Discovery of disubstituted cyclohexanes as a new class of CC chemokine receptor 2 antagonists

Article abstract of DOI:10.1021/jm701488f

We describe the design, synthesis, and evaluation of novel disubstituted cyclohexanes as potent CCR2 antagonists. Exploratory SAR studies led to the cis-disubstituted derivative 22, which displayed excellent binding affinity for CCR2 (binding IC₅₀

Full text of DOI:10.1021/jm701488f

J. Med. Chem. 2008, 51, 721–724
721
Scheme 1
Discovery of Disubstituted Cyclohexanes as a
New Class of CC Chemokine Receptor 2
Antagonists
Robert J. Cherney,* Ruowei Mo, Dayton T. Meyer,
David J. Nelson, Yvonne C. Lo, Gengjie Yang,
Peggy A. Scherle, Sandhya Mandlekar, Zelda R. Wasserman,
Heather Jezak, Kimberly A. Solomon, Andrew J. Tebben,
Percy H. Carter, and Carl P. Decicco
Research and DeVelopment, Bristol-Myers Squibb Company,
Princeton, New Jersey 08543-4000
ReceiVed NoVember 29, 2007
Abstract: We describe the design, synthesis, and evaluation of novel
disubstituted cyclohexanes as potent CCR2 antagonists. Exploratory
SAR studies led to the cis-disubstituted derivative 22, which displayed
excellent binding affinity for CCR2 (binding IC50 ) 5.1 nM) and potent
functional antagonism (calcium flux IC50 ) 18 nM and chemotaxis
IC50 ) 1 nM). Site-directed mutagenesis studies with 22 suggest the
compound is binding near the key receptor residue Glu291, however,
22 is not reliant on Glu291 for its binding affinity.
Figure 1. Overlay of Teijin/CombiChem derivative 1 (yellow) and
compound 2 (cyan).
case, we hypothesized that the two highlighted hydrogens (see
Scheme 1) of the pyrrolidine might project toward solvent,
hence, identifying an area that could accommodate a new
constraining factor. If a cyclohexane was selected as the
constraining factor, and the pyrrolidine ring was excised along
the dotted lines, this would reveal a trans-1,2-disubstituted
cyclohexane 2. As shown by the overlay of 1 and 2 (Figure 1),
the two basic amines come in close proximity to one another;
however, the benzyl component appears to be in need of
optimization.
Chemokines are small molecular weight proteins implicated
in inflammatory and autoimmune processes through their role
as leukocyte chemoattractants.1 To date, over 50 chemokines
have been identified, with the majority belonging to two major
families (CC and CXC).2 We have been interested in a CC
chemokine family member, monocyte chemoattractant protein-1
(MCP-1a or CCL2),3 which is produced by a variety of cells in
response to inflammatory stimuli. MCP-1 elicits a functional
response by binding to its receptor, CC chemokine receptor 2
(CCR2), which is a member of the G-protein coupled receptor
family (GPCR).4 The CCR2/MCP-1 pair plays a major role in
the activation and migration of monocytes from the circulation
to sites of inflammation within the tissues. Both MCP-1 and
CCR2 have been implicated in a host of diseases that are
characterized by monocyte accumulation, including rheumatoid
arthritis,5 atherosclerosis,6 and multiple sclerosis,7 and insulin
resistance.8 As a result, there has been much interest in the
design and synthesis of CCR2 antagonists as potential thera-
peutics.9 In this communication, we describe the discovery of
disubstituted cyclohexane derivatives as novel and potent CCR2
antagonists.
As members of the GPCR family, chemokine receptors
contain the common 7-transmembrane (7-TM) core as the main
structural unit. However, chemokine receptors are unique in that
they contain a conserved glutamic acid (Glu) in TM7.10 In
CCR2, this is Glu291, which has been shown to be critical for
small molecule antagonist binding via site-directed mutagen-
esis.11 Included in this research11a was a Teijin/CombiChem
compound 1,12 which was shown to have a large reliance on
Glu291 for CCR2 binding, presumably through an interaction
with the basic pyrrolidine nitrogen. We became interested in
the binding of 1 and considered several binding modes. In one
To test our hypothesis, we evaluated several trans-disubsti-
tuted cyclohexanes using a radiolabeled MCP-1 displacement
assay in peripheral blood mononuclear cells (PBMCs) to assess
their binding affinity for CCR2 (see Table 1).13 We were
interested in selective CCR2 antagonists and, hence, used a
CCR3 binding assay14 for an initial assessment of selectivity
over a chemokine family member. The trans-disubstituted core
2 did show activity in the CCR2 binding assay as the
2,4-dimethyl derivative 3 had a CCR2 IC50 of 0.54 µM and
displayed good selectivity versus CCR3. However, conversion
of the secondary amine to the more basic tertiary amine 4 was
not advantageous for the CCR2 affinity, contrary to what one
would expect for a pyrrolidine mimetic. Modifying the 2,4-
dimethyl substitution pattern was met with some difficulty, as
the addition of a third methyl to the 2,4,6-derivative 5 gave a
2-fold loss in activity relative to 3. Likewise, the 2,4-dichloro
derivative 6 and the 4-chloro derivative 7 both lost affinity for
CCR2 as compared to 3. Not surprisingly, the amide 8 lost
substantial affinity (12-fold) for CCR2 when compared to 7.
We focused our optimization on the benzylamine portion of
the molecule and next explored the racemic cis-disubstituted
cyclohexanes shown in Table 2. The cis-2,4-dimethyl derivative
9 was 4-fold less active for CCR2 relative to the same trans-
derivative 3. However, exploration of monosubstitution at the
4-postion was promising, as several derivatives (10-13)
displayed good affinity for CCR2 with promising (∼20-fold)
selectivity versus CCR3. The best compound of the series was
the 4-isopropyl derivative 13 with a CCR2 binding IC50 of
0.36 µM.
* To whom correspondence should be addressed. Phone: 609-252-3066.
Fax: 609-252-7446 . E-mail: robert.cherney@bms.com.
a
Abbreviations: MCP-1, monocyte chemoattractant protein-1; CCL2,
chemokine (C-C motif) ligand 2; CCR-2, CC chemokine receptor 2; GPCR,
G-protein coupled receptor; 7-TM, 7-transmembrane; CTX, chemotaxis.
10.1021/jm701488f CCC: $40.75
2008 American Chemical Society
Published on Web 01/31/2008

722 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Table 1. Evaluation of trans-Disubstituted Derivatives
Letters
pound 16 was also tested in the chemotaxis assay13 and it was
found to have good activity (CTX IC50 ) 160 nM). Continuing
at the 4-position, both the 4-thiomethyl derivative 17 and the
4-sulfonylmethyl derivative 18 lost affinity for CCR2, whereas
the sulfonamide derivative 19 gained some binding affinity as
compared to 16. To achieve additional affinity, we turned our
attention to the 2-position of the glycinamide benzamide.12 The
2-amino compound 20 did not enhance the binding affinity for
CCR2 as compared to 19, but it did improve the potency in the
chemotaxis assay by 2-fold and the calcium assay by 8-fold
relative to 19. In fact, we found that large groups in this position
could have a dramatic enhancing effect on chemotaxis potency.16
For example, the cyclohexyl amide derivative 21 had a 2-fold
increase in CCR2 binding affinity as compared with 20, but its
chemotaxis potency increased 126-fold.17 Likewise, the urea
derivative 22 had a 4-fold increase in binding affinity relative
to 20, while its chemotaxis potency increased by 63-fold.
CCR2 binding
IC50 (µM)
CCR3 binding
a
cmpd
R
X
R1
H
% inh at 10 µMb
3
4
5
6
7
8
2,4-diMe
2,4-diMe
2,4,6-triMe H,H
2,4-diCl
4-Cl
H,H
0.54 ( 0.24 (2)
H,H Me 3.01 ( 3.14 (2)
54%
NTc
NT
NT
50%
NT
H
H
H
H
1.19 ( 0.84 (2)
7.86 ( 1.78 (2)
0.97 ( 0.65 (3)
12 (1)
H,H
H,H
O
4-Cl
a IC50 values (n) are displayed as mean ( SD (n ) 2) and mean ( SEM
(n > 2). b CCR3 % inhibition are n ) 1. c NT ) not tested.
Table 2. Evaluation of cis-Disubstituted Derivativesa
As nonbasic antagonists, these compounds are rare in the
CCR2 field,18 given the previously mentioned reliance on
Glu291 for antagonist binding.10,11 To probe the reliance of our
compounds for Glu291 and obtain information on their place-
ment within the receptor, we investigated the binding of three
compounds with single-site receptor mutants of CCR2 (Table
4). The first receptor mutant studied was the well characterized
Glu291 to Ala291 (E291A).19 Pyrrolidine 1 had a large reliance
on Glu291 for binding and showed a 100-fold shift in binding
affinity between wild-type and the E291A mutant.11a Our benzyl
amine compound 10 displayed a negligible change in binding
affinity (8-fold) when compared in wild-type and the E291A
mutant, as did the amide 14. Our most potent compound 22
was tested in the E291A mutant and it did not exhibit a shift in
the binding affinity, indicating that even 22 had no reliance on
Glu291 for CCR2 binding. Hence, the cis-amides did not have
a reliance on Glu291. As Glu291 is conserved across the
chemokine receptors, we felt this was a positive step toward
achieving selectivity over other chemokine family members. It
also differentiated our compounds from pyrrolidine 1, however,
it did not offer any information as to the placement of our
compounds within the receptor. For this reason, we also tested
compound 22 in a second mutant Thr292 to Ala292 (T292A),
which is the residue adjacent to Glu291. This mutant was
previously described,11a and pyrrolidine 1 was reported to have
a 30-fold binding shift when tested in the T292A mutant as
CCR2 binding
IC50 (µM)
CCR3 binding
b
cmpd
R
% inh at 10 µMc
9
2,4-diMe
4-Cl
4-OCF3
4-CF3
2.17 (1)
54
24
50
47
55
10
11
12
13
0.79 ( 0.46
0.53 ( 0.0042
0.40 ( 0.096
0.36 ( 0.20
4-i-Pr
a Compounds are racemic, one enantiomer is displayed for illustrative
purposes. b IC50 values are displayed as mean ( SD (n ) 2) c CCR3 %
inhibition are n ) 1.
As in the trans-series, we subsequently made cis-amides (see
Table 3), and compound 14 displayed weak CCR2 activity
(CCR2 binding IC50 ) 4.13 µM). Surprisingly, the 4-methyl
derivative 15 provided a 7-fold gain in affinity, and the 4-iodo
derivative 16 gave a 100-fold increase in affinity for CCR2 when
compared to 14. As a result of this activity, compound 16 was
tested in the calcium flux assay (a functional assay based in
PBMCs)13 and gave an IC50 of 30 nM in the presence of MCP-
1. Calcium flux inhibition was unchanged when the incubation
with 16 was performed without MCP-1 (in the presence of 16
only), thus validating compound 16 as an antagonist.15 Com-
Table 3. Evaluation of cis-Diamide Derivativesa
b
IC50 (µM)
CCR3 binding
cmpd
R
R1
CCR2 binding
4.13 (1)
0.55 ( 0.16 (2)
0.037 ( 0.0071 (2)
0.064 ( 0.023 (2)
0.087 ( 0.051 (2)
0.021 (1)
0.023 ( 0.0045 (6)
0.009 ( 0.0027 (6)
0.0051 ( 0.0036 (2)
Ca flux
CTX mono
% inh at 10 µMc
14
15
16
17
18
19
20
21
22
2,4-diMe
4-Me
4-I
H
H
H
H
H
H
NH2
NTd
NT
NT
NT
NT
57
27
31
3
39
6
10
37
0.030 (1)
0.215 ( 0.035 (2)
0.16 ( 0.0081 (2)
1.2 (1)
4-SMe
4-SO2Me
4-SO2NH2
4-SO2NH2
4-SO2NH2
4-SMe
NT
NT
0.042 ( 0.0028 (2)
0.0048 ( 0.0011 (4)
0.0037 ( 0.0017 (3)
0.018 ( 0.00071 (2)
0.13 ( 0.16 (2)
0.063 ( 0.033 (2)
0.0005 ( 0.0001 (4)
0.001 ( 0.0002 (2)
NHCOC6H11
NHCONHi-Pr
a Compounds are racemic, one enantiomer is displayed for illustrative purposes. b IC50 values (n) are displayed as mean ( SD (n ) 2) and mean ( SEM
(n > 2). c CCR3 % inhibition are n ) 1. d NT ) not tested.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 723
Table 4. Evaluation of Derivatives Versus Mutant CCR2 Receptors
Glu291 pocket. Hence, the CCR2 affinity has been optimized
for these compounds without utilizing an interaction to Glu291.
This strategy has not been widely described, as nonbasic CCR2
antagonists are uncommon.
E291A
fold
change
T292A
fold
change
a
a
a
WT CCR2 IC50 E291A IC50
T292A IC50
(nM)
cmpd
(nM)
(nM)
10
14
22
268 (1)
4130 ( 1089 (2) 7847 (1)
7.5 ( 1.9 (3) 3.7 (1)
2117.2 (1)
7.9
1.9
0.5
NTb
NT
303.7 (1)
NT
NT
40.5
Acknowledgment. The authors thank Dr. Joel C. Barrish
for a critical review of the manuscript.
a IC50 values (n) are displayed as mean ( SD (n ) 2) and mean ( SEM
(n > 2). b NT ) not tested.
Supporting Information Available: Compound characterization
data for all target compounds and experimental details for the
representative synthesis of Scheme 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
Scheme 2. Representative Synthesisa
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Scheme 2 for the compounds of Table 1. The purchased diamine
23 was selectively protected as the tert-butyl carbamate 24.20
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JM701488F