Discovery of 3,5-bis(trifluoromethyl)benzyl l-arylglycinamide based potent CCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.04.045

Systematic modification of a screening lead yielded a class of potent glycinamide based CCR2 antagonists. The best compound (55, (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-{[2-(1-piperidinyl)ethyl]amino}-2-(3-thienyl)acetamide) displayed good binding affinity (IC₅₀ = 30 and 39 nM) toward human monocytes and CHO cell expressing human CCR2b, respectively. Functionally, it blocked MCP-1 (CCL2)-induced calcium mobilization (IC₅₀ = 50 nM) and chemotaxis mediated through the CCR2 receptor (9.6 nM). It is selective against other chemokine receptors tested.

Full text of DOI:10.1016/j.bmcl.2006.04.045

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3735–3739
Discovery of 3,5-bis(trifluoromethyl)benzyl
L-arylglycinamide based potent CCR2 antagonists
Lihu Yang,* Changyou Zhou, Liangqin Guo, Gregori Morriello, Gabor Butora,
Alexander Pasternak, William H. Parsons, Sander G. Mills, Malcolm MacCoss,
Pasquale P. Vicario, Hans Zweerink, Julia M. Ayala, Shefali Goyal,
William A. Hanlon, Margaret A. Cascieri and Marty S. Springer
Merck Research Laboratories, Rahway, NJ 07065-0900, USA
Received 30 March 2006; revised 19 April 2006; accepted 19 April 2006
Available online 15 May 2006
Abstract—Systematic modification of a screening lead yielded a class of potent glycinamide based CCR2 antagonists. The best
compound (55, (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-{[2-(1-piperidinyl)ethyl]amino}-2-(3-thienyl)acetamide) displayed good
binding affinity (IC50 = 30 and 39 nM) toward human monocytes and CHO cell expressing human CCR2b, respectively. Function-
ally, it blocked MCP-1 (CCL2)-induced calcium mobilization (IC50 = 50 nM) and chemotaxis mediated through the CCR2 receptor
(9.6 nM). It is selective against other chemokine receptors tested.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Chemokines or chemotactic cytokines are a large family
of small (ꢀ8–15 kDa) structurally related proteins that
play an important role in leukocyte migration and acti-
vation.1–5 Most members of this family belong to one of
two major subfamilies which are distinguished based on
the arrangement of the first two conserved cysteines in
the sequence: the CC family which contains adjacent
cysteines and the CXC family in which the cysteines
are separated by a single intervening amino acid.
Chemokines mediate their effects through activation of
specific cell-surface seven-transmembrane spanning
G-protein coupled receptors. Monocyte chemoattrac-
tant protein (MCP-1/CCL2) belongs to the CC chemo-
kine subfamily and binds to CC chemokine receptor 2
(CCR2), which is expressed on the majority of blood
born monocytes.6 Interruption of the MCP-1/CCR2 axis
in rodent models of inflammatory and autoimmune dis-
eases by genetic deletion of either MCP-17 or CCR28–10
and use of peptidyl CCR2 antagonists,11 or anti-MCP-1
antibodies12 suggests that inhibition of CCR2 may pro-
vide potential therapies for a variety of diseases includ-
ing rheumatoid arthritis,11,12 multiple sclerosis13–15, and
atherosclerosis.10,16–18 These prospects have prompted
the search for small molecule MCP-1/CCR2 antagonists
in many research laboratories.19 Several examples have
been reported in the scientific and patent literature as
shown in Figure 1. Compound 1 was among the first
reported CCR2 antagonists to exhibit micromolar bind-
ing affinity to the receptor and functional antagonism in
the chemotaxis (CTX) assay.20 Compound 2 showed
higher affinity to the receptor but had much reduced
functional antagonism.21 Compound 3, which bears
structural resemblance to earlier reported CCR1 antag-
onists, was reported to have potent binding and func-
tional activities.22 A series of indolyl piperidine based
CCR2b antagonists which also bind to 5-HT was report-
ed (compound 4).23,24 In addition, a class of potent
CCR5 antagonists have been reported to have low nano-
molar binding to CCR2 and act as functional antago-
nists of the CCR2 receptor.25,26 All of these are
piperidine based with at least two aromatic rings as their
pharmacophore. Two novel classes of CCR2 antago-
nists as represented by compounds 5 and 6 have also
been disclosed. Compound 5 is a carboxylic acid which
was reported to be a CCR2 antagonist with good bind-
ing affinity to the receptor.27 The acyl urea 6, however,
inhibits MCP-1-induced chemotaxis of human mono-
cytes in vitro and in vivo even though it does not
displace the binding of MCP-1 to CCR2.28,29 All these
Keywords: CCR2; CCR2b; MCP-1; CCL2; Antagonist; Chemokine;
Chemotaxis; GPCR.
*
Corresponding author. Tel.: +1 732 594 4660; fax: +1 732 594
3007; e-mail: lihu_yang@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.045

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L. Yang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3735–3739
Figure 1. CCR2 antagonists.
compounds suffer from various problems ranging from
low binding affinity and/or low functional activity to
poor selectivity. Herein we report a new class of glycin-
amide based potent CCR2 antagonists with good selec-
tivity over other chemokine receptors.
bicarbonate in refluxing ethanol provided the desired
compound. The stereochemistry of the amino acid was
retained during the synthesis.
A second approach from a-bromo-phenylacetamide is
depicted in Scheme 2. The desired a-bromo-phenylace-
tamide was prepared through the coupling of a-bromo-
acetic acid with a benzylamine as described earlier.
Treatment of the bromo phenylacetamide with neat
diamines, amino thiols, and amino alcohols gave the de-
sired compounds. This two-step synthesis allowed the
facile preparation of compounds incorporating sulfur
and oxygen as well as nitrogen at the backbone of the
lead.
Screening of the Merck sample collection resulted in a
single hit 7 (IC50 = 770 nM, CCR2), which was original-
ly prepared for the neurokinin antagonist program
(IC50 = 650 nM, NK1). A modular approach was taken
to develop this lead, namely identification of an optimal
central amino acid, modification or replacement of the
diamines, and investigation of the importance of the
amide.
The synthetic approaches to these phenyl glycine deriv-
atives were carried out either through the alkylation of
the glycine or displacement of a-bromo-phenylaceta-
mide. Scheme 1 shows a typical route starting from an
N-protected phenyl glycine. EDC coupling with a benzyl
amine provided the corresponding amide. After acidic
deprotection, treatment of the resulting amine with an
amino ethyl chloride in the presence of sodium
Compounds were initially evaluated for their ability to
inhibit human 125I-MCP-1 binding to the CCR2b recep-
tor, stably expressed on CHO cells.30 A binding assay
based on human monocytes was later established and
the two binding assays generally agree well in this lead
class. Functional antagonism and efficacy of key com-
pounds were studied in a calcium flux or chemotaxis
assays, both using human primary monocytes.31,32
O
O
O
H
BocHN
H2N
CF3
N
CF3
OH
N
N
N
H
H
a, b
c
CF3
CF3
F
F
F
7
8
Scheme 1. Reagents and conditions: (a) EDC, HOBT, 3,5-bis(trifluoromethyl)benzyl amine, CH2Cl2, 0 ꢁC; (b) HCl, EtOAc; (c) dimethylaminoethyl
chloride HCl, NaHCO3, EtOH, 80 ꢁC.

L. Yang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3735–3739
3737
O
O
O
Br
Br
CF3
X
CF3
a
b
OH
N
N
N
H
H
CF3
CF3
10
9
Scheme 2. Reagents and conditions: (a) EDC, HOBT, 3,5-bis(trifluoromethyl)benzyl amine, CH2Cl2, 0 ꢁC; (b) Me2N(CH2)2XH (X = NH, O, S;
neat).
Table 2. Binding affinity to human CCR2 (CHO).
Structural optimization was carried out on each subunit
of the screening lead 7. The central 4-fluorophenyl
O
X
H
glycine has the L or (S)-stereochemistry, while its D or
(R)-enantiomer 11 is inactive (Table 1). Deletion of the
aromatic ring also resulted in an inactive compound
12. A survey of aromatic amino acids demonstrated that
the aryl group is important for hCCR2 activity. The par-
ent phenyl glycine derivative 13 is equipotent to the lead,
but its homolog phenylalanine 14 was inactive. As a
result, much of the early SAR was carried out with
phenyl at this position for easier access of starting mate-
rials. A fully saturated cyclohexyl glycine 15 did not
bind to the receptor suggesting very tight SAR in this
region. Most substitutions on the phenyl group with
the exception of small groups at the 4 position reduced
potency as demonstrated by compounds 16–20. The
4-fluorophenyl group can be replaced by a variety of
small heteroaryl groups that retain or improve hCCR2
binding affinity. For example, the thiophene derivative
22 and furan analog 23 are more potent than the
original lead.
N
N
N
H
R
Compound
X
R
Binding IC50 (nM)
24
25
26
27
28
13
H
2-CF3
3-CF3
4-CF3
1%
5%
H
H
7%
0%
H
3,5-DiMe
3,5-DiCF3
3,5-DiCF3
Me
H
28%
1000
% inhibition at 1 lM when no IC50’s were measured.
Table 3. Binding affinity to human CCR2 (CHO)
O
H
N
CF3
N
N
H
X
The bis-trifluoromethylbenzyl group is extremely sensi-
tive to modification (Table 2). Both of the CF3 groups
are critical for activity. Attempts to replace the bis-triflu-
oromethylbenzyl group with other substituted benzyl
groups resulted in inactive compounds (24–27) as shown
in Table 2. The introduction of a methyl at the benzylic
position is a way of restricting the number of low-energy
conformations at this region, potentially favoring a
CF3
Compound
X
Binding IC50 (nM)
13
29
30
31
32
33
34
35
CONHCH2
1000
20%
0%
CONMeCH2
CH2NHCO
CH2NHSO2
CH2NMeSO2
CH2NAcCH2
CH2NMsCH2
CH2OCH2
0%
0%
4%
0%
Table 1. Binding affinity to human CCR2 (CHO)
6%
O
H
% inhibition at 1 lM when no IC50’s were measured.
N
CF3
N
N
H
R
more active conformation. Unfortunately, in this
instance it greatly reduced the binding of compound
28 as compared with the parent 13.
CF3
Compound
Stereo
R
Binding IC50 (nM)
7
11
12
13
14
15
16
17
18
19
20
21
22
23
L
4-F–Ph
4-F–Ph
H
770
8%
D
Likewise, the secondary amide is also critical for recep-
tor binding (Table 3). Replacement of the amide linker
with N-methyl amide, reversed amide, sulfonamide, or
ether linkers results in total loss in activity as shown in
Table 3.
—
L
0%
Ph
4-F–PhCH2
c-Hex
1000
1%
L
L
9%
DL
DL
DL
DL
L
3,4-DiF–Ph
4-Cl–Ph
4-Br–Ph
3-Br–Ph
4-MeO–Ph
33%
1100
25%
17%
47%
865
424
592
The effects of substitution and replacement of the
glycine amine (NH) were investigated (Table 4). Simple
methylation to convert it to a tertiary amine (36)
abolished the binding affinity, as did acetylation (37).
The imine (38), ether (39), thioether (40), and methylene
(41) replacements all gave compounds with reduced
binding affinity.
L
2-Thiophene
3-Thiophene
2-Furan
L
DL
% inhibition at 1 lM when no IC50’s were measured.

3738
L. Yang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3735–3739
Table 4. Binding affinity to human CCR2 (CHO)
Table 7. Receptor selectivity profile of compound 55
Receptor Binding IC50 (nM) Receptor Binding IC50 (nM)
O
X
CF3
CCR1
CCR3
CCR4
CCR5
>1000 (À19%)
>1000 (2%)
>2000 (6%)
>1000 (49%)
CCR8
NK1
NK2
NK3
>1000 (15%)
>100 (31%)
>1000 (20%)
>1000 (10%)
N
N
H
CF3
Compound
X
Binding IC50 (nM) (%)
3-thiopheneglycine derivative 55 showed potent binding
affinity to the human CCR2 receptor. For comparison,
the phenylglycine and 4-F-phenylglycine analogs were 4-
fold less active. Furthermore, compound 55 showed sim-
ilar binding affinities to human monocytes with an IC50 of
30 nM. It inhibited MCP-1-induced calcium flux in hu-
man monocytes with an IC50 of 50 nM. More important-
ly, it inhibited MCP-1-induced chemotaxis of human
monocytes with an IC50 of 9.6 nM. In addition, com-
pound 55 shows good selectivity against other chemokine
receptors as well as neurokinin receptors (Table 7). These
data indicate that 55 is a CCR2-specific antagonist.
36
37
38
39
40
41
MeN
AcN
–N@
O
8
0
10
0
S
CH2
25
13
% inhibition at 1 lM when no IC50’s were measured.
The dimethylamine unit on the left side of the molecule
is essential for binding as replacement with non-basic
groups generates inactive compounds (44, 45, Table 5).
Replacement of the dimethylamine group with other
amino groups is tolerated and can lead to improved
potency, with the piperidine group being optimal (47–
50). Substitution on the piperidine group did not im-
prove activity (51–53). Prolongation of the linker be-
tween the two amines reduced potency (54).
The PK profile of compound 55 was evaluated in rats.
Oral bioavailability was low (F = 4.5%), clearance was
high (Clp = 80 mL/min/kg), and half-life was modest
(t1/2 = 1.8 h). A study was carried out to see if the low
oral bioavailability was due to high first pass metabo-
lism. It was orally administered to rats (3 mg/kg), and
portal and systemic levels are measured at several time
points. It exhibited good oral absorption as demonstrat-
ed by high portal levels (over 300 ng/mL at 1 h), but lim-
ited systemic exposure (<20 ng/mL) suggests that first
pass metabolism is responsible for the low oral
bioavailability.
Having established the optimal moieties at each position,
we incorporated them into one molecule (Table 6). The
Table 5. Binding affinity to human CCR2 (CHO)
O
H
N
CF3
R
N
H
In conclusion, we have described SAR studies that have
resulted in the identification of compound 55 as a novel
and potent non-peptidyl CCR2 antagonist with compa-
rable binding (cloned receptor and human monocyte)
and functional chemotaxis and calcium flux activities.
Compound 55 shows remarkable selectivity toward
other chemokine receptors as well as the neurokinin
receptors. This lead has served as a starting point for
the design and synthesis of additional CCR2 antago-
nists. Progress in this area will be reported in due course.
CF3
Compound Stereo
R
Binding IC50 (nM)
42
43
44
45
46
47
48
49
50
51
52
53
54
DL
DL
DL
DL
DL
DL
DL
L
H2N
Me2N
i-Pr
14%
1477
0%
MeO
Et2N
0%
678
521
342
400
575
838
756
822
1-pyrrolidinyl
1-piperidinyl
1-homopiperidinyl
1-morpholinyl
DL
L
2-Me-1-piperidinyl
4-Me-1-piperidinyl
4-Ph-1-piperidinyl
References and notes
L
L
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1-piperidinylmethyl 26%
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Table 6. Binding affinity to human CCR2 (CHO)
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H
N
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55
56
57
3-Thiophene
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