Novel potential antifungal agents based on thiazolidine-2,4-dione and a triazole

Abstract

The invention relates to organic chemistry, pharmacology and medicine and pertains to a novel chemical compound characterized by high antifungal activity, and more particularly to the compound (Z)-(5-(4-chlorobenzylidene)-3-(2-(4-((2R,3R)-3-(2,4-difluorophenyl)-hydroxy-4-(1Н-1,2,4-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thaizolidine-2,4-dione of formula (I) and pharmaceutically acceptable salts thereof. Experiments have demonstrated that the proposed compound is active against many fungal strains, including strains resistant to commercial azole-based drugs.

Description

[0001] NEW POTENTIAL ANTIFUNGAL AGENTS BASED ON THIAZOLIDINE-2, 4-DIONE AND TRIAZOLE

[0002] Field of technology

[0003] The invention relates to the chemistry of organic compounds, pharmacology and medicine and concerns a new chemical compound characterized by high antifungal activity, which, in particular, can be used for the prevention and treatment of infectious diseases in a subject.

[0004] State of the art

[0005] At the end of 2022, WHO published a list of the most dangerous fungal pathogens posing a threat to humanity and classified the creation of new therapeutic agents for the treatment of invasive infections as the most in-demand category. Among the listed pathogens, the first line is occupied by such fungal strains as C. auris, C. tropicalis, C. parapsilosis, Mucorales [WHO fungal priority pathogens list to guide research, development and public health action, https: / / www.who. int / publications / i / item / 9789240060241 , (accessed October 2022, ns^ / www.who.inW ngy^ / jtem / 25:10:2022-whg-re]easgs-Hretgygrdistof"hgajth-threatgr;jnfl-- fungi].

[0006] One of the most alarming changes in the epidemiology of invasive candidiasis is the worldwide emergence of a particularly dangerous new multidrug-resistant strain of C. auris, capable of transmitting infection via patient-to-patient contact with objects.

[0007] The pathogen is characterized by high virulence parameters, which include the ability to form biofilms with a tendency to colonize, leading to transmission in health care settings and the development of resistance to existing antifungal drugs. Most isolates studied had high-level resistance to fluconazole (minimum inhibitory concentration, MIC > 64 g / mL). In addition, up to 30% of isolates demonstrated reduced susceptibility to amphotericin B. More than 5% may be resistant to echinocandins, which are currently recommended first-line antifungals for candidemia. Risk factors in intensive care unit patients include: previous antifungal use, vascular surgery, hospital stay, underlying pulmonary disease, and indwelling urinary tract catheterization. Although C.auris is implicated in a minority of cases of candidemia, with mortality associated with C. auris reaching 60% in some studies [J. Fungi, 2020, 6, 91 .doi: 10.3390 / jof60091 ].

[0008] Invasive candidiasis is a serious infection that primarily affects critically ill and immunocompromised patients. Candida albicans, which is usually susceptible to fluconazole, has long been the most common species associated with invasive candidiasis. However, with the increasing use of antifungal agents and new diagnostic techniques, the epidemiology of Candida infections is changing, resulting in an increase in infections caused by species with less predictable susceptibility to antifungal drugs.

[0009] Established in 1997, the SENTRY antifungal surveillance program tracks the global epidemiology of invasive Candida infections, including species distribution and antifungal resistance. The program recently released its first 20 years of data, which included more than 20,000 clinical isolates collected through passive surveillance in 39 countries.

[0010] Although C. albicans remains the most common species causing invasive candidiasis, the overall proportion of infections caused by C. albicans decreased from 57.4 to 46.4% over a 20-year follow-up period [Pfaller, MA; Diekema, DJ; Turnidge, JD; Castanheira, M.; Jones, RN Twenty years of the SENTRY Antifungal Surveillance Program: Results for Candida Species from 1997-2016. In Open Forum Infectious Diseases; Oxford University Press: Oxford, UK, 2019].

[0011] In the United States, more than 30% of candidemia cases are now caused by C. glabrata, a worrying trend given the increasing rates of antifungal resistance associated with this species [Lamoth, F.; Lockhart, SR; Berkow, EL; Calandra, T. Changes in the epidemiological landscape of invasive candidiasis. J. Antimicrob. Chemother. 2018, 1 , i4-i13].

[0012] In some centres, resistance to echinocandins mediated by mutations in FKS1 occurs in 10% of C. glabrata isolates, and of these echinocandin-resistant strains, resistance to current commercial azole agents occurs in 20% of strains. In contrast, in the UK, C. glabrata isolates were rarely resistant to echinocandins (0.55%). C. parapsilosis is also more common. This species accounts for 15% of candidemia cases in the USA and 20% in Russia, and in South Africa, C. parapsilosis competes with C. albicans as the leading cause of invasive disease [Friedman, & Schwartz. (2019). Emerging Fungal Infections: New Patients, New Patterns, and New Pathogens. Journal of Fungi, 5(3), 67. doi:10.3390 / jof5030067].

[0013] Candida species have undergone taxonomic reclassification with the emergence of new classes, genera, and species over the past decade. However, C. albicans remains the most common causative agent of candidiasis.

[0014] A serious problem in the treatment of oncohematological patients is associated with the poor efficiency of the applied therapy and, consequently, high mortality. Treatment of opportunistic fungal infections remains a problem, despite significant advances in supportive therapy. They are still one of the most common causes of death in immunosuppressed patients. In addition to the detection of fungi of the genus Aspergillus species, zygomycosis, which is the second most common filamentous fungus in patients with hematological neoplasms, is increasingly reported [Gleissner, B., Schilling, A., Anagnostopolous, I., Siehl, I., & Thiel, E. (2004). Improved Outcome of Zygomycosis in Patients with Hematological Diseases? Leukemia & Lymphoma, 45(7), 1351-1360. doi:10.1080 / 10428190310001653691].

[0015] Zygomycosis is an umbrella term, previously called mucormycosis, that includes all mycotic diseases caused by fungi of the genus Zygomycetes.

[0016] The major known human pathogens belong to the order Mucorales, family Mucoraceae, which includes the genera Rhizopus, Absidia, Mucor, Rhizomucor, and Apophysomyces. Most human diseases are caused by members of the genus Mucorales. Although the most common causes are Rhizopus spp. Zygomycosis due to Mucorales usually occurs in immunocompromised hosts as an opportunistic infection. Risk factors include diabetes mellitus, neutropenia, long-term immunosuppressive therapy, chronic prednisone use, iron chelation therapy, broad-spectrum antibiotic use, and a primary disruption of the skin barrier such as trauma, surgical wounds, needle sticks, or burns. Mucorales are associated with angioinvasive disease, often resulting in thrombosis, infarction of involved tissues, and tissue destruction mediated by a variety of fungal proteases, lipases, and mycotoxins. If diagnosis is not made early, dissemination often occurs.Therapy, if effective, must be started early and requires a combination of antifungal drugs, surgery, and elimination of underlying risk factors.

[0017] Therefore, there remains a high need to develop new effective antifungal agents for the treatment of a wide range of diseases.

[0018] Disclosure of invention

[0019] The objective of the present invention is to develop and create a new effective antifungal agent that is promising for use in clinical practice for the treatment and / or prevention of infectious diseases caused by fungal infection.

[0020] The technical result of the invention consists in developing a new compound characterized by highly effective antifungal activity of a wide spectrum of action, including with respect to zygomycetes, and which is promising for use in the therapy of infectious diseases in a subject caused by fungal infections (including mold fungi), for example, caused by candidal and filamentous pathogens, in particular C. auris, C. albicans, C. tropicalis, C. krusei, C. parapsilosis, Cryptococcus neoformans, A. niger, A. fumigates and others, for the treatment, in particular, of disseminated mycoses, vaginal candidiasis, candidal stomatitis, endocarditis, as well as diseases caused by mold zygomycetes (mucormycosis), and other diseases of humans and animals. In addition, the compounds according to the invention are characterized by high antimicrobial activity against fungal strains resistant to the commercial azole drugs used.

[0021] The specified technical result is achieved by means of a new compound (Z)-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-1,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2,4-dione of the following formula: or a pharmaceutically acceptable salt thereof.

[0022] The specified compound is characterized by:

[0023] - m / z 617.07.

[0024] The achievement of the specified technical result is also ensured by using the compound according to the invention or its pharmaceutically acceptable salt as an antifungal agent.

[0025] The achievement of the specified technical result is also ensured by using the compound according to the invention or its pharmaceutically acceptable salt to suppress the growth and / or complete elimination of fungal pathogens.

[0026] In some embodiments, the pathogen is a pathogen of the genus Candida spp., Rhizopus spp., Aspergillus spp., Microsporum spp., Trichophyton spp. and / or Epidermophyton spp.

[0027] In particular embodiments of the invention, the pathogen is Candida auris, Candida albicans, Candida non albicans (such as Candida krusei, Candida glabrata, Candida parapsilosis), Aspergillus fumigates, Aspergillus niger and / or Rhizopus stoloniferw others.

[0028] The achievement of the specified technical result is also ensured as a result of using the compound according to the invention or its pharmaceutically acceptable salt to obtain a pharmaceutical composition with antifungal activity for the treatment and / or prevention of an infectious disease of microbial etiology in a subject caused by a fungal infection.

[0029] Achieving the specified technical result is also ensured by a pharmaceutical composition with antifungal activity for the treatment and / or prevention of an infectious disease of microbial etiology in a subject caused by a fungal infection, containing an effective amount of a compound according to the invention and / or its pharmaceutically acceptable salt and at least one pharmaceutically acceptable auxiliary substance.

[0030] In particular embodiments of the invention, the pharmaceutically acceptable excipient is a carrier, filler and / or solvent.

[0031] In particular embodiments of the invention, the subject is a human or an animal.

[0032] In particular embodiments of the invention, the disease is a disease of the skin, nails and / or internal organs.

[0033] In particular embodiments of the invention, the disease is disseminated candidiasis, disseminated zygomycosis, dermatophytosis, superficial mycosis, candidiasis of the skin and / or nails, invasive candidiasis, vaginal candidiasis, candidal stomatitis, endocarditis, or mucormycosis.

[0034] The present invention also relates to a method for inhibiting the growth and / or completely eliminating fungal pathogens, in particular pathogens of the genus Candida spp., Rhizopus spp., Aspergillus spp., Microsporum spp., Trichophyton spp. and / or Epidermophyton spp., comprising administering to a subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

[0035] The invention also includes a method for treating and / or preventing a disease of microbial etiology in a subject caused by a fungal infection, comprising administering a therapeutically or prophylactically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof, to the body of a subject in need of treatment and / or prevention of such diseases.

[0036] In particular embodiments of the invention, the method involves administering a compound of the invention or a pharmaceutically acceptable salt thereof as monotherapy or in combination with one or more therapeutic agents.

[0037] The present invention also includes the preparation of compounds and / or compositions of the invention.

[0038] Detailed Disclosure of the Invention Definitions and Terms For a better understanding of the present invention, some terms used in the present description of the invention are provided below. The following definitions apply herein unless otherwise expressly stated.

[0039] In the description of the present invention, the terms "includes", "including", etc., as well as "contains", "comprising", etc. are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be construed as "consists only of".

[0040] The term "and / or" means one, more, or all of the listed elements.

[0041] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0042] The term "optional" or "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but need not, occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0043] The compound that is the essence of this invention may exist in a radioisotope-labeled form, i.e., said compound may contain one or more atoms whose atomic mass or mass number differs from the atomic mass or mass number of the most common natural isotopes. Radioisotopes of hydrogen, carbon, phosphorus, chlorine include 3 H, 14 WITH, 32 R, 35 S, and 36 C1, respectively. The compound of the present invention which contains such radioisotopes and / or other radioisotopes of other atoms are within the scope of the present invention. Tritiated, i.e. 3H and carbon, i.e. 14 Radioisotopes are particularly preferred due to their ease of preparation and detection.

[0044] A radiolabeled compound of the present invention can be prepared using methods well known to those skilled in the art. The labeled compound can be prepared using the procedures described herein by simply replacing the unlabeled reagents with the appropriate labeled reagents.

[0045] The compound of the present invention may exist in free form or, if desired, in the form of a pharmaceutically acceptable salt or other derivative. The term "pharmaceutically acceptable salt" as used herein refers to those salts which, within the limits of medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, etc., and meet a reasonable benefit-risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates and other types of compounds are well known in the medical field. Salts may be prepared in situ during the isolation or purification of the compounds of the invention, and may also be prepared separately by reacting a free acid or a free base of a compound of the invention with a suitable base or acid, respectively.Examples of pharmaceutically acceptable, non-toxic acid salts include salts of the amino group formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or organic acids such as acetic, oxalic, maleic, tartaric, succinic or malonic acids, or obtained by other methods used in the art, such as ion exchange.Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undecanoate, valeriate, and the like. Typical alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and others.In addition, pharmaceutically acceptable salts may contain, if desired, non-toxic ammonium, quaternary ammonium and amine cations derived from such counterions as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0046] The compound of the present invention may exist as a prodrug. In the context of the present application, the term "prodrug" refers to a precursor or derivative form of a compound of the invention, which may have improved properties, such as better solubility, reduced cytotoxicity or increased bioavailability, compared to the parent compound or drug, and is capable of being activated or converted into a more active parent form. "Prodrug group" means a group that is converted by reaction with an enzyme, gastric acid or the like under physiological conditions in vivo, to produce a compound of the invention, i.e., a group that is converted to produce a compound of the invention by hydrolysis or the like caused by gastric acid or the like.

[0047] By "therapeutically effective amount" is meant that amount of the compound administered or delivered to a patient which will most likely produce the desired response to treatment. The exact amount required may vary from subject to subject depending on the age, body weight, and general condition of the patient, the severity of the disease, the method of administration, combination therapy with other drugs, etc. By "prophylactically effective amount" is meant that amount of the compound administered or delivered to a patient which will most likely produce the desired response to the prophylaxis of infectious diseases caused by fungal infection. The exact amount required may vary from subject to subject depending on the age, body weight, and general condition of the patient, the severity of the disease, the method of administration, combination therapy with other drugs, etc.For prophylactic treatment, a therapeutically or prophylactically effective amount is that amount that will be effective in preventing a fungal infection.

[0048] The term "patient" ("subject") includes all mammalian species, preferably humans, that utilize the compounds of the invention either by self-administration or by administration to the patient by another person for the treatment and / or prevention of a disease or medical condition.

[0049] The terms "treatment" and "therapy" cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) blocking (stopping) the course of the disease, b) alleviating the severity of the disease, i.e. inducing regression of the disease.

[0050] The term "prophylaxis", "prevention", "preventive therapy" covers the elimination of risk factors, as well as preventive treatment of subclinical stages of the disease in humans, aimed at reducing the likelihood of the clinical stages of the disease. Patients for preventive therapy are selected based on factors that, based on known data, entail an increased risk of developing clinical stages of the disease compared to the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prevention is defined as preventive treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of recurrence of the same or a similar clinical state of the disease.

[0051] The term risk reduction refers to therapy that reduces the incidence of clinical disease. Examples of risk reduction include primary and secondary disease prevention.

[0052] The term "administering" a compound of the present invention to an organism includes delivering to a recipient a compound described in the present invention, a prodrug, or other pharmacologically acceptable derivative of such a compound using any acceptable preparations or routes of administration to the organism well known in the art. The term "MIC" as used herein means the minimum inhibitory concentration.

[0053] The term "MIC" in this document means minimum inhibitory concentration.

[0054] References to techniques used in describing the present invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0055] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0056] Preparation of compounds according to the invention

[0057] The compounds of the present invention can be prepared using the following synthetic methods. The methods listed are not exhaustive and allow for reasonable modifications. The reactions should be carried out using suitable solvents and materials. It should be understood that these and all examples provided in the application materials are not limiting and are provided only to illustrate the present invention.

[0058] The compound according to the invention can be obtained by one of the methods presented in the diagrams below.

[0059] Scheme 2. Preparation of ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2П,ЗП)-3-(2,4-difluorophenyl)-hydroxy-4-(1Н-1 ,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2, 4-dione by method B.

[0060] The starting materials in methods A and B are 2-(2,4-difluorophenyl)-4-(piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)butan-2-ol (1) and a commercially available compound, the 3-substituted acetic acid 5-(4-chlorobenzylidene)thiazolidine-2,4-dione (CAS 423153-08-6).

[0061] Scheme 3. Preparation of ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2П,ЗП)-3-(2,4-difluorophenyl)~ hydroxy-4-(1Н-1 ,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2, 4-dione by method B.

[0062] Method B uses 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl)piperazin-1-yl (2) and a commercially available methyl-substituted oxirane (CAS 127000-90-2) as starting materials.

[0063] Obtaining the compound (1).

[0064] The preparation of compound (1) is described in the prior art [e.g. US6153616; Bull. Korean Chem. Soc. 2010]. For example, in the cited source it was obtained as a result of prolonged (48 hours) refluxing of methyloxirane with piperazine in acetonitrile in the presence of a LiCIC catalyst with a yield of 50 to 78%:

[0065] Scheme 4. Preparation of compound (1) (2-(2,4-difluorophenyl)-4-(piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)butan-2-ol).

[0066] In order to optimize the synthesis conditions - to reduce the time and increase the yield - the acetonitrile solvent was replaced with dimethylformamide or dimethyl sulfoxide and magnesium chloride salt MgCh was added to the LiCIC catalyst, which made it possible to reduce the time to achieve the maximum yield of the target compound to 12 hours and increase the yield.

[0067] Obtaining the compound (2).

[0068] Scheme 5. Preparation of the compound tert-butyl 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl)piperazine-1-carboxylate.

[0069] Boc-piperazine (2.5 g, 13.42 mmol, 1 equiv) was suspended in methylene chloride (40 ml), then triethylamine (4.07 g, 40.27 mmol, 3 equiv) was added. The reaction mixture is cooled with ice, then a solution of 2-(5-(4-(chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid chloride, obtained in situ (5.09 g, 16.11 mmol, 1.2 equiv) in methylene chloride (20 ml) is added dropwise with stirring. After the addition of the acid chloride solution is complete, the cooling bath is removed and the reaction mixture is stirred for 16 hours at room temperature. Then the reaction mixture is washed with a saturated aqueous solution of citric acid until an acidic reaction is achieved. The organic layer is separated, dried over sodium sulfate and evaporated on a rotary evaporator. The substance is used further without additional purification. The yield is quantitative (6.25 g), yellowish powder. C21H24CIN3O5S. m / z 465.5. LCMS [M+1] + =466 + Individuality confirmed chromatographically.

[0070] 1H NMR (400 MHz, dmso) b 7.91 (d, J = 23.9 Hz, 1 H), 7.63 (dd, J = 22.6, 7.7 Hz, 5H), 4.62 (s, 2H), 3.37 (s, 16H), 1.40 (s, 9H), 1.01 (d, J = 5.9 Hz, 3H). 13 C NMR (101 MHz, dmso) 6 167.25, 165.60, 163.78, 154.17, 135.84, 132.66, 132.27, 132.17, 132.09, 129.91, 129.84, 122.13, 79.69, 44.27, 43.19, 41.87, {40.54, 40.33, 40.12, 39.91, 39.70, 39.49, 39.29}, 28.46, 25.92

[0071] Scheme 6. Preparation of 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl)piperazin- 1 -yl.

[0072] Tert-butyl 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl) piperazine-1-carboxylate (4.65 g, 10.0 mmol, 1 equiv) was dissolved in 50 mL of absolute ethyl acetate, then 50 mL of a saturated solution of hydrogen chloride in ethyl acetate was added. The resulting precipitate was stirred overnight at room temperature, then filtered, washed with ethyl acetate (30 mL) and diethyl ether (50 mL). The resulting substance was transferred to a flask and dried on a rotary evaporator to constant weight. The obtained salt - 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl)piperazin-1-yl hydrochloride (3.28 g) - was suspended in water (40 ml), BiOH (60 ml) was added and ArCO3 (2.0 g) was carefully added, stirred at room temperature for 30 minutes, then the organic phase was separated, and the aqueous portion was re-extracted with BiOH (20 ml). The organic extracts were combined and evaporated in vacuo.The residue was dissolved in EtOHAc (50 ml), filtered and evaporated to give the base (2.99 g, 82%) as a white powder. C16H16CIN3O3S. m / z 365.4. LCMS [M+1]. + =366 + Individuality confirmed chromatographically.

[0073] 1 Н NMR (400 MHz, dmso) b 7.94 (s, 1 Н), 7.62 (dd, J = 23.2, 8.5 Hz, 4H), 4.59 (s, 2H), 3.43 (d, J = 30.8 Hz, 5H), 2.79 (s, J = 34.9 Hz, 2H), 2.71 (s, 1 H).

[0074] 13 C NMR (101 MHz, dmso) 6 167.25, 165.62, 163.47, 135.83, 132.61, 132.25, 132.18, 129.91, 122.18, 45.59, 45.19, 43.17, 42.56.

[0075] Preparation of a compound according to the invention - ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-1 ,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2,4-dione (L 363).

[0076] Method A

[0077] 3-substituted acetic acid 5-(4-chlorobenzylidene)thiazolidine-2,4-dione (5.94 g, 20 mmol, 1 equiv) and 2,4-difluorophenyl derivative of triazole (1) (6.74 g, 20 mmol, 1 equiv) were dissolved in methylene chloride (50 ml), then the calculated amount of triethylamine (847 mg, 5 equiv) was added. The resulting solution was stirred for 30 min, then TBTLI (6.45 g, 20.1 mmol, 1.2 equiv) was added. After pH control (pH should be at least 8), the reaction mixture was stirred at room temperature for 16 h. Then the organic layer was washed with water, methylene chloride was separated and dried over sodium sulfate. Methylene chloride was evaporated on a rotary evaporator. The obtained substance was purified by flash chromatography on silica gel (methylene chloride - 4% methanol solution in methylene chloride system). 5.06 g of substance were obtained as a dry foam, yield 40.08%. C28H27CIF2N6O4S. m / z 617.07. LCMS [M+1]+=618+.

[0078] Method B

[0079] 2-(2,4-Difluorophenyl)-4-(piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)butan-2-ol (1) (3.37 g, 10.0 mmol, 1 equiv) was suspended in methylene chloride (40 mL), then triethylamine (253 mg, 3 equiv) was added. The reaction mixture was cooled with an ice bath, then a solution of 5-(4-chlorobenzylidene)thiazolidine-2,4-dione acetic acid chloride* in 2 mL of methylene chloride (4.04 g, 12 mmol, 1.2 equiv) was added dropwise. After the addition of the acid chloride, the pH of the mixture was checked; it should be no less than 8. The reaction mixture was gradually heated to room temperature and stirred overnight at room temperature. Then it was washed with a saturated solution of citric acid until an acidic reaction was obtained, the organic layer was separated, dried over sodium sulfate, and methylene chloride was distilled off on a rotary evaporator. The resulting residue was dissolved in methylene chloride and the substance was purified by chromatography on silica gel, the system methylene chloride - 3% solution of methanol in methylene.Individuality confirmed by chromatography. Yield 3.33 g 54%, as a yellowish foam. LCMS [M+1]+=618+.

[0080] *5-(4-chlorobenzylidene)thiazolidine-2,4-dione acetic acid chloranhydride was obtained immediately before the reaction by treating (4-chlorobenzylidene)thiazolidine-2,4-dione 3-substituted acetic acid with an equimolecular amount of thionyl chloride upon heating. The yield is quantitative. The obtained product was used without purification.

[0081] Method B

[0082] A mixture of methyl oxirane: (2P,38)-2-(2,4-difluorophenyl)-3-methyl-[(1H-1 ,2,4-triazol-1-yl)methyl]oxirane (5.3 g, 20 mmol, 1 equiv.), 4-(2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)acetyl)piperazin-1-yl (7.3 g, 20 mmol, 1 equiv.) and diisopropylethylamine (4.0 g, 3 equiv.) is refluxed in DMSO with the addition of lithium perchlorate and magnesium chloride for 16 hours. The solvent is evaporated on a rotary evaporator under vacuum and the residue is dissolved in methylene chloride and washed with aqueous citric acid until an acidic reaction, then with water. The organic phase is dried No. 2804 and evaporated. The product in the form of foamed oil crystallized upon standing. Yield 7.16 g (58%). Individuality confirmed by chromatography. LCMS [M+1]+=618+.

[0083] 1Н NMR (600 MHz, DMSO-d6) б 8.29 (s, 1 Н), 7.96 (s, 1 Н), 7.66 (d, J = 8.8 Hz, ЗН), 7.63 - 7.59 (m, 2H), 7.32 (td, J = 9.0, 6.8 Hz, 1 H), 7.10 (ddd, J = 1 1.9, 9.0, 2.6 Hz, 1 H), 6.90 (td, J = 8.5, 2.6 Hz, 1 H), 5.54 (s, 1 H), 4.91 (d, J = 14.8 Hz, 1 H), 4.81 (d, J = 14.8 Hz, 1 H), 4.61 (s, 2H), 3.59 (d, J = 16.8 Hz, 4H), 3.48 (s, 2H), 3.15 (q, J = 7.0 Hz, 1 H), 2.92 (s, OH), 2.81 (s, 1 H), 2.44 (s, 1 H), 0.75 (d, J = 6.9 Hz, 3H).

[0084] 13 C NMR (151 MHz, DMSO-d6) 6 166.92 , 165.29 , 162.96 , 161.74 (dd, J = 246.0, 12.5 Hz), 158.48 (dd, J = 246.6, 12.1 Hz), 150.60 , 144.76 , 135.49 , 132.29 , 131.91 , 131.85 , 130.29 (dd, J = 9.5, 6.1 Hz), 129.56 , 125.69 (dd, J = 13.0, 3.6 Hz), 121.85 , 1 10.84 (d, J = 20.5 Hz), 103.92 (dd, J = 28.7, 25.6 Hz), 78.84 (d, J = 6.0 Hz), 63.24 (d, J = 4.4 Hz), 55.78 (d, J = 5.0 Hz), 50.42 , 44.82 , 42.85 , 42.46 , 39.61 (dp, J = 42.1 , 21 .0 Hz), 7.21 .

[0085] Examples of the preparation of pharmaceutically acceptable salts of the compound of the invention

[0086] Due to the presence of nitrogen atoms in the structure of the compound, the ability to attach a proton under the action of strong acids is realized, which leads to the formation of a salt. This is confirmed, for example, by the NMR spectrum of the tosylate salt, which contains signals in the region of 2.28-2.44 ppm, corresponding to 2 equivalents of the methyl group of the sulfone residue and chemical shifts of the protons of the phenyl ring at 7.50-7.47 (4H) and 7.14-7.13 (4H) ppm.

[0087] Obtaining hydrochloride salt

[0088] ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-1,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2, 4-dione hydrochloride (1:2), C28H29CI3F2N6O4S, m / z 689.997.

[0089] To a solution of ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-1 ,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2,4-dione (6.17 g, 0.01 mol) in 50 ml of dry ethyl acetate is added 50 ml of ethyl acetate solution, pre-treated with gaseous hydrogen chloride. Upon stirring for 2 hours, a suspension is obtained, which is filtered and washed successively with dry ethyl acetate and ether. The yield is quantitative. The resulting powder is dried at room temperature in a vacuum and stored in a desiccator to prevent sorption of atmospheric moisture.

[0090] 1N NMR (700 MHz, DMSO-d6) 6 10.39 (s, 1 H), 10.30 (s, 1 H), 8.83 (s, 1 H), 7.99 (s, 1 H), 7.97 (s, 1 H), 7.69 - 7.66 (m, 2 H), 7.63 - 7.59 (t, 2 H), 7.38 (s, 1 H), 7.26 (t, J = 10.6 Hz, 1 H), 7.03 (t, J = 8.6 Hz, 1 H), 6.85 (s, 3 H), 5.54 (d, J = 14.7 Hz, 1 H), 5.01 (d, J = 14.5 Hz, 1 H), 4.77 (s, 1 H), 4.63 (s, 1 H), 4.31 (s, 1 H), 4.07 (s, 2H), 3.86 - 3.68 (m, H), 3.66 - 3.50 (m, 2H), 3.49 - 3.39 (m, H), 3.38 - 3.27 (m, H), 3.22 - 3.07 (m, H), 2.49 (p, J = 1.9 Hz, 4H), 1.15 - 1.06 (m, 3H).

[0091] 13 C NMR (176 MHz, DMSO-d6) 6 167.28, 165.63, 163.85, 163.49, 162.12, 149.82, 145.47, 135.89, 132.74, 132.32, 132.23 ,130.41, 129.97, 124.04, 122.18, 111.89, 111.77, 104.96,104.81, 104.66, 77.28, 67.13, 55.39, 52.83, 49.24, 43.06, 9.99.

[0092] Obtaining tosylate

[0093] ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-

[0094] 1,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2, 4-dione 4-methylbenzenesulfonate (1:2), C42H43CIF2N6O10S3, m / z 961.47.

[0095] This salt was obtained in a similar manner to the method described above using suitable reagents.

[0096] 1 N NMR (700 MHz, DMSO-d6) b 9.40 (s, 1 H), 8.58 (s, 1 H), 8.09 (s, 1 H), 7.96 (s,

[0097] 1 N), 7.69 - 7.66 (m, 2H), 7.64 - 7.60 (m, 2H), 7.50 - 7.47 (m, 4H), 7.47 - 7.40 (m, 1 N), 7.29 (s, 1 N), 7.11 (d, J = 7.9 Hz, 5H), 5.10 (d, J = 14.6 Hz, 1 H), 4.93 (d, J= 14.7 Hz, 1 H), 4.77 (s, 1 H), 4.63 (s, 1 H), 4.31 (s, 1 H), 4.07 (s, 2H), 3.86 - 3.68 (m, OH), 3.66 - 3.50 (m, 2H), 3.49 - 3.39 (m, 1 H), 3.38 - 3.27 (m, 1 H), 3.22 - 3.07 (m, 1 H), 2.28 (s, 6H), 1 .16 (t, J = 7.1 Hz, 3H).

[0098] 13C NMR (176 MHz, DMSO-d6) 6 167.29, 165.61, 163.81, 150.84, 145.77, 138.37, 135.91, 132.75, 132.33, 132.22, 130.30 , 129.98, 128.62, 125.96, 124.09, 122.19, 112.05, 105.09, 77.10, 55.27, 52.80, 48.95, 43.06, 21.26, 9.64.

[0099] Characteristic of the biological activity of the compound according to the invention Comparative study of the antifungal activity of the compound according to the invention on clinical isolates of Candida spp., Rhizopus spp., Aspergillus spp. by the micromethod of double serial dilutions in vitro

[0100] The comparative antifungal activity of the compound of the invention, the prior art analogue (compound 9 from document RU2703997) and microbiological standards of fluconazole and voriconazole was studied for the spectrum of antifungal action against clinical isolates of Candida spp. and Rhizopus spp. using the micromethod of double serial dilutions in RPMI 1640 nutrient broth against clinical isolates and standard strains of Candida spp. The activity was assessed in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI), a global non-profit organization for the development of standards and recommendations in medicine. Methods M27-A3 (Clinical and Laboratory Standards Institute.Approved standard third edition M27-A3, Wayne, PA, USA, 2008) and ISO 16256:2012 Clinical laboratory testing and in vitro diagnostic test systems — Reference method for testing the in vitro activity of antimicrobial agents against yeast fungi involved in infectious diseases (IDT) to determine the minimum inhibitory concentration (MIC) value, using a micro serial dilution method in RPMI 1640 medium supplemented with glucose to a concentration of 0.2%. The results are presented in Table 1 below.

[0101] Table 1. Comparison of the antifungal activity of fluconazole suspension, compound 9 from RU2703997 and the compound of the invention L 363 on clinical strains of C. albicans and C. non albicans spp., including fluconazole-resistant strains. significantly less control without impact

[0102] From the results presented in Table 1 it is evident that the compound according to the invention (L 363) is characterized by a 2-fold higher activity on clinical strains of C. albicans and C. non albicans spp. compared to the activity of the analogue from RU2703997, as well as tens of times higher activity compared to the microbiological standard - the commercial drug fluconazole.

[0103] Table 2. Comparison of MIC values ​​of the substances of commercial preparations fluconazole (Flu), compound 9 from RU2703997 and compound L 363 according to the invention on clinical strains of Aspergillus spp.

[0104] From the results presented in Table 2, it is evident that the compound according to the invention is characterized by 4 times higher activity compared to the activity of the analogue from RU2703997 and tens of times higher activity compared to the microbiological standard fluconazole.

[0105] Moreover, comparative studies have shown that the compound of the invention is also much more effective against the highly dangerous pathogen of the Rhisopus spp. species known as "Black Fungus" (outbreak of infection in 2020-21 in India, 10 thousand affected) compared to the analog compound from document RU2703997 and the commercial drug voriconazole. The results of the studies are presented in Table 3.

[0106] Table 3. Distribution of MIC values ​​among Rhizopus spp. to the substances of the compound of the invention, compound 9 from RU2703997n voriconazole.

[0107] Thus, the results of the conducted studies showed that the compound according to the invention is characterized by a significantly higher level of activity against the particularly dangerous pathogen of the Rhisopus spp. species compared to the known analogue compound from the prior art, as well as compared to the antifungal drug voriconazole, namely, the MIC level of the compound according to the invention is at least 33 times lower than that of the analogue compound from RU2703997, and at least 266 times lower compared to voriconazole. Further, comparative studies were also conducted in relation to strains

[0108] C.auris. The results are presented in Table 4.

[0109] Table 4. Distribution of MIC values ​​among C. auris strains to the substances of the compound of the invention, compound 9 from RU2703997 and voriconazole (Vori). The results of the studies showed that the compound of the invention L 363 is characterized by a significantly higher level of activity against C.auris strains compared to the known analogue compound from the prior art (RU2703997), namely, the MIC level of the compound of the invention is 2 times lower than that of the analogue compound from RU2703997 and 8 times higher than the activity of the commercial drug voriconazole against the C.auris 2019-731 strain resistant to it.

[0110] Comparative studies were also conducted on strains of dermatophyte fungi: Microsporum canis, Epidermophyton spp., Trichophyton spp., the results are presented in Table 5.

[0111] Table 5. Distribution of MIC values ​​among strains of dermatophyte fungi to the substance of compounds: 9 from RU2703997, L 363 according to the invention and commercial preparations of itraconazole and amphotericin B (Ab B).

[0112] As can be seen from the data provided, the results of the studies conducted showed that the activity of the inhibitory effect of the compound according to the invention L 363 in relation to strains of dermatophyte fungi is several times higher in comparison with the comparison drugs (itraconazole and amphotericin B) and compound 9 from RU2703997.

[0113] Thus, the proposed compounds are of interest to medicine and can find application in the treatment and prevention of infectious diseases, in particular those caused by various fungal infections (including particularly dangerous pathogens), for example, such diseases as mucormycosis, invasive candidiasis, vaginal candidiasis, candidal stomatitis, endocarditis, and other diseases of humans and animals.

[0114] PHARMACEUTICAL COMPOSITIONS

[0115] The invention also relates to pharmaceutical compositions that comprise a compound of the invention (or a prodrug, a pharmaceutically acceptable salt or another pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and / or excipients that can be administered to a patient together with the compound that is the essence of this invention, do not reduce the pharmacological activity of this compound and are not toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0116] The pharmaceutical compositions referred to in this invention comprise the compounds of this invention together with pharmaceutically acceptable carriers, which may include any solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives, binders, lubricants, etc., suitable for the particular dosage form.Materials that can serve as pharmaceutically acceptable carriers include mono- and oligosaccharides and their derivatives; gelatin; talc; auxiliary substances such as cocoa butter and suppository wax; oils such as peanut, cottonseed, sesame, olive, corn and soybean oils and others; glycols such as propylene glycol and kohl; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; depyrogenated water; isotonic solution, Ringer's solution; alcohol and phosphate buffer solutions. The composition may also contain other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, film agents, sweeteners, flavorings and aromas, preservatives and antioxidants.

[0117] The subject of the present invention is also dosage forms - a class of pharmaceutical compositions, the structure of which is optimized for a specific method of administration into the body in a therapeutically effective dose, for example, for administration into the body intravenously, intramuscularly, orally, subcutaneously, by inhalation, intranasally, sublingually, rectally, or by other generally accepted methods in recommended dosages.

[0118] The dosage forms of the present invention may contain structures obtained by methods of using liposomes, microencapsulation methods, methods of obtaining nanoforms of a drug, or other methods known in pharmaceuticals.

[0119] In preparing a composition, for example in the form of a tablet, the active ingredient is mixed with one or more pharmaceutical excipients such as gelatin, starch, lactose, magnesium stearate, talc, silicon dioxide, acacia, mannitol, microcrystalline cellulose, hypromellose or similar compounds.

[0120] The tablets may be coated with sucrose, cellulose derivatives or other suitable coating agents. The tablets may be prepared by various methods, such as direct compression, dry or wet granulation or hot hot alloying.

[0121] A pharmaceutical composition in the form of a gelatin capsule can be obtained by mixing the active ingredient with a solvent and filling soft or hard capsules with the resulting mixture.

[0122] For parenteral administration, aqueous suspensions, isotonic saline solutions or sterile solutions for injections and infusions are used.

[0123] Examples of obtaining a composition according to the invention

[0124] Method A: Granulation (P. Bhatt. Handbook of pharmaceutical technology practical. P. Bhatt, A.Kumar Eds. Pharmatech, 2021).

[0125] The compound according to the invention, finely ground powders of lactose, starch, talc are thoroughly mixed in a mortar and starch paste is added to the resulting mixture as a binding ingredient and mixed. The resulting mixture is sifted through a sieve. The resulting granules are dried in an oven at a temperature of 60°C for 35 minutes.

[0126] Method B: Tableting

[0127] Granules obtained by method A are lubricated with magnesium stearate and baked in an oven at 60°C until completely dry (P. Bhatt. Handbook of pharmaceutical technology practical. P. Bhatt, A.Kumar Eds. Pharmatech 2021). Then the dried granules are formed into tablets using an eccentric (pressing) tablet machine (Menshutina N.V., Alves S.V., Mishina Yu.V. Innovative technologies and equipment for pharmaceutical production. BINOM, 2012).

[0128] METHODS OF THERAPEUTIC APPLICATION

[0129] The compound of the present invention and its pharmaceutically acceptable salts are an antifungal agent, i.e. a useful agent for the therapy and / or prevention of diseases or conditions caused by fungal infections, including pathogens of the genus Candida spp., Rhizopus spp., Aspergillus spp., Microsporum spp., Trichophyton spp. Due to its antifungal activity, the compound of the present invention can be used for the prevention, treatment and / or reduction of the risk of developing an infectious disease of microbial etiology in a subject caused by a fungal infection.

[0130] These infectious diseases are well characterized in humans, but also present in other mammals with similar etiology, and can be treated with the pharmaceutical compositions of the present invention. For therapeutic use, the compound of the invention can be administered using a pharmaceutical composition in any pharmaceutical dosage form by any route of administration. Dosage forms typically include a pharmaceutically acceptable carrier suitable for the specific dosage form selected. In particular, the compound of the invention can be administered to a patient daily for a period of time necessary for the treatment and / or prevention of diseases, including a course of therapy lasting days, months, years. Routes of administration include, but are not limited to: intravenously, intramuscularly, orally, subcutaneously, by inhalation, intranasally and sublingually. Preferred routes of administration are oral and intravenous.

[0131] The invention also relates to a pharmaceutical composition containing a daily dose of said compound in the form of a fixed dosage unit, and to a combination containing said pharmaceutical composition or said compound. In a preferred embodiment, said composition for use according to the invention is administered one or more times a day in a dosage of 1 mg or more of the compound of the invention. The preferred dosage is 1-1500 mg. The most preferred dosage is 10-1000 mg.

[0132] One or more additional pharmacologically active agents may be administered in combination with the compound of the invention. In general, any additional single or multiple active agents other than the compound of the invention may be used in any combination with the compound of the invention in a single or separate dosage form that allows for simultaneous or sequential therapeutic action of the active agents. Such additional one or more agents may be administered simultaneously, or as part of a pharmaceutical composition, or at separate times.

[0133] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be considered as limiting the scope of the invention in any way. It will be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

Invention formula 1. Compound ( )-(5-(4-chlorobenzylidene)-3-(2-(4-((2H,3H)-3-(2,4-difluorophenyl)-hydroxy-4-(1 H-1 ,2,4-triazol-1-yl)butan-2-yl)piperazin-1-yl)-2-oxoethyl)thiazolidine-2, 4-dione, having the following structural formula: or a pharmaceutically acceptable salt thereof.

2. Use of the compound according to claim 1 as an antifungal agent.

3. Use of a compound according to claim 1 for the preparation of a pharmaceutical composition with antifungal activity for the treatment and / or prevention of an infectious disease of microbial etiology in a subject caused by a fungal infection.

4. Use according to paragraph 3, where the disease is caused by a pathogen of the genus Candida spp., Rhizopus spp., Aspergillus spp., Microsporum spp., Trichophyton spp. and / or Epidermophyton spp.

5. Use according to paragraph 3, where the disease is caused by a pathogen that is Candida auris, Candida albicans, Candida non albicans Aspergillus fumigatus, Aspergillus niger and / or Rhizopus stolonifer.

6. The use according to claim 5, wherein the Candida non albicans is C. krusei, C. glabrata and / or C. parapsillosis.

7. A pharmaceutical composition with antifungal activity for the treatment and / or prevention of an infectious disease of microbial etiology in a subject caused by a fungal infection, containing an effective amount of a compound according to claim 1 and at least one pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 6, wherein the disease is caused by a pathogen of the genus Candida spp., Rhizopus spp., Aspergillus spp., Microsporum spp., Trichophyton spp. and / or Epidermophyton spp.

9. The pharmaceutical composition according to claim 6, wherein the disease is caused by a pathogen that is Candida auris, Candida albicans, Candida non albicans, Aspergillus fumigatus, Aspergillus niger and / or Rhizopus stolonifer.

10. The pharmaceutical composition according to claim 9, wherein the Candida non albicans is C. krusei, C. glabrata and / or C. parapsillosis.

11. A pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable excipient is a carrier, filler and / or solvent.

12. The pharmaceutical composition according to claim 6, wherein the subject is a human or an animal.

13. The pharmaceutical composition according to claim 6, wherein the disease is a disease of the skin, nails and / or internal organs.

14. The pharmaceutical composition according to claim 6, characterized in that the disease is disseminated candidiasis, disseminated zygomycosis, dermatophytosis, superficial mycosis, candidiasis of the skin and / or nails, invasive candidiasis, vaginal candidiasis, candidal stomatitis, endocarditis or mucormycosis.

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