Structure-Affinity Relationships and Structure-Kinetic Relationships of 1,2-Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.7b00861

We report on the synthesis and biological evaluation of a series of 1,2-diarylimidazol-4-carboxamide derivatives developed as CB₁ receptor antagonists. These were evaluated in a radioligand displacement binding assay, a [³⁵S]GTPγS binding assay, and in a competition association assay that enables the relatively fast kinetic screening of multiple compounds. The compounds show high affinities and a diverse range of kinetic profiles at the CB₁ receptor and their structure-kinetic relationships (SKRs) were established. Using the recently resolved hCB₁ receptor crystal structures, we also performed a modeling study that sheds light on the crucial interactions for both the affinity and dissociation kinetics of this family of ligands. We provide evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting new hCB₁ receptor antagonists in the early phases of drug discovery.

Full text of DOI:10.1021/acs.jmedchem.7b00861

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Article
Structure-affinity Relationships and Structure-kinetic
Relationships of 1,2-Diarylimidazol-4-carboxamide
Derivatives as Human Cannabinoid 1 Receptor Antagonists
Lizi Xia, Henk de Vries, Eelke B. Lenselink, Julien Louvel, Michael J. Waring,
Leifeng Cheng, Sara Pahlén, Maria J Petersson, Peter Schell, Roine I.
Olsson, Laura H. Heitman, Robert J Sheppard, and Adriaan P. IJzerman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00861 • Publication Date (Web): 07 Nov 2017
Downloaded from http://pubs.acs.org on November 7, 2017
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Structureꢀaffinity Relationships and Structureꢀ
kinetic Relationships of 1,2ꢀDiarylimidazolꢀ4ꢀ
carboxamide Derivatives as Human Cannabinoid 1
Receptor Antagonists
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Lizi Xia , Henk de Vries , Eelke B. Lenselink, Julien Louvel† , Michael J. Waring , Leifeng
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a
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Cheng , Sara Pahlén , Maria J. Petersson , Peter Schell , Roine I. Olsson , Laura H.
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2*
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Heitman , Robert J. Sheppard , and Adriaan P. IJzerman
1
Division of Medicinal Chemistry, LACDR, Leiden University, the Netherlands
2
Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United
Kingdom
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Medicinal Chemistry, Cardiovascular and Metabolic Diseases, IMED Biotech Unit,
AstraZeneca, Gothenburg, Sweden
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a
Current address: Northern Institute for Cancer Research, School of Chemistry, Bedson
Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom
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Current address: Caltiora Consulting, Gothenburg, Sweden.
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Current address: Global Product & Portfolio Strategy, Business Development Operations,
AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden
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Current address: Respiratory, Inflammation & Autoimmunity, IMED Biotech Unit,
AstraZeneca, Gothenburg, Sweden
†
We wish to dedicate this study to the memory of Dr, Julien Louvel, who passed away on Nov.
5, 2017.
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ABSTRACT
We report on the synthesis and biological evaluation of a series of 1,2ꢀdiarylimidazolꢀ4ꢀ
carboxamide derivatives developed as CB receptor antagonists. These were evaluated in a
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radioligand displacement binding assay, a [ S]GTPγS binding assay, and in a competition
association assay that enables the relatively fast kinetic screening of multiple compounds. The
compounds show high affinities and a diverse range of kinetic profiles at the CB receptor, and
1
their structureꢀkinetic relationships (SKRs) were established. Using the recently resolved hCB1
receptor crystal structures, we also performed a modelling study that sheds light on the crucial
interactions for both the affinity and dissociation kinetics of this family of ligands. We provide
evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting
new hCB receptor antagonists in the early phases of drug discovery.
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INTRODUCTION
Within the endocannabinoid system (ECS) two human cannabinoid receptor subtypes have been
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identified: the human CB (hCB ) receptor and the human CB (hCB ) receptor. They are
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members of the rhodopsinꢀlike class A Gꢀproteinꢀcoupled receptors (GPCRs), and are primarily
activated by endogenous cannabinoids (endocannabinoids, ECs), including anandamide (or Nꢀ
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, 2
arachidonylethanolamine, AEA) and 2ꢀarachidonoylglycerol (2ꢀAG).
The hCB and hCB
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receptors show 44% overall sequence homology, and display different pharmacological profiles.
The hCB receptor is present in the central nervous system (CNS) and is widely distributed in the
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, 4
peripheral nervous system (PNS) and peripheral tissues,
including heart, liver, lung,
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gastrointestinal tract, pancreas and adipose tissue. The presence of the hCB receptor within
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both the CNS and PNS mediates neurotransmitter release and controls various cognitive, motor,
emotional and sensory functions. Furthermore, activation in the peripheral tissues contributes to
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ꢀ9
energy balance and metabolic processes.
The broad presence of the hCB receptor in a variety of complex physiological systems
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provides numerous opportunities for therapeutic intervention. In the particular case of obesity,
the ECS, including the hCB receptor, is overactive with increased levels of endocannabinoids in
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plasma, both in central and peripheral tissues. Therefore, blockade of the hCB has been
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explored for the treatment of obesity. With this in mind, rimonabant (SR141716A, Figure 1a), a
hCB receptor inverse agonist, was developed by SanofiꢀAventis and introduced in Europe in
1
2006. However, it was quickly withdrawn from the market due to unacceptable psychiatric side
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effects.
Many other hCB receptor antagonists entered into clinical trials, such as taranabant
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(
MKꢀ0364, Figure 1b) and otenabant (CP945598, Figure 1c). However, they were not
developed further due to similar psychiatric side effects, despite their diverse chemical
structures.
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In order to avoid the CNS side effects, peripherally acting hCB receptor antagonists with
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physicochemical features that reduce brain penetration have been developed. Another approach
has been the development of hCB receptor neutral antagonists, because it has been postulated
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7ꢀ19
that the CNS side effects of rimonabant were due to its inverse agonism.
Drug target binding kinetic parameters are receiving increasing attention, alongside classical
affinity (K ) and potency (IC ) values, as has been discussed for several other class A GPCRs. In
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particular the receptorꢀligand residence time (RT) is emerging as an additional parameter to
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assess the therapeutic potential of drug candidates with respect to drug efficacy and safety.
In
the research field of GPCRs, a number of structureꢀkinetic relationship (SKR) studies have been
published, and the results suggest that the strategic combination of SKR with classic structureꢀ
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affinity relationships (SAR) can improve the resulting decision process.
By doing so, ligandꢀ
receptor interactions can be better understood, as together they not only comprise the equilibrium
state of a ligandꢀreceptor interaction but also its metastable intermediates and/or transition
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states. The binding kinetics driven drug discovery approach for the hCB receptor has been
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validated in some aspects already by its application in the development of allosteric modulators
28, 29
of the hCB receptor.
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In the current study we report the synthesis and evaluation of 1,2ꢀdiarylimidazolꢀ4ꢀ
carboxamide derivatives (Figure 1d), as human CB receptor antagonists with more polar
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characteristics than rimonabant.
Together with rimonabant they were evaluated in a
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radioligand displacement assay, a [ S]GTPγS binding assay, and a dualꢀpoint competition
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association assay that enables the relatively fast kinetic screening of compounds. Selected
compounds were progressed to a full competition association assay. The compounds show high
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affinities and a diverse range of kinetic profiles at the hCB receptor, which allowed their
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structureꢀkinetic relationships (SKRs) to be established. Their putative binding mode was
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analyzed using the recently resolved crystal structures of the hCB receptor,
shedding light
1
on key structural features of the receptor binding site that are involved in ligand recognition and
dissociation. Thus we provide evidence that, in additional to affinity, knowledge of binding
kinetics is useful for selecting new hCB receptor antagonists in the early phases of drug
1
discovery.
RESULTS AND DISCUSSION
Chemistry. The synthesis of the 1,2ꢀdiarylimidazolꢀ4ꢀcarboxamide scaffold commenced from
commercially available 4ꢀ(benzyloxy)aniline 1, which was converted to the 2,4ꢀ
dichlorobenzamidine 2 (Scheme 1). After a oneꢀpot condensation and cyclization sequence, the
coreꢀimidazole 3 was obtained. Afterwards, either saponification of the ethyl ester or acidic
hydrolysis of the benzyl ether of 3 led to intermediates 4 and 5, respectively. Subsequently,
Mitsunobu reaction on intermediate 5 yielded monoꢀ and triꢀfluoropropyl ether derivatives 6a
and 6b. After saponification of the ethyl esters of 6a and 6b, the corresponding carboxylic acids
(
7a and 7b) were transformed to acid chlorides and reacted with piperidinꢀ1ꢀamine to yield the
corresponding amides (8a and 8b). Alternatively, the rest of the series was produced from
intermediate 4 by first introducing the piperidinꢀ1ꢀamide. Lewis acidꢀcatalyzed cleavage of
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benzyl ether 9 followed by substitution of the released alcohol 10 with various alkyl halides gave
the corresponding ethers 11aꢀ11h, completing the “left arm” series of antagonists (Table 1).
The synthesis of the “right arm” series of antagonists was started from intermediate 4 (Scheme
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2
). Using various amines and the aforementioned acid chloride introduction/ amide formation
sequence, amides 12aꢀ12h were obtained, as well as racemic (±) 20. Deprotection of the
aromatic alcohol on 12aꢀ12h and subsequent sulfonylation using 3,3,3ꢀtrifluoropropaneꢀ1ꢀ
sulfonylchloride gave compounds 14aꢀ14h. After deprotection of racemic (±) 20 however, it was
found that direct substitution was not possible, therefore a series of protecting group
manipulations was executed on (±) 21 to end up with (±) 22. Towards (±) 25, (±) 20 was first diꢀ
methylated and subsequently debenzylated and sulfonylated giving (±) 25. Exploring alternative
synthesis routes, compound 19 was synthesized, with a few extra steps, by first esterifying 4 with
2,2,2ꢀtrichloroethanol, followed by deprotection of the aromatic alcohol. Sulfonylation of the
released alcohol, saponification of the trichloroethylester, acid chloride formation and
subsequent amide formation gave 19. To obtain trifluoromethylpyridine derivative 28,
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conventional methods as described for the industrial production of rimonabant were applied,
starting with the direct amidation of ethyl ether 3 followed by debenzylation and sulfonylation.
Biology. All 1,2ꢀdiarylimidazolꢀ4ꢀcarboxamide derivatives were evaluated as antagonists in an
3
5
in vitro [ S]GTPγS binding assay on HEKꢀ293 cells membrane fractions overexpressing the
human CB receptor. We also determined the functional activity of nine representative
1
antagonists on the human CB receptor. The data in Table 1 and S1 shows that all compounds
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tested had higher functional activity for the human CB receptor over the human CB receptor,
1
2
with approximately 110 to 570ꢀfold selectivity.
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Likewise they were also tested in a [ H]CP55940 radioligand displacement assay on membrane
fractions of CHO cells overexpressing the recombinant human CB receptor. These results are
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reported in Tables 1 and 2. We found that, although using different cellular background and
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assay systems, there is a significant correlation (r = 0.49, P = 0.0001) between the affinity (pK )
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values from the radioligand binding assay and the potencies (pIC ) determined in the
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[
S]GTPγS binding assay (Figure 2). We subsequently determined the binding kinetics of the
,2ꢀdiarylimidazolꢀ4ꢀcarboxamide derivatives in a competition association assay with
1
3
[ H]CP55940 as the probe after a validation step.
3
[
H]CP55940 binding kinetic assay. Receptor association and dissociation rate constants of
3
[
H]CP55940 were directly determined in classic radioligand association and dissociation
o
3
experiments at 30 C. The binding of [ H]CP55940 approached equilibrium after approximately
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ꢀ1 ꢀ1
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5 min (Figure 3), yielding a kon (k ) value of (1.4 ± 0.08) x 10 M s . Binding of the
1
radioligand was reversible after the addition of rimonabant (10 ꢁM), although the dissociation
was rather slow. Even 240 min after the addition of rimonabant residual receptor binding (~15%)
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of [ H]CP55940 was observed. The dissociation rate constant, k (k ), of [ H]CP55940 from the
off
2
ꢀ4
ꢀ1
3
hCB receptor was (1.5 ± 0.2) x 10 s . The kinetic K value (k /k ) of [ H]CP55940 was 0.12
1
D
off on
3
±
0.03 nM (Table 3). The residence time (RT) of [ H]CP55940 was calculated as 114 ± 16 min.
3
Validation of the [ H]CP55940 competition association assay for human CB receptor. With
1
3
the k (k ) and k (k ) values of [ H]CP55940 binding established from classical association
on
1
off
2
and dissociation experiments, k (k ) and k (k ) of unlabeled CP55940 were determined by
on
3
off
4
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fitting the values based on the mathematical model as described in the experimental. In this
validation experiment we tested three different concentrations of unlabeled CP55940,
corresponding to IC , IC and IC75 (Figure 4a). Values for kon and koff determined by this
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ꢀ1 ꢀ1
ꢀ4 ꢀ1
competition association method were (1.2 ± 0.1) x 10 M ·s and (6.5 ± 1.0) x 10 s ,
respectively. The k value was in good agreement with the k (k ) value determined in the
on
on
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classical association experiment (Table 3). The koff value obtained by this method was also
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similar to that found in the classical kinetic dissociation experiments with [ H]CP55940, with
just a fourꢀfold difference between the values (Table 3). In order to confirm the robustness of the
assay with unlabeled human CB receptor antagonists, an experiment was performed using
1
rimonabant (Figure 4b, Table 4). The kon and koff values determined by this competition
5
ꢀ1 ꢀ1
ꢀ3 ꢀ1
association method were (2.3 ± 0.3) x 10 M ·s and (1.4 ± 0.2) x 10 s , respectively,
demonstrating that rimonabant behaves as a short residence time antagonist (14 ± 2.0 min), in
3
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good agreement with findings reported earlier.
Screening of hCB receptor antagonists using the dualꢀpoint competition association assay. The
1
competition association assay described above is quite laborious and timeꢀconsuming. Therefore,
a soꢀcalled “dualꢀpoint competition association assay” for the hCB receptor was developed,
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according to the concept that we had previously established for the adenosine A receptor. To
1
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this end, [ H]CP55940 and unlabeled antagonists were coꢀincubated at concentrations equal to,
3
or 2 to 3ꢀfold higher than, their K /IC values which had been determined in the [ H]CP55940
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displacement assay. The soꢀcalled kinetic rate index (KRI) was calculated by dividing the
specific radioligand binding at 30 min (t ) by the binding at 240 min (t ). Antagonists with a KRI
1
2
3
value larger than 1 indicate a slower dissociation rate, and thus a longer RT, than [ H]CP55940,
and vice versa. Furthermore, it was observed that the KRI values of the hCB receptor
1
antagonists had no obvious correlation with their affinities (Figure 5a).
Structure−Affinity Relationships (SARs) versus Structure−Kinetic Relationships (SKRs).
The 1,2ꢀdiarylimidazolꢀ4ꢀcarboxamide derivatives are rimonabant bioisosteres, in which the 2,4ꢀ
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dichlorophenyl, amide, aryl, and methyl moieties are maintained on an alternative heterocyclic
diazo core (Figure 1a and 1d). The derivatives included in this study differ in their substituents at
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the R and R positions, which are at the “left” and “right” arms of the scaffold, respectively
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(Figure 1d).
We were conscious that compound polarity may influence the activity parameters being
studied, so polarity was determined by both calculated and experimental methods. Calculated
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methods included Polar Surface Area (PSA), ACDlogD7.4 with pK correction and
a
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AZlogD7.4, which were supplemented with experimentally determined LogD values. A PSA of
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0 Å has been described as a threshold value below which penetration of the blood–brain barrier
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is more likely, and thus serves as an indicator for potential to have CNS activity. The calculated
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PSA values (Tables S2 and S3) of most of the compounds in this study were above 90 Å ,
suggesting that they would have low bloodꢀbrain barrier penetration, and be better suited for
peripheral antagonism of the hCB receptor. We observed that neither affinities nor KRI values
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of the CB receptor antagonists in this study had any obvious linear correlation with their
1
lipophilicity or PSA values (Figures S1 and S2).
“
Left arm” optimization. Fixing the right arm as a piperidine moiety, as in rimonabant, various
ethers with different carbon chain lengths were introduced on the left arm (Table 1). Extension of
the trifluoromethylalkyl chain from three carbons (8a, 1.26 nM) to four atoms (11a, 0.32 nM)
increased affinity by about fourꢀfold. Reducing the level of fluorination on the terminal carbon of
the linear ether sideꢀchain from three atoms (8a, 1.26 nM) to one atom (8b, 0.34 nM) also
increased the affinity. By contrast, the analogue possessing a benzyl substituent on the left arm
(9, 6.28 nM) displayed the weakest affinity of the analogues studied. The aforementioned
modifications did not seem to have a drastic effect on KRI, with all compounds giving values
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around unity (0.80 to 1.09). As part of a strategy to increase PSA a sulfonylꢀcontaining sideꢀ
chain was introduced. The ligand bearing an nꢀpropylꢀsulfonyl moiety (11b) displayed a good
affinity of 0.28 nM and a rather low KRI value of 0.59. Monoꢀfluorinating the terminal position
led to no change in affinity (11c, 0.32 nM). In contrast to the ether substituents, trifluorination
resulted in an almost threeꢀfold increase (11d, 0.11 nM) relative to the monoꢀfluoro analogue. A
slight increase in affinity was observed when the linear sulfonyl sideꢀchain was extended from
three carbon atoms (11b, 0.28 nM) to four (11e, 0.18 nM). Combination of this chain length with
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trifluoroꢀsubstitution, to give the side chain found in the CB receptor agonist (ꢀ)ꢀ(R)ꢀ3ꢀ(2ꢀ
1
43, 44
hydroxymethylindanylꢀ4ꢀoxy)phenylꢀ4,4,4ꢀtrifluoroꢀ1ꢀsulfonate (BAY 38ꢀ7271),
led to a
very potent antagonist of the human CB receptor (11f, 62 pM). Branching the chain from nꢀ
1
butyl to iꢀpentyl did not change the affinity (11g vs. 11e), while introducing an additional methyl
group led to a decrease in affinity (11h, tꢀhex chain, 0.60 nM). None of these ligands had a KRI
value higher than 1, indicating their dissociation from the hCB receptor was faster than
1
CP55940. The analogue with the lowest KRI value (11b, 0.59) was selected for fullꢀcurve
measurement (Figure 6, Table 4). As expected, its residence time (78 min) was shorter than that
of CP55940 (114 min, see above) (Table 4). This result also serves as evidence that a KRI value
seems to reliably reflect the corresponding dissociation rate constant.
All the linear sideꢀchain antagonists had high affinities in the nanomolar to subꢀnanomolar
range, with 11f (60 pM) as the most potent derivative. However, from the perspective of drugꢀ
target kinetic studies, despite giving a range of KRIs (0.59ꢀ1.09), none of these antagonists
showed a KRI value significantly higher than 1, suggesting that none had longer residence times
than CP55940.
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“
Right arm” optimization. To explore the “right arm” of the 1,2ꢀdiarylimidazolꢀ4ꢀcarboxamides,
we chose to fix the “left arm” as a trifluoropropyl sulfonyl moiety (11d), since this group
delivered high affinity (0.11 nM) and demonstrated a residence time similar to CP55940 (KRI =
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1.02, Table 1). Introducing a hydroxyl at the 3ꢀposition of the piperidine ring yielded a ligand
with lower affinity and KRI value (14a, K = 0.27 nM, KRI = 0.71) than 11d (Table 2).
i
Efforts then focused on a series of ligands bearing cyclohexyl substituents instead of a
piperidine. A carbocyclic analogue of 14a, bearing a transꢀhydroxyl on the 3ꢀposition of the
cyclohexyl ring 14b (racemic), delivered an approximately threeꢀfold improvement in affinity
and a slightly larger KRI value relative to the piperidine 14a (Table 2). Moving the hydroxyl to
the 4ꢀposition gave 4ꢀhydroxycyclohexyl analogue (19), as a mixture of cis and trans
diastereoisomers in a ratio of 0.3:1, and resulted in an approximately fourꢀfold reduction in
affinity (0.37 nM), whilst the KRI was unchanged (0.88); having a mixture does not allow any
further conclusions, though. Interestingly, the cisꢀ and transꢀ2ꢀhydroxycyclohexyl antagonists
(14d and 14c, respectively) showed a substantial 10ꢀfold difference in affinity, while their KRI
values were quite similar. The more potent cisꢀisomer (14d, (+)) displayed an affinity of 27 pM
and a KRI value close to unity. Switching the 2ꢀsubstituent of the cyclohexane ring to an amine
was detrimental, resulting in ligands with lower affinities. However, it is of note that the
unsubstituted cisꢀamino group (22, (±), 0.52 nM) was less detrimental to affinity than a cisꢀ
dimethylamino substituent (25, (±), 3.3 nM), whilst the dissociation rates were very similar, as
judged by their KRI values (Table 2). At this stage, on the basis of affinity alone, 14d with an
affinity of 27 pM seems an even better lead than 11f with an affinity of 62 pM.
Last but not least, we found that by introducing an aromatic moiety, the compounds retain
affinity in the subꢀnanomolar range and, more importantly, their kinetic profiles were rather
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diverse. The analogue which bears a 4ꢀtrifluoromethoxyphenyl substituent (14e) showed high
affinity (0.22 nM) and its KRI value was one of the highest measured (Table 2). Introduction of a
pyridine moiety was then studied. The 3ꢀpyridyl analogues 14f and 14g, bearing a 6ꢀfluoro or
trifluoromethyl group, respectively, showed similar affinities (0.13 nM vs. 0.31 nM,
respectively), although the latter had a much higher KRI value (1.12 vs. 0.70, respectively). This
effect on KRI was increased further when the position of the nitrogen atom in the ring was
switched to give the 5ꢀsubstituted 2ꢀpyridyl analogue (28, KRI = 1.39), which displayed the
highest KRI value of all the compounds presented in this study. Finally, defluorinating this latter
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compound did not change the affinity, but gave rise to a marked reduction in KRI (14h, K = 0.14
i
nM, KRI = 0.92).
The compounds with high (28) and low (11b and 14f) KRI values were tested in a full
competition association assay to determine their association and dissociation rate constants
(Figure 6 and Table 4). According to the full curves, the compound with KRI > 1 (28) displayed
an “overshoot” in the competition association curve, indicating its slow dissociation and yielding
the longer residence time of 260 min, as compared to 114 min of the radioligand. By contrast, the
compounds with KRI < 1 produced gradually ascending curves, suggesting faster dissociation
and consequently shorter residence times of 78 min (11b) and 62 min (14f) (Figure 6, Table 4).
Additionally, we determined their affinities on the human CB receptor. From Table 1 and S1 it
2
shows that they all had higher affinity for the human CB receptor, where approximately 12 to
1
1
25ꢀfold selectivity over human CB receptors was observed.
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Functional assays. As mentioned above, the antagonism in the [ S]GTPγS binding assay
3
compares quite well with the affinities derived from the [ H]CP55940 displacement studies
(
Figure 2), while the KRI values of the compounds did not show any meaningful correlation with
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the pIC values from the GTPγS binding assay (Figure 5b). Since 28 showed slow dissociation,
5
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3
5
we decided to study this compound further in a more elaborate [ S]GTPγS binding experiment,
in which its functional activity in the inhibition of CP55940 action was characterized and
compared with rimonabant. Pretreatment of CHOK1 hCB receptor membranes with rimonabant
1
for 1h, prior to stimulation by the CB receptor agonist CP55940 for 30 min, induced
1
surmountable antagonism (a rightward shift of the agonist curve with little suppression of the
4
5
maximum effect) as reported before. In the case of 28 insurmountable antagonism was
observed; the agonist concentrationꢀeffect curve was shifted to the right with a concomitant
decrease (~50%) in its maximal response (Figure 7). In both cases inverse agonism by the
compounds alone (in the absence of CP55940) was also apparent (negative values at Yꢀaxis in
Figure 7).
Computational studies. Finally, we investigated the ligandꢀreceptor interactions using the
recently disclosed Xꢀray crystal structure of hCB in complex with 29 [4ꢀ(4ꢀ(1ꢀ(2,4ꢀ
1
dichlorophenyl)ꢀ4ꢀmethylꢀ3ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)phenyl)butꢀ3ꢀynyl
32
nitrate, AM6538], crystal structure code: PDB:5TGZ). By docking 28 into the hCB receptor it
1
can be seen that, like 29, it lies quite deep in the binding pocket of hCB in the docked pose,
1
6
.48
immediately above the conserved Trp356
(Figures 8a and b). The main scaffold of the
imidazole core and the 2,4ꢀdichlorophenyl ring form a πꢀπ interaction with the side chains of
Nꢀterm
2.57
Phe102
and Phe170
respectively (Figure 8b). Unsurprisingly, and consistent with the
SAR reported in Table 1, the “left arm” of our ligand docks into the same place as “Arm 2” of 29
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in the crystal structure. This “left arm” extends into a long, narrow, and highly lipophilic channel
formed by helices III, V, VI and ECL2 (Figure 8a). By contrast, the “right arm” of our ligands,
which resemble “Arm 3” of 29 dock into an open cavity formed by various hydrophobic amino
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acid residues, irrespective of whether a cyclohexyl, piperidine, or pyridine moiety is present. In
the case of a pyridine moiety (14eꢀ14h and 28), the crystal structure suggests that there may be a
2.65
πꢀπ stacking interaction with His178 . Further support for the docked pose of 28 comes from
3
4
the higher resolution xꢀray structure of taranabant bound to hCB1 (PDB: 5U09), since both
compounds share a trifluoromethylpyridine moiety on their “right arm”.
Using the crystal structure of the hCB ꢀ29 complex, we performed WaterMap calculations to
1
try and understand the differences in residence times observed for the ligands studied, with the
46ꢀ48
hypothesis that unfavorable hydration might provide an explanation.
We focused on the
pyridine ring substituents on the “right arm”, and ligands 14f and 28 in particular, because of
their similar binding affinities but differing residence times. The smaller of the two ligands (14f,
ꢀF substitution, relatively short RT) was docked into the hCB receptor, and a WaterMap was
1
calculated for the complex. Around the –F substituent we found unstable water molecules (41,
4
9
6
9, 72, 81 and 88 in Figure 8c ); these water molecules are coined unhappy waters. By contrast,
ligand 28 was able to displace these water molecules with its larger ꢀCF substituent, a process
3
which might raise the energy of the transition state for dissociation. We postulate that this
destabilization of the transition state may contribute to the prolonged residence time observed
with this compound.
CONCLUSIONS
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We have demonstrated that, in addition to affinity, knowledge of binding kinetics is useful for
selecting and developing new hCB receptor antagonists in the early phases of drug discovery. In
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the specific case of the hCB receptor, a long residence time compound may be beneficial for a
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peripherally selective antagonist. We explored SAR and SKR parameters in a series of 1,2ꢀ
diarylimidazolꢀ4ꢀcarboxamide derivatives by examining the influence of substitutions at both
“arms” of the molecules.
By introducing more polar linear sulfonyl side chains on the “left arm”, affinity could be
modulated, however the KRI values indicative for the compounds’ kinetic properties were less
than or similar to CP55940. Substitution of the “right arm” maintained or increased affinity, and
with the introduction of an aromatic ring system KRI values >1 were obtained. With a residence
time of 260 min, which is substantially longer than CP55940 (114 min.) or rimonabant (14 min.),
4ꢀ(2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ((5ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)carbamoyl)ꢀ1Hꢀ
imidazolꢀ1ꢀyl)phenyl 3,3,3ꢀtrifluoropropaneꢀ1ꢀsulfonate (28) stood out from the ligands studied.
This slowly dissociating hCB receptor antagonist also showed insurmountability in a functional
1
GTPγS binding assay. Using the recently resolved hCB crystal structures we analyzed the
1
putative interactions of 28 with the receptor, from which we speculate that displacement of
‘unhappy’ water molecules may provide a plausible explanation for its slow dissociation.
Therefore, compound 28, or derivatives with similar characteristics, may be a useful tool to test
whether prolonged blockade of the (peripheral) hCB receptor has a beneficial effect on CB1
1
receptor related disorders, such as obesity.
EXPERIMENTAL SECTION
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Chemistry. All solvents and reagents were purchased from commercial sources and were of
analytical grade. Demineralized water is simply referred to as water or H O, as was used in all
2
cases unless stated otherwise (i.e., brine). Thinꢀlayer chromatography (TLC) was routinely
consulted to monitor the progress of reactions, using aluminumꢀcoated Merck silica gel F254
plates. Purification was performed on a semiꢀpreparative high performance liquid
chromatography (HPLC) with a mass triggered fraction collector, Shimadzu QP 8000 single
quadrupole mass spectrometer equipped with 19 x 100 mm C8 column. The mobile phase used
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was, if nothing else is stated, acetonitrile and buffer (aqueous NH OAc (0.1 M) : acetonitrile 95 :
4
5
). For isolation of isomers, a Kromasil CN E9344 (250 x 20 mm i.d.) column was used. A
mixture of heptane/ethyl acetate/diethylamine 95 : 5 : 0.1 was used as mobile phase (1 mL/min).
Fraction collection was guided using a UVꢀdetector (330 nm). Analytical purity of the final
products was determined by Waters Acquity Iꢀclass ultraꢀperformance liquid chromatography
(
UPLC) consisting of a binary solvent system, ultraꢀviolet (UV) photoꢀdiode array (PDA)
detector, column temperature control manager and sample manager modules, coupled with inꢀ
line and mass spectrometry detection. The sample was injected onto, and separated by, a Waters
Acquity BEH (C18) 1.7 mm (150x3 mm) UPLC column maintained at 40°C and eluted with
0
.1% ammonium hydroxide in water (A) and acetonitrile (B) at a flow rate of 1 mL/min, using a
linear gradient. Initial conditions started at 3% B, which was increased to 97% over 1.3 min,
maintained for 0.2 min before returning to initial conditions over 0.2 min prior to the next
injection. Eluent containing UPLCꢀseparated analytes then flowed via the UV PDA detector
scanning between 220ꢀ320 nm wavelengths at a resolution of 1.2 nm sampling at 40 points/s,
into a Waters SQD single quadrupole mass spectrometer (MS) fitted with an electrospray source.
All MS analyses were acquired for a total run time of 2 min, with mass scanning from 100ꢀ1000
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u in both positive and negative ion modes alternately, using electrospray ionization (ESI).
Typical MS settings included capillary voltage ꢀ 1kV, cone voltage ꢀ 25V, source temperature ꢀ
150°C, and desolvation temperature ꢀ 350°C. The data were acquired via a PC running
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MassLynx v4.1 in open access mode and processed and reported via OpenLynx software
application. For each sample the purity is determined by integration of the UV absorption
chromatogram. All final compounds show a single peak and are at least 95% pure.
1
H NMR measurements were performed on either a Varian Mercury 300 or a Varian Inova 500,
1
operating at H frequencies of 300 and 500 MHz respectively at ambient temperature. Chemical
shifts are reported in parts per million (ppm), are designated by δ, and are downfield to the
internal standard tetramethylsilane (TMS) in CDCl . Coupling constants are reported in Hz and
3
are designated as J. Highꢀresolution mass spectra were recorded on either a Micromass ZQ
single quadrupole or a Micromass LCZ single quadrupole mass spectrometer both equipped with
a pneumatically assisted electrospray interface (LCꢀMS). Melting points were determined on a
Reichert melting point microscope and are uncorrected.
Nꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2,4ꢀdichlorobenzamidine (2). Compound 1 (5.0 g, 21.2 mmol) was
added dropwise to a solution of ethyl magnesium bromide (44.5 mL, 1 M in THF, 44.5 mmol) in
dry THF (25 mL) under a nitrogen atmosphere. After stirring for 20 minutes a solution of 2,4ꢀ
dichlorobenzonitrile (3.65 g, 21.2 mmol) in THF (25 mL) was added. The reaction mixture was
stirred for 20 hours at r.t.. Water (50 mL) was carefully added. Extraction with EtOAc (2 x 100
mL), drying (Na SO ), filtration and evaporation to dryness afforded the crude title compound
2
4
(7.7 g, 98%).
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Ethyl
1ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀcarboxylate
(
3). To a solution of compound 2 (6.88 g, 18.5 mmol) in THF (50 mL) was added potassium
carbonate (2.56 g, 18.5 mmol) and the suspension was stirred for 10 minutes. Ethylꢀ3ꢀbromoꢀ2ꢀ
oxobutanoate (4.65 g, 22.2 mmol) was added dropwise over 1 hour, and the mixture was stirred
for 66 hours at r.t.. The solution was filtered and evaporated to dryness. The residue was
dissolved in AcOH and refluxed for 1 hour. The mixture was cooled to r.t., water (100 mL)
added and the product extracted with EtOAc (2 x 200 mL). The combined organic phases were
washed with saturated aqueous sodium hydrogen carbonate, dried (Na SO ), filtered, and
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concentrated in vacuo. Flash chromatography (silica, 30ꢀ40% EtOAc in hexane) afforded the title
1
compound (5.75 g, 65%) as a pale yellow solid. H NMR (CDCl ): δ 7.50ꢀ7.20 (m, 8H), 7.10ꢀ
3
6.90 (m, 4H), 5.10 (s, 2H), 4.50 (q, 2H), 2.5 (s, 3H), 1.5 (t, 3H).
1
ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀcarboxylic acid (4).
To a suspension of compound 3 (3.62 g, 7.5 mmol) in MeOH (60 mL) was added potassium
hydroxide (4.05 g, 72 mmol) in water (20 mL), and the reaction mixture heated to reflux. After 2
h the mixture was cooled to r.t., acidified to pH~2 with HCl (1 M) and extracted with ethyl
acetate (2 x 200 mL). The combined organic phases were dried (Na SO ), filtered and
2
4
concentrated in vacuo to give the crude title compound (3.38 g, 99%).
Ethyl 2ꢀ(2,4ꢀdichlorophenyl)ꢀ1ꢀ(4ꢀhydroxyphenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀcarboxylate (5).
compound 3 (4.82 g, 10 mmol) was dissolved in HBr (33% in AcOH, 80 mL) and stirred
overnight at r.t. with exclusion of light. The solvents were evaporated and the residue coꢀ
evaporated with EtOH. The residue was dissolved in EtOH, HCl (4 M in dioxane, 5 mL) and
MgSO were added, and the resulting mixture heated under reflux for 2.5 h. The reaction mixture
4
was cooled to r.t., filtered, and concentrated in vacuo. The residue was dissolved in EtOAc and
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washed with water basified with triethylamine and then brine. The organic layer was dried over
Na SO and concentrated in vacuo to give the crude title compound (4.74 g) as a brown, viscous
2
4
oil of sufficient purity for the next step.
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Ethyl 2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1ꢀ(4ꢀ(3,3,3ꢀtrifluoropropoxy)phenyl)ꢀ1Hꢀimidazoleꢀ4ꢀ
carboxylate (6a). A solution of compound 5 (978 mg, 2.5 mmol), 3,3,3ꢀtrifluoroꢀ1ꢀpropanol
(428 mg, 3.75 mmol) and triphenylphosphine (984 mg, 3.75 mmol) in anhydrous THF (12 mL)
were treated with DEAD (40% in toluene, 1.72 mL, 3.75 mmol). The resulting mixture was
stirred at r.t. for 30 h, then heated to 50 °C overnight. After cooling to r.t., additional 3,3,3ꢀ
trifluoroꢀ1ꢀpropanol (428 mg, 3.75 mmol) and triphenylphosphine (984 mg, 3.75 mmol) were
added, followed by diꢀtertꢀbutylazodicarboxylate (863 mg, 3.75 mmol), and the resulting mixture
stirred at r.t. overnight. Again, additional 3,3,3ꢀtrifluoroꢀ1ꢀpropanol (428 mg, 3.75 mmol) and
triphenylphosphine (984 mg, 3.75 mmol) were added, followed by diꢀtertꢀbutyl azodicarboxylate
(863 mg, 3.75 mmol), and the resulting mixture stirred at r.t. overnight. The mixture was
concentrated in vacuo and the residue purified by column chromatography (silica gel, 10ꢀ50%
EtOAc in hexanes) to yield the title compound (880 mg, 68%) as a yellowish foam of sufficient
1
purity for the next transformation. H NMR (500 MHz, CDCl ) δ 7.22ꢀ7.16 (m, 3H), 7.01 (d, J =
3
8
.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 4.22ꢀ4.10 (m, 2H), 2.66ꢀ2.54 (m,
H), 2.40 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H).
2
Ethyl
carboxylate (6b). A solution of compound 5 (978 mg, 2.5 mmol), 3ꢀfluoropropanꢀ1ꢀol (293 mg,
.75 mmol) and triphenylphosphine (984 mg, 3.75 mmol) in anhydrous THF (9 mL) were treated
2ꢀ(2,4ꢀdichlorophenyl)ꢀ1ꢀ(4ꢀ(3ꢀfluoropropoxy)phenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀ
3
with DEAD (40% solution in toluene, 1.72 mL, 3.75 mmol). The resulting mixture was stirred at
r.t. overnight. The residue was purified by column chromatography (silica gel, 20ꢀ40% EtOAc in
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hexanes). The product containing fractions were combined and concentrated in vacuo. The
residue was dissolved in CH Cl , then an equal amount of hexane was added. The resulting solid
2
2
was filtered off, and the filtrate concentrated in vacuo to yield the title compound (1.07 g, 85%)
10
11
12
13
14
15
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as a colorless foam of ca. 90% purity which was used in the next transformation without further
1
purification. H NMR (500 MHz, CDCl ) δ 7.35ꢀ7.20 (m, 3H), 7.03 (d, J = 8.7 Hz, 2H), 6.87 (d,
3
J = 8.7 Hz, 2H), 4.73ꢀ4.60 (m, 2H), 4.44 (q, J = 7.1 Hz, 2H), 4.11ꢀ4.07 (m, 2H), 2.44 (s, 3H),
2.24ꢀ2.13 (m, 2H), 1.44 (t, J = 7.1 Hz, 3H).
2
ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ1ꢀ(4ꢀ(3,3,3ꢀtrifluoropropoxy)phenyl)ꢀ1Hꢀimidazoleꢀ4ꢀ
carboxylic acid (7a). A stirred solution of compound 6a (880 mg, 1.72 mmol), in a mixture of
THF (15 mL) and EtOH (15 mL), was treated with KOH (1.07 g, 19 mmol) dissolved in water
(10 mL) and the resulting mixture stirred at 50 °C. After 3 h 30 min the reaction mixture was
cooled to r.t. then concentrated in vacuo. The residue was partitioned between CH Cl and HCl
2
2
(
1 M) and, after phase separation, the aqueous layer was extracted two more times with CH Cl .
2 2
The combined organic extracts were dried over MgSO and concentrated in vacuo to give the
4
1
title compound (714 mg, 90%) as a yellowish foam. H NMR (500 MHz, CDCl ) δ 7.32ꢀ7.18 (m,
3
3H), 7.00 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.18ꢀ4.14 (m, 2H), 2.66ꢀ2.55 (m, 2H),
2.42 (s, 3H).
2
ꢀ(2,4ꢀDichlorophenyl)ꢀ1ꢀ(4ꢀ(3ꢀfluoropropoxy)phenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀcarboxylic
acid (7b). A solution of compound 6b (1.07 g, 2.13 mmol, ca. 90% pure), in a mixture of THF
(20 mL) and EtOH (20 mL), was treated with KOH (1.40 g, 25 mmol) dissolved in water (10
mL) and the resulting mixture stirred at 50 °C. After 3 h 30 min the reaction mixture was cooled
to r.t. then concentrated in vacuo. The residue was partitioned between CH Cl and HCl (1 M)
2
2
and, after phase separation, the aqueous layer extracted with CH Cl and twice with EtOAc. The
2
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combined organic extracts were dried over MgSO and concentrated in vacuo to give the title
4
1
compound (856 mg, 95%) as a yellowish foam which was sufficiently pure for the next step. H
NMR (500 MHz, CDCl ) δ 7.35ꢀ7.22 (m, 3H), 7.04 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H),
3
0
1
2
3
4
5
6
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9
0
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9
0
4
.72ꢀ4.60 (m, 2H), 4.12ꢀ4.09 (m, 2H), 2.46 (s, 3H), 2. 25ꢀ2.14 (m, 2H).
2
ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀNꢀ(piperidinꢀ1ꢀyl)ꢀ1ꢀ(4ꢀ(3,3,3ꢀtrifluoropropoxy)phenyl)ꢀ1Hꢀ
imidazoleꢀ4ꢀcarboxamide (8a). A solution of compound 7a (643 mg, 1.4 mmol) in CH Cl (10
2
2
mL) was treated with oxalyl chloride (200 µL, 2.36 mmol), followed by 10 µL DMF. The
resulting mixture was stirred for 90 min at r.t., then concentrated in vacuo. The residue was dried
under vacuum as a yellowish foam which was used without further purification. Subsequently, to
a mixture of piperidinꢀ1ꢀamine hydrochloride (0.3 mmol) and pyridine (100 µL) in CH Cl (1
2
2
mL) was added a portion of crude intermediate (2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1ꢀ(4ꢀ(3,3,3ꢀ
trifluoropropoxy)phenyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarbonyl chloride (96 mg, 0.2 mmol)) in CH Cl (1
2
2
mL) and the resulting mixture stirred at r.t. for 2 h 30 min. The reaction mixture was washed
with saturated aqueous NaHCO (2 mL) and, after phase separation, filtered through a phase
3
separator. The solvents were evaporated and the residue purified by preparative HPLC eluting on
a reverseꢀphase column (5ꢀ100% acetonitrile in aqueous NH OAc (0.1 M)) to give the title
4
1
compound (45 mg, 41%) as a colorless solid. H NMR (500 MHz, CDCl ) δ 7.90 (s, 1H), 7.35
3
(d, J = 1.9 Hz, 3H), 7.29 (d, J = 8.3 Hz, 1H), 7.23 (dd, J = 1.9, 8.3 Hz, 1H), 7.03 (d, J = 8.9 Hz,
2H), 6.87 (d, J = 8.9 Hz, 2H), 4.19 (t, J = 6.6 Hz, 2H), 2.94ꢀ2.81 (m, 4H), 2.69ꢀ2.60 (m, 2H),
2.47 (s, 3H), 1.82ꢀ1.73 (m, 4H), 1.49ꢀ1.41 (m, 2H); HRMS Calcd for [C H Cl F N O +H]:
25 25 2 3 4 2
5
41.1385.
Found:
541.1366.
HPLC:
100%.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ1ꢀ(4ꢀ(3ꢀ
(8b).
solution of compound 7b (732 mg, 1.55 mmol) in CH Cl (20 mL) was treated with oxalyl
fluoropropoxy)phenyl)ꢀ5ꢀmethylꢀNꢀ(piperidinꢀ1ꢀyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxamide
A
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chloride (200 µL, 2.36 mmol), followed by DMF (10 µL). The resulting mixture was stirred for
90 min at r.t., then concentrated in vacuo. The residue was dried under vacuum as a yellowish
foam which was used without further purification. Subsequently, to a mixture of piperidinꢀ1ꢀ
amine hydrochloride (0.39 mmol) and pyridine (100 µL) in CH Cl (2 mL) was added a portion
10
11
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53
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56
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59
60
2
2
of
crude
2ꢀ(2,4ꢀdichlorophenyl)ꢀ1ꢀ(4ꢀ(3ꢀfluoropropoxy)phenyl)ꢀ5ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀ
carbonyl chloride (115 mg, 0.26 mmol) in CH Cl (2 mL) and the resulting mixture was stirred at
2
2
r.t. for 2 h. The reaction mixture was washed with saturated aqueous NaHCO (2 mL) and, after
3
phase separation, filtered through a phase separator. The solvents were evaporated and the
residue purified by preparative HPLC eluting on a reverseꢀphase column (5ꢀ100% CH CN in
3
1
aqueous NH OAc (0.1 M)) to give the title compound (74 mg, 56%) as a colorless solid. H
4
NMR (500 MHz, CDCl ) δ 7.90 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.23
3
(dd, J = 2.0, 8.2 Hz, 1H), 7.01 (d, J = 8.9 Hz, 2H), 6.86 (d, J = 8.9 Hz, 2H), 4.66 (dt, J = 5.7,
4
1
5
7.0 Hz, 2H), 4.09 (t, J = 6.1 Hz, 2H), 2.95ꢀ2.82 (m, 4H), 2.47 (s, 3H), 2.25ꢀ2.13 (m, 2H), 1.81ꢀ
.73 (m, 4H), 1.49ꢀ1.40 (m, 2H); HRMS Calcd for [C H Cl FN O +H]: 505.1573. Found:
2
5
27
2
4
2
05.1572. HPLC: 100%.
1ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀ(2,4ꢀdichlorophenyl)ꢀ5ꢀmethylꢀNꢀ
(piperidinꢀ1ꢀyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxamide (9). To a solution of compound 4 (3.38 g, 7.5
mmol) in CH Cl (60 mL) were added 3 drops of DMF, followed by oxalyl chloride (1.3 mL,
2
2
14.9 mmol). The mixture was refluxed for 2 hours, then cooled to r.t. and evaporated to dryness.
The residue was dissolved in CH Cl (50 mL) and cooled to 0 °C. Triethylamine (2.1 mL, 14.9
2
2
mmol) was added, followed by piperidinꢀ1ꢀamine (0.9 mL, 8.2 mmol), and the mixture stirred at
r.t. for 2 hours. Water (300 mL) was added and the mixture extracted with CH Cl (3 x 100 mL).
2
2
The organic extracts were dried (Na SO ), filtered, and concentrated in vacuo. Flash
2
4
chromatography (silica, 66ꢀ100% EtOAc in hexane) afforded the title compound (2.94 g, 74%)
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5
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6
1
as a white solid. H NMR (400 MHz, CDCl ) δ 7.71 (d, J = 8.3 Hz, 1H), 7.42ꢀ7.32 (m, 7H), 7.29
3
(
dd, J = 1.9, 8.3 Hz, 1H), 7.24 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 5.04 (s, 2H), 4.05ꢀ
.52 (m, 4H), 2.54 (s, 3H), 2.29ꢀ2.16 (m, 4H), 1.78ꢀ1.57 (m, 2H); HRMS Calcd for
C H Cl N O +H]: 535.1667. Found: 535.1667. HPLC: 96.9%.
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2
2
ꢀ(2,4ꢀDichlorophenyl)ꢀ1ꢀ(4ꢀhydroxyphenyl)ꢀ5ꢀmethylꢀ
Nꢀ(piperidinꢀ1ꢀyl)ꢀ1Hꢀimidazoleꢀ4ꢀ
carboxamide (10). A solution of compound 9 (2.78 g, 5.2 mmol) in CH Cl (80 mL) was cooled
2
2
to 0 °C then treated dropwise with boron tribromide (1 M in CH Cl , 10.4 mL, 10.4 mmol). The
2
2
reaction mixture was stirred at r.t. for 1 hour then treated with water (200 mL). The mixture was
extracted with EtOAc (3 x 200 mL). The combined organic phases were dried (Na SO ), filtered,
2
4
and concentrated in vacuo. Flash chromatography (silica, 75ꢀ100% EtOAc in hexane) afforded
1
the title compound (1.34 g, 58%) as a white solid. H NMR (400 MHz, CDCl ) δ 8.66 (br s, 1H),
3
7
.94 (br s, 1H), 7.31 (d, J = 1.9 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.18 (dd, J = 1.9, 8.3 Hz, 1H),
6
.92ꢀ6.85 (m, 4H), 2.90ꢀ2.67 (m, 4H), 2.43 (s, 3H), 1.69ꢀ1.56 (m, 4H), 1.43ꢀ1.30 (m, 2H).
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀNꢀ(piperidinꢀ1ꢀyl)ꢀ1ꢀ(
4ꢀ(4,4,4ꢀtrifluorobutoxy)ꢀphenyl)ꢀ1Hꢀ
imidazoleꢀ4ꢀcarboxamide (11a). A suspension of compound 10 (351 mg, 0.79 mmol) and K CO
2
3
(
218 mg, 1.58 mmol) in acetone (50 mL) was treated dropwise with 1ꢀiodoꢀ4,4,4ꢀtrifluorobutane
(376 mg, 1.58 mmol). The reaction mixture was refluxed overnight then cooled, filtered, and
concentrated in vacuo. Flash chromatography (silica, hexane : EtOAc 1:2) afforded the title
1
compound (200 mg, 46%) as a white solid. H NMR (400 MHz, CDCl ) δ 7.91 (br s, 1H), 7.32
3
(d, J = 1.9 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 7.21 (dd, J = 2.0, 8.3 Hz, 1H), 7.00 (d, J = 8.9 Hz,
2
H), 6.83 (d, J = 8.9 Hz, 2H), 3.99 (t, J = 6.0 Hz, 2H), 3.13ꢀ2.67 (m, 4H), 2.45 (s, 3H), 2.38ꢀ2.23
m, 2H), 2.10ꢀ2.00 (m, 2H), 1.84ꢀ1.71 (m, 4H), 1.50ꢀ1.38 (m, 2H); MS m/z 578 (M+Na); HRMS
Calcd for [C H Cl F N O +H]: 555.1541. Found: 555.1504. HPLC: 100%.
(
2
6
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ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl
propaneꢀ1ꢀsulfonate (11b). A solution of compound 10 (320 mg, 0.72 mmol) in CH Cl (10 mL)
2
2
was cooled to 0 °C. Et N (100 µL, 0.72 mmol) was added followed by 1ꢀpropanesulfonyl
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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chloride (81 µL, 0.72 mmol) and the reaction mixture was stirred at room temperature overnight.
Water was added, the mixture extracted with CH Cl (3 x 20 mL), dried (Na SO ), filtered and
2
2
2
4
concentrated. Flash chromatography (silica, hexane : EtOAc 1 : 2) afforded the title compound
1
(
220 mg, 56%) as a white solid. H NMR (400 MHz, CDCl ) δ 7.82 (br s, 1H), 7.29ꢀ7.15 (m,
3
5H), 7.10ꢀ7.03 (m, 2H), 3.23ꢀ3.14 (m, 2H), 2.90ꢀ2.70 (m, 4H), 2.42 (s, 3H), 2.01ꢀ1.88 (m, 2H),
13
1
.75ꢀ1.65 (m, 4H), 1.41ꢀ1.31 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H); C NMR (126 MHz, CDCl ) δ
3
160.8, 149.0, 142.3, 136.8, 135.3, 135.0, 133.8, 133.4, 130.6, 129.9, 129.1, 128.2, 127.4, 123.1,
57.2, 52.9, 25.4, 23.3, 17.5, 13.0, 10.9; HRMS Calcd for [C H Cl N O S+H]: 551.1287.
2
5
28
2
4
4
Found: 551.1313. HPLC: 100%.
4
ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl 3ꢀ
fluoropropaneꢀ1ꢀsulfonate (11c). A suspension of compound 10 (200 mg, 0.45 mmol) in dry
CH Cl (3 mL) was treated with Et N (45 mg, 0.45 mmol) at r.t.. The resulting mixture was
2
2
3
cooled to ꢀ78 °C and 3ꢀfluoropropaneꢀ1ꢀsulfonyl chloride (72 mg, 0.45 mmol) in dry CH Cl (0.5
2
2
mL) was added dropwise. After 1 h 40 min at ꢀ78 °C was added 3ꢀfluoropropaneꢀ1ꢀsulfonyl
chloride (72 mg, 0.45 mmol) and after a total of 4 h 40 min was added Et N (55 mg, 0.54 mmol).
3
The reaction was allowed to reach r.t. overnight. It was then cooled to 0 °C and Et N (55 mg,
3
0.54 mmol) was added, followed by 3ꢀfluoropropaneꢀ1ꢀsulfonyl chloride (72 mg, 0.45 mmol)
after a total of 19 h. After 1 h the reaction mixture was washed with water and concentrated in
vacuo. The product was purified by HPLC (30ꢀ100% CH CN in aqueous NH OAc (0.1 M) over
3
4
1
4
0 min) to yield the title compound as a white solid (160 mg, 63%). H NMR (400 MHz, CDCl )
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2
2
2
2
2
2
2
2
3
3
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3
3
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3
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6
δ 7.88 (br s, 1H), 7.39ꢀ7.17 (m, 5H), 7.11 (d, J = 8.8 Hz, 2H), 4.58 (dt, J = 5.5, 46.8 Hz, 2H),
3
1
1
.53ꢀ3.33 (m, 2H), 2.92ꢀ2.71 (m, 4H), 2.45 (s, 3H), 2.40ꢀ2.23 (m, 2H), 1.83ꢀ1.62 (m, 4H), 1.46ꢀ
.33 (m, 2H). HRMS Calcd for [C H Cl FN O S+H]: 569.119. Found: 569.1192. HPLC:
2
5
27
2
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
00%.
4
ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl
3,3,3ꢀtrifluoropropaneꢀ1ꢀsulfonate methanesulfonic acid salt (11d). A solution of compound 10
(
0.89 g, 2.00 mmol) in CH Cl (20 mL) was cooled to 0 °C then treated with Et N (0.35 mL, 2.4
2
2
3
mmol), followed by 3,3,3ꢀtrifluoropropanesulfonyl chloride (prepared by an analogous method
to that described in WO00/010968 for the butyl homologue) (0.35 mL, 2.40 mmol). The reaction
mixture was stirred at r.t. overnight. TLC showed remaining starting material and so another
portion of Et N and 3,3,3ꢀtrifluoropropanesulfonyl chloride was added and the reaction mixture
3
stirred for additional 2 h. Water was added and the product was extracted with CH Cl , dried
2
2
(
Na SO ), filtered, and concentrated in vacuo. Flash chromatography (33ꢀ100% EtOAc in
2 4
hexane) followed by recrystallisation (hexane : EtOAc) afforded the title compound (700 mg,
1
5
9%) as a colorless solid. H NMR (400 MHz, CDCl ) δ 7.92 (s, 1H), 7.34ꢀ7.24 (m, 5H), 7.20ꢀ
3
7.13 (m, 2H), 3.54ꢀ3.48 (m, 2H), 3.00ꢀ2.82 (m, 4H), 2.84ꢀ2.73 (m, 2H), 2.50 (s, 3 H), 1.83ꢀ1.72
(
m, 4 H), 1.49ꢀ1.39 (m, 2H); HRMS Calcd for [C H Cl F N O S +H]: 605.1004. Found:
26 29 2 3 4 7 2
605.1012. HPLC: 100%.
4ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl
butaneꢀ1ꢀsulfonate (11e). A solution of compound 10 (320 mg, 0.72 mmol) in CH Cl (10 mL)
2
2
was cooled to 0 °C. Et N (100 µL, 0.72 mmol) was added followed by 1ꢀbutanesulfonyl chloride
3
(93 µL, 0.72 mmol) and the reaction mixture was stirred at r.t. overnight. Water was added and
the mixture extracted with CH Cl (3 x 20 mL), dried (Na SO ), filtered, and concentrated in
2
2
2
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vacuo. Flash chromatography (silica, hexane : EtOAc 1:2) afforded the title compound (230 mg,
1
5
7%) as a white solid. H NMR (400 MHz, CDCl ) δ 7.82 (br s, 1H), 7.27ꢀ7.16 (m, 5H), 7.09ꢀ
3
7.04 (m, 2H), 3.23ꢀ3.17 (m, 2H), 2.92ꢀ2.68 (m, 4H), 2.42 (s, 3H), 1.93ꢀ1.84 (m, 2H), 1.74ꢀ1.66
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(
m, 4H), 1.50ꢀ1.40 (m, 2H), 1.40ꢀ1.33 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H) ; MS m/z 588 (M+Na);
HRMS Calcd for [C H Cl N O S+H]: 565.1443. Found: 565.1450. HPLC: 100%.
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4
4
4
ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)ꢀphenyl
,4,4ꢀtrifluorobutaneꢀ1ꢀsulfonate (11f). A solution of compound 10 (0.49 g, 1.20 mmol) in
CH Cl (20 mL) was cooled to 0 °C and treated with Et N (0.67 mL, 4.8 mmol), followed by
4
2
2
3
4
,4,4ꢀtrifluorobutaneꢀ1ꢀsulfonyl chloride (prepared as described in WO00/010968) (0.38 g, 1.80
mmol). The reaction mixture was stirred at r.t. for 3 h. TLC showed remaining starting material
so another portion of Et N and 4,4,4ꢀtrifluorobutaneꢀ1ꢀsulfonyl chloride was added and the
3
reaction mixture stirred overnight. Water was added, then the mixture was extracted with
CH Cl . The organic extracts were dried (Na SO ), filtered, and concentrated in vacuo. Flash
2
2
2
4
chromatography (33ꢀ100% EtOAc in hexane) followed by recrystallisation (hexane : EtOAc)
1
afforded the title compound (0.45 g, 61%) as a colorless solid. H NMR (400 MHz, CDCl ) δ
3
7
.92 (br s, 1H), 7.34ꢀ7.22 (m, 5H), 7.15 (d, J = 8.7 Hz, 2H), 3.38 (t, J = 7.3 Hz, 2H), 3.12ꢀ2.74
m, 4H), 2.49 (s, 3H), 2.43ꢀ2.32 (m, 2H), 2.32ꢀ2.22 (m, 2H), 1.82ꢀ1.74 (m, 4H), 1.50ꢀ1.40 (m,
H); HRMS Calcd for [C H Cl F N O S+H]: 619.1160. Found: 619.1148. HPLC: 96.9%. 4ꢀ
(
2
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3
4
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(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl
3ꢀ
methylbutaneꢀ1ꢀsulfonate (11g). A solution of compound 10 (50 mg, 0.11 mmol) in CH Cl (3
2
2
mL) was cooled to 0 °C then treated with Et N (20 µL, 0.13 mmol). The resulting mixture was
3
cooled to ꢀ78 °C, then 3ꢀmethylbutaneꢀ1ꢀsulfonyl chloride (23 mg, 0.13 mmol) carefully added.
The reaction was stirred at ꢀ78 °C for 1.5 h. Water was added, then the mixture was extracted
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with CH Cl . The organic extracts were dried, filtered, and concentrated in vacuo to give a
2
2
1
residue which was purified by HPLC to deliver the title compound (46 mg, 71%) as a solid. H
NMR (400 MHz, CDCl ) δ 7.86 (s, 1H), 7.31ꢀ7.20 (m, 5H), 7.14ꢀ7.08 (m, 2H), 3.27ꢀ3.20 (m,
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0
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2H), 2.89ꢀ2.76 (m, 4H), 2.46 (s, 3H), 1.87ꢀ1.79 (m, 2H), 1.78ꢀ1.68 (m, 5H), 1.44ꢀ1.36 (m, 2H),
0.93 (d, J = 6.5 Hz, 6H); HRMS Calcd for [C H Cl F N O S+H]: 579.1600. Found: 579.1584.
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3
4
4
HPLC: 100%.
4
ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀmethylꢀ4ꢀ(piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)phenyl 3,3ꢀ
dimethylbutaneꢀ1ꢀsulfonate (11h). A solution of compound 10 (50 mg, 0.11 mmol) in CH Cl (3
2
2
o
mL) was cooled to 0 C and treated with Et N (20 µL, 0.13 mmol). The resulting mixture was
3
cooled to ꢀ78 °C and 3,3ꢀdimethylbutaneꢀ1ꢀsulfonyl chloride (25 mg, 0.13 mmol) was carefully
added. The reaction was stirred at ꢀ78 °C for 2 h. Water was added, then the mixture extracted
with CH Cl . The organic extracts were dried, filtered, and concentrated in vacuo to give a
2
2
residue which was purified by preparative HPLC to deliver the title compound (46 mg, 69%) as a
1
solid. H NMR (400 MHz, CDCl ) δ 7.85 (s, 1H), 7.32ꢀ7.17 (m, 5H), 7.11ꢀ7.09 (d, J = 8.7 Hz,
3
2
1
5
H), 3.26ꢀ3.15 (m, 2H), 2.92ꢀ2.74 (m, 4H), 2.46 (s, 3H), 1.87ꢀ1.78 (m, 2H), 1.77ꢀ1.68 (m, 5H),
.46ꢀ1.34 (m, 2H), 0.92 (s, 9H); HRMS Calcd for [C H Cl N O S+H]: 593.1756. Found:
28 34
2
4
4
93.1755. HPLC: 100%.
racemic 1ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀ(2,4ꢀdichlorophenyl)ꢀNꢀ(3ꢀhydroxypiperidinꢀ1ꢀyl)ꢀ5ꢀmethylꢀ
Hꢀimidazoleꢀ4ꢀcarboxamide (12a). Compound 4 (752 mg, 1.66 mmol) and SOCl (33.2 mmol)
1
2
were mixed and the resulting mixture was refluxed for 1.5 h. Excess SOCl was removed under
2
reduced pressure and the residue was azeotroped with toluene. 3ꢀHydroxyꢀ1ꢀaminopiperidine
(
6.64 mmol) was mixed with CH Cl (15 mL) and THF (2 mL) and Et N (13.28 mmol). The
2 2 3
mixture was cooled to ꢀ20 °C under a nitrogen atmosphere. A THF (5 mL) mixture of the acid
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chloride from above was added dropwise during 20 minutes. The resulting mixture was allowed
to slowly warm to r.t. and stirred overnight. Aqueous NaOH (1 M, 5 mL) and EtOH (15 mL)
were added and the mixture was heated to 40 °C for 15 minutes. The reaction mixture was then
diluted to 50 mL with CH Cl and washed with water (2 x 20 mL) and brine (20 mL). The
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organic layer was dried (MgSO ), filtered, and concentrated in vacuo. The residue was purified
4
by flash chromatography (8% EtOH in CH Cl ) and then by reverse phase HPLC (Kromasil C8,
2
2
6
0% CH CN in aqueous NH OAc (0.1 M)). The product fraction was concentrated in vacuo and
3 4
then dissolved in CH Cl and washed with water several times and then brine. The organic layer
2
2
was dried (MgSO ), filtered and concentrated in vacuo to give the title compound (160 mg, 17%
4
1
yield). H NMR (400 MHz, CDCl ) δ 7.99 (s, 1H), 7.33ꢀ7.19 (m, 6H), 7.18ꢀ7.07 (m, 2H), 6.90
3
(d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 5.18 (s, 1H), 4.92 (s, 2H), 3.94ꢀ3.85 (m, 1H), 3.06ꢀ
2.97 (m, 1H), 2.85ꢀ2.66 (m, 3H), 2.34 (s, 3H), 1.87ꢀ1.77 (m, 1H), 1.63ꢀ1.50 (m, 2H), 1.46ꢀ1.34
(m, 1H); MS m/z 551 (M+H).
racemic
1ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀ(2,4ꢀdichlorophenyl)ꢀNꢀ(3ꢀhydroxycyclohexyl)ꢀ5ꢀmethylꢀ
1
Hꢀimidazoleꢀ4ꢀcarboxamide (12b). A suspension of compound 4 (2.00 g, 4.41 mmol) in CH Cl
2
2
(50 mL) was treated with oxalyl chloride (2.80 g, 22.1 mmol) at r.t., followed by one drop of
DMF. The mixture was stirred at r.t. for 15 min after which the solvent was removed in vacuo.
The acid chloride was suspended in CH Cl (10 mL) and added dropwise to a mixture of 3ꢀ
2
2
aminocyclohexanol (610 mg, 5.29 mmol), aqueous NaOH (1 M, 30 mL) and CH Cl (30 mL).
2
2
After stirring at r.t. for 2 h, adding more 3ꢀaminocyclohexanol after 1 h 25 min (67 mg, 0.58
mmol) and 1 h 45 min (58 mg, 0.50 mmol), water and CH Cl were added and the phases
2
2
separated. The organic phase was washed with aqueous HCl (10%) and brine, then dried
1
(
MgSO ), filtered, and concentrated in vacuo to yield the crude title compound (2.79 g). H
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