Discovery and synthesis of novel Wogonin derivatives with potent antitumor activity in vitro

Article abstract of DOI:10.1016/j.ejmech.2017.09.046

Phenotypic screening of high quality compound library is one of the most effective strategy to obtain novel bioactive compounds. Recently, our group have constructed a Wogonin-scaffold library with substituents diversity and successfully obtained a series

Full text of DOI:10.1016/j.ejmech.2017.09.046

Accepted Manuscript
Discovery and synthesis of novel Wogonin derivatives with potent antitumor activity in
vitro
Jubo Wang, Raoling Ge, Xiaqiu Qiu, Xi Xu, Libin Wei, Zhiyu Li, Jinlei Bian
PII:
S0223-5234(17)30761-4
10.1016/j.ejmech.2017.09.046
EJMECH 9761
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 July 2017
Revised Date: 4 September 2017
Accepted Date: 21 September 2017
Please cite this article as: J. Wang, R. Ge, X. Qiu, X. Xu, L. Wei, Z. Li, J. Bian, Discovery and synthesis
of novel Wogonin derivatives with potent antitumor activity in vitro, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.046.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery and synthesis of novel Wogonin derivatives with potent antitumor
activity in vitro
Jubo Wang a, Raoling Ge a, Xiaqiu Qiu a, Xi Xu a, Libin Wei b, Zhiyu Li a*, Jinlei Bian a*
a.Jiangsu Key Laboratory of Drug Design and Optimization, School of Pharmacy, China PharmaceuticalUniversity, 24 Tongjiaxiang,
Nanjing, 210009, China.
.
b. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, Department of Basic
Medicine, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009,
China.
*Corresponding author. zhiyuli@cpu.edu.cn (Z.Y. Li), bianjl@cpu.edu.cn (J.L. Bian); Tel: + 86-25-83271216
Abstract
Phenotypic screening of high quality compound library is one of the most effective strategy to
obtain novel bioactive compounds. Recently, our group have constructed a Wogonin-scaffold library
with substituents diversity and successfully obtained a series of potent compounds. Herein, we
reported the synthesis of these compounds and evaluated the in vitro antitumor activity against a
panel of human tumor cell lines. Most of them showed good activity with a broad spectrum and
preliminary structure-activity relationship for the substitutions were obtained. Further biological
assays showed that the most potent compounds 18n and 20b could significantly enhance the
intracellular ROS level and induce the cell apoptosis at low micromole level. Through similarity
searching, CDK9 was identified as the potential target for 20b, which could be a start point for next
structure-based drug design.
Keywords: Wogonin, antitumor, ROS, Phenotypic screening, target fishing
.

ACCEPTED MANUSCRIPT
1. Introduction
Cancer remains the leading cause of death worldwide though much important progress has been
achieved recently. It is expected that annual cancer cases will rise to 22 million in the next two
decades [1]. Treatments for cancers include surgery, radiation therapy, and chemotherapy. However,
surgery is usually the treatments of choice for early stage cancers, for patients with unrespectable
disease, local control can be achieved with radiation therapy combined with chemotherapy,
molecular-targeted drugs, or some combination [2,3]. Currently, chemotherapy is still the main type
of treatment for cancer, and phytochemicals have been a crucial part of antineoplastic drugs [4].
Indeed, more than half of anticancer drugs approved are either natural products or developed based
on the knowledge gained form natural products. Moreover, about 75 percent of plant-derived drugs
used today in the clinic come from traditional medicines [4].
The dry root of Scutellaria, also named HuangQin in China, is one of the most popular and
multi-purpose herb used in China. In the traditional Chinese medicines (TCM), extracts from the
Scutellaria radix are widely used for clinical treatment of hyperlipemia, atherosclerosis,
hypertension, dysentery, common cold and inflammatory diseases. In addition, recently, Scutellaria
alone or in combination with other herbs has been shown to possess cytostatic effect on several
cancer cells in vitro [5,6] and also in vivo in mouse tumor models [7,8]. To identify the antitumor
components of Scutellaria, many researches have been focused on separating the effective
components. It has been proved that the antitumor constituents of Scutellaria are flavonoids. Among
these flavonoids, Wogonin (1) has been reported to selectively kill cancer with little to no toxicity to
normal cells at concentrations that are lethal to tumor cells.5 And it has been approved by the China
Food and Drug Administration (CFDA) for phase I/II clinical studies since 2014 [9]. However, 1 and
its simple derivatives have often been reported at pharmacologically nonachievable high micromolar
concentrations (20 to 200 µM) against cancer cells [10]. Hence, medicinal chemistry strategies are
necessary to optimize the drug-like profiles of these compounds to ensure that the full clinical
applications for flavonoids are realized.
Fig. 1. The structure of Wogonin (1).
2

ACCEPTED MANUSCRIPT
Phenotypic screening is an important approach in drug discovery and attracts great interests
recently [11]. As an alternative method to target-based screening, phenotypic screening discovers
drug hits or leads based on assessing a molecule’s effects on the whole-cell level. Thus, phenotypic
screening is one of the drug discovery strategy for identifying initial starting points that are
pharmaceutically relevant and potentially available for medicinal chemistry optimization [12].
Analysis of the origins of new antitumor drugs approved by the US Food and Drug Administration
(FDA) in recent years, a significant number were identified from phenotypic screening approaches
[13-14]. Thus, our group planned to employ the phenotypic screening method to discover efficient
derivatives of 1. The success and efficiency of phenotypic screening is largely dependent on high
quality compounds libraries [15]. In order to develop high quality compound library for phenotypic
screening, our laboratory recently synthesized plenty of Wogonin derivatives to construct a library
contains more than two hundred compounds. Interestingly, preliminary in vitro antitumor assay
identified a series of compounds which showed efficient antitumor activity for several types of
human cancer cell lines [16]. In this work, we selected and reported the most potent compounds in
the library and all of the selected compounds were further screened using four different types of
cancer cells to clarify the influence of the substitutions at different positions on the antitumor
activity. Among them, the most two potent compound 18n and 20b showed good antitumor activity
against all tested cancer cell lines and could effectively induce the apoptosis through ROS mediated
pathways in HepG2 cancer cells. Through similarity searching, CDK9 was identified as the potential
target for 20b, which could be a start point for next structure-based drug design of Wogonin
derivatives.
2. Chemistry
The common synthetic routes of the target compounds were outlined in Scheme 1-4. Compounds
12a-12j were synthesized as shown in Scheme 1. Benzylation of 2 was conducted by reaction with
benzyl bromide in K2CO3 to afford intermediate 3. Oxidation of 3 using 65% HNO3 provided the
quinone mediate 4, which was further reduced by sodium thiosulfate to the phenol 5. Methylation of
4 with dimethylsulfate obtained intermediate 6, which then carried the debenzylation reaction to give
compound 7 using hydrogen/palladium-carbon system. Followed by Friedel-Crafts acylation with
cinnamoyl chloride derivatives using BF3-Et2O as catalyst provided the intermediate 8. Subsequently,
o
the intermediate 8 was cyclized to the flavones 9 mediated by iodine at 120 C. And the flavone 9
was then introduced a four-carbon linker to 7-OH to form the key intermediate 10. Compound 10
3

ACCEPTED MANUSCRIPT
was obtained through demethylation of intermediate 11. Finally, the target compounds 12a-12j were
obtained from 11 with N-methylpiperazine by reflux in acetonitrile.
Scheme 2 described the synthesis of target compounds 18a-18n. These compounds were
synthesized in five steps from chrysin (13). Tetramethylammonium hydroxide reacted with 13
resulted in the intermediate 14 in 30% yield. The benzylation of 14 was conducted by reaction with
benzyl bromide in the presence of potassium carbonate to afford intermediate 15. Then compound 16
was obtained by acidified 15 in the presence of hydrochloric acid. Followed by a two to four-carbon
linker to 8-OH to form intermediate 17, which then reacted with various amines to give the target
compounds 18a-18n.
Based on the compound 18n, compounds 20a-20f were obtained as outlined in Scheme 3.
Debenzylation of 18n give rise to the compound 19, which was subsequently reacted with
corresponding amines to provide target compounds 20a-20f in 30-60% yield (Scheme 3). The
1
structures of all compounds were confirmed by H NMR and electrospray ionization mass
spectrometry (ESI-MS). Before these compounds were used in biological experiments, they were
purified by silica gel column chromatography and HPLC was used to determine their purity (all >
95%).
Scheme 1. Synthesis of compounds 12a-12j. Reagents and conditions: (i) PhCH2Br, K2CO3, acetone, reflux, 48 h,
○
87%; (ii) AcOH, 65% HNO3, 40 C, 4 h, 45%; (iii) Na2S2O4, CH3COOC2H5/H2O, r.t., 1 h, 37%; (iv) (CH3)2SO4,
4

ACCEPTED MANUSCRIPT
NaOH, C2H5OH, r.t., 3 h, 85%; (v) H2, Pd/C, MeOH, r.t., 8 h, 90%; (vi) Cinnamoyl chloride derivatives, BF3-Et2O,
CHCl3, reflux, 1.5-6 h, 35-80%; (vii) I2, DMSO, 120 ○C, 5-8 h, 30-40%; (viii) AlCl3, CH3CN, reflux, 8 h, 65-85%.
(viii) Br(CH2)4Br, K2CO3, acetone, reflux, 8 h, 60-75%; (ix) AlCl3, CH3CN, reflux, 6 h, 70-90%; (x)
N-methylpiperazine, dry CH3CN, reflux, 5 h, 50-64%.
Scheme 2. Synthesis of compounds 18a-18n. Reagents and conditions: (i) K2S2O8, (CH3)4NOH, H2O, r.t., 7 h, 30%;
(ii) PhCH2Br, K2CO3, DMF, 50 oC, 6 h; (iii) 6 M HCl, r.t., 8 h, 40%; (iv) Br(CH2)nBr, K2CO3, acetone, reflux, 8 h,
75%; (v) substituted amino derivatives, CH3CN, reflux, 1–5 h, 30–60%.
N
N
N
N
N
N
R1
R1
=
=
=
R1
=
=
20d
20e
20a
S
O
20b R1
20c R1
NC
O
O
O
N
S
O
O
O
HO
O
O
O
O
O
R1
i
ii
20f R1
=
O
N
OH
OH
OH
18n
19
20
Scheme 3. Synthesis of compounds 20a-20f. Reagents and conditions: (i) Pd/C, H2, THF, r.t., 4 h, 42%; (ii) R-Cl,
base, CH3CN, reflux, 1–5 h, 30–60%.
3. Results and Discussion
3.1. In vitro antitumor activity.
The results of in vitro antitumor activity of all compounds against four cancer cell lines were
summarized in Table 1. Notably, most compounds showed good antitumor activity against all the
four cancer cell lines. Namely, these compounds exhibited a broad spectrum of inhibition on human
cancer cells, with relatively low IC50 values ranging from 0.6 µM to 10 µM. In addition, they were
generally more active than the control 1, carboplatin and 5-fluorouracil (5-Fu). As for the first series,
5

ACCEPTED MANUSCRIPT
compounds 12a-12j with substituents at B-ring, showed the similar activity toward all cancer cells.
Among compounds 18a-18n, 18n showed the most potent activity, especially for HepG2. Based on
the structure of 18n, we synthesized compounds 20a-20f, and the activity of these compounds
against the four cancer cells were in the same level with 18n. In addition, it was worth mentioning
that nearly all the compounds possessed significant better selectivity against HepG2 cancer cell line
than other three cancer cell lines, indicating the good potency of these structures for the treatment of
human hepatocarcinoma. Among these compounds, two most promising compounds 18n and 20b
exhibited the highest antitumor activity against A549, MCF-7, B16 and HepG2 with IC50 values of
2.91, 2.14 µM (A549), 2.93, 2.42 µM (MCF-7), 2.37, 3.70 µM (B16), and 1.50, 0.59 µM (HepG2),
respectively. These two compounds showed approximately 2-20-fold more potent than carboplatin
and 5-Fu in this biological assay.
3.2. Selective killing effect of representative compounds 20b in cancer cells.
In an effort to obtain antitumor drugs with efficacy and safety, representative compound 20b was
further assessed versus other cancer cells and normal cells L02. As shown in Fig. 2A, the compound
treatment markedly induced cell death in all cancer cells with concentration-dependent trend. And
this compound showed antitumor activity against all cancer cells with IC50 about 1 µM, shifting the
selectivity ratio 4.3-fold to 27-fold (the fold is the ratio of the IC50 of normal cell L02 to the IC50 of
cancer cells). Among these cancer cell lines, HepG2 was the most sensitive cancer cell line for 20b,
with the selectivity ratio 27-fold. When the normal cells L02 were incubated with this compound for
48 h, there were little apparent reduction in cell viability under the concentration of 15 µM.
However, the inhibitory curve was jumped at the concentration of 30 µM, indicated that this
compound would be toxic at high concentrations towards normal cells. The results represented that
compound 20b could show an exposure time window but with a relatively narrow concentration
range in vitro. However, compared with the control Wogonin, this compound had a
cancer-cell-selective killing property, which showed a more potent profile toward cancer cells to that
of control.
Table 1
IC50 values for Wogonin derivatives against A549, MCF-7, B16 and HepG2 cancer cell lines.
6

ACCEPTED MANUSCRIPT
A549
MCF-7
B16
IC50 (µM)
4.10
5.43
2.09
4.99
2.89
9.30
4.32
4.33
3.44
4.52
3.69
4.37
5.42
4.90
6.89
7.98
10.0
3.76
3.22
4.88
3.76
3.21
5.41
2.37
3.29
3.70
3.53
2.12
2.08
2.78
26.4
25.1
1.19
HepG2
Mean
Entry
No.
12a
12b
12c
12d
12e
12f
12g
12h
12i
1
2
3
4
5
6
7
8
9
2.32
3.81
1.43
2.89
3.21
6.79
2.22
2.13
2.98
2.09
6.40
4.41
2.91
3.62
3.15
4.72
8.91
3.94
1.09
3.91
4.35
1.10
6.60
2.91
3.20
2.14
3.60
2.10
3.87
1.06
20.1
11.1
8.9
3.98
4.02
3.21
2.98
4.09
5.43
3.86
3.21
3.09
2.89
6.07
4.43
3.21
3.21
4.23
2.23
8.72
1.02
3.21
1.92
1.09
1.89
9.90
2.93
2.22
2.42
2.77
3.18
4.98
4.85
28.0
12.2
14.7
4.20
1.00
3.75
2.40
2.62
2.33
2.81
2.32
2.13
2.58
3.17
2.89
4.95
3.01
3.55
2.66
2.43
3.11
2.09
1.65
3.14
4.66
3.29
1.50
5.09
0.59
4.11
4.56
4.11
5.44
19.5
25.7
10.4
3.65
3.57
2.62
3.32
3.20
5.96
3.30
3.00
2.91
3.02
4.83
4.02
4.12
3.69
4.46
4.40
7.52
2.96
2.40
3.09
3.09
2.72
6.30
2.42
3.45
2.21
3.50
2.99
3.76
3.53
23.5
18.5
8.80
10
12j
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
20a
20b
20c
20d
20e
20f
1
carboplatin
5-Fu
Fig. 2. (A) Cell death of cancer cells (LNCap, MCF-7, Hela, HepG-2, A549 and B16) and normal hepatic cells
(L02) treated with compound 20b. (B) Cell viability with different concentrations (1, 5, 10 µM) of compound 20b
or 20b with NAC. The selectivity ratio of cell viability between 20b treated with NAC and 20b were calculated as
7

ACCEPTED MANUSCRIPT
2.7-fold (1 µM), 5.5-fold (5 µM) and 7.0-fold (10 µM).
3.3. Induction of ROS accumulation in HepG2 cells by 18n and 20b.
Several Wogonin derivatives have been reported to exert their antitumor activity by accumulating
ROS in cancer cells [17], we next determined the effect of the representative compounds 18n and
20b on cellular ROS level in HepG2 cancer cells by flow cytometry. To demonstrate the effects of
18n and 20b on ROS induction, we determined the production of ROS with a probe
2’,7’-dichlorfluorescein diacetate (DCFH-DA), which is converted to a green fluorescent product via
oxidation. After being uptaken by cells, DCFH-DA is hydrolyzed by cellular esterases to
dichlorodihydrofluorescein (DCFH), which is trapped within the cell. The DCFH is then oxidized to
fluorescent dichlorofluorescent by action of cellular ROS. As shown in Fig.3, a rapid production of
ROS occurred after the exposure of HepG2 cells to compounds 18n and 20b. After treating with
these two compounds for 24 h, a marked increase in ROS levels was caused with increasing
concentration of each compound (2 µM, 4 µM or 6 µM). The results indicated that these two
compounds induced ROS production in dose-dependent manners in cancer cells. In addition, we
examined whether ROS made effect on the cancer cell viability. HepG2 was incubated with these
two compounds or compounds with 5 mM N-acetylcysteine (NAC) for 48 h, respectively. As
expected, the NAC treated group got higher cell viability (the selectivity ratio between the NAC
treated group and without NAC treated group was shown in Fig. 2), which indicated that these
compounds 20b might enhance the intracellular ROS level and induce the cell apoptosis (Fig. 2B).
8

ACCEPTED MANUSCRIPT
Fig. 3. Effects of 18n (A,B) and 20b (C,D) on the ROS of of HepG2 cells. The level of intracellular reactive
oxygen species (ROS) in HepG2 cells was measured by flow cytometry/ Data were shown as mean ± SEM (n = 3).
3.4. Apoptosis induced by compounds 18n and 20b in HepG2 cells.
It has been previously reported that Wogonin and its several derivatives exerted antitumor
activity of by inducing a mitochondrial-mediated apoptosis [18]. The representative compounds 18n
and 20b were employed to investigate the apoptosis effect. The apoptotic cell counts were measured
using AnnexinV-FITC/PI by flow cytometry. HepG2 cells were treated with 2 µM, 4 µM and 6 µM
of compounds 18n and 20b for 24 h. The DMSO (0.1%) was used as vehicle control. As shown in
Fig.4, exposure to 6 µM of compounds 18n and 20b resulted in 20.7% and 30.7% of cell apoptosis
respectively, compared with 3.4% and 2.6% of DMSO-treated group, while lower doses treatment
showed weaker effect. These data indicated that this series of compounds could induce HepG2
apoptosis in a dose-dependent manner.
Fig. 4. (A) Apoptosis effect of compounds 18n (2, 4, 6 µM) and 20b (2, 4, 6 µM) in HepG2 cells by Annexin
V-FITC/PI assay for 24 h. (B) Quantification of apoptosis rate of compound 18n and 20b.
3.5. Ligand similarity search for exploring the potential target of 20b.
9

ACCEPTED MANUSCRIPT
According to the preliminary in vitro biological results, compounds 18n and 20b could be
promising anticancer candidates. It has been shown that these two compounds induced apoptosis
through increasing cellular ROS level. However, to further design compounds with better drug-like
properties and pharmacological activity based on the scaffold of them, identifying the specific
protein which could be a potential target for this series of compounds is especially urgent to be study.
In silico target fishing is an emerging field that aims at predicting biological targets of molecules
based on their chemical structure [19]. These in silico method complement much more expensive
biological experimental approaches to drug design and have been integrated into virtually all modern
drug-discovery programs. Among the in silico methods, similarity searching is an efficient method to
discover the target. The fundamental idea underlying ligand-based approaches is that two similar
ligands are possible to possess similar target-binding profiles. Target information on a unique
collection of 267 anticancer compounds was extracted from the Cambridge cancer compound library
in Selleck Co. For 27 molecules, no target information could be obtained which were excluded from
further analysis. The remaining 240 molecules were assigned to 220 targets that were reviewed
manually. Fig. 5 showed the distribution of the compounds for these 220 targets. Remarkably, to 203
targets only one drug is assigned in the database, indicating the target richness of these compounds.
Fig. 5. (A) The number of drugs belong to different to number of targets. Most of these compounds belong to the
specific target. (B) The ROCs model developed based on the scaffold of 20b.
Table 2
The screened compounds and potential targets based on the method of similarity search.
ShapeTanimo
No.
Compound
Structure
Target
to Score
LDE225
(NVP-LDE225,
Erismodegib)
0.58
Smoothened
1
10

ACCEPTED MANUSCRIPT
BMS 794833
0.57
0.55
c-Met
EGFR
2
3
Gefitinib
(Iressa)
BMS 777607
NSC 23766
0.55
0.54
0.52
c-Met
Rac
4
5
6
PCI-32765
(Ibrutinib)
Src
JNJ-38877605
LY294002
0.49
c-Met
7
0.46
0.45
PI3K
8
9
Amuvatinib
(MP-470)
c-Met
Flavopiridol
(Alvocidib)
0.45
CDK
10
Based on the representative scaffold of 20b, a shape query using ROCs was developed to screen the
database. Compounds with top ten ShapeTanimoto score were selected, and these compounds were
assigned to 7 targets. All of the selected compounds were localized in the similarity range of
0.45-0.58 (Table 2), which means that the selected compounds had certain diversity with the
reference ligands. Notably, among the top ten compounds, four of them targeting c-Met which may
be a potential target for these series of compounds. Then, we decided to determine the biological
functions of the representative compound 20b targeting the screened enzyme. Contrary to
expectations, the in vitro enzymatic assays showed that the compound 20b was not a good inhibitor
of c-Met with IC50 at 35 µM. However, it was a potential selective inhibitor of CDKs with IC50 at
0.87 µM against CDK9, while IC50 at 6.7 µM against CDK2. And the compound did not show
inhibiting activity against other screened enzymes. We also docked the compound 20b into the
crystal structure of CDK9 (PDB ID: 3BLR), it binds to CDK9 at the ATP-binding pocket in a similar
manner to the binding of the native ligand flavopiridol. There are hydrogen bonds form the 20b O4
oxygen and O5 hydroxyl to the residues Cys106 and Phe105. The scaffold also formed the H…π
stacking interactions with Asp167 and Phe103. The long side chain at 8-position towards the solvent
11

ACCEPTED MANUSCRIPT
area. The docking study further proved that this series of compounds were potential CDK9 inhibitors.
Thus, according to the results, our group further designed another series of compounds which
targeting CDK9 base on compound 20b through the structure-based drug design. And several
compounds selectively inhibited CDK9 with IC50 at nanomolar concentration were discovered and
the detailed results would be reported in the near future. In addition, the relationship between the
inhibitory of CDK9 enzyme and the enhancement of the intracellular ROS level are also studying in
our lab.
Fig. 6. (A) Docked conformation of compound 20b into active site of CDK9. The interaction mode was obtained
through molecular docking (PDB ID: 3BLR) and depicted using MOE 2016.09. The carbon atoms of the
compounds and the key residues in the active site of CDK9 were colored in yellow and gray, respectively. The
H-bonds were shown as dot lines. (B) The 2D ligand interactions of 20b with key residues in the active site of
CDK9.
12

ACCEPTED MANUSCRIPT
4. Conclusions
In summary, inspired by the flavonoid antitumor scaffold, a series of derivatives were
synthesized and screened for antitumor activity. Among them, the potent thirty novel flavonoid
derivatives were selected and reported in this paper. Most of the selected compounds possessed good
in vitro antitumor activity with a broad spectrum. Within this series of compounds, 18n and 20b
exhibited the highest antitumor activity against A549, MCF-7, B16 and HepG2 with IC50 values of
2.91, 2.14 µM (A549), 2.93, 2.42 µM (MCF-7), 2.37, 3.70 µM (B16), and 1.50, 0.59 µM (HepG2),
respectively. Further biological assays showed that HepG2 was the most sensitive cancer cell line for
20b. Compounds 18n and 20b could significantly enhance the intracellular ROS level and induce the
cell apoptosis at 6 µM. Further through similarity searching, CDK9 was identified as the potential
target for 20b, which could be a start point for next structure-based drug design. Taken together, the
findings disclosed herein endorse the therapeutic potential of these novel Wogonin derivatives and
deserve further studies to explore their potential utility as antitumor agents.
5. Experimental Section
5.1. Chemistry
5.1.1. General Experimental Methods
Reactions were monitored by thin-layer chromatography on silica gel plates (60F-254) visualized
under UV light. Melting points were determined on a Mel-TEMP II melting point apparatus without
1
correction. HNMR and 13CNMR spectra were recorded in CDCl3 on a Bruker Avance 300 MHz
spectrometer at 300 MHz and 75 MHz, respectively. Chemical shifts (δ) are reported in parts per
million (ppm) from tetramethylsilane (TMS) using the residual solvent resonance (CDCl3: 7.26 ppm
1
for H NMR, 77.16 ppm for 13C NMR. Multiplicities are abbreviated as follows: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet). IR spectra were recorded on a Nicolet iS10 Avatar
FT-IR spectrometer using KBr film. MS spectra were recorded on a LC/MSD TOF HR- MS
Spectrum. Flash column chromatography was performed with 100-200 mesh silica gel and yields
refer to chromatographically and spectroscopically pure compounds.
All chemicals purchased from commercial suppliers were used as received unless otherwise
stated. Reactions and chromatography fractions were monitored by Merck silica gel 60 F-254 glass
TLC plates.
13

ACCEPTED MANUSCRIPT
5.1.2. Synthesis
1,2,3-Tribenzyloxybene (3). To a solution of gallic acid (100 g, 0.8 mol) in acetone (1000 mL),
were added KI (26.7 g, 0.16 mol), K2CO3 (220 g, 0.16 mol) and benzyl bromide (265 mL, 2.4 mol)
successively. The reaction mixture was heated to reflux. After the end of the reaction (monitored by
TLC (PE : EA = 10 : 1)), the mixture was filtered and the cake was washed with dichloromethane
(100 mL × 4). The filtrate was evaporated under vacuum to afford the crude product, which was
purified through recrystallization with ethanol (1000 mL) to give 3 as a white solid (275 g, 87.5%).
o
m.p. 68-70 C. 1H-NMR (300 MHz, CDCl3):
δ 7.30-7.27 (m, 15H, ArH), 6.92 (t, 1H, J = 8.4 Hz,
ArH), 6.64 (d, 2H, J = 8.4 Hz, ArH), 5.1 (s, 4H, -OCH2Ph), 5.07 (s, 2H, -OCH2Ph) ppm. EI-MS
(m/z): 91, 350, 396 [M]+.
2,6-Dibenzyloxy-p-benzoqunine (5). 3 (60 g, 0.126 mol) was dissolved in acetic acid (100 mL),
o
and the mixture was stirred at 40 C for 30 min. Then, 65% HNO3 (3 mL) was added dropwise and
the mixture was stirred for an 4 h. After that, the mixture was filtered, and 10 mL HNO3 was added
to the filtrate to stir for another 4 h. The mixture was filtered, and crude 4 (40 g) was obtained as the
cake without further purification. Then, to a solution of 4 (30 g) in EA (260 mL) was added excess
sodium hydrosulphite portionwise and the mixture was stirred for 40 minutes. Then the mixture was
filtered and the organic layer was separated, washed with dilute hydrochloric acid (100 mL × 2) and
saturated sodium chloride solution (100 mL × 2), successively and dried over Na2SO4. The mixture
was filtered, concentrated under reduced pressure to afford the crude 5, which was recrystallized
o
1
with toluene to give 5 as a pale-white solid (14 g, 34%). m.p. 115-117 C. H-NMR (300 MHz,
CDCl3): 7.43-7.26 (m, 10H, Ar-H), 6.15 (s, 2H, ArH), 5.16 (s, 1H, ArOH), 5.10 (s, 4H, -OCH2Ph),
4.36 (s, 1H, ArOH) ppm. MS-EI: 323 (M+H)+, 322 (M)+.
δ
1,4-dimethoxy-2,6-dibenzyloxybenzene
(6).
To
a
stirred
solution
of
2,6-dibenzyloxy-p-benzoqunine (5) (27g, 0.084 mol) in ethanol (214 mL) was added dimethyl
sulfate (20 mL) and then 28% NaOH was added dropwise to adjust pH to 8-9. The reaction mixture
was stirred for 2 h at room temperature. After cooling, the suspension was filtered. The precipitate
1
was washed with water to neutral, and dried to obtain 6 as a white solid (25.1 g, 85.5%). H-NMR
(300 MHz, CDCl3):
δ 7.46-7.26 (m, 10H, ArH), 6.19 (s, 2H, ArH), 5.12 (s, 4H, -OCH2Ph), 3.84 (s,
3H, -OCH3), 3.68 (s, 3H, -OCH3) ppm.
14

ACCEPTED MANUSCRIPT
2,6-dimethoxy-benzene-1,4-diol (7). A mixture of 6 (10 g, 0.029 mol) and Pd/C (10%, 0.43 g)
in MeOH (70 mL) was pumped with H2 for 8 h. After the end of the reaction, the solution was
filtered through a pad Celite. The filtrate was concentrated to afford 7 as a yellow oil (4.86 g, 99%).
Formula: C8H10O4 MS(EI) m/z:127,155,170[M]+.
(E)-1-(2,4-dihydroxy-3,6-dimethoxyphenyl)-3-(4-methoxyph enyl)prop-2-en-1-one (8a). To
a stirred solution of p-methoxyl ethyl cinnamyl chloride (3.89 g, 0.02 mol) and (7) (3.37 g, 0.02 mol)
in dry diethyl ether (10 mL) was added BF3•Et2O (5.1 mL) and the reaction mixture was stirred for
1.5 h at 40 oC. After cooling, the mixture was filtered and the precipitate was washed with methanol
1
(15 mL × 3). Then 8a was obtained as a red solid (3.2 g, 51%). H-NMR (300MHz, DMSO-d6):
δ
8.33 (d, 1H, J = 15.3 Hz, -CH=CH-CO), 7.91 (d, 1H, J = 15.3 Hz, -CH=CH-CO), 7.65 (d, 2H, J =
8.7 Hz, Ar-H), 6.98 (d, 2H, J = 8.7 Hz, Ar-H), 6.10 (s, 1H, Ar-H), 4.00 (s, 3H, -OCH3), 3.98 (s, 3H,
-OCH3), 3.89 (s, 3H, -OCH3) ppm. IR (KBr): 3420 (OH), 2943(CH3), 1597(C=O), 1511, 1383, 1242,
1176, 1131, 1018 cm-1. MS-EI (m/z) (m/z):331[M+1] +.
7-Hydroxy-5,8-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (9a). To a stirred
solution of Compound 8 (6.45 g, 0.0195 mol) in dry DMSO (18 mL) was added catalytic amount of
o
I2, and the reaction mixture was heated to 140 C for 5 h. Then the mixture was cooled to room
temperature, filtered and the precipitate was washed with methanol (8 mL × 3). Then 9a was
1
obtained as a yellow solid (2.75 g, 35%). m.p. 242-245 oC; H-NMR (300 MHz, DMSO-d6):
δ
7.87
(d, 2H, J = 8.7 Hz, Ar-H), 7.04 (d, 2H, J = 8.7 Hz, Ar-H), 6.67 (s, 1H, Ar-H), 6.53 (s, 1H, -C=CH),
4.04 (s, 3H, -OCH3), 3.94 (s, 3H, -OCH3 ), 3.89 (s, 3H, -OCH3 ) ppm. IR (KBr): 3441(OH),
3111(C-H, Aromatic), 1647(C=O), 1595(C=C), 1576, 1356, 1253, 1114, 1044, 746 cm-1; MS-EI
(m/z):285, 313, 328[M]+.
7-(4-Bromobutoxy)-5,8-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
(10a).
A
mixture of 9a (143 mg, 0.44 mmol), K2CO3 (361 mg, 2.61 mmol) and 1,4-dibromobutane (0.21 mL,
1.74 mmol) in acetone (71 mL) was refluxed for 8 h. Then the reaction mixture was cooled to room
temperature and filtered. The filtrate was concentrated under reduced pressure, and the residual
obtained was slurried in PE (20 mL) to obtain 10a as a white solid (140 mg, 69.7%). m.p.124-126
1
oC; H-NMR (300 MHz, CDCl3):
δ
7.88 (d, 2H, J = 8.7 Hz, Ar-H), 7.02 (d, 2H, J = 8.7 Hz, Ar-H),
15

ACCEPTED MANUSCRIPT
6.60 (s, 1H, Ar-H), 6.42 (s, 1H, -C=CH), 4.20 (t, 2H, J = 5.8 Hz, BrCH2-), 3.97 (s, 3H, -OCH3 ), 3.95
(s, 3H, -OCH3 ), 3.89 (s, 3H, -OCH3 ), 3.55 (t, 2H, J = 6.1 Hz, -CH2O-), 2.11 (m, 4H,
-CH2CH2CH2CH2-) ppm. IR (KBr): 3441(OH), 2948(CH3), 2843(CH2), 1649(C=O), 1599(C=C),
1510, 1341, 1258, 1185, 1114, 1046, 831 cm-1; MS-EI (m/z): 463[M-1]+, 465[M+1]+;
7-(4-Bromobutoxy)-5-hydroxy-8-methoxy-2-(4-methoxphen yl )-4H-chromen-4-one (11a).
To a solution of 10a (107 mg, 0.23 mmol) in dry acetonitrile (53 mL) was added AlCl3 (245 g, 1.84
mmol). The rection mixture was refluxed for 6 h. After cooling, duilted HCl (5.7 mL, 4 mol/L) was
added and the mixture was extracted with DCM (20 mL × 3). The organic layer was washed with
brine, dried over MgSO4, and concentrated to afford the crude 11a as a yellow solid (100 mg,
o
1
97.1%). m.p.185-187 C; H-NMR (300 MHz, CDCl3):
δ 7.92 (d, 2H, J = 8.9 Hz, Ar-H), 7.04 (d,
2H, J = 8.9 Hz, Ar-H), 6.58 (s, 1H, Ar-H), 6.39 (s, 1H, -C=CH), 4.13 (t, 2H, J = 5.9 Hz, BrCH2-),
3.93(s,3H, -OCH3 ), 3.90 (s, 3H, -OCH3 ), 3.53 (t, 2H, J = 6.1 Hz, -CH2O-), 2.10 (m, 4H,
-CH2CH2CH2CH2-) ppm. MS-EI (m/z): 449[M]+;
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(4-methoxyphenyl)-5-hydroxy-8-methoxy-4H-chro
men-4-one (12a). To a stirred solution of 11a (120 mg, 0.26 mmol) in anhydrous acetonitrile (60
mL) was added N-methylpiperazine (0.15 mL, 0.13 mmol), and the reaction mixture was heated to
reflux. After the end of the reaction (monitored by TLC (PE: EA: Triethylamine = 1: 1: 0.01)), the
mixture was concentrated under reduced pressure, and purified by chromatography to give 12a as a
o
1
yellow solid (80 mg, 64 %). m.p. 145 - 147 C. H-NMR (300 MHz, CDCl3):
δ 12.63 (s, 1H,
5-Ar-OH), 7.90 (d, 2H, J = 8.8 Hz, Ar-H), 7.04 (d, 2H, J = 8.8 Hz, Ar-H), 6.58 (s, 1H, Ar-H), 6.44
(s, 1H, -C = CH), 4.13 (t, 2H, J = 6.4, -O-CH2-), 3.92 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 2.80 (m,
10H, -N((CH2)2)2)N-CH2-), 2.53 (s, 3H, -NCH3), 1.92 (m, 2H, -OCH2-CH2-CH2-), 1.78 (t, 2H,
-OCH2CH2CH2CH2N-) ppm. IR (KBr): 3427(OH), 2938(CH2), 2784, 2678, 2595, 1656(C=O),
1603(C=C), 1507, 1426, 1377, 1263, 1182, 1120, 1029, 835 cm-1. ESI-HRMS m/z calculated for
C26H33N2O6 [M + H]+ 469.2333, found 469.2330.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(4-methylphenyl)-5-hydroxy-8-methoxy-4H-chrom
en-4-one (12b). The mixture of 11b (100 mg, 0.23 mol), anhydrous acetonitrile (50 mL) and
N-methylpiperazine (100 mg, 0.23 mmol) was heated to reflux under stirring. After the end of the
16

ACCEPTED MANUSCRIPT
reaction (monitored by TLC (PE: EA: Triethylamine = 1: 1: 0.01)), the mixture was concentrated
under reduced pressure, and purified by chromatography to give 12b as a yellow solid (50 mg, 48
o
1
%). m.p.145-147 C. H-NMR (300 MHz, CDCl3):
δ 12.58 (s, 1H, 5-Ar-OH), 7.84 (d, 2H, J = 8.1
Hz, Ar-H), 7.33 (d, 2H, J = 8.1 Hz, Ar-H), 6.63 (s, 1H, Ar-H), 6.42 (s, 1H, -C=CH), 4.11 (t, 2H, J =
6.2 Hz, -O-CH2-), 3.92 (s, 3H, -OCH3), 2.41-2.44 (m, 13H, N(CH2CH2)2 and ArCH3), 2.30 (s, 3H,
NCH3), 1.90 (m, 2H, NCH2CH2CH2CH2O), 1.71 (m, 2H, NCH2CH2CH2CH2O) ppm. IR (KBr): 3410
(OH), 2932(CH2), 2789, 1661(C=O), 1608(C=C), 1586, 1436, 1337, 1277, 1191, 1037, 820 cm-1.
ESI-HRMS m/z calculated for C26H33N2O5 [M + H]+ 453.2384, found 453.2387.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(4-chlorophenyl)-5-hydroxy-8-methoxy-4H-chrome
n-4-one (12c). The mixture of 11c (102 mg, 0.22 mmol), anhydrous acetonitrile (50 mL) and
N-methylpiperazine (0.12 mL, 1.1 mmol) was heated to reflux under stirring. After the end of the
reaction (Monitored by TLC (PE: EA: Triethylamine = 1: 1: 0.01)), the mixture was concentrated
under reduced pressure, and purified by chromatography to give 12c as a yellow solid (60 mg, 56.6
o
1
%). m.p. 195-198 C. H-NMR (300 MHz, CDCl3):
δ 12.49 (s, 1H, ArOH),7.88 (d, 2H, J = 8.7 Hz,
Ar-H), 7.52 (d, 2H, J = 8.7 Hz, Ar-H), 6.64 (s, 1H, Ar-H), 6.46 (s, 1H, -C=CH), 4.14 (t, 2H, J = 5.7
Hz, BrCH2-), 3.92 (s, 3H, -OCH3), 2.85 (m, 8H, N(CH2CH2)2N), 2.61 (m, 2H,
-OCH2CH2CH2CH2N-), 1.92 (m, 2H, -OCH2CH2CH2CH2N-), 1.79 (m, 2H, -OCH2CH2CH2CH2N-)
ppm. IR (KBr): 3441 (OH), 2938 (CH2), 2677, 1658 (C=O), 1612 (C=C), 1586, 1416, 1376, 1337,
1276, 1033, 830 cm-1. ESI-HRMS m/z calculated for C25H30ClN2O5 [M + H]+ 473.1838, found
473.1837.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(4-(trifluoromethyl)phenyl)
-5-hydroxy-8-methoxy-4H-chromen-4-one (12d). The mixture of 11d (118 mg, 0.24 mmol),
anhydrous acetonitrile (59 mL) and N-methylpiperazine (0.13 mL, 1.2 mmol) was heated to reflux
under stirring. After the end of the reaction (monitored by TLC (PE: EA: Triethylamine = 1: 1:
0.01)), the mixture was concentrated under reduced pressure, and purified by chromatography to give
12d as a yellow solid (100 mg, 82.6 %). m.p. 81-83 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.47 (s, 1H,
5-Ar-OH), 8.05 (d, 2H, J = 8.1 Hz, Ar-H), 7.80 (d, 2H, J = 8.1 Hz, Ar-H), 6.78 (s, 1H, Ar-H), 6.45
(s, 1H, -C=CH), 4.13 (t, 2H, J = 6.8 Hz, -O-CH2-), 3.99 (s, 3H, -OCH3), 2.53 (m, 8H,
N(CH2CH2)2N), 2.38 (s, 3H, NCH3), 1.90 (m, 2H, NCH2CH2CH2CH2O), 1.71 (m, 4H,
17

ACCEPTED MANUSCRIPT
NCH2CH2CH2CH2O) ppm. IR (KBr): 3442 (OH), 2935 (CH2), 2790, 1661 (C=O), 1593 (C=C),
1422, 1289, 1170, 1119, 1032, 839, 806 cm-1. ESI-HRMS m/z calculated for C26H30F3N2O5 [M + H]+
507.2101, found 507.2097.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(4-nitrophenyl)-5-hydroxy-8-methoxy-4H-chromen
-4-one (12e). The mixture of 11e (130 mg, 0.28 mmol), anhydrous acetonitrile (65 mL) and
N-methylpiperazine (0.15 mL, 1.4 mmol) was heated to reflux under stirring. After the end of the
reaction (monitored by TLC (PE: EA: Triethylamine = 1: 1: 0.01)), the mixture was concentrated
under reduced pressure, and purified by chromatography to give 12e as a yellow solid (70 mg, 51.8
%). m.p. 80–84 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.33 (s, 1H, 5-Ar-OH), 8.41 (d, 2H, J = 8.4 Hz,
Ar-H), 8.12 (d, 2H, J = 8.4 Hz, Ar-H), 6.76 (s, 1H, Ar-H), 6.46 (s, 1H, -C=CH), 4.13 (t, 2H, J = 6.4
Hz, -O-CH2-), 3.93 (s, 3H, -OCH3), 2.50 (m, 10H, N(CH2CH2)2NCH2-), 2.34 (s, 3H, NCH3), 1.92
(m, 2H, NCH2CH2CH2CH2O), 1.75 (m, 2H, NCH2CH2CH2CH2O) ppm. IR (KBr): 3426 (OH), 2934
(CH2), 2795, 1655 (C=O), 1604 (C=C), 1523 (N=O), 1345 (N=O), 1114, 1030, 848, 689 cm-1.
ESI-HRMS m/z calculated for C25H30N3O7 [M + H]+ 484.2078, found 484.2076.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(2-methoxyphenyl)-5-hydroxy-8-methoxy-4H-chro
men-4-on (12f). The mixture of 11f (70 mg, 0.15 mmol), anhydrous acetonitrile (35 mL) and
N-methylpiperazine (0.08 mL, 0.75 mmol) was heated to reflux under stirring. After the end of the
reaction (monitored by TLC (PE: EA: Triethylamine = 1: 1: 0.01)) , the mixture was concentrated
under reduced pressure, and purified by chromatography to give 12f as a yellow solid (45 mg, 61.7
%). m.p. 82-83 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.65 (s, 1H, Ar-H), 7.98 (dd, 1H, J1 = 1.5 Hz; J2
= 7.9 Hz, Ar-H), 7.50 (td, J1 = 1.5 GHz; J2 = 8.0 Hz, 1H, Ar-H), 7.13 (t, 1H, J = 7.5 Hz, Ar-H), 7.07
(m, 2H, Ar-H), 6.41 (s, 1H, -C=CH), 4.11 (t, 2H, J = 6.4, -O-CH2-), 3.95 (s, 3H, -OCH3 ), 3.90 (s,
3H, -OCH3), 2.44 (m, 10H, -N((CH2)2)2)N-CH2-), 2.29 (s, 3H, -NCH3), 1.90 (m, 2H,
-OCH2-CH2-CH2-), 1.71 (m, 2H, -OCH2-CH2-CH2-CH2N-) ppm. IR(KBr): 3434 (OH), 2932 (CH2),
2794, 2755, 1657 (C=O), 1609 (C=C), 1583, 1507, 1400, 1376, 1256, 1165, 1131, 1018, 856, 821,
753 cm-1. ESI-HRMS m/z calculated for C26H33N2O6 [M + H]+ 469.2333, found 469.2334.
7-(4-(4-Methylpiperazin-1-yl)butoxy)-2-(2-fluorophenyl)-5-hydroxy-8-methoxy-4H-chrome
n-4-on (12g).
18

ACCEPTED MANUSCRIPT
The mixture of 11g (1.1 g, 2.5 mmol), anhydrous acetonitrile (100 mL) and N-methylpiperazine
(1.3 g, 12.6 mmol) was heated to reflux under stirring. After the end of the reaction (monitored by
TLC(PE: EA = 2 : 1)), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure, and purified by chromatography to give 12g as a yellow solid (680 mg, 61.8%).
o
m.p. 195-197 C. 1H-NMR (300 MHz, DMSO-d6):
δ 12.62 (s, 1H, ArOH), 8.15 (d, 2H, J = 8.7 Hz,
Ar-H), 7.44 (d, 2H, J = 8.7 Hz, Ar-H), 7.04 (s, 1H, Ar-H), 6.61 (s, 1H, -C=CH), 4.18-4.15
(m,2H,-CH2-O-Ar), 3.85 (s, 3H, -OCH3), 3.36 (s, 2H, -OCH2CH2CH2CH2N-), 2.35-2.06 (m, 8H,
-N(CH2CH2)2N-), 2.19-2.13 (m, 3H, -NCH3), 1.91-1.80 (m, 2H, -OCH2CH2CH2CH2N-), 1.61-1.56
(m, 2H, -OCH2CH2CH2CH2N-) ppm. 13C-NMR (75 MHz, DMSO-d6):
δ 182.2 (-CO-), 165.9
(-O-CH=), 162.6, 158.0, 156.6, 148.8, 129.1, 129.0, 128.6, 127.4, 127.3, 120.3, 104.9, 103.9
(R-O-C6H-OH), 96.7 (=CH-CO-), 68.8 (-O-CH2), 67.2 (-O-CH3), 61.1, 59.3, 56.8, 54.1
(-N(CH2CH2)2N-), 51.9, 45.0 (-N-CH3), 26.3, 22.4 (-O-C4H8-N) ppm. IR(KBr): 3448 (OH), 3128
(C-H, aromatic), 2941 (CH2), 2341, 1661 (C=O), 1637(C=C), 1618(C=C), 1598 (C=C), 1508, 1421,
1400, 1336, 1285, 1235, 1115, 1035, 836, 811, 595 cm-1. ESI-HRMS m/z calculated for
C25H30FN2O5 [M + H]+ 457.2133, found 469.2135.
7-[4-(4-Methylpiperazin-1-yl)butoxy]-2-(4-bromophenyl)-5-hydroxy-8-methoxy-4H-chrom
en-4-one (12h). To a stirred solution of 11h (1.97 g, 4 mmol) in anhydrous acetonitrile (100 mL)
was added N–methylpiperazine (1.90 g, 20 mmol) with nitrogen passing through the mixture. Then
the reaction mixture was heated to reflux. After the end of the reaction (monitored by TLC (PE: EA:
= 2: 1)), the mixture was filtered immediately, and the filtrate was concentrated under reduced
pressure, and purified by chromatography to give 12h as a yellow solid (1.18 g, 59.0 %).
o
1
m.p.191-192 C. H NMR (300 MHz, DMSO-d6):
δ 12.57 (s, 1H, ArOH), 8.01 (d, 2H, J = 8.6 Hz,
Ar-H), 7.83 (d, 2H, J = 8.5 Hz, Ar-H), 7.07 (s, 1H, Ar-H), 6.61 (s, 1H, -C=CH), 4.16 (t, 2H, J = 6.3
Hz, -CH2-O-Ar), 3.85 (s, 3H, -OCH3), 2.38 (m, 10H, -N(CH2CH2)2N-, -OCH2CH2CH2CH2N-), 2.20
(s, 3H, -N-CH3), 1.79 (t, 2H, J = 6.6 Hz, -OCH2CH2CH2CH2N-), 1.60 (t, 2H, J = 6.8 Hz,
-OCH2CH2CH2CH2N-) ppm. 13C-NMR (75 MHz, DMSO-d6):
δ 182.6 (-CO-), 162.7 (-O-CH=),
158.5, 157.1, 149.3, 132.8, 130.5, 128.7, 126.4(-C6H4-Br), 105.8, 104.5 (R-O-C6H-OH), 97.3
(=CH-CO-), 69.3(-O-CH2), 61.6 (-O-CH3), 57.5 (-N(CH2CH2)2N-), 54.9, 52.7, 45.8 (-N-CH3), 26.8,
22.9 (-O-C4H8-N) ppm. IR (KBr): 3448 (OH), 3128 (C-H, aromatic), 2933 (CH2), 2361, 1660
19

ACCEPTED MANUSCRIPT
(C=O), 1638 (C=C), 1617 (C=C), 1583 (C=C), 1534, 1421, 1400, 1376, 1274, 1224, 1167, 1033,
1010, 825, 810, 583 cm-1. ESI-HRMS m/z calculated for C25H30BrN2O5 [M + H]+ 517.1333, found
524.1123.
7-[4-(4-Methylpiperazin-1-yl)butoxy]-2-(4-fluoro-2-bromophenyl)-5-hydroxy-8-methoxy-4
H-chromen-4-one (12i). To a stirred solution of 11i (1.15 g, 11.1 mol) in anhydrous acetonitrile (60
mL) was added N–methylpiperazine (1.07 g, 55.5mmol) with nitrogen passing through the mixture.
Then the reaction mixture was heated to reflux. After the end of the reaction (Monitored by TLC
(PE: EA: = 2: 1)), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure, and purified by chromatography to give 12i as a yellow solid (0.73 g, 61.3 %).
m.p.173–176 oC. 1H-NMR (300 MHz, DMSO-d6):
δ 7.86 (s, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 6.66 (s,
1H, Ar-H), 6.58 (s, 1H, -C=CH), 4.17 (s, 2H, -CH2-O-Ar), 3.76 (s, 3H, -OCH3), 2.41 (s, 3H,
-N-CH3), 2.29 (s, 10H, -OCH2CH2CH2CH2N-, -N(CH2CH2)2N-), 1.79 (t, 2H, J = 6.6 Hz,
13
-OCH2CH2CH2CH2N-), 1.60 (t, 2H, J = 6.8 Hz, -OCH2CH2CH2CH2N-) ppm. C-NMR (75 MHz,
DMSO-d6):
δ 181.9 (-CO-), 163.6 (-O-CH=), 158.2, 156.7, 149.2, 133.5, 129.9, 128.6, 122.2, 121.0,
115.7 (-C6H4-Br), 110.5, 103.9, 96.9 (=CH-CO-), 68.8 (-O-CH2), 61.2 (-O-CH3), 56.7, 54.0
(-N(CH2CH2)2N-), 51.3, 45.9 (-N-CH3), 26.2, 22.3 (-O-C4H8-N) ppm. IR (KBr): 3448 (OH), 3134
(C-H, aromatic), 2724, 2361, 1655 (C=O), 1617 (C=C), 1460, 1400, 1371, 1278, 1224, 1179, 1111,
1002, 853, 553 cm-1. ESI-HRMS m/z calculated for C25H29BrFN2O5 [M + H]+ 535.1238, found
535.1246.
7-[4-(4-Methylpiperazin-1-yl)butoxy]-2-(3-fluorophenyl)-5-hydroxy-8-methoxy-4H-chrome
n-4-one (12j). To a stirred solution of 11j (0.93 g, 0.0025 mol) in anhydrous acetonitrile (50 mL)
was added N–methylpiperazine (1.07 g, 0.0106 mol) with nitrogen passing through the mixture.
Then the reaction mixture was heated to reflux. After the end of the reaction (monitored by TLC (PE:
EA: = 2: 1)), the mixture was filtered immediately, and the filtrate was concentrated under reduced
pressure, and purified by chromatography to give 12j as a yellow solid (0.48 g, 49.5%). m.p.194–195
oC. 1H-NMR (300 MHz, DMSO-d6):
δ 12.54 (s, 1H, ArOH), 7.92 (t, 2H, J = 7.6 Hz, Ar-H), 7.67 (t,
1H, J = 6.2 Hz, Ar-H), 7.50 (t, 1H, J = 6.9 Hz, Ar-H), 7.12 (s, 1H, Ar-H), 6.62 (s, 1H, -C=CH), 4.16
(t, 2H, J = 6 Hz, -CH2-O-Ar), 3.86 (s, 3H, -OCH3), 2.45 (m, 10H, -OCH2CH2CH2CH2N-,
-N(CH2CH2)2N-), 2.24 (s, 3H, -N-CH3), 1.79 (t, 2H, J = 6.6Hz, -OCH2CH2CH2CH2N-), 1.60 (t, 2H,
20

ACCEPTED MANUSCRIPT
J = 6.8 Hz, -OCH2CH2CH2CH2N-) ppm. 13C-NMR (75 MHz, DMSO-d6):
δ 181.8 (-CO-), 164.7
(-O-CH=), 161.3, 158.3, 156.7, 149.2, 133.4, 129.9, 122.2, 121.0, 115.6 (-C6H4-Br), 110.5, 103.9),
96.9 (=CH-CO-), 69.0 (-O-CH2), 61.2 (-O-CH3), 56.6, 54.3 (-N(CH2CH2)2N-), 45.9 (-N-CH3), 51.0,
26.3, 23,3 (-O-C4H8-N) ppm. IR (KBr): 3448 (OH), 3128 (C-H, aromatic), 2935 (CH2), 2787, 2363,
1659 (C=O), 1593 (C=C), 1508, 1400, 1337, 1264, 1227, 1119, 1034, 812, 697 cm-1. ESI-HRMS
m/z calculated for C25H30BrFN2O5 [M + H]+ 457.2133, found 457.2127.
7-(Benzyloxy)-8-(2-(bis(2-hydroxyethyl)amino)ethoxy)-5-hydroxy-2-phenyl-4H-chromen-4-
one (18a). The mixture of 17a (0.5 g, 1.1 mmol), diethanolamine (1 g), K2CO3 (0.3 g, 2.2 mmol) and
anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. The mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 20: 1) to give 18a as a yellow solid (0.25 g, 37.46 %). m.p. 94-96 oC.
1H-NMR (300 MHz, CDCl3):
δ 12.5 (s, 1H, 5-OH), 7.93 (m, 2H, , Ph-H), 7.55 (m, 3H, J = 1.65
Hz,Ph-H), 7.44 (m, 5H, Ph-H), 6.65 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.22 (s, 2H, ArOCH2 ), 4.12
(t, 2H, J = 5.1 Hz,O-CH2), 3.66 (t, 4H, J = 5.2 Hz, 2 × CH2OH), 3.04 (t, 2H, J = 5.1 Hz, N-CH2),
2.77 (t, 4H, J = 5.2 Hz ,2 × N-CH2) ppm. IR (KBr): 3395 (OH), 3061 (C-H, aromatic), 3026, 2943
(CH2), 2861 (CH2), 2814, 1660 (C=O), 1610 (C=C), 1586 (C=C), 1508, 1451, 1419, 1374, 1339,
1276, 1226, 1210, 1191, 1123, 1072, 1030, 846, 766, 700 cm-1. ESI-HRMS m/z calculated for
C28H30NO7 [M + H]+ 492.2017, found 492.2025.
7-(Bnzyloxy)-5-hydroxy-8-(2-(4-methylpiperazin-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one
(18b). The mixture of 17b (0.5 g, 1.1 mmol), mthylpiperazine (216 mg, 2.2 mmol), K2CO3 (0.3 g,
2.2 mmol) and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of
the reaction (monitored by TLC), the mixture was filtered immediately, and the filtrate was
concentrated under reduced pressure. The residual obtained was dissolved in DCM (100mL), washed
by water (50 mL × 3) and saturated sodium chloride solution (50 mL × 3), successively, and dried
over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure and
purified by chromatography (EA: MeOH = 50: 1) to give 18b as a yellow solid (0. 3 g, 59.76 %).
21

ACCEPTED MANUSCRIPT
m.p. 142-144 oC; 1H-NMR (300 MHz, CDCl3):
δ
12.55 (s, 1H, 5-OH), 7.95 (m, 2H, Ph-H), 7.55 (m,
3H, Ph-H), 7.43 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.17 (s, 2H, ArOCH2), 4.16
(t, 2H, J = 5.7 Hz, O-CH2), 2.84 (t, 2H, J = 5.7 Hz, N-CH2), 2.80 - 2.37 (m, 8H, 4 × N-CH2), 2.2 (s,
3H, N-CH3) ppm. IR: 3464 (OH), 3115 (C-H, aromatic), 2973 (CH3), 2932 (CH2), 2792, 2684, 1655
(C=O), 1613 (C=C), 1585 (C=C), 1503, 1449, 1435, 1372, 1337, 1272, 1201, 1115, 1030, 1009, 816,
695 cm-1. ESI-HRMS m/z calculated for C29H31N2O5 [M + H]+ 487.2227, found 487.2231.
7-(Benzyloxy)-8-(3-(dimethylamino)propoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one
(18c). The mixture of 17c (0.53 g, 1.05 mmol), dimethylamine hydrochloride (1 g), K2CO3 (0.6 g,
4.4 mmol) and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of
the reaction (monitored by TLC), the mixture was filtered immediately, and the filtrate was
concentrated under reduced pressure and purified by chromatography (EA: MeOH = 10: 1) to give
18c as a yellow solid (0. 25 g, 47.40 %). M.p. 208–210 oC; 1H-NMR (300 MHz, DMSO):
δ 12.65 (s,
1H, 5-OH), 8.09 (m, 2H, Ph-H), 7.83 (m, 3H, Ph-H), 7.53 (m, 5H, Ph-H), 7.05 (s, 1H, Ar-H), 6.82 (s,
1H, Ar-H), 5.30 (s, 2H, ArOCH2 ), 4.23 (t, 2H, J = 5.82 Hz, O-CH2), 3.06 (t, 2H, J = 5.82 Hz,
N-CH2), 2.56 (s, 6H, 2×O-CH3) ppm. IR: 3461 (OH), 3124 (C-H, aromatic), 2943 (CH2), 2677, 1663
(C=O), 1610 (C=C), 1509, 1451, 1397, 1374, 1339, 1273, 1227, 1118, 832, 688 cm-1. ESI-HRMS
m/z calculated for C27H28NO5 [M + H]+ 446.1962, found 446.1969.
7-(Benzyloxy)-8-(3-(diethylamino)propoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (18d).
The mixture of 17d (0.53 g, 1.1 mmol), diethylamine (0.15 g, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol)
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure and purified by chromatography (EA: MeOH = 20: 1) to give 18d as a yellow solid
o
1
(0. 25 g, 52.32 %). m.p. 172–173 C. H-NMR (300 MHz, CDCl3):
δ 12.6 (s, 1H, 5-OH), 7.89 (m,
2H, Ph-H), 7.58 (m, 3H, Ph-H), 7.46 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.54 (s, 1H, Ar-H), 5.11 (s,
2H, ArOCH2), 4.14 (t, 2H, J = 5.01 Hz, O-CH2), 3.13 (m, 6H, 3 × N-CH2), 2.38 (m, 2H,
O-CH2-CH2), 1.45 (t, 6H, J = 2.67 Hz, 2 × CH2-CH3) ppm. IR (KBr): 3416 (OH), 3221, 3061 (C-H,
aromatic), 2976 (CH3), 2943 (CH2), 2737, 2652, 2489, 1656 (C=O), 1616 (C=C), 1506, 1450, 1399,
1374, 1340, 1272, 1221, 1121, 1044, 1010, 809, 770, 694, 640 cm-1. ESI-HRMS m/z calculated for
C29H32NO5 [M + H]+ 474.2275, found 474.2287.
22

ACCEPTED MANUSCRIPT
7-(Benzyloxy)-8-(3-(bis(2-hydroxyethyl)amino)propoxy)-5-hydroxy-2-phenyl-4H-chromen-
4-one (18e). The mixture of 17e (0.53 g, 1.1 mmol), diethanolamine (1 g), K2CO3 (0.3 g, 2.2 mmol)
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. Then the mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 20: 1) to give 18e as a yellow solid (0.3 g, 55.22 %). m.p. 125–126
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.55 (s, 1H, 5-OH), 7.93 (m, 2H, Ph-H), 7.56 (m, 3H, Ph-H),
7.54 (m, 5H, Ph-H), 6.61 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.19 (s, 2H, ArOCH2), 4.12 (t, 2H, J =
6.00 Hz, O-CH2), 3.39 (t, 4H, J = 6.6 Hz, 2 × HO-CH2), 2.01 (m, 6H, 3 × N-CH2) ppm. IR (KBr):
3469 (OH), 3162, 3073 (C-H, aromatic), 3038, 2958 (CH2), 2873 (CH2), 2779, 1660 (C=O), 1613
(C=C), 1587, 1508, 1450, 1717, 1389, 1374, 1333, 1226, 1208, 1187, 1118, 1030, 1017, 1006, 839,
807, 698, 684 cm-1. ESI-HRMS m/z calculated for C29H32NO7 [M + H]+ 506.2173, found 506.2172.
7-(Benzyloxy)-5-hydroxy-2-phenyl-8-(3-(pyrrolidin-1-yl)propoxy)-4H-chromen-4-one (18f).
The mixture of 17f (0.53 g, 1.1 mmol), pyrrolidine (150 mg, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol)
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100 mL), washed by water (50 mL
× 3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. The mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 30: 1) to give 18f as a yellow solid (0.43 g, 81.22 %). m.p. 186–188
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.54 (s, 1H, 5-OH), 7.95 (m, 2H, Ph-H), 7.55 (m, 3H, Ph-H),
7.45 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.4 (s, 1H, Ar-H), 5.19 (s, 2H, ArOCH2), 4.13 (t, 2H, J = 6.3
Hz, O-CH2), 2.71 (t, 2H, J = 6.3 Hz, N-CH2), 2.47 (br, 4H, 2 × N-CH2), 2.09 (m, 2H,
O-CH2-CH2-CH2), 1.76 (br, 4H, N-CH2-CH2-CH2) ppm. IR (KBr): 3469 (OH), 3162, 3073 (C-H,
aromatic), 3038, 2958 (CH2), 2873 (CH2), 2779, 1660 (C=O), 1613 (C=C), 1587, 1508, 1450, 1389,
1374, 1272, 1226, 1208, 1187, 1118, 1030, 1017, 1006, 839, 807, 765, 755 cm-1. ESI-HRMS m/z
calculated for C29H 30NO5 [M + H]+ 472.2118, found 472.2125.
23

ACCEPTED MANUSCRIPT
7-(Benzyloxy)-5-hydroxy-8-(3-morpholinopropoxy)-2-phenyl-4H-chromen-4-one (18g). The
mixture of 17g (0.53 g, 1.1 mmol), morpholine (186 mg, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol) and
anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. Then the mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 20: 1) to give 18g as a yellow solid (0. 46 g, 83.42 %). m.p. 144–146
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.54 (s, 1H, 5-OH), 7.94 (m, 2H, Ph-H), 7.55 (m, 3H, Ph-H),
7.47 (m, 5H, Ph-H), 6.72 (s, 1H, Ar-H), 6.67 (s, 1H, Ar-H), 5.17 (s, 2H, ArOCH2), 4.12 (t, 2H, J =
6.1 Hz, O-CH2), 3.68 (t, 4H, J = 6.3 Hz, 2 × O-CH2), 2.58 (t, 2H, N-CH2, J = 10.1 Hz), 2.39 (br, 4H,
2 × N-CH2), 2.04 (m, 2H, O-CH2-CH2-CH2) ppm. IR (KBr): 3461 (OH), 3115(C-H, aromatic), 2954
(CH2), 2879, 2867 (CH2), 2813, 2761, 1661 (C=O), 1613 (C=C), 1587, 1451, 1419, 1374, 1274,
1230, 1210, 1189, 1117, 1029, 1006, 999, 766, 757 cm-1. ESI-HRMS m/z calculated for C29H30NO6
[M + H]+ 488.2068, found 488.2068.
7-(Benzyloxy)-5-hydroxy-8-(3-(4-methylpiperazin-1-yl)propoxy)-2-phenyl-4H-chromen-4-o
ne (18h). The mixture of 17h (0.53 g, 1.1 mmol), ethylpiperazine (216 mg, 2.2 mmol), K2CO3 (0.3 g,
2.2 mmol) and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of
the reaction (Monitored by TLC), the mixture was filtered immediately, and the filtrate was
concentrated under reduced pressure. The residual obtained was dissolved in DCM (100mL), washed
by water (50 mL × 3) and saturated sodium chloride solution (50 mL × 3), successively, and dried
over anhydrous sodium sulfate. Then the mixture was filtered, concentrated under reduced pressure
and purified by chromatography (EA: MeOH = 20: 1) to give 18h as a yellow solid (0.40 g, 69.33
%). m.p. 161–163 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.47 (s, 1H, 5-OH), 7.94 (m, 2H, Ph-H), 7.55
(m, 3H, Ph-H), 7.44 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.18 (s, 2H, ArOCH2),
4.13 (t, 2H, J = 6.3 Hz, O-CH2), 2.62 (t, 2H, J = 7.2 Hz, N-CH2), 2.57 (br, 6H, J = 10.1 Hz, 3 ×
N-CH2), 2.30 (s, 3H, N-CH3), 2.04 (m, 2H, O-CH2-CH2-CH2) ppm. IR (KBr): 3472 (OH), 3116 (C-H,
aromatic), 2933 (CH2), 2785, 1663 (C=O), 1614 (C=C), 1587, 1508, 1451, 1375, 1336, 1274, 1228,
24

ACCEPTED MANUSCRIPT
1187, 1166, 1031, 1008, 998, 764, 638 cm-1. ESI-HRMS m/z calculated for C30H33N2O5 [M + H]+
501.2384, found 501.2390.
7-(Benzyloxy)-8-(4-(dimethylamino)butoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (18i).
The mixture of 17i (0.55 g, 1.1 mmol), dimethylamine hydrochloride (1 g), K2CO3 (0.6 g, 4.4 mmol)
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure and purified by chromatography (EA: MeOH = 10: 1) to give 18i as a yellow solid
o
1
(0. 29 g, 56.22 %). m.p. 138–140 C. H-NMR (300 MHz, CDCl3):
δ 12.54 (s, 1H, 5-OH), 7.93 (m,
2H, Ph-H), 7.56 (m, 3H, Ph-H), 7.46 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.17 (s,
2H, ArOCH2), 4.08 (t, 2H, J = 5.4 Hz, O-CH2), 2.50 (m, 2H, N-CH2), 2.27 (s, 6H, 2 × N-CH3), 1.83
(m, 4H, O-CH2-CH2-CH2) ppm. IR (KBr): 3453 (OH), 3061 (C-H, aromatic), 2943 (CH2), 2861
(CH2), 2808, 2755, 1661 (C=O), 1613 (C=C), 1587, 1507, 1450, 1419, 1374, 1335, 1273, 1226,
1209, 1188, 1119, 1031, 1008, 998, 807, 765, 699, 685 cm-1. ESI-HRMS m/z calculated for
C30H33N2O5 [M + H]+ 460.2118, found 460.2123.
8-(4-(Dimethylamino)butoxy)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one
(18j).
The
mixture of 17j (0.55 g, 1.1 mmol), diethylamine (0.15 g, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol) and
anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure and purified by chromatography (EA: MeOH = 20: 1) to give 18j as a yellow solid
(0.3 g, 56.86 %). m.p. 125-126 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.52 (s, 1H, 5-OH), 7.95 (m, 2H,
Ph-H), 7.55 (m, 3H, Ph-H), 7.46 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.19 (s, 2H,
ArOCH2 ), 4.08 (t, 2H, J = 5.43 Hz, O-CH2), 2.50 (m, 6H, 3 × N-CH2), 1.85 (m, 2H,
O-CH2-CH2-CH2), 1.65 (m, 4H, O-CH2-CH2-CH2) ppm. IR: 3455 (OH), 3109 (C-H, aromatic), 2966
(CH3), 2941 (CH2), 2867 (CH2), 2790, 1662 (C=O), 1613 (C=C), 1587, 1507, 1450, 1374, 1336,
1274, 1229, 1211, 1188, 1118, 1030, 1008, 999, 837, 807, 765, 700 cm-1. ESI-HRMS m/z calculated
for C30H34NO5 [M + H]+ 488.2431, found 488.2436.
7-(Benzyloxy)-8-(4-(bis(2-hydroxyethyl)amino)butoxy)-5-hydroxy-2-phenyl-4H-chromen-4
-one (18k). The mixture of 17k (0.55 g, 1.1 mmol), diethanolamine (1 g), K2CO3 (0.3 g, 2.2 mmol)
25

ACCEPTED MANUSCRIPT
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. Then the mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 10: 1) to give 18k as a yellow solid (0. 36 g, 63.42 %). m.p. 111–113
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.52 (s, 1H, 5-OH), 7.92 (m, 2H, Ph-H), 7.56 (m, 3H, Ph-H),
7.46 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.19 (s, 2H, ArOCH2 ), 4.08 (t, 2H, J =
5.8 Hz, O-CH2), 3.61 (t, 4H, J = 5.3 Hz, 2 × CH2-OH), 2.61 (m, 6H, 3 × N-CH2), 2.13 (br, 2H, 2 ×
CH2-OH), 1.82 (m, 2H, O-CH2-CH2-CH2), 1.73 (m, 2H, O-CH2-CH2-CH2) ppm. IR (KBr): 3143
(C-H, aromatic), 2958 (CH2), 1654 (C=O), 1579, 1400, 1275, 1191, 1101, 1010, 841, 767, 674, 558
cm-1. ESI-HRMS m/z calculated for C30H34NO7 [M + H]+ 520.2330, found 520.2336.
7-(Benzyloxy)-5-hydroxy-2-phenyl-8-(4-(pyrrolidin-1-yl)butoxy)-4H-chromen-4-one (18l).
The mixture of 17l (0.55 g, 1.1 mmol), pyrrolidine (150 mg, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol)
and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. The mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 10: 1) to give 18l as a yellow solid (0. 43 g, 81.63 %). m.p. 132–134
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.53 (s, 1H, 5-OH), 8.22 (m, 2H, Ph-H), 7.93 (m, 3H, Ph-H),
7.46 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.48 (s, 1H, Ar-H), 5.19 (s, 2H, ArOCH2), 4.09 (t, 2H, J =
6.4 Hz, O-CH2), 2.46 (m, 6H, 3 × N-CH2), 1.89 (m, 2H, CH2-CH2), 1.75 (br, 6H, 3 × CH2- CH2)
ppm. IR (KBr): 3469 (OH), 3162, 3097 (C-H, aromatic), 3032, 2946 (CH2), 2873 (CH2), 2772, 2678,
1662 (C=O), 1613 (C=C), 1587, 1507, 1451, 1420, 1374, 1335, 1273, 1228, 1211, 1188, 1030, 1008,
998, 837, 808, 765, 699 cm-1. ESI-HRMS m/z calculated for C30H32NO5 [M + H]+ 486.2275, found
486.2285.
7-(Benzyloxy)-5-hydroxy-8-(4-morpholinobutoxy)-2-phenyl-4H-chromen-4-one (18m). The
mixture of 17m (0.55 g, 1.1 mmol), morpholine (186 mg, 2.2 mmol), K2CO3 (0.3 g, 2.2 mmol) and
26

ACCEPTED MANUSCRIPT
anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of the reaction
(Monitored by TLC), the mixture was filtered immediately, and the filtrate was concentrated under
reduced pressure. The residual obtained was dissolved in DCM (100mL), washed by water (50 mL ×
3) and saturated sodium chloride solution (50 mL × 3), successively, and dried over anhydrous
sodium sulfate. The mixture was filtered, concentrated under reduced pressure and purified by
chromatography (EA: MeOH = 10: 1) to give 18m as a yellow solid (0.45 g, 82.56 %). m.p. 136–138
1
oC. H-NMR (300 MHz, CDCl3):
δ 12.53 (s, 1H, 5-OH), 7.94 (m, 2H, J = 5.8 Hz, Ph-H), 7.53 (m,
3H, J = 5.8 Hz, Ph-H), 7.46 (m, 5H, J = 1.7 Hz, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.19
(s, 2H, ArOCH2), 4.09 (t, 2H, J = 6.1 Hz, O-CH2), 3.67 (t, 4H, J = 4.4 Hz, 2 × O-CH2), 2.35 (m, 6H,
3 × N-CH2), 1.87 (m, 2H, O-CH2-CH2-CH2), 1.75 (m, 2H, J = 5.8 Hz, O-CH2 -CH2-CH2) ppm. IR
(KBr): 3463 (OH), 3097 (C-H, aromatic), 3026, 2946 (CH2), 2867 (CH2), 2802, 2761, 1661 (C=O),
1613 (C=C), 1587, 1507, 1450, 1419, 1373, 1335, 1274, 1230, 1214, 1187, 1116, 1029, 1007, 999,
865, 806, 765, 701, 685 cm-1. ESI-HRMS m/z calculated for C30H32NO6 [M + H]+ 502.2224, found
502.2225.
7-(Benzyloxy)-5-hydroxy-8-(4-(4-methylpiperazin-1-yl)butoxy)-2-phenyl-4H-chromen-4-on
e (18n). The mixture of 17n (0.55 g, 1.1 mmol), methylpiperazine (216 mg, 2.2 mmol), K2CO3 (0.3
g, 2.2 mmol) and anhydrous acetonitrile (25 mL) was heated to reflux under stirring. After the end of
the reaction (Monitored by TLC), the mixture was filtered immediately, and the filtrate was
concentrated under reduced pressure. The residual obtained was dissolved in DCM (100mL), washed
by water (50 mL × 3) and saturated sodium chloride solution (50 mL × 3), successively, and dried
over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure and
purified by chromatography (EA: MeOH = 20: 1) to give 18n as a yellow solid (0. 41 g, 72.75 %).
m.p. 166–168 oC. 1H-NMR (300 MHz, CDCl3):
δ 12.53 (s, 1H, 5-OH), 7.94 (m, 2H, Ph-H), 7.53 (m,
3H, Ph-H), 7.43 (m, 5H, Ph-H), 6.67 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.19 (s, 2H, ArOCH2), 4.09
(t, 2H, J = 6.2 Hz, O-CH2), 2.42 (m, 8H, 4 × N-CH2), 2.28 (s, 3H, N-CH3), 1.87 (m, 2H,
O-CH2-CH2-CH2), 1.75 (m, 2H, O-CH2-CH2-CH2) ppm. IR (KBr): 3451 (OH), 3097 (C-H,
aromatic), 3056, 3032, 2935 (CH2), 2861 (CH2), 2789, 1661 (C=O), 1613 (C=C), 1587, 1507, 1450,
1374, 1333, 1273, 1225, 1187, 1165, 1119, 1008, 997, 837, 805, 764, 701, 684 cm-1. ESI-HRMS m/z
calculated for C30H35N2O5 [M + H]+ 515.2540, found 515.2544.
27

ACCEPTED MANUSCRIPT
7-(4-Methyl-benzyloxy)-8-(4-(4-methylpiperazin-1-yl)butoxy)-5-hydroxy-2-phenyl-4H-chro
men-4-one (20a). The mixture of 19a (0.21 g, 0.5 mmol), 2-chloromethylfuran (0.08 g, 0.6 mmol),
o
K2CO3 (0.17 g, 1.3 mmol) and anhydrous acetonitrile (22 mL) was heated to 60 C under stirring.
After the end of the reaction (Monitored by TLC), the mixture was filtered immediately to remove
K2CO3, and the filtrate was concentrated under reduced pressure and purified by chromatography
(DCM: MeOH = 30: 1) to give 20a as a yellow solid (0.1 g, 20 %). m.p. 104 - 106 oC. 1H-NMR (300
MHz, CDCl3):
δ 12.54 (s, 1H, Ar-OH), 7.93 (q, 2H, J = 2.4 Hz, Ph-H), 7.57 (t, 2H, J = 2.4 Hz,
Ph-H), 7.33 (d, 2H, Ar-H), 7.24 (t, 2H, Ar-H), 6.68 (s, 1H, -C=CH), 6.50(s, 1H, -C=CH), 5.15 (s,
2H, O-CH2-Ar), 4.08 (t, 2H, O-CH2-R), 2.60 (s, 8H, N-(CH2CH2)2-N), 2.39 (s, 6H, CH3-N/CH3-Ar)
ppm. 13C-NMR (75 MHz, CDCl3):
δ 182.1 (-CO-), 163.4 (-O-C=), 156.8, 138.0, 131.4, 128.9, 127.6,
127.1, 126.9, 125.8, 104.9, 96.7 (=CH-CO-), 80.5, 76.9, 76.5, 76.0 (N-(CH2CH2)2-N), 73.4, 70.5,
57.4, 46.1 (N-(CH2)4-O) ppm. IR (KBr): 3414 (OH), 3075 (C-H, aromatic), 2931 (CH2), 2750, 1655
(C=O), 1617 (C=C), 1452, 1374, 1333, 1274, 1181, 1107, 1014, 811, 692, 579. ESI-HRMS m/z
calculated for C32H37N2O5 [M + H]+ 529.2697, found 529.2718.
7-(4-Cyano-benzyloxy)-8-(4-(4-methylpiperazin-1-yl)butoxy)-5-hydroxy-2-phenyl-4H-chro
men-4-one (20b). The mixture of 19b (0.21 g, 0.5 mmol), 4-nitrobenzyl bromide (0.21 g, 0.6 mmol),
o
K2CO3 (0.29 g, 1.3 mmol) and anhydrous acetonitrile (22 mL) was heated to 55 C under stirring.
After the end of the reaction (Monitored by TLC), the mixture was filtered immediately to remove
K2CO3, and the filtrate was concentrated under reduced pressure and purified by chromatography
o
1
(DCM: MeOH = 30: 1) to give 20b as a yellow solid (0.13 g, 43 %). m.p. 116–118 C. H-NMR
(300 MHz, CDCl3): 12.56 (s, 1H, Ar-OH), 7.94 (q, 2H, J = 2.9 Hz, Ph-H), 7.55 (t, 3H, J = 2.9 Hz,
δ
Ph-H), 7.48 (d, 1H, Ar-H), 6.69 (s, 1H, -C=CH), 6.57 (s, 1H, -C=CH), 6.51 (d, 1H, Ar-H), 6.41 (q,
1H, Ar-H), 5.15 (s, 2H, O-CH2), 4.05 (t, 2H, N-CH2), 2.34 (s, 3H, N-CH3) ppm. 13C-NMR (75 MHz,
CDCl3):
δ 182.2 (-CO-), 163.4 (-O-C=), 156.9, 156.6, 142.9, 131.4, 130.9, 128.6, 125.8, 110.2,
110.1, 104.9, 96.8 (=CH-CO-), 76.9, 76.5, 76.1, 73.6 (N-(CH2CH2)2-N), 62.7 (O-CH2-Ar), 57.6,
54.4, 45.8 (-N-CH3), 27.7, 22.8 (N-(CH2)4-O) ppm. IR (KBr): 3568, 3432 (OH), 3118 (C-H,
aromatic), 2937 (CH2), 2792, 1654 (C=O), 1617 (C=C), 1589, 1451, 1400, 1374, 1334, 1109, 1004,
918, 683, 601, 568 cm-1. ESI-HRMS m/z calculated for C32H34N3O5 [M + H]+ 540.2493, found
540.2508.
28