Design, synthesis and SAR of piperidyl-oxadiazoles as 11β- hydroxysteroid dehydrogenase 1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.12.059

The potential roles of 11β-HSD1 inhibitors in metabolic syndrome, T2D and obesity were well established and currently several classes of 11β-HSD1 inhibitors have been developed as promising agents against metabolic diseases. To find potent compounds with good pharmacokinetics, we used the bioisosterism approach, and designed the compound 2 and 3 bearing an 1,2,4-oxadiazole ring to replace the amide group in compound 1. Guided by docking study, we then transformed compound 3 into a potent lead compound 4a by changing sulfonamide group to amide. To elaborate this series of piperidyl-oxadiazole derivatives as human 11β-HSD1 inhibitors, we explored the structure-activity relationship of several parts of the lead compound. Based on their potency toward human 11β-HSD1 two compounds 4h and 4q were advanced to pharmacokinetic study. It was found that 4h and 4q are potent and selective human 11β-HSD1 inhibitors with better pharmacokinetic properties than those of the original piperidine-3-carboxamide compound 1, and suitable for further in vivo preclinical study in primate model.

Full text of DOI:10.1016/j.ejmech.2012.12.059

European Journal of Medicinal Chemistry 62 (2013) 1e10
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and SAR of piperidyl-oxadiazoles
as 11 -hydroxysteroid dehydrogenase 1 inhibitors
b
,
,
,
Guangxin Xia a b, Xiaodi You a b, Lin Liu a b, Haiyan Liu b, Jianfa Wang b,
Yufang Shi b, Ping Li b, Bing Xiong a , Xuejun Liu , Jingkang Shen
,
b
a,b,
*
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, China
b Central Research Institute, Shanghai Pharmaceutical Holding Co., Ltd., 898 Halei Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The potential roles of 11
and currently several classes of 11
metabolic diseases. To find potent compounds with good pharmacokinetics, we used the bioisosterism
approach, and designed the compound 2 and 3 bearing an 1,2,4-oxadiazole ring to replace the amide
group in compound 1. Guided by docking study, we then transformed compound 3 into a potent lead
compound 4a by changing sulfonamide group to amide. To elaborate this series of piperidyleoxadiazole
b
-HSD1 inhibitors in metabolic syndrome, T2D and obesity were well established
-HSD1 inhibitors have been developed as promising agents against
Received 23 October 2012
Received in revised form
17 December 2012
Accepted 23 December 2012
Available online 10 January 2013
b
derivatives as human 11
parts of the lead compound. Based on their potency toward human 11
were advanced to pharmacokinetic study. It was found that 4h and 4q are potent and selective human
11 -HSD1 inhibitors with better pharmacokinetic properties than those of the original piperidine-3-
carboxamide compound 1, and suitable for further in vivo preclinical study in primate model.
Ó 2013 Elsevier Masson SAS. All rights reserved.
b
-HSD1 inhibitors, we explored the structureeactivity relationship of several
Keywords:
11b-HSD1 inhibitor
Oxadiazole
Bioisostere
SAR
b
-HSD1 two compounds 4h and 4q
b
Docking
1. Introduction
highly expressed in liver and adipose tissue, predominantly reduces
cortisone to cortisol, the type 2 isoform (11
expressed in kidney, oxidizes cortisol to cortisone [6]. The potential
roles of 11 -HSD1 inhibitors in metabolic syndrome, T2D and
obesity were established using transgenic mice, and accumulation
b-HSD2), primarily
Metabolic syndrome is defined as a collection of metabolic ab-
normalities including central obesity, insulin resistance, athero-
genic dyslipidemia, hyperglycemia, and hypertension [1,2]. The
prevalence of metabolic syndrome among the US adult population
is more than 20%, and about 80% of type 2 diabetics (T2D) meet the
criteria for the diagnosis of this disorder [1]. As a key risk factor for
cardiovascular disease and T2D, metabolic syndrome has attracted
numerous investigations on genes, proteins, pathways associated
with this disease. Among them, glucocorticoid receptor (GR) sig-
naling plays significant role in metabolic regulation [3e5]. GR sig-
naling depends not only on the circulating cortisol levels but also
on the intracellular production of cortisol through reduction of
cortisone, the inactive glucocorticoid. The enzymes catalyzing the
b
of these findings has made 11
b
-HSD1 a promising target for met-
abolic disease [7e9].
In the past decade, several classes of 11
b-HSD1 inhibitors have
been published, including sulfonamides (e.g., BVT-14225), amides
(e.g., PF-877423), triazoles (e.g., Merck 544), and thiazolones (e.g.,
AMG-221) [10e13]. In a phase II clinical trial, 11b-HSD1 inhibitor
INCB-13739 (structure undisclosed) significantly improved insulin
sensitivity in T2D patient who failed on metformin treatment and
lowered triglyceride and cholesterol levels of patients with hyper-
lipidemia and hypertriglyceridemia [14]. Currently, other drugs
such as PF-915275, MK-0916, and AZD-4071 are being evaluated in
phase I/II trials for potential oral treatment of metabolic diseases
[15e17].
conversion between cortisone and cortisol are 11
b
-hydroxysteroid
-HSD1),
dehydrogenases (11 -HSDs). While the type 1 isoform (11
b
b
Recently, we disclosed a series of sulfonamides represented
by 1 (shown in Fig. 1), which showed high inhibitory activity
* Corresponding authors. State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, China. Tel.: þ86 21 50806600x5407;
fax: þ86 21 50807088.
against 11
b
-HSD1 but with poor liver microsome stability (HLM
Clint 209
m
L/min/mg protein) and low oral bioavailability in rats
(F ¼ 8%) [18]. As part of our continuing efforts on optimizing
E-mail addresses: bxiong@mail.shcnc.ac.cn (B. Xiong), jkshen@mail.shcnc.ac.cn
(J. Shen).
pharmacokinetics properties of this series, we incorporated the
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.059

2
G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
Fig. 1. Design and identification of piperidyl-oxadiazole 11b-HSD1 inhibitors.
readily constructed 1,2,4-oxadiazole ring, a known amide bio-
isostere [19], into the molecule to form compound 2. Although 2
displayed very weak activity against human 11b-HSD1, interest-
ingly, a simple replacement of cyclohexyl group of 2 with phenyl
group led to compound 3 with moderate inhibition against human
11
b
-HSD1 (IC50 ¼ 763 nM).
Utilizing 3 as a starting point, we designed and elaborated
a series of piperidyleoxadiazoles as 11 -HSD1 inhibitors with the
b
approaches of docking and bioisosterism. In the present work, we
report the lead identification and StructureeActivity Relationship
(SAR) studies of this series.
Fig. 2. The docking results of compound 3 (A) and 4a (B). The crystal structure 3G49
was used as the template for docking study. The protein was shown in Ribbon scheme,
and colored green. The NADPH, compound 3 and 4a were shown in stick model,
colored green, pink and cyan respectively. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
2. Results and discussion
2.1. Docking study and lead identification
To investigate the binding mode of compound 3 with 11b-HSD1,
the docking program Vina [20] was applied by using the crystal
structure 3G49 as the template [21]. As obtained from docking
although 13 has similar scaffold to 4a, 4a remains the best one in
terms of potency toward human 11b-HSD1. As depicted in Table 2,
introducing 4-methyl on triazole dramtically decreased the activity
(11 vs. 10).
To explore the extensive SAR of 4a series, more piperidyl-
oxadiazole analogs have been prepared according to Schemes
1 and 2.
study (shown in Fig. 2), compound 3 bound to 11b-HSD1 with the
extended conformation. The 3-chloro-2-methyl benzene group is
located in a subpocket lined with the residues Val-96, Leu-101 and
ꢀ
Tyr-158. The oxygen atom of sulfonamide group is about 4.1 A away
from the oxygen atom of the side chain of Tyr-158. This docking
study suggested that substitution of the sulfonamide group with its
bioisostere amide group may render the ligand into linear shape,
and may introduce a hydrogen bond between the ligand and
Tyr-158. Therefore, we constructed a model compound and docked
Syntheses of compounds with various R1 groups were depicted
in Scheme 1. (S)-Ethyl piperidine-3-carboxylate 15 was condensed
with 3-chloro-2-methylbenzoic acid to produce ester 16, which
was hydrolysized to acid 17. Various nitriles (18aeh) were
reacted with hydroxylamine hydrochloride to afford N0-hydrox-
yimidamides 19aeh. Target molecules (14aeh) were obtained via
two consecutive steps: condensation of 19aeh with 17 and intra-
molecular cyclization of intermediates 20aeh.
it into the binding site of 11b-HSD1. The result showed that it
ꢀ
formed a hydrogen bond about 3.0 A with the side chain of Tyr-158.
To verify this, we synthesized the compound 4a, a bioisostere of the
compound 3. Through the enzymatic inhibition assay, inhibitor 4a
(IC50 ¼ 27 nM) is 28-fold more potent than compound 3. Thus 4a
was selected as a promising hit of 11b-HSD1 for further exploring
Table 1
its structureeactivity relationship.
Human 11
core part.
b-HSD1 activities of regionisomers and enantiomers on the piperidine
2.2. Optimization and SAR study
Since we changed sulfonamide group to amide group, the ligand
may interact to 11 -HSD1 enzyme with a different binding con-
N
b
N
N
O
Cl
formation. Therefore, we performed a systematic scan of 4a type
structure by varying its possible regio- and stereo-isomers. This
yielded compounds 5e8 (Table 1). In consistent with previous
studies, 4a displayed the highest inhibition ratio of 77% at the
ligand concentration of 100 nM, while other isomers showed
considerably low inhibition ratios and were not considered for
further SAR studies.
O
Compound
Substitution position and
Inhibition ratioa
stero-configuration
4a
5
6
3S
3R
4
77%
2%
22%
44%
36%
7
8
2S
2R
Next, we conducted a scaffold hopping on oxadiazole ring to
prepare other five-member heterocycle linkers, including triazoles,
tetrazole, and two other oxadiazoles. It is interested to found that,
a
Note: The inhibition ratio is measured at ligand concentration 100 nM.

G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
3
Table 2
Human 11
Next, we modify the left hand moiety with various acyl groups,
and the synthetic route is described in Scheme 2. N0-hydrox-
ybenzimidamide 22, obtained from benzonitrile 21 was reacted
with hydroxylamine hydrochloride and condensed with Boc-(S)-
piperidine-3-carboxylic acid to provide intermediate 23. Intra-
molecular cyclization of 23 formed oxadiazole 24, which was
deprotected under acidic condition to afford the common inter-
mediate 25. Target compounds (4aew) were produced by acylation
of 25 with acyl chlorides or carboxylic acids.
b
-HSD1 activities of the scaffold hopping toward oxadiazole core L.
N
L
Cl
O
Compound
L
IC50 (nM)
1316
∗
∗
O
∗
9
The inhibitory activity toward human 11b-HSD1 of compounds
N
N
4aex is summarized in Table 4. Among the benzoic acid derivatives
(4aei), compound 4a and 4g had comparable activity. As indicated
by docking study, the left hand moiety of 4a is located in a hydro-
phobic sub-pocket, and the interaction between the benzene ring
N
H
N
∗
10
11
945
9%a
and 11b-HSD1 is matched very well. Interchange methyl and
N
chloro-group was tolerated and showed no difference in potency
(4g vs. 4a). While substituting the chlorine of 4a with iodine dra-
matically decreased the inhibitory activity (4i), compound 4h with
bromine substitution instead of chlorine displayed the best activity
in this series with IC50 ¼ 3.3 nM. However, regarding to the het-
eroaryl carboxylic acid derivatives (4jeo), all of them exhibited low
activity with IC50 > 100 nM. In addition to this, the saturated ring
derivatives were also investigated. There is no clear correlation
between the ring size and the in vitro potency, and among them the
cyclohexyl group compound 4q was the best with the IC50 71 nM.
Taking together of the SAR studies on analogs of 4a, compound
4h and 4q were selected for further in vitro and in vivo evaluation.
Compounds 4h and 4q displayed more than 1000 fold selectivity
∗
∗
∗
N
∗
∗
N
N
N
N
N
12
4a
594
27
N
N
∗
∗
O
N
for human 11
at the concentration 10
concentration 100 M vs. HSD2). Besides, both compounds were
species-dependent inhibitors and showed less than 30% inhibitory
b
-HSD1 over human 11b-HSD2 (4h: <50% inhibition
mM vs. HSD2; 4q: <50% inhibition at the
∗
N
m
13
107
N
O
activity against rodent 11b-HSD1 at a concentration of 1 mM.
a
Note: The inhibition ratio is measured at ligand concentration 100 nM.
2.3. DMPK study
Inhibitory activities of the target compounds against human
11
substitution at various positions of benzene moiety decreased the
activity against 11 -HSD1, especially at para-position (14d and
14h). Changing the phenyl ring to cyclohexane ring or adamantly
group also decreased the inhibitory activity (14a and 14b). These all
indicated that the benzene group at right hand moiety may fit well
into the binding pocket.
b
-HSD1 were determined [18]. As shown in Table 3, halogen
To further assess the druglike properties of this series, we
performed primary DMPK study on 4h and 4q, and the results were
listed in Table 5. By comparing with the compound 1, it was found
that compounds 4h and 4q both showed good in vitro properties,
including very weak inhibition to three P450 enzymes 3A4, 2D6
b
and 2C9 at the concentration 10 mM and better stability in human
liver microsomes. The pharmacokinetic profile of 4h and 4q are also
Cl
COOH
b
N
O
N
OH
HN
15
O
Cl
Cl
a
O
O
O
O
O
17
16
NH2
N
c
CN
OH
R1
R1
d
18a-h
19a-h
e
H
N
N
R1
N
N
Cl
Cl
R1
O
O
N
O
O
N
HO
14a-h
20a-h
Scheme 1. Reagents and conditions: (a) EDCI$HCl, HOBT, DCM, rt, overnight, 86%; (b) 3 N aq. NaOH rt, 30 min, 99%; (c) NH2OH$HCl, TEA, EtOH, 70 ꢀC, 48 h, 74e100%; (d) EDCI$HCl,
HOBT, dioxane, rt, overnight; (e) dioxane, 80 ꢀC, overnight, 11e32% for two-steps.

4
G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
H
N
NH2
N
N
b
CN
Boc
COOH
a
OH
Boc
N
O
N
c
HO
23
21
22
N
N
O
N
d
e
N
N
O
N
O
N
HN
N
O
Boc
R
4a-w
25
24
Scheme 2. Reagents and conditions: (a) NH2OH$HCl, TEA, EtOH, 75 ꢀC, 12 h, 78%; (b) EDCI$HCl, HOBT, 1,4-dioxane, rt; (c) 1,4-dioxane, refluxed, overnight, 59% (two steps);
(d) 4 N HCl-dioxane, DCM, rt, overnight, 86%; (e) RCOOH, EDCI$HCl, HOBT, DCM, rt, overnight, 51e87%.
Table 4
Human 11
moiety.
b
-HSD1 activities of compounds with different R2 group at left hand
Table 3
Human 11
group.
b
-HSD1 activities of compounds with variable right hand moiety at R1
R2
N
N
N
O
O
R1
IC50 (nM)
27
Compound
R2
IC50 (nM) or inhibition ratioa
Compound
4a
4a
27
∗
∗
∗
∗
Cl
14a
14b
14c
14d
621
4b
4c
2373
435
∗
∗
>1000
>1000
956
S
∗
∗
∗
4d
853
∗
Cl
F
4e
4f
741
277
∗
∗
Cl
Cl
14e
14f
277
825
Cl
∗
∗
∗
4g
28
∗
Br
Cl
4h
4i
3.3
14g
14h
310
∗
Br
I
Br
19%
>1000
Br
∗

G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
5
Table 4 (continued )
evaluated in rat. As shown in Table 5, 4h and 4q have higher
bioavailability, longer half life, and slower clearance than
compound 1. The T1/2 of 4h is 2.1 h, about 5 times longer than
compound 1. Compound 4h and 4q are now being advanced to
further PK/PD studies in primate models.
Compound
R2
IC50 (nM) or inhibition ratioa
4j
3%
∗
N
O
N
3. Conclusion
4k
659
715
In conclusion, utilizing the concept of bioisosterism, we
designed and elaborated a series of piperidyl-oxadiazole de-
rivatives as human 11b-HSD1 inhibitors. On the basis of docking
and primary SAR studies, compounds 4h and 4q were selected for
pharmacokinetics study. It was found that 4h and 4q were potent
∗
∗
4l
and selective human 11b-HSD1 inhibitors with better pharmaco-
kinetic properties than those of the original piperidine-3-
carboxamide 1, and suitable for further in vivo preclinical study in
primate model.
Br
4m
19%
Br
N
∗
4. Experimental
N
4.1. General
4n
4o
141
20%
The reagents (chemicals) were purchased from Acros, Aldrich,
Alfa-Aesar, TCI, Shanghai Chemical Reagent Company (SCRC) and
Labpartner, and used without further purification. All non-aqueous
reactions were performed in dried glassware under an atmosphere
of Ar, unless otherwise specified. Yields were not optimized.
Melting points were measured in capillary tube on a SGW X-4
melting point apparatus without correction. Nuclear magnetic
resonance (NMR) spectra were performed on Varian Mercury-300
or 400 spectrometer. Chemical shifts were reported in parts per
∗
N
∗
4p
4q
1484
71
million (ppm, d) downfield from tetramethylsilane. Proton coupling
∗
patterns were described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), doublet of doublets (dd), and broad (br).
Elemental analyses were performed on an Elementar vario EL I
analyzer. The LC-MS was carried out on Thermo Finnigan LCQDE-
CAXP. Low-resolution mass spectra (LRMS) were produced by
Finnigan MAT-95 and Finnigan LCQ Deca spectrometers and high
resolution mass spectra (HRMS) were measured on Finnigan MAT
95 and MicroMass Q-Tof ultima mass spectrometers.
∗
4r
4s
326
92
∗
4.2. General procedure for the synthesis of target compounds 14aeh
∗
4.2.1. (S)-Ethyl 1-(3-chloro-2-methylbenzoyl)piperidine-3-
carboxylate (16)
To a stirred solution of 3-chloro-2-methylbenzoic acid (0.75 g,
4.4 mmol) in DCM (30 mL) was added (S)-ethyl piperidine-3-
carboxylate (15, 0.63 g, 4.0 mmol), HOBT (0.54 g, 4.0 mmol) and
EDCI$HCl (1.5 g, 8.0 mmol). The heterogeneous mixture was stirred
at room temperature overnight, and washed successively with
water (20 mL) and brine (20 mL ꢁ 2). The organic layer was dried
over anhydrous Na2SO4, and concentrated to give the crude prod-
uct, which was purified by flash chromatography (hexane/
EtOAc ¼ 1:1) afforded the ester intermediate as a colorless oil
4t
793
4%
∗
4u
∗
4v
15%
(1.07 g, 86%). 1H NMR (300 MHz, CDCl3):
d
¼ 7.37 (d, J ¼ 9.0 Hz, 1H),
∗
7.17 (dd, J ¼ 9.0, 8.9 Hz,1H), 7.07 (d, J ¼ 8.9 Hz,1H), 4.18 (q, J ¼ 7.1 Hz,
2H), 3.51e2.98 (m, 4H), 2.34e2.30 (m, 4H), 2.13e2.08 (m,1H),1.89e
1.62 (m, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H); EI-MS : m/z 311 [M þ H]þ.
4w
21%
17%
∗
4.2.2. (S)-1-(3-Chloro-2-methylbenzoyl)piperidine-3-carboxylic
acid (17)
The obtained ester 16 (0.6 g) was dissolved in EtOH (15 mL),
which was treated with aq. NaOH (3 N, 15 mL) and stirred at room
temperature overnight. The solvent was evaporated at reduced
pressure, the residue was dissolved in H2O (25 mL), washed with
4x
N
∗
a
Note: The inhibition ratio is measured at ligand concentration 100 nM.

6
G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
Table 5
In vitro and in vivo DMPK properties of selected compounds.
Compound
Microsomal
stability
clint (mL/min/
%inhibition@10
3A4
m
M
Rat PKa
2D6
2C9
iv CL
L/min/kg)
po AUC0-t
(mM min)
po T1/2 (h)
F (%)
mg protein)
4h
4q
1
63
44
209
22
7.7
73
0
12
34
0
16
16
0.016
0.024
0.130
34.1
25.3
11.3
2.1
1.3
0.4
25
36
8
a
Note: iv dose of 3 mg/kg, po dose of 6 mg/kg, po vehicle: 1% CMC-Na in deionized water (suspension).
EtOAc (20 mL), acidified with aq. HCl (2 N), and extracted with DCM
(50 mL ꢁ 3). The combined organic phase was dried over anhydrous
Na2SO4 and evaporated to give product 17 as a white solid (0.54 g,
4.2.8. (S)-(3-Chloro-2-methylphenyl)(3-(3-(4-fluorophenyl)-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (14d)
White solid (110 mg, yield 14%). 1H NMR (300 MHz, CDCl3):
yield 99%).1H NMR (300 MHz, CDCl3):
d
¼ 7.38 (d, J ¼ 9.0 Hz,1H), 7.17
d
¼ 8.11e7.97 (m, 2H), 7.37 (m,1H), 7.21e7.06 (m, 4H), 4.85, 4.62 and
(dd, J ¼ 9.0, 8.8 Hz, 1H), 7.05 (d, J ¼ 8.8 Hz, 1H), 3.51e2.66 (m, 4H),
4.35 (3ꢁ m,1H), 3.74 and 3.46 (2ꢁ m, 2H), 3.29 and 3.07 (2ꢁ m, 2H),
2.38e2.26 (m, 4H), 2.04e1.48 (m, 3H); LC/MS (ESI): m/z 400.0
[M þ H]þ; Anal. Calcd. for C21H19ClFN3O2: C, 63.08; H, 4.79; N,10.51;
Found: C, 63.27; H, 4.84; N, 10.65.
2.64e2.21 (m, 5H), 1.86e1.65 (m, 3H); EI-MS m/z: 282 [M þ H]þ.
4.2.3. Amide oxime 19aeh
N0-Hydroxyimidamides (or amide oxime) 19aeh was prepared
from nitriles 18aeh according to the same procedure described for
compound 22 (see below). The crude products were confirmed by
LC/MS (ESI) and used for the next step without further purification.
4.2.9. (S)-(3-Chloro-2-methylphenyl)(3-(3-(2-chlorophenyl)-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (14e)
White solid (102 mg, yield 12%). 1H NMR (300 MHz, CDCl3):
d
¼ 7.91 (m, 1H), 7.55 (m, 1H), 7.42e7.38 (m, 3H), 7.20e7.10 (m, 2H),
4.2.4. General method of condensation and cyclization for 14aeh
A solution of 17 (0.55 g, 2.0 mmol) in dry 1,4-dioxane (20 mL) at
room temperature was charged with HOBt (0.27 g, 2.0 mmol), DIPEA
(3.0 mmol) and EDCI$HCl (0.58 g, 3.0 mmol). After 30 min, an amide
oxime 19aeh (2.0 mmol) was added and the mixture was stirred
until17 hadcompletely disappeared asmonitored by LC-MS analysis.
Then, the reaction mixture was refluxed overnight. After the solvent
was removed, the solid residue was partitioned between EtOAc and
water (1:1, 60 mL). The organic layer was separated, washed with
water (15 mL) and 1 N NaOH (15 mL), dried with anhydrous Na2SO4
and concentrated under reduced pressure to give the crude product,
which was purified by flash chromatography (hexane/EtOAc ¼ 2:1)
provided target oxadiazole (14aeh) in 11e32% yield.
4.88, 4.67 and 4.36 (3ꢁ m, 1H), 3.77 and 3.46 (2ꢁ m, 2H), 3.35 and
3.12 (2ꢁ m, 2H), 2.38e2.28 (m, 4H), 2.01e1.54 (m, 3H); LC/MS (ESI):
m/z 415.8 [M
þ
H]þ; HRMS-ESI: m/z [M
þ
H]þ calcd for
C21H20Cl2N3O2þ: 416.0927, found: 416.0934.
4.2.10. (S)-(3-(3-(2-Bromophenyl)-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)(3-chloro-2-methylphenyl)methanone (14f)
White solid (100 mg, yield 11%). 1H NMR (400 MHz, CDCl3):
d
¼ 7.87e7.71 (m, 2H), 7.44e7.34 (m, 3H), 7.20e7.10 (m, 2H), 4.88,
4.67 and 4.33 (3ꢁ m, 1H), 3.79e3.03 (m, 4H), 2.38e2.27 (m, 4H),
2.04e1.59 (m, 3H); LC/MS (ESI): m/z 459.7 [M þ H]þ; HRMS-ESI: m/
z [M þ Na]þ calcd for C21H19BrClN3C21H19BrClN3NaO2þ: 482.0241,
found: 482.0221.
4.2.5. (S)-(3-Chloro-2-methylphenyl)(3-(3-cyclohexyl-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (14a)
4.2.11. (S)-(3-(3-(3-Bromophenyl)-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)(3-chloro-2-methylphenyl)methanone (14g)
White solid (113 mg, yield 20%). 1H NMR (300 MHz, CDCl3):
Primrose yellow solid (289 mg, yield 31%). 1H NMR (400 MHz,
d
¼ 7.37 (d, J ¼ 8.1 Hz, 1H), 7.17 (dd, J ¼ 7.3, 8.1 Hz, 1H), 7.09 (d,
CDCl3):
d
¼ 8.22 (m, 1H), 7.99 (m, 1H), 7.64 (m, 1H), 7.40e7.32
J ¼ 7.3 Hz, 1H), 4.82, 4.66 and 4.37 (3ꢁ m, 1H), 3.67 and 3.45e3.15
(2ꢁ m, 3H), 3.03 (m,1H), 2.75 (m,1H), 2.36e2.26 (m, 4H), 2.03e1.91
(m, 3H), 1.85e1.64 (m, 5H), 1.57e1.25 (m, 5H); LC/MS (ESI): m/z
388.0 [M þ H]þ; Anal. Calcd. for C21H26ClN3O2: C, 65.02; H, 6.76; N,
10.83; Found: C, 65.23; H, 6.85; N, 10.76.
(m, 2H), 7.20e7.08 (m, 2H), 4.86, 4.67 and 4.31 (3ꢁ m, 1H), 3.75
and 3.48 (2ꢁ m, 2H), 3.32 and 3.11 (2ꢁ m, 2H), 2.38e2.27 (m, 4H),
2.04e1.54 (m, 3H); LC/MS (ESI): m/z 459.7 [M þ H]þ; HRMS-ESI: m/
z [M þ H]þ calcd for C21H20BrClN3O2þ: 460.0422, found: 460.0420.
4.2.12. (S)-(3-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)(3-chloro-2-methylphenyl)methanone (14h)
4.2.6. (S)-(3-Chloro-2-methylphenyl)(3-(3-(adamant-1-yl)-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (14b)
Primrose yellow solid (290 mg, yield 31%). 1H NMR (400 MHz,
White solid (140 mg, yield 32%). 1H NMR (400 MHz, CDCl3):
CDCl3):
d
¼ 7.96 (m, 1H), 7.88 (m, 1H), 7.65e7.59 (m, 2H), 7.38 (m,
d
¼ 7.38 (d, J ¼ 7.7 Hz, 1H), 7.18 (dd, J ¼ 7.3, 7.7 Hz, 1H), 7.10 (d,
1H), 7.22e7.06 (m, 2H), 4.84, 4.68 and 4.33 (3ꢁ m, 1H), 3.72 and
3.47 (2ꢁ m, 2H), 3.31 and 3.10 (2ꢁ m, 2H), 2.38e2.27 (m, 4H), 2.02e
1.63 (m, 3H); LC/MS (ESI): m/z 459.8 [M þ H]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C21H20BrClN3O2þ: 460.0422, found: 460.0417.
J ¼ 7.3 Hz, 1H), 4.82, 4.66 and 4.33 (3ꢁ m, 1H), 3.66 and 3.48e3.17
(2ꢁ m, 3H), 3.04 (m, 1H), 2.36e2.13 (m, 4H), 2.09e1.95 (m, 10H),
1.81e1.59 (m, 8H); LC/MS (ESI): m/z 439.9 [M þ H]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C25H31ClN3O2þ: 440.2099, found: 440.2105.
4.3. General procedure for the synthesis of target compounds 14aew
4.2.7. (S)-(3-Chloro-2-methylphenyl)(3-(3-(thiophen-2-yl)-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (14c)
4.3.1. N0-Hydroxybenzimidamide (22)
White solid (95 mg, yield 12%). 1H NMR (300 MHz, CDCl3):
Triethylamine (42 mL, 0.3 mol) was added to a solution of ben-
zonitrile (21, 10.3 g, 0.1 mol), hydroxylamine hydrochloride (20.9 g,
0.3 mol) and ethanol (150 mL) and stirred for 12 h at 75 ꢀC. The
reaction mixture was cooled to room temperature and evaporated
to dryness, extracted with DCM (300 mL)/water (100 mL). The
organic layer dried with MgSO4, filtered and evaporated to dryness
d
¼ 7.75 (m, 1H), 7.49 (m, 1H), 7.36 (m, 1H), 7.19e7.06 (m, 3H), 4.84,
4.63 and 4.26 (3ꢁ m, 1H), 3.71 and 3.45 (2ꢁ m, 2H), 3.29 and 3.07
(2ꢁ m, 2H), 2.38e2.24 (m, 4H), 2.05e1.45 (m, 3H); LC/MS (ESI): m/z
388.0 [M þ H]þ; Anal. Calcd. for C19H18ClN3O2S: C, 58.83; H, 4.68; N,
10.83; Found: C, 58.66; H, 4.64; N, 10.59.

G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
7
yielding the desired product 22 as a primrose yellow liquid (10.6 g,
yield 78%). 1H NMR (300 MHz, DMSO-d6):
(m, 2H), 7.32e7.37 (m, 3H), 5.77 (s, 2H); LC/MS (ESI): m/z 137
[M þ H]þ.
4.3.7. (S)-(3-(3-Phenyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)(o-
d
¼ 9.59 (s,1H), 7.62e7.67
tolyl)methanone (4c)
Colorless oil (112 mg, yield 65%). 1H NMR (400 MHz, CDCl3):
d
¼ 8.10 (m, 1H), 8.00 (m, 1H), 7.50e7.43 (m, 3H), 7.29e7.20 (m, 4H),
4.93, 4.66 and 4.45 (3ꢁ m, 1H), 3.79 and 3.46 (2ꢁ m, 2H), 3.30 and
3.10 (2ꢁ m, 2H), 2.37e2.26 (m, 4H), 2.04e1.58 (m, 3H); LC/MS (ESI):
m/z 347.9 [M þ H]þ; HRMS-ESI: m/z [M þ Na]þ calcd for
C21H21N3NaOþ2 : 370.1526, found: 370.1524.
4.3.2. (S)-tert-Butyl 3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidine-1-
carboxylate (24)
A
solution
of
(S)-1-(tert-butoxycarbonyl)piperidine-3-
carboxylic acid (5.05 g, 22 mmol) in dry 1,4-dioxane (50 mL) at
room temperature was charged with HOBt (2.97 g, 22 mmol) and
EDCI$HCl (6.34 g, 33 mmol). After 30 min, 22 (3.0 g, 22 mmol) was
added and the mixture was stirred until starting material had
completely disappeared as monitored by LC-MS analysis. Then, the
reaction mixture was refluxed overnight. After the solvent was
removed, the solid residue was partitioned between EtOAc and
water (1:1, 150 mL). The organic layer was separated, washed with
water (50 mL) and 1 N NaOH (50 mL), dried over anhydrous Na2SO4
and concentrated under reduced pressure to give the crude prod-
uct, which was purified by flash chromatography (hexane/
4.3.8. (S)-(2-Chlorophenyl)(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4d)
White solid (148 mg, yield 81%). 1H NMR (400 MHz,
CDCl3):
d
¼ 8.11 (m, 1H), 8.03 (m, 1H), 7.55e7.44 (m, 3H), 7.41
(m, 1H), 7.34e7.26 (m, 3H), 5.06, 4.97, 4.83 and 4.39 (4ꢁ m, 1H),
3.80e2.95 (m, 4H), 2.36 (m, 1H), 2.04e1.68 (m, 3H); LC/MS (ESI):
m/z 367.9 [M þ H]þ; HRMS-ESI: m/z [M þ Na]þ calcd for
C20H18ClN3NaO2þ: 390.0980, found: 390.0973.
4.3.9. (S)-(3-Chlorophenyl)(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4e)
EtOAc ¼ 2:1) provided 24 as a white solid (4.28 g, yield 59%).
20
[
d
a
]
¼ þ76.6 (c ¼ 1.0 in CH2Cl2); 1H NMR (400 MHz, CDCl3):
Colorless oil (115 mg, yield 63%). 1H NMR (400 MHz, CDCl3):
D
¼ 8.08e8.06 (m, 2H), 7.49e7.46 (m, 3H), 4.28 (br s, 1H), 3.98
d
¼ 8.06 (m, 2H), 7.50e7.46 (m, 3H), 7.43e7.30 (m, 4H), 4.82e3.96
(m, 1H), 3.33 (br s, 1H), 3.15 (m, 1H), 2.98 (m, 1H), 2.25 (m, 1H),
(m, 2H), 3.68e3.13 (m, 3H), 2.34 (m, 1H), 2.03e1.62 (m, 3H);
LC/MS (ESI): m/z 367.9 [M þ H]þ; HRMS-ESI: m/z [M þ Na]þ calcd
for C20H18ClN3NaO2þ: 390.0980, found: 390.0979.
1.92e1.81 (m, 2H), 1.60 (m, 1H), 1.46 (s, 9H); 13C NMR (100 MHz,
CDCl3):
d
¼ 179.93, 168.25, 154.52, 131.17, 128.84 (2C), 127.46 (2C),
126.81, 80.06, 46.73 (br s, 1C), 43.68 (br s, 1C), 34.87, 28.59, 28.41
(3C), 23.98 (br s, 1C); LC/MS (ESI): m/z 352 [M þ Na]þ.
4.3.10. (S)-(4-Chlorophenyl)(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4f)
4.3.3. (S)-3-Phenyl-5-(piperidin-3-yl)-1,2,4-oxadiazole (25)
White solid (135 mg, yield 74%). 1H NMR (300 MHz, CDCl3):
To a solution of 24 (3.7 g, 11 mmol) in DCM (30 mL) was added
4 N HCl in dioxane (10 mL). The resulting mixture was stirred at rt
overnight. Water (50 mL) was added, and the mixture was washed
with DCM (30 mL ꢁ 2). The aqueous phase was basified with 2 N aq.
NaOH to pH ¼ 9w10, and extracted with EtOAc (100 mL ꢁ 2). The
combined organic phase was dried over anhydrous MgSO4, and
concentrated under reduced pressure to afford the product as
a primrose yellow oil (2.2 g, yield 86%), which was used for the next
step without further purification. LC/MS (ESI): m/z 230 [M þ H]þ.
d
¼ 8.05 (m, 2H), 7.50e7.44 (m, 3H), 7.42e7.34 (m, 4H), 7.43e7.32
(m, 4H), 4.82e3.73 (m, 2H), 3.57e3.16 (m, 3H), 2.33 (m, 1H),
2.03e1.64 (m, 3H); LC/MS (ESI): m/z 367.9 [M þ H]þ; HRMS-ESI: m/z
[M þ Na]þ calcd for C20H18ClN3NaO2þ: 390.0980, found: 390.0965.
4.3.11. (S)-(2-Chloro-3-methylphenyl)(3-(3-phenyl-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (4g)
White solid (130 mg, yield 86%). 1H NMR (300 MHz, CDCl3):
¼ 8.08 (m, 1H), 8.00 (m, 1H), 7.49e7.41 (m, 3H), 7.24e7.02 (m, 3H),
d
5.02, 4.95, 4.77 and 4.37 (4ꢁ m, 1H), 3.75e3.10 (m, 3H) 2.41e2.27
(m, 4H), 2.03e1.64 (m, 3H); LC/MS (ESI): m/z 382.0 [M þ 1]þ;
Anal. Calcd. for C21H20ClN3O2: C, 66.05; H, 5.28; N, 11.00. Found: C,
66.12; H, 5.19; N, 10.86.
4.3.4. General method of condensation for target compounds 4aew
To a stirred solution of 25 (115 mg, 0.5 mmol) in DCM (10 mL)
was added carboxylic acid (0.5 mmol), HOBT (68 mg, 0.5 mmol) and
EDCI$HCl (144 mg, 0.75 mmol). The mixture was stirred at room
temperature overnight, and washed successively with water (5 mL)
and brine (10 mL ꢁ 2). The organic layer was dried over anhydrous
Na2SO4, and concentrated to give the crude product, which was
purified by flash chromatography (hexane/EtOAc ¼ 3:1e1:1)
afforded 4aew.
4.3.12. (S)-(3-Bromo-2-methylphenyl)(3-(3-phenyl-1,2,4-
oxadiazol-5-yl)piperidin-1-yl)methanone (4h)
White solid (156 mg, yield 82%). 1H NMR (400 MHz, CDCl3):
¼ 8.09 (m, 1H), 8.01 (m, 1H), 7.60e7.46 (m, 4H), 7.18e7.03 (m, 2H),
d
4.88, 4.65 and 4.33 (3ꢁ m, 1H), 3.75 and 3.47 (2ꢁ m, 2H), 3.33 and
3.12 (2ꢁ m, 2H), 2.43e2.29 (m, 4H), 2.08e1.73 (m, 3H); LC/MS (ESI):
m/z 425.8 [M þ H]þ; HRMS-ESI: m/z [M þ Na]þ calcd for
C21H20BrN3NaO2þ: 448.0631, found: 448.0621.
4.3.5. (S)-(3-Chloro-2-methylphenyl)(3-(3-phenyl-1,2,4-oxadiazol-
5-yl)piperidin-1-yl)methanone (4a)
20
White solid (139 mg, 73%). [
a
d
]
¼ þ65.8 (c ¼ 0.35 in
D
CH2Cl2); 1H NMR (300 MHz, CDCl3):
¼ 8.09 (m, 1H), 8.01 (m, 1H),
4.3.13. (S)-(3-Iodo-2-methylphenyl)(3-(3-phenyl-1,2,4-oxadiazol-
5-yl)piperidin-1-yl)methanone (4i)
7.52e7.44 (m, 3H), 7.37 (m, 1H), 7.21e7.07 (m, 2H), 4.89e4.33
(m, 1H), 3.79e3.08 (m, 4H), 2.39e2.28 (m, 4H), 2.04e1.70 (m,
3H); LC/MS (ESI): m/z 381.9 [M þ H]þ; HRMS-ESI: m/z [M þ Na]þ
calcd for C21H20ClN3NaO2þ: 404.1136, found: 404.1161.
White solid (205 mg, yield 87%).1H NMR (300 MHz, CDCl3):
d
¼ 8.09
(d, J ¼ 7.9 Hz, 1H), 8.00 (m, 1H), 7.85 (dd, J ¼ 7.8, 7.9 Hz, 1H), 7.51e7.46
(m, 3H), 7.20e7.15 (dd, J ¼ 7.8, 8.4 Hz,1H), 6.89e6.96 (m,1H), 4.88, 4.63
and 4.32 (3ꢁ m, 1H), 3.75e3.07 (m, 4H), 2.45e2.33 (m, 4H), 2.04e1.71
(m, 3H); EI-MS m/z: 474 [M þ H]þ; Anal. Calcd. for C21H20IN3O2: C,
53.29; H, 4.26; N, 8.88; Found: C, 53.44; H, 4.31; N, 8.63.
4.3.6. (S)-Phenyl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)
methanone (4b)
Colorless oil (93 mg, yield 56%). 1H NMR (400 MHz, CDCl3):
d
¼ 8.07 (m, 2H), 7.49e7.47 (m, 3H), 7.42 (m, 5H), 4.91 and 4.44 (2ꢁ
4.3.14. (S)-(3-(3-Phenyl-1,2,4-oxadiazol-5-yl)piperidin-1-
yl)(pyridin-2-yl)methanone (4j)
m, 1H), 4.08 and 3.78 (2ꢁ m, 1H), 3.50e3.17 (m, 3H), 2.35 (m, 1H),
2.04e1.68 (m, 3H); LC/MS (ESI): m/z 333.9 [M þ H]þ; HRMS-ESI: m/z
[M þ Na]þ calcd for C20H19N3NaO2þ: 356.1369, found: 356.1367.
White solid (140 mg, yield 84%). 1H NMR (300 MHz, CDCl3):
d
¼ 8.71 (m, 2H), 8.09e8.00 (m, 2H), 7.50e7.45 (m, 3H), 7.33 (m,

8
G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
2H), 4.78 and 4.20 (2ꢁ m, 1H), 3.89e3.16 (m, 4H), 2.72 (m, 1H), 2.34
(m, 1H), 2.06e1.71 (m, 2H); 13C NMR (100 MHz, CDCl3):
C21H27N3O2: C, 71.36; H, 7.70; N, 11.89; Found: C, 71.10; H, 7.93; N,
11.73.
d
¼ 179.20
and 178.74 (1C), 168.33, 168.09, 150.41 (2C), 150.31, 143.30, 131.33,
128.90 (2C), 127.45 (2C), 126.65, 121.11, 49.64 and 47.72 (1C), 44.45
and 42.34 (1C), 35.15 and 34.46 (1C), 28.46 and 28.38 (1C), 24.71
and 23.33 (1C); LC/MS (ESI): m/z 334.9 [M þ H]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C19H19N4O2þ: 335.1503, found: 335.1502.
4.3.21. (S)-Cyclohexyl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)methanone (4q)
White solid (108 mg, yield 64%). 1H NMR (400 MHz, CDCl3):
d
¼ 8.08e8.04 (m, 2H), 7.49 (m, 3H), 4.86 and 4.23 (2ꢁ m, 1H), 4.11
and 3.91 (2ꢁ m, 1H), 3.69e3.05 (m, 3H), 2.57 (m, 1H), 2.30 (m, 1H),
2.03e1.49 (m, 13H); 13C NMR (100 MHz, CDCl3):
¼ 179.72 and
4.3.15. (S)-Furan-2-yl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)methanone (4k)
d
179.42 (1C), 174.86 and 174.79 (1C), 168.35 and 168.22 (1C), 131.34
and 131.16 (1C), 128.85 (2C), 127.46 (2C), 126.80 and 126.59 (1C),
47.98 and 45.68 (1C), 44.32 and 41.96 (1C), 40.60 and 40.35 (1C),
35.68 and 34.79 (1C), 29.65 and 29.51 (1C), 29.42 and 29.29 (1C),
28.86 and 28.65 (1C), 25.88 and 25.84 (3C), 25.32 and 23.77 (1C);
LC/MS (ESI): m/z 340.0 [M þ H]þ; HRMS-ESI: m/z [M þ H]þ calcd for
C20H26N3O2þ: 340.2020, found: 340.2022.
Primrose yellow oil (111 mg, yield 69%). 1H NMR (400 MHz,
CDCl3):
d
¼ 8.07e8.05 (m, 2H), 7.50e7.47 (m, 4H), 7.05 (d, J ¼ 3.4 Hz,
1H), 6.49 (dd, J ¼ 1.8, 3.4 Hz, 1H), 4.77 (m, 1H), 4.44 (m, 1H), 3.51 (br
s, 1H), 3.34e3.18 (m, 2H), 2.38 (m, 1H), 2.03e1.90 (m, 2H), 1.75 (m,
1H); 13C NMR (100 MHz, CDCl3):
d
¼ 179.42, 168.31, 159.42, 147.86,
143.79, 131.19, 128.82 (2C), 127.48 (2C), 126.74, 116.63, 111.34, 47.31
(br s,1C), 45.22 (br s,1C), 35.27, 28.89, 24.55; LC/MS (ESI): m/z 323þ.9
[M þ H]þ; HRMS-ESI: m/z [M þ H]þ calcd for C18H18N3O3
:
4.3.22. (S)-Cyclobutyl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)methanone (4r)
324.1343, found: 324.1345.
White solid (88 mg, yield 56%). 1H NMR (400 MHz, CDCl3):
4.3.16. (S)-(1-Methyl-1H-pyrrol-2-yl)(3-(3-phenyl-1,2,4-oxadiazol-
d
¼ 8.09e8.06 (m, 2H), 7.52e7.47 (m, 3H), 4.84 and 4.33 (2ꢁ m, 1H),
5-yl)piperidin-1-yl)methanone (4l)
3.98 and 3.71 (2ꢁ m, 1H), 3.49e2.94 (m, 4H), 2.43e2.13 (m, 5H),
White solid (105 mg, yield 63%). 1H NMR (300 MHz, CDCl3):
2.01e1.82 (m, 4H), 1.57 (m, 1H); 13C NMR (100 MHz, CDCl3):
d
¼ 8.07e8.04 (m, 2H), 7.49e7.45 (m, 3H), 6.71 (m, 1H), 6.38 (m,
d
¼ 179.71 and 179.36 (1C), 173.27, 168.36 and 168.25 (1C), 131.35
J ¼ 2.2 Hz, 1H), 6.10e6.08 (dd, J ¼ 2.1, 3.5 Hz, 1H), 4.73 (d,
J ¼ 12.3 Hz, 1H), 4.36 (d, J ¼ 12.3 Hz, 1H), 3.77 (s, 3H), 3.53 (m, 1H),
3.40e3.21 (m, 2H), 2.36 (m,1H), 2.07e1.89 (m, 2H),1.73 (m,1H); LC/
MS (ESI): m/z 336.9 [M þ H]þ; HRMS-ESI: m/z [M þ H]þ calcd for
C19H21N4O2þ: 337.1659, found: 337.1664.
and 131.18 (1C), 128.90 and 128.86 (2C), 127.46 (2C), 126.80 and
126.60 (1C), 47.75 and 45.27 (1C), 44.42 and 42.01 (1C), 37.38 and
37.25 (1C), 35.60 and 34.73 (1C), 28.84 and 28.60 (1C), 25.56 and
25.32 (1C), 25.04, 24.84 and 23.82 (1C), 17.97; EI-MS m/z: 312
[M þ H]þ; LC/MS (ESI): m/z 312.0 [M þ H]þ; HRMS-ESI: m/z
[M þ Na]þ calcd for C18H21N3NaO2þ: 334.1526, found: 334.1534.
4.3.17. (S)-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-[3-(3-phenyl-
[1,2,4]oxadiazol-5-yl)-piperidin-1-yl]-methanone (4m)
4.3.23. (S)-Cyclopropyl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4s)
Primrose yellow solid (126 mg, yield 51%). 1H NMR (300 MHz,
CDCl3):
d
¼ 8.06e8.03 (m, 2H), 7.54e7.44 (m, 3H), 6.44 (s, 1H), 4.51
White solid (78 mg, yield 53%). 1H NMR (400 MHz, CDCl3):
¼ 8.09e8.06 (m, 2H), 7.50e7.48 (m, 3H), 4.87 and 4.36 (2ꢁ m, 1H),
(m, 1H), 4.10 (m, 1H), 3.70 (s, 3H), 3.67 (m, 1H), 3.38 (m, 1H), 3.24
(m, 1H), 2.32 (m, 1H), 2.06 (m, 1H), 1.91 (m, 1H), 1.71 (m, 1H); Anal.
Calcd. for C19H18Br2N4O2: C, 46.18; H, 3.67; N, 11.34; Found: C,
46.06; H, 3.81; N, 11.42.
d
4.21 (m, 1H), 4.12 and 3.80 (2ꢁ m, 1H), 3.28e3.09 (m, 2H), 2.33 (m,
1H), 2.07e1.78 (m, 4H), 1.00e0.98 (m, 2H), 0.80e0.78 (m, 2H); 13C
NMR (100 MHz, CDCl3):
d
¼ 179.66 and 179.49 (1C), 172.20, 168.28,
131.26, 128.87, 127.46, 127.35 and 126.64 (1C), 48.23 and 45.90 (1C),
44.96 and 42.58 (1C), 35.45 and 34.67 (1C), 28.89 and 28.52 (1C),
25.15 and 23.61 (1C), 11.11, 7.56 (2C); LC/MS (ESI): m/z 298þ.0
4.3.18. (S)-(1-Methyl-1H-indol-4-yl)-[3-(3-phenyl-[1,2,4]
oxadiazol-5-yl)-piperidin-1-yl]-methanone (4n)
White solid (146 mg, yield 76%). 1H NMR (300 MHz, CDCl3):
¼ 8.06e8.02 (m, 2H), 7.53e7.41 (m, 3H), 7.37 (d, J ¼ 8.3 Hz, 1H), 7.24
[M þ H]þ; HRMS-ESI: m/z [M þ Na]þ calcd for C17H19N3NaO2
:
d
320.1369, found: 320.1370.
(dd, J¼ 7.1, 8.3 Hz, 1H), 7.15 (d, J¼ 7.1 Hz, 1H), 7.09 (d, J¼ 2.6 Hz,1H),6.47
(d, J ¼ 2.6 Hz,1H), 5.00 and 4.66 (2ꢁ m,1H), 4.22e4.01 (m,1H), 3.80 (s,
3H), 3.46e3.10 (m, 3H), 2.32 (m, 1H), 2.03e1.89 (m, 2H), 1.74 (m, 1H);
LC-MS (ESI): m/z 386.9 [M þ 1]þ; Anal. Calcd. for C23H22N4O2: C, 71.48;
H, 5.74; N, 14.50; Found: C, 71.75; H, 5.87; N, 14.24.
4.3.24. (S)-(3-(3-Phenyl-1,2,4-oxadiazol-5-yl)piperidin-1-
yl)(2,2,3,3-tetramethylcyclopropyl)methanone (4t)
White solid (92 mg, yield 52%). 1H NMR (300 MHz, CDCl3):
¼ 8.08e8.06 (m, 2H), 7.49 (br s, 3H), 4.80 and 3.96 (2ꢁ m,1H), 4.27
d
(m, 1H), 3.53 and 2.92 (2ꢁ m, 1H), 3.23e3.05 (m, 2H), 2.31 (m, 1H),
2.03e1.78 (m, 3H), 1.60 (m, 1H), 1.17 (s, 12H); LC/MS (ESI): m/z 354.0
[M þ H]þ; Anal. Calcd. for C21H27N3O2: C, 71.36; H, 7.70; N, 11.89;
Found: C, 71.56; H, 7.91; N, 11.68.
4.3.19. (S)-(1-Methyl-1H-indol-3-yl)(3-(3-phenyl-1,2,4-oxadiazol-
5-yl)piperidin-1-yl)methanone (4o)
White solid (125 mg, yield 81%). 1H NMR (300 MHz, CDCl3):
d
¼ 8.06e8.03 (m, 2H), 7.71 (d, J ¼ 4.5 Hz, 1H), 7.48e7.41 (m, 4H),
7.34e7.19 (m, 3H), 4.68 (d, J ¼ 12.0 Hz, 1H), 4.27 (d, J ¼ 13.3 Hz, 1H),
3.78 (s, 3H), 3.50 (dd, J1 ¼ 10.3 Hz, J2 ¼ 12.7 Hz, 1H), 3.33e3.20 (m,
2H), 2.33 (m, 1H), 2.05e1.83 (m, 2H), 1.70 (m, 1H); LC-MS (ESI): m/z
387.0 [M þ 1]þ; Anal. Calcd. for C23H22N4O2: C, 71.48; H, 5.74; N,
14.50; Found: C, 71.61; H, 5.66; N, 14.39.
4.3.25. Bicyclo[2.2.1]heptan-2-yl((S)-3-(3-phenyl-1,2,4-oxadiazol-
5-yl)piperidin-1-yl)methanone (4u)
White solid (120 mg, yield 68%). 1H NMR (300 MHz,
CDCl3):
d
¼ 8.08e8.02 (m, 2H), 7.51e7.43 (m, 3H), 5.02e4.31 (m, 1H),
4.24e3.88 (m, 1H), 3.61e3.20 (m, 1H), 3.18e2.88 (m, 3H), 2.38e2.25
(m, 3H), 2.03e1.74 (m, 3H), 1.57e1.27 (m, 8H); Anal. Calcd. for
C21H25N3O2:C,71.77;H, 7.17;N,11.96;Found:C,71.63;H, 7.22;N,11.76.
4.3.20. (S)-Cycloheptyl(3-(3-phenyl-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)methanone (4p)
White solid (138 mg, yield 78%). 1H NMR (300 MHz, CDCl3):
4.3.26. (S)-(1-Methylcyclohexyl)(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4v)
d
¼ 8.08e8.05 (m, 2H), 7.49 (br s, 3H), 4.84 and 4.21 (2ꢁ m,1H), 4.09
and 3.88 (2ꢁ m, 1H), 3.69e3.04 (m, 3H), 2.72 (m, 1H), 2.30 (m, 1H),
White solid (94 mg, yield 53%). 1H NMR (300 MHz, CDCl3):
2.05e1.46 (m, 15H); LC-MS (ESI): 354.0 [M þ 1]þ; Anal. Calcd. for
d
¼ 8.09e8.06 (m, 1H), 7.49e7.46 (m, 3H), 4.70 (d, J ¼ 12.6 Hz, 1H),

G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
9
4.37 (d, J ¼ 12.6 Hz, 1H), 3.28e3.11 (m, 2H), 2.99 (dd, J1 ¼ 11.1 Hz,
J2 ¼ 13.3 Hz,1H), 2.18 (m, 1H), 2.07e1.85 (m, 4H),1.68e1.31 (m, 9H);
Anal. Calcd. for C21H27N3O2: C, 71.36; H, 7.70; N, 11.89; Found: C,
71.50; H, 7.84; N, 11.98.
The amount of [3H] cortisol generated by 11
b
-HSD1 was captured
on the beads and measured in a microplate liquid scintillation
counter. Percent inhibition was calculated relative to noninhibited
control. Data were obtained from at least three independent
experiments. IC50 values were calculated by using Prism Version 4
(GraphPad Software, San Diego, CA).
4.3.27. (S)-(Adamant-1-yl)(3-(3-phenyl-1,2,4-oxadiazol-5-yl)
piperidin-1-yl)methanone (4w)
Microsomes (Human microsome: Xenotech, Lot No.H0610;
Rat microsome: Xenotech, Lot No. R1000) (0.5 mg/mL) were
White solid (160 mg, yield 73%). 1H NMR (300 MHz, CDCl3):
d
¼ 8.09e8.06 (m, 2H), 7.53e7.48 (m, 3H), 4.82 (d, J ¼ 9.6 Hz, 1H),
preincubated with 1 m
M test compound for 5 min at 37 ꢀC in 0.1 M
4.50 (d, J ¼ 13.5 Hz, 1H), 3.18e3.14 (m, 2H), 2.98 (m, 1H), 2.34 (m,
phosphate buffer (pH 7.4) with 1 mM EDTA, and 5 mM MgCl2. The
reactions were initiated by adding prewarmed cofactors (1 mM
NADPH). After 0, 5, 10, and 30 min incubations at 37 ꢀC, the
reactions were stopped by adding an equal volume of cold aceto-
nitrile. The samples were vortexed for 10 min and then centrifuged
at 10,000 g for 10 min. Supernatants were analyzed by LC/MS/MS
for the amount of parent compound remaining, and the corre-
sponding loss of parent compound also determined by LC/MS/MS.
The CYP enzymatic activities were characterized based on their
probe reactions: CYP3A4 (midazolam 1-hydroxylation), CYP2D6
(dextromethorphan O-demethylation), and CYP1A2 (phenacetin O-
deethylation). Incubation mixtures were prepared in a total volume
1H), 2.05e1.88 (m, 10H), 1.77e1.59 (m, 8H); 13C NMR (100 MHz,
CDCl3):
d
¼ 179.66, 176.02, 168.27, 131.20, 128.86 (2C), 127.48 (2C),
126.77, 47.98, 45.98, 39.14 (3C), 36.64 (3C), 35.42, 29.72, 28.89,
28.51 (3C), 24.94; LC/MS (ESI): m/z 392.0 [M þ H]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C24H30N3O2þ: 392.2333, found: 392.2339.
4.4. Docking study
The crystal structure 3G49 was downloaded from Protein Data
Bank (PDB) database [21]. Then, Autodock toolkit was used to
prepare the models for docking study [20]. For that, the crystal
water was deleted, hydrogen atoms were added, and Gasteiger
charge model was used for protein and NAD. Totally, two mono-
mers and one NAD molecule were included in receptor model for
docking. The ligand 3 and 4a were built and added the Gasteiger
charges. Then the receptor and ligands were saved in PDBQT format
for Vina program. The default parameters of Vina were used for
docking simulation, except for the parameter exhaustiveness was
set to 11 to enable the Vina to explore more search space. And
finally, the lowest energy binding conformations were analyzed
and illustrated with Pymol program.
of 100
Xenotech, Lot No.H0610), NADPH (1 mM), 100 mM phosphate
buffer (pH 7.4), probe substrates cocktail (midazolam 5 M, dex-
tromethorphan 5 M, phenacetin 100 M) and 10 M tested
compound or positive control cocktail (ketoconazole 10 M, qui-
nidine 10 M, -naphthoflavone 10 M or negative control (PBS)).
ml as follows: 0.2 mg/mL microsome (Human microsome:
m
m
m
m
m
m
a
m
The final concentration of organic reagent in incubation mixtures
was less than 1% v/v. There was a 5 min preincubation period at
37 ꢀC before the reaction was initiated by adding a NADPH-
generating system. Reactions were conducted for 20 min for
CYP3A4, CYP2D6 and CYP1A2. For each probe drug, the percentage
of metabolite conversion was less than 20% of substrate added. The
inhibition rate was calculated as: (The formation of the metabolite
4.5. Biological assays
Enzymatic activities of compounds against h11
m11 -HSD1 and h11 -HSD2 were determined by the scintillation
proximity assay (SPA) using microsomes containing 11 -HSDs ac-
cording to previous studies [18]. Briefly, the full-length cDNAs of
human or mouse 11 -HSDs were isolated from cDNA libraries
provided by NIH Mammalian Gene Collection and cloned into
pcDNA3 expression vectors (Invitrogen, Carlsbad, CA, USA) by PCR.
HEK-293 cells were transfected with the pcDNA3-derived expres-
b
-HSD1 and
of probe substrates with 10 mM tested compound)/(The formation
b
b
of the metabolite of probe substrates with PBS) ꢁ 100%.
b
The samples for the time-dependent inactivation screening
assay were preincubated for 0, 5, 10, 20 min at 37 ꢀC with
b
0.2 mg/mL human microsome (Xenotech, Lot No.H0610) and 10
test compound or positive control (troleandomycin 10 M, parox-
etine 10 M and furafylline 10 M) with or without 1 mM NADPH.
mM
m
m
m
The percentage of remaining activity of CYP3A4, CYP2D6, and
CYP1A2 was measured by the formation of the metabolite of
their marker substrates: midazolam, dextromethorphan and phe-
nacetin at single concentration approximating their Km values
sion plasmids and selected by cultivation at the presence of 700
mL G418. The microsomal fraction over-expressing 11 -HSDs was
prepared from the HEK-293 cells stably transfected with 11 -HSDs
mg/
b
b
and used as the enzyme source for SPA. The assay was performed in
a 96-well microtiter plate. Compounds with different concentra-
(midazolam 5 mM, dextromethorphan 5 mM, phenacetin 100 mM).
The percentage of remaining activity of microsomes preincubated
with NADPH was compared with that of microsomes without
preincubation.
tions were added, followed by adding 80
pH 7.4 containing 25 nM cortisone [1,2-3H(N)] (Amersham, Buck-
inghamshire, UK) and 1.25 mM NADPH (for 11 -HSD1 assay) or
12.5 nM cortisol [1,2,6,7-3H(N)] (Amersham, Buckinghamshire, UK)
and 0.625 mM NAD (for 11 -HSD2 assay).
mL of 50 mM HEPES buffer,
b
Acknowledgments
b
We thank Dr. David Burk for critical reading the manuscript and
4.6. In vitro DMPK evaluation
Prof. Ying Leng of SIMM for the 11b-HSD1 assay. We are grateful for
financial support from New Drug Creation Project of the National
Science and Technology Major Foundation of China (project
2010ZX09401-404), to Shanghai Pharmaceutical Holding, Shanghai
Postdoctoral Sustentation Fund, China (grant No. 07R214213 to GX.
Xia) and Program of Excellent Young Scientist of Chinese Academy
of Sciences (Grant to BX: KSCX2-EW-Q-3-01).
Reactions were initiated by the addition of enzyme preparations
as microsome fractions from HEK293 cells in a final concentration
of 80
reaction was stopped by adding 35
SPA beads (GE, Piscataway, NJ, USA) suspended in SuperblockÒ
Blocking Buffer (Pierce, Rockford, IL) with 3 g/mL of murine
monoclonal cortisol antibody (East Coast Biologics, North Berwick,
Maine, USA)and 314 M glycyrrhetinic acid (SigmaeAldrich, St.
mg/mL for 11b
-HSD1. Following 60 min incubation at 37 ꢀC, the
mL of 10 mg/mL protein A-coated
m
Appendix A. Supplementary data
m
Louis, MO). The plates were incubated under plastic film on an
orbital shaker for 120 min at room temperature before counting.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.059.

10
G. Xia et al. / European Journal of Medicinal Chemistry 62 (2013) 1e10
[13] M.H. Veéniant, C. Hale, R.W. Hungate, K. Gahm, M.G. Emery, J. Jona, S. Joseph,
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