Discovery of pyrazolo[1,5-a]pyrimidine-based Pim inhibitors: A template-based approach

Article abstract of DOI:10.1016/j.bmcl.2013.08.110

The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a] pyrimidine-derived hit 5 to the identification of a series of potent, pan-Pim inhibitors such as 11j are described.

Full text of DOI:10.1016/j.bmcl.2013.08.110

Accepted Manuscript
Discovery of Pyrazolo[1,5-a]pyrimidine-based Pim Inhibitors: A Template-
Based Approach
Michael P. Dwyer, Kartik Keertikar, Kamil Paruch, Carmen Alvarez, Marc
Labroli, Cory Poker, Thierry O. Fischmann, Rosemary Mayer-Ezell, Richard
Bond, Yan Wang, Rita Azevedo, Timothy J. Guzi
PII:
S0960-894X(13)01059-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.110
BMCL 20848
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
10 June 2013
26 August 2013
29 August 2013
Please cite this article as: Dwyer, M.P., Keertikar, K., Paruch, K., Alvarez, C., Labroli, M., Poker, C., Fischmann,
T.O., Mayer-Ezell, R., Bond, R., Wang, Y., Azevedo, R., Guzi, T.J., Discovery of Pyrazolo[1,5-a]pyrimidine-based
Pim Inhibitors: A Template-Based Approach, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://
dx.doi.org/10.1016/j.bmcl.2013.08.110
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Discovery of Pyrazolo[1,5-a]pyrimidine Pim inhibitors: A
Template-Based Approach.
Michael P. Dwyer,a,* Kerry Keertikar,a,* Kamil Paruch,a Carmen Alvarez,a Marc Labroli,a Cory Poker,a Thierry
O. Fischmann,a Rosemary Mayer-Ezell,b Richard Bond,b Yan Wang,b Rita Azevedo,c and Timothy J. Guzib
aDepartment of Chemical Research, Merck Research Laboratories, 2015 Galloping Hill Rd. Kenilworth, NJ 07033.
bDepartment of Tumor Biology, Merck Research Laboratories, 2015 Galloping Hill Rd. Kenilworth NJ 07033.
cMolecular Design and Informatics, Oss, Netherlands 5340 BH.
The synthesis and SAR development of a class of pyrazolo[1,5-a]pyrimidine-based Pim kinase inhibitors are detailed from an early HTS
hit to a pan-Pim inhibitor such as 11j .
O
N
N
H
11j
Pim-1 IC50 = 1.3 nM
Pim-2 IC50 = 7.3 nM
Pim-3 IC50 = 1.8 nM
N
N
H2N
N
N

Discovery of Pyrazolo[1,5-a]pyrimidine-based Pim Inhibitors: A
Template-Based Approach
Michael P. Dwyer,a,* Kartik Keertikar,a,* Kamil Paruch,a Carmen Alvarez,a Marc Labroli,a
Cory Poker,a Thierry O. Fischmann,a Rosemary Mayer-Ezell,b Richard Bond,b Yan
Wang,b Rita Azevedo,c and Timothy J. Guzia
aDepartment of Chemical Research, bDepartment of Tumor Biology, Merck Research Laboratories, Kenilworth NJ 07033.
cMolecular Design and Informatics, Oss, Netherlands 5340 BH
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000
Abstract—The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a]pyrimidine-derived hit 5 to the identification of a
series of potent, pan-Pim inhibitors such as 11j are described. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.
The Pim kinases are a family of serine/threonine kinases
in the CAMK (calmodulin-dependent protein kinase)
family consisting of three members Pim-1, Pim-2, and
Pim-3.1 There is a high level of sequence homology
between these family members which is suggestive of
functional redundancy.2 Both Pim-1 and Pim-2 have
been shown to be overexpressed in a number of human
hematopoietic malignancies such as myeloid leukemia
and lymphomas.3
In addition, DNA microarray
analyses demonstrated that Pim-1 is overexpressed in
human prostate cancer and correlates with disease
outcome.4 In animal models, overexpression of Pim-1
and Pim-2 in human prostate cancer cells enhances their
growth in nude mice.5 These observations suggest that
Pim-1 and/or Pim-2 inhibitor may be effective in the
treatment for hematologic malignancies.
Initial HTS screening of our internal compound
collection identified pyrazolo[1,5-a]pyrimidines 5 and
615 as early Pim program hits (Figure 2). These initial
hits possessed sub-micromolar potency for Pim-1, and 5
showed good selectivity versus both CDK2 and CHK1.
We found this chemotype was of particular interest due
to our long standing efforts utilizing the pyrazolo[1,5-
a]pyrimidine core for the identification of selective cell-
cycle kinase inhibitors of CDK (Dinaciclib)16 and
CHK1 (SCH 900776)17 which are currently under
clinical evaluation. With these initial screening hits in
hand (Figure 2), efforts were initiated to conduct
systematic modifications around the pyrazolo[1,5-
a]pyrimidine core to enhance in-vitro potency against
Pim-1 as well as other family members such as Pim-2.
It was not clear at the outset of the program whether a
Pim isoform selective inhibitor or a pan-Pim inhibitor
would be more desireable. The ability to access
compounds that possessed either profile was of interest
for biology validation studies.
In 2005, Knapp and coworkers described the first small
molecule Pim inhibitors6 represented by 1 (Figure 1).
Since these initial reports, several additional small
molecule Pim inhibitor classes7-13 have been reported and
several excellent reviews of recent Pim inhibitor
chemotypes have appeared.14 In addition, several X-ray
structures of Pim-1 co-crystallized with inhibitors point
to some very unique structural features for this family of
kinases: the hinge region. The unique hinge region for
Pim-1 contains an extra residue, proline-123, which
allows for the formation of only a single hydrogen
bond with either ATP or ATP mimetics. With this
information in hand, we set about identifying novel Pim
inhibitors that may serve as effective agents for the
treatment of hematologic malignancies.
Figure 1. Representative Pim inhibitors 1-4.
Figure 2. Pyrazolo[1,5-a]pyrimidine-based Pim Hits and pyrazolo[1,5-
a]pyrimidine-based cell-cycle kinase inhibitors.

environment.
This modification resulted in the
identification of extremely selective Pim-1 compounds
such as compounds 9m and 9n.
1
R
N
N
R
N
N
5, 9a-n
Table 1. Pim-1 and Pim-2 in-vitro activity of N5 pyrazolo[1,5-
a]pyrimidines 5, 9a-n.
Pim-1 IC50
Pim-2 IC50
Compd
NRR1
(nM)a
(nM)a
5
87
na
na
9a
1527
Initial chemistry efforts focused upon structural
modifications of the C5 position of naphthyl derivative
5 to explore the SAR around this region. The synthetic
preparation of the C5 derivatives is depicted in Scheme
1. Condensation of 3-aminopyrazole with 1,3-dimethyl
uracil18,19 followed by treatment with POCl3 and
iodination afforded compound 7. Treatment with
sodium thiomethoxide followed by Suzuki coupling
with 2-naphthyl boronic acid under standard conditions
afforded 8. Oxidation of the thiomethyl moiety with
mCPBA afforded roughly a 1:1 mixture of the
intermediate sulfoxide/sulfone which was treated
directly with amines in NMP to afford the title
compounds 9a-e,k,m-n found in Table 1. The Boc
protected diamine intermediates were treated with TFA
to afford final compounds 9f-j,l (Scheme 1).
9b
9c
804
718
na
na
9d
9e
na
18
na
na
9f
3.1
2.9
190
157
9g
9h
9i
6.8
1.5
25
1462
87
9j
781
9k
1618
na
9l
5.1
5.0
2302
na
9m
9n
1.6
4308
Scheme 1. a) 1,3-dimethyluracil, NaOMe; b) POCl3; c) NIS; 55% (3
steps) d) NaSMe, THF; e) 2-naphthyl boronic acid, PdCl2(dppf), K3PO4,
DME/H2O 65% (2 steps) f) mCPBA, CH2Cl2; g) HNRR1, NMP, 20-62%
(two steps): h) TFA, CH2Cl2; 85-96%.
aValues are the mean of two (n =2) runs. See ref 20 for assay details.
bna = not active at >5000 nM
As depicted in Table 1, simple shortening of the chain
or elimination of the pyridyl functionality from
compound 5 resulted in a dramatic loss in both Pim-1
and Pim-2 potency. While simple alkyl or cycloalkyl
functionality (9c,d) did not retain potency versus either
Pim isoform, incorporation of polar functionality
particularly a primary amine moiety resulted in a
dramatic improvement in potency for both Pim-1 and
Pim-2 as demonstrated by compound 9f. While the
pendant amino functionality could be moved around and
retain reasonable Pim-1 activity, the Pim-2 activity
seemed to be very sensitive towards the steric
Owing to our interest in obtaining a pan-Pim inhibitor
for a tool compound for biological studies, we chose to
further explore SAR around compound 9i which
represented the best pan-Pim profile in Table 1 as
measured by the Pim-1 and Pim-2 in-vitro assays. With
compound 9i in hand, efforts were directed towards the
exploration of additional C3 modifications, particularly
around bicyclic and biaryl motifs.21 The preparation of
these C3 analogs is shown in Scheme 2. Treatment of
iodide
7
with tert-butyl(2-aminoethyl)carbamate
followed by subsequent Boc protection afforded 10.
Using 10 as a common starting material, Suzuki

coupling with various boronic acids/boronates under
standard conditions followed by TFA-mediated Boc
deprotection afforded the title compounds 11a-o (Table
2).
11b
11c
11d
7
212
19
7.2
9.4
I
I
Boc
24
N
N
Cl
N
a, b
BocHN
N
N
N
N
10
11e
11f
247
10
na
7
R
H
N
N
d,e
H N
2
137
N
N
11a-o
Scheme 2. a) tert-butyl(2-aminoethyl)carbamate, NMP; b) Boc2O,
DMAP; 60% (two steps) c) RB(OH)2, PdCl2(dppf), K3PO4, DME/H2O; d)
TFA, CH2Cl2, 40-65% (two steps).
11g
834
na
As summarized in Table 2, simple replacement of the
napthyl motif with either a benzothiophene (11a) or
benzothiazole (11b) resulted in very comparable Pim in-
11h
11i
3511
4.5
1.3
na
na
89
7.3
na
vitro profiles to the compound 9i.
incorporation of polar functionality such as
However,
a
benzimidazolone (11c) or indazole (11d) resulted in 3-4
fold improvement in Pim-2 activity while retaining
11j
11k
good Pim-1 potency.
Systematic exploration of
placement of biaryl functionality demonstrated that
meta-substituted derivatives (11f) were far superior to
both ortho and para-substituted derivatives.
The
pendant ring of the biaryl motif of 11f was found to be
tolerant of certain heterocyclic rings such as a 1,2,4-
oxadiazole (11j) and furan (11i) while the 2-imidazole
derivative (11k) was completely devoid of Pim activity,
illustrating the stringent structural requirements for this
ring. In this vein, attempted replacement of the pendant
ring with heteroatom linked rings or O-tethered
functionality led to a loss in Pim potency while
replacement of the proximal ring of 11f was generally
not well tolerated as illustrated by compounds 11n and
11o.
11m
136
886
11n
11o
162
834
na
na
aValues are the mean of two (n =2) runs. See ref 20 for assay details.
bna = not active at >5000 nM
As depicted in Table 2, compound 11j emerged as a
lead pan-Pim inhibitor based upon the single digit
nanomolar potency it demonstrated versus both Pim-1
and Pim-2. Further evaluation of this compound across
a battery of kinases summarized in Table 3 reinforces
the pan Pim activity (including Pim-3) of 11j while
demonstrating excellent selectivity versus several other
kinases. This compound was found to be clean in both
CYP P450 evaluation (3A4, 2D6, and 2C9 > 20 μM)
and hERG ion channel evaluation (11% I @ 10 μM).
Table 2. Pim-1 and Pim-2 in-vitro activity of C3 pyrazolo[1,5-
a]pyrimidines 9i, 11a-n.
Pim-1 IC50
Pim-2 IC50
Compd
R
(nM)a
(nM)a
9i
1.5
2
87
11a
114
Table 3. Kinase selectivity of Compound 11j

IC50
(nM)
1.8
7100
580
>3000
>3000
950
Kinase
Pim-3
CHK1
CDK2
PI3K
mTor
CK2
In order to better understand the SAR trends observed in
Tables 1 and 2 for this class of compounds, X-ray co-
crystal structures of two compounds (11p22 and 9g)
bearing different functionality at C3 and C5 bound to
Pim-1 (shown in Figures 3 and 4) were obtained.
In Figure 4, the larger C3 naphthyl group in 9g induces
a flip from the canonical binding mode observed for
11p shown in Figure 3. The N1 moiety of the
pyrazolo[1,5-a]pyrimidine acts as a H-bond acceptor for
Lys57 with the C3 napthyl group oriented parallel to
the hinge region of the protein. In addition, the primary
amine on the cyclohexyl ring of 9g makes a H-bond
contact with the oxygen of Glu171. The binding mode
observed for 9g is consistent with an X-ray structure for
a structurally related imidazo[1,2-b]pyridazine bound to
In Figure 3, compound 11p does not make contact with
the hinge area of the Pim-1 protein but makes only a
single hydrogen bond to a water molecule found in the
kinase specificity pocket. The canonical binding mode
observed for 11p in Pim-1 is consistent with the
orientation observed for most other pyrazolo[1,5-
a]pyrimidine derivatives from both our CDK and CHK1
programs.16,17
Pim-1.6
While a X-ray co-crystal structure of
compound 11j bound to Pim-1 could not be obtained, it
can be rationalized this compound might bind in a
similar fashion to 9g with the large C3 group oriented
along the hinge region.
Figure 4. X-ray of crystal structure of 9g in Pim-1.24
Figure 3. X-ray of crystal structure of 11p bound to Pim-1.23
In summary, systematic optimization of both the C3 and
C5 position of pyrazolo[1,5-a]pyrimidine Pim hit 5 led
to the discovery of potent, pan Pim inhibitors
represented by 11j. Modifications of either the C3 or
C5 functionality of this series were shown to play a
critical role in determining the ultimate selectivity
versus the various Pim isoforms. In addition, single X-
ray crystallography of several members of the
pyrazolo[1,5-a]pyrimidine series bound to Pim-1

illustrated several unique binding modes which seem to
be driven by the size of the C3 substituent. This work
futher validates the utility of the pyrazolo[1,5-
a]pyrimidine core toward the development of additional
classes of kinase inhibitors. Additional evaluation of
this class of Pim inhibitors25 including compound 11j
will be reported in due course.
15. The first pyrazolo[1,5-a]pyrimidine-based Pim
inhibitor was reported by Knapp and coworkers: See ref 6.
16. (a) Dwyer, M. P.; Paruch, K.; Alvarez, C.; Doll, R. J.;
Keertikar, K.; Duca, J.; Fischmann, T. O.; Hruza, A.;
Madison, V.; Lees, E.; Parry, D.; Seghezzi, W.;
Sgambellone, N.; Shanahan, F.; Wiswell, D.; Guzi, T. J.
Bioorg. Med. Chem. Lett. 2007, 17, 6216. (b) Paruch, K.;
Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll,
R. J.; Keertikar, K.; Knutson, C.; McKittrick, B.; Rivera,
J.; Rossman, R.; Tucker, G.; Fischmann, T.; Hruza, A.;
Madison, V.; Nomeir, A. A.; Wang, Y.; Lees, E.; Parry,
D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan,
F.; Wiswell, D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar,
V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T. J.
Bioorg. Med. Chem. Lett. 2007, 17, 6220. (c) Paruch, K.;
Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan, T.-Y.; Doll,
R. J.; Keertikar, K.; Knutson, C.; McKittrick, B.; Rivera,
J.; Rossman, R.; Tucker, G.; Fischmann, T. O.; Hruza, A.;
Madison, V.; Nomeir, A. A.; Wang, Y.; Lees, E.; Parry,
D.; Sgambellone, N.; Seghezzi, W.; Schultz, L.; Shanahan,
F.; Wiswell, D.; Xu, X.; Zhou, Q.; James, R. A.; Paradkar,
V. M.; Park, H.; Rokosz, L. R.; Stauffer, T. M.; Guzi, T. J.
ACS Med. Chem. Lett. 2010, 1, 204-208.
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a]pyrimidine. More recent examination of this reaction by
Gavrin and coworkers (Reference 19) found this
cyclization in fact affords the 5-OH pyrazolo[1,5-
a]pyrimidine instead of the 7-OH analog first reported by
Kraft, A. S Mol. Cancer Res. 2005, 3, 443.
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Cutler and coworkers.
Independent X-ray analysis
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core as proposed by Gavrin and coworkers.
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STK1 (Pim2 & 3), anti-phospho Ser/Thr Cryptate and
streptavidin-XL-665 in Corning black LVP 384 well plates
(Fisher Catalog # 3676). (Pim 1 and 3 are from Millipore
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catalog #s 14-573 and 14-738, Pim 2 is from Invitrogen
catalog # PV4036). Briefly, 0.75 ng/well enzyme was
added in 7 μl kinase buffer (10 mM HEPES pH 7, 0.02%
sodium azide, 0.01% BSA, 0.1 mM Orthovanidate, 1mM
DTT, 10mM MgCl2) followed by 3 μl of compound diluted
from DMSO 1:6 with kinase buffer. After a 30 min
preincubation, the reaction was started by adding 2 μl of
6X ATP/STK mix (final concentrations for Pim 1: 25 μM
ATP and 200 nM STK3; Pim 2: 5 μM ATP and 700 nM
STK1; Pim 3: 10 μM ATP and 300 nM STK1). Reactions
were run at room temperature for 45 min (Pim 1) or 60 min
(Pim 2 and 3) and stopped by addition of 10 μl detection
buffer containing 50 nM, 175 nM, or 75 nM SA-XL665 for
Pims 1, 2, and 3 respectively and anti-phospho Ser/Thr
Cryptate at 1:100. Fluorescense at 620 nM (Cryptate) and
665 nM (XL665) were measured on a Pherastar (BMG
Labtech) and the 665/620 ratio x 10,000 was used for data
analysis (ABASE). Dose–response curves were generated
from duplicate 8 point serial dilutions of inhibitory
compounds. IC50 values were derived by nonlinear
regression analysis.
21. Early SAR efforts around the C3 monocyclic aryl
motifs as found in compound 6 did not yield significant
improvements in the in-vitro potency (unpublished
results).
22. Compound 11p was prepared according to the
synthetic route outlined in Scheme 2 by substituting
dimethylethylamine and Bocpyrazole boronic acid to
afford the final compound. The binding assay data for 11p
is Pim-1 IC50 = 7 nM and Pim-2 IC50 = 185 nM.
23. The X-ray coordinates for compound 11p bound to
Pim-1 have been deposited in the ProteinDataBank (PDB
ID code: 4MBI).
24. The X-ray coordinates for compound 9g bound to
Pim-1 have been deposited in the ProteinDataBank (PDB
ID code: 4MBL).
25. A series of potent 3-carboxamido pyrazolo[1,5-
a]pyrimidine Pim inhibitors has been recently disclosed:
Wang, X.; Magnuson, S.; Pastor, R.; Fan, E.; Hu, H.; Tsui,
V.; Deng, W.; Murray, J.; Steffek, M.; Wallweber, H.;
Moffat, J.; Drummond, J.; Chan, G.; Harstad, E.; Ebens, A.
J. Bioorg. Med. Chem. Lett. 2013, 23, 3149.
*Corresponding Author (MPD) tel: (732) 594-1733;
michael.dwyer@merck.com