Novel, orally bioavailable γ-aminoamide CC chemokine receptor 2 (CCR2) antagonists

Article abstract of DOI:10.1021/jm060439n

Through modification of a screening hit we have discovered a structurally distinct new lead, (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluorophenyl)-4- (4-phenylpiperidin-1-yl)butanamide (11), which has subsequently served as the departure point for an

Full text of DOI:10.1021/jm060439n

© Copyright 2006 by the American Chemical Society
Volume 49, Number 16
August 10, 2006
Letters
Novel, Orally Bioavailable γ-Aminoamide CC
Chemokine Receptor 2 (CCR2) Antagonists
Alexander Pasternak,*,† Dominick Marino,†
Pasquale P. Vicario,‡ Julia Marie Ayala,‡
Margaret A. Cascierri,‡ William Parsons,† Sander G. Mills,†
Malcolm MacCoss,† and Lihu Yang†
Department of Medicinal Chemistry and Department of
Immunology/Rheumatology, Merck Research Laboratories,
Rahway, New Jersey 07065
ReceiVed April 12, 2006
Abstract: Through modification of a screening hit we have discovered
a structurally distinct new lead, (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-
2-(4-fluorophenyl)-4-(4-phenylpiperidin-1-yl)butanamide (11), which
has subsequently served as the departure point for an ongoing program
targeting CCR2 antagonists. Optimization of 11 leading to antagonists
26 and 37 is described. Antagonist 26 was shown to have good oral
bioavailability in rats. Antagonist 37 had a CCR2 IC50 of 59 nM and
excellent potency in a functional assay measuring inhibition of MCP-1
induced monocyte chemotaxis (IC50 of 41 nM).
Figure 1. Leads 1 and 2 and target analogues 3 and 4.
this elevated expression can be reduced by administration of
antiarthritic drugs.4b Relating to atherosclerosis, studies con-
ducted with MCP-1 and CCR2 knockout mice maintained on
high-fat diets showed reductions in aortic lesion size, macroph-
age content, and necrosis.5 Consequently, the therapeutic
potential of CCR2 antagonists for treating inflammatory diseases
has stimulated considerable interest. Several CCR2 antagonists
have already been described in the literature.6 Most of these
have one or more disadvantages, such as poor binding affinity
and/or functional activity,6e undisclosed pharmacokinetic
properties,6b-e and off-target activities.6b,c This communication
describes the discovery of a new class of potent, orally
bioavailable CCR2 antagonists based on a γ-aminoamide core
4 (Figure 1).
Screening of the Merck sample collection identified 1 as a
potential lead with a CCR2 IC50 of 720 nM.7 Subsequent
modification of 1 revealed that incorporation of a piperidine
moiety in place of N,N-dimethylamine results in improved
binding affinity (2, IC50 ) 180 nM).6a Further optimization of
2 led to additional potency enhancements, but pharmacokinetic
studies in rats revealed rapid clearance as an issue for this
series.6a To address this concern, we set out to explore
modifications to the backbone of 2 by replacing the R-amine
with a carbon atom (3a) or by removing it entirely with an
associated shortening of the amide to amine chain length (4a).
In addition, because of the structural similarity of the resulting
analogues with CCR5 antagonists studied within our laborato-
Chemokines (chemotactic cytokines) are structurally related
small proteins that play an important role in leukocyte migration
and activation.1 Most chemokines are classified as belonging
to one of two major subfamilies that are distinguished on the
basis of the arrangement of the first two conserved cysteines in
their sequences: the CC family that contains adjacent cysteines;
the CXC family in which the cysteines are separated by a single
intervening amino acid. Monocyte chemoattractant protein-1
(MCP-1, CCL2), included within the CC subfamily of chemok-
ines, mediates chemotaxis of monocytes to inflammatory sites
primarily through interactions with its CC chemokine receptor
2 (CCR2).2 CCR2 is a member of the G-protein-coupled seven-
transmembrane receptor superfamily and is expressed on most
blood borne monocytes. Recent studies have linked MCP-1 and
CCR2 to various inflammatory diseases3 including rheumatoid
arthritis (RA), atherosclerosis, and multiple sclerosis. Notably,
MCP-1 expression is elevated in RA inflamed synovium,4 and
* To whom correspondence should be addressed. Phone: 732-594-3847.
Fax: 732-594-3007. E-mail: alexander_pasternak@merck.com.
† Department of Medicinal Chemistry.
‡ Department of Immunology/Rheumatology.
10.1021/jm060439n CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/11/2006

4802 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Letters
Table 2. Binding Affinities7 for Analogues of 11
Scheme 1. Preparation of Three-Carbon- and
Four-Carbon-Chain Analoguesa
a (a) 3,5-Bis(trifluoromethyl)benzylamine, EDC, HOBt, DIEA, DCM,
room temp; (b) O3, DCM, -78 °C; DMS, -78 °C to room temp; (c) BH3,
THF; NaBO3; (d) (COCl)2, DMSO, Et3N, DCM, -78 °C; (e) HNR2,
NaB(OAc)3H, 4 Å molecular sieves powder, room temp.
Table 1. Binding Affinities7 for 1, 2, and Carbon Chain Analogs with
and without Piperidine 4-Substitution
a SD given when IC50 is an average of three or more determinations.
Otherwise one to two determinations were performed. b % inhibition at 1
µM. c A mixture of two diastereomers.
cessor 4a. No potency enhancement was observed in the four-
carbon chain length series (12), having the same chain length
as our original lead 1. Similarly, incorporation of 4-phenylpi-
peridine into the diamine lead decreased potency relative to
simple piperidine analogue 2, suggesting a different SAR pattern
(15). Both analogues prepared using benzyl allyl(piperidin-4-
yl)carbamate (13, 14) were inactive, implying divergent SAR
from the CCR5 program. Interestingly, despite the structural
similarity of 11 with some of the CCR5 compounds studied at
Merck, no hits from the CCR5 program were found in our
original screening for CCR2. Finally, we evaluated the impor-
tance of stereochemistry R to the amide carbonyl by preparing
16 (Table 1) and found that, as with the original lead, the
preferred absolute stereochemistry is S.
We explored variations of 11, focusing on the phenylpiperi-
dine moiety (Table 2). Compounds 17-19 show that the
piperidine ring size is best. The closely related piperazine-based
20 was inactive; however, installation of a 3-methyl group on
the piperazine partially restored potency (21). The fully saturated
form of 11 was inactive (22). Some analogues with heterocycles
(23) were tolerated. Analogues in which the 4-phenyl group of
11 was relocated (24, 25) were less potent. The conformationally
locked spiroindenylpiperidine analogue 26 was 2-fold more
potent than 11. The closely related spiroindanylpiperidine 27
was less potent. The conformationally locked trans-1,2,3,4,-
4a,5,6,10b-octahydrobenzo[f]isoquinoline10 analogue 28 was
inactive.
Concurrent with the phenylpiperidine SAR described above,
we explored incorporation of methyl groups in positions along
the γ-aminoamide backbone and on the piperidine ring to probe
the effect of conformational changes (Table 3). Incorporation
of methyl groups in all positions of the γ-aminoamide backbone
(29-31) led to decreases in potency, even when taking into
account the presence of multiple analogue isomers. When a
methyl was incorporated into the 3-position of the piperidine,
the potency depended on the relative stereochemistry; the
mixture of two cis isomers (32) was less potent than 11, but
the mixture of two trans isomers (33) was at least equally potent
to 11.
a SD given when IC50 is an average of three or more determinations.
Otherwise one to two determinations were performed. b H in place of F
has little effect on IC50; replacing F with H in 2 gives an IC50 of 0.22 µM.
c % inhibition at 1 µM.
ries, we decided to incorporate several of the optimized
substituted piperidines found in those antagonists.8
Analogues were prepared as described in Scheme 1. The
requisite scalemic acid (S)-5 was synthesized according to
known methodology.9 Standard peptide coupling of (S)-5 with
3,5-bis(trifluoromethyl)benzylamine provided amide 6. Elabora-
tion into homologous aldehydes 7 and 8 was accomplished by
ozonolysis and by a hydroboration-oxidation, Swern oxidation
sequence, respectively. Compounds 7 and 8 are unstable and
slowly decompose, even when stored in a freezer. Reaction of
7 and 8 with various amines in the presence of sodium
triacetoxyborohydride gave the target analogues 9 and 10 in
modest yields.
Neither 3a nor 4a (Table 1), which directly correspond to
lead 2 with four- and three-carbon amine to amide chain lengths,
respectively, was as potent as 2. Two optimized 4-substituted
piperidines from our CCR5 program (4-phenylpiperidine8a and
benzyl allyl(piperidin-4-yl)carbamate8b) were incorporated in
both chain length series to make analogues 11-14. Only 11,
with 4-phenylpiperidine in the three-carbon chain length series,
had improved potency over its unsubstituted piperidine prede-

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4803
Scheme 2a
Table 3. Incorporation of Methyl at Various Positions
a (a) Ethanolamine (8 equiv) 0 °C to room temp; (b) Boc2O, DCM, room
temp; (c) MsCl, Et3N, DMAP (cat.), DCM, 0 °C; (d) LHMDS (2.1 equiv),
THF, 0 °C to room temp; (e) recrystallization from hot hexanes; (f)
anhydrous 4 N HCl/dioxane, room temp.
a SD given when IC50 is an average of three or more determinations.
Otherwise one to two determinations were performed. b Racemate. c Mixture
of all four diastereomers. d cis-Piperidine, mixture of two diastereomers.
e trans-Piperidine, mixture of two diastereomers. f % inhibition at 1 µM.
Table 5. CCR2 and CCR515 Affinities of 11, 26, and 37
Table 4. Spiroindenylpiperidine Analoguesa
40. Following Boc removal, piperidine 41 was incorporated
according to Scheme 1 to make analogue 37. The requisite
piperidine for analogue 36 was prepared in an identical fashion
starting from (S)-(-)-propylene oxide.
Table 4 shows that the (R,R,S)-isomer 37 was more potent
in our binding assay, but its diastereomer 36 retained significant
potency. Table 4 also shows that milestone analogues 11, 26,
and 37 are all functional antagonists of CCR2 in that they inhibit
MCP-1 stimulated recruitment of monocytes.13 Furthermore, in
the progression from 11 to 26 and 37, the potency enhancements
are even more pronounced when measured in the chemotaxis
functional assay. Compound 11 inhibits chemotaxis by only 40%
at 1 µM, while optimized 37 inhibits chemotaxis with an IC50
of 41 nM. Later-generation analogues incorporating spiropip-
eridine 41 and its (S,S)-isomer confirmed that CCR2 antagonists
containing the 41-derived subunit are consistently more potent
in binding and functional assays.14
Because our original lead 1 came from the Merck NK1
antagonist program, we wanted to evaluate if analogues 11, 26,
and 37 retained NK1 binding affinity. Table 4 shows that all
three retain significant potency in the NK1 assay. Encouragingly,
in the progression from 11 to 26 and 37, the selectivity shifts
from much greater potency in NK1 to more evenly balanced
activities. Subsequent reports from our laboratories will describe
how we have tuned out the NK1 activity from later-generation
analogues of 37.14
a SD given when IC50 is an average of three or more determinations.
Otherwise one to two determinations were performed. b Inhibition of MCP-1
stimulated monocyte chemotaxis.13 c A mixture of two trans diastereomers.
Another consideration about selectivity involves the possible
binding affinity of 11, 26, and 37 to other chemokine receptors.
To evaluate this, we screened these three compounds against
other chemokine receptors and found them to be highly selective
against all the receptors tested (CCR1, CCR3, CXCR3, CCR4,
CXCR4, CCR8) except CCR5, where they demonstrated
significant potency (Table 5).15 The difficulty in separating
parallel CCR2 and CCR5 binding affinities is precedented.6b,c
Although our aim was to find a completely selective antagonist
of CCR2, a dual CCR2/CCR5 antagonist has intriguing pos-
sibilities in that both receptors are thought to play a role in RA
pathogenesis.4c Later publications from our group will detail
modifications of 37 leading to improved selectivity over
CCR5.14
We then decided to combine the two potency enhancing/
retaining discoveries outlined above by incorporating the known
trans-3-methyl-4,4′-spiroindenylpiperidine.11 The resulting 34
(Table 4) was approximately 2-fold more potent than 26 and
4-fold more potent than lead 11, suggesting an additive effect
of combining the spiroindenylpiperidine with the trans-3-methyl
substituent. Increasing the steric bulk at the piperidine 3-position
(35) reduced potency.12 The single enantiomers of trans-3-
methyl-4,4′-spiroindenylpiperidine were prepared (Scheme 2)
and incorporated into final target analogues 36 and 37 to
determine the importance of absolute stereochemistry. According
to Scheme 2, (R)-(+)-propylene oxide was treated with an
excess of ethanolamine. The resulting ring-opened product was
protected (Boc2O) to give 38. Conversion to bis-mesylate 39
followed by reaction with indene in the presence of 2 equiv of
LHMDS gave 40 as a 3:1 mixture of trans/cis isomers (53%
two steps). Crystallization from hot hexanes provided pure trans-
Of the three milestone CCR2 antagonists described herein,
only one has been evaluated in a rat pharmacokinetic model.
Compound 26 had good oral bioavailability in rats at 3 mpk

4804 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Letters
(6) For reviews covering published and patented CCR2 antagonists, see
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Morriello, G.; Butora, G.; Pasternak, A.; Parsons, W. H.; Mills, S.
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S.; Hanlon, W. A.; Cascieri, M. A.; Springer, M. S. Discovery of
3,3-bis(trifluoromethyl)benzyl L-arylglycinamide based potent CCR2
antagonists. Bioorg. Med. Chem. Lett., in press. (b) Forbes, I. T.;
Cooper, D. G.; Dodds, E. K.; Hickey, D. M. B.; Ife, R. J.; Meeson,
M.; Stockley, M.; Berkhout, T. A.; Gohil, J.; Groot, P. H. E.; Moores,
K. CCR2b receptor antagonists: conversion of a weak HTS hit to a
potent lead compound. Bioorg. Med. Chem. Lett. 2000, 10, 1803-
1806. (c) Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.;
Sawada, H.; Iizawa, Y.; Shuraishi, M.; Aramaki, Y.; Okonogi, K.;
Ogawa, Y.; Meguro, K.; Fulino, M. A small-molecule, nonpeptide
CCR5 antagonist with highly potent and selective anti-HIV-1 activity.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5698-5703. (d) Kettle, J.
G.; Faull, A. W.; Barker, A. J.; Davies, D. H.; Stone, M. A.
N-Benzylindole-2-carboxylic acids: potent functional antagonists of
the CCR2b chemokine receptor. Bioorg. Med. Chem. Lett. 2004, 14,
405-408. (e) Mirzadegan, T.; Diehl, F.; Ebi, B.; Bhakta, S.; Polsky,
I.; McCarley, D.; Mulkins, M.; Weatherhead, G. S.; Lapierre, J.-M.;
Dankwardt, J.; Morgans, D., Jr.; Wilhelm, R.; Jarnagin, K. Identifica-
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(7) Radioligand competition binding assay: CHO cells expressing human
CCR2b. Assay experimental details provided in Supporting Informa-
tion.
(8) (a) Finke, P. A.; Meurer, L. C.; Oates, B.; Mills, S. G.; MacCoss,
M.; Malkowitz, L.; Springer, M. S.; Daugherty, B. L.; Gould, S. L.;
DeMartino, J. A.; Siciliano, S. J.; Carella, A.; Carver, G.; Holmes,
K.; Danzeinsen, R.; Hazuda, D.; Kessler, J.; Lineberger, J.; Miller,
M.; Schleif, W. A.; Emini, E. A. Antagonists of the human CCR5
receptor as anti-HIV-1 agents. Part 2: structure-activity relationships
for substituted 2-aryl-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-
(piperidin-1-yl)butanes. Bioorg. Med. Chem. Lett. 2001, 11, 265-
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Carella, A.; Carver, G.; Holmes, K.; Danzeinsen, R.; Hazuda, D.;
Kessler, J.; Lineberger, J.; Miller, M.; Schleif, W. A.; Emini, E. A.
Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part
4: synthesis and structure-activity relationships for 1-[N-(methyl)-
N-(phenylsufonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxy-
carbonyl)amino)piperidin-1-yl)butanes. Bioorg. Med. Chem. Lett.
2001, 11, 2475-2479.
and excellent overall pharmacokinetic characteristics (F ) 38%,
AUCpo ) 1.8 µM h kg mg-1, Cl ) 6 mL min-1 kg-1, t1/2 ) 8
h).
In summary, through modification of a screening lead from
our sample collection, we have discovered a structurally distinct
new lead, 11, which has subsequently served as the departure
point for an ongoing program targeting CCR2 antagonists.
Compound 11 was optimized by modifications to the 4-phe-
nylpiperidine moiety to spiroindenylpiperidine analogue 26 and
ultimately to trans-3-methyl-4,4′-spiroindenylpiperidine ana-
logue 37, which was potent in our CCR2 binding assay (IC50
) 59 nM) and in a functional assay measuring inhibition of
MCP-1 induced monocyte chemotaxis (IC50 ) 41 nM). CCR2
antagonists 11, 26, and 37 were selective against all other
chemokine receptors assayed with the exception of the CCR5
receptor where they had similar potency. All three compounds
also showed binding affinity for the NK1 receptor. Compound
26 had good oral bioavailability in rats. Subsequent reports from
our laboratories will detail further SAR studies in this series
that address enhancing potency and selectivity against NK1 and
CCR5.14
Acknowledgment. We thank Marc M. Kurtz and Kwei-Lan
C. Tsao for performing the NK1 assays on 11, 26, and 37, and
Bernard Kartsen Choi for HRMS analyses.
Supporting Information Available: Experimental details for
the synthesis and characterization of representative CCR2 antago-
nists 11, 26, and 37. This material is available free of charge via
the Internet at http://pubs.acs.org.
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JM060439N