Design and synthesis of cannabinoid receptor 1 antagonists for peripheral selectivity

Article abstract of DOI:10.1021/jm201731z

Antagonists of cannabinoid receptor 1 (CB1) have potential for the treatment of several diseases such as obesity, liver disease, and diabetes. Recently, development of several CB1 antagonists was halted because of adverse central nervous system (CNS) related side effects observed with rimonabant, the first clinically approved CB1 inverse agonist. However, recent studies indicate that regulation of peripherally expressed CB1 with CNS-sparing compounds is a viable strategy to treat several important disorders. Our efforts aimed at rationally designing peripherally restricted CB1 antagonists have resulted in compounds that have limited blood-brain barrier (BBB) permeability and CNS exposure in preclinical in vitro and in vivo models. Typically, compounds with high topological polar surface areas (TPSAs) do not cross the BBB passively. Compounds with TPSAs higher than that for rimonabant (rimonabant TPSA = 50) and excellent functional activity with limited CNS penetration were identified. These compounds will serve as templates for further optimization.

Full text of DOI:10.1021/jm201731z

Article
pubs.acs.org/jmc
Design and Synthesis of Cannabinoid Receptor 1 Antagonists for
Peripheral Selectivity
Alan Fulp, Katherine Bortoff, Herbert Seltzman, Yanan Zhang, James Mathews, Rodney Snyder,
Tim Fennell, and Rangan Maitra*
Discovery Sciences, Research Triangle Institute, 3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park,
North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: Antagonists of cannabinoid receptor 1 (CB1)
have potential for the treatment of several diseases such as
obesity, liver disease, and diabetes. Recently, development of
several CB1 antagonists was halted because of adverse central
nervous system (CNS) related side effects observed with
rimonabant, the first clinically approved CB1 inverse agonist.
However, recent studies indicate that regulation of peripherally
expressed CB1 with CNS-sparing compounds is a viable
strategy to treat several important disorders. Our efforts aimed
at rationally designing peripherally restricted CB1 antagonists
have resulted in compounds that have limited blood−brain barrier (BBB) permeability and CNS exposure in preclinical in vitro
and in vivo models. Typically, compounds with high topological polar surface areas (TPSAs) do not cross the BBB passively.
Compounds with TPSAs higher than that for rimonabant (rimonabant TPSA = 50) and excellent functional activity with limited
CNS penetration were identified. These compounds will serve as templates for further optimization.
INTRODUCTION
■
The endocannabinoid system (ECS) consists of receptors,
transporters, endocannabinoids, and the enzymes involved in
synthesis and degradation of endocannabinoids.1 There have
been two cannabinoid receptors (CBRs) identified to date,
CB1 and CB2. CB1 and CB2 are both G-protein-coupled
receptors (GPCRs), and their primary function is to activate
inhibitory G proteins (Gi/o).1,2 The ECS is responsible for
many important physiological processes, and regulation of these
processes holds promise for the treatment of several diseases.
ECS components are under evaluation for the treatment of
obesity, liver disease, diabetes, pain, and inflammation.2
Figure 1. CB1 antagonists.
The CB1 receptor is expressed throughout the body; however,
it is found at much greater concentrations in the central nervous
system (CNS). There has been great interest in the use of CB1
antagonists for the treatment of metabolic disorders, such as
obesity and diabetes. Rimonabant (SR141716A, 1, Figure 1), a
potent and selective CB1 inverse agonist/antagonist, was clinically
approved to treat obesity in Europe. Unfortunately, 1 produced
serious CNS-related side effects such as anxiety, depression, and
suicidal ideation in patients, leading to its withdrawal from
European markets and denial of approval in the United States.3
Upon discovery of rimonabant’s side effects, several other CB1
antagonists, such as taranabant, otenabant (2), and ibipinabant,
were pulled from development because of regulatory concerns.4
A strategy to take advantage of the therapeutic potential of
CB1 antagonism and avoid the CNS-related adverse effects is
to generate CB1 antagonists that do not cross the blood−brain
barrier (BBB). This strategy is being pursued by several groups,
and a small set of CB1 antagonists that do not cross the BBB
have been reported (3−6, Figure 2).5 However, none of these
peripherally restricted CB1 antagonists have been fully
characterized or their efficacy demonstrated clinically.
Our group has pursued a two-pronged strategy to develop
peripherally restricted CB1 antagonists. The first strategy involved
development of CB1 antagonists that have a permanent charge.
Charged compounds do not normally cross the BBB unless they
are acted upon by a transporter.6 Results for this strategy have
been previously reported.7 The second strategy was to target
compounds with high topological polar surface areas (TPSAs). It
has been shown that compounds with higher TPSAs have lower
Received: December 22, 2011
Published: February 28, 2012
© 2012 American Chemical Society
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Figure 3. Design of compound 11.
(Table 1). Compound 10 was only moderately active (Ke =
1170 nM). However, examination of the structure of 2 revealed a
primary amide at the 4-position of the piperdine ring (Figure 1).
This amide was in a similar position as the carboxylic acid
functionality of compound 10, leading to the decision to convert
carboxylic acid 10 to amide 11 (Figure 3). Amide 11 lacked the
charged nature of a carboxylic acid, but it did have hydrogen
bonding ability that could lower its permeability into the CNS.
Compound 11 was found to be a potent CB1 antagonist having a
Ke of 0.44 nM and was also highly selective for CB1 over CB2
(CB2/CB1 of 1600). Interestingly, the 4-phenylpiperidine-4-
carboxamide group was also reported on a closely related pyrrole
scaffold.5c Compound 11 was advanced into Madin−Darby canine
kidney cells transfected with the human MDR1 gene (MDCK-
mdr1) model of BBB penetration, which is widely used to predict
in vivo permeability of compounds.9 The potency, selectivity, and
relatively low permeability of compound 11 across the MDCK-
mdr1 cells (apical (A) to basal (B), 8%) made it an interesting
starting point for further modifications toward designing potent
and selective CB1 antagonists that do not cross the BBB.
Compound 11 served as a starting point for several modi-
fications to the amine portion of the pyrazole C-3 carboxamide
(Figure 4, Table 2). One of the first modifications of compound
11 studied was the replacement of the phenyl group. Compound
12 was targeted because it represented a hybrid of compounds 11
and 2. Compound 12 also closely resembles the Sanofi-Aventis
compound 5. However, compound 12’s potency (Ke(CB1) =
91 nM) and selectivity (ratio CB2/CB1 of 28.3) did not warrant
further investigation of this compound. Next, the reversal of the
primary amide of compound 11 became of interest. To realize
this, both compounds 13 and 14 needed to be synthesized as
precursors of reverse amide compound 15. However, both
compounds 13 and 14 were interesting in their own right.
Compound 13 added the additional functionality of a carbamate;
this was a functionality that had not been pursued in our
laboratory, and it also had good potency (Ke(CB1) = 20.2 nM)
and selectivity (ratio CB2/CB1 of ∼50). Compound 14 was of
interest because it replaced the primary amide of 11 with a
primary amine. This maintained the possibility of hydrogen
bonding and increased the basicity of the molecule. This amine
group proved to be detrimental to potency (Ke(CB1) = 485 nM).
However, the amine group in compound 14 also allowed for the
introduction of different functionalities. The amine group of
compound 14 was used to make the reverse amide compound 15
Figure 2. Reported CB1 antagonists that are selective for the
periphery.
permeability into the CNS.8 Higher TPSAs can be achieved by
adding polar groups, such as sulfonamide or sulfamide, or by
replacing existing functional groups with more polar functional
groups. This strategy led to the identification of compounds 7 and
8 that we have previously reported. While these compounds were
promising, their selectivity for CB1 over CB2 was only modest.
Here we describe our ongoing efforts toward designing
peripherally restricted CB1 antagonists with improved properties.
This study evolved to include a more empirical approach that was
based more on SAR than on computational parameters. We have
been able to design, synthesize, and characterize highly selective
CB1 antagonists that appear to be peripherally restricted.
RESULTS
■
Ligand Design and Pharmacological Characterization.
Compounds were synthesized, purified, characterized, and
tested as has been described in the “Experimental Section”. All
compounds were tested in vitro as antagonists using a calcium
mobilization assay as has been previously described.7 The
ability of each compound to antagonize functional activation of
CB1 was quantitatively measured and expressed as its apparent
dissociation affinity constant (Ke). Compounds that were found
to be potent (Ke ≈ 100 nM or less) using the functional assay
were subsequently characterized using radioligand displacement
of either [3H]SR141716A or [3H]CP55940 at CB1 and CB2.
Equilibrium dissociation constant (Ki) values were determined
at each receptor.
During our studies of charged compounds, carboxylic acids
were examined because they are negatively charged at the phys-
iological pH. Around the same time, carboxylic acid 9 (Figure 3)
was reported by another group to be a CB1 antagonist.5d This
finding led to the synthesis and evaluation of carboxylic acid 10
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Table 1. Pharmacological Assessment of Compound 11 with Rimonabant 1 and Otenabant 2
Ki (nM)
Ke (nM)
a
compd
TPSA
50
CB1
CB1 [3H]SR141716 (1)
CB1 [3H]CP55940
6.2
CB2 [3H]CP55940
313
CB2/CB1
50.6
MDCK-mdr1 (%)
1
1.1
15
90
2
8.7
10
11
75
81
1170
0.4
0.4
3.4
5504
1600
8
a
Compound’s permeability was measured from apical to basal sides of the membrane.
Figure 4. Ligand design around compound 11.
which was only weakly active (Ke(CB1) = 201 nM). Sulfonamide
16, which was also made from amine 14, had higher TPSA
compared to compound 11 and was potent but only moderately
selective for CB1 over CB2 (Ke(CB1) = 3.5 nM, ratio of CB2/CB1
of 5.64). Finally, amine 14 also allowed for the synthesis of urea
17a. Urea 17a proved to be a potent CB1 antagonist (Ke(CB1) =
2.4 nM) and had good selectivity against CB2 (ratio CB2/CB1 of
∼425). Compound 17a was advanced into the in vitro model of
BBB permeability (MDCK-mdr1, apical to basal) and was predicted
not to cross the BBB (Table 2, <1% transported). These results
spawned the synthesis of a small library of ureas 17b−k, which had
potencies (Ke) ranging from 0.5 to >10000 nM against CB1. Several
of these compounds were very selective with 5 of the 10 com-
pounds being over 100-fold selective for CB1 over CB2 (Table 2).
The positive results for compound 17a also led to the
exploration of 4- and 3-aminopiperdine and cyclohexylamides
as different spacers between the pyrazole amide and their polar
functionality (Figure 5, Table 3). Structurual series 18 was
chosen because it offered similar spacing to compound 17a and
presented the opportunity for rapid derivatization off the
piperidine nitrogen. Because of the positive results observed for
compounds 18a−l (7 out of the 12 compounds had Ke < 100 nM
against CB1, and 4 analogues had CB2/CB1 ratios greater than
100), other aminopiperidine linkers were explored. The 1,3-
disubstituted aminopiperdine series 19 and 20 were used to
explore the importance of the effect of an alternative
juxtaposition of substituents and the introduction of stereo-
chemistry versus the 4-aminopiperdine linker of structurual
series 18. Both enantiomers were explored to examine the
effect of chirality on potency and selectivity. Positive results
were obtained with compounds 19a−j, with 3 of the 10
analogues having Ke values below 100 nM at CB1, and two
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Table 2. Pharmacological Assessment of Analogues of Compound 11
Ki (nM)
Ke (nM)
CB1
a
compd
TPSA
CB1 [3H]CP55940
CB2 [3H]CP55940
CB2/CB1
MDCK-mdr1 (%)
12
93
76
64
67
93
79
79
103
79
82
79
79
79
79
79
79
91
78.4
42.3
104
62.6
7.3
2217
2110
1127
214
28.3
49.9
10.8
3.4
13
20.2
485
201
3.5
<1
14
15
16
41
5.6
3
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
2.4
47.1
43
20000
17126
424.6
398
<1
<1
2.1
>10000
89
614
1489
38.8
13.5
15
20000
16289
2414
4914
182
33
264
0.5
11
62
<1
<1
0.7
364
12
10.8
0.4
7.6
293
39
12
792
15.5
20000
2760
25
0.4
178
a
Compound’s permeability was measured from apical to basal sides of the membrane.
Figure 5. Exploration of different spacers.
analogues were over 100-fold selective for CB1 versus CB2.
Compounds 20a−j were also of interest with 3 of the 10
analogues having Ke(CB1) less than 30 nM. In addition 3 of the
10 analogues were over 100-fold selective for CB1 vs CB2.
Finally, since sulfonamide 7 and sulfamide 8 have been found
to be potentially useful in the development of periphery
restricted CB1 antagonists,7 ureas of structure 21 were targeted.
In general these compounds were only weakly active, and
compounds 21a−e were not pursued further.
Synthesis. Compound 10 (Scheme 1) was prepared by first
making the acid chloride of the readily available acid 22.10 Acid
22 was treated with oxalyl chloride and a catalytic amount of
dimethylformamide (DMF) in dichloromethane to form the
desired acid chloride. This acid chloride was then treated with
amino acid 23 in the presence of triethylamine to yield
compound 10 in 67% yield. Carboxylic acid 10 was converted
to amide 11 by the use of benzotriazol-1-yloxytris-
(dimethylamino)phosphonium hexafluorophosphate (BOP),
triethylamine, and ammonium chloride in 46% yield.
Aminoamide 12 was made by reacting acid 22 with
commercially available piperidine 24 under BOP coupling
conditions, and this reaction produced compound 12 in 46%
yield (Scheme 2). The protected amine 13 was made by the
coupling of acid 22 and readily available amine 25.11 The Boc
group of compound 13 was removed using 30% trifluoroacetic
acid (TFA) in dichloromethane to yield amine 14 in 87% yield.
Amine 14 was used as a common intermediate for compounds
15, 16, and 17a−k. Acetylamide 15 was made by reacting
amine 14 with acetic anhydride and pyridine in 71% yield.
Sulfonamide 16 was formed by the reaction of 14 with
methanesulfonyl chloride and triethylamine in 65% yield. Urea
17a was synthesized in 45% yield by reacting amine 14, tert-
butyl isocyanate, and triethylamine at 40 °C in THF. Synthesis
of ureas 17b−k was accomplished by reacting amine 14, the
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Table 3. Pharmacological Assessment of Different Spacers and Functional Groups
Ki (nM)
Ke (nM)
a
compd
TPSA
CB1
CB1 [3H]CP55940
2.9
CB2 [3H]CP55940
2510
CB2/CB1
877.6
MDCK-mdr1 (%)
<1
18a
18b
18c
18d
18e
18f
76
59
67
93
119
79
93
79
79
79
79
76
76
58
67
93
79
79
79
79
79
76
76
58
67
93
79
79
79
79
79
76
88
88
88
88
88
4.7
5115
195
269
592
78
65.3
14.7
2236
3349
34.2
227.8
<1
14
<1
18g
18h
18i
4097
20.5
16.7
66.5
78.1
3
165
86
5693
9791
8459
8721
2997
35
114
46
18j
184
187
9.2
18k
18l
47
326
19a
19b
19c
19d
19e
19f
842
1879
86
39.1
77.9
4350
111.3
140
14
<1
62
10893
280
265
70
19g
19h
19i
271
7795
29
230
206
75
19j
65.5
22.3
5420
6720
83
<1
<1
20a
20b
20c
20d
20e
20f
52
302
1174
530
7
11.84
1248
105.5
<1
171
31.3
27
97
4135
3090
3031
43
23
24
20g
20h
20i
134
125
118
268
10.4
135
351
486
274
150
20j
17.5
2507
143
<1
21a
21b
21c
21d
21e
a
Compound’s permeability was measured from apical to basal sides of the membrane.
a
Scheme 1. Synthesis of Compound 11
a
Reagents and conditions: (a) (1) oxalyl chloride, DMF (cat.), dichloromethane, (2) 23, triethylamine, dichloromethane; (b) benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), triethylamine, ammonium chloride, THF.
appropriate isocyanate, and triethylamine in tetrahydrofuran
(THF) at room temperature. These reactions proceeded in
yields that ranged from 58% to 74%.
Compounds 18a, 19a, and 20a were synthesized by the
coupling of the appropriate commercially available amine to
acid 22 in the presence of BOP and triethylamine in yields
ranging from 88% to 96% (Scheme 3). Treatment of
compounds 18a, 19a, and 20a with trifluoroacetic acid in
dichloromethane produced the amines 18b, 19b, and 20b. The
amines 18b, 19b, and 20b were reacted with acetic anhydride
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a
Scheme 2. Synthesis of Analogues of Compound 11
a
Reagents and conditions: (a) amine, BOP, triethylamine, THF; (b) 30% TFA in dichloromethane; (c) acetic anhydride, pyridine; (d)
methanesulfonyl chloride, triethylamine, dichloromethane; (e) isocyanate, triethylamine, THF, rt or 40 °C.
and pyridine to produce amides 18c, 19c, and 20c, respectively,
in yields ranging from 81% to 87%. Sulfonamides 18d, 19d, and
20d were made by reacting amines 18b, 19b, and 20b with
methanesulfonyl chloride and triethylamine in THF. Ureas
18f,h−l, 19e−j, and 20e−j were made by reacting the
appropriate amine with the appropriate isocyanate in dichloro-
methane or THF. The N-tert-butylpiperidinecarboxamide 18f
was converted to the unsubstituted piperidine carboxamide 18g
in 81% yield by stirring 18f in 50% TFA in dichloromethane.12
The sulfamide 18e was synthesized from the reaction of 18b
with excess sulfamide in dioxane at 90 °C in 67% yield (Scheme 4).7
Ureas 21a−e were made as a mixture of cis/trans isomers from
the previously described amine 26 in yields ranging from 71%
to 98%.7
vitro metabolic stability (Table 4). Stability was measured in
human plasma and human hepatic S9 fractions to gauge any
metabolic liabilities that might be present with these
compounds. All compounds tested had good stability in
plasma. Stabilities of compounds in S9 fractions were more
variable than stabilities in plasma. However, all compounds
except 17b displayed metabolic stabilities similar to or greater
than compound 1.
Compounds were prioritized and progressed into in vivo
experiments in mice for analysis of brain penetration (Table 5).
Compound 13 was not progressed because of its relatively low
selectivity compared with other compounds found in Table 4,
and compound 17b was not progressed because of its relatively
low stability in S9 fractions. Ureas 17a and 18f along with
carbamate 18a were chosen and evaluated in vivo. Urea 17a
was bioavailable with either oral or intraperitoneal (ip) dosing.
Brain levels of 17a were below the lower limit of quantitation
when dosed orally, and its brain to plasma ratio was 0.03 with ip
In Vitro Metabolic Stability and in Vivo Evaluation of
Brain Penetration. A small set of compounds that were
potent, selective, and predicted not to penetrate the CNS as
determined using the MDCK-mdr1 assay were tested for in
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a
Scheme 3. Synthesis of Compounds with Different Spacers and Functional Groups
a
Reagents and conditions: (a) amine, BOP, triethylamine, THF; (b) 30% TFA in dichloromethane; (c) acetic anhydride, pyridine; (d)
methanesulfonyl chloride, triethylamine, THF; (e) isocyanate, triethylamine, THF, rt; (f) ethyl chloroformate, triethylamine, THF; (g) 50% TFA in
dichloromethane.
dosing at 1 h. Carbamate 18a was also bioavailable with either
oral or ip dosing. When dosed by ip injection, carbamate 18a
had a brain to plasma ratio of 0.02 at 1 h. Urea 18f was also
bioavailable with oral or intraperitoneal dosing. However, brain
to plasma ratios for urea 18f were 0.16 with oral dosing at 1 h
and were 0.38 with intraperitoneal dosing at 1 h. Since
unperfused brains were examined and because the volume of
blood in the unperfused brain is ∼2−4%,13 these promising
results indicated that 17aand 18a had little to no permeability
into the brain as expected while 18f was not selective for the
periphery.
DISCUSSION
■
In this publication we report our ongoing efforts to produce
peripherally selective CB1 antagonists that may be useful in
treating a wide range of clinical indications. We have now
identified several highly selective CB1 antagonists that are
metabolically stable with limited oral bioavailability. The
addition of polarity to the 3-carboxamide position has been
found to be advantageous. Sulfonamide 7 came out of our
efforts to synthesize CB1 antagonists that had high TPSAs.7
Those efforts focused on compounds that contained
sulfonamide and sulfamide functionality because of the
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a
Scheme 4. Synthesis of Compounds with Different Spacers and Functional Groups Continued
a
Reagents and conditions: (a) sulfamide, dioxane, 90 °C; (b) isocyanate, triethylamine, THF, rt.
Table 4. Pharmacological Assessment of Select Compounds
in for in Vitro Metabolic Stability
Ke(CB1) = 20.5 nM). The enhancing effects of lipophilicity on
potency at CB1 could be also seen in compounds 13−17k.
The shape of the functional group seemed to have impact on
potency. The linear (4-aminopiperdine) linker seemed to be
favored over the bent (3-aminopiperidine) linker for potency.
Structure 18 was found to be the most potent analogue in 6 out
of the 10 examples where structures 18−20 possessed the same
substituent. However, those six analogues were all ureas or
carbamates. Of the analogues that favored the bent (3-
aminopiperidine) linker only one contained a urea (20g).
Amine (19b and 20b), amide (19c), and sulfonamide (20d)
substituents favored the 3-aminopiperdine linker. Of the two
bent (19, (3S)-3-aminopiperdine; 20, (3R)-3-aminopiperdine)
linkers, the R enantiomer (20) was the most favored for activity
at CB1. Analogues of structure 20 were found to be more
potent than their corresponding analogues of structure 19 in 7
out of 10 times. The 4-amino-4-phenylpiperidine linker present
in compounds 13−17k was by far the most potent linker
tested. However, it was difficult to determine if the improved
potency observed with the 4-amino-4-phenylpiperidine linker
was due to shape or the greater lipophilicity present with this
linker.
While maintaining a desirable profile in the MDCK-mdr1
assay, which is predictive of brain penetration, gains were made
in selectivity over our previously reported sulfonamide 7.
Fifteen compounds with over 100-fold selectivity (CB1/CB2 Ki
vs CP55940) have been identified. These compounds, at least
in part, were designed to increase the TPSA over currently
known CB1 antagonists in hopes of limiting exposure to the
CNS. They were found to have limited permeability (<1%) in
the MDCK-mdr1 permeability assay, which serves as an in vitro
measure of CNS permeability. On the basis of data from the
MDCK-mdr1 permeability assay, a set of compounds were
chosen for both plasma and metabolic stability studies in
human plasma and hepatic S9 fractions. These studies
demonstrated that most compounds tested were at least as
in vitro metabolic
stability
MDCK-
S9 (%
plasma (%
Ke (nM) CB2/CB1 [3H] mdr1 A to remaining, remaining,
compd TPSA CB1
CP55940
B (%) 120 min) 60 min)
1
50
1.1
50.6
16
15
<1
<1
<1
<1
<1
<1
<1
47
37
62
88
88
18
>90
67
>90
>90
>90
>90
>90
82
7
101
127
76
113
106
20.2
2.4
8
17
13
49.9
424.6
398
17a 79
17b 79
18a 76
2.09
4.7
877.6
227.8
>90
71
18f
79
78
relatively large TPSA for these functional groups. Continued
efforts along those lines have yielded more positive results,
including two new sulfonamides 19d and 20d that are more
selective than 7. Sulfonamide 7 demonstrated modest
selectivity for CB1 versus CB2 (16-fold), but both 19d and
20d demonstrated over 100-fold selectivity. This improvement
in selectivity came with an increase in potency as well;
sulfonamide 20d is 16-fold more potent than 7.
Sulfonamides and sulfamides were not the only polar
functionalities to be utilized. Amides, ureas, and carbamates
were also synthesized to increase the polarity. Examples of all
three have been found to be potent and selective. However,
polar groups caused a loss of activity unless they were
accompanied by additional lipophilicity. This is best demon-
strated by comparing 18g to 18h. The unsubstituted
piperidinecarboxamide 18g contained no additional lipophil-
icity and had poor activity at CB1 (18g, Ke(CB1) = 4097 nM).
With the addition of an ethyl group, such as found in
compound 18h, the activity was significantly increased (18h,
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Table 5. In Vivo Evaluation of Select Compounds
a
b
LOQ: below limit of quantitation. NA: not applicable.
coupling constants (J) are reported in hertz (Hz). Thin-layer
chromatography (TLC) was performed on EMD precoated silica gel
60 F254 plates, and spots were visualized with UV light or I2 detection.
Low-resolution mass spectra were obtained using a Waters Alliance
HT/Micromass ZQ system (ESI). All test compounds were greater
than 95% pure as determined by HPLC on an Agilent 1100 system
using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 μm column
with gradient elution using the mobile phases (A) H2O containing
0.05% CF3COOH and (B) methanol. A flow rate of 1.0 mL/min was
used.
1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxylic Acid
(10). A 2 M solution of oxalyl chloride in dichloromethane (3
equiv, 0.19 mL, 0.377 mmol) was added to 5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (22) (1
equiv, 48 mg, 0.126 mmol) in dichloromethane (5 mL). Next, 2
drops of anhydrous N,N-dimethylformamide was added, and the
mixture was stirred for 2 h. The mixture was concentrated in vacuo.
The reaction mixture was dissolved in dichloromethane (5 mL).
Triethylamine (3 equiv, 0.05 mL, 0.377 mmol) and 4-carboxy-
4-phenylpiperidin-1-ium chloride (23) (1.5 equiv, 45.7 mg, 0.189
mmol) was added, and the mixture was stirred for 16 h. The mixture
was concentrated in vacuo. The crude reaction material was then
purified by silica gel column chromatography using 0−10% methanol/
dichloromethane with 1% acetic acid to yield pure desired product
(10) (48 mg, 67%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.87−
2.09 (m, 2 H), 2.15 (s, 3 H), 2.61 (t, J = 16.18 Hz, 2 H), 3.21 (t, J =
12.03 Hz, 1 H), 3.47 (t, J = 11.94 Hz, 1 H), 4.26 (d, J = 13.61 Hz,
1 H), 4.57 (d, J = 13.56 Hz, 1 H), 7.05 (d, J = 8.34 Hz, 2 H), 7.12−
7.45 (m, 10 H); [M + H]+ 568.4.
stable as 1 with low loss of the parent molecule even after 2 h of
incubation. Further evidence that some of these compounds do
not penetrate the CNS was seen in an in vivo pharmacokinetics
(PK) assay on compounds 17a, 18a, and 18f. Of these, 17a and
18a had little to no CNS penetration as demonstrated by a very
low brain/plasma ratio. Further, both compounds demon-
strated limited but clearly detectable oral absorption. Future
studies will be aimed at improving the oral bioavailability of this
class of compounds as well as exploring other scaffolds. Efficacy
studies in disease models where these compounds may be
useful are being planned as are more detailed PK studies to
establish compound half-lives and dosing regimens.
In conclusion, a series of highly potent and selective CB1
antagonists were synthesized and evaluated leading to the
identification of two compounds with limited brain penetration.
These compounds will serve as templates for further refinement
to enhance their oral bioavailability and to examine the role of
peripheral CB1 receptors in various diseases such as obesity,
liver fibrosis, and diabetes.
EXPERIMENTAL SECTION
■
Compound Synthesis and Characterization. Chemistry.
Reactions were conducted under N2 atmosphere using oven-dried
glassware. All solvents and chemicals used were reagent grade.
Anhydrous tetrahydrofuran, dichloromethane, and N,N-dimethylfor-
mamide (DMF) were purchased from Aldrich and used as such.
Unless otherwise mentioned, all reagents and chemicals were
purchased from commercial vendors and used as received. Flash
column chromatography was carried out on a Teledyne ISCO
CombiFlash Companion system using RediSep Rf prepacked columns.
Purity and characterization of compounds were established by a
combination of HPLC, TLC, and NMR analytical techniques
1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxamide
(11). 1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxylic acid (22) (1
equiv, 12.7 mg, 0.024 mmol), ammonium chloride (10 equiv, 12.7 mg,
0.24 mmol), benzotriazole-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) (1 equiv, 10.5 mg, 0.024 mmol), and
triethylamine (10.1 equiv, 0.03 mL, 0.024 mmol) were stirred in
tetrahydrofuran (5 mL) for 3 days. The mixture was concentrated in
vacuo. The crude reaction material was then purified by silica gel
1
described below. H and 13CNMR spectra were recorded on a Bruker
Avance DPX-300 (300 MHz) spectrometer and were determined in
CHCl3-d or MeOH-d4 with tetramethylsilane (TMS) (0.00 ppm) or
solvent peaks as the internal reference unless otherwise noted.
Chemical shifts are reported in ppm relative to the solvent signal, and
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column chromatography using 0−100% ethyl acetate/hexane to yield
pure desired product (11) (6 mg, 44%). 1H NMR (300 MHz,
chloroform-d) δ ppm 1.92−2.31 (m, 5 H), 2.46 (d, J = 13.94 Hz, 2 H),
3.65 (t, J = 10.36 Hz, 1 H), 3.75−3.90 (m, 1 H), 4.02 (d, J = 13.38 Hz,
1 H), 4.23 (d, J = 13.00 Hz, 1 H), 5.24 (br s, 2 H), 7.07 (d, J = 8.38
Hz, 2 H), 7.12−7.20 (m, 1 H), 7.20−7.49 (m, 9 H); [M + H]+ 567.5.
1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-(ethylamino)piperidine-4-carboxa-
mide (12). 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (22) (1 equiv, 20 mg, 0.052 mmol),
triethylamine (3 equiv, 0.02 mL, 0.157 mmol), 4-(ethylamino)-
4-piperidinecarboxamide (1 equiv, 9 mg, 0.052 mmol), and ben-
zotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophos-
phate (BOP) (1 equiv, 23 mg, 0.052 mmol) were stirred in
tetrahydrofuran (5 mL) for 16 h. The mixture was concentrated in
vacuo. The crude reaction material was then purified by silica gel
column chromatography using 0−100% CMA 80 (chloroform,
methanol, ammonium hydroxide 80:18:2)/ethyl acetate and precipi-
tated from ethyl acetate with hexane to yield pure desired product
ophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-
amine (14) (1 equiv, 36.5 mg, 0.068 mmol), methanesulfonyl chloride
(2 equiv, 0.01 mL, 0.135 mmol), and triethylamine (3 equiv, 0.03 mL,
0.203 mmol) were stirred in tetrahydrofuran for 16 h. The mixture was
concentrated in vacuo. The crude reaction material was then purified
by silica gel column chromatography using 0−100% ethyl acetate/
1
hexane to yield pure desired product (16) (27 mg, 65%). H NMR
(300 MHz, chloroform-d) δ ppm 2.18 (d, J = 4.52 Hz, 6 H), 2.21−
2.37 (m, 2 H), 2.39−2.61 (m, 2 H), 3.65 (t, J = 10.69 Hz, 1 H), 3.86
(t, J = 10.93 Hz, 1 H), 4.07−4.20 (m, 2 H), 4.29 (d, J = 13.75 Hz, 1
H), 5.30 (s, 1 H), 7.03−7.11 (m, 2 H), 7.15−7.21 (m, 1 H), 7.21−7.38
(m, 4 H), 7.38−7.47 (m, 2 H), 7.47−7.55 (m, 2 H); [M + H]+ 617.3.
3-tert-Butyl-1-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-
yl)urea (17a). 1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14) (1
equiv, 39.3 mg, 0.073 mmol), tert-butyl isocyanate (1.5 equiv, 0.013
mL, 0.109 mmol), and triethylamine (3.0 equiv, 0.03 mL, 0.218 mmol)
were stirred in dichloromethane (5 mL) for 16 h. Next,
tetrahydrofuran (5 mL) and an additional 0.02 mL of tert-butyl
isocyanate were added, and the mixture was stirred for 16 h. Finally,
the mixture was heated to 40 °C for 16 h. The mixture was
concentrated in vacuo. The crude reaction material was then purified
by silica gel column chromatography using 0−100% ethyl acetate/
1
(12) (13 mg, 46%). H NMR (300 MHz, chloroform-d) δ ppm 1.10
(t, J = 6.97 Hz, 3 H), 1.61−1.80 (m, 2 H), 2.08−2.26 (m, 5 H), 2.45−
2.63 (m, 2 H), 3.68 (td, J = 8.85, 4.43 Hz, 2 H), 3.96−4.19 (m, 2 H),
5.40 (br s, 1 H), 7.07 (d, J = 8.29 Hz, 2 H), 7.12−7.19 (m, 1 H),
7.20−7.36 (m, 3 H), 7.44 (d, J = 1.98 Hz, 1 H); [M + H]+ 534.5.
tert-Butyl N-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-
carbamate (13). 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-carboxylic acid (22) (1 equiv, 201 mg, 0.53 mmol),
triethylamine (3 equiv, 0.22 mL, 0.157 mmol), tert-butyl N-(4-
phenylpiperidin-4-yl)carbamate (25) (1 equiv, 146 mg, 0.53 mmol),
and benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) (1 equiv, 233 mg, 0.53 mmol) were stirred
in tetrahydrofuran (10 mL) for 16 h. The mixture was concentrated in
vacuo. The crude reaction material was then purified by silica gel
column chromatography using 0−100% ethyl acetate/hexane to yield
pure desired product (13) (295 mg, 87%). 1H NMR (300 MHz,
chloroform-d) δ ppm 1.37 (br s, 9 H), 2.11 (dd, J = 12.67, 4.00 Hz, 2
H), 2.21 (s, 3 H), 2.24−2.34 (m, 1 H), 2.34−2.56 (m, 1 H), 3.26 (t,
J = 12.01 Hz, 1 H), 3.56 (t, J = 12.39 Hz, 1 H), 4.32 (d, J = 13.75 Hz,
1 H), 4.64 (d, J = 13.56 Hz, 1 H), 4.96 (br s, 1 H), 7.08 (d, J = 8.38
Hz, 2 H), 7.13−7.49 (m, 10 H); [M + H]+ 639.7.
1
hexane to yield pure desired product (17a) (21 mg, 45%). H NMR
(300 MHz, chloroform-d) δ ppm 1.15 (s, 9 H), 1.93−2.17 (m, 4 H),
2.20 (s, 3 H), 2.42 (br s, 1 H), 3.13−3.35 (m, 1 H), 3.58 (br s, 1 H),
4.25 (br s, 1 H), 4.44 (s, 1 H), 4.52−4.69 (m, 1 H), 5.13 (s, 1 H),
7.03−7.10 (m, 2 H), 7.13−7.37 (m, 7 H), 7.38−7.46 (m, 3 H); [M +
H]+ 638.7.
1-Benzyl-3-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)urea
(17b). Amine 14 (1 equiv, 26.1 mg, 0.048 mmol), benzyl isocyanate
(1.5 equiv, 9.7 mg, 0.073 mmol), and triethylamine (3.0 equiv,
0.02 mL, 0.145 mmol) were stirred in THF (2 mL) for 16 h. The
mixture was concentrated in vacuo. The crude reaction material was
then purified by silica gel column chromatography using 0−100% ethyl
acetate/hexane to yield 17b (24 mg, 74%). 1H NMR (300 MHz,
chloroform-d) δ ppm 1.79−2.13 (m, 3 H), 2.17 (s, 3 H), 2.53 (br s,
1 H), 3.13 (br s, 1 H), 3.48 (br s, 1 H), 4.21 (d, J = 5.75 Hz, 3 H), 4.49
(d, J = 13.47 Hz, 1 H), 5.24 (br s, 1 H), 5.53 (s, 1 H), 6.94−7.49 (m,
17 H); [M + H]+ 672.4.
1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14). tert-
Butyl N-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)carbamate (13)
(1 equiv, 243 mg, 0.380 mmol) was stirred in dichloromethane
(7 mL) and trifluoroacetic acid (3 mL) for 16 h. The mixture was
concentrated in vacuo. The crude reaction material was then purified
by silica gel column chromatography using 0−50% CMA 80/ethyl
3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-(4-
cyanophenyl)urea (17c). Following a procedure similar to the
preparation of 17b, 17c was obtained from 14 and the appropriate
1
isocyanate in 74% yield. H NMR (300 MHz, chloroform-d) δ ppm
1.98 (br s, 2 H), 2.11−2.41 (m, 4 H), 2.87 (d, J = 13.47 Hz, 1 H), 3.40
(br s, 1 H), 3.64 (br s, 1 H), 3.97−4.24 (m, 1 H), 4.58 (d, J =
13.19 Hz, 1 H), 6.79 (s, 1 H), 6.90−7.52 (m, 16 H), 8.56 (br s, 1 H);
[M + H]+ 683.8.
1
acetate to yield pure desired product (14) (178 mg, 87%). H NMR
(300 MHz, chloroform-d) δ ppm 1.64−1.95 (m, 2 H), 2.10−2.29 (m,
5 H), 3.50−3.66 (m, 1 H), 3.70−3.88 (m, 1 H), 4.03−4.19 (m, 1 H),
4.42 (d, J = 13.28 Hz, 1 H), 7.04−7.10 (m, 2 H), 7.13−7.20 (m, 1 H),
7.20−7.40 (m, 6 H), 7.40−7.50 (m, 3 H); [M + H]+ 539.4.
3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-(4-
fluorophenyl)urea (17d). Following a procedure similar to the
preparation of 17b, 17d was obtained from 14 and the appropriate
N-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)acetamide
(15). 1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14) (1 equiv, 35.3
mg, 0.066 mmol) was stirred in a mixture of acetic anhydride (2 mL)
and pyridine (2 mL) for 16 h. The mixture was concentrated in vacuo.
The crude reaction material was then purified by silica gel column
chromatography using 0−100% ethyl acetate/hexane to yield pure
desired product (15) (27 mg, 71%). 1H NMR (300 MHz, chloroform-
d) δ ppm 2.01 (s, 3 H), 2.05−2.19 (m, 2 H), 2.21 (s, 3 H), 2.34 (d, J =
14.60 Hz, 1 H), 2.66 (d, J = 13.85 Hz, 1 H), 3.15−3.34 (m, 1 H), 3.52
(t, J = 11.68 Hz, 1 H), 4.26 (d, J = 13.75 Hz, 1 H), 4.54 (d, J = 13.75
Hz, 1 H), 6.10 (s, 1 H), 7.03−7.11 (m, 2 H), 7.14−7.19 (m, 1 H),
7.19−7.41 (m, 8 H), 7.44 (d, J = 2.17 Hz, 1 H); [M + H]+ 581.0.
N-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-
methanesulfonamide (16). 1-{[5-(4-Chlorophenyl)-1-(2,4-dichlor-
1
isocyanate in 67% yield. H NMR (300 MHz, chloroform-d) δ ppm
1.95 (br s, 2 H), 2.08−2.34 (m, 4 H), 2.83 (d, J = 13.37 Hz, 1 H),
3.23−3.41 (m, 1 H), 3.58 (t, J = 12.29 Hz, 1 H), 4.21 (d, J = 13.56 Hz,
1 H), 4.58 (d, J = 13.28 Hz, 1 H), 6.31 (br s, 1 H), 6.82 (t, J = 8.62 Hz,
2 H), 6.93−7.48 (m, 14 H), 7.78 (br s, 1 H); [M + H]+ 676.3.
3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-[4-
(dimethylamino)phenyl]urea (17e). Following a procedure similar
to the preparation of 17b, 17e was obtained from 14 and the
appropriate isocyanate in 58% yield. 1H NMR (300 MHz, chloroform-
d) δ ppm 1.86−2.15 (m, 3 H), 2.21 (s, 3 H), 2.62 (d, J = 13.47 Hz,
1 H), 2.90 (s, 6 H), 3.11 (br s, 1 H), 3.47 (br s, 1 H), 4.29 (d, J = 13.37
Hz, 1 H), 4.59 (d, J = 13.37 Hz, 1 H), 5.40 (br s, 1 H), 6.50 (br s, 1 H),
6.66 (d, J = 8.67 Hz, 2 H), 6.94−7.49 (m, 14 H); [M + H]+ 701.6.
1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-hexylurea
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(17f). Following a procedure similar to the preparation of 17b, 17f was
obtained from 14 and the appropriate isocyanate in 72% yield. H
was stirred in dichloromethane (4 mL) and trifluoroacetic acid (1 mL)
for 16 h. The mixture was concentrated in vacuo. The crude reaction
material was then purified by silica gel column chromatography using
0−100% CMA 80/ethyl acetate to yield pure desired product (18b)
1
NMR (300 MHz, chloroform-d) δ ppm 0.84 (t, J = 6.78 Hz, 3 H),
1.04−1.38 (m, 8 H), 1.95−2.17 (m, 3 H), 2.17−2.25 (m, 3 H), 2.51
(br s, 1 H), 2.90−3.11 (m, 2 H), 3.24 (br s, 1 H), 3.55 (br s, 1 H),
4.28 (d, J = 13.56 Hz, 1 H), 4.48−4.65 (m, 2 H), 5.18−5.38 (m, 1 H),
6.96−7.51 (m, 12 H); [M − H]− 666.8.
1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-(propan-
2-yl)urea (17g). Following a procedure similar to the preparation of
17b, 17g was obtained from 14 and the appropriate isocyanate in 73%
yield. 1H NMR (300 MHz, chloroform-d) δ ppm 0.82−1.03 (m, 6 H)
1.94−2.24 (m, 6 H) 2.46 (d, J = 13.47 Hz, 1 H) 3.26 (t, J = 11.26 Hz,
1 H) 3.56 (t, J = 12.10 Hz, 1 H) 3.68−3.85 (m, 1 H) 4.16−4.37 (m,
2 H) 4.59 (d, J = 13.47 Hz, 1 H) 5.05 (s, 1 H) 6.94−7.49 (m, 12 H);
[M + H]+ 624.7.
1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-ethylurea
(17h). Following a procedure similar to the preparation of 17b, 17h
was obtained from 14 and the appropriate isocyanate in 73% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 0.78−1.04 (m, 3 H), 1.92−
2.25 (m, 6 H), 2.51 (d, J = 13.56 Hz, 1 H), 2.98−3.15 (m, 2 H), 3.24
(br s, 1 H), 3.55 (br s, 1 H), 4.29 (br s, 1 H), 4.44−4.68 (m, 2 H),
5.23 (s, 1 H), 6.90−7.49 (m, 12 H); [M + H]+ 610.1.
1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-propylur-
ea (17i). Following a procedure similar to the preparation of 17b, 17i
was obtained from 14 and the appropriate isocyanate in 71% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 0.74 (t, J = 7.39 Hz, 3 H),
1.21−1.40 (m, 2 H), 2.05−2.29 (m, 6 H), 2.35 (br s, 1 H), 3.02 (q, J =
6.72 Hz, 2 H), 3.28 (br s, 1 H), 3.57 (br s, 1 H), 4.07 (t, J = 5.27 Hz,
1 H), 4.34 (d, J = 13.66 Hz, 1 H), 4.63 (d, J = 14.32 Hz, 1 H), 4.74 (s,
1 H), 7.00−7.54 (m, 12 H); [M + H]+ 624.8.
1
(415 mg, 95%). H NMR (300 MHz, chloroform-d) δ ppm 1.44 (qd,
J = 11.81, 3.86 Hz, 2 H), 1.92−2.08 (m, 2 H), 2.37 (s, 3 H), 2.64−2.85
(m, 2 H), 2.98−3.21 (m, 2 H), 3.92−4.19 (m, 1 H), 6.85 (d, J =
8.29 Hz, 1 H), 7.06 (d, J = 8.38 Hz, 2 H), 7.28 (s, 4 H), 7.43 (s, 1 H);
[M + H]+ 463.5.
N-(1-Acetylpiperidin-4-yl)-5-(4-chlorophenyl)-1-(2,4-dichlor-
ophenyl)-4-methyl-1H-pyrazole-3-carboxamide (18c). 5-(4-
Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-
1H-pyrazole-3-carboxamide (18b) (1 equiv, 34 mg, 0.073 mmol) was
stirred in pyridine (1 mL) and acetic anhydride (1 mL) for 16 h. The
mixture was concentrated in vacuo. The crude reaction material was
then purified by silica gel column chromatography using 0−100% ethyl
acetate/hexane to yield pure desired product (18c) (30 mg, 81%). 1H
NMR (300 MHz, chloroform-d) δ ppm 1.28−1.49 (m, 2 H), 1.87−
2.13 (m, 5 H), 2.30 (s, 3 H), 2.62−2.82 (m, 1 H), 3.05−3.24 (m, 1 H),
3.75 (d, J = 13.56 Hz, 1 H), 3.98−4.25 (m, 1 H), 4.49 (d, J = 13.37 Hz,
1 H), 6.80 (d, J = 8.01 Hz, 1 H), 6.99 (d, J = 8.48 Hz, 2 H), 7.13−7.29
(m, 4 H), 7.36 (d, J = 1.51 Hz, 1 H); [M + H]+ 505.5.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-methane-
sulfonylpiperidin-4-yl)-4-methyl-1H-pyrazole-3-carboxamide
(18d). Methanesulfonyl chloride (2 equiv, 0.01 mL, 0.15 mmol) was
added to 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(pi-
peridin-4-yl)-1H-pyrazole-3-carboxamide (18b) (1 equiv, 35 mg, 0.076
mmol) and triethyamine (3 equiv, 0.03 mL, 0.227 mmol) in
tetrahydrofuran (2 mL). The mixture was stirred for 16 h. The
mixture was concentrated in vacuo. The crude reaction material was
then purified by silica gel column chromatography using 0−100% ethyl
acetate/hexane to yield pure desired product (18d) (36 mg, 87%). 1H
NMR (300 MHz, chloroform-d) δ ppm 1.53−1.78 (m, 2 H), 2.06−
2.22 (m, 2 H), 2.37 (s, 3 H), 2.67−3.00 (m, 5 H), 3.82 (d, J = 12.24 Hz,
2 H), 4.01−4.17 (m, 1 H), 6.88 (d, J = 8.01 Hz, 1 H), 7.07 (s, 2 H),
7.19−7.36 (m, 4 H), 7.43 (d, J = 1.70 Hz, 1 H); [M + H]+ 543.6.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-
sulfamoylpiperidin-4-yl)-1H-pyrazole-3-carboxamide (18e). 5-
(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-
yl)-1H-pyrazole-3-carboxamide (18b) (1 equiv, 38 mg, 0.082 mmol)
and sulfamide (5 equiv, 39 mg, 0.41 mmol) were heated to 90 °C in
dioxane (2 mL) for 16 h. The mixture was concentrated in vacuo. The
crude reaction material was then purified by silica gel column
chromatography using 0−100% ethyl acetate/hexane to yield desired
3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-cyclohex-
ylurea (17j). Following a procedure similar to the preparation of 17b,
17j was obtained from 14 and the appropriate isocyanate in 69% yield.
1H NMR (300 MHz, chloroform-d) δ ppm 0.78−0.98 (m, 2 H), 1.06
(d, J = 9.89 Hz, 1 H), 1.16−1.35 (m, 2 H), 1.50 (d, J = 8.76 Hz, 3 H),
1.73 (d, J = 10.83 Hz, 2 H), 2.00−2.27 (m, 6 H), 2.43 (d, J = 13.56 Hz,
1 H), 3.26 (br s, 1 H), 3.37−3.66 (m, 2 H), 4.15−4.40 (m, 2 H), 4.62
(br s, 1 H), 4.99 (s, 1 H), 6.90−7.55 (m, 12 H); [M + H]+ 664.9.
3-Butyl-1-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)urea
(17k). Following a procedure similar to the preparation of 17b, 17k
was obtained from 14 and the appropriate isocyanate in 71% yield. 1H
NMR (300 MHz, chloroform-d) δ ppm 0.76−0.86 (m, 3 H), 1.08−
1.21 (m, 2 H), 1.28 (dq, J = 14.40, 7.10 Hz, 2 H), 1.94−2.26 (m, 6 H),
2.50 (d, J = 13.47 Hz, 1 H), 3.04 (q, J = 6.56 Hz, 2 H), 3.15−3.32 (m,
1 H), 3.55 (t, J = 12.15 Hz, 1 H), 4.27 (d, J = 13.56 Hz, 1 H), 4.48−
4.74 (m, 2 H), 5.33 (s, 1 H), 6.98−7.49 (m, 12 H); [M + H]+ 638.6.
tert-Butyl 4-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (18a).
Benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) (1 equiv, 490 mg, 1.11 mmol) was added to a
solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (22) (1 equiv, 422 mg, 1.11 mmol), tert-
butyl 4-amino-1-piperidinecarboxylate (1 equiv, 222 mg, 1.11 mmol),
and triethylamine (3 equiv, 0.46 mL, 3.32 mmol) in tetrahydrofuran
(5 mL). The reaction mixture was stirred for 16 h. The mixture was
concentrated in vacuo. The crude reaction material was then purified
by silica gel column chromatography using 0−100% ethyl acetate/
1
product (18e) (30 mg, 67%). H NMR (300 MHz, chloroform-d)
δ ppm 1.60−1.78 (m, 2 H), 2.07−2.21 (m, 2 H), 2.37 (s, 3 H), 2.86 (t,
J = 10.83 Hz, 2 H), 3.74 (d, J = 12.24 Hz, 2 H), 4.00−4.16 (m, 1 H),
4.37 (s, 2 H), 6.87 (d, J = 7.91 Hz, 1 H), 7.06 (d, J = 8.38 Hz, 2 H),
7.20−7.37 (m, 4 H), 7.43 (d, J = 1.32 Hz, 1 H); [M + H]+ 542.7.
1-N-tert-Butyl-4-C-5-(4-chlorophenyl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazole-3-piperidine-1,4-diamido (18f). 5-(4-
Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-
1H-pyrazole-3-carboxamide (18b) (1 equiv, 38 mg, 0.082 mmol), tert-
butyl isocyanate (1.5 equiv, 0.014 mL, 0.123 mmol), and triethylamine
(3 equiv, 0.034 mL, 0.246 mmol) were stirred in dichloromethane for
16 h. The mixture was concentrated in vacuo. The crude reaction
material was then purified by silica gel column chromatography using
0−100% ethyl acetate/hexane to yield pure desired product (18f)
(42 mg, 91%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.27 (d, J =
7.16 Hz, 9 H), 1.40−1.73 (m, 2 H), 2.05 (s, 2 H), 2.37 (s, 2 H), 2.92
(t, J = 11.44 Hz, 2 H), 3.87 (d, J = 13.37 Hz, 2 H), 4.00−4.18 (m, 1
H), 4.33 (s, 1 H), 6.84 (d, J = 7.91 Hz, 1 H), 7.05 (d, J = 8.48 Hz, 2
H), 7.20−7.35 (m, 4 H), 7.42 (s, 1 H); [M + H]+ 562.4.
1
hexane to yield pure desired product (18a) (548 mg, 88%). H NMR
(300 MHz, chloroform-d) δ ppm 1.33−1.51 (m, 9 H), 1.93−2.10 (m,
2 H), 2.37 (s, 3 H), 2.91 (t, J = 11.82 Hz, 2 H), 3.89−4.23 (m, 2 H),
6.84 (d, J = 8.19 Hz, 1 H), 7.00−7.12 (m, 2 H), 7.19−7.36 (m, 4 H),
7.43 (d, J = 1.32 Hz, 1 H); [M + H]+ 563.6.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(pi-
peridin-4-yl)-1H-pyrazole-3-carboxamide (18b). tert-Butyl 4-[5-
(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
amido]piperidine-1-carboxylate (18a) (1 equiv, 531 mg, 0.941 mmol)
4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-piperidine-1,4-diamido (18g). 1-N-tert-Butyl-4-
C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
piperidine-1,4-diamido (18f) (1 equiv, 33 mg, 0.059 mmol) was stirred
in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) overnight.
The mixture was concentrated in vacuo. The crude reaction material
was then purified by silica gel column chromatography using 0−100%
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CMA 80/ethyl acetate to yield pure desired product (18g) (24 mg,
81%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.41−1.60 (m, 2 H),
1.99−2.12 (m, 2 H), 2.37 (s, 3 H), 3.01 (t, J = 11.77 Hz, 2 H), 3.94 (d,
J = 13.09 Hz, 2 H), 4.03−4.24 (m, 1 H), 4.65 (br s, 2 H), 6.90 (d, J =
7.91 Hz, 1 H), 7.06 (d, J = 8.38 Hz, 2 H), 7.22−7.37 (m, 4 H), 7.43 (s,
1 H); [M + H]+ 506.4.
Following a procedure similar to the preparation of 18c, 19c was
obtained from 19b in 87% yield. H NMR (300 MHz, chloroform-d)
δ ppm 1.62 (s, 4 H), 2.01−2.08 (m, 3 H), 2.30 (s, 3 H), 3.10−3.29 (m,
2 H), 3.81 (d, J = 13.19 Hz, 2 H), 4.00 (d, J = 6.59 Hz, 1 H), 6.86 (d,
J = 7.06 Hz, 1 H), 6.99 (d, J = 8.38 Hz, 2 H), 7.15−7.28 (m, 4 H),
7.36 (s, 1 H); [M + H]+ 505.6.
1
4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-1-N-ethylpiperidine-1,4-diamido (18h). Follow-
ing a procedure similar to the preparation of 17b, 18h was obtained
from 18b and the appropriate isocyanate in 97% yield. 1H NMR
(300 MHz, chloroform-d) δ ppm 1.15 (t, J = 7.21 Hz, 3 H), 1.35−1.60
(m, 2 H), 1.93−2.14 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J = 11.54 Hz,
2 H), 3.16−3.38 (m, 2 H), 3.94 (d, J = 13.47 Hz, 2 H), 4.04−4.25 (m,
1 H), 4.46 (br s, 1 H), 6.87 (d, J = 8.01 Hz, 1 H), 7.08 (s, 2 H), 7.23−
7.37 (m, 4 H), 7.44 (d, J = 1.22 Hz, 1 H); [M + H]+ 534.4.
4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,4-diamido (18i).
Following a procedure similar to the preparation of 17b, 18i was
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(3S)-1-metha-
nesulfonylpiperidin-3-yl]-4-methyl-1H-pyrazole-3-carboxa-
mide (19d). Following a procedure similar to the preparation of 18d,
19d was obtained from 19b in 73% yield. 1H NMR (300 MHz,
chloroform-d) δ ppm 1.58−1.73 (m, 2 H), 1.83 (d, J = 10.46 Hz, 2 H),
2.29 (s, 3 H), 2.74 (s, 3 H), 2.99−3.25 (m, 3 H), 3.45 (dd, J = 11.73,
3.16 Hz, 1 H), 4.24 (br s, 1 H), 6.84−7.11 (m, 3 H), 7.14−7.28 (m,
4 H), 7.35 (s, 1 H); [M + H]+ 543.5.
(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-ethylpiperidine-1,3-diamido (19e). Fol-
lowing a procedure similar to the preparation of 17b, 19e was
1
obtained from 19b and the appropriate isocyanate in 80% yield. H
1
obtained from 18b and the appropriate isocyanate in 99% yield. H
NMR (300 MHz, chloroform-d) δ ppm 1.04 (t, J = 7.39 Hz, 3 H), 1.55
(d, J = 7.44 Hz, 3 H), 1.91 (br s, 1 H), 2.29 (s, 3 H), 2.95−3.30 (m, 4
H), 3.60 (br s, 1 H), 3.97 (br s, 1 H), 4.63 (br s, 1 H), 6.90 (d, J = 6.97
Hz, 1 H), 6.99 (d, J = 8.38 Hz, 2 H), 7.15−7.29 (m, 4 H), 7.35 (s,
1 H); [M − H]− 534.4.
NMR (300 MHz, chloroform-d) δ ppm 1.16 (d, J = 6.50 Hz, 6 H),
1.49 (dd, J = 11.68, 3.11 Hz, 2 H), 1.98−2.11 (m, 2 H), 2.38 (s, 3 H),
2.95 (t, J = 11.68 Hz, 2 H), 3.86−4.02 (m, 3 H), 4.11 (dd, J = 13.70,
6.92 Hz, 1 H), 4.27 (d, J = 7.16 Hz, 1 H), 6.86 (d, J = 7.91 Hz, 1 H),
7.07 (d, J = 8.38 Hz, 2 H), 7.25−7.36 (m, 4 H), 7.44 (s, 1 H);
[M + H]+ 548.5.
(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,3-diamido
(19f). Following a procedure similar to the preparation of 17b, 19f was
4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-1-N-propylpiperidine-1,4-diamido (18j). Follow-
ing a procedure similar to the preparation of 17b, 18j was obtained
from 18b and the appropriate isocyanate in 95% yield. 1H NMR
(300 MHz, chloroform-d) δ ppm 0.86−0.98 (m, 3 H), 1.43−1.60 (m,
4 H), 1.94−2.13 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J = 11.77 Hz, 2 H),
3.20 (q, J = 6.69 Hz, 2 H), 3.94 (d, J = 13.37 Hz, 2 H), 4.09 (d, J =
6.78 Hz, 1 H), 4.51 (br s, 1 H), 6.87 (d, J = 8.01 Hz, 1 H), 7.07 (d, J =
8.29 Hz, 2 H), 7.23−7.37 (m, 4 H), 7.44 (s, 1 H); [M + H]+ 548.6.
1-N-Butyl-4-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-piperidine-1,4-diamido (18k). Following
a procedure similar to the preparation of 17b, 18k was obtained from
18b and the appropriate isocyanate in 76% yield. 1H NMR (300 MHz,
chloroform-d) δ ppm 0.94 (t, J = 7.16 Hz, 3 H), 1.28−1.41 (m, 2 H),
1.42−1.58 (m, 4 H), 1.95−2.12 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J =
12.24 Hz, 2 H), 3.16−3.33 (m, 2 H), 3.94 (d, J = 13.37 Hz, 2 H),
4.03−4.23 (m, 1 H), 4.47 (br s, 1 H), 6.86 (d, J = 7.91 Hz, 1 H), 7.07
(d, J = 8.29 Hz, 2 H), 7.23−7.36 (m, 4 H), 7.44 (s, 1 H); [M + H]+
562.4.
1
obtained from 19b and the appropriate isocyanate in 78% yield. H
NMR (300 MHz, chloroform-d) δ ppm 1.01 (d, J = 6.50 Hz, 3 H),
1.07 (d, J = 6.50 Hz, 3 H), 1.41−1.66 (m, 3 H), 1.88 (d, J = 6.40 Hz, 1
H), 2.30 (s, 3 H), 3.10−3.27 (m, 2 H), 3.40−3.61 (m, 2 H), 3.87 (d,
J = 6.59 Hz, 1 H), 3.93−4.04 (m, 1 H), 4.43 (d, J = 7.06 Hz, 1 H),
6.81−7.05 (m, 3 H), 7.10−7.28 (m, 4 H), 7.35 (s, 1 H); [M − H]−
548.7.
(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-propylpiperidine-1,3-diamido (19g). Fol-
lowing a procedure similar to the preparation of 17b, 19g was obtained
from 19b and the appropriate isocyanate in 78% yield. 1H NMR (300
MHz, chloroform-d) δ ppm 0.71−0.87 (m, 3 H), 1.35−1.47 (m, 2 H),
1.48−1.63 (m, 3 H), 1.90 (br s, 1 H), 2.29 (s, 3 H), 2.95−3.27 (m,
4 H), 3.58 (br s, 2 H), 3.98 (d, J = 6.69 Hz, 1 H), 4.68 (br s, 1 H),
6.86−7.05 (m, 3 H), 7.12−7.29 (m, 4 H), 7.35 (s, 1 H); [M + H]+
548.6.
(3S)-1-N-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (19h). Fol-
lowing a procedure similar to the preparation of 17b, 19h was
Ethyl 4-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-amido]piperidine-1-carboxylate (18l). Follow-
ing a procedure similar to the preparation of 20j, 18l was obtained
from 18b and ethyl chloroformate in 76% yield. 1H NMR (300 MHz,
chloroform-d) δ ppm 1.27 (d, J = 14.22 Hz, 3 H), 1.47 (dd, J = 11.63,
3.44 Hz, 2 H), 1.93−2.14 (m, 2 H), 2.38 (s, 3 H), 2.98 (br s, 2 H),
4.14 (q, J = 6.97 Hz, 4 H), 6.86 (d, J = 8.10 Hz, 1 H), 7.06 (s, 2 H),
7.22−7.38 (m, 4 H), 7.44 (d, J = 1.51 Hz, 1 H); [M + H]+ 535.3.
tert-Butyl (3S)-3-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate
(19a). Following a procedure similar to the preparation of 18a, 19a
1
obtained from 19b and the appropriate isocyanate in 85% yield. H
NMR (300 MHz, chloroform-d) δ ppm 0.74−0.94 (m, 3 H), 1.09−
1.32 (m, 2 H), 1.33−1.46 (m, 2 H), 1.46−1.63 (m, 3 H), 1.90 (br s,
1 H), 2.29 (s, 3 H), 3.05−3.21 (m, 4 H), 3.58 (t, J = 14.32 Hz, 2 H),
3.84−4.06 (m, 1 H), 4.65 (br s, 1 H), 6.90 (d, J = 6.88 Hz, 1 H), 6.99
(d, J = 8.38 Hz, 2 H), 7.16−7.29 (m, 4 H), 7.35 (s, 1 H); [M − H]−
562.5.
(3S)-1-N-tert-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (19i).
Following a procedure similar to the preparation of 17b, 19i was
1
was obtained from 22 and an appropriate amine in 96% yield. H
1
obtained from 19b and the appropriate isocyanate in 77% yield. H
NMR (300 MHz, chloroform-d) δ ppm 1.31 (s, 9 H), 1.49 (dd, J =
12.62, 5.84 Hz, 1 H), 1.65 (d, J = 5.09 Hz, 2 H), 1.82 (d, J = 9.04 Hz,
1 H), 2.29 (s, 3 H), 3.32 (br s, 3 H), 3.48−3.73 (m, 1 H), 3.93−4.14
(m, 1 H), 6.99 (d, J = 8.38 Hz, 3 H), 7.12−7.27 (m, 4 H), 7.33 (s,
1 H); [M + H]+ 563.4.
NMR (300 MHz, chloroform-d) δ ppm 1.07−1.29 (m, 9 H), 1.42−
1.60 (m, 2 H), 1.66 (s, 1 H), 1.80−1.95 (m, 1 H), 2.30 (s, 3 H), 3.10−
3.31 (m, 2 H), 3.46 (d, J = 2.45 Hz, 2 H), 3.99 (br s, 1 H), 4.51 (s,
1 H), 6.99 (d, J = 8.29 Hz, 3 H), 7.15−7.31 (m, 4 H), 7.34 (s, 1 H);
[M + H]+ 562.4.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[(3S)-
piperidin-3-yl]-1H-pyrazole-3-carboxamide (19b). Following a
procedure similar to the preparation of 18b, 19b was obtained from
Ethyl (3S)-3-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (19j).
Following a procedure similar to the preparation of 20j, 19j was
1
19a in >99% yield. H NMR (300 MHz, chloroform-d) δ ppm 1.42−
1
obtained from 19b and ethyl chloroformate in 67% yield. H NMR
1.95 (m, 4 H), 2.26 (s, 3 H), 2.86 (dd, J = 12.15, 8.76 Hz, 2 H), 3.09
(d, J = 12.81 Hz, 1 H), 3.40 (dd, J = 12.20, 2.97 Hz, 1 H), 4.13−4.32
(m, 1 H), 6.99 (s, 2 H), 7.09−7.28 (m, 5 H), 7.33 (s, 1 H); [M + H]+
463.7.
(300 MHz, chloroform-d) δ ppm 1.01−1.20 (m, 3 H), 1.40−1.75 (m,
3 H), 1.91 (br s, 1 H), 2.30 (s, 3 H), 3.16 (br s, 2 H), 3.47−3.64 (m, 1
H), 3.81 (d, J = 10.93 Hz, 1 H), 3.93−4.17 (m, 3 H), 6.88 (d, J = 8.01
Hz, 1 H), 6.99 (d, J = 8.38 Hz, 2 H), 7.15−7.28 (m, 4 H), 7.35 (s,
1 H); [M + H]+ 535.5.
N-[(3S)-1-Acetylpiperidin-3-yl]-5-(4-chlorophenyl)-1-(2,4-di-
chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (19c).
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tert-Butyl (3R)-3-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate
(20a). Following a procedure similar to the preparation of 18a, 20a
(3R)-1-N-tert-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (20i).
Following a procedure similar to the preparation of 17b, 20i was
1
1
was obtained from 22 and an appropriate amine in 89% yield. H
obtained from 20b and the appropriate isocyanate in 70% yield. H
NMR (300 MHz, chloroform-d) δ ppm 1.28−1.42 (m, 9 H), 1.47−
1.61 (m, 1 H), 1.70 (d, J = 5.18 Hz, 2 H), 1.87 (d, J = 8.95 Hz, 1 H),
2.35 (s, 3 H), 3.38 (br s, 3 H), 3.60 (br s, 1 H), 3.95−4.21 (m, 1 H),
7.04 (d, J = 8.38 Hz, 3 H), 7.19−7.33 (m, 4 H), 7.39 (s, 1 H); [M +
H]+ 563.3.
NMR (300 MHz, chloroform-d) δ ppm 1.22−1.38 (m, 9 H), 1.61 (d,
J = 8.01 Hz, 2 H), 1.71−1.79 (m, 1 H), 1.89−2.04 (m, 1 H), 2.38 (s,
3 H), 3.23−3.37 (m, 2 H), 3.54 (d, J = 2.73 Hz, 2 H), 4.07 (d, J = 6.78
Hz, 1 H), 4.59 (s, 1 H), 6.94−7.13 (m, 3 H), 7.20−7.39 (m, 4 H), 7.43
(s, 1 H); [M + H]+ 562.5.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
[(3R)-piperidin-3-yl]-1H-pyrazole-3-carboxamide (20b). Follow-
ing a procedure similar to the preparation of 18b, 20b was obtained
Ethyl (3R)-3-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (20j).
Amine 20b (1.0 equiv, 19.2 mg, 0.041 mmol), ethyl chloroformate
(1.5 equiv, 6.7 mg, 0.062 mmol), and triethyamine (3.0 equiv, 0.02
mL, 0.124 mmol) were stirred in THF (2 mL) at room temperature
for 16 h. The mixture was concentrated in vacuo. The crude reaction
material was then purified by silica gel column chromatography using
1
from 20b in >99% yield. H NMR (300 MHz, chloroform-d) δ ppm
1.54−2.01 (m, 4 H), 2.33 (s, 3 H), 2.80−3.00 (m, 2 H), 3.17 (d, J =
12.62 Hz, 1 H), 3.38−3.57 (m, 1 H), 4.22−4.41 (m, 1 H), 6.50 (br s,
2 H), 7.06 (d, J = 8.38 Hz, 2 H), 7.18−7.35 (m, 5 H), 7.41 (s, 1 H);
[M + H]+ 463.7.
1
0−100% ethyl acetate/hexane to yield 20j (15 mg, 68%). H NMR
N-[(3R)-1-Acetylpiperidin-3-yl]-5-(4-chlorophenyl)-1-(2,4-di-
chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (20c).
Following a procedure similar to the preparation of 18c, 20c was
(300 MHz, chloroform-d) δ ppm 1.08−1.22 (m, 3 H), 1.42−1.74 (m,
3 H), 1.91 (br s, 1 H), 2.30 (s, 3 H), 3.17 (br s, 2 H), 3.56 (d, J =
13.19 Hz, 1 H), 3.82 (d, J = 10.74 Hz, 1 H), 3.94−4.15 (m, 3 H), 6.88
(d, J = 7.91 Hz, 1 H), 6.94−7.04 (m, 2 H), 7.15−7.28 (m, 4 H), 7.36
(s, 1 H); [M + H]+ 535.4.
1
obtained from 20b in 87% yield. H NMR (300 MHz, chloroform-d)
δ ppm 1.72 (br s, 4 H), 2.11−2.17 (m, 3 H), 2.38 (s, 3 H), 3.18−3.37
(m, 2 H), 3.89 (d, J = 13.19 Hz, 2 H), 4.09 (d, J = 6.59 Hz, 1 H), 6.95
(d, J = 7.06 Hz, 1 H), 7.08 (d, J = 8.29 Hz, 2 H), 7.20−7.40 (m, 4 H),
7.44 (s, 1 H); [M + H]+ 505.5.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(4-
{[(ethylcarbamoyl)amino]methyl}cyclohexyl)methyl]-4-meth-
yl-1H-pyrazole-3-carboxamide (21a). Following a procedure
similar to the preparation of 17b, 21a was obtained from 26 and
the appropriate isocyanate in 91% yield. 1H NMR (300 MHz,
chloroform-d) δ ppm 1.01−1.12 (m, 3 H), 1.21−1.63 (m, 8 H), 1.72
(br s, 2 H), 2.29 (s, 3 H), 2.94 (br s, 1 H), 3.04 (br s, 1 H), 3.09−3.23
(m, 2 H), 3.30 (t, J = 6.73 Hz, 1 H), 4.48 (br s, 2 H), 6.85−7.06 (m,
3 H), 7.15−7.27 (m, 4 H), 7.36 (s, 1 H); [M + H]+ 576.6.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-{[4-
({[(propan-2-yl)carbamoyl]amino}methyl)cyclohexyl]methyl}-
1H-pyrazole-3-carboxamide (21b). Following a procedure similar
to the preparation of 17b, 21b was obtained from 26 and the
appropriate isocyanate in 84% yield. 1H NMR (300 MHz, chloroform-
d) δ ppm 1.07 (d, J = 6.40 Hz, 6 H), 1.25−1.61 (m, 8 H), 1.64−1.78
(m, 2 H), 2.30 (s, 3 H), 2.93 (br s, 1 H), 3.04 (br s, 1 H), 3.16−3.24
(m, 1 H), 3.25−3.36 (m, 1 H), 3.77 (br s, 1 H), 4.21 (br s, 1 H), 4.38
(br s, 1 H), 6.81−7.06 (m, 3 H), 7.12−7.28 (m, 4 H), 7.36 (s, 1 H);
[M + H]+ 590.5.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(3R)-1-metha-
nesulfonylpiperidin-3-yl]-4-methyl-1H-pyrazole-3-carboxa-
mide (20d). Following a procedure similar to the preparation of 18d,
20d was obtained from 20b in 78% yield. 1H NMR (300 MHz,
chloroform-d) δ ppm 1.68−1.82 (m, 2 H), 1.91 (d, J = 10.36 Hz, 2 H),
2.38 (s, 3 H), 2.83 (s, 3 H), 3.11−3.34 (m, 3 H), 3.53 (dd, J = 11.73,
3.06 Hz, 1 H), 4.33 (br s, 1 H), 7.00−7.19 (m, 3 H), 7.23−7.38 (m,
4 H), 7.44 (s, 1 H); [M − H]− 541.6.
(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-ethylpiperidine-1,3-diamido (20e). Fol-
lowing a procedure similar to the preparation of 17b, 20e was
1
obtained from 20b and the appropriate isocyanate in 76% yield. H
NMR (300 MHz, chloroform-d) δ ppm 1.06−1.21 (m, 3 H), 1.49−
1.74 (m, 3 H), 1.99 (br s, 1 H), 2.38 (s, 3 H), 3.04−3.34 (m, 4 H),
3.57−3.79 (m, 2 H), 4.07 (d, J = 6.50 Hz, 1 H), 4.72 (br s, 1 H), 6.98
(d, J = 6.97 Hz, 1 H), 7.07 (d, J = 8.38 Hz, 2 H), 7.23−7.36 (m, 4 H),
7.44 (s, 1 H); [M + H]+ 534.5.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[(4-
{[(propylcarbamoyl)amino]methyl}cyclohexyl)methyl]-1H-pyr-
azole-3-carboxamide (21c). Following a procedure similar to the
preparation of 17b, 21c was obtained from 26 and the appropriate
(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,3-diamido
(20f). Following a procedure similar to the preparation of 17b, 20f was
1
1
obtained from 20b and the appropriate isocyanate in 69% yield. H
isocyanate in 84% yield. H NMR (300 MHz, chloroform-d) δ ppm
NMR (300 MHz, chloroform-d) δ ppm 1.09 (d, J = 6.50 Hz, 3 H),
1.17 (s, 3 H), 1.51−1.78 (m, 3 H), 1.98 (br s, 1 H), 2.38 (s, 3 H),
3.18−3.41 (m, 2 H), 3.63 (dd, J = 13.47, 2.73 Hz, 2 H), 3.95 (d, J =
6.59 Hz, 1 H), 4.07 (d, J = 6.78 Hz, 1 H), 4.51 (d, J = 7.16 Hz, 1 H),
6.90−7.14 (m, 3 H), 7.21−7.37 (m, 4 H), 7.43 (s, 1 H); [M + H]+
548.6.
0.78−0.89 (m, 3 H), 1.25−1.53 (m, 10 H), 1.71 (br s, 2 H), 2.29 (s,
3 H), 2.94 (br s, 1 H), 3.05 (br s, 3 H), 3.19 (s, 1 H), 3.29 (t, J = 6.73
Hz, 1 H), 4.49 (br s, 2 H), 6.82−7.05 (m, 3 H), 7.12−7.28 (m, 4 H),
7.35 (s, 1 H); [M + H]+ 590.4.
N-[(4-{[(Butylcarbamoyl)amino]methyl}cyclohexyl)methyl]-
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zole-3-carboxamide (21d). Following a procedure similar to the
preparation of 17b, 21d was obtained from 26 and the appropriate
(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-1-N-propylpiperidine-1,3-diamido (20g). Fol-
lowing a procedure similar to the preparation of 17b, 20g was obtained
from 20b and the appropriate isocyanate in 87% yield. 1H NMR (300
MHz, chloroform-d) δ ppm 0.81 (t, J = 7.44 Hz, 3 H) 1.36−1.47 (m,
2 H), 1.48−1.65 (m, 3 H), 1.82−1.96 (m, 1 H), 2.29 (s, 3 H), 2.98−
3.24 (m, 4 H), 3.46−3.68 (m, 2 H), 3.86−4.09 (m, 1 H), 4.68 (t, J =
4.99 Hz, 1 H), 6.91 (d, J = 6.97 Hz, 1 H), 6.99 (d, J = 8.38 Hz, 2 H),
7.15−7.28 (m, 4 H), 7.35 (s, 1 H); [M + H]+ 548.8.
1
isocyanate in 98% yield. H NMR (300 MHz, chloroform-d) δ ppm
0.69−0.91 (m, 3 H), 1.08−1.62 (m, 12 H), 1.64−1.84 (m, 2 H), 2.29
(s, 3 H), 2.94 (s, 1 H), 2.99−3.13 (m, 3 H), 3.19 (s, 1 H), 3.29 (t, J =
6.73 Hz, 1 H), 4.47 (d, J = 5.56 Hz, 2 H), 6.81−7.06 (m, 2 H), 7.13−
7.29 (m, 4 H), 7.36 (s, 1 H); [M + H]+ 604.6.
N-[(4-{[(tert-Butylcarbamoyl)amino]methyl}cyclohexyl)-
methyl]-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxamide (21e). Following a procedure similar
to the preparation of 17b, 21e was obtained from 26 and the
appropriate isocyanate in 71% yield. 1H NMR (300 MHz, chloroform-
d) δ ppm 1.34 (d, J = 2.07 Hz, 9 H), 1.38−1.66 (m, 8 H), 1.82 (br s,
2 H), 2.38 (s, 3 H), 2.98 (br s, 1 H), 3.09 (br s, 1 H), 3.28 (s, 1 H),
3.38 (t, J = 6.78 Hz, 1 H), 4.25 (br s, 2 H), 6.93−7.12 (m, 3 H), 7.23−
7.37 (m, 4 H), 7.44 (s, 1 H); [M + H]+ 604.6.
(3R)-1-N-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (20h). Fol-
lowing a procedure similar to the preparation of 17b, 20h was
1
obtained from 20b and the appropriate isocyanate in 72% yield. H
NMR (300 MHz, chloroform-d) δ ppm 0.82 (t, J = 7.16 Hz, 3 H) 1.24
(dd, J = 14.93, 7.21 Hz, 2 H), 1.32−1.45 (m, 2 H), 1.47−1.64 (m,
3 H), 1.90 (br s, 1 H), 2.29 (s, 3 H), 2.95−3.26 (m, 4 H), 3.42−3.70
(m, 2 H), 3.98 (d, J = 6.69 Hz, 1 H), 4.65 (br s, 1 H), 6.90 (d, J =
6.88 Hz, 1 H), 6.98 (d, J = 8.38 Hz, 2 H), 7.14−7.28 (m, 4 H), 7.35 (s,
1 H); [M + H]+ 562.3.
Calcium Mobilization and Radioligand Displacement As-
says. Each compound was pharmacologically characterized using a
functional fluorescent CB1 activated Gαq16-coupled intracellular
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Journal of Medicinal Chemistry
Article
calcium mobilization assay in CHO-K1 cells as has been described in
our previous publications, and apparent affinity (Ke) values were
determined.7 Briefly, CHO-K1 cells were engineered to coexpress
human CB1 and Gqα16. Activation of CB1 by an agonist then leads to
generation of inositol phospahatase 3 (IP3) and activation of IP3
receptors, which leads to mobilization of intracellular calcium. Calcium
flux was monitored in a 96-well format using the fluorescent dye
calcein-4 AM in an automated plate reader (Flexstation, Molecular
Devices). The antagonism of a test compound was measured by its
ability to shift the concentration response curve of the synthetic CB1
agonist CP55940 rightwards using the equation
taken from all rats at 1 h postdose. At 30 min postdose, tail vein blood
was collected only from rats dosed orally.
Samples were prepared and analyzed as follows: Plasma (50 μL)
was mixed with 10 μL of internal standard, reserpine (1 μg/mL),
10 μL of acetonitrile, and 300 μL of acetonitrile, vortexed, and centrifuged
at 9000g for 5 min. Supernatant, (100 μL) was mixed with 900 μL of
50:50 methanol/water in autosampler vials. For 30 min plasma samples,
the supernatant was injected without dilution. The left lobe of the brain
was homogenized with 50:50 ethanol/water (3:1, v/v) using a Potter−
Elvehjem type homogenizer. Homogenate (50 μL) was mixed with 10 μL
of internal standard, reserpine (1 μg/mL), 10 μL of acetonitrile, and 300
μL of acetonitrile, vortexed, and centrifuged at 9000g for 5 min.
Supernatant was transferred to inserts and injected without dilution.
Standards were prepared as above for each compound in blank plasma,
blank liver homogenate, and blank brain homogenate. Standards used
were within 15% of nominal except for 20% at LOQ. Compounds for
LC−MS/MS analyses were supplied at 1 mg/mL in methanol. The stock
solutions were further diluted to ∼100 ng/mL. The 100 ng/mL solutions
were used to optimize the mass spectrometer for MRM transitions and
mass spectrometer parameters. Infusion and flow injection optimization
were also performed.
K = [ligand]/[DR − 1]
e
where DR is the EC50 ratio of CP55940 in the presence or absence of a
test agent.14
Further characterization of select compounds was performed using
radioligand displacement of [3H]SR141716, and equilibrium dissoci-
ation constant (Ki) values were determined as has been described
previously.7,15 Selectivity of these compounds at CB1 versus CB2 was
also determined by obtaining Ki values at either receptor using
displacement of [3H]CP55940 in membranes of CHO-K1cells
overexpressing either receptor. Data reported are average values
from three to six measurements.
ASSOCIATED CONTENT
* Supporting Information
HPLC data of target compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
MDCK-mdr1 Permeability Assays. MDCK-mdr1 cells obtained
from The Netherlands Cancer Institute were grown on transwell type
filters (Corning) for 4 days to confluence in DMEM/F12 medium
containing 10% fetal bovine serum and antibiotics as has been
described previously.7 Compounds were added to the apical side at
3.16 μM in a transport buffer comprising 1× Hank’s balanced salt
solution and 25 mM D-glucose and buffered with HEPES to pH 7.4.
Samples were incubated for 1 h at 37 °C and carefully collected from
both the apical and basal sides of the filters. Compounds selected for
MDCK-mdr1 cell assays were infused on an Applied Biosystems API-
4000 mass spectrometer to optimize for analysis using multiple
reaction monitoring (MRM). Flow injection analysis was also
conducted to optimize for mass spectrometer parameters. Samples
from the apical and basolateral side of the MDCK cell assay were dried
under nitrogen on a Turbovap LV. The chromatography was
conducted with an Agilent 1100 binary pump with a flow rate of 0.5
mL/min. Mobile phase solvents were A, 0.1% formic acid in water, and
B, 0.1% formic acid in methanol. The initial solvent conditions were
10% B for 1 min, then a gradient was used by increasing to 95% B over
5 min, then returning to initial conditions. Data reported are average
values from two to three measurements.
In Vitro Stability Testing. In vitro testing for metabolic stability
was conducted in pooled samples of mixed gender human plasma from
BioChemed Services, Winchester, VA, and human mixed gender
pooled hepatic S9 fraction supplied by Xenotech, LLC, Lenexa, KS.
Identities of the donors were unknown.
For the hepatic S9 metabolism studies, all samples were tested at
10 μM final concentration in a 1 mL volume containing 1 mg/mL S9.
Samples were incubated in a buffer containing 50 mM potassium
phosphate, pH 7.4, with 3 mM MgCl2 and a NADPH regeneration
system comprising NADP (1 mM), glucose 6-phosphate (5 mM), and
glucose 6-phosphate dehydrogenase (1 unit/mL). Triplicate samples
were incubated for 0, 15, 30, 60, and 120 min. Reactions were
terminated by addition of 3 volumes of acetonitrile and processed as
described for the MDCK-mdr1 assays, but standard curves were
prepared in blank matrix for each compound for quantitative
assessment.
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 919-541-6795. Fax: 919-541-8868. Email: rmaitra@rti.
■
org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Ann Gilliam for performing the binding
assays, and Sherry Black and Purvi Patel for help with metabolic
stability studies. The authors also express gratitude to the
NIDA drug supply program for providing radiolabeled probes
and to Dr. Brian Thomas for supplying the CB1 cells. This
research was funded by Research Grants 1R21AA019740-01
and 1R03AA017514-01 to R.M. from NIAAA.
ABBREVIATIONS USED
■
CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2;
CNS, central nervous system; BBB, blood−brain barrier; TPSA,
topological polar surface area; ECS, endocannabinoid system;
CBR, cannabinoid receptor; Ke, apparent affinity constant;
MDCK-mdr1, Madin−Darby canine kidney cells transfected
with the human MDR1 gene; A, apical; B, basal; BOP,
benzotriazole-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate; CHO-K1, Chinese hamster ovary cell; IP3,
inositol phospahatase 3; MRM, multiple reaction monitoring;
LOQ, below limit of quantitation; NA, not applicable
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Reactions were terminated by addition of acetonitrile and analyzed as
described above.
Evaluation of Compounds in Vivo. Male Sprague−Dawley rats
aged 7−8 weeks at time of dosing were acquired from Charles River
Laboratories and were dosed by two routes: ip and oral. Oral doses
were formulated in corn oil, and ip doses were formulated in 1:1:18
ethanol/cremophor/saline, both at 10 mg/kg. Plasma and brain were
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