RU2623087C1 - Method for 2,3,16,17,18,19-hexahydrooligomycin a and its application for inhibiting growth of candida yeasts - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to a new derivative of macrolide antibiotic oligomycin A - 2,3,16,17,18,19-hexahydrooligomycin A, its structure and its preparation method, as well as its application for treatment of diseases caused by Candida yeast fungi, since this compound has selective activity against a number of Candida spp. strains, and also a low (in comparison with the original oligomycin A) cytotoxicity and corresponds to formula

.

EFFECT: increased effeciency of compound application.

5 cl, 2 tbl, 2 ex

Description

Technical field

The invention relates to the pharmaceutical industry and relates to a method for producing a new semi-synthetic derivative of macrolide antibiotic oligomycin A and its medical use for inhibiting the growth of yeast-like fungi of the genus Candida.

State of the art

Oligomycins belong to the class of macrolide antibiotics, the structure of which is a 26-membered α, β-unsaturated lactone, articulated with a bicyclic spiroketal structure. The most important representatives of this group are oligomycin A (1), oligomycin B (2), oligomycin C (3) [Carter G.T. J. Org. Chem., 1986, 51, 4264].

It is well known that the mechanism of action of oligomycins is associated with the inhibition of the work of F ₀ F ₁ -ATP synthase in pro- and eukaryotic cells. In submicromolar concentrations, oligomycin binds to the F ₀ subunit, blocking the work of OSCP (the oligomycin sensitivity conferring protein), which disrupts ATP synthesis [Jonckeere AL et al. J. Inherit. Metab. Dis., 2012, 35, 211]. Due to the suppression of oxidative phosphorylation and disruption of energy metabolism, oligomycins exhibit antifuntal, antimycotic and antiproliferative activity, however, gram-positive and gram-negative bacteria are resistant to their action [Bibikova MV and other Antibiotics and chemotherapy, 2003, 48, 33]. The main disadvantage of antibiotics of this series, limiting their use in clinical practice, is the high toxicity to mammalian cells.

Chemical modification of natural antibiotics is one of the main ways to obtain semi-synthetic derivatives with improved pharmacological properties. However, the preparation of semisynthetic oligomycins is complicated by the poor knowledge of the chemical properties of this class of antibiotics, the complexity of their structure and the high lability of oligomycin in an alkaline environment [Lysenkova L.N. et al. J. Antibiot., 2014, 67, 153]. Previously described methods for the chemical transformation of oligomycin A both along the side chain (33 position) [Lysenkova L.N. et al., Bioorg. Med. Chem., 2013, 21, 2918; Lysenkova L.N. et al. Macroheterocycles, 2015, 8, 424], and along the macrolactone ring [Lysenkova L.N. et al. J. Antibiot., 2010, 63, 17; Lysenkova L.N. et al. J. Antibiot., 2012, 65, 223]. It was established that a change in the structure of macrolactone significantly reduces the activity of the antibiotic.

In the literature there is evidence of attempts to restore oligomycin. Thus, it was shown that under the action of sodium borohydride, oligomycin A keto groups at positions 7 and 11 are reduced to the corresponding hydroxyl groups without changing the macrolactone cycle (formula 4) [Rfmires F. et al. Eur. J. Biochem., 1982, 121, 275]. Reduced compound 4, like the original antibiotic 1, inhibits ADP-dependent respiration and proton transport, but does not affect other respiration-related activities in intact rat liver mitochondria. Recently, microbiological reduction of oligomycin A to 2,3-dihydro-oligomycin A has been described (5) and it has been shown that restoration of the double bond at positions 2, 3 of the macrocycle leads to a slight increase in activity against S. cerevisiae yeast, therefore 2,3-dihydro-oligomycin A is patented as an antifungal agent [Patent KR 2012016713, 2012].

Disclosure of invention

The authors of the present invention unexpectedly found that by catalytic hydrogenation of oligomycin A (1), a derivative of 2,3,16,17,18,19-hexahydro-oligomycin (6) is formed, which has selective activity against yeast-like fungi of the genus Candida, and is less toxic to mammalian cells than the original antibiotic.

The reaction described in the application description is carried out without heating, preferably at 20-25 ° C, at a pressure of not more than 14 psi, a preferred pressure range of 3-6 psi.

The production method claimed in the invention 2,3,16,17,18,19-hexahydro-oligomycin (6) is based on the hydrogenation of oligomycin A (1) on a heterogeneous metal catalyst. In a preferred embodiment, the hydrogenation is carried out on a Pd-containing catalyst, more preferably on palladium supported on activated carbon (Pd / C). Hydrogenation is carried out in a suitable inert organic solvent, preferably in a protic solvent, for example methanol or ethanol or a mixture of solvents (Scheme 1).

Scheme 1

Biological studies of the semi-synthetic antibiotic 6 claimed in the invention revealed that hydrogenation of double bonds of the macrocycle unexpectedly leads to an increase in the selectivity of the antibiotic in the inhibition of a number of Candida spp strains. Therefore, the present invention relates to the use of compound 6 for the inhibition of yeast-like fungi Candida spp, which are the causative agents of a number of human fungal diseases

Starting antibiotic 1, catalysts and solvents are commercial chemical compounds supplied by firms such as Aldrich Chemical Co.

Unless otherwise specified, the terms used in the description of the application and in the claims have the meanings indicated below.

“Heterogeneous metal catalyst” means a catalyst forming an independent solid phase, separated by a phase boundary from the liquid phase of the reacting substances. The catalyst is a sponge metal, for example palladium, platinum, nickel, or a metal, for example palladium, platinum, nickel, supported on an inert carrier, for example, activated carbon, barium sulfate, alumina.

"Inert organic solvent" means a solvent inert under the conditions described in the text of the reaction, for example benzene, toluene, tetrahydrofuran, methanol, ethanol, propanol, isopropanol, tert-butanol.

"Protonic solvent" means a solvent capable of dissociating to form a hydrogen ion H ⁺ , for example methanol, ethanol, propanol, acetic acid, water.

Examples describing the preparation and identification of 2,3,16,17,18,19-hexahydro-oligomycin (6), which is the subject of the present invention, as well as its physicochemical properties, antifungal activity and cytotoxicity for mammalian cells, are given below.

Example 1

2,3,16,17,18,19-hexahydro-oligomycin A (6)

To a solution of 60.0 mg (0.075 mmol) of oligomycin A (1) in 4.0 ml of methanol, 48.0 mg (0.0225 mmol) of 5% Pd / C, previously suspended in a mixture of 0.5 ml of MeOH and 0.05 ml of H ₂ O _dist. and hydrogenated under a pressure of 0.4 atm at room temperature for 1 h. The reaction mixture was filtered, the catalyst was washed with methanol and concentrated in vacuo. The residue was purified by column chromatography (hexane: acetone 10: 3 → 10: 4, chloroform: methanol 10: 0.2 → 10: 0.3). Yield 42.0 mg (69%). Amorphous white powder. T. pl. 83 ° C.

Found: m / z ESI 797.5593 (100%) [M + H] ⁺ . C ₄₅ H ₈₀ O ₁₁ .

Calculated 796.5701. IR (film) ν _max cm ^-1 : 3416 s, 2971 sr, 2928 sr, 2857 sr, 1731 s, 1698 s, 1458 s, 1382 sr, 1340 s, 1264 s, 1222 s, 1191 s, 1169 s, 1135 sr, 1089 sr, 1048 sr, 982 s, 882 sr, 844 sl, 802 sl. UV (methanol) λmax (logε) nm: 207 (3.7), 268 (2.8). [α] ²⁰ _D (c 0.1, methanol) -80 °. HPLC: Rt = 23.2 (A-H ₂ O, B-MeCN; conc. B - 80% → 95% 10 min, 95% - 30 min), purity 95.2%. ¹ H NMR spectrum (600 MHz, CD ₃ OD) δ, ppm, J (Hz): 4.99 (1H, dq, J ¹ = 11.8, J ² = 4.5); 4.16 (1H, dd, J ¹ = 7.4, J ² = 4.0); 4.03 (1H, doublet of doublets, J ¹ = 10.1, J ² = 2.8); 3.93 (1H, ddq, J ¹ = 9.3, J ² = 3.7 J ³ = 6.2); 3.90 (1H, d, J = 2.0); 3.85 (1H, ddd, J ¹ = 5.9, J ² = 0.9 J ³ = 2.0); 3.65 (1H, tq, J ¹ = 6.3, J ² = 4.7); 3.51 (1H, doublet of doublets, J ¹ = 7.1, J ² = 6.9); 3.02 (1H, doublet of doublets, J ¹ = 6.9, J ² = 6.7); 2.71 (1H, qq, J ¹ = 4.0, J ² = 7.1); 2.46 (1H, ddd, J ¹ = 15.7, J ² = 6.6, J ³ = 0.8); 2.37 (1H, ddd, J ¹ = 15.7, J ² = 6.6, J ³ = 0.8); 2.16 (1H, m); 2.11 (1H, doublet of doublets, J ¹ = 2.0, J ² = 0.9, J ³ = 7.2); 1.93 (1H, m); 1.78-1.77 (3H, m); 1.56-1.23 (24H, m); 1.21 (3H, s); 1.19 (3H, d, J = 6.2); 1.14 (3H, d, J = 7.1); 1.14 (3H, d, J = 6.9); 1.13 (3H, d, J = 6.9); 0.97 (3H, d, J = 6.9); 0.94 (3H, d, J = 6.6); 0.94 (3H, d, J = 6.9); 0.93 (3H, d, J = 6.9); 0.91 (3H, d, J = 7.2); 0.89 (3H, t, J = 7.1). ¹³ C NMR spectrum (150 MHz, CD ₃ OD) δ, ppm: 221.5 (C = O); 218.4 (C = O); 175.1 (O = CO); 100.6 (CCA); 84.5 (C-O); 77.7 (CH); 75.9 (CH); 75.8 (CH); 72.9 (CH); 70.6 (CH); 68.8 (CH); 65.4 (CH); 49.9 (CH); 48.3 (CH); 44.1 (CH); 43.7 (CH ₂ ); 40.0 (CH); 39.3 (CH); 36.7 (CH); 36.5 (CH); 36.2 (CH ₂ ); 34.8 (CH); 33.1 (CH ₂ ); 32.6 (CH ₂ ); 31.8 (CH); 30.5 (CH ₂ ); 30.1 (CH ₂ ); 29.2 (CH ₂ ); 28.9 (CH ₂ ); 27.8 (CH ₂ ); 27.5 (CH ₂ ); 27.6 (CH ₂ ); 27.1 (CH ₂ ); 27.0 (CH ₂ ); 25.1 (CH ₃ ); 22.4 (CH ₃ ); 14.8 (CH ₃ ); 14.4 (CH ₃ ); 14.2 (CH ₃ ); 13.3 (CH ₃ ); 12.3 (CH ₃ ); 12.1 (CH ₃ ); 11.9 (CH ₃ ); 11.0 (CH ₃ ); 6.4 (CH ₃ ).

Examples of biological activity

Example 2

Antifungal activity against Candida spp. and filamentous mushrooms.

A comparative study of the antifungal activity of oligomycin 1 and its perhydroderivative 6 was carried out on control strains of yeast cultures Candida albicans ATCC 24433, Candida parapsilosis ATCC 22019, filamentous fungi Aspergillus niger 137a, dermatophytes Microsporum canis B-200, and Trichophyton spp. one). When choosing strains, the sensitivity of Candida spp was taken into account. to antifungal drugs. So, C krusei has natural resistance to fluconazole, used for systemic and local treatment of fungal infections. Most clinical isolates of C. albicans are susceptible to fluconazole, C tropicalis is intermediate, and 15% of C. glabrata isolates are resistant to fluconazole.

Assessment of the activity of the test samples was carried out in accordance with the standard [GOST R ISO 16256-2015] and the recommendations of the [Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standart, 3rd ed., M27-A3; Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standart, 2rd ed., M38-A2]. To determine the value of the minimum inhibitory concentration (MIC), a serial dilution micromethod was used in RPMI 1640 medium with the addition of glucose to a concentration of 0.2% (with glutamine and not containing carbonate, manufactured by PanEco).

To obtain basic solutions of the tested antibiotics 1 and 6 with a concentration of 10,000 μg / ml, weighed samples were dissolved in dimethyl sulfoxide (DMSO). To obtain working solutions with a concentration of 64 μg / ml, basic solutions in the amount of 0.064 ml were transferred into 9.93 ml of RPMI 1640 nutrient broth with glucose 0.2%, the final concentration of DMSO did not exceed 0.3%.

Candida spp. Inoculum suspension was prepared in RPMI medium with 0.2% glucose according to the 0.5 McFarland turbidity standard (~ 5 × 10 ⁶ CFU / ml for yeast cultures), evaluated densitometrically (Densimat, Biomerieux), diluted 1: 1000 to ~ 5 × 10 ³ CFU / ml in RPMI medium with 0.2% glucose. For each test strain of filamentous fungi, the inoculum was obtained by grinding a portion of the colony in saline in a test tube with glass beads. Conidia and spores were pipetted through a gauze filter. Conidia and spores were counted using a Goryaev camera. Each inoculum was adjusted to a working titer in RPMI medium. The result was a suspension containing 1.5-3.2 × 10 ⁴ CFU / ml. To control the titer of viable colony forming units (CFU), 10 μl of the inoculum was transferred to Saburo agar medium.

We used 96 well plates for immunological studies (Medpolymer, St. Petersburg). 100 μl of a suspension of yeast cultures in a nutrient medium and solutions of the test compounds in the concentration range of 32-0.25 μg / ml were added to the plate wells. Each sample was analyzed in triplicate.

To maintain the accuracy of the procedure for determining the values of the minimum inhibitory concentration (MIC), fluconazole (Sinergin Active Ingredients (PI) LTD) was used as the internal standard, the activity range of which against the Candida parapsilosis ATCC 22019 reference strain is 1-4 μg / ml [GOST R ISO 16256-2015]. To control growth, all test cultures were seeded in culture medium without samples.

Sensitivity was assessed visually, after incubation at 35 ° C for 24 and 48 hours for Candida spp and 48-96 hours for A. niger, M. canis B, T. rubrum, comparing with the growth density in the control without the drug. MIC was taken as the lowest concentration of the sample, at which the visible growth was suppressed by at least 80% in comparison with the growth control.

As can be seen from the above data, the activity of perhydro derivative 6, which is the subject of the present invention, with respect to Candida spp. It is worth noting that perhydro-oligomycin 6, like the original antibiotic 1, is active against the resistant strain of C. krusei resistant to the action of fluconazole.

Example 3

Antiproliferative activity against human cell line

A comparative study of cytotoxicity for mammalian cells of oligomycin 1 and its derivative 6 was carried out on human leukemia cells K-562 using the MTT test [Shchekotikhin A.E. et al. Eur. J. Med. Chem., 2011, 46, 213].

Comparison of inhibitory concentrations (IC ₅₀ ) of oligomycin A (1) and its derivative (6) shows that the reduction of double bonds leads to a 8.5-fold decrease in cytotoxicity (Table 2).

As can be seen from the data presented, the new semi-synthetic antibiotic 6, which is the subject of this invention, has a specific activity against strains of yeast of the genus Candida, which in some cases is superior to the comparison drug fluconazole. The modification of the macrocycle claimed in the present invention not only increases the selectivity of action on Candida spp., But also causes a decrease in the high cytotoxicity for mammalian cells characteristic of oligomycin A.

Claims (6)

1. Derived oligomycin A - 2,3,16,17,18,19-hexahydro-oligomycin A, corresponding to the formula

2. The method of obtaining 2,3,16,17,18,19-hexahydro-oligomycin A according to claim 1, which consists in the catalytic hydrogenation of oligomycin A in an inert solvent on a heterogeneous catalyst containing a metal selected from platinum, palladium or nickel. 3. The method according to claim 2, characterized in that palladium supported on activated carbon is used as a hydrogenation catalyst. 4. The method according to p. 2, characterized in that methanol is used as a solvent. 5. The use of 2,3,16,17,18,19-hexahydro-oligomycin A according to claim 1 for the treatment of diseases caused by yeast of the genus Candida.