A novel series of N-(azetidin-3-yl)-2-(heteroarylamino)acetamide CCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.12.017

The inflammatory response associated with the activation of C-C chemokine receptor CCR2 via it's interaction with the monocyte chemoattractant protein-1 (MCP-1, CCL2) has been implicated in many disease states, including rheumatoid arthritis, multiple sclerosis, atherosclerosis, asthma and neuropathic pain. Small molecule antagonists of CCR2 have been efficacious in animal models of inflammatory disease, and have been advanced into clinical development. The necessity to attenuate hERG binding appears to be a common theme for many of the CCR2 antagonist scaffolds appearing in the literature, presumably due the basic hydrophobic motif present in all of these molecules. Following the discovery of a novel cyclohexyl azetidinylamide CCR2 antagonist scaffold, replacement of the amide bond with heterocyclic rings was explored as a strategy for reducing hERG binding and improving pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2012.12.017

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel series of N-(azetidin-3-yl)-2-(heteroarylamino)acetamide
CCR2 antagonists
⇑
Nalin L. Subasinghe , James Lanter, Thomas Markotan, Evan Opas, Sandra McKenney, Carl Crysler,
Cuifen Hou, John O’Neill, Dana Johnson, Zhihua Sui
Janssen Pharmaceutical Research & Development, L.L.C., Welsh & McKean Roads, Spring House, PA 19477, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The inflammatory response associated with the activation of C–C chemokine receptor CCR2 via it’s inter-
action with the monocyte chemoattractant protein-1 (MCP-1, CCL2) has been implicated in many disease
states, including rheumatoid arthritis, multiple sclerosis, atherosclerosis, asthma and neuropathic pain.
Small molecule antagonists of CCR2 have been efficacious in animal models of inflammatory disease,
and have been advanced into clinical development. The necessity to attenuate hERG binding appears
to be a common theme for many of the CCR2 antagonist scaffolds appearing in the literature, presumably
due the basic hydrophobic motif present in all of these molecules. Following the discovery of a novel
cyclohexyl azetidinylamide CCR2 antagonist scaffold, replacement of the amide bond with heterocyclic
rings was explored as a strategy for reducing hERG binding and improving pharmacokinetic properties.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 29 August 2012
Revised 26 November 2012
Accepted 10 December 2012
Available online xxxx
Keywords:
CCR2 inhibitor
Monocyte chemoattractant protein-1
MCP-1
CCL2
CCR2
Chemokine antagonist
CCR2 antagonist
GPCR
Activation of the C–C chemokine receptor CCR2 via its interac-
tion with the monocyte chemoattractant protein-1 (MCP-1, CCL2)
leads to monocyte/macrophage recruitment and the resulting
inflammatory response. Studies using rodent models have impli-
cated the inflammatory response associated with MCP-1/CCR2
interaction to many disease states, including rheumatoid arthritis,
multiple sclerosis, atherosclerosis, asthma and neuropathic pain.1–
10 Mouse models have also demonstrated that adipose tissue mac-
rophage accumulation via MCP-1 interaction with CCR2 leads to
the development of insulin resistance related with obesity.
Small-molecule CCR2 antagonist strategies to ameliorate inflam-
mation-mediated disease has been extensively pursued over the
last decade and several compounds have been advanced into clin-
ical development.11 Our CCR2 antagonist program has explored
several chemotypes over the last few years. Following our discov-
ery of the (1-cyclohexylazetidin-3-yl)-N-acylglycineamide CCR2
antagonist scaffold 1,12 an ongoing effort has been to identify het-
erocyclic replacements for the N-acylglycine moiety with the
intention of improving the in vivo pharmacokinetic profile. Contin-
uing this amide bond replacement strategy, compound 2 was iden-
tified as a potent CCR2 antagonist, but with significant affinity at
the hERG channel. Here we describe some of our SAR studies
geared towards reducing hERG binding while attempting to
maintain a favorable pharmacokinetic profile.
F
F
F
F
F
F
N
N
N
N
N
N
N
O
O
N
O
2
1
Initially, the cyclohexyl portion of compound 2 was modified
while maintaining the rest of the molecule constant. These
compounds (Table 1) were synthesized according to Scheme 1.
The commercially available amine 19 was coupled to Cbz-glycine
and subjected to hydrogenolysis to give the amine 20. 6-(Trifluoro-
methyl)-quinazolin-4-ol13 was converted to chloroquinazoline 21,
which was coupled to amine 20 to give compound 27.
The BOC-group was removed and the resulting amines, 28 were
subjected to reductive amination conditions in the presence of var-
ious ketones,12 29 to give compounds 2–18. The cis and trans iso-
mers were separated by silica column chromatography (8 N NH3/
⇑
Corresponding author. Tel.: +1 215 628 7824; fax: +1 215 540 4612.
E-mail address: NSUBASIN@its.jnj.com (N.L. Subasinghe).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.017
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

2
N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 1
Arylcyclohexyl modifications
were synthesized according to Scheme 1, using heteroaryl chlo-
rides, 22–25.17
F
The cyanoquinoline analog 34 was also prepared according to
Scheme 1; however instead of displacing a chloride to form the
glycine to quinoline bond, the hydroxyl residue on quinoline 26
was activated with bromo-tris-pyrrolidinophosphonium-hexa-
F
F
W
R
N
N
N
N
fluoro-phosphate (PyBrOP) to form
a phosphonium leaving
O
N
group.18 Chloro quinazolines 22 and 23 were prepared by treating
the corresponding quinazolinone with oxalyl chloride and catalytic
DMF. Phthalazine, 24 was synthesized according to Scheme 2. Lac-
tone 39 was nitrated and reduced to give the aniline 40, which was
diazotized in the presence of KI to give the iodide 41. Copper cat-
alyzed trifluoromethylation of 41 gave compound 42. Compound
42 was brominated and heated in the presence of hydrazine to give
the phthalazine 44, which was converted to the chloride 24.
Dihydronaphthyridine 45 (Scheme 3) was oxidized to the lac-
tam, 46. DDQ oxidation gave 1,6-naphthyridinone 48, which was
converted to the chloro naphthyridine 25. Cyanoquinoline 26
was prepared according to Scheme 4. Commercially available 4-hy-
droxyl quinoline ester 49 was protected as the p-methoxybenzyl
ether and converted to the amide 51.
Compound
R
W
CCR2
binding
CTX
hERG
a,b
IC50
binding
a
c
IC50 (nM)
(nM)
IC50
(lM)
2
3
H
15
4
5.2
OH 15
OH 10
OH 22
OH 40
18
16.9
S
4
5
6
8
>50
28.4
>50
N
S
N
24
18
N
S
S
S
Dehydration of 51 with trifluoroacetic anhydride provided the
nitrile 52, which was deprotected to give 4-hydroxy cyanoquino-
line 26. Compound 33 was prepared according to Scheme 5. Reduc-
tive amination of ketone 53 with Boc-aminoazetidine, followed by
deprotection gave amine 55, which was coupled to Boc-gly to give
compound 56. Compound 56 was deprotected and coupled to 4-
hydroxyquinoline, 58 to give compound 33.
Compound 35 was synthesized according to Scheme 6. 4-Ami-
noquinoline 59 was CBz-protected and the carbamide was alkyl-
ated with bromoacetic acid to give compound 61. The ester was
hydrolyzed and the acid was coupled to 1-Boc-amino azetidine
to give the amide 62. Both protecting groups were removed and
the resulting azetidine amine was coupled to the ketone 53 give
compound 35. Chloroisoquinoline20 64 (Scheme 7) was converted
to the iodide 56 by halogen metal exchange followed by treatment
with iodine. Copper catalyzed trifluoromethylation gave
compound 66, which was converted to the chloroisoquinoline 67.
Initially an unsuccessful attempt was made to couple chloroiso-
quinoline 67 to amine 20 in a manner similar to that in Scheme 1,
which led us to the rather lengthy route to compound 36 described
here.
N
7
8
9
OH 27
OH 22
OH 19
11
6
>50
N
37.5
28.5
O
4
N
10
11
OH 56
65
13.4
46
N
O
N
H
H
36
7
NTd
H2N
O
N
12
2
20.2
N
13
14
H
H
5
7
7
6
16.0
12.9
OH
Chloroisoquinoline 67 was converted to the phenoxy derivative
by heating in molten phenol in the presence of KOH. The phenox-
ide 68 was heated in molten ammonium acetate to give the amino-
isoquinoline 69. The amine was protected as its Cbz derivative and
alkylated with bromoacetic acid methyl ester. Methyl ester was
hydrolyzed and the acid was coupled to amine 55. Deprotection
via catalytic hydrogenation gave compound 36.
N
N
15
H
17
5
8.3
H2N
16
17
H
H
5
7
2
2
7.5
6.6
N
Our previous work12 had suggested that introducing a hydroxyl
residue on the cyclohexane ring as in compound 3 attenuated
hERG binding in cyclohexylazetidinyl CCR2 inhibitors. While com-
pound 3 did have reduced hERG binding, increasing hydrophilicity
by replacing the phenyl ring with various thiazoles (4, 6, 7) and iso-
thiazole 5 resulted in a much more significant reduction in hERG
binding while maintaining good CCR2 potency. Increasing the
hydrophobicity of the thiazole ring (8), results in an increase in
hERG binding. However, comparing compounds 6, 7 and 8 lipo-
philic substitutions at the 2-possition of the thiazole ring does
seem to improve CCR2 potency. While 2-methoxypyridin-5-yl sub-
stitution (9) also reduced hERG binding significantly, 2-methoxy-
pyridin-6-yl substitution (10) was comparable to phenyl
substitution. Although thiazole carbinol analogs (4, 6, 7) provided
the necessary reduction in hERG binding while maintaining good
potency their pharmacokinetic properties were not optimal. Based
on our observation that des-hydroxy analogs in this series had
HO
NC
18
H
21
43
2.1
a
IC50 values are reported as the average of at least two separate determinations if
IC50 <100 nM with a typical variation of less than 25%. MCP-1 receptor binding
assay in THP-1 cells (Ref. 14).
b
MCP-1 induced chemotaxis in THP-1 cells (Ref. 15).
hERG 3H-astemizole binding activity on HEK-293 cell (Ref. 16).
c
d
NT = not tested.
MeOH in EtOAc). The trans isomers have marginal potency towards
CCR2.
Various heterocyclic replacements for the quinazoline ring
(Table 2) were also synthesized. Compounds 31, 32, 37 and 38
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
N
O
N
O
NH2
BocN
NH2
N
N
21, 24
22, 23
25
26
: c;
: d; : e; : f
a, b
CF3
Z1
Z2
N
R2
Boc
Boc
R1
Z3 Z4
Z1
Z2 R1
Z3
20
27
19
F3C
Z4
21
: R1 = H, R2 = Cl, Z1 = N, Z2 = C, Z3 = N, Z4 = C
22: R1 = CH3, R2 = Cl, Z1 = N, Z2 = C, Z3 = N, Z4 = C
23: R1 = CF3, R2 = Cl, Z1 = N, Z2 = C, Z3 = N, Z4 = C
24
: R2 = Cl, Z1 = N, Z2 = N, Z3 = C, Z4 = C
25: R1 = H, R2 = Cl, Z1 = N, Z2 = C, Z3 = C, Z4 = N
26: R1 = CN, R2 = OH, Z1 = C, Z2 = C, Z3 = N, Z4 = C
N
O
N
O
N
HN
N
N
R4
g
CF3
CF3
h
Z1
Z2
Z1
Z2
R3
R3
R4
R1
Z3 Z4
R1
Z3
Z4
O
R3 = H, OH
30
28
29
Scheme 1. Reagents and conditions: (a) CbzGly-OH, DIEA, EDCI, HOBT; (b) Pd/C H2; (c) TEA, iPrOH, 90 °C, 1 h; (d) TEA, THF, reflux 18 h; (e) TEA, diglyme, 120 °C 18 h; (f)
PyBrOP, TEA; (g) 50% TFA, DCM; (h) Na(OAc)3BH, DCM.
Table 2
Heterocyclic amide bond replacements
O
N
N
Z
HO
N
S
N
hERG patchb % inh @ 3
lM (sol con.)
a
a,c
d
Compound
Z
CCR2 binding IC50 (nM)
CTX IC50 (nM)
hERG binding IC50
(l
M)
N
6
40
18
>50
10.8 2.1 (6.5 3.9)
N
CF3
N
N
CF3
31
32
33
8
39
50
7
25.7
32.7
36.1
NTe
N
F3C
F3C
N
N
N
17
64
NT
CF3
25(17)
CF3
N
34
35
23
72
25
59
35.1
11.2
9.6 1.9 (6.7 3.9)
CF3
N
NT
CF3
CF3
N
36
37
20
19
8
32.9
NT
NT
NT
12.4 2.4(7.5 3.7)
(continued on next page)
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

4
N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 2 (continued)
a
a,c
d
Compound
Z
CCR2 binding IC50 (nM)
CTX IC50 (nM)
hERG binding IC50
(l
M)
hERG patchb % inh @ 3
lM (sol con.)
N
N
CF3
CF3
N
38
38
NT
>50
8.5 1.7 (7.5 3.7)
N
a
IC50 values are reported as the average of at least two separate determinations if IC50 <100 nM with a typical variation of less than 25%.
The membrane K+ current IKr in HERG-transfected HEK293 cells (solvent control), see Ref. 19.
MCP-1 induced chemotaxis in THP-1 cells (Ref. 15).
b
c
d
e
hERG 3H-astemizole binding activity on HEK-293 cell (Ref. 16).
NT = not tested.
O
O
O
O
I
CF3
NH2
c
a, b
d
O
O
O
O
MeO2C
F
SO2F
39
42
40
41
F
Cl
OH
O
CF3
CF3
CF3
N
N
N
N
f
g
e
O
Br
43
24
44
Scheme 2. Reagents and conditions: (a) H2SO4/KNO3; (b) Fe, AcOH; (c) NaNO2/HCl, KI; (d) CuI, DMF, 100 °C, 18 h; (e) NBS, AIBN, CCl4, 65 °C, 3 h; (f) NH2NH2, EtOH, 60 °C, 18 h;
(g) POCl3, 100 °C, 2 h.
O
O
CF3
CF3
CF3
BocN
BocN
HN
a
b
N
O
N
N
45
46
47
Cl
CF3
CF3
HN
N
d
c
N
N
25
48
Scheme 3. Reagents and conditions: (a) CCl4/CH3CN (10:1), NaIO4 aq, RuCl3.H2O, 12 h; (b) TFA, DCM; (c) DDQ, 100 °C, 6 h; (d) POCl3, CH3CN, 90 °C, 2 h.
OH
N
OPMB
OPMB
CF3
CF3
CF3
a
b
MeO2C
H2NOC
N
MeO2C
N
49
50
51
OPMB
OH
CF3
CF3
d
c
NC
N
NC
N
26
52
Scheme 4. Reagents and conditions: (a) Cs2CO3, p-MeO-PhCH2Cl; (b) NH3; (c) TFAA, TEA; (d) TFA, DCM.
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
O
NHR
HO
NHGlyBoc
N
N
c
a
HO
S
BocGly-OH
N
HN
S
NHBoc
S
N
HO
55
54
: R = H
b
N
56
53
: R = Boc
N
O
N
HO
S
S
N
d
N
N
b
N
NHGlyNH2
CF3
O
N
HO
CF3
33
57
F3C
F3C
N
58
Scheme 5. Reagents and conditions: (a) Na(OAc)3BH, DCM; (b) TFA, DCM; (c) EDCI, HOBT, TEA, DCM; (d) PyBrOP, DIEA, dioxane, rt, 3 days.
O
O
NHCbz
NH2
CF3
CF3
a
c,d
b
CbzN
CF3
N
N
N
BocN
60
59
61
N
Boc
N
N
O
N
N
O
N
N
O
N
N
S
CbzN
g
e, f
N
CF3
CF3
CF3
S
HO
O
N
N
N
OH
N
53
35
63
62
Scheme 6. Reagents and conditions: (a) CbzCl, NaHCO3; (b) Methyl bromoacetate, Cs2CO3, DMF, rt, 18 h; (c) LiOH, THF/MeOH, 3 h; (d) EDCI, HOBT, TEA, DCM, 18 h; (e) Pd/C
H2; (f) TFA, DCM; (g) Na(OAc)3BH, NMM, DCM.
F3C
I
F3C
Br
a
c
b
MeO2C
F
N
SO2F
Cl
Cl
e
N
MeO
N
N
Cl
d
66
65
67
F
64
F3C
N
Cbz
71
F3C
F3C
H2N
F3C
g
f
O
N
PhO
HN
Cbz
MeO
N
N
N
68
69
70
F3C
S
N
NH2
O
N
1.
F3C
h
HO
j
55
N
Cbz
72
i
HO
N
O
2.
S
HN
NH
N
N
N
HO
36
Scheme 7. Reagents and conditions: (a) nBuLi, I2; (b) CuI, DMF, 100 °C, 18 h; (c) POCl3, 150 °C, 18 h; (d) PhOH, KOH, 100 °C; (e) NH4OAc, 160 °C; (f) CbzCl, NMM; (g) Methyl
bromoacetate, NaH; (h) NaOH; (i) EDC, HOBT, TEA; (j) Pd/C H2.
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

6
N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 3
better PK properties than their hydroxyl counterparts we exam-
ined the possibility of identifying des-hydroxy analogs with re-
duced hERG binding by replacing the phenyl ring in compound 2
with more hydrophilic aryl residues. Out of the several pyridyl,
amino and hydroxyl-pyridyl analogs synthesized (11–16), only
the 3-aminopyridyl analog 11 showed significant reduction in
hERG binding. Hydrophilic phenyl substitutions such as 2-hydroxy,
(17) or 2-cyano (18) did not produce analogs with significantly re-
duced hERG binding.
Reduced hERG activity for compound 6 was confirmed using a
hERG patch clamp study. Minimal Inhibition of hERG-mediated
K+ current in HEK293 cells was observed at 3 lM (Table 2). Reduc-
ing hydrophilicity and basicity were considered as two potential
approaches to improve the poor oral bioavailability of compound
6. However, we were aware that increasing lipophilicity within this
series could result in greater hERG affinity.
In vivo rat pharmacokinetic parameters for compounds 6 and 34
Compound t1/2 AUClast (h ng/mL) Vss (L/kg) Cl (mL/min/kg) F (%)
6
34
1.8
1.8
10.6
6.0
8.5
13.0
89
148
0.3
0.5
IV: 2 mg/kg in 20% HPbCD; PO: 10 mg/kg in 0.5% Methocel.
Quinoline analog 35 has increased hERG binding compared to
the quinoline analogs 33–34, and quinazoline 6. Here again in-
creased basicity21 of the unsubstituted quinoline might be a con-
tributing factor. In contrast the less basic isoquinoline 36, has
significantly reduced hERG binding. Compared to quinazoline 6,
the more basic quinoline 35 has significantly reduced CCR2 binding
and functional activity, where as the less basic isoquinoline 36 has
improved binding and functional CCR2 potency.
Using compound 6 as a template with significantly reduced
hERG binding and good CCR2 potency we continued to explore var-
ious heterocyclic replacements for the quinazoline ring (Table 2).
These compounds had good CCR2 binding and functional activity.
Increased lipophilicity due to the 2-methyl substitution on the qui-
nazoline ring (31), while improving CCR2 potency, increased hERG
binding as well. Introducing an electron withdrawing trifluoro-
methyl group that is also lipophillic instead of the 2-methyl resi-
due provides reduced hERG binding, possibly due to reduced
basicity of the quinazoline ring.21 Replacing the quinazoline ring
with a similarly substituted quinoline ring provides comparable
hERG binding. In comparison with the qinazoline analog 6,
compound 33 has significantly increased inhibition in the hERG
patch assay. Replacing the trifluoromethyl residue with a cyano
group provides a compound (34) with good potency and similar
hERG binding to compound 33, but with significantly reduced
inhibition in the patch clamp assay.
While reducing the basicity of the quinoline ring by introducing
a trfluoromethyl residue (33) has little effect on CCR2 potency, 2-
cyano substitution (34) significantly improves both CCR2 binding
and functional potency. The more hydrophilic and less basic
phthalazine 37 and naphthyridine 38, with comparable CCR2 activ-
ity to quinazoline 6, are clean in the hERG patch assay.
Compound 34 was evaluated for efficacy in a thioglycollate-in-
duced peritoneal leukocyte infiltration model in mouse CCR2
knock-out/human CCR2 knock-in mice22 (Fig 1). When compound
34 was dosed orally at 3, 10 and 30 mpk, a dose dependent inhibi-
tion of peritonitis was observed. At the 30 mpk dose >50% inhibi-
tion of T-lymphocyte, Monocyte/macrophage and total leukocyte
infiltration was observed.
In summary, we have evaluated a series of heterocyclic replace-
ments for the aryl amide potion of (1-cyclohexylazetidin-3-yl)-N-
acylglycineamide, CCR2 antagonist. While compounds devoid of
Figure 1. Effect of compound 34 on thioglycollate-induced peritoneal leukocyte infiltration in hCCR2KI mice (Ref. 22). Compound 34 dosed po, BID. Positive control
dexamethasone (DEX) dosed 5 mg/kg, po, QD. Statistical significance was determined using unpaired t-test. In all analyses, p <0.05 was taken to indicate statistical
significance. ⁄p <0.05, ⁄⁄p <0.01 and ⁄⁄⁄p <0.001 versus control.
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017

N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
7
15. MCP-1 induced chemotaxis in THP-1 cells: MCP-1 induced chemotaxis was run
in a 24-well chemotaxis chamber. MCP-1 (0.01 g/mL) was added to the lower
chamber and 100
hERG activity and with good CCR2 binding and functional activity
were identified (6, 34, 37, 38), none of these compounds possessed
l
l
L of THP-1 cells (1 Â 107 cell/mL) was added to the top
a
suitable pharmacokinetic profile (Table 3) for further
chamber. Varying concentrations of test compound were added to the top and
bottom chambers. Cells were allowed to chemotax for 3 h at 37 °C and 5% CO2.
An aliquot of the cells which had migrated to the bottom chamber was taken
and counted then compared to vehicle.
advancement.
Acknowledgments
16. hERG [3H]-astemizole binding experiment: this assay is a 384well in-plate
vacuum filtration binding assay. Assay reagents are added into a prepared/
blocked 384 well assay plate in the following order: (1) hERG membrane
diluted in assay buffer; (2) test compound; and (3) 3H astemizole diluted in
assay buffer. Assay reagents are incubated in the filter plate for 1 h and then
washed six times with ice-cold wash buffer. Plates are allowed to dry overnight
at room temperature. The following morning, plates are sealed and scintillant
is added to each well. Following a 2 h incubation with scintillant, plates are
placed on the TopCount and counted 1 min per well. Data is calculated using
raw CPM. Where applicable, IC50 values are calculated using raw CPM values.
Curves are fitted individually from singlet 11 point dosing curves +1% DMSO
control.
The authors thank the High Output Synthesis, ADME/PK, Sec-
ondary Pharmacology and Lead Generation Biology teams and
the Cardiovascular Center of Excellence for their contributions to
this work.
References and notes
1. Gong, J. H.; Ratkay, L. G.; Waterfield, J. D.; ClarkLewis, I. J. Exp. Med. 1997, 186,
131.
2. Ogata, H.; Takeya, M.; Yoshimura, T.; Takagi, K.; Takahashi, K. J. Pathol. 1997,
182, 106.
17. Xue, C.; Zheng, C.; Cao, G.; Feng, H.; Xia, M.; Anand, R.; Glenn, J.; Metcalf, B. U.S.
Patent Appl. Publ. US20050267146.
18. Kang, F.; Sui, Z.; Murray, W. Eur. J. Org. Chem. 2009, 4, 461.
19. Patch express experiment: experiments were performed using HEK293 cells
stably expressing the HERG potassium channel. Cells were grown at 37 °C and
5% CO2 in culture flasks in MEM medium supplemented with 10% heat
3. Huang, D. R.; Wang, J. T.; Kivisakk, P.; Rollins, B. J.; Ransohoff, R. M. J. Exp. Med.
2001, 193, 713.
4. Izikson, L.; Klein, R. S.; Charo, I. F.; Weiner, H. L.; Luster, A. D. J. Exp. Med. 2000,
192, 1075.
inactivated fetal bovine serum, 1%
L-glutamine–penicillin–streptomycin
solution, 1% nonessential amino acids (100x), 1% sodium pyruvate (100 mM)
and 0.8% geneticin (50 mg/ml). Before use the cells were subcultured in MEM
medium in the absence of 5 ml L-glutamine–penicillin–streptomycin. For use
in the automated patch-clamp system PatchXpress 7000A (Axon Instruments)
cells were harvested to obtain cell suspension of single cells. Extracellular
solution contained (mM): 150 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, five
glucose (pH 7.4 with NaOH). Pipette solution contained (mM): 120 KCl, 10
HEPES, 5 EGTA, 4 ATP–Mg2, 2 MgCl2, 0.5 CaCl2 (pH 7.2 with KOH). Patch-clamp
experiments were performed in the voltage-clamp mode and whole-cell
currents were recorded with an automated patch-clamp assay utilizing the
PatchXpress 7000A system (Axon Instruments). Current signals were amplified
and digitized by a Multiclamp amplifier, stored and analyzed by using the
PatchXpress, DataXpress software and Igor 5.0 (Wavemetrics). The holding
potential was À80 mV. The HERG current (K+-selective outward current) was
determined as the maximal tail current at À40 mV after a 2 s depolarization to
+60 mV. Pulse cycling rate was 15 s. Before each test pulse a short pulse (0.5 s)
from the holding potential to À60 mV was given to determine (linear) leak
current. After establishing whole-cell configuration and a stability period, the
vehicle was applied for 5 min followed by the test substance by increasing
concentrations of 3 Â 10À6 M, 10À5 M and 3 Â 10À5 M. Each concentration of
the test substance was applied twice. The effect of each concentration was
determined after 5 min as an average current of three sequential voltage
pulses. To determine the extent of block the residual current was compared
with vehicle pre-treatment. Data are presented as mean values standard
error of the mean (SEM).
5. Fife, B. T.; Huffnagle, G. B.; Kuziel, W. A.; Karpus, W. J. J. Exp. Med. 2000, 192,
899.
6. Dawson, T. C.; Kuziel, W. A.; Osahar, T. A.; Maeda, N. Atherosclerosis 1999, 143,
205.
7. Kanda, H.; Tateya, S.; Tamori, Y.; Kotani, K.; Hiasa, K. I.; Kitazawa, R.; Kitazawa,
S.; Miyachi, H.; Maeda, S.; Egashira, K.; Kasuga, M. J. Clin. Investig. 2006, 116,
1494.
8. Weisberg, S. P.; Hunter, D.; Huber, R.; Lemieux, J.; Slaymaker, S.; Vaddi, K.;
Charo, I.; Leibel, R.; Ferrante, A. W. J. Clin. Investig. 2006, 116, 115.
9. Kamei, N.; Tobe, K.; Suzuki, R.; Ohsugi, M.; Watanabe, T.; Kubota, N.; Ohtsuka-
Kowatari, N.; Kumagai, K.; Sakamoto, K.; Kobayashi, M.; Kadowaki, T. J. Biol.
Chem. 2006, 281, 26602.
10. White, F. A.; Feldman, P.; Miller, R. J. Mol. Interv. 2009, 9, 188.
11. (a) Xia, M.; Sui, Z. Expert Opin. Ther. Pat. 2009, 19, 295; (b) Xue, C.; Wang, A.;
Han, Q.; Zhang, Y.; Cao, G.; Feng, H.; Huang, T.; Zheng, C.; Xia, M.; Zhang, K.;
Kong, L.; Glenn, J.; Anand, R.; Meloni, D.; Robinson, D.; Shao, L.; Storace, L.; Li,
M.; Hughes, R.; Devraj, R.; Morton, P.; Rogier, J.; Covington, M.; Scherle, P.;
Diamond, S.; Emm, T.; Yeleswaram, S.; Contel, N.; Vaddi, K.; Newton, R.; Hollis,
G.; Metcalf, B. ACS Med. Chem. Lett. 2011, 2, 913; (c) Xue, C.; Feng, H.; Cao, G.;
Huang, T.; Glenn, J.; Anand, R.; Meloni, D.; Zhang, K.; Kong, L.; Wang, A.; Zhang,
Y.; Zheng, C.; Xia, M.; Chen, L.; Tanaka, H.; Han, Q.; Robinson, D.; Modi, D.;
Storace, L.; Shao, L.; Sharief, V.; Li, M.; Galya, L.; Covington, M.; Scherle, P.;
Diamond, S.; Emm, T.; Yeleswaram, S.; Contel, N.; Vaddi, K.; Newton, R.; Hollis,
G.; Friedman, S.; Metcalf, B. ACS Med. Chem. Lett. 2011, 2, 450.
12. Zhang, Xuqing; Hufnagel, Heather; Markotan, Thomas; Lanter, James; Cai,
Chaozhong; Hou, Cuifen; Singer, Monica; Opas, Evan; McKenney, Sandra;
Crysler, Carl; Johnson, Dana; Sui, Zhihua Bioorg. Med. Chem. Lett. 2011, 21, 5577.
13. Cherney, Robert J.; Carter, Percy; Duncia, John V.; Gardner, Daniel S.; Santella,
Joseph B. PCT Int. Appl. WO 2004071460.
20. Fish, P.; Barber, C.; Brown, D.; Butt, R.; Collis, M.; Dickinson, R.; Henry, B.;
Horne, V. A.; Huggins, J.; King, E.; O’Gara, M.; McCleverty, D.; McIntosh, F.;
Phillips, C.; Webster, R. J. Med. Chem. 2007, 50, 2341.
21. Calculated (measured) Pka of heterocycles: quinazolin-4-amine = 4.84 (5.73);
2-methylquinazolin-4-amine = 5.76;
2-(trifluoromethyl)quinazolin-4-
(7.62, Guenter Grethe,
Sons, Feb 20, 1981); quinolin-4-
2-(trifluoromethyl)quinolin-4-amine = 6.35; 4-
14. MCP-1 receptor binding assay in THP-1 cells: THP-1 cells were obtained from
American Type Culture Collection (Manassas, VA, USA). The THP-1 cells were
amine = 3.06;
Isoquinolines, Vol. 1, John Wiley
amine = 9.07
isoquinolin-1-amine = 4.22
&
grown in RPMI-1640 supplemented with 10% fetal bovine serum in
a
(9.08);
humidified 5% CO2 atmosphere at 37 °C. The cell density was maintained at
0.5 Â 106 cells/mL. THP-1 cells were incubated with 0.5 nM 125I labeled MCP-
1 (Perkin–Elmer Life Sciences Inc., Boston, MA) in the presence of varying
concentrations of either unlabeled MCP-1 (R&D Systems, Minneapolis, MN) or
test compound for 2 h at 30 °C in a 96 well plates. Cells were then harvested
aminoquinoline-2-carbonitrile = 5.34.
22. Thiolycollate-induced peritonitis in mice: animals were intraperiponeally
injected with sterile thioglycollate (25 mL/kg, ip, Sigma) for induction of
peritonitis. Animals were orally dosed with vehicle or CCR2 antagonists at 3, 10
or 30 mg/kg twice daily. At the 72-hour time point, peritoneal cavities were
lavaged with 10 mL of sterile saline. Total cell counts in the peritoneal lavage
onto a filter plate, dried, and 20
lL of Microscint 20 was added to each well.
Plates were counted in TopCount NXT, Microplate Scintillation
a
&
fluid were performed using
a microscope and cell differentiation was
Luminescence Counter (Perkin–Elmer Life Sciences Inc., Boston, MA, USA).
Blank values (buffer only) were subtracted from all values and drug treated
performed using cytospinanalysis after Giemsa staining (Hema Tek 2000).
Percent inhibition of the thioglycollate induced peritonitis was calculated by
comparing the change in number of leukocytes of CCR2 antagonist treated
mice to the vehicle-treated mice.
values were compared to vehicle treated values. 1
nonspecific binding.
lM cold MCP-1 was used for
Please cite this article in press as: Subasinghe, N. L.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.017