RU2800457C1 - Synthesis of esters of flavonoids naringenin, quercetin, hesperetin - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing esters of naringenin, quercetin, hesperetin. The synthesis of flavonoid esters is described at an equimolar ratio of alcohol:acid — 0.002:0.003, where the alcohol component of the esters is either naringenin or hesperetin, or benzoic acid, or salicylic, or cinnamic acid, or nicotinic acid, also the alcohol component of esters is quercetin, either benzoic acid or nicotinic acid is used as acids, they are dissolved in 50 ml of acetone, the reaction mixture is stirred for 6 hours at a temperature of 50°C at a speed of 120 rpm, changing the direction of stirring after 15 minutes, the synthesis process is controlled by HPLC, after the reaction is completed, the immobilized enzyme is separated by filtration, the reaction medium is purified from excess unreacted acids and diesters by column chromatography, using a mixture of ethyl alcohol 70% as eluent and acetonitrile in a volume ratio of 7:3, the resulting esters are dried for 2 hours at a pressure of 50 mm Hg and a temperature of 60°C and sealed in dark glass ampoules.

EFFECT: obtaining the synthesis of new esters of flavonoids, such as naringenin, quercetin and hesperetin.

1 cl, 12 ex

Description

The invention relates to pharmacy, in particular to the chemical and pharmaceutical industry and relates to a method for obtaining esters of naringenin, quercetin, hesperetin and can be used to obtain drugs based on these compounds.

Flavonoids are classified as natural polyphenols with a wide range of biological effects. The most well-known and studied type of their activity is antioxidant [Flavonoids as Antioxidants / Slobodan V. Jovanovic, Steen Steeden, Mihajlo Tosic, Budimir Marjanovic, Michael G. Simic // J. Am. Chem. SOC. 1994,116, 4846-4851. Predictive model of the relationship between antiradical activity and the ionization potential of molecules and ions of flavonoids / Belaya N.I., Belyi A.V., Shcherbakov I.N. // Kinetics and catalysis. 2020. V. 61. No. 3. S. 334-342]. For flavonoids, antimutagenic, hepatoprotective, anticancer, capillary-strengthening, antiviral and antibacterial actions have also been established and confirmed [Chemistry and biological activities of flavonoids: an overview / Kumar S, Pandey AK. // Scientific World Journal. 2013. 2013; 162750. Flavonoids with inhibitory activity against SARSCoV-2 3CLpro / Seri Jo, Suwon Kim, Dae Yong Kim, Mi-Sun Kim, Dong Hae Shin // Journal of Enzyme Inhibition and Medicinal Chemistry. 2020, 35 (1); 1539-1544 Flavonoid compounds of Artemisia glabella Kar. et Kir., syntheses based on them and their biological activity / Baisarov G.M., Zhumataeva A.R., Mukusheva G.K., Shults E.E., Seidakhmetova R.B., Adekenov S.M. // Chemistry of plant raw materials. 2018. №3. pp. 215-222. United States Patent US 10765660 B2].

However, the widespread use of flavonoids in medicine and pharmacy is limited, and this is primarily due to the fact that they are easily oxidized and have low bioavailability due to low lipophilicity. There is an inverse correlation between the number of hydroxyl groups and the lipophilicity of flavonoids [The uncoupling efficiency and affinity of flavonoids for vesicles / C van Dijk1, A J Driessen, K Recourt // Biochem Pharmacol. 2000, 60(11); 1593-600]. One of the most effective ways to enhance the pharmacological activity and increase the bioavailability of biologically active compounds is the targeted chemical modification of their structure. In particular, an increase in the lipophilization of flavonoids is possible due to the transfer of hydroxyl groups to esters [Enzyme-Mediated Preparation of Flavonoid Esters and Their Applications / Jana Viskupicova, Miroslav Ondrejovic and Tibor Maliar // Biochemistry, 2011. DOI: 10.5772/34174]. The esterification of flavonoids is carried out by traditional chemical synthesis methods or in the presence of biocatalysts [Flavonoids: Chemistry, Biochemistry and Applications. Edited by Øyvind M. Andersen Kenneth R. Markham, 2006, Taylor & Francis Group, LLC. 1212 r.].

Most of the works related to this topic are devoted to the esterification of flavonoids at the hydroxyl groups of the sugar residue. For example, in [Synthesis of naringin 6"-ricinoleate using immobilized lipase / Verônica M Almeida, Carla RC Branco, Sandra A Assis, Ivo JC Vieira, Raimundo Braz-Filho, Alexsandro Branco // Almeida et al. Chemistry Central Journal. 2012 6:41; 2-7.] describes the esterification reaction of naringin with castor oil containing ricinoleic acid, which is the main fatty acid of castor oil.The authors carried out the synthesis using a biocatalyst - immobilized lipase B from Candida antarctica .

A known method for producing esters of rutin, naringin, hesperidin, which are glycosides of quercetin, naringenin and hesperetin, respectively. Esterification was carried out with aliphatic acids, for example, pentanoic and undecylic acids at the hydroxyl groups of the sugar residues of rutin, naringin, hesperidin in the presence of lipase B from Candida antarctica (CALB) [Biocatalytic Synthesis of Flavonoid Esters by Lipases and Their Biological Benefits / Maria Elisa MB de Araújo, Yollanda EM Franco, Marcia CF Messias, Giovanna B. Longato, João A. Pamphile, Patricia de O. Carvalho // Planta Med. 2017; 83:7-22].

However, the literature describes several methods for the synthesis of flavonoid esters based on the acylation of phenolic groups of flavonoid aglycones - quercetin and naringenin [Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones and rapid product screening / E. Kyriakou, A. Primikyri, P. Charisiadis, M. Katsoura, I.P. Gerothanassis, H. Stamatis, A.G. Tzakos, Org. Biomol. Chem., 2012, 10, 1739-1742]. The synthesis was carried out with the participation of lipase B from Candida antarctica (CALB), vinyl acetate or vinyl butyrate was used as the acids of the constituent esters.

In [Ester-Based Precursors to Increase the Bioavailability of Quercetin / Lucia Biasutto, Ester Marotta, Umberto De Marchi, Mario Zoratti, Cristina Paradisi // J. Med. Chem. 2007, 50, 241-253] the results of obtaining pentaacetylquercetin in acetic anhydride and pyridine at reflux for 5 hours are given.

It should be noted that even small changes in the structure of flavonoids lead to the manifestation of new properties, for example, antiviral, anti-inflammatory and anti-cancer effects have been established for quercetin monomethyl esters. The preparation of esters of naringin and hesperedin with butanoic and decanoic acids resulted in the potentiation of their antifungal activity. The introduction of an oleic acid residue into the rutin molecule enhanced its antioxidant properties, the ester of isoquercetin and eicosapentaenoic acid showed more pronounced anti-inflammatory properties [Biocatalytic Synthesis of Flavonoid Esters by Lipases and Their Biological Benefits / Maria Elisa M. B. de Araújo, Yollanda E. M. Franco, Marcia C. F. Messias, Giovanna B. Longato, João A. Pamphile, Patricia de O. Carvalho // Planta Med. 2017; 83:7-22]. We assume that the preparation of esters of naringenin, quercetin, and hesperetin will most likely reduce their high metabolic activity, increase lipophilicity, and lead to potentiation of their angioprotective and vasodilating activities, for example, upon esterification with nicotinic acid.

The following were chosen as prototypes: 1) a method for obtaining esters of rutin and naringin [Regioselective acylation of flavonoids catalyzed by lipase in low toxicity media / Athina Kontogiannia, Vasso Skouridoua, Vasiliki Seretia, Haralambos Stamatisb, Fragiskos N.Kolisis/ Eur. J. Lipid Sci. Technol. 103 (2001) 655-660], according to which the esters of naringin and rutin and fatty acids (octanoic, decanoic and dodecanoic) were obtained in the presence of lipase B from Candida antarctica (Novozyme 435), using water and tert-butanol as solvents with the addition of salts of lithium chloride, magnesium chloride, cobalt chloride, potassium nitrate, and sodium nitrate; .3) (volume percent) in the case of esters of naringin and chloroform/methanol/water (8/2/0.3) (volume percent) in the case of esters of rutin. 2) a method for the synthesis of esters of quercetin, naringenin and vinyl acetate or vinyl butyrate [Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones and rapid product screening / E. Kyriakou, A. Primikyri, P. Charisiadis, M. Katsoura, IP Gerothanassis, H. Stamatis, A. G. Tzakos // Org. Biomol. Chem., 2012, 10, 1739-1742] in acetonitrile medium, in the presence of lipase B from Candida antarctica (CALB), for 72 hours, at a temperature of 60°C.

The claimed invention aims at the synthesis of esters of flavonoids, in particular esters:

naringenin and benzoic acid;

naringenin and salicylic acid;

naringenin and cinnamic acid;

naringenin and nicotinic acid;

hesperitin and benzoic acid;

hesperitin and salicylic acid;

hesperitin and cinnamic acid;

hesperitin and nicotinic acid;

quercetin and benzoic acid;

quercetin and salicylic acid;

quercetin and cinnamic acid;

quercetin and nicotinic acid.

The essential distinguishing features of the invention are the following features:

esters were obtained for agnikone flavonoids, namely for naringenin, hesperitin, quercetin;

esters obtained using benzoic acid, salicylic acid, cinnamic acid, nicotinic acid;

mixing speed - 120 rpm, change of direction after 15 minutes;

process temperature - 50°C;

drying the obtained compounds - for 2 hours, at a pressure of 50 mm Hg. Art., temperature 60°C;

purification of target compounds by column chromatography with eluent - a mixture of ethyl alcohol 70% and acetonitrile in a volume ratio of 7:3.

Establishment of the structure of the obtained esters was carried out by NMR and mass spectrometry:

Naringenin 4'-benzoate (4-(5,7-dihydroxy-4-oxochroman-2-yl)phenyl benzoate) (1). Yield 0.655 g (87%), m.p. 229-232°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 2.9, 17.0), 3.25 dd (1H, eq -H ³ , J = 12.9, 17.1), 5.44 dd (1H, H ² , J = 3.0, 12.7), 5.92 m (2H, H ^6.8 ), 6.88 d (2H, H ^3',5' , J = 8.8), 7.33 d (2H, H ^{2 ',6'} , J = 8.4), 7.45 d (2Н, Н ^3'',5'' , J = 8.5), 7.58 t (1Н, Н ^4'' , J = 7.3), 8.07 d (2Н, Н ^{2'', 6''} , J = 2.0), 10.78 s (1H, OH ⁷ ), 12.15 s (1H, OH ⁵ ). Mass spectrum, m/z: 377.09 [M + H] ⁺ (calculated for C ₂₂ H ₁₆ O ₆ H ⁺ : 377.10).

Naringenin 4'-salicylate (4-(5,7-dihydroxy-4-oxochroman-2-yl)phenyl 2-hydroxybenzoate) (2). Yield 0.670 g (86%), m.p. 251-253°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 2.9, 17.0), 3.25 dd (1H, eq -H ³ , J = 12.9, 17.1), 5.44 dd (1H, H ² , J = 3.0, 12.7), 5.92 m (2H, H ^6.8 ), 6.88 d (2H, H ^3',5' , J = 8.8), 6.91 t (1H, H ^{5 ''} , J = 7.4), 6.97 d (1H, H ^3'' , J = 7.5), 7.33 d (2H, H ^2',6' , J = 8.4), 7.53 t (1H, H ^4'' , J = 7.3), 7.82 d (1H, H ^6'' , J = 7.7), 10.78 s (1H, OH ⁷ ), 11.53 s (1H, OH ^2'' ), 12.15 s (1H, OH ⁵ ). Mass spectrum, m/z: 393.08 [M + H] ⁺ (calculated for C ₂₂ H ₁₆ O ₇ H ⁺ : 393.09).

Naringenin 4'-cinnamate (4-(5,7-dihydroxy-4-oxochroman-2-yl)phenyl cynamate) (3). Yield 0.685 g (85%), m.p. 234-237°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 2.9, 17.0), 3.25 dd (1H, eq -H ³ , J = 12.9, 17.1), 5.44 dd (1H, H ² , J = 3.0, 12.7), 5.92 m (2H, H ^6.8 ), 6.45 d (1H, H ^2''' , J = 15.9), 6.88 d (2H, H ^3',5' , J = 8.8), 7.33 d (2H, H ^2',6' , J = 8.4), 7.40 t (2Н, Н ^3'',5'' , J = 7.9), 7.45 d (1Н, H ^4'' , J = 7.3), 7.55 d (2H, H ^2'',6'' , J = 16.0), 7.81 d (1H, H ^3''' , J = 15.9), 10.78 s (1H, OH ⁷ ), 12.15 s (1H, OH ⁵ ). Mass spectrum, m/z: 403.09 [M + H] ⁺ (calculated for C ₂₄ H ₁₈ O ₆ H ⁺ : 403.11).

Naringenin 4'-nicotinate (4-(5,7-dihydroxy-4-oxochroman-2-yl)phenyl nicotinate) (4). Yield 0.664 g (88%), m.p. 245-247°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 2.9, 17.0), 3.25 dd (1H, eq -H ³ , J = 12.9, 17.1), 5.44 dd (1H, H ² , J = 3.0, 12.7), 5.92 m (2H, H ^6.8 ), 6.88 d (2H, H ^3',5' , J = 8.8), 7.33 d (2H, H ^{2 ',6'} , J = 8.4), 7.57 t (1H, H ^5'' , J = 8.0), 8.31 d (1H, H ^4'' , J = 7.9), 8.81 d (1H, H ^6'' , J = 5.0), 9.11 s (1Н, ^Н2'' ), 10.78 s (1H, OH ⁷ ), 12.15 s (1H, OH ⁵ ). Mass spectrum, m/z: 378.07 [M + H] ⁺ (calculated for C ₂₁ H ₁₅ NO ₆ H ⁺ : 378.09).

Hesperetin 4'-benzoate (5-(5,7-dihydroxy-4-oxochroman-2-yl)-2-methoxyphenyl benzoate) (9). Yield 0.655 g (87%), m.p. 229-232°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 3.0, 17.0), 3.24 dd (1H, eq -H ³ , J = 12.9, 17.2), 3.78 s (3H, -OCH ₃ ), 5.42 dd (1H, H ² , J = 3.0, 12.7), 5.88 m (2H, H ^6.8 ), 6.96 d (1H, H ^5' , J = 8.5), 7.02 dd (1H, H ^6' , J = 8.3), 7.11 d (1H, H ^2' , J = 2.5), 7.45 d (2H, H ^3'',5'' , J = 8.5), 7.58 t ( 1H, H ^4'' , J = 7.3), 8.05 d (2H, H ^{2'', 6''} , J = 2.0), 10.71 s (1H, OH ⁷ ), 12.12 s (1H, OH ⁵ ). Mass spectrum, m/z: 407.10 [M + H] ⁺ (calculated for C ₂₃ H ₁₈ O ₇ H ⁺ : 407.11).

Hesperetin 4'-salicylate (5-(5,7-dihydroxy-4-oxochroman-2-yl)-2-methoxyphenyl 2-hydroxybenzoate) (10). Yield 0.701 g (83%), m.p. 251-253°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 3.0, 17.0), 3.24 dd (1H, eq -H ³ , J = 12.9, 17.2), 3.78 s (3H, -OCH ₃ ), 5.42 dd (1H, H ² , J = 3.0, 12.7), 5.88 m (2H, H ^6.8 ), 6.96 d (1H, H ^5' , J = 8.5), 7.02 dd (1H, H ^6' , J = 8.3), 7.11 d (1H, H ^2' , J = 2.5), 7.45 d (2H, H ^3'',5'' , J = 8.5), 7.58 t ( 1Н, Н ^4'' , J = 7.3), 7.79 d (1Н, Н ^6'' , J = 7.7), 10.71 s (1H, OH ⁷ ), 11.53 s (1H, OH ^2'' ), 12.12 s ( 1H,OH ⁵ ). Mass spectrum, m/z: 423.07 [M + H] ⁺ (calculated for C ₂₃ H ₁₈ O ₈ H ⁺ : 423.10).

Hesperetin 4'-cinnamate (5-(5,7-dihydroxy-4-oxochroman-2-yl)-2-methoxyphenyl cinnamate) (11). Yield 0.727 g (84%), m.p. 231-233°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 3.0, 17.0), 3.24 dd (1H, eq -H ³ , J = 12.9, 17.2), 3.78 s (3H, -OCH ₃ ), 5.42 dd (1H, H ² , J = 3.0, 12.7), 5.88 m (2H, H ^6.8 ), 6.45 d (1H, H ^2''' , J = 15.9 ), 6.96 d (1H, H ^5' , J = 8.5), 7.02 d (1H, H ^6' , J = 8.3), 7.11 d (1H, H ^2' , J = 2.5), 7.45 d (2H, H ^3'',5'' , J = 8.5), 7.58 t (1H, H ^4'' , J = 7.3), 7.81 d (1H, H ^3''' , J = 15.9), 8.07 d (2H, H ^{2'', 6''} , J = 2.0), 10.71 s (1H, OH ⁷ ), 12.12 s (1H, OH ⁵ ). Mass spectrum, m/z: 433.11 [M + H] ⁺ (calculated for C ₂₅ H ₂₀ O ₇ H ⁺ : 433.12).

Hesperetin 4'-nicotinate 5-(5,7-dihydroxy-4-oxochroman-2-yl)-2-methoxyphenyl benzoate) (9). Yield 0.694 g (85%), m.p. 244-246°C. 1Н NMR spectrum, δ, ppm (J, Hz): 2.69 dd (1H, ax -H ³ , J = 3.0, 17.0), 3.24 dd (1H, eq -H ³ , J = 12.9, 17.2), 3.78 s (3H, -OCH ₃ ), 5.42 dd (1H, H ² , J = 3.0, 12.7), 5.88 m (2H, H ^6.8 ), 6.96 d (1H, H ^5' , J = 8.5), 7.02 dd (1H, H ^6' , J = 8.3), 7.11 d (1H, H ^2' , J = 2.5), 7.57 t (1H, H ^5'' , J = 8.0), 8.31 d (1H, H ^{4 ''} , J = 7.9), 8.81 d (1Н, Н ^6'' , J = 5.0), 9.11 s (1Н, Н ^2'' ), 10.71 s (1H, OH ⁷ ), 12.12 s (1H, OH ⁵ ). Mass spectrum, m/z: 408.08 [M + H] ⁺ (calculated for C ₂₂ H ₁₇ NO ₇ H ⁺ : 408.10).

Quercetin 4'-benzoate (2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl benzoate) (5). Yield 0.715 g (88%), m.p. 289-292°C. 1Н NMR spectrum, δ, ppm (J, Hz): 6.19 d (1H, H ⁶ , J = 2.0), 6.41 d (1H, H ⁸ , J = 2.0), 6.88 d (1H, H ^5' , J = 8.4), 7.45 d (2H, H ^3'',5'' , J = 8.5), 7.54 dd (1H, H ^6' , J = 2.2, J = 8.4), 7.58 t (1H, H ^{4 ''} , J = 7.3), 7.67 d (1H, H ^2' , J = 2.0), 8.07 d (2H, H ^{2'', 6''} , J = 2.0), 9.35 s (1H, OH ^3' ) , 9.55 s (1H, OH ³ ), 10.76 s (1H, OH ⁷ ), 12.48 s (1H, OH ⁵ ). Mass spectrum, m/z: 407.04 [M + H] ⁺ (calculated for C ₂₂ H ₁₄ O ₈ H ⁺ : 407.07).

Quercetin 4'-salicylate (2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl 2-hydroxybenzoate) (6). Yield 0.717 g (85%), m.p. 322-324°С. 1Н NMR spectrum, δ, ppm (J, Hz): 6.19 d (1H, H ⁶ , J = 2.0), 6.41 d (1H, H ⁸ , J = 2.0), 6.88 d (1H, H ^5' , J = 8.4), 6.91 t (1Н, Н ^5'' , J = 7.4), 6.97 d (1Н, Н ^3'' , J = 7.5), 7.54 dd (1H, H ^6' , J = 2.2, J = 8.4), 7.58 t (1H, H ^4'' , J = 7.3), 7.67 d (1H, H ^2' , J = 2.0), 8.01 d (1H, H ^6'' , J = 7.7), 9.35 s (1H, OH ^3' ), 9.55 s (1H, OH ³ ), 10.76 s (1H, OH ⁷ ), 11.53 s (1H, OH ^2'' ), 12.48 s (1H, OH ⁵ ). Mass spectrum, m/z: 423.06 [M + H] ⁺ (calculated for C ₂₂ H ₁₄ O ₉ H ⁺ : 423.06).

Quercetin 4'-cinnamate (2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl cinnamate) (7). Yield 0.760 g (88%), m.p. 297-299°С. 1Н NMR spectrum, δ, ppm (J, Hz): 6.19 d (1H, H ⁶ , J = 2.0), 6.41 d (1H, H ⁸ , J = 2.0), 6.45 d (1H, H ^2''' , J = 15.9), 6.88 d (1H, H ^5' , J = 8.4), 7.45 d (2H, H ^3'',5'' , J = 8.5), 7.54 d (1H, H ^6' , J = 2.2, J = 8.4), 7.58 t (1H, H ^4'' , J = 7.3), 7.67 d (1H, H ^2' , J = 2.0), 7.81 d (1H, H ^3''' , J = 15.9), 8.07 d (2Н, Н ^{2'', 6''} , J = 2.0), 9.35 s (1H, OH ^3' ), 9.55 s (1H, OH ³ ), 10.76 s (1H, OH ⁷ ) , 12.48 s (1H, OH ⁵ ). Mass spectrum, m/z: 433.07 [M + H] ⁺ (calculated for C ₂₄ H ₁₆ O ₈ H ⁺ : 433.09).

Quercetin 4'-nicotinate (2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl nicotinate) (8). Yield 0.685 g (84%), m.p. 308-310°С. 1Н NMR spectrum, δ, ppm (J, Hz): 6.19 d (1H, H ⁶ , J = 2.0), 6.41 d (1H, H ⁸ , J = 2.0), 6.88 d (1H, H ^5' , J = 8.4), 7.54 dd (1H, H ^6' , J = 2.2, J = 8.4), 7.57 t (1H, H ^5'' , J = 8.0), 7.67 d (1H, H ^2' , J = 2.0), 8.31 d (1Н, Н ^4'' , J = 7.9), 8.81 d (1Н, Н ^6'' , J = 5.0), 9.11 s (1Н, Н ^2'' ), 9.35 s (1H, OH ^3' ), 9.55 s (1H, OH ³ ), 10.76 s (1H, OH ⁷ ), 12.48 s (1H, OH ⁵ ). Mass spectrum, m/z: 408.07 [M + H] ⁺ (calculated for C ₂₁ H ₁₃ NO ₈ H ⁺ : 408.06).

The proposed method includes the following steps:

1) equimolar weights of flavonoid (or naringenin, or hesperitin, or quercetin) and acid (or benzoic, or salicylic, or cinnamic, or nicotinic) are dissolved in 50 ml of acetone;

2) Novozyme 435 catalyst (Novozyme 435) (Cat. No. L4777 Sigma-Aldrich) is added to the reaction medium;

3) the reaction medium is heated to 50°C;

4) reaction time - 6 hours, stirring speed 120 rpm, change of stirring direction after 15 minutes;

5) after 6 hours, the immobilized enzyme is separated by filtration;

6) the resulting solution is purified from excess unreacted acids and diesters on a column filled with Silica gel 60, a mixture of ethyl alcohol 70% and acetonitrile in a volume ratio of 7:3 is used as an eluent

7) the purity of the fractions is controlled by HPLC, determining the quantitative content of the reaction products and the residual content of the starting components;

8) a fraction containing either naringenin 4'-benzoate, or naringenin 4'-salicylate, or naringenin 4'-cinnamate, or naringenin 4'-nicotinate, or hesperetin 4'-benzoate, or hesperetin 4'-salicylate, or hesperetin 4 '-cinnamate, or hesperetin 4'-nicotinate, or quercetin 4'-benzoate, or quercetin 4'-salicylate, or quercetin 4'-cinnamate, or quercetin 4'-nicotinate, dried for 2 hours, at a pressure of 50 mm Hg . Art. and temperature 60°С

9) the obtained substances are sealed in dark glass ampoules.

An example of obtaining naringenin 4'-benzoate : 2 mmol of naringenin (0.544 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.366 g) of benzoic acid 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 ° With a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing naringenin 4'-benzoate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of naringenin 4'-salicylate : naringenin (0.544 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.414 g) of salicylic acid and 0.5 g of Novozyme 435 were added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 ° C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing naringenin 4'-salicylate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of naringenin 4'-cinnamate : 2 mmol of naringenin (0.544 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.444 g) of cinnamic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing naringenin 4'-cinnamate (at least 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of naringenin 4'-nicotinate: 2 mmol of naringenin (0.544 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.369 g) of nicotinic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing naringenin 4'-nicotinate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of hesperetin 4'-benzoate : 2 mmol of hesperetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.366 g) of benzoic acid and 0.5 g of Novozyme 435 were added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing hesperetin 4'-benzoate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of hesperetin 4'-salicylate : 2 mmol of hesperetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.414 g) of salicylic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing hesperetin 4'-salicylate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of hesperetin 4'-cinnamate : 2 mmol of hesperetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.444 g) of cinnamic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing hesperetin 4'-cinnamate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of the preparation of hesperetin 4'-nicotinate : 2 mmol of hesperetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.369 g) of nicotinic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing hesperetin 4'-nicotinate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

An example of obtaining quercetin 4'-benzoate : 2 mmol of quercetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.366 g) of benzoic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing quercetin 4'-benzoate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

Example of obtaining quercetin 4'-salicylate : 2 mmol of quercetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.414 g) of salicylic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing quercetin 4'-salicylate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

Example of obtaining quercetin 4'-cinnamate : 2 mmol of quercetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.444 g) of cinnamic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing quercetin 4'-cinnamate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

Example of obtaining quercetin 4'-nicotinate : 2 mmol of quercetin (0.604 g) was dissolved in 50 ml of acetone, an excess of 3 mmol (0.369 g) of nicotinic acid and 0.5 g of Novozyme 435 was added. The resulting reaction mixture was stirred for 6 hours at a temperature of 50 °C at a speed of 120 rpm, changing the direction of mixing after 15 minutes. After completion of the reaction, the immobilized enzyme was separated by filtration. The resulting solution was purified from excess unreacted acids and diesters on a column filled with Silica gel 60. A mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 was used as an eluent; the purity of the fraction was controlled by HPLC. The resulting fraction containing quercetin 4'-nicotinate (not less than 99.0%) was dried for 2 hours at a pressure of 50 mm Hg. Art. and a temperature of 60°C. The resulting substance was sealed into dark glass ampoules.

The proposed method for the synthesis of esters of naringenin, quercetin, hesperetin with model acids (benzoic, salicylic, cinnamic, nicotinic) can later be used to obtain new original domestic drugs.

Claims (1)

Synthesis of flavonoid esters at an equimolar ratio of alcohol : acid - 0.002:0.003, where the alcohol component of the esters is either naringenin or hesperetin, either benzoic acid, or salicylic, or cinnamic acid, or nicotinic acid is used as acids, and the alcohol component of the esters is quercetin, either benzoic acid or nicotinic acid is used as acids, which are dissolved in 50 ml of acetone, the reaction mixture is stirred for 6 hours at a temperature of 50 ° C at a speed of 120 rpm, changing the direction of stirring after 15 minutes, the synthesis process is controlled by HPLC, after the reaction is completed, the immobilized enzyme is separated by filtration, the reaction medium is purified from excess unreacted acids and diesters by column chromatography, using a mixture of 70% ethyl alcohol and acetonitrile in a volume ratio of 7:3 as an eluent, the obtained esters are dried for 2 h at a pressure of 50 mmHg Art. and a temperature of 60°C and sealed in dark glass ampoules.