RU2523284C9 - Method for modification of macrolide antibiotic oligomycin a by reaction of [3+2]-dipolar cycloconnection of azide and alkines 33-deoxy-33-(triazol-1-yl)-oligomycins a and their biological activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and concerns new derivatives of the antitumour antibiotic oligomycin A and a method for preparing them by regioselective [3+2]-dipolar cycloconnection of an azido group of 33-deoxy-33-azidooligomycin A(1) to monosubstituted alkines. The new derivatives of the antibiotic oligomycin A described by formula:

, wherein R represents 1,4-disubstituted 1,2,3-triazoles, namely a. - (phenyl-triazol-1-yl), b. - (4-carboxy-triazol-1-yl), c. - (4-4-methoxycarbonyl-triazol-1-yl), d. - (4-dimethylaminoethylamidocarboxytriazol-1-yl) possess manifested antitumour activity and higher solubility as compared to the initial oligomycin A.

EFFECT: preparing the new derivatives of the antitumour antibiotic.

3 cl, 4 dwg, 3 tbl

Description

FIELD OF THE INVENTION

The invention relates to the pharmaceutical industry and relates to new derivatives of the target-specific macrolide antibiotic oligomycin A and the method for their preparation.

State of the art

Macrolide antibiotics are highly effective natural drugs with antifungal and antibacterial effects. The most important representatives of this group are oligomycin A (formula 1, R ₁ = CH ₃ , R ₂ = CH ₃ , R ₃ = H, R ₄ = OH), oligomycin B (formula 1 R ₁ = C = O, R ₂ = H , R ₃ = CH ₃ , R ₄ = OH), oligomycin C (formula 1, R ₁ = CH ₂ , R ₂ = H, R ₃ = CH _3, R ₄ = H) ₂ , R ⁴ = H), rutamycin A (formula 2, R ⁴ = OH), rutamycin B (formula 2, R = H) (Fig. 1).

Oligomycins and rutamycins are 26-membered α, β-unsaturated lactones linked to a bicyclic spiroketal structure.

The mechanism of action of macrolide antibiotics of the oligomycin group is based on a violation of the relationship between the F ₀ and F ₁ factors of mitochondrial ATP synthase and the inhibition of phosphorylation in mitochondria. Oligomycin is specific for ATP synthase and, in micromolar concentrations, blocks proton transport through the F ₀ subunit. The enzyme complex F ₀ F ₁ ATP synthase is considered as the molecular target of new drugs for the treatment of tumors and infections (Shchepina et al. Oligomycin, inhibitor of the FO part of H + -ATP-synthase, suppresses the TNE-incklced apoptosis, Oncogene (2002) 21, 8149-8157). Due to the specific suppression of mitochondrial ATP synthase, oligomycins exhibit antibacterial, antifungal, and antitumor effects. Antibiotics of the oligomycin group along with valuable properties have several disadvantages, the main of which are: high toxicity and low solubility.

Chemical modification of macrolide antibiotics of the oligomycin group of highly active ATP synthase inhibitors is aimed at obtaining new derivatives that have selective antitumor or anti-infectious effects and have advantages over the original antibiotics. At the same time, the chemical modification of other macrolides (erythromycin) has led to highly active selectively acting antibacterial drugs that currently dominate the pharmaceutical market. One of the important synthetic approaches in macrolide chemistry is the complete synthesis of certain antibiotics of this group and the preparation of their analogues (Evans et al., Total synthesis of oligomycin CJ Org. Chem. 1998. 63. 4572-4576; Panek. Nareshkumar. Total synthesis of rutamycin B and oligomycin CJ Org. Chem., 2001, 66, 2747 2756; White et al., Total synthesis of rutamycin B, a macrolide antibiotic from Streptomyces aureofaciens. J. Org. Chem., 2001, 66, 5217 5231). As part of these syntheses, synthetic approaches have been developed to the synthesis of macrolactone cycles and the glycosylation of the resulting aglycones with various, including non-natural, sugars. These approaches were most fully used in the synthesis of antibiotics of the apoptolidine family and their derivatives (Wehlan et al. Apoptolidin A: total synthesis and partially glycosylated analogues. Chemistry - Eur. 7., 2006, 12, 7378-7397; Ghidu et al., Synthesis and evaluation of the cytotoxicity of apoptolidinones A and DJ Org. Chem. 2008, 73, 4949-4955).

The preparation of semisynthetic oligomycins is difficult due to the complexity of the structure of the molecule — the presence of a large number of asymmetric atoms, functional groups and the lability of the antibiotic, which is unstable even under slightly acid and alkaline conditions. A serious problem is not only the choice of selective modification methods, but also the strict identification of the derivatives obtained. The most accessible for modification are oxygen-containing groups (hydroxy- and carbonyl groups. Partial acetylation of oligomycin A is described, which led to the formation of 5,9,33-tri-O-acetyl and 5,9,13,33-tetraacetyl derivatives. These derivatives are not showed inhibitory activity when tested on Aspergillus niger spores (Szilagyi et al., Structure elucidation of two acetylated derivatives of oligomycin A, Spectroscopy Lett., 1995, 28, 699-707). Oligomycin A contains several keto groups whose reactivity has been studied (Ramirez et al., Effects of borohydride-treated oligomycins on processes of energy transduction in mitocho ndria. Eur. J. Biochem., 1982, 121, 2275-2279). The authors showed that the gradual addition of sodium borohydride in an ethanol solution of rutamycin and oligomycin restores the keto groups at the C-7 and C-11 positions of the macrolide aglycon to the corresponding hydroxyl groups without further alteration of the lactone cycle Reduced compounds, like the original antibiotics, inhibit ADP-dependent respiration and proton emission during ATP hydrolysis, but do not affect other respiration-related processes in rat liver mitochondria.

Methods for the chemical modification of oligomycin A are described that selectively affect the 7-keto group and the C-2, C-3 double bond. The interaction of oligomycin A with hydroxylamine led to the formation of a 6-membered nitron annelated with the starting antibiotic at positions 3, 4, 5, 6, 7. The reaction with 1-aminopyridinium iodide gave pyrazolo [1,5-a] pyridines conjugated to oligomycin A according to the provisions of C-2 and C-3, followed by spontaneous oxidation of the product of addition on a double bond C2 C3. The derivatives obtained are less toxic than the original antibiotic and, unlike the latter, do not exhibit antifungal activity.

The structures of the compounds obtained were confirmed by NMR and mass spectrometry, including compounds labeled with the ¹⁵ N isotope (Lysenkova et al. The first examples of chemical modification of oligomycin AJ Antibiot. (Tokyo), 2010, 63, 17 22). Oligomycin A can be brominated with N-bromosuccinimide under neutral conditions, forming a monobromo derivative (Lysenkova et al. Synthesis and properties of a novel brominated oligomycin A derivative. J. Antibiot. (Tokyo), 2012. doi: 10.1038 / ja.2012.4. Detailed NMR- the study showed that the semisynthetic derivative obtained has the structure of a 6-membered pyran annelated with a macrocycle (Fig. 2). When reactions are carried out in the presence of bases, oligomycin A forms a stable compound with a split macrocycle (Lysenkova et al., A novel acyclic oligomycin A derivative formed via retro-aldol rearrangement of oligomycin AJAntibiot. (Tokyo), 2012, p dia).

Preliminary studies show that changes in the structure of the macrolactone cycle reduce, to varying degrees, the activity of the semisynthetic antibiotics described above with respect to ATP synthase, however, the substitution of the hydroxyl group at C-33 for the position of oligomycin A (1) (Scheme 1) with other functional groups - methanesulfonyl (4 ) and azido (5), did not interfere with the interaction of the antibiotic with the target ATP synthase. The cytotoxicity of the obtained compounds 4 and 5 is close to that of the initial antibiotic [Patent on the invention No. 2454420 from 06.27.2012? Bull. No. 18] (Fig. 3).

Disclosure of invention

Compared to the semi-synthetic derivatives described above, in the compounds of the present invention, the azido group of 33-azido-oligomycin A is transformed into 1,4-disubstituted 1,2,3-triazoles.

The invention includes a method for producing new semi-synthetic analogues of the antibiotic oligomycin A (formula 3), which consists in regioselective [3 + 2] dipolar cycloaddition of a 33-azido group to monosubstituted alkynes (Scheme 2). The cycloaddition of 33-deoxy-33-azido-oligomycin (formula 1) to alkynes (formula 2) was carried out in a mixture of tert-butanol: water (1: 1) both in the presence of a catalyst (salts of monovalent copper) and without a catalyst.

Examples of the identification of oligomycin A derivatives of the present invention and the study of their antibacterial and antitumor activity

Example 1. 33-Deoxy-33- (4-phenyl-triazol-1-yl) oligomycin (formula 3a).

Oligomycin azide (1) (0.05 g, 0.06 mmol) was placed in a round bottom flask, dissolved in 5 ml of a mixture of t-butanol: water (1: 1), phenylacetylene (0.6 mmol) was added, sodium ascorbate was added in 0.018 g (0.06 mmol), Cu 0.06 g (0.95 mmol) and CuI 0.05 g (0.26 mmol) in powder form and stirred at 60-65 ° C for 18 hours, the reaction mixture was purged argon. The reaction was monitored by thin layer chromatography on Merck plates in a hexane: acetone system (1: 1). At the end of the reaction, the product was extracted with ethyl acetate from the reaction mixture diluted with water twice in 15 ml each, the extract was filtered, dried with sodium sulfate, and the solvent was removed on a rotary evaporator. Phenyltrizole oligomycin was purified by silica gel column chromatography in hexane: acetone (4: 1) system. Obtained 0.022 g of phenyltriazole oligomycin in the form of an amorphous white powder. Yield 39%. Gross formula C ₅₃ H ₇₉ N ₃ O ₁₀ , mol. weight 917.5765; ion 940.5684 (M + Na) ⁺ was observed in the mass spectrum.

UV (MeOH) λ max (ε) 205 (60,000), 225 pl. (54000), 232 (56000), 241 pl. (45000).

IR ν (cm ^-1 ) 3466, 2971, 2931, 2870, 1699, 1643, 1456, 1384, 1278, 1226, 1191, 1097, 1049.

[α] D ²⁴ -65 ° (s - 0.184, CH ₂ OH).

R _T 16.053 (A - H ₂ O; B - MeCN), A-20%, B-80%.

Example 2. 33-Deoxy-33- (4-carboxytriazol-1-yl) oligomycin (formula 3b). Oligomycin azide (1) (0.05 g, 0.06 mmol) was placed in a round bottom flask, dissolved in 5 ml of a mixture of t-butanol: toluene (1: 1), 0.2 ml (0.2 mmol) of acetylene carboxylic acid was added. , the reaction mixture was stirred at 45 ° C for 24 hours, 0.05 ml (0.05 mmol) of acetylene carboxylic acid was added every 6 hours. The reaction was monitored by thin layer chromatography on Merck plates in the chloroform: methanol: acetic acid system (volume ratio 10: 2: 0.1). The reaction mixture was evaporated from toluene, the reaction product was extracted with ethyl acetate from the reaction mixture twice with 15 ml of water, the extract was washed with 1% NaHCO ₃ solution from excess acetylene carboxylic acid, filtered, dried with sodium sulfate, and the solvent was removed on a rotary evaporator. Carboxytriazole oligomycin was purified by silica gel column chromatography in the chloroform: methanol: acetic acid system (volume ratio 20: 2: 0.1), the pure fractions were washed with water, dried with sodium sulfate, concentrated in vacuo, and oligomycin hexane carboxitriazole was precipitated. Received 0.027 g of carboxytriazole oligomycin in the form of an amorphous white powder. Yield 50%. Gross formula C ₄₈ H ₇₅ N ₃ O ₁₂ , mol. weight 885.5350; ion 908.5281 (M + Na) ⁺ was observed in the mass spectrum.

UV (MeOH) λ max (ε) 213-223 (58571), 232 pl. (51428), 241 pl. (31428).

IR ν (cm ^-1 ) 3462, 2972, 2936, 2810, 1704, 1644, 1548, 1456, 1385, 1280, 1226, 1193, 1135, 1033, 1048, 986, 922, 880.

[α] D ²⁴ -91 ° (s - 0.176, CH ₃ OH).

R _T 10.30 (A - H ₃ PO ₄ 0.01 M pH 2.6; B - MeCN), A-30%, B-70.

Example 3. 33-Deoxy-33- (4-methoxycarbonyltriazol-1-yl) oligomycin (formula 3c).

Oligomycin azide (1) (0.06 g, 0.075 mmol) was placed in a round bottom flask, dissolved in 5 ml of a mixture of t-butanol: water (1: 1 volume ratio), 0.03 ml of acetylene carboxylic acid methyl ester was added (0.37 mmol), 0.06 g (0.95 mmol) of powder was sprinkled with Cu in the form of a powder and stirred at room temperature for 6 hours. The reaction was monitored by thin layer chromatography on Merck plates in a toluene: ethyl acetate system (2: 1). At the end of the reaction, the product was extracted with ethyl acetate from the reaction mixture diluted with water twice in 15 ml each, the extract was filtered, dried with sodium sulfate, and the solvent was removed on a rotary evaporator. Carboxytriazole oligomycin methyl ester was purified by silica gel column chromatography using toluene: ethyl acetate (25: 1). Obtained 0.034 g (0.037 mmol) of carboxytriazole oligomycin methyl ester in the form of an amorphous white powder. Yield 52%.

Gross formula C ₄₉ H ₇₇ N ₃ O ₁₂ , mol. weight 899.5072, ion 922.5406 (M + Na) ⁺ was observed in the mass spectrum.

UV (Meon) λ max (ε) 223 (86364), 232 pl. (73636), 239 pl. (43536).

IR ν (cm ^- ) 3486, 2972, 2934, 1704, 1456.1385, 1278, 1226, 1099, 1046, 987.

[α] D ²⁴ -66 ° (s - 0.18, CH ₃ OH).

R _T 14.274 (A - H ₃ PO ₄ 0.01 M pH 2.6; B - MeCN), A-30%, B-70%.

Example 4. 33-Deoxy-33 - [(4-dimethylaminoethylamidocarbonyl) triazol-1-yl] oligomycin A (formula 3g).

Carboxitriazole oligomycin (36) (0.05 g, 0.056 mmol) was placed in a round bottom flask, dissolved in 5 ml of dimethylformamide, 0.05 g (0.1 mmol) was added to PyBOP, and N, N-dimethylethylenediamine 0.012 ml (0.1) was added. mmol) and was stirred for 24 hours. The reaction was monitored by thin layer chromatography on Merck plates in the chloroform: methanol: acetic acid system (10: 3: 0.2).

The reaction mixture was diluted with water, NaCl was added, extracted with ethyl acetate twice in 15 ml, the extract was washed with 1% NaHCO ₃ solution and water, dried with sodium sulfate, the solvent was removed on a rotary evaporator. Carboxytriazole oligomycin amide was purified by silica gel column chromatography in the chloroform: methanol: acetic acid system (10: 0.1), the pure fractions were washed with 1% NaHCO ₃ solution, concentrated in vacuo, then the carboxytriazole amide was purified on Sephadex LH-20 in methanol. Received 0.008 g of carboxytriazole amide oligomycin in the form of an amorphous white powder. Yield 15%.

Gross formula C ₅₂ H ₈₅ O ₁₁ , mol. weight 955.6245; ion 956.6328 (M + H) ⁺ was observed in the mass spectrum.

Example 5. NMR spectra of oligomycin A and its triazole derivatives (δ _C , δ _H , ppm).

Example 6

A comparison of the ability of oligomycin A and new derivatives to inhibit FoFi-ATP synthase was evaluated by suppressing the growth of a strain of Streptomyces fradiae sensitive to very low (in the picomolar range) concentrations of oligomycin A.

Testing of the sensitivity of bacteria to inhibition of FoFi-ATP synthase was carried out by the method of disc stacking and it consisted in determining the growth suppression zone of the Streptomyces fradiae strain, seeded with a lawn on an agar medium, around paper disks containing oligomycin A and oligomycin A azide in various concentrations. To prepare the lawn, the spore suspension was mixed with MG-2 agar medium (0.7% agar) in a ratio of 2 × 10 ⁶ -1 × 10 ⁷ spores per Petri dish and plates with MG-1 agar medium (2% agar) were seeded. After the agarized spore suspension solidified, paper discs containing the test compounds were applied to the plates. The lawn was grown for 40-48 hours at 28 ° C. Media with the general name MG contains malt extract, yeast extract, NaCl, MgSO ₄ , K ₂ HPO ₄ , FeSO ₄ , KNO ₃ , glucose.

The test results are presented in table 1.

Table 1 Compound Concentration, nmol / disk 0.001 0.1 one 10 oligomycin A 9 26 32 - 1 (oligomycin A azide) no no 7.5 10 3a no 7.5 13 twenty 3c no no no 8 3G no* no 8 eleven * diameter (mm) of the growth inhibition zone of Streptomyces fradiae.

Thus, in the Streptomyces fradiae test system, oligomycin azide and compound 3g ~ are 1000 times less active than oligomycin A, 3a ~ 100 times less active, 3b ~ 10,000 times lower.

Example 7

The study of the ability of oligomycin A and new derivatives to cause the death of tumor cells was carried out according to the following method. Adhesion cells (HCT116, human colon adenocarcinoma) were detached from the substrate by versene-trypsin, thoroughly pipetted to form a unicellular suspension, 50 μl of the suspension were added to 450 μl of physiological buffer, pipetted and counted in a Goryaev chamber. Human leukemia cells (line K562) growing in suspension added 50 μl of the suspension to 450 μl of physiological buffer, pipetted and counted in a Goryaev chamber. Used cultures in the logarithmic phase of growth. 190 ml of cell suspension were added to the wells of a 96-well plate and left in a CO ₂ incubator for 24 hours. Stock solutions of the studied preparations (DMSO solvent) with a concentration of 10 mM were prepared, from which serial dilutions of the preparations were carried out in cell culture medium (Dulbecco's modified Eagle medium with the addition of 5% fetal calf serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin). DMSO solutions in the same dilutions as the studied solutions were used to control the effect of the solvent. The volumes of solvent and the change in the total volume of the medium in the well were neglected. Received the following concentrations of drugs in the well: 0.1 μm; 0.2 μM; 0.4 μM; 0.8 μM; 1.6 μM; 3.2 μM, 6.4 μM; 12.5 μM; 25 μM; 50 μm. Each concentration of the study drug was studied in triplicate. Tablets with added compounds were placed in a CO ₂ incubator at 37 ° C for 72 hours. 10 μl MTT aqueous solution (5 mg / ml) was added to the wells. Incubated 1-4 hours before the development of intense dark violet staining of formazan inside the cells. The medium was selected without affecting the cells. 100 μl of DMSO was added to the wells, pipetted until homogeneous and shaken on a shaker for 2 minutes. The optical density (OD) of the resulting solutions was measured at 540 nm, and cell survival curves were constructed. When constructing them, the OD of ₅₄₀ control wells was taken as 100%, to which the OD of wells with a particular concentration of the studied drug was attributed. A survival concentration of 50% of cells (IC ₅₀ ) was noted as critical.

Table 2 shows the results of a study of the cytotoxicity of oligomycin A and new derivatives for leukemia cells (line K562) and colon cancer (line HCT116).

table 2 ICs ₅₀ μm * Compound K562 HCT116 oligomycin A 0.18: 1: 0.02 1.00 ± 0.20 33-deoxy-33-azido-oligomycin A (1) 0.12 ± 0.02 0.70 ± 0.12 33-deoxy-33- (4-methoxycarbonyl-triazol-1-yl) -oligomycin (3c) 0.15 ± 0.01 - 33-deoxy-33- (4-phenyl-triazol-1-yl) -oligomycin A (3a) 0.15 ± 0.02 - 33-deoxy-33 - [(4-dimethylaminoethylamidocarboxy) triazol-1-yl) oligomycin A (3g) 0.22 ± 0.03 0.70 ± 0.11 33-deoxy-33- (4-carboxy-triazol-1-yl) -oligomycin A (3b) 25.0 ± 5.0 > 25.0 * The concentration of the compound, causing the death of 50% of the cells after 72-hour continuous incubation.
- The indicator was not determined.

Thus, the proposed new method for modifying the antibiotic oligomycin A allows to obtain new triazole derivatives of oligomycin A, which have advantages over the original antibiotic oligomycin A and its semisynthetic analogue 33-azido oligomycin, namely, high cytotoxicity for tumor cells and good solubility.

Claims (3)

1. The method of producing semi-synthetic derivatives of the antibiotic oligomycin A (3a-g), which consists in regioselective [3 + 2] dipolar cycloaddition of the azido group of 33-deoxy-33-azido oligomycin A (1) to monosubstituted alkynes 2a-b as in the presence of a catalyst (mixture of copper powder and salts of monovalent halides), and in its absence, upon preparation of compound 3g, the carboxytriazole oligomycin (3b) is amidated with N, N-dimethylethylenediamine.

2. Derivatives of the antibiotic oligomycin A 3a-g, corresponding to the General formula:

where R represents a 1,4-disubstituted 1,2,3-triazole: a. - (phenyl-triazol-1-yl), b. - (4-carboxy-triazol-1-yl), c. - (4-4-methoxycarbonyl-triazol-1-yl), g. - (4-dimethylaminoethylamidocarboxytriazol-1-yl). 3. The use of compounds of formula 3a-g of claim 1 as antitumor agents.

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