Nitrogen-containing derivatives of O-tetramethylquercetin: Synthesis and biological profiles in prostate cancer cell models

Article abstract of DOI:10.1016/j.bioorg.2019.03.047

Forty-eight nitrogen-containing quercetin derivatives were synthesized from readily available rutin or quercetin for the in vitro evaluation of their biological profiles. The WST-1 cell proliferation assay data indicate that thirty-nine out of the forty-eight derivatives possess significantly improved antiproliferative potency as compared with quercetin and fisetin, as well as the parent 3,3′,4′,7-O-tetramethylquercetin toward both androgen-sensitive (LNCaP) and androgen-insensitive (PC-3 and DU145) human prostate cancer cell lines. 5-O-Aminoalkyl-3,3′,4′,7-O-tetramethylquercetins were established as a better scaffold for further development as anti-prostate cancer agents. Among them, 5-O-(N,N-dibutylamino)propyl-3,3′,4′,7-O-tetramethylquercetin (44) was identified as the optimal derivative with IC₅₀ values of 0.55–2.82 μM, being over 35–182 times more potent than quercetin. The flow cytometry-based assays further demonstrate that 44 effectively activates PC-3 cell apoptosis.

Full text of DOI:10.1016/j.bioorg.2019.03.047

Accepted Manuscript
Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and Bi-
ological Profiles in Prostate Cancer Cell Models
Pravien Rajaram, Ziran Jiang, Guanglin Chen, Alyssa Rivera, Alison Phasakda,
Qiang Zhang, Shilong Zheng, Guangdi Wang, Qiao-Hong Chen
PII:
S0045-2068(18)30979-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.047
YBIOO 2873
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 September 2018
3 February 2019
15 March 2019
Please cite this article as: P. Rajaram, Z. Jiang, G. Chen, A. Rivera, A. Phasakda, Q. Zhang, S. Zheng, G. Wang, Q-
H. Chen, Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and Biological Profiles in Prostate
Cancer Cell Models, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.047
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Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and
Biological Profiles in Prostate Cancer Cell Models
a
a
a
a
a
b,c
Pravien Rajaram , Ziran Jiang , Guanglin Chen , Alyssa Rivera , Alison Phasakda , Qiang Zhang ,
b,c
b,c
a,
ShilongZheng , Guangdi Wang , and Qiao-Hong Chen *
a
Department of Chemistry, California State University, Fresno, 2555 E. San Ramon Avenue, M/S SB70,
Fresno, CA 93740, USA
b
c
Department of Chemistry and RCMI Cancer Research Center, Xavier University of Louisiana, 1
Drexel Drive, New Orleans, LA 70125, USA
ABSTRACT: Forty-eight nitrogen-containing quercetin derivatives were synthesized from readily
available rutin or quercetin for the in vitro evaluation of their biological profiles. The WST-1 cell
proliferation assay data indicate that thirty-nine out of the forty-eight derivatives possess significantly
improved antiproliferative potency as compared with quercetin and fisetin, as well as the parent
3
,3',4',7-O-tetramethylquercetin toward both androgen-sensitive (LNCaP) and androgen-insensitive
(PC-3 and DU145) human prostate cancer cell lines. 5-O-Aminoalkyl-3,3',4',7-O-tetramethylquercetins
were established as a better scaffold for further development as anti-prostate cancer agents. Among
them, 5-O-(N,N-dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44) was identified as the
optimal derivative with IC50 values of 0.55-2.82 µM, being over 35-182 times more potent than
quercetin. The flow cytometry-based assays further demonstrate that 44 effectively activates PC-3 cell
apoptosis.
Key words: Quercetin derivative, synthesis, antiproliferative activity, prostate cancer, cell apoptosis
_
___________
Corresponding author.
E-mail: qchen@csufresno.edu. Phone: (+1)5592782394. Fax: (+1)5592784402.
*
1

1
.
Introduction
3
,3',4',5,7-Pentahydroxyflavone (1, Figure 1), commonly known as quercetin, is a dietary flavonoid
ubiquitously distributed in a variety of diets especially in fruits and vegetables [1]. Several
epidemiologic studies led to the general conclusion that an inverse correlation exists between the
incidence of prostate cancer and the flavonoid-enriched diets in the East Asian [2-6]. This conclusion
inspired several research groups to assess the potential of quercetin, a most common and abundant
naturally occurring flavonoid, in treating and preventing various cancers including prostate cancer
alone or in combination with other dietary natural products [6-9]. Quercetin was revealed to suppress
cell proliferation in five prostate cancer cell line models with IC values ranging from 15.5 to 64 µM
5
0
[
10-13]. As summarized in our review article [7], the underlying mechanism of antiproliferative effects
of quercetin in prostate cancer cells was reported to be associated with the regulation of cell cycle, cell
apoptosis, and androgen receptor. Quercetin was also demonstrated to have significant in vivo anti-
prostate tumor efficacy either as a diet supplement or as a pure compound in three different animal
models [14-16]. Additionally, the safety profile of quercetin has been confirmed by a rat model and two
clinical studies [17-19]. The above-described cell-based and animal-based studies, as well as clinical
studies, corroborate the notion that quercetin warrants further exploration as an anti-prostate cancer
agent. However, its moderate potency, as evidenced by its IC values measured in in vitro studies and
5
0
the high dose (200 mg per kg body weight) needed for the in vivo animal studies, hinders its further
development as a drug candidate for prostate cancer. This study aims to engineer quercetin derivatives
with improved potency for the treatment of prostate cancer through chemical manipulations. Our
previous studies suggest that introduction of one substituent group, especially lengthy and/or bulky
group to a phenolic hydroxyl group might be the best strategy for modification of quercetin as anti-
prostate cancer agents [20]. Incorporation of an appropriate amino moiety to the 3-OH of 3',4'-
dimethoxyflavonol through a three-carbon linker led to the optimal derivative 2 with up to 292-fold
increase in potency [21] in three prostate cancer cell lines. Also, modification of 5-OH of 2,3-
2

dehydrosilybin (another group of flavonoids) resulted in an optimal derivative 3 that can consistently
inhibit prostate cancer cell proliferation [22]. Encouraged by these positive results, this study was
planned to explore the possibility of enhancing the antiproliferative potency of quercetin as anti-
prostate cancer agents through modification on its 3-OH or 5-OH group. Consequently, twenty-four 3-
O-aminoalkyl-3',4',5,7-O-tetramethylquercetins (11-34) and twenty-four 5-O-aminoalkyl-3,3',4',7-O-
tetramethylquercetins (38-61) have been designed and synthesized for the evaluation of their biological
profiles in prostate cancer cell models. 3',4',5,7-O-Tetramethylquercetin (6) and 3,3',4',7-O-
tetramethylquercetin (7) were chosen as the parent compounds because they have grounds to
selectively incorporate a linker at either 3-OH or 5-OH and were expected to overcome, to some
degree, pharmacokinetic limitations caused by the phenolic hydroxyl groups in quercetin.
Figure 1. Flavonoids as anti-prostate cancer agents
2
.
Results and discussion
2
.1 Chemistry
As shown in Scheme 1, 3',4',5,7-O-tetramethylquercetin (6) was synthesized from readily available
rutin through global methylation followed by glycoside hydrolysis according to the reported procedure
23]. 3,3',4',7-O-Tetramethylquercetin (7) was prepared from quercetin by selective tetramethylation of
[
quercetin using the procedure as described by Manthey and Guthrie (Scheme 2) [24-25]. As illustrated
in Scheme 3, twenty-four 3-O-aminoalkyl-3',4',5,7-O-tetramethylquercetins (11-34) have been
3

prepared by a two-step transformation including O-alkylation of 3',4',5,7-O-tetramethylquercetin (6)
with the appropriate dibromoalkanes followed by N-alkylation of the consequent 3-O-bromoalkyl-
3
',4',5,7-O-tetramethylquercetins (8-10) with the suitable amines. In both alkylation reactions,
potassium carbonate was used as the base and N,N-dimethylformamide (DMF) was used as the polar
aprotic solvent. Similarly, twenty-four 5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-61) have
been synthesized from 3,3',4',7-O-tetramethylquercetin (7, Scheme 4). It is worth noting that each of
crude 3-O-bromoalkyl-3',4',5,7-O-tetramethylquercetins (8-10) was pure enough for the follow-up N-
alkylation reaction. However, the O-alkylation of 3,3',4',7-O-tetramethylquercetin (7) cannot reach
1
00% conversion rate even under harsher reaction conditions including prolonged reaction time (48
o
hours at room temperature) or increased reaction temperature (70 C). Consequently, each of 5-O-
bromoalkyl-3,3',4',7-O-tetramethylquercetins (35-37) has to be purified by either column
chromatography or preparative thin layer chromatography (PTLC) over silica gel prior to being used
for the follow-up N-alkylation reaction. All pure 3-O-aminoalkyl-3',4',5,7-O-tetramethylquercetins (11-
3
4) and 5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-61) were obtained by PTLC purification
over silica gel. The mixture of dichloromethane:diethylamine (100:3, v/v) is the optimal solvent to
retrieve the desired nitrogen-containing products from the PTLC silica gel.
Scheme 1. Synthesis of 3',4',5,7-O-tetramethylquercetin (6)
4

Scheme 2. Synthesis of 3,3',4',7-O-tetramethylquercetin (7)
Scheme 3. Synthesis of 3-O-aminoalkyl-3',4',5,7-O-tetramethylquercetins (11-34). For the specific
structure for each of 11-34, see Table 1. R-H represents different amines. The structures for all R-H
were shown in the box.
5

Scheme 4. Synthesis of 5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-61). For the specific
structure for each of 38-61, see Table 2. R-H represents different amines. The structures for all R-H
were shown in the box.
2
.2 Antiproliferative activity towards prostate cancer cells
The in vitro antiproliferative activity of twenty-four 3-O-aminoalkyl-3',4',5,7-O-
tetramethylquercetins (11-34) and twenty-four 5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-
1) against a panel of prostate cancer cell lines (PC-3, DU145, and LNCaP) was determined using
6
WST-1 cell proliferation assay according to the procedure described in the Experimental Section.
Fisetin and quercetin were used as positive controls, and dimethyl sulfoxide (DMSO) was used as
negative control. As illustrated in Table 1, all of the twenty-four 3-O-aminoalkyl-3',4',5,7-O-
tetramethylquercetins (11-34) have greater antiproliferative potency than quercetin and fisetin as
indicated in their IC values in PC-3 and LNCaP human prostate cancer cell lines. Among them, only
5
0
fifteen derivatives are significantly more potent than the two positive controls in the DU145 prostate
cancer cell line. Derivatives 20 and 21 were identified as two optimal 3-O-aminoalkyl-3',4',5,7-O-
tetramethylquercetins with IC50 values of 1.73-6.53 µM. In contrast, all of the twenty-four 5-O-
aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-61, Table 2) are more potent than fisetin, quercetin,
6

and 3,3',4',7-O-tetramethylquercetin (7) against three prostate cancer cell lines. 5-O-(N,N-
Dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44) is established as the optimal derivative with
IC values of 0.55 µM, 2.82 µM, and 1.16 µM, respectively, towards PC-3, DU145, and LNCaP
5
0
prostate cancer cell lines.
The structure-antiproliferative activity relationship of O-aminoalkyl-O-tetramethylquercetins can be
summarized as below:
-
-
-
-
It can be clearly concluded that modification on 3-OH of 3',4',5,7-O-tetramethylquercetins and
-OH of 3,3',4',7-O-tetramethylquercetin did significantly improve their potency.
5
5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins are generally more potent than 3-O-
aminoalkyl-3',4',5,7-O-tetramethylquercetins towards prostate cancer cells.
N,N-dipentylamino moiety in 20 and 21 is the optimal nitrogen-containing group for the
antiproliferative activity of 3-O-aminoalkyl-3',4',5,7-O-tetramethylquercetins.
N,N-dibutylamino moiety in 44, and N,N-dipentylamino moiety in 47 & 48 serve as the most
preferred nitrogen-containing group for the enhanced in vitro antiproliferative potency of 5-O-
aminoalkyl-3,3',4',7-O-tetramethylquercetins.
-
All forty-eight derivatives are generally more effective in suppressing PC-3 and LNCaP cell
proliferation than in inhibiting the DU145 cell proliferation.
Table 1. Antiproliferative activity of 3-O-aminoalkyl-3',4',5,7-O-tetramethylquercetins (11-34)
7

a
IC (µM)
5
0
Compd
b
c
d
R
PC-3
>100
> 50
DU145
>100
> 50
LNCaP
Quercetin
Fisetin
-
45.5 ± 1.3
34.1 ± 7.7
-
1
1
1
1
2
3
(N,N-diethylamino)propyl
(N,N-diethylamino)butyl
(N,N-diethylamino)pentyl
16.62 ± 0.24
22.73 ± 7.56
18.33 ± 6.80
> 50
17.63 ± 1.04
19.44 ± 3.83
11.39 ± 2.27
> 50
> 50
1
1
1
1
1
4
5
6
7
8
(N,N-dipropylamino)propyl
(N,N-dipropylamino)butyl
(N,N-dipropylamino)pentyl
(N,N-dibutylamino)propyl
(N,N-dibutylamino)butyl
11.59 ± 1.98
11.05 ± 4.06
20.12 ± 3.71
10.70 ± 2.30
7.00 ± 2.11
25.41 ± 3.68
> 50
9.57 ± 1.97
10.99 ± 2.38
13.80 ± 3.88
7.14 ± 0.78
6.54 ± 1.45
34.81 ± 3.25
22.92 ± 4.56
15.12 ± 2.29
1
2
2
2
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
8
9
(N,N-dibutylamino)pentyl
(N,N-dipentylamino)propyl
(N,N-dipentylamino)butyl
(N,N-dipentylamino)pentyl
morpholinopropyl
7.60 ± 1.87
4.00 ± 0.63
3.81 ± 0.45
6.97 ± 1.09
20.38 ± 2.28
19.94 ± 2.37
13.19 ± 3.77
12.57 ± 4.27
31.28 ± 2.04
21.04 ± 2.21
20.13 ± 5.14
4.74 ± 0.51
6.53 ± 0.16
9.21 ± 0.21
> 50
7.18 ± 0.79
1.73 ± 0.34
3.45 ± 0.23
6.74 ± 0.79
27.34 ± 4.61
13.29 ± 2.71
8.48 ± 0.62
15.06 ± 2..48
21.49 ± 3.84
13.49 ± 3.37
morpholinobutyl
28.88 ± 4.26
18.05 ± 2.13
41.12 ± 3.70
> 50
morpholinopentyl
piperidinopropyl
piperidinobutyl
piperidinopentyl
27.99 ± 8.32
(4-methylpiperazin-1-
yl)propyl
3
2.01 ± 2.41
51.92 ± 4.67
> 50
22.39 ± 1.96
12.09 ± 3.24
14.62 ± 4.10
3
3
0
1
(4-methylpiperazin-1-yl)butyl 12.46 ± 0.84
(4-methylpiperazin-1-
19.89 ± 3.18
25.95 ± 0.12
yl)pentyl
3
3
3
2
3
4
pyrrolidinopropyl
pyrrolidinobutyl
pyrrolidinopentyl
24.01 ± 5.44
22.51 ± 1.82
22.87 ± 2.92
39.38 ± 7.72
> 50
18.58 ± 0.81
13.32 ± 2.90
11.37 ± 2.62
22.64 ± 2.37
8

a
IC is the compound concentration effective in inhibiting 50% of the cell viability measured by
5
0
WST-1 cell proliferation assay after 3 days exposure. The data were presented as the mean ± SD
from n = 3.
Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor
Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor
Human androgen-sensitive prostate cancer cell line derived from lymph node metastasis of prostate
b
c
d
tumor
Table 2. Antiproliferative activity of 5-O-aminoalkyl-3,3',4',7-O-tetramethylquercetins (38-61)
a
IC (µM)
5
0
Compd
b
c
d
R
PC-3
>100
> 50
>50
DU145
>100
> 50
LNCaP
Quercetin
Fisetin
7
-
45.5 ± 1.3
34.1 ± 7.7
>50
-
H
>50
3
3
4
8
9
0
(N,N-diethylamino)propyl
(N,N-diethylamino)butyl
(N,N-diethylamino)pentyl
16.38 ± 4.23
13.94 ± 1.34
17.06 ± 0.68
3.37 ± 1.23
4.71 ± 0.60
10.20 ± 1.14
0.55 ± 0.16
3.35 ± 0.84
4.78 ± 0.61
1.85 ± 0.29
2.10 ± 0.38
4.11 ± 1.11
12.15 ± 3.84
12.40 ± 3.17
11.81 ± 3.81
36.19 ± 3.83
14.94 ± 1.52
13.58 ± 2.23
14.38 ± 2.41
8.54 ± 2.64
6.79 ± 0.21
4
4
4
4
4
4
4
4
4
5
5
5
1
2
3
4
5
6
7
8
9
0
1
2
(N,N-dipropylamino)propyl
(N,N-dipropylamino)butyl
(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
morpholinopropyl
8.98 ± 1.57
8.02 ± 2.43
16.29 ± 4.71
2.82 ± 0.27
9.61 ± 2.03
12.97 ± 2.46
5.44 ± 0.24
5.33 ± 0.92
8.48 ± 0.50
27.95 ± 1.05
26.02 ± 3.66
22.93 ± 373
2.94 ± 0.54
2.53 ± 0.73
5.80 ± 0.80
1.16 ± 0.18
2.50 ± 0.42
3.14 ± 0.28
2.67 ± 0.38
1.23 ± 0.35
1.32 ± 0.04
12.82 ± 0.75
9.45 ± 2.13
6.87 ± 1.13
morpholinobutyl
morpholinopentyl
9

5
5
5
5
3
4
5
6
piperidinopropyl
piperidinobutyl
piperidinopentyl
(4-methylpiperazin-1-
yl)propyl
4.07 ± 2.12
10.31 ± 2.46
12.61 ± 0.65
9.09 ± 0.28
9.96 ± 1.97
14.98 ± 1.53
4.07 ± 0.33
6.54 ± 2.39
7.83 ± 1.77
4
.47 ± 0.92
15.70 ± 2.17
17.42 ± 3.54
20.46 ± 3.35
4.33 ± 0.74
2.97 ± 0.71
8.46 ± 0.78
5
5
7
8
(4-methylpiperazin-1-yl)butyl 12.34 ± 1.83
(4-methylpiperazin-1-
1
7.17 ± 2.12
yl)pentyl
5
6
6
9
0
1
pyrrolidinopropyl
pyrrolidinobutyl
pyrrolidinopentyl
5.80 ± 2.15
9.99 ± 2.22
15.87 ± 5.23
14.63 ± 5.95
13.92 ± 0.62
18.67 ± 5.52
9.42 ± 0.23
7.86 ± 1.80
10.28 ± 2.29
a
IC is the compound concentration effective in inhibiting 50% of the cell viability measured by
5
0
WST-1 cell proliferation assay after 3 days exposure. The data were presented as the mean ± SD
from n = 3.
Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor
Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor
Human androgen-sensitive prostate cancer cell line derived from lymph node metastasis of prostate
b
c
d
tumor
2
.3 Docetaxel Resistance Susceptibility
Additionally, the optimal derivative 44 was selected for further investigation on its antiproliferative
activity towards docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX) (Table 3).
The resistant cells were confirmed to have significantly lost their sensitivity to docetaxel treatment, as
evidenced in the 1232-fold (PC-4/DTX) and 7150-fold (DU-145/DTX) increase in IC50 values. In
contrast, compound 44 exhibited comparable antiproliferative activity in both docetaxel-sensitive and
docetaxel-resistant cells, and were slightly more potent than docetaxel in PC3/DXT and DU145/DTX
cell lines (Table 3).
Table 3. Antiproliferative activity of 44 against docetaxel-resistant prostate cancer cells.
a
d
a
c
Compd
IC : (nM)
R/S
IC : (nM)
R/S
5
0
50
b
c
e
f
PC-3
PC-3/DTX
DU145
DU145/DTX
1
0

Docetaxel 1.9 ± 0.6
550 ± 160
2340 ± 250
1700 ± 670
1232
3.1
1.2 ± 0.3
8580 ± 390
2290 ± 190
7150
0.8
4
4
2820 ± 270
a
IC is the compound concentration effective in inhibiting 50% of the cell viability measured by
5
0
WST-1 cell proliferation assay after 3 days exposure. The data were presented as the mean ± SD
from n = 3.
Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor
Docetaxel-resistant PC-3 prostate cancer cell line
b
c
d
The relative resistance of the two cell lines obtained by dividing the IC value of the resistance cell
5
0
line by the parental cell line
e
Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor
Docetaxel-resistant DU145 prostate cancer cell line
f
2
.4 Effects of 5-O-(N,N-dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44) on PC-3 cell cycle
progression and apoptosis
2
6
Quercetin was reported to induce PC-3 cell cycle arrest at G /M and S phase. To evaluate the effect
2
of 5-aminoalkyl group on the PC-3 prostate cancer cell cycle regulation, 5-O-(N,N-
dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44), the optimal derivative, was selected for
further assessment using flow cytometry analysis with propidium iodide DNA staining. When PC-3
cells were treated with 44 at 10 and 20 µM, a different cell cycle regulation was observed. Specifically,
4
4 causes slight accumulation of PC-3 cells in the G /G phase from 26% for control cells at 16 hours
0 1
to 28% (10 µM) and 30% (20 µM) for the compound-treated cells. The data imply that PC-3 cell
proliferation of 5-O-(N,N-Dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin may be associated
with its cell cycle regulation in the G /G phase.
0
1
The cell proliferation inhibition of quercetin was revealed to be associated with its cell apoptosis
activation in several prostate cancer cell models [12,26-29]. To evaluate the effect of 5-O-substitution
on the PC-3 cell apoptosis, 5-O-(N,N-dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin as the most
promising derivative, was chosen for F2N12S and SYTOX AADvanced double staining flow
cytometry-based assay at 0-50 µM dosing range. The results (Figures 2 and 3) imply that treatment of
PC-3 cells with 44 for 16 h led to PC-3 cells dying from apoptosis rather than necrosis in a dose-
1
1

responsive manner. Specifically, treatment with 10 µM of derivative 44 resulted in 32% early apoptotic
cells and only 4% late apoptotic/necrotic cells; 20 µM of derivative 44 significantly activated apoptosis
as well, with 50% early apoptotic cells and 11% late apoptotic/necrotic cells. Both apoptotic and
necrotic cell populations increased in response to increasing concentration of 44 (0-50 µM
concentration range). The apoptotic cell population reached maximum when exposure PC-3 cancer
cells to 44 at 30 µM concentration.
Apoptotic Effect of 44 against PC-3 Cells
1
1
20.0
00.0
9
6.5
9
5.1
91.0
89.9
8
0.0
8
6
4
2
0.0
0.0
0.0
0.0
6
4.0
6
2.5
2
5
0.1
3
8.1
32.0
8.0
1
9.1
11.1
9
.2
5
.2
6.9
2.6
2.6
3
.7
1
.6
0.8
50 μM
1
.1
1.4
3.0
0
.0
Control
DMSO
1 μM
5 μM
10 μM
20 μM
30 μM
Concentration of compound 44
Viable Cell
Apoptotic Cell
Necrotic Cell
Figure 2. Evolution of viable, apoptotic, and necrotic PC-3 cell populations in response to 44
Control
44-(10 μM)-treated
1
2

4
4-(20 μM)-treated
44-(30 μM)-treated
Figure 3. Apoptosis in PC-3 cells treated with 44 at 10, 20, and 40 µM (by F2N12S and SYTOX
AADvanced double staining)
3
. Conclusion
In summary, forty-eight quercetin derivatives containing a nitrogen group at 3-OH of 3',4',5,7-O-
tetramethylquercetin and 5-OH of 3,3',4',7-O-tetramethylquercetin were synthesized from rutin or
quercetin. The WST-1 cell proliferation assay data indicate that thirty-nine out of the forty-eight
derivatives possess significantly improved antiproliferative potency as compared with quercetin,
fisetin, and 3,3',4',7-O-tetramethylquercetin against both androgen-sensitive (LNCaP) and androgen-
insensitive prostate cancer cell lines (PC-3 and DU145). 5-O-aminoalkyl-3,3',4',7-O-
tetramethylquercetins serve as a better scaffold for further development of anti-prostate cancer agents.
Among them, 5-O-(N,N-Dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44) was identified as
the optimal derivative with IC values of 0.55-2.82 µM, being over 35- to 182-fold more potent than
5
0
quercetin. The flow cytometry-based assays furhter demonstrate that 44 can significantly induce PC-3
cell apoptosis.
4
. Experimental Section
4
.1. General Procedures
High resolution mass spectrometry (HRMS) was obtained on an Orbitrap mass spectrometer with
electrospray ionization (ESI). Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker
1
3

Fourier 300 spectrometer in CDCl . The chemical shifts are given in ppm referenced to the respective
3
solvent peak, and coupling constants are reported in Hz. All reagents and solvents were purchased from
commercial sources and were used without further purification. Silica gel column chromatography was
performed using silica gel (32-63 µ). Preparative thin-layer chromatography (PTLC) separations were
carried out on thin layer chromatography plates loaded with silica gel 60 GF254 (EMD Millipore
Corporation, MA, USA). 3',4',5,7-O-Tetramethylquercetin (6) was synthesized according to the
reported procedure [23]. Quercetin was purchased from Fisher Scientific (Alfa Aesar, Cat# A1580722).
4
.2. Synthesis of 3,3',4',7-O-tetramethylquercetin (7) [24-25]
This compound was synthesized by a modified method of Manthey and Guthrie [24]. Quercetin (3.0
g, 10 mmol) was added to a solution of acetone (150 mL), water (75 mL) and 30% aqueous KOH
solution (6 mL) in a three-neck round bottom flask. The reaction mixture was refluxed for 10 min.
Dimethyl sulfate (2.4 mL) was added, and the mixture was refluxed for 20 min. KOH solution (3 mL)
was added, producing a dark brown solution. An additional 2.4 mL of dimethyl sulfate was added, and
the solution was again refluxed for 20 min. KOH (3.0 mL) was added, followed by dimethyl sulfate
(0.6 mL). An additional aliquot of dimethyl sulfate (3.0 mL) was added and the mixture was refluxed
for 1.5 h and allowed to cool down and stand at room temperature overnight. Yellow crystals (1.82 g)
were collected and directly used for the next step reaction.
4
.3 The general procedure for the synthesis of 3-O-dialkylaminoalkyl-3',4',5,7-O-tetramethylquercetins
11-34)
The solution of 3',4',5,7-O-tetramethylquercetin (6, 60 mg, 0.17 mmol) and potassium carbonate (70
(
mg, 0.51 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
stirred at room temperature for 12 h until the starting material was fully consumed as indicated by TLC.
The reaction mixture was diluted with a mixture of ethyl acetate and diethyl ether (100 mL, 1:1, v/v),
1
4

which was rinsed with water (10 mL × 1) and brine (10 mL × 4). The subsequent organic layer was
dried over anhydrous sodium sulfate and concentrated in vacuo to generate a crude product (8, 9, or
1
0). To the solution of this crude product in DMF (0.5 mL, 0.3 M) was added potassium carbonate (70
mg, 0.51 mmol, 3 equiv.), and the mixture was stirred for 10 min. The appropriate amine (0.51 mmol, 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 reaction
mixture was diluted with a mixture of ethyl acetate and diethyl ether (100 mL, 1:1, v/v), which was
rinsed with water (10 mL × 1) and brine (10 mL × 4). The subsequent organic layer was dried over
anhydrous sodium sulfate and concentrated in vacuo to generate a crude product, which was subjected
to PTLC purification using dichloromethane/methanol (90/10, v/v) as eluent. The respective 3-O-
dialkylaminoalkyl-3',4',5,7-O-tetramethylquercetin was retrieved from PTLC silica gel by washing
with dichloromethane/diethylamine (100/3, v/v).
4
.3.1. 3-O-(N,N-Diethylamino)propyl-3',4',5,7-O-tetramethylquercetin (11)
Yield, 69%; yellow wax. IR (film) νmax: 2965, 2836, 1622, 1600, 1513, 1457 cm . H NMR (300
MHz, CDCl ) : 7.70-7.59 (overlapped, 1H), 7.65 (s, 1H), 6.92 (d, J = 9.0 Hz, 1H), 6.44 (s, 1H), 6.28
-1 1
3
(s, 1H), 4.01 (t, J = 6.3 Hz, 2H), 3.91 (s, 9H), 3.85 (s, 3H), 2.56 (t, J = 7.5 Hz, 2H), 2.47 (q, J = 7.2 Hz,
1
3
4
1
5
4
H), 1.86 (quin, J = 7.8 Hz, 2H), 0.95 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.1, 163.9,
3
61.0, 158.8, 152.8, 150.8, 148.6, 140.3, 123.5, 121.8, 111.4, 110.8, 109.5, 95.8, 92.5, 71.0, 56.4, 56.1,
6.0, 55.8, 49.8, 46.9, 27.8, 11.5. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 472.2335; found
2
6
34
7
72.2335.
4
.3.2. 3-O-(N,N-Diethylamino)butyl-3',4',5,7-O-tetramethylquercetin (12)
-1 1
Yield, 39%; yellow wax. IR (film) νmax: 2937, 1624, 1602, 1514 cm . H NMR (300 MHz, CDCl )
3
: 7.71 (s, 1H), 7.69 (dd, J = 6.9, 2.1 Hz, 1H), 6.97 (d, J = 9.3 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 6.34
(d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.96 (s, 9H), 3.90 (s, 3H), 2.59-2.49 (overlapped, 6H),
1
5

1
3
1
1
7
4
.73 (quin, J = 6.9 Hz, 2H), 1.65-1.53 (m, 2H), 1.03 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) :
3
74.1, 164.1, 161.0, 159.0, 153.4, 151.1, 148.8, 139.9, 123.2, 122.0, 111.2, 111.1, 109.4, 96.0, 92.7,
0.8, 56.6, 56.3, 56.2, 56.0, 51.3, 46.6, 27.5, 20.7, 9.0. HR-MS (ESI) m/z: calcd for C H NO [M+H]:
2
7
36
7
86.2492; found 486.2492.
4
.3.3. 3-O-(N,N-Diethylamino)pentyl-3',4',5,7-O-tetramethylquercetin (13)
-1 1
Yield, 61%; yellow oil. IR (film) νmax: 2937, 1622, 1601, 1513 cm . H NMR (300 MHz, CDCl ) :
3
7
1
.68 (s, 1H), 7.66 (d, J = 11.1 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 1.8 Hz, 1H), 6.30 (d, J =
.8 Hz, 1H), 3.98 (t, J = 6.6 Hz, 2H), 3.92 (s, 9H), 3.86 (s, 3H), 2.55 (q, J = 6.9 Hz, 4H), 2.42 (t, J = 7.2
1
3
Hz, 2H), 1.72 (quin, J = 7.5 Hz, 2H), 1.50-1.35 (m, 4H), 1.01 (t, J = 7.2 Hz, 6H). C NMR (75 MHz,
CDCl ) : 174.2, 163.9, 161.1, 158.9, 152.9, 150.8, 148.6, 140.4, 123.6, 121.8, 111.5, 110.8, 109.5,
3
9
5.8, 92.5, 72.3, 56.4, 56.1, 56.0, 55.9, 52.6, 46.8, 30.2, 26.1, 24.1, 11.2. HR-MS (ESI) m/z: calcd for
C H NO [M+H]: 500.2648; found 500.2645.
28
38
7
4
.3.4. 3-O-(N,N-Dipropylamino)propyl-3',4',5,7-O-tetramethylquercetin (14)
Overall yield for two steps, 22%; yellow syrup. IR (film) vmax: 2957, 2872, 2361, 1601 cm . H
NMR (300 MHz, CDCl ) δ 7.68 (dd, J = 7.5, 1.8 Hz, 1H), 7.66 (s, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.49
-1 1
3
(d, J = 2.1 Hz, 1H), 6.33 (d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 3.94 (s, 6H), 3.89 (s,
3
H), 2.70 (t, J = 6.6 Hz, 2H), 2.43 (t, J = 7.5 Hz, 4H), 1.94 (quin, J = 6.3 Hz, 2H), 1.46 (sextet, J = 7.5
1
3
Hz, 4H), 0.83 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) δ 174.2, 164.0, 161.1, 158.9, 153.0,
3
1
4
50.9, 148.7, 140.3, 123.6, 121.9, 111.5, 110.9, 109.5, 95.9, 92.6, 70.8, 56.5, 56.2, 56.1, 56.0, 51.2,
7.7, 27.6, 19.6, 11.9. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 500.2648; found 500.2637.
2
8
38
7
4
.3.5. 3-O-(N,N-Dipropylamino)butyl-3',4',5,7-O-tetramethylquercetin (15)
-1 1
Yield, 92%; yellow oil. IR (film) νmax: 2954, 1622, 1601, 1513 cm . H NMR (300 MHz, CDCl ) :
3
7
.67 (s, 1H), 7.66 (d, J = 11.1 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.44 (s, 1H), 6.27 (s, 1H), 3.99 (t, J =
1
6

6
7
.6 Hz, 2H), 3.91 (s, 9H), 3.84 (s, 3H), 2.40 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.5 Hz, 4H), 1.69 (quin, J =
1
3
.2 Hz, 2H), 1.52 (quin, J = 6.9 Hz, 2H), 1.38 (sextet, J = 7.5 Hz, 4H), 0.80 (t, J = 7.2 Hz, 6H). C
NMR (75 MHz, CDCl ) : 174.1, 163.9, 161.0, 158.8, 152.7, 150.7, 148.5, 140.3, 123.6, 121.7, 111.4,
3
1
10.7, 109.4, 95.7, 92.4, 72.4, 56.4, 56.1, 56.0, 56.0, 55.8, 53.8, 28.4, 23.3, 20.0, 11.9. HR-MS (ESI)
m/z: calcd for C H NO [M+H]: 514.2805; found 514.2801.
2
9
40
7
4
.3.6. 3-O-(N,N-Dipropylamino)pentyl-3',4',5,7-O-tetramethylquercetin (16)
-1 1
Yield, 38%; yellow oil. IR (film) νmax: 2939, 1620, 1600, 1513 cm . H NMR (300 MHz, CDCl ) :
3
7
1
3
6
1
1
.69 (dd, J = 8.7, 2.1 Hz, 1H), 7.65 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 2.1 Hz,
H), 6.34 (d, J = 2.1 Hz, 1H), 3.97 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 3H), 3.94 (s, 3H), 3.89 (s,
H), 2.96-2.86 (overlapped, 6H), 1.87-1.74 (m, 8H), 1.53 (quin, J = 7.5 Hz, 2H), 0.96 (t, J = 7.5 Hz,
1
3
H). C NMR (75 MHz, CDCl ) : 174.2, 164.1, 161.1, 159.0, 153.2, 151.1, 148.8, 140.2, 123.5,
3
22.0, 111.4, 111.0, 109.5, 96.0, 92.7, 71.5, 56.5, 56.3, 56.2, 56.0, 54.4, 52.6, 29.5, 23.7, 22.8, 17.3,
1.4. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 528.2961; found 528.2961.
3
0
42
7
4
.3.7. 3-O-(N,N-Dibutylamino)propyl-3',4',5,7-O-tetramethylquercetin (17)
Overall yield for two steps, 33%; yellow syrup. IR (film) vmax: 3408, 2960, 1601, 1515 cm . H
NMR (300 MHz, CDCl ) : 7.67 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.50 (d, J =
-1 1
3
1
.8 Hz, 1H), 6.34 (d, J = 1.5 Hz, 1H), 4.00-3.90 (overlapped, 2H), 3.94 (s, 9H), 3.88 (s, 3H), 3.46-3.41
(m, 2H), 3.04-2.96 (m, 4H), 2.36-2.21 (m, 2H), 1.76 (quin, J = 7.5 Hz, 4H), 1.44-1.32 (m, 4H), 0.94 (t,
1
3
J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.2, 164.3, 161.0, 159.0, 153.7, 151.3, 149.0, 139.6,
3
1
1
22.9, 122.1, 111.2, 111.0, 109.2, 96.1, 92.7, 69.1, 56.6, 56.4, 56.2, 56.0, 52.8, 51.4, 25.5, 25.3, 20.3,
3.7. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 528.2961; found 528.2963.
3
0
42
7
4
.3.8. 3-O-(N,N-Dibutylamino)butyl-3',4',5,7-O-tetramethylquercetin (18)
1
7

-1 1
Yield, 68%; yellow wax. IR (film) νmax: 2932, 2870, 1622, 1601, 1513, 1489, 1457 cm . H NMR
(
300 MHz, CDCl ) : 7.69 (s, 1H), 7.67 (d, J = 9.3 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.46 (s, 1H), 6.30
3
(s, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.92 (s, 9H), 3.86 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 7.8 Hz,
4
H), 1.71 (quin, J = 6.9 Hz, 2H), 1.54 (quin, J = 6.9 Hz, 2H), 1.36 (quin, J = 7.2 Hz, 4H), 1.24 (sextet,
1
3
J = 6.9 Hz, 4H), 0.86 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.2, 163.9, 161.1, 158.9,
3
1
52.8, 150.8, 148.6, 140.4, 123.6, 121.8, 111.5, 110.8, 109.5, 95.8, 92.5, 72.4, 56.4, 56.1, 56.0, 55.8,
5
3.8, 53.7, 28.8, 28.4, 23.1, 20.8, 14.1. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 542.3118;
3
1
44
7
found 542.3116.
4
.3.9. 3-O-(N,N-Dibutylamino)pentyl-3',4',5,7-O-tetramethylquercetin (19)
-1 1
Yield, 51%; yellow oil. IR (film) νmax: 2932, 2860, 1622, 1601, 1513 cm . H NMR (300 MHz,
CDCl ) : 7.70 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.47 (d, J = 1.5 Hz, 1H), 6.32
3
(s, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.94 (s, 9H), 3.88 (s, 3H), 2.42 (t, J = 7.5 Hz, 6H), 1.71 (quin, J = 6.9
1
3
Hz, 2H), 1.78 – 1.24 (m, 12 H), 0.88 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.2, 163.9,
3
1
5
61.1, 158.9, 152.8, 150.8, 148.6, 140.5, 123.7, 121.8, 111.6, 110.8, 109.6, 95.8, 92.5, 72.5, 56.5, 56.2,
6.1, 55.9, 54.0, 53.8, 30.3, 28.8, 26.4, 24.1, 20.8, 14.2. HR-MS (ESI) m/z: calcd for C H NO
7
3
2
46
[
M+H]: 556.3274 ; found 556.3272.
4
.3.10. 3-O-(N,N-Dipentylamino)propyl-3',4',5,7-O-tetramethylquercetin (20)
Yield, 70%; yellow oil. IR (film) νmax: 2929, 2855, 1620, 1597, 1514, 1458 cm . H NMR (300
MHz, CDCl ) : 7.70 (dd, J = 8.7, 2.1 Hz, 1H), 7.58 (d, J = 1.8 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.53
-1 1
3
(d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 4.04 (t, J = 4.2 Hz, 2H), 3.98 (s, 9H), 3.91 (s, 3H), 3.56-
3
6
.45 (m, 2H), 3.08-2.99 (m, 4H), 2.42-2.31 (m, 2H), 1.89-1.77 (m, 4H), 1.41-1.20 (m, 8H), 0.91 (t, J =
13
.6 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.2, 164.0, 161.2, 159.0, 152.9, 150.9, 148.7, 140.4,
3
1
8

1
2
23.7, 121.9, 111.5, 110.8, 109.6, 95.9, 92.6, 71.1, 56.6, 56.2, 56.1, 55.9, 54.2, 51.2, 29.9, 28.0, 26.5,
2.8, 14.2. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 556.3274; found 556.3273.
3
2
46
7
4
.3.11. 3-O-(N,N-Dipentylamino)butyl-3',4',5,7-O-tetramethylquercetin (21)
-1 1
Yield, 54%; yellow oil. IR (film) νmax: 2931, 1624, 1603, 1514 cm . H NMR (300 MHz, CDCl ) :
3
7
1
.69 (d, J = 1.5 Hz, 1H), 7.65 (dd, J = 8.4, 1.8 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 1.8 Hz,
H), 6.27 (d, J = 2.1 Hz, 1H), 3.99 (t, J = 6.9 Hz, 2H), 3.91 (s, 6H), 3.89 (s, 3H), 3.84 (s, 3H), 2.36 (t, J
=
7.2 Hz, 2H), 2.31 (t, J = 7.8 Hz, 4H), 1.69 (quin, J = 7.2 Hz, 2H), 1.48 (quin, J = 6.0 Hz, 2H), 1.35
1
3
(
quin, J = 7.5 Hz, 4H), 1.28-1.15 (overlapped, 8H), 0.83 (t, J = 7.2 Hz, 6H). C NMR (75 MHz,
CDCl ) : 174.1, 163.9, 161.0, 158.8, 152.6, 150.7, 148.6, 140.4, 123.6, 121.7, 111.5, 110.7, 109.4,
3
9
5.7, 92.4, 72.5, 56.4, 56.1, 56.0, 55.8, 54.1. 53.9, 29.8, 28.4, 26.6, 23.3, 22.7, 14.2. HR-MS (ESI) m/z:
calcd for C H NO [M+H]: 570.3431; found 570.3429.
3
3
48
7
4
.3.12. 3-O-(N,N-Dipentylamino)pentyl-3',4',5,7-O-tetramethylquercetin (22)
-1 1
Yield, 45%; yellow oil. IR (film) νmax: 2931, 1624, 1603, 1514 cm . H NMR (300 MHz, CDCl ) :
3
7
.70 (s, 1H), 7.69 (dd, J = 8.7, 2.1 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.49 (d, J = 2.1 Hz, 1H), 6.34 (d, J
=
2.1 Hz, 1H), 4.00 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 6H), 3.89 (s, 3H), 2.63-2.50 (overlapped,
6
7
1
2
H), 1.75 (quin, J = 7.5 Hz, 2H), 1.62-1.38 (overlapped, 6H), 1.34-1.18 (overlapped, 10H), 0.88 (t, J =
13
.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.2, 164.0, 161.2, 159.0, 152.9, 150.9, 148.7, 140.4,
3
23.7, 121.9, 111.6, 110.9, 109.6, 95.9, 92.6, 72.3, 56.5, 56.3, 56.1, 55.9, 53.7, 53.6, 31.1, 30.2, 29.7,
5.5, 24.0, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 584.3587; found 584.3586.
3
4
50
7
4
.3.13. 3-O-Morpholinopropyl-3',4',5,7-O-tetramethylquercetin (23)
Overall yield for two steps, 36%; yellow solid. IR (film) vmax: 2937, 2847, 1621, 1600, 1513 cm .
-1
1
H NMR (300 MHz, CDCl ) δ 7.68 (dd, J = 8.7, 2.1 Hz, 1H), 7.66 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H),
3
6
.50 (d, J = 2.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.95 (s, 9H), 3.90 (s, 3H),
1
9

13
3
.76 (t, J = 3.6 Hz, 4H), 2.77-2.63 (m, 2H), 2.63-2.48 (m, 4H), 2.00 (quin, J = 6.9 Hz, 2H). C NMR
(
75 MHz, CDCl ) δ 174.2, 164.1, 161.2, 159.0, 153.2, 151.0, 148.8, 140.2, 123.5, 122.0, 111.5, 110.9,
3
1
09.5, 96.0, 92.6, 70.4, 66.4, 56.6, 56.3, 56.2, 56.0, 56.0, 53.5, 27.0. HR-MS (ESI) m/z: calcd for
C H NO [M+H]: 486.2128; found 486.2127.
26
32
8
4
.3.14. 3-O-Morpholinobutyl-3',4',5,7-O-tetramethylquercetin (24)
Yield, 89%; yellow oil. IR (film) νmax: 2943, 1623, 1602, 1514, 1456 cm . H NMR (300 MHz,
CDCl ) : 7.64 (d, J = 9.0 Hz, 1H), 7.60 (s, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 1.2 Hz, 1H), 6.31
-1 1
3
(s, 1H), 4.00-3.85 (overlapped, 6H), 3.91 (s, 9H), 3.86 (s, 3H), 2.99 (t, J = 6.3 Hz, 2H), 2.99-2.83 (m
1
3
4
1
6
5
H), 1.95 (quin, J = 5.8 Hz, 2H), 1.76 (quin, J = 6.0 Hz, 2H). C NMR (75 MHz, CDCl ) : 174.2,
3
64.1, 160.9, 158.9, 153.3, 151.0, 148.7, 139.9, 123.2, 121.9, 111.2, 110.9, 109.3, 95.9, 92.6, 71.0,
4.8, 57.8, 56.5, 56.2, 56.1, 55.9, 52.4, 27.5, 21.3. HR-MS (ESI) m/z: calcd for C H NO [M+H]:
2
7
34
8
00.2284; found 500.2275.
4
.3.15. 3-O-Morpholinopentyl-3',4',5,7-O-tetramethylquercetin (25)
-1 1
Yield, 58%; yellow oil. IR (film) νmax: 2937, 2857, 1621, 1600, 1512 cm . H NMR (300 MHz,
CDCl ) : 7.70 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.47 (s, 1H), 6.31 (s, 1H),
3
3
6
1
6
.99 (t, J = 6.3 Hz, 2H), 3.93 (s, 9H), 3.87 (s, 3H), 3.75-3.64 (m, 4H), 2.50-2.33 (m, 4H), 2.28 (t, J =
13
.3 Hz, 2H), 1.72 (quin, J = 6.9 Hz, 2H), 1.56-1.34 (m, 4H). C NMR (75 MHz, CDCl ) : 174.2,
3
64.0, 161.1, 158.9, 152.9, 150.8, 148.6, 140.4, 123.7, 121.8, 111.6, 110.8, 109.5, 95.8, 92.5, 72.3,
6.9, 59.1, 56.5, 56.2, 56.1, 55.9, 53.8, 30.3, 26.2, 24.0. HR-MS (ESI) m/z: calcd for C H NO
8
2
8
36
[
M+H]: 514.2441; found 514.2447.
4
.3.16. 3-O-Piperidinopropyl-3',4',5,7-O-tetramethylquercetin (26)
Yield for two steps, 80%; yellow/orange solid. IR (film) vmax: 2935, 1624, 1600, 1513 cm . H NMR
300 MHz, CDCl ) δ 7.65 (d, J = 5.7 Hz, 1H), 7.64 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.45 (s, 1H), 6.29
-1 1
(
3
2
0

(s, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.92 (s, 9H), 3.86 (s, 3H), 2.45 (t, J = 7.2 Hz, 2H), 2.38-2.28 (m, 4H),
13
1
.90 (quin, J = 6.3 Hz, 2H), 1.56 (quin, J = 5.1 Hz, 4H), 1.45-1.31 (m, 2H). C NMR (75 MHz,
CDCl ) δ 174.1, 163.9, 161.0, 158.9, 152.9, 150.8, 148.6, 140.2, 123.5, 121.9, 111.5, 110.8, 109.5,
3
9
5.8, 92.5, 71.0, 56.4, 56.4, 56.2, 56.0, 55.8, 54.6, 27.6, 25.8, 24.3. HR-MS (ESI) m/z: calcd for
C H NO [M+H]: 484.2335; found 484.2331.
27
34
7
4
.3.17. 3-O-Piperidinobutyl-3',4',5,7-O-tetramethylquercetin (27)
Yield, 29%; yellow oil. IR (film) νmax: 2945, 1622, 1601, 1514, 1457 cm . H NMR (300 MHz,
CDCl ) : 7.66 (d, J = 8.7 Hz, 1H), 7.62 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.48 (s, 1H), 6.32 (s, 1H),
-1 1
3
4
2
1
2
.00-3.90 (overlapped, 2H), 3.92 (s, 9H), 3.87 (s, 3H), 2.93 (t, J = 8.1 Hz, 2H), 2.90 (t, J = 6.6 Hz, 4H),
1
3
.04-1.73 (m, 10H). C NMR (75 MHz, CDCl ) : 174.2, 164.1, 161.0, 158.9, 153.3, 151.1, 148.8,
3
40.0, 123.3, 122.0, 111.3, 111.0, 109.4, 96.0, 92.6, 71.2, 57.7, 56.5, 56.3, 56.1, 55.9, 53.5, 27.7, 23.6,
2.9, 21.5. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 498.2492; found 498.2490.
2
8
36
7
4
.3.18. 3-O-Piperidinopentyl-3',4',5,7-O-tetramethylquercetin (28)
-1 1
Yield, 40%; yellow oil. IR (film) νmax: 2934, 1623, 1602, 1514 cm . H NMR (300 MHz, CDCl ) :
3
7
6
1
1
2
.68 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.47 (s, 1H), 6.31 (s, 1H), 3.97 (t, J =
.3 Hz, 2H), 3.93 (s, 9H), 3.87 (s, 3H), 2.56-2.42 (m, 4H), 2.37 (t, J = 7.5 Hz, 2H), 1.74-1.54 (m, 8H),
1
3
.41-1.35 (m, 4H). C NMR (75 MHz, CDCl ) : 174.1, 163.8, 161.0, 158.8, 152.8, 150.7, 148.5,
3
40.3, 123.5, 121.7, 111.4, 110.7, 109.4, 95.7, 92.4, 72.1, 58.9, 56.4, 56.1, 56.0, 55.8, 54.2, 30.0, 25.7,
5.1, 23.94, 23.88. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 512.2648; found 512.2643.
29
38
7
4
.3.19. 3-O-(4-Methylpiperazin-1-yl)propyl-3',4',5,7-O-tetramethylquercetin (29)
2% overall yield for two steps as a yellow wax. IR (film) vmax: 2938, 2796, 2360, 1624, 1602, 1513
5
-1 1
cm . H NMR (300 MHz, CDCl ) δ 7.65 (d, J = 6.6 Hz, 1H), 7.64 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H),
3
6
.47 (d, J = 2.1 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 3.93 (s, 9H), 3.87 (s, 3H),
2
1

1
3
2
1
7
.68-2.66 (m, 2H), 2.53-2.46 (m, 8H), 1.90 (quin, J = 6.3 Hz, 2H). C NMR (75 MHz, CDCl ) δ
3
74.2, 164.0, 161.1, 158.9, 153.0, 150.9, 148.7, 140.3, 123.6, 121.9, 111.5, 110.8, 109.5, 95.9, 92.5,
0.8, 56.5, 56.2, 56.1, 55.9, 55.3, 54.9, 53.0, 45.9, 27.7. HR-MS (ESI) m/z: calcd for C H N O
7
2
7
35
2
[
M+H]: 499.2444; found 499.2437.
4
.3.20. 3-O-(4-Methylpiperazin-1-yl)butyl-3',4',5,7-O-tetramethylquercetin (30)
Yield, 76%; yellow oil. IR (film) νmax: 2936, 2801, 1621, 1601, 1513, 1456, 1421 cm . H NMR
-1 1
(
300 MHz, CDCl ) : 7.67 (s, 1H), 7.65 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.46 (s, 1H), 6.30
3
(s, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.92 (s, 9H), 3.86 (s, 3H), 2.56-2.38 (m, 8H), 2.33 (t, J = 7.5 Hz, 2H),
1
3
2
1
7
.26 (s, 3H), 1.70 (quin, J = 6.9 Hz, 2H), 1.57 (quin, J = 6.3 Hz, 2H). C NMR (75 MHz, CDCl ) :
3
74.2, 164.0, 161.1, 158.9, 152.9, 150.8, 148.6, 140.3, 123.6, 121.8, 111.6, 110.8, 109.5, 95.8, 92.5,
2.2, 58.2, 56.5, 56.2, 56.1, 55.9, 54.9, 52.9, 45.9, 28.3, 23.2. HR-MS (ESI) m/z: calcd for C H N O
7
2
8
37
2
[
M+H]: 513.2601; found 513.2601.
4
.3.21. 3-O-(4-Methylpiperazin-1-yl)pentyl-3',4',5,7-O-tetramethylquercetin (31)
-1 1
Yield, 53%; yellow oil. IR (film) νmax: 2937, 2803, 1622, 1602, 1514 cm . H NMR (300 MHz,
CDCl ) : 7.67 (s, 1H), 7.64 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 1.8 Hz, 1H), 6.29
3
(s, 1H), 3.96 (t, J = 6.6 Hz, 2H), 3.91 (s, 9H), 3.85 (s, 3H), 2.62-2.57 (m, 8H), 2.31 (t, J = 7.5 Hz, 2H),
1
3
2
1
5
.29 (s, 3H), 1.70 (quin, J = 7.2 Hz, 2H), 1.53-1.30 (m, 4H). C NMR (75 MHz, CDCl ) : 174.2,
3
63.9, 161.0, 158.9, 152.8, 150.8, 148.6, 140.3, 123.6, 121.7, 111.5, 110.7, 109.5, 95.8, 92.5, 72.2,
8.4, 56.4, 56.1, 55.8, 54.6, 52.7, 45.7, 30.2, 26.4, 24.0. HR-MS (ESI) m/z: calcd for C H N O
7
2
9
39
2
[
M+H]: 527.2757; found 527.2757.
4
.3.22. 3-O-Pyrrolidinopropyl-3',4',5,7-O-tetramethylquercetin (32)
Overall yield for two steps, 27%; yellow wax. IR (film) vmax: 2935, 2793, 1622, 1600. H NMR (300
MHz, CDCl ) δ 7.67 (dd, J = 6.9, 2.1 Hz, 1H), 7.65 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.48 (d, J = 2.4
1
3
2
2

Hz, 1H), 6.32 (d, J = 2.4 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H), 3.939 (s, 3H), 3.936 (s, 3H), 3.93 (s, 3H),
.88 (s, 3H), 2.74 (t, J = 9.0, 2H), 2.66-2.55 (m, 4H), 1.99 (quin, J = 6.6 Hz, 2H), 1.80 (quin, J = 3.6
3
1
3
Hz, 4H). C NMR (75 MHz, CDCl ) δ 174.2, 164.0, 161.1, 158.9, 153.1, 150.9, 148.7, 140.2, 123.5,
3
1
21.9, 111.5, 110.9, 109.5, 95.9, 92.6, 70.6, 56.5, 56.3, 56.1, 55.9, 54.2, 53.4, 29.4, 23.5. HR-MS (ESI)
m/z: calcd for C H NO [M+H]: 470.2179; found 470.2179.
2
6
32
7
4
.3.23. 3-O-Pyrrolidinobutyl-3',4',5,7-O-tetramethylquercetin (33)
Yield, 98%; yellow oil. IR (film) νmax: 2937, 2794, 1621, 1600, 1512 cm . H NMR (300 MHz,
CDCl ) : 7.64 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.42 (s, 1H), 6.26 (s, 1H),
-1 1
3
1
3
3
.97 (t, J = 6.0 Hz, 2H), 3.89 (s, 9H), 3.83 (s, 3H), 2.50-2.34 (m, 6H), 1.79-1.49 (m, 8H). C NMR (75
MHz, CDCl ) : 174.1, 163.9, 160.9, 158.8, 152.7, 150.7, 148.5, 140.3, 123.5, 121.7, 111.4, 110.7,
3
1
09.4, 95.7, 92.4, 72.2, 56.4, 56.2, 56.1, 56.0, 55.8, 54.1, 28.4, 25.3, 23.4. HR-MS (ESI) m/z: calcd for
C H NO [M+H]: 484.2335; found 484.2332.
27
34
7
4
.3.24. 3-O-Pyrrolidinopentyl-3',4',5,7-O-tetramethylquercetin (34)
-1 1
Yield, 80%; yellow oil. IR (film) νmax: 2938, 1625, 1604, 1514 cm . H NMR (300 MHz, CDCl ) :
3
7
.69 (dd, J = 9.9, 2.1 Hz, 1H), 7.68 (s, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.34 (d, J
=
2.4 Hz, 1H), 3.98 (t, J = 6.0 Hz, 2H), 3.98 (s, 3H), 3.97 (s, 3H), 3.96 (s, 3H), 3.90 (s, 3H), 3.12-2.98
1
3
(
m, 4H), 2.89-2.74 (m, 2H), 2.02-1.97 (m, 4H), 1.82-1.72 (m, 4H), 1.56-1.45 (m, 2H). C NMR (75
MHz, CDCl ) : 174.1, 163.9, 160.9, 158.8, 152.9, 150.8, 148.6, 140.2, 123.4, 121.8, 111.4, 110.8,
3
1
09.4, 95.8, 92.5, 71.7, 56.4, 56.1, 56.0, 55.8, 55.8, 53.7, 29.7, 26.5, 23.6, 23.4. HR-MS (ESI) m/z:
calcd for C H NO [M+H]: 498.2492; found 498.2493.
2
8
36
7
4
.4. General procedure for the synthesis of 5-O-bromoalkyl-3,3',4',7-tetramethylquercetins (35-37)
The solution of 3,3',4',7-O-tetramethylquercetin (60 mg, 0.17 mmol) and potassium carbonate (72
mg, 0.52 mmol, 3 equiv.) in DMF (0.5 mL, 0.3 M) was stirred at room temperature for 10 min before
2
3

an appropriate alkyl dibromide (0.52 mmol, 3 equiv.) was added through a syringe. The reaction was
o
stirred at room temperature for three days at room temperature (or for overnight at 70 C). The reaction
mixture was diluted with a mixture of ethyl acetate and diethyl ether (100 mL, 1:1, v/v), which was
rinsed with water (10 mL × 1) and brine (10 mL × 4). The subsequent organic layer was dried over
anhydrous sodium sulfate, and concentrated in vacuo to generate a crude product, which was subjected
to PTLC purification eluting with 10% methanol in dichloromethane. The desired compound was
retrieved from PTLC by washing with ethyl acetate.
4
.4.1. 5-O-(3-Bromopropyl)-3,3',4',7-O-tetramethylquercetin (35)
1
Yield, 73%; yellow solid. H NMR (300 MHz, CDCl ) : 7.70 (dd, J = 7.2, 2.1 Hz, 1H), 7.68 (s,
3
1
2
H), 6.97 (d, J = 9.0 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 4.19 (t, J = 5.7 Hz,
H), 3.952 (s, 3H), 3.949 (s, 3H), 3.89 (s, 3H), 3.82 (s, 3H), 2.43 (t, J = 6.0 Hz, 2H).
4
.4.2. 5-O-(4-Bromobutyl)-3,3',4',7-O-tetramethylquercetin (36)
1
Yield, 58%; yellow solid. H NMR (300 MHz, CDCl ) : 7.68 (dd, J = 7.2, 2.1 Hz, 1H), 7.67 (s,
3
1
2
2
H), 6.95 (d, J = 9.0 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.07 (t, J = 6.0 Hz,
H), 3.94 (s, 3H), 3.93 (s, 3H), 3.87 (s, 3H), 3.81 (s, 3H), 3.54 (t, J = 6.3 Hz, 2H), 2.25-2.16 (m, 2H),
.11-2.06 (m, 2H).
4
.4.3. 5-O-(5-Bromopentyl)-3,3',4',7-O-tetramethylquercetin (37)
1
Yield, 58%; yellow solid. H NMR (300 MHz, CDCl ) : 7.71 (dd, J = 9.0, 2.1 Hz, 1H), 7.70 (d, J =
3
2
.1 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 4.08 (t, J = 6.3
Hz, 2H), 3.96 (s, 6H), 3.90 (s, 3H), 3.84 (s, 3H), 3.47 (t, J = 6.9 Hz, 2H), 1.98 (quin, J = 7.5 Hz, 4H),
1
.74 (quin, J = 6.6 Hz, 2H).
4
.5 The general procedure for the synthesis of 5-O-dialkylaminoalkyl-3,3',4',7-O-tetramethylquercetins
(38-61):
2
4

To the solution of the appropriate 5-O-bromoalkyl-3,3',4',7-O-tetramethylquercetin (0.17 mmol) in
DMF (0.5 mL, 0.3 M) was added potassium carbonate (70 mg, 0.51 mmol, 3 equiv.), and the mixture
was stirred for 10 min. The appropriate amine (0.51 mmol, 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 reaction mixture was diluted with a mixture of ethyl
acetate and diethyl ether (100 mL, 1:1, v/v), which was rinsed with water (10 mL × 1) and brine (10
mL × 4). The subsequent organic layer was dried over anhydrous sodium sulfate, and concentrated in
vacuo to generate a crude product, which was subjected to PTLC purification using
dichloromethane/methanol (90/10, v/v) as eluent. The respective 5-O-dialkylaminoalkyl-3,3',4',7-O-
tetramethylquercetin
was
retrieved
from
PTLC
silica
gel
by
washing
with
dichloromethane/diethylamine (100/3, v/v).
4
.5.1. 5-O-(N,N-Diethylamino)propyl-3,3',4',7-O-tetramethylquercetin (38)
-1 1
Yield, 40%; yellow wax. IR (film) νmax: 2939, 2837, 1625, 1603, 1515 cm . H NMR (300 MHz,
CDCl ) : 7.68 (dd, J = 6.9, 2.1 Hz, 1H), 7.67 (s, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.49 (d, J = 2.1 Hz,
3
1
3
H), 6.33 (d, J = 2.1 Hz, 1H), 4.17 (t, J = 5.7 Hz, 2H), 3.93 (s, 6H), 3.87 (s, 3H), 3.79 (s, 3H), 3.22-
1
3
.09 (m, 2H), 3.00-2.83 (m, 4H), 2.34-2.21 (m, 2H), 1.22 (t, J = 7.2 Hz, 6H). C NMR (75 MHz,
CDCl ) : 174.1, 164.2, 159.9, 158.8, 153.2, 151.1, 148.8, 141.1, 123.3, 121.8, 111.3, 111.0, 109.4,
3
9
6.8, 93.0, 67.6, 60.1, 56.2, 56.1, 56.0, 49.9, 47.5, 25.4, 10.4. HR-MS (ESI) m/z: calcd for C H NO
26 34 7
[
M+H]: 472.2335; found 472.2334.
4
.5.2. 5-O-(N,N-Diethylamino)butyl-3,3',4',7-O-tetramethylquercetin (39)
Yield, 92%; brown syrup. IR (film) νmax: 2934, 2836, 1623, 1601, 1514, 1438 cm . H NMR (300
MHz, CDCl ) : 7.68 (dd, J = 9.0, 2.1 Hz, 1H), 7.67 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.46
-1 1
3
(
d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.4 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.933 (s, 3H), 3.931 (s, 3H), 3.86
s, 3H), 3.81 (s, 3H), 2.70-2.62 (overlapped, 6H), 1.95 (quin, J = 6.6 Hz, 2H), 1.85-1.76 (m, 2H), 1.08
(
2
5

1
3
(
t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.0, 163.9, 160.4, 158.9, 152.7, 150.9, 148.8,
3
1
1
41.2, 123.5, 121.7, 111.3, 110.9, 109.7, 96.8, 92.6, 69.3, 60.1, 56.2, 56.1, 55.9, 52.4, 46.8, 27.0, 23.0,
1.1. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 486.2492; found 486.2491.
2
7
36
7
4
.5.3. 5-O-(N,N-Diethylamino)pentyl-3,3',4',7-O-tetramethylquercetin (40)
Yield, 86%; yellow oil. IR (film) νmax: 2924, 2853, 1625, 1602, 1514 cm . H NMR (300 MHz,
CDCl ) : 7.61 (dd, J = 9.0, 1.8 Hz, 1H), 7.67 (s, 1H), 6.95 (d, J = 9.3 Hz, 1H), 6.47 (d, J = 2.4 Hz,
-1 1
3
1
2
H), 6.30 (d, J = 2.1 Hz, 1H), 4.05 (t, J = 6.3 Hz, 2H), 3.94 (s, 6H), 3.85 (s, 3H), 3.81 (s, 3H), 2.81-
1
3
.67 (overlapped, 6H), 1.98-1.93 (m, 2H), 1.73-1.60 (m, 4H), 1.15 (t, J = 7.2 Hz, 6H). C NMR (75
MHz, CDCl ) : 174.0, 164.0, 160.5, 158.9, 152.8, 150.9, 148.8, 141.2, 123.5, 121.8, 111.3, 110.9,
3
1
09.7, 96.7, 92.6, 69.3, 60.1, 56.2, 56.1, 55.9, 52.3, 46.7, 28.7, 25.2, 24.1, 10.4. HR-MS (ESI) m/z:
calcd for C H NO [M+H]: 500.2648 ; found 500.2642.
2
8
38
7
4
.5.4. 5-O-(N,N-Dipropylamino)propyl-3,3',4',7-O-tetramethylquercetin (41)
Yield, 88%; brown syrup. IR (film) νmax: 2957, 2872, 1623, 1601, 1514, 1439 cm . H NMR (300
MHz, CDCl ) : 7.69 (dd, J = 9.0, 2.1 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H), 6.96 (d, J = 9.3 Hz, 1H), 6.47
-1 1
3
(d, J = 2.1 Hz, 1H), 4.13 (t, J = 6.3 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.84 (t, J = 6.9 Hz,
2
H), 2.50 (t, J = 6.9 Hz, 4H), 2.13 (quin, J = 6.6 Hz, 2H), 1.53 (quin, J = 7.8 Hz, 4H), 0.85 (t, J = 7.5
1
3
Hz, 6H). C NMR (75 MHz, CDCl ) : 174.0, 164.0, 160.5, 158.8, 152.7, 150.9, 148.8, 141.2, 123.5,
3
1
21.8, 111.3, 110.9, 109.6, 96.7, 92.6, 67.7, 60.1, 56.2, 56.2, 56.1, 55.9, 50.6, 26.4, 19.8, 12.0. HR-MS
(
ESI) m/z: calcd for C H NO [M+H]: 500.2648; found 500.2636.
2
8
38
7
4
.5.5. 5-O-(N,N-Dipropylamino)butyl-3,3',4',7-O-tetramethylquercetin (42)
Yield, 81%; yellow wax. IR (film) νmax: 2933, 2872, 1623, 1601, 1514, 1439 cm . H NMR (300
MHz, CDCl ) : 7.69 (d, J = 6.3 Hz, 1H), 7.68 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.48 (s, 1H), 6.31 (s,
-1 1
3
1
H), 4.08 (t, J = 5.7 Hz, 2H), 3.95 (s, 6H), 3.88 (s, 3H), 3.83 (s, 3H), 2.79-2.66 (m, 2H), 2.54 (t, J = 6.6
2
6

13
Hz, 4H), 2.00-1.90 (m, 2H), 1.90-1.78 (m, 2H), 1.61-1.45 (m, 4H), 0.88 (t, J = 7.2 Hz, 6H). C NMR
(
75 MHz, CDCl ) : 174.0, 164.0, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.8, 111.3, 110.9,
3
1
09.8, 96.8, 92.7, 69.3, 60.1, 56.2, 56.1, 55.9, 55.7, 53.5, 26.9, 22.9, 19.4, 12.0. HR-MS (ESI) m/z:
calcd for C H NO [M+H]: 514.2805; found 514.2800.
2
9
40
7
4
.5.6. 5-O-(N,N-Dipropylamino)pentyl-3,3',4',7-O-tetramethylquercetin (43)
-1 1
Yield, 84%; yellow oil. IR (film) νmax: 2937, 1622, 1600, 1514 cm . H NMR (300 MHz, CDCl ) :
3
7
.68 (dd, J = 9.3, 2.1 Hz, 1H), 7.66 (s, 1H), 6.95 (d, J = 9.3 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.30 (d, J
=
=
2.4 Hz, 1H), 4.05 (t, J = 6.3 Hz, 2H), 3.934 (s, 3H), 3.931 (s, 3H), 3.87 (s, 3H), 3.81 (s, 3H), 2.72 (t, J
7.5 Hz, 2H), 2.63 (t, J = 8.1 Hz, 4H), 1.95 (quin, J = 7.2 Hz, 2H), 1.77-1.55 (m, 8H), 0.89 (t, J = 7.5
1
3
Hz, 6H). C NMR (75 MHz, CDCl ) : 174.0, 164.0, 160.5, 158.9, 152.8, 150.9, 148.8, 141.2, 123.5,
3
1
1
21.7, 111.3, 110.9, 109.7, 96.8, 92.6, 69.3, 60.1, 56.2, 56.1, 55.9, 55.3, 53.5, 28.6, 25.2, 24.0, 18.8,
1.8. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 528.2961; found 528.2960.
3
0
42
7
4
.5.7. 5-O-(N,N-Dibutylamino)propyl-3,3',4',7-O-tetramethylquercetin (44)
Yield, 17%; yellow syrup. IR (film) νmax: 2954, 2930, 2870, 1624, 1601, 1514, 1489 cm . H NMR
300 MHz, CDCl ) : 7.70 (dd, J = 9.0, 2.1 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 6.98 (d, J = 9.3 Hz, 1H),
-1 1
(
3
6
3
.48 (d, J = 2.4 Hz, 1H), 6.35 (d, J = 2.1 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 3.96 (s, 6H), 3.89 (s, 3H),
.84 (s, 3H), 2.84-2.72 (m, 2H), 2.55-2.43 (m, 4H), 2.17-2.07 (m, 2H), 1.50-1.40 (m, 4H), 1.33-1.21
1
3
(
m, 4H), 0.87 (t, J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.1, 164.2, 159.7, 158.8, 153.3,
3
1
5
5
51.2, 148.9, 141.2, 123.4, 121.9, 111.3, 111.0, 109.4, 96.9, 93.3, 67.0, 60.1, 56.3, 56.2, 56.1, 53.4,
1.2, 25.9, 24.9, 20.5, 13.9. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 528.2961; found
3
0
42
7
28.2958.
4
.5.8. 5-O-(N,N-Dibutylamino)butyl-3,3',4',7-O-tetramethylquercetin (45)
2
7

-1 1
Yield, 72%; yellow wax. IR (film) νmax: 2931, 2870, 1624, 1602, 1514, 1438 cm . H NMR (300
MHz, CDCl ) : 7.70-7.67 (overlapped, 2H), 6.96 (d, J = 9.0 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.31 (d,
3
J = 1.8 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.61 (t, J = 7.2 Hz,
2
7
H), 2.49 (t, J = 7.5 Hz, 4H), 1.95 (quin, J = 7.2 Hz, 2H), 1.76 (quin, J = 6.9 Hz, 2H), 1.42 (quin, J =
13
.5 Hz, 4H), 1.28 (sextet, J = 7.5 Hz, 4H), 0.88 (t, J = 7.5 Hz, 4H), 0.88 (t, J = 7.5 Hz, 6H). C NMR
(
75 MHz, CDCl ) : 174.0, 163.9, 160.5, 158.9, 152.7, 150.9, 148.8, 141.2, 123.6, 121.7, 111.3, 110.9,
3
1
09.8, 96.8, 92.6, 69.4, 60.1, 56.2, 56.1, 55.9, 53.7, 53.6, 28.7, 27.0, 23.1, 20.8, 14.2. HR-MS (ESI)
m/z: calcd for C H NO [M+H]: 542.3118; found 542.3118.
3
1
44
7
4
.5.9. 5-O-(N,N-Dibutylamino)pentyl-3,3',4',7-O-tetramethylquercetin (46)
Yield, 80%; yellow oil. IR (film) νmax: 2934, 2872, 1621, 1600, 1514 cm . H NMR (300 MHz,
CDCl ) : 7.68 (dd, J = 6.6, 2.1 Hz, 1H), 7.67 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.46 (d, J = 2.1 Hz,
-1 1
3
1
3
1
1
5
H), 6.30 (d, J = 2.4 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.87 (s, 3H), 3.81 (s,
H), 2.64 (t, J = 6.9 Hz, 2H), 2.59 (t, J = 7.5 Hz, 4H), 1.51 (quin, J = 8.4 Hz, 2H), 1.95-1.47 (m, 8H),
13
.29 (sextet, J = 7.5 Hz, 4H), 0.89 (t, J = 7.5 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.0, 164.0,
3
60.5, 158.9, 152.7, 150.9, 148.8, 141.2, 123.5, 121.7, 111.3, 110.9, 109.7, 96.7, 92.6, 69.4, 60.1, 56.2,
6.1, 55.9, 53.6, 53.4, 28.7, 27.8, 25.6, 24.1, 20.7, 10.0. HR-MS (ESI) m/z: calcd for C H NO
7
3
2
46
[
M+H]: 556.3274; found 556.3269.
4
.5.10. 5-O-(N,N-Dipentylamino)propyl-3,3',4',7-O-tetramethylquercetin (47)
Yield, 9%; brown wax. IR (film) νmax: 2929, 2858, 1623, 1601, 1515, 1439 cm . H NMR (300
-1 1
MHz, CDCl ) : 7.70 (dd, J = 9.0, 2.1 Hz, 1H), 7.70 (s, 1H), 6.98 (d, J = 9.3 Hz, 1H), 6.51 (d, J = 2.4
3
Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 4.18 (t, J = 5.8 Hz, 2H), 3.96 (s, 6H), 3.89 (s, 3H), 3.82 (s, 3H), 2.82-
1
3
2
.66 (m, 4H), 2.35-2.21 (m, 2H), 1.71-1.56 (m, 4H), 1.35-1.24 (m, 12H), 0.89 (t, J = 6.6 Hz, 6H). C
NMR (75 MHz, CDCl ) : 174.1, 164.2, 160.0, 158.9, 153.1, 151.1, 148.9, 141.2, 123.4, 121.9, 111.4,
3
2
8

1
11.0, 109.5, 96.8, 93.0, 67.3, 60.1, 56.2, 56.1, 56.0, 53.9, 50.9, 31.1, 29.9, 29.5, 22.6, 14.1. HR-MS
(
ESI) m/z: calcd for C H NO [M+H]: 556.3274; found 556.3271.
3
2
46
7
4
.5.11. 5-O-(N,N-Dipentylamino)butyl-3,3',4',7-O-tetramethylquercetin (48)
Yield, 96%; brown wax. IR (film) νmax: 2929, 2857, 1623, 1601, 1514, 1439 cm . H NMR (300
MHz, CDCl ) : 7.69 (dd, J = 9.0, 2.1 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.47
-1 1
3
(d, J = 2.1 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 3.94 (s, 3H), 3.87 (s,
3
H), 3.83 (s, 3H), 2.56 (t, J = 7.2 Hz, 2H), 2.44 (t, J = 7.8 Hz, 4H), 1.95 (quin, J = 6.3 Hz, 2H), 1.73
1
3
(
quin, J = 6.9 Hz, 2H), 1.44 (quin, J = 7.2 Hz, 4H), 1.33-1.16 (m, 8H), 0.86 (t, J = 6.6 Hz, 6H). C
NMR (75 MHz, CDCl ) : 174.0, 164.0, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.8, 111.4,
3
1
10.9, 109.8, 96.8, 92.6, 69.4, 60.1, 56.2, 56.1, 55.9, 53.9, 53.5, 29.8, 27.0, 26.7, 22.8, 22.7, 14.2. HR-
MS (ESI) m/z: calcd for C H NO [M+H]: 570.3431; found 570.3430.
33
48
7
4
.5.12. 5-O-(N,N-Dipentylamino)pentyl-3,3',4',7-O-tetramethylquercetin (49)
-1 1
Yield, 18%; yellow oil. IR (film) νmax: 2930, 2858, 1623, 1602, 1514 cm . H NMR (300 MHz,
CDCl ) : 7.71 (dd, J = 9.3, 2.1 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 6.50 (d, J =
3
2
2
.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.97 (s, 6H), 3.90 (s, 3H), 3.85 (s, 3H),
.62-2.53 (overlapped, 6H), 1.98 (quin, J = 6.9 Hz, 2H), 1.72-1.47 (m, 8H), 1.34-1.24 (m, 8H), 0.89 (t,
1
3
J = 7.2 Hz, 6H). C NMR (75 MHz, CDCl ) : 174.1, 164.0, 160.6, 158.9, 152.8, 150.9, 148.8, 141.3,
3
1
2
5
23.6, 121.8, 111.3, 110.9, 109.8, 96.8, 92.6, 69.5, 60.2, 56.24, 56.17, 56.15, 55.9, 53.8, 29.8, 28.8,
5.9, 25.8, 24.1, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C H NO [M+H]: 584.3587; found
3
4
50
7
84.3587.
4
.5.13. 5-O-Morpholinopropyl-3,3',4',7-O-tetramethylquercetin (50)
Yield, 15%; yellow syrup. IR (film) νmax: 2932, 2851, 1623, 1601, 1514, 1489 cm . H NMR (300
MHz, CDCl ) : 7.71 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.50
-1 1
3
2
9