Novel pyrrolidine ureas as C-C chemokine receptor 1 (CCR1) antagonists

Article abstract of DOI:10.1021/jm801416q

Monocyte infiltration is implicated in a variety of diseases including multiple myeloma, rheumatoid arthritis, and multiple sclerosis. C-C chemokine receptor 1 (CCR1) is a chemokine receptor that upon stimulation, particularly by macrophage inflammatory protein 1a (MIP-1a) and regulated on normal T-cell expressed and secreted (RANTES), mediates monocyte trafficking to sites of inflammation. High throughput screening of our combinatorial collection identified a novel, moderately potent CCR1 antagonist 3. The library hit 3 was optimized to the advanced lead compound 4. Compound 4 inhibited CCR1 mediated chemotaxis of monocytes with an IC50 of 20 nM. In addition, the compound was highly selective over other chemokine receptors. It had good microsomal stability when incubated with rat and human liver microsomes and showed no significant cytochrome P450 (CYP) inhibition. Pharmacokinetic evaluation of the compound in the rat showed good oral bioavailability.

Full text of DOI:10.1021/jm801416q

J. Med. Chem. 2009, 52, 1295–1301
1295
Novel Pyrrolidine Ureas as C-C Chemokine Receptor 1 (CCR1) Antagonists
J. Robert Merritt,* Jinqi Liu, Elizabeth Quadros, Michelle L. Morris, Ruiyan Liu, Rui Zhang, Biji Jacob, Jennifer Postelnek,
Catherine M. Hicks, Weiqing Chen, Earl F. Kimble, W. Lynn Rogers, Linda O’Brien, Nicole White, Hema Desai,
Shalini Bansal, George King, Michael J. Ohlmeyer, Kenneth C. Appell, and Maria L. Webb
Pharmacopeia, Inc., 3000 Eastpark BouleVard, Cranbury, New Jersey 08512
ReceiVed NoVember 10, 2008
Monocyte infiltration is implicated in a variety of diseases including multiple myeloma, rheumatoid arthritis,
and multiple sclerosis. C-C chemokine receptor 1 (CCR1) is a chemokine receptor that upon stimulation,
particularly by macrophage inflammatory protein 1R (MIP-1R) and regulated on normal T-cell expressed
and secreted (RANTES), mediates monocyte trafficking to sites of inflammation. High throughput screening
of our combinatorial collection identified a novel, moderately potent CCR1 antagonist 3. The library hit 3
was optimized to the advanced lead compound 4. Compound 4 inhibited CCR1 mediated chemotaxis of
monocytes with an IC50 of 20 nM. In addition, the compound was highly selective over other chemokine
receptors. It had good microsomal stability when incubated with rat and human liver microsomes and showed
no significant cytochrome P450 (CYP) inhibition. Pharmacokinetic evaluation of the compound in the rat
showed good oral bioavailability.
Introduction
respectively (Figure 1). Although efficacy was not observed in
these trials, CCR1 still remains as an attractive therapeutic target
for the treatment of a number of human diseases. Improvement
of compound potency and physical/chemical properties repre-
sents a viable approach to identify new CCR1 antagonists
suitable for clinical evaluation. In this study, we identified novel,
selective, small molecule CCR1 antagonists with high oral
bioavailability and potent activities in inhibiting receptor binding
and monocyte chemotaxis. These compounds with clinically
desired attributes represent a new chemical series for CCR1
antagonism and therapeutic intervention.
Upon screening of our combinatorial compound collection,
we identified the pyrrolidine amide 3 as an initial lead. This
compound was a moderate inhibitor of the CCR1 receptor with
an IC50 of 660 nM. In addition, the compound possessed good
stability in vitro with rat liver microsomes as well as good
permeability as assessed in a Caco-2 assay. These latter results
suggested that this class of compound might exhibit acceptable
bioavailability and warrant further optimization.
Chemokines, interacting through their respective receptors,
are involved in mediating recruitment and activation of immune
cells in response to physiological and pathological stimuli.
Dysregulation of this process has been implicated in the
initiation and progression of a number of important diseases
including rheumatoid arthritis (RAa), multiple sclerosis (MS),
allograft rejection, atherosclerosis, tissue fibrosis, airway hyper-
responsiveness, remodeling, and mucus production. CCR1,
which is expressed on human monocytes, T lymphocytes,
dendritic cells, eosinophils and basophils, plays a key role in
the development and progression of different diseases by
interacting with its major ligands including C-C chemokine
ligand (CCL) 3 (MIP1-R), CCL5 (RANTES), and CCL15
(leukotactin-1). Studies with CCR1 deficient mice, anti-MIP1-
R, anti-RANTES, or small molecule inhibitors of CCR1 have
demonstrated significant reduction of clinical scores in various
animal disease models, such as experimental autoimmune
encephalomyelitis,1 adjuvant induced arthritis,2,3 graft versus
host disease,4-6 renal fibrosis,7 interleukin-13-induced lung
fibrosis and mucus production,8 and airway hyper-responsive-
ness and remodeling in response to infectious agents.9 Taken
together, accumulating evidence suggests that CCR1 is a key
player in mediating leukocyte infiltration and activation and
contributes to the pathogenesis of different diseases. As a result,
small molecule antagonists of CCR1 may have therapeutic
potential for the treatment of different immunological disorders
without eliciting a general immunosuppressive effect. Thera-
peutic applications of CCR1 antagonists were further evaluated
in clinical trials conducted by Pfizer with 1 (CP-481715)10 and
Berlex/Schering AG with 2 (BX471)11 for RA and MS,
Chemistry
Synthesis of these pyrrolidine derivatives was straightforward
following the general procedures outlined in Schemes 1, 2, and
3. The amine derivatives 5a-o were either commercially
available or readily prepared as described. Amides were prepared
by coupling of respective amines to the appropriate pyrrolidine
carboxylic acid using N-(3-dimethylaminopropyl)-N′-ethylcar-
bodiimide (EDC)/1-hydroxybenzotriazole (HOBt) or benzot-
riazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP). Urea formation proceeded either through coupling
of the commercially available isocyanate or via treatment of
the corresponding aniline with triphosgene to form the isocy-
anate in situ.
* To whom correspondence should be addressed. Phone: (609)-452-3702.
Fax: (609)-452-3699. E-mail: bmerritt@pcop.com.
a Abbreviations: CCL, C-C chemokine ligand; CCR1, C-C chemokine
receptor 1; CYP, cytochrome P450; DIEA, N,N-diisopropylethylamine;
EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; HOBt, 1-hydroxy-
benzotriazole; MIP-1R, macrophage inflammatory protein 1R; MS, multiple
sclerosis; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexaflu-
orophosphate; RA, rheumatoid arthritis; RANTES, regulated on normal
T-cell expressed and secreted; SAR, structure-activity relationships; TFA,
trifluoroacetic acid.
Results and Discussion
In Vitro Structure-Activity Relationships (SAR). Initial
SAR exploration focused on the alkylamide portion of the
molecule. Truncation of the hydroxyethyl moiety to give the
NH amide 8 resulted in complete loss of activity (Table 1).
Replacement of hydroxyethyl with n-propyl 9 resulted in
10.1021/jm801416q CCC: $40.75
2009 American Chemical Society
Published on Web 01/30/2009

1296 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Merritt et al.
Figure 1
Scheme 1a
a Reagents and conditions: (a) EDC, HOBt, CH2Cl2; or PyBOP, CH2Cl2; (b) trifluoroacetic acid (TFA), CH2Cl2; or 4 M HCl in dioxane; (c) 4-chlorophenyl
isocyanate, N,N-diisopropylethylamine (DIEA), CH2Cl2.
significant loss of potency. Smaller alkyl groups however were
well tolerated. The ethyl and methyl amides 10 and 11 were
slightly more potent than the original hydroxyethyl lead.
Next our focus turned to the benzylic region. Shifting the
chloro group to the 2- and 3-positions, 12 and 13, was not
tolerated. Also not tolerated was replacement of 4-chloro with
4-cyano 14, 4-methoxy 15, or 4-trifluoromethyl 16. Replacement
of 4-chloro with 4-fluoro 17 or 4-methyl 18 resulted in
significant loss of potency. Further substitution of the 4-chlo-
robenzylamide with 2-methyl 19 and 3-methyl 20 resulted in
loss or reduction of activity. Methyl substitution at the R-ben-
zylic position, 21, also reduced potency.
SAR around the 4-chlorobenzylamide appeared to be tightly
constrained; thus, we shifted our investigations to the phenylurea
portion of the molecule. Replacement of 4-chloro with 4-methyl
22, 4-fluoro 23, 4-cyano 24, and 4-methoxy 25 resulted in
modest loss of potency (Table 2). Substitution with 4-trifluoro-
methyl 4, however, resulted in a compound with improved
potency. This compound was also a potent inhibitor of MIP-
1R-induced chemotaxis. Compound 4 was not as potent as the
standard compound 2 which, when tested in the same assays,
bound to CCR1 with IC50 ) 5.5 ( 0.3 nM and inhibited
chemotaxis with IC50 ) 1.5 ( 0.2 nM.
Expansion to the six-membered ring racemate 31 dramatically
reduced potency (Figure 2). Contraction to the four-membered
ring gave compound 32, which was inactive at CCR1. Inversion
of stereochemistry gave the S-pyrrolidine amide 33, which also
had poor activity.
Further Testing. Having identified five reasonably potent
compounds, we next assessed in vitro metabolic stability,
permeability, and in vivo pharmacokinetics in rat. All of the
compounds exhibited good stability in the presence of human
and rat microsomes and good permeability in Caco-2 assays.
When dosed orally in rats, compound 4 had the best exposure
(Table 3). Further substitution at the 2-position with halogen
or methyl resulted in decreased exposure. The most potent
compound 30 also had the lowest exposure of the five.
Since it had good potency and acceptable oral exposure in
the rat, compound 4 was selected for further study. The
compound was found to have good pharmacokinetics in rat with
45% absolute oral bioavailability, t1/2 of 4.4 h, and low plasma
clearance of 4.2 (mL/min)/kg. No significant CYP inhibition
was observed for 3A4, 2D6, or 1A2 up to 20 µM. The
compound showed no significant activity against a broad panel
of glycoprotein coupled receptors and ion channel receptors.
In addition compound 4 was inactive versus other chemokine
receptors including CCR2b, CCR4, CCR5, CXCR1, CXCR2,
and CX3CR1. Pyrrolidine ureas such as 4 comprise a novel
chemotype for antagonism of CCR1 and appear to be suitable
for further pharmaceutical development. Further optimization
of this series has been achieved and will be reported in due
course.
Since substitution at the 3-position was not well-tolerated,
as exemplified by compound 26, we retained the 4-trifluorom-
ethylphenylurea and investigated further substitution in the 2-
and 6- positions. 2-Chloro substitution 27 had no impact on
potency. Substitution of the 2-position with fluoro 28 and methyl
29 resulted in improved binding potency but showed no
significant improvement for inhibition of chemotaxis. Only the
2,6-dimethyl-4-trifluorophenylurea 30 gave improved inhibition
of chemotaxis with an IC50 of 8 nM.
Experimental Section
Mass spectrometry was conducted using a Thermo Finnigan LCQ
classic ion trap or a PE SCIEX API 150EX single quadrupole.
Liquid chromatography-mass spectrometry was conducted using
With more optimal substituents now in place, we investigated
the effect of changes in core ring size and stereochemistry.

Pyrrolidine Ureas
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1297
Scheme 3
Scheme 2a
Table 1
a Reagents and conditions: EDC, HOBt, CH2Cl2; or PyBOP, CH2Cl2.
compd
R1
R2
R3
R4
IC50 (nM)
a Waters 2690 with a 996 photodiode array detector linked to a
Thermo Finnigan LCQ classic ion trap. High resolution mass
spectrometry was conducted using a Micromass LCT oa-TOF with
a Waters 2795 HPLC system and Waters 996 PDA. 1H NMR
spectroscopy was conducted using a Varian 300 MHz Gemini 2000
FT NMR. Elemental analyses were carried out by Robertson
Microlit Laboratories. TLC was performed on silica gel plates
purchased from Analtech Co. Flash column chromatography was
performed using silica gel-60 (230-400 mesh). Preparative HPLC
was carried out on a Rainin Dynamax system with Sunfire Prep
C18 OBD, 5 µm, 19 mm × 100 mm column. All tested target
compounds possessed a purity of at least 95% as determined by
elemental analysis, when listed, or by HPLC-MS. HPLC for purity
determinations were conducted using a Waters 2690 HPLC with a
996 photodiode array detector at wavelengths of 215 and 254 nm
and a Phenomenex, Columbus, C18, 5 µm, 100 mm × 2.00 mm
column. Solvent and gradient conditions for HPLC purity deter-
minations were as follows: solvent A, 0.05% TFA in water; solvent
B, 0.05%TFA in acetonitrile; flow rate ) 0.4 mL/min; gradient,
3
8
9
CH2CH2OH
H
n-Pr
Et
Me
CH2CH2OH
CH2CH2OH
Me
Me
Me
Me
Me
Me
Me
Me
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2-Cl
3-Cl
4-CN
4-OMe
4-CF3
4-F
H
H
H
H
H
H
H
H
H
H
H
H
2-Me
3-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
540 ( 210
>75000
3600 ( 710
270 ( 160
270 ( 20
10
11
12
13
14
15
16
17
18
19
20
21
43000 ( 2800
>100000
>13,000
>77,000
>100000
1300 ( 700
5700 ( 3300
>100000
4-Me
4-Cl
4-Cl
4-Cl
4000 ( 280
4700 ( 700
0-0.50 min at 10% B, 0.50-5.50 min at 10% B f 90% B (linear),
5.50-7.50 min at 90% B, 7.50-8.00 min at 90% B f 10% B
(linear), 8.00-10.00 min at 10% B; total run time ) 10 min.

1298 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Merritt et al.
Table 2
3.52-3.72 (m, 2H), 4.52-4.64 (m, 2H), 4.76-4.82 (m, 1H),
7.22-7.28 (m, 4H), 7.34-7.43 (m, 4H). HRMS (ESI) m/z calcd
for C22H26Cl2N3O2 [M + H]+: 434.1402. Found: 434.1394.
(R)-N2-(4-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-ethylpyrro-
lidine-1,2-dicarboxamide (10). 10 was prepared from 5d according
1
to general procedure B. Yield: 3%. H NMR (300 MHz, CD3OD)
δ 1.07 (t, 1H), 1.25 (t, 2H), 1.80-2.40 (m, 4H), 3.42-3.72 (m,
4H), 4.50-4.64 (m, 2H), 7.26 (m, 5H), 7.38 (m, 3H). HRMS (ESI)
m/z calcd for C21H24Cl2N3O2 [M + H]+: 420.1246. Found:
420.1235.
compd
R5
R6 R7 binding IC50 (nM) chemotaxis IC50 (nM)
(R)-N2-(4-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (11). 11 was prepared from 6e and
4-chlorophenyl isocyanate by the same procedure described for 4.
Yield: 65%. 1H NMR (300 MHz, DMSO-d6) δ 1.66 (m, 1H), 1.79
(m, 2H), 1.96-2.20 (m, 1H), 2.79-2.89 (m, 3H), 3.45 (m, 2H),
4.29-4.77 (m, 3H), 7.12-7.20 (m, 4H), 7.27 (m, 1H), 7.33 (m,
1H), 7.46 (m, 2H), 8.28 (m, 1H). Anal. (C20H21Cl2N3O2) C, H, N.
MS calcd for C20H22Cl2N3O2: 406.1 (M + H)+. Found: 405.8.
22
23
24
25
4
26
27
28
29
30
4-Me
4-F
H
H
H
H
H
H
Cl
F
Me
H
H
H
H
H
H
H
H
H
680 ( 90
700 ( 70
720 ( 330
290 ( 20
140 ( 10
2950 ( 210
120 ( 7
ND
ND
ND
440 ( 00
22 ( 9
ND
35 ( 15
22 ( 7
16 ( 4
6 ( 3
4-CN
4-OMe
4-CF3
3-CF3
4-CF3
4-CF3
4-CF3
4-CF3
23 ( 4
50 ( 10
11 ( 00
(R)-N2-(2-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-(2-hydroxy-
ethyl)pyrrolidine-1,2-dicarboxamide (12). 12 was prepared from
Me Me
1
5f according to general procedure C. Yield: 5%. H NMR (300
(R)-N2-(4-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-(2-hydroxy-
ethyl)pyrrolidine-1,2-dicarboxamide (3). To a solution of 6a in
CH3CN (2 mL) was added Et3N (132 mg) and 4-chlorophenyl
isocyanate (132 mg). The reaction mixture was stirred at room
temperature for 16 h. The mixture was concentrated and purified
MHz, CD3OD) δ 1.93-2.36 (m, 4H), 3.53-3.76 (m, 6H),
4.61-4.88 (m, 2H), 5.05 (m, 1H), 7.25 (m, 4H), 7.30-7.45 (m,
4H). HRMS (ESI) m/z calcd for C21H24Cl2N3O3 [M + H]+:
436.1195. Found: 436.1208.
(R)-N2-(3-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-(2-hydroxy-
ethyl)pyrrolidine-1,2-dicarboxamide (13). 13 was prepared from
1
by preparative HPLC to give pure compound (140 mg, 61%). H
1
NMR (300 MHz, CDCl3) δ 1.88-2.04 (m, 3H), 2.22 (m, 2H), 3.13
(m, 1H), 3.51 (m, 1H), 3.70 (m, 2H), 3.87 (m, 2H), 4.05 (m, 1H),
4.63-4.99 (m, 1H), 5.03-5.07 (m, 1H), 5.19 (m, 1H), 7.13-7.39
(m, 8H). Anal. (C21H23Cl2N3O3) C, H. N: calcd, 9.63. Found, 7.22.
HRMS (ESI) m/z calcd for C21H24Cl2N3O3 [M + H]+: 436.1195.
Found: 436.1191.
(R)-N2-(4-Chlorobenzyl)-N2-methyl-N1-[4-(trifluoromethyl)phe-
nyl]pyrrolidine-1,2-dicarboxamide (4). To a solution of 6e (50
mg, 0.2 mmol) in dichloromethane (2 mL) were added 4-(trifluo-
romethyl)phenyl isocyanate (0.030 mL, 0.2mmol) and DIEA (0.050
mL). The reaction mixture was stirred for 12 h and then evaporated
to dryness. Purification by silica gel column chromatography (ethyl
acetate/hexane) afforded the desired product as a white amorphous
solid (40.9 mg, 47%). 1H NMR (300 MHz, DMSO-d6) δ 1.66 (m,
1H), 1.84 (m, 2H), 2.12 (m, 1H), 2.91 (s, 3H), 3.50 (m, 2H), 4.30
(m, 1H), 4.47 (m, 1H), 4.71 (m, 1H), 7.24 (m, 4H), 7.49 (d, 2H),
7.63 (m, 2H), 8.58 (s, 1H). Anal. (C21H21ClF3N3O2) C, H, N. MS
calcd: 440.1 (M + H)+. Found: 439.8.
5g according to general procedure B. Yield: 8%. H NMR (300
MHz, CD3OD) δ 1.82-2.40 (m, 4H), 3.37-3.76 (m, 6H),
4.56-4.82 (m, 2H), 5.05 (m, 1H), 7.26 (m, 4H), 7.30-745 (m,
4H). HRMS (ESI) m/z calcd for C21H24Cl2N3O3 [M + H]+:
436.1795. Found: 436.1181.
(R)-N2-(4-Cyanobenzyl)-N1-(4-chlorophenyl)-N2-methylpyrro-
lidine-1,2-dicarboxamide (14). 14 was prepared from 5h according
1
to general procedure B. Yield: 6%. H NMR (300 MHz, CD3OD)
δ 1.85-2.40 (m, 4H), 2.90-3.12 (m, 3H), 3.56-3.72 (m, 2H),
4.52-4.67 (m, 1H), 4.80-4.96 (m, 2H), 7.24-7.26 (d, 2H),
7.40-7.76 (m, 6H). HRMS (ESI) m/z calcd for C21H22ClN4O2 [M
+ H]+: 397.1431. Found: 397.1441.
(R)-N2-(4-Methoxybenzyl)-N1-(4-chlorophenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (15). 15 was prepared from 5i ac-
1
cording to general procedure C. H NMR (300 MHz, CD3OD) δ
1.82-2.34 (m, 4H), 2.86-3.01 (m, 3H), 3.55-3.70 (m, 2H), 3.74
(m, 3H), 4.53 (m, 1H), 4.90 (m, 2H), 6.83-6.95 (m, 2H), 7.16-7.32
(m, 4H), 7.39-7.42 (m, 2H). HRMS (ESI) m/z calcd for
C21H25ClN3O3 [M + H]+: 402.1584. Found: 402.1584.
(R)-N2-(4-Chlorobenzyl)-N1-(4-chlorophenyl)pyrrolidine-1,2-
dicarboxamide (8). 8 was prepared from 6b and 4-chlorophenyl
isocyanate by the same procedure described for 4. Yield: 48%. 1H
NMR (300 MHz, DMSO-d6) δ 1.94-2.03 (m, 3H), 2.21-2.26 (m,
1H), 3.57 (m, 1H), 3.75 (m, 1H), 4.38 (m, 2H), 4.47 (m, 1H),
7.38-7.48 (m, 6H), 7.67-7.72 (m, 2H), 8.48 (s, 1H), 8.57 (t, 1H).
Anal. (C19H19Cl2N3O2) C, H, N. MS calcd for C19H20Cl2N3O2: 392.1
(M + H)+. Found: 391.9.
General Procedure B for Amide Formation. To a solution of
(R)-1-[(4-chlorophenyl)carbamoyl]pyrrolidine-2-carboxylic acid (1
equiv) and the appropriate amine 5 (1 equiv) in dichloromethane
was added EDC hydrochloride (2.5 equiv) and catalytic HOBt. The
mixture was stirred at room temperature overnight, then filtered
and concentrated to give the crude product which was purified by
flash chromatography.
General Procedure C for Amide Formation. To a solution of
(R)-1-[(4-chlorophenyl)carbamoyl]pyrrolidine-2-carboxylic acid (1
equiv) and the appropriate amine 5 (1 equiv) in dichloromethane
was added PyBOP (1.5 equiv). The mixture was stirred at room
temperature overnight, then concentrated to give the crude product
which was purified by flash chromatography.
(R)-N2-(4-Chlorobenzyl)-N1-(4-chlorophenyl)-N2-propylpyr-
rolidine-1,2-dicarboxamide (9). 9 was prepared from 5c according
to general procedure B. Yield: 6%. H NMR (300 MHz, CD3OD)
(R)-N2-[4-(Trifluoromethyl)benzyl]-N1-(4-chlorophenyl)-N2-
methylpyrrolidine-1,2-dicarboxamide (16). 16 was prepared from
1
5j according to general procedure C. Yield: 5%. H NMR (300
MHz, CD3OD) δ 1.86-2.42 (m, 4H), 2.92-3.06 (m, 3H),
3.56-3.74 (m, 2H), 4.56-4.82 (m, 2H), 4.92-4.98 (m, 1H),
7.24-7.27 (m, 2H), 7.45 (m, 4H), 7.60-7.63 (m, 2H). HRMS (ESI)
m/z calcd for C21H22ClF3N3O2 [M + H]+: 440.1353. Found:
440.1345.
(R)-N2-(4-Fluorobenzyl)-N1-(4-chlorophenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (17). 17 was prepared from 6k and
4-chlorophenyl isocyanate by the same procedure described for 4.
Yield: 52%. 1H NMR (300 MHz, DMSO-d6) δ 1.65 (m, 1H), 1.85
(m, 2H), 1.95-2.20 (m, 1H), 2.63-2.89 (m, 3H), 3.41-3.50 (m,
2H), 4.29-4.47 (m, 2H), 4.74 (m, 1H), 7.00-7.20 (m, 4H), 7.35
(m, 1H), 7.46 (m, 2H), 8.29 (s, 1H). Anal. (C20H21ClFN3O2) C, H,
N. MS calcd for C20H22ClFN3O2: 390.1 (M + H)+. Found: 389.9.
(R)-N2-(4-Methylbenzyl)-N1-(4-chlorophenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (18). 18 was prepared from 5l ac-
1
cording to general procedure B. Yield: 9%. H NMR (300 MHz,
CD3OD) δ 1.83-2.14 (m, 4H), 2.30-2.32 (m, 3H), 2.88-3.04 (m,
3H), 3.56-3.73 (m, 2H), 4.48-4.88 (m, 2H), 4.92 (m, 1H),
7.13-7.25 (m, 6H), 7.40-7.42 (m, 2H). HRMS (ESI) m/z calcd
for C21H25ClN3O2 [M + H]+: 386.1680. Found: 386.1688.
1
δ 0.82-0.94 (m, 3H), 1.46-2.40 (m, 6H), 3.03-3.46 (m, 2H),

Pyrrolidine Ureas
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1299
Figure 2
Table 3
(C21H21ClN4O2) C, H, N. MS calcd for C21H22ClN4O2: 397.1 (M
+ H)+. Found: 396.8.
HLM
RLM
AUC(0-∞)
Cmax
(R)-N2-(4-Chlorobenzyl)-N1-(4-methoxyphenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (25). 25 was prepared from 6e and
4-methoxyphenyl isocyanate by the same procedure described for
compd (% remaining) (% remaining) po (ng/(mL ·h)) (ng/mL)
4
78
73
93
58
74
74
68
76
81
84
9390 ( 1590
2060 ( 391a
1240 ( 452a
2590 ( 428a
611 ( 221
834 ( 299
437 ( 53
291 ( 89
699 ( 153
195 ( 33
27
28
29
30
1
4. Yield: 87%. H NMR (300 MHz, DMSO-d6) δ 1.61-1.67 (m,
1H), 1.79-1.94 (m, 2H), 2.08-2.15 (m, 1H), 2.64-2.89 (m, 3H),
3.23 (s, 3H), 3.39-3.51 (m, 2H), 4.30-4.75 (m, 3H), 6.73 (d, 2H),
7.13-7.33 (m, 6H), 7.97 (s, 1H). Anal. (C21H24ClN3O3) C, H, N.
MS calcd for C21H25ClN3O3: 402.1 (M + H)+. Found: 401.8.
(R)-N2-(4-Chlorobenzyl)-N2-methyl-N1-(3-(trifluoromethyl)phe-
nyl)pyrrolidine-1,2-dicarboxamide (26). 26 was prepared from
6e and 3-(trifluoromethyl)phenyl isocyanate by the same procedure
a AUC(0-6)
.
(R)-N2-(4-Chloro-2-methylbenzyl)-N1-(4-chlorophenyl)-N2-
methylpyrrolidine-1,2-dicarboxamide (19). 19was prepared from
5m according to general procedure C. Yield: 9%. H NMR (300
1
1
described for 4. Yield: 74%. H NMR (300 MHz, DMSO-d6) δ
MHz, CD3OD) δ 1.83-2.40 (m, 7H), 1.80-2.46 (m, 4H),
2.92-3.05 (m, 3H), 3.55-3.70 (m, 2H), 4.59 (m, 2H), 4.92-4.96
(m, 1H), 7.14-7.26 (m, 5H), 7.38-7.42 (m, 2H). HRMS (ESI)
m/z calcd for C21H24Cl2N3O2 [M + H]+: 420.1246. Found:
420.1249.
1.67 (m, 1H), 1.82-2.18 (m, 3H), 2.65-2.91 (m, 3H), 3.47-3.51
(m, 2H), 4.28-4.77 (m, 3H), 7.14-7.40 (m, 6H), 7.65-7.73 (m,
1H), 7.88 (d, 1H), 8.50 (d, 1H). Anal. (C21H21ClF3N3O2) C, H, N.
MS calcd for C21H22ClF3N3O2: 440.1 (M + H)+. Found: 439.8.
(R)-N2-(4-Chlorobenzyl)-N1-[2-chloro-4-(trifluoromethyl)phe-
nyl]-N2-methylpyrrolidine-1,2-dicarboxamide (27). 27 was pre-
pared from 6e and 2-chloro-4-(trifluoromethyl)phenyl isocyanate
by the same procedure described for 4. Yield: 99%. 1H NMR (300
MHz, DMSO-d6) δ 1.70 (m, 1H), 1.88 (m, 2H), 1.98-2.22 (m,
1H), 2.65-2.90 (m, 3H), 3.51 (m, 2H), 4.30-4.88 (m, 3H), 7.13
(m, 1H), 7.27-7.35 (m, 3H), 7.57 (d, 1H), 7.76 (m, 1H), 7.99 (d,
1H). Anal. (C21H20Cl2F3N3O2) H, N. C: calcd, 53.17. Found: 54.27.
HRMS (ESI) m/z calcd for C21H21Cl2F3N3O2 [M + H]+: 474.0963.
Found: 474.0965.
(R)-N2-(4-Chloro-3-methylbenzyl)-N1-(4-chlorophenyl)-N2-
methylpyrrolidine-1,2-dicarboxamide (20). 20 was prepared from
1
5n according to general procedure C. Yield: 6%. H NMR (300
MHz, CD3OD) δ 1.83-2.39 (m, 7H), 2.88-3.07 (m, 3H),
3.53-3.74 (m, 2H), 4.40-4.74 (m, 2H), 4.80 (m, 1H), 7.04-7.36
(m, 5H), 7.40-7.44 (m, 2H). HRMS (ESI) m/z calcd for
C21H24Cl2N3O2 [M + H]+: 420.1246. Found: 420.1244.
(2R)-N1-(4-Chlorophenyl)-N2-[1-(4-chlorophenyl)ethyl]-N2-
methylpyrrolidine-1,2-dicarboxamide (21). 21 was prepared from
6o and 4-chlorophenyl isocyanate by the same procedure described
for 4. Yield: 34%. 1H NMR (300 MHz, CD3OD) δ 1.47-1.70 (m,
3H), 1.80-2.46 (m, 4H), 2.56-2.64 (m, 1H), 2.77-2.80 (m, 2H),
3.55-3.73 (m, 4H), 5.36-5.90 (m, 1H), 7.21-7.49 (m, 8H). Anal.
(C21H23Cl2N3O2) C, H, N. MS calcd: 420.1 (M + H)+. Found:
419.9.
(R)-N2-(4-Chlorobenzyl)-N1-[2-fluoro-4-(trifluoromethyl)phe-
nyl]-N2-methylpyrrolidine-1,2-dicarboxamide (28). 28 was pre-
pared from 6e and 2-fluoro-4-(trifluoromethyl)phenyl isocyanate
by the same procedure described for 4. Yield: 22%. 1H NMR (300
MHz, CDCl3) δ 1.96-2.28 (m, 4H), 2.95-3.03 (m, 3H), 3.56 (m,
1H), 3.76 (m, 1H), 4.48-4.67 (m, 2H), 4.82-4.94 (m, 1H), 6.69
(m, 1H), 7.16-7.36 (m, 5H), 8.35 (m, 1H). Anal. (C21H20ClF4N3O2)
H, N. C: calcd, 55.08. Found, 54.16. HRMS (ESI) m/z calcd for
C21H21ClF4N3O2 [M + H]+: 458.1258. Found: 458.1260.
(R)-N2-(4-Chlorobenzyl)-N2-methyl-N1-p-tolylpyrrolidine-1,2-
dicarboxamide (22). 22 was prepared from 6e and 4-methylphenyl
isocyanate by the same procedure described for 4. Yield: 69%. 1H
NMR (300 MHz, DMSO-d6) δ 1.67 (m, 1H), 1.78-2.13 (m, 6H),
2.64-2.89 (m, 3H), 3.47 (m, 2H), 4.29-4.75 (m, 3H), 6.94 (m,
2H), 7.13 (m, 2H), 7.23-7.33 (m, 4H), 8.02 (s, 1H). Anal.
(C21H24ClN3O2) C, H, N. MS calcd for C21H25ClN3O2: 386.1 (M
+ H)+. Found: 385.9.
(R)-N2-(4-Chlorobenzyl)-N2-methyl-N1-[2-methyl-4-(trifluo-
romethyl)phenyl]pyrrolidine-1,2-dicarboxamide (29). 2-Methyl-
4-(trifluoromethyl)phenyl isocyanate 7i was generated in situ by
dropwise addition of triphosgene (44.5 mg, 0.15 mmol) in CH2Cl2
(1 mL) to a solution of 2-methyl-4-(trifluoromethyl)benzenamine
(79 mg, 0.45 mmol) in dichloromethane (1 mL). The mixture was
stirred at room temperature for 3 h. The resulting suspension was
added dropwise to a solution of 6e (116 mg, 0.40 mmol) and DIEA
(0.175 mL, 1.0 mmol) in dichloromethane (2 mL), stirred for 10
min, and purified by preparative HPLC to give the title product as
(R)-N2-(4-Chlorobenzyl)-N1-(4-fluorophenyl)-N2-methylpyr-
rolidine-1,2-dicarboxamide (23). 23 was prepared from 6e and
4-fluorophenyl isocyanate by the same procedure described for 4.
Yield: 77%. 1H NMR (300 MHz, DMSO-d6) δ 1.65 (m, 1H),
1.81-2.16 (m, 3H), 2.63-2.89 (m, 3H), 3.40-3.48 (m, 2H),
4.29-4.76 (m, 3H), 6.97 (t, 2H), 7.13 (d, 1H), 7.25 (d, 1H),
7.37-7.42 (m, 4H), 8.17 (s, 1H). Anal. (C20H21ClFN3O2) C, H, N.
MS calcd for C20H22ClFN3O2: 390.1 (M + H)+. Found: 389.9.
(R)-N2-(4-Chlorobenzyl)-N1-(4-cyanophenyl)-N2-methylpyrro-
lidine-1,2-dicarboxamide (24). 24 was prepared from 6e and
4-cyanophenyl isocyanate by the same procedure described for 4.
Yield: 71%. 1H NMR (300 MHz, DMSO-d6) δ 1.68 (m, 1H),
1.82-1.88 (m, 2H), 1.98-2.19 (m, 1H), 2.65-2.91 (m, 3H),
3.45-3.56 (m, 2H), 4.28-4.78 (m, 3H), 7.13 (d, 1H), 7.24 (d, 1H),
7.34 (t, 2H), 7.43 (d, 2H), 7.62-7.65 (m, 2H), 8.56 (s, 1H). Anal.
1
light-yellow oil (57.3 mg, 32%). H NMR (300 MHz, CDCl3) δ
1.97-2.02 (m, 1H), 2.10-2.14 (m, 1H), 2.24-2.35 (m, 5H), 2.99
(s, 1H), 3.06 (s, 2H), 3.54 (m, 1H), 3.70 (m, 1H), 4.59-4.67 (m,
2H), 4.89-4.98 (m, 1H), 7.17 (d, 1H), 7.24-7.38 (m, 3H), 7.44
(d, 2H), 7.81 (t, 1H). Anal. (C22H23ClF3N3O2) C, H, N. MS calcd:
454.1 (M + H)+. Found: 453.9.
(R)-N2-(4-Chlorobenzyl)-N1-[2,6-dimethyl-4-(trifluorometh-
yl)phenyl]-N2-methylpyrrolidine-1,2-dicarboxamide (30). To a
solution of 7j in dichloromethane was added 6e (45 mg, 0.18 mmol)

1300 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Merritt et al.
and DIEA (0.063 mL, 0.36 mmol) in dichloromethane (0.5 mL),
in one portion, and the mixture was stirred at room temperature
overnight. The mixture was concentrated and purified by HPLC to
afford 17.3 mg of the desired product. Yield: 24%. 1H NMR (300
MHz, CDCl3) δ 2.01-2.46 (m, 10H), 3.02-3.09 (m, 3H),
3.61-3.68 (m, 1H), 3.78-3.85 (m, 1H), 4.54-4.79 (m, 2H),
4.89-5.03 (m, 1H), 7.21-7.41 (m, 6H). Anal. (C23H25ClF3N3O2)
H, N. C: calcd, 59.03. Found, 58.03. HRMS (ESI) m/z calcd for
C23H26ClF3N3O2 [M + H]+: 468.1666. Found: 468.1662.
in assay buffer (RPMI plus 0.1% BSA) and then added to the
bottom wells of the chamber. THP1 cells with a density of 2 ×
106 cells/mL were added to the top chamber in the presence or
absence of various concentrations of compounds. The apparatus
was incubated for 3 h in a 5% CO2-humidified incubator at 37 °C.
After the incubation period, the migrating cells from the bottom
chamber were quantified by transferring equal volumes of cells into
CellTiter-Glo and read on PerkinElmer Victor multilable counter
using luminescence protocol.
Rat and Human Liver Microsome Stability Assays. Assay
mixtures typically contained rat or human microsomes (300 nM
cytochrome P450, BD Gentest, Woburn, MA), phosphate buffer
(pH 7.4), 1 µM test compound, 1 mM NADPH in a final assay
volume of 0.1 mL. Incubations were for 30 min at 37 °C and were
terminated by the addition of 0.2 mL of acetonitrile. Samples were
centrifuged at 2000g and analyzed by LC/MS. The percentage of
compound remaining at 30 min was calculated.
Caco-2 Permeability. Caco-2 cells were obtained from ATCC
(Rockville, MD). The cells were seeded onto BIOCOAT insert
plates (BD Biosciences, San Jose, CA) at a density of 250 000 cells/
well in basal seeding medium containing MITO+ serum extender
for 3 days followed by the treatment with Entero-STIM medium
containing MITO+ serum extender for 2 days. Permeability studies
were performed with monolayers cultured for 5 days. The cell
monolayer integrity was monitored by transepithelial electrical
resistance and by permeation of Lucifer yellow. Permeability studies
were initiated by the addition of test compound (20 µM) in Hank’s
buffered salt solution to either the apical or basolateral side of the
monolayer to measure apical to basolateral or basolateral to apical
transport, respectively. Plates were incubated at 37 °C and samples
taken from the donor solution at time zero and 90 min and from
the basolateral solution at 90 min. The concentration of test
compound was determined by HPLC/UV.
Cytochrome P450 Inhibition. Human recombinant cytochrome
P450 isoenzymes (BD Gentest, Woburn, MA) were tested (CYP1A2,
2C9, 2C19, 2D6, and 3A4). Each isoenzyme converts a specific
fluorogenic substrate to a fluorescent product. Test compound (up
to 20 µM) was added, and the fluorescence was read, after a defined
reaction time, using a Victor V2 Wallac 1420 multilabel counter.
The percentage inhibition at each concentration was calculated, and
the IC50 was determined by fitting the plot of percent inhibition vs
logarithmic concentration values (µM).
N2-(4-Chlorobenzyl)-N2-methyl-N1-[4-(trifluoromethyl)phe-
nyl]piperidine-1,2-dicarboxamide (31). 1-(tert-Butoxycarbon-
yl)piperidine-2-carboxylic acid (2.29 g, 10 mmol) and N-(4-
chlorobenzyl)-N-methylamine (1.56 g, 10 mmol) were subjected
to the procedure described for 6e to afford N-(4-chlorobenzyl)-N-
methylpiperidine-2-carboxamide. Yield: 55%. The product (53 mg,
0.2 mmol) was combined with DIEA (50 µL), 4-(trifluorometh-
yl)phenyl isocyanate (30 µL, 0.21 mmol), and dichloromethane (2
1
mL) and treated as described for 4. Yield: 29%. H NMR (300
MHz, DMSO-d6) δ 1.80-2.00 (m, 6H), 3.12 (s, 3H), 3.66 (m, 1H),
4.04 (m, 1H), 4.52-4.68 (m, 2H), 5.24-5.36 (m, 1H), 7.36 (m,
1H), 7.44 (m, 2H), 7.70-7.82 (m, 4H), 8.00 (m, 1H), 9.03 (s, 1H).
Anal. (C22H23ClF3N3O2) C, H, N. MS calcd for Na+C22H23-
ClF3N3O2: 476.1 (M + Na)+. Found: 475.9.
(R)-N2-(4-Chlorobenzyl)-N2-methyl-N1-[4-(trifluoromethyl)phe-
nyl]azetidine-1,2-dicarboxamide (32). (R)-1-(tert-Butoxycarbo-
nyl)azetidine-2-carboxylic acid (100 mg, 0.49 mmol) and N-(4-
chlorobenzyl)-N-methylamine (76 mg, 0.49 mmol) were subjected
to the procedure described for 6e to afford (R)-N-(4-chlorobenzyl)-
N-methylazetidine-2-carboxamide. Yield: 51%. The product (30 mg,
0.13 mmol) was combined with DIEA (44 µL), 4-(trifluorometh-
yl)phenyl isocyanate (19 µL, 0.13 mmol), and CH2Cl2 (2 mL) and
treated as described for 4. Yield: 62%. 1H NMR (300 MHz, CDCl3)
δ 2.41-2.55 (m, 2H), 2.86-2.98 (m, 3H), 3.72-3.79 (m, 1H),
4.02-4.14 (m, 1H), 4.35-4.46 (m, 1H), 4.74-4.79 (d, 1H), 5.04
(t, 1H), 7.09-7.37 (m, 4H), 7.44-7.51 (m, 4H). Anal.
(C20H19ClF3N3O2) H, N. C: calcd, 56.40. Found: 55.28. HRMS
(ESI) m/z calcd for C20H20ClF3N3O2 [M + H]+: 426.1196. Found:
426.1193.
(S)-N2-(4-Chlorobenzyl)-N2-methyl-N1-[4-(trifluoromethyl)phe-
nyl]pyrrolidine-1,2-dicarboxamide (33). 33 was prepared from
(S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid by the
same procedure described for 4. Yield: 71%. 1H NMR (300 MHz,
DMSO-d6) δ 1.65 (m, 1H), 1.82-2.16 (m, 3H), 2.64-2.90 (m,
3H), 3.50 (m, 2H), 4.27-4.78 (m, 3H), 7.11-7.37 (m, 4H), 7.48
(m, 2H), 7.63 (m, 2H), 8.56 (s, 1H). Anal. (C21H21ClF3N3O2) C,
H, N. MS calcd for C21H22ClF3N3O2: 440.1 (M + H)+. Found:
439.8.
Biological Assays: Materials and Methods. Recombinant
membranes were prepared by cloning human CCR1 into an
episomal expression vector (Horlick-2000)12 and stably expressing
in HEK293 cells. [125I]MIP1R was obtained from Perkin-Elmer
(Waltham, MA), and SPA beads were obtained from GE healthcare
(Piscataway, NJ). The human monocytic cell line, THP-1, was
purchased from ATCC (Manassas, VA). Recombinant human
MIP1R was obtained from R&D Systems (Minneapolis, MN). Plates
used for chemotaxis in 96-well format (5 µm) were obtained from
Corning (Corning, NY). CellTiter-Glo was obtained from Promega,
Madison WI.
Pharmacokinetics Studies in the Rat. In preliminary screening
studies, test compounds were dosed orally by gavage (5 mg/kg in
0.5% methyl cellulose) to three to four male Sprague-Dawley rats,
either as single compounds or as part of a cassette. Serial blood
samples were collected from an indwelling arterial cannula up to 6
or 24 h. For bioavailability studies, test compound (1 mg/kg) was
also administered intravenously via an indwelling catheter. Plasma
was separated, and samples were prepared by protein precipitation
and analyzed for parent compound by LC/MS/MS. Pharmacokinetic
parameters were determined using noncompartmental analysis
(WinNonlin, Pharsight Corp., Mountain View, CA).
Supporting Information Available: Details of intermediate
compound synthesis and elemental analysis data. This material is
available free of charge via the Internet at http://pubs.acs.org.
Receptor and Ligand Binding Assay. The receptor binding
assay was conducted in 384-well plates by preparing membrane
and SPA bead mixture in assay buffer (130 mM NaCl, 5 mM KCl,
1 mM MnCl2, 50 mM Tris HCl, 7.4, 0.1% BSA) at 50 µg/mL
membrane and 10 mg/mL SPA beads. An amount of 10 µL of the
membrane/SPA mixture was transferred to the assay plate along
with 10 µL of different concentrations of testing compounds. Then
5 µL of [125I]MIP1R was added at a final concentration of 0.1 nM.
The plate was spun at 2500 rpm for 2 min and incubated for 4 h
and read (1 min/well count time) on a MicroBeta TriLux (Perkin-
Elmer).
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