FIELD OF THE INVENTION

The present invention relates to the field of organic chemistry and pharmacology, and relates to a novel gossypol polymer derivative, namely to gossypol covalently bound to dialdehyde polysaccharides, and a process of preparing thereof .

BACKGROUND OF THE INVENTION

Gossypol-2, 2 'bis (1, 6, 7-trihydroxy-5-isopropyl-3- methyl-8-formylnaphtalene) (C ₃ oH ₃ o0 ₈ ) is a natural polyphenol extracted from a cotton plant and cotton seed oil, and has the following formula:

^30^30^8

Gossypol molecule

A gossypol molecule consists of two identical moieties connected through the single C-C bond, and form a dihedral angle φ close to on average 90°, between the planes of naphthalene cycles. Rotation around the C-C bond is sterically hindered, and gossypol extracted from cotton is therefore a racemic mixture of (-) and (+) separable enantiomers :

Chemical and (+)/(-) enantiomeric structure of gossypol

(-) Enantiomer of gossypol is more active and toxic than (+) enantiomer and a racemic mixture.

In addition to enantiomerism, gossypol is also characterized by symmetric and asymmetric tautomerism. Depending on a solvent, it can exist in three tautomeric forms: aldehyde, lactol and ketone forms:

aldehyde form lactol form

I

ketone form

III

Structure of tautomers and numbering of atoms in a gossypol molecule

The aldehyde . form dominates in undissolved solid gossypol and in nonpolar solvents, such as chloroform and dichloromethane . Ketone tautomer exists in aqueous alkaline solutions and is unstable in neutral and acidic solvents where it can transit into the aldehyde form. In polar solvents, gossypol is in equilibrium between lactol and aldehyde forms, and the equilibrium is shifted toward the lactol tautomer when the solvent nucleophilicity increases, for example in dimethylsulfoxide . Symmetrical ketone tautomer of gossypol forms stable complexes with ions of two- and three-valent iron and zinc. Cations of nickel and copper shift the equilibrium towards the formation of the lactol form [Juanjuan Yin, Chemical modification and biological activity exploration of the natural product- gossipol, PhD Dissertation, Clemson University, 2010] .

Gossypol is a highly active substance and has a number of pharmacological activities. Studies of antiviral, antiproliferative, antioxidant and interferon-inducing activity have been reported. In patent publications, gossypol and derivatives thereof are proposed to be used as anticancer agents, agents for the treatment of child autism, herpes, as well as in medicaments for the treatment of human immunodeficiency virus (HIV) , hepatitis C, and generalized fungal infections. Patent publication [RU2270708] discloses the use of gossypol and derivatives thereof as antiviral agents which are effective in the treatment of diseases caused by influenza viruses A and B virus, as well as of adenoviral infections.

Despite its unique set of pharmacological properties, gossypol is highly toxic, so, along with high antiviral activity, gossypol-based drugs have a number, of side effects. Gossypol inhibits male fertility and is highly cytotoxic for rapidly dividing cells. One of the most effective approaches to reduce the toxicity of physiologically active compounds is to attach them to a polymer carrier, which allows, in some cases, the use of highly active substances for the treatment, while avoiding side effects associated with systemic toxic action.

The literature describes polymer derivatives of vaccination-type pharmacologically active compounds (PAC) containing a drug covalently bound to a biodegradable polysaccharide polymer carrier [N.A. Plate, A.N. Vasilev, Physiologically active polymers. Moscow, Chemistry, 1986, 296] . The binding of pharmacologically active substances (PAC) to a polymer carrier through a bond helps to reduce toxicity and improve solubility, and, in some cases, allows targeted delivery of the resulting polymer drug to a target organ. Such polymers contain, as a rule, peptide drugs attached by the peptide amino group to form an azomethine bond between the molecule of the physiologically active substance and the polymer carrier. A gossypol molecule has no amine groups and cannot be therefore bound through the azomethine bond.

Patent literature [UZ IAP 04811] also describes a gossypol polymer derivative having antiviral properties, which ^' can be considered as an analogue, wherein said gossypol polymer derivative is bound with carboxymethylcellulose to form a carboxymethylcellulose- gossypol copolymer in the form of a sodium salt. The copolymer is described to comprise, the following units: sodium 2, 3-diethoxy-6-0-carboxymethyl (1→4) - β-D-oxyglucose- diethoxy-gossypolate (C46PH57017Na) ; 2-ethoxy ( 1→4 ) - ( β-D- oxyglucose-diethoxy-gossypolate (C42H50O12) ; 2-0- carboxymethyl- (1→4) - β-D-glucose . (C8H1107Na) ; 2,6- dicarboxymethyl- (1-.4) -β-D-glucose (C10H12O9Na2 ) . The chemical structure of the copolymer according to said analogue cannot be determined due to non-nomenclature names of the compounds, used to describe thereof. In the description, the authors use both nomenclature and trivial (oxyglucose, ethoxy-gossypolate ) formulas in any combination, as well as the trademark (TselAgrip) . It can be assumed that, the gossypol molecule according to the prototype comprises an ethyl alcohol residue, and is bound to a polysaccharide derived from carboxymethylcellulose. Thus, in the above-mentioned invention, other chemical compounds, rather than gossypol, are bound to carboxymethylcellulose .

An antiviral conjugate of gossypol with sodium carboxymethylcellulose having a molecular weight of 780- 180000 Da is an analogue to the present invention, wherein the gossypol : sodium carboxymethylcellulose ratio is (1- 5) : (99-95) wt.%, and the content of a low molecular-weight fraction is up to 20%, and the content of a high molecular- weight fraction of 780-1500 Da is up to 80% [RU 2499002] .

The closest prior art to the proposed invention is an antiviral sodium salt of a carboxymethylcellulose-gossypol copolymer of _. the following formula:

where

a:b:c = 1: (3-6) : (5-7) ,

with a molecular weight of 120 , 000-130 , 000 , and of the following empirical formula:

[ (C38H39NaOi ₅ ) a(C6Hio0 ₅ ) b(G8HuNa07) c ] n,

as disclosed in [RU 22707087] . Another publication [RU 2453559] describes an improved process for preparing said gossypol polymer derivative and the use thereof in complex therapy of patients with autistic disorders. Known processes for preparing sodium salt of the gossypol-carboxymethylcellulose copolymer, which is the closest prior art to the claimed invention, comprise reacting gossypol with oxidized dialdehyde carboxymethylcellulose under various conditions. However, the oxidized cellulose has not been isolated and described. It is known that oxidation of cellulose under the same conditions can result in the formation of various products: dialdehyde carboxymethylcellulose, corresponding oxyacid and diacid. The direction of the reaction depends on many factors difficult to control. They include light intensity that affects the radical process of the Malaprade periodate oxidation, the rate of molecular iodine removal, as well as trace contaminants in the original carboxymethylcellulose. Furthermore, both trace contaminants in gossypol and the presence of oxidants - air oxygen dissolved in the medium can affect the oxidation and addition of gossypol.

SUMMARY OF THE INVENTION

The object of the present invention is to extend the range of pharmacologically active gossypol polymer derivatives which exhibit the pharmacological activity inherent in gossypol and have a controlled toxicity level depending on a method of chemical modification of the aldehyde groups in gossypol. Extension of the current range of antiviral agents is a regular work aimed to prevent addiction and development of new forms of infections resistant to existing drugs.

The present invention relates to a novel gossypol polymer derivative having the following general formula

(I) :

wherein: R ¹ is Sach; R ¹¹ is Sach or H;

Sach is an oxidized hexose or pentose unit in a polysaccharide having one of the following formulas:

wherein R is -CH ₂ OH; -CH (OH) CH ₂ OH; -CH2OR ¹ ; CH (OH) CH2OR ¹ ; R ¹ is -CH2CH2OH ; -CH ₂ CH ₂ NH ₂ ; -CH ₂ C(0)OH; - SO3H , -OSO3H ; -CH ₂ C (0) OR ¹¹ ; and

R ¹¹ is Alk,

and a molecular weight of from 1 to 2000 kDa, preferably from 3 to 80 kDa.

Another object of the present invention is a process for preparing a gossypol polymer derivative of general formula (I), comprising oxidizing a polysaccharide with iodic acid or a salt thereof to form an oxidized polysaccharide containing oxygenated cycles closed by an acetal or azomethine bond, followed by isolation of the formed oxidized polysaccharide, and reacting said oxidized polysaccharide with gossypol or a derivative thereof at a pH value of from 3.5 to 14 and a molar ratio of gossypol to the oxidized polysaccharide unit of from 10:1 to 1:100.

Still another object of the invention is a product having antiviral activity, prepared by the above process.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a ¹³ C-NMR spectrum of initial carboxymethylcellulose in a D ₂ 0 solution with assignment of the signals.

Figure 2 shows fragments of ¹³ C NMR spectra of (1)- carboxymethylcellulose; ( 2 ) -dialdehyde carboxymethylcellulose with an oxidation degree of 65%; and (3) -dialdehyde carboxymethylcellulose with, an oxidation degree of 80%.

Figure 3 shows ¹³ C NMR spectrum of a gossypol polymer derivative covalently bound to DACMC prepared in an excess of gossypol.

Figure 4 shows the ¹³ C NMR spectrum of a gossypol polymer derivative covalently bound to dialdehyde dextrane (DAD) prepared in an excess of gossypol.

DETAILED DESCRIPTION OF THE INVENTION

Gossypol-containing raw material used in the invention includes both gossypol in the form of a single compound and any derivatives thereof, preferably gossypol-acetic acid clathrate referred hereinafter to as gossypol-acetic acid or GAA.

A carrier polymer used in the invention is a polysaccharide. The polysaccharide is pentosane and hexosane, as well as a heteropolysaccharide, i.e. poly (pyranose) , poly ( furanose ) , a mixed polysaccharide and a derivative thereof, namely glucan, xylan, mannan, galactan, fructosane, arabinan, arabinogalactan, glycosaminoglycan, glucuronxylane , glucomannan, galactoglucomannan, arabinogalactan, sulfo-polysaccharide, sulfamino, and aminopolysaccharide, which comprise, in the hydrocarbon chain, vicinal hydroxyl groups or a hydroxyl group and an amino group, which are capable of entering into the Malaprade periodate oxidation reaction [A. Berka, Ya. Vulterin, Ya . Zyka, New redox methods in analytical chemistry, trans, from the Czech., Moscow, 1968, p. 114- 127. V.Ya. Zaharans, "Izvestiya AN Latviiskoi SSR, Ser. Khim. . " , 1982, No.l, pp.3-18] to form dialdehyde polysaccharide, cyclic hemiacetal or cyclic azomethine thereof, according to the following scheme:

Scheme of oxidation of polysaccharides to form cyclic emiacetal and azomethine

wherein R is -CH ₂ OH, -CH (OH) CH ₂ 0H, -CH ₂ OR ¹ , CH (OH) CH ₂ OR ¹ ;

R ¹ is -CH ₂ CH ₂ OH, -CH ₂ CH ₂ NH ₂ , -CH ₂ C(0)OH, -S0 ₃ H, -OSO3H , CH ₂ C (0)OR ,·

R ¹¹ is Alk; and

n is polymerization degree.

Polysaccharides and derivatives thereof used in the invention are linear and branched, as well as polymorph (heterounit) polysaccharides and derivatives thereof, which comprise in one macromolecule different types of monosaccharide molecules bound through different types of glycosidic bonds. Examples of the polysaccharides include polysaccharides, such as carboxymethylcellulose, oxyethylcellulose, hemicellulose , dextran and derivatives thereof, alginic acid, starch and derivatives thereof, such as oxyethyl starch, carboxymethyl starch, aminoethyl starch, carrageenan, chitosan, ficoll and derivatives thereof .

The above-mentioned polysaccharides are hardly suitable for covalent binding of gossypol before the periodate oxidation and require a chemical modification to produce reactive derivatives. The reaction of activation of a polymer carrier, used in the invention includes the reaction of periodate oxidation to form cyclic dialdehyde polysaccharides, namely dialdehyde polysaccharides, wherein oxidized units are closed by a hemiacetal or azomethine bond to form a 5-, 6- or 7-membered cycle, as shown in the above "Scheme of oxidation of polysaccharides to form cyclic hemiacetals and azomethines" .

For this purpose, in the invention they are oxidized with periodic acid or a salt thereof, preferably sodium or potassium metaperiodate in an aqueous or aqueous-organic medium. Two methods are used: homophase oxidation with sodium metaperiodate in an aqueous solution or heterophase oxidation by passing a polysaccharide solution through an anion-exchange resin in the I0 ₄ ^~ form. Homophase oxidation is preferred under laboratory and semi-preparative conditions. It allows easy control of the oxidation degree by changing the ratio of reactants. The heterophase method is suitable under plant conditions because it avoids a step of purification of the product from salts and residual- amounts of periodate. Both the proposed methods lead to the formation of polymers having similar chemical structures and properties.

The number of oxidized units depends on thepolysaccharide : periodate ratio, reaction time, and structure of the units of a polysaccharide backbone. Thus, if the unit structure, like in carboxymethylcellulose (CMC) , comprises only one pair of vicinal hydroxyl groups capable of entering to the Malaprade reaction, then one mole of periodate is consumed in the oxidation of one mole of the units, and the amount of oxidized units is equivalent to the amount of periodate, in the following reaction:

Scheme of reaction of the periodate oxidation of carboxymethylcellulose to form dialdehyde carboxymethylcellulose

If the unit structure has three vicinal hydroxyl groups, like, for example, in dextran, then two molecules of periodate can be consumed to its oxidation, and the dependence of the oxidation degree on the amount of periodate is more complex (see Table 1) . The oxidation degree was defined as the number of oxidized units in 100 sugar moieties of the polysaccharide, regardless of their structure. In the case of CMC, the oxidation degree was calculated by summarizing both carboxymethylated residual non-carboxymethylated glucose units. In the case of dextran, the calculation was made by summarizing all units formed during the oxidation, regardless of their chemical structure. The oxidation degree of dialdehyde polysaccharides according to the invention is within a range of from 0.1 units per 100 sugar units of the polysaccharide (γ = 0.1) to 100 units per 100 sugar units of the polysaccharide (γ = 100), which is equivalent to the content of from 1 oxidized unit per 1000 saccharide units of the polysaccharide to the _. completely oxidized polysaccharide .

A dextran molecule has three vicinal hydroxyl groups in its anhydroglucose unit (AGU) . Depending on the dextran : periodate ratio, oxidation can proceed in two steps to form products of three types: A, B and C, according to the following scheme:

Scheme of the periodate oxidation of dextran to form dialdehyde dextran

One mole of periodate is consumed in the first step, wherein during the oxidation, the C(3)-C(4) or C(2)-C(3) bond disrupted to form A and B units, respectively. The second mole of periodate is consumed in the second step, wherein the oxidation of the formed dialdehyde unit is accompanied by the rupture of the C(2)-C(3) bond in the product A or C(3)-C(4) bond in the product B, respectively, and the release of formic acid and formation of unit C. The rate of the oxidation reaction in the second step is known to be approximately equal to that in the first step, and the resulting polysaccharide can therefore simultaneously contain all three types of the units. The units do not exist in the open form and immediately form seven-membered ( and B) and six-membered (C) cyclic hemiacetals, respectively, as shown in the "Scheme of the periodate oxidation of dextran to form dialdehyde dextran".

The ratio of the A, B and C units after completion of the reaction depends on the ratio of periodate :AGU .

Table 1. Dependence of the oxidation degree of dextran on the periodate : dextran ratio

Addition of gossypol to a polymer carrier occurs at a pH value of between 3.5 and 14.0, preferably between 7.0 and 11.0, most preferably at 8.5. The medium is an aqueous or aqueous-organic medium where gossypol is soluble and the activated polysaccharide is dissolved or swollen. Examples of the medium are alkaline borate, phosphate, and other ammonium-free buffer solutions which do not comprise amines, as well as alkaline aqueous-organic mixtures comprising alcohols (methanol, ethanol and isopropanol) , ketones (acetone, methylethyl ketone), cellosolves, acetonitrile, dioxane, tetrahydrofuran, dimethyl sulfoxide, as well as other solvents miscible with water, or mixtures thereof- in various combinations. Preferably, the amount of an organic solvent in the aqueous-organic medium ranges from 1% to 85% by weight. The ^" reaction is carried out at a temperature of from 0°C up to the boiling point of a solvent, preferably at a temperature close to 18°C. The reaction time is not limited and depends on the temperature of the reaction, and is preferably from 15 minutes to 5 days, most preferably 8 hours. The chemical structure of the resulting polymer compound is determined by the relative amount of gossypol in the reaction, and more particularly by the molar ratio between gossypol and oxidized polysaccharide units, hereinafter referred to as gossypol : oxidized unit or GSP:OU. In particular embodiments, this ratio is described as a molar ratio between gossypol-acetic acid (GAA) clathrate that is used as a raw material, and oxidized polysaccharide units of a particular polysaccharide in the reaction, hereinafter referred to as GAA:DACMC for the reaction between gossypol and dialdehyde carboxymethylcellulose, or as GAA: DAD for the reaction between gossypol and dialdehyde dextran.

The molar ratio of gossypol : oxidized unit (GSP:OU) in the reaction according to the invention is between 10:1 and 1:100, preferably between 10:1 and 5:1 for polymers comprising gossypol, one aldehyde group of which is involved into the reaction, and between preferably 1:2 and 1:100 for polymers comprising gossypol, both aldehyde groups of which are involved into the reaction.

A large excess of gossypol in the reaction and a ratio of GSP:OU of 10:1 result in a compound, wherein gossypol is bound to a polymer carrier through only one of the symmetrical parts of the molecule (in general formula (I), R ¹ is Sach; R ¹¹ is H) , wherein only one of two aldehyde groups of gossypol enters into the reaction. The second reactive aldehyde group is not chemically modified.

A large excess of oxidized units of the polysaccharide and a ratio of GSP:OU of from 1:10 to 1:100 result in a compound, wherein gossypol is bound to a polymer carrier via both symmetrical parts of the molecule, and both aldehyde groups of gossypol are therefore chemically modified (in general formula (I), R ¹ is Sach; R ¹¹ is Sach). All other molar ratios of. gossypol : oxidized unit (GSP:OU), and under conditions close to equally molar, lead to the formation of inseparable mixtures of the above mono- and di-substituted gossypol derivatives present in different ratios.

In all the cases described, any (but only one) of two hydroxyl groups of the cyclic hemiacetal or cyclic azomethine enters into the reaction, without disruption of the integrity of the cycle.

The content of oxidized units in the polysaccharide according to the invention is between one oxidized unit per 1000 anhydrosaccharide units of the polysaccharide and completely oxidized polysaccharide. In the polymer carrier, all oxidized units, in the case of low-oxidized polymers, and only a part of them can enter into the reaction to form an inseparable mixture of macromolecules , comprising non- oxidized units, oxidized gossypol-unsubstituted units and oxidized gossypol-substituted units. In each macromolecule , both all oxidized units and only a part of them can comprise a gossypol molecule bound thereto.

Controlled chemical modification of the . aldehyde groups of gossypol according to the invention is used to regulate the toxicity of gossypol covalently bound to the polymer carrier. This is achieved by varying the amount ratio of mono- and disubstituted gossypol derivatives in an inseparable polymer mixture, wherein mono- and disubstituted gossypol molecules can be bound to the same or different polysaccharide macromolecules.. The higher the amount of a monosubstituted derivative and a fraction of unsubstituted . aldehyde groups, the higher general toxicity of the polymer is.

The reaction product is isolated from the reaction mixture according to the invention by any suitable process, preferably by one of the above-mentioned processs: precipitation in a corresponding precipitator followed by reprecipitation or without it; dialysis; chromatography through sorbents or ion-exchange resins followed by precipitation or removal of the solvent by evaporation, lyophilization or spray drying.

Examples of specific embodiments

Characteristics of the compounds used

Na-carboxymethy1 cellulose

manufactured by Ashland (US) , brand - "Blanose" 7MF, batch 070411/2, is a linear polymer, a cellulose derivative. A degree of carboxymethylation is 65%, Mw = 712 kDa . Light yellow powder was used without further purification.

Gossypol-acetic acid

manufactured in Uzbekistan, "0 ' ZATANDART" agency, "AXBOROT-MA' LUMOT VARKAZ" U ITAR enterprise. A homogeneous fine crystalline powder of from light yellow to dark yellow color with a greenish tinge was used without further purification.

Dextran

Clinical blood substitutes " Poliglyukin" and "Reopoliglyukin" of "Krasfarma" company were desalted by dialysis and lyophilized. Bright white loose fibrous powder.

Study methods

Nuclear magnetic resonance spectroscopy

The chemical structure of synthesized polymers was studied by ¹³ C nuclear magnetic resonance spectroscopy.

Samples were prepared in the form of solutions in

DMSO-d ₆ , D ₂ 0 and D ₂ 0 with the addition of NaOH. ¹³ C NMR spectra were recorded in the mode of suppression in proton splitting with gated decoupling, as well as in the DEPT mode on Bruker CXP-200 apparatuses at a working frequency of 50.32 MHz at 297 K.

Spectra were also recorded on a Bruker Avance-600 apparatus at a working frequency of 150.94 MHz at 297 K in the same modes.

The spectra were recorded in the laboratory of nuclear magnetic resonance of the Institute of Elementary Organic Compounds of the Russian Academy of Sciences.

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to study the chemical structure of oxidized units in samples of dialdehyde carboxymethylcellulose, GAA, as well as GAA- DACMC conjugate and GAA-DAD conjugate. IR spectra were recorded by using a Nicolet 380 device in a range of from 400 to 4000 cm ^"1 with a resolution of 2 cm ^""1 , at room temperature from KBr tablets. The spectra were processed by using the "OMNIC" software of the "Thermo Scientific" company and "ACD labs" 10.0. The sensitivity was increased by a peak deconvolution method with approximation by Gaussian and Lorenz curves.

Ultraviolet spectrophotometry

Ultraviolet spectroscopy was used to control the process and study the kinetics of the periodate oxidation of carboxymethylcellulose and dextran, and to develop a method for the quantitative determination of gossypol in a substance. The absorption spectra were recorded on a SPEX SSP-715 spectrometer in an ultraviolet spectral region of from 190 to 1050 nm. Results were processed by using the software UV-Vis analyst and ACD/SpecManager .

Gel-permeation chromatography

Gel-permeation chromatography was used to determine the molecular weight, molecular weight distribution and the composition of the initial carboxymethylcellulose, obtained samples of dialdehyde carboxymethylcellulose, dialdehyde dextran and the products of addition of gossypol to dialdehyde carboxymethylcellulose and dialdehyde dextran. Analyses were performed on Agilent 1200 and Waters liquid chromatographs equipped with two detectors sequentially connected with a refractometric detector and a multiwave UV detector.

The analysis was performed on columns Ultrahydrogel Linear 1000, 250, 120 (manufactured by "Waters"), as well as on a pair of columns Ultrahydrogel 1000 and 120 sequentially connected, at a temperature of the columns of 25°C, with water and air thermostatic control. Eluents: 0.2 M ammonium acetate buffer (pH=8.4), 0.1 M borate buffer (pH=8), and B 0,1 M borate buffer (pH=11.0), a flow rate of 0.5 ml/min. Standard polyacrylamides , polyethylene oxides and pullulans of narrow molecular weight distribution were used as standards for calibration. Calibration curves were approximated by a 3 ^rd degree polynomial function.

Molecular weight characteristics of the polymer were calculated by using a universal calibration and software "Millenium" and "Breeze 2".

Periodate oxidation of a polysaccharide comprising only ^' two vicinal hydroxyl groups

Said polysaccharide is carboxymethylcellulose. During its periodate ^" oxidation the C-C bond between the C(2) and C(3) carbon atoms is disrupted to form cyclic oxide units of only one type.

Synthesis of dialdehyde carboxymethylcellulose (DACMC) in an aqueous solution

A solution containing 4.28 g of NaI0 ₄ in 200.0 ml of water was added to a solution containing 4.00 g of sodium carboxymethylcellulose (CMC) in 400.0 ml of distilled water. The resulting solution was kept in an open necked flask at room temperature in a dark place under stirring (Table 2) . After completion of the reaction, the mixture was dialyzed against distilled water for four days with the change of dialysis water for four days, wherein dialysis water was replaced four times up to a negative reaction of dialysate to the I0 ₃ ^~ ion on iodine-starch paper. The resulting product was lyophilized and precipitated in ethanol, washed, and dried under vacuum without - heating .

Synthesis dialdehyde carboxymethylcellulose (DACMC) in a aqueous-organic medium

4.00 g of sodium carboxymethylcellulose (CMC) were added to 50.0 ml of an equivoluminar mixture of water and acetone under · vigorous stirring, · and the mixture was allowed to swell under stirring for 24 hours. A solution comprising 4.28 g of NaI0 ₄ in 50.0 ml of water was added to the swollen mixture under stirring. The mixture was stirred in an open necked flask at room temperature in a dark place. The weight ratio of the initial reaction products and the oxidation conditions are shown in Table 2. After completion of the reaction, a solution of 1.5 g of KJ in 50.0 ml of water was added to the mixture to remove traces of iodine and purified by dialysis or four-time discontinuous washing with aqueous acetone up to a negative reaction of dialysate or wash water to the I0 ₃ ^~ ion on iodine-starch paper-. The resulting product was lyophilized or precipitated in ethanol, washed, and dried under vacuum without heating.

Determination of the oxidation degree of dialdehyde carboxymethylcellulose (DACMC) and dialdehyde dextran (DAD) by a iodometric back titration method

Four 20.0 mg samples of dialdehyde carboxymethylcellulose (dialdehyde dextran) , weighed with an accuracy of 0.1 mg were placed into four 50.0 ml flat- bottomed flasks. 10 ml of 0.1 N NaOH were added to each sample and stirred for 5 minutes; then 20.0 ml of 0.1 N I ₂ were added and stirred for 0.5 hour. After that, 12.0 mL of 0.1N HC1 were poured into each sample and titrated with COIN Na ₂ S ₂ 0 ₃ solution by using starch as an indicator. A blank test was concurrently performed, wherein non-oxidized carboxymethylcellulose (dextran) was used instead dialdehyde carboxymethylcellulose (dialdehyde dextran) .

The oxidation degree of was calculated according to formula ( 1 ) :

wherein

₌ V(I ₂ ) · N(Na ₂ S ₂ 0 ₃ )

V'(Na ₂ S ₂ 0 ₃ )contr ' '

V (N S ₂ 0 ₃ ) con tr is a correction factor obtained by titration of sodium thiosulfate with iodine.

V(Na ₂ S ₂ 0 ₃ ) = V(Na ₂ S ₂ 0 ₃ )col - V(Na ₂ S ₂ 0 ₃ )contr, (3)

V(Na ₂ S ₂ C>3) col is a volume consumed for the titration of carboxymethylcellulose (dextran) with sodium thiosulfate;

V(Na ₂ S ₂ 0 ₃ ) contr is a volume consumed for the titration of dialdehyde carboxymethylcellulose (dialdehyde dextran) with sodium thiosulfate.

d is a weighed quantity of polymer, mg

M(IOOAGU) = 24700 - for CMC, brand Blanoza 7 MF,

M(IOOAGU) = 16200 - for dextran.

Table 2. Synthesis of dialdehyde carboxymethylcellulose

Structure of oxidized units of dialdehyde carboxymethylcellulose (DACMC)

Analysis of the ¹³ C N R spectra of the initial CMC (see Fig.l), as well as samples of dialdehyde carboxymethylcellulose of various oxidation degrees, confirmed a conclusion that the aldehyde groups in oxidized units exist in the form of cyclic hemiacetal, but not in the free form. The spectra do not have the signals of aldehyde groups within a range of 195-200 ppm. An assumption that in an aqueous solution, aldehyde groups exist in a hydrated form is not confirmed: these signals are not detected in the spectra (see Figure 2) . The signal in 102.77 ppm corresponds to C(l) carbon atom of the anhydroglucose unit of CMC. The C(7) methylene group in the carboxyl group has a chemical shift of 71.79 ppm. The C(8) carbonyl signals are shown in a weak field and have chemical shifts of 179.21 ppm for the free acid and of 178.37 ppm for the carboxylate anion. Referring to the literature, a high-intensity signal at 60.55 ppm is referred to the C (6) signal of the anhydroglucose unit in an unmodified cellulose atom, whereas a modified _. C (6) atom has a chemical shift equal to 70.98 ppm. A chemical shift of the C(5) carbon atom is equal to 82.56 ppm. A low- intensity peak at 150.65 ppm corresponds to the signal of sodium carbonate impurity formed in the reaction of carbon dioxide with alkali.

Analysis of the spectra DACMC of various oxidation degrees (see. Figure 2) showed that with an increase in the oxidation degree, the intensity of signals at 74.62 and 73.58 ppm decreases. Those are C(2) and C(3) signals of a non-oxidized anhydroglucose units in DACMC. The higher the oxidation degree, the lower their intensity is and, correspondingly, the lower the content of oxidized units in the polymer.

A signal at 75.43 ppm corresponds to the C (4) carbon atom during the transition from low-oxidized to highly oxidized polymers, wherein its intensity remains almost unchanged, and it does not participate in the oxidation.

When the oxidation degree of carboxymethylcellulose increases, both the number and the intensity of the peaks within a range of 85-100 ppm increase. They are signals of the hemiacetal carbon atoms C-O-C. An increase in the number of signals is associated with an increase in the number of potential diastereomeric hemiacetal structures.

In a highly oxidized polymer, the signal at 99.89 and 99.62 ppm corresponds to C"(4) atom that is shifted towards a weak field because the adjacent C"(3) carbon atom acquires a more electronegative substituent. The peaks at 96.25, 95.51, and 94.76 ppm relate to the forth carbon atom in side-chains of 1-6 oxidized carboxymethylcellulose. The signals at 91.95, 91.20, and 90.45 ppm belong to atom C"(2),. and the signals at 89.67, 88.59, 88.30, and 87.99 ppm are signals of C"(3) atom.

Synthesis of gossypol polymer derivative covalently bound to dialdehyde carboxymethylcellulose in a gossypol excess

(See. Table 3)

1.000 g DACMC (γ _οχ =40, 44%) was placed in a flat- bottomed 100 ml flask. Then, 20.0 ml of a borate buffer (pH = 8.5) was added to the flask and stirred on a magnetic stirrer until fully dissolved.

Gossypol (1.543 g) was placed in a 250.0 ml conical flask and dissolved in the minimum amount of 0. IN NaOH (90.0 ml) under stirring in a magnetic stirrer until fully dissolved. Then, a borate buffer (90.0 ml, pH = 8.5) was added under stirring to the resulting solution in a thin stream by using a burette. The pH value was checked with litmus paper (pH = 8.5) .

The solution of DACMC was slowly poured into the gossypol solution under vigorous stirring on the magnetic stirrer. The combined solution was stirred for 195 minutes, and after completion of the reaction, the solution was placed in a dialysis bag. The product was dialyzed against the borate buffer until the wash water is colorless, then it was dialyzed four times against distilled water.

Synthesis of a gossypol polymer derivative covalently bound to dialdehyde carboxymethylcellulose in an excess of oxidized polysaccharide (see. Table 3)

1.000 g DACMC (γ _οχ =40, 44%) was placed in a flat- bottomed 100 ml flask. Then, 20.0 ml of a borate buffer (pH = 8.5) was added to the flask and stirred on a magnetic stirrer until fully dissolved.

Gossypol (1.000 g) was placed in a 50.0 ml conical flask and dissolved in the minimum amount of 0. IN NaOH (10.0 ml) under stirring in a magnetic stirrer until fully dissolved. Then, a borate buffer (10.0 ml, pH = 8.5) was added under stirring to the resulting solution in a thin stream by using a burette. The pH value was checked with litmus paper (pH = 8.5) .

The solution of gossypol was slowly poured into the solution of DACMC under vigorous stirring on the magnetic stirrer. The combined solution was stirred for 160 minutes, and after completion of the reaction, the solution was placed in a dialysis bag. The product was dialyzed against the borate buffer until the wash water is colorless, then it was dialyzed four times against distilled water.

Table 3. Synthesis of gossypol covalently bound to dialdehyde carboxymethylcellulose

The . structure of the gossypol polymer derivative covalently bound to dialdehyde carboxymethylcellulose

The ¹³ C NMR spectrum of the product obtained in the reaction with a slight excess of gossypol (see. Fig.3) comprises signals of carbons of the aromatic rings of gossypol within a range of 120-170 ppm and signals of DACMC within a range of 55-110 ppm and at 190 ppm (carbon of the DACMC carboxyl group) . The signal at 169 ppm belongs to the carbon of the aldehyde group of monosubstituted gossypol in the enol form. The lack of the signals of unbound gossypol in the non-enol form at 146 ppm means that its content in the product does not exceed 5.7 mol%. A complete assignment of the signals is given in the spectra.

Given the oxidized units in dialdehyde carboxymethylcellulose, which are formed from non- carboxylated cellulose cycles, in the chemical structure of the product obtained with an excess of a polysaccharide comprises three types of gossypol-containing units:

30

Synthesis of a gossypol polymer derivative covalently bound to dialdehyde dextran (DAD) obtained in an excess of gossypol

DAD (0.2154 g = 1 mmol eq. ; γ _οχ = 71.05% and [η] = 0,0858) and borate buffer (5 ml; pH = 8.5) were placed in a 100 ml flat-bottomed flask and stirred on a magnetic stirrer until fully dissolved.

GAA (0.518 g = 1 mmol eq. ) was dissolved in 5 mL of 0.1N NaOH. A fourfold excess of 0.1 M borate buffer (pH = 8.5) was added to the resulting solution. The DAD solution was slowly added to the resulting solution. The mixture was stirred for a day at room temperature and purified by dialysis followed by lyophilization . The weight ratios of the initial reaction products and conditions of the reaction are shown in Table 4.

Table 4. Synthesis of gossypol covalently bound aldehyde dextran

The C N R spectrum of the product obtained in the reaction with an excess of gossypol (polysaccharide deficiency), shown in Figure 4, comprises signals of carbons of the aromatic rings of gossypol within a range of 120-160 ppm, and the signals of the anhydroglucose unit of DADS within a range of 55-110 ppm and at 190 ppm (carbon of the DAD carboxyl group) . The signal at 162 ppm belongs to a carbon atom of monosubstituted gossypol. The lack of signals of unbound gossypol at 146 ppm means that its content in the product does not exceed 5-7 mol%.

Determination of the cytotoxic properties of gossypol covalently bound to dialdehyde carboxymethylcellulose

Cells in an amount of 10, 000 cells per a well in 100 μΐ of a complete medium containing 10% fetal bovine serum (FBS) were placed in 96-well flat-bottomed test-plates. Cells were cultured for a day in the dark in a laminar flow hood. Then the liquid medium was removed from the wells of the test plates with a pipette, leaving immobilized living cells. 100 μΐ of dilutions of the test drugs prepared on complete medium were introduced into the wells. Each dilution was duplicated in 3-4 replicates. On the plate 4 control wells were prepared that were further filled with 100 μΐ of fresh complete medium containing no drugs to be tested. The plate was placed in the laminar flow hood, and the cells were cultured for 3 days. Then, the liquid contents were removed from the wells, 100 μΐ of the complete medium and 20 μΐ of FBS were added thereto, stirred through the pipette nozzle, and the plate is incubated for 3.5 hours in the laminar flow hood in the dark, after that a relative absorbance of the solution in each well was measured on a TECAN . spectrophotometer at 492 nm relative to its absorption at a reference wavelength of 620 nm. Cytotoxicity CC ₅₀ was calculated by the standard method: Terry L. Riss and Richard A. Moravec "Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays". ASSAY and Drug Development Technologies Volume 2, Number 1, 2004, pp.51-62.

Table 5. Determination of cytotoxicity (CC50) of the preparations having different contents of gossypol on the cell line A549* Amount of gossypol in a Percentage of CC ₅₀ , mg/ml medicament based on unsubstituted

gossypol-acetic acid, aldehyde groups

wt . %

100.0 100 0.01

0.06 0 >50.00

0.22 0-5 8.49

0.67 0-10 3.36

10.22 25-50 0.25

In vitro study of the antiviral action of gossypol polymer derivatives on different influenza A virus strains List of abbreviations:

TCD50 - tissue cytopathic dose causing death in 50% of the cell monolayer

CPE - cytopathic effect

TD50 - toxic dose of a drug, causing death in 50% of the cell monolayer

ED50 - effective dose in which 50% of cells in the monolayer survived

Technique

Test drugs in serial dilutions were added to MDCK cells reached monolayer confluence in 96-well culture plates. Thereafter, the cells were infected with influenza virus in a dose of 10TCD50/well. To determine TD50, cells with the same concentrations of the drugs were not infected. The infected cells were incubated for 72 hours, while CPE reaches in the control up to 100%. Then the cells were stained with MTS dye, and the absorbance was read on a plate reader. Accurate TD50 and ED50 values were determined by plotting a non-linear regression curves. The chemotherapeutic index was calculated according to the formula: CTI = TD50/ED50. Viral strain Ratio of ED50 CTI Cell GSP:OU [mg/ml] viability

[%]*

H3N2 1:1 0.0064 327.8 100

A/Aichi/1/68 1:10 0.002 333.6 100

H1N1 1:1 0.0031 670 100

A/Brisbane/59/07 1:10 <ED50** _ * * * 42

H3N2 1:1 <ED50 - 47

A/Pert/16/09 1: 10 0.0125 53.16 56

H3N2 1:1 «0.01 209.7 100

A/Brisbane/10/07 1: 10 <ED50 - 32

H1N1 1:1 «0.013 161.3 69

A/Moscow/01/09 1: 10 <ED50 - 31

* - Maximum cell survival achieved for each compound and the corresponding influenza virus strain

** - the value of 50% cell survival has not been not reached

*** - index was not calculated due to the lack of a ED50 value

The experiments conducted have surprisingly shown a difference in the specific antiviral activity of the studied polymer derivatives, depending on the virus strain, which depends on the GSP:OU ratio in a polymer derivative. The use of a mixture of polymer derivatives will broaden the range of antiviral activity. Thus, we have found that, depending on the binding of gossypol with an oxidized unit of the polysaccharide (by one or two bonds) , one can change and regulate antiviral activity and broaden the spectrum of antiviral agents.