Design, Synthesis, and Antitumor Evaluation of 4-Amino-(1H)-pyrazole Derivatives as JAKs Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00247

Abnormalities in the JAK/STAT signaling pathway lead to many diseases such as immunodeficiency, inflammation, and cancer. Herein, we designed and synthesized a series of 4-amino-(1H)-pyrazole derivatives as potent JAKs inhibitors for cancer treatment. Res

Full text of DOI:10.1021/acsmedchemlett.6b00247

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Letter
Design, Synthesis and Antitumor Evaluation of 4-
Amino-(1H)-pyrazole Derivatives as JAKs Inhibitors
Xuewu Liang, Jie Zang, Mengyuan Zhu, Qianwen Gao, Binghe Wang, Wenfang Xu, and Yingjie Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00247 • Publication Date (Web): 23 Aug 2016
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Design, Synthesis and Antitumor Evaluation of 4-Amino-(1H)-
pyrazole Derivatives as JAKs Inhibitors
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Xuewu Liang†, Jie Zang†, Mengyuan Zhu ‡, Qianwen Gao†, Binghe Wang*, ‡, Wenfang Xu† and Yingjie Zhang*, †
†, Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong, 250012, P. R. China
‡Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303,
USA
ABSTRACT: Abnormalities in the JAK/STAT signaling pathway lead to many diseases such as immunodeficiency, inflammation
and cancer. Herein, we designed and synthesized a series of 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives as potent JAKs inhibitors for cancer
treatment. Results from in vitro protein kinase inhibition experiments indicated that compounds 3a-f and 11b are potent JAKs inꢀ
hibitors. For example, the IC50 values of compound 3f against JAK1, JAK2 and JAK3 were 3.4 nM, 2.2 nM and 3.5 nM, respecꢀ
tively. In cell culture experiments, compound 3f showed potent antiꢀproliferative activity against various cell lines (PCꢀ3, HEL,
K562, MCFꢀ7 and MOLT4) at low micromolar levels, while compound 11b showed selective cytotoxicity at submicromolar levels
against HEL (IC50: 0.35 ꢁM) and K562 (IC50: 0.37 ꢁM) cell lines. It is worth noting that both 3f and 11b showed more potent antiꢀ
proliferative activities than the approved JAKs inhibitor Ruxolitinib.
KEYWORDS: JAKs, Inhibitors, 4-Amino-(1H)-pyrazole, Anticancer
The JAK/STAT signaling pathway plays critical roles in
immunity, hematopoiesis as well as cell growth.1 Abnormaliꢀ
ties in the JAK/STAT signaling pathway lead to many diseasꢀ
es such as immunodeficiency, inflammation and cancers.2
Constitutive activations of JAKs are correlated to oncogenesis.
Dysregulation of JAK2 is discovered in patients with
myeloproliferative neoplasms and childhood T cell acute lymꢀ
phoblastic leukemia.3 Several hematologic malignancies inꢀ
cluding malignant lymphoma and myeloproliferative disorders
are associated with mutations of JAK2.4 Some cytokines and
growth factors bind to their receptors and then stimulate JAKs
for the phosporylation of STAT3, which is a potential target
for antiꢀcancer therapy.5 Persistent activation of the
JAK/STAT3 signaling promotes proliferation and survival of
tumor cells.6 Thus, the JAK/STAT signaling pathway is a
promising antitumor target.
fectively inhibit castrationꢀresistant growth of prostate cancer.8
JAK2 inhibitor TG101348 blocks JAK/STAT signaling leadꢀ
ing to suppression of proliferation and induction of apoptosis,
and is used for the treatment of myelofibrosis.9 JAKs inhibiꢀ
tors Momelotinib and Pacritinib are both developed for the
treatment of myelofibrosis.10
Inhibitors of JAKs are widely explored for treatment of
immunodeficiency, inflammation and cancer. Among the synꢀ
thetic JAK inhibitors for the treatment of cancer identified to
date (Figure 1), Ruxolitinib (Incyte’s Jakafi) was approved by
the US Food and Drug Administration (FDA) in 2013 for the
treatment of myelofibrosis, a rare bone marrow cancer. Beꢀ
sides, it is in phase III clinical trials for the treatment of metaꢀ
static pancreatic cancer and phase II clinical trials for the
treatment of multiple myeloma, leukemia and colon cancer.7
There are several other JAKs inhibitors in clinical trials for
cancer treatment (Figure 1). JAK2 inhibitor AZD1480 can
inhibit STAT5 signaling in prostate cancer cells and then efꢀ
Figure 1. Approved or clinical JAK inhibitors for cancer treatꢀ
ment
In our previous work, we discovered a series of 4ꢀ
aminopyrazole derivatives as novel and potent JAK inhibitors
(Figure 2).11 Structureꢀactivity relationship (SAR) studies
showed that R1 group modifications in these analogs did not
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influence their JAK inhibition significantly.11 Such results
Quinazolineꢀbased 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives 6a-f
indicate that the R1 side chain is not crucial to the interaction
between these 4ꢀaminopyrazole derivatives and JAKs proteins.
Herein, using 1 as a lead, a series of 4ꢀaminoꢀ(1H)ꢀpyrazole
derivatives were designed, synthesized and evaluated as potent
JAKs inhibitors. Firstly, removing the R1 side chain of comꢀ
pound 1 led to compound 3a, which exhibited improved JAKs
kinase inhibition at 10 ꢁM. Then further structural modificaꢀ
were synthesized as shown in Scheme 2. The synthetic routes
of derivatives 6a-f were similar to that of Scheme 1. Intermeꢀ
diates 5a-f were obtained from reaction of 2,4ꢀ
dichloroquinazoline with various amines under basic condition
of CH3COONa.13 Then, 5a-f reacted with 1Hꢀpyrazolꢀ4ꢀamine
at high temperature to give target molecules 6a-f.
3
4
5
6
7
8
9
tions of 3a led to pyrimidineꢀbased 4ꢀaminoꢀ(1
derivatives quinazolineꢀbased 4ꢀaminoꢀ(1 )ꢀpyrazole derivaꢀ
tives and 7 ꢀpyrrolo[2,3ꢀd] pyrimidineꢀbased 4ꢀaminoꢀ(1 )ꢀ
H)ꢀpyrazole
Scheme 2. Synthesis of Quinazoline-based 4-Amino-
(1H)-pyrazole Derivatives 6a-f.
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,
H
H
H
pyrazole derivatives (Figure 2).
R1
HN
N
H
N
N
N
NH
N
HN
remove
R1 side
chain
Cl
N
N
NH
Cl
N
N
N
heteroaromatic
ring
H
Cl
3a
N
H
Conditions and reagents: (a), CH3COONa, THF/H2O=3:1, r.tꢀ
60 oC, overnight. (b) nꢀBuOH, 120 oC, 1 h, microwave (MW
irradiation).
compound 1
(17n)
Fused ring
µM
nM
20
10
µM
10
JAK1: 100% JAK1: 79%
JAK2: 100% JAK2: 77%
JAK3: 100% JAK3: 44%
JAK1: 90%
JAK2: 92%
JAK3: 94%
five member ring
HN
N
HN
N
HN
N
NH
7
Hꢀpyrrolo[2,3ꢀd] pyrimidineꢀbased 4ꢀaminoꢀ(1
H)ꢀpyrazole
NH
NH
R4
R3
N
N
derivatives 11a-d were synthesized as shown in Scheme 3.
N
N
N
N
( )n
( )n
N
The starting material 2,4ꢀdichloroꢀ7Hꢀpyrrolo[2,3ꢀd] pyrimiꢀ
N
N
HN
R3
H
H
H
dine was protected by 4ꢀmethylbenzenesulfonyl chloride14 to
give compound 8, which was reacted with various amines,
leading to intermediates 9a-d, under basic conditions at high
temperature. Intermediates 9a-d reacted with 1Hꢀpyrazolꢀ4ꢀ
amine using TFA as catalyst at high temperature to produce
intermediates 10a-d. Ts-Deprotection of 10a-d yielded target
molecules 11a-d.15
R1
R2
R1
R1
R3
R2
R2
pyrimidine-based 4-amino-
(1H)-pyrazole derivatives
quinazoline-based 4-amino-
(1H)-pyrazole derivatives
7H-pyrrolo[2,3-d] pyrimidine-based
4-amino-(1H)-pyrazole derivatives
Figure 2. Design of 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives as
JAKs inhibitor
Pyrimidineꢀbased 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives 3a-m
were synthesized as described in Scheme 1. Generally, the
reaction of 5ꢀsubstitutedꢀ2,4ꢀdichloropyrimidine with various
aromatic amines under acidic (HCl) or basic (DIPEA, Et3N,
Na2CO3) conditions led to the intermediates 2a-m, which was
then reacted with 1Hꢀpyrazolꢀ4ꢀamine using TFA as a catalyst
at high temperature to give target molecules 3a-m.12
Scheme 3. Synthesis of 7
H-pyrrolo[2,3-d] pyrimidine-
based 4-Amino-(1 )-pyrazole Derivatives 11a-d.
H
Scheme 1. Synthesis of Pyrimidine-based 4-Amino-(1H)-
pyrazole Derivatives 3a-m.
Conditions and reagents: (a), TsCl, Et3N, DMAP, CH2Cl2. r.t.
5 h. (b) DIPEA, nꢀBuOH, 100 oC, overnight. (c)TFA, nꢀBuOH,
120 oC. 1 h. microwave. (MW irradiation). (d) Cs2CO3,
H2O:CH3OH: dioxane=1:3:3（V/V/V）, 80 oC, 6 h.
Conditions and reagents: (a). For compounds 2a, 2b and 2d,
DIPEA, DMF, r.t. 16 h. For compound 2c, Na2CO3, EtOH, r.t.
overnight. For compound 2e, DIPEA, NMP, 100 oC, 12 h. For
compounds 2f, 2g, Et3N, EtOH, 50ꢀ80 oC, overnight. For comꢀ
pound 2h, HCl, H2O, r.t. 5 days. For compounds 2i, 2l, HCl,
H2O, 50 oC, 5 h. For compound 2j, DIPEA, iꢀ PrOH, 60 oC,
overnight. For compounds 2k, 2m, H2O/CH3OH=3:1, 50 oC, 5
h. (b). TFA, nꢀBuOH, 120 oC, 1 h, microwave (MW irradiaꢀ
tion).
The 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives were screened for
their in vitro kinase inhibitory activities towards JAK1, JAK2,
and JAK3 at 10 ꢁM, 1 ꢁM, 0.1 ꢁM, 40 nM, and 20 nM. Beꢀ
cause we are only interested in compounds with nM inhibition
activities, the final screening was done at 20 nM. Staurosporꢀ
ine (a prototypical ATPꢀcompetitive kinase inhibitor, IC50:
JAK1 3 nM, JAK2 2 nM, JAK3 1 nM) and Ruxolitinib (an
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Figure 4. In vitro inhibitory activity against JAK1, JAK2 and
JAK3.
approved JAK inhibitor, inhibition at 20 nM: JAK1 97%,
JAK2 99%, JAK3 95%) were used as positive controls.16 All
the inhibition results were shown in Figures 3ꢀ6.
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8
Results in Figure 3 showed that compounds 3a-3f exhibited
remarkable inhibitory activities against JAK1, JAK2 and
JAK3 at 20 nM with the exception of compound 3d, which
was not active against JAK3 at 20 nM. For example, at 20 nM,
compound 3f inhibited protein kinase activities by 88%, 80%
and 79% against JAK1, JAK2 and JAK3 respectively. Further
evaluation revealed that the IC50 values of 3f against JAK1,
JAK2 and JAK3 were 3.4 nM, 2.2 nM and 3.5 nM, respectiveꢀ
ly. Generally, different substituents on the phenyl ring were
well tolerated.
From the data shown in Figure 5, we could see that quinazoꢀ
lineꢀbased 4ꢀaminoꢀ(1 )ꢀpyrazole derivatives 6a-f almost
completely lost their inhibitory activities towards JAKs at 20
nM. This indicated that a large fused ring, such as quinazoline,
was not beneficial to binding with JAKs.
H
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Figure 5. In vitro inhibitory activity against JAK1, JAK2 and
JAK3.
Comparing the compounds in Figure 6 with compounds in
Figure 3, we could see that 7Hꢀpyrrolo[2,3ꢀd] pyrimidineꢀ
based 4ꢀaminoꢀ(1
moderate JAKs inhibition at 20 nM, but less potent than pyꢀ
rimidineꢀbased 4ꢀaminoꢀ(1 )ꢀpyrazole derivatives (Figure 3).
H)ꢀpyrazole derivatives (Figure 6) showed
Figure 3. In vitro inhibitory activity against JAK1, JAK2 and
JAK3.
H
For example, compounds 11a, 11b and 11c in Figure 6 were
less potent than 3a, 3b and 3d in Figure 3, respectively.
Results in Figure 4 showed that replacing the Cl group on
pyrimidine ring with other groups, such as H or F could lead
to reduced JAKs inhibition. For example, compound 3g and
3k were much less potent than 3a (Figure 3). Taking the reꢀ
sults in Figure 3 and Figure 4 together, we conclude that R1
group on pyrimidine ring contributed much more to JAKs
inhibition than R2, R3 and R4 groups on the phenyl ring.
Figure 6. In vitro inhibitory activity against JAK1, JAK2 and
JAK3.
It was reported that mutation in the JH2 pseudokinase doꢀ
main of the Janus kinase 2 gene (JAK2 V617F) existed in
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HEL (human erythroleukemia) cell line.17 Therefore, all target
IC50 a (ꢁM)
Compound
compounds were screened against HEL cell line at the concenꢀ
tration of 5 ꢁM to evaluate their in vitro anticancer activities.
Results in Figure 7 showed that among these analogs, comꢀ
pounds 3a-f and 11a-d exhibited superior antiꢀproliferative
activities against HEL cell line (indicated by the red column)
than the other compounds we synthesized. These data were
generally consistent with their JAKs inhibitory potency.
PC-3
MCF-7
HEL
K562
MOLT4
3a
2.57±0.22
1.93±0.02
1.53±0.15
5.93±0.01
1.18±0.15
1.76±0.24
1.24±0.19
1.08±0.06
3.96±1.05
0.35±0.07
ND
1.70±0.27
1.37±0.23
>5
3b
5.38±0.62
1.03±0.25
2.30±0.98
1.13±0.08
1.08±0.05
10.38±0.97
4.47±1.29
13.52±1.98
ND
3.66±1.29
1.87±0.01
ND c
>8.3 b
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3c
1.86±0.29
2.08±0.33
1.29±0.21
0.77±0.05
3.79±0.86
0.37±0.11
3.72±0.71
>8.3
3.28±0.45
ND
3d
3e
3f
1.10±0.01
1.33±0.42
ND
1.26±0.15
1.61±0.35
ND
3k
11b
>5
>5
Figure 7. Activity screening against HEL cell line at the conꢀ
centration of 5 ꢁM. The plates containing compounds and cells
were incubated for 48 h in MTT assay.
11d
>5
>5
Considering their potent JAKs inhibitory activities and antiꢀ
proliferative potency against the HEL cell line, ten compounds
(3a-f, 3k, 11b, 11d, and 6d) were chosen for further antiꢀ
proliferative evaluation against human prostate cancer PCꢀ3,
human breast cancer MCFꢀ7, human erythroleukemia HEL,
human myelogenous leukemia K562 and human lymphoid
leukemia MOLT4 cell lines. Ruxolitinib was used as a positive
control. The results in Table 1 showed that most of the ten
compounds possessed potent antiꢀcancer activity in vitro.
Among these compounds, 3a, 3c, 3e and 3f were cytotoxic to
all five tested cell lines, while 11b exhibited remarkably selecꢀ
tive cytotoxicity to HEL (IC50: 0.35 ꢁM) and K562 (0.37 ꢁM).
It is worth emphasizing that though less potent than Ruxꢀ
olitinib in JAK inhibition, most of our compounds exhibited
more potent cytotoxicity than Ruxolitinib (Table 1), indicating
that our compounds might have offꢀtarget effects. Therefore,
representative compounds 3f and 11b were evaluated against
fourteen other cancer related kinases. The results in Figure 8
showed that at 20 nM, compound 3f was active against a numꢀ
ber of kinases including Fltꢀ3, VEGFRꢀ2, PDGFRα and TYK2,
while compound 11b exhibited very good selectivity against
JAK2 and JAK3 over the other tested kinases. These results
could explain why 3f were cytotoxic to all five cell lines while
11b was more selective against JAK/STAT pathway promoted
cell lines, such as HEL18,19 and K562.20,21,22 However, our kiꢀ
nase panel screening results still could not explain why 11b
were more cytotoxic than Ruxolitinib. Further anticancer
mechanism research of 11b is warranted.
6d
>5
9.71±0.99
2.62±0.19
ND
Ruxolitinib
>5
>5
10.3±0.3
15.8±1.4
a IC50 are mean of two or three experiments and standard deviation
is given.
b > 8.3 or > 5 in this figure means the IC50 value of this compound
is larger than 8.3 or 5.
c ND, not determined
Table 1. The Inhibitory Activities of Compounds Against Tu-
mor Cell Lines.
Figure 8. Selectivity profile of compounds 3f and 11b on
14 protein kinases at 20 nM.
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To investigate the binding mode of these 4ꢀaminoꢀ(1H)ꢀ
4
5
6
7
8
9
pyrazole derivatives in JAK2, the most potent compound 3f
was docked into the ATP binding pocket of JAK2 using
SYBYLꢀX 2.0. (Figure 9). The results showed in Figure 8
suggested that the –NH and =N moieties of pyrazole in comꢀ
pound 3f could form hydrogen bonds with GLU930 and
LEU932. These hydrogen interactions were the crucial part for
the protein kinase inhibition. The –NH moiety of pyrazole as
hydrogen donor was necessary for the improvement of binding
to JAKs. This maybe the reason why the kinase inhibitory
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activities of 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives were much
potent than their parent 4ꢀaminoꢀpyrazole derivatives. For
example, compound 3a reported here is much more potent
than its parent 17n (compound 1) reported in our previous
research.11 Additional docking with the compound that have a
fused phenyl ring (compound 6d) was also studied. Docking
result showed compounds 3f and 6d share a similar binding
mode. To understand why compound 6d showed weak activity,
a molecular dynamics (MD) simulation and a molecular meꢀ
chanics Poisson–Boltzmann surface area (MM/PBSA) energy
decomposition calculation experiment were conducted. Our
result showed that the fused phenyl ring and the sidechain of
ASP939 gave a 1.50kcal/mol negative energy contribution in
binding (Figure 10). Besides, MD simulation indicated that the
fused ring position is solvent accessible: it is exposed to water
(Figure 11). These two reasons may explain why adding an
additional large hydrophobic ring, such as a fused phenyl ring,
does not favor binding.
Figure 11. Fused phenyl ring is exposed to water in MD simuꢀ
lation. Water molecules within 4Å of the compound 6d were seꢀ
lected for visualization (spheres).
In summary, a series of 4ꢀaminoꢀ(1H)ꢀpyrazole derivatives
as potent JAKs inhibitors was designed, synthesized and evalꢀ
uated. In enzyme inhibition screenings, many of the comꢀ
pounds reached IC50 values below 20 nM. In cellꢀbased assays,
compound 3f showed potent cytotoxicity against a wide varieꢀ
ty of cell lines at micromolar levels. Compound 11b possessed
very potent cytotoxicity against HEL and K562 cell lines with
IC50 values in the subꢀmicromolar range, which were over tenꢀ
fold lower than in PCꢀ3, MCFꢀ7 and MOLT4 cell lines. Furꢀ
ther kinase panel screening results revealed that compound 3f
is a panꢀkinase inhibitor, while 11b is a highly selective JAK2
and JAK3 inhibitor, which could be used as lead compound
for further structural optimizations to find more potent and
selective JAKs inhibitors. Moreover, considering the discrepꢀ
ancy between JAKs inhibition and cytotoxicity when comꢀ
pared with Ruxolitinib, more detailed mechanistic studies are
warranted for 11b.
ASSOCIATED CONTENT
Supporting Information
Experimental section and characterization data (HRMS, 1HꢀNMR)
for new compounds.
Figure 9. The docking of compound 3f with JAK2 (PDB code
3FUP)
AUTHOR INFORMATION
Corresponding Author
*wang@gsu.edu
*zhangyingjie@sdu.edu.cn
Funding Sources
This work was supported by the National HighꢀTech R&D Proꢀ
gram of China (863 Program) (Grant No. 2014AA020523), Naꢀ
tional Natural Science Foundation of China (Grant Nos.
21302111, 81373282, 21172134), China Postdoctoral Science
Foundation funded project (Grant No. 2014T70654), Young
Scholars Program of Shandong University (YSPSDU), Major
Project of Science and Technology of Shandong Province
(2015ZDJS04001).
Figure 10. The docking of compound 6d with JAK2 (PDB
code 3FUP).
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tetraazatetracyclo[14.3.1.1(3),
1(20),3(22),4,6,9(21),10,12,16,18ꢀnonaene macrocycles.
Chem. 2012, 55, 449ꢀ64.
(7).1(9),(1)(3)]docosaꢀ
Acknowledgement
J
Med
This work used the Extreme Science and Engineering Disꢀ
covery Environment (XSEDE), which is supported by Naꢀ
tional Science Foundation grant number ACIꢀ1053575. We
also gratefully acknowledge the use of Orion that is supꢀ
ported by Georgia State University's Research Solutions.
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ABBREVIATIONS
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JAK, Janus kinase; STAT, Signal transducers and activators of
transcription; DIPEA, N, NꢀDiisopropylethylamine; DMAP, 4ꢀ
dimethylaminopyridine; TFA, trifluoroacetic acid; DMF, Dimeꢀ
thylformamide; NMP, Nꢀmethylꢀ2ꢀpyrrolidone.
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