Design, Synthesis, and Molecular Docking Study of Novel Heterocycles Incorporating 1,3,4-Thiadiazole Moiety as Potential Antimicrobial and Anticancer Agents

A new series of 5-(3,5-dinitrophenyl)-1,3,4-thiadiazole derivatives were prepared and evaluated for their in vitro antimicrobial, antitumor, and DHFR inhibition activity. Compounds 9, 10, 13, and 16 showed strong and broad-spectrum antimicrobial activity comparable to Amoxicillin and Fluconazole as positive antibiotic and antifungal controls, respectively. Compounds 6, 14, and 15 exhibited antitumor activity against four human cancer cell lines, CCRF-CEM leukemia, HCT-15 colon, PC-3 prostate, and UACC-257 melanoma cell lines using Doxorubicin as a reference drug. Compounds 10, 13, 14, and 15 proved to be the most active DHFR inhibitors with an IC50 range of 0.04 ± 0.82–1.00 ± 0.85 µM, in comparison with Methotrexate (IC50 = 0.14 ± 1.38 µM). The highly potent DHFR inhibitors shared a similar molecular docking mode and made a critical hydrogen bond and arene‒arene interactions via Ser59 and Phe31 amino acid residues, respectively.


Introduction
Dihydrofolate reductase (DHFR) is a prevalent enzyme that is present in all prokaryotic and eukaryotic cells. It has a key role in folate metabolism and subsequently DNA and RNA synthesis [1,2]. Inhibition of DHFR revealed importance in the development of therapeutic agents against anticancer drugs as well as bacterial and parasitic infections [3][4][5][6][7]. DHFR inhibitors exhibit a vital role in clinical medicine, like the use of Methotrexate in neoplastic diseases, inflammatory bowel diseases, rheumatoid arthritis, psoriasis, and asthma [8].
Considering the findings above and as a continuation of our efforts towards the development of biologically active heterocyclic compounds [24][25][26][27][28][29][30][31][32][33][34], we undertook the design and synthesis of some novel 1,3,4-thiadiazole prototypes that possess the advantages of pharmacophores (I, II), as outlined in Figure 2. All the newly hybrid compounds comprising the 1,3,4-thiadiazole motif were evaluated for their antimicrobial and anticancer activities through a study of their in vitro inhibitory activity enzyme against DHFR enzyme, followed by molecular docking studies to get insight into the interactions and binding modes in the active site of this enzyme.
Thus, when compound 1 was treated with carbon disulfide and potassium hydroxide, followed by stirring with hydrazine hydrate, thiosemicarbazide derivative 2 was produced.
Compound 2 was used as a precursor to construct different heterocyclic ring systems such as thiadiazole, thiazole, and pyrimidine through the reaction with different reagents.
Thus, when compound 1 was treated with carbon disulfide and potassium hydroxide, followed by stirring with hydrazine hydrate, thiosemicarbazide derivative 2 was produced.
Compound 2 was used as a precursor to construct different heterocyclic ring systems such as thiadiazole, thiazole, and pyrimidine through the reaction with different reagents.

Scheme 1. Synthesis route for compounds 2-6.
Compound 3 was formed via a nucleophilic attack of the NH2 group of thiosemicarbazide on the carbonyl group of phenylalanine through tetrahedral mechanism, followed by elimination of the water molecule; then ring closure takes place via the elimination of another water molecule from the thiol and OH of the carboxylic group. However, in the case of compound 4 the carboxylic group is converted into acid chloride and as a result a thiadiazole ring is formed by the elimination of water and HCl molecules. In compound 5, a nucleophilic attack of NH2 group of thiosemicarbazide on the C=S group was followed by ring closure through the elimination of the H2S molecule.
As proof of the proposed structure of compound 4, its IR spectrum displayed a stretching frequency of 3265 (NH) and the 1H-NMR spectrum had peaks at δ 7.33-7.66 (m, 5H, Ph-H) and 10.22 (s, 1H, NH). For compound 5, the IR spectrum showed two characteristic absorption bands at 3261 (NH) and 2561 (SH), which indicates the presence of the compound in the thiol form, besides the 1H-NMR peaks at δ 10.13 and 12.44 corresponding to the NH and SH groups. The IR spectrum of compound 6 lacked the band of the SH group and showed bands at 3269 (NH) and 2921 (CHaliph.), and the 1 H-NMR spectrum displayed peaks at δ 4.96 (s, 2H, S-CH2-CO), 7.37-7.69 (m, 5H, Ph-H) and 10.29 (s, 1H, NH).
Furthermore, thiosemicarbazide 2 reacted with bis-methylthiomethylene-barbituric acid (7), chloroacetic acid/sodium acetate, and/or malonic acid in the presence of acetyl chloride to give barbituric, thiazolidin-4-one, and thiobarbituric derivatives 8-10, respectively (Scheme 2). Compound 3 was formed via a nucleophilic attack of the NH 2 group of thiosemicarbazide on the carbonyl group of phenylalanine through tetrahedral mechanism, followed by elimination of the water molecule; then ring closure takes place via the elimination of another water molecule from the thiol and OH of the carboxylic group. However, in the case of compound 4 the carboxylic group is converted into acid chloride and as a result a thiadiazole ring is formed by the elimination of water and HCl molecules. In compound 5, a nucleophilic attack of NH 2 group of thiosemicarbazide on the C=S group was followed by ring closure through the elimination of the H 2 S molecule.
As proof of the proposed structure of compound 4, its IR spectrum displayed a stretching frequency of 3265 (NH) and the 1H-NMR spectrum had peaks at δ 7.33-7.66 (m, 5H, Ph-H) and 10.22 (s, 1H, NH). For compound 5, the IR spectrum showed two characteristic absorption bands at 3261 (NH) and 2561 (SH), which indicates the presence of the compound in the thiol form, besides the 1H-NMR peaks at δ 10.13 and 12.44 corresponding to the NH and SH groups. The IR spectrum of compound 6 lacked the band of the SH group and showed bands at 3269 (NH) and 2921 (CH aliph .), and the 1 H-NMR spectrum displayed peaks at δ 4.96 (s, 2H, S-CH 2 -CO), 7.37-7.69 (m, 5H, Ph-H) and 10.29 (s, 1H, NH).
Furthermore, thiosemicarbazide 2 reacted with bis-methylthiomethylene-barbituric acid (7), chloroacetic acid/sodium acetate, and/or malonic acid in the presence of acetyl chloride to give barbituric, thiazolidin-4-one, and thiobarbituric derivatives 8-10, respectively (Scheme 2). The mechanism for the formation of compound 8 could be as follows: first, an Michael addition reaction between the NH2 of the thiosemicarbazide 2 and the bis-methyl-thiomethylene barbituric acid (7), resulting in the elimination of the CH3SH group to give intermediate (A), which underwent intramolecular cyclization through the elimination of the second CH3SH group to furnish the pyrimidine-2,4-6-trione derivative 8.
Compound 9 could be formed via nucleophilic attack of the nitrogen atom of thiosemicarbazide on the carbonyl group of chloroacetic acid with elimination of the H2O molecule; then ring closure takes place through nucleophilic attack of the sulfur atom (in the thiol form) on the carbon atom (attached to Cl), synchronized with elimination of the HCl molecule.
The attack of two NH groups of thiosemicarbazide on the two carbonyl groups of malonic acid resulted in ring closure with elimination of two water molecules to produce compound 10, accompanied by acylation of the NH2 group.
Meanwhile, compound 1 was allowed to react with phenylisothiocyante in dioxane to produce the thiadiazole derivative 11, which may be formed as a result of the nucleophilic attack of the NH2 on the carbon atom of the isothiocyant derivative (Scheme 3). The mechanism for the formation of compound 8 could be as follows: first, an Michael addition reaction between the NH 2 of the thiosemicarbazide 2 and the bis-methyl-thiomethylene barbituric acid (7), resulting in the elimination of the CH 3 SH group to give intermediate (A), which underwent intramolecular cyclization through the elimination of the second CH 3 SH group to furnish the pyrimidine-2,4-6-trione derivative 8.
Compound 9 could be formed via nucleophilic attack of the nitrogen atom of thiosemicarbazide on the carbonyl group of chloroacetic acid with elimination of the H 2 O molecule; then ring closure takes place through nucleophilic attack of the sulfur atom (in the thiol form) on the carbon atom (attached to Cl), synchronized with elimination of the HCl molecule.
The attack of two NH groups of thiosemicarbazide on the two carbonyl groups of malonic acid resulted in ring closure with elimination of two water molecules to produce compound 10, accompanied by acylation of the NH 2 group.
Meanwhile, compound 1 was allowed to react with phenylisothiocyante in dioxane to produce the thiadiazole derivative 11, which may be formed as a result of the nucleophilic attack of the NH 2 on the carbon atom of the isothiocyant derivative (Scheme 3). Compound 11 was supported by its spectral data: its IR spectrum displayed stretching absorption bands at 3329 and 3256, corresponding to two NH groups. At the same time, 1 H-NMR revealed peaks at δ 10.49, 11.33 (2s, 2H, 2NH), and δ 7.45-7.89 (m, 5H, Ar-H).
The reactivity of compound 11 was explored through the reaction with phenacyl bromide in the presence of triethylamine as a catalyst, and/or malonic acid/acetyl chloride to give thiazole and thiobarbituric derivatives 12 and 13, respectively (Scheme 3).
The formation of compound 12 could be via a nucleophilic attack of NH on the carbonyl group, resulting in the elimination of a water molecule; then, the sulfur in the thiol form attacks the methylene group, leading to ring closure, accompanied by elimination of HBr molecule to produce the thiazole derivative 12.
Compound 13 was coupled with benzene diazonium chloride in the presence of sodium acetate and/or condensation with benzaldehyde in the presence of piperidine to produce phenyl hydrazone and thiadiazole derivatives 14 and 15, respectively (Scheme 3).
The present investigation was extended to demonstrate the reactivity of dimethyl carbon-imidodithioate derivative 16 towards some amine derivatives in order to synthesize different heterocyclic systems. Compound 16 was prepared through the treatment of the 2-aminothiadiazole derivative 1 with carbon disulfide in a basic medium followed by S-methylation using methyl iodide.
The reactivity of compound 11 was explored through the reaction with phenacyl bromide in the presence of triethylamine as a catalyst, and/or malonic acid/acetyl chloride to give thiazole and thiobarbituric derivatives 12 and 13, respectively (Scheme 3).
The formation of compound 12 could be via a nucleophilic attack of NH on the carbonyl group, resulting in the elimination of a water molecule; then, the sulfur in the thiol form attacks the methylene group, leading to ring closure, accompanied by elimination of HBr molecule to produce the thiazole derivative 12.
Compound 13 was coupled with benzene diazonium chloride in the presence of sodium acetate and/or condensation with benzaldehyde in the presence of piperidine to produce phenyl hydrazone and thiadiazole derivatives 14 and 15, respectively (Scheme 3).
The present investigation was extended to demonstrate the reactivity of dimethyl carbon-imidodithioate derivative 16 towards some amine derivatives in order to synthesize different heterocyclic systems. Compound 16 was prepared through the treatment of the 2-aminothiadiazole derivative 1 with carbon disulfide in a basic medium followed by S-methylation using methyl iodide.
When compound 16 was allowed to react with amines, namely o-aminophenol, o-aminothiophenol, o-phenylenediamine, and/or ethylene diamine in dimethylformamide, benzo-oxazole, benzothiazole, benzoimidazole, and dihydroimidazole derivatives 17-20 were produced, respectively (Scheme 4).  The formation of compounds 17-20 was proposed to take place via the nucleophilic attack of the amino and HX groups on the carbon atom of thioacetal followed by elimination of two MeSH molecules. This can be shown as a speculated mechanistic pathway in Scheme 5.  The formation of compounds 17-20 was proposed to take place via the nucleophilic attack of the amino and HX groups on the carbon atom of thioacetal followed by elimination of two MeSH molecules. This can be shown as a speculated mechanistic pathway in Scheme 5.  The formation of compounds 17-20 was proposed to take place via the nucleophilic attack of the amino and HX groups on the carbon atom of thioacetal followed by elimination of two MeSH molecules. This can be shown as a speculated mechanistic pathway in Scheme 5.

Antimicrobial Sensitivity Assay
All the synthesized compounds 1-20 were screened for their antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and yeast-like pathogenic fungi (Aspergillus niger and Candida albicans). The antimicrobial screening was carried out using a standard well agar diffusion assay according to Cheesbrough et al. [35]. The broad-spectrum antibiotic Amoxicillin and the antifungal Fluconazole were used at a concentration of 100 µg/mL as positive controls. The obtained results (Table 1) revealed that the tested compounds 9, 10, 13, and 16 showed the highest antimicrobial activity. Compound 13 is the most potent one since it demonstrated antimicrobial activity higher than that of the standard drugs (Figures 3 and 4). The rest of the tested compounds had antimicrobial activity that ranged from moderate to weak.

Antimicrobial Sensitivity Assay
All the synthesized compounds 1-20 were screened for their antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and yeast-like pathogenic fungi (Aspergillus niger and Candida albicans). The antimicrobial screening was carried out using a standard well agar diffusion assay according to Cheesbrough et al. [35]. The broad-spectrum antibiotic Amoxicillin and the antifungal Fluconazole were used at a concentration of 100 µg/mL as positive controls. The obtained results (Table 1) revealed that the tested compounds 9, 10, 13, and 16 showed the highest antimicrobial activity. Compound 13 is the most potent one since it demonstrated antimicrobial activity higher than that of the standard drugs (Figures 3 and 4). The rest of the tested compounds had antimicrobial activity that ranged from moderate to weak.

Structure-Activity Relationship for Antimicrobial Activity
By analysis of the previous results and concerning the structural modifications that occurred only at position-2 (p-2) of the parent 1,3,4-thiadiazole scaffold, it was found that: a 1,3,4-Thiadiazole derivative 1 having free amino group at p-2 showed moderate inhibitory activity against all tested strains. Substitution of NH2 group with thiocarbohydrazide in compound 2 produced excellent activity, while its phenyl congener 11 displayed moderate activity. Furthermore, substitution of the amino group with dimethyl carbonimidodithioate in compound 16 exhibited high inhibitory activity and was approximately equipotent with the reference.
The existence of a new 1,3,4-thiadiazole ring via the NH linker revealed various levels of antimicrobial activity: Direct attachment of the thiazole moiety achieved excellent potency in compound 9, while attachment via an imine group led to a drop in activity in compound 12.
Moreover, direct attachment of a thioxopyrimidine-4,6-dione scaffold led to excellent activity with substitution at N-1 either with phenyl as in 13 or with acetyl acetamide as in 10. It was noted that insertion of 2-phenyl hydrazone in compound 14 or benzylidine in compound 15, to p-5 of the newly formed 2-thioxopyrimidine-4,6-dione of 10 and 13, decreased the activity to a moderate level.
Upon attachment of an imidazole moiety via a NH linker there was significant antimicrobial activity in compound 20, followed by a drastic drop due to cyclization forming benzo [

In Vitro Anticancer Activity
All synthesized compounds 1-20 were investigated for their in vitro cytotoxic activity against four human carcinoma cell lines, CCRF-CEM, HCT-15, PC-3, and UACC-257, using Doxorubicin as a standard anticancer drug. The anticancer activity was expressed as IC50 values (the concentration of test compounds required to kill 50% of the cell population) in (µM) ± SEM from three replicates.
The results depicted in Table 2 revealed that the most potent compounds are 15 > 14 > 6, in descending order, against all tested cell lines. Compounds 8 and 12 exhibited moderate activity

Structure-Activity Relationship for Antimicrobial Activity
By analysis of the previous results and concerning the structural modifications that occurred only at position-2 (p-2) of the parent 1,3,4-thiadiazole scaffold, it was found that: a 1,3,4-Thiadiazole derivative 1 having free amino group at p-2 showed moderate inhibitory activity against all tested strains. Substitution of NH 2 group with thiocarbohydrazide in compound 2 produced excellent activity, while its phenyl congener 11 displayed moderate activity. Furthermore, substitution of the amino group with dimethyl carbonimidodithioate in compound 16 exhibited high inhibitory activity and was approximately equipotent with the reference.
The existence of a new 1,3,4-thiadiazole ring via the NH linker revealed various levels of antimicrobial activity: Direct attachment of the thiazole moiety achieved excellent potency in compound 9, while attachment via an imine group led to a drop in activity in compound 12.
Moreover, direct attachment of a thioxopyrimidine-4,6-dione scaffold led to excellent activity with substitution at N-1 either with phenyl as in 13 or with acetyl acetamide as in 10. It was noted that insertion of 2-phenyl hydrazone in compound 14 or benzylidine in compound 15, to p-5 of the newly formed 2-thioxopyrimidine-4,6-dione of 10 and 13, decreased the activity to a moderate level.
Upon attachment of an imidazole moiety via a NH linker there was significant antimicrobial activity in compound 20, followed by a drastic drop due to cyclization forming benzo [

In Vitro Anticancer Activity
All synthesized compounds 1-20 were investigated for their in vitro cytotoxic activity against four human carcinoma cell lines, CCRF-CEM, HCT-15, PC-3, and UACC-257, using Doxorubicin as a standard anticancer drug. The anticancer activity was expressed as IC 50 values (the concentration of test compounds required to kill 50% of the cell population) in (µM) ± SEM from three replicates.
The results depicted in Table 2 revealed that the most potent compounds are 15 > 14 > 6, in descending order, against all tested cell lines. Compounds 8 and 12 exhibited moderate activity about half that of the reference drug. The rest of the tested compounds showed weak cytotoxic activity. Comparing the IC 50 values obtained for the synthesized derivatives against CCRF-CEM, HCT-15, PC-3, and UACC-257 with those obtained against non-tumorigenic MCF-10A cells, we can conclude that the synthesized derivatives have much less toxicity against normal cells.

Dihydrofolate Reductase (DHFR) Inhibition
The synthesized compounds 1-20 were evaluated as inhibitors of bovine liver DHFR using a reported procedure [36]. Results were summarized as IC 50 values in Table 3 and Methotrexate was used as a positive control. As illustrated in Table 3, compounds 10, 13, 14, and 15 proved to be the most active inhibitors with an IC 50 range from 0.04 ± 0.82 to 1.00 ± 0.85 µM, in comparison with Methotrexate (IC 50 = 0.14 ± 1.38 µM). However, the rest of the tested compounds showed moderate to weak inhibitory activity, with an IC 50 range of 8.46 ± 0.13-36.48 ± 0.72 µM. The inhibitory activity of the tested derivatives could be correlated to structure variation and modifications. By investigating the variation in selectivity of the highly potent compounds 10, 13, 14, and 15 over the DHFR enzyme, it was revealed that the existence of thioxopyrimidine-4,6-dione moiety at p-2 of the parent 1,3,4-thiadiazole scaffold led to enhanced activity, and the potency order was 15 > 14 > 13 > 10. Structure-activity relationships in these compounds demonstrated that compounds with substitution at p-5 of the newly formed 2-thioxopyrimidine-4,6-dione (14, 15, with excellent anticancer activity) showed more potent inhibitory activity against the DHFR enzyme than those having no substituents (10, 13, with excellent antimicrobial activity).

Molecular Modeling Studies
To gain a better understanding of the potency of the studied compounds and guide further SAR studies, we proceeded to examine the interaction of compounds 10, 13, 14, and 15 via the X-ray crystallographic structure of DHFR (PDB ID: 1DLS) [37]. The co-crystallized ligand Methotrexate was redocked into the pocket sites of DHFR and revealed docking score energies of −11.4 kcal/mol at a root mean square deviation (RMDS) value of 9.1. The molecular docking was performed by inserting compounds 10, 13, 14, and 15 into the ATP binding site of DHFR. All docking runs applied the LigandFit Dock protocol of Molecular Operating Environment (MOE, 10.2008) software [38,39]. The docking scores for compounds 10, 13, 14, and 15 were all in the range −15.6 to −12.3 kcal/mol. Representations of the docking results of these compounds and DHFR are given in Figures 5 and 6. hydrogen bonding network was involved between the nitrogen atom and the NH proton of the phenylhydrazone moiety, and the side chains of Ser59 and Thr56, respectively (distance: 2.39 and 2.42 Å, respectively). The other oxygen of thioxopyrimidine-4,6-dione in compound 15 mediated a strong hydrogen bond acceptor with the side chains of Thr56 (distance: 2.49 Å) ( Figure 5). From the docking results, it is evident that the thioxopyrimidine-4,6-dione moiety linked to the 1,3,4-thiadiazole scaffold contributes to the activity of compounds 10, 13, 14, and 15 through H-bonds to Ser59, which considerably strengthens the binding interaction ( Figure 6). Inspection of the binding modes demonstrated that all compounds were potently bound to the ATP binding site of DHFR via arene-arene interaction between the centroids of Phe31 and 3,5-dinitrophenyl moiety, and a hydrogen bond acceptor between the sidechain of Ser59 and one oxygen of thioxopyrimidine-4,6-dione.
Furthermore, compound 10 was stabilized by another hydrogen bond acceptor between the oxygen of acetamide moiety and the side chain of Tyr121 (distance: 2.95 Å). In compound 14, a dual hydrogen bonding network was involved between the nitrogen atom and the NH proton of the phenylhydrazone moiety, and the side chains of Ser59 and Thr56, respectively (distance: 2.39 and 2.42 Å, respectively). The other oxygen of thioxopyrimidine-4,6-dione in compound 15 mediated a strong hydrogen bond acceptor with the side chains of Thr56 (distance: 2.49 Å) ( Figure 5).
From the docking results, it is evident that the thioxopyrimidine-4,6-dione moiety linked to the 1,3,4-thiadiazole scaffold contributes to the activity of compounds 10, 13, 14, and 15 through H-bonds to Ser59, which considerably strengthens the binding interaction ( Figure 6). From the docking results, it is evident that the thioxopyrimidine-4,6-dione moiety linked to the 1,3,4-thiadiazole scaffold contributes to the activity of compounds 10, 13, 14, and 15 through H-bonds to Ser59, which considerably strengthens the binding interaction ( Figure 6).

Chemistry
All solvents, reagents, and chemicals were obtained from Alfa Aesar (Ward Hill, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA). All melting points are not corrected and were measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). Infrared spectra were recorded on a Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan), using KBr discs and are reported as ν cm −1 . A Bruker Avance III spectrometer (Bruker Corporation, Rheinstetten, Germany) was used to record 1 H-NMR spectra at 400 MHz using TMS as an internal standard and DMSO-d6 as a solvent. 13 C-NMR spectra were recorded on the same spectrometer at 100 MHz using the same solvent. MS spectra were measured on a Shimadzu GC-MS-QP-1000 EX mass spectrometer instrument operating at 70 eV. The elemental analyses of the new compounds were recorded on a Perkin-Elmer CHN-2400 analyzer (Waltham, MA, USA) and carried out at the Microanalytical Centre, Cairo University, Cairo, Egypt. The microanalysis showed that the observed values were within ±0.4% of theoretical values. The homogeneity of the compounds and the progress of the chemical reactions were monitored by TLC silica gel plates (60F254, Merck, Munchen, Germany). The biological evaluation was conducted at the Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt. Compound 1 [40] was prepared using a previously reported method [41] (m.p. 217-219 °C, Let. 215 °C).

Chemistry
All solvents, reagents, and chemicals were obtained from Alfa Aesar (Ward Hill, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA). All melting points are not corrected and were measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). Infrared spectra were recorded on a Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan), using KBr discs and are reported as ν cm −1 . A Bruker Avance III spectrometer (Bruker Corporation, Rheinstetten, Germany) was used to record 1 H-NMR spectra at 400 MHz using TMS as an internal standard and DMSO-d6 as a solvent. 13 C-NMR spectra were recorded on the same spectrometer at 100 MHz using the same solvent. MS spectra were measured on a Shimadzu GC-MS-QP-1000 EX mass spectrometer instrument operating at 70 eV. The elemental analyses of the new compounds were recorded on a Perkin-Elmer CHN-2400 analyzer (Waltham, MA, USA) and carried out at the Microanalytical Centre, Cairo University, Cairo, Egypt. The microanalysis showed that the observed values were within ±0.4% of theoretical values. The homogeneity of the compounds and the progress of the chemical reactions were monitored by TLC silica gel plates (60F 254 ; Merck, Munchen, Germany). The biological evaluation was conducted at the Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt. Compound 1 [40] was prepared using a previously reported method [41] (m.p. 217-219 • C, Let. 215 • C).

Antimicrobial Sensitivity Assay
The antimicrobial activities of the synthesized compounds were evaluated against four bacterial strains, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and two fungal strains, Aspergillus niger and Candida albicans, using a standard well agar diffusion assay according to Cheesbrough et al. [35]. Plates containing nutrient agar medium and sabouraud dextrose agar medium (for bacteria and fungi, respectively) were surface-inoculated with 106 CFU/mL of freshly prepared microorganisms. Using a 6-mm sterile cork borer, wells were punched in the agar and filled separately with 100 µL of the tested compounds (100 µg/mL in DMSO). The plates were left in a refrigerator for 2 h to allow diffusion of the tested compounds. After that, the plates were incubated for 24 h at 37 • C for bacteria and for 72 h at 28 • C for fungi, then the inhibition zones surrounding the wells were measured in millimeters. Amoxicillin and Fluconazole were used as the standard against bacteria and fungi, respectively, using the same concentration (100 µg/mL).

In Vitro Anticancer Activity
The newly synthesized heterocyclic compounds were evaluated for their in vitro cytotoxicity against four cancer cell lines, CCRF-CEM (leukemia), HCT-15 (human colon carcinoma), PC-3 (prostate cancer) and UACC-257 (melanoma, skin cancer) cell lines, which were obtained from Sigma-Aldrich Chemical Company, USA. DOX (Doxorubicin) was utilized as a reference drug according to a previously reported MTT method [38,39].

Dihydrofolate Reductase (DHFR) Inhibition
The in vitro DHFR enzyme inhibition assessment was carried out in a confirmatory diagnostic unit, Vacsera, Egypt. All synthesized derivatives 1-20 were screened against DHFR using Methotrexate as a reference according to a previously reported method [36]. The results are reported as IC50 values of enzymatic activity in Table 3.

Molecular Modeling Studies
The docking study was performed using Molecular Operating Environment (MOE ® ) 2008.10 software [38,39]. The X-ray crystal structure of the dihydro-folate reductase enzyme was downloaded from the Protein Data Bank website (PDB ID: 1DLS) [37]. Regularization and optimization for ligand and protein were done. The performance of the docking method was evaluated by re-docking the crystal ligand into the assigned active DHFR enzyme to evaluate a root-mean-square deviation value. Then, the molecular docking was applied for compounds 10, 13, 14, and 15 into ATP binding site of DHFR according to the reported method [38,39].

Conclusions
In summary, a series of 1,3,4-thiadiazole derivatives 2-20 incorporating with different heterocyclic systems was designed and synthesized. All synthesized compounds were examined for their in vitro antimicrobial, antitumor, and DHFR inhibition activity. The antimicrobial results exhibited the ability of the compounds 9, 10, 13, and 16 to inhibit the growth of a panel of six strains with higher inhibition zones in comparison with the reference drugs. In addition, the cytotoxic activity against four cell lines illustrated that compounds 6, 14, and 15 have mostly prevented cell growth with lower IC 50 values. Based on the data obtained from the DHFR inhibition study, compounds 10, 13, 14, and 15, containing a thioxopyrimidine-4,6-dione moiety at p-2 of the parent 1,3,4-thiadiazole ring, were the most potent derivatives in comparison with Methotrexate. Moreover, the docking study indicated that compounds 10, 13, 14, and 15 showed good fitting and caused favorable contacts in the binding site of DHFR enzyme. Therefore, the obtained mark points have been proposed as an explanation for the unique activity of such derivatives and could be used as a template for further development and future optimization of new antimicrobial and anticancer agents via DHFR inhibition.