Synthesis, structure-activity relationship and in vivo antiinflammatory efficacy of substituted dipiperidines as CCR2 antagonists

Article abstract of DOI:10.1021/jm070902b

A series of substituted dipiperidine compounds have been synthesized and identified as selective CCR2 antagonists. Combining the most favorable substituents led to the discovery of remarkably potent CCR2 antagonists displaying IC₅₀ values in th

Full text of DOI:10.1021/jm070902b

J. Med. Chem. 2007, 50, 5561-5563
5561
Synthesis, Structure-Activity Relationship and in
Vivo Antiinflammatory Efficacy of Substituted
Dipiperidines as CCR2 Antagonists
Mingde Xia,* Cuifen Hou, Duane E. DeMong,
Scott R. Pollack, Meng Pan, James A. Brackley,
Nareshkumar Jain, Chrissy Gerchak, Monica Singer,
Ravi Malaviya, Michele Matheis, Gil Olini,
Druie Cavender, and Michael Wachter
Figure 1.
Table 1. Functional Group Effect on CCR2 Binding Affinity
Drug DiscoVery, Johnson & Johnson Pharmaceutical Research and
DeVelopment, L.L.C., 8 Clarke DriVe, Cranbury, New Jersey 08512
ReceiVed July 25, 2007
compd
R
CCR2 IC50 (nM)
1a
1b
1c
1d
CO2Me
CONH2
H
3300 ( 400
1800 ( 100
470 ( 170
5 ( 2
Abstract: A series of substituted dipiperidine compounds have been
synthesized and identified as selective CCR2 antagonists. Combining
the most favorable substituents led to the discovery of remarkably potent
CCR2 antagonists displaying IC50 values in the nanomolar range.
Compound 7a had outstanding selectivity over CCR1, CCR3, CCR4,
CCR5, CCR6, CCR7, and CCR8 and showed excellent efficacy in
adjuvant-induced arthritis model, collagen-induced arthritis model, and
allergic asthma model.
CO2H
Scheme 1. Synthesis of Carboxylic Acid Analoguesa
Monocyte chemoattractant protein-1 (MCP-1) is a major
chemoattractant for monocytes and memory T cells through
binding to its specific cell-surface receptor, CC-chemokine
receptor-2 (CCR2). Animal model studies of chronic inflam-
matory diseases have demonstrated that inhibition of binding
between MCP-1 and CCR2 by an antagonist suppresses the
inflammatory response. MCP-1 and its receptor CCR2 have been
implicated in inflammatory disease pathologies such as uveitis,
rheumatoid arthritis, multiple sclerosis, allergic rhinitis, chronic
obstructive pulmonary disease (COPD), allergic asthma, and
solid tumors.1-8
Monocyte migration is inhibited by MCP-1 antagonists (either
antibodies or soluble, inactive fragments of MCP-1) that have
been shown to inhibit the development of arthritis, asthma, and
uveitis. MCP-1 and CCR2 knockout (KO) mice have demon-
strated that monocyte infiltration into inflammatory lesions is
significantly decreased.9,10 In addition, such knockout mice are
resistant to the development of experimental allergic encepha-
lomyelitis (EAE, a murine model of human multiple sclerosis),
cockroach allergen-induced asthma, atherosclerosis, and uveitis.
Rheumatoid arthritis and Crohn’s disease patients have dem-
onstrated a reduction in symptoms during the treatment with
TNF-R antagonists (e.g., monoclonal antibodies and soluble
receptors) at dose levels that correlated with decreases in MCP-1
expression and the number of infiltrating macrophages.11
MCP-1 has been implicated in the pathogenesis of seasonal
and chronic allergic rhinitis, having been found in the nasal
mucosa of most patients with dust mite allergies. MCP-1 has
also been found to induce histamine release from basophils in
vitro. During allergic conditions, allergens and histamines have
been shown to trigger (i.e., to up-regulate) the expression of
MCP-1 and other chemokines in the nasal mucosa of people
with allergic rhinitis, suggesting the presence of a positive
feedback loop in such patients.
a (i) LHMDS, TMSCl, -78 °C, then Br2, 77%; (ii) LiOH, 66%; (iii)
substituted indolepiperidine, CH3CN, TEA, reflux, 27-70%; (iv) (a) HCl,
(b) ArCHn ) CHnCOCl or ArNCO, 60-90%.
regarding the discovery of CCR2 antagonists have been
published to date.12-24
Recently, we disclosed a series of potent phenylpiperidine-
based CCR2 antagonists. Those compounds demonstrated good
selectivity over CCR1, CCR3, and 5-HT, an excellent cyto-
chrome P450 profile, and reasonable pharmacokinetics.25
Our search for more potent CCR2 antagonists led to the
discovery of a series of substituted dipiperidines exemplified
by the structure shown in Figure 1. In this communication, we
report the synthesis, structure-activity relationships, and anti-
inflammatory activities of these compounds.
Initial SAR studies on the CH2 linker between the two
piperidine moieties revealed that carboxylic acid derivative 1d
showed a marked improvement in binding affinity to the human
CCR2 receptor relative to the unsubstituted, amide, and ester
analogues (Table 1). These results encouraged us to further
explore the SAR surrounding the carboxylic acid-methylene-
linked bispiperidine series. The synthesis of these carboxylic
acid analogues is outlined in Scheme 1.
Boc-protected piperidin-4-ylacetic acid methyl ester 2 was
converted to the R-bromoacid 4 through bromination (LHMDS/
TMSCl, Br2) and ester hydrolysis. Bromoacid 4 was refluxed
with the desired substituted indole piperidine in acetonitrile to
afford 5, which was deprotected and then reacted with a suitable
acid chloride or isocyanate to give substituted dipiperidineacetic
acid analogues 6a-z.
There remains a need for small-molecule CCR2 antagonists
for preventing, treating, or ameliorating a CCR2-mediated
inflammatory syndrome, disorder, or disease. Many reports
* To whom correspondence should be addressed. Phone: (609) 409-
3485. Fax: (609) 655-6930. E-mail: mxia@prdus.jnj.com.
10.1021/jm070902b CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/11/2007

5562 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Table 2. CCR2 Binding Affinities of Carboxylic Acid Analogues
Letters
antagonized the MCP-1-induced effect with an IC50 of 2 nM.
A similar IC50 was observed when the MCP-1-induced flux of
Ca2+ ions was measured instead of chemotaxis. This compound
did not significantly inhibit the binding of relevant chemokines
to CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, or CCR8 at 25
µM and showed excellent specificity for CCR2. To further
investigate the specificity, 7a was tested for its ability to inhibit
the binding of relevant ligands to many other GPCRs, and to
various ion channels, in the Cerep panel. At the screening
concentration of 1 µM (∼250 times its IC50 for CCR2), it did
not inhibit binding to any receptor by >30% except for the H1
receptor (41%), the 5-HT1B receptor (39%), the 5-HT2A
receptor (39%), and the NK3 receptor (31%).
b
compd
R1
Xa
R2
IC50
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
H
H
H
H
H
H
H
5-OH
2-CO2H
6-F
6-Cl
5-CH3O
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
NH
4-CH3O
4-NO2
4-CH3
660 ( 230
210 ( 90
115 ( 55
80 ( 9
20 ( 10
15 ( 5
4 ( 0.5
15 ( 5
15%c
20 ( 6
7.7 ( 1.3
16.7 ( 3.3
50 ( 10
6.3 ( 1.3
4 ( 0.6
2.5 ( 0.5
4 ( 0.5
44%c
H
3,5-diF
4-CF3
3,4-diCl
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,4,5-triF
3,5-diF
3,5-diF
2,3-diCl
H
Consistent with the relatively low degree of sequence
homology between mouse and human MCP-1 and between
mouse and human CCR2, 7a was significantly less potent when
tested in binding assays using 125I-mouse MCP-1 and either
mouse peripheral blood monocytes or a mouse monocytic cell
line (WEHI-265.1). Compound 7a had IC50 of 2 µM in these
assays, much higher than its IC50 in the hCCR2 membrane
binding assay and the human cellular-based assays. A similar
phenomenon was observed in the rat assay (IC50 ) 2 µM).
These species differences became critical when the compound
was used for in vivo studies in rodents. The compound was
dosed ip rather than po in order to attain the high systemic drug
levels necessary to overcome the relatively low affinity of the
compound for mouse and rat CCR2. The bioavailability of 7a
in rats was 15% with po (t1/2 ) 7 h) and 100% with ip at 10
mg/kg dosage (vehicle of 20% Solutol, 30% PEG400, and 50%
0.1 N NaHCO3).
5-F
5-CH3SO2NH
5-NH2
7-CH3O
2-CH3
1-CH3
1-CH3CO
H
H
H
H
H
H
H
3400
6800
1100
720
6t
6u
6v
6w
6x
6y
6z
NH
NH
NH
NH
NH
CH2CH2
3,5-diCl
4-CF3
3,4-diCl
4-OCF3
3,4-diCl
120
25 ( 5
10 ( 1
320
a All CHdCH are trans. b Data in nM. c At 25 µM.
Compound 7a was used for in vivo studies in the adjuvant-
induced arthritis and the collagen-induced arthritis models. In
the adjuvant-induced arthritis model, 7-week-old male Lewis
rats were injected in the right hind footpad with a mixture of
heat-killed Mycobacterium butyricum (0.5 mg) in liquid paraffin
oil (50 µL). An increase in volume of the contralateral
(noninjected) hind paw was used as a measure of arthritis
severity. Body weight and hind paw volume (as measured by
mercury plethysmography volume displacement) were typically
recorded on days 0, 3, 7, 10, 12, 14, and 16. Rats were dosed
with test compound 7a (ip, bid, 100 mg/kg) or with vehicle
alone from day 7 to day 14. Under these conditions, 7a inhibited
swelling of the contralateral paws by 94%. In a collagen-induced
arthritis model in mice, DBA1 mice were immunized with
bovine type II collagen on day 0, injected (sc) with lipopoly-
saccharide (LPS) on day 21, and dosed (ip, bid) with a test
compound at 25, 50, or 100 mg/kg from day 20 to day 35. Body
weight was monitored and clinical disease score recorded every
2-3 days starting on day 20. Compound 7a inhibited the
development of arthritis (clinical disease score on day 35) by
23%, 50%, and 79% at the 25, 50, and 100 mg/kg doses,
respectively.
Table 3. CCR2 Binding Affinities of the Different Enantiomers
compd
R1
R2
CCR2 IC50 (nM)
7a
7b
H
CO2H
CO2H
H
4 ( 2
210 ( 100
Table 2 lists the CCR2 binding affinities for carboxylic acid
analogues 6a-z shown in Scheme 1. Substitution of the indole
nitrogen (6r, 6s) was not tolerated, while the 2-position tolerated
a methyl group in preference to a carboxyl group (6q, 6i).
Generally, the 5, 6, and 7 positions on the indole ring tolerated
various functional groups (6j-p). Halogen or trifluoromethyl
substitution (6e-h) on the cinnamoylphenyl ring was preferred.
Urea analogues (6u, 6x) had lower affinity than the correspond-
ing cinnamoyl compounds (6d, 6g). Cinnamoyl (6g) and urea
(6x) analogues were more potent than the corresponding
phenylpropionamide compound (6z). The ortho position of the
phenyl ring (6t) was much less tolerant of substitution than the
meta or para position (6v, 6x).
The carboxylic acid series had one chiral center. The
racemates of key analogues were separated into enantiomers
by chiral preparative HPLC. For example, racemate 1d was
separated into (S)-enantiomer 7a and (R)-enantiomer 7b with a
Chiralpak AD column (eluent 85/15 CH3CN/CH3OH). The
absolute configuration was determined by X-ray crystallography.
Compound 7a had higher binding affinity than compound 7b
(Table 3).
Compound 7a was also tested in a mouse model of allergic
asthma for therapeutic effect on asthmatic response as a function
of airway inflammation and hyperresponsiveness. Airway
responsiveness was measured in unrestrained mice by non-
invasive whole body plethysmography using a BioSystem
plethysmography instrument. Airway inflammation was mea-
sured in terms of total cellular influx in the bronchoalveolar
lavage fluids (BALF). The result for the mice treated with 7a
(ip, 100 mg/kg, vehicle 20% of Solutol, 30% PEG400, and 50%
0.1 N NaHCO3) represents an average of 36% reduction in
airway hyperresponsiveness and 83% reduction in the total
cellular influx in the BALF.
Compound 7a was selected for further evaluation. In a
chemotaxis assay using the THP-1 cell line, 7a effectively

Letters
In summary, substituted dipiperidine compounds have been
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5563
(12) 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. Diaryl substituted pyrazoles as potent
CCR2 receptor antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 807-
813.
(13) 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. R-Aminothiazole-γ-aminobutanoic amides as potent, small mol-
ecule CCR2 receptor antagonists. Bioorg. Med. Chem. Lett. 2007,
17, 309-314.
(14) Pasternak, A.; Marino, D.; Vicario, P. P.; Ayala, J. M.; Cascierri,
M. A.; Parsons, W.; Mills, S. G.; MacCoss, M.; Yang, L. Novel,
orally bioavailable γ-aminoamide CC chemokine receptor 2 (CCR2)
antagonists. J. Med. Chem. 2006, 49, 4801-4804.
synthesized and identified as selective CCR2 antagonists.
Carboxylic acid analogues 6a-z exhibited remarkable affinity
in a human CCR2 binding assay. They had much higher affinity
than the corresponding ester (1a) or amide (1b). Compound 7a
had excellent selectivity for CCR2 over CCR1, CCR3, CCR4,
CCR5, CCR6, CCR7, CCR8 and showed significant in vivo
efficacy in adjuvant-induced arthritis, collagen-induced arthritis,
and allergic asthma models in rats and mice. An in depth
biological profile of 7a and systematic SAR studies of the
dipiperidine scaffold will be reported in due course.
(15) Yang, L.; Zhou, C.; Guo, L.; Morriello, G.; Butora, G.; Pasternak,
A.; Parsons, W. H.; Mills, S. G.; MacCoss, M.; Vicario, P. P.;
Zweerink, H.; Ayala, J. M.; Goyal, S.; Hanlon, W. A.; Cascieri, M.
A.; Springer, M. S. Discovery of 3,5-bis(trifluoromethyl)benzyl
l-arylglycinamide based potent CCR2 antagonists. Bioorg. Med.
Chem. Lett. 2006, 16, 3735-3739.
(16) Brodmerkel, C. M.; Huber, R.; Covington, M.; Diamond, S.; Hall,
L.; Collins, R.; Leffet, L.; Gallagher, K.; Feldman, P.; Collier, P.;
Stow, M.; Gu, X.; Baribaud, F.; Shin, N.; Thomas, B.; Burn, T.;
Hollis, G.; Yeleswaram, S.; Solomon, K.; Friedman, S.; Wang, A.;
Xue, C. B.; Newton, R. C.; Scherle, P.; Vaddi, K. Discovery and
pharmacological characterization of a novel rodent-active CCR2
antagonist, INCB3344. J. Immunol. 2005, 175, 5370-5378.
(17) Feria, M.; Diaz-Gonzalez, F. The CCR2 receptor as a therapeutic
target. Expert Opin. Ther. Pat. 2006, 16, 49-57.
(18) Van Lommen, G.; Doyon, J.; Coesemans, E.; Boeckx, S.; Cools, M.;
Buntinx, M.; Hermans, B.; Van Wauwe, J. 2-Mercaptoimidazoles, a
new class of potent CCR2 antagonists. Bioorg. Med. Chem. Lett.
2005, 15, 497-500.
(19) 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. Small molecule antagonists of the
CCR2b receptor. Part 2: discovery process and initial structure-
activity relationships of diamine derivatives. Bioorg. Med. Chem.
Lett. 2004, 14, 5413-5416.
Acknowledgment. We thank Dr. William Murray for
support and Dr. Mark Macielag, Dr. Peter Connolly, and Dr.
Zhihua Sui for helpful discussions.
Supporting Information Available: Experimental details of
the synthesis and characterization of representative CCR2 antago-
nists. This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Carulli, M. T.; Ong, V. H.; Ponticos, M.; Xu, S.; Abraham, D. J.;
Black, C. M.; Denton, C. P. Chemokine receptor CCR2 expression
by systemic sclerosis fibroblasts: evidence for autocrine regulation
of myofibroblast differentiation. Arthritis Rheum. 2005, 52, 3772-
3782.
(2) Weisberg, S. P.; Hunter, D.; Huber, R.; Lemieux, J.; Slaymaker, S.;
Vaddi, K.; Charo, I.; Leibel, R. L.; Ferrante, A. W., Jr. CCR2
modulates inflammatory and metabolic effects of high-fat feeding.
J. Clin. InVest. 2006, 116, 115-124.
(3) Charo, I. F.; Taubman, M. B. Chemokines in the Pathogenesis of
vascular disease. Circ. Res. 2004, 95, 858-866.
(4) Kitagawa, K.; Wada, T.; Furuichi, K.; Hashimoto, H.; Ishiwata, Y.;
Asano, M.; Takeya, M.; Kuziel, W. A.; Matsushima, K.; Mukaida,
N.; Yokoyama, H. Blockade of CCR2 ameliorates progressive fibrosis
in kidney. Am. J. Pathol. 2004, 165, 237-246.
(5) Dawson, J.; Miltz, W.; Mir, A. K.; Wiessner, C. Targeting monocyte
chemoattractant protein-1 signalling in disease. Expert Opin. Ther.
Targets 2003, 7, 35-48.
(6) Rollins, B. J. Monocyte chemoattractant protein 1: a potential
regulator of monocyte recruitment in inflammatory disease. Mol. Med.
Today 1996, 2, 198-204.
(7) Ogilvie, P.; Thelen, S.; Moepps, B.; Gierschik, P.; da Silva Campos,
A. C.; Baggiolini, M.; Thelen, M. Unusual chemokine receptor
antagonism involving a mitogen-activated protein kinase pathway.
J. Immunol. 2004, 172, 6715-6722.
(8) Belvisi, M. G.; Hele, D. J.; Birrell, M. A. New anti-inflammatory
therapies and targets for asthma and chronic obstructive pulmonary
disease. Expert Opin. Ther. Targets 2004, 8, 265-285.
(9) Tsutsumi, C.; Sonoda, K.; Egashira, K.; Qiao, H.; Hisatomi, T.;
Nakao, S.; Ishibashi, M.; Charo, I. F.; Sakamoto, T.; Murata, T.;
Ishibashi, T. The critical role of ocular-infiltrating macrophages in
the development of choroidal neovascularization. J. Leukocyte Biol.
2003, 74, 25-32.
(10) Conrad, S. M.; Strauss-Ayali, D.; Field, A. E.; Mack, M.; Mosser,
D. M. Leishmania-derived murine monocyte chemoattractant protein
1 enhances the recruitment of a restrictive population of CC
chemokine receptor 2-positive macrophages. Infect. Immun. 2007,
75, 653-665.
(11) Seitz, M.; Loetscher, P.; Dewald, B.; Towbin, H.; Rordorf, C.; Gallati,
H.; Gerber, N. J. Interleukin 1 (IL-1) receptor antagonist, soluble
tumor necrosis factor receptors, IL-1.beta., and IL-8-markers of
remission in rheumatoid arthritis during treatment with methotrexate.
J. Rheumatol. 1996, 23, 1512-1516.
(20) 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.
(21) Gao, Z.; Metz, W. A. Unraveling the chemistry of chemokine receptor
ligands. Chem. ReV. 2003, 103, 3733-3752.
(22) Witherington, J.; Bordas, V.; Cooper, D. G.; Forbes, I. T.; Gribble,
A. D.; Ife, R. J.; Berkhout, T.; Gohil, J.; Groot, P. H. E. Conforma-
tionally restricted indolopiperidine derivatives as potent CCR2B
receptor antagonists. Bioorg. Med. Chem. Lett. 2001, 11, 2177-2180.
(23) 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.
(24) 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. Identification of the
binding site for a novel class of CCR2b chemokine receptor
antagonists: binding to a common chemokine receptor motif within
the helical bundle. J. Biol. Chem. 2000, 275, 25562-25571.
(25) Xia, M.; Hou, C.; Pollack, S.; Brackley, J.; DeMong, D.; Pan, M.;
Singer, M.; Matheis, M.; Olini, G.; Cavender, D.; Wachter, M.
Synthesis and biological evaluation of phenyl piperidine derivatives
as CCR2 antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 5964-
5968.
JM070902B