Synthesis and evaluation of cis-3,4-disubstituted piperidines as potent CC chemokine receptor 2 (CCR2) antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.07.123

A series of cis-3,4-disubstituted piperidines was synthesized and evaluated as CC chemokine receptor 2 (CCR2) antagonists. Compound 24 emerged with an attractive profile, possessing excellent binding (CCR2 IC₅₀ = 3.4 nM) and functional antagonism (calcium flux IC₅₀ = 2.0 nM and chemotaxis IC₅₀ = 5.4 nM). Studies to explore the binding of these piperidine analogs utilized a key CCR2 receptor mutant (E291A) with compound 14 and revealed a significant reliance on Glu291 for binding.

Full text of DOI:10.1016/j.bmcl.2008.07.123

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5063–5065
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of cis-3,4-disubstituted piperidines as potent CC
chemokine receptor 2 (CCR2) antagonists
*
Robert J. Cherney , David J. Nelson, Yvonne C. Lo, Gengjie Yang, Peggy A. Scherle, Heather Jezak,
Kimberly A. Solomon, Percy H. Carter, Carl P. Decicco
Research and Development, Bristol-Myers Squibb Company, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cis-3,4-disubstituted piperidines was synthesized and evaluated as CC chemokine receptor 2
(CCR2) antagonists. Compound 24 emerged with an attractive profile, possessing excellent binding (CCR2
IC50 = 3.4 nM) and functional antagonism (calcium flux IC50 = 2.0 nM and chemotaxis IC50 = 5.4 nM).
Studies to explore the binding of these piperidine analogs utilized a key CCR2 receptor mutant
(E291A) with compound 14 and revealed a significant reliance on Glu291 for binding.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 July 2008
Revised 29 July 2008
Accepted 31 July 2008
Available online 5 August 2008
Keywords:
CCR2
CCR2 antagonist
Chemokine
GPCR
Chemokines are a large family of proteins involved in the acti-
vation and migration of leukocytes.1 Our interest in chemokines
has focused on a CC family member, monocyte chemoattractant
protein-1 (MCP-1 or CCL2),2 which is upregulated in many autoim-
mune and inflammatory conditions.3 MCP-1 binds to its exclusive
receptor, CC chemokine receptor 2 (CCR2),4 which is a member of
the G-protein coupled receptor (GPCR) family. The CCR2/MCP-1
pair has been implicated in a variety of diseases, including rheuma-
toid arthritis,5 atherosclerosis,6 multiple sclerosis,7 and insulin
resistance.8 This has generated a great deal of interest in small
molecule antagonists of CCR2 as potential therapeutic agents.9 In
this letter, we describe a novel series of disubstituted piperidines
as potent CCR2 antagonists.
Recently, we described a series of disubstituted cyclohexanes 1
(Fig. 1) as selective CCR2 antagonists (see also derivative 2 in Table
1).10 Looking to improve these antagonists, we became interested
in the addition of a nitrogen within the cyclohexane to give a
piperidine 3. These piperidines were synthesized in racemic form,
starting from dihydropyridine 4, as shown in Scheme 1. Racemic
epoxide 5 was produced via m-CPBA and was opened with sodium
azide to give the regiomeric pair 6 and 7.11 This mixture was sub-
jected to a hydrogenolysis (H2, Pd/C) followed by carbamate forma-
tion and chromatography to afford the major isomer 8. The
secondary alcohol was then converted to an azide via a Mitsunobu
reaction to yield 9. This, in turn, was reduced to the amine 10 be-
Figure 1. Exploration of piperidines as CCR2 antagonists.
fore it was coupled to the glycinamide 11 to give 12. Hydrogenol-
ysis gave the primary amine 13, which was taken to either a target
amine 14 or a target amide 15. As shown in Scheme 2, for piperi-
dine nitrogen substitution, 12 was treated with TFA to provide
amine 16. Selective alkylation with crotyl bromide (or other elec-
trophiles) and subsequent hydrogenolysis gave 17. As before, tar-
get amines 18 were produced via a reductive amination, and
amides 19 were produced by a coupling reaction.
The newly synthesized piperidines were evaluated using a radi-
olabeled MCP-1 displacement assay in peripheral blood mononu-
clear cells (PBMCs).12 We were interested in selective CCR2
antagonists, and hence used a CCR3 binding assay13 for an initial
assessment of selectivity. Highly active compounds were also eval-
uated in two functional assays, the calcium flux assay12 based on
PBMCs and a chemotaxis assay12 based on monocytes. With these
highly active compounds, we also verified that the compounds did
not induce calcium flux when incubated alone in PBMCs (without
MCP-1 present), thus validating the compounds as antagonists and
* Corresponding author. Tel.: +1 609 252 3066; fax: +1 609 252 7446.
E-mail address: robert.cherney@bms.com (R.J. Cherney).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.123

5064
R. J. Cherney et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5063–5065
Table 1
Evaluation of benzylamine piperidines derivativesa
R1
IC50 (nM)
CCR3 binding % inh. at 10 l
Md
b
Compound
R
X
CCR2 binding
Ca flux
CTX monoc
2
4-SMe
4-SMe
4-CI
CH2
NH
NH
NH
NH
NMe
NPr
NBu
NPr
NPr
NPr
NAc
NBoc
2-NH2
2-NH2
2-NH2
2-NH2
H
2-NH2
2-NH2
2-NH2
2-NHCONHMe
2-NHCOpyrrolidine
3-NH2
H
H
1180 130 (3)
6.3 5.2 (2)
21.5 7.7 (2)
123 21.2 (2)
97 48.1 (2)
0.95 0.2 (2)
3.4 2.3 (2)
7.9 2.7 (2)
3.8 1.1 (2)
8.7 0.3 (2)
6.1 1.1 (2)
463 117.4 (2)
NT
NT
NT
47.3
90
NT
46
56
47
75
5.5 3.9 (2)
38.2 0.8 (2)
49.3
NT
NT
14
20
21
22
23
24
18
25
26
27
28
29
7.0 1.4 (2)
19 (1)
NT
285 3.5 (2)
NT
NT
4-SEt
4-SMe
4 SMe
4-SMe
4 SMe
4-SMe
4 SMe
4-SMe
4 SMe
4-SMe
510 70.7 (2)
5.0 (1)
2.0 (1)
13.0 (1)
3.0 (1)
21 (1)
3.0 (1)
NT
NT
27.5 12 (2)
5.4 0.5 (2)
19.0 (1)
8.0 2.9 (2)
4.8 0.6 (2)
26.0 21.6 (3)
NT
0% at 1
l
M
NT
NT
a
Compounds are racemic, one enantiomer is displayed for illustrative purposes.
IC50 values (n) are displayed as mean SD (n = 2) and mean SEM (n > 2).
CTX mono, chemotaxis in monocytes.
b
c
d
CCR3 % inhibition are n = 1, unless otherwise noted. NT, not tested. 2-NHCOpyrrolidine =
.
Scheme 1. Reagents and conditions: (a) m-CPBA, CH2Cl2, 0 °C; (b) NaN3, NH4Cl, MeOH/H2O,
D; (c) i—H2, Pd/C, MeOH; ii—Cbz2O, TEA, THF/H2O; iii—separation of
diastereomers; (d) HN3, DEAD, PPh3, THF/PhH, 5 °C; (e) PPh3, H2O, THF, 65 °C; (f) 11, NMM, HATU, DMF; (g) H2, Pd/C, MeOH; (h) i—4-methylthiobenzaldehyde, NaBH3CN,
ZnCl2, MeOH; ii—TFA; (i) i—4-methylthiobenzoic acid, NMM, HATU, DMF; ii—TFA.
not partial agonists. As shown in Table 1, the addition of a nitrogen
to form the piperidine was very beneficial. When compared to the
cyclohexyl derivative 2,10 piperidine 14 displayed a 180-fold
improvement in binding affinity for CCR2. Piperidine 14 also had
excellent activity in the calcium flux assay (IC50 = 7 nM) and the
chemotaxis assay (28.5 nM), along with good selectivity over
CCR3. In order to optimize this CCR2 activity, we examined other
areas of piperidine 14. The methyl sulfide substituent was opti-
mized in the cyclohexyl series,10 and again proved to be sensitive
toward substitution as shown with the chloro-derivative 20
(CCR2 IC50 = 21.5 nM) and the ethyl sulfide 21 (CCR2
IC50 = 123 nM). The 2-amino substituent14 of the benzamide was
known to be important for binding affinity and functional activ-
ity,10,15 and its removal in compound 22 (CCR2 IC50 = 97 nM)
proved to be detrimental when compared to 14. As shown by
derivatives 18, 23, and 24, the CCR2 affinity was retained by substi-
tuting the piperidine nitrogen with small alkyl groups. This could
be effectively matched with urea substitutions on the benzamide
(derivatives 25 and 26) or the incorporation of a 3-amino group
(derivative 27). However, conversion of the piperidine nitrogen
to a non-basic moiety was unfavorable as observed for acetamide
28 (CCR2 IC50 = 463 nM) and carbamate 29 (CCR2 IC50 > 1000 nM).

R. J. Cherney et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5063–5065
5065
Table 3
Evaluation of 14 and 15 versus the E291A CCR2 mutant
a
a
Compound
WT CCR2 IC50 (nM)
E291A IC50 (nM)
E291A fold change
14
15
6.1
12
749.3
380.8
123
32
a
IC50 values are n = 1.
Glu291 (32-fold shift, Table 3). This represents a departure from
the cyclohexyl derivatives, which did not have a large reliance on
Glu291 when examined in the E291A mutant, as reported
previously.10
In conclusion, we have described the synthesis and evaluation
of novel piperidine derivatives as potent CCR2 antagonists. As ob-
served for the comparison of cyclohexane 2 to piperidine 14, addi-
tion of the piperidine nitrogen alone can afford a significant
enhancement in CCR2 affinity. From a binding study with the
E291A CCR2 mutant, this enhanced affinity appears to be attrib-
uted to an interaction with Glu291 within the CCR2 receptor.
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2; (b) i—crotyl bromide, K2CO3,
CH3CN; ii—H2, Pd/C, MeOH; (c) 4-methylthiobenzaldehyde, NaBH3CN, ZnCl2, MeOH;
(d) 4-methylthiobenzoic acid, NMM, HATU, DMF.
Unlike the piperidine nitrogen, the benzyl amine could be con-
verted to an amide and retain significant CCR2 affinity. As shown in
Table 2, the unsubstituted piperidine derivative 15 lost only 4-fold
in CCR2 affinity when compared to its benzyl amine analog 14. The
amide derivatives with small N-alkyl piperidines were essentially
equipotent to their benzyl amine counterparts as shown by N-bu-
tyl 19, N-methyl 30, and N-propyl 31. As before, these derivatives
lost all CCR2 activity when a carbamate was installed on the piper-
idine nitrogen (see derivative 32).
Acknowledgments
We thank Mr. Dayton T. Meyer and Ms. Ruowei Mo for several
of the benzoic acids used in this work. We also thank Dr. Joel
C. Barrish for a critical review of the manuscript.
References and notes
Given the 180-fold enhancement in CCR2 binding affinity of
piperidine 14 as compared to the cyclohexyl derivative 2, it was
suspected that the piperidine nitrogen of 14 was making a new
contact within the CCR2 receptor. In order to gain additional in-
sight into this interaction, we examined the binding of piperidine
14 in a CCR2 receptor mutant. Of the GPCRs, chemokine receptors
are unique in that they contain a conserved glutamic acid (Glu) in
transmembrane domain-7 (TM-7).16 In CCR2, this is Glu291, which
has been shown to be critical for small molecule antagonist bind-
ing via site-directed receptor mutagenesis with the Glu291 to
Ala291 mutant (E291A).17 Piperidine 14 was examined in
wild-type CCR2 and the E291A mutant in order to assess the
involvement of Glu291 in its binding (Table 3). As evident from
the 123-fold change in binding between wild-type CCR2 and the
E291A mutant, piperidine 14 had a significant reliance on Glu291
for its binding affinity. A second derivative, 15, was examined in
the E291A mutant, and it also displayed a large reliance on
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b
Compound
X
R1
IC50 (nM)
Ca flux
CCR3
binding %
inh. at
CCR2
binding
CTX monoc
10 l
Md
15
19
30
31
32
NH
2-NH2
28 7.1 (2) 75 0.7 (2) 109 58.0 (2) 19.7
NBu 2-NH2
NMe 2-NH2
NPr 3-NH2
4.3 0.73 (3) 13 (1)
1.8 0.3 (2) 2 (1)
6.2 0.9 (2) 6.0 (1)
17.5 2.1 (2) 77
38.5 9.2 (2) 43.1
25.0 19.8 (2) NT
NBoc 2-NHBoc 0% at 1
lM
NT
NT
NT
a
Compounds are racemic, one enantiomer is displayed for illustrative purposes.
IC50 values (n) are displayed as mean SD (n = 2) and mean SEM (n > 2).
CTX mono, chemotaxis in monocytes.
b
c
d
CCR3 % inhibition are n = 1. NT, not tested.