Structure-activity relationship and pharmacokinetic studies of 3-O-substitutedflavonols as anti-prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.08.047

Thirty-eight 3-O-substituted-3′,4′-dimethoxyflavonols and twenty-five 3-O-substituted-3′,4′,7-trimethoxyflavonols have been synthesized for systematic investigation on the structure-activity relationships of 3-O-substituted-3′,4′-dimethoxyflavonols in thr

Full text of DOI:10.1016/j.ejmech.2018.08.047

Accepted Manuscript
Structure-activity relationship and pharmacokinetic studies of 3-O-substitutedflavonols
as anti-prostate cancer agents
Xiang Li, Changde Zhang, Shanchun Guo, Pravien Rajaram, Maizie Lee, Guanglin
Chen, Ryan Fong, Aaron Gonzalez, Qiang Zhang, Shilong Zheng, Guangdi Wang,
Qiao-Hong Chen
PII:
S0223-5234(18)30717-7
10.1016/j.ejmech.2018.08.047
EJMECH 10658
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 June 2018
Revised Date: 15 August 2018
Accepted Date: 17 August 2018
Please cite this article as: X. Li, C. Zhang, S. Guo, P. Rajaram, M. Lee, G. Chen, R. Fong, A. Gonzalez,
Q. Zhang, S. Zheng, G. Wang, Q.-H. Chen, Structure-activity relationship and pharmacokinetic studies
of 3-O-substitutedflavonols as anti-prostate cancer agents, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.08.047.
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ACCEPTED MANUSCRIPT
Table of Contents Graphic
Structure-Activity Relationship and Pharmacokinetic Studies of 3-O-Substitutedflavonols as
Anti-Prostate Cancer Agents, Xiang Li, Changde Zhang, Shanchun Guo, Pravien Rajaram, Maizie
Lee, Guanglin Chen, Qiang Zhang, Shilong Zheng, Guangdi Wang, and Qiao-Hong Chen
1

ACCEPTED MANUSCRIPT
Structure-Activity Relationship and Pharmacokinetic Studies of 3-O-Substitutedflavonols as
Anti-Prostate Cancer Agents
a
b,c
b,c
a
a
a
Xiang Li , Changde Zhang , Shanchun Guo , Pravien Rajaram , Maizie Lee , Guanglin Chen , Ryan
a
a
b,c
b,c
b,c
a,
Fong , Aaron Gonzalez , Qiang Zhang , Shilong Zheng , 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
_
___________
Corresponding author.
E-mail: qchen@csufresno.edu. Phone: (+1)5592782394. Fax: (+1)5592784402.
*
1

ACCEPTED MANUSCRIPT
ABSTRACT:
Thirty-eight 3-O-substituted-3',4'-dimethoxyflavonols and twenty-five 3-O-substituted-3',4',7-
trimethoxyflavonols have been synthesized for systematic investigation on the structure-activity
relationships of 3-O-substituted-3',4'-dimethoxyflavonols in three human prostate cancer cell models.
Our findings indicate that incorporation of an appropriate amino group to 3-OH of 3',4'-
dimethoxyflavonol and 3',4',7-trimethoxyflavonol through a 3- to 5-carbon linker can substantially
improve the in vitro antiproliferative potency in three human prostate cancer cell models, but not in two
non-neoplastic human epithelial cell models (MCF 10A and PWR-1E). 1-Methylpiperazine,
pyrrolidine, and dibutylamine are optimal terminal amine groups that, in combination with a 3- to 5-
carbon linker, are notably beneficial to the anti-proliferative potency of 3-O-substituted-3',4'-
dimethoxyflavonols. It is worth noting that 3-O-(4-methylpiperazin-1-yl)propyl-3',4',7-
trimethoxyflavonol (76) induces PC-3 cell death in a completely different way from 3-O-
pyrrolidinopentyl-3',4',7-trimethoxyflavonol (81) even though they belong to 3-O-substituted-3',4',7-
trimethoxyflavonols and exhibit similar potency in inhibiting PC-3 cell proliferation, suggesting that
the mechanism of action for each specific 3-O-substitutedflavonol varies with different amino moiety.
3
-O-(N,N-Dibutylamino)propyl-3',4'-dimethoxyflavonol (42) emerged as the most promising derivative
due to its substantially improved potency in cell models, superior bioavailability in rats, and good
selectivity of inhibiting prostate cancer cell proliferation over non-neoplastic human epithelial cell
proliferation.
Keywords: Flavonol; Prostate Cancer; Structure-activity relationship; Pharmacokinetic study; Cell
apoptosis
2

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1
.
INTRODUCTION
Flavonoids are structurally characteristic of two aromatic rings and a central heterocyclic ring. They are
widely distributed in a variety of dietary plants, displaying various health and medicinal benefits. The
prime attention of researchers to the potential of flavonoids in preventing and treating prostate cancer is
mainly due to an inverse association between the intake of flavonoid-enriched diet and the incidence of
prostate cancer [1-8]. Flavonols are classified as one of the largest subgroups of flavonoids, structurally
featuring a hydroxyl group at C-3 [9-10].
Both naturally occurring and synthetic flavonols, as exemplified by quercetin (1), fisetin (2), and
3
',4',5'-trimethoxyflavonol (3) (Fig. 1), have been demonstrated by in vitro cell-based and in vivo
animal experiments to possess potential in treating prostate cancer [1,11-16]. However, the reported in
vitro anti-prostate cancer potency is moderate, typically with IC values in the range of 15-64 µM.
5
0
The synthetic 3',4',5'-trimethoxyflavonol (3) exhibits 5- to 16-fold greater potency than quercetin (1)
and fisetin (2), but only possesses very moderate in vivo antitumor efficacy even at the highest dose
(
0.73 mmol/kg) in two prostate cancer xenografts in nude mice [16] and poor bioavailability [17]. The
moderate potency and poor bioavailability have hindered the further advancement of these flavonols as
chemotherapeutic agents. Poor bioavailability of naturally occurring flavonols is mainly due to their
polyphenolic structures that are highly susceptible to first-pass metabolism of glucuronidation and
sulfation [18]. We envision that modifications on the phenolic groups of flavonols may lead to
simultaneous improvement of potency and bioavailability. Specifically, we are interested in the
chemical manipulations on 3-OH, the unique hydroxyl group for all flavonols, while converting all
other phenolic hydroxyl groups into methoxyl groups.
Our earlier WST-1 cell proliferation assay in three prostate cancer cell models suggests that the anti-
proliferative potency of 3',4'-dimethoxyflavonol (4) and 3',4',5'-trimethoxyflavonol (3) could be
appreciably enhanced through chemical modifications on its 3-OH group [19-20]. 3',4'-
3

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Dimethoxyflavonol (4) and 3',4',5'-trimethoxyflavonol (3) were chosen as our first two lead flavonols
because they contain two or three methoxyl groups instead of phenolic hydroxyl groups on ring B.
These lead flavonols were expected to overcome, to some degree, pharmacokinetic limitations caused
by the phenolic hydroxyl groups and to provide an efficient approach to incorporation of a basic
nitrogen-containing group to the only hydroxyl group at C-3. Encouraged by these preliminary data, the
present study aims to further investigate the in-depth structure-activity relationships of 3-O-substituted-
3
',4'-dimethoxyflavonols and to explore the pharmacokinetic profiles for the optimal derivatives. The
reason that 3',4',5'-trimethoxyflavonols were not selected for in-depth investigation is because 3-O-
substituted-3',4',5'-trimethoxyflavonols did not display significantly improved potency as compared
with the corresponding 3-O-substituted-3',4'-dimethoxyflavonols.
Figure 1. Structures of representative flavonols
2
2
.
RESULTS AND DISCUSSION
.1 Chemistry. 3',4'-Dimethoxyflavonol (4) and 3',4',7-trimethoxyflavonol (5) were synthesized
through a well-known two-step procedure, including Claisen-Schmidt condensation and Algar-Flynn-
Oyamada (AFO) reactions as illustrated in Scheme 1, that is the general approach to the synthesis of
flavonols [21-23]. Specifically, the intermediates 3',4'-dimethoxychalcone (6) and 3',4',7-
trimethoxychalcone (7) were prepared by Claisen-Schmidt condensation of 2'-hydroxyacetophenone
(
8) or 2'-hydroxy-4'-methoxyacetophenone (9) with 3,4-dimethoxybenzaldehyde (10), which were
directly subjected to the AFO reaction without further purification to furnish 3',4'-dimethoxyflavonol
4) and 3',4',7-trimethoxyflavonol (5) in 34% and 64% overall yields for two steps, respectively.
(
4

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As shown in Scheme 2, ten 3-O-alkyl-3',4'-dimethoxyflavonols (11-20) and ten 3-O-alkyl-3',4',7-
trimethoxyflavonols (21-30) have been synthesized in 11-99% yields by O-alkylating 3',4'-
dimethoxyflavonol (4) and 3',4',7-trimethoxyflavonol (5) with the appropriate alkyl halide using
potassium carbonate as base and DMF as an aprotic solvent. Twenty eight 3-O-aminoalkyl-3',4'-
dimethoxyflavonols (36-63) and fifteen 3-O-aminoalkyl-3',4',7-trimethoxyflavonols (67-81) have been
synthesized from 3',4'-dimethoxyflavonol (4) and 3',4',7-trimethoxyflavonol (5) in 27-94% yields
through intermediates 31-35 and 64-66 via two sequential alkylation reactions as illustrated in Scheme
3
. Potassium carbonate was used as the base and DMF as the polar aprotic solvent in both O-alkylation
and N-alkylation reactions. Each crude 3-O-bromoalkylflavonol (31-35 or 64-66) was directly used,
without further purification, for the follow-up N-alkylation. All synthesized 3-O-substitutedflavonols
were purified by preparative thin layer chromatography (PTLC) over silica gel and the nitrogen-
containing final compounds were retrieved by washing the PTLC silica gel with dichloromethane-
diethylamine (100:3, v/v).
Scheme 1. Synthesis of 3',4'-dimethoxyflavonol (4) and 3',4',7-trimethoxyflavonol (5)
5

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Scheme 2. Synthesis of 3-O-alkyl-3',4'-dimethoxyflavonols (11-20) and 3-O-alkyl-3',4',7-
trimethoxyflavonols (21-30). For the specific structure for each of 11-30, see Tables 1 and 2.
OCH3
OCH3
OCH3
Br(CH ) Br
2
2
n
R -H
R1
O
O
R1
K CO
R1
O
O
2
3
O
O
OCH3
K2CO3
OCH3
OCH3
Br
DMF
DMF
R2
OH
O
O
(
CH2)n
(CH2)n
1
1
4
, R = H
31-35, R = H; n = 3,4,5,6,7
1
3
6-63, R = H; n = 3,4,5,6,7
1
1
5
, R = OMe
64-66, R = OMe; n = 3,4,5
1
6
7-81, R = OMe; n = 3,4,5
2
R -H
N
N
H
N
H
N
H
H
diethylamine
dibutylamine
dipropylamine
dipentylamine
H
N
H
N
N
HN
N
H
O
morpholine
piperidine
1-methylpiperazine
pyrolidine
Scheme 3. Synthesis of 3-O-aminoalkyl-3',4'-dimethoxyflavonols (36-63) and 3-O-aminoalkyl-3',4',7-
trimethoxyflavonols (67-81). For the specific structure for each of 36-63, see Table 1. For the specific
structure for each of 67-81, see Table 2.
6

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2
.2 Antiproliferative activity and structure-activity relationships in three prostate cancer cell
models. The in vitro antiproliferative activity of thirty-eight 3-O-substituted-3',4'-dimethoxyflavonols
11-20, 36-63) and twenty-five 3-O-substituted-3',4',7-trimethoxyflavonols (21-30, 67-81) against a
(
panel of prostate cancer cell lines (PC-3, DU145, and LNCaP) was assessed using WST-1 cell
proliferation assay according to the procedure described in the Experimental Section. The PC-3,
DU145, and LNCaP cell lines are the most common cell-based models for in vitro assessment of
potency and efficacy of anti-prostate cancer agents. The androgen-insensitive PC-3 and DU145 cell
lines are derived from bone and brain metastasis of prostate tumor, respectively, which cannot express
prostate-specific antigen [24-25]. The androgen-sensitive LNCaP cell line is derived from lymph node
metastasis of prostate tumor and capable of expressing prostate-specific antigen. Two naturally
occurring flavonols, fisetin and quercetin, and two parent flavonols, 3',4'-dimethoxyflavonol (4) and
3
',4',7-trimethoxyflavonol (5), were used as positive controls. DMSO was used as negative control. The
IC values, as summarized in Tables 1 and 2, were calculated from the dose-response curves based on
5
0
at least five dosages for each compound. Our findings indicated that 3',4'-dimethoxyflavonol (4) is
notably less potent than fisetin and quercetin; in contrast, 3',4',7-trimethoxyflavonol (5) is appreciably
more potent than fisetin and quercetin. This suggests that the overall size of a flavonol may be critical
to the antiproliferative potency. Among sixty-three 3-O-substitutedflavonols tested, forty-three
displayed markedly improved ability in suppressing the human prostate cancer cell proliferation, as
compared with two naturally occurring flavonols (quercetin and fisetin).
As illustrated in Table 1, twenty-eight out of thirty-eight 3-O-substituted-3',4'-dimethoxylflavonols (11-
2
0, 36-63) are significantly more potent than the parent 3',4'-dimethoxylflavonol (4), quercetin (1), and
fisetin (2). Alkylation of 3-OH in parent 3',4'-dimethoxylflavonol (4) with an appropriate alkyl group
leads to a significant increase in the inhibitory activity against prostate cancer cell proliferation. The
optimal alkyl groups include ethyl, propyl, butyl, pentyl, and hexyl. The potency of 3-O-alkyl-3',4'-
7

ACCEPTED MANUSCRIPT
dimethoxylflavonols is however dramatically decreased with increasing length or branching of the
alkyl group (e.g. 17, 18, 19, 20), especially towards androgen-insensitive PC-3 and DU145 prostate
cancer cell lines. Introduction of a suitable amino moiety (i.e. N,N-dibutylamino, N,N-dipentylamino
morpolinopentyl, 4-methylpiperazin-1-yl, and pyrrolidinopropyl) to 3-OH of 3',4'-dimethoxylflavonol
through a three- to five-carbon linker bestows up to 818-fold enhancement in antiproliferative effect in
prostate cancer cell models. The potency of 3-O-aminoalkyl-3',4'-dimethoxylflavonol is seriously
diminished if the three- to five-carbon linker is substituted by a six- or seven-carbon linker (e.g. 57, 58,
6
2, 63).
As shown in Table 2, 3',4',7-trimethoxylflavonol (5) is more potent than 3',4'-dimethoxylflavonol (4),
but 3-O-substituted-3',4'-dimethoxylflavonols (11-20, 36-63) exhibit similar potency as the
corresponding 3-O-substituted-3',4',7-trimethoxylflavonols (21-30, 67-81). Eighteen out of twenty-five
3
-O-substituted-3',4',7-trimethoxylflavonols possess improved potency towards the PC-3 prostate
cancer cell line, as compared with the parent 3',4',7-trimethoxylflavonol (5). Twenty-one out of twenty-
five 3-O-substituted-3',4',7-trimethoxylflavonols have enhanced potency against LNCaP prostate
cancer cells, as compared with the parent flavonol 5. The common structure-activity relationship of 3-
O-substituted-3',4'-dimethoxylflavonols and 3-O-substituted-3',4',7-trimethoxylflavonols can be
concluded as below:
‹
‹
‹
The in vitro antiproliferative potency of 3',4'-dimethoxylflavonol and 3',4',7-trimethoxylflavonol
can be substantially ameliorated by an appropriate chemical modification on 3-OH.
N,N-dibutylamino,
N,N-dipentylamino
morpolinopentyl,
4-methylpiperazin-1-yl,
and
pyrrolidinopropyl serve as the optimal amino groups for the activity against prostate cancer cells.
Three- to five-carbon are the optimal length for the linker, between 7-OH of flavonoid and the
amino moiety, that is beneficial to the potency.
8

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‹
‹
The PC-3 and LNCaP prostate cancer cells are generally more susceptible than the DU145 cells to
the 3-O-substituted-flavonols.
Several optimal derivatives (56, 61, 76, 77, 79, and 81) reveal submicromolar IC values against
5
0
PC-3 prostate cancer cells.
Table 1. Anti-Proliferative Activity of 3-O-Substituted-3',4'-dimethoxyflavononols (11-20; 36-63)
OMe
O
OMe
OR
O
a
IC50 (µM)
Compd
R
H
b
c
d
PC-3
DU145
LNCaP
4
4
3
09.0 ± 1.8
0.0 ± 15.0
174.6 ± 31.6
46.1 ± 28.5
39.2 ± 11.4
14.6 ± 0.3
26.7 ± 7.6
38.9 ± 6.1
19.4 ± 5.7
111.9 ± 71.1
38.0 ± 1.6
> 50
202.5 ± 32.2
16.2 ± 9.4
9.8 ± 0.6
1
1
2
3
Methyl
Ethyl
1
1
1
3.7 ± 9.3
Propyl
1
0.1± 3.6
6.8 ± 6.7
6.8 ± 4.6
4.0 ± 6.5
6.4 ± 2.2
1
1
1
1
1
1
2
3
3
3
3
4
4
4
4
5
6
7
8
9
0
6
7
8
9
0
1
2
Butyl
Pentyl
1
8.1 ± 3.1
1
1
14.2 ± 7.6
12.6 ± 5.8
43.9 ± 3.3
10.1 ± 4.3
10.9 ± 2.9
13.1 ± 5.3
13.2 ± 6.4
40.5 ± 16.1
18.9 ± 7.4
13.5 ± 6.3
13.2 ± 6.5
2.7 ± 2.1
Hexyl
Heptyl
8
6.3 ± 21.2
Isopropyl
3
3.0 ± 7.7
Sec-Butyl
4
4.2 ± 19.6
Pentan-2-yl
2
8.1 ± 5.7
7.2 ± 5.0
0.7 ± 5.6
2.0 ± 2.4
6.0 ± 7.5
5.7 ± 6.1
3.1 ± 4.0
> 50
(N,N-Diethylamino)propyl
(N,N-Diethylamino)butyl
(N,N-Diethylamino)pentyl
(N,N-Dipropylamino)propyl
(N,N-Dipropylamino)butyl
(N,N-Dipropylamino)pentyl
(N,N-Dibutylamino)propyl
5
49.8 ± 1.2
> 50
6
3
2
2
1
48.1 ± 10.2
38.6 ± 6.7
24.3 ± 11.4
14.1 ± 6.5
7.6 ± 1.7
1
.4 ± 0.2
2.4 ± 1.5
9

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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
(N,N-Dibutylamino)butyl
(N,N-Dibutylamino)pentyl
(N,N-Dipentylamino)propyl
(N,N-Dipentylamino)butyl
(N,N-Dipentylamino)pentyl
Morpholinopropyl
2
4
2
7
4
.7 ± 0.8
.0 ± 1.0
.4 ± 0.8
.7 ± 2.2
.9 ± 1.5
16.3 ± 5.5
11.9 ± 3.4
12.8 ± 3.0
26.9 ± 1.6
14.4 ± 4.3
38.6 ± 4.3
44.0 ± 1.3
25.9 ± 4.1
> 50
4.3 ± 1.3
5.0 ± 1.3
4.8 ± 1.3
10.2 ± 2.7
7.8 ± 2.1
14.9 ± 12.1
8.5 ± 6.2
7.7 ± 3.5
36.0 ± 14.4
24.1 ± 8.9
26.5 ± 4.4
12.2 ± 0.1
6.4 ± 1.0
3.9 ± 0.7
12.2 ± 3.5
4.0 ± 2.4
10.6 ± 2.1
4.5 ± 1.6
4.9 ± 1.0
10.3 ± 2.6
7.4 ± 3.7
45.5 ± 1.3
34.1 ± 7.7
1
0.9 ± 4.7
Morpholinobutyl
9
.1 ± 1.8
.5 ± 1.0
Morpholinopentyl
4
Piperidinopropyl
>
50
3.4 ± 3.6
8.6 ± 6.5
.3 ± 0.5
.1 ± 0.8
.5 ± 0.4
9.8 ± 3.1
Piperidinobutyl
1
> 50
Piperidinopentyl
3
> 50
(4-Methylpiperazin-1-yl)propyl
(4-Methylpiperazin-1-yl)butyl
(4-Methylpiperazin-1-yl)pentyl
(4-Methylpiperazin-1-yl)hexyl
(4-Methylpiperazin-1-yl)heptyl
Pyrrolidinopropyl
3
> 50
1
31.6 ± 0.7
20.3 ± 8.4
24.6 ± 0.6
8.3 ± 3.2
27.1 ± 6.7
13.0 ± 7.1
29.2 ± 2.2
17.3 ± 5.5
8.5 ± 4.3
>100
0
1
6
2
1
0
.2 ± 0.6
.0 ± 1.9
.5 ± 0.6
.7 ± 0.5
Pyrrolidinobutyl
Pyrrolidinopentyl
Pyrrolidinohexyl
1
1
7.4 ± 3.5
3.5 ± 2.2
Pyrrolidinoheptyl
Quercetin
Fisetin
-
>100
-
>
50
> 50
a
IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days of
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 tumor
b
c
d
Table 2. Anti-Proliferative Activity of 3-O-Substituted-3',4',7-trimethoxyflavononols (21-30; 67-81)
1
0

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a
IC50 (µM)
Compd
b
c
d
R
H
PC-3
DU145
LNCaP
5
19.9 ± 3.4
14.9 ± 4.1
8.5 ± 1.7
24.9 ± 12.0
30.8 ± 17.1
59.8 ± 5.2
24.3 ± 8.9
26.3 ± 6.5
33.2 ± 1.0
64.4 ± 12.3
127.2 ± 45.6
103.1 ± 13.0
> 50
24.5 ± 4.5
15.6 ± 4.6
30.2 ± 12.2
6.2 ± 2.9
2
2
2
2
2
2
2
2
2
3
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
1
2
3
4
5
6
7
8
9
0
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Methyl
Ethyl
Propyl
10.0 ± 7.0
14.1 ± 6.6
20.3 ± 1.7
42.3 ± 4.5
25.7 ± 13.0
38.0 ± 8.0
22.0 ± 3.0
39.0 ± 9.1
Butyl
9.4 ± 2.4
Pentyl
17.7 ± 1.2
19.6 ± 3.4
22.9 ± 5.7
27.1 ± 2.2
13.5 ± 3.2
12.2 ± 1.8
5.5 ± 1.1
Hexyl
Heptyl
Isopropyl
Sec-Butyl
Pentan-2-yl
> 50
(N,N-Dibutylamino)propyl
(N,N-Dibutylamino)butyl
(N,N-Dibutylamino)pentyl
Morpholinopropyl
1.9 ± 0.4
3.2 ± 0.2
5.2 ± 1.8
12.4 ± 5.9
9.1 ± 3.3
9.5 ± 1.3
24.5 ± 5.6
16.3 ± 4.4
8.9 ± 1.2
0.79 ± 0.17
0.62 ± 0.10
1.6 ± 0.8
0.82 ± 0.10
4.2 ± 0.5
0.54 ± 0.31
>100
11.8 ± 1.7
16.5 ± 1.0
26.0 ± 2.8
41.3 ± 13.6
119.9 ± 5.5
34.6 ± 4.6
> 50
7.1 ± 1.3
12.4 ± 0.6
32.2 ± 12.3
10.0 ± 4.5
11.1 ± 2.8
30.7 ± 10.7
16.1 ± 6.3
11.0 ± 3.2
6.0 ± 0.4
Morpholinobutyl
Morpholinopentyl
Piperidinopropyl
Piperidinobutyl
> 50
Piperidinopentyl
27.1 ± 7.2
20.1 ± 6.1
34.7 ± 9.7
22.1 ± 3.1
21.3 ± 2.1
25.0 ± 2.6
15.4 ± 5.8
>100
(4-Methylpiperazin-1-yl)propyl
(4-Methylpiperazin-1-yl)butyl
(4-Methylpiperazin-1-yl)pentyl
Pyrrolidinopropyl
4.6 ± 0.7
5.7 ± 1.3
4.5 ± 2.2
Pyrrolidinobutyl
7.4 ± 1.3
Pyrrolidinopentyl
6.3 ± 1.4
Quercetin
-
45.5 ± 1.3
1
1

ACCEPTED MANUSCRIPT
Fisetin
-
> 50
> 50
34. 1 ± 7.7
a
IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days of
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 tumor
b
c
d
2
.3 Anti-proliferative activity towards MCF 10A and PWR-1E non-neoplastic human epithelial
cell lines
One parent flavonol, 5, and three 3-O-substitutedflavonols, 42, 76, and 81, together with two naturally
occurring flavonols, quercetin (1) and fisetin (2), were selected for further evaluation against MCF 10A
non-neoplastic human mammary epithelial cell line and PWR-1E non-neoplastic human prostate
epithelial cells. It is arguable whether the MCF 10A cells can truly represent normal human mammary
cells [26] and it was believed by Rhim and co-workers [27] that PWR-1E immortalized human prostate
epithelial cell model stands for an early stage in tumor progression. They are therefore called non-
neoplastic cell models rather than normal cell models. Four synthetic flavonols (5, 42, 76, and 81) were
chosen based on the following grounds: 5 represents the more potent parent flavonol; 42 is the most
potent 3-O-substituted-3',4'-dimethoxyflavonol considering its overall potency towards three prostate
cancer cell models; 76 and 81 are the most promising 3-O-substituted-3',4',7-trimethoxyflavonols. As
shown in Table 3, two natural flavonols, fisetin (2) and quercetin (1), demonstrate significantly higher
capability of suppressing non-neoplastic cell (MCF 10A and PWR-1E) proliferation than prostate
cancer cell proliferation. The parent flavonol, 3-O-3',4',7-trimethoxyflavonol (5), did not exhibit
significantly differential responses to prostate cancer cells and to non-neoplastic MCF 10A cells.
However, it doesn’t demonstrate apparent antiproliferative activity towards PWR-1E non-neoplastic
epithelial cells up to 50 µM concentration. Interestingly, the 3-O-substituted-3',4'-dimethoxyflavonol
4
2 and the 3-O-substituted-3',4',7-trimethoxyflavonols 76 and 81 illustrate good selectivity of inhibiting
prostate cancer cell proliferation over non-neoplastic MCF 10A and PWR-1E human epithelial cell
1
2

ACCEPTED MANUSCRIPT
proliferation (Table 3), suggesting that modification at 3-OH of 3',4'-dimethoxyflavonol and 3',4',7-
trimethoxyflavonol improves the antiproliferative potency only towards prostate cancer cells but not
non-neoplastic epithelial cells.
Table 3. Antiproliferative activity of selected derivatives against MCF-10A and PWR-1E
a
compound
IC50 (µM)
PC-3
> 50
DU145
> 50
LNCaP
MCF 10A
PWR-1E
Fisetin
34.1 ± 7.7 2.44 ± 0.27
45.5 ± 1.3 11.97 ± 1.49
5.39 ± 1.16
15.08 ± 1.41
Quercetin > 100
> 100
5
8
7
19.9 ± 3.4
24.9 ± 12.0 24.5 ± 4.5 23.95 ± 5.05 > 50
1
6
2
0.54 ± 0.3 15.4 ± 5.8
0.79 ± 0.2 20.1 ± 6.1
6.3 ± 1.4
6.0 ± 0.4
2.4 ± 1.5
35.69 ± 7.69
33.75 ± 14.22 > 50
20.93 ± 0.81 > 50
> 50
4
1.4 ± 0.2
7.6 ± 1.7
a
IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days of
exposure. The data were presented as the mean ± SD from n = 3.
2
.4 Cell Cycle Regulation and Cell Apoptosis Induction:
It has been demonstrated that quercetin and fisetin, as two naturally occurring flavonols, possess cell
cycle perturbation properties in the androgen-insensitive PC-3 human prostate cancer cell line by
2
arresting cell cycle at G /M phase [12,15]. 3-O-Substitued-3',4'-dimethoxyflavonol 42 can induce PC-3
cell cycle arrest at the G2/M phase as well, as reported in our earlier study [19]. The effect of 3',4',7-
trimethoxyflavonol 5 and two 3-O-substituted-3',4',7-trimethoxyflavonols 76 and 81 at 10 µM and 20
µM on the PC-3 cell cycle was assessed using flow cytometric analysis with propidium iodide DNA
staining. 3',4',7-trimethoxyflavonol (5) arrests PC-3 cell cycle at the G /G phase in 16 hours by
0
1
increasing PC-3 cell population. In contrast, two 3-O-substituted-3',4',7-trimethoxyflavonols 76 and 81
induce PC-3 cell cycle at the S phase in 16 hours. The above-mentioned data imply that modification of
3
-OH of 3',4',7-trimethoxyflavonol can alter its regulatory effect on PC-3 cell cycle.
1
3

ACCEPTED MANUSCRIPT
The PC-3 cell proliferation inhibition of quercetin and fisetin, two naturally occurring flavonols, has
been revealed to be associated with cell apoptosis promotion [28-30]. The F2N12S and SYTOX
AADVanced double staining assay in a flow cytometer was used for the discrimination between early
apoptotic PC-3 cells and late apoptotic/necrotic PC-3 cells when treated with one parent flavonol 5 and
three 3-O-substitutedflavonols (42, 76, and 81) at 0, 5, 10 and 20 µM concentrations for 16 h. The
known apoptotic inducer staurosporine was used as the positive control in apoptotic experiments. As
illustrated in Figure 2, 3-O-substituted-3',4'-dimethoxyflavonol 42, 3',4',7-trimethoxyflavonol (5), and
3
-O-pyrrolidinopentyl-3',4',7-trimethoxyflavonol (81) 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. Specifically, treatment of 42, 5, and 81 at 5 µM can lead to 45%, 37%, and 59% of PC-3
cells in early phase of apoptosis as compared with control cells; exposure of PC-3 cells to 42, 5, and 81
at 10 µM induces 80%, 79%, and 77% early apoptotic cells together with 5%, 2%, and 3% late
apoptotic/necrotic cells; 20 µM of 42, 5, and 81 activates notable apoptosis as well, with 93%, 90%,
and 92% early apoptotic cells and 4%, 2%, and 4% late apoptotic/necrotic cells. All these three
flavonols (42, 5, and 81) promote androgen-insensitive PC-3 cell apoptosis much more effectively than
the known apoptotic inducer staurosporine (Figure 2). In contrast, treatment of the PC-3 cells with 3-O-
(
4-methylpiperazin-1-yl)propyl-3',4',7-trimethoxyflavonol (76) up to 100 µM high concentration only
led to 7% early apoptotic cells. It is worth noting that 3-O-(4-methylpiperazin-1-yl)propyl-3',4',7-
trimethoxyflavonol (76) induces PC-3 cell death in a completely different way from 3-O-
pyrrolidinopentyl-3',4',7-trimethoxyflavonol (81) even though they both belong to 3-O-substituted-
3
',4',7-trimethoxyflavonols and exhibit similar potency in inhibiting PC-3 cell proliferation, suggesting
that the action of mechanism for each specific 3-O-substitutedflavonol varies with different amino
moiety.
1
4

ACCEPTED MANUSCRIPT
4
2-control
42-(5 µM)-treated
4
2-(10 µM)-treated
42-(20 µM)-treated
5 (20 µM)-treated
5
-control
5-(5 µM)-treated
5
(10 µM)-treated
7
6-(5 µM)-treated
76 (10 µM)-treated
76 (20 µM)-treated
7
6-control
8
1 -control
81 (20 µM)-treated
8
1 (5 µM)-treated
81 (10 µM)-treated
strauroporine -control
strauroporine (5 µM) -treated
strauroporine (10 µM)-treated
strauroporine (20 µM)-treated
Figure 2. Apoptosis in PC-3 cells treated with 42, 5, 76, and 81 at 5 µM, 10 µM, and 20 µM (by
F2N12S and SYTOX AADvanced double staining)
1
5

ACCEPTED MANUSCRIPT
2
.5 In vivo pharmacokinetic studies
To evaluate if the 3-O-substituted-flavonols with significantly improved in vitro anticancer activities
could also possess greater bioavailability, we selected three of the most promising derivatives, 42, 76,
and 81, together with one naturally occurring flavonol (fisetin), for pharmacokinetic studies in rats.
Sprague Dawley rats, a species that are more suited for bioavailability study, were chosen in this study
as the animal models for the pharmacokinetic studies. The animals administered with fisetin, 42, 76, or
8
1, via oral gavage at a single dose of 10 mg/kg, and blood samples were collected at 1, 3, 6, 8, and 24
h after oral administration. Plasma was prepared from the blood samples and was analyzed by HPLC-
MS/MS for determination of drug concentration as described in the Experimental Section. The PK
parameters for fisetin cannot be calculated because its plasma concentrations can barely be achieved at
1
h time point, implying the poor pharmacokinetic profile of fisetin. Summarized in Table 4 are the
pharmacokinetic parameters for three 3- -substituted flavonols (42, 76, or 81), which lead to the
O
conclusion that they gain the obvious improvement in their bioavailability with the peak concentration
at 5773 ng/mL (12.36 µM), 894 ng/mL (1.92 µM), and 172 ng/mL (0.37 µM), respectively. Among the
three 3-O-substituted-flavonols, 42 gains the greatest improvement in its bioavailability; its peak
concentration at 5773 ng/mL (12.36 µM) is far exceeding its IC50 values ranging from 1.4-7.6 µM in
three human prostate cancer cell lines. It is thus logical to conclude that the excellent bioavailability of
4
2, as demonstrated by its high peak plasma concentration and AUC value, will provide the therapeutic
efficacy necessary to suppress in vivo tumor growth.
1
6

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Table 4.
Pharmacokinetic parameters for 42, 76, and 81
Parameter
Lambda_z
t1/2
Unit
1/h
h
42
0.18
76
0.079
81
0.074
3.87
8.83
8
9.39
3
Tmax
h
1
Cmax
ng/ml
5773.00
0
893.99
0
171.81
0
Tlag
h
Clast_obs/Cmax
AUC 0-t
0.00044
51805.98
51820.33
1.00
0.044
0.036
2947.61
3032.42
0.97
ng/ml*h
ng/ml*h
16599.23
17101.39
0.97
AUC 0-inf_obs
AUC 0-t/0-inf_obs
AUMC 0-inf_obs
MRT 0-inf_obs
Vz/F_obs
ng/ml*h^2
343077.41
6.62
270875.20
15.84
39656.44
13.08
0.045
h
(mg/kg)/(ng/ml)
0.0011
0.0074
(
mg/kg)/(ng/ml)/
Cl/F_obs
h
0.00019
0.00058
0.0033
3
.
CONCLUSION
To further investigate the in-depth structure-activity relationships of 3-
three 3- -substituted-3',4'-dimethoxyflavonols and 3-
been synthesized for anti-proliferative evaluation in both androgen-sensitive (LNCaP) and androgen-
insensitive (PC-3 and DU145) prostate cancer cell lines. Forty-six out of sixty-three 3-
substitutedflavonols are significantly more potent than naturally occurring flavonols quercetin and
fisetin, with the optimal 3- -substitutedflavonol being up to 808-fold more potent than fisetin and
O
-substitutedflavonols, sixty-
O
O-substituted-3',4',7-trimethoxyflavonols have
O
-
O
quercetin. The acquired structure-activity relationship data indicated that i) 1-methylpiperazine,
pyrrolidine, and dibutylamine serve as optimal amino groups for notably improved in vitro potency;
1
7

ACCEPTED MANUSCRIPT
and ii) a 3-carbon to 5-carbon linker contributes to substantially improved antiproliferative effect in
prostate cancer cell models. Interestingly, incorporation of an appropriate amino group to 3-OH of 3',4'-
dimethoxyflavonol and 3',4',7-trimethoxyflavonol through a 3- to 5-carbon linker can substantially
improve the in vitro antiproliferative potency in three human prostate cancer cell models, but not in two
non-neoplastic human epithelial cell models (MCF 10A and PWR-1E). It is worth noting that 3-
methylpiperazin-1-yl)propyl-3',4',7-trimethoxyflavonol (76) induces PC-3 cell death in a completely
different way from 3- -pyrrolidinopentyl-3',4',7-trimethoxyflavonol (81) even though they belong to
-substituted-3',4',7-trimethoxyflavonols and exhibit similar potency in inhibiting PC-3 cell
proliferation, suggesting that the action of mechanism for each specific 3- -substitutedflavonol varies
O-(4-
O
3-O
O
with different amino moiety. Pharmacokinetic studies in rat model identified compound 42 possesses
the excellent bioavailability, as demonstrated by its high peak plasma concentration and AUC value. 3-
O-(N,N-Dibutylamino)-propyl-3',4'-dimethoxyflavonol (42) has been established as the most promising
derivative at this point because of its substantially improved potency in cell models, superior
bioavailability in rats, and good antiproliferative selectivity of prostate cancer cells over non-neoplastic
human epithelial cells.
4
4
. EXPERIMENTAL SECTION
.1. General Procedures.
HRMS were obtained on an Orbitrap mass spectrometer with electrospray ionization (ESI). IR
spectra were recorded on a Nicolet Nexus 470 FTIR spectrophotometer. NMR spectra were obtained
on a Bruker Fourier 300 spectrometer in CDCl . The chemical shifts are given in ppm referenced to the
3
respective 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 µm). Preparative thin-layer chromatography
1
8

ACCEPTED MANUSCRIPT
(
PTLC) separations were carried out on thin layer chromatography plates loaded with silica gel 60
GF254 (EMD Millipore Corporation, MA, USA). The purities of biologically tested compounds are
≥
9
5% as determined by HPLC. Specifically, the major peak accounted for 95% of the combined total
≥
peak area when monitored by a Diode Array Detector (DAD) at 325 ± 100 nm. The HPLC analyses
were performed on an Agilent Hewlett Packard 1100 Series HPLC DAD system using a 5 µM C18
reversed phase column (4.6 mm × 250 mm) and a Diode Array Detector.
4
.2. The general procedure for the synthesis of 3-O-alkyl-3',4'-dimethoxyflavonols (11-20).
The solution of 3',4'-dimethoxyflavonol (50 mg, 0.17 mmol) and potassium carbonate (70 mg, 0.51
mmol, 3.0 equiv.) in DMF (0.5 mL) was stirred for 10 min at R.T.. To this solution was added an
appropriate alkyl iodide or alkyl bromide (0.51 mmol, 3.0 equiv.) through syringe, the reaction was
kept at room temperature over 12 h until the starting material was consumed as detected by TLC
(
dichloromethane/methanol, 100/3, v/v). The mixture was poured into ice water (3.0 mL), and the
solution 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 purified by PTLC eluting with dichloromethane/methanol
(
100/3, v/v) to furnish the respective 3-O-alkyl-3',4'-dimethoxyflavonols. The yields and spectral data
for compounds 11, 12, 13, 14, 15, 16, 17, and 18 have been reported in the supporting information of
our previous Letter report [19]. HPLC purity for 4 97.5% (30 min run of 45-80% CH CN in H O, with
3
2
1
5 min gradient, 1.0 mL/min). HPLC purity for 11 is 95.7% (30 min run of 45-80% CH CN in H O,
3
2
with 15 min gradient, 1.0 mL/min). HPLC purity for 12 is 99.5% (30 min run of 45-80% CH
3
CN in
H
2
O, with 15 min gradient, 1.0 mL/min). HPLC purity for 13 is 96.7% (30 min run of 45-80% CH
in H O, with 15 min gradient, 1.0 mL/min). HPLC purity for 14 is 98.8% (30 min run of 45-80%
CH CN in H O, with 15 min gradient, 1.0 mL/min). HPLC purity for 15 is 95.1% (30 min run of 45-80%
3
CN
2
3
2
CH CN in H O, with 15 min gradient, 1.0 mL/min). HPLC purity for 16 is 95.1% (30 min run of 45-80%
3
2
1
9

ACCEPTED MANUSCRIPT
CH CN in H O, with 15 min gradient, 1.0 mL/min). HPLC purity for 17 is 99.7% (30 min run of 45-80%
3
2
CH CN in H O, with 15 min gradient, 1.0 mL/min). HPLC purity for 18 is 97.6% (30 min run of 45-80%
3
2
CH
3
CN in H
2
O, with 15 min gradient, 1.0 mL/min).
4
.2.1. 3-O-Sec-butyl-3',4'-dimethoxyflavonol (19)
This derivative was achieved in 67% yield as a syrup. IR (film)
ν
max: 2965, 2931, 1634, 1614, 1614,
= 8.0, 1.4 Hz, 1H), 7.84 (d, = 1.9
= 8.4, 6.9, 1.5 Hz, 1H), 7.53 (d, = 8.3 Hz, 1H),
= 6.3 Hz, 1H), 3.971 (s, 3H), 3.965 (s,
-
1 1
1
598, 1557, 1509 cm . H NMR (300 MHz, CDCl
= 8.5, 2.0 Hz, 1H), 7.66 (ddd,
= 7.2 Hz, 1H), 6.99 (d, = 8.6 Hz, 1H), 4.58 (sextet,
H), 1.80 – 1.65 (m, 1H), 1.57 – 1.45 (m, 1H), 1.12 (d,
3
)
δ
8.25 (dd,
J
J
Hz, 1H), 7.77 (dd,
J
J
J
7
3
.39 (t,
J
J
J
13
J
= 6.2 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). C
NMR (75 MHz, CDCl3)
22.5, 118.0, 112.5, 110.6, 79.3, 56.1, 56.1, 29.7, 19.4, 9.9. HR-MS (ESI) m/z: calcd for C H O
5
δ 175.5, 156.2, 155.3, 151.0, 148.4, 138.8, 133.3, 125.9, 124.6, 124.2, 124.2,
1
2
1
22
+
[
M+H] : 355.1545; found 355.1540. HPLC purity 96.9% (30 min run of 45-80% CH CN in H O, with
3 2
1
5 min gradient, 1.0 mL/min).
4
.2.2. 3-O-(Pentan-2-yl)-3',4'-dimethoxyflavonol (20)
This derivative was achieved in 20% yield as a syrup. IR (film)
ν
max: 2957, 2930, 2870, 1634, 1614,
= 8.0, 1.6 Hz, 1H), 7.84 (d, = 2.0
= 8.6, 7.1, 1.7 Hz, 1H), 7.53 (d, = 8.0 Hz, 1H),
= 8.7 Hz, 1H), 4.67 (sextet, = 6.1 Hz, 1H), 3.97 (s, 3H),
.96 (s, 3H), 1.77 – 1.60 (m, 1H), 1.53 – 1.34 (m, 3H), 1.10 (d, = 6.2 Hz, 3H), 0.88 (t, = 7.2 Hz,
175.5, 156.1, 155.3, 151.0, 148.4, 138.8, 133.2, 125.9, 124.6,
-
1 1
1
598, 1558, 1509 cm . H NMR (300 MHz, CDCl
= 8.5, 2.1 Hz, 1H), 7.66 (ddd,
= 8.0, 7.1, 1.0 Hz, 1H), 6.98 (d,
3
)
δ
8.24 (dd,
J
J
Hz, 1H), 7.77 (dd,
J
J
J
7
3
3
1
.38 (ddd,
J
J
J
J
J
1
3
H). C NMR (75 MHz, CDCl3)
δ
24.3, 124.2, 122.4, 118.0, 112.5, 110.7, 77.8, 56.1, 56.0, 39.2, 19.91, 18.87, 14.3. HR-MS (ESI) m/z
:
+
calcd for C H O [M+H] : 369.1702; found 369.1695. HPLC purity 95.0% (30 min run of 45-80%
2
2
24
5
3 2
CH CN in H O, with 15 min gradient, 1.0 mL/min).
2
0

ACCEPTED MANUSCRIPT
4
.3. The general procedure for the synthesis of 3-O-aminoalkyl-3',4'-dimethoxyflavonols (36-63).
The solution of 3',4'-dimethoxyflavonol (4, 50 mg, 0.17 mmol) and potassium carbonate (70 mg,
.51 mmol, 3 equiv.) in DMF (0.5 mL) was stirred at room temperature for 10 min before an
0
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 (dichloromethane/methanol, 100/3, v/v). The reaction mixture was poured into ice
water (3.0 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 (0.5 ml) was added potassium carbonate (0.51 mmol, 3 equiv.), 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 (dichloromethane/methanol, 100/3, v/v). The mixture was poured into
ice water (3.0 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 eluent. The respective 3-
trimethoxyflavonol was retrieved from PTLC silica gel by washing with dichloromethane/diethylamine
100/3, v/v). The yields and spectral data for compounds 42, 43, 44, 48, 49, and 50 have been reported
O-dialkylaminoalkyl-3',4',5'-
(
in the supporting information of our previous Letter [19]. HPLC purity for 42 is 95.3% (30 min run
of 15-75% CH CN in aqueous solution of ammonium formate (1.26 g ammonium formate in
3
1
000 mL water), with 15 min gradient, 1.0 mL/min). HPLC purity for 43 is 95.8% [30 min run of
5-75% CH CN in aqueous ammonium formate solution (1.26 g per 1000 mL water), with 15 min
gradient, 1.0 mL/min]. HPLC purity for 44 is 97.9% (30 min run of 45-80% CH CN in in
1
3
3
2
1

ACCEPTED MANUSCRIPT
aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with
1
5 min gradient, 1.0 mL/min). HPLC purity for 48 is 95.6% (30 min run of 15-75% CH CN in
3
aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with
5 min gradient, 1.0 mL/min). HPLC purity for 50 is 97.0% (30 min run of 45-80% CH CN in
1
3
aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with
5 min gradient, 1.0 mL/min). HPLC purity for 3-O-Mopholinobutyl-3',4'-dimethoxyflavonol
49) is 96.5% (30 min run of 45-80% CH CN in aqueous solution of ammonium formate (1.26
1
(
3
g ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.1. 3-O-(N,N-Diethylamino)propyl-3',4'-dimethoxyflavonol (36)
This derivative was achieved in 88% overall yield for two steps as a syrup. IR (film) νmax: 2964,
-
1 1
2
7
8
2
835, 1635, 1614, 1599, 1559, 1510 cm . H NMR (300 MHz, CDCl )
δ
8.22 (dd,
J = 8.0, 1.5 Hz, 1H),
3
.74 (dd,
.2 Hz, 1H), 7.36 (br.d,
.54 (t, = 7.5 Hz, 2H), 2.47 (q,
C NMR (75 MHz, CDCl3)
J
= 8.5, 2.0 Hz, 1H), 7.70 (d,
= 7.2 Hz, 1H), 6.97 (d,
= 7.2 Hz, 4H), 1.87 (quin,
J
= 1.9 Hz, 1H), 7.64 (ddd,
J
= 8.6, 7.1, 1.6 Hz, 1H), 7.50 (d, J =
J
J
= 8.5 Hz, 1H), 4.07 (t,
J
= 6.5 Hz, 2H), 3.94 (s, 6H),
J
J
J
= 7.2 Hz, 2H), 0.96 (t, J = 7.1 Hz, 6H).
1
3
δ
175.1, 155.8, 155.2, 151.2, 148.7, 140.1, 133.3, 125.8, 124.6, 124.2,
1
23.6, 122.4, 118.0, 111.7, 110.8, 71.4, 56.1, 56.0, 49.8, 46.9, 28.0, 11.6. HR-MS (ESI) m/z: calcd for
+
C H NO [M+H] : 412.2124; found 412.2118. HPLC purity 95.3% (30 min run of 45-80% CH CN in
24
29
5
3
aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with 15 min
gradient, 1.0 mL/min).
4
.3.2. 3-O-(N,N-Diethylamino)butyl-3',4'-dimethoxyflavonol (37)
This derivative was achieved in 90% overall yield for two steps as a syrup. IR (film) νmax: 2963,
-
1 1
2
7
797, 1635, 1614, 1599, 1560, 1511 cm . H NMR (300 MHz, CDCl
3
)
δ
8.22 (d,
J = 8.0 Hz, 1H), 7.75-
.73 (overlapped, 2H), 7.70 – 7.61 (m, 1H), 7.51 (d, = 8.4 Hz, 1H), 7.37 (t,
J
J
= 7.5 Hz, 1H), 6.98 (d,
J
2
2

ACCEPTED MANUSCRIPT
=
8.3 Hz, 1H), 4.04 (t,
.72 (quin, = 7.2 Hz, 2H), 1.53 (quin,
175.1, 155.8, 155.2, 151.2, 148.6, 140.2, 133.3, 125.9, 124.7, 124.2, 123.6, 122.4, 118.0,
J
= 6.6 Hz, 2H), 3.95 (s, 6H), 2.49 (q,
J
= 7.2 Hz,4H), 2.43 (t,
J
= 7.8 Hz, 2H),
1
3
1
J
J
= 7.2 Hz, 2H), 0.98 (t,
J = 7.2Hz, 6H). C NMR (75 MHz,
3
CDCl ) δ
1
11.7, 110.8, 72.6, 56.1, 56.1, 52.5, 46.8, 28.4, 23.2, 11.6. HR-MS (ESI) m/z: calcd for C25H31NO
5
+
[
M+H] : 426.2280; found 426.2273. HPLC purity 95.1% [30 min run of 45-80% acetonitrile in
ammonium formate solution (1.26 g ammonium formate in 1000 mL water), with 15 min gradient, 1.0
mL/min].
4
.3.3. 3-O-(N,N-Diethylamino)pentyl-3',4'-dimethoxyflavonol (38)
This derivative was achieved in 85% overall yield for two steps as a syrup. IR (film) νmax: 2933,
-
1 1
1
–
6
2
6
1
636, 1614, 1599, 1559, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.23 (dd,
= 8.3 Hz, 1H), 7.37 (t,
= 6.7 Hz, 2H), 3.95 (s, 3H), 3.94 (s, 3H), 2.50 (q,
= 8.1 Hz,2H), 1.74 (quin,
175.1, 155.8, 155.2, 151.2, 148.6, 140.2, 133.3, 125.9, 124.7, 124.3,
J = 8.0, 1.2 Hz, 1H), 7.80
3
7.70 (m, 2H), 7.64 (ddd,
J
= 8.7, 6.9, 1.5 Hz, 1H), 7.51 (d,
J
J
J
= 7.2 Hz, 1H),
= 7.2 Hz, 4H),
.98 (d,
.38 (t,
J
= 9.1 Hz, 1H), 4.01 (t,
J
J
J = 7.2 Hz, 2H), 1.47-1.34(overlapped, 4H), 0.99 (t, J = 7.2 Hz,
1
3
3
H). C NMR (75 MHz, CDCl ) δ
23.7, 122.3, 118.0, 111.8, 110.8, 72.7, 56.1, 56.1, 52.7, 46.9, 30.3, 26.6, 24.1, 11.5. HR-MS (ESI) m/z
:
+
calcd for C H NO [M+H] : 440.2437; found 440.2428. HPLC purity 97.2% (30 min run of 45-80%
2
6
33
5
CH CN in aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water),
3
with 15 min gradient, 1.0 mL/min).
4
.3.4. 3-O-(N,N-Dipropylamino)propyl-3',4'-dimethoxyflavonol (39)
This derivative was achieved in 44% overall yield for two steps as a syrup. IR (film) νmax: 2955,
-
1 1
2
7
8
871, 1636, 1614, 1599, 1559, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.24 (dd,
= 8.6, 7.1, 1.6 Hz, 1H), 7.52 (d,
= 8.5 Hz, 1H), 4.07 (t, = 6.4 Hz, 2H), 3.96
= 6.3 Hz, 4H),1.93 (quin, = 7.2 Hz, 2H), 1.44 (sextet, = 7.5 Hz,
J = 8.0, 1.4 Hz, 1H),
3
.75 (dd,
J
= 8.5, 2.0 Hz, 1H), 7.71 (d,
J
= 1.9 Hz, 1H), 7.66 (ddd,
J
J =
.0 Hz, 1H), 7.39 (ddd,
J
= 7.8, 7.2, 0.9 Hz, 1H), 6.99 (d,
J
J
(
s, 6H), 2.63 (br.s, 2H), 2.40 (t,
J
J
J
2
3

ACCEPTED MANUSCRIPT
1
3
4
1
1
H), 0.83 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ 175.1, 155.9, 155.3, 151.3, 148.8, 140.1,
3
33.4, 125.9, 124.7, 124.3, 123.6, 122.5, 118.0, 111.7, 110.9, 71.3, 56.2, 56.1, 56.0, 51.1, 27.8, 19.8,
+
1.9. HR-MS (ESI) m/z: calcd for C26
H33NO
5
[M+H] : 440.2437; found 440.2429. HPLC purity 97.8%
(
30 min run of 15-70% CH
3
CN in aqueous solution of ammonium formate (1.26 g ammonium formate
in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.5. 3-O-(N,N-Dipropylamino)butyl-3',4'-dimethoxyflavonol (40)
This derivative was achieved in 64% overall yield for two steps as a syrup. IR (film) νmax: 2954,
-
1 1
2
7
7
2
2
1
5
870, 1636, 1614, 1599, 1560, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.24 (dd,
= 8.7, 6.9, 1.5 Hz, 1H), 7.52 (d,
= 8.3 Hz, 1H), 4.04 (t, = 6.6 Hz, 2H), 3.962 (s, 3H), 3.957 (s, 3H),
= 8.3 Hz, 4H), 1.74 (quin, = 7.3 Hz, 2H), 1.62-1.57 (overlapped,
= 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) 175.2, 155.8,
J
= 8.0, 1.4 Hz, 1H),
3
.78-7.74 (overlapped, 1H), 7.74 (s, 1H), 7.66 (ddd,
J
J
= 8.2 Hz, 1H),
.38 (t,
.49 (t,
J
J
= 7.5 Hz, 1H), 6.99 (d,
= 7.6 Hz, 2H), 2.39 (t,
J
J
J
J
1
3
H), 1.44 (sextet,
J
= 7.4 Hz, 4H), 0.84 (t,
J
δ
3
55.3, 151.2, 148.7, 140.2, 133.4, 125.9, 124.7, 124.3, 123.7, 122.4, 118.0, 111.8, 110.9, 72.6, 56.2,
+
6.1, 56.0, 53.7, 28.3, 23.2, 19.9, 12.0. HR-MS (ESI) m/z: calcd for C27
H35NO
5
[M+H] : 454.2593;
3
found 454.2581. HPLC purity 96.7% (30 min run of 45-80% CH CN in aqueous solution of
ammonium formate (1.26 g ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.6. 3-O-(N,N-Dipropylamino)pentyl-3',4'-dimethoxyflavonol (41)
This derivative was achieved in 69% overall yield for two steps as a syrup. IR (film) νmax: 2932,
-
1 1
2
869, 2797, 1636, 1614, 1599, 1560, 1511 cm . H NMR (300 MHz, CDCl
= 8.6, 7.2, 1.6 Hz, 1H), 7.52 (d,
= 9.0Hz, 1H), 4.03 (t, = 6.7 Hz, 2H), 3.96 (s, 3H), 3.95
= 6.9 Hz, 2H), 1.50 – 1.33 (overlapped, 8H), 0.85 (t,
175.1, 155.8, 155.3, 151.2, 148.7, 140.2, 133.3, 125.9,
24.7, 124.3, 123.7, 122.4, 118.0, 111.8, 110.8, 72.7, 56.2, 56.14, 56.08, 54.1, 30.3, 26.6, 24.0, 20.1,
3
)
δ
8.24 (dd,
J = 8.0, 1.4
Hz, 1H), 7.78 – 7.73 (overlapped, 2H), 7.65 (ddd,
.38 (ddd, = 8.6, 6.9, 0.9 Hz, 1H), 6.99 (d,
s, 3H), 2.37 (q, = 7.5 Hz, 6H), 1.74 (quin,
.4 Hz, 6H). C NMR (75 MHz, CDCl3)
J
J
= 8.2 Hz, 1H),
7
J
J
J
(
J
J
J =
1
3
7
1
δ
2
4

ACCEPTED MANUSCRIPT
+
1
2.0. HR-MS (ESI) m/z: calcd for C H NO [M+H] : 468.2750; found 468.2739. HPLC purity 97.3%
2
8
37
5
(
30 min run of 45-80% CH CN in aqueous solution of ammonium formate (1.26 g ammonium formate
3
in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.7. 3-O-(N,N-Diamylamino)propyl-3',4'-dimethoxyflavonol (45)
This derivative was achieved in 46% overall yield for two steps as a syrup. IR (film) νmax: 2952,
-
1 1
2
929, 2858, 1637, 1615, 1600, 1560, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.24 (dd,
= 8.4, 7.2, 1.8 Hz, 1H), 7.52 (d,
= 8.5 Hz, 1H), 4.08 (t, = 6.5 Hz, 2H), 3.96 (s, 6H),
= 8.1 Hz, 4H), 1.89 (quin, = 6.9 Hz, 2H), 1.56 – 1.12 (m, 12H),
= 6.8 Hz, 6H). C NMR (75 MHz, CDCl 175.1, 155.8, 155.2, 151.2, 148.7, 140.1, 133.3,
J = 8.0, 1.4
3
Hz, 1H), 7.76 (dd,
J
= 8.7, 1.8 Hz,1H), 7.72 (d,
= 8.2 Hz, 1H), 7.38 (t, = 7.1 Hz, 1H), 6.98 (d,
= 6.6 Hz. 2H), 2.37 (t,
J = 1.8 Hz), 7.65 (ddd, J
J
J
J
J
2
0
1
2
4
.56 (t,
.85 (t,
J
J
J
J
1
3
3
) δ
25.9, 124.7, 124.3, 123.6, 122.4, 118.0, 111.7, 110.8, 71.4, 56.1, 56.1, 54.1, 52.2, 51.1, 35.6, 29.8,
+
8.0, 26.6, 26.5, 22.8, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C H NO [M+H] : 496.3063; found
3
0
41
5
96.3055. HPLC purity 95.3% (30 min run of 15-75% CH CN in aqueous solution of ammonium
3
formate (1.26 g ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.8. 3-O-(N,N-Diamylamino)butyl-3',4'-dimethoxyflavonol (46)
This derivative was achieved in 55% overall yield for two steps as a syrup. IR (film) νmax: 2929,
-
1 1
2
7
7
859, 1636, 1615, 1599, 1560, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.24 (dd,
= 8.4, 6.9, 1.8 Hz, 1H), 7.52 (d,
= 8.5 Hz, 1H), 4.04(t, = 6.5 Hz, 2H), 3.95(s, 6H), 2.42 (t,
= 7.5 Hz, 2H), 1.58– 1.12 (m, 12H), 0.85 (t, = 6.8 Hz,
J
= 8.0, 1.4 Hz, 1H),
= 8.2 Hz, 1H),
= 8.4
3
.77 (dd,
.38 (t,
J
= 7.5, 2.1 Hz,1H), 7.75 (s, 1H),7.65 (ddd,
J
J
J
= 7.1 Hz, 1H), 6.98 (d,
J
J
J
Hz, 2H), 2.36 (t,
J
= 7.2 Hz, 4H), 1.73 (quin,
J
J
1
3
7
1
2
9
H). C NMR (75 MHz, CDCl ) δ 175.1, 155.8, 155.2, 151.2, 148.7, 140.1, 133.3, 125.9, 124.7, 124.3,
3
23.6, 122.4, 118.0, 111.7, 110.8, 71.4, 56.1, 56.1, 54.1, 52.2, 51.1, 35.6, 29.8, 28.0, 26.6, 26.5, 22.8,
+
2.7, 14.2. HR-MS (ESI) m/z: calcd for C31
5.0% (30 min run of 45-80% CH
H43NO
5
[M+H] : 510.3219; found 510.3213. HPLC purity
3
CN in aqueous solution of ammonium formate (1.26 g ammonium
2
5

ACCEPTED MANUSCRIPT
formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.9. 3-O-(N,N-Diamylamino)pentyl-3',4'-dimethoxyflavonol (47)
This derivative was achieved in 46% overall yield for two steps as a syrup. IR (film) νmax: 2929,
-
1 1
2
1
859, 1636, 1615, 1599, 1560, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.23 (dd,
= 8.6, 7.1, 1.6 Hz, 1H), 7.51 (d, = 8.3 Hz, 1H), 7.37
= 6.7 Hz, 2H), 3.96 (s, 3H), 3.95 (s,
J = 8.0, 1.3 Hz,
3
H),7.75 (dd,
= 7.8, 7.2, 0.9 Hz, 1H), 6.98 (d,
H), 2.38 (t, = 7.8 Hz, 6H), 1.75 (quin,
C NMR (75 MHz, CDCl3) 175.1, 155.7, 155.2, 151.2, 148.6, 140.2, 133.3, 125.9, 124.7, 124.3,
J
= 4.3, 2.5 Hz, 2H), 7.65 (ddd,
J
J
(
ddd,
J
J
= 9.1 Hz, 1H), 4.02 (t, J
3
J
J = 6.9 Hz, 2H), 1.60 – 1.12 (m, 16H), 0.91 – 0.81 (m, 6H).
1
3
δ
1
2
23.7, 122.3, 118.0, 111.8, 110.8, 72.7, 56.1, 56.1, 54.1, 54.0, 52.2, 35.6, 30.3, 29.9, 26.7, 26.6, 26.5,
+
4.1, 22.9, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C33
H47NO
5
[M+H] : 538.3532; found 538.3529.
HPLC purity 95.0% (30 min run of 15-75% CH CN in aqueous solution of ammonium formate (1.26 g
3
ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.10. 3-O-Piperidinopropyl-3',4'-dimethoxyflavonol (51)
This derivative was achieved in 75% overall yield for two steps as a syrup. IR (film) νmax: 2937,
-
1 1
2
847, 2779, 1635, 1611, 1598, 1558, 1513 cm . H NMR (300 MHz, CDCl
= 1.8 Hz, 1H), 7.64 (ddd,
= 6.9 Hz, 1H), 6.97 (d, = 8.5 Hz, 1H), 4.06 (t,
= 7.5 Hz, 2H), 2.36 (br.s, 3H), 1.93 (quin, = 6.6 Hz, 2H), 1.55 (quin,
175.1, 155.9, 155.2, 151.2, 148.7, 140.1,
33.4, 125.8, 124.7, 124.2, 123.6, 122.5, 118.0, 111.7, 110.8, 71.3, 56.2, 56.1, 54.6, 27.6, 25.8, 24.3.
3
)
δ
8.22 (dd,
= 8.7, 7.2, 1.5 Hz,
= 6.4 Hz, 2H),
= 5.7
J = 8.0, 1.3
Hz, 1H), 7.73 (dd, J = 8.4, 2.1 Hz, 1H), 7.69 (d,
J
J
1
3
H),7.51 (d,
J
= 8.2 Hz, 1H), 7.37 (t,
J
J
J
.95 (s, 6H), 2.45 (t,
J
J
J
1
3
Hz, 4H), 1.43-1.36 (m, 2H). C NMR (75 MHz, CDCl )
δ
3
1
+
HR-MS (ESI) m/z: calcd for C H NO [M+H] : 424.2124; found 424.2119. HPLC purity 99.0% (30
2
5
29
5
min run of 45-80% CH
3
CN in aqueous solution of ammonium formate (1.26 g ammonium formate in
1
000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.11. 3-O-Piperidinobutyl-3',4'-dimethoxyflavonol (52)
2
6

ACCEPTED MANUSCRIPT
This derivative was achieved in 84% overall yield for two steps as a syrup. IR (film) νmax: 2931,
-
1 1
1
634, 1614, 1599, 1558, 1511 cm . H NMR (300 MHz, CDCl )
δ
8.24 (dd,
= 8.4, 0.5 Hz, 1H), 7.38 (ddd,
J = 6.4 Hz, 2H), 3.963 (s, 3H), 3.957 (s, 3H), 2.30-
J
= 8.0, 1.4 Hz, 1H), 7.74
3
(
overlapped, 2H), 7.66 (ddd,
J
= 8.6, 7.1, 1.7 Hz, 1H), 7.52 (dd,
= 8.4 Hz, 1H), 4.04 (t,
.45 (overlapped, 6H), 1.78-1.56 (overlapped, 8H), 1.48– 1.37 (m, 2H). C NMR (75 MHz, CDCl
J
J
= 8.1,
7
2
.1, 1.1 Hz, 1H), 6.99 (d,
J
1
3
3
)
δ175.1, 155.9, 155.3, 151.3, 148.7, 140.1, 133.4, 125.9, 124.7, 124.3, 123.6, 122.5, 118.0, 111.7, 110.9,
+
7
4
2.2, 58.5, 56.2, 56.1, 54.2, 28.2, 25.1, 23.9, 22.6. HR-MS (ESI) m/z: calcd for C H NO [M+H] :
2
6
31
5
38.2280; found 438.2272. HPLC purity 95.2% (30 min run of 45-80% CH CN in aqueous solution of
3
ammonium formate (1.26 g ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.12. 3-O-Piperidinopentyl-3',4'-dimethoxyflavonol (53)
This derivative was achieved in 91% overall yield for two steps as a syrup. IR (film) νmax: 2931,
-1 1
2
852, 2763, 1635, 1614, 1599, 1560, 1510 cm . H NMR (300 MHz, CDCl )
δ
8.23 (dd,
J = 8.0, 1.4
3
Hz, 1H), 7.44 (dd,
J
= 7.2, 1.8 Hz, 1H), 7.73 (s, 1H), 7.65 (ddd,
= 7.2 Hz, 1H), 6.99 (d, = 9.1 Hz, 1H), 4.01 (t,
.95 (s, 3H), 2.42 (s, 4H), 2.31 (t, = 7.8 Hz, 2H), 1.74 (quin,
J
= 8.4, 7.2, 1.5 Hz, 1H), 7.51 (d, J =
8
3
4
1
5
4
.2 Hz, 1H), 7.38 (t,
J
J
J
= 6.6 Hz, 2H), 3.96 (s, 3H),
J
J
= 7.0 Hz, 2H), 1.61 (quin, J = 5.4 Hz,
1
3
H), 1.56 – 1.47 (m, 2H), 1.44-1.33 (overlapped, 4H). C NMR (75 MHz, CDCl )
δ 175.1, 155.8,
3
55.3, 151.2, 148.7, 140.2, 133.4, 125.9, 124.7, 124.3, 123.7, 122.4, 118.0, 111.8, 110.8, 72.6, 59.1,
+
6.2, 56.1, 54.4, 30.2, 26.1, 25.5, 24.2, 24.1. HR-MS (ESI) m/z: calcd for C H NO [M+H] :
2
7
33
5
52.2437; found 452.2428. HPLC purity 96.0% (30 min run of 45-80% CH CN in aqueous solution of
3
ammonium formate (1.26 g ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.13. 3-O-(4-Methylpiperazin-1-yl)propyl-3',4'-dimethoxyflavonol (54)
This derivative was achieved in 77% overall yield for two steps as a syrup. IR (film) νmax: 2931,
-1 1
2
852, 2763, 1635, 1614, 1599, 1560, 1510 cm . H NMR (300 MHz, CDCl 8.20 (dd,
3
)
δ
J = 8.0, 1.4
Hz, 1H), 7.70 (dd,
J
= 8.7, 1.8 Hz,1H), 7.65 (d, = 1.5 Hz, 1H), 7.62 (ddd, J = 8.4, 7.2, 2.1 Hz, 1H),
J
2
7

ACCEPTED MANUSCRIPT
7
3
.48 (d,
.92 (s, 6H), 2.69 – 2.27 (overlapped, 10H), 2.23 (s, 3H), 1.88 (quin,
175.0, 155.8, 155.2, 151.2, 148.7, 140.0, 133.3, 125.8, 124.7, 124.2, 123.6, 122.4,
J
= 8.3 Hz, 1H), 7.34 (t,
J
= 7.5 Hz, 1H), 6.95 (d,
J
= 8.5 Hz, 1H), 4.05 (t,
J = 6.4 Hz, 2H),
1
3
J
= 6.0 Hz, 2H). C NMR (75
3
MHz, CDCl ) δ
1
17.9, 111.8, 110.8, 71.1, 56.1, 56.0, 55.2, 55.0, 53.0, 46.0, 27.7. HR-MS (ESI) m/z: calcd for
+
C
25
H
30
N
2
O
5
[M+H] : 439.2233; found 439.2218. HPLC purity 98.8% (30 min run of 45-80% CH
3
CN
in aqueous solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with 15 min
gradient, 1.0 mL/min).
4
.3.14. 3-O-(4-Methylpiperazin-1-yl)butyl-3',4'-dimethoxyflavonol (55)
This derivative was achieved in 80% overall yield for two steps as a syrup. IR (film) νmax: 2931,
-
1 1
2
852, 2763, 1635, 1614, 1599, 1560, 1510 cm . H NMR (300 MHz, CDCl )
δ
8.21 (dd,
= 8.6, 7.1, 1.6 Hz, 1H), 7.49 (d,
= 8.5 Hz, 1H), 4.02 (t, = 6.5 Hz, 2H),
= 7.5 Hz,2H), 2.24 (s, 3H), 1.71
= 6.9 Hz, 2H), 1.64 – 1.49 (m, 2H). C NMR (75 MHz, CDCl 175.1, 155.8, 155.2, 151.2,
J
= 8.0, 1.4
3
Hz, 1H), 7.72 (dd,
J
= 8.1, 1.8 Hz,1H), 7.70 (s,1H), 7.63 (ddd,
= 8.1, 6.9, 1.2 Hz,1H), 6.96 (d,
J
J
J
= 8.0
Hz, 1H), 7.35 (ddd,7.70 (dd,
J
J
3
.94(s, 3H), 3.93 (s, 3H), 2.91 (br.s, 2H), 2.41 (br.s, 6H), 2.29 (t, J
1
3
(
quin,
J
3
) δ
1
5
48.6, 140.1, 133.3, 125.8, 124.7, 124.2, 123.6, 122.4, 117.9, 111.8, 110.8, 72.5, 58.2, 56.1, 56.0, 55.0,
+
3.0, 46.0, 28.2, 23.3. HR-MS (ESI) m/z: calcd for C H N O [M+H] : 453.2389; found 453.2383.
2
6
32
2
5
HPLC purity 95.5% (30 min run of 15-75% CH CN in aqueous solution of ammonium formate (1.26 g
3
ammonium formate in 1000 mL water), with 15 min gradient, 1.0 mL/min).
4
.3.15. 3-O-(4-Methylpiperazin-1-yl)pentyl-3',4'-dimethoxyflavonol (56)
This derivative was achieved in 63% overall yield for two steps as a syrup. IR (film) νmax: 2931,
-
1 1
2
1
6
–
852, 2763, 1635, 1614, 1599, 1560, 1510 cm . H NMR (300 MHz, CDCl )
δ
8.21 (d,
= 8.3 Hz, 1H), 7.35 (t, = 7.5 Hz, 1H),
J = 6.6 Hz, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 2.44 (br.s, 7H), 2.32
J = 8.0 Hz,
3
H), 7.73-7.71 (overlapped, 2H), 7.67 – 7.58 (m, 1H), 7.49 (d,
.96 (dd, = 9.0, 3.6 Hz, 1H), 4.00 (t,
2.11 (overlapped, 3H), 2.25 (s, 3 H), 1.72 (quin,
J
J
J
1
3
J
= 7.2 Hz, 2H), 1.53 – 1.29 (m, 4H). C NMR (75
2
8