Discovery of imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.07.008

The synthesis and structure-activity relationships (SAR) of novel, potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors are described. The X-ray crystal structure of imidazo[1,2-a]pyrazine Aurora inhibitor 1j is disclosed. Compound 10i was identified as lead compound with a promising overall profile.

Full text of DOI:10.1016/j.bmcl.2010.07.008

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5170–5174
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors
a
b
b
a
a
David B. Belanger a, , Patrick J. Curran , Alan Hruza , Johannes Voigt , Zhaoyang Meng , Amit K. Mandal ,
*
M. Arshad Siddiqui a, Andrea D. Basso c, Kimberly Gray c
a Department of Chemistry, Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, United States
b Department of Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
c Department of Oncology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationships (SAR) of novel, potent imidazo[1,2-a]pyrazine-based
Aurora kinase inhibitors are described. The X-ray crystal structure of imidazo[1,2-a]pyrazine Aurora
inhibitor 1j is disclosed. Compound 10i was identified as lead compound with a promising overall profile.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 May 2010
Revised 1 July 2010
Accepted 2 July 2010
Available online 8 July 2010
Keywords:
Aurora kinase inhibitors
Imidazo[1,2-a]pyrazine
Aurora kinases A, B, and C are cell cycle regulated serine/threo-
nine kinases expressed only during mitosis.1 Ubiquitously ex-
pressed Aurora A regulates cell cycle events such as centromere
maturation, bipolar spindle assembly, mitotic entry and exit, as
well as, kinetochore-spindle attachment.1 Aurora B phosphorylates
histone H3, regulates chromosomal remodeling, kinetochore-spin-
dle attachment, and cytokinesis.1 Aurora C is believed to have a
function related to Aurora B but has limited expression.1 Aurora
A and B are essential protein kinases and as such are required for
successful mitotic progression. siRNA depletion of Aurora A results
in G2/M delay followed by apoptotic cell death. siRNA depletion of
Aurora B causes aberrant endoreduplication (polyploidy) followed
by apoptosis. Dual Aurora A/B siRNA knockdown displays an Aur-
ora B siRNA phenotype.1 Since it was first discovered that Aurora
A kinase was over expressed in breast tumors,2 intense academic,
and pharmaceutical research has helped develop a better under-
standing of the role of Aurora kinases in cancer. Amplification or
over expression of Aurora kinases has been observed in multiple
tumor types3,4 and is often correlated with poor prognosis.5
According to recent letters, 13 Aurora inhibitors are currently in
Phase I/II clinical trials with many other companies in preclinical
research.4,6 Interestingly, a diverse set of Aurora kinase inhibitors
has progressed to clinical trials (selective Aurora A, selective Aur-
ora B, or Aurora A/B inhibitors), yet no drugs have been approved
for use. Our initial goal was to identify an Aurora A/B kinase inhib-
itor with sub-micromolar target engagement (phos-HH3) and an
in vitro DMPK profile suitable for progression to lead optimization.
Herein, we report our initial SAR findings in the imidazo[1,2-a]pyr-
azine series and the first X-ray structure of this compound class
with Aurora A.
Screening of our internal compound library against Aurora A
identified imidazo[1,2-a]pyrazine-based inhibitors (Fig. 1).14 Com-
pound 1 was a low molecular weight (MW = 311.37), modestly po-
tent Aurora A and Aurora B inhibitor (Aurora A IC50 = 0.57
lM,
Aurora B IC50 = 0.36 M). Our primary objective was to improve
l
biochemical potency and mechanism-based cell activity (phos-
HH3). Medicinal chemistry efforts commenced by simultaneously
investigating the SARs at the 3-, 6- and 8-positions.
The synthetic route to 3-, 8-disubstituted imidazo[1,2-a]pyra-
zine analogs of Aurora inhibitor 1 is shown in Scheme 1. The imi-
dazo[1,2-a]pyrazine core structure was assembled by conden
sation of chloroacetaldehyde and commercially available 2-amino-
3-chloropyrazine 2.7 Subsequent bromination with bromine in ace-
tic acid afforded the 3-bromo-8-chloroimidazo[1,2-a]pyrazine 3 in
N
N
3
6
N
N
N
1
8
NH
S
N
Aurora A IC50 = 0.57µM
Aurora BIC50 = 0.36µM
* Corresponding author. Tel.: +1 617 499 3504; fax: +1 617 499 3535.
E-mail address: david.belanger@merck.com (D.B. Belanger).
Figure 1. Aurora kinase screening hit 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.008

D. B. Belanger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5170–5174
5171
R3'
N
N
Br
Br
Br
N
a,b
c,d
e
f,g
N
N
N
N
N
N
N
NH2
N
N
N
N
N
N
Cl
O S O
NH
5
NH
1
8R
Cl
8R
2
3
4
Scheme 1. Reagents and conditions: (a) 2-chloroacetaldehyde diethyl acetal, concd HCl, water, reflux; Et2O extract, concentrate, 2-amino-3-chloropyrazine, cat. HCl, DME,
reflux, 90%; (b) AcOH, Br2, rt, 91%; (c) NaSMe, MeOH, rt, 95%; (d) m-CPBA, DCM, rt, 95%; (e) NaH, DMSO, aniline, rt; (f) Suzuki reaction; (g) 4 N HCl dioxane, if necessary.
Table 1
Table 1 (continued)
Aurora A inhibition data for imidazopyrazines 1a–l
0
Compd R8
Aurora Aa IC50 M) Aurora Ba IC50
(l (lM)
R3
R3'
S
N
N
N
1k
H
0.006
0.018
N
N
O
N
N
S
O
NH
R8
R3
0
Compd R8
Aurora Aa IC50
(l (lM)
M) Aurora Ba IC50
O
S
1l
H
0.081
0.12
O
S
N
N
1a
Me
Me
0.57
0.26
O
S
N
a
Assay conditions listed in Ref. 15.
N
1b
1c
0.64
1.6
0.70
0.20
S
N
good yield. Intermediate 3 was originally envisioned to be a vital
synthetic intermediate as we8 and others9 had shown that various
amines and anilines could be added to the 8-position. In our hands,
the reaction of dihalide 3 with anilines (e.g., 3-dimethylamino ani-
line) proceeded quite sluggishly even using forcing conditions (ex-
cess aniline, reaction temperature >150 °C). Furthermore, less
nucleophilic aminoheterocycles (e.g., 2-amino-5-methylthiazole)
failed to give any product. Auspiciously, we found that 3-bromo-8-
(methylsulfonyl)imidazo[1,2-a]pyrazine 4 was quite reactive to-
ward various anilines and aminoheterocyles such as 2-amino-5-
methylthiazole. Sulfone 4 was prepared in two steps by sequential
treatment of 3-bromo-8-chloroimidazo[1,2-a]pyrazine 3 with so-
dium methanethiolate followed by m-chloroperbenzoic acid oxida-
tion.10 Installation of groups at the 3-position was achieved by a
Suzuki reaction.
Me
Me
HN
O
S
1d
0.17
0.11
N
S
O
S
N
1e
1f
Me >10
Me >10
N/A
N/A
3.0
N
Initial SAR studies revealed that only isothiazole analogs related
to hit 1a retained Aurora A potency (Table 1, compounds 1b–d).
Structurally similar congeners derived from 8-aminothiadiazole
1e and 8-aminothiazole 1f were found to be significantly less ac-
tive than the initial screening hit 1a. Interestingly, 8-aminothioph-
ene analog 1g retained modest potency versus Aurora A, but most
S
S
N
1g
1h
Me
2.5
substituted anilines showed enzyme potency >5 lM. The distinct
N
preference for precise 8-position sulfur containing heterocycle
was clearly evident given that over 50 substituted aniline analogs
of 1a were evaluated and all showed poor biochemical potency
Me >10
N/A
(Aurora A IC50 >5 lM). In contrast, Aurora A was more receptive
O
1i
1j
Me
H
3.7
N/A
to inhibitors bearing an unsubstituted 3-(4-pyrazolo) group. For
instance, isothiazole inhibitors 1j and 1k both showed improved
enzyme potency (Aurora A IC50 6 4 nM and 6 nM, respectively)
S
O
N
S
and low micromolar target engagement (phos-HH3 IC50 = 1.3 lM).
N
60.004
60.013
Furthermore, inclusion of the 3-(4-pyrazolo) afforded inhibitors
(e.g., 8-aniline 1l) with improved Aurora A potency though sulfur
containing heterocycles were still preferred.

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D. B. Belanger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5170–5174
Figure 2. X-ray structure of inhibitor 1j in Aurora A (2.4 Å). Graphic was prepared using PyMOL.16
SEM
H
N
SEM
N
N
N
N
N
I
R6
S
Br
Br
Br
Br
N
N
a,b,c
N
d
e,f
g,h
N
N
N
N
N
N
N
N
N
N
N
NH2
NH
NH
10
S
S
Br
S
N
N
6
7
8
9
Scheme 2. Reagents and conditions: (a) 50% chloroacetaldehyde in water, IPA, relux, 89%; (b) NaSMe, MeOH, rt, 95%; (c) N-iodosuccinimide, DMF, 60 °C, 76%; (d) 5 mol %
PdCl2(dppf), K3PO4, 1,4-dioxane/water (10/1), 40 °C, 46%; (e) m-CPBA, DCM, rt, 95%; (f) NaH, 5-amino-3-methyl isothiazole hydrochloride, DMSO, rt, 56%; (g) chemistry varies;
(h) 4 N HCl dioxane in THF.
The X-ray crystal structure of inhibitor 1j in Aurora A revealed
the inhibitor 1j binds in the adenosine triphosphate (ATP) pocket
(Fig. 2).11 The imidazo[1,2-a]pyrazine core forms key donor–accep-
tor hydrogen bonds to the main chain carbonyl oxygen and amide
NH of Ala213. The ATP competitive inhibitor projected the 3-(4-
pyrazolo) group toward Asp274 with the Aurora A in the catalyti-
cally active ‘DFG-in’ conformation.12 Additionally, the hydrophobic
pocket formed between the imidazo[1,2-a]pyrazine core and 3-(4-
pyrazolo) allows the side chain of Leu194 to pack next to the inhib-
itor. The noteworthy potency disparity between N-methyl pyrazole
analog 1a and the N–H pyrazole inhibitor 1j was attributed primar-
ily to the stabilizing hydrogen bond with Asp274 and to removal of
a putative repulsive van der Waals interaction between the Asp274
and N-methyl group of inhibitor 1a.13 The X-ray also revealed the
8-aminoisothiazole group was located entirely within a hydropho-
bic region at the front of the ATP binding pocket and extended to-
ward the solvent accessible front. Presumably, the preferred
bioactive conformation of the 8-aminoisothiazole and the imi-
dazo[1,2-a]pyrazine core in potent inhibitor 1j was stabilized
through a polar interaction between the core nitrogen the isothiaz-
ole sulfur atom.14
protected pyrazole 8 in acceptable yield. The two step sequence
from 6-bromide 8 gave intermediate 9 (m-CPBA, then NaH, 5-ami-
no-3-methyl isothiazole hydrochloride).
With the crucial intermediate 9 in hand, Pd-mediated function-
alization was used to install different 6-position groups (Table 2).
The SAR showed that small, hydrophobic groups were tolerated
and preferred (e.g., 6-Me 10a, 6-Et 10d, 6-SMe 10i) over larger
groups (e.g., 6-(3-pyridyl 10h), 6-cyclopropyl 10f, 6-S-t-Bu 10n).
Substituents bearing polar (10j, 10k, and 10p) or basic functional-
ity (10b, 10e, and 10m) showed significantly less biochemical po-
tency than the parent compound 1j. Inhibitors 10a (6-Me) and 10i
(6-SMe) consistently showed better cell based potency than parent
compound 1j. Inhibitor 10i demonstrated mechanism-based cell
activity with an EC50 of 0.6 lM. Consistent with the expected phe-
notype of a pan Aurora inhibitor, at this dose 10i decreased phos-
phorylation of Histone H3 and induced >4 N DNA content as
measured by FACS. Inhibitor 10i also potently inhibited tumor cell
line growth in a panel of cells from different tissue origin and ge-
netic backgrounds (e.g., p53, PTEN, etc.).
With the identification of Aurora inhibitors with sub-micromo-
lar cell based potency, in vitro DMPK properties were evaluated.
Inhibitor 10i showed a good CYP inhibition profile (3A4, 2D6,
In order to more thoroughly investigate the imidazo[1,2-a]pyr-
azine SAR, a synthetic route was developed that enabled elabora-
tion of the 6-position.13 2-Amino-3,5-dibromopyrazine 6 was
converted to 6-bromo-3-(4-pyrazolo)-8-aminoisothiazoleimi-
dazo[1,2-a]pyrazine 9 using the sequence depicted in Scheme 2.
The key step in the sequence was a chemoselective Suzuki reaction
of 3-iodo-6-bromoimidazo[1,2-a]pyrazine 7 that afforded SEM-
and 2C9 >20
at 1.5 g/mL) and showed high human plasma protein binding
l
M), displayed a modest hERG signal (8.7% Rb efflux17
l
(99.4%). Inhibitor 10i had good measured permeability (Caco-2 api-
cal to basolateral = 633 nm/s, efflux ratio = 1.0), but suffered from
high in vitro hepatocyte clearance (human = 49.7
lL/min/million
cells). The oral bioavailability of 10i in mouse was low (%F = 4)

D. B. Belanger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5170–5174
5173
Table 2
Aurora A and B inhibition data for imidazopyrazines 1j–10p
H
N
N
R6
S
N
N
N
NH
N
Compd
R6
Aurora Aa IC50 (nM)
Aurora Ba IC50 (nM)
phos-HH3a EC50
(lM)
1j
H
Me
64
64
50
27
64
83
17
46
8
64
30
603
22
82
105
85
14
613
613
110
252
613
33
51
219
62
613
185
>3000
24
1.3
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
0.25
>10
>10
0.85
>10
2.6
>10
>10
0.6
>10
not tested
3.0
>10
5.6
CH2morpholine
CF3
Et
CH2CH2NH2
CycloPr
CN
3-Pyridyl
SMe
( )-S(O)Me
SO2Me
SEt
SCH2CH2NMe2
S-t-Bu
S-4-Fluorophenyl
N-Pyrrolidinone
81
272
129
26
2.5
9.5
a
Assay conditions listed in Ref. 15.
the preference for an unsubstituted pyrazole and aminoisothiazole
at the 3- and 8-positions, respectively. An X-ray structure has en-
abled the understanding of the inhibitor binding mode and helped
us identify opportunities for improving the characteristics (solubil-
ity, in vitro hepatocyte clearance) of these early lead compounds.
Future publications from our group will detail the optimization
of the imidazo[1,2-a]pyrazine series.
Table 3
Kinase inhibition profile of Aurora inhibitor 10i
H
N
N
S
N
N
N
Acknowledgments
NH
S
N
We thank Drs. John Piwinski, Neng-Yang Shih, Paul Kirschmeier,
and W. Robert Bishop for support of this work. We also thank Dr.
Jayaram Tagat for the preparation of compound 10b.
Kinase
IC50 (nM)
Kinase
IC50 (nM)
Aurora A
Aurora B
Akt1
Camk4
Cdk2
Chk1
CSNK1d
EGFR
64
IRAK4
Jak2
LCK
167
>10,000
468
>10,000
>10,000
>10,000
785
>10,000
1306
613
References and notes
>10,000
>10,000
2273
187
1900
cMet
MST2
PKCa
PLK3
Rock2
RSK2
TSSK2
VEGFR2
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7. We found that higher yields were achieved by using chloroacetaldehyde
freshly prepared from the corresponding diethylacetal rather than using
commercially available aqueous choroacetaldehyde.
presumably due to low aqueous solubility (25
l
M) and high first
pass metabolism (Cl = 89 mL/min/kg). The kinase inhibition profile
of compound 10i is shown in Table 3. Compound 10i is a potent
inhibitor of Aurora A and B (Aurora A TdF = 0.2 nM, Aurora B
TdF = 0.4 nM) without inhibiting a number of other kinases (e.g.,
Akt1, ERK2, JAK2). However, sub-micromolar kinase activity was
observed against Chk1, IRAK4, LCK, PLK3, and VEGFR2.
In summary, we have discovered imidazo[1,2-a]pyrazine Aur-
ora kinase inhibitors with sub-micromolar on-target cell based
activity and a promising overall profile. The initial SAR suggests
8. Guzi, T.; Paruch, K.; Dwyer, M.; Zhao, L.; Curran, P. J.; Belanger, D. B.; Hamann,
B.; Reddy, P. A.; Siddiqui, M. A. U.S. 2006/106023 (A1).

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D. B. Belanger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5170–5174
9. Sun, C. L.; Liang, C.; Huang, P.; Harris, G. D.; Guan, H. US/2004/0220189; Sun, C.
L.; Liang, C.; Huang, P.; Harris, G. D.; Guan, H. US/2005/009832.
10. 3-Bromo-8-(methylsulfonyl)imidazo[1,2-a]pyrazine 4 is a stable solid that can
be stored cold for months without decomposition.
Aurora A assay was modified and utilized 4 nM enzyme, 100 nM PKAtide, and
100 M ATP. For the Aurora B assay, each reaction consisted of 26 nM enzyme
(Aurora B, Invitrogen), 100 nM Tamra-PKAtide (Molecular Devices, 5TAMRA-
GRTGRRNSICOOH), 50 M ATP, 1 mM DTT, and kinase buffer (10 mM Tris,
l
l
11. The coordinates for compound 1j have been deposited in the RCSB Protein Data
Bank under the Accession Code 3NRM.
10 mM MgCl2, 0.01% Tween 20). Dose–response curves were plotted from
inhibition data generated in duplicate, from eight point serial dilutions of
inhibitory compounds. Concentration of compound was plotted against kinase
activity, calculated by degree of fluorescent polarization. To generate IC50
values, the dose–response curves were then fitted to a standard sigmoidal
curve and IC50 values were derived by nonlinear regression analysis.
Immunofluorescent assays: HCT-116 cells were plated at 15,000 cells per well
12. (a) Nowakowski, J.; Cronin, C. N.; McRee, D. E.; Knuth, M. W.; Nelson, C. G.;
Pavletich, N. P.; Rogers, J.; Sang, B.-C.; Scheibe, D. N.; Swanson, R. V.;
Thompson, D. A. Structure 2002, 10, 1659; (b) Zuccotto, F.; Ardini, E.; Casale,
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53, 1413.
13. Subsequent X-ray data with related imidazo[1,2-a]pyrazine inhibitors revealed
a water mediated hydrogen bond between the enzyme and the 3-(4-pyrazolo)
group. Yu, T.; Tagat, J. R.; Kerekes, A. D.; Doll, R. J.; Zhang, Y.; Xiao, Y.; Esposite,
S.; Belanger, D. B.; Curran, P. J.; Amit K. Mandal; Siddiqui, M. A.; Shih, N-Y.;
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15. Biochemical assays: Aurora A and B kinase assays were performed in low
protein binding 384-well plates. Compounds were diluted in 100% DMSO to
the desired concentrations. For the Aurora A assay, each reaction consisted of
8 nM enzyme (Aurora A, Upstate), 100 nM Tamra-PKAtide (Molecular Devices,
in poly-D-lysine coated black micro-clear 384-well tissue culture plates. For the
phos-Histone H3 assay, cells were first treated with 0.4 mg/mL nocodazole.
Sixteen hours later cells were treated in triplicate with compound (0.1% final
DMSO concentration) for 1 h. Cells were fixed with PreferÒ fixation solution
(Anatech) plus 1000 nM Hoechst 33342 dye and incubated for 30 min at room
temperature. The fixation solution was removed and cells were washed with
PBS. Cells were permeabilized with 0.2% Triton-X in PBS and incubated for
10 min. Cells were washed with PBS and incubated with PBS containing 3% FBS
for 30 min. Cells were then stained overnight at 4 °C with Phos-Histone H3
(Ser10)-Alexa Flur 488 Conjugate antibody (Cell Signaling) solution in PBS plus
3% FBS. Cells were washed with PBS and then immunofluorescence images
were captured at 10ꢀ with HT Pathway 855 automated fluorescent microscope
(BD Bioscience). Percent positive cells were quantitated using Hoechst staining
for cell number using Attovision software (BD Bioscience). To generate IC50
values, the dose–response curves were then fitted to a standard sigmoidal
curve and IC50 values were derived by nonlinear regression analysis.
16. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: Palo
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5TAMRA-GRTGRRNSICOOH), 25
Tris, 10 mM MgCl2, 0.01% Tween 20). For the high throughput screen the
l
M ATP, 1 mM DTT, and kinase buffer (10 mM
17. Sorota, S.; Zhang, X. S.; Margulis, M.; Tucker, K.; Priestley, T. Assay Drug Dev.
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