Synthesis and biological activity evaluation of azacycloheptane sulfonamide derivatives as potential orexin receptor antagonists

Article abstract of DOI:10.1039/d0ra05068g

As the orexin signaling system is crucial for the regulation of the sleep/wake cycle, inhibitors of orexin-1 and orexin-2 receptors are of significant interest in the treatment of insomnia. Herein, a series of novel azacycloheptane sulfonamide derivatives were designed and synthesized, and all the compounds were evaluated as potential orexin receptor inhibitors by FLIPR Tetra calcium assay. A majority of the tested azacycloheptane sulfonamide derivatives showed OX1R and OX2R inhibitory activity. Chloro-substituted derivatives functionalized at the C5 or C6 position of the benzoxazole group exhibited better inhibitory activity for OX1R and OX2R than unsubstituted derivatives functionalized at C5 or C6. In addition, phenyl group modification had positive effects on the inhibitory activities, and an electron-withdrawing fluorine group at the ortho or meta position of the phenyl ring improved the OX2R inhibitory activity of the derivatives. This suggests that azacycloheptane sulfonamide derivatives are promising scaffolds for the development of OX1R and OX2R antagonists.

Full text of DOI:10.1039/d0ra05068g

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Synthesis and biological activity evaluation of
azacycloheptane sulfonamide derivatives as
potential orexin receptor antagonists†
Cite this: RSC Adv., 2020, 10, 30683
a
b
b
a
*
Yi Shen and Qingeng Li
Bin Guo,
Jingya Xiu,
As the orexin signaling system is crucial for the regulation of the sleep/wake cycle, inhibitors of orexin-1 and
orexin-2 receptors are of significant interest in the treatment of insomnia. Herein, a series of novel
azacycloheptane sulfonamide derivatives were designed and synthesized, and all the compounds were
evaluated as potential orexin receptor inhibitors by FLIPR Tetra calcium assay. A majority of the tested
azacycloheptane sulfonamide derivatives showed OX1R and OX2R inhibitory activity. Chloro-substituted
derivatives functionalized at the C5 or C6 position of the benzoxazole group exhibited better inhibitory
activity for OX1R and OX2R than unsubstituted derivatives functionalized at C5 or C6. In addition, phenyl
group modification had positive effects on the inhibitory activities, and an electron-withdrawing fluorine
group at the ortho or meta position of the phenyl ring improved the OX2R inhibitory activity of the
derivatives. This suggests that azacycloheptane sulfonamide derivatives are promising scaffolds for the
development of OX1R and OX2R antagonists.
Received 9th June 2020
Accepted 12th August 2020
DOI: 10.1039/d0ra05068g
rsc.li/rsc-advances
the rst DORA, and it initially showed positive effects in the
treatment of insomnia symptoms.23,24 However, almorexant was
ultimately discontinued in 2011 because of safety concerns.10
Introduction
Orexin-1 (OX1R) and orexin-2 (OX2R) receptors are G-protein
coupled receptors (GPCRs) that were discovered in 1998.1,2
Many studies have conrmed that orexin signaling system is
crucial for the regulation of the sleep/wake cycle.3,4 Orexin
peptide-decient mice and humans with narcolepsy have
similar symptoms.5 Moreover, mice with knockout of OX1R and
OX2R showed an acute narcoleptic phenotype similar to
peptide-decient animals.6 Consistent with these observations,
rats treated with orexin peptides by direct intra-
cerebroventricular (ICV) infusions showed an increase in
arousal and a decrease in sleep.7 As a result, due to their inti-
mate involvement in insomnia diseases, orexin receptors have
become promising drug targets for treating insomnia.8–13
A number of antagonists of OX1R and OX2R, such as selec-
tive orexin-1 receptor antagonists (1-SORAs), selective orexin-2
receptor antagonists (2-SORAs) and dual orexin receptor
antagonists (DORAs), have been synthesized in the past
decade.14–22 However, many studies have found that a more
robust effect on increases REM and non-REM sleep when both
receptors are inhibited.6 So, most work has focused on DORAs.
Almorexant, developed by Actelion and GlaxoSmithKline, was
The best dual orexin antagonists reported to date are suvorexant
and lemborexant, which received approval from the FDA in 2014
and 2019, respectively.25–29 Despite these advances and the
results of orexin antagonists, adverse effects such as somno-
lence limit the application of orexin antagonists as rst-line
treatments for insomnia. Therefore, further studies are
needed to identify selective orexin antagonists and dual orexin
antagonists for potential use as sleep drugs.
In this study, we designed and synthesized a series of twenty
one novel azacycloheptane sulfonamide derivatives. The in vitro
OX1R and OX2R inhibitory activities were assayed to evaluate
the potential of these compounds as orexin antagonists.
Compound 23 showed the most potent activities in both the
OX1R and OX2R inhibition assays. The structure–activity anal-
ysis of these compounds was conducted according to their
biological activities in vitro. Then, molecular docking simula-
tions were performed to elucidate the binding mode of active
compound 23. To the best of our knowledge, this is the rst
report on azacycloheptane sulfonamide derivatives with potent
inhibitory activities against OX1R and OX2R.
Results and discussion
Chemistry
aSchool of Pharmaceutical Science, Chongqing Medical University, Chongqing 400016,
China. E-mail: 13908387568@vip.163.com
bJiangsu Nhwaluokang Pharmaceutical Research and Development Co., Ltd.,
The synthetic strategy for the prepare of target compounds 9–29
are depicted in Scheme 1. Using benzylglycine and (R)-3-ami-
nobutanoic acid as starting materials, intermediate products 1
Chongqing 400016, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra05068g
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Table 1 The IC50 values of the prepared compounds 9–14 against
OX1R and OX2R: part 1
IC50 (mM)
Compd
A-Ring
OX1R
OX2R
9
10
11
12
13
14
3-Fluorophenyl
NAa
NAa
NAa
NAa
NAa
NAa
0.14
3.80
NAa
1.77
NAa
NAa
1.79
0.15
4-Methoxyphenyl
4-tert-Butylphenyl
2,3-Dihydrobenzo[b][1,4]dioxin-6-yl
4-Fluorophenyl
4-Methylphenyl
—
Suvorexant
Scheme 1 Synthesis of the azacycloheptane sulfonamide derivatives
9–29. Reagents and conditions: (a) methanol, thionyl chloride, rt, 3
days; (b) 1,4-dioxane, H2O, sodium hydroxide, di-tert-butyl dicar-
bonate, rt, 3 days; (c) EDCI, HOBt, triethylamine, DMF, 0–5 ꢀC; (d)
hydrochloric acid, ethyl acetate, methanol, rt, 3 h; (e) sodium meth-
oxide, methanol, rt, 0.5 h; (f) LAH, tetrahydrofuran, 0–5 ꢀC; (g) trie-
thylamine, DCM, rt, 1 h; (h) 10% Pd/C, methanol, 24 h; (i) 2-
chlorobenzoxazole, potassium carbonate, DMF, 60 ꢀC, 3 h. (j) 2,6-
Dichlorobenzoxazole, potassium carbonate, DMF, 60 ꢀC, 3 h. (k) 2,5-
Dichlorobenzoxazole, potassium carbonate, DMF, 60 ꢀC, 3 h.
a
No inhibitory activity.
comparable to that of suvorexant. The OX1R inhibitory activities
of compounds 19, 24 and 25 are similar to that of suvorexant.
However, the OX1R (IC50 ¼ NA) and OX2R (IC50 ¼ NA) inhibitory
activities shown by compounds 10, 12 and 13, which contained
4-methoxyphenyl, 2,3-dihydrobenzo[b][1,4]dioxin-6-yl, or 4-u-
orophenyl, were not observed.
Structure–activity analysis revealed that a chloro substituent
at the C5 or C6 position of the benzoxazole group plays a key
role in the inhibitory activities of these azacycloheptane
sulfonamide derivatives for OX1R and OX2R. In general, the
derivatives with 6-dichlorobenzoxazole group (15–22) or 5-
dichlorobenzoxazole (23–29) group showed better OX1R and
OX2R inhibitory activities than the derivatives with unsub-
stituted benzoxazole group (9–14). The inhibitory activities of
the derivatives with benzoxazole group and various substituted
phenyl groups, such as 3-uorophenyl (9), 4-methoxyphenyl
(10), 4-tert-butylphenyl (11), 2,3-dihydrobenzo[b][1,4]dioxin-6-yl
(12), 4-uorophenyl (13) or p-methylphenyl (14) for OX1R and
and 2 were constructed. The reaction between 1 and 2 in the
presence of 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI) pro-
ceeded at 0–5 ꢀC and afforded compound 3. Treatment of 3 with
hydrochloric acid in ethyl acetate at room temperature gener-
ated compound 4, which was directly treated with sodium
methoxide to obtain compound 5. Compound 6 was obtained
from the reaction of compound 5 with lithium aluminium
hydride (LAH) in tetrahydrofuran (THF). Then, compound 6 was
reacted with a series of substituted benzenesulfonyl chloride
derivatives in dichloromethane (DCM) to afford compounds 7a–
7i. Compounds 7a–7i were reduced to the corresponding
secondary amines with hydrogen to provide compounds 8a–8i.
Finally, target compounds 9–29 were synthesized by reacting
compounds 8a–8i with the corresponding chlorine-substituted
benzoxazole derivatives in N,N-dimethylformamide (DMF) at
55–60 ꢀC. The structures of compounds 1, 2, 5, 6, 7a–7i, and 8a–
8i were qualitatively conrmed by mass spectrometry (MS). The
Table 2 The IC50 values of the prepared compounds 15–22 against
OX1R and OX2R: part 2
1
structures of compounds 9–29 were conrmed by H and 13C
NMR spectroscopy and HRMS.
IC50 (mM)
Compd
A-Ring
OX1R
OX2R
Biological activity and in vitro SAR
15
16
17
18
19
20
21
22
3-Fluorophenyl
4-Methoxyphenyl
Phenyl
4-tert-Butylphenyl
2,3-Dihydrobenzo[b][1,4]dioxin-6-yl
4-Fluorophenyl
2-Fluorophenyl
p-Acetamidophenyl
—
1.98
2.29
8.58
NAa
0.20
NAa
NAa
1.33
0.14
1.65
5.11
1.52
1.32
1.78
NAa
3.87
2.05
0.15
The inhibitory activities of compounds 9–29 for OX1R and
OX2R were tested using a previously described FLIPR Tetra
calcium assay (Tables 1–3). The IC50 values of suvorexant
against OX1R and OX2R are 0.14 mM and 0.15 mM, respectively.
A majority of the tested azacycloheptane sulfonamide deriva-
tives showed OX1R and OX2R inhibitory activities. Further-
more, compound 23 showed much more potent combined
Suvorexant
inhibitory activities for OX1R (IC50 ¼ 0.63) and OX2R (IC50
¼
a
No inhibitory activity.
0.17 mM) than did the other compounds, and its potency is
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Table 3 The IC50 values of the prepared compounds 23–29 against
OX1R and OX2R: part 3
IC50 (mM)
Compd
A-Ring
OX1R
OX2R
23
24
25
26
27
28
29
3-Fluorophenyl
4-Methoxyphenyl
Phenyl
4-tert-Butylphenyl
2,3-Dihydrobenzo[b][1,4]dioxin-6-yl
2-Fluorophenyl
p-Acetamidophenyl
—
0.63
0.21
0.57
NAa
0.75
1.58
4.52
0.14
0.17
1.80
1.09
1.15
1.20
1.48
4.89
0.15
Suvorexant
a
No inhibitory activity.
OX2R were very weak or could hardly be observed. Analysis 15–
17, 19, and 22–29, with chloro substituents at the C5 or C6
position, revealed remarkably enhanced inhibitory activities for
these derivatives against OX1R, and their affinities for OX2R
also changed. Compared to the best OX2R inhibitor, C5 and C6
unsubstituted 11, most of the chloro-substituted compounds
(15, 17–18, 23, and 25–28) showed improved inhibitory activities
against OX2R. The evaluation of the derivatives with chloro-
substituted benzoxazole groups and substituted phenyl
groups, i.e., p-methoxyphenyl 16, p-acetamidophenyl 22, p-
methoxyphenyl 24 and p-acetamidophenyl 29, revealed that the
electron-donating groups 4-methoxyphenyl and p-acet-
amidophenyl at the para position of the phenyl ring decreased
the inhibitory activity for OX2R. In general, an electron-
withdrawing uorine group at the ortho or meta position of
phenyl ring improved the inhibitory activity of the derivative for
OX2R. Phenyl group modication positively impacted the
inhibitory activities of these compounds. 19 and 24 were the
best OX1R inhibitors, and 23 was the best OX1R and OX2R dual
inhibitor.
Fig. 1 (A) 3D interaction diagram of suvorexant with OX1R. (B) 3D
interaction diagram of 23 with OX1R. (C) 3D interaction diagram of 19
with OX1R. Hydrogen bond interactions is represented by green
bonds, hydrophobic interactions by pink bonds, p–sulfur interactions
by dark blue bonds, carbon–hydrogen interactions by red bonds, p–
cation interactions by dark yellow bonds, p–sigma interactions by
purple bonds, p–p stacked interactions by dark pink bonds, and
halogen (fluorine) interactions by light blue bonds.
Molecular docking
To identify the possible binding modes of our inhibitors,
molecular docking of compounds 23 and 19 and suvorexant was
performed to elucidate the key interactions within the active
sites of OX1R and OX2R. As shown in Fig. 1, the docking of
suvorexant into the binding site of OX1R indicates one key
hydrogen bonding interaction between the O in the amide
carbonyl and Asn318. In addition, a p–cation interaction interactions formed with Ala102, Gln179 and Pro123. A p–
between the substituted phenyl group and His344 was cation interaction between the substituted phenyl group and
observed. The docking of compound 23 into the binding site of His344 was also observed. Compound 19 showed stronger
OX1R indicates hydrogen bonding interaction between the O in OX1R inhibitory activity than did compound 23 and the other
the sulfonamide and Asn318 and a cation–p interaction azacycloheptane sulfonamide derivatives. The possible reason
between the 3-uoro-substituted phenyl group and His344. The is compound 19 have stronger hydrogen bond and more carbon
docking of compound 19 into the binding site of OX1R indi- hydrogen bonding interactions. As shown in Fig. 2, the docking
cates that a hydrogen bonding interaction between the O in the of suvorexant into the binding site of OX2R indicates one key
sulfonamide and Asn318 and three carbon hydrogen bonding hydrogen bond between the O in the amide and Asn324 and
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was absent. Compared to compound 23 and suvorexant,
compound 19 shows completely different docking behavior for
OX2R. The possible reason is that the sterically hindered of 2,3-
dihydrobenzo[b][1,4]dioxin-6-yl is larger than other substituent
groups. More importantly, the substituent and the benzene ring
are rigidly connected. In summary, the result of molecular
docking are in agreement with the experimental activity result
that the OX2R inhibitory activity of compound 23 (IC50 ¼ 0.17
mM) is comparable to that of suvorexant (IC50 ¼ 0.15 mM), but
the inhibitory activity of 23 (IC50 ¼ 0.63 mM) for OX1R was
slightly weaker than that of suvorexant (IC50 ¼ 0.14 mM).
Experimental section
General information
Reagents (Energy Chemical, Shanghai, China) were used
without further purication. Preparative TLC separations were
performed using silica gel GF254 (Qingdao Hailang Chemical,
Qingdao, China). NMR spectra were recorded on a Bruker
Av500M spectrometer and a Bruker ARX 600 MHz spectrometer
(Bruker, Zurich, Swiss), and TMS was used as the internal
standard. MS data were obtained on a Waters ACQUITY QDa.
HRMS data were obtained on a Bruker impact II O-TOF mass
spectrometry. If necessary, organic solvents were dried by
standard methods before use.
Chemistry
Starting compounds 1–3 were synthesized according to previ-
ously reported methods as given below.30,31
Synthesis of (R)-4-benzyl-7-methyl-1,4-diazepane-2,5-dione
(5). To a solution of methyl (R)-N-benzyl-N-(3-((tert-butox-
ycarbonyl)amino)butanoyl)glycinate (3) (20.00 g, 54.9 mmol) in
200 ml of ethyl acetate (180 ml) and methanol (20 ml) was slowly
introduced freshly prepared hydrochloric acid with stirring. The
reaction mixture was stirred at rt for 3 h. Ethyl acetate and
methanol were removed under vacuum. The residue was dis-
solved and then concentrated to dryness under vacuum 3 times.
The obtained residue was immediately taken on to the next
step. To a solution of the obtained residue in 160 ml of CH3OH
was added sodium methoxide (19.27 g, 356.7 mmol). The
reaction mixture was stirred at rt for 0.5 h. The reaction mixture
Fig. 2 (A) 3D interaction diagram of suvorexant with OX2R. (B) 3D
interaction diagram of 23 with OX2R. (C) 3D interaction diagram of 19
with OX2R. Hydrogen bond interactions is represented by green
bonds, hydrophobic interactions by pink bonds, p–sulfur interactions
by dark blue bonds, carbon hydrogen interactions by red bonds, p–
cation interactions by dark yellow bonds, p–sigma interactions by
purple bonds, p–p stacked interactions by dark pink bonds, and
halogen (fluorine) interactions by light blue bonds.
ꢀ
was cooled to 0–5 C and quenched with 320 ml of water. The
mixture was extracted with CH2Cl2, and the organic layers were
combined, washed with water and then bine, dried over MgSO4
and concentrated to provide compound 5 (10.7 g, white solid,
yield: 83.9%). MS (ESI+) m/z calculated for C13H16N2O2 [M + H]+
233.12, found 233.10.
a p–cation interaction between the substituted phenyl group
and His350. The docking of compound 23 into the binding site
of OX2R indicates one key hydrogen bond between the O atoms
in the sulfonamide and Asn324. Furthermore, a p–cation
interaction and p–sulfur interaction were also observed. The
inhibitory activity of compound 19 towards OX2R was weaker
than those of suvorexant and compound 23. The docking of
compound 19 into the binding site of OX2R showed that the key
hydrogen bond between the O in the sulfonamide and Asn324
Synthesis of (R)-1-benzyl-5-methyl-1,4-diazepane (6). A solu-
tion of (R)-4-benzyl-7-methyl-1,4-diazepane-2,5-dione (5) (4.20 g,
120.45 mmol) in THF (42 ml) was added dropwise to a solution
of LAH (34.85 ml, 87.11 mmol) in dry THF (21 ml) cooled to 0–
5 ꢀC. Aer stirring at rt overnight, the reaction was cooled to 0–
ꢀ
5 C and quenched with water (1.5 ml), then 15% NaOH solu-
tion (1.5 ml) followed by an additional 4.5 ml of water. The
mixture was dried with MgSO4 and stirred for 0.5 h. The ltrate
was concentrated under vacuum to provide 6 (3.1 g, light-yellow
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oil, yield: 83.9%). MS (ESI+): m/z calculated for [M + H]+ 205.16, 1H), 4.06–3.91 (m, 2H), 3.69–3.59 (m, 1H), 3.50 (dd, J ¼ 14.4,
found 205.12.
7.5 Hz, 1H), 3.32–3.22 (m, 1H), 2.30–2.20 (m, 1H), 1.75–1.67 (m,
General procedure for the preparation of compounds 7a–7i. 1H), 1.31 (d, J ¼ 18.8 Hz, 9H), 1.00 (d, J ¼ 6.4 Hz, 3H); 13C NMR
A mixture of 6 (1.00 g, 4.89 mmol), the appropriate substituted (151 MHz, DMSO-d6) d: 162.05, 156.16, 148.94, 143.73, 138.52,
benzenesulfonyl chloride derivative (4.16 mmol) and triethyl- 126.92, 126.52, 124.33, 120.59, 116.06, 109.26, 51.22, 48.82,
amine (9.78 mmol) in DCM was stirred at rt for 1 h. Then, the 43.80, 42.46, 35.21, 34.88, 31.16, 17.85; HRMS (ESI (+)) m/z
reaction mixture was washed with water and brine, dried over calculated for C23H29N3O3S [M + H]+: 428.2002, found 428.2001.
1
Na2SO4, ltered, and concentrated under vacuum to afford
12. Yield: 35.7%, white solid; H NMR (600 MHz, CDCl3) d:
compounds 7a–7i. The obtained compounds (7a–7i) were 7.38 (s, 1H), 7.36–7.29 (m, 2H), 7.26–7.22 (m, 1H), 7.18 (d, J ¼
immediately taken into the next step without purication. The 6.7 Hz, 1H), 7.04 (d, J ¼ 6.7 Hz, 1H), 6.89 (d, J ¼ 8.0 Hz, 1H), 4.23
structures of compounds 7a–7i were conrmed by MS.
(d, J ¼ 12.8 Hz, 4H), 4.12 (d, J ¼ 12.2 Hz, 1H), 4.04 (d, J ¼ 7.8 Hz,
General procedure for the preparation of compounds 8a–8i. 1H), 3.94 (d, J ¼ 15.0 Hz, 1H), 3.63 (d, J ¼ 12.0 Hz, 1H), 3.54 (s,
Compounds 7a–7i (4.89 mmol) were dissolved in methanol (10 1H), 3.32–3.19 (m, 1H), 2.28 (d, J ¼ 6.3 Hz, 1H), 1.76–1.64 (m,
ml). Aer adding a portion of 10% Pd/C (100 mg), the reaction 1H), 1.26 (s, 1H), 1.02 (d, J ¼ 5.8 Hz, 3H); 13C NMR (151 MHz,
was stirred for 24 h under a H2 atmosphere at rt. The reaction CDCl3) d: 161.19, 148.45, 147.25, 143.59, 133.75, 124.35, 121.01,
mixture was ltered, and the ltrate was concentrated under 120.54, 117.78, 116.44, 115.95, 108.93, 64.46, 64.14, 51.31,
vacuum to provide 8a–8i. The obtained compounds were 49.57, 44.99, 43.52, 35.52, 18.32; HRMS (ESI (+)) m/z calculated
immediately taken into the next step without purication. The for C21H23N3O5S [M + H]+: 430.1431, found 430.1430.
structures of compounds 8a–8i were conrmed by MS.
13. Yield: 40.8%, white solid; 1H NMR (500 MHz, CDCl3)
General procedure for the preparation of compounds 9–14. A d 7.83 (s, 2H), 7.34 (d, J ¼ 7.6 Hz, 1H), 7.30–7.21 (m, 1H), 7.14
mixture of compounds 8a, 8b–8f, or 8h (1.77 mmol), 2-chlor- (dt, J ¼ 15.5, 7.4 Hz, 3H), 7.02 (t, J ¼ 7.5 Hz, 1H), 4.24 (d, J ¼
obenzoxazole (1.42 mmol) and potassium carbonate (2.30 6.6 Hz, 1H), 4.12 (d, J ¼ 14.3 Hz, 1H), 4.06–3.88 (m, 2H), 3.64
mmol) was dissolved in DMF (7.5 ml). The mixture was stirred at (dd, J ¼ 13.3, 11.1 Hz, 1H), 3.52 (dd, J ¼ 14.5, 7.6 Hz, 1H), 3.33–
60 ꢀC for 3 h. The reaction was then quenched with water, and 3.23 (m, 1H), 2.36–2.20 (m, 1H), 1.73 (s, 1H), 1.00 (d, J ¼ 6.4 Hz,
the mixture was extracted with ethyl acetate. The organic layers 3H); 13C NMR (151 MHz, DMSO-d6) d: 165.47, 163.81, 162.02,
were combined and washed with water and then bine, dried 148.92, 143.67, 137.84, 130.06, 129.82, 126.98, 124.34, 120.64,
over MgSO4 and concentrated under vacuum. The residue was 117.00, 116.85, 116.06, 109.26, 51.50, 48.76, 44.04, 42.68, 34.84,
puried by preparative TLC and then recrystallized from 17.86; HRMS (ESI (+)) m/z calculated for C19H20FN3O3S [M + H]+:
petroleum ether (60–90) to afford 9–14.
390.1282, found 390.1281.
1
9. Yield: 42.5%, off-white solid; H NMR (500 MHz, CDCl3)
14. Yield: 45.3%, white solid; 1H NMR (500 MHz, CDCl3)
d 7.61 (d, J ¼ 7.7 Hz, 1H), 7.54 (d, J ¼ 7.4 Hz, 1H), 7.48–7.39 (m, d 7.74 (s, 2H), 7.37 (s, 1H), 7.29 (d, J ¼ 20.4 Hz, 3H), 7.21 (s, 1H),
1H), 7.34 (d, J ¼ 7.8 Hz, 1H), 7.26 (s, 1H), 7.22–7.12 (m, 2H), 7.02 7.06 (s, 1H), 4.30 (s, 1H), 4.13 (d, J ¼ 14.0 Hz, 1H), 4.01 (d, J ¼
(t, J ¼ 7.7 Hz, 1H), 4.27 (dd, J ¼ 13.4, 6.6 Hz, 1H), 4.13 (d, J ¼ 14.2 Hz, 2H), 3.66 (t, J ¼ 11.6 Hz, 1H), 3.53 (s, 1H), 3.30 (t, J ¼
14.7 Hz, 1H), 4.06–3.98 (m, 1H), 3.94 (d, J ¼ 15.1 Hz, 1H), 3.68– 12.0 Hz, 1H), 2.38 (s, 3H), 2.29 (s, 1H), 1.72 (s, 1H), 1.04 (s, 3H);
3.59 (m, 1H), 3.52 (dd, J ¼ 14.6, 7.7 Hz, 1H), 3.36–3.24 (m, 1H), 13C NMR (151 MHz, DMSO-d6) d: 162.01, 148.92, 143.71, 143.37,
2.37–2.23 (m, 1H), 1.73 (dd, J ¼ 15.1, 7.6 Hz, 1H), 1.01 (d, J ¼ 138.58, 130.19, 126.98, 124.31, 120.59, 116.04, 109.25, 51.27,
6.7 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) d: 163.10, 162.02, 48.62, 43.90, 42.43, 34.76, 21.29, 17.91; HRMS (ESI (+)) m/z
161.46, 148.93, 143.68, 132.19, 124.33, 123.23, 120.62, 120.24, calculated for C20H23N3O3S [M + H]+: 386.1533, found 386.1532.
116.07, 114.22, 114.06, 109.27, 51.69, 48.81, 44.03, 42.82, 34.87,
17.85; HRMS (ESI (+)) m/z calculated for C19H20FN3O3S [M + H]+: A mixture of compound 8a–8g, or 8i (1.77 mmol), 2,6-dichlor-
390.1282, found 390.1291. obenzoxazole (1.42 mmol) and potassium carbonate (2.30
General procedure for the preparation of compounds 15–22.
10. Yield: 48.2%, off-white solid; 1H NMR (500 MHz, CDCl3) mmol) was dissolved in DMF (7.5 ml). The mixture was stirred at
d 7.74 (d, J ¼ 8.6 Hz, 2H), 7.34 (d, J ¼ 7.7 Hz, 1H), 7.24 (d, J ¼ 60 ꢀC for 3 h. Then, the reaction was quenched with water, and
7.9 Hz, 1H), 7.16 (t, J ¼ 7.6 Hz, 1H), 7.01 (t, J ¼ 7.7 Hz, 1H), 6.89 the mixture was extracted with ethyl acetate. The organic layers
(d, J ¼ 8.6 Hz, 2H), 4.24 (dd, J ¼ 13.2, 6.5 Hz, 1H), 4.09 (d, J ¼ were combined, washed with water and then bine, dried over
14.3 Hz, 1H), 3.97 (t, J ¼ 18.4 Hz, 2H), 3.78 (s, 3H), 3.62 (t, J ¼ MgSO4 and concentrated under vacuum. The residue was
12.3 Hz, 1H), 3.50 (dd, J ¼ 14.4, 7.6 Hz, 1H), 3.31–3.19 (m, 1H), puried by preparative TLC and then recrystallized from
2.30–2.19 (m, 1H), 1.69 (d, J ¼ 5.1 Hz, 1H), 1.00 (d, J ¼ 6.6 Hz, petroleum ether (60–90) to afford 15–22.
3H); 13C NMR (151 MHz, DMSO-d6) d: 162.72, 162.06, 148.93,
15. Yield: 41.1%, white solid; 1H NMR (500 MHz, CDCl3)
143.73, 133.09, 129.20, 124.32, 120.59, 116.04, 114.86, 109.25, d 7.61 (d, J ¼ 7.6 Hz, 1H), 7.54 (d, J ¼ 7.9 Hz, 1H), 7.44 (dt, J ¼
56.00, 51.16, 48.73, 43.94, 42.40, 34.86, 17.88; HRMS (ESI (+)) m/ 13.2, 6.7 Hz, 1H), 7.32–7.18 (m, 3H), 7.14 (d, J ¼ 8.3 Hz, 1H), 4.27
z calculated for C20H23N3O4S [M + H]+: 402.1482, found (dd, J ¼ 13.2, 6.6 Hz, 1H), 4.09 (d, J ¼ 14.3 Hz, 1H), 4.03–3.90 (m,
402.1481.
2H), 3.64 (dd, J ¼ 13.5, 11.1 Hz, 1H), 3.52 (dd, J ¼ 14.5, 7.6 Hz,
11. Yield: 31.9%, white solid; 1H NMR (500 MHz, CDCl3) 1H), 3.35–3.23 (m, 1H), 2.28 (dd, J ¼ 14.6, 6.8 Hz, 1H), 1.73 (dd, J
d 7.74 (d, J ¼ 7.8 Hz, 2H), 7.45 (d, J ¼ 7.6 Hz, 2H), 7.34 (d, J ¼ ¼ 15.2, 7.6 Hz, 1H), 0.99 (t, J ¼ 13.2 Hz, 3H); 13C NMR (151 MHz,
7.7 Hz, 1H), 7.26 (d, J ¼ 6.4 Hz, 1H), 7.16 (t, J ¼ 7.6 Hz, 1H), 7.01 DMSO-d6) d: 163.10, 162.54, 161.45, 149.20, 143.47, 142.85,
(t, J ¼ 7.7 Hz, 1H), 4.25 (d, J ¼ 6.7 Hz, 1H), 4.13 (d, J ¼ 14.2 Hz, 132.19, 124.42, 123.22, 120.31, 120.17, 116.68, 114.20, 114.04,
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109.94, 51.65, 48.79, 44.07, 42.60, 34.71, 17.82; HRMS (ESI (+))
21. Yield: 46.6%, light-yellow solid; 1H NMR (500 MHz,
m/z calculated for C19H19ClFN3O3S [M + H]+: 424.0892, found CDCl3) d 7.93 (t, J ¼ 7.4 Hz, 1H), 7.53 (d, J ¼ 5.1 Hz, 1H), 7.33–
424.0885.
7.20 (m, 3H), 7.15 (dd, J ¼ 10.3, 8.6 Hz, 2H), 4.22 (dd, J ¼ 13.3,
16. Yield: 39.8%, white solid; 1H NMR (500 MHz, CDCl3) 6.5 Hz, 1H), 4.18–3.97 (m, 3H), 3.60 (t, J ¼ 10.8 Hz, 2H), 3.46–
d 7.74 (d, J ¼ 8.7 Hz, 2H), 7.22 (d, J ¼ 14.8 Hz, 2H), 7.14 (d, J ¼ 3.33 (m, 1H), 2.39–2.28 (m, 1H), 1.75 (s, 1H), 0.96 (d, J ¼ 6.2 Hz,
8.3 Hz, 1H), 6.89 (d, J ¼ 8.7 Hz, 2H), 4.30–4.19 (m, 1H), 4.05 (d, J 3H); 13C NMR (151 MHz, DMSO-d6) d: 162.59, 159.20, 157.53,
¼ 14.3 Hz, 1H), 3.95 (d, J ¼ 14.7 Hz, 2H), 3.78 (d, J ¼ 15.2 Hz, 149.23, 142.88, 136.08, 130.58, 128.98, 125.58, 124.43, 117.89,
3H), 3.62 (t, J ¼ 12.4 Hz, 1H), 3.51 (dd, J ¼ 14.5, 7.7 Hz, 1H), 117.75, 116.69, 109.97, 51.62, 49.21, 44.26, 42.83, 34.88, 17.64;
3.30–3.20 (m, 1H), 2.31–2.19 (m, 1H), 1.66 (dd, J ¼ 16.0, 8.4 Hz, HRMS (ESI (+)) m/z calculated for C19H19ClFN3O3S [M + H]+:
1H), 1.01 (d, J ¼ 6.6 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) d: 424.0892, found 424.0891.
162.70, 162.56, 149.19, 142.88, 133.09, 129.17, 124.40, 116.62,
22. Yield: 30.5%, off-white solid; 1H NMR (600 MHz, CDCl3)
114.84, 109.93, 55.97, 51.12, 48.63, 43.95, 42.15, 34.67, 17.90; d: 7.73 (d, J ¼ 7.2 Hz, 2H), 7.58 (d, J ¼ 7.2 Hz, 2H), 7.24 (s, 2H),
HRMS (ESI (+)) m/z calculated for C20H22ClN3O4S [M + H]+: 7.15 (d, J ¼ 7.6 Hz, 1H), 4.28 (d, J ¼ 6.1 Hz, 1H), 4.01 (d, J ¼
436.1092, found 436.1091.
13.4 Hz, 1H), 3.94 (t, J ¼ 16.0 Hz, 2H), 3.66 (d, J ¼ 12.2 Hz, 1H),
17. Yield: 37.3%, white solid; 1H NMR (500 MHz, CDCl3) 3.55 (s, 1H), 3.32–3.20 (m, 1H), 2.32–2.10 (m, 5H), 1.73–1.63 (m,
d 7.82 (d, J ¼ 7.2 Hz, 2H), 7.49 (dd, J ¼ 22.5, 7.3 Hz, 3H), 7.26– 1H), 1.02 (d, J ¼ 5.8 Hz, 3H); 13C NMR (151 MHz, CDCl3) d:
7.18 (m, 2H), 7.14 (d, J ¼ 8.2 Hz, 1H), 4.34–4.19 (m, 1H), 4.09 (d, J 168.60, 148.67, 141.92, 135.81, 128.01, 124.57, 119.29, 116.25,
¼ 14.2 Hz, 1H), 3.98 (t, J ¼ 14.5 Hz, 2H), 3.62 (t, J ¼ 11.6 Hz, 1H), 109.74, 51.29, 49.22, 44.85, 43.10, 35.19, 24.64, 18.27; HRMS
3.50 (dd, J ¼ 12.1, 7.6 Hz, 1H), 3.32–3.20 (m, 1H), 2.35–2.19 (m, (ESI (+)) m/z calculated for C21H23ClN4O4S [M + H]+: 463.1201,
1H), 1.78–1.66 (m, 1H), 0.98 (d, J ¼ 6.6 Hz, 3H); 13C NMR (151 found 463.1196.
MHz, DMSO-d6) d: 162.57, 149.22, 142.88, 141.40, 133.11,
General procedure for the preparation of compounds 23–29.
129.82, 126.97, 124.42, 116.67, 109.96, 51.41, 48.92, 44.11, A mixture of compound 8a–8c, 8d–8g, or 8i (1.77 mmol), 2,5-
42.55, 34.82, 17.75; HRMS (ESI (+)) m/z calculated for C19H20- dichlorobenzoxazole (1.42 mmol) and potassium carbonate
ClN3O3S [M + H]+: 406.0987, found 406.0985.
(2.30 mmol) ꢀwas dissolved in DMF (7.5 ml). The mixture was
1
18. Yield: 49.3%, white solid; H NMR (500 MHz, CDCl3) d: stirred at 60 C for 3 h. Then, the reaction was quenched with
7.73 (d, J ¼ 7.7 Hz, 2H), 7.46 (d, J ¼ 7.7 Hz, 2H), 7.24 (dd, J ¼ water, and the mixture was extracted with ethyl acetate. The
18.9, 9.8 Hz, 2H), 7.13 (d, J ¼ 8.3 Hz, 1H), 4.25 (d, J ¼ 6.6 Hz, 1H), organic layers were combined, washed with water and then
4.09 (d, J ¼ 14.4 Hz, 1H), 3.97 (t, J ¼ 18.7 Hz, 2H), 3.69–3.59 (m, bine, dried over MgSO4 and concentrated under vacuum. The
1H), 3.51 (dd, J ¼ 14.4, 7.6 Hz, 1H), 3.31–3.20 (m, 1H), 2.31–2.20 residue was puried by preparative TLC and then recrystallized
(m, 1H), 1.69 (s, 1H), 1.32 (d, J ¼ 18.2 Hz, 9H), 1.00 (d, J ¼ 6.5 Hz, from petroleum ether (60–90) to afford 23–29.
3H); 13C NMR (151 MHz, DMSO-d6) d: 162.57, 156.16, 149.22,
23. Yield: 38.7%, white solid; 1H NMR (500 MHz, CDCl3)
142.90, 138.51, 126.90, 126.50, 124.43, 116.66, 109.96, 51.14, d 7.61 (d, J ¼ 7.6 Hz, 1H), 7.54 (d, J ¼ 6.9 Hz, 1H), 7.48–7.39 (m,
48.77, 43.79, 42.20, 35.19, 34.71, 31.13, 17.84; HRMS (ESI (+)) m/ 1H), 7.28 (d, J ¼ 12.8 Hz, 1H), 7.21 (t, J ¼ 8.2 Hz, 1H), 7.13 (d, J ¼
z calculated for C23H28ClN3O3S [M + H]+: 462.1613, found 8.4 Hz, 1H), 6.98 (d, J ¼ 8.4 Hz, 1H), 4.27 (d, J ¼ 6.6 Hz, 1H), 4.10
462.1611.
(d, J ¼ 14.3 Hz, 1H), 3.97 (dd, J ¼ 28.0, 16.2 Hz, 2H), 3.70–3.61
19. Yield: 29.3%, off-white solid; 1H NMR (600 MHz, CDCl3) (m, 1H), 3.53 (dd, J ¼ 14.3, 7.4 Hz, 1H), 3.34–3.22 (m, 1H), 2.37–
d: 7.36–7.28 (m, 2H), 7.24 (d, J ¼ 14.3 Hz, 2H), 7.15 (d, J ¼ 7.7 Hz, 2.25 (m, 1H), 1.79–1.67 (m, 1H), 1.01 (d, J ¼ 6.5 Hz, 3H); 13C
1H), 6.89 (d, J ¼ 8.2 Hz, 1H), 4.24 (d, J ¼ 14.0 Hz, 4H), 4.07 (d, J ¼ NMR (151 MHz, DMSO-d6) d: 163.07, 161.45, 147.74, 145.32,
13.7 Hz, 1H), 3.97 (dd, J ¼ 33.5, 12.2 Hz, 2H), 3.64 (t, J ¼ 11.4 Hz, 143.47, 132.19, 128.55, 123.22, 120.31, 120.17, 115.72, 114.20,
1H), 3.52 (t, J ¼ 20.7 Hz, 1H), 3.30–3.19 (m, 1H), 2.33–2.20 (m, 114.04, 110.32, 51.64, 48.74, 44.03, 42.56, 34.69, 17.81; HRMS
1H), 1.73–1.64 (m, 1H), 1.26 (s, 1H), 1.00 (t, J ¼ 16.6 Hz, 3H); 13
C
(ESI (+)) m/z calculated for C19H19ClN3O3S [M + H]+: 424.0892,
NMR (151 MHz, CDCl3) d: 161.86, 148.80, 147.25, 143.58, found 424.0891.
133.73, 125.86, 124.47, 120.55, 117.78, 116.37, 109.67, 64.46,
24. Yield: 44.7%, white solid; 1H NMR (500 MHz, CDCl3)
64.14, 51.25, 49.49, 44.88, 43.41, 35.47, 18.29; HRMS (ESI (+)) m/ d 7.74 (d, J ¼ 7.4 Hz, 2H), 7.28 (s, 1H), 7.13 (d, J ¼ 8.3 Hz, 1H),
z calculated for C21H22ClN3O5S [M + H]+: 464.1041, found 6.97 (d, J ¼ 8.2 Hz, 1H), 6.89 (d, J ¼ 7.4 Hz, 2H), 4.25 (d, J ¼
464.1037.
6.5 Hz, 1H), 4.05 (d, J ¼ 14.5 Hz, 1H), 3.95 (d, J ¼ 14.8 Hz, 2H),
1
20. Yield: 38.9%, white solid; H NMR (500 MHz, CDCl3) d: 3.80 (s, 3H), 3.63 (dd, J ¼ 13.5, 11.1 Hz, 1H), 3.51 (dd, J ¼ 14.3,
7.84 (d, J ¼ 5.0 Hz, 2H), 7.24 (dd, J ¼ 18.2, 9.3 Hz, 2H), 7.13 (t, J ¼ 7.7 Hz, 1H), 3.30–3.19 (m, 1H), 2.32–2.17 (m, 1H), 1.73 (s, 1H),
9.4 Hz, 3H), 4.25 (dd, J ¼ 13.0, 6.4 Hz, 1H), 4.09 (d, J ¼ 14.3 Hz, 1.01 (d, J ¼ 6.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) d: 163.06,
1H), 4.03–3.90 (m, 2H), 3.64 (dd, J ¼ 13.4, 11.0 Hz, 1H), 3.52 (dd, 162.73, 147.73, 145.36, 133.08, 129.17, 128.55, 120.13, 115.67,
J ¼ 14.5, 7.6 Hz, 1H), 3.31–3.21 (m, 1H), 2.34–2.23 (m, 1H), 1.71 114.84, 110.29, 55.97, 51.11, 48.62, 43.93, 42.14, 36.23, 34.66,
(d, J ¼ 7.8 Hz, 1H), 0.99 (d, J ¼ 6.5 Hz, 3H); 13C NMR (151 MHz, 31.25, 17.87; HRMS (ESI (+)) m/z calculated for C20H22ClN3O4S
DMSO-d6) d: 165.47, 163.80, 162.53, 149.18, 142.84, 137.82, [M + H]+: 436.1092, found 436.1091.
130.04, 126.97, 124.43, 117.00, 116.85, 116.66, 109.94, 51.46,
25. Yield: 45.0%, white solid; 1H NMR (500 MHz, CDCl3)
48.71, 44.06, 42.45, 34.67, 17.86; HRMS (ESI (+)) m/z calculated d 7.82 (d, J ¼ 7.8 Hz, 2H), 7.59–7.39 (m, 3H), 7.28 (s, 1H), 7.13 (d,
for C19H19ClFN3O3S [M + H]+: 424.0892, found 424.0892.
J ¼ 8.4 Hz, 1H), 6.97 (d, J ¼ 8.4 Hz, 1H), 4.34–4.21 (m, 1H), 4.09
(d, J ¼ 14.4 Hz, 1H), 3.98 (t, J ¼ 15.5 Hz, 2H), 3.67–3.58 (m, 1H),
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3.50 (dd, J ¼ 14.5, 7.7 Hz, 1H), 3.33–3.20 (m, 1H), 2.37–2.21 (m, serum, penicillin–streptomycin, HBSS (calcium and magne-
1H), 1.74–1.68 (m, 1H), 1.00 (dd, J ¼ 19.0, 6.7 Hz, 3H); 13C NMR sium with no phenol red) and HEPES were obtained from
(151 MHz, DMSO-d6) d: 163.06, 147.75, 145.36, 141.39, 133.12, Gibco, Ham's F-12K was obtained from HyClone, 1ꢂ PBS (pH
129.82, 128.55, 126.97, 120.16, 115.71, 110.33, 51.40, 48.87, 7.2–7.4) was obtained from Solarbio, 1ꢂ TrypLE express enzyme
44.07, 42.51, 34.80, 17.74; HRMS (ESI (+)) m/z calculated for (no phenol red) was obtained from Thermo Fisher Scientic,
C
19H20ClN3O3S [M + H]+: 406.0987, found 406.0985.
and hygromycin B gold solution was obtained from Invivogen.
1
26. Yield: 35.1%, white solid; H NMR (600 MHz, CDCl3) d: The seeded plates were incubated overnight at 37 ꢀC under 5%
7.73 (d, J ¼ 8.3 Hz, 2H), 7.45 (d, J ¼ 7.4 Hz, 2H), 7.28 (d, J ¼ CO2. Human orexin-A stock solution and test compound stock
12.7 Hz, 2H), 7.13 (dd, J ¼ 8.4, 1.3 Hz, 1H), 7.01–6.92 (m, 1H), (30 nM) solutions were prepared. Human orexin-A solution was
4.30–4.19 (m, 1H), 4.09 (d, J ¼ 13.8 Hz, 1H), 3.96 (t, J ¼ 15.0 Hz, prepared by diluting the stock solution in assay buffer (1ꢂ HBSS
2H), 3.70–3.60 (m, 1H), 3.52 (dd, J ¼ 11.8, 6.0 Hz, 1H), 3.31–3.20 and 20 mM HEPES) to a nal concentration of 8 nM for use in
(m, 1H), 2.31–2.16 (m, 1H), 1.73–1.63 (m, 1H), 1.31 (d, J ¼ the assay. The test compound solutions were prepared by
17.1 Hz, 9H), 1.00 (d, J ¼ 6.7 Hz, 3H); 13C NMR (151 MHz, CDCl3) diluting the stock solution. Then, the test compound solutions,
d: 162.51, 156.45, 147.42, 144.35, 138.13, 129.47, 126.71, 126.09, DMSO and assay buffer were added into 384-well plates in
120.40, 116.25, 109.24, 51.29, 49.59, 44.79, 43.59, 35.52, 35.07, order. The cells were washed with assay buffer and then incu-
31.02, 18.21; HRMS (ESI (+)) m/z calculated for C23H28ClN3O3S bated for 120 min at 37 ꢀC in 40 ml of assay buffer containing 1ꢂ
[M + H]+: 462.1613, found 462.1609.
component A (calcium probe) and 2.5 mM of probenecid on the
27. Yield: 33.2%, off-white solid; 1H NMR (600 MHz, CDCl3) day of the assay. Test compound solutions (10 ml) were added to
d: 7.31 (t, J ¼ 7.5 Hz, 3H), 7.14 (d, J ¼ 8.2 Hz, 1H), 6.98 (d, J ¼ the plate and incubated for 30 min at rt. Then, 10 ml of the
6.8 Hz, 1H), 6.89 (d, J ¼ 8.3 Hz, 1H), 4.24 (d, J ¼ 14.8 Hz, 5H), agonist was added. The intensity of the uorescence was
4.07 (d, J ¼ 14.0 Hz, 1H), 4.03–3.90 (m, 2H), 3.64 (t, J ¼ 11.5 Hz, measured for each well at 1 s intervals for 2 min and compared
1H), 3.54 (s, 1H), 3.30–3.20 (m, 1H), 1.73–1.64 (m, 1H), 1.26 (s, with the intensity of the uorescence induced by orexin-A (8
1H), 1.02 (d, J ¼ 6.1 Hz, 3H); 13C NMR (151 MHz, CDCl3) d: nM) instead of the antagonist. The IC50 value for each antago-
162.36, 147.29, 143.98, 143.59, 133.73, 129.55, 120.55, 117.77, nist was determined.
116.43, 116.21, 109.30, 77.21, 77.00, 76.79, 64.46, 64.14, 51.25,
49.45, 44.84, 43.41, 35.45, 18.27; HRMS (ESI (+)) m/z calculated
Docking
for C21H22ClN3O5S [M + H]+: 464.1041, found 464.1040.
1
DS soware (www.3dsbiovia.com) was used for the docking
studies. All the ligand structures were prepared by Gauss View
5.0 program. All the ligands were optimized using the Gaussian
09 program (www.gaussian.com) using the density functional
theory (DFT) method at the B3LYP/6-311G (d,p) level. For
protein preparation, the crystal structure coordinates of the
human orexin receptor were obtained from the reference and
28. Yield: 47.1%, white solid; H NMR (500 MHz, CDCl3) d:
7.93 (t, J ¼ 7.3 Hz, 1H), 7.53 (d, J ¼ 4.8 Hz, 1H), 7.35–7.22 (m,
2H), 7.15 (dd, J ¼ 11.6, 8.3 Hz, 2H), 6.97 (d, J ¼ 8.4 Hz, 1H), 4.29–
4.17 (m, 1H), 4.17–3.97 (m, 3H), 3.61 (t, J ¼ 10.7 Hz, 2H), 3.47–
3.33 (m, 1H), 2.40–2.26 (m, 1H), 1.73 (dd, J ¼ 15.1, 7.5 Hz, 1H),
0.96 (d, J ¼ 6.5 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) d: 163.09,
159.20, 157.52, 147.77, 145.36, 136.08, 130.58, 128.98, 128.56,
then prepared with AutoDockTools 4.2 and PyMOL. The
125.58, 120.17, 117.89, 117.75, 115.73, 110.34, 51.60, 49.17,
binding site coordinates of the receptors were determined by
44.22, 42.79, 34.86, 17.64; HRMS (ESI (+)) m/z calculated for
the size of the ligand in the PDB le (PDB 6TO7 and 6TPJ).32
Docking simulations were performed using CDOCKER (for
GABAAR) and LibDock (for CYP11B1) protocol in DS to predict
how the ligands bind to OX1R and OX2R. The co-crystallized
ligand (suvorexant) within the structures were dened as
a center of the binding site. Generally, ten poses were generated
for each docked ligand. Other docking options were kept as
default. To validate the docking reliability, suvorexant was rst
re-docked to the binding site. Consequently, all ligands were
docked into the same active site.
C
19H19ClFN3O3S [M + H]+: 424.0892, found 424.0892.
29. Yield: 22.6%, off-white solid; 1H NMR (600 MHz, CDCl3)
d: 7.72 (d, J ¼ 7.6 Hz, 2H), 7.66 (s, 1H), 7.58 (d, J ¼ 7.3 Hz, 2H),
7.14 (d, J ¼ 8.3 Hz, 1H), 6.99 (d, J ¼ 6.6 Hz, 1H), 4.28 (d, J ¼
5.8 Hz, 1H), 4.05–3.86 (m, 3H), 3.68 (t, J ¼ 10.7 Hz, 1H), 3.56 (d, J
¼ 7.0 Hz, 1H), 3.31–3.20 (m, 1H), 2.30–2.12 (m, 5H), 1.75–1.63
(m, 1H), 1.02 (d, J ¼ 5.7 Hz, 3H); 13C NMR (151 MHz, CDCl3) d:
168.63, 161.95, 147.03, 141.95, 135.75, 129.74, 127.97, 120.87,
119.30, 115.99, 109.54, 51.25, 49.18, 44.83, 42.97, 35.10, 24.65,
18.26, 14.17; HRMS (ESI (+)) m/z calculated for C21H23ClN4O4S
[M + H]+: 463.1201, found 463.1200.
Conclusions
Biological activity
In this study, twenty one novel azacycloheptane sulfonamide
For intracellular calcium measurements, Flp-In-CHO-OX1 and derivatives were designed and synthesized, and all the
Flp-In-CHO-OX2 stable cells were grown in Ham's F-12K compounds were evaluated as potential orexin receptor antag-
(Kaighn's modied Ham's F-12 Nutrient Mixture), 600 mg mlꢁ1 onists. Surprisingly, compound 23 showed high inhibitory
hygromycin B, 10% fetal bovine serum and 1ꢂ penicillin– activity against OX1R and OX2R, and the IC50 values of
streptomycin (PS). The OX1R and OX2R cells were seeded into compound 23 against OX1R and OX2R were 0.63 mM and 0.17
Corning black 384-well clear-bottom sterile plates at 6500 cells mM, respectively. Moreover, compounds 19 and 24 exhibited
per well and 7000 cells per well, respectively. Fetal bovine potent inhibitory activities against OX1R in vitro. The binding
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modes of suvorexant and compound 23 to OX2R indicated that
the amino acid residues Asn324 and His350 were important for
D. Smart, F. Jewitt, P. Jeffrey and N. Austin, Bioorg. Med.
Chem. Lett., 2001, 11, 1907–1910.
ligand binding via hydrogen bonding interactions, p–p stack- 15 C. J. Langmead, J. C. Jerman, S. J. Brough, C. Scott,
ing interactions, or p–s interactions, which could explain the
SAR of these compounds and is consistent with the active data.
R. A. Porter and H. J. Herdon, Br. J. Pharmacol., 2004, 141,
340–346.
Hence, azacycloheptane sulfonamide derivatives are promising 16 M. A. Steiner, J. Gateld, C. Brisbare-Roch, H. Dietrich,
scaffolds for the discovery of novel OX1R and OX2R antagonists.
A. Treiber, F. Jenck and C. Boss, ChemMedChem, 2013, 8,
898–903.
17 P. Malherbe, E. Borroni, L. Gobbi, H. Knust, M. Nettekoven,
E. Pinard, O. Roche, M. Rogers-Evans, J. Wettstein and
J.-L. Moreau, Br. J. Pharmacol., 2009, 156, 1326–1341.
18 B. A. Kummangal, D. Kumar and H. N. Mallick, Behav. Brain
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Conflicts of interest
There are no conicts to declare.
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