Design, synthesis, biological evaluation, and molecular docking of novel flavones as H3R inhibitors

Article abstract of DOI:10.1111/cbdd.12981

A series of novel flavone derivatives were designed, synthesized, and evaluated for their H₃R inhibitory activity. The results showed that four compounds exhibited significant anti-H₃R activity. Molecular docking experiments indicated that a salt bridge, hydrogen-bonding, and hydrophobic interactions all contributed to interactions between inhibitors and H₃R.

Full text of DOI:10.1111/cbdd.12981

MS. DONGMEI WANG (Orcid ID : 0000-0003-1489-0502)
Received Date : 27-Nov-2016
Revised Date : 25-Feb-2017
Accepted Date : 06-Mar-2017
Article type
: Research Article
Design, Synthesis, Biological Evaluation, and Molecular Docking of Novel
Flavones as H3R Inhibitors
Gang Wen †, Qian Liu†, Huabin Hu, Dongmei Wang *, Song Wu *
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines &
Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing,
100050
† These authors contributed equally to this work.
Corresponding author mail id: 1428724836@qq.com
Abstract: A series of novel flavone derivatives were designed, synthesized, and evaluated for
their H3R inhibitory activity. The results showed that four compounds exhibited significant
anti-H3R activity. Molecular docking experiments indicated that a salt bridge,
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12981
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hydrogen-bonding, and hydrophobic interactions all contributed to interactions between
inhibitors and H3R.
Key words: H3R inhibitor, flavone, molecular docking, homology modeling, crystal structure
Histamine is a neurotransmitter or neuromodulator that functions in the central nervous
system, and its expression is restricted to the brain (1). It exerts its biological action through
four histamine receptors (H1R, H2R, H3R, H4R) (2), of which H3R was discovered in 1983 as
a presynaptic auto-receptor (3) and a member of the superfamily of G protein-coupled
receptors (GPCRs). H3R not only modulates the synthesis and release of histamine from
histaminergic neurons, but also has a modulatory effect on other neurotransmitters including
acetylcholine (ACh) (4), norepinephrine (NE) (5), dopamine (DA) (6), serotonin (5-HT) (7),
and others. H3R is associated with neurobehaviors such as cognition, the sleep-wake cycle (8),
and anxiety (9) . Furthermore, inhibition of H3R enhances memory performance and
improves cognitive ability (10, 11). Therefore, antagonists of H3R have attracted considerable
interest from the medicinal chemistry community and are sought for the potential treatment
of a variety of disorders such as attention deficit hyperactivity disorder (12-14), Alzheimer’s
disease (15-17), diabetes (18) and sleep disorders (19).
In the current study, high-throughput screening of compound libraries (IMM, Institute of
Material Medica) revealed that compound 4, belonging to the flavone class, has a significant
anti-H3R activity (Figure 1). It was the first found that flavones could inhibit H3R. The
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molecular skeletone of flavones is the preferred structure which possess hepatoprotective,
anti-inflammatory (20, 21), anti-oxidation (22, 23), antibiosis (24) anticancer (25, 26),
antiviral (27), cardiovascular-protective (28), and anticoagulation (29) activities. To increase
the flavones inhibitory activity of H3R, further optimization and modification based on the
structure of compound 4 was conducted to develop new flavone derivatives with potent H3R
inhibitory activity. Considering structural diversity, we choose to vary the flavone nucleus by
introducing different groups onto baicalein, chrysin, luteolin, apigenin and formononetin. As
shown in Table 1, a series of novel flavone derivatives were designed, synthesized, and
evaluated for their H3R inhibitory activities. The IC50 of four compounds that possessed good
anti-H3R activity were tested and the result was showed in Figure 2. Additionally, molecular
docking studies were performed to investigate the interactions between H3R and the most
active inhibitors.
Experimental Procedures
Unless otherwise indicated, all solvents and organic reagents were obtained from
commercially available sources and were used without further purification. The reaction
process was monitored using thin layer chromatography (TLC) with silica gel plates
(thickness = 0.20 mm, GF254) under UV light. Column chromatography was performed using
a ZCX-II, (200–300 mesh) to purify the products. Melting points were determined using an
YRT-3 apparatus (Tian Jin Optical Instrument Factory, China) and are presented as
1
uncorrected values. H NMR spectra was recorded on a Varian Mercury-300 or 400 MHz
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instrument, while 13C NMR was recorded at 400MHz or 600MHz on a Varian Mercury using
1
DMSO-d6 as a solvent and tetramethylsilane (TMS) as an internal standard ( H NMR and
13C NMR were recorded in different time.). Mass spectra were obtained using a Waters
Acquity UPLC-SQD mass spectrometer. High resolution mass spectra (HRMS) were
recorded on an Agilent Technologies LC/MSD TOF spectrometer.
Chemistry
General procedure for synthesis of compounds 2–10
Baicalein (0.50 g, 1.85 mmol), 37% formalin (0.30 g, 3.70 mmol), aliphatic amine (3.50
mmol), and methanol (40 mL) were added to a three-necked flask (100 mL) and stirred at
70C. After the reaction was complete based on TLC (CH2Cl2:CH3OH = 10:1), solvent was
removed under reduced pressure. The mixture was poured into water (100 mL) and the
precipitate was filtered. The residue was purified by column chromatography using a mixture
of dichloromethane and methanol (30:1) as eluent to give the target compounds in yields of
41.091.0%.
Compounds 11–15 were synthesized from chrysin, compounds 16–17 were synthesized from
luteolin, and compound 18 was synthesized from apigenin (all using the same procedure
described above).
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General procedure for synthesis of compounds 21–26
Formononetin (0.50 g, 1.86 mmol), potassium carbonate (2.83 g, 20.46 mmol),
1,2-dibromaethane (1.61 mL, 18.64 mmol), and acetone (50 mL) were added to a
three-necked flask (100 mL) and stirred at 80C. After the reaction was complete based on
TLC (CH2Cl2:CH3OH = 10:1), solvent was removed under reduced pressure. The residue was
poured into water (150 mL) and the precipitate was filtered. The residue was purified by
recrystallization using ethanol to give the key intermediate 20 in a yield of 85%.
2-bromoethoxy formononetin (0.50 g, 1.33 mmol), potassium carbonate (3.00 g, 21.71 mmol),
aliphatic amine (2.00 mmol), and DMF (30 mL) were added to a three-necked flask (100 mL)
and stirred at 100C. After the reaction was complete based on TLC (ethyl acetate), the
mixture was poured into water (200 mL) and the precipitate was filtered. The residue was
purified by column chromatography using ethyl acetate to give the target compounds in
yields of 75–77%.
1a
1b
1c
1d
R1 = OH, R2 = R3 = H
R1 = R2 = R3 = H
2–18
R1 = H, R2 = R3 = OH
R1 = R2 = H, R3 = OH
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Scheme 1: Synthesis of compounds 2–18. Reagents and conditions: (i) NHR5R6, HCHO (aq.),
MeOH, reflux, 8 h.
19
20
21–26
Scheme 2: Synthesis of compounds 21–26. Reagents and conditions: (i) BrCH2CH2Br,
K2CO3, acetone, reflux, 8 h; (ii) NHR1R2, K2CO3, DMF, reflux, 3 h.
Title compounds were characterized as follows:
5,6,7-trihydroxy-2-phenyl-8-(piperidin-1-ylmethyl)-4H-chromen-4-one (2)
1
mp: 214C–215C; H NMR (300MHz, DMSO-d6) δ ppm: 8.04 (m, 2H), 7.57 (m, 3H), 6.84
(s, 1H), 4.16 (s, 2H), 2.84 (brs, 4H), 1.62 (brs, 4H), 1.48 (brs, 2H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 181.41, 161.53, 148.80, 144.60, 131.71, 131.54, 129.98, 129.35, 126.31,
104.66, 102.07, 97.17, 52.95, 52.59, 24.64, 22.82; HR-MS (ESI): Calcd for [M+H]+:
368.1498, found: 368.1486.
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5,6,7-trihydroxy-2-phenyl-8-(piperazin-1-ylmethyl)-4H-chromen-4-one (3)
mp: 233C–235C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.06 (m, 2H), 7.56 (m, 3H), 6.92
(s, 1H), 3.97 (s, 2H), 2.67 (brs, 4H), 2.48 (brs, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
182.35, 162.61, 155.03, 148.22, 146.03, 132.00, 131.32, 129.39, 129.17, 126.48, 104.60,
103.69, 100.27, 51.91, 51.24, 49.47, 43.30; HR-MS (ESI): Calcd for [M+H]+: 369.1450,
found: 369.1430.
5,6,7-trihydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-phenyl-4H-chromen-4-one
(4)
mp: 115C–117C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.06 (m, 2H), 7.57 (m, 3H), 6.89
(s, 1H), 4.01 (s, 2H), 3.48 (t, J = 6.1 Hz, 2H), 2.68 (brs, 4H), 2.48 (brs, 4H), 2.41 (t, J = 6.1
Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 182.19, 162.40, 156.08, 148.20, 145.77,
131.97, 131.39, 129.40, 126.46, 104.63, 103.40, 99.53, 59.91, 58.43, 52.75, 51.83; HR-MS
(ESI): Calcd for [M+H]+: 413.1713, found: 413.1686.
5,6,7-trihydroxy-8-((4-methylpiperidin-1-yl)methyl)-2-phenyl-4H-chromen-4-one (5)
mp: 185C–186C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.06 (m, 2H), 7.57 (m, 3H), 6.84
(s, 1H), 4.17 (s, 2H), 3.14 (m, 2H), 2.56 (m, 2H), 1.69 (m, 2H), 1.50 (brs, 1H), 1.23 (m, 2H),
13
0.91 (d, J = 6.4 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ ppm: 181.51, 161.67, 159.72,
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148.73, 144.85, 131.76, 131.50, 129.88, 129.35, 126.35, 104.68, 102.24, 97.23, 52.41, 51.99,
32.71, 29.20, 21.45; HR-MS (ESI): Calcd for [M+H]+: 382.1654, found: 382.1642.
5,6,7-trihydroxy-2-phenyl-8-(pyrrolidin-1-ylmethyl)-4H-chromen-4-one (6)
mp: 205C–206C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.03 (m, 2H), 7.57 (m, 3H), 6.79
(s, 1H), 4.33 (s, 2H), 3.09 (brs, 4H), 1.89 (brs, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
180.65, 163.24, 160.79, 149.74, 143.47, 131.68, 131.47, 130.39, 129.31, 126.22, 104.68,
100.79, 96.89, 52.97, 49.51, 23.14; HR-MS (ESI): Calcd for [M+H]+: 354.1341, found:
354.1309.
5,6,7-trihydroxy-8-((4-hydroxypiperidin-1-yl)methyl)-2-phenyl-4H-chromen-4-one (7)
mp: 99C–101C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.05 (m, 2H), 7.57 (m, 3H), 6.85 (s,
1H), 4.14 (s, 2H), 3.62 (brs, 1H), 3.01 (m, 2H), 2.64 (m, 2H), 1.79 (m, 2H), 1.51 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) δ ppm: 181.60, 161.75, 159.30, 148.64, 144.93, 131.78, 131.49,
129.84, 129.36, 126.36, 104.66, 102.39, 97.68, 52.29, 49.49, 33.13; HR-MS (ESI): Calcd for
[M+H]+: 384.1447, found: 384.1414.
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8-((4-benzylpiperidin-1-yl)methyl)-5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one (8)
mp: 212C–213C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.03 (m, 2H), 7.57 (m, 3H), 7.26
(m, 2H), 7.16(m, 3H), 6.84 (s, 1H), 4.13 (s, 2H), 3.09 (d, J = 12.2 Hz, 2H), 2.50 (m, 4H),
1.65 (m, 3H), 1.297 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 181.56, 161.70, 148.58,
144.84, 140.05, 131.77, 131.50, 129.83, 129.35, 129.21, 128.40, 126.34, 126.10, 104.67,
102.32, 97.50, 52.52, 51.94, 41.85, 36.18, 30.67; HR-MS (ESI): Calcd for [M+H]+: 458.1967,
found: 458.1938.
8-(((3R,5S)-3,5-dimethylmorpholino)methyl)-5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one
(9)
mp: 198C–199C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.06 (m, 2H), 7.58 (m, 3H), 6.94
(s, 1H), 3.93 (s, 2H), 3.56 (m, 2H), 2.86 (t, J = 10.8 Hz, 2H), 1.94 (t, J = 10.8 Hz, 2H), 1.04
(d, J = 6.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 182.45, 162.70, 154.58, 148.08,
146.23, 132.05, 131.31, 129.38, 129.16, 126.50, 104.60, 103.89, 100.20, 71.18, 58.14, 51.69,
19.04; HR-MS (ESI): Calcd for [M+H]+: 398.1604, found: 398.1630.
5,6,7-trihydroxy-8-((4-(hydroxymethyl)piperidin-1-yl)methyl)-2-phenyl-4H-chromen-4-one
(10)
mp: 198C–199C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.05 (m, 2H), 7.57 (m, 3H), 6.84
(s, 1H), 4.17 (s, 2H), 3.25 (m, 2H), 3.14 (d, J = 11.8 Hz, 2H), 2.56 (t, 2H), 1.74 (d, J = 12.0
Hz, 2H), 1.52 (brs, 1H), 1.25 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 181.48,
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161.61, 159.95, 148.67, 144.71, 131.74, 131.52, 129.92, 129.35, 126.33, 104.66, 102.18,
97.26, 65.34, 52.63, 51.82, 37.18, 27.70; HR-MS (ESI): Calcd for [M+H]+: 398.1604, found:
398.1568.
5,7-dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-2-phenyl-4H-chromen-4-one (11)
mp: 181C–183C; 1H NMR (300MHz, DMSO-d6) δ ppm: 12.86 (s, 1H), 8.08 (m, 2H), 7.60
(m, 3H), 6.97 (s, 1H), 6.22 (s, 1H), 3.88 (s, 2H), 2.59 (brs, 4H), 2.35 (brs, 4H), 2.15 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ ppm: 182.20, 165.19, 163.00, 160.51, 155.24, 132.21,
131.12, 129.41, 126.58, 105.19, 103.81, 100.92, 99.17, 54.66, 52.20, 51.24, 45.69; HR-MS
(ESI): Calcd for [M+H]+: 367.1658, found: 367.1625.
8-(((3R,5S)-3,5-dimethylmorpholino)methyl)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one
(12)
mp: 191C–192C; 1H NMR (300MHz, DMSO-d6) δ ppm: 12.87 (s, 1H), 8.07 (m, 2H), 7.58
(m, 3H), 6.98 (s, 1H), 6.23 (s, 1H), 3.83 (s, 2H), 3.54 (m, 2H), 2.82 (t, J = 10.7 Hz, 2H), 1.88
(t, J = 10.7 Hz, 2H), 1.03 (d, J = 6.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 182.31,
164.55, 163.13, 160.57, 155.38, 132.26, 131.09, 129.40, 126.61, 105.22, 104.06, 100.98,
99.05, 71.21, 58.44, 51.06, 19.09; HR-MS (ESI): Calcd for [M+H]+: 382.1654, found:
382.1621.
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5,7-dihydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-phenyl-4H-chromen-4-one
(13)
mp: 164C–165C; 1H NMR (300MHz, DMSO- d6) δ ppm: 12.85 (s, 1H), 8.08 (m, 2H), 7.60
(m, 3H), 6.97 (s, 1H), 6.19 (s, 1H), 3.89 (s, 2H), 3.46 (t, J = 6.3 Hz, 2H), 2.59(brs, 8H),
2.49(brs, 4H), 2.39 (t, J = 6.3 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 182.17,
165.33, 162.96, 160.56, 155.15, 132.20, 131.11, 129.40, 126.59, 126.55, 105.20, 103.79,
100.68, 99.19, 60.18, 58.68, 53.13, 52.30, 51.48; HR-MS (ESI): Calcd for [M+H]+: 397.1763,
found: 397.1764.
5,7-dihydroxy-2-phenyl-8-(pyrrolidin-1-ylmethyl)-4H-chromen-4-one (14)
mp: 179C–181C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.04 (m, 2H), 7.59 (m, 3H), 6.90
(s, 1H), 6.04 (s, 1H), 4.13 (s, 2H), 2.80 (brs, 4H), 1.80 (brs, 4H); 13C NMR (400 MHz,
DMSO-d6) δ ppm: 181.55, 168.50, 162.34, 160.77, 132.00, 131.26, 129.38, 126.47, 105.09,
102.47, 100.34, 99.86, 52.99, 49.39, 23.34; HR-MS (ESI): Calcd for [M+H]+: 338.1392,
found: 338.1383.
8-((4-benzylpiperidin-1-yl)methyl)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one (15)
mp: 175C–176C; 1H NMR (300MHz, DMSO-d6) δ ppm: 12.85 (s, 1H), 8.06 (m, 2H), 7.59
(m, 3H), 7.14 (m, 5H), 6.94 (s, 1H), 6.11 (s, 1H), 3.95 (s, 2H), 2.97 (d, J = 10.8 Hz, 2H), 2.25
13
(t, J = 10.8 Hz, 2H), 1.60 (m, 2H), 1.16 (m, 2H); C NMR (100 MHz, DMSO-d6) δ ppm:
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186.51, 166.90, 162.76, 160.62, 154.93, 140.29, 132.16, 131.15, 129.41, 129.18, 128.36,
126.53, 126.01, 105.22, 103.35, 99.82, 99.51, 52.42, 42.08, 36.80, 31.42; HR-MS (ESI):
Calcd for [M+H]+: 442.2018, found: 442.1981.
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-8-(morpholinomethyl)-4H-chromen-4-one (16)
mp: 153C–154C; 1H NMR (300MHz, DMSO-d6) δ ppm: 13.03 (s, 1H), 7.42 (m, 2H), 6.99
(d, J = 8.9 Hz, 1H), 6.68 (s, 1H), 6.21 (s, 1H), 3.81 (s, 2H), 3.57 (brs, 4H), 3.53 (brs, 4H); 13C
NMR (100 MHz, DMSO-d6) δ ppm: 182.12, 164.04, 163.86, 160.57, 155.32, 149.93, 145.98,
121.90, 119.19, 116.26, 113.63, 103.75, 102.84, 100.96, 98.79, 66.32, 52.92, 51.16; HR-MS
(ESI): Calcd for [M+H]+: 386.1240, found: 386.1226.
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-8-((2-methylpiperidin-1-yl)methyl)-4H-chromen-4-o
ne (17)
mp: 214C–216C; 1H NMR (300MHz, DMSO-d6) δ ppm: 13.02 (s, 1H), 7.40 (d, J = 3.6Hz,
2H), 7.39 (brs, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.62 (s, 1H), 6.04 (s, 1H), 4.29 (d, J = 14.8 Hz,
2H), 3.87 (d, J = 14.8 Hz, 1H), 2.90 (m, 1H), 2.72 (brs, 1H), 2.38 (m, 1H), 1.64 (m, 3H), 1.45
13
(m, 1H), 1.38 (m, 2H), 1.18 (d, J = 6.3 Hz, 3H); C NMR (150 MHz, DMSO-d6) δ ppm:
181.71, 167.17, 163.43, 160.50, 154.29, 149.89, 145.96, 121.86, 119.03, 116.22, 113.43,
102.96, 102.84, 99.50, 99.45, 56.26, 49.70, 33.35, 25.14, 22.29. HR-MS (ESI): Calcd for
[M+H]+: 398.1604, found: 398.1581.
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5,7-dihydroxy-2-(4-hydroxyphenyl)-8-((3-hydroxypiperidin-1-yl)methyl)-4H-chromen-4-one
(18)
1
mp: 228C–230C; H NMR (300MHz, DMSO-d6) δ ppm: 12.98 (brs, 1H), 7.92 (d, J = 8.7
Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.75 (s, 1H), 6.11 (s, 1H), 3.91 (s, 2H), 3.55 (brs, 1H), 2.86
(m, 1H), 2.70 (m, 1H), 2.26 (d, J = 9.3 Hz, 1H), 2.19 (d, J = 9.3 Hz, 1H),1.72 (m, 2H), 1.40
(m, 1H), 1.23 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ ppm: 181.96, 165.76, 163.45,
161.31, 160.55, 154.83, 128.58, 121.58, 116.19, 103.30, 102.84, 100.07, 99.15, 65.49, 59.96,
52.37, 52.15, 32.35, 22.45; HR-MS (ESI): Calcd for [M+H]+: 384.1447, found: 384.1460.
7-(2-bromoethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (20)
mp: 178C–180C; 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.42 (s, 1H), 8.03 (d, J = 8.9 Hz,
1H), 7.52 (d, J = 8.2 Hz, 2H), 7.20 (s, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 7.9 Hz, 2H),
4.49 (t, J = 5.1 Hz, 2H), 3.86 (m, 2H), 3.78 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
174.77, 162.40, 159.19, 153.71, 130.24, 127.27, 124.18, 123.57, 118.09, 115.15, 113.79,
101.60, 68.70, 55.32 , 31.14; HR-MS (ESI): Calcd for [M+H]+: 375.0232, found: 375.0219.
3-(4-methoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one (21)
mp: 124C–126C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.39 (s, 1H), 8.01 (d, J = 8.9 Hz,
1H), 7.52 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 2.1 Hz, 1H), 7.04 (dd, J = 8.9, 2.1 Hz, 1H), 6.96 (d,
J = 8.7 Hz, 1H), 4.20 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 2.66 (t, J = 5.8 Hz, 2H), 2.42 (m, 4H),
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1.48 (m, 4H), 1.37 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 174.81, 163.15, 159.19,
157.61, 153.67, 130.26, 127.08, 124.26, 123.53, 117.72, 115.32, 113.80, 400.32, 66.75, 57.30,
55.33, 54.56, 25.75, 24.10; HR-MS (ESI): Calcd for [M+H]+: 380.1862, found: 380.1826.
3-(4-methoxyphenyl)-7-(2-morpholinoethoxy)-4H-chromen-4-one (22)
mp: 125C–127C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.39 (s, 1H), 8.02 (d, J = 8.9 Hz,
1H), 7.51 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.9, 2.2 Hz, 1H), 6.97 (d,
J = 8.7 Hz, 1H), 4.19 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 3, 56 (m, 4H), 2.27 (t, J = 5.8 Hz, 2H),
2.48 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 174.81, 163.08, 159.19, 157.60,
153.68, 130.26, 127.11, 124.25, 123.55, 117.76, 115.31, 113.81, 400.34, 66.42, 66.35, 56.93,
55.34, 53.76; HR-MS (ESI): Calcd for [M+H]+: 382.1654, found: 382.1619.
7-(2-(4-hydroxypiperidin-1-yl)ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (23)
mp: 187C–189C; 11H NMR (300MHz, DMSO-d6) δ ppm: 8.40 (s, 1H), 8.01 (d, J = 8.9 Hz,
1H), 7.52 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 2.1 Hz, 1H), 7.08 (dd, J = 8.9, 2.1 Hz, 1H), 6.96 (d,
J = 8.6 Hz, 1H), 4.53 (d, J = 4.1 Hz, 1H), 4.20 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 3.42 (brs,
13
1H), 2.78 (m, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.12 (m, 2H), 1.67 (m, 2H), 1.38 (m, 2H); C
NMR (100 MHz, DMSO-d6) δ ppm: 174.82, 163.15, 159.20, 157.62, 153.68, 130.27, 127.10,
124.27, 123.54, 117.73, 115.33, 113.82, 400.34, 66.91, 66.39, 56.56, 55.34, 51.59, 40.25,
34.61; HR-MS (ESI): Calcd for [M+H]+: 396.1811, found: 396.1778.
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3-(4-methoxyphenyl)-7-(2-(3-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one (24)
mp: 134C–136C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.39 (s, 1H), 8.01 (d, J = 8.9 Hz,
1H), 7.50 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 2.3 Hz, 1H), 7.08 (d, J = 8.9, 2.3 Hz, 1H), 6.96 (d, J
= 8.8 Hz, 1H), 4.20 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 2.80 (m, 2H), 2.66 (t, J = 5.8 Hz, 2H),
1.91 (m, 1H), 1.60 (m, 5H), 0.82 (d, J = 8.9 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
174.81, 163.16, 159.19, 157.62, 153.67, 130.26, 127.09, 124.26, 123.54, 117.72, 115.32,
113.80, 400.33, 66.74, 62.06, 57.05, 55.33, 53.99, 39.16, 32.70, 30.84, 25.26, 19.78; HR-MS
(ESI): Calcd for [M+H]+: 394.2018, found: 394.1987.
7-(2-(4-benzylpiperidin-1-yl)ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (25)
mp: 141C–143C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.39 (s, 1H), 8.01 (d, J = 8.9 Hz,
1H), 7.53 (d, J = 8.7 Hz, 2H), 7.28 (m, 2H), 7.14 (m, 4H), 7.05 (dd, J = 8.9, 2.2 Hz, 1H), 6.97
(d, J = 8.7 Hz, 2H), 4.19 (t, J = 5.7 Hz, 2H), 3.78 (s, 3H), 2.88 (d, J = 11.2 Hz, 2H), 2.68 (t, J
= 5.7 Hz, 2H), 2.49 (m, 2H), 1.95 (t, J = 11.2 Hz, 2H), 1.51 (m, 3H), 1.19 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) δ ppm: 174.81, 163.14, 159.19, 157.60, 153.67, 140.55, 130.26,
129.16, 128.29, 127.09, 125.90, 124.26, 123.54, 117.72, 115.31, 113.80, 400.32, 66.79, 56.86,
55.34, 53.85, 42.53, 37.41, 31.90; HR-MS (ESI): Calcd for [M+H]+: 470.2331, found:
470.2300.
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3-(4-methoxyphenyl)-7-(2-(4-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one (26)
mp: 149C–150C; 1H NMR (300MHz, DMSO-d6) δ ppm: 8.41 (s, 1H), 8.02 (d, J = 8.9 Hz,
1H), 7.50 (d, J = 7.5 Hz, 2H), 7.17 (s, 1H), 7.08 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H),
4.21 (brs, 2H), 3.77 (s, 3H), 2.87 (d, J = 9.5Hz, 2H), 2.70 (brs, 2H), 1.99 (t, J = 11.4 Hz, 2H),
1.53 (d, J = 9.5 Hz, 2H), 1.11 (m, 3H), 0.86 (d, J = 6.0 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 174.82, 163.16, 159.19, 157.62, 153.68, 130.27, 127.10, 124.27, 123.55,
117.72, 115.33, 113.81, 400.33, 66.82, 56.92, 55.34, 53.96, 34.16, 30.39, 22.04; HR-MS (ESI):
Calcd for [M+H]+: 394.2018, found: 394.1986.
Biochemical methods
Cell culture
U2OS cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM; Hyclone,
Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Shanghai, China)
at 37C in a humidified atmosphere with 5% CO2. Exponentially growing cells were adjusted
to a density of 5104 cells /mL and inoculated into 32  32 wing cells plates. Cells were
incubated at 37C for 1824 h and used in subsequent experiments.
Fluorescent H3R assay
Cells were incubated in medium containing either different samples, thiazole amide (positive
control), or no sample (blank control) for 5 h. Then, LiveBLAzer-FRET B/G Substrate
(CCF4-AM) was added and incubation continued for 2 h. Plates were subjected to
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fluorescence reading from the bottom with a WALLAC Victor 2 plate reader (Perkin Elmer,
Waltham, MA, USA) equipped with 410 nm excitation and 460 nm and 530 nm emission
filters. The inhibition rate was calculated based on the fluorescence using the following
equation: % inhibition = (ModelResponse ratio - CompoundResponse ratio) / ModelResponse ratio
.
Computational methods
Homology modeling
To elucidate the interactions between compounds and H3R, we performed molecular docking
studies. And we chose the crystal structure of the human histamine 1 receptor in complex
with the doxepin antagonist as a template (PDB entry 3RZE) (30) to build a homology model
of H3R. The sequence of hH3R was downloaded from the UniProt database (Entry No.
Q9Y5N1) (31). Due to the slight difference between sequence conservation and structural
conservation, we initially aligned the target sequence to the template. The sequence identity
of the two sequences is 31.4% when the intracellular loop ICL3 (Gln222Gly404) is deleted,
which matches the sequence for T4 lysozyme (30). We then used DS MODELER (Discovery
Studio 2016) to build a 3D model of the target structure and evaluated the model according to
the PDF Total Energy and the Profile-3D procedure.
Docking
To consider the conformation of the protein and its ligands, Flexible Docking (Discovery
Studio 2016) was used for the docking procedure. The 3D model of H3R with the lowest PDF
Total Energy was chosen for docking. Water and the initial ligand (doxepin) were removed
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from the model, and hydrogen atoms were added. A 3D structure was also generated for the
compound using DS 2016.
Results and Discussion
Chemistry
Target compounds 2–18 were synthesized from compounds 1a, 1b, 1c, and 1d following the
route summarized in Scheme 1. An efficient Mannich reaction was employed for the
preparation of target compounds 2–18 according to the published procedure (32). The route
utilized 1a, 1b, 1c, and 1d as starting materials, 37% formalin, and an aliphatic amine as the
Mannich reagent.
Derivatives 21–26 were prepared from formononetin 18 by following the synthetic route
detailed in Scheme 2. First, 2-bromoethoxy formononetin 20 was synthesized by refluxing
formononetin 19 with 1,2-dibromaethane and potassium carbonate (K2CO3) in acetone (33,
34), which afforded the product in good yield. Subsequent nucleophilic substitution (35, 36)
with the aliphatic amine using K2CO3 as a base in DMF resulted in the target compounds
21–26.
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Evaluation of H3R inhibitory activity
The in vitro H3R inhibitory activity of the newly synthesized compounds was studied at a
concentration of 25 mol/mL. The percentage inhibition was calculated based on
fluorescence using the following formula: % inhibition = (ModelResponse
-
ratio
CompoundResponse ratio) / ModelResponse ratio
.
Table 1: Structure and H3R inhibition rate (% inhibition) of compounds 2–18 and 21–26 at 25
mol/mL concentration.
% inhibitonb
IC50 (M)
compound R1 R2 R3 R4
R7
H3R
Thiazole
amidea
60.92 ±
3.5
2.87
noc
2
3
4
5
OH H
OH H
OH H
OH H
H
H
H
H
41.65 ±
1.2
13.32 ±
5.1
noc
81.32 ±
4.3
8.58
noc
39.21 ±
2.9
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6
7
8
OH H
OH H
OH H
H
H
H
10.17 ±
5.6
noc
noc
noc
8.87 ±
1.9
5.88 ±
3.8
9
OH H
OH H
H
40.10 ±
4.0
noc
10
11
12
H
V
H
41.82 ±
6.0
noc
noc
noc
H
H
H
H
21.04 ±
2.3
29.63 ±
0.9
13
14
15
H
H
H
H
H
H
H
H
H
12.31 ±
4.5
noc
87.26 ±
3.5
3.40
noc
8.24 ±
1.9
16
17
H
H
OH OH
OH OH
15.47 ±
2.6
noc
89.46 ±
2.8
4.01
18
H
H
OH
37.35 ±
1.6
noc
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21
22
23
24
25
45.85 ±
1.3
noc
noc
noc
5.80
noc
9.23 ±
0.7
26.78 ±
5.2
90.12 ±
4.1
11.95 ±
2.4
26
1.12 ±
6.3
noc
Thiazole amidea, an inhibitor of H3R, was used as a positive control at 5 mol/mL.
% inhibitonb, Value represented as mean ± standard deviation of three independent
experiment ± (n = 3).
noc , IC50 without test.
Homology modeling and docking
To elucidate the interactions between compound 14 and H3R, we performed molecular
docking studies. We chose the crystal structure of the human H1R as the template, which is a
phylogenetically close neighbor of H3R. A total of ten models of H3R were generated using
DS MODELER. The best 3D model is shown in Figure 3. Then compound 14 was then
docked into the active site of H3R using the Flexible Docking function of DS (2016), and the
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results are shown in Figure 4. Protein-ligand interactions include: (1) a salt bridge between
the protonated amine of the pyrrolidine group and Glu206 in TM5; (2) a -anion interaction
between the ligand aromatic system and Asp114 in TM3; (3) hydrophobic interactions
between the hydrophobic part of the ligand and the surrounding Tyr115 and Cys118 of TM3,
Ala202 of TM5, Trp371 and Tyr374 of TM6, and Leu401 of TM7; (4) a hydrogen bond
between the ligand carbonyl group and the hydroxyl group of Tyr374 in TM6. The large
number of interactions revealed by docking results therefore explains the strong inhibitory
activity of compound 14.
Discussion
As summarized in Table 1, most of the title compounds inhibited H3R to a significant extent.
Compounds 2, 5, 9, 10, 18, and 21 exhibited only moderate inhibitory activity, while the
inhibitory activity of compounds 4, 14, 17, and 24 was much higher (IC50 :8.58 mol/mL,
3.40 mol/mL, 4.01 mol/mL, and 5.80 mol/mL, respectively). The result showed that the
flavones had a better inhibitory acticity on H3R than isoflavones. Substituent group at C8
played a significant role in the inhibitory activity. The hydroxyl at C6 also showed an
important effect by comparing compound 4 with 13.
Conclusion
In this study, a series of novel flavone derivatives were designed and synthesized, and
preliminary biological evaluation of their anti-H3R activity indicated that compounds 4, 14,
17, and 24 possessed significant anti-H3R activity. Molecular docking experiments showed
that interactions between the inhibitors and H3R involved a salt bridge, hydrogen-bonding,
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and hydrophobic interactions. In the paper, the H3R inhibitory effects of the flavone
derivatives were first reported, and these results were helpful for future research into the
development of novel H3R inhibitors.
Acknowledgments
This work was supported by Natural Science Foundation of Beijing (No. 7172141).
Conflicts of Interest
The authors declare that they have no conflicts of interest.
References
1. Lovenberg TW, Pyati J, Chang H, Wilson SJ, Erlander MG (2000) Cloning of rat
histamine H3 receptor reveals distinct species pharmacological profiles. Journal of
Pharmacology and Experimental Therapeutics;293: 771-8.
2. Hough LB (2001) Genomics meets histamine receptors: new subtypes, new receptors.
Molecular Pharmacology;59: 415-9.
3. Arrang J-M, Garbarg M, Schwartz J-C (1983) Auto-inhibition of brain histamine release
mediated by a novel class (H3) of histamine receptor.
4. Clapham J, Kilpatrick G (1992) Histamine H3 receptors modulate the release of [3H]‐
acetylcholine from slices of rat entorhinal cortex: evidence for the possible existence of H3
receptor subtypes. British journal of pharmacology;107: 919-23.
5. Schlicker E, Fink K, Hinterthaner M, Göthert M (1989) Inhibition of noradrenaline
release in the rat brain cortex via presynaptic H3 receptors. Naunyn-Schmiedeberg's archives
of pharmacology;340: 633-8.
6. Feuerstein TJ. (2008) Presynaptic receptors for dopamine, histamine, and serotonin.
Presynaptic receptors for dopamine, histamine, and serotonin. Springer: p. 289-338.
7. Schlicker E, Betz R, Göthert M (1988) Histamine H3 receptor-mediated inhibition of
serotonin release in the rat brain cortex. Naunyn-Schmiedeberg's archives of
pharmacology;337: 588-90.
8. Letavic MA, Aluisio L, Apodaca R, Bajpai M, Barbier AJ, Bonneville A, et al. (2015)
Novel Benzamide-Based Histamine H3 Receptor Antagonists: The Identification of Two
Candidates for Clinical Development. ACS medicinal chemistry letters;6: 450-4.
This article is protected by copyright. All rights reserved.

9. Yokoyama F, Yamauchi M, Oyama M, Okuma K, Onozawa K, Nagayama T, et al. (2009)
Anxiolytic-like profiles of histamine H3 receptor agonists in animal models of anxiety: a
comparative
study
with
antidepressants
and
benzodiazepine
anxiolytic.
Psychopharmacology;205: 177-87.
10. Griebel G, Pichat P, Pruniaux M-P, Beeské S, Lopez-Grancha M, Genet E, et al. (2012)
SAR110894, a potent histamine H 3-receptor antagonist, displays procognitive effects in
rodents. Pharmacology Biochemistry and Behavior;102: 203-14.
11. Medhurst AD, Atkins AR, Beresford IJ, Brackenborough K, Briggs MA, Calver AR, et
al. (2007) GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors
in Alzheimer's disease brain and improves cognitive performance in preclinical models.
Journal of Pharmacology and Experimental Therapeutics;321: 1032-45.
12. Wager TT, Pettersen BA, Schmidt AW, Spracklin DK, Mente S, Butler TW, et al. (2011)
Discovery
trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidinylmethyl) phenyl] cyclobutanecarboxamide
(PF-03654746) and trans-3-Fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)
of
Two
Clinical
Histamine
H3
Receptor
Antagonists:
phenyl]-N-(2-methylpropyl) cyclobutanecarboxamide (PF-03654764). Journal of medicinal
chemistry;54: 7602-20.
13. Letavic MA, Aluisio L, Atack JR, Bonaventure P, Carruthers NI, Dugovic C, et al. (2010)
Pre-clinical characterization of aryloxypyridine amides as histamine H 3 receptor antagonists:
identification of candidates for clinical development. Bioorganic & medicinal chemistry
letters;20: 4210-4.
14. Esbenshade T, Browman K, Bitner R, Strakhova M, Cowart M, Brioni J (2008) The
histamine H3 receptor: an attractive target for the treatment of cognitive disorders. British
journal of pharmacology;154: 1166-81.
15. Gemkow MJ, Davenport AJ, Harich S, Ellenbroek BA, Cesura A, Hallett D (2009) The
histamine H3 receptor as a therapeutic drug target for CNS disorders. Drug discovery
today;14: 509-15.
16. Brioni JD, Esbenshade TA, Garrison TR, Bitner SR, Cowart MD (2011) Discovery of
histamine H3 antagonists for the treatment of cognitive disorders and Alzheimer's disease.
Journal of Pharmacology and Experimental Therapeutics;336: 38-46.
17. Singh M, R Jadhav H (2013) Histamine H3 receptor function and ligands: recent
developments. Mini reviews in medicinal chemistry;13: 47-57.
18. Rao AU, Shao N, Aslanian RG, Chan T-Y, Degrado SJ, Wang L, et al. (2011) Discovery
of a potent thiadiazole class of histamine H3 receptor antagonist for the treatment of diabetes.
ACS medicinal chemistry letters;3: 198-202.
19. Mignot E, Taheri S, Nishino S (2002) Sleeping with the hypothalamus: emerging
therapeutic targets for sleep disorders. nature neuroscience;5: 1071-5.
20. Bryan MC, Biswas K, Peterkin TA, Rzasa RM, Arik L, Lehto SG, et al. (2012)
Chromenones as potent bradykinin B1 antagonists. Bioorganic & medicinal chemistry
letters;22: 619-22.
21. Williams DA, Zaidi SA, Zhang Y (2014) 5-Hydroxy-2-(2-phenylethyl) chromone
(5-HPEC): A novel non-nitrogenous ligand for 5-HT 2B receptor. Bioorganic & medicinal
chemistry letters;24: 1489-92.
This article is protected by copyright. All rights reserved.

22. Carneiro CD, Amorim JC, Cadena SM, Noleto GR, Di Mascio P, Rocha ME, et al.
(2010) Effect of flavonoids on 2′-deoxyguanosine and DNA oxidation caused by singlet
molecular oxygen. Food and Chemical Toxicology;48: 2380-7.
23. Guo Y, Sun J, Ye J, Ma W, Yan H, Wang G (2016) Saussurea tridactyla sch. Bip.-derived
polysaccharides and flavones reduce oxidative damage in ultraviolet B-irradiated hacaT cells
via a p38MaPK-independent mechanism. Drug design, development and therapy;10: 389.
24. Xu H-X, Lee SF (2001) Activity of plant flavonoids against antibiotic-resistant bacteria.
Phytotherapy Research;15: 39-43.
25. Romagnolo DF, Selmin OI (2012) Flavonoids and cancer prevention: a review of the
evidence. Journal of nutrition in gerontology and geriatrics;31: 206-38.
26. Liu HL, Jiang WB, Xie MX (2010) Flavonoids: recent advances as anticancer drugs.
Recent patents on anti-cancer drug discovery;5: 152-64.
27. Sanchez I, Gómez‐Garibay F, Taboada J, Ruiz B (2000) Antiviral effect of flavonoids
on the dengue virus. Phytotherapy Research;14: 89-92.
28. Testai L, Martelli A, Cristofaro M, Breschi MC, Calderone V (2013) Cardioprotective
effects of different flavonoids against myocardial ischaemia/reperfusion injury in
Langendorff‐perfused rat hearts. Journal of Pharmacy and Pharmacology;65: 750-6.
29. Lin Y, Shen X, Yuan Q, Yan Y (2013) Microbial biosynthesis of the anticoagulant
precursor 4-hydroxycoumarin. Nature communications;4:
30. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, et al. (2011)
Structure of the human histamine H1 receptor complex with doxepin. Nature;475: 65-70.
31. Jain E, Bairoch A, Duvaud S, Phan I, Redaschi N, Suzek BE, et al. (2009) Infrastructure
for the life sciences: design and implementation of the UniProt website. BMC
bioinformatics;10: 1.
32. Carrasco MP, Newton AS, Gonçalves L, Góis A, Machado M, Gut J, et al. (2014)
Probing the aurone scaffold against Plasmodium falciparum: design, synthesis and
antimalarial activity. European journal of medicinal chemistry;80: 523-34.
33. Li S-Y, Wang X-B, Xie S-S, Jiang N, Wang KD, Yao H-Q, et al. (2013) Multifunctional
tacrine–flavonoid hybrids with cholinergic, β-amyloid-reducing, and metal chelating
properties for the treatment of Alzheimer's disease. European journal of medicinal
chemistry;69: 632-46.
34. Liang Y, Xiang B, Liu C, Zhou X, Wang D, Song S, et al. (2014) Ultraviolet light
absorber with low surface energy: synthesis and characterization. Tetrahedron;70: 6585-93.
35. Zhou D, Tuo W, Hu H, Xu J, Chen H, Rao Z, et al. (2013) Synthesis and activity
evaluation of tilorone analogs as potential anticancer agents. European journal of medicinal
chemistry;64: 432-41.
36. Ma Z, Pan Y, Huang W, Yang Y, Wang Z, Li Q, et al. (2014) Synthesis and biological
evaluation of the pirfenidone derivatives as antifibrotic agents. Bioorganic & medicinal
chemistry letters;24: 220-3.
Figure legends
Figure 1: The structure of compound 4.
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Figure 2：The IC50 of the four componds showed good anti-H3R activity.
Figure 3: The best homology model of H3R generated by DS MODELER.
Figure 4: (A) The best H3R homology and (B) the conformation of compound 14 bound to
the H3R homology model. H-bonds are shown as dotted lines. (C) 2D schematic
representation of the interactions between compound 14 and the H3R homology model.
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