Discovery of N-propylurea 3-benzylpiperidines as selective CC chemokine receptor-3 (CCR3) antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.01.059

The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented. Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT_2A receptor. Chiral resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas with CCR3 binding IC₅₀s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged from high picomolar to low nanomolar EC₅₀s and correlated well with antagonist binding IC₅₀s.

Full text of DOI:10.1016/j.bmcl.2004.01.059

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1645–1649
Discovery of N-propylurea 3-benzylpiperidines as selective CC
chemokine receptor-3 (CCR3) antagonists
Jeffrey G. Varnes, Daniel S. Gardner, Joseph B. Santella, III, John V. Duncia,
Melissa Estrella, Paul S. Watson, Cheryl M. Clark, Soo S. Ko, Patricia Welch,
Maryanne Covington, Nicole Stowell, Eric Wadman, Paul Davies, Kimberley Solomon,
Robert C. Newton, George L. Trainor, Carl P. Decicco and Dean A. Wacker*
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08542-5400, USA
Received 11 December 2003; revised 21 January 2004; accepted 22 January 2004
Abstract—The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented.
Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT2A receptor. Chiral
resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas
with CCR3 binding IC50s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged
from high picomolar to low nanomolar EC50s and correlated well with antagonist binding IC50s.
# 2004 Elsevier Ltd. All rights reserved.
Asthma is one of the most common chronic diseases in
industrialized nations.1,2 Clinical studies have linked
symptom severity in asthma patients to the gross accu-
mulation of eosinophils in the lungs.3 Eosinophils are
recruited and directed to sites in the body via chemoat-
traction by the chemotactic cytokine (chemokine)
eotaxin. Eotaxin binds exclusively to the seven trans-
membrane G protein-coupled receptor CC chemokine
receptor-3 (CCR3), which is predominantly expressed
on eosinophils and basophils, suggesting that eotaxin is
a specific chemoattractant for these leukocytes.4 Sup-
ported by studies using monoclonal antibodies for the
CCR3 receptor5 and animal eotaxin knockout models,6
we have targeted antagonism of the CCR3 receptor as a
mechanism of inhibiting eosinophil recruitment to the
lungs by eotaxin.
potential for adverse side effects stemming from interac-
tions with receptors of the central nervous system required
that we improve the selectivity of our antagonists.
We hoped that the known sensitivity of some G protein-
coupled receptors to structural changes in the piperidine
functionality could be used to improve the selectivity of
our antagonists for the CCR3 receptor. To that end we
synthesized a series of 4-benzylpiperidine analogues,
which are represented by entries 7–12 of Table 2. The
Table 1. 4-Benzylpiperidine CCR3 IC50 versus serotonin 5HT2A bind-
ing Ki
We previously reported the design and synthesis of
potent antagonists of the CCR3 receptor (Table 1).7,8
The acetyl- and (1-methyl-tetrazol-5-yl)phenyl ureas (5–
6)9 were among the most potent antagonists discovered.
However, our compounds also demonstrated affinity for
several serotonin receptors, particularly 5HT2A. The
Compd
X
R
CCR3
IC50, nM
5HT2A
Ki, nM
1
2
3
4
5
6
H
H
F
F
F
F
MeO
CN
MeO
CN
Ac
300Æ100
200Æ100
30Æ10
20Æ10
10Æ4
613
770Æ290
224Æ64
167Æ31
193Æ91
—
* Corresponding author. Tel.: +44-609-818-5381; fax: +44-609-818-
6570; e-mail: dean.wacker@bms.com
1-Me-tetrazol-5-yl
5Æ3
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.059

1646
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1645–1649
Table 2. Unsubstituted 3-benzylpiperidines and morpholine analo-
guesa
effect of ring size. As shown by 3-(4-fluorobenzyl)-
pyrrolidine 19, this resulted in a 10-fold decrease in
selectivity for 5HT2A
.
Incorporating the para-fluoro moiety, we synthesized a
number of structurally diverse and conformationally
restricted 3-benzylpiperidine analogues to further eluci-
date the binding requirements for the CCR3 receptor
(Table 4). Heteroatom incorporation resulted in com-
plete (20) loss of activity, similar to previous results for
the 2-benzylmorpholines (11–12). Bicyclo[3.2.1]octane
21, with its benzyl moiety in an equatorial orientation,
had a CCR3 IC50 of 7.4 nM and exhibited greater than
800-fold selectivity for the CCR3 receptor (6400Æ424 nM
5HT2A Ki). In contrast, the same bicyclo[3.2.1]octane
with the benzyl group in an axial orientation (22) was
approximately 6-fold less potent than 21. Several bicyc-
lic and tricylcic head pieces (23–28) were also prepared,
three of which (25, 27–28) were comparable in potency
to the simple benzylpiperidine 17.
Compd
X
n
R
CCR3%
Inh.b
CCR3
IC50, nM
5HT2A
Ki, nM
7
8
9
10
11
12
CH2
CH2
CH2
CH2
O
1
1
2
2
1
1
MeO
CN
MeO
CN
MeO
CN
55Æ9
41Æ22
787Æ4
913Æ88
2940Æ354
84
66
14
36
O
a All compounds are racemic.
b Percent inhibitions determined using 5 mM antagonist.
3-methoxy and 3-cyanophenyl urea 3-benzylpiperidines
(7–8) were approximately 5-fold more potent for the
CCR3 receptor than their corresponding 4-benzylpiper-
idine analogues (1–2) and were 16- and 70-fold more
selective, respectively, for CCR3 than 5HT2A. In con-
trast, increasing the distance between the aromatic and
piperidine rings of the 3-benzylpiperidine moiety (9–10)
or incorporation of an oxygen atom into the piperidine
ring (11–12) led to decreased binding affinity.
Table 4. Conformationally constrained benzylpiperidine analoguesa,b
Compd
X
CCR3%
Inh.
CCR3
IC50, nM
Aromatic substitution of the 3-benzylpiperidine moiety
was examined (Table 3) to try and find a substitution
pattern that offered improved selectivity and/or potency
for the CCR3 receptor. Of particular interest is the
para-fluorobenzylpiperidine, one of the most potent
pharmacophores tested in the 4-benzylpiperidine series.
We were encouraged that the 4-fluoro- and 2,4-difluoro-
benzylpiperidines (17–18) exhibited slightly greater
binding affinities than the nonfluorinated 3-benzylpi-
peridine parent compound (8) and gave modestly better
selectivity for CCR3 over 5HT2A. However, in contrast
to the 8- to 10-fold increase in binding observed for the
4-benzylpiperidines, fluorine incorporation at the 4-
position in the 3-benzylpiperidines gave only a modest
(ꢀ2-fold) boost in potency. A small number of 3-(4-
fluoro)benzylpyrrolidines were also prepared to test the
20
21
22
0c
—
7.4Æ3.8
47Æ8
23
24
25
802Æ239
169Æ6
39Æ9
Table 3. 3-Benzylpiperidine aromatic ring substitutionsa
26
27
28
68d
—
Compd
R
n
CCR3%
Inh.b
CCR3
IC50, nM
5HT2A
Ki, nM
49Æ23
56Æ21
13
14
15
16
17
18
19
4-CF3
3-MeO
3-F
2-F
4-F
1
1
1
1
1
1
0
11
31
243Æ94
38Æ15
29Æ9
2235Æ304
2310Æ354
2355Æ1351
178Æ30
a All compounds are racemic.
2,4-F
4-F
19Æ7
b Structures of configurational isomers were determined by nOe
experiments.
31Æ5
a All compounds are racemic.
b Percent inhibitions determined using 1 mM antagonist.
c Percent inhibition determined using 5 mM antagonist.
d Percent inhibition determined using 1 mM antagonist.

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1645–1649
1647
To make the appropriate comparison between the 3-
cyanophenyl ureas of Table 3 and the 3-acetylphenyl
ureas of Table 4, we synthesized enantiomerically pure
3-(4-fluorobenzyl)-piperidine acetyl ureas 29 and 30.
Both oxadiazole 31 and 1-iPr-tetrazol-5-yl 34 were
slightly less active than 30, while the 1-imidazole (32)
and 5-oxazole (33) phenyl ureas were less active by
approximately 3-fold. In contrast, the 1-Et- and 1-Me-
tetrazol-5-yl phenyl ureas (35–36) were approximately
twice as active as the 3-acetylphenyl urea. Further test-
ing of 36 gave calcium mobilization and chemotaxis
EC50’s of 1.3Æ1.1 and 3.2Æ1.6 nM, respectively. The
close correlation between these three in vitro results was
an improvement over data for the acetyl urea (30),
which had a chemotaxis EC50 that was 10-fold less
potent than the corresponding CCR3 IC50.
The stereochemistry for each benzylpiperidine was
assigned based on the crystal structure of the camphor
sulfonamide derived from the (+)-enantiomer of 3-(p-
fluorobenzyl)-piperidine. The (S)-enantiomer (30) was
approximately 16-times more potent than the (R)-enan-
tiomer (29) and was 4-fold more potent than 5. Also, 30
had 540-fold selectivity for CCR3 over 5HT2A. Data for
compound 30 in eotaxin-induced calcium mobilization10
and eotaxin chemotaxis assays were in reasonable
correlation with CCR3 binding and proved that 30 is a
functional antagonist of the CCR3 receptor.
We proposed that a 3,5-substitution pattern might offer
an advantage over monosubstitution because di-
substitution could increase the likelihood of maintain-
ing binding interactions with the CCR3 receptor even
with rotation about the N-phenyl bond of the urea. To
test this hypothesis, several 3,5-disubstituted phenyl
ureas were prepared, the most potent of which are
shown in Table 5 (37–45). The 3,5-disubstituted bis(a-
cetyl)phenyl urea (37) gave a CCR3 IC50 of 1.2 nM and
an EC50 for chemotaxis that agreed well with CCR3
binding data. Some improvement in binding was also
observed for disubstituted analogues of the 3-(1-Me-
tetrazol-5-yl)phenyl urea (36) when small polar func-
tionalities (38–39) were introduced at the 5-position. In
contrast, hydrophobic groups (40–42, 44) at the 5-posi-
tion were slightly less potent than the parent 3-(1-Me-
tetrazol-5-yl)phenyl urea (36). The tetrazole-acet-
ylphenyl urea (43) was less active than 36, while the 3,5-
bis(1-Me-tetrazol-5-yl)phenyl urea (45) had a CCR3
IC50 of 0.7 nM. This was an approximate 2-fold
improvement over 36 and was matched in potency only
by 38 and 39. In addition, bistetrazole 45 had an EC50
of 0.4 nM in the chemotaxis assay and is the most
potent compound reported to date.
During the course of optimizing the 3-benzylpiperidine
head piece, we also reexamined the 3-substituent of the
phenyl urea to improve potency and selectivity (Table 5;
31–36). Several 3-substituted ureas were designed based
on the premise that aromatic heterocycles could be used
to mimic or improve the binding interactions of the
acetyl group of the 3-acetylphenyl urea (30). We focused
on the 3-position of the phenyl urea because this sub-
stitution pattern in the 4-benzylpiperidine series gave
the greatest binding affinities.7 A number of different
heterocycles were tested, but only a few compounds
exhibited binding affinities comparable to that of 30.
Table 5. Structure–activity relationships for phenyl urea substitutiona,b
Compd
R1
R2
CCR3
IC50, nMa
Ca2+
EC50, nMa
Chemotaxis
EC50, nM
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Ac
Oxadiazol-2-yl
Imidazol-1-yl
Oxazol-5-yl
1-iPr-Tet
1-Et-Tet
H
H
H
H
H
H
H
2.5Æ1.2
3.6Æ1.7
7.1Æ3.7
7.9Æ0.3
3.1Æ1.9
1.2Æ1.6
0.6Æ0.4
1.2Æ0.4
0.9Æ0.3
0.7Æ0.4
1.5Æ1.0
1.2Æ0.2
2.4Æ1.0
2.6Æ1.4
3.1Æ1.0
0.7Æ0.5
2.4
45Æ34
25
1-Me-Tet
Ac
1.3Æ1.1
3.2Æ1.6
Ac
3
1-Me-Tet
1-Me-Tet
1-Me-Tet
1-Me-Tet
1-Me-Tet
1-Me-Tet
1-Me-Tet
1-Me-Tet
CH2OH
CONHCH3
Me
0.6
0.9Æ0.7
<1
1.0Æ0.4
CF3
Ph
Ac
Br
3.4Æ1.5
<1
0.4Æ0.4
1-Me-Tet
0.76Æ0.03
a Tet=tetrazol-5-yl.
b All compounds were >100-fold selective versus 5HT2A
.

1648
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1645–1649
The 3-benzylpiperidines and their analogues were
prepared according to Scheme 1, which depicts the pre-
paration 3-benzylpiperidin-1-yl-n-propylureas from com-
mercially available 3-hydroxypiperidine hydrochloride
(46, Scheme 1). Protection of the piperidine nitrogen
with Boc2O and oxidation with tetrapropylammonium
perruthenate (TPAP)11 afforded the 3-piperidone tert-
butyl carbamic acid (47). A Wittig reaction between 47
and an appropriately substituted benzyltriphenylphos-
phonium bromide resulted in olefin 48; reduction then
gave racemic Boc-protected benzylpiperidine 49. Chiral
3-(4-fluorobenzyl)-piperidines were prepared upon chiral
resolution of 49.
antagonists with binding affinities under 5 nM. This was
accomplished by replacing the 4-(4-fluorobenzyl)piper-
idine of our molecules with a 3-(4-fluorobenzyl)piper-
idine discovered in an effort to improve selectivity for
the CCR3 receptor. Further improvements in binding
were found by substituting the 5-position of the 3-(1-
Me-tetrazol-5-yl)phenyl urea of these molecules with
small polar functional groups. This resulted in a CCR3
antagonist with a binding IC50 and in vitro EC50 bind-
ing data for calcium mobilization and eotaxin chemo-
taxis assays in the mid to high picomolar range. Having
demonstrated that our compounds are functional
antagonists of the CCR3 receptor, we have focused on
determining the in vivo efficacy of these molecules, the
results of which will be reported in a future publication.
Deprotection of 49 under acid conditions and reductive
amination with Boc-protected 3-aminopropanal (52)
and sodium triacetoxyborohydride was followed by
acid-mediated cleavage of the resulting Boc-carbamate
to give hydrochloride 53. Urea 54 was obtained by
reacting 53 with commercially available 3-substituted
phenyl isocyanates in the presence of triethylamine.
Where the desired 3-substituted phenyl isocyanates were
not commercially available, the corresponding phenolic
carbamates were prepared from the appropriately sub-
stituted aniline (C6H5OCOCl, Et3N, CH2Cl2, 90%) and
used instead.
Acknowledgements
We gratefully acknowledge the help of A. J. Mical, K.
A. Rathgeb, D.-R., Wu, and N. C. Caputo, of the
Separations Group; M. X. Xia, G. A. Cain, and C.
Teleha, of the Chemical Synthesis Group; and T. H.
Scholz and G. A. Nemeth of the Spectroscopy Group.
References and notes
The 3-phenethylpiperidines were prepared by substituting
phenethyltriphenylphosphonium bromide12 for benzyl-
triphenylphosphonium bromide in Scheme 1. The 2-
benzylmorpholines were prepared according to the
procedure of Brown et al.13 Piperazine 20 was prepared
from commercially available 2-tert-butoxycarbonylamino-
3-(4-fluorophenyl)propionic acid using the procedure of
Hendrix et al.14 The strategies for preparing compounds
21–28 have been previously reported.15 The 3-tetra-
zolephenyl ureas were prepared from the corresponding
benzoic acid.16 Ureas 31 and 33 were prepared from
phenolic carbamates derived from commercially avail-
able nitro benzenes. Imidazole 32 was prepared from
commercially available 1-bromo-3-nitrobenzene.17
1. Asthma; Barnes, P. J., Grunstein, M. M., Leff, A. R.,
Woolcock, A. J., Eds.; Vol. 1; Lippincott-Raven Publish-
ers: New York, 1997.
2. Ramshaw, H. S.; Woodcock, J. M.; Bagley, C. J.;
McClure, B. J.; Hercus, T. R.; Lopez, A. F. Immunol. Cell
Biol. 2001, 79, 154.
3. Egan, R. W.; Umland, S. P.; Cuss, F. M.; Chapman,
R. W. Allergy 1996, 51, 71.
4. Kitaura, M.; Nakajima, T.; Imai, T.; Harada, S.; Com-
badiere, C.; Tiffany, H. L.; Murphy, P. M.; Yoshie, O. J.
Biol. Chem. 1996, 271, 7725.
5. Heath, H.; Qin, S.; Rao, P.; Wu, L.; LaRosa, G.; Kassam,
N.; Ponath, P. D.; Mackay, C. R. J. Clin. Invest. 1997, 99,
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6. Hogan, S. P.; Mishra, A.; Brandt, E. B.; Royalty, M. P.;
Pope, S. M.; Zimmermann, N.; Foster, P. S.; Rothenberg,
M. E. J. Immunol. 2001, 2, 353.
In conclusion, we have taken potent non-selective 4-
benzylpiperidines (5–6) and prepared selective CCR3
7. Wacker, D. A.; Santella, J. B., III; Gardner, D. S.;
Varnes, J. G.; Estrella, M.; DeLucca, G. V.; Ko, S. S.;
Tanabe, K.; Watson, P. S.; Welch, P. K.; Covington, M.;
Stowell, N. C.; Wadman, E. A.; Davies, P.; Solomon,
K. A.; Newton, R. C.; Trainor, G. L.; Friedman, S. M.;
Decicco, C. P.; Duncia, J. V. Bioorg. Med. Chem. Lett.
2002, 12, 1785.
8. De Lucca, G. V.; Kim, Ui. T.; Johnson, C.; Vargo, B. J.;
Welch, P. K.; Covington, M.; Davies, P.; Solomon, K. A.;
Newton, R. C.; Trainor, G. L.; Decicco, C. P.; Ko, S. S. J.
Med. Chem. 2002, 45, 3794.
9. The binding assay was carried out using 150 pM 125I
labeled human eotaxin, 5Â105 CHO cells, and 0.0001–1
mM compound in 150 mL of binding buffer (0.5% bovine
serum albumen, 10 mM HEPES buffer and 5 mM mag-
nesium chloride in RPMI 1640 media) in 96-well filtration
plates (Millipore) pretreated with 5 mg/mL protamine in
phosphate buffered saline, pH 7.2. The assay was incu-
bated at room temperature for 30 min. The plates were
vacuum filtered and the remaining cells were washed three
times with binding buffer containing 0.5 M NaCl added.
Scheme 1. (a) Boc2O, NaHCO3, THF, 14 h, 90%; (b) TPAP, NMO,
CH2Cl2/CH3CN, 4A MS, 1 h, 60–80%; (c) R-C6H4CH2P+Ph3BrÀ, n-
˚
BuLi, THF, À78 ꢁC, 6–8 h, 60–70%; (d) H2 (40 psi), Pd/C, MeOH, 12
h, quant; (e) 4M HCl, diox, 3 h, quant.; (f) 52, NaBH(OAc)3,
ClCH2CH2Cl, 14 h, 80%; (g) 3-R1-5-R2-C6H4NCO, Et3N, CH2Cl2, 5
min, 60–85%; (h) 3-R1-5-R2-C6H4NHC(O)OC6H5, CH3CN, 50 ꢁC,
1 h, 55–85%.

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1645–1649
1649
Radiolabel binding was quantified upon filter removal via
liquid scintillation counting.
12. Thakur, S. K.; Hosangadi, B. D. Tetrahedron 1996, 52,
8755.
10. The Ca+2 mobilization assay was carried out using 10 nM
eotaxin, 2Â105 eosinophils preloaded with Fluo-3AM
(Molecular Probes) and 0.0001–1 mM compound in 200
mL of buffer (0.1% bovine serum albumen, 20 mM
HEPES buffer and 2.5 mM Probenecid in RPMI 1640
media) in 96-well plates in a fluorescent imaging plate
reader. Data was generated as arbitrary fluorescence units
and compound dependent inhibition was calculated as a
percentage of the response of eotaxin alone.
13. Brown, G. R.; Forster, G.; Foubister, A. J.; Stribling, D. J.
Pharm. Pharmacol. 1990, 42, 797.
14. Hendrix, J.; Strupczewski, J. T.; Bordeau, K. J.; Brooks,
S.; Hemmerle, H.; Urmann, M.; Zhao, X.-Y.; Mueller, P.
J. World Patent Appl. WO 02066468 A2.
15. Wacker, D.; Duncia, J. V. World Patent Appl. WO
0129000.
16. Thomas, E. W. Synthesis 1993, 767.
17. Tanouchi, T.; Kawamura, M.; Ohyama, I.; Kajiwara, I.;
Iguchi, Y.; Okada, T.; Taniguchi, K.; Miyamoto, T.;
Hayashi, M. J. Med. Chem. 1981, 24, 1139.
11. Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P.
Synthesis 1994, 7, 639.