4-(adamantan-1-yl)-n-(1-(3-(n',n'-diethylamino)-propylamino)-3-(1-h-indol-3-yl)-1-oxopropan-2-yl)benzamide and its pharmaceutically acceptable salts with antifungal activity

Abstract

FIELD: medicine.SUBSTANCE: 4-(adamantan-1-yl)-N-(1-(3-(N',N'-diethylamino)propylamino)-3-(1-H-indol-3-yl)-1-oxopropan-2-yl)benzamide of formula (I) and its pharmaceutically acceptable salts, which have pronounced antifungal activity, as well as additional antibacterial activity against gram-positive and gram-negative bacteria.EFFECT: production of new compounds that can be used in medicine and veterinary science for antifungal therapy.1 cl, 1 tbl, 4 ex

Description

[0001] The invention relates to new chemical compounds, namely 4-(adamantan-1-yl)-N-(1-(3-(N',N'-diethylamino)propylamino)-3-(1-H-indol-3-yl)-1-oxopropan-2-yl)benzamide (I) and its pharmaceutically acceptable salts, which have pronounced antifungal activity, which can be used in medicine and veterinary medicine for antifungal therapy. Additionally, according to the invention, the claimed compounds have antibacterial activity against gram-positive and gram-negative bacteria.

[0002]

[0003] The most common fungal infection, mainly localized on the skin or mucous membranes, is candidiasis or "yeast" infection caused by Candida fungi, most often Candida albicans (Ruping MJ, Vehreschild JJ, Cornely OA Patients at high risk of invasive fungal infections / / Drugs. 2008. Vol. 68. N 14. P. 1941. doi: 10.2165 / 00003495-200868140-00002). The risk of developing invasive candidiasis is considered especially dangerous for a large number of patients, including those with hematological malignancies, neutropenia, age less than one month or over 65 years, recent abdominal surgery, etc.

[0004] In this regard, due to the limited spectrum of available antifungal drugs and the development of drug resistance to them, as well as due to the increasing number of cases of immunosuppressive state in patients, an urgent problem is the search for new alternative strategies of antifungal therapy, which include the use of natural and synthetic cationic antimicrobial peptides (AMPs) with a molecular weight of 1.5-10 kDa (Buda De Cesare G., Cristy SA, Garsin DA, Lorenz MC Antimicrobial peptides: a new frontier in antifungal therapy / / mBio. 2020. Vol. 11. N6. e02123-20. doi: 10.1128 / mBio.02123-20; Mani Chandrika KVS, Sharma S. Promising antifungal agents: a minireview / / Bioorg. Med. Chem. 2020. Vol. 28. Iss. 7. 115398. doi 10.1016 / j.bmc.2020.115398). Despite their high activity, antimicrobial peptides have a number of disadvantages, in particular, high cost, limited stability, low oral bioavailability, and immunogenicity.

[0005] Currently, ultrashort peptide mimetics and amino acid derivatives have high potential for use in clinical practice as antifungal agents, since they are highly active, have a significantly lower molecular weight compared to AMPs (<1000 Da), are easier to synthesize and can be more proteolytically stable (Su M., Su Y. Recent Advances in Amphipathic Peptidomimetics as Antimicrobial Agents to Combat Drug Resistance / / Molecules. 2024. Vol. 29. N 11. P. 2492. doi 10.3390 / molecules29112492; Svenson J., Molchanova N., Schroeder CI Antimicrobial Peptide Mimics for Clinical Use: Does Size Matter? / / Front. Immunol. 2022. Vol. 13. 915368. doi 10.3389 / fimmu.2022.915368).These compounds mimic the structure and functions of natural AMPs and also have a pronounced amphipathic structure, with hydrophobic properties provided, in particular, by the introduction of residues of amino acids such as tryptophan and additional highly lipophilic fragments, for example, adamantane, and hydrophilic properties by the introduction of one or more amine fragments.

[0006] N-isopropyl-, N-hexyl- and N-(2-phenylethyl)amides of L-arginyl-L-2,5,7-tri(tert-butyl)tryptophanyl-L-arginine are known to exhibit antifungal activity against the strain Candida albicans (ATCC 10231) with a minimum inhibitory concentration (MIC) of 8-32 μg / ml and a low risk of resistance development (Stensen W., Turner R., Brown M., Kondori N., Svendsen JS, Svenson J. Short cationic antimicrobial peptides display superior antifungal activities towards candidiasis and onychomycosis in comparison with terbinafine and amorolfine / / Mol. Pharmaceutics. 2016. Vol. 13. Iss. 10. P. 3595. doi 10.1021 / acs.molpharmaceut.6b00654). The disadvantage of these compounds is the multi-stage synthesis (RU 2804780 C1, 2023) and, as a consequence, the high labor intensity of production and the cost of the final product.

[0007] N-benzylamide of L-tryptophanyl-L-2-(adamantan-1-yl)histidine is known to exhibit antifungal activity against the strain Candida albicans (ATCC 10231) with a minimum inhibitory concentration (MIC) of 50 μg / ml (Sharma RK, Reddy RP, Tegge W., Jain R. Discovery of Trp-His and His-Arg Analogues as New Structural Classes of Short Antimicrobial Peptides / / J. Med. Chem. 2009. Vol. 52. Iss. 23. P. 7421. doi 10.1021 / jm900622d). The disadvantage of this compound is the multi-stage synthesis and relatively low yields of some individual stages of synthesis, as well as moderate antifungal activity.

[0008] The objective of the present invention was to obtain an original low molecular weight AMP mimetic containing a structural combination of hydrophobic tryptophan and adamantane fragments and a hydrophilic amine fragment, which can be used for antifungal therapy, and which can be relatively easily synthesized in a small number of steps with high yields.

[0009] The technical result is achieved using a new compound, 4-(adamantan-1-yl)-N-(1-(3-(N',N'-diethylamino)propylamino)-3-(1- / / -indol-3-yl)-1-oxopropan-2-yl)benzamide (I) and its pharmaceutically acceptable salts, which exhibit pronounced antifungal activity.

[0010] An additional aspect of the invention is the presence of antibacterial activity in the claimed compounds against gram-positive and gram-negative bacteria, which demonstrates the possibility of using these compounds as a broad-spectrum antimicrobial agent.

[0011] According to the invention, the claimed compound (I) is in the form of a racemate.

[0012] For the implementation of the invention, compound (I) may be in the form of pharmaceutically acceptable salts at the tertiary nitrogen atom with organic or inorganic acids. Examples of inorganic acids include hydrohalic acids and sulfuric acid, and organic acids include acetic, lactic, sorbic, malic, tartaric, and the like.

[0013] The claimed compound (I) and its salts are not described in the literature.

[0014] According to the invention, the method for obtaining compound (I) consists in using one of the methods of liquid-phase peptide synthesis, well known from the existing state of the art, from 2-(4-(adamantan-1-yl)phenylcarboxamido)-3-(1-H-indol-3-yl)propanoic acid, a method for obtaining which with a high yield was previously developed by us (RU 2417988 C2, 2011), and commercially available 3-(N, N-diethylamino)propylamine in a suitable solvent, followed by isolation of the product from the reaction mixture and purification. N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC) in combination with or without 1-hydroxybenzotriazole (HOBt), etc. can be used as a reagent for activating the carboxyl group (El-Faham A., Albericio F. Peptide Coupling Reagents, More than a Letter Soup / / Chem. Rev. 2011. Vol. 111. P. 6557. doi 10.1021 / crl00048w).

[0015] Pharmaceutically acceptable salts of compound (I) can be obtained by directly reacting it with an inorganic or organic acid in a solvent and then isolating it.

[0016] The following examples of the method of obtaining and evaluating biological activity illustrate, but do not limit, the proposed technical solution.

[0017] The structure and purity of the compounds obtained in the invention were confirmed by a number of physical and physicochemical analytical methods. IR spectra were recorded on a Spectrum RX1 FTIR spectrometer (PerkinElmer) using the attenuated total internal reflection (ATR) method in the frequency range of 400-4000 cm -1 NMR spectra 1H was recorded on a Varian VXR-400 apparatus (operating frequency 400 MHz) in DMSO-d6- solution. Melting point was determined on a Buchi Melting Point M-560 apparatus. Elemental analysis was performed. The reaction progress and homogeneity of the obtained compounds were monitored by thin-layer chromatography using Sorbfil PTSKh-AF-A-UV plates, eluent of n-hexane / toluene / acetone (8 / 8 / 5, v / v / v), development under UV irradiation. Flash chromatography was performed using silica gel (200-400 mesh, 40-75 µm) and an eluent similar in composition to the thin-layer chromatography method.

[0018] Example 1 (Synthesis of Compound (I) Using CDI)

[0019] 0.18 g (1.1 mmol) of CDI was added to a solution of 0.55 g (1 mmol) of (2S,R)-(4-(adamantan-1-yl)phenylcarboxamido)-3-(1-H-indol-3-yl)propanoic acid in 10 ml of dried tetrahydrofuran and the mixture was stirred for 1 h at 60-65°C. Then 0.14 g (0.17 ml, 1.1 mmol) of 3-(N,N-diethylamino)propylamine was added to the reaction mixture and the mixture was stirred for another 3 h. After this, the mixture was evaporated to 1 / 3 of the original volume and diluted with 15 ml of water, and the precipitated crystals were filtered, washed with water, and then dried. The resulting product was recrystallized from a mixture of EtOH and hexane (1 / 4, v / v), filtered, and air-dried. 0.47 g of compound (I) with the (S,R)-configuration was obtained as white crystals. Yield 85%, mp 106-110°C, R f 0.31. IR, ν, cm -1 : 3259 (NH), 2903 (CH2), 1620 (C=O), 1513 (NH), 1506 (Ar), 1205, 740. NMR 1H, 5, m.d.: 10.77 (m, 1H,), 8.36 (d, J8.0 Hz, 1H), 8.03 (t, J8.0 Hz, 1H), 7.77 (d, J 8.4 Hz, 2H), 7.65 (d, J 8.0 Hz, 1H), 7.65 (d, J 8.0 Hz, 1H, 40), J. Hz, 2H), 7.30 (d, J 8.0 Hz, 1H), 7.18 (m, 1H), 7.04 (t, J8.0 Hz, 1H), 6.98 (t, J 8.0 Hz 1H), 4.62-4.70 (m, 1H), 3.0-4.4.4, (m, 1H). 2.02-2.08 (m, 3H), 1.82-1.88 (m, 6H), 1.68-1.78 (m, 6H), 1.44-1.52 (m, 2H), 0.90 (t, J 7.0 Hz, 6H). Found, %: C 75.92; H 8.43; N 10.17. C 35 H 46 N4O2. Calculated, %: C 75.78; H 8.36; N 10.10.

[0020] Example 2 (synthesis of compound (I) using DCC and HOBt)

[0021] A solution of 0.55 g (1 mmol) of (2S,R)-(4-(adamantan-1-yl)phenylcarboxamido)-3-(1- / 7-indol-3-yl)propanoic acid in 10 ml of dried tetrahydrofuran was cooled using an ice-water bath to 0-5°C. Then 0.21 g (1 mmol) of DCC and 0.14 g (1 mmol) of HOBt were added and stirred for 48 h at room temperature. After completion of the reaction, the mixture was filtered to remove the precipitate of N',N'-dicyclohexylurea, then evaporated to 1 / 3 of the original volume and diluted with 15 ml of water. The precipitated crystals were filtered and washed with water. Purification was performed by flash chromatography. The obtained product was air-dried. 0.49 g of compound (I) with the (S-configuration) was obtained as white crystals. Yield 90%, mp 107-110°C, R f 0.31. IR, ν, cm -1 3260 (NH), 2910 (CH2), 1619 (C=O, 1513 (NH), 1506 (Ar, 1206, 741. NMR 1H, δ, MD: 10.76 (m, 1H,), 8.25 (d, J8.0 Hz, 1H), 7.91 (t, J 8.0 Hz, 1H), 7.77 (d, J 8.4 Hz, 2H), 7.65 (d, J 8.4 Hz, 2H), 7.65 (d, J 8.0 Hz, 1H, 7.48). Hz, 2H), 7.30 (d, J 8.0 Hz, 1H), 7.18 (m, 1H), 7.04 (t, J 8.0 Hz, 1H), 6.98 (t, J 8.0 Hz 1H), 4.60-4.70 (m, 1H), 3.0-4.4, (3.4H), (m, 1H). 2.00-2.08 (m, 3H), 1.81-1.88 (m, 6H), 1.68-1.77 (m, 6H), 1.44-1.51 (m, 2H), 0.90 (t, J7.0 Hz, 6H). Found, %: C 75.87; H 8.44; N10.15. C 35 H 46 N4O2. Calculated, %: C 75.78; H 8.36; N 10.10.

[0022] Example 3 (synthesis of hydrochloride compound (I))

[0023] To a suspension of 0.28 g (0.5 mmol) of compound (I), obtained in Example 1, in aqueous ethyl alcohol (50% vol.) were added 85 μl (0.1 mmol) of concentrated hydrochloric acid (36.5% by weight) and stirred for 0.5 h at room temperature. Then the resulting mixture was evaporated to 1 / 3 of the initial volume, cooled and the formed crystals were filtered off, which were dried in air. 0.28 g (95%) of (3-((2S,R)-2-((4-(adamantan-1-yl)phenyl)foramido)-3-(1H-indol-3-yl)propanamido)propyl)-diethylaminium chloride were obtained. NMR 1 H, δ, ppm: 10.77 (m, 1H), 10.35 (br s, 1H), 8.36 (d, J 8.0 Hz, 1H), 8.23 ​​(t, J 8.0 Hz, 1H), 7.77 (d, J 8.4 Hz, 2H), 7.67 (d, J 8.0 Hz, 1H), 7.41 (d, J 8.4 Hz, 2H), 7.32 (d, J 8.0 Hz, 1H), 7.18 (m, 1H), 7.05 (t, J 8.0 Hz, 1H), 6.99 (t, J 8.0 Hz 1H), 4.64-4.72 (m, 1H), 3.06-3.16 (m, 4H), 2.01-2.08 (m, 3H), 1.90-1.95 (m, 2H), 1.82-1.89 (m, 6H), 1.67-1.79 (m, 6H), 1.20 (t, J 7.0 Hz, 6H).

[0024] Antifungal activity, as well as additionally antibacterial activity, were determined in vitro by serial dilution using a turbidimetric method for monitoring the growth of microorganisms in accordance with known recommendations (CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing Filamentous Fungi, Approved Standard, 2nd ed., CLSI document M38-A2, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2008; Determination of the sensitivity of microorganisms to antibacterial drugs (MUK 4.2.1890-04) / / Klin. microbiol. antimicrob, chemother. 2004. Vol. 6. No. 4. P. 306; CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard, 9th ed., CLSI document M07-A9, Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012).Sensitive strains of fungi (Candida albicans ATCC 10231), gram-negative (Escherichia coli CI, Pseudomonas fluorescens Al), and gram-positive bacteria (Staphylococcus aureus ATCC-25923, Micrococcus luteus 2665) were used as test cultures. The broad-spectrum antimicrobial drug pefloxacin mesylate dihydrate was used as a positive control in all tests. An ALLSHENG FlexA-200 Microplate Reader spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd.) and Nest plastic sterile 96-well plates were used to study antimicrobial activity.

[0025] Example 4 (antifungal and antibacterial activity)

[0026] For the initial cultivation of test culture strains, which was carried out in sterile test tubes and flasks with cotton-gauze stoppers, Petri dishes with aeration at a temperature of 37 ° C overnight, LB Broth was used in all cases. For screening, a solution containing 5.12 mg of compound (I) hydrochloride, 350 μl of distilled water and 150 μl of dimethyl sulfoxide, 5 μl were added to 200 μl wells of a 96-well plate, followed by a two-fold dilution: 256 -128 - 64 - 32 - 16-8-2 μg / ml. Then, the prepared daily culture of the microorganism (0.5 units according to McFarland, diluted 100 times in nutrient broth, final concentration 10 6CFU / ml) in a volume of 100 μl. The total volume of liquid added to all wells was 200 μl. After adding all the components, the plates were covered with sterile lids, then the optical density was measured using a FlexA-200 Microplate Reader photometer (wavelength 625 nm) and then incubated for 20 hours in a thermostat at 37°C. At the end of the incubation, the growth of fungi and bacteria was recorded using a photometer. The average values ​​of the optical density of the suspension were calculated minus the initial light transmittance of the solution (before incubation) in each test cuvette: 1) positive growth control of the working suspension containing the reference antimicrobial drug at a concentration equal to the minimum concentration of the substances being studied; 2) control containing a bacterial suspension with a concentration of microorganisms of 5*10 5CFU / ml; 3) each concentration of the test substance. A plot of the activity versus drug concentration was constructed using the obtained optical density data. The minimum inhibitory concentration (MIC) was defined as the drug concentration at which the average transmittance of the suspension did not significantly exceed 1% of the average growth control value and / or the point at which the curve reached a plateau. The results of determining the antifungal and antibacterial activity of compound (I) hydrochloride are presented in Table 1.

[0027] Thus, compound (I) hydrochloride exhibits pronounced antifungal activity against Candida albicans (ATCC 10231) with an MIC value of 8 μg / ml, which is comparable to or superior to the activity of existing structural analogs. A confirmed additional effect of this compound is its antibacterial activity against both gram-positive and gram-negative bacteria. Furthermore, a significant advantage of compound (I) hydrochloride over analogs is the ease of its synthesis, as it involves fewer steps and produces a high yield of target products.

[0028]

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