Discovery and lead optimization of a novel series of CC chemokine receptor 1 (CCR1)-selective piperidine antagonists via parallel synthesis

Article abstract of DOI:10.1021/jm300896d

A series of novel, potent CCR1 inhibitors was developed from a moderately active hit using an iterative parallel synthesis approach. The initial hit (composed of three subunits: an amine, a central amino acid, and an N-terminal cap) became the basis for a series of parallel chemical libraries designed to generate SAR data. Libraries were synthesized that explored each of the three subunits; the CCR1 binding data obtained revealed the following: (1) changes to the amine are not well tolerated; (2) small alkylamino acids are preferred in the center of the molecule; (3) substitutions at the N-terminus are generally well tolerated. These data were used to drive the optimization of the series, ultimately providing a lead with a CCR1 binding IC₅₀ of 28 nM (48). This lead demonstrates high selectivity for CCR1 over other CCR-family members, high microsomal stability, and good pharmacokinetics in mice.

Full text of DOI:10.1021/jm300896d

Article
pubs.acs.org/jmc
Discovery and Lead Optimization of a Novel Series of CC Chemokine
Receptor 1 (CCR1)-Selective Piperidine Antagonists via Parallel
Synthesis
Cullen L. Cavallaro,* Stephanie Briceno, Jing Chen, Mary Ellen Cvijic, Paul Davies, John Hynes, Jr.,
Rui-Qin Liu, Sandhya Mandlekar, Anne V. Rose, Andrew J. Tebben, Katy Van Kirk, Andrew Watson,
Hong Wu, Guchen Yang, and Percy H. Carter
Research and Development, Bristol-Myers Squibb Company, Route 206 and Provinceline Road, Princeton, New Jersey 08540, United
States
S
* Supporting Information
ABSTRACT: A series of novel, potent CCR1 inhibitors was developed from a moderately active hit using an iterative parallel
synthesis approach. The initial hit (composed of three subunits: an amine, a central amino acid, and an N-terminal cap) became
the basis for a series of parallel chemical libraries designed to generate SAR data. Libraries were synthesized that explored each of
the three subunits; the CCR1 binding data obtained revealed the following: (1) changes to the amine are not well tolerated; (2)
small alkylamino acids are preferred in the center of the molecule; (3) substitutions at the N-terminus are generally well
tolerated. These data were used to drive the optimization of the series, ultimately providing a lead with a CCR1 binding IC50 of
28 nM (48). This lead demonstrates high selectivity for CCR1 over other CCR-family members, high microsomal stability, and
good pharmacokinetics in mice.
mechanism was obtained with 2 in regards to blocking cellular
migration,13,14 the demonstration of clinical efficacy was not
achieved in a phase 2 trial in patients with rheumatoid arthritis.8
Likewise, 4 failed in its phase 2 rheumatoid arthritis trial.15
Failed phase 2 trials have also been reported for 1 in multiple
sclerosis16 and 3 in chronic obstructive pulmonary disease.17
Although these phase 2 clinical experiences have been
disappointing, it remains clear that CCR1 plays a role in the
migration of cells involved in the pathogenesis of human
rheumatoid arthritis.13,18 Indeed, a number of authors have
suggested that the problems with previous trials may stem from
the fact that earlier compounds did not achieve complete
blockade of CCR1 for the course of the trial.4,18 On the basis of
this hypothesis, Chemocentryx and GSK have continued to
advance a compound19 in phase 2 trials20,21 and recently
INTRODUCTION
■
CC Chemokine receptor-1 (CCR1), a family A G protein-
coupled receptor,1 is expressed on the surface of monocytes/
macrophages, osteoclasts, T-cells, and neutrophils. It serves as a
receptor for 11 different chemokines, including MIP-1α,
RANTES, and leukotactin-1. Through its interactions with
these ligands, CCR1 helps mediate leukocyte activation and
migration, processes that are critical for the progression of
inflammatory diseases.
As with many other chemokine receptors,2−4 extensive
preclinical studies in mice have suggested that CCR1 should
represent a viable target for the development of therapies for
inflammatory disorders.5 Accordingly, the drug discovery
community has expended a considerable amount of effort in
identifying small molecule antagonists of CCR1.6,7 Indeed, a
number of candidates have been advanced to clinical trials
(Figure 1).8 The first four candidates with publically disclosed
clinical efficacy data were 1 (BX-471),9 2 (CP-481715),10
3
Received: June 25, 2012
Published: October 17, 2012
(AZD-4818),11 and 4 (MLN-3897).12 Although proof-of-
© 2012 American Chemical Society
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Figure 1. Structures of CCR1 antagonists with reported data from phase 2 clinical trials.
Figure 2. Screening hit 5 and its elimination product 6.
disclosed favorable results from a 12-week phase 2 study in
patients with rheumatoid arthritis.22
Scheme 1. Synthesis of 4-(4-Chlorophenyl)-4-
hydroxypiperidine Derivatives
a
We had likewise been encouraged by the initial validation
data for CCR1, especially for the treatment of rheumatoid
arthritis, and set out to identify a potent, selective, and orally
bioavailable antagonist of this target. A screen of our
proprietary compound collection identified 5 as a moderately
potent CCR1 antagonist (Figure 2). Further evaluation of this
hit revealed it to be a mixture of compounds containing 5 and
its elimination product 6. The mixture had an IC50 of 332 nM
in the CCR1 binding assay. Testing of the two isolated
compounds showed that both were active against CCR1 but
that 6 (CCR1 binding IC50 = 370 nM) was more potent than 5
(IC50 = 2832 nM) and therefore likely responsible for most of
the activity of the original hit mixture. We were encouraged by
the potency of these compounds and their structural differ-
entiation from other CCR1 series (Figure 1).6 Accordingly,
they became the starting point for an iterative series of parallel
synthetic libraries designed to improve CCR1 potency, explore
structural tolerance, generate on-target SAR, and identify
compound-related profiling liabilities.
a
Reagents and conditions: (i) R2NHCH(R1)COOH, EDC, HOBt,
DIEA, DMF.
a
Scheme 2. Synthesis of Bz-D,L-valine Derivatives
a
Reagents and conditions: (i) R3R4NH, EDC, HOBt, DIEA, DMF.
CHEMISTRY
Scheme 3 for a list of side chains) with 4-(4-chlorophenyl)-
piperidine (9). These intermediates were deprotected with HCl
in dioxane to provide 11a−i which could then be coupled with
the appropriate reagents to provide the final products as shown
in Scheme 3. Amide derivatives 24−36 and 43−80 were
synthesized from the corresponding acids using EDC,
carbamates 37 and 38 were made from the carbamoyl chlorides,
ureas 40−42 were made by reaction with isocyanates in
dioxane, and sulfonamides (such as 39) were made by reaction
with sulfonyl chlorides (Scheme 3). Compounds 43 and 48
were made from L- and D-Boc-valine, respectively, by EDC
coupling with 9, followed by deprotection with HCl in dioxane
and EDC coupling with benzoic acid. This method was found
■
Libraries of potential inhibitors were synthesized by the general
methods provided in Schemes 1, 2, and 3. Libraries containing
compounds 12−16 were made by coupling 4-(4-chlorophen-
yl)-4-hydroxypiperidine (7) with various benzoyl- or Cbz-
protected amino acid derivatives as shown in Scheme 1;
screening hits 5 and 6 were also resynthesized by the same
method. Libraries containing compounds 17−23 were
synthesized from benzoyl-D,L-valine and a variety of amines
(see Table 2 for examples of the final products) as shown in
Scheme 2. For libraries containing compounds 24−80, the Boc-
protected intermediates (10a−i) were first made by EDC
coupling of the appropriate Boc-protected amino acid (see
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a
Scheme 3. Synthesis of 4-(4-Chlorophenyl)piperidine Derivatives
a
Reagents and conditions: (i) BocNHCH(R5)COOH, EDC, HOBt, DIEA, DMF; (ii) 4 M HCl in dioxane; (iii) R6COOH, EDC, HOBt, DIEA,
DMF; (iv) R6OC(O)Cl, DIEA, THF; (v) R6NCO, dioxane; (vi) R6SO2Cl, DIEA, THF, DCM. Side chains (R5) for intermediates 10 and 11 are
defined as follows: (a) n-Pr; (b) i-Pr; (c) i-Bu; (d) t-Bu; (e) (R)-Et; (f) (R)-i-Pr; (g) (S)-i-Pr; (h) (R)-CH2(Me)Et; (i) (R)-cyclohexyl.
to minimize racemization, which was observed in direct
couplings between enantiopure N-benzoyl-D-valine and 9.
drops in activity were observed when the N-terminal benzoyl
group was replaced with Cbz (cf., 12 versus 16) or acetyl (data
not shown).
RESULTS AND DISCUSSION
Efforts to replace the piperidine unit were more challenging,
and only minor changes were tolerated in that portion of the
molecule. Several libraries were synthesized from benzoyl-D,L-
valine to explore this area, and although more than 70 amine
replacements were investigated, the active compounds (Table
2) all closely resembled the original piperidine. Replacing the
chlorine in 12 with fluorine (17) resulted in a significant drop
in potency, but replacement with bromine resulted in an
equipotent compound (18). Removing the 4-hydroxyl group
from the piperidine led to a 3-fold improvement in potency
(21); the fluoro analogue 20 was also active but not as potent
as the chloro compound. The unsaturated piperidine 19 was
nearly equipotent to the hydroxypiperidine derivative 12. The
other tolerated change was the replacement of the piperidine
with a piperazine, and although this resulted in almost a 3-fold
loss in potency (22), some of the potency could be regained
with the addition of a second chlorine on the phenyl ring (23).
All attempts to replace the aryl halogens with hydrogen,
regardless of the nature of the heterocycle used, resulted in a
complete loss in CCR1 activity (data not shown). These results
are consistent with those published for other CCR1 chemo-
types in which a haloaromatic group appears to be necessary for
tight binding to the receptor.23
■
The initial libraries built using 4-(4-chlorophenyl)-4-hydrox-
ypiperidine (7) as a core revealed that a number of amino acid
substitutions could be made in the center of the molecule that
preserved CCR1 binding (Table 1). Although benzoyl-D,L-
valine was favored (12, 180 nM), other amino acids with alkyl
side chains also showed reasonable activity, for example in
compounds 6 and 14. Although less potent, n-propyl (13) and
(methylthio)ethyl (15) side chains were tolerated. Significant
Table 1. CCR1 Binding Data for 4-(4-Chlorophenyl)-4-
hydroxypiperidine Derivatives
compound
R1
R2
Bz
CCR1 IC50 (nM)
5
CH(OH)Me
=CHMe
i-Pr
2832
370
180
521
324
539
854
6
Bz
Bz
Bz
Bz
Bz
Cbz
12
13
14
15
16
Pr
4-(4-Chlorophenyl)piperidine (9) became the basis for a
library designed to generate additional SAR for the amino acid
portion of the molecule (Table 3). A set of amino acids with
small alkyl side chains was examined (racemic norvaline, valine,
i-Bu
CH2CH2SMe
i-Pr
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Table 2. CCR1 Binding Data for Benzoyl-D,L-valine
Derivatives
Table 4. Comparison of CCR1 Binding Data for a Set of 4-
(4-Chlorophenyl)piperidine Derivatives with Varying N-
Terminal Capping Groups
compound
X-R6
*
CCR1 IC50 (nM)
34
35
36
37
38
39
40
41
42
C(O)CH2Ph
D,L
D,L
D
40
35
C(O)CH2CH2Ph
C(O)CH2CH2CH2Ph
C(O)OPh
95
D,L
D,L
D,L
D
265
1077
303
616
180
543
C(O)OCH2Ph
SO2CH2Ph
C(O)NHPh
C(O)NHCH2Ph
C(O)NHCH2CH2Ph
D
D
amide analogues (34, 35, and 36). Alkyl and aryl amides
provided compounds with good potency against CCR1.
The synthesis of both benzoyl valine stereoisomers 43 and
48 revealed the preferred D-stereochemistry at the amino acid
position (Figure 3) and the synthesis of a two-dimensional D-
amino acid library (Table 5) reconfirmed earlier work in which
the valine analogues were found to be the most potent.
Isoleucine and aminobutyric acid analogues also showed very
good activity.
Table 3. CCR1 Binding Data for 4-(4-
Chlorophenyl)piperidine Derivatives
The data from the analogues listed in Table 5 led to the
synthesis of a series of libraries designed around a 4-(4-
chlorophenyl)piperidine-D-valine core, which produced a large
amount of SAR data for the N-terminal amides (Table 6).
Alkyl, aryl, and heteroaryl substituents all provided compounds
with acceptable CCR1 binding, with the alkyl series providing
the most active compound (61). Small changes to this molecule
affected binding negatively, but the resulting compounds still
showed CCR1 binding of less than 200 nM. For example,
changing the cyclohexyl ring in 61 to cyclopentyl (60) resulted
in a 2.5-fold loss in binding, and a switch to cyclopropyl (59)
resulted in about a 6-fold loss in binding; however, these
compounds were still 20 and 46 nM, respectively. In the
benzoic acid series, the binding seemed to stay in a tight range
between 14 nM and 32 nM for most of the derivatives shown
(48 and Table 6, 62−68), although there was a significant loss
in potency for the 4-fluoro, 2,3-dimethyl, and 2,6-dimethyl
analogues. The substituted phenylacetic acid analogues, in
general, were less potent than the unsubstituted parent 52
except for the 2-fluoro compound 69 which showed a 2.5-fold
improvement. In this series the addition of a 3-fluoro or 3-
chloro substituent led to a slight loss in binding from the
unsubstituted analogue 52 and more than a 9-fold loss when a
4-chloro or 4-methyl group was added. Activity was maintained
upon further chain extension (74−78). As was observed with
the phenylacetic acid analogues, 2-substitution also seemed to
be preferred in the phenethyl series (74 and 76) and led to
binding better than that of the unsubstituted analogue 55, in
addition to being superior to other substituted derivatives. Two
heterocyclic compounds were also made, and the binding for
benzothiazole derivative 79 was 18 nM; however, when the
compound
R5
R6
i-Pr
CCR1 IC50 (nM)
24
25
21
26
27
28
29
30
31
32
33
i-Pr
388
120
55
n-Pr
i-Pr
Ph
Ph
i-Bu
t-Bu
n-Pr
i-Pr
Ph
130
259
152
36
Ph
2-MePh
2-MePh
2-MePh
2-MePh
2-naphth
2-naphth
i-Bu
t-Bu
n-Pr
i-Pr
88
262
191
80
tert-butyl glycine, and leucine) in combination with a set of
acids to cap the N-terminus of the amino acid. The data from
the library revealed that the valine analogues had superior
potency in all cases (see 21, 29, and 33).
Additional libraries were synthesized to generate SAR data
for the N-terminus of the 4-(4-chlorophenyl)piperidine-valine
core (Table 4). It is of interest to note that capping the N-
terminus of this particular scaffold with the sulfonamide (39),
carbamates (37 and 38), or ureas (40, 41, and 42) that are
shown reduced CCR1 binding relative to the corresponding
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Figure 3. CCR1 binding data for the stereoisomers of 21.
isopropyl group, Tyr113 and Tyr114 from TM3, and Leu185
from EL2. Mutagenesis studies have shown Tyr113 and Tyr114
to be critical contacts for the CCR1 antagonist 1.26 In our own
studies, when tested against the CCR1 mutant receptors Y113A
and Y114A, the binding affinity of 48 was reduced 113-fold and
64-fold, respectively (data not shown). The terminal benzoyl
moiety falls into the third region, a less defined hydrophobic
surface between TM5 and TM6 open to solvent with key
contacts to Leu203 and Ile259 from TM5 and TM6,
respectively. Although the benzoyl group is packed against
TM5, the pocket is large enough to tolerate a variety of groups,
as was observed in the SAR described (Table 6). In contrast to
other CCR1 antagonists, no interaction is observed with the
conserved glutamic acid on TM7, Glu287.
The amino acids predicted to be in contact with 48 and their
counterparts in CCR2, CCR3, and CCR5, as well as the mouse,
rat, and rabbit CCR1 orthologues, are tabulated in Table 7.
Comparison of these residues provides some insight into the
selectivity of this compound against CCR2, CCR3, and CCR5.
The most substantial variation is found within the predicted
ligand binding site of CCR2 where the key changes are as
follows: Leu109 to phenylalanine (TM3), occluding the
chlorophenyl pocket; Leu203 to arginine (TM5), disrupting
the hydrophobic contact with the benzoyl moiety; contact with
the isopropyl moiety by replacement of Tyr114 with the more
polar histidine, and Leu185 to proline (EL2), altering the
conformation of this loop and resulting in the loss of a
hydrophobic interaction. The other CC receptors as well as the
rat and mouse CCR1 have more subtle changes but, with the
exception of rabbit CCR1, do show variability in position 185.
The retention of activity in human and rabbit CCR1 suggests
that Leu185 may be a critical interaction for the binding of 48
and a key determinant for specificity among the CC receptors.
Further mutagenesis studies will be required to assess the
contribution of this and additional residues to the binding of
this series of compounds.
Table 5. Comparison of CCR1 Binding Data in a Series of 4-
(4-Chlorophenyl)piperidine Derivatives with Varying
Central Amino Acids
compound
R5
R6
CCR1 IC50 (nM)
44
45
46
47
48
49
50
51
52
53
54
55
56
Et
i-Bu
i-Bu
i-Bu
Ph
152
32
i-Pr
(R)-C(Me)Et
34
Et
121
28
i-Pr
Ph
(R)-C(Me)Et
cyclohexyl
Et
Ph
45
Ph
121
50
CH2Ph
i-Pr
CH2Ph
30
(R)-C(Me)Et
Et
CH2Ph
38
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
85
i-Pr
40
(R)-C(Me)Et
66
benzothiazole was replaced with a benzothiophene (80), the
binding IC50 value rose to 58 nM.
Additional data were obtained for compound 48 and showed
that it was highly selective for CCR1, relative to CCR2, CCR3,
and CCR5 (Table 8). No inhibition was detected in the
cytochrome P450 (CYP) panel (3A4, 2C9, 2C19, and 2D6).
Compound 48 also showed reasonable stability to human,
mouse, and rat liver microsomes and exhibited a high species
selectivity, as it was potent against the human receptor, less
active against the rabbit, and inactive in the murine and rodent
assays. Species selectivity has been observed for other CCR1
chemotypes24 and is believed to result from a number of small
changes in the amino acid sequences of the receptors that cause
subtle changes to the binding site.25
To understand the origins of selectivity for this series, a
binding model of 48 in the orthosteric site of a CCR1
homology model built from a CXCR4 template (3OE9) was
constructed. The model shows three sites of interaction
corresponding to the three pharmacophoric elements of the
small molecules. The first is an aromatic box composed of
Phe89, Trp90, and Tyr93 from transmembrane region 2
(TM2), and Trp99 from extracellular loop 1 (EL1), and capped
by a conserved cysteine, Cys106, on EL2. As would be expected
from the SAR, in which the halophenyl moiety was found to be
essential for CCR1 affinity, it fits tightly into this box, forming
multiple contacts with these residues (Figure 4). The second
region is a set of hydrophobic interactions between the
Overall, the chemical libraries that were synthesized provided
significant SAR for all three areas of the initial lead molecule
(amine, amino acid, and N-terminal cap) and numerous
submicromolar CCR1 binders were discovered. Of these
compounds, 48 proved to be the most viable lead. This
compound was shown to be an antagonist of the CCR1 ligands
it was tested against and demonstrated good functional activity
in a cell migration assay (IC50 78 nM, Table 8).
Mouse pharmacokinetic values for 48 are listed in Table 9. A
clearance of 12.5 mL/min/kg, distribution volume of 4.8 L/kg,
AUC of 5933 nM·h, and a half-life of 6.9 h were observed for an
intravenous dose of 2.5 mg/kg. An orally administered dose of
5.2 mg/kg provided a Cmax of 3129 nM, AUC of 6640 nM·h,
half-life of 5.2 h, and a bioavailability of 52%.
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Table 6. CCR1 Binding Data for 4-(4-Chlorophenyl)piperidine D-Valine Amide Derivatives
revealed the following: (1) changes to the amine are not well
tolerated; (2) small alkylamino acids are preferred in the center
of the molecule, with valine providing the broadest and most
consistently active compounds tested; (3) alterations at the N-
terminus are often well tolerated. Optimization led to
CONCLUSIONS
■
A series of novel, potent CCR1 inhibitors was developed from
the moderately potent lead 6 using an iterative parallel
synthesis strategy. SAR data generated for the three sections
of the lead series (amine, amino acid, and N-terminal cap)
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Table 8. Additional Data for Compound 48
48
CCR1 binding IC50, nM (MIP-1α)
CCR1 Ca flux IC50, nM (MIP-1α)
CCR1 Ca flux IC50, nM (CK-8β)
CCR1 chemotaxis IC50 (MIP-1α), nM
CCR2 binding IC50, nM
28
15
85
5
a
a
b
78 10
>20 000
>30 000
>20 000
100
CCR3 binding IC50, nM
CCR5 binding IC50, nM
human LM (% remaining)
mouse LM (% remaining)
73
rat LM (% remaining)
83
mouse CCR1 binding IC50, nM (MIP-1α)
rabbit CCR1 binding IC50, nM (MIP-1α)
rat CCR1 binding IC50, nM (MIP-1α)
CYP IC50, nM (3A4, 2C9, 2C19, 2D6)
hERG patch clamp IC50, nM
>25 000
550 125
>30 000
>40 000
7000
c
solubility, pH 6.5 buffer (mg/mL)
0.001
a
b
c
Based on a single determination. n = 11 determinations. n = 5
determinations.
a
Table 9. Pharmacokinetic Data for Compound 48 in Mice
Figure 4. Extracellular view of the binding model of 48 (red tube) in
the orthosteric site of a homology model of CCR1. Contact residues
are shown as small sticks and labeled with a single letter amino acid
code and sequence position. Helices are denoted by TMx and
extracellular loop 2 labeled as EL2.
iv, 2.5 mg/kg
po, 5.2 mg/kg
CL (mL/min/kg)
Vss (L/kg)
Cmax (nM)
AUC (nM h)
T1/2 (h)
12.5
4.8
NA
NA
3100
6600
5.2
NA
5900
6.9
compound 48, which inhibited CCR1 binding with an IC50 of
28 nM, was a functional antagonist of CCR1-mediated cellular
migration, was selective against a variety of related CCR-family
members, and showed good oral bioavailability.
F%
NA
52
a
Vehicle: 10% DMA, 30% PEG400, 60% HCl. 48 was insoluble in the
dosing vehicle at 1 mg/mL; n = 3 mice.
EXPERIMENTAL SECTION
■
spectrometer or a Bruker Avance. Miniblocks were obtained from
Mettler-Toledo AutoChem (Columbia, MD). Reagents and solvents
were obtained from commercial suppliers and used without further
purification. Unless otherwise indicated, all biological assays were run
in duplicate.
General Procedure A for the Synthesis of Amide Libraries
from an Acid Core. A Miniblock XT was equipped with 16 × 100
mm glass reaction tubes. To each tube were added 120 μL of a 0.5 M
stock solution of 8 in a 1:3 mixture of DIEA/DMF, 75 μL of a 1 M
solution of HOBt in DMF, and 300 μL of a 0.25 M solution of EDC in
DMF. A different amine was then added to each well (300 μL of a 0.25
M solution of amine in DMF). The reactions were agitated overnight
on a platform shaker at room temperature. Heterogeneous reaction
mixtures were filtered, and the crude mixtures were purified by
preparative LC-MS without further workup.
Materials, Methods, and Equipment. Analytical LC-MS: data
for purity and compound identification were obtained with a Shimadzu
Discovery VP system equipped with a Waters ZQ mass spectrometer
using the following column and conditions: Waters SunFire C18
column (4.6 × 50 mm, 5 um particle size); 10 μL injection volume; 4
mL/min flow rate; 4 min gradient from 0 to 100% B, where A is 1:9
MeOH/H2O with 0.1% TFA, and B is 9:1 MeOH/H2O with 0.1%
TFA; UV detection at 220 nM; electrospray ionization (positive).
Unless otherwise indicated, all compounds tested were of at least 95%
purity when tested by this method. Preparative LC-MS: unless
otherwise indicated, all compounds were purified with a Shimadzu
Discovery VP preparative system using the following column and
conditions: Waters SunFire C18 OBD column (19 × 100 mm, 5 μm
particle size); 2 mL injection volume; 20 mL/min flow rate; 10 min
gradient from 20% to 100% B, where A is 1:9 MeOH/H2O with 0.1%
TFA, and B is 9:1 MeOH/H2O with 0.1% TFA; UV detection at 220
nM; mass triggered collection by electrospray ionization (positive). 1H
NMR: 400 MHz spectra were taken using a Bruker DRX-400
spectrometer; 500 MHz spectra were taken using a JEOL Eclipse
General Procedure B for the Synthesis of Amide Libraries
from an Amine Core. A Miniblock XT was equipped with 16 × 100
mm glass reaction tubes. To each tube were added a different acid
(300 μL of a 0.25 M solution of acid in DMF), 75 μL of a 1 M solution
of HOBt in DMF, and 300 μL of a 0.25 M solution of EDC in DMF. A
Table 7. Contact Residues from the CCR1 Model
receptor
CCR1
89
90
93
99
109
113
114
183
185
203
259
287
F
F
F
F
F
F
F
W
W
W
W
W
W
W
Y
Y
Y
Y
S
W
W
W
W
W
W
W
L
L
L
L
F
L
L
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
H
H
F
C
C
C
C
C
C
C
L
P
P
L
P
A
S
L
L
L
L
R
M
I
I
E
E
E
E
E
E
E
CCR1 rat
CCR1mouse
CCR1 rabbit
CCR2
V
V
L
I
CCR3
Y
Y
I
CCR5
L
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solution of 11b was then added to each well (300 μL of a 0.25 M
solution in 1:5 mixture of DIEA/DMF). The reactions were agitated
overnight on a platform shaker at room temperature. Heterogeneous
reaction mixtures were filtered, and the crude mixtures were purified
by preparative LC-MS without further workup.
General Procedure C for the Synthesis of Sulfonamide
Libraries. A Miniblock XT was equipped with 16 × 100 mm glass
reaction tubes. To each tube were added 75 μmol of a different
sulfonyl chloride, 50 μL of DIEA, 300 μL of DCM, and 300 μL of a 0.2
M solution of 11b in THF. The reactions were agitated overnight on a
platform shaker at room temperature. The reaction mixtures were
diluted with 1 mL of MeOH, and the crude mixtures were purified by
preparative LC-MS without further workup.
General Procedure D for the Synthesis of Carbamate
Libraries. A Miniblock XT was equipped with 16 × 100 mm glass
reaction tubes. To each tube was added 75 μmol of a different
carbamoyl chloride, 250 μL of a 0.2 M solution of 11f in THF, 250 μL
of THF, and 50 μL of DIEA. The reactions were agitated overnight on
a platform shaker at room temperature. The reaction mixtures were
diluted with 1 mL of MeOH, and the crude mixtures were purified by
preparative LC-MS without further workup.
General Procedure E for the Synthesis of Urea Libraries. A
Miniblock XT was equipped with 16 × 100 mm glass reaction tubes.
To each tube were added 500 μL of a 0.2 M solution of an isocyanate
in dioxane and 250 μL of a 0.2 M solution of 11f in dioxane. The
reactions were agitated overnight on a platform shaker at room
temperature. The reaction mixtures were diluted with 1 mL of MeOH,
and the crude mixtures were purified by preparative LC-MS without
further workup.
1.59 (m, 2H), 0.87−1.01 (m, 6H); LC-MS (ESI) 398.20 m/z (M +
H)+.
(R)-2-Amino-1-(4-(4-chlorophenyl)piperidin-1-yl)-3-methyl-
butan-1-one (11f, R5 = iPr). HOBt (1.1 equiv), EDC (1.1 equiv),
DIEA (3 equiv), and 9 (1.1 equiv) were added to a solution of Boc-D-
valine (1300 mg, 6.0 mmol) in chloroform (8 mL). The mixture was
stirred for 16 h at rt and then was washed with 2% aqueous HCl and
saturated aqueous NaHCO3. The organic portion was dried over
MgSO4, concentrated, and dried in vacuo to provide a pale yellow
gum, which was treated with 20 mL of 4 M HCl in dioxane. The
solution was stirred for 2 h at rt, concentrated, and dried in vacuo to
provide a waxy yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 7.34−
7.41 (m, 2H), 7.24−7.31 (m, 2H), 4.57 (br d, J = 7.93 Hz, 1H), 3.81−
3.94 (m, 1H), 3.08−3.20 (m, 1H), 2.78−2.91 (m, 1H), 2.62−2.75 (m,
1H), 1.76−1.94 (m, 3H), 1.36−1.60 (m, 2H), 0.96 (dd, J = 6.69, 11.64
Hz, 3H), 0.87 (dd, J = 6.94, 17.83 Hz, 3H); LC-MS (ESI) 295.15 (M
+ H)+.
(R)-N-(1-(4-(4-Chlorophenyl)piperidin-1-yl)-3-methyl-1-oxo-
butan-2-yl)benzamide (48). HOBt (1.1 equiv), DIEA (3 equiv),
and benzoic acid (1.1 equiv) were added to a solution of 11f (41 mg,
0.13 mmol) in 1 mL of DMF. After 5 min of stirring, EDC (1.1 equiv)
was added and the solution was stirred for 16 h. The solution was
1
purified by preparative LC-MS without any workup. H NMR (400
MHz, CDCl3) δ 7.82−7.92 (m, 2H), 7.44−7.59 (m, 3H), 7.26−7.37
(m, 2H), 7.15 (dd, J = 12.7, 8.6 Hz, 2H), 5.15 (ddd, J = 10.4, 8.6, 6.4
Hz, 1H), 4.75−4.88 (m, 1H), 4.25−4.37 (m, 1H), 3.27 (qd, J = 13.0,
2.3 Hz, 1H), 2.68−2.87 (m, 2H), 1.87−2.24 (m, 3H), 1.54−1.82 (m,
2H), 0.98−1.13 (m, 6H); LC-MS (ESI) 399.17 m/z (M + H)+.
CCR1 Binding Assay. For radioligand competition studies, a final
concentration of 1 × 105 THP-1 monocytic leukemia cells were
combined with 100 μg of LS WGA PS beads (Amersham cat. no.
RPNQ 0260) in 40 μL of assay buffer (RPMI 1640 without phenol
red, 50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, 0.1% BSA). The
THP-1 cell/bead mixture was added to each well of a 384-well assay
plate (PerkinElmer cat. no. 6007899) containing test compound in 3-
fold serial dilution, with final concentrations ranging from 8 μM to 140
pM. A final concentration of 0.1 nM [125I]-MIP-1α (PerkinElmer cat.
no. NEX298) in 20 μL of assay buffer was added to the reaction.
Sealed assay plates were incubated at room temperature for 12 h and
then analyzed by LEADseeker. Each experiment was run in duplicate.
The competition data of the test compound over a range of
concentrations was plotted as percentage inhibition of radioligand
specific bound in the absence of test compound (percent of total
signal). After correction for nonspecific binding, IC50 values were
determined. The IC50 value is defined as the concentration of test
compound needed to reduce radioligand specific binding by 50% and
is calculated using the four-parameter logistic equation to fit the
normalized data. Assay variability was 15% and was measured using a
standard control.
N-(1-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-3-hy-
droxy-1-oxobutan-2-yl)benzamide (5). This compound was
resynthesized by the method that follows: HOBt (1.25 equiv), EDC
(1.25 equiv), DIEA (3 equiv), and 7 (1.25 equiv) were added to a
solution of N-benzoyl-L-threonine (56 mg, 0.25 mmol) in 2 mL of 1:1
DCE/DMF. The mixture was stirred for 16 h at rt and then was
purified by preparative LC-MS. The fraction containing 5 was
concentrated, treated with saturated aqueous NaHCO3, and extracted
with CHCl3. The organic portion was then concentrated and dried in
vacuo. The crude material was dissolved in MeOH and purified by
1
preparative HPLC (UV collection). H NMR (400 MHz, CD3OD) δ
7.81−8.00 (m, 2H), 7.43−7.67 (m, 5H), 7.26−7.41 (m, 2H), 5.06−
5.20 (m, 1H), 4.53 (br d, J = 12.72 Hz, 1H), 4.06−4.32 (m, 2H),
3.58−3.77 (m, 1H), 3.13−3.28 (m, 1H), 1.91−2.20 (m, 2H), 1.71−
1.90 (m, 2H), 1.22−1.39 (m, 3H); purity 93%; LC-MS (ESI) 417.23
m/z (M + H)+.
N-(1-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-oxo-
but-2-en-2-yl)benzamide (6). Isolated as a byproduct from the
1
synthesis 5. H NMR (500 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.90−
8.03 (m, 2H), 7.45−7.61 (m, 6H), 7.36−7.44 (m, 2H), 5.51−5.63 (m,
1H), 5.20−5.30 (m, 1H), 4.15−4.27 (br m, 1H), 4.02−4.12 (br m,
1H), 3.40−3.55 (m, 1H), 3.14−2.98 (br m, 1H), 2.01−2.15 (br m,
1H), 1.72−1.79 (m, 3H), 1.47−1.64 (m, 2H); LC-MS (ESI) 399.26
m/z (M + H)+.
CCR1 Calcium Flux Assay. THP-1 cells were labeled with calcium
fluorophore (fluo-3 a.m., Molecular Probes cat. no. F-1241),
resuspended in assay buffer containing probenecid, and transferred
to plates. Calcium flux was monitored in a FLIPR (Molecular Devices,
CA) following addition of MIP-1α (R&D 270-LD, 20 nM). Effects of
compound were determined as changes in the base-to-peak excursion
relative to control.
N-(1-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-3-meth-
yl-1-oxobutan-2-yl)benzamide (12). Prepared from commercially
1
available 7 and 8 according to general procedure A. H NMR (500
CCR1 Chemotaxis Assay. THP-1 cells were fluorescently labeled
with calcein-AM (10 μg/mL; Molecular Probes, Eugene, OR). The
labeled cells were then washed and resuspended in chemotaxis buffer
and added to the top chamber of a chemotaxis plate (Neuroprobe cat.
no. 101-8; 8 μm pore size) with human MIP-1α (R&D 270-LD, 1 nM)
in the bottom well. Compounds were added to upper and lower wells.
The plate was incubated for 60 min at 37 °C after which fluorescence
in the lower wells was read at 485 nm/530 nm excitation/emission.
CCR2 Binding Assay. Radioligand competition binding assays
were used for assessment of binding affinity of test compounds to the
CCR2 receptor. For radioligand competition studies, 100 μL
containing 2.5 × 105 THP-1 cells/well (in assay buffer containing
50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, and 0.5% BSA)
were added to 96-well assay plates containing the test compounds in 3-
MHz, DMSO-d6) δ 8.45−8.58 (m, 1H), 7.88−7.94 (m, 2H), 7.33−
7.57 (m, 7H), 5.29 (d, J = 11.39 Hz, 1H), 4.74−4.82 (m, 1H), 4.38 (br
d, J = 12.39 Hz, 1H), 4.01−4.19 (m, 1H), 3.40−3.53 (m, 1H), 2.92−
3.09 (m, 1H), 2.15−2.28 (m, 1H), 1.80−1.94 (m, 1H), 1.63−1.80 (m,
2H), 1.49−1.63 (m, 1H), 0.89−1.01 (m, 6H); LC-MS (ESI) 437.24
m/z (M + Na)+.
N-(1-(4-(4-Chlorophenyl)piperidin-1-yl)-3-methyl-1-oxobu-
tan-2-yl)benzamide (21). Prepared from commercially available 8
and 9 according to general procedure A. 1H NMR (500 MHz, DMSO-
d6) δ 8.45−8.55 (m, 1H), 7.84−7.96 (m, 2H), 7.50−7.60 (m, 1H),
7.42−7.50 (m, 2H), 7.30−7.40 (m, 2H), 7.28 (d, J = 8.92 Hz, 1H),
7.21 (d, J = 8.42 Hz, 1H), 4.72−4.85 (m, 1H), 4.59 (br d, J = 12.88
Hz, 1H), 4.24−4.40 (m, 1H), 3.08−3.21 (m, 1H), 2.78−2.88 (m, 1H),
2.60−2.76 (m, 1H), 2.13−2.27 (m, 1H), 1.72−1.87 (m, 2H), 1.35−
9650
dx.doi.org/10.1021/jm300896d | J. Med. Chem. 2012, 55, 9643−9653

Journal of Medicinal Chemistry
Article
fold serial dilution, with final concentrations ranging from 5 μM to 100
pM. Subsequently, 50 μL 125I-MCP-1 radioligand at a final
concentration of 0.2 nM in assay buffer was added to the reaction.
After a 90 min incubation period at room temperature, the binding
reaction was terminated by harvesting on GF/B filter plates
(PerkinElmer cat. no. 6005177) followed by washing with ice-cold
wash buffer (50 mM HEPES, pH 7.4, 0.1% BSA, 0.5 M NaCl) to
remove unbound ligand. After washing, the plate was dried for 45 min
at 60 °C followed by addition of 40 μL MicroScint 20 scintillation
fluid, sealed, and analyzed by a Packard TopCount reader.
CCR3 Binding Assay. Ligand binding to the human CCR3
receptor was determined by a scintillation proximity assay (SPA)
established using a CHO CCR3/2 chimeric cell line. Cells were grown
in F-10 cell culture medium (Invitrogen cat. no. 11550-043), 10% fetal
bovine serum (Invitrogen cat. no. 26140-122), 1% penicillin/
streptomycin (Invitrogen cat. no. 15140-122), and 600 μg/mL
Geneticin (Invitrogen cat. no. 10131-027). For competition studies,
a 96-well Corning plate (Costar cat. no. 3912) containing compounds
of interest at concentration−response curves with final concentrations
ranging from 40 μM to 0.7 nM (3-fold, 11-point serial dilution) was
prepared. A 100 μL volume of assay buffer (25 mM HEPES, pH 7.6, 1
mM CaCl2, 5 mM MgCl2, 120 mM NaCl, 0.5% BSA) containing 2.0 ×
105 CHO CCR3/2 cells and 200 μg of PVT-WGA-PS beads
(Amersham cat. no. RPNQ0001) were then added to each well of
the assay plate. Subsequently, 50 μL of 125I-Eotaxin (PerkinElmer cat.
no. NEC314) at a 0.2 nM final concentration was added to the
reaction. The assay plate was sealed and incubated for 3 h at room
temperature followed by analysis on a Packard TopCount. The control
for nonspecific binding was a 100 nM final concentration of Eotaxin
(R&D Systems cat. no. 320-EO-020). Each experiment was run in
duplicate.
CCR5 Binding Assay. The human CCR5 ligand binding
scintillation proximity assay (SPA) was established using the Athersys
HT1080 human fibrosarcoma cell line, which stably expresses a
RAGE-activated human CCR5 receptor, and 125I-MIP-1β was used as
tracer ligand. HT1080-CCR5 cells were grown in alpha MEM
(Mediatech cat. no. 15-012-CV) supplemented with 10% dialyzed
fetal bovine serum, 2% penicillin/streptomycin/glutamine (Invitrogen
cat. no. 0378-016), and 500 μg/mL hygromycin B (Invitrogen cat. no.
25300-062). For CCR5 cell membrane preparation, 1 × 108 cells/
pellet were suspended in buffer (50 mM HEPES, 5 mM MgCl2, 1 mM
CaCl2), homogenized on ice with a Polytron homogenizer, and
centrifuged at 48 000g for 10 min at 4 °C. The cell pellet was
resuspended in buffer and protein concentration determined by the
Bradford assay. For competition studies, a 96-well Corning plate
(Costar cat. no. 3912) containing compounds of interest at
concentration−response curves with final concentrations ranging
from 40 μM to 0.7 nM (3-fold, 11-point serial dilution) was prepared.
Ten micrograms of membrane preparation protein/well and 200 μg/
well PVT-WGA-PS beads (Amersham cat. no. RPNQ0001) were
mixed in 100 μL total volume and then added to each well of the assay
plate. Subsequently, 50 μL of 125I-MIP-1β at 0.1 nM final
concentration was added to the reaction. The assay plate was sealed
and incubated for 4 h at room temperature followed by analysis on a
Packard TopCount.
(Woburn, MA). A 2.5 mL aliquot of a prewarmed 2-fold-concentrated
mixture of appropriate fluorogenic substrate and P450 enzyme in
potassium phosphate assay buffer was added to each well of the assay-
ready plates (containing 10 nL of test substance). Plates were then
prewarmed at 37 °C for 30 min. Reactions were initiated by the
addition of 2.5 mL of prewarmed 2-fold-concentrated NADPH-
regenerating system in the same assay buffer. Assay plates were
incubated at 37 °C. Following incubation, reactions were quenched/
stopped by the addition of 3 mL of a quench buffer (80% acetonitrile,
20% 0.5 M TRIS-base).
hERG Patch Clamp Assay. A whole-cell patch-clamp was used to
directly measure hERG tail currents in HEK-293 cells stably expressing
the cloned hERG potassium channel α subunit. Effects of compounds
were calculated by measuring inhibition of peak tail current.
Experiments were carried out using an aqueous buffer with pH 7.4
at room temperature. There was no protein in the assay buffer.
Solubility. Aqueous solubility was determined using a thermody-
namic equilibrium solubility assay. A solution of 0.5 to 0.7 mg of the
compound of interest was dissolved in 5 mL of methanol (or DMSO/
methanol if required), diluted in a serial fashion, and used to create a
standard calibration curve from the UV data obtained by HPLC
analysis at 210 or 254 nM. A test sample was created by adding 1 mL
of aqueous potassium phosphate (50 mM, pH 6.5) to 1 mL of the
standard solution. This solution was vortexed, sonicated for 30 s, and
then agitated on an orbital shaker for 15−24 h at room temperature.
The sample was then centrifuged for 2 min at 10 000 rpm and
analyzed by HPLC with the same method as that used for the
standard. Solubility was determined by comparison of the result with
the standard calibration curve.
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of intermediate compound synthesis and analytical data
(1H NMR and LC-MS) for the remaining compounds from the
tables. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (609) 252-4170; e-mail: cullen.cavallaro@bms.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Gerry Everlof for providing solubility data,
Bob Grafstrom for his contributions to the mutagenesis work,
and Mian Gao for the plasmid production performed in
support of the mutagenesis effort.
ABBREVIATIONS USED
■
CCR1, CC chemokine receptor 1; CHO, Chinese hamster
ovary; DIEA, N,N-diisopropylethylamine; EDC, N-(3-dimethy-
laminopropyl)-N′-ethylcarbodiimide; FLIPR, fluorometric
imaging plate reader; HEPES, 4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid; HOBt, 1-hydroxybenzotriazole; LM,
liver microsomes; MCP, monocyte chemotactic protein; MIP,
macrophage inflammatory protein; RANTES, regulated upon
activation normal T-cell expressed and secreted, also known as
CC chemokine ligand 5 (CCL5)
Liver Microsome Assays. Compound metabolic stability was
determined in human, rat, and mouse liver microsomes by a previously
reported method.27
Cross Species Assays. Binding was assessed using human 125I-
MIP-1α (NEN/Perkin-Elmer cat. no. NEX298) diluted to a final
concentration of 0.08 nM in assay buffer. Nonspecific binding was
determined in the presence of excess unlabeled ligand. Plates were
incubated for 60 min at room temperature and washed. After air-
drying, filters were removed and gamma-counted. Cell sources for the
assays were as follows: mouse, WEHI 274.1 cell line (ATCC cat. no.
CRL-1679); rat, thioglycollate-elicited peritoneal cells; rabbit,
peripheral blood mononuclear cells.
REFERENCES
■
CYP Assays. Cyp inhibition potential was assayed against a panel
of recombinant CYP microsomes (Supersomes) derived from
baculovirus-infected insect cells obtained from BD Biosciences
(1) Allen, S. J.; Crown, S. E.; Handel, T. M. Chemokine:receptor
structure, interactions, and antagonism. Annu. Rev. Immunol. 2007, 25,
787−820.
9651
dx.doi.org/10.1021/jm300896d | J. Med. Chem. 2012, 55, 9643−9653

Journal of Medicinal Chemistry
Article
(2) Viola, A.; Luster, A. D. Chemokines and their receptors: drug
targets in immunity and inflammation. Annu. Rev. Pharmacol. Toxicol.
2008, 48, 171−197.
(3) Horuk, R. Chemokine receptor antagonists: overcoming
developmental hurdles. Nat. Rev. Drug Discovery 2009, 8, 23−33.
(4) Schall, T. J.; Proudfoot, A. E. Overcoming hurdles in developing
successful drugs targeting chemokine receptors. Nat. Rev. Immunol.
2011, 11, 355−363.
(5) Horuk, R. Targeting CCR1. Methods Princ. Med. Chem. 2011, 46
(Chemokine Receptors as Drug Targets), 323−338.
(6) Review: Cheng, J.-F.; Jack, R. CCR1 antagonists. Mol. Diversity
2008, 12, 17−23.
(7) For leading references to patent and public literature since that
described in ref 6, see: (a) Carter, P. H.; Hynes, J. N-aryl pyrazoles,
indazoles and azaindazoles as antagonists of CC chemokine receptor
1: patent cooperation treaty applications WO2010/036632, WO2009/
134666 and WO2009/137338. Expert Opin. Ther. Pat. 2010, 20,
1609−1618. (b) Merritt, J. R.; James, R.; Paradkar, V. M.; Zhang, C.;
Liu, R.; Liu, J.; Jacob, B.; Chiriac, C.; Ohlmeyer, M. J.; Quadros, E.;
Wines, P.; Postelnek, J.; Hicks, C. M.; Chen, W.; Kimble, E. F.;
O’Brien, L.; White, N.; Desai, H.; Appell, K. C.; Webb, M. L. Novel
pyrrolidine heterocycles as CCR1 antagonists. Bioorg. Med. Chem. Lett.
2010, 20, 5477−5479.
(17) Kerstjens, H. A.; Bjermer, L.; Eriksson, L.; Dahlstrom, K.;
Vestbo, J. Tolerability and efficacy of inhaled AZD4818, a CCR1
antagonist, in moderate to severe COPD patients. Respir. Med. 2010,
104, 1297−1303.
(18) Lebre, M. C.; Vergunst, C. E.; Choi, I. Y. K.; Aarrass, S.;
Oliveira, A. S. F.; Wyant, T.; Horuk, R.; Reedquist, K. A.; Tak, P. P.
Why CCR2 and CCR5 blockade failed and why CCR1 blockade might
still be effective in the treatment of rheumatoid arthritis. PLoS One
2011, 6, e21772.
(19) The structure has not been formally disclosed, but several patent
applications from Chemocentryx claim a single compound. For
example, see: Li, L.; Pennell, A. M. K.; Zhang, P. 3-(Imidazolyl)-
pyrazolo[3,4b]-pyridines. US Patent application US 2010/0173911
A1.
(20) Dairaghi, D. J.; Zhang, P.; Wang, Y.; Seitz, L. C.; Johnson, D. A.;
Miao, S.; Ertl, L. S.; Zeng, Y.; Powers, J. P.; Pennell, A. M.; Bekker, P.;
Schall, T. J.; Jaen, J. C. Pharmacokinetic and pharmacodynamic
evaluation of the novel CCR1 antagonist CCX354 in healthy human
subjects: Implications for selection of clinical dose. Clinical Pharm.
Ther. 2011, 89, 726−734.
(21) CCX354 is listed as in an active clinical trial on www.
clinicaltrials.gov (accessed Aug 10, 2011). See trial identifier
NCT01242917, “A Study to Evaluate the Safety and Efficacy of
CCX354-C in Subjects With Rheumatoid Arthritis Partially
Responsive to Methotrexate Therapy (CARAT-2)”.
(22) Tak, P. P.; Balanescu, A.; Tseluyko, V.; Bojin, S.; Drescher, E.;
Dairaghi, D.; Miao, S.; Marchesin, V.; Jaen, J.; Bekker, P.; Schall, T. J.
Safety and efficacy of oral chemokine receptor 1 antagonist CCX354-C
in a phase 2 rheumatoid arthritis study. Presented at the ACR/ARHP
Scientific Meeting, Chicago, IL, November 5−9, 2011; Presentation
L11.
(23) (a) Xie, Y. F.; Sircar, I.; Lake, K.; Komandla, M.; Ligsay, K.; Li,
J.; Xu, K.; Parise, J.; Schneider, L.; Huang, D.; Liu, J.; Sakurai, N.;
Barbosa, M.; Jack, R. Identification of novel series of human CCR1
antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 2215−2221. (b) Merritt,
J. R.; Liu, J.; Quadros, E.; Morris, M. L.; Liu, R.; Zhang, R.; Jacob, B.;
Postelnek, J.; Hicks, C. M.; Chen, W.; Kimble, E. F.; Rogers, W. L.;
O’Brien, L.; White, N.; Desai, H.; Bansal, S.; King, G.; Ohlmeyer, M.
J.; Appell, K. C.; Webb, M. L. Novel pyrrolidine ureas as C-C
chemokine receptor 1 (CCR1) antagonists. J. Med. Chem. 2009, 52,
1295−1301.
(24) For chemotypes that show selectivity for the human receptor
over the murine receptor, see: (a) Naya, A.; Sagara, Y.; Ohwaki, K.;
Saeki, T.; Ichikawa, D.; Iwasawa, Y.; Noguchi, K.; Ohtake, N. Design,
synthesis, and discovery of a novel CCR1 antagonist. J. Med. Chem.
2001, 44, 1429−1435. (b) Xie, Y. F.; Lake, K.; Ligsay, K.; Komandla,
M.; Sircar, I.; Nagarajan, G.; Li, J.; Xu, K.; Parise, J.; Schneider, L.;
Huang, D.; Liu, J.; Dines, K.; Sakurai, N.; Barbosa, M.; Jack, R.
Structure-activity relationships of novel, highly potent, selective, and
orally active CCR1 antagonists. Bioorg. Med. Chem. Lett. 2007, 17,
3367−3372. Chemotypes are also known that show little selectivity
between the human and murine receptors; for example, see: Revesz,
L.; Bollbuck, B.; Buhl, T.; Eder, J.; Esser, R.; Feifel, R.; Heng, R.;
Hiestand, P.; Jachez-Demange, B.; Loetscher, P.; Sparrer, H.;
Schlapbach, A.; Waelchli, R. Novel CCR1 antagonists with oral
activity in the mouse collagen induced arthritis. Bioorg. Med. Chem.
Lett. 2005, 15, 5160−5164.
(8) Gladue, R. P.; Brown, M. F.; Zwillich, S. H. CCR1 antagonists:
what have we learned from clinical trials. Curr. Top. Med. Chem. 2010,
10, 1268−1277.
(9) Liang, M.; Mallari, C.; Rosser, M.; Ng, H. P.; May, K.; Monahan,
S.; Bauman, J. G.; Islam, I.; Ghannam, A.; Buckman, B.; Shaw, K.; Wei,
G. P.; Xu, W.; Zhao, Z.; Ho, E.; Shen, J.; Oanh, H.; Subramanyam, B.;
Vergona, R.; Taub, D.; Dunning, L.; Harvey, S.; Snider, R. M.;
Hesselgesser, J.; Morrissey, M. M.; Perez, H. D. Identification and
characterization of a potent, selective, and orally active antagonist of
the CC chemokine receptor-1. J. Biol. Chem. 2000, 275, 19000−19008.
(10) Gladue, R. P.; Tylaska, L. A.; Brissette, W. H.; Lira, P. D.; Kath,
J. C.; Poss, C. S.; Brown, M. F.; Paradis, T. J.; Conklyn, M. J.;
Ogborne, K. T.; McGlynn, M. A.; Lillie, B. M.; DiRico, A. P.; Mairs, E.
N.; McElroy, E. B.; Martin, W. H.; Stock, I. A.; Shepard, R. M.;
Showell, H. J.; Neote, K. CP-481,715, a potent and selective CCR1
antagonist with potential therapeutic implications for inflammatory
diseases. J. Biol. Chem. 2003, 278, 40473−40480.
(11) Structure suggested from a patent analysis, as described in:
Norman, P. AZD-4818,
a
chemokine CCR1 antagonist:
WO2008103126 and WO2009011653. Expert Opin. Ther. Pat. 2009,
19, 1629−1633.
(12) Lu, C.; Balani, S. K.; Qian, M. G.; Prakash, S. R.; Ducray, P. S.;
von Moltke, L. L. Quantitative prediction and clinical observation of a
CYP3A inhibitor-based drug-drug interactions with MLN3897, a
potent C-C chemokine receptor-1 antagonist. J. Pharm. Exp. Ther.
2010, 332, 562−568.
(13) Haringman, J. J.; Kraan, M. C.; Smeets, T. J. M.; Zwinderman,
A. H.; Tak, P. P. Chemokine blockade and chronic inflammatory
disease: proof of concept in patients with rheumatoid arthritis. Ann.
Rheum. Dis. 2003, 62, 715−721.
(14) Borregaard, J.; Skov, L.; Wang, L.; Ting, N.; Wang, C.; Beck, L.
A.; Sonne, J.; Clucas, A. Evaluation of the effect of the specific CCR1
antagonist CP-481715 on the clinical and cellular responses observed
following epicutaneous nickel challenge in human subjects. Contact
Dermatitis 2008, 59, 212−219.
(15) Vergunst, C. E.; Gerlag, D. M.; von Moltke, L.; Karol, M.;
Wyant, T.; Chi, X.; Matzkin, E.; Leach, T.; Tak, P. P. MLN3897 plus
methotrexate in patients with rheumatoid arthritis: safety, efficacy,
pharmacokinetics, and pharmacodynamics of an oral CCR1 antagonist
in a phase IIa, double-blind, placebo-controlled, randomized, proof-of-
concept study. Arthritis Rheum. 2009, 60, 3572−3581.
(16) Zipp, F.; Hartung, H.-P.; Hillert, J.; Schimrigk, S.; Trebst, C.;
Stangel, M.; Infante-Duarte, C.; Jakobs, P.; Wolf, C.; Sandbrink, R.;
Pohl, C.; Filippi, M. CCR1 Antagonist Study Group. Blockade of
chemokine signaling in patients with multiple sclerosis. Neurology
2006, 67, 1880−1883.
(25) (a) Liang, M.; Rosser, M.; Ng, H. P.; May, K.; Bauman, J. G.;
Islam, I.; Ghannam, A.; Kretschmer, P. J.; Pu, H.; Dunning, L.; Snider,
R. M.; Morrissey, M. M.; Hesselgesser, J.; Perez, H. D.; Horuk, R.
Species selectivity of a small molecule antagonist for the CCR1
chemokine receptor. Eur. J. Pharmacol. 2000, 389, 41−49. (b) Onuffer,
J.; McCarrick, M. A.; Dunning, L.; Liang, M.; Rosser, M.; Wei, G.-P.;
Ng, H.; Horuk, R. Structure function differences in nonpeptide CCR1
antagonists for human and mouse CCR1. J. Immunol. 2003, 170,
1910−1916.
(26) Vaidehi, N.; Schlyer, S.; Trabanino, R. J.; Floriano, W. B.; Abrol,
R.; Sharma, S.; Kochanny, M.; Koovakat, S.; Dunning, L.; Liang, M.;
Fox, J. M.; Lopes de Mendonca, F.; Pease, J. E.; Goddard, W. A., III;
9652
dx.doi.org/10.1021/jm300896d | J. Med. Chem. 2012, 55, 9643−9653

Journal of Medicinal Chemistry
Article
Horuk, R. Predictions of CCR1 chemokine receptor structure and BX
471 antagonist binding followed by experimental validation. J. Biol.
Chem. 2006, 281, 27613−27620.
(27) Wu, S. C.; Yoon, D.; Chin, J.; van Kirk, K.; Seethala, R.; Golla,
R.; He, B.; Harrity, T.; Kunselman, L. K.; Morgan, N. N.; Ponticiello,
R. P.; Taylor, J. R.; Zebo, R.; Harper, T. W.; Li, W.; Wang, M.; Zhang,
L.; Sleczka, B. G.; Nayeem, A.; Sheriff, S.; Camac, D. M.; Morin, P. E.;
Everlof, J. G.; Li, Y.-X.; Ferraro, C. A.; Kieltyka, K.; Shou, W.; Vath, M.
B.; Zvyaga, T. A.; Gordon, D. A.; Robl, J. A. Discovery of 3-hydroxy-4-
cyano-isoquinolines as novel, potent, and selective inhibitors of human
11β-hydroxydehydrogenase 1 (11β-HSD1). Bioorg. Med. Chem. Lett.
2011, 21, 6693−6698.
9653
dx.doi.org/10.1021/jm300896d | J. Med. Chem. 2012, 55, 9643−9653