Synthesis and biological evaluation of pyrazolo[1,5- A ]pyrimidine compounds as potent and selective pim-1 inhibitors

Article abstract of DOI:10.1021/ml500300c

Pim-1 has emerged as an attractive target for developing therapeutic agents for treating disorders involving abnormal cell growth, especially cancers. Herein we present lead optimization, chemical synthesis and biological evaluation of pyrazolo[1,5-a]pyri

Full text of DOI:10.1021/ml500300c

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Biological Evaluation of Pyrazolo[1,5‑a]pyrimidine
Compounds as Potent and Selective Pim‑1 Inhibitors
Yong Xu,* Benjamin G. Brenning, Steven G. Kultgen, Jason M. Foulks, Adrianne Clifford, Shuping Lai,
Ashley Chan, Shannon Merx, Michael V. McCullar, Steven B. Kanner, and Koc-Kan Ho
Astex Pharmaceuticals, Inc., 4140 Dublin Boulevard, Suite 200, Dublin, California 94568 United States
S
* Supporting Information
ABSTRACT: Pim-1 has emerged as an attractive target for developing therapeutic agents for treating disorders involving
abnormal cell growth, especially cancers. Herein we present lead optimization, chemical synthesis and biological evaluation of
pyrazolo[1,5-a]pyrimidine compounds as potent and selective inhibitors of Pim-1 starting from a hit from virtual screening.
These pyrazolo[1,5-a]pyrimidine compounds strongly inhibited Pim-1 and Flt-3 kinases. Selected compounds suppressed both
the phosphorylation of BAD protein in a cell-based assay and 2-dimensional colony formation in a clonogenic cell survival assay
at submicromolar potency, suggesting that cellular activity was mediated through inhibition of Pim-1. Moreover, these Pim-1
inhibitors did not show significant hERG inhibition at 30 μM concentration. The lead compound proved to be highly selective
against a panel of 119 oncogenic kinases, indicating it had an improved safety profile compared with the first generation Pim-1
inhibitor SGI-1776.
KEYWORDS: Pyrazolo[1,5-a]pyrimidine, Pim-1 inhibitor, BAD phosphorylation, 2-dimensional colony formation, hERG inhibition,
kinase selectivity
he Pim (provirus insertion site of Moloney murine
leukemia virus) genes represent a small family of proto-
mice dramatically suppressed PC-3 tumor progression.8
Inhibitors of Pim-1 kinase have recently been reviewed in the
literature,3 including both the promiscuous kinase inhibitors
and various selective Pim-1 inhibitors. Moreover, the lack of
any overt phenotypes in Pim-1 knockout mice indicated that a
Pim-1 inhibitor may have a low toxicity profile.2 Given the
strong correlation of Pim-1 activity and its roles in tumor cell
survival and proliferation, Pim-1 kinase is an attractive target for
developing therapeutic agents effective for treating disorders
involving abnormal cell growth, especially malignancies.
As a continuing effort to discover novel molecules as potent
and selective Pim-1 inhibitors, here we describe the synthesis
and evaluation of pyrazolo[1,5-a]pyrimidine compounds as
novel inhibitors of Pim-1 kinase. We employed a virtual
screening strategy for the generation and identification of hit
compounds as Pim-1 kinase inhibitors. Initial virtual screening
of a large number of library compounds identified a
pyrazolo[1,5-a]pyrimidine as one type of early Pim program
hit.9 As shown in Scheme 1, this initial pyrazolo[1,5-
a]pyrimidine hit compound possessed 52 μM potency against
T
oncogenes that are encoded by three different serine/threonine
protein kinases, Pim-1, Pim-2, and Pim-3, which are highly
conserved in vertebrates.1,2 Unlike many other protein kinases,
Pim kinases do not have a regulatory domain and therefore do
not require upstream phosphorylation for activation and are
constitutively active once transcribed. The induction of Pim
kinase activity is largely regulated at the transcriptional and
translational levels. The expression of Pim kinases is relevant
for growth factor-induced cell survival and proliferation and is
induced by a range of cells; Pim kinases regulate numerous
oncogenic processes including cancer cell survival, cytokines,
growth factors, and mitogenic stimuli in a large variety of cell
types, particularly in cancer apoptosis, migration, and tumori-
genesis.3,4 As the first discovered and identified Pim kinase,
Pim-1 is frequently overexpressed in a wide range of human
tumors, including hematological malignancies such as lympho-
mas and leukemias,5,6 and solid tumors, including prostate,
bladder, and oral cancers.7
The therapeutic potential of Pim-1 inhibition has been
demonstrated by siRNA and small molecule inhibitors.
Silencing of the Pim-1 gene with siRNA resulted in decreased
cell proliferation, inhibition of cell cycle progression, and
induction of apoptosis in the PC-3 prostate cancer cell line. In
addition, the intratumoral injection of Pim-1 siRNA in nude
Special Issue: New Frontiers in Kinases
Received: July 25, 2014
Accepted: October 22, 2014
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
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position chloride on 5-chloropyrazolo[1,5-a]pyrimidine 6
proceeded selectively with good yield under basic conditions.
Despite the presence of a secondary alcohol in trans-4-
aminocyclohexanol, the amination reaction was highly selective,
and no O-linked product was detected and separated.16 The
nuclear magnetic resonance (NMR) and mass spectrometry
(MS) spectrum unambiguously confirmed the structure of an
N-linked compound.
Scheme 1. Generation and Identification of Pyrazolo[1,5-
a]pyrimidine Hit Compound 1
Given that the new scaffold pyrazolo[1,5-a]pyrimidine
(compound 1) was active against Pim-1 with an IC50 = 45
nM, we investigated the optimization of the substituents at the
3- and 5-positions. According to our previous structure−activity
relationship (SAR) information, the 5-position of the
substituent was critical for potency, particularly the hydrogen
bonding interaction with residues Asp-128 and Asp-131 in Pim-
1. To maintain this key hydrogen bonding interaction, we
replaced the initial (1-methylpiperidin-4-yl)methanamine with
various oxygen and nitrogen containing moieties as shown in
Table 1, including trans-1,4-diaminocyclohexane. Considering
that basic amine moieties may induce strong hERG inhibition
(observed in compound SGI-1776), we also explored various
neutral functional groups at the terminal position of the 5-
position substituent, including hydroxyl, ether, and sulfone
functional groups. As shown in Table 1, the amino compound 7
Pim-1. However, the pyrazolo[1,5-a]pyrimidine chemotype was
of particular interest to us as the core structure is different from
the imidazo[1,2-b]pyridazine chemotype compounds (SGI-
1776, etc.), which were identified as selective and potent Pim-1
inhibitors, but with significant hERG and CYP inhibition.10−13
Starting from this initial hit compound and the imidazo[1,2-
b]pyridazine candidate SGI-1776, we combined the pyrazolo-
[1,5-a]pyrimidine core with the substituents at the 3,5-
positions of SGI-1776 and generated compound 1, which had
an IC50 = 45 nM for Pim-1 kinase. On the basis of the structure
of compound 1, we conducted systematic modifications around
the pyrazolo[1,5-a]pyrimidine core to improve in vitro potency
against Pim-1, as well as other critical physicochemical
properties.
Table 1. SAR of 5-Substituents on Pyrazolo[1,5-
a]pyrimidine
The synthetic route utilized to build pyrazolo[1,5-a]-
pyrimidines with aryl substitution at the 3-position and a
halogen leaving group in the 5-position was a cascade
cyclization. Specifically, aryl-substituted acetonitrile (Scheme
2) was treated with N,N-dimethylformamide dimethyl acetal to
Scheme 2. Synthesis of Pyrazolo[1,5-a]pyrimidine
a
Compounds
a
Reagents and conditions: (a) N,N-dimethylformamide dimethyl
acetal, reflux, 4 h, 50%; (b) N2H4, HOAc, ethanol, reflux, 16 h,
97%; (c) N-methyl uracil, C2H5ONa, ethanol, reflux, 3 h, 62%; (d)
POCl3, reflux, 3 h, 67%; (e) DIPEA, iPrOH, 130 °C, microwave
irradiation, 16 h, 80%.
give the corresponding 3-(dimethylamino)-2-(phenyl)-
acrylonitrile. The 4-phenyl-1H-pyrazol-5-amine was prepared
almost quantitatively by the treatment of acrylonitrile 3 with
hydrazine and glacial acetic acid in ethanol.14 The cyclization of
4-phenyl-1H-pyrazol-5-amine with N-methyl uracil as a masked
Michael acceptor in ethanol in the presence of sodium ethoxide
provided the key building block 3-phenylpyrazolo[1,5-a]-
pyrimidinone 5 in good yield.15 Chlorination of the pyrimidone
5 using POCl3 without solvent gave penult compound 5-
chloro-3-phenylpyrazolo[1,5-a]pyrimidine, which was used for
synthesis of final compounds with different amino groups on
the 5-position of 3-phenylpyrazolo[1,5-a]pyrimidine. The final
amination reaction of trans-4-aminocyclohexanol with the 5-
a
Values represent the average of at least two separate experiments, and
the standard deviation is less than 10% of the mean.
B
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ACS Medicinal Chemistry Letters
Letter
exhibited the most potent Pim-1 inhibition compared with
hydroxyl compounds 9−11, ether compounds 12 and 13, or
sulfone compound 14. Compounds with a hydroxyl group (9−
11) displayed potent inhibition of Pim-1. Ether compounds 12
and 13 and sulfone compound 14, which do not have an active
proton, showed slightly reduced potency on Pim-1. In addition,
the ketone compound 16b, was also active on Pim-1 and Pim-2,
which exhibited similar potency compared with ether and
sulfone compounds. The SAR of the presence of an active
proton enhances Pim-1 inhibition, consistent with the modeling
result.
Table 2. SAR of Substituents on Pyrazolo[1,5-a]pyrimidine
The 3-substituent aromatic ring bearing a meta-position
electron-withdrawing group was essential to maintain potency,
according to the previous SAR information during the
discovery of SGI-1776. Hence, we investigated −OCF3,
−CF3, and −Cl at this position. As shown in Tables 2 and 3,
all compounds had nanomolar IC50 activity on Pim-1.
Specifically, compounds with −CF3 and −Cl substituents had
similar Pim-1 inhibition but had improved Pim-1 inhibition
compared to compounds with −OCF3. It was noteworthy that
compound 15a, which has a shorter chain compared with 14a,
lost potency completely, suggesting that the length of the linker
is crucial to maintain hydrogen bonding with Asp-128 and Asp-
131 in Pim-1 and potency. The inactivity of compound 15a also
suggested that the 5-position substituent was more important
than the 3-position substituent in the pyrazolo[1,5-a]-
pyrimidine for Pim-1 inhibition. Indeed, our analysis of
fragment activity also supported this observation. As shown
in Scheme 3, the fragment 3-bromo-5-chloropyrazolo[1,5-
a]pyrimidine 17 was inactive against Pim-1 completely, the 3-
substituted 3-aryl-5-chloropyrazolo[1,5-a]pyrimidine 18 ex-
hibited 5 μM IC50 activity, while the 5-substituted fragment
19 achieved 294 nM IC50, and the 3,5-disubstituted compound
9 had a 27 nM IC50. Apparently, installation of a trans-4-
aminocyclohexanol group at the 5-position of pyrazolo[1,5-
a]pyrimidine increased the potency 100-fold, while the
introduction of an aryl group at the 3-position only increased
the potency 10-fold.
We also tested all compounds against the Pim-2 kinase. All
compounds showed relatively weak inhibition of Pim-2
compared with Pim-1, where most of them had low micromolar
IC50 values against Pim-2, indicating that these pyrazolo[1,5-
a]pyrimidine compounds were selective between Pim-1 and
Pim-2.
Subsequently, we evaluated selected compounds for their
effect on hERG potassium channels by using an automated
patch clamp assay (QPatchHTX). As expected, the amino
compound 1, which has a basic tertiary amino group, exhibited
strong hERG inhibition with an IC50 = 1.94 μM. Compounds
with neutral terminal hydroxyl moieties, trans-4-aminocyclohex-
anol (compounds 9 and 9a) and 2-(trans-4-aminocyclohexyl)-
propan-2-ol (compounds 11a and 11b), did not show
observable hERG inhibition at 30 μM. This demonstrated
that the potent hERG inhibition of both compound 1 and SGI-
1776 were induced by the terminal basic moieties, either the
primary amino group or the tertiary amino group.17 Removing
the basic moiety completely addressed the hERG inhibition in
this series of compounds. We also tested selected compounds
for Flt-3 IC50, and the data are shown in Table 3. These
selected pyrazolo[1,5-a]pyrimidine compounds strongly in-
hibited both Pim-1 and Flt-3 as dual inhibitors; however, these
compounds (9, 9a, 11a, and 11b) were 2−15-fold more potent
against Pim-1 than Flt-3.
a
Values represent the average of at least two separate experiments, and
b
the standard deviation is less than 10% of the mean. NA, not active.
The cellular potency of the pyrazolo[1,5-a]pyrimidine
compounds was evaluated by measuring their effects on
baseline phosphorylation of BAD (BCL-2 antagonist of cell
death) protein on serine 112, a known substrate of Pim-1. All 4
tested compounds (9a/b and 11a/b) showed 68−77%
inhibition of BAD phosphorylation (normalized to nontreated
cells, see Supporting Information) at 1 μM concentration for 45
min (see Supporting Information Figure 1). This demonstrated
that these tested compounds exhibited potent Pim-1 cellular
activity (sub-μM IC50).
Our recent studies demonstrated that Pim-1 mRNA was
significantly reduced using independent shRNAs targeting Pim-
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Table 4). (The complete kinase selectivity profiling is available
in the Supporting Information.) We conclude that the
Table 3. hERG IC50 and Flt-3 IC50 values of selected
compounds
a
a
Comp. No.
hERG IC50, μM
Flt-3 IC50, nM
Table 4. Kinase Selectivity Profile of Compound 11b
b
1
1.9
ND
a
b
selectivity score S(50)
0.14
SGI-1776
<1.0
>30
>30
>30
>30
ND
9
157
53
>95% inhibition at 1.0 μM
Pim-1, TRKC
9a
95% to >80% inhibition at
1.0 μM
Flt-3, TRKB, LCK, Haspin, MUSK, TRKA,
HIPK4
11a
11b
271
125
80% to >50% inhibition at
1.0 μM
YES1, CK2a, HIPK2, Pim-2, FYN, JAK2,
FMS, c-Src
hERG IC50 was determined by WuXi AppTec using the QPatchHTX
functional hERG assay as described in the experimental section. The
Flt-3 IC50 of reference compound staurosporine was less than 1 nM.
a
S(50) indicates the ratio of kinases in a panel (119 total) inhibited by
>50% at 1 μM concentration.
a
Values represent the average of at least two separate experiments, and
b
the standard deviation is less than 10% of the mean. ND, not
determined.
pyrazolo[1,5-a]pyrimidine core interacts selectively with Pim-
1. Although the scaffold pyrazolo[1,5-a]pyrimidine has been
reported as a Pim-1 inhibitor in the literature,23−25 herein we
demonstrated that the pyrazolo[1,5-a]pyrimidine scaffold was a
highly selective Pim-1 inhibitor against a comprehensive kinase
panel (119 oncogenic kinases). In addition, this is the first
report that the hERG issue has been successfully addressed
without compromise of potency for Pim-1 inhibition, which
could be useful to guide hERG optimization for another series
of Pim-1 inhibitors, as well as other kinase inhibitors.
Scheme 3. Analysis of Fragment Activity
In conclusion, we present the chemical synthesis and SAR
studies of novel pyrazolo[1,5-a]pyrimidine compounds as
selective and potent Pim-1 and Flt-3 kinase inhibitors. A
convenient synthesis of the 3-aryl-5-amino-pyrazolo[1,5-a]-
pyrimidine scaffold was achieved to readily access diversely
substituted analogues. Most of the tested pyrazolo[1,5-
a]pyrimidine compounds exhibited nanomolar inhibitory
activity of Pim-1. In addition, four compounds (9a/b and
11a/b) showed strong inhibition of BAD phosphorylation in a
cell-based assay at 1 μM concentration, suggesting the potent
cellular activity may be induced through the inhibition of Pim-
1. Compound 11b also potently inhibited 2D cell colony
formation in clonogenic cell survival assays mimicking Pim-1
knockdown by shRNA, further confirming the cellular activity
of this compound was mediated through inhibition of Pim-1.
Finally, we determined that the pyrazolo[1,5-a]pyrimidine
compounds not only have a pharmaceutically acceptable hERG
profile (>30 μM) but also possess an excellent kinase selectivity
profile, critical properties for the development of a therapeutic
kinase inhibitor. The in vivo efficacy studies in tumor xenografts
using this series of Pim-1 inhibitors will be disclosed separately.
1 compared to the nontarget shRNA control in UM-UC-3
epithelial bladder carcinoma cells.18 Further, Pim-1 protein was
reduced using Pim-1 shRNA compared to nontarget shRNA,
and UM-UC-3 2-dimensional colony growth was markedly
reduced with Pim-1 knockdown.18 In this study, we also
evaluated compound 11b’s cytostatic effect on colony growth
of UM-UC-3 and HSC-3 (human oral squamous carcinoma
cells) cells. The clonogenic cell survival assay reflects long-term
cytostatic effects caused by anticancer agents and is considered
a standard for measuring long-term cell viability since it reveals
multiple modes of cell death or arrest.19 The clonogenic cell
survival data (Supporting Information Figure 2) indicated that
compound 11b inhibited and reduced the growth of both UM-
UC-3 and HSC-3 with EC50 values of 0.914 and 0.600 μM,
respectively, further confirming that the cellular activity of
compound 11b was mediated through inhibition of Pim-1.
Given that most kinase inhibitors interact with the hinge
motif within the ATP pocket, which is highly conserved
between members of this family, the kinase selectivity profile of
any kinase inhibitor is particularly important for predicting and
interpreting the effects of the inhibitor in both research and
clinical settings. The lack of kinase selectivity is one of the
major hurdles to discover and develop a kinase inhibitor with
an acceptable therapeutic window and minimal side
effects.20−22 Therefore, the kinase selectivity of compound
11b was investigated by screening it against a panel of 119
oncogenic kinases (oncoKP, Reaction Biology Corporation,
PA, USA) at a single concentration (1 μM). It was observed
that Pim-1 was the most potently inhibited kinase, with
inhibition of >98% at 1 μM. The only other kinase with more
than 95% inhibition at 1 μM concentration of 11b was TRKC
(96% inhibition at 1 μM). In addition, compound 11b only
inhibited three kinases (TRKC, Flt-3, and TRKB) with more
than 90% inhibition and 16 kinases with 50% inhibition at 1
μM concentration. The overall selectivity was determined by
calculating the selectivity score for this molecule (S(50) = 0.14,
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures for chemical synthesis and biological
evaluation, spectra, and kinase selectivity profile. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Fax: 925-560-0101. E-mail: yxu22@hotmail.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
Pim, provirus insertion site of Moloney murine leukemia virus;
BAD, Bcl-2-associated death promoter; hERG, the human
■
̀
ether-a-go-go-related gene
D
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