Optimization of the heterocyclic core of the quinazolinone-derived CXCR3 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.11.060

A series of six-six and six-five fused heterocyclic CXCR3 antagonists has been synthesized and their activities evaluated in an [¹²⁵I]-IP-10 displacement assay and an ITAC mediated in vitro cell migration assay. The pharmacokinetic properties o

Full text of DOI:10.1016/j.bmcl.2007.11.060

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 688–693
Optimization of the heterocyclic core
of the quinazolinone-derived CXCR3 antagonists
An-Rong Li, Michael G. Johnson, Jiwen Liu, Xiaoqi Chen, Xiaohui Du, Jeffrey T. Mihalic,
Jeffrey Deignan, Darin J. Gustin, Jason Duquette, Zice Fu, Liusheng Zhu,
Andrew P. Marcus, Phillipe Bergeron, Lawrence R. McGee, Jay Danao,
Bryan Lemon, Teresa Carabeo, Timothy Sullivan, Ji Ma, Liang Tang, George Tonn,
Tassie L. Collins and Julio C. Medina*
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
Received 18 October 2007; revised 14 November 2007; accepted 15 November 2007
Available online 21 November 2007
Abstract—A series of six–six and six–five fused heterocyclic CXCR3 antagonists has been synthesized and their activities evaluated
in an [125I]-IP-10 displacement assay and an ITAC mediated in vitro cell migration assay. The pharmacokinetic properties of several
top compounds have also been studied. This effort led to the discovery of compounds with increased potency and improved phar-
macokinetic properties that could serve as useful tools to study the role of the CXCR3 receptor in vivo.
Ó 2007 Elsevier Ltd. All rights reserved.
The recent development of various biologic agents such
as RaptivaÒ (efalizumab)1,2 and TysabriÒ (natal-
izumab)3,4 demonstrates the therapeutic value of block-
ing T cell recruitment into inflamed tissue. These agents
are efficacious in the treatment of autoimmune diseases
such as psoriasis, multiple sclerosis (MS), and inflamma-
tory bowel disease (IBD), and thus there is great interest
in novel mechanisms that inhibit T cell recruitment.
Chemokine receptors and their ligands play an impor-
tant role in mediating leukocyte trafficking. CXCR3 is
a chemokine receptor primarily expressed on activated
CD4+ and CD8+ T cells with the Th1 phenotype.5,6
The ligands for CXCR3, MIG (CXCL9), IP-10
(CXCL10), and ITAC (CXCL11), mediate migration
of CXCR3-expressing cells and are found at increased
concentrations in inflamed tissue from patients suffering
from IBD, MS, rheumatoid arthritis, and transplant
rejection.7–18 It has been hypothesized that blockade of
CXCR3 will prevent inflammatory cells from reaching
sites of inflammation, thereby alleviating a variety of
inflammatory and autoimmune diseases.
Several research groups, including ours, have been in-
volved in the discovery of small molecule antagonists
of CXCR3.5,19–22 We previously reported that a series
of 2,3-disubstituted-quinazolinone and 2,3-disubsti-
tuted-8-aza-quinazolinone derivatives, typified by
AMG 487, are potent and selective CXCR3 antago-
nists.19 AMG 487 inhibits binding of [125I]-IP-10 and
[
125I]-ITAC to CXCR3 with IC50 values of 8.0 and
8.2 nM, respectively, and inhibits CXCR3-mediated
in vitro cell migration by IP-10 (IC50 = 8 nM), ITAC
(IC50 = 15 nM), and MIG (IC50 = 36 nM). In this arti-
cle, we report the replacement of the 8-aza-quinazoli-
none core of AMG 487 with alternative bicyclic-ring
systems to evaluate the role that the core has on the
affinity of these antagonists for the CXCR3 receptor.
Since the 8-aza-quinazolinone-derivative 1 exhibits
potencies similar to those of AMG 487 in the ligand dis-
placement assays ([125I]-IP-10 IC50 in buffer = 11 nM;
Keywords: Chemokine; CXCR3; Mig; ITAC; IP10; CXCL9; CXCL10;
CXCL11.
*
Corresponding author. Tel.: +1 6502442425; fax: +1 6502442015;
e-mail: medinaj@amgen.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.060

A.-R. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 688–693
689
IC50 in plasma = 25 nM) and the in vitro cell migration
assay (ITAC IC50 = 115 nM), but is devoid of the time-
dependent CYP 3A4 inactivation that results from O-
deethylation of AMG 487, we decided to explore the
structure activity relationship of the core with reference
to 1. Compounds were evaluated in a [125I]-IP-10 ligand
replacement assay run both in the presence and in the
absence of plasma.23 A selected group of compounds
was also evaluated in an ITAC-induced in vitro migra-
tion assay using activated human peripheral blood
mononuclear cells (PBMC) in the presence of plasma.23
Additionally, the pharmacokinetic profile in the rat was
determined for several compounds.
Scheme 2. Reagents and conditions: (a) LHMDS, THF, ꢀ78 °C to rt,
20–30% over three steps; (b) tert-butoxy-N,N,N0,N0-tetramethylme-
thanediamine, THF, 60 °C; (c) O3, DCM, ꢀ78 °C; (d) benzene-1,2-
diamine, EtOH, AcOH, reflux; (e) Pd(PPh3)4, CuI, NaCN, MeCN,
microwave, 120 °C, 80–90% (f) PdCl2, Et3SiH, TEA, DCM, rt, 85%;
(g) ethylsulfonylethene, TEA, MeOH; (h) 4-fluoro-3-trifluoromethyl-
phenylacetic acid, EDC, HOBt, NMM, DMF, rt, 80–90% two steps.
We demonstrated previously that the majority of the
CXCR3 activity in this series resides with the (R)-stereo-
isomers.19 Here we assayed enantiomerically enriched
compounds (>95% ee) obtained either by synthesis from
optically pure starting materials or by separation of the
enantiomers from racemic product mixtures via chiral
phase HPLC. When enantiomers were separated by
HPLC the (R)-stereochemistry was putatively assigned
to the stereoisomer with higher binding affinity in the
in vitro [125I]-IP-10 displacement assay.
In the case of quinoxaline 9 (Scheme 2), the intermediate
3-Cbz-amino-1-(4-iodophenyl)butan-2-one obtained in
Scheme 1 was reacted with tert-butoxy-N,N,N0,N0-
tetramethylmethanediamine and then oxidized with
ozone to generate intermediate dione 7, which cyclized
with 1,2-diaminobenzene to furnish Cbz-protected (R)-
1-(3-(4-iodophenyl)quinoxalin-2-yl)ethanamine. Subse-
quent conversion of the iodide to the nitrile and removal
of the Cbz group provided intermediate 8. Compound 9
was synthesized by Michael addition to ethylsulfony-
lethene and acylation with 4-fluoro-3-(trifluoro-
methyl)phenylacetic acid as described in Scheme 1.
Quinoline 5 and 1,8-naphthyridine 6 were obtained as
shown in Scheme 1. Treatment of 2-(4-iodophenyl) ace-
tic acid with Cbz-protected alanine methyl ester in the
presence of lithium hexamethyldisilazide led to the
formation of the Cbz-protected 3-amino-1-(4-iodo-
phenyl)butan-2-one,24 which cyclized with 2-amino-
nicotinaldehyde or 2-aminobenzaldehyde to afford
com-pound 2 or 3. Michael addition of 2 or 3 to
ethylsulfonylethene, followed by phenylacylation with
4-fluoro-3-(trifluoromethyl)phenylacetic acid and
palladium-mediated transformation of the iodophenyl
group into a cyanophenyl group, afforded the racemic
products 5 and 6. Optically pure 5 and 6 were isolated
by chiral phase HPLC.
Quinoline 12 was synthesized from 2-aminobenzalde-
hyde and 4-butyrylbenzonitrile in eight steps according
to Scheme 3. Cyclization to form the quinoline ring sys-
tem 10 was carried out as in Scheme 1. During the cycli-
zation the nitrile was hydrolyzed to the acid and a
two-step amide formation from the acid followed by
dehydration to the desired nitrile was required to afford
4-(3-ethylquinolin-2-yl)benzonitrile. Installation of the
amino group was achieved by radical bromination fol-
lowed by azide substitution of the bromide and
reduction of the azide to the primary amine 11, which
Scheme 1. Reagents and conditions: (a) LHMDS, THF, ꢀ78 °C to rt,
20%; (b) 2-aminonicotinaldehyde or 2-aminobenzaldehyde, KOH,
EtOH, reflux, 47%. (c) ethylsulfonylethene, TEA, MeOH, reflux; (d) 4-
fluoro-3-trifluoromethylphenylacetic acid, EDC, HOBt, NMM, DMF,
rt, 80–90% two steps. (e) Pd(PPh3)4, CuI, NaCN, MeCN, microwave,
120 °C, 80–90%. Enantiomerically pure products were isolated by
chiral phase HPLC.
Scheme 3. Reagents and conditions: (a) KOH, EtOH, 80 °C, 90%; (b)
oxalyl chloride, DCM, then NH4OH,; (c) Tf2O, TEA, DCM, two step
36%; (d) NBS, CCl4, benzoylperoxide, reflux, 97%; (e) NaN3, DMF,
85 °C (f) PPh3, DMF/H2O, 40 °C, 85% over two steps; (g) ethylsulf-
onylethene, TEA, MeOH, 99%; (h) 4-fluoro-3-trifluoromethylphenyl-
acetic acid, EDC, HOBt, NMM, DMF, rt, 61% over two steps.
Enantiomerically pure products were isolated by chiral phase HPLC.

690
A.-R. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 688–693
Scheme 4. Reagents and conditions: (a) H2, 10% Pd/C, 90%; (b)
paraformaldehyde, Na(OAc)3BH; ClCH2CH2Cl, rt, 60%; (c) BBr3,
DCM, rt, 71%; (d) Tf2O, 2,6-dimethylpyridine, DCM, rt, 83%; (e)
Pd(PPh3)4, CuI, NaCN, MeCN, microwave, 120 °C, 80–90%.
Scheme 7. Reagents and conditions: (a) EtOH, reflux; (b) NIS,
CH3CN, rt, 80–90%; (c) 4-cyanophenylboronic acid, Pd(PPh3)4,
Na2CO3, 80 °C, DME, 90%; (d) BBr3, DCM, ꢀ10 °C to rt, 92%; (e)
ethylsulfonylethene, TEA, MeOH; (f) 4-fluoro-3-trifluoromethylphe-
nylacetic acid, EDC, HOBt, NMM, DMF, rt, 80–90% two steps.
was converted to racemic 12 in two steps as pre-
viously described. Enantiopure 12 was isolated by chiral
HPLC.
Tetrahydropyridopyrimidinone 16 was readily obtained
starting from the 8-aza-quinazolinone 13 as shown in
Scheme 4. Reduction of 13 followed by reductive amina-
tion with paraformaldehyde provided compound 14,
which was deethylated and transformed to the aryl tri-
flate 15 before conversion to racemic final product by
palladium catalyzed cyanation.
Pyridopyrimidine 19 was prepared as described in
Scheme 5. Treatment of 2-amino-pyridine with methyl
3-oxopentanoate followed by bromination and then Su-
zuki coupling to introduce the 3-(40-cyanophenyl) group
generated intermediate 17. The amino group in 18 was
installed in three steps via bromination of 17, nucleo-
philic displacement of the bromide with phthalimide,
Scheme 5. Reagents and conditions: (a) EtOH, reflux; (b) NBS,
MeCN, rt, 50% over two steps; (c) 4-cyanophenylboronic acid,
Pd(PPh3)4, Na2CO3, 80 °C, DME, 95%; (d) NBS, CCl4, benzoylper-
oxide, reflux; (e) K2CO3, phthalimide; (f) NH2NH2, 91% over two
steps; (g) ethylsulfonylethene, TEA, MeOH; (h) 4-fluoro-3-trifluorom-
ethylphenylacetic acid, EDC, HOBt, NMM, DMF, rt, 80–90% two
steps. Enantiomerically pure products were isolated by chiral phase
HPLC.
Scheme 8. Reagents and conditions: (a) K2CO3, air, DMF; (b) 3N
NaOH, MeOH; (c) H2SO4/H2O, 70 °C, 48% over three steps; (d) NIS,
AcOH, rt; (e) 4-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, DME,
80 °C, 37% over two steps; (f) NBS, CCl4, benzoylperoxide, reflux, 97%;
(g) NaN3, DMF, 85 °C; (h) 10% Pd/C, MeOH, rt, 80% over three steps; (i)
ethylsulfonylethene, TEA, MeOH, reflux; (j) 4-fluoro-3-trifluoromethyl-
phenylacetic acid, EDC, HOBt, NMM, DMF, rt, 80–90% two steps.
Enantiomerically pure products were isolated by chiral phase HPLC.
Scheme 6. Reagents and conditions: (a) SnCl2, EtOAc, reflux, 95%; (b)
(R)-2-(tert-butoxycarbonyl)propanoic acid, EDC, HOBt, NMM, rt,
85%; (c) AcOH, 80 °C, 81%; (d) TFA, DCM, rt, 75%; (e) ethylsulf-
onylethene, TEA, MeOH; (f) 4-fluoro-3-trifluoromethylphenylacetic
acid, EDC, HOBt, NMM, DMF, rt, 80–90% two steps. (g) Pd(PPh3)4,
CuI, NaCN, MeCN, microwave, 120 °C, 92%.

A.-R. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 688–693
691
and hydrolysis of the phthalimide with hydrazine. The
transformation of 18 to racemic 19 was achieved using
the same methods as described in Scheme 1. Enantio-
pure 19 was isolated by chiral HPLC.
group. Michael addition and phenylacylation described
in Scheme 1 afforded 28 and 29.
The synthesis of compound 33 is described in Scheme 8.
Treatment of 1-aminopyridinium iodide with ethyl pent-
2-ynoate followed by decarboxylation afforded 2-ethyl-
pyrazolo[1,5-a]pyridine 30. Subsequent iodination and
Suzuki coupling led to formation of compound 31. Rad-
ical bromination, followed by azide displacement of the
bromide and reduction of the azide, afforded the primary
amine 32. Racemic final product 33 was obtained after
Michael addition with ethylsulfonylethene, followed by
acylation with 4-fluoro-3-(trifluoromethyl)phenylacetic
acid as previously described in Scheme 1. Enantiomeri-
cally enriched 33 was obtained by chiral HPLC.
The synthesis of benzimidazole 23 is outlined in Scheme
6. Reduction of nitroaryl 20 followed by coupling with
Cbz-protected D-alanine yielded 21, which was cyclized
in acetic acid and deprotected to afford 22. Compound
23 was obtained by employing the two-step Michael
addition and phenylacylation sequence described in
Scheme 1, followed by palladium-catalyzed cyanation.
The intermediates 26 and 27 to H-imidazo1–24 pyridine
28 and imidazo[1,2-a]pyrimidine 29 were obtained in
four steps starting from aminopyridine or aminopyrim-
idine as described in Scheme 7. The cyclization with (R)-
3-amino-1-bromobutan-2-one followed by iodination
yielded 24 and 25. Compounds 26 and 27 were obtained
via Suzuki coupling followed by removal of the Cbz
Initially, we designed a series of six–six fused heterobicy-
clic derivatives such as quinoline 5, 1,8-naphthyridine 6,
and quinoxaline 9 to determine the influence of the car-
bonyl and the 3-position nitrogen atom of the 8-aza-qui-
Table 1. In vitro activities of six–six fused heterocyclic ring systemsa
b
Binding IC50 (nM)
e
Lymphocyte migration IC50 (nM)
ID
Bufferc
11
Plasmad
1
25
115
72
5
0.80
2.0
9.0
5.0
4.0
2.0
21
6
27
48
9
121
115
60
n.d.
n.d.
88
12
16
19
17
41
a Values are means of three experiments, standard deviation is 30% (see Ref. 23).
b Displacement of [125I]-IP-10 from the CXCR3 receptor expressed on activated PBMC.
c RPMI-1640 buffer supplemented with 0.5% BSA.
d EDTA-anti-coagulated human plasma to 50%.
e ITAC mediated migration of PBMC in the presence of 100% human plasma.

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A.-R. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 688–693
nazolinone lead 1 on binding and functional activities.
These compounds were evaluated in [125I]-IP-10 dis-
placement and ITAC-mediated lymphocyte migration
assays (Table 1). Relative to 1, compounds 5, 6, and 9
exhibited similar or improved affinities for the CXCR3
receptor as evident from their IC50’s in the [125I]-IP-10
displacement assay run in buffer.23 Clearly the carbonyl
and the 3-position nitrogen atom of the parental 8-aza-
quinazolinone lead 1 are not essential for binding of
these compounds to the receptor. It was also found that
quinoline 5 showed a greater than 10-fold increase in
affinity for the CXCR3 receptor in buffer compared to
1. However, the increase in potency was negligible when
taking into consideration protein binding as demon-
strated by the [125I]-IP-10 ligand displacement and lym-
phocyte migration assays run in the presence of human
plasma. Compound 16 was designed to evaluate the
necessity of a fully aromatic bicyclic ring system for high
binding affinity. This compound had similar potency to
the lead compound 1 across all assays, confirming that
an extensive aromatic bicyclic ring system is not essen-
tial for potent binding or functional activity in the
in vitro assays.
IC50 = 115 nM). While the benzoimidazole 22 and the
imidazopyrimidine 29 exhibited strong affinity for the
receptor in the [125I]-IP-10 ligand displacement assay
in buffer, these compounds afforded only modest activity
in the binding and migration assays run in the presence
of human plasma.
The pharmacokinetic properties of selected compounds
were determined in the rat (Table 3). In general com-
pounds evaluated showed good oral absorption in the
rat. In addition, compound 19 showed lower clearance
after i.v. administration compared to the 8-azaquinaz-
olinone lead 1, while compounds 5 and 6 had compara-
ble pharmacokinetic profiles to 1.
In summary, we have discovered that a variety of six–six
fused heterocycles, such as quinoline, 1,8-naphthyridine,
and quinoxaline, and six–five fused heterocycles, such as
benzoimidazole, imidazopyridine, and imidazopyrimi-
dine, yield viable replacements of the 8-azaquinazoli-
none core featured in AMG 487 and 1. We have
demonstrated that the carbonyl and the 3-position nitro-
gen atom of the parental 8-aza-quinazolinone lead are
not essential for the binding of these compounds to
We also investigated six–five fused heterocycles in place
of the 8-aza-quinazolinone core (Table 2). Benzoimidaz-
ole 22, imidazopyridine 28, imidazopyrimidine 29, and
pyrozolopyridine 33 afforded good affinity for the
CXCR3 receptor as evident from the [125I]-IP-10 ligand
displacement assay in buffer. The imidazopyridine-de-
rived compound 28 afforded the highest affinity for the
receptor ([125I]-IP-10 IC50 in buffer = 3 nM; IC50 in plas-
ma = 21 nM), while the pyrazolopyridine-derivative 33
had the weakest binding affinity ([125I]-IP-10 IC50 in buf-
fer = 18 nM; IC50 in plasma = 523 nM). Compound 28
also had improved CXCR3 activity in the migration as-
say (ITAC IC50 = 72 nM) relative to the lead 1 (ITAC
Table 3. Pharmacokinetic profiles of select analogs in rat
ID
Cls(L/h/Kg)a
MRT(h)a
F(%)b
1
2.38
2.53
2.38
3.78
0.24
2.18
0.53
1.5
47
17
5
6
0.37
0.35
1.4
31
16
19
28
n.d.
24
0.26
24
a Following i.v. dosing in rat at 0.5 mg/kg.
b Following p.o. dosing in rat at 2 mg/kg.
Table 2. In vitro activities of six–five fused heterocyclic ring systemsa
b
Binding IC50 (nM)
e
Lymphocyte migration IC50 (nM)
ID
Bufferc
6.0
Plasmad
22
28
29
33
35
521
72
3.0
21
10
79
643
n.d.
18
523
a Values are means of three experiments, standard deviation is 30% (see Ref. 23).
b Displacement of [125I]-IP-10 from the CXCR3 receptor expressed on activated PBMC.
c RPMI-1640 buffer supplemented with 0.5% BSA.
d EDTA-anti-coagulated human plasma to 50%.

A.-R. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 688–693
693
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23. [125I]-IP-10 binding assay in buffer: human peripheral
blood mononuclear cells (PBMC) were activated with
anti-CD3 monoclonal antibody and recombinant human
IL-2 for 14 days. Cells were co-incubated with CXCR3
antagonists and recombinant human [125I]-IP-10 (50 pM)
for 2 h at rt. The assay buffer used was RPMI-1640
(without phenol red), supplemented with 0.5% BSA. Cells
were harvested onto 96-well filter plates and radioactivity
was counted on a scintillation counter. Assay values were
means of three experiments. [125I]-IP-10 binding assay in
plasma: conditions were the same as the [125I]-IP10
binding assay in buffer with the exception that EDTA-
anti-coagulated human plasma (‘‘plasma’’) was added to
the assay buffer, to 50% final assay concentration. ITAC
in vitro cell migration assay: 100 ng/ml of human ITAC
resuspended in 100% plasma was added to the lower
chamber of a migration plate. Human PBMC (prepared as
above) were resuspended in 100% plasma and placed in
the upper chamber of the migration plate. Compounds
were measured for their ability to inhibit CXCR3 medi-
ated migration of cells into the lower chamber, in response
to ITAC.
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