Synthesis, biological evaluation, and molecular docking of dihydroflavonol derivatives as anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-019-02340-6

A series of dihydroflavonol derivatives (4a–4l) were synthesized from chalcones via classical Algar–Flynn–Oyamada (AFO) reaction and characterized on the basis of spectroscopic analyses. All synthesized compounds were evaluated for their inhibitory activity against the pro-inflammatory-inducible TNF-alpha, IL-1beta, and IL-6 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell lines and showed various efficiency. Furthermore, compounds 4d and 4k were selected to examine their in vivo anti-inflammatory activity by using two classical models. Herein compound 4k showed maximum anti-inflammatory activity of 32.98% inhibition in mice ear-swelling model and 40.06% inhibition at the 2 h intervals in rat paw edema model in comparison to the two references: aspirin and meloxicam. Similar effect was observed at a lower dose. In addition, the compound 4k was docked against cyclooxygenases-2 to validate the attained pharmacological data and provide understandable evidence for the observed anti-inflammatory activity.

Full text of DOI:10.1007/s00044-019-02340-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02340-6
ORIGINAL RESEARCH
Synthesis, biological evaluation, and molecular docking of
dihydroflavonol derivatives as anti-inflammatory agents
1
Yuanhang Xiang1 Chunling Hu
Yuejie Zhang1 Xiaochuan Ye1
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Received: 28 November 2018 / Accepted: 29 March 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A series of dihydroflavonol derivatives (4a–4l) were synthesized from chalcones via classical Algar–Flynn–Oyamada (AFO)
reaction and characterized on the basis of spectroscopic analyses. All synthesized compounds were evaluated for their
inhibitory activity against the pro-inflammatory-inducible TNF-alpha, IL-1beta, and IL-6 in lipopolysaccharide (LPS)-
stimulated RAW 264.7 cell lines and showed various efficiency. Furthermore, compounds 4d and 4k were selected to
examine their in vivo anti-inflammatory activity by using two classical models. Herein compound 4k showed maximum
anti-inflammatory activity of 32.98% inhibition in mice ear-swelling model and 40.06% inhibition at the 2 h intervals in rat
paw edema model in comparison to the two references: aspirin and meloxicam. Similar effect was observed at a lower dose.
In addition, the compound 4k was docked against cyclooxygenases-2 to validate the attained pharmacological data and
provide understandable evidence for the observed anti-inflammatory activity.
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●
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Keywords Inflammation Dihydroflavonol Molecular docking Cytokine
Introduction
et al. 2016), anti-plasmodial (Muiva-Mutisya et al. 2018),
and anti-proliferative (Sun et al. 2018) activities. Of which,
the anti-inflammatory activity is one of the most important
and versatile functions of dihydroflavonols (Ding et al.
2018; Sun et al. 2014; Topal et al. 2016)
Inflammation is a condition of body immunity associated
with damaged tissue or organ. The scientific community has
always been interested in the treatment of inflammation
(Cooke and Ferrari 2018). Many trials have been under-
taken to identify compounds from natural resources that
suppress or mitigate the progress of inflammation, espe-
cially in herbal medicine. Flavonoids are often designated
as active constituents in many traditional medicinal plants
(Devi et al. 2015).
Nowadays, naturally-occurring flavonoids are emerging
as a vibrant chemical backbone that could help in the fight
against several human diseases (Singh et al. 2014). Among
which, dihydroflavonols have received more and more
attention for novel structure and excellent pharmacological
activities and been reported to have anti-allergic, anti-
inflammation (Ayoub et al. 2018), anti-microbial (Karim
In previous studies, we found a simple and effective
synthetic route of dihydroflavonols by optimizing the
reaction conditions (Hu et al. 2018). Herein, we synthesized
a series of dihydroflavonol derivatives with 3,5,7-trihy-
droxy-2-phenylchroman-4-one skeleton by introducing dif-
ferent groups in the B ring as described in Scheme 1 and
evaluated their anti-inflammatory activities in vitro and
in vivo. In order to get insights into the binding modes of
these molecules with hypothetical protein target, Autodock
Vina was performed to dock the compounds into the active
site of COX-2 (PDB:4M11). The anti-inflammatory effect
of dihydroflavonol derivatives was duly supported by
molecular docking studies.
Materials and methods
* Chunling Hu
huchunling2007@hbtcm.edu.cn
Chemistry
1
Key Laboratory of Chinese Medicine Chemistry and Chinese
All chemicals used for the synthesis of compounds were
purchased from Sinopharm Chemical Reagent Co. Ltd.
Herbal Compound of Hubei Province, Hubei University of
Traditional Chinese Medicine, Wuhan 430065, China

Medicinal Chemistry Research
Scheme 1 Synthesis of dihydroflavonol derivatives. Reagents and conditions. a DIPEA, MOMCl, CH2Cl2, 0 °C; b NaH, MOMCl, THF, 0 °C; c
substituted benzaldehydes, NaOH, r.t.; d H2O2, NaOH, MeOH, then HCl, 55 °C
(China). All synthesized compounds were checked by thin
layer chromatography (TLC) performed on Silica gel 60
GF254 coated plates. The FT-IR spectra were recorded with
Shimadzu FTIR-8400S spectrometer (Tokyo, Japan).
Nuclear magnetic resonance (NMR) spectra were processed
in DMSO-d6 on a Bruker NMR spectrophotometer operat-
MHz) δ 7.44 (1H, s, H-6), 7.29 (1H, d, J = 8.5 Hz, H-7),
7.18 (1H, d, J = 16.0 Hz, H-8), 7.11 (1H, d, J = 8.4 Hz, H-
6), 6.91 (1H, d, J = 16.1 Hz, H-5), 6.52 (2H, s, H-3′,5′),
5.24 (4H, s, 2′6′-OCH2O-), 5.21 (2H, s, 4′-OCH2O-), 5.14
(4H, s, 3,4-OCH2O-), 3.41 (3H, s, 4′-OCH3), 3.39 (6H, d,
J = 2.6 Hz, 3,4-OCH3), 3.27 (6H, d, J = 1.0 Hz, 2′6′-
OCH3); 13C NMR (DMSO-d6, 100 MHz) δ 193.20 (C=O),
158.74 (C, C-4′), 154.99 (C, C-2′,6′), 149.17 (C, C-3),
146.75 (C, C-4), 144.51 (CH, C-8), 128.30 (C, C-1), 127.51
(CH, C-7), 123.82 (CH, C-6), 116.42 (CH, C-5), 116.17
(CH, C-2), 114.41 (C, C-1′), 96.55 (CH, C-3′,5′), 94.63
(CH2, 4-OCH2O-), 94.41 (CH2, 3-OCH2O-), 94.12 (CH2,
4′-OCH2O-), 93.90 (CH2, 2′,6′-OCH2O-), 55.96 (CH3, 4-
OCH3), 55.85 (CH3, 3-OCH3), 55.83 (CH3, 4′-OCH3),
55.76 (CH3, 2′,6′-OCH3); EIMS m/z: 508.0 [M]+.
1
ing at 400 MHz for H and 100 MHz for 13C. Mass spec-
trometer was used for the measurement of molecular masses
of compounds.
General synthesis of dihydroflavonol derivatives
(4a–4l)
MOM-protected chalcone derivatives (1.0 mmol) and aqu-
eous NaOH solution (5 M, 10 ml) were added to methanol
(15.0 ml). To the above reaction mixture, 30% H2O2 (0.3
ml, 3.0 mmol) was added and stirred for 3 h at room tem-
perature. After completion of the reaction (as indicated by
TLC), the reaction was quenched by saturated sodium sul-
fite and extracted with EtOAc. Then the epoxide was dis-
solved in methanol solution and treated with HCl at 55 °C.
The crude material was purified via silica gel flash chro-
matography to give corresponding dihydroflavonol deriva-
tives (4a–4l).
(E)-3-(4-fluorophenyl)-1-(2,4,6-tris(methoxymethoxy)
phenyl)prop-2-en-1-one (3k)
Light yellow solid; yield: 85%; mp 141–143 °C; IR (KBr)
νmax 2956, 2908, 1653, 1602, 1508, 1449, 1414, 1231,
1
1155, 1110, 1048, 922, 834 cm−1; H NMR (DMSO-d6,
400 MHz) 7.79 (2H, dd, J = 8.6, 5.6 Hz, H-2,4), 7.28 (1H,
dd, J = 16.2, 2.7 Hz, H-8), 7.24 (2H, d, J = 8.8, 1.6 Hz, H-
3,5), 7.01 (1H, dd, J = 16.1, 2.2 Hz, H-7), 6.53 (2H, s, H-
3′,5′), 5.21 (2H, s, 4′-OCH2-), 5.14 (4H, s, 2′,6′-OCH2O-),
3.41 (3H, s, 4′-OCH3), 3.27 (6H, s, 2′,6′-OCH3); 13C NMR
(DMSO-d6, 100 MHz) δ 193.19 (C=O), 163.38 (d, J =
249.1 Hz, C-F), 158.87 (C, C-4′), 155.10 (C, C-3′,5′),
143.24 (CH, C-8), 130.94 (CH, C-2,6), 130.88 (C, C-1),
128.75 (CH, C-7), 116.11 (CH, C-3), 115.97 (CH, C-5),
(E)-3-(3,4-bis(methoxymethoxy)phenyl)-1-(2,4,6-tris
(methoxymethoxy)phenyl)-prop-2-en-1-one (3d)
Light yellow solid; yield: 78%; mp 131–132 °C; IR (KBr)
νmax 2956, 2918, 1649, 1603, 1509, 1434, 1395, 1259,
1152, 1048, 922, 822, 755 cm−1; 1H NMR (DMSO-d6, 400

Medicinal Chemistry Research
114.29 (C, C-1′), 96.59 (CH, C-3′,5′), 94.11 (CH2, 4′-
OCH2O-), 93.99 (CH2, 2′,6′-OCH2O-), 55.96 (CH3, 4′-
OCH3), 55.77 (CH3, 2′,6′-OCH3); EIMS m/z: 406.0 [M]+.
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one
(4d)
White solid; yield: 30%; mp 229–230 °C; IR (KBr) νmax
3,5,7-Trihydroxy-2-phenylchroman-4-one (4a)
3356, 2926, 2854, 1640, 1468, 1283, 1162, 1086, 996, 811,
1
777, 665 cm−1; H NMR (DMSO-d6, 400 MHz): δ = 11.89
White solid; yield: 40%; mp 177–180 °C; IR (KBr) νmax
(1H, s, OH-5), 10.80 (1H, s, OH-7), 9.02 (1H, s, OH-3′),
8.96 (1H, s, OH-4′), 6.85 (1H, s, H-2′), 6.75–6.70 (2H, m,
H-5′,6′), 5.86 (2H, dd, J = 29.2, 2.1 Hz, H-6,8), 5.74 (1H,
d, J = 6.2 Hz, OH-3), 4.96 (1H, d, J = 11.2 Hz, H-2), 4.48
(1H, dd, J = 11.2, 6.2 Hz, H-3); 13C NMR (DMSO-d6, 100
MHz): δ = 198.2 (C=O, C-4), 167.2 (C, C-7), 163.7 (C, C-
5), 163.0 (C, C-9), 146.2 (C, C-3′), 145.3 (C, C-4′), 128.4
(C, C-1′), 119.8 (CH, C-6′), 115.7 (CH, C-5′), 115.5 (CH,
C-2′), 100.9 (C, C-10), 96.4 (CH, C-6), 95.4 (CH, C-8),
83.4 (CH, C-2), 71.9 (CH, C-3); EIMS m/z: 305.1 [M]+.
1
3351, 2925, 1640, 1451, 1300, 1042, 820 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.91 (1H, s, OH-5), 10.88 (1H,
s, OH-7), 7.53 (2H, d, J = 5.7 Hz, H-2′,6′), 7.42 (3H, d,
J = 7.2 Hz, H-3′,4′,5′), 5.92 (2H, dd, J = 16.1, 2.1 Hz, H-
6,8), 5.88 (1H, d, J = 6.3 Hz, OH-3), 5.19 (1H, d, J = 11.4
Hz, H-2), 4.64 (1H, dd, J = 11.4, 6.0 Hz, H-3); 13C NMR
(DMSO-d6, 100 MHz): δ = 198.0 (C=O, C-4), 167.3 (C, C-
7), 163.8 (C, C-5), 162.91 (C, C-9), 137.7 (C, C-1′), 129.1
(CH, C-4′), 128.6 (C, C-3′,5′), 128.5 (C, C-2′,6′), 100.9 (C,
C-10), 96.6 (CH, C-6), 95.5 (CH, C-8), 83.3 (CH, C-2),
71.9 (CH, C-3); EIMS m/z: 272.0 [M]+.
3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-4-one
(4e)
3,5,7-Trihydroxy-2-(3-hydroxyphenyl)chroman-4-one (4b)
White solid; yield: 25%; mp 234–236 °C; IR (KBr) νmax
1
White solid; yield: 55%; mp 201–203 °C; IR (KBr) νmax
3335, 2911, 1640, 1465, 1288, 1094, 825 cm−1; H NMR
1
3333, 2913, 1637, 1442, 1280, 1037, 842 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 12.10 (1H, s, OH-5), 10.89 (1H,
s, OH-7), 9.45 (1H, s, OH-4′), 8.89 (1H, s, OH-3′), 8.86
(1H, s, OH-5′), 7.11 (2H, s, H-2′,6′), 5.89 (2H, dd, J = 9.8,
2.4 Hz, H-6, 8), 5.73 (1H, d, J = 6.1 Hz, OH-3), 5.01 (1H,
d, J = 11.2 Hz, H-2), 4.48 (1H, dd, J = 11.2, 6.2 Hz, H-3);
13C NMR (DMSO-d6, 100 MHz): δ = 197.9 (C, C-4), 167.2
(C, C-7), 164.8 (C, C-5), 164.2 (C, C-9), 158.0 (C, C-4′),
145.9 (C, C-3′), 133.8 (C, C-5′), 131.4 (C, C-1′), 116.5
(CH, -2′, 6′), 101.5 (C, C-10), 96.7 (CH, C-6), 96.1 (CH, C-
8), 81.9 (CH, C-2), 72.4 (CH, C-3); EIMS m/z: 320.2 [M]+.
(DMSO-d6, 400 MHz): δ = 12.02 (1H, s, OH-5), 10.91 (1H,
s, OH-7), 10.91 (1H, s, OH-3′), 7.14 (1H, t, J = 7.4 Hz, H-
5′), 7.02 (1H, d, J = 7.0 Hz, H-6′), 6.93–6.84 (2H, m, H-
2′,4′), 5.91 (2H, dd, J = 20.8 Hz, 1.4 Hz, H-6,8), 5.72 (1H,
d, J = 6.2 Hz, OH-3), 5.01 (1H, d, J = 11.6 Hz, H-2), 4.48
(1H, dd, J = 11.6 Hz, H-3); 13C NMR (DMSO-d6, 100
MHz): δ = 198.1 (C=O, C-4), 168.0 (C, C-7), 164.4 (C, C-
5), 163.6 (C, C-9), 158.7 (C, C-3′), 134.9 (CH, C-4′), 130.6
(C, C-1′), 119.9 (CH, C-6′), 116.0 (CH, C-5′), 115.5 (CH,
C-2′), 101.2 (C, C-10), 96.7 (CH, C-6), 96.0 (CH, C-8),
83.2 (CH, C-2), 72.6 (CH, C-3); EIMS m/z: 288.0 [M]+.
3,5,7-Trihydroxy-2-(3-methoxyphenyl)chroman-4-one (4f)
3,5,7-Trihydroxy-2-(4-hydroxyphenyl)chroman-4-one (4c)
White solid; yield: 37%; mp 134–136 °C; IR (KBr) νmax
1
3325, 2918, 1651, 1455, 1286, 1084, 817 cm−1; H NMR
White solid; yield: 35%; mp 219–220 °C; IR (KBr) νmax
(DMSO-d6, 400 MHz): δ = 11.93 (1H, s, OH-5), 10.98 (1H,
s, OH-7), 7.35 (1H, t, J = 7.8 Hz, H-5′), 7.12 (2H, d, J =
8.0 Hz, H-2′,6′), 6.97 (1H, d, J = 5.8 Hz, H-4′), 5.96 (2H, d,
J = 13.1 Hz, H-6,8), 5.88 (1H, s, OH-3), 5.17 (1H, d, J =
11.3 Hz, H-2), 4.65 (1H, d, J = 11.3 Hz, H-3), 3.79 (3H, s,
O-CH3); 13C NMR (DMSO-d6, 100 MHz): δ = 197.9
(C=O, C-4), 167.3 (C, C-7), 163.8 (C, C-5), 162.8 (C, C-9),
159.6 (C, C-3′), 139.2 (C, C-1′), 129.7 (CH, C-5′), 120.7
(CH, C-6′), 114.4 (CH, C-4′), 114.2 (CH, C-2′), 100.9 (C,
C-10), 96.6 (CH, C-6), 95.6 (CH, C-8), 83.3 (CH, C-2),
72.0 (CH, C-3), 55.5 (CH3, O-CH3); EIMS m/z: 302.0
[M]+.
1
3327, 2902, 1630, 1462, 1281, 1088, 813 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.92 (1H, s, OH-5), 10.84 (1H,
s, OH-7), 9.56 (1H, s, OH-4′), 7.32 (2H, d, J = 8.1 Hz, H-
2′,6′), 6.80 (2H, d, J = 8.1 Hz, H-3′,5′), 5.90 (2H, d, J =
21.4 Hz, H-6,8), 5.76 (1H, d, J = 6.1 Hz, OH-3), 5.06 (1H,
d, J = 11.3 Hz, H-2), 4.59 (1H, dd, J = 11.4, 5.6 Hz, H-3);
13C NMR (DMSO-d6, 100 MHz): δ = 198.3 (C=O, C-4),
167.2 (C, C-7), 163.7 (C, C-5), 163.0 (C, C-9), 158.2 (C, C-
4′), 129.9 (CH, C-2′,6′), 128.0 (C, C-1′), 115.3 (CH, C-
3′,5′), 100.9 (C, C-10), 96.5 (CH, C-6), 95.4 (CH, C-8),
83.1 (CH, C-2), 71.9 (CH, C-3); EIMS m/z: 288.2 [M]+.

Medicinal Chemistry Research
3,5,7-Trihydroxy-2-(4-methoxyphenyl)chroman-4-one (4g)
(DMSO-d6, 400 MHz): δ = 11.88 (1H, s, OH-5), 10.84 (1H,
s, OH-7), 7.10–7.44 (5H, m, OPh-H), 6.88–7.04 (4H, m, H-
2′, 3′, 5′, 6′), 5.95 (2H, dd, J = 2.4, H-6, 8), 5.75 (1H, d,
J = 6.4 Hz, OH-3), 4.95 (1H, d, J = 11.5 Hz, H-2), 4.43
(1H, dd, J = 11.5 Hz, H-3); 13C NMR (DMSO-d6, 100
MHz): δ = 197.9 (C, C-4), 167.2 (C, C-7), 164.29 (C, C-5),
163.53 (C, C-9), 159.50 (C, C-4′), 155.2 (C, OPh-C-1),
129.8 (C, C-1′),129.1 (CH, OPh-C-3,5), 124.49 (CH, C-2′,
6′), 123.9 (CH, OPh-C-4), 120.1 (CH, OPh-C-2,6), 118.69
(CH, C-3′, 5′), 101.20 (C, C-10), 96.77 (CH, C-6), 96.05
(CH, C-8), 84.50 (CH, C-2), 72.98 (CH, C-3); EIMS m/z:
364.4 [M]+.
White solid; yield: 17%; mp 140–142 °C; IR (KBr) νmax
1
3327, 2907, 1653, 1463, 1286, 1092, 827 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.90 (1H, s, OH-5), 11.01 (1H,
s, OH-7), 7.40–7.44 (2H, m, H-2′, 6′), 6.92–7.14 (2H, m,
H-3′, 5′), 6.01 (2H, d, J = 9.6 Hz, H-6, 8), 5.73 (1H, s, OH-
3), 4.93 (1H, d, J = 11.2 Hz, H-2), 4.42 (1H, d, J = 11.2 Hz,
H-3), 3.80 (3H, s, O-CH3); 13C NMR (DMSO-d6, 100
MHz): δ = 197.9 (C, C-4), 167.2 (C, C-7), 164.0 (C, C-5),
163.4 (C, C-9), 159.5 (C, C-4′), 129.6 (C, C-1′), 128.1 (CH,
C-2′, 6′), 113.9 (CH, C-3′, 5′), 101.2 (C, C-10), 96.7 (CH,
C-6), 96.0 (CH, C-8), 84.1 (CH, C-2), 73.1 (CH, C-3), 55.9
(CH3, O–CH3); EIMS m/z: 302.2 [M]+.
2-(4-Fluorophenyl)-3,5,7-trihydroxychroman-4-one (4k)
2-(3,4-Dimethoxyphenyl)-3,5,7-trihydroxychroman-4-one
(4h)
White solid; yield: 55%; mp 213–215 °C; IR (KBr) νmax
3337, 2925, 2854, 1639, 1152, 1470, 1230, 1158, 1086,
1
995, 832, 642 cm−1; H NMR (DMSO-d6, 400 MHz): δ =
White solid; yield: 15%; mp 167–170 °C; IR (KBr) νmax
11.89 (1H, s, OH-5), 10.88 (1H, s, OH-7), 7.58 (2H, dd,
J = 8.6, 5.6 Hz, H-2′,6′), 7.26 (2H, t, J = 8.9 Hz, H-3′,5′),
5.94 (1H, d, J = 2.1 Hz, H-6), 5.89 (1H, d, J = 2.1 Hz, H-8),
5.88 (1H, d, J = 6.2 Hz, OH-3), 5.20 (1H, d, J = 11.5 Hz,
H-2), 4.63 (1H, dd, J = 11.6, 6.1 Hz, H-3); 13C NMR
(DMSO-d6, 100 MHz): δ = 197.6 (C=O, C-4), 166.8 (C, C-
7), 163.2 (C, C-5), 162.3 (C, C-9), 161.4 (C, C-4′), 133.6
(C, C-1′), 130.2 (CH, C-2′, 6′), 115.1 (CH, C-3′, 5′), 100.4
(C, C-10), 96.2 (CH, C-6), 95.1 (CH, C-8), 82.0 (CH, C-2),
71.5 (CH, C-3); EIMS m/z: 291.1 [M]+.
1
3333, 2905, 1650, 1460, 1280, 1094, 825 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.90 (1H, s, OH-5), 11.01 (1H,
s, OH-7), 7.11 (1H, s, H-2′), 6.90–7.02 (2H, m, H-5′,6′),
5.85 (2H, dd, J = 2.1 Hz, H-6, 8), 5.65 (1H, d, J = 6.1 Hz,
OH-3), 4.97 (1H, d, J = 11.4 Hz, H-2), 4.49 (1H, dd, J =
11.4 Hz, H-3), 3.89 (3H, s, O-CH3), 3.86 (3H, s, O-CH3);
13C NMR (DMSO-d6, 100 MHz): δ = 197.6 (C, C-4), 167.2
(C, C-7), 164.2 (C, C-5), 163.4 (C, C-9), 148.6 (C, C-3′),
148.2 (C, C-4′), 129.5 (C, C-1′), 120.5 (CH, C-6′), 111.1
(CH, C-5′), 111.0 (CH, C-2′), 101.2 (C, C-10), 96.7 (CH,
C-6), 96.0 (CH, C-8), 83.6 (CH, C-2), 72.4 (CH, C-3), 56.0
(CH3, O–CH3); EIMS m/z: 332.3 [M]+.
2-(4-Chlorophenyl)-3,5,7-trihydroxychroman-4-one (4l)
White solid; yield: 57%; mp 189–192 °C; IR (KBr) νmax
1
3,5,7-Trihydroxy-2-(4-isopropylphenyl)chroman-4-one (4i)
3331, 2930, 1647, 1450, 1230, 1083, 829 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.89 (1H, s, OH-5), 10.90 (1H,
s, OH-7), 7.57 (2H, d, J = 8.3 Hz, H-2′,6′), 7.50 (2H, d,
J = 8.3 Hz, H-3′,5′), 5.97 (1H, s, H-6), 5.93 (1H, s, H-8),
5.91 (1H, s, OH-3), 5.22 (1H, d, J = 11.5 Hz, H-2), 4.61
(1H, d, J = 10.7 Hz, H-3); 13C NMR (DMSO-d6, 100
MHz): δ = 197.7 (C=O, C-4), 167.3 (C, C-7), 163.8 (C, C-
5), 162.7 (C, C-9), 136.7 (C, C-4′), 133.7 (C-1′), 130.3
(CH, C-2′, 6′), 128.6 (CH, C-3′, 5′), 100.9 (C-10), 96.7
(CH, C-6), 95.6 (CH, C-8), 82.5 (CH, C-2), 72.0 (CH, C-3);
EIMS m/z: 306.7 [M]+.
White solid; yield: 45%; mp 156–157 °C; IR (KBr) νmax
1
3320, 2901, 1648, 1462, 1282, 1095, 827 cm−1; H NMR
(DMSO-d6, 400 MHz): δ = 11.91 (1H, s, OH-5), 10.86 (1H,
s, OH-7), 7.44 (2H, d, J = 8.0 Hz, H-2′,6′), 7.29 (2H, d, J =
7.9 Hz, H-3′,5′), 5.93 (1H, d, J = 2.1 Hz, H-6), 5.88 (1H, d,
J = 2.1 Hz, H-8), 5.85 (1H, d, J = 6.3 Hz, OH-3), 5.14 (1H,
d, J = 11.4 Hz, H-2), 4.62 (1H, dd, J = 11.4, 6.3 Hz, H-3),
2.92 (1H, hept, J = 7.0 Hz, CH3–CH–CH3), 1.23 (6H, s,
CH3-CH-CH3); 13C NMR (DMSO-d6, 100 MHz): δ = 198.1
(C=O, C-4), 167.2 (C, C-7), 163.7 (C, C-5), 162.9 (C, C-9),
149.3 (C, C-4′), 135.2 (C, C-1′), 128.5 (CH, C-2′, 6′), 126.6
(CH, C-3′, 5′), 100.9 (C, C-10), 96.5 (CH, C-6), 95.5 (CH,
C-8), 83.2 (CH, C-2), 73.11 (CH, C-3), 33.7 (CH, CH3–CH–
CH3), 24.3 (CH3, CH3–CH–CH3); EIMS m/z: 314.1 [M]+.
In vitro anti-inflammatory activities
Cell culture
RAW 264.7 macrophage cells were obtained from Cell
Bank of Academy of Sciences in Huazhong University of
Science and Technology (China). The macrophage cells
were incubated in Dulbecco′s modified Eagle′s medium
(HyClone, USA) supplemented with 10% fetal bovine
3,5,7-Trihydroxy-2-(4-phenoxyphenyl)chroman-4-one (4j)
White solid; yield: 39%; mp 99–101 °C; IR (KBr) νmax
1
3334, 2933, 1651, 1455, 1233, 1088, 838 cm−1; H NMR

Medicinal Chemistry Research
serum (Bovine, China), 100 U/ml penicillin, and 100 μg/ml
streptomycin. All cells were incubated at 37 °C in a 5% CO2
atmosphere during the whole experiment.
administered with compounds 4d, 4k at a dose of 100 mg/
kg body weight. After consecutive administration for
7 days, ear swelling was induced by smearing 20 μL para-
xylene to each side of right ears. After 1 h, the mice were
sacrificed. Both ears at the same position were cut down and
weighed. Swelling degree and swelling inhibition were
calculated according to the following equations:
Cell viability assay
Mouse RAW 264.7 macrophages were used to analyze the
toxicity of dihydroflavonol derivatives. About 8 × 104 cells/
well were seeded into 96-well plates and treated with
vehicle control or various concentrations of samples for 24
h. 20 μL MTT solution (5 mg/ml) was added to each well
and incubated at 37 °C in a humidified atmosphere of 5%
CO2 for 4 h. After MTT removal, 150 μL of dimethyl
sulfoxide (DMSO) was added to each well and the assay
plate was read at a wavelength of 490 nm using a micro-
plate reader. Three independent experiments were con-
ducted and samples were analyzed in triplicates for each
experiment.
Swelling degree ðmgÞ ¼ weight of the right ear À weight of the left ear
Swelling inhibition ð%Þ ¼ ½Wc À Wd=WcŠ  100
Herein, Wd represents the average swelling degree of
dosage group and Wc represents the average swelling degree
of control group.
Egg-white induced rat paw edema
The anti-inflammatory activity of compounds 4d, 4k was
further evaluated using egg-white induced paw edema in
rats (Chen et al. 2015). Male Sprague–Dawley rats (150–
170 g) were divided into five groups (n = 6). The negative
control group was intragastrically administrated with 0.5%
carboxymethylcellulose sodium solution. Positive control
groups were intragastrically administered with standard
drug aspirin (100 mg/kg) and meloxicam (10 mg/kg). The
remaining test groups were intragastrically administered
with compounds 4d, 4k at a dose of 100 mg/kg body
weight. Acute paw edema was induced by sub-plantar
injection of 0.1 ml freshly prepared egg-white of each rat
after 1 h of administrating the test samples. Right hind paw
volume was measured with the help of digital plethysm-
ometer through time intervals (0, 30, 60, 120, 180, and 240
min) after induction of inflammation. The percent anti-
inflammatory activity was calculated according to the for-
mula given below:
Cytokines assay
RAW264.7 cells were cultured at 8 × 104/well in 96-well
plates and preincubated for 12 h. Then, cells were stimu-
lated with LPS (1 μg/ml) for 24 h and the tested compounds
in 0.1% DMSO were given at 20 μM for another 24 h. TNF-
α, IL-1β, and IL-6 levels were quantified by ELISA kit
(eBioScience, Inc.) according to the manufacturer′s
instructions.
In vivo anti-inflammatory activities
All animals were purchased from Hubei Province Academy
of Preventive Medicine. The animal studies were approved by
the Hubei University of Traditional Chinese Medicine. All
animal studies were conducted according to protocols
approved by the Animal Ethics Committee of Hubei Province
Hospital of Traditional Chinese Medicine. Animals were
housed in separate cages at room temperature 25 2 °C, and
given free access to rodent chow, water and maintained at a
12 h light/dark cycle.
Anti À inflammatory activity ð%Þ ¼ ½Vc À Vt=VcŠ  100
Herein, Vt represents the mean increase in paw volume of
rats treated with the test sample and Vc represents the mean
increase in paw volume of rats in the control group.
Xylene induced ear edema
Molecular docking study
The in vivo anti-inflammatory activity was evaluated using
para-xylene-induced mice ear swelling model in Kunming
mice (Li et al. 2017). Kunming mice (22–25 g) were divi-
ded into the control group and dosage groups (n = 10 for
each group).
The negative control group was intragastrically admini-
strated with 0.5% carboxymethylcellulose sodium solution.
The positive control group was intragastrically administered
with standard drug aspirin (100 mg/kg) and meloxicam
(10 mg/kg). The remaining test groups were intragastrically
Protein preparation
The crystal protein structure of COX-2 (PDB:4M11) (Silva
et al. 2018) was downloaded from the Protein Data Bank
(http://www.rcsb.org/pdb/). The Autodock Tools (Morris
et al. 2009) graphics interface was employed for manual
preparation of the protein by adding non-polar hydrogen,
Gasteiger charges and saved as pdbqt format. 4M11 retains
only A chain.

Medicinal Chemistry Research
Circular dichroism spectra of Taxifolin
0.18
0.1
CD[mdeg]
0
-0.06
281
275
HT[V]
270
267
250 260
280
300
320
Wavelength [nm]
Circular dichroism spectra of 4d
-0.5
-0.6
-0.8
-1
CD[mdeg]
HT[V]
-1.2
281
275
270
267
250 260
280
300
320
Wavelength [nm]
Fig. 1 Circular dichroism spectra of Taxifolin and 4d
Ligands preparation
Result and discussion
Trans isomers were sketched in BIOVIADraw. Openbabel
2.4.1 (O′Boyle et al. 2011) was used to transform 2D into
3D structure by genetic algorithm and MMFF94 force
field. Hydrogen was added at pH 7.4 and saved as pdbqt
format.
The synthesis of dihydroflavonol derivatives
Synthesis of the intermediates and the target compounds
(4a–4l) were accomplished according to the steps depicted
in Scheme 1. Protection of 2,4,6-trihydroxy acetophenone
was achieved with MOMCl to give compound 2. Then
chalcone derivatives (3a–3l) were produced by condensa-
tion reaction of compounds 2 and corresponding benzal-
dehydes. Finally, the target compounds (4a–4l) were
obtained through Algar–Flynn–Oyamada (AFO) reaction
by H2O2 and hydrochloric acid.
Molecular docking
After the protein and ligand files had been prepared,
docking analysis was performed using AutoDock Vina
(Trott and Olson 2009). The crystallographic protein
structure of COX-2 was selected for docking procedure
validation by re-docking approach and also to know the
standard docking energy of binding site.
Docking parameters remained the default. Docking out-
put was obtained in the form of affiliate energy (kcal/mol)
and binding pose file for each ligand–protein binding con-
formation. The selected pose was visualized in Pymol v2.1
(Schrödinger 2015). The interaction data were graphically
analyzed by Lig-Plus v1.4.5 (Laskowski and Swindells
2011).
In order to obtain the configuration of synthetic dihy-
droflavonols, compound 4d was selected to determine the
configuration by proton nuclear magnetic resonance (1H-
1
NMR) and circular dichroism (CD). By H-NMR analysis
of 4d, the coupling constant of 11.2 Hz between 2H and 3H
indicated that 4d was trans configuration. By CD analysis of
4d, there was only negative cotton effect at 290–330 nm
(Fig. 1). However, there was not only a negative cotton
effect at 290–300 nm, but also a positive cotton effect at
320–330 nm for natural Taxifolin (2R,3R), which had the

Medicinal Chemistry Research
same chemical structure as 4d. Above results indicated that
the synthetic compound 4d was racemic mixture. Since it is
very difficult to separate these isomers (Jiang et al. 2015)
and there was no significant difference in the anti-
inflammatory activity between the natural product (Taxi-
folin) and synthetic product (4d) (Hu et al. 2018), we used
racemates to further study their anti-inflammatory activities
in this paper. All target compounds were characterized by
In vivo anti-inflammatory activity
In order to investigate the in vivo anti-inflammatory efficacy
of compounds 4d, 4k, two different models were employed:
xylene induced ear edema in mice and egg-white induced
paw edema in rats.
Xylene induced ear-swelling
1
IR, H-NMR, 13C-NMR, and mass spectroscopy.
Ear swelling induced by xylene in mice was used to eval-
uate the anti-inflammatory activity of compounds 4d, 4k. In
xylene induced ear edema test, mediators of inflammation
are released after following stimulation. This leads to the
dilation of arterioles and venules and to increase vascular
permeability. As shown in Table 1, the topical application
of xylene on the right ear of the control group produced an
increase in the average of the ear weight. The administration
of 4k significantly suppressed the ear edema at 32.98%
inhibition when compared to the control group (P < 0.001),
which was nearly the same compared to the 31.99% inhi-
bition delivered by Meloxicam. However, 4d did not exhibit
a considerable anti-inflammatory potential in this model.
In vitro anti-inflammatory activity
Cell viability
To investigate the effect of dihydroflavonol derivatives on
RAW 264.7 cell viability, the cells were treated with dif-
ferent concentrations of derivatives (320, 160, 80, 40, 20,
10, and 5 μM) with 1 μg/ml LPS for 24 h. As the results
shown in Fig. 2, no cytotoxicity was found after treatment
with dihydroflavonol derivatives (4a–4l) and Taxifolin at 5,
10, 15, 20, 40 μM/ml for 24 h compared to control group,
which demonstrated that cell viability was not significantly
affected by up to 20 μM/ml (Fig. 2). Thus, concentrations of
20 μM/ml could be used for follow-up tests.
Inhibition of pro-inflammatory cytokines in RAW 264.7 cell
lines
100
IL-1beta
IL-6
80
60
40
20
0
All compounds were evaluated for their in vitro IL-1β, IL-6,
TNF-α inhibition level in LPS induced RAW 264.7 cell
lines by ELISA method. Meloxicam was used as positive
control. All compounds were screened for their in vitro anti-
inflammatory activity, of which 4d, 4k showed desirable
suppression of IL-1β, IL-6, TNF-α compared to Meloxicam
as the results shown in Fig. 3. According to the result, we
chose 4k and 4d for further in vivo anti-inflammatory
studies.
TNF-alpha
4i
4j
4l
4a
4c
4e
4f
4b
4d
4g
4h
4k
Taxifolin
Meloxicam
Compounds
Fig. 3 In vitro IL-1β, IL-6, TNF-α inhibition of all compounds
Control
Table 1 Anti-inflammatory effect of 4d and 4k on xylene induced ear
edema in mice
DMSO
Taxifolin
4a
100
4b
Compound Dose (mg/kg) Swelling degree (mg)a Inhibition (%)
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
80
60
40
Control
Aspirin
–
10.18 0.53
–
100
5.93 0.24***
6.93 0.61***
8.91 0.50
41.73
31.99
12.52
32.98
Meloxicam 10
4d
4k
100
100
6.82 0.29***
5
80
40
20
10
aThe results were expressed as mean SEM (n = 10), one-way
ANOVA test was performed
320
160
concentration (μM)
Fig. 2 Cell viability of 4a–4l
***P < 0.001 was compared with control

Medicinal Chemistry Research
Table 2 The anti-inflammatory
activity of 4d, 4k, and reference
drugs in egg-white induced rat
paw edema assay
Compound Dose (mg/kg) Mean paw volume (ml) SEM and (% of inhibition)a
0.5 h 1 h 2 h 3 h
4 h
Control
Aspirin
–
1.052 0.062 1.180 0.035 1.165 0.055 0.992 0.050 0.842 0.049
1.058 0.081 1.058 0.116 0.807 0.037 0.602 0.068 0.483 0.068
100
(0.00)
1.008 0.054 1.022 0.072 0.832 0.136 0.695 0.110 0.605 0.092
(4.12) (13.42) (28.61)** (29.92)* (28.12)
0.983 0.078 0.933 0.053 0.850 0.065 0.740 0.117 0.515 0.022
(6.50) (20.90)* (27.04)** (25.38)* (38.81)**
0.855 0.063 0.718 0.077 0.698 0.092 0.585 0.086 0.553 0.049
(18.70) (39.12)*** (40.06)*** (41.01)*** (34.26)*
0.815 0.084 0.855 0.136 0.760 0.113 0.637 0.086 0.532 0.093
(22.50) (27.54)** (34.76)*** (35.80)** (36.83)**
0.908 0.048 1.073 0.044 0.795 0.080 0.682 0.076 0.537 0.102
(13.63) (9.04) (31.76)** (31.26)** (36.24)**
(10.31)
(30.76)**
(39.33)***
(42.57)**
Meloxicam 20
4d
4k
4k
4k
100
100
75
50
aOne-way ANOVA using Dunnett′s test is applied for statistical analysis. Values are expressed as mean
SEM (ml) and percentage inhibition of inflammation
*The mean difference is significant at the P < 0.05 level
**The mean difference is significant at the P < 0.01 level
***The mean difference is significant at the P < 0.001 level
Egg-white induced rat paw edema
was found (Fig. 4a). Of which, the oxygen atom on the
hydroxyl group of Meloxicam and the oxygen atom on the
carbonyl group of 4k (2R,3R isomer) formed two hydrogen
bonds with Ser530 at the same distance of 3.05 Å (Fig. 4b,
c). However, the oxygen atom on carbonyl group of 4k
(2S,3S isomer) formed a hydrogen bond with Arg120 at the
distance of 2.56 Å (Fig. 4d). It was interesting that the F
atom of isomers of 4k (2R,3R and 2S,3S) in the B ring both
entered the hydrophobic pocket formed by Val349 and
Ala527. Moreover, the binding energy of the 2R,3R isomer
(−8.9 kcal/mol) was nearly the same as the 2S,3S isomer
(−8.6 kcal/mol). Above results gave a possible mechanistic
explanation for the potent activity of 4k.
The in vivo anti-inflammatory activity of compounds 4d, 4k
was further evaluated by the egg-white induced paw edema
method. As shown in Table 2, the synthesized compounds
4d, 4k produced a significant reduction at interval time 1–4
h at 100 mg/kg when compared with the control group. At
the interval time 2–4 h, only 4k showed a higher effect than
reference drugs, which indicated that 4k had the best anti-
inflammatory activity. Thus, we further investigated the
anti-inflammatory effect of 4k at lower doses and found the
lowest dose of 4k (50 mg/kg) evidently decreased the per-
centage inhibition of paw edema at the interval time 2–4 h.
Molecular docking study
Conclusion
In order to gain a better understanding of the potency of the
studied compounds, we continued to examine the interaction
of the active compound 4k with COX-2 crystal structure. The
molecular docking was performed by inserting the compound
4k into the binding site of COX-2. Herein, molecules were
deeply embedded into the active site pocket and occupy the
same position like Meloxicam (shown in yellow color) and
the superimposition of Meloxicam and potent 4k dock poses
in the active pocket are shown in Fig. 4.
The molecular basis interactions between target enzyme
and synthesized ligands can be understood with the help of
docking analysis and interactions. By superposed docking
pose of 4k and meloxicam, almost the same binding mode
We synthesized 12 compounds with 3,5,7-trihydroxy-2-
phenylchroman-4-one skeleton by classical AFO reaction
and examined their anti-inflammatory activities. All the
synthesized compounds are subjected to the evaluation of
in vitro TNF-alpha, IL-1beta, IL-6 production inhibition in
RAW 264.7 cell lines. Furthermore, the hit compounds 4d,
4k were selected to investigate the in vivo anti-inflammatory
efficacy by two classical models: xylene induced ear edema
and egg-white induced paw edema. Among all, compound
4k exhibited potent anti-inflammatory activities.
Molecular docking studies also suggested that active
compound 4k interacts with COX-2 enzyme more

Medicinal Chemistry Research
Fig. 4 Meloxicam and 4k
docked into the binding site of
COX-2. a Superposition of
meloxicam (yellow) and 4k
(2R,3R in red and 2S,3S in
green). b Interaction between
Meloxicam and COX-2. c
Interaction between 4k (2R,3R
isomer) and COX-2. d
Interaction between 4k (2S,3S
isomer) and COX-2
efficiently and is well agreement with anti-inflammatory
activity. Therefore, the compound 4k may be considered as
a promising candidate for the development of new anti-
inflammatory agent.
Besides, their structure–activity relationships were dis-
cussed preliminarily. The results showed that (1) The
hydroxyl/methoxy groups substituted in the B ring increases
anti-inflammation activity, but two substitutions are better
than three substitutions. (2) The introduction of a large
sterically hindered group has a slight impact on anti-
inflammatory activity. (3) When the 4′ position of B-ring
substituted by the fluorine atom, the anti-inflammatory
activity of compound (4k) was the best.
Acknowledgements The authors would like to extend their sincere
appreciation to the National Natural Science Foundation of China for
funding this research group (No. 31370378).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

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