III. Identification of novel CXCR3 chemokine receptor antagonists with a pyrazinyl-piperazinyl-piperidine scaffold

Article abstract of DOI:10.1016/j.bmcl.2011.09.120

The SAR of a novel pyrazinyl-piperazinyl-piperidine scaffold with CXCR3 receptor antagonist activity was explored. Optimization of the DMPK profile and reduction of hERG inhibition is described. Compound 16e with single-digit CXCR3 affinity, good rat PK a

Full text of DOI:10.1016/j.bmcl.2011.09.120

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6982–6986
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
III. Identification of novel CXCR3 chemokine receptor antagonists
with a pyrazinyl–piperazinyl–piperidine scaffold
Seong Heon Kim a, , Gopinadhan N. Anilkumar a, Lisa Guise Zawacki b, Qingbei Zeng a, De-Yi Yang a,
⇑
Yuefei Shao b, Guizhen Dong b, Xiaolian Xu b, Wensheng Yu a, Yueheng Jiang a, Chung-Her Jenh a,
James W. Hall III b, Carolyn DiIanni Carroll b, Doug W. Hobbs b, John J. Baldwin b, Brian F. McGuinness b,
Stuart B. Rosenblum a, Joseph A. Kozlowski a, Bandarpalle B. Shankar a, Neng-Yang Shih a
a Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Ligand Pharmaceuticals, 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The SAR of a novel pyrazinyl–piperazinyl–piperidine scaffold with CXCR3 receptor antagonist activity
was explored. Optimization of the DMPK profile and reduction of hERG inhibition is described. Compound
16e with single-digit CXCR3 affinity, good rat PK and hERG profiles has been identified as a lead for fur-
ther study.
Received 24 August 2011
Revised 27 September 2011
Accepted 28 September 2011
Available online 5 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
CXCR3
Chemokine
GPCR
O
Chemokines are a family of cytokines that are produced during
inflammation and are able to induce chemotaxis of immune cells.1
CXCR3 is a chemokine receptor that binds CXCL10 (IP-10), CXCL9
(MIG), and CXCL11 (I-TAC) ligands to facilitate the recruitment of
Th1 cells to the site of inflammation.2 CXCR3 has been implicated
in inflammatory diseases (psoriasis and inflammatory bowel dis-
ease), autoimmune diseases (multiple sclerosis and rheumatoid
arthritis), type I diabetes, and acute cardiac allograft rejection.
Therefore small molecule antagonists of the CXCR3 receptor have
attracted significant interest and may provide therapeutic tools
for the treatments of such diseases.3
Compounds exemplified by 1 (Fig. 1) have been reported as po-
tent CXCR3 antagonists.4 However, compounds in this series
exhibited only modest rat PK and undesirable hERG K+ channel
activity in a Rb efflux assay. As part of the efforts to identify a
new chemotype, the pyridine ring of 1 has been replaced by a pyr-
azine (2). The systematic SAR around the core motif including vari-
ations on pyrazine, piperidine, and piperazine rings to generate
compounds with good CXCR3 activity as well as improved hERG
and PK profiles are described.
X
N
Cl
N
1
(X = CH)
N
H
hCXCR3 IC50 = 0.3 nM
.
µ
rat AUC = 0.4 M h
N
Cl
N
2
(X = N)
Figure 1. Lead compound in pyridine core series.
compounds listed inTables 1 and 2 was accomplished by the meth-
od outlined in Scheme 1. The 2-chlorine of commercially available
pyrazine 3 was regioselectively displaced with (S)(+)-2-ethylpiper-
azine in the presence of a Pd-catalyst. Subsequent reductive ami-
nation and deprotection gave key tricyclic amine intermediate 5,
which reacted with aldehydes or acid chlorides to afford the benzyl
amine and amide derivatives (6), respectively. Modification of the
pyrazine amide (R1) was accomplished by conversion of the methyl
ester 6 or the acid 7 with amines to afford compounds 8a–r.
Table 1 summarizes the selected SAR of 5-carboxamide analogs
with variations on R2. Many of the carboxamide derivatives
exhibited sub-nanomolar human CXCR3 binding affinity. Primary
amides displayed good to excellent activities when R2 was a ben-
zylic aromatic derivative (8a–d) whereas the corresponding amide
analog (8e) exhibited weaker binding affinity. Fixing the R2 substi-
tuent as a 4-chlorobenzyl, the SAR at R1 was explored. Alkyl and
The initial efforts were focused on 6-amino-3-chloropyrazine
scaffold 8 which was derived from the reported optimization on
2-(S)-ethyl piperazinyl–piperidine fragment.4b Preparation of the
⇑
Corresponding author.
E-mail address: seong.heon.kim@merck.com (S.H. Kim).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.120

S. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6982–6986
6983
Table 1
Binding affinity of 6-amino-3-chloropyrazine-5-carboxamides
O
N
N
Cl
N
1
R HN
H N
2
N
N
2
R
Compd
R1
H
R2
hCXCR3 IC50a, nM
0.8
Cl
8a
OCF
CN
Cl
3
8b
8c
H
H
3.7
8.5
8db
8e
H
1.9
76
OH
Cl
H
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
8f
8g
8h
8i
Me
0.3
0.8
0.4
0.3
8.4
0.9
1.9
6.4
CF3CH2
HO
HO
HO
8j
O
2
S
8k
8l
F C
N
H
3
O
S
2
H N
2
N
H
S
8m
O
2
O
H
8n
1.5
O
a
See reference 10.
Single diastereomer.
b
hydroxyalkyl amides (8f–i) maintained good CXCR3 affinity
whereas the tertiary hydroxyl analog (8j) displayed moderate
activity. Polar groups such as sulfones (8k–m) and lactone (8n)
maintained moderate to good CXCR3 affinities as well. Although
we identified several compounds with sub-nanomolar CXCR3
(8q) which contains primary amide on the R2 aryl moiety also
improved the hERG profile and maintained good binding affinity.
A similar result was obtained with the 6-amino-2-chloropyri-
dine-5-carboxamide8 derivative (8r). In addition to the hERG
improvement, compound 8r also showed good rat exposure
activity, many compounds including 8a5 (rat AUC = 5.1
l
MÁh) were
(AUC = 5.9
l
MÁh) in comparison with the analogs in Table 1. Based
found to have undesired hERG K+ channel inhibition (80–90%
inhibition measured by Rb efflux assay).6
upon the profile of 8r R2 was fixed as the 6-amino-2-chloropyri-
dine-5-carboxamide while attention was concentrated toward
optimization of other areas of the molecule.
Our strategy to improve the hERG profile was to further explore
polar substituents7 in R1 and add polar groups to R2 (Table 2).
Sulfonamide substitution at R1 did suppress hERG inhibition and
maintained moderate binding affinity (8o and 8p). Compound
As the previous pyridine series4 demonstrated good binding to
the receptor without the amino motif on pyrazine 6-position, we
next investigated the effect of different R1 groups with elimination

6984
S. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6982–6986
Table 2
Binding affinity and hERG inhibition of 6-amino-3-chloropyrazine-5-carboxamides
O
N
Cl
N
1
R HN
H N
N
2
N
N
2
R
Compd
R1
R2
hERGa%
4
hCXCR3 IC50b, nM
O
S
F
2
H N
2
N
H
8o
9
F
O
S
Cl
2
8p
8q
39
14
4.4
4.1
N
H
Cl
H
HO
CONH
Cl
2
HO
N
2
8r
17
3.4
O
NH
a
% Inhibition measured by Rb efflux assay at 10
See reference 10.
lM,
b
O
O
O
N
N
Cl
N
N
Cl
N
MeO
N
Cl
Cl
MeO
b, c
a
MeO
H2N
H2N
N
H2N
N
N
NH
5
NH
4
3
O
O
N
N
Cl
N
N
N
Cl
N
R1HN
R3O
H2N
f
d
H2N
N
N
N
N
R2
R2
8
6, R3 = Me
e
3
7,
R = H
Scheme 1. Reagents and conditions: (a) (S)(+)-2-ethylpiperazine, Pd(OAc)2, Cs2CO3, DBPD, dioxane, 90 °C; (b) N-Boc-piperidin-4-one, Ti(OiPr)4, NaBH3CN, 1,2-dichloroethane,
60 °C; (c) TFA, CH2Cl2, RT; (d) ArCHO, NaBH(OAc)3, 1,2-dichloroethane, or ArCOCl, TEA, THF, or ArCO2H, EDCI, HOBt, DMF, RT; (e) LiOH, THF-MeOH, RT; (f) with 6, R1NH2,
MeOH, 60–80 °C, with 7, R1NH2, EDCI, HOBt, DMF-THF, RT.
of the pyrazine amino group (Table 3). The synthesis of des-6-ami-
nopyrazine is described in Scheme 2. The diazonium salt of amino-
Scheme 3. The bromide 13 obtained from 12 via NBS bromination
was subjected to trifluoromethylation conditions8 affording 14
(R4 = CF3). Followingthe routes describedin Scheme1 afforded com-
pound 15a–c. The trifluoromethyl analogs (15a–c, Table 4) were
well tolerated with significantly improved hERG profiles with the
exception of cyclopropyl amide (15c). In addition to the good affinity
and hERG, compounds 15a and 15b also displayed good rat PK pro-
files. The methyl analogs, 15d–f, were prepared by the methods de-
scribed in Scheme 3 in which bromopyrazine 13 was converted to
the methylated compound 14 (R4 = Me) via Suzuki coupling with
methylboronic acid. Replacement of trifluoromethyl by methyl
group was well tolerated. Primary and methyl amides (15d and
15e) had similar CXCR3 affinities and hERG profiles to the corre-
sponding trifluoromethyl analogs, however, the rat PK profiles were
significantly improved by the methyl replacement. The cyclopropyl
amide (15f) demonstrated significant improvements in rat PK and
hERG profiles while maintaining the similar potency as compared
to 15c. The maintained 3-methyl substitution unreactive the hERG
pyrazine
4 was converted to the bromide 9 followed by
hydrogenolysis to provide the des-amino intermediate 10. Subse-
quent structural manipulation as in Scheme 19 afforded com-
pounds 11a–f.
As shown in Table 3, primary and alkyl amides (11a–f) exhibited
similar CXCR3 affinity and maintained good hERG profile with
exception of isopropyl amide (11d). While the elimination of 6-
amino motif of pyrazine produced several good compounds, no sig-
nificant improvements on CXCR3 affinity and hERG profile were
achieved as illustrated in direct comparison of the compound 11e
to 8r. These results demonstrated that the 6-amino motif was not
necessary for the positive ligand-protein interactions while
eliminating it may increase the liability of 3-chlorine of pyrazine.
Next we investigated the SAR with variations at R1 and R4 to
replace the 3-chlorine with a more stable surrogate. The trifluoro-
methyl compounds were synthesized by the methods described in

S. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6982–6986
6985
Table 3
Table 4
Binding affinity and hERG inhibition of 6-desamino-3-chloropyrazine-5-carboxam-
ides
Effects of pyrazine C3-substituents of 6-amino-2-chloropyridine amide derivatives
O
N
N
R4
N
O
R1HN
N
N
Cl
N
R1HN
H
N
Cl
Cl
N
N
N
N
N
O
NH2
O
NH2
Compd
R1
R4
hERGa%
hCXCR3 IC50b, nM
AUCc
lMÁh
Compd
R1
hERGa%
7
hCXCR3 IC50b, nM
15a
15b
H
Me
CF3
CF3
0
0
1.9
1.5
15.3
6.4
11a
H
2.5
11b
11c
13
19
1.5
1.2
15c
CF3
53
1.8
3.6
15d
15e
H
Me
Me
Me
0
5
4.0
2.4
30.2
12.1
11d
11e
11f
56
11
17
0.9
2.9
2.9
15f
Me
16
1.9
22.0
a
b
c
HO
HO
% Inhibition measured by Rb efflux assay at 10
See reference 10.
Rat PK (10 mpk, po, 6 h, MC).
lM.
a
% Inhibition measured by Rb efflux assay at 10
See reference 10.
lM.
b
Table 5
Effects of pyrazine C3-substituents of 4-chlorobenzamide derivatives
O
O
O
H
N
N
Me
N
R1HN
N
N
Cl
N
N
Cl
MeO
MeO
Scheme 1
b
a
11
4
Br
N
N
NH
NH
Cl
N
10
9
N
Scheme 2. Reagents and conditions: (a) Br2, NaONO, 48% HBr, AcOH, 0 °C; (b) H2
(1 atm), 5% Pd/C, THF, RT.
O
Compd
R1
hERGa
%
hCXCR3 IC50b, nM
profile of the 3-chloro analogs and compounds with good overall
profile examplified by 15d have been identified.
15g
15h
15i
H
Me
Et
6
41
44
18
3.6
2.0
After optimization of pyrazine ring, we revisited the investiga-
tion of the 4-chlorobenzamide at R2 to address the potential
reactivity of 6-amino-2-chloropyridine motif. Table 5 summarizes
the selected 4-chlorobenzamide analogs with variations at R1.
The primary amide 15g displayed fourfold reduced CXCR3 affinity
as compared to 15d demonstrating the effect of 6-amino-2-
chloropyridine motif on the affinity. However, if compared to the
compound with same R2 exemplified by 8e (Table 1), 15g showed
significantly improved binding affinity while maintaining good
hERG profile. Increasing the size of R1 alkyl improved affinity sig-
nificantly as illustrated with 15g–j, but compromised the hERG
profile. Particularly the cyclopropyl amide analog 15j displayed
excellent combination of CXCR3 activity and rat PK (AUC = 8.4 vs
15j
74
1.1
a
% Inhibition measured by Rb efflux assay at 10
See reference 10.
lM
b
R1 to improve hERG profile without compromising binding affinity
and PK profiles. First, primary amides of 2-ethyl-5-alkyl-pipera-
zines11,12 were tested and were tolerated when R6 was (R)-Me as
summarized in Table 6 (16a–c). Compound 16a also maintained
good hERG profile. Thus, the 2-ethyl-5-(R)-methyl-piperazine scaf-
fold was incorporated with other R1 analogs (16d and 16e) and the
cyclopropyl amide 16e showed significant improvement of hERG
profile while maintaining good CXCR3 affinity as compared to
15j. Furthermore compound 16e displayed good PK profile as well
2.9
l
MÁh of 15i) and was selected for further optimization to im-
prove the hERG profile.
Finally, our efforts were focused on exploration of substitutions
of the piperazine ring. Based on our previous findings in pyridine
series4b small alkyl groups (R6) were explored with variations of
(rat AUC 9.8
l
MÁh at 10 mpk, po, 6 h, MC).
O
O
O
N
N
R4
N
N
N
Br
N
N
N
b, c MeO
MeO
MeO
Scheme 1
a
15
N
NH
N
N
Boc
Boc
14
12
13
Scheme 3. Reagents and conditions: (a) NBS, DMF, RT; (b) for R4 = CF3, ClCF2CO2Me, KF, CuI, DMF, 110 °C, sealed tube, for R4 = Me, MeB(OH)2, K2CO3, Pd(PPh3)2Cl2, DMF, 85 °C;
(c) TFA, CH2Cl2, RT.

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S. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6982–6986
Table 6
References and notes
Effects of 5-alkyl substitutions of piperazine ring
1. (a) Liu, L.; Callahan, M. K.; Huang, D.; Ransohoff, R. M. Curr. Top. Dev. Biol. 2005,
O
68, 149; (b) Turner, J. E.; Steinmetz, O. M.; Stahl, R. A.; Panzer, U. Mini-Rev. Med.
Chem. 2007, 7, 1089.
2. (a) Loetscher, M. et al J. Exp. Med. 1996, 184, 963; (b) Cole, K. E. et al J. Exp. Med.
Me
N
N
N
R6
R1HN
´
1998, 187, 2009; (c) Szczucinski, A.; Losy, J. Acta Neurol. Scand. 2007, 115, 137.
3. For reviews of small molecule CXCR3 antagonists see: (a) Wijtmans, M.; Verzijl,
D.; Leurs, R.; de Esch, I. J. P.; Smit, M. J. Chem. Med. Chem. 2008, 3, 861; (b)
Collins, T. L.; Johnson, M. G.; Medina, J. C. Chem. Biol. Basic Res. Clin. Appl. 2007,
2, 79; (c) Medina, J. C.; Johnson, M. G.; Collins, T. L. Annu. Rep. Med. Chem. 2005,
40, 215.
4. (a) McGuinness, B. F.; Carroll, C. D.; Zawacki, L. G.; Dong, Z.; Yang, C.; Hobbs, D.
W.; Biji, J.-S.; Hall, J. W., III; Jenh, C.-H.; Kozlowski, J. A.; Anilkumar, G. N.;
Rosenblum, S. B. Bioorg. Med. Chem. Lett. 2009, 19, 5205; (b) Shao, Y.;
Anilkumar, G. N.; Carroll, C. D.; Dong, G.; Hall, J. W., III; Hobbs, D. W.; Jiang,
Y.; Jenh, C.-H.; Kim, S. H.; Kozlowski, J. A.; McGuinness, B. F.; Rosenblum, S. B.;
Schulman, I.; Shih, N.-Y.; Shu, Y.; Wong, M. K. C.; Yu, W.; Zawacki, L. G.; Zeng, Q.
Bioorg. Med. Chem. Lett. 2011, 21, 1527.
N
Cl
N
O
Compd
R1
R6
hERGa
%
hCXCR3 IC50b, nM
16a
16b
16c
16d
H
H
H
Me
(R)-Me
(R)-Et
(S)-Me
(R)-Me
9
5.4
13
na
30
235
>1000
4.2
5. Compound 8a has been selected for the proof-of-concept (POC) evaluation in a
rat EAE model. Jenh, C.-H., et al., manuscript in preparation.
16e
(R)-Me
7
1.3
6. Vaz, R. J.; Li, Y.; Rampe, D. Prog. Med. Chem. 2005, 43, 1.
7. Blum, C. A.; Zheng, X.; De Lombaert, S. J. Med. Chem. 2004, 47, 2318.
8. Zeng, Q.; Yang, D.-Y.; Rosenblum, S. B.; Wong, M. K. C.; Anilkumar, G.; Kim, S.
H.; Yu, W.; Kozlowski, J. A.; Shih, N.-Y.; McGuinness, B. F.; Zawacki, L. G.; Hobbs,
D. W. WO Patent 2006/091428.
a
% Inhibition measured by Rb efflux assay at 10
See reference 10.
l
M.
b
9. R2 was installed under amide coupling condition (EDCI, HOBt, DMF, RT) with
sodium 6-amino-2-chloropyridine carboxylate.8 R1 variations were performed
using the conditions described in Scheme 1.
In summary, we have discovered a new chemotype, pyrazinyl–
piperazinyl–piperidine, as a potent CXCR3 antagonist. Extensive
SAR around the core, R1, and R2 fragments of the initial lead 8a ad-
dressed the undesired hERG inhibition and identified compound
16e which exhibited good human CXCR3 activity and improved
hERG profile. Particularly the pyrazine core compound 16e demon-
strated significantly improved rat exposure as compared to pyri-
dine core compound 1 (Fig. 1). Further optimization of these
analogs will be reported in due course.
10. The CXCR3 binding affinity was measured by displacement of 125I-human
CXCL10 from recombinant Ba/F3 cells expressing human CXCR3. IC50’s are the
mean values (n = 2) with SD of ꢀ13%. For reference see: Jenh, C.-H.; Cox, M. A.;
Hipkin, W.; Lu, T.; Pugliese-Sivo, C.; Gonsiorek, W.; Chou, C.-C.; Narula, S. K.;
Zavodny, P. J. Cytokine 2001, 15, 113.
11. 1-Boc-2(S)-ethyl-5(R)-methylpiperazine
and
1-Boc-2(S)-ethyl-5(S)-
methylpiperazine were prepared by the method described in reference 8.
12. 2(S)-Ethyl-5(R)-ethylpiperazine was purchased from the commercial source.