3-Amino-1-alkyl-cyclopentane carboxamides as small molecule antagonists of the human and murine CC chemokine receptor 2

Article abstract of DOI:10.1016/j.bmcl.2007.04.053

A series of low molecular weight antagonists of both the human and murine CC chemokine receptor 2, containing a 1-alkyl-3-(3-methyl-4-spiroindenylpiperidine)-substituted cyclopentanecarboxamide, is described. A SAR study of the C₁ substituent r

Full text of DOI:10.1016/j.bmcl.2007.04.053

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3636–3641
3-Amino-1-alkyl-cyclopentane carboxamides as small molecule
antagonists of the human and murine CC chemokine receptor 2
Gabor Butora,* Richard Jiao, William H. Parsons, Pasquale P. Vicario, Hong Jin,
Julia M. Ayala, Margaret A. Cascieri and Lihu Yang
Merck Research Laboratories, Rahway, NJ 07065, USA
Received 14 March 2007; revised 10 April 2007; accepted 16 April 2007
Available online 25 April 2007
Abstract—A series of low molecular weight antagonists of both the human and murine CC chemokine receptor 2, containing a
1-alkyl-3-(3-methyl-4-spiroindenylpiperidine)-substituted cyclopentanecarboxamide, is described. A SAR study of the C1 substitu-
ent revealed that short, branched alkyl groups such as isopropyl, isobutyl, or cyclopropyl are optimal for both human and murine
CCR2 binding activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Chemokines (chemotactic cytokines) are important
mediators of inflammation and host defense.1–3 These
low molecular weight proteins are secreted by pro-
inflammatory cells, leukocytes, and endothelial cells in
response to certain stimuli or insult to the immune sys-
tem.4–6 Chemokines can be divided into two major (CC
and CXC) and two minor (CX3C and C) groups based
on the number and spacing of the two conserved cys-
teine residues near the N-terminus of the molecule and
the type of leukocytes they attract.7
our own effort, high throughput screening of the Merck
Sample Collection produced the initial lead compound 1
(IC50 = 720 nM), which was elaborated into the piperi-
dine analog 2 with approximately three fold improved
binding affinity, Figure 1.22 Introduction of a 3-
methyl-4-spiroindenyl piperidine moiety afforded the
second generation lead 3 (IC50 = 67 nM).23 Additional
refinement of the central region led to the discovery of
cyclopropyl derivative 4 with outstanding binding affin-
ity (IC50 = 4 nM), as well as oral bioavailability.24
The Monocyte Chemoattractant Protein 1 (MCP-1), a
member of the CC family of chemokines, binds to and
activates a seven transmembrane domain receptor
known as CC chemokine receptor 2 (CCR2).8 It has
been strongly implicated in several inflammatory dis-
eases, including rheumatoid arthritis9 and atherosclero-
Attempts to improve the pharmacological properties
through cyclization within the central region of the scaf-
fold afforded cyclopentane carboxamides such as 5a,
with
a
surprisingly
high
binding
affinity
(hIC50 = 20 nM), Figure 1.25 Unfortunately, none of
these exhibited sufficient affinity toward the murine
CCR2 receptor,26 (e.g., 5a, mCCR2, 27% inhibition at
1 lM). A small molecule antagonist of both human
and murine CCR2 activity would be highly desirable
as it would greatly facilitate target validation and other
studies in various rodent models.
sis.10,11 Studies with CCR2À/À and MCP-1À/À
12
13,14
mice as well as with peptide MCP-1 antagonists15 sug-
gest that blocking the interaction of CCR2 and MCP-
1 may be a viable approach for treatment of rheumatoid
arthritis and atherosclerosis.16
Recently, a number of small molecule non-peptide
CCR2 receptor antagonists from Roche,17 Takeda,18,19
and SmithKline20,21 have been described. As part of
Herein, we wish to describe the structure–activity studies
which led to discovery of novel CCR2 receptor antago-
nists with excellent binding and functional activity at
both the human as well as the murine derived CCR2
receptor.
Keywords: CCR2 receptor antagonists; Chemokines.
*
Target compounds 5 and 6 (except for the cyclopropyl
substituted cases 5k and 6k) were synthesized via one
Corresponding author. Tel.: +1 732 594 8563; fax: +1 732 594
3007; e-mail: gabor_butora@merck.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.053

G. Butora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3636–3641
3637
of the four principal routes depicted in Scheme 1.
According to Route A (compounds 5a, 6b, Scheme 1),
methyl acrylate (7, R = H) or methyl methacrylate (7,
R = Me) and 3-(trimethylsilyl)methyl-2-propen-1-yl ace-
tate were subjected to a palladium-catalyzed 3 + 2 cyclo-
addition.27,28 The double bond in esters 8 and 9 was
cleaved with ozone and the crude ozonides were sub-
jected to a reductive amination with enantiomerically
pure 3-methyl-4-spiroindenyl-piperidine23 without isola-
tion of the respective ketones. A base-catalyzed hydroly-
sis of the esters 10 yielded the respective acids 11, which
were coupled with 3,5-bistrifluoromethylbenzylamine
(12) or 3-fluoro-5-trifluoromethylbenzylamine (13) to
yield the final amides as a mixture of four diastereoiso-
mers. The enantiomerically pure29 compounds 5a and 6b
were obtained by an HPLC separation of the diastereo-
isomers using either a Chiralcel OD or Chiralpak AD
column.30
O
N
O
N
H
H
N
CF3
N
CF3
N
N
H
H
CF3
CF3
F
F
1,
h
CCR2 IC50 = 720 nM
2,
h
CCR2 IC50 = 180 nM
O
N
CF3
N
H
R
CF3
3, R = 4-fluorophenyl-,
4, R = cyclopropyl-,
h
h
CCR2 IC50 = 67 nM
CCR2 IC50 = 4 nM
The majority of the target compounds (5c, d, f, h–j, and
6b–i) were synthesized following Route B. Thus, the
enolate formed from the ester 8 using lithium diisopro-
pylamide (LDA) was alkylated with the respective halo-
alkane to yield esters 9, which were subjected to
ozonolysis and a reductive amination as described
above. Once again, EDC mediated amide formation fol-
lowed by an HPLC separation of the isomers completed
the synthesis.
O
N
CF3
N
H
CF3
5a,
h
CCR2 IC50 = 20 nM
CCR2 IC50 > 1 μM (27% @ 1μM)
m
Figure 1. CCR2 antagonists.
Route C (compounds 5b, e, and g) involved an early
installation of the amide: the methyl 1-alkyl-3-methyl-
Route A, B
O
O
O
a
b,c
O
O
N
OR1
R
R
R
10, R1 = Me
11, R1 = H
8, R = H
9, R = alkyl
7, R = H, Me
g
d
X
CF3
e,f
X
CF3
Route C
O
O
O
b,c,f
OR1
e
N
H
N
N
R
H
R
R
9, R1 = Me
14, R1 = H
15, X = CF3, or F
5, X = CF3, 6, X = F
d
c,f
Route D
O
O
O
O
h
CF3
OR1
OR1
O
O
N
H
O
R
R
17, R = H, R1 = Me
18a, R = alkyl, R1 = Me
18b, R = alkyl, R1 = H
16
X
19, X = CF3 or X = F
i
d
Scheme 1. Reagents and conditions: (a) methyl acrylate, or methyl methacrylate, 3-(trimethylsilyl)methyl-2-propen-1-yl acetate, Pd(AcO)2, (i-
˚
PrO)3P, toluene, reflux; (b) O3, CH2Cl2, À78 ꢁC; (c) 3-methyl-4-spiroindenylpiperidine hydrochloride, NaHB(OAc)3, DIEA, 4 A molecular sieves,
CH2Cl2; (d) LiOH or NaOH, H2O, rt or 80 ꢁC; (e) 12 or 13 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxy-7-
azabenzotriazole (HOAT), CH2Cl2; (f) Chiralcel OD or Chiralpak AD preparative column, EtOH/Hexanes at 9.0 mL/min; (g) alkyl halide, LDA,
THF, À78 to 0 ꢁC; (h) TMOF, pTSA, CH2Cl2; (i) chloromethyl methyl sulfide or dimethyl disulfide, LDA, THF, À78 to 0 ꢁC; (j) TFA, CH2Cl2.

3638
G. Butora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3636–3641
enecyclopentane carboxylates 9 were subjected to a
base-catalyzed hydrolysis and the resulting acids 14 were
coupled with amine 12 or 13 to yield amides 15. As de-
scribed in Route A, the double bond was cleaved with
ozone, and the crude ozonides were subjected to a reduc-
tive amination.
The target compounds containing sulfur in the side chain
(5l, m, 6l, m) were synthesized via Route D. Thus, 3-oxo-
cyclopentanecarboxylic acid31 (16) was converted to the
ester acetal 17 with trimethyl orthoformate (TMOF).
The respective lithium enolate was alkylated with chloro-
methyl methyl sulfide (5l, 6l) or dimethyl disulfide (5m,
6m). The esters 18a were hydrolyzed, and the resulting
acids were coupled to amines 12 or 13. The acetal protect-
ing group was removed under acidic conditions and the
keto amides 19 were reductively aminated as described
above to yield the final products. The respective isomers
were separated by HPLC.
Cl
CN
CN
CN
O
a
b
+
Cl
20
21
c
O
CN
For the synthesis of the cyclopropyl substituted target
compounds 5k and 6k an alternative strategy (Route
E) had to be adopted as shown in Scheme 2. The com-
mercially available cyclopropylacetonitrile was alkylated
with 1,4-dichloro-cis-2-butene in the presence of lithium
bis(trimethylsilyl)amide (LHMDS) to form the cyclo-
pentenecarbonitrile 20. Hydroboration, followed by a
pyridinium chlorochromate (PCC) oxidation yielded
the ketone 21, which was subjected to reductive amina-
tion with 3-methyl-4-spiroindenyl piperidine. The
aminonitrile 22 was hydrolyzed, and a standard amide-
forming step installed the 3,5-bistrifluoromethylbenzyla-
N
N
d
OH
23
22
O
e,f
CF3
N
N
H
X
5, X = CF3, 6, X = F
Scheme 2. Reagents and conditions: (a) LHMDS, DME, 1,3-dimethyl-
3,4,5,6-tetrahydro-2(1H)-pyrimidinone, (10:1); (b) B2H6, À78 ꢁC,
THF; PCC, MgSO4, CH2Cl2; (c) 3-methyl-4-spiroindenylpiperidine
mide-
and
3-fluoro-5-trifluoromethylbenzylamide
groups, respectively. The single enantiomers were ob-
tained via a HPLC separation, as described above.
˚
hydrochloride, NaB(OAc)3H, DIEA, 4 A molecular sieves, dichloro-
ethane; (d) NaOH, H2O, dioxane, 90 ꢁC; (e) 12 or 13, EDC, HOAT,
CH2Cl2; (f) Chiralcel OD or Chiralpak AD preparative column,
EtOH/Hexanes at 9.0 mL/min.
As illustrated in Table 1, a substituent attached at po-
sition C1 of the cyclopentane ring has beneficial effect
Table 1. Human and murine binding affinities in the 3,5-bistrifluoromethylbenzylamide series
O
CF3
N
N
H
R
CF3
5
Entry
Substituent R
Synthetic method
Binding IC50 (nM) or % at 1 lM (SEM, n)34
hCCR2a
mCCR2b
5a
5b
5cc
5d
5e
H–
Me–
A
C
B
B
C
B
C
B
B
B
E
D
D
20.0 ( 1.41, 2)
9.0 (1)
10.0 (1)
27 % (n/a)
126.0 ( 33.08, 3)
17.5 ( 2.12, 2)
15.0 (1)
Et-
i-Pr–
7.6 ( 2.19, 1)
16.0 (1)
8.0 (1)
n-Pr–
i-Bu–
44.3 ( 23.5, 3)
26.0 ( 1.41, 2)
63.5 ( 19.1, 2)
64.0 (1)
5f
5gc
5h
5ic
5jc
5kc
5l
c-PrCH2–
c-BuCH2–
CH3(CH2)5–
CH3OCH2–
c-Pr–
20.0 (1)
19.0 ( 8.49, 2)
231.0 ( 167.6, 2)
15.5 ( 0.71, 2)
6.5 ( 2.12, 2)
13.0 (1)
279.0 (1)
93.0 (1)
45.0 (1)
65.5 ( 21.9, 2)
351.0 (1)
CH3SCH2–
CH3S–
5m
28.0 (1)
a Human CHO cell.
b Mouse CHO cell.
c Mixture of two 1,3-cis-cyclopentane isomers.

G. Butora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3636–3641
3639
on the hCCR2 activity as evidenced by an approxi-
mate twofold increase of the human CCR2 binding
affinity when a hydrogen was replaced by a methyl
group (entries 5a vs 5b, Table 1). However, the same
change induced a dramatic increase in murine CCR2
binding. In this case, the affinity improved from inhi-
bition of 27% at 1 lM concentration to IC50 of
126 nM. The observed activities after an increase in
chain-length from methyl (5b) to ethyl (5c), n-propyl
(5e), and n-hexyl (5i) suggest that the optimal chain
Table 2. Human and murine binding affinities in the 3-fluoro-5-trifluoromethylbenzylamide series
O
CF3
N
N
H
R
F
6
Entry
Substituent R
Synthetic method
Binding IC50 (nM) or % at 1 lM (SEM, n)34
h-CCR2a
m-CCR2b
6b
Me–
Et–
B
B
B
B
B
B
B
B
B
E
D
D
4.0 (1)
57.0 ( 29.7, 2)
3.5 ( 0.71, 2)
5.0 (1)
6cc
6d
3.3 ( 2.48, 2)
3.1 ( 1.56, 2)
5.0 (3)
i-Pr–
n-Pr–
i-Bu–
6ec
17.0 (1)
7.5 ( 3.01, 10)
15.0 (1)
6f
3.9 ( 2.97, 2)
5.5 ( 0.71, 2)
6.3 ( 1.06, 2)
27.0 (1)
6gc
6hd
6ie
6je
6k
6l
c-PrCH2–
c-BuCH2–
CH3(CH2)5–
CH3OCH2–
c-Pr–
28.0 ( 5.66, 2)
128.0 (1)
74.0 (1)
8.5 ( 0.71, 2)
1.9 ( 1.41, 2)
3.5 ( 2.12, 2)
67.0 (1)
8.0 (1)
17.0 ( 1.71, 2)
193.0 (1)
CH3SCH2–
CH3S–
6m
a Human CHO cell.
b Mouse CHO cell.
c Mixture of two 1,3-cis-cyclopentane isomers.
d The binding affinities of the remaining three isomers were determined to be 32, 135, and 229 nM for the human-, and 147, 559 nM, and 49% (at
1 lM) for the murine derived CCR2 receptor.
e Mixture of four 1,3-cis/trans-cyclopentane isomers.
Table 3. Functional activities of selected compounds
Entry
Substituent R
Chemotaxisa IC50 (nM) (SEM,n)
Ca2+ Fluxb IC50 (nM) (SEM, n)34
6d
6f
i-Pr–
i-Bu–
c-Pr–
0.15 ( 0.1096, 2)
0.32 ( 0.2302, 39)
0.23 ( 0.1768, 2)
0.71 (1)
2.04 ( 0.198, 2)
0.63 (1)
6k
a Human MCP-1 monocytes.
b Human monocytes.
Table 4. Pharmacokinetic properties of the isopropyl derivative 6d (male Sprague–Dawley rats)
O
CF3
N
N
H
6d
F
Route
Dose (mg/kg)
AUCn (lM h)
Clearance (mL/min/kg)
19.4
Vol. distrib. (L/kg)
2.99
t1/2 (h)
Cmax (lM)
Tmax (h)
F (%)
iv
po
1.00
3.00
1.637
0.492
2.5
2.0
0.414
0.83
32

3640
G. Butora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3636–3641
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length is two to three carbon atoms. The presence of a
heteroatom within the chain (5j, 5l, and 5m) did not
seem to have a dramatic influence on the binding
affinity. Conversely, branching in the side chain (5d
or 5f) improved both the murine as well as human
CCR2 activities. With additional restriction of the ali-
phatic substituent (5k, cyclopropyl, and 5g, cyclopro-
pylmethyl) both the human and murine affinities
remained approximately the same.
12. Boring, L. F.; Gosling, J. A.; Cleary, M. D.; Charo, F.
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A similar overall trend was observed in the analogous 5-
fluoro-3-trifluoromethylbenzyl series (compounds 6a–m,
Table 2), except that both the human and murine activ-
ities were approximately twofold higher. Within this ser-
ies, isopropyl (6d), isobutyl (6f), and cyclopropyl (6k)
substitution yielded compounds with activities in the
nanomolar range.
Compounds which were found to be the most active in
the respective binding assays were also the most potent
in the chemotaxis32 and calcium flux33 functional assays
as shown in Table 3. In fact, the shorter, branched alkyl
substituted cyclopentanes 6d, f, and k were generally
subnanomolar in the monocyte chemotaxis and calcium
flux assays.
The pharmacokinetic properties of selected compounds
were evaluated in Sprague–Dawley Rats. For example,
the isopropyl derivative 6d exhibited excellent drug lev-
els after both intravenous (1.0 mg/kg, AUCn =
1.637 lM) and oral (3.0 mg/kg, AUCn = 0.492 lM, nor-
malized value) administration. The compound showed a
moderate clearance rate of 19.4 mL/min/kg, low volume
of distribution (2.99 L/kg), and good oral bioavailability
of 32%, Table 4.
20. Forbes, I. T.; Cooper, D. G.; Dodds, E. K.; Hickey, D.
M.; Ife, R. J.; Meeson, M.; Stockley, M.; Berkhout, T. A.;
Gohil, J.; Groot, P. H.; Moores, K. Bioorg. Med. Chem.
Lett. 2000, 10, 1803.
21. Witherington, J.; Bordas, V.; Cooper, D. G.; Forbes, I. T.;
Gribble, A. D.; Ife, R. J.; Berkhout, T.; Gohil, J.; Groot,
P. H. Bioorg. Med. Chem. Lett. 2001, 11, 2177.
22. Yang, L. H.; Zhou, C. Y.; Guo, L. Q.; 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.
Bioorg. Med. Chem. Lett. 2006, 16, 3735.
23. Pasternak, A.; Marino, D.; Vicario, P. P.; Ayala, J. M.;
Cascierri, M. A.; Parsons, W.; Mills, S. G.; MacCoss, M.;
Yang, L. H. J. Med. Chem. 2006, 49, 4801.
In conclusion, systematic variation of the aliphatic side
chain attached at C1 of the cyclopentane core in lead
structure 5 and 6 yielded compounds with low nanomo-
lar affinities for both the human and murine CCR2
receptor. Some of these compounds, especially those
belonging to the 3-fluoro-5-trifluoro-methylbenzamide
series (e.g., 6d, 6f, and 6k) were ideally suited for target
validation and other biological studies requiring a ro-
dent model.
24. Butora, G.; Morriello, G. J.; Kothandaraman, S.; Guia-
deen, D.; Pasternak, A.; Parsons, W. H.; MacCoss, M.;
Vicario, P. P.; Cascieri, M. A.; Yang, L. H. Bioorg. Med.
Chem. Lett. 2006, 16, 4715.
25. Yang, L., Butora, G., Jiao, R. X., Pasternak, A., Zhou, C.,
Parsons, W. H., MacCoss, M., Vicario, P. P., Ayala, M. J.,
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26. CHO cells (5 · 104) expressing either human CCR2B I40L
or murine CCR2B receptor were incubated with
125I-hMCP-1 (20–25 pM, 2200 Ci/mmol; Dupont/New
England Nuclear) or 125I-JE (20–25 pM, 2200 Ci/mmol;
Dupont/New England Nuclear) at room temperature for
45 min in buffer containing Hepes (50 mM), MgCl2
(5 mM), CaCl2 (1 mM), pH 7.4, 0.5% BSA, and protease
inhibitor cocktail. The reactions were terminated by
filtration over GF/B filters that had been presoaked in
0.10% polyethyleneimine using a Packard Cell Harvester
to separate bound from free ligand. The filters were
washed with 25 mM Hepes, pH 7.5, containing 500 mM
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34. Standard Error Margins were not calculated when the
datapoints were obtained from the same experiment using
different dilutions. In this instance the number of exper-
iments (n) is considered 1 and the IC50 value is calculated
as an average of two datapoints.
30. Chiral Technologies, Inc. 730 Springdale Drive, PO Box
564, Exton, PA 19341.