Imidazo-pyrazine derivatives as potent CXCR3 antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.021

A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was identified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is evaluated in a mouse lung inflammation model.

Full text of DOI:10.1016/j.bmcl.2009.07.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5200–5204
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazo-pyrazine derivatives as potent CXCR3 antagonists
*
Xiaohui Du , Darin J. Gustin, Xiaoqi Chen, Jason Duquette, Lawrence R. McGee, Zhulun Wang,
Karen Ebsworth, Kirk Henne, Bryan Lemon, Ji Ma, Shichang Miao, Emmanuel Sabalan, Timothy J. Sullivan,
George Tonn, Tassie L. Collins, Julio C. Medina
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was iden-
tified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved
pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is
evaluated in a mouse lung inflammation model.
Received 8 May 2009
Revised 22 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Published by Elsevier Ltd.
Keywords:
CXCR3 antagonist
Imidazo-pyrazine
CXCR3, the receptor for the chemokines CXCL9 (MIG), CXCL10
(IP-10), and CXCL11 (I-TAC), is preferentially expressed on acti-
vated Th1 T cells and is responsible for the trafficking of T cells
to inflamed tissues.1 There is accumulating evidence suggesting
that CXCR3 and its chemokines play a pivotal role in a variety of
inflammatory diseases,2 such as multiple sclerosis,3 psoriasis,4
rheumatoid arthritis,5 allograft rejection,6 and inflammatory bowel
disease.7 The therapeutic potential of CXCR3 antagonists in treat-
ing theses diseases has triggered great interest in recent years. Sev-
eral series of small molecule CXCR3 antagonists, including the
camphor sulfonamides,8 the benzetimide derivatives,9 2-imino-
benzimidazoles,10 the 2-amino-4-(tropinyl)quinolines11 have been
recently described in the literature.
with excellent in vivo efficacy in a mouse lung inflammation
model.
Both, compounds 2 (2.4 L/h/kg) and 3 (3.7 L/h/kg) had lower
clearance in rats, after iv administration, compared to compound
1 (5.5 L/h/kg). However, compounds 2 and 3 were not as potent
as desired. In addition, the pharmacokinetics of both compounds
needed further improvement, as evidenced by the high in vivo
clearance in rats. One of the key metabolisms of compound 2
shown in rat liver microsome is the oxidation by aldehyde oxidase
at C7, which led to the formation of the corresponding pyridone
CN
OEt
O
We have previously reported on a quinazolinone-based12 series
as potent CXCR3 antagonists, and our optimization efforts led to
the discovery of AMG 487, a compound that was selected for eval-
uation in clinical tirals.13 We have also demonstrated that C4-
substituted-imidazole derivatives afford potent and selective
CXCR3 antagonists. As shown in Figure 1, phenyl-substituted imid-
azole 1 was one of the most potent CXCR3 antagonists, with an IC50
of 0.7 nM in 125I-IP-10 displacement assay.14 We also reported that
the 8-azaquinazolinone core in 2 could be substituted by several
bicyclic-heterocyclic ring systems, such as an imidazo-pyrimidine
core as in 3 (Fig. 1). In this Letter, we report our efforts to further
improve the potency of CXCR3 antagonists featuring hetero-bicy-
clic core structures, which led to the discovery of a potent new ser-
ies15 with an imidazo-pyrazine core that afforded a compound
1
5
4
N
N
O
N
3
N
N
N
N
N
S
O
O
O
F3C
O
F
125I-IP10 = 8 nM
125I-IP10 = 0.7 nM
CN
CF3
AMG 487
1
CN
O
3
N
1
N
N
N
7
N
7
N
N
1
N
S
O O
S
O
O
O
O
F
F
125I-IP10 = 11 nM
3
2
CF3
125I-IP10 10 nM
CF3
* Corresponding author. Tel.: +1 650 2442685.
E-mail address: xdu@amgen.com (X. Du).
Figure 1. Lead CXCR3 antagonists.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.021

X. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5200–5204
5201
Table 2
C7-substitution on imidazo-pyrimidine core
CN
R
N
N
N
N
S
O O
O
F
CF3
Entry
#
R
IP-10 buffer
bindinga,b
(nM)
IP-10 plasma
bindinga,b
(nM)
ITAC plasma
migrationa,c
(nM)
IV CLd
(L/h/kg)
Figure 2. Comparison of the structures of compounds 1, 2, and 3 by modeling. The
compound structures were built and overlaid in Quanta (Accelrys). Each compound
was minimized using CHARMm Minimization module in Quanta. Magenta:
compound 1; turquoise: compound 2; green: compound 3. Color codes for atoms:
red: oxygen; blue: nitrogen, yellow: sulfur.
3
11
H
Me
10
2
79
22
643
100
3.7
5.1
a
Values are means of three experiments. Standard deviation is 30%.
b
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
See Ref. 13b for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Ref. 13b for assay protocol.
metabolite. Comparison of imidazole 1, 8-azaquinazolinone 2 and
imidazo-pyrimidine 3 by modeling (Fig. 2) indicates that there is
space available for both compounds 2 and 3 to extend further into
the area occupied by the phenyl group at C4 of compound 1. We
postulated that substitution at C7 of the 8-azaquinazolinone and
the imidazo-pyrimidine could fill the hydrophobic pocket occupied
by the phenyl moiety of 1 and possibly improve the potency of the
antagonists. In addition, C7 substitution might prevent the forma-
tion of the aforementioned oxidative metabolite at C7 of 2.
Indeed, small substituents such as methyl and methoxy at C7 of
the azaquinazolinone core improved the potency in both the IP-10
binding and ITAC migration assays (4, 5, and 10 in Table 1). But
larger groups such as trifluoromethyl, ethoxy and isopropoxy were
less well-tolerated. Likewise, methyl substitution at C7 of the
d
Clearance in the rat after a 0.5 mg/kg IV dose.
imidazo-pyrimidine (11 in Table 2) also improved the potency
significantly.
The C7 substitution on the azaquinazolinone core significantly
improved the in vitro stability of compounds 4 and 5 as assessed
by using rat liver microsome. Compound 1 had 27% remaining after
an hour in the presence of rat liver microsomes while compounds 4
and 5 had 86% and 99% remaining, respectively. However, this did
not translate to improvements in the in vivo rat clearance of these
molecules (Table 1). In addition, 11 had higher clearance than the
parent 3 in the imidazo-pyrimidine series.
Table 1
Table 3
C7-substitution on 8-azaquinazolinone core
C8-substitution on imidazo-pyrazine corea
CN
O
CN
N
N
N
N
R
N
N
R
N
S
N
O
O
S
O
O O
G
O
F3C
F
Entry
#
R
G$
IP-10 buffer
bindinga,b
(nM)
IP-10 plasma
bindinga,b
(nM)
ITAC plasma
migrationa,c
(nM)
IV CLd
(L/h/kg)
Entry
#
R
IP 10 buffer
bindinga,b
(nM)
IP 10 plasma
bindinga,b
(nM)
ITAC plasma
migrationa,c
(nM)
IV CLd
(L/h/kg)
2
4
5
H
A
A
A
A
A
A
B
B
11
4
4
142
>1000
29
10
3
25
12
10
457
>1000
149
23
115
30
22
2.4
3.0
1.8
Me
OMe
OEt
OiPr
CF3
H
12
H
Cl
OMe
OMe
Me
15
3
2
0.9
1.6
1.1
0.9
1.4
44
60
20
17
6
10
11
16
27
371
ND
57
18
7
44
12
17
45
ND
6*
7*
8*
9
NDe
ND
ND
129
18
13*
14*
15
16
17
2.5
1.2
10
OMe
7
Et
18
Cyclo-propyl
NMe2
OH
a
Values are means of three experiments. Standard deviation is 30%.
19*
20*
b
Displacement of 125I-labeled IP-10 from the CXCR3 receptor expressed on PBMC
with or without human plasma. See Ref. 13b for assay protocol.
c
a
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Values are means of three experiments. Standard deviation is 30%.
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
b
Ref. 13b for assay protocol.
d
Clearance in the rat after a 0.5 mg/kg IV dose.
Potency not measured.
(A) 4-F, 3-CF3; (B) 3-F, 4-CF3.
See Ref. 13b for assay protocol.
e
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
$
Ref. 13b for assay protocol.
*
d
Racemic. Usually the racemic compound was about half as potent as the pure
Clearance in the rat after a 0.5 mg/kg IV dose.
*
enantiomer.
Racemic.

5202
X. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5200–5204
CN
N
N
Cl
Br
N
N
N
a
N
O
N
N
b
c
d
N
O
N
N
29
NH2
Cl
27
28
CN
CN
CN
CN
N
O
N
N
N
O
N
N
N
N
N
N
O
N
N
or
e
or
O
N
N
NH2
NH2
S
S
30
O
O
31
O
O
F3C
O
O
F3C
14
15
F
F
CN
CN
CN
N
N
N
N
N
g
f
N
R
N
h
HO
N
Cl
N
N
S
N
O
O
S
N
O
S
O
O
F3C
O
O
O
O
20
F3C
F
13
F3C
F
F
12, 16-19
18
CN
e
CN
q
Br
N
N
N
p
c,d
N
N
N
N
N
N
27
NH2
NH2
32
33
34
Scheme 1. Reagents and conditions: (a) 1-bromobutan-2-one, tBuOH, heat, 44%; (b) (i) NaOMe, MeOH, reflux, 93%; (ii) NBS, HOAc, 88%; (c) 4-cyanophenylboronic acid,
Pd2(dba)3, S-phos, K3PO4, THF, 69%; (d) for 30: (i) NBS, CCl4; (ii) NaN3, DMF, 80 °C; (iii) PPh3, DMF, H2O, 40 °C; 70% for three steps; for 31: (IV) chiral HPLC of 30, 44%; (e) (i)
from 30 to 14: ethyl vinyl sulfone, Et3N, MeOH, 50 °C, 94%; (ii) 2-(3-fluoro-4-(trifluoromethyl)phenyl)acetic acid, EDC, HOBt, NMM, DMF, 83%; compound 15 was obtained
from 31 with same condition; (f) Concd HCl, dioxane, 87%; (g) POCl3, reflux, 94%; (h) for 16, R = Me: (i) methyl boroxine, PdCl2dppf, Na2CO3, microwave, 41%; (ii) chiral HPLC,
35%; for 17, R = Et: (i) BEt3, Pd2(dba)3, S-phos, K3PO4, THF, 77%; (ii) chiral HPLC, 40%; for 19, R = N,N dimethyl: (i) N,N-dimethylamine, THF, pressure tube, 80 °C, 85%; for 18,
R = cyclopropyl and for 12, R = H: (i) The active enantiomer of compound 13 was used as starting material, cyclopropylboronic acid, PdCl2dppf, Na2CO3, microwave, 88% for
12, 11% for 18; (p) (i) cyclopropylboronic acid, Pd(PPh3)4, K3PO4, toluene, 105 °C; (ii) NBS, HOAc, 55% for two steps; (q) chiral HPLC, 50%.
This prompted us to look into antagonists with nitrogen at a dif-
ferent position on the left side of the bicyclic core structure. One of
such possibility was the antagonists with an imidazo-pyrazine core
(Table 3). Their synthesis is illustrated in Scheme 1.
3-Chloropyrazin-2-amine and 1-bromobutan-2-one were
reacted to form 8-chloro-2-ethylimidazo-pyrazine 27. After dis-
placing the chloro group with sodium methoxide, bromination
led to 28 and Suzuki coupling with the bromide yielded 29. The
Table 4
Cyclic sulfone analogs of the imidazo-pyrazine core
CN
N
N
N
O
S
O
N
R
O
Entry #
R
IP-10 buffer bindinga,b (nM)
IP-10 plasma bindinga,b (nM)
ITAC plasma migrationa,c (nM)
PXR at 2 l
M (% pos.)d
21
22
23
24
25
26
4-CF3, 3-F
4-OCF3
4-F,3-CF3
3-CF3
4-CF3
3-OCF3
0.9
1.1
0.9
1.7
1.3
1.1
7.3
13
21
29
16
18.9
34
25
36
22
6%
9%
29%
144%
7%
25
75
ND
a
Values are means of three experiments. Standard deviation is 30%.
b
c
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC. See Ref. 13b for assay protocol.
ITAC mediated migration of PBMC in the presence of 100% human plasma. See Ref. 13b for assay protocol.
Percent of positive control, rifampin.
d

X. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5200–5204
5203
amino group in 30 was introduced through a three-step sequence
Table 5
Pharmacokinetic profiles of 21 and 22 in rat and mouse.
of bromination, azide formation and reduction of the azide. Mi-
chael addition of the amine to ethyl vinyl sulfone and amide cou-
pling furnished the racemic 14. Chiral separation of 30 was
accomplished by HPLC with a chiral stationary phase. The enanti-
omerically pure amine 31 was carried through similar sequences
to afford compound 15. Deprotection of the methoxy group in 14
led to compound 20. The hydroxyl group in 20 was converted to
a chloro group in compound 13 by refluxing in POCl3. Compound
13 served as a common intermediate to 12 and 16–19. Methyl
(16), ethyl (17) and cyclopropyl groups (18) were introduced
through Suzuki coupling; while the N,N-dimethyl amino group
(19) was formed by nucleophilic replacement of the chloro group
with N,N-dimethylamine. Compound 12 was obtained as a side
product of the reaction to form 18.
Larger amounts of 18 were synthesized through an earlier intro-
duction of the cyclopropyl group by a higher-yielding Suzuki cou-
pling of the chloride 27 with cyclopropyl boronic acid. After chiral
separation of 33, the chiral amine 34 was carried to compound 18
through Michael addition to ethyl vinyl sulfone and acid coupling.
Consistent with the potency enhancement by small group sub-
stitution at C7 of compounds with azaquinazolinone and imidazo-
pyrimidine cores, small groups such as methyl (16), methoxy (15),
chloro (13), ethyl (17), cyclopropyl (18) and dimethyl amino (19),
at the 8-position of the imidazo-pyrazine core, led to significant
improvements in potency compared to the non-substituted com-
pound 12. The methoxy (15) and cyclopropyl (18) substitutions
boosted the IP-10 binding potency by more than 15-fold. However,
15 had poor microsomal stability with 7% remaining after incuba-
tion in rat liver microsomes for 30 min., while methyl-substituted
16 displayed improved stability with 64% remaining after the same
incubation time. The ethyl-derivative, 17, had similar stability as
16 with 68% remaining, while the cyclopropyl-substituted com-
pound 18 was the most stable in rat microsomes with 85%
remaining.
Entry #
IVa
21 (rat)a
21 (mouse)b
22 (rat)c
22 (mouse)d
CL (L/h/kg)
AUC g h/L)
MRT (h)
1.0
658
2.9
0.87
1910
1.4
0.84
617
7.5
0.53
1900
1.3
l
POa
AUC g h/L)
MRT (h)
F (%)
l
428
5.3
21
2290
2.6
40
499
4.5
20
2570
3.4
27
a
IV dose: 0.65 mg/kg; PO dose: 2.0 mg/kg. n = 2.
IV dose: 1.66 mg/kg; PO dose: 5.0 mg/kg. n = 1.
IV dose: 0.5 mg/kg; PO dose: 2.0 mg/kg. n = 2.
IV dose: 1.0 mg/kg; PO dose: 5.0 mg/kg. n = 1.
b
c
d
played an IC50 of 21 nM and 13 nM, respectively. In addition, com-
pound 21 was evaluated in an in vivo mouse model of lung
inflammation. An asymmetric synthetic route was developed to
facilitate the synthesis of 21 in large scale (Scheme 2).
Starting from 2,3-dichloropyrazine, amination of one of the
chloride generated compound 35. Cyclopropanation of 35 by
Suzuki coupling and deprotection furnished 3-cyclopropylpyra-
zin-2-amine 36. Compound 36 was reacted with (R)-benzyl
4-bromo-3-oxobutan-2-ylcarbamate to form 37 with the imi-
dazo-pyrazine core. The chiral amine 34 was obtained after bro-
mination of 37, Suzuki coupling to introduce the cyanophenyl
moiety, and subsequent deprotection of the CBZ group. Reductive
amination of the amine to introduce the cyclic sulfone and amide
coupling completed the synthesis of 21.
Compound 21 was tested in the bleomycin-induced lung
inflammation model in mouse.17 The test result was shown in
Figure 3. In this experiment, 0.1 unit of bleomycin is introduced
in the lungs of C57BL/6J mice through intra-tracheal instillation
after tracheostomy. This triggered inflammation response in the
lung as shown in changes in bronchoalveolar lavage (BAL) fluid
with increased total cell count and an increase in lymphocytes
infiltration. AMG 487 was used as a positive control in this exper-
iment. AMG 487 and compound 21 were dosed subcutaneously at
Consistent with the stability in rat liver microsomes, compound
16 had a clearance in rat of 2.5 L/h/kg, while 18 afforded a lower
clearance of 1.2 L/h/kg and a MRT of 3.6 h after IV dosing and a
71% oral bioavailability after PO dosing (2 mg/kg).
a flow rate of 0.5 ll/h with the use of an Alzet osmotic mini-
Compound 18, however, displayed significant PXR activation in
a reporter gene assay. Activation of the nuclear hormone receptor,
Pregnane X receptor (PXR), induces the formation of CYP3A4, lead-
N
N
Cl
N
N
Cl
Cl
N
N
O
a
ing to potentially harmful drug–drug interactions in vivo. At 2
lM
b
concentration, compound 18 had 35% of activation relative to the
positive control, rifampin, in the PXR-luciferase assay. Several imi-
dazo-pyrazines with the less flexible cyclic sulfone moiety were
synthesized and evaluated for PXR transactivation potential (Table
4). Compound 21 had a significant lower PXR activation potential
compared to 18. Modifications to the amide side chain also had
an effect on the PXR transactivation capability of these molecules.
In addition to compound 21, the 4-trifluoromethoxy (22) and 4-tri-
fluormethyl (25) derivatives displayed reduced PXR activation,
while the 3-trifluoromethyl substituted compound (24) had a
marked increase in PXR activation.
Based on their CXCR3 potency and lack of PXR activation, com-
pounds 21 and 22 were evaluated for their pharmacokinetics prop-
erties in rat (Table 5). Since our in vivo efficacy model was in
mouse, the mouse pharmacokinetic properties of both compounds
were also evaluated. Both compounds displayed low clearance in
rat and mouse with moderate oral bioavailability. Since compound
21 was more potent in the ITAC plasma migration assay, it was
evaluated further.
N
H
NH2
O
36
CN
35
O
N
N
N
N
N
d
Br
c
N
NHCbz
NHCbz
CN
NHCbz
N
38
37
CN
N
N
e
f
N
N
O
S
O
N
N
NH2
O
34
21
F3C
F
Scheme 2. Reagents and conditions: (a) DMBA, quantitative; (b) (i) cyclopropylbo-
ronic acid, Pd2(dba)3, S-phos, K3PO4, toluene, 100 °C, 5 h, 97%; (ii) TFA, DCM, 16 h,
85%; (c) 3-cyclopropylpyrazin-2-amine, K2CO3, CH3CN, 75 °C, 16 h, 68%; (d) (i)
DBDMH, 5 min, quantitative; (ii) 4-cyanophenylboronic acid, Pd2(dba)3, S-phos,
K3PO4, THF, reflux, 68%; (e) TFA, 75 °C, 1.5 h, 82%; (f) (i)cyclic sulfone-4-carbalde-
hyde, AcOH, NaBH(OAc)3, DMC, 0–20 °C, 1 h, 60%; (ii) 2-(3-fluoro-4-(trifluoro-
methyl)phenyl)acetic acid, EDC, HOBt, NMM, DMF, 70%, >99%ee.
Compound 21 was potent in a mouse IP-10 binding assay with a
10 nM IC50. Compound 21 was also evaluated in a human and
mouse whole blood assay16 that measure its ability to displace
ITAC binding to the CXCR3 receptors at 500 ng/ml of ITAC and dis-

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PBS Veh
10
1
Veh
10
3
1
--------------
AMG487
(mg/mL)
------------------------
Compound 21
(mg/mL)
Exposure (nM)
AMG487
205
37
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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.
Compound 21
291
182
86
Figure 3. Compound 21 was efficacious in mouse lung bleomycin model. *p <0.005
as determined by Student’s t-tests.
12. 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. Bioorg. Med.
Chem. Lett. 2007, 17, 3339.
13. (a) 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.; 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; (b) 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.
pump18 on the same day of bleomycin challenge for six days. On
day six, the number of cells collected in BAL fluid was counted.
Plasma samples were also collected at the end of the experiment.
The efficacy observed with AMG 487 in this experiment is consis-
tent with previous reports. Compound 21 inhibited infiltration of
cells into the lung at a steady state plasma concentration of
P86 nM.
In conclusion, substitution alpha to the left ring nitrogen of the
fused hetero-bicyclic cores such as the 8-azaquinazolinone, the
imidazo-pyrimidine, and the imidazo-pyrazine core, generally im-
proved the potency of the CXCR3 antagonists. In particular, the
imidazo-pyrazines had superior pharmacokinetic properties in
addition to its potency. Compound 21 demonstrated in vivo effi-
cacy in blocking the leukocyte migration into the lung in response
to bleomycin.
14. See Ref. 13b for assay protocol.
15. All compounds were characterized by 1H NMR and LC/MS and were >95% in
purity by reverse phase HPLC. Chiral purity was analyzed with ChiralTech AD
column. The enantiomeric purity was higher than 95% ee.
16. This assay measures cell-surface CXCR3 occupancy by FACS analysis of
fluorescent using
a fluorescently-tagged anti-CXCR3 antibody specific to
CXCR3. Addition of the CXCR3 ligand, ITAC, results in the loss of fluorescent
signal of antibody due to the competing ligand binding. The antagonist blocks
ITAC binding against receptor, but does not compete against anti-CXCR3
antibody binding to CXCR3 receptor. The fluorescent signal of the antibody was
restored with the addition of the antagonists. An ITAC concentration of 500 ng/
ml was used in the assay.
References and notes
17. It has been shown that CXCR3 plays an important role in noninfectious lung
injury induced by bleomycin instilled intratracheally into CXCR3-deficient
C57BL/6J mice background and WT littermate controls. Examination of the
inflammatory response after bleomycin-induced lung injury revealed an
insignificant reduction in total inflammatory cells in the bronchoalveolar
lavage (BAL) fluid from CXCR3-deficient animals as compared with WT
animals. Differential counts of the BAL cells revealed fewer total lymphocytes
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) Lazzeri, E.; Romagnani, P. Curr. Drug Targets Immune Endocr. Metabol. Disord.
2005, 5, 109; (b) Collins, T. L.; Johnson, M. G.; Medina, J. C. Antagonists of
CXCR3: a Review of Current Progress. In Chemkine Biology—Basic Research and
Clinical Information; Neote, K., Letts, G. L., Moser, B., Eds.; Birkhäuser Verlag
Publishers, 2007; Vol. II, p 79. 2.
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) 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.
in CXCR3-deficient mice after bleomycin-induced lung injury at day
7
compared with WT controls. See: Jiang, D.; Liang, J.; Hodge, J.; Lu, B.; Zhu,
Z.; Yu, S.; Fan, J.; Gao, Y.; Yin, Z.; Homer, R.; Gerard, C.; Noble, P. W. J. Clin.
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18. The 10 mg/ml group used 4.8 mg/kg/day of compound; 3 mg/ml used 1.44 mg/
kg/day of compound; and 1 mg/ml used 0.48 mg/kg/day of compound. For
more information of Alzet osmotic mini-pump, please visit http://
www.alzet.com/downloads/TIM.pdf.