α-Aminothiazole-γ-aminobutanoic amides as potent, small molecule CCR2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.10.059

A series of racemic and homochiral α-aminothiazole-γ-aminobutyroamides that display high affinities for human and murine CCR2 and functional antagonism by inhibition of monocyte recruitment are described. A representative example is (2S)-2-[2-(acetylamino

Full text of DOI:10.1016/j.bmcl.2006.10.059

Bioorganic & Medicinal Chemistry Letters 17 (2007) 309–314
a-Aminothiazole-c-aminobutanoic amides as potent,
small molecule CCR2 receptor antagonists
Changyou Zhou,a,* Liangqin Guo,a William H. Parsons,a Sander G. Mills,a
Malcolm MacCoss,a Pasquale P. Vicario,b Hans Zweerink,b Margaret A. Cascieri,b
Martin S. Springerb and Lihu Yanga
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 28 August 2006; revised 11 October 2006; accepted 23 October 2006
Available online 25 October 2006
Abstract—A series of racemic and homochiral a-aminothiazole-c-aminobutyroamides that display high affinities for human and
murine CCR2 and functional antagonism by inhibition of monocyte recruitment are described. A representative example is (2S)-
2-[2-(acetylamino)-1,3-thiazol-4-yl]-N-[3-methyl-5-(trifluoromethyl)benzyl]-4-(4-phenylpiperidin-1-yl)butanamide, which shows
5 nM affinity for human monocytes and CHO cells expressing the human CCR2b receptor. It also inhibited MCP-1 initiated che-
motaxis of human monocytes with an IC50 of 0.69 nM.
Ó 2006 Elsevier Ltd. All rights reserved.
Chemokines are a family of small (70–200 amino acids)
proinflammatory cytokines with potent chemotactic
activities. Chemokine receptors have been implicated
as important mediators of inflammatory and immuno-
regulatory disorders and diseases, including asthma, rhi-
nitis, and allergic diseases, as well as autoimmune
pathologies such as rheumatoid arthritis and atheroscle-
rosis.1 A subset of chemokines are potent chemoattrac-
tants for monocytes and macrophages. The best
characterized of these is MCP-1 (monocyte chemoat-
tractant protein-1 (CCL2)), whose primary receptor is
CCR2.2,3 MCP-1 specifically attracts monocytes and
In earlier reports,8 we described the discovery of our first
screening CCR2 antagonist lead 1 with micromolar
binding affinity (IC50 = 0.7 lM) and identified several
more potent analogs by replacement of 4-fluorophenyl
group with 3-thienyl or 3-furanyl groups. However,
there were concerns that the molecules lacked sufficient
selectivity over the NK-1 receptor (compound 1: NK-1
IC50 = 700 nM).
After systematic modification of the backbone, we im-
proved the binding affinities to the nanomolar range
(compound 2a: IC50 = 34 nM). Replacement of 4-fluor-
ophenyl ring with small aliphatic rings such as cyclopro-
pyl resulted in more potent analogs (compound 2b:
IC50 = 4 nM). Unfortunately the compounds in this ser-
ies lacked binding affinity toward murine CCR-2, which
prevented evaluation in in vivo animal models. Accord-
ingly, a strategy was put in place to replace the aryl/ali-
phatic groups with heterocycles in the hope of finding
additional interactions with the receptor to further en-
hance potency. In this paper, we report the details of
our effort on the introduction of the aminothiazole
and its derivatives at the a-position of the c-aminobuty-
roamide scaffold leading to a series of novel CCR2
antagonists with sub-nanomolar binding and functional
affinities. In addition, some of these compounds showed
potent antagonism of the mouse CCR2 receptor for the
first time in this series Figure 1.
4
memory T cells. Its expression occurs in a variety of
diseases characterized by mononuclear cell infiltration,
and there is substantial biological and genetic evidence
for its essential role in inflammation.5 CCR2 antago-
nism is recognized as an approach for the treatment of
autoimmune diseases such as rheumatoid arthritis and
multiple sclerosis.6 Thus, the discovery and development
of small molecule CCR2 antagonists has been regarded
as an important pharmaceutical goal.7
Keywords: CCR2; Antagonist; Aminothiazole; Chemotaxis;
Monocyte.
*
Corresponding author. Tel.: +1 7325945693; fax: +1 7325949556;
e-mail: changyou_zhou@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.059

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C. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 309–314
O
H
N
CF3
O
N
N
H
N
CF3
N
H
CF3
2a: IC50 = 34 nM
CF3
F
1: IC50 = 700 nM
F
O
O
R1
N
CF3
N
CF3
N
H
N
H
N
CF3
S
F
NHCOR2
3
2b: IC50 = 4 nM
Figure 1. CCR2 antagonists.
The key intermediate c-amino ester 8 was prepared
according to the procedure described by Kang et al.9
(Scheme 1). Commercially available ethyl (2-amino-
1,3-thiazol-4-yl)acetate 4 was protected as its bis-Boc
ester 5. Subsequent allylation was accomplished by
went various standard derivatizations to give the final
compounds 3, and 12–18.
It was found that racemic 10 could be easily separat-
ed into two single enantiomers on chiral HPLC in
most cases. TFA-mediated deprotection then gave
the corresponding enantiomerically pure intermediates
11, which could be converted into the final com-
pounds 19–25 as single enantiomers. In some cases,
direct separation of racemic final products was possi-
ble; for examples, almost all the compounds from the
spiroindenyl-piperidine series were separable on chiral
HPLC (OD Column, eluting with 10% EtOH/
hexane).
n
treatment of 5 with BuLi at À78 °C, followed by allyl
bromide. The resulting crude alkylated intermediate
was selectively hydrolyzed to mono-Boc protected ester
6 with 30% aq citric acid. The c-aldehyde ester 7 was
prepared by oxidation of the alkene ester 6. Reductive
aminations of the aldehyde 7 with amines gave the
c-amino ester 8.
Hydrolysis of the c-amino ester 8 (Scheme 2) was carried
out in aqueous MeOH with lithium hydroxide. The
crude reaction mixture was passed through a silica gel
column, eluting with methanol/methylene chloride to
give the amino acid 9. The coupling of the amino acid
with bis-trifluoromethyl benzylamine hydrochloride salt
was mediated by EDAC without added base, affording
the Boc-protected a-aminothiazole-c-aminobutanoic
amide 10. Removal of the Boc group in 10 with neat
TFA yielded the free aminothaizole 11, which under-
All the compounds prepared above were evaluated for
their ability to inhibit MCP-1 binding to membranes
of human monocytes stably expressing CCR2 in the
presence of 0.5% BSA. For functional studies, inhibition
of MCP-1-stimulated chemotaxis in freshly isolated
peripheral human monocytes was evaluated (CTX as-
say). The binding displacement data and the chemotaxis
data are expressed as IC50 values.
O
O
O
b, c
a
O
O
O
Boc
N
N
S
N
S
S
NH2
NHBoc
NBoc
6
5
4
O
N
O
R
O
N
O
O
d
e
N
S
S
NHBoc
NHBoc
8
7
Scheme 1. Reagents and conditions: (a) Boc2O (2 equiv)/DMSO, 5 days, the product precipitated out of the solution, 82%; (b) nBuLi (1.1 equiv)/
THF/À78 °C, allyl bromide (2.0 equiv)/À78 °C to RT; (c) 30% aq citric acid, RT, overnight, 67% (two steps); (d) NMO (1.1 equiv)/OsO4 (cat.)/
acetone/water (1:1), then NaIO4 (1.2 equiv)/MeOH/water, 52% (two steps); (e) amine (1.2 equiv)/DIEA (1.0 equiv)/NaBH(OAc)3 (1.2 equiv),
Molecular Sieves (4A)/DCM, overnight.

C. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 309–314
311
O
R
O
N
CF3
R
N
N
H
OH
g
f
N
8
N
CF3
S
S
NHBoc
10
NHBoc
9
O
O
N
R
R
N
CF3
N
CF3
N
H
N
H
h
i, j, k, l,m
N
CF3
S
CF3
S
NHR
NH2
3, 12-18
11
Scheme 2. Reagents and conditions: (f) LiOHÆH2O (1.5 equiv)/MeOH, reflux, 2 h; (g) 3,5-bis-trifluoromethylbenzyl amine hydrochloride (1.0 equiv)/
EDACÆHCl (2.0 equiv)/DCM; (h) 50% TFA/DCM; (i) for the synthesis of amide: (RCO)2O (1.2 equiv)/Py/DCM; (j) for the synthesis of urea:
RN@C@O (1.2 equiv)/DCM; (k) for the synthesis of guanidine: pyrrolyl–C(@NH)NH2 (2 equiv), 220 °C, 30 min; (l) for the synthesis of carbamide:
ClCOOR (1.2 equiv)/Py/DCM; (m) for the synthesis of sulfonamide: (RSO2)O (1.5 equiv)/Py/DCM.
Table 1. Binding affinity of compounds at hCCR210,11
R1
N
O
N
CF3
R2
N
H
CF3
S
NHR
NR1R2
Compound
IC50 (nM) R = H
20
Compound
IC50 (nM) R = COMe
5.2
11a
3a
N
11b
11c
9.1
3b
3c
1.8
3.0
F
N
N
N
N
28.6
N
11d
11e
7.3
1.3
3d
3e
1.7(0.7)a
3.0(0.6)a
N
N
11f
70%b
182.7
3f
32.7
14.5
F
N
11g
3g
N
N
b
11h
75%
3h
13
N
a IC50: Chemotaxis, human MCP-1 Monocyte.
b Inhibition at 1 lM.

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C. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 309–314
Our initial effort starting from 4-phenylpiperidine led to
the synthesis of the precursor 10a which displayed sim-
ilar binding affinity toward hCCR2 as that of the lead
compound 2. After removal of the protecting Boc
group, more active 11a (IC50 = 20 nM) was obtained.
When compound 11a was converted into its acetamide
3a, the binding affinity improved to 5.2 nM (a 4-fold in-
crease). Encouraged by these results, various amino-
thiazoles 11b–11h and acetamidothiazoles 3b–3h were
prepared by incorporating other piperidines and pipera-
zines into the gamma position. The results are summa-
rized in Table 1.
when amides were incorporated, providing pharmaco-
logical tools for in vivo animal model studies. The re-
sults are summarized in Table 3.
The spiroindenylpiperidine part of the compounds 12–
18 seems important for the murine CCR2 activities
too. Much weaker mCCR2 antagonism (IC50 > 1 lM)
was observed for the compounds without spiro-
indenylpiperidine substructure.
As expected, the binding affinity of one of the two
enantiopure isomers was much higher than the other
one. Table 4 lists a few examples of single enantiomers
purified by preparative chiral HPLC. About a 2-fold
increase in potency was observed in most cases when
compared to the racemic analogs.
The head-to-head comparison between the free amino-
thiazoles 11 and acetamidothiazoles 3 showed that the
binding affinities of 11 were much weaker than those
of 3. The most potent compounds were obtained when
phenylpiperidines and spiroindenepiperidines were used
as the amine moieties. Although the aminothiazoles
from piperazines such as 11f–11g generally showed only
micromolar binding affinities, their respective aceta-
mides 3f–3g were quite potent. The potency of com-
pounds 11d and 11e was further confirmed by the
CTX assay (IC50 = 0.7 and 0.6 nM, respectively). The ef-
fect of acetylation was also quite obvious in the simple
piperidine cases (11h and 3h). The above results may im-
ply a binding pocket of the CCR2 receptor being
reached by acetyl group.
Table 3. Binding Affinity of compounds at hCCR2 and mCCR210
O
N
CF3
N
H
N
CF3
S
NHCOR
Compound
R
IC50 (nM)
hCCR-2 mCCR-2
A more detailed study involving derivatization of the
free aminothiazole 11a showed that most of the amides,
ureas, and even guanidines displayed similar potent
antagonism toward the hCCR2 receptor. In the amide
series, smaller groups gave higher potency. For example,
larger amides were less potent than smaller ones
(COtBu < COPh < COiPr < COcPr < COEt). In addi-
tion, methanesulfonamide 3s was much less active com-
pared with the amide 3a. These results are summarized
in Table 2.
12
13
Ph
CH2Ph
17
19
952
165
H2C
S
N
14
15
23
81
H2C
13
79
16
17
3-Pyridyl
CHMePh
14
74
186
517
One exception to the structure-activity trend was that no
binding affinity improvement was observed when com-
pound 11e was converted into amide 3e. None-the-less,
murine CCR2 antagonism was observed in this series
18
36
111
Table 2. Binding affinity of compounds at hCCR210
O
N
N
CF3
N
H
CF3
S
NHR2
Compound
Compound
R2
IC50 (nM)
R2
IC50 (nM)
3a
3i
COMe
CHO
5.2
4.0
3.5
3.9
7.4
18
3n
3o
3p
3q
3r
3s
COPh
16
CONHMe
CONMe2
COOMe
C(@NH)NH2
SO2Me
3.0
2.3
2.6
1.3
36
3j
COEt
COcPr
COiPr
COtBu
3k
3l
3m

C. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 309–314
Table 4. Binding affinity of enantiopure compounds at hCCR210,11
313
O
O
R1
R1
+
N
CF3
N
CF3
N
N
H
H
Chiral HPLC
N
N
CF3
S
CF3
S
10%EtOH/Hex
OD Column
NHR2
NHR2
A
B (Slow Isomer)
(Fast Isomer)
Compound
R2
IC50 (nM)
R1
A
B
N
19
20
21
22
23
Boc
H
14.3
13.3
N
13.3
229
42
N
N
N
N
COMe
1.8 (0.7)a
COOMe
CONHMe
3.5
85% at 1 lM
0.43 (1.8)a
30(87)a
N
N
24
25
COMe
1.4 (5.3)a
6.5(27)a
CONHMe
0.8
5.8
a IC50: Chemotaxis, human MCP-1 Monocyte.
The selectivity of these new CCR2 antagonists over
other chemokine receptors was generally quite good.
In addition, the NK-1 selectivity was greatly improved
compared with our original lead 1. The selectivity of
compound 21 is shown in Table 5.
Compound 21 can be orally absorbed but it was cleared
rapidly. In general, these compounds suffered from poor
pharmacokinetic profiles. The PK data in rats and dogs
for compound 21 are shown in Table 6.
In summary, we have prepared a series of novel
a-aminothiazole-c-aminobutyroamides that show good
hCCR2 and/or mCCR2 binding potency, excellent che-
motaxis inhibitory activity, and selectivity over other
chemokine receptors. Studies on further optimization
of in vitro and in vivo properties will be reported in
due course.
Table 5. Binding affinities of compound 21 over other chemokine
receptors
CCR CCR-1 CCR-3 CCR-4 CXCR4 CCR-5 NK-1
a
IC50
66%
7%
86%
27%
44%
56%
a % inhibition at 1 lM (IC50 not determined).
Table 6. Pharmacokinetic profiles of compound 21
PK
Fa (%)
T1/2 (h)
CL (b)
Vd (l/kg)
AUCN po/iv (lM h)
Cmax (lM)
Tmax (h)
Rat
Dog
17
1.6
1.4
2.28
52.5
21.6
5.3
4.0
0.13/0.71
0.03/1.79
0.064
0.018
2.2
0.5
a Doses: male rat Sprague–Dawley (iv: 1.0 mpk; oral: 3 mpk); Male Beagle Dog (iv: 0.20 mpk; oral: 3.0 mpk).
b ml/min/kg.

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C. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 309–314
MacCoss, M.; Vicario, P. P.; Zweerink, H.; Ayala, J. M.;
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presence of 0.5% BSA.
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