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ISSN 1070-3632, Russian Journal of General Chemistry, 2016, Vol. 86, No. 11, pp. 2534–2540. © Pleiades Publishing, Ltd., 2016. Original Russian Text © E.N. Fedoseeva, V.F. Ur’yash, N.Yu. Kokurina, V.B. Fedoseev, 2016, published in Zhurnal Obshchei Khimii, 2016, Vol. 86, No. 11, pp. 1893–1900.

Synthesis of Chemical Compounds of Chitosan with Betulin under Nearly Homogeneous Conditions E. N. Fedoseevaa*, V. F. Ur’yasha, N. Yu. Kokurinaa, and V. B. Fedoseeva,b a

Research Institute of Chemistry, Lobachevskii State University of Nizhny Novgorod, pr. Gagarina 23, Nizhny Novgorod, 603950 Russia *e-mail: [email protected] b

Razuvaev Institute of Organometallic Chemistry, Russian Academy of Sciences, ul. Tropinina 49, Nizhny Novgorod, 603137 Russia Received May 10, 2016

Abstract—Chemical compounds of chitosan with betulin have been synthesized with the use of chitosan gels in alcoholic and acetic acid dispersions under nearly homogeneous conditions. The resulting biphilic compounds contain up to one betulin residue per 2–3 chitosan units. The formation of chemical compounds of chitosan with betulin has been confirmed by several physicochemical methods. The procedure developed for the preparation of chitosan gels with alcohol-containing dispersion medium makes it possible to carry out chitosan reactions with compounds poorly soluble in aqueous and acetic acid media. Keywords: chitosan, betulin, gel synthesis, biphilic derivatives, physicochemical properties

DOI: 10.1134/S1070363216110219 Natural triterpenoids of the lupane series are interesting as potential therapeutic agents exhibiting antitumor, hepatoprotective, antimalarial, and other kinds of activity [1]. The lack of toxicity and accessibility of these compounds makes them attractive for practical applications. Betulin [lup-20(29)-ene-3β,28-diol] is a pentacyclic triterpenoid present in birch bark as one of the main components and exhibiting a broad spectrum of pharmacological activity [1, 2]. However, the use of betulin in medicine and veterinary is strongly limited by its low bioavailability due to its very poor solubility in aqueous media. The solubility of betulin can be improved by known methods, in particular by using solvent mixtures, preparation of inclusion compounds, e.g., with cyclodextrins [3], complexation with amphiphilic polymers, incorporation of betulin into liposomes and fat emulsions [4, 5], and synthesis of betulin composites with water-soluble polymers by mechanochemical treatment [6]. The hydrophilicity of compounds can also be increased by introduction of polar substituents into hydrophobic molecules [7]. However, success of such reactions is largely determined by proper choice of the solvent. In this work we used chitosan as hydrophilic modifier.

The goal of the present study was to obtain betulin compounds with chitosan under nearly homogeneous conditions and examine their physicochemical properties. It was difficult to find conditions for the synthesis of betulin compounds with chitosan in a homogeneous medium. Betulin is soluble in alcohols and somewhat lower soluble in glacial acetic acid. Chitosan is readily soluble in acidic aqueous medium, but in the presence of alcohols its solubility considerably decreases up to precipitation [8]. Therefore, homogeneous system necessary for efficient chemical reaction cannot be obtained by direct mixing of an alcoholic betulin solution with aqueous chitosan. In order to achieve a homogeneous system containing both reactants, we tried various procedures for the preparation of chitosan gels where the dispersion medium would dissolve betulin. Scheme 1 shows the operation sequence for the preparation of chitosan gels containing betulin and chemical synthesis. While preparing chitosan gels with a high acetic acid content we used the phase diagram given in [9] for the threecomponent system chitosan–acetic acid–water [9]. Gels with ethanol as dispersion medium were prepared with account taken of the data reported in [10].

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SYNTHESIS OF CHEMICAL COMPOUNDS OF CHITOSAN WITH BETULIN Scheme 1. Moist chitosan b

a

Benzoic acid

Glacial acetic acid

Ethanol Gel Betulin

Initiating system

2535

this case, a solution of sodium nitrite was added to a preliminarily prepared acetic acid chitosan gel with dispersed betulin. Decomposition of chitosan according to the Van Slyke reaction affords 2,5-anhydromannose terminal unit [12, 13] possessing a reactive aldehyde group. Nucleophilic addition of hydroxy groups of betulin to that aldehyde group gives rise to a covalent bond. We also analyzed a mixture of betulin and chitosan, which was obtained by extraction of excess betulin from the gel not treated with an initiating agent. The extraction was assumed to be complete when there was no dry residue after removal of the solvent. The weight of all isolated fractions was ~66–67 wt % of the initially added betulin amount, i.e., a considerable part (~33–34 wt %) of betulin remained in the chitosan gel. Repeated extraction with alcohol until no dry residue was also used to purify the reaction products from excess betulin and by-products.

The resulting gel absorbed an appreciable amount of betulin. The concentration of the latter was 0.02 to 0.2–0.25 mol per mole of chitosan constitutional repeating units (CRU). Betulin partially dissolved in the gel, and excess betulin formed a stable dispersion therein, whose sedimentation was hampered by the high viscosity of the gel.

The compounds isolated by extraction were identified by their melting points. For instance, the melting point (mp) of the dry residue isolated by extraction from the gel not treated with an initiating agent was 252°C, which corresponded to the melting point of betulin used in the synthesis and is close to the melting point of pure betulin (mp = 257°C [14]).

Two different versions were tested to initiate a chemical reaction between functional groups of betulin and chitosan. These versions conformed to the reaction centers present in betulin molecules and capable of forming covalent bonds with chitosan, i.e., isopropenyl group and two hydroxy groups. Also, the ability of glycoside bonds of chitosan to undergo homolytic dissociation by the action of radical species (generated, e.g., by thermal decomposition of hydrogen peroxide or by reaction of hydrogen peroxide with ascorbic acid [11]) was taken into consideration. It was presumed that the double bond of betulin can be reactive toward free radicals. Covalent bond between the isopropenyl group of betulin and C1 atom of chitosan can be formed as a result of disproportionation. Thus, the first version is radical initiation. In our experiments, hydrogen peroxide or redox system hydrogen peroxide– ascorbic acid (C6H8O6) was added to a chitosan gel with preliminarily dispersed therein and partially dissolved betulin.

The initial compounds, reaction products, and betulin/chitosan mixture inseparable by extraction were examined by FTIR spectroscopy and differential thermal analysis (DTA) [15]. In addition, the concentration of amino groups was determined by potentiometric titration, and samples were analyzed for nitrogen by the Kjeldahl method (Table 1). Betulin shows in the IR spectrum an absorption band with its maximum at 3080 cm–1, which may be assigned to C–H stretching vibrations of the =CH2 group [16]. No such band was observed in the IR spectrum of the product, indicating that the terminal double bond of betulin was involved in chemical reaction. Similarity of the other IR absorption bands of the compounds under study did not allow us to draw more detailed conclusions regarding change of their structure during the reaction.

An alternative version of the gel synthesis of betulin compounds with chitosan involved the hydroxy groups of betulin through a non-radical mechanism. In

The nitrogen content of the product (sample no. 2, Table 1) was lower than that of initial chitosan (6.3 and 8.2%, respectively). These values correspond to one betulin molecule per 8 CRUs of chitosan. Unlike the initial compounds, the product was insoluble in ethanol and aqueous acetic acid. However, addition of a co-

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FEDOSEEVA et al.

Table 1. Component ratios in the reaction systems Gel preparation method

Amount of betulin per arbitrary mole of chitosan, mol

1.0×10

5

1

2

0.6×10

5

3а 4

а

5

а

Sample no. 1

6 a

а

Molecular weight of chitosan, Da

Amount of initiating system per arbitrary mole of chitosan, mol H2O2

C6H8O6

NaNO2

0.25

–

–

–

3

0.02

0.20

0.20

–

1.0×105

1

0.25

1.00

1.00

–

1.0×10

5

1

0.25

0.10

0.10

–

1.0×10

5

2

0.19

–

–

0.02

1.0×10

5

2

0.53

0.42

0.24

–

Samples were subjected to differential thermal analysis.

solvent (ethanol to aqueous acetic acid and vice versa) resulted in swelling of the solid and formation of a colloidal dispersion. Titrimetric determination of the amino group content (degree of deacetylation) also revealed a difference between initial chitosan and sample no. 2. Calculation of the degree of deacetylation of chitosan by formula (1) gave a value of 79% for sample no. 2 against 93% for chitosan. This may be related to increase of the average CRU weight of chitosan modified with betulin. It should be noted that sample no. 2 remained dissolved even at pH ≥ 9. Initial chitosan precipitated at pH ~6.5. Sample nos. 1 and 3–5 (Table 1) obtained in acetic acid solution were subjected to differential thermal analysis (Table 2). Crab chitosan with a molecular weight of 6.0 × 105 Da (which is close to the molecular weight of chitosan used in this work) showed several relaxation transitions on the thermogram (tγ = –33°C, tβ = 47°C, tg1 = 74°C, tg2 = 140°C), as well as two exothermic peaks corresponding to decomposition (tdec1 = 237°C, tdec2 = 284°C) [17, 18]. The data in Table 2 show that neither γ-transition nor tg1 were observed for sample no. 1 and that β-transition (44°C) and tg2 = 138°C appeared. The exothermic peak was observed at 275°C. Thus, betulin present in sample no. 1 (18.5–19 wt %) affects the behavior of chitosan by suppressing its γ-transition and first vitrification and changing its thermal decomposition pattern. On the other hand, chitosan affects the properties of betulin which melts at 244°C (endothermic peak). This value is lower by 13°C than that given in [14]. It is known [14, 19] that betulin is capable of crystallizing in different forms, depending on the conditions and

solvent nature. Presumably, sample no. 1 contained betulin crystals different from those described in [14]. The fact that both components displayed their intrinsic (though somewhat changed) properties indicated that sample no. 1 consisted of two phases. Samples nos. 3 and 4 (Table 2) were synthesized under radical initiation with different ratios of the components of the H2O2–C6H8O6 redox system. According to the DTA data, these samples showed no phase transitions typical of betulin [ttr(CrII↔CrI) 228°C, mp 257°C] [14], and changed processes typical of chitosan were observed. The DTA curve for sample no. 3 revealed γ-transition (tγ –28°C) and two devitrification ranges (tg1 = 65°C, tg2 = 146°C). There were no β-transition and exothermic decomposition. Sample no. 4 displayed on the DTA curve only the second devitrification (tg2 = 139°C) and exothermic peak at 289°C; the latter is close to the second exothermic peak of chitosan decomposition (284°C [17, 18]). Decomposition of samples nos. 3 and 4 at 330 and 326°C, respectively, was accompanied by heat absorption. The weight loss from sample no. 4 was larger by a factor of ~1.5 than that from sample no. 3 (Table 2). Air-dried sample no. 4 was also characterized by the highest adsorbed water content, which is likely to be responsible for its devitrification at tg2 = 51°C. Herein, water acts as plasticizer on sample no. 4. The above results indicate formation of chemical compounds between betulin and chitosan in samples nos. 3 and 4. It should be noted that the use of concentrated acetic acid as dispersion medium for the synthesis of betulin-modified chitosan is likely to favor transformation of betulin to the corresponding diacetate [20]. During purification of the product, from the alcoholic


SYNTHESIS OF CHEMICAL COMPOUNDS OF CHITOSAN WITH BETULIN

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Table 2. Temperatures of phase, relaxation transitions, and decomposition for betulin-modified chitosan samplesa Sample

no. 1

no. 3

no. 4

no. 5

Sample weight, g

0.2278

0.3475

0.3156

0.1897

First heating tγ, °C

–

–

–

–20

tβ, °C

47.5

–

–

–

tg1, °C

–

–

–

42

tg2, °C

–

–

51

–

129

135

142

142

9

4

15

6

tvap(H2O), °C Water content, wt %

Second heating tγ, °С

–

–24

–

–22

tβ, °С

43

–

–

–

tg1, °С

–

62

–

68.5

tg2, °С

136

148

138

–

Third heating tγ, °С

–

–32

–

–20

tβ, °С

45

–

–

–

tg1, °С

–

68

tg2, °С

140

144

140

–

mp (betulin), °C

244↓

–

–

–

tdec1, °C

275↑

–

289↑

226↑

tdec2, °C

–

330↓

326↓

326↓

41.5

36.6

53.5

33.0

Dry weight loss, % a

73.5

“↓” stands for endothermic effect, and ”↑”, for exothermic effect.

extract we isolated a solid with mp 212–213°C which is close to the published value 222°C [21]. Kjeldahl determination of nitrogen in sample no. 6 (which was obtained at the largest concentration of betulin on the reaction mixture) showed that the product contained one betulin residue per 2–3 chitosan units. This compound formed very stable foam which persisted for several days. Dried sample no. 5 (prepared via Van Slyke reaction) displayed γ-transition at tγ = –21°C), first devitrification at tg1 = 71°C, and exothermic decomposition peak at 226°C (Table 2) which approaches the first exothermic decomposition peak of chitosan (237°C [17, 18]). Plasticizing effect of water was also observed for sample no. 5. Its vitrification temperature

decreases to tg1 = 42°C. The γ-transition temperature does not change under the action of water (Table 2), which is characteristic of small-scale γ- and βtransitions [17, 18]. In addition, an endothermic decomposition peak was observed at 326°C. These findings indicated that sample no. 5 is a chemical compound of betulin with chitosan. Thus, chitosan gels with ethanol and aqueous acetic acid as dispersion media provide nearly homogeneous conditions necessary for efficient initiation of a chemical reaction between chitosan and betulin. Enhanced mutual compatibility of the reactants made it possible to synthesize biphilic compounds with a high concentration of betulin units. The formation of chemical compounds of betulin with chitosan was


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confirmed by IR spectroscopy, titrimetric and gravimetric analysys, and differential thermal analysis. Samples obtained by different methods are characterized by the lack of transition temperatures typical of betulin and by changed transition temperatures intrinsic to chitosan. A compound containing one betulin residue per 2–3 chitosan units can be obtained by the gel synthesis. EXPERIMENTAL The IR spectra (4000–400 cm–1) were recorded in KBr on a Shimadzu IR Affinity-1 spectrometer. The melting points were determined in capillaries on a Stuart SMP10 melting point apparatus with a resolution of 1°C; maximum temperature 300°C. Differential thermal analysis was performed in the range from –190 to 400°C; the instrument and experimental procedure were described in [15]; samples and standard (quartz), 0.2–0.4 g were placed into open aluminum crucibles, which were heated in a helium atmosphere at a rate of 5 deg/min (deviation from linearity did not exceed 1%). The temperature of a sample and the temperature difference between a sample and standard were measured with an accuracy of ±0.5°C using a chromel–copel thermocouple which was calibrated against a platinum resistance thermometer and reference substances throughout the entire temperature range. The DTA setup operation was checked by determining the melting point of nheptane (analytical standard) and vitrification temperature (tg) of pure glycerol. The results coincided with published data for n-heptane within 0.2°C [22], and for glycerol, within 1°C [23]. The relaxation transition temperature (tg, tβ, tγ) was assumed to be the mid value of the corresponding temperature range, for it was characterized by the best reproducibility [24]. Three successive cooling–heating cycles were performed. In the first cycle, samples were heated up to ~150°C, and vaporized adsorbed water was removed by evacuation directly from the DTA setup chamber. The adsorbed water content was then determined by weighting. In the second cycle, dried samples were heated to ~155°C, and in the third cycle, to 400°C. Initial chitosan samples with molecular weights of 0.6 × 105 and 1.0 × 105 Da and a degree of deacetylation of 93% were used without additional purification; the weight fraction of insoluble substances was 0.25%, and the dry residue after calcination was 0.3 wt %. The molecular weight of chitosan was determined with an

Ubbelode viscometer at 21°C from a 0.33 M solution in aqueous acetic acid containing 0.3 mol/L of NaCl. The viscosity average molecular weight was calculated by the Mark–Houwink equation: [η] = kM ¯ να, k = 3.41 × –5 10 , α = 1.01 [25]. The degree of deacetylation of chitosan was determined by potentiometric titration of its solution in 0.1 M aqueous HCl with a 0.1 M solution of potassium hydroxide using an Ekotest-2000pH-m pH meter equipped with an Ekom-pH-kom combined pH electrode. The weight fraction of amino groups (ω, wt %) was calculated by formula (1): ω = (VKOHMKOH10)/0.62 m.

(1)

Here, VKOH is the titrant volume, mL; MKOH is the concentration of alkali, M; m is the sample weight, g; and 0.62 is the number of glucosamine unit moles in 100 g of completely deacetylated chitosan. For analysis, sample no. 2 was dissolved in a mixture of 0.1 M aqueous HCl and ethanol (4 : 3 by volume). The nitrogen content of chitosan and reaction products was determined by the modified Kjeldahl method (treatment of nitrogen-containing compounds with concentrated sulfuric acid until quantitative formation of ammonium sulfate). Betulin (mp 252°C) containing 93% of the main substance was used without additional purification. For DTA study, betulin was purified by triple recrystallization from chloroform [14]; after purification, it contained 99.5 wt % of the main substance. Benzoic, glacial acetic, and ascorbic acids were of chemically pure grade. A 0.1 M solution of HCl was prepared from the corresponding analytical standard. Hydrogen peroxide (30% solution in water) was of analytical grade. Rectified ethanol (GOST R 516522000), propan-2-ol (GOST 9805-84), and reagent grade sodium nitrite were also used. Preparation of chitosan gels. a. Distilled water, 1 mL, was added to 1 g of air-dried chitosan placed in a flask with a ground stopper, and the mixture was kept for 24 h until complete absorption of water. Glacial acetic acid, 10 mL, was then added, and the mixture was heated for 30 min at 50°C on a water bath and left to stand for 24 h. The resulting gel contained 8.3 wt % of chitosan, and the dispersion medium was 90.9% aqueous acetic acid. b. Glacial acetic acid, 5 mL, was added to 1 g of chitosan. After 2 h, an additional portion of glacial acetic acid, 4 mL, was added, and the mixture was heated for 30 min at 50°C on a water bath. Distilled


SYNTHESIS OF CHEMICAL COMPOUNDS OF CHITOSAN WITH BETULIN

water, 6 mL, was then added to the swollen chitosan. An additional portion of water was added in no less than 2 h. The resulting gel contained 6.25 wt % of chitosan, and the dispersion medium was 60% aqueous acetic acid. c. Distilled water, 1.5 mL, was added to 1 g of chitosan, and the mixture was kept for 24 h until complete water absorption. Crystalline benzoic acid, 0.757 g (1 mol per arbitrary mole of chitosan), was then added to the moist chitosan, the mixture was kept for 1 h, and 4.8 g of ethanol was added in portions. The resulting gel contained 13.4 wt % of chitosan, and the dispersion medium was ~73% aqueous ethanol. Synthesis of betulin compounds with chitosan. A weighed amount of betulin was added under stirring to a chitosan gel to a required betulin–chitosan molar ratio (Table 1). Initiating system was then added in an amount indicated in Table 1. Isolation and identification of the products. Samples nos. 1 and 6. After 72 h, the resulting suspension was diluted with a large amount of distilled water to adjust it to pH 4. The mixture was filtered, the solid phase was washed with distilled water and dried, and its melting point was measured. The solvent was distilled off from the filtrate, and the residue was Soxhlet extracted with propan-2-ol. Sample no. 6 treated in this way was analyzed for nitrogen by the Kjeldahl method. Sample no. 2. After 140 h, the solid and liquid phases were separated by filtration. The filtrate was diluted with distilled water, and the precipitate was filtered off, washed with distilled water, and dried. It melted at 250°C (melting point of betulin 257°C [14]). The solid phase was purified by repeated extraction first with ethanol and then with propan-2-ol, and the extracts were evaporated. The dry residue was poorly soluble in water to form an acidic solution. It melted at 115°C and was identified as benzoic acid (mp 122.05°C [26]). The extraction was performed until the extract contained no dissolved substances. The purified product was examined by IR spectroscopy, and the nitrogen content thereof was determined by the Kjeldahl method. Sample no. 3. After 72 h, the solid phase was separated from the resulting suspension by filtration, washed with distilled water, and dried, and its melting point was measured (247°C). The filtrate was made alkaline (pH 11) by adding aqueous sodium hydroxide (35 wt %), and the solvent was distilled off.

2539

Sample no. 4. After 72 h, the suspension was diluted with propan-2-ol, and the precipitate was filtered off and purified by Soxhlet extraction with propan-2-ol. Sample no. 5. In 48 h after addition of NaNO2, the mixture was neutralized to pH 6 with aqueous sodium hydroxide (20 wt %). The resulting suspension was separated by centrifugation. The solid phase was washed with distilled water and dried. Its melting point (252°C) corresponded to the melting point of initial betulin, although the sample turned dark on melting. The liquid fraction was adjusted to pH 10 by adding a solution of sodium hydroxide. The moist precipitate was extracted with propan-2-ol and washed with six portions of distilled water. The product was dried and Soxhlet extracted with several portions of propan-2-ol until the extract contained no dissolved substances. ACKNOWLEDGMENTS This study was performed under partial financial support by the Russian Foundation for Basic Research (project no. 15-43-02 603 r_povolzh’e_a). REFERENCES 1. Tolstikova, T.G., Sorokina, I.V., Tolstikov, G.A., Tolstikov, A.G., and Flekhter, O.B., Russ. J. Bioorg. Chem., 2006, vol. 32, no. 1, p. 37. doi 10.1134/ S1068162006010031 2. Tolstikov, G.A., Flekhter, O.B., Shul’ts, E.E., Baltina, L.A., and Tolstikov, A.G., Khim. Interesakh Ustoich. Razvit., 2005, vol. 13, no. 1, p. 1. 3. Cerga, O., Borcan, F., Ambrus, R., and Popovici, I., J. Agroaliment. Processes Technol., 2011, vol. 17, no. 4, p. 405. 4. Son L.B., Kaplun, A.P., Shpilevskii, A.A., AndiyaPravdivyi, Yu.E., Alekseeva, S.G., Grigor’ev, V.B., and Shvets, V.I., Russ. J. Bioorg. Chem., 1998, vol. 24, no. 10, p. 700. 5. Karlina, M.V., Pozharitskaya, O.N., Shikov, A.N., Makarov, V.G., Mirza, S., Miroshnyk, I., and Hiltunen, R., Pharm. Chem. J., 2010, vol. 44, no. 9, p. 501. doi 10.1007/s11094-010-0502-x 6. Shakhtshneider, T.P., Kuznetsova, S.A., Mikhailenko, M.A., Malyar, Yu.N., and Boldyrev, V.V., Zh. Sib. Fed. Univ., Ser. Khim., 2012, vol. 5, no. 1, p. 52. 7. Khimiya privitykh poverkhnostnykh soedinenii (Chemistry of Grafted Surface Compounds), Lisichkin, G.V., Ed., Moscow: Fizmatlit, 2003. 8. Arzamastsev, O.S., Artemenko, S.E., Abdullin, V.F., and Arzamastsev, S.V., Vestn. Saratov. Gos. Tech. Univ., 2011, no. 4 (60), no. 2, p. 112.


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