Discovery, optimization, and pharmacological characterization of novel heteroaroylphenylureas antagonists of C-C chemokine ligand 2 function

Article abstract of DOI:10.1021/jm1012903

Through the application of TRAP (target-related affinity profiling), we identified a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-dependent and selective with regard to other chemokines. The compounds, however, did not antagonize the binding of ¹²⁵I-labeled CCL2 to the CCR2 receptor nor did they block CCR2-mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC₅₀ = 80 nM) with excellent oral bioavailability in rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.

Full text of DOI:10.1021/jm1012903

ARTICLE
pubs.acs.org/jmc
Discovery, Optimization, and Pharmacological Characterization
of Novel Heteroaroylphenylureas Antagonists of C-C Chemokine
Ligand 2 Function†
Edgardo Laborde,*,‡ Robert W. Macsata,‡ Fanying Meng,‡ Brian T. Peterson,‡ Louise Robinson,‡
Steve R. Schow,‡ Reyna J. Simon,‡ Hua Xu,‡ Kunihisa Baba,§ Hideaki Inagaki,§ Yoshiro Ishiwata,§
Takahito Jomori,§ Yukiharu Matsumoto,§ Atsushi Miyachi,§ Takashi Nakamura,§ Masayuki Okamoto,§
Tracy M. Handel,|| and Claude C. A. Bernard^
‡Telik, Inc., 700 Hansen Way, Palo Alto, California 94304, United States
§Sanwa Kagaku Kenkyusho Co., Ltd., Mie Research Park, 363 Shiosaki, Hokusei-Cho, Inabe-Shi, Mie, 511-0406, Japan
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla,
California 92093, United States
^Immunology and Stem Cell Laboratories, Monash University, Building 75, Wellington Road, Clayton, Victoria 3800, Australia
S Supporting Information
b
ABSTRACT: Through the application of TRAP (target-related affinity profiling), we identifi-
ed a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of
monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-
dependent and selective with regard to other chemokines. The compounds, however, did not
antagonize the binding of 125I-labeled CCL2 to the CCR2 receptor nor did they block CCR2-
mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic
parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC50 = 80 nM) with excellent oral bioavailability in
rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease
onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.
’ INTRODUCTION
member of the C-C chemokine subfamily and acts primarily
through the C-C chemokine receptor 2 (CCR2) to recruit
monocytes, basophils, and T-cells to sites of inflammation.13
CCL2 has been implicated as an important mediator of
several diseases, including multiple sclerosis,14-16 rheumatoid
arthritis,17-19 and atherosclerosis.20-22 Therefore, drugs that
selectively block CCL2-induced leukocyte recruitment in these
diseases may hold significant therapeutic potential.23-25 This
premise prompted the search in recent years for small-molecule
CCR2 antagonists that would compete with CCL2 for binding to
the receptor without inducing a functional response. These
efforts identified several potent compounds, some of which
showed significant therapeutic activity in animal models of
inflammation. A number of these agents have advanced to clinical
trials in humans, where they have met with various degrees of
success.26 We report herein the discovery and optimization of a
series of heteroaroylphenylureas that inhibit CCL2-induced
chemotaxis of monocytes/macrophages both in vitro and in vivo
but do not antagonize the binding of CCL2 to the CCR2
receptor. In particular, compound 17 was identified as a potent
and selective chemotaxis inhibitor with significant oral efficacy in
animal models of rheumatoid arthritis and multiple sclerosis.
The migration of leukocytes from blood vessels into diseased
tissues is an important process in the initiation of normal inflam-
matory responses to certain stimuli or insults to the immune system.
However, this process is also involved in the onset and progression
of life-threatening inflammatory and autoimmune diseases, suggest-
ing that blocking leukocyte recruitment in these disease states may
be an effective therapeutic strategy.
The mechanism by which leukocytes leave the bloodstream
and accumulate at sites of inflammation encompasses at least
three distinct steps: (1) rolling along the vasculature, (2) arrest
and firm adhesion, and (3) transendothelial migration.1-5 The
second and third steps are mediated by proinflammatory cyto-
kines, adhesion molecules, and chemokines (chemoattractant
cytokines), low-molecular-weight proteins secreted by proin-
flammatory cells at the site of damage or infection that stimulate
directed cell migration or chemotaxis.6-8 The biological actions
of chemokines are mediated through their binding to specific
G-protein-coupled receptors (GPCRs) on the leukocyte surface.
This binding activates signaling pathways that lead to an increase
in leukocyte adhesion to the endothelium and subsequent
transmigration into the affected tissue.9,10
C-C chemokine ligand 2 (CCL2, previously referred to as
monocyte chemoattractant protein 1 or MCP-1) was one of the
first chemokines to be characterized biologically.11,12 It is a
Received: October 6, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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Table 1. Effect of Substitution on Ring A and Acylurea Linker
compd
R1
R2
R3
Cl
R4
chemotaxis IC50 (μM)a
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
Me
OH
H
3.9
H
Cl
1.6
H
Cl
Cl
H
1.5
H
CF3
OiPr
Ph
H
1.5
H
H
1.0
H
H
>10
>10
>10
>10
H
Ph
H
Me
Me
Cl
Figure 1. Lead optimization strategy.
Cl
H
a Inhibition of hCCL2-induced chemotaxis of THP-1 cells. Cells were
induced to migrate in response to hCCL2 in the presence of various
concentrations of compound. The concentration that reduced migration
by 50% (IC50) is shown.
3.9 μM and showed no cytotoxicity (CC50 > 50 μM) against this
cell line.
Following the discovery of 1, a medicinal chemistry program
was launched to assess the effect of modifying each of the three
structural components of the molecule, namely, ring A, the
acylurea linker, and the bicyclic ring system B-C. Small lipo-
philic substituents (e.g., Cl, CF3, O-iPr) on position 3 and/or
position 4 of ring A were well tolerated and provided a 2- to
4-fold improvement in potency in the chemotaxis assay; larger
groups (e.g., Ph) resulted in the loss of inhibitory activity, as did
alkylation of the urea nitrogens (Table 1). Replacement of the
acylurea linker by an acylguanidine (cf. 10, Figure 3) or a
conformationally rigid 1,2,4-oxadiazol-3-amine (cf. 11) also
suppressed activity, as did the transposition of the A and B-C
rings (cf. 12). This last observation suggested the existence of a
unidirectional mode of binding of these molecules relative to the
substituents on either side of the acylurea linker.
Replacement of the benzo[d][1,3]dioxole ring system B-C
with different heterocycles led to the identification of the
pyrazolo[3,4-b]pyridine derivative 13 (Figure 4) as a submicro-
molar inhibitor of CCL2-induced chemotaxis (IC50 = 0.7 μM)
with no cytotoxicity (CC50 > 50 μM). Interestingly, 13 possesses
structural features also present in other small-molecule CCR2
antagonists, specifically, a basic heterocyclic ring connected by a
spacing moiety (the acylurea linker) to a lipophilic aromatic
group.29
Figure 2. Structure of compound 1.
’ LEAD DISCOVERY AND OPTIMIZATION
Most of the research programs aimed at discovering small-
molecule CCR2 antagonists have been initiated by screening
compounds for their ability to disrupt binding of the human
CCL2 ligand to the human CCR2 receptor. Although this
strategy identified potent antagonists in vitro, several of these
compounds later showed low selectivity over other GPCRs, poor
correlation between binding affinity and functional activity, or
lack of pharmacological action in animal models of inflammation.
To circumvent these difficulties, we used a functional assay
involving inhibition of CCL2-induced chemotaxis as our initial
screening platform and tested compounds in vivo as expedi-
tiously as possible to determine whether functional activity
correlated with pharmacological activity. The lead optimization
strategy implemented throughout our program is shown in
(Figure 1).
Compounds active in the chemotaxis assay (IC50 < 1 μM)
were screened for cytotoxicity to distinguish between inhibition
of cell migration and overt cell toxicity and for selectivity toward
other chemokines. Those compounds with a good safety index
(defined as the ratio of CC50 to IC50) and high selectivity were
then tested in vivo to assess their pharmacokinetic-drug meta-
bolism (PKDM) and toxicity profiles and finally in animal
models of inflammation.
Examination of 13 in vivo revealed three hydroxylated meta-
bolites, which were assigned as 14, 15, and 16 on the basis of
LC-MS data (Figure 5).
With this information at hand, we focused our lead optimiza-
tion efforts on introducing structural modifications to 13 that
would prevent the formation of these phenolic metabolites.
Different combinations of substituents at the 3- and/or 4-posi-
tion of phenyl ring A were explored to preclude the formation of
14. The most effective solution involved removal of the 4-F
substituent and replacement of the electron-withdrawing 3-CF3
group by an electron-donating group of similar lipophilicity, such
as an isopropoxy. To prevent hydroxylation at the 6-position of
the pyrazolopyridine ring leading to 15, a salt-forming group was
The initial lead, compound 1 (Figure 2), was discovered using
TRAP (target-related affinity profiling), Telik’s highly efficient
drug discovery technology.27 As TRAP identifies druglike leads with
the screening of as few as 200 compounds, it is well suited to
complex biological assays such as the inhibition of cell migration.
Selected compounds were tested for their ability to inhibit hCCL2-
induced chemotaxis of THP-1 cells, a human monocytic leukemia
cell line that shares many properties with human monocyte-derived
macrophages.28 Compound 1 inhibited chemotaxis with an IC50 of
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Figure 3. Effect of modifying the acylurea linker or transposing rings A and B-C.
Figure 4. Structure and properties of compounds 13 and 17.
Figure 5. Metabolites of compound 13 in rat.
introduced at this position not only to block metabolic oxidation
but also to increase the aqueous solubility of the resulting
molecule. Among the several analogues prepared, 17
(Figure 4) showed improved solubility and a nearly 10-fold
increase in chemotaxis inhibitory potency (IC50 = 80 nM)
relative to 13.
was converted into the corresponding acyl isocyanate by reaction
with oxalyl chloride, and the acyl isocyanate was immediately
reacted with an aniline to give the final acylurea. In method B, the
carboxamide was reacted with an aryl isocyanate to afford the
acylurea directly. In cases where the isocyanate was not commer-
cially available, it was prepared by reaction of the corresponding
aniline with triphosgene. The acylureas were isolated by pre-
cipitation from the reaction media and further purified by
preparative HPLC or recrystallization.
’ CHEMISTRY
The aroyl- and heteroaroylphenylureas were synthesized by
either of two methods depending on the commercial availability
of the starting materials (Scheme 1). In method A, a carboxamide
Compound 17, in particular, was prepared according to the
sequence shown in Scheme 2. Commercially available 2,5-dimethyl-
2H-pyrazol-3-ylamine, 18, was converted into pyrazolo[3,4-b]
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pyridine-5-carbonitrile 19 via a Vilsmeier-Haack reaction fol-
lowed by condensation of the resulting aldehyde with ethyl
cyanoacetate and intramolecular cyclization to form the bicyclic
ring system. Treatment of 19 with phenylphosphonyl dichloride
followed by N,N,N0-trimethylethane-1,2-diamine and subse-
quent hydrolysis of the nitrile group provided carboxamide 20,
which was then reacted with 3-isopropoxyphenyl isocyanate to
afford 17 as the hydrochloride salt after treatment with HCl in
dioxane.
signals through CCR5, a receptor closely related to CCR2. It is
worth noting that a number of small-molecule CCR2 antagonists
reported in the literature also bind to CCR5 with a range of
relative affinities.30,31
Receptor Binding Studies. Compounds 1 and 13 were
evaluated for their ability to antagonize the binding of hCCL2
to CCR2 expressed in THP-1 or HEK293 cells. Surprisingly,
despite their potent and fairly selective chemotaxis inhibitory
activity, neither compound competed with the binding of 125I-
labeled CCL2 to the receptor (<10% inhibition of control-
specific binding at 10 μM). This observation suggests the
possibility that the compounds may be acting as noncompetitive
allosteric inhibitors of CCR2, a mode of action that has been
reported for other chemokine receptor antagonists.32,33
Compound 13 was also examined for its ability to bind to other
chemokine receptors (CCR4 and CXCR1/2), as well as to the
complement component 5a receptor (C5aR), all of which are also
potent mediators of leukocyte migration during inflammation. As
expected based on the chemotaxis data, the compound did not
inhibit binding (<10% inhibition at 10 μM) of 125I-CCL17
(TARC) to human recombinant CCR4 expressed in CHO-K1
cells or of 125I-CXCL8 (IL-8) to CXCR1/2 expressed in human
neutrophils or of 125I-hC5a to hC5aR expressed in human
recombinant CHO cells.
CCL2 Binding Studies. The possibility that the compounds
were binding to CCL2 rather than to CCR2 was examined using
two-dimensional heteronuclear shift correlation NMR spectroscopy.
Compound 17 was incubated with isotopically labeled 15N-CCL2
(ratio CCL2/compound = 1:1 and 2:1), and a two-dimensional
1H-15N heteronuclear single quantum coherence (HSQC) spec-
trum was recorded. No evidence of compound binding to CCL2 was
observed.
’ IN VITRO PHARMACOLOGY
Chemotaxis. All new compounds were tested for their ability
to inhibit hCCL2-induced migration of THP-1 cells. Briefly, the
cells were incubated with different concentrations of compound
in a modified Boyden chamber and induced to migrate in
response to 12.5 nM hCCL2. The extent of cell migration was
quantified by counting stained cells, and the concentration that
reduced migration by 50% was reported as the IC50. Represen-
tative data are shown in Figure 6.
Selectivity. To assess the degree of selectivity toward CCL2/
CCR2, representative compounds were tested for their ability to
inhibit chemotaxis mediated by the binding of other chemokines
to their cognate receptors. As shown in Table 2, compounds 13
and 17 were mostly selective for CCL2, as they did not
significantly inhibit chemotaxis induced by CCL3 (MIP-1R),
CCL5 (RANTES), CCL11 (eotaxin), CCL17 (TARC), or
CXCL8 (IL-8). However, both compounds did inhibit chemo-
taxis of human peripheral blood mononuclear cells induced by
CCL4 (MIP-1β), albeit with weaker potency. This chemokine
Scheme 1. Preparation of Aroyl- and
Heteroaroylphenylureas
Calcium Mobilization Studies. The binding of chemokines
to their receptors is transduced into a variety of biochemical and
physiological changes, including inhibition of cAMP synthesis,
mobilization of calcium from intracellular pools into the cytosol,
up-regulation of adhesion proteins, receptor desensitization and
internalization, and cytoskeletal rearrangements leading to
chemotaxis.34 The molecular details of the chemokine-receptor
interactions responsible for inducing signaling pathways that lead
to each of these physiological changes are still being elucidated.
Notwithstanding the complexity of these events, the rapid
increase in cytosolic free calcium (Ca2þ) concentration upon
CCR2 activation by CCL2 is frequently used as a functional assay
for measuring the activity of CCR2 antagonists. Compound 1 was
tested in this assay where it displayed a concentration-dependent
inhibition of CCL2-stimulated calcium flux in THP-1 cells.
However, this inhibition was weak and not significantly different
from that of the vehicle control even at 50 μM (Figure 7).
Scheme 2. Synthesis of Compound 17a
a Reagents: (a) DMF, POCl3; (b) NaOH, EtOH; (c) ethyl cyanoacetate; (d) PhPOCl2; (e) N,N,N0-trimethylethane-1,2-diamine, THF; (f) H2SO4; (g)
3-(Isopropoxy)phenylisocyanate, Et3N, toluene; (h) HCl, dioxane.
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Species Specificity. A significant number of the CCR2
antagonists disclosed in the literature have been optimized for
the human receptor and show little or no cross-reactivity to
rodent CCR2, precluding their pharmacological evaluation in
rodent models of inflammation. For this reason, we evaluated
select compounds for their ability to inhibit chemotaxis mediated
by mouse and/or rat CCL2. Although most compounds inhib-
ited chemotaxis regardless of CCL2’s origin, relative potencies
varied from one compound to another. For example, 13 inhibited
chemotaxis mediated by human and mouse CCL2 with similar
potency, whereas 17 showed a significant difference in chemo-
taxis IC50 values between human and mouse or rat CCL2
(Table 3). In spite of this difference, 17 proved to be highly
efficacious in rodent models of rheumatoid arthritis and multiple
sclerosis (vide infra).
CYP450 Inhibition. Compound 17 was tested for inhibition
of five major CYP450 isoenzymes at 1 and 100 μM. Very low
(e11%) or no inhibition was observed at the lower concentra-
tion. At 100 μM, the compound was found to be a moderate
inhibitor of CYP3A4, CYP2C9, and CYP2C19 and a stronger
inhibitor of CYP2D6 (Table 4). The clinical relevance of these
data with regard to potential drug-drug interaction, however, is
considered to be low.
Inhibition of Macrophage Recruitment. Thioglycollate-
Induced Peritonitis Model. The thioglycollate-induced perito-
nitis model represents a rapid screen for anti-inflammatory
activity in vivo. In this model, thioglycollate is injected in the
peritoneal cavity of mice to induce local recruitment of leuko-
cytes. Neutrophil accumulation usually reaches a peak at 6 h and
declines rapidly, while the number of monocytes/macrophages
gradually increases and reaches a peak at 72 h.35 Several studies
using CCR2 knockout or CCL2-deficient mice have demon-
strated a selective reduction of monocyte influx in this
model.36-38 CCL2 knockout mice were specifically unable to
recruit monocytes 72 h after intraperitoneal thioglycollate
administration.39 To ascertain whether our compounds were
able to inhibit monocyte migration in vivo, we tested 13 in this
model. Mice were injected with 3% Brewer’s thioglycollate broth
into the peritoneal cavity. Compound 13 was administered
subcutaneously 3 h prior to and immediately after the injection
of thioglycollate and then again 3 h after injection. An anti-CCL2
antibody was used as a positive control. Three days after the
thioglycollate administration, the mice were sacrificed and the
total number of elicited cells and MOMA-2 positive cells in the
peritoneal cavity were analyzed by flow cytometry.40 As shown in
Table 5, compound 13 at a dose of 5 mg/kg significantly reduced
the accumulation of total elicited cells and MOMA-2 positive
cells in the peritoneal exudate. This result demonstrated that the
in vitro chemotaxis inhibitory effect of 13 is also exhibited in vivo.
’ IN VIVO PHARMACOLOGY
Pharmacokinetics. Compound 17 was examined for oral
bioavailability in rat. It exhibited a long apparent half-life after
oral administration, suggesting that absorption may be a limiting
factor (Figure 8). The extrapolated oral bioavailability, however,
was quite good (F = 60%).
Figure 7. Effect of compound 1 on hCCL2-stimulated calcium flux in
THP-1 cells. Inhibition achieved by increasing concentration of test
compound was calculated as a percentage of the compound-free DMSO
control.
Figure 6. Inhibition of THP-1 cell chemotaxis by compounds 13 and
17: /, p < 0.05; //, p < 0.01 (two-tailed t test).
Table 2. Inhibition of Chemotaxis Mediated by Different Chemokine-Receptor Systems
chemotaxis IC50 (μM) vs the indicated chemokine and cell type
chemokine
chemokine receptor(s)
cell type
13
17
CCL2
CCL2
CCL3
CCL4
CCL5
CCL11
CCL17
CXCL8
CCR2
THP-1
PBMC
PBMC
PBMC
PBMC
0.7
0.08
0.2
CCR2
NT
>10
2.9
CCR1, CCR5
CCR5
>10
0.4
CCR1, CCR3, CCR5
CCR1, CCR3
CCR4
NT
NT
>10
>10
>10
>10
>10
>10
eosinophil
CCR4-transfected THP-1
PMN
CXCR1, CXCR2
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Table 3. Inhibition of Chemotaxis Mediated by Human, Mouse, and Rat CCL2a
compd
hCCL2 (THP-1 cells)
mCCL2 (mouse spleen cells)
rCCL2 (rat spleen cells)
13
17
0.70
0.08
1.4
NT
12.0
10.0
a Chemotaxis IC50 (μM) values vs the indicated chemokine and cell type: h = human; m = mouse; r = rat.
Table 4. CYP450 Inhibition by Compound 17
tissues both peak at approximately 2 weeks after immunization.
Injection of a neutralizing monoclonal antibody (mAb) against
rat CCL2 significantly decreased the number of exudate macro-
phages in the lesions and reduced swelling by 30% compared
with controls.43 Similarly, blockade of CCR2 with mAbs during
the first 2 weeks after immunization markedly improved clinical
signs and histological scores measuring leukocyte infiltration,
synovial hyperplasia, and bone and cartilage erosion. CCR2
blockade from days 21 to 36, on the other hand, aggravated
clinical and histological signs of arthritis and increased the
humoral immune response against collagen.44
Lewis female rats were immunized on day 0 and then again on
day 7 with bovine collagen type II. Vehicle, compound 17 (2, 10, or
30 mg/kg, b.i.d.), or methotrexate (MTX, 0.2 mg/kg, 3 ꢀ week)
was given orally for 28 days starting on day 0 immediately after the
first immunization. The degree of footpad swelling was measured
on days 12, 14, 21, and 28 after immunization. As shown in
Figure 10, treatment with 17 resulted in a dose-dependent inhibi-
tion of footpad swelling. On day 29, the animals were sacrificed
and radiographs of the hind paws were taken to assess the extent
of bone destruction. Radiological evaluation was performed by a
blinded independent observer using an arbitrary scale (Figure 11).
Treatment with 17 at 30 mg/kg b.i.d. significantly inhibited bone
resorption in the joints of these animals (Figure 12).
The concentration of 17 in plasma, tarsal homogenates, and bone
marrow (thigh bone) was measured 5 h after the last dosing on day
28 of the CIA study. As shown in Figure 13, compound 17
accumulated preferentially in the tarsal joints and bone marrow,
where it reached concentrations well above its IC50 against rat CCL2.
Adjuvant-Induced Arthritis Model of Rheumatoid Arthri-
tis. Compound 17 was also evaluated in the rat adjuvant-induced
arthritis (AIA) model using a therapeutic dosing schedule. AIA is a
form of (sub)chronic arthritis. Strains of rats have a varying genetic
susceptibility to AIA, whereas mice generally are not susceptible.
The disease is a T-cell-mediated autoimmune arthritis frequently
used to study immunological aspects of RA and as a model for
testing anti-inflammatory drugs.45 Increased expression of CCL2
and CCR2 has been observed in the knee joints of rats with AIA,
suggesting a role for this chemokine/receptor system in triggering
the mechanisms involved in the pathogenesis of AIA.46,47 Further-
more, postonset treatment of AIA-bearing rats with an inhibitor of
endogenous CCL2 (P8A-CCL2) improved clinical signs of arthritis
and histological scores measuring joint destruction, synovial lining,
macrophage infiltration, and bone erosion.48
Following a typical protocol, Lewis female rats were immu-
nized on day 0 by intradermal injection of Freund’s complete
adjuvant into the footpad of the right hind paw. Oral dosing with
vehicle, compound 17 (10 or 30 mg/kg, b.i.d.), or methotrexate
(MTX, 0.2 mg/kg, 3 ꢀ week) started on day 10, when macro-
phages had already begun to infiltrate the joint, and continued for
an additional 18 days. Paw swelling was assessed by measuring
the thickness of the footpad. Significant inhibition of footpad
swelling was achieved with 17 at 30 mg/kg b.i.d. (Figure 14).
EAE Model of Multiple Sclerosis. Several studies have
provided evidence that CCL2-dependent leukocyte chemotaxis
% inhibition
compd concn (μM) CYPA12 CYP3A4 CYP2C9 CYP2C19 CYP2D6
17
1
-12
-2
4
11
36
-8
-5
100
49
34
73
Suppression of DTH Response. The delayed-type hypersensi-
tivity (DTH) response is a well characterized model frequently
used as an indicator of cell-mediated immune status.41 The
model is dependent upon both T helper 1 (Th1) driven
responses and recruitment and chemotaxis to a local site. As a
result, the DTH functional response may be influenced by
disruption of Th1-driven, antigen-dependent T-cell develop-
ment, or mobilization of sensitized T-cells to a local site. The
model is usually performed by first injecting small quantities of an
antigen during the sensitization (or immunization) phase and
then again during the challenge (or inflammatory) phase. A
hallmark response is elicited, which includes induration, swelling,
and monocytic infiltration into the site of the lesion within 24-
72 h. CCR2 knockout or CCL2-deficient mice have significant
defects in both delayed-type hypersensitivity responses and
production of Th1-type cytokines.37,39 To determine the effect
of 17 in the murine DTH reaction, mice were first immunized
with sheep red blood cells (SRBC) and 4 days later challenged
with SRBCs administered intradermally in their footpad. Vehicle
or varying doses of 17 were given orally 1 h prior to and 2 h after
challenge, and the footpad thickness was measured on day 5. As
shown in Figure 9, treatment with 17 resulted in a dose-
dependent suppression of footpad swelling.
Collagen-Induced Arthritis Model of Rheumatoid Arthritis.
Compound 17 was evaluated prophylactically in the rat col-
lagen-induced arthritis (CIA) model. CIA is an animal model of
rheumatoid arthritis (RA) widely used to study disease patho-
genesis and to test potential antiarthritic agents. Arthritis is
normally induced in mice or rats by immunization with homo-
logous or heterologous type II collagen in an adjuvant. Suscept-
ibility to CIA is associated with major histocompatibility
complex class II genes, and the development of arthritis is
accompanied by a strong T- and B-cell response. The main
pathological features of CIA include a proliferative synovitis with
infiltration of polymorphonuclear and mononuclear cells, pan-
nus formation, marked cartilage destruction associated with
immune complex deposition on articular surfaces, bone resorp-
tion, and fibrosis. As in RA, proinflammatory cytokines such as
tumor necrosis factor R (TNF-R) and interleukin-1 β (IL-1β)
are highly expressed in the arthritic joints of mice and rats with
CIA, and blockade of these cytokines results in a reduction of
disease severity.42
Both CCL2 and CCR2 have been implicated in the pathogen-
esis of CIA in rats, with CCR2 reportedly having a dual role
during initiation and progression of the disease. CCL2 concen-
trations in the joint lavages and CCL2 mRNA levels in the joint
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Figure 8. Pharmacokinetics of compound 17 after a single intravenous or oral dose to nonfasted rats. Because of the long half-life of the compound,
AUC0-24 values are estimated.
Table 5. Inhibition of Macrophage Recruitment by
Compound 13 in the Thioglycollate-Induced Peritonitis
Model in Mice
been reported to be markedly resistant to developing EAE,
although susceptibility to EAE in different mice strains lacking
CCR2 has also been observed.50-53 Treatment of EAE-bearing
mice with an anti-CCL2 antibody or a small-molecule CCR2
antagonist, on the other hand, has been shown to reduce CNS
macrophage accumulation and decrease the clinical severity of
the disease.54,55 Therefore, we chose EAE as a disease model to
further evaluate the efficacy of compound 17.
cell number (ꢀ106)a
treatment
dose (mg/kg)
total cells
MOMA-2 positive cells
no treatment
vehicle
2.1 ( 0.3**
24.4 ( 1.1
19.7 ( 1.6*
12.3 ( 1.8**
1.2 ( 0.2**
18.5 ( 0.9
14.3 ( 1.2*
8.8 ( 1.2**
C57BL/6 mice were immunized subcutaneously with an ence-
phalitogenic emulsion of the myelin oligodendrocyte glycoprotein-
13
5
1
56,57
derived peptide MOG33-55
.
Starting on the day of the im-
anti-CCL2 Ab
munization (day 0), mice were given vehicle or 17, once or twice a
day orally, and were monitored daily for disease onset and severity of
clinical symptoms. As shown in Table 6, treatment with compound
17 at 30 mg/kg b.i.d. significantly delayed the onset of EAE.
Neurological impairment was quantified on an arbitrary scale
as follows: 0 = no detectable impairment; 1 = flaccid tail; 2 = hind
a Values are the mean ( SD: /, p < 0.05; //, p < 0.01 (two-tailed t test).
is a critically important event for the development of experi-
mental autoimmune encephalomyelitis (EAE), a mouse model of
chronic neuroinflammation resembling multiple sclerosis
(MS).49 Knockout mice lacking either CCL2 or CCR2 have
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limb weakness; 3 = hind limb paralysis; 4 = hind limb paralysis
and ascending paralysis; 5 = moribund or deceased. Figure 15
shows the mean clinical score for the vehicle and the 17-treated
groups as a function of the number of days after immunization.
Compound 17 ameliorated the symptoms of EAE in a dose-
dependent manner and essentially produced a complete resolu-
tion of clinical symptoms at 30 mg/kg b.i.d.
Histological examination of brain, cerebellum, and spinal cord
showed a significant decrease in inflammatory cell infiltrates
following treatment with 17 (Figure 16).
’ TOXICOLOGY STUDIES
Compound 17 was examined for overt toxicity in 6-week-old
male ICR mice after single (acute) and repeated (2-week) oral
dosing. In the acute study, no signs of toxicity were observed at
doses up to 100 mg/kg. Hypoactivity was observed at 500 mg/kg,
whereas CNS excitation (tonic convulsions, Straub tail) and
increased feeding behavior were observed at 1000 mg/kg.
In the 2-week repeated-dose study, mice were dosed orally
once a day for 14 days with vehicle or with 30, 100, or 300 mg/kg
of compound 17. All the animals survived the study, and no
abnormal behavior or significant body weight changes were
observed at any of the doses tested. Elevated liver enzymes
(AST and ALT) were observed at the highest dose tested (300
mg/kg). Histological examination showed signs of liver and
kidney toxicity (i.e., vacuolar degeneration) at this dose.
Figure 9. Effect of compound 17 on the DTH response in mice: /, p <
0.05; //, p < 0.01 (two-tailed t test).
’ SUMMARY AND CONCLUSIONS
Through the application of TRAP, we discovered a new class
of heteroaroylphenylureas that inhibit chemotaxis of monocytes/
macrophages induced by the chemokine CCL2. Medicinal
chemistry optimization of the initial lead for potency and
metabolic stability resulted in the identification of 17, a potent
small-molecule inhibitor of human CCL2-induced chemotaxis.
This inhibition was selective for CCL2, since 17 did not inhibit
chemotaxis induced by other chemokines except for CCL4,
which was inhibited with a >5-fold lower potency. In spite of
this selectivity, the new class of compounds did not compete with
the binding of radiolabeled CCL2 to the CCR2 receptor nor
did they significantly inhibit biochemical processes (e.g., Ca2þ
mobilization) that are triggered upon CCR2 activation by CCL2.
The in vivo pharmacologic profile of these compounds, however,
Figure 10. Effect of compound 17 on footpad swelling in the CIA
prophylactic model. MTX = methotrexate.
Figure 11. Bone destruction scoring in the CIA model. Scores are as follows: 0 = normal; 1 = slight damage; 2 = mild damage; 3 = obvious damage; 4 =
marked destruction.
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closely resembles the CCR2 or CCL2 knockout phenotypes in
classical models of macrophage trafficking and parallels that of
other selective CCR2 antagonists described in the literature.55
Indeed, the reduction of monocyte/macrophage infiltration in
the thioglycolate-induced peritonitis model observed with 13
and the impaired delayed-type hypersensitivity response seen
with 17 mirror effects reported for CCR2 or CCL2 knockout
mice, suggesting that the compounds block the pharmacological
actions mediated by this chemokine/receptor interaction. Inter-
estingly, a previous DTH study using CCL2 knockout mice
showed a reduction in the number of F4/80-positive macro-
phages in the ears of mice undergoing the DTH reaction, but the
swelling response was normal.39 The result of our DTH study
with 17 has more similarities with that reported for a small-
molecule CCR2 antagonist which reduced not only monocyte
influx but also swelling.55 It is well established that macrophages
accumulate at the DTH site and become activated through the
CD4 Th1 cell-cytokine-macrophage axis, producing various
proinflammatory cytokines and chemotactic factors that lead to
local inflammation.
Figure 12. Effect of compound 17 on bone resorption in the CIA
prophylactic model. Radiographs of both hind paws were taken and
bone condition graded from 0 = normal to 4 = marked destruction. The
average of both legs was calculated and assigned the score shown. NT =
no treatment. MTX = methotrexate. /, p < 0.05; //, p < 0.01 (two-tailed
t test).
The efficacy observed in the rat CIA model following pro-
phylactic dosing of 17 is of particular relevance given that
different outcomes were reported in this disease model when
different methods and schedules of CCL2 inhibition were used.
Blockade of CCR2 with an anti-CCR2 antibody before the onset
of CIA resulted in disease amelioration. In contrast, blockade
with the same antibody after disease onset (i.e., therapeutic
administration) markedly aggravated the clinical and histological
signs of arthritis.44 Similarly, CCL2(9-76), a CCL2 antagonist
that blocks CCL2 binding to CCR2, prevented the spontaneous
onset of arthritis in the MRL-lpr mouse model but was less
effective when given only after the disease had already
developed.58 Compound 17 was also efficacious in the EAE
mouse model of MS when administered prophylactically, that is,
under conditions that replicate previous studies using CCR2
knockout or CCL2-null mice.50-52 On the other hand, thera-
peutic dosing of 17 in the AIA model resulted in significant
reduction of footpad swelling, in agreement with the effects
observed with other small-molecule CCR2 antagonists when
dosed after the onset of the disease.55 These observations suggest
Figure 13. Concentration of compound 17 (logarithmic scale) in
plasma, tarsal joints, and bone marrow on day 28 of the CIA prophylactic
model study.
Figure 14. Effect of compound 17 on footpad swelling in the AIA therapeutic model. MTX = methotrexate.
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that the outcome of inhibiting CCL2 or blocking CCR2 depends
on the disease model, the nature of the inhibitor, and the timing
of its administration.
recognized by a variety of small-molecule ligands. Allosteric
modulators may even offer certain advantages over classic
orthosteric ligands as therapeutic agents, including the potential
for greater GPCR-subtype selectivity and safety.61,62
Although we cannot rule out other mechanisms of action to
explain the in vitro and in vivo pharmacologic profile of
compounds such as 17, the data presented herein support the
hypothesis that these novel acylureas are antagonists of CCL2/
CCR2 signaling and that they exert this action by binding to the
receptor in an allosteric manner. Indeed, it has been shown that
specific features of CCL2 can induce different conformations in
CCR2 that are coupled to separate postreceptor signaling path-
ways. For example, certain mutations of surface-exposed CCL2
residues provided peptides that were agonists of pathways
involving inhibition of cAMP synthesis and Ca2þ mobilization
but antagonists of chemotaxis. These results demonstrate that
the former two events are not sufficient to drive chemotaxis and
that different ligands may be able to trap CCR2 in conformations
that are unable to stimulate the pathways required for
chemotaxis.59 On the other hand, site-directed mutagenesis of
CCR2 provided mutants with normal CCL2 binding but that
were unable to elicit Ca2þ mobilization or inhibit adenylate
cyclase at any CCL2 concentration. Some of these mutants,
however, showed lamellipodia formation and were capable of
inducing chemotaxis.60 These examples illustrate the observation
that most classic GPCR antagonists bind to the receptor’s
orthosteric site, that is, the site recognized by the endogenous
agonist. However, it has become evident that GPCRs possess
additional, extracellular, allosteric binding sites that can be
Several of the CCR2 antagonists reported in the literature lack
rodent cross-reactivity, precluding their pharmacological evalua-
tion in animal models of inflammation. Compound 17 proved to
be a more potent inhibitor of chemotaxis induced by human
CCL2 than by mouse or rat CCL2, but it was nonetheless
efficacious in rodent models of arthritis and multiple sclerosis,
most likely because of accumulation at the sites of inflammation.
We can only speculate that, based on the chemotaxis data, it
should be even more efficacious in man.
’ EXPERIMENTAL SECTION
General. All reactions were performed under an atmosphere of dry
nitrogen. Commercial reagents were used without further purification.
All final compounds were purified by preparative HPLC on a Varian
PrepStar SD-1 system or by recrystallization from the indicated solvent.
Final purity was determined by RP-HPLC analysis on a Varian Prostar
210 system using a Microsorb R0089200C5 column (250 mm ꢀ 4.6
mm, 3.5 μm), linear gradient elution with H2O/MeCN containing
0.05% TFA, and UV detection at 254 nm. Purity and retention time [e.g.,
HPLC: tR = 14.6 min (97% purity)] are given for each compound.
Unless otherwise noted, all tested compounds had g95% purity.
Molecular parent ion identity was determined by tandem LC-MS
analysis on an Agilent 1100 LC/MSD instrument using a Zorbax Eclipse
XDB-C8 column (2.1 mm ꢀ 50 mm, 3.5 μm), linear gradient elution
with 10 mM aqueous ammonium acetate/MeCN containing 0.1%
AcOH, UV detection at 254 nm, and atmospheric-pressure electrospray
ionization (ESI) in either a negative (-) or positive (þ) mode. 1H NMR
spectra were recorded at 400 MHz on a Varian Mercury-400 spectro-
meter; chemical shifts (δ) are reported in ppm downfield from internal
tetramethylsilane (TMS) and coupling constants (J) in hertz (Hz).
The synthesis of representative compounds is given below.
Table 6. Effect of Compound 17 on the Development of
Acute EAE
treatment
dose (mg/kg)
day of disease onseta
vehicle
17.3 ( 1.6
17
30 (b.i.d.)
32.7 ( 11.1*
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-chloro-4-hydro-
xyphenyl)urea (1). Aqueous ammonium hydroxide (28-30% am-
monia, 30 mL) was added dropwise to ice-cooled piperonyloyl chloride
a Values are the mean ( SD: /, p < 0.05 (two-tailed t test with unequal
variance).
Figure 15. Effect of compound 17 on the clinical symptoms of acute EAE. Animals in the vehicle and 17 10 mg/kg group were sacrificed on days 22 and
26, respectively, because of severe EAE.
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4.60 (q, 1H, J= 6.0 Hz), 6.16 (s, 2H), 6.66 (d, 1H, J=8.0Hz), 7.05(m, 2H),
7.22 (m, 2H), 7.59 (s, 1H), 7.68 (d, 1H, J = 8.2 Hz), 10.82 (s, 2H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-phenylphenyl)
urea (6). White solid (39% yield). HPLC: tR = 17.6 min (98%
1
purity). MS ESI(-) = 359 (M - H). H NMR (DMSO-d6) δ: 6.16
(s, 2H), 7.07 (d, 1H, J = 8.2 Hz), 7.14 (m, 2H), 7.49 (m, 3H,), 7.61 (s,
2H), 7.68 (m, 3H), 7.85 (s, 1H,), 10.86 (s, 1H), 10.96 (s, 1H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(4-phenylphenyl)
urea (7). White solid (80% yield). HPLC: tR = 18.2 min (96%
1
purity). MS ESI(-) = 359 (M - H). H NMR (DMSO-d6) δ: 6.16
(s, 2H), 7.07 (d, 1H, J = 8.2 Hz), 7.34 (t, 1H, J = 7.3 Hz), 7.46 (t, 2H, J =
7.8 Hz), 7.61 (s, 2H), 7.68 (m, 6H,), 10.89 (s, 1H), 10.94 (s, 1H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-chlorophenyl)-
1-methylurea (8). Lithium bis(trimethylsilyl)amide (0.38 mL of 1
M solution in THF) was added to a solution of 2 (0.10 g, 0.31 mmol) in
DMF (5 mL), and the mixture was stirred for 30 min. Methyl iodide
(69 μL, 1.1 mmol) was added, and the resulting solution was stirred at
room temperature for an additional 4 h. Water (15 mL) was added, and
the solution was filtered. The filtrate was collected, and the solvents
were removed in vacuo. The resulting solid was triturated with water
(30 mL), filtered, and dried under high vacuum to give 8 (16 mg, 16%
yield). HPLC: tR = 17.0 min (85% purity). MS ESI(-) = 331 (M - H).
1H NMR (DMSO-d6) δ: 3.32 (s, 3H), 6.07 (s, 2H), 6.90 (d, 1H, J = 8.1
Hz), 7.01 (s, 1H), 7.09 (d, 1H, J = 8.1 Hz), 7.24 (m, 2H), 7.32 (m, 1H),
7.73 (s, 1H), 11.41 (s, 1H).
Figure 16. Effect of compound 17 on cellular infiltrates following acute
EAE. Histological scores are as follows: 0 = no inflammation; 1 = very
small cellular infiltrate in the perivascular areas and meninges; 2 = mild
cellular infiltrate; 3 = moderate cellular infiltrate; 4 = severe cellular
infiltrate; 5 = very large and extensive cellular infiltrate. /, p < 0.05; //, p
< 0.01 (two-tailed t test).
(3.02 g, 16.36 mmol). The ice bath was removed, and the mixture was
stirred at room temperature for 1 h. The resulting solid was collected by
filtration, washed with water, and dried under high vacuum to yield
benzo[d]1,3-dioxolene-5-carboxamide as a white powder. A portion of
this material (0.199 g, 1.20 mmol) was suspended in anhydrous CH2Cl2
(6 mL) and treated with a 2 M solution of oxalyl chloride in CH2Cl2
(0.91 mL, 1.82 mmol). The mixture was heated at gentle reflux for 16 h
and then cooled to room temperature. The solvent was removed under
reduced pressure to give benzo[d][1,3]dioxole-5-carbonyl isocyanate,
which was immediately dissolved in anhydrous THF and added to an
ice-cooled solution of 4-amino-2-chlorophenol (0.174 g, 1.21 mmol) in
anhydrous THF (2 mL). The ice bath was removed, and the mixture was
stirred at room temperature for 2 h. The resulting solid was collected by
filtration, washed with CH2Cl2, and dried under high vacuum to afford
0.183 g (46% yield) of 1 as a white powder. HPLC: tR = 14.6 min (97%
purity). MS ESI(-) = 333** (M - H). 1H NMR (DMSO-d6): δ 6.15 (s,
2H), 6.94 (d, 1H, J = 8.8 Hz), 7.06 (d, 1H, J = 8.2 Hz), 7.23 (d, 1H, J = 8.8
Hz), 7.64 (m, 2H), 10.02 (s, 1H), 10.68 (s, 1H), 10.82 (s, 1H).
Compounds 2-7 were prepared following a similar procedure.
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-chlorophenyl)
urea (2). White powder (38% yield). HPLC: tR = 17.4 min (95%
purity). MS ESI(-) = 317 (M - H). 1H NMR (DMSO-d6) δ: 6.16 (s,
2H), 7.06 (d, 1H, J = 8.2 Hz), 7.15 (d, 1H, J = 7.8 Hz), 7.35 (t, 1H, J = 8.0
Hz), 7.44 (d, 1H, J = 8.3 Hz), 7.59 (d, 1H, J = 1.6 Hz), 7.67 (d, 1H, J = 8.2
Hz), 7.83 (d, 1H, J = 2.0 Hz), 10.93 (s, 1H), 10.95 (s, 1H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3,4-dichlorophenyl)
urea (3). White solid (41% yield). HPLC: tR = 18.5 min (96% purity).
MS ESI(-) = 351 (M -H). 1H NMR (DMSO-d6) δ: 6.16 (s, 2H), 7.07(d,
1H, J = 7.8 Hz), 7.59 (t, 3H, J = 8.1 Hz), 7.68 (d, 1H, J = 7.9 Hz), 8.01 (s,
1H,), 10.96 (s, 1H), 10.97 (s, 1H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-(trifluoromethyl)
phenyl)urea (4). White solid (88% yield). HPLC: tR = 16.3 min (98%
purity). MS ESI(þ) = 353 (M þ H). 1H NMR (DMSO-d6) δ: 6.15 (s,
2H), 7.06 (d, 1H, J = 8.2 Hz), 7.44 (d, 1H, J = 7.3 Hz), 7.59 (m, 2H), 7.69
(d, 1H, J = 8.2 Hz), 7.78 (d, 1H, J = 7.7 Hz), 8.01 (s, 1H,), 10.94 (s, 1H),
11.05 (s, 1H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-chlorophenyl)-
1,3-dimethylurea (9). A solution of 2 (0.098 g, 0.307 mmol) in
DMF (5 mL) was treated with sodium hydride (0.025 g, 0.625 mmol).
and the mixture was stirred at room temperature for 30 min. Methyl
iodide (39 μL, 0.626 mmol) was added and the resulting solution was
stirred for an additional 3 h. The reaction was quenched with water
(50 mL), and the mixture was extracted with CH2Cl2 (3 ꢀ 50 mL). The
organic layers were combined, washed with water, dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on silica gel (1:1 EtOAc/Hex) to give 9
(13 mg) as a colorless oil. HPLC: tR = 14.9 min (97% purity). MS
ESI(þ) = 347 (M þ H). 1H NMR (DMSO-d6) δ: 3.20 (s, 3H), 3.30 (s,
3H), 6.04 (s, 2H), 6.41 (s, 1H), 6.64 (d, 1H, J = 7.5 Hz), 6.69 (s, 1H),
6.77 (d, 1H, J = 8.2 Hz), 6.89 (d, 1H, J = 7.8 Hz), 7.22 (m, 2H).
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-chlorophenyl)
guanidine (10). Cyanamide (100 mg, 2.38 mmol) was dissolved in
water (1 mL) and treated with piperonyl chloride (440 mg, 2.38
mmol). Aqueous NaOH (190 μL of a 12.5 N solution) was added, and
the mixture was stirred for 2 h. 3-Chloroaniline (288 mg, 240 mmol)
was added, and the pH of the solution was adjusted to pH 3 by
addition of concentrated HCl. The mixture was diluted with water
(5 mL), refluxed for 1 h, and then cooled to room temperature. The
resulting solid was collected by filtration, dissolved in 30% aqueous
NaOH (1 mL), and the solution was diluted with water (50 mL). The
precipitated solid was filtered and dried under high vacuum to afford
10 as a white solid (106 mg, 14% yield). HPLC: tR = 3.04 min (90%
purity). MS ESI(-) = 316 (M - H). 1H NMR (DMSO-d6) δ: 6.09 (s,
2H), 6.95 (d, 1H, J = 8.1 Hz), 7.14 (d, 1H, J = 7.2 Hz), 7.36 (m, 2H),
7.53 (s, 1H), 7.69 (d, 1H, J = 8.2 Hz), 7.91 (s, 1H), 9.31 (s, 1H).
5-(Benzo[d][1,3]dioxol-5-yl)-N-(3,4-dichlorophenyl)-1,2,4-
oxadiazol-3-amine (11). Cyanamide (82 mg, 1.95 mmol) was
dissolved in water (1 mL) and treated with piperonyl chloride
(369 mg, 1.95 mmol). Aqueous sodium hydroxide (160 μL of a 12.5 N
solution) was added, and the mixture was stirred for 2 h. 3,4-Dichloroani-
line (290 mg, 1.79 mmol) was added, and the pH of the solution adjusted
to pH 3 by addition of concentrated HCl. The mixture was diluted with
water (5 mL), refluxed for 1 h, and then cooled to room temperature.
The resulting solid was collected by filtration, dissolved in 30% aqueous
NaOH (1 mL) and the solution diluted with water (50 mL). The
1-(Benzo[d][1,3]dioxole-6-carbonyl)-3-(3-isopropoxyphenyl)
urea(5). White solid (64% yield). HPLC: tR = 16.7 min (99% purity). MS
ESI(-) = 341 (M - H). 1H NMR (DMSO-d6) δ: 1.27 (d, 6H, J = 6.0 Hz),
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precipitated solid was collected by filtration and dried under high
vacuum. This solid was dissolved in 3:1 CH2Cl2/MeOH (4 mL) and
the solution treated with 13% NaOCl (4 mL). The resulting biphasic
solution was stirred at room temperature for 2 h. The aqueous layer was
discarded, and the organic layer was treated with Na2SO3 (50 mg in 2 mL
water) and methanol (5 mL). The resulting solution was refluxed for
16 h. The solvents were removed under reduced pressure and the residue
was chromatographed on silica gel (1:1 EtOAc/Hex) to give 11 (16 mg)
as a white solid. HPLC: tR = 9.25 min (97% purity). MS ESI(-) = 348
(M - H). 1H NMR (DMSO-d6) δ: 6.20 (s, 2H), 7.17 (d, 1H, J = 8.2 Hz),
7.45 (d, 1H, J = 8.8 Hz), 7.50 (s, 1H), 7.58 (d, 1H, J = 8.9 Hz), 7.66 (d,
1H, J = 8.2 Hz), 7.75 (s, 1H), 10.40 (s, 1H).
1-(Benzo[d][1,3]dioxol-5-yl)-3-(3-chlorobenzoyl)urea (12).
To a solution of 3,4-(methylene)dioxyaniline (82.6 mg, 0.60 mmol) in
THF (2 mL) was added 3-chlorobenzoyl isocyanate (1 mL of a 0.60 M
solution in THF). The solution was stirred at room temperature for 1 h.
The precipitated solid was collected by filtration, washed with CH2Cl2, and
dried under high vacuum to afford 12(85.0 mg, 44% yield) as a white solid.
HPLC: tR = 16.5 min (88% purity). MS ESI(-) = 317 (M - H). 1H NMR
(DMSO-d6) δ: 6.02(s, 2H), 6.98 (m, 2H), 7.28 (s, 1H), 7.57 (t, 1H, J =7.9
Hz), 7.73 (d, 1H, J = 9.0 Hz), 7.95 (d, 1H, J = 7.8 Hz), 8.05 (s, 1H), 10.56
(s, 1H), 11.08 (s, 1H).
1-(1,3-Dimethyl-1H-pyrazolo[3,4-b]pyridine-5-carbonyl)-
3-(4-fluoro-3-(trifluoromethyl)phenyl)urea (13). 4-Chloro-
1,3-dimethylpyrazolo[5,4-b]pyridine-5-carboxamide (3.5 g, 18.4 mmol)
was dissolved in hot toluene (30 mL) and azeotroped for 1 h. The
solution was allowed to cool to room temperature and treated with
4-fluoro-3-(trifluoromethyl)phenyl isocyanate (4.6 g, 22.1 mmol). The
mixture was refluxed for 16 h and then cooled to room temperature. The
precipitated solid was filtered, washed with MeOH, and dried under
high vacuum to give 13 (3.6 g, 49% yield) as a white solid. HPLC: tR =
16.2 min (99% purity). MS ESI(-) = 394 (M - H). 1H NMR (DMSO-
d6) δ: 2.55 (s, 3H), 4.01 (s, 3H), 7.51 (t, 1H, J = 9.7 Hz), 7.90 (m, 1H),
8.12 (d, 1H, J = 6.3 Hz), 8.90 (s, 1H), 9.08 (s, 1H), 10.93 (s, 1H), 11.25
(s, 1H).
for 16 h and then cooled to room temperature. The solvent was removed
under reduced pressure, and the residue was taken up in THF (100 mL) and
treated with HCl (4 N in dioxane, 7 mL). The resulting solid was filtered, dried
under high vacuum, and then recrystallized from MeCN to give 17 (2.55 g,
74% yield) as a white solid. HPLC: tR = 10.5 min (99% purity). MS ESI(-)
=
466 (M - H). 1H NMR (DMSO-d6) δ: 1.27 (d, 6H, J = 6.0 Hz), 2.41 (s,
3H), 2.53 (t, 2H, J = 6.4 Hz), 2.83 (s, 6H), 2.98 (s, 3H), 3.82 (s, 3H), 3.92 (t,
2H, J=6.4Hz),4.60(q,1H,J= 6.0 Hz), 6.68 (m, 1H), 7.05 (m, 1H), 7.22 (m,
2H), 8.24 (s, 1H), 10.58 (s, 1H), 11.09 (s, 1H).
Biological Assays and Procedures. Chemotaxis Assay. Cell
migration experiments were performed in a 48-well modified Boyden
microchemotaxis chamber with a 5 μm pore size PVP-coated polycarbo-
nate filter membrane (Neuro Probe Inc., Cabin John, MD). Compounds
were prepared as 10 mM stock solution in DMSO and serially diluted with
medium. THP-1 cells were washed with RPMI 1640 medium supplemen-
ted with 0.5% (w/v) bovine serum albumin (BSA) and 25 mM HEPES,
pH 7.4, and suspended at a density of 4 ꢀ 106 cells/mL in the same
medium. A 150 μL aliquot of this suspension was treated with an equal
volume of test compound solution and the mixture incubated at 37 ꢀC for
15 min. The lower chamber was loaded with 26 μL of a 2.5 nM solution of
hCCL2 (PeproTech) in medium. The filtermembrane was placedover the
lower chamber, followed by a silicone rubber gasket and the upper
chamber. A 50 μL aliquot of the THP-1 cell suspension containing the
test compound was added to the upper chamber and the assembly
incubated in a 5% CO2 atmosphere at 37 ꢀC for 2 h. The chamber was
then disassembled, and the cells remaining on the upper surface of the filter
were scraped off with a rubber scraper. The filter was fixed with methanol,
stainedwith Diff-Quik solution, and mounted on a glass slide. The cells that
had migrated across the filter onto the lower surface were then counted by
microscopic observation. Chemotaxis studies using other chemokines and
cell types were performed similarly.
Receptor Binding Assay. In vitro radioligand receptor binding assays
were performed by Cerep (Poitiers, France; CCR2 ref no. 0362; C5a ref
no. 0042) and Ricerca (formerly MDS Pharma Services, Taipei, Taiwan;
CCR4 catalog no. 217660; CXCR1/2 - IL-8 nonselective catalog no.
244300).
1-(6-((2-(Dimethylamino)ethyl)(methyl)amino)-1,3-dimethyl-
1H-pyrazolo[3,4-b]pyridine-5-carbonyl)-3-(3-isopropoxyphenyl)
urea Hydrochloride (17). A solution of 5-amino-1,3-dimethylpyrazole-4-
carbaldehyde (6.15 g) in ethyl cyanoacetate (10 mL) was stirred at 185 ꢀC for
3 h. The mixture was allowed to cool to room temperature, and the
precipitated solid was filtered, washed with EtOAc, and dried under high
vacuum to give 6-hydroxy-1,3-dimethylpyrazolo[5,4-b]pyridin-5-carbonitrile
as a white powder. A portion of this material (2.18 g) was dissolved in
phenylphosphonic dichloride (10 mL) and stirred at 150 ꢀC for 17 h. The
solution was cooled to room temperature and poured into water. The mixture
was extracted with EtOAc. The organic layer was decanted, washed with
saturated NaHCO3 solution, dried over anhydrous Na2SO4, and filtered. The
solvent was removed under reduced pressure to give a white solid. This
material (2.16 g) was dissolved in THF (50 mL) and treated with N,N,N0-
trimethylethane-1,2-diamine (10.6 mL). The mixture was heated at reflux for
20 h, cooled to room temperature, and extracted with EtOAc. The organic
extracts were dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to afford 6-{[2-(dimethylamino)ethyl]methylamino}-1,3-
dimethylpyrazolo[5,4-b]pyridine-5-carbonitrile as a brown oil. This material
(2.96 g) was dissolved in concentrated H2SO4 (10 mL), and the solution was
stirred at 60 ꢀC for 15 h. The mixture was then neutralized with saturated
NaHCO3 solution and extracted with CH2Cl2. The extracts were washed with
NaCl solution, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by recrystallization from EtOAc-hexanes
to give 6-{[2-(dimethylamino)ethyl]methylamino}-1,3-dimethylpyrazolo-
[5,4-b]pyridine-5-carboxamide as a white solid. A portion of this material
(2.0 g, 6.8 mmol) was dissolved in hot toluene (20 mL) and azeotroped for
2 h. The solution was cooled to room temperature and treated with
3-isopropoxyphenyl isocyanate (1.7 g, 9.6 mmol). The mixture was refluxed
Binding to CCL2 Studies. Compound 17 was incubated with iso-
topically labeled 15N-CCL2 (250 μM) in either a 1:1 or a 1:2 CCL2-to-
compound ratio, and the 2D 1H/15N shift correlation NMR spectrum
(HSQC spectrum) was recorded.
Calcium Mobilization Assay. Intracellular calcium flux was mea-
sured as the increase in fluorescence emitted by the calcium-binding
fluorophore Fluo-3 when preloaded cells were stimulated with CCL2.
THP-1 cells were resuspended in serum-free RPMI 1640 medium at a
density of 2 ꢀ 106 cells/mL. Cells were loaded with 3 μM of Fluo-3AM
(Sigma-Aldrich) and 0.1% F127 and incubated at 37 ꢀC for 30 min. The
cells were washed twice to remove excess fluorophore, resuspended in
serum-free RPMI 1640 medium at a density of 5 ꢀ 106 cells/mL, and
kept at room temperature. Cells were pretreated with either DMSO or
test compound for 30 min. To detect intracellular calcium fluxed by flow
cytometry, pretreated cells were collected for 15 s and then continuously
from 20 to 120 s after adding 100 ng/mL hCCL2, and the fluorescence
shift was monitored. The inhibition achieved by increasing the concen-
tration of test compound was calculated as a percentage of the
compound-free DMSO control.
Thioglycolate-Induced Peritonitis in Mice. Six-week-old male ICR
mice (n = 5) were injected intraperitoneally with 2 mL of 3% Brewer’s
thioglycollate broth (Difco, Detroit, MI). The test compounds were
administered subcutaneously immediately after the thioglycollate injec-
tion and then again at 3 and 6 h. An anti-CCL2 antibody was used as a
positive control. Three days after the thioglycollate administration, the
mice were sacrificed and the total number of elicited cells and MOMA2
positive cells in the peritoneal exudates were analyzed using a Beckman
Coulter Epics XL flow cytometer.
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Delayed-Type Hypersensitivity (DTH) Response in Mice. Six-week-
old male ICR mice (n = 8) were first immunized by intravenous
administration of 1 ꢀ 106 sheep red blood cells (SRBC) and 4 days
later challenged with 2 ꢀ 108 SRBC administered intradermally on their
footpad. Vehicle or test compound was given orally 1 h prior to and 2 h
after challenge, and the footpad thickness was measured on day 5 using a
plethysmometer. Swelling was quantified as the mean percentage of
footpad thickness relative to that of the vehicle-treated group.
’ ASSOCIATED CONTENT
S
Supporting Information. Analytical data (HPLC, MS,
b
1
and H NMR) for compounds 8, 10, and 12; AST and ALT
values and histological changes in liver and kidney observed with
17 at 300 mg/kg. This material is available free of charge via the
Internet at http://pubs.acs.org.
Collagen-Induced Arthritis (CIA) in Rat. CIA was induced in 6-week-
old female Lewis SPF rats (n = 8/group) by intradermal injection at the
base of the tail of 900 μg of bovine tail tendon type II collagen (Collagen
Gijutsu Kenshukai, Tokyo, Japan) emulsified in 600 μL of Freund’s
complete adjuvant (Difco, Detroit, U.S.) containing 4 mg/mL Myco-
bacterium tuberculosis on day 0 and boosted with another injection of
450 μg/300 μL of the collagen emulsion on day 7. Rats were dosed twice
daily by gavage with the test compound suspended in 5% (w/v) aqueous
gum arabic (GA) or an equal volume of GA solution; methotrexate,
given 3 times per week, was used as a positive control. The volume of the
rat’s hind limbs was measured with a plethysmometer on days 0, 12, 14,
21, and 28, and inflammation was quantified as the mean percentage of
increased volume over that at day 0. On day 29, the animals were
sacrificed and radiographs of the hind paws were taken to assess the
extent of bone destruction. Assessment was made in a blind manner
using the following scale: 0, normal; 1, slight damage; 2, mild; 3, obvious
damage; 4, marked bone destruction. The average of both legs was
calculated and assigned as the score of the bone destruction.
Adjuvant-Induced Arthritis (AIA) in Rat. AIA was induced in
6-week-old female Lewis SPF rats (n = 8/group) by intradermal
injection into the footpad of the right hind paw of 100 μL of PBS-
emulsified Freund’s complete adjuvant (Difco, Detroit, U.S.) contain-
ing 10 mg/mL Mycobacterium butyricum. Starting on day 10 after the
immunization, rats were dosed twice daily by gavage with the test
compound suspended in 5% (w/v) aqueous gum arabic (GA) or an
equal volume of vehicle; methotrexate, given 3 times per week, was
used as a positive control. The volume of the left hind limbs of the rats
was measured with a plethysmometer on days 0, 8, 15, 21, and 28, and
inflammation was quantified as the mean percentage of increased
volume over that at day 0.
Experimental Autoimmune Encephalomyelitis (EAE) in Mice. EAE
was induced in C57BL/6 mice (n = 10/group) by subcutaneous
injection into the flanks of 150 μg of encephalitogenic peptide
MOG35-55 (MEVGWYRSPFSRVVHLYRNGK; Auspep, Melbourne,
Australia) emulsified in Freund’s complete adjuvant (Difco, Detroit,
U.S.) containing 4 mg/mL of Mycobacterium tuberculosis. Mice were
then immediately injected intravenously with 350 ng of pertussis
vaccine (List Biological Laboratories, Campbell, U.S.) and again 48 h
later. Compounds were dissolved in 3% aqueous Tween 80 at
4 mg/mL and stored at 4 ꢀC. Starting on the day of immunization
(day 0), mice were dosed b.i.d. with test compound or an equal volume
of water given orally and monitored daily for neurological impairment.
Clinical scoring was based on an arbitrary scale of 0-5 as follows: 0, no
detectable impairment; 1, flaccid tail; 2, hind limb weakness; 3, hind
limb paralysis; 4, hind limb paralysis and ascending paralysis; 5,
moribund or dead. A semiquantitative assessment of histological
lesions was performed at the end of the study by staining sections of
the brain and spinal cord with hematoxylin and eosin and scoring them
using the following scale: 0, no inflammation; 1, few very small cellular
infiltrate in the perivascular areas and meninges; 2, mild cellular
infiltrate; 3, moderate cellular infiltrate; 4, severe cellular infiltrate; 5,
very large and extensive cellular infiltrate.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 650-845-7752. Fax: 650-845-7800. E-mail: elaborde@
telik.com.
’ ACKNOWLEDGMENT
We thank Steven Anuskiewicz and Lisa Meng for their
assistance with statistical analyses and Carrol Strain for her
invaluable help proofreading and formatting the original docu-
ment. T.M.H. acknowledges grant support from the National
Institutes of Health (Grant RO1-AI37113).
’DEDICATION
†We dedicate this manuscript to the memory of Dr. Reinaldo
(“Rey”) Gomez.
’ ABBREVIATIONS USED
AIA, adjuvant-induced arthritis; CIA, collagen-induced arthritis;
CCL2, C-C chemokine ligand 2; CCR2, C-C chemokine
receptor 2; DTH, delayed-type hypersensitivity; EAE, experi-
mental autoimmune encephalomyelitis; GPCR, G-protein-
coupled receptor; IU/L, international units per liter; MCP-1,
monocyte chemoattractant protein 1; MOG, myelin oligoden-
drocyte glycoprotein; MS, multiple sclerosis; RA, rheumatoid
arthritis; SD, standard deviation; TRAP, target-related affinity
profiling
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