Bioisosteric replacement of the pyrazole 3-carboxamide moiety of rimonabant. A novel series of oxadiazoles as CB1 cannabinoid receptor antagonists

Article abstract of DOI:10.1039/b807648k

Based on the bioisosteric replacement of the pyrazole C3-carboxamide of rimonabant with a 5-alkyl oxadiazole ring, a novel class of oxadiazole derivatives with promising biological activity towards CB1 receptors was discovered. Among them, compounds with an alkyl linker containing a strong electron-withdrawing group (e.g., CF₃) and a sterically favorable bulky group (e.g., t-butyl) exhibited excellent CB1 antagonism and selectivity, and thus might serve as potential candidates for further development as anti-obesity agents.

Full text of DOI:10.1039/b807648k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Bioisosteric replacement of the pyrazole 3-carboxamide moiety of rimonabant.
A novel series of oxadiazoles as CB1 cannabinoid receptor antagonists†
Cheng-Ming Chu,‡ Ming-Shiu Hung,‡ Min-Tsang Hsieh, Chun-Wei Kuo, Suja T. D., Jen-Shin Song,
Hua-Hao Chiu, Yu-Sheng Chao and Kak-Shan Shia*
Received 6th May 2008, Accepted 20th June 2008
First published as an Advance Article on the web 23rd July 2008
DOI: 10.1039/b807648k
Based on the bioisosteric replacement of the pyrazole C3-carboxamide of rimonabant with a 5-alkyl
oxadiazole ring, a novel class of oxadiazole derivatives with promising biological activity towards CB1
receptors was discovered. Among them, compounds with an alkyl linker containing a strong
electron-withdrawing group (e.g., CF3) and a sterically favorable bulky group (e.g., t-butyl) exhibited
excellent CB1 antagonism and selectivity, and thus might serve as potential candidates for further
development as anti-obesity agents.
as N-acyl-phosphatidyl-ethanolamine-selective phospholipase D
(NAPE-PLD) and the sn-1-selective diacylglycerol (DAG) lipases.
Introduction
Obesity, a cause of major morbidity and mortality,1–3 has become
During the past two decades, intensive studies on the
a global epidemic and is recognized as a chronic disease by the
cannabinoid-CB1 receptor axis have been fulfilled, showing that
World Health Organization (WHO). Accordingly, obesity and its
appetite control and weight loss in animals could be significantly
induced through CB1 blockers,15–18 leading to the discovery of
SR141716A (Fig. 1; rimonabant, AcompliaTM), the first anti-
obesity drug behaving as a selective CB1 inverse agonist19 and
launched in Europe in 2006. Although rimonabant has been shown
to afford weight reduction and improvements in cardiometabolic
risk factors after one year and two years of treatment,20–23 a higher
incidence of psychiatric adverse events was observed with patients
of depressive illness. Most recently, however, the two-year studies
of RIO-Europe (rimonabant in obesity and related metabolic
disorders-Europe) demonstrated that with rimonabant therapy,
the clinically significant weight loss and improvements in several
cardiometabolic risk factors, including triglycerides, high-density
lipoprotein cholesterol, insulin resistance and fasting glucose
levels, were sustained over two years with favorable tolerability and
safety for overweight/obese patients without a history of severe
depressive disorder or anxiety.24
associated co-morbidities, such as type 2 diabetes, hypertension,
dyslipidemia, and cancers, pose a serious threat to the public
health, and constitute a substantial healthcare burden in modern
society. Therefore, finding effective pharmacological therapeutics
to treat obesity is becoming an urgent necessity, especially
considering that the current two major anti-obesity drugs, Orlistat
and Sibutramine, only meet with moderate success in weight
reduction, but the accompanying adverse effects are many.4–8
The endocannabinoid signaling system has been shown to be
involved in both neuronal and peripheral physiological processes,
including feeding behavior, intestinal motility, nociception, lo-
comotive activity, and immune responses.9 These activities are
mediated by the combined action of three components such
as receptors, lipid-like endocannabinoids and proteins involved
in the production and removal of endocannabinoids.10 Two
cannabinoid receptors CB111 and CB212, which belong to the
G protein-coupled receptor family, have been identified as re-
sponding to endocannabinoids, plant derived cannabinoids (e.g.
D9-tetrahydrocannabinol) or synthetic ligands (e.g. CP55,940 and
R-(+)-WIN55,212). CB1 receptors are predominantly expressed
in neurons to inhibit the release of neurotransmitters, and CB2
receptors are abundantly expressed in immune cells and potentially
participate in cytokine release and function.9 Anandamide (AEA)
and 2-arachidonoylglycerol (2-AG) derived from phospholipid
precursors are the two most studied endocannabinoids.13,14 Phys-
iologically, these endocannabinoids are not prestored in secre-
tary vesicles, but are synthesized on demand by enzymes such
Fig. 1 SR141716A (rimonabant, AcompliaTM).
Division of Biotechnology and Pharmaceutical Research, National Health
Research Institutes, Miaoli County 350, Taiwan, R. O. C. E-mail: ksshia@
nhri.org.tw; Fax: 886-37-586456; Tel: 886-37-246166-35709
† Electronic supplementary information (ESI) available: Table of Ki values
of all compounds, including 4a–k, 8a–h, and SR141716A, toward CB1 and
CB2 receptors.30 See DOI: 10.1039/b807648k
The aforementioned encouraging results stimulated our long-
lasting efforts in pursuing a new generation of rimonabant-
mimicking molecules as anti-obesity agents with less CNS
toxicity.25 Herein, we wish to report that the bioisosteric
‡ These authors contributed equally to this work.
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replacement of the pyrazole C3-carboxamide of rimonabant with
an oxadiazole moiety has been envisioned and experimentally
realized to afford an array of novel analogues, several of which
exhibited high binding affinity and potent activity towards CB1
receptors with excellent CB2/1 selectivity. Detailed description
of the synthetic design and structure–activity relationship (SAR)
studies of these newly developed compounds is presented as
follows.
Scheme 2
Results and discussion
with the required acid chloride in refluxing toluene to generate the
desired 5-alkyl oxadiazoles 8a–h in 40–52% yields over four steps.
Synthesis
The procedures employed in our laboratory for the synthesis of
3-alkyl oxadiazoles, as represented by the generic formula 4, are
depicted in Scheme 1. The synthesis began with carboxylic acid
1, readily accessible through a four-step sequence reported in the
literature (Scheme 2).26 Under treatment with thionyl chloride,
intermediate 1 was converted to the corresponding acyl chloride 2
in 98% yield which, without purification, was coupled with various
Biological studies
All the oxadiazole compounds, including 4a–k and 8a–h, were sub-
jected to biological evaluations towards CB1 and CB2 receptors,
the results of which are compiled in Table 1.
As shown, though the initial test compound, 3-methyl oxa-
diazole 4a, exhibited poorer binding affinity and less potency
toward the CB1 receptor (IC50 = 509.6 nM; EC50 = 552.1 nM)
than rimonabant (IC50 = 13.6 nM; EC50 = 17.3 nM), which
was constantly employed as the reference in our assay systems,
the submicromolar activities observed with 4a implied that when
a hydrophobic substituent was appropriately positioned, the
oxadiazole ring might serve as a potential bioisostere for the
pyrazole C3-carboxamide moiety. Based on the binding mode
of rimonabant in the proposed CB1-receptor homology model
(Fig. 2),18a further structural modifications aiming to enhance the
hydrophobic interaction by extending the alkyl chain were then
carried out. When the methyl side chain was replaced with an
ethyl or n-propyl group, the corresponding 3-ethyl oxadiazole 4b
(IC50 = 225.1 nM; EC50 = 75.5 nM) and 3-propyl oxadiazole
4c (IC50 = 291.2 nM; EC50 = 59.1 nM) resulted in considerable
improvements in the CB1 biological activities, with particularly
remarkable increases of ca. 10-fold in the functional activity
(EC50). However, further extension of the linear alkyl chain to
=
amide oximes (R(C NOH)NH2) followed by thermal annulation
to afford the desired products 4a–k (Table 1) in 30–80% yields
over the two steps. The required amide oximes were previously
prepared in moderate to good yields (50–80%) by treatment of the
corresponding alkyl nitriles with hydroxylamine.27 Alternatively,
acid chloride 2 was individually treated with the deprotonated
amides, formed in situ by treatment with LiHMDS, to afford the
corresponding imides which in turn underwent condensation with
hydroxylamine to generate the desired 3-alkyl oxadiazoles in 36–
60% yields over the two steps.
For the synthesis of 5-alkyl oxadiazoles, as represented by the
generic formula 8, the synthetic sequence illustrated in Scheme 3
was fulfilled. Carboxylic acid 1 was first converted to the primary
amide 5 by coupling with ammonia in the presence of EDCI
and HOBT as a pair of coupling reagents. The compound
5 thus obtained was subjected to dehydration under standard
conditions to give nitrile 6, which was sequentially reacted with
hydroxylamine to afford amide oxime 7, followed by treatment
Scheme 1 Reagents and conditions: (i) SOCl2, toluene, reflux, 2 h; (ii) RC(NOH)NH2, toluene, reflux, 2 h; (iii) RCONH2, LiHMDS, THF, −78 ◦C then
−10 ◦C, 2 h; (iv) NH2OH then pyridine, reflux, 4 h.
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Table 1 Biological evaluation of 3-alkyl oxadiazoles and 5-alkyl oxadiazoles on hCB1 and hCB2 receptors
a,c
Compd
R
EC50
hCB1 IC50a,b/nM
hCB2 IC50a,b/nM
Selectivity (hCB2/hCB1)
4a
4b
4c
4d
4e
4f
CH3
CH2CH3
552.1 109.6
75.5 12.1
59.1 23.1
141.1 8.8
128.5 36.3
55.3 8.5
509.6 101.3
225.1 44.9
291.2 87.5
255.8 13.7
220.7 27.9
80.2 19.5
62.2 14.7
64.7 8.6
114.5 9.3
317.6 9.2
435.1 204.1
924.8 106.1
270.7 47.6
160.2 40.9
77.8 24.1
55.1 17.6
30.5 8.7
>10000
> 20
29
26
> 39
> 45
86
56
80
59
29
6507.5 509.8
7518.1 1047.0
>10000
CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)3CH3
CH(CH3)2
CH(C2H5)2
C(CH3)3
>10000
6924.8 724.9
3490.8 1273.9
5198.5 95.5
6794.7 122.4
9207.0 833.6
9926.7 824.8
>10000
4g
4h
4i
39.1 19.3
25.8 6.2
c-C3H5
95.0 77.7
95.8 32.7
155.5 17.6
435.5 21.7
365.3 38.2
163.9 18.3
150.1 69.3
131.5 61.1
56.1 24.1
64.6 5.1
4j
CH2CH(CH3)2
CH2(c-C3H5)
CH3
4k
8a
8b
8c
8d
8e
8f
21
> 11
> 37
62
116
24
321
> 269
247
121
CH2CH3
>10000
CH2CH2CH3
CH(CH3)2
CH(C2H5)2
C(CH3)3
CF3
C(CH3)2CF3
SR141716A
9965.6 398.3
9021.7 1316.9
1329.5 168.4
9790.6 268.7
>10000
3544.5 1374.4
1643.7 107.0
8g
8h
37.1 5.0
14.2 4.0
13.6 1.0
8.2 3.9
17.3 1.8
a Data are expressed as the mean SD of at least three independent experiments. b The binding affinity determined by inhibition of [3H]-CP55940 binding
to the hCB1 or hCB2-transfected HEK 293 membrane is expressed as IC50. c The functional activity determined by inhibition of Eu-GTP binding to
hCB1-transfected HEK 293 membrane is expressed as EC50.
Scheme 3 Reagents and conditions: (i) EDCI, HOBT, CH2Cl2, r.t., 40 min, then NH3(aq.), r.t., 30 min; (ii) TFAA, Et3N, CH2Cl2, 0 ◦C, 1h; (iii) 50%
NH2OH(aq.), MeOH, 40 ◦C, 16 h; (iv) RCOCl, toluene, reflux, 2 h.
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highly expected to behave the same as compounds 8g and 8h
with desirable receptor–ligand interactions. Unfortunately, these
efforts turned out to be fruitless due to the labile intermediates
formed following the synthetic Scheme 1. Also, following the
protocol reported in the literature,28 the intrinsic properties of all
the compounds in Table 1 were identified, leading to the finding
that these rimonabant-mimicking molecules, without exception,
behaved as inverse agonists as does rimonabant.
Fig. 2 The binding mode of rimonabant in the proposed CB1-receptor
homology model.
3 and 4 methylene units gave rise to compounds 4d and 4e,
respectively, with no improvements in either the binding affinity
or the potency towards the CB1 receptor, indicating that the
hydrophobic cavity of the CB1 receptor around the pyrazole C-3
substitution might be shallow.
As such, analogues with a branched alkyl group, instead of
a linear linker, were then explored. As illustrated in Table 1,
compounds 4f–i thus obtained apparently supported the afore-
mentioned inference of CB1 receptor with a shallow cavity
surrounding the C-3 position, leading to an enhancement of both
the functional activity and the binding affinity by up to 20- and
8-fold (i.e., 4h), respectively. Further expanding the dimensions
of the branched alkyl group seemed not to be tolerated, as
demonstrated by 4j–k whose biological activities are inferior to
those of 4f–i. Though the array of 3-alkyl oxadiazoles described
above revealed great potential as bioisosteres of rimonabant,
all its compounds share the liability of poor CB2/1 selectivity
(CB2/1 = 20∼86). For comparison purposes, attempts were also
made to explore 5-alkyl oxadiazoles, in which a 1,2-transposition
of the heteroatoms O and N was implemented. As a result,
not unexpectedly, the corresponding compounds 8a–f displayed
similar binding and functional behaviors towards CB1 receptors
as did the 3-alkyl oxadiazoles 4a–c and 4f–h, suggesting that the
binding mode against CB1 receptors was little influenced by this
minor structural modification. However, it was observed that the
CB2/1 selectivity of compound 8f (CB2/1 = 321) was dramatically
enhanced as compared to 4h (CB2/1 = 80). This finding is hard to
rationalize by the current results and is worth studying further.
Also encouraging is the finding that when the methyl group
was substituted with a trifluoromethyl moiety, the resulting 8g
(IC50 = 37.1 nM; EC50 = 64.6 nM; CB2/1 = >269) exhibited a
Conclusions
In conclusion, based on the bioisosteric replacement of the
pyrazole C3-carboxamide of rimonabant with a 5-alkyl oxadiazole
ring, a novel class of oxadiazole derivatives with promising biolog-
ical activity towards CB1 receptors was discovered. Among them,
compounds with an alkyl linker containing a strong electron-
withdrawing group (e.g., CF3) and a sterically favorable bulky
group (e.g., t-butyl) have shown potent CB1 antagonism and
excellent selectivity, and thus might serve as potential candidates
for further development as anti-obesity agents. Further structural
modifications and in vivo efficacy studies on the series are currently
under active investigation and the results will be reported elsewhere
in due course.
Experimental
General methods for chemistry
All reactions were performed in oven-dried glassware and some
reactions were carried out under a positive pressure of argon
or nitrogen when the reactions were sensitive to moisture or
oxygen. Analytical thin layer chromatography was performed with
E. Merck silica gel 60F glass plates and flash chromatography
by the use of E. Merck silica gel 60 (230–400 mesh). Fourier
transform infrared spectra (IR) were recorded on a Perkin-Elmer
spectrum RX-1. 1H and 13C NMR spectra were recorded on
a Bruker Aavance EX 400 FT NMR or a Bruker DMX-600.
Chloroform-d was used as the solvent and TMS (d = 0.00 ppm)
as an internal standard. Chemical shift values are reported in ppm
relative to the TMS in delta (d) units. Multiplicities are recorded
as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet),
sext (sextet), sept (septet), dd (doublet of doublets), dt (doublet
of triplets), br (broadened) and m (multiplet). Coupling constants
(J) are expressed in Hz. MS and HRMS were measured by JEOL
JMS-D300 and JEOL JMS-HX110 spectrometers, respectively;
electrospray mass spectra (ESMS) were recorded using an Agilent
1100MSD spectrometer. Spectral data were recorded as m/z
values.
substantial improvement relative to 8a (IC50 = 924.8 nM; EC50
=
435.5 nM; CB2/1 = >11) in all biological aspects, including
binding affinity, potency, and selectivity towards CB1 receptors,
presumably due to the enhancement of the Asp366–Lys192 salt
bridge-stabilizing capacity in the presence of a strong electron-
withdrawing group (see Fig. 2).18a By this inference, a hybrid
with an alkyl chain combining both the favored trifluoromethyl
and bulky t-butyl motifs was designed, leading to the desired
compound 8h (IC50 = 14.2 nM; EC50 = 8.2 nM; CB2/1 = 247), the
biological profile of which is comparable to SR141716A and which
might serve as an advanced lead for further development for the
treatment of obesity. Based on these encouraging results, attempts
were also made to synthesize compounds 4l and 4m, which are
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General procedure for the synthesis of compounds 4a, 4b, 4f, 4g, 4h
and 4k
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)-3-(pentan-3-yl)-1,2,4-oxadiazole (4g). Compound
4g was obtained by a similar procedure to that used for compound
4a. Treatment of acid 1 (0.150 g, 0.39 mmol) with thionyl chlo-
ride (0.17 mL, 2.36 mmol), and 2-ethyl-N-hydroxybutanamidine
(0.153 g, 1.18 mmol) gave compound 4g (0.086 g, 46%): mp 173–
174 ◦C; IR (CH2Cl2, cast) mmax 3087, 2964, 2932, 2876, 1606, 1568,
1496, 1486 cm−1; 1H NMR (400 MHz, CDCl3) d 7.41–7.27 (m, 5H),
7.13 (d, J = 8.4 Hz, 2H), 2.82 (quint, J = 5.5 Hz, 1H), 2.44 (s, 3H),
1.89–1.73 (m, 4H), 0.89 (t, J = 7.4 Hz, 6H); 13C NMR (100 MHz,
CDCl3) d 173.3, 170.5, 143.1, 138.6, 136.1, 135.7, 135.2, 132.9,
130.7, 130.6, 130.1, 128.9, 127.8, 126.7, 117.6, 40.6, 25.9, 11.7, 9.5;
MS (EI, 70 eV) m/z (% intensity) 474.0 (M+, 25.4); HRMS calcd
for C23H21Cl3N4O 474.0781, found 474.0786.
The general procedure is illustrated immediately below with
compound 4a as a specific example.
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H -
pyrazol-3-yl)-3-methyl-1,2,4-oxadiazole (4a). To a stirred solu-
tion of the acid 1 (0.150 g, 0.39 mmol) in toluene (5 mL) was
added thionyl chloride (0.17 mL, 2.36 mmol) at room temperature.
The mixture was heated to reflux for 2 h and then cooled to room
temperature. This was followed by concentration in vacuo to afford
the crude carbonyl chloride 2 (0.153 g, 98% yield) as a white
solid. To a stirred solution of N-hydroxyacetimidamide (0.087 g,
1.18 mmol) in toluene (2 mL) was added the carbonyl chloride 2 in
toluene (2 mL) in one portion. The resulting mixture was heated
to reflux for 2 h and then cooled to room temperature. Water
(10 mL) was added to quench the reaction. The aqueous layer
was separated and extracted with dichloromethane (3 × 10 mL);
the combined organic extracts were washed with brine, dried
over anhydrous magnesium sulfate, filtered, and concentrated in
vacuo to afford the crude product, which was purified by flash
chromatography on silica gel with EtOAc–n-hexane (1 : 12) to
give compound 4a (0.042 g, 31% yield) as a white solid: mp 92–
93 ◦C; IR (CH2Cl2, cast) mmax 3085, 2958, 2927, 2856, 1605, 1569,
3-tert-Butyl-5-(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazol-3-yl)-1,2,4-oxadiazole (4h). Compound 4h
was obtained by a similar procedure to that used for compound
4a. Treatment of acid 1 (0.150 g, 0.39 mmol) with thionyl chloride
(0.17 mL, 2.36 mmol), and N-hydroxypivalamidine (0.137 g,
1.18 mmol) gave compound 4h (0.143 g, 79% yield) as a white
solid: mp 155–156 ◦C; IR (CH2Cl2, cast) mmax 3086, 2970, 2930,
2870, 1608, 1569, 1497, 1487 cm−1; 1H NMR (400 MHz, CDCl3)
d 7.41–7.28 (m, 5H), 7.12 (d, J = 8.4 Hz, 2H), 2.44 (s, 3H), 1.46
(s, 9H); 13C NMR (100 MHz, CDCl3) d 177.8, 170.3, 143.0, 138.5,
136.1, 135.6, 135.1, 132.9, 130.7, 130.6, 130.0, 128.9, 127.7, 126.6,
117.6, 32.4, 28.3, 9.5; MS (EI, 70 eV) m/z (% intensity) 460.0 (M+,
15.4); HRMS calcd for C22H19Cl3N4O 460.0624, found 460.0634.
1
1497, 1487 cm−1; H NMR (400 MHz, CDCl3) d 7.42–7.30 (m,
5H), 7.13 (d, J = 8.2 Hz, 2H), 2.51 (s, 3H), 2.43 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 170.6, 167.5, 143.1, 138.3, 136.2, 135.6, 135.2,
132.8, 130.8, 130.5, 130.2, 129.0, 127.8, 126.6, 117.6, 11.6, 9.5; MS
(EI, 70 eV) m/z (% intensity) 417.9 (M+, 28.3); HRMS calcd for
C19H13Cl3N4O 418.0155, found 418.0143.
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)-3-(cyclopropylmethyl)-1,2,4-oxadiazole (4k). Com-
pound 4k was obtained by a similar procedure to that used for
compound 4a. Treatment of acid 1 (0.155 g, 0.40 mmol) with
thionyl chloride (0.18 mL, 2.50 mmol), and 2-cyclopropyl-N-
hydroxyacetamidine (0.120 g, 1.20 mmol) gave compound 4k
(0.104 g, 56% yield) as a white solid: mp 120–121 ◦C; IR (CH2Cl2,
cast) mmax 3082, 2959, 2925, 2871, 1605, 1569, 1496, 1486 cm−1; 1H
NMR (400 MHz, CDCl3) d 7.43–7.27 (m, 5H), 7.14 (d, J = 8.4 Hz,
2H), 2.76 (d, J = 7.0 Hz, 2H), 2.45 (s, 3H), 1.25 (m, 1H), 0.58 (m,
2H), 0.32 (m, 2H); 13C NMR (100 MHz, CDCl3) d 170.5, 170.4,
143.1, 138.4, 136.1, 135.6, 135.2, 132.8, 130.7, 130.5, 130.1, 128.9,
127.8, 126.6, 117.6, 30.8, 9.4, 8.71, 4.70; MS (EI, 70 eV) m/z
(% intensity) 458.0 (M+, 28.6); HRMS calcd for C22H17Cl3N4O
458.0468, found 458.0457.
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)-3-ethyl-1,2,4-oxadiazole (4b). Compound 4b was
obtained by a similar procedure to that used for compound 4a.
Treatment of acid 1 (0.155 g, 0.40 mmol) with thionyl chloride
(0.18 mL, 2.50 mmol), and N-hydroxypropionimidamide (0.105 g,
1.20 mmol) gave compound 4b (0.064 g, 38% yield) as a white
solid: mp 129–130 ◦C; IR (CH2Cl2, cast) mmax 3052, 2959, 2928,
2871, 1604, 1569, 1498, 1490 cm−1; 1H NMR (400 MHz, CDCl3)
7.45–7.27 (m, 5H), 7.13 (d, J = 8.4 Hz, 2H), 2.88 (q, J = 7.6 Hz,
2H), 2.44 (s, 3H), 1.40 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 171.9, 170.5, 143.1, 138.4, 136.1, 135.6, 135.2, 132.8,
130.7, 130.5, 130.1, 128.9, 127.8, 126.6, 117.5, 19.7, 11.4, 9.5; MS
(EI, 70 eV) m/z (% intensity) 432.0 (M+, 33.8); HRMS calcd for
C20H15Cl3N4O 432.0311, found 432.0298.
General procedure for the synthesis of compounds 4c, 4d, 4e, 4i
and 4j
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H -
pyrazol-3-yl)-3-isopropyl-1,2,4-oxadiazole (4f). Compound 4f
was obtained by a similar procedure to that used for compound
4a. Treatment of acid 1 (0.155 g, 0.40 mmol) with thionyl chloride
(0.18 mL, 2.50 mmol), and N-hydroxyisobutyramidine (0.122 g,
1.20 mmol) gave compound 4f (0.084 g, 46% yield) as a white
solid: mp 130–132 ◦C; IR (CH2Cl2, cast) mmax 3086, 2972, 2932,
2874, 1606, 1569, 1497, 1486 cm−1; 1H NMR (400 MHz, CDCl3)
d 7.47–7.25 (m, 5H), 7.13 (d, J = 8.2 Hz, 2H), 3.21 (quint, J =
6.9 Hz, 1H), 2.45 (s, 3H), 1.43 (d, J = 6.9 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d 175.3, 170.4, 143.1, 138.5, 136.1, 135.6, 135.1,
132.8, 130.7, 130.6, 130.0, 128.9, 127.7, 126.6, 117.6, 26.7, 20.4,
9.4; MS (EI, 70 eV) m/z (% intensity) 446.0 (M+, 36.9); HRMS
calcd for C21H17Cl3N4O 446.0468, found 446.0481.
The general procedure is illustrated immediately below with
compound 4c as a specific example.
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)-3-propyl-1,2,4-oxadiazole (4c). To a stirred solu-
tion of the acid 1 (0.150 g, 0.39 mmol) in toluene (5 mL) was
added thionyl chloride (0.17 mL, 2.36 mmol) at room temperature.
The mixture was heated to reflux for 2 h and then cooled to room
temperature. This was followed by concentration in vacuo to afford
the crude carbonyl chloride 2 (0.153 g, 98% yield) as a white
solid. A stirred solution of butyramide (0.072 g, 0.83 mmol) in
THF (5 mL) was treated with lithium bis(trimethylsilyl)amide
(1 M solution in THF, 1.0 mL, 1.00 mmol) at −78 ◦C. The
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mixture was stirred for 50 min then carbonyl chloride 2 in THF
(5 ml) was added dropwise. The resulting mixture was allowed
to warm to −10 ◦C and stirred for 2 h. Water (10 mL) was
added and the aqueous layer was separated and extracted with
dichloromethane (3 × 10 mL). The combined organic extracts
were washed with brine, dried over anhydrous magnesium sulfate,
filtered, and concentrated in vacuo to afford the crude imide
product, which in turn, without purification, was treated with
hydroxylamine hydrochloride (125 mg, 1.81 mmol) in pyridine
(3 mL) under reflux conditions for 4 h. This was followed by a
usual work-up procedure to afford the crude oxadiazole product,
which was purified by flash chromatography on silica gel with
EtOAc–n-hexane (1 : 12) to give compound 4c (0.073 g, 41% yield)
as a white solid: mp 98–99 ◦C; IR (CH2Cl2, cast) mmax 3086, 2964,
4c. Treatment of acid 1 (0.150 g, 0.39 mmol) with thionyl chloride
(0.17 mL, 2.36 mmol), lithium bis(trimethylsilyl)amide (1.0 mL,
1.00 mmol) and cyclopropane-carboxamide (0.070 g, 0.83 mmol)
gave compound 4i (0.063 g, 36% yield) as a white solid: mp 110–
111 ◦C; IR (CH2Cl2, cast) mmax 3087, 2959, 2928, 2871, 1605, 1569,
1
1497, 1487 cm−1; H NMR (400 MHz, CDCl3) d 7.41–7.28 (m,
5H), 7.11 (d, J = 8.2 Hz, 2H), 2.40 (s, 3H), 2.25–2.15 (m, 1H),
1.23–1.05 (m, 4H); 13C NMR (100 MHz, CDCl3) d 172.8, 170.4,
143.2, 138.5, 136.2, 135.7, 135.3, 133.0, 130.8, 130.7, 130.2, 129.0,
127.8, 126.7, 117.7, 9.6, 7.8, 6.9; MS (EI, 70 eV) m/z (% intensity)
443.9 (M+, 27.8); HRMS calcd for C21H15Cl3N4O 444.0311, found
444.0309.
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-3-isobutyl-1,2,4-oxadiazole (4j). Compound 4j was
obtained by a similar procedure to that used for compound 4c.
Treatment of acid 1 (0.150 g, 0.39 mmol) with thionyl chloride
(0.17 mL, 2.36 mmol), lithium bis(trimethylsilyl)amide (1.0 mL,
1.00 mmol) and 3-methylbutanamide (0.084 g, 0.83 mmol) gave
compound 4j (0.067 g, 37% yield) as a white solid: mp 126–127 ◦C;
IR (CH2Cl2, cast) mmax 3093, 2960, 2926, 2855, 1607, 1569, 1497,
1
2933, 2874, 1605, 1570, 1497, 1487 cm−1; H NMR (400 MHz,
CDCl3) d 7.50–7.29 (m, 5H), 7.13 (d, J = 8.4 Hz, 2H), 2.82 (t, J =
7.6 Hz, 2H), 2.44 (s, 3H), 1.88 (sext, J = 7.6 Hz, 2H), 1.03 (t, J =
7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 170.8, 170.5, 143.1,
138.5, 136.2, 135.7, 135.2, 132.8, 130.7, 130.6, 130.1, 128.9, 127.8,
126.7, 117.6, 27.9, 20.4, 13.6, 9.5; MS (EI, 70 eV) m/z (% intensity)
445.9 (M+, 32.2); HRMS calcd for C21H17Cl3N4O 446.0468, found
446.0469.
1
1487 cm−1; H NMR (400 MHz, CDCl3) d 7.43–7.28 (m, 5H),
7.13 (d, J = 8.4 Hz, 2H), 2.72 (d, J = 6.8 Hz, 2H), 2.44 (s, 3H),
2.26 (m, J = 6.8 Hz, 1H), 1.03 (d, J = 6.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d 170.46, 170.16, 143.12, 138.50, 136.18,
135.68, 135.22, 132.84, 130.77, 130.60, 130.14, 128.97, 127.82,
126.69, 117.61, 34.72, 27.02, 22.30, 9.54; MS (EI, 70 eV) m/z
(% intensity) 460.0 (M+, 31.8); HRMS calcd for C22H19Cl3N4O
460.0624, found 460.0640.
3-Butyl-5-(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl)-1,2,4-oxadiazole (4d). Compound 4k was ob-
tained by a similar procedure to that used for compound 4c.
Treatment of acid 1 (0.155 g, 0.40 mmol) with thionyl chloride
(0.18 mL, 2.50 mmol), lithium bis(trimethylsilyl)amide (1.0 mL,
1.00 mmol) and pentanamide (0.084 g, 0.83 mmol) gave compound
4d (0.071 g, 38% yield) as a white solid: mp 136–137 ◦C; IR
(CH2Cl2, cast) mmax 3085, 2959, 2931, 2872, 1605, 1569, 1497,
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zole-3-carboxamide (5). To a solution of acid 1 (1.290 g,
3.38 mmol) in CH2Cl2 (20 mL) was added 1-hydroxybenzo-
triazole (HOBT, 0.550 g, 4.06 mmol) and 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (EDCI, 0.900 g,
5.07 mmol) in sequence. The resulting mixture was stirred at
room temperature for 40 min and then ammonia (30% in H2O,
10 eq.) was added dropwise. The reaction mixture was stirred for
an additional 30 min at room temperature. The combined organic
extracts were washed with brine (10 mL), dried over anhydrous
magnesium sulfate, filtered, and concentrated in vacuo to afford
the crude product, which was purified by flash chromatography
on silica gel with EtOAc–n-hexane (3 : 10) gave carboxamide 5
(1.160 g, 90% yield) as a white solid: mp 185–186 ◦C; IR (CH2Cl2,
cast) mmax 3458, 3287, 1678, 1592, 1573, 1494, 1484 cm−1; 1H NMR
(600 MHz, CDCl3) d 7.39 (d, J = 2.0 Hz, 1H), 7.27–7.23 (m, 4H),
7.04–7.02 (m, 2H), 6.82 (br s, 1H), 5.80 (br s, 1H), 2.33 (s, 3H);
13C NMR (150 MHz, CDCl3) d 164.7, 144.2, 143.0, 135.9, 135.7,
134.8, 132.8, 130.7, 130.4, 130.2, 128.8, 127.8, 127.0, 117.9, 9.4;
ESMS m/z: 380.1 (MH+).
1
1487 cm−1; H NMR (400 MHz, CDCl3) d 7.42–7.28 (m, 5H),
7.13 (d, J = 8.2 Hz, 2H), 2.84 (t, J = 7.5 Hz, 2H), 2.44 (s, 3H),
1.82 (quint, J = 7.5 Hz, 2H), 1.45 (sext, J = 7.5 Hz, 2H), 0.96
(t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 170.9, 170.4,
143.0, 138.4, 136.1, 135.6, 135.1, 132.8, 130.7, 130.5, 130.0, 128.9,
127.7, 126.6, 117.5, 29.0, 25.6, 22.1, 13.5, 9.4; MS (EI, 70 eV)
m/z (% intensity) 460 (M+, 55.0); HRMS calcd for C22H19Cl3N4O
460.0619, found 460.0636.
5 - (5 - (4 - Chlorophenyl) - 1 - (2,4 - dichlorophenyl) - 4 - methyl-
1H-pyrazol-3-yl)-3-pentyl-1,2,4-oxadiazole (4e). Compound 4e
was obtained by a similar procedure to that used for compound
4c. Treatment of acid 1 (0.150 g, 0.39 mmol) with thionyl chloride
(0.17 mL, 2.36 mmol), lithium bis(trimethylsilyl)amide (1.0 mL,
1.00 mmol) and hexanamide (0.095 g, 0.83 mmol) gave compound
4e (0.110 g, 59% yield) as a white solid: mp 150–152 ◦C; IR
(CH2Cl2, cast) mmax 3086, 2957, 2930, 2860, 1605, 1568, 1497,
1
1486 cm−1; H NMR (400 MHz, CDCl3) d 7.41–7.27 (m, 5H),
7.13 (d, J = 8.4 Hz, 2H), 2.83 (t, J = 7.5 Hz, 2H), 2.44 (s, 3H),
1.86–1.80 (m, 2H), 1.50–1.26 (m, 4H), 0.91 (t, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) d 170.9, 170.5, 143.1, 138.4, 136.1,
135.6, 135.2, 132.8, 130.7, 130.5, 130.1, 128.9, 127.7, 126.6, 117.5,
31.2, 26.6, 25.9, 22.2, 13.8, 9.5; MS (EI, 70 eV) m/z (% intensity)
474.0 (M+, 31.8); HRMS calcd for C23H21Cl3N4O 474.0781, found
474.0769.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zole-3-carbonitrile (6). To a stirred solution of carboxamide 5
(1.160 g, 3.05 mmol) in CH2Cl2 (20 mL) was added triethylamine
(1.28 mL, 9.15 mmol) and trifluoroacetic anhydride (0.85 mL,
6.10 mmol) at 0 ◦C. The resulting solution was stirred at 0 ◦C
for 1 h. Water (15 mL) was added to quench the reaction. The
aqueous layer was separated and extracted with dichloromethane
(3 × 10 mL). The combined organic extracts were washed with
brine, dried over anhydrous magnesium sulfate, filtered, and
5-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl)-3-cyclopropyl-1,2,4-oxadiazole (4i). Compound 4i
was obtained by a similar procedure to that used for compound
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concentrated in vacuo to afford the crude product, which was
purified by flash chromatography on silica gel with EtOAc–n-
hexane (1 : 10) gave carbonitrile 6 (1.050 g, 95% yield) as a white
solid: mp 90–92 ◦C; IR (CH2Cl2, cast) mmax 3089, 2239, 1604, 1560,
d 180.4, 164.0, 142.8, 140.6, 136.0, 135.7, 134.8, 133.0, 130.8,
130.7, 129.9, 128.8, 127.6, 127.2, 116.3, 20.2, 10.8, 9.8; ESMS
m/z: 433.1 (MH+).
1
1496, 1487 cm−1; H NMR (600 MHz, CDCl3) d 7.40 (d, J =
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-propyl-1,2,4-oxadiazole (8c). Compound 8c was
obtained by a similar procedure to that used for compound 8a.
Treatment of carboximidamide 7 (0.16 g, 0.41 mmol) with butyryl
chloride (0.057 g, 0.53 mmol) gave compound 8c (0.133 g, 74%
yield) as a white solid: mp 163–165 ◦C; IR (CH2Cl2, cast) mmax
2.0 Hz, 1H), 7.30–7.23 (m, 4H), 7.04–7.02 (m, 2H), 2.22 (s, 3H);
13C NMR (150 MHz, CDCl3) d 142.1, 136.5, 135.5, 135.1, 132.6,
130.4, 130.3, 130.2, 129.1, 127.6, 125.9, 120.9, 113.1, 8.7; ESMS
m/z: 362.0 (MH+).
(Z)-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-Nꢀ -hydroxy-4-
methyl-1H-pyrazole-3-carboximidamide (7). To a stirred solu-
tion of compound 6 (0.863 g, 2.28 mmol) in MeOH (20 ml) was
added 50% aqueous hydroxylamine solution (4.56 ml, 2.99 mmol)
at room temperature. The mixture was heated to 40 ◦C and
stirred for 16 h. The resulting solution was then cooled to room
temperature and concentrated in vacuo to afford the crude product,
which was purified by flash chromatography on silica gel with
EtOAc–n-hexane (1 : 1) to give carboximidamide 7 (0.753 g, 80%
yield) as a white solid: mp 218–221 ◦C; IR (CH2Cl2, cast) mmax 3501,
3390, 1644, 1567, 1496, 1485 cm−1; 1H NMR (400 MHz, CDCl3)
d 7.54 (br s, 1H), 7.32–7.25 (m, 5H), 7.08 (d, J = 8.3 Hz, 2H),
5.26 (br s, 2H), 2.25 (s, 3H); 13C NMR (100 MHz, CDCl3) d 148.9,
143.8, 142.7, 136.1, 135.6, 134.7, 133.0, 130.8, 130.6, 130.2, 128.8,
127.7, 127.5, 114.6, 10.3; ESMS m/z: 395.0 (MH+).
1
3085, 2985, 2942, 2881, 1583, 1567, 1496, 1486 cm−1; H NMR
(600 MHz, CDCl3) d 7.37–7.34 (m, 2H), 7.28–7.25 (m, 3H),
7.09 (d, J = 8.4 Hz, 2H), 2.92 (t, J = 7.6 Hz, 2H), 2.36 (s, 3H),
1.90–1.85 (m, 2H), 1.00 (t, J = 7.5 Hz, 3H); 13C NMR (150 MHz,
CDCl3) d 180.4, 164.0, 142.8, 140.6, 136.0, 135.7, 134.8, 133.0,
130.8, 130.7, 129.9, 128.8, 127.6, 127.2, 116.3, 20.2, 10.8, 9.8;
ESMS m/z: 447.0 (MH+).
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-isopropyl-1,2,4-oxadiazole (8d). Compound 8d was
obtained by a similar procedure to that used for compound
8a. Treatment of carboximidamide 7 (0.15 g, 0.38 mmol) with
isobutyryl chloride (0.052 g, 0.49 mmol) gave compound 8d
(0.125 g, 74% yield) as a white solid: mp 155–157 ◦C; IR (CH2Cl2,
cast) mmax 2977, 2934, 2876, 1580, 1567, 1497, 1486 cm−1; 1H NMR
(600 MHz, CDCl3) d 7.38–7.34 (m, 2H), 7.29–7.25 (m, 3H), 7.09
(d, J = 8.3 Hz, 2H), 3.31 (quint, J = 6.9 Hz, 1H), 2.36 (s, 3H),
1.44 (d, J = 6.8 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 183.6,
163.9, 142.8, 140.7, 136.0, 135.7, 134.8, 133.0, 130.9, 130.8, 129.9,
128.8, 127.6, 127.2, 116.4, 27.5, 20.1, 9.8; ESMS m/z: 447.0
(MH+).
General procedure for the synthesis of compounds 8a–8f
The general procedure is illustrated immediately below with
compound 8a as a specific example.
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-methyl-1,2,4-oxadiazole (8a). To a stirred solution
of carboximidamide 7 (0.150 g, 0.38 mmol) in anhydrous
toluene (5 mL) was added a solution of acetyl chloride (0.036 g,
0.49 mmol). The resulting mixture was heated to reflux and
stirred for 2 h. The reaction was subsequently cooled to room
temperature and water (5 mL) was added. The aqueous layer
was separated and extracted with dichloromethane (3 × 10 mL).
The combined organic extracts were washed with brine, dried
over anhydrous magnesium sulfate, filtered, and concentrated in
vacuo to afford the crude product, which was purified by flash
chromatography on silica gel with EtOAc–n-hexane (1 : 6) to
give compound 8a (0.111 g, 70% yield) as a white solid: mp
112–115 ◦C; IR (CH2Cl2, cast) mmax 3085, 2975, 2931, 2870, 1579,
1561, 1497, 1486 cm−1; 1H NMR (600 MHz, CDCl3) d 7.36–7.34
(m, 2H), 7.28–7.24 (m, 3H), 7.09 (d, J = 8.3 Hz, 2H), 2.63 (s, 3H),
2.35 (s, 3H); 13C NMR (150 MHz, CDCl3) d 176.2, 164.0, 142.7,
140.4, 135.9, 135.7, 134.8, 132.9, 130.7, 129.9, 128.8, 127.6, 127.1,
116.2, 12.2, 9.8; ESMS m/z: 419.0 (MH+).
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-(pentan-3-yl)-1,2,4-oxadiazole (8e). Compound 8e
was obtained by a similar procedure to that used for compound
8a. Treatment of carboximidamide 7 (0.155 g, 0.39 mmol) with
2-ethylbutanoyl chloride (0.066 g, 0.49 mmol) gave compound 8e
(0.137 g, 76% yield) as a white solid: mp 195–197 ◦C; IR (CH2Cl2,
cast) mmax 3084, 2966, 2933, 2876, 1578, 1567, 1497, 1486 cm−1;
1H NMR (600 MHz, CDCl3) d 7.39–7.34 (m, 2H), 7.29–7.25 (m,
3H), 7.09 (d, J = 8.4 Hz, 2H), 3.00–2.95 (m, 1H), 2.37 (s, 3H),
1.91–1.85 (m, 2H), 1.81–1.76 (m, 2H), 0.88 (t, J = 7.4 Hz, 6H);
13C NMR (150 MHz, CDCl3) d 182.5, 163.9, 142.8, 140.7, 136.0,
135.7, 134.8, 133.0, 130.9, 130.7, 129.9, 128.8, 127.6, 127.2, 116.4,
41.8, 26.1, 11.7, 9.9; ESMS m/z: 475.1 (MH+).
5-tert-Butyl-3-(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl)-1,2,4-oxadiazole (8f). Compound 8f was
obtained by a similar procedure to that used for compound
8a. Treatment of carboximidamide 7 (0.155 g, 0.39 mmol) with
pivaloyl chloride (0.059 g, 0.49 mmol) gave compound 8f (0.146 g,
75% yield) as a white solid: mp 178–180 ◦C; IR (CH2Cl2, cast)
mmax 3088, 2962, 2928, 2853, 1587, 1566, 1497, 1486 cm−1; 1H
NMR (400 MHz, CDCl3) d 7.41–7.35 (m, 2H), 7.29–7.24 (m,
3H), 7.09 (d, J = 8.0 Hz, 2H), 2.36 (s, 3H), 1.49 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 183.6, 163.8, 142.8, 140.7, 136.0, 135.7,
134.8, 133.1, 130.9, 130.8, 129.9, 128.8, 127.6, 127.2, 116.4, 33.6,
28.4, 9.9; ESMS m/z: 461.1 (MH+).
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-ethyl-1,2,4-oxadiazole (8b). Compound 8b was
obtained by a similar procedure to that used for compound
8a. Treatment of carboximidamide 7 (0.16 g, 0.41 mmol) with
propionyl chloride (0.048 g, 0.53 mmol) gave compound 8b
(0.126 g, 71% yield) as a white solid: mp 145–147 ◦C; IR (CH2Cl2,
cast) mmax 3086, 2977, 2934, 2876, 1580, 1567, 1497, 1486 cm−1;
1H NMR (600 MHz, CDCl3) d 7.37–7.35 (m, 2H), 7.29–7.25 (m,
3H), 7.09 (d, J = 8.3 Hz, 2H), 2.98 (q, J = 7.7 Hz, 2H), 2.36
(s, 3H), 1.43 (t, J = 4.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
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General procedure for the synthesis of compounds 8g and 8h
was centrifuged for another 30 minutes at 43000g at 4 ◦C. The
final pellet was resuspended in buffer A and stored at −80 ◦C.
The general procedure is illustrated immediately below with
compound 8g as a specific example.
Radioligand binding assay
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-(trifluoromethyl)-1,2,4-oxadiazole (8g). To a stirred
solution of 2,2,2-trifluoroacetic acid (0.061 g, 0.53 mmol)
in anhydrous CH2Cl2 (0.5 mL) was added oxalyl chloride
(0.06 mL, 0.68 mmol). The resulting solution was stirred at room
temperature for 2 h and concentrated in vacuo to afford the crude
carbonyl chloride. To a stirred solution of carboximidamide 7
(0.100 g, 0.25 mmol) in toluene (2 mL) was added the above crude
carbonyl chloride in toluene (2 mL). The mixture was heated
to reflux for 2 h and then cooled to room temperature. Water
(15 mL) was added to quench the reaction. The aqueous layer
was separated and extracted with dichloromethane (3 × 10 mL).
The combined organic extracts were washed with brine, dried
over anhydrous magnesium sulfate, filtered, and concentrated in
vacuo to afford the crude product, which was purified by flash
chromatography on silica gel with EtOAc–n-hexane (1 : 10) to
give compound 8g (0.111 g, 70% yield) as a white solid: mp
128–129 ◦C; IR (CH2Cl2, cast) mmax 3090, 2969, 2930, 2876, 1582,
1569, 1497, 1486, 1178 cm−1;1H NMR (400 MHz, CDCl3) d
7.41–7.30 (m, 5H), 7.14–7.12 (m, 2H), 2.41 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 165.9 (q, JC-F = 44 Hz), 164.8, 143.3, 138.8,
136.2, 135.7, 135.2, 133.0, 130.8, 130.7, 130.1, 129.0, 127.8, 126.7,
117.3 (q, JC-F = 272 Hz), 117.0, 9.8; ESMS m/z: 474.0 (MH+).
0.2–8 lg of the purified membrane was incubated with 0.75 nM
[3H] CP55,940 and the compound of interest in the incubation
buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, 0.3% BSA,
pH 7.4). The non-specific binding was defined in the presence
of 1 lM of CP55,940. The reactions were incubated for one
◦
and a half hours at 30 C in Multiscreen microplates (Millipore
Corp., Billerica, MA). The reactions were terminated by manifold
filtration and washed four times with ice-cold wash buffer (50 mM
Tris, pH 7.4, 0.25% BSA). The radioactivity bound to the filters
was measured by Topcount (PerkinElmer Inc., Waltham, MA).
The IC50 was determined as the concentration of compound
required to inhibit 50% of the binding of [3H] CP55,940 and
calculated by non-linear regression (GraphPad software, San
Diego, CA).29 Alternatively, the Ki was measured according to
the standard protocol reported in the literature30; these binding
affinity values are compiled in Table 2 as exhibited in the ESI.†
Eu-GTP binding assay
The Eu-GTP binding assay was performed using the DELFIA
Eu-GTP binding kit (Perkin Elmer Inc., Waltham, MA) based on
methods developed by Frang et al.,31 with minor modifications
as described in the following: 1–4 lg of purified membrane was
incubated with the compound of interest and 20 nM CP55,940 in
the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 100 lg
mL−1 saponin, 5 mM MgCl2, 2 lM GDP, 0.5% BSA) at 30 ◦C for
60 minutes in acroplates (Pall Life Sciences, Ann Arbor, Mich.).
Following the addition of Eu-GTP and incubation for 30 minutes
at 30 ◦C, the assay was terminated by washing four times with the
washing buffer provided in the kit. The fluorescence signal of the
Eu-GTP was determined with a Victor 2 multilabel reader (Perkin
Elmer Inc., Waltham, MA). The EC50 of the tested compounds
at inhibiting 50% of the CP55,940-stimulated Eu-GTP binding
was determined from the dose–response curves using non-linear
regression (GraphPad software, San Diego, CA).
3-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zol-3-yl)-5-(1,1,1-trifluoro-2-methylpropan-2-yl)-1,2,4-oxadiazole
(8h). Compound 8h was obtained by a similar procedure
to that used for compound 8g. Treatment of 3,3,3-trifluoro-
2,2-dimethylpropanoic acid (0.078 g, 0.5 mmol) with oxalyl
chloride (0.06 mL, 0.65 mmol) and carboximidamide 7 (0.090 g,
0.23 mmol) gave compound 8h (0.071 g, 60% yield) as a white
solid: mp 176–177 ◦C; IR (CH2Cl2, cast) mmax 3094, 2929, 2870,
1588, 1566, 1498, 1486, 1215, 1178, 1157 cm−1; 1H NMR
(400 MHz, CDCl3) d 7.42–7.37 (m, 2H), 7.34–7.28 (m, 3H),
7.13 (d, J = 8.4 Hz, 2H), 2.39 (s, 3H), 1.76 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 177.7, 164.2, 143.0, 140.1, 135.9, 135.9,
135.0, 133.1, 130.8, 130.8, 130.0, 128.9, 127.7, 127.3 (q, JC-F
280 Hz), 127.1, 116.8, 43.6 (q, JC-F = 28 Hz), 20.7 (× 2, q, JC-F
6 Hz), 9.8; ESMS m/z: 516.0 (MH+).
=
Acknowledgements
=
We are grateful to the National Health Research Institutes and
National Science Council of the Republic of China (NSC 95–
2323-B-400–001) for financial support.
General methods for biological evaluation
Establishment of human CB1 (hCB1) and CB2 (hCB2) stable cell
lines and membrane purification: either hCB1 cDNA tagged with
Flag at the N terminus or hCB2 cDNA was subcloned into the
pIRES2-EGFP vector (Clontech Laboratories, Inc., Mountain
View, CA). After being transfected into HEK 293 cells, clones
stably expressing either hCB1 or hCB2 were selected by GFP and
G418 sulfate, and maintained in DMEM supplemented with 10%
fetal bovine serum and 0.5 mg ml−1 G418 sulfate under 5% CO2
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