Synthesis and In-silico Simulation of Some New Bis-thiazole Derivatives and Their Preliminary Antimicrobial Profile: Investigation of Hydrazonoyl Chloride Addition to Hydroxy-Functionalized Bis-carbazones

2.1 Reaction chemistry

To kick-start our synthetic strategy, the common bis-thiosemicarbazone precursor 3 was constructed when one equivalent of hydroxyl-tagged bis-aldehyde 1 was condensed with two equivalents of thiosemicarbazide 2 in refluxing ethanol, acidified with HCl, for 2 h to give the new hydroxyl-bearing bis-thiosemicarbazone 3 as depicted in Scheme 1. The bis-aldehyde 1 itself was synthesized via reacting two equivalents of salicylaldehyde with epichlorohydrin in NaOH solution as previously reported by Bin, et al. (Zhao et al., 1996) (Scheme 1).

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Scheme 1

Synthesis of bis-thiosemicarbazone3.

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The chemical reactivity of the new hydroxyl-bearing bis-thiosemicarbazone 3 towards a host of hydrazonoyl chlorides 4 was evaluated to construct a new group of bis-thiazoles. Therefore, reaction of bis-thiosemicarbazone 3 with hydrazonoyl chlorides 4a-g in refluxing dioxane in the presence of catalytic triethylamine delivered the target bis-thiazoles 6a-g (Scheme 2). Elucidation of all the new bis-thiazole derivatives was executed using various spectral analyses. For example, the IR spectrum of compound 6f revealed typical –NH absorption at the expected wave number v 3410 cm⁻¹. Also, ¹H NMR spectrum of 6f revealed four typical singlets for CH₃, CH-OH, CH = N, and NH groups at δ 2.60, 5.36, 8.51, and 11.36 ppm, respectively. The characteristic multiplet for the two CH₂ groups is visible at δ 4.19–4.25 ppm, in addition to the C-H multiplet shown at δ 5.40–5.45 ppm.

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Scheme 2

Synthesis of bis-thiazolesderivatives 6a-g.

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In a similar route, hydroxyl-tagged bis-thiosemicarbazone 3 reacted with N-aryl-2-oxopropanehydrazonoyl halides 7a-e (the ester counterparts of compounds 4a-e) in refluxing dioxane and catalytic triethylamine, leading to the formation of the expected bis-thiazole derivatives 9a-e (Scheme 3). Elucidation of the new bis-heterocycles 9a-e was substantiated typically by extensive spectral analysis. For example, the IR spectrum of derivative 9c showed the presence of –NH absorption at 3396 and 3281 cm^-1characteristic for the four –NH groups. Further structural confirmation of the isolated bis-thiazole derivatives 9a-e was based on their ¹H NMR spectra which contained four singlets for the CH-OH, CH = N and 2NH groups near δ 5.36, 8.50, 9.42, and 11.37 ppm, respectively.

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Scheme 3

Synthesis of bis-thiazole derivatives 9a-e.

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In a similar fashion, the reaction of compound 3 with phenacyl bromides 10a-c in dioxane under reflux using catalytic triethylamine, afforded bis-thiazoles 12a-c (Scheme 4). Structural analysis of the new bis-thiazoles was established using both spectral and elemental analyses. For example, the IR spectrum of product 12c revealed –NH absorption at 3412 cm⁻¹. Moreover, the ¹H NMR spectra of the isolated compounds 12a-c showed the characteristic four singlets at δ 3.78, 5.59, 8.93, and 11.35 ppm due to 2OCH₃, CH-OH, thiazole-CH and 2NH, respectively. Also, two multiplets were shown at δ4.21–4.24 and 6.43–6.45 corresponding to the 2CH₂ and the CH-OH groups, respectivly.

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Scheme 4

Synthesis of bis-thiazole derivatives 12a-c.

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2.2 Molecular geometries

The molecular geometries of the studied compounds were subjected to energy minimization by implementing the B3LYP/6-311G level of theory. The obtained optimized structures and their corresponding frontier molecular orbitals (FMOs) are illustrated in Fig. 1. The calculated total energies are −83,764.15, −221,680.97, −83,579.12, −108,592.06 and −213,585.72 eV for 6c, 6f, 9b, 9d, and 12c, respectively. Based on their relative energy values, compounds stability follows the following order: 6f > 12c > 9d > 6c > 9b. The quantum mechanical descriptors of the investigated molecules are collected in Table 1.

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Fig. 1

Optimized molecular geometries of compounds 6c, 6f, 9b, 9d and 12c and their corresponding MOs. The isovalue used for visualization of the MOs is 0.02. Color codes: C: grey, O: red, H: white, N: blue, Cl: green, Br: brown, S: yellow.

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From the DFT energy calculations, some quantum chemical descriptors (Table 1) can be obtained that give information about the reactivity, complex formation with transition metals, the ability to donate/accept electrons, etc. For instance, ionization energy (I) and electron affinity (A) are two properties that can indicate the reactivity of the molecules. They can be calculated as follows: I = − E_HOMO and A = − E_LUMO (Abo Dena et al., 2019). Moreover, the complexation with transition metals requires having information about the softness (S) and/or hardness (η) of the molecules. Hardness is calculated from η = (I − A)/2 and softness is calculated as the reciprocal of hardness. Furthermore, during charge-transfer reactions, chemical potential (μ = − (I + A)/2), Mullikan electronegativity (χ = (I + A)/2) and electrophilicity index (ω = μ²/2η) measure the lowering in energy that result from the maximal electron flow from the donor molecule to the acceptor molecule (Abo Dena and Abdel Gaber, 2017; Abo Dena and Hassan, 2016; Hassan and Abo Dena, 2014).

In order to make it easier to predict the chemical behavior/reactivity of the investigated organic molecules, the electrostatic potential maps (EPMs) were visualized (Fig. 2). In EPMs, areas of low electrostatic potential energy are shown in red color (nucleophilic sites), while blue areas indicate parts of low electron density (electrophilic sites). The compounds of interest contain areas susceptible for electrophilic addition, for instance the N atom of the thiazole rings and the OH groups. These are considered as electron-rich areas due to the localization of electron lone pairs on nitrogen and oxygen atoms. On the other hand, common functional groups or molecular parts that act as suitable sites for nucleophilic attack are represented by all aromatic and/or aliphatic hydrogens due to the strong electron attraction by the attached aromatic groups.

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Fig. 2

Electrostatic potential maps of compounds 6c, 6f, 9b, 9d, and 12c. The isocalue of the electrostatic potntial ssurfaces is 0.0004.

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Interestingly, Frontier molecular orbitals are not only used for calculating the above-mentioned quantum mechanical descriptors, but also they can give information about light excitability and electron transfer through the molecules. HOMO refers to the highest occupied molecular orbital, while LUMO is an abbreviation for the lowest unoccupied molecular orbital. It is clear from the data illustrated in Table 1 that compound 6f (ΔE = 2.94 eV) has the highest light excitability as well as the highest heterogeneous electronic charge transfer among all of the investigated candidate compounds. Meanwhile, compound 12c (ΔE = 3.9) demonstrates the lowest light excitability and the lowest ability to catalyze heterogeneous electronic charge transfer. Due to the bilateral symmetry of the molecules, HOMOs are always delocalized in an opposite side to LUMOs of the same molecule. According to the above-mentioned ΔE values, it is expected that compound 12c will absorb higher light energy (i.e. shorter wavelength) than compound 6f.

2.3 XTT assay results

The cell proliferation kit II (XTT) assay was performed to find the minimum amounts (a.k.a. minimum inhibitory concentration, MIC) of the compounds that inhibit cell viability/metabolism of P. aeruginosa, S. aureus and C. albicans. The results obtained from the XTT assay are summarized in Table 2. Obviously, compound 9b has excellent antibacterial (P. aeruginosa, MIC: 0.12 – 1.95 μg/mL) and antifungal (C. albicans, MIC: 0.98 – 3.90 μg/mL) activities, as reflected by the MIC values, compared to the standard antibiotic molecules, vancomycin, amikacin, and amphotericin B. In addition, molecule 12c demonstrated an acceptable antibacterial activity against S. aureus (MIC: 0.98 – 1.95 μg/mL).

Table 2 showed that:

• Most of the studied molecules demonstrated greater antimicrobial activities against bacteria than fungi.

• All molecules showed different degrees of biological activities towards antibiotic-resistant Staphylococcus aureus (MRSA) TCC BAA-1720. The activity of compound 9b (0.49 µg/mL) was greater than the standard antibiotic (0.98 µg/mL); however, compounds 6c, 6f, 9d and 12c showed the same effect (1.95 µg/mL).

• The activity of compound 9b against the antibiotic-sensitive Pseudomonas aeruginosa ATCC 10145 and the antibiotic-resistant Pseudomonas aeruginosa ATCC BAA-2108 was greater than that of vancomycin that showed an MIC value for the antibiotic-resistant strain similar to that of compound 9d.

• On the other hand, all compounds did not have antimicrobial activity against Azole- resistant Candida albicans ATCC10231 fungus except compound 9b.

• Compounds 9b demonstrated a high antimicrobial effect against the antibiotic-resistant P. aeruginosa ATCC BAA-2108, and compound 9d shows an acceptable antimicrobial activity in case of P. aeruginosa and S. aureus (MIC: 0.12 and 0.49 μg/mL, respectively).

• What’s more, the antimicrobial activity of molecule 3 is very powerful as revealed from its very low MIC value. The compound was able to act against all resistant bacterial (P. aeruginosa and S. aureus, MIC: 0.49 and 3.9 μg/mL, respectively) and fungal (C. albicans, MIC: ND μg/mL) strains.

• Most of the synthesized molecules show very strong (MIC < 10 µg/mL) biological activity and few of them demonstrate either strong (MIC 10–25 µg/mL) or good (MIC 26–125 µg/mL) bioactivity according to the ranking reported elsewhere (O’Donnell et al., 2010).

2.4 Molecular docking

Molecular docking is a molecular modeling technique that helps in studying how two or more molecules fit together; for instance, a drug and an enzyme. It represents a potent tool in drug design and discovery of lead molecules. In the present study, this technique was applied to the investigated compounds to predict their biological activities. Reportedly, thiazoles and bis-thiazoles usually have powerful biological activities against C. albicans and S. aureus. Accordingly, those microorganisms were significantly influenced by treatment with the synthesized bis-thiazoles. The investigated compounds showed high toxicity to Pseudomonas aeruginosa. Consequently, we have chosen the most suitable microbial receptor proteins that can be influenced by bis-thiazole ligands based on a thorough literature review (Mahmoud et al., 2020).

Secreted Aspartic Proteinase (SAP) plays a significant role as an important virulence factor in C. albicans disseminated/mucosal infections. The attachment/invasion of different Candida species, particularly C. albicans, is thought to be attributed to this protein, and hence it plays a crucial role in their pathogenicity. That is why SAPs can be a suitable target for drug intervention for candidiasis (Cutfield et al., 1995).

S. aureus is a common Gram-positive microbe which infects wounds resulting in increased incidence of staphylococcal scalded skin syndrome (a reaction of the skin to a staphylococcal exotoxin that can be absorbed into the blood stream) (Priyadarshi et al., 2010). Enoyl-[acyl-carrier-protein] reductase (FabI) is an enzyme of the FAS II system (a group of fatty acid synthases that catalyze fatty acids synthesis many types of bacteria and plants).

This enzyme is also crucial for other types of bacteria such as Pseudomonas aeruginosa. Accordingly, SAP and FabI were selected for with the studied molecules in order to relate the in-silico results with those of the experimental antibacterial tests.

Docking results indicated that the theoretical binding energies of the ligands are consistent with the experimental findings. For SAP (C. albicans), compound 9b showed the best binding as indicated by the calculated energy of binding (-10.304 kcal/mol). However, compound 12c demonstrated the highest binding energy (B.E., −8.736 kcal/mol) with the S. aureus protein, (FabI).

Finally, FabI receptor of P. aeruginosa shows the best affinity to compound 9b with a calculated B.E. of −9.583 kcal/mol. It is worth mentioning that the arene-H dominated the types of receptor-ligand interactions in all of the above-mentioned cases, Fig. 3.

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Fig. 3

Best receptor-ligand interaction poses (a, c and e) and ligand interaction diagrams (b, d and f) of compounds 9b (with SAP of C. albicans), 12c (with FabI of S. aureus) and 9b (with FabI of P. aeruginosa), respectively.

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Clearly, the empirical MIC values of the investigated compounds are in good agreement with the theoretically computed binding energies. Consequently, compound 9b may be suitable for inhibiting C. albicans and P. aeruginosa growth, while compound 12c is suitable for retarding the growth of S. aureus.

3.1 Synthesis and characterization

Melting points were determined on a Gallenkamp apparatus and are uncorrected. IR spectra were recorded via the KBr-disc method using a Pye-Unicam SP300 spectrophotometer. ¹H and ¹³C NMR spectra were recorded in deuterated DMSO‑d₆ using a Varian Gemini 300 NMR spectrometer (300 MHz for ¹H NMR and 75 MHz for ¹³C NMR) and the chemical shifts were related to that of the solvent DMSO‑d₆. Mass spectra were recorded with the aid of a GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spectrometers by applying an ionizing voltage of 70 eV. Elemental analyses were performed at the Microanalytical Centre, Faculty of Science, Cairo University, Giza, Egypt.

3.2 Synthesis of Bis-thiosemicarbazone derivative 3

A mixture of Hydroxy-bis-aldeyde1(3.0031 g, 0.01 mol), thiosemicarbazide(0.01 mol) and a few drops of conc. HCl in ethanol (30 mL) was refluxed for 3 h. The solid product which formed after cooling was collected and recrystallized from ethanol/dioxane as yellow crystals,72% yield; mp 229–230 °C; IR (KBr): v 3426 (OH), 3356,3252, 3147 (NH₂ and NH), 3062, 2986 (C-H) cm⁻¹; ¹H NMR (DMSO‑d₆):δ4.19–4.25 (m, 5H, 2CH₂, CH-OH), 5.39 (s, 1H, OH),6.96–6.98 (t, 2H, Ar-H), 7.10–7.12 (d, 2H, Ar-H), 7.35–7.39 (t, 2H, Ar-H), 7.92 (s, 2H, 2NH), 8.05–8.07 (d, 2H, Ar-H), 8.12 (s, 2H, 2NH), 8.50 (s, 2H, 2CH = N-), 11.37 (s, 2H, 2NH) ppm; ¹³C NMR (DMSO‑d₆): δ68.2 (CH-OH), 69.7(CH₂O), 113.1, 121.2, 122.8, 126.5, 131.7, 139.0, 157.5 (Ar-C and N = C), 178.2 (C = S) ppm; MS m/z (%): 446 (M⁺, 16), 435 (33), 410 (1 0 0), 373 (51), 320 (78), 299 (35), 210 (46), 126 (58), 60 (66)ppm; Anal. Calcd for C₁₉H₂₂N₆O₃S₂(446.54): C, 51.11; H, 4.97; N, 18.82. Found: C, 51.45; H, 4.65; N, 18.57%.

3.3 In-vitro XTT assay

XTT assay (Al-Bakri and Afifi, 2007; Kuhn et al., 2003; Liang et al., 2012; Monteiro et al., 2012) is a non-radioactive spectrophotometric assay usually used for measuring cell viability and proliferation via the assessment of the cellular metabolic activity. The assay depends on the chemical reduction of the XTT dye (a yellow tetrazolium salt) to an orange formazan dye by living cells. The minimal inhibitory concentration values represent the lowest concentrations of test or standard drugs (vancomycin and amikacin for bacteria, and amphotericin B for fungi) that completely inhibit microbial growth. For determining the MIC values, the microdilution method was applied. Briefly, the microbial inoculum was prepared and the concentrations of the suspensions were adjusted to 10⁶ CFU/mL. Solutions of the standard and test molecules were prepared in dimethyl sulfoxide (DMSO) and subsequent two-fold dilutions were carried out in a 96-well microplate. Each well of the microplate included 40 μL of the Brain Heart Infusion (BHI) growth medium, 10 μL of the microbial inoculum and 50 μL of the investigated molecules diluted to final concentrations of 1000–0.12 µg/mL. Pure DMSO was used as a blank. The plates were incubated for 24 h at 37 °C; thereafter, 40 μL of XTT tetrazolium salt were added. Consequently, the plates were incubated at 37 °C in a dark environment for 1 h, and then the color change was measured spectrophotometrically at 492 nm using a microtiterplate reader (Tecan Sunrise absorbance reader; Tecan UK, Reading, United Kingdom). MIC values were determined as the minimum concentrations that caused the largest color change compared to the blank.

3.4 Theoretical calculations

Geomtery optimizations were implemented, using DFT algorithms at the B3LYP/6-311G level of theory, with the aid of Gaussian 09 W package (Gomha et al., 2016a). The obtained molecular geometries and their corresponding molecular orbitals were visualized using GaussView 5.0.9 package. Becke’s three-parameter hybrid method (B3LYP) demonstrates more accurate results than the pure Hartree-Fock method (Gomha et al., 2016b). B3LYP represents the combinaton of the correlation functional of Lee, Yang and Parr with Becke’s three parameters (Lee et al., 1988).

3.5 Molecular docking

Molecular docking was performed using Molecular Operating Environment (MOE) 2014 software (Group, 2014). Compounds that showed the lowest MIC values in the XTT assay were selected and docked with the corresponding target proteins. The compounds were subjected to energy minimzation using the MMFF94x force field prior to docking. High resolution 3D molecular structures of the receptors (Table 3) were obtained from the Protein Data Bank (PDB).