1-Cycloalkanecarbonyl-substituted thioureas and thiosemicarbazides as effective dihydrofolate reductase inhibitors with antibacterial activity

Aim. Search for new antibacterial agents with dihydrofolate reductase-inhibitory activity among N -(R-carbamothiol)cycloalkylcarboxamides using in silico and in vitro methodology, SAR analysis to optimize the synthesis of new potential antinicrobials. Methods. Molecular docking, in vitro DHFR inhibition assay, antimicrobial evaluation, SAR analysis, statistical methods. Results. According to the results of molecular docking to the active center of dihydrofolate reductase (DHFR), namely affinity, the main types of interactions and arrangement in the active center of the enzyme, several N -(R-carbamothioyl)cycloalkylcarboxamides were selected for their inhibitory effect. Based on in vitro screening, few promising compounds with high ability to inhibit DHFR were identified. It was found, that diacylsemicarbazides are more effective inhibitors of DHFR compared to acylthioureas. The studies on antibacterial activity have revealed several promising compounds, namely N -(2-R-hydrazine-1-carbonothioyl)cy-cloalkanecarboxamides, as highly active antimicrobial agents against E. сoli and St. aureus (MIC 3.125–25.0 μg/ml) with high DHFR-inhibitory effect, the activity of which competes with the comparison drug “Nitrofurazone”. This justifies the continuation of systematic research in this direction. Conclusions. A well-founded search among N -(R-carbamothiol)cycloalkyl-carboxamides for new antibacterial agents with dihydrofolate reductase-inhibitory activity, using in silico and in vitro methodology, established relationship between the chemical structure and activity aimed at further design of new potential drug agents.


Introduction
Pathogenic microorganisms are a danger to humanity since its inception, being the leading cause of morbidity and mortality. During the 20 th century, antibacterial drugs (ADs) have played an important role in the treatment of infectious diseases, however, in the last decade O. V. Kholodniak there has been an increase in the number of dangerous microorganisms resistant to ADs. The resistance to ADs could be natural or acquired caused by various mechanisms. The natural resistance is characterized by the absence of target microorganisms or inaccessibility of the target due to low permeability or enzymatic inactivation of drugs. In the presence of bacteria of natural resistance, ADs are clinically ineffective. The acquired stability is the property of individual strains of microorganisms to remain viable at the concentrations of ADs that suppress the bulk of the microbial population. The situations are possible when only a part of the microbial population shows acquired resistance. The appearance of acquired resistance in bacteria is not necessarily accompanied by a decrease in the clinical efficacy of the antibiotic. The formation of resistance in all cases is due to genetics: the acquisition of new genetic information or changes in the level of expression of their own genes. Overcoming such a problem is a difficult task. The causes of resistance to ADs include irresponsible or excessive use of drugs in the areas such as medicine, veterinary medici ne and agriculture. Moreover, the range of new ADs is constantly depleted, whereas the resistance of microorganisms increased. One of the mechanisms of ADs activity is the effect on folate metabolism, which was long ago recognized as an attractive chemotherapeutic target. Progress in understanding the biochemical basis of the mechanisms responsible for the enzyme selectivity and antiproliferative effects, the discovery of aminopterin and methotrexate, a detailed study of their metabolism, were the impetus for the introduction of a group of "classical" antifolates (Raltitrexed, Pralatrexate, Leucovorin, Pemetrexed, etc.) and "non-classical" antifolates (Trimetrexate, Piritrexim, Nolatrexed, Talotrexime, Trimethoprim, etc.) into medical practice [1][2][3][4][5][6][7]. These drugs are widely used as antitumor, antibacterial, antimalarial, antifungal agents, etc. [1][2][3][4][5][6][7].
Nowadays, more than 60 structures of antibacterial agents with DHFR inhibitory activity are known, but Trimethoprim is the "gold" standard for the treatment of urinary tract infections (UTIs) in women caused by E. coli, Proteus mirabilis, Klebsiella spp. and other microorganisms (Enterobacteriaceae and Staphylococcus saprophyticus) [8]. Interes tingly, the combination of trimethoprim and sulfamethoxazole ("Co-trimoxazole") is synergistic and is used to treat airways (bronchitis, pneumonia, pulmonary abscess, pulmonary abscess, empyema) meningitis, brain abscess, genitourinary system (pyelonephritis, urethritis, salpingitis, prostatitis), including gonorrhea, gastrointestinal (typhoid, paratyphoid, diarrhea), skin and soft tissues (pyoderma, furunculosis and others). However, the search for antibacterial agents has not been very successful in recent years, only a small number of effective antimicrobials with this mechanism of activity was introduced in the last 20 years. "Trimethoprim" was highly effective against E. coli, S. aureus, St. pneumonia [9] and against methicillin, trimethoprim-and vancomycin-resistant strains of S. aureus.
However, despite the proven effectiveness, confirmed mechanism of activity and identified biotargets, the design and search for antibacterial agents continue among various classes of compounds. We paid attention to functionalized acylthioureas and acylthiosemicarbazides, among which the compounds with antibacterial, fungicidal, antitumor and other activities were found [10].
The aim of the study is to search for new antibacterial agents with the dihydrofolate reductase-inhibitory activity among N-(Rcarbamothiol)cycloalkylcarboxamides, using in silico and in vitro methodology, to establish relationship between chemical structure and antimicrobial activity and to optimize the synthesis of new potential substances.
Molecular docking. The research was conducted by flexible molecular docking, as an approach of finding the molecules with affinity to a specific biological target. The macro-molecules from Protein Data Bank (PDB) were used as biological targets, namely DHFR (PDB ID -1RG7) (Protein Data Bank. http://www. rcsb.org/pdb/home/home.do. Accessed Marсh, 2022). The choice of biological targets was due to the literature about the mechanism of antibacterial drugs activity [14].
Ligand preparation. Substances were drawn using MarvinSketch 20.20.0 and saved in mol format (MarvinSketch version 20.20.0, ChemAxon http://www.chemaxon.com). After that they were optimized by program Chem3D, using molecular mechanical MM2 algorithm, and saved as pdb-files. Molecular mechanics were used to produce more realistic geometry values for most organic mo le cules, owing to the fact of being highly para me terized. Using AutoDockTools-1.5.6, the pdb files were converted into PDBQT, the number of active torsions was set as default [15].
Protein preparation.

Reagents
Dihydrofolate Reductase Assay Kit (Sigma-Aldrich, Catalog Number CS0340, Batch Number 067M4065V) was used for evaluation of DHFR-inhibitory activity of synthesized compounds. The protein content in supplied dihydrofolate reductase was 0.032 mg/ml and the activity of enzyme was 3.75 U/mgP.

The procedure of estimation of DHFRinhibitory activity of studied compounds
To the microcentrifuge tube (volume 2 ml) 966 µl of diluted 1:10 assay buffer we added. Then sequentially 13 µl of dihydrofolate reductase and 10 µl of 100 µM solution of studied compound in DMSO were added. The tube was sealed, intensively shaken, and the formed mixture was transferred to the 1.4 ml quartz cuvette. 6 µl of 10 mM solution of the NADPH were added to the formed mixture, the cuvette was sealed by parafilm and shaken. 5 µl of 10 mM solution of dihydrofolic acid were added to the formed mixture, the cuvette was sealed by parafilm, shaken, and immediately transferred to spectrophotometer ULab 108 UV. The absorption of sample at 340 nm was measured each 15 second[s] during 150 seconds. The activity of enzyme was calculated according to the following formula: Activity (Units/mg P) = = ∆OD/min sample -∆OD/min blank 12.3 × 0.013 × 0.032 where: ∆OD/min blank = ∆OD/min for the blank, from the spectrophotometer readings; ∆OD/min sample = ∆OD/min. for the reaction, from the spectrophotometer readings; 12.3 = extinction coefficient (ε, mM -1 *cm -1 ) for the DHFR reaction at 340 nm; 0.013 = enzyme volume in ml (the volume of enzyme used in the assay); 0.032 = enzyme concentration of the original sample.
The value of DHFR-inhibitory activity in % was calculated according to the formula: DHFR -inhibitory activity (%) = 3.75 -Activity (Units/mg P) × 100 % 3.75 Methotrexate was used as a reference compound.
Antimicrobial activity. The sensitivity of the microorganisms to the synthesized compounds was evaluated according to the described methods [16]. The assay was conducted on Mueller-Hinton agar by two-fold serial dilution of the compound in 1 ml. After this, 0.1 ml of microbial seeding (10 6 cells/ ml) was added. Minimal inhibition concentration of the compound was determined by the absence of visual growth in the test tube with a minimal concentration of the substance. Minimal bactericide/fungicide concentration was determined by the absence of growth on agar medium after inoculation of the microorganism from the transparent test-tubes. DMSO was used as a solvent, initial solution concentration was 1 mg/ml. For preliminary screening of the abovementioned standard the test cultures were used: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853. All test strains were received from bacteriological laboratory in Zaporizhzhia Regional Laboratory Center of State Sanitary and Epidemiological Service of Ukraine. Nitrofural ((E)-2-((5-nitrofuran-2-yl)methylene)hydrazine-1-carboxamide) was used as reference compound with proved antibacterial activity. Additional quality control of the culture media and solvents was conducted by commonly used methods [16].

Results and Discussion
Molecular docking was used as a tool to determine the affinity, the main types of interactions of N-(R-carbamothioyl)cycloalkylcarboxamides (1, 2) to the active site of DHFR and to predict the ability to inhibit it. The results of molecular docking of more than 50 compounds showed that compounds 1 and 2 do not have a high affinity for the enzyme, in this case lower than Methotrexate (Table 1). Noteworthy, the affinity for the enzyme in 20 selected compounds 1 and 2 is practically independent of the size of the cycloalkane fragment, and is mostly determined by the nature of the arylthiourea or aryl-(hetaryl-)thiosemicarbazide residue. A more detailed analysis showed that the compounds containing acyl residues with donor properties should be more effective DHFR inhibitors.
Subsequently, we determined a probable mechanism of activity of this class of compounds. To do this, we visualized the molecular docking of several compounds (compounds 2.2, 2.3, 2.6) and ligand (MT) to DHFR. Visualization of MT interaction in the active Note: * -data of compounds whose affinity is> -6.9 kcal/mol; # -MT -Methotrexate center of DHFR showed ( Visualization of the interactions of compound 2.3 showed that the replacement of the oxygen atom in the molecule (compound 2.2) by sulfur did not lead to other interactions (Fig. 2). These interactions occur due to hy- Notes: * -а -Conventional Hydrogen Bond; b -Carbon Hydrogen Bond, c -Pi-Donor Hydrogen Bond, d -Hydrophobic (π-σ); e -Hydrophobic (Alk); f -Hydrophobic (π-Alk); g -Hydrophobic (π-Orbitals); h -Hydrophobic (amide-π stacked); i -Hydrophobic (π-π T-shaped), j -Electrostatic, k -π-Sulfur Alk-interaction of the cyclopropane cycle with LEU A: 28 (5.00 Å) and ILE A: 50 (4.16 Å) ( Fig. 2.2). Thus, it is found that compounds 2.2, 2.3 and 2.6 in most cases have similar interactions with the amino acid residues as the classical inhibitor of MT and this leads to their close location in the active site of the enzyme. Undoubtedly, this is a clear argument for their further in vitro study of the ability to inhibit DHFR.
In vitro studies have shown ( Table 3) that  compounds 1.4, 1.6-1.9, 1.11 do not inhibit  DHFR, and compounds 1.1-1.3, 1.5, 1.10, 1.12 do not show a significant inhibitory effect on the enzyme (3.16-20.59 %). However, compounds 2.1-2.6 were found to be more active and inhibited DHFR by 28.34-90.32 %, except for N-(2-isonicotinoylhydrazine-1-carbothioyl) cyclopropanecarboxamide (2.7). The analysis of the structure-activity relationship has shown that diacylsemicarbazides (2) are more effective inhibitors of DHFR than acylthioureas (1). It is important that the modification of cyclopropanecarbonyl isothiocyanates with acylhydrazide moieties (2.1-2.6) has a positive effect on the enzyme inhibition and, importantly, in most cases is determined by the nature (donoracceptor properties) of acyl residues. Thus, the introduction of cyclopropane (2.1), phenoxy-(2.2), phenylthiol-(2.3) and furyl (2.6) fragments as electron donors leads to high acti vity (Table 3). However, the replacement of the above fragments by phenyl-(2.4), o-aminophenyl-(2.5) and isonicotinic-(2.7, 2.8) substituents as electron acceptors leads to a significant loss of the ability to inhibit DHFR. An increase in the size of the cycloalkyl moiety from cyclopropane to cyclohexane has a positive effect on the DHFR-inhibitory activity with the additional introduction of a "pharmacophore" furan cycle (2.6). Compound 2.6 inhibits the enzyme by 90.32 %, exceeding the reference compound "Methotrexate" (82.57 %). The disubstituted thioureas (1), regardless of the size of the cycloalkanecarboxamide moiety and the nature of the aryl group, are not effective DHFR inhibitors. The highest inhibitory activity among the compounds of this class is characteristic of N-[(4-(methylphenyl(carbamothioyl))]cyclopropanecarboxamide (2.2, 20.59 %), which contains a donor substituent in the thioureide group.
The antimicrobial activity was investigated for the compounds that had a high inhibitory effect on DHFR (Table 4). It was found, that N-substituted cycloalkanecarbonylthioureas (1.1-1.3, 1.5, 1.10, 1.12 An important issue of this study is to establish the relationship of antibacterial activity   (Tables 3,  4). It is true that compounds 2.1-2.6, which showed a high enzyme-inhibiting effect at the level of 45.77-90.32 %, inhibit bacterial growth with MIC of 3.125-100 μg/ml. Thus, the studies on the antibacterial activi ty revealed several promising compounds, namely N-(2-R-hydrazine-1-carbonothioyl) cycloalkanecarboxamides (2.1-2.6), as highly active antimicrobial agents against E. сoli and St. aureus (MIC 3.125-25.0 μg/ml) with the DHFR-inhibitory action, which justifies the continuation of systemic studies in this direction.

Conclusion
Using in silico and in vitro methodology, the well-founded search for new antibacterial agents with dihydrofolate reductase-inhibitory activity was performed among N-(R-carba mothiol)cycloalkylcarboxamides, the regularities between chemical structure and antimicrobial activity were established to optimize chemical synthesis. A few N-(R-carbamothioyl)cycloalkylcarboxamides were selected for their inhibitory effect, according to the results of molecular docking to the active center of dihydrofolate reductase (DHFR), namely affinity, the main types of interactions in the active center of the enzyme. Based on traditional in vitro screening, several promising compounds with high ability to inhibit DHFR have been identified. It was found, that diacylsemicarbazides are more effective inhibitors of DHFR compared to acylthioureas. The studies on antibacterial activity have revealed several promising compounds, namely N-(2-Rhydrazine-1-carbonotioyl)cycloalkanecarboxamides to be highly active antimicrobial agents against Es. coli and St. aureus (MIC 3.125-25.0 μg/ml) with high DHFR-inhibitory activi ty, which competes with the compared drug "Nitrofurazone". This justifies the continuation of systematic research in this direction.