Design and optimization of imidazole derivatives as potent CXCR3 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.11.072

A series of imidazole derivatives have been designed and optimized for CXCR3 antagonism, pharmacokinetic properties, and reduced formation of glutathione conjugates. Our efforts led to the discovery of potent CXCR3 antagonists with good pharmacokinetic properties. These compounds are useful tools for in vivo studies of CXCR3 function.

Full text of DOI:10.1016/j.bmcl.2007.11.072

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 608–613
Design and optimization of imidazole derivatives
as potent CXCR3 antagonists
Xiaohui Du, Xiaoqi Chen, Jeffrey T. Mihalic, Jeffrey Deignan, Jason Duquette,
An-Rong Li, Bryan Lemon, Ji Ma, Shichang Miao, Karen Ebsworth, Timothy J. Sullivan,
George Tonn, Tassie L. Collins and Julio C. Medina*
Amgen Inc. 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
Received 25 October 2007; revised 16 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—A series of imidazole derivatives have been designed and optimized for CXCR3 antagonism, pharmacokinetic properties,
and reduced formation of glutathione conjugates. Our efforts led to the discovery of potent CXCR3 antagonists with good phar-
macokinetic properties. These compounds are useful tools for in vivo studies of CXCR3 function.
Ó 2007 Elsevier Ltd. All rights reserved.
The CXCR3 chemokine receptor and its ligands, Mig
(CXCL9), IP10 (CXCL10), and ITAC (CXCL11), are
believed to play major roles in the recruitment of
Type-1 T helper (Th1) cells from the peripheral blood
into inflamed tissues, thus aggravating the inflammatory
response.1 This hypothesis is supported by the increased
numbers of infiltrating cells that express CXCR3 and
the increased levels of Mig, IP10, and ITAC observed
in disease tissue from patients suffering from inflamma-
tory bowel disease,2 multiple sclerosis,3 psoriasis,4 rheu-
matoid arthritis,5 and solid organ transplant rejection.6
Further support for the key role that CXCR3 plays in
mediating immune disorders comes from evaluation of
CXCR3 deficient mice in cardiac allograft experiments
where the CXCR3 knock-out mice show increased allo-
graft tolerance when compared to wild-type mice.7 Like-
wise, transplant experiments with antibodies to the
CXCR3 ligands, Mig and IP10, enhance allograft sur-
vival.7 Furthermore, cardiac allografts from mice lack-
ing IP10 show prolonged allograft survival time when
transplanted into a wild-type mice.8 Additional reports
of in vivo transplant models of lung, small bowel, and
islet further validate the key role that CXCR3 plays in
cellular recruitment and allograft survival.9 Therefore,
there is great interest in discovering small molecule
CXCR3 antagonists1a,10a–d for the treatment of autoim-
mune and inflammatory diseases.
We have previously demonstrated11 that 2,3-substituted-
quinazolin-4-one derivatives such as compound 1 are
potent and selective CXCR3 antagonists. We also re-
ported that the quinazolin-4-one core could be replaced
by several bicyclic–heterocyclic ring systems.12
Our previous work demonstrated that significant modi-
fications of the quinazolinone core moiety in 1 are toler-
OEt
O
3
N
core
structure
1
N
a
IC50 = 1 nM
N
N
1
O
F
CF3
OEt
OEt
1
5
N
O
N
4
N
N
3
N
N
N
N
O
F
F
CF3
CF3
IC50a = 11 nM
IC50a = 1.8 μM
Keywords: CXCR3; IP10; ITAC; Mig; CXCL9; CXCL10; CXCL11;
Imidazole; Chemokine.
3
2
*
Figure 1. Imidazole derivatives afford potent CXCR3 antagonists.
aIC50 for 125I-IP10 ligand displacement in buffer.
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.2007.11.072

X. Du et al. / Bioorg. Med. Chem. Lett. 18 (2008) 608–613
609
OEt
OEt
OEt
ated and we postulated that the main role of this moiety
is to hold the peripheral groups in the correct orienta-
tion. We hypothesized that an imidazole ring system
would be a good replacement for the quinazolinone core
since the imidazole ring system contains nitrogens in a
1,3-relationship similar to the quinazolinone core struc-
ture. The resulting compound 2, however, displayed sig-
nificant loss of CXCR3 affinity compared to
quinazolinone 1. It was postulated that adding a lipo-
philic moiety resembling the rest of the phenyl moiety
on the quinazolinone core was necessary. Indeed, addi-
tion of a phenyl group at the 4-position of the imidazole
ring (3) significantly increased the compound affinity for
the CXCR3 receptor, indicating that a lipophilic moiety
connected to the imidazole core structure was preferred
for increased potency (Fig. 1). In this article, we report
the systematic optimization of the imidazole-derived
CXCR3 antagonists that led to compounds13 with sig-
nificantly improved potency and good pharmacokinetic
properties. The affinity of these compounds for the
CXCR3 receptor was evaluated using an 125I-IP10 li-
gand displacement assay to IL-2 activated human
peripheral blood mononuclear cells (PBMC) in the pres-
ence or absence of human plasma.14 Furthermore, the
antagonistic activity of these compounds was studied
using an ITAC-induced cellular migration assay of
PBMC.14
N
O
N
O
N
a,b,c
d
Ph
Ph
Ph
N
N
N
N
N
NH2
N
Me
N
H
F3C
F
F3C
F
8
13 *(S)
14 *(R)
11 *(S)
12 *(R)
OEt
OEt
OEt
e
N
O
N
Ph
N
Ph
f
N
Ph
N
N
N
F3C
HN
18
OEt
NH2
OEt
8
F
15
OEt
OEt
h
OEt
g
N
O
N
Ph
N
Ph
Ph
Ph
N
N
N
N
R2
N
Et
HN
Et
S
S
NH2
O
O
O
O
19
8
16, 21-28
OEt
OEt
OEt
i
N
N
Ph
j,k
SEt
N
Ph
N
O O
S
N
N
HN
20
F3C
F
Et
NH2
17
O
8
Scheme 2. Reagents and conditions: (a) tert-butyl 2-formylpyrrolidine-
1-carboxylate, NaBH3CN, HOAc, DCE; (b) 4-fluoro-3-trifluorometh-
ylphenylacetic acid, EDC, HOBt, NMM, DMF; (c) TFA, DCM, 11:
27% for 3 steps; 12: 62% for 3 steps; (d) formaldehyde, NaBH(OAc)3,
ClCH2CH2Cl, 13: 100%; 14: 82%; (e) BrCH2CH2OEt, DMF, K2CO3,
NaI, 80 °C, 42%; (f) trifluoromethoxyphenylacetic acid, EDC, HOBt,
NMM, DMF, 83%; (g) ethyl vinyl sulfone, Et3N, MeOH, 50 °C, 18 h,
52%; (h) for 16: 4-trifluoromethoxyphenylacetic acid, EDC, HOBt,
NMM, DMF, 96%; for 21–28: various phenylacetic acid
R2CH2COOH, BOPCl, Et3N, THF, 29–94%; (i) 3-(ethylthio)propanal,
NaBH3CN, HOAc, DCM, 48%; (j) 4-trifluoromethoxyphenylacetic
acid, EDC, HOBt, NMM, DMF, 81%; (k) MCPBA, DCM, 72%.
Previous work performed on the related quinazolinone
series suggested that optimization of the peripheral
groups surrounding the heterocyclic core could further
increase affinity for the receptor. Therefore, a series of
imidazole derivatives with N-alkyl modifications was
prepared as single enantiomers (Schemes 1 and 2).
Alkylation of 4-ethoxyaniline with 2-bromo-1-phenyl-
ethanone 4 generated 2-(4-ethoxyphenylamino)-1-phe-
nylethanone 5. Subsequent coupling with the mixed
anhydride of Boc-protected D-alanine and cyclization
of compound 6 with ammonium acetate furnished imid-
azole 7. Deprotection of the Boc group led to amine 8.
Compound 8 was a common intermediate that led to
various CXCR3 antagonists in Tables 1 and 2. Com-
pounds 3, 9, and 10 were prepared through reductive
amination of 8 and coupling with 4-fluoro-3-trifluor-
ophenylacetic acid. The syntheses of compounds 11–14
from compound 8 involved additional manipulations
of the pyrrolidine moiety. Deprotection of the Boc
group gave compounds 11 and 12, while introduction
of a methyl group via reduction amination gave 13
and 14. Alkylation of compound 8 with 1-bromo-2-eth-
oxyethane led to amine 18. Standard amide coupling of
18 generated antagonist 15. Michael addition of com-
pound 8 with ethyl vinyl sulfone followed by coupling
with phenylacetic acid furnished compound 16. With a
different coupling reagent, BOPCl, compounds 21–28
can be prepared from the same intermediate 19. A pro-
pyl sulfone side chain could be installed through reduc-
NHCbz
O
O
O
H
N
O
a
b
OEt
Br
Ph
Ph
c
N
Ph
4
5
tive
amination
of
compound
8
with
3-
6
OEt
N
(ethylthio)propanal, amide coupling, and oxidation of
the thio ether to sulfone 17. With minor modifications,
this route was efficient and reproducible in preparing
compounds 29–34 with substitutions at the 4-position
of the imidazole ring. The enantiomeric purity of the fi-
nal compounds was assessed by chiral HPLC.15
OEt
OEt
e
OEt
N
Ph
N
f,g
Ph
Ph
N
N
N
R1
N
O
3, R1=3-Py
9, R1=4-Py
F3C
F
NHBoc
NH2
7
8
10, R1=2-Py
In addition, a series of cyclopropyl-substituted imidaz-
ole derivatives were prepared as described in Scheme
3. Iodination at the 5-position of the imidazole ring of
antagonist 32 resulted in iodide 35, which was converted
to compound 36 via Stille coupling. Saturation of the
double bond in 36 led to compound 37. On the other
hand, ozonolysis of the alkene moiety in 36 and reduc-
Scheme 1. Reagents and conditions: (a) 4-ethoxyaniline, Et2O, 16 h,
67%; (b) BOC-D-alanine, isobutylchloroformate, NMM, DCM,
À20 °C to rt, then 5, 92%; (c) NH4OAc, HOAc, 90 °C, 2 h, 96%; (d)
TFA, DCM, 100%; (f) various pyridyl aldehyde, NaBH3CN, HOAc,
DCE; (g) 4-fluoro-3-trifluoromethylphenylacetic acid, EDC, HOBt,
NMM, DMF. Yields for steps f and g combined: 3: 48%; 9: 47%; 10:
9%.

610
X. Du et al. / Bioorg. Med. Chem. Lett. 18 (2008) 608–613
Table 1. SAR of amide N-substituents
CN
CN
CN
I
OEt
N
O
N
O
N
O
a
b
c
N
N
N
N
N
N
N
SO2Et
SO2Et
SO2Et
CN
N
F
F
F
N
36
35
R
CF3
CF3
32
CF3
O
CN
CN
F
OH
N
O
F
CF3
N
N
d
e
N
N
N
Compound R Group
125I-IP10 binding
Buffera,b (nM) Plasmaa,b (nM)
N
N
N
SO2Et
SO2Et
SO2Et
O
O
O
F
F
F
38
39
45
CF3
CF3
CF3
CN
CN
3
11
35
80
46
11
14
6.4
91
N
N
N
O
N
f
N
N
N
N
9
129
1179
36
SO2Et
CN
SO2Et
CN
O
F
F
F
F
37
35
CF3
F
CF3
CF3
CF3
O
F
10
11
12
13
14
N
N
N
e
N
N
N
N
SO2Et
CN
SO2Et
CN
N
H
O
O
H
H
H
H
F
45
40
CF3
R
I
20
N
N
H
N
O
g,h,i
R=CF3 41
CN 42
Ph 44
N
N
N
N
SO2Et
CN
SO2Et
CN
O
81
N
N
F
35
CF3
Cl
31
N
O
j
N
O
N
N
N
N
SO2Et
SO2Et
15
16
17
11
202
4.5
18
O
S
F
F
32
CF3
CF3
43
0.4
2.4
Scheme 3. Synthesis of 4-substituted imidazole analogs. Reagents and
conditions: (a) NIS, CH2Cl2, 95%; (b) n-Bu3SnCHCH2, Pd(PPh3)4,
toluene, 79%; (c) O3, CH2Cl2–MeOH, À78 °C, then Me2S, 31%; (d)
NaBH4, MeOH, 60%; (e) Deoxo-Fluor, CH2Cl2, 81% for 39 and 40%
for 40; (f) H2, 10% Pd/C, 90%; (g) 41: FSO2CF2COOMe, CuBr, PdCl2,
150 °C, lW, 55%; (h) 42: CuCN, DMF, 200 °C, lW, 60%; (i) 44:
Bu3SnPh, PdCl2(PPh3)2, 180 °C, lW, 78%; (j) SO2Cl2, ClCH2CH2Cl,
35%.
O
O
O O
S
a Values are means of three experiments. Standard deviations are
30%.
b Displacement of 125I-labeled IP10 from the CXCR3 receptor
expressed on PBMC. See Ref. 14 for assay protocol.
tion of the resulting aldehyde 45 generated alcohol 38.
Fluorination of 38 and 45 generated compounds 39
and 40. A few additional antagonists could be prepared
from iodide 35, such as trifluoromethyl-substituted com-
pound 41, compound 42 through cyanation, and com-
pound 44 through Stille coupling. Chlorination of 32
afforded compound 43.
of polar groups. It was also noted that, the potency of
some of the antagonists did not shift much in the pres-
ence of plasma in the 125I-IP10 binding assay such as
compounds 11 and 12, while the potency of the ethoxy-
ethyl-substituted compound 15 decreased about 20-fold
in the presence of plasma. In addition, sulfone groups
(16 and 17) were identified as potent replacements of
the 3-pyridyl moiety. Subsequent studies were carried
out by using the ethyl sulfone of 16 as the amide N-
substituents.
The compounds in Table 1 demonstrated that a variety
of substitutions at the amide-N-alkyl moiety were well
tolerated. It was interesting to observe that, while a 3-
pyridyl moiety was preferred for CXCR3 activity over
the 2- or 4-substituted pyridine derivatives, the fact that
all regioisomers had significant activity suggested that
the position of the nitrogen atom is not critical for activ-
ity (3, 9, and 10). Furthermore, it was observed that
amines (11–14) and alkoxy groups (15) also had good
potency which suggested that this site tolerated a variety
In an attempt to identify more polar molecules, the
phenylacetic acid side chain was replaced with a variety
of bicyclic side chains, such as imidazole-phenyl, tria-
zole-phenyl, and tetrazole-phenyl (Table 2). Unfortu-
nately, these were uniformly less potent than the
parent 16, although most only by an order of magnitude
(22–26). The bicyclic replacements with smaller lipo-

X. Du et al. / Bioorg. Med. Chem. Lett. 18 (2008) 608–613
611
Table 2. Replacing amide side chain with polar groups led to potent
CXCR3 antagonists
group (32–34) resulted in reduced activity (Table 3).
However, evaluation of the pharmacokinetic proper-
ties of 29 and 32 (Table 4) demonstrated that 29
was cleared faster than 32 following intravenous
administration in rats. Furthermore, 32 had signifi-
cantly improved solubility and permeability compared
to 29. Therefore, it was decided to study further ana-
logs of 32 bearing a cyclopropyl substituent at the 4-
position of imidazole.
OEt
N
N
N
S
O
R
O
O
Compound
R Group
125I-IP10 binding
Buffera,b Plasmaa,b
(nM)
(nM)
Table 3. Effect of substitutions at the 4-position of the imidazole ring
CN
F
16
21
22
23
24
25
26
27
28
0.4
7.1
4.8
5.6
9.4
5
4.5
F3C
N
R
N
O
F3C
N
45
S
O
O
O
F
CF3
N
24
N
N
Compound R Group
125I-IP10 binding
Buffera,b Plasmaa,b
ITAC plasma
migrationa,c
(nM)
N
N
49
(nM)
(nM)
29
0.7
14
10
91
33
53
54
100
N
N
N
N
69
30
2.3
4.0
6.6
2.6
2.5
29
43
37
19
21
N
N
N
47
F
31
N
N
N
N
32
33
34
N
N
2.1
44
19
F3C
N
N
N
1659
1124
N
N
a Values are means of three experiments. Standard deviations are
30%.
N
N
40
F3C
b Displacement of 125I-labeled IP10 from the CXCR3 receptor
expressed on PBMC. See Ref. 14 for assay protocol.
c One hundred nanograms per milliliter of ITAC was used in the
presence of 100% human plasma. See Ref. 14 for assay protocol.
a Values are means of three experiments. Standard deviations are
30%.
b Displacement of 125I-labeled IP10 from the CXCR3 receptor
expressed on PBMC. See Ref. 14 for assay protocol.
Table 4. Pharmacokinetic profiles in rat and physical properties of the
CN-substituted antagonists 29 and 32
philic groups such as cyclopropyl 27 or trifluoromethyl
28 all resulted in loss of potency.
Compound
29
32
Metabolism studies of the 4-ethoxy-phenyl-substituted
analogs with human liver microsomes demonstrated
the formation of phenolic metabolites that are time-
dependent inhibitors of CYP3A4. Therefore, replace-
ment of the ethoxy group was desired. Based on previ-
ous SAR studies in the quinazolinone series,11 the
cyano group was found to be a suitable replacement of
the ethoxy group. Compound 29 had an IC50 of
0.7 nM in the 125I-IP10 binding assay in buffer com-
pared to 0.4 nM of 16. Therefore, the cyano group
was used in the design of subsequent compounds.
IVa
CL (L/h/kg)
AUC (lg h/L)
MRT (h)
5.5
92
3.3
149
0.3
95
1.0
11
POa
AUC (lg h/L)
MRT (h)
F (%)
0.9
3
2.4
16
Solubilityb (lg/mL)
0.5
0.9
32
5.1
Permeabilityc (10À6 cm/s)
a IV dose: 0.5 mg/kg; PO dose: 2.0 mg/kg. In all cases: n = 2.
b The solubility assay measures a compound’s concentration in the
supernatant after a 10 mM DMSO stock solution is added into the
assay buffer pH 7.4 with the final DMSO concentration ꢀ1%.
c Permeability assay is based on the PAMPA technology (parallel
artificial membrane permeability assay) using a phospholipid lipid
bilayer. It measures the passive diffusion of the compound across the
lipid bilayer.
Exploration of substitutions at the 4-position of the
imidazole ring demonstrated that replacement of the
phenyl moiety by pyridyl (30–31) or a small alkyl

612
X. Du et al. / Bioorg. Med. Chem. Lett. 18 (2008) 608–613
Table 5. The effect of substitutions at the 5-position of the imidazole ring
CN
R
N
N
N
S
O
O
O
F
CF3
Compound
R
125I-IP10 binding
ITAC plasma migrationa,c (nM)
GSH adduct percentage (%)
Buffera,b (nM)
Plasmaa,b (nM)
32
36
37
38
39
40
41
42
43
44
–H
–CHCH2
–Et
6.6
3.8
3.2
10
37
22
53
45
44
8
14
16
33
29
–CH2OH
–CH2F
–CHF2
–CF3
–CN
113
119
20
1
8
12
28
18
N.D.d
0
19
72
45
268
N.D.d
80
N.D.d
0
276
24
0
–Cl
–Ph
7.8
752
2
10,000
N.D.d
a Values are means of three experiments.
b Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC. See Ref. 14 for assay protocol.
c One hundred nanograms per milliliter of ITAC was used in the presence of 100% human plasma. See Ref. 14 for assay protocol.
d N.D. = not determined.
Table 6. Pharmacokinetic profiles of 39 and 42 in rat
Metabolic studies with 32 using human liver microsome
indicated a significant amount of glutathione (GSH)
conjugates formed primarily at the imidazole ring.
Therefore, a number of analogs of 32 with additional
substitution at the 5-position of the imidazole ring were
synthesized and studied with the aim of reducing meta-
bolic activation and improving metabolic stability.
Compound
40
43
IVa
CL (L/h/kg)
AUC (lg h/L)
MRT (h)
2.2
237
0.9
2.3
219
1.3
POa
AUC (lg h/L)
MRT (h)
F (%)
456
2.4
48
225
5.4
26
Small alkyl (36, 37, and 40) and chloro (43) substituents
at the 5-position of the imidazole core maintained affin-
ity for the CXCR3 receptor (Table 5). However, some
loss in potency was observed when a strong electron-
withdrawing group such as a trifluoromethyl group
(41) and a cyano group (42) or a bulkier phenyl group
(44) was added to the this position. It was also noted
that substitutions at the 5-position of the imidazole ring
significantly decreased the formation of GSH conju-
gates. Electron-withdrawing groups such as difluoro-
methyl (40), trifluoromethyl (41), cyano (42), chloro
(43) group substitution completely prevented the forma-
tion of such adducts.
a IV dose: 0.5 mg/kg; PO dose: 2.0 mg/kg. In all cases: n = 2.
potency and pharmacokinetic properties, while reducing
the potential for metabolic bioactivation. Our optimiza-
tion efforts have led to the discovery of compounds 40
and 43 as potent CXCR3 antagonists with oral bioavail-
ability and acceptable pharmacokinetics in rodent, mak-
ing them useful tool compounds for in vivo studies of
CXCR3 function.
References and notes
Compounds 40 with a difluoromethyl substituent and 43
with a chloro substituent not only maintained potency
against the CXCR3 receptor and displayed decreased
formation of GSH conjugates, but also had improved
pharmacokinetic properties when compared to the
parental compound 32 (Table 6).
1. (a) Medina, J.; Johnson, M. G.; Collins, T. L. Ann. Rep.
Med. Chem. 2005, 40, 215; (b) Baggiolini, M. Nature 1998,
392, 565.
2. (a) Yuan, Y. H.; ten Hove, T.; The, F. O.; Slors, J. F.; van
Deventer, S. J.; te Velde, A. A. Inflamm. Bowel Dis. 2001,
7, 281; (b) Uguccioni, M.; Gionchetti, P.; Robbiani, D. F.;
Rizzello, F.; Peruzzo, S.; Campieri, M.; Baggiolini, M.
Am. J. Pathol. 1999, 155, 331.
3. (a) Mahad, D. J.; Howell, S. J.; Woodroofe, M. N.
J. Neurol. Neurosurg. Psychiatry 2002, 72, 498; (b) Mahad,
D. J.; Lawry, J.; Howell, S. J.; Woodroofe, M. N. Mult.
Scler. 2003, 9, 189; (c) Sorensen, T. L.; Tani, M.; Jensen,
J.; Pierce, V.; Lucchinetti, C.; Folcik, V. A.; Qin, S.;
Rottman, J.; Sellebjerg, F.; Strieter, R. M.; Frederiksen, J.
L.; Ransohoff, R. M. J. Clin. Invest. 1999, 103, 807; (d)
In conclusion, we have discovered that the quinazoli-
none ring in our first-generation series can be effectively
replaced by substituted imidazole derivatives to give a
new class of potent CXCR3 antagonists. An efficient
synthetic route to this class of compounds was devel-
oped. It was discovered that disubstitution of the imid-
azole core with lipophilic groups improved both

X. Du et al. / Bioorg. Med. Chem. Lett. 18 (2008) 608–613
613
Sorensen, T. L.; Trebst, C.; Kivisakk, P.; Klaege, K. L.;
Majmudar, A.; Ravid, R.; Lassmann, H.; Olsen, D. B.;
Strieter, R. M.; Ransohoff, R. M.; Sellebjerg, F.
J. Neuroimmunol. 2002, 127, 59.
J. W. G.; Owen, D. A.; Watson, R. J. Bioorg. Med. Chem.
Lett. 2007, 17, 697; (c) Storelli, S.; Verzijl, D.; Al-Badie, J.;
Elders, N.; Bosch, L.; Timmerman, H.; Smit, M. J.; De
Esch, I. J. P.; Leurs, R. Arch. Pharm. 2007, 340, 281; (d)
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.
4. (a) Gottlieb, A. B.; Luster, A. D.; Posnett, D. N.; Carter,
D. M. J. Exp. Med. 1988, 168, 941; (b) Gottlieb, A. B.;
Krueger, J. G.; Wittkowski, K.; Dedrick, R.; Walicke, P.
A.; Garovoy, M. Arch. Dermatol. 2002, 138, 591; (c)
Rottman, J. B.; Smith, T. L.; Ganley, K. G.; Kikuchi, T.;
Krueger, J. G. Lab. Invest. 2001, 81, 335.
11. Johnson, M.; Li, A.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.;
Wang, X.; Xu, Q.; Huang, A.; Marcus, 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. Biol. Org. Med. Chem. Lett. 2007,
17, 3339.
5. Patel, D. D.; Zachariah, J. P.; Whichard, L. P. Clin.
Immunol. 2001, 98, 39.
6. (a) Melter, M.; Exeni, A.; Reinders, M. E.; Fang, J. C.;
McMahon, G.; Ganz, P.; Hancock, W. W.; Briscoe, D. M.
Circulation 2001, 104, 2558; (b) Segerer, S.; Cui, Y.;
Eitner, F.; Goodpaster, T.; Hudkins, K. L.; Mack, M.;
Cartron, J. P.; Colin, Y.; Schlondorff, D.; Alpers, C. E.
Am. J. Kidney Dis. 2001, 37, 518; (c) Agostini, C.;
Calabrese, F.; Rea, F.; Facco, M.; Tosoni, A.; Loy, M.;
Binotto, G.; Valente, M.; Trentin, L.; Semenzato, G. Am.
J. Pathol. 2001, 158, 1703.
7. (a) Hancock, W. W.; Lu, B.; Gao, W.; Csizmadia, V.;
Faia, K.; King, J. A.; Smiley, S. T.; Ling, M.; Gerard, N.
P.; Gerard, C. J. Exp. Med. 2000, 192, 1515; (b) Hancock,
W. W.; Wang, L.; Ye, Q.; Han, R.; Lee, I. Curr. Opin.
Immunol. 2003, 15, 479.
8. Hancock, W. W.; Gao, W.; Csizmadia, V.; Faia, K. L.;
Shemmeri, N.; Luster, A. D. J. Exp. Med. 2001, 193, 975.
9. (a) Hancock, W. W.; Lu, B.; Gao, W.; Csizmadia, V.;
Faia, K.; King, J. A.; Smiley, S. T.; Ling, M.; Gerard, N.
P.; Gerard, C. J. Exp. Med. 2000, 192, 1515; (b) Belperio,
J. A.; Keane, M. P.; Burdick, M. D.; Lynch, J. P., 3rd;
Zisman, D. A.; Xue, Y. Y.; Li, K.; Ardehali, A.; Ross, D.
J.; Strieter, R. M. J. Immunol. 2003, 171, 4844; (c) Zhang,
Z.; Kaptanoglu, L.; Tang, Y.; Ivancic, D.; Rao, S. M.;
Luster, A.; Barrett, T. A.; Fryer, J. Gastroenterology 2004,
126, 809; (d) Baker, M. S.; Chen, X.; Rotramel, A. R.;
Nelson, J. J.; Lu, B.; Gerard, C.; Kanwar, Y.; Kaufman,
D. B. Surgery 2003, 134, 126.
10. (a) Collins, T. L.; Johnson, M. G.; Medina, J. C.
Antagonists of CXCR3: a Review of Current Progress.
In Chemokine Biology-Basic Research and Clinical Appli-
cation, Neote, K.; Letts, G. L.; Moser, B., Eds.; Birkha-
user Verlag Publishers, 2007; Vol. II, 2, pp 79; (b) Allen,
D. R.; Bolt, A.; Chapman, G. A.; Knight, R. L.; Meissner,
12. Li, A.; 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.;
Bryan, L.; Carabeo, T.; Sullivan, T.; Ma, J.; Tang, J.;
Tonn, G.; Collins, T.; Medina, J. C. Biol. Org. Med.
Chem. Lett. 2008, 18, 688.
1
13. All compounds were characterized by H NMR and LC/
MS and their purity was determined to be >95% by reverse
phase HPLC.
14. 125I-IP10 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 antagonist
and recombinant human 125I-IP10 for 2 h at room
temperature. 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-IP10 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 (from frozen stocks) was used
instead of the RPMI buffer. ITAC in vitro cell migration
assay: 100 ng/ml of ITAC was used in the presence of
100% human plasma. Compounds were measured by their
ability to inhibit CXCR3 mediated cell migration in
response to ITAC.
15. Chiral purity was analyzed with ChiralTech AD column
with hexanes and isopropanol as solvents. The enantio-
meric purity was higher than 95% ee with this synthetic
route.