3-O-Substituted-3',4',5'-trimethoxyflavonols: Synthesis and cell-based evaluation as anti-prostate cancer agents

Article abstract of DOI:10.1016/j.bmc.2017.07.022

Twenty-two 3-O-substituted-3',4',5'-trimethoxyflavonols have been designed and synthesized for their anti-proliferative activity towards three human prostate cancer cell lines. Our results indicate that most of them are significantly more potent than the parent 3',4',5'-trimethoxyflavonol in inhibiting the cell proliferation in PC-3 and LNCaP prostate cancer cell models. 3-O-Substituted-3',4',5'-trimethoxyflavonols have generally higher potency towards PC-3 and LNCaP cell lines than the DU145 cell line. Incorporation of an ethyl group to 3-OH of 3',4',5'-trimethoxyflavonol leads to 3-O-ethyl-3',4',5'-trimethoxyflavonol as the optimal derivative with up to 36-fold enhanced potency as compared with the corresponding lead compound 3',4',5'-trimethoxyflavonol, but with reversed PC-3 cell apoptotic response. Introduction of a dipentylaminopropyl group to 3-OH increases not only the antiproliferative potency but also the ability in activating PC-3 cell apoptosis. Our findings imply that modification on 3-OH of trimethoxyflavonol can further enhance its in vitro anti-proliferative potency and PC-3 cell apoptosis induction.

Full text of DOI:10.1016/j.bmc.2017.07.022

Accepted Manuscript
3-O-Substituted-3',4',5'-trimethoxyflavonols: synthesis and cell-based evalua-
tion as anti-prostate cancer agents
Xiang Li, Maizie Lee, Guanglin Chen, Qiang Zhang, Shilong Zheng, Guangdi
Wang, Qiao-Hong Chen
PII:
S0968-0896(17)31178-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.022
BMC 13859
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2017
8 July 2017
11 July 2017
Please cite this article as: Li, X., Lee, M., Chen, G., Zhang, Q., Zheng, S., Wang, G., Chen, Q-H., 3-O-Substituted-3',
4',5'-trimethoxyflavonols: synthesis and cell-based evaluation as anti-prostate cancer agents, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.022
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3-O-Substituted-3',4',5'-trimethoxyflavonols: synthesis and cell-based evaluation as
anti-prostate cancer agents
Xiang Lia, Maizie Leea, Guanglin Chena, Qiang Zhangb,c, Shilong Zhengb,c,
Guangdi Wangb,c, Qiao-Hong Chena,*
aDepartment of Chemistry, California State University, Fresno, 2555 E. San Ramon Avenue, M/S SB70, Fresno, CA 93740,
USA.
bRCMI Cancer Research Center, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
cDepartment of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
ABSTRACT ― Twenty-two 3-O-substituted-3',4',5'-trimethoxyflavonols have been designed and
synthesized for their anti-proliferative activity towards three human prostate cancer cell lines. Our
results indicate that most of them are significantly more potent than the parent 3',4',5'-
trimethoxyflavonol in inhibiting the cell proliferation in PC-3 and LNCaP prostate cancer cell models.
3-O-Substituted-3',4',5'- trimethoxyflavonols have generally higher potency towards PC-3 and LNCaP
cell lines than the DU145 cell line. Incorporation of an ethyl group to 3-OH of 3',4',5'-
trimethoxyflavonol leads to 3-O-ethyl-3',4',5'-trimethoxyflavonol as the optimal derivative with up to
36-fold enhanced potency as compared with the corresponding lead compound 3',4',5'-
trimethoxyflavonol, but with reversed PC-3 cell apoptotic response. Introduction of a
dipentylaminopropyl group to 3-OH increases not only the antiproliferative potency but also the ability
in activating PC-3 cell apoptosis. Our findings imply that modification on 3-OH of trimethoxyflavonol
can further enhance its in vitro anti-proliferative potency and PC-3 cell apoptosis induction.
1 | P a g e

1. Introduction:
Flavonoids are nutraceuticals with various health and medicinal benefits. Flavonols belong to a large
subgroup of flavonoids that are characteristic of a hydroxyl group at C-3 and are ubiquitously
distributed in dietary foods.1-2 The anti-prostate cancer activity of several naturally occurring flavonols,
as exemplified by quercetin (1) and fisetin (2) (Fig. 1), has been explored by in vitro and in vivo
experiments.3-8 3',4',5'-Trimethoxyflavonol (3, Fig. 1) is a synthetic flavonol and a promising anti-
prostate cancer lead compound that exhibits 5- to 16-fold greater potency than the naturally occurring
quercetin (1) and fisetin (2) in suppressing cell growth in four prostate cancer cell models.3,9 The in
vitro anti-prostate cancer potency of 3',4',5'-trimethoxyflavonol (3) has been translated into its in vivo
antitumor efficacy in two prostate cancer xenografts (the murine TRAMP C2 and human-derived
22Rv1) in nude mice, but the efficacy is very moderate even at the highest dose (0.731 mmol/kg).9
Additionally, oral administration of a single dose of 3',4',5'-trimethoxyflavonol (3) at 240 mg/kg
yielded a Cmax of only 18.4 nM in the mice prostate at 6 hours after administration.10 These data
suggest that either potency, bioavailability, or both of 3',4',5'-trimethoxyflavonol (3) need to be further
improved prior to being considered as a drug candidate for the treatment of prostate cancer. Our earlier
WST-1 cell proliferation assay indicates that incorporation of a dibutylamino group to the 3-OH group
of 3',4'-dimethoxyflavonol (4, Fig. 1) through a three- to five-carbon linker leads to the optimal
derivatives (e.g. 5, Fig. 1) with up to 292-fold enhanced potency as compared with the parent 3',4'-
dimethoxyflavonol (4) in human prostate cancer cell models.11 Encouraged by these promising results,
the present study aims to explore the possibility of further enhancing the anti-proliferative potency of
3',4',5'-trimethoxyflavonol (3) as anti-prostate cancer agents through chemical manipulations on its 3-
OH group. Consequently, ten 3-O-alkyl-3',4',5'-trimethoxyflavonols (one known and nine new) and
twelve new 3-O-dialkylaminoalkyl-3',4',5'-trimethoxyflavonols have been synthesized and evaluated
for their ability in suppressing prostate cancer cell proliferation.
2 | P a g e

Figure 1. Flavonols as anti-prostate cancer agents
2. Results and Discussion:
2.1. Chemistry
The parent compound, 3',4',5'-trimethoxyflavonol (3), has been synthesized through a well-known two-
step procedure for the synthesis of flavonols,12,13 including formation of chalcone via a Claisen-
Schmidt condensation of 2'-hydroxylacetophenone (6) with 3,4,5-trimethoxylbenzaldehyde (7)14 and
cyclization of chalcone through Algar-Flynn-Oyamada (AFO) reaction.15 This two-step procedure has
been widely applied to the synthesis of flavonols, but with tedious workup and purification processes,
as well as moderate overall yields. To overcome these limitations, Ozturk and co-workers have
developed a two-step one-pot procedure that has been successfully applied to the efficient syntheses of
3 | P a g e

several other flavonols in 35-79% yields.16 We envisioned the yield could be further improved by
lowering reaction temperature from refluxing to room temperature (or below) but prolonging reaction
time. Consequently, we synthesized 3',4',5'-trimethoxyflavonol (3), from 2'-hydroxylacetophenone (6)
and 3,4,5-tryimethoxylbenzaldehyde (7), in 47% overall yield (Scheme 1) through a four-day one-pot
procedure with facile work-up at room temperature as described in the experimental section.
As shown in Scheme 2, ten 3-O-alkyl-3',4',5'-trimethoxyflavonols (8-17) have been prepared in 54-99
% yields by O-alkylation of 3',4',5'-trimethoxyflavonol (3) with the appropriate alkyl halide using
potassium carbonate as the base and DMF as the aprotic solvent. Twelve new 3-O-aminoalkyl-3',4',5'-
trimethoxyflavonols (19-30) have been synthesized from 3',4',5'-trimethoxyflavonol (3) in 32-88%
yield via a two-step transformation as illustrated in Scheme 3. Potassium carbonate was used as the
base and DMF was used as the polar aprotic solvent in both O-alkylation and N-alkylation reactions.
Three crude 3-O-bromoalkyl-3',4',5'-trimethoxyflavonols (18) were directly used, without further
purification, for the follow-up N-alkylation reaction. The pure 3-O-dialkylaminoalkyl-3',4',5'-
trimethoxyflavonols (19-30) from the N-alkylation reaction were obtained by preparative thin layer
chromatography (PTLC) over silica gel. It is worth noting that the mixture of
dichloromethane:diethylamine (100:3, v/v) is the optimal solvent to retrieve the desired nitrogen-
containing products from the preparative thin layer silica gel.
Scheme 1. Synthesis of 3',4',5'-trimethoxyflavonol (3)
4 | P a g e

Scheme 2. Synthesis of 3-O-alkyl-3',4',5'-trimethoxyflavonols (8-17)
Scheme 3. Synthesis of 3-O-aminoalkyl-3',4',5'-trimethoxyflavonols (19-30)
2.2. Anti-proliferative effects toward three prostate cancer cell lines
5 | P a g e

To determine the in vitro ability of the synthesized 3-O-substituted-3',4',5'-trimethoxyflavonols in
suppressing prostate cancer cell proliferation, the anti-proliferative potency of the twenty-two 3-O-
substituted-3',4',5'-trimethoxyflavonols (8-17, 19-30) towards both androgen-sensitive (LNCaP) and
androgen-insensitive (PC-3 and DU145) human prostate cancer cell lines has been assessed by WST-
1cell proliferation assay. Quercetin (1), fisetin (2), and 3',4',5'-trimethoxyflavonols (3) were used as
positive controls. The IC50 values were calculated from the dose-response curves and summarized in
Table 1. Our data show that 3',4',5'-trimethoxyflavonol (3), with IC50 values of 32.1 µM, 27.2 µM, and
14.7 µM against PC-3, DU145, and LNCaP cells, respectively, is significantly more potent than
quercetin and fisetin. These results are consistent with those reported in the literature.9 It is worth
noting that the IC50 values in this study for 3',4',5'-trimethoxyflavonol (3) are higher than those
reported in the literature, which can be probably attributed to the different days of drug exposure (we
used three days; while the literature used six days). The PC-3 and LNCaP cancer cells are generally
more sensitive than DU145 cells to the 3-O-substituted-3',4',5'-trimethoxyflavonols (8-17, 19-30).
Most of the synthetic 3-O-substituted-3',4',5'-trimethoxyflavonols are more potent in inhibiting PC-3
and LNCaP prostate cancer cell proliferation than the parent flavonol, 3',4',5'-trimethoxyflavonol (3)
and the naturally occurring flavonols, quercetin (1) and fisetin (2), as determined by the IC50 values
acquired in this study. Incorporation of an ethyl group to 3-OH of 3',4',5'-trimethoxyflavonol (3) results
in the optimal derivative 9 with 36 times enhanced potency in suppressing PC-3 prostate cancer cell
proliferation. 3-O-Alkyl-3',4',5'-trimethoxyflavonols with the α-branched alkyl groups (derivatives 16-
17) or with relatively lengthy alkyl groups (derivatives 13-14) are similar or less potent than 3. The
introduction of a basic dialkylamino moiety to 3-OH of 3',4',5'-trimethoxyflavonol (3) through a 3- to
5-carbon linker can typically bestow 2-12 folds enhancement in anti-proliferative potency toward
androgen-insensitive PC-3 prostate cancer cells (derivatives 19-30, Table 1). These 3-O-
dialkylaminoalkyl-3',4',5'-trimethoxyflavonols (19-30) have slightly improved potency in inhibiting
LNCaP prostate cancer cell proliferation.
6 | P a g e

Table 1. Anti-proliferative activity of 3-O-substituted-3',4',5'-trimethoxyflavonols
Compound
R
IC50 (µM) a
DU-145c
27.2 8.9
>50
PC-3b
LNCaPd
14.7 2.1
24.7 8.3
5.4 1.1
5.7 1.9
8.9 3.8
15.4 0.3
19.6 7.6
>50
H
32.1 13.1
19.1 7.2
0.9 0.5
11.8 3.5
12.8 3.6
22.2 6.8
29.2 3.5
>50
3
methyl
ethyl
8
6.2 0.7
2.8 6.3
>50
9
propyl
10
11
12
13
14
15
16
17
19
20
21
22
23
butyl
pentyl
>50
hexyl
>50
heptyl
>50
isopropyl
21.2 5.9
38.6 21.9
49.3 17.0
13.6 1.7
13.7 4.2
6.6 1.5
16.8 5.1
11.6 1.3
> 50
8.8 3.2
9.3 0.5
15.1 1.6
37.6 8.8
13.5 2.2
10.0 0.9
17.9 4.9
16.8 5.5
sec-butyl
>50
pentan-2-yl
>50
(N,N-diethylamino)propyl
(N,N-diethylamino)butyl
(N,N-diethylamino)pentyl
(N,N-dipropylamino)propyl
(N,N-dipropylamino)butyl
> 50
13.3 2.2
33.4 3.2
>50
35.8 3.5
7 | P a g e

(N,N-dipropylamino)pentyl
(N,N-dibutylamino)propyl
(N,N-dibutylamino)butyl
(N,N-dibutylamino)pentyl
(N,N-dipentylamino)propyl
(N,N-dipentylamino)butyl
(N,N-dipentylamino)pentyl
-
2.6 0.4
7.1 2.2
5.3 0.9
6.8 1.7
5.8 1.2
7.9 2.7
9.6 2.4
> 100
24.2 3.9
15.7 4.4
15.3 6.3
>50
7.6 1.5
9.0 2.1
7.8 1.2
7.8 0.9
5.1 0.4
9.7 0.8
7.9 0.4
45.46 1.31
34.1 7.7
24
25
26
27
12.3 2.2
12.5 2.1
11.7 2.6
> 100
28
29
30
Quercetin (1)
Fisetin (2)
-
> 50
> 50
aIC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell
proliferation assay after 3 days exposure. The data were presented as the mean standard deviation
of the mean from three independent experiments (n = 3).
bHuman androgen-insensitive prostate cancer cell line derived from bond metastasis of prostate tumor.
cHuman androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor.
dHuman androgen-sensitive prostate cancer cell line.
2.3. Anti-proliferative activity of 3 and its derivatives 9 and 28 towards PWR-1E non-neoplastic
human prostate epithelial cell line
In this study, we observed the IC50 values for 3, 9, and 28 are >50, 8.05 0.85, and 14.83 1.83 µM
towards PWR-1E non-neoplastic human prostate epithelial cell line. As shown in Figure 2, there are
significantly differential responses of these three flavonols to PC-3 androgen-insensitive prostate
cancer cells and PWR-1E benign human prostate epithelial cells. Specifically, all three flavonols (3, 9,
and 28) are more effective in inhibiting PC-3 cancer cell proliferation than PWR-1E non-neoplastic
human prostate epithelial cell proliferation. It is worth noting that modification at 3-OH of flavonol 3
also improves the antiproliferative potency towards PWR-1E non-neoplastic human prostate epithelial
8 | P a g e

cell model that is an immortalized cell line and was believed by Rhim and co-workers to represent an
early stage in tumor progression.17
60
50
40
30
20
10
0
PC-3
PWR-1
3
9
28
Comopound
Figure 2: Comparison of IC50 values (µM) for 3, 9, and 28 in PC-3 cell model with those in the PWR-
1E cell model. The data were presented as the mean standard deviation of the mean from three
independent experiments (n = 3). Statistical significance was established at P < 0.05. Note that no error
bar for the IC50 values for 3 on PC-3 model is expressed because they are greater than 50 µM.
2.4. Effects of 3, 9, and 28 on PC-3 cell apoptosis and cell cycle progression
Quercetin (1) and fisetin (2), as well as one synthetic 3-O-substituted-3,4-dimethyoxyflavonol (5),
have shown cell cycle perturbation properties in the androgen-insensitive PC-3 human prostate cancer
cell line by arresting cell cycle at G2/M phase.4,11,18 Trimethoxyflavonol (3) has been revealed to arrest
cell cycle at the G2/M phase in murine TRAMP C2 prostate cancer cells, but have no effect on human
22Rv1 cell cycle distribution.9 No report was available so far on PC-3 cell cycle perturbation activity
of trimethoxyflavonol (3). Consequently, the effect of parent compound 3, and derivatives 9 and 28, at
10 µM and 20 µM on the PC-3 cell cycle was evaluated using flow cytometric analysis with propidium
iodide DNA staining. It is worth noting that compound 28 was chosen as the representative aminoalkyl
derivative for further investigation on its PC-3 cell cycle regulation and cell apoptosis induction even
though it (IC50 = 5.8 µM) is slightly less potent than compound 24 (IC50 = 2.6 µM) in the PC-3 cell
9 | P a g e

model. This is because compound 28 shows greater potential to be further developed as an anti-
prostate cancer agent due to its slightly greater overall potency towards three human prostate cancer
cell lines, as compared with compound 24. As illustrated in Table 2, modification of 3-OH can change
its regulatory effect on PC-3 cell cycle. Specifically, trimethoxyflavonol (3) induces PC-3 cell cycle
arrest at the S phase at 16 h, but at the G2/M phase at 24 h. 3-O-Ethyl-3',4',5'-trimethoxyflavonol (9)
induces cell cycle arrest at the G0/G1 phase at 16 h, and at both G0/G1 & S phase at 24 h by increasing
PC-3 cell population in the G0/G1 and S phase, while fewer cells were observed in the G2/M phase
(Table 2). Interestingly, derivative 28 with a dipentylaminopropyl group at 3-OH of 3',4',5'-
trimethoxyflavonol consistently induces PC-3 cell cycle arrest at the G0/G1 phase at 10 µM and 20 µM,
and at 16 h and 24 h.
The naturally occurring flavonols quercetin (1) and fisetin (2), and the synthetic flavonol derivative 5
have been demonstrated to promote androgen-insensitive PC-3 human prostate cancer cell
apoptosis.11,19-21 Additionally, trimethoxyflavonol (3) has been reported to induce mouse prostate
carcinoma TRAMP C2 cell apoptosis in vitro as detected by annexin V staining, but not in human
prostate carcinoma 22Rv1 cells.9 No apoptosis activation of trimethoxyflavonol (3) in human
androgen-insensitive PC-3 prostate cancer cell line has been reported. The F2N12S and SYTOX
AADVanced double staining assay in a flow cytometer was used in this study for the discrimination
between early apoptotic PC-3 cells and late apoptotic/necrotic PC-3 cells when treated with
trimethoxyflavonol (3), and derivatives 9 and 28 at the concentrations specified in Figures 3 for 16 h.
The data, as listed in Figures 3-4, indicate that trimethoxyflavonol (3) and derivative 28 can
significantly activate apoptotic cell death in the androgen-insensitive PC-3 prostate cancer cell line in a
dose-dependent manner after a 16-hour treatment. The data also suggest that derivative 28 with an
amino group at 3-OH has much greater capability in the induction of PC-3 cell apoptosis. Specifically,
as shown in Figures 3-4, exposure of PC-3 cells to 5 µM and 10 µM of trimethoxyflavonol (3) with its
free 3-OH group resulted in 11 1% and 39 13% early apoptotic cells, respectively; while treatment
10 | P a g e

of PC-3 cells with 5 µM and 10 µM of derivative 28 led to 51 20% and 93 1% early apoptotic
cells, respectively. In contrast, derivative 9 with an ethyl group at 3-OH in 3',4',5'-trimethoxyflavonol
did not promote significant levels of PC-3 cell apoptotic death even at a high dose of 100 µM after a
16-hour treatment as compared with control cells. These findings imply that modification on 3-OH of
trimethoxyflavonol can change not only the in vitro anti-proliferative potency but also its associated
mechanism of action.
Table 2. Cell cycle analysis of PC-3 prostate cancer cells. PC-3 cancer cells were untreated or treated
with 3, 9, and 28, respectively, at 10 µM or 20 µM. Cells were harvested after 16 h, fixed, stained, and
analyzed for DNA content.
Cell Cycle
G0/G1
S
G2/M
Treatment Time
Control (DMSO)
3 (10 µM)
16 hours 24 hours 16 hours 24 hours 16 hours 24 hours
34.1
31.2
33.3
55.7
59.0
51.3
40.8
37.5
34.6
33.8
40.8
48.6
52.6
59.8
8.0
32.3
33.0
26.6
22.2
24.5
28.6
34.8
9.9
27.8
33.3
31.2
19.3
17.9
25.4
26.4
6.0
12.3
11.7
7.7
3 (20 µM)
6.0
9 (10 µM)
18.7
11.8
3.7
9 (20 µM)
4.6
28 (10 µM)
28 (20 µM)
4.5
6.5
1.9
11 | P a g e

a) Apoptotic Effect of 3 against PC-3 Cells
ViableCell
ApoptoticCell
NecroticCell
120
100
80
60
40
20
0
*
81
95
90
*
66
***
61
***
73
***
54
***
50
***
39
***
47
***
43
***
45
***
24
*
11
2
6
3
3
2
2
2
2
2
2
Control 1 μM 5 μM 10 μM 20 μM 30 μM 50 μM 100 μM
Concentration of compound 3
b) Apoptotic Effect of 9 against PC-3 Cells
ViableCell
ApoptoticCell
95
93
NecroticCell
120
100
80
60
40
20
0
95
95
94
94
92
91
6 6
5 5
5 5
3 3
3 3
3 3
3 3
3 3
Control 1 μM 5 μM 10 μM 20 μM 30 μM 50 μM 100 μM
Concentration of compound 9
c) Apoptotic Effect of 28 against PC-3 Cells
ViableCell
ApoptoticCell
NecroticCell
120
100
80
***
94
***
93
***
92
95
*
87
***
72
***
65
***
51
*
46
60
***
*
35
27
40
**
10
20
***
3
8
***
0
***
0
***
0
6
***
0
4
2
3
2
2
0
Control 1 μM 5 μM 10 μM 20 μM 30 μM 50 μM 100 μM
(20)
Concentration of compound 28
Figure 3. Evolution of viable, apoptotic, and necrotic PC-3 cell populations in response to increasing
dosages of flavonols a) 3, b) 9; and c) 28. Shown are the mean SD from three independent
experiments. Significance is expressed as (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 versus control.
12 | P a g e

3-control
3 (5 µM)-treated
9 (5 µM)-treated
28 (5 µM)-treated
3 (10 µM)-treated
9 (10 µM)-treated
9 -control
28 (10 µM)-treated
28 -control
Figure 4. Apoptosis in PC-3 cells treated with 3, 9, and 28 at 5 µM and 10 µM (by F2N12S and
CYTOX AADvanced double staining).
13 | P a g e

3. Conclusion
Comparing the IC50 value, as well as cell cycle regulatory and cell apoptosis inductory ability, of
parent compound with those of flavonol derivatives, we can conclude that i) most 3-O-alkyl-3',4',5'-
trimethoxyflavonols and 3-O-dialkylaminoalkyl-3',4',5'-trimethoxyflavonols are more potent than 3 in
suppressing cell proliferation of PC-3 and LNCaP prostate cancer cell lines, ii) incorporation of an
ethyl group to 3-OH leads to the optimal derivative 9 with up to 36-fold enhanced potency as
compared with 3, but reverse PC-3 cell apoptotic response; iii) introduction of a dipentylaminopropyl
group at 3-OH of 3 has appreciably improved antiproliferative potency and capability in inducing PC-3
cell apoptosis, as compared with parent compound 3, and iv) 3-O-substituted-3',4',5'-
trimethoxyflavonols have higher potency towards PC-3 and LNCaP cell lines than DU145 cell line.
These findings imply that structural modification on 3-OH of trimethoxyflavonol can further enhance
its in vitro anti-proliferative potency in three human prostate cancer cell lines and its ability to activate
PC-3 cell apoptosis.
4. Experiments
4.1. General synthetic procedures: NMR spectra were obtained on a Bruker Fourier 300
spectrometer in CDCl3. The chemical shifts are given in ppm referenced to the solvent peak,
and coupling constants are reported in Hz. Anhydrous THF and dichloromethane were purified
by PureSolv MD 7 Solvent Purification System from Innovative Technologies (MB-SPS-800).
All other reagents and solvents were purchased from commercial sources and were used
without further purification. Silica gel column chromatography was performed using silica gel
14 | P a g e

(32-63 µm). Preparative thin-layer chromatography (PTLC) separations were carried out on
thin layer chromatography plates preloaded with silica gel GF254.
4.2. Synthesis of 3',4',5'-trimethoxyflavonol (3). The mixture of 2'-hydroxyacetophenone
(0.15 g, 1.08 mmol), 3',4',5'-trimethoxybenzaldehyde (0.20 g, 1.03 mmol), and potassium
o
hydroxide (0.19 g, 3.31 mmol, 3 equiv.) in ethanol (3 mL) was stirred at 0 C (ice bath) for 45
min. The reaction was then allowed to proceed at room temperature for 48 hours. The red
solution (3',4',5'-trimethoxychalcone) was obtained, to which aqueous solution of sodium
hydroxide (1 M, 0.8 mL, 0.8 mmol) and hydrogen peroxide (30%, 0.5 mL, 4.9 mmol) were
added at 0 °C (ice bath). After standing in cold room (4 °C) for 48 h, the resulting mixture was
stirred at room temperature until the starting material was fully consumed, as detected by TLC.
The reaction mixture was acidified with HCl solution (3M) to pH 2 to yield a precipitate, which
was filtered off and rinsed with 95% ethanol to give 3',4',5'-trimethoxyflavonol
(
3
) as a yellow
= 8.1, 1.2 Hz,
= 7.5
173.4, 155.3,
solid in 47% yield. M.p. 166-168
°
C; 1H NMR (300 MHz, CDCl3)
δ 8.23 (dd, J
1H), 7.69 (ddd,
J
= 8.6, 6.9, 1.5 Hz, 1H), 7.57 (d,
J
= 8.4 Hz, 1H), 7.52 (s, 2H), 7.40 (t,
J
Hz, 1H), 7.14 (br.s., 1H), 3.95 (s, 6H), 3.93 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ
153.3, 144.8, 140.0, 138.3, 133.7, 126.3, 125.5, 124.6, 120.7, 118.3, 105.5, 61.1, 56.4; HRMS
(ESI, m/z) calcd. for C18H17O6 [M+H]+ 329.1025, found 329.1016; IR (film) 3267, 2929, 2828,
1616, 1606, 1582, 1562, 1505 cm−1.
4.3. The general procedure for the synthesis of 3-O-alkyl-3',4',5'-trimethoxyflavonols (8-17). The
solution of 3',4',5'-trimethoxyflavonol (3, 50 mg, 0.15 mmol) and potassium carbonate (60 mg, 0.43
mmol) in DMF (0.5 mL, 0.3 M) was stirred for 10 min at room temperature. To this solution was
added an appropriate alkyl iodide or alkyl bromide (0.75 mmol) through syringe, the subsequent
reaction mixture was stirred at room temperature over 12 h until the starting material was completely
consumed as detected by TLC. The reaction mixture was poured into ice water (3 mL), and the
15 | P a g e

subsequent mixture was extracted with ethyl acetate three times. The combined extracts were
sequentially rinsed with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate,
and concentrated in vacuo to give a crude mass, which was subjected to PTLC purification using
hexane/ethyl acetate (100/3, v/v) as eluent to furnish the respective 3-O-alkyl-3',4',5'-
trimethoxyflavonol (8-17).
o
4.3.1. 3-O-Methyl-3',4',5'-trimethoxyflavonol (8).Yellow solid, 99% yield, mp. 88-90 C; H NMR
1
(300 MHz, CDCl3) δ 8.25 (dd, J = 8.1, 1.5 Hz, 1H), 7.68 (ddd, J = 8.6, 7.2, 1.8 Hz, 1H), 7.54 (d, J =
13
8.2 Hz, 1H), 7.42 – 7.37 (overlapped, 3H), 3.94 (s, 9H), 3.89 (s, 3H). C NMR (75 MHz, CDCl3) δ
175.1, 155.4, 155.2, 153.2, 141.4, 140.5, 133.6, 126.0, 125.92, 124.8, 124.2, 118.05, 106.2, 61.1, 60.2,
56.4; HRMS (ESI, m/z) calcd. for C19H19O6 [M+H]+ 343.1181, found 343.1177; IR (film) 2941,
2836,1644,1601, 1584, 1562, 1504 cm−1.
o
1
4.3.2. 3-O-Ethyl-3',4',5'-trimethoxyflavonol (9). Yellow solid, 81% yield, mp. 56-58 C; H NMR
(300 MHz, CDCl3) δ 8.25 (dd, J = 7.8, 1.5 Hz, 1H), 7.68 (ddd, J = 8.6, 7.2, 1.8 Hz, 1H), 7.54 (dd, J =
8.0, 0.5 Hz, 1H), 7.46 (s, 2H), 7.40 (ddd, J = 7.8, 7.2, 0.6 Hz, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.94 (s,
13
9H), 1.36 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, CDCl3) δ 175.2, 155.4, 155.2, 153.2, 140.4, 140.2,
133.4, 126.2, 125.8, 124.7, 124.1, 118.0, 106.2, 68.4, 61.0, 56.3, 15.8.; HRMS (ESI, m/z) calcd. for
C20H21O6 [M+H]+ 357.1338, found 357.1331; IR (film) 2982, 2937, 2836, 1627,1615, 1598, 1557,
1505 cm−1.
o
4.3.3. 3-O-Propyl-3',4',5'-trimethoxyflavonol (10). Yellow solid, 98% yield, mp. 84-86 C;1H NMR
(300 MHz, CDCl3) δ 8.25 (dd, J = 8.0, 1.4 Hz, 1H), 7.67 (ddd, J = 8.6, 6.9, 1.4 Hz, 1H), 7.53 (d, J =
8.3 Hz, 1H), 7.42 (s, 2H), 7.39 (t, J = 7.2 Hz, 1H), 4.00 (t, J = 6.9 Hz, 2H), 3.94 (s, 9H), 1.76 (sextet, J
13
= 7.2 Hz, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.1,
140.7, 140.3, 133.5, 126.3, 125.9, 124.8, 124.2, 118.0, 106.4, 74.6, 61.1, 56.4, 23.7, 10.5; HRMS (ESI,
16 | P a g e

m/z) calcd. for C21H23O6 [M+H]+ 371.1494, found 371.1487; IR (film) 2927, 2872, 1627, 1615, 1601,
1583, 1558, 1503 cm−1.
o
1
4.3.4. 3-O-Butyl-3',4',5'-trimethoxyflavonol (11). Yellow solid, 92% yield, m.p. 89-91 C. H NMR
(300 MHz, CDCl3) δ 8.23 (dd, J = 8.0, 1.5 Hz, 1H), 7.65 (ddd, J = 8.6, 6.9, 1.5 Hz, 1H), 7.52 (d, J =
8.4 Hz, 1H), 7.40 (s, 2H), 7.40-7.34 (overlapped, 1H), 4.02 (t, J = 6.7 Hz, 2H), 3.92 (s, 9H), 1.70 (quin,
J = 6.9 Hz, 2H), 1.41 (sextet, J = 7.5 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
175.2, 155.4, 155.2, 153.0, 140.6, 140.2, 133.4, 126.2, 125.8, 124.7, 124.2, 118.0, 106.3, 72.7, 61.1,
56.3, 32.4, 19.2, 13.9; HRMS (ESI, m/z) calcd. for C22H25O6 [M+H]+ 385.1651, found 385.1645; IR
(film) 2927, 2872, 1627, 1615, 1601, 1583, 1558, 1503 cm−1.
4.3.5. 3-O-Pentyl-3',4',5'-trimethoxyflavonol (12). Yellow solid, 89% yield, m.p. 71-73 oC. 1H NMR
(300 MHz, CDCl3) δ 8.23 (dd, J = 8.0, 1.5 Hz, 1H), 7.66 (ddd, J = 8.6, 6.9, 1.5 Hz, 1H), 7.52 (dd, J =
8.4, 0.5 Hz, 1H), 7.31 (s, 2H), 7.38 (ddd, J = 8.1, 7.2, 1.2 Hz, 1H), 4.02 (t, J = 6.7 Hz, 2H), 3.930 (s,
6H), 3.926 (s, 3H), 1.72 (quin, J = 7.2 Hz, 2H), 1.40 – 1.21 (m, 4H), 0.84 (t, J = 7.1 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.0, 140.6, 140.2, 133.4, 126.3, 125.9, 124.7, 124.2, 118.0,
106.3, 73.0, 61.1, 56.3, 30.1, 28.2, 22.5, 14.1; HRMS (ESI, m/z) calcd. for C23H27O6 [M+H]+ 399.1807,
found 399.1797; IR (film) 3000, 2928, 2872, 2835, 1630, 1611, 1600, 1583, 1562, 1504cm−1.
o
1
4.3.6. 3-O-Hexyl-3',4',5'-trimethoxyflavonol (13). Yellow solid, 89% yield, m.p. 54-56 C. H NMR
(300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.6 Hz, 1H), 7.66 (ddd, J = 8.7, 7.2, 1.2 Hz, 1H), 7.53 (d, J =
7.9 Hz, 1H), 7.40 (s, 2H), 7.39 (ddd, J = 8.0, 7.2, 0.9 Hz, 1H), 4.03 (t, J = 6.7 Hz, 2H), 3.934 (s, 6H),
3.932 (s, 3H), 1.71 (quin, J = 7.8 Hz, 2H), 1.40 – 1.30 (m, 2H), 1.30 – 1.19 (m, 4H), 0.84 (t, J = 6.8
13
Hz, 3H). C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.1, 140.7, 140.3, 133.5, 126.3, 125.9,
124.8, 124.2, 118.0, 106.4, 73.1, 61.1, 56.4, 31.7, 30.4, 25.8, 22.7, 14.1; HRMS (ESI, m/z) calcd. for
17 | P a g e

C24H29O6 [M+H]+ 413.1964, found 413.1962; IR (film) 2990, 2917, 2837, 1630, 1613, 1599, 1557,
1504 cm−1.
1
4.3.7. 3-O-Heptyl-3',4',5'-trimethoxyflavonol (14). Syrup, 96% yield. H NMR (300 MHz, CDCl3) δ
8.25 (dd, J = 8.0, 1.1 Hz, 1H), 7.68 (t, J = 6.9 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.41 (s, 2H), 7.39 (t, J
= 7.2 Hz, 1H), 4.04 (t, J = 6.7 Hz, 2H), 3.94 (s, 9H), 1.73 (quin, J = 6.6 Hz, 2H), 1.42 – 1.13
13
(overlapped, 8H), 0.85 (t, J = 6.6 Hz, 3H). C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.1,
140.7, 140.3, 133.4, 126.3, 125.9, 124.7, 124.2, 118.0, 106.4, 73.1, 61.1, 56.4, 31.8, 30.4, 29.2, 26.0,
22.6, 14.1; HRMS (ESI, m/z) calcd.for C25H31O6 [M+H]+ 427.2121, found 427.2113; IR (film) 2927,
2855, 1637, 1614, 1602 cm−1.
1
4.3.8. 3-O-Isopropyl-3',4',5'-trimethoxyflavonol (15). Syrup, 54% yield. H NMR (300 MHz,
CDCl3) δ 8.25 (dd, J = 8.0, 1.4 Hz, 1H), 7.67 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.54 (dd, J = 8.5, 0.6 Hz,
1H), 7.47 (s, 2H), 7.40 (ddd, J = 8.0, 6.9, 0.9 Hz, 1H), 4.76 (septet, J = 6.3 Hz, 1H), 3.944 (s, 6H),
13
3.940 (s, 3H), 1.22 (d, J = 6.2 Hz, 6H). C NMR (75 MHz, CDCl3) δ 175.6, 155.9, 155.3, 152.9,
140.2, 139.2, 133.4, 126.7, 126.0, 124.8, 124.1, 118.0, 106.7, 74.7, 61.2, 56.4, 22.7; HRMS (ESI, m/z)
calcd.for C21H23O6 [M+H]+ 371.1494, found 371.1488; IR (film) 2971, 2934, 2835, 1634, 1614, 1599,
1582, 1558, 1502 cm−1.
1
4.3.9. 3-O-Sec-Butyl-3',4',5'-trimethoxyflavonol (16). Syrup, 84% yield. H NMR (300 MHz,
CDCl3) δ 8.24 (dd, J = 8.0, 1.6 Hz, 1H), 7.67 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H),
7.45 (s, 2H), 7.39 (ddd, J = 7.8, 7.1, 0.9 Hz, 1H), 4.60 (sextet, J = 6.6 Hz, 1H), 3.93 (s, 9H), 1.79-1.67
13
(m, 1H), 1.57 – 1.44 (m, 1H), 1.12 (d, J = 6.2 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). C NMR (75 MHz,
CDCl3) δ 175.5, 155.8, 155.2, 152.9, 140.2, 139.2, 133.4, 126.7, 125.9, 124.7, 124.1, 118.0, 106.8,
79.3, 61.1, 56.4, 29.7, 19.4, 9.9; HRMS (ESI, m/z) calcd.for C22H25O6 [M+H]+ 385.1651, found
385.1644; IR (film) 2966, 2934, 2836, 1635, 1613, 1599, 1582, 1559, 1502 cm−1.
18 | P a g e

1
4.3.10. 3-O-(Pentan-2-yl)-3',4',5'-trimethoxyflavonol (17). Syrup, 78% yield. H NMR (300 MHz,
CDCl3) δ 8.23 (dd, J = 8.0, 1.6 Hz, 1H), 7.66 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H),
7.45 (s, 2H), 7.38 (ddd, J = 8.1, 6.9, 0.9 Hz, 1H), 4.68 (sextet, J = 6.1 Hz, 1H), 3.93 (s, 9H), 1.75 –
1.60 (m, 1H), 1.49-1.35 (m, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 175.5, 155.8, 155.2, 152.8, 140.2, 139.1, 133.3, 126.7, 125.9, 124.7, 124.1, 118.0, 106.7,
77.8, 61.1, 56.4, 39.2, 20.0, 18.8, 14.2.; HRMS (ESI, m/z) calcd.for C23H27O6 [M+H]+ 399.1807, found
399.1802; IR (film) 2932, 2871, 1635, 1614, 1599, 1582, 1559, 1502 cm−1.
4.4. The general procedure for the synthesis of 3-O-dialkylaminoalkyl-3',4',5'-
trimethoxyflavonols (19-30): The solution of 3',4',5'-trimethoxyflavonol (50 mg, 0.15 mmol) and
potassium carbonate (60 mg, 0.43 mmol, 3 equiv.) in DMF (0.5 mL, 0.3 M) was stirred at room
temperature for 10 min before an appropriate alkyl dibromide (0.51 mmol, 3 equiv.) was added
through a syringe. The reaction was allowed to proceed at room temperature over 12 h until the starting
material was fully consumed as indicated by TLC. The reaction mixture was poured into ice water (3
mL), and the subsequent mixture was extracted with ethyl acetate three times. The combined organic
extracts were sequentially rinsed with saturated sodium bicarbonate and brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo to generate a crude product. To the solution of this crude
product in DMF (0.1 M) was added potassium carbonate (3 equiv.), and the mixture was stirred for 10
min. The appropriate amine (3 equiv.) was added to the reaction mixture through a syringe, and the
subsequent reaction mixture was stirred at room temperature over 12 h until no starting material was
detected by TLC. The mixture was poured into ice water (3 mL), and the mixture was extracted with
ethyl acetate three times. The combined extracts were sequentially rinsed with saturated sodium
bicarbonate and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a crude
product, which was subjected to PTLC purification using dichloromethane/methanol (100/3, v/v) as
19 | P a g e

eluent. The respective 3-O-dialkylaminoalkyl-3',4',5'-trimethoxyflavonol was retrieved from PTLC
silica gel by washing with dichloromethane/diethylamine (100/3, v/v).
4.4.1. 3-O-(3-(Diethylamino)propyl)-3',4',5'-trimethoxyflavonol (19). Syrup, 84% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.24 (d, J = 7.3 Hz, 1H), 7.67 (t, J = 7.1 Hz, 1H), 7.53 (d, J =
8.4 Hz, 1H), 7.40-7.38 (overlapped, 1H), 7.38 (s, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.93 (s, 9H), 2.58-2.46
13
(overlapped, 6H), 1.96 – 1.81 (m, 2H), 0.97 (t, J = 7.1 Hz, 6H). C NMR (75 MHz, CDCl3) δ 175.1,
155.7, 155.2, 153.1, 140.5, 140.4, 133.5, 126.2, 125.9, 124.8, 124.2, 118.0, 106.5, 71.4, 61.1, 56.5,
49.9, 46.9, 27.8, 11.2; HRMS (ESI, m/z) calcd. for C25H32NO6 [M+H]+ 442.2230, found 442.2224; IR
(film) 2922, 2848, 1627, 1614, 1601, 1582, 1559, 1504 cm−1.
4.4.2. 3-O-(3-(Diethylamino)butyl)-3',4',5'-trimethoxyflavonol (20). Syrup, 69% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.3 Hz, 1H), 7.67 (ddd, J = 9.9, 8.1, 1.5 Hz,
1H), 7.53 (d, J = 8.3 Hz, 1H), 7.42-7.37 (overlapped, 1H), 7.41 (s, 2H), 4.06 (t, J = 6.6 Hz, 2H), 3.94
(s, 9H), 2.55-2.44 (overlapped, 6H), 1.74 (quin, J = 7.1 Hz, 2H), 1.56 (quin, J = 7.4 Hz, 2H), 1.00 (t, J
13
= 7.1 Hz, 6H). C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.3, 153.1, 140.6, 140.4, 133.5, 126.3,
126.0, 124.8, 124.3, 118.1, 106.4, 72.7, 61.1, 56.4, 52.5, 46.9, 28.4, 23.2, 11.5. HRMS (ESI, m/z)
calcd. For C26H34NO6 [M+H]+ 456.2386, found 456.2383; IR (film) 2916, 2848, 2793, 1625, 1614,
1600, 1581, 1557, 1504cm−1.
4.4.3. 3-O-(3-(Diethylamino)pentyl)-3',4',5'-trimethoxyflavonol (21). Syrup, 88% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 8.0 Hz, 1H), 7.66 (dd, J = 8.3, 7.1 Hz, 1H), 7.52
(d, J = 8.5 Hz, 1H), 7.39 (s, 2H), 7.39-7.35 (overlapped, 1H), 4.02 (t, J = 6.1 Hz, 2H), 3.92 (s, 9H),
2.51 (q, J = 6.9 Hz, 4H), 2.39 (t, J = 6.1 Hz, 2H), 1.82 – 1.67 (m, 2H), 1.51 – 1.31 (m, 4H), 0.99 (t, J =
13
7.2 Hz, 6H). C NMR (75 MHz, CDCl3) δ 175.1, 155.5, 155.2, 153.1, 140.6, 140.4, 133.4, 126.2,
125.9, 124.7, 124.2, 118.0, 106.5, 72.8, 61.1, 56.4, 52.7, 46.9, 30.3, 26.5, 24.1, 11.4; HRMS (ESI, m/z)
20 | P a g e

calcd. for C27H36NO6 [M+H]+470.2542, found 470.2538; IR (film) 2935, 1636, 1614, 1601, 1582,
1561, 1503 cm−1.
4.4.4. 3-O-(3-(Dipropylamino)propyl)-3',4',5'-trimethoxyflavonol (22). Syrup, 69% overall yield for
two steps. 1H NMR (300 MHz, CDCl3) δ 8.21 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.50 (d, J =
8.4 Hz, 1H), 7.36 (s, 2H), 7.38-7.33 (overlapped, 1H), 4.06 (t, J = 6.5 Hz, 2H), 3.91 (s, 9H), 2.50 (t, J =
6.8 Hz, 2H), 2.29 (t, J = 7.2 Hz, 4H), 1.92 – 1.78 (m, 2H), 1.43 – 1.27 (m, 4H), 0.78 (t, J = 7.3 Hz,
13
6H). C NMR (75 MHz, CDCl3) δ 175.1, 155.5, 155.2, 153.0, 140.4, 140.3, 133.4, 126.2, 125.8,
124.7, 124.2, 118.0, 106.3, 71.5, 61.0, 56.3, 56.1, 51.0, 28.1, 20.0, 11.9; HRMS (ESI, m/z) calcd.for
C27H36NO6 [M+H]+ 470.2542, found 470.2535; IR (film) 2945, 2866, 2803, 1634, 1615, 1603, 1583,
1563, 1504 cm−1.
4.4.5. 3-O-(3-(Dipropylamino)butyl)-3',4',5'-trimethoxyflavonol (23). Syrup, 68% overall yield for
two steps;1H NMR (300 MHz, CDCl3) δ 8.23 (dd, J = 8.0, 1.2 Hz, 1H), 7.66 (ddd, J = 8.4, 6.9, 1.5 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.40 (s, 2H), 7.38 (t, J = 7.2 Hz, 1H), 4.04 (t, J = 6.8 Hz, 2H), 3.92 (s,
9H), 2.39 (t, J = 7.3 Hz, 2H), 2.30 (t, J = 7.5 Hz, 4H), 1.73 (quin, J = 7.8 Hz, 2H), 1.58 – 1.45 (m, 2H),
1.45 – 1.31 (m, 4H), 0.81 (t, J = 7.3 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 175.2, 155.4, 155.2, 153.0,
140.6, 140.3, 133.5, 126.2, 125.9, 124.8, 124.2, 118.0, 106.3, 72.9, 61.1, 56.4, 56.1, 53.8, 28.4, 23.4,
20.2, 12.0; HRMS (ESI, m/z) calcd. for C28H38NO6 [M+H]+ 484.2699, found 484.2685; IR (film) 2998,
2925, 2872, 2795, 1626, 1601, 1583, 1561, 1503 cm−1.
4.4.6. 3-O-(3-(Dipropylamino)pentyl)-3',4',5'-trimethoxyflavonol (24). Syrup, 72% overall yield for
two steps;1H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.52 (d, J =
8.2 Hz, 1H), 7.40 (s, 2H), 7.40-7.35 (overlapped, 1H), 4.02 (t, J = 6.3 Hz, 2H), 3.92 (s, 9H), 2.38-2.26
(overlapped, 6H), 1.81 – 1.66 (m, 2H), 1.46-1.31 (overlapped, 8H), 0.83 (t, J = 7.1 Hz, 6H). 13C NMR
(75 MHz, CDCl3) δ 175.1, 155.4, 155.2, 153.0, 140.6, 140.3, 133.4, 126.2, 125.9, 124.7, 124.2, 118.0,
21 | P a g e

106.3, 72.9, 61.1, 56.3, 56.2, 54.1, 30.3, 26.8, 24.0, 20.1, 12.0; HRMS (ESI, m/z) calcd. for C29H40NO6
[M+H]+ 498.2856, found 498.2842; IR (film) 2933, 2869, 1638, 1614, 1602, 1582, 1562, 1503 cm−1.
4.4.7. 3-O-(3-(Dibutylamino)propyl)-3',4',5'-trimethoxyflavonol (25). Syrup, 69% overall yield for
two steps;1H NMR (300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.3 Hz, 1H), 7.67 (ddd, J = 8.4, 6.9, 1.5 Hz,
1H), 7.53 (d, J = 8.3 Hz, 1H), 7.40 (s, 2H), 7.40 (t, J = 7.2 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.94 (s,
9H), 2.62-2.52 (m, 2H), 2.39 (t, J = 6.9 Hz, 4H), 1.90 (quin, J = 7.5 Hz, 2H), 1.43 – 1.30 (m, 4H), 1.24
13
(quin, J = 7.1 Hz, 4H), 0.86 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl3) δ 175.2, 155.6, 155.3,
153.1, 140.5, 140.4, 133.5, 126.2, 125.9, 124.8, 124.3, 118.1, 106.4, 71.6, 61.1, 56.4, 53.9, 51.1, 28.9,
28.1, 20.8, 14.1; HRMS (ESI, m/z) calcd. for C29H40NO6 [M+H]+ 498.2855, found 498.2840; IR (film)
2930, 2870, 1637, 1614, 1602, 1582, 1562, 1503 cm−1.
4.4.8. 3-O-(3-(Dibutylamino)butyl)-3',4',5'-trimethoxyflavonol (26). Syrup, 60% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.4 Hz, 1H), 7.67 (ddd, J = 8.6, 6.9, 1.5 Hz,
1H), 7.53 (d, J = 8.1 Hz, 1H), 7.42 (s, 2H), 7.39 (t, J = 7.2 Hz, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.94 (s,
9H), 2.41 (t, J = 6.9 Hz, 2H), 2.36 (t, J = 7.5 Hz, 4H), 1.75 (quin, J = 7.5 Hz, 2H), 1.60 – 1.46 (m, 2H),
13
1.45 – 1.30 (m, 4H), 1.23 (sextet, J = 7.0 Hz, 4H), 0.87 (t, J = 7.2 Hz, 6H). C NMR (75 MHz,
CDCl3) δ 175.1, 155.4, 155.2, 153.0, 140.6, 140.3, 133.4, 126.2, 125.9, 124.7, 124.2, 118.0, 106.3,
72.9, 61.0, 56.3, 53.8, 53.7, 29.2, 28.4, 23.4, 20.8, 14.1; HRMS (ESI, m/z) calcd. for C30H42NO6
[M+H]+ 512.3012, found 512.3007; IR (film) 2930, 2870, 1637, 1614, 1602, 1582, 1562, 1503 cm−1.
4.4.9. 3-O-(3-(Dibutylamino)pentyl)-3',4',5'-trimethoxyflavonol (27). Syrup, 56% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 8.0 Hz, 1H), 7.66 (dd, J = 8.4, 7.1 Hz, 1H), 7.52
(d, J = 8.4 Hz, 1H), 7.40 (s, 2H), 7.38 (t, J = 7.8 Hz, 1H), 4.03 (t, J = 6.7 Hz, 2H), 3.93 (s, 9H), 2.37 (t,
J = 7.2 Hz, 6H), 1.75 (quin, J = 6.9 Hz, 2H), 1.49 – 1.31 (m, 8H), 1.25 (sextet, J = 7.2 Hz, 4H), 0.87 (t,
J = 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.1, 140.6, 140.4, 133.5, 126.3,
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125.9, 124.8, 124.2, 118.0, 106.4, 72.9, 61.1, 56.4, 54.1, 53.9, 30.4, 29.1, 26.8, 24.1, 20.8, 14.2; HRMS
(ESI, m/z) calcd. for C31H44NO6 [M+H]+ 526.3169, found 526.3165; IR (film) 2931, 2860, 1638, 1614,
1601, 1582, 1562, 1503 cm−1.
4.4.10. 3-O-(3-Dipentylamino)propyl)-3',4',5'-trimethoxyflavonol (28). Syrup, 32% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 7.9 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.54 (d, J =
8.4 Hz, 1H), 7.40 (s, 2H), 7.40 (t, J = 7.4 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.94 (s, 9H), 2.65-2.55 (m,
2H), 2.40 (t, J = 7.2 Hz, 4H), 2.01 – 1.84 (m, 2H), 1.40-1.19 (overlapped, 12H), 0.86 (t, J = 6.5 Hz,
13
6H). C NMR (75 MHz, CDCl3) δ 175.2, 155.6, 155.3, 153.1, 140.5, 140.5, 133.5, 126.2, 125.9,
124.8, 124.3, 118.1, 106.4, 71.5, 61.1, 56.5, 54.1, 51.1, 29.8, 22.8, 22.7, 14.2; HRMS (ESI, m/z) calcd.
for C31H44NO6 [M+H]+ 526.3169, found 526.3165; IR (film) 2928, 2858, 1638, 1614, 1602, 1582,
1562, 1504 cm−1.
4.4.11. 3-O-(3-(Dipentylamino)butyl)-3',4',5'-trimethoxyflavonol (29). Syrup, 43% overall yield for
two steps; 1H NMR (300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.3 Hz, 1H), 7.66 (ddd, J = 8.7, 7.2, 1.8 Hz,
1H), 7.53 (d, J = 8.4 Hz, 1H), 7.42 (s, 2H), 7.38 (t, J = 7.2 Hz, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.93 (s,
9H), 2.40 (t, J = 7.8 Hz, 2H), 2.34 (t, J = 7.8 Hz, 4H), 1.74 (quin, J = 7.5 Hz, 2H), 1.60 – 1.13 (m,
14H), 0.86 (t, J = 6.3 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 175.2, 155.4, 155.2, 153.1, 140.6, 140.4,
133.5, 126.3, 125.9, 124.8, 124.2, 118.0, 106.4, 72.9, 61.1, 56.4, 54.1, 53.8, 29.9, 28.5, 26.7, 26.6,
23.4, 22.9, 22.7, 14.2; HRMS (ESI, m/z) calcd. for C32H46NO6 [M+H]+ 540.3325, found 540.3317; IR
(film) 2929, 2859, 1639, 1614, 1602, 1582, 1563, 1504 cm−1.
4.4.12. 3-O-(3-(Dipentylamino)pentyl)-3',4',5'-trimethoxyflavonol (30). Syrup, 47% overall yield
for two steps; 1H NMR (300 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.3 Hz, 1H), 7.67 (ddd, J = 8.4, 6.9, 1.5
Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.41 (s, 2H), 7.39 (t, J = 7.2 Hz, 1H), 4.04 (t, J = 6.7 Hz, 2H), 3.93
(s, 9H), 2.37 (t, J = 7.8 Hz, 6H), 1.75 (quin, J = 6.9 Hz, 2H), 1.51 – 1.15 (m, 16H), 0.91 – 0.82
23 | P a g e

(overlapped, 6H). 13C NMR (75 MHz, CDCl3) δ 175.2, 155.5, 155.2, 153.1, 140.6, 140.4, 133.5, 126.3,
125.9, 124.8, 124.3, 118.0, 106.4, 72.9, 61.1, 56.4, 54.2, 54.1, 30.4, 29.9, 26.8, 26.7, 26.6, 24.1, 22.9,
22.7, 14.2; HRMS (ESI, m/z) calcd. for C33H48NO6 [M+H]+ 554.3481, found 554.3465; IR (film) 2930,
2859, 1639, 1614, 1602, 1582, 1562, 1504 cm−1.
4.5. Cell culture. All cell lines were initially purchased from American Type Culture Collection
(ATCCTM). The PC-3 and LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640
medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a
o
high humidity environment supplemented with 5% carbon dioxide at a temperature of 37 C. The
DU145 prostate cancer cells were routinely cultured in Eagle’s Minimum Essential Medium (EMEM)
supplemented with 10% FBS and 1% penicillin/spreptomycin. The PWR-1E non-neoplastic prostate
epithelial cells were routinely cultured in Keratinocyte serum free medium (K-SFM) supplemented
with bovine pituitary extract and human recombinant epidermal growth factor.
4.6. WST-1 cell proliferation assay. PC-3, LNCaP, DU145, or PWR-1E cells were plated in 96-well
plates at a density of 3,200 each well in 200 µL of culture medium. The cells were then treated with
quercetin, or synthesized flavonols at different doses for 3 days, while equal treatment volumes of
DMSO were used as vehicle control. The cells were cultured in a CO2 incubator at 37 oC for three days.
10 µL of the premixed WST-1 cell proliferation reagent (Clontech) was added to each well. After
mixing gently for one minute on an orbital shaker, the cells were incubated for additional 3 hours at 37
oC. To ensure homogeneous distribution of color, it is important to mix gently on an orbital shaker for
one minute. The absorbance of each well was measured using a microplate-reader (Synergy HT,
BioTek) at a wavelength of 430 nm. The IC50 value is the concentration of each compound that inhibits
cell proliferation by 50% under the experimental conditions and is the average from at least triplicate
determinations that were reproducible and statistically significant. For calculating the IC50 values, a
linear proliferative inhibition was made based on at least five dosages for each compound.
24 | P a g e

4.7. F2N12S and CYTOX AADvanced double staining assay: PC-3 cells were plated in 24-well plates
at a density of 200,000 each well in 400 µL of culture medium. After 3 hours of cell attachment, the
cells were then treated with the test compound at different concentrations in CO2 incubator at 37 °C for
16 hours, while equal treatment volumes of DMSO were used as vehicle control. Both attached and
floating cells were collected in a centrifuge tube by centrifugation at rcf value of 450 g for 5 to 6
minutes. The collected cells were re-suspended with 500 µL HBSS to remove proteins which may
affect flow signal. After centrifugation and discarding the supernatant, the collected cells were re-
suspended with 300 µl HBSS and stained with 0.3 µL of F2N12S for 3-5 minutes followed by 0.3 µL
SytoxAAdvanced for an additional 5 minutes. The fluorescence intensity of the two probes was further
measured in individual PC-3 cells using an Attune flow cytometer (Life Technologies) 0.5 to 1 hour
after staining.
4.8. Cell cycle analysis: PC-3 cells were plated in 24-well plates at a density of 200,000 each well in
400 µL of culture medium. After 3 hours of cell attachment, the cells were then treated with the test
derivative at 10 µM and 20 µM for 16 hours and 24 hours, respectively, in CO2 incubator at 37oC,
while equal treatment volumes of DMSO were used as vehicle control. Both attached and floating cells
were collected in a centrifuge tube by centrifugation at rcf value 450 g for 5 minutes. After discarding
the supernatant, the collected cells were re-suspended with 500 µL 80% cold ethanol to fix for 30
minutes in 4°C. The fixed cells could be stored in -20 °C for one week. After fixation, the ethanol was
removed after centrifuging and the cells were washed with PBS. The cells were then re-suspended with
100 µL of 100 mg/mL ribonuclease and were cultured at 37 °C for 30 minutes to degrade all RNA. The
cells were stained with 200 µL of 50 µg/mL propidium iodide stock solution for 30 minutes at -20 °C,
and then the fluorescence intensity of PI was detected in individual PC-3 cells using an Attune flow
cytometer (Life Technologies) within 0.5 to 1 hour after staining.
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4.9. Statistical analysis: All data are represented as the mean standard deviation (S.D.) for the
number of experiments indicated. Other differences between treated and control groups were analyzed
using the Student’s t-test. A p-value < 0.05 was considered statistically significant.
Acknowledgments
This study was financially supported by California State University (CSU)-Fresno. The HRMS data
were supported by NIH RCMI program at Xavier University of Louisiana through Grant
2G12MD007595 (G.W.). We are also grateful to the Graduate Net Initiative and the Division of
Graduate Studies at CSU-Fresno for (i) 2015-2016 Graduate Conference Travel Award (to X.L.), (ii)
Travel grant award (to X.L.), and (iii) Graduate Student Research and Creative Activities Support
Award (to X.L.).
Supplementary data
Supplementary data (comprised of copies of 1H and 13C NMR spectra for the final products)
associated with this article can be found, in the online version, at http://dx.doi.org/.
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GRAPHICAL ABSTRACT
Leave this area blank for abstract info.
3-O-Substituted-3',4',5'-
trimethoxyflavonols: synthesis and
cell-based evaluation as anti-prostate
cancer agents
Xiang Li, Maizie Lee, Guanglin Chen, Qiang Zhang, Shilong Zheng, Guangdi Wang, and Qiao-Hong Chen*
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
O
O
O
O
O
O
OCH3
OCH3
N
OCH3
O
OH
O
IC50 = 5.8 M (PC-3)
Arrest cell cycle at G0/G1 phase
Induce 93% apoptotic cells at 10
IC50 = 0.9 M (PC-3)
Arrest cell cycle at S and G0/G1 phase
Induce 3% apoptotic cells at 10
IC50 = 32.1 M (PC-3)
Arrest cell cycle at S and G2/M phase
Induce 39% apoptotic cells at 10
M
M
M
Key Words: Trimethoxyflavonol derivatives; Prostate cancer; Cell proliferation, Cell apoptosis; Cell
cycle regulation.
*Corresponding author: Tel: +1 559 278 2394; fax: +1 559 278 4402; e-mail: qchen@csufresno.edu
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