Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3

Article abstract of DOI:10.1016/j.bmcl.2007.03.106

A series of quinazolinone-derived inhibitors of the CXCR3 receptor have been synthesized and their affinity for the receptor evaluated. Compounds were evaluated in a ¹²⁵I-IP10 displacement assay and in in vitro cell migration assays to IP10, IT

Full text of DOI:10.1016/j.bmcl.2007.03.106

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3339–3343
Discovery and optimization of a series of
quinazolinone-derived antagonists of CXCR3
Michael Johnson,a An-Rong Li,a Jiwen Liu,a Zice Fu,a Liusheng Zhu,a
Shichang Miao,a Xuemei Wang,a Qingge Xu,a Alan Huang,a, Andrew Marcus,a,
Feng Xu,a, Karen Ebsworth,a Emmanuel Sablan,a Jay Danao,a Jeff Kumer,a,
Dan Dairaghi,b Chris Lawrence,a, Tim Sullivan,a George Tonn,a Thomas Schall,b
Tassie Collinsa and Julio Medinaa,*
aAmgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
bChemoCentryx, 850 Maude Avenue, Mountain View, CA 94043, USA
Received 23 February 2007; revised 30 March 2007; accepted 30 March 2007
Available online 6 April 2007
Abstract—A series of quinazolinone-derived inhibitors of the CXCR3 receptor have been synthesized and their affinity for the recep-
tor evaluated. Compounds were evaluated in a 125I-IP10 displacement assay and in in vitro cell migration assays to IP10, ITAC, and
MIG using human peripheral blood mononuclear cells.
Ó 2007 Elsevier Ltd. All rights reserved.
CXCR3 is a chemokine receptor primarily expressed on
activated CD4+ and CD8+ T cells with a Th1 pheno-
type;1 it is also expressed on B cells,2 natural killer
(NK) cells,3 malignant T cells4, and astrocytes.5 The
ligands for CXCR3 are Mig (CXCL9), IP-10 (CXCL10),
and ITAC (CXCL11). These ligands are induced pri-
marily by IFN-c and are produced by macrophages as
well as other cell types in inflamed tissue.6 CXCR3
and its ligands have been implicated in a number of
inflammatory diseases including rheumatoid arthritis,7
multiple sclerosis,8 inflammatory bowel diseases,9 psori-
asis,10 and transplant rejection.11 Therefore, it has been
postulated that blockade of CXCR3 may play a benefi-
cial role in the treatment of these diseases.12
expressed on activated peripheral blood lymphocytes
with an IC50 of 0.25 lM (Table 1). In addition, 1 also
inhibited calcium mobilization induced by MIG with
IC50 of 0.50 lM. Pharmacokinetic studies with 1 in rats
revealed high clearance (2.8 L/kg/h) after a 0.5 mg/kg iv
dosing. The bioavailability after oral gavage dosing at
2 mg/kg is very low (1.5%). In this report, we describe
our effort in improving the potency and pharmacoki-
netic properties of compound 1 that led to the identifica-
tion of AMG 487 (47), which was selected for evaluation
in clinical studies.
A series of quinazolinone derivatives were prepared as
described in Scheme 1.14 The critical intermediates 2
were easily synthesized by treatment of 2-aminobenzoic
acid and the appropriate BOC-protected amino acid
with triphenyl phosphite and pyridine, followed by reac-
tion with anilines in one pot.15 Deprotection of 2 using
TFA provided the primary amines, which were subse-
quently converted to secondary amine 3 by reductive
amination reactions with aldehydes. In the case where
R2 is dimethylamino methyl, the corresponding alde-
hyde was prepared by heating dimethylamino acetalde-
hyde dimethyl acetal in concentrated HCl at 100 °C
for 1 h and neutralizing with 10 N NaOH at 0 °C (The
prepared aldehyde in aqueous solution was used in the
next step without purification.). Final functionalization
High-throughput screening led to the discovery of struc-
ture (1) as a moderate CXCR3 antagonist.13 Compound
1 inhibited binding of 125I-IP10 to CXCR3 receptors
Keywords: CXCR3; IP10; ITAC; MIG; Chemokine receptor.
*
Corresponding author. Tel.: +1 6502442425; fax: +1 6502442015;
e-mail: medinaj@amgen.com
Alan Huang, Gilead Sciences, Inc., Foster City, CA 94404; Andrew
Marcus, University of California-Berkeley, Berkeley, CA 94720; Feng
Xu, XenoPort Inc., Santa Clara, CA 94404; Jeff Kumer, Sunesis
Pharmaceuticals Inc., South San Francisco, CA 94080; Chris Law-
rence, Xencor, Monrovia, CA, 91016.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.106

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M. Johnson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3339–3343
R1
Table 1. Exploration of the stereocenter
O
O
c
F
c
N
O
O
c
O
N
N
H
a, b
b
b
a
NH
HO
a
N
NHBOC
NHBOC
NHBOC
O
5
4
R1
R1
O
CH3(CH2)8
N
O
c
d, e
f, g
N(Me)2
N
O
c
N
b
a
N
O
b
a
N
N
R2
NH2
1
R3
125I -IP10 IC50 (lM)
a,b
6
46-50
Compound
1
0.25
0.146
>10
Scheme 2. Reagents and conditions: (a) isobutylchloro- formate,
NMM, DCM, À25 °C, 1 h; (b) 2-amino aza-benzene carboxylic acid,
À25 °C, 12 h; (c) anilines, DCM, À25 °C, 2 h; (d) isobutylchloro-
formate, DCM, À25 to À15 °C, 6 h, 30% four steps; (e) TFA, DCM,
rt, 2 h, >90%; (f) aldehydes, Na(OAc)3BH, ClCH2CH2Cl, rt, 2 h,
ꢀ80%; (g) acetic acids, EDC, HOBt, DMF, rt, 1 h, 50–90%.
1(R-enantiomer)
1(S-enantiomer)
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. 16 for assay protocol.
with the appropriate carboxylic acids afforded com-
pounds 1, 7–9, 14–45. Tertiary amines 10 and 11 were
obtained by a second reductive amination of 3 with
the respective aldehydes. Formation of sulfonamides
12 and 13 was achieved by treatment of 3 with the sul-
fonyl chlorides.
significant difference in activty between the enantiomers
of 1. Enantioselective synthesis established that the R
stereoisomer is the more active of the two enantiomers.
Our initial effort focused on identifying replacements for
the long alkyl chain and the dimethylamine groups of 1
which were identified as the major metabolic sites in
stability studies with microsomes. These groups were
presumed to be responsible in part for the high clearance
of the compound in vivo.
In addition, a series of aza-quinazolinones were synthe-
sized according to Scheme 2. The cyclization method
from Scheme 1 resulted in significant racemization in
the case of 2-amino aza-benzene carboxylic acid. Thus,
the oxazinones 4 were obtained by addition of the
appropriate 2-amino aza-benzene carboxylic acid to a
solution of the amino acid pretreated with isobutylchlo-
roformate. Ring opening by anilines provided bisamides
5, which upon treatment with isobutylchloroformate fol-
lowed by deprotection with TFA afforded the amine 6.
Amine 6 was converted to compounds 46–50 using
transformations described in Scheme 1.
When evaluating replacements to the long alkyl-amide
moiety, it was found that the amide carbonyl was impor-
tant for activity (Table 2). Replacement of the amide by
an amine 10–11 or a sulfonamide 12–13 resulted in sig-
nificant loss of activity. However, it was discovered that
the long alkyl amide group could be effectively replaced
Table 2. Optimization of the amide moiety
The lead optimization was primarily guided by a 125I-
IP10 ligand replacement assay.16 Table 1 shows the
F
O
N
N
N
R1
R
N(Me)2
O
O
Compound
R
125I -IP10 IC50 (lM)
a,b
O
N
R
a, b
R
R'
OH
R'
1
7
–CO(CH2)8CH3
–CO(CH2)10CH3
–CO(CH2)6CH3
–CO(CH2)4CH3
–(CH2)8CH3
0.146
0.154
0.375
>10
+
HO
N
NHBOC
NH2
NHBOC
42%
8
2
9
R1
R1
10
11
12
13
14
15
16
17
18
19
0.710
0.79
O
N
O
–(CH2)7CH3
e
c, d
N
N
–S(O)2(CH2)9CH3
–S(O)2(CH2)7CH3
–COCH2Ph-4-Ph
–COPh-4-Ph
0.587
1.4
R
R
R'
R'
N
HN
R2
N
R2
0.075
>10
>10
R3
3
1, 7-45
–COCH2Ph
–COCH2Ph-4-CH3
–COCH2Ph-4-CF3
–COCH2Ph-4-OCF3
>10
0.088
0.156
Scheme 1. Reagents and conditions: (a) P(OPh)3, pyridine, 70 °C, 3 h;
(b) anilines, 55 °C, 1 h, ꢀ50% for two steps; (c) TFA, DCM, rt, 2 h,
>90%; (d) R2CHO, Na(OAc)3BH, ClCH2CH2Cl, rt, 2 h, 50–80%; (e)
for amides: acetic acid, EDC, HOBt, DMF, rt, 1 h, 50–90%; for
sulfonamides: sulfonyl chloride, pyridine, rt, 3 h, 60%; for amines:
aldehydes, Na(OAc)3BH, ClCH2CH2Cl, rt, 2 h, ꢀ80%.
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. 16 for assay protocol.

M. Johnson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3339–3343
Table 4. Exploration of 4-phenyl substitutions
3341
Table 3. Exploration of N-substituents
F
R
O
N
N
O
5
8
4
3N
6
7
2
1
N
N
N
N
R
O
O
F3C
F3C
a,b
125I -IP10 IC50 (lM)
Compound
R
125I –IP10
Compound
R
a,b
IC50 (lM)
29
30
31
32
33
34
35
36
37
38
39
40
–H
–F
0.299
0.012
0.022
0.025
0.014
0.006
0.004
0.007
0.011
10
18
20
21
22
23
24
25
26
27
28
–(CH2)2N(CH3)2
–(CH2)2OMe
–(CH2)2OEt
0.088
0.30
0.04
–Cl
–CH3
–OCH3
–(CH2)2CH3
>10
–CH2-2-thiazolyl
–CH2-2-imidazoyl
–CH2-4-imidazoyl
0.10
0.23
–OCH2CH3
–C„CCH3
–NO2
0.65
0.24
–CH2-4-(1-methyl-imidazoyl)
–CH2-2-pyridyl
–CH2-3-pyridyl
–CN
0.073
0.013
–SO2Me
–COOH
–NHCOMe
1.37
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. 16 for assay protocol.
25
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. 16 for assay protocol.
by a biphenyl-acetyl moiety (14). It was also discovered
that the orientation of the biphenyl group played an
important role in maintaining CXCR3 antagonistic
activity. Removal of the methylene group from the
biphenyl-acetamide moiety resulted in complete loss of
activity (15). In addition, the importance of the terminal
phenyl group was established with phenyl acetamide 16,
which exhibited significantly lower activity. The series of
para-substituted phenyl acetamides 17–19 established
that CF3 and OCF3 groups, but not CH3 (17), could
serve as a replacement for the terminal phenyl ring.
Table 5. Exploration of the stereocenter
OEt
O
N
R2
N
R1
N
OEt
O
Compound
R1
R2
125I -IP10 IC50 (lM)
a,b
Evaluation of the dimethylamine moiety demonstrated
that while this group is important for activity, it could
be substituted by other polar groups (Table 3). Substitu-
tion of the dimethylamine group by a methyl group, as
in 22, resulted in significant loss of activity. However,
the dimethylamine moiety could be effectively replaced
by alkoxy (20–21) or a heterocycle (23–28).
41
42
43
44
45
H
H
2.3
H
Me
Me
Et
0.075
>10
0.009
4
Me
H
H
Ph
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. 16 for assay protocol.
Substitutions on the 3-N-phenyl were also explored
(Table 4). Alkoxy and propargyl groups (33–35) were
preferred for activity, however, the propargyl analogs
exhibited significant CYP 3A4 inhibition. Fluoro,
chloro, and cyano were also well tolerated (30, 31, 37),
while inclusion of groups such as carboxylic acid,
sulfone or amide resulted in dramatic reduction in
potency (38–40).
In order to increase polarity we explored the effect that
introducing nitrogen atoms in the central quinazolinone
core have in potency (Table 6). It was discovered that
quinazolinone 46 and 8-azaquinazolinone (47, AMG
487) derivatives were similar in activity, while the 5-,
7-azaquinazolinone and 5,8-diazaquinazolinone deriva-
tives (48–50) were significantly less potent.
Investigation of the 2-alkylamino group is illustrated in
Table 5 with the N-ethoxyethyl-biphenylacetyl scaffold.
Efforts to remove the stereo center by replacing the
methyl with hydrogen (41) or adding an additional
methyl group to the stereocenter (43) resulted in signif-
icant loss of CXCR3 activity (Table 5). The methyl
group attached to the stereocenter can be replaced by
an ethyl group (44) while maintaining potency. How-
ever, replacement of the methyl group by phenyl (45)
resulted in decreased activity.
Further evaluation of AMG 487 (47) demonstrated that,
in addition of inhibiting 125I-IP-10 binding, AMG 487
also inhibits binding of 125I-ITAC to CXCR3 with an
IC50 value of 8.2 nM. Evaluation of AMG 487 in
in vitro functional assays demonstrated that it inhibits
CXCR3-mediated cell migration by the three CXCR3
chemokines (IP-10 IC50 = 8 nM, ITAC IC50 = 15 nM,
and MIG IC50 = 36 nM). Furthermore, AMG 487

3342
M. Johnson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3339–3343
Table 6. Replacement of the quinazolinone core
Cellular Infiltration (BAL)
OEt
3
2
1
0
O
N
c
N
b
n.s.
Vehicle
a
N
N
AMG 487
*
*
O
F3CO
CXCR3 KO
125I -IP10 IC50 (lM)
a,b
*
Compound
a
b
c
46
47
48
49
50
C
N
C
C
N
C
C
N
C
C
C
C
C
N
N
0.006
0.008
0.144
1.40
*
*
*
0.480
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. 16 for assay protocol.
V
0. 03
0. 1
0. 3
1. 0
3. 0
10
KO
AMG 487 SC, BID (mg/kg)
Table 7. Pharmacokinetic profile of AMG 487 (47)
Figure 1. Evaluation of AMG 487 in a mouse bleomycin model of
cellular recruitment. *p < 0.005 as determined by Student’s t tests.
Parameter
Rat
Dog
Cyno
CL (L/h/kg)a
t1/2 (h)a
Fpo (%)b
1.6
9.3
1.1
0.4
85
0.12
7.6
12–57
19
moiety and the dimethylaminoethyl moieties of the lead
compound, led to the discovery of 47 (AMG 487) which
exhibited good potency in binding and functional assays
and good in vivo pharmacokinetic properties across spe-
cies. This compound was shown to inhibit in vitro cell
migration mediated by IP-10, Mig, and ITAC. Further-
more, this compound is a potent inhibitor of cellular
recruitment in vivo using a bleomycine-mouse model.
a Following iv dosing in rat at 0.5 mg/kg and dog and cyno at 1 mg/kg.
b Following po dosing in rat at 2 mg/kg, dog at 2.5 and cyno at 5 mg/kg.
inhibits calcium mobilization in response to ITAC
(IC50 = 5 nM).
The pharmacokinetic parameters of AMG 487 were
markedly improved relative to (1). AMG 487 showed
moderate to low clearance after iv dosing in rat, dog
or cyno and was well absorbed after oral administration
in rat and dog (Table 7).
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