Structure-kinetic relationships - An overlooked parameter in hit-to-lead optimization: A case of cyclopentylamines as chemokine receptor 2 antagonists

Article abstract of DOI:10.1021/jm4011737

Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (K_i = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.

Full text of DOI:10.1021/jm4011737

Article
pubs.acs.org/jmc
Structure−Kinetic RelationshipsAn Overlooked Parameter in Hit-
to-Lead Optimization: A Case of Cyclopentylamines as Chemokine
Receptor 2 Antagonists
Maris Vilums,†,‡ Annelien J. M. Zweemer,†,‡ Zhiyi Yu,‡ Henk de Vries,‡ Julia M. Hillger,‡
Hannah Wapenaar,‡ Ilse A. E. Bollen,‡ Farhana Barmare,§ Raymond Gross,§ Jeremy Clemens,§
Paul Krenitsky,§ Johannes Brussee,‡ Dean Stamos,§ John Saunders,§ Laura H. Heitman,‡
and Adriaan P. IJzerman*,‡
‡Division of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Post Office Box 9502, 2300 RA
Leiden, Netherlands
§Vertex Pharmaceuticals, Incorporated, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Preclinical models of inflammatory diseases
(e.g., neuropathic pain, rheumatoid arthritis, and multiple
sclerosis) have pointed to a critical role of the chemokine
receptor 2 (CCR2) and chemokine ligand 2 (CCL2).
However, one of the biggest problems of high-affinity
inhibitors of CCR2 is their lack of efficacy in clinical trials.
We report a new approach for the design of high-affinity and
long-residence-time CCR2 antagonists. We developed a new
competition association assay for CCR2, which allows us to
investigate the relation of the structure of the ligand and its
receptor residence time [i.e., structure−kinetic relationship (SKR)] next to a traditional structure−affinity relationship (SAR). By
applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as
CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence
time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound
22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.
classes. However, there are still no selective CCR2 antagonists
on the market for the treatment of inflammatory diseases.
Clinical trials, thus far, have failed mostly because of the lack of
efficacy, including the one for the CCR2 antagonist MK-0812
(Figure 1).10
It has been suggested that binding kinetics, especially the
lifetime of the ligand−receptor complex, can be used as a
predictor for drug efficacy and safety.11,12 The concept of
binding kinetics is often overlooked in the early phase of drug
discovery; however, incorporation of this parameter could help
INTRODUCTION
■
Chemokines are a class of chemoattractant cytokines, and their
main action is to control the trafficking and activation of
leukocytes and other cell types for a range of inflammatory and
non-inflammatory conditions. One of these, monocyte chemo-
tactic protein-1 [MCP-1/chemokine ligand 2 (CCL2)], acts on
monocytes, memory T cells, and basophils.1 It creates a
chemotactic gradient and activates the movement of immune
cells to the site of inflammation by binding to its cell-surface
receptor, chemokine receptor 2 (CCR2).2 This CCL2/CCR2
pair is overexpressed in several inflammatory conditions, in
which excessive monocyte recruitment is observed. CCR2 and
CCL2 knockout mice and CCR2 or CCL2 antibody-treated
rodents show decreased recruitment of monocytes and produce
considerably decreased inflammatory responses.3 This indicates
CCR2 as a potential target for the treatment of several
immune-based inflammatory diseases and conditions, such as
multiple sclerosis,4 atherosclerosis,5 rheumatoid arthritis,6
diabetes,7 asthma,8 and neuropathic pain.9
Figure 1. CCR2 antagonist MK-0812.
Received: August 1, 2013
In the past decade, there has been an increasing interest in
the development of small-molecule antagonists of the CCR2
receptor, resulting in the disclosure of many different chemical
© XXXX American Chemical Society
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Figure 2. RT and affinity values are both pharmacological parameters that may, however, suggest different lead structures.
a
Scheme 1. Synthesis of CCR2 Antagonists
a
Reagents and conditions: (a) 4 M LiOH aqueous, MeOH, reflux, 4 h, 91%; (b) 3,5-bis(trifluoromethyl)benzylamine, PyBrOP, N,N-
diisopropylethylamine (DiPEA), N,N-dimethylaminopyridine (DMAP), DCM, room temperature, 24 h, 83%; (c) TFA, DCM, room temperature, 1
h, 85%; (d) corresponding ketone, (AcO)3BHNa, AcOH, DCE, room temperature, 18 h, 21−86% (compounds 1, 2, and 11, 12); (e) corresponding
alkylating agent, DiPEA, CH3CN, 60 °C, 2 h, 14−54% (compounds 7−9); (f) for array synthesis, corresponding ketone, 5-ethyl-2-methylpyridine
borane (PEMB), AcOH, N-methylpyrrolidone (NMP), 65 °C, 24 h (compounds 10 and 13−32).
to decrease the attrition rate in later stages of drug
development.13 In this concept of kinetics, an additional
pharmacological parameter, the ligand−receptor residence time
(RT; the reciprocal of the dissociation rate constant koff), is
defined,14 which is a measure for the duration that a ligand is
bound to its target.
In this study, we first evaluated several reference CCR2
antagonists using a recently developed competition association
assay for CCR2 that yielded the respective association and
dissociation rate constants. As our starting point, we chose
compound 1, which was also the lead compound in the process
that led to the development of MK-0812 by the Merck group.10
The determination of the binding kinetics of several known
structures with this particular scaffold subsequently allowed us
to generate a new series of high-affinity and long-residence-time
CCR2 antagonists based on structure 2, which was previously
abandoned by other groups in optimization steps because of its
modest binding affinity (Figure 2).15
with trifluoroacetic acid (TFA) in dichloromethane (DCM)
produced amine 6. Reductive amination with different ketones
under NaBH(AcO)3 conditions afforded the desired products
1, 2, and 11, 12. Compounds 7−9 were synthesized by
alkylating amine 6 with different alkylating agents. Compounds
10 and 13−32 were generated from amine 6 and an array of
different ketones with 5-ethyl-2-methylpyridine borane com-
plex (PEMB) under conditions reported by Burkhardt and
Coleridge (Scheme 1).17
Biology. To determine the binding affinity, all compounds
were tested in a 125I-CCL2 displacement assay on human bone
osteosarcoma (U2OS)−CCR2 membrane preparations as
described previously by our group.18 Several methods can be
used to determine ligand binding kinetics [e.g., a kinetic
radioligand binding assay,19 surface plasmon resonance
(SPR),20 “two-step” competition binding assay,21 and “Tag-
lite” Cisbio22]. Most of these assays require special
modifications of the target protein or the ligand. Therefore,
we chose to use the competition association assay, because this
assay allowed us to determine the kinetics of unlabeled ligands
to the receptor expressed in membrane preparations. In our
hands, this is the most robust and accurate assay to measure
kinetics of unlabeled ligands.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of (1S,3R)-methyl-3-((tert-
butoxycarbonyl)amino)-1-isopropylcyclopentanecarboxylate
(3) was achieved following the synthetic approach reported by
Kothandaraman et al.15 The desired N-tert-butyloxycarbonyl
(Boc)-protected ester 3 was saponified to yield acid 4.
Subsequently, acid 4 was used in the peptide-coupling reaction
with 3,5-bis(trifluoromethyl)benzylamine to yield amide 5
under bromo-tris-pyrrolidino phosphoniumhexafluorophos-
phate (PyBroP) conditions.16 Removal of the N-Boc group
Validation of the [3H]INCB3344 Competition Associ-
ation Assay for CCR2. A competition association assay was
set up to determine the kinetic parameters of unlabeled
ligands.23 For this assay, we used the radiolabeled small-
molecule CCR2 antagonist [3H]INCB3344,24 instead of the
endogenous agonist protein radioligand 125I-CCL2. Because of
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Figure 3. Competition association assay with [3H]INCB3344 at 25 °C in the absence or presence of 1.1, 3.7, and 11 nM unlabeled INCB3344.
the large size of CCL2 (8600 Da), there is at best only a partial
overlap in the binding site with small-molecule antagonists.
Because the theoretical model of the competition association
assay is based on the assumption that unlabeled and
radiolabeled ligands should compete for the same binding
site, we decided to use [3H]INCB3344 in our assay. This
radioligand bears considerable chemical resemblance to the
compounds reported in this study. We first validated this
method by measuring the competition association of [3H]-
INCB3344 in the absence and presence of three different
concentrations of INCB3344 (1-, 3-, and 10-fold its Ki) (Figure
3). This resulted in kon and koff values for unlabeled INCB3344
radioligand binding at 50 min (t1) by the binding at 240 min
(t2). In this assay, antagonists with a slower dissociation rate
and, therefore, a longer RT than [3H]INCB3344 would result
in a KRI > 1 (Figure 4).
Structure−Affinity Relationships (SARs) versus Struc-
ture−RT Relationships. The 3-amino-1-isopropylcyclopenta-
necarboxamide scaffold has been extensively evaluated on the
basis of binding affinities for CCR2 and selectivity against other
̀
chemokine receptors and the human ether-a-go-go-related gene
(hERG) channel.15,26,27 Therefore, we decided to resynthesize
several reported derivatives of compound 115,28 and determine
their binding affinity in radioligand displacement assays (Table
2). Introduction of the benzyl group yielded compound 7 with
an affinity of 437 nM. When the spacer length between the
phenyl ring and basic nitrogen was extended to ethyl, binding
was almost lost (compound 8; Ki = 2400 nM). Prolonging the
chain to propyl allowed us to regain affinity (compound 9; Ki =
134 nM). Combining the knowledge of compounds 7 and 9 in
one structure yielded the indane derivative compound 2 with
even more improved affinity (Ki = 50 nM). Expanding the ring
system to tetrahydronaphthalene resulted in an additional
increase in affinity (compound 10; Ki = 33 nM). Removal of
aromatics yielded compound 11 with a cyclohexane ring, which
showed a decrease in affinity (Ki = 110 nM), but incorporation
of heteroatoms in the 4 position regained affinity (compounds
1 and 12; Ki = 6.8 and 31 nM, respectively) as described by
Kothandaraman et al.15 On the basis of affinity alone,
compound 1 would be the logical choice for lead optimization,
which yielded the clinical candidate MK-0812 in the case of the
Merck research group.10 However, the kinetic evaluation of
these known structures in a competition association assay
allowed us to use an additional parameter, RT. In this assay, the
best affinity compound 1 had a RT of 2.4 min, while compound
2 had a 4-fold longer RT of 9.5 min (Table 2). Structurally
closely related compound 10 had a RT of 5.6 min, which
convinced us to continue with compounds 2 and 10, because
they had a longer RT.
of 0.035
0.010 nM−1 min−1 and 0.024
0.002 min−1,
respectively, at 25 °C (Table 1). The corresponding RT was 43
Table 1. Comparison of Equilibrium Binding and Kinetic
Parameters of INCB3344 Determined Using Different
a
Methods
kon
assay
KD/Ki (nM)
(nM−1 min−1
)
koff (min−1
)
b
c
saturation binding
0.90 0.03
1.2 0.1
NA
NA
NA
d
displacement
NA
association and
0.23 0.04
0.054 0.002
0.013 0.002
e
dissociation
competition
association
0.72 0.19
0.035 0.010
0.024 0.002
f
a
Data are presented as means standard error of the mean (SEM) of
b
three independent experiments performed in duplicate. Saturation
c
binding of 1−45 nM [3H]INCB3344 to CCR2 at 25 °C. NA = not
d
applicable. Displacement of 3.5 nM [3H]INCB3344 from CCR2 at
e
25 °C. Association and dissociation of [3H]INCB3344 measured in
f
standard kinetic assays at 25 °C. Association and dissociation of
INCB3344 measured in competition association assays at 25 °C.
2 min. These results were in good agreement with kon and koff
values of [3H]INCB3344 binding from “traditional” association
and dissociation experiments, 0.054 0.002 nM−1 min−1 and
0.013 0.002 min−1, respectively17 (Table 1).
Screening of CCR2 Antagonists Using the Dual-Point
Competition Association Assay. The competition associa-
tion assay described above is laborious and time-consuming,
and hence, we developed a so-called dual-point competition
association assay for CCR2, according to principles that we
recently established for the adenosine A1 receptor.25 To this
end, we co-incubated [3H]INCB3344 with unlabeled antago-
nists at a concentration equal to their Ki value that was
determined in the 125I-CCL2 displacement assay. The so-called
kinetic rate index (KRI) was calculated by dividing the specific
Using a number of commercially available indanones, we
introduced different substituents on the indane ring (Table 3)
to cover chemical space as broadly as possible. The SAR
exploration on the 4 position showed that H-bond-accepting
and hydrophilic groups are tolerated. The 4-NH2 group led to a
minor increase (compound 13; Ki = 43 nM), but 4-OH and 4-
CN groups showed a decrease in affinity (compounds 14 and
15; Ki = 86 and 70 nM, respectively). 4-Me (compound 16)
and 4-MeO (compound 17) were not tolerated on this position
(26 and 30% displacement at 1 μM, respectively). On the 5
position, methoxy and hydroxyl groups improved the affinity,
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Figure 4. Representative competition association assay curves of control and long-residence-time compound 22a. Bt , specific radioligand binding at
1
the first time point (t1 = 50 min); Bt , specific radioligand binding at the second time point (t2 = 240 min). KRI is defined as Bt /Bt , which equaled
2
1
2
1.2 for compound 22a.
Table 2. Binding Affinities and RT of Compounds 1, 2, and
7−12
Table 3. Binding Affinities and KRI of Indenyl Derivatives 2
and 13−28
compound
number
Ki (nM) SEM
(n = 3)
R
KRI (n = 2)
2
H
50
43
86
8
7
8
0.7 (0.7/0.7)
0.8 (0.7/0.8)
0.6 (0.5/0.8)
0.8 (0.7/0.8)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
4-NH2
4-OH
4-CN
4-Me
4-OMe
5-OMe
5-OH
5-F
70 11
a
26%
a
30%
6.1 0.7
0.6 (0.6/0.6)
0.7 (0.7/0.8)
29
30%
18
2
a
5-Cl
1
1.1 (1.1/1.2)
1.1 (1.0/1.1)
5-Br
7.2 0.5
a
6-Cl
28%
6-Me
6-CN
55
54
2
4
6
0.8 (0.8/0.8)
0.6 (0.6/0.6)
4;5-di-OMe
130
5;6-di-OMe
3.9 0.3
0.7 (0.7/0.7)
0.6 (0.6/0.7)
5;6-(−OCH2O−)
6.3 0.8
a
Percent displacement at 1 μM 125I-CCL2.
previously reported as the best substituent in arylpiperidine
analogues by Pasternak et al.,27 resulted in a dramatic decrease
in affinity in the case of the indenyl derivative (compound 20;
30% displacement at 1 μM). 5-Cl substitution yielded better
affinity than 5-F (compound 21; 18 nM), and 5-Br was better
than 5-Cl (compound 22; 7.2 nM). 6-Cl (compound 23) led to
a dramatic decrease in affinity (28% displacement at 1 μM).
However, 6-Me and 6-CN groups were tolerated, having similar
affinities to the unsubstituted indane ring (compounds 24 and
25; Ki = 55 and 54 nM, respectively).
We continued the investigation with the analysis of
disubstitution, learning that the combination of 4,5 substitution
resulted in more than a 2-fold decrease in affinity (compound
26; Ki = 130 nM). On the contrary, the 5,6-dimethoxy group
yielded compound 27 with a high affinity of 3.9 nM.
Connecting the dimethoxy groups into a dioxolane ring yielded
a small decrease in affinity (compound 28; Ki = 6.3 nM).
which had also been suggested for other CCR2 antagonists.27,29
The methoxy group (compound 18) showed an 8-fold increase
in affinity (6.1 nM), while the hydroxyl group (compound 19)
displayed a less than 2-fold increase compared to the
unsubstituted indenyl derivative (29 and 50 nM, respectively).
On the contrary, the introduction of fluorine, which was
D
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Using the knowledge of the best position for substitution, we
continued the investigation on the 1,2,3,4-tetrahydronaphtha-
lene ring by introducing substituents on the 5 position (Table
4). Electron-donating groups showed very similar results to
Apparently, the stereochemistry of the indane ring system has
a major impact on the compound association rate to the
receptor, while the dissociation is not affected.
CONCLUSION
■
Table 4. Binding Affinities and KRI of
Tetrahydronaphthalene Derivatives 10 and 29−32
We have demonstrated that, next to affinity, additional
knowledge of the RT is useful for selecting and developing
new CCR2 antagonists. (1S,3R)-N-(3,5-Bis(trifluoromethyl)-
benzyl)-3-((5-bromo-2,3-dihydro-1H-inden-1-yl)amino)-1-iso-
propylcyclopentanecarboxamide (22a) had a RT of 135 min. In
comparison to the best affinity compound from the first SAR
screening, i.e., compound 1 (Table 1), compound 22a had a
56-fold increased RT while having similar affinity. This
indicates that affinity and RT do not correlate; moreover,
while SAR driven hit-to-lead optimizations often fail in later
stages of drug development because of the lack of efficacy (e.g.,
MK-0812), it has been shown on other targets that RT is linked
to the duration of the in vivo antagonist effect.30−32 Compound
22a may thus be a useful tool to test whether prolonged
blockade of CCR2 has a beneficial effect on CCR2-related
disorders, such as neuropathic pain.
compound
number
R
Ki (nM) SEM (n = 3)
KRI
10
29
30
31
32
H
33
27
35
2
1
2
0.6 (0.6/0.5)
0.7 (0.7/0.8)
0.8 (0.7/0.8)
5-OMe
5-OH
a
5-Br
48%
a
5-COOH
0%
a
Percent displacement at 1 μM 125I-CCL2.
EXPERIMENTAL SECTION
■
what we found for the indenyl moiety. Compounds 29 and 30
showed good affinity (27 and 35 nM, respectively), while
electron-withdrawing groups showed a decrease or complete
lack of affinity (compounds 31 and 32).
After SAR evaluation, the higher affinity compounds were
screened in our kinetic assay to determine their KRI value25
(see also Figure 2). A KRI value of <1 indicates that the RT of a
tested compound is shorter than the RT of the radioligand (less
than 43 min in this particular case). A KRI value of >1 reflects a
RT of more than 43 min.
Compound 2 in the screen showed a KRI value of 0.7 (RT =
9.5 min). However, compounds 21 and 22 had higher KRI
values (1.1 for both compounds). These compounds were
tested in a full competition association assay to determine their
association and dissociation rate constants (Table 5). Increasing
the size of the substituent, change from 5-Cl to 5-Br
(compound 21 versus compound 22), also yielded longer
RTs (56 and 94 min, respectively). Compound 22 was
separated in two diastereomers by preparative supercritical
fluid chromatography (SFC) using a Phenomenex Lux-4
column (Phenomenex, Inc.). The first compound to elute
(22a) had an affinity of 3.6 nM. However, the second
compound (22b) to elute had a 100-fold decreased affinity
(Ki = 289 nM). These separated diastereomers had very similar
Chemistry. All solvents and reagents were purchased from
commercial sources and were of analytical grade. Demineralized
water is simply referred to as H2O, because it was used in all cases,
unless stated otherwise (i.e., brine). 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker AV 400 liquid
spectrometer (1H NMR, 400 MHz; 13C NMR, 100 MHz) or using a
Bruker 500 MHz Avance III NMR spectrometer (compounds 22a and
22b) at ambient temperature. Chemical shifts are reported in parts per
million (ppm), are designated by δ, and are downfield to the internal
standard tetramethylsilane (TMS). Coupling constants are reported in
hertz and are designated as J. Analytical purity of the final compounds
was determined by high-performance liquid chromatography (HPLC)
with a Phenomenex Gemini 3 μm C18 110A column (50 × 4.6 mm, 3
μm), measuring UV absorbance at 254 nm. The sample preparation
and HPLC method for compounds 1, 2, 7−9, and 11, 12 were as
follows: 0.3−0.8 mg of compound was dissolved in 1 mL of a 1:1:1
mixture of CH3CN/H2O/t-BuOH and eluted from the column within
15 min at a flow rate of 1.0 mL. The elution method was set up as
follows: 1−4 min isocratic system of H2O/CH3CN/1% TFA in H2O,
80:10:10; from the 4th min, a gradient was applied from 80:10:10 to
0:90:10 within 9 min, followed by 1 min of equilibration at 0:90:10
and 1 min at 80:10:10. All compounds showed a single peak at the
designated retention time and are at least 95% pure. High-resolution
mass spectral analyses (HRMS) were performed on LTQ-Orbitrap
FTMS operated in a positive ionization mode with an electrospray
ionization (ESI) source, with the following conditions: mobile phase
A, 0.1% formic acid in water; mobile phase B, 0.08% formic acid in
CH3CN; gradient, 10−80% B in 26 min; and flow rate, 0.4 mL/min.
Preparative HPLCs (for compounds 10 and 13−32) were performed
koff rates (Table 5), which translated to similar RTs (compound
22a, RT = 135 min; compound 22b, RT = 77 min), but a
significant difference was observed for their kon rates.
Table 5. Kinetic Data of Compounds 21, 22, 22a, and 22b
compound number
R
Ki (nM) SEM (n = 3)
18
kon (nM−1 min−1
)
koff (min−1
)
RT (min)
56 14
21
5-Cl
5-Br
5-Br
5-Br
1
0.0027 0.0006
0.010 0.002
0.020 0.004
0.011 0.0002
0.0074 0.0004
0.015 0.004
22
7.2 0.5
3.6 0.9
289 94
94
3
8
22a
22b
0.0053 0.0007
0.00030 0.00007
135
77 18
E
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on a Waters AutoPurification HPLC−ultraviolet (UV) system with a
diode array detector using a Luna C18 Phenomenex column (75 × 30
mm, 5 μm), and a linear gradient from 1 to 99% of mobile phase B was
applied. Mobile phase A consisted of 5 mM HCl solution, and mobile
phase B consisted of acetonitrile. The flow rate was 50 mL/min.
Liquid chromatography−mass spectrometry (LC−MS) analyses were
performed using an Onyx C18 monolithic column (50 × 4.6 mm, 5
μm), and a linear gradient from 1 to 99% mobile phase B was applied.
Mobile phase A consisted of 0.05% TFA in water, and mobile phase B
consisted of 0.035% TFA in acetonitrile. The flow rate was 12 mL/
min. Separations of enantiomers were accomplished using chiral SFC.
The column was Phenomenex Lux-4 (250 × 10 mm, 5 μm). The
mobile phase condition of 10% MeOH with 20 mM NH3 and 90%
CO2 was applied at a flow rate of 10.0 mL/min. Optical rotations were
measured on a Perkin-Elmer polarimeter in CHCl3 at 20 °C. Thin-
layer chromatography (TLC) was routinely consulted to monitor the
progress of reactions, using aluminum-coated Merck silica gel F254
plates. Purification by column chromatography was achieved by use of
Grace Davison Davisil silica column material (LC60A, 30−200 μm).
The procedure for a series of similar compounds is given as a general
procedure for all within that series, annotated by the numbers of the
compounds.
reaction tube, and the corresponding ketone (1 equiv) was added.
Sequentially, acetic acid (1 equiv) and sodium triacetoxyborohoydride
(1.5 equiv) were added. The reaction mixture was stirred for 18 h at
room temperature and then washed with 1 M NaOH and H2O. The
organic layer was dried with MgSO4, filtered, and evaporated. The
product was purified by column chromatography (0−100% ethyl
acetate in DCM) to give the desired product.
(1S,3R)-N-(3,5-Bis(trifluoromethyl)benzyl)-1-isopropyl-3-((tetra-
hydro-2H-pyran-4-yl)amino)cyclopentanecarboxamide (1). Yield =
1
21%. H NMR (400 MHz, CDCl3) δ: 9.16 (s, 1H), 7.76−7.73 (m,
3H), 4.56−4.53 (m, 2H), 3.98−3.89 (m, 2H), 3.57−3.53 (m, 1H),
3.43−3.28 (m, 2H), 2.66−2.61 (m, 1H), 2.36−2.30 (m, 1H), 2.03−
1.80 (m, 2H), 1.78−1.6 (m, 5H), 1.49−1.40 (m, 1H), 1.31−1.20 (m,
3H), 0.93−0.89 (m, 6H). 13C NMR (400 MHz, CDCl3) δ: 179.1,
142.4, 131.8, 131.54, 131.2, 130.9, 127.7, 127.3, 124.6, 121.8, 121.0,
119.2, 66.9, 66.9, 57.5, 54.8, 51.9, 42.6, 37.1, 35.1, 34.3, 33.7, 33.6,
33.3, 19.5, 17.0. LC−MS: 481+; tR = 7.01 min.
(1S,3R)-N-(3,5-Bis(trifluoromethyl)benzyl)-3-((2,3-dihydro-1H-
inden-1-yl)amino)-1-isopropylcyclopentanecarboxamide (2). Yield
1
= 25% (mixture of diastereomers). H NMR (400 MHz, CDCl3) δ:
9.48 (s, 1H), 7.76−7.74 (m, 3H), 7.22−7.05 (m, 4H), 4.60−4.50 (m,
2H), 4.28−4.22 (m, 1H), 3.70−3.60 (m, 1H), 3.00−2.90 (m, 1H),
2.87−2.78 (m, 1H), 2.70−2.34 (m, 3H), 2.1−1.53 (m, 6H), 0.93−0.89
(m, 6H). 13C NMR (400 MHz, CDCl3) δ: 179.4, 144.5, 144.4, 144.6,
131.8, 131.5, 127.9, 127.6, 126.3, 126.2, 125.0, 123.5, 123.5, 122.0
121.9, 61.3, 58.0, 56.6, 42.6, 37.2, 36.0, 34.5, 33.9, 33.7, 33.4, 19.6,
17.0. LC−MS: 513+; tR = 8.12 min.
General Procedure for the Synthesis of Compounds 7−9. Amine
6 (1 equiv) was disolved in 4 mL of acetonitrile, and coresponding
alkylating agent (1.2 equiv) was added. Sequentually, DiPEA (1.2
equiv) was added. The reaction mixture was stirred in a microwave for
2 h at 60 °C and purified with column chromatography (60%
ethylacetate, 20% DCM, 20% petroleum ether, and 0−3% triethyl-
amine in ethyl acetate).
(1S,3R)-3-(Benzylamino)-N-(3,5-bis(trifluoromethyl)benzyl)-1-iso-
propylcyclopentane-1-carboxamide (7). Yield = 27% (as HCl salt).
1H NMR (400 MHz, CDCl3) δ: 9.40 (br s, 1H), 7.73 (s, 1H), 7.66 (s,
2H), 7.30−7.24 (m, 3H), 7.16−7.13 (m, 2H), 4.44 (d, J = 4.8 Hz,
2H), 3.73 (d, J = 2.4 Hz, 2H), 3.46−3.41 (m, 1H), 2.41−2.33 (m,
1H), 2.02−1.90 (m, 4H), 1.85−1.78 (m, 2H), 1.59−1.52 (m, 1H),
0.91 (dd, J1 = 10.8 Hz, J2 = 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ: 179.4, 142.6, 139.0, 132.0, 131.7, 131.3, 131.0, 128.6, 127.8, 127.5,
127.3, 124.6, 121.9, 120.8, 58.8, 58.7, 57.3, 51.9, 42.5, 35.3, 33.5, 33.1,
19.5, 16.9. LC−MS: 487+; tR: 7.40 min.
General Procedure for the Synthesis of Compounds 10 and 13−
32. To a series of 1.5 mL glass tubes was added amine 6 in NMP (0.95
M, 0.095 mmol), followed by solutions of different ketones (0.5 M, 0.1
mmol) in NMP, and these mixtures were subsequently treated with
acetic acid (0.1 mmol), followed by 5-ethyl-2-methyl-pyridine borane
(PEMB) (0.2 mmol). The reaction mixture was heated at 65 °C on a
reaction block for 24 h. The reaction mixtures were purified directly
using an automated mass-guided reverse-phase HPLC, and product-
containing fractions were concentrated to give final products of >90%
purity, as judged by LC−MS (average of 220 and 254 nm traces).
Biology. Chemicals and Reagents. 125I-CCL2 (2200 Ci/mmol)
was purchased from Perkin-Elmer (Waltham, MA). INCB3344 was
synthesized as described previously.33,34 [3H]INCB3344 (specific
activity of 32 Ci mmol−1) was custom-labeled by Vitrax (Placentia,
CA), for which a dehydrogenated precursor of INCB3344 was
provided. Tango CCR2-bla U2OS cells stably expressing human
CCR2 were obtained from Invitrogen (Carlsbad, CA).
Synthesis of (1S,3R)-methyl-3-((tert-butoxycarbonyl)amino)-1-iso-
propylcyclopentanecarboxylate (3) was achieved following the
synthetic approach reported by Kothandaraman et al.15
(1S,3R)-3-(tert-Butoxycarbonylamino)-1-isopropylcyclopentane-
carboxylic Acid (4). A solution of ester 3 (4.20 g, 14.72 mmol) in
EtOH (30 mL) and 4 M aqueous lithium hydroxide (LiOH aqueous,
40 mL) was refluxed for 4 h. After concentration in vacuum, the
solution was acidified with aqueous hydrochloric acid and extracted
with DCM/H2O. The organic layer was dried over MgSO4 and, after
concentration in vacuum, yielded the desired product as a yellow
powder (3.62 g, 91%). 1H NMR (400 MHz, CDCl3) δ: 10.75 (s, 1H),
6.53a (s, 0.5H), 5.05b (s, 0.5H), 3.98 − 3.78 (m, 1H), 2.25 − 1.50 (m,
7H), 1.40 (d, J = 16.8 Hz, 9H), 0.86 (d, J = 6.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3): δ 182.9a, 181.7b, 157.6b, 155.6a, 80.4b, 79.1a, 56.9,
52.8b, 51.7a, 38.6b, 38.2a, 35.0b, 34.5a, 33.2a, 32.9b, 32.1a, 31.8b, 28.3,
18.7, 18.2b, 18.0a. a and b are indicated for different rotamers.
tert-Butyl(3-((3,5-bis(trifluoromethyl)benzyl)carbamoyl)-3-isopro-
pylcyclopentyl) Carbamate (5). Compound 4 (1.53 g, 5.65 mmol)
was dissolved in 50 mL of DCM. To this mixture 3,5 bis-
(trifluoromethyl) benzylamine (1.89 g, 5.65 mmol) was added with
DiPEA (2.95 mL, 16.9 mmol), PyBrOP (2.64 g, 5.65 mmol), and
DMAP (0.55 g, 4.5 mmol). The reaction mixture was stirred for 24 h
at room temperature. The product was extracted with DCM/citric acid
solution in water and then with DCM/1 M NaOH. The organic layer
was dried with MgSO4 and evaporated. The product was purified by
column chromatography (0−100% ethyl acetate in DCM) to give the
product as a yellow oil (2.33 g, 83%). 1H NMR (400 MHz, CDCl3) δ:
7.69 (s, 3H), 7.25 (br s, 1H), 5.17 (br.s, 1H), 4.51−4.49 (m, 2H), 3.81
(br s, 1H), 1.99−1.90 (m, 4H), 1.69−1.72 (m, 2H), 1.50−1.58 (m,
1H), 1.36 (s, 9H), 0.74−0.77 (m, 6 H). 13C NMR (100 MHz, CDCl3)
δ: 178.6, 155.6, 142.1, 132.2, 131.8, 131.5, 131.2, 127.4, 127.3, 124.5,
121.8, 121.0, 119.1, 78.9, 57.6, 51.6, 42.8, 36.3, 34.6, 33.3, 32.6, 28.2,
18.7, 17.5.
3-Amino-N-(3,5-bis(trifluoromethyl)benzyl)-1-isopropylcyclopen-
tanecarboxamide (6). Trifluoroacetic acid (20 mL) was added to a
solution of compound 5 (2.33 g, 4.6 mmol) in 50 mL of DCM. The
reaction mixture was stirred for 1 h at room temperature. The reaction
mixture was neutralized with 1 M NaOH and extracted with DCM.
The organic layer was dried with MgSO4, filtered, and evaporated to
1
give the product as a yellow crystal (1.55 g, 85%). H NMR (400
Cell Culture and Membrane Preparation. U2OS cells stably
expressing the human CCR2 receptor (Invitrogen, Carlsbad, CA) were
cultured in McCoys5a medium supplemented with 10% fetal calf
serum, 2 mM glutamine, 0.1 mM non-essential amino acids (NEAAs),
25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),
1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL
streptomycin, 100 μg/mL G418, 50 μg/mL hygromycin, and 125
μg/mL zeocin in a humidified atmosphere at 37 °C and 5% CO2. Cell
MHz, CDCl3) δ: 9.16 (br s, 1H), 7.70−7.67 (m, 3H), 4.50−4.39 (m,
2H), 3.61−3.60 (m, 1H), 2.22−2.15 (m, 1H), 2.02−1.95 (m, 1H),
1.85−1.64 (m, 3H), 1.42−1.37 (m, 2H), 0.82−0.80 (m, 6H). 13C
NMR (100 MHz, CDCl3) δ: 179.4, 142.5, 131.8, 131.5, 131.2, 130.9,
127.3, 127.2, 124.6, 121.9, 120.4, 119.2, 57.3, 52.2, 42.4, 39.7, 35.3,
33.9, 33.6, 18.8, 16.9.
General Procedure for the Synthesis of Compounds 1, 2, and 11,
12. Amine 6 was dissolved in 4 mL of dichloroethane in a 5 mL
F
dx.doi.org/10.1021/jm4011737 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
culture and membrane preparation were performed as described
previously.18
KA = k1[L] + k2
KB = k3[I] + k4
S = (KA − KB)2 + 4k1k3LI × 10−18
KF = 0.5(KA + KB + S)
125I-CCL2 Displacement Assay. Binding assays were performed as
described previously.18
[3H]INCB3344 Competition Association Assay. The kinetic
parameters of unlabeled ligands at 25 °C were determined using the
competition association assay described by Motulsky and Mahan.23 At
different time points, 10 μg of U2OS−CCR2 membranes was added to
1.8 nM [3H]INCB3344 in a total volume of 100 μL of assay buffer in
the absence or presence of competing ligand. To validate the assay,
three concentrations of INCB3344 (1-, 3-, and 10-fold its Ki value of
[3H]INCB3344 displacement) were used. This validation showed that
using a single concentration (that equals the Ki) of unlabeled ligand
was sufficient to accurately measure kon and koff. Incubation was
terminated by dilution with ice-cold 50 mM tris(hydroxymethyl)-
aminomethane (Tris)-HCl buffer supplemented with 0.05% 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).
Separation of bound from free radioligand was performed by rapid
filtration through a 96-well GF/B filter plate pre-coated with 0.25%
polyethylenimine (PEI) using a Perkin-Elmer FilterMate harvester
(Perkin-Elmer, Groningen, Netherlands). Filters were washed 10 times
with ice-cold wash buffer. A total of 25 μL of Microscint scintillation
cocktail (Perkin-Elmer, Waltham, MA) was added to each well, and
the filter-bound radioactivity was determined by scintillation
spectrometry using the P-E 1450 Microbeta Wallac Trilux scintillation
counter (Perkin-Elmer). Kinetic parameters of unlabeled ligands were
calculated using eq 3, as mentioned below in the Data Analysis section.
[3H]INCB3344 Dual-Point Competition Association Assay. KRI
values of unlabeled ligands were determined using the dual-point
competition association assay as described previously, in which
radioligand binding was determined at two different time points.25
Time point t1 represents the time at which radioligand binding reached
99.5% of total binding at equilibrium.
KS = 0.5(KA + KB − S)
Bmaxk1L × 10−9
Q =
KF − KS
⎛
⎜
⎝
⎞
k4(KF − KS)
k4 − KF
KF
k4 − KS
KS
(−KFX)
(−KSX)
Y = Q
+
e
−
e
⎟
⎠
KFKS
(3)
where X is the time (min), Y is the specific binding [disintegrations per
minute (DPM)], k1 is kon (M−1 min−1) of [3H]INCB3344
predetermined in association experiments, k2 is koff (min−1) of
[3H]INCB3344 predetermined in dissociation experiments, L is the
concentration of [3H]INCB3344 used (nM), Bmax is the total binding
(DPM), and I is the concentration of unlabeled ligand (nM). Fixing
these parameters into eq 3 allows for the following parameters to be
calculated: k3 is kon (M−1 min−1) of the unlabeled ligand, and k4 is koff
(min−1) of the unlabeled ligand. The association and dissociation rates
were used to calculate the “kinetic KD” as follows:
KD = koff /kon
(4)
The RT was calculated according to the formula RT = 1/koff.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data of compounds 8−32. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Fax: +31-71-527-4565. E-mail: ijzerman@lacdr.leidenuniv.nl.
■
t1 = 8t1/2,association
(1)
Author Contributions
The second time point (t2) was arbitrarily set at 4 h, where little but
reliably measurable, specific binding remained. A total of 10 μg of
U2OS−CCR2 membranes were incubated for 50 min (t1) or 240 min
(t2) in a total volume of 100 μL of assay buffer with 1.8 nM
[3H]INCB3344 in the absence or presence of unlabeled ligands at 25
°C. The amount of radioligand bound to the receptor was measured
after co-incubation of the unlabeled ligands at 1-fold their respective Ki
value in the 125I-CCL2 displacement assay. Incubations were
terminated, and samples were obtained as described under the
[3H]INCB3344 Competition Association Assay section. KRI values of
unlabeled ligands were calculated using eq 2, as mentioned below in
the Data Analysis section.
†Maris Vilums and Annelien J. M. Zweemer contributed equally
to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was financially supported by the Dutch Top
Institute Pharma (Project D1-301). We thank Dr. Julien Louvel
and Jacobus van Veldhoven for their input in analytical data
analysis. We acknowledge Dong Guo and Prof. Dr. Martine
Smit (Vrije Universiteit, Amsterdam, Netherlands) for helpful
comments and suggestions.
Data Analysis. All experiments were analyzed using the nonlinear
regression curve fitting program Prism 5 (GraphPad, San Diego, CA).
For radioligand displacement data, Ki values were calculated from IC50
values using the Cheng and Prusoff equation.35
ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; CCL2, chemokine ligand 2; CCR2,
chemokine receptor 2; CHAPS, 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate; DCM, dichlorome-
thane; DiPEA, N,N-diisopropylethylamine; DMAP, N,N-
dimethylaminopyridine; DPM, disintegrations per minute;
HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid;
Data of the dual-point competition association assay were analyzed
as described previously.25
KRI values were calculated by dividing the specific radioligand
binding measured at t1 (Bt ) by its binding at t2 (Bt ) in the presence of
1
2
unlabeled competing ligand as follows:
̀
hERG, human ether-a-go-go-related gene; HPLC, high-
KRI = B /B
performance liquid chromatography; HRMS, high-resolution
mass spectral analyses; KRI, kinetic rate index; NEAA, non-
essential amino acid; NMP, N-methylpyrrolidone; NMR,
nuclear magnetic resonance; PEI, polyethylenimine; PyBrOP,
bromo-tris-pyrrolidino phosphoniumhexafluorophosphate; RT,
t1
t2
(2)
Association and dissociation rates for unlabeled ligands were
determined by nonlinear regression analysis of the competition
association data as described by Motulsky and Mahan23
G
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Journal of Medicinal Chemistry
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(18) Zweemer, A. J. M.; Nederpelt, I.; Vrieling, H.; Hafith, S.;
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residence time; SAR, structure−affinity relationship; SFC,
supercritical fluid chromatography; SKR, structure−kinetic
relationship; TFA, trifluoroacetic acid; TLC, thin-layer
chromatography; Tris, tris(hydroxymethyl)aminomethane;
U2OS, human bone osteosarcoma
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