Synthesis, Biological and In Silico Studies of Griseofulvin and Usnic Acid Sulfonamide Derivatives as Fungal, Bacterial and Human Carbonic Anhydrase Inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho-/physiological conditions. A series of griseofulvin and usnic acid sulfonamides were synthesized and tested as possible CA inhibitors. Since β- and γ- classes are expressed in microorganisms in addition to the α- class, showing substantial structural differences to the human isoforms they are also interesting as new antiinfective targets with a different mechanism of action for fighting the emerging problem of extensive drug resistance afflicting most countries worldwide. Griseofulvin and usnic acid sulfonamides were synthesized using methods of organic chemistry. Their inhibitory activity, assessed against the cytosolic human isoforms hCA I and hCA II, the transmembrane hCA IX as well as β- and γ-CAs from different bacterial and fungal strains, was evaluated by a stopped-flow CO2 hydrase assay. Several of the investigated derivatives showed interesting inhibition activity towards the cytosolic associate isoforms hCA I and hCA II, as well as the three γ-CAs and Malassezia globosa (MgCA) enzyme. Six compounds (1b–1d, 1h, 1i and 1j) were more potent than AAZ against hCA I while five (1d, 1h, 1i, 1j and 4a) showed better activity than AAZ against the hCA II isoform. Moreover, all compounds appeared to be very potent against MgCA with a Ki lower than that of the reference drug. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds within the active site of human CAs.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of metalloenzymes implicated in pH buffering of extra-and intracellular spaces by catalyzing the reversible hydration of carbon dioxide (a cellular waste product) to bicarbonate and a proton [1][2][3][4]. This family of enzymes to date, is divided in eight independent gene families (i.e., α, β, γ, δ, ζ, η, θ and ι-classes) Among the 15 known humans (h) CA isoforms belonging to class α, the cytosolic isoforms hCA I and hCA II are omnipresent in the body and represent the targets for anticonvulsant, diuretic, and anti-glaucoma drugs. On the other hand, the transmembrane isoforms hCA IX and XII are linked with some types of cancers as they are overexpressed by tumor hypoxia, and have become a good target for anti-cancer drug design. Moreover, a sulfonamide derivative called SLC-0111, a selective CA IX/XII inhibitor is in Phase II clinical trials for the treatment of primary tumors/metastases, which overexpress these enzymes [5].

Preliminary Docking Studies
Computational methods are an important part of the drug design process and they are presently used to get a deeper understanding of the drug-enzyme interactions. Previously, we published an article [16,17] on the antimicrobial activity of new derivatives of griseofulvin and usnic acid. According to PASS prediction [45] these derivatives showed carbonic anhydrise inhibitory activity with Pa up to 0.646 and 0.572 for antibacterial and antifungal activities, respectively. On the other hand, as already mentioned above, the sulfonamide fragment is part of a large number of antibiotic drugs, as well as in the composition of many molecules that are carbonic anhydrase inhibitors [40][41][42][43]. The idea of choosing derivatives of griseofulvin and usnic acid containing sulfonamide fragments is based on the so-called search on hybrid molecules containing simultaneously modified natural backbone and sulfonamide moiety with potential properties as specific carbonic anhydrase inhibitors and antibiotic targets. Consequently, herein we present the synthesis of griseofulvin and usnic acid sulfonamide derivatives (1a-j,4a,b) and the evaluation of their inhibitory activity against three human CAs (I, II, IX) as well as β and γ CA from different bacterial and one fungal strains.

Preliminary Docking Studies
Computational methods are an important part of the drug design process and they are presently used to get a deeper understanding of the drug-enzyme interactions. Molecular docking studies were performed in a series of designed griseofulvin and usinc aisid derivatives bearing sulfamoyl moiety. As presented in Table S1 from all compounds only the ones highlighted in red exhibited good to excellent binding energy and were selected for further studies.

Prediction of Toxicity
Predicting the toxicity of a compound is a critical step in the development of new drug candidates, making in silico toxicity studies a faster and cheaper procedure than in vivo animal toxicity testing or in vitro testing in cell lines. It also helps significantly reduce the number of animals used in experimental assays. There are several online programs that access toxicities that use in silico models to predict mean lethal dose, carcinogenicity, mutagenicity, and more.
The Pro-Tox II web server [46] predicts the mean lethal dose (LD50) in rodents. According to this program, all compounds can be classified into six GHS (Globally Harmonized System of Classification and Labeling of Chemicals) Categories [47] according to their toxicity and LD50 value.
Toxicity classes are defined according to the globally harmonized system of classification of labeling of chemicals (GHS). LD50 values are given in [mg/kg]: The griseofulvin derivatives 1e,f was synthesized by condensation of griseofulvic acid 2 with the corresponding amine. Excess triethyl orthoformate was used as solven wherein the final products were obtained in 47-51% yields (Scheme 2). The griseofulvin amide derivatives 1g-j were synthesized from corresponding es ters 3. Starting griseofulvic esters 3 were prepared via a multicomponent reaction of grise ofulvic acid, aldehydes, and Meldrum's acid using a previously described protocol [22] The obtained products were synthesized in 68-86% yields (Scheme 3). Note that starting compounds 3 and target amides 1g-j exist as a mixture of diastereomers. Scheme 1. Synthesis of compounds 1a-d.
The griseofulvin derivatives 1e,f was synthesized by condensation of griseofulvic acid 2 with the corresponding amine. Excess triethyl orthoformate was used as solvent wherein the final products were obtained in 47-51% yields (Scheme 2).

Chemistry
Griseofulvin derivatives 1a-d were obtained by the reaction of griseofulvic acid 2 and corresponding amine in refluxing acetic acid. The starting material griseofulvic acid 2 was synthesized from griseofulvin by known procedures [48]. This method allows one to prepare the target products 1a-d in 42-66% yields (Scheme 1).

Scheme 1. Synthesis of compounds 1a-d.
The griseofulvin derivatives 1e,f was synthesized by condensation of griseofulvic acid 2 with the corresponding amine. Excess triethyl orthoformate was used as solven wherein the final products were obtained in 47-51% yields (Scheme 2). The griseofulvin amide derivatives 1g-j were synthesized from corresponding es ters 3. Starting griseofulvic esters 3 were prepared via a multicomponent reaction of grise ofulvic acid, aldehydes, and Meldrum's acid using a previously described protocol [22] The obtained products were synthesized in 68-86% yields (Scheme 3). Note that starting compounds 3 and target amides 1g-j exist as a mixture of diastereomers. The griseofulvin amide derivatives 1g-j were synthesized from corresponding esters 3. Starting griseofulvic esters 3 were prepared via a multicomponent reaction of griseofulvic acid, aldehydes, and Meldrum's acid using a previously described protocol [22]. The obtained products were synthesized in 68-86% yields (Scheme 3). Note that starting compounds 3 and target amides 1g-j exist as a mixture of diastereomers.
The usnic acid derivatives 4a,b were obtained by the previously described method from (R)-usnic acid 5 and appropriate amines in refluxing ethanol [17]. The yields of synthesized products were 54-67% (Scheme 4).
The structure of all compounds (Table 1) was characterized by 1 H-NMR, 13 C-NMR, and elemental analysis and presented in the experimental part. The usnic acid derivatives 4a,b were obtained by the previously described method from (R)-usnic acid 5 and appropriate amines in refluxing ethanol [17]. The yields of synthesized products were 54-67% (Scheme 4). The structure of all compounds (Table 1) was characterized by 1 H-NMR, 13 C-NMR, and elemental analysis and presented in the experimental part. The usnic acid derivatives 4a,b were obtained by the previously described from (R)-usnic acid 5 and appropriate amines in refluxing ethanol [17]. The y synthesized products were 54-67% (Scheme 4). The structure of all compounds (Table 1) was characterized by 1 H-NMR, 13 C and elemental analysis and presented in the experimental part. The usnic acid derivatives 4a,b were obtained by the previously described method from (R)-usnic acid 5 and appropriate amines in refluxing ethanol [17]. The yields of synthesized products were 54-67% (Scheme 4). The structure of all compounds (Table 1) was characterized by 1 H-NMR, 13 C-NMR, and elemental analysis and presented in the experimental part.
The usnic acid derivatives 4a,b were obtained by the previously described method from (R)-usnic acid 5 and appropriate amines in refluxing ethanol [17]. The yields of synthesized products were 54-67% (Scheme 4). The structure of all compounds (Table 1) was characterized by 1 H-NMR, 13 C-NMR, and elemental analysis and presented in the experimental part.

Evaluation of CA Inhibitory Activity
All compounds 1a-j, 4a,b were evaluated for their inhibitory activity against three human CA isoforms, namely: hCA I, hCA II, hCA IX, and CAs from some bacterial and fungal pathogenic species, such as Malassezia globosa (β-CA from a fungus involved in dandruff formation [49]), MgCA, as well as βand γ-CAs from the following bacteria: Porphyromonas gingivalis (PgiCA); Streptococcus mutans (SmuCA); Burkholderia pseudomallei (BpsCA) and Colwellia psychrerythraea, a non-pathogenic Antarctic bacterium (CpsCA) [50][51][52][53][54]. The results are shown in Table 2. The evaluation revealed that all compounds exhibited carbonic anhydrase inhibitory activity against all isoforms tested, but with different ranges of inhibition constants. For example, in the case of hCA I the K i value of tested compounds arethe in range from 15.3 to 2986 nM, while for hCA II and hCA IX the Ki ranged from 4.9 to 4052 nm and 22.5 to 709 nm, respectively. According to the data in Table 2 it is obvious that tested compounds are more potent against the hCA I isoform, with seven out of twelve compounds being more active inhibitors than acetazolamide, used as a reference drug. The compound's order of activity can be presented as follows: The highest activity was achieved by compound 1i with K i value of 15.3 nM compared with AAZ (K i = 250 nM). The lowest activity was observed for compound 1g with a K i value of 2986 nM.
As far as the inhibitory activity of compounds against the hCAII isoform is concerned five compounds (1d, 1h, 1i, 1j, and 4a) appeared to be more potent than the reference drug. According to the activity order of compounds against hCA II which is: demonstrating compound 1d is the most active with K i 4.9 nM and a selectivity index (SI) towards hCA I and hCA IX of 5.88 and 6.00 respectively. On the other hand, derivative 1g showed the lowest activity with a K i of 4052 nM. It should be mentioned that the most selective derivative among all compounds tested was 4a with a SI towards hCA I and hCA IX of 43.9 and 7.96, respectively.
Regarding the inhibition activity against the hCA IX isoform, in general, all compounds showing moderate to low activity in the following order: . Thus, the most potent appeared to be compound 1j with K i of 22.5 nM, followed by 1i (K i of 23.3 nM), while the lowest activity was observed for compound 1g with K i of 709.6 nM. The comparison of the activity towards three human CA isoforms revealed that compounds 1d, 1i, and 1j were among the most active against all isoforms, whereas compound 1g showed the lowest activity in all cases. Table 2. Inhibition data of human CA isoforms I, II, IX, and β, γ from some bacterial and fungal strains with titled compounds and AAZ by a stopped-flow CO 2 hydrase assay. The study of structure-activity relationships revealed that the presence of (4-(methylthio)phenyl)-N-(4-sulfamoylphenethyl)propionamide (1i) in position 3 of griseofulvin seems to be beneficial for hCA I inhibitory activity.
Regarding the structure-activity relationships in the case of the hCA II isoform, despite the first four the most active compounds, as already mentioned being the same, the influence of substituent's is different. Thus, the presence of 4-amino-3-hydroxybenzenesulfonamide in position 4 of the main core of compound (1d) was favorable for hCA II inhibitory activity, while the introduction of (4-(methylthio)phenyl)-N-(4-sulfamoylphenethyl)propionamide to position 3 of griseofulvin core led to compound 1i, which exhibited the lowest activity among the four (1d, 1h, 1i, 1j) most active ones. The presence of N-(2-oxo-2-((4sulfamoylphenyl)amino)ethyl)propionamide (1j) showed the same influence on activity as in the case of hCA I, being the second active one, while replacement of 4-methylthiophenyl group by 4-hydroxy-3-methoxyphenyl (1g) in position 3 of griseofulvin moiety was detrimental as in case of hCA I.
From all mentioned above it can be concluded that the same substitution at position 3 of the main core of compounds (1 S,6 R)-7-chloro-2 -hydroxy-4,6-dimethoxy-6 -methyl-3 -(4-(methylthio)benzyl)-3H-spiro[benzofuran-2,1 -cyclohex [2]ene]-3,4 -dione play different role dependent on hCA isoform. Furthermore, we investigated the activity of our compounds towards three beta (PgiCAβ, SmuCA, and MgCA) and three gamma CAs (BpsCAγ, PgiCAγ, CspCAγ) from different microorganisms. It was found that the compounds showed inhibitory activity against all the bacterial CAs examined but to carrying extents. Only in the case of PgiCAγ, CspCAγ and St.mutans some compounds excited the activity of reference drug. Thus, compound 1a exhibited excellent activity against PgiCAγ with K i at 9.1 nM and SI 8.5 towards PgiCAβ being 3.5 fold more active than acetazolamide. Three compounds, 1e, 1g and 1j were found to be more potent than the reference drug against CpsCAγ. Among them, the highest activity was achieved for compound 1j with K i at 176.8 nM compared to acetazolamide (K i of 502 nM). This compound appeared to be selective towards PgiCAγ, S. mutans, and BpsCAγ with SI 13, 6.9, and 4, respectively. Finally, one compound 1b (K i at 293.7 nM) was the only which exceeded the activity of the reference drug against S. mutans (K i of 344 nM and SI 3 towards all other bacterial strains. In addition, our compounds showed very good activity in the case of the fungal isoform from Malassezia globosa (MgCA), being all twelve compounds more potent than acetazolamide with K i in the range of 475.6-4346 nM compared to reference drug (K i of 40,000 nM). The order of activity can be presented as: 1b > 1a > 1c > 1d > 1e > 1i > 1j > 4b > 1g > 4a > 1f > 1h, with the best activity exhibiting by compound 1b with K i at 475.6 nM followed by compound 1a (K i of 544 nM). The less potent appeared to be compound 1h with Ki at 4346 nM. The comparison of activity towards MgCA with hCA isoforms revealed that among the most active compounds, only one is common (1d). Besides, compound 1h is between the four most active compounds in the case of hCA isoforms ranking from first to fourth place in the activity order, whereas in the case of MgCA it is the less active one.
According to structure-activity relationship studies the presence of the 4-aminobenzensulfonamide group in the main core (1b) is beneficial for Mg CA inhibitory activity, while its replacement by 4-(aminomethyl)benzenesulfonamide decreased activity leading to compound 1c. The introduction of 4-amino-3-hydroxybenzenesulfonamide resulted in a less active compound (1d) compared to the previous one (3), while the presence of N-(4-sulfamoylbenzyl)acetamide (1h) substituent played a negative role to activity.
Thus, according to obtained results, it is obvious that substituents and their position play an important role in CA inhibitory activity, human and microbial.

Molecular Docking Studies in Human CAs Isoforms
As representatives of the whole set of compounds, 1c, 1g, 1f, and 1k were chosen for docking studies in order to predict possible inhibition mechanisms.
All human CAs isoforms contain conserved residues His94, His96, and His119 in their active sites. In this way, these residues act as zinc ligands. In addition, all isoforms have two additional conserved residues at the active site, Thr199 and Glu105, that serve as 'gatekeepers' [55][56][57]. Nevertheless, these isoforms differ mainly between their middle and exit residues in the active site cavity.
Molecular docking results for the tested compounds on hCA I, II, and IX isoforms are shown in Table 3. Based on these results, all compounds chelating the Zn (II) ion are anions (negative nitrogen of the sulfonamide group) that bind the enzymes in the same manner [57]. Docking results reveal that the selectivity profiles and inhibition modes of some compounds depend on variations in enzyme active sites. The nature of the amino acids in the enzyme's active site as well as the substitution of each compound determines the conformation that compounds adopt within the active site of the enzyme and how they interact with it.
Taking all these into account, comparing the docking poses in the hCA II enzyme of compounds 4a and 4b with Ki values for the hCA II enzyme of 8.4nM and 755.3 nM respectively, we can say that the para substitution of sulphonamide group in compound 4a plays an important role in the inhibition profile of this compound compared to compound 4b. hCA II enzyme has a hydrophobic residue Phe131 in the active site that provides a bulky environment for the compound to freely enter the active site. Compound 4a having a para substitution of sulphonamide group provides a more line structure that can have the flexibility and enables it to avoid the steric hindrance of the bulky residue Phe131 hCA II isoform, increasing the inhibition potency ( Figure 2).
As it is illustrated in Figure 2 this compound (4a) inserts to the active site of the enzyme freely, the negative nitrogen of the sulphonamide group chelates the Zn (II) ion and forms hydrogen bonds. Moreover, the oxygen atom of the sulphonamide group forms a hydrogen bond with residue Thr199 (distance 1.73). Furthermore, the benzene moiety is interacting hydrophobically with residues Val121, Leu131, Thr200, and Leu198. These interactions further stabilize the complex and explain its high inhibition potency (Figure 2A).
On the other hand, compound 4b probably because of the presence of meta substitution in the benzene ring cannot fully enter the active site of the enzyme and reach the Zn ion to chelate resulting in its low inhibition potency ( Figure 2B,C).
in the enzyme's active site as well as the substitution of each compound determines the conformation that compounds adopt within the active site of the enzyme and how they interact with it.
Taking all these into account, comparing the docking poses in the hCA II enzyme of compounds 4a and 4b with Ki values for the hCA II enzyme of 8.4nM and 755.3 nM respectively, we can say that the para substitution of sulphonamide group in compound 4a plays an important role in the inhibition profile of this compound compared to compound 4b. hCA II enzyme has a hydrophobic residue Phe131 in the active site that provides a bulky environment for the compound to freely enter the active site. Compound 4a having a para substitution of sulphonamide group provides a more line structure that can have the flexibility and enables it to avoid the steric hindrance of the bulky residue Phe131 hCA II isoform, increasing the inhibition potency ( Figure 2).
As it is illustrated in Figure 2 this compound (4a) inserts to the active site of the enzyme freely, the negative nitrogen of the sulphonamide group chelates the Zn (II) ion and forms hydrogen bonds. Moreover, the oxygen atom of the sulphonamide group forms a hydrogen bond with residue Thr199 (distance 1.73). Furthermore, the benzene moiety is interacting hydrophobically with residues Val121, Leu131, Thr200, and Leu198. These interactions further stabilize the complex and explain its high inhibition potency (Figure 2A).
On the other hand, compound 4b probably because of the presence of meta substitution in the benzene ring cannot fully enter the active site of the enzyme and reach the Zn ion to chelate resulting in its low inhibition potency ( Figure 2B,C). The flexible structure of compound 4i can also explain its inhibition potency towards hCA I, hCA II, and hCA IX enzymes with Ki values of 15.3, 8.1, and 23.3 nM respectively. Indeed, the superposition of this compound bound to hCA I in comparison to hCA II and hCA IX (Figure 3) shows that it can adopt a conformation that favors the interactions with all active sites of the isoforms, avoiding the steric hindrance of the bulky residue Phe131 of hCA II isoform and increasing the stability of each complex and subsequently the inhibition potency of the compound. Indeed, the superposition of this compound bound to hCA I in comparison to hCA II and hCA IX (Figure 3) shows that it can adopt a conformation that favors the interactions with all active sites of the isoforms, avoiding the steric hindrance of the bulky residue Phe131 of hCA II isoform and increasing the stability of each complex and subsequently the inhibition potency of the compound.
In particular, in all isoform structures, the negative nitrogen of the sulphonamide group chelates the Zn (II) ion and forms hydrogen bonds ( Figure 3A). In all isoforms, the one oxygen atom of the sulphonamide group forms a hydrogen bond with residue Thr199. Furthermore, in isoform hCA I, the Cl atom of the benzene ring is forming a halogen bond with residue His64. Additionally, the benzene ring is interacting hydrophobically with Val121 and Leu198 ( Figure 3A). In isoform hCA IX there are except the aforementioned H-bond with residue Thr199, another three hydrogen bonds formed with residues His119, Gln67, and Gln92. These interactions can probably explain the high ki value of compound 1i against all isoforms and especially its superiority over AAZ in isoform hCA IX. The same conclusion can be made and for compounds 1h and 1j with a similar structure to compound 1i. group chelates the Zn (II) ion and forms hydrogen bonds ( Figure 3A). In all isoforms, one oxygen atom of the sulphonamide group forms a hydrogen bond with resi Thr199. Furthermore, in isoform hCA I, the Cl atom of the benzene ring is formin halogen bond with residue His64. Additionally, the benzene ring is interacting hyd phobically with Val121 and Leu198 ( Figure 3A). In isoform hCA IX there are except aforementioned H-bond with residue Thr199, another three hydrogen bonds form with residues His119, Gln67, and Gln92. These interactions can probably explain high ki value of compound 1i against all isoforms and especially its superiority o AAZ in isoform hCA IX. The same conclusion can be made and for compounds 1h 1j with a similar structure to compound 1i.

Drug Likeneess
All tested compounds were evaluated for their Drug-likeness and bioavailability scores and the results of the prediction are shown in Table 4. According to prediction, the bioavailability score of most of the compounds was about 0.55 except for compounds 1g, 1h, 1j, 4a and 4b with 0.17 values Only five compounds showed 2 violations of Lipinski's rule of five and in combination with their excellent Drug-likeness scores ranging from −0.13 to 1.24, it can be concluded that they have good oral bioavailability (Figure 4).

CA Inhibition Assay
An applied photophysics stopped-flow instrument was used for assaying the CAcatalyzed CO 2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) for α-class as a buffer, 20 mM TRIS (pH 8.3) for βand γ-class as a buffer, and 20 mM Na 2 SO 4 (for maintaining constant ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. The non-catalyzed CO 2 hydration was not subtracted from these curves and accounts for the remaining observed activity even at a high concentration of inhibitor, being in the range of 16-25%. However, the background activity from the uncatalyzed reaction is always subtracted when IC 50 values are obtained by using the data analysis software for the stopped-flow instrument. Enzyme concentrations ranged between 5 and 10 nM. For each inhibitor, at least six traces of the initial 5-10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the inhibitor (0.1 mM) were prepared in distilled-deionized water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant proteins obtained in-house, as reported earlier [58][59][60] and their concentrations in the assay system were in the range of 6.2-14.8 nM.

Molecular Docking Studies
Molecular modeling studies were performed using the software AutoDock 4.2 (The Scripps Research Institute, La Jolla, CA, USA) [61]. Protein Data Bank was also used in order to obtain the he crystal structures ofhCA I (PDB code 3W6H) and hCA II (PDB code 3HS4) cytosolic isoforms as well as hCA IX (PDB code 3IAI) transmembrane tumor-associated isoform [62]. All the procedure was carried out as in our previous works [41,42].

Drug Likeness
The study was performed as described in our previous paper [41].

Conclusions
In conclusion, we synthetized and investigated a novel griseofulvin and usnic acid sulfonamides for their effective inhibition against three relevant human carbonic anhydrase isoforms, such as the ubiquitous hCA I and hCA II isoforms and the tumor-associated isoforms hCA IX, which are involved in many diseases such as glaucoma, retinitis pigmentosa, epilepsy, and tumors. Furthermore, the inhibitory activity against βand γ-CAs from three different bacterial and one fungal strain was evaluated. Six compounds (1b-1d, 1h, 1i and 1j) were more potent than AAZ against hCA I while five (1d, 1h, 1i, 1j and 4a) showed better activity than AAZ against the hCA II isoform. It should be mentioned that compound 1d demonstrated the best activity with Ki 4.9 nm and selectivity index (SI) towards hCA I and hCA IX isoforms 5.88 and 6.00 respectively. The investigation of CA inhibitory activity against bacterial stains (Burkholderia pseudomallei-BpsCAγ, Porphyromonas gingivalis (PgiCAβ and PgiCAγ), Colwellia psychrerythraea (CpsCA) as well as Streptococcus mutans, Malassezia globosa) revealed that only in case of PgiCAγ, CspCAγ and St. mutans some compounds excited the activity of reference drug Moreover, all compounds appeared to be very potent against the MgCA with a Ki lower than that of the reference drug. The comparison of activity towards MgCA with hCA isoforms revealed that among the most active compounds, only one is common (1d. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds against hCA isoforms, and the obtained results were in agreement with the experimental data.