white solid, yield 70%, m.p. 212.9-214.1℃ , 1H NMR (400 MHz,
DMSO-d6), δ(ppm): 9.14 (1H, brs), 7.76 (2H, d, J = 8.2 Hz), 7.61 (2H, ddm, J = 9.1, 5.0
Hz), 7.25 (2H, dm, J = 8.2 Hz), 7.13 (2H, dt, J = 9.1, 8.8 Hz), 5.89 (1H, s), 4.62 (2H, m),
4.07 (1H, m), 2.96 (2H, m), 2.34 (3H, s), 2.15 (3H, s), 1.85 (2H, m), 1.50 (2H, m), 13C NMR
(100MHz, DMSO-d6): δ (ppm): 165.9, 165.5, 161.5, 161.4, 157.7(238.3 Hz), 141.3, 137.3,
132.3, 129.2, 129.2, 127.7, 127.7, 121.5 (7.4 Hz), 121.5 (7.4 Hz), 115.6 (22.3 Hz), 115.6
(22.3 Hz), 94.8, 47.5, 43.4, 43.4, 31.7, 31.7, 24.4, 21.4, HER-ESI-MS m/z 420.2193 M+H+
(calcd. For C24H27FN5O2, 420.2200).
4.3 Enzymatic assays
The CDKs inhibitory activity assay was performed using ADP-GloTM Kinase assay kit
(Promega, Catalog: V4489) according to the manufacturer's instructions.24 Recombinant
human CDK2/Cyclin E, CDK4/Cyclin D1, CDK6/Cyclin D3 and CDK7/ Cyclin H / MNAT1
were incubated with substrates, compounds and ATP in a final buffer of 25 mM HEPES
(pH 7.4), 10 mM MgCl2, 0.01% Triton X-100, 100 µ mL -1 BSA, 2.5 mM DTT in 384-well
plate with the total volume of 10 µL, respectively. Then the ADP-Glo™ kinase assay was
performed in two steps once the kinase reaction is complete. Subsequently, 5 µL ADP-Glo
Reagent was added to stop the kinase reaction and deplete the unconsumed ATP. Only

ADP and very low background of ATP were left. Then the mixture was incubated at room
temperature for 40 min and added 10 µL of Kinase detection reagent to convert ADP to
ATP and introduced luciferase and luciferin to detect ATP. At last, the mixture was
incubated at room temperature for 30 - 60 min and measured the luminescence with a
plate-reading luminometer. The signal was correlated with the amount of ATP present in
the reaction and was inversely correlated with the kinase activity. Each IC50 value was
calculated from the plot of percentage of inhibition relative to controls versus inhibitor
concentration using GraphPad Software.
4.4 Molecular docking
The discovery Studio 3.5 docking program was adopted here.25 The preparation of
protein structure, including adding hydrogen atoms, removing water molecules, and
assigning Charmm forcefield. Goldscore was selected as the score function, and the
other parameters were set as default. For each docking study, a total of 10 docking
poses were retained. The root-mean-square deviation (RMSD) between docking poses
were calculated.
4.5 Molecular dynamics (MD) simulations
In the following molecular mechanics (MM) minimizations and MD simulations, the
AMBER99 force field and the general AMBER force field (gaff) were used to establish the
potential of receptors and ligands, respectively. An appropriate number of sodium
counter ions were placed around CDK2-ligand complex to neutralize the charges of the
systems. Finally, the whole system was solvated in a cubic periodic box of TIP3P water
molecules, and the distance between the edges of the water box and the closest atom of
the solutes was at least 10 Å. To avoid edge effects, periodic boundary conditions were
applied during the whole molecular dynamics (MD) simulation. For this system, energy
minimization and MD simulation were performed by using the Sander module of the
AMBER 12.0. Prior to MD simulations, the entire system was subjected to energy
minimization in two stages to remove bad contacts between the complex and the solvent
molecules. Firstly, the water molecules and counterions were minimized by freezing the
solute using a harmonic constraint of the strength of 100 kcal mol-1 Å-2. Secondly, the

entire system was minimized without restriction. Each stage consisted of a 5000-step
steepest descent and a 2000-step conjugate gradient minimization. Finally, 10 ns MD
simulations were carried out for each system in an isothermal-isobaric ensemble (NPT)
under periodic boundary conditions.26
4.6 Cell culture, antibodies and reagents
MDA-MB-468 cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). Cells were cultured in DMEM with 10% fetal bovine serum and
incubated with 5% CO2.
Thr160
Antibodies used in this study were as follows: CDK2 (2546, CST), p-CDK2
(2561, CST), p-Cyclin E Thr62 (4136, CST), Cyclin E (EP435E, Abcam), Bax (2774, CST),
Bim (2819, CST), Caspase3 (9662, CST), PARP (9532, CST), β-actin (60008-1-Ig,
Proteintech). MTT (M2128) was purchased from Sigma- Aldrich (St. Louis, MO, USA).
Recombinant human CDK2 /cyclin E (Abcam, ab85639), Recombinant Human CDK4
/ Cyclin D1 protein (ab55695), Recombinant human CDK6 / CCND 3 protein (ab84557),
and Recombinant human CDK7 / Cyclin H / MNAT1 protein (ab64303) were purchased
from Abcam.
4.7 Cell viability assay
Cells were dispensed in 96-well plates at a density of 5 × 104 cells/ml. After 24 h, 48h,
and 72h incubation, cells were treated with different concentrations of compounds for the
indicated time periods. Cell viability was measured by the MTT assay.
4.8 Cell cycle analysis
Cells were treated with 15 µM compound 25 for 24h. Afterward, the cells were
harvested and fixed in 70% ethanol for 30min on ice. After washing with phosphate buffer
saline (PBS), the cells were labeled with propidium iodide (PI, 0.05mg/mL) in the
presence of RNaseA (0.5 mg/mL) and incubated at room temperature in the dark for 30
min. DNA contents were analyzed using the flow cytometer (CytoFLEX S, Beckman
Coulter, USA). 27
4.9 Morphological assay
The cells were placed on a 24-well chamber slide at a density of 2× 106 cells/slide

and treated with compound 15 µM 25 for 6, 12 and 24h. The cells were fixed in
formaldehyde with 40g/L in PBS for 20min followed by Hoechst 33258(10mg/L) staining
for 30min in the dark at 37℃. The cells in the slides were then inspected using
fluorescence microscopy (OBSERVER D1/AX10 cam HRC, Zeiss, GER). 28
4.10 Apoptosis assays by flow cytometry
Compound 25 induced apoptosis in MDA-MB-468 was determined by flow cytometry
using the Annexin V-FLUOS Staining Kit (11988549001, Roche) following the
manufacturer’s instructions. Briefly, 1.5×105 cells/mL were treated with 15 µM compound
25 for 6, 12 and 24h. Afterward, the cells were washed twice with ice-cold PBS, and then
5 µL of Annexin V-FITC and 1 µL of PI (mg/mL)were applied to stain cells. The stained
cells were analyzed using a flow cytometer (CytoFLEX S, Beckman Coulter, USA). 29
4.11 Western blot
Cells were treated with 15 µM Compound 25 for indicated times. Both adherent and
floating cells were collected. Western blot analysis was carried out. Briefly, the cell pellets
were resuspended in lysis buffer consisting of Hepes 50 mM pH 7.4, Triton-X-100 1%,
sodium orthovanadate 2 mM, sodium fluoride 100 mM, edetic acid 1 mM, PMSF 1 mM,
aprotinin (Sigma, MO, USA) 10 mg/L and leupeptin (Sigma) 10 mg/L and lysed at 4°C for
1 h. After 12,000 rpm centrifugation for 15 min, the protein content of the supernatant was
determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA).
Equal amounts of the total protein were separated by 10-15% SDS-PAGE and transferred
to PVDF membranes, the membranes were soaked in blocking buffer (5% skimmed milk
or BSA). Proteins were detected using primary antibodies, followed by HRP-conjugated
secondary antibody and visualized by using ECL as the HRP substrate. Quantification of
immunoblot was performed by Quantity One 4.4.
4.12 Statistical analysis
All the presented data and results were confirmed by at least three independent
experiments. The data are expressed as means SEM and analyzed with GraphPad
Prism 7.0 software. Statistical comparisons were made by One-way ANOVA and
Student’s t-test. P < 0.05 was considered statistically significant.

Acknowledgments
This work was supported by grants from National Natural Science Fund
(Nos.81560566) and National Science and Technology Major Project for “Significant New
Drugs Creation” (Nos.2017ZX090305005).
Author Contributions
H-Y. W., B. L., and J-H. W. conceived and designed the experiments; Y -T. W., Y-M. C.
performed the experiments; X-L. C. and K. Z. contributed reagents/materials/analysis
tools; Y -T. W and H-Y. W. wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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