Lead identification of 2-iminobenzimidazole antagonists of the chemokine receptor CXCR3

Article abstract of DOI:10.1016/j.bmcl.2008.02.049

Modification of a 2-iminobenzimidazole series derived from an HTS hit resulted in compounds with improved in-vitro species selectivity. Incorporation of an 8-quinoline amide and conformational rigidification of an aliphatic tether furnished potent compounds suitable for further lead optimization.

Full text of DOI:10.1016/j.bmcl.2008.02.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2414–2419
Lead identification of 2-iminobenzimidazole antagonists
of the chemokine receptor CXCR3
Martin E. Hayes,* Eric C. Breinlinger, Grier A. Wallace, Pintipa Grongsaard,
Wenyan Miao, Michael J. McPherson, Robert H. Stoffel,
David W. Greenà and Gregory P. Roth§
Abbott Bioresearch Center, Medicinal Chemistry, 381 Plantation Street, Worcester, MA 01605, USA
Received 29 January 2008; revised 19 February 2008; accepted 21 February 2008
Available online 26 February 2008
Abstract—Modification of a 2-iminobenzimidazole series derived from an HTS hit resulted in compounds with improved in-vitro
species selectivity. Incorporation of an 8-quinoline amide and conformational rigidification of an aliphatic tether furnished potent
compounds suitable for further lead optimization.
Ó 2008 Elsevier Ltd. All rights reserved.
Chemokines are chemotactic cytokines that mediate leu-
kocyte migration and recruitment in response to pro-
inflammatory cytokines1,2 They bind to 7-transmem-
brane G-protein coupled receptors on the surface of T
cells, macrophages, and mononuclear cells, and the
expression of these receptors is correlated with immune
diseases such as multiple sclerosis. The receptor CXCR3
is found primarily on T cells and is specific for the che-
mokine ligands MIG (monokine induced by
c-interferon; CXCL9), IP-10 (c-interferon-inducible
protein 10; CXCL10), and I-TAC (interferon-inducible
T cell a-attractant; CXCL11). MIG, IP-10 and I-TAC
are not known to be ligands for any other chemokine
receptors besides CXCR3. Binding of any one of these
ligands to CXCR3 activates signaling pathways that re-
sult in actin polymerization, cytoskeletal rearrange-
ments, adhesion, and ultimately cell activation and
chemotaxis.
sclerosis patients.3 Recent studies using CXCR3 (À/À)
mice have demonstrated a critical role for CXCR3 and
CXCL10 in regulating leukocyte effector functions such
as IFN-c production in an autoimmune disease model.4
Several small molecule antagonists of CXCR3 have been
reported including 4-N-aryl-[1,4]diazepine ureas,5 1-aryl-
3-piperidin-4-yl-ureas,6 aminotropane derivatives,7 N-
arylimidazoles,8 and
a
quinazolinone antagonist,
AMG-487, which has been progressed into clinical trials
for both psoriasis and rheumatoid arthritis.9
In our screen for inhibitors of CXCR3, we identified a
benzimidazole hit (1) with moderate molecular weight
and low cLogP. Through modifications of the heterocy-
cle and benzenoid ring, we were able to identify tractable
SAR for the pharmacophore (see Fig. 1) and address
CXCR3 has been of interest as a therapeutic target due
to its role in T cell chemotaxis and the presence of
CXCR3+ cells and CXCL10 in lesions from multiple
Keywords: Chemokine; Chemokine antagonist; CXCR3; IP-10; CXC-
L10; Multiple sclerosis; Benzimidazole; 2-Iminobenzimidazole.
*
Corresponding author. Tel.: +1 508 688 8081; fax: +1 508 688
8100; e-mail: martin.hayes@abbott.com
Present address: Wyeth Research, Cambridge, MA 02140, USA.
Present address: Amgen Inc., Cambridge, MA 02139, USA.
Present address: Burnham Institute for Medical Research at Lake
Nona, Orlando, FL 32819, USA.
à
§
Figure 1. SAR identified during initial hit-to-lead efforts.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.049

M. E. Hayes et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2414–2419
2415
partial solubility associated with the core in order to ob-
tain complete dose response values at the higher concen-
trations of the competition-binding assay.10 Our interest
then turned toward the impact of further substitution at
the N-3 position of benzimidazoles such as 3.
with an appropriate amine as shown in Scheme 1.11
Reduction of the nitro group was effected with iron in
protic acid12 and formation of benzimidazoles such as
5 was achieved using an excess of BrCN in acetonitrile.13
Alkylation with a bromoacetophenone under neutral
conditions furnished benzimidazoles of type 6.14 Com-
pounds such as 9 were prepared in the same manner
using an amino ester nucleophile. To generate amide
substrates of the type 11, conversion of the ester to an
Substituents at the N-3 position of benzimidazoles such
as 6 were introduced through nucleophilic aromatic sub-
stitution of an o-flouro- or an o-chloronitrobenzene (4)
Scheme 1. Synthetic routes to N-3 substituted CXCR3 antagonists 6a–c, 9a, 11a–b, and 14a–n. Yields given in the scheme are for compounds 6a, 9a,
11b, and 14f. Reagents and conditions: (a) R2NH2, EtOH, reflux, 16 h; (b) Pd/C, NH4CO2H, EtOH, rt, 16 h; or Fe, AcOH, rt, 16–48 h; (c) BrCN,
Na2CO3, MeCN, rt, 8–16 h; (d) ArC(O)CH2Br, DMF, rt, 4–12 h; (e) NaOH, EtOH/H2O (2:1), rt, 15 h; (f) NH2R6R7, HOBt, DCC, THF, rt, 15 h; (g)
TFA, CH2Cl2, rt, 2 h; (h) ClCOR4, TEA, CH2Cl2, rt, 2 h.

2416
M. E. Hayes et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2414–2419
amide moiety prior to benzimidazole formation was nec-
essary in order to avoid a competing intramolecular
reaction. For the preparation of reversed amides such
as 13, a mono-Boc protected diamine was employed in
the aromatic substitution step. We reasoned that a Boc
protecting group would be stable to the subsequent
reduction and cyanogen bromide condensation steps in
order to facilitate a late-stage diversification strategy.
Several analogs of type 14, derived from a variety of
mono-Boc protected diamines were prepared in this
manner.
pounds that were essentially equipotent with 2 in the
binding assay, the functional antagonism, as measured
by a FLIPR assay, showed notable improvement with
the incorporation of an extended amide group (compare
entries 14c and 14d).
The improvement in functional activity with incorpora-
tion of N-benzylamides came with the addition of con-
siderable molecular weight. We hoped to offset this
substantial weight increase through optimization of
binding activity associated with the distal amide moiety.
We quickly crossed over the newly identified N-benzyla-
mides onto our core with a chloro substituent at the C-4
position (entries 14e–g). We reasoned that a chloro
group could provide an increase in potency (compare
entries 14f and 14h) through potential interaction with
a putative hydrophobic pocket of the receptor while
avoiding potential metabolic liabilities that could be
associated with small aliphatic groups such as methyl
or ethyl. Variation of the tether identified a three-atom
chain as optimal for the placement of the amide (entry
14f). We then executed a focused library of suitably di-
verse aryl, heteroaryl, and aliphatic amides to probe
for a more optimal amide substituent. Several aryl
groups with heteroatoms at the ortho position were
identified as potent, functional antagonists at the human
receptor, as shown in Table 2.
Compounds were initially evaluated for their ability to
inhibit [125I]-labeled CXCL10 binding to membranes
of CHO cells stably expressing human CXCR3.15 Func-
tional antagonism was also measured in CHO cells using
a FLIPR-based calcium mobilization assay.16
Our earlier work on the benzimidazole series identified
small aliphatic substituents at the C-4 position to be
an important element of the pharmacophore.10 How-
ever, we chose to examine substitution at the N-3 posi-
tion with less potent analogs devoid of this C-4
substitution in order to better gauge the impact of mod-
ifications. Initial replacement of the N-3 methyl with
increasingly larger aliphatic groups showed no signifi-
cant impact on the binding potency (entries 6a–c), as
shown in Table 1. It is notable, though, that the receptor
was tolerant of groups with increasing steric demand at
this position. We next examined substitution at N-3 with
more functionalized groups in order to identify an addi-
tional point of interaction of the receptor–ligand com-
plex (entries 9a, 11a, 11b, 14a–d). While incorporation
of increasingly larger, tethered-amides provided com-
We were also interested in screening for potency at
the mouse receptor since species differentiation is com-
monly observed with small molecule inhibitors of che-
mokines17 and one of the early milestones for the
project included in-vivo efficacy in a rodent model
of multiple sclerosis. Compound 14n stood out
Table 1. SAR of N-3 substituted analogs
R1
R2
huRLB IC50 (lM)
huFLIPR IC50 (lM)
muRLB IC50 (lM)
a
a
a
Entry
6a
6b
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Et
Me
Et
0.7
0.7
1.1
1.2
2.2
0.4
1.3
0.5
0.3
0.4
0.4
0.3
0.4
0.2
9
3.5
—
6c
9a
Propyl
CH2CH2CO2Et
2.5
—
11a
11b
14a
14b
14c
14d
14e
14f
14g
14h
CH2C(O)N(Me)CH2Ph
CH2CH2C(O)N(Me)CH2Ph
CH2CH2N(Me)C(O)Me
CH2CH2N(Me)C(O)C6H11
CH2CH2N(Me)C(O)Ph
CH2CH2N(Me)C(O)CH2Ph
CH2CH2N(Me)C(O)CH2Ph
CH2(CH2)2N(Me)C(O)CH2Ph
CH2(CH2)3N(Me)C(O)CH2Ph
CH2(CH2)2N(Me)C(O)CH2Ph
0.2
3.4
0.9
2.3
0.8
0.3
0.07
0.4
0.1
1.5
1.4
1.8
a IC50 values are an average of two runs.

M. E. Hayes et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2414–2419
Table 2. Library of tethered amides at N-3 position
2417
a
a
a
Compound
R
huRLB IC50 (lM)
huFLIPR IC50 (lM)
muRLB IC50 (lM)
MLM T1/2 (min)
14i
0.6
0.4
14J
14k
14l
0.015
0.3
0.04
0.5
0.8
0.15
0.2
9.5
0.02
0.02
0.02
0.05
0.03
5.4
14m
14n
0.02
0.02
0.05
15
a IC50 values are an average of two runs.
amongst the various amide-tethered compounds for its
potency at both the human and murine receptors
(compare entries 14n with 14l and 14m). However,
this compound, as well as several amide-tethered sub-
strates exhibited rapid transformation upon exposure
to mouse liver microsomes (entries 14j, 14l, 14n).
Mass spectral analysis of the microsome homogenates
(data not shown) indicated that N-demethylation was
the primary metabolic event in-vitro. To address this
potential liability, we examined the impact of varying
the N-methyl substituent. However, incorporation of
larger aliphatic groups, such as the N-cyclopropyl
moiety (Table 3, entry 14s), provided compounds that
maintained potency at the human receptor but were
less potent at the mouse receptor.
Replacement of the aliphatic tether with a piperidine
ring, as shown in Table 3, was well-tolerated in-vitro
showing no substantial impact on functional activity
or binding potency across species (entry 14o). Each
enantiomer was independently prepared (entries 14p–
q) and no discernable preference for either stereoiso-
mer was observed. Additional ring-containing tethers
were explored and most were well tolerated with a
2-pyrrolidine amide (entry 14r) demonstrating excel-
lent potency and functional activity across species.
However, when compound 14r was exposed to mouse
liver microsomes, rapid modification (T1/2 = 7.5 min)
to an unidentified metabolite was observed.
In summary, through systematic modification of the
N-3 position of compound 3, we identified an n-pro-
pyl tethered amide containing an ortho-heteroatom
motif, as exemplified by an 8-quinoline amide, as a
key element of our pharmacophore. This group im-
parts substantial binding potency at both the human
and mouse receptors. Through modification of the al-
kyl tether we also identified a number of cyclic amides
We considered another strategy to address the poten-
tial metabolic liabilities of an N-methyl amide tether.
Constraining the n-propyl linker into a ring would
provide a blocking group to minimize N-dealkylation
while also reducing the number of rotatable bonds
to enhance the drug-like properties of the molecule.18

2418
M. E. Hayes et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2414–2419
Table 3. Alternate tethers of 8-quinoline amide analogs
a
a
a
Compound
R
huRLB IC50 (lM)
huFLIPR IC50 (lM)
muRLB IC50 (lM)
14o
0.015
0.02
0.08
14p
0.015
0.02
0.06
14q
0.016
0.02
0.07
14r
14s
0.008
0.02
0.02
0.01
0.04
0.10
a IC50 values are an average of two runs.
Frederiksen, J. L.; Ransohoff, R. M. J. Clin. Invest. 1999,
103, 807.
with excellent potency and functional activity that rep-
resent good candidates for further lead optimization.
4. Liu, L.; Huang, D.; Matsui, M.; He, T. T.; Hu, T.;
DeMartino, J.; Lu, B.; Gerard, C.; Ransohoff, R. M.
J. Immunol. 2006, 176, 4399.
5. Cole, A. G.; Stroke, I. L.; Brescia, M-R.; Simhadri, S.;
Zhang, J. J.; Hussain, Z.; Snider, M.; Haskell, C.; Ribeiro,
S.; Appell, K. C.; Henderson, I.; Webb, M. L. Bioorg.
Med. Chem. Lett. 2006, 16, 200.
6. (a) Allen, D. R.; Bolt, A.; Chapman, G. A.; Knight, R. L.;
Meissner, J. W. G.; Owen, D. A.; Watson, R. J. Bioorg.
Med. Chem. Lett. 2007, 17, 697; (b) Watson, R. J.; Allen,
D. R.; Birch, H. L.; Chapman, G. A.; Hannah, D. R.;
Knight, R. L.; Meissner, J. W. G.; Owen, D. A.; Thomas,
E. J. Bioorg. Med. Chem. Lett. 2007, 17, 6806.
Acknowledgments
The authors would like to thank Lisa Alder and Lor-
raine McGuinness for generating radio-ligand binding
and FLIPR assay data and Brian Bettencourt for
obtaining in-vitro stability data.
References and notes
1. Onuffer, J. J.; Horuk, R. Trends Pharmacol. Sci. 2002, 23,
459.
2. Carter, P. H. Curr. Opin. Chem. Biol. 2002, 6, 510.
3. Sorenson, T.; Tani, M.; Jensen, V.; Lucchinetti, C.; Folcik,
V. A.; Qin, S.; Rottman, J.; Sellebjerg, F.; Strieter, R. M.;
7. (a) Watson, R. J.; Allen, D. R.; Birch, H. L.; Chapman, G.
A.; Galvin, F. C.; Jopling, L. A.; Knight, R. L.; Meier, D.;
Oliver, K.; Meissner, J. W. G.; Owen, D. A.; Thomas, E.
J.; Tremayne, N.; Williams, S. C. Bioorg. Med. Chem.
Lett. 2008, 18, 147; (b) Knight, R. L.; Allen, D. R.; Birch,

M. E. Hayes et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2414–2419
2419
H. L.; Chapman, G. A.; Galvin, F. C.; Jopling, L. A.;
Lock, C. J.; Meissner, J. W. G.; Owen, D. A.; Raphy, G.;
Watson, R. J.; Williams, S. C. Bioorg. Med. Chem. Lett.
2008, 18, 629.
S. H.; Sham, H. L. Bioorg. Med. Chem. Lett. 2005, 15,
2918.
12. Shepherd, T.; Smith, D. M. J. Chem. Soc. Perkin Trans.
1987, 1, 501.
8. (a) Du, X.; Chen, X.; Mihalic, J. T.; Deignan, J.; Duquette,
J.; Li, A.-R.; Lemon, B.; Ma, J.; Miao, S.; Ebsworth, K.;
Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina, J. C.
Bioorg. Med. Chem. Lett. 2008, 18, 608; (b) Li, A.-R.;
Johnson, M. G.; Liu, J.; Chen, X.; Du, X.; Mihalic, J. T.;
Deignan, J.; Gustin, D. J.; Duquette, J.; Fu, Z.; Zhu, L.;
Marcus, A. P.; Bergeron, P.; McGee, L. R.; Danao, J.;
Lemon, B.; Carabeo, T.; Sullivan, T.; Ma, J.; Tang, L.;
Tonn, G.; Collins, T. L.; Medina, J. C. Bioorg. Med. Chem.
Lett. 2008, 18, 688.
13. Valdez, J.; Cedillo, R.; Hernandez-Campos, A.; Yepez, L.;
Hernandez-Luis, F.; Navarrete-Vazquez, G.; Tapia, A.;
Cortes, R.; Hernandez, M.; Castillo, R. Bioorg. Med.
Chem. Lett. 2002, 12, 2221.
14. Anisimova, V. A.; Spasov, A. A.; Kosolapov, V. A.;
Kucheryavenko, A. F.; Ostrovskii, O. V.; Larionov, N. P.;
Libinzon, R. E. Pharm. Chem. J. 2005, 39, 476.
15. Radioligand binding assays were performed in CHO
cells expressing human or murine CXCR3. All
compounds were dissolved in DMSO and assays
9. (a) Berry, K. et al. Inflamm. Res. 2004, Suppl. 3, S197,
Abstract A089.; (b) Johnson, M. G.; Li, A.; Liu, J.; Marcus,
A. P.; Huang, A. X.; Medina, J. C. Abstracts ofPapers, 231st
National Meeting of the American Chemical Society,
Atlanta, GA, March 26–30, 2006;; (c) Johnson, M.; Li,
A.-R.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.;
Huang, A.; Matcus, A.; Xu, F.; Ebsworth, K.; Sablan, E.;
Danao, J.; Kumer, J.; Dairaghi, D.; Lawrence, C.; Sullivan,
T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Bioorg. Med.
Chem. Lett. 2007, 17, 3339.
run at
[
a final DMSO concentration of 1% (v/v).
125I]-labeled CXCL10 was purchased from PerkinEl-
mer and used at 50 pM per assay.
16. Calcium flux assays were performed in CHO cells
expressing human CXCR3 and the Ga16 coupling protein
using a FLIPR instrument (Molecular Devices). All
compounds were dissolved in DMSO and assays run at
a final DMSO concentration of 1% (v/v). Human CXCL10
was purchased from Peprotech and used at a final assay
concentration of 30 nM.
10. Hayes, M. E.; Wallace, G. A.; Grongsaard, P.; Bischoff,
A.; George, D. M.; Miao, W.; McPherson, M. J.; Stoffel,
R. H.; Green, D. W.; Roth, G. P. Bioorg. Med. Chem.
Lett. 2008, 18, 1573.
17. (a) Ribeiro, S.; Horuk, R. In Leukocyte Trafficking;
Hamann, A., Engelhardt, B., Eds.; Wiley-VCH: Wein-
heim, 2005; p 371; (b) Godessart, N. Ann. N.Y. Acad. Sci.
2005, 1051, 647.
11. Li, Q.; Li, T.; Woods, K. W.; Gu, W.; Cohen, J.; Stoll,
V. S.; Galicia, T.; Hutchins, C.; Frost, D.; Rosenberg,
18. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.;
Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615.