Optimization of a series of quinazolinone-derived antagonists of CXCR3

Article abstract of DOI:10.1016/j.bmcl.2009.07.032

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoi

Full text of DOI:10.1016/j.bmcl.2009.07.032

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5114–5118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of a series of quinazolinone-derived antagonists of CXCR3
*
Jiwen Liu , Zice Fu, An-Rong Li, Michael Johnson, Liusheng Zhu, Andrew Marcus, Jay Danao,
Tim Sullivan, George Tonn, Tassie Collins, Julio 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:
The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of
an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series
that led to the discovery of compounds devoid of the formation of the active metabolite that was seen
with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic
properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruit-
ment induced by bleomycin.
Received 16 May 2009
Revised 24 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Keywords:
CXCR3
Ó 2009 Elsevier Ltd. All rights reserved.
IP10
ITAC
MIG
CXCL9
CXCL10
CXCL11
Chemokine receptor
GPCR
Antagonist
Quinazolinone
SAR
Chemokine receptors and their ligands play an important role in
mediating cell trafficking. CXCR3 is a chemokine receptor primarily
expressed on activated CD4+ T cells with the Th1 phenotype.1,2 The
ligands for CXCR3, Mig (CXCL9), IP10 (CXCL9), 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.3–15
Furthermore, recent reports point to potential roles for CXCR3
and its ligands in viral infection16 and tumor metastasis.17 There-
fore, several groups18–25 including ours have been interested in
discovering small molecules inhibitors of CXCR3.
of compounds with improved CXCR3 potency relative to AMG
487 and devoid of any major active metabolite formation.
The compounds reported in this Letter were synthesized follow-
ing our previously reported route18 (Scheme 1). The oxazinones 1
were obtained by addition of the appropriate 2-amino benzene
Table 1
OEt
N
OEt
N
O
N
O
N
N
O
N
O
We previously reported18 the optimization efforts that led to
the discovery of the CXCR3 antagonist AMG 487 from a series of
quinazolinone derivatives identified by high-throughput screen-
ing. The evaluation of AMG 487 in the clinic was complicated by
the discovery of significant circulating levels of a pyridine-N-oxide
active metabolite26 (Table 1). This active metabolite is approxi-
mately four fold more potent than AMG 487 in the in vitro cell
migration assay in response to ITAC in the presence of human plas-
ma. In this Letter, we report our efforts towards the identification
N
N
N
N
O
F3CO
F3CO
AMG 487
AMG 487 N-oxide
Compound
AMG 487
125I -IP10 binding in
ITAC migration in
a,b
a,c
buffer IC50
(lM)
plasma IC50
(lM)
0.005
0.004
0.24
0.058
AMG 487 N-Oxide
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. 28 for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
* Corresponding author.
E-mail address: jiwenl@amgen.com (J. Liu).
Ref. 28 for assay protocol.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.032

J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5114–5118
5115
R1
Br
OH
a, b
O
O
N
O
N
N
z
x
F3C
F3C
O
z
x
N
O
H
c
a, b
43
44
d
y
HO
y
NH
NHBOC
NHBOC
Scheme 5. Reagents and conditions: (a) 2-t-butoxy-2-oxoethyl zinc chloride,
Pd(PPh3)4, THF, 120 °C, microwave 5 min, 40%; (b) TFA, DCM, rt, 2 h, >90%.
NHBOC
O
1
2
R1
R1
g
R1
e, f
O
O
O
N
N
3,3,3-trifluoro-1-iodopropane at 100 °C under basic conditions
and subsequent ester hydrolysis using lithium hydroxide.
Benzimidazole acetic acid 40 was synthesized in three steps
(Scheme 3). Diaminobenzene 38 was condensed with malonic acid
mono ester to afford the amide, which was cyclized in acetic acid
to give benzimidazole ester 39. Hydrolysis of the ester afforded
acid 40.
Imidazole acetic acid 42 was prepared via alkylation of 4-triflu-
oromethylimidazole (41) with t-butyl bromoacetate, followed by
conversion of the t-butyl ester to the acid using TFA (Scheme 4).
Pyridyl acetic acid 44 was synthesized via palladium catalyzed
coupling27 of the Reformatsky reagent with 5-trifluoromethyl-2-
bromopyridine 43 and subsequent ester hydrolysis (Scheme 5).
The lead optimization was primarily guided by 125I-IP10 ligand
displacement assay in buffer28 and ITAC migration assay in plas-
ma.28 IC50 of a compound in the 125I-IP10 ligand displacement
assay reflects the compound’s intrinsic binding affinity to the
receptor, while IC50 of a compound in the ITAC migration assay
in plasma reveals the compound’s antagonistic activity and its
non-specific plasma protein binding affinity.
The quinazolinone core was used for initial SAR studies, instead
of the 8-azaquinazolinone core as in AMG 487, because quinazoli-
none derivatives and their corresponding 8-azaquinazolinone
compounds are equally potent18 and using readily accessible
quinazolinone derivatives could speed up optimization. Modifica-
tions of the acetamide moiety are shown in Table 2 and 3. As pre-
viously reported,18 the trifluoromethyl group of the acetamide
moiety in compound 5 and AMG 487 is a desirable substituent
since it significantly improves potencies and microsomal stability.
z
z
x
z
x
N
O
N
y
y
y
x
N
N
N
R3
R2
HN
NHBOC
R2
3
4
x, y, z = CH or N
5-35
Scheme 1. Reagents and conditions: (a) isobutylchloroformate, NMM, DCM,
À25 °C, 1 h; (b) 2-amino benzene or pyridine carboxylic acids, À25 °C, 12 h; (c)
anilines, DCM, À25 °C, 2 h; (d) isobutylchloroformate, DCM, À25 to À15 °C, 6 h,
ꢀ30% four steps; (e) TFA, DCM, rt, 2 h, >90%; (f) for compounds 5–32, aldehydes or
ketones, Na(OAc)3BH, ClCH2CH2Cl, rt, 2 h, ꢀ80%; For compounds 33–35, ethylsulf-
onylethene, TEA, MeOH, reflux, 90%; (g) acetic acids, EDC, HOBt, DMF, rt, 1 h, 50–
90%.
or pyridine carboxylic acids to a solution of the Boc-protected
amino acids pretreated with isobutylchloroformate. Ring opening
with the appropriate anilines provided bisamides 2, which upon
treatment with isobutylchloroformate afforded the Boc-protected
amines 3.
After the removal of the Boc group with TFA, the resulting
primary amines were alkylated using one of two methods. For
the synthesis of compounds 5–32, the primary amines were alkyl-
ated using reductive amination with various aldehydes or ketones.
For compounds 33–35, Michael addition of ethylsulfonylethene to
the primary amines afforded the alkylated secondary amines 4. The
secondary amines 4 were finally acylated using various substituted
acetic acids to yield compounds 5–35.
The syntheses of the heterocyclic-acetic acids 37, 40, 42, and 44
used in the synthesis of compounds 6–9, respectively, are shown in
Scheme 2–5. Piperazine acetic acid 37 was prepared from
compound 36 in two steps (Scheme 2) via alkylation with
Table 2
Modification of phenyl acetamide phenyl ring
OEt
O
OEt
OH
a, b
N
N
N
N
O
O
HN
N
F3C
36
37
N
N
R
Scheme 2. Reagents and conditions: (a) 3,3,3-trifluoro-1-iodopropane, potassium
carbonate, DMF, 100 °C, 1.5 day, 80%; (b) LiOH, MeOH/THF/water, 25 °C, 2 h, 90%.
O
Compd
R
125I -IP10 binding in
ITAC migration in
a,b
a,c
buffer IC50
(l
M)
plasma IC50
(lM)
NH2
NH2
OEt
N
a, b
F3C
5
6
7
0.006
0.35
NH
39
OH
O
F3C
F3C
N
F3C
F3C
38
N
0.69
0.21
ND
ND
N
c
NH
O
N
40
NH
F3C
Scheme 3. Reagents and conditions: (a) malonic acid mono ethyl ester, EDC, DCM,
25 °C, overnight; (b) HOAc, 70 °C, 12 h, 50% for two steps; (c) LiOH, MeOH/THF/
water, 25 °C, 2 h, 90%.
N
F3C
8
0.007
0.013
0.45
0.50
N
9
N
F3C
OH
a, b
NH
N
F3C
F3C
a
O
Values are means of three experiments, standard deviation is 30%.
N
N
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
b
41
42
See Ref. 28 for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Scheme 4. Reagents and conditions: (a) t-butyl bromoacetate, potassium carbon-
ate, DMF, 25 °C, 12 h, 80%; (b) TFA, DCM, rt, 2 h, >90%.
Ref. 28 for assay protocol.

5116
J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5114–5118
Table 3
Table 4
Substitutions of phenyl acetamide phenyl ring
Quinazolinone and azaquinazolinone cores
OEt
OEt
O
N
N
O
z
x
N
y
N
n
F3C
F
N
N
N
N
R
O
O
Compd
n
R
125I -IP10 binding
ITAC migration in
Compd
x
y
z
125I -IP10 binding
ITAC migration
in plasma IC50
a,b
a,c
a,b
a,c
in buffer IC50
(l
M)
plasma IC50
(lM)
in buffer IC50
(l
M)
(lM)
5
10
11
12
13
14
15
16
17
18
19
20
1
2
1
1
1
1
1
1
1
1
1
1
4-CF3
4-CF3
3-CF3
4-OCF3
4-CN
0.006
2.09
0.011
0.007
0.021
0.77
0.35
ND
ND
0.14
ND
ND
ND
ND
0.017
0.22
0.075
0.020
20
21
22
23
24
CH
N
CH
CH
N
CH
CH
N
CH
CH
CH
CH
CH
N
0.003
0.005
0.032
0.11
0.020
0.023
ND
ND
ND
N
0.042
4-SO2Me
3,4-F
a
Values are means of three experiments, standard deviation is 30%.
0.04
b
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
3,4,5-F
3,5-CF3
3-F-4-OCF3
3-F-4-CF3
4-F-3-CF3
0.008
0.007
0.009
0.002
0.003
See Ref. 28 for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Ref. 28 for assay protocol.
a
Values are means of three experiments, standard deviation is 30%.
azaquinazolinones (22–24) are much less potent, with the
5-azaquinazolinone (23) being the least potent. In order to increase
polarity, we chose to use the 8-azaquinazolinone core for further
optimization.
b
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
See Ref. 28 for assay protocol.
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Ref. 28 for assay protocol.
c
The amide N-alkyl moiety was studied with 8-azaquinazolinone
core and 3-trifluoromethyl-4-fluorophenylacetamide moiety in the
molecules (Table 5). As previously reported, this area tolerated
many changes.18 Alkoxy ethyl, amino ethyl and various heterocy-
clic methyl all produced compounds with good potencies.
3-Pyridyl methyl group as in AMG 487 was the optimal moiety
discovered in the early optimization efforts. Table 5 shows other
potent modifications discovered later and a couple modifications
(26 and 30) provided compounds that are more potent than the
compound (21) with 3-pyridyl methyl. This data, in combination
with the previously reported data, suggest that polar groups are
preferred in the area, but the position of the heteroatoms is not
critical and many types of polar groups are tolerated in this region.
We eventually decided to use the ethylsulfone-ethyl moiety (33),
because of the improvement in potency and metabolic stability.
In addition, compounds with the ethylsulfone moiety avoid major
active metabolite formation as demonstrated by our in vitro
metabolism studies.
Substitutions of the 3-N-phenyl ring on the quinazoline core
were studied extensively in the early lead optimization.18 It was
discovered that small substituents such as ethoxy or halogen
atoms at the para position afforded compounds with good poten-
cies. Deethylation of the ethoxy group in compound 33 was one
of the metabolic routes observed in vitro, so in order to decrease
this metabolism, 3,3,3-trifluoroethoxy and cyano were introduced
to the para position of the 3-N-phenyl moiety (Table 6). The com-
pound with the 3,3,3-trifluoroethoxy group (34) is as potent as the
ethoxy analog (33). However, the cyano derivative (35) is not as
potent as the ethoxy derivative (33), even though in the early opti-
mization the cyano and ethoxy analogs have similar potencies.18
Apparently, with changes in the core, the acetamide, and N-alkyl
moieties, the cyano group is not as desirable as ethoxy group for
potency.
Therefore, when we modified the acetamide moiety, we always
tried to keep trifluoromethyl group in the region. In Table 2, we
examined the replacement of the phenylacetamide phenyl group
of compound 5 with various heterocycles. The imidazole (8) and
pyridine (9) were similar in potency to the phenyl derivative (5),
while other replacements yielded loss of potency. Overall, the
examined heterocyclic replacements did not provide any improve-
ment in potency and other modifications in the area provided
significant improvement (see below), so the heterocyclic replace-
ments were not adopted for further studies.
The substitutions of the phenylacetamide phenyl ring and the
linker between the carbonyl and the phenyl ring were also
explored (Table 3). The methylene between the carbonyl and the
phenyl seems to be optimal, because the ethylene linker (10)
significantly decreases the potency and removal of the methylene
also results in dramatic loss in potency.18 In order to investigate
whether the improvement in potency observed with the trifluoro-
methyl group (5) is due to its electron-withdrawing ability, the
cyano (13) and methyl sulfone (14) derivatives were evaluated.
Compounds 13 and 14 yielded decreases in potency. Therefore,
the data suggests that the electron-withdrawing ability of trifluo-
romethyl group cannot fully account for the improvement in
potency afforded by the trifluoromethyl group. It is known that
fluorine can have several kinds of interactions with proteins, such
as hydrophobic, stacking, edge-on, and multipolar interactions.29,30
Therefore, it was hypothesized that fluorine atoms in the phenyl-
acetamide area may have favorable interactions with the receptor
and additional fluorine atoms in the region may provide additional
improvement on potency. It was rewarding to see that analogs
with additional fluorine indeed improved potency in several cases,
such as in compounds 16 versus 15, 19 versus 5, and 20 versus 11.
The 3-trifluoromethyl-4-fluorophenyl acetamide moiety in com-
pound 20 is one of the best in the area and it was used for further
SAR studies.
Compound 34 was chosen for further evaluation. It was found
that it is a potent antagonist of cell migrations mediated by CXCR3
in response to IP10, ITAC or MIG (Table 7).
After optimization of the phenylacetamide moiety, the correla-
tion between the quinazolinone core and other azaquinazolinone
cores was re-examined. It was encouraging to observe that, as pre-
viously noted,18 quinazolinone 20 and the corresponding 8-azaqui-
nazolinone compound 21 are equally potent (Table 4). Other
The ability of compound 34 to inhibit the CXCR3 receptor across
several species was demonstrated using several 125I-IP10 displace-
ment assays (Table 7). The rat, mouse, dog, and rhesus monkey
receptors were evaluated and 34 displayed similar activity in these
species as in the human.

J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5114–5118
5117
Table 5
Table 7
Modification of the amide N-alkyl moiety
Activities of compound 34 in human and other species
OEt
CF3
O
O
O
N
O
N
O
N
N
N
N
F3C
F
N
F3C
F
N
R
SO2Et
34
Compd
R
125I -IP10 binding
in buffer IC50
ITAC migration
in plasma IC50
a,b
a,c
(
lM)
(lM)
Assay (IC50
,
lM)
In buffer
In plasma
Human 125I -IP10 bindinga,b
Human 125I -ITAC bindinga,b
Human IP10 migrationa,c
Human ITAC migrationa,c
Human MIG migrationa,c
Rat 125I -IP10 bindinga,b
Mouse 125I -IP10 bindinga,b
Dog 125I -IP10 bindinga,b
Rhesus 125I -IP10 bindinga,b
0.001
0.001
0.0004
0.0008
0.0006
0.004
0.003
0.005
0.002
0.006
ND
ND
0.015
ND
ND
ND
ND
ND
21
25
0.005
0.023
N
N
O
0.002
0.002
0.059
0.010
26
27
N
N
a
0.005
0.080
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. 28 for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
28
29
0.002
0.002
0.020
0.020
N
H
Ref. 28 for assay protocol.
NMe
Table 8
Pharmacokinetic profile of compound 34
30
0.001
0.006
0.005
0.131
N
N
O
Parameter
Rat
Mouse
Dog
Cyno
CL (L/h/kg)a
MRT (h)a
3.9
0.65
2.5
26
0.78
1.1
0.8
84
0.45
2.5
1.1
25
0.87
1.6
1.4
14
Vdss (L/kg)a
Fpo (%)b
Me
31
32
O
a
Following iv dosing in rat and mouse at 0.5 mg/kg and dog and cyno at 1 mg/kg.
Following po dosing in rat and mouse at 2 mg/kg, dog at 2.5 and cyno at 5 mg/
b
0.005
0.001
0.162
0.022
kg.
33
SO2Et
a
Values are means of three experiments, standard deviation is 30%.
inhibition toward CYP 1A2, 3A4, 2C9, and 2D6 and it is selective
against other chemokine receptors in MDS receptor screen.
The in vivo efficacy of 34 was studied in a mouse model of
cellular recruitment induced by bleomycin. In this mouse model
study, bleomycin was introduced in the lungs of mice via intra-tra-
cheal instillation after tracheostomy.31 Compound 34 and AMG
b
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
See Ref. 28 for assay protocol.
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Ref. 28 for assay protocol.
c
Table 6
Substitutions of 3-N-phenyl ring
R
Cellular Infiltration (BAL)
O
3.5E+05
N
3.0E+05
n.s.
N
N
F3C
F
N
2.5E+05
SO2Et
*
*
O
*
2.0E+05
1.5E+05
1.0E+05
5.0E+04
0.0E+00
*
*
Compd
R
125I -IP10 binding
in buffer IC50
ITAC migration
in plasma IC50
*
a,b
a,c
*
(l
M)
(lM)
33
34
35
–OEt
–OCH2CF3
–CN
0.001
0.001
0.011
0.022
0.015
0.115
a
Values are means of three experiments, standard deviation is 30%.
Displacement of 125I-labeled IP10 from the CXCR3 receptor expressed on PBMC.
b
Veh.
AMG 487
Compound 34
See Ref. 28 for assay protocol.
c
ITAC mediated migration of PBMC in the presence of 100% human plasma. See
Dose (mg/mL)
Blood levels (ng/mL)
10
104
3
26
1
19
10
35
3
18
1
11
0.3 0.1
6
Ref. 28 for assay protocol.
2
AMG 487 N-oxide
The pharmacokinetic properties of 34 were evaluated across
several species (Table 8). In mouse, dog, and cyno, the compound
has low to moderate clearance and decent to good oral bio-
availability. This compound also demonstrated low to moderate
Blood levels (ng/mL)
(70) (19) (14)
Figure 1. Evaluation of AMG 487 and compound 34 in a mouse bleomycin model of
cellular recruitment. p <0.01 as determined by Student’s t-tests.
*

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J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5114–5118
15. Rosenblum, J. M.; Zhang, Q. W.; Siu, G.; Collins, T.; Sullivan, T.; Dairaghi, D.;
Medina, J.; Fairchild, R. L. Transplantation 2009, 87, 360.
487 were each dosed subcutaneously via Alzet osmotic mini-
pump32 for six days after the bleomycin challenge. Six days after
bleomycin challenge, a bronchoalveolar lavage (BAL) was per-
formed and the number of cells collected in the BAL were counted
using a hemocytometer. As shown in Figure 1, compound 34 was
able to achieve the same degree of inhibition of cell infiltration
as AMG 487 with lower blood levels. If the blood level of the
N-oxide of AMG 487 is also taken into consideration, the potency
of compound 34 is even more impressive. AMG 487 N-oxide is
more potent than AMG 487 (Table 1). At a blood concentration of
35 ng/mL, compound 34 provided the same inhibition of cell infil-
tration as AMG 487 with a blood concentration of 104 ng/mL and
its N-oxide at a concentration of 70 ng/mL.
In summary, further optimization of the quinazolinone-derived
CXCR3 antagonists led to the discovery of compounds that avoid
the formation of the pyridine-N-oxide active metabolite seen with
AMG 487 in the clinic. Compound 34 was selected for extensive
evaluation. This compound displays increased potency in vitro
and in vivo compared to AMG 487 and possesses good pharmaco-
kinetic profile across several species. This compound represents a
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32. Alzet osmotic mini-pump with
subcutaneously. The infusion rate was 1
visit http://www.alzet.com/downloads/TIM.pdf.
a
volume of 0.2 mL was implanted
lL/h. For more information, please