Design, synthesis and anti-inflammatory activity of dihydroflavonol derivatives

Article abstract of DOI:10.1007/s00044-017-2054-z

Thirty dihydroflavonol derivatives (D1–D30) were designed and synthesized, meanwhile the synthesized compounds were characterized on the basis of spectroscopic analyzes. Their inhibitory activity against the pro-inflammatory inducible interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages were evaluated and showed various efficiency. Compounds D1–D30 showed no toxic effects on RAW 264.7 cells at the concentration 20 μM; among them, compounds D9, D13, and D19 exhibited best anti-inflammatory activity through decreasing IL-1β, IL-6, and TNF-α. Furthermore, their structure–activity relationships were discussed preliminarily.

Full text of DOI:10.1007/s00044-017-2054-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2054-z
ORIGINAL RESEARCH
Design, synthesis and anti-inflammatory activity of
dihydroflavonol derivatives
Chunling Hu1 Zongbao Zhou1 Yuanhang Xiang1 Xiaoying Song1 Hong Wang1
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Kaiqi Tao1 Xiaochuan Ye1
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Received: 4 June 2017 / Accepted: 28 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Thirty dihydroflavonol derivatives (D1–D30)
were designed and synthesized, meanwhile the synthesized
compounds were characterized on the basis of spectro-
scopic analyzes. Their inhibitory activity against the
pro-inflammatory inducible interleukin-1β (IL-1β),
interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)
in lipopolysaccharide (LPS)-stimulated murine RAW 264.7
macrophages were evaluated and showed various effi-
ciency. Compounds D1–D30 showed no toxic effects on
RAW 264.7 cells at the concentration 20 μM; among them,
compounds D9, D13, and D19 exhibited best anti-
inflammatory activity through decreasing IL-1β, IL-6, and
TNF-α. Furthermore, their structure–activity relationships
were discussed preliminarily.
novel structure and excellent pharmacological activities, in
which the inflammatory response plays an important
role in the pathological processes of many diseases,
including cancer, cardiovascular disorder and inflammation
(Mantovani et al. 2008; Ragab and Raafat 2016; Swami-
nathan et al. 2014). During the inflammatory process, the
mononuclear cells usually differentiate into macrophages,
these cells release a plethora of inflammatory mediators,
including interleukin-1β (IL-1β), interleukin-6 (IL-6),
and tumor necrosis factor-α (TNF-α), that play an important
role in promoting the inflammatory response and
pathological processes (Raison et al. 2006; Siemieniuch
et al. 2016). Inhibition of the expression of inflammatory
cytokines by small molecules or antagonism of their
actions by antibodies has been considered to be effective
strategies for the treatment of inflammation-related diseases
(Bengmark 2006; Zhang et al. 2014). In nature, many
dihydroflavonols such as dihydromyricetin, astilbin and
silybin are found and possess outstanding bioactivity,
which constitute the core of various natural products and
play a unique role in drug discovery history (Terrier et al.
2009).
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Keywords Dihydroflavonol derivatives Synthesis Anti-
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inflammatory activity Structure–activity relationship
Introduction
Taxifolin (Fig. 1) known as dihydroquercetin, which
belongs to dihydroflavonols, is widely found in medicinal
plants and has a wide range of biological activities (Mani-
gandan et al. 2015; Marín et al. 2011). Many research
showed that taxifolin had a significant anti-inflammatory
effect and could inhibit LPS-induced IL-1β, IL-6, and
TNF-α production by cytokine assay (Kim et al. 2008).
Recently, our group isolated taxifolin from Smilax
China L. and proved to be the best anti-inflammatory
activity in Smilax China L (Xie et al. 2012). However, the
natural source of taxifolin is very limited and extractive
procedures are very costly, which restricts the application
of taxifolin widely. To obtain better anti-inflammatory
Flavonoids are phenolic substances isolated from a wide
range of plants, including over 8000 individual compounds
known (Vezza et al. 2016). Among the flavonoids, dihy-
droflavonols have received more and more attention for
Chunling Hu and Zongbao Zhou contributed equally to this work.
* Xiaochuan Ye
hzyyxc2017@163.com
1
Key Laboratory of Chinese Medicine Chemistry and Chinese
Herbal Compound of Hubei Province, Hubei University of
Traditional Chinese Medicine, Wuhan 430065, China

Med Chem Res
Fig. 1 Structures of 2-phenyl
chromone, taxifolin, and
dihydroflavonol derivatives
Scheme 1 Synthetic route of
compounds (D1–D30 and
TA-SY)
activity compounds, the modification and synthesis of
taxifolin are very urgent.
Materials and methods
Experimental
Structural analysis indicates that the basic mother-
nucleus of flavonoids is 2-phenyl chromone (Fig. 1),
which usually has three aromatic rings (A, B, and C). Many
studies stated that anti-inflammatory activity of flavonoids
was closely related with the electron density distribution in
its conjugated system and substitution patterns of the three
aromatic rings (Cho et al. 2013; Lu et al. 2015). Dihydro-
flavonols as typical flavonoids are a kind of flavones, in
which 2, 3-position is a single bond and 3-position is sub-
stituted by hydroxyl group, the biological activity of dihy-
droflavonols may also be affected by these factors.
In previous studies, we found a simple and effective
synthetic route of dihydroflavonols by optimizing the
reaction conditions (Zhou et al. 2016), which is illustrated
in the Scheme 1. Herein, we synthesized a series of
dihydroflavonol derivatives by introducing different groups
as described in Fig. 2 and evaluated their anti-inflammatory
activities. Although dihydroflavonols had been reported
to suppress the expression of various inflammatory cyto-
kines, their structure–activity relationship (SAR) was not
reported (Hernández et al. 2007; Dok-Go et al. 2003). To
carry out effective modification and improve anti-
inflammatory effect, the preliminary structure–activity
relationship of dihydroflavonol derivatives was further
discussed.
All the materials and solvents were procured from Sino-
pharm Chemical Reagent Co., Ltd (China). All the
synthesized compounds were checked by thin layer chro-
matography (TLC) performed on Silica gel 60 GF254 coated
plates. Infrared spectra were recorded in the range of
4000–600 cm−1 on a (Spectrum BX) Perkin Elmer Fourier
transform infrared spectrophotometer in KBr phase. Melting
points of the compounds were checked on a Shinuo melting
point apparatus (WR-2, Shanghai), and the thermometer
was uncorrected. Nuclear magnetic resonance (NMR)
spectra were processsed in dimethyl sulfoxide (DMSO)-d6
on a Bruker nuclear magnetic resonance NMR spectro-
1
photometer operating at 400 MHz for H and 100 MHz for
13C at the Analysis and Testing Center, Huazhong Uni-
versity of Science and Technology, China. Mass spectro-
meter was used for the measurement of molecular masses of
compounds.
General synthesis of dihydroflavonol derivatives
(D1–D30)
2′-Hydroxyl chalcone derivatives (1.0 mmol) and DEA
(3.0 mmol) were added to stirred in anhydrous THF

Med Chem Res
Fig. 2 Structures of TA-SY and D1–D30
(15.0 ml). To the above reaction mixture 30% H2O2
(0.3 ml, 3.0 mmol) was added and stirred at −5 °C. After
completion of the reaction (as indicated by TLC), it was
poured into ice cold water (20.0 ml) and stirred well for 30
min. Then filtering and washing in cold water, the
crude material was recrystallized from petroleum ether and
ethyl acetate to give corresponding dihydroflavonol
derivatives.

Med Chem Res
3,5,7-trihydroxy-2-(3′,4′-dihydroxyphenyl) chroman-4-one
(TA-SY)
ppm: 12.01 (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′), 7.10 (1H,
d, J = 3.2 Hz, H-2′), 7.12 (1H, d, J = 3.2 Hz, H-6′), 5.90
(1H, d, J = 2.4 Hz, H-8), 5.87 (1H, d, J = 2.4 Hz, H-6), 5.73
(1H, s, OH-3), 5.01 (1H, d, J = 12.4 Hz, H-2), 4.48 (1H, d,
White solid; yield: 61%; M.p. 229–230 °C, IR (KBr): νmax
/
cm−1: 1649 (C=O). 1H NMR (400 MHz, DMSO-d6) δ ppm:
11.90 (1H, s, OH-5), 10.80 (1H, s, OH-7), 9.01 (1H, s, OH-
4′), 8.96 (1H, s, OH-3′), 6.98–7.14 (3H, m, H-2′, 5′, 6′), 5.90
(1H, d, J = 2.4 Hz, H-8), 5.85 (1H, d, J = 2.4 Hz, H-6), 5.74
(1H, s, OH-3), 4.97 (1H, d, J = 12.0 Hz, H-2), 4.49 (1H, d,
J = 12.0, H-3). 1C NMR (100 MHz, DMSO-d6) δ ppm:
197.16 (C-4), 167.22 (C-7), 164.24 (C-5), 163.04 (C-9),
145.99 (C-3′), 145.45 (C-4′), 128.04 (C-1′), 120.75 (C-6′),
115.38 (C-5′), 115.21 (C-2′), 101.44 (C-10), 96.17 (C-6),
96.97 (C-8), 83.05 (C-2), 72.94 (C-3). Mass spectrometry
(MS) electrospray ionization (ESI) m/z: 304.0 [M]+.
1
J = 12.4 Hz, H-3). C NMR (100 MHz, DMSO-d6) δ ppm:
197.90 (C-4), 167.28 (C-7), 164.80 (C-5), 164.27 (C-9),
158.01 (C-4′), 145.90 (C-3′), 133.89 (C-5′), 131.45 (C-1′),
119.89 (C-6′), 115.50 (C-2′), 101.55 (C-10), 96.75 (C-6),
96.10 (C-8), 81.97 (C-2), 72.46 (C-3). MS (ESI) m/z: 320.2
[M]+.
3,5,7-Trihydroxy-2-phenylchroman-4-one (D4)
White solid; yield: 72%; M.p. 177–180 °C, IR (KBr): νmax
/
cm−1: 1648 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.91 (1H, s, OH-5), 10.88 (1H, s, OH-7), 7.38–7.56
(5H, m, H-2′, 3′, 4′, 5′, 6′), 5.94 (1H, s, H-8), 5.91 (1H, s, H-
6), 5.70 (1H, s, OH-3), 5.19 (1H, d, J = 12.0 Hz, H-2), 4.64
3,5,7-Trihydroxy-2-(4′-hydroxyphenyl) chroman-4-one
(D1)
1
White solid; yield: 67%; M.p. 219–220 °C, IR (KBr): νmax
/
(1H, d, J = 12.0 Hz, H-3). C NMR (100 MHz, DMSO-d6)
cm−1: 1649 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.89 (1H, s, OH-5), 10.78 (1H, s, OH-7), 9.14 (1H, s,
OH-4′), 7.12–7.18 (2H, m, H-2′, 3′), 6.98–7.08 (2H, m, H-
5′, 6′), 5.95 (1H, d, J = 2.4 Hz, H-8), 5.89 (1H, d, J = 2.4
Hz, H-6), 5.70 (1H, s, OH-3), 4.99 (1H, d, J = 12.0 Hz, H-
δ ppm: 198.05 (C-4), 167.28 (C-7), 164.27 (C-5), 163.53
(C-9), 133.89 (C-1′), 128.66 (C-3′), 128.32 (C-5′), 127.99
(C-4′), 126.13 (C-2′), 125.98 (C-6′), 101.24 (C-10), 96.72
(C-6), 96.07 (C-8), 83.10 (C-2), 71.70 (C-3). MS (ESI) m/z:
272.0 [M]+.
1
2), 4.50 (1H, d, J = 12.0 Hz, H-3). C NMR (100 MHz,
DMSO-d6) δ ppm: 197.95 (C-4), 167.18 (C-7), 164.04 (C-
5), 163.55 (C-9), 157.77 (C-4′), 129.60 (C-1′), 128.70
(C-3′), 119.98 (C-6′), 115.61 (C-5′), 115.50 (C-2′), 101.24
(C-10), 96.72 (C-6), 96.07 (C-8), 84.18 (C-2), 73.18 (C-3).
MS (ESI) m/z: 288.2 [M]+.
3,5,7-Trihydroxy-2-(3′,4′-dimethoxyphenyl) chroman-4-one
(D5)
White solid; yield: 64%; M.p. 169–170 °C, IR (KBr): νmax
/
cm−1: 1650 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.90 (1H, s, OH-5), 11.01 (1H, s, OH-7), 6.88–7.08
(3H, m, H-2′, 5′, 6′), 5.90 (1H, s, H-8), 5.79 (1H, s, H-6),
5.65 (1H, s, OH-3), 4.97 (1H, d, J = 12.0 Hz, H-2), 4.49
(1H, d, J = 12.0 Hz, H-3), 3.89 (3H, s, OCH3), 3.86 (3H, s,
OCH3). 1C NMR (100 MHz, DMSO-d6) δ ppm: 197.64 (C-
4), 167.28 (C-7), 164.27 (C-5), 163.48 (C-9), 148.66 (C-3′),
148.21 (C-4′), 129.50 (C-1′), 120.57 (C-6′), 111.15 (C-5′),
111.05 (C-2′), 101.21 (C-10), 96.77 (C-6), 96.07 (C-8),
83.60 (C-2), 72.41 (C-3), 56.03 (O–CH3), 55.95 (O–CH3).
MS (ESI) m/z: 332.3 [M]+.
3,5,7-Trihydroxy-2-(3′-hydroxyphenyl) chroman-4-one
(D2)
White solid; yield: 58%; M.p. 201–203 °C, IR (KBr): νmax
/
cm−1: 1650 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 12.02 (1H, s, OH-5), 10.91 (1H, s, OH-7), 8.89 (1H, s,
OH-3′), 7.14 (1H, s, H-2′), 6.88–7.09 (3H, m, H-4′, 5′, 6′),
5.98 (1H, d, J = 2.4 Hz, H-8), 5.90 (1H, d, J = 2.4 Hz, H-6),
5.72 (1H, s, OH-3), 5.01 (1H, d, J = 11.6 Hz, H-2), 4.48
1
(1H, d, J = 11.6 Hz, H-3). C NMR (100 MHz, DMSO-d6)
δ ppm: 198.14 (C-4), 168.01 (C-7), 164.47 (C-5), 163.68
(C-9), 158.70 (C-3′), 134.98 (C-4′), 130.60 (C-1′), 119.89
(C-6′), 116.03 (C-5′), 115.50 (C-2′), 101.24 (C-10), 96.72
(C-6), 96.07 (C-8), 83.26 (C-2), 72.61 (C-3). MS (ESI) m/z:
288.0 [M]+.
3,5,7-Trihydroxy-2-(4′-methoxyphenyl) chroman-4-one
(D6)
White solid; yield: 58%; M.p. 140–142 °C, IR (KBr): νmax
/
cm−1: 1653 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 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′), 6.04 (1H, s,
H-8), 5.95 (1H, s, H-6), 5.73 (1H, s, OH-3), 4.93 (1H, d, J
= 12.0 Hz, H-2), 4.42 (1H, d, J = 12.0 Hz, H-3), 3.80 (3H,
3,5,7-Trihydroxy-2-(3′,4′,5′-trihydroxyphenyl) chroman-4-
one (D3)
1
White solid; yield: 41%; M.p. 234–236 °C, IR (KBr): νmax
/
s, OCH3). C NMR (100 MHz, DMSO-d6) δ ppm: 197.98
cm−1: 1658 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
(C-4), 167.21 (C-7), 164.09 (C-5), 163.43 (C-9), 159.50

Med Chem Res
(C-4′), 129.65 (C-1′), 128.10 (C-2′, 6′), 113.91 (C-3′, 5′),
101.24 (C-10), 96.72 (C-6), 96.07 (C-8), 84.18 (C-2), 73.18
(C-3), 55.35 (O–CH3), 55.95 (O–CH3). MS (ESI) m/z:
302.2 [M]+.
ppm: 12.01 (1H, s, OH-5), 10.98 (1H, s, OH-7), 7.09–7.14
(2H, m, H-2′, 6′), 6.88–7.92 (2H, m, H-3′, 5′), 5.91 (1H, s,
H-8), 5.85 (1H, s, H-6), 5.77 (1H, s, OH-3), 4.87 (1H, d, J
1
= 12.0 Hz, H-2), 4.29 (1H, d, J = 12.0 Hz, H-3). C NMR
(100 MHz, DMSO-d6) δ ppm: 198.55 (C-4), 167.47 (C-7),
164.51 (C-5), 163.53 (C-9), 134.11 (C-4′), 132.29 (C-1′),
128.75 (C-2′, 6′), 128.67 (C-3′, 5′), 100.99 (C-10), 96.25
(C-6), 95.91 (C-8), 84.86 (C-2), 73.10 (C-3). MS (ESI) m/z:
306.7 [M]+.
3,5,7-Trihydroxy-2-(3′-hydroxyphenyl) chroman-4-one
(D7)
White solid; yield: 71%; M.p. 134–136 °C, IR (KBr): νmax
/
cm−1: 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 12.02 (1H, s, OH-5), 10.98 (1H, s, OH-7), 7.22 (1H, s,
H-2′), 6.92–7.16 (3H, m, H-4′, 5′, 6′), 6.01 (1H, s, H-8),
5.98 (1H, s, H-6), 5.68 (1H, s, OH-3), 5.02 (1H, d, J = 12.0
Hz, H-2), 4.49 (1H, d, J = 12.0 Hz, H-3), 3.82 (3H, s,
OCH3). 1C NMR (100 MHz, DMSO-d6) δ ppm: 197.98 (C-
4), 167.21 (C-7), 164.09 (C-5), 163.43 (C-9), 159.50 (C-4′),
129.65 (C-1′), 128.10 (C-2′, 6′), 113.89 (C-3′, 5′), 100.94
(C-10), 96.78 (C-6), 96.04 (C-8), 84.18 (C-2), 73.21 (C-3),
55.35 (O–CH3). MS (ESI) m/z: 302.0 [M]+.
3,5,7-Trihydroxy-2-(4-isopropylphenyl) chroman-4-one
(D11)
White solid; yield: 67%; M.p. 156–157 °C, IR (KBr): νmax
/
cm−1: 1648 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.94 (1H, s, OH-5), 10.89 (1H, s, OH-7), 7.10–7.25
(2H, m, H-2′, 6′), 6.91–6.99 (2H, m, H-3′, 5′), 6.12 (1H, s,
H-8), 5.93 (1H, s, H-6), 5.70 (1H, s, OH-3), 5.01 (1H, d,
J = 12.0 Hz, H-2), 4.35 (1H, d, J = 12.0 Hz, H-3), 2.68 (1H,
1
s, CH3–CH–CH3), 1.20 (6H, s, CH3–CH–CH3). C NMR
3,5,7-Trihydroxy-2-(4′-phenoxyphenyl) chroman-4-one
(D8)
(100 MHz, DMSO-d6) δ ppm: 198.07 (C-4), 167.31 (C-7),
164.30 (C-5), 163.55 (C-9), 148.94 (C-4′), 132.67 (C-1′),
126.06 (C-2′, 6′), 125.56 (C-3′, 5′), 100.06 (C-10), 96.55
(C-6), 96.01 (C-8), 83.04 (C-2), 73.11 (C-3), 33.61 (CH3–
CH–CH3), 24.00 (CH3–CH–CH3). MS (ESI) m/z: 314.1
[M]+.
White solid; yield: 45%; M.p. 99–101 °C, IR (KBr): νmax
/
cm−1: 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.88 (1H, s, OH-5), 10.84 (1H, s, OH-7), 7.10–7.44
(5H, m, Ar–H), 6.88–7.04 (4H, m, H-2′, 3′, 5′, 6′), 6.05 (1H,
s, H-8), 5.93 (1H, s, H-6), 5.75 (1H, s, OH-3), 4.95 (1H, d,
J = 12.0 Hz, H-2), 4.43 (1H, d, J = 12.0 Hz, H-3). 1C NMR
(100 MHz, DMSO-d6) δ ppm: 197.98 (C-4), 167.21 (C-7),
164.29 (C-5), 163.53 (C-9), 159.50 (C-4′), 129.87 (C-1′),
124.49 (C-2′, 6′), 118.69 (C-3′, 5′), 101.20 (C-10), 96.77
(C-6), 96.05 (C-8), 84.50 (C-2), 72.98 (C-3). MS (ESI) m/z:
364.4 [M]+.
3,5,7-Trihydroxy-2-(naphthalen-2-yl) chroman-4-one (D12)
White solid; yield: 65%; M.p. 132–133 °C, IR (KBr): νmax
/
cm−1: 1653 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 12.00 (1H, s, OH-5), 10.88 (1H, s, OH-7), 7.88–8.01
(3H, m, Ar–H), 714–7.68 (4H, m, Ar–H), 5.99 (1H, s, H-8),
5.88 (1H, s, H-6), 5.72 (1H, s, OH-3), 4.99 (1H, d, J = 12.0
1
Hz, H-2), 4.41 (1H, d, J = 12.0 Hz, H-3). C NMR (100
3,5,7-Trihydroxy-2-(4′-fluorophenyl) chroman-4-one (D9)
MHz, DMSO-d6) δ ppm: 197.48 (C-4), 167.35 (C-7),
163.99 (C-5), 163.44 (C-9), 134.21, 133.02, 132.44,
127.73, 127.42, 126.88, 126.45, 126.11, 125.55, 101.16 (C-
10), 96.25 (C-6), 96.12 (C-8), 83.13 (C-2), 73.61 (C-3). MS
(ESI) m/z: 322.0 [M]+.
White solid; yield: 53%; M.p. 213–215 °C, IR (KBr): νmax
/
cm−1: 1652 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.89 (1H, s, OH-5), 10.78 (1H, s, OH-7), 7.02–7.10
(2H, m, H-2′, 6′), 6.88–7.92 (2H, m, H-3′, 5′), 5.94 (1H, s,
H-8), 5.83 (1H, s, H-6), 5.79 (1H, s, OH-3), 4.97 (1H, d,
J = 12.0 Hz, H-2), 4.45 (1H, d, J = 12.0 Hz, H-3). 1C NMR
(100 MHz, DMSO-d6) δ ppm: 198.05 (C-4), 167.28 (C-7),
164.27 (C-5), 163.57 (C-9), 161.56 (C-4′), 131.12 (C-1′),
126.95 (C-2′, 6′), 115.09 (C-3′, 5′), 101.24 (C-10), 96.72
(C-6), 96.07 (C-8), 83.56 (C-2), 73.18 (C-3). MS (ESI) m/z:
290.2 [M]+.
3,5,7-Trihydroxy-2-(pyridin-3-yl) chroman-4-one (D13)
White solid; yield: 52%; M.p. 127–130 °C, IR (KBr): νmax
/
cm−1: 1652 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.92 (1H, s, OH-5), 10.82 (1H, s, OH-7), 8.80 (1H, s,
C-2′), 8.55 (1H, d, J = 5.2 Hz, C-4′), 7.95 (1H, d, J = 8.0
Hz, C-6′), 7.43 (1H, dd, J = 5.2, 8.0 Hz, C-5′), 5.97 (1H, s,
H-8), 5.91 (1H, s, H-6), 5.74 (1H, s, OH-3), 5.02 (1H, d,
J = 12.0 Hz, H-2), 4.52 (1H, d, J = 12.0 Hz, H-3). 1C NMR
(100 MHz, DMSO-d6) δ ppm: 197.55 (C-4), 167.30 (C-7),
164.19 (C-5), 163.29 (C-9), 148.21 (C-2′), 146.79 (C-4′),
132.84 (C-1′), 131.42 (C-6′), 122.92 (C-5′), 101.21 (C-10),
3,5,7-Trihydroxy-2-(4/-chlorophenyl) chroman-4-one (D10)
White solid; yield: 58%; M.p. 189–192 °C, IR (KBr): νmax
/
cm−1: 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ

Med Chem Res
96.77 (C-6), 96.05 (C-8), 81.21 (C-2), 72.69 (C-3). MS
(C-4), 165.00 (C-9), 161.85 (C-7), 145.86 (C-3′), 145.69
(C-4′), 131.50 (C-5), 129.42 (C-1′), 120.05 (C-6′), 115.58
(C-5′), 115.41 (C-2′), 112.23 (C-10), 112.19 (C-6), 103.70
(C-8), 83.26 (C-2), 73.06 (C-3). MS (ESI) m/z: 288.2 [M]+.
(ESI) m/z: 273.0 [M]+.
3,5,7-Trihydroxy-2-(furan-2-yl) chroman-4-one (D14)
White solid; yield: 58%; M.p. 137–138 °C, IR (KBr): νmax
/
cm−1: 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.94 (1H, s, OH-5), 10.85 (1H, s, OH-7), 7.46 (1H, s,
C-3′), 6.12–6.42 (2H, m, C-4′, 5′), 6.00 (1H, s, H-8), 5.90
(1H, s, H-6), 5.72 (1H, s, OH-3), 4.92 (1H, d, J = 12.0 Hz,
3,5-Trihydroxy-2-(3′,4′-dihydroxyphenyl) chroman-4-one
(D18)
White solid; yield: 51%; M.p. 201–204 °C, IR (KBr): νmax
/
cm−1: 1644 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 12.01 (1H, s, OH-5), 9.11 (1H, s, OH-4′), 8.98 (1H, s,
OH-3′), 7.35 (1H, t, H-7), 6.58–7.02 (5H, m, H-6, 8, 2′,
5′,6′), 5.77 (1H, s, OH-3), 5.17 (1H, d, J = 12.0 Hz, H-2),
4.54 (1H, d, J = 12.0 Hz, H-3). 1C NMR (100 MHz,
DMSO-d6) δ ppm: 196.76 (C-4), 162.19 (C-9), 157.20 (C-
5), 145.86 (C-3′), 145.69 (C-4′), 135.43 (C-7), 129.42
(C-1′), 120.05 (C-6′), 115.58 (C-5′), 115.41 (C-2′), 112.26
(C-8), 111.04 (C-6), 105.45 (C-10), 83.26 (C-2), 72.61
(C-3). MS (ESI) m/z: 288.1 [M]+.
1
H-2), 4.52 (1H, d, J = 12.0 Hz, H-3). C NMR (100 MHz,
DMSO-d6) δ ppm: 199.51 (C-4), 167.89 (C-7), 164.25 (C-
5), 162.29 (C-9), 149.83 (C-1′), 142.12 (C-3′), 113.13 (C-
5′), 110.42 (C-4′), 101.22 (C-10), 96.76 (C-6), 95.65 (C-8),
81.17 (C-2), 72.41 (C-3). MS (ESI) m/z: 262.2 [M]+.
3,5,7-Trihydroxy-2-(thiophen-2-yl) chroman-4-one (D15)
White solid; yield: 66%; M.p. 147–138 °C, IR (KBr): νmax
/
cm−1: 1650 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.90 (1H, s, OH-5), 10.81 (1H, s, OH-7), 7.26 (1H, s,
H-3′), 6.13–6.45 (2H, m, H-4′, 5′), 6.04 (1H, s, H-8), 5.95
(1H, s, H-6), 5.69 (1H, s, OH-3), 5.02 (1H, d, J = 12.0 Hz,
3,5,6,7-Tetrahydroxy-2-(3′,4′-dihydroxyphenyl) chroman-4-
one (D19)
1
H-2), 4.52 (1H, d, J = 12.0 Hz, H-3). C NMR (100 MHz,
DMSO-d6) δ ppm: 199.60 (C-4), 167.31 (C-7), 164.29 (C-
5), 162.35 (C-9), 137.53 (C-1′), 126.31 (C-3′), 125.93 (C-
5′), 125.34 (C-4′), 101.31 (C-10), 96.72 (C-6), 95.68 (C-8),
79.91 (C-2), 72.59 (C-3). MS (ESI) m/z: 278.0 [M]+.
White solid; yield: 44%; M.p. 254–256 °C, IR (KBr): νmax
/
cm−1: 1653 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.99 (1H, s, OH-5), 10.89 (1H, s, OH-7), 10.14 (1H,
s, OH-6), 9.15 (1H, s, OH-4′), 8.99 (1H, s, OH-3′),
6.89–7.02 (2H, m, H-5′, 6′), 6.52 (1H, d, J = 2.4 Hz, H-8),
5.67 (1H, s, OH-3), 5.03 (1H, d, J = 12.0 Hz, H-2), 4.50
3-Hydroxy-2-(3′,4′-dihydroxyphenyl) chroman-4-one (D16)
1
(1H, d, J = 12.0 Hz, H-3). C NMR (100 MHz, DMSO-d6)
White solid; yield: 42%; M.p. 101–104 °C, IR (KBr): νmax
/
δ ppm: 195.93 (C-4), 159.34 (C-9), 154.48 (C-5), 153.94
(C-7), 145.86 (C-4′), 145.69 (C-3′), 132.72 (C-1′), 128.84
(C-6), 120.05 (C-6′), 115.58 (C-5′), 115.41 (C-2′), 101.11
(C-10), 94.85 (C-8), 83.73 (C-2), 72.35 (C-3). MS (ESI) m/
z: 320.0 [M]+.
cm−1: 1643 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 9.05 (1H, s, OH-4′), 9.00 (1H, s, OH-3′), 7.59–7.80
(2H, m, H-5, 7), 7.09–7.11 (2H, m, H-6, 8), 6.80–6.95 (3H,
m, H-2′, 3′, 6′), 5.67 (1H, s, OH-3), 5.10 (1H, d, J = 12.0
1
Hz, H-2), 4.60 (1H, d, J = 12.0 Hz, H-3). C NMR (100
MHz, DMSO-d6) δ ppm: 194.24 (C-4), 156.38 (C-9),
145.86 (C-4′), 145.69 (C-3′), 136.49 (C-7), 129.42 (C-1′),
128.38 (C-5), 121.78 (C-10), 120.05 (C-6), 118.27 (C-6′),
118.25 (C-8), 116.58 (C-5′), 115.41 (C-2′), 83.26 (C-2),
73.06 (C-3). MS (ESI) m/z: 272.2 [M]+.
3-Hydroxy-5,7-dimethoxy-2-(3′,4′-dihydroxyphenyl)
chroman-4-one (D20)
White solid; yield: 51%; M.p. 178–180 °C, IR (KBr): νmax
/
cm−1: 1642 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 8.99 (1H, s, OH-4′), 8.90 (1H, s, OH-3′), 6.68–7.00
(3H, m, H-2′, 5′, 6′), 5.95 (1H, d, J = 2.4 Hz, H-8), 5.89
(1H, d, J = 2.4 Hz, H-6), 5.72 (1H, s, OH-3), 5.03 (1H, d,
J = 12.0 Hz, H-2), 4.49 (1H, d, J = 12.0 Hz, H-3), 3.91 (3H,
s, OCH3), 3.84 (3H, s, OCH3). 1C NMR (100 MHz, DMSO-
d6) δ ppm: 189.70 (C-4), 165.13 (C-7), 163.70 (C-5),
161.70 (C-9), 145.86 (C-4′), 145.69 (C-3′), 128.84 (C-1′),
120.05 (C-6′), 115.58 (C-5′), 115.41 (C-2′), 103.70 (C-10),
93.80 (C-6), 93.10 (C-8), 83.25 (C-2), 72.50 (C-3), 56.13
(OCH3), 55.87 (OCH3). MS (ESI) m/z: 332.3 [M]+.
3, 7-Hydroxy-2-(3′,4′-dihydroxyphenyl) chroman-4-one
(D17)
White solid; yield: 32%; M.p. 189–190 °C, IR (KBr): νmax
/
cm−1: 1643 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 10.87 (1H, s, OH-7), 9.12 (1H, s, OH-4′), 9.00 (1H, s,
OH-3′), 7.88 (1H, d, J = 7.6 Hz, H-5), 6.78–7.01 (4H, m,
H-8, 2′, 5′,6′), 6.21 (1H, d, J = 7.6 Hz, H-6), 5.77 (1H, s,
OH-3), 5.07 (1H, d, J = 12.0 Hz, H-2), 4.67 (1H, d, J =
1
12.0, H-3). C NMR (100 MHz, DMSO-d6) δ ppm: 193.02

Med Chem Res
3-Hydroxy-7-methoxy-2-(3′,4′-dihydroxyphenyl) chroman-
4-one (D21)
5.79 (1H, s, OH-3), 5.38 (1H, s, NH-1), 4.57 (1H, d, J =
12.0 Hz, H-2), 4.29 (1H, d, J = 12.0, H-3). C NMR (100
1
MHz, DMSO-d6) δ ppm: 199.04 (C-4), 164.32 (C-7),
163.88 (C-5), 153.45 (C-9), 145.37 (C-4′), 145.32 (C-3′),
136.30 (C-1′), 120.53 (C-6′), 116.42 (C-5′), 115.41 (C-2′),
100.54 (C-10), 97.27 (C-6), 95.56 (C-8), 72.00 (C-2), 61.41
(C-3). MS (ESI) m/z: 303.0 [M]+.
White solid; yield: 62%; M.p. 152–155 °C, IR (KBr): νmax
/
cm−1: 1644 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 9.02 (1H, s, OH-4′), 8.97 (1H, s, OH-3′), 7.75 (1H, d,
J = 7.2 Hz, H-5), 6.60–6.99 (5H, m, H-6, 8, 2′, 5′, 6′), 5.71
(1H, s, OH-3), 5.04 (1H, d, J = 12.0 Hz, H-2), 4.51 (1H, d,
J = 12.0 Hz, H-3), 3.78 (3H, s, OCH3). 1C NMR (100 MHz,
DMSO-d6) δ ppm: 194.22 (C-4), 167.40 (C-7), 162.72 (C-
9), 145.88 (C-4′), 145.79 (C-3′), 129.42 (C-5), 129.10 (C-
1′), 120.05 (C-6′), 115.59 (C-5′), 115.11 (C-2′), 112.00 (C-
10), 111.25 (C-6), 101.25 (C-8), 83.16 (C-2), 73.16 (C-3),
55.64 (OCH3). MS (ESI) m/z: 302.2 [M]+.
3,5,7-Trihydroxy-3-methyl-2-(3′,4′-dihydroxyphenyl)
chroman-4-one (D25)
White solid; yield: 25%; M.p. 201–202 °C, IR (KBr): νmax
/
cm−1: 1642 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.99 (1H, s, OH-5), 10.82 (1H, s, OH-7), 9.08 (1H, s,
OH-4′), 8.91 (1H, s, OH-3′), 6.75–6.93 (3H, m, H-2′, 5′, 6′),
5.98 (1H, d, J = 2.4 Hz, H-8), 5.89 (1H, d, J = 2.4 Hz, H-6),
5.67 (1H, s, OH-3), 5.27 (1H, s, H-2), 1.45 (3H, s, CH3). 1C
NMR (100 MHz, DMSO-d6) δ ppm: 198.89 (C-4), 166.84
(C-7), 164.20 (C-5), 161.83 (C-9), 147.80 (C-4′), 146.19
(C-3′), 128.05 (C-1′), 121.08 (C-6′), 116.29 (C-5′), 116.20
(C-2′), 102.29 (C-10), 96.52 (C-6), 95.26 (C-8), 87.20 (C-
2), 76.59 (C-3), 19.47 (CH3). MS (ESI) m/z: 318.2 [M]+.
3-Hydroxy-5-methoxy-2-(3′,4′-dihydroxyphenyl) chroman-
4-one (D22)
White solid; yield: 58%; M.p. 172–174 °C, IR (KBr): νmax
/
cm−1: 1645 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 9.01 (1H, s, OH-4′), 8.96 (1H, s, OH-3′), 7.41 (1H, s,
H-7), 6.63–7.01 (5H, m, H-6, 8, 2′, 5′, 6′), 5.70 (1H, s, OH-
3), 5.03 (1H, d, J = 12.0 Hz, H-2), 4.48 (1H, d, J = 12.0 Hz,
1
H-3), 3.91 (3H, s, OCH3). C NMR (100 MHz, DMSO-d6)
δ ppm: 195.92 (C-4), 159.87 (C-5), 157.04 (C-9), 145.86
(C-3′), 145.69 (C-4′), 135.09 (C-7), 129.42 (C-1′), 120.05
(C-6′), 115.58 (C-5′), 115.41 (C-2′), 113.16 (C-10), 108.88
(C-6), 107.47 (C-8), 83.26 (C-2), 72.61 (C-3), 56.45
(OCH3). MS (ESI) m/z: 302.0 [M]+.
3-Hydroxy-5,7-dimethoxy-2-(3,4-dimethoxyphenyl)
chroman-4-one (D26)
White solid; yield: 75%; M.p. 209–210 °C, IR (KBr): νmax
/
cm−1: 1646 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 6.81–7.04 (3H, m, H-2′, 5′, 6′), 5.97 (1H, d, J = 2.4
Hz, H-8), 5.88 (1H, d, J = 2.4 Hz, H-6), 5.81 (1H, s, OH-3),
4.99 (1H, d, J = 12.0 Hz, H-2), 4.47 (1H, d, J = 12.0 Hz, H-
3), 3.81–3.90 (12H, m, OCH3). 1C NMR (100 MHz,
DMSO-d6) δ ppm: 189.70 (C-4), 165.13 (C-7), 163.70 (C-
5), 161.70 (C-9), 148.66 (C-4′), 148.21 (C-3′), 129.50 (C-
1′), 120.57 (C-6′), 111.15 (C-5′), 111.05 (C-2′), 103.70 (C-
10), 93.80 (C-6), 93.10 (C-8), 83.60 (C-2), 72.50 (C-3),
56.13 (OCH3), 56.03 (OCH3), 55.95 (OCH3), 55.87
(OCH3). MS (ESI) m/z: 360.3 [M]+.
3-Hydroxy-7-chloro-2-(3′,4′-dihydroxyphenyl) chroman-4-
one (D23)
White solid; yield: 68%; M.p. 166–168 °C, IR (KBr): νmax
/
cm−1: 1641 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 9.11 (1H, s, OH-4′), 9.00 (1H, s, OH-3′), 7.80 (1H, d,
J = 7.6 Hz, H-5), 7.13–7.27 (2H, m, H-6, 8), 6.71–6.93
(3H, m, H-2′, 5′, 6′), 5.70 (1H, s, OH-3), 5.06 (1H, d, J =
1
12.0 Hz, H-2), 4.51 (1H, d, J = 12.0 Hz, H-3). C NMR
(100 MHz, DMSO-d6) δ ppm: 195.39 (C-4), 155.33 (C-9),
145.86 (C-4′), 145.69 (C-3′), 135.79 (C-7), 129.42 (C-5),
127.06 (C-1′), 126.96 (C-6), 120.05 (C-6′), 119.76 (C-10),
119.12 (C-8), 115.59 (C-5′), 115.41 (C-2′), 83.26 (C-2),
73.06 (C-3). MS (ESI) m/z: 306.4 [M]+.
3-Hydroxy-2-phenylchroman-4-one (D27)
White solid; yield: 75%; M.p. 186–188 °C, IR (KBr): νmax
/
cm−1: 1643 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 7.92–7.94 (1H, m, H-7), 7.50–7.58 (6H, m, H-5, 2′, 3′,
4′, 5′, 6′), 7.08–7.18 (2H, s, H-6, 8), 5.13 (1H, d, J = 12.0
Hz, H-2), 4.62 (1H, d, J = 12.0 Hz, H-3), 3.69 (1H, s, OH-
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-2,3-
dihydroquinolin-4(1H)-one (D24)
1
White solid; yield: 61%; M.p. >300 °C, IR (KBr): νmax
/
3). C NMR (100 MHz, DMSO-d6) δ ppm: 194.23 (C-4),
cm−1: 1644 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.85 (1H, s, OH-5), 10.75 (1H, s, OH-7), 9.11 (1H, s,
OH-4′), 8.82 (1H, s, OH-3′), 6.68–6.86 (3H, m, H-2′, 5′, 6′),
5.94 (1H, d, J = 2.4 Hz, H-8), 5.85 (1H, d, J = 2.4 Hz, H-6),
161.74 (C-9), 136.92 (C-1′), 136.36 (C-7), 129.30 (C-3′, 5′),
128.69 (C-4′), 127.57 (C-5), 127.35 (C-2′, 6′), 122.11 (C-
10), 118.14 (C-6), 118.75 (C-8), 83.91 (C-2), 73.67 (C-3).
MS (ESI) m/z: 340.2 [M]+.

Med Chem Res
3,5,7-Trihydroxy-2-(pyridin-3-yl)-2,3-dihydroquinolin-4
(1H)-one (D28)
ml of penicillin (Gibco), and 100 mg/ml of streptomycin
(Gibco). Cell cultures were maintained at an atmosphere of
5% CO2 humidified incubator at 37 °C.
White solid; yield: 42%; M.p. 188–190 °C, IR (KBr): νmax
/
cm−1: 1647 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 11.89 (1H, s, OH-5), 10.91 (1H, s, OH-7), 9.10 (1H, s,
OH-4′), 8.95 (1H, s, OH-3′), 7.35–8.51 (3H, m, H-2′, 5′, 6′),
5.92 (1H, d, J = 2.4 Hz, H-8), 5.86 (1H, d, J = 2.4 Hz, H-6),
5.74 (1H, s, OH-3), 5.28 (1H, s, NH), 4.80 (1H, d, J = 12.0
Cell viability assay
The cytotoxicity of the tested compounds was evaluated by
MTT (Sigma, USA) colorimetric assay (Hanelt et al. 1994).
RAW 264.7 cells at 5 × 103 per well were seeded in 96-well
plates (Corning, NY, USA), and the testing compounds at
seven concentrations (320, 160, 80, 40, 20, 10, and 5 μM)
were incorporated into the cell culture in DMEM medium in
humidified incubator within a 5% CO2 atmosphere at 37 °C
for 24 h. Then, 20 μl MTT solutions (5 mg/ml) were added
to each well for another 4 h. The resulting crystals were
dissolved in DMSO. The optical density was measured at
490 nm. The cytotoxicity was calculated from the plotted
results using untreated cells at 100%.
1
Hz, H-2), 4.32 (1H, d, J = 12.0, H-3). C NMR (100 MHz,
DMSO-d6) δ ppm: 199.04 (C-4), 164.32 (C-7), 163.88 (C-
5), 153.45 (C-9), 145.37 (C-2′), 145.32 (C-4′), 136.30 (C-
1′), 120.53 (C-6′), 116.42 (C-5′), 100.54 (C-10), 97.27 (C-
6), 95.56 (C-8), 72.00 (C-2), 61.41 (C-3). MS (ESI) m/z:
272.2 [M]+.
2-(3,4-Dihydroxyphenyl)-3-hydroxy-2,3-dihydroquinolin-4
(1H)-one (D29)
White solid; yield: 52%; M.p. 225–228 °C, IR (KBr): νmax
/
In vitro anti-inflammatory assay
cm−1: 1645 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 10.01 (1H, s, OH-4′), 9.05 (1H, s, OH-3′), 6.68–7.40
(7H, m, H-5, 6, 7, 8, 2′, 5′, 6′), 5.89 (1H, s, OH-3), 4.74
(1H, d, J = 12.0 Hz, H-2), 4.32 (1H, d, J = 12.0 Hz, H-3).
1C NMR (100 MHz, DMSO-d6) δ ppm: 195.89 (C-4),
148.73 (C-9), 145.37 (C-4′), 145.32 (C-3′), 136.30 (C-1′),
133.30 (C-7), 128.79 (C-5), 120.67 (C-6′), 120.53 (C-6),
118.95 (C-8), 116.57 (C-5′), 116.42 (C-10), 115.41 (C-2′),
71.98 (C-2), 61.41 (C-3). MS (ESI) m/z: 271.2 [M]+.
Pro-inflammatory cytokine production by LPS in the RAW
264.7 cells was measured according to the method descri-
bed by Sudsai et al. (2014) and Wang et al. (2016). The
RAW 264.7 cells were stimulated with LPS (1 μg/ml) for
22 h. Then, the tested compounds in 0.1% DMSO were
given and the concentration was adjusted to 20 μM with the
medium for 24 h. The culture media were collected and
centrifuged (1000 rpm) at 4 °C for 10 min, and the super-
natant was collected. IL-1β, IL-6, and TNF-a levels in the
medium were determined with an ELISA kit (eBioScience,
Inc.) according to the manufacturer’s instructions.
3-Hydroxy-2-phenyl-2,3-dihydroquinolin-4(1H)-one (D30)
White solid; yield: 71%; M.p. 158–160 °C, IR (KBr): νmax
/
cm−1: 1640 (C=O). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 7.40–7.68 (4H, m, H-5, 7, 3′, 5′), 6.85–7.28 (5H, m,
H-6, 8, 2′, 4′, 6′), 4.57 (1H, d, J = 12.0 Hz, H-2), 4.29 (1H,
Result and discussion
Chemistry
1
d, J = 12.0 Hz, H-3), 3.61 (1H, s, OH-3). C NMR (100
MHz, DMSO-d6) δ ppm: 195.89 (C-4), 148.73 (C-9),
138.13 (C-1′), 133.30 (C-7), 128.81 (C-3′, 5′), 128.15 (C-
4′), 127.51 (C-5), 120.67 (C-2′, 6′), 118.95 (C-10), 116.54
(C-6), 115.75 (C-8), 71.98 (C-2), 60.76 (C-3). MS (ESI) m/
z: 239.0 [M]+.
The dihydroflavonol derivatives described in this paper
were prepared according to the synthetic route in Scheme 1.
In general, the 2′-hydroxyl chalcone derivatives were syn-
thesized via Claisen–Schmidt reaction (Sui et al. 2012;
Guan et al. 2013; Sun et al. 2012). According to Scheme 1,
treatment of 2′-hydroxyl chalcone derivatives with 30%
H2O2 and diethylamine in tetrahydrofuran under −5 °C,
afforded a white crystalline product identified as dihydro-
flavonol derivatives (D1–D30 and TA-SY). The infrared
(IR) spectrum of compounds D1–D30 and TA-SY which
showed sharp peak at around 1650 cm−1 indicates the pre-
sence of ketone (–C=O) functional group (4-position) of C
ring. NMR spectroscopy was very important for the iden-
tification of flavonoids. NMR datas of the target compounds
(D1–D30 and TA-SY) structure confirmed by the presence
Biological assays
Cell cultures
The 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 cultured in Dulbecco’s modified Eagle’s medium
(DMEM; HyClone, USA) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (Bovine, China), 100 U/

Med Chem Res
of 2, 3-position position of C ring. 1H NMR spectrum
displayed one double doublet at δH = 4.50 ppm (1H, d, H-
3) and one doublet at δH = 5.00 ppm (1H, d, H-2). The
value of coupling constant (J = 12.0 Hz) for H-2 and H-3
protons indicate that the compounds exist in the E-config-
uration, which is in complete agreement with the results
recentl reported (Slade et al. 2005). Moreover, all the aro-
matic protons were observed at 8.10–5.80 ppm. 13C NMR
spectral assigned signals in the range at near δ 80.00 ppm
and 70.00 ppm due to presence of C-2 and C-3 of C ring
(Pelter et al. 1976). Mass spectroscopy helps to find the
molecular weight of the synthesized compounds. The
dihydroflavonol derivatives showed the molecular ion peak
that equivalent to the molecular weight of proposed com-
pound. The synthesized compounds were supported by
Anti-inflammatory activity
The precision of the assays for IL-1 beta, IL-6, and TNF
alpha is mainly studied by plotting standard curves.
According to the manufacturer’s instructions, we take the
standard density (standard curves use the following con-
centrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 pg/
ml) as the abscissa, the OD value for the vertical, draw the
standard curve. Finally, the linear correlations R2 between
the sample linear regression and the expected concentration
coefficient were more than 0.995. This shows that the kits
performance is excellent according to the kits instructions.
Therefore, the data determined later is feasible. Having
secured a series of structurally diverse dihydroflavonol
derivatives, next their anti-inflammatory activity was eval-
uated. The results of the anti-inflammatory are collected in
Figs. 4, 5, and 6 respectively, Meloxicam was used as the
positive control. Taxifolin is a natural compound with a
single configuration, and TA-SY with similar structure of
taxifolin is only a synthetic dihydroflavonol compound. So
we mainly wanted to investigate whether different config-
urations of dihydroflavonol have an impact on their anti-
inflammatory activity. In Fig. 4, the results revealed that
there was no significant difference in the anti-inflammatory
activity between the natural product (taxifolin) and syn-
thetic product (TA-SY). Among all the compounds
screened, Meloxicam, compounds D9, D13, and D19
showed better inhibitory effect than Taxifolin on LPS-
induce IL-1β, IL-6, and TNF-α in Fig. 5; however, com-
pound D12 substituted by naphthaline showed the lowest
effect on LPS-induce IL-1β, IL-6, and TNF-α, which may
be due to the bad solubility.
1
spectral data. The IR, H NMR, 13C NMR, and MS results
are consistent with the proposed structures.
Cell viability
MTT assay was used to screen the influences of dihydro-
flavonol derivatives on the viability of RAW 264.7 cells.
Seven concentrations (320, 160, 80, 40, 20, 10, and 5 μM)
of Taxifolin and dihydroflavonol derivatives and the sti-
mulator LPS (1 μg/ml) were choosed to determine the cell
viability. As shown in Fig. 3, there was no significant dif-
ference between the control group (normal) and all the
treated groups. It indicated that all the compounds were no
significant proliferation effects on RAW 264.7 cells at the
concentration 20 μM. Thus, we selected the concentration
20 μM and 1 μg/ml of LPS as the effective concentration in
the next experiments.
Fig. 4 Effects of Taxifolin and TA-SY on IL-1β, IL-6 and TNF-α
production at 20 μM. Values represent the mean SD for three inde-
pendent experiments. Statistical significance relative to the DMSO
group was indicated, #p < 0.05. Statistical significance relative to the
LPS group was indicated, ***p < 0.001
Fig. 3 Cell viability of Taxifolin and dihydroflavonol derivatives on
RAW 264.7 cells at 20 μM. Cell viability was determined for Taxifolin
and tested compounds (TA-SY and D1–D30) at 20 μM. The values are
mean SEM (n = 3)

Med Chem Res
Fig. 5 Effects of dihydroflavonol derivatives on IL-1β (a), IL-6 (b),
and TNF-α (c) production at 20 μM. Values represent the mean SD
for three independent experiments. Statistical significance relative to
the DMSO group was indicated, #p < 0.05. Statistical significance
relative to the LPS group was indicated, *p < 0.05; **p < 0.01; ***p
< 0.001
As shown in the Fig. 6, There were significant differences
between the low dose groups and the high dose groups (p <
0.05). However, there were no significant differences
between the high dose groups and the medium dose groups.
At the same time, the relationship between the medium dose
groups and the low dose groups were very complicated. For
example, for compounds D9, it had an inhibitory effect on
inflammatory factors (IL-1β and TNF-α), where there was a
significant difference between the medium dose group and
the low dose group (p < 0.05). However, there was no sig-
nificant difference between the medium dose group and the
low dose group in the suppression of inflammatory factor
(IL-6). From the above results, we analyzed that it was
noteworthy that compounds D9, D13, and D19 decreased
obviously the content of IL-1β, IL-6, and TNF-α in a
concentration-dependent manner.
had a close relationship with 3′- and or 4′-substituted B-ring
(Jiang et al. 2015). In comparison of functional group
effects at 3′- or 4′-position of B-ring for inflammatory
cytokines inhibition, the rank of the orders of the inhibitory
activities for dihydroflavonol derivatives was fluoro (D9) >
phenolic hydroxyl (D1) > methoxy (D6) > isopropyl (D11)
for 4′-substituent, and phenolic hydroxyl (D2) = methoxy
(D7) for 3/- substituent, respectively. Compared to the
inhibitory activities of inflammatory cytokines on B-ring,
pyridine ring (D13) showed more potent activity than that
of several B-ring analogs (D4, D12, D14, and D15). SAR
study around 5- and/or 7-substituted A-ring gave unique
results as well. Compared to the inhibitory activities of
inflammatory cytokines for phenolic hydroxyl substituent
on A-ring, 5, 6, 7-phenolic hydroxyl substituent on A-ring
(D19) showed more potent than other analogs. However,
SAR study around 1- and 3-substituted C-ring gave bad
results (D24, D25), which suggested that the C-ring is a
The inhibitory activity of dihydroflavonols against pro-
inflammatory cytokines production in inflammatory cells

Med Chem Res
Fig. 6 Compounds D9, D13, and D19 inhibited LPS-induced IL-1β
(a), IL-6 (b), and TNF-α (c) release in a dose-dependent manner.
Values represent the mean SD for three independent experiments.
Statistical significance relative to the DMSO group was indicated, #p
< 0.05. Statistical significance relative to the LPS group was indicated,
*p < 0.05; **p < 0.01; ***p < 0.001
necessary structure for its anti-inflammatory activity. With
the above analysis, the various substituent-effects toward
potent dihydroflavonol derivatives clarified in the present
SAR study in vitro might be useful for designing further
dihydroflavonol anti-inflammatory drugs.
was like positive control and exhibited an obvious
dose–effect relationship. In summary, compounds D9, D13,
and D19 may present a potential therapeutical use as an
anti-inflammatory agent, being also useful as a precursor of
new flavonoids endowed with such activity.
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).
Conclusions
In conclusion, thirty dihydroflavonol derivatives (D1–D30)
had been designed and synthesized by the new method, and
their anti-inflammatory activities were evaluated. Besides,
their structure–activity relationships were discussed pre-
liminarily. The results showed that the A ring, B ring, and C
ring substituted by different group exhibited different anti-
inflammatory activities. When the 4′-position of B-ring
substituted by the fluorine atom or the B-ring was pyridine
ring, the anti-inflammatory activity of compounds D9, D13
were increased. With the amounts of hydroxyl group sub-
stituted on A-ring increasing, the anti-inflammatory activity
of compound D19 was better. Among these derivatives,
compounds D9, D13, and D19 showed impressive anti-
inflammatory activities at micro molar concentration, which
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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