RU2814111C1 - 5,8-dimethoxy-6,7-methylenedioxycoumarin derivatives having antiradical activity - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: invention relates to organic chemistry, particularly to a method of producing a compound of general formula: , where R is selected from: , , , , . Method is carried out by treating 5,8-dimethoxy-6,7-methylenedioxycoumarin of formula A with lead tetraacetate, and then with excess HCl/iPrOH, followed by catalytic hydrogenation of the obtained dihydroxycoumarin of formula B in a hydrogen atmosphere to obtain 6,7-dihydroxy-5,8-dimethoxychroman-2-one (C), according to the scheme:

Hydrolysis of the compound of formula C is then carried out, followed by oxidation of air with oxygen and obtaining 5,8-dimethoxy-6,7-methylenedioxycoumarin derivatives.

EFFECT: synthesis of 5,8-dimethoxy-6,7-methylenedioxycoumarin derivatives exhibiting antiradical activity.

6 cl, 2 dwg, 1 tbl, 9 ex

Description

Field of technology

The invention relates to the field of organic chemistry, in particular to medicinal chemistry, namely to the synthesis of agents of formulas (1) 3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl )propanoic acid, (2) 3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)-N-(2-(5-methoxy-1H-indole -3-yl)ethyl)propanamide, (3) N-(3,4-dihydroxyphenethyl)-3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl )propanamide, (4) 4-(3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)butanoic acid, (5) 3-(3 -(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido) propanoic acid, which has antiradical activity.

State of the art

Substances with antiradical activity (ARA) play a leading role in the body’s defense system against free radicals. The body's defense system against excess free radicals consists of the activity of oxido-reductase enzymes and various reactions of low-molecular antioxidants: chelate compounds, hormones, water- and fat-soluble vitamins, thiol-containing amino acids and polypeptides, flavonoids, carotenoids, etc. Most of these compounds prevent the development of oxidative stress by interrupting the chain reaction of free radical formation, which is why these substances are called both substances with antiradical activity and antioxidants.

The use of intracellular antioxidants is known, in particular coenzyme Q10 (ubiquinone, CoQ10), which is a component of the mitochondrial electron transport chain, and is widely used in medicine, ranging from the treatment of neurodegenerative diseases such as multiple system atrophy, to conditions such as Barth syndrome, cardiac deficiency, fibromyalgia and insulin resistance (Pastor-Maldonado, C. J., Suárez-Rivero, J. M., Povea-Cabello, S., Álvarez-Córdoba, M., Villalón-García, I., Munuera-Cabeza, M., Sánchez-Alcázar, J. A. Coenzyme Q10: Novel Formulations and Medical Trends. International Journal of Molecular Sciences, 2020, 21(22), 8432. doi:10.3390/ijms21228432). Despite the effectiveness of CoQ10 therapy, its clinical use is hampered by low bioavailability. The oral bioavailability of CoQ10 is limited by the pronounced hydrophobicity of the side chain, consisting of 10 isoprene units, as well as its photosensitivity and thermolability. All this determines the relevance of the search and development of its derivatives.

An important class of biologically active compounds are coumarins. They are part of plants as secondary metabolites and have a wide spectrum of activity. Coumarins are a class of natural organic compounds that are unsaturated aromatic lactones. A feature of coumarins is their easy solubility in aqueous solutions of alkalis (especially when heated) due to the formation of salts of hydroxycinnamic acid. Coumarins, being lactones, specifically relate to alkali (they do not interact with acids and ammonia). Under the action of hot dilute alkali, they slowly hydrolyze, and the lactone ring ruptures with the formation of yellow solutions representing a salt of coumaric acid (cis-, ortho-hydroxycinnamic). When acidifying alkaline solutions or when saturated with CO ₂ , coumarins are regenerated to their original state. The varied effects of coumarins on the body are known. One of the characteristic properties of coumarins is anticoagulating activity. This is especially pronounced in dicumarol, it prevents the formation of prothrombin. Scopoletin is distinguished by this property. Many furocoumarins have photosensitizing activity, i.e. increase the skin's sensitivity to ultraviolet rays, which results in intense skin pigmentation and severe burns. This property of furocoumarins is used to treat vitiligo (leukoderma). The most pronounced photosensitizing properties are psoralen and xanthotoxin. Furocoumarins accelerate the formation of melanin. For the treatment of leukoderma, the following drugs are used: ammifurin (a mixture of isopimpinellin and bergapten from the fruits of ammi major), beroxan (a mixture of xanthotoxin and bergapten from the fruits of parsnips), psoralen (a mixture of psoralen and angelicin from the fruits of psoralea). Derivatives of furocoumarins and pyranocoumarins have antispasmodic and coronary-dilating effects. The most active are visnadine and dihydrosamidine. The following drugs are used as antispasmodics: flowerin (from Siberian spleen), Avisan (from ammi dentifrice). Clover coumestrols have significant estrogenic activity. Coumarins have been shown to have antimitotic activity, which prompted the study of their anticancer effect. It has been established that peucedonin and xanthotoxin have this effect. They enhance the effect of antitumor drugs. A number of coumarins and furocoumarins exhibit bacteriostatic properties. Some coumarins (esculetin) have P-vitamin activity (M.M. Konopleva Textbook “Pharmacognosy: natural biologically active substances”, Vitebsk, VSMU, 2006 - p. 50).

Today, coumarin derivatives and their use as pla2 inhibitors are also known. This applies to coumarin derivatives of general formula I

in which R1 is hydrogen or C1-C6 alkyl, R2 is the remainder of OT1 or R3 is the remainder of OT2, R4 is hydrogen or C1-C6 alkyl, T1 and T2, which may be the same or different, and each is hydrogen, C1- C6-alkyl, C1-C6-alkonoyl, or T1 and T2, together with the atoms to which they are attached, form a 5-7-membered heterocyclic ring, which, if desired, can be substituted with oxo or thioxo, or Q - oxygen atom X - sulfur atom or NH group, Y is a valence bond, a C1-C6 alkylene residue, which can optionally be substituted by a hydroxy group or an amino group, a phenylene residue, which can optionally be single or multiple substituted with hydroxyl, halogen, C1-C6- alkyl or carboxyl, Z - hydrogen, halogen, carboxyl hydroxymethyl, C1-C6 alkoxycarbonyl, cyano or NR5R6 group, and R5 and R6 together with the nitrogen atom to which they are attached form a 3-7 membered heterocyclic ring, which, if desired may be substituted with oxo, hydroxy or C1-C6 alkoxy, their tautomers, as well as their salts with non-toxic acids or bases. These representatives of coumarin inhibit pla2 depending on the dose and up to 100% and are thus superior to known anti-inflammatory compounds (RU2133745, 07/27/1999).

However, the area of the antiradical effect of coumarins remains not fully studied today due to the extremely few examples of the use of this type of drugs in medical practice. In terms of antiradical activity, 5,8-dimethoxy-6,7-methylenedioxycoumarin is of interest.

The synthesis of three highly oxygenated natural coumarins, 8-methoxy-6,7-methylenedioxycoumarin, 5-methoxy-6,7-methylenedioxycoumarin and 5,8-dimethoxy-6,7-methylenedioxycoumarin, is described for the first time together with a new method for the preparation of auapine (6,7 -methylenedioxycoumarin). Comparison of spectroscopic data from synthetic tetraoxygenated coumarin a with literature spectroscopic data led to a revision of the structure of several natural coumarins. The synthesis of 5,8-dimethoxy-6,7-methylenedioxy coumarin is given in the article by Maes D. et al. Synthesis and structural revision of naturally occurring ayapin derivatives // Tetrahedron. 2005. Vol. 61, No. 9. P. 2505-2511.

The invention can be used to create new agents with antiradical activity, in particular a new analogue of ubiquinone, as well as a series of combined multifunctional molecules with two pharmacophore fragments connected through a linker, capable of binding free radicals (FR). Quinone, which is part of the structure of the compounds, can provide effective protection of cells under conditions of oxidative stress (OS), prevent mitochondrial dysfunction, acting as an analogue of coenzyme CoQ10. The lipophilicity of the quinone fragment ensures the passage of the molecule through the blood-brain barrier, thus the target structure can be represented as an effective transporter of free neurotransmitters or amino acids into the brain. The labile amide bond can be easily cleaved by enzymatic systems, which would release free neurotransmitters and amino acids and replenish their levels in the brain. The inclusion of a quinone moiety increases the overall lipophilicity of the molecule, which facilitates its efficient transport into tissues and cells, and the labile amide bond can be easily broken enzymatically, followed by release of the free neurotransmitter or amino acid.

Disclosure of the Invention

The technical result is the synthesis of new derivatives of 5,8-dimethoxy-6,7-methylenedioxycoumarin, characterization of the resulting compounds by physicochemical methods that exhibit antiradical activity, as well as a simple method for the synthesis of these compounds. The proposed invention allows us to significantly expand the range of drugs with antiradical activity.

The technical result is achieved by creating derivatives of 5,8-dimethoxy-6,7-methylenedioxy coumarin with the general formula:

, where R1= OH, R2= N-(2-(5-methoxy-1H-indol-3-yl)ethyl)propan-2-amine, R3= 4-(2-(isopropylamino)ethyl)benzene-1, 2-diol, R4= 4-(isopropylamino)butanoic acid, R5= 3-(isopropylamino)propanoic acid.

And also a compound has been created according to claim 1, characterized in that it is a compound selected from the group including:

(1) 3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanoic acid,

(2) 3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)-N-(2-(5-methoxy-1H-indol-3-yl )ethyl)propanamide,

(3) N-(3,4-dihydroxyphenethyl)-3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamide,

(4) 4-(3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)butanoic acid,

(5) 3-(3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)propanamido)propanoic acid, which has antiradical activity.

And in addition, a method has been created for the synthesis of compounds according to claim 2, characterized in that the starting compound 5,8-dimethoxy-6,7-methylenedioxy coumarin (A) is treated with lead tetraacetate, and then with an excess of HCl/iPrOH, after which catalytic hydrogenation of the resulting dihydroxycoumarin (B), in a hydrogen atmosphere to obtain 6,7-dihydroxy-5,8-dimethoxychroman-2-one (B), as a result of hydrolysis of which followed by oxidation with atmospheric oxygen, 5,8-dimethoxy-6,7- derivatives are obtained methylenedioxy coumarin, which has antiradical activity.

In a preferred embodiment, a compound of formula (1)

obtained by hydrolyzing the compound of formula B and reacting with an acidic medium at room temperature for 24 hours in an air atmosphere.

In a preferred embodiment, the compound of formula (2)

prepared by hydrolysis and reaction of the compound of formula B with 5-methoxytryptamine hydrochloride in dioxane at room temperature using atmospheric oxygen as an oxidizing agent.

In a preferred embodiment, the compound of formula (3)

prepared by hydrolysis and reaction of the compound of formula B with 3,4-bis(benzyloxy)phenethylamine in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, under a hydrogen atmosphere for 24 hours and oxidation in air for 24 hours.

In a preferred embodiment, the compound of formula (4)

prepared by hydrolysis and reaction of the compound of formula B with β-alanine benzyl ester in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, under a hydrogen atmosphere for 24 hours and oxidation in the atmosphere air within 24 hours.

In a preferred embodiment, the compound of formula (5)

prepared by hydrolysis and reaction of the compound of formula B with γ-aminobutyric acid in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, in an atmosphere of hydrogen for 24 hours and oxidation in an atmosphere of air in 24 hours.

Carrying out the invention

The primary raw material for the synthesis of the compounds under study is apiol, a readily available polyalkoxyallylbenzene isolated from parsley essential oil with a yield of 65-70% [V. V. Semenov, et.al.Russ. Chem. Bull., Int. Ed., 2007, 56, 2448 (Izv. Akad. Nauk, Ser. Khim., 2007, 2364], [A.V. Samet, et.al., Journal of Natural Products, 2019, 82, 6, 1451.].

To synthesize the product with structural formula (1-5), the following reagents were used: benzene, NaHCO ₃ , CH ₂ Cl ₂ , iPrOH, dioxane, tBuOH, Pd/C (10%), PtO ₂ ^. H ₂ O, Pb(OAc) ₄ , HCl/iPrOH, Et ₃ N, hydrochlorides of 5-methoxytryptamine (G), 3,4-bis(benzyloxy)phenethylamine (D) and benzyl ester of β-alanine (E) and γ- aminobutyric acid (A) were obtained in accordance with the described methods [Raymond B., Pierre H. Preparation of serotonine and derivatives], [Cai W., et al. Metal-assisted assembly and stabilization of collagen-like triple helices // J Am Chem Soc.2004.Vol. 126, No. 46.P. 15030-15031.], [Buttner F., et al. Cyclic beta-tetra- and pentapeptides: synthesis through on-resin cyclization and conformational studies by X-ray, NMR and CD spectroscopy and theoretical calculations // Chemistry. - 2005. - Vol. 11, No. 21. - P. 6145-58.]

The general synthesis scheme is presented in the diagram

5,8-Dimethoxy-6,7-methylenedioxy coumarin (A) was purchased from Chemical-block. Oxidation of the methylene bridge was carried out according to the described method [Nicolaou K. C., Tang Y., Wang J. Total synthesis of sporolide B // Angew Chem Int Ed Engl. 2009. Vol. 48. P. 3449-53.] Dihydroxycoumarin (B) was obtained by the action of lead tetraacetate on (A) followed by treatment with excess HCl/iPrOH. Catalytic hydrogenation (B) led to lactone (C). Compound (B) is sensitive to the action of nucleophiles, and therefore is a convenient starting compound for the synthesis of various hybrid molecules containing a hydroquinone moiety. Thus, as a result of hydrolysis (B) followed by oxidation with atmospheric oxygen, compound (1) was obtained. When (B) was treated with 5-methoxytryptamine hydrochloride and subsequent oxidation with atmospheric oxygen, amide (2) was synthesized; as a result of the reaction of (B) with 3,4-bis(benzyloxy)phenethylamine hydrochloride followed by hydrogenolysis of benzyl groups, the corresponding hydroquinone was obtained, which is easily was oxidized by atmospheric oxygen to compound (3), similarly, as a result of opening (B) with protected amino acids (E) and (G), adducts (4) and (5) were obtained.

Examples

Example 1. Synthesis of coumarin (B)

A mixture of 18.52 g (74 mmol) (A) , 50.20 g (15 mmol) Pb(OAc) ₄ (95%) and 350 ml of benzene was heated with stirring (T=80°C) for 60 hours. The lead diacetate precipitate was filtered off and washed with benzene. The filtrate was evaporated to 50-70 ml, 300 ml of water and 9.32 g of NaHCO ₃ (111 mmol) were added to the residue, the organic layer was separated, dried with magnesium sulfate, and evaporated in vacuum. The aqueous phase was additionally extracted with CH ₂ Cl ₂ (1x50 ml), dried with magnesium sulfate, and evaporated in vacuum. The orange amorphous residue was dissolved in 100 ml CH ₂ Cl ₂ and 10 ml iPrOH, then 40 ml iPrOH-HCl (9M, 360 mmol) was added. The mixture was stirred for 1 hour at 20°C, then cooled in a bath of ice and water, the precipitate was filtered, washed with iPrOH. The product was purified by recrystallization from iPrOH. A total of 14.11 g of coumarin (B) was obtained in the form of light beige crystals. Yield 80%

¹H NMR (500 MHz, DMSO-d ₆, δ, ppm) 3.80 (s, 3H, OCH₃) 3.81 (s, 3H, OCH₃), 6.21 (d,J=9.6 Hz, 1H, CH) 7.97 (d,J=9.6 Hz, 1H, CH) 9.21 (br.s., 1H, OH) 10.04 (b.s., 1H, OH) ¹³C NMR (126 MHz, DMSO-d₆, δ, ppm): 60.93, 61.46, 105.28, 111.34, 131.23, 135.65, 139.36, 139.64, 140.90, 144.75, 160.13 Mass C_elevenH₁₀O₆ M.v. 238.2: 238, 223, 209, 195, 177, 167, 152, 139, 123, 111, 106, 83, 68 T_pl=222-223°C.

Example 2. Synthesis of lactone (B)

By hydrogenating 0.476 g (2 mmol) of coumarin (B) with 0.019 g (0.08 mmol) PtO ₂ •H ₂ O in 30 ml tBuOH (12 hours at 60°C and 24 hours at 20°C) in a hydrogen atmosphere we obtained 0.479 g 6 ,7-dihydroxy-5,8-dimethoxychroman-2-one (B) in the form of beige crystals. Yield 99%

¹ H NMR (500 MHz, DMSO- d ₆ , δ, ppm) 2.69 (t, J=7.2 Hz, 2H, CH ₂ ) 2.83 (t, J=7.2 Hz, 2H, CH ₂ ) 3.66 (s , 3H, OCH ₃ ), 3.70 (s, 3H, OCH ₃ ), 8.54 (br.s., 1H, OH), 8.90 (br.s., 1H, OH). ¹³ C NMR (151 MHz, DMSO- d6 , δ, ppm) 17.60, 28.54, 60.22, 60.81, 107.12 _, 132.63, 135.62, 137.34, 138.68, 140.33, 168.33. Mass C ₁₁ H ₁₂ O ₆ M.w. 240.21: 240, 225, 211, 198, 183, 165, 155, 147, 137, 123, 112, 106, 97.T _pl = 185-188°C.

Example 3: Synthesis of quinonic acid (1)

A mixture of 0.120 g (0.5 mmol) (B) , 5 ml of water and 0.05 ml of 1M aq. HCl solution (0.05 mmol) was heated until dissolved, then stirred at room temperature in air for 24 hours until the starting material completely disappeared (TLC). Then 5 ml of CH ₂ Cl ₂ was added to the mixture, the organic layer was separated, the aqueous layer was extracted with CH ₂ Cl ₂ (3x5 ml), the organic phases were dried with magnesium sulfate, evaporated in vacuum, and 0.050 g of quinonic acid (1) was obtained in the form of red crystals. Yield 40%

¹H NMR (500 MHz, DMSO-d ₆, δ, ppm): 2.27 (t, J=7.9Hz, 2H, CH₂), 2.53 (t, J=7.9Hz, 2H, CH₂), 3.77 (s, 3H, OCH₃), 3.88 (c, 3H, OCH₃), 10.40 (br.c., 1H, OH), 12.12 (br.c., 1H, CO₂H). ¹³C NMR (151 MHz, DMSO-d ₆, δ, ppm): 18.6, 32.7, 60.1, 60.9, 128.2, 138.1, 143.3, 153.3, 173.5, 180.3, 183.3. Mass C₂₂H₂₄N₂O₇ M.v. 428.44: 428, 413, 398, 367, 352, 336, 326, 314, 296, 276, 268, 257, 240. T_pl= 65-66°C.

Example 4 Synthesis of methoxyquinone-5-methoxytryptamine (2)

0.240 g (1 mmol) B , 0.227 g (1 mmol) (D) , 6 ml of dioxane were placed in a 50 ml round-bottom flask, then 0.167 ml of triethylamine (0.121 g, 1.2 mmol) was added under an argon atmosphere. The reaction mixture was stirred in an argon atmosphere at room temperature for 24 hours until the starting material completely disappeared (control was carried out by TLC). After stirring, the reaction mixture was evaporated, the product was isolated by column chromatography on 35 g of silica gel, eluting with a mixture of CH ₂ Cl ₂ -CH ₃ OH. We obtained 0.230 g of methoxyquinone-5-methoxytryptamine ( 2) in the form of a red amorphous substance. Yield 54%

¹H NMR (500 MHz, DMSO-d ₆, δ, ppm): 2.15 (t, J=7.9Hz, 2H, CH₂), 2.54 (t, J=7.9Hz, 2H, CH₂), 2.76 (t, J=7.4Hz, 2H, CH₂), 3.30 (sq., J=6.9Hz, 2H, CH₂), 3.76 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.87 (c, 3H, OCH₃), 6.71 (dd., J=8.7Hz, J=2.3Hz, 1H, CH_Ar), 7.01 (d., J=2.3 Hz, 1H, CH_Ar), 7.09 (d., J=1.3Hz, 1H, CH_Ar), 7.22 (d., J=8.7Hz, 1H, CH_Ar), 7.93 (t, J=5.6Hz, 1H, NH), 10.81 (c., 1H, NH). ¹³C NMR (151 MHz, DMSO-d ₆, δ, ppm): 19.3, 25.2, 34.3, 55.3, 60.0, 60.9, 100.1, 111.0, 111.7, 112.0, 123.2, 127.5, 129.1, 131.4, 138.1, 143.3, 153.0 , 153.1, 170.8, 180.3, 183.3 . Mass C₂₂H₂₄N₂O₇ M.v. 428.44: 428, 413, 398, 367, 352, 336, 326, 314, 296, 276, 268, 257, 240. T_pl= 68-70°C.

Example 5 Synthesis of methoxyquinone-dopamine (3)

To a mixture of 0.480 g (2 mmol) (2) , 0.787 g (94%, 2 mmol) (B) and 12 ml of dioxane in an argon atmosphere, 0.335 ml of Et ₃ N (0.243 g, 2.4 mmol) was added. Stirred in an argon atmosphere at room temperature for 24 hours until the starting material completely disappeared (TLC). After stirring, the mixture was evaporated, the residue was dissolved in CH ₃ OH, and stirred in air for 48 hours. Upon completion, the mixture was evaporated, the residue was passed through a column of silica gel, eluting with a mixture of CH ₂ Cl ₂ -CH ₃ OH (100:2.5). The resulting red powder was stirred for 24 hours in a hydrogen atmosphere with 10% Pd/C (0.068 g) in 25 ml of CH ₃ OH, filtered from the catalyst and stirred in air for 24 hours. Then the solution was evaporated, the residue was purified by column chromatography, eluting with a mixture of CH ₂ Cl ₂ -CH ₃ OH-AcOH (100:10:1), to obtain 0.382 g of methoxyquinone-dopamine (3) in the form of a red amorphous substance. Overall yield 50%

¹ H NMR (500 MHz, Acetone- d ₆ , δ, ppm) 2.27 (t, J=7.9 Hz, 2H, CH ₂ ), 2.62 (t, J=7.4 Hz, 2H, CH ₂ ), 2.67 (t, J=7.9 Hz, 2H, CH ₂ ), 3.33 (sq., J=6.5 Hz, 2H, CH ₂ ), 3.89 (s, 3H, OCH ₃ ), 3.95 (s, 3H, OCH ₃ ), 6.52 (dd., J=8.1 Hz, J=1.8 Hz, 1H, CH _Ar ), 6.70 (d., J=1.8 Hz, 1H, CH _Ar ), 6.71 (d., J=8.1 Hz, 1H, CH _Ar ), 7.14 (t, J=4.6 Hz, 1H, NH). ¹³ C NMR (151 MHz, Acetone - d ₆ , δ, ppm) 20.38, 35.63, 35.92, 41.81, 60.60, 61.54, 116.02, 116.57, 120.82, 130.78, 132.07, 138.92, 142.85, 144.27, 145.80, 154.14 , 172.06, 181.31, 184.26 Mass C ₁₉ H ₂₁ NO ₈ M.v. 391.38: 391, 380, 363, 252, 239, 218, 197, 183, 169, 151, 136, 123, 77. T _pl = 58-60°C.

Example 6. Synthesis of methoxyquinone-GABA (4)

A 50 ml round bottom flask was filled with 1 mmol (IN), 1 mmolIN, 6 ml of dioxane, then 1.2 mmol (0.167 ml) of triethylamine was added under argon. The reaction mixture was stirred in an argon atmosphere at room temperature for 24 hours until the starting material completely disappeared (control was carried out by TLC). After stirring, the reaction mixture was evaporated, the product was isolated by column chromatography on 20 g of silica gel, eluting with a mixture of CH₂Cl₂-CH₃OH. A mixture of quinone and hydroquinone was obtained in the form of a red powder. The resulting red powder was placed in a 100 ml Schlenk flask, 0.21 mmol of 10% Pd/C (0.022 g) and 15 ml of CH were added₃OH. The reaction mixture was degassed 3 times and then filled with hydrogen. Hydrogenation was carried out for 24 hours in a hydrogen atmosphere. Upon completion of hydrogenation, the reaction mixture was filtered from the catalyst through a thin layer of celite and washed with CH₃OH, then stirred in air for 5 days. After stirring, the reaction mixture was evaporated to obtain methoxyquinone-GABA (4) in the form of red crystals, yield 47%

¹ H NMR (500 M Hz , DMSO- d ₆ , δ, ppm): 1.58 (pentet, J=7.2 Hz , 2H, CH ₂ ), 2.12 (t, J=8.0 Hz , 2H, CH ₂ ) , 2.19 (t, J=7.5 Hz , 2H, CH ₂ ), 2.47-2.52 (m, 2H, CH ₂ ), 3.01 (sq., J=6.5 Hz , 2H, CH ₂ ), 3.76 (s, 3H, OCH ₃ ), 3.85 (s, 3H, OCH ₃ ), 7.81 (t, J=5.4 Hz , 1H, NH). ¹³ C NMR (151 MHz , DMSO- d6 _, δ, ppm): 19.3, 24.6, 31.0, 34.3, 37.9, 60.0, 60.9, 129.1, 138.0, 143.2, 153.1, 170.8, 174.2, 180. 2, 183.3 . Mass C ₁₅ H ₁₉ NO ₈ M.v. 341.32: 341, 327, 310, 296, 284, 262, 254, 238, 223, 212, 197, 183, 165. T _pl = 121-123°C.

Example 7 Synthesis of methoxyquinone-β-alanine (5)

Synthesized similarly to (4) , we obtained methoxyquinone-β-alanine ( 5) in the form of red crystals, yield 64%

¹ H NMR (500 M Hz , DMSO- d ₆ , δ, ppm): 2.12 (t, J=7.9 Hz , 2H, CH ₂ ), 2.35 (t, J=7.0 Hz , 2H, CH ₂ ) , 2.50 (t, J=7.9 Hz , 2H, ² CH ₂ ), 3.20 (sq., J=6.4 Hz , 2H, CH ₂ ), 3.77 (s, 3H, OCH ₃ ), 3.86 (s, 3H, OCH ₃ ), 7.88 (t, J=5.2 Hz , 1H, NH). ¹³ C NMR (151 MHz , DMSO- d6 _, δ, ppm): 19.2, 33.8, 34.2, 34.8, 60.0, 60.9, 129.0, 138.0, 143.2, 153.1, 170.9, 172.9, 180.2, 183 .3. Mass C ₁₄ H ₁₇ NO ₈ M.v. 327.29: 327, 309, 296, 285, 278, 270, 252, 238, 223, 212, 197, 183, 173. T _pl = 159-160°C.

Example 8. Evaluation of antiradical activity.

The antiradical activity of the compounds was assessed spectrophotometrically by their ability to neutralize the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH test) (Schlesier K., 2002). Solutions of compounds 1-4, prepared in a mixture of ethanol and DMSO 9:1 in final concentrations of 100, 50, 25, 10 μM, were added to DPPH dissolved in 50% ethanol and the optical density (OD519) was measured at a wavelength of 590 nm using Amersham Biosciences Ultraspec 3300 Pro spectrophotometer. The OD519 value was recorded at fixed points in time and presented as a percentage of the initial optical density of the DPPH solution. Data were presented as the average decrease in DPPH concentration from the initial value (0 sec) to 5, 15, 30, 60, 90, 120 and 180 sec of reaction ± standard deviation (M±SD). From 4 to 6 measurements were carried out for each concentration of each test compound. The standard synthetic antioxidant Trolox was used as a comparison drug.

Table 1 shows the ability of compounds (1), (2), (3), (4), (5) at concentrations of 100, 50, 25 and 10 μM) to neutralize DPPH by 3 minutes of reaction in comparison with the standard antioxidant Trolox. The graphs show the dynamics of the development of the reaction of neutralization of the DPPH radical within 3 minutes depending on the concentration of the compounds.

Table 1. Connections Residue of DPPH radical at 3 minutes, %±SD Concentrations, µM 100 50 25 10 Trolox 4.6±0.3 4.7±0.9 35.8±1.0 74.9±0.8 (1) 36.8±1.6** 46.1±0.3** 59.8±0.7** 77.7±0.7 (2) 40.7±1.3** 58.9±0.4** 76.4±0.9** 92.0±0.5** (3) 11.0±1.1* 9.0±0.9* 28.4±1.0* 66.7±2.2* (4) 49.7±0.4** 63.5±1.8** 75.5±0.3** 88.6±0.3** (5) 52.7±0.7** 65.7±0.3** 79.3±0.7** 91.2±0.7**

*- p < 0.05, **- p < 0.001 compared to Trolox at the same time point.

Based on the data presented in Table 1 and Graphs 1 and 2, it is clear that the antiradical activity of compound (3) at concentrations of 100 and 50 μM is slightly lower than Trolox (by 6.4% and 4.3%, respectively), and at concentrations 25 and 10 µM exceed it (by 7.4% and 8.2%, respectively). The antiradical activity of compound (1), which does not contain additional substituents in the form of amino acids in the molecule, is lower than Trolox, but becomes comparable to it at the lowest concentration taken - 10 μM. Compounds (2), (4) and (5) are on average 2 times less effective in reducing the radical content in the incubation medium, but still have antiradical activity. Thus, the addition of compounds that do not have antiradical activity (alanine, GABA) to the quinone molecule reduces the antiradical activity of the molecule, and the addition of a compound that has (dopamine, in compound (3)) enhances it.

We can conclude that compounds (1) and (3) have high antiradical activity, and to a lesser extent (2), (4), (5).

Example 9. Toxicity assessment for neuronal cell culture.

The toxicity of new compounds was studied using human neuroblastoma cell culture SH-SY5Y (ATCC®, USA). Cells were grown at 37°C in DMEM/F-12 culture medium supplemented with fetal bovine serum (10%), L-glutamine (1%) and penicillin-streptomycin (1%). Cells were seeded into 96-well plates and differentiated for 7 days by adding retinoic acid (RA) to the culture medium at a final concentration of 10 μM with a gradual decrease in serum content to 1%.

Toxicity was determined by assessing cell viability using the MTT test. The method is based on the reduction of yellow 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide into blue formazan by living cells and characterizes the reduction potential of the cell cytoplasm. Then the formazan crystals were dissolved in 100 μl of DMSO and the optical density was measured at 570 and 660 nm using a SynergyH1 plate reader (BioTec, USA) (Lopachev, A. V., 2016). The substances under study were added to the culture medium at final concentrations of 500, 100, 50 and 1 µM and incubated for 24 hours.

In experiments on human neuroblastoma cell culture SH-SY5Y, differentiated according to the neuronal type, the toxicity of compounds (1), (2), (3), (4), (5) was studied. It was shown that compounds (2), (3), (4) and (5), taken at a concentration of 500 μM, do not affect cell viability (see Fig. 1). Under the influence of compound (1), culture survival is reduced by 75.5% at a concentration of 500 μM, by 19.9% at a concentration of 100 μM, by 14.8% at a concentration of 50 μM and by 12.2% at a concentration of 10 μM (cm Fig. 2). The minimum non-toxic concentration of compound (1) was 1 µM. Thus, we can conclude that compounds (2), (3), (4) and (5) have low toxicity for neuronal culture, and compound (1) has high toxicity.

In general, a comprehensive study of the antiradical activity and cytotoxicity of compounds (2), (3), (4) and (5) provides grounds for their use as antiradical agents in in vivo systems for the treatment of diseases of the central nervous system accompanied by the formation of free radicals, and also for the delivery of therapeutic amino acids to the brain and further assessment of their biological properties. Compound (1) is characterized by high antiradical activity and cytotoxicity, which can be used to assess its antitumor activity, due to the fact that tumor therapy requires the use of a combined approach, including antiradical activity to reduce the amount of ROS and cytotoxicity aimed at destroying pathological cells . In addition, since compounds (2), (3), (4) and (5) can be obtained from compound (1), it is proposed to use it as a substrate for the synthesis of quinone compounds with any amino acids of interest for delivery to the brain.

Claims (21)

1. Method for preparing a compound of the general formula: where R is selected from: , , , , , characterized in that the starting compound 5,8-dimethoxy-6,7-methylenedioxycoumarin (A) is treated with lead tetraacetate and then with an excess of HCl/iPrOH, after which the resulting dihydroxycoumarin (B) is catalytically hydrogenated in a hydrogen atmosphere to obtain 6,7 -dihydroxy-5,8-dimethoxychroman-2-one (B), according to the scheme: , with further hydrolysis of the compound of formula B, subsequent oxidation with atmospheric oxygen and the production of 5,8-dimethoxy-6,7-methylenedioxycoumarin derivatives with antiradical activity. 2. Method according to claim 1, characterized in that the compound of formula (1): prepared by hydrolyzing the compound of formula B and reacting in an acidic medium at room temperature for 24 hours in an air atmosphere. 3. Method according to claim 1, characterized in that the compound of formula (2): prepared by hydrolysis and reaction of the compound of formula B with 5-methoxytryptamine hydrochloride in dioxane at room temperature using atmospheric oxygen as an oxidizing agent. 4. Method according to claim 1, characterized in that the compound of formula (3): prepared by hydrolysis and reaction of the compound of formula B with 3,4-bis(benzyloxy)phenethylamine in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, under a hydrogen atmosphere for 24 hours and oxidation in air for 24 hours. 5. Method according to claim 1, characterized in that the compound of formula (4): prepared by hydrolysis and reaction of the compound of formula B with benzyl ester of γ-aminobutyric acid in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, in a hydrogen atmosphere for 24 hours and oxidation in air atmosphere for 24 hours. 6. Method according to claim 1, characterized in that the compound of formula (5): prepared by hydrolysis and reaction of the compound of formula B with β-alanine benzyl ester in dioxane at room temperature using atmospheric oxygen as an oxidizing agent, followed by deprotection by hydrogenation on Pd/C, under a hydrogen atmosphere for 24 hours and oxidation in the atmosphere air within 24 hours.