RU2538962C1 - Antifungal compounds based on 3,5,8-trioxabicyclo[5,1,0]octane derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to antifungal compounds based on 3,5,8-trioxabicyclo[5,1,0]octane derivatives, obtained by trans-opening of its epoxide cycle, namely 6-(arylthio)-1,3-dioxepan-5-ols in racemic and enantiopure form of the general formula Ia

and Ib

, where at R1=F, R2=H, R3=H; at R1=Br, R2=H, R3=H; at R1=H, R2=Br, R3=H; at R1=H, R2=H, R3=Br.

EFFECT: compounds of formula Ia and Ib possess low toxicity and high antifungal activity with respect to fungi Candida albicans, Aspergillus fumigatus, Epidermophyton floccosum, Mucor pusilos, Saccharomyces cerevisiae and can be applied in medicine and veterinary.

2 tbl, 13 ex

Description

The invention relates to synthetic biologically active substances of the heterocyclic series having high antifungal activity, and which are products of trans-opening of the epoxy cycle of 3,5,8-trioxabicyclo [5.1.0] octane, namely 6- (arylthio) -1,3- dioxepan-5-ol in racemic and enantiopure form of the general formula Ia and Ib:

, где:

where:

when R ₁ = F, R ₂ = H, R ₃ = H

when R ₁ = Br, R ₂ = H, R ₃ = H

when R ₁ = H, R ₂ = Br, R ₃ = H

when R ₁ = H, R ₂ = H, R ₃ = Br

The compounds of formula Ia and Ib have high antifungal activity against a background of low toxicity and can be used in medicine and veterinary medicine.

Mycosis is an urgent medical problem. The increase in the population of immunocompromised patients with a high risk of developing severe fungal infections is due to the introduction of new medical technologies into clinical practice (for example, organ and tissue transplantation, high-dose immunosuppressive therapy, invasive diagnostic and therapeutic procedures, etc.), an HIV pandemic, and successes in treatment of bacterial complications. The number of invasive mycoses is progressively increasing, while the mortality associated with them remains very high (40-90%). The main method of treating mycosis today is a chemical therapeutic method based on the use of various drugs with antifungal properties.

An important feature of mycoses at present is the high frequency of pathogens resistant to the used antimycotics, such as Fusarium spp., Scedosporium spp., Rhizopus spp., Mucor spp., Etc. In addition, pathogens of invasive candidiasis, in particular, Candida, are more and more common. glabrata and Candida krusei, characterized by resistance to first-generation triazoles and reduced sensitivity to amphotericin B. The appearance of Aspergillus spp. resistant to amphotericin B and itraconazole was noted [N. Klimko N. Invasive mycoses: new treatment options // Infections and antimicrobial therapy - 2004. - T.6, No. 2. - S. 71-74]. The development of new effective and safe antimycotics acting on previously unknown biological targets provides new opportunities for clinicians to combat these serious diseases [Lee Yu-Ting, Cui Chang-Jun, Chow Eve WL, Pue Nason, Lonhienne Thierry, Wang Jian-Guo, Fraser James A., Guddat Luke W. Sulfonylureas Have Antifungal Activity and Are Potent Inhibitors of Candida albicans Acetohydroxyacid Synthase // J. Med. Chem. - 2013 .-- V.56. - P.210-219].

From the prior art examined by the applicant, analogues of the claimed technical solution with a similar structural formula have not been identified, however, analogues for their intended purpose - preparations with high antimycotic activity have been identified.

The most common antibiotics prescribed in clinical practice are fluconazole and terbinafine.

Fluconazole exhibits high antimycotic activity and low toxicity [US Pat. No. 4,402,216. Antifungal 1,3-bis-triazolyl-2-propanol derivative I Pfizer. Inc. - Publ. - 09/13/1983].

However, as already noted, recently the number of fluconazole-resistant strains has increased significantly [Vanden Bossche H., Marichal P., Odds F. Molecular mechanisms of drug resistance in fungi // Trends Microbiol - 1994. -V 2. - P .393-400].

From the studied prior art revealed no less significant antimycotic terbinafine [patent US 5231183. Antifungal 1,3-bis-triazolyl-2-propanol derivative / Banyu Pharmaceutical Co., Ltd. - Publ. - 07/27/1993]. It should be noted that resistance to this drug in microorganisms is produced much less frequently than to drugs of the triazole class, however, the toxicity of the latter is noticeably lower.

From the investigated prior art, the applicant has identified thiolysis products of thiophenol disubstituted at the acetal carbon atom of 3,5,8-trioxabicyclo [5.1.0] octane, namely 2,2-dimethyl-3,5,8-trioxabicyclo [5.1.0] octane, which were described by Japanese authors back in 1995 [Yamada O., Ogasawara K. Lipase-mediated preparation of optically pure four-carbon di- and triols from meso-precursor // Synthesis. - 1995. - N.10. - P.1291-1294]. In this work, they used PS lipase (Amano) to separate the racemic alcohol obtained into enantiomers. However, the biological properties of the synthesized enantiomers have not been investigated in principle.

The prior art revealed the disclosure by thiophenol of another disubstituted derivative of 3,5,8-trioxabicyclo [5.1.0] octane, namely 3,5,8-trioxaspiro [bicyclo [5.1.0] octane-4,1-cyclohexane], obtained the applicant [Paveliev R.S., Klimovitsky E.N., Nikitina L.E. Synthesis of isomeric hydroxysulfides (sulfones) based on 3,5,8-trioxaspiro [bicyclo [5.1.0] octane-4,1′-cyclohexane] // Chemistry for Sustainable Development. - 2010. - T.18, No. 6. - S.775-781].

Thus, the resulting 3,5,8-trioxaspiro [bicyclo [5.1.0] octane-4,1-cyclohexane] was also not investigated by the applicant for the presence of antifungal activity.

In addition to the above, the applicant also identified his own work on the thiolysis reaction of thiophenol monophenyl substituted at the carbon atom for isomeric 3,5,8-trioxabicyclo [5.1.0] octanes [Pavelyev RS, Gnevashev SG, Vafina RM, Gnezdilov O.I., Dobrynin A .V., Lisovskaya SA, Nikitina LE, Klimovitsckii EN Synthesis and Antimycotic Properties of Hydroxy Sulfides Derived from exo- and endo-4-phenyl-3,5,8-trioxabicyclo [5.1.0] octanes // Mendeleev communications. - 2012. - V.12, No. 3. - P.127-128].

The obtained isomeric beta-hydroxysulfides (including in enantiopure form) were examined for the presence of antifungal activity, which strongly depended on the structure of the synthesized compounds. However, the minimum inhibitory concentrations of these compounds were very high and were not of practical interest from the point of view of the availability of prospects in the development of new antimycotic agents.

The 3,5,8-trioxabicyclo [5.1.0] octane itself was subjected to thiolysis with thiophenol in the presence of potash by a solvent-free reaction, during which 6- (phenylthio) -1,3-dioxepan-5-ol was obtained, which differs from the claimed compounds the absence of a halogen atom in the aromatic ring [Paveliev R.S. Synthesis of β-hydroxy (acetoxy) sulfides and sulfones based on 3,5,8-trioxabicyclo [5.1.0] octanes: Dis. ... cand. Chem. Sciences: 02.00.03: is protected 04/22/2011: approved. 07/08/2011 / Paveliev Roman Sergeevich. - Kazan, 2011 .-- 142 p. - Bibliography: p.127-142].

This alcohol showed antimycotic activity, completely inhibiting the growth of the studied fungi (Candida albicans, Aspergillus fumigatus, Epidermophyton floccosum) at a concentration of less than 1 mg / ml, but more than 0.5 mg / ml.

Also, this racemic alcohol was resolved into enantiomers using PS lipase (Amano). The antifungal properties of enantiomers have not been investigated.

It should be noted that the compounds described above as a whole, according to the applicant, can be considered as structural analogues to the claimed technical solution due to the fact that they belong to the same class of compounds - 1,3-dioscepanes and do not coincide with the claimed technical solution by the nature of the substituents in the dioscepane cycle, which determine the antimycotic properties of the claimed technical solution.

The objective of the claimed technical solution is to create new low-toxic biologically active compounds with high antifungal activity against both micellial, yeast-like and yeast fungi.

The problem is solved by synthesis of antifungal substances of the general formula Ia and Ib:

, где

where

when R ₁ = F, R ₂ = H, R ₃ = H

when R ₁ = Br, R ₂ = H, R ₃ = H

when R ₁ = H, R ₂ = Br, R ₃ = H

when R ₁ = H, R ₂ = H, R ₃ = Br

In modern medicine, antimycotics with similar structures are not used.

The inventive substances on the background of low toxicity showed high antifungal activity against strains of the fungi Candida albicans, Aspergillus fumigatus, Epidermophyton floccosum, Mucor pusilos, Saccharomyces cerevisiae and can be used in medicine and veterinary medicine.

The applicant has not identified sources that would contain information about the influence of the distinguishing features of the invention on the achieved technical result. The specified new property of the object determines, according to the applicant, the compliance of the invention with the criterion of "inventive step".

Presented in the claimed technical solution, the compounds of formula Ia and Ib were obtained according to the following scheme:

Where

R ¹ R ² R ³ Ia-1 F H H Ib-1 F H H Ia-2 Br S H Ib-2 Br H H Ia-3 H Br H Ib-3 H Br H Ia-4 H H Br Ib-4 H II Br III-1 F H H III-2 Br H H III-3 H Br H III-4 H II Br

Characteristics of the new compounds are given in examples of specific performance. The structures of the compounds obtained were confirmed by mass spectrometry, ¹ H and ¹³ C NMR spectroscopy. NMR spectra were recorded on a Bruker AVANCE-400 instrument. The chemical shift was determined relative to the signals of the residual protons of deuterated solvents ( ¹ H and ¹³ C). The reaction progress and the purity of the compounds were monitored by TLC on Sorbfil Plates plates. MALDI mass spectra were recorded on a Bruker Ultraflex III instrument equipped with a solid state laser and time-of-flight mass analyzer. Accelerating voltage 25 kV. Samples were applied to an Anchor Chip target. Spectra were recorded in the mode of positive ions. The resulting spectrum was the sum of 300 spectra obtained at different points in the sample. As matrices, 2,5-dihydroxybenzoic acid (DHB) (Acros, 99%) and n-nitroaniline (PNA) were used. Chloroform was used to prepare the matrices. The application of samples to the target was carried out by the method of "dried drops".

The angles of rotation of the beam of polarized light of optically active compounds were determined using an ADP-440 (B + S) polarimeter (Britain).

The melting point of crystalline substances was determined using an MPA-100 OptiMelt instrument (Stanford Research Systems).

To determine the enantiomeric excess (s), ¹ H NMR spectroscopy was used in the presence of a shift reagent (tris [3- (heptafluoropropylhydroxymethylene) - (+) camphor] europium (III) from ALDRICH CHEMISTRY), comparing the integrand signal area (intensity) hydrogen atoms at a carbon atom of C ² enantiomers.

Examples of specific implementation of the claimed technical solution

Example 1. Synthesis of 3,5,8-trioxabicyclo [5.1.0] octane (II). To 22.35 g (223 mmol) of 1,3-dioxacyclohept-5-ene and 89 g (1061 mmol) of NaHCO ₃ in 400 ml of fifty percent aqueous acetone at room temperature, 178 g (290 mmol) of oxone was added portionwise with stirring. The reaction mass was further stirred for 1.5 hours. The progress of the reaction was monitored by TLC. The product was extracted with methylene chloride (3 × 70 ml). Water was saturated with sodium chloride and extraction was performed again. The organic phases were combined and dried over magnesium sulfate. The solvent was evaporated. Received 19.18 g of colorless crystals with so pl. 56-57 ° C (yield 74%) [lit. Gianni M.N., Cody R., Asthana MR The role of the generalized anomeric effect in the conformational analysis of 1,3-dioxacycloalkanes. Conformational analysis of 3,5-dioxabicyclo [5.1.0] octanes and 3,5,8-trioxabicyclo [5.1.0] octanes // J. Org. Chem. - 1977. - V.42, N.2. - P.365-368].

Example 2. Synthesis of racemic 6 - ((2-fluorofeiyl) thio) -1,3-dioxepan-5-ol (Ia-1 + Ib-1). A mixture of 0.22 g (1.72 mmol) of 2-fluorothiophenol, 0.023 g (0.17 mmol) of K ₂ CO ₃ and 0.2 g (1.72 mmol) of epoxide II was heated until the first bubbles appeared, then the heating was stopped. Short-term (<1 min) boiling of the mixture occurred with a sharp increase in temperature. The mass was cooled, the product was purified by column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in the ratio 4: 1). Received 0.38 g of a colorless oily liquid (yield 90%).

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 3.07 br. s (1H, OH), 3.17 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.4 Hz, ³ J (H ⁶ H ⁷ _A ) = 5.8 Hz, ³ J (H ⁶ H ⁷ _B ) = 6.3 Hz), 3.65 m (1H, H ⁵ , ³ J (H ⁵ H ⁴ _A ) = 2.7 Hz, ³ J (H ⁵ H ⁴ _B ) = 1.9 Hz), 3.74 m (1H, H ⁴ _A , ² J (H ⁴ _A , H ⁴ _V ) = - 12.0 Hz), 3.83 dd (1H, H ⁷ _A ), 4.04 dd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.4 Hz), 4.09 dd (1H, H ⁴ _B ), 4.72 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.4 Hz), 4.73 d (1H, H ² _B ,), 7.04-7.51 m (4H, 2-FC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 54.61 (C ⁶ , ⁴ J (FC ⁶ ) = - 1.2 Hz), 65.41 (C ⁷ ), 66.44 (C ⁴ ), 71.63 (C ⁵ ), 94.20 (C ² ), 116.20 (C _Ar , ² J = -23.1 Hz), 120.02 (C _Ar , ² J = -18.0 Hz), 124.75 (C _Ar , ³ J = 3.8 Hz), 130.38 (C _Ar , ³ J = 8.1 Hz), 135.49 (C _Ar ), 162.59 (C _Ar , ¹ J = 246.0 Hz). Mass spectrum MALDI: 267 [M + Na] ⁺ , 283 [M + K] ⁺ .

Example 3. Synthesis of racemic 6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-2 + Ib-2). Synthesize and purify similarly to the compound (Ia-1 + Ib-1) from 0.2 g (1.72 mmol) of epoxide II. Obtained 0.44 g of a colorless oily liquid (yield 83%).

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 3.01 br. s (1H, OH), 3.31 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.5 Hz, ³ J (H ⁶ H ⁷ _A ) = 5.7 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.7 Hz, ⁴ J (H ⁶ H ⁴ _A ) = - 1.2 Hz), 3.72 td (1H, H ⁵ , ³ J (H ⁵ H ⁴ _A ) = 5.3 Hz, ³ J (H ⁵ H ⁴ _B ) = 1.5 Hz), 3.78 ddd (1H, H ⁴ _A , ² J (H ⁴ _A , H ⁴ _B ) = - 12.1 Hz), 3.93 dd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.6 Hz), 4.14 dd (1H, H ⁷ _B ), 4.14 dd (1H, H ⁴ _B ), 4.75 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.6 Hz), 4.76 d (1H, H ² _B ,), 7.07-7.60 m (4H, 2-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 54.17 (C ⁶ ), 65.21 (C ⁷ ), 66.51 (C ⁴ ), 71.51 (C ⁵ ), 94.42 (C ² ), 126.70 (C _Ar ), 128.12 (C _Ar ), 128.50 (C _Ar ), 132.03 (C _Ar ), 133.59 (C _Ar ), 135.14 (C _Ar ). Mass spectrum MALDI: 328 [M + Na] ⁺ , 344 [M + K] ⁺ .

Example 4. Synthesis of racemic 6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-3 + Ib-3). Synthesize and purify similarly to the compound (Ia-1 + Ib-1) from 0.2 g (1.72 mmol) of epoxide II. Obtained 0.47 g of a colorless oily liquid (yield 89%).

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 2.96 br. s (1H, OH), 3.24 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.4 Hz, ³ J (H ⁶ H ⁷ _A ) = 5.8 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.7 Hz), 3.69 td (1H, H ⁵ , ³ J (H ⁵ H ⁴ _A ) = 5.5 Hz, ³ J (H ⁵ H ⁴ _B ) = 1.5 Hz), 3.76 dd (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.0 Hz), 3.84 dd (1H, H ⁷ _A , ³ J (H ⁷ _A H ⁷ _B ) = - 12.5 Hz), 4.04 dd (1H, H ⁷ _B ), 4.06 dd (1H, H ⁴ _B ), 4.73 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.6 Hz), 4.74 d (1H, H ² _B ,), 7.13-7.56 m ( 4H, 3-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 54.75 (C ⁶ ), 65.22 (C ⁷ ), 66.39 (C ⁴ ), 71.50 (C ⁵ ), 94.27 (C ² ), 122.98 (C _Ar ), 129.86 (C _Ar ), 130.44 (C _Ar ), 130.53 (C _Ar ), 133.86 (C _Ar ). 136.13 (C _Ar ). Mass spectrum MALDI: 328 [M + Na] ⁺ , 344 [M + K] ⁺ .

Example 5. Synthesis of racemic 6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-4 + Ib-4). Synthesize and purify similarly to the compound (Ia-1 + Ib-1) from 0.2 g (1.72 mmol) of epoxide II. Obtained 0.48 g of a colorless crystalline substance T _PL = 58 ° C (yield 91%).

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 3.00 br. d (1H, OH, ³ J (OHH ⁵ ) = 3.5 Hz), 3.20 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.5 Hz, ³ J (H ⁶ H ⁷ _A ) = 5.9 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.7 Hz, ⁴ J (H ⁶ H ⁴ _A ) = 0.8 Hz), 3.75 ears. m (1H, H ⁵ , ³ J (H ⁵ H ⁴ _A ) = 5.7 Hz), 3.76 m (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.1 Hz), 3.83 dd (1H , H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.5 Hz), 4.05 t (1H, H ⁷ _B ), 4.06 t (1H, H ⁴ _B ), 4.73 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.6 Hz), 4.75 d (1H, H ² _B ,), 7.27-7.44 m (4H, 4-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 54.91 (C ⁶ ), 65.28 (C ⁷ ), 66.39 (C ⁴ ), 71.46 (C ⁵ ), 94.30 (C ² ), 121.66 (C _Ar ), 132.36 (C _Ar ), 132.74 (C _Ar ). 133.30 (C _Ar ). Mass spectrum MALDI: 328 [M + Na] ⁺ , 344 [M + K] ⁺ .

Example 6. Separation of racemic 6 - ((2-fluorophenyl) thio) -1,3-dioxepan-5-ol (Ia-1 + Ib-1) into enantiomers by enzymatic acylation using PS lipase (Amano).

To a solution of 1.00 g (4.09 mmol) of racemic 6 - ((2-fluorophenyl) thio) -1,3-dioxepan-5-ol (Ia-1 + Ib-1) in 30 ml of tetrahydrofuran or toluene was added 0.76 ml (8.19 mmol) ) vinyl acetate and 0.5 g of PS lipase immobilized on diatomite. The mixture was stirred at 30-40 ° C for two days. The lipase was filtered, the solvent was removed in vacuo. The products were separated by column chromatography (eluent is a mixture of petroleum ether and ethyl acetate in a ratio of 7: 1).

(5S, 6S) -6 - ((2-fluorophenyl) thio) -1,3-dioxepan-5-ol (Ia-1).

Obtained 0.45 g of a transparent oily substance (yield 45%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = 1.2 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

(5R, 6R) -6 - ((2-fluorophenyl) thio) -1,3-dioxepan-5-yl acetate (III-1).

Obtained 0.54 g of a colorless crystalline substance T _PL = 51-52 ° C (46% yield) with a rotation angle of the beam of polarized light α = -0.4 °; ee> 99%.

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 2.06 s (3H, CH ₃ ); 3.36 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.5 Hz, ³ J (H ⁶ H ⁷ _A ) = 6.5 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.1 Hz); 3.87 dd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.3 Hz); 3.89 dd (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.7 Hz, ³ J (H ⁴ _A H ⁵ ) = 5.6 Hz); 4.08 dd (1H, H ⁴ _B , ³ J (H ⁴ _B H ⁵ ) = 2.1 Hz); 4.15 m (1H, H ⁷ _B ); 4.75 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.4 Hz); 4.80 d (1H, H ² _B ); 4.87 td (1H, H ⁵ ); 7.08-7.54 m (4H, 2-FC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 21.15 (CH ₃ ), 51.89 (C ⁶ ), 65.23 (C ^{7 (4)} ), 65.34 (C ^{4 (7)} ), 73.82 (C ⁵ ), 94.49 (C ² ), 116.18 (C _Ar , ² J = -23.1 Hz), 120.09 (C _Ar , ² J = -17.8 Hz), 124.76 (C _Ar , ³ J = 3.7 Hz), 130.49 (C _Ar , ³ J = 8.0 Hz), 135.60 (C _Ar ), 162.69 (C _Ar , ¹ J = 246.4 Hz), 170.19 (CO). Mass spectrum MALDI: 309 [M + Na] ⁺ , 328 [M + K] ⁺ .

Example 7. Synthesis of (5R, 6R) -6 - ((2-fluorophenyl) thio) -1,3-dioxepan-5-ol (Ib-1).

To a solution of 0.5 g (1.75 mmol) of optically active acetate (III-1) in 20 ml of methanol was added 0.72 g (5.25 mmol) of potash (K ₂ CO ₃ ). The mixture was stirred for 5 hours at room temperature, then potash and the solvent were removed. The product was purified by column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a ratio of 1: 1). Obtained 0.40 g of a transparent oily substance (yield 93%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = -1.2 °; her> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

Example 8. Separation of racemic 6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-2 + Ib-2) into enantiomers by enzymatic acylation using PS lipase (Amano).

(5S, 6S) -6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-2).

Synthesize and purify analogously to compound (Ia-1) from 1 g (3.28 mmol) of racemic 6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-2 + Ib-2). Obtained 0.41 g of a transparent oily substance (yield 41%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = 1.0 °; her> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

(5R, 6R) -6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol acetate (III-2).

Obtained 0.51 g of a transparent oily substance (yield 45%) with an angle of rotation of the beam of polarized light α = 0.2 °; her> 99%.

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 2.08 s (3H, CH ₃ ); 3.46 m (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 4.6 Hz, ³ J (H ⁶ H ⁷ _A ) = 5.3 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.2 Hz, ⁴ J (H ⁶ H ⁴ _A ) = - 1.2 Hz); 3.92 ddd (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.8 Hz, ³ J (C ⁴ _A H ⁵ ) = 4.7 Hz); 3.96 ddd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.3 Hz, ⁴ J (H ⁷ _A H ⁵ ) = - 0.8 Hz); 4.13 dd (1H, H ⁴ _B , ³ J (H ⁴ _B H ⁵ ) = 2.0 Hz); 4.18 dd (1H, H ⁷ _B ); 4.77 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.4 Hz); 4.82 d (1H, H ² _B ); 4.92 m (1H, H ⁵ ); 7.09-7.60 m (4H, 2-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 21.13 (CH ₃ ), 51.72 (C ⁶ ), 64.87 (C ^{7 (4)} ), 65.47 (C ^{4 (7)} ), 73.65 (C ⁵ ), 94.47 (C ² ), 126.64 (C _Ar ), 128.05 (C _Ar ), 128.55 (C _Ar ), 132.40 (C _Ar ), 133.49 (C _Ar ), 134.97 (C _Ar ), 170.10 ( CO). Mass spectrum MALDI: 370 [M + Na] ⁺ , 386 [M + K] ⁺ .

Example 9. Synthesis of (5R, 6R) -6 - ((2-bromophenyl) thio) -1,3-dioxepan-5-ol (Ib-2).

Synthesize and purify similarly to compound (Ib-1) from 0.5 g (1.44 mmol) of optically active acetate (III-2). Obtained 0.43 g of a transparent oily substance (yield 98%) with a rotation angle of the beam of polarized light [α] ²⁴ _D = -1.0 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

Example 10. Separation of racemic 6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-3 + Ib-3) into enantiomers by enzymatic acylation using PS lipase (Amano).

(5S, 6S) -6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-3).

Synthesize and purify similarly to compound (Ia-1) from 1 g (3.28 mmol) of racemic 6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-3 + Ib-3). Obtained 0.40 g of a colorless crystalline substance T _PL = 69 ° C (yield 40%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = 1.2 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

(5R, 6R) -6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol acetate (III-3).

Obtained 0.45 g of a transparent oily substance (yield 40%) with an angle of rotation of the beam of polarized light α = -0.1 °; ee> 99%.

¹ H NMR spectrum (400 MHz, CDCl ₃ ) δ, ppm: 2.09 s (3H, CH ₃ ); 3.45 td (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.4 Hz, ³ J (H ⁶ H ⁷ _A ) = 6.2 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.3 Hz); 3.84 dd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.3 Hz); 3.87 dd (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.3 Hz, ³ J (H ⁴ _A H ⁵ ) = 5.4 Hz); 4.03 dd (1H, H ⁴ _B , ³ J (H ⁴ _B H ⁵ ) = 2.3 Hz); 4.13 dd (1H, H ⁷ _B ); 4.74 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.6 Hz); 4.80 d (1H, H ² _B ); 4.92 td (1H, H ⁵ ); 7.15-7.63 m (4H, 3-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 21.07 (CH ₃ ), 52.06 (C ⁶ ), 65.08 (C ^{7 (4)} ), 65.40 (C ^{4 (7)} ). 73.65 (C ⁵ ), 94.38 (C ² ), 122.84 (C _Ar ), 129.85 (C _Ar ), 130.37 (C _Ar ), 130.42 (C _Ar ), 133.81 (C _Ar ), 136.07 (C _Ar ), 170.04 ( CO). Mass spectrum MALDI: 370 [M + Na] ⁺ , 386 [M + K] ⁺ .

Example 11. Synthesis of (5R, 6R) -6 - ((3-bromophenyl) thio) -1,3-dioxepan-5-ol (Ib-3).

Synthesize and purify similarly to compound (Ib-1) from 0.5 g (1.44 mmol) of optically active acetate (III-3). Obtained 0.40 g of a colorless crystalline substance T _PL = 69 ° C (yield 98%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = -1.2 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

Example 12. Separation of racemic 6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-4 + Ib-4) into enantiomers by enzymatic acylation using PS lipase (Amano).

(5S, 6S) -6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol (IA-4).

Synthesize and purify similarly to compound (Ia-1) from 1 g (3.28 mmol) of racemic 6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol (Ia-4 + Ib-4). Received 0.39 g of a colorless crystalline substance T _PL = 68 ° C (yield 39%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = 1.0 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

(5R, 6R) -6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol acetate (III-4).

Obtained 0.45 g of a transparent oily substance (yield 40%) with an angle of rotation of the beam of polarized light α = -0.9 °; ee> 99%.

¹ H NMR Spectrum (400 MHz, CDCl ₃ ) δ, ppm: 2.09 s (3H, CH ₃ ); 3.32 td (1H, H ⁶ , ³ J (H ⁶ H ⁵ ) = 5.4 Hz, ³ J (H ⁶ H ⁷ _A ) = 6.1 Hz, ³ J (H ⁶ H ⁷ _B ) = 2.4 Hz); 3.82 dd (1H, H ⁷ _A , ² J (H ⁷ _A H ⁷ _B ) = - 12.3 Hz); 3.86 dd (1H, H ⁴ _A , ² J (H ⁴ _A H ⁴ _B ) = - 12.5 Hz, ³ J (H ⁴ _A H ⁵ ) = 5.3 Hz); 4.03 dd (1H, H ⁴ _B ,, ³ J (H ⁴ _B H ⁵ ) = 2.3 Hz); 4.13 dd (1H, H ⁷ _B ); 4.73 d (1H, H ² _A , ² J (H ² _A H ² _B ) = - 4.6 Hz); 4.80 d (1H, H ² _B ); 4.91 td (1H, H ⁵ ); 7.32-7.44 m (4H, 4-BrC ₆ H ₄ ). NMR ¹³ C {H} (100 MHz, CDCl ₃ ) δ, ppm: 21.13 (CH ₃ ), 52.26 (C ⁶ ), 65.23 (C ^{7 (4)} ), 65.45 (C ^{4 (7)} ), 73.66 (C ⁵ ), 94.46 (C ² ), 121.75 (C _Ar ), 132.27 (C _Ar ), 132.68 (C _Ar ), 133.48 (C _Ar ), 170.11 (CO). Mass spectrum MALDI: 370 [M + Na] ⁺ , 386 [M + K] ⁺ .

Example 13. Synthesis of (5R, 6R) - 6 - ((4-bromophenyl) thio) -1,3-dioxepan-5-ol (Ib-4).

Synthesize and purify similarly to compound (Ib-1) from 0.5 g (1.44 mmol) of optically active acetate (III-4). Received 0.37 g of a colorless crystalline substance T _PL = 68 ° C (yield 91%) with an angle of rotation of the beam of polarized light [α] ²⁴ _D = -1.0 °; ee> 99%.

¹ H NMR and ¹³ C NMR spectra of the obtained compound fully correspond to the racemic sample.

Method for the study of antifungal activity

Due to the variety of properties of microscopic fungi and their differences in adaptive capabilities, including the existence of fungi in vivo, it is necessary to use test strains of fungi belonging to different classes on representatives of other kingdoms of living creatures, including humans, and take into account their potential ability to be pathogens. For a more complete assessment of the studied substances from the point of view of their antimycotic properties, predicting the consequences of their use and identifying areas of practical use, we have proposed the most significant groups of fungi with different physiological and biochemical activity related to the departments: Zygomycota - lower fungi (Mucor pusilos), Ascomycota - higher mushrooms. In addition, since the vegetative forms of Ascomycota fungi are most often detected in laboratory conditions, a working classification is used for them, according to which this group of fungi is called deuteromycetes, which in turn are also divided into blastomycetes (Candida albicans yeast) and hyphomycetes (with septated GIFs - Aspergillus fumigatus, Epidermophyton floccosum). Data on antimycotic activity in relation to the presented fungi can be extrapolated to other representatives of the corresponding taxa due to the proximity of their structure and vital processes.

Based on the above facts, the study of antifungal activity was carried out on strains of the fungi Candida albicans, Aspergillus fumigatus, Epidermophyton floccosum, Mucor pusilos, which were isolated from people with mycoses. The cultivation of fungi was carried out on a liquid nutrient medium Saburo.

Evaluation of antifungal activity was carried out in vitro, by the method of serial dilutions based on the determination of the minimum inhibitory concentration (MIC).

To study the fungicidal activity, physiologically active two-day cultures of yeast-like fungi and six-day cultures of mycelial fungi were taken. Then a suspension of mushrooms was prepared at a concentration of 1-2 million / ml. A suspension of vegetative cells or a spore suspension was introduced into test tubes with media containing a set of concentrations of the studied compounds: from 160 mg / ml to 0.1 mg / ml. Incubated at a temperature of 28-30 ° C for 9 days. As a control, we used media in the absence of drugs. MIC (minimum inhibitory concentration) was evaluated using a photoelectric calorimeter at a wavelength of 530 nm. Values were compared with absorbance values in control tubes.

A yeast strain Saccharomyces cerevisiae No. Y-830 was also used. The yeast S. cerevisiae cells were grown on YPD medium to a stationary growth phase during the day at 37 ° C and 250 rpm. The determination of the minimum inhibitory concentration of the substance was carried out according to the standard method of micro-dilutions of the culture [Wiegand I., Hilpert K., Hancock R.E.W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances / Nature Protocols. - 2008. - V.3. - pp. 163-175]. The culture was diluted to 106 cells / ml. The stock concentration of the substance was 90 mg / ml, which was then diluted 10 times and sequentially diluted twice to 20,480 times from the initial concentration. Dilutions of the substance were added in an amount of 100 μl to 100 μl of YPD culture medium in a 96-well plate. Nutrient medium and untreated cells were used as controls. Incubation occurred at 37 ° C for 24 hours and 250 rpm. The obtained data of the optical density of the culture at a wavelength of 600 nm were compared with the controls and expressed as the ordinate axis on the polynomial graph, the abscissa axis was expressed in decimal logarithm of the concentration of the substance. According to the schedule, the minimum inhibitory concentration was found.

As reference (compounds for comparing antifungal activity) compounds, well-known modern antimycotics - fluconazole and terbinafine were chosen.

Table 1. Minimum inhibitory concentrations (mg / ml) of compounds of the general formula Ia and Ib with respect to the strains of the studied fungi Sample Saccharomyces cercvisiae Candida albicans Aspergillus fumigatus Epidermophyton floccosum Mucor pusilos Ia-1 0.16 1,5 1,5 1,5 1,5 Ib-1 1.13 2,5 2,5 2,5 2,5 Ia-2 0.1 0.5 0.5 0.9 0.9 Ib-2 0.93 0.7 one 1,5 1,5 Ia-3 0.04 0.15 0.15 0.15 0.3 Ib-3 0.26 0.6 0.7 0.7 0.9 Ia-4 0.15 one 1,5 1,5 0.3 Ib-4 0.96 1,5 2,5 2,5 0.9 Fluconazole 0.24 1,5 2,5 2,5 2 Terbinafine 0.26 0.1 0.12 0.15 0.5

From the data presented in table 1 it follows that the claimed substances have an antimycotic effect, while compounds Ia-3 and Ia-4 inhibit the growth of fungi at the level of terbinafia - one of the most powerful systemic antimycotics identified by the applicant at the date of submission of application materials, indicate its high cytotoxicity.

Table 2 shows the hypoxicity of the most active compounds Ia-2 and Ia-3 on normal dog kidney cells (MDCK) in comparison with the known antimycotic drugs fluconazole and terbinafine. The experiment was carried out according to the following procedure.

Maidin-Derby dog kidney cells (MDCK line) were cultured in polystyrene vials in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 μg / ml streptomycin and 100 units / ml penicillin in a CO ₂ incubator at 37 ° C, 5% CO ₂ . Cells were grown to achieve a monolayer and dissociated using a trypsin-EDTA solution. The density of cells in suspension was evaluated on a hemocytometer. Cytotoxicity was determined using a proliferative MTT assay. Cells were scattered in a 96-well plate with a density of 1000 cells per well and recultured for 12 hours under standard conditions. An aliquot of the solutions of the studied substances was introduced into the culture medium in phosphate-buffered saline (PBS) at different concentrations and the cells were cultured for 3 days. After incubation, 20 μl of MTT reagent with a concentration of 5 mg / ml in DMEM was added to the wells, the mixture was incubated for 2 hours, then the culture medium was replaced with DMSO and then the absorption in the wells was measured at 550 with an Infinite 200 PRO plate analyzer (Tecan). Cell viability, proportional to the absorption of the solution, was expressed as a percentage relative to the control (FSB), taken as 100%. Cytotoxicity was assessed by the concentration of the studied substances, inhibiting cell growth by 50% (inhibitory concentration, IC50).

Table 2. The results of the study of the cytotoxicity of compounds Ia-2 and Ia-3 on normal MDCK dog kidney cells (IC50, μg / ml) Compound IC50, mcg / ml Ia-2 32.2 ± 5.6 Ia-3 39.7 ± 4.3 Fluconazole 57.2 ± 6.9 Terbinafine 9.8 ± 1.0

From the data presented in table 2 it follows that the test substances are one and a half to two times more toxic than fluconazole and three to four times less toxic of terbinafine. Thus, the synthesized 6- (arylthio) -1,3-dioxepan-5-ols are of interest in the development of new low-toxic antifungal agents, since The claimed technical solution has a more acceptable therapeutic breadth of action.

The claimed technical solution meets the criterion of "novelty" presented to the invention, because the claimed combination of actions leading to the implementation of the goal is not identified from the investigated prior art.

The claimed technical solution meets the criterion of "inventive step" for inventions, because allows you to solve the problem of creating effective and safe antimycotics that is not resolved before the filing date of these application materials.

The claimed technical solution meets the criterion of "industrial applicability" to the invention, because The performed experiments showed the possibility of industrial application for the production of antimycotic agents in the industry, based on the claimed technical solution.

Claims (1)

Antifungal compounds based on derivatives of 3,5,8-trioxabicyclo [5.1.0] octane obtained by trans-opening of its epoxy cycle, namely 6- (arylthio) -1,3-dioxepan-5-ol in racemic and enantiopure general form formulas Ia and Ib

where
when R ₁ = F, R ₂ = H, R ₃ = H
when R ₁ = Br, R ₂ = H, R ₃ = H
when R ₁ = H, R ₂ = Br, R ₃ = H
when R ₁ = H, R ₂ = H, R ₃ = Br