Potency switch between CHK1 and MK2: Discovery of imidazo[1,2-a]pyrazine- and imidazo[1,2-c]pyrimidine-based kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.100

Chemistry has been developed to access both imidazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines. Small structural modifications in both series led to a switch of potency between two kinases involved in mediating cell cycle checkpoint control, CHK1 and MK2.

Full text of DOI:10.1016/j.bmcl.2013.03.100

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2863–2867
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potency switch between CHK1 and MK2: Discovery of imidazo[1,2-
a]pyrazine- and imidazo[1,2-c]pyrimidine-based kinase inhibitors
Zhaoyang Meng a, , Jeffrey P. Ciavarri a,1, Andrew McRiner a,2, Yinyan Zhao a, Lianyun Zhao a,
⇑
Panduranga Adulla Reddy a, Xingmin Zhang a, Thierry O. Fischmann b, Charles Whitehurst a,
M. Arshad Siddiqui a,3
a Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, USA
b Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemistry has been developed to access both imidazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines.
Small structural modifications in both series led to a switch of potency between two kinases involved
in mediating cell cycle checkpoint control, CHK1 and MK2.
Received 31 January 2013
Revised 19 March 2013
Accepted 25 March 2013
Available online 4 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Potency switch
CHK1
MK2
Kinase
Inhibitor
Upon DNA damage, cells are arrested at (G1, S, or G2/M) check-
points in order to initiate the DNA repair process. Compared to nor-
mal cells that have an operative G1 checkpoint enabling repair
upon DNA damage, more than half of tumor cells are defective in
tumor suppressor p53-mediated G1 arrest upon DNA damage. To
cope with damaged DNA, p53-deficient tumor cells have to rely
on the S and G2/M checkpoint.1 Checkpoint kinase 1 (CHK1), which
regulates both the S and G2/M checkpoint, is a promising target for
the development of small molecule inhibitors against cancer.2,3
Several CHK1 inhibitors have entered clinical trials including
PF-00477736,4 AZD77625 and SCH-900776.6 Recently, mitogen
activated protein kinase-activated protein kinase 2 (MK2) was
identified as a cell cycle checkpoint kinase that can also maintain
the S and G2/M checkpoints and shares similar substrate specificity
with CHK1.7 Yaffe and co-workers further demonstrated that p53-
defective cells contain two spatially and temporally distinct G2/M
checkpoint networks—an early, CHK1-dependent nuclear check-
point and a late MK2-dependent cytoplasmic checkpoint.8 Thus,
like CHK1 inhibitors, inhibitors of MK2 may provide a novel ap-
proach for cancer treatment.9 It has been well documented that
MK2 plays a crucial role in the synthesis of tumor necrosis factor
(TNF)-a as well as other inflammatory cytokines and MK2 inhibi-
tors have been proposed as possible agents for the treatment of
inflammatory disease.10
From a high throughput affinity-selective mass spectrometry-
based screen of human recombinant MK2 against a Merck internal
compound library, we identified the imidazo[1,2-a]pyrazine amide
1 (Fig. 1) as a moderate MK2 inhibitor in a follow-up enzyme assay
verification screen of hits. Interestingly, this hit was originally syn-
thesized in a CHK1 inhibitor program as part of an ATP-competitive
inhibitor series and it showed better in vitro potency for CHK1
(IC50: 0.039 lM) than MK2 (IC50: 0.65 lM). With an aim to further
improve the potency and selectivity profile toward MK2, our SAR
development included (1) exploration of the basic amine substitu-
ent at the C8 position, (2) variation of the hydrophobic group at the
C2 position of the imidazo[1,2-a]pyrazine core, and (3) replace-
ment of the imidazo[1,2-a]pyrazine with imidazo[1,2-c]pyrimi-
dine. In this Letter, we wish to report how the substituent
variation and core modification influenced the potency and selec-
tivity against CHK1 and MK2.
⇑
Corresponding author Address: Merck Research Laboratories, West Point, PA
19486, USA. Tel.: +1 215 652 6199.
E-mail address: zhaoyang.meng@merck.com (Z. Meng).
Present address: Millennium: The Takeda Oncology Company, Cambridge, MA
02139, USA.
Our initial synthetic efforts focused upon modifications of the
C8 and C2 positions of compound 1. As shown in Scheme 1, the
imidazo[1,2-a]pyrazine intermediate 4 was assembled by conden-
à
Present address: EnVivo Pharma Inc., Watertown, MA 02472, USA.
§
sation of 2-amino-3-chloropyrazine 3 with different
a-bromom-
Present address: Paraza Pharma Inc., Laval, Quebec, Canada, H7V 5B7.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.100

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Z. Meng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2863–2867
Table 1
CHK1 and MK2 inhibition data for 8a–i
HN
NH
R2
8
1
N
N
7
N
Cl
N
2
R1
N
6
N
O
5
3
H2N
O
H2N
1
CHK1 IC50 = 0.039 uM
MK2 IC50 = 0.65 uM
Compds
R1
R2
CHK1 IC50
0.039
(l
M)
MK2 IC50
0.65
(lM)
Cl
Cl
Cl
Cl
Figure 1. High throughput screening MK2 hit.
HN
1
NH
N
8a
8b
NA
10.0
4.15
NH
Cl
NHR2
HN
N
Cl
Br
N
N
0.69
a
c
N
b
N
NH
R1
R1
R1
+
N
N
5
O
NH2
N
2
NH2
4
3
8c
0.29
0.89
4.09
0.40
NH
NHR2
NHR2
N
NHR2
Cl
N
N
N
HN
e
d
N
N
N
R1
R1
R1
8d
N
N
NH
I
CN
6
H2N
H2N
H2N
H2N
O
8
Cl
Cl
Cl
8e
8f
1.08
0.31
5.07
0.060
1.90
0.82
7
NH
Scheme 1. Reagents and conditions: (a) DME, 90 °C; (b) R2NH2, K2CO3, NMP,
100 °C; (c) NIS, CHCl3; (d) Pd(PPh3)4, Zn(CN)2, DMF, 80 °C; (e) concd H2SO4, 60 °C.
NH
NH
ethylphenyl ketone 2. Various amines could be added to the 8-po-
sition by a displacement reaction. Subsequent regioselective iod-
onization with NIS occurred at the C5 position of compound 5.
Pd-catalyzed cyanation followed by hydrolysis of the nitrile group
with concentrated H2SO4 gave the desired compounds 8a–i.11
The CHK1 and MK2 potency of the analogs 8a–i are shown in
Table 1. Removal of the piperidine N–H (8a), substitution with an
alternative piperidine isomer (8b) or a more flexible acyclic amine
derivative (8c) all resulted in loss of potency against MK2. Compar-
ing 8d to 8c, an extension of the terminal amine functionality by
one more carbon led to a significant improvement in both MK2 po-
tency and selectivity over CHK1. The corresponding N-desmethyl
analog 8e proved to be the most potent MK2 inhibitor identified
8g
Cl
HN
HN
8h
8i
0.023
0.027
0.91
0.91
NH
NH
F
Table 2
Kinase selectivity of MK2 inhibitor 8e
in the imidazo[1,2-a]pyrazine series with an IC50 of 0.06 lM and
H2N
nearly 18-fold selectivity over CHK1. By contrast, the related cis
cyclohexane-1,4-diamine 8f showed 31-fold loss in MK2 potency.
Changes to the hydrophobic group at the C2 position of the mole-
cule (1 vs 8h, 8i; 8e vs 8g) resulted in a loss of potency against MK2
and did little to alter the kinase selectivity trend.
The selectivity of the most potent MK2 inhibitor 8e was inves-
tigated by screening against a panel of kinases. While 8e was selec-
tive against a number of kinases, it did show modest inhibition of
certain kinases (i.e. IRAK4, CAMK4, and LCK (Table 2)).
Having achieved reasonable potency and selectivity for MK2
with a compound in the imidazo[1,2-a]pyrazine series (8e), we ex-
tended the effort to investigate the utility of the closely related
imidazo[1,2-c]pyrimidine scaffold (Scheme 2). Heating a mixture
of 5-iodocytosine 9 and 2-bromo-1-(4-chlorophenyl)ethanone 10
at reflux followed by palladium-catalyzed cyanation with Zn(CN)2
as the cyanide source produced 12. Compound 12 was treated with
P2S5 and sulfur in pyridine to afford 13, which was then reacted
with MeI in the presence of KOtBu at room temperature to yield
14. Intermediate 15 was made by heating 14 in a sealed vial with
an excess of the appropriate amine at 150 °C. The desired com-
pounds 16a–e were obtained after treatment with concentrated
H2SO4.
NH
N
N
Cl
N
O
H2N
8e
Kinase
IC50
(
l
M)
Kinase
hIGF1R
IC50 (lM)
hIKKB
hAKT1
hMET
hERK2
hERK2
hJAK2
>30.0
>30.0
>30.0
>30.0
>30.0
13.0
12.0
12.0
12.0
1.5
1.1
1.0
hCSNK1D
hTSSK2
hIRAK4
hCAMK4
hLCK
change to the imidazo[1,2-c]pyrimidine scaffold resulted in a con-
siderable loss of potency against both CHK1 and MK2 (Table 3).
While it is recognized that the relocation of N7 and the change
in proximity of N1 to the amide may play a role in binding to the
active site, it was hypothesized that the poor potency of 16a–b is
due to the formation of an intramolecular hydrogen bond between
Assuming the amide bond is making key interactions within the
hinge region of both kinases, it is interesting to observe that

Z. Meng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2863–2867
2865
I
I
NH2 Br
N
a
b
Cl
Cl
+
HN
HN
N
HN
N
N
O
O
O
10
11
9
CN
CN
N
c
d
Cl
Cl
Cl
HN
N
N
S
O
13
12
NH2
O
N
CN
CN
N
N
N
e
f
Cl
Cl
N
N
N
N
N
NHR3
SMe
NHR3
16
14
15
Scheme 2. Reagents and conditions: (a) DMF, refluxing; (b) Pd(PPh3)4, Zn(CN)2, DMF, 90 °C; (c) P2S5 , S, Pyridine, 225 °C; (d) MeI, KOtBu, tBuOH; (e) R3NH2, EtN(iPr)2, DMSO,
150 °C; (f) concd H2SO4, 60 °C.
Table 3
CHK1 and MK2 inhibition data for 16a–e
O
N
NH2
N
Cl
N
R1
Compds
R1
CHK1 IC50
NT
(lM)
MK2 IC50
—
(l
M)
MK2 Inhibition at 10
67
lM (%)
HN
16a
N
H
HN
HN
HN
16b
50.0
4.0
—
NH2
16c
16d
42.1
50.0
—
—
56
69
N
H
N
HN
HN
16e
50.0
—
61
NT = not tested.
the NH2 of the amide and the N1 nitrogen of the imidazo[1,2-
c]pyrimidine core which would disrupt potential binding to the ki-
nase (Fig. 2). To test this hypothesis, we prepared compounds 22a–
b in which the primary amide substituent in compounds 16a–b
was moved from the C8 to C5 position. Obviously, these newly de-
signed compounds 22a–b can also be viewed as analogues of the
original imidazo[1,2-a]pyrazine series (1 and 8e) with a simple
relocation of the N7 nitrogen to the 6 position of the scaffold.
H
O
N
N
H
N
R1
R1
relocation
of N atom
reversed
analogs
1
1
8
8
N
N
N
Cl
Cl
Cl
N
N
N
N
2
2
6
5
5
R4
H2N
O
H2N
O
16a-b
22a-b
1 & 8e
Figure 2. Rationale of compounds 22a–b.

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Z. Meng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2863–2867
Br
O
Br
Br
N
NH2
a
b
Cl
Cl
HN
N
HN
N
N
+
O
O
18
10
17
Br
Br
N
N
c
d
Cl
Cl
Cl
N
N
N
N
Cl
CN
20
19
N
NHR4
NHR4
N
e
Cl
N
N
O
N
CN
H2N
22a-b
21
Scheme 3. Reagents and conditions: (a) DMF, refluxing; (b) POCl3, EtN(iPr)2,
100 °C; (c) KCN, DABCO, DMSO/H2O = 9:1, 40 °C; (d) Pd2dba3, rac-BINAP, NaOtBu,
R4NH2, toluene, 100 °C; (e) concd H2SO4, 60 °C.
Figure 3. X-ray structure of CHK1 in a complex with compound 22a.
A CHK1 inhibitor based on the imidazo[1,2-c]pyrimidine scaf-
fold has not been previously reported and the X-ray crystal struc-
ture of CHK1 in complex with compound 22a ( Fig. 3) reveals
several important binding characteristics.13 Key polar interactions
between protein and bound inhibitor are shown in red dashes. The
carbonyl and NH2 of the amide group form hydrogen bonds with
the Cys87 and Glu85 residues in the hinge region of the CHK1
ATP binding pocket. The NH2 of the amide group along with the
N6 nitrogen interacts with a conserved water molecule, which fur-
ther locks the amide confirmation. The NH of the piperidine moiety
is involved in making hydrogen bonds with Asp 148 and Glu 134.
In summary, chemistry has been developed to access both imi-
dazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines. Small struc-
tural modifications in both series led to switching of preferential
inhibitory activity between two kinases involved in mediating cell
cycle checkpoint control, CHK1 and MK2. Knowledge gained from
the X-ray structure of the binding mode of 22a in the CHK1 ATP
cleft along with the reported potency switching SAR may assist
in the development of more potent and selective ATP-competitive
MK2 inhibitors in the future.
Table 4
CHK1 and MK2 inhibition data for 22a–b
R1
N
A
B
Cl
N
O
H2N
Compds
A
B
R1
CHK1 IC50
0.039
(lM)
MK2 IC50
0.65
(lM)
H
N
1
N
CH
NH
NH
H2N
8e
N
CH
N
1.08
0.018
N.T.
0.060
0.84
1.98
NH
NH
HN
22a
22b
CH
CH
H2N
Acknowledgments
N
We thank Drs. John Piwinski and Neng-Yang Shih for support of
this work.
N.T. = not tested.
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