Solid phase synthesis of 1,5-diarylpyrazole-4-carboxamides: Discovery of antagonists of the CB-1 receptor

Article abstract of DOI:10.1021/co200147y

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor.

Full text of DOI:10.1021/co200147y

Research Article
pubs.acs.org/acscombsci
Solid Phase Synthesis of 1,5-Diarylpyrazole-4-carboxamides:
Discovery of Antagonists of the CB-1 Receptor
Annapurna Pendri,* Dharmpal S. Dodd, Jing Chen, Mary Ellen Cvijic, Liya Kang, Rose A. Baska,
Kenneth E. Carlson, Neil T. Burford, Chongqing Sun, William R. Ewing, and Samuel W. Gerritz
Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
S
* Supporting Information
ABSTRACT: We have developed a solid phase synthesis route to 1,5-
substituted pyrazole-4-carboxamides with three diversity points aimed at
the discovery of new compounds as potential G-Protein coupled receptor
(GPCR) ligands. The new chemistry involves acylation of a resin bound
secondary amine with a β-ketoester via transamidation, conversion of the
resulting β-ketoamide to the corresponding vinylogous amide, pyrazole
formation upon reaction with a aryl hydrzine, and cleavage of the product
from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used
collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar
antagonist activities at the cannabinoid subtype 1 (CB-1) receptor.
KEYWORDS: solid phase synthesis, pyrazole-4- carboxamide, GPCR ligands, privileged scaffolds
Using the reported methodology, we describe the syntheses
of multiple arrays of pyrazoles that were used collectively to
construct a library of more than 1000 analogues. This
compound collection was subsequently screened against a
range of GPCR targets, with hits being identified against
multiple GPCR receptors. Of particular interest to us was the
finding that several members of this library displayed
submicromolar antagonist activities at the cannabinoid subtype
1 (CB-1) receptor, vide infra.
INTRODUCTION
■
The solid-phase synthesis of compound libraries for general
screening has been shown to be effective for lead identification,
and can facilitate subsequent lead optimization when the solid
phase route has sufficient generality to allow access to novel
analogues of interest. However, a challenge in these exercises is
selecting a structural motif to serve as the library scaffold, given
the diversity of structures that can now be prepared on solid-
phase and the related range of available chemistries.1,2
Our efforts in this area have been guided by the concept of
“privileged scaffolds”, which are defined as structural templates
known to yield biologically active molecules for a range of
therapeutically interesting targets.3,4 In the current work, we
identified the pyrazole heterocycle as one such privileged
scaffold based on its wide-ranging biological activities.5 As
shown in Figure 1, bioactive pyrazoles include the following: G-
Protein coupled receptor (GPCR) targeted drugs such as the
serotonin 5HT2A inverse agonist Nelotanserin (1a), used for
the treatment of insomnia;6 Celecoxib (1b), a selective
cyclooxygenase-2 (COX-2) inhibitor that is marketed for the
treatment of osteoarthritis, rheumatoid arthritis, and acute
pain;7 and the N-substituted pyrazole Doramapimod (1c), an
inhibitor of p38 MAP kinase used for the treatment of cancer.8
In this current study, we report a solid phase synthesis of 1,5-
diarylpyrazole-4-carboxamides, as typified by 2 in Figure 2, and
comment on the generality of the reported chemistry. From a
library synthesis perspective, compounds of this type are
attractive because they contain three vectors (denoted R1, R2,
and Ar) that allow the introduction of significant structural
diversity. The pendant functionality is also disposed in a less
common 1,4,5-pattern, which we felt would explore a chemical
space that had not been adequately addressed in currently
disclosed structural classes.9−27
CHEMISTRY
■
The following chemistry was conducted using IRORI Micro-
Kan technology as a synthesis platform.28 The chemical
protocol developed (Scheme 1) involves the introduction of
the first diversity element (R1) by loading a primary amine
reagent chemset 3 (Figure 3) onto a 4-formyl-3-methoxyphe-
noxy (FMP) resin29 via standard reductive amination30
conditions to provide resin-bound amine chemset 4. The
subsequent transacylacetylation31 of 4 with an aromatic tert-
butyl-β-ketoester (R2) reagent chemset 5 (Figure 4) requires
heating in a resin-compatible solvent such as NMP in the
presence of catalytic DMAP, and provides corresponding resin-
bound β-ketoamide chemset 6, containing the nascent R2
substituent. The success of this step is highly dependent on
temperature, with 80 °C found to be optimal. Conversion of
the resulting β-ketoamide chemset 6 to the corresponding
vinylogous amide chemset 7 is accomplished by reaction with
dimethylformamide-dimethylacetal in DMF at 95 °C for 48 h.
Cyclization of chemset 7 to resin-bound chemset 9 can be
Received: September 7, 2011
Revised: January 27, 2012
Published: February 16, 2012
© 2012 American Chemical Society
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Figure 1. Approved trisubstituted pyrazole based drugs.
The data presented in Table 1 also demonstrates that all
three points of diversity (i.e., R1, R2, and Ar) tolerate a wide
range of substituents. With respect to the amide component, it
is apparent that primary amines of varying basicity (cf. (R1{1−
3})) react in an essentially similar fashion, and that branching
alpha to the amine group is also tolerated without a significant
impact on product yields, as is seen with analogue 2{2,1,1}. In
relation to the aryl moiety (R2{1−5}) at C5, both electron
donating and electon withdrawing groups can be accommo-
dated, without an undue impact on product yields, as noted
with analogues 2{1,2,1}, 2{1,3,1}, and 2{1,4,1}. In relation to
the hydrazine component (R3{1−4}) used in the above
protocol, electron rich and deficient hydrazines are well
tolerated, as noted with analogues 2{4,5,2} and 2{4,5,4}, and
ortho-substitution in the hydrazine moiety is accommodated as
reflected in the similar yields obtained with pyrazole 2{4,5,3}.
Figure 2. 1,5-Diaryl-4-carboxamide substituted pyrazoles.
achieved via reaction with an aryl hydrazine (Ar) reagent
chemset 8 (Figure 5), this step resulting in the introduction of
the Ar moiety. The desired 1,5-diarylpyrazole-4-carboxamide
chemset 2 are then liberated from the solid support by
treatment with 50% TFA in DCE. Interestingly, in all cases
examined, only a single regioisomer is obtained.32
Using this methodology we synthesized multiple compound
arrays that resulted in the generation of thousands of analogues.
The protocol was found to be general in scope, as can be
appreciated by the product yields and purities obtained with the
representative chemset analogues presented in Table 1. These
crude yields were determined gravimetrically, and the overall
yields were based on initial FMP resin loading of 36 μmol for
the five step sequence and ranged between 10 and 30 μmol.
LCMS analyses of these products showed the desired products
to be present in >80% purity, as determined by UV (220 nm)
and light scattering detection.
DISCUSSION
■
As part of an effort to identify novel GPCR ligands,33,34
analogues from the above compound arrays were screened in a
high throughput screening (HTS) format against a library of
GPCR targets, and hits were observed against several GPCRs of
interest. Specifically, several examples exhibited binding
affinities against the cannabinoid subtype 1 (CB-1)
GPCR.35−37 CB-1 is expressed in the central nervous system
on presynaptic terminals, and modulates neurotransmitter
release. Endogenous (endocannabinoids) and many exogenous
a
Scheme 1
a
Reagents and conditions: (a) 3, 2% HOAc in DMF/(CH3O)3CH (7:3), 0.25 M NaBH(OAc)3, 25 °C, 72 h; (b) 5, NMP, DMAP, 80 °C, 48 h; (c)
(CH3O)2CHNMe2/DMF, 95 °C, 48 h; (d) 8, NMP, DMAP, 85 °C, 72 h; (e) 50% TFA/DCM, RT, 1 h.
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Figure 3. R1 Diversity reagents 3{1−6}.
Figure 4. R2 Diversity reagents 5{1−5}.
Figure 5. Ar Diversity reagents 8{1−8}.
ligands of CB-1 have been identified, and these have been
demonstrated to modulate a number of neurological processes,
including several related to metabolic function. Correspond-
ingly, the CB-1 receptor has been identified as a target for the
treatment of obesity as well as psychiatric and neuro-
degenerative disorders,38−43 and the development of suitable
ligands for CB-1 receptors is of obvious interest. CB-1 binding
assay is used to measure the binding affinity of the library
samples toward the CB-1 receptor.44
The initial CB-1 binding results of a number of 1,5-
diarylpyrazole-4-carboxamide 2 derivatives synthesized in the
above array format were encouraging. The CB-1 IC50 values for
an active set of analogues are presented in Table 2.
not displaying any significant dependence on substitution
pattern. Finally, the naphthyl derivative 2{1,1,3} represented
the most active compound (hCB-1 IC50 = 90 nM) identified
from the compound library.
Several active samples from the library were subsequently
resynthesized and purified by preparative HPLC, and the CB-1
IC50 values were determined, and are shown in Table 3. The
close correlation observed for the IC50 values of crude and
purified samples suggests that reliable biological data can be
obtained from crude library samples of established purity and
quantity. Compound 2{6,1,1} with an IC50 value of 0.5 μM
(Entry 2) also demonstrated full functional CB-1 antagonism
(IC50 0.3 μM) in a GTP-γS assay.45
Significant structure activity relationships (SARs) can be
elucidated from the activity data presented in Table 2. In the
case of cyclopropylmethyl amides R1{5}, a range of activities
from >10 uM to approximately 300 nM is observed, with the
most active derivative being the meta-chloro Ar{1} analogue
2{5,1,1}. In the related n-butyl amide series R1{6}, similar
trends are observed, although these compounds tend to be less
active than their cyclopropylmethyl congeners. Within this
class, the meta-chloro Ar{1} analogue 2{6,1,1} is again the
most active derivative (hCB-1 IC50 = 500 nM). Interestingly, in
the case of 3-phenylpropyl amides R1{1}, the SAR at Ar
(Ar{1−4}) tends to be flat, with all compounds generally
showing enhanced levels of activity relative to other series, but
In conclusion, a novel series of CB-1 antagonists were
identified from a library of 1,5-diarylpyrazole-4-carboxamides
synthesized on solid support using IRORI MicroKan
technology. This solid phase synthesis route facilitated the
rapid synthesis of compounds for SAR exploration by providing
diversity at three points on the pyrazole scaffold. Analysis of the
CB-1 binding data from the library indicated that biphenyl
substitution at the 5-position of the pyrazole was essential for
activity. Even though the original library comprised crude
samples (>80% pure by HPLC), the activity of crude and
resynthesized samples showed excellent correlation. The results
of the subsequent lead optimization of the 5-biphenylpyrazole-
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Table 1. Product Examples Illustrative of the Generality of
the Chemistry Depicted in Scheme 1
Table 2. Compounds with hCB1 Activity Identified from
Initial Screening of Compound Library
4-carboxamide series for CB-1 antagonist activity will be
reported in a future manuscript.
EXPERIMENTAL SECTION
■
All compounds synthesized were analyzed by LC-MS with UV
(λ = 220 nm) to determine their purity: Conditions:
Phenomenex-prime S5 C-18, 4.6 × 30 mm. eluted with 0%
to 100% B, 2 min gradient. (A = 10% MeOH/H2O containing
0.1% TFA and B = 90% MeOH/H2O containing 0.1% TFA);
Flow rate at 5 mL/min. UV detection at 220 nm. The
molecular mass of the compounds was determined by MS (ES)
by the formula m/z.
General Procedure for Chemset 4. To a reaction vessel
with Microkans containing 4-formyl-3-methoxyphenoxy (FMP)
resin, (Polymer laboratories, 1.2 mmol/g, 75−150 uM mesh, 30
mg) and an Rf tag was added 2.5 mL per Microkan of 7:3
DMF/trimethyl orthoformate (TMOF), the appropriate R1
amine (15.0 equiv), acetic acid (50 μL per Microkan), and
NaBH(OAc)3 (15.0 equiv). The reaction vessel was shaken at
room temperature for 2 days, then the Microkans were filtered
and washed with DMF (4 × 3 mL/Microkan), 2:1 DMF/
MeOH (3 × 3 mL/Microkan), THF (2 × 3 mL/Microkan),
DCM (2 × 3 mL/Microkan), and dried under vacuum for 12 h
in a vacuum oven at 30 °C to afford resin-bound amine
chemset 4.
General Procedure for Chemset 6. To a reaction vessel
with Microkans containing the above resin-bound amine
chemset 4 was added 2 mL per Microkan of N-methylpyrro-
lidine (NMP), the appropriate t-butyl-β-keto ester R2 reagent
chemset 5 (5.0 equiv) and 4-dimethylaminopyridine (DMAP,
0.1%.). The reaction vessel was agitated for 48 h at 80 °C using
a turbocoil shaker, then the Microkans were filtered and washed
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Using the above procedures, the following compounds were
prepared and characterized.
Table 3. CB-1 IC50 Values for Crude and Resynthesized
Library Samples
5-(biphenyl-4-yl)-1-(3-chlorophenyl)-N-(3-phenylpropyl)-
1H-pyrazole-4-carboxamide (2{1,1,1}). LCMS [M+H]
492.4, RT 2.14 min, HPLC purity 92%, (18.4 μmol,
51% yield).
5-(biphenyl-4-yl)-1-(3-chlorophenyl)-N-(4-phenylbutan-2-
yl)-1H-pyrazole-4-carboxamide (2{2,1,1}). LCMS [M+H]
506.1, RT 1.86 min, HPLC purity 92%, (21 μmol, 58%
yield).
5-(biphenyl-4-yl)-1-(3-chlorophenyl)-N-(2-phenoxyethyl)-
1H-pyrazole-4-carboxamide (2{3,1,1}). LCMS [M+H]
494.2, RT 1.74 min, HPLC purity 96%, (18 μmol, 50%
yield).
1-(4-chloro-2-methylphenyl)-5-(3-chlorophenyl)-N-(3-phe-
nylpropyl)-1H-pyrazole-4-carboxamide (2{1,2,1}). LCMS
[M+H] 464.4, RT 1.93 min, .HPLC purity 100% (15
μmol, 42% yield).
1-(4-chloro-2-methylphenyl)-N-(3-phenylpropyl)-5-(3-
(trifluoromethyl)phenyl)-1H-pyrazole-4-carboxamide (2
{1,3,1}). LCMS [M+H] 499.17, RT 2.06 min, HPLC
purity 87%, (11 μmol, 31% yield).
1-(4-chloro-2-methylphenyl)-5-(4-(dimethylamino)phenyl)-
N-(3-phenylpropyl)-1H-pyrazole-4-carboxamide (2
{1,4,1}). LCMS [M+H] 474.3, RT 1.90 min, HPLC
purity 100%, (15 μmol, 39% yield).
N-benzyl-5-(3-chloro-4-methoxyphenyl)-1-(4-methoxyphen-
yl)-1H-pyrazole-4-carboxamide (2{4,4,2}). LCMS [M+H]
448.17, RT 1.8 min, HPLC purity 100%, (14 μmol, 39%
yield).
N-benzyl-5-(3-chloro-4-methoxyphenyl)-1-(naphthalen-1-
yl)-1H-pyrazole-4-carboxamide (2{4,4,3}). LCMS [M+H]
469.5, RT 1.74 min, HPLC purity 89%, (21 μmol, 58%
yield).
N-benzyl-5-(3-chloro-4-methoxyphenyl)-1-(3,5-dichloro-
phenyl)-1H-pyrazole-4-carboxamide (2{4,4,4}). LCMS [M
+H] 486.8, RT 2.12 min, HPLC purity 87%, (19 μmol,
53% yield).
5-(biphenyl-4-yl)-N-(cyclopropylmethyl)-1-phenyl-1H-pyra-
zole-4-carboxamide (2{5,1,5}). LCMS [M+H] 394.2, RT
1.93 min, HPLC purity 100%, (19 μmol, 53% yield).
5-(biphenyl-4-yl)-1-(3-chlorophenyl)-N-(cyclopropylmeth-
yl)-1H-pyrazole-4-carboxamide (2{5,1,1}). LCMS [M+H]
428.4, RT 1.95 min, HPLC purity 88% (23 μmol, 64%
yield).
5-(biphenyl-4-yl)-N-(cyclopropylmethyl)-1-o-tolyl-1H-pyra-
zole-4-carboxamide (2{5,1,6}). LCMS [M+H] 408.3, RT
1.96 min, HPLC purity 98%, (19 μmol, 55% yield).
5-(biphenyl-4-yl)-N-(cyclopropylmethyl)-1-o-tolyl-1H-pyra-
zole-4-carboxamide (2{5,1,7}). LCMS [M+H] 408.6, RT
1.98 min, HPLC purity 96%, (22 μmol, 61% yield).
5-(biphenyl-4-yl)-N-(cyclopropylmethyl)-1-(2,3-dimethyl-
phenyl)-1H-pyrazole-4-carboxamide (2{5,1,8}). LCMS [M
+H] 422.5, RT 1.88 min, HPLC purity 100%, (28 μmol,
78% yield).
with DMF (2 × 3 mL/Microkan), 1:1 DMF-MeOH (2 × 3
mL/Microkan), DCM (3 × 3 mL/Microkan) and dried under
vacuum for 12 h in a vacuum oven at 30 °C to afford resin-
bound amide chemset 6.
General Procedure for Chemset 7. To a reaction vessel
with Microkans containing the above resin-bound amide
chemset 6 was added 1 mL per Microkan of DMF and 1 mL
per Microkan of dimethylformamide-dimethylacetal (DMF-
DMA). The reaction vessel was agitated for 24 h at 80 °C using
a turbocoil shaker; then the Microkans were filtered and
washed with DMF (2 × 3 mL/Microkan), 1:1 DMF-MeOH (2
× 3 mL/Microkan), DCM (3 × 3 mL Microkan) and dried
under vacuum for 12 h in a vacuum oven at 30 °C to afford
resin-bound enamine chemset 7.
General Procedure for Chemset 9. To a reaction vessel
with Microkans containing the above resin-bound enamine
chemset 7 was added 2 mL per Microkan of N-methylpyrro-
lidine (NMP) and the appropriate arylhydrazine reagent
chemset 8 as Ar reagent (15.0 equiv). When the arylhydrazine
is an hydrochloride an equivalent amount of diisopropylethyl
amine is used to free base the hydrochloride The reaction vessel
was agitated for 72 h at 90 °C using a turbocoil shaker; then the
Microkans were filtered and washed with DMF (2 × 3 mL/
Microkan), 1:1 DMF-MeOH (2 × 3 mL/Microkan), DCM (3
× 3 mL/Microkan) and dried under vacuum for 12 h in a
vacuum oven at 30 °C to afford resin-bound pyrazoleamide
chemset 9.
5-(biphenyl-4-yl)-N-butyl-1-phenyl-1H-pyrazole-4-carboxa-
mide (2{6,1,5}). LCMS [M+H] 396.22, RT 2.2 min,
HPLC purity 91%, (13 μmol, 36% yield).
5-(biphenyl-4-yl)-N-butyl-1-(3-chlorophenyl)-1H-pyrazole-
4-carboxamide (2{6,1,1}). LCMS [M+H] 430.9, RT 2.14
min, HPLC purity 85%, (23 μmol, 64% yield).
General Procedure for Product 2 Cleavage. The
Microkan containing resin-bound pyrazole amide chemset 9
was treated with 1.5 mL of 50% trifluoroacetic acid (TFA) in
DCM at room temperature for 1 h, then filtered. The filtrate
was concentrated in speed vacuum to afford the crude product
chemset 2.
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124.8, 123.6, 117.6, 37.8, 30.8, 19.1, 13.2.HRMS (ESI) calcd for
C26H25ClN3O [M+H]+ 430.1681, found 430.1669.
5-(biphenyl-4-yl)-N-butyl-1-o-tolyl-1H-pyrazole-4-carboxa-
mide (2{6,1,6}). LCMS [M+H] 410.25, RT 2.27 min.,
HPLC purity 81%, (17 μmol 47% yield).
5-(biphenyl-4-yl)-N-butyl-1-m-tolyl-1H-pyrazole-4-carboxa-
mide (2{6,1,7}). LCMS [M+H] 410.5, RT 2.23 min,
HPLC purity 84% (14 μmol 55% yield).
5-(biphenyl-4-yl)-N-butyl-1-(2,3-dimethylphenyl)-1H-pyra-
zole-4-carboxamide (2{6,1,8}). LCMS [M+H] 424.6, RT
1.98 min, HPLC Purity 92%, (21 μmol, 58% yield).
5-(biphenyl-4-yl)-1-(naphthalen-1-yl)-N-(3-phenylpropyl)-
1H-pyrazole-4-carboxamide (2{1,1,3}). LCMS [M+H]
508.4, RT 2.2 min, HPLC purity 90%, (15 μmol, 42%
yield).
5-([1,1′-Biphenyl]-4-yl)-1-(2,3-dimethylphenyl)-N-(3-
phenylpropyl)-1H-pyrazole-4-carboxamide (2{1,1,8}).
1
(24 mg, 51% yield). H NMR (500 MHz, CDCl3): δ 8.14 (1
H, s), 7.53−7.58 (2 H, m), 7.49−7.53 (2 H, m), 7.41 (2 H, t, J
= 7.5 Hz), 7.34−7.37 (1 H, m), 7.30−7.34 (2 H, m), 7.10−7.21
(4 H, m), 7.00−7.08 (4 H, m), 3.27−3.34 (2 H, m), 2.45−2.52
(2 H, m), 2.22 (3 H, s), 1.86 (3 H, s), 1.71 (2 H, d). 13C NMR
(126 MHz, DMSO-d6) δ 161.6, 143.3, 141.2, 138.8, 138.6,
139.4, 137.9, 137.3, 133.6, 130.2, 130.0, 128.0, 127.7, 127.7,
127.3, 126.1, 125.5, 125.3(s, 2C), 125.2, 116.1, 37.8, 32.1, 30.5,
19.3, 13.4. HRMS (ESI) calcd for C33H32N3O [M+H]+
486.2540, found 486.2526.
5-(biphenyl-4-yl)-N-(3-phenylpropyl)-1-o-tolyl-1H-pyra-
zole-4-carboxamide (2{1,1,6}). LCMS [M+H] 472.5, RT
2.13 min, HPLC purity 96%, (12 μmol, 33% yield).
5-(biphenyl-4-yl)-N-(3-phenylpropyl)-1-m-tolyl-1H-pyra-
zole-4-carboxamide (2{1,1,7}). LCMS [M+H] 472.6, RT
2.12 min, HPLC purity 94%, (12 μmol, 33% yield).
5-(biphenyl-4-yl)-1-(2,3-dimethylphenyl)-N-(3-phenylprop-
yl)-1H-pyrazole-4-carboxamide (2{1,1,8}). LCMS [M+H]
486.6, RT 1.99 min, HPLC purity 98%, (17 μmol 47%
yield).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C NMR, HRMS and LCMS data on purified compounds
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: 203-677-7702. E-mail: annapurna.pendri@bms.com.
General Procedure for the Purification of Resynthe-
sized Compounds. Four samples from Table 3 compounds
2{5,1,1}, 2{6,1,1}, 2{1,1,1}, and 2{1,1,8} were resynthesized
on solid support using loose resin using the protocol described
above, and the reagents specified in the following below. The
resulting product was purified by chromatography (YMC
combiprep ODS-A, 30 mm × 50 mm, MeOH/H2O/0.1%
TFA) to yield the title compounds.
Notes
The authors declare no competing financial interest.
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nylpropyl)-1H-pyrazole-4-carboxamide (2{5,1,1}). (26
1
mg, 55% yield). H NMR (500 MHz, CDCl3): δ 8.14 (1 H,
s), 7.68 (3 H, d, J = 8.5 Hz), 7.55−7.61 (3 H, m), 7.42−7.48 (3
H, m), 7.35−7.42 (4 H, m), 7.24 (2 H, br. s.), 7.11−7.21 (4 H,
m), 6.98−7.05 (3 H, m), 3.25−3.33 (2 H, m), 2.43−2.51 (2 H,
m), 1.65−1.74 (2 H, m). 13C NMR (126 MHz, DMSO-d6) δ
161.7, 142.7, 141.7, 140.3, 140.3, 140.0, 139.1, 133.1, 131.1,
130.6, 129.0, 128.3, 128.3, 127.9, 127.9, 127.9, 126.1, 126.1,
125.7, 125.3, 124.1, 118.1, 38.3, 32.6, 30.9. HRMS (ESI) calcd
for C31H27ClN3O [M+H]+ 492.1837, found 492.1823.
5-([1,1′-Biphenyl]-4-yl)-1-(3-chlorophenyl)-N-(cyclo-
propylmethyl)-1H-pyrazole-4-carboxamide (2{6,1,1}).
1
(24 mg, 58% yield). H NMR (500 MHz, CDCl3): δ 8.20 (1
H, s), 7.63−7.74 (2 H, m), 7.58−7.64 (2 H, m), 7.47 (2 H, t),
7.36−7.44 (4 H, m), 7.24 (1 H, s), 7.18 (1 H, t), 7.00−7.08 (1
H, m), 3.12 (2 H, s), 0.77 (1 H, s), 0.30−0.37 (2 H, m), 0.02 (2
H, s). 13C NMR (126 MHz, DMSO-d6) δ 161.1, 142.4, 139.9,
139.8, 139.5, 138.7, 132.6, 130.7, 130.1, 128.6, 127.5, 127.4,
127.4,126.17, 125.6, 124.9, 123.6, 117.5, 42.4, 33.9, 10.5, 2.7.
HRMS (ESI) calcd for C26H23ClN3O [M+H]+ 428.1524, found
428.1511.
5-([1,1′-Biphenyl]-4-yl)-N-butyl-1-(3-chlorophenyl)-
1H-pyrazole-4-carboxamide (2{1,1,1}). (33 mg, 79% yield).
1H NMR (500 MHz, CDCl3): δ 8.19 (1 H, s), 7.70 (2 H, d, J =
13.4 Hz), 7.61 (2 H, s), 7.47 (2 H, s), 7.40 (4 H, s), 7.18 (1 H,
s), 7.02 (1 H, s), 3.26 (2 H, s), 2.69 (2 H, s), 1.30 (4 H, s),
1.28−1.35 (2 H, m), 1.11 (2 H, s), 0.80 (3 H, t). 13C NMR
(126 MHz, DMSO-d6) δ 161.1, 142.3, 139.9, 139.8, 139.5,
138.7, 132.6, 130.6, 130.1, 128.6, 127.4, 127.4, 126.2, 125.6,
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converted into % inhibition based on total and nonspecific binding and
Ki values were calculated by the Cheng-Prusoff correction (Cheng, Y.-
C.; Prusoff, W. H. Br. Pharmacol. 1973, 22, 3099−3108) using the
parameters of radioligand concentration and the Kd values determined
for each radioligand. Reported Ki values are the average of at least 3
independent determinations.
(45) CB-1 functional assay: CB-1 functional assay is used to measure
the functionality of molecules to the receptor. The [35S]GTPgammaS
binding assay is considered as a functional assay. The [35S]GTPgS
assay measures the level of G protein activation following agonist
occupation of a GPCR, by determining the binding of the
nonhydrolyzable analogue [35S]GTPgS to Ga subunits. Thus, the
assay measures a functional consequence of receptor occupancy at one
of the earliest receptor-mediated events. Compounds were diluted in
100% DMSO and then added 1:100 into 96 well plates containing 1
μg of CHO-CB-1 membrane protein in assay buffer (20 mM HEPES,
pH 7.4, 100 mM NaCl, 10 mM MgCl2, 10 μg/mL saponin, 1 mM
EDTA, 0.25% BSA, WGA-PVT beads 150 μg, cold GTPγ-S 10 μM
and 60 μM GDP). After adding [35S]-GTPγ-S to all the wells, the
plates were incubated for 1 h at room temp. The binding reaction was
terminated by centrifuging at 1000 rpm for 5 min. The plates were
then counted on a Packard TopCount Scintillation Counter
immediately.
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