DESCRIPTION

The present invention relates to the pharmaceutical/pharmacological field, and especially to the use of new antifungal agents, and specifically of agents having selective inhibition activity of the growth of fungi/yeasts belonging to the Malassezia pachydermatis strain, for topical veterinary and human use, so as to obviate the resistance problems towards the current first-choice drugs.

Antimicrobial resistance is one of the greatest threats to global health, because the etiological agent (e.g., bacteria, fungi, protozoa and viruses) of a wide range of infections no longer adequately responds to first-choice pharmacological treatment. This makes infections more difficult to treat, increases the risk of disease eradication and spread, while resulting in a Darwinian selection of strains of microorganisms with multiple resistances. In particular, some fungi can easily cause severe infectious states, or even death in subjects with significant immune system impairment.

The present invention relates to compounds containing the carbamoselenoyl and/or sulfamoyl function, usable as selective antifungal agents against Malassezia pachydermatis strains for topical veterinary and human use, having the following general structures:

Formula I Formula II

Formula III where

X= C,N,O,S,Se or a Metal (M)

R= H, SO ₂ NHR, Alkyl, Alkylaryl, Halogen, Alkoxy, NO2, NNPR, Piperonyl, Naphthyl

R ₂ = H, alkyl, aryl, heteroaryl. In particular, the following compounds have been identified and tested for each of the three structure formulas:

Formula I

R= 3-Br, 5-OCF3 X= H A-l-01

R= H X= 2-F A-l-02

R= H X= 2, 3, 5, 6 F A-l-03

R= H X= 2, 3, 4, 5, 6 F A-l-04

R= H X= 4-SO ₂ NH ₂ A-l-05

R= 3-Br, 5-OCF3 X= 4-SO ₂ NH ₂ A-l-06

R= 4-Heptyl X= 4-SO ₂ NH ₂ A-l-07

R= 4-NO ₂ X= 4-SO ₂ NH ₂ A-l-08

R= 4-F X= 4-SO ₂ NH ₂ A-l-09

R= 4-1 X= 4-SO ₂ NH ₂ A-l-10

R= 2-F, 4Br X= 4-SO ₂ NH ₂ A-l-11

R= 4-CH ₂ CI X= 4-SO ₂ NH ₂ A-l-12

R= 4-NNC ₆ H ₅ X= 4-SO ₂ NH ₂ A-l-13

R=H X= 3-SO ₂ NH ₂ A-l-14

R= 3-Br, 5-OCF3 X= 3-SO ₂ NH ₂ A-l-15

R= 4-Heptyl X= 3-SO ₂ NH ₂ A-l-16 R= 4-F X= 3-SO ₂ NH ₂ A-l-17

R= 4-1 X= 3-SO ₂ NH ₂ A-l-18

R= 4-CI X= 3-SO ₂ NH ₂ A-l-19

R= 2-F, 4Br X= 3-SO ₂ NH ₂ A-l-20

R= H X= 2-OH; 3-SO ₂ NH ₂ A-l-21

R= 4-Heptyl X= 2-OH; 3-SO ₂ NH ₂ A-l-22

R= 4-NO ₂ X= 2-OH; 3-SO ₂ NH ₂ A-l-23

R= 4-1 X= 2-OH; 3-SO ₂ NH ₂ A-l-24

R= 4-CI X= 2-OH; 3-SO ₂ NH ₂ A-l-25

R= R= 2-F, 4Br X= 2-OH; 3-SO ₂ NH ₂ A-l-26

R= 4-CH ₂ CI X= 2-OH; 3-SO ₂ NH ₂ A-l-27

R= 2-OCOCH3 X= 4-SO ₂ NH ₂ A-l-28

R= Piperonyl X= 4-SO ₂ NH ₂ A-l-29

R= H X= H A-l-30

R= 2-OCOCH3 X= 2-OH; 3-SO ₂ NH ₂ A-l-31

R= Piperonyl X= 4-SO ₂ NH ₂ A-l-32

R= 3-Br, 5-OCF3 X= 2-OH; 3-SO ₂ NH ₂ A-l-33 Formula II According to the invention, such compounds are designed to act through a dual and concomitant action which includes: a) inhibition of the metal enzyme Carbonic Anhydrase (AC; EC 4.2.1.1 ) expressed in Malassezia pachydermatis; b) the function as a carrier of the element selenium in organic form which is known to very effectively interfere with the metabolism and biosynthesis of lipid components of the target fungal organisms.

As for item a), eukaryotic cells, fungi, algae bacteria and protozoa express ACs belonging to the classes a- 0- y-, 6- q, 0- and i. These differ from one another for structural, kinetic and expression and/or distribution features in each microorganism (1 -3 and references thereof). Substantial differences can be found between enzymatic isoforms of ACs belonging to the same class and expressed in the different organisms/cells (1 -3 and references thereof). Such enzymes reversibly catalyze the hydration reaction of CO2 and are thus essential to ensure the body/cell adequate support of biologically usable carbon (HCO3 ) and protons. The reduction in the enzymatic activity of the AC expressed in Malassezia pachydermatis through specific inhibitory compounds has important repercussions on the metabolism of the pathogen up to inducing the cell death thereof (selective anti-infective/antifungal effect).

As for item b), the use of inorganic selenium (e.g., SeS2) for topical use in a maximum concentration of 1% is currently attributable to the use as an antifungal agent in the treatment of dandruff and seborrheic dermatitis of the scalp caused by fungi of the genus Malassezia spp. The use of the element Se, inserted in organic molecular structures, make it more usable by fungal microorganisms with consequent greater impact on cellular metabolism (e.g., impairment of ROS homeostasis, biosynthesis of biomembrane lipids, etc.) and induction of cell death (antifungal effect) (4, 5 and references contained therein).

BACKGROUND ART The phenomenon of antimicrobial resistance is a serious and concrete threat to the favorable outcome of drug treatment for a wide range of infections the etiological agent of which (e.g., bacteria, fungi, protozoa and viruses) no longer adequately responds to drugs. This makes infections more difficult to treat, increases the risk of disease eradication and spread, while resulting in a Darwinian selection of strains of microorganisms with multiple resistance.

In the context of the present invention:

The current first-line antifungal drug treatment comprises the use in monotherapy or mutual combination regimen of:

1 ) 14-a-demethylase enzyme inhibitors; Ketoconazole, Miconazole, Clotrimazole, Econazole, Itraconazole, Fluconazole, Posaconazole

2) Inhibitors of the enzyme 1 .3-[3-glucan synthase: cilofungin and caspofungin

3) Squalene epoxidase inhibitors: Terbinafine

4) Microtubule assembly inhibitors: Thiabendazole

5) Membrane lipid disruptors: Nystatin

6) Chitin synthesis inhibitors: Nikkomycin Z

7) Mixed mechanisms: Metronidazole SeS

However, the inappropriate, prolonged large-scale and/or incorrectly regimented use of the current drugs causes the onset of drug resistance phenomena by pathogenic organisms. Such phenomena are mainly manifested through mutations of the biological targets of interest and/or the activation of efflux and/or metabolic mechanisms. The onset of drug resistance can occur towards a single and/or various drugs with severe impairment of therapeutic efficacy and frequent increase in side effects.

The compounds l-lll of the invention are suggested as the first antifungal agents with dual action since they incorporate in a single chemical structure functional groups such as carbamoselenoyl and/or sulfamoyl, capable of specifically inhibiting the AC expressed in Malassezia pachydermatis and further acting as organic carriers of the element selenium.

Therefore, the resulting pharmacological effect is due to the concomitant action of: i) inhibition of the AC enzyme target of Malassezia pachydermatis not yet validated at the clinical veterinary/human level in the topical and/or systemic treatment of infections sustained from the aforesaid pathogen; ii) irreversible impairment of the cellular metabolism of the aforesaid pathogenic organism due to preferential intake of organo-selenated molecules with respect to the eukaryotic cells of the host.

The procedure for the insertion of selenium as well as the synthesis and structure of compounds with general formula I and II is prior art, as well as the use of some AC isoforms of mammals, parasites (genus Leishmania) and bacteria ( Vibrio cholerae) as inhibitors.

The antifungal application is described in the publication to Vullo D et al., Bioorg Med Chem 2017, in which researchers investigated the use of another class of AC inhibitors, namely dithiocarbamates for the treatment of Malassezia globosa infections. Such a paper describes the use of a wide range of compounds, however, not attributable to formulas l-lll: a) in relation to the inhibitory activity against the hydration reaction of CO ₂ catalyzed by the AC expressed by the fungus Malassezia globosa with respect to human AC isoforms; b) in relation to the MIC (minimum inhibitory concentration) against three different species of Malassezia (of both human and veterinary interest) and some Gram-positive and Gram-negative bacterial strains. Furthermore, on the best candidates, selected based on antimicrobial activity, cytotoxicity tests were performed on MDBK cells (bovine kidney), applying the MTT cell viability assessment assay.

OBJECT OF THE INVENTION

It is the task of the present invention to provide antifungal drugs with dual and concomitant action, with a profile of therapeutic efficacy and pathogen vs host selectivity which is highly superior as compared to those currently in use; this will result in a significant reduction in both the therapeutic dose and the induction of drug resistance phenomena.

DETAILED DESCRIPTION

The compounds of structure l-lll of the present invention consist of:

1 ) A component carrying a functional group which specifically interacts with the active catalytic site and/or accessory/allosteric sites of the AC expressed by Malassezia pachydermatis, so as to inhibit the hydration activity of CO ₂ and/or reactions catalyzed by it;

2) A component carrying the element selenium in organic form.

The functional groups of 1 and 2 are known in the literature and comprise, respectively: i) primary and/or substituted sulfonamides, carboxylic and boronic acids, amines, alcohols, phenols, selenols, carbamates and/or derivatives thereof including those obtained by isosteric substitution of elements and/or whole functional groups. ii) selenoureas and/or organo-selenated derivatives thereof.

According to the invention, the compounds of the present patent application consist of the components of 1 and 2 which form part of the same chemical structure in stoichiometric relationship to each other, and which are obtained through the synthetic method of Koketsu et al. for structures I and II [4], Some compounds contained in the present invention are previously reported in the literature by certain authors as inhibitors of isoforms of a-AC expressed by mammals, [3-AC by L. donovani chagasi and a-AC by Vibrio cholerae and thus potentially useful for treating infectious states and/or diseases relatable thereto [5-9].

The compounds belonging to the series l - lll were tested in vitro through the stopped-flow technique to determine the inhibitory activity thereof against the hydration reaction of CO ₂ catalyzed both by the AC expressed by the fungus of the genus Malassezia globosa (MgAC) and by the human isoforms a-AC I and II, taken as an off-target reference (Annex 2). Such a technique includes measuring the pH change rate of a solution containing the enzyme isoform of interest, incubated at room temperature with scalar amounts of ligand for a sufficient time to form the enzymesubstrate complex. The enzymes used are either commercially available recombinant (AC I and II) or obtained in house (MgAC).

Based on the data shown in the following Table I:

Table I : Stopped-flow kinetic data of reference series l-lll and AAZ compounds on hCA

* Average of 3 different dosages, with a stopped-flow technique (errors were in the range ± 5-10% of reported values).

**Data are expressed in pM

The table shows that: the compounds A-l-01 , A-l-02, A-l-03, A-l-04, A-l-30 and A-lll-08 belonging to classes I and III not containing the sulfonamide group are inactive against human ACs and from Malassezia globosa (K|S >100000 nM).

The remaining derivatives belonging to classes I - III generally have inhibitory potencies against superior human ACs with respect to MgAC. The only exceptions are A-l-35, A-l-38 and A-l-39 having favorable selectivity profiles for MgACs.

ANTIMICROBIAL ACTIVITY AND CYTOTOXICITY REPORT

Compounds series l-lll (T able 2)

The compounds of the series l-lll have been evaluated for the anti- infective activity thereof on both fungal and bacterial reference strains. The antifungal activities were evaluated against three different species of Malassezia spp.: two of human interest, Malassezia globosa ATCC MYA 4612 and Malassezia furfur ATCC 14521 , and one of veterinary interest, Malassezia pachydermatis DSMZ 6172. The antibacterial activities were evaluated against a Gram-negative strain, E coll ATCC 25922, and three Gram-positive strains, Staphylococcus aureus ATCC 25923, MRSA ATCC 43300, and Staphylococcus pseudintermedius ATCC 21284.

The evaluation of the Minimum Inhibitory Concentration (MIC) against each of the tested strains was performed following the guidelines suggested by the CLSI (CLSI, 2018b) using the appropriate culture media, in particular Mueller Hinton for the bacteria. RPMI for M. pachydermatis and modified RPMI for M. globosa and M. furfur. The antimicrobial activity of each compound was evaluated in triplicate.

On the best candidates selected based on antimicrobial activity, cytotoxicity tests were then performed on MDBK cells (bovine kidney), applying the MTT cell viability assessment assay.

ANTIMICROBIAL ACTIVITY

An overall assessment of the antimicrobial activity of all the molecules tested shows excellent inhibitory activity against fungal strains, where the MIC values are between 0.5 and 64 pg/ml for Malassezia pachydermatis. while for Malassezia furfur they are in a range between 0.5 and 16 pg/ml.

Globally, the concentrations needed to achieve an inhibition of the growth of Malassezia globosa are on average higher, in particular some compounds showed little or no inhibitory activity (>256 pg/ml) while the best candidates showed a MIC between 4.3 and 64 pg/ml.

The difference detected in antifungal activity, in particular the poor activity demonstrated towards M. globosa, could be attributable to the different constitution of the lipidome in the three fungal species. As reported in the literature (Ramirez et al. 2020), the species M. furfur and M. pachydermatis are quite similar in lipodome composition, while M. globosa mainly differs in the cholesterol and triglycerides content. Other differences relate to the content of FAHFA and PE, practically absent in M. globosa and present but in low concentration in the other two species under consideration.

As for the antibacterial activity, the tested compounds were globally active against Gram-positive bacteria. In particular, good or excellent antimicrobial activity was detected against Staphylococcus aureus ATCC 25923, MRSA ATCC 43300 and Staphylococcus pseudintermedius ATCC 21284, where the best candidates showed MIC values in the range 0.5 and 21 .3 pg/mL.

It should be noted that no inhibitory activity was detected against the Gram-negative strain tested (MIC > 256 pg/ml), configuring a particularly apparent selectivity of action against fungal strains and for some molecules also towards the Gram-positive bacteria for all the molecules tested.

The action specificities related to the class of belonging are listed below.

CLASS I COMPOUNDS:

The series I compounds lacking sulfonamide (A-l-01 , A-l-02, A-l-03, A-l-04 and A-l-30) showed action selectivity for Malassezia pachydermatis (MIC 0.8-6 pg/ml) and furfur (MIC 0.8-11 pg/ml), while they showed limited activity against M. globosa (MIC > 64 pg/ml) with the exception of A-l-30 which showed an MIC of 29.3 pg/ml.

A MIC value > 256 pg/ml was detected towards the tested bacterial strains (E. co// and Staph, aureus), with the exception of A-l-04 on Staph, aureus ATCC 25923 (MIC = 32 pg/ml).

Among the series I compounds, the best candidates (A-l-07, A-l-09, A-l-10, A-l-11 , A-l-15, A-l-16, A-l-21 , A-l-22, A-l-23, A-l-25, A-l-31 , A-l-32, A-l-33, A-l-34, A-l-36, A-l-38) showed excellent activity on M. pachydermatis (MIC 0.5-2 pc/ml) and M. furfur (MIC 0.5-10.7 pc/ml). Lower activity was instead detected for M. globosa, with MIC values in the range between 64 and >256 pg/ml for the compounds A-l-05 and A-l-30, while for the compound A-l-33 the MIC value detected against M. globosa is 64 pg/ml.

Compared to Gram-positive bacterial strains, the best candidates were the molecules referred to as A-l-15, A-l-22 and A-l-33, with MICs between 1 and 8 pg/ml, while no activity was detected against the Gramnegative strain E. coll ATCC 25922 (MIC > 256 pg/ml).

There is therefore an action selectivity against the fungal strains, in particular towards M. pachydermatis and M. furfur.

The molecules A-l-15, A-l-22 and A-l-33 are exceptions, which also show activity against Gram-positive bacteria. CLASS II COMPOUNDS:

The molecules A-ll-01 , A-ll-02, 03, A-ll-04, A-ll-05, A-ll-06, A-ll-07 and A-ll-08 belong to this class.

The best candidates are the molecules A-ll-01 , A-ll-04 and A-ll-05. They show excellent antifungal activity against Malassezia pachydermatis (MIC 1 ng/ml) and M. furfur (MIC 3.1 - 4 pg/ml) and good activity against M. globosa (MIC 32- 128 pg/ml).

No activity was detected against the Gram-negative steipe E. coli ATCC 25922 and Gram-positive Staph, aureus ATCC 25923 (MIC > 256 pg/ml).

These data therefore show an action selectivity against the fungal strains tested, including M. globosa.

CLASS III COMPOUNDS:

The molecules A-lll-01 , A-lll-02, A-lll-03, A-lll-04, A-lll-05, A-lll-06, A-lll-07, A-lll-09, A-lll-10, A-lll-11 belong to this class.

The best candidates are the molecules A-lll-03, A-lll-04, A-lll-05, A- III-06, A-lll-07, A-lll-09, A-lll-10 and A-lll-11 .

They show excellent antifungal activity against Malassezia pachydermatis (MIC 0.5 - 1 pg/ml) and M. furfur (MIC O 5-.3 pg/ml), while lower activity was detected against M. globosa (MiC 64- 256 pg/ml), with the exception of A-lll-09 and A-lll-10, for which MIC in the range from 4.3 to 9 pg/ml were detected./

No activity was detected against the Gram-negative strain E. coli ATCC 25922.

For Staph, aureus ATCC 25923 only two molecules, A-lll-06 and A- III-09. showed good activity (MIC 16 pg/ml), while the other candidates showed higher MIC values, more specifically between 21.3 and > 256 pg/ml

A-lll-09 and A-lll-06 also showed good activity against MRSA ATCC 43300 and S. pseudintermedius ATCC 21264. In this case, the MIC values ranged from 0.5 to 21 .3 pg/ml.

Again for this class of compounds there is a greater action selectivity against fungal strains, in particular towards M. pachydermatis and M. furfur.

CLASS III COMPOUNDS WITHOUT SULFONAMIDE:

Out of this class, only one molecule was tested. A-lll-08. It shows good antifungal activity in particular against M. furfur (MIC 1.9 pg/mll) and M. globose (MIC 24 pg/ml), while limited activity was detected against M. pachydermatis (MIC 64 pg/ml).

No activity was detected against the Gram-negative strain E. coll ATCC 25922 and Gram-positive Staph, aureus ATCC 25923 (MIC > 256 pg/ml), limited activity against S. pseudintermedius ATCC 21284 (MIC 85.3 pg/ml), while good activity was detected against MRSA ATCC 43300 (MIC 21.3 gg/ml).

On the best candidates selected based on antimicrobial activity, cytotoxicity tests were then performed on MDBK cells (bovine kidney), applying the MTT cell viability assessment assay.

CYTOTOXICITY:

Cytotoxicity tests were performed on MDBK bovine kidney cells and using the MTT cell viability assessment assay. Six replicates were performed per single experiment and negative controls with and without 1% DMSO were repeated for each single plate used

* The result marked with * is distorted due to an intense blue/purple coloration of the molecule. Upon observation without optical microscope staining, the cells of these wells are detached and not viable.

We considered as the best candidates the molecules that showed values of cell survival to the MTT test over 70% at a concentration equal to or greater than the MIC value of that same molecule. For this reason, in the experimental tests the molecules were not all tested at the same concentration, but two specific concentrations were tested for each molecule based on the results of the microbiological activity.

Generally, we found that cell survival was dose-dependent, with a higher survival rate at lower concentrations with respect to the higher concentrations among those tested. Below, we only report the values related to the best candidates of the different classes:

CLASS I COMPOUNDS WITHOUT SULFONAMIDE:

The best candidate is molecule A-l-01 , for which a survival of 88% at the concentrations of interest was detected, from 16 to 1 pg/ml on MDBK cells.

CLASS I COMPOUNDS:

Globally, excellent survival rates of more than 77% were detected at the lowest concentrations tested (from 2 pg/ml to 0.5 pg/ml), with the exception of molecule A-l-21 , which showed slightly higher toxicity, detecting survival rates of about 50% of the cell population at 1 pg/ml.

Globally, the best candidates in this class are A-l-09, A-l-15, A-l-16, A-l-25, A-l-33, A-l-36 and A-l-38. Specifically, the excellent tolerability of the molecule A-l-15, where a survival of 99% of the cell population at the concentration of 8 pg/ml was detected, and of the molecule A-l-36, where a survival of 76% of the cell population at the concentration of 256 pg/ml was detected, should be highlighted.

CLASS II COMPOUNDS:

Of this class, the molecules A-ll-01 , A-ll-04, A-ll-05, and A-ll-07 were tested. The best cell survival data were related to the molecule A-ll- 04, for which a survival of 64% of the cell population was detected at the concentration of 128 pg/ml and 99% at the concentration of 1 pg/ml. 76% of the cell population of the molecule A-ll-07 was found to survive at the concentration of 256 pg/ml.

CLASS III COMPOUNDS:

Of this class, the molecules A-lll-03, A-lll-04, A-lll-05, A-lll-06, A-lll- 07, A-lll-09 and A-lll-10 were tested.

The best cell survival data are for the molecules A-lll-05 (98% survival at 2 pg/ml), A-lll-07 (74% survival at 64 pg/ml) and A-lll-09 (91 .5% survival at 0.5 pg/ml).

The only molecule tested of class III without sulfonamide (A-lll-08) resulted in limited cell survival (22% at the concentration 8 pg/ml).

CONCLUSIONS

From the data obtained on the entire library of molecules tested, a marked selectivity of action against the fungal strains of M. pachydermatis and M. furfur is apparent. Such a selectivity is most apparent in the molecules belonging to classes I and III.

Alongside the excellent antifungal activity, the good results obtained in terms of antibacterial activity by the compounds A-l-15, A-l-22, A-lll-09 and A-l-33 and A-lll-06 should also be emphasized. However, the latter molecule is hardly tolerable by the cell population, having acceptable values only at extremely low concentrations (1 pg/ml).

Globally, the best results were obtained by comparing the data obtained from the antimicrobial activity and cytotoxicity tests with the molecules: class I without sulfonamide: A-l-01 class I: A-l-09, A-l-15, A-l-22, A-l-25, A-l-33, A-l-36 and A-l-38. class II: A-ll-05 A-ll-07, class III: A-lll-05, A-lll-07 and A-lll-09.

Among these, the three best candidates globally are the molecules A-lll-05 and A-lll-09 (class III) and A-l-38 (class I).

In conclusion, the kinetic data of in vitro inhibition, antimicrobial activities and cytotoxicity support the thesis that the compounds of the present application and belonging to classes l-lll act as antifungals with dual concomitant action of: i) inhibition of the AC enzyme target not currently validated at the clinical veterinary and human level for topical and/or systemic use for treating infections sustained from microorganisms of the genus Malassezia, and specifically Malassezia pachydermatis; ii) irreversible impairment of the cellular metabolism of pathogenic organisms due to the preferential intake of organo-selenated molecules by the latter with respect to the eukaryotic cells of the host.

Such an approach allows: i) validating an innovative therapeutic target (e.g., AC of Malassezia pachydermatis) never previously suggested and therefore alternative to the known biological targets and object of the current therapeutic protocols; ii) thus limiting the drug resistance phenomenon; iii) combining the new biological target (e.g., AC of Malassezia pachydermatis) with a broad-spectrum, antifungal mechanism of action induced by selenium and suggested here in the organic form most biologically usable by pathogenic organisms.

MATERIALS AND METHODS

1 Introduction

The compounds of the series l-lll were evaluated for the anti- infective activity thereof on both fungal and bacterial reference strains. The antifungal activities were evaluated against three different species of Malassezia spp.; two of human interest, Malassezia globosa ATCC MYA 4612 and Malassezia furfur ATCC 14521 , and one of veterinary interest, Malassezia pachydermatis DSMZ 6172. The antibacterial activities were evaluated against a Gram-negative strain, E coli ATCC 25922, and three Gram-positive strains, Staphylococcus aureus ATCC 25923, MRSA ATCC 43300, and Staphylococcus pseudintermedius ATCC 21284.

The evaluation of the Minimum Inhibitory Concentration (MIC) against each of the tested strains was performed following the guidelines suggested by the CLSI (CLSI, 2018b) using the appropriate culture media, in particular Mueller Hinton for the bacteria, RPMI for M. pachydermatis and modified RPMI for M. globosa and M. furfur. The antimicrobial activity of each compound was evaluated in triplicate.

On the best candidates selected based on antimicrobial activity, cytotoxicity tests were then performed on MDBK cells (bovine kidney), applying the MTT cell viability assessment assay and the evaluation of hemolytic activity, on sheep red blood cells.

1.1 Powders The powders of the compounds of the present patent were provided by UNIFI. In particular, the antimicrobial activities of the following compounds were evaluated:

1.2 Reference bacterial strains used in the experimental tests

E. coll ATCC 25922

Staphylococcus aureus ATCC 25923

MRSA ATCC 43300

Staphylococcus pseudintermedius ATCC 21284

1.3 Reference fungal strains used in the experimental tests

Malassezia globosa ATCC MYA 4612

Malassezia furfur ATCC 14521

Malassezia pachydermatis DSMZ 6172

2 Dilutions of powders of the different compounds

The Minimum Inhibitory Concentration (MIC) assays were carried out following the guidelines of the CLSI (CLSI, 2018b) with some minor changes. The powders of the different compounds of the patent were diluted to the stock concentration of 25.6 mg/ml in DMSO.

3 Evaluation of the minimum inhibitory concentration

The dilutions based on two of the stock solution, in the range 25.6- 0.05 mg/ml, were performed in 96-well microtiter plates (Greiner, Milan, Italy) in DMSO solvent. In another microtiter plate, 49 pl of the culture medium were placed in each of the 96 wells, and 1 microliter of each dilution of the compounds of the present patent was then added. The test was completed with the addition of 50 microliters of bacterial or fungal suspension containing 10 ⁶ CFU/ml, to obtain a final concentration of 5- 10 ⁵ CFU/ml. The range of final dilutions tested was therefore between 256-0.5 pg/ml. Growth and sterility controls were also prepared for each strain and for each tested compound.

The plates were incubated for 24/48 h (for the bacteria and the fungi, respectively) at 37°C under aerobic conditions for all bacteria and M. pachydermatis and M. furfur, at 33°C instead for M. globosa. The minimum inhibitory concentration is the lowest concentration of the tested compound at which no visible growth of bacteria or fungi was observed. Each assay was performed in triplicate.

3.1 Preparation of the inoculum

Four or five morphologically similar colonies from fresh cultures were inoculated in Mueller Hinton broth (MHB) culture medium at 37°/33°C (respectively as specified above) under stirring at 240 rpm for 24 hours for the bacterial strains and in RPMI for 48 hours for the fungal strains until the logarithmic growth phase was reached. The bacterial or fungal suspension was then centrifuged at 2000 rpm for 20 minutes at 4°C. The pellet thus obtained was resuspended in 10 mM pH 7 phosphate buffer (PB).

The concentration of the bacterial suspension was adjusted to obtain an optical density (OD) in the range 0.08 - 0.13 at 600 nm in a cuvette of 1 cm optical path, approximately equivalent to a bacterial concentration of 10 ⁸ CFU/ml, evaluated with the Biophotometer plus spectrophotometer (Eppendorf, Hamburg, Germany) (A = 600 nm). The bacterial suspensions thus obtained were further diluted to 1 :100 in sterile MHB.

For the fungal suspension, so as to obtain a concentration of 10 ⁶ CFU/ml of fungal cells, the turbidity of the suspension was evaluated in comparison with McFarland's 0.5 standard (CLSI, 2018b).

Fifty microliters of the bacterial or fungal suspension containing 10 ⁶ CFU/ml were then inoculated in each well, to obtain a final concentration of 5x10 ⁵ CFU/ml.

All the microbiological assays were performed within 30 minutes of the standardization of the bacterial or fungal inoculum.

4 - Evaluation of the minimum fungicidal concentration (MFC)

The minimum fungicidal concentration (MFC) was assessed following the guidelines of the CLSI (CLSI, 2018b). From the microtiter plates in which the MIC assay was performed, after 48 hours incubation, 20 pl of the suspension was taken from each well and transferred to a plate of Sabouraud agar medium and incubated for 48 hours at 37°/33°C under aerobic conditions. After incubation, for each concentration tested, the colonies were counted and the growth inhibition percentage was calculated. For each fungal strain and each compound, sterility and growth controls were also performed. The minimum fungicidal concentration is defined as the lowest concentration of antimicrobial agent capable of killing 99.9% of the initial fungal inoculum (Pfaller et al, 2004). If the MIC and MFC values are different, and the MIC value < MFC, the tested compound was classified as fungostatic, while if the MIC value > MFC, the tested compound was classified as fungicidal. Three replicates were performed for each test.

5 Cytotoxicity assay

One microliter of the compounds of the patent, dissolved in DMSO at different concentrations, was added to each well of a 96-well plate containing 100 pl of MDBK cells in DMEM medium. The plates were incubated for 24 hours at 37°C in the presence of 5% CO2. After incubation, 10 pl of the MTT reagent (3-(4,5-dimethylthiazol-2-yl)- 2,5diphenyltetrazolium bromide) was added into each well of the assay and incubated again at 37°C for 6 hours. At the end of the incubation period, 110 pl of the lysing solution (10% SDS in 0.01 M HCI) was added to each well and then incubated overnight. The MTT compound, a yellow tetrazolium salt, is reduced by the mitochondrial enzymes (succinate dehydrogenase) of metabolically active cells into insoluble crystals of formazan. In the presence of metabolically active cells, after the addition of the lysing solution which allows the release of formazan from the cells, a violet color is apparent in the medium. However, in the presence of non- viable cells, the MTT is not reduced to formazan and therefore the solution remains yellow. After incubation, the plates were read on the 1 spectrophotometer at a wavelength of 620 nm. The positive and negative controls were also prepared for each plate and for each assay, three replicates were carried out in two independent experiments (Donofrio, et al 2008).

6 Hemolysis assay

50 microliters of defibrinated sheep blood were placed in a 96-well U-bottom sterile plate, then contacted with 49 microliters of sterile saline + 1 pl of each concentration of the different compounds of the patent, and incubated for 24 hours at room temperature. Positive (100% lysis, with hemoglobin release) and negative (0% lysis) controls were also prepared with sterile water and sterile saline solution added with 1 % sterile DMSO. After incubation, the plate was centrifuged at 1400 rpm for 15 min and, after transferring the supernatant to a sterile plate, the hemolysis values were calculated by spectrophotometer reading at 450 nm. The hemolysis percentages were calculated as follows: 100 where A _com p represents the optical density of the sample at 450 nm, Ape the optical density of the positive control and ANC the optical density of the negative control. For each assay, three replicates were performed.

Advantages and improvements over the prior art

The type l-lll compounds of the present invention are suggested as dual-acting anti-infective drugs, with a largely higher efficacy and selectivity profile than those currently in use. This results in a significant reduction in the effective dose and a decrease in the induction of drug resistance phenomena with a consequent improvement in the safety profile. e. Data proving the inventive step of the invention

The compounds l-lll showed on the panel of considered microorganisms (e.g., Malassezia pachydermatis, Malassezia furfur, Malassezia globosa, E. coil, S. aureus. MRSA, S. pseudintermedius, high action efficacy against specific fungal and/or bacterial strains with MIC values in the submicromolar range, associated with poor cytotoxicity assessed on eukaryotic MDBK cells.

In general, the compounds I - III of the present invention have far superior therapeutic efficacy and selectivity profiles as compared to the drugs currently in use.

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