Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series

Article abstract of DOI:10.1016/j.bmcl.2008.02.015

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional a

Full text of DOI:10.1016/j.bmcl.2008.02.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1869–1873
Potent antagonists of the CCR2b receptor.
Part 3: SAR of the (R)-3-aminopyrrolidine series
Wilna J. Moree,a,* Ken-ichiro Kataoka,b Michele M. Ramirez-Weinhouse,a,
Tatsuki Shiota,b Minoru Imai,b Takaharu Tsutsumi,b Masaki Sudo,b,à Noriaki Endo,b
Yumiko Muroga,b Takahiko Hada,b,§ Dewey Fanning,a,– John Saunders,a,–
Yoshinori Kato,b Peter L. Myersa, and Christine M. Tarbya,àà
aDeltagen Research Laboratories (Former CombiChem, Inc.), 4570 Executive Drive, San Diego, CA 92121, USA
bTeijin Institute for Biomedical Research, 4-3-2 Asahigaoka, Hino, Tokyo, 191-8512, Japan
Received 21 December 2007; revised 4 February 2008; accepted 7 February 2008
Available online 10 February 2008
Abstract—SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis.
Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and func-
tional assays [Compound 71 CCR2b Binding IC50 3.2 nM; MCP-1-Induced Chemotaxis IC50 0.83 nM; Ca2+ Flux IC50 7.5 nM].
Ó 2008 Elsevier Ltd. All rights reserved.
Chemokines or chemotactic cytokines are small molecu-
lar weight (6–15 kD) proteins that modulate inflamma-
tory and immune responses through promotion of cell
migration, cell adhesion and transmigration.1 They are
divided into four classes dependent on the arrangement
of conserved cysteines in the N-terminal region. Mono-
cyte Chemoattractant Protein-1 (MCP-1) is a member of
the CC-chemokine family and selectively recruits leuko-
cytes from the circulation to the site of inflammation
through binding with the CCR2b receptor.2 This recep-
tor is a member of the seven transmembrane receptor
family (7-TM) and is expressed on the surface of mono-
cytes and macrophages.
Studies with CCR23 and MCP-14 knock-out mice have
indicated that antagonists of the CCR2b receptor may
exert therapeutic effects in a variety of inflammatory dis-
eases including rheumatoid arthritis,5 atherosclerosis,6
glomuleronephritis,7 and multiple sclerosis.8 This
prompted our laboratories and others,9 to develop small
molecular weight inhibitors of the interaction between
MCP-1 and the CCR2b receptor.
In our previous manuscripts,10 we described the discov-
ery of several diamine based lead compounds as CCR2b
antagonists. Compound 1, containing a (R)-3-amino-
pyrrolidine core, was identified as a key lead structure
with good activity in a [125I]-MCP-1 binding assay
(IC50 180 nM) and in a CCR2b-mediated chemotaxis as-
say (IC50 24 nM). Herein we report our structure activ-
ity relationship (SAR) studies around this lead
compound with the objective of generating potent and
selective CCR2b antagonists.
Keywords: CCR2b; Chemokine; Antagonist.
*
Corresponding author at present address: Neurocrine Biosciences,
12790 El Camino Real, San Diego, CA 92130, USA. Tel.: +1 858 617
7506; fax: +1 858 617 7925; e-mail: wmoree@neurocrine.com
Present address: Pfizer Global Research & Development, La Jolla
Laboratories, Discovery Chemistry, 10614 Science Center Drive
(CB6), San Diego, CA 92121, USA.
à
Present address: Pfizer Global Research and Development, Nagoya
Laboratories, 5-2 Taketoyo, Aichi 470-2393, Japan.
Present address: Bizen Chemical Co., Ltd, Research and Develop-
ment Department, 363-Tokutomi, Kumayama-Cho, Akaiwa-Gun,
Okayama 709-0716, Japan.
§
Three fragments were defined for optimization as illus-
trated in Figure 1; the R1 substituted N-benzyl group,
the amino acid, and the R2 substituted benzamide. In
order to conduct the lead optimization studies in the
(R)-3-aminopyrrolidine series, both solution and solid-
phase methodologies were developed. Using solution
–
Present address: Vertex, 11010 Torreyana Road, San Diego, CA
92121, USA.
Present address: LEAD Therapeutics, Inc., 999 Bayhill Drive, Suite
130, San Bruno, CA 94066, USA.
àà
Present address: Bristol-Myers Squibb Company, PO Box 4000,
Princeton, NJ 08543, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.015

1870
W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1869–1873
Cl
Cl
O
a,b,c,b
N
HN
NH2
R3
H
N
O
NHBoc
N
N
N
N
CF3
6
N
H
7
H
O
Cl
d
H
R2
1
O
N
N
H
R3
O
R1
8
H
N
R3
O
R2
Scheme 2. Reagents: (a) p-chlorobenzyl chloride, K2CO3; (b) TFA; (c)
N-Boc-CH(R3)-CO2H, EDCI, HOBt, iPr2NEt; (d) R2PhCO2H, EDCI,
N
H
O
i
HOBt, Pr2NEt.
2
Figure 1. Lead optimization plan for Compound 1.
O
O
R2
synthesis, the R1 substituted N-benzyl group was exam-
ined independently of the amino acid and R2 substituted
benzamide (Scheme 1). (R)-1-Benzyl-3-aminopyrrolidine
3 was converted to 4 through the following sequence:
EDCI-mediated coupling with N-Boc-glycine, removal
of the N-Boc-protecting group, acylation with m-trifluo-
romethyl benzoyl chloride, and debenzylation by
hydrogenolysis. High-throughput alkylation or reduc-
tive amination chemistry afforded the desired com-
pounds 5, which were purified using acid–base
extraction.
Fmoc
a,b
N
Fmoc
N
N
R3
NH2
R3
N
H
N
H
10
O
9
c,d,e
2
= AMEBA Resin
CHO
Scheme 3. Reagents and conditions: (a) AMEBA resin, NaBH(OAc)3,
1% HOAc-DMF; (b) R2PhCO2H, HBTU, iPr2NEt, DMF, 1 h; (c) 20%
piperidine-DMF, 10 min; (d) R1PhCHO, NaBH(OAc)3, 1% HOAc-
DMA; (e) 95% TFA.
The amino acid portion of the molecule and the R2
substituted benzamide were examined in concert, while
maintaining the 4-chlorobenzyl substitution on the pyr-
rolidine nitrogen (Scheme 2). (R)-3-N-Boc-pyrrolidine 6
was alkylated with p-chlorobenzyl chloride, followed by
the TFA deprotection of N-Boc group. A variety of
commercially available N-Boc-protected natural and
unnatural amino acids were used and coupled to the 3-
amino group of the pyrrolidine. Following N-deprotec-
tion, the resulting intermediates 7 were coupled in a
high-throughput fashion to a variety of acids. The cou-
pling conditions employed allowed for extractive purifi-
cation of the final product. These products were
typically produced in >90% purity as determined by
HPLC.
duced onto acid sensitive methoxybenzaldehyde (AME-
BA) resin11 by reductive amination. Coupling with a
variety of benzoic acids provided 10. Following removal
of the Fmoc-protecting group, reductive amination with
aromatic aldehydes provided the fully functionalized fi-
nal products, which were cleaved from resin with TFA
and purified using ion exchange chromatography. The
purities of all compounds synthesized by solid and solu-
tion phase chemistries were assessed by HPLC and com-
pounds were re-purified via mass-triggered reverse phase
HPLC12 if found to be less than 85% pure.
The compounds were evaluated in a radioligand binding
assay using [125I]-MCP-1 and THP-1 cells.13 Potent
compounds were then subsequently tested in a MCP-1-
induced cell chemotaxis assay using THP-1 as the che-
motactic cell line.14 The results for selected compounds
are summarized in Tables 1–3.
In order to effectively vary the R1 substituted N-benzyl
group and the R2 substituted benzamide, simulta-
neously, a solid phase synthesis route was devised
(Scheme 3). Fmoc-protected (R)-3-aminopyrrolidine
preloaded with an amino acid of interest (9) was intro-
We first sought to optimize the R1 substituted benzyl
group (Table 1). An initial set of compounds (1, 12–
20), was prepared based on the Topliss optimization
tree.15 The resulting SAR indicated a clear preference
for p-substitution on the benzyl ring. The p-substituted
compounds (1, 19–20) were 4- to 8-fold more potent
than 12 (R = H) and the o-substituted variants (13–15)
and at least 25-fold more potent than m-substituted
compounds (16–18). Based on this observation, the
range of substituents at the 4-position of the benzyl ring
was expanded. Small electron donating groups (19–23)
were preferred over electron withdrawing substituents
(28–30). Additionally 2,4- and 3,4-substitution patterns
were tolerated and either maintained or slightly
improved activity (31–33). The functional activities of
H
N
O
a,b,c,d
N
HN
N
CF3
N
H
NH2
R1
O
4
3
H
O
e or f
N
CF3
N
O
H
5
Scheme 1. Reagents and conditions: (a) N-Boc-Glycine, EDCI, HOBt,
TEA; (b) 3 M HCl-CH3OH; (c) m-CF3-PhCOCl, TEA; (d) H2,
Pd(OH)2-C, MeOH; (e) R1PhCH2Cl or R1PhCH2Br, (piperidino-
methyl)polystyrene, CH3CN, 50°C; (f) R1PhCHO, NaBH3CN, 2.5%
aq, AcOH, MeOH, 60 °C.

W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1869–1873
1871
Table 1. Binding affinity to CCR2b and CCR2-mediated chemotaxis
activity for (R)-3-amino-pyrrolidine series
Table 2. Variation of R2 in (R)-3-aminopyrrolidine series
Cl
R1
R2
H
N
O
H
N
O
N
N
CF3
N
H
N
H
O
O
Compound
R2
Binding
a
IC50 (nM)
Chemotaxis
a
IC50 (nM)
Compound
R1
Binding
a
IC50 (nM)
Chemotaxis
a
IC50 (nM)
35
36
37
38
39
40
1
H
>10000
>10000
>10000
>10000
2380
ndb
nd
12
13
14
15
16
17
18
1
H
690
630
418
418
127
ndb
nd
2-CH3
2-Cl
2-Cl
2-CH3
nd
nd
960
2-CF3
3-CH3
3-Cl
2-OCH3
3-Cl
1360
>5000
3100
3170
180
nd
nd
2400
180
3-CH3
3-OCH3
4-Cl
nd
nd
3-CF3
4-CH3
4-Cl
24
nd
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
10000
>5000
>5000
>10000
4330
24
12.4 (0.3)
35
nd
nd
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
4-CH3
4-OCH3
4-Et
116 (31)
114
59
4-CF3
3-CN
3-F
nd
nd
9.4
21
4-Br
166
121
3-Br
785
489
473 (163)
360
nd
4-Vinyl
4-CH3S
4-OH
6.2
21
3-OCF3
3-NH2
3-OCH3
3-NO2
2-NH2
204 (108)
226
305
>10000
>10000
832
5.5
nd
nd
4-NHAc
4-OCF3
4-F
445
nd
610
960
103
121
nd
>10000
208
730
2-NH2, 5-NO2
2-NH2, 5-Cl
2-NH2, 5-Br
2-NH2, 5-I
43
nd
4-NO2
4-CN
1550
2650
54
nd
16
643
308
nd
22
2,4-(CH3)2
2,4-Cl2
300 (223)
153
759
286
77
2-NH2, 5-OCF3
2-NH2, 5-CF3
88
26
43
3.6
4-OH, 3-OCH3
2-Naphthyl
74
a IC50 values were derived from dose response curves generated from
duplicate data points. Values with standard deviations (in paren-
theses) are means of at least three independent experiments.
b nd = not determined.
a IC50 values were derived from dose response curves generated from
duplicate data points. Values with standard deviations (in paren-
theses) are means of at least three independent experiments.
b nd = not determined.
Table 3. Binding affinity to CCR2b and CCR2-mediated chemotaxis
activity for (R)-3-amino-pyrrolidine series
the compounds, assessed in a chemotaxis assay, demon-
strated a loose correlation with the binding data.
CF3
R1
O
H
N
We next examined a variety of R3 amino acid spacers
simultaneously with replacements for the R2 substituted
benzamide functionality. Although many different a and
ß-amino acids were screened, only the glycine spacer
consistently afforded compounds with an appropriate le-
vel of CCR2 binding activity for further optimization.16
Likewise the incorporation of aliphatic amides instead
of R2 substituted benzamides completely abolished
CCR2 activity and will not be discussed further in this
manuscript. Systematic optimization of the substituents
on the benzamide ring (R2) indicating meta-substitution
on the aromatic ring was favored as exemplified by the
comparison of compound 1, 39 and 40 with 35–38 and
41–43 (Table 2). Using a Craig plot guided optimiza-
tion,17 we observed a preference for substituents with
positive Hammett r/Hansch p constants. This is illus-
trated by the comparison of active compounds 1
(CF3), 46 (Br), 47 (OCF3) and 50 (NO2) with the inac-
tive 44 (CN), 48(NH2), and 49 (OCH3). The parent tri-
fluoromethyl group was the most active single
substituent identified for the ring. Further improve-
ments in activity required incorporation of an additional
functionality on this ring. Introduction of an aniline
N
N
H
O
NH2
Compound
R1 (substituted
phenyl)
Binding
a
IC50 (nM)
Chemotaxis
a
IC50 (nM)
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
4-Cl
4-Br
26
3.6
2.6
3.1
0.89
3.9
11
116
20
4-CH3
4-Et
11 (6)
19
20
4-Vinyl
4-OCH3
4-OH
42
5.1
11
4-Cl, 3-NH2
4-CH3, 3-NH2
4-OCH3, 3-NH2
4-OH, 3-NH2
4-OCH3, 3-OH
4-OH, 3-OCH3
2,4-(CH3)2
2,4-Cl2
4.1
6.1
5.3
14
12
8.8
18
53
39
9.2
14
3.2 (2.6)
96
0.83
ndb
a IC50 values were derived from dose response curves generated from
duplicate data points. Values with standard deviations (in paren-
theses) are means of at least three independent experiments.
b nd = not determined.

1872
W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1869–1873
group to afford the 2,5-substituted benzamides provided
a 3- to 7-fold improvement in binding activity across the
series. The effect was most dramatic in compound 57 (2-
NH2,5-CF3) where a 7-fold improvement in binding
activity and functional activity over 1 was observed.
To add evidence that these compounds exerted their ef-
fects on THP-1 cells specifically through CCR2, com-
pound 57 was further evaluated in an [125I]-MCP-1
binding assay using HEK293 cells transfected with the
hCCR2b receptor. The observed binding IC50 of
45 nM was consistent with the binding IC50 observed
in the THP-1 cell line. The effect of compound 57 on
CCR1 was also measured using [125I]-MIP-1a and
THP-1 cells showing 25% inhibition at 1 lM
(IC50 > 1lM), indicating more than 50-fold selectivity
over the related chemokine receptor.
did not elicit a Ca2+ flux and therefore were not partial
agonists, but antagonists (data not shown).
In conclusion, high-throughput parallel synthesis al-
lowed us to rapidly explore the SAR of all three compo-
nents (R1 substituted benzyl group, R3 amino acid
spacer and R2 substituted benzamide) of the (R)-3-ami-
no pyrrolidine series 2 in a systematic fashion. Signifi-
cant improvements in binding affinity and functional
activity were achieved and compound 71 (binding IC50
3.2 nM, chemotaxis IC50 0.83 nM) showed a >60-fold
improvement over lead compound 1. The potent
CCR2b antagonists described herein will be useful in
the studies on the role of MCP-1 and CCR2 in diseases
where monocyte trafficking plays a role.
Acknowledgments
Re-optimization of R1 substitution on the benzyl group
in the presence of preferred amino acid spacer glycine
and 5-substituted 2-anthranilimides afforded extremely
potent CCR2b antagonists. Guided by the SAR devel-
oped in earlier compounds disclosed in Table 1, a num-
ber of para-substituted benzyl groups were examined
with the new anthranilamides. Not unexpectedly, the
most active compounds were found to contain the 5-tri-
fluoromethyl-2-anthranilamide for R2, which are listed
in Table 3. A relatively flat SAR was obtained for the
incorporation of a single electron-donating substituent
in the 4-position at R1 (Compounds 58–64, binding
IC50 11–42 nM). An improvement in activity could be
achieved by the incorporation of an additional elec-
tron-donating substituent in the 2- or 3- position of
the benzyl ring. Several of these 2,4- and 3,4-disubsti-
tuted compounds exhibited single-digit nanomolar bind-
ing affinity for the CCR2b receptor and potently
inhibited MCP-1-induced chemotaxis (Compounds 65–
72). Compound 71 was the most active compound in
this series and was a potent inhibitor of MCP-1 binding
(IC50 3.2 nM) as well as a sub-nanomolar inhibitor of
MCP-1 induced chemotaxis (IC50 0.83 nM).
The authors wish to acknowledge Dr. Erin Bradley and
Dr. David Spellmeyer for computational support on this
program, Dr. Dan Kassel, Dr. Lu Zeng and Ms. Xiaoli
Wang for their technical expertise in setting up systems
for mass triggered HPLC purification, Ms. Akiko
Takeuchi for her expertise on the Ca2+ flux inhibition
assay and Dr. Takeshi Hara and Dr. Seizi Kurozumi
for general support.
References and notes
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Several compounds were chosen for further evaluation
in an additional functional assay: the MCP-1 induced
18
Ca2+ flux assay.
Inhibition of the release of Ca2+
was observed for all compounds tested and a close cor-
relation between the binding IC50 and the inhibition of
Ca2+ release was observed (see Table 4). Compounds
incubated with THP-1 cells in the absence of MCP-1
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Binding
a
IC50 (nM)
Chemotaxis
a
IC50 (nM)
Ca2+ Flux
b
IC50 (nM)
19
25
58
61
71
116 (31)
226
26
12.4 (0.3)
5.5
3.6
64
365
23.1
5.3
7.5
11 (6)
3.2 (2.6)
0.89
0.83
a IC50 values were derived from dose response curves generated from
duplicate data points. Values with standard deviations (in paren-
theses) are means of at least three independent experiments.
b IC50 values are averages from dose response curves generated from
duplicate data points from two independent experiments.

W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1869–1873
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were cultured in HBSS loading buffer supplemented with
1% fetal bovine serum (FBS) and 20 mM HEPES. After
centrifugation, cells were loaded for 30 min with the Ca2+
sensitive fluorescent dye Fluo-3 AM (2 million cells/mL in
RPMI medium containing 4 lM Fluo-3 AM, 20 mM
HEPES, 0.1% bovine serum albumin (BSA), and 5 mM
probenecid). Excess dye was removed by 3-fold washing
with buffer (5 mM HEPES, 140 mM NaCl, 1 mM MgCl2,
5 mM KCl, 10 mM glucose, 2.5 mM probenecid, 1.25 mM
CaCl2, and 0.1% BSA; all further incubations were done in
this matter). Cells were plated at a density of 150,000 cells/
well in dark-wall 96-well plates and sedimented by
centrifugation (1 min). The cells were pre-incubated for
3 min with test compound. Then 10À7 M human MCP-1
was added. Changes in intracellular free Ca2+ concentra-
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Reader (FLIPR). Fluorescence was recorded in two
consecutive sequences. Each sequence was comprised of
60 s (recordings at 1 s intervals) followed by 120 s
(recordings at 6 s intervals). Test compound was added at
10 s into the first sequence. MCP-1 was added at 10 s into
the second sequence. After addition in each sequence,
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Methods 1980, 33, 239.
15. Topliss, J. G. J. Med. Chem. 1977, 20, 463.
16. For example, replacement of Gly in compound 1 with
D/L-Ala resulted in a MCP-1 binding IC50 of 22 lM,
replacement with b-Ala gave an IC50 of 10 lM.