PL239358B1 - New polymeric derivatives of betulin and their application - Google Patents

Description

Description of the invention

The present invention relates to novel polymeric betulin derivatives and their use. These compounds can be used in the pharmaceutical industry.

Betulin belongs to the pentacyclic triterpenes, a convenient source for its preparation is the outer layer of birch bark (Alakurtti S. et al., Eur. J. Pharm. Sci., 29.1,2006, 1-13). Moreover, betulin and readily derived derivatives exhibit a broad spectrum of biological activity, such as: anti-inflammatory (Patocka Jiri, J. Appl. Biomed., 1.1,2003, 7-12), antiviral (Muhammad A. et al., J Microbiol . Biotechnol, 14.5, 2004, 1086-1088), antitumor (e.g., Alakurtti S. et al., Eur. J. Pharm. Sci., 29.1, 2006, 1-13; P.390998). Due to the proven numerous directions of pharmacological activity, including the ones quoted above, betulin derivatives can be used as new potential therapeutic substances.

Betulin disuccinate (DBB) exhibits biological activity, is known to have antitumor, anti-leishmanosis (Chowdhury S. et al., Antimicrob. Agents Chemother., 58.4, 2014, 2186-2201) and also hypolipidemic (Pegova RA et al., Medicinskj al'manah, 3, 2015, 216-219), fungicidal (Krasutsky PA et al., US Patent, WO2002026761 A1,2002), bactericidal (Krasutsky PA et al., US Patent 20020119935 A1, 2002) and antiviral, in including against Epstein-Barr virus (Muhammad A. et al., J. Microbiol. Biotechnol, 14.5, 2004, 1086-1088) and against HIV (Sun, I-Ch., et al., J. Med. Chem., 41.23, 1998, 4648-4657).

Betulin disuccinate, having two carboxyl groups, is an excellent raw material to obtain, as a result of polycondensation, the polyanhydride, which is the polymeric form of this compound.

Polyanhydrides are a group of polymers that have an anhydride bond in their backbone. Obtained by the polycondensation of compounds containing two carboxyl groups. The simplest method of their preparation is the two-stage polycondensation with acetic anhydride. These polymers undergo hydrolytic degradation to the corresponding dicarboxylic acids. The degradation time depends largely on the type of diacid from which the polyanhydride is formed and ranges from several days to several years. Due to their properties, such as biocompatibility and appropriate release kinetics of active substances, they are used mainly in medicine, both as drug carriers and biomaterials (Domb, AJ, Kumar, N., Ezra, A., Wiley & Sons Inc., Hoboken, New Jersey, 2011).

Polymer prodrugs are one type of controlled drug release system, the so-called DDS (Drug Delivery Systems). These systems are characterized by a gradual release of the active substance in the body, which prolongs the therapeutic effect. In addition, the use of this form of the drug gives a greater possibility of introducing the drug into the body (e.g. in the form of polymer microspheres or nanospheres, implants, etc.) and its distribution, and in the case of drugs that are poorly soluble in aqueous solutions (such as, for example, betulin disuccinate), it also has a positive effect. on bioavailability and biological activity due to the gradual release of the active substance.

From American descriptions US2003 / 0059469 A1, US6,613,807 B2 it is known to use an anhydride bond to obtain polymer prodrugs from active substances containing one carboxyl group in the molecule and another functional group: amino, thiol, hydroxyl or phenol, which is used to connect two molecules active substances, using the linker - (CH2) n-, thus obtaining a dicarboxylic compound that can be used to obtain a polyanhydride. This method is used to obtain polymer prodrugs in the form of polyanhydrides, based on derivatives of salicylic acid, which is an active substance, and which has a carboxyl and hydroxyl group in the molecule (Erdmann, L., et al. Biomaterials, 21, 2000, p. 1941) -1946). Betulin disuccinate, which contains two carboxyl groups in the molecule, can be directly used in a polycondensation reaction to obtain a polyanhydride. The polyanhydride obtained from betulin disuccinate is a polymer prodrug because betulin disuccinate, which is an active substance, is chemically bound to the polymer chain, and its therapeutic properties are obtained by hydrolysis of the polymer under physiological conditions. Both betulin and betulin disuccinate are non-toxic both in vitro and in vivo, polymers based on these substances can also be used in the pharmaceutical industry as matrices in systems for the controlled release of drugs into the body. Polymeric forms of betulin have been described in the literature so far, such as: polyesters (Jeromenok, Jekaterina, et al., Macromol. Rapid Commun., 2011, 32.22: 1846-1851), polyurethanes (Jeromenok, Jekaterina, et al., ChemistryOpen, 2013, 2.1 : 17-20) and polyethylene oxide conjugates (J. Zhao,

PL 239 358 B1

H. Schlaad, S. Weidner, M. Antonietti, Polym. Chem. 2011). They are proposed as highly microporous materials for use in gas adsorption. However, there are no reports on the preparation of polyanhydrides based on betulin or its derivatives.

The aim of the invention is to present new polymeric betulin derivatives of formula 1 and formula 2, which will be polymer prodrugs that release the active substance betulin disuccinate as a result of hydrolysis under physiological conditions. The use of poly (ethylene glycol) as a comonomer is to increase the hydrophilicity of the polymeric forms of betulin.

This aim was achieved by linking betulin disuccinate and dicarboxylic derivatives of poly (ethylene glycol) with an anhydride bond as a result of the polycondensation reaction of the carboxyl groups present in the starting compounds.

The essence of the invention is a new polymeric betulin derivative of the formula I consists of a betulin derivative having succinyl groups in the C-3 position and in the C-28 position (betulin disuccinate), the free carboxyl groups of which are linked into polymeric linear structures by an anhydride bond.

The new polymeric betulin derivative of formula 2 consists of a betulin derivative having a succinyl group in the C-3 position and in the C-28 position (betulin disuccinate) and a dicarboxylic derivative of poly (ethylene glycol), in which the number of repeating units x is from 3 to 43, preferably x = 10 for poly (ethylene glycol) 600, the free carboxyl groups of which are linked to polymeric linear structures by anhydride bonds.

Preferably, the weight proportion of betulin disuccinate in the polymeric betulin derivative represented by formula 2 is from 20% to 80%.

The use of new compounds of formula 1 as polymer prodrugs and matrices in drug controlled release systems.

The use of new compounds of formula 2 as polymer prodrugs and matrices in drug controlled release systems.

The compounds of the invention are new and have not yet been described in the literature.

The obtained polymers under conditions similar to physiological (37 ° C, pH = 7.4 and pH = 5.5) undergo hydrolytic degradation to the starting reagents: betulin disuccinate (DBB), whose biological activity is known and confirmed, and polyethylene glycol diacids ) that are physiologically acceptable. Therefore, the use of these polymers in biological systems will lead to the release of DBB, controlled by the rate of polymer degradation. The described polymeric betulin derivatives, obtained according to Examples 1 and 2, were subjected to the hydrolytic degradation tests according to Example 3, in conditions similar to physiological (at a temperature of 37 ° C, in a phosphate buffer at pH = 7.4 and in distilled water at pH = 5). , 5). The degradation process was investigated on the basis of changes in the mass of the samples subjected to degradation (weight loss was determined) and on the content of anhydride bonds in the sample remaining after the degradation, which was determined on the basis of 1H NMR spectra. Additionally, the degradation products dissolved in the degradation medium were also investigated. For this purpose, the aqueous solutions were freeze-dried and then the residue was subjected to spectroscopic analysis. Betulin disuccinate and carboxylic derivatives of polyethylene glycols have been identified in the degradation products. There was no hydrolysis of ester groups in betulin disuccinate under the conditions and at the time of the study. The obtained polymers were characterized by a different rate of hydrolytic degradation, which is closely related to the participation of the hydrophilic component, polyethylene glycol. The polyanhydride obtained according to example 1 from betulin disuccinate (polyDBB) degrades relatively slowly due to its high hydrophobicity. PolyDBB degrades completely in 14 days under similar physiological conditions. During this time, the anhydride bonds in the sample exposed to the aqueous solution are completely lost, both in the medium with pH = 5.5 and pH = 7.4 (Fig. 1). In the in vitro conditions under which the test was performed (according to Example 3), the test sample was not completely dissolved in the buffer during this time. After 14 days, the weight loss of polyDBB was 20% w / w.

The copolymers obtained according to example 2, with the participation of polyethylene glycols, due to their hydrophilicity, show greater susceptibility to hydrolytic degradation in relation to polyDBB. The degradation rate of the copolymers, determined on the basis of the disappearance of the anhydride bonds and the weight loss, increases with the increase in the content of poly (ethylene glycol) in the copolymer. Copolymers prepared with 80% of PEG 600 completely degrade under physiological conditions within two days. During this time, the anhydride bonds in the sample are completely lost

After exposure to an aqueous solution, both in the medium at pH = 5.5 and pH = 7.4 (Fig. 2). At the same time, over 90% weight loss of this polymer is observed.

The polymeric betulin derivatives, which are the subject of the invention, were tested to determine their cytotoxic activity in relation to selected tumor cell lines. These studies were carried out according to example 4, in which the cultures of pure tumor cell lines were used: cervix, breast, lung, liver, central nervous system and nasopharynx. On the basis of the tests carried out for various concentrations, the IC50 parameter was determined - the concentration that inhibits the growth of cells in culture by 50%. The IC50 numerical values obtained for the compounds prepared according to Examples 1 and 2 are shown in Table 1 - Fig. 3.

The obtained results of cytotoxicity studies, expressed by IC50, presented in relation to selected new polymeric betulin derivatives, according to the invention, indicate their effectiveness in inhibiting the development of neoplastic cells, with the simultaneous lack of cytotoxicity in relation to normal cells. The presented results also indicate a higher cytotoxic activity of the polyanhydrides obtained with PEG 600, which is related to faster hydrolytic degradation and the release of more betulin disuccinate during the experiment.

Due to the intended use, on the one hand, it is advantageous to obtain copolymers containing as much DBB as possible (having antitumor activity), but on the other hand, a higher content of polyethylene glycol increases the hydrophilicity of the copolymer, allowing for faster release of the active substance and increases its bioavailability. For this reason, polymeric betulin derivatives obtained from betulin disuccinate and polyethylene glycol 600, in which the content of polyethylene glycol 600 is up to 40%, have been found to be particularly preferred. Such polyanhydrides contain up to 60 wt. DBB (active substance) and at the same time their hydrophilicity allows for a quick release of the active substance and increases its bioavailability, which is confirmed by the results of tests of cytostatic activity of the obtained polymers in relation to selected tumor cell lines (Table 1).

The following examples illustrate the invention.

P r z k ł a d 1

Preparation of betulin disuccinate polyanhydride - polyDBB.

Betulin disuccinate (5 g) is dissolved in a 10-fold excess (w / v) of acetic anhydride (50 ml) and heated to the reflux temperature of the solvent for 40 min. under nitrogen atmosphere. After this time, the excess acetic anhydride and the acetic acid formed in the reaction are distilled off on a rotary vacuum evaporator. The betulin disuccinate diacyl derivative remaining after distillation is subjected to polycondensation under vacuum (0.1 mm Hg) at 150 ° C for 2 h. %. The spectroscopic data of the obtained betulin disuccinate polyanhydride of formula 1 are as follows:

¹ H NMR (600 MHz, CDCl 3) δ: 4.69 '(1H,' d, C29-Ha); 4.59 (1H, d, C29-Hb), 4.50 (1H, t, C3-H '); 4.30 (1H, d, C28-Ha); 3.89 (1H, d, C28-Hb); 2.82-2.77 (4H, m, CH2C (O) OC (O)); 2.71-2.64 (4H, m, CH2C (O) O); 2.44-2.40 (1H, td, C19-H); 1.68 (3H, s, C30-H3); 0.83-0.85; 0.97 and 1.02 (5 x 3H, 5 xs, 5 CH3 groups).

¹³ C NMR (150 MHz, CDCl 3) δ: 171.93 (Cq, C (O) O); 167.96 (Cq, C (O) OC (O)); 150.03 (Cq, C-20); 109.93 (CH2. C-29); 81.74 (CH. C-3); 63.36 (CH2. C-28), 55.40 (CH, C-5); 50.27 (CH. C-9); 48.81 (CH, C-18), 47.71 (CH, C-19); 46.43 (Cq. C-17); 42.71 (Cq, C-14); 40.91 (Cq, C-8); 38.36 (CH2. C-1); 37.86 (Cq. C-4); 37.61 (CH. C-13); 37.07 (Cq, C-10); 34.52 (CH2. C-22); 34.11 (CH2. C-7); 30.38-30.32 (CH2C (O) OC (O)); 29.72 (CH2. C-21); 29.57 (CH2. C-16); 28.62 (CH2C (O) O); 27.97 (CH3. C-23); 27.04 (CH2. C-15); 25.17 (CH2. C-12); 23.65 (CH2. C-2); 20.81 (CH2. C-11); 19.14 (CH3. C-30); 18.16 (CH2. C-6); 16.53 (CH3. C-24); 16.14 (CH3. C-25); 16.04 (CH3. C-26); 14.76 (CH3. C-27).

P r z k ł a d 2

Preparation of betulin disuccinate polyanhydride and poly (ethylene glycol) 600.

Betulin disuccinate (1 g) and polyethylene glycol 600 dicarboxylate (4 g), are dissolved in a 10-fold excess (w / v) of acetic anhydride (50 ml) and heated to the reflux temperature of the solvent for 40 min. under nitrogen atmosphere. After this time, the excess acetic anhydride and the acetic acid formed in the reaction were distilled off on a rotary vacuum evaporator. The prepolymer remaining after evaporation is subjected to polycondensation under vacuum (0.1 mm Hg) at 150 ° C for 2 hours.

PL 239 358 Β1

The spectroscopic data of the obtained betulin disuccinate copolymer with polyethylene glycol 600 diacid of formula 2 are as follows:

¹ H NMR (600 MHz, CDCl ₃ ) δ: 4.68 (1H, d, C ₂₉ -H _a ); 4.59 (1H, d, C ₂₉ -H _b ), 4.50 (1H, t, C ₃ -H _a ); 4.30 (1H, d, C ₂ s-Ha and CH ₂ C (O) OC (O) w PEG), 3.89 (1H, d, C ₂₈ -H _b ); 2.82-2.78 (4H, m, CH ₂ C (O) OC (O)); 2.71-2.64 (4H, m, CH ₂ C (O) O); 2.44-2.40 (1H, td, C19-H); 1.68 (3H, s, C30-H3); 0.83-0.85; 0.97 and 1.02 (5 × 3H, 5 × s, 5 CH3 groups).

¹³ C NMR (75 MHz, CDCl 3) δ: 171.95 (C _q , C (O) O); 167.94 (C _q , C (O) OC (O)); 150.03 (C _q , C-20); 110.0 (CH ₂ , C-29); 81.73 (CH. C-3); 71.12-70.56 ((-O-CH ₂ -CH ₂ -) _n in PEG); 63.32 (CH _2. C-28), 55.36 (CH. C-5); 50.25 (CH. C-9); 48.78 (CH, C-18), 47.68 (CH, C-19); 46.40 (C _q , C-17); 42.68 (C _q , C-14); 40.88 (C _q , C-8); 38.33 (CH ₂ , C-1); 37.84 (C _q , C-4); 37.58 (CH. C-13); 37.03 (C _q , C-10); 34.48 (CH ₂ , C-22); 34.07 (CH _2. C-7); 30.35-30.18 (CH ₂ C (O) OC (O)); 29.70 (CH ₂ , C-21); 29.54 (CH _2. C-16); 28.89 and 28.60 (CH ₂ C (O) O); 27.95 (CH ₃ , C-23); 27.0 (CH _2. C-15); 25.13 (CH _2. C-12); 23.62 (CH _2. C-2); 20.78 (CH _2. C-11); 19.12 (CH ₃ , C-30); 18.13 (CH _2. C-6); 16.51 (CH ₃ , C-24); 16.13 (CH _3. C-25); 16.0 (CH _3. C-26); 14.74 (CH ₃ , C-27).

Example 3

In vitro hydrolytic degradation test under similar to physiological conditions.

The hydrolytic degradation was carried out in phosphate buffer at pH = 7.4 (PBS) and in distilled water (pH = 5.5) at the temperature of 37 ° C. Its course was monitored by determining the weight loss of the tested samples after different residence times in the degradation medium.

About 0.1 g of the tested polymers, weighed with an accuracy of 0.0001 g, were introduced into glass vials and poured with 15 cm ^{3 of} PBS or distilled water. The vials were sealed and placed in an incubator at 37 ° C. The vials were withdrawn after the specified time had elapsed (1 hour to 14 days). The degradation medium was decanted from the part of the sample remaining after degradation and subjected to lyophilization. Lyophilisates were tested ¹ H-NMR, in order to examine the degradation products. Remaining after degradation of the sample, rinsed with distilled water, then dried to constant weight in a vacuum oven, and after drying weighed to 0.0001 g and was tested for its ¹ H-NMR, in order to calculate the anhydride to the ester groups.

The weight loss of the sample during degradation was calculated using the following relationship:

Δτη =. 100% where:

mi - sample weight before degradation [g], m ₂ - sample weight after degradation [g]

The ratio of anhydride to ester groups in the polyanhydrides was calculated using the formula:

_ ^ SAc E ~ I _E where:

Isac - intensity of methylene protons signals at the anhydride moiety with a chemical shift δ = 2.82 - 2.77 ppm

Ie - intensity of methylene protons signals at the ester group with a chemical shift δ = 2.74 - 2.64 ppm

Fig. 1 and Fig. 2 illustrate the progress of the hydrolytic degradation of betulin polymeric forms as expressed by the loss of anhydride bonds during hydrolytic degradation performed in phosphate buffer (pH = 7.4) or distilled water (pH = 5.5).

Example 4

Investigation of the cytostatic activity of betulin disuccinate polyanhydrides.

The cytostatic activity of the synthesized compounds was tested against the following human tumor cell lines: cervix, breast, lung, liver, central nervous system and nasopharynx, and two normal cell lines. Laboratory cultures of pure cell lines were used for the research.

PL 239 358 B1

The cell suspension at a density of 2000 cells / 0.1 ml was seeded into wells of a 96-well plate containing the growth medium: Eagle's fluid supplemented with 10% calf serum and antibiotics. After incubation for 24 hours at 37 ° C, 5% CO 2 and 95% humidity, cells in log phase growth were treated with test compounds. The test substances were dissolved in a mixture of DMSO and water, and the resulting solutions (with concentrations ranging from 0.1-100.0 micrograms / ml) were added to the cultures of individual tumor cell lines, while preparing the corresponding control samples without the test substance. All samples were incubated for 72 hours at 37 ° C, 5% CO2 and 95% humidity, and then the number of viable cells was determined using the spectrophotometric method using sulforhodamine B as a dye that selectively binds to the proteins of proliferating cells .

On this basis, the change in the number of viable cells in the culture as a result of the test substance was determined by relating it to the corresponding control. The result was expressed as a percentage as the degree of growth inhibition caused by the action of the tested cytostatics. Based on the tests carried out for various concentrations, the IC50 parameter was determined for each test substance - the concentration that inhibits the growth of cells in the culture by 50%. The results of the cytotoxic activity, expressed by IC50, are presented in the table in Fig. 3.

Claims (5)

1. The new polymeric betulin derivative of formula I consists of a betulin derivative having a succinyl group in the C-3 position and in the C-28 position (betulin disuccinate), the free carboxyl groups of which are linked into polymeric linear structures by an anhydride bond. 2. The new polymeric betulin derivative of formula 2 consists of a betulin derivative having a succinyl group in the C-3 position and in the C-28 position (betulin disuccinate) and a dicarboxylic derivative of poly (ethylene glycol), in which the number of repeat units x is from 3 to 43, preferably x = 10 for poly (ethylene glycol) 600, the free carboxyl groups of which are linked to polymeric linear structures by anhydride bonds. 3. A polymeric betulin derivative according to claim 1, The method of claim 2, characterized in that the weight proportion of betulin disuccinate in the polymeric betulin derivative represented by formula 2 is from 20% to 80%. 4. The use of new compounds as defined in claim 1 1 as polymer prodrugs and matrices in drug controlled release systems. 5. The use of new compounds as defined in claim 1 2 as polymer prodrugs and matrices in drug controlled release systems.