Synthesis and cytotoxic activity of 7,4’- O -modified genistein amino acid derivatives

Abstract

Fifteen 7,4’-O-modified genistein amino acid derivatives are synthesized through nucleophilic substitution and hydrolysis, followed by condensation with diverse amino acid esters. The antiproliferative activity of all the synthesized compounds is evaluated against three cancer cell lines (MGC-803, HeLa, HCT-116) and one normal cell line (HUVEC) using 5-fluorouracil (5-Fu) as the positive control. The results show that methyl [2-({5-hydroxy-3-[4-(2-{[3-(4-hydroxyphenyl)-1-methoxy-1-oxopropan-2-yl]amino}-2-oxoethoxy)phenyl]-4-oxo-4H-chromen-7-yl}oxy)acetyl] tyrosinate exhibits significant antiproliferative activity against the MGC-803 cell line with an IC₅₀ value of 8.52 µM, and its inhibitory effects on HeLa and HCT-116 cancer cells are stronger than that of the positive control drug 5-Fu.

Keywords

amino acid derivatives cytotoxic activity human gastric carcinoma cells tyrosine

Introduction

Genistein, also known as 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one, is a type of isoflavone widely found in leguminous plants.¹ A large number of studies have reported that genistein possesses a wide spectrum of pharmacological activities such as estrogenic, anti-inflammatory, antitumor, antihypertension, antidiabetic activity, and so on.^2–6 The extensive antitumor activities of genistein have received significant attention.^7–9 Growing evidence suggests that genistein exhibits inhibitory effects on human prostate cancer cells,¹⁰ colon cancer cells,¹¹ and human ovarian cancer cells,¹² and the mechanism of its action may be associated with its effect on the cell cycle,¹³ cellular invasion and metastasis,¹⁴ and apoptosis.¹⁵ However, due to its poor solubility and bioavailability, genistein has not been widely used in the medical field.^16,17 In order to overcome this shortcoming and to enhance the anticancer activity of genistein, structural modification based on genistein is required.

Amino acids, as the basic components of proteins, are characterized by diverse structures, good safety, stability and water solubility, as well as improved sustained release and enhanced drug targeting property.^18,19 Studies have shown that amino acids have been widely used in prodrug design and can improve oral availability of parent drugs with poor solubility and permeability.^20–22 For instance, Lee et al.²³ reported that introducing 11 amino acids into the structure of the antitumor compound DW22822 improved its water solubility, and compounds conjugated with L-valine and L-leucine showed significant antiproliferative activity against human colon cancer cells (SW620). Mattarei et al.²⁴ showed that the metabolic stability of resveratrol derivatives linked with isoleucine was improved, because the introduction of amino acids could shield them from the first-pass effect through the liver.

In our previous work, we discovered that genistein bearing an alanine chain at the C7 position showed better antiproliferative activity than the parent scaffold genistein against the MGC-803 cell line.²⁵ In order to further explore the structure-activity relationship, fifteen genistein derivatives with amino acid chains at both C7 and C4’ were synthesized and evaluated for their cytotoxicity against three cancer cell lines (MGC-803, HeLa, HCT-116) and one normal cell line (HUVEC) by using the MTT assay.

Results and discussion

The general synthetic route for 7,4’-O-modified genistein amino acid derivatives 7a–9e is outlined in Scheme 1. Genistein first underwent nucleophilic substitution with ethyl bromoacetate in the presence of KOH to afford intermediate 1. Next, the ester groups were transformed into free carboxyl groups using a solution of KOH in CH₃OH/H₂O.²⁶ Finally, the carboxyl groups were condensed with different L-amino acids in the presence of EDCI, HOBT, DIPEA, and DMAP in DMF at room temperature to afford the target compounds 7a–e.²⁵ Other target compounds 8a–e and 9a–e could be obtained by the reaction of genistein with ethyl 4-bromobutyrate and ethyl 6-bromohexanoate, respectively, under the same conditions.

Scheme 1.

Synthetic route to 7,4’-O-modified genistein amino acid derivatives 7a–9e.

All the synthesized compounds were screened for their antitumor activity against three cancer cell lines (MGC-803, HeLa, HCT-116) and one normal cell line (HUVEC) by the MTT assay with 5-fluorouracil (5-Fu) as the positive control,^27–30 and the results are showed in Table 1. From Table 1, the following conclusions can be made: (1) genistein modified with an alanine group (7b) showed a better inhibitory effect on the MGC-803 cancer cell line than the parent genistein skeleton. However, the cytotoxicity decreased with an increasing the linker length between genistein and alanine (7b vs 8b and 9b); (2) genisteins bearing a tyrosine group (7d, 8d and 9d) exhibited significant antiproliferative effects on MGC-803 cancer cell line. In particular, the antitumor activity of compound 7d against HeLa and HCT-116 cancer cell lines was superior to the positive control drug 5-Fu. In addition, its cytotoxic activity against HUVEC cells was far lower than those against the other three cancer cell lines, suggesting that compound 7d had a good selectivity; (3) introducing glycine (7a), phenylalanine (7c), or tryptophan (7e) into genistein showed hardly any enhancement of antitumor activity against the three cancer cell lines.

Table 1.

Antiproliferative activity of 7,4’-O-modified genistein amino acid derivatives.

Compound	IC₅₀ (μM)^a
	MGC-803	HeLa	HCT-116	HUVEC
7a	>200	>200	>200	ND
7b	11.29 ± 1.65	217.2 ± 1.46	>200	>200
7c	>200	>200	>200	>200
7d	8.52 ± 0.13	41.61 ± 1.39	55.45 ± 2.15	>200
7e	ND	>200	>200	>200
8a	>200	>200	>200	>200
8b	101.8 ± 0.56	161.2 ± 3.12	189.3 ± 1.88	>200
8c	>200	>200	>200	>200
8d	14.14 ± 0.62	>200	>200	ND
8e	ND	ND	>200	>200
9a	176.3 ± 2.25	>200	>200	ND
9b	>200	>200	>200	>200
9c	ND	>200	27.4 1± 1.22	>200
9d	14.69 ± 1.92	ND	>200	>200
9e	>200	ND	ND	>200
genistein	>200	ND	ND	>200
5-Fu	0.13 ± 0.75	>200	142.9 ± 2.21	>200

Data are the mean ± SD of three estimations.

In conclusion, 15 novel 7,4’-O-modified genistein amino acid ester derivatives have been synthesized and evaluated for their antiproliferative activity by MTT assays. Genistein bearing a tyrosine group (7d) exhibited excellent antiproliferative activity against the MGC-803 cell line with an IC₅₀ value of 8.52 µM, which could serve as a potential antitumor candidate for further investigations.

Experimental

Commercially available chemicals were obtained from commercial suppliers and used without further purification unless otherwise stated. Proton NMR (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker Avance III 400 MHz (Ascend TM 500 MHz) NMR spectrometer. The following abbreviations are used for the multiplicities: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br s: broad singlet for proton spectra. Coupling constants (J) are reported in Hertz (Hz). High-resolution mass spectra (HRMS) were recorded on a BRUKER VPEXII spectrometer with electron ionization (EI) and electrospray ionization (ESI) mode unless otherwise stated. Melting points (uncorrected) were determined on an automatic melting point apparatus (ZRD-1) from Tianjin optical instrument factory. Analytical thin-layer chromatography was performed on Polygram SIL G/UV₂₅₄ plates. Visualization was accomplished with shortwave UV light, or by KMnO₄ staining followed by heating. Flash column chromatography was performed using silica gel (200–300 mesh) from Qingdao Haiyang Chemical Co., Ltd, with solvents distilled prior to use.

General procedure for the synthesis of compounds 1–3

To a solution of genistein (1 g, 3.70 mmol) in DMF (20 mL) was added KOH (829 mg, 14.78 mmol) and the mixture was stirred at 60 °C for 4 h. Next, ethyl bromoacetate, ethyl 4-bromobutyrate or ethyl 6-bromohexanoate (14.80 mmol) and KI (61.4 mg, 0.37 mmol) were added. After the reaction was complete (monitored by TLC), the reaction mixture was poured into distilled H₂O (80 mL) and then extracted with EtOAc (3 × 30 mL). The combined organic phase was dried over anhydrous Na₂SO₄ and concentrated in vacuum. The residue was purified by column chromatography on silica gel (PE/EtOAc = 6:1, v/v) to afford the target compounds 1–3.

Ethyl 2-{4-[7-(2-ethoxy-2-oxoethoxy)-5-hydroxy-4-oxo-4H-chromen-3-yl]phenoxy} acetate (1): yellow solid; yield: 41%; m.p.: 125–127 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.91 (s, 1H), 8.47 (s, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 2.3 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 4.96 (s, 2H), 4.83 (s, 2H), 4.23–4.16 (m, 4H), 1.24 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 180.3, 168.6, 168.0, 163.5, 161.7, 157.3, 157.5, 154.9, 130.1, 123.4, 122.1, 114.3, 105.8, 98.5, 93.2, 65.0, 64.6, 60.9, 60.7, 14.1, 14.0; HRMS (ESI): calcd for C₂₃H₂₃O₉ [M + H]⁺: 443.1336; found: 443.1333.

Ethyl 4-{4-[7-(4-ethoxy-4-oxobutoxy)-5-hydroxy-4-oxo-4H-chromen-3-yl]phenoxy} butanoate (2): yellow solid, yield: 60%; m.p.: 73–75 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 12.91 (s, 1H), 8.44 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 6.65 (d, J = 2.2 Hz, 1H), 6.39 (d, J = 2.1 Hz, 1H), 4.13–4.02 (m, 8H), 2.47 (td, J = 7.3, 1.7 Hz, 4H), 2.02–1.96 (m, 4H), 1.19 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 180.2, 172.5, 172.4, 164.4, 161.7, 158.4, 157.4, 154.7, 130.1, 122.8, 122.1, 114.2, 105.4, 98.39, 92.8, 67.5, 66.5, 60.0, 59.9, 30.1, 29.9, 24.2, 23.9, 14.1; HRMS (ESI): calcd for C₂₇H₃₀O₉Na [M + Na]⁺: 521.1782; found: 521.1779.

Ethyl 6-(4-{7-[(6-ethoxy-6-oxohexyl)oxy]H-chromen-3-yl}phenoxy) hexanoate (3): yellow solid, yield: 56%; m.p.: 65–67 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 12.91 (s, 1H), 8.42 (s, 1H), 7.49 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 6.62 (d, J = 2.3 Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H), 4.08–4.03 (m, 6H), 3.98 (t, J = 6.5 Hz, 2H), 2.31 (td, J = 7.4, 1.8 Hz, 4H), 1.76–1.70 (m, 4H), 1.63–1.56 (m, 4H), 1.46–1.36 (m, 4H), 1.18 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 180.2, 172.8, 164.6, 161.7, 158.6, 157.4, 154.6, 130.1, 122.6, 122.1, 114.2, 105.3, 98.3, 92.8, 68.3, 67.3, 59.6, 33.5, 33.4, 28.3, 28.0, 25.0, 24.9, 24.2, 24.1, 14.1; HRMS (ESI): calcd for C₃₁H₃₈O₉Na [M + Na]⁺: 577.2408; found: 577.2402.

General procedure for the synthesis of compounds 4–6

To a stirred solution of compound 1–3 (0.5 mmol) in MeOH (5.0 mL) and H₂O (2.0 mL) was added KOH (224.4 mg, 4.0 mmol). The reaction was stirred at room temperature for 30 min and monitored by TLC. After the reaction was complete, 1 M HCl was added to adjust the pH to 2–3. A white precipitate formed and was filtered from the solution. The precipitate was dried in vacuo to afford compounds 4–6 as white solids. Compounds 4–6 were directly used in the next step without further purification.

General procedure for the synthesis of compounds 7a–9e

To a solution of compound 4–6 (0.5 mmol) in DMF (8.0 mL) was added EDCI·HCl (383.4 mg, 2.0 mmol) and HOBT·H₂O (270.3 mg, 2.0 mmol) at room temperature. After 4 h, DIPEA (258.5 mg, 2.0 mmol), DMAP (122.2 mg, 1.0 mmol), and the corresponding L-amino acid methyl ester (4.0 mmol) were added. The reaction mixture was stirred at room temperature for 24 h and then poured into ice-cold H₂O (20 mL). A yellow or white precipitate formed and was filtered from the solution. The precipitate was dried and purified by column chromatography on silica gel (CH₂Cl₂/EtOAc = 10:1 to 1:1, v/v) to afford compounds 7a–9e.

Methyl (2-{[5-hydroxy-3-(4-{2-[(2-methoxy-2-oxoethyl)amino]H-chromen-7-yl]oxy}acetyl)glycinate (7a): white solid, yield: 49%; m.p.: 197–199 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.91 (s, 1H), 8.64 (t, J = 5.9 Hz, 1H), 8.56 (t, J = 6.0 Hz, 1H), 8.48 (s, 1H), 7.54 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 6.69 (d, J = 2.3 Hz, 1H), 6.47 (d, J = 2.3 Hz, 1H), 4.72 (s, 2H), 4.60 (s, 2H), 3.94–3.92 (m, 4H), 3.65 (s, 6H);¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 170.1, 170.0, 168.2, 167.5, 163.4, 161.6, 157.5, 157.3, 154.9, 130.1, 123.5, 122.1, 114.6, 105.8, 98.4, 93.3, 67.0, 66.8, 51.8, 51.7, 40.3; HRMS (ESI): calcd for C₂₅H₂₅N₂O₁₁ [M + H]⁺: 529.1453; found: 529.1452.

Methyl (2-{[5-hydroxy-3-(4-{2-[({S}-1-methoxy-1-oxopropan-2-yl)amino]H-chromen-7-yl]oxy}acetyl)-L-alaninate (7b): white solid, yield: 72%; m.p.: 157–159 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.91 (s, 1H), 8.61 (d, J = 7.3 Hz, 1H), 8.54 (d, J = 7.3 Hz, 1H), 8.48 (s, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 2.2 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 4.74–4.54 (m, 4H), 4.39 (m, 2H), 3.64 (s, 6H), 1.34 (d, J = 7.3 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.7, 172.6, 167.5, 166.8, 163.6, 161.6, 157.7, 157.3, 154.9, 130.1, 123.4, 122.1, 114.5, 105.7, 98.7, 93.3, 66.8, 66.6, 52.0, 47.4, 47.3, 16.8; HRMS (ESI): calcd for C₂₇H₂₉N₂O₁₁ [M + H]⁺: 557.1766; found: 557.1764.

Methyl (2-{[5-hydroxy-3-(4-{2-[({S}-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino]H-chromen-7-yl]oxy}acetyl)-L-phenylalaninate (7c): white solid, yield: 33%; m.p.: 108–110 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 12.92 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.48 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.30–7.17 (m, 10H), 6.95 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 2.3 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 4.70–4.52 (m, 6H), 3.64 (s, 6H), 3.14–3.10 (m, 2H), 3.05–2.98 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 171.7, 171.6, 167.7, 167.0, 163.5, 161.6, 157.6, 157.2, 154.9, 137.1, 137.0, 130.1, 129.1, 129.0, 128.3, 128.2, 126.6, 126.5, 123.4, 122.1, 114.5, 105.8, 98.7, 93.1, 66.8, 66.5, 53.3, 53.1, 52.0, 36.4, 36.3; HRMS (ESI): calcd for C₃₉H₃₇N₂O₁₁ [M + H]⁺: 709.2392; found: 709.2383.

Methyl (2-{[5-hydroxy-3-(4-{2-[({S}-3-{4-hydroxyphenyl}-1-methoxy-1-oxopropan-2-yl)amino]H-chromen-7-yl]oxy}acetyl)-L-tyrosinate (7d): white solid, yield: 42%; ¹H NMR (500 MHz, DMSO-d₆): δ 12.92 (s, 1H), 9.24 (d, J = 12.0 Hz, 2H), 8.52 (d, J = 7.9 Hz, 1H), 8.46 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.7 Hz, 2H), 7.00 (dd, J = 8.4, 3.4 Hz, 4H), 6.94 (d, J = 8.8 Hz, 2H), 6.65 (dd, J = 12.2, 8.3 Hz, 4H), 6.57 (d, J = 2.3 Hz, 1H), 6.43 (d, J = 2.2 Hz, 1H), 4.70–4.62 (m, 2H), 4.55–4.46 (m, 4H), 3.63 (s, 6H), 3.01–2.96 (m, 2H), 2.92–2.86 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆): δ 180.3, 171.8, 171.7, 167.6, 167.0, 163.5, 161.6, 157.6, 157.3, 156.0, 154.8, 130.1, 130.0, 129.9, 127.1, 127.0, 123.4, 122.2, 115.1, 115.0, 114.4, 105.8, 98.7, 93.1, 66.8, 66.5, 53.6, 53.4, 51.8, 35.7, 35.6; HRMS (ESI): calcd for C₃₉H₃₇N₂O₁₃ [M + H]⁺: 741.2290; found: 741.2283.

Methyl (2-{4-[7-(2-{[(S)-3-{1H-indol-3-yl}-1-methoxy-1-oxopropan-2-yl]amino}-2-oxoethoxy)-5-hydroxy-4-oxo-4H-chromen-3-yl]phenoxy}acetyl)-L-tryptophanate (7e): white solid, yield: 66%; m.p.: 153–155 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 10.89 (d, J = 2.4 Hz, 2H), 8.49 (d, J = 7.8 Hz, 1H), 8.46 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.54–7.50 (m, 4H), 7.36 (dd, J = 8.1, 3.4 Hz, 2H), 7.18 (dd, J = 11.7, 2.3 Hz, 2H), 7.08 (td, J = 7.5, 3.2 Hz, 2H), 7.04–6.92 (m, 4H), 6.60 (d, J = 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 4.65 (dd, J = 14.6, 5.0 Hz, 4H), 4.55 (s, 2H), 3.64 (d, J = 1.2 Hz, 6H), 3.31–3.16 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.0, 171.9, 167.6, 166.9, 163.4, 161.6, 157.6, 157.3, 154.9, 136.1, 130.1, 127.1, 127.0, 123.84, 123.81, 123.4, 122.1, 121.0, 118.5, 118.4, 118.0, 117.9, 114.5, 111.5, 109.3, 109.2, 105.8, 98.7, 93.2, 66.8, 66.5, 52.9, 52.7, 52.0, 26.7; HRMS (ESI): calcd for C₄₃H₃₈N₄O₁₁Na [M + Na]⁺: 809.2429; found: 809.2417.

Methyl (4-{[5-hydroxy-3-(4-{4-[(2-methoxy-2-oxoethyl)amino]H-chromen-7-yl]oxy}butanoyl)glycinate (8a): white solid, yield: 45%; m.p.: 163–165 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 8.44 (s, 1H), 8.37–8.34 (m, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 2.2 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.12 (t, J = 6.4 Hz, 2H), 4.03 (t, J = 6.5 Hz, 2H), 3.85 (dd, J = 5.9, 1.3 Hz, 4H), 3.63 (s, 6H), 2.35–2.31 (m, 4H), 2.01–1.94 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.3, 172.1, 170.5, 164.5, 161.7, 158.5, 157.5, 154.7, 130.2, 122.7, 122.2, 114.3, 105.4, 98.4, 92.9, 67.8, 66.8, 51.7, 40.6, 31.3, 31.1, 24.7, 24.4; HRMS (ESI): calcd for C₂₉H₃₃N₂O₁₁ [M + H]⁺: 585.2079; found: 585.2073.

Methyl (4-{[5-hydroxy-3-(4-{4-[({S}-1-methoxy-1-oxopropan-2-yl)amino]H-chromen-7-yl]oxy}butanoyl)-L-alaninate (8b): white solid, yield: 78%; m.p.: 156-158 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 8.45 (s, 1H), 8.34 (dd, J = 7.2, 2.2 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 6.65 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.3 Hz, 1H), 4.31–4.24 (m, 2H), 4.11 (t, J = 6.4 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.62 (d, J = 1.3 Hz, 6H), 2.31 (td, J = 7.4, 1.8 Hz, 4H), 2.04–1.90 (m, 4H), 1.27 (d, J = 7.3 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.2, 173.2, 171.6, 171.5, 164.5, 161.7, 158.5, 157.5, 154.6, 130.1, 122.7, 122.1, 114.2, 105.3, 98.4, 92.8, 67.8, 66.8, 51.8, 47.5, 31.2, 31.0, 24.7, 24.4, 16.9; HRMS (ESI): calcd for C₃₁H₃₇N₂O₁₁ [M + H]⁺: 613.2392; found: 613.2385.

Methyl (4-{[5-hydroxy-3-(4-{4-[({S}-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino]H-chromen-7-yl]oxy}butanoyl)-L-phenylalaninate (8c): white solid, yield: 79%; m.p.: 158-160 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.93 (s, 1H), 8.46 (s, 1H), 8.39 (dd, J = 7.8, 2.5 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.29–7.18 (m, 10H), 6.97 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 4.52–4.50 (m, 2H), 4.00 (t, J = 6.4 Hz, 2H), 3.91 (t, J = 6.4 Hz, 2H), 3.61 (s, 6H), 3.04 (dd, J = 13.8, 5.4 Hz, 2H), 2.89 (dd, J = 13.7, 9.6 Hz, 2H), 2.27–2.24 (m, 4H), 1.91–1.87 (m, 4H); ¹³C NMR (126 MHz, DMSO-d₆): δ 180.3, 172.2, 172.1, 171.7, 171.6, 164.5, 161.7, 158.4, 157.4, 154.7, 137.3, 130.1, 129.0, 128.21, 128.20, 126.5, 122.7, 122.1, 114.2, 105.3, 98.4, 92.8, 67.6, 66.7, 53.5, 53.4, 51.8, 36.6, 31.2, 31.0, 24.6, 24.3; HRMS (ESI): calcd for C₄₃H₄₅N₂O₁₁ [M + H]⁺: 765.3018; found: 765.3008.

Methyl (4-{[5-hydroxy-3-(4-{4-[({S}-3-{4-hydroxyphenyl}-1-methoxy-1-oxopropan-2-yl) amino]H-chromen-7-yl]oxy}butanoyl)-L-tyrosinate (8d): white solid, yield: 80%; m.p.: 173–175 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.93 (s, 1H), 9.21 (d, J = 7.4 Hz, 2H), 8.45 (s, 1H), 8.32 (dd, J = 7.9, 2.9 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.00–6.97 (m, 6H), 6.67–6.63 (m, 5H), 6.39 (d, J = 2.2 Hz, 1H), 4.44–4.38 (m, 2H), 4.04 (t, J = 6.5 Hz, 2H), 3.94 (t, J = 6.5 Hz, 2H), 3.59 (s, 6H), 2.91 (dd, J = 13.8, 5.6 Hz, 2H), 2.78 (dd, J = 13.8, 9.2 Hz, 2H), 2.30–2.25 (m, 4H), 1.92–1.88 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.4, 172.3, 171.7, 171.6, 164.5, 161.7, 158.4, 157.5, 155.9, 154.7, 130.1, 129.9, 127.3, 127.2, 122.7, 122.1, 115.0, 114.2, 105.4, 98.4, 92.8, 67.7, 66.8, 53.9, 51.7, 36.0, 31.3, 31.1, 24.7, 24.4; HRMS (ESI): calcd for C₄₃H₄₅N₂O₁₃ [M + H]⁺: 797.2916; found: 797.2910.

Methyl (4-{4-[7-(4-{[(S)-3-{1H-indol-3-yl}-1-methoxy-1-oxopropan-2-yl]amino}-4-oxobutoxy)-5-hydroxy-4-oxo-4H-chromen-3-yl]phenoxy}butanoyl)-L-tryptophanate (8e): white solid, yield: 71%; m.p.: 132-135 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.93 (s, 1H), 10.86 (s, 2H), 8.44 (s, 1H), 8.36 (dd, J = 7.6, 2.6 Hz, 2H), 7.51 (d, J = 8.8 Hz, 4H), 7.35 (dd, J = 8.1, 2.8 Hz, 2H), 7.16 (d, J = 2.3 Hz, 2H), 7.07 (td, J = 7.6, 3.1 Hz, 2H), 7.01–6.96 (m, 4H), 6.62 (d, J = 2.2 Hz, 1H), 6.39 (d, J = 2.2 Hz, 1H), 4.58–4.52 (m, 2H), 4.04 (t, J = 6.5 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 3.59 (s, 6H), 3.17 (dd, J = 14.5, 5.7 Hz, 2H), 3.05 (dd, J = 14.6, 8.5 Hz, 2H), 2.36–2.26 (m, 4H), 1.93–1.90 (m, 4H); ¹³C NMR (126 MHz, DMSO-d₆): δ 180.3, 172.6, 172.5, 171.7, 171.6, 164.5, 161.7, 158.4, 157.5, 154.6, 136.1, 130.1, 127.0, 123.6, 122.7, 122.1, 120.9, 118.4, 118.0, 114.2, 111.4, 109.6, 109.5, 105.3, 98.4, 92.8, 67.7, 66.8, 53.1, 51.8, 31.3, 31.1, 27.1, 27.0, 24.6, 24.3; HRMS (ESI): calcd for C₄₇H₄₇N₄O₁₁ [M + H]⁺: 843.3236; found: 843.3226.

Methyl (6-{[5-hydroxy-3-(4-{[6-({2-methoxy-2-oxoethyl}amino)-6-oxohexyl]H-chromen-7-yl]oxy}hexanoyl)glycinate (9a): white solid, yield: 58%; m.p.: 123–125 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 8.44 (s, 1H), 8.27 (t, J = 5.9 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 3.82 (d, J = 5.9 Hz, 4H), 3.63 (s, 6H), 2.17 (t, J = 7.3 Hz, 4H), 1.77–1.70 (m, 4H), 1.62–1.54 (m, 4H), 1.46–1.38 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.2, 172.7, 172.6, 170.5, 164.6, 161.7, 158.6, 157.5, 154.6, 130.1, 122.6, 122.1, 114.2, 105.3, 98.4, 92.8, 68.4, 67.4, 51.6, 40.5, 34.9, 34.8, 28.4, 28.1, 25.1, 24.97, 24.95, 24.8; HRMS (ESI): calcd for C₃₃H₄₁N₂O₁₁ [M + H]⁺: 641.2705; found: 641.2698.

Methyl (6-{[5-hydroxy-3-(4-{[6-({[S]-1-methoxy-1-oxopropan-2-yl}amino)-6-oxohexyl]H-chromen-7-yl]oxy}hexanoyl)-L-alaninate (9b): white solid, yield: 77%; m.p.: 152–154 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 8.44 (s, 1H), 8.25 (d, J = 7.0 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.29–4.22 (m, 2H), 4.08 (t, J = 6.5 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 3.62 (s, 6H), 2.14 (t, J = 7.3 Hz, 4H), 1.77–1.70 (m, 4H), 1.60–1.53 (m, 4H), 1.45–1.38 (m, 4H), 1.26 (d, J = 7.3 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.2, 173.2, 172.1, 172.0, 164.6, 161.7, 158.6, 157.5, 154.6, 130.1, 122.6, 122.1, 114.2, 105.3, 98.4, 92.8, 68.4, 67.4, 51.7, 47.4, 34.8, 34.7, 28.4, 28.1, 25.1, 25.0, 24.9, 24.8, 16.9; HRMS (ESI): calcd for C₃₅H₄₄N₂O₁₁Na [M + Na]⁺: 691.2837; found: 691.2824.

Methyl (6-{[5-hydroxy-3-(4-{[6-({[S]-1-methoxy-1-oxo-3-phenylpropan-2-yl}amino)-6-oxohexyl]H-chromen-7-yl]oxy}hexanoyl)-L-phenylalaninate (9c): white solid, yield: 66%; m.p.: 130–132 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.93 (s, 1H), 8.45 (s, 1H), 8.30 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.29–7.17 (m, 10H), 6.99 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.52–4.46 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 3.61 (s, 6H), 3.06–3.01 (m, 2H), 2.91–2.85 (m, 2H), 2.09 (t, J = 7.2 Hz, 4H), 1.71–1.64 (m, 4H), 1.48 (p, J = 7.4 Hz, 4H), 1.33–1.23 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.3, 172.2, 164.7, 161.7, 158.6, 157.5, 154.7, 137.3, 130.2, 129.0, 128.2, 126.5, 122.6, 122.2, 114.2, 105.3, 98.4, 92.8, 68.4, 67.4, 53.4, 51.8, 36.7, 34.9, 34.8, 28.4, 28.1, 25.0, 24.9, 24.8; HRMS (ESI): calcd for C₄₇H₅₂N₂O₁₁Na [M + Na]⁺: 843.3463; found: 843.3451.

Methyl (6-{[5-hydroxy-3-(4-{[6-({[S]-3-[4-hydroxyphenyl]-1-methoxy-1-oxopropan-2-yl}amino)-6-oxohexyl]H-chromen-7-yl]oxy}hexanoyl)-L-tyrosinate (9d): white solid, yield: 75%; m.p.: 168–170 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (s, 1H), 9.22 (d, J = 2.0 Hz, 2H), 8.44 (s, 1H), 8.21 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 6H), 6.66 (dd, J = 8.4, 1.9 Hz, 5H), 6.40 (d, J = 2.2 Hz, 1H), 4.43–4.37 (m, 2H), 4.05 (t, J = 6.5 Hz, 2H), 3.96 (t, J = 6.5 Hz, 2H), 3.59 (s, 6H), 2.93–2.88 (m, 2H), 2.79–2.73 (m, 2H), 2.10 (t, J = 7.2 Hz, 4H), 1.73–1.65 (m, 4H), 1.54–1.46 (m, 4H), 1.36–1.28 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.2, 172.3, 172.2, 172.1, 164.6, 161.7, 158.6, 157.5, 155.9, 154.6, 130.1, 129.9, 127.2, 122.6, 122.1, 114.9, 114.2, 105.3, 98.3, 92.8, 68.4, 67.4, 53.7, 51.7, 36.0, 34.9, 34.8, 28.3, 28.1, 25.0, 24.9, 24.8; HRMS (ESI): calcd for C₄₇H₅₃N₂O₁₃ [M + H]⁺: 853.3542; found: 853.3532.

Methyl (6-{4-[7-({6-[({S}-3-[1H-indol-2-yl]-1-methoxy-1-oxopropan-2-yl)amino]-6-oxohexyl}oxy)-5-hydroxy-4-oxo-4H-chromen-3-yl]phenoxy}hexanoyl)-L-tryptophanate (9e): white foamy solid, yield: 79%; m.p.: 64–66 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 12.93 (s, 1H), 10.86 (s, 2H), 8.43 (s, 1H), 8.25 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 8.2 Hz, 4H), 7.34 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 2.3 Hz, 2H), 7.07 (t, J = 7.5 Hz, 2H), 7.01–6.98 (m, 4H), 6.64 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.56–4.51 (m, 2H), 4.03 (t, J = 6.5 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 3.59 (s, 6H), 3.16 (dd, J = 14.6, 5.7 Hz, 2H), 3.04 (dd, J = 14.6, 8.6 Hz, 2H), 2.13 (t, J = 7.3 Hz, 4H), 1.72–1.65 (m, 4H), 1.54–1.47 (m, 4H), 1.37–1.29 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆): δ 180.3, 172.6, 172.3, 172.2, 164.7, 161.7, 158.6, 157.5, 154.6, 136.1, 130.1, 127.1, 123.6, 122.6, 122.2, 121.0, 118.4, 118.0, 114.2, 111.4, 109.6, 105.3, 98.4, 92.8, 68.4, 67.4, 53.0, 51.8, 34.9, 34.8, 28.4, 28.1, 27.1, 25.0, 24.94, 24.90, 24.8; HRMS (ESI): calcd for C₅₁H₅₅N₄O₁₁ [M + H]⁺: 899.3862; found: 899.3850.

MTT assay

All the cells lines were seeded in 96-well plates at a volume of 100 μL/well (1 × 10⁴ cells/well) and incubated under a humidified atmosphere of 5% CO₂ at 37 °C for 24 h. Then different concentrations (0, 5, 10, 20, 40, 80, 160 μM) of tested compounds were added and incubated for 48 h. After 48 h, the supernatant was discarded, and then DMEM (90 μL) and MTT (10 μL, 5 mg/mL) were added. The plates were incubated for another 4 h. The suspension was discarded and DMSO (110 μL) was added to dissolve the crystal. The absorbance of the solution was measured at 490 nm by a microplate reader. All experiments were performed in triplicate. IC₅₀ values were obtained using Prism software.

Supplemental Material

SI – Supplemental material for Synthesis and cytotoxic activity of 7,4’-O-modified genistein amino acid derivatives

Supplemental material, SI for Synthesis and cytotoxic activity of 7,4’-O-modified genistein amino acid derivatives by Yao-Fu Zeng, Yu-Qin Duan, Lanqing Liao, Xiaokang Long, Cheng Gao and Xianghao Wen in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the “Double First-Class” Discipline Construction Foundation of the University of South China, the Undergraduate Research Learning and Innovative Experiment Project (2018XJXZ191), and the Foundation of Hu’nan Educational Committee (09C830, 18C0476).

ORCID iD

Yao-Fu Zeng

Supplemental material

Supplemental material for this article is available online.

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