3-Amino-5-(indol-3-yl)methylene-4-oxo-2-thioxothiazolidine Derivatives as Antimicrobial Agents: Synthesis, Computational and Biological Evaluation

Herein we report the design, synthesis, computational, and experimental evaluation of the antimicrobial activity of fourteen new 3-amino-5-(indol-3-yl) methylene-4-oxo-2-thioxothiazolidine derivatives. The structures were designed, and their antimicrobial activity and toxicity were predicted in silico. All synthesized compounds exhibited antibacterial activity against eight Gram-positive and Gram-negative bacteria. Their activity exceeded those of ampicillin and (for the majority of compounds) streptomycin. The most sensitive bacterium was S. aureus (American Type Culture Collection ATCC 6538), while L. monocytogenes (NCTC 7973) was the most resistant. The best antibacterial activity was observed for compound 5d (Z)-N-(5-((1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-hydroxybenzamide (Minimal inhibitory concentration, MIC at 37.9–113.8 μM, and Minimal bactericidal concentration MBC at 57.8–118.3 μM). Three most active compounds 5d, 5g, and 5k being evaluated against three resistant strains, Methicillin resistant Staphilococcus aureus (MRSA), P. aeruginosa, and E. coli, were more potent against MRSA than ampicillin (MIC at 248–372 μM, MBC at 372–1240 μM). At the same time, streptomycin (MIC at 43–172 μM, MBC at 86–344 μM) did not show bactericidal activity at all. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain. Antifungal activity of all compounds exceeded those of the reference antifungal agents bifonazole (MIC at 480–640 μM, and MFC at 640–800 μM) and ketoconazole (MIC 285–475 μM and MFC 380–950 μM). The best activity was exhibited by compound 5g. The most sensitive fungal was T. viride (IAM 5061), while A. fumigatus (human isolate) was the most resistant. Low cytotoxicity against HEK-293 human embryonic kidney cell line and reasonable selectivity indices were shown for the most active compounds 5d, 5g, 5k, 7c using thiazolyl blue tetrazolium bromide MTT assay. The docking studies indicated a probable involvement of E. coli Mur B inhibition in the antibacterial action, while CYP51 inhibition is likely responsible for the antifungal activity of the tested compounds.


Introduction
Infectious diseases affect large populations and cause significant morbidity and mortality [1]. They represent a global indirect load on public health security and an impact on socio-economic stability worldwide. Bacterial, fungal, and viral infections have monopolized the dominant factors of death and disability of millions of humans for centuries. They are presently plaguing and even ravaging populations worldwide each year with performances far surpassing wars [2].
It should be mentioned that several dozen new infections have grown and affected the health of billions of people over the world, mainly in developing countries [3]. Unfortunately, there are no successful pharmaceuticals or vaccines for many of these new infections [3].
The treatment of infectious disease is still an imperative and demanding problem due to the growing number of multi-drug resistant pathogens, especially Gram-positive bacteria. Due to this, the lack of effective antimicrobial drugs, morbidity, and mortality notably increased [4].
Drug resistance causes vast human suffering, and now it is one of the most significant challenges of the twenty-first century. Species such as the methicillin-resistant S. aureus and vancomycin-resistant enterococci have emerged due to the irrational or overuse of antimicrobial agents [5].
The pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. which also called ESCAPE pathogens, are of particular importance since they play a significant role affecting several human organs including the lung and urinary system. Besides, they exhibited increased resistance to clinically used antibiotics [6].
Numerous of these pathogens are Gram-negative bacteria, which are of specific concern due to their resistance of up to 50% against carbapenems that have been reported in some developing countries [6]. Despite the availability of some new antibiotics against Gram-positive pathogens, no treatment of these pathogens with a new class of compounds has been introduced in the last 40 years. Therefore, to overcome the resistance, the discovery of safer and more effective antimicrobial agents with a different mechanism of action is still an urgent need [7].
The interest in thiazolidine-based compounds attracted the attention of medicinal chemists, and a plethora of them have been studied to evaluate pharmacological properties [8][9][10]. Despite the appearance of some controversial opinions regarding the analysis of the molecular mechanism of their action, prominent representatives among the developed drug-like molecules are thiazolidinone derivatives [11,12] since they are a valuable source of building blocks for the development of novel molecules [13][14][15].
The combination of the thiazolidinone ring with other pharmacologically promising heterocycles has been a warranted approach for developing new "drug-like" molecules with the desired activity profile [29][30][31]. Our previous studies showed that thiazolidinone core with indole fragment in one molecule gave the compounds with high antimicrobial activity [19].
On the other hand, indole derivatives represent another scaffold widely spread in nature with a broad spectrum of biological activities.
The indole ring was found not only in natural compounds but also in diverse semisynthetic and synthetic drug-like molecules [32,33]. They exhibit antimicrobial [34][35][36][37][38][39], anti-inflammatory [40,41], COX inhibitory [42,43] anticancer [44][45][46], antiviral [47,48], anti-HIV [49,50], and antidiabetic [51] activities. Among the natural compounds containing the indolene fragment, several imidazoline and imidazolidine alkaloids are known, which have a wide spectrum of biological activity, including antibacterial. Thus, indole-containing azahydantoins 1-6 from sponges and streptomycetes have a potent antibacterial and antiseptic action ( Figure 1) [52][53][54]:  It is also known that synthetic thiohydantoin (rhodanine) analogs 7, 8 ( Figure 2), exhibit pronounced antibacterial properties [55]. Therefore, the design and development of hybrid molecules combining thiazolidinone and indole cores in the same structure is a promising approach. Taking into account all issues mentioned above and encouraging results obtained in our earlier studies [19], in this paper, we present the synthesis and biological evaluation of new (1H-indole-3-yl-methylene)-4-oxo-2-thioxothiazolidin derivatives with potent antimicrobial activity.  It is also known that synthetic thiohydantoin (rhodanine) analogs 7, 8 ( Figure 2), exhibit pronounced antibacterial properties [55].  It is also known that synthetic thiohydantoin (rhodanine) analogs 7, 8 ( Figure 2), exhibit pronounced antibacterial properties [55]. Therefore, the design and development of hybrid molecules combining thiazolidinone and indole cores in the same structure is a promising approach. Taking into account all issues mentioned above and encouraging results obtained in our earlier studies [19], in this paper, we present the synthesis and biological evaluation of new (1H-indole-3-yl-methylene)-4-oxo-2-thioxothiazolidin derivatives with potent antimicrobial activity. Therefore, the design and development of hybrid molecules combining thiazolidinone and indole cores in the same structure is a promising approach. Taking into account all issues mentioned above and encouraging results obtained in our earlier studies [19], in this paper, we present the synthesis and biological evaluation of new (1H-indole-3-yl-methylene)-4-oxo-2-thioxothiazolidin derivatives with potent antimicrobial activity.

Antibacterial Activity
Using AntiBac-Pred [56] one of the predictive web services of Way2Drug platform [57], activity against at least one strain of bacteria was predicted for each of the fourteen designed Pharmaceuticals 2020, 13, 229 4 of 24 compounds with Pa-Pi values in the range from 0.001 to 0.309. According to the prediction results, the highest probability of antibacterial activity against the Bacillus subtilis subsp. subtilis str. 168 was estimated for derivatives 7a and 5b (Pa-Pi values are 0.309 and 0.305, respectively).
Similarly, we estimated in silico the probability of antibacterial activity for the reference drugs streptomycin and ampicillin. For both reference drugs, wide antibacterial action was predicted. For the top-10 predictions of streptomycin Pa-Pi values vary from 0.905 to 0.947; for ampicillin-from 0.712 to 0.989. Contrary, for relatively new antibacterial agent trifolirhizin, which structure was disclosed only on July 7, 2020 (Clarivate Analytics Integrity [58]), the top-10 predictions Pa-Pi values vary from 0.369 to 0.552.

Antifungal Activity
Using web service AntiFun-Pred [59], activity against at least one of the fungal species was predicted for six of the fourteen studied compounds with Pa-Pi values ranging from 0.001 to 0.112. The results show that among the studied compounds, derivatives 5a (Pa-Pi against Trichophyton mentagrophytes equals 0.112) and 7a (Pa-Pi against Candida equals 0.101) have better chances to be found active in biological evaluation of the antifungal activity.
The results of in silico antimicrobial activity assessment are given in the supplementary file PASSweb_results_13mols.xlsx. Small Pa-Pi values reflect the novelty of the analyzed compounds compared to those included in the PASS training set.
Similarly, for the reference drug ketoconazole wide antifungal action was predicted with Pa-Pi values in the range 0.622-0.812 (top-10 predictions), while for the new antifungal agent drimenin disclosed on 12 June 2020 (Clarivate Analytics Integrity [58]), only two antifungal activity were predicted with Pa-Pi values 0.007 and 0.030.

Acute Rat Toxicity
Using web service based on GUSAR software [60,61], acute rat toxicity with regards to different administration routes was estimated for the studied compounds. LD50 values and toxicity classes are given in Table 1. Most of the predictions indicate that the studied compounds belong to the fifth or fourth rodent toxicity classes.

Chemistry
The starting N-(4-oxo-2-thioxothiazolidin-3-yl) -carbamides 3a-d was prepared by reacting the acid hydrazides 1a-d with trithiocarbonyl diglycolic acid (Scheme 1). The reaction was carried out in a medium of boiling aqueous alcohol. The yield of the products was 83-97%.

Chemistry
The starting N-(4-oxo-2-thioxothiazolidin-3-yl) -carbamides 3a-d was prepared by reacting the acid hydrazides 1a-d with trithiocarbonyl diglycolic acid (Scheme 1). The reaction was carried out in a medium of boiling aqueous alcohol. The yield of the products was 83-97%.  All compounds were characterized by IR, 1 H and 13 C NMR spectroscopy. In the IR spectra of compounds 3а-d, 5а-k, and 7а, 7с, the carbonyl group of the 4-thiazolidone ring absorbs at 1753.21-

Chemistry
The starting N-(4-oxo-2-thioxothiazolidin-3-yl) -carbamides 3a-d was prepared by reacting the acid hydrazides 1a-d with trithiocarbonyl diglycolic acid (Scheme 1). The reaction was carried out in a medium of boiling aqueous alcohol. The yield of the products was 83-97%.  All compounds were characterized by IR, 1 H and 13 C NMR spectroscopy. In the IR spectra of compounds 3а-d, 5а-k, and 7а, 7с, the carbonyl group of the 4-thiazolidone ring absorbs at 1753.21- All compounds were characterized by IR, 1 H and 13 C NMR spectroscopy. In the IR spectra of compounds 3a-d, 5a-k, and 7a, 7c, the carbonyl group of the 4-thiazolidone ring absorbs at 1753.21-1690.53 cm −1 , and the thiocarbonyl group-at 1608.56-1556.48 cm −1 . The absorption band of the carbonyl group of the amide fragment of 3a-d and 5a-k is located at 1689.56-1654.84 cm −1 .
In the starting 3-substituted 2-thione-4-thiazolidones, the amide proton NH-CO of the compounds 3a-d appears as a singlet in the range 11.95-10.91 ppm, and the cyclic methylene group resonates as a singlet or quartet at 4.55-4.48 ppm. etc. In the target products 5a-k, the amide proton is in the range of 11.85-11.12 ppm. The 5-methylidene proton CH = of compounds 5a-k and 7a, 7c resonates in the form of a singlet at 8.20-7.94 ppm, which, according to the literature [9,62], is characteristic of the Z isomer. The singlet NH of the protons of the indole ring appeared in the range 12.31-12.06 ppm.

Antibacterial Activity
Compounds 5a-k and 7a-c were evaluated for antibacterial activity, by microdilution method to determine the minimal bacteriostatic and bactericidal concentrations. As reference compounds, we used ampicillin and streptomycin, which are both broad-spectrum antibiotics commonly applied to treat different conditions. Antibacterial activity of tested compounds is shown in Table 2 with MIC values in the range of 36.5-211.5 µM and MBC at 73.3-282.0 µM. According to the order of activity which can be presented as: It is worth to notice that all compounds appeared to be more potent than ampicillin against all bacteria used and more active than streptomycin against all bacteria except B. cereus and S. typhimurium (ATCC 13,311).
The structure-activity studies revealed that the most beneficial for antibacterial activity is the presence of hydroxybenzamide (5d) on the N-atom of (Z)-5-((5-methoxy-1H-indol-3-yl)methylene)-3morpholino-2-thioxothiazolidin-4-one. Introduction of the 5-methoxy group to indole ring and replacement of hydroxybenzamide by nicotinamide (5g) decreased a little activity while shifting of methoxy group from position 5 to position 6 of indole ring and replacement of nicotinamide by isonicotinamide led to less active compound 5k compared to compound 5g.
Thus, it can be concluded that the most favorable effect on the antibacterial activity of the target compounds is provided by the introduction into the molecule of an unsubstituted indolidene and 6-methoxyindolidene fragment. In addition, the nature of the substituent at position 3 of the thiazolidine ring has a direct influence on the enhancement of the antibacterial action. An increase in the antibacterial effect is observed from the use of 4-hydroxybenzamide and isonicotinamide substitutes.
From all mentioned above, it is evident that the antibacterial activity of these compounds depends not only on substituent and its position in the indole ring but also on substituent on the N-atom of 2-thioxothiazolidin-4-one ring.  Three most active compounds were also evaluated against the resistant strains, including MRSA, P. aeruginosa, and E. coli, (Table 3). From the obtained results, it is evident that all three compounds were more active against MRSA than ampicillin, while streptomycin did not show any bactericidal activity. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain.

Antifungal Activity
All compounds also showed antifungal activity with MIC values ranging from 9.7 to 347.4 µM and MFC at 19.5-694.8 µM.The antifungal activity of compounds is shown in Table 4 and follows the order: It should be mentioned that all compounds appeared to be more potent than ketoconazole and bifonazole. Only compound 7a against A. fumigatus (human isolate) was less active than bifonazole.
According to the analysis of the structure-activity relationships, the most beneficial for antifungal activity is the presence of the 5-methoxy group in indole ring as well as nicotinamide as a substituent of the side chain (5g). In contrast, the presence of isonicotinamide in methylindole (5i) derivative appeared to be detrimental. Shifting of 5-OMe of compound 5g to position 6 of indole and replacement of nicotinamide by 2-hydroxybenzamide resulted in compound 5c with decreased activity. Removal of methoxy group and introduction of morpholino moiety to the N atom of thioxothiazolidinone (7a) decreased more activity.
In indole derivatives (5d, 5e, 5h), the presence of 4-hydroxybenzamide was favorable for antifungal activity, while isonicotinamide substituent had a negative effect. On the contrary, for methylindole derivatives (5a, 5f, 5i), the negative impact was observed with the presence of 2-hydroxybenzamide, while in the case of the 5-methoxy indole derivatives (5b, 5j) it was the opposite. Finally, for the derivatives with morpholino moiety, the best activity was observed with the presence of the 5-methoxy group in the indole ring. The indole derivative was one of the less potent. Thus, as in the case of antibacterial activity, antifungal activity depends not only on substitution in the indole ring but also on substituent on the N-atom of the 2-thioxothiazolidinone ring. In the series of (Z)-5-((5-methoxy-1H-indol-3-yl)methylene)-3-morpholino-2-thioxothiazolidin-4-one derivatives the most important structural features which enhanced the antifungal activity are again 4-hydroxybenzamide and 1H-indole moiety as well as nicotinamide and 5-and 6-methoxyindole moieties. On the other hand, in the series of indole 3-methylene morpholino-2-thioxothiazolidin-4-one derivatives, the presence of the 5-OCH3 group in the indole ring enhance the antifungal activity.

Cytotoxicity Assessment
Low toxicity and selectivity of action of antimicrobial compounds is a crucial pre-requisite for further development. Thus, we studied the cytotoxicity of the most active compounds. MTT analysis was performed on the HEK-293 human embryonic kidney cell line. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. The cells were inoculated into a 96-well plate at a concentration of 5 . 10 4 /mL (5 . 10 3 per well, 100 µL each). After one day of culture, compound preparations were added, and the results were obtained after a 72 h culture period. The compounds were added at four concentrations (25,50,100, and 250 µM). Since compound solutions contained DMSO, control cultures containing only DMSO at the final concentration obtained when the appropriate volume of compound solution was added were performed.
Although the compounds do not exhibit statistically significant concentration-dependent toxicity up to 100 µM (Figure 3), they show some toxicity at higher concentrations. The average CC 50 values obtained from three different experiments are given in Tables 5 and 6. The SI index is also shown in Tables 5 and 6.
Compound 5g and 7c exhibited the best SI index for anti-fungal activity while compound 5d exhibited the best SI index for anti-bacterial activity.
We compared the CC 50 values of compounds 5d, 5k, 5g, 7c with cytotoxicity of the reference drugs obtained in the HEK-293 human embryonic kidney cell line. For antibacterials streptomycin, ampicillin and antifungal bifonazole CC 50 exceeded 100 µM [63,64]; for antifungal ketoconazole CC 50 = 60 µM [65]. Thus, cytotoxicity of the most active compounds in our study is comparable or lower than cytotoxicity of the reference antimicrobial drugs.

Docking Studies
Since the mechanism of antimicrobial action of our compounds is not known, to choose the proteins as potential targets, we based on the literature. It was found that benzothiazole derivatives are mentioned as Gyrase inhibitors [66][67][68]. On the other hand, according to the literature, thiazolidinones act as MurB inhibitors [69][70][71][72]. Furthermore, prediction of the mechanism of action by computer program PASS indicated Thymidylate kinase as the probable antibacterial target. On the other hand, several publications mentioned thiazolidinone and indole derivatives as 14 α -lanosterol demethylase inhibitors [73][74][75]. Thus, taking all these into account, we proposed E. coli DNA Gyrase, Thymidylate kinase, and E. coli MurB enzymes as antibacterial targets, with CYP51 as the antifungal target.

Docking to Antibacterial Targets
The docking studies revealed that estimated binding energy to E. coli DNA Gyrase (−2.59 to −6.54 kcal/mol) as well as to thymidylate kinase (−1.55 to −4.12 kcal/mol), were higher than that to E. coli MurB (−7.07 to −10.93 kcal/mol). Therefore, it may be resolved that E. coli MurB is the most suitable enzyme where binding scores were consistent with biological activity (Table 7). The docking pose of the most active compound 5d in E. coli MurB enzyme showed two favorable hydrogen bond interactions. The first one is between the oxygen atom of the C=O group of the compound and the hydrogen of the side chain of Ser228. The second one between the oxygen atom of -OH group of the compound and the side chain of Arg326 (distances 2.17 Å and 1.99 Å, respectively). The fused rings interact hydrophobically with the residues Tyr189, Asn232, Leu289, Ala123, Leu217, and Arg213, while the benzene ring interacts hydrophobically with the residues Asn50, Ser115, Ile118, Ile121, Gln119 and Glu324 (Figure 4). These interactions stabilize the complex compound-enzyme and play a crucial role in the increased inhibitory activity of compound 5d Moreover, the hydrogen bond formation with the residue Ser228 is essential for the inhibitory action of the compounds; thus, this residue takes part in the proton transfer at the second stage of peptidoglycan synthesis [76].
The second most active compound, 5g, also forms the hydrogen bond interaction with the residue Ser228 that explains its high inhibitory action (Figure 2). Detailed analysis of the docking pose of the two most active compounds showed that they similarly bind MurB, and they insert deeper to the binding center of the enzyme than FAD, forming a hydrogen bond with the residue Ser228 ( Figure 5). the binding center of the enzyme than FAD, forming a hydrogen bond with the residue Ser228 ( Figure  5). The same behavior was observed in the case of docking of the most active compound among 5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkane carboxylic acids [19] and 5adamantane thiadiazole-based thiazolidinones [70]. Again, the formation of the hydrogen bond between the C=O group and Ser228 was observed. Thus, the obtained results support previous data [69][70][71][72] that MurB maybe is the most appropriate target for the antibacterial activity for this chemical series.

Docking to Lanosterol 14α-demethylase of C. albicans
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans (Table 8). The same behavior was observed in the case of docking of the most active compound among 5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkane carboxylic acids [19] and 5-adamantane thiadiazole-based thiazolidinones [70]. Again, the formation of the hydrogen bond between the C=O group and Ser228 was observed. Thus, the obtained results support previous data [69][70][71][72] that MurB maybe is the most appropriate target for the antibacterial activity for this chemical series. the binding center of the enzyme than FAD, forming a hydrogen bond with the residue Ser228 ( Figure  5). The same behavior was observed in the case of docking of the most active compound among 5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkane carboxylic acids [19] and 5adamantane thiadiazole-based thiazolidinones [70]. Again, the formation of the hydrogen bond between the C=O group and Ser228 was observed. Thus, the obtained results support previous data [69][70][71][72] that MurB maybe is the most appropriate target for the antibacterial activity for this chemical series.

Docking to Lanosterol 14α-demethylase of C. albicans
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans (Table 8).

Docking to Lanosterol 14α-demethylase of C. albicans
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans (Table 8). Docking results showed that all the synthesized compounds might bind to CYP51 Ca close to those of the reference drug ketoconazole. Compound 5g is located inside the enzyme alongside to heme group, interacting with the Fe of the heme group of CYP51 Ca throughout its atom N of the pyridine ring. Moreover, compound 5g forms a hydrogen bond between the oxygen of -OCH 3 substituent and the hydrogen of the side chain of Tyr132. Hydrophobic interactions were detected between residues Thr122, Phe126, Tyr132, and Ile131 and the fused rings of the compound 5g, also between Leu376, Thr311 and the benzene ring of the compound. Furthermore, compound 5g interacts hydrophobically throughout its benzene ring with the heme group of the enzyme, and also it forms a positive ionizable bond with it ( Figure 6). Interaction with the heme group was also observed with the benzene ring of ketoconazole, which forms positive ionizable interactions (Figures 6 and 7). However, compound 5g forms a more stable complex of the ligand with enzyme indicating its interaction with the Fe, which is probably why compound 5g showed high antifungal activity.    It should be mentioned that the tested compounds interact more strongly with the heme group of the enzyme CYP51Ca because the heme's Fe is involved in this interaction. In the case of our previous work [19], the most active compound interacts with the heme but throughout its benzene ring and the -NO2 group, forming pi and negative ionizable interactions with the heme group, respectively. In the case of 5-adamantane thiadiazole-based thiazolidinones [72], again, the most active compound form positive interactions between the heme group and heterocyclic rings of the compound. Thus, it can be concluded that thiazolidinone derivatives, in general, can interact with the heme of CYP51Ca in the same way as ketoconazole interacts.

Materials and Methods
All starting materials were purchased from Merck and used without purification. NMR spectra were determined with Varian Mercury VX-400" (Varian Co., Palo Alto, CA, USA) and AM-300 Bruker 300 MHz. spectrometers in DMSO-d6. MS (ESI) spectra were recorded on an LC-MS system -HPLC Agilent 1100 (Agilent Technologies Inc., Santa, Clara, CA USA) equipped with a diode array detector Agilent LC\MSD SL. Parameters of analysis: Zorbax SB -C18 column (1.8 μm, 4.6-15 mm, PN 821975-932), solvent water -acetonitrile mixture (95:5), 0.1% of aqueous trifluoroacetic acid; eluent flow 3 It should be mentioned that the tested compounds interact more strongly with the heme group of the enzyme CYP51 Ca because the heme's Fe is involved in this interaction. In the case of our previous work [19], the most active compound interacts with the heme but throughout its benzene ring and the -NO 2 group, forming pi and negative ionizable interactions with the heme group, respectively. In the case of 5-adamantane thiadiazole-based thiazolidinones [72], again, the most active compound form positive interactions between the heme group and heterocyclic rings of the compound. Thus, it can be concluded that thiazolidinone derivatives, in general, can interact with the heme of CYP51 Ca in the same way as ketoconazole interacts.

In Silico Biological Activity Evaluation
Antimicrobial activity and toxicity of the designed compounds have been estimated in silico using web services available on the Way2Drug portal [56]. These services are based on the PASS (Prediction of Activity Spectra for Substances) and GUSAR (General Unrestricted Structure-Activity Relationships) software, which is described in detail elsewhere [60,61]. It is essential to mention that PASS-based services provide the assessments of the compound's activity as the difference between the probabilities for the chemical compound with a particular structure to display activity (Pa) and do not display this activity (Pi). By default, in PASS, all activities with Pa > Pi are considered as probable. High Pa-Pi values reflect the high structural similarity of the analyzed compound to the structures included in the training set with those activities. Since our goal was not finding close analogs of the earlier discovered antimicrobial agents, we considered compounds with small Pa-Pi values as the promising hits for experimental testing. If the experiment will confirm their activity, there is a chance to find a New Chemical Entity. GUSAR-based service [60,61] provides the quantitative assessment of acute rat toxicity expressed as LD 50 values for four routes of administration: intraperitoneal (IP), intravenous (IV), oral, and subcutaneous (SC).

Chemistry
3.2.1. General Procedure for the Preparation of N-(4-oxo-2-thioxothiazolidin-3-yl) carbamides 3a-d In a round-bottom flask equipped with a reflux condenser, 0.05 mol of trithiocarbonyl diglycolic acid, 0.05 mol of the corresponding hydrazide and alcohol-water mixture (1:1) were placed and boiled for 3 h. The reaction mixture is cooled, the precipitate is filtered off and recrystallized. In a round-bottom flask equipped with a reflux condenser, 2.5 mmol of 3-substituted 2-thioxo-4-oxothiazolidine 3a-d or 6, 3.3 mmol of the corresponding aldehyde 1a-d, 2.5 mmol of ammonium acetate and 5 mL of acetic acid are placed. The reaction mixture is boiled for 2 h, cooled, the precipitate is filtered off, washed with acetic acid and water, dried and recrystallized.