Polybenzyls from Gastrodia elata, their agonistic effects on melatonin receptors and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2019.06.008

Gastrodia elata is a famous traditional Chinese herb with medicinal and edible application. In this study, nine polybenzyls (1?9), including six new ones (2?5, 7 and 9), were isolated from the EtOAc extract of G. elata. Five compounds 1, 3, 4, 6 and 8 wer

Full text of DOI:10.1016/j.bmc.2019.06.008

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Polybenzyls from Gastrodia elata, their agonistic effects on melatonin
receptors and structure-activity relationships
Si-Yue Chena,b,c, Chang-An Genga,b, Yun-Bao Maa,b, Xiao-Yan Huanga,b, Xiao-Tong Yanga,b,c
,
Li-Hua Sua,b,c, Xiao-Feng Hea,b,c, Tian-Ze Lia,b, Zhen-Tao Denga,b,c, Zhen Gaoa,b,c
,
⁎
Xue-Mei Zhanga,b, Ji-Jun Chena,b,c,
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
b Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, China
c University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Gastrodia elata is a famous traditional Chinese herb with medicinal and edible application. In this study, nine
polybenzyls (1−9), including six new ones (2−5, 7 and 9), were isolated from the EtOAc extract of G. elata. Five
compounds 1, 3, 4, 6 and 8 were found to activate melatonin receptors. Especially, compound 1 showed ago-
nistic effects on MT1 and MT2 receptors with EC50 values of 237 and 244 μM. For better understanding their
structure-activity relationships (SARs), ten polybenzyl analogs were further synthesized and assayed for their
activities on melatonin receptors. Preliminary SARs study suggested that two para-hydroxy groups were the key
pharmacophore for maintaining activity. Molecular docking simulations verified that compound 1 could
strongly interact with MT2 receptor by bonding to Phe 118, Gly 121, His 208, Try 294 and Ala 297 residues.
Gastrodia elata
Polybenzyls
Melatonin receptors
Structure-activity relationships
Docking study
1. Introduction
sedation and hypnosis, of which NHBA took effects by activating ade-
nosine receptors.19 However few literatures reported about the psy-
Melatonin receptors, consisting of MT1 and MT2 subtypes, are vital
targets for accommodating sleep disorder via regulating circadian
rhythms.1–4 Several melatonin receptor-mediated drugs have been on
the market for treating insomnia and depression, involving prolonged
release melatonin (Circadin®, Neurim, Israel, UK),5 ramelteon (Ro-
zerem®, Takeda Pharmaceutical Co, Osaka, Japan)6 and tasimelteon
(Hetlioz®, Vanda Pharmaceuticals, USA).5 The currently published
melatonin receptor agonists are almost synthetic compounds with
highly structural and metabolic similarity to melatonin.7–9 In contrast,
only a few melatonin natural agonists, e.g. magnolol, tropine, dimeric
isoechinulin-type alkaloid, peaoveitols and gramnie derivatives have
been reported.10–15 Regarding to the importance of natural products in
drug development,16 it is meaningful to discover new scaffolds of
melatonin agonists from natural sources.
choactive effects of polybenzyls, a characteristic type of compounds in
G. elata.
In this research, the EtOAc extract of G. elata was first found to
activate MT2 receptor with an agonistic rate of 105.4% at the con-
centration of 102.2 μg/mL. In order to characterize the active con-
stituents, bioactivity-guided isolation yielded nine polybenzyls (1−9,
Fig. 1). Compounds 1, 6 and 8 showed agonistic effect on MT2 receptor,
among which compound 1 could obviously stimulate MT1 and MT2
receptors with EC50 values of 237 and 244 μM. For better understanding
the structure-activity relationships, ten derivatives (10−19, Fig. 2)
were further synthesized. Herein, we report their isolation, structural
modification and agonistic effects on MT receptors.
2. Material and methods
Gastrodia elata is a famous traditional Chinese medicine widely used
for the treatment of hypertension, headache, convulsions and neuro-
degenerative diseases. Recent study manifested that gastrodin,17 N6-(4-
hydroxybenzyl) adenosine (NHBA) and N6-(3-methoxyl-4-hydro-
xybenzyl) adenine riboside (B2)18 from G. elata had the potency of
2.1. General instruments and chemical reagents
NMR spectra were undertaken by Avance III-400 and III-600 spec-
trometers (Bruker, Bremerhaven, Germany). LCMS-IT-TOF (Shimadzu,
⁎ Corresponding author at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650201, China.
E-mail address: chenjj@mail.kib.ac.cn (J.-J. Chen).
https://doi.org/10.1016/j.bmc.2019.06.008
Received 6 March 2019; Received in revised form 30 May 2019; Accepted 5 June 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Si-YueChen,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.06.008

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 1. Structures of compounds 1−9.
Fig. 2. Structures of synthetic compounds 10−19.
Kyoto, Japan) was used for detecting molecular mass spectra. IR spectra
were obtained on NICOLET iS10 (Thermo scientific, Waltham, USA) or
Bio-Rad FTS-135 (Hercules, California, USA) spectrometers using ATR
ITX-DIAMOND mode or KBr pellets. UV spectra were recorded on a UV-
2401 equipment (Shimadzu, Kyoto, Japan). Thin-layer chromatography
(TLC) analyses were performed on silica gel GF254 plates (Jiangyou,
Chemical Co. Ltd., Yantai, China). Compounds were purified by silica
gel (200–300 mesh, Qingdao Makall group Co. Ltd., Qingdao, China)
and Sephadex LH-20 (Amersham Bioscience, Sweden) column chro-
matography. MPLC was performed on a Dr-Flash II apparatus (Lisui,
Suzhou, China) using a CHP20P MCI gel column (Mitsubishi, Tokyo,
Sweden), and HPLC was conducted on a Shimadzu LC-CBM-20 system
(Shimadzu, Kyoto, Japan) with an Agilent XDB-C18 column
(9.4 × 250 mm, Agilient, California, USA). Bioactive assay was con-
ducted on a FlexStation3 Benchtop Multi-Mode Microplate Reader
(Molecular Devices, Sunnyvale, California, USA). Acetonitrile (chro-
matographic grade, Merk Co. Ltd., Darmstadf, Germany) and formic
acid (chromatographic grade, Aladdin Chemistry Co. Ltd., Shanghai,
China) were used for HPLC. Chemical reagents for modification were
purchased from J&K Co. Ltd. (Shanghai, China). Melatonin as the po-
sitive control was obtained from Damas-beta Co. Ltd. (Shanghai,
China).
2.2. Plant material
Gastrodia elata Bl. was bought from Zhaotong in Yunnan Province of
China, in September 2014, and was identified by Prof. Li-Gong Lei,
Kunming Institute of Botany, CAS. A voucher specimen (No. 20141107)
was deposited at the Laboratory of Anti-virus and Natural Medicinal
Chemistry, Kunming Institute of Botany, CAS.
2.3. Extraction and isolation
Fresh rhizomes of G. elata (45.0 kg) were cut into slices and ex-
tracted with 90% aqueous ethanol (45 L × 3) for three times at room
temperature. The combined extract was concentrated under reduced
pressure and partitioned between H2O and EtOAc. The EtOAc extract
(88 g) was separated by silica gel column chromatography (CC) using
gradient elution with EtOAc-CHCl3 (0:100, 5:95, 10:90, 20:80, 40:60,
100:0 v/v) as the mobile phase to yield five fractions (Fr. 1-Fr. 5). Based
on the bioassay, Fr. 2 (22 g) was separated by silica gel CC with EtOAc-
petroleum ether system (5:95, 10:90, 20:80, 40:60, 100:0) to yield five
fractions (Fr. 2.1-Fr. 2.5). Then, Fr. 2.4 (3.4 g) was further separated by
silica gel CC (Me2CO-petroleum ether, 3:97, 5:95, 10:90, 20:80, 50:50)
to give five fractions (Fr. 2.4.1-Fr. 2.4.5). Fr. 2.4.4 was isolated by
Sephadex LH-20 (MeOH-CHCl3, 50:50) and silica gel CC (Me2CO-
CHCl3, 10:90) to give compound 1 (1 g). Compounds 2 (12 mg), 5
2

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(6 mg) and 3 (120 mg) were purified from Fr. 2.4.5 by HPLC on an
Agilent XDB-C18 column with the elution of MeCN-H2O (40:60). Fr. 3
was subjected to silica gel CC (Me2CO-petroleum ether, 5:95, 10:90,
20:80, 40:60, 100:0) to yield five fractions (Fr. 3.1-Fr. 3.5). Further
purified by silica gel CC, Sephadex LH-20 CC (MeOH-CHCl3 50:50) and
semi-preparative HPLC to yield compound 4 (1 g, MeCN-H2O, 23:77,
tR = 11.5 min) from Fr. 3.1, compounds 6 (700 mg, MeCN-H2O 45:55,
v/v, tR = 19 min) and 7 (11 mg, MeCN-H2O, 45:55, tR = 26 min) from
Fr. 3.2, and compounds 8 (35 mg, MeCN-H2O, 50:50, tR = 20 min) and
9 (27 mg, MeCN-H2O, 33:67, tR = 20 min) from Fr. 3.5.
Table 2
13C NMR (150 MHz, CD3OD) data of compounds 2–5 and 7.
No
2
3
4
5
7
1
132.6
129.3
114.7
155.1
114.7
129.3
39.7
–
132.8
129.4
114.8
155.0
114.8
129.4
39.8
–
133.5
131.0
116.1
156.4
116.1
131.0
35.8
–
133.5
130.5
116.1
154.9
125.3
131.3
35.9
69.4
66.3
15.6
129.8
156.1
115.9
128.5
130.3
131.9
73.9
66.9
15.5
–
133.9
130.9
116.0
156.4
116.0
130.9
36.0
–
2
3
4
5
6
7
8
9
–
–
–
–
2.4. Spectroscopic data
10
1′
2′
3′
4′
5′
6′
7′
8′
9′
1′'
2′'
3′'
4′'
5′'
6′'
7′'
8′'
9′'
–
–
–
–
134.2
129.3
114.5
157.2
114.5
129.3
–
132.9
128.9
114.8
153.4
123.9
129.6
67.9
65.5
14.1
–
129.8
156.0
115.9
128.5
130.3
131.8
73.8
66.4
15.5
–
129.4
154.3
116.0
128.6
133.4
132.3
35.8
–
The known compounds 1, 6 and 8 were determined as bisphenol F
(1),20 2,4-bis(4-hydroxybenzyl)phenol (6)21 and gastrol (8)22 by com-
paring their MS and NMR data with the previous reports.
2.4.1. Compound 2
–
White powder; UV (MeOH) λmax (log ε): 277 (3.59), 252 (2.91), 230
(4.38), 215 (1.21) nm; IR (KBr) νmax: 3427, 1613, 1513, 1455, 1340,
1306, 1234, 1171 cm−1; HRMS (ESI) m/z 305.1168 [M−H]− (calcd for
305.1183). 1H NMR and 13C NMR see Tables 1 and 2.
–
–
128.2
129.1
114.8
156.9
114.8
129.1
69.7
–
129.9
156.0
115.8
128.4
130.3
131.8
73.9
66.4
15.5
–
–
–
–
–
–
–
–
–
–
–
–
2.4.2. Compound 3
–
–
–
White powder; UV (MeOH) λmax (log ε): 281 (3.51), 252 (2.61), 229
(4.08), 220 (4.04) nm; IR (KBr) νmax: 3424, 1614, 1598, 1512, 1444,
1553, 1248, 1076 cm−1; HRMS (ESI) m/z 257.1171 [M−H]− (calcd for
257.1183). 1H NMR and 13C NMR see Tables 1 and 2.
–
–
–
–
–
–
–
–
–
–
1H NMR and 13C NMR see Tables 1 and 2.
2.4.3. Compound 4
Pale yellow powder; UV (MeOH) λmax (log ε): 280 (3.35), 251
(2.50), 225 (3.96), 217 (3.92) nm; IR (KBr) νmax: 3405, 1613, 1512,
1441, 1352, 1261, 1232, 1067 cm−1; HRMS (ESI) m/z 257.1166
[M−H]− (calcd for 257.1183). 1H NMR and 13C NMR see Tables 1 and
2.
2.4.5. Compound 7
Pale yellow colloidal solid; UV (MeOH) λmax (log ε): 281 (3.77), 253
(2.98) nm; IR (KBr) νmax: 3425, 1613, 1511, 1440, 1373, 1257, 1228,
1099, 1068 cm−1; HRMS (ESI) m/z 363.1588 [M−H]− (calcd for
363.1602). 1H NMR and 13C NMR see Tables 1 and 2.
2.4.4. Compound 5
White powder; UV (MeOH) λmax (log ε): 281 (3.45), 251 (2.68) nm;
IR (KBr) νmax: 3420, 1631, 1614, 1509, 1441, 1373, 1352, 1266, 1095,
1074 cm−1; HRMS (ESI) m/z 315.1577 [M−H]− (calcd for 315.1602).
2.4.6. Compound 9
Pale yellow colloidal solid; UV (MeOH) λmax (log ε): 281 (3.84), 253
(3.12) nm; IR (KBr) νmax: 3440, 1632, 1615, 1513, 1443, 1236, 1072,
Table 1
1H NMR (600 MHz, J in Hz, CD3OD) data of compounds 2–5 and 7.
No
2
3
4
5
7
2
6.98 (1H, d, 8.5)
6.70 (1H, d, 8.5)
6.70 (1H, d, 8.5)
6.98 (1H, d, 8.5)
3.79 (2H, s)
6.97 (1H, d, 8.6)
7.03 (1H, d, 8.6)
6.98 (1H, m)
6.99 (1H, d, 8.5)
3
6.72 (1H, d, 8.6)
6.68 (1H, d, 8.6)
6.68 (1H, d, 8.2)
6.65 (1H, d, 8.5)
5
6.72 (1H, d, 8.6)
6.68 (1H, d, 8.6)
6.65 (1H, d, 8.5)
6
6.97 (1H, d, 8.6)
7.03 (1H, d, 8.6)
7.10 (1H, d, 2.1)
6.99 (1H, d, 8.5)
7
3.74 (2H, s)
3.82 (2H, s)
3.83 (2H, s)
3.78 (2H, s)
8
–
–
–
4.50 (2H, s)
–
9
–
–
3.54 (2H, q, 7.0)
–
10
2′
3′
4′
5′
6′
7′
8′
9′
2′'
3′'
4′'
5′'
6′'
7′'
8′'
9′'
–
–
–
1.19 (3H, t, 7.0)
–
7.06 (1H, d, 8.5)
6.92 (1H, dd, 8.2, 2.1)
–
–
–
6.87 (1H, d, 8.5)
6.73 (1H, d, 8.2)
6.75 (1H, d, 8.1)
6.74 (1H, d, 8.0)
6.66 (1H, d, 8.1)
–
–
6.98 (1H, dd, 8.1, 2.2)
6.99 (1H, m)
6.85 (1H, dd, 8.1, 2.0)
6.87 (1H, d, 8.5)
–
–
–
–
7.06 (1H, d, 8.5)
7.04 (1H, d, 2.1)
6.95 (1H, d, 2.2)
6.96 (1H, d, 2.1)
6.86 (1H, brs)
–
4.50 (2H, s)
4.29 (2H, s)
4.33 (2H, s)
3.77 (2H, s)
–
3.53 (2H, q, 7.0)
3.45 (2H, q, 7.0)
3.47 (2H, q, 7.0)
–
–
1.19 (3H, t, 7.0)
1.15 (3H, t, 7.0)
1.16 (3H, t, 7.0)
–
7.25 (1H, d, 8.5)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6.79 (1H, d, 8.5)
6.73 (1H, d, 8.1)
6.97 (1H, dd, 8.1, 2.1)
–
–
6.79 (1H, d, 8.5)
7.25 (1H, d, 8.5)
6.89 (1H, d, 2.1)
4.30 (2H, s)
3.45 (2H, q, 7.0)
1.15 (3H, t, 7.0)
4.89 (2H, s)
–
–
3

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 3
10 mL CH2Cl2 was added HBr (2 mL, 2 M in AcOH) dropwise at 0 °C.
After being stirred for 4 h, the mixture was pour into ice-cold water and
extracted with CH2Cl2. The organic layer was washed with saturated
sodium bicarbonate, brine, dried over anhydrous sodium sulfate and
concentrated under reduced pressure to yield of crude bromide. 4,4′-
Thiobisphenol (109 mg, 0.5 mmol) was dissolved in potassium carbo-
nate (10 mL, 2 M) and stirred for 30 min. Then, the mixture was added
to the solution of crude bromide and TBAB (322 mg, 1 mmol) in CH2Cl2
(10 mL) and stirred for 3 h at room temperature. The resulting mixture
was quenched with 5% HCl and extracted with EtOAc (10 mL × 3). The
combined organic layers were washed with brine, dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The crude
was purified by silica gel CC (acetone-petroleum ether 20:80) to yield of
compounds 13 and 14.
1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data of com-
pound 9.
No
δH (J in Hz)
δC
No
δH (J in Hz)
δC
1
–
133.2
130.9
116.2
156.5
116.2
130.9
36.2
1′'
2′'
3′'
4′'
5′'
6′'
7′'
1′''
2′''
3′''
4′''
5′''
6′''
7′''
–
–
132.9
132.2
132.6
154.3
116.0
128.6
36.2
2
7.03 (1H, d, 8.5)
6.86 (1H, d, 2.1)
3
6.70 (1H, d, 8.5)
–
4
–
–
5
6.70 (1H, d, 8.5)
6.70 (1H, d, 8.5)
6
7.03 (1H, d, 8.5)
6.85 (1H, dd, 8.5, 2.1)
7
3.88 (2H, s)
3.84 (2H, s)
1′
2′
3′
4′
5′
6′
7′
8′
9′
–
130.4
153.2
132.9
129.6
130.7
129.7
73.8
–
133.8
131.0
116.0
156.3
116.0
131.0
35.9
–
7.01 (1H, d, 8.5)
–
6.67 (1H, d, 8.5)
6.81 (1H, d, 1.8)
–
–
6.67 (1H, d, 8.5)
6.76 (1H, d, 1.8)
4.25 (2H, s)
3.43 (2H, q, 7.0)
1.13 (3H, t, 7.0)
7.01 (1H, d, 8.5)
2.5.2.1. 4-S-(4-Phenol) phenol 1-O-penta-O-acetyl-β-D-glucopyranoside
(13). White powder, 11% yield. HRMS (ESI) m/z 547.1296 [M−H]−
(calcd for 547.1280). UV (MeOH) λmax (logε): 251 (4.13), 238 (4.04),
230 (4.07), 219 (4.01) nm; IR (KBr) νmax: 3424, 1751, 1742, 1633,
1601, 1584, 1492, 1431, 1371, 1262, 1231, 1077, 1051 cm−1. 1H NMR
(400 MHz, CD3OD) δH: 7.25 (2H, d, J = 8.5 Hz), 7.15 (2H, d,
J = 8.5 Hz), 6.94 (2H, d, J = 8.5 Hz), 6.79 (2H, d, J = 8.5 Hz),
5.38−4.05 (7H, m), 2.01−2.04 (12H,COCH3); 13C NMR (100 MHz,
CD3OD) δC: 170.9–169.7 (COCH3), 157.6, 155.5, 134.5, 130.2, 132.3,
123.6, 117.2, 116.0, 98.3, 72.7−61.6, 19.2 (COCH3).
3.80 (2H, s)
66.4
–
–
–
15.6
–
–
1047 cm−1; HRMS (ESI) m/z 469.2024 [M−H]− (calcd for 469.2020).
1H NMR and 13C NMR see Table 3.
2.5. Schemes of synthetic compounds
The synthesis of compounds 12−19 were depicted in Schemes 1–3.
In the first attempt, the methylene groups was replaced by different
hetero atoms to speculate the influence of the methylene on activity
(compound 12, Scheme 1). Next, glycosylation was applied to see the
influence of a hydroxy group (compounds 13−15, Scheme 2). Addi-
tional phenyls were introduced into the structure to assess the effect of
enlarged skeleton (compounds 16−19, Scheme 3).
2.5.2.2. 4-S-(4-Phenol)
phenol
1,4′-O-di-penta-O-acetyl-β-D-
glucopyranoside (14). White powder, 10% yield. HRMS (ESI) m/z
923.2300 [M + COOH]− (calcd for 923.2285). UV (MeOH) λmax (log
ε): 252 (4.12), 225 (4.20), 239 (4.05), 219 (4.18) nm; IR νmax: 1745,
1491, 1367, 1224, 1075, 1054, 1034 cm−1
.
1H NMR (400 MHz,
CD3OD) δH: 7.32 (4H, d, J = 8.7 Hz), 7.07 (4H, d, J = 8.7 Hz),
5.45–4.06 (14H), 2.02−1.79 (24H, COCH3); 13C NMR (100 MHz,
CD3OD) δC: 169.8−168.8 (COCH3), 156.5, 132.7, 129.7, 117.7, 98.2,
72.4–61.8, 19.7−18.5 (COCH3).
2.5.1. Compound 12
To a stirred solution of 4,4′-dimethoxy diphenylamine (115 mg,
0.5 mmol) in DMSO (2 mL), sodium hydroxide (1 mL, 1 M) was added
and the reaction mixture was stirred at room temperature for 30 min.
Then methyl iodide was added and stirred for additional 1 h. The re-
action was diluted with H2O (10 mL) and extracted with ethyl acetate
(10 mL × 3). The organic layer was washed with brine, dried over an-
hydrous sodium sulfate and concentrated under vacuum. To a solution
of the residue in dry CH2Cl2 (10 mL) at −78 °C, BBr3 solution (2 mL,
5 M in THF) was added dropwise. The reaction was stirred for 1 h and
slowly warmed to 0 °C for 3 h. The crude solution was quenched with
saturated sodium bicarbonate and extracted with EtOAc (10 mL × 3).
The organic phase was washed with brine, dried over anhydrous so-
dium sulfate, and concentrated under reduced pressure. Column chro-
matography of the residue on silica gel CC (acetone-petroleum ether
20:80) provided compound 12.
2.5.3. Compound 15
Sodium hydroxide (5 mL, 1 M) was added to the solution of 4-S-(4-
phenol) phenol 1-O-penta-O-acetyl-β-D-glucopyranoside (27 mg,
0.05 mmol) in DMSO (3 mL) at room temperature. After 1 h, the reac-
tion mixture was quenched with 5% HCl and extracted with EtOAc
(10 mL × 3). The combined organic layers were washed with brine,
dried over anhydrous sodium sulfate and concentrated under reduced
pressure. Compound 15 was purified by silica gel CC (MeOH-CHCl3
10:90).
2.5.3.1. 4-S-(4-Phenol) phenol 1-O-β-D-glucopyranoside (15). White
powder, 79% yield. HRMS (ESI) m/z 379.0866 [M−H]− (calcd for
379.0857). UV (MeOH) λmax (log ε): 251 (4.19), 231 (4.16), 239 (4.12),
219 (4.07) nm; IR (KBr) νmax
: 3442, 1634, 1490, 1227, 1068,
2.5.1.1. 4-(4-Methylimino) bisphenol (12). Pale purple powder, 66%
yield. HRMS (ESI) m/z 214.0878 [M−H]− (calcd for 214.0874). UV
(MeOH) λmax (log ε): 282 (3.78), 261 (3.70), 247 (3.77), 230 (3.67) nm;
IR (KBr) νmax: 3424, 1636, 1509, 1451, 1235 cm−1. 1H NMR (400 MHz,
CD3OD) δH: 6.72 (4H, d, J = 8.5 Hz), 6.43 (4H, d, J = 8.5 Hz), 3.2 (3H,
s); 13C NMR (100 MHz, CD3OD) δC: 148.2, 142.1, 122.4, 116.8, 37.8.
1037 cm−1
.
1H NMR (400 MHz, CD3OD) δH: 7.24 (2H, d, J = 8.6 Hz),
7.17 (2H, d, J = 8.6 Hz), 7.03 (2H, d, J = 8.6 Hz), 6.78 (2H, d,
J = 8.6 Hz), 3.90−3.42 (7H, m); 13C NMR (100 MHz, CD3OD) δC:
157.4, 156.6, 134.0, 130.7, 130.8, 124.3, 117.1, 115.9, 100.9,
76.7−61.1.
2.5.4. Compounds 16 and 17
To a stirred solution of 4,4′-oxybisphenol (404 mg, 2 mmol) or 4,4′-
thiobisphenol (437 mg, 2 mmol) in 1,4-dioxane (7.5 mL) and toluene
(2.5 mL) at room temperature was added sequentially p-hydroxybenzyl
alcohol (124 mg, 1 mmol) and TsOH (0.4 mg, 0.2 mmol). The mixture
was heated at 60 °C for 3 h. The reaction mixture was quenched with
saturated sodium bicarbonate and extracted with EtOAc (10 mL × 3).
The organic layers were washed with brine, dried over anhydrous so-
dium sulfate and concentrated under reduced pressure. The residues
were purified by silica gel CC (EtOAc-petroleum ether, 10:90) and
2.5.2. Compounds 13 and 14
To a solution of the β-D-glucose penta-acetate (390 mg, 1 mmol) in
Scheme 1. Synthesis of compound 12. (a) CH3I, DMSO, rt, 2 h; (b) BBr3,
CH2Cl2, −78 °C to 0 °C, 4 h.
4

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 2. Synthesis of compounds 13−15. (a) K2CO3, TBAB, CH2Cl2, rt, 3 h; (b) NaOH, DMSO, rt, 1 h.
delivered compounds 16 and 17.
yellow powder, 23% yield. HRMS (ESI) m/z 379.1706 [M−H]−
(calcd for 379.1704). UV (MeOH) λmax (log ε): 281 (3.21), 253 (2.61)
nm; IR (KBr) νmax: 3398, 1613, 1607, 1511, 1451, 1252, 1237, 1219,
2.5.4.1. 2-(4-Hydroxybenzyl)-4-O-(4-phenol)-1,4-bisphenol (16). Pale
yellow powder, 19% yield. HRMS (ESI) m/z 307.0963 [M−H]−
(calcd for 307.0976). UV (MeOH) λmax (log ε): 285 (3.76), 260
(3.20), 227 (4.27), 220 (4.25) nm; IR (KBr) νmax: 3520, 3424, 1613,
1200, 1096 cm−1
.
1H NMR (400 MHz, CD3OD) δH: 7.26−6.66 (16H,
m), 3.90 (2H, s, H-7′), 3.85 (2H, s), 3.69 (2H, s); 13C NMR (100 MHz,
CD3OD) δC: 154.9, 152.8, 141.6, 139.0, 138.5, 133.0, 132.8, 130.6,
129.3, 128.5, 128.3, 128.0, 127.6, 127.0, 125.5, 114.6, 41.0, 39.7,
35.2.
1500, 1441, 1357, 1206, 1174 cm−1
.
1H NMR (400 MHz, CD3OD) δH:
7.03 (2H, d, J = 8.4 Hz), 6.76–6.68 (8H, m), 6.61(1H, brs), 3.77 (2H, s);
13C NMR (100 MHz, CD3OD) δC: 155.0−150.3, 131.7, 129.7, 129.4,
120.2, 118.9, 116.4, 115.5, 115.1, 114.6, 34.4.
2.6. Molecular docking
The docking model of MT2 receptor from Rivara23 was chosen in
this research because the crystal structure of melatonin receptors was
not released yet, and docking model was validated by Rivara. Among
these models, RhoACT model with ligand 7a, a melatonin agonist,24 was
chosen as a docking pocket for its high EF2% and ever usage in previous
study.23,25 AutoDock Vina, a free software (http://vina.scripps.edu),
was applied for molecular docking26 since the algorithms of AutoDock
Vina for scoring function were widely applied.26,27 In docking simula-
tion study, the position of the original compound in RhoACT model was
firstly redefined as a binding site of docking pocket. Then compound 1
and melatonin were docked into the model by AutoDock Vina. After
docking, binding affinities were calculated by AutoDock Vina as a
standard parameter to estimate whether compound 1 could bind to the
central location as that of melatonin. The interactions between protein
and compounds were analyzed by Discovery Studio 4.5.
2.5.4.2. 2-(4-Hydroxybenzyl)-4-S-(4-phenol) phenol (17). Pale yellow
powder, 13% yield. HRMS (ESI) m/z 323.0748 [M−H]− (calcd for
323.0747). UV (MeOH) λmax (log ε): 277 (3.82), 273 (3.82), 228 (4.24),
221 (4.22) nm; IR (KBr) νmax: 3442, 1636, 1615, 1513, 1492, 1413,
1239 cm−1
.
1H NMR (400 MHz, CD3OD) δH: 7.10 (2H, d, 8.4),
7.01–7.00 (4H, m), 7.73−6.67 (5H, m), 3.78 (2H, s); 13C NMR
(100 MHz, CD3OD) δC: 156.5, 155.0, 154.4, 133.3, 130.0, 132.3,
131.6, 129.5, 129.4, 126.4, 125.8, 115.6, 114.6, 115.2, 34.2.
2.5.5. Compounds 18 and 19
A mixture of 4,4′-methylenebisphenol (200 mg, 1 mmol), benzyl
alcohol (0.2 mL, 2 mmol) and phosphoric acid (1 mL, 17 mmol) in to-
luene (10 mL) was kept at 100 °C for 6 h. The reaction was quenched
with saturated sodium bicarbonate and extracted with EtOAc
(10 mL × 3). The organic layers were washed with brine, dried over
anhydrous sodium sulfate and concentrated under reduced pressure.
Purification of the crude by silica gel CC (EtOAc-petrleum ether 10:90)
provided compounds 18 and 19.
2.7. Agonistic assay on MT1 and MT2 receptors
Agonistic activities of MT1 and MT2 receptors were assayed on
HEK293 cell line in accordance with the previous study.28,29 Cells were
cultivated in 5% CO2 incubator at 37 °C by adding Dulbecco's modified
eagle medium, G418 (400 μg/mL) and fetal bovine serum (10%). Cells
were put at a density of 4 × 104/well in a Matrigel® coated 96-well
black plate and proliferated in CO2 for 24 h. Subsequently, cells were
dyed by Wash Free Fluo-8 Calcium Assay Kit (HD Biosicences Co. Ltd.,
Hd03-0010) and placed in CO2 incubator for 1 h. Tested compounds
and positive control were dissolved in 10 μL DMSO and 990 μL HBSS
Buffer, respectively, and a volume of 100 μL/well was put in another
96-well plate. Then two 96-well plates were put into Flex Station 3
2.5.5.1. 2-Benzyl-4-(4-hydroxybenzyl) phenol (18). Pale yellow powder,
22% yield. HRMS (ESI) m/z 289.1214 [M−H]− (calcd for 289.1234).
UV (MeOH) λmax (log ε): 281 (3.63), 253 (2.90) nm; IR (KBr) νmax
:
3443, 1636, 1615, 1511, 1444, 1252 cm−1
.
1H NMR (400 MHz,
CD3OD) δH: 7.24–7.10 (5H, m), 6.94 (2H, d, J = 8.5 Hz), 6.83 (1H,
brs), 6.81 (1H, m), 6.71−6.66 (3H, m), 3.90 (2H, s), 3.71 (2H, s); 13C
NMR (100 MHz, CD3OD) δC: 155.0, 152.9, 141.1, 132.9, 132.8, 130.6,
129.3, 128.4, 127.7, 127.4, 127.0, 125.2, 114.6, 114.5, 39.7, 35.2.
2.5.5.2. 2-(2-Benzyl)benzyl-4-(4-hydroxybenzyl)
phenol
(19). Pale
Scheme 3. Synthesis of compounds 16−19. (a)
TsOH, 1,4-dioxane-toluene, 60 °C, 3 h (compounds
16–17) or H3PO4, toluene, 100 °C, 6 h (compounds
18−19).
5

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Benchtop Multi-Mode Micro plate Reader (Molecular Devices, Sunny-
vale, California, USA), and compounds or positive control was added a
volume of 50 μL/well in 96-well plate with cells automatically. Data
were recorded and read via Flex Station 3 Benchtop Multi-Mode Micro
plate Reader at room temperature using specified settings (excitation
wave length: 485 nm, emission wave length, 525 nm, emission cut-off,
515 nm). EC50 values were calculated by GraphPad Prism 5 software.
(calcd for 257.1183). Detailed analysis of their NMR data manifested
the differences of δC 39.8 (C-7) and 67.9 (C-7′) in 3 vs δC 35.8 (C-7) and
73.8 (C-7′) in 4. This analysis suggested that the hydroxyl group at C-4′
in 3 might migrate to C-2′ in 4. Further evidence was established by the
HMBC correlations of H-7 (δH 3.82, 2H, s) with C-2′ (δC 156.0), and of
H-7′ (δH 4.29, 2H, s) with C-4′ (δC 128.5). Finally, compound 4 was
established as gastropolybenzylol C.
3. Results and discussion
3.1.4. Compound 5
The molecular formula of 5 was determined to be C19H24O4 with
eight degrees of unsaturation from the [M−H]− ion at m/z 315.1577
(calcd for 315.1602). Compared with the NMR spectra of 4, compound
5 possessed one more ethoxymethyl: δH 4.50 (2H, s, H-8), 3.54 (2H, q,
H-9) and 1.19 (3H, t, H-10); δC 69.4 (C-8), 66.3 (C-9) and 15.6 (C-10).
The position of ethoxymethyl group was located at C-5 (δC 125.3) by
the HMBC correlation of H-8 (δH 4.50, s) with C-5 (δC 125.3), C-4
(156.4) and C-6 (131.3). As a result, compound 5 was elucidated as
gastropolybenzylol D.
3.1. Structural elucidation
3.1.1. Compound 2
Compound 2 had a molecular formula of C20H18O3 with twelve
degrees of unsaturation, which was deduced from negative HR-ESI-MS
at m/z 305.1168 (calcd. for 305.1183). In the 1H NMR spectrum, three
sets of AB coupled protons at δH 7.25 (2H, d, J = 8.5 Hz) and 6.79 (2H,
d, J = 8.5 Hz), 7.06 (2H, d, J = 8.5 Hz) and 6.87 (2H, d, J = 8.5 Hz),
and 6.98 (2H, d, J = 8.5 Hz) and 6.70 (2H, d, J = 8.5 Hz) indicating the
presence of three para-substituted phenyls. In the shielded region, two
methylenes at δH 4.89 (2H, s) and 3.79 (2H, s) were recognized.
Compared with 1, compound 2 had an additional 4-hydroxybenzyl
moiety: δH 7.25 (2H, d, J = 8.5 Hz, H-2′' and H-6′'), 6.79 (2H, d,
J = 8.5 Hz, H-3′' and H-5′') and 4.89 (2H, s, H-7′'); δC 156.9 (C-4′'),
129.1 (C-2′' and C-6′'), 128.2 (C-1′'), 114.8 (C-3′' and C-5′') and 69.7 (C-
7′'). Based on the HMBC correlation from H-7′' (δH 4.89) to C-4′ (δC
157.2), the ether linkage between C-7′' and C-4′ was established
(Fig. 3). Thus, compound 2 was elucidated as gastropolybenzylol A.
3.1.5. Compound 7
Compound 7 was assigned a molecular formula of C23H24O4 with 12
degrees of unsaturation based on the [M−H]− ion at m/z 363.1588
(calcd for 363.1602). Its 1H NMR indicated the existence of two sets of
ABX coupled protons [δH 6.97 (1H, dd, J = 8.1, 2.1 Hz), 6.89 (1H, d,
J = 2.1 Hz) and 6.73 (1H, d, J = 8.1 Hz); 6.86 (1H, brs), 6.85 (1H, dd,
J = 8.1, 2.0 Hz) and 6.66 (1H, d, J = 8.1 Hz)], one set of AB coupled
protons [δH 6.99 (2H, d, J = 8.5 Hz) and 6.65 (2H, d, J = 8.5 Hz)] and
four sets of methylenes [δH 4.30 (2H, s), 3.78 (2H, s), 3.77 (2H, s) and
3.45 (2H, q)], implying 7 with one more 4-hydroxybenzyl moiety than
4. The HMBC correlations of H-7 (δH 3.78, 2H, s) with C-2 (δC 130.9), C-
6 (δC 130.9), C-2′ (δC 154.3) and C-6′ (δC 132.3) proved that a 4-hy-
droxybenzyl moiety was attached to C-1′ (δC 129.4) (Fig. 3). As a result,
compound 7 was determined as gastropolybenzylol E.
3.1.2. Compound 3
Compound 3 had a molecular formula of C16H18O3 with eight de-
grees of unsaturation, which was speculated from [M−H]− ion at m/z
257.1171 (calcd for 257.1183). Compound 3 had similar NMR data
with 1 besides an extra ethoxymethyl: δH 4.50 (2H, s, H-7′), 3.53 (2H, q,
H-8′) and 1.19 (3H, t, H-9′); δC 67.9 (C-7′), 65.5 (C-8′), 14.1 (C-9′). The
alkylation position at C-5′ (δC 123.9) was proved by the key HMBC
correlations of H-7′ (δH 4.50, 2H, s) with C-4′ (δC 153.4), C-6′ (δC 129.6)
3.1.6. Compound 9
Compound 9 was assigned a molecular formula of C30H30O5 with 16
degrees of unsaturation according to the [M−H]− ion at m/z 469.2024
(calcd for 469.2020). The 1H NMR showed an ethoxyl [δH 3.43 (2H, q)
and 1.13 (3H, t)] and five methylenes [δH 4.25(2H, s), 3.88 (2H, s), 3.84
(2H, s), 3.80 (2H, s) and 3.43 (2H, q)]. The remaining signals were all
aromatic protons with two sets of AB coupled protons [δH 7.03 (2H, d,
J = 8.5 Hz) and 6.70 (2H, d, J = 8.5 Hz); 7.01(2H, d, J = 8.5 Hz) and
6.67 (2H, d, J = 8.5 Hz)], one ABX coupled protons [δH 6.86 (1H, d,
and C-8′ (δC 65.5). Thus, compound
3 was defined as gastro-
polybenzylol B.
3.1.3. Compound 4
Compound 4 showed a molecular formula of C16H18O3 with eight
degrees of unsaturation according to the [M−H]− ion at m/z 257.1166
7
1
1'
7'
6'
8'
7''
7
6'
7
7'
1
1'
1
1'
HO
O
1'
O
O
4'
8'
1''
9'
9'
6
OH
HO
OH
HO
7''
HO
2
3
4
6'
9
8
6
6'
7'
7'
7
6''
7
1
1
1''
1'
O
O
O
8'
9'
8''
10
9''
HO
HO
5
HO
HO
HO
7
OH
3'
7'''
7
7''
1
1'' 3''
1'''
1H-1H COSY
HMBC
HO
OH
OH
5'
7'
O
9
Fig. 3. Key 1H−1H COSY and the HMBC correlation of compounds 2−5, 7 and 9.
6

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 4
3.2. Agonistic activity and structure-activity relationships
Agonistic rates of compounds 1–19 on MT1 and MT2 receptors (0.5 mM).
Agonistic activities of all compounds on melatonin receptors were
evaluated at the concentration of 0.5 mM (Table 4). Compound 1
showed potent activities on MT1 and MT2 receptors with agonistic rates
Comp.
Agonistic rates (%)
Comp.
Agonistic rates (%)
2
MT1
MT2
MT1
MT2
of 80.3
14.4% and 103.0
7.1%. Compounds 10, 11 and 12
1
2
3
4
5
6
7
8
9
80.3
−5.6
37.0
3.3
14.4
1.6
103.0
15.3
7.1
0.3
16.0
13.3
10
11
12
13
14
15
16
17
18
19
45.9
44.6
−9.5
0.3
18.0
6.4
67.1
94.1
44.7
8.5
13.0
54.7
7.6
containing heteroatoms showed decreased activities, indicating the
methylene moiety were crucial to the activity. When one of the hy-
droxyl group in 1 was etherified by 4-hydroxybenzyl moiety, the ob-
tained derivative 2 lost activity. The same results were observed in
13−15 by comparing with 11, which suggested that two hydroxyls
were crucial for maintaining activity. Compounds 4 and 5 with an
adjacent hydroxyl group (C-2′) were inactive in this tests, indicating the
importance of two para-hydroxyl groups (C-4′). Based on the above
results, two para-hydroxy groups were supposed as the vital site in
binding to melatonin receptors. The trimer 6 showed activities on MT1/
18.1
117.1
38.3
0.8
0.3
1.8
1.8
−11.2
46.5
−5.7
25.1
−5.3
1.7
4.5
5.3
4.4
−0.4
−2.3
37.1
31.3
−8.6
−2.3
3.0
1.7
−3.6
−2.2
51.2
48.4
−6.6
−6.7
1.7
1.9
134.2
−10.4
59.4
31.5
1.0
0.5
28.8
16.4
1.7
7.7
16.8
2.6
4.1
33.0
1.3
29.49
1.6
0.4
6.0
The agonistic activities were expressed as Mean
SD (n = 3). Melatonin was
used as the positive control with EC50 values of 1.0 nM (MT1) and 25.0 nM
(MT2).
receptors with agonistic rates of 46.5
4.5% and 134.9
31.5%,
2
while the agonistic activities of 16 and 17 decreased when heteroatom
was introduced into the structure. The tetramers 9 and 19 showed no
activity, which might be due to the steric hindrance. The most active 1
was further investigated for the dose-response relationship on MT1 and
MT2 receptors, which provided the EC50 values of 237 μM and 244 μM,
respectively (Fig. 4).
3.3. Potential mechanisms of compound 1 for activating MT2 receptor
Binding affinity could verify the interaction between compounds
and receptors. The binding affinities of compound 1 and melatonin with
MT2 receptor were −6.9 kcal/mol and −6.4 kcal/mol, respectively,
suggesting that 1 could interact with MT2 receptor. To further predict
interaction between the MT2 receptor and ligands, pharmacophore
analysis was performed by Discovery Studio 4.5. Compound 1 showed
interaction with Phe 118, Gly 121, His 208, Try 294 and Ala 297 re-
sidues (Fig. 5), similar to melatonin (Fig. 6). Furthermore, two para-
hydroxyl groups could form hydrogen bonds with the His 208 and Phe
118 residues of MT2 receptor.
Fig. 4. The dose-dependent effects of compound 1 on MT1 and MT2 receptors,
which provided the EC50 values of 237 μM and 244 μM, respectively. The
agonistic activities were expressed as Mean
SD (n = 3).
J = 2.1 Hz), 6.85 (1H, dd, J = 8.5, 2.1 Hz) and 6.70 (1H, d,
J = 8.5 Hz)] and a 1,2,3,5-subtituted aromatic protons [δH 6.81 (1H, d,
J = 1.8 Hz) and 6.76 (1H, d, J = 1.8 Hz)]. Compared with the NMR
spectra of 7, compound 9 had one more 4-hydroxybenzyl moiety: δH
7.03 (2H, d, J = 8.5 Hz, H-2 and H-6), 6.70 (2H, d, J = 8.5 Hz, H-3 and
H-5) and 3.88 (2H, s, H-7); δC 156.5 (C-4), 133.2 (C-1), 130.9 (C-2 and
C-6), 116.2 (C-3 and C-5) and 36.2 (C-7). The HMBC correlations of H-7
(δH 3.88, 2H, s) and H-7′' (δH 3.84, 2H, s) with C-2′ (δC 153.2) verified
the connection. Accordingly, compound 9 was defined as gastro-
polybenzylol F.
4. Conclusion
In this research, polybenzyls were first revealed with agonistic ef-
fects on melatonin receptors. Especially, compound 1 showed fasci-
nating effects on MT1 and MT2 receptors with EC50 values of 237 and
244 μM. For better understanding their structure-activity relationships,
both synthesis and docking simulation were conducted, which sug-
gested that two para-hydroxy groups were the key pharmacophore for
maintaining activity. Compound 1 was considered as a potential can-
didate for melatonergic agonists.
Fig. 5. Interaction between compound 1 (yellow) and MT2 receptor in 3D diagram (a: left) and 2D diagram (b: right). Hydrogen bonds and hydrophobic effects were
marked as green and pink balls, respectively. The binding affinity was −6.9 kcal/mol.
7

S.-Y. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 6. Interaction between melatonin (yellow) and MT2 receptor in 3D diagram (a: left) and 2D diagram (b: right). Hydrogen bonds and hydrophobic effects were
marked as green and pink balls, respectively. The binding affinity was −6.4 kcal/mol.
Acknowledgments
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This work was supported by the National Natural Science
Foundation of China (No. 81773612), the Program of Yunling Scholar,
the Youth Innovation Promotion Association, CAS (2013252), and the
Applied Basic Research Programs of Yunnan Province (2017FB137).
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Biochem Behav. 2014;126:146–151.
Supplementary data (1D, 2D NMR, UV, IR, HRESIMS spectra for
compounds 2-5, 7 and 9; 1D NMR, UV, IR, HRESIMS spectra for the
derivatives 12-19) to this article can be found online at https://doi.org/
10.1016/j.bmc.2019.06.008.
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trimethylsilylation, and carbethoxylation of some polycyclic aromatic hydrocarbons
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