Evaluation of (4-Arylpiperidin-1-yl)cyclopentanecarboxamides As High-Affinity and Long-Residence-Time Antagonists for the CCR2 Receptor

Article abstract of DOI:10.1002/cmdc.201500058

Animal models suggest that the chemokine ligand 2/CC-chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We prev

Full text of DOI:10.1002/cmdc.201500058

DOI: 10.1002/cmdc.201500058
Full Papers
Evaluation of (4-Arylpiperidin-1-yl)-
cyclopentanecarboxamides As High-Affinity and Long-
Residence-Time Antagonists for the CCR2 Receptor
Maris Vilums,[a] Annelien J. M. Zweemer,[a] Arian Dilanchian,[a] Jacobus P. D. van Veldhoven,[a]
Henk de Vries,[a] Johannes Brussee,[a] John Saunders,[b] Dean Stamos,[b] Laura H. Heitman,[a]
and Adriaan P. IJzerman*[a]
Animal models suggest that the chemokine ligand 2/CC-che-
mokine receptor 2 (CCL2/CCR2) axis plays an important role in
the development of inflammatory diseases. However, CCR2 an-
tagonists have failed in clinical trials because of a lack of effica-
cy. We previously described a new approach for the design of
CCR2 antagonists by the use of structure–kinetics relationships
(SKRs). Herein we report new findings on the structure–affinity
relationships (SARs) and SKRs of the reference compound MK-
0483, its diastereomers, and its structural analogues as CCR2
antagonists. The SARs of the 4-arylpiperidine group suggest
that lipophilic hydrogen-bond-accepting substituents at the 3-
position are favorable. However, the SKRs suggest that a lipo-
philic group with a certain size is desired [e.g., 3-Br: Ki =
2.8 nm, residence time (tres)=243 min; 3-iPr: Ki =3.6 nm, tres
=
266 min]. Alternatively, additional substituents and further op-
timization of the molecule, while keeping a carboxylic acid at
the 3-position, can also prolong tres; this was most prominently
observed in MK-0483 (Ki =1.2 nm, tres =724 min) and a close
analogue (Ki =7.8 nm) with a short residence time.
Introduction
Chemotactic cytokines (chemokines) play a vital role in the ac-
tivation and regulation of leukocyte trafficking[1] and are in-
volved in immunomodulation and host-defense mechanisms.[2]
Their chemoattractant activity is mediated through activation
of cell-surface seven-transmembrane spanning G protein-cou-
pled receptors (GPCRs). It seems that most of the chemokines
are promiscuous in their actions, as they can bind to numerous
receptors.[3] However, monocyte chemoattractant protein-1/
chemokine ligand 2 (MCP-1/CCL2) only binds and activates the
CC-chemokine receptor 2 (CCR2), and the axis of CCL2/CCR2
has been suggested to be involved in various autoimmune
and inflammation-associated diseases (e.g., multiple sclerosis,
atherosclerosis, asthma, diabetes, and neuropathic pain).[4]
As a consequence, there has been increasing interest in ad-
vancing CCR2 receptor antagonists into clinical studies. How-
ever, thus far, high-affinity CCR2 antagonists have failed to
show efficacy in phase 2 clinical trials. Recently, several re-
views[5] have suggested to incorporate an additional parameter
known as the drug–target residence time (tres) in the early
drug-discovery process; tres is thought to be correlated to drug
efficacy and could serve as an early criterion to diminish the at-
trition rate in later stages of drug development. We previously
described how the implementation of this additional parame-
ter in the hit-to-lead optimization process helped us to distin-
guish between different structures for optimization.[6] Instead
of proceeding with the highest affinity hit (i.e., compound 1,
Figure 1) having a very short tres, a close analogue of MK-0812
(Ki =0.55 nm, tres =92 min)[7] that failed to show efficacy in clini-
cal trials, we continued with a structure having moderate affin-
ity only but longer tres on the CCR2 receptor.
In the optimization process, we improved both parameters
simultaneously to yield a high-affinity and longer tres CCR2 an-
tagonist (i.e., compound 2). However, the Merck research
group also reported on the discovery of MK-0483 (3c) a potent
and orally bioavailable CCR2 and CCR5 dual antagonist having
a very slow dissociation half-life from the CCR2 receptor (t1/2
>
9 h).[8] Struthers and Pasternak[9] described MK-0483 (3c) as the
backup compound of the clinical candidate MK-0812; however,
after the failure of MK-0812 in clinical trials there was no infor-
mation on any further advancements of MK-0483 (3c), despite
its slow dissociation kinetics.
[a] Dr. M. Vilums, Dr. A. J. M. Zweemer, A. Dilanchian, J. P. D. van Veldhoven,
H. de Vries, Dr. J. Brussee, Dr. L. H. Heitman, Prof. A. P. IJzerman
Division of Medicinal Chemistry
Leiden Academic Centre for Drug Research
Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands)
E-mail: ijzerman@lacdr.leidenuniv.nl
In this study, we used knowledge obtained from our previ-
ous findings[6] to evaluate the binding kinetics of MK-0483
(3c), its diastereomers (e.g., compounds 3a, 3b, and 3d), and
structural analogues that we synthesized (e.g., compounds 4–
27) and to determine the structure–kinetics relationships
(SKRs) on the CCR2 receptor for this class of compounds. In
[b] Dr. J. Saunders, Dr. D. Stamos
Vertex Pharmaceuticals Inc.
11010 Torreyana Road, San Diego, CA 92121 (USA)
Supporting Information (analytical data of compounds 5–14, 16, 21–25,
27, and 36–46) for this article is available on the WWW under http://
dx.doi.org/10.1002/cmdc.201500058.
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substituents on the aryl group
by using this synthetic route re-
sulted in dehalogenation prod-
ucts during the hydrogenation
step. We argued that the hydro-
genation could be more selec-
tive if less bulky structures were
used. Therefore, we decided to
reverse the synthetic route and
to modify the piperidine side of
the molecules prior to amide for-
mation at the 3-position of the
cyclopentane ring (Scheme 2).
The synthesis of (1S,3R)-
methyl-3-[(tert-butoxycarbonyl)a-
mino]-1-isopropylcyclopentane-
carboxylate (47) was achieved
Figure 1. Structures of CCR2 antagonists 1, 2,[6] MK-0812 (Merck’s clinical candidate)[7] and MK-0483 (Merck’s back-
up clinical candidate).[8]
a step-by-step manner, we classi-
fied substituents at the 4-posi-
tion of the piperidine ring to
assess their importance in bind-
ing kinetics. In addition, we eval-
uated different amide substitu-
ents at the 3-position of the cy-
clopentane ring for the same
purpose.
Results and Discussion
Chemistry
Synthesis of MK-0483 (3c) and
its diastereomers (e.g., com-
pounds 3a, 3b, and 3d) was
achieved by following the syn-
thetic approach reported by Pas-
ternak et al.[8] For the synthesis
of MK-0483 analogues 4–18, we
designed a new synthetic route
Scheme 1. Reagents and conditions: a) K2CO3, EtOH/H2O (3:1), reflux, 5 h, 25–72%; b) 1. LDA, dry THF, À788C!
À208C, 2. N-phenyl-bis(trifluoromethanesulfonimide), À788C!RT, 21–42%; c) corresponding arylboronic acid,
LiCl, 2m aq Na2CO3, Pd(PPh3)4, DME, 908C, 3.5 h, 20–99%; d) Pd/C, Pd(OAc)2, H2 (1 atm), MeOH or THF, 9–70%;
e) ester 16, 4m aq LiOH, EtOH, 508C, 1.5 h, 53%.
that allowed us to incorporate
different substituents directly on
the cis-cyclopentane isomer in
the penultimate step of the syn-
thesis (Scheme 1).
We started with a sequenced Hoffman elimination and con-
jugate addition[10] of N,N-ethylmethyl-4-oxopiperidinium
iodide[11] (28) to amines 29 and 30, which were obtained fol-
lowing the synthetic approaches described earlier by our
group.[6] Piperidones 31 and 32 were deprotonated with lithi-
um diisopropylamide (LDA) and subsequently treated with N-
phenylbis(trifluoromethanesulfonimide) to generate triflates 33
and 34. These triflates were used in a Suzuki coupling with dif-
ferent arylboronic acids, and subsequent hydrogenation of
formed olefins 17 and 35–46 resulted in final compounds 4–
14, 16, and 18 with the desired groups at the 4-position of the
piperidine ring. Additional saponification of compound 16
yielded benzoic acid 15. Any attempts to introduce halogen
by following the synthetic approach reported by Kothandara-
man et al.[12] Trifluoroacetic acid (TFA)-mediated N-tert-
butyloxycarbonyl (Boc) deprotection yielded amine 48, which
was used in a coupling reaction with piperidone salt 28. The
coupling was performed in several parallel smaller batches
(4 mmol scale) because in large-scale (40 mmol) reactions poor
yields were obtained. Subsequently, triflate 50 was generated
from piperidone 49 under the same conditions used to pre-
pare compounds 33 and 34. Triflate 50 was coupled with the
corresponding boronic acids, and the obtained olefins were
hydrogenated by using PtO2. In this case, only trace amounts
of the dehalogenated products were observed. Next, saponifi-
cation of the esters and peptide coupling under bromotripyr-
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by our research group.[14] Next
to all four MK-0483 diastereo-
mers, compounds with affinities
lower than or equal to 100 nm
were subsequently screened in
a [3H]INCB3344 dual-point com-
petition association assay on
U2OS-CCR2 membrane prepara-
tions to determine their kinetic
rate index (KRI),[15] which served
as an indicator for the magni-
tude of tres. To this end, we co-in-
cubated [3H]INCB3344 with unla-
beled antagonists at a concentra-
tion equal to their Ki value that
was determined in the 125I-CCL2
displacement assay. The KRI was
Scheme 2. Reagents and conditions: a) TFA, CH2Cl2, 2 h, RT, 98%; b) K2CO3, EtOH/H2O (3:1), reflux, 3 h, 56%;
c) 1. LDA, dry THF, À1008C!À408C, 2. N-phenyl-bis(trifluoromethanesulfonimide), À808C!RT, 67%; d) corre-
sponding arylboronic acid, LiCl, 2m aq Na2CO3, Pd(PPh3)4, DME, 908C, 3.5 h; e) PtO2, H2 (2 atm), THF, RT, 10–
15 min; f) 4m aq LiOH, H2O/EtOH (1:10), reflux, 3 h; g) 7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline hydrochlo-
ride, PyBrOP, DIPEA, DMAP, CH2Cl2, RT, 48 h, 0.8–9% (four steps).
rolidinophosphonium hexafluorophosphate (PyBroP) condi-
tions yielded final compounds 20, 24, and 25. However, this
approach gave poor overall yields and was abandoned for fur-
ther synthesis. Recently, Allwood et al.[13] described a new,
metal-free reductive coupling of saturated heterocyclic sulfo-
nylhydrazones with boronic acids as an alternative to the tedi-
ous step-by-step route depicted in Scheme 1. Analogous to
the method described by Allwood et al., tosylhydrazone 52
was obtained in quantitative yield from piperidone intermedi-
ate 32 and sulfonylhydrazide 51 (Scheme 3).[13] Subsequently,
reductive coupling of the corresponding arylboronic acids and
tosylhydrazone 52 resulted in the desired products, which
were purified by preparative HPLC to yield final compounds
19, 21–23, and 27 as their TFA salts. Methyl ester 27 was sapo-
nified to give carboxylic acid derivative 26 as a white HCl salt.
calculated by dividing the specific radioligand binding at
50 min (t1), at which the radioligand binding in the absence of
competitor had attained full equilibrium,[14,15] by the binding at
240 min (t2). In this assay, antagonists with a slower dissocia-
tion rate and, therefore, a longer tres than [3H]INCB3344 had
a KRI>1. Compounds with a KRIꢀ1 were finally tested in the
full competition association assay to determine their tres values,
as described previously by our group.[6]
Structure–affinity relationships and structure–kinetics rela-
tionships
The 3-piperidinylcyclopentanecarboxamide scaffold has been
extensively evaluated by the Merck research group for CCR2
receptor binding.[16] All these efforts resulted in the discovery
of MK-0483, which, upon tritia-
tion, served as a radioligand.
This [3H]MK-0483 bound to mon-
ocytes and appeared to have
very slow receptor dissociation
kinetics.[8] We decided to resyn-
thesize MK-0483 and its diaste-
reomers and to evaluate these
four compounds in our various
binding assays on the CCR2 re-
ceptor to determine the structur-
al components responsible for
both the high affinity and the
long tres of MK-0483. Following
the synthetic approach and sep-
aration methodology reported
by Pasternak et al.,[8] we were
able to generate the same four
diastereomers and assigned
them 3a–d according to the se-
Scheme 3. Reagents and conditions: a) MeOH, RT, 4 h, 100%; b) corresponding boronic acid, Cs2CO3, dry 1,4-diox-
ane, 1008C, 18 h, 8–23%; c) ester 27, 4m aq LiOH, H2O/EtOH (2:5), 508C, 2 h, 65%.
Pharmacology
quence of elution from preparative HPLC on a chiral stationary
phase. First, we determined their affinity in a 125I-CCL2 compe-
tition displacement assay in which trans-cyclopentane isomers
3a and 3b showed only moderate affinity (Ki =59 and 383 nm,
respectively; Table 1). However, cis-cyclopentane isomers 3c
To determine their binding affinities, all compounds were
tested in an 125I-labeled chemokine ligand 2 (125I-CCL2) radioli-
gand displacement assay on human bone osteosarcoma cells
(U2OS)–CCR2 membrane preparations, as described previously
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of structures. In the first array, we generated the more flexible
(at the 3-position of the cyclopentane ring) bis(trifluorome-
thyl)benzyl derivatives with different substituents on the aro-
matic ring connected to the piperidine ring (i.e., compounds
4–16, Table 2).
Table 1. Binding affinities, association and dissociation rate constants,
and residence times of MK-0483 (3c) and its diastereomers.[a]
Table 2. Binding affinities and kinetic rate index values of compounds 4–
16.
Compd
R
X
Ki [nm][a]
KRI[b]
4
5
6
7
8
9
10
11
12
13
14
15
16
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
1.5Æ0.1
28Æ2
0.7 (0.6/0.7)
0.6 (0.5/0.7)
0.7 (0.6/0.8)
0.8 (0.7/0.9)
0.6 (0.5/0.6)
0.6 (0.6/0.7)
–
2-OMe
3-OMe
4-OMe
3,4-di-OMe
3,4-OCH2O-
4-OCF3
4-tBu
4-OH
H
4-OMe
3-COOH
3-COOMe
5.9Æ1.7
29Æ2
Compd
Ki [nm]
kon [nmÀ1 minÀ1
]
koff [minÀ1]
tres [min]
48Æ1
3a
3b
3c
3d
59Æ2
0.00044Æ0.0004
0.0028Æ0.0004
357Æ51
270Æ51
17Æ3
383Æ19
0.000058Æ0.000007 0.0037Æ0.0007
122Æ20
1.2Æ0.1 0.020Æ0.002
0.0014Æ0.00004 724Æ20
7%[c]
–
11 Æ 3
0.013Æ0.001
0.018Æ0.002
56Æ6
17Æ2
0.6 (0.6/0.6)
0.6 (0.6/0.6)
0.5 (0.5/0.6)
0.8 (0.8/0.8)
0.6 (0.5/0.7)
[a] Data represent the mean ÆSEM (n=3).
5.4Æ0.9
76Æ14
22Æ6
N
CH
CH
7.9Æ1.6
and 3d had high affinity for the CCR2 receptor (Ki =1.2 and
11 nm, respectively) which is in accordance with the reported
values.[8] Next, we evaluated all four diastereomers in the com-
petition–association assay, as this could help us to understand
the importance of the 3D configuration for SKRs. Surprisingly,
trans isomers 3a and 3b, despite their moderate affinity, both
had similar and very slow dissociation characteristics, which
translated into residence times longer than 4 h (tres =357 and
270 min, respectively; Table 1), whereas the association rate
constants displayed a sevenfold difference (kon =0.00044 and
0.000058 nmÀ1 minÀ1, respectively). The opposite was observed
in the case of cis isomers 3c and 3d. For these compounds,
the association rate constants were similar and the dissociation
rate constants showed a more than 12-fold difference, which
yielded residence times of 724 min for 3c and 56 min for 3d;
this is also in line with the half-life value for MK-0483 (3c) re-
ported by the Merck researchers.[8]
[a] Data are the meanÆSEM (n=3). [b] n=2. [c] Percent displacement at
1 mm 125I-CCL2.
Unsubstituted phenyl compound 4 had an affinity of 1.5 nm
for CCR2. Incorporation of a 2-methoxy group to give 5 result-
ed in a decrease in the affinity (Ki =28 nm). The affinity was
slightly recovered for compound 6 with a 3-methoxy group
(Ki =5.9 nm, correlating with the reported values).[16c] However,
4-methoxy derivative 7 afforded a fivefold decrease in the af-
finity (Ki =29 nm). Compound 8 with double substitution with
a 3,4-dimethoxy group resulted in an even bigger decrease in
the affinity. Closing the methoxy groups to yield 9 with a ben-
zo[1,3]dioxole ring resulted in a minor improvement in the af-
finity. Introduction of lipophilic groups at the 4-position (e.g.,
R=4-OCF3, 4-tBu) resulted in an even bigger decrease in the
affinity (for compound 10, R=4-OCF3) or a complete loss of af-
finity (for compound 11, R=4-tBu). Compound 12 with the hy-
drophilic 4-hydroxy group had affinity similar to that of com-
pound 7 with the 4-methoxy substituent. 3-Pyridine derivative
13 had slightly lower affinity than phenyl derivative 4 (Ki =
5.4 nm, correlating with the reported values).[16c] Compound
14, with a 4-methoxypyridin-3-yl group, yielded a 50-fold de-
crease in affinity relative to that of 4. Apparently, any substitu-
tion on the aryl ring is not favorable, as it results in a decrease
in affinity relative to that of a simple unsubstituted phenyl ring
(i.e., compound 4), although 3-substituents are tolerated more
than others. The evaluation of direct analogues of MK-0483,
such as 3-carboxylic acid 15, resulted in a 14-fold decrease in
affinity (Ki =22 nm, in agreement with the reported values)[16c]
relative to that of 4, whereas methyl ester 16 had an even
Apparently, the cis isomers have the best conformation for
the eventual binding state on the receptor (affinity); they
reach that state quickly, as gauged from their relatively fast kon
values, and they stay there for prolonged periods of time
(Table 1). The only difference for the trans isomers is their rela-
tively slow association rate, but they are still capable of bind-
ing to the CCR2 receptor, and they also display long residence
times.
Bis(trifluoromethyl)benzyl derivatives 4–16
From the above, we chose to continue with the cis analogues
of MK-0483 and decided to synthesize several new analogues
of MK-0483 and a small number of known[8,16c] compounds to
describe the SARs and SKRs for the CCR2 receptor in this series
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better affinity than the acid (Ki =7.9 nm). However, the dual-
point competition–association assay for all compounds of this
series yielded a KRI below unity, indicative of a short tres.
tably, compounds 20–23 had longer tres values than the actual
incubation time of the assay (150 min). As a consequence, the
affinities for these compounds are best described as “appar-
ent”, as full equilibrium had not been reached for these (and
other tres >150 min) compounds. Their “true” affinities may be
slightly higher than those reported here. In our efforts to in-
crease the tres, we also explored the 4-position by introducing
lipophilic 4-chloro (i.e., compound 24) and 4-trifluoromethyl
(i.e., compound 25) groups. However, despite the good affinity,
both compounds had KRI values below unity (KRI=0.8 for
both).
7-(Trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline derivatives
17–27
Rigidification on the carboxamide side into the 7-(trifluoro-
methyl)-1,2,3,4-tetrahyroisoquinoline group provided com-
pounds 17–27. Unsubstituted phenyl olefin 17 had an affinity
that was 14-fold lower than that of saturated analogue 18 and
a tres that was twofold shorter than that of 18 (Table 3), which
indicates that the phenyl ring should be positioned at an
angle to the piperidine ring for optimal binding.
Compound 26, which is a direct analogue of MK-0483 with
an acid group at the 3-position, showed only a minor decrease
in affinity (comparable to the reported values);[8] however, the
KRI value was below unity (KRI=0.7), which is suggestive of
a short tres. Apparently, the additional methyl group at the 3-
position of the piperidine ring, the oxygen atom in the 3,4-di-
hydro-2H-benzo[e][1,3]oxazine ring, or the combination of
both is responsible for the long residence time of MK-0483.
Compound 27 with a methyl ester group yielded the highest
affinity in this study, although the KRI suggests a short tres (Ki =
0.91 nm, KRI=0.8).
Table 3. Binding affinities, kinetic rate index values, and residence times
of compounds 17–27.
Conclusions
We evaluated the structure–affinity and structure–kinetics rela-
tionships of MK-0483, its diastereomers, and its structural ana-
logues as CCR2 antagonists. A 3-carboxamide group on the cy-
clopentane ring linked to a rigid 7-(trifluoromethyl)-1,2,3,4-tet-
rahydroisoquinoline moiety yielded better affinity than the
more flexible bis(trifluoromethyl)benzyl substituent and gener-
ally prolonged the drug–target residence time. The SARs of the
phenyl ring adjacent to the piperidine moiety suggest that lip-
ophilic hydrogen-bond-accepting substituents at the 3-posi-
tion (e.g., R=3-OMe, 3-COOMe) are vital for improved affinity.
However, the SKRs suggest that to have a long residence time
(tres), a lipophilic group with a certain size is desired (e.g., R=
3-Br, 3-iPr) or alternatively (in the case of MK-0483) a carboxylic
acid group can be used. However, the acid group per se does
not provide the long tres for these structures. Possibly the addi-
tional methyl group on the piperidine ring (as observed with
MK-0483) provides additional interaction to keep the molecule
bound to the receptor. Likewise, the additional oxygen atom
in the 3,4-dihydro-2H-benzo[e][1,3]oxazine ring of MK-0483
could make an additional hydrogen bond and cause its long
tres, as the hydrogen bond would be shielded by the ring
system itself. We reported similar observations in another
series of CCR2 antagonists;[7] moreover, a similar idea was put
forward by Schmidtke et al.[17] in calculations on hydrogen-
bond shielding. In conclusion, this study contributes to one of
the first detailed structure–kinetics relationships for CCR2 an-
tagonists, and it may help to develop better drug candidates.
Moreover, the value of the long tres drugs has already been
proven on other targets, although only in retrospect.[5a] From
this perspective, MK-0483 would be a suitable candidate to be
advanced into clinical studies owing to its long tres. If it failed
as a result of a lack of efficacy, one could argue that blockade
Compd
R
Ki [nm][a]
70Æ25
KRI[b]
tres [min][a]
17
18
19
20
21
22
23
24
25
26
27
–
H
1.1 (1.1/1.0)
1.1 (1.2/1.0)
0.9 (0.8/0.9)
1.0 (0.9/1.2)
1.0 (1.0/1.1)
1.0 (1.0/0.9)
1.0 (0.9/1.1)
0.8 (0.8/0.8)
0.8 (0.8/0.9)
0.7 (0.7/0.7)
0.8 (0.8/0.8)
38Æ4
4.4Æ0.7
0.95Æ0.22
2.6Æ0.3
2.8Æ0.2
3.6Æ0.4
5.3Æ0.3
2.0Æ0.3
7.2Æ0.2
7.8Æ1.4
0.91Æ0.25
91Æ25
3-OMe
3-Cl
3-Br
3-iPr
3-CF3
4-Cl
4-CF3
3-COOH
3-COOMe
–
200Æ29
243Æ45
266Æ48
158Æ35
–
–
–
–
[a] Data are the meanÆSEM (n=3). [b] n=2.
From the bis(trifluoromethyl)benzyl series (Table 2), we
learned that substituents at the 3-position were better tolerat-
ed; therefore, we focused on this position in this array of com-
pounds. 3-Methoxy derivative 19 had excellent affinity (Ki =
0.95 nm), but the KRI was below unity. 3-Chloro derivative 20
has a slightly lower affinity; however, the compound had a tres
of 200 min. 3-Bromo derivative 21 had affinity and tres similar
to that of 3-chloro compound 20. We previously reported
a similar correlation in another series of CCR2 antagonists, for
which methoxy substituents yielded the best affinity com-
pounds, whereas the halogen derivatives had longer tres
values.[6] Compound 22 with a 3-isopropyl substituent that is
similar in size to the bromine substituent but with electron-do-
nating properties yielded only a minor decrease in the affinity,
whereas the tres stayed unchanged, which thus suggests the
importance of the space-filling properties of the substituents.
3-Trifluoromethyl derivative 23 (a bioisostere of chlorine)
showed results similar to those obtained with 20 (R=3-Cl). No-
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6H); 13C NMR (101 MHz, CDCl3): d=176.8, 161.5, 161.2, 142.6,
141.7, 131.9, 131.6, 129.2, 129.0, 127.9, 127.4, 126.6, 124.7, 122.0,
117.5, 67.5, 57.3, 52.9, 51.8, 43.3, 40.3, 34.8, 32.5, 31.9, 30.5, 27.9,
18.6, 17.8 ppm; MS: m/z: 541 [H+]; HPLC: tR =8.9 min.
of CCR2 (alone) is not sufficient to treat the many immunologi-
cal disorders that were mentioned in the Introduction.
Experimental Section
3-{1-[(1R,3S)-3-{[3,5-Bis(trifluoromethyl)benzyl]carbamoyl}-3-iso-
propylcyclopentyl]piperidin-4-yl}benzoic acid (15): In a 50 mL
round-bottomed flask, compound 16 (0.058 g, 0.128 mmol) was
dissolved in EtOH (10 mL). Subsequently, a solution of 4m LiOH in
H2O (0.3 mL, 1.2 mmol) was added, and the mixture was stirred for
1.5 h at 508C. EtOH was evaporated under vacuum. The mixture
was partitioned between brine and chloroform (15 mL/15 mL) and
the solution was adjusted to pH 7 with 1m HCl (aq). The organic
layer was collected, dried (MgSO4), and concentrated under
Chemistry
All solvents and reagents were purchased from commercial sources
and were of analytical grade. Demineralized water is simply re-
ferred to as H2O, because it was used in all cases, unless stated
1
otherwise (i.e., brine). H NMR and 13C NMR spectra were recorded
with a Bruker AV 400 liquid-state spectrometer (1H NMR, 400 MHz;
13C NMR, 100 MHz) at room temperature. Chemical shifts are re-
ported in parts per million (ppm) downfield from the internal stan-
dard tetramethylsilane (TMS). Analytical purity of the final com-
pounds was determined by HPLC with a Phenomenex Gemini
3 mm C18 110A column (50”4.6 mm, 3 mm) by measuring UV ab-
sorbance at l=254 nm. Preparation of the samples and the HPLC
method were as follows: the compound (0.3–0.8 mg) was dissolved
in a mixture of CH3CN/H2O/tBuOH (1:1:1, 1 mL) and eluted from
the column within 15 min at a flow rate of 1.3 mLminÀ1. The elu-
tion method was set up as follows: 1–4 min isocratic system of
H2O/CH3CN/1% TFA in H2O (80:10:10), from minute 4, 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 com-
pounds showed a single peak at the designated retention time
and were at least 95% pure. Preparative HPLC was performed with
a Shimadzu HPLC/UV system by using a Gemini C18 Phenomenex
column (100”10 mm, 5 mm); a linear gradient from 10 to 90% of
mobile phase B was applied, keeping mobile phase C constant at
10%. Mobile phase A consisted of H2O, mobile phase B consisted
of acetonitrile, and mobile phase C consisted of 1% TFA solution in
H2O. The flow rate was 5 mLminÀ1. LC–MS analyses were per-
formed by using a Thermo Finnigan Surveyor - LCQ Advantage
Max LC–MS system and a Gemini C18 Phenomenex column (50”
4.6 mm, 3 mm). The elution method was set up as follows: 1-4 min
isocratic system of H2O/CH3CN/1% TFA in H2O (80:10:10), from
minute 4, 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. Microwave reactions were done by using a Biotage Initia-
tor microwave synthesizer. TLC was routinely consulted to monitor
the progress of reactions by using aluminum-coated Merck silica
gel F254 plates. Purification by column chromatography was ach-
ieved by use of Grace Davison Davisil silica column material
(LC60A, 30–200 mm). The procedure for a series of similar com-
pounds is given as a general procedure for all within that series,
annotated by the numbers of the compounds.
1
vacuum to yield 15·HCl (0.042 g, 53%). H NMR (400 MHz, CDCl3):
d=9.34 (s, 1H), 8.04 (s, 1H), 7.76–7.75 (m, 3H), 7.64 (s, 1H), 7.22–
7.20 (m, 2H), 4.64–4.58 (m, 1H), 4.27–4.22 (m, 1H), 3.88 (d, J=
11.2 Hz, 1H), 3.78 (d, J=11.2 Hz, 1H), 3.49–3.42 (m, 1H), 3.14–3.12
(m, 1 h), 2.90–2.65 (m, 4H), 2.36–2.32 (m, 1H), 2.19–1.96 (m, 5H),
1.93–1.88 (m, 2H), 1.69–1.68 (m, 1H), 0.93–0.89 ppm (m, 6H);
13C NMR (101 MHz, CDCl3): d=176.3, 173.1, 142.9, 142.5, 136.6,
131.4, 131.2, 131.0, 128.2, 128.0, 127.9, 125.2, 124.7, 122.0, 120.6,
68.1, 57.6, 53.5, 52.7, 43.2, 40.2, 35.2, 34.3, 32.8, 30.6, 29.4, 26.9,
19.3, 17.8 ppm; MS: m/z: 585 [H+]; HPLC: tR =9.2 min.
{(1S,3R)-1-Isopropyl-3-[4-phenyl-3,6-dihydropyridin-1(2H)-yl]cy-
clopentyl}[7-(trifluoromethyl)-3,4-dihydroisoquinolin-2(1H)-yl]-
methanone (17): In a 5 mL microwave tube, a mixture of triflate
34 (0.06 g, 0.1 mmol), phenylboronic acid (0.018 g, 1.5 mmol), LiCl
(0.012 g, 0.3 mmol), 2m Na2CO3 in H2O (0.15 mL, 0.3 mmol), and
tetrakis(triphenylphosphine)palladium(0) (5 mol%) was dissolved in
dimethoxyethane (DME, 1 mL) under a nitrogen atmosphere. The
mixture was heated under microwave irradiation at 908C for 5.5 h.
The mixture was partitioned between CH2Cl2 and H2O (15 mL/
15 mL). The organic layer was dried (MgSO4) and concentrated
under vacuum. The product was purified by preparative HPLC to
yield 17·TFA (0.014 g, 23%). 1H NMR (400 MHz, CD3CN): d=7.50–
7.28 (m, 8H), 6.00 (s, 1H), 4.90–4.60 (m, 3H), 4.30 (d, J=16.0 Hz,
0.5Ha), 4.20 (d, J=16.0 Hz, 0.5Hb), 3.90–3.55 (m, 4H), 3.34 (brs,
0.5Ha), 3.20–2.90 (m, 3.5H), 2.80–2.65 (m, 3H), 2.40–1.95 (m, 4H),
1.72 (brs, 1H), 0.93 (d, J=6.4 Hz, 3Ha), 0.86 ppm (d, J=6.4 Hz,
3Hb); superscripts a and b indicate different rotamers; MS: m/z:
497 [H+]; HPLC: tR =8.7 min.
[(1S,3R)-1-Isopropyl-3-(4-phenylpiperidin-1-yl)cyclopentyl][7-(tri-
fluoromethyl)-3,4-dihydroisoquinolin-2(1H)-yl]methanone (18):
In
a 10 mL round-bottomed flask, compound 17 (6.6 mg,
0.011 mmol) was dissolved in THF (5 mL). 5 wt% Pd/C (5 mol%)
and palladium acetate (1.5 mol%) were added. The mixture was
flushed with hydrogen and kept under a hydrogen atmosphere by
using a hydrogen balloon. The mixture was heated at 508C for
30 s and stirring was continued at room temperature for 2 h. Upon
completion of the reaction, the mixture was filtered over Celite,
and the product was purified by preparative HPLC to yield 18·TFA
(4.5 mg, 67%). 1H NMR (400 MHz, CD3CN): d=7.45–7.18 (m, 8H),
4.85–4.68 (m, 2H), 3.90–3.60 (m, 3H), 3.40 (brs, 1H), 3.00–1.80 (m,
17H), 1.64 (brs, 1H), 0.91 (d, J=6.4 Hz, 3H), 0.82 ppm (d, J=
6.4 Hz, 3H); MS: m/z: 499 [H+]; HPLC: tR =8.7 min.
The synthesis of MK-0483 (3c) and diastereomers 3a, 3b, and 3d
was achieved by following the synthetic approach reported by Pas-
ternak et al.[8]
General procedure for the synthesis of compounds 4–14 and
16: In
a round-bottomed flask, a mixture of olefin 35–46
(0.25 mmol), 5wt% Pd/C (5 mol%), and palladium acetate
(1.5 mol%) was dissolved in MeOH (15 mL), and the mixture was
stirred overnight under an atmosphere of hydrogen. The mixture
was filtered through Celite and purified by preparative HPLC.
(1S,3R)-N-[3,5-Bis(trifluoromethyl)benzyl]-1-isopropyl-3-(4-phe-
nylpiperidin-1-yl)cyclopentane-1-carboxamide TFA salt (4): Yield:
69%. H NMR (400 MHz, CDCl3): d=10.62 (s,1H), 7.76–7.74 (m, 3H),
7.60 (s, 1H), 7.37–7.17 (m, 5H), 4.60–4.43 (m, 2H), 3.76-3.65 (m,
2H), 3.43 (s, 1H), 2.92–2.71 (m, 3H), 2.61–2.56 (m, 1H), 2.29–2.01
(m, 8H), 2.92–2.85 (m, 1H), 1.81–7.23 (m, 1H), 0.88–0.87 ppm (m,
General procedure for the synthesis of compounds 19, 21–23,
and 27: An oven-dried glass tube was charged with tosylhydra-
zone 52 (140 mg, 0.23 mmol, 1 equiv), the boronic acid
(0.34 mmol, 1.5 equiv), and Cs2CO3 (110 mg, 0.34 mmol, 1.5 equiv,
dried at 1008C overnight), and the tube was sealed with a crimp
top with a septum. High vacuum was applied for 30 min, after
1
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Full Papers
which the tube was backfilled with nitrogen and dry 1,4-dioxane
(1 mL) was added. Subsequently, the mixture was degassed (4”)
by applying a vacuum and a nitrogen atmosphere sequentially.
The mixture was heated at 1008C for 18 h. TLC showed full conver-
sion of the tosylhydrazone (EtOAc, KMnO4 spray to visualize). The
cooled mixture was quenched with an aqueous saturated solution
of NaHCO3 (2 mL), extracted with CH2Cl2 (3”10 mL), dried (MgSO4),
and concentrated under vacuum. The crude yellow oil was pre-pu-
rified by column chromatography (silica gel, EtOAc) followed by
preparative HPLC to give final compounds 19, 21–23, and 27 as
TFA salts as colorless solidified oils.
(brs, 1H), 0.89 ppm (brs, 6H); MS: m/z: 533 [H+]; HPLC: tR =
8.82 min.
4-(3-Carboxyphenyl)-1-{(1R,3S)-3-isopropyl-3-[7-(trifluorometh-
yl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl]cyclopentyl}piperi-
din-1-ium chloride (26): Ester 27 (25 mg, 0.045 mmol, 1.0 equiv)
was dissolved in a mixture of EtOH (5 mL) and water (2 mL), and
4m LiOH in H2O (113 mL, 0.45 mmol, 10.0 equiv) was added. After
2 h at 508C, 27 was completely consumed, as shown by TLC
(EtOAc, KMnO4 spray to visualize). Ethanol was evaporated, and the
pH was adjusted to pH 1 with 3m HCl. The white precipitate was
filtered off, rinsed with water (2”2 mL), and dried under vacuum
1
1-{(1R,3S)-3-Isopropyl-3-[7-(trifluoromethyl)-1,2,3,4-tetrahydro-
isoquinoline-2-carbonyl]cyclopentyl}-4-(3-methoxyphenyl)piperi-
din-1-ium 2,2,2-trifluoroacetate (19): Yield: 10%; 1H NMR
(400 MHz, CD3CN): d=10.66 (brs, 1H, NH), 7.53 (s, 1H), 7.47 (d, J=
8.4 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.23 (td, J=7.2, 2.0 Hz, 1H),
6.82–6.70 (m, 3H), 4.78–4.68 (m, 2H), 3.78–3.65 (m, 6H), 3.60 (d,
J=12.0 Hz, 1H), 3.40–3.28 (m, 1H), 2.95–2.86 (m, 4H), 2.81–2.74
(m, 1H), 2.57–2.52 (m, 1H), 2.48–2.35 (m, 2H), 2.11–1.98 (m, 6H),
1.72–1.56 (m, 2H), 0.89 (d, J=6.4 Hz, 3H), 0.75 ppm (d, J=6.4 Hz,
3H); LC–MS: m/z: 529 [H+]; HPLC: tR =8.09 min.
to yield 26·HCl (17 mg, 65%). H NMR (400 MHz, CD3CN): d=7.88–
7.84 (m, 2H), 7.52 (s, 1H), 7.50–7.45 (m, 2H), 7.42 (t, J=7.6 Hz, 1H),
7.33 (d, J=8.0 Hz, 1H), 4.80–4.70 (m, 2H), 3.90–3.63 (m, 3H), 3.58
(d, J=12.8 Hz, 1H), 3.40–3.29 (m, 1H), 3.01–2.85 (m, 5H), 2.70–2.40
(m, 3H and water), 2.24–2.05 (m, 6H), 1.82–1.75 (m, 1H), 1.67–1.59
(m,1H), 0.88 (d, J=6.4 Hz, 3H), 0.76 ppm (d, J=6.4 Hz, 3H); LC–
MS: m/z: 543 [H+]; HPLC: tR =8.10 min.
1-Ethyl-1-methyl-4-oxopiperidin-1-ium iodide (28): In a 20 mL mi-
crowave tube,
a solution of 1-methyl-4-piperidone (2.2 mL,
17.9 mmol) in acetone (15 mL) was mixed with ethyl iodide
(1.43 mL, 17.9 mmol) under a nitrogen atmosphere. The mixture
was stirred in the microwave at 608C for 5 h to form a yellow
solid. The solid was filtered, washed with acetone (2”25 mL), and
dried under vacuum to yield 28 (4.35 g, 90%) as a yellow salt. The
compound was used in the next step without further purification.
General procedure for synthesis of compounds 20, 24, and 25:
In a 5 mL microwave tube, a solution of 50 (0.5 mmol, 1 equiv) in
DME (3 mL) was mixed with the arylboronic acid (0.65 mmol,
1.3 equiv), LiCl (1.5 mmol, 3 equiv), 2m Na2CO3 in H2O (1.5 mmol,
3 equiv), and Pd(PPh3)4 (5 mol%). The mixture was heated under
microwave irradiation at 908C for 30 min to 2.5 h. The mixture was
partitioned between CH2Cl2 and H2O (15 mL/15 mL). The organic
layer was dried (MgSO4) and concentrated under vacuum. The
product was purified by column chromatography [silica gel,
CH2Cl2 +EtOAc (0–50%, gradient)]. The olefin was dissolved in THF
(1S,3R)-3-Amino-N-[3,5-bis(trifluoromethyl)benzyl]-1-isopropylcy-
clopentane-1-carboxamide (29): Prepared by following the syn-
thetic approach previously reported by our group.[6]
[(1S,3R)-3-Amino-1-isopropylcyclopentyl][7-(trifluoromethyl)-3,4-
dihydroisoquinolin-2(1H)-yl]methanone (30): Prepared by follow-
ing the synthetic approach previously reported by our group.[7]
(5 mL) and transferred into
a 20 mL microwave tube. PtO2
(20 wt%) and acetic acid (3 equiv) were added. The tube was
capped, flushed with H2 gas, and additionally pressurized with H2
gas (20 mL) by using a 20 mL syringe. The progress of the reaction
was monitored by TLC/MS. The reaction was complete after 10 to
15 min. In the case of halogen substituents, if longer reaction
times were used dehalogenation was observed. Next, the ester
was dissolved in EtOH (20 mL) and transferred into a 50 mL round-
bottomed flask. 4m LiOH in H2O (10 equiv) was added, and the
mixture was heated at reflux for 3 h. EtOH was evaporated under
vacuum. The mixture was acidified with 1m HCl to pH 1, and the
product was extracted with CH2Cl2 (3”15 mL). The organic layer
was dried (MgSO4) and concentrated under vacuum. The corre-
sponding acid was dissolved in CH2Cl2 (5 mL) and transferred into
a 10 mL round-bottomed flask. 7-Trifluoromethyl-1,2,3,4-tetrahy-
droisoquinoline·HCl (2 equiv) was added, which was followed by
the addition of N,N-diisopropylethylamine (DIPEA, 12 equiv),
PyBrOP (3 equiv), N,N-dimethylaminopyridine (DMAP, 1.5 equiv),
and several beads of 4 Š molecular sieves. The mixture was stirred
at room temperature for 48 h. The mixture was partitioned be-
tween CH2Cl2/H2O (15 mL/15 mL) and extracted with CH2Cl2 (3”
5 mL), dried (MgSO4), and concentrated under vacuum. The prod-
uct was purified by preparative HPLC. All intermediates were
checked by MS and were advanced to the next step without fur-
ther analysis.
(1S,3R)-N-[3,5-Bis(trifluoromethyl)benzyl]-1-isopropyl-3-(4-oxop-
iperidin-1-yl)cyclopentane-1-carboxamide (31): In 250 mL round-
bottomed flask, a solution of amine 29 (2.0 g, 5.0 mmol) dissolved
in ethanol (75 mL) and H2O (25 mL) was stirred at 408C. Compound
28 (2.02 g, 7.5 mmol) dissolved in H2O (30 mL) was added dropwise
over 5 min. Then, K2CO3 (1.4 g, 10 mmol) was added, and the mix-
ture was heated at reflux for 5 h, which was followed by an addi-
tional 12 h stirring at room temperature. Ethanol was removed
under vacuum, and the mixture was partitioned between CH2Cl2/
H2O (150 mL/150 mL). The organic layer was dried (MgSO4) and
concentrated under vacuum. Compound 31 was purified by
column chromatography [silica gel, CH2Cl2 +EtOAc (50–100%, gra-
1
dient)]. Yield: 1.7 g, (72%). H NMR (400 MHz, CDCl3): d=7.77-7.70
(m, 3H), 7.49 (s, 1H), 4.56 (d, J=5.6 Hz, 2H), 2.83–2.76 (m, 5H),
2.38–2.27 (m, 4H), 2.19–1.90 (m, 5H), 1.70–1.53 (m, 2H), 1.15 (s,
1H), 0.92–0.89 ppm (m, 6H).
1-{(1R,3S)-3-Isopropyl-3-[7-(trifluoromethyl)-1,2,3,4-tetrahydroi-
soquinoline-2-carbonyl]cyclopentyl}piperidin-4-one
(32):
In
a 20 mL microwave tube, a solution of amine 30 (0.71 g, 2.0 mmol)
in ethanol (10 mL) and H2O (3 mL) was stirred at 408C. Compound
28 (0.81 g, 3 mmol) dissolved in H2O (3 mL) and added dropwise
over 5 min. Then, K2CO3 (0.56 g, 4 mmol) was added, and the mix-
ture was heated under microwave irradiation at 1008C for 3 h. Eth-
anol was removed under vacuum, and the mixture was extracted
with CH2Cl2/1m NaOH (50 mL/25 mL). The organic layer was dried
(MgSO4) and concentrated under vacuum. Compound 32 was puri-
fied by column chromatography [silica gel, CH2Cl2 +EtOAc (50–
100%, gradient)]. Yield: 0.22 g (25%). 1H NMR (400 MHz, CDCl3):
{(1S,3R)-3-[4-(3-Chlorophenyl)piperidin-1-yl]-1-isopropylcyclo-
pentyl}[7-(trifluoromethyl)-3,4-dihydroisoquinolin-2(1H)-yl]me-
thanone TFA salt (20): Overall yield: 9%; 1H NMR (400 MHz,
CD3CN): d=8.51 (brs, 1H), 7.50–7.40 (m, 2H), 7.30–7.16 (m, 4H),
7.09 (d, J=6.8 Hz, 1H), 4.81 (brs, 2H), 4.00–3.65 (m, 4H), 3.52 (brs,
1H), 3.15–2.75 (m, 5H), 2.60–2.40 (m, 2H), 2.35–2.00 (m, 7H), 1.79
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d=7.40–7.30 (m, 2H), 7.21 (d, J=8 Hz, 1H), 4.80–4.60 (m, 2H), 3.77
(brs, 2H), 2.88 (brs, 2H), 2.72 (brs, 3H), 2.63–2.53 (m, 2H), 2.41–
2.38 (m, 3H), 2.24–2.18 (m, 1H), 2.05–1.80 (m, 4H), 1.60–1.30 (m,
3H), 0.89 (d, J=6.8 Hz, 3H), 0.73 ppm (d, J=6,8 Hz, 3H).
(1S,3R)-N-[3,5-Bis(trifluoromethyl)benzyl]-1-isopropyl-3-[4-
phenyl-3,6-dihydropyridin-1(2H)-yl]cyclopentane-1-carboxamide
(35): Yield: 40%; MS: m/z: 539 [H+].
(1S,3R)-Methyl-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclo-
pentanecarboxylate (47): Prepared by following the synthetic ap-
proach reported by Kothandaraman et al.[12]
1-[(1R,3S)-3-{[3,5-Bis(trifluoromethyl)benzyl]carbamoyl}-3-isopro-
pylcyclopentyl]-1,2,3,6-tetrahydropyridin-4-yl trifluoromethane-
sulfonate (33): In a 100 mL round-bottomed flask, a mixture of 2m
LDA in heptanes (2.2 mL, 2.88 mmol) in dry THF (20 mL) was
cooled down to À788C under a nitrogen atmosphere. Ketone 31
(0.55 g, 1.15 mmol) dissolved in dry THF (15 mL) was added drop-
wise to the mixture. Vigorous stirring was necessary. The mixture
was stirred for 1 h at À788C. After 1 h, the mixture was slowly
warmed to À208C and kept at this temperature for 1 h. Subse-
quently, the mixture was cooled down to À788C and N-phenylbis(-
trifluoromethanesulfonimide) (0.65 g, 2.3 mmol) dissolved in dry
THF (8 mL) was added dropwise. After the addition was complete,
the mixture was slowly brought to room temperature and stirred
overnight. The mixture was quenched with EtOH (15 mL), concen-
trated under vacuum, and partitioned between CH2Cl2/H2O (50 mL/
50 mL). The organic layer was dried (MgSO4) and concentrated
under vacuum. The product was purified by column chromatogra-
phy [silica gel, CH2Cl2 +EtOAc (0–30%, gradient)] to yield triflate 33
Methyl (1S,3R)-3-amino-1-isopropylcyclopentane-1-carboxylate
(48): In a 100 mL round-bottomed flask, compound 47 (11.4 g,
40 mmol) was dissolved in a mixture of CH2Cl2 (30 mL) and TFA
(20 mL). The mixture was stirred at room temperature for 2 h.
Upon completion of the reaction, the mixture was basified to
pH 14 with 2m NaOH and extracted with CH2Cl2 (3”50 mL). The
organic layer was dried (MgSO4) and concentrated under vacuum.
Yield: 7.3 g (98%). The crude product was used in the next step
1
without further purification. H NMR (400 MHz, CDCl3): d=3.57 (s,
3H), 3.25–3.18 (m, 1H), 2.20–2.12 (m, 1H), 1.90–1.70 (m, 4H), 1.48–
1.35 (m, 2H), 1.28–1.15 (m, 2H), 0.80–0.71 ppm (m, 6H).
Methyl (1S,3R)-1-isopropyl-3-(4-oxopiperidin-1-yl)cyclopentane-
1-carboxylate (49): In seven separate 100 mL round-bottomed
flasks, a solution of amine 48 (0.74 g, 4.0 mmol) dissolved in etha-
nol (20 mL) and H2O (8 mL) was stirred at 408C. Compound 28
(1.61 g, 6 mmol) dissolved in H2O (8 mL) was added dropwise over
5 min. Then, K2CO3 (1.12 g, 8 mmol) was added, and the mixture
was heated at reflux for 3 h. The contents of all flasks were com-
bined, and ethanol was removed under vacuum. The mixture was
then partitioned between CH2Cl2/H2O (250 mL/250 mL). The organ-
ic layer was dried (MgSO4) and concentrated under vacuum. The
product was purified by column chromatography [silica gel,
CH2Cl2 +EtOAc (0–100%, gradient)]. Combined yield: 4.19 g (56%).
1H NMR (400 MHz, CDCl3): d=3.55 (s, 3H), 2.63 (t, J=6.4 Hz, 4H),
2.59–2.50 (m, 1H), 2.29 (t, J=6.4 Hz, 4H), 2.16–2.12 (m, 1H), 2.01–
1.95 (m, 1H), 1.87–1.74 (m, 3H), 1.37 (t, J=4.8 Hz, 2H), 0.74 (d, J=
6.8 Hz, 3H), 0.71 ppm (d, J=6.8 Hz, 3H).
1
(0.3 g, 42%). H NMR (400 MHz, CDCl3): d=8.08 (t, J=5.6 Hz, 1H),
7.76–7.72 (m, 3H), 5.68 (s, 1H), 4.49 (d, J=5.6 Hz, 2H), 3.20–3.06
(m, 2H), 2.89–2.82 (m, 2H), 2.67–2.60 (m, 1H), 2.40–2.20 (m, 3H),
2.04–1.79 (m, 4H), 1.73–1.59 (m, 2H), 0.91(d, J=6.8 Hz, 3H),
0.88 ppm (d, J=6.8 Hz, 3H).
1-[(1R,3S)-3-{[3,5-Bis(trifluoromethyl)benzyl]carbamoyl}-3-isopro-
pylcyclopentyl]-1,2,3,6-tetrahydropyridin-4-yl trifluoromethane-
sulfonate (34): In a 20 mL microwave tube, a mixture of 2m LDA
in heptanes (0.33 mL, 0.65 mmol) in THF (5 mL) was cooled down
to À788C under a nitrogen atmosphere. Ketone 32 (0.22 g,
0.5 mmol) dissolved in dry THF (5 mL) was added dropwise to the
mixture. Vigorous stirring was necessary. The mixture was stirred
for 1 h at À788C. After 1 h, the mixture was slowly warmed to
À208C and kept at this temperature for 1.5 h. Subsequently, the
mixture was cooled down to À788C and N-phenylbis(trifluorome-
thanesulfonimide) (0.23 g, 0.65 mmol) dissolved in THF (5 mL) was
added dropwise. After the addition was complete, the mixture was
slowly brought to room temperature and stirred overnight. The
mixture was quenched with EtOH (5 mL), concentrated under
vacuum, and partitioned between CH2Cl2/H2O (25 mL/25 mL). The
organic layer was dried (MgSO4) and concentrated under vacuum.
The product was purified by column chromatography [silica gel,
CH2Cl2 +EtOAc (0–20%, gradient)] to yield triflate 34 (0.06 g, 21%).
1H NMR (400 MHz, CDCl3): d=7.44 (d, J=8 Hz, 1H), 7.38 (s, 1H),
7.27 (d, J=8.0 Hz, 1H), 5.71 (s, 1H), 4.90–4.57 (m, 2H), 3.82 (brs,
2H), 3.17 (s, 2H), 2.93 (d, J=5.2 Hz, 2H), 2.80–2.60 (m, 4H), 2.43
(brs, 2H), 2.25–2.15 (m, 1H), 2.08–1.82 (m, 3H), 1.60–1.38 (m, 2H),
0.95 (d, J=6.4 Hz, 3H), 0.80 ppm (d, J=6.4 Hz, 3H).
Methyl (1S,3R)-1-isopropyl-3-[4-{[(trifluoromethyl)sulfonyl]oxy}-
3,6-dihydropyridin-1(2H)-yl]cyclopentane-1-carboxylate (50): In
a 250 mL round-bottomed flask, a mixture of 2m LDA in heptanes
(11 mL, 22 mmol) in dry THF (100 mL) was cooled down to À1008C
under a nitrogen atmosphere. Ketone 49 (4.19 g, 15.6 mmol) dis-
solved in dry THF (20 mL) was added dropwise keeping the tem-
perature below À788C. Vigorous stirring was necessary. The mix-
ture was stirred for 3 h, slowly rising the temperature to À408C.
Subsequently, the mixture was cooled down to À808C and N-phe-
nylbis(trifluoromethanesulfonimide) (7.86 g, 22 mmol) dissolved in
dry THF (10 mL) was added dropwise to the mixture keeping the
temperature of the reaction below À808C. After the addition was
complete, the mixture was slowly brought to room temperature
and stirred overnight. The mixture was quenched with EtOH
(50 mL), concentrated under vacuum, and partitioned between
CH2Cl2/H2O (250 mL/250 mL). The organic layer was dried (MgSO4)
and concentrated under vacuum. The product was purified by
column chromatography [silica gel, CH2Cl2 +EtOAc (0–10%, gradi-
General synthesis of compounds 35–46: In a 5 mL microwave
tube, a mixture of triflate 33 (0.25 mmol, 1 equiv), arylboronic acid
(0.35 mmol, 1.4 equiv), LiCl (0.75 mmol, 3 equiv), 2m Na2CO3 in H2O
(0.75 mmol, 3 equiv), and tetrakis(triphenylphosphine)palladium(0)
(5 mol%) was dissolved in DME (4 mL) under a nitrogen atmos-
phere. The mixture was heated under microwave irradiation at
908C for 3.5 h. DME was evaporated under vacuum, and the mix-
ture was partitioned between 2m Na2CO3/CH2Cl2 (25 mL/25 mL).
The organic layer was dried (MgSO4) and concentrated under
vacuum. The product was purified by column chromatography
[silica gel, CH2Cl2 +EtOAc (0–50%, gradient)]. LC–MS was used to
confirm the product before it was used in the next step.
1
ent)] to yield triflate 50 (4.2 g, 67%). H NMR (400 MHz, CDCl3): d=
5.73 (s, 1H), 3.70 (s, 3H), 3.25–3.12 (m, 2H), 2.80–2.67 (m, 3H), 2.46
(brs, 2H), 2.35–2.25 (m, 1H), 2.13–1.85 (m, 4H), 1.60–1.48 (m, 2H),
0.89 (d, J=6.4 Hz, 3H), 0.86 ppm (d, J=6.4 Hz, 3H).
N’-(1-{(1R,3S)-3-Isopropyl-3-[7-(trifluoromethyl)-1,2,3,4-tetrahy-
droisoquinoline-2-carbonyl]cyclopentyl}piperidin-4-ylidene)-4-
methoxybenzenesulfonohydrazide (52): Sulfonylhydrazide 51
(365 mg, 1.80 mmol, 1.05 equiv) was slurried in MeOH (3.5 mL). Pi-
peridone 32 (750 mg, 1.72 mmol, 1.00 equiv) was added at room
temperature, which resulted in a homogeneous mixture. After 4 h,
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Full Papers
piperidone was completely consumed, as shown by TLC (EtOAc/
petroleum ether 1:1). Methanol was removed under vacuum, and
the solidified oil (1.06 g, 100%) was used in the next reaction with-
out purification. 1H NMR (400 MHz, CDCl3): d=7.88 (d, J=8.0 Hz,
2H), 7.42 (d, J=8.0 Hz, 1H), 7.36 (s, 1H), 4.90–4.60 (m, 2H), 3.87 (s,
3H), 3.85–3.60 (m, 2H), 3.46 (s, 1H), 2.90 (s, 2H), 2.60–2.41 (m, 6H),
2.33 (brs, 3H), 2.19–2.14 (m, 1H), 2.00 (brs, 1H), 1.88–1.78 (m, 2H),
1.54–1.43 (m, 1H), 1.36–1.25 (m, 1H), 0.92 (d, J=6.4 Hz, 3H),
0.75 ppm (d, J=6.4 Hz, 3H).
Student’s t-test.
Bt
Bt
1
KRI ¼
ð1Þ
2
Association and dissociation rates for unlabeled ligands were de-
termined by nonlinear regression analysis of the competition asso-
ciation data as described by Motulsky and Mahan [Eq. (2)], for
which X is the time [min], Y is the specific binding [disintegrations
per minute (DPM)], k1 is kon [mÀ1 minÀ1] of [3H]INCB3344 predeter-
mined in association experiments, k2 is koff [minÀ1] 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].
Pharmacology
Chemicals and reagents. 125I-CCL2 (2200 CimmolÀ1) was purchased
from PerkinElmer (Waltham, MA). INCB3344 was synthesized as pre-
viously described.[18] [3H]INCB3344 (specific activity 32 CimmolÀ1
)
KA ¼ k1½LŠ Á 10À9 þ k2
was custom-labeled by Vitrax (Placentia, CA) for which a dehydro-
genated precursor of INCB3344 was provided. Tango CCR2-bla
U2OS cells stably expressing human CCR2 were obtained from Invi-
trogen (Carlsbad, CA).
KB ¼ k3½IŠ Á 10À9 þ k4
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
S ¼ ðKA À KBÞ þ 4 Á k1 Á k3 Á L Á I Á 10À18
KF ¼ 0:5ðKA þ KB þ SÞ
Cell culture and membrane preparation. U2OS cells stably express-
ing the human CCR2 receptor (Invitrogen, Carlsbad, CA) were cul-
tured in McCoys5a medium supplemented with 10% fetal calf
serum, 2 mm glutamine, 0.1 mm nonessential amino acids (NEAA),
25 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
1 mm sodium pyruvate, 100 IUmLÀ1 penicillin, 100 mgmLÀ1 strepto-
mycin, 100 mgmLÀ1 G418, 50 mgmLÀ1 hygromycin, and 125 mgmLÀ1
zeocin in a humidified atmosphere at 378C and 5% CO2. Cell cul-
ture and membrane preparation were performed as previously de-
scribed.[14]
ð2Þ
KS ¼ 0:5ðKA þ KB À SÞ
Bmax Á k1 Á L Á 10À9
Q ¼
KF À KS
k4 Á ðKF À KSÞ k4 À KF
k4 À KS
ðÀK ÁXÞ À
e
ðÀK ÁXÞÞ
F
S
Y ¼ Q Á ð
þ
e
KF Á KS
KF
KS
Fixing these parameters into Equation (3) allows for the following
parameters to be calculated: k3 is kon [mÀ1 minÀ1] of the unlabeled
ligand, and k4 is koff [minÀ1] of the unlabeled ligand. The association
and dissociation rates were used to calculate the “kinetic KD”, and
the tres was calculated according to Equation (4).
125I-CCL2 displacement assay. Binding assays were performed as pre-
viously described.[14] Briefly, the U2OS-CCR2 membrane (15 mg) was
incubated for 150 min with 0.1 nm 125I-CCL2 in the absence or pres-
ence of competing ligand at 378C.
koff
kon
ð3Þ
ð4Þ
KD
¼
¼
[3H]INCB3344 dual point competition association assay. Kinetic rate
index (KRI) values of unlabeled ligands were determined by using
the dual-point competition association assay as previously de-
scribed.[6] Briefly, the U2OS-CCR2 membrane (10 mg) was incubated
for 50 min (t1) or 240 min (t2) with 1.8 nm [3H]INCB3344 in the ab-
sence or presence of unlabeled ligands at 258C. The amount of
radioligand bound to the receptor was measured after co-incuba-
tion of the unlabeled ligands at onefold their respective Ki value in
the 125I-CCL2 displacement assay. KRI values of unlabeled ligands
were calculated by using Equation (1), as show below in the sec-
tion entitled Data Analysis.
1
koff
tres
Acknowledgements
This study was supported financially by the Top Institute Pharma
(The Netherlands), project number D1-301. The authors thank Dr.
Julien Louvel (Leiden University) for helpful comments and sug-
gestions on the synthesis and for his input on analytical data
analysis. The authors also acknowledge Prof. Dr. Martine Smit
(Vrije Universiteit, The Netherlands) for helpful comments and
suggestions.
[3H]INCB3344 competition association assay. The kinetic parameters
of unlabeled ligands were determined by using the competition
association assay described earlier by our group.[6] Briefly, at differ-
ent time points, U2OS-CCR2 membrane (10 mg) was added to
1.8 nm [3H]INCB3344 in a total volume of 100 mL of assay buffer in
the absence or presence of competing ligand at 258C. Kinetic pa-
rameters of unlabeled ligands were calculated by using Equa-
tion (2), as shown below in the section entitled Data Analysis.
Keywords: antagonists · cytokines · kinetics · structure–affinity
relationships · structure–kinetics relationships
Data analysis. All experiments were analyzed by using the nonlin-
ear regression curve fitting program Prism 5 (GraphPad, San Diego,
CA). For radioligand displacement data, Ki values were calculated
from IC50 values by using the Cheng–Prusoff equation.[19] KRI values
were calculated by dividing the specific radioligand binding mea-
sured at t1 (Bt ) by its binding at t2 (Bt ) in the presence of unlabeled
competing ligand according to Equation (1). Data presented are
the mean ÆSEM of at least three experiments performed in dupli-
cate. Statistical analysis was performed with a two-tailed unpaired
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Received: February 7, 2015
Revised: March 21, 2015
Published online on June 1, 2015
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