Synthesis,cytotoxicity,and molecular docking of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives as novel antitumor agents

Abstract

A series of novel methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives is synthesized by utilizing the click reaction. Evaluation of their cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) using the MTT assay shows that most of these compounds exhibit weak cytotoxicity. It is observed that compound 12 shows the highest activity against A-549 cells with an IC₅₀ value of 10.27 ± 0.90 μM. Molecular docking results suggested that this compound 12 has a higher binding affinity for epidermal growth factor receptor than for tubulin. Our findings support the utility of compound 12 as a novel compound for the further development of anticancer agents.

Introduction

Natural products are valuable materials with various structures, unique biological activities, and specific selectivity that can aid in the discovery of anticancer drugs. ¹ However, many potential drugs lack tumor selectivity and often display significant toxic side effects, which hampers their development for clinical use.^2,3 In order to reduce cytotoxicity and maintain activity of these natural compounds, appropriate structural modification can be adopted in order to develop drugs. Podophyllotoxin (PPT, 1) (Figure 1), one of the naturally occurring aryltetralin lignins, isolated from Podophyllum peltatum and Podophyllum hexandrum,^4,5 shows strong cytotoxic activity against various cancer cells by inhibiting tubulin through binding with part of the colchicine domain.^6,7 Unfortunately, PPT shows high toxicity wards to non-carcinoma cells, which hinders its development as an antitumor agent, ⁸ but its semisynthetic derivative etoposide (2) (Figure 1) is in clinical use for the treatment of a variety of malignancies, including small cell lung cancer, testicular carcinoma, lymphoma, and Kaposi’s sarcoma,^9,10 and it has fewer side effects. ¹¹ Meanwhile, these reports have inspired us to search for novel anticancer PPT analogues with improved therapeutic efficacy and fewer side effects by targeting tubulin.

Figure 1.

Chemical structures of podophyllotoxin (PPT, 1), etoposide (VP-16, 2), (–)-epicatechin (EC, 3), (–)-epicatechin-3-gallate (ECG, 4), (–)-epigallocatechin (EGC, 5), and (–)-epigallocatechin-3-gallate (EGCG, 6).

Green tea (Camellia sinensis leaves) is an extremely popular drink, ¹² and the major catechins found in green tea are (–)-epicatechin (EC, 3), (–)-epicatechin-3-gallate (ECG, 4), (–)-epigallocatechin (EGC, 5), and (–)-epigallocatechin-3-gallate (EGCG, 6) (Figure 1), ¹³ which are the most important active compounds. EGCG is one of the most abundant and biologically active compounds in tea catechins, and has been reported to inhibit tumor incidences such as skin, lung, liver, breast, prostate, and stomach. ¹⁴ There is considerable evidence that EGCG inhibits tumorigenesis, signal transduction pathways, cell invasion, angiogenesis, and metastasis.^15–19 Moreover, many studies have reported that EGCG inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR).^20–23 However, EGCG also has several limitations, such as poor stability, rapid poor metabolism, membrane permeability, and degradation in aqueous solutions.^24–26 Thus, it would be beneficial to improve the stability of EGCG and maintain its toxicity against tumor cells.

Typically, tubulin inhibitors with a trimethoxyphenyl (TMP) moiety, which is essential for interactions with tubulin, are interesting compounds. In our previous studies, ²⁷ we reported a group of (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin conjugates as a new class of antitumor compounds. Herein, we report the chemical synthesis of a series of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives from the natural compounds PPT and EGCG by click reactions, and have evaluated their in vitro anticancer activity against five human cancer cell lines: including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer). Besides, molecular docking analyses of these compounds were studied and correlated with their anticancer activity.

Results and discussion

Chemistry

As shown in Scheme 1, the first part of the synthesis of the methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives required the preparation of the terminal alkynes 8 and 9. EGCG-1-propyne (7) was prepared in 30% yield by treatment of EGCG (6) with sodium hydride (NaH) and propargyl bromide. ²¹ To obtain methylated terminal alkynes 8, compound 7 was permethylated with dimethylsulfate [(CH₃)₂SO₄] and potassium carbonate (K₂CO₃) in anhydrous acetone under reflux (overall yield 98%), which was subsequently hydrolyzed with sodium hydroxide (NaOH) in CH₃OH to afford tetramethylated terminal alkyne 9 in 70% yield. ²⁸ To introduce the azido functionality for the click-reaction, podophyllotoxin was readily converted into 4β-azido-4-deoxypodophyllotoxin (10) and 4β-azido-4-deoxy-4’-demethypodophyllotoxin (11) according to known procedures^29–31 (see the Supporting Information). Finally, 4β-podophyllotoxins 10 and 11 were allowed to react with the above terminal alkynes 8 and 9 in the presence of CuSO₄·5H₂O, sodium ascorbate in THF, and ^tBtOH-H₂O (1:1) at room temperature for 2 h to give methylated catechin-4β-triazolopodophyllotoxin derivatives 12–15 in 78%–84% yield (Scheme 2).

Scheme 1.

Synthesis of the terminal alkynes 7–9. Reagents and conditions: (a) NaH, DMF, 0 °C, propargyl bromide, then, 80 °C, 12 h, 30%; (b) (CH₃)₂SO₄, acetone, K₂CO₃, reflux, 2 h, 98%; and (c) NaOH, CH₃OH, rt, 2 h, 70%.

Scheme 2.

Synthesis of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives 12–15. Reagents and conditions: (a) CuSO₄·5H₂O, sodium ascorbate, THF, ^tBuOH-H₂O (1:1), 2 h, rt, and 78%–84%.

All the synthesized compounds were characterized by ¹H NMR, ¹³C NMR, and ESI-MS analytical data. The formation of the triazole ring was confirmed by the resonance of its C¹⁴-H signal (7.8–8.2 ppm) in the aromatic region in the ¹H NMR spectra, and which was further supported by two characteristic carbon signals at around 145 and 126 ppm in the ¹³C NMR spectra.

In vitro antiproliferative activity

The antiproliferative activity of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives 12–15 against HL-60 (leukemia), A-549 (lung cancer), MCF-7 (breast cancer), SMMC-7721 (hepatoma), and SW480 (colon cancer) cells were screened in vitro using the MTT assay. PPT, EGCG, and cisplatin (DDP) were used as positive control compounds. The compounds were evaluated in the concentration range of 10.27–39.90 μM, and the calculated IC₅₀ values were given in Table 1. As shown in Table 1, compound 12 showed better growth inhibition against the five cancer cell lines, exhibiting the most potent cytotoxicity against A-549 cells with an IC₅₀ value of 10.27 ± 0.90 μM. Interestingly, all of compounds 12–15 showed antiproliferative activities against A-549 cells, with IC₅₀ values ranging from 10.27 to 37.61 μM.

Table 1.

In vitro anticancer activity (IC₅₀, μM) of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives 12–15.

Compounds	IC50 (μM) a
	HL-60	SMMC-7721	A-549	MCF-7	SW480	BEAS-2B
12	15.33 ± 1.24	21.75 ± 0.78	10.27 ± 0.90	23.10 ± 1.10	35.56 ± 0.56	>40
13	29.61 ± 0.47	36.98 ± 0.34	27.12 ± 0.12	32.11 ± 0.87	>40	>40
14	18.58 ± 0.71	29.43 ± 1.01	19.90 ± 1.10	30.06 ± 0.13	>40	>40
15	39.90 ± 0.51	>40	37.61 ± 0.07	> 40	>40	>40
PPT (1)	<0.064	<0.064	<0.064	<0.064	<0.064	2.24 ± 0.09
EGCG (6)	>40	>40	>40	>40	>40	>40
DDP	0.766 ± 0.03	1.93 ± 0.43	4.72 ± 0.35	14.20 ± 0.93	10.20 ± 1.20	12.86 ± 0.25

a

Values are mean values of three independent experiments.

Molecular docking studies

To further investigate the potential binding between tubulin (PDB code: 3UT5)/EGFR (PDB code: 3IKA) and compound 12, molecular docking was performed. Detailed molecular docking studies are shown in Figures S1 and S2 (see the Supporting Information). The docking results showed that the interaction energy of tubulin binding with 12 was −2.08 kJ/mol, while that of EGFR was −12.21 kJ/mol, suggesting that compound 12 had a higher binding affinity than EGFR. The binding modes of compound 12 at the active pocket of tubulin and EGFR are shown in Figure 2. It can be seen from Figure 2(a) and (c) that the complex of compound 12 and tubulin was stabilized by two hydrogen bonds (with ASP251 and LYS254) and six hydrophobic interactions with LYS254, ASN258, VAL315, ALA354, and GLN247. The results presented in Figure 2(b) and (d) show the presence of two hydrogen bonds (with MET793 and CYS797) and two hydrophobic interactions (with ASP855 and ASP800). In addition, the key residues are labeled and the important molecular interactions including the hydrogen bonds and hydrophobic interactions are summarized in Table 2. We believe that the presence of more hydrophobic effects in compound 12 binding with EGFR seems to be the key factor for its high activity against A-549 cells.

Figure 2.

Binding sits of proteins and compound 12. (a) and (b) 3D modes of compound 12 docking into the binding site of tubulin and EGFR: the proteins are shown as lines; ligand is shown as sticks (ligand color: C, black; N, blue; O red; and polar hydrogen). Hydrogen bonds are shown as yellow dotted lines; hydrophobic interactions are shown as blue dotted lines. (c) and (d) 2D modes of compound 12 docking into the binding site of tubulin and EGFR: ligand is shown as lines (ligand color: C, black; N blue; O, red).

Table 2.

The residues for molecular interaction between tubulin/12 and EGFR/12.

Tubulin				EGFR
Nature of interaction	Molecular interaction	Distance (Å)	∆G kcal/mol	Nature of interaction	Molecular interactions	Distance (Å)	∆G kcal/mol
Hydrogen bond	ASP251: H┈Lig: O	3.04	−2.08	Hydrogen bond	MET793: H┈Lig: O	3.09	−10.21
	LYS254: H┈Lig: O	2.81			LYS797: H┈Lig: O	3.40
Hydrophobic	LYS254┈Lig	4.43		Hydrophobic	ASP855┈Lig	2.99
	ASN258┈Lig	2.57			ASP800┈Lig	3.54
	ASN258┈Lig	3.00
	VAL315┈Lig	3.53
	ALA354┈Lig	3.76
	GLN247┈Lig	2.89

Conclusion

In summary, a series of novel (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives has been synthesized by utilizing the click-reaction, and the cytotoxicity of these compounds has been screed against a panel of five human cancer cells (HL-60, SMMC-7721, A-549, MCF-7, and SW480) using the MTT assay. Compound 12 showed the highest cytotoxicity against A-549 cells with an IC₅₀ value of 10.27 ± 0.90 μM. Molecular docking studies showed that the presence of more hydrophobic effects in compound 12 binding with EGFR seemed to be the key factor for its high activity against A-549 cells. Therefore, compound 12 may represent a novel compound for the development of anticancer agent.

Experimental

General

All cancer cells (HL-60, SMMC-7721, A-549, MCF-7, and SW480) were obtained from the Shanghai Cell Bank, China. (–)-Epigallocatechin-3-gallate and podophyllotoxin were purchased from Chengdu Proifa Technology Development Co., Ltd (Chengdu, China); 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents were commercially available and were used without further purification unless indicated otherwise. Melting points were measured with an X-4 melting point apparatus and are uncorrected. MS data were obtained in the ESI mode on an API Qstar Pulsar instrument; HRMS data were obtained in the ESI mode on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan) instrument. ¹H NMR and ¹³C NMR spectra were recorded on Bruker AVANCE III 500 MHz (Bruker BioSpin GmbH, Rheinstetten, Germany) instrument, using tetramethylsilane (TMS) as an internal standard: chemical shifts (δ) are given in ppm, coupling constants (J) in Hz, and the solvent signals were used as references (CDCl₃: δ_C = 77.2; residual CHCl₃ in CDCl₃: δ_H = 7.26; CD₃OD: δ_C = 49.0; residual CH₃OH in CD₃OD: δ_H = 4.78). Column chromatography (CC) was performed using silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd.; Qingdao; China). All reactions were monitored by thin-layer chromatography (TLC), and samples were visualized under ultraviolet light (254 nm) and/or using 10% phosphomolybdic acid/EtOH.

(–)-Epigallocatechin-3-gallate-1-propyne (7)

(–)-Epigallocatechin-3-gallate (2.3 g, 5.0 mmol) was dissolved in N,N-dimethylformamide (DMF) (20 mL), and then sodium hydride (300 mg, 7.5 mmol) was added at 0 °C under nitrogen and the mixture was stirred at room temperature for 0.5 h. Propargyl bromide (0.5 mL, 5.0 mmol) was quickly added, and the reaction was stirred at 80 °C for 12 h. After cooling, the mixture was concentrated under vacuum and the resulting residue was purified by silica gel chromatography with CHCl₃/CH₃OH, (9:1 → 4:1) to afford the product 7 (685 mg, 30%). ¹H NMR (500 MHz, CD₃OD): δ 6.90 (s, 2H, C^2''-H, C^6'-H), 6.50 (s, 2H, C^2'-H, C^6'-H), 5.96 (s, 2H, C⁶-H, C⁸-H), 5.53 (s, 1H, C³-H), 4.97 (s, 1H, C²-H), 4.78 (d, 2H, J = 2.4 Hz, OCH₂), 3.29 (t, 1H, J = 1.6 Hz, C≡CH), 2.96 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_a), 2.85 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_b); ¹³C NMR (125 MHz, CD₃OD): δ 167.0 (C=O), 157.9 (C-7), 157.8 (C-9), 157.2 (C-5), 151.9 (C-3^', C-5^'), 146.7 (C-3^'', C-5^''), 138.4 (C-4^''), 133.8 (C-4^'), 130.7 (C-1^'), 127.1 (C-1^''), 110.1 (C-2^'', C-6^''), 106.8 (C-2^', C-6^'), 99.3 (C-10), 96.5 (C-8), 95.9 (C-6), 80.4 (C-2), 79.5 (C≡CH), 78.5 (C≡CH), 76.7 (C-3), 60.0 (OCH₂), 26.8 (C-4); MS (ESI): m/z = 495 [M − H]⁻.

Methylated (–)-epigallocatechin-3-gallate-1-propyne (8)

To a solution of (–)-epigallocatechin-3-gallate-1-propyne (7) (496 mg, 1.0 mmol) in dry acetone (5 mL), (CH₃)₂SO₄ (1.0 mL, 10 mmol) and K₂CO₃ (1.4 g, 10 mmol) were added and resulting mixture was refluxed for 2 h with stirring until TLC showed the reaction to be complete. After removal of inorganic salts by filtration, the filtrated was concentrated and the residue was purified by CC (petroleum ether/ethyl acetate = 4:1) to afford compound 8 (582 mg, 98%). ¹H NMR (500 MHz, CD₃OD): δ 7.17 (s, 2H, C^2''-H, C^6''-H), 6.69 (s, 2H, C^2'-H, C^6'-H), 6.24 (d, 1H, J = 2.2 Hz, C⁶-H), 6.12 (d, 1H, J = 2.2 Hz, C⁶-H), 5.66 (s, 1H, C³-H), 5.08 (s, 1H, C²-H), 4.76 (d, 2H, J = 2.4 Hz, OCH₂), 3.96 (s, 1H, C≡CH), 3.81 (s, 9H, 3 × OCH₃), 3.78 (s, 6H, 2 × OCH₃), 3.71 (s, 6H, 2 × OCH₃), 3.05 (d, 2H, J = 3.2 Hz, C4-CH₂); ¹³C NMR (125 MHz, CD₃OD): δ 165.1 (C=O), 157.8 (C-7), 158.9 (C-5), 155.5 (C-9), 153.2 (C-3^', C-5^'), 153.1 (C-3^'', C-5^''), 139.6 (C-4^''), 137.9 (C-4^'), 133.4 (C-1^'), 125.9 (C-1^''), 107.0 (C-2^'', C-6^''), 103.9 (C-2^', C-6^'), 100.1 (C-10), 93.2 (C-6), 91.9 (C-8), 78.8 (C-2), 77.8 (C≡CH), 75.2 (C≡CH), 68.7 (C-3), 60.8 (OCH₃), 59.9 (OCH₂), 56.3 (OCH₃), 56.0 (OCH₃), 55.4 (OCH₃), 26.0 (C-4); MS (ESI): m/z = 617 [M + Na]⁺.

Methylated (–)-epigallocatechin-1-propyne (9)

To a solution of methylated (–)-epigallocatechin-3-gallate-1-propyne (8) (1.2 g, 2.0 mmol) in CH₃OH (15 mL) was added NaOH (80 mg, 2 mmol). The reaction mixture was stirred at room temperature for 2 h until TLC showed the reaction to be complete. The solvent was evaporated in vacuo, and the residue was purified by CC (petroleum ether/ethyl acetate = 2:1) to afford compound 9 (565.6 mg, 70%). ¹H NMR (500 MHz, CD₃OD): δ 6.83 (s, 2H, C^2'-H, C^6'-H), 6.16 (d, 1H, J = 2.4 Hz, C⁶-H), 6.14 (d, 1H, J = 2.4 Hz, C⁸-H), 4.99–4.86 (m, 2H, C²-H, C³-H), 4.58 (s, 2H, OCH₂), 4.23 (brs, 1H, C≡CH), 3.85 (s, 6H, 2 × OCH₃), 3.78 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 2.90 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_a), 2.77 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_b); ¹³C NMR (125 MHz, CD₃OD): δ 161.1 (C-7), 160.6 (C-5), 156.9 (C-9), 154.2 (C-3^', C-5^'), 138.3 (C-4^'), 136.7 (C-1^'), 105.2 (C-2^', C-6^'), 102.1 (C-10), 94.6 (C-8), 92.6 (C-6), 80.1 (C-2), 67.3 (C≡CH), 61.1 (C≡CH), 56.6 (C-3), 55.9 (OCH₂), 55.9 (OCH₂), 55.7 (OCH₂), 29.5 (C-4); MS (ESI): m/z = 423 [M + Na]⁺.

General procedure for the synthesis of methylated catechin-4β-triazolopodophyllotoxin derivatives 12–15

To a solution of methylated catechin-1-propyne 8 or 9 (0.1 mmol) and 4β-azido-podophyllotoxin 10 or 11 (0.1 mmol) in THF (1.0 mL) and ^tBtOH-H₂O (1.0 mL, 1:1) at room temperature were added copper(II) sulfate pentahydrate (0.1 mmol) and sodium ascorbate (0.05 mmol). The reaction mixture was then stirred room temperature for 2 h until TLC showed the reaction to be complete. Removal of the solvents gave a residue which was chromatographed on silica gel (CHCl₃/CH₃OH = 9:1) to afford the product.

Methylated (–)-epigallocatechin-3-gallate-[4β-(1,2,3-trizaol-1-yl-4-deoxypodophyll-otoxin)] ether (12)

White amorphous powder; yield 82%; m.p. = 180–182 °C; ¹H NMR (500 MHz, CD₃OD): δ 7.92 (s, 1H, C¹⁴-H), 6.98 (s, 2H, C^2'', C^6''-H), 6.82 (s, 2H, C^2', C^6'-H), 6.68 (s, 1H, C^5'''-H), 6.65 (s, 1H, C^8'''-H), 6.30 (s, 2H, C^2'''', C^6''''-H), 6.23 (d, 1H, J = 2.3 Hz, C⁶-H), 6.21 (d, 1H, J = 2.3 Hz, C⁸-H), 6.17 (d, 1H, J = 2.3 Hz, C³-H), 6.02-5.97 (m, 2H, OCH₂O), 5.55 (s, 1H, C²-H), 5.25 (s, 2H, OCH₂), 5.03 (d, 1H, J = 3.0 Hz, C^4'''-H), 4.71 (d, 1H, J = 5.0 Hz, C^1'''-H), 4.27-4.25 (m, 2H, C^11'''-CH₂), 3.72 (s, 6H, C^3'', C^5''-OCH₃), 3.71 (s, 3H, C7-3OCH), 3.66-3.60 (m, 21H, C^3', C^4', C^5', C^4'', C^3''''', C^4'''', C^5''''-OCH₃), 3.40 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_a), 3.03 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_b), 2.89-2.86 (m, 1H, C^3'''-H), 2.83-2.81 (m, 1H, C^2'''-H); ¹³C NMR (125 MHz, CD₃OD): δ 173.5 (C-12^'''), 164.3 (C-11), 159.4 (C-7), 158.4 (C-5), 155.1 (C-9), 153.0 (C-3^'''', C-5^''''), 152.4 (C-3^'', C-5^''), 152.1 (C-3^', C-5^'), 148.2 (C-7^'''), 147.0 (C-6^'''), 142.9 (C-4^''), 140.0 (C-14), 137.0 (C-4^''''), 136.5 (C-4^'), 135.2 (C-1^''''), 133.8 (C-9^'''), 132.9 (C-10^'''), 126.0 (C-1^'), 125.3 (C-13), 125.1 (C-1^''), 109.8 (C-5^'''), 108.7 (C-8^'''), 108.1 (C-2^'''', C-6^''''), 106.5 (C-2^'', C-6^''), 103.8 (C-2^', C-6^'), 101.7 (OCH₂O), 99.5 (C-10), 93.4 (C-8), 91.8 (C-6), 76.6 (C-2), 69.0 (OCH₂), 67.1 (C-3, C-11^'''), 65.0 (C-4^'''), 60.2 (4^''''-OCH₃), 60.0 (4^''-OCH₃), 59.9 (4^'-OCH₃), 57.3 (7-OCH₃), 56.0 (3^''''-OCH₃), 55.8 (5^''''-OCH₃), 55.7 (3^''-OCH₃), 55.6 (5^''-OCH₃), 55.4 (3^'-OCH₃), 55.2 (5^'-OCH₃), 42.9 (C-1^'''), 40.7 (C-2^'''), 36.4 (C-3^'''), 25.3 (C-4); MS (ESI): m/z = 1034 [M + H]⁺.

Methylated (–)-epigallocatechin-3-gallate-[4β-(1,2,3-trizaol-1-yl-4-deoxy-4^’-demethylpodophyllotoxin)] ether (13)

White amorphous powder; yield 78%; m.p. = 185–186 °C; ¹H NMR (500 MHz, CD₃OD): δ 7.91 (s, 1H, C¹⁴-H), 6.09 (s, 2H, C^2'', C^6''-H), 6.82 (s, 2H, C^2', C^6'-H), 6.65 (d, 1H, J = 2.3 Hz, C^5'''-H), 6.64 (d, 1H, J = 2.3 Hz, C^8'''-H), 6.26 (s, 2H, C^2'''', C^6''''-H), 6.23 (d, 1H, J = 2.3 Hz, C⁶-H), 6.21 (d, 1H, J = 2.3 Hz, C⁸-H), 6.17 (d, 1H, J = 2.3 Hz, C³-H), 6.02-5.97 (m, 2H, OCH₂O), 5.55 (brs, 1H, C²-H), 5.25 (s, 2H, OCH₂), 5.03 (d, 1H, J = 3.0 Hz, C^4'''-H), 4.66 (d, 1H, J = 5.0 Hz, C^1'''-H), 4.27-4.25 (m, 2H, C^11'''-CH₂), 3.74 (s, 6H, C^3'', C^5''-OCH₃), 3.72 (s, 3H, C7-3OCH), 3.71-3.60 (m, 18H, C^3', C^4', C^5', C^4'', C^3''''', C^4'''', C^5''''-OCH₃), 3.34-3.31 (m, 1H, C⁴-H_a), 3.04-3.01 (m, 1H, C⁴-H_b), 2.89-2.87 (m, 1H, C^3'''-H), 2.83-2.81 (m, 1H, C^2'''-H); ¹³C NMR (125 MHz, CD₃OD): δ 173.5 (C-12^'''), 164.3 (C-11), 159.4 (C-7), 158.4 (C-5), 155.1 (C-9), 153.0 (C-3^'''', C-5^''''), 152.7 (C-3^'', C-5^''), 152.1 (C-3^', C-5^'), 148.1 (C-7^'''), 147.3 (C-6^'''), 142.9 (C-4^''), 140.0 (C-14), 137.0 (C-4^''''), 134.9 (C-4^'), 133.8 (C-9^'''), 133.3 (C-10^'''), 129.6 (C-1^''''), 126.0 (C-1^'), 125.3 (C-13), 125.1 (C-1^''), 109.8 (C-5^'''), 108.6 (C-8^'''), 108.4 (C-2^'''', C-6^''''), 106.5 (C-2^'', C-6^''), 103.8 (C-2^', C-6^'), 101.6 (OCH₂O), 99.5 (C-10), 93.4 (C-8), 91.8 (C-6), 76.6 (C-2), 69.0 (OCH₂), 67.0 (C-3, C-11^'''), 65.0 (C-4^'''), 60.2 (4^''-OCH₃), 60.0 (4^'-OCH₃), 57.4 (7-OCH₃), 56.00 (3^''''-OCH₃), 55.9 (5^''''-OCH₃), 55.8 (3^''-OCH₃), 55.7 (5^''-OCH₃), 55.5 (3^'-OCH₃), 55.2 (5^'-OCH₃), 42.8 (C-1^'''), 40.8 (C-2^'''), 36.3 (C-3^'''), 25.3 (C-4); MS (ESI): m/z = 1029 [M + H]⁺.

Methylated (–)-epigallocatechin-[4β-(1,2,3-trizaol-1-yl-4-deoxypodophyllotoxin)] ether (14)

White amorphous powder; yield 84%; m.p. = 182–184 °C; ¹H NMR (500 MHz, CD₃OD): δ 7.58 (s, 1H, C¹⁴-H), 6.78 (s, 2H, C^2'', C^6''-H), 6.65 (s, 1H, C^5'''-H), 6.62 (s, 1H, C^8'''-H), 6.40 (s, 2H, C^2', C^6'-H), 6.22 (d, 1H, J = 2.3 Hz, C⁶-H), 6.17 (d, 1H, J = 2.3 Hz, C⁸-H), 6.14 (d, 1H, J = 2.3 Hz, C³-H), 5.97-5.96 (m, 2H, OCH₂O), 5.25-5.20 (m, 3H, C²-H, OCH₂), 5.13 (d, 1H, J = 3.0 Hz, C^4'''-H), 4.74 (d, 1H, J = 5.0 Hz, C^1'''-H), 4.27-4.25 (m, 2H, C^11'''-CH₂), 3.78 (s, 3H, C7-3OCH), 3.78-3.72 (m, 18H, C^3', C^4', C^5', C^3''''', C^4'''', C^5''''-OCH₃), 3.29-3.27 (m, 1H, C⁴-H_a), 3.10-3.08 (m, 1H, C⁴-H_b), 2.88-2.86 (m, 1H, C^3'''-H), 2.84-2.82 (m, 1H, C^2'''-H); ¹³C NMR (125 MHz, CD₃OD): δ 175.9 (C-12^'''), 161.1 (C-7), 160.6 (C-5), 156.9 (C-9), 154.6 (C-3^'''', C-5^''''), 154.0 (C-3^', C-5^'), 150.6 (C-7^'''), 149.3 (C-6^'''), 138.3 (C-4^''''), 137.3 (C-14), 136.7 (C-1^''''), 135.7 (C-4^'), 134.7 (C-9^'''), 134.7 (C-10^'''), 126.7 (C-13), 126.4 (C-1^'), 111.2 (C-5^'''), 109.9 (C-8^'''), 109.4 (C-2^'''', C-6^''''), 105.4 (C-2^', C-6^'), 103.3 (OCH₂O), 102.1 (C-10), 94.7 (C-8), 92.5 (C-6), 80.0 (C-2), 68.9 (OCH₂), 67.3 (C-3, C-11^'''), 65.7 (C-4^'''), 61.1 (4^''''-OCH₃), 59.8 (4^'-OCH₃), 56.8 (7-OCH₃), 56.7 (3^''''-OCH₃), 56.6 (5^''''-OCH₃), 55.9 (3^'-OCH₃), 55.8 (5^'-OCH₃), 44.9 (C-1^'''), 42.5 (C-2^'''), 38.6 (C-3^'''), 29.4 (C-4); MS (ESI): m/z = 840 [M + H]⁺.

Methylated (–)-epigallocatechin-[4β-(1,2,3-trizaol-1-yl-4-deoxy-4’-demethylpodophyllotoxin)] ether (15)

White amorphous powder; yield 80%; m.p. = 187–188 °C; ¹H NMR (500 MHz, CD₃OD): δ 7.58 (s, 1H, C¹⁴-H), 6.77 (s, 2H, C^2'', C^6''-H), 6.62 (d, 2H, J = 3.0 Hz, C^5''', C^8'''-H), 6.36 (s, 2H, C^2', C^6'-H), 6.20 (d, 1H, J = 2.3 Hz, C⁶-H), 6.17 (d, 1H, J = 2.3 Hz, C⁸-H), 6.14 (d, 1H, J = 2.3 Hz, C³-H), 5.96-5.95 (m, 2H, OCH₂O), 5.25-5.20 (m, 3H, C²-H, OCH₂), 5.13 (s, 1H, C^4'''-H), 4.74 (d, 1H, J = 5.0 Hz, C^1'''-H), 4.36-4.33 (m, 2H, C^11'''-CH₂), 3.78 (s, 3H, C7-3OCH), 3.78-3.72 (m, 15H, C^3', C^4', C^5', C^3''''', C^5''''-OCH₃), 3.18 (dd, 1H, J = 4.6, 12.0 Hz, C⁴-H_a), 3.08-3.06 (m, 1H, C⁴-H_b), 2.85-2.83 (m, 1H, C^3'''-H), 2.82-2.80 (m, 1H, C^2'''-H); ¹³C NMR (125 MHz, CD₃OD): δ 176.0 (C-12^'''), 161.1 (C-7), 160.6 (C-5), 156.9 (C-9), 154.5 (C-3^'''', C-5^''''), 150.6 (C-7^'''), 149.2 (C-6^'''), 148.7 (C-3^', C-5^'), 137.3 (C-14), 136.9 (C-4^''''), 136.0 (C-4^'), 136.7 (C-1^''''), 135.7 (C-1^'), 135.1 (C-9^'''), 131.3 (C-10^'''), 126.7 (C-13), 111.3 (C-5^'''), 109.8 (C-8^'''), 109.3 (C-2^'''', C-6^''''), 105.4 (C-2^', C-6^'), 103.3 (OCH₂O), 102.1 (C-10), 94.7 (C-8), 92.6 (C-6), 80.0 (C-2), 68.9 (OCH₂), 67.3 (C-3, C-11^'''), 65.7 (C-4^'''), 59.8 (4^'-OCH₃), 56.8 (7-OCH₃), 56.7 (3^''''-OCH₃), 56.6 (5^''''-OCH₃), 55.9 (3^'-OCH₃), 55.8 (5^'-OCH₃), 44.7 (C-1^'''), 42.7 (C-2^'''), 38.6 (C-3^'''), 29.4 (C-4); MS (ESI): m/z = 826 [M + H]⁺.

Cytotoxicity assays

The cytotoxicity of all the compounds against human myeloid leukemia (HL-60), lung cancer (A-549), breast cancer (MCF-7), colon cancer (SW480), and hepatocellular carcinoma (SMMC-7721) cell lines was measured using the MTT method. ³² Adherent cells (100 μL) were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 10⁵ cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h. After the incubation, MTT (100 μg) was added to each well, and incubation was continued for 4 h at 37 °C. The cells were lysed with SDS (200 μL) after removal of 100 μL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). IC₅₀ values were calculated by Reed’s method. ³³

Molecular docking studies

In this study, the crystal structure of tubulin (PDB ID: 3UT5) ³⁴ and EGFR (PDB ID: 3IKA) ³⁵ was obtained from the in Protein Data Bank (https://www.rcsb.org/). DiscoveryStudio 4.0 software was used for the modeling of the ligand and receptor. AutoDock Tools v1.56 was used for grid and docking according to the literature. ³⁶ Docking parameters were set as the defaults values, except The Number of GA Runs, which was set to 20 and the maximum number of medium was set to 5,000,000 on AutoGrid v4.2 and AutoDock v4.2 (http://autodock.scripps.edu). Docking conformations were classified into different clusters by binding energy, and the cluster with the lowest binding energy was selected. In the selected cluster, conformations with the lowest binding energy and root mean square deviation (RMSD) (< 2.0 Å) were finally chosen to analyze the receptor–ligand interactions.

Supplemental Material

sj-pdf-1-chl-10.1177_17475198211027328 – Supplemental material for Synthesis, cytotoxicity, and molecular docking of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives as novel antitumor agents

Supplemental material, sj-pdf-1-chl-10.1177_17475198211027328 for Synthesis, cytotoxicity, and molecular docking of methylated (–)-epigallocatechin-3-gallate-4β-triazolopodophyllotoxin derivatives as novel antitumor agents by Cheng-Ting Zi, Ze-Hao Wang, Jing Shi, Bo-Ya Shi, Ning Zhang, Yi-Long Wu, Yin-Rong Xie, Lu Zhou, Chun Xiao, Xuan-Jun Wang and Jun Sheng in Journal of Chemical Research