Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.08.008

The discovery, the lead optimization, and the structure-activity relationship of N,N′-disubstituted homopiperazines are reported. N,N′-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the σ factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.

Full text of DOI:10.1016/j.bmcl.2004.08.008

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5407–5411
Small molecule inhibitors of the CCR2b receptor. Part 1:
Discovery and optimization of homopiperazine derivatives
a
a
b,
Minoru Imai,a, Tatsuki Shiota, Ken-ichiro Kataoka, Christine M. Tarby,
*
Wilna J. Moree,b,à Takaharu Tsutsumi,a Masaki Sudo,a,§
Michele M. Ramirez-Weinhouse,b,– Daniel Comer,b,k Chung-Ming Sun,b,**
Shinsuke Yamagami,a Hiroko Tanaka,a Takuya Morita,a Takahiko Hada,a,
Jonathan Greene,b Doug Barnum,b John Saunders,b,à
Peter L. Myers,b,àà Yoshinori Katoa and Noriaki Endoa
aTeijin Institute for Bio-medical Research, 4-3-2 Asahigaoka, Hino, Tokyo 191-8512, Japan
bDeltagen Research Laboratories,§§ 4570 Executive Drive, Suite 400, San Diego, CA 92121, USA
Received 6 October 2003; accepted 5 August 2004
Available online 15 September 2004
Abstract—N,N0-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors
with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an
important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the
r factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphen-
ylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to
search for alternative substructures is also described.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Chemokines, also known as chemotactic cytokines, are a
family of structurally related molecules that mainly reg-
ulate inflammatory responses such as leukocyte recruit-
ment, cellular activation, and inflammatory mediator
release. They act through interaction with chemokine
receptors, which are members of the G-protein coupled
receptor family.1 Monocyte Chemoattractant Protein-1
(MCP-1) is a member of the CC chemokine subfamily
and is produced by macrophages, smooth muscle cells,
fibroblasts, and vascular endothelial cells.
*
Corresponding author. Tel.: +81 3 3506 4556; fax: +81 3 3506 4080;
e-mail: min.imai@teijin.co.jp
Present address: Bristol–Myers Squibb Co., PO Box 4000, Princeton,
NJ 08543, USA.
MCP-1 causes cell migration and cell adhesion of mono-
cytes, memory T-cells and natural killer cells, as well as
mediation of histamine release by basophils.2 In addi-
tion, high expression of MCP-1 has been observed at
sites of inflammation, therefore MCP-1 is thought to
play an important role in inflammatory diseases such
as rheumatoid arthritis,3 atherosclerosis,4 glomerulone-
phritis,5 and multiple sclerosis.6 For reasons described
above, many groups7 including ours, have developed
programs to discover CCR2b antagonists. Among the
hit compounds found from broad screening of the Teijin
small molecule library and known GPCR antagonists,
homopiperazine 1 was recognized as a suitable structure
for traditional optimization (Fig. 1). In this paper, the
hit-to-lead studies around the initial screening hit are
described.
à Present address: Neurocrine Biosciences, 12790 El Camino Real, San
Diego, CA 92130, USA.
§ Present address: Pfizer Global Research and Development, Nagoya
Laboratories, 5-2 Taketoyo, Aichi 470-2393, Japan.
– Present address: Pfizer Global Research and Development, La Jolla
Laboratories, 3550 General Atomics Court, San Diego, CA 92121,
USA.
k Present address: Neurogenetics Inc., 11085 North Torrey Pines Road,
Suite 300, La Jolla, CA 92121, USA.
** Present address: Department of Chemistry, National Dong Hwa
University, Shou-Feng, Hualien 974, Taiwan.
Present address: Bizen Chemical Co., Ltd, Research and Develop-
ment Department, 363-Tokutomi, Kumayama-Cho, Akaiwa-Gun,
Okayama 709-0716, Japan.
àà Present e-mail address: myerspl@mindspring.com
§§Formerly CombiChem, Inc.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.008

5408
M. Imai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5407–5411
Grignard reaction generated compounds 6 (Scheme 2).
SO2CH3
Independent substitution of each benzhydryl ring was
accomplished in a similar fashion. Michael addition
products 7 or 8, formed by reaction of phenyl vinyl
ketone and 4, were prepared and immediately used with-
out purification due to their tendency to undergo retro-
Michael reactions. The addition of Grignard reagents to
the freshly prepared ketones afforded the unsymmetrical
compounds 9 or 10, respectively (Scheme 3). In order to
obtain 10, desilylation with tetrabutylammonium fluo-
ride (TBAF) in THF was carried out after mono-aryl-
ation. All the compounds described in this paper were
purified using silica gel column chromatography. Puri-
ties were checked with reverse phase HPLC under iso-
cratic condition and the structures were confirmed
N
N
1i
Figure 1. N-(3,3-Diphenylpropyl)-N0-(4-methylsulfonylbenzyl)homo-
piperazine (1i).
a
+
HN
NH
HN
NH
HN
NH
2HCl
HCl
2
1
c
b
with H NMR.
N
NH
N
N
R1
We envisioned that our computational methodology
could assist in the prioritization of compounds for syn-
thesis, either within the homopiperazine series or around
alternative scaffolds. This computational methodology
utilizes an ensemble of 2–4 point pharmacophores to
refine the molecular space to areas where active com-
pounds likely reside. While pharmacophore models have
been employed by others to identify active compounds,
our strategy differs in that only those pharmacophores
capable of discriminating between active and inactive
molecules become part of the model. Typically, the
ensemble contains 50–100 pharmacophores. Once
selected, the ensemble is then used to assist in the prior-
itization of compounds for synthesis. Since nearly all of
the molecules in the initial dataset were closely related to
1, locating pharmacophores that were selective for active
3
1
Scheme 1. Reagents and conditions: (a) EtOH; (b) (Ph)2CHCH2-
CH2OMs, K2CO3, EtOH, heat, 93% yield; (c) R1Cl or R1Br, MeCN,
heat, 20–70% yield.
The initial goal of the program was to optimize the
homopiperazine series to potent selective CCR2b antag-
onists. As a separate goal, we also sought to probe for
information around the lead series such that a computa-
tional model of activity could be generated and
employed.8 This paper will also discuss the identification
of several informer molecules used to begin the process
of lead evolution (using the pharmacophoric informa-
tion from one series of molecules to identify new chem-
ical scaffolds with similar or improved activities).
SO2Me
Derivatives of N-(3,3-diphenylpropyl)homopiperazine 1
were synthesized by mono-alkylation of homopiperazine
monohydrochloride 2, prepared in situ from equimolar
amounts of free diamine and dihydrochloride salt, with
3,3-diphenylpropyl methanesulfonate, followed by a
subsequent alkylation of the other nitrogen (Scheme 1).
a
4
N
N
A
O
7, A=H
8, A=OSiMe2-t-Bu
SO2Me
OH
R1
b
N
N
A
In order to synthesize the intermediate for left-hand side
modification, monobenzylation was carried out with
homopiperazine monohydrochloride 2 and 4-(methyl-
sulfonyl)benzyl methanesulfonate to obtain 1-[4-(methyl-
sulfonyl)benzyl]homopiperazine 4 (Scheme 2). Alkyl-
ation of the second nitrogen with methyl 3-bromoprop-
ionate gave intermediate 5, followed by a double
9, A = H
10, A = OH
Scheme 3. (a) PhCOCH@CH2 (neat) or (i) 3-TBSO–PhCOCH@CH2,
CH2Cl2, (ii) concentrate in vacuo; (b) (i) R1MgBr (1equiv), Et2O or
THF; (ii) H3O+, (iii) TBAF, THF for 10, 8–62% yield for two steps.
SO2Me
b
SO2Me
a
2
+
HN
N
MsO
4
SO2Me
OH
SO2Me
c
R1
N
N
MeO
N
N
R1
O
5
6
Scheme 2. Reagents and conditions: (a) EtOH, heat, 85% yield; (b) BrCH2CH2CO2Me, K2CO3, MeCN, heat, 59% yield; (c) (i) R1MgBr (2equiv),
Et2O or THF, (ii) H3O+, 10–84% yield.

M. Imai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5407–5411
5409
designed to query the pharmacophoric space around
the template in an overlapping fashion. Since this library
was designed to procure information, the compounds
from the set are called Ôinformer moleculesÕ. The com-
pounds were prepared as shown in Scheme 4. N-Boc-
homopiperazine was heated with alkyl halide in the pres-
ence of K2CO3 to introduce the first R group. Removal
of the Boc group followed by either a second alkylation
reaction or coupling to a carboxylic acid under standard
conditions, afforded the fully functionalized molecules.
R1
a
b, c
N
N
N
N
Boc
NH
N
R
Y
R
Boc
Y = CH2 or C=O
Scheme 4. Reagents and conditions: (a) RX (1.1equiv), K2CO3
(5equiv), CH3CN, 70ꢁC; (b) 3M HCl–EtOAc; (c) R1CH2X (1.1equiv),
K2CO3 (5equiv), CH3CN, 70ꢁC or R1COOH (1.2equiv), EDCI
(1.3equiv), HOBt (1.3equiv), i-Pr2NEt (3equiv), DMF, rt.
molecules over inactive ones was very difficult. There-
fore, to improve the quality of the early computational
model, a library of homopiperazine molecules was
Inhibitory activity of compounds was determined
against human 35S-MCP-1 or 125I-MCP-1 binding to
THP-1 cells.9,10 The results of selected compounds are
summarized in Tables 1–5. In Table 1, the structure
activity relationships around the R1 group in 1 are
shown. Compounds with simple alkyl groups (1a) or
unsubstituted benzyl groups (1b) have no affinity for
CCR2b. Introduction of 4-pyridylmethyl at the R1 posi-
tion (1c) improved activity for the receptor. A tendency
of a preference for the 4-pyridyl ring was observed, and
the 2- and 3-pyridyl systems exhibited reduced affinities
(1d and 1e). A similar preference for 4-substitution was
observed for the nitrobenzyl derivatives 1f–h. Moving
the nitro group to the 2- or 3-position resulted in loss
of activity. Other electron-withdrawing groups such as
methylsulfonyl (1i) and cyano (1j) increased activity,
while electron-donating groups like methoxy (1k) de-
creased activity. Substituents that have moderate Ham-
metÕs rp values in range of 0.2–0.6 also resulted in low
affinity (1l–n).
Table 1. Binding inhibitory activity of N-(3,3-diphenylpropyl)homo-
piperazine derivatives
N
N
R1
1a - 1n
Compounds
R1
% Inhibition
at 100lMa
IC50, lMa
1a
1b
15
24
Ndb
Nd
N
N
1c
1d
87
69
11
Nd
Compound1i, 1-(4-methylsulfonylbenzyl)-4-(3,3-diphen-
ylpropyl) homopiperazine (IC50 = 13lM) with an EC50
of 5.8lM (n = 1) in a chemotaxis assay11 was chosen
1e
1f
35
87
Nd
19
N
NO2
NO2
Table 2. Effect of substituent on phenyl groups
SO2Me
1g
1h
35
7
Nd
Nd
R
OH
N
N
NO2
R
6a - 6n
% Inhibition at 100lMa IC50 lMa
SO2Me
CN
1i
91
74
7
13c
43
Compounds
R
H
6a
6b
6c
6d
6e
6f
104
39
0
4.1 (n = 2)
Ndb
Nd
4-t-Bu
4-Ph
4-CF3
1j
OMe
CF3
38
Nd
45
1k
1l
Nd
Nd
Nd
Nd
4-NMe2 68
4-OH
3-OH
3-OMe
2-OMe
2-Me
4-F
83
108
38
14
0
21
6g
6h
6i
1.5c
Nd
25
8
Nd
Nd
Cl
6j
1m
1n
6k
6l
100
75
94
28
7.0
34
3-F
4-Cl
CO2Me
1
6m
6n
11
Nd
3-Cl
a Assayed with 125I-MCP-1 (n = 1, unless indicated otherwise).
b Not determined.
c SEM = 0.16lM (n = 5).
a Assayed with 35S-MCP-1 (n = 1 unless indicated otherwise).
b Not determined.
c SEM = 1.8lM (n = 5).

5410
M. Imai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5407–5411
Table 3. Derivatives of 1i that have unsymmetrical left hand structures
shown). Therefore, the lead optimization from 1i was
carried out by structural development of the benzhydryl
group. Table 2 shows the activity of symmetrical 3,3-
diphenyl-3-hydroxypropyl derivatives 6. Introduction
of a hydroxy group at 3-position of the propyl chain
in 1i (6a) led to a 3-fold increase in binding affinity
and resulted in the first compound with a low micromo-
lar IC50 value. A range of activities was observed for 4-
monosubstituted phenyl derivatives. Lipophilic substitu-
tion (6b–d) reduced activity, while dimethylamino (6e)
and hydroxyl (6f) preserved inhibitory activity. More-
over, transfer of the hydroxyl group to the 3-position
(6g) improved binding affinity, though introduction of
a 3-methoxy substituent (6h) was not effective in aug-
menting activity. Compounds 6i and 6j, functionalized
in the 2-position, showed no significant activity. The
halogen-substituted compounds 6k–n exhibited an
opposite SAR as compared with the hydroxy deriva-
tives, in that the 4-halo compounds were more active
than 3-halo compounds.
SO2Me
OH
N
N
R
9a - 9g
% Inhibition at 100lMa IC50, lMa
Compounds
R
9a
9b
9c
9d
9e
9f
3-OH
105
0.71 (n = 2)
3-CH2OH 100
3-NH2 102
3-NHMe 87
4.0
4.2
5.0
7.9
12
3-OMe
3-F
3-Me
97
95
92
9g
16
a Assayed with 125I-MCP-1 (n = 1, unless indicated otherwise).
Table 4. Unsymmetrical derivatives of 1i with 3-hydroxyphenyl group
SO2Me
In the series of compounds 9 (Table 3) and 10 (Table 4)
the two phenyl rings of the benzhydryl moiety were
independently substituted.
OH
N
N
HO
As seen in the symmetrical derivatization (Table 2), the
3-hydroxyphenyl derivative 9a exhibited the best activ-
ity, with an IC50 value of 0.71lM (Table 3). Introduc-
tion of hydroxymethyl (9b), amino (9c), methylamino
(9d), or methoxy (9e), to the 3-position of the phenyl
ring decreased activity relative to 9a, but still resulted
in low lM affinities for the receptor. A decrease in activ-
ity was observed when fluoro (9f) or methyl (9g) were
introduced into the same position. In comparing the
halo-substituted derivatives 10 (Table 4), as seen in Ta-
ble 3, 4-substitution was preferred over 3-substitution
(10a–d). Compounds 10c and 10d had the same activity
as 6g but were less active than 9a. The 3,5-difluoro
R
10a - 10e
Compounds
R
% Inhibition at 100lMa IC50, lMa
10a
10b
10c
10d
10e
3-F
104
99
2.4
4.5
1.5
1.5
9.3
3-Cl
4-F
105
104
4-Cl
3,5-DiF 107
a Assayed with 125I-MCP-1 (n = 1).
as the lead compound for further optimization, because
of its metabolic stability and low cytotoxicity (data not
Table 5. Optimization of the benzenesulfonyl group and identification of informer molecules
OH
Cl
N
N
HO
R1
N
N
R1
Class I
Class II
R1
Class I
Class II
Compound
IC50, lMa
Compound
IC50, lMa
SO2CH3
SO2NH2
9a
0.71
14
>100
11
12
13
1.5
6.4
28
15
16
17
Ndb
7.4
30
O
O
S
H
N
CH3
N
H
O
S
H
N
N
H
O
a Assayed with 125I-MCP-1 (n = 1).
b Not determined.

M. Imai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5407–5411
5411
J. Immunol. 1994, 24, 3233; (h) Alam, R.; Lett-Brown, M.
A.; Forsythe, P. A.; Anderson-Walters, D. J.; Kenamore,
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723.
derivative (10e) exhibited decreased activity as com-
pared to the mono-fluorinated compound (10a). In addi-
tion, since all the compounds with an unsymmetrical
benzhydryl moiety 9 and 10 were synthesized as racemic
mixtures, resolution to optically active isomers should
afford improvements in activity over that reported in
Tables 3 and 4.
3. (a) Ogata, H.; Takeya, M.; Yoshimura, T.; Takagi, K.;
Takahashi, K. J. Pathol. 1997, 182, 106; (b) Gong, J.-H.;
Ratkay, L. G.; Waterfield, J. D.; Clark-Lewis, I. J. Exp.
Med. 1997, 186, 131.
4. (a) Boring, L.; Gosling, J.; Cleary, M.; Charo, I. F. Nature
1998, 394, 894; (b) Dawson, T. C.; Kuziel, W. A.; Osahar,
T. A.; Maeda, N. Atherosclerosis 1999, 143, 205.
5. Lloyd, C. M.; Minto, A. W.; Dorf, M. E.; Proudfoot, A.;
Wells, T. N. C.; Salant, D. J.; Gutierrez-Ramos, J.-C.
J. Exp. Med. 1997, 185, 1371.
Interestingly, some of the molecules from the informer
library (Table 5) exhibited activities in the range of the
initial leads. It was found that in the hydrazido series,
16 and 17, a para-chlorobenzyl substituent could be used
as a suitable replacement for the 3,3-diphenylpropyl
group. This observation did not carry over to the benz-
enesulfonyl series (e.g. 14). As such, compounds 16
(EC50 of 9.5lM (n = 1) in a chemotaxis assay) and 17
constituted a new series of MCP-1R inhibitors.
6. Kennedy, K. J.; Strieter, R. M.; Kunkel, S. L.; Luckacs,
N. W.; Karpus, W. J. J. Neuroimmunol. 1998, 92, 98.
7. (a) Mirzadegan, T.; Diehl, F.; Ebi, B.; Bhakta, S.; Polsky,
I.; McCarley, D.; Mulkins, M.; Weatherhead, G. S.;
Lapierre, J.-M.; Dankwardt, J.; Morgans, D., Jr.; Wil-
helm, R.; Jarnagin, K. J. Biol. Chem. 2000, 275, 25562; (b)
Forbes, I. T.; Cooper, D. G.; Dodds, E. K.; Hickey, D. M.
B.; Ife, R. J.; Meeson, M.; Stockley, M.; Berkhout, T. A.;
Gohil, J.; Groot, P. H. E.; Moores, K. Bioorg. Med. Chem.
Lett. 2000, 10, 1803; (c) Witherington, J.; Bordas, V.;
Cooper, D. G.; Forbes, I. T.; Gribble, A. D.; Ife, R. J.;
Berkhout, T.; Gohil, J.; Groot, P. H. E. Bioorg. Med.
Chem. Lett. 2001, 11, 2177; For short reviews see: (d)
Trivedi, B. K.; Low, J. E.; Carson, K.; LaRosa, G. J.
Annu. Rep. Med. Chem. 2000, 35, 191; (e) Howard, O. M.
Z.; Yoshimura, T. Expert Opin. Ther. Pat. 2001, 11(7),
1147.
In conclusion, within the N-(3,3-diphenylpropyl)-N0-
(benzyl)homopiperazine series, electron-withdrawing
groups in the 4-position of the benzyl moiety such as
the methylsulfonyl and the nitro substituents are fav-
ored, while electron-donating groups decrease activity.
Furthermore, introduction of a hydroxyl group to the
methyne carbon of the benzhydryl group as well as 3-
position of one phenyl ring increased the activity of
the compounds.
We also synthesized a series of informer molecules from
which a new series of CCR2b inhibitors were identified
(16, 17). These results provided the information used
to initiate the lead evolution program, which will be
the subject of our next letter.
8. For a treatise on the use of informative libraries for drug
discovery programs see: Myers, P. L.; Greene, J. W.;
Saunders, J.; Teig, S. L. TodayÕs Chemist at Work
1997(Jul/Aug), 47.
9. Preparation method of 35S-MCP-1 expressed from bacu-
lovirus with culture medium containing 35S-Met refer to:
Ishii, K.; Yamagami, S.; Tanaka, H.; Motoki, M.; Suwa,
Y.; Endo, N. Biochem. Biophys. Res. Commun. 1995, 206,
955.
Acknowledgements
10. Recombinant baculovirus carrying the human MCP-1
gene was constructed such that the protein could be
expressed and subsequently labeled (Amersham). The
THP-1 cells suspended in 50lL assay buffer (RPMI-1640
containing 0.1% BSA and 25mM HEPES, pH7.4,
1 · 107 cells/mL) were treated with compound solution
(25lL) and labeled ligand (25lL, 1mCi/mL). After
incubation and washing, the radioactivity retained by the
cells was measured by scintillation counting. Dose curves
of each compound with 4–6 concentration points in
triplicate were generated to determine IC50 values.
11. THP-1 cells suspended in assay buffer (RPMI-1640
containing 10% FCS) were placed in the upper chamber
The authors gratefully acknowledge Dr. Takeshi Hara
and Dr. Seizi Kurozumi for general support.
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TM
(200lL) of a 96-well micro-chemotaxis chamber , and
MCP-1 in a same solution at a final concentration of
20ng/mL was placed in the lower chamber, with polycar-
bonate filter placed between the two chambers. After
incubation at 37ꢁC for 2h in 5% CO2, the filter was
removed, and the cells, which had migrated to the
underside of the filter were fixed, stained using Diff Quick
and then quantitated using a plate reader at a wavelength
of 550nm to determine the index of cell migration as a
mean of three cells. Test compounds were placed in the
upper and lower chambers along with THP-1 and MCP-1,
respectively. Dose curves of each compound with 6–8
concentration points in triplicate were generated to
determine EC50 values.