4-Amino-2-alkyl-butyramides as small molecule CCR2 antagonists with favorable pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2006.07.011

A systematic examination of the central aromatic portion of the lead (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluorophenyl)-4-(1′H-spiro[indene-1,4′-piperidin]-1′-yl)butanamide (9) led to the discovery of a novel class of CCR2 receptor antagonists, which carry small alicyclic groups such as cyclopropyl, cylobutyl, or cyclopropylmethyl attached at C₂ of the carbon backbone. The most potent compound discovered, namely (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,3′R)-3′-methyl-1′H-spiro[indene-1,4′-piperidin]-1′-yl]butanamide (29), showed very high binding affinity (IC₅₀ = 4 nM, human monocyte) and excellent selectivity toward other related chemokine receptors. The excellent pharmacokinetic profile of this new lead compound allows for extensive in vivo evaluation.

Full text of DOI:10.1016/j.bmcl.2006.07.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4715–4722
4-Amino-2-alkyl-butyramides as small molecule CCR2 antagonists
with favorable pharmacokinetic properties
Gabor Butora,* Gregori J. Morriello, Shankaran Kothandaraman,
Deodialsingh Guiadeen, Alexander Pasternak, William H. Parsons, Malcolm MacCoss,
Pasquale P. Vicario, Margaret A. Cascieri and Lihu Yang
Merck Research Laboratories, Rahway, NJ 07065, USA
Received 12 May 2006; revised 26 June 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—A systematic examination of the central aromatic portion of the lead (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluoro-
phenyl)-4-(10H-spiro[indene-1,40-piperidin]-10-yl)butanamide (9) led to the discovery of a novel class of CCR2 receptor antagonists,
which carry small alicyclic groups such as cyclopropyl, cylobutyl, or cyclopropylmethyl attached at C2 of the carbon backbone. The
most potent compound discovered, namely (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,30R)-30-methyl-10H-spi-
ro[indene-1,40-piperidin]-10-yl]butanamide (29), showed very high binding affinity (IC50 = 4 nM, human monocyte) and excellent
selectivity toward other related chemokine receptors. The excellent pharmacokinetic profile of this new lead compound allows
for extensive in vivo evaluation.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Chemokines (Chemotactic cytokines) are small molecu-
lar weight (8–10 kDa) water-soluble proteins composed
of 340–380 amino acids that play a key role in immuno-
modulation and host-defense.1,2 Their multiple effects
are mediated through binding to a variety of related
7-transmembrane domain G-protein coupled receptors.3
The observation that some chemokines and their respec-
tive receptors are upregulated during both chronic and
acute inflammatory diseases, as well as in response to
challenge of the immune system, suggested that they
may present a viable target for drug intervention.4,5
platform for therapeutic intervention.12 As a result of
intensive research a number of small molecule CCR2
antagonists have been described in the scientific litera-
ture13 and some representative examples from Take-
da,14,15 SmithKline,16,17 Roche,18 Teijin,19 Johnson
and Johnson,20 and Astra-Zeneca21 are shown in
Figure 1.
High throughput screening of the Merck Sample Collec-
tion produced the initial lead 7 with a binding affinity22
of 720 nM, Figure 2. Its optimization led to the discov-
ery of the piperidine analog 8 with threefold improved
potency.23 Subsequently it was established that the basic
nitrogen present in the backbone of the molecule can be
omitted without significant loss of activity.24 Intensive
synthetic effort focusing on the amine portion of the lead
structure produced further refinement of the piperidine
ring. The pertinent SAR study revealed that introduc-
tion of a (substituted) phenyl ring at position 4 of the
piperidine greatly enhances the observed binding affini-
ties. Particularly interesting was the discovery of the spi-
roindene structural motif such as in 9, which, albeit only
slightly more potent than 8, showed acceptable oral
bioavailability.25,26
Chemokines are classified as CC, CXC, CX3C, and XC
based on the sequential relationship of the first two of
four conserved cysteine residues.6 Most of them appear
to have redundant specificity since they bind to numer-
ous receptors within the same family.7 Monocyte Che-
moattractant Protein 1 (CCL2/MCP-1), a member of
the CC-subfamily, binds and activates the CC-chemo-
kine receptor 2 (CCR2).8 A large body of evidence accu-
mulated in the recent past strongly suggests the
involvement of the CCL2/CCR2 axis in arthritis,9 multi-
10
ple sclerosis, and vascular disease11 offering a logical
Herein, we wish to describe the results of an SAR study
focusing on the central, aromatic portion of the lead
structure 9. This study was initiated by the observation
Keywords: CCR2; Chemokines; Antagonists.
*
Corresponding author. Tel.: +1 732 594 8563; fax: +1 732 594
9556; e-mail: gabor_butora@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.011

4716
G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
O
placed methyl group, such as in compound 13, led to al-
most full restoration of the activity, Figure 3.
NH
O
NH
We were interested to find out if the binding and func-
tional activity of 13 could be further improved by the
introduction of a pharmacophore with size and electron-
ic characteristics between those of the 4-fluorophenyl
group and a hydrogen atom.
Cl
N
Cl
HO
O
Cl- N+
N
H
The synthetic sequence utilized to access the desired
compounds in a racemic form is illustrated for the exam-
ple of the 2-cyclopropyl derivative 18, Scheme 1.
1 (Takeda)
IC50 = 27 nM (CCR2b)
2 (SmithKline Beecham)
Ki = 50 nM (CCR2b)
O
H
O
N
N
F3C
NH
The dianion of the commercially available cyclopropyl
acetic acid (14) was alkylated with 2,2-dimethoxyethyl
bromide. The amide was introduced using a 1-[(3-di-
methylamino)propyl]-3-ethyl-carbodiimide (EDC) med-
iated coupling of acid 15 and the commercially
available 3,5-bis(trifluoromethyl)-benzyl amine. The
acetal was cleaved under acidic conditions at ambient
temperature, and the liberated aldehyde was reductively
aminated with spiro[indene-1,40-piperidine].27 Unfortu-
nately, this sequence was only poorly reproducible as
the intermediate aldehyde 17 readily underwent an
intramolecular cyclization (19) followed by elimination
of water (20) and decomposition.
O
N
N
HN
O
Cl
O
3 (Roche)
IC50 = 89 nM (CCR2)
4 (Teijin)
IC50 = 180 nM(CCR2b)
O
F
O
N
MeO
SH
MeO
N
N
OH
O
Cl
Cl
Cl
Cl
In an attempt to block the lactam formation as well as
facilitate access to single enatiomers, the acid was react-
ed via its pivaloyl anhydride with (4R)-4-benzyl-1,3-
oxazolidin-2-one.28 The two diastereoisomers were read-
ily separated by flash column chromatography (silica
gel, ethyl acetate–hexanes/3:7). Unfortunately, an at-
tempt at hydrolytical cleavage of the oxazolidone failed
to produce the acid 22, as it readily underwent a c-lact-
onization during the workup (23, 24). To remedy this
problem, the amine was introduced before the removal
of the auxiliary, Scheme 2. Accordingly, the acetal was
5 (Johnson and Johnson)
pIC50 = 8 nM (Ca-flux)
6 (Astra Zeneca)
IC50 = 56 nM (CCR2b)
Figure 1. CCR2 antagonists.
O
H
N
CF3
N
N
H
CF3
F
7, IC50 = 720 nM
O
H
N
N
H
CF3
O
N
N
CF3
N
H
CF3
CF3
F
8, IC50 = 180 nM
F
9, IC50 = 80 nM
R
O
O
N
CF3
N
N
CF3
H
N
H
CF3
12, IC50 = 570 nM
CF3
F
9, R = H, IC50 = 80 nM
10, R = Me, IC50 67 nM
11, R = Et, IC50 = 437 nM
=
O
N
N
H
CF3
Figure 2. Lead evolution, CHO binding assay.
CF3
13, IC50 = 173 nM
that omission of the 4-fluorophenyl pharmacophore in
9 had caused a less than expected decrease in binding
affinity, approximately sevenfold. Furthermore, a properly
Figure 3. Central region SAR: initial observations, CHO binding
assay.

G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
4717
O
O
O
O
O
CF3
CF3
CF3
OH
i
OH
N
H
ii
O
O
H
CF3
CF3
CF3
14
15
16
iii
O
O
O
N
CF3
N
H
N
iv
H
CF3
18
17
v
O
O
CF3
N
v
N
OH
CF3
20
19
Scheme 1. Reagents and conditions: (i) 2,2-dimethoxyethyl bromide, LDA, THF, ꢀ78 to +40 ꢁC; (ii) 3,5-(bistrifluoromethyl)benzylamine, EDC,
HOAT, DMF; (iii) aq TFA, rt 10 min; (iv) spiroindene, NaHB(OAc)3, dichloroethane; (v) spontaneous.
O
O
O
O
O
O
O
O
iii
OH
O
O
24
22
23
ii
O
O
O
O
O
O
O
OH
N
O
N
O
i
+
O
O
O
15
20
21
iv
iv
O
O
O
O
O
O
N
O
O
N
O
v
N
O
N
O
H
H
27
25
26
ii
O
O
N
N
CF3
vi
OH
N
H
CF3
28
29
Scheme 2. Reagents and conditions: (i) tBuCOCl, DIEA, THF, lithium (4R)-4-benzyl-1,3-oxazolidin-2-one; (ii) LiOOH, 0 ꢁC, THF, water; (iii)
spontaneous; (iv) aq TFA; (v) (1R,30R)-30-methyl-spiro[indene-1,40-piperidine], NaHB(OAc)3, dichloroethane; (vi) 3,5-bis(trifluoromethyl)benzyl-
amine, EDC, HOAT, DCM.
cleaved (93% yield) and aldehydes 25 and 26 were reduc-
tively aminated with (1R,30R)-30-methyl-spiro[indene-
1,40-piperidine].27 The auxiliary was removed under
standard conditions and the target compound 29 was
obtained in an EDC-mediated amide forming step,
Scheme 2.
would further streamline the synthesis. This strategy is
illustrated in Scheme 3, which describes the preparation
of the methylene homolog of 29, compound 37.
To this end, the commercially available 4-pentenoic acid
(30) was coupled with (4S)-4-benzyl-1,3-oxazolidin-2-
one (vide ante) and the cyclopropyl group introduced via
a palladium-catalyzed cyclo-propanation of the olefin
with diazomethane.30 The LDA mediated allylation of
32 produced stereoselectively the (4S)-4-benzyl-3-[(2S)-
2-(cyclopropylmethyl)pent-4-enoyl]-1,3-oxazolidin-2-
The chiral oxazolidinone is uniquely suited to control
absolute stereochemistry at the newly formed C2-cen-
ter.29 We envisioned that replacing the 2,2-dimethoxy-
ethyl bromide with the more reactive allyl chloride

4718
G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
O O
O
O
O
OH
N
O
N
O
ii
i
31
30
32
iii
O
O
O
O
CF3
N
H
OH
N
O
v
iv
CF3
35
34
33
vi
O
O
CF3
vii
O
N
N
CF3
O
H
N
O
H
CF3
36
37
CF3
Scheme 3. Reagents and conditions: (i) tBuCOCl, DIEA, THF, lithium (4R)-4-benzyl-1,3-oxazolidin-2-one; (ii) CH2N2, Pd(OAc)2, Et2O; (iii) allyl
bromide, LDA, ꢀ78 to ꢀ15 ꢁC, THF; (iv) LiOOH, THF, water, 0 ꢁC; (v) 3,5-(bistrifluoromethyl)benzyl-amine, EDC, HOAT, DCM; (vi) O3,
ꢀ78 ꢁC, DCM; (vii) crude ozonide 36, (1R,30R)-30-methylspiro[indene-1,40-piperidine], NaHB(OAc)3, dichloroethane.
O
O
O
O
O
one (33). Hydrolytic cleavage of the oxazolidone with lith-
ium hydroperoxide afforded, albeit in low yield (27%), the
desired acid 34. The facile introduction of the amide (35)
was followed by an oxidative cleavage of the olefin (O3,
ꢀ78 ꢁC). The intermediate ozonide could be reduced to
the respective aldehyde under standard conditions (e.g.,
with dimethyl sulfide), however the reaction was slow
allowing sufficient time for the unwanted intramolecular
cyclization (such as 19, Scheme 1) to prevail. On the other
hand, when the ozonide reduction was performed in the
presence of the amine component (sodium triacetoxy-
borohydride, ambient temperature) the formed aldehyde
was trapped as an enamine, and the subsequent in situ
reductive amination completed the synthesis.
X
OH
N
O
N
O
v
iii
iv
38, X=Br
39, X=CN
40, X=COOH
41
42
43
vi
i
ii
O
O
CF3
CF3
O
N
H
N
vii
O
H
O
45
CF3
44
CF3
viii
O
N
CF3
N
H
The low-yielding lithium hydroperoxide mediated auxil-
iary cleavage was addressed during the synthesis of the
cyclobutyl derivative 46, Scheme 4. According to this,
the commercially available cyclobutyl bromide was
reacted with potassium cyanide in DMF at elevated
temperature, and the nitrile 39 was hydrolyzed. The chi-
ral auxiliary was attached under the usual conditions
and the LDA derived anion was allylated at ꢀ16 ꢁC
overnight. Saponification of the oxazolidone 42 with less
nucleophilic potassium hydroxide required somewhat
forceful conditions, and we were pleased to find that
no scrambling at the C2-chiral center had occurred.
The synthesis of the cyclobutyl analog 46 was then com-
pleted as described above.
CF3
46
Scheme 4. Reagents and conditions: (i) KCN, DMF, 80 ꢁC; (ii) aq
KOH, 80 ꢁC; (iii) tBuCOCl, DIEA, THF, lithium (4R)-4-benzyl-1,3-
oxazolidin-2-one; (iv) allyl bromide, LDA, ꢀ78 to ꢀ15 ꢁC, THF;
(v) aq KOH, EtOH 60 ꢁC; (vi) 3,5-(bistrifluoromethyl)benzyl-
amine, EDC, HOAT, DCM; (vii) O3, ꢀ78 ꢁC, DCM; (viii) crude
ozonide 45, (1R,30R)-30-methylspiro[indene-1,40-piperidine], NaHB
(OAc)3, dichloroethane.
reductive amination followed by a base-catalyzed ester
hydrolysis completed the synthesis of 54.
The synthesis of the hydroxyethyl derivative 54 utilized
a Lemieux–Johnson oxidation of the double bond in
51 to unmask the aldehyde group, Scheme 5. Due to
the presence of an intramolecular hydrogen bond be-
tween the ester oxygen and the amide nitrogen, no c-lac-
tam formation was observed and the aldehyde 52 was
isolated in nearly quantitative yield. An uneventful
The target compounds in which the C2-substituent is
linked to the main backbone of the lead scaffold through
a nitrogen linker are depicted in Scheme 6. The commer-
cially available 1-amino-4-pentenoic acid (55) was
reacted with phthalanhydride and esterified with dia-
zomethane (57). The olefin was oxidatively cleaved,
and the ozonide was reduced at ambient temperature

G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
4719
O
O
O
O
well, with the monocyte assay indicating approximately
a fourfold higher binding affinity.
O
O
O
O
OH
i
ii
iii
O
HO
HO
AcO
The observation that compound 12 retained some activ-
ity (IC50 = 570 nM, Table 1), seemed to suggest that less
prominent, perhaps metabolically more robust groups
could take up the role of the aromatic ring in the 4-flu-
orophenyl derivative 9.
47
48
49
50
O
CF3
CF3
OHC
AcO
N
H
v
N
H
iv
AcO
CF3
CF3
52
51
We were pleased to find that attachment of a simple
methyl group to the carbon-chain backbone at C2 in-
creased the binding activity by approximately twofold
(63, IC50 = 231 nM). Comparison of the activities of
the two stereoisomeric methyl derivatives 63 and 64
(Table 1) confirmed that the substituent has to be of
the same absolute orientation (R) as in the lead scaffold.
Further increasing the chain length to an allyl (65,
IC50 = 193 nM), or early branching (isopropyl, 66,
IC50 = 204 nM), had only a marginal effect. Similarly,
a lipophilic cyclohexyl group (69, IC50 = 160 nM, race-
mate) did not seem to offer an advantage.
vi
O
N
CF3
N
H
XO
53, X=Ac
54, X= H
vii
CF3
Scheme 5. Reagents and conditions: (i) NaBH4, EtOH; (ii) allyl
bromide, LDA, ꢀ78 to ꢀ15 ꢁC, THF; (iii) Ac2O, Py; (iv) TFA, DCM,
anisole; (v) 3,5-(bistrifluoromethyl)benzyl-amine, PyBroP, DCM; (vi)
OsO4, NaIO4, THF, MeOH; (vii) (1R,30R)-30-methylspiro[indene-1,40-
piperidine], NaHB(OAc)3, dichloroethane; (viii) aq K2CO3, MeOH.
Introduction of polar groups seemed to decrease poten-
cy regardless of the size. The activity of the small amino
analog 61 (Table 2) was quite low (IC50 > 1 lM) and
similarly ineffective were the small heterocycles. Only
the two smallest sulfonamides 74 (methanesulfonamide,
IC50 = 244 nM, racemic) and 75 (trifluoromethanesulf-
onamide, IC50 = 125 nM, racemic) showed some
activity.
with dimethyl sulfide. A routine reductive amination
(59) was followed by ester hydrolysis and the amide
formed as described above. The phthalide protecting
group in 60 was cleaved with hydrazine at elevated tem-
perature, and 2-amino intermediate 61 was used then as
a synthetic relay to access the respective amides and/or
sulfonamides.
The synthesized target compounds were initially as-
sessed by their capacity to displace radiolabeled MCP-
1 from CCR2b receptors expressed in a CHO cell line.
Later, a human monocyte-based assay was employed.
The two respective data sets parallel each other quite
On the other hand, small cycloalkyl groups, particularly
cyclopropyl (68, IC50 = 98 nM, Table 1), increased the
binding potency dramatically. These small alicycles, par-
ticularly in conjunction with the strategically placed
methyl group (30R on spiro[indene-1,40-piperidine]),
O
O
O
O
OH
OH
O
OMe
O
OHC
O
OMe
O
i
ii
iii
NH2
N
N
N
O
O
55
56
57
58
iv
O
O
CF3
N
N
N
v,vi
OMe
O
H
O
N
N
O
O
CF3
60
59
O
vii
CF3
N
N
H
NHX
61, X=H
62, X= Acyl, or RSO2
viii
CF3
Scheme 6. Reagents and conditions: (i) phthalanhydride, toluene, reflux, 6 h; (ii) CH2N2, Et2O; (iii) O3, DCM, ꢀ78 ꢁC, then DMS, rt; (iv)
spiro[indene-1,40-piperidine], NaHB(OAc)3, dichloroethane; (v) aq LiOH, dioxane; (vi) 3,5-(bistrifluoromethyl)benzyl-amine, EDC, HOAT, DCM;
(vii) NH2NH2, EtOH, reflux, 30 min; (viii) acyl chloride or anhydride or RSO2Cl, DIEA, DCM.

4720
G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
Table 1. Spiroindene class: binding affinities
produced some of the most active antagonists of the
CCR2b receptor described to date, Table 4.
R2
O
The C2-cyclopropyl analog 29 exhibited remarkable
activity of 14 nM (IC50, CHO assay) and it appeared
to be even more active (IC50 = 4 nM) in the hMonocyte
assay. Slightly less active were the respective cyclobutyl
(46, IC50 = 28 nM, CHO, IC50 = 11 nM hMonocyte)
N
CF3
N
H
R1
CF3
Compound R1
(stereo)
R2
CCR2 IC50, nM
CHO hMonocyte
Table 3. 4-Phenylpiperidine class: binding affinities
12
–H
H
H
H
H
H
H
H
H
H
570
231
605
193
204
349
69
80%a
37
R2
63R
64S
Methyl–
Methyl–
Allyl–
86
37
O
65R
66S
N
CF3
iPropyl–
iPropyl–
cPropyl–
cPropyl–
cHexyl–
47
72
N
H
R1
67R
18R/S
68S
38
15
CF3
CCR2 IC50, nM
98
160
69R/S
73
Compound
R1
R2
70R/S
71R/S
H
783
70%a
CHO
hMonocyte
MeO2SN
AcN
82S
83S
cPropyl
cPropyl
nPropyl
Allyl
F
280
285
126
642
42a
38a
n/a
72
129
42
H
H
H
H
H
F
84R
85R
86S
H
H
660
298
80
216
n/a
n/a
462
MeOCOCH2–
MeOCOCH2–
1-Hydroxyethyl–b
87R
88R/S
72R/S
13
29S
Benzyl–
H
232
Methyl 173
93
4
a % Inhibition at 1 lM.
cPropyl–
cPropyl–
cButyl–
Methyl
Methyl
Methyl
Methyl
14
80
28
25
b Both R and S isomers present within the side chain.
73R
46S
13
12
9
37S
54R/S
cPropylmethyl–
1-Hydroxyethyl–b Methyl n/a
48
Table 4. Functional activities of selected compounds
a % Inhibition at 1 lM.
R2
b Both R and S isomers present within the side chain.
O
N
CF3
Table 2. Spiroindene class: C2-amides, binding affinities
N
H
R1
O
CF3
N
CF3
N
R1
R2
Ca-Flux IC50 (nM)
H
Compound
NHX
29S
46S
37S
cPropyl–
cButyl–
cPropylmethyl–
Methyl
Methyl
Methyl
4
11
3
CF3
Compounda
X
CCR2 IC50 (nM) CHO assay
61
74
75
76
77
H
33%b
244
125
MeSO2–
CF3SO2–
MeOCO–
–CH@O
Table 5. Selectivity profile of the cyclopropyl analog 29
805
25%b
O
20%b
45%b
SO2-
78
79
N
CF3
S
N
H
N
SO2-
S
CF3
AcHN
SO2-
Receptor
Inhibition (%)
Concentration (lM)
80
81
7%b
hCCR1
hCCR3
hCCR4
ꢀ5
3
1
4
2
1
1
1
HN
CO-
ꢀ18
8
525
N
O
hCXCR4
CCR5 (MIP1alpha)
CCR8
H
38
9
a All compounds in this series were racemic.
b % Inhibition at 1 lM.

G. Butora et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4715–4722
4721
Table 6. Pharmacokinetic properties of the cyclopropyl derivative 29 (Sprague–Dawley rats)
O
N
CF3
N
H
CF3
Route
Dose (mg/kg)
AUCn (lM h)
Clearance (mL/min/kg)
7.36
Vol. Distrib. (L/kg)
2.19
t1/2 (h)
Cmax (lM)
Tmax (h)
F (%)
iv
p.o.
1.00
3.00
4.17
4.08
4.11
1.58
4.67
98
2. Onuffer, J. J.; Horuk, R. Trends Pharmacol. Sci. 2002, 23,
459.
and cyclopropylmethyl (37, IC50 = 25 nM, CHO,
IC50 = 9 nM hMonocyte) analogs.
3. Bendall, L. Histol. Histopathol. 2005, 20, 907.
4. Wells, T. N.; Power, C. A.; Shaw, J. P.; Proudfoot, A. E.
Trends Pharmacol. Sci. 2006, 27, 41.
5. Pease, J. E.; Williams, T. J. Br. J. Pharmacol. 2006, 147,
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6. Laing, K. J.; Secombes, C. J. Dev. Comp. Immunol. 2004,
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7. Tarrant, T. K.; Patel, D. D. Pathophysiology 2006, 13, 1.
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Cinque, P.; Poli, G.; Sica, A.; Mantovani, A. J. Leukocyte
Biol. 1997, 62, 30.
The effectiveness of such small aliphatic substituents was
also evaluated in simpler 4-phenyl piperidines, Table 3.
In this series, the activities of the synthesized com-
pounds did not reach the level of those obtained in the
spiroindene class. The most potent compound in this
series (propyl, 84, IC50 = 126 nM, CHO, IC50 = 42 nM
hMonocyte) was approximately ten-fold less active, than
the best spiroindenes.
9. Quinones, M. P.; Estrada, C. A.; Kalkonde, Y.; Ahuja, S.
K.; Kuziel, W. A.; Mack, M.; Ahuja, S. S. J. Mol. Med.
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The functional activity of selected compounds was eval-
uated using the calcium flux based FLIPR assay.31 The
results are summarized in Table 4. Once again, in agree-
ment with the binding data, the cyclopropyl derivative
29 and its methylene homolog 37 exhibited particularly
high functional activities.
The cyclopropyl analog 29 also showed quite remark-
able selectivity against other closely related chemokine
receptors, Table 5. Except for the CCR5 receptor, which
it showed an inhibition of 38% at a concentration of
1 mM, affinities to all other chemokine receptors were
negligible.
13. Feria, M.; Diaz-Gonzalez, F. Expert Opin. Ther. Pat.
2006, 16, 49.
14. Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.;
Sawada, H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.;
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16. Forbes, I. T.; Cooper, D. G.; Dodds, E. K.; Hickey, D.
M.; Ife, R. J.; Meeson, M.; Stockley, M.; Berkhout, T. A.;
Gohil, J.; Groot, P. H.; Moores, K. Bioorg. Med. Chem.
Lett. 2000, 10, 1803.
17. Witherington, J.; Bordas, V.; Cooper, D. G.; Forbes, I. T.;
Gribble, A. D.; Ife, R. J.; Berkhout, T.; Gohil, J.; Groot,
P. H. Bioorg. Med. Chem. Lett. 2001, 11, 2177.
18. Mirzadegan, T.; Diehl, F.; Ebi, B.; Bhakta, S.; Polsky, I.;
McCarley, D.; Mulkins, M.; Weatherhead, G. S.; Lapi-
erre, J. M.; Dankwardt, J.; Morgans, D., Jr.; Wilhelm, R.;
Jarnagin, K. J. Biol. Chem. 2000, 275, 25562.
19. 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,
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The cyclopropyl derivative 29 was also evaluated
in a rat pharmacokinetic model. Excellent drug levels
were observed after both intravenous (1.0 mg/kg,
AUCn = 4.17 lM) as well as oral (3.0 mg/kg, AUCn =
4.08 lM) administration. The compound showed a slow
clearance rate of 7.36 mL/min/kg, low volume of distri-
bution (2.19 L/kg), and an outstanding oral bioavail-
ability of 98%, Table 6.
A systematic examination of the central part of the lead
structure 10 demonstrated that the aromatic ring can be
successfully replaced with small, alicyclic groups. The
present study led to the discovery of (2S)-N-[3,5-bis
(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,30R)-30-meth-
yl-10H-spiro[indene-1,40-piperidin]-10-yl]butanamide (29), a
high affinity antagonist of the CCR2b receptor with an
excellent selectivity profile and outstanding pharmacokinet-
ic properties.
21. Kettle, J. G.; Faull, A. W.; Barker, A. J.; Davies, D. H.;
Stone, M. A. Bioorg. Med. Chem. Lett. 2004, 14, 405.
22. Initially, the binding affinities were evaluated in the CHO
assay, later the Human Monocyte-based assay was used.
Radioligand Competition Binding Assays: Human mono-
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(5 · 104) were incubated with 125I-hMCP-1 (20–50 pM) and
various concentrations of unlabeled chemokines in binding
buffer for 60 min at room temperature. The binding 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 for @
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. All data represent mean values for at least two
separate experiments.
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Pasternak, A.; Parsons, W. H.; Mills, S. G.; MacCoss, M.;
Vicario, P. P.; Zweerink, H.; Ayala, M. J.; Goyal, S.;
Hanlon, W. A.; Cascieri, M. A.; Springer, M. S. Bioorg.
Med. Chem. Lett. 2006, 16, 3735.
24. Yang, L.; Butora, G.; Jiao, R. X.; Pasternak, A.; Zhou, C.;
Parsons, W. H.; MacCoss, M.; Vicario, P. P.; Ayala, M. J.;
Cascieri, M. A. Discovery of 3-Piperidinyl-1-cyclopentan-
ecarboxamide as a Novel Scaffold for Highly Potent CCR2
Receptor Antagonists, 226th National Meeting of the
American Chemical Society, New York, NY, September 9,
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September 9, 2003.
31. CHO cells expressing hCCR2B (4 · 104) were incubated in
cell culture medium containing Fluo-3, AM fluorescent
dye (5 lg/mL, Molecular Probes), and probenicid (710 lg/
mL, Sigma) for 1 h at 37 ꢁC. Cells were washed with
Hanks buffer containing HEPES (20 mM), BSA (0.1%),
and probenicid, and then were resuspended in 90 lL of the
same buffer. A ligand addition plate was prepared by
adding 135 lL of chemokines at various concentrations
for the titration. To test inhibition of calcium release by
TAK-779, 2 lL of this antagonist at various concentra-
tions was added to the cells upon resuspension and 20 nM
hMCP-1 was used to activate CCR2B receptors. Both
plates were placed into the Fluorescence Imaging Plate
Reader (FLIPR, Coherent, Inc.). Chemokines were dis-
pensed from addition plate into the cell plate and calcium
release was measured by the Argon Laser at an excitation
wavelength of 488 nm and an emission wavelength of
530 nm.