RU2790377C1 - Application of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4,7-dione as an antifungal agent against cryptococcus neoformans - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the use of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4,7-dione (1) as an antifungal agent against Cryptococcus neoformans.

EFFECT: pronounced antifungal activity against Cryptococcus neoformans in the absence of cytotoxic and hemolytic action, provided by an agent based on 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6- diazaspiro[4.4]nona-2,8-diene-4,7-dione.

1 cl, 2 tbl, 3 ex

Description

The invention relates to the field of organic chemistry and medicine, namely to an individual derivative of 1-thia-3,6-diazaspiro[4.4]nonane 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl -1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4,7-dione, which has antifungal activity against Cryptococcus neoformans, which can be used in pharmacology, medicine and veterinary medicine.

The micromycetes C. neoformans are important human pathogens encountered in clinical practice. They cause severe meningoencephalitis and disseminated infections mainly in patients with AIDS and other immunodeficiencies [Kuzikova I.L., Medvedeva N.G. // Human ecology. 2021. №3. pp. 4-14. DOI 10.33396 /1728-0869-2021-3-4-14].

Known structural analogue (2) of the claimed compound (1) - 4-(4,5-diphenyl-1-(thiazol-2-yl)-1H-imidazol-2-yl)-2-methoxyphenol, exhibiting an antifungal effect against C neoformans (MNC ₈₀ =5 µg/ml) [A. P. G. Nikalje, S. V. Tiwari, A. P. Sarkate, K. S. Karnik // Med. Chem. Res., 2018, Vol.27, PP. 592-606. DOI: 10.1007/s00044-017-2085-5]. The formula of the structural analog (2) is shown in the diagram:

The objective of the present invention is to develop a new antifungal agent against C. neoformans micromycetes that does not have cytotoxic and hemolytic activities.

This task is achieved by studying 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4, 7-dione for the presence of antifungal activity against fungi C. neoformans, as well as for the presence of cytotoxic and hemolytic activities.

The claimed compound (1) is synthesized by the interaction of 3-benzoyl-8-chloropyrrolo[2,1-c][1,4]benzoxazine-1,2,4-trione (2) with thiobenzamide in anhydrous toluene, followed by isolation of the target product by known methods [A.I. Kobelev, E.E. Stepanova, M.V. Dmitriev, E.S. Denislamova, A.N. Maslivets II Russ. J. Org. Chem., 2018, Vol.54, No. 5, PP. 766-770, DOI 10.1134/S 1070428018050159; A.H. Maslivets, A.I. Kobelev, E.E. Stepanova, S.Yu. Balandina // RF Patent 2 707 195, 11/25/2019] according to the scheme:

EFFECT: discovery of a new compound having antifungal activity against C. neoformans and not having cytotoxic and hemolytic activities.

The invention is illustrated by the following examples.

Example 1 Preparation of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4, 7-dione (1).

A suspension of 1.0 mmol of 3-benzoyl-8-chloropyrrolo[2,1-c][1,4]benzoxazine-1,2,4-trione (2) and 1.0 mmol of thiobenzamide in 5 ml of anhydrous toluene was refluxed for 10 min, cooled to room temperature temperature, the precipitate of compound (1) was filtered off. Yield 91%, m.p. 201-202°С (dec., toluene). Compound (1) C ₂₅ H ₁₅ ClN ₂ O ₅ S.

Found, %: С 61.21; H 3.17; N 5.61.

Calculated, %: С 61.17; H 3.08; N 5.71.

Compound (1) is a yellow high-melting crystalline substance, melting with decomposition, easily soluble in DMSO and DMF, soluble in water-alcohol mixtures, acetone, chloroform, 1,2-dichloroethane, 1,4-dioxane, ethyl acetate, sparingly soluble in aromatic hydrocarbons, carbon tetrachloride, insoluble in alkanes and water, giving a positive test (cherry color) for the presence of an enol hydroxyl group with an alcoholic solution of iron (III) chloride. Stable under normal storage conditions.

IR spectrum (PE Spectrum Two), v, cm ^-1 : 3236 (OH), 1744, 1709 (C ⁴ =O, C ⁷ =O), 1669 (COPh). ^1H NMR spectrum (Bruker Avance III HD 400, 400 MHz, DMSO-d ₆ ) δ, ppm: 6.95 m (2Н, _Нarom ), 7.29 m (1Н, _Нarom ), 7.50-7.65 m (5Н , H _arom ), 7.76–7.82 m (3H, H _arom ), 8.03 m (2H, H _arom ), 10.23 br.s (OH _phenol ). ^13C NMR spectrum (Bruker Avance III HD 400, 100 MHz, DMSO-d ₆ ) δ, ppm: 82.7 (C ⁵ ), 116.8 (C ⁹ ), 118.6, 121.4, 121.9, 128.2 (2C), 128.6 (2С), 128.7, 128.9 (2С), 129.6 (2С), 130.8, 131.2, 132.9, 136.1, 137.1, 153.9 ( _Сarom ), 154.1 ( ^С7 ), 165.3 ( ^С2 ), 187.3 ( ^С4 ) , 187.7 (COPh), 194.3 ( ^С8 ).

Example 2 Primary study of the antimicrobial and antifungal activities of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8 -diene-4,7-dione (1) at a concentration of 32 µg/ml.

Antimicrobial screening of compound (1) was carried out under the CO-ADD program (The Community for Antimicrobial Drug Discovery, Institute of Molecular Biology of the University of Queensland, Australia), funded by the Wellcome Trust (UK) and the University of Queensland (Australia), on five bacterial strains: Escherichia coli ATCC 25922 (Ee), Klebsiella pneumoniae ATCC 700603 (Kp), Acinetobacter baumannii ATCC 19606 (Ab), Pseudomonas aeruginosa ATCC 27853 (Pa), and Staphylococcus aureus ATCC 43300 (Sa). Antifungal activity was determined in the same place on two fungal strains: Candida albicans ATCC 90028 (Ca) and Cryptococcus neoformans var. grubii ATCC 208821 (Cn).

Primary screening for activity was performed by cell proliferation inhibition tests using samples at a single concentration (32 μg/mL) in duplicate. An aliquot of each sample in DMSO was placed in a 384-well plate and diluted with the appropriate culture of microorganisms. Microbial growth inhibition was determined spectrophotometrically at 600 nm on a Tecan M1000 Pro monochrome microplate reader. Percent growth inhibition was calculated for each well using a negative control (medium only) and a positive control (bacteria without inhibitors) on the same plate. If growth inhibition ≥80% was observed one or both times, the compound was considered active (Table 1).

Primary screening revealed fungicidal activity in 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8 -diene-4,7-dione (1) in relation to the fungal culture of C. neoformans. For compound (1), the minimum inhibitory concentration was determined in relation to the above cultures, and cytotoxic and hemolytic activities were also studied.

Example 3 In-depth study of the antimicrobial, antifungal, cytotoxic and hemolytic activities of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]none -2,8-diene-4,7-dione (1) in the concentration range 0.250-32 µg/ml.

In-depth studies of compound (1) were also carried out under the CO-ADD program.

The minimum inhibitory concentration (MIC ₈₀ , µg/ml) was set in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI, USA), determining the lowest concentration at which complete inhibition of bacteria or fungi was found. Screening was carried out by the method of serial double dilutions. Samples were prepared in DMSO at a test concentration of 32 µg/mL. All microorganisms were cultured on Mueller-Hinton agar at 37°C overnight. A sample of ^each culture was then diluted 40 times and incubated at 37°C for 1.5-3 hours. 50 µl). All plates were covered and incubated at 37°C for 18 hours without shaking. Microbial growth inhibition was determined by measuring absorbance at 600 nm using a Tecan M1000 Pro monochrome microplate reader. Percent growth inhibition was calculated for each well using a negative control (medium only) and a positive control (bacteria without inhibitors) on the same plate. Inhibition values were determined using modified Z scores calculated using the median and median absolute deviation (MAD) of the samples (no control) on the same plate. Samples with an inhibition value greater than 80% and a Z-score greater than 2.5 for both replicates were classified as active substances. The tests were carried out in duplicate.

The cytotoxic effect was determined on the human embryonic kidney cell line HEK293 (Hk) by determining the concentration causing 50% cell death (CC ₅₀ ). Growth inhibition of HEK293 cells was determined by measuring fluorescence after adding 5 μl of 25 μg/ml resazurin (final concentration 2.3 μg/ml) and after incubation for another 3 h at 37°C in 5% CO ₂ . Fluorescence intensity was measured using a Tecan M1000 Pro monochrome microplate reader using automatic gain calculation. The compound was considered toxic at CC ₅₀ ≤ 32 μg/mL or CC ₅₀ ≤ 10 μM.

Hemolytic activity (HC ₁₀ - concentration at 10% hemolysis) was determined by measuring the absorbance at 405 mm supernatant - the supernatant formed after incubation for 1 h at 37°C plates containing samples of compounds with added washed human blood cells (Hm ), followed by centrifugation at 1000 rpm for 10 min. Absorbance was measured using a Tecan M1000 Pro monochrome microplate reader. HC ₁₀ > 32 µg/ml (maximum concentration tested) indicates samples without hemolytic activity. Samples with hemolytic activity were characterized at HC ₁₀ ≤ 32 µg/ml.

Colistin and Vancomycin were used as positive bacterial inhibition standards for Gram-negative and Gram-positive bacteria, respectively. "Fluconazole" was used as the standard fungal inhibitor for C. albicans and C. neoformans. "Tamoxifen" was used as a positive standard for cytotoxicity. "Melittin" was used as a positive hemolytic standard.

A methodology for testing antimicrobial, fungicidal, cytotoxic, and hemolytic activity in vitro of compounds is also available at http://www.coadd.org.

Sample of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4,7-dione (1) at a concentration of ≤ 0.250 μg / ml showed fungicidal activity, inhibiting the growth and reproduction of > 80% of the fungal culture of C. neoformans, which indicates a stronger antifungal effect of compound (1) than that of the reference drug - "Fluconazole" (Table 2 ). Compound (1) does not show hemolytic and cytotoxic activity even at the maximum tested concentration > 32 μg/ml (Table 2).

Thus, the compound (1) exhibits fungicidal activity against the pathogenic fungal culture of C. neoformans and does not have cytotoxic and hemolytic effects at the maximum tested concentration.

Claims (3)

Application of 9-benzoyl-8-hydroxy-6-(2-hydroxy-5-chlorophenyl)-2-phenyl-1-thia-3,6-diazaspiro[4.4]nona-2,8-diene-4,7-dione (1)

as an antifungal agent against Cryptococcus neoformans.