Synthesis and Biological Studies of a Novel CB1 Antagonist

Article abstract of DOI:10.1055/s-0035-1548848

This paper describes the synthesis, early process development, salt selection strategies and pre clinical evaluation of novel, potent and selective CB1 antagonist, 8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]azulene-3-carboxyli

Full text of DOI:10.1055/s-0035-1548848

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
Synthesis and Biological Studies of a Novel CB1
Antagonist*
Authors
K. Banerjee1, 2, M. Jain1, A. Vallabh2, B. Srivastava1, A. Joharapurkar1, H. Patel1
Affiliations
1 Zydus Research Centre, Ahmedabad, India
2 Department of Chemistry, Faculty of Science, M. S. University of Baroda, Vadodara, India
Key words
were found to be suitable for further develop-
ment. The amorphous form of the compound 1 is
found to be better suited. In vivo efficacy study of
the amorphous form of compound 1 in 5%
sucrose solution intake model in female Zucker
fa/fa rats at single oral dose of 10mg/kg demon-
strates better reduction in the sucrose solution
consumption than the corresponding crystalline
form. The plasma concentration Cmax at AUC
exposure of the amorphous form of the com-
pound 1 is significantly improved and better than
the Cmax of the corresponding crystalline form of
the compound 1. On the basis of the efficacy,
pharmacokinetic and toxicological evaluations,
the compound 1 in the amorphous form is
selected as a pre-clinical lead candidate.
Abstract
▶
●
cannabinoid CB1 and CB2
receptors
▼
This paper describes the synthesis, early process
development, salt selection strategies and pre
clinical evaluation of novel, potent and selective
CB1 antagonist, 8-Chloro-1-(2,4-dichloro-phenyl)-
4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]azu-
lene-3-carboxylic acid piperidin-1-ylamide 1.
The CB1 antagonism of compound 1 is also con-
firmed by reversal of CB1 agonist-induced hypo-
thermia in Swiss albino mice. The process for the
preparation of the compound 1 as a crystalline
solid is also described. The crystalline form of the
compound is found to be low bioavailable, there-
fore attempts have been made to improve its bio-
availability through polymorphic transformation
and salt formation. None of the salts prepared
▶
neutral antagonists
anti-obesity drugs
piperidin-1-ylamide
derivatives
●
▶
●
▶
●
Introduction
generally produce marked or sustainable weight
loss [7,8], whereas psychological therapies are
difficult to deliver on a mass scale [9] and long-
term results are disappointing. Hence, pharma-
cotherapy for obesity has become a popular
choice, especially among the younger genera-
tions [10].
The role of cannabinoid receptor system in the
regulation of appetite and food intake in animals
and humans intrigued the scientific community
to develop drugs targeted towards cannabinoid
▼
Obesity is widely recognised as the largest and
fastest growing public health problem in the
developed and developing countries [1] and con-
sidered as a global epidemic. The International
Obesity Task Force estimates that more than 300
million individuals world wide are obese and an
additional 800 million are over-weight [2,3].
Sedentary life style, advent of junk foods, mod-
ern lifestyles with their various stresses and
increasing disposable income play a part in this
problem [4]. Obesity is associated with substan-
tial increases in morbidity, premature mortality,
impaired quality of life and large healthcare costs
[5]. The problem with obesity are compounded
due to the major comorbidities associated with it
that include type 2 diabetes, metabolic syn-
drome, hypertension, dyslipidaemia, myocardial
infarction, stroke, certain types of cancers, sleep
apnea and osteoarthritis [6]. Lifestyle changes in
the form of dieting and/or exercise per se do not
received 02.10.2014
accepted 23.03.2015
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1548848
Published online: 2015
Drug Res
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 2194-9379
receptor type 1 (CB1) [11–13]. Rimonabant hydro-
▶ꢀ
chloride (Compound-2, Fig. 1) has been the
●
first therapeutically relevant, potent and selec-
tive CB1 receptor inverse agonist, which was
approved in Europe as an anti-obesity drug [14].
However, reports of serious psychiatric problems
(such as anxiety, depression and suicide) led to
withdrawal of Rimonabant and termination of
several other CB1 receptor-antagonist-based anti-
obesity drug development programmes (such as
taranabant, otenabant, surinabant and ibipina-
bant) [15,16].
Correspondence
K. Banerjee
Zydus Research Centre
Sarkhej-Bavla N.H 8A Moraiya
Ahmedabad-382210
India
Tel.: +91/2717/665 555
Fax: +91/2717/665 355
kaushik.banerji@zyduscadila.
com
*ZRC communication 465
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
Fig. 1 CB1 receptor antagonist with diverse
chemical structures.
Researchers have tried to develop several classes of CB1 receptor
antagonist with diverse chemical structures [17–19]. Thus, for
example, Solvay Pharmaceuticals has disclosed the 3,4-diaryl
amide bond formation chemistry yielded 8-Chloro-1-(2,4-
dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]
azulene-3-carboxylic acid piperidin-1-ylamide as an oil which
was treated with ethereal HCl to get the HCl salt 10 as a brown
solid . Trituration with ethyl acetate and subsequent drying
yielded the pure compound 10 as a white solid. The neutraliza-
tion of 10 with NaOH provided the compound 1 in crystalline
form. The crystalline compound 1 is dissolved in dimethyl for-
mamide by heating at 40°C and the resulting solution is cooled
to 27–30°C over a period of 48h to give needle shaped crystals
of the DMF solvate 11. The different salts of 1 were prepared by
dissolving 1 in acetone and adding appropriate mineral/organic
acids at 40–50°C [12 (a–e)]. Stirring the compound 1 in dichlo-
romethane at room temperature (27–30°C), followed by removal
of solvents under reduced pressure, afforded the amorphous
form of the compound 1.
▶ꢀ
dihydropyrazole class of compounds (Compound-3, Fig. 1) as a
●
CB1 antagonist, which has elicited potent in vitro [20] and in
vivo activities [21]. 8-Chloro-1-(2,4-dichlorophenyl)-N-piperi-
din-1-yl-1,4,5,6-tetrahydrobenzo-[6, 7]-cyclohepta-[1, 2–c]-
▶ꢀ
pyrazole-3-carboxamide (Compound-4, Fig. 1) was found to
●
be a very potent CB1 antagonist in cell-based in vitro assays, and
ex vivo screens [22]. However, the compound 4 had poor in vivo
efficacy and oral bioavailability [23]. Despite the withdrawal of
Rimonabant and the demise of several CB1 receptor antagonist
development programmes, researchers believe that one has not
yet reached the end of the line for anti-obesity treatments tar-
geting the CB1 receptor [24,25]. It is now believed that neutral
antagonists might retain the weight loss advantages and will be
devoid of adverse effects [26]. Another promising approach is to
develop agents which restricts their function through the
periphery and do not reach to brain [27]. In continuation of our
drug-discovery efforts to develop novel therapeutic agents to
Results and Discussions
▼
treat obesity, we have discovered a series of novel compounds as
The compound 1 in its crystalline form is synthesized according
▶ꢀ
▶ꢀ
CB1 modulators [28] and compound 1 ( Fig. 1) has been
to the scheme described in Fig. 2. The crystalline nature of the
●
●
▶ꢀ
●
selected as a lead compound for pre-clinical evaluation. Though
the compound 1 showed good pharmacological profile, it had
poor bioavailability. The aim of the present study is to develop a
new synthetic process for the preparation of compound 1 in high
yield suitable for pre-clinical development and improve its bio-
availability through salt selection strategies as well as through
polymorphic transformations. Herein, the efficacy, pharmacoki-
netic evaluations of novel CB1 antagonist, 8-Chloro-1-(2,4-
dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]
azulene-3-carboxylic acid piperidin-1-ylamide 1 are described.
compound 1 was confirmed by XRD ( Fig. 3b). The binding
affinity of compound 1 was tested in an in vitro cAMP assay. The
compound 1 exhibits an EC50 14.5μM in human CB1 receptor
forskolin-induced cAMP assay. In the human CB2 receptor (can-
nabinoid receptor 2) binding assay, the compound 1 showed Ki
3.19μM, IC50 4.75μM at 30μM concentration, indicating favora-
ble CB1 selectivity. In vitro CB1 antagonism was measured using
a binding assay in CHO cells expressing human CB1 receptors
(hCB1). Interestingly, the compound 1 did not change the forsko-
lin-stimulated cAMP accumulation in CB1-transfected HEK cells
up to 10µM concentration indicating the possible neutral anta-
gonist nature. The CB1 antagonism of compound 1 was also con-
firmed by reversal of CB1 agonist-induced hypothermia in Swiss
albino mice [29].
However, the crystalline form of the compound 1 like other
promising molecules in the CB1 class suffers from poor oral bio-
availability and pharmacokinetic profile. In order to improve the
oral bioavailability of compound 1, attempts were made to
change the polymorphic form of the compound 1. However,
these attempts were limited due to poor solubility of compound
1 in most of the organic solvents. In one of the attempts, the
compound 1 was dissolved in large excess of dichloromethane,
heated to reflux and the volume of the resulting solution was
reduced to minimum, which upon cooling to 27–30°C over a
period of 48h did not yield any solid. In another attempt, the
compound 1 was dissolved in large excess of acetone, heated to
reflux and the volume of the solution reduced to minimum. The
residue obtained was cooled to 27–30°C and kept over a period
of 48h, but did not yield any solid. The compound 1 was found to
Chemistry
▼
▶ꢀ
●
The compound 1 is synthesized as described in
Fig. 2.
4-(3-chlorophenyl)-butanoic acid 5 is reacted with oxalyl chlo-
ride in dichloromethane at room temperature (27–30°C) fol-
lowed by Friedel-craft’s acylation to provide 8-Chloro-3,4-
dihydro-2H-benzo[b]oxepin-5-one 6. Treatment of 6 with die-
thyl oxalate in ethanol followed by 2N HCl afforded 8-Chloro-
5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepin-4-yl)-oxo-acetic acid
ethyl ester 7. The reaction of 7 with 2,4-Dichlorophenyl hydra-
zine hydrochloride in ethanol at 5–7°C provided 8-Chloro-1-
(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-
benzo[e]azulene-3-carboxylic acid ethyl ester 8, which upon
hydrolysis using KOH in methanol yielded 8-Chloro-1-(2,4-
dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]
azulene-3-carboxylic acid 9. The reaction of 9 with SOCl2/tolu-
ene followed by reaction with 1-Amino piperidine under usual
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
Fig. 2 Synthetic scheme for preparing compound
1. Reagents and conditions: a (COCl)2, anhydrous
CH2Cl2, stirred at 0°C for 30min and 26–28°C
for 1.5h; b anhydrous AlCl3, CH2Cl2, stirred at
0–5°C for 20–25 min and at 26–27°C for 30min;
c diethyl oxalate, Na metal, ethanol; d 2N HCl,
CHCl3; eꢀethanol,ꢀIPA/HCl,ꢀrefluxedꢀatꢀ75–77ꢀ°Cꢀ
for 2 h; f KOH/MeOH; g SOCl2,ꢀtoluene,ꢀrefluxedꢀ
at 107–108°C for 30 min; h anhydrous methanol,
ethereal HCl, 0–5°C; i NaOH; j CH2Cl2,ꢀrefluxꢀunderꢀ
reduced pressure at 50°C; k DMF, heat at 40–50°C
for 2h, cooled and kept for 48h; l acetone, heat at
40°C till clear solution, added acids corresponding
toꢀXꢀ=ꢀClꢀ−,ꢀHSO4ꢀ−,ꢀ(COO)2−2ꢀ&ꢀ−ꢀCH3I in acetone,
and the mixture was cooled when necessary and
filtered.
be more soluble in solvents such as dimethyl formamide (DMF)
and dimethyl sulfoxide (DMSO). The hot solution of compound 1
in DMSO upon cooling at 27–30°C over a period of 48h afforded
a pasty material, which was found to be the solvate of DMSO. In
a subsequent attempt, the solution of compound 1 was prepared
in DMF by heating and subsequent cooling at 27–30°C over a
period of 48h yielded crystals. The crystals of compound 1 in
DMF were characterized as solvates of DMF 11. The acute expo-
sure to dimethyl formamide is reported to cause liver toxicity in
humans [30], thus the crystalline form of the DMF solvate of
compound 1 was not evaluated further in any pharmacological
studies.
be associated with accumulation in tissues after acute exposure,
thereby limiting their use for repeated dose medication.
Development of amorphous form of pharmaceutical compounds
represents both an opportunity and a necessity in pharmaceuti-
cal development. The opportunity arises from the potential of
the amorphous form to improve the pharmacokinetic properties
and bioavailability rather than the corresponding crystalline
form.
The compound 1 was made into amorphous form and confirmed
▶ꢀ
through XRD spectra ( Fig. 3a). The amorphous form of com-
●
pound 1 was evaluated for its pharmacokinetic properties and
its efficacy was evaluated using sucrose (5% w/v) consumption
Furthermore, we synthesized the stable oxalate 12b, benzene-
sulfonate 12c, bisulfate 12d, and methyl iodide 12e salts of com-
pound 1. None of the salts altered the in vitro binding mode,
potency and selectivity as initially exhibited by the crystalline
form of compound 1. The hydrochloride salt 12a upon storage
model in Zucker fa/fa rats.
The results are provided in Tables 1 and ꢀ2:
The crystalline form of the compound 1 showed significant
appetite suppression in rodent model ( Table 1), however its
▶ꢀ
●
▶ꢀ
●
▶ꢀ
●
pharmacokinetic profile was poor ( Table 2).
gets hydrolysed wherein the piperidine ring gets cleaved to form
The in vivo efficacy study of the amorphous form of compound
▶ꢀ
the corresponding methyl ester 13, Fig. 4. The structure of
1 in 5% sucrose solution intake model in female Zucker fa/fa rats
●
▶ꢀ
compound 13 was confirmed through single crystal analysis.
The oxalate 12b, benzenesulfonate 12c, bisulfate 12d, and
methyl iodide 12e salts of compound 1 significantly improved
the pharmacokinetic parameters; however, they were found to
at single oral dose of 10mg/kg ( Table 1) demonstrates better
●
reduction in the sucrose solution consumption than the corre-
sponding crystalline form.
The pharmacokinetic parameters of both the crystalline and
▶ꢀ
amorphous form of compound 1 were evaluated ( Table 2). The
●
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
plasma concentration Cmax at AUC exposure of the amorphous
form of the compound 1 is significantly improved and better
than the Cmax of the corresponding crystalline form of the com-
pound 1. Further, compound 1 exhibited acceptable safety pro-
file upto a dose 30 times of the efficacy dose. No changes in body
weight and weights of the organs (kidney, heart and liver) were
observed upto 300mg/kg dose in a 14 day repeat dose study. The
compound also did not bind to hERG upto 3 micromolar concen-
trations.
Based on the impressive in vitro, in vivo and pharmacokinetic
parameters of the amorphous form of the compound 1 as a pos-
sible neutral CB1 antagonist, the compound has been selected
for further development.
In order to prepare sufficient quantities of the compound 1 to
support the development program a novel process for prepara-
▶ꢀ
tion of the compound 1 was developed as described in Fig. 2.
●
▶ꢀ
●
The process described in Fig. 2 is scalable and gives the com-
pound 1 in high yield (77.6%).
Conclusion
▼
Early process development, synthesis and identification of the
amorphous form of the potent CB1 antagonist compound 1 is
described, The improved process described herein is scalable,
gives the stable amorphous form in high yield and is suitable for
meeting the needs of long term development. The compound 1
demonstrated favourable safety profile in rodent models. Initial
pharmacological data of compound 1 are indicative that the
compound may be a neutral antagonist and it may not be accom-
panied by behavioral signs of nausea etc. during the feeding sup-
pression induced by compound 1. Additional research will be
necessary to confirm this hypothesis.
Experimental Section
▼
Synthesis
Synthetic materials and methods
Reagents and solvents were obtained from commercial suppliers
and used without further purification. Flash chromatography
was performed using commercial silica gel (230–400 mesh).
Melting points were determined on a capillary melting point
apparatus and are uncorrected. IR spectra were recorded on a
Shimadzu FT IR 8300 spectrophotometer (Vmax in cm− 1, using
Fig. 3 XRD patterns of amorphous form a and crystalline form b of
compound 1.
1
KBr pellets or Nujol). The H NMR spectra were recorded on a
Brucker Avance-300 spectrometer (300MHz). The chemical
shifts (δ) are reported in parts per million (ppm) relative to TMS,
either in CDCl3 or DMSO-d6 solution. Signal multiplicities are
represented by s (singlet), d (doublet), dd (doublet of doublet), t
(triplet), q (quartet), bs (broad singlet), and m (multiplet).
13CNMR spectra were recorded on Brucker Avance-400 at
100MHz either in CDCl3 or DMSO-d6 solution. Mass spectra
(ESI-MS) were obtained on Shimadzu LC-MS 2010-A spectrom-
eter. HPLC analysis were carried out at λmax 220nm using col-
umn ODS C-18, 150nm×4.6nm×4μ on AGILENT 1100 series.
8-Chloro-3,4-dihydro-2H-benzo[b]oxepin-5-one(6): 4-(3-chloro-
phenyl)-butanoic acid (128.0g, 596.74mmol) was taken in a
round bottom flask and anhydrous dichloromethane (1L) was
Fig. 4 Structure of compound 13. Reagents and conditions: a acetone,
heat at 40°C till clear solution, HCl.
Table 1ꢀ Inꢀvivoꢀefficacyꢀofꢀtheꢀ
crystalline and amorphous form
of the compound 4 in 5% sucrose
solution intake model in female
Zucker fa/fa rats at single oral
dose of 10mg/kg.a
in-vivo (in 5% sucrose solution intake)
Compound
Total consumption
in 4h
% Change vs. Control
(Sucrose intake)
5% Change vs. Control
(per 100g body wt.)
Control
48.00±2.20
8.4±3.3
Rimonabant
ꢀ−ꢀ66.4ꢀ±ꢀ11.0
ꢀ−ꢀ38.7ꢀ±ꢀ12.2
ꢀ−ꢀ54.7ꢀ±ꢀ11.5
ꢀ−ꢀ62.0ꢀ±ꢀ15.3
ꢀ−ꢀ30.2ꢀ±ꢀ16.4
ꢀ−ꢀ24.2ꢀ±ꢀ15.3
Crystalline Form of 1
Amorphous Form of 1
32.2±6.4
31.9±5.2
a Values indicate Mean±SEM for n=6 in 4h
Banerjee K et al. Novel CB1 Antagonist… Drug Res

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Table 2 Mean pharmacokinetic parameters of the crystalline and amorphous form of the 1 in fasted female Zucker fa/fa rats p.o. at 10mg/kg.
Compd.
Route
dose (mg/kg)
Tmax (h)
Cmax (ng/mL)
T1/2 (h)
AUC(0–∞) (h.ng/mL)
1 (crystalline)
1 (Amorphous)
Oral
10
10
4.5±0.7
4.2±1.2
223±13
575±32
23.9±6.8
22.3±2.9
3241±125
6338±234
a Values indicate mean±SD for n=6
added to it. The solution was stirred and cooled to −20°C. To this
solution oxalyl chloride (74.06mL, 835.43mmol, 1.4 eq) was
added drop wise at −20°C over a period of 15–20min. The
resulting solution was stirred at 0°C for 30min and 26–28°C for
1.5h. The progress of the reaction was monitored by TLC until all
starting material was consumed. The reaction was quenched
with ethanol and TLC was checked using mobile phase 5% meth-
anol in chloroform. The solvents were removed on a rotatory
evaporator under reduced pressure to afford brown oil. Sepa-
rately, in a 4-necked round bottomed flask, anhydrous AlCl3
(95.6g, 716.08mmol, 1.2 eq) was taken and to it was added
anhydrous dichloromethane (1 L). The suspension was stirred
and cooled to 0–5°C. To this cooled suspension, a solution of acid
chloride obtained above in anhydrous CH2Cl2 (200mL) was
added dropwise at 0–5°C over a period of 20–25min. The result-
ing solution was stirred at 26–27°C for 30min. The progress of
the reaction was monitored by TLC using mobile phase 5%
methanol in chloroform. The reaction mixture was poured into
mixture of demineralized water (DM water) and crushed ice
(3L) in 5L round bottom flask followed by CH2Cl2 (1L). The mix-
ture was stirred at 26–27°C for 16h. The organic layer was sepa-
rated, washed with DM water (3×1L). The organic layer was
separated, dried over anhydrous Na2SO4 and treated with acti-
vated charcoal (3-tea spoon) at 38–39°C for 15–20min and fil-
tered hot through Hyflow. The solvents were removed on a
rotatory evaporator under reduced pressure to yield a brown oil
(118g, 100%).
IR (KBr cm− 1) 3417, 3109, 1830, 1714, 1614, 1595, 1544, 1207,
1089, 661, 617, 565, 447; 1H NMR (400MHz, CDCl3) δ (ppm)
7.93 (d, J=9.3Hz, 1H), 7.26 (m, 2H), 4.37 (m, 2H), 2.88 (m, 2H).
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-dia-
za-benzo[e]azulene-3-carboxylic acid ethyl ester (8): (8-Chloro-
5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepin-4-yl)-oxo-acetic acid
ethyl ester (22.0g, 74.199mmol) 7 was taken in a round bottom
flask and to it was added anhydrous ethanol (225mL). The sus-
pension was stirred and cooled to 5–7°C. To this suspension,
2,4-Dichlorophenyl
hydrazine
hydrochloride
(17.90g,
83.845mmol, 1.13 eq) was added portion wise at 5–7°C. The
resulting suspension was stirred at 5–7°C for 15–20min and
brought to 25–26°C. Isopropyl alcohol: HCl (2.2mL) was added
to this mixture and refluxed at 75–77°C for 2h. The progress of
the reaction was monitored by TLC using mobile phase 20%
ethyl acetate in petroleum ether. The reaction mixture was
cooled to 25–27°C and the solid separated out was filtered on a
Buchner funnel under reduced pressure and dried to afford
orange solid (38.0g, 100%).
IR (KBr cm− 1) 3425, 1724, 1679, 1564, 1535, 1454, 1083, 906,
813; 1H NMR (400MHz, CDCl3) δ (ppm) 7.40 (t, 1H), 7.34 (d,
J=2.25Hz, 2H), 6.83 (dd, J=8.58Hz, 2H), 6.66 (d, J=8.58Hz, 1H),
4.50-4.31 (m, 4H), 3.46 (d, 4H), 1.44 (t, 3H); ESI-MS m/z (Relative
intensities) (+ve mode) 458.5 (M+H)+ .
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylicacid(9): 8-Chloro-1-(2,4-
dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-diaza-benzo[e]azu-
lene-3-carboxylic acid ethyl ester (38.0g, 86.857mmol) (8) was
taken in a round bottom flask and to it was added methanol
(300mL). To this, a solution of KOH (9.73g, 173.174mmol, 2.0
eq) in methanol: water (1:1, 200mL) was added and reaction
mixture was refluxed at 65–68°C for 2.0h. The progress of the
reaction was monitored by TLC using 10% methanol in chloro-
form as mobile phase. The reaction mixture was cooled to
25–26°C, poured into ice cold water and acidified to pH 4 using
10% HCl solution. The solid separated out was filtered on a Buch-
ner funnel under suction, washed with water, dried under suc-
tion. The solid was further taken in isopropyl alcohol (IPA),
stirred for 10–15min and filtered to afford an off white solid
(20.2g, 56.8%).
IR (KBr cm− 1): 2970, 2887, 1685, 1595, 1087, 821, 767; 1H NMR
(400MHz, CDCl3) δ (ppm) 7.70 (dd, J=8.72Hz, J=0.77, 1H), 7.06
(m, 2H), 4.25 (t, J=6.60Hz, 2H), 2.88 (t, J=6.97Hz, 2H), 2.22 (m,
2H); ESI-MS m/z (Relative intensities) (+ve mode) 256.8
(M+K)+ .
(8-Chloro-5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepin-4-yl)-oxo-
acetic acid ethyl ester (7): Anhydrous ethanol (1500mL) was
taken in a round bottom flask and small pieces of sodium metal
(31.10g, 1526.71mmol, and 2.0 eq) were added to it with stirring.
The solution was stirred till all Na metal was dissolved. The solu-
tion was cooled to 25–27°C. To this diethyl oxalate (103.67mL,
763.335mmol, 1.0 eq) was added drop wise at 25–27°C over a
period of 15–20min and stirred for 25–30min. at the same tem-
perature. To this was added a solution 8-Chloro-3,4-dihydro-
2H-benzo[b]oxepin-5-one (150.0g, 763.335mmol) in ethanol
(2000mL) drop wise at 25–27°C over a period of 15–20min. The
resulting yellow solution was stirred at 25–26°C for 3–4h and
the progress of reaction was monitored by TLC using mobile
phase 10% ethyl acetate in petroleum ether. The reaction mix-
ture was diluted with DM water (2500mL), acidified with 2 N
HCl to pH 4 and extracted with CHCl3. The chloroform layer was
separated, washed with DM water, dried over anhydrous Na2SO4
and evaporated on a rotatory evaporator under reduced pressure
to get yellow oil, which solidifies upon standing. The solid was
slurried in diethyl ether (1L) and filtered to get yellow solid
(110g, 48.6%).
IR (KBr cm− 1) 3450, 3082, 1687, 1596, 1568, 1431, 1386, 1035,
985, 567, 549, 455; 1H NMR (400MHz, DMSO-d6) δ (ppm) 13.08
(bs, 1H), 7.88 (d, J=2.04Hz, 1H), 7.72-7.64 (m, 2H), 7.23 (d,
J=2.13Hz, 1H), 7.01 (dd, J=2.19Hz, J=8.58Hz, 1H), 6.71 (d,
J=8.58Hz, 1H), 4.04 (m, 4H); ESI-MS m/z (Relative intensities)
(+ve mode) 410.5 (M+H)+ .
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-dia-
za-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide (1),
crystalline: 8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-
1H-6-oxa-1,2-diaza-benzo[e]azulene-3-carboxylic acid (5.0g,
12.25mmol) was taken in a round bottom flask and toluene
(30mL) was added to it. To this solution, thionyl chloride (2.91g,
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
24.5mmol) was added and the mixture was refluxed at 107–
108°C for 30min. The complete conversion of acid to acid chlo-
ride was confirmed by TLC. Subsequently, the reaction mixture
was cooled to 30–35°C and transferred into single neck round
bottom flask. The solvents were evaporated on a rotatory evapo-
rator under reduced pressure to afford an oil. The oil was further
diluted in anhydrous CH2Cl2 (20mL), cooled to 0–5°C in an ice
bath and treated with 1-amino piperidine (1.83g, 18.38mmol).
The resulting mixture was stirred at 25–26°C for 15–20min. The
progress of the reaction was monitored by TLC using mobile
phase 50% EtOAc in hexane. The reaction mixture was diluted
with DM water (200mL) and extracted with toluene. The organic
layer was separated, washed with DM water (100mL), brine
solution (100mL), dried over anhydrous Na2SO4 and solvents
were removed on a rotatory evaporator under reduced pressure
to afford an oil. The oil was diluted with anhydrous methanol
(25mL), cooled to 0–5°C in an ice bath and treated with ethereal
HCl (5–6mL). The solvents were evaporated on a rotatory evapo-
rator under reduced pressure to afford brown solid. The crude
solid was triturated in ethyl acetate, filtered on a Buchner funnel
under suction and dried to afford an off white solid (5.0g, 77.6%).
1HNMR: (CDCl3, 300MHz): δ 10.35 (s, 1H), 7.51 (d, J=1.74Hz,
1H), 7.40 (m, 2H), 7.34 (m, 1H), 7.14 (d, J=1.95Hz, 1H), 6.62 (d,
J=8.55Hz, 1H), 4.38 (m, 2H), 4.06 (bs, 3H), 3.48 (bs, 3H), 3.26 (m,
2H), 3.11 (m, 2H), 1.67(bs, 4H), 1.41(m, 1H), 1.18 (m, 1H);
1HNMR: (DMSO-D6, 300MHz): δ 10.53 (s, 1H), 7.89 (d, J=2.01Hz,
1H), 7.78 (d, J=8.49Hz, 1H), 7.68 (dd, J=10.47, 1.95Hz, 1H), 7.26
(d, J=1.95Hz, 1H), 7.03 (dd, J=10.56, 1.95Hz, 1H), 6.72 (d,
J=8.61Hz, 1H), 4.45 (bs, 1H), 4.25 (bs, 1H), 3.23 (bs, 4H), 3.06 (m,
3H), 1.72 (bs, 4H), 1.42(bs, 2H), 1.16(t, 1H), 0.98 (t, 1H).
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
oxalate {12 b}: Compound 1 (0.500g (1.017mmol) was taken
in a round bottomed flask, followed by 10mL acetone. The sus-
pension was warmed upto 45–50°C to get clear solution. To the
solution of 1 in acetone a solution of oxalic acid (0.075g,
1.017mmol) in 3mL acetone was added slowly. The resulting
clear solution was cooled to 0–5°C and stirred for 35–40min.
The solid separated out which was filtered, washed with acetone
and dried under vacuum to afford white solid (0.267g, 45.9%).
IR (KBr cm− 1) 3412, 2951, 1899, 1691, 1597, 1564, 1492, 1236,
817, 704;
1H NMR (400MHz, DMSO-d6) δ (ppm) 9.19 (s, 1H), 7.90 (d,
J=2.4Hz, 1H), 7.80 (d, J=8.4Hz, 1H), 7.71 (dd, J=8.4Hz, J=2.4Hz,
1H), 7.27 (d, J=2.4Hz, 1H), 7.04 (dd, J=8.6Hz, J=2.4Hz, 1H), 6.74
(d, J=8.4Hz, 1H), 4.49 (bs, 1H), 4.26 (bs, 1H), 3.40 (bs, 2H), 3.28
(bs, 2H), 2.82-2.80 (m, 4H), 1.64-1.58 (m, 4H), 1.4-1.3 (m, 2H).
ESI-MS m/z (Relative intensities) (+ve mode) 493.05 (M+H)+
(80%).
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
benzene sulfonate salt {12c}: Placed 1 (0.500g, 1.017mmol)
in a round bottomed flask and to it was added acetone (10mL).
The suspension was warmed upto 45–50°C to get clear solution.
To the solution of 1 in acetone, benzene sulfonic acid (0.160g,
1.017mmol) in acetone (3mL) was added slowly. The resulting
clear solution was cooled to 0–5°C and stirred for 35–40min.
The solid separated out was filtered, washed with acetone and
dried under vacuum to afford a white solid (0.350g, 53%).
IR (KBr cm− 1) 3383, 3153, 2945, 1896, 1701, 1697, 1606, 1552,
1219, 1151, 981, 956, 896, 731; 1H NMR (400MHz, DMSO-d6) δ
(ppm) 11.05 (bs, 1H), 7.94 (d, J=2.4Hz, 1H), 7.83 (d, J=8.4Hz,
1H), 7.73 (dd, J=8.6Hz, J=2.4Hz, 1H), 7.65-7.61 (m, 2H), 7.37-
7.30 (m, 4H), 7.07 (dd, J=8.6Hz, J=2.4Hz, 1H), 6.77 (d, J=8.8Hz,
1H), 4.55-4.52 (m, 1H), 4.30-4.29 (m, 1H), 3.46-3.43 (m, 1H),
3.33-3.32 (m, 5H), 1.83-1.80 (m, 4H), 1.5-1.4 (m, 2H). ESI-MS
m/z (Relative intensities) (+ve mode) 493.0 (M+H)+ (100%).
The basification of the HCl salt of compound 1 with NaOH gave
the compound of 1 in the crystalline form.
1HNMR: (CDCl3, 300MHz) δ 7.8 (d, J=7.5Hz, 2H), 7.4 (d, J=7.5Hz,
2H), 7.39 (d, J=8.4Hz, 1H), 7.34 (d, J=1.8Hz, 1H), 7.3 (m, 2H), 7.0
(d, J=8.4Hz, 1H), 6.5 (d, J=5.4Hz, 1H), 3.27 (d, J=5.1Hz, 2H),
3.18 (d, J=5.1Hz, 1H), 2.6 (m, 3H), 1.5 (s, 3H); Melting point (by
DSC): Onset=195.3°C, peak=196.6°C.
XPRD: 6.97, 16.42, 17.53, 18.19, 18.35, 19.8, 20.93, 21.4, 23.69,
27.62°±0.2° degrees 2θ.
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
bisulfate {12d}: Placed 1 (1.000g, 2.034mmol) in a round bot-
tomed flask and to it was added acetone (15mL). The suspension
was warmed upto 45–50°C to get clear solution. To the solution
of 1 in acetone was added conc. H2SO4 (0.108mL, 2.034mmol)
dropwise. The resulting clear solution was cooled to 0–5°C and
stirred for 15–20min. The solid separated was filtered, washed
with acetone and dried under vacuum to afford a White solid
(1.03 g, 85.87%) IR (KBr cm− 1) 3404, 3153, 2951, 1708, 1599,
1550, 1369, 1215, 1049, 987, 958, 868, 812;
1H NMR (400MHz, DMSO-d6) δ (ppm) 10.85 (bs, 1H), 7.93 (d,
J=2.4Hz, 1H), 7.83 (d, J=8.8Hz, 1H), 7.71 (dd, J=8.6Hz, J=2.4Hz,
1H), 7.29 (d, J=2.4Hz, 1H), 7.07 (dd, J=8.6Hz, J=2.4Hz, 1H), 6.76
(d, J=8.8Hz, 1H), 4.55-4.52 (m, 1H), 4.29-4.28 (m, 1H), 3.45-
3.3.42 (m, 1H), 3.33-3.31 (m, 1H), 3.35-3.15 (m, 4H), 1.80-1.77
(m, 4H), 1.5-1.4 (m, 2H). ESI-MS m/z (Relative intensities) (+ve
mode) 493.0 (M+H)+ (99%).
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
(1), dimethyl formamide solvate (11): Placed crystalline com-
pound 1 (0.50g, 1.017mmol) into round bottom flask, followed
by addition of dimethyl formamide (15mL). The solution was
heated to 60–70°C for 2h to obtain a clear solution. The solution
was cooled to 27–30°C for 48h whereby white crystals were
obtained.
Weight of White solid substance=0.258g
IR (KBr cm− 1) 3412, 2941, 1678, 1595, 1564, 1492, 1290, 1265,
1209, 1153, 1026, 983, 956, 869, 817, 810;
1H NMR (400MHz, CDCl3) δ (ppm) 8.01 (s, 1H), 7.63 (s, 1H), 7.53
(d, J=2.4Hz, 1H), 7.39 (dd, J=8.6Hz, J=2.0Hz, 1H), 7.33 (dd,
J=9.2Hz, J=4.4Hz, 1H), 7.14 (d, J=2.0Hz, 1H), 6.79 (dd, J=8.8Hz,
J=2.0Hz, 1H), 6.60 (d, J=8.4Hz, 1H), 4.38 (bs, 2H), 3.49 (bs, 2H),
2.95 (s, 3H), 2.85(s, 3H), 2.95-2.85 (m, 4H), 1.78-1.72 (m, 4H),
1.45-1.43 (m, 2H).
ESI-MS m/z (Relative intensities) (+ve mode) 493.0 (M+H)+
(100%).
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
methyl iodide {13e}: Placed 1 (5.000g, 10.17mmol) in a round
bottomed flask and to it was added acetone (15mL). The suspen-
sion was warmed to 45–50° to get clear solution. To this solution
methyl iodide (14.435g, 6.322mmol) was added. The resulting
clear solution was refluxed over a period of 16h. The solid pre-
cipitated was dissolved in distilled acetone and resulting solu-
tion was concentrated under reduced pressure at 45°C to get a
yellow solid. The solid was filtered, washed with acetone and
dried under vacuum to yield off-white solid (5.3g, 82.81%) IR
(KBr cm− 1) 3419, 2958, 1593, 1564, 1496, 1309, 1294, 1209,
1030, 954, 920, 869, 812; 1H NMR (400MHz, CDCl3) δ (ppm)
7.83 (d, J=2.4Hz, 1H), 7.62 (dd, J=8.6Hz, J=2.0Hz, 1H), 7.57 (d,
J=8.8Hz, 1H), 7.18 (d, J=2.0Hz, 1H), 6.96 (dd, J=8.8Hz, J=2.0Hz,
1H), 6.66 (d, J=8.4Hz, 1H), 4.40 (bs, 1H), 4.22 (bs, 1H), 4.12 (bs,
2H), 3.41 (s, 3H), 3.22-3.14 (m, 4H), 2.01-1.98 (m, 2H), 1.66-1.58
(m, 3H), 1.42-1.36 (m, 1H). ESI-MS m/z (Relative intensities)
(+ve mode) 507.0 (M+H)+ (100%).
Animal Ethical Committee approved all the study protocols.
Female Zucker fa/fa rats (age of 10–12 weeks and 300–350g of
weight) were used for in vivo experiments. Compounds were
suspended with 0.5% carboxymethyl cellulose sodium salt in
distilled water. The test compounds were administered at the
dose of 10mg/kg and by oral route in a volume of 2mL/kg body
weight. The obese Zucker fa/fa rats were housed individually
and subjected to training for consuming 5% sucrose solution
over a period of 4h, by allowing access to the 5% sucrose solu-
tion in the bottles. Food and water were withdrawn during this
time. This training was given for 6 consecutive days, at the same
time of the day. On seventh day, the animals were randomized
into groups of 6 animals each and treated with the test com-
pounds. After 1h of treatment, the animals were exposed to the
5% sucrose solution for 4h as that of the training schedule. The
amount of sucrose solution consumed by each animal was cal-
culated. Difference between the control and treatment groups
were analyzed by performing one way ANOVA followed by Dun-
nett’s test on sucrose solution consumption using Graph pad
Prism software.
8-Chloro-1-(2,4-dichloro-phenyl)-4,5-dihydro-1H-6-oxa-1,2-
diaza-benzo[e]azulene-3-carboxylic acid piperidin-1-ylamide
(1), amorphous: The crystalline 8-Chloro-1-(2,4-dichloro-
phenyl)-4,5-dihydro-1H-6-oxa-diaza-benzo[c]azulene-3-car-
boxylic acid-piperidin-1-ylamide (5g, 10.17mmol) was placed
in a round bottom flask and added of dichloromethane (100mL).
The resulting solution was stirred at 27–29°C for 10min. The
solvents were removed under reduced pressure at 50°C to afford
white solid. The solid obtained was analyzed by XRD pattern,
established it to be in the amorphous form. The DSC thermo-
gram showed 2 peaks one at 182.9°C and the other at 196.3°C.
Pharmacokinetics experiment
Pharmacokinetics of the test compound was studied via per-oral
route of administration in Zucker fa/fa rats of 8–10 weeks of age
(6 animals). Animals were fasted for 18h and food was supplied
after 4h of administration of the test compound. There was free
access to water throughout the study. A homogenous suspension
of the test substance was prepared in 0.5% w/v CMC in normal
saline and a per-oral dose of 10mg/kg was administered. After
the administration of the test compounds, blood samples were
withdrawn at various time intervals through retro-orbital
plexus and collected into heparinized micro centrifuge tubes.
Plasma was separated by centrifugation at 4000rpm for 5min at
ambient temperature and analyzed immediately. Remaining
samples were stored at −20°C until analyzed.
Analysis was carried out by taking an aliquots of 180µL plasma
and 20µL of internal standard (Atorvastatin) and was extracted
with 2.5mL of extracting solvent (ethyl acetate: acetonitrile
80:20, v/v) in glass test-tube by vortexing with spinix vortex
mixture for a minute. This was then centrifuged at 2000rpm for
2.0min. The supernatant was transferred to another glass test-
tube and the solvent was evaporated under nitrogen using
Zymark evaporator at 40°C. Finally, the tubes were reconstituted
with 0.1mL diluent (acetonitrile: methanol: water 40:40:20,
v/v/v). The reconstituted samples were analyzed on Agilent
1100 Series HPLC system with a mobile phase of 0.05% v/v trif-
luoroacetic acid in water: acetonitrile (32:68, v/v); flowing at a
flow rate of 1.0mL/min through a Kromasil 250mm×4.6mm×5µ
column maintained at 30°C. Chromatographic separation was
achieved within 15min. Agilent software version Chemstation
Rev.A.09.01. (1206) was used to acquire and process all chroma-
tographic data. Quantification was based on a series of calibra-
tors ranging from 0.031 to 32µg/mL, prepared by adding test
compound to drug free rat plasma. Quality control samples were
analyzed in parallel to verify that the system performs in con-
trol. Pharmacokinetic parameters namely; maximum plasma
concentration (Cmax), time point of maximum plasma concen-
tration (tmax), area under the plasma concentration−time curve
from 0h to infinity (AUC0-∞) and half-life of drug elimination
during the terminal phase (t1/2) were calculated from plasma
concentration vs. time data, by standard non-compartmental
In vitro cAMP assay
Fatty acid-free BSA, IBMX (isobutyl methyl xanthine), RO20-
1724 {4-[(3-butoxy-4-methoxyphenyl) methyl]-2-imidazololid-
inone}, forskolin and DMSO (hybrimax) were purchased from
Sigma Chemical Co. cAMP detection ELISA kit was from Assay
Designs, USA. Tissue culture reagents were purchased from
Sigma and Hi-media. Other reagents used were all of analytical
grade. The cAMP assay was carried out in Chinese Hamster
Ovarian (CHO) cells (CHOK1) stably expressing human CB1
receptor following the method of Rinaldi-Carmona et al [31].
Cells grown to 80% confluence were maintained in HAM’S F12
medium containing 10% heat inactivated dialyzed fetal bovine
serum and 0.8mg/mL G-418. Cells were seeded at a density of
50000 cells/well in 24-well plate, grown for 16–18h, washed
once with PBS and incubated for 30min at 37°C in plain
HAM’SF12 containing 0.25% free fatty acid BSA, IBMX (0.1mM)
and RO20-1724 (0.1mM). IBMX, the pan phosphodiesterase
inhibitor and RO20-1724, the specific phosphodiesterase-4
inhibitor were added to restore cAMP up to the detection limit.
After 5min incubation with the drugs, forskolin was added at a
final concentration of 10mM and incubation was carried out for
another 20min at 37°C. The reaction was terminated by wash-
ing once with PBS and adding 200 lL lysis buffer comprising 0.1
N HCl and 0.1% Trition X-100. The lysates were centrifuged and
aliquotes from supernatants were used for detection of cAMP by
ELISA as per the manufacturer’s protocol.
5% Sucrose solution intake in Zucker fa/fa rats
All the animals used in the study were procured from the Ani-
mal Breeding Facility of Zydus Research Center. Institutional
Banerjee K et al. Novel CB1 Antagonist… Drug Res

DrugRes/2014-10-0878/10.4.2015/MPS
Original Article
14 Sorbera LA, Castaner J, Silvestre JS. Rimonabant Hydrochloride. Drugs
Future 2005; 30: 128–137
15 http://www.ema.europa.eu/docs/en_GB/document_library/Press_
release/2009/11/WC500014774.pdf
methods, using the WinNonLin software version 4.0.1 procured
from Pharsight Corporation, USA.
16 Plieth J. Obesity: what next after the CB1 antagonists’ failure. Scrip
2008; 44–47
17 Hertzog DL. Recent advances in the cannabinoids. Expert Opin Ther
Patents 2004; 14: 1435–1452
18 Lange JHM, Kruse CG. Medicinal chemistry strategies to CB1 cannabi-
noid receptor antagonists. Drug Discov Today 2005; 10: 693–702
19 Muccioli GG, Lambert DM. Current Knowledge on the Antagonists and
Inverse Agonists of Cannabinoid Receptors. Curr Med Chem 2005;
12: 1361–1394
Acknowledgement
▼
Authors are grateful to the management of Zydus Group as well
as the faculty of Chemical Sciences, MS University, Baroda, for
encouragement, and the Medicinal Chemistry and Analytical
Departments of Zydus Research Centre for support.
20 Lange JHM, Coolen HKAC, van Stuivenberg HH et al. Synthesis, Biological
Properties, and Molecular Modeling Investigations of Novel 3,4-Dia-
rylpyrazolines as Potent and Selective CB1 Cannabinoid Receptor
Antagonists. J Med Chem 2004; 47: 627–643
21 Need AB, Davis RJ, Alexander-Chacko JT et al. The relationship of in
vivo central CB1 receptor occupancy to changes in cortical monoam-
ine release and feeding elicited by CB1 receptor antagonists in rats.
Psychopharmacology 2006; 184: 26–35
Conflict of Interest
▼
None
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