Diaryl substituted pyrazoles as potent CCR2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.10.060

We have identified and synthesized a series of diaryl substituted pyrazoles as potent antagonists of the chemokine receptor subtype 2. Structure-activity relationship studies directed toward improving the potency led to the discovery of 23 (IC₅₀

Full text of DOI:10.1016/j.bmcl.2006.10.060

Bioorganic & Medicinal Chemistry Letters 17 (2007) 807–813
Diaryl substituted pyrazoles as potent CCR2 receptor antagonists
Anthony B. Pinkerton,a,* Dehua Huang,a, Rowena V. Cube,a,à John H. Hutchinson,a,§
Mary Struthers,b Julia M. Ayala,b Pasquale P. Vicario,b Sima R. Patel,b
Thomas Wisniewski,b Julie A. DeMartinob and Jean-Michel Verniera,–
aDepartment of Medicinal Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court,
San Diego, CA 92121, USA
bDepartment of Immunology/Rheumatology, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 5 October 2006; revised 17 October 2006; accepted 23 October 2006
Available online 25 October 2006
Abstract—We have identified and synthesized a series of diaryl substituted pyrazoles as potent antagonists of the chemokine recep-
tor subtype 2. Structure–activity relationship studies directed toward improving the potency led to the discovery of 23
(IC50 = 6 nM).
ꢀ 2006 Elsevier Ltd. All rights reserved.
Chemokines1 (chemotactic cytokines) are a class of small
molecular weight peptides that are released by a wide
variety of cells to attract cells such as monocytes, mac-
rophages, T cells, eosinophils, basophils, and neutro-
phils to sites of inflammation.2 In particular, monocyte
chemoattractant protein-1 (MCP-1), which is included
within the CC class of chemokines, is a key monocyte
chemoattractant3 acting primarily through its receptor,
CCR2,4 which is a member of the G-protein coupled
receptor (GPCR) superfamily.5 The critical role of
MCP-1 and CCR2 in monocyte migration to sites of
inflammation has been highlighted by the MCP-16 and
CCR27 knockout mice. Numerous studies have impli-
cated the importance of MCP-1 and CCR2b in a variety
of inflammatory diseases,8 including rheumatoid arthri-
tis,9 atherosclerosis,10 multiple sclerosis,11 glomerulone-
phritis12 and stroke.13 Therefore, the therapeutic
potential of CCR2 antagonists in treating a wide range
of inflammatory diseases has attracted considerable
interest, including the disclosure in the literature of a
number of small molecule CCR2 antagonists.14–23 This
paper outlines the discovery and optimization of a new
class of CCR2 antagonists based on a diarylpyrazole
core which originated from screening of the Merck sam-
ple collection. This screen identified diarylpyrazole 1 as
a potential lead with a CCR2 IC50 of 221 nM. Our goal
then became to optimize the potency of this lead
structure.
NH
H
N
N
N
H2N
N
H
O
OCH3
O
Cl
Keywords: Chemokines; CCR2; MCP-1; GPCR.
1
Cl
*
Corresponding author at present address: Kalypsys, Inc., 10420
Wateridge Circle, San Diego, CA 92121, USA. Tel.: +1 858 754 3457;
fax: +1 858 754 3301; e-mail: apinkerton@kalypsys.com
Present address: Celgene, Inc., 4550 Towne Centre Court, San Diego,
CA 92121, USA.
The compounds described herein were synthesized as
outlined in Schemes 1–4.24 For the diarylpyrazoles with
a carbon linkage, synthesis began with reaction of a
ketoester (2) and a hydrazine (3) in neat acetic acid
to form cyclic products (4) in moderate yields
(Scheme 1). These were converted to bromopyrazoles 5
à
§
Present address: Merck Research Laboratories, West Point, PA
19486, USA.
Present address: Amira Pharmaceuticals, 9535 Waples St., Suite 100,
San Diego, CA 92121, USA.
–
Present address: Valeant, Inc., 3300 Hyland Ave., Costa Mesa, CA
92626, USA.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.060

808
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
R1
R1
O
O
a
N
b
N
c
N
N
O
R2
Br
O
R1
NH2
R2
R2
N
H
2
HO
5
4
6
3
R1
N
R1
N
R1
N
H
f
N
d,e
N
N
R3
N
R3 NH2
HO
R2
HO
R2
O
O
R2
9
7
8
10
Scheme 1. Reagents and conditions: (a) HOAc (neat), 130 ꢁC, 40–65%; (b) PBr3, CH3CN, microwave, 150 ꢁC, quant; (c) Pd(PPh3)4, CuBr, Et3N,
70 ꢁC, 65–85%; (d) Pd/C, H2, EtOAc, rt, 70–90%; (e) PDC, DMF, rt, 55–80%; (f) EDCI, DMAP, CH2Cl2, rt, 75–95%.
R1
N
R1
N
b
a
N
TBSO
O
N
O
R2
R1
TBSO
Br
R2
11
4
12
R1
N
N
N
N
c
HO
O
HO
O
R2
R2
O
14
13
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 60 ꢁC, 85–95%; (b) TBAF, THF, rt, quant; (c) PDC, DMF, rt, 65–70%.
R1
N
O
R1
N
b
a
H
N
N
NC
R2
EtO
N
CO2Et
R1
NH2
R2
H2N
Br
N
H
O
15
R2
3
17
16
R1
N
c
H
N
N
HO
R2
O
18
Scheme 3. Reagents and conditions: (a) PhCH3, microwave, 130 ꢁC, 85–95%; (b) K2CO3, NaI, DMF, microwave, 200 ꢁC, 65–72%; (c) LiOH, MeOH/
THF 9/1, 70 ꢁC, quant.
via reaction with phosphorous tribromide under micro-
wave irradiation in quantitative yields. Bromopyrazoles
5 were then coupled to 3-butyn-1-ol (6) under Sonagash-
ira conditions to give alkyne-pyrazoles 7. Reduction of
the triple bond followed by oxidation of the pendant
alcohol gave acids 8, which were then coupled with the
appropriate amines (9) under standard conditions to
give the desired compounds. In this manner the com-
pounds in Tables 1 and 4 were synthesized.
For nitrogen substituted diarylpyrazoles, synthesis
began with cyanoketones 15 and hydrazines 3 (Scheme
3). Reaction under microwave irradiation gave
aminopyrazoles 16 in excellent yields. Alkylation with
3-bromoethylpropionate gave esters 17, which were
hydrolyzed to give acids 18 and further reacted as
described above to give the desired compounds. Com-
pounds 35 and 37 were synthesized via this scheme.
Analogs 23 and 39–43 were synthesized in the fashion
shown in Scheme 4. Oxidation and addition of isopro-
pylmagnesium bromide to monoprotected 1,4-butanedi-
ol (19) gave alcohol 20 which was converted to amine 21
in good yield using a three-step procedure (tosylation,
displacement with sodium azide, and reduction). Cou-
pling of 21 with acid 14 (Scheme 2) gave pyrazole 22.
Deprotection, followed by oxidation of the primary
alcohol, gave an aldehyde which was reductively
For the diarylpyrazoles with an oxygen linker, synthesis
began with the same intermediate (4) as described in
Scheme 1. These compounds were alkylated with bromide
11 to give oxopyrazoles 12 in good yields (Scheme 2).
After removal of the protecting group, oxidation gave
acids 14, which were reacted as above using standard cou-
pling conditions to give the desired compounds. Com-
pounds in Tables 2 and 3 were synthesized in this fashion.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
809
OTBDPS
c,d,e
a,b
OTBDPS
HO
HO
19
21
20
f
N
N
HO
O
+
OTBDPS
H2N
Cl
O
14
Cl
H
N
N
TBDPSO
O
g,h,j
N
N
N
N
HN
O
O
Cl
Cl
O
22
23
Cl
Cl
˚
Scheme 4. Reagents and conditions: (a) TPAP, NMO, 4 Amol sieves, CH2Cl2/CH3CN 3/1, rt, quant; (b) i-PrMgBr, THF, 0 ꢁC, 72%; (c) Ts-Cl,
pyridine, 0 ꢁC; (d) NaN3, DMF, 40 ꢁC; (e) Pd/C, H2, MeOH/EtOAc 1/1, rt, 70% over 3 steps; (f) EDCI, DMAP, DMF, 40 ꢁC, 65%; (g) TBAF, THF,
rt, quant; (h) Dess–Martin, CH2Cl2, rt; (j) diethylamine, NaBH3CN, AcOH, THF, rt, 55% over 2 steps.
Table 1. Effect of pendant amide group on binding affinity
H
N
N
R3
N
O
Cl
Cl
R3
Chemotaxis IC50 (nM)
a
CCR2 IC50 (nM)
Compound
NH
H2N
N
1
221
185
H
O
OCH3
HN
24
25
26
2840
9332
1123
NTb
NTb
1400
N
O
OCH3
N
H
N
N
27
28
29
1803
1225
108
NTb
NTb
209
N
N
N
a Value represents mean of two or more experiments. The standard deviation limits are within 25% of the reported values.
b NT, not tested.

810
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
Table 2. Linker modifications
inhibit the MCP-1 induced migration of monocytes
was used.26
We initially examined the replacement of the arginine
group from the lead structure (Table 1). A number of
other amino acid esters were incorporated, but only his-
tidine 24 was found to be moderately active with an IC50
of 2840 nM. Other amino acid esters such as glycine,
alanine, phenylalanine, tyrosine, and glutamate methyl
ester all gave inactive compounds, as did the enantiomer
of compound 1 (data not shown). We then examined a
large number of other amine containing side chains,
mainly focusing on accessing the same binding site as
the guanidine group. Most of these attempts led to inac-
tive compounds, although analog 25 did have weak
activity at 9332 nM. We then turned to a number of sim-
pler amine containing side chains. Some success was
achieved in this area with pyridine 26 and dimethyl-
amine 27 with IC50s of 1123 and 1803 nM, respectively.
Extending the alkyl chain to a diethyl amino group as in
28 slightly improved the activity (simple cyclic amines
such as pyrrolidine and piperidine in this position were
less active—data not shown). A large boost in potency
was observed when a methyl group was incorporated a
to the amide to give compound 29 with an IC50 of
108 nM as well as good activity in the chemotaxis assay
(IC50 = 209 nM). This result points to the importance of
a group a to the amide to give potent compounds in this
series, an effect that was examined further (vide infra).
NH
H
N
N
N
LINKER
H2N
N
H
O
Cl
OCH3
Cl
Compound LINKER
CCR2 IC50 Chemotaxis IC50
(nM)a
(nM)
1
221
185
O
30
4741
NAb
NTc
NTc
O
31
32
NAb
NTc
O
O
33
62
7601
43
118
NTc
68
O
We next turned to the effect of the linker between the
diaryl pyrazole group and the arginine side chain on
activity (Table 2). It appears that the SAR is relatively
tight for modifications in this area. For example, short-
ening the chain one carbon, as in 30, leads to a precipi-
tous drop in activity to 4741 nM. Analog 31 highlights
the importance of the central amide for potency—re-
moval of the carbonyl gives a compound that is inactive.
Likewise, constraining the linker as in phenyl analog 32
gives an inactive compound. Better results were achieved
when an additional heteroatom was incorporated next
to the pyrazole. Both oxygen analog 33 and nitrogen
analog 35 gave a 4- to 5-fold boost over the original lead
with IC50s of 62 and 43 nM, respectively. Furthermore,
these analogs were also more potent in the functional
chemotaxis assay (IC50 = 118 nM for 33 and 68 nM
for 35). Further modifications were not as well tolerated.
Extending analog 33 by one atom to 34 leads to over a
100-fold drop in potency. Likewise, incorporating an
additional amide as in analog 36 gave an inactive
compound.
O
34
O
H
N
35
O
H
N
36
NAb
NTc
O
O
a Value represents mean of two or more experiments. The standard
deviation limits are within 25% of the reported values.
b NA denotes not active <10 lM concentration.
c NT, not tested.
aminated with diethylamine to give analog 23 in moder-
ate yield. Compounds 39–43 were synthesized in the
same fashion using the appropriate Grignard reagent.
In order to investigate the SAR and improve the poten-
cy of compound 1 four areas were addressed: (1)
replacement of the arginine of the lead compound with
a variety of other amino acids as well as simple amines,
(2) investigation of the linker between the diarylpyrazole
moiety and the amine group, (3) substitution on a potent
diethylamine scaffold, and (4) examination of other aryl
groups around the pyrazole. All compounds described
herein were synthesized and tested as racemates unless
otherwise noted. Two in vitro assays were used to drive
the SAR. The primary assay monitored inhibition of
A combination of the diethylamino side chain with var-
ious linker combinations was then explored (Table 3).27
Incorporation of nitrogen (37) or oxygen (38) led to a
boost in potency as was observed with the arginine
analogs. We then focused on a series of oxygen linked
compounds wherein the group a to the amide (R4) was
varied. Increasing the size of the group from methyl to
ethyl (39) led to a large boost in potency both in the
binding (IC50 = 9 nM) and functional (IC50 = 83 nM)
assays. Increasing the size to an n-propyl (40) group
gave a compound that was similar in potency to methyl
[
125I]MCP-1 binding to primary human blood mono-
cytes which endogenously express CCR2.25 Second, a
functional chemotaxis assay that detected the ability to

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
Table 3. Dialkylamino containing pyrazoles
811
H
N
N
X
N
N
R4
O
Cl
Cl
Compound
R4
X
Chemotaxis IC50 (nM)
a
CCR2 IC50 (nM)
29
37
38
39
40
–CH3
–CH3
–CH2–
–NH–
–O–
108
30
66
9
209
54
–CH3
–CH2CH3
–CH2CH2CH3
280
83
–O–
–O–
44
154
CH3
23
41
–O–
–O–
6
32
CH3
H3C
CH3
74
178
H3C
42
43
–O–
–O–
107
225
CH3
–CH2(CH2)2CH3
52 at 1 lM
NTb
a Value represents mean of two or more experiments. The standard deviation limits are within 25% of the reported values.
b NT, not tested.
analog 38 (IC50 = 44 nM vs 66 nM). An isopropyl
group (23) appeared to be the optimal substituent in
this series, with an IC50 of 6 nM in the binding assay
and an IC50 of 32 nM in the chemotaxis assay. This
compound represents one of the more potent CCR2
antagonists reported to date. Increasing the size
Table 4. Pyrazole substitution
R1
H
N
N
N
N
O
R2
R1
R2
Chemotaxis IC50 (nM)
a
CCR2 IC50 (nM)
Compound
29
44
45
46
47
48
49
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
3,5-Dichlorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
3-Methoxyphenyl
3-Fluoro phenyl
108
209
NTb
69% at 10 lM
168
442
57 at 300 nM
NA at 300 nM
NTb
NTb
NTb
2391
522
3-Trifluoromethylphenyl
207
CF3
50
51
52
2-Naphthyl
Phenyl
634
NTb
NTb
NTb
CF3
3,5-Dichlorophenyl
69% at 10 lM
6475
CF3
3,5-Dichlorophenyl
CF3
a Value represents mean of two or more experiments. The standard deviation limits are within 25% of the reported values.
b NT, not tested.

812
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
Table 5. Rat pharmacokinetic parameters for selected compounds
References and notes
Compound
Cla (mL/min/kg)
Vd (L/kg)
t1/2 (h)
%Fa
1. Bachmann, M. F.; Kopf, M.; Marsland, B. J. Nat. Rev.
Immunol. 2006, 6, 159.
1
29
8.3
28.1
0.98
13.0
2.49
6.52
0
5
2. (a) Ward, S. G.; Westwick, J. Biochem. J. 1998, 333, 457;
(b) Baggiolini, M.; Dewald, B.; Moser, B. Annu. Rev.
Immunol. 1997, 15, 675; (c) Epstein, F. H. N. Engl. J. Med.
1998, 338, 436; (d) Power, C. A.; Wells, T. N. C. Trends
Pharmacol. Sci. 1996, 17, 209; (e) Schwarz, M. K.; Wells,
T. N. C. Expert Opin. Ther. Patents 1999, 9, 1471.
3. Saunders, J.; Tarby, C. M. Drug Discov. Today 1999, 4, 80.
4. Feria, M.; Diaz-Gonzalez, F. Expert Opin. Ther. Patents
2006, 16, 49.
5. Charo, I. F.; Myers, S. J.; Herman, A.; Franci, F.;
Connolly, A. J.; Coughlin, S. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 2751.
6. Lu, B.; Rutledge, B. J.; Gu, L.; Fiorillo, J.; Lukacs, N. W.;
Kunkel, S. L.; North, R.; Gerard, C.; Rollins, B. J. J. Exp.
Med. 1998, 187, 601.
a Dosed 1 mpk iv (n = 2) and 2 mpk po (n = 3) in Sprague–Dawley rats.
beyond isopropyl led to a rapid drop off in potency, as
seen in analogs 41–43.
Lastly, we investigated the effect of different aryl substitu-
ents around the pyrazole ring. In general, modifications of
the original scaffold (29) led to compounds with consider-
ably lower activity.27 Monochloro analogs at R2 were
examined first, with only the 3-chlorophenyl compound
45 giving binding activity comparable to 29 (68 nM vs
108 nM), indicating that at least one substituent at the 3
position was optimal for good activity. Other 3-substitut-
ed aryl groups were examined, including methoxy (47),
fluoro (48), and trifluoromethyl (49), but in general activ-
ity was diminished. Finally, a trifluoromethyl analog (50)
of compound 29 gave 6-fold diminished potency com-
pared to the lead. Thus, the 3,5-dichlorophenyl moiety
appeared to be the optimal group. Attempts to replace
the 2-naphthyl group at R1 were less successful, with both
a phenyl (51) and a bistrifluoromethyl (52) group giving
dramatically lower activity.
7. Kuziel, W. A.; Morgan, S. J.; Dawson, T. C.; Griffin, S.;
Smithies, O.; Ley, K.; Maeda, N. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 12053.
8. Rollins, B. J. Mol. Med. Today 1996, 2, 198.
9. (a) Koch, A. E.; Kunkel, S. L.; Harlow, L. A.; Johnson,
B.; Evanoff, H. L.; Haines, G. K.; Burdick, M. D.; Pope,
R. M.; Strieter, R. M. J. Clin. Invest. 1992, 90, 772; (b)
Loetscher, P.; Wewald, B.; Bagglioni, M.; Seitz, M.
Cytokine 1994, 6, 162; (c) Ruth, J. H.; Rottman, J. B.;
Katschke, K. J., Jr.; Qin, S.; Wu, L.; LaRosa, G.; Ponath,
P.; Pope, R. M.; Koch, A. E. Arthritis Rheum. 2001, 44,
2750.
Two of the diarylpyrazoles described above were exam-
ined for their pharmacokinetic properties in rat. As
shown in Table 5, compound 1 has moderate rat PK fol-
lowing iv dosing with low clearance, albeit with no oral
bioavailability. Compound 29 had a somewhat higher
clearance but slightly better oral bioavailability.
10. (a) Gosling, J.; Slaymaker, S.; Gu, L.; Tseng, S.; Zlot, C.
H.; Young, S. G.; Rollins, B. J.; Charo, I. F. J. Clin.
Invest. 1999, 103, 773; (b) Boring, L.; Gosling, J.; Cleary,
M.; Charo, I. F. Nature 1998, 394, 894; (c) Dawson, T. C.;
Kuziel, W. A.; Osahar, T. A.; Maeda, N. Atherosclerosis
1999, 143, 205.
11. Lloyd, C. M.; Minto, A. W.; Dorf, M. E.; Proudfoot, A.;
Wells, T. N. C.; Salant, D. J.; Gutierrez-Ramos, J.-C.
J. Exp. Med. 1997, 185, 1371.
12. (a) Kennedy, K. J.; Strieter, R. M.; Kunkel, S. L.; Lukacs,
N. W.; Karpus, W. J. J. Neuroimmunol. 1998, 92, 98; (b)
Kurihara, T.; Warr, G.; Loy, J.; Bravo, R. J. Exp. Med.
1997, 186, 1757; (c) Boring, L.; Gosling, J.; Chensue, S.
W.; Kunkel, S. L.; Farese, R. V., Jr.; Broxmeyer, H. E.;
Charo, I. F. J. Clin. Invest. 1997, 100, 2552.
13. Hughes, P. M.; Allegrini, P. R.; Rudin, M.; Perry, V. H.;
Mir, A. K.; Wiessner, C. J. Cereb. Blood Flow Metab.
2002, 22, 308.
Selectivity of compounds for CCR2 over the most close-
ly related CC chemokine receptors, CCR1 and CCR5,
was determined. All the compounds described above
were selective against CCR1. CCR2 and CCR5 have a
higher degree of sequence homology (71% identity be-
tween CCR2b and CCR5),28 and therefore separation
of activity at these two receptors might be anticipated
to be more difficult. For the series described here, selec-
tivity could be achieved for some compounds. For
example, although compounds 1 (CCR2 IC50 = 221 nM,
CCR5 IC50 = 63 nM) and 29 (CCR2 IC50 = 108 nM,
CCR5 IC50 = 220 nM) were comparable in activity,
compound 23 had considerable selectivity for CCR2
(CCR2 IC50 = 6 nM, CCR5 IC50 = 1610 nM).
14. Forbes, I. T.; Cooper, D. G.; Dodds, E. K.; Hickey, D. M.
B.; Ife, R. J.; Meeson, M.; Stockley, M.; Berkhout, T. A.;
Gohil, J.; Groot, P. H. E.; Moores, K. Bioorg. Med. Chem.
Lett. 2000, 10, 1803.
15. Witherington, J.; Bordas, V.; Cooper, D. G.; Forbes, I. T.;
Gribble, A. D.; Ife, R. J.; Berkhout, T.; Gohil, J.; Groot,
P. H. E. Bioorg. Med. Chem. Lett. 2001, 11, 2177.
16. Kettle, J. G.; Faull, A. W.; Barker, A. J.; Davies, D. H.;
Stone, M. A. Bioorg. Med. Chem. Lett. 2004, 14, 405.
17. Imai, M.; Shiota, T.; Kataoka, K.; Tarby, C. M.; Moree,
W. J.; Tsutsumi, T.; Sudo, M.; Ramirez-Weinhouse, M.
M.; Comer, D.; Sun, C.-M.; Yamagami, S.; Tanaka, H.;
Morita, T.; Hada, T.; Greene, J.; Barnum, D.; Saunders,
J.; Myers, P. L.; Kato, Y.; Endo, N. Bioorg. Med. Chem.
Lett. 2004, 14, 5407.
In conclusion, a new class of diaryl pyrazole containing
compounds that are potent antagonists at the CCR2
receptor has been developed from a screening lead.
These compounds are among the most potent CCR2
antagonists yet described and should be useful pharma-
cological tools to further elucidate the role of CCR2 in
inflammatory diseases.
18. Moree, W. J.; Kataoka, K.; Ramirez-Weinhouse, M. M.;
Shiota, T.; Imai, M.; Sudo, M.; Tsutsumi, T.; Endo, N.;
Muroga, Y.; Hada, T.; Tanaka, H.; Morita, T.; Greene, J.;
Barnum, D.; Saunders, J.; Kato, Y.; Myers, P. L.; Tarby,
C. M. Bioorg. Med. Chem. Lett. 2004, 14, 5413.
Acknowledgment
We thank Merryl Cramer for assistance in measuring
the PK parameters for 1 and 29.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 807–813
813
19. Lommen, G. V.; Doyon, J.; Coesemans, E.; Boeckx, S.;
Cools, M.; Buntinx, M.; Hermans, B.; VanWauwe, J.
Bioorg. Med. Chem. Lett. 2005, 15, 497.
20. Pasternak, A.; Marino, D.; Vicario, P. P.; Ayala, J. M.;
Cascierri, M. A.; Parsons, W.; Mills, S. G.; MacCoss, M.;
Yang, L. J. Med. Chem. 2006, 49, 4801.
21. Yang, L.; Zhou, C.; Guo, L.; Morriello, G.; Butora, G.;
Pasternak, A.; Parsons, W. H.; Mills, S. G.; MacCoss, M.;
Vicario, P. P.; Zweerink, H.; Ayala, J. M.; Goyal, S.;
Hanlon, W. A.; Cascieri, M. A.; Springer, M. S. Bioorg.
Med. Chem. Lett. 2006, 16, 3735.
22. Butora, G.; Morriello, G. J.; Kothandaraman, S.; Guia-
deen, D.; Pasternak, A.; Parsons, W. H.; MacCoss, M.;
Vicario, P. P.; Cascieri, M. A.; Yang, L. Bioorg. Med.
Chem. Lett. 2006, 16, 4715.
trations of unlabeled compounds in binding buffer for
60 min at room temperature. The buffer contains 50 mM
Hepes, 5 mM MgCl2, and 1 mM CaCl2, pH 7.4.
125I-hMCP-1 was purchased from Perkin-Elmer Life
Sciences, Inc., with a specific activity of 2200 Ci/mmol.
The assay was terminated by filtration of the reaction
mixture through GF/B filter plates (presoaked in 0.1%
polyethyleneimine) using a Packard Cell Harvester. The
filter plates were washed with 25 mM Hepes, pH 7.5,
containing 500 mM NaCl and dried in an incubator at
37 ꢁC for 30 min. The plates were loaded with Microscint
0 (Packard) and counted in a Topcount NXT (Packard).
The software program Prism (GraphPad) was used for all
calculations.
26. Ayala, J. M.; Goyal, S.; Liverton, N. J.; Claremon, D. A.;
O’Keefe, S. J.; Hanlon, W. A. J. Leukoc. Biol. 2000, 67,
869.
27. These compounds were tested as racemates. The effect, if
any, of stereochemistry on activity was not investigated.
28. Raport, C. J.; Gosling, J.; Schweickart, V. L.; Gray, P. W.;
Charo, I. F. J. Biol. Chem. 1996, 29, 17161.
23. Expert Opin. Ther. Patents 2003, 13, 115.
24. All final compounds displayed spectral data (NMR, MS)
that was consistent with the assigned structure.
25. Human peripheral blood monocytes were isolated as
described in reference.26 Monocytes (2 · 105) were incu-
bated with 125I-hMCP-1 (20–50 pM) and various concen-