When structure-affinity relationships meet structure-kinetics relationships: 3-((Inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.01.063

Chemokine ligand 2 (CCL2) mediates chemotaxis of monocytes to inflammatory sites via interaction with its G protein-coupled receptor CCR2. Preclinical animal models suggest that the CCL2-CCR2 axis has a critical role in the development and maintenance of inflammatory disease states (e.g., multiple sclerosis, atherosclerosis, insulin resistance, restenosis, and neuropathic pain), which can be treated through inhibition of the CCR2 receptor. However, in clinical trials high-affinity inhibitors of CCR2 have often demonstrated a lack of efficacy. We have previously described a new approach for the design of high-affinity CCR2 antagonists, by taking their residence time (RT) on the receptor into account. Here, we report our findings on both structure-affinity relationship (SAR) and structure-kinetic relationship (SKR) studies for a series of 3-((inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists. SAR studies showed that this class of compounds tolerates a vast diversity of substituents on the indenyl ring with only small changes in affinity. However, the SKR is affected greatly by minor modifications of the structure. The combination of SAR and SKR in the hit-to-lead process resulted in the discovery of a new high-affinity and long-residence-time CCR2 antagonist (compound 15a, K_i Combining double low line 2.4 nM; RT Combining double low line 714 min).

Full text of DOI:10.1016/j.ejmech.2015.01.063

European Journal of Medicinal Chemistry 93 (2015) 121e134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
When structureeaffinity relationships meet structureekinetics
relationships: 3-((Inden-1-yl)amino)-1-isopropyl-cyclopentane-1-
carboxamides as CCR2 antagonists
Maris Vilums a, Annelien J.M. Zweemer a, Farhana Barmare b, Anouk M.F. van der Gracht a,
Dave C.T. Bleeker a, Zhiyi Yu a, Henk de Vries a, Raymond Gross b, Jeremy Clemens b,
Paul Krenitsky b, Johannes Brussee a, Dean Stamos b, John Saunders b, Laura H. Heitman a,
,
*
Adriaan P. IJzerman a
a Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
b Vertex Pharmaceuticals Incorporated, 11010 Torreyana Rd., San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemokine ligand 2 (CCL2) mediates chemotaxis of monocytes to inflammatory sites via interaction with
its G proteinecoupled receptor CCR2. Preclinical animal models suggest that the CCL2-CCR2 axis has a
critical role in the development and maintenance of inflammatory disease states (e.g., multiple sclerosis,
atherosclerosis, insulin resistance, restenosis, and neuropathic pain), which can be treated through in-
hibition of the CCR2 receptor. However, in clinical trials higheaffinity inhibitors of CCR2 have often
demonstrated a lack of efficacy. We have previously described a new approach for the design of high
eaffinity CCR2 antagonists, by taking their residence time (RT) on the receptor into account. Here, we
report our findings on both structureeaffinity relationship (SAR) and structureekinetic relationship
(SKR) studies for a series of 3-((inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2
antagonists. SAR studies showed that this class of compounds tolerates a vast diversity of substituents on
the indenyl ring with only small changes in affinity. However, the SKR is affected greatly by minor
modifications of the structure. The combination of SAR and SKR in the hit-to-lead process resulted in the
Received 28 August 2014
Received in revised form
29 January 2015
Accepted 31 January 2015
Available online 2 February 2015
Keywords:
CC chemokine receptor 2
CC chemokine receptor antagonists
Long residence time
Structureekinetics relationships
discovery of
a new higheaffinity and longeresidenceetime CCR2 antagonist (compound 15a,
Ki ¼ 2.4 nM; RT ¼ 714 min).
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
proteins that act through activation and recruitment of leukocytes
and other cell types in a range of inflammatory and non-
inflammatory conditions. However, inappropriate overexpression
Chemokines are
a class of endogenous pro-inflammatory
Abbreviations: Boc, tert-butyloxycarbonyl; CCL2, chemokine ligand 2; CCR2, chemokine receptor 2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate; DBU, 1,8-diazabicycloundec-7-ene; DEA, diethylamine; DEAD, diethyl azodicarboxylate; DCM, dichloromethane; DiPEA, N,N-diisopropylethylamine;
DMAP, N,N-dimethylaminopyridine; DMF, dimethylformamide; DPM, disintegrations per minute; EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionisation; FTMS,
ꢀ
fourier transform mass spectrometer; G418, geneticin; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; hERG, human Ether-a-go-go-Related Gene; HPLC, high-
performance liquid chromatography; HRMS, high resolution mass spectral analyses; INCB3344, N-[2-[[(3S,4S)-1-[4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-
ethoxypyrrolidin-3-yl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide; IPA, iso-propanol; KRI, kinetic rate index; LDA, lithium diisopropylamide; LC-MS, liquid chroma-
tography e mass spectrometer; NEAA, non-essential amino acids; NMP, N-methylpyrrolidone; NMR, nuclear magnetic resonance; PEI, polyethylenimine; PEMB, 5-ethyl-2-
methylpyridine borane; PyBrOP, bromo-tris-pyrrolidino phosphoniumhexafluorophosphate; RT, residence time; SAR, structureeaffinity relationships; SFC, supercritical fluid
chromatography; SKR, structureekinetic relationships; TBDMS-Cl, tert-butyldimethylsilyl chloride; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chroma-
tography; TMS, tetramethylsilane; Tris, tris(hydroxymethyl)aminomethane; U2OS, human bone osteosarcoma cells; UPLC, ultra performance liquid chromatography; UV,
ultraviolet.
* Corresponding author.
E-mail address: ijzerman@lacdr.leidenuniv.nl (A.P. IJzerman).
http://dx.doi.org/10.1016/j.ejmech.2015.01.063
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

122
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
ligandereceptor complex could be used as a possible indicator for
drug efficacy and safety [10,11].
To probe this concept for CCR2 antagonists we determined the
residence time of the failed clinical candidate MK-0812. In our
assays it had a Ki value of 0.55 nM and a residence time of 92 min
(see Fig. S4 and Table S1 in supporting materials). Previously we
have generated higheaffinity and longereresidenceetime CCR2
antagonists
based
on
a
(1S,3R)-3-amino-N-(3,5-
bis(trifluoromethyl)benzyl)-1-isopropylcyclopentane-1-
Fig. 1. Structure of MK-0812 [5].
carboxamide scaffold [12]. We explored different substituents
and ring systems on the 3-amino group of the scaffold and
observed that the longest residence time was found with an
indane ring system. Although potent and longeresidenceetime
compounds were identified in that study they were still very
close to the residence time of MK-0812, thus we sought to pro-
long the RT and define more detailed structureekinetics re-
lationships for the CCR2 receptor. In the present study we
evaluated different amide groups for the 3-((inden-1-yl)amino)-
1-isopropyl-cyclopentane-1-carboxamide scaffold based on their
RT and explored a broad chemical space around the indane ring
system to define the SAR and SKR for 3-((inden-1-yl)amino)-1-
isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists.
of such proteins is implicated in a variety of disease conditions [1].
Both CeC Chemokine Ligand 2 (CCL2) and its cognate receptor CeC
Chemokine Receptor 2 (CCR2) are involved in various autoimmune
or inflammation-associated diseases. Blockade of the CCL2-CCR2
axis via either genetic or pharmacologic intervention has proven
efficacious in animal models of multiple sclerosis, atherosclerosis,
insulin resistance, restenosis, and neuropathic pain [2e4].
Fuelled by such promising preclinical data, there has been an
increasing interest in advancing antagonists of CCR2 into clinical
trials. Historically, small molecules tested have failed in clinical
trials because of lack of efficacy, e.g., MK-0812 (orthosteric CCR2
antagonist for the treatment of rheumatoid arthritis and multiple
sclerosis, Fig. 1) [5] and AZD-2423 (potent, orally bioavailable, non-
competitive, negative allosteric modulator of the CCR2 chemokine
receptor for treatment of neuropathic pain) [6]. A humanized anti-
CCR2 antibody (MLN-1202) did not show efficacy either in patients
with rheumatoid arthritis and multiple sclerosis [7]. However,
administration of the antibody reduced the numbers of circulating
monocytes in peripheral blood [8]. Moreover, a study of MLN-1202
in patients with risk factors for atherosclerosis demonstrated that
treatment was able to reduce Cereactive protein levels [9]. This
shows that CCR2 antagonism can have important biological effects
in humans.
2. Results and discussion
2.1. Chemistry
The synthesis of N-(3,5-bis(trifluoromethyl)benzyl)-3-((2,3-
dihydro-1H-inden-1-yl)amino)-1-isopropylcyclopentane-1-
carboxamide
isopropylcyclopentane-1-carboxylic acid
1
and (1S,3R)-3-((tert-butoxycarbonyl)amino)-1-
was achieved
2
following the approach reported earlier by our group [12]. From
acid 2 and 1-(4-(trifluoromethyl)pyridin-2-yl)piperazine or 7-(tri-
fluoromethyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride via a
peptide coupling reaction were generated amides 3 and 4 under
Perhaps, to be efficacious in treatment of CCR2erelated diseases,
higheaffinity antagonism is not enough. Moreover, blockade of
CCR2 can cause an increase in endogenous CCL2 levels [8] which
could compete again with the administered drug. In recent years
more and more attention has been devoted to an additional
parameter in the drug discovery pipeline, i.e. drugetarget resi-
dence time. It has been proposed that the lifetime of the
bromo-tris-pyrrolidino
phosphoniumhexafluorophosphate
(PyBroP) conditions [13]. Subsequently, a solution of TFA in DCM
(1:1) was used to remove the N-Boc protecting group which yielded
amines 5 and 6. Compounds 7, 9e13 and 17a e 22 were generated
from amines 5 and 6 using an array of different indanones with the
5-ethyl-2-methylpyridine borane complex (PEMB) under condi-
tions reported by Burkhardt and Coleridge (Scheme 1) [14]. In the
Scheme 1. Reagents and conditions: a) 1-(4-(trifluoromethyl)pyridin-2-yl)piperazine or 7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride, PyBrOP, DIPEA, DMAP,
DCM, room temperature, 24 h, 82e90%; b) TFA, DCM, room temperature, 1 h, 90e98%; c) for array synthesis e corresponding indanone (1.05 equiv), 5-ethyl-2-methylpyridine
borane (PEMB) (2.1 equiv), AcOH (1.05 equiv), NMP, 65 ꢀC, 24 h, (compounds 7; 9e13 and 17e22); d) i) corresponding indanone (1,1 eq.), Ti(O-i-Pr)4 (3 eq.), THF, 48 h, ii)
NaBH4, EtOH 16 h, room temperature, 8e41% (compounds 8; 14e16 and 36e40); e) array synthesis - compound 15, corresponding aryl boronic acid, Na2CO3, Pd(PPh3)4, toluene/
NMP/H2O, 80 ꢀC, 24 h, (compounds 23e35).

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
123
Scheme 2. Reagents and conditions: a) i) NaCl, AlCl3, melt at 130 ꢀC, ii) 40-bromo-4-
chloro-butyrophenone, 180 ꢀC, 30 min, 91%.
case of the 5-CF3 derivative (compound 17) diastereomers were
separated during the purification, however, only the first diaste-
reomer to elute (17a) had a sufficient purity to be tested in bio-
assays. Compounds 8,14e16 and 36e40 were synthesized from 6 in
a Ti(Oi-Pr)4 promoted reductive amination reaction with different
indanones. Compounds 23e35 were synthesized via Suzukiecou-
pling of compound 15 with the corresponding arylboronic acids.
The synthesis of 3-Me; 5-Br indanone 42 was achieved by
intramolecular cyclization of commercially available 1-(4-
bromophenyl)-4-chlorobutan-1-one 41 following a procedure re-
ported in the patent literature (Scheme 2) [15].
Scheme 4. Reagents and conditions: a) (R)-(þ)-2-methyl-CBS-oxazaborolidine cata-
lyst, N,N,diethylaniline borane, toluene, 3 h, room temperature, 97%; b) (S)-(þ)-2-
methyl-CBS-oxazaborolidine catalyst, N,N, diethylaniline borane, toluene, 3 h, room
temperature, 99%.
were subsequently screened in
a
[3H]INCB3344 dual point
competition association assay on U2OSeCCR2 membrane prepa-
rations to determine their kinetic-rate-index (KRI), which served as
an indicator for the magnitude of the RT. Compounds with a KRI > 1
were finally tested in the full competition association assay to
determine the RT, as described previously by our group [12].
The 3-alkyl; 5-Br; 6-OMe indanones were synthesized as shown
in Scheme 3. The reaction of 5-Br; 6-OMe indanone 43 with tert-
butyldimethylchlorosilane and DBU in benzene gave ((6-bromo-5-
methoxy-1H-inden-3-yl)oxy)
(tert-butyl)dimethylsilane
(44).
Deprotonation of 44 with n-BuLi and reaction of the lithium salt
with methyl iodide or ethyl iodide in THF and subsequent
quenching of the reaction mixture with 12 M HCl resulted in 5-
bromo-6-methoxy-3-alkyl-indanones (45, 46).
2.2.1. Structureeaffinity relationships and structureekinetics
relationships
In the past few years several distinctly different amide groups
have been disclosed for the general CCR2 scaffold of 3-amino-1-
isopropylcyclopentanecarboxamides, with many final compounds
displaying high and often very similar affinities (Fig. 2) [19e21]. In
the current study we decided to keep the 3-((inden-1-yl)amino)-1-
isopropyl-cyclopentane-1-carboxamide scaffold that was central in
our previous report [12], and investigated the effect on affinity and
RT of three different amide groups. When we changed the 3,5-
bis(trifluoromethyl)benzyl group (compound 1) (Table 1) to a 1-
(4-(trifluoromethyl)pyridin-2-yl)piperazine group (compound 7)
the affinity was improved 3-fold (p < 0.05, Student's t-test), while a
rigidification of the benzyl group into the 7-(trifluoromethyl)-
1,2,3,4-tetrahydroisoquinoline group (compound 8) yielded an
even higher, 20-fold increase in affinity (p < 0.005, Student's t-test)
(compounds 1, 7 and 8; Ki ¼ 50 nM, 15 nM and 2.2 nM, respectively)
(Fig. S2A in supporting materials). In kinetic tests of these com-
pounds (kinetic rate index (KRI) [22] and RT) we learned that for
longer receptor occupancy the smaller and less flexible tetrahy-
droisoquinoline was preferred (KRI ¼ 0.8, 0.5 and 1.0; RT ¼ 9.1, 8.3
and 21 min, for 1, 7 and 8, respectively) (Fig. S3A in supporting
materials).
The (1S)-5-bromo-3-methyl-2,3-dihydro-1H-indanol (47) and
(1R)-5-bromo-3-methyl-2,3-dihydro-1H-indanol (48) were pre-
pared via catalytic enantioselective reduction of racemic 5-bromo-
3-methyl-2,3-dihydro-1H-indanone (42) using the (R)-methyl-
CBS-oxazaborolidine and (S)-methyl-CBS-oxazaborolidine cata-
lysts, respectively, with N,N-diethylaniline borane as reducing
agent providing excellent enantioselectivity (Scheme 4) [16].
To prepare for the subsequent coupling, amine 6 was protected
as the 2-nitrobenzenesulfonamide (49) (Scheme 5) [17]. The
respective alcohols (47) and (48) when treated with (49) under
FukuyamaeMitsunobu conditions, resulted in N-alkylation to
afford the 2-nitrobenzenesulfonamides (50) and (51) (Scheme 6).
Selective deprotection of the 2-nitrobenzenesulfonamides (50) and
(51) with thiophenol and K2CO3 gave the desired diastereomers
37a, 37b, 37c and 37d (Scheme 7).
2.2. Pharmacology
To determine their binding affinity all compounds were tested
in a 125I-CCL2 radioligand displacement assay on U2OS-CCR2
membrane preparations as described previously by our group
[18]. Compounds with affinities better than or equal to 100 nM
Encouraged by these results we decided to continue with
compound 8 and investigate different substituents on the indenyl
Scheme 3. Reagents and conditions: a) TBDMS-Cl, DBU, 0 ꢀC / room temperature, 99%; b) i)LDA, 1 h, e78 ꢀC / ꢁ35 ꢀC, 1 h / ꢁ78 ꢀC, corresponding alkyliodide, 2 h; ii) 12 M HCl,
82%.

124
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
Scheme 5. Reagents and conditions: a) 2-nitrobenzenesulfonyl chloride, DIPEA, CH2Cl2, 1 h, room temperature, 98%.
Scheme 6. Reagents and conditions: a) and b) DEAD, PPh3, THF, e78 ꢀC to room temperature.
Scheme 7. Reagents and conditions: a) and b) PhSH, K2CO3, DMF, room temperature.

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
125
Fig. 2. CCR2 antagonists from Merck and Pfizer based on a 3-amino-1-isopropylcyclopentanecarboxamide scaffold with different amide groups [19e21].
group (Table 2). The rigidification of the right-hand side of the
structure improved the affinity in general, while the order of sub-
stituents on the indenyl group was hardly affected when compared
to our previous findings on the 3,5-bis(trifluoromethyl)benzyla-
mide derivatives (compound 1) [12]. Substitution on the 4-position
decreased the affinity. Introduction of 4-Me (compound 9) led to a
15-fold decrease (p < 0.001, Student's t-test). However, more polar
groups were better tolerated. The 4-NH2 substituent (compound
10) maintained the affinity, while 4-CN and 4-OH groups were less
tolerated (compounds 11 and 12; Ki ¼ 15 and 21 nM, respectively).
Halogen substituents on the 5 position (compounds 13, 14, 15 and
16) had little effect on affinity. Also an introduction of a CF3 group
(compound 17a) - often considered as a bioisostere of chlorine e
was tolerated. On the 6 position an electron donating methyl group
(compound 18) resulted in a decrease in affinity (p < 0.05, Student's
t-test), while the electron withdrawing cyano (compound 19) or the
6-Cl (compound 20), both substituents were tolerated. However,
the highest affinity compound 21 was obtained by double
Table 1
Binding Affinities, KRI and Residence Time of compounds 1; 7 and 8.
Compound number
R
Ki (nM) SEM (n ¼ 3)
50 8
KRI (n ¼ 2)
RT (min) SEM (n ¼ 3)
1
0.8 (0.7/0.8)
9.1 1.7
7
15
1
0.5 (0.5/0.5)
8.3 2.8
8
2.2 0.6
1.0 (0.9/1.0)
21
3

126
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
Table 2
Binding Affinities, KRI and Residence Time of compounds 8e22.
Compound number
R
Ki (nM) SEM (n ¼ 3)
KRI (n ¼ 2)
RT (min) SEM (n ¼ 3)
8
9
H
2.2 0.6
1.0 (0.9/1.0)
0.7 (0.7/0.7)
0.7 (0.7/0.8)
0.6 (0.6/0.6)
0.7 (0.7/0.7)
0.9 (0.9/0.9)
1.2 (1.0/1.3)
1.3 (1.3/1.3)
1.3 (1.3/1.2)
1.4 (1.5/1.3)
0.6 (0.6/0.5)
0.6 (0.6/0.7)
0.6 (0.6/0.6)
1.0 (1.1/0.9)
0.8 (0.8/0.8)
21
e
3
4-Me
4-NH2
4-CN
4-OH
5-F
5-Cl
5-Br
5-I
5-CF3
6-Me
6-CN
6-Cl
5; 6-di-OMe
4,7-di OMe
31
2
10
11
12
13
14
15
16
17a
18
19
20
21
22
4.6 1.0
e
e
e
15
21
5
3
4.9 1.6
1.6 0.7
2.3 0.6
4.4 0.9
55
100 20
213 32
103
667 222
e
e
e
6
9
13
23
13
5
6
8
7.9 2.0
1.2 0.3
63
e
5
49
7
substitution on the 5 and 6 positions with methoxy groups. The
corresponding regioisomer with 4,7-di-OMe (compound 22) dis-
played a 40-fold decrease in affinity compared to compound 21
(p < 0.005, Student's t-test).
Testing these compounds in the highethroughput dualepoint
competition association assay showed that the abovementioned
rigidification on the rightehand side of the molecule affects RT only
for 5-substituted indenyl derivatives, with most of them having KRI
values higher than unity. These compounds were tested in a full
competition association assay and the highest affinity compound
21 had a RT of 63 min. Halogen substituents had sizeedependent
effects on KRI values. When tested for RT, indeed, increasing size
correlated with longer residence times except for 5-I 16 where we
observed a decrease in RT, as was the case for its affinity. However,
compound 17a (single diastereomer) displayed longer RTcompared
to its bioisostere the 5-Cl compound (14). All other compounds
showed KRI values below unity and thus showed a behavior com-
parable to the benzyl derivatives reported earlier [12].
Next, we explored the 5 position by incorporating an additional
aromatic system. Previously Xue et al. [23] had shown this
approach to be successful in a pyrrolidine series of CCR2 antago-
nists. However, for our structures, an added unsubstituted phenyl
ring (compound 23) resulted in a dramatic decrease of affinity
(Table 3). Adding a 2-Me group (compound 24) yielded a small
increase while 3-Me (compound 25) did not improve the affinity
compared to unsubstituted 23. Incorporation of a cyano group on
Table 3
Binding Affinities, KRI and Residence Time of compounds 23e30.
Compound number
R
Ki (nM) SEM (n ¼ 3)
KRI (n ¼ 2)
23
24
25
26
27
28
29
30
H
28%a
e
2-Me
3-Me
3-CN
4-CN
2-OMe
3-OMe
4-OMe
46%a
e
24%a
e
55 14
11 4.7
2%a
0.7 (0.7/0.7)
0.9 (0.8/1.0)
e
39
6
0.8 (0.8/0.8)
0.9 (0.9/0.9)
75 23
a
Percent displacement at 1m
M125I-CCL2.

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
127
Table 4
Binding Affinities, KRI and Residence Time of compounds 31e35.
Compound number
31
R
Ki (nM) SEM (n ¼ 3)
10 5
KRI (n ¼ 2)
0.7 (0.6/0.7)
32
41
8
0.8 (0.7/0.8)
33
10
4
0.5 (0.4/0.6)
34
23
7
0.6 (0.6/0.6)
35
12
1
0.8 (0.8/0.8)
the 3 or 4 position (compounds 26 and 27) resulted in a regain of
affinity into the nanomolar range. The same effect was observed
with a methoxy group (compounds 29 and 30). However, 2-OMe
(compound 28) was detrimental for affinity. These findings sug-
gest that the space filling and hydrogeneaccepting properties are
more important for binding than the electronic properties of the
substituents. Possible hydrogen bonding may also play a role when
the phenyl ring was exchanged for 3-pyridine (compound 31;
Ki ¼ 10 nM) (Table 4). Incorporation of a 2-OMe group (compound
32) resulted in a decrease of affinity, with an affinity comparable to
its phenyl analog 29. Extending the substituent to an ethoxy group
(compound 33) is in accordance with the idea of space filling
properties, as it yielded a gain in affinity compared to 32 (p < 0.05,
Student's t-test). Changing the location of the nitrogen atom in the
pyridine ring to the 4-position (compound 34) was beneficial vs 32.
An additional gain in affinity was reached by incorporating a
fluorine atom on the 5 position (compound 35). In general, this
series of compounds suggests there is enough space in the binding
pocket to accommodate another aromatic ring with preferably
hydrogen bond accepting properties. However, when these com-
pounds were tested in the dual-point competition association assay
none of them showed KRI values above 1.
Another approach to investigate SAR and SKR in more detail was
based on the superimposition of structure 8 with the structure of
MK-0483, which has been reported as a CCR2 antagonist with a
receptor dissociation time (T1/2) of over 9 h (Fig. 3) [24].

128
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
Fig. 3. Superimposition of MK-0483 (yellow) with compound 8 (cyan) using “ICM-Pro 3.7b, Molsoft LLC”. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
In the superimposition, the 3-position of the indane ring of
compound 8 overlaps with the 3 position of the piperidine ring of
MK-0483. So we decided to incorporate a methyl group on the 3
position of the indenyl system to yield compound 36 (Table 5). This
had a minor effect on the affinity, but caused a significant decrease
in KRI value. However, the combination of a 3-Me and 5-Br sub-
stituent (compound 37) yielded an increase in RT while affinity
remained unchanged. This is an indication that the 3-Me group per
se is not in direct contact with the receptor binding site. However, it
could play an important role in shielding of a sub-pocket in com-
bination with 5-Br; a similar idea was put forward by Schmidtke
et al. [25] in calculations on hydrogen bond shielding. Intrigued by
our findings, we decided to combine the substituents of the long RT
compound 15 (5-Br) and highest affinity compound 21 (6-OMe) in
one structure 38. This combination yielded additional prolongation
of the RT, while the affinity was not affected. Our next step was to
make a hybrid of compounds 37 and 38 to incorporate 3-Me; 5-Br;
6-OMe substituents on the indenyl group in one compound 39 but
it had no significant effect on the affinity. Next to this, we also
extended the methyl group into an ethyl group (compound 40).
Unfortunately, these changes yielded 40-fold decreases in affinity
(p < 0.0001, Student's t-test). However, the RT of the hybrid mol-
ecules were not affected when compared to 5-Br (compound 15).
We then decided to separate compound 15 into diastereomers
(Table 6) by preparative supercritical fluid chromatography (SFC).
Similar to our previous findings [12] the first compound to elute
also showed a higher affinity. However, the difference in affinity
between the diastereomers was only 10-fold in the case of com-
pounds 15a and 15b (Fig. S2A in supporting materials). In addition,
compound 37 was resynthesized using a different method to yield
all four diastereomers, which were separated (37a-d). R-di-
astereomers 37a and 37b retained high affinity, while S-di-
astereomers 37c and 37d had only sub-micromolar affinity values
(Ki ¼ 1.7, 4.6, 199 and 137 nM, respectively) (Fig. S2B in supporting
materials).
In the RT measurements, a distinct difference was observed for
Table 5
Binding Affinities, KRI and Residence Time of compounds 36e40.
Compound number
R
Ki (nM) SEM (n ¼ 3)
KRI (n ¼ 2)
RT (min) SEM (n ¼ 3)
36
37
38
39
40
3-Me
5-Br, 3-Me
5-Br, 6-OMe
5-Br, 3-Me, 6-OMe
5-Br, 3-Et, 6-OMe
3.4 0.5
2.0 0.2
4.5 1.0
0.7 (0.6/0.7)
1.3 (1.3/1.2)
1.3 (1.3/1.2)
1.5 (1.6/1.3)
1.3 (1.3/1.3)
e
345 48
323 10
238 11
179 10
13
83
4
5

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
129
Table 6
Binding Affinities, KRI and Residence Time of separated diastereomers. (15, 15a, 15b, 37, 37aed).
Compound number
R
Ki (nM) SEM (n ¼ 3)
KRI (n ¼ 2)
RT (min) SEM (n ¼ 3)
15
5-Br
5-Br
5-Br
5-Br, 3-Me
5-Br, 3-Me
5-Br, 3-Me
5-Br, 3-Me
5-Br, 3-Me
2.3 0.6
2.4 1.2
1.3 (1.3/1.3)
1.7 (1.7/1.7)
0.8 (0.7/0.9)
1.3 (1.3/1.2)
1.4 (1.4/1.4)
1.2 (1.2/1.1)
e
213 32
714 153
15a
15b
37
37a
37b
37c
37d
24
9
15
4
2.0 0.2
1.7 0.1
4.6 0.1
199 47
137 15
345 48
588 208
208 35
e
e
e
the different diastereomers in the case of 7-(trifluoromethyl)-
1,2,3,4-tetrahydroisoquinoline compounds on the amide part, quite
the opposite of our previous findings for the flexible benzylamide
derivatives [12]. The diastereomers of compound 15 had very
different dissociation rate constants (p < 0.05, Student's t-test)
while the association rate constants were similar (Table 7) (Fig. S3B
in supporting materials). We succeeded in crystalizing compound
15a for singleecrystal X-ray diffraction analysis. Based on the
crystallographic analysis, the absolute configuration of compound
15a was the R-isomer (see Fig. S1 in supporting information).
Apparently, the rigidification stabilizes specific interactions of the
R-isomer in the binding site of the CCR2, which results in smaller
dissociation rate constants. This is also in accordance with 37a and
37b, however, the additional methyl group on the indane ring
should be positioned in the 3R-conformation (37a) resulting in a
better affinity and longer RT than the 3S-diastereomer (37b)
(Fig. S3C in supporting materials). The stereochemistry of 37aed
was assigned using NOE experiments.
antagonists. On the right-hand side of the molecule the 7-(tri-
fluoromethyl)-1,2,3,4-tetrahydroisoquinoline group is optimal to
increase residence time. On the left-hand side, the indane ring can
accommodate very different substituents, as many compounds
maintain nanomolar affinity. However, lipophilic and electron-
withdrawing substituents (e.g. Cl, Br, I, CF3) on the 5-position of
the indane ring are crucial for long residence time. Moreover a
comparison between e.g., compounds 8 and 15a demonstrates that
affinity and residence time do not necessarily correlate and it
stresses the importance of additional screening for residence time
in the early stages of drug discovery. In addition, small changes of
the structure can have a big impact on residence time, e.g., the
incorporation of a 5-Br (15a) substituent yielded a more than 30-
fold increase in residence time (714 min). From this perspective
compound 15a should be evaluated in CCR2erelated disease ani-
mal models to assess the usefulness of prolonged inhibition of
CCR2. In general, this work provides methodology to retrieve the
kinetic parameters from the vast number of high affinity com-
pounds in the early stages of the drug discovery process, which
could help to provide better drug candidates for the later stages of
drug development.
To confirm that our compounds are antagonists of CCR2 we
performed a G protein-dependent functional assay. We used a [35S]
GTP
measured G protein activation by CCL2 in the absence and presence
of 10 M of a selection of antagonists (Fig. 4). Compounds 15a, 15b,
gS binding assay on U2OS-CCR2 membranes, where we
m
4. Experimental section
17a, 37a and 37b all inhibited CCL2-induced G protein activation,
hence they are antagonists of CCR2.
4.1. Chemistry
3. Conclusion
All solvents and reagents were purchased from commercial
sources and were of analytical grade. Demineralized water is sim-
ply referred to as H2O, because it was used in all cases, unless stated
otherwise (i.e., brine). 1H and 13C NMR spectra were recorded on a
We have evaluated the SAR and SKR of 3-((inden-1-yl)amino)-1-
isopropyl-cyclopentane-1-carboxamide derivatives as CCR2
Table 7
Kinetic data of 15a, b, 17a and 37a, b compounds.
Compound
number
Ki (nM) SEM (n ¼ 3)
kon (nMꢁ1 minꢁ1
)
SEM (n ¼ 3)
koff (minꢁ1
)
SEM (n ¼ 3)
RT (min) SEM (n ¼ 3)
15a
15b
17a
37a
37b
2.4 1.2
0.0080 0.0011
0.0059 0.001
0.0032 0.0004
0.0044 0.0003
0.0026 0.0006
0.0014 0.0003
0.066 0.017
0.0015 0.0005
0.0017 0.0006
0.0048 0.0008
714 153
24
13
9
5
15
4
667 222
588 208
208 35
1.7 0.1
4.6 0.1

130
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
spectrometry (LCeMS) analyses were performed using an Onyx C18
m), and a linear gradient from
monolithic column (50 ꢂ 4.6 mm, 5
m
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 1.2 mL/min. Separations of enan-
tiomers were accomplished using chiral SFC. The column was
Phenomenex Lux-4 (250 ꢂ 10 mm, 5
mm). The mobile phase con-
dition of 10% MeOH with 20 mM NH3 and 90% CO2 was applied at a
flow rate of 10 mL/min. Optical rotations were measured in ethanol
at 20 ꢀC on a PerkineElmer polarimeter (Wavelength ¼ 589 nm).
The single crystal X-ray diffraction studies were carried out on a
Bruker Kappa APEX-II CCD diffractometer equipped with Mo Ka
radiation (
l
¼ 0.71073 Å). Thin-layer chromatography (TLC) was
routinely consulted to monitor the progress of reactions, using
aluminum-coated Merck silica gel F254 plates. Purification by col-
umn chromatography was achieved by use of Grace Davison Davisil
silica column material (LC60A, 30e200 mm). 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.
Fig. 4. G protein-activation by 10 nM CCL2 measured in a [35S]GTP
U2OS-CCR2 membranes in the absence and presence of 10
compounds.
gS binding assay on
mM of a selection of
Synthesis
of
(1S,3R)-3-(tert-butoxycarbonylamino)-1-
isopropylcyclopentanecarboxylic acid (2) was achieved following
the synthetic approach reported earlier by our group [12].
4.1.1. General procedure for the synthesis of compounds 3 and 4
Compound 2 (1 equiv) was dissolved in 25 mL DCM. To this
mixture the corresponding amine (1 equiv) was added and subse-
quently DiPEA (3 equiv), PyBrOP (1 equiv) and DMAP (0.8 equiv).
The reaction mixture was stirred for 24 h at room temperature. The
product was partitioned between DCM and 1 M 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 (0e100% ethyl acetate in DCM).
Bruker AV 400 liquid spectrometer (1H NMR, 400 MHz; 13C NMR,
100 MHz) or on a Bruker 500 MHz Avance III NMR spectrometer
(compounds 15a, b and 37a e d) at ambient temperature. Chemical
shifts are reported in parts per million (ppm), are designated by d,
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
mm C18 110A column (50 ꢂ 4.6 mm, 3
mm), measuring UV
absorbance at 254 nm. The sample preparation and HPLC method
for compounds 8; 14e16 and 36e40 were as follows: 0.3e0.8 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
1.3 mL/min. The elution method was set up as follows: 1e4 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. Enantiomeric excess was
accomplished using chiral SFC. For 47 and 48 the column was
4.1.1.1. tert-Butyl ((1R,3S)-3-isopropyl-3-(4-(4-(trifluoromethyl)pyr-
idin-2-yl)piperazine-1-carbonyl)cyclopentyl)carbamate (3) [20].
Yield ¼ 90%. 1H NMR (400 MHz, CDCl3)
: 8.22 (d, J ¼ 4.8 Hz, 1H),
d
6.77e6.72 (m, 2H), 4.94 (br s, 1H), 3.82 (br s,1H), 3.70e3.61 (m, 4H),
3.59e3.45 (m, 4H), 2.11e1.90 (m, 3H), 1.80e1.61 (m, 4H), 1.31 (s,
9H), 0.81e0.72 (m, 6H) ppm.
4.1.1.2. tert-Butyl
((1R,3S)-3-isopropyl-3-(7-(trifluoromethyl)-
1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclopentyl)carbamate (4)
[26]. Yield ¼ 82%. 1H NMR (400 MHz, CDCl3)
: 7.45 (d, J ¼ 7.6 Hz,
d
1H), 7.40 (s, 1H), 7.28 (d, J ¼ 7.6, 1H), 4.97e4.61 (m, 3H), 4.00e3.73
(m, 3H), 2.94 (br s, 2H), 2.30e2.05 (m, 3H), 1.88e1.67 (m, 3H), 1.59
(br s, 1H), 1.42 (s, 9H), 0.91e0.84 (m, 6H) ppm.
Chiralpak AD-H (250 ꢂ 4.6 mm), 5
mm. The mobile phase condition
of 10% MeOH with 20 mM NH3 and 90% CO2 was applied at a flow
rate of 3.0 mL/min at 254 nm. For 37a and 37b the column was
Phenomenex Lux-4 (250 ꢂ 4.6 mm), 5
m
m. The mobile phase
4.1.2. ((1S,3R)-3-Amino-1-isopropylcyclopentyl) (4-(4-
condition of 20% i-propanol (IPA) with 1.0% DEA and 80% CO2 was
applied at a flow rate of 3.0 mL/min at 254 nm. For 37c and 37d the
(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methanone (5) [20]
Trifluoroacetic acid (4 mL) was added to a solution of compound
3 (1.20 g, 2.48 mmol) in 10 mL of DCM. The reaction mixture was
stirred for 2 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 give the
product as yellow crystals (0.86 g, 90%). 1H NMR (400 MHz, CDCl3)
column was Regis RR-Whelko (250 ꢂ 4.6 mm), 5
mm. The mobile
phase condition of 25% IPA with 1.0% diethylamine (DEA) and 75%
CO2 was applied at a flow rate of 3.0 mL/min at 254 nm. High-
eresolution 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, 10e80% B in 26 min; and flow rate,
0.4 mL/min. Preparative HPLC (for compounds 7; 9e13 and 18e35)
was performed on a Waters Auto Purification HPLCeultraviolet
(UV) system with a diode array detector using a Luna C18 Phe-
d
: 8.20 (d, J ¼ 4.8 Hz, 1H), 6.77e6.72 (m, 2H), 3.72e3.38 (m, 8H),
3.20e3.10 (m, 1H), 2.45e2.30 (m, 1H), 2.07e1.90 (m, 2H), 1.80e1.35
(m, 4H), 0.83e0.70 (m, 6H) ppm.
4.1.3. ((1S,3R)-3-Amino-1-isopropylcyclopentyl) (7-
(trifluoromethyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (6)
[26]
Trifluoroacetic acid (10 mL) was added to a solution of com-
pound 4 (3.27 g, 7.2 mmol) in 10 mL of DCM. The reaction mixture
was stirred for 1 h at room temperature. The reaction mixture was
nomenex column (75 ꢂ 30 mm, 5
mm), 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 chromatographyemass

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
131
neutralized with 1 M NaOH and extracted with DCM. The organic
layer was dried with MgSO4, filtered, and evaporated to give the
product as yellow crystals (2.50 g, 98%). 1H NMR (400 MHz, CDCl3)
and 17b ¼ 8.0%, 83% purity, UPLC-MS: 539þ; tR: 1.48 min 17a: 1H
NMR (400 MHz, CDCl3)
d: 10.09 (s, 1H), 9.24 (s, 1H), 7.85 (d,
J ¼ 8.0 Hz, 1H), 7.57 (s, 1H), 7.50 (d, J ¼ 7.7 Hz, 1H), 7.46e7.38 (m,
1H), 7.35 (s, 1H), 7.29e7.20 (m, 1H), 4.90e4.62 (m, 3H), 3.84 (s, 1H),
3.68 (t, J ¼ 8.7 Hz, 1H), 3.48 (q, J ¼ 7.8, 7.0 Hz, 1H), 2.93 (ddt, J ¼ 19.3,
11.4, 5.0 Hz, 3H), 2.68 (d, J ¼ 14.1 Hz, 1H), 2.64e2.37 (m, 3H),
2.18e1.97 (m, 3H), 1.92 (dd, J ¼ 14.2, 7.4 Hz, 1H), 1.80 (s, 1H), 1.68 (d,
J ¼ 12.1 Hz, 1H), 0.84 (dd, J ¼ 20.0, 6.6 Hz, 6H) ppm.
d
: 7.40 (d, J ¼ 7.6 Hz, 1H), 7.36 (s, 1H), 7.24 (d, J ¼ 7.6, 1H), 4.76 (s,
2H), 3.81 (br s, 2H), 3.32e3.22 (m, 1H), 2.91 (br s, 2H), 2.53e2.45
(m, 1H), 2.18e1.73 (m, 4H), 1.69e1.60 (m, 1H), 1.40e1.35 (m, 1H),
0.91e0.84 (m, 6H) ppm.
4.1.4. General procedure for the synthesis of compounds 7, 9e13
and 18e23
4.1.7. General procedure for the synthesis of compounds 23e35
To a series of 1.5 mL glass tubes was added 15 in toluene
(0.1 mmol) followed by solutions of different aryl boronic acids
(0.5 M, 0.2 mmol) in NMP and these mixtures were subsequently
treated with Na2CO3 solution (1 M, 0.2 mmol) followed by
Pd(PPh3)4 in toluene (0.05 eq, 0.005 mmol). The reaction mixtures
(0.15 M) were capped and heated at 80 ꢀC on a reaction block
overnight. The reaction mixtures were purified directly using an
automated masseguided reverse phaseeHPLC, and product con-
taining fractions were concentrated to give final products >90%
purity as judged by LC-MS (average of 220 nm and 254 nm traces).
To a series of 1.5 mL glass tubes was added amine 5 or 6 in NMP
(0.95 M, 0.095 mmol), followed by solutions of different indanones
(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 LCeMS (average of
220 and 254 nm traces).
4.1.5. General procedure for the synthesis of compounds 8, 14e16
and 36e40
4.1.8. 5-bromo-3-methyl-2,3-dihydro-1H-inden-1-one (42)
[15] In a 50 mL round-bottom flask NaCl (3.7 g, 63.0 mmol) and
AlCl3 (15.0 g, 115 mmol) were loaded and heated at 130 ꢀC until
completely melted. Next, 40-bromo-4-chloro-butyrophenone (41)
(3.0 g, 11.5 mmol) was added and the reaction mixture was heated
at 180 ꢀC for 30 min. After cooling to room temperature, the reac-
tion mixture was slowly poured on an ice/1 N HCl mixture e an
exothermic reaction was observed. The reaction mixture was
extracted with DCM, dried with MgSO4 and the organic solvent was
evaporated to yield 2.35 g (yield 91%, with 90% purity) of light
brown crystals which were used in the next step without purifi-
In a 5 mL glass tube, amine 6 (1 equiv) dissolved in 1 mL of dry
THF, and the corresponding indanone (1.2 equiv) dissolved in 1 mL
of dry THF were loaded. Mixture was flushed with nitrogen gas and
the tube was capped. Through the septa Ti(O-i-Pr)4 (3 equiv) was
added and the reaction mixture was stirred for 48 h at room tem-
perature. Then the tube was decapped and NaBH4 (5 equiv) and
0.5 mL of absolute EtOH were added, and stirred for 16 h. The re-
action mixture was quenched with H2O and resulting inorganic
precipitate was filtered off and washed with DCM. The filtrate was
extracted with DCM/H2O. The organic layer was dried with MgSO4
and evaporated. The product was purified by column chromatog-
raphy (40% ethyl acetate in DCM).
cation. 1H NMR (400 MHz, CDCl3)
d
: 7.70 (s, 1H), 7.61 (d, J ¼ 8.4 Hz,
1H), 7.54 (d, J ¼ 8.4 Hz, 1H), 3.50e3.40 (m, 1H), 2.96 (ABX,
J ¼ 19.2 Hza, J ¼ 7.6 Hzb, 1H), 2.30 (ABX, J ¼ 18.8 Hza, J ¼ 3.6 Hzb, 1H),
1.43 (d, J ¼ 7.2 Hz, 3H) ppm.
4.1.5.1. ((1S,3R)-3-((2,3-dihydro-1H-inden-1-yl)amino)-1-
isopropylcyclopentyl)
(7-(trifluoromethyl)-3,4-dihydroisoquinolin-
4.1.9. ((6-Bromo-5-methoxy-1H-inden-3-yl)oxy) (tert-butyl)
dimethylsilane (44)
2(1H)-yl)methanone (8). Yield ¼ 34%. 1H NMR (400 MHz, CDCl3)
d:
7.57e7.37 (m, 2H), 7.37e7.10 (m, 5H), 4.91e4.75 (m, 2H), 4.37e4.20
(m, 1H), 3.85 (s, 2H), 3.36e3.20 (m, 1H), 3.09e2.88 (m, 3H),
2.88e2.74 (m, 1H), 2.58 (br s, 1H), 2.49e2.33 (m, 1H), 2.25e1.87 (m,
4H), 1.87e1.72 (m, 2H), 1.72e1.56 (m, 1H), 1.48e1.35 (m, 1H),
1.01e0.79 (m, 6H) ppm. LCeMS: 471þ; tR: 9.79 min.
In a 25 mL roundebottom flask 5-bromo-6-methoxy-2,3-
dihydro-1H-inden-1-one (43) (0.96 g, 4.0 mmol) was dissolved in
8 mL of toluene and cooled to 0 ꢀC. TBDMS-Cl solution in toluene
(1.95 mL, 6.5 mmol) was added, followed by dropwise addition of
DBU (1.12 mL, 7.5 mmol). The reaction mixture was stirred at 0 ꢀC
for 10 min and continued to be stirred at room temperature for 7
days. The reaction mixture was extracted with Et2O/H2O, dried with
MgSO4, and evaporated. Yield ¼ 1.40 g (98%), which was used in the
4.1.6. ((1S,3R)-1-isopropyl-3-((5-(trifluoromethyl)-2,3-dihydro-
1H-inden-1-yl)amino)cyclopentyl) (7-(trifluoromethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)methanone (17a)
next step without purification. 1H NMR (400 MHz, CDCl3)
d: 7.55 (s,
In a 50 mL roundebottom flask, to a solution of 1 equivalent of
amine 6 in anhydrous methanol was added 1 equivalent of 5-(tri-
fluoromethyl)-2,3-dihydro-1H-inden-1-one which was subse-
quently treated with 2 equivalents of acetic acid, followed by 4
equivalents of 5-ethyl-2-methyl-pyridine borane (PEMB). The re-
action mixture was heated at 65 ꢀC for 24 h. Reaction mixture was
monitored by reverse phase UPLC (tR: 0.53) and was carefully
quenched with concentrated HCl, then water was added to the
reaction mixture and extracted with dichloromethane. The com-
bined organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. Reraction mixture was purified by flash
chromatography (0e20 % CH2Cl2/MeOH) to afford product 17
(mixture of diastereomers). Further purification by reverse-phase
HPLC using a gradient from 1 to 99% mobile phase B (mobile
phase A ¼ 0.1% HCl in water, mobile phase B ¼ 0.1% HCl in CH3CN)
resulted in the separation of the two diastereomers 17 a and 17 b as
HCl salt. Yield: 17a ¼ 19.5%, 96% purity,UPLC-MS: 539þ; tR: 1.45 min
1H), 6.94 (s, 1H), 5.47 (t, J ¼ 2.4 Hz, 1H), 3.96 (s, 3H), 3.22 (d,
J ¼ 2.4 Hz, 2H), 1.05 (s, 9H), 0.29 (s, 6H) ppm.
4.1.10. 5-bromo-6-methoxy-3-methyl-2,3-dihydro-1H-inden-1-one
(45)
In a 20 mL reaction tube a 2 M solution of LDA (0.6 mL,1.2 mmol)
in THF/heptane/ethylbenzene was mixed with 5 mL of dry THF. The
reaction mixture was cooled down to ꢁ78 ꢀC and compound 44
(0.35 g, 1 mmol) (dissolved in 1 mL of dry THF) was added. The
reaction mixture was warmed to ꢁ35 ꢀC during 1 h and then cooled
back to ꢁ78 ꢀC. Methyl iodide (0.08 mL, 1.3 mmol) was added and
the reaction mixture was warmed to room temperature over 2 h
and left stirring overnight. 37% HCl in water (0.27 mL, 3.2 mmol)
was added and this mixture was stirred for 3 h. The reaction
mixture was extracted with DCM/H2O, dried with MgSO4, and
evaporated. The product was purified by column chromatography
with DCM as eluent. Yield ¼ 0.21 g (82%). 1H NMR (400 MHz, CDCl3)

132
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
d
: 7.61 (s,1H), 7.03 (s, 1H), 3.83 (s, 3H), 3.35e3.25 (m, 1H), 2.85 (ABX,
6.6 Hz, 1H), 1.49 (ddd, J ¼ 12.7, 8.9, 7.7 Hz, 1H), 1.34 (d, J ¼ 6.8 Hz,
3H),1.26 (d, J ¼ 7.0 Hz, 2H) ppm. HPLC (Chiralpak AD-H column,10%
MeOH with 20 nM NH3, 90% CO2, 3.0 mL/min, 254 nm, ee ¼ 96.2%.
UPLC-MS: 209þ, 211þ; tR: 1.92, 1.95 min.
J ¼ 18.8 Hza, J ¼ 7.6 Hzb, 1H), 2.18 (ABX, J ¼ 19.2 Hza, J ¼ 3.2 Hzb, 1H),
1.29 (d, J ¼ 7.2 Hz, 3H) ppm.
4.1.11. 5-bromo-6-methoxy-3-ethyl-2,3-dihydro-1H-inden-1-one
(46)
4.1.14. N-((1R,3S)-3-isopropyl-3-(7-(trifluoromethyl)-1,2,3,4-
tetrahydroisoquinoline-2-carbonyl)cyclopentyl)-2-
nitrobenzenesulfonamide (49)
In a 20 mL reaction tube a 2 M solution of LDA (0.6 mL,1.2 mmol)
was dissolved in 5 mL of dry THF. The reaction mixture was cooled
down to ꢁ78 ꢀC and compound 44 (0.35 g, 1 mmol) (dissolved in
1 mL of dry THF) was added. The reaction mixture was warmed
to ꢁ35 ꢀC during 1 h and then cooled back to ꢁ78 ꢀC. Ethyl iodide
(0.10 mL, 1.3 mmol) was added and the reaction mixture was
warmed to room temperature during 2 h and left stirring overnight.
37% HCl in water (0.27 mL, 3.2 mmol) was added and stirred for 3 h.
The reaction mixture was extracted with DCM/H2O, dried with
MgSO4, and evaporated. The product was purified by column
chromatography with DCM as eluent. Yield ¼ 0.24 g (89%). 1H NMR
In a 50 mL round bottom flask, to a solution of amine 6 (1.00 g,
2.82 mmol) in anhydrous CH2Cl2 (10 mL) was added 2-
nitrobenzenesulfonyl chloride (0.75 mg, 3.38 mmol) followed by
DIPEA (1.70 mL, 9.73 mmol) and stirred at room temperature
(25 ꢀC) for 1 h. The reaction was monitored by reverse phase UPLC
(tR: 0.77). The reaction mixture was concentrated in vacuo to ob-
tained the crude product (yellow oil). The product was purified by
flash chromatography (40e80 % ethyl acetate/hexanes) to afford
the light yellow foamy solid 49. Yield ¼ 1.50 g (98%).1H NMR
(400 MHz, CDCl3)
d
: 7.67 (s,1H), 7.11 (s, 1H), 3.88 (s, 3H), 3.35e3.25
(400 MHz, CDCl3) d: 8.16e8.09 (m, 1H), 7.82e7.75 (m, 1H),
(m, 1H), 2.81 (ABX, J ¼ 19.2 Hza, J ¼ 7.2 Hzb, 1H), 2.32 (ABX,
J ¼ 19.2 Hza, J ¼ 3.2 Hzb, 1H), 1.95e1.85 (m, 1H), 1.55e1.45 (m, 1H),
0.93 (t, J ¼ 7.2 Hz, 3H) ppm.
7.75e7.64 (m, 2H), 7.48e7.40 (m,1H), 7.36 (s, 1H), 7.27 (d, J ¼ 4.9 Hz,
1H), 6.05 (s, 1H), 4.81 (s, 1H), 4.71 (d, J ¼ 16.4 Hz, 1H), 3.94 (d,
J ¼ 16.7 Hz,1H), 3.81 (dq, J ¼ 8.7, 4.3 Hz,1H), 3.76e3.64 (m,1H), 2.90
(tt, J ¼ 16.6, 6.4 Hz, 2H), 2.40 (d, J ¼ 12.9 Hz, 1H), 2.14 (pent.,
J ¼ 6.7 Hz, 1H), 1.82 (d, J ¼ 16.1 Hz, 3H), 1.67 (dd, J ¼ 8.6, 4.5 Hz, 1H),
1.58e1.40 (m, 1H), 0.87 (d, J ¼ 6.7 Hz, 3H), 0.76 (d, J ¼ 6.7 Hz, 3H)
ppm. UPLC-MS: 540þ; tR: 2.85 min.
4.1.12. (1S)-5-bromo-3-methyl-2,3-dihydro-1H-inden-1-ol (47)
In a 50 mL round bottom flask under N2 atmosphere, (R)-(þ)-2-
methyl-CBS-oxazaborolidine catalyst (73.9 mg, 0.27 mmol, 1 M in
toluene) in anhydrous toluene (5 mL) was added to N,N-dieth-
ylaniline borane (1.58 mL, 8.88 mmol) at room temperature (25 ꢀC).
To this solution 42 (0.50 g, 2.22 mmol) in toluene (20 mL) was
added dropwise over 5 h, and the resulting mixture was stirred
overnight at room temperature (25 ꢀC). Reaction was monitored by
reverse phase UPLC (tR: 0.59). The reaction mixture was carefully
quenched with methanol (5 mL) and 1 M HCl (1 mL) and extracted
with ethyl acetate (4 ꢂ 50 mL). The combined organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The white residue was purified by flash chromatography (0e30 %
ethyl acetate/hexanes) to afford the white crystalline solid 47 as a
racemic mixture of diastereomers in a ratio 3:5. Yield ¼ 0.49 g,
4.1.15. N-((1R)-5-bromo-3-methyl-2,3-dihydro-1H-inden-1-yl)-N-
((1R,3S)-3-isopropyl-3-(7-(trifluoromethyl)-1,2,3,4-
tetrahydroisoquinoline-2-carbonyl)cyclopentyl)-2-
nitrobenzenesulfonamide (50)
In a 100 mL round-bottom flask, to a cooled solution (ꢁ78 ꢀC,
acetone/dry ice bath) of PPh3 (1.23 g, 4.69 mmol) in anhydrous THF
(20 mL) was added DEAD (0.82 g, 4.69 mmol) and stirred for
30 min, followed by addition of 47 (0.40 g, 1.76 mmol). The pink
colored reaction mixture was allowed to stir for 30 min maintaining
the temperature at ꢁ78 ꢀC. To this solution, 49 (0.63 g, 1.17 mmol)
was added. The reaction mixture was stirred for 6 h at ꢁ78 ꢀC and
allowed to warm up to room temperature overnight. The reaction
was monitored by reverse phase UPLC (tR: 0.93). The reaction
mixture was concentrated in vacuo to obtained a pink-colored
residue. The crude residue was purified by flash chromatography
(0e10 % CH2Cl2/MeOH) to afford the light pink foamy solid 50
(purity 80%). Yield ¼ 1.00 g (<100%). UPLC-MS: 540þ; tR: 3.53 min.
Without further purification, 50 was taken forward in the following
deprotection step.
2.16 mmol (97%). 1H NMR (400 MHz, CDCl3)
d: 7.41e7.32 (m, 3H),
7.29e7.22 (m, 3H), 5.20 (dd, J ¼ 6.4, 3.1 Hz, 1H), 5.16e5.08 (m, 1H),
3.43 (h, J ¼ 7.0 Hz, 1H), 3.05 (h, J ¼ 7.1 Hz, 1H), 2.76 (dt, J ¼ 12.7,
7.1 Hz, 1H), 2.25 (ddd, J ¼ 13.6, 7.4, 3.1 Hz, 1H), 1.98 (dt, J ¼ 13.4,
6.6 Hz, 1H), 1.49 (ddd, J ¼ 12.7, 8.9, 7.6 Hz, 1H), 1.34 (d, J ¼ 6.8 Hz,
3H), 1.26 (dd, J ¼ 7.1, 2.4 Hz, 2H) ppm. HPLC (Chiralpak AD-H col-
umn, 10% MeOH with 20 nM NH3, 90% CO2, 3.0 mL/min, 254 nm,
ee ¼ 95.8%. UPLC-MS: 209þ, 211þ; tR: 1.91 min.
4.1.13. (1R)-5-bromo-3-methyl-2,3-dihydro-1H-inden-1-ol (48)
In a 50 mL round bottom flask under N2, (S)-(þ)-2-methyl-CBS-
oxazaborolidine catalyst (73.9 mg, 0.27 mmol, 1 M in toluene) in
anhydrous toluene (5 mL) was added to N,N-diethylaniline borane
(1.58 mL, 8.88 mmol) at room temperature (25 ꢀC). To this solution
42 (0.50 g, 2.22 mmol) in toluene (20 mL) was added dropwise over
5 h, and the resulting mixture was stirred overnight at room tem-
perature (25 ꢀC). The reaction was monitored by reverse phase
UPLC (tR: 0.58). The reaction mixture was carefully quenched with
methanol (5 mL) and 1 N HCl (1 mL) and then extracted with ethyl
acetate (4 ꢂ 50 mL). The combined organic layer washed with
brine, dried over Na2SO4, and concentrated in vacuo. The white
residue was purified by flash chromatography (0e30 % ethyl ace-
tate/hexanes) to afford the white crystalline solid 48 as a racemic
mixture in a ratio (3:5). Yield ¼ 0.50 g, 2.20 mmol (99%). 1H NMR
4.1.16. N-((1S)-5-bromo-3-methyl-2,3-dihydro-1H-inden-1-yl)-N-
((1R,3S)-3-isopropyl-3-(7-(trifluoromethyl)-1,2,3,4-
tetrahydroisoquinoline-2-carbonyl)cyclopentyl)-2-
nitrobenzenesulfonamide (51)
In a 100 mL round-bottom flask, to a cooled solution (ꢁ78 ꢀC,
acetone/dry ice bath) of PPh3 (1.23 g, 4.69 mmol) in anhydrous THF
(20 mL) was added DEAD (0.82 g, 4.69 mmol) and stirred for
30 min, followed by addition of 48 (0.40 g, 1.76 mmol). The pink
colored reaction mixture was allowed to stir for 30 min maintaining
the temperature at ꢁ78 ꢀC. To this solution, 49 (0.63 g, 1.17 mmol)
was added. The reaction mixture was stirred for 6 h at ꢁ78 ꢀC and
allowed to warm up to room temperature overnight. The reaction
was monitored by reverse phase UPLC (tR: 0.93). The reaction
mixture was concentrated in vacuo to obtained a pink color residue.
The crude residue was purified by flash chromatography (0e10 %
CH2Cl2/MeOH) to afford the light pink foamy solid 55 (purity 80%).
Yield ¼ 1.00 g (<100%). UPLC-MS: 540þ; tR: 3.53 min. Without
further purification, 51 was taken forward in the following depro-
tection step.
(400 MHz, CDCl3)
d
: 7.37 (dddt, J ¼ 8.4, 5.6, 2.8, 1.2 Hz, 4H),
7.28e7.22 (m, 2H), 5.20 (dd, J ¼ 6.4, 3.1 Hz, 1H), 5.12 (t, J ¼ 7.3 Hz,
1H), 3.43 (h, J ¼ 7.0 Hz, 1H), 3.12e2.97 (m, 1H), 2.76 (dt, J ¼ 12.7,
7.1 Hz, 1H), 2.25 (ddd, J ¼ 13.6, 7.4, 3.1 Hz, 1H), 1.98 (dt, J ¼ 13.4,

M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
133
4.1.17. (1S,3R)-N-(3,5-bis(trifluoromethyl)benzyl)-3-(((1S,3R)-5-
bromo-3-methyl-2,3-dihydro-1H-inden-1-yl)amino)-1-
isopropylcyclopentanecarboxamide (37a) and (1S,3R)-N-(3,5-
bis(trifluoromethyl)benzyl)-3-(((1S,3S)-5-bromo-3-methyl-2,3-
dihydro-1H-inden-1-yl)amino)-1-
4.2.2. Cell culture and membrane preparation
U2OS cells stably expressing the human CCR2 receptor (Invi-
trogen, Carlsbad, CA) were cultured in McCoys5a medium supple-
mented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM non-
essential amino acids (NEAA), 25 mM HEPES, 1 mM sodium pyru-
isopropylcyclopentanecarboxamide (37b)
vate, 100 IU/mL penicillin, 100 g/mL streptomycin, 100
m
mg/mL
In a 20 mL scintillation vial, to a solution of 50 (1.00 g,
1.34 mmol) in DMF (10 mL) was added K2CO3 (0.55 g, 4.01 mmol)
and benzenethiol (274.4 mL, 2.67 mmol), and the reaction mixture
G418, 50 mg/mL hygromycin and 125 mg/mL zeocin in a humidified
atmosphere at 37 ꢀC and 5% CO2. Cell culture and membrane
preparation were performed as described previously [18].
was stirred at room temperature for 30 min. The reaction was
monitored by reverse phase UPLC (tR: 0.73 and 0.74). To the reac-
tion mixture was added water (20 mL) followed by extraction with
CH2Cl2 (4 ꢂ 100 mL). The combined organic layer was washed with
water, brine, dried over Na2SO4, and concentrated in vacuo. The
brown residue was purified by flash chromatography (0e10%
CH2Cl2/MeOH) to afford 37a (0.10 g) and 37b (0.10 g) with 80%
diastereomeric excess purity by SFC analysis. Further purification
was carried out by reverseephase preparatory HPLC-UV with a
gradient from 1 to 99% mobile phase B (mobile phase A ¼ 0.1% HCl
in water, mobile phase B ¼ 0.1% HCl in CH3CN) resulting in pure 37a
HCl salt and 37b HCl salt as a white solid. Yield: 37a ¼ 14.7 mg, 1.8%,
UPLC-MS: 563þ, 565þ; tR: 2.45 min and 37b ¼ 17.3 mg, 2.1%, UPLC-
MS: 563þ, 565þ; tR: 2.49 min.
4.2.3. 125I-CCL2 displacement assay
Binding assays were performed as described previously [18].
Briefly, 15
mg of U2OSeCCR2 membranes were incubated for
150 min with 0.1 nM 125I-CCL2 in the absence or presence of
competing ligand at 37 ꢀC.
4.2.4. [3H]INCB3344 dual point competition association assay
Kinetic rate index (KRI) values of unlabeled ligands were
determined using the dual-point competition association assay as
described previously [12]. Briefly, 10 mg of U2OSeCCR2 membranes
were incubated for 50 min (t1) or 240 min (t2) 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. KRI values
of unlabeled ligands were calculated using eq. (1), as mentioned
below in the Data Analysis section.
4.1.18. (1S,3R)-N-(3,5-bis(trifluoromethyl)benzyl)-3-(((1R,3R)-5-
bromo-3-methyl-2,3-dihydro-1H-inden-1-yl)amino)-1-
isopropylcyclopentanecarboxamide (37c) and (1S,3R)-N-(3,5-
bis(trifluoromethyl)benzyl)-3-(((1R,3S)-5-bromo-3-methyl-2,3-
dihydro-1H-inden-1-yl)amino)-1-
isopropylcyclopentanecarboxamide (37d)
In a 20 mL scintillation vial, to a solution of 51 (1.00 g,
1.34 mmol) in DMF (10 mL) was added K2CO3 (0.55 g, 4.01 mmol)
4.2.5. [3H]INCB3344 competition association assay
The kinetic parameters of unlabeled ligands were determined
using the competition association assay described earlier by our
group [12]. Briefly, at different time points, 10
mg of U2OSeCCR2
membranes was added to 1.8 nM [3H]-INCB3344 in a total volume
and benzenethiol (274.4 mL, 2.67 mmol), and the reaction mixture
of 100 mL of assay buffer in the absence or presence of competing
was stirred at room temperature for 30 min. The reaction was
monitored by reverse phase UPLC (tR: 0.76). To the reaction mixture
was added water (20 mL) followed by extraction with CH2Cl2
(4 ꢂ 100 mL). The combined organic layer was washed with water,
brine, dried over Na2SO4, and concentrated in vacuo. The brown
residue was purified by flash chromatography (0e10% CH2Cl2/
MeOH) to afford 37c (50 mg) and 37d (90 mg) both with 50% dia-
stereomeric excess purity. Further purification was carried out by
reverseephase preparatory HPLC-UV with a gradient from 1 to 99%
mobile phase B (mobile phase A ¼ 0.1% HCl in water, mobile phase
B ¼ 0.1% HCl in CH3CN) resulting in 37c HCl salt and 37d HCl salt as
a white solid, both again with 70% diastereomeric excess purity.
Therefore further purification was carried out on a Gilson purifi-
cation instrument with a normal phase silica column and a diode
array detector using a Luna Phenomenex column (50 mm ꢂ 21 mm,
ligand at 25 ꢀC. Kinetic parameters of unlabeled ligands were
calculated using eq. (2), as mentioned below in the Data Analysis
section.
4.2.6. [35S]GTP
Ten micrograms of membranes were diluted in 100
buffer containing 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2,
100 mM NaCl, 1 mM EDTA, 0.05% bovine serum albumin, 10
GDP, and 10 g of saponin per assay point. The membranes were
preincubated with 10
M of antagonist for 30 min at 25 ꢀC. Then
gS binding assay
mL of assay
mM
m
m
CCL2 (10 nM) was added, followed by another incubation of 30 min;
finally, the mixture was incubated for 90 min after the addition of
[
35S]GTP
gS (0.3 nM). The incubation was terminated by dilution
with ice-cold 50 mM Tris-HCl buffer supplemented with 5 mM
MgCl2. Separation of bound from free radioligand was performed
by rapid filtration through Whatman GF/B filters using a Brandel
harvester. Filters were subsequently washed three times with ice-
cold buffer. Filter-bound radioactivity was measured by scintilla-
tion spectrometry (LKB Wallac, 1219 Rackbeta) after addition of
3.5 mL of Packard Emulsifier Safe.
5 mm), and a linear gradient from 1 to 10% (CH2Cl2/MeOH) of mobile
phase was applied. Mobile phase A consisted of CH2Cl2 and mobile
phase B consisted of 10% MeOH/CH2Cl2. Yield: 37c ¼ 13.1 mg, 1.7%,
UPLC-MS: 563þ, 565þ; tR: 2.52 min 37d ¼ 12.7 mg, 1.7%, UPLC-MS:
563þ, 565þ; tR: 2.52 min.
4.2. Pharmacology
4.2.7. 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 [29]. KRI values were
calculated by dividing the specific radioligand binding measured at
t1 (Bt1) by its binding at t2 (Bt2) in the presence of unlabeled
competing ligand as follows:
4.2.1. Chemicals and reagents
125I-CCL2 (2200 Ci/mmol) was purchased from PerkineElmer
(Waltham, MA). INCB3344 was synthesized as described previously
[27,28]. [3H]INCB3344 (specific activity 32 Ci/mmol) was custom-
labeled by Vitrax (Placentia, CA) for which a dehydrogenated pre-
cursor of INCB3344 was provided. Tango™ CCR2-bla U2OS cells
stably expressing human CCR2 were obtained from Invitrogen
(Carlsbad, CA).
KRI ¼ Bt1=Bt2
(1)

134
M. Vilums et al. / European Journal of Medicinal Chemistry 93 (2015) 121e134
for the treatment of autoimmune diseases, Expert Opin. Inv Drug 17 (2008)
Data presented are the mean S.E.M. of at least 3 experiments
1267e1279.
performed in duplicate. Statistical analysis was performed with a
two-tailed unpaired Student's t-test.
Association and dissociation rates for unlabeled ligands were
determined by nonlinear regression analysis of the competition
association data as described by Motulsky and Mahan,
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KA ¼ k1½Lꢃ$10ꢁ9 þ k2
KB ¼ k3½Iꢃ$10ꢁ9 þ k4
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
S ¼ ðKA ꢁ KBÞ2 þ 4$k1$k3$L$I$10ꢁ18
KF ¼ 0:5ðKA þ KB þ SÞ
KS ¼ 0:5ðKA þ KB ꢁ SÞ
B
max$k1$L$10ꢁ9
KF ꢁ KS
Q ¼
ꢀ
ꢁ
k4$ðKF ꢁ KSÞ k4 ꢁ KF
k4 ꢁ KS
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methylpyridine borane, Tetrahedron Lett. 49 (2008) 5152e5155.
ðꢁK $XÞ ꢁ
eðꢁK $XÞ
F
S
Y ¼ Q$
þ
e
KF$KS
KF
KS
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(2)
where X is the time (min), Y is the specific binding (disintegrations
per minute (DPM)), k1 is kon (Mꢁ1 minꢁ1) of [3H]-INCB3344 pre-
determined in association experiments, k2 is koff (minꢁ1) 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 (Mꢁ1 minꢁ1) of the unlabeled ligand, and k4
is koff (minꢁ1) of the unlabeled ligand. The association and disso-
ciation rates were used to calculate the “kinetic KD” as follows:
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.
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KD ¼ koff kon
(3)
The RT was calculated according to the formula RT ¼ 1/koff
.
Acknowledgment
This study was financially supported by the Dutch Top Institute
Pharma, project number D1-301. We thank Dr. Julien Louvel and
Jacobus van Veldhoven for their input in analytical data analysis
and helpful comments. We acknowledge Dong Guo and Prof. Dr.
Martine Smit (Vrije Universiteit, Amsterdam, The Netherlands) for
helpful comments and suggestions.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.063.
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