Design, synthesis and biological evaluation of ligustrazine-flavonoid derivatives as potential anti-tumor agents

Article abstract of DOI:10.3390/molecules23092187

In the clinic some anti-tumor drugs have shown damage to normal blood vessels, which could lead to vascular diseases. Therefore, it is necessary to evaluate the effects of anti-tumor drugs on normal blood vessels at the beginning of the drug design process. In this study, ligustrazine (TMP) and flavonoids were selected as raw materials. Sixteen novel TMP-flavonoid derivatives were designed and synthesized. Interestingly, compounds 14 and 16 were obtained by hydrolysis of a dihydroflavone to a chalcone under alkaline conditions. The cytotoxicity of the TMP-flavonoid derivatives was evaluated on five human tumor cell lines and one classical type of normal endothelial cell lines (HUVEC-12) by an MTT assay. Part of the derivatives showed better anti-tumor activities than the corresponding raw materials. Among them, compound 14 exhibited the closest activity to the positive control against the Bel-7402 cell line (IC₅₀ = 10.74 ± 1.12 μM; DDP IC₅₀ = 6.73 ± 0.37 μM) and had no toxicity on HUVEC-12 (IC₅₀ > 40 μM). Subsequently, fluorescence staining and flow cytometry analysis indicated that compound 14 could induce apoptosis of Bel-7402 cell lines. Moreover, the structure-activity relationships of these derivatives were briefly discussed.

Full text of DOI:10.3390/molecules23092187

molecules
Article
Design, Synthesis and Biological Evaluation of
Ligustrazine-Flavonoid Derivatives as Potential
Anti-Tumor Agents
Hui Wang 1, Wenxi Zhang 1, Yatao Cheng 2, Xinyu Zhang 1, Nannan Xue 1, Gaorong Wu 1,
Meng Chen 1, Kang Fang 1, Wenbo Guo 1, Fei Zhou 1, Herong Cui 1 ID , Tao Ma 1,*,
Penglong Wang 1,* and Haimin Lei 1,
*
ID
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
15652387323@163.com (H.W.); 20170931820@bucm.edu.cn (W.Z.); 20160931837@bucm.edu.cn (X.Z.);
18810612758@163.com (N.X.); gaorongwu09@163.com (G.W.); 18702267252@163.com (M.C.);
18364166994@163.com (K.F.); wb_guo@126.com (W.G.); zf116318@163.com (F.Z.);
zhenzhu7696@163.com (H.C.)
2
International Cooperation Division, China Sinopharm International Corporation, Beijing 100102, China;
chengyatao@sinopharm.com
*
Correspondence: mosesmatao@163.com (T.M.); wpl581@126.com (P.W.); hm_lei@126.com (H.L.);
Tel.: +86-10-8473-8640 (P.W.); +86-10-8473-8645 (H.L.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 July 2018; Accepted: 21 August 2018; Published: 30 August 2018
Abstract: In the clinic some anti-tumor drugs have shown damage to normal blood vessels, which
could lead to vascular diseases. Therefore, it is necessary to evaluate the effects of anti-tumor drugs
on normal blood vessels at the beginning of the drug design process. In this study, ligustrazine
(TMP) and flavonoids were selected as raw materials. Sixteen novel TMP-flavonoid derivatives
were designed and synthesized. Interestingly, compounds 14 and 16 were obtained by hydrolysis
of a dihydroflavone to a chalcone under alkaline conditions. The cytotoxicity of the TMP-flavonoid
derivatives was evaluated on five human tumor cell lines and one classical type of normal endothelial
cell lines (HUVEC-12) by an MTT assay. Part of the derivatives showed better anti-tumor activities
than the corresponding raw materials. Among them, compound 14 exhibited the closest activity to the
positive control against the Bel-7402 cell line (IC50 = 10.74
±
1.12
µ
M; DDP IC50 = 6.73
±
0.37 µM) and
had no toxicity on HUVEC-12 (IC50 > 40 M). Subsequently, fluorescence staining and flow cytometry
µ
analysis indicated that compound 14 could induce apoptosis of Bel-7402 cell lines. Moreover, the
structure-activity relationships of these derivatives were briefly discussed.
Keywords: TMP-flavonoid derivatives; anti-tumor; human tumor cell lines; HUVEC-12; fluorescence
staining; flow cytometry
1. Introduction
As the world0s population ages and grows, the global burden of cancer continues to increase.
Cancer is the leading cause of death and a major public health problem around the world [
Chemotherapy is commonly used for cancer treatment. In recent years, many chemotherapeutic
drugs with high cytotoxicy have been discovered and used as first-line clinical drugs [ ]. However,
many anti-tumor drugs, such as cisplatin, paclitaxel and bleomycin [ ], have serious side effects,
including lethal hemoptysis and anemia [ ]. The reasons are that the new tumor blood vessels were
1,2].
3,4
5–7
8,9
usually produced by an existing vascular system and considered to be the same as normal blood
vessels. When drugs work, they have the same inhibition on normal endothelial cells (NECs) and
tumor endothelial cells (TECs) [10–12]. Therefore, it is necessary to evaluate the effects of drugs on
Molecules 2018, 23, 2187; doi:10.3390/molecules23092187
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2187
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NECs while evaluating their anti-tumor activities. Compared with other drugs, Traditional Chinese
Medicines (TCMs) have the advantages of lower toxicity and more targets. Therefore, it is possible to
obtain high efficiency and low toxicity anti-tumor leading compounds by combining the anti-tumor
ingredients from TCM.
In the clinic, TCMs with similar activities are often used together to realize synergistic effects.
In the same way, it was possible to obtain more active compounds by synthesis of ingredients
with similar effects [13,14]. TMP and flavonoids are two such ingredients [15]. Ligustrazine (TMP,
2,3,5,6-tetramethylpyrazine) is one of the active ingredients of Ligusticum Chuanxiong Hort, and it
is widely used in occlusive cerebrovascular diseases. Previous studies have found that TMP and
its derivatives could not only inhibit growth, invasion and metastasis of tumor cells, but also could
reverse the multi-drug resistence and act as potential reversal drugs on P-gp [16
used in the synthesis of leading compounds and a lot of compounds with significant effects have
been obtained [20 22]. Flavonoids are a class of polyphenolic compounds widely distributed in
the plant kingdom and abundantly consumed in the daily diet. They display potential therapeutic
benefits in cancer treatment and may be considered as candidates for tumor therapeutic agents [23 25].
–19]. It is widely
–
–
Flavonoids have been reported to be correlated to G1 and G2 cell cycle arrest and apoptosis promotion.
Moreover, the flavonoids can reverse the multidrug resistance (MDR) of tumors and affect the
expression and function of efflux transporters [26–28]. The nine flavonoids selected in this study are
shown in Table 1. The main types of flavones were chosen, including flavones, flavonols, isoflavones
and dihydroflavones.
Table 1. The structures of the nine flavonoid compounds.
Classification
Flavone
Flavonoids’ Structure
Flavonoid Compounds (1–9)
0
0
Luteolin (1)
Baicalein (2)
Apigenin (3)
Chrysin (4)
5, 7, 3 , 4 —OH
5, 6, 7—OH
0
5, 7 ,3 —OH
5, 7—OH
0 0
5, 7, 3 , 4 —OH
Quercetin (5)
Fisetin (6)
Flavonol
0
0
7, 3 , 4 —OH
0
5, 7, 4 —OH
Genistein (7)
Daidzein (8)
Isoflavone
Flavanone
0
7, 4 —OH
0
Naringenin (9)
5, 7, 4 —OH

Molecules 2018, 23, 2187
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In this study, we conjugated TMP and flavonoids, the main components of blood-activating
drugs with anti-tumor activities, into the one molecule via ether bonds, constructing a number of new
TMP-flavonoid derivatives. A variety of tumor cell lines were selected to evaluate the anti-tumor
activities of these compounds, and human umbilical vein endothelial cells (HUVEC-12) as the NECs
in vitro toxicity measurement model.
2. Results
2.1. Chemical Synthesis
As shown in Scheme 1, we first synthesized the intermediate TMP-Br via a free radical reaction.
Then, TMP-Br was linked to flavonoids via ether bonds to obtain TMP-flavonoid derivatives. Finally,
end-products were obtained by crystallization. All TMP-flavonoid derivatives were synthesized by
adopting a similar alkaline catalyst strength, which should be strictly controlled, as the flavanone
structure is unstable under strong alkaline conditions. The C-O bond of C ring of naringenin derivatives
was disconnected to form a chalcone structure (compounds 14, 16). Treatment of TMP-Br with daidzein
in dry acetone at 75 ◦C for 2 h, afforded compound 10. We had tried to synthesize more compounds,
but the yield was too low. We could not obtain more compounds except these 16 derivatives (Table 2)
and they are new compounds that had not been previously reported. All TMP-flavonoid derivatives
were characterized by 1H-NMR, 13C-NMR and HRMS.
Scheme 1. Synthesis routes to ligustrazine-flavonoid derivatives (10–25).
Table 2. The structures of ligustrazine-flavonoid derivatives 10–25.
No.
10
Structure
No.
Structure
11

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Table 2. Cont.
No.
12
Structure
No.
Structure
13
15
17
14
16
18
20
19
21
22
23

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Table 2. Cont.
No.
24
Structure
No.
Structure
25
2.2. Biological Activities
2.2.1. Cytotoxicity Assay Using Five Tumor Cell Lines
All the synthesized compounds were tested for their cytotoxicities on five tumor cell lines (HepG-2,
Bel-7402, HT-29, MCF-7 and HeLa) using the standard MTT assay with DDP as positive control.
The IC50 values of these compounds are summarized in Table 3. After combination, the activity of
most synthesized compounds had not been improved significantly compared to the parent flavonoids.
Among them, compounds 14 and 19 displayed lower IC50 values than the rest of compounds against
Bel-7402, and MCF-7, respectively. As shown in Figure 1, the activity of compound 14 was significantly
better than that of naringenin on all cell lines. Furthermore, compound 14 had the strongest inhibitory
effect on Bel-7402 and slightly weaker than that of DDP. In the same way, the activity of compound 19
was superior to that of daidzein, and was shown to be slightly weaker than that of DDP against MCF-7.
Table 3. IC50 values of TMP-flavonoid derivatives and raw materials on HepG-2, Bel-7402, HT-29,
MCF-7 and HeLa for 72 h. Data were expressed as inhibitory ratio
experiments (n = 3).
±
SD based on three independent
IC50 (µM)
Compound
HepG-2
Bel-7402
HT-29
MCF-7
HeLa
Luteolin
Baicalein
Apigenin
Chrysin
Quercetin
Fisetin
Genistein
Daidzein
10
11.83 ± 0.44
20.35 ± 2.07
16.11 ± 1.52
15.56 ± 0.30
13.57 ± 0.73
17.39 ± 1.04
19.76 ± 1.31
21.81 ± 1.53
>40
13.37 ± 2.50
17.31 ± 0.47
20.62 ± 1.39
>40
14.51 ± 0.68
21.59 ± 3.68
28.22 ± 0.38
35.00 ± 1.46
30.39 ± 2.51
25.46 ± 1.54
34.66 ± 0.63
>40
19.24 ± 1.17
19.20 ± 2.72
23.44 ± 1.89
30.31 ± 1.96
27.00 ± 0.47
23.88 ± 0.81
31.13 ± 0.26
>40
16.99 ± 0.31
37.88 ± 1.52
29.71 ± 1.43
32.06 ± 2.78
>40
38.10 ± 1.83
35.62 ± 1.38
36.82 ± 1.48
>40
21.28 ± 1.12
22.76 ± 3.54
30.58 ± 0.71
>40
20.83 ± 4.04
20.88 ± 1.65
34.61 ± 2.52
>40
26.67±0.34
17.31 ± 1.07
25.11 ± 1.80
30.50 ± 2.62
>40
14.31 ± 1.17
16.48 ± 1.79
4.76 ± 0.41
>40
>40
13
14
16
17
19
20
DDP
26.13 ± 0.11
10.74 ± 1.12
>40
22.42 ± 0.35
31.88 ± 1.96
34.39 ± 2.32
10.67 ± 1.35
11.72 ± 1.29
10.90 ± 2.30
5.83 ± 0.59
>40
29.79 ± 2.18
16.54 ± 0.45
>40
>40
14.49 ± 0.48
28.87 ± 0.49
25.16 ± 1.72
6.73 ± 0.37
10.43 ± 1.23
>40
>40
4.14 ± 0.16
6.75 ± 0.57
IC50 values: when the IC50 values of the compounds were higher than 40 for all cells, the groups of data were not
listed in the table.
In addition, it was observed that different types of flavonoids could lead to different cytotoxicities.
The activity of derivatives with flavones, isoflavones and chalcone as mother nucleus was better than
other derivatives, such as compound 14 (IC50 was 17.31
±
0.47
µ
M, 10.74
±
1.12
µ
M, 31.88
±
1.96 µM,
29.79 2.18 M, 25.11 1.80 M aginst HepG-2, Bel-7402, HT-29, MCF-7 and HeLa respectively)
±
µ
±
µ

Molecules 2018, 23, 2187
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and compound 19 (IC50 was 14.49
±
0.48
µ
M, 28.87
±
0.49
µ
M, 11.72
±
1.29
µ
M, 10.43
±
1.23 µM,
14.31
±
1.17 µM aginst HepG-2, Bel-7402, HT-29, MCF-7 and HeLa respectively). Because of the limited
number of derivatives, the relationships between the number and position of introduced TMP moieties
and the activities of TMP-flavonoid derivatives need to be further explored.
Figure 1. The IC50 values against HepG-2, Bel-7402, HT-29, MCF-7, HeLa. (
a) Naringenin, compound
14, DDP. ( ) Daidzein, compound 19, DDP. ** p < 0.001, *** p < 0.001, vs. Naringenin, Daidzein. The red
b
mark indicated that the IC50 value of the compounds to the cells were closest to that of the DDP.
2.2.2. Cytotoxicity Assay Using HUVEC-12 Cell
In order to further investigate the effects of compounds with clear anti-tumor activities
on NECs, MTT colorimetry was applied using human umbilical vein endothelial (HUVEC-12)
cells. As shown in Table 4, when the concentration was 40 µM, compound 14 presented a little
cytotoxicity. However, it showed a certain role in promoting cell proliferation and a concentration
dependence at most concentrations. On the contrary, compound 19 appeared to have weak damaging
effects and DDP had definite cytotoxic effects on cells (compounds 14 and 19 IC50 > 40
µM;
DDP IC50 = 9.11 ± 0.54
µ
M) [29,30]. The results of compound 14 were consistent with expectations
that the compounds had no effect on NECs. Therefore, compound 14 with the greater difference was
selected for further mechanistic study.
Table 4. Proliferation rates of TMP-flavonoid derivatives on HUVEC-12 for 24 h. Data were expressed
as inhibitory ratio ± SD based on three independent experiments (n = 3).
Proliferation Rate (%)
Compound
IC50 (µM)
2.5 µM
5 µM
10 µM
20 µM
40 µM
14
19
DDP
1.62 ± 0.75
1.99 ± 0.23
14.84 ± 1.41
16.71 ± 0.29
−2.32 ± 1.37
>40
>40
9.11 ± 0.54
−8.75 ± 0.38
−
−
19.35
31.38
±
±
0.35
1.46
−
−
19.49
58.24
±
±
2.17
0.82
−
−
24.22
66.58
±
±
3.19
1.75
−
−
25.07
82.69
±
±
1.02
2.19
−
23.93
±
0.75
2.2.3. Morphological Analysis Using Giemsa and DAPI Staining
To confirm the apoptopic morphological changes induced by compound 14, Bel-7402 cells were
treated with 0, 10, 20 and 40 M of compound 14 for 72 h and then Giemsa staining and DAPI staining
were performed. As shown in Figure 2I, the number and morphological changes of Bel-7402 cells
significantly increased with increasing concentrations of compound 14. When the dose reached 40 M,
µ
µ
there were mainly cell fragments rather than complete cells. In the same way, as we can see from
Figure 2II, the cells treated with compound 14 showed morphological features of apoptosis, including
a number decrease, nuclear fragmentation, cytoplasmic shrinkage, and the formation of apoptotic
bodies with irregular shape while untreated cells displayed normal.

Molecules 2018, 23, 2187
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Figure 2. Gimesa (
concentrations: (100
observed under the fluorescence microscope. The most representative fields are shown.
I
) and DAPI (II) staining on Bel-7402 cells induced by compound 14 with different
×
): (a) control group; (b) 10 M; (c) 20 M; (d) 40 M. The cell morphology was
µ
µ
µ
2.2.4. Apoptosis Analysis by Flow Cytometric Using Annexin V-FITC/Propidium Iodide (PI) Staining
The effects of compound 14 on apoptosis of Bel-7402 cells were further determined by flow
cytometric analysis. The cells were treated with compound 14 at four concentrations of 0, 5, 10,
20 µM and then stained with both annexin Annexin V-FITC and PI. The flow cytometry observed
four quadrant images: necrotic (Q1; Annexin−/PI+), late apoptotic (Q2; Annexin+/PI+), intact (Q3;
Annexin−/PI−), and early apoptotic (Q4; Annexin+/PI−) cells. The results are shown in Figure 3,
where the apoptosis ratios (including the early and late apoptosis ratios) increased to 33.7% (5
µM),
45.5% (10 M), 54.6% (20 M) while that of control was 5.4%. Futhermore, the results indicated that
µ
µ
compound 14 could induce Bel-7402 cells apoptosis in a concentration-dependent manner.
Figure 3. Apoptosis analysis of Bel-7402 cells induced by compound 14 using AnnexinV-FITC/PI
staining: (a) control group; (b) 5 µM; (c) 10 µM; (d) 20 µM.

Molecules 2018, 23, 2187
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3. Discussion
In this study, although chalcone was not selected, compounds 14 and 16 with chalcone as
the mother nucleus were obtained via synthesis. This was due to the fact the dihydroflavones
hydrolyzed back into chalcone under alkaline conditions [31]. In addition, we analyzed the effects
of the TMP-flavonoid derivatives on five tumor cell lines. The flavonoids selected showed clear
anti-tumor activity, especially luteolin (IC50 was 11.83
±
0.44
µ
M, 14.51
±
0.68
µ
M, 19.24
±
1.17 µM,
16.99 0.31 M, 21.28 1.12 M aginst HepG-2, Bel-7402, HT-29, MCF-7 and HeLa, respectively).
±
µ
±
µ
The results confirmed the anti-tumor potential of luteolin in tumor therapy [32]. Most of the
flavonoids had better inhibition on HepG-2 than other cell lines, which was consistent with previous
studies [27,33]. After TMP was introduced, the selectivity of the derivatives changed. For example,
compound 14 showed the strongest inhibitory effect on Bel-7402. Among all compounds, the
compounds with isoflavones and chalcone as the mother nucleus had better anti-tumor activities
than other compounds. Furthermore, the retention of 5, 7-OH could increase the inhibition of the
compounds on tumor cells, such as compound 10
> 11, compound 17 > 18, compound 19 > 20. That is
to say, isoflavones, chalcone and its derivatives with 5, 7-OH substituents have great potential in
tumor therapy [26]. Since vascular diseases caused by anti-tumor drugs are common, it is necessary to
consider the effect of antitumor drugs on normal blood vessels at the beginning of the design plan.
In this study, we considered not only the inhibition of drugs on tumor cell lines, but also the effects
of drugs on normal vascular cells. The design idea of this study was from the perspective of clinical
application. While compound 14 had a definite inhibition on Bel-7402, it also had a little proliferation
on HUVEC-12. The selectivity was not detected for other samples, including positive ones. The results
of staining and flow cytometric also demonstrated that compound 14 promoted the apoptosis of
Bel-7402. The results are consistent with previous studies [26]. The flavonoids selected have been
shown to be correlated to a G1 and G2 cell cycle arrest [27,28], so it is worth performing further studies
on the effect of TMP-flavonoid derivatives in cell cycle determination. Based on the above, compound
14 showed bright prospects and should be further studied.
4. Materials and Methods
4.1. Materials and Instruments
Ligustrazine, luteolin, baicalein, apigenin, chrysin, quercetin, fisetin, genistein, daidzein,
naringenin, and DDP were purchased from Aladdin Bio-Chem Technology Co., Ltd., (Shanghai,
China) and Alfa Aesar Chemical Co., Ltd. (Tianjin, China). The purity of all the materials was more
than 98%. All reagents were used without any further purification. Reagents of analytical reagent
1
grade were provided by Beijing Chemical Plant (Beijing, China). H-NMR and 13C-NMR spectra were
recorded on a DRX-500 spectrometer at 500/125 MHz (Bruker, Fällanden, Switzerland). ESI–MS were
recorded on a Thermo Scientific TM LTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies,
New York, NY, USA). Reactions were monitored by thin-layer chromatography (TLC) on precoated
silica gel G-254 plates, which visualized in UV light (254 nm). Flash column chromatography was
performed using 200–300 mesh silica gel (Qingdao Haiyang Chemical Co., Qingdao, China). Cellular
morphologies were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan).
The yields were calculated based on the last step reaction.
Fetal bovine serum (FBS) and RPMI 1640 (DMEM) medium, Penicillin and streptomycin were
obtained from Thermo Technologies. Gimesa, 6-diamidino-2-phenylindole (DAPI) were obtained
from Molecular Probes/Invitrogen Life Technologies (Carlsbad, CA, USA). Five kinds of tumor cell
lines were provided by the Institute of Peking Union Medical College: Hepatocellular cancer cell lines
(HepG-2, Bel-7402), human breast cancer cell lines (MCF-7), human colon cancer cells (HT-29), human
cervical cancer cells (HeLa), human umbilical vein endothelial cells (HUVEC-12).

Molecules 2018, 23, 2187
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4.2. Chemical Syntheses
2-(Bromomethyl)-3,5,6-trimethylpyrazine (TMP-Br): TMP
·
3H2O (0.26 mol) was dissolved in benzene
(120 mL) and refluxed for 10 h. After concentrating the solution, white solids (TMP) were obtained.
TMP (0.15 mol) was dissolved in carbon tetrachloride (100 mL) and NBS (0.12 mol) was added in
portions. The mixture was refluxed and illuminated by four 60 W tungsten light bulbs. After 12 h, the
brown liquid was filtered and dried in vacuum. A brown crude oily product was obtained (TMP-Br),
which was not purified further, as it caused a strong mucous membrane irritation. Yield: 70%.
5-Hydroxy-2-phenyl-7-((3,5,6-trimethylpyrazin-2-yl)methoxy)-4H-chromen-4-one, (10). TMP-Br (2.81 mmol)
and chrysin (1.97 mmol) were dissolved in dry DMF (30 mL), then K2CO3 (10.87 mmol) was added
◦
in portions. The mixture was refluxed and kept at 75 C for 2 h under a nitrogen atmosphere,
the brown solution was filtered and concentrated under vacuum. The product was separated
by flash chromatography with dichloromethane-acetone (10:1) as eluent and recrystallized from
1
dichloromethane. White powder, m.p.: 156.2–157.1 ◦C, yield 58%. H-NMR (CDCl3) (ppm):
δ 12.72
(s, 1H), 7.88–7.87 (m, 2H), 7.53–7.52 (m, 2H), 6.66 (s, 1H), 6.64 (s, 1H), 6.47 (s, 1H), 5.22 (s, 2H), 2.53 (s,
6H), 2.47 (s, 3H); 13C-NMR (CDCl3) (ppm)
182.61, 164.59, 164.18, 162.35, 157.83, 151.92, 150.09, 148.98,
δ
144.71, 131.99, 131.42, 129.23, 126.42, 106.05, 99.07, 93.72, 70.47, 21.88, 21.55, 21.33, 20.74. MS (ESI) m/z:
[M + H]+ 389.1510, calcd. for C23H20N2O4 388.1423.
2-Phenyl-5,7-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)-4H-chromen-4-one, (11). TMP-Br (5.63 mmol) and
chrysin (1.97 mmol) were dissolved in dry DMF (30 mL), then K2CO3 (10.87 mmol) was added
◦
in portions. The mixture was refluxed and kept at 75 C for 2 h under a nitrogen atmosphere;
the brown solution was filtered and concentrated under vacuum. The product was separated
by flash chromatography with dichloromethane-◦acetone (10:1) as eluent and recrystallized from
1
dichloromethane. White powder, m.p.: 223.1–224.2 C, yield 60%. H-NMR (CDCl3) (ppm)
δ
7.83–7.81
(m, 2H), 7.47–7.46 (m, 3H), 6.718 (d, J = 5.0 Hz, 2H), 6.58 (s, 1H), 5.30 (s, 2H), 5.20 (s, 2H), 2.69 (s, 3H),
2.57 (s, 3H), 2.51 (s, 6H), 2.49 (s, 3H), 2.47 (s, 3H); 13C-NMR (CDCl3) (ppm)
177.14, 162.73, 160.76,
δ
159.75, 159.60, 151.94, 151.24, 150.88, 150.09, 149.02, 148.28, 145.37, 144.77, 131.72, 131.30, 129.07, 126.08,
110.09, 109.21, 98.65, 95.21, 71.30, 70.42, 21.87, 21.69, 21.57, 21.45, 20.82, 20.73. MS (ESI) m/z: [M + H]+
523.2359, calcd. for C31H30N4O4 522.2267.
5-Hydroxy-2-phenyl-6,7-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)-4H-chromen-4-one, (12).
TMP-Br
(5.29 mmol) and baicalein (1.85 mmol) were dissolved in dry D◦MF (30 mL), then K2CO3 (10.87 mmol)
was added in portions. The mixture was refluxed and kept at 75 C for 2 h under a nitrogen atmosphere;
the brown solution was filtered and concentrated under vacuum. The product was separated by flash
chromatography with petroleum ether-acetone (8:1) as eluent and recrystallized from dichloromethane.
1
Yellow powder, m.p.: 232.9–233.8 ◦C, yield 65%. H-NMR (CDCl3) (ppm)
δ 12.67 (s, 1H), 7.93–7.83
(m, 2H), 7.62–7.45 (m, 3H), 6.81 (s, 1H), 6.67 (s, 1H), 5.25 (s, 2H), 5.14 (s, 2H), 2.61 (s, 3H), 2.57 (s, 3H),
2.53 (s, 6H), 2.49 (s, 3H), 2.45 (s, 3H); 13C-NMR (CDCl3) (ppm)
182.83, 164.16, 158.21, 153.89, 153.54,
δ
151.94, 150.87, 150.54, 150.34, 148.81, 148.56, 146.40, 144.56, 132.00, 131.66, 131.47, 129.27, 126.40, 106.70,
105.83, 92.34, 74.26, 71.03, 21.91, 21.73, 21.54, 21.46, 20.83, 20.50. MS (ESI) m/z: [M + H]+ 539.2303, calcd.
for C31H30N4O5 538.2216.
2-Phenyl-5,6,7-tris((3,5,6-trimethylpyrazin-2-yl)methoxy)-4H-chromen-4-one, (13). TMP-Br (7.93 mmol) and
baicalein (1.85 mmol) were dissolved in dry DMF (30 mL), then K2CO3 (10.87 mmol) was added in
portions. The mixture was refluxed and kept at 75 ◦C for 2 h under a nitrogen atmosphere; the brown
solution was filtered and concentrated in vacuum. The product was separated by flash chromatography
with petroleum ether-acetone (8:1) as eluent and recrystallized from dichloromethane. White powder,
◦
1
m.p.: 185.4–186.9 C, yield 55%. H-NMR (CDCl3) (ppm)
1H), 6.67 (s, 1H), 5.26 (s, 2H), 5.24 (s, 2H), 5.02 (s, 2H), 2.66 (s, 3H), 2.56 (s, 3H), 2.53 (s, 6H), 2.46 (s, 6H),
2.40 (s, 6H), 2.29 (s, 3H); 13C-NMR (CDCl3) (ppm)
177.14, 161.32, 157.01, 154.77, 152.02, 151.79, 150.67,
150.24, 149.78, 148.88, 148.47, 146.52, 146.24, 144.40, 139.88, 131.73, 131.46, 129.16, 126.13, 113.69, 108.54,
δ 7.92–7.85 (m, 2H), 7.58–7.47 (m, 3H), 7.12 (s,
δ

Molecules 2018, 23, 2187
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98.39, 76.27, 75.13, 70.92, 21.92, 21.73, 21.53, 21.41, 20.77, 20.36. MS (ESI) m/z: [M + H]+ 673.3163, calcd.
for C39H40N6O5 672.3060.
(E)-1-(2-Hydroxy-4,6-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)
methoxy)phenyl)prop-2-en-1-one (14). TMP-Br (7.88 mmol) and naringenin (1.84 mmol) were dissolved in
dry DMF (30 mL), then K2CO3 (7.24 mmol) was added in portions. The mixture was refluxed and kept
◦
at 75 C for 2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under
vacuum. The product was separated by flash chromatography with dichloromethane-acetone (10:1)
as eluent and recrystallized from dichloromethane. Yellow powder, m.p.: 128.1–129.1 ◦C, yield 70%.
1H-NMR (CDCl3) (ppm)
δ
14.39 (s, 1H), 7.62 (s, 2H), 7.02 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H),
6.26 (dd, J = 13.0, 2.1 Hz, 2H), 5.20 (s, 2H), 5.17 (d, J = 1.6 Hz, 4H), 2.60 (s, 6H), 2.53 (s, 18H), 2.49 (s, 3H);
13C-NMR (CDCl3) (ppm)
192.83, 168.48, 164.93, 161.57, 160.26, 152.03, 151.80, 150.03, 149.26, 149.10,
δ
145.46, 144.95, 144.75, 142.25, 129.96, 128.68, 125.56, 115.04, 106.73, 95.71, 92.57, 70.58, 70.12, 70.12, 22.01,
21.83, 21.80, 21.59, 20.71, 20.67. MS (ESI) m/z: [M + H]+ 675.3306, calcd. for C39H42N6O5 674.3217.
5,7-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)-2-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)chroman-
4-one (15), TMP-Br (7.88 mmol) and naringenin (1.84 mmol) were dissolved in dry DMF (30 mL),
then K2CO3 (14.49 mmol) was added in portions. The mixture was refluxed and kept at 75 ◦C for
2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under vacuum.
The product was separated by flash chromatography with dichloromethane-acetone (10:1) as eluent
1
and recrystallized from dichloromethane. White powder, m.p.: 176.4–177.6 ◦C, yield 52%. H-NMR
(CDCl3) (ppm)
δ 7.36 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.45 (d, J = 1.5 Hz, 1H), 6.24 (d,
J = 1.7 Hz, 1H), 5.33 (dd, J = 13.0, 2.0 Hz, 1H), 5.23 (s, 2H), 5.16 (s, 2H), 5.12 (s, 2H), 2.99 (dd, J = 16.4,
13.4 Hz, 1H), 2.71 (dd, J = 16.5, 2.5 Hz, 1H), 2.66 (s, 3H), 2.58 (s, 3H), 2.55 (s, 3H), 2.53 (s, 3H), 2.52
(s, 12H), 2.49 (s, 3H); 13C-NMR (CDCl3) (ppm)
δ 188.86, 164.88, 164.66, 161.01, 159.01, 151.85, 151.49,
151.35, 150.79, 150.14, 150.04, 148.98, 148.77, 148.29, 145.63, 145.29, 144.82, 131.43, 127.85, 115.19, 106.66,
95.50, 95.20, 79.06, 70.98, 70.21, 70.15, 45.65, 21.88, 21.83, 21.78, 21.56, 21.49, 20.83, 20.76, 20.72. MS (ESI)
m/z: [M + H]+ 675.3302, calcd. for C39H42N6O5 674.3217.
(E)-3-(4-((3,5,6-Trimethylpyrazin-2-yl)methoxy)phenyl)-1-(2,4,6-tris((3,5,6-trimethylpyrazin-2-yl)-methoxy)
phenyl)prop-2-en-1-one (16), TMP-Br (10.51 mmol) and naringenin (1.84 mmol) were dissolved in dry
DMF (30 mL), then K2CO3 (14.49 mmol) was added in portions. The mixture was refluxed and kept at
75 ◦C for 2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under
vacuum. The product was separated by flash chromatography with dichloromethane-acetone (10:1)
◦
as eluent and recrystallized from dichloromethane. Yellow powder, m.p.: 61.6–62.3 C, yield 58%.
1H-NMR (CDCl3)(ppm)
δ
7.32 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 16.0 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.69
(d, J = 16.0 Hz, 1H), 6.54 (s, 2H), 5.16 (s, 4H), 5.09 (s, 4H), 2.61 (s, 3H), 2.58 (s, 3H), 2.54 (s, 3H), 2.53 (s,
3H), 2.52 (s, 6H), 2.43 (s, 6H), 2.41 (s, 12H); 13C-NMR (CDCl3) (ppm)
194.08, 160.90, 160.46, 157.39,
δ
151.71, 151.66, 151.32, 150.63, 150.20, 150.14, 148.85, 148.23, 145.35, 144.73, 130.10, 127.99, 127.40, 115.22,
112.90, 93.83, 70.97 (2C), 70.29, 70.10, 21.90, 21.89, 21.77, 21.62, 21.59, 21.45, 20.81, 20.79, 20.73. MS (ESI)
m/z: [M + H]+ 809.4166, calcd. for C47H52N8O5 808.4061.
5-Hydroxy-7-((3,5,6-trimethylpyrazin-2-yl)methoxy)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)-phenyl)-4H-
chromen-4-one (17). TMP-Br (5.29 mmol) and genistein (1.85 mmol) were dissolved in dry DMF (30 mL),
then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at 75 ◦C for
2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under vacuum.
The product was separated by flash chromatography with dichloromethane-acetone (10:1) as eluent
and recrystallized from dichloromethane. White powder, m.p.: 134.6–135.4 ◦C, yield 67%, 1H-NMR
(CDCl3) (ppm)
J = 1.9 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 5.21 (s, 2H), 5.19 (s, 2H), 2.60 (s, 3H), 2.59 (s, 3H), 2.53 (s,
12H); 13C-NMR (CDCl3) (ppm)
180.82, 164.40, 162.75, 158.80, 157.86, 152.79, 151.75, 151.26, 149.91,
148.94, 148.76, 145.66, 144.62, 130.28, 130.14, 123.65, 123.43, 115.69, 115.02, 106.56, 99.00, 93.34, 70.32,
δ 12.82 (s, 1H), 7.86 (s, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H), 6.54 (d,
δ

Molecules 2018, 23, 2187
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69.99, 40.15, 28.35, 21.71, 21.60, 21.43, 20.57. MS (ESI) m/z: [M + H]+ 539.2296, calcd. for C31H30N4O5
538.2216.
5,7-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-4H-
chromen-4-one (18). TMP-Br (7.93 mmol) and genistein (1.85 mmol) were dissolved in dry DMF (30 mL),
then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at 75 ◦C for
2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated in vacuum.
The product was separated by flash chromatography with dichloromethane-acetone (10:1) as eluent
1
and recrystallized from dichloromethane. White powder, m.p.: 188.2–189.1 ◦C, yield 53%. H-NMR
(CDCl3) (ppm) δ 7.74 (s, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.77 (d, J = 2.1 Hz, 1H),
6.62 (d, J = 2.1 Hz, 1H), 5.31 (s, 2H), 5.23 (s, 2H), 5.18 (s, 2H), 2.67 (s, 3H), 2.61 (s, 4H), 2.60 (s, 3H), 2.56
(s, 7H), 2.54 (s, 6H), 2.51 (s, 3H), 2.50 (s, 3H); 13C-NMR (CDCl3) (ppm) δ 174.88, 162.41, 160.09, 159.65,
158.44, 151.82, 151.21, 150.89, 150.05, 149.97, 148.88, 148.60, 148.08, 145.73, 145.16, 144.63, 130.39, 126.00,
124.91, 115.42, 114.76, 110.50, 98.36, 94.72, 71.20, 70.28, 69.97, 21.76, 21.66, 21.62, 21.45, 21.42, 21.33,
20.85, 20.61. MS (ESI) m/z: [M + H]+ 673.3164, calcd. for C39H40N6O5 672.3060.
7-Hydroxy-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-4H-chromen-4-one (19). TMP-Br (2.81 mmol)
and daidzein (1.97 mmol) were dissolved in dry acetone (30 mL), then K2CO3 (10.87 mmol) was added
in portions. The mixture was refluxed for 2 h under a nitrogen atmosphere; the brown solution was
filtered and concentrated under vacuum. The product was separated by flash chromatography with
petroleum ether-acetone (10:1) as eluent and recrystallized from dichloromethane. White powder, m.p.:
1
213.1–214.2 ◦C, yield 51%. H-NMR (DMSO-d6) (ppm)
δ
9.52 (s, 1H), 8.33 (s, 1H), 7.98 (d, J = 8.9 Hz,
1H), 7.35 (d, J = 8.5 Hz, 2H), 7.09 (dd, J = 8.9, 2.3 Hz, 1H), 6.77 (d, J = 8.6 Hz, 2H), 5.27 (s, 2H), 2.46 (s,
3H), 2.40 (s, 6H); 13C-NMR (DMSO-d6) (ppm)
δ
175.15, 163.08, 157.72, 153.67, 151.81, 149.86, 148.94,
145.00, 130.54, 127.48, 124.19, 122.80, 118.35, 115.44, 114.92, 102.06, 70.38, 21.76, 21.46, 20.62. MS (ESI)
m/z: [M + H]+ 389.1506, calcd. for C23H20N2O4 388.1423.
7-((3,5,6-Trimethylpyrazin-2-yl)methoxy)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-4H-chromen-
4-one (20). TMP-Br (5.63 mmol) and daidzein (1.97 mmol) were dissolved in dry DMF (30◦mL), then
K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at 75 C for 2 h
under a nitrogen atmosphere; the brown solution was filtered and concentrated under vacuum.
The product was separated by flash chromatography with petroleum ether-acetone (10:1) as eluent
◦
and recrystallized from dichloromethane. White solid, m.p.: 185.4–186.9 C, yield 62%. 1H NMR
(CDCl3) (ppm) 8.223 (d, J = 9 Hz, 1H), 7.94 (s, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.09 (dd, J = 8.9, 2.3 Hz,
1H), 6.77 (d, J = 8.6 Hz, 2H), 5.27 (s, 2H), 5.21 (s, 2H), 2.62 (s, 3H), 2.61 (s, 3H), 2.56 (s, 6H), 2.55 (s, 6H);
13C NMR (CDCl3) (ppm)
175.77, 162.80, 158.57, 157.77, 152.16, 151.83, 151.25, 149.98, 148.90, 148.68,
δ
δ
145.70, 144.61, 130.16, 127.91, 124.84, 124.71, 118.75, 114.93, 114.88, 101.45, 70.38, 69.98, 21.76, 21.66,
21.45, 21.44, 20.62. MS (ESI) m/z: [M + H]+ 523.2351, calcd. for C31H30N4O4 522.2267.
2-(3,4-Dihydroxyphenyl)-3,5,7-tris((3,5,6-trimethylpyrazin-2-yl)methoxy)-4H-chromen-4-one (21). TMP-Br
(7.09 mmol) and quercetin (1.66 mmol) were dissolved in dry DMF (30 mL), then K2CO3 (10.87 mmol)
◦
was added in portions. The mixture was refluxed and kept at 75 C for 2 h under a nitrogen
atmosphere; the brown solution was filtered and concentrated under vacuum. The product was
separated by flash chromatography with dichloromethane-acetone (15:1) as eluent and recrystallized
1
from dichloromethane. Yellow powder, m.p.: 121.2–122.3 ◦C, yield 65%. H-NMR (CDCl3) (ppm)
δ
12.63 (s, 1H), 7.48 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 1.6 Hz, 1H), 6.45
(d, J = 1.5 Hz, 1H), 5.21 (s, 2H), 5.19 (s, 2H), 5.18 (s, 2H), 2.58 (s, 3H), 2.57 (s, 3H), 2.56 (s, 3H), 2.52 (s,
9H), 2.51 (s, 3H), 2.42 (s, 3H), 2.40 (s, 3H) ; 13C-NMR (CDCl3) (ppm)
δ 178.92, 164.45, 162.18, 156.94,
156.85, 151.92, 151.87, 151.13, 150.03, 149.95, 149.49, 149.02, 148.82, 148.64, 148.35, 148.24, 145.45, 145.13,
144.73, 137.59, 126.04, 121.31, 117.19, 116.91, 106.50, 98.73, 93.23, 73.15, 72.00, 70.38, 21.85, 21.68, 21.55,
21.37, 21.06, 20.71, 20.59, 20.34. MS (ESI) m/z: [M + H]+ 705.3043, calcd. for C39H40N6O7 704.2958.

Molecules 2018, 23, 2187
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2-(3,4-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)phenyl)-3,5,7-tris((3,5,6-trimethylpyrazin-2-yl)-methoxy)-4H-
chromen-4-one (22). TMP-Br (11.83 mmol) and quercetin (1.66 mmol) were dissolved in dry DMF
(30 mL), then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at
75 ◦C for 2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under
vacuum. The product was separated by flash chromatography with dichloromethane-acetone (15:1)
as eluent and recrystallized from dichloromethane. White powder, m.p.: 179.4–180.1 ◦C, yield 67%.
1H-NMR (CDCl3) (ppm)
δ 7.75 (s, 1H), 7.63 (dd, J = 8.6, 1.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 6.74 (s,
1H), 6.70 (d, J = 1.8 Hz, 1H), 5.37 (s, 2H), 5.27 (s, 2H), 5.24 (s, 2H), 5.22 (s, 2H), 5.01 (s, 2H), 2.74 (s, 3H),
2.65 (s, 3H), 2.61 (s, 3H), 2.55 (s, 9H), 2.53 (s, 4H), 2.52 (s, 15H), 2.50 (s, 3H), 2.39 (s, 3H), 2.32 (s, 3H);
13C-NMR (CDCl3) (ppm)
δ 173.50, 162.49, 159.57, 158.53, 153.02, 151.84, 151.31, 151.18, 151.12, 150.77,
150.29, 150.18, 150.07, 149.98, 148.88, 148.49, 148.11, 148.02, 145.98, 145.54, 145.35, 145.19, 144.61, 139.67,
123.86, 122.56, 114.44, 113.34, 110.02, 98.20, 94.63, 72.83, 71.24, 71.05, 70.91, 70.32, 21.78, 21.71, 21.68,
21.52, 21.47, 21.39, 21.37, 21.29, 20.86, 20.73, 20.63, 20.57. MS (ESI) m/z: [M + H]+ 973.4751, calcd. for
C55H60N10O7 972.4646.
2-(3,4-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)phenyl)-5-hydroxy-7-((3,5,6-trimethylpyrazin-2-yl)-methoxy)-
4H-chromen-4-one (23). TMP-Br (7.49 mmol) and luteolin (1.75 mmol) were dissolved in dry DMF
(30 mL), then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at
75 ◦C for 2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under
vacuum. The product was separated by flash chromatography with dichloromethane-acetone (10:1)
as eluent and recrystallized from dichloromethane. Yellow powder, m.p.: 163.8–164.7 ◦C, yield 55%.
1H-NMR (CDCl3) (ppm)
δ 12.79 (s, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H), 7.17 (d,
J = 8.6 Hz, 1H), 6.63 (d, J = 2.1 Hz, 1H), 6.56 (s, 1H), 6.46 (d, J = 2.1 Hz, 1H), 5.28 (s, 2H), 5.26 (s, 2H),
5.22 (s, 2H), 2.59 (s, 3H), 2.57 (s, 3H), 2.57 (s, 3H), 2.53 (s, 9H), 2.51 (s, 3H), 2.51 (s, 3H), 2.50 (s, 3H);
13C-NMR (CDCl3) (ppm)
δ 182.50, 164.45, 163.90, 162.35, 157.72, 151.97, 151.88, 151.63, 151.56, 150.36,
150.26, 150.08, 149.00, 148.74, 145.42, 145.18, 144.78, 124.32, 120.58, 114.00, 112.64, 106.02, 104.90, 98.98,
93.65, 71.36, 71.04, 70.48, 21.84, 21.56, 21.53, 20.74, 20.70. MS (ESI) m/z: [M + H]+ 689.3098, calcd. for
C39H40N6O6 688.3009.
5,7-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)-2-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-4H-
chromen-4-one (24). TMP-Br (7.93 mmol) and apigenin (1.85 mmol) were dissolved in dry DMF (30 mL),
then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at 75 ◦C for
2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under vacuum.
The product was separated by flash chromatography with dichloromethane-acetone (8:1) as eluent
1
and recrystallized from dichloromethane. White powder, m.p.: 180.9–181.5 ◦C, yield 55%. H-NMR
(CDCl3) (ppm) δ 7.78 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 10.4 Hz, 2H), 6.51 (s, 1H),
5.31 (s, 2H), 5.22 (s, 4H), 2.71 (s, 3H), 2.59 (s, 3H), 2.59 (s, 3H), 2.53 (s, 6H), 2.52 (s, 6H), 2.51 (s, 3H), 2.49
(s, 3H); 13C-NMR (CDCl3) (ppm) δ 177.13, 162.59, 161.10, 160.65, 159.65, 159.55, 151.91, 151.67, 151.18,
150.86, 150.08, 149.00, 148.93, 148.27, 145.43, 145.25, 144.79, 127.73, 124.48, 115.36, 110.00, 107.94, 98.59,
95.18, 71.28, 70.39, 70.17, 21.86, 21.81, 21.67, 21.56, 21.44, 20.80, 20.72. MS (ESI) m/z: [M + H]+ 673.3158,
calcd. for C39H40N6O5 672.3060.
2-(3,4-bis((3,5,6-Trimethylpyrazin-2-yl)methoxy)phenyl)-3,7-bis((3,5,6-trimethylpyrazin-2-yl)-methoxy)-4H-
chromen-4-one (25). TMP-Br (9.99 mmol) and fisetin (1.75 mmol) were dissolved in dry DMF (30 mL),
then K2CO3 (10.87 mmol) was added in portions. The mixture was refluxed and kept at 75 ◦C for
2 h under a nitrogen atmosphere; the brown solution was filtered and concentrated under vacuum.
The product was separated by flash chromatography with dichloromethane-acetone (10:1) as eluent
1
and recrystallized from dichloromethane. White powder, m.p.: 162.7–163.1 ◦C, yield 48%. H-NMR
(CDCl3) (ppm)
5.29 (s, 2H), 5.27 (s, 2H), 5.22 (s, 2H), 2.68 (s, 3H), 2.61 (s, 3H), 2.53 (s, 9H), 2.51 (s, 12H), 2.48 (s, 3H),
2.41 (s, 3H), 2.33 (s, 3H); 13C-NMR (CDCl3) (ppm)
174.55, 163.03, 156.86, 155.57, 151.97, 151.45, 151.25,
151.05, 150.75, 150.28, 150.18, 150.12, 149.03, 148.68, 148.24, 145.97, 145.70, 145.46, 144.74, 139.42, 127.34,
δ 8.17 (d, J = 8.6 Hz, 1H), 7.81 (s, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.11 (s, 2H), 7.05 (s, 1H),
δ

Molecules 2018, 23, 2187
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124.11, 123.02, 121.32, 118.52, 117.92, 116.91, 114.88, 114.82, 113.54, 101.45, 72.99, 71.27, 71.04, 70.54,
21.88, 21.81, 21.77, 21.64, 21.57, 21.51, 21.43, 20.74, 20.68. MS (ESI) m/z: [M + H]+ 823.3950, calcd. for
C47H50N8O6 822.3853.
4.3. Bio-Evaluation Methods
4.3.1. Drugs
Ligustrazine, luteolin, baicalein, apigenin, chrysin, quercetin, fisetin, genistein, daidzein,
naringenin, DDP and TMP-flavonoid derivatives (compounds 10–25). All samples were dissolved in
DMSO and prepared to 104 µM. The final DMSO concentration did not exceed 0.1% (V/V).
4.3.2. Cell Culture
Five kinds of tumor cell lines and one normal endothelial cell were routinely cultured respectively
in RPMI 1640 medium supplemented with 10% (V/V) FBS and 1% (V/V) penicillin-streptomycin,
incubated at 37 ◦C in a 5% CO2 containing incubator.
4.3.3. Cytotoxicity Assay Using Five Tumor Cell Lines
The five tumor cell lines were plated onto 96-well sterile plates in 100
µ
L/well of medium at a
◦
density of 3.5
×
103 cells per well and incubated at 37 C with 5% CO2 for 24 h. Then luteolin, baicalein,
apigenin, chrysin, quercetin, fisetin, genistein, daidzein, naringenin, DDP and compounds 10
added at various concentrations (5, 10, 20, 40, 50 M). Each plate contained control group, blank group,
drug group and positive group (DDP). After 72 h, 20 L MTT in phosphate buffered saline (PBS,
5 mg/mL) was added to each well and the plates were incubated at 37 ◦C for 4 h, then removing the
liquid and adding dimethyl sulfoxide (DMSO, 100 L) to dissolve the MTT formazan. The optical
–25 were
µ
µ
µ
density (OD) for each well was measured on a BIORAD 550 spectrophotometer plate reader (Bio-Gene
Technology Ltd., Guangzhou, China) at a wavelength of 490 nm. All tests were carried out three times
in parallel. The proliferation inhibition rates of tumor cells were calculated by {1
group) OD490 (Blank group)]/[OD490 (Control group) OD490 (Blank group)]}
values were defined as the concentration of compounds that produced a 50% proliferation inhibition
−
[OD490 (Drug
100%; the IC50
−
−
×
of surviving cells and calculated using the following equation: logIC50 = Xm − log2 × ( P − 0.5),
∑
Where Xm = logCmax
,
P = sum of proliferation inhibition rates, the number 0.5 is an empirical
∑
constant. We first tested the activities of TMP-flavonoid derivatives with concentrations of 10 and
20 M respectively. When compounds’ inhibition rate was not beyond 35% at a concentration of 20 M,
µ
µ
the compounds would not be considered to study further.
4.3.4. Cytotoxicity Assay Using HUVEC-12 Cell
The HUVEC-12 cell were seeded in 96-well plates (6.0
×
103 cells/ well), and incubated at 37 ◦C
in a 5% CO2 containing incubator. After 24 h, the medium was replaced with fresh medium containing
the compounds to be tested. Compounds 14 19 and DDP were added in final concentrations ranging
from 2.5 to 40 M. Each plate contained control group, blank group and drug group. After 24 h, 20
MTT was added to each well and the plates were incubated at 37 ◦C for 4 h, then removing the liquid
and adding dimethyl sulfoxide (DMSO) (100 L) to dissolve the MTT formazan. The optical density
,
µ
µL
µ
(OD) for each well was measured on a BIORAD 550 spectrophotometer plate reader at a wavelength of
490 nm. All tests were carried out three times in parallel. The proliferation inhibition rates of tumor
cells were calculated by {1
OD490 (Blank group)]} × 100%.
−
[OD490 (Drug group)
−
OD490 (Blank group)]/[OD490 (Control group)
−
4.3.5. Morphological Analysis Using Giemsa and DAPI Staining
The Bel-7402 cells were plated onto 12-well sterile plates at a density of 2.4
and incubated at 37 C in a 5% CO2 containing incubator. Then the cells were incubated in the presence
×
104 cells per well
◦

Molecules 2018, 23, 2187
14 of 16
of compound 14 at various concentrations (0, 10, 20, 40
µM) for 72 h. Then cells were washed with PBS
twice, fixed with cold ethanol and 4% paraformaldehyde (pH = 7.4) for 10 min respectively and washed
with PBS again. Then fixed cells were stained with 6% Giemsa solution or DAPI at the concentration of
1 mg/mL for 5 min in the dark. Finally, the cells washed with water and dried. The cell morphological
changes were observed by fluorescent microscopy and images were captured by digital camera [34,35].
4.3.6. Apoptosis Analysis by Flow Cytometric Using Annexin V-FITC/Propidium Iodide (PI) Staining
◦
The Bel-7402 cells were plated onto 6-well sterile plates (4.8
with 5% CO2 for 24 h. Then compound 14 at various concentrations (0, 5, 10, 20
×
104 cells/well) and placed at 37 C
µ
M) was added.
After 72 h, all cells were collected respectively with the right amount of trypsin (without EDTA)
digestion. Then the cells were washed with cold PBS twice and centrifuged at 1000 rpm for 5 min.
The harvested cells were resuspended in 200 µL binding buffer, which contained 10 µL Annexin V-FITC
and PI. After avoided light reaction for 15 min, the cells were analyzed with a flow cytometer [36].
4.4. Statistical Analysis
All data were expressed as the means
±
standard deviation (SD) of three replications.
The statistical analysis was performed by SPSS software (Version 20.0, International Business Machines
Corp. New York, NY, USA) to analyze the variance. One-way analysis of variance (ANOVA) followed
by the least significant difference (LSD) post-hoc test for multiple comparisons. A p-value of less than
0.05 was considered significant.
5. Conclusions
Using TMP and flavonoids as starting materials, we have designed and synthesized sixteen novel
TMP-flavnoid derivatives through conjugation of anti-tumor bioactive compounds via ether bonds.
Among them, compounds 14 and 16 were obtained via dihydroflavone hydrolysis to chalcones under
alkaline conditions. As the flavovoids and TMP showed broad anti-tumor activities, we chose five
different human tumor cell lines to evaluate the TMP-flavonoid derivatives. In vitro chemosensitivity
testing showed that most of compounds had certain anti-tumor activity against the HeLa, MCF-7,
HT-29, HepG-2, Bel-7402 cell lines. Part of derivatives showed better anti-tumor activities than the
raw materials. Besides, compounds 14 and 19 exhibited activities close to that of the positive control
(DDP) against the Bel-7402 and MCF-7 cell lines, respectively (IC50 = 10.49
±
1.12, 10.43
±
1.23 µM;
DDP IC50 = 6.73 ± 0.37, 6.75
±
0.57 M). Then, the effects of compounds 14 and 19 on NECs were
µ
determined using HUVEC-12 cells. Compound 14 showed pro-proliferation effects while compound
19 appeared to cause minor damage on cells and DDP had similar cytotoxic effects on HUVEC-12
and tumor cells (compounds 14 and 19 IC50 > 40
µ
M; DDP IC50 = 9.11
±
0.54 µM). Subsequently,
the results of fluorescence staining and flow cytometry analysis indicated that compound 14 could
induce apoptosis in the Bel-7402 cell line. All results suggested that compound 14 has bright prospects.
In addition, the discussion of structure-activity relationships indicated that isoflavones, chalcone and
its derivatives with 5, 7-OH substituents have great potential on tumor therapy. Moreover, the attempt
to apply structure combination to discover more efficient and multi-effective anti-tumor leading
compounds from TCM formulations is viable.
Author Contributions: H.W., T.M., P.W. and H.L. conceived and designed the experiments and discussed the data;
H.W. and G.W. performed chemistry experiment; H.W., N.X. and H.C. performed biological activity experiments;
X.Z., W.G., Y.C. and F.Z. analyzed the pharmacological data, M.C., K.F. elaborated the cell morphology and conduct
data analysis and statistics; H.W., W.Z. wrote the paper and modified the language of the paper. All authors read
and approved the final manuscript.
Acknowledgments: This study was financially supported by the National Natural Science Foundation of China
(No. 81173519 and 81603256), supported by the Fundamental Research Funds for the Central Universities
project of “acid-base complexation” and “structure combination” principle to discover lead compounds from
Chinese traditional Chinese medicine (BUCM, 2018–2019), Beijing Key Laboratory for Basic and Development
Research on Chinese Medicine (Beijing, 100102), the Fundamental Research Funds for the Central Universities

Molecules 2018, 23, 2187
15 of 16
(BUCM-2019-JCRC002), the Innovation Team Project Foundation of Beijing University of Chinese Medicine
(Lead Compounds Discovering and Developing Innovation Team Project Foundation, No. 2011-CXTD-15)and
Graduate Independent Topics of Beijing University of Chinese Medicine (2018-JYBZZ-XS062).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 10–25 are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).