Microwave-Assisted One Pot Three-Component Synthesis of Novel Bioactive Thiazolyl-Pyridazinediones as Potential Antimicrobial Agents against Antibiotic-Resistant Bacteria

Pyridazine and thiazole derivatives have various biological activities such as antimicrobial, analgesic, anticancer, anticonvulsant, antitubercular and other anticipated biological properties. Chitosan can be used as heterogeneous phase transfer basic biocatalyst in heterocyclic syntheses. Novel 1-thiazolyl-pyridazinedione derivatives were prepared via multicomponent synthesis under microwave irradiation as ecofriendly energy source and using the eco-friendly naturally occurring chitosan basic catalyst with high/efficient yields and short reaction time. All the prepared compounds were fully characterized by spectroscopic methods, and their in vitro biological activities were investigated. The obtained results were compared with those of standard antibacterial/antifungal agents. DFT calculations and molecular docking studies were used to investigate the electronic properties and molecular interactions with specific microbial receptors.

Continuously over the years, it has been noticed that interesting biological activities [9,10] were associated with thiazole derivatives. Recently, thiazoles have had a wide range of applications in drug development for the treatment of allergies, inflammation, schizophrenia, bacterial diseases, hypnotics and more recently for the treatment of pain, as fibrinogen receptor antagonists with antithrombotic activity and as new inhibitors of bacterial DNA gyrase B [11][12][13][14][15][16].
Multi-component reactions (MCRs) are one-pot processes with at least three components to form a single product, which incorporates most or even all of the starting materials [17][18][19][20]. The huge interest in such MCRs during the last years has been oriented towards developing combinatorial chemistry procedures, because of their high efficiency and convenience in comparison to multistage procedures. Additionally, the utility of MCRs under microwave irradiation (MWI) in the synthesis of heterocyclic compounds enhanced reaction rates and improved regioselectivity [21][22][23][24].
Chitosan, a biocompatible and biodegradable naturally occurring polysaccharide, is a copolymer containing both glucosamine and N-acetylglucosamine units. It can be used as a heterogeneous phase transfer basic biocatalyst in heterocyclic syntheses, such as enantioselective syntheses of asymmetric products with chiral center(s) [25,26], Michael addition reactions [27][28][29], as well as transition metal support for the preparation of heterogeneous catalysts [30]. The presence of amino groups is responsible for the basic nature of chitosan. Keeping this in mind, in continuation of our previously reported works on the synthesis of new biologically active agents [31][32][33][34][35][36][37], we present herein an efficient synthesis of novel 1-thiazolyl-pyridazinedione derivatives as antimicrobial agents, which have not been reported hitherto in a multicomponent synthesis under MWI as an ecofriendly energy source and using the eco-friendly naturally occurring chitosan catalyst.

Synthesis
In continuation of our previous work to synthesize bioactive heterocyclic compounds under mild conditions [38][39][40][41][42][43], we wish to report herein mild and efficient procedures for the synthesis of some novel 1-thiazolyl-pyridazinedione derivatives via the threecomponent reaction of maleic anhydride 1, thiosemicarbazide 2 and the appropriate 2-oxo-N-arylpropanehydrazonoyl chlorides 3a-f in ethanol in the presence of chitosan under MWI at 500 W and 150 • C for 4-8 min. as monitored by TLC (Scheme 1). Scheme 1. Synthesis of arylazothiazole derivatives 5a-f.
The structure of 5a-f was confirmed by their spectral data (IR, MS and 1 H-NMR), elemental analyses and alternative synthetic routes. For example, the 1 H NMR spectra of compounds 5a-f exhibited singlet signals at δ~2.56 ppm (CH 3 ) and one D 2 O exchangeable peaks at δ~10.71 ppm corresponding to NH-phenyl, in addition to the expected signals for the aromatic protons and the two doublet signals of the CH=CH protons. The IR spectra of product 6 revealed in each case three absorption bands in the regions υ~1654, 1668 and 3435 cm −1 due to the two carbonyl groups and NH group. The mass spectra of products 5a-f revealed a molecular ion peak for each one, which is consistent with their respective molecular weights.
In the light of the foregoing results, the mechanism outlined in (Scheme 1) seems to be the most plausible pathway for the formation of compounds 5a-f from the reaction of the 1 + 2 + 3. The reaction involves initial formation of thiohydrazonate intermediate 4 via S-alkylation, with removal of HCl, which underwent dehydrative cyclization to afford the final product 5.
Compound 5a was alternatively synthesized by reacting carbothioamide 6 (prepared separately via condensation of maleic anhydride 1 and thiosemicarbazide 2) with 2-oxo-Nphenylpropanehydrazonoyl chloride (3a) in ethanol containing catalytic amount of chitosan under MWI (Scheme 1). The obtained product was found to be identical with 5a in all respects (TLC, mp. and IR spectrum), which affords further evidence to all structures 5a-f.
The structure of compounds 9a-e was proved based on spectral data, elemental analyses and chemical transformations. The spectroscopic information confirmed the reaction product 9 via intermediate 8 with elimination of EtOH molecule (Scheme 2).
Coupling of thiazolone 11 (prepared separately from reaction of carbothioamide derivative 6 with ethyl bromoacetate 10 in ethanol/chitosan under reflux) with PhN 2 Cl in pyridine yielded a product was found to be identical to 9a in all regards (mp., TLC and IR spectrum), providing an additional evidence to all 9a-e structures.
From literature reports [44][45][46][47] we found that compounds bearing more than one thiazole ring unit also exhibit good biological activities. For example, Myxothiazol is an inhibitor of the mitochondrial cytochrome bc1 complex and Bleomycin is an anticancer agent, containing 2,4 -bis thiazole system. From the above findings, we thought it is useful to synthesize a heterocyclic ring system carrying bis-thiazole moiety associated with pyridazine ring. This aim was achieved via the reaction of bis-hydrazonoyl chlorides 12a and 12b with two moles of maleimide 1 and two moles of thiosemicarbazide 2 under MWI in presence of chitosan to afford the respective bis-thiazoles 13 and 14 in a good yield (Scheme 3). The structure of compounds 13 and 14 was proven based on spectral data and elemental analyses (Experimental part).

XTT Assay Results
The minimum inhibitory concentration (MIC) of the tested compounds on cell metabolism/viability of S. aureus, P. aeruginosa and C. albicans was determined using XTT assay compared to the standard counterparts (vancomycin and amphotericin B).
The results presented in Table 1 depict that most of the investigated compounds have higher activities towards bacterial strains than fungal ones. Compound 9d has a low MIC and acts against all resistant bacterial (P. aeruginosa and S. aureus, MIC: 0.42 and 1.84 g/mL, respectively) and fungal (Candida albicans, MIC: 2.17 g/mL) strains, indicating that it has a significant antibacterial and antifungal activity. Compounds 5a, 5b, 5c, 5e, 5f, 9b, 9c, and 13, on the other hand, show no effect on the azole-resistant C. albicans ATCC10231 fungus. The majority of the studied molecules demonstrate different degrees of activity towards the resistant S. aureus (MRSA) TCC-BAA-1720. Compounds 5d, 9b, and 14 appeared to be the most effective. Compound 5d was more effective than the reference drug vancomycin against the sensitive Pseudomonas aeruginosa ATCC 10145 and resistant Pseudomonas aeruginosa ATCC BAA-2108. Compounds 5c, 5d, 9b, 9c and 13 showed no activity against the resistant Pseudomonas aeruginosa ATCC BAA-2108. In addition, compound 14 shows a good antimicrobial activity against S. aureus and P. aeruginosa (MIC: 1.13 and 1.49 µg/mL, respectively). In order to correlate the in silico results with those of the experimental antibacterial testing, SAP and FabI receptors were chosen for docking with the tested compounds.

Molecular Modeling
At the B3LYP/6-311G level of theory, the geometries of the synthesized molecules that demonstrated the greatest biological activity in the XTT experiments (5d, 5e, 9c, and 9d) were investigated ( Figure 1). The findings revealed that the molecules under investigation are nearly planar. The highest occupied molecular orbitals (HOMO) are noticed on the substituted phenyl and thiazole rings in all of the investigated compounds, whereas the lowest unoccupied molecular orbitals (LUMO) are found on the pyridazine-3,6-dione rings. Molecular orbital analysis can give information about the reactivity and excitability of the studied molecules. From HOMO/LUMO analysis, it can be concluded that molecules with narrow energy gaps (e.g., 5d and 9c) may show better reactivity/excitability than those having wide energy gaps (e.g., 5e and 9d).
The quantum mechanical descriptors of the picked molecules are summarized in Table 2. The energy gaps between HOMO and LUMO were discovered to be in the range of 2.87 to 3.06 eV, with 5d having the smallest energy gap.
Molecular docking was used to study the ligand-receptor interactions that may result in the obtained biological activities of the studied molecules. Thiazole derivatives have been reported to exhibit strong antibacterial activity against Staphylococcus aureus and Candida albicans. As a result, the studied candidate chemicals have strong antibacterial activity against these two pathogens. Furthermore, antibacterial activity against Pseudomonas aeruginosa was established by the substances under investigation. Accordingly, we chose the most appropriate receptors from the organisms mentioned above for molecular docking investigations.
During disseminated/mucosal infections of Candida albicans, secreted aspartic proteinase (SAP) plays a key function as a virulence factor. This receptor is assumed to be involved in the fungus' attachment and invasion, and so plays a role in its pathogenicity. As a result, SAPs may be useful as pharmacological target receptors for candidiasis treatment [48].
Staphylococcus aureus is a common Gram-positive bacterium that can cause wound infections and staphylococcal scalded skin syndrome (a cutaneous reaction to a staphylococcal exotoxin absorbed into the circulation) [49]. One of the essential components of the FAS II system (a group of fatty acid synthases used by most of bacteria and plants to catalyze fatty acid synthesis) is enoyl-[acyl-carrier-protein] reductase (FabI). Other bacteria, such as Pseudomonas aeruginosa, require this enzyme as well. In order to correlate the in silico results with those obtained from the experimental antibacterial tests, SAP and FabI were chosen for docking with the compounds of interest. The quantum mechanical descriptors of the picked molecules are summarized in Table 2. The energy gaps between HOMO and LUMO were discovered to be in the range of 2.87 to 3.06 eV, with 5d having the smallest energy gap. Molecular docking was used to study the ligand-receptor interactions that may result in the obtained biological activities of the studied molecules. Thiazole derivatives have been reported to exhibit strong antibacterial activity against Staphylococcus aureus and Candida albicans. As a result, the studied candidate chemicals have strong antibacterial activity  were chosen for docking with the compounds of interest. Figure 2 and Figure 3 depict the layouts of the receptors under investigation. and their interactions with the studied ligands. Molecular docking revealed that compounds 5d, 9c, and 9d are the best ligands for SAP2 of Candida albicans, FabI of S. aureus, and FabI of P. aeruginosa, respectively. The calculated docking scores were found to be −11.35, -11.30 and −11.36 kcal/mol for SAP2 of C. albicans/5d, FabI of S. aureus/9c and FabI of P. aeruginosa/9d, respectively.   silico results with those obtained from the experimental antibacterial tests, SAP and FabI were chosen for docking with the compounds of interest. Figure 2 and Figure 3 depict the layouts of the receptors under investigation. and their interactions with the studied ligands. Molecular docking revealed that compounds 5d, 9c, and 9d are the best ligands for SAP2 of Candida albicans, FabI of S. aureus, and FabI of P. aeruginosa, respectively. The calculated docking scores were found to be −11.35, -11.30 and −11.36 kcal/mol for SAP2 of C. albicans/5d, FabI of S. aureus/9c and FabI of P. aeruginosa/9d, respectively.   The in silico studies revealed that the of interaction of ligand 5d with SAP2 of C. albicans occurs via the hydrogen-aryl interaction between the aryl group of the ligand and the Asp 218 residue, and between the quinoid ring of the ligand and the Ile 119 amino acid. Whereas ligand 9c interacts with FabI of S. aureus through hydrogen bonding with Val D67 amino acid residue. In addition, compound 9d interacts with FabI of P. aeruginosa by aryl interaction with Tyr D149 and via the formation of a hydrogen bond with Tyr D159.
By comparing the results of in vitro XTT assay with those of the molecular docking study, it can be obviously noticed that there is an excellent agreement between them. For instance, compound 5d, which shows the best docking score with SAP2 of C. albicans, is active against both C. albicans and S. aureus as shown in Table 1. In addition, ligand 9c, which demonstrated the best binding to Fab I of S. aureus, was found to be inactive against all microorganisms but S. aureus, as indicated from the XTT assay; thus, confirming the accuracy of the docking studies. Furthermore, compound 9d interestingly demonstrated a better antimicrobial activity against P. aeruginosa (MIC = 0.24 µg/mL) than the standard molecule, vancomycin (MIC = 0.49 µg/mL). This agrees with the activity predicted from docking which revealed that ligand 10d has the best docking score (−11.36 kcal/mol) amongst all the theoretically studied ligands.

General Experimental Procedures
Melting points were measured with an Electrothermal IA 9000 series digital melting point apparatus. IR spectra were recorded in potassium bromide discs on PyeUni-camSP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz ( 1 HNMR) and run in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyzes were measured by using a German made ElementarVario LIII CHNS analyzer. Irradiation was done in an ultrasonicator, (Electric supply: 230 v, A.C. 50 Hz, 1phase; Ultrasonic frequency: 36 KHz; Ultrasonic power: 100 W). Maleic anhydride 1, thiosemicarbazide 2, chitosan, aniline and pyridine were purchased from Sigma Aldrich Kingdom of Saudi Arabia and were used without further purification. Hydrazonoyl halides 3a-f, 7a-e and bis-hydrazonoyl halides 12a,b were prepared according to the reported methods [50,51].

Synthesis of Thiazole Derivatives 5a-f, and 9a-e
An equivalent amount of glacial acetic acid (0.5 mL) was added to a solution of maleic anhydride 1 (0.98 g, 1 mmol), thiosemicarbazide 2 (0.92 g, 1 mmol) in ethanol (20 mL). The reaction mixture was heated in microwave oven at 500 W and 150 • C for 2 min. Then, the appropriate hydrazonoyl halides 3a-f or 7a-e and chitosan (0.1 g) were added, the reaction mixture was further heated in microwave oven at 500 W and 150 • C until all the starting material was consumed (4-8 min. as monitored by TLC). The hot solution was filtered to remove chitosan and excess solvent was removed under reduced pressure. The reaction mixture was triturated with methanol and the product separated was filtered, washed with methanol, dried and recrystallized from EtOH or DMF to give products 5a-f and 9a-e, respectively. The analytical and spectral data of the products 5a-f and 9a-e are listed below.

Reaction of 6 with 3a
Equimolar amounts of carbothioamide 7 (0.171 g, l mmol) and 2-oxo-N-phenylpropane hydrazonoyl chloride 3a (0.196 g, mmol) in ethanol (15 mL) containing an equivalent amount of chitosan (0.1 g) was heated in a microwave oven at 500 W and 150 • C for 5 min. as monitored by TLC. The hot solution was filtered to remove chitosan and excess solvent was removed under reduced pressure, gave product identical in all respects (m.p., mixed m.p. and IR spectra) with compounds 5a.

Coupling of 11
To a solution of each of compound 10 (0.211 g, 1 mmol) with sodium acetate trihydrate in ethanol (10 mL) was added benzenediazonium chloride solution, (prepared as usual by diazotizing aniline (1 mmol) in hydrochloric acid (1 mL, 6 M) with sodium nitrite (0.07 g, 1 mmol) in 10 mL water) portion wise with stirring and cooling. After complete addition, the reaction mixture was left for 12 h. in the refrigerator. The precipitate formed was collected by filtration, washed with water, dried and then recrystallized from EtOH to give the respective product identical in all respects with 9a.

Synthesis of Bis-Thiazole 13 and Bis-Thiazolone 14
To a solution of maleic anhydride 1 (0.196 g, 2 mmol), thiosemicarbazide 2 (0.184 g, 2 mmol) in ethanol (20 mL), an equivalent amount of glacial acetic acid (1 mL) was added. The reaction mixture was heated in microwave oven at 500 W and 150 • C for 2 min. Then, the appropriate bis-hydrazonoyl halides 12a,b (1 mmol for each) and chitosan (0.2 g) were added, the reaction mixture was further heated in microwave oven at 500 W and 150 • C until all the starting material was consumed (8 min as monitored by TLC). The hot solution was filtered to remove chitosan and excess solvent was removed under reduced pressure. The reaction mixture was triturated with methanol and the product separated was filtered, washed with methanol, dried and recrystallized from ethanol to give products 13 and 14, respectively.

In Vitro XTT Assay
XTT assay, a non-radioactive colorimetric assay system, is usually used for measuring cell viability, proliferation and cytotoxicity through the measurement of cellular metabolic activity. This test depends on the reduction of a yellow tetrazolium salt (XTT dye) to an orange formazan dye by metabolically active cells. The minimal inhibitory concentration (MIC) values, which represent the lowest concentrations of samples or standard drugs (Vancomycine for bacteria and Amphotricine B for fungi) that completely inhibit the microbial growth. MICs were determined using the microdilution method. The bacterial inoculum was prepared, and the suspensions were adjusted to 10 6 CFU/mL. The samples under investigation and the standard drugs were prepared in dimethyl sulfoxide (DMSO) and subsequent twofold dilutions were performed in a 96-well plate. Each well of the microplate included 40 µL of the growth medium (Brain Heart Infusion, BHI), 10 µL of the inoculum and 50 µL of the investigated compounds diluted to final concentrations of (1000-0.12 µg/mL), and DMSO was used as a negative control. The plates were incubated at 37 • C for 24 h. Thereafter, 40 µL of tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-car-boxanilide (XTT)} were added. The plates were incubated in dark for 1 h at 37 • C, after which colorimetric change in the XTT reduction assay was measured using a microtiter plate reader (Tecan Sunrise absorbance reader; Tecan UK, Reading, United Kingdom) at 492 nm. The MIC was detected as the lowest concentration capable of causing the largest color change compared to the negative control [52].

In Silico Studies
The electronic properties of the synthesized derivatives that demonstrated the best biological activities in the in vitro XTT assay were investigated with density functional theory calculations. The calculations were carried out with the aid of Gaussian 09 [53]. The geometry of the studied molecules was fully optimized using B3LYP/6-311G functional and the obtained molecular orbitals were visualized.
Molecular docking was used to investigate the interaction of the best biologically active molecules with the microbial receptors. We selected the most probable bacterial/fungal proteins that can be affected by the synthesized thiazole ligands based on the results previously reported in the literature.

Conclusions
In summary, we have developed a new green methodology and synthesized several novel 1-thiazolylpyridazine derivatives by MWI in high, efficient yields and short reaction time. Additionally, the antimicrobial activities of the candidate lead molecules were tested against S. aureus, P. aeruginosa and C. albicans using the XTT assay and compounds with the highest activity in terms of MIC were docked with the corresponding microorganisms' receptors. The results depict that compound 5d shows comparable biological activities to these of the standard antibacterial/antifungal drugs in case of S. aureus and C. albicans. In addition, compound 9d demonstrated the highest activity against P. aeruginosa.