Discovery of (R)-5-((5-(1-methyl-1H-pyrazol-4-yl)-4-(methylamino)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile, a novel CHK1 inhibitor for hematologic malignancies

Article abstract of DOI:10.1016/j.ejmech.2019.03.062

Through virtual screening, we identified the lead compound MCL1020, which exhibited modest CHK1 inhibitory activity. Then a series of 5-(pyrimidin-2-ylamino)picolinonitrile derivatives as CHK1 inhibitors were discovered by further rational optimization. One promising molecule, (R)-17, whose potency was one of the best, had an IC₅₀ of 0.4 nM with remarkable selectivity (>4300-fold CHK1 vs. CHK2). Compound (R)-17 effectively inhibited the growth of malignant hematopathy cell lines especially Z-138 (IC₅₀: 0.013 μM) and displayed low affinity for hERG (IC₅₀ > 40 μM). Moreover, (R)-17 significantly suppressed the tumor growth in Z-138 cell inoculated xenograft model (20 mg/kg I.V., TGI = 90.29%) as a single agent with body weight unaffected. Taken together, our data demonstrated compound (R)-17 could be a promising drug candidate for the treatment of hematologic malignancies.

Full text of DOI:10.1016/j.ejmech.2019.03.062

Accepted Manuscript
Discovery of (R)-5-((5-(1-methyl-1H-pyrazol-4-yl)-4-(methylamino)pyrimidin-2-
yl)amino)-3-(piperidin-3-yloxy)picolinonitrile, a novel CHK1 inhibitor for hematologic
malignancies
Lexian Tong, Pinrao Song, Kailong Jiang, Lei Xu, Tingting Jin, Peipei Wang, Xiaobei
Hu, Sui Fang, Anhui Gao, Yubo Zhou, Tao Liu, Jia Li, Yongzhou Hu
PII:
S0223-5234(19)30296-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.062
EJMECH 11232
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 March 2019
Revised Date: 29 March 2019
Accepted Date: 30 March 2019
Please cite this article as: L. Tong, P. Song, K. Jiang, L. Xu, T. Jin, P. Wang, X. Hu, S. Fang, A. Gao, Y.
Zhou, T. Liu, J. Li, Y. Hu, Discovery of (R)-5-((5-(1-methyl-1H-pyrazol-4-yl)-4-(methylamino)pyrimidin-2-
yl)amino)-3-(piperidin-3-yloxy)picolinonitrile, a novel CHK1 inhibitor for hematologic malignancies,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.062.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of (R)-5-((5-(1-Methyl-1H-pyrazol-4-
yl)-4-(methylamino)pyrimidin-2-yl)amino)-3-
(piperidin-3-yloxy)picolinonitrile, a Novel CHK1
Inhibitor for Hematologic Malignancies
Lexian Tonga,1, Pinrao Songb,1, Kailong Jiangc,d,1, Lei Xuc,d,e, Tingting Jina, Peipei Wangc,d,
Xiaobei Huc,d, Sui Fangd, Anhui Gaoc,d, Yubo Zhouc,d, Tao Liu*,a, Jia Li*,c,d,e,f, Yongzhou Hu*,a
aZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-
Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, People's Republic of China
bShanghai Haini Pharmaceutical Co. Ltd., Yangtze River Pharmaceutical Group, Shanghai
201318, People's Republic of China
cNational Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic
of China
dUniversity of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People's
Republic of China
eShanghaiTech University, shanghai 201210, People's Republic of China
f Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory
for Marine Science and Technology (Qingdao), 1 Wenhai Road, Aoshanwei, Jimo, Qingdao,
266237, China
*Corresponding authors: Tao Liu, E-mail: Lt601@zju.edu.cn; Jia Li, E-mail: jli@simm.ac.cn;
Yongzhou Hu, huyz@zju.edu.cn
1Equal contribution
1

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Abstract
Through virtual screening, we identified the lead compound MCL1020, which exhibited modest
CHK1 inhibitory activity. Then a series of 5-(pyrimidin-2-ylamino)picolinonitrile derivatives as
CHK1 inhibitors were discovered by further rational optimization. One promising molecule, (R)-
17, whose potency was one of the best, had an IC50 of 0.4 nM with remarkable selectivity
(>4300-fold CHK1 vs. CHK2). Compound (R)-17 effectively inhibited the growth of malignant
hematopathy cell lines especially Z-138 (IC50: 0.013 µM) and displayed low affinity for hERG
(IC50 > 40 µM). Moreover, (R)-17 significantly suppressed the tumor growth in Z-138 cell
inoculated xenograft model (20mg/kg I.V., TGI = 90.29%) as a single agent with body weight
unaffected. Taken together, our data demonstrated compound (R)-17 could be a promising drug
candidate for the treatment of hematologic malignancies.
Keywords: CHK1 kinase inhibitor; selectivity; monotherapy; hematologic malignancies
1. Introduction
The genomic integrity within eukaryotic cells is threatened from both endogenous and
exogenous sources of damage. In response to DNA damage, a complex series of interactions
sense and mediate this process. A central protein involved in the DNA damage response (DDR)
is Checkpoint Kinase 1 (CHK1), a serine/threonine kinase [1]. It responds to replication fork
abnormalities via ATR-dependent phosphorylation at two sites, Ser-345 and Ser-317, through
which it activates an array of downstream events to provoke cells to undergo S or G2/M phase
cell-cycle arrest, preserve replication fork viability, activate DNA repair mechanisms, and
terminate the checkpoint to resume cell division cycle [2-4].
In general, cancer cells harboring a deficient tumor p53 pathway lack efficient G1 checkpoint
and maintaining the G2/M checkpoint fully depend on checkpoint kinases [5, 6]. Therefore, the
pharmacological inhibition of checkpoint kinases in combination with the DNA damaging
chemotherapy or radiotherapy will selectively sensitize p53 deficient cancer cells to
chemotherapeutics and induce mitotic catastrophe and cell death [7-9]. Such combined therapy
results in mild side-effects towards healthy cells while efficiently eradicating the cancer cells.
Currently, quite a number of diverse CHK1 inhibitors have been disclosed and evaluated in
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clinical trials [10-13]. Early CHK1 inhibitors such as AZD7762, XL844, and PF477736, often
showed low selectivity [12, 14], and most of them have been halted after early phase clinical
trials because of being associated with unfavorable toxicity profile or limited efficacy. In an
attempt to overcome these drawbacks, potent and selective CHK1 inhibitors have received
significant attentions. The CHK1 inhibitors developed in recent years including SRA-737 (1),
GDC-0575 (2), and LY2880070 (3), were likely to benefit from increased selectivity and
potency [15, 16].
Recent researches demonstrated the single agent efficacy for CHK1 inhibitors in specific cancer
types, which could significantly enhance the clinical benefit of future drugs in this class. Intrinsic
DNA damage and the activation of CHK1 signaling resulting from high endogenous replication
stress appear to underlie the effectiveness of the inhibitors in many cases [12]. Preclinical data
reported suggested that inhibition of CHK1 may result in caspase-dependent apoptosis of MYC-
overexpressing cells, so CHK1 inhibitors should be evaluated as potential drugs against MYC-
driven malignancies such as certain B-cell lymphoma/leukemia, neuroblastoma, and some breast
cancers [17, 18]. Indeed some of CHK1 inhibitors such as SRA-737(1) and LY2606368 (4),
were reported could be as single agents in treating malignancy and now these two compounds
are in phase II clinical trials. The benefit of CHK1 inhibition remains to be tested, so the studies
on small-molecule CHK1 inhibitors have aroused great concern from a large number of research
groups including ours [19-24]. Herein, we described the discovery and development of a novel
class of CHK1 inhibitors which contained 2-amino pyrimidine scaffold.
In order to discover novel scaffolds of CHK1 inhibitors, we screened the Chembridge database
and our in-house compound library that led to the identification of MCL1020 (Fig. 2) with 2-
amino pyrimidine scaffold as a lead. With structure guided efforts toward optimization of a 2-
amino pyrimidine class of CHK1 inhibitors, we identified potent and highly selective CHK1
inhibitor (R)-17 which shown strong single agent activity in Z-138 cell inoculated xenograft
model.
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HN
HN
N
O
NH2
O
N
N
HN
F3C
CN
Br
N
N
N
H
N
H
1
2
N
NH2
N
O
N
N
N
N
NH
O
N
HN
O
N
N
N
H
H
O
NH
3
4
Figure 1. Structures of representative CHK1 inhibitors SRA-737 (1), GDC-0575 (2), LY2880070 (3),
LY2606368 (4).
2. Results and discussion
2.1. Virtual screening
Several CHK1 crystal structures have recently been published, which allows structure-based
drug design to be used in the search for novel classes of CHK1 inhibitors. We explored a
compound library from Chembridge database which contains about 60000 small molecules and
our in-house compound library containing about 3000 small molecules. These compounds were
docked into the active site of CHK1 kinase (PDB code 2YM8) [25], using the Discovery Studio
(version 2.1)/Ligandfit suite, in which six scoring functions (LigScore1, LigScore2, -PLP1, -
PLP2, Jain, and -PMF) were utilized [26, 27]. Statistical quality of the models was evaluated by
enrichment factor (EF) metrics and receiver operating curve (ROC) analysis [28]. The results
revealed that LigScore2 outperformed all other scoring functions. Therefore, 10 compounds were
finally selected based on balanced scores from LigScore2 and druglike properties (Table S2).
Details of the workflows, screening compound numbers and filters used for the virtual screening,
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and postprocessing analyses with scoring functions are detailed in the Supporting Information
(Section 1).
Figure 2. Schematic showing the discovery of new CHK1 inhibitor MCL1020.
2.2. Novel CHK1 inhibitors design strategy
Among these 10 compounds, MCL1020 grabbed our attention because it was the only one
derived from our internal compound library harboring novel skeleton. And it was intriguing that
MCL1020 could be regarded as scaffold hopping from SRA-737 (1)，thus we initially tested the
CHK1 inhibitory activity of MCL1020 in vitro. Gratifyingly, MCL1020 exhibited modest CHK1
inhibitory activity (IC50 = 1.61 µM). To further understand binding characteristics of MCL1020
with CHK1, docking of MCL1020 into CHK1 X-ray crystal structure (PDB code 2YM8) was
performed (Fig. 3). The docking result indicated that the MCL1020 properly occupied the ATP
pocket by interacting with a diverse range of the sites of the kinase. The kinase hinge-binding 2-
amino pyrimidine core was predicted to form two hydrogen-bonded polar contacts with the
Cys87 and Glu85 residues. The N of pyridine accepted a hydrogen bond from the nearest of the
three waters and 2-cyano of pyridine accepted a hydrogen bond from the protonated catalytic
lysine (Lys38). The area around the ribose region was unexplored, and our initial effort
concentrated on probing this area. It appears that key residues in ribose pocket including Glu91,
Glu134, and Asn135, a polar fragment usually vector toward this pocket [29, 30].
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(a)
(b)
Figure 3. (a) Docking of MCL1020 to CHK1 (PDB code 2YM8, van der Waals surfaces); MCL1020 is shown
as sticks colored green for carbon, blue for nitrogen; (b) Docking of MCL1020 to CHK1 (PDB code 2YM8);
CHK1 interacting residues are shown as sticks colored pink for carbon, blue for nitrogen, red for oxygen;
MCL 1020 is shown as sticks colored green for carbon, blue for nitrogen; Figure prepared with Discovery
Studio (version 2.1)
2.3. Chemistry
All target compounds were prepared by routes outlined in Schemes 1−3. The synthesis of 2-
amino pyrimidine derivatives 5−15 is shown in Scheme 1. The synthesis started from
commercially available pyrimidin-2-amine 38, which was initially brominated with NBS to
obtain 5-bromopyrimidin-2-amine 39, followed by Suzuki−Miyaura coupling to give 40a−j.
Commercially available 41 reacted with 1-Boc-3-hydroxypiperidine 44 to provide intermediate
42a [31]. Buchwald–Hartwig cross-coupling of 42a with 40a−j and subsequent acidic
deprotection afforded 6−15 (Scheme 1) [32]. Intermediate 43 was obtained by
Buchwald−Hartwig cross-coupling of 39 and 5-bromopicolinonitrile. Compound 44 was
transformed into 45 by methyl sulfonylation with MSCl. The nucleophilic substitution of 45 with
2-bromophenol was performed in the presence of appropriate base to obtain compound 46.
Intermediate 47 was obtained by Suzuki−Miyaura coupling of 46 and bis(pinacolato)diboron in
dry atmosphere. Then compound 5 was obtained by Suzuki−Miyaura coupling and subsequent
acidic deprotection.
The synthesis of 2-amino pyrimidine derivatives 16−31 is shown in Scheme 2. Commercially
available 48 reacted with sodium methylate or methylamine to give 49 and 50, respectively.
Nucleophilic displacement of 49 or 50 with ammonium hydroxide afforded the corresponding 2-
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amino pyrimidine derivatives 51 or 52 [33]. Compounds 16−30 were obtained by 51 or 52 from
sequential Suzuki-Miyaura coupling and Buchwald-Hartwig cross-coupling reactions, followed
by acidic deprotection, respectively. Commercially available 4-methylthiazole 55 was converted
to lithium-bearing compound 56 through reaction with triisopropyl borate in the presence of
appropriate base n-butyllithium. Compound 31 was obtained by 52 under reaction of Buchwald–
Hartwig coupling then Suzuki−Miyaura coupling, followed by acidic deprotection.
The synthesis of 2-amino pyrimidine derivatives (R)-17, (S)-17, 32−37 is shown in Scheme 3.
Intermediate 42b-j were obtained in the same method as 42a. Compound (R)-17, (S)-17, 32−37
were formed in the same method as 17.
Br
Br
CN
N
N
d
N
N
NH2
N
N
H
43
39
HN
BocN
BocN
Boc
Boc
N
O
N
g
i,e
O
f
h
CN
O
N
O
MsO
HO
B
O
N
N
N
H
5
45
44
Br
46
47
Scheme 1. Synthetic routes of compounds 5−15. Reagents and conditions: (a) NBS, MeCN, rt (97%); (b) R1-
boronic acid/ester, Pd(dppf)Cl2, 1 N Na2CO3, DME, reflux (79%−87%); (c) 44, NaH, THF, 0°C−rt, 3 h (79%);
(d) 40a−j for synthesizing 6−15, 5-bromopicolinonitrile for synthesizing 43, Pd2(dba)3, Cs2CO3, Xantphos,
7

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dioxane, reflux, 5 h, (70%−87%); (e) CF3COOH, CH2Cl2, rt (80%); (f) MsCl, TEA, DMAP, CH2Cl2, 0°C, 1 h
(78%); (g) 2-bromophenol, Cs2CO3, DMF, 70°C, 5 h (43%); (h) Bis(pinacolato)diboron, Pd(dppf)Cl2, TEA,
dioxane, reflux (77%); (i) 43, Pd(dppf)Cl2, 1 N Na2CO3, DME, reflux (65%).
Scheme 2. Synthetic routes of compounds 16−31. Reagents and conditions: (a) Na, MeOH, rt, 3 h (90%) ; (b)
CH3NH2, MeOH, rt, 3 h (88%); (c) NH3·H2O, dioxane, 160°C, 3 h (78%); (d) 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl2, 1 N Na2CO3, DME, reflux (80%); (e) 42a, Pd2(dba)3,
Cs2CO3, Xantphos, dioxane, reflux, 5 h (75%−84%); (f) CF3COOH, CH2Cl2, rt (80%); (g) R1-boronic
acid/ester, Pd(dppf)Cl2, 1 N Na2CO3, DME, reflux (65%−84%); (h) Triisopropyl borate, n-butyllithium,
isopropanol, dry toluene/ THF (v/v = 4:1), −78°−0°C; (i) 56, Pd(dppf)Cl2, CuCl, ZnCl2, Cs2CO3, DMF, 100°C
(70%).
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Scheme 3. synthetic routes of compounds (R)-17, (S)-17, 32−37. Reagents and conditions: (a) NaH, R2-
OH,THF, 0°C−rt, 3 h (51%−90%); (b) 54a, Pd2(dba)3, Cs2CO3, Xantphos, dioxane, reflux, 5 h (74%−84%); (c)
CF3COOH, CH2Cl2, rt (80%).
2.4. Biological evaluation
2.4.1. CHK1 inhibition and structure activity relationship (SAR) studies
We hypothesized that introducing a polar fragment directed toward the ribose region could not
only enhance affinity for CHK1 by interacting with additional residues near the ribose region but
also improve physicochemical property of molecule. Furthermore we analyzed the complex of
MCL1020 and CHK1 and found the C-ring (phenyl) directed toward solvent-exposed surface
and B-ring (pyridine) substituted a small substituent where could adopt a large group (Fig. 3), so
the polar fragment was introduced on these two rings. We were pleased to find that the potency
of the optimized compound 6, the hydrophilic segment located in the B-ring, was significantly
improved with IC50 11 nM (Table 1), while the hydrophilic segment located in the C-ring as
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shown in table 1, compound 5 had no inhibitory effect on CHK1. Taking compound 6 as our new
lead, we conducted further structure modification to explore the structure−activity relationships.
Table 1. SAR exploration of the location of hydrophilic segment and C-ring
Cpd.
C-ring
-
IC50 (nM)a
6053.5±1198.55
5
10.95±0.57
14.30±0.85
21.19±1.70
15.92±2.93
8.34±0.67
99.58±3.00
58.94±2.87
1.96±0.65
12.71±0.67
3.75±0.94
phenyl
3-fluorophenyl
4-fluorophenyl
3-methoxyphenyl
4-methoxyphenyl
pyridin-3-yl
6
7
8
9
10
11
12
13
14
15
pyridin-4-yl
thiophen-2-yl
furan-2-yl
1-methyl-1H-pyrazol-4-yl
aValues are means of three experiments.
The modification toward the C-ring (5-phenyl ring) in compound 6 was to install different
groups in the para- or meta- positions to give compounds 7−10. Subsequently, aromatic
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heterocyclic rings were introduced to replacement of C-ring. The results demonstrated that
introduction of electron-withdrawing (7 and 8) or donating (9 and 10) groups in the either para-
or meta- position of phenyl ring had a little effect on the potencies against CHK1. Replace
phenyl ring with pyridine ring yielded compound 11 and 12 that demonstrated a relatively
modest drop in potencies, while the potencies of five-membered heteroaromatic ring analogues
were kept or increased. Thiophen-2-yl 13 and 1-methyl-1H-pyrazol-4-yl 15 shown the best
potency in this series, regrettably, the poor aqueous solubility of 13 was assumed to be limiting
its further development.
Further analysis of ribose region suggests that there is still some space at 4-position of B-ring
where can adopt small substituent, thus, we add a small substituent at this position to see whether
the potency would be affected. At first, methoxy group or methylamino group was introduced to
the 4-position of B-ring of the compound 15 giving two new compounds 16 and 17. A sharp drop
in potency was observed when introducing methoxy group. In contrast, the potency of 4-
methylamino pyrimidine analogue 17 increased about 4 times relative to that of compound 15.
Encouragingly, a set of 4-methylamino pyrimidine analogues were synthesized (18-31, Table 2).
As expected, the potencies of these newly prepared analogues presented in Table 2 were
maintained or slightly increased compared with compounds presented in Table 1. Introduction of
a substituent in ortho-position of C-ring may cause unfavorable interactions with residues
surrounding the ATP-binding pocket and weaken the potency against CHK1 (20 vs 21, 23 vs
24). In this turn, 1-methyl-1H-pyrazol-4-yl 17 (IC50 = 0.9 nM) shown the best potency, was
chosen as the basis for further structural optimization.
H
N
X
O
B
C
CN
N
A
N
N
N
H
Table 2. SAR exploration of X at 4-position of A-ring and C-ring
Cpd.
C-ring
X
O
IC50 (nM)a
1-methyl-1H-pyrazol-4-yl
1406.0±199.40
16
11

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1-methyl-1H-pyrazol-4-yl
N
0.89±0.06
2.56±0.55
17
18
phenyl
N
N
N
N
N
N
N
N
N
N
N
N
N
N
4.49±0.94
8.76±0.62
3-fluorophenyl
4-fluorophenyl
19
20
21
22
23
24
25
26
27
28
29
30
31
29.02±0.29
3.62±0.71
2-fluorophenyl
3-methoxyphenyl
4-methoxyphenyl
2,4-dimethoxyphenyl
pyridin-3-yl
19.43±18.34
365.25±14.78
15.14±1.68
4.72±1.51
pyridin-4-yl
1.43±0.09
thiophen-2-yl
2.58±0.34
furan-2-yl
1.80±0.17
5-methylthiophene-2-carboxylate
1-methyl-1H-pyrazol-5-yl
4-methylthiazol-2-yl
517.35±73.19
4.72±1.51
a Values are means of three experiments.
Table 3. SAR exploration of R at 3-position of B-ring
Cpd.
R
IC50 (nM)a
0.4±0.0
(R)-17
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(S)-17
8.88±0.45
0.5±0.1
1.3±0.3
32
33
6.44±0.23
34
2.43±0.26
26.30±8.32
26.33±6.84
35
36
(R)-37
59.49±11.12
10.4±5.2
0.7±0.4
(S)-37
LY2603618
LY2606368
-
-
a Values are means of three experiments.
Having identified 1-methyl-1H-pyrazol-4-yl as a preferred C-ring, our efforts focused on
exploring the R substituent. Various amine substitutions were introduced on the B-ring (Table 3).
As was observed in table 3, two pairs of enantiomers (R)-17 and (S)-17, (R)-37 and (S)-37
together shown that the potencies of compounds with R configuration were superior to S. The
potency of compound 32 that contained piperidine was retained (IC50 = 0.5 nM). When
methylation of the piperidine of 32 gave compound 33, potency reduced by 2.6-fold, indicating
that the substituents holding the secondary amine were more suitable than tertiary amine.
Subsequent introduction of piperidin-4-ylmethyl led 34, which was more than 12-fold less potent
than 32. Five-member heterocyclic analogue 35 exhibited a 6-fold decrease in activity compared
with six-member heterocyclic analogue (R)-17. On the other hand, introduction of linear
aminoalkylamines giving compounds 36-37 resulted in dramatically drop of potency compared
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with 33. In this series, cyclic substituents introduced were more suitable than linear substituents,
and compound (R)-17 was deemed as the most potent compound.
2.4.2. Kinase selectivity profile of (R)-17
Selectivity of inhibition for a particular kinase is a well-known issue, as well as CHK1. The
chemical modifications toward compound MCL1020 resulted in one promising molecule (R)-17.
Thus, compound (R)-17 was selected to assess for selectivity against 15 kinases belonged to four
kinase subfamilies (CHK1, CHK2, AMPKα2β1γ1, AMPKα1β1γ1, PIM1, PIM3 from CAMK
subfamily; CDK1, CDK2 from CMGC subfamily; p70S6k, PKC, PKA from AGC subfamily;
FLT3, BLK, LCK, LYN from TK subfamily). The results were summarized in Table 4.
Compound (R)-17 displayed excellent selectivity against CHK2 (>4300-fold) and was
significantly more selective than that of positive control LY2606368 (58-fold CHK1 vs. CHK2).
Cyclin-dependent kinases, such as CDK1 and CDK2, important in regulating the cell cycle, were
also not significantly inhibited by (R)-17. Compared with LY2606368, (R)-17 displayed a little
bit stronger binding against PIM1 and PIM3 and showed similar kinase inhibition against other
selected kinases. These results indicated the (R)-17 had good kinase selectivity profiles and was
almost equal to LY2606368.
Table 4. Kinase inhibition profile of compound (R)-17Ac against selected protein kinases
(R)-17Aca
IC50(nM)
LY2606368
IC50(nM)
(R)-17 Aca
IC50(nM)
LY2606368
IC50(nM)
382.18±17.75
n.t.b
Kinase
Kinase
CHK1
CHK2
0.4±0.0
0.56±0.16
32.57±2.13
p70S6k
PKC
625.68±124.89
＞10 µM
1729.0±60.8
AMPKα2β1γ1
AMPKα1β1γ1
PIM1
91.11±9.59
107.50±12.42
511.80±73.31
735.53±176.37
＞10µM
30.09±5.09
101.62±9.92
＞10µM
PKA
FLT3
BLK
LCK
LYN
＞10 µM
n.t.b
385.98±22.95
4409.00±686.79
>10µM
100.45±9.35
2713.75±209.85
3156.00±334.56
4297.50±437.58
PIM3
＞10µM
CDK1
＞10µM
>10µM
CDK2
＞10µM
＞10µM
a (R)-17Ac is acetate of (R)-17
b n.t. indicates not tested.
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2.4.3. In vitro antiproliferative activities of (R)-17
On the basis of potency and kinase selectivity profile, in particular the selectivity over CHK2,
(R)-17 was progressed to more detailed studies. Recent evidence suggested that cell lines derived
from malignant hematopathy cell lines exhibited greater sensitivity to the CHK1 inhibitor than
cell lines derived from solid tumors [34, 35]. So the growth inhibitory potencies of (R)-17
against six malignant hematopathy cell lines were examined, and the results were presented in
table 5. Among these six malignant hematopathy cell lines, (R)-17 seemed to be even more
selective for Jeko-1, MV4-11 and Z-138 cell lines (IC50 <40 nM). In particular, (R)-17 inhibited
the proliferation of Z-138 with IC50 values of 0.013
μM, which displayed the best activity over
all six cell lines. This encouraging cellular assay data led to further profiling of (R)-17.
Table 5. Cellular activities of the (R)-17
IC50(µM)a
Cpd.
RPMI8226
Mino
Romas
Jeko-1
MV4-11
Z-138
(R)-17
1.990
4.546
0.045
0.155
0.254
1.110
0.095
0.533
0.004
0.036
0.920
0.004
0.039
0.869
0.004
0.013
0.364
0.033
LY2603618
LY2606368
a Values are means of three experiments. For SD values see Table S3.
2.4.4. In vivo PK/PD evaluation
Given the encouraging biological profile, pharmacokinetic (PK) properties of compound (R)-17
was further evaluated. Key PK parameters are summarized in Table 6. After I.V. administration
of 15 mg/kg compound (R)-17, the area under the concentration−time curve (AUC0−∞), the half-
life (T1/2), peak concentration (Cmax), and plasma clearance (CL) of (R)-17 were 370 ng/mL·h,
2.23 h, 849 ng/mL, and 135 mL/min/kg, respectively. Compound (R)-17 exhibited reasonable
PK properties in mice.
Table 6. PK parameters of (R)-17 in mice
Kel
T1/2
(h)
Cmax
AUC(0-t)
AUC(0-∞)
Vdss
CL
MRT
(h)
(L/kg)
(ng/mL)
(ng/mL·h)
(ng/mL·h)
(L/kg)
(mL/min/kg)
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IV(15mg/kg)
0.365
2.23
849
363
370
6.42
135
0.80
The antitumor efficacy of compound (R)-17 as a single agent was tested in Z-138 cell inoculated
xenograft mouse model. When tumors had reached an average volume of 100 mm3, the mice
were grouped and treated intravenously with 10 or 20 (mg/kg)/d of compound (R)-17Ac ((R)-
17Ac is acetate of (R)-17) , Cytarabine (AraC, as a standard drug) or vehicle (5% Mannitol) 5
times every week for 3 weeks. The results of this study (Fig. 4) showed that compound (R)-17Ac
administered on this schedule led to a dose-dependent inhibition of tumor growth. Tumor growth
inhibitions of 78.64% and 90.29% were observed at doses of 10 and 20mg/kg, respectively.
Notably, no significant body weight loss and no adverse effects were observed for compound
(R)-17Ac during the in vivo studies. All of the studies presented here support (R)-17 as a novel
candidate and deserve further research and development.
(a)
(b)
Vehicle
AraC 40mg/kg
20
15
10
5
(R)-17Ac 20mg/kg
(R)-17Ac 10mg/kg
0
0
5
10
15
20
25
day
(c)
4
2
0
1.74
**
TGI =60.95%
**
*
0.37
TGI =90.29% TGI =78.64%
0.68
0.17
16

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Figure 4. In vivo tumor growth inhibition for (R)-17Ac. Female nu/nu mice bearing established human Z-
138 inoculated xenograft were administered I.V. with either vehicle (5% Mannitol), AraC at 40 mg/kg or (R)-
17Ac at 10 and 20mg/kg body weight 5 times every week for 3 weeks. (a) Relative tumor volume. Initial
tumor size was set as 1. (b) Body weight of mice. (c) Tumor weight measurements from Z-138 xenograft mice
after 21 days compound administration. Numbers in red indicate the mean tumor weight in each group (*
P<0.05，** P<0.01 vs vehicle).
2.4.4. The hERG inhibition of the compound (R)-17
CHK1 inhibitors XL-844 and AZD7762 halted after early phase clinical trials because of being
associated with cardiotoxicity [12], which to some extent incurred by certain affinity for the
hERG ion channel, thus hERG inhibition of (R)-17 was investigated initially. As shown in figure
5, compound (R)-17 displayed low affinity for hERG (IC50 > 40 µM), which was less potent in
inhibiting hERG compared with positive controls LY2603618, LY2606368 (IC50 = 28.1 µM,
IC50 = 20 µM, respectively). The hERG model classified compounds as hERG-inactive if their
percentage inhibition was less than 40% @ 10 µM [15], so compound (R)-17 could be seen as
hERG-inactive and the wider therapeutic index against hERG of (R)-17 was suitable for
preclinical evaluation.
(b)
(a)
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(c)
Figure 5. (a) The hERG inhibition of the compound (R)-17. (b) The hERG inhibition of the positive control
compound LY2603618. (c) The hERG inhibition of the positive control compound LY2606368.
2.5. Molecular modeling of compound (R)-17 with CHK1
Compound (R)-17 shows optimal CHK1 inhibitory activities, hence, the binding mode of
compound (R)-17 within CHK1 were elucidated using a docking model (Fig. 6). As is shown in
Figure 6, compound (R)-17 binds to the ATP-binding site of CHK1 in an orientation similar to
that of MCL1020 (Fig. 3b). The 2-amino pyrimidin skeleton of compound (R)-17 forms two
conserved hydrogen bonds with the hinge region of CHK1, one between the N1 of pyrimidin and
Cys87 and the other between the NH of amino and Glu85. The N of pyridine accepts a hydrogen
bond from the nearest of the three waters and 2-cyano of pyridine accepts a hydrogen bond from
the protonated catalytic lysine (Lys38), related to selectivity. The piperidine NH forms additional
polar interactions with the side chains of Glu134. In general, the docking results further confirm
the rationality of our design strategy.
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Figure 6. Compound (R)-17 docked into CHK1 kinase (PDB code 2YM8). CHK1 interacting residues are
shown as sticks colored green for carbon, blue for nitrogen, red for oxygen; (R)-17 is shown as sticks colored
light blue for carbon, dark blue for nitrogen; Figure prepared with Discovery Studio (version 2.1)
3. Conclusions
Through virtual screening and rational drug design, we finally identified (R)-17 bearing 2-amino
pyrimidine scaffold as a novel CHK1 inhibitor. Compound (R)-17 is a selective inhibitor, which
potently inhibited CHK1 with IC50 value of 0.4 nM and exhibited selectivity >4300-fold against
CHK2. Additionally, (R)-17 significantly inhibited the growth of malignant hematopathy cell
lines (IC50 ＜40 nM) such as Z-138, MV4-11, Jeko-1. Ultimately, the antitumor efficacy of
compound (R)-17 as a single agent was tested in Z-138 cell inoculated xenograft mouse model.
Pleasingly, tumors arising in these animals underwent significant growth inhibition in response
to treatment with (R)-17 (20mg/kg I.V., TGI = 90.29%) with mice body weight unaffected.
Meanwhile, (R)-17 displayed low affinity for hERG (IC50 > 40 µM), could be seen as hERG-
inactive and its wider therapeutic index against hERG was suitable for preclinical evaluation. In
summary, all these reported results illustrated that CHK1 inhibitor (R)-17 could be as potential
drug candidate to treat hematologic malignancies.
4. Experimental section
19

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Reagents and solvents were obtained from commercial suppliers and used without further
purification. All reactions were monitored by TLC, using silica gel plates with fluorescence F254
and UV light visualization was used as the visualizing agent. Column chromatography was
performed using 200-300 mesh silica gel supplied by Qingdao Marine Chemical Factory,
Qingdao, China. Melting points were determined on a Büchi melting point B-540 apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker AVANCE III spectrometer
at 500 MHz or Bruker AM 400 at 400 MHz. All NMR spectra were calibrated using residual
1
13
1
solvents as internal references (for CDCl3: H NMR = 7.26, C NMR = 77.16; DMSO-d6: H
NMR = 2.50, 13C NMR = 39.52). Coupling constants (J) are expressed in hertz (Hz). All
chemical shifts were reported in parts per million (ppm). The following abbreviations were used
to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
Electrospray ionization mass spectroscopy (ESI-MS) spectra were obtained with a Shimadzu
LCMS-2020 mass spectrometer with mobile phases as methanol and water containing 0.1%
formic acid. High resolution mass spectra (HRMS) were measured on an Agilent 6224 TOF
LC/MS spectrometer using ESI-TOF (electrospray ionization-time of flight). HPLC analysis was
performed on Agilent 1260 infinity equipped with Cosmosil 5C18-AR- column (4.6 mm × 250
mm), eluting at 1.0 mL/min using a listed gradient: (mobile phase A, 0.1%CF3COOH in H2O;
mobile phase B, MeCN). Purity of all biologically tested compounds was determined by HPLC
to be > 95%.( Supporting Information (Section 4 and 5).).
4.1. 5-bromopyrimidin-2-amine (39). A stirred solution of 2-aminopyridine (2.5 g, 12 mmol) in
MeCN (25 ml) cooled in an ice water bath was treated with N-bromosuccinimide (4.6 g, 28
mmol). The resulting solution was stirred in the dark at room temperature overnight. The solvent
was evaporated under reduced pressure. Water (100mL) added, and the precipitate filtered and
washed with ice water. The crude product was dried in a vacuum desiccator to obtain 39 as an
off white powder (4.4 g, 97%). m.p.: 241−243 °C.
4.2. General Procedure A for the Synthesis of Compounds (40a-j). A mixture of appropriate
aryl bromide (10 mmol), appropriate boronic acid/ester (12 mmol), Pd(dppf)Cl2 ( 0.50 mmol), aq
Na2CO3 (1 M; 20 mL) in DME (80 mL) was heated 85 °C overnight under nitrogen. The cooled
reaction mixture was absorbed onto silica gel, and solvent was removed by evaporation. The
20

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residue obtained was purified by column chromatography (eluent gradient CH2Cl2: MeOH =
90:1), gave 40a−j.
4.3. 5-phenylpyrimidin-2-amine (40a). Compound 40a was prepared according to general
1
procedure A. Yield: 86%; m.p.: 159−161 °C; HNMR (400 MHz, DMSO-d6): δ 8.57 (s, Ar-H,
2H), 7.62 (d, J = 7.6 Hz, Ar-H, 2H), 7.45 (t, J = 7.2 Hz, Ar-H, 2H), 7.34 (t, J = 7.2 Hz, Ar-H,
1H), 6.77 (s, NH, 2H); ESI-MS: m/z = 172 [M+1]+.
4.4. 5-(3-fluorophenyl)pyrimidin-2-amine (40b). Compound 40b was prepared according to
1
general procedure A. Yield: 83%; m.p.: 168−170 °C; HNMR (400 MHz, DMSO-d6): δ 8.62 (s,
Ar-H, 2H), 7.53–7.43 (m, Ar-H, 3H), 7.16−7.11 (m, Ar-H, 1H), 6.86 (s, NH2, 2H); ESI-MS: m/z
= 190 [M+1]+ .
4.5. 5-(4-fluorophenyl)pyrimidin-2-amine (40c). Compound 40c was prepared according to
1
general procedure A. Yield: 85%; m.p.: 172−174 °C; HNMR (400 MHz, DMSO-d6): δ 8.58 (s,
Ar-H, 2H), 7.68−7.64 (m, Ar-H, 2H), 7.29 (t, J = 8.8 Hz, Ar-H, 2H), 6.78 (s, NH2, 2H); ESI-MS:
m/z = 190 [M+1]+.
4.6. 5-(3-methoxyphenyl)pyrimidin-2-amine (40d). Compound 40d was prepared according to
1
general procedure A. Yield: 87%; m.p.: 133−135 °C; HNMR (400 MHz, DMSO-d6): δ 8.58 (s,
Ar-H, 2H), 7.36 (t, J = 8.0 Hz, Ar-H, 1H), 7.18 (d, J = 7.6 Hz, Ar-H, 2H), 6.90 (dd, J = 8.4 Hz,
1.6 Hz, Ar-H, 1H), 6.78 (s, NH2, 2H), 3.81 (s, CH3, 3H); ESI-MS: m/z = 202 [M+1]+.
4.7. 5-(4-methoxyphenyl)pyrimidin-2-amine (40e). Compound 40e was prepared according to
1
general procedure A. Yield: 82%; m.p.: 165−167 °C; HNMR (400 MHz, DMSO-d6): δ 8.51 (s,
Ar-H, 2H), 7.55 (d, J = 8.8 Hz, Ar-H, 2H), 7.01 (d, J = 8.8 Hz, Ar-H, 2H), 6.67 (s, NH2, 2H),
3.78 (s, CH3, 3H); ESI-MS: m/z = 202 [M+1]+.
4.8. 5-(pyridin-3-yl)pyrimidin-2-amine (40f). Compound 40f was prepared according to
general procedure A. Yield: 79%; m.p.: 183–185 °C; 1HNMR (400 MHz, DMSO-d6): δ 8.87 (s,
Ar-H, 1H), 8.64 (s, Ar-H, 2H), 8.53 (br, Ar-H, 1H), 8.05 (d, J = 7.6 Hz, Ar-H, 1H), 7.46 (d, J =
4.0 Hz, Ar-H, 1H), 6.89 (s, NH2, 2H); ESI-MS: m/z = 173 [M+1]+.
21

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4.9. 5-(pyridin-4-yl)pyrimidin-2-amine (40g). Compound 40g was prepared according to
general procedure A. Yield: 87%; m.p.: 196–198 °C; 1HNMR (400 MHz, DMSO-d6): δ 8.75 (s,
Ar-H, 2H), 8.58 (br, Ar-H, 2H), 7.70 (d, J = 4.8 Hz, Ar-H, 2H), 7.06 (s, NH2, 2H); ESI-MS: m/z
= 173 [M+1]+.
4.10. 5-(thiophen-2-yl)pyrimidin-2-amine (40h). Compound 40h was prepared according to
1
general procedure A. Yield: 84%; m.p.: 156−158 °C; HNMR (400 MHz, DMSO-d6): δ 8.53 (s,
Ar-H, 2H), 7.49 (d, J = 4.8 Hz, Ar-H, 1H), 7.38 (d, J = 2.8 Hz, Ar-H, 1H), 7.12 (t, J = 4.4 Hz,
Ar-H, 1H), 6.87 (s, NH2, 2H); ESI-MS: m/z = 178 [M+1]+.
4.11. 5-(furan-2-yl)pyrimidin-2-amine (40i). Compound 40i was prepared according to general
procedure A. Yield: 813%; m.p.: 156−158 °C; 1HNMR (400 MHz, DMSO-d6): δ 8.57 (s, Ar-H,
2H), 7.69 (s, Ar-H, 1H), 6.88 (s, NH, 2H), 6.78 (d, J = 2.0 Hz, Ar-H, 1H), 6.56 (s, Ar-H, 1H);
ESI-MS: m/z = 162 [M+1]+.
4.12. 5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (40j). Compound 40j was prepared
1
according to general procedure A. Yield: 85%; m.p.: 174−176 °C; HNMR (400 MHz, DMSO-
d6): δ 8.46 (s, Ar-H, 2H), 8.03 (s, Ar-H, 1H), 7.78 (s, Ar-H, 1H), 6.57 (s, NH2, 2H), 3.84 (s, CH3,
3H); ESI-MS: m/z = 176 [M+1]+.
4.13. General Procedure B for the Synthesis of Compounds (42a-k). A mixture of sodium
hydrogen (7.5 mmol), appropriate alcohol (5.0 mmol) in dry THF (100 mL) was cooled at 0 °C,
then the mixture was stirred at room temperature for 30 min. Adding the reaction liquid to the
mixture of 5-bromo-3-nitropicolinonitrile (5.0 mmol) in 50 THF (50 mL) dropwise at 0 °C then
the mixture was stirred at rt for 3 h. The reaction solution was treated with saturated NH4Cl and
was extracted with EtOAc. The organic layer was collected, washed with brine, and dried over
MgSO4, concentrated. The residue obtained was purified by column chromatography (eluent
gradient 10−20% petroleum ether in EtOAc), gave 42a−k.
4.14. Tert-butyl 3-((5-bromo-2-cyanopyridin-3-yl)oxy)piperidine-1-carboxylate (42a).
Compound 42a was prepared according to general procedure B. Yield: 79%; as light yellow oil;
1HNMR (500 MHz, CDCl3): δ 8.26 (s, Ar-H, 1H), 7.53 (s, Ar-H, 1H), 4.38 (br, CH, 1H), 3.63
22

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(br, CH, 1H), 3.51 (br, CH, 1H), 3.38 (br, CH, 2H), 1.98−1.96 (m, CH, 1H), 1.91−1.85 (m, CH,
2H), 1.51 (br, CH, 1H), 1.36 (s, CH3×3, 9H); ESI-MS: m/z = 382 [M+1]+.
4.15. Tert-butyl (R)-3-((5-bromo-2-cyanopyridin-3-yl)oxy)piperidine-1-carboxylate (42b).
Compound 42b was prepared according to general procedure B. Yield: 76%; as light yellow oil;
1HNMR (500 MHz, CDCl3): δ 8.26 (s, Ar-H, 1H), 7.53 (s, Ar-H, 1H), 4.38 (br, CH, 1H), 3.63
(br, CH, 1H), 3.51 (br, CH, 1H), 3.38 (br, CH, 2H), 1.98−1.96 (m, CH, 1H), 1.91−1.85 (m, CH,
2H), 1.51 (br, CH, 1H), 1.36 (s, CH3×3, 9H); ESI-MS: m/z = 382 [M+1]+.
4.16. Tert-butyl (S)-3-((5-bromo-2-cyanopyridin-3-yl)oxy)piperidine-1-carboxylate (42c).
Compound 42c was prepared according to general procedure B. Yield: 78%; as light yellow oil;
1HNMR (500 MHz, CDCl3): δ 8.26 (s, Ar-H, 1H), 7.53 (s, Ar-H, 1H), 4.38 (br, CH, 1H), 3.63
(br, CH, 1H), 3.51 (br, CH, 1H), 3.38 (br, CH, 2H), 1.98−1.96 (m, CH, 1H), 1.91−1.85 (m, CH,
2H), 1.51 (br, CH, 1H), 1.36 (s, CH3×3, 9H); ESI-MS: m/z = 382 [M+1]+.
4.17. Tert-butyl 4-((5-bromo-2-cyanopyridin-3-yl)oxy)piperidine-1-carboxylate (42d).
Compound 42d was prepared according to general procedure B. Yield: 80%; m.p.: 97−99 °C;
1HNMR (500 MHz, CDCl3): δ 8.34 (s, Ar-H, 1H), 7.51 (s, Ar-H, 1H), 4.66−4.62 (m, CH, 1H),
3.69−3.63 (m, CH2, 2H), 3.52−3.46 (m, CH2, 2H), 1.99−1.92 (m, CH2, 2H), 1.90−1.82 (m, CH2,
2H), 1.47 (s, CH3×3, 9H); ESI-MS: m/z = 382 [M+1]+.
4.18. 5-bromo-3-((1-methylpiperidin-4-yl)oxy)picolinonitrile (42e). Compound 42e was
prepared according to general procedure B. Yield: 51%; as light yellow oil; 1HNMR (500 MHz,
CDCl3): δ 8.44 (s, Ar-H, 1H), 8.28 (s, Ar-H, 1H), 4.81−4.78 (m, CH, 1H), 2.57 (br, CH2, 2H),
2.32−2.28 (m, CH2, 2H), 2.21 (s, CH3, 3H), 1.96−1.92 (m, CH2, 2H), 1.75−1.69 (m, CH2, 2H);
ESI-MS: m/z = 296 [M+1]+.
4.19. Tert-butyl4-(((5-bromo-2-cyanopyridin-3-yl)oxy)methyl)piperidine-1-carboxylate(42f).
Compound 42f was prepared according to general procedure B. Yield: 80%; as light yellow oil;
1HNMR (500 MHz, CDCl3): δ 8.34 (s, Ar-H, 1H), 7.51 (s, Ar-H, 1H), 4.20 (d, J = 11.5 Hz, CH2,
2H), 3.95 (br, CH2, 2H), 2.80 (br, CH2, 2H), 2.14−2.05 (m, CH, 1H), 1.89 (br, CH2, 2H), 1.47 (s,
CH3×3, 9H), 1.33−1.26 (m, CH2, 2H)；ESI-MS: m/z = 206 [M+1]+.
23

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4.20. Tert-butyl (R)-3-((5-bromo-2-cyanopyridin-3-yl)oxy)pyrrolidine-1-carboxylate (42g).
Compound 42g was prepared according to general procedure B. Yield: 75%; as light yellow oil;
1HNMR (500 MHz, DMSO-d6): δ 8.48 (d, J = 2.0 Hz, Ar-H, 1H), 8.29 (s, Ar-H, 1H), 5.32 (br,
CH, 1H), 3.62−3.55 (m, CH, 1H), 3.49−3.44 (m, CH2, 2H), 3.39−3.33 (m, CH2, 1H), 2.21−2.16
(m, CH2, 1H), 2.13−2.09 (m, CH2, 1H), 1.41 (s, CH3×3, 9H); ESI-MS: m/z = 368 [M+1]+.
4.21. 5-bromo-3-(2-(dimethylamino)ethoxy)picolinonitrile (42h). Compound 42h was
prepared according to general procedure B. Yield: 77%; as light yellow oil; 1HNMR (500 MHz,
CDCl3): δ 8.27 (s, Ar-H, 1H), 7.49 (s, Ar-H, 1H), 4.16 (t, J = 7.0 Hz, CH2, 2H), 2.78 (t, J = 7.0
Hz, CH2, 2H), 2.31 (s, CH3×2, 6H); ESI-MS: m/z = 270 [M+1]+.
4.22. (R)-5-bromo-3-((1-(dimethylamino)propan-2-yl)oxy)picolinonitrile (42i). Compound
1
42i was prepared according to general procedure B. Yield: 70%; as light yellow oil; HNMR
(500 MHz, CDCl3): δ 8.31 (s, Ar-H, 1H), 7.62 (s, Ar-H, 1H), 4.62−4.55 (m, CH, 1H), 2.76−2.71
(m, CH2, 1H), 2.54−2.49 (m, CH2, 1H), 2.31 (s, CH3×2, 6H), 1.40 (d, J = 8.0 Hz, CH3, 3H); ESI-
MS: m/z = 284 [M+1]+.
4.23. (S)-5-bromo-3-((1-(dimethylamino)propan-2-yl)oxy)picolinonitrile (42j). Compound
1
42j was prepared according to general procedure B. Yield: 74%; as light yellow oil; HNMR
(500 MHz, CDCl3): δ 8.31 (s, Ar-H, 1H), 7.62 (s, Ar-H, 1H), 4.62−4.55 (m, CH, 1H), 2.76−2.71
(m, CH2, 1H), 2.54−2.49 (m, CH2, 1H), 2.31 (s, CH3×2, 6H), 1.40 (d, J = 8.0 Hz, CH3, 3H); ESI-
MS: m/z = 284 [M+1]+.
4.24. General Procedure C for the Synthesis of Compounds (6-15). A mixture of appropriate
aryl bromides (0.50 mmol), appropriate arylamine (0.50 mmol), Pd2 (dba)3 ( 0.0050 mmol),
Xantphos ( 0.012 mmol), Cs2CO3(1.0 mmol) in dry dioxane (10 mL) was heated 101 °C for 5 h
under nitrogen. The cooled reaction mixture was absorbed onto silica gel, and solvent was
removed by evaporation. The residue obtained was purified by column chromatography (eluent
gradient 4-5% EtOH in CH2Cl2), gave intermediate (79%−87%). CF3COOH (2.2 mmol) mL)
was added in a mixture of intermediate (0.36 mmol) in CH2Cl2 (2.2 mL). The reaction mixture
was stirred at room temperature for 2 h. Then the solvent was removed by evaporation, the
residue obtained was purified by column chromatography (eluent CH2Cl2: MeOH: TEA =10:1:1),
gave 6−15.
24

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4.25. 5-((5-phenylpyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile (6). Compound
1
6 was prepared according to general procedure C. Yield: 75%; m.p.: 84−86 °C; HNMR (500
MHz, DMSO-d6): δ 10.55 (s, NH, 1H), 8.99 (s, Ar-H, 2H), 8.65 (d, J = 2.0 Hz, Ar-H, 1H), 8.39
(d, J = 2.0 Hz, Ar-H, 1H), 7.78 (d, J = 7.0 Hz, Ar-H, 2H), 7.52 (t, J = 7.5 Hz, Ar-H, 2H), 7.43 (t,
J = 7.0 Hz, Ar-H, 1H), 4.42−4.37 (m, CH, 1H), 3.19 (d, J = 12.0 Hz, 2.0 Hz, CH2, 1H),
2.81−2.77 (m, CH2, 1H), 2.66−2.62 (m, CH2, 1H), 2.56−2.53 (m, CH2, 1H), 2.14−2.11 (m, CH2,
1H), 1.76−1.71 (m, CH2, 1H), 1.65−1.58 (m, CH2, 1H), 1.54–1.46 (m, CH2, 1H). 13C NMR (125
MHz, DMSO-d6): δ 158.33, 157.24, 155.92, 141.82, 134.17, 134.03, 129.13, 127.91, 125.97,
116.20, 113.83, 109.24, 74.69, 49.64, 45.28, 29.82, 24.26; ESI-MS: m/z = 373 [M+1]+. ESI-
HRMS m/z calcd for C21H21N6O+ 373.1777, found 373.1779 [M + H] +. HPLC purity 97%.
4.26. 5-((5-(3-fluorophenyl)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile (7).
Compound 7 was prepared according to general procedure C. Yield: 72%; m.p.: 107−109 °C;
1HNMR (500 MHz, DMSO-d6): δ 10.59 (br, NH, 1H), 9.03 (s, Ar-H, 2H), 8.65 (d, J = 1.5 Hz,
Ar-H, 1H), 8.37 (s, Ar-H, 1H), 7.70 (d, J = 10.5 Hz, Ar-H, 1H), 7.65 (d, J = 8.0 Hz, Ar-H, 1H),
7.56 (dd, J = 14.0 Hz, 8.0 Hz, Ar-H, 1H), 7.26 (td, J = 8.5 Hz, 2.5 Hz, Ar-H, 1H), 4.42−4.37 (m,
CH, 1H), 3.19 (dd, J = 12.0 Hz, 2.0 Hz, CH2, 1H), 2.82−2.78 (m, CH2, 1H), 2.67−2.63 (m, CH2,
1H), 2.57−2.52 (m, CH2, 1H), 2.14−2.11 (m, CH2, 1H), 1.77−1.71 (m, CH2, 1H), 1.66−1.59 (m,
CH2, 1H), 1.54−1.47 (m, CH2, 1H)；13C NMR (125 MHz, DMSO): δ 163.73, 161.79, 158.62,
157.25, 156.23, 141.75, 136.54, 136.48, 134.24, 131.17, 131.10, 124.71, 122.02, 121.99, 116.20,
114.71, 114.54, 113.97, 112.85, 112.67, 109.39, 74.64, 49.55, 45.24, 29.77, 24.17. ESI-HRMS
m/z calcd for C21H20FN6O+ 391.1683, found 391.1687 [M + H] +. HPLC purity 98%.
4.27. 5-((5-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile (8).
Compound 8 was prepared according to general procedure C. Yield: 78%; m.p.: 182−184 °C;
1HNMR (500 MHz, DMSO-d6): δ 10.52 (br, NH, 1H), 8.94 (d, J = 1.0 Hz, Ar-H, 2H), 8.63 (d, J
= 2.0 Hz, Ar-H, 1H), 8.35 (d, J = 2.0 Hz, Ar-H, 1H), 7.87 – 7.74 (m, Ar-H, 2H), 7.32 (t, J = 9.0
Hz, Ar-H, 2H), 4.37 (dt, J = 8.5, 4.5 Hz, CH, 1H), 3.17 (ddd, J = 12.0, 4.0, 1.5 Hz, CH2, 1H),
2.78 (dt, J = 12.5, 4.5 Hz, CH2, 1H), 2.63 (dd, J = 12.0, 8.0 Hz, CH2, 1H), 2.56–2.51 (m, CH2,
1H), 2.15–2.08 (m, CH2, 1H), 1.73 (dp, J = 13.0, 4.0 Hz, CH2, 1H), 1.65–1.55 (m, CH2, 1H),
1.54–1.43 (m, CH2, 1H).13C NMR(125 MHz, DMSO-d6)：δ 163.08, 161.13, 158.26, 157.24,
155.89, 141.80, 134.14, 130.58, 130.56, 128.16, 128.10, 125.14, 116.20, 116.08, 115.91, 113.83,
25

ACCEPTED MANUSCRIPT
109.20, 74.69, 49.65, 45.28, 29.82, 24.27. ESI-HRMS m/z calcd for C21H20FN6O+ 391.1683,
found 391.1684 [M + H] +. HPLC purity 98%.
4.28. 5-((5-(3-methoxyphenyl)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile (9).
Compound 9 was prepared according to general procedure C. Yield: 74%; m.p.: 102−104 °C;
1HNMR (500 MHz, DMSO-d6): δ 10.53 (br, NH, 1H), 8.97 (s, Ar-H, 2H), 8.64 (d, J = 2.0 Hz,
Ar-H, 1H), 8.35 (d, J = 2.0 Hz, Ar-H, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.35–7.27 (m, Ar-H, 2H),
6.96 (ddd, J = 8. 5, 2.5, 1.0 Hz, Ar-H, 1H), 4.38 (tt, J = 8.0, 4.0 Hz, CH, 1H), 3.83 (s, CH3, 3H),
3.17 (ddd, J = 12.0,4.0, 1.5 Hz, CH2, 1H), 2.78 (dt, J = 12.5, 4.5 Hz, CH2, 1H), 2.64 (dd, J = 12.0,
8.0 Hz, CH2, 1H), 2.57–2.51 (m, CH2, 1H), 2.17–2.06 (m, CH2, 1H), 1.73 (ddt, J = 13.5, 9.0, 4.5
13
Hz, CH2, 1H), 1.68–1.56 (m, CH2, 1H), 1.55–1.43 (m, CH2, 1H). C NMR(125 MHz, DMSO-
d6): δ 159.91, 158.39, 157.24, 156.02, 141.82, 135.42, 134.17, 130.22, 125.85, 118.18, 116.22,
113.83, 113.64, 111.36, 109.21, 74.66, 55.22, 49.63, 45.29, 29.81, 24.24. ESI-HRMS m/z calcd
+
for C22H23N6O2 403.1882, found 403.1881 [M + H] +. HPLC purity 97%.
4.29.
5-((5-(4-methoxyphenyl)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile
(10). Compound 10 was prepared according to general procedure C. Yield: 75%; m.p.:
218−220 °C; 1HNMR (400 MHz, DMSO-d6): δ 10.46 (s, NH, 1H), 8.90 (s, Ar-H, 2H), 8.62 (d, J
= 2.0 Hz, Ar-H, 1H), 8.35 (d, J = 2.0 Hz, Ar-H, 1H), 7.88–7.48 (m, Ar-H, 2H), 7.21–6.88 (m,
Ar-H, 2H), 4.37 (dt, J = 8.8, 4.4 Hz, CH, 1H), 3.80 (s, CH3, 3H), 3.24–3.11 (m, CH, 1H), 2.78
(dt, J = 12.4, 4.2 Hz, CH, 1H), 2.63 (dd, J = 12.2, 8.0 Hz, CH, 1H), 2.57–2.51 (m, CH, 1H),
2.17–2.05 (m, CH, 1H), 1.75–1.70 (m, CH, 1H), 1.65–1.56 (m, CH, 1H), 1.53–1.45 (m, CH,
1H).13C NMR(100 MHz, DMSO-d6): δ 159.24, 157.85, 157.25, 155.33, 141.91, 134.07, 127.19,
126.29, 125.84, 116.24, 114.60, 113.64, 109.00, 74.69, 55.20, 49.69, 45.31, 29.84, 24.31; ESI-
+
HRMS m/z calcd for C22H23N6O2 403.1882, found 403.1882 [M + H] +. HPLC purity 99%.
4.30. 3-(piperidin-3-yloxy)-5-((5-(pyridin-3-yl)pyrimidin-2-yl)amino)picolinonitrile (11).
Compound 11 was prepared according to general procedure C. Yield: 75%; m.p.: 221–223°C;
1HNMR (500 MHz, DMSO-d6): δ 10.60 (br, NH, 1H), 9.05 (s, Ar-H, 2H), 8.99 (dd, J = 2.5, 1.0
Hz, Ar-H, 1H), 8.66 (d, J = 2.0 Hz, Ar-H, 1H), 8.61 (dd, J = 4.5, 1.5 Hz, Ar-H, 1H), 8.36 (d, J =
2.0 Hz, Ar-H, 1H), 8.22–8.16 (m, Ar-H, 1H), 7.52 (ddd, J = 8.0, 5.0, 1.0 Hz, Ar-H, 1H), 4.40 (tt,
J = 8.0, 4.0 Hz, CH, 1H), 3.18 (ddd, J = 12.0, 4.0, 1.5 Hz, CH, 1H), 2.79 (dt, J = 12.5, 4.5 Hz,
CH, 1H), 2.66 (dd, J = 12.0, 8.0 Hz, CH, 1H), 2.55 (ddd, J = 12.5, 9.5, 3.0 Hz, CH, 1H), 2.17–
26

ACCEPTED MANUSCRIPT
2.08 (m, CH, 1H), 1.77–1.71 (m, CH, 1H), 1.66–1.58 (m, CH, 1H), 1.55–1.45 (m, CH, 1H). 13C
NMR (125 MHz, DMSO) δ 158.73, 157.28, 156.31, 148.93, 147.03, 141.76, 134.29, 133.51,
130.00, 124.00, 123.30, 116.23, 114.03, 109.47, 74.69, 49.61, 45.29, 29.82, 24.23. ESI-HRMS
m/z calcd for C20H20N7O+ 374.1729, found 403.1882 [M + H] +. HPLC purity 95%.
4.31. 3-(piperidin-3-yloxy)-5-((5-(pyridin-4-yl)pyrimidin-2-yl)amino)picolinonitrile (12).
Compound 12 was prepared according to general procedure C. Yield: 71%; m.p.: 232–234°C;
1HNMR (400 MHz, DMSO-d6): δ 10.67 (br, NH, 1H), 9.12 (s, Ar-H, 2H), 8.67 (d, J = 4.4 Hz,
Ar-H, 2H), 8.65 (s, Ar-H, 1H), 8.35 (s, Ar-H, 1H), 7.84 (d, J = 6.0 Hz, Ar-H, 2H), 4.41-4.37 (m,
CH, 1H), 3.20 (d, J = 12.0 Hz, CH2, 1H), 3.82-2.79 (m, CH2, 1H), 2.68-2.63 (m, CH2, 1H), 2.57-
2.52 (m, CH2, 1H), 2.14-2.12 (m, CH2, 1H), 1.76-1.73 (m, CH2, 1H), 1.67-1.58 (m, CH2, 1H),
1.55-1.46 (m, CH2, 1H). 1H NMR (400 MHz, DMSO-d6) δ 10.66 (br, NH, 1H), 9.10 (s, Ar-H,
2H), 8.80–8.54 (m, Ar-H, 3H), 8.33 (d, J = 2.0 Hz, Ar-H, 1H), 7.91–7.74 (m, Ar-H2, H), 4.38 (dq,
J = 8.4, 4.2 Hz, CH, 1H), 3.24–3.10 (m, CH, 1H), 2.79 (dt, J = 12.4, 4.4 Hz, CH, 1H), 2.64 (dd, J
= 12.2, 8.0 Hz, CH, 1H), 2.59–2.52 (m, CH, 1H), 2.11 (dd, J = 12.4, 5.2 Hz, CH, 1H), 1.76–1.70
(m, CH, 1H), 1.65–1.56 (m, CH, 1H), 1.54–1.45 (m, CH, 1H). 13C NMR (100 MHz, DMSO-d6):
159.23, 157.17, 156.46, 150.27, 141.50, 141.31, 134.32, 122.96, 120.08, 116.10, 114.25, 109.67,
74.71, 49.63, 45.28, 29.80, 24.24; ESI-HRMS m/z calcd for C20H20N7O+ 374.1729, found
374.1728 [M + H] +. HPLC purity 97%.
4.32. 3-(piperidin-3-yloxy)-5-((5-(thiophen-2-yl)pyrimidin-2-yl)amino)picolinonitrile (13).
Compound 13 was prepared according to general procedure C. Yield: 74%; m.p.: 212–214 °C;
1HNMR (500 MHz, DMSO-d6): δ 10.57 (br, NH, 1H), 8.92 (s, Ar-H, 2H), 8.59 (d, J = 2.0 Hz,
Ar-H, 1H), 8.34 (d, J = 2.0 Hz, Ar-H, 1H), 7.71–7.50 (m, Ar-H, 2H), 7.18 (dd, J = 5.0, 3.5 Hz,
Ar-H, 1H), 4.38 (tt, J = 8.0, 4.0 Hz, CH, 1H), 3.16 (ddd, J = 12.0, 4.0, 1.5 Hz, CH, 1H), 2.78 (dt,
J = 12.5, 4.5 Hz, CH, 1H), 2.64 (dd, J = 12.0, 8.0 Hz, CH, 1H), 2.54 (td, J = 9.5, 5.0 Hz, CH,
1H), 2.15–2.08 (m, CH, 1H), 1.77–1.68 (m, CH, 1H), 1.67–1.56 (m, CH, 1H), 1.55–1.44 (m, CH,
1H); 13C NMR(125 MHz, DMSO-d6): δ 157.99, 157.24, 154.62, 141.66, 136.45, 134.17, 128.54,
126.14, 124.27, 121.12, 116.20, 113.90, 109.23, 74.64, 56.01, 49.62, 45.30, 29.81, 24.26; ESI-
HRMS m/z calcd for C19H19N6OS+ 379.1341, found 379.1342 [M + H] +. HPLC purity 98%.
4.33. 5-((5-(furan-2-yl)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile (14).
Compound 14 was prepared according to general procedure C. Yield: 70%; m.p.: 200–202 °C;
1HNMR (400 MHz, DMSO-d6): δ 10.56 (br, NH, 1H), 8.93 (s, Ar-H, 2H), 8.57 (d, J = 2.0 Hz,
27

ACCEPTED MANUSCRIPT
Ar-H, 1H), 8.33 (d, J = 2.0 Hz, Ar-H, 1H), 7.79 (d, J = 2.0 Hz, Ar-H, 1H), 7.03 (d, J = 3.6 Hz,
Ar-H, 1H), 6.63 (dd, J = 3.6, 2.0 Hz, Ar-H, 1H), 4.37 (td, J = 8.0, 4.0 Hz, CH, 1H), 3.16 (dt, J =
12.0, 2.8 Hz, CH, 1H), 2.78 (dt, J = 12.4, 4.4 Hz, CH, 1H), 2.63 (dd, J = 12.4, 8.0 Hz, CH, 1H),
2.58–2.51 (m, CH, 1H), 2.11 (dd, J = 12.4, 5.2 Hz, CH, 1H), 1.75–1.70 (m, CH, 1H), 1.65–1.56
(m, CH, 1H), 1.53–1.44 (m, CH, 1H). 13C NMR (100 MHz, DMSO-d6): δ 157.75, 157.22,
153.17, 148.22, 143.35, 141.61, 134.16, 117.89, 116.18, 113.88, 112.07, 109.23, 106.18, 74.66,
+
49.63, 45.30, 29.81, 24.27; ESI-HRMS m/z calcd for C19H19N6O2 363.1569, found 363.1569 [M
+ H] +. HPLC purity 97%.
4.34.
5-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-3-(piperidin-3-
yloxy)picolinonitrile (15). Compound 15 was prepared according to general procedure C. Yield:
74%; m.p.: 227–229 °C; 1HNMR (500 MHz, DMSO-d6): δ 10.43 (s, NH, 1H), 8.87 (s, Ar-H, 2H),
8.58 (d, J = 2.0 Hz, Ar-H,1H), 8.35 (d, J = 2.0 Hz, Ar-H, 1H), 8.23 (s, Ar-H, 1H), 7.97 (d, J =
1.0 Hz, Ar-H, 1H), 4.37 (tt, J = 8.0, 4.0 Hz, Ar-H, 1H), 3.88 (s, CH3, 3H), 3.17 (ddd, J = 12.0,
4.0, 1.5 Hz, CH , 1H), 2.79 (dt, J = 12.5, 4.5 Hz, CH, 1H), 2.64 (dd, J = 12.0, 8.0 Hz, CH, 1H),
2.55 (dd, J = 9.5, 3.0 Hz, CH, 1H), 2.13 (dt, J = 11.0, 5.0 Hz, CH, 1H), 1.74 (dp, J = 13.0, 4.5,
4.0 Hz, CH, 1H), 1.61 (dtd, J = 12.5, 9.5, 9.0, 4.0 Hz, CH, 1H), 1.50 (dddd, J = 16.5, 13.0, 8.5,
5.0 Hz, CH, 1H). 13C NMR (125 MHz, DMSO-d6): δ 157.30, 154.28, 142.00, 135.77, 134.01,
127.60, 119.79, 116.31, 115.48, 113.43, 108.73, 74.63, 49.71, 45.33, 38.77, 29.85, 24.30. ESI-
HRMS m/z calcd for C19H21N8O+ 377.1838, found 377.1837 [M + H] +. HPLC purity 99%.
4.35. 5-((5-bromopyrimidin-2-yl)amino)picolinonitrile (43). Compound 43 was prepared
according to general procedure C. Yield: 70%; 1H NMR (400 MHz, DMSO-d6): δ 10.64 (s, NH,
1H), 8.98 (s, Ar-H, 1H), 8.76 (s, Ar-H, 2H), 8.43 (d, J = 11.0 Hz, Ar-H, 1H), 7.98 (d, J = 10.5
Hz, Ar-H, 1H); ESI-MS: m/z = 276 [M+1]+.
4.36.
Tert-butyl
3-((methylsulfonyl)oxy)piperidine-1-carboxylate
(45).
1-N-tert-
butyloxycarbonyl-3-hydroxypiperidine (1.4 g, 7.0 mmol), methanesulfonyl chloride (1.2 g, 10.5
mmol) and DMAP (171 mg, 1.4 mol) with triethylamine (1.8 g, 1.8 mol) suspend in 14 mL
CH2Cl2. The reaction mixture was stirred at 0 °C for 1 h, then the mixture was partitioned
between aqueous citric acid (5%, 20 mL) and CH2Cl2 three times. The combined organic layers
28