Discovery of novel non-peptide CCR1 receptor antagonists

Article abstract of DOI:10.1021/jm990316l

Ligands for the CCR1 receptor (MIP-1α and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1α and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7- TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Full text of DOI:10.1021/jm990316l

4680
J . Med. Chem. 1999, 42, 4680-4694
Discover y of Novel Non -P ep tid e CCR1 Recep tor An ta gon ists
Howard P. Ng,†,1 Karen May,*,† J ohn G. Bauman,† Ameen Ghannam,† Imadul Islam,† Meina Liang,†
Richard Horuk,‡ J oseph Hesselgesser,‡ R. Michael Snider,† H. Daniel Perez,‡ and Michael M. Morrissey†
Departments of Discovery Research and Immunology, Berlex Biosciences, 15049 San Pablo Avenue, P.O. Box 4099,
Richmond, California 94804-0099
Received J une 21, 1999
Ligands for the CCR1 receptor (MIP-1R and RANTES) have been implicated in a number of
chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis.
Because these ligands share a common receptor, CCR1, we sought to discover antagonists for
this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines
has been discovered by high throughput screening (HTS) which potently inhibits the binding
of MIP-1R and RANTES to the recombinant human CCR1 chemokine receptor. The structure-
activity relationships of various segments of this template are described as the initial HTS
lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound
has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human
7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro
functional assays demonstrate the functional antagonism of compound 6s and structurally
related analogues against the CCR1 receptor in a concentration dependent manner. The
discovery and optimization of potent and selective CCR1 receptor antagonists represented by
compound 6s potentially represent a novel approach to the treatment of chronic inflammatory
diseases.
In tr od u ction
of very important disease states including multiple
sclerosis,6-8 rheumatoid arthritis,9-11 allograft rejec-
Chemokines are chemotactic cytokines that belong to
a large family of chemoattractant molecules involved
in the directed migration of immune cells.2 The physi-
ological role of chemokines in the immune process is to
elicit mobilization of immune cells against pathogenic
organisms by direct recruitment and activation.
Chemokines are small proteins that are divided into
two main classes, based on the position of the first two
cysteines. They are the C-X-C and C-C families of
chemokines2 (two smaller branches of this family have
been described, the C3 and CX3C4 subfamilies). To date
over 40 chemokines have been identified and character-
ized. The chemokines all act by direct interaction with
cell surface receptors, known as chemokine receptors.
Chemokine receptors are members of the superfamily
of seven transmembrane domain proteins that signal
across the cellular membrane through coupled G pro-
teins.5 At last count these G-protein coupled receptors
(GPCR) number well over 600. Fifteen chemokine
receptors have thus far been cloned and identified: five
C-X-C, eight C-C, one CX3C, and one C receptor. They
produce their biological effect through a cascade of
intracellular events that begin with binding of the
chemokines to their respective receptors and lead
ultimately to activation and/or directed migration of the
cell.
tion,12,13 atherosclerosis,14 asthma,15-17 and AIDS.18
A
number of pharmaceutical companies have reported or
disclosed significant efforts in discovering chemokine
receptor antagonists.19-24
Our interest in chemokines was an extension of our
interest in the treatment of multiple sclerosis (MS).
Multiple sclerosis is a debilitating disease affecting over
200 000 people in the United States. It primarily afflicts
young adults, temporarily paralyzing parts of their body
with remission of disease usually followed by relapses
of greater severity and duration resulting ultimately in
permanent disability in many cases. One of the hall-
marks of MS disease is the infiltration and activation
of peripheral blood leukocytes into the brain. This along
with central nervous system immune cell activation lead
to active demyelination of the central nervous system.
To date no approved orally available small molecule
therapy exists for the treatment of MS. However, the
possibility of utilizing antagonism of binding of a
chemokine to its receptor as a novel treatment for
inflammatory diseases such as MS was recently dem-
onstrated.7
Studies by Karpus7 show strong evidence for a role
of several chemokines in a mouse experimental autoim-
mune encephalomyelitis (EAE) model of MS. More
specifically, they reported that a CC chemokine mac-
rophage inflammatory protein (MIP-1R) played a key
role in the development and progression of rodent EAE
disease. Investigators treated the mice with antibodies
to MIP-1R and found that both the initial and relapsing
paralytic aspects of the disease were significantly
reduced. This reduction of clinical disease correlated
Since the first chemokines were discovered and their
receptors cloned, they have been implicated in a number
* To whom correspondence should be addressed: Berlex Biosciences,
Department of Discovery Research, 15049 San Pablo Ave., Richmond,
CA 94806. E-mail: karen•may@berlex.com.
† Department of Discovery Research.
‡ Department of Immunology.
10.1021/jm990316l CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/08/1999

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4681
Ch a r t 1
with reduction in infiltration of mononuclear cells into
the central nervous system. These results led the
authors to conclude that MIP-1R plays an important role
in this T cell mediated disease model. In addition
Godiska8 demonstrated that the lesions and spinal fluid
isolated from mice from a similar mouse model of EAE
contained unregulated levels of mRNA for a number of
chemokines, including MIP-1R. The major receptor for
MIP-1R, CCR1, therefore represented a potential target
for the discovery of a new therapy for MS.
In addition to MS, CCR1 has been implicated in other
chronic inflammatory disorders, most notably rheuma-
toid arthritis (RA). Rheumatoid arthritis is character-
ized by memory T lymphocyte and monocyte infiltration
into synovial tissues, resulting in inflammation, dis-
comfort, and ultimately loss of joint function. Examina-
tion of synovial fibroblasts of RA patients revealed that
the mRNA and RANTES protein were upregulated.9,10
In contrast, osteoarthritis tissue does not express
RANTES mRNA.10 Additional evidence implicating
RANTES in the pathophysiology of RA was recently
reported in a study utilizing anti-RANTES antibodies
in an in vivo animal study.11 In this study, rats from
an adjuvant-induced arthritis (AIA) model of RA were
treated with antibodies to RANTES, resulting in greatly
reduced clinical scores for RA relative to untreated
animals. This and other studies suggest that RANTES
could play a major role in attracting T cells and
monocytes into joints during onset of AIA, thereby
making the main receptor for RANTES, CCR1, a
candidate for involvement in the inflammatory and
destructive processes culminating in rheumatoid ar-
thritis.
moiety to the 4-hydroxy-4-phenylpiperidine, was also in
the sample collection employed for the CCR1 HTS assay.
Compound 1b was not identified as a potent inhibitor
of MIP-1R binding to the CCR1 receptor in the initial
screen. Subsequent re-testing of 1b confirmed that this
compound does not significantly inhibit binding of MIP-
1R to the CCR1 receptor at 5.0 µM.
Ch em istr y
Analogues of compound 1 were synthesized as out-
lined in Scheme 1. Aryl acetonitriles 2 were deproto-
nated and reacted with dihaloalkanes to give interme-
diate 3. Compound 3 was either reacted with commer-
cially available piperidine 5 or with the protected
piperidone (1,4-dioxa-8-azaspiro[4,5]decane) followed by
deprotection to piperidone 4 before reacting with Grig-
nard or lithium reagents to give 6s through 6a o. In
some cases R1 and R2 were components of a tricyclic
Herein, we present the discovery of a family of novel
compounds by high throughput screening and their
subsequent optimization into potent and selective an-
tagonists of the human CCR1 receptor. The functional
antagonism and functional selectivity of compound 6s
has been demonstrated through a number of in vitro
experiments and previously reported by this group.25
28,29
moiety leading to analogues of 6a through 6r .
Analogues of 2 having an ester in place of the cyano
group were treated as outlined in Scheme 1 to afford
ester analogues 6w and 6ck . Ester functional groups
were hydrolyzed to the corresponding carboxylic acid 6x
using LiOH while benzyl groups were removed using
catalytic hydrogenation with palladium on carbon. The
ester 6w was reduced using LAH to give the alcohol 6y.
Sulfoxides and amine N-oxides were prepared by per-
oxide and/or peracid oxidation of the sulfide or tertiary
amine (6p ,q,a q). Compounds with quaternary am-
monium salts of the piperidine group (6a r , 6a t, 6a s)
were generated by reaction of 6s, 6a u , and 8 with
methyl iodide, respectively.
Lea d Discover y of Non -P ep tid e CCR1 Recep tor
An ta gon ists by HTS
A stable cell line with the human CCR1 receptor26
overexpressed in human embryonic kidney (HEK-293)
cells was developed and employed in a competitive
receptor binding assay with 125I-labeled human MIP-
1R.25 This assay was used to screen approximately
200000 members of the Berlex HTS compound library.27
The initial screen produced over 250 hits that inhibited
binding by at least 50% at an initial compound concen-
tration of 5 µM. These compounds were re-screened in
a dose-responsive fashion and their structures verified
The preparation of the des-nitrile analogue, compound
8, was accomplished as shown in Scheme 2. Addition of
3-benzyloxypropyltriphenylphosphonium bromide to ben-
zophenone gave intermediate 7. Subsequent reduction
by catalytic hydrogenation both reduced the olefinic
group as well as removed the benzyl group. The result-
ing alcohol was converted to a mesylate and displaced
with 4-(4-chlorophenyl)-4-hydroxy piperidine (5s) to give
8. Complete experimental procedures for all compounds
described in this paper can be found in the Experimental
Section.
1
by analysis (MS, H and 13C NMR) and in some cases
re-synthesis. Of these compounds, eight showed Ki
values under 500 nM and several appeared to belong
to the same structural family. The most potent com-
pound from this series is represented by compound 1
(Chart 1) which potently inhibited the binding of 125I-
labeled human MIP-1R to the CCR1 receptor (Ki ) 44
nM). It is interesting to note that the structurally
related analogue 1b (Chart 1), which differs by one
methylene in the chain connecting the dibenzothiepine

4682 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ng et al.
Sch em e 1a
a
Conditions: (a) NaH, DMF or LDA, THF, 18 h; (b) DMF, 18 h; (c) 6 N HCl, MeOH, 24 h; (d) R3Li or R3MgX, THF, 4 h; (e) Cs2CO3,
DMF, 16 h.
Sch em e 2a
Ta ble 1. SAR of the 4-Phenylpiperidine in the Dibenzothiepine
Series
cmpd
R
R′
4-F-Ph
Ki (nM)a,b
c
1
OH
OH
OH
OH
OH
OH
OH
H
44 ( 19
6a
6b
6c
6d
6e
6f
6g
6h
6i
Ph
203 ( 28
164 ( 13
2-Me-Ph
3-CF3-Ph
3-CF3, 4-Cl-Ph
4-Br-Ph
4-Cl-Ph
Ph
Ph
Ph
CH2Ph
C(Ph)2OH
623 ( 132
224 ( 41
22 ( 3
24 ( 10
19% @3 µM ( 5%
0% @ 3 µM
0% @ 3 µM
405 ( 111
0% @ 3 µM
Conditions: (a) BnO(CH3)3PPh3+Br-, KOtBu, THF, 70 °C, 6
a
h; (b) 10% Pd/C, H2 (50 psi), EtOH; (c) MsCl, Et3N, CH2Cl2; (d) 5,
Cs2CO3, DMF, 16 h.
CN
Resu lts a n d Discu ssion
C(O)CH3
6j
6k
OH
H
Compounds were screened for their potential to
competitively inhibit binding of radiolabeled human
MIP-1R in cells expressing the CCR1 receptor in a dose
range, and these are reported as Ki values.25 Beginning
with compound 1 as the starting point for lead optimi-
zation, substituents at the 4-position of the piperidine
ring were examined (Table 1). In general, the 4-phenyl
group appeared to be very sensitive to changes in
regiochemistry, sterics, and electronics. For example,
removal of the 4-halogen substituent (6a ) resulted in a
10-fold loss of potency. While a methyl group introduced
at the 2-position (6b) did not significantly change the
binding affinity of this template, the 3-trifluoromethyl
derivative (6d ) was significantly less potent. This loss
of potency can be partially restored by inclusion of a
chloro in the 4-position of the phenyl ring (6d ). Within
the group of 4-halophenylpiperidinyl compounds, the
4-bromo (6e) and 4-chloro (6f) proved to be superior to
the 4-fluoro derivative (1).
Alternative pharmacophores were explored to poten-
tially replace the 4-hydroxy-4-phenylpiperidine motif
because this same structural motif is contained in
haloperidol and raised neurotoxicity concerns.30 Re-
moval of the hydroxyl group proved detrimental to
potency (6g), and replacement with nitrile or methyl
carbonyl (6h , 6i) was not successful for maintaining
binding affinity. Alternatives to the 4-phenyl group were
also investigated. While the benzyl (6j) was only 2-fold
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
where noted). c This value is the average of n ) 71 ( SD.
Compound 1 was used as a standard for all Ki determinations.
less potent than the phenyl analogue, more significant
changes such as diphenylhydroxy methyl were not
tolerated (6k ).
Variations to the tricyclic moiety were also pursued
to lower the possibility of cross-reactivity with related
CNS receptors.31 As shown in Table 2, the compound
with a single oxygen bridge (6l) was less potent than
any of the representative two-atom bridged tricyclics
(6m -r ). Among the two-atom-bridged compounds tested,
the thiomethylene (6f) proved optimal. Oxidation of this
compound produced two diastereomeric sulfoxides, 6p
and 6q, which were separated and tested; both exhibited
comparable potency to 6f. Interestingly, substitution of
the thiomethylene bridge with isosteric replacements
including oxomethylene (6o), ethyl (6m ), ethylene (6n ),
or amide functionality (6r ) provided potent compounds
as well, indicating that a high degree of flexibility is
allowed in this part of the template. Finally, the acyclic
analogue 6s, which contains no bridging functionality,
demonstrated a similar level of inhibitory potency. With
this discovery, the focus of the optimization effort was

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4683
Ta ble 2. SAR of Tricyclic Bridge Atoms
Ta ble 4. SAR of Substitution at the 4-Position on
4-Hydroxypiperidine
cmpd
R
Ki (nM)a,b
52 ( 5c
1188 ( 252
1020 ( 10
906 ( 86
715 ( 28
206 ( 19
6s
6v
4-Cl-Ph
cmpd
R
Ki (nM)a,b
Ph
6f
6l
CH2S
O
24 ( 10
234 ( 41
45 ( 1
6a a
6a b
6a c
6a d
6a e
6a f
6a g
6a h
6a i
6a j
6a k
6a l
6a m
6a n
2-thienyl
2-pyridyl
2-naphthyl
3-pyridyl
4-diphenyl
n-butyl
6m
6n
6o
6p
6q
6r
CH2CH2
CHdCH
CH2O
CH2S(O)
CH2S(O)
N(CH3)C(O)
H, H
56 ( 3
34 ( 2
30% @ 10 µM ( 4%
4010 ( 610
233 ( 22
54 ( 5
71 ( 3
4-I-Ph
3-Cl-Ph
32 ( 10
52 ( 3
385 ( 23
6s
3,5-Cl, Cl-Ph
4-MeO-Ph
4-CN-Ph
4-CF3-Ph
4-NH2-Ph
4-Me2N-Ph
1330 ( 30
347 ( 64
a
Ki values are derived from competitive binding on CCR1 with
92 ( 30
153 ( 8
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
where noted).
b
33% @ 10 µM ( 3%
1155 ( 105
Ta ble 3. Comparison of SAR Trends between the
Dibenzothiepine Series and the Benzyhydryl Series
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
where noted). c n ) 4 ( SD.
affinity. From these results it was concluded that the
SAR discovered in the tricyclic series could be applied
to the related acyclic series, and other variations to the
piperidine ring were investigated.
cmpd
R′
OH
OH
OH
OH
OH
OH
OH
R′′
R
Ki (nM)a,b
1
4-F-Ph
Ph
CH2S
CH2S
CH2S
CH2S
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
44 ( 19c
More extensive modification of the 4-phenyl group of
the piperidine was explored to complement the data in
Tables 1 and 3. Other aryl groups (Table 4) appeared
to be tolerated in this position, including 2-thienyl (6a a ),
2-pyridyl (6a b), and 2-naphthyl (6a c), all of which
showed similar binding affinities to the unsubstituted
phenyl derivative (6v). Interestingly the 3-pyridyl group
(6a d ) was actually more potent than the unsubstituted
phenyl group (6v). The 4-biphenyl (6a e) and the n-butyl
(6a f) groups lowered potency.
In addition to the previously discussed 4-halophenyl
compounds 6s, 6t, and 6u (Table 3), other analogues of
the 4-halophenyl group were investigated. The halogen
series was completed with synthesis of the 4-iodophenyl
analogue (6a g), which showed lower potency. Moving
the halogen to the 3-position of the phenyl ring (6a h )
or bis-halogenation (6a i) decreased binding affinities as
well, compared to the 4-chlorophenyl (6s). Interestingly,
when a methoxy group was substituted in the 4-position
of the phenyl ring (6a j) lower potency than the corre-
sponding 4-chloro compound (6s) but higher potency
than the unsubstituted ring (6v) was observed. This
result is somewhat surprising because the methoxy
group possesses similar steric and electronic properties
to the chloro group. Substitution at the 4-position of the
phenyl ring with electron-withdrawing groups such as
nitrile (6a k ) or trifluoromethyl (6a l) also lowered po-
tency. An amino group in this position (6a m ) eliminated
most of the binding affinity, while its dimethyl analogue
(6a n ) was less detrimental. Based on these data further
optimization of the template was performed with 4-chlo-
rophenyl analogues.
6a
6e
6f
6s
6t
6u
6v
6w
6x
6y
6z
203 ( 28
4-Br-Ph
4-Cl-Ph
4-Cl-Ph
4-F-Ph
4-Br-Ph
Ph
22 ( 3
24 ( 10
52 ( 5d
136 ( 13
29 ( 9
OH
1188 ( 252
62% @ 10 µM ( 8%
15% @10 µM ( 4%
7500e
CO2Me
CO2H
CH2OH
OH
Ph
Ph
Ph
H
20% @10 µM ( 8%
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the average of n ) 2
b
(except where noted). c This value is the average of n ) 71 ( SD.
Compound 1 was used as a standard for all Ki determinations.d
) 4 ( SD. e n ) 1.
n
shifted to the benzylhydryl compound represented by
6s (Chart 1) because it was equipotent with the diben-
zothiepine series represented by 1 (Chart 1) but was
both potentially less cross-reactive with CNS receptors
and more accessible synthetically.
Analogues were first synthesized to confirm that the
SAR discovered in the dibenzothiepine series was
applicable to this new series. As shown in Table 3, the
same SAR trends for substitution on the 4-phenylpi-
peridine moiety were observed. Halogen substitution at
the 4-position of the phenyl ring proved superior to
compounds without this substituent, as demonstrated
by the higher binding affinities of 6t, 6s, 6u , 1, 6f, and
6e as compared to 6v and 6a . Once again, the chloro
(6s, 6f) and bromo (6u , 6e) compounds were more potent
than the fluoro (6t, 1). Finally, substitution for the
hydroxyl group (6w , 6x, 6y) or removal of one phenyl
group (6z) resulted in a large decrease in binding
Further synthetic efforts focused on addressing the
effect of changing the regiochemistry of substituents on

4684 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ta ble 5. SAR of Heterocyclic Ring Position and Size
Ng et al.
Ta ble 7. SAR of Alkyl Linker Chain Length
cmpd
m
n
Ki (nM)a,b
cmpd
m
Ki (nM)a,b
6t
6a o
6a p
2
1
1
1
2
1
136 ( 13
0% @10 µM
6a v
6s
6a w
6a x
1
2
3
4
4060 ( 800
52 ( 5c
78 ( 4
22% @10 µM ( 4%
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
99 ( 5
where noted).
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
Ta ble 6. Activity of Quaternary Ammonium Salts vs
Nonquaternized Compounds
where noted). c n ) 4 ( SD.
Ta ble 8. SAR of Benzhydryl Replacements
cmpd
Ar
R
R′
n
Ki (nM)a,b
cmpd
R
Ki (nM)a,b
6s
Ph
Ph
Ph
Ph
H
H
Ph
Ph
CN
CN
CN
CN
H
0
1
0
1
0
1
52 ( 5c
8 ( 2
6a r
6a u
6a t
8
6a y
6a z
6bb
6bc
6bd
6be
6bf
6bg
6bh
6bi
Ph
54 ( 5
2-naphthyl
2-naphthyl
Ph
206 ( 28
22 ( 1
3-Br, 4-MeO-Ph
2-CN-Ph
2,3,4,5,6-F5-Ph
3-OH-Ph
74 ( 8
98 ( 35
116 ( 41
6 ( 1
330 ( 85
6a s
Ph
H
156 ( 50
a
2-F-Ph
4-F-Ph
371 ( 41
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
533 ( 84
where noted). c n ) 4 ( SD.
2-Br-Ph
4-Cl-Ph
2-Cl-Ph
2-CF3-Ph
2-BnO-Ph
3-BnO-Ph
4-BnO-Ph
3-PhO-Ph
2-HO-Ph
366 ( 37
463 ( 90
306 ( 93
the piperidine ring, the size of the ring, and the
substitution of the piperidine ring nitrogen itself. Mov-
ing the hydroxy and phenyl substituents to the 3-posi-
tion on the piperidine (6a o) or replacement of the
piperidine with a pyrrolidine (6a p ) drastically reduced
the binding affinity (Table 5). On the basis of these
results, substitution on the 4-position of the piperidine
ring (6t) appeared optimal. In addition, oxidation of the
piperidine nitrogen to the corresponding N-oxide (Chart
1, 6a q) produced a 7-fold loss of potency. This indicated
the need for a basic nitrogen at this position.
An interesting trend was observed for the binding
affinities of quaternary ammonium salts derived from
this series. Although interest in these salts as clinical
candidates was limited due to known pharmacokinetic
liabilities (i.e., poor oral absorption and rapid clearance
in vivo), a surprisingly large number of simple quater-
nary ammonium salts were found to demonstrate
significant inhibitory activity in the original CCR1 HTS
screen of our compound library. Consequently, the
quaternary ammonium salts (6a r , 6a t, 6a s) were syn-
thesized and demonstrated a 5- to 20-fold increase in
binding inhibition in comparison to the parent piperi-
dine derivatives 6s, 6a u , and 8 (Table 6).
6bj
6bk
6bl
25% @ 10 µM ( 7%
46% @ 10 µM ( 4%
0% @ 10 µM
3430 ( 80
908 ( 89
6bm
6bn
6bo
6bp
6bq
6br
6bs
6bt
6bu
6bv
6a u
6bw
6bx
6by
6bz
790 ( 220
975 ( 35
4-HO-Ph
3,4,5-(MeO)3-Ph
3-NO2-Ph
4-MeO-Ph
4-EtO-Ph
2-thienyl
497 ( 66
510 ( 13
359 ( 38
1425 ( 65
128 ( 36
3-thienyl
236 ( 53
206 ( 29
2-naphthyl
1-naphthyl
2-pyridyl
3-pyridyl
2-pyrrole(N-Me)
51% @ 10 µM ( 4%
584 ( 163
1000 ( 20
429 ( 231
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
where noted).
suggests that the optimal length of the linker is three
atoms and corresponds nicely with the results obtained
from initial high throughput screening.
With the SAR on the linker and the piperidine
established, focus was shifted to chemical optimization
of the benzhydryl nitrile moiety to further modify the
physicochemical properties of this structural series. As
shown in Table 8, removal of one of the two phenyl rings
did not result in a drop in potency (6a y vs 6s). Only
specific substitutions on the single phenyl ring were
tolerated as in 6a z, 6bb. However the majority of the
substituents examined on this ring had a negative effect
The effect of changing the length of the linker between
the piperidine and benzhydryl groups was also explored
systematically. As shown in Table 7, compound 6s, with
a three-atom linker, was much more potent than
compound 6a v, which contains a two-atom linker, and
slightly more potent than compounds 6a w and 6a x,
which contain four and five atoms, respectively. This

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4685
Ta ble 10. SAR of Substitution of Nitrile in Benzhydryl Series
Ta ble 9. SAR of Replacement of Phenyl Ring
cmpd
R
Ki (nM)a,b
cmpd
R
Ki (nM)a,b
6s
Ph
CO2Et
CO2Me
CH2CH2CH2Cl
Me
Et
CH2CN
c-hex
n-piperidyl
H
52 ( 5c
33 ( 3
6s
8
6ck
6cl
6cm
CN
H
52 ( 5c
116 ( 42
406 ( 51
244 ( 81d
9 ( 8d,e
6cc
6cd
6ce
6cf
6cg
6ch
6ci
6cj
6a y
53 ( 8
CO2Me
CH2NH2
CH2NHC(O)NH(4-piperidyl)
51 ( 7
460 ( 60
93 ( 8
125 ( 17
400 ( 48
130 ( 14
54 ( 5
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
where noted). c n ) 4 ( SD. n ) 3 ( SD. e The Ki value in our
d
assay is significantly different from the value (5 µM) reported by
Takeda for MIP-1R binding (refs 20, 32).
a
Ki values are derived from competitive binding on CCR1 with
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
b
Ta ble 11. Comparison of Binding Assay Data with Functional
Assay Data
where noted). c n ) 4 ( SD.
inhibition of 125I-MIP-1R inhibition of MIP-1R-induced
on the binding inhibition of this template (6bc, 6bd ,
6be, 6bf, 6bg, 6bh , 6bi, 6bj, 6bk -bq, 6br , 6bo, 6bp ).
Steric factors also appear to play a role in determining
potency as demonstrated by the significant difference
in binding affinity between the 4-methoxy (6bs) and the
4-ethoxy (6bt) analogues.
Additional analogues were synthesized in which both
phenyl groups were removed and replaced with other
aromatic ring systems. As shown in Table 6, only three
analogues, the 2-thienyl (6bu ), 3-thienyl (6bv), and the
2-naphthyl (6a u ) showed comparable binding affinities
to the monophenyl compound (6a y). Compound 6b u ,
with a 2-thienyl group, even had slightly increased
potency. The fact that the 2-naphthyl isomer (6a u ) was
much better tolerated than the 1-naphthyl (6bw ) indi-
cates that this portion of the template interacts fairly
closely with the CCR1 receptor. The lower binding
affinities of the corresponding pyridyl (6bx and 6by)
and pyrrole (6bz) analogues were also noted.
Replacement of one phenyl ring of the benzhydryl
system of 6s with other groups was also investigated
(Table 9). The most effective replacements were found
to be the ethyl carboxylate (6cc), methyl carboxylate
(6cd ), and followed by the 3-chloropropyl group (6ce).
Other alkyl groups (6cf, 6cg, and 6ch ) as well as
carbocyclic (6ci) and heterocyclic groups (represented
by 6cj) were clearly inferior to the phenyl group (6s),
but several were better than 6a y, which lacks a sub-
stituent at this position.
The SAR of the nitrile functionality was investigated
last (Table 10). Removal of the cyano group (8) or
replacement of the nitrile with an ester (6ck ) or amino-
methyl functionality (6cl) resulted in significant loss of
potency compared to the cyano-containing 6s. However,
researchers at Takeda Chemical Industries, Ltd., re-
cently disclosed in a published patent application19 that
further elaboration at this position was fruitful. These
workers disclosed that compound 6cm , which is a direct
derivative of the aminomethyl derivative 6cl, inhibited
binding of RANTES to CCR1 with an IC50 of 6 nM and
binding of MIP-1R to CCR1 with an IC50 of 5 µM. This
compound was synthesized, screened in our binding
binding to human CCR1
intracellular calcium
cmpd
(Ki, nM)a,b
mobilization (IC50, µM)c
1
6f
6m
6n
6o
44 ( 19d
24 ( 10
45 ( 1
3.0 ( 1.0
7.0 ( 0.8
7.7 ( 0.4
5.3 ( 0.4
7.7 ( 0.4
3.3 ( 1.8
2.5 ( 0.7
10.9 ( 1.6
0.4 ( 0.1
0.7 ( 0.2
1.8 ( 0.3
7.1 ( 0.4
1.6 ( 0.6
5.6 ( 0.4
1.9 ( 0.9
5.8 ( 1.8
9.0 ( 2.8
8.4 ( 0.3
9.8 ( 3.2
7.0 ( 4.2
8.3 ( 1.5
3.2 ( 1.6
4.8 ( 1.1
8.7 ( 1.8
56 ( 3
34 ( 2
6r
6s
32 ( 10
52 ( 5e
92 ( 30
8 ( 2
6a k
6a v
6a t
6a s
6a u
6a w
6a x
6a z
6bb
6bd
6bu
6bv
6cc
6ce
6ch
6cj
8
22 ( 1
6 ( 1
206 ( 28
78 ( 4
89 ( 14
74 ( 8
98 ( 35
156 ( 50
128 ( 36
236 ( 53
33 ( 3
51 ( 7
125 ( 17
130 ( 14
116 ( 41
a
Ki values are derived from competitive binding on CCR1 with
b
125I MIP-1R (ref 25). Values represent the mean of n ) 2 (except
d
where noted). c Values represent the mean of n ) 2 ( SD. This
value is the average of n ) 71 ( SD. Compound 1 was used as a
standard for all Ki determinations.
assay, and confirmed as a highly potent inhibitor of
MIP-1R binding to the CCR1 receptor (Ki ) 9 nM).32
Finally, a selected group of analogues described in this
paper were characterized in a MIP-1R-induced intra-
cellular calcium mobilization assay using FLIPR (fluo-
rometric imaging plate reader) technology with HEK293
cells that overexpress the human CCR1 receptor (Table
11). These analogues demonstrate functional antago-
nism against the human CCR1 receptor with an IC50
in the sub-micromolar to mid-micromolar range and
show no significant agonist activity at the highest
concentration tested (5 µM). SAR results from this assay
suggest that a positively charged nitrogen plays an
important role in functional potency, since the ana-
logues which contain a quaternary ammonium moiety

4686 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ng et al.
6H), 7.25 (m, 3H), 7.40 (m, 1H), 7.88 (m, 2H). Anal. (C30H32N2-
OS‚0.7SiO2) C, H, N.
(6a r , 6a s, 6a t) demonstrate the best binding affinity
and functional activity.
11-[3-[4-Hyd r oxy-4-(3-tr iflu or om eth ylp h en yl)-1-p ip er i-
d in yl]p r op yl]-6,11-d ih yd r od iben zo[b,e]th iep in -11-ca r bo-
n itr ile (6c) was prepared in the same manner as 1 starting
with 3a (200 mg, 0.56 mmol) and 4-(3-trifluoromethylphenyl)-
4-hydroxy piperidine to afford 6c after flash chromatography
as a colorless oil (180 mg, 61% yield). 1H NMR (300 MHz,
CDCl3) δ (TMS) 1.70 (m, 6H), 2.20 (m, 2H), 2.50 (m, 5H), 2.80
(m, 3H), 3.05 (m, 1H), 4.15 (d, 1H), 4.55 (d, 1H), 7.15 (m, 3H),
7.30 (m, 3H), 7.50 (m, 2H), 7.70 (m, 1H), 7.90 (m, 3H). Anal.
(C30H29F3N2OS‚0.36SiO2) C, H, N.
11-[3-[4-Hydr oxy-4-(3-tr iflu or om eth yl-4-ch lor oph en yl)-
1-p ip er id in yl]p r op yl]-6,11-d ih yd r od iben zo[b,e]t h iep in -
11-ca r bon itr ile, h yd r och lor id e (6d ) was prepared in the
same manner as 1 starting with 3a (200 mg, 0.56 mmol) and
4-(3-trifluoromethyl-4-chlorophenyl)-4-hydroxy piperidine to
afford 6d after flash chromatography as a colorless oil (180
mg, 57% yield). The hydrochloride salt was prepared by
dissolving the freebase in ethyl acetate and adding an excess
of 1 N HCl in ether. The product precipitated as a solid and
was isolated by filtration. 1H NMR (300 MHz, CDCl3) δ (TMS)
1.20 (m, 2H), 1.90 (m, 4H), 2.60 (m, 4H), 3.15 (m, 4H), 4.10 (d,
1H), 4.50 (d, 1H), 5.20 (s, 2H), 7.20 (m, 8H), 7.70 (m, 2H), 7.90
(s, 1H). Anal. (C30H38ClF3N2OS‚HCl‚0.25H2O) C, H, N.
11-[3-[4-(4-Br om op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
p yl]-6,11-d ih yd r od iben zo[b,e]th iep in -11-ca r bon itr ile (6e)
was prepared in the same manner as 1 starting with 3a (200
mg, 0.56 mmol) and 4-(4-bromophenyl)-4-hydroxy piperidine
to afford 6e as a white solid (220 mg, 73% yield). 1H NMR
(300 MHz, CDCl3) δ (TMS) 1.70 (m, 6H), 2.10 (m, 2H), 2.45
(m, 3H), 2.75 (m, 2H), 3.08 (m, 1H), 4.10 (d, 1H), 4.55 (d, 1H),
7.13 (m, 4H), 7.25 (m, 2H), 7.38 (d, 2H), 7.43 (d, 2H), 7.88 (m,
2H). Anal. (C29H29BrN2OS‚0.35SiO2) C, H, N.
Con clu sion
We have discovered a series of potent and selective
non-peptide antagonists of the human CCR1 receptor
through HTS and subsequent chemical optimization
studies. Upon evaluation of numerous analogues in this
series, we have determined that the piperidine and
linker subunits are optimal as discovered in the HTS
lead 1. In addition, SAR efforts on the benzhydryl and
nitrile components of this template resulted in an
increase in inhibitory activity in MIP-1R binding to
human CCR1. These compounds have been shown to
be both potent and selective in competitive binding
assays25 and demonstrated functional activity in a MIP-
1R calcium mobilization assay. These antagonists may
represent a novel approach to the treatment of diseases
mediated by the CCR1 chemokine receptor.
The most interesting SAR result is the significant
increased activity of the quaternary ammonium com-
pounds (6a r -a t) over their nonquaternized analogues
(6s, 6a u , and 8). This finding suggests that the binding
site favors positively charged species. Although these
compounds do not meet the criteria for a drug candidate
(i.e., poor oral absorption and rapid clearance in vivo),
they do help elucidate the binding site requirements and
can influence inhibitor design. Further efforts directed
toward additional improvements of potency and selec-
tivity as well as in vivo data will be presented in future
publications.
11-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p iper id in yl]p r o-
p yl]-6,11-d ih yd r od iben zo[b,e]th iep in -11-ca r bon itr ile (6f)
was prepared in the same manner as 1 starting with 3a and
4-(4-chlorophenyl)-4-hydroxy piperidine to afford 6f as a yellow
solid (184 mg, 6.3% yield). 1H NMR (300 MHz, CDCl3) δ (TMS)
1.75 (m, 6H), 2.05 (m, 2H), 2.45 (m, 3H), 2.79 (m, 3H), 3.10
(m, 1H), 4.10 (d, 1H), 4.55 (d, 1H), 7.15 (m, 2H), 7.28 (m, 6H),
7.42 (d, 2H), 7.85 (m, 2H). Anal. (C29H29ClN2SO‚1.0H2O) C,
H, N.
Exp er im en ta l Section
NMR spectra were obtained on a 300 MHz Varian XL-300
or a 400 MHz Varian UP-400 as noted and were consistent
with the assigned structures. Electrospray mass spectra were
recorded on
a SCIEX API III plus using flow injection.
Elemental analyses were performed by Robertson Microlit
Laboratories, Madison, NJ , and results were within 0.4% of
calculated values except where noted. Silica gel for flash
chromatography was E. Merck grade (230-400 mesh). Pre-
parative HPLC was run on a C18 Dynamax column (Ca. #80-
240-C8), eluting with a linear gradient of 80:20 to 30:70 (0.1%
aqueous TFA:0.1% TFA in MeCN) over 30 min.
11-[3-[4-(4-Flu or op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
p yl]-6,11-d ih yd r od iben zo[b,e]th iep in -11-ca r bon itr ile (1).
The preparation of 1 was previously described by Sindelar et
al.28 1H NMR (300 MHz, CDCl3) δ (TMS) 1.75 (m, 5H), 2.14
(m, 2H), 2.51 (m, 4H), 2.80 (m, 3H), 2.08 (m, 1H), 4.55 (d, 1H),
7.04 (t, 2H), 7.16 (m, 3H), 7.28 (m, 3H), 7.46 (m, 2H), 7.84 (m,
2H). Anal. (C29H29FN2OS‚0.53SiO2) C, H, N.
6,11-Dih yd r o-11-[3-(4-p h en yl-1-p ip er id in yl)p r op yl]-
d iben zo[b,e]th iep in -11-ca r bon itr ile (6g) was prepared in
the same manner as 1 starting with 3a (200 mg, 0.56 mmol)
and 4-phenylpiperidine to afford 6g as a colorless oil (184 mg,
1
73% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.80 (m, 6H),
2.10 (m, 2H), 2.50 (m, 3H), 2.80 (m, 1H), 3.10 (m, 3H), 4.10 (d,
1H), 4.60 (d, 1H), 7.25 (m, 11H), 7.85 (m, 2H). Anal. (C29H30N2S‚
0.5SiO2) C, H, N.
1-[3-(11-Cya n o-6,11-d ih yd r od iben zo[b,e]th iep in -11-yl)-
p r op yl]-4-p h en yl-4-p ip er id in eca r bon it r ile, h yd r och lo-
r id e sa lt (6h ) was prepared in the same manner as 6d
starting with 3a (200 mg, 0.56 mmol) and 4-cyano-4-phenylpi-
1
peridine to afford 6h as a yellow solid (86 mg, 30% yield). H
NMR (300 MHz, CDCl3) δ (TMS) 1.80 (m, 3H), 2.10 (m, 3H),
3.05 (m, 6H), 3.70 (m, 1H), 4.2 (m, 1H), 4.65 (m, 1H), 7.25 (m,
9H), 7.60 (d, 2H), 7.88 (m, 2H). Anal. (C30H29N3S‚1.0HCl‚
0.75H2O) C, H, N.
6,11-Dih yd r o-11-[3-(4-h yd r oxy-4-p h en yl-1-p ip er id in yl)-
p r op yl]d iben zo[b,e]th iep in -11-ca r bon itr ile (6a ) was pre-
pared in the same manner as 1 starting from 11-(3-bromopro-
pyl)-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile (3a ) (200
mg, 0.56 mmol) and 4-hydroxy-4-phenyl piperidine to afford
11-[3-(4-Acetyl-4-p h en yl-1-p ip er id in yl)p r op yl]-6,11-d i-
h yd r od ib en zo[b,e]t h iep in -11-ca r b on it r ile, h yd r och lo-
r id e sa lt (6i) was prepared in the same manner as 6d starting
with 3a (200 mg, 0.56 mmol) and 4-acetyl-4-phenylpiperidine
to afford 6i as a white solid (156 mg, 52% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.95 (s, 3H), 2.20 (m, 2H), 2.70 (m, 5H),
3.05 (m, 3H), 3.50 (m, 2H), 4.10 (d, 1H), 4.55 (d, 1H), 7.30 (m,
11H), 7.80 (m, 2H). Anal. (C31H32N2OS‚1.6HCl) C, H, N.
6,11-Dih yd r o-11-[3-[4-h yd r oxy-4-(p h en ylm eth yl)-1-p i-
p er id in yl]p r op yl]d iben zo[b,e]th iep in -11-ca r bon itr ile (6j)
was prepared in the same manner as 1 starting with 3a (200
mg, 0.56 mmol) and 4-hydroxy-4-phenylmethyl piperidine to
1
6a as a colorless oil (190 mg, 72% yield). H NMR (300 MHz,
CDCl3) δ (TMS) 1.75 (m, 6H), 2.15 (m, 2H), 2.45 (m, 3H), 2.78
(m, 2H), 3.10 (m, 1H), 4.10 (d, 1H), 4.58 (d, 1H), 7.15 (m, 4H),
7.30 (m, 5H), 7.50 (d, 2H), 7.85 (m, 2H). Anal. (C29H30N2OS‚
0.2SiO2) C, H, N.
11-[3-[4-Hydr oxy-4-(2-m eth ylph en yl)-1-piper idin yl]pr o-
pyl]-6,11-dih ydr odiben zo[b,e]th iep in -11-ca r bon itr ile (6b)
was prepared in the same manner as 1 starting with 3a (358
mg, 1.0 mmol) and 4-(2-methylphenyl)-4-hydroxy piperidine
to afford 6b after flash chromatography as a colorless oil (430
1
mg, 90% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.70 (m,
4H), 1.95 (m, 2H), 2.20 (m, 2H), 2.50 (m, 3H), 2.65 (s, 3H),
2.80 (m, 2H), 3.15 (m, 1H), 4.10 (d, 1H), 4.60 (d, 1H), 7.15 (m,
1
afford 6j as a white foam (200 mg, 43% yield). H NMR (300
MHz, CDCl3) δ (TMS) 1.50 (d, 2H), 1.75 (m, 4H), 2.25 (m, 2H),

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4687
2.45 (m, 2H), 2.70 (m, 2H), 2.80 (s, 3H), 3.05 (m, 1H), 4.10 (d,
1H), 4.55 (d, 1H), 7.25 (m, 11H), 7.85 (m, 2H). Anal. (C30H32N2-
OS‚0.16SiO2) C, H, N.
2 is described by Akermann et al.29 Compound 6r is prepared
from 2r (150 mg, 0.60 mmol) in a manner similar to 1 to afford
6r after HPLC purification as a white powder (101 mg, 25%
1
yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.73 (m, 3H), 2.06
6,11-Dih yd r o-11-[3-[4-(h yd r oxyd ip h en ylm et h yl)-1-p i-
per idin yl]pr opyl]diben zo[b,e]th iepin -11-car bon itr ile (6k)
was prepared in the same manner as 1 starting with 3a (200
mg, 0.56 mmol) and 4-hydroxydiphenylmethyl piperidine to
afford 6k as a yellow solid (25 mg, 4% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.60 (m, 2H), 1.95 (m, 2H), 2.15 (m, 3H),
2.60 (m, 6H), 2.85 (m, 1H), 3.20 (m, 2H), 4.05 (m, 1H), 4.60
(m, 1H), 7.30 (m, 16H), 7.88 (m, 2H). Anal. (C36H36N2OS‚
1.0H2O‚1.3HCl) C, H, N.
(m, 3H), 2.36 (m, 1H), 2.82 (m, 1H), 3.20 (m, 2H), 3.32 (m,
2H), 3.52 (s, 3H), 3.58 (m, 2H), 7.32 (m, 2H), 7.48 (m, 12H),
9.11 (br s, 0.5H), 9.40 (br s, 0.5H). Anal. (C30H30 ClN3O2‚
0.55TFA‚0.25C4H8O2‚0.5H2O) C, H, Cl, F, N.
A Gen er a l P r oced u r e for th e P r ep a r a tion of Ta r get
Com p ou n d s 6 Usin g Com m er cia lly Ava ila ble 4-Hyd r oxy-
4-p h en ylp ip er id in es. 4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-
d ip h en yl-1-p ip er id in ep en ta n en itr ile (6s). Diphenylaceto-
nitrile (10 g, 52 mmol) and 1,3-dibromopropane (52 g, 259
mmol) were dissolved in anhydrous DMF (125 mL) under
nitrogen. The solution was chilled to 5 °C in an ice bath, and
sodium hydride (2.48 g, 62.1 mmol, 60% dispersion in oil) was
added portionwise. The ice bath was removed, and the mixture
was stirred for 18 h at ambient temperature. The reaction was
quenched by pouring into water (1 L). The product was
extracted into ethyl acetate (3 × 250 mL). The combined
organic extract was washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo to give an oil. Flash
9-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
p yl]-9H-xa n th en e-9-ca r bon itr ile (6l) was prepared from
commercially available 9-cyanoxanthene in a manner similar
1
to 1. H NMR (300 MHz, CDCl3) δ (TMS) 1.41 (m, 2H), 2.65
(m, 4H), 2.05 (m, 4H), 2.3 (m, 4H), 2.40 (d, 2H), 7.30 (m, 10H),
7.63 (d, 2H). Anal. (C28H27ClN2O2‚0.33H2O) C, H, N.
5-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
p yl]-10,11-d ih yd r o-5H -d ib en zo[a ,d ]cycloh ep t en e-5-ca r -
bon itr ile (6m ) was prepared from commercially available
dibenzosuberyl chloride in a manner similar to 1. 1H NMR (300
MHz, CDCl3) δ (TMS) 1.48 (m, 2H), 1.68 (d, 2H), 2.05 (m, 2H),
2.35 (m, 4H), 2.60 (m, 4H), 3.05 (m, 2H), 3.44 (m, 2H), 7.23
(m, 8H), 7.42 (d, 2H), 8.00 (d, 2H). Anal. (C30H31ClN2O‚
0.25H2O) C, H, N.
chromatography on silica gel with
a gradient of 0-25%
methylene chloride in hexanes afforded 3s, R-(3-bromopropyl)-
R-phenyl-benzeneacetonitrile, as a white solid (8.7 g, 51%). 1H
NMR (300 MHz, CDCl3) δ (TMS) 2.00 (m, 2H), 2.58 (m, 2H),
3.42 (t, 2H), 7.38 (m, 10H). To a solution of 3s (200 mg, 0.6
mmol) in anhydrous DMF (6 mL) under nitrogen was added
4-(4-chlorophenyl)-4-hydroxypiperidine, 5s, (142 mg, 0.67 mmol)
and cesium carbonate (300 mg, 0.9 mmol). The resulting
solution was stirred for 16 h at ambient temperature. The
reaction was quenched with water (30 mL) and extracted with
ethyl acetate (3 × 30 mL). Combined organic extract was
washed with water and brine, dried over magnesium sulfate,
filtered, and evaporated in vacuo to an oil (130 mg). The oil
was purified by flash chromatography on silica gel eluting with
0-100% ethyl acetate in methylene chloride to give 6s as a
colorless foam (200 mg, 73%). 1H NMR (300 MHz, CDCl3) δ
(TMS) 1.70 (m, 4H), 1.82 (br s, 1H) 2.16 (m, 2H), 2.40 (t, 2H),
2.45 (t, 4H), 2.72 (d, 2H), 7.38 (m, 14H). Anal. (C28H29ClN2O‚
0.3H2O) C, H, N.
5-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
pyl]-5H-diben zo[a ,d]cycloh epten e-5-car bon itr ile (6n ) was
prepared from commercially available dibenzosuberenol in a
manner similar to 1 to give 6n in a 34% yield from dibenzo-
subernol. 1H NMR (300 MHz, CDCl3) δ (TMS) 1.38 (m, 2H),
1.65 (m, 2H), 2.04 (m, 2H), 2.35 (m, 6H), 2.64 (d, 2H), 7.00 (s,
2H), 7.38 (m, 10H), 8.04 (d, 2H). Anal. (C30H29ClN2O‚0.85H2O)
C, H, N.
11-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]pr o-
p yl]-6,11-d ih yd r od iben zo[b,e]oxep in -11-ca r bon itr ile (6o)
was prepared in a manner similar to 1 starting with phenol
(1.23 g, 13.1 mmol) to afford 6o as white solid (130 mg, 2.1%
yield over eight steps). 1H NMR (300 MHz, CDCl3) δ (TMS)
1.41 (m, 1H), 1.70 (m, 3H), 2.10 (m, 2H), 2.39 (m, 5H), 2.68
(m, 2H), 2.85 (m, 1H), 5.05 (d, 1H), 5.42 (d, 1H), 7.2 (m, 10H),
7.82 (d, 1H), 8.0 (d, 1H). Anal. (C29H29ClN2O2‚0.3H2O) C, H,
N.
11-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]pr o-
pyl]-6,11-dih ydr odiben zo[b,e]th iepin -11-car bon itr ile,5-Ox-
id e (6p a n d 6q). Compound 3a (186 mg, 0.52 mmol) was
dissolved in 30% aqueous hydrogen peroxide (0.43 mL) and
acetic acid (3 mL) and stirred for 1 h. The reaction was then
poured into water, neutralized with sodium carbonate, and
extracted with ethyl acetate. The ethyl acetate was washed
with brine, dried over magnesium sulfate, and concentrated
in vacuo to give the sulfoxide as an oil (190 mg, 97%). NMR of
the oil showed two diastereomers in a 4:6 ratio. 1H NMR (300
MHz, CDCl3) δ (TMS) 2.04 (m, 2H), 2.44 (m, 0.8H), 2.65 (m,
1.2H), 2.84 (m, 2H), 3.3 (m, 4H), 4.28 (m, 1.6H), 4.75 (m, 2.4H),
7.20 (d, 2H), 7.41 (m, 4H), 7.62 (m, 4H), 8.08 (m, 4H). This
mixture was treated as described for the preparation of 6f to
afford 6p and 6q as a mixture of diastereomers. The diaster-
omers were separated by flash chromatography on silica gel,
eluting with a gradient of 0-5% methanol in ethyl acetate.
The first diasteromer to elute (6p ) was converted to the
hydrochloride salt as described for 6d . The compound was
isolated as a white solid (60 mg, 20% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.95 (m, 7H), 2.55 (m, 1H), 3.20 (m, 6H),
4.22 (d, 1H), 4.51 (d, 1H), 7.37 (m, 7H), 7.62 (m, 2H), 8.15 (m,
3H). Anal. (C29H29ClN2O2S‚1.6HCl) C, H, N.
A Gen er a l P r oced u r e for th e P r ep a r a tion of Ta r get
Com p ou n d s 6 Usin g Com m er cia lly Ava ila ble Gr ign a r d
a n d Lith iu m Rea gen ts. 4-(4-F lu or op h en yl)-4-h yd r oxy-
r,r-d ip h en yl-1-p ip er id in ep en ta n en itr ile (6t). To a solu-
tion of 3s (6.8 g, 21.6 mmol) in anhydrous DMF (50 mL) under
nitrogen was added 1,4-dioxa-8-azaspiro[4.5]decane (7.8 g, 54.5
mmol). The resulting solution was stirred for 18 h at ambient
temperature. The reaction was quenched with water and
extracted into ethyl acetate. The organic extract was washed
with brine, dried over magnesium sulfate, and filtered, and
the solvent was removed in vacuo to yield a oil (9.6 g). The oil
was dissolved in methanol (35 mL), and 6 N HCl (190 mL)
was added. The solution was stirred at ambient temperature.
After 24 h the reaction was made basic with aqueous KOH
and extracted with ethyl acetate. The organic extract was
washed with brine, dried over magnesium sulfate, filtered, and
evaporated in vacuo to a solid which was purified by flash
chromatography on silica gel eluting with 0-20% ethyl acetate
in methylene chloride to give 4-oxo-R,R-diphenyl-1-piperidine-
pentanenitrile (4t) as a white solid (6.9 g, 96%). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.75 (m, 4H), 2.47 (m, 6H), 2.66 (t, 4H),
7.36 (m, 10H). To a 0 °C solution of 4t (125 mg, 0.36 mmol) in
anhydrous THF (3 mL) under nitrogen was added 4-fluoro-
phenyl magnesium bromide (0.22 mL, 2.0 M in diethyl ether,
0.43 mmol). The ice bath was removed, and the solution was
stirred for 4 h at ambient temperature. The solution was cooled
to 0 °C and quenched with water, acidified with 1 N HCl (1
mL), and then neutralized with 1 N KOH. The solution was
buffered to pH 9 with saturated sodium bicarbonate and the
product extracted into ethyl acetate (2 × 20 mL). The combined
organic extracts were washed with water and brine, dried over
magnesium sulfate, filtered, and evaporated in vacuo to a
crude product (150 mg). The crude product was purified by
The second diasteromer to elute (6q) was isolated also as a
1
colorless oil (90 mg, 35% yield). H NMR (300 MHz, CDCl3) δ
(TMS) 1.78 (m, 5H), 2.04 (m, 2H), 2.38 (m, 4H), 7.65 (m, 4H),
4.65 (d, 1H), 4.84 (d, 1H), 7.35 (m, 7H), 7.56 (m, 2H), 7.98 (m,
3H). Anal. (C29H29ClN2SO2‚1.36Si2O) C, H, N.
11-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
pyl]-6,11-dih ydr o-5-m eth yl-6-oxo-5H-diben zo[b,e]azepin e-
11-ca r bon itr ile (6r ). The preparation of the corresponding

4688 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ng et al.
flash chromatography on silica gel eluting with 0-100% ethyl
acetate in methylene chloride to give 6t as a colorless foam
(110 mg, 71%). 1H NMR (300 MHz, CDCl3) δ (TMS) 1.78 (d,
4H), 2.05 (m, 2H), 2.48 (m, 6H), 2.75 (d, 2H), 7.04 (t, 2H), 7.42
(m, 12H). Anal. (C28H29FN2O‚0.2H2O) C, H, N.
s, 2H), 2.16 (d, 2H), 2.60 (m, 4H), 3.32 (m, 4H), 3.64 (d, 2H),
3.90 (s, 1H), 7.38 (m, 10H), 7.52, (m, 3H), 7.86 (m, 4H). Anal.
(C32H32N2O‚1.0HCl‚0.5H2O) C, H, N.
4-Hyd r oxy-r,r-d ip h en yl-4-(3-p yr id in yl)-1-p ip er id in e-
p en ta n en itr ile (6a d ) was prepared in a manner similar to
6a j starting with 4t (500 mg, 1.5 mmol) and 3-bromopyridine
to afford 6a d as a foam (120 mg, 19% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.70 (m, 4H), 2.10 (m, 2H), 2.44 (m, 6H),
2.76 (d, 2H), 7.34 (m, 10H), 7.1 (m, 1H), 8.46 (m, 1H), 8.74 (d,
1H). Anal. (C27H29N3O‚0.1C4H8O2‚0.5H2O) C, H, N.
4-[1,1′-Bip h en yl]-4-yl-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ep en ta n en itr ile (6a e) was prepared in a manner similar
to 6a j starting with 4t (500 mg, 1.5 mmol) and 4-bromobi-
phenyl to afford 6a e as a solid (50 mg, 8% yield). 1H NMR
(300 MHz, CDCl3) δ (TMS) 1.70 (m, 4H), 1.96 (m, 2H), 2.46
(m, 6H), 2.76 (m, 2H), 7.38 (m, 13H), 7.59 (m, 6H). ES/MS (M
+ H) ) 487. Anal. (C34H34N2O) C, H, N.
4-Bu tyl-4-h yd r oxy-r,r-d ip h en yl-1-p ip er id in ep en ta n e-
n itr ile, h yd r och lor id e sa lt (6a f) was prepared in a manner
similar to 6t starting with 4t (500 mg, 1.5 mmol) and
butyllithium (2.5 M in hexanes) to afford 6a f, after chroma-
tography and salt formation, as a solid (94 mg, 16% yield). 1H
NMR (400 MHz, CDCl3) δ (TMS) 0.85 (t, 3H), 1.28 (m, 4H),
1.60 (m, 4H) 1.95 (m, 2H), 2.36 (m, 2H), 2.64 (t, 2H), 3.00 (m,
4H), 3.17 (m, 2H), 7.36 (m, 10H), 11.50 (br s, 1H). Anal.
(C26H34N2O‚1.4HCl‚1.0H2O) C, H, N.
4-(4-Br om op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er i-
d in ep en ta n en itr ile (6u ) was prepared in a manner similar
to 6s starting with 3s (600 mg, 1.85 mmol) and 4-(4-bromo-
phenyl)-4-hydroxy piperidine to afford 6u as a white solid (320
1
mg, 35% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.68 (m,
6H), 2.08 (m, 2H), 2.42 (m, 5H), 2.70 (m, 2H), 7.38 (m, 14H).
Anal. (C28H29BrN2O) C, H, N.
4-H yd r oxy-r,r- 4-t r ip h en yl-1-p ip er id in ep en t a n en i-
tr ile, h yd r och lor id e sa lt (6v) was prepared in a manner
similar to 6s starting with 3s (600 mg, 1.85 mmol) and
4-hydroxy-4-phenyl piperidine. The hydrochloride salt was
prepared as described in 6d . The solid was isolated by filtration
and dried in vacuo to afford 6v as a white solid (300 mg, 36%
1
yield). H NMR (300 MHz, DMSO-d6) δ 1.78 (d, 2H), 2.32 (t,
2H), 2.52 (s, 2H), 2.64 (t, 2H), 3.20 (m, 3H), 3.35 (m, 3H), 5.48
(s, 1H), 7.42 (m, 15H) 10.17 (br s, 1H). Anal. (C28H30N2O‚
1.0HCl) C, H, N.
1-(4-Cya n o-4,4-d ip h en ylbu tyl)-4-p h en yl-4-p ip er id in e-
ca r boxylic a cid , m eth yl ester , h yd r och lor id e sa lt (6w )
was prepared in a manner similar to 6s starting with 3s (250
mg, 0.76 mmol) and 4-phenyl-4-piperidinecarboxylic acid,
methyl ester to afford 6w as a white solid (220 mg, 47% yield).
1H NMR (300 MHz, CDCl3) δ (TMS) 1.70 (m, 2H), 2.00 (m,
2H), 2.70 (m, 8H), 3.10 (m, 1H), 3.50 (m, 2H), 3.75, (s, 3H),
7.40 (m, 15H). Anal. (C30H32N2O2‚1.3HCl) C, H, N.
1-(4-Cya n o-4,4-d ip h en ylbu tyl)-4-p h en yl-4-p ip er id in e-
ca r boxylic a cid (6x) was prepared by hydrolysis of 6w with
lithium hydroxide followed purification by preparative HPLC
to afford 6x as a white solid (80 mg, 74% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.20 (br s, 1H), 1.75 (m, 3H), 1.90 (m,
2H), 2.25 (m, 1H), 2.50 (m, 2H), 2.70 (m, 2H), 2.90 (m, 1H),
3.10 (m, 2H), 355 (d, 1H), 7.35 (m, 15H), 9.20 (br s, 1H). Anal.
(C29H30N2O2‚1.15TFA) C, H, N.
4-Hydr oxy-4-(4-iod oph en yl)-r,r-d ip h en yl-1-p ip er id in e-
p en ta n en itr ile (6a g) was prepared in a manner similar to
6a j starting with 4t (500 mg, 1.5 mmol) and 1,4-diiodobenzene.
The crude product was purified by flash chromatography to
1
afford 6a g as a foam (45 mg, 6% yield). H NMR (300 MHz,
CDCl3) δ (TMS) 1.78 (d, 4H), 2.05 (t, 2H), 2.50 (m, 6H), 2.75
(d, 2H), 7.39 (m, 12H), 7.75 (d, 2H). Anal. (C28H30IN2O) C, H,
N.
4-(3-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ep en ta n en itr ile, h yd r och lor id e sa lt (6a h ) was pre-
pared in a manner similar to 6a j starting with 4t (500 mg,
1.5 mmol) and 3-bromochlorobenzene to give a white solid. The
solid was further purified by crystallization from methanol to
afford 6a h as a white solid (180 mg, 24% yield). 1H NMR (300
MHz, DMSO-d6) δ 1.74 (d, 4H), 2.30 (t, 2H), 2.50 (m, 2H), 2.61
(m, 2H), 3.20 (m, 4H), 5.61 (s, 1H), 7.40 (m, 14H), 9.95 (br s,
1H). ES/MS M + H ) 445. Anal. (C28H29ClN2O‚0.85HCl‚
0.15HBr‚1.0H2O) C, H, N, Cl, Br.
4-(H yd r oxym et h yl)-r,r-4-t r ip h en yl-1-p ip er id in ep en -
ta n en itr ile (6y) was prepared from 6w by reduction with
1
LAH to 6y as a colorless oil (72 mg, 34% yield). H NMR (300
MHz, CDCl3) δ (TMS) 1.20 (t, 3H), 1.90 (m, 2H), 2.00 (s, 1H),
2.50 (m, 10H), 2.85 (br s, 1H), 3.50 (m, 4H), 4.10 (q, 2H), 7.35
(m, 15H), 11.40 (br s, 1H). Anal. (C29H32N2O‚0.9HCl‚1.0C4H8O2)
C, H, N.
4-(3,5-Dich lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p i-
p er id in ep en ta n en itr ile (6a i) was prepared in a manner
similar to 6a j starting with 4t (500 mg, 1.5 mmol) and 3,5-
dichlorobromobenzene to afford 6a i as a white solid (310 mg,
38% yield). 1H NMR (300 MHz, DMSO-d6) δ 1.78 (d, 4H), 2.15
(t, 2H), 2.48 (s, 2H), 2.62 (m, 2H), 3.25 (M, 4H), 5.63 (s, 1H),
7.40 (m, 14H), 10.00 (br s, 1H). Anal. (C28H28Cl2N2O‚1.1HCl‚
1.6H2O) C, N, Cl; H: calcd, 5.94; found, 5.26.
4-Hydr oxy-r,r-diph en yl-1-piper idin epen tan en itr ile (6z)
was prepared in a manner similar to 6s starting with 3s (200
mg, 0.61 mmol) and 4-hydroxy piperidine to afford 6s as a
white solid (160 mg, 75% yield). 1H NMR (300 MHz, CDCl3) δ
(TMS) 1.58 (m, 4H), 1.83 (m, 2H), 2.05 (t, 2H), 2.40 (m, 4H),
2.68 (d, 2H), 3.65 (m, 1H), 7.36 (m, 10H). Anal. (C22H26N2O‚
0.4H2O) C, H, N.
4-Hyd r oxy-r,r-d ip h en yl-4-(2-th ien yl)-1-p ip er id in ep en -
ta n en itr ile (6a a ) was prepared in a manner similar to 6t
starting with 4t (450 mg, 1.35 mmol) and commercially
available 2-thienyllithium (1 M in THF) to afford 6a a after
chromatography as a foam (235 mg, 42% yield). 1H NMR (300
MHz, CDCl3) δ (TMS) 1.66 (m, 2H), 1.72 (d, 3H), 2.14 (t, 2H),
2.44 (m, 5H), 2.64 (d, 2H), 6.98 (m, 2H), 7.21 (d, 1H), 7.36 (m,
10H). Anal. (C26H28N2OS‚0.5H2O) C, H, N, S.
A Gen er a l P r oced u r e for th e P r ep a r a tion of Ta r get
Com p ou n d s 6 Usin g Lith iu m Rea gen ts P r ep a r ed by
Ha logen Exch a n ge. 4-Hyd r oxy-4-(4-m eth oxyp h en yl)-r,r-
diph en yl-1-piper idin epen tan en itr ile, Hydr och lor ide Salt
(6a j). A solution of 4-bromoanisole (309 mg, 1.65 mmol) in
anhydrous ether (5 mL) was chilled to -80 °C under nitrogen.
tert-Butyllithium (1.95 mL, 1.7 M in pentane, 3.3 mmol) was
added over 10 min, and the mixture was stirred for 1 h at -80
°C. A second solution of 4t (500 mg, 1.5 mmol) in anhydrous
THF (5 mL) was prepared and chilled under nitrogen to -70
°C. The litho-anisole solution was added via cannula to the
solution of 4t over 10 min. The mixture was stirred for 30 min
at -70 °C and then allowed to come to ambient temperature
over 30 min. The reaction was quenched by the addition of 1
N HCl (5 mL). Ether was added, and the product precipitated
as the salt. The white solid was isolated by filtration and dried
in vacuo to afford 6a j (620 mg, 81%). 1H NMR (300 MHz,
DMSO-d6) δ 1.74 (d, 4H), 2.26 (t, 2H), 2.62 (t, 2H), 3.26 (m,
6H), 3.74 (s, 3H), 5.40 (s, 1H), 6.90 (d, 2H), 7.4 (m, 12H), 10.02
(br s, 1H). Anal. (C29H32N2O2‚0.7HCl‚0.3HBr‚1.0H2O) C, H, N,
Cl, Br, Li.
4-Hyd r oxy-r,r-d ip h en yl-4-(2-p yr id in yl)-1-p ip er id in e-
p en ta n en itr ile (6a b) was prepared in a manner similar to
6a j starting with 4t (500 mg, 1.5 mmol) and 2-bromopyridine
to afford 6a b, after chromatography as a white solid (288 mg,
1
47% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.64 (m, 4H),
2.02 (t, 2H), 2.45 (m, 6H), 2.78 (d, 2H), 5.24 (s, 1H), 7.2 (m,
1H), 7.34 (m, 11H), 7.71 (t, 1H), 8.52 (d, 1H). Anal. (C27H29N3O)
C, H, N.
4-H yd r oxy-4-(2-n a p h t h a len yl)-r,r-d ip h en yl-1-p ip er -
id in ep en ta n en itr ile, h yd r och lor id e sa lt (6a c) was pre-
pared in a manner similar to 6a j starting with 4t (400 mg,
1.2 mmol), 2-bromonaphthalene, and n-butyllithium (1.6 M in
hexanes) to afford 6a c after salt formation as a solid (65 mg,
1
11% yield). H NMR (300 MHz, CDCl3/TFA) δ (TMS) 2.00 (br

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4689
4-(4-Cyan oph en yl)-4-h ydr oxy-r,r-diph en yl-1-piper idin e-
p en ta n en itr ile (6a k ) was prepared in a manner similar to
6a j starting with 4t (500 mg, 1.5 mmol) and 4-bromobenzoni-
trile to afford 6a k after chromatography as a white solid (98
mg, 15% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.70 (d,
6H), 2.10 (m, 2H), 2.44 (m, 4H), 2.76 (d, 2H), 7.36 (m, 10H),
brown oil (400 mg, 0.92 mmol). The hydrochloride salt was
prepared in a manner previously described. 1H NMR (300
MHz, CDCl3) δ (TMS) 1.68 (m, 2H), 2.22 (m, 2H), 2.53 (m, 6H),
2.95 (m, 1H), 3.10 (m, 1H), 7.02 (m, 2H), 7.35 (m, 12H). Anal.
(C27H27FN2O‚1.3HCl‚0.5H2O) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ep en ta n en itr ile, N-oxid e (6a q) was prepared from 6s
(200 mg, 0.44 mmol) in 30% aqueous hydrogen peroxide (3 mL)
and acetic acid (2 mL) at 110 °C for 1 h. The product was
isolated by dilution with acetonitrile and was purified by
preparative HPLC to afford 6a q as a white solid (140 mg, 54%
yield): 1H NMR (300 MHz, CDCl3) δ (TMS) 1.95 (d, 2H), 2.12
(m, 2H), 2.60 (m, 4H), 3.70 (m, 2H), 3.88 (m, 4H), 7.38 (m,
12H). Anal. (C28H29ClN2O2‚1.15TFA) C, H, N. LC/MS M + H+
) 461.
1
7.64 (s, 4H). Anal. (C29H29N3O‚0.4H2O) C, H, N.
4-Hyd r oxy-r,r-d ip h en yl-4-[4-(tr iflu or om eth yl)p h en yl]-
1-p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6a l) was
prepared in a manner similar to 6a j starting with 4t (500 mg,
1.5 mmol) and 4-bromobenzotrifluoride. The crude product was
purified by flash chromatography, and the hydrochloride salt
formed to give 6a l as a white solid (180 mg, 25% yield). 1H
NMR (300 MHz, DMSO-d6) δ 1.78 (d, 4H), 2.44 (m, 2H), 2.64
(t, 2H), 3.20, (m, 4H), 3.36 (m, 2H), 5.76 (s, 1H), 7.42 (m, 10H),
7.70 (m, 4H), 10.60 (br s, 1H). Anal. (C29H29F3N2O‚1.0HCl) C,
H, N.
A Gen er a l P r oced u r e for th e P r ep a r a tion of Qu a ter -
n a r y Am m on iu m Sa lt s. 4-(4-Ch lor op h en yl)-4-h yd r oxy-
1-[5-cya n o-5-(1,5-cycloh exa d ien -1-yl)-5-p h en ylp en tyl]-1-
m eth yl-p ip er id in iu m , Iod id e (6a r ). To a solution of 6s (400
mg, 0.9 mmol) in methylene chloride (4 mL) was added methyl
iodide (0.5 mL, 8.0 mmol). The solution was stirred at ambient
temperature for 16 h. Ether was added to the solution to
precipitate the product as an oil. The solvent was decanted
and the oil redissolved in methylene chloride. Ether was added
to this solution dropwise and the product crystallized. The
product was isolated by filtration as a yellow solid (270 mg,
4-(4-Am in op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ep en ta n en itr ile (6a m ) was prepared in
a manner
similar to 6a j starting with 4t (1.0 g, 3.0 mmol) and 4-bromo-
N,N-bistrimethylsilylaniline to afford 6a m , after chromatog-
raphy, as a solid (445 mg, 35% yield). 1H NMR (300 MHz,
CDCl3) δ (TMS) 1.70 (m, 4H), 2.06 (t, 2H), 2.41 (m, 6H), 2.68
(d, 2H), 3.65 (br s, 2H), 6.67 (d, 2H), 7.34 (m, 12H). Anal.
(C28H31N3O‚0.1H2O) C, H, N.
4-[4-(Dim eth yla m in o)p h en yl]-4-h yd r oxy-r,r-d ip h en yl-
1-p ip er id in ep en ta n en itr ile (6a n ) was prepared in a man-
ner similar to 6a j starting with 4t (500 mg, 1.5 mmol) and
N,N-dimethyl-4-bromoaniline to afford 6a n , after chromatog-
raphy, as a foam (250 mg, 37% yield). 1H NMR (400 MHz,
CDCl3) δ (TMS) 1.65 (m, 2H), 1.74 (d, 2H), 2.06 (t, 2H), 2.41
(m, 6H), 2.66 (d, 4H), 2.72 (s, 6H), 6.71 (d, 2H), 7.34 (m, 12H).
Anal. (C30H35N3O‚0.4H2O) C, H, N.
3-(4-F lu or op h e n yl)-r-(1,5-cycloh e xa d ie n -1-yl)-3-h y-
d r oxy-r-p h en yl-1-p ip er id in eh exa n en itr ile, h yd r och lo-
r id e sa lt (6a o) was prepared in a manner similar to 6a p
starting from 1-(phenylmethyl)-3-piperidinone (1.1 g, 3.85
mmol) to afford 6a o freebase as a colorless oil (390 mg, 0.88
mmol). 1H NMR (300 MHz, CDCl3) δ (TMS) 1.68 (m, 6H), 1.95
(m, 2H), 2.10 (d, 2H), 2.45 (m, 4H), 2.60 (d, 1H), 2.85 (m, 1H),
3.84 (br s, 1H), 7.08 (m, 2H), 7.40 (m, 12H). Anal. (C28H29FN2O‚
1.5HCl) C, H, N.
3-(4-F lu or op h e n yl)-r-(1,5-cycloh e xa d ie n -1-yl)-3-h y-
d r oxy-r-p h en yl-1-p yr r olid in eh exa n en itr ile, Hyd r och lo-
r id e Sa lt (6a p ). A solution of 1-(phenylmethyl)-3-pyrrolidi-
none (500 mg, 2.9 mmol) in anhydrous THF (15 mL) was cooled
to 0 °C under nitrogen and treated with 4-fluorophenyl
magnesium bromide (1.7 mL, 2.0 M in ether, 3.4 mmol). The
ice bath was removed and the mixture allowed to stir at
ambient temperature for 18 h. The solution was cooled to 0
°C, quenched with water, acidified with 1 N HCl then neutral-
ized with 1 N KOH. The solution was buffered to pH 9 with
saturated sodium bicarbonate and the product extracted with
ethyl acetate (2 × 50 mL). The combined organic extracts were
washed with water and then brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo to a brown oil (800
mg). The oil was purified by flash chromatography on silica
gel eluting with 0-100% ethyl acetate in methylene chloride
to give a clear yellow oil (600 mg). The oil was dissolved in
methanol (50 mL), 10% Pd/C (100 mg) was added, and the
mixture was shaken under 50 psi H2 for 16 h at ambient
temperature. The mixture was filtered through Celite and the
filtrate concentrated in vacuo to give an orange oil (640 mg,
quantitative). This material was dissolved in anhydrous DMSO
(20 mL) and treated with 3s (600 mg, 2.2 mmol), followed by
diisopropylethylamine (600 mg, 4.4 mmol). The resulting
brown solution was heated to 60 °C for 27 h. The mixture was
cooled and poured into water (100 mL), and the product was
isolated by extraction with ethyl acetate (3 × 50 mL). The
combined organic extracts were washed with water and then
brine, dried over magnesium sulfate, filtered, and concentrated
in vacuo to a brown oil. The product was purified by flash
chromatography on silica gel eluting with a 0-40% ethyl
acetate in methylene chloride gradient to afford the 6a p as a
1
51% yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.98 (m, 2H),
2.17 (m, 4H), 2.70 (m, 2H), 3.14 (s, 3H), 3.44 (m, 2H), 3.82 (m,
4H), 7.40 (m, 14 H). Anal. (C29H32ClIN2O‚0.8H2O) C, H, N, Cl,
I. ES/MS m/z 459.
4-(4-Ch lor op h en yl)-4-h yd r oxy-1-[5-(1,5-cycloh exa d ien -
1-yl)-5-ph en ylpen tyl]-1-m eth yl-piper idin iu m , iodide (6as)
was prepared in a manner similar to 6a r starting with 8 (90
mg, 0.21 mmol) to afford 6a r as a white solid after purification
by HPLC (40 mg, 33% yield). 1H NMR (300 MHz, CDCl3) δ
(TMS) 1.70 (m, 4H), 2.21 (m, 4H), 3.00 (s, 3H), 3.30 (m, 2H),
3.48 (m, 3H), 4.05 (t, 1H), 7.15 (m, 2H), 7.30 (m, 8H), 7.39 (m,
2H), 7.57 (m, 2H). Anal. (C28H33ClNO‚1.05TFA‚0.8H2O) C, H,
N, Cl, I.
4-(4-Ch lor op h en yl)-4-h yd r oxy-1-[5-cya n o-5-(2-n a p h -
th a len yl)p en tyl]-1-m eth yl-p ip er id in iu m , iod id e (6a t) was
prepared in a manner similar to 6a r starting with 6a u (200
mg, 0.4 mmol) to afford 6a t as a white solid (120 mg, 50%
yield). 1H NMR (300 MHz, DMSO-d6) δ 1.78 (m, 2H), 2.00 (m,
4H), 2.31 (m, 2H), 3.08 (s, 3H), 3.42 (m, 6H), 4.50 (m, 1H),
5.60 (s, 1H), 7.40 (m, 2H), 7.59 (m, 5H), 7.98 (m, 4H). Anal.
(C27H30ClIN2O‚0.33H2O) C, H, N, Cl, I.
4-(4-Ch lor oph en yl)-4-h ydr oxy-r-(2-n aph th alen yl)- 1-pi-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6a u ) was
prepared in a manner similar to 6bh starting with 2-naph-
thylacetonitrile (840 mg, 5.0 mmol) to afford 25a as a white
solid (610 mg, 26% yield). 1H NMR (300 MHz, DMSO-d6) δ
1.80 (m, 4H), 2.05 (m, 2H), 2.38 (m, 2H), 3.20 (m, 4H), 3.40
(m, 2H), 4.50 (t, 1H), 5.60 (s, 1H), 7.42 (m, 4H), 7.55 (m, 4H),
7.95 (m, 5H), 10.50 (br s, 1H). Anal. (C26H27ClN2O‚1.0HCl‚
0.5H2O) C, H, Cl, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ebu ta n en itr ile, h yd r och lor id e sa lt (6a v) was prepared
as described in the preparation of 6s starting with 4-bromo-
2,2-diphenyl butyronitrile (200 mg, 0.67 mmol) and 4-(4-
chlorophenyl)-4-hydroxypiperidine (155 mg, 0.73 mmol) to
afford 6a v, after chromatography and salt formation, as a
1
white solid (65 mg, 20% yield). H NMR (300 MHz, CDCl3) δ
(TMS) 1.75 (br s, 1H), 1.90 (d, 2H), 2.78 (m, 2H), 3.10 (m, 4H),
3.35 (m, 3H), 7.40 (m, 12H). Anal. (C27H27ClN2O‚1.2HCl) C,
H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in eh exa n en itr ile (6a w ) was prepared in the same manner
as 6s starting with phenylacetonitrile (600 mg, 3.1 mmol) and
1,4-dibromobutane (3.3 g, 15 mmol) followed by substitution
with 4-(4-chlorophenyl)-4-hydroxypiperidine to afford 6a w ,
after chromatography, as a white solid (780 mg, 48% yield).
1H NMR (300 MHz, CDCl3) δ (TMS) 1.62 (m, 7H), 2.10 (m,

4690 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ng et al.
2H), 2.39 (m, 5H), 2.79 (d, 2H), 7.35 (m, 14H). Anal. (C29H31
ClN2O) C, H, N.
-
ylacetonitrile (840 mg, 4.3 mmol) to afford 6bg as a white solid
(380 mg, 15% yield). 1H NMR (300 MHz, DMSO-d6) δ 1.95 (m,
8H), 3.18 (m, 4H), 3.40 (m, 2H), 4.38 (m, 1H), 5.60 (s, 1H),
7.55 (m, 8H), 9.00 (br s, 1H). Anal. (C22H24BrClN2O‚1.0HI) C,
H, N, Br, Cl, I.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in eh ep ta n en itr ile, h yd r och lor id e sa lt (6a x) was pre-
pared in a manner similar to 6s. Phenylacetonitrile (600 mg,
3.1 mmol) was reacted with 1,5-dibromopentane (3.6 g, 15
mmol) followed by the substitution with 4-(4-chlorophenyl)-4-
hydroxypiperidine to afford 6a x, after chromatography and
A Gen er a l P r oced u r e for th e P r ep a r a tion of Ta r get
Com p ou n d 6 fr om Com m er cia lly Ava ila ble Su bstitu ted
Acet on it r iles. r,4-Bis(4-ch lor op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, Hyd r och lor id e Sa lt (6bh ). A
solution of LDA (2.0 mL, 1.5 N in cyclohexane, 3.0 mmol) was
added by syringe over a 5 min period to a stirred solution
(cooled in a dry ice/acetone bath) of 4-chlorobenzylcyanide (0.45
g, 3.0 mmol) and 1-chloro-3-iodopropane (0.32 mL, 3.0 mmol)
in anhydrous THF (6.0 mL) under nitrogen. After 5 min the
bath was removed, and after warming for 1 h, 4-(4-chlorophen-
yl)-4-hydroxypiperidine (0.95 g, 4.5 mmol) was added. The
mixture was stirred until homogeneous and then heated
overnight at 65 °C. The mixture was partitioned between water
(50 mL) and ethyl ether (50 mL). The ethyl ether layer was
washed successively with 25 mL portions of water, 10%
aqueous K2CO3, and brine. The ethyl ether solution was dried
over MgSO4, filtered, and concentrated in vacuo. Flash chro-
matography on silica gel (20 g) with a gradient of 0-10%
2-propanol in 4:1 dichloromethane/ethyl acetate afforded 0.51
g of 6bh as its free base. To prepare the hydrochloride salt,
the free base was dissolved in methanol (5 mL) and concen-
trated HCl (0.15 mL). Ethyl ether (20 mL) was added slowly
with stirring. The resulting precipitate was collected by
filtration, rinsed with ethyl ether (2 × 5 mL), and dried at 50
°C under vacuum to obtain 6bh as a white solid (310 mg, 23%
yield). 1H NMR (300 MHz, DMSO-d6) δ 1.78 (m, 4H), 1.98 (m,
2H), 2.35 (m, 2H), 3.18 (m, 4H), 3.37 (m, 2H), 4.39 (t, 1H),
7.48 (m, 8H), 10.4 (br s, 1H). Anal. (C22H24Cl2N2O‚1.0HCl) C,
H, N, Cl.
1
salt formation, as a white solid (630 mg, 39% yield). H NMR
(300 MHz, CDCl3) δ (TMS) 1.41 (m, 4H), 1.92 (m, 4H), 2.40
(m, 2H), 2.75 (m, 4H), 3.23 (m, 4H), 7.21 (m, 14H). Anal.
(C30H33ClN2O‚1.0HCl‚0.5H2O) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-p h en yl-1-p ip er id in e-
p en ta n en itr ile, h yd r och lor ic sa lt (6a y) was prepared in a
manner similar to 6bh starting with phenylacetonitrile (300
mg, 2.56 mmol) to afford 6a y as a white solid (330 mg, 28%
yield). 1H NMR (300 MHz, CDCl3-TFA) δ (TMS) 1.75 (m, 6H),
2.35 (m, 4H), 2.81 (m, 2H), 3.00 (m, 3H), 3.75 (m, 1H), 7.15
(m, 9H), 10.80 (br s, 1H). Anal. (C22H25ClN2O‚1.2HCl) C, H,
N.
r-(3-Br om o-4-m et h oxyp h en yl)-4-(4-ch lor op h en yl)-4-
h yd r oxy-1-p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt
(6a z) was prepared in a manner similar to 6bh starting with
3-bromo-4-methoxyphenylacetonitrile (678 mg, 3.0 mmol) to
1
afford 6a z as a white solid (620 mg, 38% yield). H NMR (300
MHz, DMSO-d6) δ 1.77 (m, 4H), 1.90 (m, 2H), 2.35 (m, 2H),
3.17 (m, 4H), 3.39 (m, 2H), 3.85 (s, 3H), 4.29 (m, 1H), 5.59 (s,
1H), 7.17 (d, 1H), 7.44 (m, 5H), 7.65 (d, 1H), 10.32 (br s, 1H).
Anal. (C23H26BrClN2O2‚1.0HCl‚0.65C4H10O) C, H, N, Br, Cl.
4-(4-Ch lor op h en yl)-r-(2-cya n op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bb) was
prepared in a manner similar to 6bh starting with 2-cy-
anophenylacetonitrile (426 mg, 3.0 mmol) to afford 6bb as an
off-white solid (320 mg, 25% yield). 1H NMR (300 MHz, DMSO-
d6) δ 1.83 (m, 4H), 2.03 (m, 2H), 2.34 (m, 2H), 3.25 (m, 4H),
3.38 (m, 2H), 4.58 (m, 1H), 5.58 (s, 1H), 7.45 (m, 4H), 7.60 (m,
r-(2-Ch lor op h en yl)-4-(4-ch lor op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bi) was
prepared in a manner similar to 6bh starting with 2-chlo-
rophenylacetonitrile (455 mg, 3.0 mmol) to afford 6bi as a
1H), 7.74 (m, 1H), 7.95 (m, 1H), 10.38 (br s, 1H). Anal. (C23H24
ClN3O‚1.0HCl) C, H, N, Cl.
-
1
white solid (360 mg, 27% yield). H NMR (300 MHz, DMSO-
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(2,3,4,5,6-p en t a flu o-
r oph en yl)-1-piper idin epen tan en itr ile, h ydr och lor ide salt
(6bc) was prepared in a manner similar to 6bh starting with
2,3,4,5,6-pentafluorophenylacetonitrile (830 mg, 4.0 mmol) to
afford 6bc as a white solid (170 mg, 8.6% yield). 1H NMR (300
MHz, DMSO-d6) δ 1.95 (m, 4H), 2.08 (m, 2H), 2.35 (m, 2H),
3.15 (m, 4H), 3.40 (m, 2H), 4.71 (m, 1H), 5.60 (s, 1H), 7.45 (m,
4H), 10.40 (br s, 1H). Anal. (C22H20ClF5N2O‚1.0HCl‚0.2C4H10O)
C, H, N, Cl, F.
d6) δ 1.93 (m, 6H), 2.31 (m, 2H), 3.18 (m, 4H), 3.36 (m, 2H),
4.55 (m, 1H), 5.60 (s, 1H), 7.44 (m, 6H), 7.58 (m, 2H), 10.24
(br s, 1H). Anal. (C22H24Cl2N2O‚1.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-[2-(tr iflu or om eth yl)-
p h en yl]-1-p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt
(6bj) was prepared in a manner similar to 6bh starting with
2-trifluoromethylphenylacetonitrile (370 mg, 2.0 mmol) to
afford 6bh as a white solid (420 mg, 44% yield). 1H NMR (300
MHz, DMSO) δ 1.82 (m, 5H), 2.15 (m, 1H), 2,25 (t, 2H), 3.21
(m, 4H), 3.42 (m, 1H) 4.38 (m, 1H) 5.60 (s, 1H) 7.42 (m, 5H)
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(3-h yd r oxyp h en yl)-1-
p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bd ) was
prepared from 6bl (320 mg, 0.67 mmol) by catalytic hydroge-
7.62 (m, 1H), 7.82 (m, 3H), 10.15 (br s, 1H). Anal. (C23H24
-
1
ClF3N2O‚1.0HCl) C, H, N, Cl, F.
nation to afford 6bd as a white solid (120 mg, 43% yield). H
NMR (300 MHz, DMSO-d6) δ 1.84 (m, 6H), 2.31 (m, 2H), 3.15
(m, 4H), 3.38 (m, 2H), 4.21 (m, 1H), 5.58 (s, 1H), 6.76 (m, 3H),
7.20 (m, 1H), 7.40 (m, 4H), 9.68 (s, 1H), 10.11 (br s, 1H). Anal.
(C22H25ClN2O2‚1.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-[2-(p h en ylm et h oxy)-
p h en yl]-1-p ip er id in ep en ta n en itr ile (6bk ) was prepared in
a manner similar to 6bh starting with 2-(phenylmethoxy)-
phenylacetonitrile (4.47 g, 20 mmol) to afford 6bk as a white
solid (830 mg, 9% yield). H NMR (300 MHz, CDCl3) δ (TMS)
1.6 (m, 6H), 2.01 (m, 4H), 2.18 (m, 4H), 2.72 (t, 2H), 4.35 (t,
1
4-(4-Ch lor op h en yl)-r-(2-flu or op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6be) was
prepared in a manner similar to 6bh starting with 2-fluo-
rophenylacetonitrile (400 mg, 3.0 mmol) to afford 6be as a
1H), 5.10 (s, 2H), 6.98 (m, 2H), 7.40 (m, 11H). Anal. (C29H31
ClN2O2) C, H, N, Cl.
-
1
white solid (280 mg, 22% yield). H NMR (300 MHz, DMSO-
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-[3-(p h en ylm et h oxy)-
p h en yl]-1-p ip er id in ep en ta n en itr ile (6bl) was prepared in
a manner similar to 6bh starting with 3-(phenylmethoxy)-
phenylacetonitrile (4.47 g, 20 mmol) to afford 6bl as a white
solid (2.08 g, 20% yield). 1H NMR (300 MHz, DMSO) δ 1.8 (m,
3H), 2.0 (m, 3H) 2.2 (t, 2H), 3.18 (m, 4H), 3.4 (m, 2H), 4.23 (t,
1H), 5.10 (s, 2H), 5.60 (s, 1H), 6.92 (d, 2H), 7.05 (s, 1H), 7.40
(m, 10H), 10.4 (br s, 1H). Anal. (C29H31ClN2O2‚1.0HCl) C, H,
N, Cl.
d6) δ 1.95 (m, 6H), 2.38 (m, 2H), 3.18 (m, 4H), 3.40 (m, 2H),
4.45 (m, 1H), 5.60 (s, 1H), 7.28 (m, 2H), 7.45 (m, 6H), 10.50
(br s, 1H). Anal. (C22H24ClFN2O‚1.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-r-(4-flu or op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bf) was
prepared in a manner similar to 6bh starting with 4-fluo-
rophenylacetonitrile (400 mg, 3.0 mmol) to afford 6bf as a
1
white solid (190 mg, 15% yield). H NMR (300 MHz, DMSO-
d6) δ 1.95 (m, 6H), 2.38 (m, 2H), 3.18 (m, 4H), 3.40 (m, 2H),
4.38 (m, 1H), 5.60 (s, 1H), 7.25 (m, 2H), 7.45 (m, 6H), 10.45
(br s, 1H). Anal. (C22H24ClFN2O‚1.0HCl) C, H, N, Cl.
r-(2-Br om op h en yl)-4-(4-ch lor op h en yl)-4-h yd r oxy-1-p i-
p er id in ep en ta n en itr ile, h yd r oiod id e sa lt (6bg) was pre-
pared in a manner similar to 6bh starting with 2-bromophen-
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-[4-(p h en ylm et h oxy)-
p h en yl]-1-p ip er id in ep en ta n en itr ile (6bm ) was prepared
from 6bp (147 mg, 0.35 mmol) with benzyl bromide (81 µL)
and cesium carbonate (340 mg) in anhydrous DMF (2 mL) to
afford 6bm as a white solid (59 mg, 33% yield). H NMR (300
MHz, DMSO-d6) δ 1.83 (m, 6H), 2.33 (m, 2H), 3.14 (m, 4H),
1

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4691
3.40 (m, 2H), 4.23 (m, 1H), 5.10 (s, 2H), 5.58 (s, 1H), 7.06 (d,
1H), 7.35 (m, 11H), 10.27 (br s, 1H). Anal. (C29H31ClN2O2‚
1.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(3-t h ien yl)-1-p ip er -
id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bv) was pre-
pared in a manner similar to 6bh starting with 3-thienyl-
acetonitrile (370 mg, 3.0 mmol) to afford 6bv as a gum (80
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(3-p h en oxyp h en yl)-1-
p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bn ) was
prepared in a manner similar to 6bh starting with 3-phenox-
yphenylacetonitrile (950 mg, 4.5 mmol) to afford 6bn as a
1
mg, 6% yield). H NMR (300 MHz, DMSO-d6) δ 1.80 (m, 4H),
1.95 (m, 2H), 2.30 (m, 2H), 3.10 (m, 4H), 3.40 (m, 2H), 4.40 (t,
1H), 5.60 (s, 1H), 7.15 (m, 1H), 7.40 (m, 4H), 7.58 (s, 1H), 7.65
(m, 1H), 10.10 (br s, 1H). Anal. (C20H23ClN2OS‚1.0HCl‚
0.3ether) C, H, N, Cl.
1
white solid (270 mg, 12% yield). H NMR (300 MHz, DMSO)
δ 1.82 (m, 6H), 2.40 (t, 2H), 3.15 (m, 4H), 3.40 (m, 2H), 4.32
(t, 1H), 5.58 (s, 1H), 6.95 (m, 1H), 7.08 (m, 3H), 7.19 (m, 2H),
7.40 (m, 7H) 10.29 (br s, 1H). Anal. (C28H29ClN2O2‚1.0HCl) C,
H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(1-n a p h th a len yl)-1-p i-
p er id in ep en ta n en itr ile (6bw ) was prepared in a manner
similar to 6bh starting with 1-naphthalenylacetonitrile (360
mg, 3.0 mmol) to afford 6bw as a off-white solid (170 mg, 12%
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(2-h yd r oxyp h en yl)-1-
p ip er id in ep en ta n en itr ile, tr iflu or oa cetic a cid sa lt (6bo)
was prepared from 6bk by (475 mg, 1 mmol) by catalytic
hydrogenation using 10% Pd/C and purified by preparative
HPLC to afford 6bo as a white solid (81 mg, 15% yield). 1H
NMR (300 MHz, DMSO-d6) δ 1.81 (m, 6H), 2.15 (m, 2H), 3.20
(M, 4H), 3.42 (m, 2H), 4.34, (m, 1H), 5.40 (s, 1H), 6.92 (m, 2H),
7.19 (m, 1H), 7.25 (d, 1H), 7.44 (s, 4H), 9.10 (br s, 1H), 10.08
(s, 1H). Anal. (C22H25ClN2O2‚1.25TFA‚0.5H2O) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(4-h yd r oxyp h en yl)-1-
p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bp ) was
prepared in a manner similar to 6bk starting with 4-hydrox-
yphenylacetonitrile (4.93 mg, 37 mmol) which was protected
using tert-butyldimethylsilyl chloride.33 The protecting group
was removed with TBAF to afford 6bp as a white solid (450
mg, 3% yield). 1H NMR (300 MHz, DMSO) δ 1.80 (m, 6H), 2.38
(t, 2H), 3.20 (m, 4H), 3.41 (m, 2H), 4.20 (t, 1H), 5.58 (s, 1H),
6.80 (d, 2H), 7.20 (d, 2H), 7.42 (m, 4H), 9.62 (s, 1H), 10.21 (br
s, 1H). Anal. (C22H25ClN2O2‚1.0HCl) C, H, N, Cl.
1
yield). H NMR (300 MHz, DMSO-d6) δ 1.75 (m, 2H), 2.0 (m,
4H), 2.35 (m, 2H), 3.20 (m, 4H), 3.40 (m, 2H), 5.10 (t, 1H),
5.60 (s, 1H), 7.05 (m, 1H), 7.40 (m, 4H), 7.60 (m, 4H), 8.00 (m,
4H), 8.20 (d, 2H), 10.30 (br s, 1H). Anal. (C26H27ClN2O‚1.0HCl)
C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(2-p yr id in yl)-1-p ip er -
id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bx) was pre-
pared in a manner similar to 6bh starting with 2-pyridinyl-
acetonitrile (360 mg, 3.0 mmol) to afford 6bx as an off-white
solid (170 mg, 12% yield). 1H NMR (300 MHz, DMSO-d6) δ
1.80 (m, 4H), 2.0 (m, 2H), 2.38 (m, 2H), 3.10 (m, 4H), 3.40 (m,
2H), 4.50 (t, 1H), 7.45 (m, 6H), 7.90 (m, 1H), 8.40 (d, 2H), 10.40
(br s, 1H). Anal. (C21H24ClN3O‚1.5HCl) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(3-p yr id in yl)-1-p ip er -
id in ep en ta n en itr ile, d ih yd r och lor id e sa lt (6by) was pre-
pared in a manner similar to 6bh starting with 3-pyridinyl-
acetonitrile (360 mg, 3.0 mmol) to afford 6by as an off-white
solid (170 mg, 12% yield). 1H NMR (300 MHz, DMSO-d6) δ
1.80 (m, 4H), 2.0 (m, 2H), 2.40 (m, 2H), 3.20 (m, 4H), 3.40 (m,
2H), 4.60 (t, 1H), 7.45 (m, 4H), 7.80 (m, 1H), 8.30 (d, 1H), 8.70
(d, 1H), 8.90 (d, 1H), 10.80 (br s, 1H). Anal. (C21H24ClN3O‚
2.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(1-m eth yl-1H-p yr r ol-
2-yl)-1-piper idin epen tan en itr ile, h ydr och lor ide salt (6bz)
was prepared in a manner similar to 6bh starting with (1-
methyl-1H-pyrrol-2-yl)acetonitrile (360 mg, 3.0 mmol) to afford
6bz as a off-white solid (170 mg, 12% yield). 1H NMR (300
MHz, DMSO-d6) δ 1.80 (m, 4H), 2.0 (m, 2H), 2.38 (m, 2H), 3.20
(m, 4H), 3.35 (m, 2H), 3.60 (s, 3H), 5.60 (s, 1H), 5.95 (m, 1H),
6.10 (s, 1H), 6.78 (s, 1H), 7.45 (m, 4H), 10.40 (br s, 1H). Anal.
(C21H26ClN3O‚1.0HCl‚1.0tBuOH) C, H, N, Cl.
4-(4-Ch lor op h e n yl)-4-h yd r oxy-r-(3,4,5-t r im e t h oxy-
p h en yl)-1-p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt
(6bq) was prepared in a manner similar to 6bh starting with
3,4,5-trimethoxyphenylacetonitrile (1.04 g, 5 mmol) to afford
1
6bq as a white solid (290 mg, 12% yield). H NMR (300 MHz,
DMSO) δ 1.80 (m, 4H), 1.98 (m, 2H), 2.30 (m, 2H), 3.20 (m,
4H), 3.40 (m, 2H), 3.65 (s, 3H), 3.80 (s, 6H), 4.20 (t, 1H), 5.60
(s, 1H), 6.78 (s, 2H) 7.42 (m, 4H), 10.20 (br s, 1H). Anal. (C25H31
-
ClN2O4‚1.0HCl) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(3-n itr op h en yl)-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6br ) was
prepared in a manner similar to 6bh starting with 3-nitro-
phenylacetonitrile (1.62 mg, 10 mmol) to afford 6br as a tan
solid (250 mg, 6% yield). 1H NMR (300 MHz, DMSO-d6) δ 1.77
(m, 4H), 2.01 (m, 2H), 2.30 (m, 2H), 3.18 (m, 4H), 3.38 (m,
2H), 4.60 (m, 1H), 5.58 (s, 1H), 7.44 (m, 4H), 7.58 (t, 1H), 7.93
(d, 1H), 8.24 (d, 1H), 8.31 (s, 1H), 10.13 (br s, 1H). Anal.
(C22H24ClN3O3‚1.0HCl) C, H, N.
4-(4-Ch lor op h en yl)-r-cya n o-4-h yd r oxy-r-p h en yl-1-p i-
p er id in ep en ta n oic a cid , eth yl ester , h yd r och lor id e (6cc)
was prepared in the same manner as 6ch starting with ethyl
phenylcyanoacetate (2.0 g, 10.5 mmol) to afford 6cc as a white
1
solid (2.2 g, 44% yield). H NMR (300 MHz, CDCl3) δ 1.22 (t,
3H), 2.00 (m, 4H), 2.25 (m, 2H), 2.55 (m, 3H), 3.20 (m, 2H),
3.37 (m, 1H), 3.61 (m, 2H), 4.15 (M, 2H), 7.4 (m, 9H). Anal.
(C25H29ClN2O3‚1.0HCl) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(4-m eth oxyp h en yl)-1-
p ip er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bs) was
prepared from 6bp (180 mg, 0.47 mmol) and methyl iodide to
1
afford 6bs as a white solid (40 mg, 19% yield). H NMR (300
4-(4-Ch lor op h en yl)-r-cya n o-4-h yd r oxy-r-p h en yl-1-p i-
p er id in ep en ta n oic Acid , Meth yl Ester , Hyd r och lor id e
Sa lt (6cd ). To a solution of benzyl cyanide (11.8 g, 100 mmol)
and dimethyl carbonate (45.0 g, 500 mmol) in toluene (80 mL)
at 0 °C was added sodium methoxide (5.40 g, 54.0 mmol). The
resulting slurry was stirred at ambient temperature for 30 min
and then warmed to 80 °C for 20 min. As the solution cooled
to ambient temperature a precipitate formed. The solid was
isolated by filtration to give benzeneacetic acid, R-cyano methyl
ester, as its sodium salt. This compound (1.0 g, 5.1 mmol) was
treated as described in the preparation of 6bh to afford 6cd
as a white solid (170 mg, 37% yield). 1H NMR (300 MHz,
DMSO) δ 1.78 (m, 4H), 2.25 (m, 3H), 2.50 (m, 2H), 3.20 (m,
MHz, DMSO) δ 1.80 (m, 6H), 2.31 (t, 2H), 3.20 (m, 4H), 3.40
(m, 2H), 3.80 (m, 3H), 4.22 (t, 1H), 5.58 (s, 1H) 7.01 (d, 2H)
7.38 (d, 2H) 7.48 (m, 4H), 10.08 (br s, 1H). Anal. (C23H27ClN2O2‚
1.0HCl‚0.25H2O) C, H, N, Cl.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(4-eth oxyp h en yl)-1-p i-
p er id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bt) was
prepared in a manner similar to 6bs starting with 6bp (147
mg, 0.35 mmol) and ethyl iodide to afford 6bt as a white solid
(46 mg, 28% yield). 1H NMR (300 MHz, DMSO) δ 1.25 (m, 3H),
1.81 (m, 6H), 2.30 (t, 2H), 3.18 (m, 4H), 4.0 (q, 2H), 4.25 (t,
1H), 5.60 (s, 1H), 6.95 (d, 2H), 7.38 (d, 2H), 7.49 (m, 4H), 10.20
(br s, 1H). Anal. (C24H29ClN2O2‚1.0HCl‚0.25H2O) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-(2-t h ien yl)-1-p ip er -
id in ep en ta n en itr ile, h yd r och lor id e sa lt (6bu ) was pre-
pared in a manner similar to 6bh starting with 2-thienyl-
acetonitrile (370 mg, 3.0 mmol) to afford 6bu as a tan solid
(140 mg, 10% yield). 1H NMR (300 MHz, DMSO-d6) δ 1.80 (m,
4H), 2.0 (m, 2H), 2.38 (m, 2H), 3.20 (m, 4H), 3.40 (m, 2H), 4.75
(t, 1H), 5.40 (s, 1H), 7.05 (m, 1H), 7.20 (d, 1H), 7.45 (m, 4H),
7.58 (d, 2H), 10.40 (br s, 1H). Anal. (C20H23ClN2OS‚1.0HCl‚
0.6C4H10O) C, H, N.
4H), 3.40 (br t, 2H), 3.78 (s, 3H), 7.49 (m, 9H). Anal. (C24H27
-
ClN2O3‚1.0HCl) C, H, N.
4-(4-Ch lor op h en yl)-r-(3-ch lor op r op yl)-4-h yd r oxy-r-
p h en yl-1-p ip er id in ep en ta n en itr ile (6ce). A solution of
LDA (2.0 mL, 1.5 N in cyclohexane, 3.0 mmol) was added by
syringe over a 5 min period to a stirred solution of benzylcya-
nide (0.35 g, 3.0 mmol) and 1-chloro-3-iodopropane (0.64 mL,
6.0 mmol) in anhydrous THF (12.0 mL) under nitrogen cooled
in a dry ice/acetone bath (EXOTHERMIC!). After 5 min the

4692 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Ng et al.
bath was removed and the mixture was allowed to warm to
ambient temperature. The mixture was again cooled in a dry
ice/acetone bath and additional LDA (2.0 mL, 1.5 N in
cyclohexane, 3.0 mmol) was added by syringe over a 5 min
period. After 5 min the bath was removed. After being stirred
overnight, the mixture was partitioned between ethyl ether
(100 mL) and 1 N HCl (50 mL). The ether layer was washed
sequentially with 1 N HCl (50 mL) and brine (25 mL), then
dried over magnesium sulfate, filtered, and concentrated in
vacuo. The residue (0.8 g) was dissolved in isooctane (20 mL)
and concentrated in vacuo. Chromatography on silica gel (15
g) with a gradient of 30-70% methylene chloride in hexane
afforded 0.24 g of an oil which was mostly R,R-bis(3-chloro-
propyl)benzylcyanide. 1H NMR (300 MHz, CDCl3) δ (TMS) 1.59
(m, 2H), 1.96 (m, 2H), 2.14 (m, 4H), 3.48 (t, 4H), 7.4 (m, 4H).
This oil was dissolved in anhydrous DMF (3 mL) and 4-(4-
chlorophenyl)-4-hydroxypiperidine (0.56 g, 2.66 mmol) and NaI
(27 mg, 0.18 mmol) were added. The mixture was heated
overnight at 50 °C then partitioned between ethyl ether (75
mL) and 5% aqueous K2CO3 (25 mL). The ethyl ether layer
was washed successively with 25 mL portions of water (3×)
and brine, then dried over magnesium sulfate, filtered, and
concentrated in vacuo. TLC (4:1:1:1 dichloromethane:ethyl
acetate:2-propanol:methanol visualized with I2) showed three
components at Rf ) 0.78, 0.67, and 0.16. The first of these was
isolated by chromatography over silica gel (15 g) with a
gradient of 0-25% 2-propanol in 4:1 methylene chloride:ethyl
acetate to obtain 0.12 g of 6ce as an oil. This oil was dissolved
in methanol (5 mL) and concentrated HCl (0.15 mL), concen-
trated in vacuo and crystallized from 2-propanol/ethyl ether.
The resulting precipitate was collected by filtration, rinsed
with ethyl ether (2 × 5 mL), and dried in vacuo to obtain 6ce
as a white solid (70 mg, 4% yield). 1H NMR (300 MHz, DMSO-
d6) δ 1.41 (m, 2H), 1.79 (m, 2H), 2.12 (m, 4H), 2. 29 (t, 2H),
3.10 (m, 4H), 3.30 (m, 2H), 3.61 (t, 2H), 5.55 (s, 1H), 7.4 (m,
9H), 10.2 (br s, 1H). Anal. (C25H30Cl2N2O‚1.0HCl) C, H, N, Cl.
(250 mg, 14% yield). 1H NMR (300 MHz, DMSO) δ 1.50 (m,
3H), 1.80 (m, 2H), 2.16 (m, 2H), 2.28 (m, 4H), 2.50 (m, 2H),
3.35 (s, 1H), 3.58 (m, 2H) 4.82 (s, 1H), 7.25 (d, 2H), 7.52 (m,
7H). Anal. (C24H26ClN3O) C, H, N.
4-(4-Ch lor op h en yl)-r-cycloh exyl-4-h yd r oxy-r-p h en yl-
1-p ip er id in ep en ta n en itr ile (6ci) was prepared in a manner
similar to 6bh starting with cyclohexylphenylacetonitrile (1.0
g, 5.0 mmol) to afford 6ci as a foam (160 mg, 7% yield). 1H
NMR (300 MHz, CDCl3) δ (TMS) 1.20 (m, 8H), 1.75 (m, 7H),
2.20 (m, 9H), 2.69 (t, 2H), 7.35 (m, 9H). Anal. (C28H35ClN2O‚
0.4H2O) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-p h en yl-r-(1-p ip er id i-
n yl)-1-p ip er id in ep en t a n en it r ile, d ih yd r och lor id e sa lt
(6cj) was prepared in a manner similar to 6bh starting with
1-(R-cyanobenzyl)piperidine (1.0 g, 5 mmol) to afford 6cj as a
1
white solid (30 mg, 1.1% yield). H NMR (300 MHz, CDCl3) δ
(TMS) 1.6 (m, 9H), 1.8-2.4 (m, 14H), 3.25 (m, 1H), 4.18 (m,
1H), 7.39 (m, 14H). Anal. (C27H34ClN3O‚2.0HCl) C, H, N.
4-(4-Ch lor op h en yl)-4-h yd r oxy-r,r-d ip h en yl-1-p ip er -
id in ep en ta n oic a cid , m eth yl ester (6ck ) was prepared in
a similar manner as 6s starting with diphenylacetic acid (600
mg, 2.65 mmol) to afford 6ck as a white solid (210 mg, 17%
1
yield). H NMR (300 MHz, CDCl3) δ (TMS) 1.32 (m, 2H), 1.65
(d, 4H), 2.08 (m, 2H), 2.38 (m, 6H), 3.70 (s, 3H), 7.30 (m, 12H),
7.41 (m, 2H). Anal. (C29H32ClNO3‚0.33SiO2) C, H, N.
1-(5-Am in o-4,4-d ip h en ylp en tyl)-4-(4-ch lor op h en yl)-4-
p ip er id in ol, Bistr iflu or a cetic Acid Sa lt (6cl). LAH (1.7 g,
45 mmol) was slurried in anhydrous THF (200 mL), and the
solution was cooled in an ice bath to 10 °C under nitrogen.
AlCl3 (6 g, 45 mmol) was dissolved in anhydrous THF (200
mL) (CAUTION: EXOTHERMIC!). The AlCl3 solution was
added to the LAH slurry via an addition funnel over 15 min.
Compound 6s (10 g, 22.5 mmol) was dissolved in anhydrous
THF (100 mL). This solution was added to the LAH/AlCl3
mixture at a rate that maintained the internal temperature
at >25 °C. The resulting mixture was stirred at ambient
temperature for 1 h and then heated to reflux for 8 h. The
reaction was cooled to 0 °C and quenched (CAUTION: EXO-
THERMIC! H2 evolution!) with water (4 mL), 15% aqueous
NaOH (4 mL), and water (12 mL) then filtered, and the filtrate
was concentrated in vacuo to a residue. The residue was
dissolved in methylene chloride and washed with NaHCO3
(saturated) and then brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo to a foam. The foam was
crystallized from 1:15:15, methanol:ether:petroleum ether to
afford 7 as a white solid (6.4 g, 63%). The foam was further
4-(4-Ch lor op h en yl)-4-h yd r oxy-r-m eth yl-r-p h en yl-1-p i-
p er id in ep en ta n en itr ile (6cf) was prepared in a manner
similar to 6bh starting with methylphenylacetonitrile (600 mg,
4.6 mmol) to afford 6bf as a foam (480 mg, 27% yield). 1H NMR
(300 MHz, CDCl3) δ (TMS) 1.78 (m, 8H), 2.05 (m, 4H), 2.38
(m, 4H), 2.75 (m, 2H), 7.39 (m, 9H). Anal. (C23H27ClN2O) C,
H, N.
4-(4-Ch lor op h en yl)-r-et h yl-4-h yd r oxy-r-p h en yl-1-p i-
p er id in ep en ta n en itr ile (6cg) was prepared in a manner
similar to 6bh starting with ethylphenylacetonitrile (1.0 g, 6.9
1
1
mmol) to afford 6cg as a foam (430 mg, 16% yield). H NMR
purified by preparative HPLC to give the TFA salt. H NMR
(300 MHz, CDCl3) δ (TMS) 0.95 (t, 3H), 1.35 (m, 1H), 1.78 (m,
3H), 2.10 (m, 6H), 2.37 (m, 4H), 2.70 (m, 2H), 7.38 (m, 9H).
Anal. (C24H29ClN2O‚0.2H2O) C, H, N.
(300 MHz, DMSO-d6) δ (TMS) 1.1 (m, 2H), 1.48 (m, 2H), 1.82
(m, 2H), 2.25 (m, 6H), 2.4 (m, 2H), 3.18 (m, 2H), 7.25 (m, 14H).
Anal. (C28H33 ClN2O‚2.25TFA‚0.5H2O) C, H, N, Cl, F.
2-[3-[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]p r o-
p yl]-2-p h en ylbu ta n ed in itr ile (6ch ). A solution of phenyl-
succinylnitrile (1.56 g, 10 mmol) in anhydrous DMF (20 mL)
was added dropwise to a 0 °C solution of sodium hydride (420
mg, 10.5 mmol) in anhydrous DMF under nitrogen. The
mixture was stirred at 0 °C for 1 h then 3-chloro-1-iodopropane
(1.18 mL, 11 mmol) was added, and the mixture was stirred
for 16 h at ambient temperature. The reaction mixture was
poured into water (150 mL), and the chloropropyl intermediate
was extracted into ethyl acetate (3 × 50 mL). The combined
organic extracts were washed with water and then brine, dried
over magnesium sulfate, filtered, and evaporated in vacuo to
an oil which was purified by flash chromatography (10-40%
ethyl acetate in hexanes). The purified chloropropyl intermedi-
ate, (1.02 g, 44% yield) was dissolved in anhydrous DMF (25
mL) along with diisopropylethylamine (1.13 g, 8.7 mmol)
sodium iodide (10 mg) and 5s (0.92 g, 4.38 mmol). The reaction
mixture was heated to 60 °C for 2 h then to 90 °C for 12 h.
The reaction was quenched by pouring into water (150 mL)
and extracting with ethyl acetate (2 × 100 mL). The combined
organic extracts were washed with water and then brine, dried
over magnesium sulfate, filtered, and evaporated to an oil
which was purified by flash chromatography (0-100% ethyl
acetate in methylene chloride) to afford 6ch as a white solid
4-(4-Ch lor op h en yl)-4-h yd r oxy-1-[5-(1,5-cycloh exa d ien -
1-yl)-5-p h en ylp en tyl]p ip er id in e (8). To a solution of (3-
benzyloxypropyl)triphenylphosphonium bromide (4.9 g, 10
mmol) in anhydrous THF (60 mL) under nitrogen was added
potassium tert-butoxide (1.34 g, 11 mmol). The solution was
heated to 70 °C for 3 h and then cooled to ambient tempera-
ture. Benzophenone (1.82 g, 10 mmol) was then introduced,
and the reaction solution was reheated to 70 °C for 6 h. After
cooling to room temperature, the reaction was quenched by
pouring into water (200 mL). The product was extracted into
ethyl acetate (2 × 200 mL). The combined organic extracts
were washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo to give a solid which was
purified by flash chromatography (ethyl acetate/hexanes) to
give an amber oil (2.17 g). This oil was dissolved in ethanol
(25 mL), treated with 10% Pd/C (200 mg), and hydrogenated
at 50 psi to give 4,4-diphenylbutan-1-ol as an oil (1.31 g). The
alcohol was dissolved in methylene chloride (50 mL) with
triethylamine (0.41 g, 4.0 mmol) and chilled to -5 °C. Meth-
anesulfonyl chloride (0.46 g, 4.0 mmol) was added dropwise
over 10 min. The reaction mixture was stirred at 0 °C for 30
min and then at ambient temperature for 2 h, poured into a
separatory funnel, and washed with water (50 mL) and brine
(50 mL), then dried over magnesium sulfate, filtered, and

Discovery of Non-Peptide CCR1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4693
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Rossi, D.; et al. A new class of membrane-bound chemokine with
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concentrated to an oil (1.32 g). This oil was dissolved in DMF
and treated with diisopropyl ethylamine and 4-(4-chlorophen-
yl)-R-hydroxy piperidine to afford 8 as an offwhite solid (350
mg, 8% yield). 1H NMR (300 MHz, CDCl3) δ (TMS) 1.5 (m,
2H), 1.72 (d, 2H), 2.12 (m, 4H), 2.40 (m, 4H), 2.78 (br d, 2H),
6.60 (t, 1H), 7.18 (m, 2H), 7.31 (m, 10H), 7.42 (d, 2H). Anal.
(C27H30ClNO‚0.25H2O) C, H, N.
Biologica l Meth od s. Radioactivity in binding studies was
determined on a Topcount scintillation counter (Packard
Instruments). Unlabeled chemokines were from Peprotech
(Rocky Hill, NJ ). 125I-Labeled chemokines were from NEN Life
Science Products.
Ch em ok in e Bin d in g Stu d ies. The binding assays were
performed by filtration methods. HEK 293 cells expressing the
human CCR1 receptor (KD ) 1-3 nM and Bmax ) (2-3) × 106
sites/cell) were used as the source of the CCR1 receptor. HEK
293 cells expressing the CCR1 receptor were detached by
shaking, washed once in PBS, and resuspended in the assay
buffer (130 mM NaCl, 5 mM KCl, 1 mM MnCl2, 50 mM Tris,
30 µg/mL bacitracin, 0.1% BSA, pH 7.4) to about 1.1 × 105
cells/mL. Cells (approximately 8000 cells/assay) were incu-
bated with 125I-MIP-1R, specific activity 2200 Ci/mmol, (ap-
proximately 15-20000 cpm/assay), in the presence and ab-
sence of varying concentrations of compounds at room
temperature for 30-40 min. The reactions were carried out
in 96-well v-bottom microtiter plates (Nunc, polypropylene) in
a total volume of 100 µL and terminated by harvesting through
a GF/B filter plate (Packard) presoaked with 0.3% PEI (Sigma
#P-3143) plus 0.5% BSA and washed five times with cold PBS.
The radioactivity in each well was determined by scintillation
counting in a Topcount (Packard) following addition of 50 µL
of scintillation fluid, Scint-20. The nonspecific binding was
defined by the binding in the presence of 100 nM of unlabeled
MIP-1R. The dose curves for each compound with six concen-
tration points were generated, and IC50 values were deter-
mined by fitting the data to the log-logit equation (linear) with
an EXCEL spreadsheet. The Ki values were then calculated
by dividing the IC50 by 1.025.
Mea su r em en t of In t r a cellu la r Ca lciu m Level Usin g
F LIP R (F lu or om etr ic Im a gin g P la te Rea d er ). HEK-293
cells that express human CCR1 receptor at high levels were
plated in the poly-D-lysine coated 96-well black wall plates
(Becton Dickinson and Company, Franklin Lakes, NJ ) at a
density of 80 000 cells/well. After a day in the culture, cells
were loaded at 37 °C for 1 h with calcium-sensitive fluorescence
dye, Fluo-3, in Hank’s buffered salt solution (without phenol
red) containing 20 mM Hepes, 3.2 mM CaCl2, 1% fetal bovine
serum, 2.5 mM probenecid, 0.04% pluronic acid, and 4 µM
Fluo-3. The excess extracellular dye was removed by gently
washing four times with Hank’s buffered salt solution contain-
ing 20 mM Hepes, 0.1% BSA, and 2.5 mM probenecid using
Denley cell washer. Cells were pretreated with compounds at
various concentrations at 37 °C for 10 min and then stimulated
with 30 nM MIP-1R (R&D systems). The change in fluores-
cence intensity was monitored over a 3 min time period at 37
°C using FLIPR. The dose curves of each compound with six
concentrations were generated, and the data was exported to
EXCEL file. IC50 values were calculated by fitting the data to
the log-logit equation (linear) with an EXCEL spreadsheet.
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