Synthesis and biological evaluation of 3-aminopyrrolidine derivatives as CC chemokine receptor 2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.02.072

Novel 3-aminopyrrolidine derivatives were synthesized and evaluated for their antagonistic activity against human chemokine receptor 2. Structure-activity studies on 3-aminopyrrolidine incorporating heteroatomic carbocycle moieties led to piperidine compo

Full text of DOI:10.1016/j.bmcl.2010.02.072

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2099–2102
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 3-aminopyrrolidine derivatives as CC
chemokine receptor 2 antagonists
a
Jee Woong Lim a,e, , Youna Oh , Jong-Hoon Kim a,b, Min-Ho Oak c, Yongho Na a, Jung-Ok Lee c,
*
Seung-Woo Lee a, Heeyeong Cho d, Woo-Kyu Park d, Gildon Choi d, Jongmin Kang e,
*
a Yangji Chemicals, Gyeonggi Bio-Center, Suwon 443-766, Republic of Korea
b Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
c Research and Development Center, Hanwha Pharma, Chuncheon 200-921, Republic of Korea
d Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
e Department of Chemistry, Sejong University, Seoul 143-747, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 3-aminopyrrolidine derivatives were synthesized and evaluated for their antagonistic activity
against human chemokine receptor 2. Structure–activity studies on 3-aminopyrrolidine incorporating
heteroatomic carbocycle moieties led to piperidine compound 19, and piperazine compounds 42, 47
and 49 as highly potent hCCR2 antagonists.
Received 29 October 2009
Revised 15 January 2010
Accepted 17 February 2010
Available online 20 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CCR2
Antagonist
3-Aminopyrrolidine
Monocyte chemoattractant protein-1 (MCP-1) is one of the che-
mokines of CC family and has been known to play an important
role in the upregulation of immune cells in the biological system.1,2
CCR2 (CC chemokine receptor 2), a receptor of MCP-1, belongs to
the G-protein coupled receptor (GPCR) family, and the blocking
of the interaction between MCP-1 and CCR2 results in variation
of physiological activities including the abatement of inflamma-
tion. It has been suggested that antagonism mediated by monoclo-
nal antibody or small molecule could be applied to the
development of pharmaceutical candidates targeting on various
inflammation-related diseases.3–7 Rheumatoid arthritis, athero-
sclerosis, multiple sclerosis and type 2 diabetes mellitus have been
tried as interesting therapeutic areas, and many literatures re-
vealed the proof of concept through clinical trials and animal stud-
ies. Recently, diverse scaffolds as small molecule CCR2 antagonists
have been explored mainly by pharmaceutical companies. Some
examples are shown in Figure 1.8–10
acids got rid of the CCR2 antagonism. Likewise, the substitution on
the benzene ring failed to provide improved in vitro efficacy com-
pared with the prior structure, except for 2-amino-5-trifluoro-
methyl substitution.11 We synthesized related compounds such
as 2,2-dimethylglycine adduct, trifluoromethylbenzenesulfona-
mide, and N-alkylamide derivatives. These compounds showed
poor in vitro activity as CCR2 antagonist. Therefore, we focused
on the variation of N-substitution of pyrrolidine. We here report
their in vitro activities and properties as CCR2 antagonists, includ-
ing the structure–activity relationship. The representative struc-
tures are shown in Figure 2.
Synthetic method of these compounds is shown in Schemes 1
and 2.12 A major intermediate 9 could be easily prepared by ami-
dation with glycine, followed by the amide coupling with benzyl-
protected pyrrolidine and deprotection with hydrogenolysis using
palladium catalyst. On the purpose to synthesize 5 and 6 series
compounds, we prepared separately N-Boc-protected piperidine
and piperazine derivatives that contained mesylated alcohol group
as a leaving group linked with ethylene. After N-pyrrolidine substi-
tution reaction, deprotection under acidic condition of Boc group
was performed to prepare useful intermediates 10. The obtained
piperidine and piperazine moieties could be easily derivatized with
reactions by use of benzoyl halides, benzyl halide, alkyl halide or
aryl isocyanate to offer final compounds 11.
Research groups of several pharmaceutical companies have
been interested in 3-aminopyrrolidine derivatives substituted with
glycine-linked 3-trifluoromethylbenzamide such as compound 1.
Centering around the pyrrolidine structure, derivatization of 3-
(R)-substituted moiety showed rather restricted results. Instead
of the glycine spacer, introduction of various a-substituted amino
We tried to introduce some small-sized alkyl side chains on eth-
ylene linker, based on the idea that the ethylene linker between
* Corresponding authors. Tel.: +82 31 888 6618; fax: +82 31 888 6620 (J.W.L.).
E-mail address: srhnys@hwpharm.com (J.W. Lim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.072

2100
J. W. Lim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2099–2102
O
H
N
O
H
N
O
i), ii)
iii)
CF3
CF3
CF3
Cl
N
H
N
H
HN
N
O
O
8
9
7
iv), v)
O
R1
W
H
N
H
N
O
vi)
HN
X
N
CF3
X
CF3
N
H
N
N
H
N
O
O
11
10
Scheme 1. Synthesis of piperidine and piperazine analogs. Reagents and conditions: (i) glycine, 2 N NaOH aq, CH3CN; (ii) (3R)-1-benzyl-3-aminopyrrolidine, NMM, isobutyl
chloroformate, THF; (iii) H2 gas, Pd(OH)2 cat., MeOH; (iv) 4-(2-methanesulfonyloxyethyl)-N-Boc-piperidine or 4-(2-methanesulfonyloxyethyl)-N-Boc-piperazine, K2CO3,
CH3CN; (v) HCl satd EtOH; (vi) any proper reagents to prepare final products.
R2
R4
R4
i)
R2
Cl
+
N
N
O
R3
O
N
NH
O
O
R3
12
13
14
ii)
R4
R2
O
H
N
N
N
CF3
O
N
H
N
R3
O
15
iv), v)
O
R2
R2
H
N
O
HO
iii)
HO
CF3
or
R2
R3
N
H
Cl
N
R3
16
R3
O
17
18
Scheme 2. Synthesis of piperazine analogs. Reagents and conditions: (i) K2CO3, CH3CN; (ii) compound 9, NaBH(OAc)3, THF; (iii) compound 9, K2CO3, CH3CN; (iv)
methanesulfonyl chloride, TEA, MC; (v) compound 12, K2CO3, CH3CN.
O
H
N
NH
O
R
CF3
S
N
H
H
N
O
O
CF3
HN
N
H
N
O
HN
O
1 : Teijin
2 : BMS
IC50 = 5.1nM (CCR2 binding)
1a : R = 4-Cl, IC50 = 180nM (CCR2 binding)
1b : R = 2,4-di-Me, IC50 = 54nM (CCR2 binding)
HO
N
NH
O
F
O
F
F
CF3
CF3
N
H
N
O
HN
3 : Merck
4 : J&J
IC50 = 1.2nM (CCR2 binding)
IC50 = 0.1nM (chemotaxis)
IC50 = 2.4nM (CCR2 binding)
Figure 1. CCR2 antagonists.

J. W. Lim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2099–2102
2101
Table 3
R1
W
R2
O
Evaluation of piperazine derivatives
H
N
N
X
CF3
O
R2
O
N
H
N
H
N
R3
N
N
O
CF3
N
H
N
R4
R3
O
5 : X = C
6 : X = N
Compound R2
R3
R4
IC50 (lM)
Ca2+
flux
hCCR2b
binding
Chemotaxis
W = CO, SO2, CH2
R1 = aryl
R2, R3 = H, alkyl
42
43
44
45
46
47
48
49
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.99
4.44
3.52
3.46
>10
1.12
1.88
3.04
0.018
0.12
0.17
0.11
1.10
0.0017
0.0026
0.0037
0.009
2-Me
3-Me
4-Me
4-F
2-Cl
2-F
Figure 2. 4-Piperidinoethyl and piperazinoethyl pyrrolidine analogs.
0.023
>0.10
0.010
Table 1
2,5-Di-
Evaluation of piperidine derivatives
F
R1
W
50
51
52
53
Me
H
H
H
H
H
H
H
>10
>10
>10
>10
0.71
1.86
>10
>10
H
N
O
Me
Et
H
N
CF3
N
H
N
O
Et
Compound R1
W
IC50 (lM)
hCCR2b
bindinga
Ca2+
flux
Chemotaxis
0.023
was carried out by two methods as shown in Scheme 2. Starting
from commercially available or in-house synthesized reagents,
various ketone analogs 14 were made, and reductive amination
with intermediate 9 using mild reductive agent such as sodium tri-
acetoxyborohydride offered the target compounds 15. Addition-
ally, chloroethanol derivatives or 2-/3-mono substituted epoxide
reagents were used to get our final compounds 15 via mesylated
alcohol intermediates. In these cases, proper synthetic methods
were chosen according to ease-of-synthesis.
Primary human CCR2b (hCCR2b) protein binding inhibitory
activity of all the synthesized compounds was monitored against
human [125I]MCP-1 binding to THP-1 cells.13 The functional activ-
ity of selected compounds was then measured using the calcium
flux (Flexstation, HEK293/CCR2b cells) and chemotaxis (human
MCP-1, monocytes) inhibition assays.14,15 To compare the activity
of our compounds with previously published compounds, N-ben-
zylpyrrolidine derivatives 1a, 1b which contain similar structure
with our compounds were co-assayed as the references. In the
hCCR2b protein binding experiments, the reference compounds
showed weak inhibition compared to their activities reported pre-
viously. But in the cell-based functional assays, they showed the
close level of activities to literature data. Therefore we integrative-
ly evaluated the activities of the synthesized compounds from
three in vitro assays, inhibition of protein binding affinity, calcium
flux and chemotaxis.
19
20
21
22
Ph
C@O 0.58
C@O 56%
C@O 53%
C@O 21%
0.030
3-Pyridyl
4-Pyridyl
2-
Naphthyl
Ph
Ph
n-Butyl
PhO–
23
24
25
26
CH2
SO2
15%
41%
C@O 32%
C@O 39%
C@O 22%
3.20
27
PhNH–
1ab
1bb
0.98
0.30
>0.30
0.070
0.61
a
% inhibition at 10
Reference compounds (Fig. 1).
l
M when no IC50 was measured.
b
Table 2
Evaluation of N-benzoyl piperidine derivatives
O
H
O
N
N
CF3
N
H
N
R5
O
Compound
R5
IC50 (lM)
Ca2+ fluxb
hCCR2b bindinga
Chemotaxis
>0.10
28
29
30
31
32
33
34
35
36
37
38
39
40
41
2-F
3-F
4-F
2,5-Di-F
2-Cl
1.13
8.33
6.60
1.41
1.31
3.16
7.52
2.21
2.88
1.18
1.00
1.74
1.73
48%
0.029
0.16
25%
0.011
0.012
31%
Table 4
Evaluation of drug candidates
>0.10
0.009
Cytotoxicityd (GI50
, lM)
4-Cl
Compound
IC50
(lM)
3,4-Di-Cl
4-CF3
2-Me
3-Me
4-Me
4-Et
41%
hERG Ka
CYP450b
GPCRc
0.66
0.52
0.75
0.62
47%
19
28
31
32
42
47
48
49
>10
>10
;10
>10
>10
>10
>10
>10
>10
>10e
>10e
>10e
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10f
>10
>10
>10
>10
>10
>10
>10
>10
>10
4-n-Pr
3,5-Di-Me
35%
a
% Inhibition at 10
% Inhibition at 1
l
M when no IC50 was measured.
lM when no IC50 was measured.
b
a
Human ERG potassium channel binding inhibition/[3H]astemizole.
Human recombinant enzyme: 1A2, 2C9, 2C19, 2D6 and 3A4 by P450-Glo
b
method.
two ring systems might tolerate some hydrophobic group substitu-
tion. Moreover, it was expected to provide steric constraint to flex-
ible ethylene linker. This idea was also supported by the in-house
modeling study (data not shown). Synthesis of these compounds
c
Receptors: 5-HT1a, 5-HT2a, 5-HT2c, 5-HT6, 5-HT7, D2, D3 and D4.
d
e
f
Test cell line: HepG2, NIH 3T3, CHO-K1, HEK 293 and HUVEC by MTT assay.
Except for 3A4 subtype.
Except for 5-HT6 and D4.

2102
J. W. Lim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2099–2102
As shown in Table 1, introduction of various N-substituents of
49 possessed excellent functional activities related to hCCR2
antagonism. Various pharmacological assays demonstrated the
usefulness of these compounds as drug candidates. Further studies
are being conducted to characterize the in vivo efficacy and phar-
macokinetics of these series of hCCR2 antagonists.
ethylene-linked piperidine resulted in the validity of benzoyl
group, compound 19, although alkylcarbonyl, heteroarylcarbonyl,
benzenesulfonyl, arylcarbonate and arylcarbamate groups showed
poor inhibition activity. In optimizing the substituents on the ben-
zoyl group of compound 19 (Table 2), halogenated derivatives 28,
31 and 32 showed comparable or slightly improved activities in
the functional calcium flux assay, and compound 32 was proved
to be the most active among them in the chemotaxis assay
(IC50 = 9 nM). However, alkyl substitution lowered the activity of
compounds regardless of the site on benzene ring. Furthermore,
as the alkyl groups became bulkier, the compounds were less ac-
tive. After all our effort of the replacement of other various func-
tional groups, it is evident that polar or bulky nonpolar
substituents were limited. For the halogen substitution, 2-position
was preferred, while 4-position was slightly preferred for methyl
substitution.
Further optimization was carried out on the piperidine ring,
replacing 4-carbon with nitrogen to offer piperazine analogs (Table
3). In the calcium flux and chemotaxis assay, compound 42 re-
sulted in improved activity compared with compound 19. As is
the case of piperidine series, halogenation on the benzoyl group
enhanced the functional activities. In the calcium assay compound
47, 48 and 49 showed excellent inhibitory activity (IC50 = 1.7, 2.6
and 3.7 nM, respectively), and compound 47 and 49 have also good
potency in the chemotaxis assay (IC50 = 23 and 10 nM, respec-
tively). The presence of small alkyl side chain on ethylene linker
did not, however, yield more active compounds. Despite the possi-
bility of spacial margin in the protein binding based on the com-
parison with published similar molecules and our molecular
modeling study, the introduction of alkyl group on the ethylene
linker probably disturbed the effective interaction of benzoylpiper-
azine moiety in the binding site of CCR2 protein.
References and notes
1. Schwarz, M. K.; Wells, T. N. Nat. Rev. Drug Disc. 2002, 1, 347.
2. Horuk, R.; Ng, H. P. Med. Res. Rev. 2000, 20, 155.
3. Libby, P. Nature 2002, 420, 868.
4. Feria, M.; Diaz-Gonzalez, F. Expert Opin. Ther. Patents 2006, 16, 49.
5. Boer, W. I. Drug Discovery Today 2005, 10, 93.
6. Das, A. M.; Brodmerkel, C. M. Drug Discovery Today: Ther. Struct. 2006, 3, 381.
7. Charo, I. F.; Ransohoff, R. M. N. Eng. J. Med. 2006, 354, 610.
8. 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. Bioorg. Med. Chem.
Lett. 2004, 14, 5413.
9. Kothandaramen, S.; Donnely, K. L.; Butora, G.; Jiao, R.; Pasternak, A.; Morriello,
G. J.; Goble, S. D.; Zhou, C.; Mills, S. G.; MacCoss, M.; Cascieri, M. A.; Yang, L.
Bioorg. Med. Chem. Lett. 2009, 19, 1830.
10. Xia, M.; Hou, C.; DeMong, D.; Pollack, S.; Pan, M.; Brackley, J.; Singer, M.;
Matheis, M.; Cavender, D.; Wachter, M. Bioorg. Med. Chem. Lett. 2008, 18, 3562.
11. Moree, W. J.; Kataoka, K.; Ramirez-Weinhouse, M. M.; Shiota, T.; Imai, M.;
Sudo, M.; Tsutsumi, T.; Endo, N.; Muroga, Y.; Hada, T.; Fanning, D.; Saunders, J.;
Kato, Y.; Myers, P. L.; Tarby, C. M. Bioorg. Med. Chem. Lett. 2008, 18, 1869.
12. All final compounds displayed spectral data (NMR, MS) that were consistent
with the assigned structures.
13. Membranes from stable HEK293-EBNA cell line expressing the human
chemokine CCR2b receptor (PerkinElmer Life and Analytical Sciences, Boston,
USA) were used. For CCR2b receptor binding assay, cell membrane (8
lg/well),
0.03 nM [125I]-MCP-1 (PerkinElmer) and appropriate concentrations of test
compounds were added to 0.25 ml of 25 mM Hepes (pH 7.2) buffer containing
2 mM CaCl2, 1 mM MgCl2 and 0.2% BSA. The mixture was incubated for 60 min
at 27 °C, and the reaction was terminated by rapid filtration using a cell
harvester (Inotech, Switzerland) through Filtermat A GF/C glass fiber filter
presoaked in 0.3% polyethylenimine. The filter was covered with MeltiLex,
sealed in a sample bag followed by drying in a microwave oven, and counted
by MicroBeta Plus (Wallac, Finland). Nonspecific binding was determined in
the presence of 0.05 lM human MCP-1. Competition binding studies were
carried out with 7–8 varied concentrations of the test compounds run in
duplicate tubes, and isotherms from three assays were calculated by
computerized nonlinear regression analysis (GraphPad Prism Program, San
Diego, USA) to yield inhibition values (IC50).
Selected compounds which were found to be active in the bind-
ing and the functional assays were evaluated further using several
in vitro assays (Table 4). In the human ERG potassium inhibition
assay and the cytotoxicity test, no toxicity was observed for most
compounds. However, halogenated analogs, 28, 31 and 32, exhib-
ited moderate inhibitory activities in CYP450 enzyme assay
14. FlexStation (Molecular Devices, Inc.) was used to measure the intracellular
calcium concentration in HEK293/CCR2b cells. The cells were seeded at 40,000
cells/well in
a 96-well, black-wall, clear-bottom, tissue culture treated
polystyrene plate (Costar 3603, Corning, NY) and grown at 37 °C in an
incubator with 5% CO2 for 24 h. After removing the media completely, the cells
were dye-loaded with Fluo-4 NW (Invitrogen) containing 1.25 mM probenecid
for 50 min at 37 °C. Subsequently, the cells were treated with various
concentrations of compounds and incubated further for 10 min at 37 °C.
Activation of CCR2b receptor was achieved by the addition of MCP-1 (final
concd = 20 nM) to the cells and the changes in relative fluorescence unit due to
the increase in the intracellular calcium concentration were monitored in the
FlexStation.
(IC50 = 0.3–0.8 lM for 3A4 subtype). Selectivity of compounds for
other GPCRs was initially determined in 5-HT and dopaminergic
receptors. All compounds showed no significant binding, except
for compound 48 which slightly bound to 5-HT6 and D4 receptors
(IC50 = 3.6 and 3.8 lM, respectively).
In conclusion, new series of ethylene-linked piperidine and
piperazine-3-aminopyrrolidine derivatives were synthesized.
Piperidine compound 19 and piperazine compounds 42, 47 and
15. For a description of the chemotaxis assay, see: Han, K. H.; Tangirala, R. K.;
Green, S. R.; Quehenberger, O. Arterioscler. Thromb. Vasc. Biol. 1998, 18, 1983.