Asymmetric synthesis and biological evaluation of N-cyclohexyl-4-[1-(2,4- dichlorophenyl)-1-(p-tolyl)methyl]piperazine-1-carboxamide as hCB1 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2011.08.030

We recently discovered and reported a novel series of benzhydrylpiperazine derivatives bearing an asymmetric carbon atom that are potent and selective hCB1 inverse agonists. In the present study, we used Davis-Ellmann-type sulfonamide chemistry to asymmet

Full text of DOI:10.1016/j.ejmech.2011.08.030

European Journal of Medicinal Chemistry 46 (2011) 5310e5316
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Asymmetric synthesis and biological evaluation of N-cyclohexyl-4-[1-(2,4-
dichlorophenyl)-1-(p-tolyl)methyl]piperazine-1-carboxamide as hCB1
receptor antagonists
,
b
,
,
1
,
a
,
a
a
a
a
Linghuan Gaoa 1, Min Lia , Tao Menga , Hongli Peng , Xin Xie , Yongliang Zhang , Yu Jin , Xin Wang ,
*
*
Libo Zoub, Jingkang Shena
,
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
b Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shengyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently discovered and reported a novel series of benzhydrylpiperazine derivatives bearing an
asymmetric carbon atom that are potent and selective hCB1 inverse agonists. In the present study, we
used Davis-Ellmann-type sulfonamide chemistry to asymmetrically synthesize two enantiomers of the
most potent racemic N-cyclohexyl-4-[1-(2,4-dichlorophenyl)-1-(p-tolyl)methyl]piperazine-1-carbo-
xamide [14]. Enantiomer separation and configuration assignment were carried out. Our results indi-
cate that the R-configuration is the more active enantiomer, displaying enhanced antagonistic activity for
hCB1 receptor, better oral bioavailability, and greater efficacy in the reduction of body weight in diet-
induced obese mice.
Received 17 May 2011
Received in revised form
19 July 2011
Accepted 20 August 2011
Available online 1 September 2011
Keywords:
Anti-obesity
CB1 receptor
Antagonist
Ó 2011 Elsevier Masson SAS. All rights reserved.
Asymmetric synthesis
Enantiomer
1. Introduction
has been suggested to be relevant in these behaviors [7]. On the
other hand, CB1 is also found in the adipose tissue, skeletal muscle,
Endocannabinoids increase food intake through their interaction
with the cannabinoid receptor 1 (CB1) expressed in the brain.
Pharmacological blockade of CB1 has shown promising results in
the treatment of obesity, type 2 diabetes, and metabolic syndromes
[1e3]. Rimonabant (SR141716, AcompliaÔ, Fig. 1), a potent and
selective CB1 receptor antagonist/inverse agonist, was launched by
Sanofi-Aventis in Europe in 2006 for the treatment of obesity and
associated risk factors [4]. However, it was associated with psychi-
atric side effects such as depression, anxiety and stress disorders [5],
which led to its withdrawal from the European market in 2008, and
also the withdrawal of several other CB1 inverse agonists including
taranabant and otenabant from phase III clinical trials [6]. Although
the exact role of the endocannabinoid system in the control of mood
and anxiety-like behaviors is not clear, CB1 modulation in the brain
peripheral nerves, and digestive system (gastrointestinal tract,
pancreas and liver) [8,9], where its activation contributes to obesity-
related metabolic and hormonal abnormalities [9]. It has been re-
ported that liver-specific CB1 knockout (LCB1ꢀ/ꢀ) mice are resistant
to diet-induced obesity [10], indicating that activation of CB1 in
peripheral liver tissue contributes to the diet-induced steatosis and
associated metabolic changes. Chronic treatment of obese mice or
rats with a CB1 antagonist induces sustained weight loss and
protects rodents from this detrimental metabolic phenotype, even
though it causes only a transient reduction in food intake [11].
Clinical trials also showed that rimonabant can significantly
improve glycemic index and dyslipidemia in type 2 diabetic
patients, and the profile of several other metabolic risk factors
including the reduction of visceral and hepatic fat in overweight and
obese patients [12,13]. These findings indicate that CB1 can directly
affect peripheral energy metabolism by mechanisms unrelated to
their effect on appetite, suggesting that selective targeting of
peripheral CB1 may result in an improved metabolic profile in
obesity without the untoward behavioral effects observed following
treatment with brain-acting CB1 antagonists.
* Corresponding authors. Tel.: þ86 21 50806600/5407; fax: þ86 21 50807088.
E-mail addresses: tmeng@mail.shcnc.ac.cn (T. Meng), penghl@mail.shcnc.ac.cn
(H. Peng), jkshen@mail.shcnc.ac.cn (J. Shen).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.030

L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
5311
compound 13. Herein, we wish to report our efforts on the asym-
metric synthesis of compounds 13S and 13R via Davis-Ellmann-
type sulfonamide chemistry [18], the pharmacokinetic profiles
and the in vitro and in vivo activities of the enantiomers.
2. Results and discussion
2.1. Chemistry
Fig. 1. CB1 receptor inverse agonists.
Chiral resolution is commonly used for the separation of racemic
compounds into their enantiomers. Opalka et al [19] has described
Recently, we have developed a novel series of benzhydryl-
piperazine derivatives, via privileged structure-based approach,
leading to the identification of compound 13 (Fig. 1) as a highly
potent and selective hCB1 inverse agonist (KiCB1 ¼ 0.15 nM;
EC50 ¼ 0.87 nM) [14]. Similar analogs were also reported by Song
et al. [15]. We found that, although compound 13 exhibited a lower
brain exposure compared to rimonabant (the brain/plasma ratios
are 1.0 and 4.4 for compound 13 and rimonabant, respectively), it
displayed comparable body weight-loss efficacy in diet-induced
obese (DIO) rats [14] (rimonabant data not shown). This strongly
supported the concept mentioned above that efficacious CB1
antagonists for the treatment of obesity are practicable without
central nervous system (CNS) liability. All the benzhydrylpiperazine
analogs examined in our previous structure-activity relationships
(SAR) studies featured a stereocenter. It is well known for chiral
drugs that their enantiomers can differ in pharmacodynamic
properties and/or biological activities, and in most cases only one of
the enantiomers, the eutomer, is responsible for therapeutic effect
[16]. Since it has been reported that there was a 2- to 10-fold
difference in affinity to hCB1 between the anti- and syn-diaste-
reomers of CB1R inverse agonist, MK-0364 (Fig. 1) [17], it was
necessary to undertake a further investigation of the stereochem-
ical consequences for pharmacokinetic profiles and bioactivities of
a
separation of
a
(ꢁ)-4-chlorobenzhydrylamine by co-
recrystallization with
resolve a racemic (2,4-dichlorophenyl)(p-tolyl)methanamine into
L
(þ)-tartaric acid. Initially, we tried to
its enantiomers (7a and 7b) by fractional crystallization of its
L-
(þ)-tartaric acid or -(ꢀ)-tartaric acid salts using a similar proce-
D
dure reported by Opalka [19]. However, after six times of co-
recrystallization from the water-ethanol or water-methanol
mixture, the enantiomeric (2,4-dichlorophenyl)(p-tolyl)methan-
amine is obtained in only up to 20% enantiomer excess determined
by chiral HPLC. This approach is inapplicable for the preparation of
the intermediates of enantiomers 7a and 7b. Therefore, asymmetric
synthesis of the major diastereomers of 5a and 5b (Scheme 1) was
investigated. Davis-Ellmann-type sulfonamide chemistry, as
a practical asymmetric process for the preparation of chiral non-
racemic amines, has been applied to the asymmetric synthesis of
levocetirizine [20e23]. Sulfinimines have also shown to be efficient
chiral auxiliaries for the preparation of chiral di-substituted benz-
hydrylamines [22,23]. As depicted in Scheme 1, Ti(O-iPr)4-medi-
ated synthesis of (R)/(S)-N-tert-butanesulfinyl aldimines(2, 3) from
(R)-t-butyl sulfonamide [(R)-TBS] or (S)-t-butyl sulfonamide [(S)-
TBS] and 2,4-dichlorobenzaldehyde proceeded in high yields.
With the addition of arylmagnesium bromides to R)/(S)-N-tert-
Scheme 1. Asymmetric synthesis route of the enantiomers of compounds 12 and 13. Reagents and conditions: (i) for compound 2: (R)-t-butyl sulfonamide, Ti(O-iPr)4, THF, 93%; for
compound 3: (S)-t-butyl sulfonamide, Ti(O-iPr)4, THF, 92%; (ii) ArMgBr, THF/toluene, ꢀ20 ꢂC-0 ꢂC, 5 h, 74%w81%; (iii) saturated HCl in MeOH, 2 h, 96%w98%; (iv) N,N-bis(2-
chloroethyl)-2-nitrobenzenesulfonamide, DIPEA, 130 ꢂC, 4 h, 66%w74%; (v) PhSH, K2CO3, acetonitrile, 30 ꢂC, 6 h, 65%w75%; (vi) for compound 12S and 12R: 1-aminopiperidine,
1,10-carbonyldiimidazole (CDI), THF, rt, 82% and 78%, respectively; for compound 13S, 13R: cyclohexyl isocyanate, CH2Cl2, rt, 89% and 86%, respectively.

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L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
butanesulfinyl aldimines(2, 3) at ꢀ20 ꢂC, followed by aging at room
temperature in toluene, the diastereomers of intermediates 5a and
5b were obtained with approximate 85:15 diastereoselectivity
observed by HPLC analysis, which was similar to the results re-
ported by Pflum et al. [22]. Since compounds 5a and 5b are oil-like
products, it is difficult to determine the absolute configuration of
the newly formed stereocenters. Thus, the corresponding analogs
without methyl group, the diastereomers of 4a and 4b, were
synthesized and crystallized from petroleum ether to provide dia-
stereopure material in over 70% yield. Single crystal X-ray analysis
shown in Fig. 2 confirmed that the absolute configuration of the
newly formed stereocenter in compound 4a was (S). Thereafter, the
absolute stereochemistry of analogs with methyl group (7a/b, 11a/
b, 13a/b) can be established by comparing their optical rotatory
dispersion with that of the corresponding S(ꢀ)/R(þ)-analogs
without methyl group (6a/b, 10a/b, 12a/b) [24].
Scheme 2. Initial proposed synthesis of compound 10a and 11a. Reagents and
conditions: (i) N,N-bis(2-chloroethyl)-4-methylbenzenesulfonamide, DIPEA, 170 ꢂC,
3 h; (ii) 40% HBr in HOAc (various phenols used in deprotection: 10% phenol/20 ꢂC, or
10% 4-hydroxybenzoic acid/20 ꢂC, or 10% phenol/80 ꢂC (HPLC yield: trace w 5%).
(Scheme 1), indicating that there was no epimerization of the
stereocenter during the acidic hydrolysis [24].
2.2. In vitro assay
After removal of the chiral auxiliary by a mild hydrolysis
The target compounds were evaluated in vitro on the hCB1 and
hCB2 receptors, stably expressed in Chinese Hamster Ovary (CHO)
cells, utilizing radioligand binding studies. hCB1 receptor antago-
nism was measured using a CP-55940 (an agonist of the CBRs)
induced Ca2þ increase functional assay in CHO cells co-expressing
condition (saturated HCl in methanol), the products with
L
(þ)-
tartaric acid or
D
(ꢀ)-tartaric acid were further diastereomerically
crystallized to afford intermediates S(ꢀ)-amine 6a and R(þ)-amine
6b, as well as 7a and 7b with higher enantiomerical purity deter-
mined by optical rotatory mearurement. According to the asym-
metric synthesis route of levocetirizine, the ring closure reaction
product of the (ꢀ)4-chlorobenzhydrylamine and N,N-bis(2-
chloroethyl)-4-methylbenzenesulfonamide was subjected to
a standard N-tosyl deprotection procedure with 40% HBr in acetic
acid solution to afford piperazine product [18,21]. However, we
found that the N-tosyl protecting group could not be removed from
N-tosyl protected piperizines 14 and 15 with HBr/HOAc (Scheme 2).
Thus, N-nosyl was used as the amine protecting group to replace
the tosyl group. The bisalkylation of (S)-amine 6a, 7a or (R)-amine
6b, 7b with N,N-bis(2-chloroethyl)-2-nitrobenzenesulfonamide,
followed by mild deprotection condition, afforded 10a, 11a or 10b,
11b. The piperazine products were reacted with cyclo-
hexylisocyanate and 1-aminopiperidine, respectively, to give 12S/
12R in over 96% ee, the desired products 13S/13R in over 95% ee
hCB1 receptor and Ga15/16.
A comparison between the two enantiomers 13R and 13S and
the corresponding racemate 13 showed that the two single enan-
tiomers 13R and 13S differed in their binding affinities for hCB1 by
5-fold, whereas the R-enantiomer was the more active form (13R,
KiCB1 ¼ 0.2 nM; 13S, KiCB1 ¼ 1.1 nM) and had a potency comparable
to that of the racemic compound 13 (KiCB1 ¼ 0.15 nM, Table 1).
Similar results were observed in the antagonistic activities by
a functional Ca2þ assay, in which again, the enantiomer 13R is the
more active form and displayed 3-fold improvement in the inhi-
bition of CP-55940-induced Ca2þ increase. Meanwhile, the enan-
tiomer 13S exhibited a significant 8-fold decrease in Ca2þ inhibitory
potency, compared to the racemic compound 13 (Table 1).
2.3. Pharmacokinetic and in vivo studies
The pharmacokinetic profiles of the racemic compounds 13,
and its enantiomers 13R and 13S after oral administration were
evaluated in SpragueeDawley rats. As shown in Table 2, it was
Table 1
Assay results of enantiomers of the racemate 13.a
Cpd.
Absolute configuration Ki hCB1
Ki hCB2 (nM) IC50 hCB1 Ca2þ
(nM)
(optical rotation)
(nM)
13 [14]
13S
13R
/
0.15 ꢁ 0.04
329 ꢁ 71
242.4 ꢁ 119.0 21.5 ꢁ 8.3
0.2 ꢁ 0.03 226.8 ꢁ 45.9 0.9 ꢁ 0.4
2.7 ꢁ 0.5
S (þ4.3ꢂ)
1.1 ꢁ 0.1
R (ꢀ4.3ꢂ)
a
Ki and IC50 (mean ꢁ SEM) (n ¼ 3 independent experiments) were calculated
from doseeresponse curves. hCB1: human CB1 receptor. hCB2: human CB2
receptor.
Table 2
Pharmacokinetic studies of the racemic compound 13, and its enantiomers 13S and
13R in SpragueeDawley rats after 10 mg/kg p.o. administration.a
Cpd.
13
13S
13R
tmax (h)
Cmax (ng/mL)
t1/2 (h)
AUC0eN (ng h/mL)
F (%)
2.4 ꢁ 0.2
179.5 ꢁ 31.3
2.3 ꢁ 0.1
883 ꢁ 112
8%
1.3 ꢁ 0.3
128.5 ꢁ 10.5
3.4 ꢁ 1.0
695 ꢁ 34
6%
1.2 ꢁ 0.3
115.7 ꢁ 19.3
3.8 ꢁ 0.6
655 ꢁ 68
15%
B/P @ 9 h postdose
0.91 ꢁ 0.07
1.42 ꢁ 0.17
0.88 ꢁ 0.06
a
Values represent the mean
ꢁ
SEM for at least three experiments. Cmax:
Fig. 2. X-Ray single crystal structure of (R,S)-sulfinamide-4a (major disastereomer
from PhMgBr addition process).
maximum plasma concentration of drug; tmax: time taken to reach Cmax; t1/2: half-
life; AUC0ꢀN: area under the curve; F: oral bioavailability.

L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
5313
greater efficacy under in vivo. The relationship between activity and
stereochemistry provided us with useful information in our search
for peripherally acting CB1 antagonists, which will be reported in
due course.
4. Experiment procedure
4.1. Chemistry
All non-aqueous reactions were performed in flame-dried
glassware under an atmosphere of dry Ar, unless otherwise speci-
fied. Tetrahydrofuran (THF) was distilled from sodium-
benzophenone ketyl under an atmosphere of Ar immediately
prior to use; Dichloromethane (DCM) was freshly distilled from
calcium hydride under Ar. All other solvents were reagent grade.
Petroleum ether refers to a mixture of alkanes with the boiling
range 60e90 ꢂC 1H and 13C NMR spectra were recorded on Varian
Mercury-300 and Varian Mercury-400 spectrometers. Chemical
shifts are reported in ppm from tetramethylsilane with the solvent
resonance as the internal standard. The following abbreviations
were used to explain the multiplicities: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, dd ¼ doublet of doublets, m ¼ multiplet,
br ¼ broadened. Infrared spectra were recorded on a Nicolet 750FT-
IR. Optical rotations were measured on a Perkin-Elmer model 341
polarimeter. The LC-MS were carried out on Thermo Finnigan
LCQDECAXP and low-resolution mass spectrometry was performed
on Finnigan MAT 95 and Finnigan LCQ Deca spectrometers, high
resolution mass spectra were measured on Finnigan MAT 95 and
MicroMass Q-Tof ultimaÔ mass spectrometers. Optical rotations
were determined on a PerkineElmer 341 polarimeter.
Fig. 3. Chronic effects after oral administration of the enantiomers 13S and 13R in DIO
mice. A: weekly body weight; B: visceral fat mass at end of study.*P < 0.05 vs. vehicle,
two-way ANOVA followed by Bonferroni posttests. Data represent mean
ꢁ
SE
(n ¼ 8e10 mice/group).
4.1.1. (R)-N-(2,4-dichlorophenyl)methylene-2-methylpropane-2-
sulfinamide (2)
found that, in addition to displaying similar in vitro biological
activities, the enantiomer 13R (t1/2 ¼ 3.8 h; brain/plasma ¼ 0.88)
and its racemic compounds 13 (t1/2 ¼ 2.3 h; brain/plasma ¼ 0.91)
also had similar pharmacokinetic profiles such as halflife (t1/2) and
brain exposure, but the enantiomer 13R was found to have almost
twofold improvement in oral bioavailability (F% ¼ 15% vs. F% ¼ 8%
for 13R and 13, respectively). The enantiomer 13S displayed poor
pharmacokinetic profiles as decreased oral bioavailability (F
% ¼ 6%) and increased brain exposure (brain/plasma ¼ 1.42).
Based on the above results, the enantiomer 13R would account for
the majority of the inhibitory potency of its racemate 13, and is
expected to have greater in vivo efficacy than the enantiomer 13S.
This was indeed the case, as seen in Fig. 3 where the effects of 13R
and 13S on body weight were evaluated in DIO mice. Long-term
applications of 13R (10 mg/kg, p.o.) once daily for 35 days led to
a steady reduction of body weight and caused a significance body
weight loss at the end of the 35-day study compared to vehicle
treatment (Fig. 3A), while 10 mg/kg of 13S group displayed no
effect on body weight reduction. The 13R group also showed
a reduction of the visceral fat mass of DIO mice (Fig. 3B), although
it did not achieve statistical significance. Overall food intake did
not differ between 13R and 13S groups in this long-term experi-
ment (data not shown).
A
solution of Ti(O-iPr)4 (247 mL, 0.83 mol) and 2,4-
dichlorobenzaldehyde (72.2 g, 0.41 mol) in 500 mL of THF was
prepared under a N2 atmosphere. Then, (R)-t-butyl sulfonamide
(50.0 g, 0.41 mol) was added and the reaction mixture was stirred at
40 ꢂC for 6 h (conversion was followed by TLC). And the mixture
cooled immediately upon completion. Once at room temperature,
the mixture was poured into 500 mL of brine while rapidly stirring.
The resulting suspension was filtered through a plug of Celite, and
the filter cake was washed with EtOAc. The filtrate was transferred
to a separatory funnel where the organic layer was washed with
brine. The brine layer was extracted once with a small volume of
EtOAc, and the combined organic portions were dried over Na2SO4,
filtered, and concentrated. Compound 2 was obtained as white
solid form the residue by recrystallization from petroleum ether
and ethyl acetate (106.3 g, 93%). ½a D20
ꢃ
¼ ꢀ178.7 (c 1.0, CHCl3). 1H
NMR (300 MHz, CDCl3)
d
8.97(s, 1H), 8.01(d, J ¼ 8.5 Hz, 1H), 7.48(d,
J ¼ 2.1 Hz, 1H), 7.34 (ddd, J ¼ 8.5, 2.0, 0.7 Hz, 1H), 1.28 (s, 9H).
LRMS(EI, 70 eV): m/z 277 [Mþ].
4.1.2. (S)-N-(2,4-dichlorophenyl)methylene-2-methylpropane-2-
sulfinamide (3)
Compound 3 was prepared from 2,4-dichlorobenzaldehyde
and (S)-t-butyl sulfonamide in 92% yield as white solid by the
3. Conclusions
same procedure as described for intermediate 2. ½a D20
¼ þ177.6
ꢃ
(c 1.0, CHCl3), other physical and chemical data were identical
In an important extension of previous work, we have demon-
strated a practical process for the assymetric synthesis of benzhy-
drylpiperazines 13S and 13R as potential CB1 antagonists. The
pharmacokinetic profiles and the in vitro and in vivo activities of the
enantiomers 13S and 13R had been evaluated, and 13R was
recognized as the more potent form. It also displayed enhanced
antagonistic activity for hCB1, better oral bioavailability, and
with those described above for the opposite enantiomer 2.
4.1.3. (R)-N-[(S)-(2,4-dichlorophenyl)(phenyl)methyl]-2-
methylpropane-2-sulfinamide (4a)
To a solution of the sulfinimine 2 (30.0 g, 0.11 mol) in 200 mL
of toluene was added 50 mL of 2.8 M phenylmagnesium bromide
tetrahydrofuran solution at ꢀ20 ꢂC. The mixture was stirred at

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L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
0
ꢂC for 3 h and then was allowed to warm to room temperature
4.1.10. (R)-(2,4-dichlorophenyl)(p-tolyl)methanamine (7b)
and stirred overnight. The reaction mixture was quenched with
10 mL of saturated ammonium chloride aqueous solution and the
aqueous layer was extracted with ethyl acetate (2 ꢄ 250 mL). The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated to provide crude product, which
was recrystallized from petroleum ether to give the pure 4a as
Compound 7b was prepared from 5b in 96% yield by the same
procedure as described for 11a. ½a D20
¼ þ10.1 (c 1.0, EtOH), other
ꢃ
physical and chemical data were identical with those described
above for the opposite enantiomer.
4.1.11. 1-[(S)-(2,4-dichlorophenyl)(phenyl)methyl]-4-[(2-
nitrophenyl)sulfonyl]piperazine (8a)
white solid (28.3 g, 74%). mp 104e106 ꢂC ½a D20
ꢃ
¼ ꢀ47.0 (c 1.0,
CHCl3). 1H NMR (300 MHz, CDCl3)
d
7.57 (d, J ¼ 8.5 Hz, 1H), 7.38 (d,
Tothefreebaseof(S)-(2,4-dichlorophenyl)(phenyl)methanamine
(6a, 44 g, 0.18 mol) was added N,N-diisopropylethylamine (33.5 mL,
0.19 mol), to this mixture was added N,N-bis(2-chloroethyl)-2-
nitrobenzenesulfonamide (57 g, 0.18 mol, prepared from bis(2-
chloroethyl)amine hydrochloride and 2-nitrobenzenesulfonyl chlo-
ride in 96% yield), carefully in portions to ensure mixing. After the
addition of the bis-alkylating agent was complete, the flask was then
equipped with a reflux condenser and drying tube, and the mixture
was stirred at 130 ꢂC for 4 h. The reaction mixture turned fromyellow
to brown, and was allowed to cool to room temperature. The reaction
mixture was redissolved in 300 mL of DCM, the solutionwas washed
with H2O (3 ꢄ 100 mL), then with brine (100 mL), dried over Na2SO4
and evaporated under vacuum to afford the crude product 8a (65.3 g,
74%), which was used in the next step without purification, LC-MS
(major peak): 506.1[Mþ1].
J ¼ 2.2 Hz, 1H), 7.28e7.34 (m, 6H), 6.07 (d, J ¼ 2.8 Hz, 1H), 3.67(d,
J ¼ 2.8 Hz, 1H, NH), 1.26 (s, 9H). LRMS(EI, 70 eV): m/z 355 [Mþ],
235 (100%).
4.1.4. (R)-N-[(S)-(2,4-dichlorophenyl)(p-tolyl)methyl]-2-
methylpropane-2-sulfinamide (5a)
Compound 5a was prepared from sulfinimine 2 and p-tol-
ylmagnesium bromide in 81% yield as yellow oil by the same
procedure as described for 4a. ½a D20
ꢃ
¼ ꢀ44.9 (c 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3)
d
7.58 (d, J ¼ 8.4 Hz, 1H), 7.36 (d, J ¼ 2.2 Hz, 1H),
7.29 (dd, J ¼ 8.4 Hz, 2.1 Hz,1H), 7.11e7.23 (m, 4H), 6.03 (d, J ¼ 2.8 Hz,
1H), 3.64 (d, J ¼ 2.4 Hz, 1H), 2.31 (s, 3H), 1.25 (s, 9H). LRMS(EI,
70 eV): m/z 369[Mþ], 249 (100%).
4.1.5. (S)-N-[(R)-(2,4-dichlorophenyl)(phenyl)methyl]-2-
methylpropane-2-sulfinamide (4b)
4.1.12. 1-[(R)-(2,4-dichlorophenyl)(phenyl)methyl]-4-[(2-
nitrophenyl)sulfonyl]piperazine (8b)
Compound 8b was prepared from 6b in 70% yield by the same
procedure as described for 8a. LC-MS (major peak): 506.1[Mþ1].
Compound 4b was prepared from sulfinimine 3 and phenyl-
magnesium bromide in 76% yield as white solid by the same
procedure as described for 4a. ½a D20
¼ þ47.3 (c 1.0, CHCl3), other
ꢃ
physical and chemical data were identical with those of 4a.
4.1.13. 1-[(S)-(2,4-dichlorophenyl)(p-tolyl)methyl]-4-[(2-
nitrophenyl)sulfonyl]piperazine (9a)
Compound 9a was prepared from 7a in 66% yield by the same
procedure as described for 8a. LC-MS (major peak): 520.1[Mþ1].
4.1.6. (S)-N-[(R)-(2,4-dichlorophenyl)(p-tolyl)methyl]-2-
methylpropane-2-sulfinamide (5b)
Compound 5b was prepared from sulfinimine 3 and p-tol-
ylmagnesium bromide in 80% yield as yellow oil by the same
procedure as described for 4a. ½a D20
ꢃ
¼ þ45.5 (c 1.0, CHCl3), other
4.1.14. 1-[(R)-(2,4-dichlorophenyl)(p-tolyl)methyl]-4-[(2-
nitrophenyl)sulfonyl]piperazine (9b)
physical and chemical data were identical with those of 5a.
Compound 9b was prepared from 7b in 71% yield by the same
procedure as described for 8a. LC-MS (major peak): 520.1[Mþ1].
4.1.7. (S)-(2,4-dichlorophenyl)(phenyl)methanamine (6a)
Compound 4a (20.0 g, 54 mmol) was treated with a saturated
HCl in methanol (200 mL) at room temperature for 2 h. The
mixture was concentrated to dryness and precipitated with
diethyl ether. The precipitate was collected by filtration and
washed with diethyl ether to give the amine hydrochloride of
sufficient purity such that no other purification was required. The
amine hydrochloride was converted to the free amine by treat-
ment with 1 N NaOH(aq.) (100 mL), and was extracted with CH2Cl2
(2 ꢄ 150 mL), the combined organic layer was washed with H2O
and brine successively, dried over Na2SO4 and evaporated under
vacuum to get the product as pale yellow oil (13.8 g, 98%).
4.1.15. 1-[(S)-(2,4-dichlorophenyl)(phenyl)methyl]piperazine (10a)
To a solution of the crude 1-[(S)-(2,4-dichlorophenyl)(phenyl)
methyl]-4-[(2-nitrophenyl)sulfonyl] piperazine (6a, 60.0 g, approx.
0.12 mol) in acetonitrile (150 mL) at 0 ꢂC was added thiophenol
(14.6 mL, 0.14 mol) and potassium carbonate (50 g, 0.36 mol), the
mixture was stirred at 50 ꢂC for 2 h, then the solvent was removed
in vacuum. The residue was dissolved in 300 mL ethyl acetate and
150 mL water, the organic layer was washed 100 mL water, fol-
lowed by 1 N HCl (200 mL), then the aqueous phase was washed
ethyl acetate (3 ꢄ 80 mL), and the combined organic phase was
discarded, the aqueous phase was basified with 3 N NaOHaq.
(80 mL) to pH ¼ 10, then the aqueous solution was extracted with
CH2Cl2 (3 ꢄ 100 mL). The combined organic phase was washed
successively with water, brine, dried over Na2SO4 and evaporated
under vacuum to provide the title compound 10a (27.3 g, 72%) as
½
a 2D0
ꢃ
¼ ꢀ7.6 (c 1.0, EtOH). 1H NMR (400 MHz, CDCl3)
d 7.52 (d,
J ¼ 8.4 Hz, 1H), 7.23e7.38 (m, 7H), 5.60 (s, 1H). LRMS(EI, 70 eV): m/
z 251[Mþ].
4.1.8. (S)-(2,4-dichlorophenyl)(p-tolyl)methanamine (7a)
Compound 7a was prepared from 5a in 97% yield by the same
yellow oil. ½a 2D0
ꢃ
¼ - 4.6 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3)
procedure as described for 11a. ½a D20
ꢃ
¼ ꢀ10.2 (c 1.0, EtOH). 1H NMR
d
7.74 (d, J ¼ 8.4 Hz, 1H), 7.37e7.40 (m, 2H), 7.18e7.31 (m, 5H), 4.80
(300 MHz, CDCl3)
d
7.52 (d, J ¼ 8.4 Hz, 1H), 7.33 (d, J ¼ 2.2 Hz, 1H),
(s, 1H), 3.02e3.08 (m, 3H), 2.26e2.61 (m, 5H). LRMS(EI, 70 eV): m/z
7.21e7.25 (m, 3H), 7.10e7.13 (br d, J ¼ 7.9 Hz, 2H), 5.55 (s, 1H), 2.32
320 [Mþ].
(s, 3H). LRMS(EI, 70 eV): m/z 265[Mþ].
4.1.16. 1-[(S)-(2,4-dichlorophenyl)(p-tolyl)methyl]piperazine (11a)
Compound 11a was prepared from 9a in 65% yield by the same
4.1.9. (R)-(2,4-dichlorophenyl)(phenyl)methanamine (6b)
Compound 6b was prepared from 4b in 98% yield by the same
procedure as described for 10a. ½a D20
ꢃ
¼ - 5.5 (c 1.0, CHCl3). 1H NMR
procedure as described for 11a. ½a D20
ꢃ
¼ þ7.6ꢂ (c 1.0, EtOH), other
(300 MHz, CDCl3)
d
7.77 (d, J ¼ 8.4 Hz, 1H), 7.22e7.29 (m, 4H),
physical and chemical data were identical with those described
above for the opposite enantiomer.
7.07e7.09 (m, 2H), 4.71 (s, 1H), 2.28e2.95 (m, 4H), 2.39 (br s, 4H),
2.28 (s, 3H). LRMS(EI, 70 eV): m/z 334 [Mþ].

L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
5315
4.1.17. 1-[(R)-(2,4-dichlorophenyl)(phenyl)methyl]piperazine (10b)
Compound 10b was prepared from 8b in 73% yield by the same
data were identical with those described above for the opposite
enantiomer 13S.
procedure as described for 10a. ½a D20
¼ þ 4.6 (c 1.0, CHCl3), other
ꢃ
physical and chemical data were identical with those described
4.2. Biology
above for the opposite enantiomer 10a.
4.2.1. Whole cell binding assay
4.1.18. 1-[(R)-(2,4-dichlorophenyl)(p-tolyl)methyl]piperazine (11b)
Compound 11b was prepared from 9b in 75% yield by the same
The assay was performed using Chinese hamster ovarian (CHO)
cells stably expressing hCB1 or hCB2 receptors (CHO-hCB1/hCB2
cells). Briefly, CHO-hCB1/hCB2 cells were starved in serum-free F12
medium for 3 h and pretreated with different concentrations of test
compounds for 10 min before the addition of [3H]-CP-55940. After
3 h incubation at 37 ꢂC, cells were washed 5 times with pre-
warmed phosphate-buffered saline (PBS) and lysed by PBS with
1% SDS. The lysed pellets were transferred into 96-well isoplate
(PerkinElmer, Waltham, MA, USA) and bound radioligand was
measured with 1450 microbeta liquid scintillation luminescence
counter (PerkinElmer, Waltham, MA, USA). Assays were performed
in triplicate. The Ki values were analyzed by nonlinear regression
analysis performed using the GraphPad Prism 4.0 software
(GraphPad Software, San Diego), and calculated by the Chenge-
Prusoff equation: Ki ¼ IC50/(1 þ L/Kd).
procedure as described for 10a. ½a D20
¼ þ 5.4 (c 1.0, CHCl3), other
ꢃ
physical and chemical data were identical with those described
above for the opposite enantiomer 11a.
4.1.19. 4-[(S)-(2,4-dichlorophenyl)(phenyl)methyl]-N-piperidin-1-
ylpiperazine-1-carbox-amide (12S)
Compound 12S was prepared from N,N’-carbonyl diimidazole
(CDI), 1-aminopiperidine and 10a in 82% yield as white solid by
the same procedure as described previously [14]. ½a D20
¼ þ 2.8 (c
ꢃ
1.0, EtOH), >99% ee (analyzed on an ‘Chiralpak AD-H’ column,
flow rate 1.0 mL/min, temperature 30 ꢂC,
PrOH, 97:3 with 0.1% diethylamine, tR
l
¼ 254 nm, hexane/i-
¼ 29.2 min, minor
(major)
one is not observed). 1H NMR (400 MHz, CDCl3)
d 7.77 (d,
J ¼ 8.4 Hz, 1H), 7.38e7.42 (m, 2H), 7.19e7.31 (m, 5H), 4.97 (s, 1H,
NH), 4.74 (s, 1H), 3.44e3.47 (m, 4H), 2.65 (br s, 4H), 2.35e2.38 (m,
4.2.2. Calcium concentration assay
4H), 1.58e1.66 (m, 4H), 1.36 (m, 2H). 13C NMR (CDCl3)
d
158.28,
Intracellular Ca2þ concentration was measured in CHO cells co-
140.20, 138.54, 134.34, 132.93, 129.73, 129.43, 128.56 2,
ꢄ
transfected with hCB1 receptors and G
a
15/16 (CHO-hCB1-G
a15/16).
128.30 ꢄ 2, 127.52, 127.47, 69.93, 57.62 (2 ꢄ CH2), 51.66 (2 ꢄ CH2),
44.71 (2 ꢄ CH2), 25.70 (2 ꢄ CH2), 23.21 (CH2). LRMS(EI, 70 eV): m/
z 446 [Mþ].
CHO-hCB1-G 15/16 cells were loaded with 2 m
a
M fluo-4 AM in
Hanks balanced salt solution (HBSS, containing 5.4 mM KCl, 0.3 mM
Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM
MgCl2, 0.6 mM MgSO4, 137 mM NaCl, 5.6 mM D-glucose and 250 mM
4.1.20. N-cyclohexyl-4-[(S)-(2,4-dichlorophenyl)(p-tolyl)methyl]
piperazine-1-carboxamide (13S)
sulfinpyrazone, pH 7.4) at 37 ꢂC for 50 min. After removal of the
loading buffer, cells were washed with HBSS once and incubated
Compound 13S was prepared from 11a and cyclohexyl isocya-
nate in 89% yield as white solid by the same procedure as described
with 50
compounds or DMSO (negative control, final concentration 1%).
After 10 min incubation at room temperature, 25 L CP55940 were
dispensed into the well by FlexStation II micro-plate reader
(Molecular Devices, Sunnyvale, CA, USA), and intracellular calcium
change was recorded with an excitation wavelength of 485 nm and
emission wavelength of 525 nm. Assays were performed in
triplicate.
mL HBSS containing various concentrations of test
previously [14]. ½a D20
ꢃ
¼ þ 4.3 (c 1.0, EtOH), 98% ee (analyzed on an
m
‘Chiralpak AD-H’ column, flow rate 1.0 mL/min, temperature 30 ꢂC,
l
¼ 254 nm, hexane/i-PrOH, 97:3 with 0.1% diethylamine, tR
¼ 35.0 min, tR (minor) ¼ 39.3). 1H NMR (400 MHz, CDCl3)
d 7.76
(major)
(d, J ¼ 8.4 Hz,1H), 7.23e7.29 (m, 4H), 7.07e7.09 (m, 2H), 4.70 (s,1H),
4.21 (d, J ¼ 7.7 Hz, 1H, NH), 3.29e3.32 (m, 4H), 2.35e2.37 (m, 4H),
2.29 (s, 3H), 1.91e1.95 (m, 2H), 1.66e1.71 (m, 2H), 1.59e1.62 (m,
2H), 1.31e1.38 (m, 2H), 1.03e1.14 (m, 2H). 13C NMR (CDCl3)
d
157.04,
4.2.3. In vivo studies
138.75, 137.22, 137.07, 134.27, 132.83, 129.57, 129.43, 129.27 ꢄ 2,
128.22 ꢄ 2, 127.50, 69.58, 51.38 (2 ꢄ CH2), 49.31, 43.80 (2 ꢄ CH2),
33.93 (2 ꢄ CH2), 25.63 (CH2), 25.01 (2 ꢄ CH2), 21.05. HRMS(EI) m/z
calcd for C25H31Cl2N3O, 459.1844; found, 459.1837.
In vivo pharmacological studies in diet-induced obese (DIO)
mice were carried out using male C57BL/6J mice (Shanghai SLAC
Laboratory Animals Co., Shanghai, China). All animal care and
experimental procedures carried out in accordance with guidelines
of the Laboratory Animal Science Center at Shanghai Institute of
Materia Medica. Mice were maintained in a 12 h/12 h lightedark
cycle with free access to food and water in group housing condi-
tions in a temperature controlled environment (25 ꢂC). To induce
obesity, mice were fed a high fat diet (HFD) containing 40.2%
sucrose, 23.6% saturated fat (lard), and 23.6% casein, with equal
quantities of fiber and minerals as in the mouse regular diet
(Shanghai SLAC Laboratory Animals Co., Shanghai, China) from
weaning. The HFD supplied 44.6% of calories as fat and 35.4% of
calories as carbohydrate comprised of cornstarch and sucrose. Food
intake (FI) and body weight (BW) were measured weekly. DIO mice
were used at 20e24 weeks of age and conditioned to oral gavage of
water for 5 days before experiments.
For 35-day BWassay, DIOmice (n ¼ 9e10 mice/group) were caged
in plastic cages in group housing conditions and dosed orally with
vehicle(0.1%Tween80)orcompounds(10 mg/kg)onceperdayfor35
days. All DIO mice were exposed to a HFD through the whole
experiment. One mouse in vehicle group died on day 32. Mice were
dosed 1 h before the dark cycle. FI and BW were measured weekly.
Statistical analysis was performed for the BW data using t-test.
4.1.21. 4-[(R)-(2,4-dichlorophenyl)(phenyl)methyl]-N-piperidin-1-
ylpiperazine-1-carboxamide (12R)
Compound 12R was prepared from CDI, 1-aminopiperidine and
10b in 78% yield as white solid by the same procedure as described
previously [14]. ½a D20
¼ ꢀ2.7 (c 1.0, EtOH), 96% ee (analyzed on an
ꢃ
‘Chiralpak AD-H’ column, flow rate 1.0 mL/min, temperature 30 ꢂC,
l
¼ 254 nm, hexane/i-PrOH, 97:3 with 0.1% diethylamine, tR
¼ 30.6 min, tR (major) ¼ 33.7). Other physical and chemical
(minor)
data were identical with those described above for the opposite
enantiomer 12S.
4.1.22. N-cyclohexyl-4-[(R)-(2,4-dichlorophenyl)(p-tolyl)methyl]
piperazine-1-carboxamide (13R)
Compound 13R was prepared from 11b and cyclohexyl isocya-
nate in 86% yield as white solid by the same procedure as described
previously [14]. ½a D20
¼ - 4.1 (c 1.0, EtOH), 95% ee (analyzed on an
ꢃ
‘Chiralpak AD-H’ column, flow rate 1.0 mL/min, temperature 30 ꢂC,
l
¼ 254 nm, hexane/i-PrOH, 97:3 with 0.1% diethylamine, tR
¼ 36.2 min, tR (major) ¼ 41.3). Other physical and chemical
(minor)

5316
L. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 5310e5316
[3] F.X. Pi-Sunyer, L.J. Aronne, H.M. Heshmati, J. Devin, J. Rosenstock, JAMA- J. Am.
4.2.4. Pharmacokinetic assays
Med. Assoc. 295 (2006) 761e775.
Male SpragueeDawley rats were dosed orally at 10 mg/kg after
overnight fasting for pharmacokinetic (PK) evaluations. The blood
samples were collected at various time points into lithium heparin
tubes and centrifuged. The plasma samples were kept at ꢀ20 ꢂC
until analysis. For brain penetration study, rats were administered
orally at 10 mg/kg and sacrificed at 3, 5, and 9 h postdose,
respectively. The whole brains and the blood samples were
collected and stored at ꢀ20 ꢂC. The plasma samples were extracted
by protein precipitation and analyzed by LC/MS/MS. The whole rat
brain was homogenized, and was extracted by protein precipitation
prior to LC/MS/MS analysis.
[4] D. Jones, Nat. Rev. Drug Discov. 7 (2008) 961e962.
[5] R. Christensen, P.K. Kristensen, E.M. Bartels, H. Blidda, A. Astrup, Lancet 370
(2007) 1706e1713.
[6] D.R. Janero, A. Makriyannis, Expert Opin. Emerg. Dr 14 (2009) 43e65.
[7] B.S. Basavarajappa, Curr. Neuropharmacol 5 (2007) 81e97.
[8] A.C. Howlett, F. Barth, T.I. Bonner, G. Cabral, P. Casellas, W.A. Devane,
C.C. Felder, M. Herkenham, K. Mackie, B.R. Martin, R. Mechoulam,
R.G. Pertwee, Pharmacol. Rev. 54 (2002) 161e202.
[9] B. Yuce, M. Kemmer, G. Qian, M. Muller, A. Sibaev, Y. Li, M.E. Kreis, M. Storr,
Neurogastroent. Motil. 22 (2010) 672ee205.
[10] D. Osei-Hyiaman, J. Liu, L. Zhou, G. Godlewski, J. Harvey-White, W.I. Jeong,
S. Batkai, G. Marsicano, B. Lutz, C. Buettner, G. Kunos, J. Clin. Invest. 118 (2008)
3160e3169.
[11] A. Serrano, I. Del Arco, F. Javier Pavon, M. Macias, V. Perez-Valero, F. Rodriguez
de Fonseca, Neuropharmacology 54 (2008) 226e234.
[12] M.A. Carai, G. Colombo, P. Maccioni, G.L. Gessa, CNS Drug Rev. 12 (2006)
91e99.
Acknowledgments
[13] J.P. Despres, Curr. Pharm. Des. 15 (2009) 553e570.
[14] T. Meng, J. Wang, H.L. Peng, G.H. Fang, M. Li, B. Xiong, X. Xie, Y.L. Zhang,
X. Wang, J.K. Shen, Eur. J. Med. Chem. 45 (2010) 1133e1139.
[15] K.S. Song, S.H. Lee, H.J. Chun, J.Y. Kim, M.E. Jung, K. Ahn, S.U. Kim, J. Kim, J. Lee,
Bioorg. Med. Chem. 16 (2008) 4035e4051.
[16] E. Francotte, Chirality in Drug Research. Wiley-VCH, Weinheim, 2006.
[17] L.S. Lin, T.J. Lanza Jr., J.P. Jewell, P. Liu, S.K. Shah, H. Qi, X. Tong, J. Wang,
S.S. Xu, T.M. Fong, C.P. Shen, J. Lao, J.C. Xiao, L.P. Shearman, D.S. Stribling,
K. Rosko, A. Strack, D.J. Marsh, Y. Feng, S. Kumar, K. Samuel, W. Yin,
L.H. Van der Ploeg, M.T. Goulet, W.K. Hagmann, J. Med. Chem. 49 (2006)
7584e7587.
We are indebted to Dr. Oleg Khorev for critical review of this
manuscript. This work was supported by 863 Program (the National
High Technology Research and Development Program of China, No.
2007AA02Z308), and the National Natural Science Foundation of
China (Grants 81001355, 30873162), and Shanghai Commission of
Science and Technology (08JC1407701).
[18] For selected reviews on Davis-Ellmann-type sulfonamide chemistry, see: (a)
J.A. Ellman, T.D. Owens, T.P. Tang, Acc. Chem. Res. 35 (2002) 984e995;
(b) P. Zhou, B.C. Chen, F.A. Davis, Tetrahedron 60 (2004) 8003e8030;
(c) G.Q. Lin, M.H. Xu, Y.U. Zhong, X.W. Sun, Acc. Chem. Res. 41 (2008)
831e840.
[19] C.J. Opalka, T.E. Dambra, J.J. Faccone, G. Bodson, E. Cossement, Synthesis-
Stuttgart (1995) 766e768.
[20] G.C. Liu, D.A. Cogan, T.D. Owens, T.P. Tang, J.A. Ellman, J. Org. Chem. 64 (1999)
1278e1284.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.08.030. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
[21] N. Plobeck, D. Powell, Tetrahedron-Asymmetr. 13 (2002) 303e310.
[22] D.A. Pflum, D. Krishnamurthy, Z.X. Han, S.A. Wald, C.H. Senanayake, Tetra-
hedron Lett. 43 (2002) 923e926.
References
[23] Z.X. Han, D. Krishnamurthy, P. Grover, Q.K. Fang, D.A. Pflum, C.H. Senanayake,
Tetrahedron Lett. 44 (2003) 4195e4197.
[24] G.R. Clemo, C. Gardner, R. Raper, J. Chem. Soc. (1939) 1958e1960.
[1] V. Di Marzo, S.K. Goparaju, L. Wang, J. Liu, S. Batkai, Z. Jarai, F. Fezza, G.I. Miura,
R.D. Palmiter, T. Sugiura, G. Kunos, Nature 410 (2001) 822e825.
[2] R.I. Wilson, R.A. Nicoll, J. Neurosci. 296 (2002) 678e682.