Research Articles

2017  |  Vol: 3(3)  |  Issue: 3(May-June)
Chemical Modification of Tetrahydroisoquinolines and their Cytotoxic Activity

Еkaterina О. Тerentevaa*Zaynat SKhashimovab, Еlena АTsayaSherzod NZhurakulova, Аbdusalom ShSaidovcValentina IVinogradovaaShakhnoz SAzimovaa

aInstitute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, M. Ulugbek str. 77, 100170 Tashkent, Uzbekistan; 

bInstitute of the Bioorganic Chemistry, Uzbek Academy of Sciences, M. Ulugbek str. 83, 100125 Tashkent, Uzbekistan; 

cSamarkand State University, Str. University Boulevard, 15, 140104 Samarkand, Uzbekistan.

*Corresponding author

Еkaterina О. Тerenteva

Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, M. Ulugbek str. 77, 100170 Tashkent, Uzbekistan


Abstract

Objective: The wide variety of potent biological activities of natural and synthetic isoquinoline alkaloids encouraged us to develop novel cytotoxic isoquinoline compounds. A variety of differently functionalized 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines were synthesized. Materials and methods: We employed classic intramolecular Bischler-Napieralski cyclodehydration to generate the isoquinoline core. All the structures were characterized by nuclear magnetic resonance and mass spectrometry. The cytotoxic activities against three human cancer cell lines and primary culture of healthy hepatocyte cells were evaluated for all the synthesized compounds and structure-activity relationships were established by MTT asay. Results: It was shown that 8a has demonstrated selective cytotoxicity against breast adenocarcinoma (IC50: 43.3 µM), 4d, 3f against laryngeal adenocarcinoma (IC50: 18.0 and 2.3 µM respectively) with the absence of toxicity to healthy cells. Bis compounds exhibit greater cytotoxic effect than mono-series compounds. Among bis samples 8f showed the greatest cytotoxic properties with an IC50 value of 3.1-4.0 µM. The all conjugate compounds completely lacked cytotoxicity toward cancer cell lines in this study. 10 b,d demonstrated proliferative activity. Conclusions: Thus, the most promising compounds for further study in vitro and in vivo methods are dibasic compound 8d, 8e. Later these substances can be offered as a basis for drugs with anti-tumor properties.

Keywords: 1,2,3,4-tetrahydroisoquinoline derivatives, Bischler-Napieralski cyclization, cancer cells, cytotoxicity


Introduction

Isoquinoline alkaloids have a special place among the many nitrogen-containing heterocyclic compounds. This is one of the largest groups of alkaloids. Natural isoquinolines and their numerous derivatives have high activity and they are the subject of attention of medical chemistry (Abe et al, 2005; Galán et al, 2013; Seo et al, 2008). This line of organic chemistry at the present time is one of the leading trends. 

The transformation of natural metabolites and the synthesis of the compounds containing natural pharmacophore groups are used as an additional source of natural drugs.

The subject of our research is directed synthesis of individual representatives of isoquinolines various groups and study of their cytotoxic activity. Using of available homoveratrilamine as the parent compound made it possible to synthesize various ranks of isoquinoline derivatives. 

Materials and methods

IR spectra were recorded from KBr pellets on a System 2000 FTIR instrument (Perkin-Elmer); mass spectra, on a Kratos M90; NMR spectra, on Varian Unity-400+ spectrometer (CDCl3 or MeOH-d4 Py, DMSO-d6 solvents, HMDS internal standard). Melting points of all synthesized compounds were measured on a Boetius microstage.

General Method for Preparing Substituted Tetrahydroisoquinolines

A solution of 3,4-dimethoxyphenylethylamine (1, 5.05 g, 0.028  mol) in C6H6 (30 mL) was treated with substituted aldehydes (2a-f, 0.028 mol) and refluxed with a Dean–Stark trap until water separation was complete (1–2 h). The solvent was distilled off. The resulting imine was treated with CF3CO2H (10 mL), heated on a water bath for ~2 h, cooled, and made basic with NH4OH to pH 9–10. The amine was extracted exhaustively with CHCl3. The crude product was purified by preparing the hydrochloride or by column chromatography over silica gel using CHCl3: MeOH (100:1, 100:10). The resulting product was crystallized from Me2CO.

6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (3а), C11H15O2N.  

Prepared from 1 (5.05 g, 0.028 mol) and formalin (2a, 3 mL, 0.01 mol). Yield 4.84 g (90%), mp of hydrochloride 263–266°C (Me2CO).

1H NMR spectrum (400 MHz, CDCl3, δ, ppm): 1.99(1Н, br. s, NH), 2.65(2Н, t, J = 5.9, Н-4), 3.05(2Н, t, J = 5.9, Н-3), 3.77(3Н, s, 7-ОСН3), 3.78(3Н, s, 6-ОСН3), 3.88(2H, s, H-1), 6.45(1Н, s, Н-8), 6.51(1Н, s, Н-5).

1-Phenyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3b), C17H19O2N.

Prepared from 1 (1.81 g, 0.01 mol) and benzaldehyde (2b, 1.06 g, 0.01 mol). Yield 2.17 g (81%), mp 118–121°C (Me2CO), mp 102–103°C (Me2CO).

1H NMR spectrum (CDCl3, δ, ppm): 2.68 (1H, m, H-4), 2.85 (2H, m, H-3, 4), 3.25 (1H, m, H-3), 3.42* (3H, s, 7-OCH3), 3.67* (3H, s, 6-OCH3), 4.85 (1H, s, H-1), 6.14 (1H, s, H-8), 6.67 (1H, s, H-5), 7.20 (5H, m, Ar-H).

1-(3,4-Dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3c), C19H23O4N.

 Prepared from 1 (1.81 g, 0.01 mol) and veratrylaldehyde (2c, 1.66 g, 0.01 mol). Yield 2.38 g (72%), mp 72–74°C (Me2CO).

1H NMR spectrum (DMSO-d6, δ, ppm): 2.65 and 2.80 (3H, 2m, 2H-4, H-3), 3.24 (1H, m, H-4), 3.44* (3H, s, 7-OCH3), 3.62* (3H, s, 3´-OCH3), 3.66* (6H, s, 6, 4´-OCH3), 4.78 (1H, s, H-1), 6.18 (1H, s, H-8), 6.77 (1H, s, H-5), 6.62, 6.68, 6.82 (each 1H, br.d, H-2´, 5´, 6´).

1-(3,4-Methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3d). C18H19O4N.

Prepared from 1 (1.66 g, 0.009 mol) and 3,4-methylenedioxybenzaldehyde (2d, 1.37 g, 0.009 mol). Yield 2.06 g (72%), mp of hydrochloride 254-257 °C (Me2CO).

1H NMR spectrum (DMSO-d6, δ, ppm): 2.5–3.0 (4H, m, H-3, 4), 3.45* (3H, s, 7-OCH3), 3.66* (3H, s, 6-OCH3), 4.86 (1H, s, H-1), 5.92* (2H, s, 3´-OCH2O-4´), 6.17 (1H, s, H-8), 6.62–6.87 (4H, m, H-5, Ar-H).

1-(6-Bromo-3,4-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3e). C18H18O4NBr.

Prepared from 1 (2.54 g, 0.014 mol) and 6-bromo-3,4-methylenedioxybenzaldehyde (2e, 3.21 g, 0.014 mol). Yield 4.95 g (90%), mp 201–203°C (Me2CO).

Mass spectrum (70 eV) m/z (Irel,%): 393/391 (M+, 100, Br-79/81), 379/377 (24, Br-79/81), 363/361 (14, Br-79/81), 193 (22), 192 (65).

1H NMR (DMSO-d6, δ, ppm): 2.98 and 3.05 (2H, m, H-4), 3.31 and 3.43 (2H, m, H-3), 3.53* (3H, s, 7-OCH3), 3.73* (3H, s, 6-OCH3), 5.78 (1H, s, H-1), 6.07* (2H, s, 3´-OCH2O-4´), 6.15 (1H, s, H-8), 6.65 (1H, s, H-5), 6.82 (1H, s, H-2´), 7.30 (1H, s, H-5´).

1-(6-Chloro-3,4-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3f). C18H18O4NCl.

Prepared from 1 (2.2 g, 0.012 mol) and 6-chloro-3,4-methylenedioxybenzaldehyde (2f, 2.24 g, 0.012 mol). Yield 1.79 g (42%), mp 107–108°C (Me2CO).

1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 2.8–2.85 (2H, ddd, J = 16.4, 6.2, 5.5, H-4), 3.03–3.11 (2H, dt, J = 12.1, 6.2, 6.2, H-3), 3.71* (3H, s, 7-OCH3), 3.88* (3H, s, 6-OCH3), 5.48 (1H, s, H-1), 5.92* (1H, d, J = 0.4, 3´-OCH2O-4´), 5.93* (1H, d, J = 0.4, 3´-OCH2O-4´), 6.27 (1H, s, H-8), 6.44 (1H, s, H-5), 6.63 (1H, s, H-2´), 6.88 (1H, s, H-5´).

General Method for Preparing Alkaloids 4c, 4d.

 A solution of 1-(3´,4´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (1.0 g, 0.003 mol) in MeOH (20 mL) was treated with formalin (0.08 mL, 0.003 mol, 27%), refluxed for 1.5 h (TLC monitoring), cooled in ice, and reduced by NaBH4 (1 g). The solvent was distilled off. The solid was dissolved in H2O (10 mL) and extracted exhaustively with CHCl3. The crude product was purified by producing the hydrochloride or by chromatography over SiO2 using CHCl3: MeOH (100:1           100:10). The product was crystallized from Me2CO or MeOH.

1-(3, 4-Dimethoxyphenyl)-2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (4c,  cryptostylin II). C20H22O4N.

Prepared from 1-(3´,4´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3c, 1.0 g, 0.003 mol) and formalin (0.08 mL, 0.003 mol, 27%). Yield 0.84 g (81%), mp of hydrochloride 206–209°C (Me2CO).

NMR spectral data corresponded to those for cryptostylin II.

1-(3, 4-Methylenedioxyphenyl)-2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (4d, cryptostylin I). C19H21O4N.

Prepared from 1-(3´,4´-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3d, 0.7 g, 0.002 mol) and formalin (0.06 mL, 0.002 mol, 27%). Yield 0.47 g (64%), mp 115–117°C (MeOH).

NMR spectral data corresponded to those for cryptostylin I  (Zhurakulov et al, 2013).

General Method for Preparation of Hydroxyethyl Derivatives of 1-(Aryl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 5 c-f.

A solution of 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3 c-f, 0.0015 mol) in EtOH (30 mL) was treated with ethylenechlorohydrin (0.0045 mol) and K2CO3 (0.0045 mol) and refluxed for 4–6 h (TLC monitoring). The mixture was worked up analogously as described above.

1-(3,4-Dimethoxyphenyl)-2β-hydroxyethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (5c). C21H27NO5.

Prepared as the hydrochloride from 3c (0.5 g, 0.0015 mol) and ethylenechlorohydrin (0.3 mL 0.0045 mol). Yield 0.35 g (62%), mp of hydrochloride 201–203°C (Me2CO).

Prepared as the hydrochloride from 3c (1.98 g, 0.006 mol), ethylenechlorohydrin (1.1 mL, 0.016 mol), and KOH (0.68 g, 0.012 mol). Yield 2.04 g (91%).

IR spectrum (KBr, νmax,cm-1): 3265 (OH), 2944, 2585, 1609 (C=C), 1523 (Ar–H), 1466 (CH2), 1264, 1241 (C–O) 1162 (C–N).

1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 2.63 (1H, m, H-9), 2.71 (3H, m, H-4, 9), 3.11 and 3.34 (each 1H, m, H-3), 3.57 (1H, s, 7-OCH3), 3.55 and 3.70 (each 1H, m, H-10), 3.77* (3H, s, 3´-OCH3), 3.80* (3H, s, 6-OCH3), 3.81* (3H, s, 4´-OCH3), 4.69 (1H, s, H-1), 6.17 (1I, s, H-8), 6.57 (1H, s, H-5), 6.72 (1H, dd, J = 2, 8, H-6´), 6.75 (1H, d, J = 8, H-5´), 6.84 (1H, br. signal, H-2´).

1-(3,4-Methylenedioxyphenyl)-2b-hydroxyethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (5d). C20H23NO5.

Prepared as the hydrochloride from 3d (1.5 g, 0.005 mol), ethylenechlorohydrin (0.9 mL, 0.013 mol), and K2CO3 (0.8 g, 0.006 mol). Yield 1.34 g (78%), mp of hydrochloride 149–152°C (Me2CO).

1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 2.56 (1H, dt, J = 4.4, 4.4, 12.9, He-9), 2.67 (1H, ddd, J = 4.6, 9.0, 12.0, Ha-3), 2.77 (1H, ddd, J = 5.4, 9.0, 16.0, He-4), 2.90 (1H, ddd, J = 4.4, 8.3, 12.9, Ha-9), 2.96 (1H, dt, J = 5.3, 5.3, 16.0, Ha-4), 3.19 (1H, dt, J = 5.5, 5.5, 12.0, He-3), 3.48 (1H, ddd, J = 4.4, 9.4, 11.1, He-10), 3.58 (3H, s, 7-OCH3), 3.64 (1H, ddd, J = 4.4, 8.3, 11.1, Ha-10), 3.78 (3H, s, 6-OCH3), 4.57 (1H, s, H-1), 5.85, 5.86 (each 1H, d, J = 1.5, 3´-OCH2O-4´), 6.17 (1H, s, H-8), 6.55 (1H, s, H-5), 6.63 (1H, s, H-6´), 6.67 (1H, s, H-5´), 6.67 (1H, s, H-2´).

1-(6-Bromo-3,4-methylenedioxyphenyl)-2β-hydroxyethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (5e). C20H22BrNO5.

Prepared from 3e (1.5 g, 0.004 mol), ethylenechlorohydrin (0.78 mL, 0.012 mol), and K2CO3 (0.8 g, 0.006 mol). Yield 1.17 g (77%), mp 143–144°C (MeOH).

IR spectrum (KBr, νmax, cm-1): 3425 (OH), 1610 (C=C), 1515 (Ar–H), 1476 (CH2), 1251, 1225 (C–O), 1131 (C–N), 1040 (C–Br), 939, 928 (O–CH2–O), 859, 804.

1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 2.47 (1H, dt, J = 3.5, 3.5, 12.7, He-9), 2.58 (1H, ddd, J = 4.0, 9.5, 12.0, Ha-3), 2.69 (1H, dt, J = 4.6, 4.2, 16.0, He-4), 2.76 (1H, ddd, J = 4.8, 9.8, 12.8, Ha-9), 2.92 (1H, ddd, J = 4.6, 9.5, 15.5, Ha-4), 3.16 (1H, dt, J = 4.5, 4.5, 11.8, He-3), 3.39 (1H, ddd, J = 3.6, 4.8, 11.0, He-10), 3.61 (3H, s, 7-OCH3), 3.64 (1H, ddd, J = 3.5, 9.8, 11.0, Ha-10), 3.79 (3H, s, 6-OCH3), 4.99 (1H, s, H-1), 5.86 (2H, s, 3´-OCH2O-4´), 6.16 (1H, s, H-8), 6.50 (1H, s, H-2´), 6.54 (1H, s, H-5), 6.95 (1H, s, H-5´).

1-(6-Chloro-3,4-methylenedioxyphenyl)-2β-hydroxyethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (5f). C20H22ClNO5.

Prepared from 3f (1.5 g, 0.004 mol), ethylenechlorohydrin (0.9 mL, 0.013 mol), and K2CO3 (0.8 g, 0.006 mol). Yield 1.46 g (87%), mp 157–159°C (MeOH).

IR spectrum (KBr, νmax, cm-1): 3503 (OH), 1613 (C=C), 1505 (Ar–H), 1474 (CH2), 1248, 1225 (C–O), 1128 (C–N), 1033 (C–Cl), 929 (O–CH2–O), 877, 859.

IR spectrum in vaseline oil (νmax, cm-1): 3501 (OH free), 3159 (intramolecular H-bond), 1611 (C=C), 1499 (Ar–H), 1306, 1222 (C–O), 1127 (C–N), 1032 (C–Cl), 928 (O–CH2–O).

1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 2.49 (1H, dt, J = 3.7, 13.0, He-9), 2.58 (1H, ddd, J = 4.1, 9.3, 12,1, Ha-3), 2.70 (1H, dt, J = 4.1, 5.1, 15.7, He-4), 2.73 (1H, ddd, J = 4.9, 9.6, 13.0, Ha-9), 2.89 (1H, ddd, J = 5.1, 9.3, 15.7, Ha-4), 3.15 (1H, dt, J = 5.1, 5.1, 12.1, He-3), 3.41 (1H, ddd, J = 3.7, 4.9, 11.1, He-10), 3.61 (3H, s, 7-OCH3), 3.63 (1H, ddd, J = 3.7, 9.6, 11.1, Ha-10), 3.79 (3H, s, 6-OCH3), 5.02 (1H, s, H-1), 5.86 (2H, s, 3´-OCH2O-4´), 6.14 (1H, s, H-8), 6.47 (1H, s, H-2´), 6.54 (1H, s, H-5), 6.78 (1H, s, H-5´).

N-(3, 4-Dimethoxy-β-phenethyl)heptanamide (7a). C17H27NO3.

A mixture of homoveratrylamine (1, 1.84 g, 0.01 mol) and heptanoic acid (6a, acid 7:0, 1.32 g, 0.01 mol). in MeOH (5 mL) underwent spontaneous heating. The mixture was heated on an oil bath for 2 h at 178°C, treated with CHCl3 (100 mL), and washed with HCl solution (3%), NaOH solution (2%), and H2O until neutral. The CHCl3 was evaporated. The residue was crystallized from Me2CO or hexane. The resulting crystals were filtered off. Yield 84% (2.47 g), mp 55–57°C (hexane).

IR spectrum (KBr, νmax, cm-1): 3310 (NH), 2933 (Ar-CH), 1645 (N-C=O).

1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 0.81 (3H, t, J = 7.3, CH3), 1.21 (6H, br. s, 3CH2), 1.52 (2H, m, H-2´), 2.05 (2H, t, J = 7.3, H-1´), 2.70 (2H, t, J = 7, Hα), 3.43 (2H, q, J = 7, Hβ), 3.795 (3H, s, OCH3), 3.801 (3H, s, OCH3), 5.45 (1H, m, NH), 6.65 (1H, H-2), 6.67 (1H, H-6), 6.74 (1H, H-5).

1-Hexyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (8a). C17H27NO2.

A mixture amide 7a (1 g, 3.4 mmol) anhydrous benzene (30 mL), and POCl3 (0.7 mL) was refluxed for 1 h. The course of the reaction was monitored by TLC. The benzene and POCl3 were distilled off. The residue was dissolved in MeOH (30 mL) and treated in portions at 0–5°C with NaBH4 (0.02 mol). The MeOH was distilled off. The residue was dissolved in H2O and extracted with CHCl3. The CHCl3 was removed. Isoquinoline 8a were crystallized from Me2CO. Yield 74% (0.7 g), mp of hydrochloride 188–191°C (Me2CO).

IR spectrum (KBr, νmax, cm-1): 3450, 2930, 1611, 1519, 1450, 1264.

1H NMR spectrum (400 MHz, CD3OD, δ, ppm, J/Hz): 0.86 (3H, m, J = 6, CH3), 1.30 (6H, d, J = 6, 3CH2), 1.38 and 1.44 (each 1H, m, CH2), 1.84 and 2.05 (each 1H, m, H-1´), 2.96 (2H, m, H-4), 3.47 (2H, q, J = 6, H-3), 3.76 (6H, s, 2OCH3), 4,40 (1H, m, H-1), 6.74 (2H, s, H-5, 8).

General Method for Preparing Diamides 7bh.

A mixture of homoveratrylamine (1, 0.02 mol) and dibasic acid (0.01 mol) was dissolved in MeOH (10 mL). The mixture underwent self-heating. The MeOH was distilled off. The resulting salt was heated on an oil bath for 1–2 h at 175–178°C, dissolved in CHCl3 (100 mL), and washed with HCl solution (3%), NaOH solution (2%), and H2O until neutral. The CHCl3 was distilled off. The solid was crystallized from Me2CO. The resulting crystals were filtered off.

N,N-(3,4-Dimethoxy-β-phenethyl)-adipoyl diamide (7b), C26H36N2O6, was prepared from 1 (4.6 g, 0.03 mol) and adipic acid (1.95 g, 0.013 mol). Yield 77% (4.64 g), mp 173–175°C (Me2CO).

IR spectrum (KBr, νmax, cm-1): 3313 (NH), 1634 (CO). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.54 (4H, m, H-3, 4), 2.07 (4H, m, H-2, 5), 2.69 (4H, t, J = 6.95, H-α), 3.42 (4H, q, J = 7.14, H-β), 3.791 (6H, s, OCH3), 3.797 (6H, s, OCH3), 5.66 (2H, t, J = 5.6, NH), 6.65 (2H, d, J = 1.9, H-2), 6.66 (2H, dd, J = 8.7, 1.9, H-6), 6.74 (2H, d, J = 8.7, H-5).

N,N-(3,4-Dimethoxy-β-phenylethyl)-pimeloyl diamide (7c), C27H38N2O6, was prepared from 1 (1.87 g, 0.01 mol) and pimelic acid (0.9 g, 0.005 mol). Yield 62.4% (1.56 g), mp 138–141°C (Me2CO).

IR spectrum (KBr, νmax, cm-1): 3307 (NH), 1643 (CO). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.20 (2H, m, H-4), 1.52 (4H, m, H-3´, 5´), 2.04 (4H, t, J = 7.5, H-2´, 6´), 2.70 (4H, t, J = 7.0, H-α), 3.43 (4H, q, J = 7.0, H-β), 3.793 (6H, s, OCH3), 3.796 (6H, s, OCH3), 5.51 (2H, br.t, J = 5.6, NH), 6.65 (2H, d, J = 2, H-2), 6.66 (2H, dd, J = 2, 8.8, H-6), 6.74 (2H, d, J = 8.8, H-5).

N,N-(3,4-Dimethoxy-β-phenethyl)-azelaoyl diamide (7d), C29H42N2O6, was prepared from 1 (1.58 g, 0.009 mol) and azelaic acid (0.9 g, 0.005 mol). Yield 66% (1.48 g), mp 141–145°C (Me2CO).

IR spectrum (KBr, νmax, cm-1): 3306 (NH), 1642 (CO). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.20 (6H, s, H-4´, 5´, 6´), 1.51 (4H, t, J = 6.7, H-3´, 7´), 2.04 (4H, t, J = 7.5, H-2´, 8´), 2.69 (4H, t, J = 6.96, H-α), 3.43 (4H, q, J = 6.4, H-β), 3.79 (12H, s, OCH3), 5.52 (2H, t, NH), 6.65 (2H, s, H-2), 6.64 (2H, d, J = 2, 8.5, H-6), 6.74 (2H, d, J = 8.5, H-5).

N,N-(3,4-Dimethoxy-β-phenethyl)-sebacoyl diamide (7e), C30H44N2O6, was prepared from 1 (1.59 g, 0.009 mol) and sebacic acid (0.98 g, 0.004 mol). Yield 65% (1.5 g), mp 152–156°C (Me2CO). IR spectrum (KBr, νmax, cm-1): 3310 (NH), 1639 (CO). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.182 (8H, s, H-4´, 5´, 6´, 7´), 1.503 (4H, t, J = 6.9, H-3´, 8´), 2.04 (4H, t, J = 7.4, H-2´, 9´), 2.68 (4H, t, J = 6.9, H-α), 3.41 (4H, q, J = 6.8, H-β), 3.78 (12H, s, OCH3), 5.59 (2H, t, J = 5.5, NH), 6.64 (2H, s, H-2), 6.65 (2H, d, J = 8.5, H-6), 6.73 (2H, d, J = 8.5, H-5).

N,N-bis(3,4-Dimethoxy-β-phenethyl)-brassyloyl diamide (7f), C33H50N2O6, was prepared from 1 (0.8 g, 4.4 mmol)  and brassylic acid (0.58 g, 2.4 mmol). Yield 91% (1.15 g), mp 160-162C (Me2CO). IR spectrum (film, v, см-1): 3304 (NH), 2919, 2850 (Ar-C), 1639 (N –C=О), 1547, 1519, 1470 (С=С). Спектр ЯМР 1Н (400 PMR spectrum (400 MHz, СD3OD, δ, ppm, J/Hz): 0.63 (2H, m, H-7); 1.22 (10H, br. s, H-4,5,6,8,9,10); 1.50 (4H, t, J=7, H-3,11); 2.07 (4H, t, J=7.6, H-2,12); 2.67 (4H, t, J=7, H-α); 3.32 (4H, kv, J=7, H-β); 3.73 (6H, s, OCH3); 3.76 (6H, s, OCH3); 6.68 (2H, dd, J=2, 8, H-6); 6.76 (2H, d, J=2, H-2); 6.79 (2H, d, J=8, H-5).

N1,N13-bis(3,4-Methylenedioxy-β-phenethyl)-brassyloyl diamide  (7g), C31H42N2O6. was prepared from 1 (0.9 g, 5.4 mmol)   and brassylic acid (0.6 g 2.4 mmol). Yield 69% (0.9g), mp. 172-174° C (Me2CO). IR spectrum (film, v, см-1): 3430, 3305 (NH), 2922, 2849 (Ar-C), 1637 (N -C=О), 1543, 1503, 1491 (С=С). 1H NMR spectrum (400 MHz, СD3OD, δ, ppm, J/Hz):  1.18 (14H, br. s, H-4′′,5′′,6′′,7′′,8′′,9′′,10′′); 1.51 (4H, t, J=7.4, H-3′′,11′′); 2.04 (4H, t, J=7.8, H-2′′,12′′); 2.65 (4H, t, J=6.8, H-α); 3.39 (4H, kv, J=6.7, H-β); 5.37 (2H, br. s, NH); 5.86 (4Н, s, ОСН2О); 6.55 (2H, dd, J=1.5, 7.8, H-6, 6); 6.61 (2H, d, J=1.5, H-2, 2); 6.67 (2H, d, J=7.8, H-5,5).

N,N-bis(3,4-Dimethoxy-β-phenethyl)isophtalamide (7h), C28H32N2O6. Method A. A mixture of homoveratrylamine (1, 3.06 g, 0.017 mol) and isophthalic acid (1.5 g, 0.009 mol) dissolved in MeOH (5 mL) was heated on an oil bath at 170–180°C for 2–5 h, dissolved in CHCl3 (100 mL), and washed with HCl solution (3%), NaOH solution (2%), and H2O until neutral. The CHCl3 was distilled off. Yield 29% (1.2 g), mp 132–134°C (Me2CO).

Method B. A mixture of homoveratrylamine (2.1 g, 0.011 mol) CHCl3 (30 mL), and NaOH solution (30 mL, 10%) was treated dropwise over 1 h with a solution of and isophthalic acid chloride (1.2 g, 0.006 mol). in C6H6 (10 mL) at 5°C. The resulting precipitate (7h) was separated and crystallized from Me2CO The resulting crystals were filtered off.Yield 82% (2.33 g).

IR spectrum (KBr, νmax, cm-1): 3316 (NH), 2938 (Ar-C), 1671, 1634 (N-C=O), 1542, 1516 (Ar-H). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 2.82 (4H, t, J = 7, Hα, α´), 3.64 (4H, q, J = 7, Hβ, β´), 3.78 (6H, s, 3, 3´-OCH3), 3.80 (6H, s, 4, 4´-OCH3,), 6.18 (2H, m, NH), 6.68 (2H, d, J = 2, H-2, 2´), 6.69 (2H, dd, J = 2, 8, H-6, 6´), 6.76 (2H, d, J = 8, H-5, 5´), 7.40 (1H, t, J = 7.7, H-5´´), 7.75 (2H, dd, J = 2, 8, H-6´´, 4´´), 8.01 (1H, t, J = 2, H-2´´).

General Method for Preparing bis-Tetrahydroisoquinolines 8bg. A mixture of dibasic acid amide (0.006 mol), anhydrous benzene (30 mL), and POCl3 (0.05 mol) was refluxed for 2 h. The course of the reaction was monitored by TLC. Benzene and POCl3 were distilled off. The residue was dissolved in MeOH (30 mL). The resulting solution was cooled to 0–5°C and treated in portions with NaBH4 (0.02 mol). The MeOH was distilled off. The residue was dissolved in H2O and extracted with CHCl3. The CHCl3 was removed. The solid was crystallized from Me2CO.

1,4-bis(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)butane (8b), C26H36N2O4, was prepared from 7b (3 g, 0.006 mol) and POCl3 (7 mL). Yield 76% (2.09 g), mp 112–116°C (Me2CO). IR spectrum (KBr, νmax, cm-1): 3340 (NH), 1610, 1519, 1464. 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.38–1.52 (4H, m, H-3´, 4´), 1.65–1.78 (4H, m, H-2´, 5´), 2.60 (2H, m, H-4), 2.66 (2H, m, H-4), 2.89 (2H, m, H-3), 3.15 (2H, m, H-3), 3.776 (6H, s, OCH3), 3.780 (6H, s, OCH3), 3.82 (2H, m, H-1), 6.50* (2H, s, H-8), 6.54* (2H, s, H-5).

1,5-bis(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)pentane (8c), C27H38N2O4, was prepared from 7c (0.5 g, 0.001 mol) and POCl3 (1.5 mL). Yield 80% (0.39 g), mp of dihydrochloride 220–224°C (Me2CO). IR spectrum (KBr, νmax, cm-1): 3423 (NH), 1613, 1519, 1460. PMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.35–1.50 (6H, 2m, H-3´, 4´, 5´), 1.65–1.75 (4H, m, H-2´, 5´), 2.60 (2H, m, H-4), 2.68 (2H, m, H-4), 2.90 (2H, m, H-3), 3.16 (2H, m, H-3), 3.770 (6H, s, OCH3), 3.774 (6H, s, OCH3), 3.86 (2H, m, H-1), 6.49* (2H, s, H-8), 6.53* (2H, s, H-5).

1,7-bis-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)heptane (8d), C29H42N2O4, was prepared from 7d (0.5 g, 0.001 mol) and POCl3 (1.5 mL). Yield 67.4% (0.31 g), mp of dihydrochloride 163–166°C (Me2CO). IR spectrum (KBr, νmax, cm-1): 3429 (NH), 1613, 1519, 1464. 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.3–1.5 (10H, 2m, H-3´, 4´, 5´, 6´, 7´), 1.65–1.85 (4H, m, H-2´, 8´), 2.65 (2H, m, H-4), 2.80 (2H, m, H-4), 2.95 (2H, m, H-3), 3.25 (2H, m, H-3), 3.78 (6H, s, OCH3), 3.77 (6H, s, OCH3), 3.98 (2H, m, H-1), 4.67 (2H, s, NH), 6.50* (2H, s, H-8), 6.53* (2H, s, H-5).

1,8-bis(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)octane (8e), C30H44N2O4, was prepared from 7e (0.5 g, 0.001 mol) and POCl3 (1.5 mL). Yield 70% (0.33 g), mp of dihydrochloride 240–244°C (Me2CO). 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.10–1.24 (12H, m, H-3´, 4´, 5´, 6´, 7´, 8´), 1.6–1.8 (4H, m, H-2´, 9´), 2.55–2.75 (4H, m, H-4), 2.90 (2H, m, H-3), 3.15 (2H, m, H-3), 3.776 (6H, s, OCH3), 3.783 (6H, s, OCH3), 3.8–3.9 (2H, m, H-1), 6.50* (2H, s, H-8), 6.54* (2H, s, H-5).

1,11-bis(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)undecan (8f) C30H44N2O4 was prepared from 7f (0.5 g, 0.88 mmol) and POCl3 (1.5 mL). Yield 87 % (0.41 g), Rf 0.44, mp 208-211C (Me2CO). IR spectrum (KBr, νmax, cm-1): 3456, 2929, 2854, 2786, 1613, 1519, 1463. 1H NMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.25-1.37 (14H, m, H-4,5,6,7,8,9,10); 1.46 (4H, m, H-3,11); 1.88 (4H, m, H-2,12); 2.86-3.05 (4H, m, H-4); 3.25, 3.48 (each 2H, m, H-3); 3.79 (12H, s, OCH3); 4.33 (2H, t, J=6, H-1); 6.57*(2H, s, H-8); 6.59*(2H, s, H-5).

1,11-bis-(6,7-Methylenedioxy-1,2,3,4-tetrahydroisoquinolin-1-yl)undecan (8g), C31H42N2O4. was prepared from 7g (0.6 g, 1.1 mmol) and POCl3 (1.5 mL). Yield 97 % (0.548), mp 115-117°C (Me2CO), IR spectrum (KBr, νmax, cm-1): 3431, 3314, 2923, 2851, 1507, 1481. PMR spectrum (400 MHz, CDCl3, δ, ppm, J/Hz): 1.20 (18H, br. s, H-3′′,4′′,5′′,6′′,7′′,8′′,9′′,10′′,11′′); 1.60-1.69 (4H, m, H-2′′, 12′′); 2.61 (4H, ttt, J=2.3, 5.2, 6.7, 8.6, H-4); 2.86 (2H, dt, J=5.6, 6.4, H-3); 3.12(2H, dt, J=5.4, 6.4, H-3); 3.78 (2H, dt, J=1.9, 8.9, H-1,1); 5.81 (2H, s, ОСН2О); 6.47*(2H, s, H-8,8); 6.54*(2H, s, H-5,5).

1,3-bis-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)benzene (8h), C28H32N2O4. A mixture of diamide 7h (1.2 g, 2.4 mmol), anhydrous 7i C6H6 (30 mL), and POCl3 (7.4 mL) was refluxed for 3 h. The course of the reaction was monitoredby TLC. The C6H6 and POCl3 were distilled off. The residue was dissolved in MeOH (60 mL), cooled to 0–5°C, and treated in portions with NaBH4 (0.05 mol). The MeOH was distilled off. The residue was dissolved in H2O and extracted withCHCl3. The CHCl3 was removed. The residue was crystallized from Me2CO. Yield 71% (0.8 g), mp of hydrochloride 230–233°C (Me2CO). IR spectrum (KBr, v, cm-1): 3376 (NH), 2952 (Ar-C), 1613 (Ar-H), 1519 (Ar-H), 1460 (Ar-H). 1H NMR spectrum (400 MHz, CD3OD, 50°C, δ, ppm, J/Hz): 3.05 (2H, m, H-4). 3.19* (2H, m, H-4), 3.37 (4H, m, H-3), 3.58* (3H, s, 6-OCH3), 3.59* (3H, s, 6-OCH3), 3.79** (6H, s, 7-OCH3), 3.81** (6H, s, 7-OCH3), 5.68 (2H, s, H-1), 6.32 (2H, s, H-8), 6.81 (2H, s, H-5), 7.36–7.38 (2H, d, J = 7.4, H-4′′, 6′′), 7.45 (1H, s, H-2′′), 7.51 (1H, t, J = 7.4, H-5′′).

Reaction of amines with DHQ (9). General Method. A solution of amine (0.667 mmol) in i-PrOH (5 mL) was stirred, treated drop wise over 10 min with a solution of DHQ (0.2 g, 0.667 mmol) in i-PrOH (5 mL), held at 20–25°C for 0.5 h, and treated drop wise with formalin solution (30%, 0.06 mL, 0.667 mmol, d = 1.092). A precipitate began to form immediately. The mixture was left for 12 h. The course of the reaction was monitored by TLC. The precipitate was filtered off and rinsed with i-PrOH, hexane–Et2O (1:1), CHCl3–Et2O (1:1), and CHCl3. The precipitates polymerized in benzene and dioxane. Equimolar amounts of DHQ (0.2 g, 0.667 mmol) and formalin solution (30%, 0.06 mL, 0.667 mmol) were used in the reactions described below.

2-[(3,4-dimetoxyphenethylino)-methyl]-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one (10а).

3,4-dimetoxyphenylethylamine (1a, 0.16 g, 0.88 mmol) afforded DHQ 9, (0.27 g, 0.88 mmol). Yield 0.33 g (75%), mp >360°C (i-PrOH). IR spectrum (KBr, v, см-1):  3431 (ОН), 1645, 1636 (С=О), 1517, 1456 (С=С), 1265 (С-О).

2-[(3,4-Methylenedioxyphenethylino)-methyl]-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one (10b).

3,4-methylenedioxyphenylethylamine (1b, 0.15 g, 0.9 mmol) afforded DHQ 9, (0.27 g, 0.9 mmol). Yield 0.30 g (68%), mp 360°C (i-PrOH). IR spectrum (KBr, vmax, см-1):  3409 (ОН), 1645 (С=О), 1503, 1489, 1446 (С=С), 1364, 1283, 1249 (С-О).

1H NMR spectrum (400 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment: 3.89 (2Н, s, N-СН2), 4.27 (1Н, d, J=11.5, Н-3), 4.92 (1Н, d, J=11.1, Н-2), 5.38 (1Н, s, Н-8), 6.82 (3Н, s, Н-5´, 6´, Н-2´), b) 3,4-methylenedioxyphenylethylamine fragment: 2.69 (1Н, m, На-7´´), 2.80 (1Н, m, На-8´´), 2.90 (1Н, m, Не-7´´), 2.96 (1Н, m, Не-8´´), 5.96 (2Н, s, 3´´-ОСН2О-4´´), 6.69 (3Н, s, Ar-Н);  13С NMR spectrum; (δ, ppm): а) DHQ fragment: 47.65 (СН2–N), 71.29 (С-3), 82.59 (С-2), 95.79 (С-8), 99.97 (С-6), 100.89 (С-8а), 115.15 (С-2'), 115.29 (С-5'), 119.29 (С-6'), 128.91 (C-1'), 145.03 (C-3'), 145.65 (C-4'), 161.64 (C-4а), 161.81 (C-5), 178.05 (C-7), 193.02 (C-4); b) 3,4-methylenedioxyphenylethylamine fragment:  25.57 (С-7´´), 32.10 (С-8´´) 108.42 (10-OCH2O), 109.15 (C-2´´), 121.79 (С-5´´), 131.57 (С-6´´), 145.69 (С-1´´), 147.42 (С-3´´), 147.45 (С-4´´).

2-(Dihydroxyphenyl)-3,5,7-trihydroxy-6-[(1-hydroxy-1phenylpropan-2-il-aminomethyl)methyl]-chroman-4-one (10c).

Psevdoephydryne (1c, 0.11 g, 0.66 mmol) afforded DHQ 9, (0.20 g, 0.66 mmol). Yield 0.16 g (52%), mp 133-137°C (i-PrOH).

IR spectrum (KBr, vmax, см-1):  3430 (ОН), 1643 (С=О), 1456 (С=С), 1365, 1282 (С-О). 1H NMR spectrum (400 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment: 3.60 (2Н, s, N-СН2), 4.48 (1Н, d, J=11.0, Н-3), 4.96 (1Н, d, J=10.7, Н-2), 5.76 (1Н, br. s, 7-ОН or 7´´-ОН), 5.85 (1Н, br. s, Н-8), 6.17 (1Н, s, Н-2´), 6.68 (1Н, s, Н-5´), 7.22 (1Н, s,  6´), 8.97, 9.03 (each 1Н, br. s, 3´, 4´-ОН), 12.34 (1Н, br. s, 5-ОН); b) Psevdoephydryne fragment: 1.91 (1Н, s. Н-8´´), 2.50 (3Н, s, N-СН3), 3.49 (3Н, s, СН3-8´´), 4.94 (1Н, d, J=11.1, Н-7´´), 7.73 (5Н, m, Ar-Н).

2-(Dihydroxyphenyl)-6-[1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2(1H)-yl)methyl]-3,5,7-trihydroxychroman-4-one (10d).

6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3a, 0.13 g, 0.67 mmol) afforded DHQ 9 (0.2 g, 0.67 mmol).  Yield 0.32 г (91 %), mp 187-190 оС (i-PrOH). IR spectrum (KBr, vmax, см-1):  3418 (ОН), 1640 (С=О), 1519, 1452 (С=С), 1262 (С–О). 1H NMR spectrum (400 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment: 3.72 (2Н, resonance overlapped, N-СН2), 4.42 (1Н, d, J=11.0, Н-3), 4.90 (1Н, d, J=11.0, Н-2), 5.74 (1Н, s, Н-8), 6.69 (2Н, s, Н-5´, 6´), 6.82 (1Н, s, Н-2´); b) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 2.73 (2Н, m, Н-4), 2.83 (2H, m, Н-3), 3.63 (3Н, s, 7-ОСН3), 3.65 (3Н, s, 6-ОСН3),  3.80 (2Н, s, Н-1), 6.62 (1Н, s, Н-8), 6.65 (1Н, s, Н-5); 13С NMR spectrum; (δ, ppm): а) DHQ fragment: 48.66 (СН2–N), 71.92 (С-3), 83.39 (С-2), 96.21 (С-8), 99.51 (С-6), 100.21 (С-4а), 115.55 (С-2'), 115.80 (С-5'), 119.81 (С-6'), 128.64 (C-1'), 145.38 (C-3'), 146.18 (C-4'), 160.99 (C-8а), 162.10 (C-5), 171.54 (C-7), 197.40 (C-4); b) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 20.06 (С-4), 24.55 (С-1), 42.78 (C-3), 55.11 (6-ОСН3), 55.14 (7-ОСН3), 111.56 (С-8), 115.40 (C-5), 124.50 (С-8а), 127.85 (С-4а), 145.76 (С-6), 146.89 (С-7).

2-(Dihydroxyphenyl)-)6-[1-(6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2(1H)-yl)methyl]-3,5,7-trihydroxychroman-4-one (10e).

Salsolidyn (3b, 0.25 g, 1.2 mmol) afforded DHQ 9 (0.367 g, 1.2 mmol).  Yield 0.49 г (78 %), mp 198-201 оС (i-PrOH).

IR spectrum (KBr, vmax, см-1):  3418 (ОН), 1639 (С=О), 1519, 1446 (С=С), 1281 (С-О). 1H NMR spectrum (400 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment:  3.89 (2Н, s, N-СН2), 4.41 (1Н, d, J=11.0, Н-3), 4.90 (1Н, d, J=11.0, Н-2), 5.69 (1Н, s, Н-8), 6.68 (2Н, s, Н-5´, 6´), 6.81 (1Н, s, Н-2´), 8.95 (each 1Н, br. s, 3´, 4´-ОН); б) salsolidyn fragment:   1.32 (3Н, d, J=6.6, 1-CH3), 2.58 (1Н, m, На-4), 2.83 (2Н, m, Н е-4, Hа-3), 3.11 (2H, m, Не-3, Н-1), 3.65 (3Н, s, 7-ОСН3), 3.66 (3Н, s, 6-ОСН3), 6.65 (1Н, s, Н-8), 6.66 (1Н, s, Н-5).

2-(3,4-Dihydroxyphenyl)-6-{[1-(3,4-dimethoxyphenyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]methyl}-3,5,7-trihydroxychroman-4-one (10f).

1-(3´,4´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3c, 0.2 g, 0.67 mmol) afforded DHQ (3e, 0.2 g, 0.67 mmol). Yield 0.35 g (84%), mp 174–178°C (i-PrOH). IR spectrum (KBr, vmax, cm–1): 3434 (OH), 1641 (C=O), 1516, 1462, 1449 (C=C), 1261 (C–O). 1H NMR spectrum (500 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment: 3.70 (2H, resonance overlapped, N–CH2), 4.48 (1H, dd, J = 3.6, 11.0, H-3), 4.95 (1H, t, J = 10.9, H-2), 5.82 (1H, s, H-8), 6.74 (2H, s, H-5´, 6´), 6.87 (1H, s, H-2´), 8.98 (2H, br.s, 3´, 4´-OH), 12.36 (1H, br.s, 5-OH); b) 1-(3´,4´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 2.68 (1H, m, Ha-3), 2.85 (2H, m, H-4), 3.03 (1H, m, He-3), 3.51 (3H, s, 7-OCH3), 3.69 (3H, s, 6-OCH3), 3.74 (3H, s, 3´-OCH3), 3.75 (1H, s, 4´-OCH3), 4.47 (1H, d, J = 1.9, H-1), 6.28 (1H, s, H-8), 6.67 (2H, dd, J = 1.8, 8.2, H-6´), 6.77 (1H, s, H-5), 6.80 (1H, d, J = 1.8, H-2´), 6.91 (1H, d, J = 8.2, H-5´); 13C NMR spectrum (δ, ppm): a) DHQ fragment: 48.36 (CH2–N), 71.78 (C-3), 83.24 (C-2), 95.44 (C-8), 100.11, 101.18 (C-4a, 6), 115.35 (C-2´), 115.58 (C-5´), 119.59 (C-6´), 128.24 (C-1´), 145.17 (C-3´), 146.00 (C-4´), 160.84 (C-8a), 161.72 (C-5), 168.60 (C-7), 197.95 (C-4); b) 1-(3´,4´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 21.44 (C-4), 45.07 (C-3), 55.65 (6-OCH3), 55.67 (7-OCH3), 55.67 (3´-OCH3), 55.79 (4´-OCH3), 65.96 (C-1), 111.56 (C-8), 111.67 (C-5), 112.12 (C-6´), 113.27 (C-5´), 122.26 (C-2´), 125.99 (C-1´), 127.97 (C-8a), 133.88 (C-4a), 147.37 (C-6), 148.02 (C-7), 148.54 (C-3´), 148.78 (C-4´).

2-(3,4-Dihydroxyphenyl)-6-{[1-(3,4-methylenedioxyphenyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]methyl}-3,5,7-trihydroxychroman-4-one (10g).

1-(3,4-Methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (3d, 0.21 g, 0.67 mmol) afforded DHQ (9, 0.20 g, 0.67 mmol. Yield 0.34 g (83%), mp 176–180°C (i-PrOH). IR spectrum (KBr, vmax, cm–1): 3204 (OH), 1639 (C=O), 1516, 1488 (C=C), 1252 (C-O). 1H NMR spectrum (400 MHz, DMSO-d6, δ, ppm, J/Hz): a) DHQ fragment: 3.67 (1H, dd, J = 3.3, 14.3, N–CH2), 3.79 (1H, d, J = 14.3, N–CH2), 4.49 (1H, dd, J = 3.9, 11.1, H-3), 4.96 (1H, dd, J = 7.8, 11.1, H-2), 5.83 (1H, s, H-8), 6.74 (2H, s, H-5´, 6´), 6.87 (1H, s, H-2´), 9.00 (2H, br.s, 3´, 4´-OH), 12.33 (1H, br.s, 5-OH); b) 1-(3´4´-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 2.65 (1H, m, Ha-3), 2.82 (2H, m, H-4), 2.99 (1H, m, He-3), 3.52 (3H, s, 7-OCH3), 3.74 (3H, s, 6-OCH3), 4.77 (1H, s, H-1), 6.01 (2H, d, J = 3.4, 3´-OCH2O-4´), 6.27 (1H, s, H-8), 6.68 (1H, s, H-2´), 6.69 (1H, d, J = 6.4, H-6´), 6.76 (1H, s, H-5), 6.88 (1H, d, J = 8.5, H-5´); 13C NMR spectrum (δ, ppm): a) DHQ fragment: 47.87 (CH2–N), 71.51 (C-3), 82.99 (C-2), 95.20 (C-8), 99.93, 101.09 (C-4a, 6), 115.09 (C-2´), 115.33 (C-5´), 119.37 (C-6´), 127.69 (C-1´), 144.92 (C-3´), 145.76 (C-4´), 160.67 (C-8a), 161.48 (C-5), 168.08 (C-7), 197.83 (C-4); b) 1-(3´4´-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline fragment: 25.49 (C-4), 44.53 (C-3), 55.40 (6-OCH3), 55.48 (7-OCH3), 65.60 (C-1), 101.09 (C-7´), 107.78 (C-5´), 109.36 (C-2´), 111.37 (C-8), 111.64 (C-5), 123.28 (C-6´), 125.80 (C-1´), 127.99 (C-8a), 135.32 (C-4a), 146.68 (C-6), 147.12 (C-7), 147.31 (C-3´), 147.74 (C-4´).

Cell culture

Normal rat hepatocyte cells and three human cancer cell lines cervical carcinoma cells (HeLa, collection of Russian Academy of Science), laryngeal adenocarcinoma cells (HEp-2, ATCC: CCL-23) and breast carcinoma cells (HBL-100, ATCC: HTB-124) were used for cytotoxicity screening of the 1,2,3,4–tetrahydroisoquinoline derivatives. The verified cell lines were purchased from the Bank of Cell Cultures in the Institute of Cytology of Russian Federation. The primary culture of hepatocytes was obtained from the liver of rats as adult animals (Tseomashko et al, 2015). Cell lines were cultured in advanced DMEM and RPMI-1640 (SIGMA, USA)  supplemented with 10% inactivated FBS (SIGMA, USA) and 2 mM L-glutamine and 1% antibiotic-antimycotic solution, and grown at 37°C in a humidified atmosphere of 5% CO2 in air. To subculture the cells were detached with Versen solution.

MTT assay

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay developed by (Mosmann T., 1983) was used to screen for cytotoxic activity of the 1,2,3,4–tetrahydroisoquinoline derivatives.

The all substances were dissolved in DMSO. Briefly, the cells were seeded in 96-well plates at a density from 2x103 to 5x103 cells per well. Following 24-h incubation and attachment, the cells were treated with substances concentrations 1 - 100 µg/ml for 24 h. Following incubation with MTT solution (5 mg/mL) for 3.5 h, cells were lysed with DMSO. The absorbance was measured after 30 min using the «EnSpire 2600» microplate reader (Perkin Elmer, USA) at a wavelength of 620 nm. Intact cells with solvent in the culture medium served as the control. The effect was compared with antineoplastic drug «Ciplatin» (Fresenius Kabi, India) with cis-diamminedichloroplatinum as active ingredient and bioflavonoid dihydroquercetin (DHQ).

Cell viability was determined by the ratio of viable cells exposed to the test substance to the amount of living cells in the control. The results are generated from three independent experiments; each experiment was performed in triplicate. 

Statistical analysis

Statistical processing and graphing were performed using the program «Origin 5». Results were considered significant at p ≤ 0.05.

Results

At first this is to obtain natural alkaloids analogues such as cryptostylines and salsolidine (3а-f, 4с,d and 8а); at the second  is alkylation at N atom (5c-f); at the third is synthesis of biomolecular compounds 1,4-bis(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)alkanes and 1,2,3,4-tetrahydroisoquinolin-2(1H)-yl)methyl]-3,5,7-trihydroxychroman-4-ones (8b-h and 10 d-g). 

For isoquinolines 3a-f Pictet-Spengler reaction has been used by (Zhurakulov et al, 2013). Target isoquinolines 3a-f were synthesized after interaction of amine 1 with aldehydes 2a-f through a Schiff base and following their cyclization by the action SF3COOH. Target isoquinolines methylation by Craig resulted alkaloids cryptostyline I and II (4c,d). Hydroxyethyl ariltetrahydroisoquinoline derivatives 5c-f were obtained by condensation 3c-f with ethylenechlorohydrine. (Zhurakulov et al, 2014) (Scheme 1). The KOH use instead of K2CO3 in the final stage resulted in increase in output a 5c from 62% to 91%.

Scheme 1. The obtaining of hydroxyethyl ariltetrahydroisoquinoline derivatives 5c-f by condensation 3c-f with ethylenechlorohydrine.

Derivatization 8a-h

Simple synthetic scheme was used for the synthesis of 8a-h target products: preparation of amides from ghomoveratrilamine (1) and the corresponding acids– 6a (7:0 heptanoic acid), 6b-h dicarboxylic acids (adipic, sebacic, tridecanoic, brassylic and izoftaleic acids) and their subsequent Bischler-Napieralski cyclization to 3.4-dihydroisoquinolines (Scheme 2).

Scheme 2 The synthesis of amides from ghomoveratrilamine (1) with the corresponding acids 6a-h and their subsequent Bischler-Napieralski cyclization to 3.4-dihydroisoquinolines

Using the obtained salt of the corresponding acid and amine 1 instead of a substances mixture in the first step yielded high yield of 7a-g amides. 7a-g amides are almost easy by heating the salt to 178ºS for 2 hours. In the second step POCl3 as a condensing agent was used. a-h target isoquinolines  by reduction 3,4-dihydroisoquinolines with NaBH4 were prepared. Isophthalic acid (6g) gave 7h amide in 29% yield. Interaction of amine 1 with isophthalic acid 6h chloroanhydride proved more effective method (Method B). Thereby 7h amide was isolated with 82% yield (Scheme 3).

Scheme 3 The synthesis of 8b-h target products

The structure of the synthesized compounds was confirmed by IR and H1 NMR spectroscopy. PMR spectra of 7a-h compounds contain proton signals of all structural fragments. Proton signal H-1 in 8a-h NMR spectra appeared at 4.40, 3.82 - 3.98, 4.33, 3.78 and 5.68 ppm, respectively.

Conjugates dihydroquercetin receiption

Flavonoids like alkaloids from a practical point of view are of particular interest as medicine drugs. One bioflavonoid dihydroquercetin (DHQ) has powerful antioxidant, hepatoprotective, anti-tumor, immunomodulatory properties (Babkin et al, 2011; Nifant’ev et al, 2013). Unfortunately, flavonoids are not produced by the human body and have low solubility in water. These properties determine the interest of chemists from leading research centers to modificate  quercetin and dihydroquercetin.

We obtained conjugates dihydroquercetine (9) from the primary 1,1a and secondary 1b, 3a, 3c, 3d amines. Reactions were carried out in isopropyl alcohol at room temperature (25-30 °C) applying the ratio of DHA / amine / formaldehyde = 1: 1: 1. Wherein 10a-g monosubstituted products were synthesized (Scheme 4). 

The structure of the synthesized compounds was confirmed by IR, 1H and 13C NMR spectroscopy.

Scheme 4 The obtaining of conjugates dihydroquercetine (9) from the primary 1,1a and secondary 1b, 3a, 3c, 3d amines and 10a-g monosubstituted products.

Cytotoxic activity

The literature suggests a wide range of physiological activity of 1,2,3,4-tetrahydroisoquinoline derivatives. So, substances were found to exhibit properties of central nervous system depressants (Ott at all, 1968) and NMDA receptor blockers (Wanner at al, 1998). A biological activity of 1,2,3,4-tetrahydroisoquinoline grounds includes antitumor properties (Debono at al,  2014; Mihoubi at al,  2015; Patil at al, 2014; Uesawa at al, 2011).

In the present study, we investigated the cytotoxic activity of three types of substances: monobasic tetrahydroisoquinoline derivatives, dibasic tetrahydroisoquinolines and synthetic complexes of alkaloids, amides and their derivatives with a natural bioflavonoid dihydroquercetin (DHQ). Monohydric tetrahydroisoquinoline data presented in Table 1.

Table 1. Cytotoxic activity of tetrahydroisoquinoline derivatives (alive cells, %)

Several monobasic tetrahydroisoquinolines have different effects of cell growth suppression at concentration of 10 µg/ml. Thus, a sample 4d showed the most selective cytotoxic activity among the derivatives, which suppressed growth of larynx cell on 66.9%, while its toxicity to normal cells was only 26.1% compared with the control.

Compounds 3a, 3b, 3e, 3f and 8a were less active, where the cell growth inhibition ranged from 56.2 to 40.9%. It is interesting to note that with regard 8a was only active to breast adenocarcinoma - at doses of 1 and 10 µg/ml of cell growth inhibition was 44.8 and 43.7% respectively with the absence of toxicity to hepatocytes.

Cytotoxic activity of dibasic tetrahydroisoquinolines presented in Table 2.

Table 2. Cytotoxic activity of bis-tetrahydroisoquinoline derivatives (alive cells, %)

Bis compounds exhibit greater cytotoxic effect than mono-series compounds. There was a tendency for activity growth of dibasic compounds for continuous cell cultures with increasing length of the hydrocarbon chain between the two isoquinoline molecules: the longer hydrocarbons chain, the derivative more active inhibits cell growth. The sample 8f showed the greatest cytotoxic activity among bis-substances: at a concentration of 10 µg/ml the sample has caused the 100% death of cell cultures. The sample 8d showed highly selective inhibitory activity against cervical adenocarcinoma (93.7%). Another substance 8h containing the benzene ring between two isoquinoline molecules exhibits mild cytotoxic effect against HEp-2 and HBL-100 (62-63% inhibition of cell growth at 10 µg/ml).

Cytotoxic activity of synthetic complexes of alkaloids and derivatives with DHQ is presented in Table 3.

The all compounds completely lacked cytotoxicity toward cancer cell lines in this study. Some of them have shown pronounced proliferation (table 3). Sample 10b demonstrated the most proliferative activity on HEp-2 cells: 184.1 and 148.7 % at 10 and 1 µg/ml, respectively. 10d had similar proliferative effects on laryngeal adenocarcinoma cells: 149.2 and 146.1% cell growth compared with the control.

Table 3. Cytotoxic activity of synthetic products of alkaloid with DHQ (alive cells, %)

It is interesting to note that the DHQ reference drug showed a pronounced proliferation only in healthy liver cells.

Compounds showing cytotoxic activity were further tested at additional concentrations to calculate the IC50 values (table 4).

Table 4. IC50 value (µM)

Discussion

In studying the relationship between the structure of the investigated compounds and their ability to inhibit the growth of cancer cells was detected following: in a series of 1-ariltetrahydroisoquinoline absence of ring C (sample 3a) shows a weak activity of the compound, and the presence of the benzene ring slightly increases its cytotoxicity. Dimethoxy- and methylenedioxy- groups in ring A at the tetrahydroisoquinoline molecule in this concentration range are functionally significant, because the inhibitory effect is manifested in different ways (3с, 3d). 

N-alkylation of these compounds leads to a double effect: the presence of the methylenedioxy group (4d) shows the manifestation of the selectivity of the molecule, which is enhanced with the introduction of halogens. At the same time isoquinoline with a dimethoxy group (samples 4c5c) loses its cytotoxic properties completely. Halogen nature is essential for cytotoxicity: inhibitory effect of the substance is manifested more in the presence of Cl atom than of Br (3e, 3f). Hexyl group in tetrahydroisoquinoline (8a) maintains the high activity of the substance.

With regard to the bis-tetrahydroisoquinolines, the introduction into their structure of the second molecule greatly enhances the cytotoxic effect of substance. Methylene chain length between molecules is important.

Possibly, a long chain between the isoquinoline molecules helps substance to cling specific receptors of cancer cells. It leads to the suppression of their growth. Because of which healthy cells less susceptible to the influence of the cytotoxic derivatives than transformed. A similar effect is described in the scientific literature. For example, substances derived from plants of the family Cruciferaceae, have the ability to "stick" to faulty proteins located on the surface of cancer cells and cause their death by apoptosis. It is noteworthy that the normal cells are resistant to these compounds, since their surface is not defective proteins specific to tumor cells (Cavell et al, 2011; Wang et al, 2008).

Synthetic complexes of tetrahydroisoquinoline alkaloid derivatives with DHQ demonstrated another interesting result. Complex compounds are interest to researchers to broaden their spectrum of activity and reduce the toxicity. 

In this paper, we studied the cytotoxic activity of synthetic derivatives containing the 1-ariltetrahydroisoquinoline (3c3d), their amines and alkaloids salsolidyn, homoveratril, pseudoephedrine. All individual compounds have little overwhelming effect. DHQ provided with weak proliferative effect on cancer cells cultures and the average - to normal.
Substances 10f, salsolidin, pseudoephedrine and 10a in conjunction with DHQ were totally inactive against all cell cultures. Complexes containing the remaining compounds, started to show proliferative properties such as on the individual cell lines (10d10g), and almost all cultures (10b). Probably, a similar effect of the samples is connected with the manifestation of the DHQ activity and the weakening of the alkaloid action. 

Thus, the most promising compounds for further study in vitro and in vivo methods are dibasic compound 8d8e. Later these substances can be offered as a basis for drugs with anti-tumor properties.

Acknowledgement

This work is done according to the fundamental research FA-F6-T198 of Academy of Sciences of Uzbekistan with funding from the state of Uzbekistan.

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