Overcoming hERG activity in the discovery of a series of 4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.06.080

A series of 4-azetidinyl-1-aryl-cyclohexanes as potent CCR2 antagonists with high selectivity over activity for the hERG potassium channel is discovered through divergent SARs of CCR2 and hERG.

Full text of DOI:10.1016/j.bmcl.2011.06.080

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5577–5582
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Overcoming hERG activity in the discovery of a series
of 4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists
⇑
Xuqing Zhang , Heather Hufnagel, Thomas Markotan, James Lanter, Chaozhong Cai, Cuifen Hou,
Monica Singer, Evan Opas, Sandra McKenney, Carl Crysler, Dana Johnson, Zhihua Sui
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, Welsh & McKean Roads, Box 776, 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:
A series of 4-azetidinyl-1-aryl-cyclohexanes as potent CCR2 antagonists with high selectivity over
activity for the hERG potassium channel is discovered through divergent SARs of CCR2 and hERG.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 2 May 2011
Revised 15 June 2011
Accepted 17 June 2011
Available online 29 June 2011
Keywords:
CCR2
hERG
Azetidine
Chemokines are a family of chemoattractant cytokines (CC)
whose main function is to regulate the trafficking and activation of
leukocytes and other cell types under a variety of inflammatory
and noninflammatory conditions. CCL2, commonly referred to as
monocyte chemotactic protein-1 (MCP-1), is one of the primary
molecules controlling the influx of mononuclear leukocytes into
sites of inflammation. MCP-1 is a potent chemotactic factor for
monocytes and memory T lymphocytes, and stimulates the move-
ment of those cells along a chemotactic gradient following binding
to its cell-surface receptor, CC chemokine receptor-2 (CCR2). This li-
gand/receptorpair is overexpressed in numerous inflammatory con-
ditions wherein excessive monocyte recruitment is observed, such
as atherosclerosis and rheumatoid arthritis.1–5 An antagonist of
the binding of MCP-1 to its receptor CCR2 may be an effective treat-
ment for any inflammatory disease in which monocytes, mast cells,
or basophils play major roles. Such diseases include, among others,
asthma, obesity, insulin resistance, multiple sclerosis, atherosclero-
sis, inflammatory bowel diseases, anterior uveitis, gingivitis, peri-
odontitis, allergic rhinitis, allergic conjunctivitis, inflammatory
acne, psoriasis, and atopic dermatitis.6–15
antagonists fall into a general class with a ‘sandwich’ structure
consisting of a central basic amine flanked by two hydrophobes.
The dominance of this chemotype is consistent with the impor-
tance of the interaction between glutamic acid-291 on CCR2 resi-
dues and ligands. MK-0812, INCB3344, JNJ lead and BMS lead are
representative examples for this chemotype (Fig. 1).
As part of our intensive efforts on CCR2 antagonists, we started
to optimize our lead (JNJ lead in Fig. 1) for eliminating the zwitter
ion form and improving solubility. Through synthesis and evalua-
tion of a large number of templates containing various heterocyclic
substitutions built around a central basic amine, we arrived at
O
OH
O
O
H
N
CF3
MeO
O
N
H
N
O
N
N
N
H
O
O
MK-0812
CF3
INCB3344
Over the past several years, there has been intensive research
interest in small molecule antagonists of the CCR2 receptor result-
ing in the disclosure of many distinct chemical series.16–20 As the
work described within this paper was being carried out, literature
examples of CCR2 antagonists have been reported within a range of
‘chemotypes’.21–33 Of all reported chemical series, many CCR2
CF3
N
O
HOOC
O
N
H
N
HN
F
F
N
N
N
NH
F
O
N
H
BMS Lead
JNJ Lead
⇑
Corresponding author.
E-mail address: xzhang5@prdus.jnj.com (X. Zhang).
Figure 1. Representative examples of potent CCR2 antagonists.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.080

5578
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5577–5582
Table 1
compound 1 as cis–trans cyclohexyl mixtures bearing azetidinyl
feature with sub M CCR2 binding affinity. We were particularly
l
interested in this scaffold since it eliminated the zwitter ion and
incorporated a soluble bis-amide side chain, which could provide
compounds with amendable pharmaceutical properties and syn-
thetic expedience.34 However, compound 1 was also a potent li-
R
N
NH
O
O
HN
CF3
gand for the hERG channel displaying IC50 of 2.4 lM (Fig. 2).
A
There was no separation between CCR2 and hERG activities. Hence,
the challenge was to improve selectivity over hERG ion channel.
Herein we describe the SAR and optimization of this azetidine ser-
ies to the identification of lead compounds with potent CCR2
antagonistic activity and high selectivity versus hERG.
The synthesis of target compounds in this series is straightfor-
ward. A general synthetic scheme is shown in Scheme 1. The
left-side building blocks 4 were constructed by Suzuki coupling
of starting material aryl bromides 2 and vinyl boronic ester 335
in 55–80% yield, from which the final compounds 8 (cis-isomer
of 1,4-substitutions on the cyclohexanyl ring) and 9 (trans-isomer)
in ratio of 3:1 to 1:1 and total 65–90% yield were synthesized by
simple reductive amination with NaBH(OAc)3 of the azetidine-con-
taining building blocks 7. Intermediates 7 were prepared by amide
coupling (EDCI and HOEt) of glycine acid derivatives 5 and amino
azetidine 6 in 78–85% yield. The structure of cis-isomer was deter-
mined by 2D NMR (COSY and NOSY).
ID
R
CCR2 bindinga IC50 (nM)
O
O
O
O
8a
5b
1,800c
O
9a
10
11
O
O
O
1,000c
>25,000c
N
a
b
c
MCP-1 receptor binding assay in THP-1 Cells, see Reference 36.
IC50 value is reported as the average of five separate determinations.
IC50 values are reported from single determination.
Target compounds were screened by CCR2 binding assays to as-
sess their ability to inhibit MCP-1 binding to human peripheral
blood monocytes (MCP-1 assay)36 and by a CCR2 functional assay
to determine their ability to inhibit MCP-1 stimulated chemotaxis
in human peripheral blood monocytes (CTX assay).37 Table 1 shows
the initial examples on modifications of the cyclohexanyl ring. The
cis-isomer 8a is a potent CCR2 inhibitor with an IC50 of 5 nM in the
MCP-1 binding assay while the trans isomer 9a is only moderately
group substantially reduced or completely abolished CCR2 activity,
as shown in 10 (IC50 of 1.0 M) and 11 (IC50 of >25 M), which indi-
cated the cis geometry of 1-aryl and 4-azetidinyl substitutions on
the cyclohexanyl ring is essential for effective ligand/receptor
binding.
l
l
Having established cis 4-azetidinyl-1-aryl cyclohexanyl moiety
as the key template which possesses encouraging CCR2 activity,
we initiated SAR studies on both phenyl rings on the left and right
sides of the molecule. Initial modification on the right side aryl ring
indicated that meta-CF3 might be the optimal substituent (Table 2).
As illustrated by 8c and 8d, either removal of meta-CF3 or moving
CF3 group from meta to para position resulted in dramatic loss of
CCR2 binding affinity. We then turned our attention to the left-
end aryl ring. Electron rich phenyl ring attached to 1-position of
the cyclohexanyl ring had beneficial effect on the CCR2 activity
as evidenced by an approximate three to sevenfold increase of
CCR2 binding affinity when a 4-methoxy or 3,4-methylenedioxy
group was installed (8a and 8e vs 8b). The observed high potency
of 8a and 8e was also translated into excellent levels of activity in
the CCR2 chemotaxis assay with IC50 of 15 nM and 25 nM, respec-
tively. In contrast, electron withdrawing groups such as carboxyl
active in the same assay (IC50 of 1.8 lM). Replacement of the cis
cyclohexanyl group with either the cyclohexenyl or the piperidinyl
O
H
N
CF3
N
H
N
O
HN
1
hCCR2 binding, IC50 < 1 μM
μ
hERG binding, IC50 2.4
M
Figure 2. Initial hit.
O
B
O
1), 2), 3)
O
O
Ar
O
ArBr
+
2
3
4
6)
O
R
H
N
O
H
HN
4), 5)
HN
N
+
BocN
NH2
HO
R
O
O
6
7
5
O
O
Ar
N
NH HN
O
Ar
N
NH HN
O
+
R
R
8
9
Scheme 1. Reagents: (i) Pd(Ph3P)4, Na2CO3; (ii) H2, 10% Pd/C, 50 psi; (iii) HCl, acetone; (iv) EDCI, HOBt; (v) H2SO4 or TFA; (vi) NaBH(Ac)3, silica gel column separation 5% 7 N
NH3/MeOH in DCM.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5577–5582
5579
Table 2
Ar
H
N
NH
O
O
HN
R
B
CTXb IC50 (nM)
35
hERG bindingc IC50
4.0
(l
M)
hERG patchd% @3
97
lM
a
ID
Ar
R
CCR2 binding IC50 (nM)
8b
3-CF3
35
O
8a
8c
8d
3-CF3
H
5
390
15
nte
nt
2.8
2.8
1.5
95
nt
nt
O
O
O
O
O
4-CF3
1200
MeO
HO
8e
8f
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
10
21
80
70
61
25
3.0
4.1
99
75
91
35
95
42
MeO2C
8g
8h
8i
nt
1.3
HO2
HN
C
1300
240
23.0
7.9
N
N
8j
3-CF3
27
21
2.5
88
a
IC50 value are reported as the average of at least two separate determinations.
MCP-1 induced chemotaxis in THP-1 cells (Ref. 37).
b
c
hERG 3H-astemizole binding activity on HEK-293 cell (Ref. 38).
d
e
The membrane K+ current IKr in HERG-transfected HEK293 cells using the PATCHXPRESS planar patch-clamp instrument (Ref. 39).
nt: not tested.
significantly reduced CCR2 binding as indicated with 8g with IC50
of 80 nM. It is well known that structures containing a central basic
amine flanked by two hydrophobes are generally good binders for
hERG ion channel. It is not surprising that our early analogs
showed no selectivity against hERG. Divergent SAR of CCR2 and
hERG is required to achieve sufficient cardiovascular safety win-
dow. To increase throughput, we used high throughput hERG bind-
ing screen38 followed by hERG patch express39 assays. This strategy
allowed us to evaluate large number of compounds in a rapid fash-
ion. While CCR2 activity could be well maintained in this scaffold
when steric bulkiness of the substitutions on the aryl ring was al-
tered as indicated in Table 2, hERG liability seems inseparable. For
example, compound 8i was a potent binder for the hERG channel
on attenuating hERG binding affinity. Replacement of the pyridinyl
with more polar pyrazinyl (12e) or pyrimidinyl (12f) group failed
to dial out hERG activity. In contrast to their substituted phenyl
analogs in Table 2, pyridines with polar substituents proved to
be effective in suppressing hERG in both binding and functional as-
says. Hydroxy (12g and 12h) or amino pyridines (12i and 12j) re-
duced hERG binding affinity significantly with maintained CCR2
activity. Encouraged by this finding, we decided to move polar
functional groups to 1-poisition of cyclohexanyl ring since it would
be located at the C-2 symmetry and hence does not increase com-
plexity for stereochemistry. This strategy was well accomplished
as 14a (Fig. 3) was found to reduce hERG activity dramatically
compared with its des-oxy analog 12a (Table 3).40 However, it also
substantially reduced CCR2 potency. Our next goal was to improve
CCR2 activity to achieve good selectivity over hERG. We decided to
switch the left side pyridinyl ring back to phenyl ring and examine
the impact of the substitution at 1-position of the cyclohexanyl
ring on both CCR2 and hERG activity. We prepared a series of 1-
substituted analogs according to the synthetic routes in
Scheme 2.39 Addition of aryl lithium generated from compounds
2 with 1,4-cyclohexanedione monoethylene ketal followed by de-
protection of the ketal group afforded hydroxyl ketones 13 in about
60% yield for two steps. Ketones 13 were then coupled with amine
7 by reductive amination to give a pair of isomers 14 and 15 in
about 3:1 to 1:1 ratio in ꢀ70% yield. The mixtures were then trea-
ted with DAST at À78 °C to form a pair of fluorinated azetidines 16
(cis-isomer) and 17 (trans-isomer) in 80% yield, Reaction of the ke-
tone with t-Bu-sulfinamide by Ti(OEt)4 afforded the corresponding
sulfinimide 18, which was then coupled with aryl Grignard
reagent generated through metal–halogen exchange followed by
displaying IC50 of 7.9
affinity of the compound was further confirmed with 95% of inhi-
bition in hERG patch express assay when screened at 3 M. To dial
lM in hERG binding assay. The strong hERG
l
out the unwanted hERG signal of this series we initially developed
a strategy of increasing the polarity of the molecules. From the ini-
tial set of analogs in Table 2, it was apparent that regardless of the
polar groups introduced, including mono-substitution (8f) and
fused ring (8j), not much hERG selectivity was achieved except
8h, a zwitter ion. Unfortunately, beside its weak functional activity
in chemotaxis (IC50, 1.3 lM), it did not have amendable pharma-
ceutical properties for further development.
We then turned our attention to dial out hERG by replacing the
left-end phenyl ring with more polar pyridinyl ring. As illustrated
in Table 3, while pyridinyl analog 12a maintained good CCR2 bind-
ing affinity (IC50, 45 nM), it still carried strong hERG binding and
functional activity. Installation of methoxy group onto the pyridi-
nyl ring (12b, 12c and 12d) did not show significant improvement

5580
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5577–5582
Table 3
N
ArHet
N
NH
O
N
NH
O
HO
H
O
O
HN
HN
CF3
CF3
C
14a
CCR2 binding IC50, 190 nM
μΜ
μΜ
hERG binding IC50, 35
hERG Patch, 22% @ 3
ID
HetAr
CCR2
CTX
IC50
(nM)
hERG
binding IC50 patch% @
M)
hERG
a
binding IC50
a
(nM)
(l
3 lM
Figure 3. Hydroxy azetidine.
12a
12b
45
7
52
3.8
2.8
92
97
N
As illustrated in Table 4, installation of OH group at the 1-position
of the cyclohexanyl ring (14b) still maintained good CCR2 activity
(IC50, 36 nM) but reduced five folds of hERG binding affinity. More-
over, compound 14b presented relatively clean hERG functional
MeO
11
N
OMe
12c
32
28
8.2
nt
N
activity with only 34% inhibition at 3 lM. Other substituents of
N
either F or NH2 resulted in significant loss of CCR2 activity as
shown in 16a and 21a. Incorporation of a cynao, carboylic acid or
carbinol group (23–25) abolished CCR2 activity.
12d
12e
25
44
170
nt
6.2
12
nt
MeO
N
87
Substituent effect on the phenyl ring of 14b was further exam-
ined (Table 5). Similar to their des-oxy analogs the electronic effect
of the substituents on the left-end aryl ring had great impact on
CCR2 activity. Electron donating groups such as methoxy (14f),
dimethylamino (14e), methylenedioxy (14g) and dihydrofuran
(14h) all improved CCR2 binding and functional activities. Elec-
tron-withdrawing groups such as F and CN caused at least 10-fold
drop of CCR2 binding affinity. Steric hinderance seemed to aban-
don CCR2 activity as compound 14i with 2,6-dimethyl substitution
on the phenyl ring was inactive. In terms of hERG profile, hydroxy
azetidine 14e was particularly promising with hERG binding affin-
N
N
Me2N
12f
31
13
nt
9.8
10
76
36
N
N
12g
14
HO
OH
12h
12i
52
57
23
28
N
N
43
85
35
nt
44
37
nt
NH2
N
ity of 25
lM and low hERG blockade in the functional assay (22%
12j
25
H2N
inhibition at 3
l
M; solvent background, 15%). We were pleased
a
to establish a large in vitro cardiovascular safety margin against
IC50 value are reported as the average of at least two separate determinations.
hERG finally.
In summary, we have identified a novel series of CCR2 antago-
nists based on the azetidine scaffold. The discovery process is high-
lighted by divergent SARs on CCR2 and hERG activities, which
enabled us to dial out hERG affinity in the scaffold and generated
de-protection of the ketal group to give amino ketone 20 in about
40% yield.41 Compound 20 was then reacted with 7 under reduc-
tive amination conditions to afford amino azetidines 21 and 22.
O
H
N
ArBr
or
HN
1), 2)
O
O
Ar
HO
CF3
HN
+
O
+
O
O
ArH
7
13
2
O
O
O
3)
4)
Ar
HO
N
NH HN
O
Ar
HO
N
N
NH HN
+
CF3
O
CF3
CF3
14
15
O
Ar
NH HN
O
Ar
N
NH HN
O
+
F
F
CF3
17
16
O
6), 7)
O
O
5)
Ar
H2N
S
O
ArI
+
O
O
N
O
20
19
18
7
O
O
3)
Ar
H2N
N
NH HN
O
Ar
H2N
N
NH HN
O
+
CF3
CF3
22
21
Scheme 2. Reagents and conditions: (i) n-BuLi, 78 °C, then 2; (ii) HCl, acetone; (iii) NaBH(OAc)3; (iv) DAST, À78 °C; (v) t-BuS(O)NH2, Ti(OEt)4, (vi) i-PrMgBr, 19; (vii) HCl, THF.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5577–5582
5581
Table 4
5. Castellani, M. L.; Bhattacharya, K.; Tagen, M.; Kempuraj, D.; Perrella, A.; Delutis,
M.; Boucher, W.; Conti, P.; Theoharides, T. C.; Cerulli, G.; Salini, V.; Neri, G. Int. J.
Immunopathol. Pharmacol. 2007, 20, 447.
6. Gong, J. H.; Ratkay, L. G.; Waterfield, J. D.; ClarkLewis, I. J. Exp. Med. 1997, 186,
131.
R
N
NH
O
O
HN
7. Ogata, H.; Takeya, M.; Yoshimura, T.; Takagi, K.; Takahashi, K. J. Pathol. 1997,
182, 106.
CF3
D
8. Huang, D. R.; Wang, J. T.; Kivisakk, P.; Rollins, B. J.; Ransohoff, R. M. J. Exp. Med.
2001, 193, 713.
9. Izikson, L.; Klein, R. S.; Charo, I. F.; Weiner, H. L.; Luster, A. D. J. Exp. Med. 2000,
192, 1075.
ID
R
CCR2 binding
IC50 (nM)
CTX IC50 hERG binding
hERG patch%
@ 3 lM
a
(nM)
IC50
(lM)
10. Fife, B. T.; Huffnagle, G. B.; Kuziel, W. A.; Karpus, W. J. J. Exp. Med. 2000, 192,
899.
11. Dawson, T. C.; Kuziel, W. A.; Osahar, T. A.; Maeda, N. Atherosclerosis 1999, 143,
205.
12. 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. Invest. 2006, 116,
1494.
13. Weisberg, S. P.; Hunter, D.; Huber, R.; Lemieux, J.; Slaymaker, S.; Vaddi, K.;
Charo, I.; Leibel, R.; Ferrante, A. W. J. Clin. Invest. 2006, 116, 115.
14. 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.
8b
H
35
36
110
240
35
55
170
160
nt
2.0
10
3.3
6.2
nt
97
34
nt
nt
nt
nt
nt
14b OH
16a
21a NH2
23
24
25
F
CN
>25,000b
>25,000b
COOH
nt
nt
nt
nt
CH2OH >25,000b
a
IC50 value are reported as the average of at least two separate determinations.
IC50 values are reported by single determination.
b
15. Higgins, P. J.; Schwartz, C. E.; Nicolas, J.-M. Chemokine Biol.–Basic Res. Clin. Appl.
2007, 2, 115.
16. Struthers, M.; Pasternak, A. Curr. Top. Med. Chem. 2010, 10, 1278.
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543.
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Hermans, B.; VanWauwe, J. Bioorg. Med. Chem. Lett. 2005, 15, 497.
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Hanlon, W. A.; Cascieri, M. A.; Springer, M. S. Bioorg. Med. Chem. Lett. 2006, 16,
3735.
Table 5
HO
R
N
NH
O
O
HN
CF3
E
ID
R
CCR2
binding
IC50 (nM)
CTX
IC50
(nM)
hERG
binding IC50 patch%@
M)
hERG
23. Butora, G.; Morriello, G. J.; Kothandaraman, S.; Guiadeen, D.; Pasternak, A.;
Parsons, W. H.; MacCoss, M.; Vicario, P. P.; Cascieri, M. A.; Yang, L. Bioorg. Med.
Chem. Lett. 2006, 16, 4715.
a
(
l
3 lM
14b
14c 4-F
H
36
55
nt
nt
22
67
27
10
34
nt
nt
22
37
33
24. Pasternak, A.; Marino, D.; Vicario, P. P.; Ayala, J. M.; Cascierri, M. A.; Parsons,
W.; Mills, S. G.; MacCoss, M.; Yang, L. J. Med. Chem. 2006, 49, 4801.
25. Zhou, C.; Guo, L.; Parsons, W. H.; Mills, S. G.; MacCoss, M.; Vicario, P. P.;
Zweerink, H.; Cascieri, M. A.; Springer, M. S.; Yang, L. Bioorg. Med. Chem. Lett.
2007, 17, 309.
26. Pinkerton, A. B.; Huang, D.; Cube, R. V.; Hutchinson, J. H.; Struthers, M.; Ayala, J.
M.; Vicario, P. P.; Patel, S. R.; Wisniewski, T.; DeMartino, J. A.; Vernier, J.-M.
Bioorg. Med. Chem. Lett. 2007, 17, 807.
210
410
15
20
22
2.8
1.5
25
8.9
13.5
14d 3-CN
14e 3-NMe2
14f
14g 3,4-
Methylenedioxy
14h 3,4-
4-OMe
25
52
nt
11.7
7.6
41
nt
27. Butora, G.; Jiao, R.; Parsons, W. H.; Vicario, P. P.; Jin, H.; Ayala, J. M.; Cascieri, M.
A.; Yang, L. Bioorg. Med. Chem. Lett. 2007, 17, 3636.
Dihydrofuran
2,6-Di-Me
14i
>25,000b
28. Lagu, B.; Gerchak, C.; Pan, M.; Hou, C.; Singer, M.; Malaviya, R.; Matheis, M.;
Olini, G.; Cavender, D.; Wachter, M. Bioorg. Med. Chem. Lett. 2007, 17, 4382.
29. Carter, P. H.; Brown, G. D.; Friedrich, S. R.; Cherney, R. J.; Tebben, A. J.; Lo, Y. C.;
Yang, G.; Jezak, H.; Solomon, K. A.; Scherle, P. A.; Decicco, C. P. Bioorg. Med.
Chem. Lett. 2007, 17, 5455.
a
IC50 value are reported as the average of at least two separate determinations.
IC50 values are reported by single determination.
b
30. Xia, M.; Hou, C.; Pollack, S.; Brackley, J.; DeMong, D.; Pan, M.; Singer, M.;
Matheis, M.; Olini, G.; Cavender, D.; Wachter, M. Bioorg. Med. Chem. Lett. 2007,
17, 5964.
31. Yang, L.; Butora, G.; Jiao, R. X.; Pasternak, A.; Zhou, C.; Parsons, W. H.; Mills, S.
G.; Vicario, P. P.; Ayala, J. M.; Cascieri, M. A.; MacCoss, M. J. Med. Chem. 2007, 50,
2609.
promising selectivity for CCR2 over hERG which is likely to lead to
a suitable cardiovascular safety margin.
32. Xia, M.; Hou, C.; DeMong, D. E.; Pollack, S. R.; Pan, M.; Brackley, J. A.; Jain, N.;
Gerchak, C.; Singer, M.; Malaviya, R.; Matheis, M.; Olini, G.; Cavender, D.;
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Acknowledgments
33. Xue, C.-B.; Wang, A.; Meloni, D.; Zhang, K.; Kong, L.; Feng, H.; Glenn, J.; Huang,
T.; Zhang, Y.; Cao, G.; Anand, R.; Zheng, C.; Xia, M.; Han, Q.; Robinson, D. J.;
Storace, L.; Shao, L.; Li, M.; Brodmerkel, C. M.; Covington, M.; Scherle, P.;
Diamond, S.; Yeleswaram, S.; Vaddi, K.; Newton, R.; Hollis, G.; Friedman, S.;
Metcalf, B. Bioorg. Med. Chem. Lett. 2010, 20, 7473.
We thank Lead Generation Biology, Center of Excellence for
Cardiovascular Safety Research and HOPS at J&J PRD for their tech-
nical assistance.
34. Moree, W. J.; Kataoka, K.; Ramirez-Weinhouse, M. M.; Shiota, T.; Imai, M.;
Sudo, M.; Tsutsumi, T.; Endo, N.; Muroga, Y.; Hada, T.; Tanaka, H.; Morita, T.;
Greene, J.; Barnum, D.; Saunders, J.; Kato, Y.; Myers, P. L.; Tarby, C. M. Bioorg.
Med. Chem. Lett. 2004, 14, 5413.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.080.
35. For preparation of this building block, see: Birch, A. M.; Bowker, S. S.; Butlin, R.
J.; Donald, C. S.; McCoull, W.; Nowak, T.; Plowright, A. PCT Int. Appl. WO
2006064189 A1, 2006; Chem Abstr. 2006, 145, 83352.
36. MCP-1 receptor binding assay in THP-1 cells:
References and notes
THP-1 cells were obtained from American Type Culture Collection (Manassas,
VA, USA). The THP-1 cells were grown in RPMI-1640 supplemented with 10%
fetal bovine serum in a humidified 5% CO2 atmosphere at 37 °C. The cell
density was maintained at 0.5Á106 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 plate. Cells
1. Charo, I. F.; Ransohoff, R. M. N. Engl. J. Med. 2006, 354, 610.
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3. Okuma, T.; Terasaki, Y.; Kaikita, K.; Kobayashi, H.; Kuziel, W. A.; Kawasuji, M.;
Takeya, M. J. Pathol. 2004, 204, 594.
were then harvested onto a filter plate, dried, and 20
added to each well. Plates were counted in a TopCount NXT, Microplate
lL of Microscint 20 was
4. Navratilova, Z. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 2006,
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5582
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5577–5582
Scintillation & Luminescence Counter (Perkin–Elmer Life Sciences, Inc., Boston,
MA). Blank values (buffer only) were subtracted from all values and drug
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, 5 Glucose (pH 7.4 with NaOH). Pipette solution contained
(mM): 120 KCl, 10 HEPES, 5 EGTA, 4 ATP-Mg2, 2 MgCl2, 0.5 CaCl2 (pH7.2 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).
treated values were compared to vehicle treated values. 1
used for nonspecific binding.
lV cold MCP-1 was
37. MCP-1 induced chemotaxis in THP-1 cells:
MCP-1 induced chemotaxis was run in a 24-well chemotaxis chamber. MCP-1
(0.01 lg/mL) was added to the lower chamber and 100 lL of THP-1 cells
(1 Â 107 cell/mL) was added to the top 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.
38. hERG [3H]-astemizole binding experiment: This assay is
a 384well in-plate
vacuum filtration binding assay. Assay reagents are added into a prepared/
blocked 384well assay plate in the following order: (1) hERG Membrane
diluted in Assay Buffer; (2) Test Compound; (3) 3H Astemizole diluted in Assay
Buffer. Assay reagents are incubated in the filter plate for 1 hour and then
washed 6Â 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 two-hour 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.
40. For discovery of a pyrrolidine-based CCR2 antagonist, see: Xue, C.-B.; 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. J.;
Modi, D.; Storace, L.; Shao, L.; Sharief, V.; Li, M.; Galya, L. G.; 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.
39. 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-
inactivated fetal bovine serum, 1%
L-Glutamine–Penicillin–Streptomycin-
solution, 1% non-essential amino acids (100Â), 1% sodium pyruvate
(100 mM) and 0.8% Geneticin (50 mg/ml). Before use the cells were
41. Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 268.
subcultured in MEM medium in the absence of 5 ml L-Glutamine–Penicillin–