First Total Synthesis and in Vitro Cytotoxicities of Flavesines G and J.

The first total synthesis of flavesines G and J, natural products exhibiting antiviral activity against hepatitis B virus, is described. A robust, protecting-group-free route starting from commercially available natural product 9-azajulolidine allowed us to obtain the title compounds in a four- and five-step sequence accordingly. Flavesines G and J exhibit micromolar cytotoxicity in A549, MCF-7, HepG2, PANC-1, and HL-60 cancer cell lines.

Introduction

Flavesines G (7) and J (10) have been found in the roots of Sophora flavescens and were first studied in 2018 by Wang and co-workers.[1] These natural products belong to the broad family of matrine-type alkaloids and are the first family representatives with an open-loop ring D and an unsaturated ring C (Figure ). Flavesines A–F (1–10) exhibit antiviral activities against hepatitis B virus in comparable potencies as the parent natural product—matrine (11).[1,2] Several studies have shown that matrine-type alkaloids are the main components responsible for the various biological activities of the roots of S. flavescens(3) extensively used in traditional Chinese medicine for the treatment of pruritus, eczema, dysentery, pyogenic infections of the skin, and trichomonas vaginitis.[4] More recent reports also reveal an antitumor,[5] antiviral,[6] antibacterial,[7] and anti-inflammatory[8] properties of the matrine-type alkaloids.

Figure 1

Flavesines A–J (1–10) and matrine (11).

During our studies on natural product analogues as new leads in anticancer drug discovery, we identified flavesine G/J core structure as a promising molecular scaffold for further development because structurally similar matrine analogues have shown considerable cytotoxicity in various cancer cell lines.[9] Furthermore, the total syntheses of flavesine subfamily alkaloids have not been reported to date. Herein, we report the first protecting-group-free total synthesis of flavesines G (7) and J (10) as well as the cytotoxicity data of these natural products.

Results and Discussion

The total syntheses of matrine-type alkaloids are usually conducted from linear precursors by stepwise cyclization of A–D rings.[10] In contrast, our approach was based on a functionalization of another natural product 9-azajulolidine (12) by introducing the necessary side chain (Scheme ).

Scheme 1

Total Synthesis of Flavesines G (7) and J (10)

The total synthesis of flavesines G (7) and J (10) was initiated with a lithiation/halogenation sequence developed by Fort and co-workers.[11] A treatment of 9-azajulolidine (12) with BuLi–LiDMAE reagent followed by quenching of the resulting lithiated species with iodine smoothly furnished the halogenated dimethylaminopyridin analogue 13. Next, a Sonogashira coupling of 13 with homopropargylic alcohol enabled an efficient introduction of the C4 side chain. In the next step, the alkyne triple bond was saturated, and the alcohol moiety was subsequently oxidized by employing the CrO3/HIO4 mixture. Depending on the final chromatographic purification conditions, flavesine G was isolated in either a nonionic (7) or zwitterionic form (16). By using the amine-containing mobile phase, flavesine G was obtained in a zwitterionic form (16) with a characteristic carbonyl shift of 181.2 ppm and a C15 proton resonance at 2.22 ppm. In contrast, when the acid-containing mobile phase was used, flavesine G was isolated in a nonionic form (7) with a carbonyl shift of 176.3 ppm and a C15 proton shift at 2.43 ppm, respectively. The NMR spectrum of flavesine G in its zwitterionic form is in good agreement with the literature, suggesting that it was originally isolated in its zwitterionic form (16) not the nonionic form as initially proposed. Finally, flavesine J (10) was synthesized by a peptide coupling of flavesine G (16) with piperidine. Also, in this case, the analytical data of the synthetic sample were in good agreement with literature values, and the designed synthetic route allowed obtaining sufficient amounts of the target natural products for further cytotoxicity evaluation.

The cytotoxicity of flavesines G (16) and J (10) was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in six commonly used cancer cell lines: HeLa, A549, MCF-7, HepG2, PANC-1, and HL-60 following a conventional testing protocol (Table ). Cytotoxicity was represented as EC50 values, the concentration at which 50% of the cells were killed.

Table 1

Cytotoxicity of Flavesines G (7) and J (10) on Various Cancer Cell Lines

	EC50 (μM)
compound	HeLa	A549	MCF-7	HepG2	PANC-1	HL-60
doxorubicina	3.1 ± 0.3	0.35 ± 0.06	1.1 ± 0.3	0.92 ± 0.21	0.72 ± 0.08	0.032 ± 0.008
matrine (11)	>4000	>4000	4000 ± 153	2396 ± 75	2479 ± 133	1972 ± 85
flavesine G (16)	>4000	2988 ± 112	>4000	3192 ± 112	4000 ± 124	1134 ± 46
flavesine J (10)	>4000	1515 ± 55	2477 ± 85	249 ± 12	621 ± 32	204 ± 10

Positive control.

The cytotoxicity studies revealed that flavesine J (10) is considerably more potent than flavesine G (16) and the parent natural product matrine (11). Flavesine G (16) exhibited weak cytotoxicity against A549, HepG2, PANC-1, and HL-60 cell lines with the EC50 values ranging from 4000 to 1134 μM; however, it turned out to be inactive against HeLa and MCF-7 cell lines (EC50 > 4000 μM). In contrast, flavesine J (10) showed cytotoxicity on A549, MCF-7, HepG2, PANC-1, and HL-60 cell lines with the EC50 values ranging from 2477 down to 204 μM, being inactive only in HeLa cell lines.

Conclusions

In summary, the first total synthesis of flavesines G (16) and J (10) was achieved starting from another natural product 9-azajulolidine (12). The synthesis was performed in a protecting-group-free fashion and features a lithiation/iodination sequence, followed by Sonogashira coupling for efficient installation of the side chain. Both natural products exhibit micromolar cytotoxicity on A549, MCF-7, HepG2, PANC-1, and HL-60 cancer cell lines with flavesine J (10) being considerably more potent than flavesine G (16).

Experimental Section

General Experimental Details

All reactions were performed under an atmosphere of argon unless otherwise indicated. Reagents and starting materials were obtained from commercial sources and used as received. The solvents were purified and dried by standard procedures prior to use; petroleum ether of boiling range 60–80 °C was used. Flash chromatography was carried out using Merck Kieselgel (230–400 mesh). NMR spectra were recorded on Varian Mercury (400 MHz) and Bruker (300 MHz) spectrometers. Chemical shift values are referenced against residual protons in the deuterated solvents, and multiplicity is given as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. Infrared spectra were recorded in the range of 4000–500 cm–1 as a film. High-resolution mass spectrometry (HRMS) spectra were recorded on a Micromass AutoSpec Ultima Magnetic sector mass spectrometer. Melting points were determined using a Stanford Research System MPA100 Automated Melting Point Apparatus and are uncorrected.

1-Iodo-5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-ij][1,6]naphthyridine (13)

A solution of freshly distilled 2-(dimethylamino)ethanol (0.56 mL, 5.567 mmol, 2.0 equiv) in dry hexane (8 mL) was cooled in an ice bath under a nitrogen atmosphere, and BuLi (2.5 M) in hexane (4.45 mL, 11.134 mmol, 4.0 equiv) was added dropwise. After 45 min of stirring at 0 °C, 9-azajulolidine (0.50 g, 2.783 mmol, 1.0 equiv) was added in one portion. After 1 h of stirring at 0 °C, the reaction mixture was cooled at −78 °C, and a solution of I2 (1.766 g, 6.959 mmol, 2.5 equiv) in hexane/toluene mixture (1:1, v/v, 35 mL) was added dropwise over 1 h. The reaction mixture was warmed to the ambient temperature overnight. The volatiles were evaporated in vacuo, and the residue was partitioned between Et2O and water at 0 °C. The organic layer was separated, and the aqueous phase was extracted with Et2O (4 × 20 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure to give the title compound as a brown solid (0.70 g, 84%). 1H NMR (CDCl3, 300 MHz): δ 7.54 (s, 1H), 3.23 (dt, J = 11.1, 5.7 Hz, 4H), 2.64 (q, J = 5.5, 4.8 Hz, 4H), 1.94 (dq, J = 11.8, 5.9 Hz, 4H); 13C NMR (CDCl3, 100 MHz): δ 148.6, 146.4, 122.5, 118.6, 116.0, 49.4, 31.9, 24.2, 21.3, 20.7; IR (neat): 2932, 2854, 1646, 1575, 1511, 1388, 1327, 1244, 1073, 1039, 899, 32; HRMS (ESI+) m/z: 301.0208 [M + H]+ (calcd for C11H14N2I, 301.0202); Rf = 0.58, 95:5 (v/v) MeOH/Et3N, mp 127–128 °C.

4-(5,6,9,10-Tetrahydro-4H,8H-pyrido[3,2,1-ij][1,6]naphthyridin-1-yl)but-3-yn-1-ol (14)

A mixture containing 13 (0.396 g, 1.31 mmol, 1.0 equiv), 3-butyn-1-ol (0.149 mL, 1.97 mmol, 1.5 equiv), Pd(PPh3)2Cl (46.30 mg, 0.066 mmol, 5 mol %), and CuI (25.0 mg, 0.132 mmol, 10 mol %) in dry dimethylformamide (DMF) (5 mL) was heated for 10 min at 60 °C under the argon atmosphere, and then Et3N (0.735 mL, 5.277 mmol, 4.00 equiv) was added. The reaction mixture was stirred for further 30 min at the same temperature and then cooled to room temperature (rt). After removal of DMF under reduced pressure, the residue was diluted with DCM (10 mL) and filtered through the Celite pad. The filtrate was poured into H2O (5 mL), and the aqueous layer was extracted with DCM (3 × 10 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography using EtOAc/MeOH (9:1, v/v) as an eluent to furnish the title compound 14 as a yellow solid (239 mg, 74%): 1H NMR (CDCl3, 400 MHz): δ 7.61 (s, 1H), 3.92 (t, J = 5.6 Hz, 2H), 3.47 (q, J = 6.7 Hz, 4H), 2.85 (t, J = 6.4 Hz, 2H), 2.77–2.70 (m, 4H), 2.05–1.98 (m, 4H); 13C NMR (CDCl3, 100 MHz): δ 152.3, 134.6, 129.4, 118.1, 115.0, 103.6, 72.3, 59.3, 50.3, 50.0, 24.6, 24.2, 23.7, 19.6, 19.4; IR (neat): 3400, 2924, 2854, 2236, 1627, 1554, 1456, 1324, 1042; HRMS (ESI+) m/z: 243.1499 [M + H]+ (cald for C15H19N2O, 243.1497); Rf 0.43, 95:5 (v/v) MeOH/Et3N, mp 198–199 °C.

4-(5,6,9,10-Tetrahydro-4H,8H-pyrido[3,2,1-ij][1,6]naphthyridin-1-yl)butan-1-ol (15)

A mixture containing 14 (1.16 g, 4.781 mmol, 1.0 equiv), MeOH (18 mL), AcOH (6 mL), and 10% Pd/C (0.382 mg, 3.590 mmol, 0.75 equiv) was stirred under the H2 atmosphere (5 bar) for 72 h at rt. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was taken up in DCM (50 mL) and washed with sat. aq K2CO3, dried over Na2SO4, filtered, and evaporated under reduced pressure to furnish the title compound 15 as a thick yellowish oil (1.09 g, 92%): 1H NMR (CDCl3, 400 MHz): δ 7.72 (s, 1H), 3.65 (t, J = 6.1 Hz, 2H), 3.21–3.16 (m, 4H), 2.65 (q, J = 6.9, 6.4 Hz, 6H), 1.94 (dt, J = 12.4, 6.2 Hz, 4H), 1.77 (p, J = 7.0 Hz, 2H), 1.64 (dt, J = 13.6, 6.5 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ 156.3, 148.1, 145.2, 113.8, 112.6, 62.2, 49.7, 48.9, 33.6, 32.3, 24.6, 24.5, 23.2, 21.4; HRMS (ESI+) m/z: 247.1812 [M + H]+ (cald for C15H23N2O, 247.1810); Rf 0.56, 95:5 (v/v) MeOH: Et3N.

Flavesine G (16)

A mixture containing periodic acid (0.420 g, 1.847 mmol, 3.5 equiv), CrO3 (0.00147 g, 0.0147 mmol, 0.28 equiv), and wet MeCN (4.2 mL wetted by adding 0.75% of water, v/v) was vigorously stirred for 1 h to achieve dissolution. This solution was then further added to a mixture containing 15 (0.130 g, 0.527 mmol, 1.0 equiv) and MeCN (6 mL) at rt over 10 min. The resulted mixture was stirred at rt for 2 h. Then, the formed precipitate was filtered off, and the filtrate was evaporated under reduced pressure. The residue was taken up in MeCN (4 mL) and filtered again. The filtrate was evaporated under reduced pressure, and the residue was purified by reversed-phase flush column chromatography using MeCN (0.01% Et2NH): H2O (0.01% Et2NH) (5:95 to 95:5 v/v) as an eluent to furnish flavesine G (16) as pale yellow oil (0.085 g, 62%). 1H NMR (methanol-d4, 400 MHz): δ 7.62 (s, 1H), 3.503–3.42 (m, 4H), 2.80–2.70 (m, 6H), 2.22 (t, J = 7.0 Hz, 2H), 2.03–1.93 (m, 4H), 1.92–1.82 (m, 2H); 13C NMR (methanol-d4, 100 MHz): δ 181.2, 153.8, 149.7, 135.1, 116.8, 115.3, 51.2, 50.6, 37.8, 31.1, 26.3, 25.0, 23.1, 20.8, 20.7; HRMS (ESI+) m/z: 261.1601 [M + H]+ (calcd for C15H21N2O2, 261.1603).

Flavesine J (10)

A vial was charged with flavesine G (16) (0.025 g, 0.096 mmol, 1.0 equiv), piperidine (0.010 mL, 0.105 mmol, 1.10 equiv), EDCI·HCl (0.027 g, 0.144 mmol, 1.50 equiv), HOBT·H2O (0.007 g, 0.048 mmol, 0.50 equiv), and dry DCM (1 mL), and the obtained slurry was stirred for 30 min. Then, N-methylmorpholine (0.010 mL, 0.0964 mmol, 1.0 equiv) was added, and the reaction mixture was stirred for 14 h at rt and then evaporated in vacuo. The residue was partitioned between EtOAc (5 mL) and sat. aq K2CO3 (5 mL). The organic layer was separated, and the aqueous phase was extracted with EtOAc (4 × 5 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by reversed-phase flush column chromatography using MeCN (0.05% formic acid)/H2O (0.05% formic acid) (9:1 v/v) as an eluent to furnish flavesine J (10) as pale yellow oil (76 mg, 55%). 1H NMR (methanol-d4, 400 MHz): δ 7.58 (s, 1H), 3.55–3.51 (m, 2H), 3.47–3.43 (m, 2H), 3.30–3.25 (m, 4H), 2.72–2.62 (m, 6H), 2.42 (t, J = 8.0 Hz, 2H), 1.99–1.91 (m, 4H), 1.88–1.81 (m, 2H), 1.70–1.64 (m, 2H), 1.60–1.50 (m, 4H); 13C NMR (methanol-d4, 100 MHz): δ 173.3, 155.1, 150.6, 143.5, 115.7, 114.5, 50.7, 50.0, 48.0, 43.9, 34.2, 33.6, 27.6, 26.8, 25.9, 25.5, 25.4, 24.0, 22.0, 21.9; HRMS (ESI+) m/z: 328.2378 [M + H]+ (cald for C20H30N3O, 328.2389); Rf 0.56, 95:5 (v/v) MeOH/Et3N.

Cytotoxicity Assay against Cancer Cell Lines

The inhibitory effects of compounds on the proliferation of HeLa (human cervix epitheloid adenocarcinoma ATCCCCL-2), HepG2 (human hepatocyte carcinoma ATCCHB-8065), A549 (human lung carcinoma ATCCCCL-185), PANC-1 (human pancreatic carcinoma ATCCCRL-1469), MCF-7 (human breast adenocarcinoma ATCCHTB-22), and HL-60 (human promyelocytic leukemia ATCCCCL-240) cell lines were evaluated in vitro by the MTT assay. The cancer cell lines HeLa, HepG2, A549, PANC-1, and MCF-7 were cultured using Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, and HL-60 was cultured using Iscove’s modified Dulbecco’s medium supplemented with 20% fetal bovine serum at 37 °C and 5% CO2 with 95% humidity. Cells were seeded into 96-well microtiter plates (2000 cells per well for HeLa, HepG2, A549; 5000 cells per well for PANC-1, 4000 cells per well for MCF-7, and 40,000 cells per well for HL-60) with 100 μL of fresh medium. After 24 h incubation, the tested compounds (100 μL, final concentrations of 1.28, 6.4, 32, 160, 800, or 4000 μM in the culture medium) were added to each well, and continuous culturing was performed for another 48 h. Afterward, 2 mg/mL of MTT (20 μL) was added to each well, which was then cultured for another 3 h under similar conditions. Finally, the medium was removed, and dimethylsulfoxide (200 μL) was added to each well. The survival rate of the cancer cells was evaluated by measuring the optical density (OD) on a microplate reader (TECAN, Infinite M1000) at 540 nm. The OD was calculated using the formula: OD (treated cells) × 100/OD (control cells). The EC50 values were calculated using the programme Graph Pad Prism 3.0.