Design, Synthesis, and Biological Evaluation of Orally Bioavailable CHK1 Inhibitors Active against Acute Myeloid Leukemia

Article abstract of DOI:10.1002/cmdc.202000882

Checkpoint kinase 1 (CHK1) is a central component in DNA damage response and has emerged as a target for antitumor therapeutics. Herein, we describe the design, synthesis, and biological evaluation of a novel series of potent diaminopyrimidine CHK1 inhibitors. The compounds exhibited moderate to potent CHK1 inhibition and could suppress the proliferation of malignant hematological cell lines. The optimized compound 13 had a CHK1 IC₅₀ value of 7.73±0.74 nM, and MV-4-11 cells were sensitive to it (IC₅₀=0.035±0.007 μM). Furthermore, compound 13 was metabolically stable in mouse liver microsomes in vitro and displayed moderate oral bioavailability in vivo. Moreover, treatment of MV-4-11 cells with compound 13 for 2 h led to robust inhibition of CHK1 autophosphorylation on serine 296. Based on these biochemical results, we consider compound 13 to be a promising CHK1 inhibitor and potential anticancer therapeutic agent.

Full text of DOI:10.1002/cmdc.202000882

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Design, Synthesis, and Biological Evaluation of Orally
Bioavailable CHK1 Inhibitors Active against Acute Myeloid
Leukemia
+
[a]
+ [b]
[a]
[b, c]
[d]
Tingting Jin , Peipei Wang , Xiubing Long, Kailong Jiang,
Pinrao Song,*
[b, c]
[b]
[b, c, e]
[b, c, e]
[a]
Wenbiao Wu,
Gaoya Xu, Yubo Zhou,*
Jia Li,*
and Tao Liu*
Checkpoint kinase 1 (CHK1) is a central component in DNA
damage response and has emerged as a target for antitumor
therapeutics. Herein, we describe the design, synthesis, and
biological evaluation of a novel series of potent diaminopyr-
imidine CHK1 inhibitors. The compounds exhibited moderate to
potent CHK1 inhibition and could suppress the proliferation of
malignant hematological cell lines. The optimized compound
were sensitive to it (IC =0.035�0.007 μM). Furthermore,
5
0
compound 13 was metabolically stable in mouse liver micro-
somes in vitro and displayed moderate oral bioavailability
in vivo. Moreover, treatment of MV-4-11 cells with compound
13 for 2 h led to robust inhibition of CHK1 autophosphorylation
on serine 296. Based on these biochemical results, we consider
compound 13 to be a promising CHK1 inhibitor and potential
anticancer therapeutic agent.
1
3 had a CHK1 IC50 value of 7.73�0.74 nM, and MV-4-11 cells
Introduction
network ensures that DNA repair pathways are in response to
DNA damage, avoiding mitotic catastrophe and resultant cell
[5,6]
Checkpoint kinase 1 (CHK1) is a serine/threonine kinase that
plays an important role in the DNA damage response (DDR).
Upon damage by genotoxic agents, ionic irradiation or faults in
the DNA replication process, CHK1 is activated by upstream
kinase ataxia telangiectasia and Rad3-related (ATR), and in turn
phosphorylates downstream protein CDC25 resulting in cell-
death or apoptosis. Whilst normal cells arrest at S, G1/S and
G2/M checkpoints for DNA repair, cancer cells harbor functional
defects in p53 and are dependent upon S and G2/M
checkpoints to repair DNA damage. Previous studies have
shown that the inhibition of CHK1 can abrogate S and G2/M
[
1]
[7–9]
checkpoints, promoting cancer cell death.
CHK1 inhibitors
[2–4]
cycle arrest in the S or G2/M phase.
This complex signaling
therefore hold promise as a promising class of anticancer
therapeutic agents.
+
Considerable efforts have been made to identify CHK1
inhibitors and several molecules have reached early clinical
trials (Figure 1), but only prexasertib (LY2606368) and SRA737
[
a] T. Jin, X. Long, Prof. T. Liu
College of Pharmaceutical Sciences
ZJU-ENS Joint Laboratory of Medicinal Chemistry
Zhejiang Province Key Laboratory of Anti-Cancer Drug Research
Zhejiang University
(CCT245737) have entered phase II clinical trials. First-gener-
Hangzhou 310058 (P. R. China)
E-mail: Lt601@zju.edu.cn
ation CHK1 inhibitors such as AZD-7762, PF00477736 and
SCH900776 show low selectivity, limited efficacy and unfavor-
able toxicity, further clinical developments of these molecules
2
1619053@zju.edu.cn
+
[
b] P. Wang, Dr. K. Jiang, W. Wu, G. Xu, Dr. Y. Zhou, Dr. J. Li
National Center for Drug Screening
State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
[10–12]
have been halted.
Moreover, they are administered intra-
venously. To overcome these limitations, second-generation
Shanghai 201203 (P. R. China)
E-mail: ybzhou@simm.ac.cn
jli@simm.ac.cn
[
c] Dr. K. Jiang, W. Wu, Dr. Y. Zhou, Dr. J. Li
University of Chinese Academy of Sciences
No. 19 A Yuquan Road, Beijing 100049 (P. R. China)
d] Dr. P. Song
[
Shanghai Jemincare Pharmaceuticals Co. Ltd
Jemincare Group Research Institute
1118 Halei Road, Shanghai, 201203 (P. R. China)
E-mail: songpinrao@jemincare.com
e] Dr. Y. Zhou, Dr. J. Li
Zhongshan Institute of Drug Discovery
Institution for Drug Discovery Innovation
Chinese Academy of Science
[
[
Zhongshan, 528400 (P. R. China)
+
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000882
Figure 1. Structures of the clinical candidate checkpoint kinase inhibitors.
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CHK1 inhibitors have been synthesized. SRA737, LY2880070 and
GDC-0575 show an increased selectivity, potency and safety,
and all are orally bioavailable, which would provide more
stable. Based on SAR exploration (Figure 3), we expanded the
library of diaminopyrimidine derivatives and assayed their
inhibitory activity against CHK1. We further evaluated the anti-
proliferative activity of the compounds against a range of
malignant hematological cell lines, and explored their metabolic
stability in vitro and pharmacokinetics in vivo. Moreover, we
evaluated the effects of representative compounds on the
inhibition of CHK1 signaling.
[13,14]
flexible administration.
In addition, a great amount of CHK1
inhibitors in combination with various DNA-damaging agents
have been evaluated of their therapeutic effect against different
types of solid tumors in early clinical trials, including non-small-
cell lung cancer (NCT01139775), head and neck cancer
[15–17]
(NCT02555644), melanoma (NCT00779584) and others.
Until recently, few evidences suggested that CHK1 inhibitors
exhibited single-agent activity in inducing cell death in human Results and Discussion
leukemia and lymphoma cells, including B-cell lymphoma,
[18–20]
chronic lymphocytic leukemia, and acute myeloid leukemia.
Chemistry
Thus, there still remains a strong need for novel CHK1 inhibitors
with oral bioavailability and inhibitory activity against leukemia
and lymphomas.
The synthesis of the compounds 2–9 are outlined in Scheme 1.
Initially, the commercially available 5-bromo-2, 4-dichloropyr-
imidine 17 was treated with tert-butyl 4-aminopiperidine-1-
carboxylate in acetonitrile to produce 18, which was subse-
quently aminated to afford 19. Suzuki cross-coupling of
compound 19 was then performed with various boronic acids/
esters to provide 20–25, which subsequently underwent
Buchwald-Hartwig reactions with 5-bromo-2-cyanopyridine fol-
lowed by TFA deprotection to give the final products 2–7.
Commercially available 5-bromo-2-cyanopyridine was installed
via a Buchwald–Hartwig reaction with 19 to afford 26. The Boc
group was then removed through treatment with TFA to yield
the final product 8. Suzuki cross-coupling of 26 with lithium
triisopropoxy(4-methylthiazol-2-yl)borate was performed and
followed by TFA deprotection to give the final product 9.
The synthesis of compounds 10–16 are shown in Scheme 2.
Treatment of compound 17 with various commercially available
amines gave 27–32, which were subsequently aminated to
produce the corresponding intermediates 33–38. These inter-
mediates were then subjected to Suzuki cross-coupling with 1-
methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole to produce intermediates 39–44. The Buchwald–
Hartwig reaction of 39–43 followed by TFA deprotection
afforded the final products 10–14. Buchwald-Hartwig reaction
of 44 directly provided the final compounds 15 and 16.
Our group has been continuously developing anticancer
drugs especially small molecule protein kinase inhibitors and
established an in-house compound library. In our previous
[21]
work
lead compound MCL-1020 (1) with moderate CHK1
inhibition (IC =1.61 μM) was found through virtual screening
50
in-house compound library. Further SAR exploration at 3-
position of pyridine led to the identification of a series of 2-
amino pyrimidine derivatives, these compounds showed potent
CHK1 inhibition. However, the most promising compound
suffered from poor pharmacokinetic profile and was adminis-
tered intravenously. Oral compounds provided an advantage of
flexibility in administration. To obtain oral compounds, we
refocused on our initial screening lead compound MCL-1020.
From potential binding mode of MCL-1020 with CHK1 (Fig-
ure 2), we found that they could form hydrogen bonds in the
hinge region and specificity pocket. However, MCL-1020 lacked
interactions with polar residues as Glu91, Glu134 and Asn135 in
the ribose region of CHK1, which had been proved important
for CHK1 inhibition. We explored whether amine substituents
on the C-4 of pyrimidine are located in this region and make
interactions with CHK1. Introduction of a piperidin-4-amine at
the C-4 position of pyrimidine led to compound 2. We are
encouraged that compound 2 showed an increased CHK1
inhibitory potency. Considering phenyl group at the C-5
position of pyrimidine of MCL-1020 was easily hydroxylated to
result in poor pharmacokinetic profile, we planned to modify
the C-5 substituents through replacement phenyl group with
five-membered heterocycles which were more metabolically
Figure 2. Binding characteristics of MCL-1020 (1).
Figure 3. Design of novel diaminopyrimidine CHK1 inhibitors.
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Scheme 2. Synthetic route of compounds 10–16. a) corresponding amine,
ACN, RT, 82–95%; b) NH
3
·H
2
O, ethanol, 100°C, 78–85%; c) 1-methyl-4-
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl
2
CO3, DME, reflux, 77–84%; d) 5-bromo-2-cyanopyridine or 5-bromopyr-
2
, 1N
Na
imidine-2-carbonitrile, Pd
e) TFA, CH Cl , 65–80%.
2 3 2 3
(dba) , Xantphos, Cs CO , dioxane, reflux, 70–85%;
2
2
Scheme 1. Synthetic route of compounds 2–9. a) tert-Butyl 4-aminopiper-
idine-1-carboxylate, ACN, RT, 82%; b) NH
corresponding boronic acid/ester, Pd(dppf)Cl
0%; d) 5-bromo-2-cyanopyridine, Pd (dba)
reflux, 70–85%; e) TFA, CH Cl
ylthiazol-2-yl)borate, Pd(dppf)Cl
3
·H
2
O, ethanol, 100°C, 80%; c)
Table 1. CHK1 inhibitory activity of compounds 2–9.
2
, 1N Na CO3, DME, reflux, 77–
2
9
2
3 2 3
, Xantphos, Cs CO , dioxane,
2
2
, 65–80%; f) lithium triisopropoxy(4-meth-
, DMF, 100°C, 45%.
2
, CuCl, ZnCl
2
, Cs
2
CO
3
[a]
In vitro structure-activity relationships of CHK1 inhibition
Cpd
2
R
CHK1 IC50 [nM]
139.40�22.63
19.59�10.68
Newly synthesized compounds 2–16 were assessed in ADP-
Glo™ Kinase Assays to investigate their inhibitory activity
against CHK1. Compound 2 was synthesized by introduction of
a piperidin-4-amine at the C-4 position of pyrimidine. It was
encouraging to find that compound 2 displayed increased
CHK1 inhibition (IC =139.40�22.63 nM) compared to 1. This
3
4
5
6
43.22�9.10
7.81�1.10
50
indicated that C-4 substituent of pyrimidine probably made
interactions with CHK1. Whereas, phenyl group was easily
hydroxylated, we further explored C-5 substituents of pyrimi-
dine through replacement phenyl group with five-membered
heterocycles. As shown in Table 1, the five-membered hetero-
cycles (3, 4, 5 and 9) showed excellent CHK1 inhibitory potency.
Interestingly, whilst ester substitutions on the thiophene or
furan ring (compounds 6 and 7) resulted in a loss of CHK1
89.07�21.26
7
8
9
32.99�5.15
45.32�8.27
3.31�1.22
Br
[
a] IC50 values are the mean of three independent experiments.
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approximately 5-fold loss of potency. Finally, replacement of
the pyridine with pyrimidine showed a similar CHK1 potency,
suggesting that no additional contacts with the CHK1 protein
were made (16 vs. 15).
Table 2. CHK1 inhibitory activity of compounds 10–16.
[
a]
Cpd
R
X
C
CHK1 IC50 [nM]
In vitro cytotoxic activity
1
0
1
35.32�8.26
Based on their highly potent CHK1 inhibitory activity, com-
pounds 5, 9, and 13 were selected for assessment of their
growth inhibitory activity against five malignant hematological
cell lines (RPMI8226, Mino, Ramos, Jeko-1, and MV-4-11) and
peripheral blood mononuclear cells (PBMC) from normal
donors. As shown in Table 3, compounds 5, 9 and 13 displayed
low inhibitory activity against RPMI8226 cells with IC50 values
1
C
C
46.70�0.81
1
2
169.20�14.57
>
1 μM. For Mino and Jeko-1 cells, three compounds showed
moderate inhibitory potency. And for Ramos cells, compounds
and 13 showed moderate inhibitory potency. Excitingly, all
1
1
3
4
C
C
7.73�0.74
5
315.00�252.58
241.25�19.87
three compounds strongly inhibited MV-4-11 cells with IC50
values of 0.044�0.001 μM, 0.035�0.006 μM and 0.035�
1
1
5
6
C
0
.007 μM, respectively. Meanwhile, all three compounds
showed low inhibitory potency on the PBMC. Compounds 5
and 9 showed excellent inhibitory potency but modestly lower
aqueous solubility compared to 13 (see the Supporting
Information). Based on the potency and solubility data,
compound 13 was selected for further studies.
N
358.00�125.72
1.20�0.05
staurosporine
[a] IC50 values are the mean of three independent experiments.
inhibition (compare 6 vs. 4; 7 vs. 5). Bromo-derivative 8 and
five-membered heterocycle 4 showed similar CHK1 inhibition.
Though compounds 5 and 9 showed potent inhibition of
CHK1 activity (IC <10 nM), both had lower aqueous solubility
Pharmacokinetic parameters of compound 13
As compound 13 showed potent CHK1 inhibition and strongly
inhibited the growth of MV-4-11 cells, it was selected for further
pharmacokinetic evaluation in rats. After oral dosing of 20 mg/
kg, plasma samples were collected at defined time points
(0.083, 0.25, 0.5, 1, 2, 4, 6, 24, 30 h). As shown in Table 4,
compound 13 showed moderate absorption and exposure (t1/
50
compared to 3 (see the Supporting Information). To obtain a
broader range of compounds, several other amines were added
to the C-4 position whilst 1-methylpyrazole substitution of
compound 3 was kept constant at the C-5 position (Table 2). As
was observed upon inserting a methylene between the amine
and piperidine (10), decreased potency was observed compared
to 3. Analogs 11–14 were synthesized by reducing the distance
of the two nitrogen atoms on substitutions at the C-4 position.
Compounds 10 and 11 showed similar CHK1 inhibition. The pair
of enantiomers 12 and 13 demonstrated that the S config-
uration was better than R, whilst compound 13 was 20 times
more potent than 12. It illustrated that chirality was significantly
important to anticancer activities. Surprisingly, the (R)-piperidin-
=5.3 h, Cmax =195.31 ng/mL, AUC0-t =1372.75 hng/mL). In ad-
2
dition, metabolic stability of compound 13 was evaluated in
mouse liver microsomes in vitro. Compound 13 showed high
levels of stability in mouse liver microsomes. These data
suggested that compound 13 was worth of further develop-
ment.
3
-amine (14) showed a pronounced loss of activity, suggesting
that a secondary amine next to the pyrimidine is required. The
replacement of amino (11) by morpholine (15) resulted in an
Table 3. In vitro growth inhibition of hematological cell lines with 5, 9, and 13.
[b]
Cpd
IC50[μM]
RPMI8226
Mino
Ramos
Jeko-1
MV-4-11
PBMC
5
9
1
3.339�0.316
1.149�0.031
3.597�0.287
0.409�0.035
0.708�0.156
1.954�0.294
0.608�0.082
0.185�0.032
0.536�0.074
0.342�0.015
0.269�0.008
0.253�0.050
0.048�0.013
0.044�0.001
0.035�0.006
0.035�0.007
0.036�0.005
3.551�0.832
1.148�0.187
7.874�1.015
>10
[
a]
NT
3
0.401�0.040
AZD7762
0.137�0.025
[a] NT indicates not-tested. [b] IC50 values are the mean of three independent experiments.
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Table 4. Pharmacokinetic profiles of compound 13.
Parameters
In rat
20 mg/kg, i.g.)
(
tmax (h)
t1/2 (h)
2.17
5.30
Cmax (ng/mL)
AUC0-t (hng/mL)
AUC0-1 (hng/mL)
MRT0-1 (h)
195.31
1372.75
1411.60
6.15
liver microsome stability
93
(%, remaining percent @ 0.5 h) [mouse]
Figure 5. Binding mode of compound 13 with CHK1 kinase (PDB ID: 2YM8).
Residues of CHK1 (green) that interact with 13 (magenta) are highlighted.
Images were prepared using Discovery Studio (version 2.1).
Effects of compound 13on the inhibition of CHK1 signaling
To fully understand the mechanism through which compound
1
3 inhibited the proliferation of MV-4-11 cells, we explored the
phosphorylation status of CHK1 through western blot analysis,
including CHK1 autophosphorylation on Ser296, and DNA
damage-mediated CHK1 phosphorylation on Ser345 by its
upstream kinase ataxia telangiectasia and Rad3-related (ATR).
The phosphorylation of serine 296 (pSer296) of CHK1 is the
formed two hydrogen bonds with Glu85 and Cys87 in the hinge
region of CHK1 which significantly contributed to its interaction.
The N of 2-cyano of pyridine formed a hydrogen bond with
Lys38 whilst the NH of pyridine formed a hydrogen bond with
[22]
autophosphorylation site of CHK1,
indicate the strength of different CHK1 inhibitors.
and its level can well
three conservative H O molecules. Moreover, the NH of
2
[
19,23–26]
The
piperidine formed hydrogen bonds with Glu91 and a water.
This docking highlighted the feasibility of design strategy.
phosphorylation of serine 345 (pSer345) of CHK1 is the key site
of CHK1 phosphorylation found in the C-terminal regulatory
domain of CHK1. Different from pSer296, CHK1 inhibitors
[27–29]
induce and enhance the level of pSer345 CHK1.
As shown Conclusion
in Figure 4, in the absence of compound 13, the autophosphor-
ylation of CHK1 was evident in MV-4-11 cells. Following
treatment of MV-4-11 cells with increasing concentrations of
compound 13 for 2 h, CHK1 autophosphorylation on Ser296
was strongly inhibited in a concentration-dependent manner,
particularly at 160 nM. Moreover, DNA damage-mediated ATR-
CHK1 signaling was also activated by compound 13 in a
concentration-dependent manner.
In this study, our efforts toward development of new
diaminopyrimidine derivatives of CHKI inhibitors with oral
bioavailability for the treatment of leukemia led to the
compound 13. Compound 13 exhibited potent CHK1 inhibitory
activity with IC50 value of 7.73�0.74 nM. Meanwhile, 13 showed
excellent cellular growth inhibitory activity, most notably in MV-
4-11 cells (IC =0.035�0.007 μM). Moreover, compound 13
5
0
was metabolically stable in mouse liver microsomes in vitro and
showed moderate oral bioavailability (AUC0-t =1372.75 hng/mL)
in vivo. Treatment of MV-4-11 cells with compound 13 for 2 h
also strongly inhibited the autophosphorylation of CHK1 on
S296. These biochemical data demonstrate that compound 13
represents a promising CHK1 inhibitor and is now worthy of
further exploration as an anticancer therapeutic agent.
Molecular modeling of compound 13 with CHK1
Since compound 13 displayed potent CHK1 inhibition and
suppressed the growth of cancer cell lines, it was docked into
the ATP-binding site of CHK1 to analyze its potential binding
mode. As shown in Figure 5, the 2-aminopyrimidine core
Experimental Section
Chemistry
Reagents and solvents were purchased from commercial sources
and used without further purification. Column chromatography was
performed on silica gel (200–300 mesh). Melting points were
determined on Büchi Melting Point B-540 apparatus without
correction. The mass spectrometer was operated in Shimadzu
LCMS-2020 mass spectrometer with methanol and water containing
1
13
0
.1% formic acid as mobile phases. H NMR and C NMR spectra
were measured on a Bruker Avance III spectrometer 500 MHz or
Bruker AM 400 MHz with CDCl3 or [D ]DMSO as the solvent.
6
Figure 4. Inhibition of CHK1 signaling in MV-4-11 cells treated with the
indicated concentrations of compound 13 for 2 h.
Chemical shifts were reported in parts per million (ppm) relative to
internal TMS. Coupling constants (J values) were reported in Hertz
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(Hz) and proton coupling patterns were designated as single (s),
tert-Butyl 4-((2-amino-5-(1-methyl-1H-pyrazol-4-yl)
doublet (d), double doublet (dd), triplet (t), quartet (q) and
multiplet (m). All assayed compounds were determined by high
performance liquid chromatography on an Agilent 1260 infinity
equipped with a COSMOSIL 5C18-AR-II column (4.6ID×250 mm).
Absorbance values were measured at 254 nm with a UV detector. A
flow rate of 1.0 mL/min was used with a mobile phase A, 0.1%
CH COONH , 0.1% HCOOH in H O and mobile phase B, CH OH. The
pyrimidin-4-yl)amino)piperidine-1-carboxylate (21)
1
General procedure A. Yield: 81%; H NMR (500 MHz, CDCl ): δ=7.66
3
(
4
1
s, 1H), 7.49 (s, 1H), 7.37 (s, 1H), 4.89 (s, 2H), 4.83 (d, J=8.0 Hz, 1H),
.15–4.08 (m, 1H), 4.04 (br, 2H), 3.96 (s, 3H), 2.93 (t, J=12.5 Hz, 2H),
.99–1.97 (m, 2H), 1.46 (s, 9H), 1.33–1.25 (m, 2H); LC-MS (ESI): m/z
+
calcd for C H N O : 374.22 [M+H] ; found: 374.23.
18 27
7
2
3
4
2
3
specific rotation data were determined by SGW®-533 at 589 nm
[α]D).
(
tert-Butyl 4-((2-amino-5-(thiophen-2-yl)pyrimidin-4-yl)amino)
piperidine-1-carboxylate (22)
tert-Butyl 4-((5-bromo-2-chloropyrimidin-4-yl)amino)
piperidine-1-carboxylate (18)
1
General procedure A. Yield: 80%; H NMR (500 MHz, [D ]DMSO): δ=
7
6
6
.73 (s, 1H), 7.51 (d, J=6.0 Hz, 1H), 7.13–7.11 (m, 1H), 7.08 (s, 1H),
.22 (br, 2H), 5.91 (d, J=10.0 Hz, 1H), 4.15–4.09 (m, 1H), 3.91–3.88
m, 2H), 2.82 (m, 2H), 1.80–1.78 (m, 2H), 1.45–1.39 (m, 2H), 1.38 (s,
A mixture of 5-bromo-2,4-dichloropyrimidine (0.5 mmol), tert-butyl
(
9
3
4
-aminopiperidine-1-carboxylate (0.55 mmol), and trimethylamine
+
H); LC-MS (ESI): m/z calcd for C H N O S: 376.17 [M+H] ; found:
18 25 5 2
(0.10 mL) in acetonitrile (11 mL) was stirred at room temperature
76.18.
for 2 h. Completion of the reaction was monitored by thin-layer
chromatography (TLC) on silica gel. The reaction mixture was
evaporated under reduced pressure. The residue was then purified
by column chromatography on silica gel (petroleum ether/ethyl
tert-Butyl 4-((2-amino-5-(furan-2-yl)pyrimidin-4-yl)amino)
1
piperidine-1-carboxylate (23)
acetate=4:1–3:1) to give compound 18. Yield: 82%; H NMR
(
4
1
500 MHz, [D ]DMSO): δ=8.26 (s, 1H), 7.38 (d, J=10.0 Hz, 1H), 4.17–
.08 (m, 1H), 3.98–3.95 (m, 2H), 2.82–2.79 (m, 2H), 1.76–1.72 (m, 2H),
1
6
General procedure A. Yield: 80%; H NMR (500 MHz, [D ]DMSO): δ=
6
7
1
.98 (s, 1H), 7.66 (s, 1H), 6.54 (m, 2H), 6.28 (s, 2H), 6.12 (d, J=
0.0 Hz, 1H), 4.22–4.15 (m, 1H), 3.93–3.91 (m, 2H), 2.84 (s, 2H), 1.86–
1.83 (m, 2H), 1.49–1.40 (m, 2H), 1.40 (s, 9H); LC-MS (ESI): m/z calcd
for C H N O : 360.20 [M+H] ; found: 360.22.
.61–1.51 (m, 2H), 1.41 (s, 9H); LC-MS (ESI): m/z calcd for
+
C H BrClN O : 391.05 [M+H] ; found: 391.06.
1
4
20
4
2
+
1
8
25
5
3
tert-Butyl 4-((2-amino-5-bromopyrimidin-4-yl)amino)
piperidine-1-carboxylate (19)
tert-Butyl 4-((2-amino-5-(5-(methoxycarbonyl)thiophen-2-yl)
pyrimidin-4-yl)amino)piperidine-1-carboxylate (24)
Compound 18 (1.23 mmol) was dissolved in ethanol (5 mL) in a
sealed tube. The reaction mixture was then added NH ·H O (5 mL)
1
3
2
General procedure A. Yield: 82%; H NMR (400 MHz, [D ]DMSO): δ=
6
and heated at 100°C for 12 h. The completion of the reaction was
monitored by TLC on silica gel and the reaction mixture was
evaporated under reduced pressure. After cooling to room temper-
ature, the residue was purified by column chromatography on silica
7
2
3
.80 (s, 1H), 7.77 (d, J=2.8 Hz, 1H), 7.16 (d, J=2.8 Hz, 1H), 6.39 (s,
H), 6.22 (d, J=6.4 Hz, 1H), 4.20–4.12 (m, 1H), 3.93–3.91 (m, 2H),
.82 (s, 3H), 2.80 (m, 2H), 1.80–1.78 (m, 2H), 1.49–1.41 (m, 2H), 1.39
+
(s, 9H); LC-MS (ESI): m/z calcd for C H N O S: 434.18 [M+H] ;
2
0
27
5
4
gel (petroleum ether/ethyl acetate=2:1) to give compound 19.
found: 434.20.
1
Yield: 80%; H NMR (500 MHz, [D ]DMSO): δ=7.78 (s, 1H), 6.22 (d,
6
J=10.5 Hz, 1H), 6.19 (s, 2H), 4.15–4.05 (m, 1H), 3.97–3.94 (m, 2H),
2
.88–2.75 (m, 2H), 1.77–1.73 (m, 2H), 1.53–1.43 (m, 2H), 1.41 (s, 9H);
tert-Butyl 4-((2-amino-5-(5-(methoxycarbonyl)furan-2-yl)
pyrimidin-4-yl)amino)piperidine-1-carboxylate (25)
+
LC-MS (ESI): m/z calcd for C H BrN O : 372.10 [M+H] ; found:
14
22
5
2
372.09.
1
General procedure A. Yield: 80%; H NMR (400 MHz, [D ]DMSO): δ=
6
8
.21 (s, 1H), 7.39 (d, J=3.2 Hz, 1H), 6.82 (d, J=2.8 Hz, 1H), 6.55 (s,
2H), 6.51 (d, J=6.0 Hz, 1H), 4.27–4.20 (m, 1H), 3.83 (m, 2H), 3.81 (s,
H), 3.02 (m, 2H), 1.90–1.87 (m, 2H), 1.47–1.44 (m, 2H), 1.41 (s, 9H);
General procedure A for the synthesis of compounds 20–25
3
A mixture of compound 19 (1.43 mmol), corresponding boronic
+
LC-MS (ESI): m/z calcd for C H N O : 418.20 [M+H] ; found:
2
0
27
5
5
acid/ester (1.72 mmol), Pd(dppf)Cl2 (0.07 mmol), and 1N Na CO
2
3
418.19.
(2.8 mL) in DME (14 mL) were heated to reflux overnight. The
completion of the reaction was monitored by TLC on silica gel.
After cooling to room temperature, the residue was purified by
column chromatography on silica gel (CH Cl /ethanol=25:1) to
General procedure B for the synthesis of compounds 2–7
2
2
A mixture of the appropriate arylamines (0.986 mmol), 5-bromo-2-
cyanopyridine (0.986 mmol), Pd (dba) (0.00986 mmol), Xantphos
give the product.
2
3
(
0.02 mmol), and Cs CO (1.48 mmol) in 1,4-dioxane (6 mL) were
2 3
tert-Butyl 4-((2-amino-5-phenylpyrimidin-4-yl)amino)
piperidine-1-carboxylate (20)
heated to reflux for 5 h. The completion of the reaction was
monitored by TLC on silica gel. The reaction mixture was then
evaporated under reduced pressure and the residue was purified
by column chromatography on silica gel (CH Cl /ethanol=30:1) to
1
General procedure A. Yield: 80%; H NMR (500 MHz, CDCl ): δ=8.09
3
2
2
(
s, 1H), 7.52 (s, 2H), 7.51 (s,2H), 7.40 (s, 1H), 5.51 (s, 1H), 4.76 (s, 2H),
.18–4.09 (m, 1H), 4.02 (m, 2H), 2.93 (m, 2H), 1.99–1.97 (m, 2H), 1.45
give intermediates. The intermediates were then dissolved in 10 mL
4
CH Cl and cooled at 0°C. The mixture was then added TFA
2
2
(s, 9H), 1.32–1.27 (m, 2H); LC-MS (ESI): m/z calcd for C H N O :
2
0
27
5
2
(5.0 mL) at room temperature for 3 h and the reaction mixture was
evaporated under reduced pressure. The residue was then purified
by column chromatography on silica gel (CH Cl /ethanol (NH )=
+
3
70.22 [M+H] ; found: 370.22.
2
2
3
100:3) to give the product.
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5
-((5-Phenyl-4-(piperidin-4-ylamino)pyrimidin-2-yl)amino)
Methyl 5-(2-((6-cyanopyridin-3-yl)
amino)-4-(piperidin-4-ylamino)pyrimidin-5-yl)
furan-2-carboxylate (7)
picolinonitrile (2)
1
General procedure B. Yield: 78%; m.p. 239–242°C; H NMR
1
(
(
7
1
1
3
500 MHz, [D ]DMSO): δ=10.01 (s, 1H), 9.08 (d, J=2.5 Hz, 1H), 8.43
dd, J=8.5, 2.5 Hz, 1H), 7.92 (d, J=8.5 Hz, 1H), 7.88 (s, 1H), 7.50–
.47 (m, 2H), 7.27–7.23 (m, 2H), 7.22–7.17 (m, 1H), 6.61 (d, J=7.5 Hz,
H), 4.14–4.04 (m, 1H), 3.16–3.07 (m, 2H), 2.81–2.68 (m, 2H), 1.96–
.87 (m, 2H), 1.60–1.54 (m, 2H); LC-MS (ESI): m/z calcd for C H N :
General procedure B. Yield: 74%; m.p. 226–228°C; H NMR
6
(500 MHz, [D ]DMSO): δ=10.22 (s, 1H), 9.07 (d, J=2.0 Hz, 1H), 8.46
6
(s, 1H), 8.41 (dd, J=8.5, 2.5 Hz, 1H), 7.93 (d, J=8.5 Hz, 1H), 7.44 (d,
J=3.5 Hz, 1H), 7.01 (d, J=3.5 Hz, 1H), 6.91 (d, J=7.0 Hz, 1H), 4.15–
2
1
21
7
4.03 (m, 1H), 3.84 (s, 3H), 3.05–2.95 (m, 2H), 2.70–2.58 (m, 2H), 1.98–
+
13
72.19 [M+H] ; found: 372.20.
1.89 (m, 2H), 1.49–1.37 (m, 3H). C NMR (100 MHz, [D ]DMSO): δ=
6
1
58.28, 158.13, 156.91, 155.06, 153.65, 141.89, 141.60, 140.61,
1
29.18, 124.14, 123.28, 119.96, 118.16, 107.48, 100.85, 51.76, 48.34,
4.62, 32.35; LC-MS (ESI): m/z calcd for C H N O : 420.17 [M+H] ;
+
5
-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(piperidin-4-ylamino)
4
2
1
21
7
3
pyrimidin-2-yl)amino)picolinonitrile (3)
found: 420.17.
1
General procedure B. Yield: 82%; m.p. 220–222°C; H NMR
(
(
500 MHz, [D ]DMSO): δ=10.00 (s, 1H), 9.05 (d, J=2.5 Hz, 1H), 8.42
dd, J=8.5, 2.5 Hz, 1H), 7.93 (s, 2H), 7.91 (s, 1H), 7.61 (s, 1H), 6.52 (d,
6
tert-Butyl 4-((5-bromo-2-((6-cyanopyridin-3-yl)amino)
pyrimidin-4-yl)amino)piperidine-1-carboxylate (26)
J=7.0 Hz, 1H), 4.18–4.07 (m, 1H), 3.88 (s, 3H), 3.32–3.29 (m, 2H),
1
3
3
.06–2.95 (m, 2H), 2.11–2.01 (m, 2H), 1.95–1.85 (m, 2H). C NMR
A mixture of compound 19 (1.0 mmol), 5-bromo-2-cyanopyridine
(
1
4
100 MHz, [D ]DMSO): δ=159.32, 157.97, 154.20, 141.75, 141.70,
37.98, 129.80, 129.68, 123.84, 122.89, 118.82, 114.28, 104.93, 48.99,
(1.0 mmol), Pd (dba) (0.01 mmol), Xantphos (0.02 mmol) and
6
2
3
Cs CO (1.50 mmol) in 1,4-dioxane (8 mL) were heated to reflux for
2 3
5.60, 39.14, 32.60; LC-MS (ESI): m/z calcd for C H N : 376.19 [M+
5 h. The completion of the reaction was monitored by TLC on silica
gel. The reaction mixture was evaporated under reduced pressure.
19
21
9
+
H] ; found: 376.19.
The residue was purified by column chromatography on silica gel
1
(
CH Cl /ethanol=30:1) to give compound 26. Yield: 77%; H NMR
2
2
5
-((4-(Piperidin-4-ylamino)-5-(thiophen-2-yl)pyrimidin-2-yl)
(
500 MHz, [D ]DMSO): δ=10.06 (s, 1H), 8.95 (d, J=2.5 Hz, 1H), 8.42
6
amino)picolinonitrile (4)
(dd, J=9.0 Hz, 2.5 Hz, 1H), 8.16 (s, 1H), 7.97 (d, J=9.0 Hz, 1H), 6.91
d, J=8.0 Hz, 1H), 4.17–4.09 (m, 1H), 4.03 (s, 2H), 2.83 (s, 2H), 1.85–
.82 (m, 2H), 1.61–1.52 (m, 2H), 1.43 (s, 9H); LC-MS (ESI): m/z calcd
(
1
General procedure B. Yield: 80%; m.p. 160–162°C; H NMR
1
(
(
400 MHz, [D ]DMSO): δ=10.05 (s, 1H), 9.07 (d, J=2.5 Hz, 1H), 8.43
dd, J=8.5, 2.5 Hz, 1H), 7.98 (s, 1H), 7.89 (d, J=8.5 Hz, 1H), 7.60 (t,
6
+
for C H BrN O : 474.12 [M+H] ; found: 474.13.
2
0
24
7
2
J=4.5 Hz, 1H), 7.22 (d, J=3.0 Hz, 1H), 7.19–7.16 (m, 1H), 6.36 (d, J=
8
.0 Hz, 1H), 4.10–3.96 (m, 1H), 2.95–2.93 (m, 2H), 2.60–2.55 (m, 2H),
5
-((5-Bromo-4-(piperidin-4-ylamino)pyrimidin-2-yl)amino)
13
1
.88–1.80 (m, 2H), 1.49–1.41 (m, 2H). C NMR (125 MHz, [D ]DMSO):
6
picolinonitrile (8)
δ=158.60, 158.05, 154.99, 141.41, 141.01, 135.61, 129.19, 128.19,
26.38, 126.01, 123.74, 122.82, 118.27, 105.78, 48.82, 45.22, 32.38;
1
Compound 26 (0.5 mol) was dissolved in 4 mL CH Cl and cooled at
2
2
+
LC-MS (ESI): m/z calcd for C H N S: 378.14 [M+H] ; found: 378.15.
0°C. The mixture was then added TFA (2 mL) at room temperature
for 3 h. The reaction mixture was evaporated under reduced
pressure. The residue was purified by column chromatography on
silica gel (CH Cl /ethanol (NH )=100:3) to give compound 8. Yield:
19
19
7
5
-((5-(Furan-2-yl)-4-(piperidin-4-ylamino)pyrimidin-2-yl)
2
2
3
1
amino)picolinonitrile (5)
70%; m.p. 228–230°C; H NMR (500 MHz, [D ]DMSO): δ=10.01 (s,
6
1
1H), 9.01 (d, J=2.0 Hz, 1H), 8.35 (dd, J=8.5, 3.0 Hz, 1H), 8.13 (s, 1H),
General procedure B. Yield: 77%; m.p. 188–190°C; H NMR
7
.90 (d, J=9.0 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 4.06–3.91 (m, 1H),
(
(
1
1
1
1
1
400 MHz, [D ]DMSO): δ=10.07 (s, 1H), 9.06 (d, J=2.4 Hz, 1H), 8.42
dd, J=8.4, 2.4 Hz, 1H), 8.27 (s, 1H), 7.88 (d, J=11.0 Hz, 1H), 7.77 (s,
H), 6.76 (d, J=3.0 Hz, 1H), 6.66–6.60 (m, 1H), 6.55 (d, J=7.4 Hz,
H), 4.14–3.98 (m, 1H), 3.03–2.94 (m, 2H), 2.68–2.55 (m, 2H), 1.96–
6
3.01–2.96 (m, 2H), 2.58–2.51 (m, 2H), 1.83–1.74 (m, 2H), 1.57–1.47
1
3
(
m, 2H). C NMR (100 MHz, [D ]DMSO): δ=157.54, 157.34, 155.99,
41.41, 140.83, 129.16, 123.81, 122.95, 118.20, 94.71, 49.24, 45.43,
2.38; LC-MS (ESI): m/z calcd for C H BrN : 374.07 [M+H] ; found:
74.08.
6
1
3
3
+
13
15 16
7
.85 (m, 2H), 1.57–1.41 (m, 2H). C NMR (100 MHz, [D ]DMSO): δ=
6
57.72, 157.00, 153.52, 148.82, 142.35, 141.41, 140.89, 129.16,
23.75, 122.88, 118.24, 111.55, 106.22, 102.45, 48.68, 45.16, 32.57;
+
LC-MS (ESI): m/z calcd for C H N O: 362.17 [M+H] ; found: 362.18.
19
19
7
5
-((5-(4-Methylthiazol-2-yl)-4-(piperidin-4-ylamino)
pyrimidin-2-yl)amino)picolinonitrile (9)
Methyl 5-(2-((6-cyanopyridin-3-yl)
amino)-4-(piperidin-4-ylamino)pyrimidin-5-yl)
thiophene-2-carboxylate (6)
A mixture of compound 26 (0.211 mmol), lithium triisopropyl (4-
methylthiazol-2-yl)borate (0.422 mmol), Pd(dppf)Cl2 (0.011 mmol),
1 N Cs CO (0.422 mmol), CuCl (0.021 mmol), and ZnCl2
2
3
(
0.211 mmol) in DMF (10 mL) was heated to 100°C overnight. The
1
General procedure B. Yield: 71%; m.p. 183–185°C; H NMR
completion of the reaction was monitored by TLC on silica gel. The
reaction mixture was treated with water and CH Cl . The organic
(
400 MHz, [D ]DMSO): δ=10.12 (s, 1H), 9.08 (s, 1H), 8.42 (d, J=
6
2
2
8
1
1
2
4
.8 Hz, 1H), 8.05 (s, 1H), 7.92 (d, J=8.4 Hz, 1H), 7.83 (d, J=4.0 Hz,
H), 7.29 (d, J=3.6 Hz, 1H), 6.75 (d, J=7.6 Hz, 1H), 4.10–3.95 (m,
H), 3.84 (s, 3H), 3.05–2.90 (m, 2H), 2.63–2.50 (m, 2H), 1.87–1.78 (m,
H), 1.52–1.42 (m, 2H); LC-MS (ESI): m/z calcd for C H N O S:
layer was collected, washed with brine, and dried over Na SO ,
2
4
evaporated under reduced pressure. The residue was dissolved in
3
mL CH Cl and cooled at 0°C. The mixture was added TFA
2
2
21
21
7
2
(
1.5 mL) at room temperature for 3 h. The reaction mixture was
evaporated under reduced pressure. The residue was purified by
+
36.15 [M+H] ; found: 436.16.
column chromatography on silica gel (CH Cl /ethanol (NH )=
2
2
3
1
1
00:3) to give compound 9. Yield: 56%; m.p. >250°C; H NMR
(500 MHz, [D ]DMSO): δ=10.28 (s, 1H), 9.61 (d, J=7.5 Hz, 1H), 9.08
6
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(
d, J=2.5 Hz, 1H), 8.56 (s, 1H), 8.44 (dd, J=8.5, 2.5 Hz, 1H), 7.93 (d,
5-Bromo-2-chloro-N-(2-morpholinoethyl)pyrimidin-4-amine
(32)
J=9.0 Hz, 1H), 7.23 (d, J=1.0 Hz, 1H), 4.17–4.08 (m, 1H), 3.01–2.95
(
(
1
m, 2H), 2.71–2.63 (m, 2H), 2.42 (s,3H), 2.02–1.95 (m, 2H), 1.49–1.41
1
13
General procedure C. Yield: 95%; H NMR (500 MHz, CDCl ): δ=8.12
3
m, 2H). C NMR (125 MHz, [D ]DMSO): δ=163.95, 158.31, 156.81,
6
(
s, 1H), 6.46 (s, 1H), 3.76 (s, 4H), 3.58 (s, 2H), 2.66 (s, 2H), 2.55 (s, 4H);
55.36, 151.02, 141.66, 140.61, 129.24, 124.25, 123.36, 118.18,
+
LC-MS (ESI): m/z calcd for C H BrClN O: 321.00 [M+H] ; found:
1
0
14
4
1
11.58, 103.55, 47.74, 44.42, 32.57, 16.64; LC-MS (ESI): m/z calcd for
+
321.03.
C H N S: 393.15 [M+H] ; found: 393.16.
1
9
20
8
General procedure D for the synthesis of compounds 33–38
General procedure C for the synthesis of compounds 27–32
Appropriate aryl chlorides (1.23 mmol) were dissolved in ethanol
A mixture of 5-bromo-2,4-dichloropyrimidine (0.44 mmol), corre-
sponding amines (0.48 mmol), and trimethylamine (0.08 mL) in
acetonitrile (10 mL) were stirred at room temperature for 2 h. The
completion of the reaction was monitored by TLC on silica gel. The
reaction mixture was evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel
(
5 mL) in sealed tube. The reaction mixture was added NH ·H O
5 mL) and was heated at 100°C for 12 h. The completion of the
3 2
(
reaction was monitored by TLC on silica gel. The reaction mixture
was evaporated under reduced pressure. After cooling to room
temperature, the residue was purified by column chromatography
on silica gel (petroleum ether/ethyl acetate=2:1) to give the
product.
(petroleum ether/ethyl acetate=4:1–3:1) to give the product.
tert-Butyl 4-(((5-bromo-2-chloropyrimidin-4-yl)amino)methyl)
piperidine-1-carboxylate (27)
tert-Butyl 4-(((2-amino-5-bromopyrimidin-4-yl)amino)methyl)
piperidine-1-carboxylate (33)
1
General procedure C. Yield: 92%; H NMR (500 MHz, CDCl ): δ=8.12
3
1
General procedure D. Yield: 85%; H NMR (500 MHz, CDCl ): δ=7.87
(
2
s, 1H), 5.64 (t, J=11.0 Hz, 1H), 4.14–4.10 (m, 2H), 3.46 (t, J=6.0 Hz,
H), 2.74 (t, J=11.0 Hz, 2H), 1.85–1.76 (m, 1H), 1.73–1.71 (m, 2H),
3
(
(
s, 1H), 5.27 (t, J=11.0 Hz, 1H), 4.81 (s, 2H), 4.13 (s, 2H), 3.35–3.33
m, 2H), 2.70 (s, 2H), 1.78–1.74 (m, 1H), 1.72–1.69 (m, 2H), 1.46 (s,
1
.46 (s, 9H), 1.25–1.16 (m, 2H); LC-MS (ESI): m/z calcd for
+
9H), 1.20–1.14 (m, 2H); LC-MS (ESI): m/z calcd for C H BrN O :
3
C H BrClN O : 405.06 [M+H] ; found: 405.08.
15 24
5
2
1
5
22
4
2
+
86.11 [M+H] ; found: 386.12.
tert-Butyl (2-((5-bromo-2-chloropyrimidin-4-yl)amino)ethyl)
carbamate (28)
tert-Butyl (2-((2-amino-5-bromopyrimidin-4-yl)amino)ethyl)
carbamate (34)
1
General procedure C. Yield: 94%; H NMR (500 MHz, [D ]DMSO): δ=
6
1
General procedure D. Yield: 81%; H NMR (500 MHz, CDCl ): δ=7.78
3
8
.24 (s, 1H), 7.68 (t, J=5.0 Hz, 1H), 6.96 (t, J=5.5 Hz, 1H), 3.42–3.38
(
s, 1H), 6.94 (t, J=5.5 Hz, 1H), 6.57 (t, J=5.5 Hz, 1H), 6.22 (s, 2H),
(
m, 2H), 3.16–3.13 (m, 2H), 1.37 (s, 9H); LC-MS (ESI): m/z calcd for
C H BrClN O : 351.01 [M+H] ; found: 351.03.
+
3.37 (dd, J=11.5 Hz, 6.0 Hz, 2H), 3.14 (dd, J=12.0 Hz, 6.0 Hz, 2H),
1
1
16
4
2
+
1
.38 (s, 9H); LC-MS (ESI): m/z calcd for C H BrN O : 332.06 [M+H] ;
11 18 5 2
found: 332.08.
tert-Butyl (R)-3-((5-bromo-2-chloropyrimidin-4-yl)amino)
piperidine-1-carboxylate (29)
tert-Butyl (R)-3-((2-amino-5-bromopyrimidin-4-yl)amino)
piperidine-1-carboxylate (35)
1
General procedure C. Yield: 85%; H NMR (500 MHz, [D ]DMSO): δ=
6
8
.24 (s, 1H), 7.35 (d, J=10.0 Hz, 1H), 4.13–4.06 (m, 1H), 3.94–3.91 (m,
1
General procedure D. Yield: 79%; H NMR (500 MHz, [D ]DMSO): δ=
2H), 2.81–2.75 (m, 2H), 1.73–1.69 (m, 2H), 1.60–1.49 (m, 2H), 1.39 (s,
6
+
7.82 (s, 1H), 6.24 (s, 2H), 5.99 (br, 1H), 3.99 (m, 1H), 3.62–3.57 (m,
9H); LC-MS (ESI): m/z calcd for C H BrClN O : 391.05 [M+H] ;
14
20
4
2
2
H), 3.00 (m, 2H), 1.83–1.80 (m, 1H), 1.68–1.63 (m, 2H), 1.41–1.37 (m,
found: 391.06.
1H), 1.37 (s, 9H); LC-MS (ESI): m/z calcd for C H BrN O : 372.10 [M
1
4
22
5
2
+
+
H] ; found: 372.10.
tert-Butyl (S)-3-((5-bromo-2-chloropyrimidin-4-yl)amino)
piperidine-1-carboxylate 30
tert-Butyl (S)-3-((2-amino-5-bromopyrimidin-4-yl)amino)
piperidine-1-carboxylate (36)
1
General procedure C. Yield: 82%; H NMR (500 MHz, [D ]DMSO): δ=
6
8
.24 (s, 1H), 7.35 (d, J=10.0 Hz, 1H), 4.13–4.06 (m, 1H), 3.94–3.91 (m,
1
General procedure D. Yield: 75%; H NMR (500 MHz, [D ]DMSO): δ=
6
2H), 2.81–2.75 (m, 2H), 1.73–1.69 (m, 2H), 1.60–1.49 (m, 2H), 1.39 (s,
+
7.82 (s, 1H), 6.24 (s, 2H), 5.99 (br, 1H), 3.99 (m, 1H), 3.62–3.57 (m,
9H); LC-MS (ESI): m/z calcd for C H BrClN O : 391.05 [M+H] ;
14
20
4
2
2
H), 3.00 (m, 2H), 1.83–1.80 (m, 1H), 1.68–1.63 (m, 2H), 1.41–1.37 (m,
found: 391.06.
1H), 1.37 (s, 9H); LC-MS (ESI): m/z calcd for C H BrN O : 372.10 [M
1
4
22
5
2
+
+
H] ; found: 372.10.
tert-Butyl (R)-(1-(5-bromo-2-chloropyrimidin-4-yl)
piperidin-3-yl)carbamate (31)
tert-Butyl (R)-(1-(2-amino-5-bromopyrimidin-4-yl)
piperidin-3-yl)carbamate (37)
1
General procedure C. Yield: 81%; H NMR (500 MHz, [D ]DMSO): δ=
6
8
.01 (s, 1H), 3.60–2.56 (m, 1H), 3.31–3.06 (m, 2H), 2.98–2.94 (m, 2H),
1
General procedure D. Yield: 72%; H NMR (500 MHz, CDCl ): δ=7.97
1.85–1.60 (m, 2H), 1.53–1.43 (m, 2H), 1.38 (s, 9H); LC-MS (ESI): m/z
3
+
(s, 1H), 6.89 (d, J=8.0 Hz, 1H), 6.49 (s, 2H), 3.95–3.88 (m, 2H), 2.77–
calcd for C H BrClN O : 391.05 [M+H] ; found: 391.05.
14
20
4
2
2
1
.67 (m, 2H), 1.87–1.84 (m, 1H), 1.76–1.73 (m, 1H), 1.61–1.52 (m, 1H),
.39 (s, 9H), 1.38–1.34 (m, 2H); LC-MS (ESI): m/z calcd for
+
C H BrN O : 372.10 [M+H] ; found: 372.12.
1
4
22
5
2
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4
5
-Bromo-N -(2-morpholinoethyl)pyrimidine-2,4-diamine (38)
1.39 (s, 9H), 1.32–1.21 (m, 2H); LC-MS (ESI): m/z calcd for C18
H
27
N
O
7
2
:
+
374.22 [M+H] ; found: 374.22.
1
General procedure D. Yield: 78%; H NMR (500 MHz, CDCl ): δ=7.87
3
(
s, 1H), 5.96 (br, 1H), 4.79 (s, 2H), 3.75 (t, J=4.5 Hz, 4H), 3.50 (dd, J=
1
1.0 Hz, 6.0 Hz, 2H), 2.62 (t, J=6.0 Hz, 2H), 2.52–2.51 (m, 4H); LC-MS
4
5
-(1-Methyl-1H-pyrazol-4-yl)-N -(2-morpholinoethyl)
+
(
ESI): m/z calcd for C H BrN O: 302.05 [M+H] ; found: 302.06.
10 16 5
pyrimidine-2,4-diamine (44)
1
General procedure E. Yield: 76%; H NMR (500 MHz, CDCl ): δ=7.70
3
General procedure E for the synthesis of compounds 39–44
(s, 1H), 7.56 (s, 1H), 7.42 (s, 1H), 5.75 (m, 1H), 4.91 (s, 2H), 3.97 (s,
3
H), 3.68 (t, J=4.0 Hz, 4H), 3.49 (t, J=6.0 Hz, 2H), 2.57 (t, J=6.0 Hz,
A mixture of appropriate aryl bromides (1.50 mmol), 1-methyl-4-
2H), 2.46 (s, 4H); LC-MS (ESI): m/z calcd for C H N O: 304.18 [M+
1
4
21
7
(
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
+
H] ; found: 304.20.
1.72 mmol), Pd(dppf)Cl (0.075 mmol), and 1N Na CO (3.0 mL) in
2
2
3
DME (15 mL) were heated to reflux overnight. The completion of
the reaction was monitored by TLC on silica gel. After cooling to
room temperature, the residue was then purified by column
chromatography on silica gel (CH Cl /ethanol=25:1) to give the
General procedure F for the synthesis of compounds 10–14
A mixture of the appropriate arylamines (1.0 mmol), 5-bromo-2-
2
2
product.
cyanopyridine (1.0 mmol), Pd
(dba)
2
3
(0.01 mmol), Xantphos
(
0.02 mmol) and Cs CO (1.50 mmol) in 1,4-dioxane (8 mL) were
2
3
heated to reflux for 5 h. The completion of the reaction was
monitored by TLC on silica gel. The reaction mixture was then
evaporated under reduced pressure and the residue was purified
by column chromatography on silica gel (CH Cl /ethanol=30:1) to
tert-Butyl 4-(((2-amino-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-4-yl)amino)methyl)piperidine-1-carboxylate (39)
2
2
1
General procedure E. Yield: 70%; m.p. 174–176°C; H NMR
give intermediates. Then the intermediates were dissolved in 10 mL
CH Cl and cooled at 0 C. The mixture was then added to TFA
(
500 MHz, CDCl ): δ=7.67 (s, 1H), 7.50 (s, 1H), 7.38 (s, 1H), 5.01 (t,
3
°
2
2
J=5.5 Hz, 1H), 4.81 (s, 2H), 4.10 (s, 2H), 3.96 (s, 3H), 3.31 (t, J=
(
5 mL) at room temperature for 3 h and the reaction mixture was
evaporated under reduced pressure. The residue was then purified
6
.0 Hz, 2H), 2.68 (m, 2H), 1.76–1.69 (m, 1H), 1.67–1.64 (m, 2H), 1.45
(s, 9H), 1.17–1.09 (m, 2H); LC-MS (ESI): m/z calcd for C H N O :
1
9
29
7
2
by column chromatography on silica gel (CH Cl /ethanol (NH )=
2
2
3
+
3
88.24 [M+H] ; found: 388.25.
100:3) to give the product.
tert-Butyl (2-((2-amino-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-4-yl)amino)ethyl)carbamate (40)
5
-((5-(1-Methyl-1H-pyrazol-4-yl)-4-((piperidin-4-ylmethyl)
amino)pyrimidin-2-yl)amino)picolinonitrile (10)
1
General procedure E. Yield: 80%; H NMR (500 MHz, [D ]DMSO): δ=
1
6
°
General procedure F. Yield: 79%; m.p. 231–233 C; H NMR
7
.78 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 6.92 (t, J=4.5 Hz, 1H), 6.05 (t,
J=4.5 Hz, 1H), 5.97 (s, 2H), 3.85 (s, 3H), 3.36–3.35 (m, 2H), 3.13 (m,
H), 1.35 (s, 9H); LC-MS (ESI): m/z calcd for C H N O : 334.19 [M+
(
(
6
1
1
500 MHz, [D ]DMSO): δ=9.93 (s, 1H), 9.03 (d, J=2.5 Hz, 1H), 8.46
dd, J=8.5, 2.5 Hz, 1H), 7.93–7.91 (m, 2H), 7.89 (s, 1H), 7.61 (s, 1H),
.81 (t, J=6.0 Hz, 1H), 3.89 (s, 3H), 3.42–3.38 (m, 2H), 3.24 (d, J=
2.5 Hz, 2H), 2.82–2.72 (m, 2H), 2.03–1.93 (m, 1H), 1.84–1.76 (m, 2H),
.46–1.35 (m, 2H); LC-MS (ESI): m/z calcd for C H N : 390.21 [M+
6
2
15
23
7
2
+
H] ; found: 334.20.
2
0
23
9
+
H] ; found: 390.22.
tert-Butyl (R)-3-((2-amino-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-4-yl)amino)piperidine-1-carboxylate (41)
1
5-((4-((2-Aminoethyl)amino)-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-2-yl)amino)picolinonitrile (11)
General procedure E. Yield: 81%; H NMR (500 MHz, [D ]DMSO): δ=
6
7
3
1
.75 (s, 1H), 7.62 (s, 1H), 7.47 (s, 1H), 5.98 (s, 2H), 5.49 (m, 1H), 4.05–
.99 (m, 1H), 3.71–3.43 (m, 2H), 3.03 (m, 2H), 1.81–1.78 (m, 1H),
.59–1.56 (m, 2H), 1.42–1.33 (m, 1H), 1.33 (s, 9H); LC-MS (ESI): m/z
calcd for C H N O : 374.22 [M+H] ; found: 374.20.
1
General procedure F. Yield: 80%; m.p. 175–177°C; H NMR
(
(
500 MHz, [D ]DMSO): δ=9.93 (s, 1H), 9.03 (d, J=2.5 Hz, 1H), 8.49
dd, J=9.0, 2.5 Hz, 1H), 7.93 (s, 1H), 7.92–7.89 (m, 2H), 7.63 (s, 1H),
6
+
18
27
7
2
6
2
1
1
.63 (t, J=5.0 Hz, 1H), 3.88 (s, 3H), 3.39 (dd, J=12.0, 6.5 Hz, 2H),
1
3
.76 (t, J=6.5 Hz, 2H). C NMR (125 MHz, [D ]DMSO): δ=159.78,
6
tert-Butyl (S)-3-((2-amino-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-4-yl)amino)piperidine-1-carboxylate (42)
57.58, 153.24, 141.31, 141.19, 137.52, 129.28, 123.30, 122.28,
18.35, 113.90, 104.54, 43.67, 40.50, 38.65. LC-MS (ESI): m/z calcd for
+
1
C H N : 336.16 [M+H] ; found: 336.20.
General procedure E. Yield: 78%; H NMR (500 MHz, [D ]DMSO): δ=
16 17
9
6
7
3
1
.75 (s, 1H), 7.62 (s, 1H), 7.47 (s, 1H), 5.98 (s, 2H), 5.49 (m, 1H), 4.05–
.99 (m, 1H), 3.71–3.43 (m, 2H), 3.03 (m, 2H), 1.81–1.78 (m, 1H),
(
R)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(piperidin-3-ylamino)
.59–1.56 (m, 2H), 1.42–1.33 (m, 1H), 1.33 (s, 9H); LC-MS (ESI): m/z
+
pyrimidin-2-yl)amino)picolinonitrile (12)
calcd for C H N O : 374.22 [M+H] ; found: 374.21.
18
27
7
2
2
0
General procedure F. Yield: 78%; m.p. 145–148°C; [α] = +58.00
D
1
(
9
c=0.5 in CH OH); H NMR (500 MHz, [D ]DMSO): δ=9.91 (s, 1H),
3
6
tert-Butyl (R)-(1-(2-amino-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-4-yl)piperidin-3-yl)carbamate (43)
.04 (d, J=2.5 Hz, 1H), 8.47 (dd, J=8.5, 2.5 Hz, 1H), 7.91 (s, 1H), 7.90
(s, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.62 (s, 1H), 6.16 (d, J=8.0 Hz, 1H),
4.09–4.02 (m, 1H), 3.89 (s, 3H), 2.95 (dd, J=11.5, 3.0 Hz, 1H), 2.75–
1
General procedure E. Yield: 68%; H NMR (500 MHz, [D ]DMSO): δ=
6
2
1
.66 (m, 1H), 2.62–2.52 (m, 2H), 1.84–1.74 (m, 1H), 1.69–1.62 (m, 1H),
7.75 (s, 1H), 7.62 (s, 1H), 7.56 (s, 1H), 6.93 (d, J=8.0 Hz, 1H), 3.88 (s,
3H), 3.64–3.61 (m, 1H), 3.44–3.41 (m, 1H), 2.71–2.69 (m, 1H), 2.62–
2.60 (m, 1H), 1.81–1.79 (m, 1H), 1.63–1.61 (m, 1H), 1.57–1.54 (m, 1H),
.61–1.54 (m, 1H), 1.48–1.41 (m, 1H). LC-MS (ESI): m/z calcd for
+
C H N : 376.19 [M+H] ; found: 376.20.
19 21
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(
S)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(piperidin-3-ylamino)
Determination of compound potency (IC50 values)
pyrimidin-2-yl)amino)picolinonitrile (13)
Human recombinant CHK1 protein, which was fused with GST (aa1-
476) was purchased from Sino Biological Inc. The enzymic activity
was detected by the HTRF® KinEASE™ STK S1(Cisbio). The CHK1
protein and peptide substrates were diluted in HTRF kinase buffer
2
0
General procedure F. Yield: 73%; m.p. 206–208°C; [α] =À 59.00
D
1
(
c=0.5 in CH OH); H NMR (500 MHz, [D ]DMSO): δ=9.90 (s, 1H),
3
6
9
.03 (d, J=2.0 Hz, 1H), 8.46 (dd, J=9.0, 2.5 Hz, 1H), 7.90 (s, 2H), 7.87
(d, J=8.5 Hz, 1H), 7.63 (s, 1H), 6.15 (d, J=8.0 Hz, 1H), 4.09–4.04 (m,
(1X Kinase buffer, 5 mM MgCl , 1 mM DTT), and then compounds of
2
1
H), 3.89 (s, 3H), 2.74–2.67 (m, 1H), 2.62–2.53 (m, 2H), 1.83–1.76 (m,
different concentrations, CHK1 protein and peptide substrates were
added to the 384 plates (ProxiplateTM-384 Plus, PerkinElmer). The
final volume of the reactions is 10 μl (2% DMSO, 0.075 ng/μl CHK1,
1 μM STK1, 50 μM ATP). The antibody was added after the kinase
reactions incubated at room temperature for 1 h. The phosphor-
ylation activity of CHK1 can be determined by detecting the ratio of
fluorescence signals at 665 and 620 nm. The IC50 value of the
compound was calculated using the software GraphPad Prism 5,
and the equation “sigmoidal dose-response (variable slope)” was
chosen for curve fitting.
1
3
1
H), 1.69–1.62 (m, 1H), 1.58 (m, 1H), 1.49–1.40 (m, 1H). C NMR
(125 MHz, [D ]DMSO): δ=158.84, 157.62, 153.49, 141.27, 141.18,
6
1
4
3
37.31, 129.23, 129.16, 123.31, 122.35, 118.32, 113.91, 104.43, 50.81,
7.07, 45.98, 38.69, 29.50, 24.13. LC-MS (ESI): m/z calcd for C H N :
1
9
21
9
+
76.19 [M+H] ; found: 376.20.
(
R)-5-((4-(3-Aminopiperidin-1-yl)-5-(1-methyl-1H-pyrazol-4-yl)
pyrimidin-2-yl)amino)picolinonitrile (14)
1
General procedure F. Yield: 70%; m.p. 182–184°C; H NMR
(
(
500 MHz, [D ]DMSO): δ=10.09 (s, 1H), 9.03 (d, J=2.5 Hz, 1H), 8.44
dd, J=8.5, 2.5 Hz, 1H), 8.11 (s, 1H), 7.93–7.89 (m, 2H), 7.63 (s, 1H),
6
In vitro cytotoxic activities
3
.87 (s, 3H), 3.79–3.74 (m, 1H), 3.68–3.63 (m, 1H), 2.76–2.67 (m, 2H),
.87–1.80 (m, 1H), 1.66–1.57 (m, 2H), 1.51–1.42 (m, 1H), 1.20–1.11
A 90 μL of cells were plated into 96-well microculture plates and
then added with 10 μL of different concentrations of compounds
1
13
(m, 1H). C NMR (100 MHz, [D ]DMSO): δ=162.88, 157.45, 156.83,
for 72 h at 37°C in 5% CO
. Then, 20 μL of MTS was added to
2
6
1
1
41.23, 141.05, 136.68, 129.32, 128.15, 123.47, 122.57, 118.28,
incubate for 3 h at 37°C and then recording absorbance at 490 nm
(background subtraction at 690 nM)with SpectraMAX340. The
inhibition rate of the compound on the growth of cancer cells was
calculated as follows. Growth inhibitory ratio=(AcontrolÀ Asample)/
Acontrol ×100% The IC50 value of the compound was calculated using
the software GraphPad Prism 5.
16.91, 107.29, 55.62, 47.46, 47.36, 38.58, 33.66, 23.15. LC-MS (ESI):
+
m/z calcd for C H N : 376.19 [M+H] ; found: 376.21.
19
21
9
5
-((5-(1-Methyl-1H-pyrazol-4-yl)-4-((2-morpholinoethyl)amino)
pyrimidin-2-yl)amino)picolinonitrile (15)
A mixture of compound 44 (1.0 mmol), 5-bromo-2-cyanopyridine
Metabolic stability in vitro assays
(
1.0 mmol), Pd (dba)3 (0.01 mmol), Xantphos (0.02 mmol), and
2
Mouse liver microsome was purchased from Research Institute for
Liver Diseases (Shanghai) Co., Ltd. Mouse liver microsome (1.0 mg)
was diluted in PBS buffer (1 mL), and followed mixed with 1 mM
NADPH solution. Microsome-NADPH solution was prewarmed at
2
3
37
°
C for 5 min. 10 μL of a 100 μg/mL compound was then added
to Microsome-NADPH solution and then divided into 3 aliquots.
The mixture was kept at 37
°
C and 10 μL aliquots from each aliquot
were taken at 30 min. The reaction was quenched with 490 μL of
methanol containing 1 μg/mL internal standard compound. After
quenching, the mixtures were vortexed and centrifuged. 10 μL of
the supernatant was injected into an LC/MS system to test peak
areas of compound 13.
In vivo PK evaluation
5
-((5-(1-Methyl-1H-pyrazol-4-yl)-4-((2-morpholinoethyl)amino)
This study was performed in strict accordance with the Laboratory
Animal Management Regulations (State Scientific and Technolog-
ical Commission Publication No. 8–27 Rev. 2017) and was approved
by Shanghai Institute of Materia Medica (Shanghai, Chian). SD rats
were purchased from the JOINN Laboratories (Suzhou). After oral
dosing of 20 mg/kg of compound 13 (5% DMSO, 5% Solutol, 90%
(0.5%MC), Blood samples were collected at nine time points (0.083,
0.25, 0.5, 1, 2, 4, 6, 24, 30 h). Plasma was separated by centrifugation
pyrimidin-2-yl)amino)pyrimidine-2-carbonitrile (16)
A mixture of compound 44 (1.0 mmol), 5-bromopyrimidine-2-
carbonitrile
(1.0 mmol),
Pd (dba)3
2
(0.01 mmol),
Xantphos
(
0.02 mmol), and Cs CO3 (1.5 mmol) in 1,4-dioxane (8 mL) were
2
heated to reflux for 5 h. The completion of the reaction was
monitored by TLC on silica gel. The reaction mixture was
evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel (CH Cl /ethanol=30:1) to
at 5000 rpm for 10 min at 4
°
C and analyzed by LC-MS/MS (Waters
2
2
1
give compounds 16. Yield: 80%; m.p. 170–172°C;
H
NMR
XEVO TQÀ S). Pharmacokinetic parameters were analyzed by
(
(
500 MHz, [D ]DMSO): δ=10.10 (s, 1H), 9.34 (s, 2H), 7.95 (s, 1H), 7.92
s, 1H), 7.64 (d, J=0.6 Hz, 1H), 6.63 (t, J=5.3 Hz, 1H), 3.90 (s, 3H),
Masslynx® V4.1.
6
3
2
1
1
.60–3.55 (m, 4H), 3.51 (dd, J=12.3, 6.5 Hz, 2H), 2.54 (t, J=6.7 Hz,
13
Western blotting
H), 2.42 (s, 4H). C NMR (125 MHz, [D ]DMSO): δ=160.00, 157.59,
6
53.62, 146.23, 139.23, 137.89, 134.43, 129.63, 117.18, 114.21,
The primary antibodies Chk1 (catalog number: sc-8408) was from
Santa Cruz Biotechnology, Beta-actin (catalog number: AM1021B)
was from Abgent, P-Chk1(Ser345) (catalog number: 2341 s) and P-
Chk1(Ser296) (catalog number: 2349 s) were from Cell Signaling
05.40, 66.78, 56.72, 53.54, 39.16, 37.93; LC-MS (ESI): m/z calcd for
+
C H N O: 407.20 [M+H] ; found: 407.21.
1
9
22 10
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(
Cell Signaling Technology, Inc). Cells treated as indicated were
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lysed, then separated on SDS-PAGE after electrophoresis. Proteins
were transferred to Nitrocellulose Blotting Membrane (GE Health-
care). The membranes were placed in primary antibody for 12 h at
4
°C. Primary antibody was detected using horseradish peroxidase
linked secondary antibodies (Jackson ImmunoResearch Laborato-
ries, Inc). Immunoreactive bands were detected by Clarity Western
ECL Substrate (BIO-RAD; for details see the Supporting Information).
602.
Molecular docking
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Lainchbury, N. Brown, M. I. Walton, P. D. Eve, K. J. Boxall, A. Hayes, A. T.
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The molecular docking was performed using Discovery Studio
(
version 2.1) with the default option. The X-ray crystal structure of
CHKI protein (PDB ID: 2YM8) was applied as the docking template.
The CHK1 protein was prepared by removing crystallographic
waters and ligands, adding missing hydrogen bond in CHARMM
force field. The 3D conformation of compound 13 was prepared by
Discovery Studio 2.1/Diverse conformation generation, then whose
energy minimized by DS 2.1/Minimize molecule. For the docking
calculation of compound 13, Discovery Studio 2.1/C-DOCKER was
used to generate the conformations. The graphical image was
analyzed by PyMOL v0.99.
[
[
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1
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Acknowledgements
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We thank Jianyang Pan (Research and Service Center, College of
Pharmaceutical Sciences, Zhejiang University) for performing NMR
spectrometry for structure elucidation. We also thank Huazhou
Ying (College of Pharmaceutical Sciences, Zhejiang University) for
performing mass spectrometry for structure elucidation. This work
was supported by grant from the National Natural Science
Foundation of China (21772174, 81673466).
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Conflict of Interest
[
The authors declare no conflict of interest.
Keywords: CHK1 inhibitor · diaminopyrimidine · DNA damage
response · oral bioavailability
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Manuscript received: November 11, 2020
Revised manuscript received: January 2, 2021
Version of record online: February 16, 2021
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