Synthesis and evaluation of C15 triene urushiol derivatives as potential anticancer agents and HDAC2 inhibitor

Article abstract of DOI:10.3390/molecules23051074

A series of C15 triene urushiol derivatives were synthesized and evaluated for their anti-HepG2 aggregation in vitro. The results indicated that all compounds had an effective anti-HepG2 vitality. Compound 1 was a potent inhibitor of HepG2 with IC_50<

Full text of DOI:10.3390/molecules23051074

molecules
Article
Synthesis and Evaluation of C15 Triene Urushiol
Derivatives as Potential Anticancer Agents and
HDAC2 Inhibitor
Zhiwen Qi 1,2,3 ID , Chengzhang Wang 1,2,* and Jianxin Jiang 3
1
Institute of Chemical Industry of Forest Products, CAF, Nanjing 210042, China; angelkissgod@163.com
Key and Open Laboratory on Forest Chemical Engineering, SFA, Nanjing 210042, China
College of Material Science and Technology, Beijing Forestry University, Beijing 100083, China;
2
3
jiangjx@bjfu.edu.cn
*
Correspondence: wangczlhs@sina.com; Tel.: +86-139-5194-5535
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 March 2018; Accepted: 27 April 2018; Published: 3 May 2018
Abstract: A series of C15 triene urushiol derivatives were synthesized and evaluated for their
anti-HepG2 aggregation in vitro
anti-HepG2 vitality. Compound
.
The results indicated that all compounds had an effective
was a potent inhibitor of HepG2 with IC50 of 7.886 M and
increased the apoptosis of HepG2. Compound ’s thiol
1
µ
150
µM against LO2. Moreover, compound
1
1
sulfur formed hydrogen bonding interactions with Gly154 and Tyr308, respectively, and made it
bound more closely to HDAC2. In addition, it also formed hydrophobic interactions with the residues
His33, Pro106, Val107, Gly154, Phe155, and His183, and was provided with a strong van der Waals
force by the hydrophobic action.
Keywords: urushiol; derivatives; antitumor; HDAC2; molecular docking
1. Introduction
As a natural alkyl phenol, urushiol is similar to SAHA which is efficient for tumor HDACs,
but has mutagenic, carcinogenic and genotoxic potential [
of SIRT inhibitors [ ] and prevent lung cancer cell-A549 proliferation [
effect on Helicobacter pylori [ ], and stimulates the activity of certain cells of mice, reducing the risk
of fatty liver in mice and changing microbial morphology [5,7].
1]. Urushiol is able to modulate the activity
2
3]. It has a good antibacterial
4–6
Our research has indicated that the urushiol derivatives have excellent binding force to HDAC2 [8,9],
which are a group of zinc metalloenzymes, regulating chromatin remodeling and gene transcription by
catalyzing the removal of an acetyl group moiety from the ε-amino groups of lysine residues on the amino
terminal tails of the core histones [10]. HDACs play a pivotal role in the regulation of gene expression, cell
growth, and proliferation [11]. Overexpression of HDACs has been linked to the development of cancers
in humans [12]. This evidence suggests that urushiol has potential antitumor effect.
Modification is a usual method to improve product qualities. For example, common catechols are
mainly modified by methylation (sulfonation) [13], halogenation [14
and boric esterification [19] which is moderately stable in air [20] due to its high affinity for diols [21],
and is frequently used for recognizing carbohydrate [22 24]. Besides, some highly fluorinated
,15], Pechmann reaction [16–18],
–
catechol compounds have excellent cytotoxic activity against prostate cancer cells, and decrease their
viability [25], giving rise to a gene delivery ability [26]. They also have excellent organic electronics
property [27], and are used as probes [28].
Hence, based on clinical molecule simulation principle [8], we tried to lower the urushiol toxicity
and enhance its efficacy. In this study, we chemically modified the C15 triene urushiol to reduce its
Molecules 2018, 23, 1074; doi:10.3390/molecules23051074
www.mdpi.com/journal/molecules

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toxicity by applying the strategies mentioned above. The best bioactive compound to inhibit HepG2
was evaluated by FCM-flow cytometry, Western blot and molecule docking analysis.
2. Results
2.1. Chemistry
C15 triene urushiol was extracted and isolated from the lacquer (Toxicodendron vernicifluum),
and purified by silica gel column chromatography. According to the general pathway outlined in
Scheme 1, target compounds 1–16 mentioned in this research were synthesized in a one-step reaction.
Since the ionization constant of 2-position phenolic hydroxyl is ten times higher than that of 1-position
of benzene ring [29], the halogenation reaction of phenol hydroxyl group at 2-position is more likely
to take place under the condition of alkalinity such as K2CO3, and produce compounds
and 11. The phenolic hydroxyl groups of urushiol Pechmann derivative are easy to react with similar
reaction to form compounds 14 15 and 16. In addition, the boric acid esterification reaction between
the two phenolic hydroxyl groups of urushiol is easy to occur, forming compounds 10
12 and 13. The detailed synthetic processes are described in the Materials and Methods Section.
1, 3, 8, 9,
,
2,
4,
5,
6
,
,
Compounds 1–16 were purified by silica gel column chromatography and their chemical structures
were characterized by using 1H NMR, 13C NMR, and ESI-MS.
Scheme 1. Synthetic routes for C15 triene urushiol derivatives via phenol hydroxide protection.
Reagents and conditi◦ons: (
a) K2CO3, acetone, reflux, 24 h; (b) p-Toluene, ethyl acetoacetate, r.t., 8 h;
and (c) DCM/EA, 60 C, 2 h, in a pressure tube.
2.2. Anti-Tumor Activity
As shown in Figure 1 and Table 1, compounds 1–16 were evaluated for their inhibitory effects
on the proliferation of HepG2 and LO2 cells by MTT assay. The structure–activity relationship of
the above-mentioned urushiol derivatives showed that, with the thiol-containing long-chain alkane,
phenylboronic acid and amino sulfoxide, urushiol as well as urushiol Pechmann derivative played an
effective role in attenuating and increasing the efficiency. It provided the experimental basis for the
subsequent synthesis of attenuating and improving urushiol derivatives.
Table 1. Urushiol derivatives inhibitory activity against HepG2 and LO2 in vitro (IC50, µM, 72 h).
Compound
1
2
3
4
5
6
7
8
HepG2 a/IC50
LO2/IC50
7.88
150.59
197.94
>200
15.33
180.78
180
>200
>200
>200
89.66
>200
37.25
180.51
>200 b
>200
Compound
HepG2 a/IC50
LO2/IC50
9
10
28.75
198.36
11
>200
>200
12
50.57
120.54
13
65.43
150.84
14
78.66
>200
15
67.04
>200
16
150.62
>200
15.01
120.81
a
HepG2: human liver hepatocellular carcinoma cells; b No inhibitory activity at 200 µM.

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100
90
80
70
60
50
40
30
20
10
0
0.78μM
12.5μM
100 μM
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
compound
(a)
45
30
15
0
100μM
25μM
0.78μM
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
compound
(b)
Figure 1. Compounds
and ( ) the inhibitory rate on LO2. Statistical treatment: SPSS 17.0 Statistical software for data
processing and statistical scoring Analysis. The experimental data are expressed as mean standard
deviation (mean SD). Normality and variance homogeneity test are carried out to satisfy the normal
1–16 inhibitory on HepG2 and LO2 in vitro: (a) the inhibitory rate on HepG2;
b
±
±
distribution and variance. Univariate ANOVA was used to compare the homogeneity. p < 0.05.
There was significant difference between groups.
We found that the inhibitory activity of compounds 1, 4 and 9 on HepG2 cells proliferation was
strong. The IC50 values of all compounds were obtained in gradient experiment. The inhibitory activity
of compound 1 on HepG2 cells was 7.886 µM.
Compounds
group, a benzene borate bond, and an amino sulfoxide, which increased their anti-HepG2 activity to
7–15 M and reduced the sensitization of urushiol. Introducing a long-chain alkane chloride into the
phenolic hydroxyl group of 2-position urushiol, compound ’s anti-HepG2 activity slightly decreased
to 37 M, but its toxic effect on normal liver cells LO2 significantly reduced to 180 M. Compound 10,
1, 4, and 9, respectively, introduced a thiol group on the 2-position phenolic hydroxyl
µ
8
µ
µ
a urushiol Pechmann derivative, was resistant to activity of HepG2, essentially maintaining at 28 µM.
However, its toxic effect on normal liver cell LO2 was reduced by 3–4 fold, reaching 180 µM.

Molecules 2018, 23, 1074
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In addition, the introduction of pyridine borate on the phenolic hydroxyl group reduced the
activity of anti-HepG2 (from 50 to 67 M). At the same time, the toxic effect on the normal cells LO2 of
liver cancer was also weakened (>120 M). The derivative introduced a molecule of 2-fluoropyridine
borate on the phenolic hydroxyl group, but its activity against hepatoma cells decreased dramatically
(almost > 200 M), and the damage to the normal liver cell LO2 also drastically decreased (all > 200 M).
µ
µ
µ
µ
2.3. Inhibition of Cell Migration
As shown in Figure 2, morphological observation showed that proliferation of HepG2 cells was
gradually inhibited by addition of the compound . The mortality rate of HepG2 cells was increasing
faster on higher concentration of compound . The inhibition of cell proliferation strengthened over
time. The negative control group showed uniform cytoplasm, clear nucleoli, full cells and good
spindle shape. Besides, with the increase of compound ’s concentration, the morphological changes
1
1
1
of cells were obvious, and the number of cells decreased significantly. The cells became round,
the volume became smaller and the refraction decreased. When the cells were treated with high
concentration of compound
1, the cells were suspended and shed. The cells no longer adhered to the
wall. They gradually became round, and the membrane ruptured, shrank and finally lost vitality.
(a)
(b)
(c)
(d)
Figure 2. Proliferation of HepG2 cells by compound
control; (b) 6.25 µM; (c) 25 µM; and (d) 50 µM.
1
at different concentrations: (a) HepG2 negative
2.4. Western Blot Results
The total protein was extracted from the logarithmic growth phase of hepatoma HepG2 cell
line, and the expression of HDAC2 was detected by Western blot method with urushiol derivative
compound
with correct molecular weight. Compared with normal hepatocyte LO2 group, the expression of
HDAC2 protein in urushiol derivative compound group was significantly lower than that in
1(see Figure 3). The Western blot analysis of HDAC2 in HepG2 cells showed clear bands
1

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hepatoma cell group (p < 0.05). The results showed that phenol derivative
1
could down-regulate
the expression of hepatoma cells and alleviate the pathological changes of HCC. The mechanism was
probably related to the inhibition of HDAC2 expression.
Figure 3. The protein expression of HDAC2 in different groups ((a) LO2 cells; (b) HepG2 cells; and (c)
Compound 1 on HepG2 cells).
2.5. FCM-Flow Cytometry Analysis
Based on the preliminary screening results, HepG2 cells were incubated with various
concentrations of compound to investigate the effect of PI Single staining assay for cell cycle,
1
Annexin-V FITC/PI Double staining for apoptosis, JC-1 Staining assay mitochondrial membrane
potential, and Calcium content detection (methods details are shown in the Supplementary Materials).
2.5.1. PI Single Staining Assay for Cell Cycle
After the cells were tested with three different doses of Compound
1 (1, 3, and 12 µM), they were
collected and detected by flow cytometry. HepG2 cells changed significantly in the distribution of cell
cycle with the increase of drug concentration. Compared with the blank control group, the proportion
of cells in G0/G1 phase of each dose of compound
stage cells and the proportion of cells in G2/M stage decreased to some extent. Compound
1
increased gradually, but both the percentage of S
could
1
block cells in G0/G1 phase. Compared with blank group, each dose group was significantly different
(p < 0.05) (see Table 2 and Figure 4).
Table 2. Compound 1 effect on cell cycle of HepG2 cells (x ± s, n = 7).
Compound
Group/µM
G1 (%)
S (%)
G2 (%)
CON
1
3
48.06
54.48
60.17
65.80
35.8
29.72
26.19
20.40
16.14
15.79
13.64
13.79
1
12
p < 0.01, compared with control group.
(a)
(b)
Figure 4. Cont.

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(c)
(d)
Figure 4. Compound
12 µM.
1
effect on cell cycle of HepG2 cells: (a) control group; (b) 1 µM; (c) 3 µM; and (d)
2.5.2. Annexin-V FITC/PI Double Staining for Apoptosis
After treating HepG2 cells with three concentrations of compound
was used to detect the apoptosis rate changes. The apoptosis rate of 12
1
for 72 h, flow cytometry
µ
M group was significantly
higher than that of the control group in a dose-dependent manner. The apoptosis rates of the three
concentrations were 15.73%, 32.60% and 47.87% (see Table 3 and Figure 5).
Table 3. Effect of compound 1 on the apoptosis rate of HepG2 cells.
Compound
1
Group
UL (%)
UR (%)
LL (%)
LR (%)
Apoptosis (%)
CON
1 µm
3 µm
12 µm
0.63
1.56
0.07
3.24
2.44
6.35
16.18
10.19
94.78
82.71
67.33
48.91
2.16
9.38
16.42
37.68
4.6
15.73
32.60
47.87
(a)
(b)
(c)
on the apoptosis rate of HepG2 cells: (
(d)
Figure 5. Compound
and (d) 12 µM.
1
a
) control group; (b) 1 µM; (c) 3 µM;

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2.5.3. JC-1 Staining Assay Mitochondrial Membrane Potential
As shown in Table 4 and Figure 6, the cells in the control group (UR: 94.20%, LR: 5.8%) and 12 µM
experimental group (UR: 46.18%, LR: 53.84%) were stimulated by compound
decrease of membrane potential and cell apoptosis.
1, which reflected the
Table 4. Effect of compound 1 on mitochondrial membrane potential in HepG2 Cells.
Compound
1
Group
Green Fluorescence (%)
CON
1 µm
3 µm
12 µm
5.80
16.72
28.14
53.82
(a)
(b)
(c)
Figure 6. Effect of compound
(d)
on mitochondrial membrane potential in HepG2 Cells: (a) control
1
group; (b) 1 µM; (c) 3 µM; and (d) 12 µM.
2.5.4. Calcium Content Detection
As shown in Table 5 and Figure 7, compared with the blank control group, the cells treated with
compound induced apoptosis. They also showed a right-shifted peak of calcium ion concentration,
1
which indicated that the intracellular calcium concentration was increased. This also means that the
cell was undergoing apoptosis.
Table 5. Effect of compound 1 on calcium in HepG2 Cells.
Compound
1
Group
Mean
CON
1 µm
3 µm
12 µm
31.00
65.01
94.39
136.80

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(a)
(c)
(b)
(d)
Figure 7. Effect of compound
and (d) 12 µM.
1
on calcium in HepG2 Cells: (
a
) control group; (b) 1 µM; (c) 3 µM;
2.6. Molecular Docking
Our previous research indicated that the molecular docking technique was used to study the
binding mode of urushiol derivatives to HDAC2 receptor [ ]. The HDAC2 X-ray crystal structure
8,9
(PDB No. 4LXZ) was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org) with a
resolution of 1.85 Å. The UCSF Chimera software [30] established the three-dimensional structure of
the ligand and performed energy optimization. Hydrogen atoms were added by using the Dock Prep
module, and the AMBER ff14SB force field and AM1-BCC charge were added [31,32]. The molecular
surface of the receptor was generated with a 1.4 Å radius probe by using the DMS tool in Chimera.
The X-ray crystal structure showed a reasonable binding site for which the sphgen module was used to
generate spheres surrounding the active site. A grid file was used to generate a grid file for grid-based
energy rating evaluation. Semi-flexible docking was performed by using the DOCK 6.8 program [33,34],
which generated 10,000 different conformational orientations and obtained electrostatic and van der
Waals interactions between the ligand molecules and the binding sites, and thus the grid score was
calculated. By cluster analysis (RMSD threshold of 2.0 Å), the best scoring conformation was obtained.
Finally, images were generated by using PyMOL (Schrödinger, LLC. The PyMOL molecular graphics
system, version 1.8, 2015).
2.6.1. Conjunction Conformation Score
The DOCK 6.8 program was used to predict the pattern of binding of the compound
1 in HDAC2.
The calculation results showed that the binding sites had multiple docking conformations, and the
scoring conditions are illustrated in Table 6. According to the scoring and combining mode, we chose
the first docking conformation to analyze the combining mode.
Table 6. Docking of ligand with receptor HDAC2 (unit: kcal/mol).
Compound
Pose
Grid Score
Grid_vdw
Grid_es
Int_energy
1
2
−54.809010
−53.249386
−47.656796
−46.243793
−7.152214
−7.005592
13.308063
15.280022
Ligand 1

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2.6.2. Binding Pattern Analysis
As shown in Figure 8, thiol sulfur atoms on compound
1
formed hydrogen bonding interactions
with Gly154 and Tyr308 respectively, which made compound
1 bind more closely to HDAC2. At the
same time, compound also formed hydrophobic interactions with the residues His33, Pro106,
1
Val107, Gly154, Phe155, and His183, and it was provided with a strong van der Waals force by the
hydrophobic action.
Figure 8. Molecular docking modeling of compound
1 in the active site of HDAC2. In the picture,
the ligands (green) and the key residues (wheat) are denoted by sticks, and the protein smudge color
by cartoon. A yellow dotted line represents a hydrogen bond.
3. Materials and Methods
3.1. General
All AR solvents were used without special treatment. All the reactions were monitored by TLC
on silica gel F254 plates (Qingdao Haiyang Inc., Qingdao, China) for detection of the spot. Silica-gel
column chromatography was performed for purification of the products. MS data were obtained on
an Agilent 7890 mass spectrometer. Nuclear magnetic resonance (NMR) spectra were measured on
Bruker AV-300 or AV-400 spectrometers. Chemical shifts were expressed in delta (δ) units and coupling
constants in Hz. The chemical shifts were reported in parts per million (ppm) and the following
abbreviations were used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and doublet of
doublets (dd). The purity of all compounds (urushiol and its derivatives) was determined by HPLC
(Shimadzu Co., Tokyo, Japan) and found to be in the 95–99% range.
3.2. Plant Material
The lacquer was harvested from Lichuan, Enshi city, Hubei Province, China and identified by
our laboratory team. All lacquers were deposited in a vacuum bag for preservation. The crude
extract of urushiol was isolated from the lacquer above. Then the C15 triene urushiol was purified
by silica gel column chromatography and characterized by spectroscopic and chemical methods.
Rf = 0.81(PE:EA = 1:1).
3.3. Extraction and Isolation
At 99.99% N2 atmosphere, the raw lacquer (triene urushiol content was more than 50% in the total
content of lacquer) was dissolved in MeOH with ultrasonic auxiliary dissolution; the 200~300 mesh
gel was used in column, and the mass ratio of silica gel to concentrated enamel was 3–4:1; the eluent
was methanol (the volume ratio of raw lacquer quality to methanol was 50:200–400 (g:mL). The eluent
were EA and PE, the total volume was 500 mL, and the volume of EA was 10–50 mL. The flow rate

Molecules 2018, 23, 1074
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of mixed elution was limited between 10 and 15 mL/min, while the EA volume varied from 10 mL
to 50 mL. The ratio of EA to PE was 10:490 when the pink ribbon was 10 cm, near to the column
sand core. The color change of column silica gel ribbon was a reference for the experiment with the
eluent polarity changing from large to small. The chromatography column colors were black-dark,
brown-dark, deep yellow, yellow, light red, and light pink (from top to bottom). The first outflow
of the pink eluent was collected, and the eluent was concentrated to produce the high purity triene
urushiol (85%); then, the column was eluted with a C18 silica gel. PE/EA (4:1) was used as eluent to
obtain four saturation urushiols which were later concentrated under reduced pressure to generate a
crude residue with more than 95% purity for cryopreserving.
3.4. synthesis of Triene Uruhsiol Hydroxyl Protection Compounds
Synthesis of 1, 2, and 3 [14]: A solution of triene urushiol (0.3 mmol) and K2CO3 (1.2 mmol) and
propargyl bromide (110 mg, 0.7 mmol) was dissolved in dry acetone (1 mL). The mixture was stirred
◦
at 30 C for 24 h and quenched with H2O (30 mL) in an ice bath. The solution was evaporated to
remove acetone and extracted with CH2Cl2 (2
NaHCO3 and brine, dried over MgSO4, filtered, and concentrated. The residue was purified by
column chromatography with CHCl3 MeOH (100:1) to afford compound . The synthesis methods of
compounds 1 and 2 followed the path above.
×
30 mL). The organic layer was washed with saturated
−
3
Compound δ 7.52 (-OH), 7.29 (s, 1H),
: yield 57%; brown oil; Rf = 0.90; 1H-NMR (400 MHz, CDCl3)
1
7.00 (s, 1H), 6.71 (s, 1H), 6.36 (t, J = 4.36 Hz, 1H), 6.00 (t, J = 4.06 Hz, 1H), 5.65 (t, J = 9.65 Hz, 1H),
5.46–5.37 (m, 5H), 3.52 (s, 1H), 2.61 (t, J = 3.98 Hz, 2H), 0.90 (t, J =2.65 Hz, 1H). 13C-NMR (101 MHz,
CDCl3)
δ 143.16, 142.09, 136.86, 132.34, 131.11, 129.97, 129.41, 126.83, 125.57, 124.34, 121.99, 120.01,
112.87, 58.77, 50.83, 27.18, 19.18, 14.12, 13.31. ESIMS m/z 167.0 [C5H13O2SSiH2]+.
Compound 2 δ 7.65 (m, 1H), 7.45 (m, 1H),
: yield 45%; brown oil; Rf = 0.91; 1H-NMR (400 MHz, CDCl3)
7.28 (s, 1H), 5.31–5.28 (d, J = 15.4 Hz, 2H), 1.18 (m, 8H). 13C-NMR (101 MHz, CDCl3)
13.71. ESIMS m/z 479.3 [C26H42O4SSi]+.
δ 167.70, 132.33,
Compound 3: yield 67%; brown oil; Rf = 0.82; 1H-NMR (400 MHz, CDCl3) δ 7.52 (-OH), 7.00 (m, 1H),
6.79–6.77 (m, 3H), 6.35 (t, J = 4.16 Hz, 1H), 5.98 (t, J = 4.46 Hz, 1H), 5.64 (m, 1H), 5.44–5.35 (m, 3H),
4.73 (d, J = 2.3 Hz, 2H), 3.73 (d, J = 7.0 Hz, 1H). 13C-NMR (101 MHz, CDCl3)
δ 150.44, 144.44, 143.90,
132.30, 131.10, 129.90, 129.39, 126.78, 125.55, 124.28, 123.40, 119.09, 110.16, 78.32, 76.73, 57.07, 30.62,
29.73, 29.71, 29.63, 29.49, 29.42, 29.25, 27.18. ESIMS m/z 352.3 [M-H]+.
Synthesis of compounds
phenylboronic acid (1.0 eq) was dissolved in DCM/EA (0.5 mL) and heated to 60 C for 2 h in a
4
–
7
[
19]: A solution of triene urushiol (314 mg, ◦1 mmol) and
pressure tube. After being concentrated in a vacuum, pure compound
Repeat the step above to get compounds , and , and their yields were 70–80%. The synthesis of
compounds 14 15, and 16 were also the same as the synthesis step of compound , and at r.t. for 2 h in
a pressure tube.
4 (400 mg, 100%) was obtained.
5,
6
7
,
4
Compound δ 7.42 (d, J = 3.06 Hz, 1H),
: yield 75%; brown oil; Rf = 0.75; 1H-NMR (400 MHz, CDCl3)
4
7.34 (s, 1H), 7.17 (m, 1H), 7.04 (d, J = 2.80 Hz, 1H), 6.94 (t, J = 3.86 Hz, 1H), 6.87 (d, J = 2.85 Hz, 1H),
6.59 (s, 2H), 6.26 (t, J = 8.81 Hz, 1H), 6.15 (s, 2H), 5.89 (t, J = 2.65 Hz, 1H), 5.34–5.26 (m, 2H). 13C-NMR
(101 MHz, CDCl3)
δ 148.13, 146.81, 145.74, 132.30, 131.08, 131.06, 129.93, 129.41, 129.22, 127.90, 126.85,
125.57, 124.30, 123.18, 122.33, 121.40, 119.24, 114.70, 114.08, 13.29. ESIMS m/z 449.4 [C27H34BNO2]+.
Compound 9.69 (s, 1H), 7.98 (s, 1H),
: yield 72%; brown oil; Rf = 0.74; 1H-NMR (400 MHz, CDCl3)
7.49 (s, 1H), 7.27, 7.20, 7.17, 6.97 (s, 6H), 5.27, 5.19, 3.39. 13C-NMR (101 MHz, CDCl3)
173.89, 173.44,
5
δ
δ
148.25, 148.18, 147.66, 130.74, 130.56, 130.26, 130.24, 129.43, 128.65, 128.47, 125.01, 124.55, 119.69, 34.63,
32.49, 30.29, 29.91, 27.75, 25.43, 24.44, 23.26, 14.67.ESIMS m/z 479.2 [M+C6H7]+.

Molecules 2018, 23, 1074
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Compound
6
: yield 80%; brown oil; Rf = 0.70; 1H-NMR (400 MHz, CDCl3)
δ
9.73 (s, 1H), 8.01 (s, 1H),
7.52 (d, J = 2.80 Hz, 1H), 7.33 (d, J = 1.88 Hz, 1H), 7.30 (d, J = 1.50 Hz, 1H), 7.23 (s, 1H), 7.10 (m, 1H),
7.00 (s, 1H), 5.30–5.23 (m, 6H), 4.25 (d, J = 8.78 Hz, 8H), 4.11 (m, 2H). 13C-NMR (101 MHz, CDCl3)
δ
172.32, 171.87, 146.68, 146.61, 146.09, 129.17, 128.99, 128.69, 128.67, 127.85, 127.07, 126.90, 123.44, 122.97,
118.12, 33.05, 30.92, 28.72, 28.33, 26.18, 23.85, 22.87, 21.69, 13.09. ESIMS m/z 419.3 [M]+.
1
Compound
7
: yield 70%; brown oil; Rf = 0.79; H-NMR (400 MHz, CDCl3)
δ
8.06 (s, 1H), 7.96 (d,
J = 0.80 Hz, 1H), 7.52 (m, 1H), 7.36 (t, J = 2.80 Hz, 1H), 6.70 (t, J = 5.80 Hz, 3H), 5.82(m, 1H), 5.3–5.34 (m,
3H), 4.97 (m, 1H), 1.61 (m, 5H), 1.33, 1.26. ESIMS m/z 448.1 [M + H]+.
Synthesis of compound 8 [15]: Triene urushiol (1.0 mmol), K2CO3 (3.5 eq.) and 1,3-dibromopropane
(1.3 eq.) were added to EtOH (1 mL) in turn, and then the mixture was heated at reflux for 5 h.
The resulting mixture was washed with H2O (5 mL) and organic layer was concentrated in vacuum to
acquire the crude product, which was purified by column chromatography to afford the corresponding
compound 8 with PE–EA (4:1, v/v) as eluents.
Compound
1H), 6.00 (m, 1H), 5.63 (m, 1H), 5.40–5.35 (m, 3H), 4.18 (m, 2H), 3.58 (t, J = 1.80 Hz, 2H), 2.02, 1.74 (t,
J = 1.10 Hz, 4H). 13C-NMR (101 MHz, CDCl3)
151.66, 149.70, 126.81, 126.78, 122.70, 122.68, 119.22,
119.20, 77.34, 70.48, 70.47, 27.23, 27.15, 14.10, 13.30. ESIMS m/z 405.1 [M]+.
8 δ 6.83–6.75 (m, 3H), 6.34 (m,
: yield 70%; brown oil; Rf = 0.81; 1H-NMR (400 MHz, CDCl3)
δ
Synthesis of compound
13]: To a solution of triene urushiol (1.0 mmol) in EA (1 mL) at 0 ◦C
9 [
was added TEA (5.06 g, 50.0 mmol) and MsNH2 (25 mg), successively. After the addition of MsNH2,
the mixture was vigorously stirred for 2 h. To the slurry was then added H2O (10 mL). The two-phase
mixture was separated. The organic layer was washed with H2O (25 mL) and dried (MgSO4). Removal
of solvent under vacuum, the corresponding pure compound 9 was obtained.
Compound
3H), 6.34 (t, J = 4.80 Hz, 1H), 5.98 (t, J = 3.00 Hz, 1H), 5.63 (m, 1H), 5.42–5.35 (m, 3H), 3.71 (s, 3H).
13C-NMR (101 MHz, CDCl3)
143.08, 141.96, 132.31, 131.07, 129.39, 129.37, 126.83, 125.56, 124.33,
122.06, 120.06, 58.63, 13.30. ESIMS m/z 436.3 [M + 2Na]+.
9 δ 6.71–6.70 (t, J = 4.45 Hz,
: yield 30%; brown oil; Rf = 0.80; 1H-NMR (400 MHz, CDCl3)
δ
Syntheses of Compound 10 16]: In a pressure tube (25 mL), p-Toluene (1 eq.) was added into
[
the mixture of triene urushiol (1 mmol) and ethyl acetoacetate (3 eq.), and the reaction mixture was
vigorously stirred at ambient temperature for 8 h. The progress of reaction was monitored by TLC
(eluent, PE:EA = 4:1). After the completion of the reaction, water (10.0 mL) was added into the reaction
mixture. Evaporate the solvent to afford the pure compound 10 in excellent yields, and 10 was regarded
as the reagent for compounds 12–16.
Compound 10 (8-hydroxy-4-methyl-7-((8Z,11E,13Z)-pentadeca-8,11,13-trien-1-yl)-2H-chro men -2-one)
yield 90%; Yellow liquid; Rf = 0.83; 1H-NMR (400 MHz, CDCl3)
δ
6.88–6.48 (m, 5H), 6.49(d, J = 5.60 Hz,
1H), 6.22 (s, 1H), 6.02–5.88 (m, 3H), 2.94 (m, 2H), 2.68 (s, 2H),2.43(t, J = 0.98 Hz, 4H), 2.11(d, J = 7.01 Hz,
2H), 1.94(m, 2H), 1.50 (m, 2H), 1.36 (m, 2H), 1.27 (m, 3H). 13C-NMR (101 MHz, CDCl3)
167.46, 165.57,
δ
164.44, 161.42, 155.03, 143.34, 142.18, 124.42, 122.44, 120.20, 119.95, 112.86, 112.07, 110.26, 106.90, 50.10,
30.13, 28.45, 28.28, 27.07, 25.15, 21.19, 19.51, 15.53, 14.11, 14.04. ESIMS m/z 448.4 [M + SiC3H9]+.
Syntheses of Compound 11: The purified triene urushiol (0.3 mmol) was etherified with methyl
iodide (0.6 mmol) in MeCN (1 mL) and the crude product was concentrated under vacuum. The crude
product was recrystallized in methanol (saturated urushiol dimethyl ether was not recrystallized) to
give pure target compound 11.
Compound 11: yield 70%; Yellow liquid; Rf = 0.81; 1H-NMR (400 MHz, CDCl3)
δ 6.94–6.65 (m, 3H),
6.34 (t, J = 4.08 Hz, 1H), 5.97 (t, J = 4.99 Hz, 1H), 5.61–5.35 (m, 3H), 3.93 (s, 3H), 3.79 (s, 3H). ESIMS m/z
341.0 [M − H]+.

Molecules 2018, 23, 1074
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Compound 12: yield 62%; Yellow liquid; Rf = 0.70; 1H-NMR (400 MHz, CDCl3)
δ
6.73 (m, 1H), 6.71 (s,
1H), 6.67 (m, 1H), 6.23 (s, 1H), 5.98 (s, 1H), 5.89 (s, 1H), 2.39 (t, J = 9.80 Hz, 3H), 2.25 (m, 3H), 1.83 (s, 3H).
13C-NMR (101 MHz, CDCl3) δ 167.30, 161.19, 154.82, 112.88, 21.21, 19.53. ESIMS m/z 421.3 [M − H]+.
Compound 13: yield 66%; Yellow liquid; Rf = 0.50; 1H-NMR (400 MHz, CDCl3)
δ 7.51 (s, 1H), 7.13 (s,
1H), 6.70 (s, 1H), 6.03 (d, J = 21.1 Hz, 1H), 5.44–5.17 (m, 6H), 4.64 (s, 2H), 3.40 (s, 1H), 2.60(t, J = 1.11 Hz,
2H), 2.40 (m, 2H), 2.24 (s, 3H), 2.05 (d, 3H),1.73 (d, 2H),1.60 (m, 2H),1.33 (m, 2H), 1.26(m, 2H), 0.88 (m,
2H). 13C-NMR (101 MHz, CDCl3)
δ 171.43, 143.17, 142.05, 132.34, 131.04, 130.02, 129.92, 129.40, 129.37,
126.84, 124.36, 121.99, 120.01, 112.85, 61.77, 60.52, 50.89, 31.98, 31.81, 30.64, 29.79, 29.52, 27.23, 22.68,
21.08, 14.20, 13.32. ESIMS m/z 432.4 [M + H]+.
Compound 14: yield 28%; Yellow liquid; Rf = 0.90; 1H-NMR (400 MHz, CDCl3)
δ 7.53 (d, J = 8.5 Hz,
1H), 7.52–7.05 (m, 3H), 6.16 (m, 1H), 5.90 (m, 1H), 5.72 (m, 1H), 5.69–5.64 (m, 3H), 3.67 (s, 1H). ESIMS
m/z 535.4 [M + 2H2O]+.
Compound 15: yield 25%; Yellow liquid; Rf = 0.85; 1H-NMR (400 MHz, CDCl3)
δ 7.72 (t, J = 8.6 Hz,
1H), 7.53 (d, J = 5.13 Hz, 1H), 7.36 (s, 1H), 7.07 (s, 1H), 6.70 (m, 8H), 6.10 (m, 1H), 5.84 (m, 1H), 5.60 (m,
1H), 5.40–5.34 (m, 3H), 3.67 (s, 2H), 2.40. ESIMS m/z 405.2 [M + H]+.
Compound 16: yield 23%; Yellow liquid; Rf = 0.87; 1H-NMR (400 MHz, CDCl3)
δ 7.79 (m, 1H), 7.52 (d,
J = 7.85Hz, 1H), 6.71 (s, 7H), 5.87 (m, 1H), 5.63 (m, 1H), 5.16 (m, 1H), 5.05–4.98 (m, 3H), 3.67 (s, 3H).
ESIMS m/z 421.4 [M + H]+.
4. Conclusions
We found that urushiol derivatives have a better inhibitory effect on HepG2 of hepatoma cells
after screening the broad-spectrum anticancer activity of urushiol derivatives, which inspired us to
select hepatoma cell HepG2 as the research object. The result showed that urushiol and its derivatives
have a good inhibitory effect on the expression of enzyme HDAC2, and their calculated results were
better than that of SAHA [8,9]. The purpose of this paper is to explain that urushiol derivative 1 had
inhibitory effect on HDAC2, but the specific inhibitory effect of urushiol on enzymes, such as HDACs,
needs more extensive research. The main significance of this paper is to synthesize urushiol derivatives
for the first time, which is beneficial for inhibiting the expression of HDAC2.
In this paper, the structure–activity relationship showed that compound
long-chain alkane, compound with the phenylboronic acid and compound
1
9
with the thiol-containing
with the amino sulfoxide
4
played an effective role in attenuating and increasing the efficiency; the same condition also applied to
the urushiol Pechmann derivative. The finding provided the experimental basis for the subsequent
synthesis of novel urushiol derivatives with reduced toxicity and enahnced efficacy.
The Grid docking studies revealed that introducing alkane thiol substituents in side chain could
increase binding affinity significantly. Molecular docking studies revealed that Zn2+ coordination,
hydrogen bonding, and hydrophobic interaction contributed to the high binding affinity of these
compounds toward HDAC2. Gly154, Tyr308, His33, Pro106, Val107, Gly154, Phe155, and His183
contributed favorably to the binding between enzyme and the compounds. In addition, the results
of binding energy decomposition clearly showed that van der Waals and electrostatics interactions
provided major contributions to the stability of these complexes. This study showed the promising
potential of urushiol and urushiol derivatives as potent HDAC2 binding agents. Further studies on
the synthesis and determination of their HDAC2 inhibitory properties are planned.
Supplementary Materials: The following are available online. Detection of cell proliferation by MTT, HDAC2
expression, Flow cytometry experimental process and compounds MS; 1H/13C NMR spectrograms.
Author Contributions: Z.Q. and C.W. conceived and designed the experiments; J.J. analyzed the data;
Z.Q. contributed reagents/materials/analysis tools and implemented experiments; and all the authors wrote
the paper.

Molecules 2018, 23, 1074
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Funding: This work was supported by National Natural Science Foundation of China (31570564), Jiangsu
Provincial Key Laboratory of Biomass Energy and Materials Basic Research Business Project (JSBEM-S-201509),
Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2018GA001) and
Introduction International Advanced Forestry Science & Technology (2015-4-46).
Conflicts of Interest: There are no conflicts of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).