Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and Biological 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

doi:10.1016/j.bioorg.2019.03.047

. Author manuscript; available in PMC: 2020 Jun 1.

Published in final edited form as: Bioorg Chem. 2019 Mar 19;87:227–239. doi: 10.1016/j.bioorg.2019.03.047

Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and Biological Profiles in Prostate Cancer Cell Models

Pravien Rajaram ^a, Ziran Jiang ^a, Guanglin Chen ^a, Alyssa Rivera ^a, Alison Phasakda ^a, Qiang Zhang ^b,^c, Shilong Zheng ^b,^c, Guangdi Wang ^b,^c, Qiao-Hong Chen ^a,^*

PMCID: PMC6538460 NIHMSID: NIHMS1525210 PMID: 30904813

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.

Keywords: Quercetin derivative, synthesis, antiproliferative activity, prostate cancer, cell apoptosis, cell cycle regulation

Graphical Abstract

graphic file with name nihms-1525210-f0001.jpg

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 IC₅₀ values ranging from 15.5 to 64 μM [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 IC₅₀ values measured in in vitro studies and 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-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.

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 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 100% conversion rate even under harsher reaction conditions including prolonged reaction time (48 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 (11-34) 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 2. — 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.

Scheme 3. — Synthesis of 3,3’,4’,7-O-tetramethylquercetin (7)

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 (38-61) against a panel of prostate cancer cell lines (PC-3, DU145, and LNCaP) was determined using 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 IC₅₀ values in PC-3 and LNCaP human prostate cancer cell lines. Among them, only 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, 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 IC₅₀ values of 0.55 μM, 2.82 μM, and 1.16 μM, respectively, towards PC-3, DU145, and LNCaP prostate cancer cell lines.

Table 1.

Antiproliferative activity of 3-O-aminoalkyl-3’,4’,5,7-O-tetramethylquercetins (11-34)


Compd	R	IC₅₀ (μM)^a
Compd	R	PC-3^b	DU145^c	LNCaP^d
Quercetin	-	>100	>100	45.5 ± 1.3
Fisetin	-	> 50	> 50	34.1 ± 7.7
11	(N,N-diethylamino)propyl	16.62 ± 0.24	> 50	17.63 ± 1.04
12	(N,N-diethylamino)butyl	22.73 ± 7.56	> 50	19.44 ± 3.83
13	(N,N-diethylamino)pentyl	18.33 ± 6.80	> 50	11.39 ± 2.27
14	(N,N-dipropylamino)propyl	11.59 ± 1.98	25.41 ± 3.68	9.57 ± 1.97
15	(N,N-dipropylamino)butyl	11.05 ± 4.06	> 50	10.99 ± 2.38
16	(N,N-dipropylamino)pentyl	20.12 ± 3.71	34.81 ± 3.25	13.80 ± 3.88
17	(N,N-dibutylamino)propyl	10.70 ± 2.30	22.92 ± 4.56	7.14 ± 0.78
18	(N,N-dibutylamino)butyl	7.00 ± 2.11	15.12 ± 2.29	6.54 ± 1.45
19	(N,N-dibutylamino)pentyl	7.60 ± 1.87	20.13 ± 5.14	7.18 ± 0.79
20	(N,N-dipentylamino)propyl	4.00 ± 0.63	4.74 ± 0.51	1.73 ± 0.34
21	(N,N-dipentylamino)butyl	3.81 ± 0.45	6.53 ± 0.16	3.45 ± 0.23
22	(N,N-dipentylamino)pentyl	6.97 ± 1.09	9.21 ± 0.21	6.74 ± 0.79
23	morpholinopropyl	20.38 ± 2.28	> 50	27.34 ± 4.61
24	morpholinobutyl	19.94 ± 2.37	28.88 ± 4.26	13.29 ± 2.71
25	morpholinopentyl	13.19 ± 3.77	18.05 ± 2.13	8.48 ± 0.62
26	piperidinopropyl	12.57 ± 4.27	41.12 ± 3.70	15.06 ± 2..48
27	piperidinobutyl	31.28 ± 2.04	> 50	21.49 ± 3.84
28	piperidinopentyl	21.04 ± 2.21	27.99 ± 8.32	13.49 ± 3.37
29	(4-methylpiperazin-1 - yl)propyl	32.01 ± 2.41	51.92 ± 4.67	22.39 ± 1.96
30	(4-methylpiperazin-1 -yl)butyl	12.46 ± 0.84	> 50	12.09 ± 3.24
31	(4-methylpiperazin-1 - yl)pentyl	19.89 ± 3.18	25.95 ± 0.12	14.62 ± 4.10
32	pyrrolidinopropyl	24.01 ± 5.44	39.38 ± 7.72	18.58 ± 0.81
33	pyrrolidinobutyl	22.51 ± 1.82	> 50	13.32 ± 2.90
34	pyrrolidinopentyl	22.87 ± 2.92	22.64 ± 2.37	11.37 ± 2.62

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IC₅₀ is the compound concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days exposure. The data were presented as the mean ± 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

Table 2.

Antiproliferative activity of 5-O-aminoalkyl-3,3’,4’,7-O-tetramethylquercetins (38-61)


Compd	R	IC₅₀ (μM)^a
Compd	R	PC-3^b	DU145^c	LNCaP^d
Quercetin	-	>100	>100	45.5 ± 1.3
Fisetin	-	> 50	> 50	34.1 ± 7.7
7	H	>50	>50	>50
38	(N,N-diethylamino)propyl	16.38 ± 4.23	36.19 ± 3.83	14.38 ± 2.41
39	(N,N-diethylamino)butyl	13.94 ± 1.34	14.94 ± 1.52	8.54 ± 2.64
40	(N,N-diethylamino)pentyl	17.06 ± 0.68	13.58 ± 2.23	6.79 ± 0.21
41	(N,N-dipropylamino)propyl	3.37 ± 1.23	8.98 ± 1.57	2.94 ± 0.54
42	(N,N-dipropylamino)butyl	4.71 ± 0.60	8.02 ± 2.43	2.53 ± 0.73
43	(N,N-dipropylamino)pentyl	10.20 ± 1.14	16.29 ± 4.71	5.80 ± 0.80
44	(N,N-dibutylamino)propyl	0.55 ± 0.16	2.82 ± 0.27	1.16 ± 0.18
45	(N,N-dibutylamino)butyl	3.35 ± 0.84	9.61 ± 2.03	2.50 ± 0.42
46	(N,N-dibutylamino)pentyl	4.78 ± 0.61	12.97 ± 2.46	3.14 ± 0.28
47	(N,N-dipentylamino)propyl	1.85 ± 0.29	5.44 ± 0.24	2.67 ± 0.38
48	(N,N-dipentylamino)butyl	2.10 ± 0.38	5.33 ± 0.92	1.23 ± 0.35
49	(N,N-dipentylamino)pentyl	4.11 ± 1.11	8.48 ± 0.50	1.32 ± 0.04
50	morpholinopropyl	12.15 ± 3.84	27.95 ± 1.05	12.82 ± 0.75
51	morpholinobutyl	12.40 ± 3.17	26.02 ± 3.66	9.45 ± 2.13
52	morpholinopentyl	11.81 ± 3.81	22.93 ± 373	6.87 ± 1.13
53	piperidinopropyl	4.07 ± 2.12	9.09 ± 0.28	4.07 ± 0.33
54	piperidinobutyl	10.31 ± 2.46	9.96 ± 1.97	6.54 ± 2.39
55	piperidinopentyl	12.61 ± 0.65	14.98 ± 1.53	7.83 ± 1.77
56	(4-methylpiperazin-1 - yl)propyl	4.47 ± 0.92	15.70 ± 2.17	4.33 ± 0.74
57	(4-methylpiperazin-1 -yl)butyl	12.34 ± 1.83	17.42 ± 3.54	2.97 ± 0.71
58	(4-methylpiperazin-1 - yl)pentyl	17.17 ± 2.12	20.46 ± 3.35	8.46 ± 0.78
59	pyrrolidinopropyl	5.80 ± 2.15	14.63 ± 5.95	9.42 ± 0.23
60	pyrrolidinobutyl	9.99 ± 2.22	13.92 ± 0.62	7.86 ± 1.80
61	pyrrolidinopentyl	15.87 ± 5.23	18.67 ± 5.52	10.28 ± 2.29

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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

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 5-OH of 3,3’,4’,7-O-tetramethylquercetin did significantly improve their potency.
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.

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.

Compd	IC50: (nM)^a		R/S^d	IC50: (nM)^a		R/S^c
Compd	PC-3^b	PC-3/DTX^c		DU145^e	DU145/DTX^f
Docetaxel	1.9 ± 0.6	2340 ± 250	1232	1.2 ± 0.3	8580±390	7150
44	550± 160	1700± 670	3.1	2820 ± 270	2290± 190	0.8

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Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor

Docetaxel-resistant PC-3 prostate cancer cell line

The relative resistance of the two cell lines obtained by dividing the IC₅₀ value of the resistance cell line by the parental cell line

Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor

Docetaxel-resistant DU145 prostate cancer cell line

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

Quercetin was reported to induce PC-3 cell cycle arrest at G₂/M and S phase.²⁶ To evaluate the effect 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, 44 causes slight accumulation of PC-3 cells in the G₀/G₁ phase from 26% for control cells at 16 hours 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 G₀/G₁ phase.

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-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.

Figure 2. — Evolution of viable, apoptotic, and necrotic PC-3 cell populations in response to 44

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 IC₅₀ values of 0.55–2.82 μM, being over 35- to 182-fold more potent than 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 Fourier 300 spectrometer in CDCl₃. The chemical shifts are given in ppm referenced to the 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 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), 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 10). 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) ν_max: 2965, 2836, 1622, 1600, 1513, 1457 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.70–7.59 (overlapped, 1H), 7.65 (s, 1H), 6.92 (d, J = 9.0 Hz, 1H), 6.44 (s, 1H), 6.28 (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, 4H), 1.86 (quin, J = 7.8 Hz, 2H), 0.95 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 163.9, 161.0, 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, 56.0, 55.8, 49.8, 46.9, 27.8, 11.5. HR-MS (ESI) m/z: calcd for C₂₆H₃₄NO₇ [M+H]: 472.2335; found 472.2335.

4.3.2. 3-O-(N,N-Diethylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (12)

Yield, 39%; yellow wax. IR (film) ν_max: 2937, 1624, 1602, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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), 1.73 (quin, J = 6.9 Hz, 2H), 1.65–1.53 (m, 2H), 1.03 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 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, 70.8, 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 C₂₇H₃₆NO₇ [M+H]: 486.2492; found 486.2492.

4.3.3. 3-O-(N,N-Diethylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (13)

Yield, 61%; yellow oil. IR (film) ν_max: 2937, 1622, 1601, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (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 = 1.8 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 Hz, 2H), 1.72 (quin, J = 7.5 Hz, 2H), 1.50–1.35 (m, 4H), 1.01 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 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, 95.8, 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 C₂₈H₃₈NO₇ [M+H]: 500.2648; found 500.2645.

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) v_max: 2957, 2872, 2361, 1601 cm⁻¹.¹H NMR (300 MHz, CDCl₃) 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 (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, 3H), 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 Hz, 4H), 0.83 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ 174.2, 164.0, 161.1, 158.9, 153.0, 150.9, 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, 47.7, 27.6, 19.6, 11.9. HR-MS (ESI) m/z: calcd for C₂₈H₃₈NO₇ [M+H]: 500.2648; found 500.2637.

4.3.5. 3-O-(N,N-Dipropylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (15)

Yield, 92%; yellow oil. IR (film) ν_max: 2954, 1622, 1601, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 = 6.6 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 = 7.2 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).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 163.9, 161.0, 158.8, 152.7, 150.7, 148.5, 140.3, 123.6, 121.7, 111.4, 110.7, 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 C₂₉H₄₀NO₇ [M+H]: 514.2805; found 514.2801.

4.3.6. 3-O-(N,N-Dipropylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (16)

Yield, 38%; yellow oil. IR (film) ν_max: 2939, 1620, 1600, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.69 (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, 1H), 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, 3H), 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, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.1, 161.1, 159.0, 153.2, 151.1, 148.8, 140.2, 123.5, 122.0, 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, 11.4. HR-MS (ESI) m/z: calcd for C₃₀H₄₂NO₇ [M+H]: 528.2961; found 528.2961.

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) v_max: 3408, 2960, 1601, 1515 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.67 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.50 (d, J = 1.8 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, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.3, 161.0, 159.0, 153.7, 151.3, 149.0, 139.6, 122.9, 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, 13.7. HR-MS (ESI) m/z: calcd for C₃₀H₄₂NO₇ [M+H]: 528.2961; found 528.2963.

4.3.8. 3-O-(N,N-Dibutylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (18)

Yield, 68%; yellow wax. IR (film) ν_max: 2932, 2870, 1622, 1601, 1513, 1489, 1457 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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, 4H), 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, J = 6.9 Hz, 4H), 0.86 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 163.9, 161.1, 158.9, 152.8, 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, 53.8, 53.7, 28.8, 28.4, 23.1, 20.8, 14.1. HR-MS (ESI) m/z: calcd for C₃₁H₄₄NO₇ [M+H]: 542.3118; found 542.3116.

4.3.9. 3-O-(N,N-Dibutylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (19)

Yield, 51%; yellow oil. IR (film) ν_max: 2932, 2860, 1622, 1601, 1513 cm⁻¹ .¹H NMR (300 MHz, CDCl₃) δ: 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 (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 Hz, 2H), 1.78 – 1.24 (m, 12 H), 0.88 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 163.9, 161.1, 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, 56.1, 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 C₃₂H₄₆NO₇ [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) ν_max: 2929, 2855, 1620, 1597, 1514, 1458 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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), 3.56–3.45 (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 = 6.6 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.0, 161.2, 159.0, 152.9, 150.9, 148.7, 140.4, 123.7, 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, 22.8, 14.2. HR-MS (ESI) m/z: calcd for C₃₂H₄₆NO₇ [M+H]: 556.3274; found 556.3273.

4.3.11. 3-O-(N,N-Dipentylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (21)

Yield, 54%; yellow oil. IR (film) ν_max: 2931, 1624, 1603, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.69 (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, 1H), 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 (quin, J = 7.5 Hz, 4H), 1.28–1.15 (overlapped, 8H), 0.83 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 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, 95.7, 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 C₃₃H₄₈NO₇ [M+H]: 570.3431; found 570.3429.

4.3.12. 3-O-(N,N-Dipentylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (22)

Yield, 45%; yellow oil. IR (film) ν_max: 2931, 1624, 1603, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 6H), 1.75 (quin, J = 7.5 Hz, 2H), 1.62–1.38 (overlapped, 6H), 1.34–1.18 (overlapped, 10H), 0.88 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.0, 161.2, 159.0, 152.9, 150.9, 148.7, 140.4, 123.7, 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, 25.5, 24.0, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C₃₄H₅₀NO₇ [M+H]: 584.3587; found 584.3586.

4.3.13. 3-O-Morpholinopropyl-3’,4’,5,7-O-tetramethylquercetin (23)

Overall yield for two steps, 36%; yellow solid. IR (film) v_max: 2937, 2847, 1621, 1600, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ 7.68 (dd, J = 8.7, 2.1 Hz, 1H), 7.66 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 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), 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).¹³C NMR (75 MHz, CDCl₃) 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, 109.5, 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 C₂₆H₃₂NO₈ [M+H]: 486.2128; found 486.2127.

4.3.14. 3-O-Morpholinobutyl-3’,4’,5,7-O-tetramethylquercetin (24)

Yield, 89%; yellow oil. IR (film) ν_max: 2943, 1623, 1602, 1514, 1456 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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 4H), 1.95 (quin, J = 5.8 Hz, 2H), 1.76 (quin, J = 6.0 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.1, 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, 64.8, 57.8, 56.5, 56.2, 56.1, 55.9, 52.4, 27.5, 21.3. HR-MS (ESI) m/z: calcd for C₂₇H₃₄NO₈ [M+H]: 500.2284; found 500.2275.

4.3.15. 3-O-Morpholinopentyl-3’,4’,5,7-O-tetramethylquercetin (25)

Yield, 58%; yellow oil. IR (film) ν_max: 2937, 2857, 1621, 1600, 1512 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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), 3.99 (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 = 6.3 Hz, 2H), 1.72 (quin, J = 6.9 Hz, 2H), 1.56–1.34 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.0, 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, 66.9, 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 C₂₈H₃₆NO₈ [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) v_max: 2935, 1624, 1600, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) 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 (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), 1.90 (quin, J = 6.3 Hz, 2H), 1.56 (quin, J = 5.1 Hz, 4H), 1.45–1.31 (m, 2H).¹³C NMR (75 MHz, CDCl₃) 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, 95.8, 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 C₂₇H₃₄NO₇ [M+H]: 484.2335; found 484.2331.

4.3.17. 3-O-Piperidinobutyl-3’,4’,5,7-O-tetramethylquercetin (27)

Yield, 29%; yellow oil. IR (film) ν_max: 2945, 1622, 1601, 1514, 1457 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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), 4.00–3.90 (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), 2.04–1.73 (m, 10H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.1, 161.0, 158.9, 153.3, 151.1, 148.8, 140.0, 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, 22.9, 21.5. HR-MS (ESI) m/z: calcd for C₂₈H₃₆NO₇ [M+H]: 498.2492; found 498.2490.

4.3.18. 3-O-Piperidinopentyl-3’,4’,5,7-O-tetramethylquercetin (28)

Yield, 40%; yellow oil. IR (film) ν_max: 2934, 1623, 1602, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (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 = 6.3 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), 1.41–1.35 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 163.8, 161.0, 158.8, 152.8, 150.7, 148.5, 140.3, 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, 25.1, 23.94, 23.88. HR-MS (ESI) m/z: calcd for C₂₉H₃₈NO₇ [M+H]: 512.2648; found 512.2643.

4.3.19. 3-O-(4-Methylpiperazin-1-yl)propyl-3’,4’,5,7-O-tetramethylquercetin (29)

52% overall yield for two steps as a yellow wax. IR (film) v_max: 2938, 2796, 2360, 1624, 1602, 1513 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ 7.65 (d, J = 6.6 Hz, 1H), 7.64 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 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), 2.68–2.66 (m, 2H), 2.53–2.46 (m, 8H), 1.90 (quin, J = 6.3 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ 174.2, 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, 70.8, 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 C₂₇H₃₅N₂O₇ [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) ν_max: 2936, 2801, 1621, 1601, 1513, 1456, 1421 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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), 2.26 (s, 3H), 1.70 (quin, J = 6.9 Hz, 2H), 1.57 (quin, J = 6.3 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 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, 72.2, 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 C₂₈H₃₇N₂O₇ [M+H]: 513.2601; found 513.2601.

4.3.21. 3-O-(4-Methylpiperazin-1-yl)pentyl-3’,4’,5,7-O-tetramethylquercetin (31)

Yield, 53%; yellow oil. IR (film) ν_max: 2937, 2803, 1622, 1602, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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), 2.29 (s, 3H), 1.70 (quin, J = 7.2 Hz, 2H), 1.53–1.30 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 163.9, 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, 58.4, 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 C₂₉H₃₉N₂O₇ [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) v_max: 2935, 2793, 1622, 1600.¹H NMR (300 MHz, CDCl₃) 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 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), 3.88 (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 Hz, 4H).¹³C NMR (75 MHz, CDCl₃) δ 174.2, 164.0, 161.1, 158.9, 153.1, 150.9, 148.7, 140.2, 123.5, 121.9, 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 C₂₆H₃₂NO₇ [M+H]: 470.2179; found 470.2179.

4.3.23. 3-O-Pyrrolidinobutyl-3’,4’,5,7-O-tetramethylquercetin (33)

Yield, 98%; yellow oil. IR (film) ν_max: 2937, 2794, 1621, 1600, 1512 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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), 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).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 163.9, 160.9, 158.8, 152.7, 150.7, 148.5, 140.3, 123.5, 121.7, 111.4, 110.7, 109.4, 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 C₂₇H₃₄NO₇ [M+H]: 484.2335; found 484.2332.

4.3.24. 3-O-Pyrrolidinopentyl-3’,4’,5,7-O-tetramethylquercetin (34)

Yield, 80%; yellow oil. IR (film) ν_max: 2938, 1625, 1604, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 163.9, 160.9, 158.8, 152.9, 150.8, 148.6, 140.2, 123.4, 121.8, 111.4, 110.8, 109.4, 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 C₂₈H₃₆NO₇ [M+H]: 498.2492; found 498.2493.

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 an appropriate alkyl dibromide (0.52 mmol, 3 equiv.) was added through a syringe. The reaction was 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)

Yield, 73%; yellow solid.¹H NMR (300 MHz, CDCl₃) δ: 7.70 (dd, J = 7.2, 2.1 Hz, 1H), 7.68 (s, 1H), 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, 2H), 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)

Yield, 58%; yellow solid.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 7.2, 2.1 Hz, 1H), 7.67 (s, 1H), 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, 2H), 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), 2.11–2.06 (m, 2H).

4.4.3. 5-O-(5-Bromopentyl)-3,3’,4’,7-O-tetramethylquercetin (37)

Yield, 58%; yellow solid.¹H NMR (300 MHz, CDCl₃) δ: 7.71 (dd, J = 9.0, 2.1 Hz, 1H), 7.70 (d, J = 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):

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)

Yield, 40%; yellow wax. IR (film) ν_max: 2939, 2837, 1625, 1603, 1515 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 1H), 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), 3.22–3.09 (m, 2H), 3.00–2.83 (m, 4H), 2.34–2.21 (m, 2H), 1.22 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 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, 96.8, 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 C₂₆H₃₄NO₇ [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) ν_max: 2934, 2836, 1623, 1601, 1514, 1438 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.4, 158.9, 152.7, 150.9, 148.8, 141.2, 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, 11.1. HR-MS (ESI) m/z: calcd for C₂₇H₃₆NO₇ [M+H]: 486.2492; found 486.2491.

4.5.3. 5-O-(N,N-Diethylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (40)

Yield, 86%; yellow oil. IR (film) ν_max: 2924, 2853, 1625, 1602, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 1H), 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), 2.81–2.67 (overlapped, 6H), 1.98–1.93 (m, 2H), 1.73–1.60 (m, 4H), 1.15 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 158.9, 152.8, 150.9, 148.8, 141.2, 123.5, 121.8, 111.3, 110.9, 109.7, 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 C₂₈H₃₈NO₇ [M+H]: 500.2648 ; found 500.2642.

4.5.4. 5-O-(N,N-Dipropylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (41)

Yield, 88%; brown syrup. IR (film) ν_max: 2957, 2872, 1623, 1601, 1514, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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, 2H), 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 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 158.8, 152.7, 150.9, 148.8, 141.2, 123.5, 121.8, 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 C₂₈H₃₈NO₇ [M+H]: 500.2648; found 500.2636.

4.5.5. 5-O-(N,N-Dipropylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (42)

Yield, 81%; yellow wax. IR (film) ν_max: 2933, 2872, 1623, 1601, 1514, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 1H), 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 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).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.8, 111.3, 110.9, 109.8, 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 C₂₉H₄₀NO₇ [M+H]: 514.2805; found 514.2800.

4.5.6. 5-O-(N,N-Dipropylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (43)

Yield, 84%; yellow oil. IR (film) ν_max: 2937, 1622, 1600, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 158.9, 152.8, 150.9, 148.8, 141.2, 123.5, 121.7, 111.3, 110.9, 109.7, 96.8, 92.6, 69.3, 60.1, 56.2, 56.1, 55.9, 55.3, 53.5, 28.6, 25.2, 24.0, 18.8, 11.8. HR-MS (ESI) m/z: calcd for C₃₀H₄₂NO₇ [M+H]: 528.2961; found 528.2960.

4.5.7. 5-O-(N,N-Dibutylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (44)

Yield, 17%; yellow syrup. IR (film) ν_max: 2954, 2930, 2870, 1624, 1601, 1514, 1489 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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), 6.48 (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), 3.84 (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 (m, 4H), 0.87 (t, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.2, 159.7, 158.8, 153.3, 151.2, 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, 51.2, 25.9, 24.9, 20.5, 13.9. HR-MS (ESI) m/z: calcd for C₃₀H₄₂NO₇ [M+H]: 528.2961; found 528.2958.

4.5.8. 5-O-(N,N-Dibutylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (45)

Yield, 72%; yellow wax. IR (film) ν_max: 2931, 2870, 1624, 1602, 1514, 1438 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 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, 2H), 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 = 7.5 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).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.5, 158.9, 152.7, 150.9, 148.8, 141.2, 123.6, 121.7, 111.3, 110.9, 109.8, 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 C₃₁H₄₄NO₇ [M+H]: 542.3118; found 542.3118.

4.5.9. 5-O-(N,N-Dibutylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (46)

Yield, 80%; yellow oil. IR (film) ν_max: 2934, 2872, 1621, 1600, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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, 1H), 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, 3H), 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), 1.29 (sextet, J = 7.5 Hz, 4H), 0.89 (t, J = 7.5 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 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, 56.1, 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 C₃₂H₄₆NO₇ [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) ν_max: 2929, 2858, 1623, 1601, 1515, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 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), 2.82–2.66 (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).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.2, 160.0, 158.9, 153.1, 151.1, 148.9, 141.2, 123.4, 121.9, 111.4, 111.0, 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 C₃₂H₄₆NO₇ [M+H]: 556.3274; found 556.3271.

4.5.11. 5-O-(N,N-Dipentylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (48)

Yield, 96%; brown wax. IR (film) ν_max: 2929, 2857, 1623, 1601, 1514, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (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, 3H), 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 (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).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.8, 111.4, 110.9, 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. HRMS (ESI) m/z: calcd for C₃₃H₄₈NO₇ [M+H]: 570.3431; found 570.3430.

4.5.12. 5-O-(N,N-Dipentylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (49)

Yield, 18%; yellow oil. IR (film) ν_max: 2930, 2858, 1623, 1602, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 = 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), 2.62–2.53 (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, J = 7.2 Hz, 6H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.0, 160.6, 158.9, 152.8, 150.9, 148.8, 141.3, 123.6, 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, 25.9, 25.8, 24.1, 22.7, 14.2. HR-MS (ESI) m/z: calcd for C₃₄H₅₀NO₇ [M+H]: 584.3587; found 584.3587.

4.5.13. 5-O-Morpholinopropyl-3,3’,4’,7-O-tetramethylquercetin (50)

Yield, 15%; yellow syrup. IR (film) ν_max: 2932, 2851, 1623, 1601, 1514, 1489 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 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 (d, J = 2.1 Hz, 1H), 6.36 (d, J = 2.1 Hz, 1H), 4.15 (t, J = 6.3 Hz, 2H), 3.96 (s, 6H), 3.89 (s, 3H), 3.83 (s, 3H), 3.75 (t, J = 4.5 Hz, 4H), 2.73 (t, J = 6.9 Hz, 2H), 2.63–2.51 (m, 4H), 2.16 (quin, J = 5.1 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.0, 160.5, 159.0, 152.9, 151.0, 148.9, 141.3, 123.6, 121.8, 111.4, 111.0, 109.8, 97.0, 92.7, 67.6, 66.9, 60.2, 56.2, 56.1, 55.9, 55.5, 53.8, 25.9. HR-MS (ESI) m/z: calcd for C₂₆H₃₂NO₈ [M+H]: 486.2128; found 486.2130.

4.5.14. 5-O-Morpholinobutyl-3,3’,4’,7-O-tetramethylquercetin (51)

Yield, 95%; light yellow solid. IR (film) ν_max: 2933, 1625, 1603, 1515, 1438 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.69–7.67 (overlapped, 1H), 7.67 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.46 (d, J = 1.8 Hz, 1H), 6.30 (d, J = 1.8 Hz, 1H), 4.07 (t, J = 6.0 Hz, 2H), 3.93 (s, 6H), 3.86 (s, 3H), 3.81 (s, 3H), 3.71 (t, J = 4.5 Hz, 4H), 2.51–2.46 (overlapped, 6H), 1.96 (quin, J = 6.9 Hz, 2H), 1.81 (quin, J = 7.5 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.5, 158.9, 152.8, 150.9, 148.8, 141.2, 123.5, 121.7, 111.3, 110.9, 109.7, 96.7, 92.5, 69.2, 66.8, 60.1, 58.5, 56.2, 56.1, 55.9, 53.7, 26.9, 22.8. HR-MS (ESI) m/z: calcd for C₂₇H₃₄NO₈ [M+H]: 500.2285; found 500.2290.

4.5.15. 5-O-Morpholinopentyl-3,3’,4’,7-O-tetramethylquercetin (52)

Yield, 66%; yellow oil. IR (film) ν_max: 2937, 1621, 1601, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.69 (dd, J = 9.0, 2.1 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 3.950 (s, 3H), 3.948 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H), 3.72 (t, J = 4.8 Hz, 4H), 2.46 (t, J = 4.2 Hz, 4H), 2.39 (t, J = 7.2 Hz, 2H), 1.96 (quin, J = 6.6 Hz, 2H), 1.59 (quin, J = 3.9 Hz, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.1, 160.2, 158.9, 153.1, 151.0, 148.8, 141.1, 123.3, 121.8, 111.2, 111.0, 109.5, 96.8, 92.8, 69.1, 63.8, 60.1, 57.7, 56.2, 56.1, 55.9, 51.8, 27.8, 24.1, 22.9. HR-MS (ESI) m/z: calcd for C₂₈H₃₆NO₈ [M+H]: 514.2441; found 514.2452.

4.5.16. 5-O-Piperidinopropyl-3,3’,4’,7-O-tetramethylquercetin (53)

Yield, 14%; yellow syrup. IR (film) ν_max: 2932, 1622, 1601, 1515 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.72 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 2.1 Hz, 1H), 6.35 (d, J = 2.1 Hz, 1H), 4.26–4.18 (m, 2H), 3.972 (s, 3H), 3.967 (s, 3H), 3.91 (s, 3H), 3.79 (s, 3H), 3.68–3.54 (m, 4H), 3.08–2.93 (m, 2H), 2.62–2.53 (m, 2H), 2.37–2.21 (m, 2H), 1.96–1.81 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.3, 164.4, 159.5, 158.8, 153.8, 151.3, 148.9, 141.1, 123.1, 122.0, 111.3, 111.0, 109.2, 97.1, 93.5, 67.1, 60.2, 56.24, 56.16, 56.11, 56.0, 53.8, 23.8, 22.7, 22.2. HR-MS (ESI) m/z: calcd for C₂₇H₃₄NO₇ [M+H]: 484.2335; found 484.2335.

4.5.17. 5-O-Piperidinobutyl-3,3’,4’,7-O-tetramethylquercetin (54)

Yield, 77%; brown wax. IR (film) ν_max: 2937, 1624, 1602, 1515, 1442 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.73–7.63 (overlapped, 2H), 6.95 (d, J = 9.0 Hz, 1H), 6.46 (s, 1H), 6.31 (s, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.46 (t, J = 7.2 Hz, 6H), 1.95 (quin, J = 7.2 Hz, 2H), 1.79 (quin, J = 7.8 Hz, 2H), 1.66–1.56 (m, 4H), 1.48–1.35 (m, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.7, 111.3, 110.9, 109.8, 96.8, 92.5, 69.4, 60.1, 58.9, 56.2, 56.1, 55.9, 54.6, 27.1, 25.8, 24.4, 23.2. HR-MS (ESI) m/z: calcd for C₂₈H₃₆NO₇ [M+H]: 498.2492; found 498.2492.

4.5.18. 5-O-Piperidinopentyl-3,3’,4’,7-O-tetramethylquercetin (55)

Yield, 90%; yellow oil. IR (film) ν_max: 2937, 1625, 1603, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.67 (dd, J = 9.6, 2.1 Hz, 1H), 7.66 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.86 (s, 3H), 3.80 (s, 3H), 2.70–2.62 (overlapped, 6H), 1.94 (quin, J = 6.9 Hz, 2H), 1.84–1.73 (m, 6H), 1.62–1.50 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.4, 158.8, 152.8, 150.9, 148.8, 141.2, 123.4, 121.7, 111.2, 110.9, 109.6, 96.7, 92.6, 69.3, 60.1, 58.4, 56.2, 56.1, 55.9, 53.9, 28.5, 25.0, 24.5, 24.2, 23.5. HR-MS (ESI) m/z: calcd for C₂₉H₃₈NO₇ [M+H]: 512.2648; found 512.2648.

4.5.19. 5-O-(4-Methylpiperazin-1-yl)propyl-3,3’,4’,7-O-tetramethylquercetin (56)

Yield, 59%; yellow syrup. IR (film) ν_max: 2932, 2836, 1623, 1600, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.69 (d, J = 7.5 Hz, 1H), 7.68 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.50 (d, J = 1.8 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 4.14 (t, J = 5.4 Hz, 2H), 3.95 (s, 6H), 3.88 (s, 3H), 3.80 (s, 3H), 3.18–2.84 (overlapped, 10H), 2.51 (s, 3H), 2.34–2.20 (m, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 164.1, 159.9, 158.9, 153.3, 151.1, 148.9, 141.2, 123.3, 121.9, 111.3, 111.0, 109.5, 97.0, 93.0, 67.1, 60.2, 56.2, 56.1, 56.0, 54.8, 53.0, 51.4, 44.8, 25.2. HR-MS (ESI) m/z: calcd for C₂₇H₃₅N₂O₇ [M+H]: 499.2444; found 499.2441.

4.5.20. 5-O-(4-Methylpiperazin-1-yl)butyl-3,3’,4’,7-O-tetramethylquercetin (57)

Yield, 85%; yellow wax. IR (film) ν_max: 2937, 2804, 1624, 1602, 1515, 1441 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 9.0, 2.1 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 6.96 (d, J = 9.3 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.31 (d, J = 2.4 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.76–2.31 (overlapped, 10H), 1.96 (quin, J = 7.5 Hz, 2H), 1.81 (quin, J = 6.9 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.5, 158.9, 152.7, 150.9, 148.8, 141.3, 123.6, 121.7, 111.3, 110.9, 109.8, 96.8, 92.6, 69.3, 60.1, 58.0, 56.2, 56.1, 55.9, 54.7, 52.8, 45.9, 26.9, 23.2. HR-MS (ESI) m/z: calcd for C₂₈H₃₇N₂O₇ [M+H]: 513.2601; found 513.2603.

4.5.21. 5-O-(4-Methylpiperazin-1-yl)pentyl-3,3’,4’,7-O-tetramethylquercetin (58)

Yield, 93%; yellow oil. IR (film) ν_max: 2936, 1621, 1600, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 8.4, 1.5 Hz, 1H), 7.66 (s, 1H), 6.95 (d, J = 9.3 Hz, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.93 (s, 6H), 3.86 (s, 3H), 3.81 (s, 3H), 2.70–2.48 (m, 8H), 2.46 (t, J = 6.3 Hz, 2H), 2.33 (s, 3H), 1.94 (quin, J = 6.6 Hz, 2H), 1.65–1.50 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 163.9, 160.5, 158.9, 152.7, 150.9, 148.8, 141.2, 123.5, 121.7, 111.3, 110.9, 109.7, 96.7, 92.5, 69.3, 60.1, 58.2, 56.2, 56.1, 55.9, 54.5, 52.6, 45.7, 28.8, 26.2, 24.0. HR-MS (ESI) m/z: calcd for C₂₉H₃₉N₂O₇ [M+H]: 527.2757; found 527.2755.

4.5.22. 5-O-Pyrrolidinopropyl-3,3’,4’,7-O-tetramethylquercetin (59)

Yield, 90%; brown syrup. IR (film) ν_max: 2934, 2796, 1623, 1601, 1514, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 9.3, 2.1 Hz, 1H), 7.67 (d, J = 1.5 Hz, 1H), 6.96 (d, J = 9.3 Hz, 1H), 6.49 (d, J = 2.1 Hz, 1H), 6.34 (d, J = 2.1 Hz, 1H), 4.16 (t, J = 6.3 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.80 (s, 3H), 3.09 (t, J = 6.6 Hz, 2H), 2.97–2.84 (m, 4H), 2.27 (quin, J = 6.6 Hz, 2H), 1.98–1.89 (m, 4H).¹³C NMR (75 MHz, CDCl₃) δ: 174.2, 164.1, 160.0, 158.8, 153.1, 151.1, 148.8, 141.2, 123.4, 121.8, 111.3, 110.9, 109.5, 96.9, 92.9, 67.7, 60.1, 56.2, 56.1, 56.0, 54.4, 53.4, 27.4, 23.6. HR-MS (ESI) m/z: calcd for C₂₆H₃₂NO₇ [M+H]: 470.2179; found 470.2177.

4.5.23. 5-O-Pyrrolidinobutyl-3,3’,4’,7-O-tetramethylquercetin (60)

Yield, 92%; brown wax. IR (film) ν_max: 2934, 2836, 1622, 1600, 1514, 1439 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 9.0, 2.1 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 4.07 (t, J = 6.0 Hz, 2H), 3.94 (s, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 2.78–2.70 (overlapped, 6H), 2.01–1.82 (overlapped, 8H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.4, 158.9, 152.7, 150.9, 148.8, 141.2, 123.5, 121.8, 111.3, 110.9, 109.7, 96.8, 92.6, 69.3, 60.1, 56.2, 56.1, 56.0, 55.9, 54.1, 26.9, 25.1, 23.6. HR-MS (ESI) m/z: calcd for C₂₇H₃₄NO₇ [M+H]: 484.2335; found 484.2334.

4.5.24. 5-O-Pyrrolidinopentyl-3,3’,4’,7-O-tetramethylquercetin (61)

Yield, 74%; yellow oil. IR (film) ν_max: 2939, 1623, 1601, 1514 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 7.68 (dd, J = 8.4, 2.1 Hz, 1H), 7.66 (s, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 4.07 (t, J = 5.7 Hz, 2H), 3.93 (s, 6H), 3.87 (s, 3H), 3.79 (s, 3H), 3.19–3.07 (m, 4H), 3.01 (t, J = 8.4 Hz, 2H), 2.05–1.90 (m, 8H), 1.72–1.62 (m, 2H).¹³C NMR (75 MHz, CDCl₃) δ: 174.0, 164.0, 160.3, 158.9 152.9, 151.0, 148.8, 141.2, 123.4, 121.8, 111.2, 110.9, 109.6, 96.7, 92.7, 69.1, 60.1, 56.2, 56.1, 55.9, 55.5, 53.6, 28.1, 25.9, 24.0, 23.5. HR-MS (ESI) m/z: calcd for C₂₈H₃₆NO₇ [M+H]: 498.2492; found 498.2494.

4.6. Cell culture

All cell lines were initially purchased from American Type Culture Collection (ATCC). The PC-3 and LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The DU-145 prostate cancer cells were routinely cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% penicillin/ streptomycin. Cultures were maintained in a high humidity environment supplemented with 5% carbon dioxide at a temperature of 37°C.

Docetaxel-resistant prostate cancer cell lines were established based on the procedure illustrated in the literature [30,31]. Docetaxel-resistant DU145 and PC-3 cell lines (DU145/DTX and PC-3/DTX) were developed over a period of one year by stepwise increased concentrations of docetaxel. Cells were continuously maintained in docetaxel, with treatments beginning at the initial IC₅₀ values of the respective parent cell lines. Media containing docetaxel will be changed every 2–3 days. As cells displayed resistance to treatments of docetaxel the concentration was subsequently increased.

4.7. WST-1 cell proliferation assay

PC-3, DU-145, LNCaP, PC-3/DTX, or DU145/DTX cells were plated in 96-well plates at a density of 3,200 each well in 200 μL of culture medium. The cells were then treated with quercetin, fisetin, or synthesized derivatives separately at different doses for 3 days, while equal treatment volumes of DMSO were used as vehicle control. The cells were cultured in a CO₂ incubator at 37 °C for three days. 10 μL of the premixed WST-1 cell proliferation reagent (Clontech) was added to each well. After mixing gently for one minute on an orbital shaker, the cells were incubated for additional 3 hours at 37 °C. To ensure homogeneous distribution of color, it is important to mix gently on an orbital shaker for one minute. The absorbance of each well was measured using a microplate-reader (Synergy HT, BioTek) at a wavelength of 430 nm. The IC₅₀ value is the concentration of each compound that inhibits cell proliferation by 50% under the experimental conditions and is the average from triplicate determinations that were reproducible and statistically significant. For calculating the IC₅₀ values, a linear proliferative inhibition was made based on at least five dosages for each compound.

4.8. Cell cycle analysis

PC-3 cells were plated in 24-well plates at a density of 200,000 each well in 400 μL of culture medium. After 3 h of cell attachment, the cells were then treated with derivative 44 at 10 μM and 20 μM, while equal treatment volumes of DMSO were used as vehicle control. The cells were cultured in CO₂ incubator at 37 °C for 16 h. Both attached and floating cells were collected in a centrifuge tube by centrifugation at rcf value of 450 g for 5 min. After discarding the supernatant, the collected cells were re-suspended with 500 μL 80% cold ethanol to fix for 30 min in 4 °C. The fixed cells could be stored at −20 °C for one week. After fixation, the ethanol was removed after centrifuging and the cells were washed with PBS. The cells were then re-suspended with 100 μL of 100 mg/mL ribonuclease and were cultured at 37 °C for 30 min to degrade all RNA. The cells were stained with 200 μL of 50 μg/mL propidium iodide (PI) stock solution for 30 min at −20 °C, and then the fluorescence intensity of PI was detected in individual PC-3 cells using an Attune flow cytometer (Life Technologies) within 0.5–1 h after staining.

4.9. F2N12S and SYTOX AADvanced double staining assay

PC-3 cells were plated in 24-well plates at a density of 200,000 each well in 400 μL of culture medium. After 3 h of cell attachment, the cells were then treated with the derivative 44 at different concentrations and cultured in CO₂ incubator at 37 °C for 16 h, while equal treatment volumes of DMSO were used as vehicle control. Both attached and floating cells were collected in a centrifuge tube by centrifugation at rcf value of 450 g for 5 min. The collected cells were re-suspended with 500 μL HBSS to remove proteins which may affect flow signal and centrifuged again. After discarding the supernatant, the collected cells were re-suspended with 300 μL HBSS and stained with 0.3 μL of F2N12S for 3–5 min followed by 0.3 μL SYTOX AADvanced for an additional 5 min. The fluorescence intensity of the two probes was further measured in individual PC-3 cells using an Attune flow cytometer (Life Technologies) within 0.5–1 h after staining.

Highlights.

48 New N-containing derivatives of O-tetramethylquercetin were synthesized.
Most of them are more potent than quercetin in three prostate cancer cell models.
5-O-(N,N-Dibutylamino)propyl-3,3’,4’,7-O-tetramethylquercetin is the optimal one.
The optimal derivative is over 35- to 182-fold more potent than quercetin.
The optimal derivative activates PC-3 cell apoptosis.

Acknowledgments

This work was financially supported by California State University (CSU)-Fresno. HRMS were supported by NIH RCMI program at Xavier University of Louisiana through Grant 2G12MD007595–064 (G. Wang) and NIGMS-NIH through grant number 1U54GM104940 (G. Wang). We are also grateful to the Undergraduate Research Grant Program at CSU-Fresno for 2017–2018 grants (to P Rajaram) and the ACS Project SEED for the support in 2017 summer (to A Phasadka).

Footnotes

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Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/.

References

1.Sampson L, Rimm E, Hollman PCH, de Vries JHM, Katan MB, Flavonol and flavone intakes in US health professionals, J. Am. Diet. Assoc 102 (2002) 1414–1420. [DOI] [PubMed] [Google Scholar]
2.Hedelin M, Klint A, Chang ET, Bellocco R, Johansson JE, Anderson SO, Heinonen SM, Adlercreutz H, Adami HO, Gronberg H, Balter KA , Dietary phytoestrogen, serum enterolactone and risk of prostate cancer: the cancer prostate Sweden study (Sweden), Cancer Causes Control 17 (2006) 169–180. [DOI] [PubMed] [Google Scholar]
3.McCann SE, Ambrosone CB, Moysich KB, Brasure J, Marshall JR, Freudenheim JL, Wilkinson GS, Graham S, Intakes of selected nutrients, foods, and phytochemicals and prostate cancer risk in western New York, Nutr. Cancer 53 (2005) 33–41. [DOI] [PubMed] [Google Scholar]
4.Bosetti C, Bravi F, Talamini R, Parpinel M, Gnagnarella P, Negri E, Montella M, Lagiou P, Franceschi S, La Vecchia C, Flavonoids and prostate cancer risk: a study in Italy, Nutr. Cancer 56 (2006) 123–127. [DOI] [PubMed] [Google Scholar]
5.Geybels MS, Verhage BA, Arts IC, Van Schooten FJ, Goldbohm RA, Van den Brandt PA, Dietary flavonoid intake, black tea consumption, and risk of overall and advanced stage prostate cancer, Am. J. Epidemiol 177 (2013) 1388–1398. [DOI] [PubMed] [Google Scholar]
6.Liu HL, Jiang WB, Xie MX, Flavonoids: recent advances as anticancer drugs, Recent Pat. Anticancer Drug Discov. 5 (2010) 152–164. [DOI] [PubMed] [Google Scholar]
7.Vue B, Zhang S, Chen Q-H, Flavonoids with therapeutic potential in prostate cancer, Anticancer Agents Med. Chem 16 (2016) 1205–1229. [DOI] [PubMed] [Google Scholar]
8.Yang F, Song L, Wang H, Wang J, Xu Z, Xing N, Quercetin in prostate cancer: chemotherapeutic and chemopreventive effects, mechanisms and clinical application potential (Review), Oncol. Rep 33 (2015) 2659–2668. [DOI] [PubMed] [Google Scholar]
9.Vue B, Zhang S, Chen Q-H, Synergistic effects of dietary natural products as anti-prostate cancer agents, Nat. Prod. Commun 10 (2015) 2179–2188. [PubMed] [Google Scholar]
10.Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM, Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model, Cancer Res. 57 (1997) 3325–3330. [PubMed] [Google Scholar]
11.Slusarz A, Shenouda NS, Sakla MS, Drenkhahn SK, Narula AS, MacDonald RS, Besch-Williford CL, Lubahn DB, Common botanical compounds inhibit the Hedgehog signaling pathway in prostate cancer, Cancer Res 70 (2010) 3382–3390. [DOI] [PubMed] [Google Scholar]
12.Shenouda NS, Zhou C, Browning JD, Andell PJ, Sakla MS, Lubahn DB, MacDonald RS, Phytoestrogens in common herbs regulate prostate cancer cell growth in vitro, Nutr. Cancer 49 (2004) 200–208. [DOI] [PubMed] [Google Scholar]
13.Aalinkeel R, Bindukumar B, Reynolds JL, Sykes DE, Mahajan SD, Chadha KC, Schwartz SA, The dietary bioflavonoid, quercetin, selectively induces apoptosis of prostate cancer cells by down-regulating the expression of heat shock protein 90, Prostate (Hoboken, NJ, United States) 68 (2008) 1773–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Wang P, Vadgama JV, Said JW, Magyar CE, Doan N, Heber D, Henning SM, Enhanced inhibition of prostate cancer xenograft tumor growth by combining quercetin and green tea, J. Nutr. Biochem 25 (2014) 73–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Pratheeshkumar P, Budhraja A, Son Y-O, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee J-C, Xu M, Chen G, Luo J, Shi X, Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR-2 regulated AKT/mTOR/P70S6K signaling pathways, PLoS one 7 (2012) e47516. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Firdous AB, Sharmila G, Balakrishnan S, RajaSingh P, Suganya S, Srinivasan N, Arunakaran J, Quercetin, a natural dietary flavonoid, acts as a chemopreventive agent against prostate cancer in an in vivo model by inhibiting the EGFR signaling pathway, Food Funct. 5 (2014) 2632–2645. [DOI] [PubMed] [Google Scholar]
17.Utesch D, Feige K, Dasenbrock J, Broschard TH, Harwood M, Danielewska-Nikiel B, Lines TC, Evaluation of the potential in vivo genotoxicity of quercetin, Mutat. Res 654 (2008) 38–44. [DOI] [PubMed] [Google Scholar]
18.Shoskes DA, Zeitlin SI, Shahed A, Rajfer J, Quercetin in men with category III chronic prostatitis: a preliminary prospective, double-blind, placebo-controlled trial, Urology 54 (1999) 960–963. [DOI] [PubMed] [Google Scholar]
19.Cialdella-Kam L, Nieman DC, Sha W, Meaney MP, Knap AM, Shanely RA, Dose-response to 3 months of quercetin-containing supplements on metabolite and quercetin conjugate profile in adults, Br. J. Nutr 109 (2013) 1923–1933. [DOI] [PubMed] [Google Scholar]
20.Al-Jabban SMR, Zhang X, Chen G, Mekuria EA, Rakotondraibe LH, Chen Q-H, Synthesis and anti-proliferative effects of quercetin derivatives, Nat. Prod. Commun 10 (2015) 2113–2118. [PubMed] [Google Scholar]
21.Li X, Chen G, Zhang X, Zhang Q, Zheng S, Wang G, Chen Q-H, A new class of flavonol-based anti-prostate cancer agents: Design, synthesis, and evaluation in cell models, Bioorg. Med. Chem. Lett 26 (2016) 4241–4245. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Vue B, Zhang X, Lee T, Nair N, Zhang S, Chen G, Zhang Q, Zheng S, Wang G, Chen Q-H, 5- or/and 20-O-alkyl-2,3-dehydrosilybins: Synthesis and biological profiles on prostate cancer cell models, Bioorg. Med. Chem 25 (2017) 4845–4854. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Rasheed A, Lathika G, Raju YP, Mansoor KP, Azeem AK, Balan N, Synthesis and pharmacological evaluation of mutual prodrugs of aceclofenac with quercetin, vanillin and L-tryptophan as gastrosparing NSAIDS, Med. Chem. Res 25 (2016) 70–82. [Google Scholar]
24.Manthey JA, Guthrie N, Antiproliferative activities of citrus flavonoids against six human cancer cell lines, J. Agric. Food Chem 50 (2002) 5837–5843. [DOI] [PubMed] [Google Scholar]
25.Nguyen TKP, P Nguyen KP, Kamounah FS, Zhang W, Hansen PE, NMR of a series of novel hydroxyflavothiones, Magn. Reson. Chem 47 (2009) 1043–1054. [DOI] [PubMed] [Google Scholar]
26.Wang G, Song L, Wang H, Xing N, Quercetin synergizes with 2-methoxyestradiol inhibiting cell growth and inducing apoptosis in human prostate cancer cells, Oncol. Rep 30 (2013) 357–363. [DOI] [PubMed] [Google Scholar]
27.Xu R, Zhang Y, Ye X, Xue S, Shi J, Pan J, Chen Q, Inhibition effects and induction of apoptosis of flavonoids on the prostate cancer cell line PC-3 in vitro, Food Chem 138 (2013) 48–53. [DOI] [PubMed] [Google Scholar]
28.Wang P, Heber D, Henning SM, Quercetin increased the antiproliferative activity of green tea polyphenol (−)-epigallocatechin gallate in prostate cancer cells, Nutr. Cancer 64 (2012) 580–587. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Vijayababu MR, Kanagaraj P, Arunkumar A, Ilangovan R, Aruldhas MM, Arunakaran J, Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression, J. Cancer Res. Clin. Oncol 131 (2005) 765–771. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.O’Neill AJ, Prencipe M, Dowline C, Fan Y, Mulrane L, Gallagher WM, O’Connor D, O’Connor R, Devery A, Corcoran C, Rani S, O’Driscoll L, Fitzpatrick JM, Watson RWG, Characterisation and manipulation of docetaxel resistant prostate cancer cell lines, Mol. Cancer 10 (2011) 126–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Shiota M, Kashiwagi E, Yokomizo A, Takeuchi A, Dejima T, Song Y, Tatsugami K, Inokuchi J, Uchiumi T, Naito S, Interaction between docetaxel resistance and castration resistance in prostate cancer: Implications of Twist1, YB-1, and androgen receptor, Prostate 73 (2013) 1336–1344. [DOI] [PubMed] [Google Scholar]

[R1] 1.Sampson L, Rimm E, Hollman PCH, de Vries JHM, Katan MB, Flavonol and flavone intakes in US health professionals, J. Am. Diet. Assoc 102 (2002) 1414–1420. [DOI] [PubMed] [Google Scholar]

[R2] 2.Hedelin M, Klint A, Chang ET, Bellocco R, Johansson JE, Anderson SO, Heinonen SM, Adlercreutz H, Adami HO, Gronberg H, Balter KA , Dietary phytoestrogen, serum enterolactone and risk of prostate cancer: the cancer prostate Sweden study (Sweden), Cancer Causes Control 17 (2006) 169–180. [DOI] [PubMed] [Google Scholar]

[R3] 3.McCann SE, Ambrosone CB, Moysich KB, Brasure J, Marshall JR, Freudenheim JL, Wilkinson GS, Graham S, Intakes of selected nutrients, foods, and phytochemicals and prostate cancer risk in western New York, Nutr. Cancer 53 (2005) 33–41. [DOI] [PubMed] [Google Scholar]

[R4] 4.Bosetti C, Bravi F, Talamini R, Parpinel M, Gnagnarella P, Negri E, Montella M, Lagiou P, Franceschi S, La Vecchia C, Flavonoids and prostate cancer risk: a study in Italy, Nutr. Cancer 56 (2006) 123–127. [DOI] [PubMed] [Google Scholar]

[R5] 5.Geybels MS, Verhage BA, Arts IC, Van Schooten FJ, Goldbohm RA, Van den Brandt PA, Dietary flavonoid intake, black tea consumption, and risk of overall and advanced stage prostate cancer, Am. J. Epidemiol 177 (2013) 1388–1398. [DOI] [PubMed] [Google Scholar]

[R6] 6.Liu HL, Jiang WB, Xie MX, Flavonoids: recent advances as anticancer drugs, Recent Pat. Anticancer Drug Discov. 5 (2010) 152–164. [DOI] [PubMed] [Google Scholar]

[R7] 7.Vue B, Zhang S, Chen Q-H, Flavonoids with therapeutic potential in prostate cancer, Anticancer Agents Med. Chem 16 (2016) 1205–1229. [DOI] [PubMed] [Google Scholar]

[R8] 8.Yang F, Song L, Wang H, Wang J, Xu Z, Xing N, Quercetin in prostate cancer: chemotherapeutic and chemopreventive effects, mechanisms and clinical application potential (Review), Oncol. Rep 33 (2015) 2659–2668. [DOI] [PubMed] [Google Scholar]

[R9] 9.Vue B, Zhang S, Chen Q-H, Synergistic effects of dietary natural products as anti-prostate cancer agents, Nat. Prod. Commun 10 (2015) 2179–2188. [PubMed] [Google Scholar]

[R10] 10.Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM, Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model, Cancer Res. 57 (1997) 3325–3330. [PubMed] [Google Scholar]

[R11] 11.Slusarz A, Shenouda NS, Sakla MS, Drenkhahn SK, Narula AS, MacDonald RS, Besch-Williford CL, Lubahn DB, Common botanical compounds inhibit the Hedgehog signaling pathway in prostate cancer, Cancer Res 70 (2010) 3382–3390. [DOI] [PubMed] [Google Scholar]

[R12] 12.Shenouda NS, Zhou C, Browning JD, Andell PJ, Sakla MS, Lubahn DB, MacDonald RS, Phytoestrogens in common herbs regulate prostate cancer cell growth in vitro, Nutr. Cancer 49 (2004) 200–208. [DOI] [PubMed] [Google Scholar]

[R13] 13.Aalinkeel R, Bindukumar B, Reynolds JL, Sykes DE, Mahajan SD, Chadha KC, Schwartz SA, The dietary bioflavonoid, quercetin, selectively induces apoptosis of prostate cancer cells by down-regulating the expression of heat shock protein 90, Prostate (Hoboken, NJ, United States) 68 (2008) 1773–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Wang P, Vadgama JV, Said JW, Magyar CE, Doan N, Heber D, Henning SM, Enhanced inhibition of prostate cancer xenograft tumor growth by combining quercetin and green tea, J. Nutr. Biochem 25 (2014) 73–80. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Pratheeshkumar P, Budhraja A, Son Y-O, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee J-C, Xu M, Chen G, Luo J, Shi X, Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR-2 regulated AKT/mTOR/P70S6K signaling pathways, PLoS one 7 (2012) e47516. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Firdous AB, Sharmila G, Balakrishnan S, RajaSingh P, Suganya S, Srinivasan N, Arunakaran J, Quercetin, a natural dietary flavonoid, acts as a chemopreventive agent against prostate cancer in an in vivo model by inhibiting the EGFR signaling pathway, Food Funct. 5 (2014) 2632–2645. [DOI] [PubMed] [Google Scholar]

[R17] 17.Utesch D, Feige K, Dasenbrock J, Broschard TH, Harwood M, Danielewska-Nikiel B, Lines TC, Evaluation of the potential in vivo genotoxicity of quercetin, Mutat. Res 654 (2008) 38–44. [DOI] [PubMed] [Google Scholar]

[R18] 18.Shoskes DA, Zeitlin SI, Shahed A, Rajfer J, Quercetin in men with category III chronic prostatitis: a preliminary prospective, double-blind, placebo-controlled trial, Urology 54 (1999) 960–963. [DOI] [PubMed] [Google Scholar]

[R19] 19.Cialdella-Kam L, Nieman DC, Sha W, Meaney MP, Knap AM, Shanely RA, Dose-response to 3 months of quercetin-containing supplements on metabolite and quercetin conjugate profile in adults, Br. J. Nutr 109 (2013) 1923–1933. [DOI] [PubMed] [Google Scholar]

[R20] 20.Al-Jabban SMR, Zhang X, Chen G, Mekuria EA, Rakotondraibe LH, Chen Q-H, Synthesis and anti-proliferative effects of quercetin derivatives, Nat. Prod. Commun 10 (2015) 2113–2118. [PubMed] [Google Scholar]

[R21] 21.Li X, Chen G, Zhang X, Zhang Q, Zheng S, Wang G, Chen Q-H, A new class of flavonol-based anti-prostate cancer agents: Design, synthesis, and evaluation in cell models, Bioorg. Med. Chem. Lett 26 (2016) 4241–4245. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Vue B, Zhang X, Lee T, Nair N, Zhang S, Chen G, Zhang Q, Zheng S, Wang G, Chen Q-H, 5- or/and 20-O-alkyl-2,3-dehydrosilybins: Synthesis and biological profiles on prostate cancer cell models, Bioorg. Med. Chem 25 (2017) 4845–4854. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Rasheed A, Lathika G, Raju YP, Mansoor KP, Azeem AK, Balan N, Synthesis and pharmacological evaluation of mutual prodrugs of aceclofenac with quercetin, vanillin and L-tryptophan as gastrosparing NSAIDS, Med. Chem. Res 25 (2016) 70–82. [Google Scholar]

[R24] 24.Manthey JA, Guthrie N, Antiproliferative activities of citrus flavonoids against six human cancer cell lines, J. Agric. Food Chem 50 (2002) 5837–5843. [DOI] [PubMed] [Google Scholar]

[R25] 25.Nguyen TKP, P Nguyen KP, Kamounah FS, Zhang W, Hansen PE, NMR of a series of novel hydroxyflavothiones, Magn. Reson. Chem 47 (2009) 1043–1054. [DOI] [PubMed] [Google Scholar]

[R26] 26.Wang G, Song L, Wang H, Xing N, Quercetin synergizes with 2-methoxyestradiol inhibiting cell growth and inducing apoptosis in human prostate cancer cells, Oncol. Rep 30 (2013) 357–363. [DOI] [PubMed] [Google Scholar]

[R27] 27.Xu R, Zhang Y, Ye X, Xue S, Shi J, Pan J, Chen Q, Inhibition effects and induction of apoptosis of flavonoids on the prostate cancer cell line PC-3 in vitro, Food Chem 138 (2013) 48–53. [DOI] [PubMed] [Google Scholar]

[R28] 28.Wang P, Heber D, Henning SM, Quercetin increased the antiproliferative activity of green tea polyphenol (−)-epigallocatechin gallate in prostate cancer cells, Nutr. Cancer 64 (2012) 580–587. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Vijayababu MR, Kanagaraj P, Arunkumar A, Ilangovan R, Aruldhas MM, Arunakaran J, Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression, J. Cancer Res. Clin. Oncol 131 (2005) 765–771. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.O’Neill AJ, Prencipe M, Dowline C, Fan Y, Mulrane L, Gallagher WM, O’Connor D, O’Connor R, Devery A, Corcoran C, Rani S, O’Driscoll L, Fitzpatrick JM, Watson RWG, Characterisation and manipulation of docetaxel resistant prostate cancer cell lines, Mol. Cancer 10 (2011) 126–128. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Shiota M, Kashiwagi E, Yokomizo A, Takeuchi A, Dejima T, Song Y, Tatsugami K, Inokuchi J, Uchiumi T, Naito S, Interaction between docetaxel resistance and castration resistance in prostate cancer: Implications of Twist1, YB-1, and androgen receptor, Prostate 73 (2013) 1336–1344. [DOI] [PubMed] [Google Scholar]

PERMALINK

Nitrogen-Containing Derivatives of O-Tetramethylquercetin: Synthesis and Biological 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

Abstract

Graphical Abstract

1. Introduction

Figure 1.

2. Results and discussion

2.1. Chemistry

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

2.2. Antiproliferative activity towards prostate cancer cells

Table 1.

Table 2.

2.3. Docetaxel Resistance Susceptibility

Table 3.

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

Figure 2.

Figure 3.

3. Conclusion

4. Experimental Section

4.1. General Procedures

4.2. Synthesis of 3,3’,4’,7-O-tetramethylquercetin (7) [24–25]

4.3. The general procedure for the synthesis of 3-O-dialkylaminoalkyl-3’,4’,5,7-O-tetramethylquercetins (11–34)

4.3.1. 3-O-(N,N-Diethylamino)propyl-3’,4’,5,7-O-tetramethylquercetin (11)

4.3.2. 3-O-(N,N-Diethylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (12)

4.3.3. 3-O-(N,N-Diethylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (13)

4.3.4. 3-O-(N,N-Dipropylamino)propyl-3’,4’,5,7-O-tetramethylquercetin (14)

4.3.5. 3-O-(N,N-Dipropylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (15)

4.3.6. 3-O-(N,N-Dipropylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (16)

4.3.7. 3-O-(N,N-Dibutylamino)propyl-3’,4’,5,7-O-tetramethylquercetin (17)

4.3.8. 3-O-(N,N-Dibutylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (18)

4.3.9. 3-O-(N,N-Dibutylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (19)

4.3.10. 3-O-(N,N-Dipentylamino)propyl-3’,4’,5,7-O-tetramethylquercetin (20)

4.3.11. 3-O-(N,N-Dipentylamino)butyl-3’,4’,5,7-O-tetramethylquercetin (21)

4.3.12. 3-O-(N,N-Dipentylamino)pentyl-3’,4’,5,7-O-tetramethylquercetin (22)

4.3.13. 3-O-Morpholinopropyl-3’,4’,5,7-O-tetramethylquercetin (23)

4.3.14. 3-O-Morpholinobutyl-3’,4’,5,7-O-tetramethylquercetin (24)

4.3.15. 3-O-Morpholinopentyl-3’,4’,5,7-O-tetramethylquercetin (25)

4.3.16. 3-O-Piperidinopropyl-3’,4’,5,7-O-tetramethylquercetin (26)

4.3.17. 3-O-Piperidinobutyl-3’,4’,5,7-O-tetramethylquercetin (27)

4.3.18. 3-O-Piperidinopentyl-3’,4’,5,7-O-tetramethylquercetin (28)

4.3.19. 3-O-(4-Methylpiperazin-1-yl)propyl-3’,4’,5,7-O-tetramethylquercetin (29)

4.3.20. 3-O-(4-Methylpiperazin-1-yl)butyl-3’,4’,5,7-O-tetramethylquercetin (30)

4.3.21. 3-O-(4-Methylpiperazin-1-yl)pentyl-3’,4’,5,7-O-tetramethylquercetin (31)

4.3.22. 3-O-Pyrrolidinopropyl-3’,4’,5,7-O-tetramethylquercetin (32)

4.3.23. 3-O-Pyrrolidinobutyl-3’,4’,5,7-O-tetramethylquercetin (33)

4.3.24. 3-O-Pyrrolidinopentyl-3’,4’,5,7-O-tetramethylquercetin (34)

4.4. General procedure for the synthesis of 5-O-bromoalkyl-3,3’,4’,7-tetramethylquercetins (35–37)

4.4.1. 5-O-(3-Bromopropyl)-3,3’,4’,7-O-tetramethylquercetin (35)

4.4.2. 5-O-(4-Bromobutyl)-3,3’,4’,7-O-tetramethylquercetin (36)

4.4.3. 5-O-(5-Bromopentyl)-3,3’,4’,7-O-tetramethylquercetin (37)

4.5. The general procedure for the synthesis of 5-O-dialkylaminoalkyl-3,3’,4’,7-O-tetramethylquercetins (38–61):

4.5.1. 5-O-(N,N-Diethylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (38)

4.5.2. 5-O-(N,N-Diethylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (39)

4.5.3. 5-O-(N,N-Diethylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (40)

4.5.4. 5-O-(N,N-Dipropylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (41)

4.5.5. 5-O-(N,N-Dipropylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (42)

4.5.6. 5-O-(N,N-Dipropylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (43)

4.5.7. 5-O-(N,N-Dibutylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (44)

4.5.8. 5-O-(N,N-Dibutylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (45)

4.5.9. 5-O-(N,N-Dibutylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (46)

4.5.10. 5-O-(N,N-Dipentylamino)propyl-3,3’,4’,7-O-tetramethylquercetin (47)

4.5.11. 5-O-(N,N-Dipentylamino)butyl-3,3’,4’,7-O-tetramethylquercetin (48)

4.5.12. 5-O-(N,N-Dipentylamino)pentyl-3,3’,4’,7-O-tetramethylquercetin (49)

4.5.13. 5-O-Morpholinopropyl-3,3’,4’,7-O-tetramethylquercetin (50)

4.5.14. 5-O-Morpholinobutyl-3,3’,4’,7-O-tetramethylquercetin (51)

4.5.15. 5-O-Morpholinopentyl-3,3’,4’,7-O-tetramethylquercetin (52)

4.5.16. 5-O-Piperidinopropyl-3,3’,4’,7-O-tetramethylquercetin (53)

4.5.17. 5-O-Piperidinobutyl-3,3’,4’,7-O-tetramethylquercetin (54)