The present invention relates to pyridine-2-thiol 1 -oxide derivatives and their use as antimicrobial agents in infectious diseases of mammals (humans and animals) caused by fungi or bacteria, in particular belonging to the Mycobacteriaceae family, especially diseases caused by Mycobacterium abscessus or Mycobacterium tuberculosis.

The Mycobacterium abscessus complex which encompasses three subspecies (M abscessus subsp. abscessus; M. abscessus subsp. bolletii and M. abscessus subsp. massihense) is a group of rapidly growing nontuberculous mycobacteria (NTM) [1], In addition to being responsible for a wide range of skin and soft tissue infections [2], these bacteria are frequent opportunistic pathogens of the lung in patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [3], The AL abscessus complex are with the Mycobacterium avium complex the NTM most frequently associated with pulmonary disease. The prevalence of M. abscessus complex in individual with CF ranges from 5% to 20% [4-6],

M. abscessus infections are difficult to treat and require long-term treatment with multidrug regimen, which is complicated by intrinsic (natural) and acquired resistance mechanisms and the lack of bactericidal activity of most of the antibiotics used [7], Other important challenges include the absence of a correlation between in vitro minimal inhibitory concentrations (MIC) of compounds and their in vivo efficacy, the ability of these bacteria to grow both intra- and extra-cellularly, and their propensity to form biofilms [8],

To date, there is no official standard drug regimen to treat AL abscessus pulmonary infections. Recommendations by the British Thoracic Society (BTS) [9] describe a 12+ month-long treatment consisting of an initial phase (including intravenous and oral antibiotics), followed by a continuation phase (including inhaled and/or oral antibiotics). The combination of antibiotics used in the management of AL abscessus infections depends on the macrolide-sensitive or macrolide-resistant phenotype of the isolates, and includes clarithromycin/azithromycin (macrolides, only in the first case), amikacin (aminoglycosides), tigecycline (tetracyclines), imipenem (P-lactam antibiotics), clofazimine (phenazines), linezolid (oxazolidinones), minocycline (tetracyclines), moxifloxacin (fluoroquinolones), and cotrimoxazole. The American Thoracic Society and the Infectious Diseases Society of America (ATS/IDSA) guidelines were updated in July 2020 [10], It is suggested to use at least 3 active drugs among patients without macrolide resistance, and at least 4 active drugs among patients with macrolide resistance in the initial treatment phase (parenteral - amikacin, imipenem (or cefoxitin), tigecycline, and oral - azithromycin (clarithromycin), clofazimine, linezolid); and at least 2-3 active drugs for the continuation phase of therapy (azithromycin (clarithromycin), clofazimine, linezolid, inhaled amikacin). However, it is noted in this guideline that the optimal drug regimens and duration of anti- abscessus pulmonary infection therapy are not known.

It is clear that the current recommended regimens for the treatment of M. abscessus pulmonary infections lack efficacy. A meta-analysis by Kwak et al. [11] revealed that the treatment success rate was only 33% for M. abscessus subsp. abscessus and about 57% for M. abscessus subsp. massiliense . The use of azithromycin, amikacin, or imipenem was associated with better success rate in patients with M. abscessus subsp. abscessus infection, while the choice of these drugs among patients with M. abscessus subsp. massiliense did not correlate with treatment success.

Similar results were presented in a retrospective analysis of the efficacy and adverse effects of different antibiotics used in combination to treat patients with AT. abscessus pulmonary infection by Chen et al. [12], The treatment success rate in patients with M. abscessus subsp. massiliense was higher (81%) compared to that among patients with AT. abscessus subsp. abscessus (33%), and this rate was 45% for all observed cases of M. abscessus complex lung infection. Treatments with drug combinations that included amikacin, imipenem, linezolid, or tigecycline were overall more successful, but these drug combinations did not exert the same beneficial effects on patients infected with M. abscessus subsp. massiliense . It was also found that clarithromycin was more commonly associated with treatment failure than treatment success (85 vs. 71%, respectively) in patients infected with M. abscessus subsp. abscessus. In addition, adverse side events were observed in the majority of patients (79% of the cases), and included gastrointestinal distress (48%) mostly caused by tigecycline, ototoxicity (23%) associated with amikacin treatment; and myelosuppression (10%) caused mainly by linezolid. The failure of currently used drugs emphasizes the urgent need for novel antibacterial agents with new mechanisms of action, as well as for new, more effective, antibiotic combinations. To date, only few molecules are in clinical development for the treatment of M. abscessus pulmonary infections. In a phase 2 open-label pilot study of inhaled NO (160 ppm) administered to CF patients with refractory M. abscessus lung infection, the treatment was well-tolerated and safe, but did not achieve statistical significance in secondary clinical outcomes, including AT. abscessus burden in airways [13], The application of high-dose nitric oxide (240 ppm) in compassionate-use treatment led to adverse symptoms in a 24-y ear-old CF patient with pulmonary AT. abscessus [14], In a Phase 2 open-label study of tigecycline-containing regimens for the treatment of M. abscessus and Mycobacterium chelonae pulmonary infections, only 16 out of 36 patients (44.4%) showed clinical improvement [15], In addition, treatment with this drug is associated with adverse events in more than 90% of participants (nausea, vomiting, fever and diarrhea being the most common). The clinicaltrials.gov website mentions two ongoing studies on the use of liposomal amikacin inhalation in the treatment of M. abscessus lung disease (ClinicalTrials.gov, NCT03038178 and NCT04163601), but results are not yet available.

There are two main strategies to enhance the pipeline of effective anti-A7. abscessus drugs. The first strategy consists in old drug repurposing, or the study of marketed drugs for new therapeutic applications. This approach is particularly interesting in the area of antibacterial where the emergence of resistance often outpaces drug development, given that the repurposing of previously approved drugs can shorten development time and lower costs. The second strategy is the common drug development process, which involves the discovery of novel chemical entities from target identification to lead optimization, full-scale preclinical studies and clinical trial. This approach is benefiting from the development of new anti -AT. tuberculosis compounds which can simultaneously be tested against other mycobacterial pathogens, including the abscessus complex.

Several recent reviews have considered the challenges and opportunities associated with drug repurposing for the treatment of AL abscessus pulmonary infections [16], Only a few articles, in contrast, discuss the novel compounds that are being developed against this emerging pathogen [17-19],

Therefore, there is an urgent need of antimicrobial drugs with specific high anti-AL abscessus activity to overcome problems concerning antibiotic resistance and drug intolerance.

The authors of the present invention now discovered a promising class of compounds with specific activity against Mycobacterium spp. that address and solve the above mentioned technical problems.

Surprisingly the compounds of the invention exhibit strong antimicrobial activity, especially against Mycobacterium abscessus or Mycobacterium tuberculosis. Thus, the compounds of the invention are useful for the treatment of microbial infections in humans and in animals.

Therefore, it is an object of the present invention a compound of formula (I): wherein R is selected from the group consisting of COOR ¹ , CONR2 ² or CF3, wherein R ¹ is a linear C1-C3 alkyl group and R ² is selected between H and C1-C3 alkyl group; wherein X is selected from the group consisting of Na, Li, K or Ca; and crystalline forms thereof; for use in the treatment or prevention of infections caused by Mycobacteria or fungi in a subject in need of such treatment.

According to a preferred embodiment of the invention, said R substituent is selected between the group consisting of 5 -methoxy carbonyl, 5-ethoxycarbonyl and 5- [(dimethylamino)carbonyl] .

In a preferred embodiment of the present invention, X is Na, preferably in combination with any of the above preferred meaning of R substituent.

In one of the most preferred embodiment of the invention the compound is sodium 5-(alkcarbonyl)pyridine-2-thiolate 1 -oxide.

In another preferred embodiment said infection caused by Mycobacteria is caused by Mycobacterium abscessus o Mycobacterium tuberculosis.

According to a further preferred embodiment said infection caused by fungi is caused by Sporobolomyces salmonicolor, Candida albicans, Penicillium notatum or Aspergillus niger.

The invention is further directed to the use of a compound of formula (I), for the treatment or prevention of infections caused by Mycobacteria or fungi in a subject in need of such treatment alone or in combination with other active principle having antimicrobial activity.

Another object of the present invention is a pharmaceutical composition comprising a compound of formula (I) as active principle together with pharmaceutically acceptable adjuvant and excipients for use in the treatment or prevention of infections caused by Mycobacteria or fungi in a subject in need of such treatment.

According to a preferred embodiment the route od administration of the pharmaceutical compositions of the invention is systemic or topical.

The compounds of formula (I) of the present invention may be formulated in a wide variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, dragee, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions. Compounds of the present invention are efficacious when administered by other routes of administration including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal, nasal, nebulizer inhalation and suppository administration, among other routes of administration.

As a preferred embodiment said pharmaceutical compositions may be in the form of saline solutions for injection, intranasal spray or nebulizer.

The compounds of the invention may be used in dosages from 0.01 - 1000 mg/kg body weight, preferably from 1-50 mg/kg body weight.

The invention further relates to the compound of formula (I) sodium 5- (alkcarbonyl)pyridine-2-thiolate 1 -oxide for use in medical field.

It is a further object of the present invention the compound of formula (I) sodium 5- (alkcarbonyl)pyridine-2-thiolate 1 -oxide for use in medical field as antibacterial or antifungal agent.

In a preferred embodiment the compound is used against infection caused by Mycobacteria (preferably by Mycobacterium abscessus or Mycobacterium tuberculosis) or fungi such as Sporobolomyces salmonicolor , Candida albicans, Penicillium notatum o Aspergillus niger. Moreover, said infection may be mediated by other bacteria such as Escherichia coli, Acinetobacter baumanii and Staphylococcus aureus (see below Table 2).

The present invention will now be described for illustrative, but non-limiting purposes, according to a preferred embodiment with particular reference to the attached figures, in which:

- Figure 1 shows the concentration in plasma of compound 2 after single 100 mg/kg per os administration;

- Figure 2 shows the results of stability study carried out with 5-(ethoxycarbonyl) pyridine-2-thiolate 1 -oxi de (base form);

- Figure 3 shows the results of stability study carried out with sodium 5-(ethoxy carbonyljpyridine -2-thiolate 1 -oxide (salt form).

The following non-limiting examples are now provided for a better illustration of the invention, in which different silk fibroin-based scaffolds were tested and compared. EXAMPLE 1 : Synthetic reaction scheme

Starting materials All reagents and solvents were purchased from commercial suppliers and used without further purification. 'H and ¹³ C spectra were measured on Bruker AC-500 (500 MHz, ¹ H) or Bruker AC-200 (75 MHz, ¹³ C).

Chemical shifts were measured in DMSO-d6 or CDCE, using tetramethyl silane as an internal standard, and reported as units (ppm) values. The following abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; brs, broad singlet; brm, broad multiplet.

Mass spectra were recorded on Finnigan MAT INCO 50 mass spectrometer (El, 70 eV) with direct injection.

The purity of the final compounds was analyzed on Agilent 1290 Infinity II HPLC system coupled to Agilent 6460 triple-quadrupole mass spectrometer equipped with an electrospray ionization source. The chromatographic separation was carried out on Agilent Eclipse Plus C18 RRHD column (2.1 x 50 mm, 1.8 pm) at 40 °C, sample injection volume was 0.2 pL. The mobile phase comprising 0.1% formic acid/water (A), and 0.1% formic acid and 85% acetonitrile/water (B) was programmed with gradient elution (0.0-3.0 min, 60% B; 3.0-4.0 min, 60% to 97% B; 4.0-6.0 min, 97% B; 6.0-6.1 min, 97% to 60% B) at a flow rate of 0.4 ml/min. The mass spectrometric detection was operated in positive ion mode. Optimal parameters were: capillary voltages of 3500 V, a nebulizer pressure of 35 psi, a gas temperature of 350°C, a gas flow rate of 12 L/min. All final compounds are > 95% pure.

Melting points were determined on Electrothermal 9001 (10°C per min).

Merck KGaA silica gel 60 F254 plates were used for analytical thin-layer chromatography.

Column chromatography was performed on Merck silica gel 60 (70-230 mesh). Yields refer to purified products.

The starting materials and the intermediates of the synthetic reaction schemes also can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data. Examples of representative compounds encompassed by the present invention and within the scope of the invention are provided in the following Examples A-L.

All compounds were synthesized according below Scheme 1 wherein corresponding 2-chloropyridine was oxidized by reacting with freshly prepared solution of H2O2/OC(NH2)2 in chloroform with formation of pyridine N-oxide, which can be transformed to 2-thiopyridine by two methods:

A) reaction of 2-chloropyridine N-oxide with large excess of sodium hydrosulfide;

B) synthesis of pyridin-2-yl imidothiocarbamate as intermediate with follow its treatment by sodium hydrogen carbonate. Pyridine-2-thiol 1 -oxide derivatives easy form corresponding salts with metal hydroxides and these final compounds are solid, white and water soluble compounds.

Scheme 1 a) H ₂ O2/OC(NH ₂ ) ₂ , CHCh; b) NaHS, EtOH, water; c) SC(NH ₂ ) ₂ , EtOH; d) NaHCOs, EtOH, water; e) XOH, MeOH, acetone.

Step of the synthesis according to Scheme 1 are herein explained in more detail:

A solution of corresponding pyridine (1 mmol) in chloroform was treated by dry UHP (H2O2/OC(NH2)2 (2 mmol) at 0°C. Trifluoroacetic acid anhydride was then slowly added to the reaction mixture (the reaction is exothermic). The reaction was followed by TLC until completion at room temperature. The reaction mixture was dissolved by water, formed precipitate was filtered off and washed by water. The desired pyridine N-oxide was recrystallized preferably from ethanol.

A solution of pyridine N-oxide derivative (1.0 mmol) in ethanol was slowly treated by drop by solution of sodium hydrosulfide (5-7 mmol) at room temperature and was stored 2-3 hours at this temperature. The reaction mixture was cooled, diluted by water and formed solid was filtered off. The thiopyridine derivative was recrystallized preferably from ethyl acetate or ethanol.

Step c) A solution of pyridine N-oxide derivative (1.0 mmol) in acetone was treated by thiourea (1/3 mmol) and refluxed for 2-3 hours. The reaction mixture was evaporated in vacuum till 14 of the volume and cooled at -18°C for 6 hours. The formed precipitate was filtered off and residue was dissolved in small volume of water. This solution was treated by water solution of sodium hydrogen carbonate and formed sediment of pyridin-2-yl imidothiocarbamate derivative was filtered off. The compound was recrystallized preferably from ethyl acetate or ethanol.

Step d) A solution of pyri din-2 -yl imidothiocarbamate derivative (1 mmol) in 20 ml of ethanol was treated by water solution of sodium hydrogencarbonate (2 mmol) at room temperature. The reaction mixture was stored for 3 hours at 60°C, cooled and treated by 0.1N hydrochloric acid water solution until pH~2. Formed residue was collected, dried and pyri dine-2 -thiol 1 -oxide derivative was recrystallized from ethanol.

Step e) A solution of pyri dine-2 -thiol 1 -oxide derivative (1 mmol) in ethanol was treated by drop by alkali hydroxide (1 mmol) solved in minimal volume of water. Sediment was collected and recrystallized from methanol or ethanol to give aim corresponding alkaline salt of 5-(R)-pyri dine-2 -thiolate 1 -oxide as an off-white solid. All compounds were obtained at a purity of not less than 98% in sufficient amounts. The confirmation of the structure and investigation of the physicochemical properties of all synthesized compounds were performed by various modem methods described in the following section referring to “Examples A-L”.

Examples A-L

A. Ethyl 6-mercaptoni cotinate 1-oxide (compound 1)

Yield 76%. Mass (El), m/z (Rel. int. (%)): 199.2298 [M] (73). Anal. Calcd. for C ₈ H ₉ NO ₃ S: (%) C, 48.23; H, 4.55; N, 7.03. Found (%) C, 48.07; H, 4.36; N, 7.18. 1H NMR (DMSO-de): 8.74 (s, 1H, CH), 7.68 (d, 1H, J= 9.8 Hz, CH), 7.59 (d, 1H, J = 9.8 Hz, CH), 4.28 (q, 2H, J= 7.2 Hz, CH ₂ ), 1.28 (t, 3H, J= 6.9 Hz, CH ₃ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 164.47, 162.64, 137.28, 134.16, 125.91, 121.08, 61.07, 14.30 ppm. B. Sodium 5-(ethoxycarbonyl)pyridine-2-thiolate 1 -oxide (compound 2)

Yield 64%. Mass (El), m/z (Rel. int. (%)): 198.2201 [M] (79). Anal. Calcd. for C ₈ H ₈ NNaO ₃ S: (%) C, 43.44; H, 3.65; N, 6.33. Found (%) C, 43.35; H, 3.67; N, 6.28. 'H NMR (DMSO-de): 8.82 (s, 1H, CH), 7.62 (d, 1H, J= 9.8 Hz, CH), 7.51 (d, 1H, J = 9.8 Hz, CH), 4.23 (q, 2H, J= 7.2 Hz, CH ₂ ), 1.17 (t, 3H, J= 6.9 Hz, CH ₃ )ppm. ¹³ C NMR (50 MHz, DMSO) 8 169.71, 164.58, 137.76, 136.33, 132.17, 125.71, 61.07, 14.31 ppm. C. Methyl 6-mercaptoni cotinate 1 -oxide (compound 3)

Yield 73%. Mass (El), m/z (Rel. Int. (%)): 185.2014 [M] (57). Anal. Calcd. for C7H7NO3S: (%) C, 45.40; H, 3.81; N, 7.56. Found (%) C, 45.17; H, 4.01; N, 7.59.

1H NMR (DMSO-d6): 8.74 (s, 1H, CH), 7.69 (d, 1H, J = 9.8 Hz, CH), 7.57 (d, 1H, J = 9.8 Hz, CH), 3.77 (s, 3H, OCH3) ppm. 13C NMR (50 MHz, DMSO) 8 164.61, 162.58, 137.79, 133.83, 125.87, 121.92, 52.23 ppm. D. Sodium 5-(methoxycarbonyl)pyridine-2-thiolate 1 -oxide (compound 4)

Yield 86%. Mass (El), m/z (Rel. Int. (%)): 184.1935 [M] (65). Anal. Calcd. for C ₇ H ₆ NNaO ₃ S: (%) C, 40.58; H, 2.92; N, 6.76. Found (%) C, 40.49; H, 3.03; N, 6.79. 'H NMR (DMSO-d ₆ ): 8.70 (s, 1H, CH), 7.68 (d, 1H, J= 9.8 Hz, CH), 7.51 (d, 1H, J = 9.8 Hz, CH), 3.75 (s, 3H, OCH ₃ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 172.82, 164.81, 138.10, 136.67, 132.17, 125.61, 52.29 ppm.

E. Sodium 5-(trifluoromethyl)pyridine-2 -thiolate 1 -oxide (compound 5)

Yield 80%. Mass (El), m/z (Rel. Int. (%)): 194.1554 [M] (79). Anal. Calcd. for C ₆ H ₃ F ₃ NNaOS: (%) C, 37.12; H, 1.56; N, 7.21. Found (%) C, 37.20; H, 1.49; N, 7.27. 'H NMR (DMSO-d ₆ ): 8.41 (s, 1H, CH), 7.97 (d, 1H, J= 9.7 Hz, CH), 7.67 (d, 1H, J= 9.7 Hz, CH) ppm. ¹³ C NMR (50 MHz, DMSO) 8 171.51, 134.12, 132.57, 132.34, 131.26, 131.08, 127.41, 123.34(q), 52.29 ppm.

F. Potassium 5-(aminocarbonyl)pyridine-2-thiolate 1 -oxide (compound 6)

Yield 93%. Mass (El), m/z (Rel. Int. (%)): 169.1822 [M] (64). Anal. Calcd. for C6H5KN2O2S: (%) C, 34.60; H, 2.42; N, 13.45. Found (%) C, 34.82; H, 2.56; N, 13.51. ¹ H NMR (DMSO-d ₆ ): 8.85 (s, 1H, CH), 8.04 (d, 1H, = 9.6 Hz, CH), 7.73 (d, 1H, J= 9.6 Hz, CH), 5.67 (br s, 2H, NH ₂ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 172.53, 170.60, 140.98, 134.61, 133.53, 129.44 ppm.

G. Potassium 5-(piperidin-l-ylcarbonyl)pyridine-2-thiolate 1-oxide (compound 7)

Yield 87%. Mass (El), m/z (Rel. int. (%)): 237.2992 [M] (60). Anal. Calcd. for C11H13KN2O2S: (%) C, 47.80; H, 4.74; N, 10.14. Found (%) C, 47.61; H, 4.61; N, 10.23. 'H NMR (DMSO-d ₆ ): 8.67(s, 1H, CH), 7.99 (d, 1H, J= 9.7 Hz, CH), 7.69 (d, 1H, J= 9.7 Hz, CH), 3.35 (m, 4H, N(CH ₂ ) ₂ ), 1.93 (m, 4H, 2CH ₂ ), 1.37 (m, 2H, CH ₂ ) ppm. ¹³ C NMR (50 MHz, DMSO) 6 170.03, 167.55, 141.99, 133.92, 132.79, 132.18, 46.70, 26.15, 24.85 ppm. H. 6-Mercapto-N,N-dimethylnicotinamide 1-oxide (compound 8)

Yield 65%. Mass (El), m/z (Rel. int. (%)): 198.2433 [M] (34). Anal. Calcd. for C8H10N2O2S: (%) C, 48.47; H, 5.08; N, 14.13. Found (%) C, 48.53; H, 5.14; N, 14.22. 'H NMR (DMSO-de): 8.67 (s, 1H, CH), 8.03 (d, 1H, J= 9.7 Hz, CH), 7.67 (d, 1H, J = 9.7 Hz, CH), 3.12 (s, 6H, N(CH ₃ ) ₂ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 167.87, 163.13, 135.71, 132.59, 131.06, 122.74, 35.62, 34.20 ppm.

I. Sodium 5-[(dimethylamino)carbonyl]pyridine-2-thiolate 1-oxide (compound 9)

Yield 79%. Mass (El), m/z (Rel. int. (%)): 197.2353 [M] (47). Anal. Calcd. for C ₈ H ₉ N ₂ NaO ₂ S: (%) C, 43.63; H, 4.12; N, 12.72. Found (%) C, 43.57; H, 4.11; N, 12.68. ¹ H NMR (DMSO-d ₆ ): 8.73 (s, 1H, CH), 8.00 (d, 1H, = 9.7 Hz, CH), 7.71 (d, 1H, J = 9.7 Hz, CH), 3.12 (s, 6H, N(CH ₃ ) ₂ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 172.41, 167.57, 142.69, 133.92, 133.45, 132.39, 37.60, 34.27 ppm.

L. Sodium 5-(isopropoxycarbonyl)pyridine-2-thiolate 1-oxide (compound 10)

Yield 63%. Mass (El), m/z (Rel. int. (%)): 212.2466 [M] (53). Anal. Calcd. for C ₉ HioNNa0 ₃ S: (%) C, 45.95; H, 4.28; N, 5.95. Found (%) C, 45.87; H, 4.33; N, 6.04. 'H NMR (DMSO-de): 8.69 (s, 1H, CH), 8.16 (d, 1H, J= 9.7 Hz, CH), 7.49 (d, 1H, J = 9.7 Hz, CH), 4.93 (m, 1H, CH), 1.14 (d, 6H, J = 4.2 Hz, 2CH ₃ ) ppm. ¹³ C NMR (50 MHz, DMSO) 8 172.82, 164.50, 137.42, 135.99, 132.17, 125.75, 69.65, 17.98 ppm.

EXAMPLE 2 In vitro activity of the compounds of formula (I)

To determine the in vitro activity of compounds (MIC90) against Mycobacterium abscessus ATCC 19977, the resazurin reduction microplate assays (REMA) is performed. Briefly, serial 2-fold dilutions of drug are prepared in 96-well microtiter plates (Fluoronunc, Thermo Fisher, Waltham, MA, LISA) in 100 pL of Middlebrook 7H9 medium (Difco), supplemented with 10% OADC without addition of Tween 80. Then, M. abscessus log-phase cultures were diluted (ODeoo= 0.02) and added in the 96-well microtiter plate. Growth controls containing no compound and sterile controls without inoculum are also included. Also, a compound known to be active against M. abscessus growth is included as positive control. After 1 day of incubation at 37°C, 10 pL of resazurin (0.025% w/v) is added to each well, and bacterial viability is assessed after a further 24 h of incubation using a Fluoroskan™ Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA; excitation= 544 nm, emission= 590 nm). Bacterial viability was calculated as a percentage of resazurin turnover in the absence of compound (MIC90). is used as positive control. Results are expressed as the average of at least three independent replicates and presented on the following Table 1.

Table 1. In vitro activity of compounds (MIC90) against Mycobacterium abscessus

ATCC 19977.

By using the same procedure, the compound according to Example B (compound 2) was tested on broad spectrum of microorganisms and the corresponding results are presented in the below Table 2. Table 2. In vitro activity of compound 2 (MIC90) on broad spectrum of microorganisms.

Antifungal agar diffusion assays were performed as previously published [20], The well diffusion test was performed using Casitone agar. The inoculum used was prepared using the Candida or Aspergillus strains from a 24-hour culture on Sabouraud dextrose agar, a suspension was made in a sterile saline solution (0.85%). The turbidity of the suspension was adjusted with a spectrophotometer at 530 nm to obtain a final concentration to match that of a 0.5 McFarland standard (0.5- 2.5 x 10 ³ ). 20 mL of Bacto-casitone were melted, cooled to 55°C and then inoculated with 1 mL of the organism suspension. The inoculated agar was poured into the assay plate (9 cm in diameter) and allowed to cool down on a leveled surface. Once the medium had solidified, well 4 mm in diameter, were cut out of the agar, and 20 pL water solution of the tested compound was placed into well and incubated at 35°C for 24 hours. The end point of the diameters of the clear zone of inhibition of growth for the azoles, it was measured after 48 hours. A dramatic effect of tested compounds was observed (see below Table 3). Table 3

EXAMPLE 3 : Acute and subacute toxicity of compound 2

Acute and subacute toxicity of compound 2 was experimentally studied for intragastric administration in white mice. Test drug is white crystals soluble in water. The compound was solubilized in saline to be administrated to animals.

Acute toxicity

Experimental animals: 35 C57BL/6 male white mice, 7 weeks old, weight 20-22 g.

The following experimental procedure was used: administration route of the compound: per os; number of doses: 5;

- dosage: 100-200-250-300-500-600-750 mg/kg;

- volume introduced into 0.3 ml/20 g (15 ml/kg); data recorded: behavior, weight changes, rectal temperature, mortality.

Results

During 14 days observation period compound 2 in doses lower than 300 mg/kg did not cause changes in the general state and behavior of the mice, it did not affect motoric and reflex activity, active and calm cycles, grooming, food consumption, there were no cases of animal death.

LDie = 396.07

LD ₅ O = 455.78±21,79

LD ₈ 4 = 515.48

LDioo = 545.33

Subacute toxicity Evaluation of subacute toxicity of compound 2 observation time after 14 days per oral administration.

Experimental animals: 14 C57BL/6 male white mice, 7 weeks old, weight 20-22 g.

The following experimental procedure was used: - administration route of the compound : per os; dosage 50 mg/kg once a day for 14 days;

- volume introduced into 0.3 ml/20 g (15 ml/kg); data recorded: behavior, weight changes, rectal temperature, mortality.

Results. 14 days of treatment with the compound 2 in a dosage of 50 mg/kg did not cause changes in the general state and behavior of the mice, did not affect motoric and reflex activity, active and calm cycles, grooming, food consumption, there were no cases of animal death. No significant differences in the mass of mice of the experimental and control groups were found (see below Table 4).

Table 4 EXAMPLE 4 Pharmacokinetic study of compound 2

The pharmacokinetics of the compound 2 has been studied white Balb/C male mice.

27 animals with average body weight of 20 g were selected for the study. The animals were kept on standard vivarium ration with dry pelleted feed before the experiment. All the test animals had free access to water but were deprived of food for 1 hour before administration of the compound. This regimen was continued for another hour after the administration.

Compound in a dose of 100 mg/kg of body weight was administered as 0.5 mL of saline solution by intragastric intubation.

The animals were euthanized by decapitation for blood and brain sampling. Blood and brain samples (3 animals per time point) were taken at 15, 30, 60, 120, 240, 360 1440 min after administration for pharmacokinetic evaluation. Blood was collected in heparinized tubes and centrifuged at 3500 RPM. Plasma was separated from formed elements and immediately frozen at -20°C in freezer. This storage continued before transferring of plasma for analysis. Serum from 6 untreated animals was used as control and for equipment calibration with compound. Mice brain was immediately frozen at -120 °C.

Compound 2 has high bioavailability and its plasma concentration is shown in Figure 1.

EXAMPLE 5: Comparative stability test of the substance

(ethoxycarbonyl)pyridine-2-thiolate 1-oxide and sodium 5-

(ethoxycarbonyl)pyridine-2-thiolate 1-oxide

The aim of the study is to investigate the physical and chemical properties of a substances at elevated temperature and to establish an experimental expiration date for the substances (saline form vs base form).

Studied samples:

- 5-(ethoxycarbonyl )pyridine-2-thiolate 1-oxide (base form)

- sodium 5-(ethoxy carbonyl )pyridine-2 -thiolate 1-oxide (compound 2 - salt form) Conditions for the study:

Storage temperature: (55 ± 2)°C. Double layer polyethylene bag. The package is placed in a bag made of laminated aluminum foil. Each package is wrapped in wrapping paper.

Intervals substance samples were analyzed after 6, 12, 18, 23 days, which is equivalent to 6, 12, 18, 24 months when stored at 5±3 °C, respectively.

The results of the analyses are presented in Tables 5 and 6 reported in Figures 2 and

3.

According to the results of the stability study, using the accelerated aging method, we can conclude that:

- 5-(ethoxycarbonyl)pyridine-2 -thiolate 1 -oxide is not stable under short-term changes in storage conditions;

- sodium 5-(ethoxycarbonyl)pyridine-2 -thiolate 1 -oxide according of the invention is stable under short-term changes in storage conditions and experimental shelf life at a temperature of 20±3 °C is about 2 years.

Salts according to the present invention are stable solid compounds; this achievement makes them suitable for certain type of drug formulations such a nebulizer or aerosol formulation which is highly important for the treatment of lung diseases caused by Mycobacteria.

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