Full text of DOI:10.1039/c8ra01972j

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4-Hydroxybenzyl alcohol derivatives and their
sedative–hypnotic activities
Cite this: RSC Adv., 2018, 8, 19539
a
Hong-yan Zhu,a Di Zhang,a Qi Zhang,b Yan Zhao,
Zhong-mei He,a
*
a
Yu-gang Gao and Lian-xue Zhanga
*
4-Hydroxybenzyl alcohol (HBA), one of the characteristic active components of Gastrodia elata, exhibits
obvious effects on the human central nervous system. In order to acquire compounds with superior
bioactivity, 10 derivatives of HBA were synthesized from HBA and carboxylic acids. The sedative effects
of the 10 HBA derivatives were evaluated using a spontaneous locomotor activity test (SLT) in mice, and
their hypnotic effects were determined to be synergistic with pentobarbital-induced sleep. The results
showed that 4-hydroxybenzyl alcohol 3-furancarboxylic acid diester (2FHBA, 10 mg kgꢀ1) exhibited the
strongest sedative–hypnotic activity among HBA and its derivatives, and 2FHBA could reverse the
insomnia caused by p-chlorophenylalanine (pCPA), flumazenil (FLU) and thiosemicarbazide (TSC).
Meanwhile, 2FHBA and 5-hydroxytryptophan (5-HTP) showed a synergistic effect. The results suggested
that 2FHBA might be a potential agent against insomnia, which might be mediated by the serotonergic
and GABAergic systems.
Received 6th March 2018
Accepted 4th May 2018
DOI: 10.1039/c8ra01972j
rsc.li/rsc-advances
Hydroxybenzyl alcohol (HBA), also named as gastrodigenin, is
the characteristic and main active component of G. elata. Gas-
1. Introduction
trodin (GAS) is another chief active ingredient of G. elata, and
studies have also reported that GAS is metabolized to HBA in
the body and then plays pharmacological roles.21 Meanwhile,
HBA was chosen as one of the standard compounds to evaluate
the quality of G. elata.22 Modern pharmacological experiments
have demonstrated that HBA apparently possesses the ability to
adjust the central nervous system, including sedation,23 anti-
convulsion,23 anti-depressant,24 and anti-neuroinammation
activities,25 facilitating memory and neuroprotection,26,27 and
it is oen used as a precursor compound through structural
modication to discover more active compounds. It has been
reported that the derivatives of HBA substituted with amino
acids, phosphate and sulfonate display excellent hep-
atoprotective, anti-cancer and inhibition of rat testicular
P-45017a activities, respectively.28–30
Carboxylic acids are usually selected to be synthesized with
compounds with hydroxyl groups to obtain better biological
activity esters. Some bioactive compounds have been synthe-
sized by our team in recent years, like ergosteryl 2-naphthoate31
and arctigenin b-indolylacetate,32 which showed excellent anti-
tumor effects. The aim of this study was to nd new
compounds with potentially sedative and hypnotic activities by
synthesizing derivatives of HBA with carboxylic acids. Behav-
ioral pharmacological tests, including the spontaneous loco-
motor activity test and the pentobarbital-induced sleep test,
were employed to evaluate their biological activities. Further-
more, the possible mechanism was also devised.
Today, almost half of human beings suffer from sleeping
disturbance all over the world, not only old people but also
young people, due to the excessive stress in their lives.1 Diffi-
culty getting to sleep, easily waking up and poor sleep quality
are all symptoms associated with insomnia, which appears to
be very prevalent and seriously threatens human health. If
there is no effective control, insomnia will develop into
a major depressive disorder. Therefore, it is necessary to
search for potent medicines with effective sedative and
hypnotic activities. Natural products have become an impor-
tant source of bioactive lead compounds for drug discovery,
especially their derivatives through synthetic transformation
or structural modication, which has occupied a leading
position recently.2
Gastrodia elata (G. elata), recorded as the stem tubers of G.
elata Blume in Chinese pharmacopoeia, has been found to
possess a wide range of biological activities, including sedative
and hypnotic,3,4 antiepileptic and anticonvulsive,5,6 anti-anxiety
and antidepressant,7,8 neuroprotective,9 anti-cardio-cerebral-
vascular disease,10 antipsychotic,11 anti-vertigo,12 anticoagulant
and antithrombotic,13 anti-atherosclerotic,14 antihypertensive,15
anti-inammatory and analgesic,16,17 memory improving and
anti-aging,18 antivirus19 and antitumor20 activities and so on. 4-
aCollege of Chinese Medicinal Materials, Jilin Agricultural University, Changchun
130118, Jilin, China. E-mail: zhaoyan@jlau.edu.cn; gaoyugang_2006@163.com;
Fax: +86 431 84533358; Tel: +86 431 84533358
bThe Fiieth Middle School of Daqing, Daqing 163000, China
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(Gangdong Sci. & Tech. Development Co., Ltd. Tianjin, China).
NMR spectra were recorded on a Varian Mercury 300 MHz NMR
spectrometer equipped with an Oxford Instruments Ltd.
superconducting magnet (Palo Alto, CA, USA). The HRMS was
measured using a Q-Exactive Electrospray Orbitrap High Reso-
lution Mass Spectrometer (Thermo-Fisher, USA). Melting point
analysis was performed in an X-4 micro melting point apparatus
(Tech Instrument Co., Ltd., Beijing, China). Specic conclu-
sions are described in detail in the Results section.
2. Materials and method
2.1. Animals
Mice weighing 18–22 g were supplied by Changchun Yisi
Experimental Animal Technology Co. Ltd. The animals were
kept under standard laboratory conditions (temperature 25 ꢁ
2 ꢂC, relative humidity 60 ꢁ 5%, 12 h light/12 h dark cycle with
light on at 7:00 a.m.) with food and tap water available ad libi-
tum for the duration of the study. Aer 1 week of acclimatiza-
tion, all mice were randomly divided into different groups. All
tests were performed in accordance with the Guide for Animal
Experimentation of Jilin Agricultural University. The protocol
was approved by the Jilin Agricultural University Institutional
Animal Care and Use Committee. Every effort was made to
minimize the number of animals used and any pain and
discomfort experienced by the subjects.
2.5. Treatment
Ten derivatives of HBA and DZP were blended with 0.05%
sodium carboxymethyl cellulose (CMC-Na)/physiological saline
and administered 30 minutes prior to the locomotor activity test
or pentobarbital administration. Sodium pentobarbital was
dissolved in physiological saline, using 28 mg kgꢀ1 (intraperi-
toneally, i.p.) as the sub-hypnotic dose and 42 mg kgꢀ1 (i.p.) as
the hypnotic dose, which could generate a 100% rate of sleep
onset. pCPA (100 mg kgꢀ1, i.p.) was administered continuously
for 4 days to obtain the mouse insomnia model. Aer the
insomnia model was successfully established, 2FHBA was
administered for observation. TSC (8 mg kgꢀ1, i.p.), diluted in
0.9% physiological saline, was injected 30 min before the
administration of 2FHBA. 5-HTP (2.5 mg kgꢀ1, i.p.) also diluted
in 0.9% physiological saline was given 15 min prior to pento-
barbital administration. FLU injection was also administrated
15 min prior to the administration of pentobarbital to obtain an
insomnia model. All the drug solutions were freshly prepared
before use.
2.2. Drugs and reagents
HBA, 3-furoic acid, 2-naphthoic acid, (Z)-9-octadecanoic acid
(oleic acid), 2,4-hexadienoic acid (sorbic acid), octadecadienoic
acid (linoleic acid), dodecanoic acid (lauric acid) and benzoic
acid were purchased from Sinopharm Chemical Reagent Co.
Ltd. (Shanghai, China). 4-Dimethylaminopyridine (DMAP) and
1-ethylethyl-3-(3-dimethyllaminopropyl) carbodiimide hydro-
chloride (EDCI) were purchased from Civi Chem Co. Ltd
(Shanghai, China). The HBA derivatives were synthesized by us.
Dichloromethane was acquired from Damao Chem Co. Ltd.
(Tianjin, China). Diazepam (DZP) was provided by Zhenxing
Pharma Co. Ltd. (Taiyuan, China). Sodium pentobarbital was
purchased from Merck Co. Ltd. (Shanghai, China). The uma-
zenil (FLU) injection was produced by Lingkang Co. Ltd.
(Hainan, China). p-Chlorophenylalanine (pCPA), thio-
semicarbazide (TSC) and 5-hydroxytryptophan (5-HTP) were all
purchased from Melone Pharma Co. Ltd. (Dalian, China). All
the reagents used in this study were of analytical grade.
2.6. Spontaneous locomotor activity test (SLT)
The sedative effects of HBA and its derivatives were assessed by
detecting the spontaneous locomotor activity of mice. Loco-
motor activity and standing numbers could be calculated using
the ZZ-6 locomotor activity tester (Taimeng Soware Co. Ltd.,
Chengdu, China), which consisted of a microcomputer vehicle
system and six separable reaction jars that possessed 36 points
of infrared array probes. 30 min aer oral administration of
HBA and its derivatives and DZP respectively, mice were placed
separately in the locomotor activity tester. Aer adapting for
5 min, the locomotor activity of each mouse was recorded for
5 min. Aer each testing session, the enclosure was thoroughly
cleaned with 70% ethanol.
2.3. Synthesis
The experimental procedure is described taking 4-hydrox-
ybenzyl alcohol 3-furoate (FHBA) and 4-hydroxybenzyl alcohol 3-
furancarboxylic acid diester (2FHBA) as an example. 3-Fur-
ancarboxylic acid (6 mmol) and EDCI (8 mmol) were dissolved
in 10 mL of dichloromethane and stirred for 10 minutes; then,
10 mL of dichloromethane solution containing HBA (4 mmol)
and DMAP (4.8 mmol) was added to the mixture. Aer disso-
lution, the mixture was heated to reux at 60 ꢂC for 6 hours and
the solvent was removed under reduced pressure. The residue
was puried by silica gel chromatography and eluted with
petroleum ether/acetone (6 : 1, v/v) to yield the product 2FHBA
and FHBA. The synthesis of the other derivatives was similar to
that of FHBA and 2FHBA.
2.7. Pentobarbital-induced sleep test
Experiments were carried out between 8:00 a.m. and 12:00 a.m.
Following sodium pentobarbital injection, each mouse was
observed for the onset of sleep. When the mice lost their
righting reex for over 60 s, they were considered to be asleep.
The loss of the righting reex was dened as a failure of the
mouse to right itself for at least 15 s aer being placed on its
back. Sleep latency was recorded as the time of pentobarbital
2.4. Structural identication
The structures of the HBA derivatives were determined by injection to the time of sleep onset, and the sleep duration was
IR, NMR and MS analysis. FTIR analysis was performed on dened as the elapsed time between the righting reex loss and
a
WGH-30/30A double beam infrared spectrophotometer recovery.
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activated carboxylic acid reacts with HBA to afford the deriva-
tives. The products were isolated using a silica gel column. IR
and NMR were used to identify the molecular structure of the
newly synthesized HBA esters. HRMS (ESI) was used to deter-
mine the molecular weight of the derivatives. In the IR spectra
of the derivatives, the characteristic peaks of the carbonyl group
from 1765 cmꢀ1 to 1731 cmꢀ1 indicated the presence of the
ester. In the 1H-NMR spectra of the new HBA esters, the signal at
5.0–6.0 (s, 1H) disappeared, indicating that the hydroxyl was
esteried; the signal at 9.0–9.5 (s, 1H) disappeared, suggesting
that the phenolic hydroxyl was esteried. In the 13C-NMR
spectra of all HBA esters, the presence of signals at 165.0–
175.0 ppm suggests the introduction of ester bonds. By
comparing the IR, 1H-NMR, 13C-NMR and HRMS spectra, it was
shown that the esterication reaction was successful. In all, we
got 4-hydroxybenzyl alcohol 3-furoate (FHBA), 4-hydroxybenzyl
alcohol 3-furancarboxylic acid diester (2FHBA), 4-hydroxybenzyl
alcohol 2-naphthoate (NHBA), 4-hydroxybenzyl alcohol 2-
naphthoic acid diester (2NHBA), 4-hydroxybenzyl alcohol
benzoate (BHBA), 4-hydroxybenzyl alcohol benzoic acid diester
(2BHBA), 4-hydroxybenzyl alcohol oleic acid diester (OHBA), 4-
hydroxybenzyl alcohol linoleic acid diester (LoHBA), 4-hydrox-
ybenzyl alcohol sorbic acid diester (SHBA), and 4-hydroxybenzyl
alcohol lauric acid diester (LHBA).
2.8. Acute toxicity test
The acute toxicity of 2FHBA was investigated according to the
Organization for Economic Cooperation and Development
guideline 423 (OECD423).33 40 mice were randomly divided into
four groups with 5 males and 5 females per group. 2FHBA was
blended with 0.05% sodium carboxymethyl cellulose (CMC-Na)/
physiological saline and given orally only once at doses of 1000,
2000 and 5000 mg kgꢀ1 for detailed assessment of signs of
toxicity (e.g., hypo-activity, breathing difficulty, convulsion and
abnormal movements). The control group only received the
same volume of vehicle. All groups were carefully observed for
mortality and changes in general behavior for the rst 24 h and
for a total of 14 days. The LD50 value was determined according
to the method described by the OECD Guideline 423.
2.9. Biochemical measurements
The brains of the mice were washed with ice-cold physiological
saline and homogenized, shaken for 10 s and centrifuged at
10 000 ꢃ g for 10 min at 4 ꢂC. The monoamine neurotrans-
mitters including 5-HT and GABA were measured according to
the instructions of competitive enzyme-linked immunosorbent
assay kits, which were purchased from US R&D Systems, Ltd.
(Minneapolis, USA).
3.1.1. 4-Hydroxybenzyl alcohol 3-furancarboxylic acid
diester (2FHBA). Yield: 51.72%, white powder, melting point:
233–234 ꢂC, HRMS (ESI) calcd for [M + H]+ C17H12O6+: 312.0634,
found: 312.0639. IR spectrum: the peak at 3148 cmꢀ1 is the
stretching vibration peak of ArC–H; that at 2980 cmꢀ1 is the
hydrocarbon stretching vibration peak (nC–H) of CH2. The
elongation vibration peak (nC]O) of the carbonyl group is shown
at 1734 cmꢀ1, suggesting the presence of the ester. The peak at
1685 cmꢀ1 is the stretching vibration peak (nC]C) of C]C. The
peaks at 1607 cmꢀ1, 1575 cmꢀ1, 1507 cmꢀ1, and 1464 cmꢀ1 are
the characteristic absorption peaks of the benzene ring.
2.10. Statistical analysis
All values were expressed as mean ꢁ S.D. The data were statis-
tically analyzed using the t-test and one-way analyses of variance
(ANOVAs), followed by Tukey's post-hoc multiple comparison
test. A value of p < 0.05 was considered statistically signicant.
3. Results
3.1. Chemistry
The HBA derivatives (Fig. 1) were obtained via the reaction of
HBA with carboxylic acids in dichloromethane at a temperature
of 60 ꢂC by reux. DMAP was used as a catalyst. EDCI reacts rst
with the acid to form an activated carboxylic acid and then this
1H-NMR (300 MHz, CDCl3) d ppm: 8.199 (s, 1H, 17-H), 8.047
(s, 1H, 12-H), 7.509 (t, 1H, J ¼ 1.5 Hz, 16-H), 7.478 (dt, 1H, J ¼
8.4, 2.7 Hz, 15-H), 7.431 (t, 2H, 3, 5-H), 7.212 (t, 1H, J ¼ 2.7 Hz,
Fig. 1 Compounds of the synthesis process and substituents.
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10-H), 7.189 (t, 1H, J ¼ 1.8 Hz, 11-H), 6.876 (d, 1H, J ¼ 1.8 Hz, 2- (ESI) calcd for [M + H]+ C31H52O4+: 488.3866, found: 488.3872.
H), 6.773 (d, 1H, J ¼ 2.1 Hz, 6-H), 5.297 (s, 2H, 7-H), 1.607–0.075 IR spectra: the peak at 2968 cmꢀ1 is the stretching vibration
(others); 13C-NMR (75 MHz, CDCl3) d ppm: 162.774 (C8), peak (nas C–H) of CH3. The peak at 2932 cmꢀ1 is the stretching
161.204 (C13), 150.275 (C16), 148.699 (C11), 147.882 (C4), vibration peak (nas C–H) of CH2, and that at 2860 cmꢀ1 is the
144.057 (C17), 143.755 (C12), 133.613 (C1), 129.469 (C2, C6), stretching vibration peak (ns C–H) of CH2. The telescopic vibra-
121.815 (C3, C5), 119.131 (C14), 118.681 (C9), 109.973 (C15), tion peaks (nC]O) of the carbonyl group are at 1761 cmꢀ1 and
109.784 (C10), 65.459 (C7).
1740 cmꢀ1. The peaks at 1602 cmꢀ1, 1554 cmꢀ1, 1506 cmꢀ1 and
3.1.2. 4-Hydroxybenzyl alcohol 3-furoate (FHBA). Yield: 1467 cmꢀ1 are the telescopic vibration peaks of the benzene
47.65%, white powder, melting point: 145–146 C, HRMS (ESI) ring. The peak at 1383 cmꢀ1 is the deformation vibration peak
ꢂ
+
calcd for [M + H]+ C12H10O4 : 218.0579, found: 218.0585. IR of methyl group (dCH ).
3
spectra: the peak at 3462 cmꢀ1 is the stretching vibration peak
1H-NMR (300 MHz, CDCl3) d ppm: 7.374 (d, 2H, J ¼ 8.4 Hz, 3,
(nOH) of the hydroxyl group; that at 3047 cmꢀ1 is the hydro- 5-H), 7.079 (d, 2H, J ¼ 8.4 Hz, 2, 6-H), 5.091 (s, 2H, 7-H), 2.572 (t,
carbon stretching vibration peak (nAr ) of the benzene ring. 2H, J ¼ 7.5 Hz, 21-H), 2.363 (t, 2H, J ¼ 7.5 Hz, 9-H), 1.771 (m, 4H,
C–H
The peak at 2884 cmꢀ1 is the hydrocarbon stretching vibration 10, 22-H), 1.251 (s, 32H, 11, 12, 13, 14, 15, 16, 17, 18, 23, 24, 25,
peak (nC–H) of CH2. The telescopic vibration peak (nC]O) of the 26, 27, 28, 29, 30-H), 0.899 (t, 6H, J ¼ 6.3 Hz, 19, 31-H); 13C-NMR
carbonyl group is at 1733 cmꢀ1. The peak at 1651 cmꢀ1 is the (75 MHz, CDCl3) d ppm: 173.463 (C8), 172.065 (C20), 150.459
stretching vibration peak (nC]C) of olen. The peaks at (C4), 133.497 (C1), 129.258 (C2, C6), 121.562 (C3, C5), 65.266
1603 cmꢀ1, 1507 cmꢀ1, and 1455 cmꢀ1 are the characteristic (C7), 34.257 (C9), 34.182 (C21), 31.788 (C17, C29), 29.475 (C13,
absorption peaks of the benzene ring.
C25), 29.330 (C14, C26), 29.211 (C15, C27), 29.127 (C12, C24),
1H-NMR (300 MHz, CDCl3) d ppm: 8.199 (m, 1H, 12-H), 7.506 29.007 (C16, C28), 28.979 (C11, C23), 24.807 (C10, C22), 22.562
(t, 1H, J ¼ 1.8 Hz, 11-H), 7.429 (dt, 1H, J ¼ 8.7, 2.1 Hz, 10-H), (C18, C30), 13.981 (C19, C31).
7.191 (dt, 2H, J ¼ 8.4, 2.4 Hz, 3, 5-H), 6.875 (dd, 2H, J ¼ 1.8,
3.1.5. 4-Hydroxybenzyl alcohol sorbic acid diester (SHBA).
0.9 Hz, 2, 6-H), 5.292 (s, 1H, 7-OH), 4.698 (s, 2H, 7-H); 13C-NMR Yield: 82.38%, light yellow solid, melting point: 132–133 ꢂC,
+
(75 MHz, CDCl3) d ppm: 161.461 (C8), 149.551 (C11), 147.881 HRMS (ESI) calcd for [M + H]+ C19H20O4 : 312.1362, found:
(C4), 143.983 (C12), 138.644 (C1), 129.417 (C2, C6), 127.942 (C3, 312.1369. IR spectra: the peak at 3047 cmꢀ1 is the hydrocarbon
C5), 121.572 (C9), 109.906 (C10), 64.373 (C7).
stretching vibration peak (nAr ) of the benzene ring. The peak
C–H
3.1.3. 4-Hydroxybenzyl alcohol linoleic acid diester at 2983 cmꢀ1 is the stretching vibration peak (nC–H) of CH2. The
(LoHBA). Yield: 87.61%, light yellow oily liquid, HRMS (ESI) peak at 2875 cmꢀ1 is the stretching vibration peak (nC–H) of the
calcd for [M + H]+ C43H68O4 : 648.5118, found: 648.5126. IR CH3; those at 1761 cmꢀ1 and 1740 cmꢀ1 are the stretching
+
spectra: the peak at 3022 cmꢀ1 is the hydrocarbon stretching vibration peaks (nC]O) of the carbonyl group; and that at
vibration peak (nAr ) of the benzene ring. The peak at 1647 cmꢀ1 is nC]C, indicating the presence of olen. The peaks
C–H
2940 cmꢀ1 is the stretching vibration peak (nas C–H) of CH2. The at 1610 cmꢀ1, 1509 cmꢀ1, and 1450 cmꢀ1 are the telescopic
peak at 2867 cmꢀ1 is the hydrocarbon stretching vibration peak vibration peaks of the benzene ring.
(ns C–H) of CH2. The peaks at 1765 cmꢀ1 and 1746 cmꢀ1 are
1H-NMR (300 MHz, CDCl3) d ppm: 7.409 (d, 2H, J ¼ 8.4 Hz,
telescopic vibration peaks (nC]O) of the carbonyl group; those at 10, 16-H), 7.260 (m, 2H, 3, 5-H), 7.132 (d, 2H, J ¼ 8.4 Hz, 2, 6-H),
1611 cmꢀ1, 1513 cmꢀ1, and 1458 cmꢀ1 are the telescopic 6.279 (m, 2H, 11, 17-H), 5.983 (d, 2H, J ¼ 15.3 Hz, 12, 18-H),
vibration peaks of the benzene ring. The peaks at 1383 cmꢀ1 5.835 (d, 2H, J ¼ 15.6 Hz, 9, 15-H), 5.170 (s, 2H, 7-H), 1.906 (dd,
and 1353 cmꢀ1 are the deformation vibration peaks of the 6H, J ¼ 5.1, 6.9 Hz, 13, 19-H); 13C-NMR (75 MHz, CDCl3) d ppm:
methyl group (dCH ).
166.835 (C14), 165.349 (C8), 150.589 (C4), 146.826 (C16),
3
1H-NMR (300 MHz, CDCl3) d ppm: 7.374 (d, 2H, J ¼ 8.4 Hz, 3, 145.358 (C10), 140.489 (C12), 139.406 (C18), 133.503 (C1),
5-H), 7.079 (d, 2H, J ¼ 8.4 Hz, 2, 6-H), 5.352 (m, 8H, 37, 35, 20, 129.651 (C11), 129.621 (C17), 129.197 (C2, C6), 121.587 (C3, C5),
19, 34, 38, 16, 17-H), 5.091 (s, 2H, 7-H), 2.794 (t, 4H, J ¼ 5.7 Hz, 118.482 (C9), 117.833 (C15), 65.273 (C7), 18.515 (C13), 18.413
18, 35-H), 2.753 (t, 2H, J ¼ 7.5 Hz, 27-H), 2.364 (t, 2H, J ¼ 7.5 Hz, (C19).
9-H), 2.058 (t, 8H, J ¼ 6.6 Hz, 15, 21, 33, 39-H), 1.773 (m, 4H, 10,
3.1.6. 4-Hydroxybenzyl alcohol benzoic acid diester
28-H), 1.301 (m, 24H, 11, 29, 13, 31, 14, 32, 22, 40, 23, 41, 24, 42- (2BHBA). Yield: 56.15%, white powder, melting point: 225–
H), 0.888 (m, 6H, 25, 43-H); 13C-NMR (75 MHz, CDCl3) d ppm: 226 ꢂC, HRMS (ESI) calcd for [M + H]+ C21H16O4 : 332.1048,
+
173.441 (C8), 172.032 (C26), 150.529 (C4), 133.566 (C1), 130.120 found: 332.1057. IR spectra: the peak at 3076 cmꢀ1 is the tele-
(C16, C20), 129.952 (C34), 129.902 (C38), 129.323 (C2, C6), scopic vibration peak of ArC–H, and the peak at 2974 cmꢀ1 is the
128.031 (C17), 127.977 (C19), 127.847 (C35, C37), 121.619 (C3, telescopic vibration peak (nC–H) of the CH2. The telescopic
C5), 65.327 (C7), 34.301 (C36), 34.221 (C9), 31.864 (C33), 31.477 vibration peaks (nC]O) of the carbonyl group are at 1737 cmꢀ1
(C39), 29.720 (C27), 29.629 (C23, C41), 29.536 (C14, C32), 29.479 and 1710 cmꢀ1. The peaks at 1602 cmꢀ1, 1507 cmꢀ1, 1496 cmꢀ1
,
(C13), 29.414 (C31), 29.276 (C22), 29.203 (C40), 29.102 (C12, and 1455 cmꢀ1 are the telescopic vibration peaks of the benzene
C30), 29.039 (C11, C29), 27.155 (C15, C21), 25.588 (C18), 24.856 ring.
(C10, C28), 22.640 (C24), 22.529 (C42), 14.059 (C25), 14.019
(C43).
1H-NMR (300 MHz, CDCl3) d ppm: 8.223 (dt, 2H, J ¼ 6.9,
3.0 Hz, 17, 21-H), 8.102 (dt, 2H, J ¼ 6.9, 3.0 Hz, 10, 14-H), 7.671
3.1.4. 4-Hydroxybenzyl alcohol lauric acid diester (LHBA). (m, 6H, 19, 12, 18, 20, 11, 13-H), 7.260 (d, 2H, J ¼ 2.1 Hz, 3, 5-H),
Yield: 91.33%, white powder, melting point: 280–281 ꢂC, HRMS 7.238 (d, 2H, J ¼ 1.8 Hz, 2, 6-H), 5.384 (s, 2H, 7-H); 13C-NMR (75
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MHz, CDCl3) d ppm: 166.197 (C8), 164.922 (C15), 150.729 (C4), 15), 135.513 (C20–21), 133.711 (C1, C10–11, C24–25), 132.401
133.602 (C19), 133.526 (C12), 132.949 (C1), 130.039 (C17, C21), (C10, C25), 131.909 (C11), 131.174 (C24), 129.565 (C2, C6),
129.904 (C16), 129.567 (C9), 129.364 (C10, C14), 129.277 (C2, 129.409 (C18), 129.313 (C13), 128.598 (C22), 128.348 (C15),
C6), 128.454 (C11, C13), 128.267 (C18, C20), 121.789 (C3, C5), 128.238 (C20), 128.138 (C9), 127.758 (C14), 127.689 (C21),
65.924 (C7).
126.787 (C12), 126.594 (C23), 125.346 (C16), 125.201 (C19),
3.1.7. 4-Hydroxybenzyl alcohol benzoate (BHBA). Yield: 121.948 (C3, C5), 66.179 (C7).
35.17%, colorless aky crystals, melting point: 139–140 ꢂC,
3.1.9. 4-Hydroxybenzyl alcohol 2-naphthoate (NHBA).
+
HRMS (ESI) calcd for [M + H]+ C14H12O3 : 228.0786, found: Yield: 38.11%, white aky crystals, melting point: 242–243 C,
ꢂ
+
228.0793. IR spectra: the peak at 3508–3102 cmꢀ1 is the HRMS (ESI) calcd for [M + H]+ C18H14O3 : 278.0943, found:
stretching vibration peak (nOH) of the hydroxyl group; that at 278.0949. IR spectra: the peak at 3394 cmꢀ1 is the stretching
3052 cmꢀ1 is the telescopic vibration peak (nAr ) of benzene. vibration peak (nOH) of the hydroxyl group; that at 3070 cmꢀ1 is
C–H
The peak at 2932 cmꢀ1 is the stretching vibration peak (nC–H) of the stretching vibration peak of ArC–H. The peak at 2878 cmꢀ1 is
the CH2. The telescopic vibration peak (nC]O) of the carbonyl the stretching vibration peak (nC–H) of the CH2. The telescopic
group is 1737 cmꢀ1; the peaks at 1602 cmꢀ1, 1587 cmꢀ1
,
vibration peak (nC]O) of the carbonyl group is at 1737 cmꢀ1. The
1507 cmꢀ1 and 1455 cmꢀ1 are the telescopic vibration peaks of peaks at 1601 cmꢀ1, 1507 cmꢀ1, and 1466 cmꢀ1 are the tele-
the benzene ring.
scopic vibration peaks of the benzene ring.
1H-NMR (300 MHz, CDCl3) d ppm: 8.219 (dt, 2H, J ¼ 7.2,
1H-NMR (300 MHz, CDCl3) d ppm: 8.783 (s, 1H, 10-H), 8.206
1.2 Hz, 10, 14-H), 7.669 (m, 3H, 12, 11, 13-H), 7.447 (dt, 2H, J ¼ (d, 2H, J ¼ 1.8 Hz, 15, 16-H), 8.177 (d, 2H, J ¼ 1.8 Hz, 14, 11-H),
7.8, 2.1 Hz, 3, 5-H), 7.254 (s, 1H, 7-OH), 7.228 (dt, 2H, J ¼ 8.4, 8.011 (m, 2H, 12, 13-H), 7.470 (dt, 2H, J ¼ 8.7, 2.4 Hz, 3, 5-H),
2.7 Hz, 2, 6-H), 4.706 (s, 2H, 7-H); 13C-NMR (75 MHz, CDCl3) 7.281 (dt, 2H, J ¼ 8.7, 2.4 Hz, 2, 6-H), 4.721 (s, 2H, 7-H); 13C-NMR
d ppm: 165.085 (C8), 150.162 (C4), 138.399 (C1), 133.452 (C12), (75 MHz, CDCl3) d ppm: 165.325 (C8), 150.324 (C4), 138.495
130.001 (C10, C14, C9), 128.399 (C2, C6), 127.948 (C11, C13), (C1), 135.722 (C14-15), 132.386 (C10–11), 131.841 (C10), 129.377
121.629 (C3, C5), 64.581 (C7).
(C11), 128.541 (13), 128.296 (C2, C6), 128.048 (C9), 127.726
3.1.8. 4-Hydroxybenzyl alcohol 2-naphthoic acid diester (C15), 126.742 (C14), 126.556 (C12), 125.326 (C16), 121.755 (C3,
(2NHBA). Yield: 58.23%, white powder, melting point: 407– C5), 64.669 (C7).
+
408 ꢂC, HRMS (ESI) calcd for [M + H]+ C29H20O4 : 432.1362,
3.1.10. 4-Hydroxybenzyl alcohol oleic acid diester (OHBA).
found: 432.1366. IR spectra: the peak at 3074 cmꢀ1 is the tele- Yield: 93.04%, light yellow oily liquid, HRMS (ESI) calcd for [M +
+
scopic vibration peak (nAr ) of benzene. The peak at 2952 cmꢀ1 H]+ C43H72O4 : 652.5431, found: 652.5437. IR spectra: the peak
C–H
is the stretching vibration peak (nC–H) of the CH2. The peaks at at 3070 cmꢀ1 is the stretching vibration peak (nAr ) of the
C–H
1740 cmꢀ1 and 1731 cmꢀ1 are the stretching vibration peaks benzene. The peak at 2860 cmꢀ1 is the stretching vibration peak
(nC]O) of the carbonyl group. The peaks at 1601 cmꢀ1
,
(nC–H) of CH2. The telescopic vibration peaks (nC]O) of the
1560 cmꢀ1, 1509 cmꢀ1, and 1466 cmꢀ1 are the telescopic carbonyl group are at 1763 cmꢀ1 and 1743 cmꢀ1. The peaks at
vibration peaks of the benzene ring.
1605 cmꢀ1, 1505 cmꢀ1, and 1467 cmꢀ1 are the telescopic
1H-NMR (300 MHz, CDCl3) d ppm: 8.793 (s, 1H, 25-H), 8.657 vibration peaks of the benzene ring. The peak at 1380 cmꢀ1 is
(s, 1H, 10-H), 8.215 (m, 6H, 20, 19, 16, 21, 15, 24-H), 8.124 (dd, the deformation vibration peak of the methyl group (dCH ).
3
2H, J ¼ 1.8, 6.9 Hz, 11, 14-H), 8.019 (m, 2H, 22, 23-H), 7.661 (m,
1H-NMR (300 MHz, CDCl3) d ppm: 7.374 (dt, 2H, J ¼ 8.4,
2H, 12, 13-H), 7.336 (d, 2H, J ¼ 2.1 Hz, 3, 5-H), 7.314 (d, 2H, J ¼ 2.7 Hz, 3, 5-H), 7.078 (dt, 1H, J ¼ 8.7, 2.7 Hz, 2, 6-H), 5.359 (m,
1.8 Hz, 2, 6-H), 5.460 (s, 2H, 7-H); 13C-NMR (75 MHz, CDCl3) 4H, 16, 17, 34, 35-H), 5.09 (s, 2H, 7-H), 2.547 (t, 2H, J ¼ 7.2 Hz,
d ppm: 166.466 (C8), 165.196 (C17), 150.921 (C4), 135.756 (C14- 27-H), 2.339 (t, 2H, J ¼ 7.5 Hz, 9-H), 2.034 (m, 8H, 15, 18, 33, 36-
Fig. 2 The sedative effect of HBA esters on spontaneous locomotor activity (A); the sedative effect of different doses of 2FHBA on spontaneous
locomotor activity (B) (10 in each group). *p < 0.05 and **p < 0.01 compared with the vehicle group.
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H), 1.748 (m, 4H, 10, 28-H), 1.300 (m, 40H, 11, 29, 13, 31, 20, 38, detected using a ZZ-6 locomotor activity tester to determine
21, 39, 14, 32, 19, 37, 12, 30, 22, 23, 24, 40, 41, 42-H), 0.888 (m, their sedative effects. The number of autonomous activities was
6H, 25, 43-H); 13C-NMR (75 MHz, CDCl3) d ppm: 173.497 (C8), recorded aer oral administration of the different HBA esters
172.093 (C26), 150.518 (C4), 133.555 (C1), 129.969 (C16), (20 mg kgꢀ1) and diazepam (3 mg kgꢀ1). Fig. 2A shows that the
129.916 (C17), 129.679 (C34), 129.634 (C35), 129.332 (C2, C6), 2FHBA, SHBA, 2NHBA, NHBA, and OHBA groups displayed
121.625 (C3, C5), 65.343 (C7), 34.313 (C9, C15, C18), 34.234 obvious sedative effects compared with the vehicle group (p <
(C27, C33, C36), 31.853 (C23, C41), 29.714 (C14, C19, C32, C37), 0.05). However, 2FHBA (20 mg kgꢀ1) exhibited the strongest
29.625 (C20, C21, C38, C39), 29.529 (C13, C31), 29.469 (C12, sedative activity. Therefore, a series of dosages (5 mg kgꢀ1
C30), 29.268 (C22, C40), 29.035 (C11, C29), 25.578 (C10), 25.479 10 mg kgꢀ1, 20 mg kgꢀ1 and 50 mg kgꢀ1) of 2FHBA were
,
(C28), 22.629 (C24), 22.519 (C42), 14.057 (C25), 14.017 (C43).
investigated, and the results (Fig. 2B) demonstrated that 10 mg
kgꢀ1 of 2FHBA showed the best sedative effect, and the sub-
sedative dose was 5 mg kgꢀ1
.
3.2. Spontaneous locomotor activity test (SLT)
Spontaneous motor activity is usually considered as an effective
index of alertness or a decrease in motor activity that leads to
sedation,34 as a result of the reduced excitability of the central
nervous system. Therefore, the spontaneous locomotor activi-
ties of mice treated with the newly synthesized HBA esters were
3.3. Screening of the HBA derivatives for hypnotic activity
Sodium pentobarbital (28 mg kgꢀ1 or 42 mg kgꢀ1) was used to
record the falling asleep rate, sleep latency and sleep duration
time. As we can see from Table 1, DZP, the positive control,
showed the strongest hypnotic activity, in which the ratio of
mice falling asleep was 100% (p < 0.01). Meanwhile, the 2FHBA
group administered in combination with sodium pentobarbital
(28 mg kgꢀ1) also performed well, with 90% of the mice falling
asleep (p < 0.01). The others did not exhibit an obvious effect.
Fig. 3 shows that 2FHBA, SHBA, 2BHBA and 2NHBA combined
with pentobarbital sodium (42 mg kgꢀ1) signicantly increased
the sleep duration in mice (Fig. 3B, p < 0.05), but the reduction
in sleep latency was not obvious except for that with 2FHBA
(Fig. 3A). From the above results, we concluded that 2FHBA
displayed the best hypnotic activity among the new esters.
Table 1 The effect of HBA esters on the sleep onset of mice treated
with a sub-hypnotic dose of pentobarbital (28 mg kgꢀ1 a
)
Dosage
No. falling
asleep/total
Sleep
onset (%)
Group
(mg kgꢀ1, i.g.)
Vehicle
DZP
HBA
—
3
1/10
10/10
1/10
9/10
1/10
1/10
1/10
1/10
0/10
0/10
2/10
0/10
0/10
10
100##
10
90##
10
10
10
10
0
20
20
20
20
20
20
20
20
20
20
20
2FHBA
FHBA
LoHBA
LHBA
SHBA
2BHBA
BHBA
2NHBA
NHBA
OHBA
3.4. Effect of 2FHBA on sleep in pentobarbital-treated mice
A series of doses of 2FHBA (5 mg kgꢀ1, 10 mg kgꢀ1, 20 mg kgꢀ1
and 50 mg kgꢀ1) were designed to evaluate its hypnotic activity
in combination with sodium pentobarbital (42 mg kgꢀ1). The
results showed that 10 mg kgꢀ1 and 20 mg kgꢀ1 of 2FHBA
signicantly prolonged the sleeping time (p < 0.05, Fig. 4B), but
only administration of 10 mg kgꢀ1 signicantly decreased the
sleep latency of the mice (p < 0.05, Fig. 4A). Furthermore, the
0
20
0
0
a
Statistical analysis by chi-square test (the c2 test); ##p < 0.01 compared
with the vehicle group.
Fig. 3 The hypnotic effect of HBA esters (2FHBA, FHBA, LoHBA, LHBA, SHBA, 2BHBA, BHBA, 2NHBA, NHBA, and OHBA) co-administered with
pentobarbital (42 mg kgꢀ1) on sleep in mice (10 in each group): the sleep latency (A) and the sleep duration (B); *p < 0.05, **p < 0.01 and ***p <
0.001 compared with the vehicle group.
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Fig. 4 The hypnotic effect of 2FHBA at different doses (5 mg kgꢀ1, 10 mg kgꢀ1, 20 mg kgꢀ1 and 50 mg kgꢀ1) co-administered with pentobarbital
(42 mg kgꢀ1) in mice (10 in each group): the sleep latency (A) and the sleep duration (B); *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the
vehicle group.
Fig. 5 Effect of 2FHBA (10 mg kgꢀ1) on pCPA-induced insomnia in pentobarbital-treated mice (10 in each group): the sleep latency (A) and sleep
duration (B); all values are presented as mean ꢁ SD of the mean. *p < 0.05 and **p < 0.01 compared with the vehicle group.
hypnotic effect of 2FHBA was dose-dependent, but this depen-
dence is not obvious. 10 mg kgꢀ1 of 2FHBA showed specic
hypnotic activity, while 5 mg kgꢀ1 represented the sub-hypnotic
dose. These two doses were used in the following mechanism
research.
3.5. Effect of 2FHBA on pCPA-induced insomnia in
pentobarbital-treated mice
Fig. 5 shows that aer 4 days of pCPA treatment (100 mg kgꢀ1
i.p.), the sleep latency of mice was increased, and the sleep
duration was reduced (p < 0.05), suggesting that a successful
insomnia model was established. Aer administration of
,
Fig. 6 Synergistic effect of 2FHBA (5 mg kgꢀ1) with 5-HTP (2.5 mg kgꢀ1) on pentobarbital-treated sleep in mice (10 in each group): the sleep
latency (A) and sleep duration (B); all values are presented as the mean ꢁ SD. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the vehicle
group; #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with the vehicle model group treated with 5-HTP.
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Fig. 7 Effect of 2FHBA (10 mg kgꢀ1) on FLU-induced insomnia in pentobarbital-treated mice (10 in each group): the sleep latency (A) and sleep
duration (B); all values are presented as mean ꢁ SD. *p < 0.05 and **p < 0.01 compared with the vehicle group; #p < 0.05 and ##p < 0.01,
compared with the diazepam group (3 mg kgꢀ1).
2FHBA (10 mg kgꢀ1), the sleep latency was signicantly short- duration. In addition, the hypnotic effect of 2FHBA (10 mg kgꢀ1
,
ened (Fig. 5A, p < 0.05), but there was no statistically signicant p < 0.05) was inhibited by FLU compared with the vehicle group,
change in sleep duration, although it was prolonged (Fig. 5B).
which was similar to the effect of DZP (3 mg kgꢀ1) co-
administered with FLU.
3.6. Synergic effects of 2FHBA and 5-HTP on sleep induced
by pentobarbital
3.8. Effect of 2FHBA on thiosemicarbazide-induced sleep
disturbance in mice
In order to determine whether the hypnotic effect of 2FHBA was
related to the serotonergic system, synergistic administration of In this study, we could see an obvious insomnia model induced
sub-hypnotic doses of 2FHBA (5 mg kgꢀ1) and 5-HTP (2.5 mg by TSC alone (8 mg kgꢀ1, p < 0.05). However, 2FHBA
kgꢀ1) was performed. As shown in Fig. 6, the sleep latency was (10 mg kgꢀ1) reversed the TSC-induced insomnia. Compared
signicantly reduced (p < 0.01), and the sleep duration was with the TSC group, the sleep latency of the co-administration
signicantly prolonged (p < 0.001). Combined with the results of group was signicantly shortened (p < 0.05, Fig. 8A) and the
the pCPA-induced model, this suggests that the serotonergic duration of sleep was signicantly prolonged (p < 0.01, Fig. 8B).
system might be involved in the hypnotic effects of 2FHBA.
The above results suggested that the hypnotic effect of 2FHBA
was related to the GABAergic system.
3.7. Effect of 2FHBA on the hypnotic-reversing action of
umazenil in pentobarbital-treated mice
3.9. Acute toxicity test results
Fig. 7 shows that administration of FLU (8 mg kgꢀ1) alone In the acute toxicity study, 2FHBA (1000, 2000 and 5000 mg
slightly prolonged the sleep latency and shortened the sleep kgꢀ1, i.g.) did not produce any mortality in the mice during the
Fig. 8 Effect of 2FHBA (10 mg kgꢀ1) on TSC-induced insomnia in pentobarbital-treated mice (10 in each group): the sleep latency (A) and sleep
duration (B); all values are presented as mean ꢁ SD. *p < 0.05 and **p < 0.01 compared with the vehicle group; #p < 0.05 and ##p < 0.01
compared with the TSC-treated group.
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Fig. 9 The content of 5-HT in the brains of mice in the 2FHBA group, pCPA group, 5-HTP group, 2FHBA + pCPA group and 2FHBA+5-HTP
group (A). The content of GABA in the brains of mice in the 2FHBA group, FLU group, TSC group, 2FHBA + FLU group and 2FHBA + TSC group (B).
The values represent the mean ꢁ SD (n ¼ 10 in each group), compared with the control group, *p < 0.05, **p < 0.01 and ***p < 0.001; compared
with the pCPA (100 mg kgꢀ1, i.g.) (FLU (B) (8 mg kgꢀ1)) group, #p < 0.05; and compared with the 5-HTP (2.5 mg kgꢀ1) (TSC (B) (8 mg kgꢀ1)) group,
&p < 0.05 and &&p < 0.01. One-way ANOVA, and a post-hoc Tukey test were used.
observation period. The 1000 and 2000 mg kgꢀ1 treated groups hypnotic activity for the rst time. As is well-known, sleep is
did not show any adverse clinical signs compared to the control a sophisticated physiological process which is affected by
group. Meanwhile, the dose of 5000 mg kgꢀ1 seemed to affect a network of neuronal systems and is hard to reproduce in
the female mice on the rst day, as this group showed a small vitro.36,37 In this study, the sedative effects of the derivatives were
decrease in their intake of food and water, but this symptom determined through articial screening using the spontaneous
gradually disappeared in the subsequent days. As the acute locomotor activity test. In addition, the classical behavioral
administration of 2FHBA did not produce death at the pharmacology method, the pentobarbital-induced sleep test, was
maximum dose of 5000 mg kgꢀ1, the LD50 could not be esti- conducted to evaluate the hypnotic effect of the newly synthesized
mated, and it was considered to be greater than 5000 mg kgꢀ1
.
HBA esters. The present study showed that 2FHBA and SHBA
The early acute toxicity study of HBA and GAS also showed non- signicantly decreased the locomotor activity and potentiated the
toxicity.35 Therefore, we considered 2FHBA to be non-toxic.
hypnotic effect of sodium pentobarbital, indicating their central
nervous system depressant activity. Ultimately, 2FHBA, which had
the strongest sedative and hypnotic activity, was selected for the
subsequent experiments. Based on the results of dose screening,
10 mg kgꢀ1 was selected as the effective hypnotic dose and 5 mg
kgꢀ1 was selected as the sub-hypnotic dose.
3.10. Effects of 2FHBA on the levels of 5-HT and GABA in
drug-treated mice
To verify whether the sedative–hypnotic effect of 2FHBA was
connected with the 5-HTergic and GABAergic systems, we
detected the levels of 5-HT and GABA in the brains of the mice
in the relevant administration groups. As Fig. 9A shows, the
content of 5-HT was obviously increased in the 2FHBA group
(p < 0.01). In the 2FHBA combined with pretreatment with pCPA
group, the 5-HT level was obviously increased (p < 0.05)
compared with that in the pCPA group. In the 5-HTP group, the
content of 5-HT was higher than that in the control group (p <
0.01). In the combined use of 2FHBA and 5-HTP group, the 5-HT
level was higher than that in the 5-HTP group (p < 0.01). Fig. 9B
shows that the levels of GABA in the brains of the mice
administered with 2FHBA were higher than the others, espe-
cially the control group (p < 0.05). The GABA level of 2FHBA
combined with FLU was higher than that with FLU alone (p <
0.05). The level of GABA in the 2FHBA combined with TSC group
was higher than that in the TSC group (p < 0.05).
The hippocampus plays an important role in sleep activity.
Since serotonergic projections in the hippocampus are diffuse
in nature,38 the local effects of 5-HT are largely dependent on
the 5-HT concentration. The hippocampus also modulates
emotional processing in the central nervous system (CNS)39 via
diffuse serotonergic projections from the midbrain raphe
nuclei.38 In fact, 5-HT imbalances are oen correlated with
psychological disorders such as insomnia, anxiety, depression,
and psychosis.40–42 Interestingly, 5-HT receptors also form
homodimers or heterodimers among themselves or other G-
protein-coupled receptors (GPCRs), respectively, which greatly
enhances the complexity of their functions.43 pCPA is an
inhibitor of 5-HT synthesis.44 In this study, the content of 5-HT
in the brain was signicantly decreased aer pCPA was used
alone, while 2FHBA increased the content of 5-HT. There was no
doubt that the increase in 5-HT level contributed signicantly to
the sedative and hypnotic effect.45 5-HTP, which is the rate-
limiting precursor in 5-HT biosynthesis, is used as an oral
supplement to enhance serotonin levels in humans.46 In this
study, co-administration of 5-HTP with 2FHBA signicantly
shortened the sleep latency and increased the duration of sleep
4. Discussion
In this study, the original chemical structure of HBA was modied
by reaction with organic acids in dichloromethane. We obtained
10 new compounds, which were screened for sedative and
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in mice, and the content of 5-HT in the brains of the mice
increased signicantly compared with that in the control group,
suggesting that 2FHBA exerted its sedative and hypnotic effect
via the 5-HT system.
DMAP 4-Dimethylaminopyridine
HBA 4-Hydroxybenzyl alcohol
2FHBA 4-Hydroxybenzyl alcohol 3-furancarboxylic acid diester
FHBA 4-Hydroxybenzyl alcohol 3-furoate
In addition to 5-HT, GABA is also considered as another
important neurotransmitter associated with sleep modula-
LoHBA 4-Hydroxybenzyl alcohol linoleic acid diester
SHBA 4-Hydroxybenzyl alcohol sorbic acid diester
tion.47 The g-aminobutyric acid-A (GABAA) receptor is a ligand- LHBA 4-Hydroxybenzyl alcohol lauric acid diester
BHBA 4-Hydroxybenzyl alcohol benzoate
gated ion channel that modulates the levels of anxiety,
insomnia, and memory.48 The GABAA receptor is a hetero-
pentameric membrane protein that forms a GABA-gated chlo-
ride channel and is composed of several classes of subunits
(a, b, g, d, and 3).49,50 Pharmacological studies suggested that
classic benzodiazepines and other GABAA receptor positive
modulators reduce anxiety and improve sleeping.51 Diazepam,
as a representative sedative–hypnotic drug, releases GABA
through binding with the GABAA-BZD receptor. In contrast,
umazenil as a specic benzodiazepine-derived (BZD) antago-
nist, can effectively induce insomnia. In this study, 2FHBA
exhibited a stable hypnotic effect. We sought to determine the
possible mechanisms of action. The hypnotic effects of 2FHBA
were blocked by the GABAA receptor antagonist umazenil,
which indicated that the hypnotic activity of 2FHBA was
attributed to the modulation of the GABAA-BZD receptor
through the GABAergic system. As a central nervous stimulant,
thiosemicarbazide is an inhibitor of glutamate decarboxylase,
and its application will cause a reduction in the GABA content
in the brain.34 From the above results, it was shown that 2FHBA
can signicantly improve insomnia caused by TSC, and 2FHBA
with TSC treatment can signicantly increase the content of
GABA in the brain, which demonstrated that the hypnotic effect
of 2FHBA was related to the GABAergic system. In order to
assess the potential druggability of 2FHBA, the acute toxicity of
2FHBA was detected, and the results showed that the mice did
not exhibit any signs of toxicity or mortality despite the dose
being up to 5000 mg kgꢀ1, so we considered 2FHBA to be safe.
2BHBA 4-Hydroxybenzyl alcohol benzoic acid diester
2NHBA 4-Hydroxybenzyl alcohol 2-naphthoic acid diester
NHBA 4-Hydroxybenzyl alcohol 2-naphthoate
OHBA 4-Hydroxybenzyl alcohol oleic acid diester
SLT
Spontaneous locomotor activity test
Diazepam
p-Chlorophenylalanine
Benzodiazepine-derived
5-Hydroxytryptamine
DZP
pCPA
BZD
5-HT
5-HTP 5-Hydroxytryptophan
GABA g-Aminobutyric acid
GABAA g-Aminobutyric acid-A
FLU
TSC
CNS
Flumazenil
Thiosemicarbazide
Central nervous system
GPCRs G-protein-coupled receptors
Acknowledgements
This work was supported by the Jilin Provincial Science and
Technology Development Project (20180520060JH), the Jilin
Provincial
Education
Department
Fund
Project
(JJKH20170321KJ), the National Key Research and Development
Project (2016YFC0500300) and the Jilin Provincial Key Scientic
and Technological Project (20170204017YY).
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5. Conclusions
In summary, 10 HBA derivatives were synthesized with simple
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The results showed that 2FHBA showed the strongest sedative–
hypnotic effects, and its hypnotic effect might be related to the
serotonergic system and GABAergic system. Overall, this study
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Conflicts of interest
The authors declare no conict of interest.
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Abbreviations
EDCI
1-Ethylethyl-3-(3-dimethyllaminopropyl) carbodiimide
hydrochloride
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