Design, Characterization, X-ray Single-crystal, Potentiometric Measurements, Molecular Modeling and Biomedical Applications of Thiosemicarbazones


 Series of thiosemicarbazone compounds ((E)-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide (TSC1), (E)-N-ethyl-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide (TSC2) and (E)-N-phenyl-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide)(TSC3) were synthesized and fully characterized by assistance of diverse physicochemical and spectroscopic tools like X-ray single-crystal, IR, mass, 1HNMR, Uv-Vis,…etc. potentiometric measurements, molecular modeling, as well as biological and antitumor activities screening. We have calculated and discussed the thermodynamics and protonation constants of TSC1 compound as a representative from the novel synthesized thiosemicarbazones. The solution speciation of different species was studied in accordance with pH. Molecular parameters of the optimized structures were calculated and discussed. The X-ray single crystal of TSC2 and TSC3 compounds have been established where TSC2 crystallizes in P21/c, a = 11.2343 (6) Å, b = 11.2575 (7) Å, c =11.8995 (8) Å, α = 90.00°, β = 94.476(7) °, γ = 90.0°, V = 1500.34 (16) Å3, Z = 4, however, TSC3 crystallizes in the space group P21/c, a = 27.958 (12) Å, b= 12.072 (5) Å, c = 9.833 (4) Å, α = 90.0°, β = 93.117(11) °, γ = 90.0°, V = 3486.75 Å3, Z = 7. Considering the antimicrobial activities and correlating structure-activity relationship for the synthesized compounds, TSC1 molecule behaves as a promising candidate as an antifungal agent versus Candida albicans. Consequently, that would be very helpful in the field of medicinal chemistry especially as antimicrobial agents. The results are of vital significance to the chemistry of antimicrobial agents.


Introduction
From the most global problems, infectious diseases represent a high burden in public health worldwide. Due to the high resistance of some Gram-positive and Gram-negative bacteria to many drugs, large number of infection diseases become a real dangerous that threaten the human life worldwide [1]. Globally, from more than 50 million infected people up to 110.000 of them die annually. By the middle of 21 st century, it is expected that the mortality rate caused by Gram-negative bacterial infection alone could possibly be increased up to ten million deaths a year [2]. Antibiotics are the main base for microbial (bacterial and fungal) infection therapy. Antibiotic overuse, indeed, has become the main cause in the appearance and spread of multi-drug resistant strains of many microbes [3]. Emergence and increasing prevalence of antibiotic resistant bacterial strains to available antibiotics urge the discovery of new therapeutic approaches [4] additionally; the available drugs are also expensive or have a lot of unwanted side effects [5]. Therefore, the necessary to get novel antimicrobial agents is of vital significance given the evidence of fast global spread of resistant clinical isolate.
Taking into consideration, the relation between bacterial infection and multi-drug resistant, the present investigation was developed to search for a new antimicrobial effective drug.
Nowadays, there is a significant concern in the medicinal chemistry of Schiff-base compound like hydrazones and thiosemicarbazones due to their wide biological activities [6,7]. The groups of C=N and N-C=S are of great interest in chemotherapy and are responsible for the pharmacological activity. Perhaps the most essential step in the metal complexes implementation is the synthesis of a novel compounds that exhibit unique properties as well as reactivity, in this regards thiosemicarbazones were a topic of interest to researchers of various profiles. Thiosemicarbazone compounds and their complexes have been widely investigated as they display very interesting properties in the field of biomedicine as well as potential medicinal applications [8][9][10][11][12] which comprise antiparasital [13], antibacterial [14] antitumor [15], antiviral [16], fungicidal [17], antineoplastic [18] and antiamebic [19] activities. Thiosemicarbazones (TSCNs) may have thione (A) and thiol (B) forms (Scheme 1). Hydrazones were a crucial class of compounds; these compounds have impressive ligation characteristics due to the existence of several coordination sites [20]. The literature also reports hydrazine and its derivatives that have anti-inflammatory, analgesic [21], antibacterial [22] and antitumor [23] activity.
Nevertheless, cadmium is a very toxic metal ion that poses both human and animal health hazards. Its toxicity is done by its easy localization inside the liver, and then by binding of metallothionein, which eventually forms a complex and is transmitted into the blood stream to be lodged in the kidney.
The cause of Cd-toxicity is the negative effect on cell enzyme systems that are the consequence of metallic ion substitution (mainly Zn(II), Cu(II), and Ca(II)) into metalloenzymes and its large affinity to thiol group compounds [24]. Zn(II) replacement with a chemically analogous Cd(II) ion usually causes apoprotein catalytic activity to break down [25,26]. Therefore, it is of paramount importance to discover novel compounds that can form stable complexes with Cd(II), because they can be used as detoxifiers. Referable to the broad scope of pharmacological properties of thiosemicarbazone compounds and their compounds, these compounds can also very well fit for this role. Recently, the experimental studies were supplemented by computational studies [27] owing to their crucial role in recognizing the likely attitudes of the compound during reactions and recognition of valuable information on the compounds under examination, such as total energy, binding energy, electronic energy, dipole moment, bond length, HOMO and LUMO [28]. With this in mind and in the perpetuation of our studies in the subject area of bioactive compounds [29,30], it seems of great interest to synthesize and identify novel compounds involving both thiosemicarbazone and hydrazo moieties. In addition, our aim is to explore biological activities of identified compounds.

Materials and reagents
All the chemicals used in this study were supplied by Aldrich Chemicals Company and used with no extra purification.

Potentiometric titrations
Through potentiometric technique using the method depicted above in the literature [36], the formation constant of complex was estimated. The standard buffer solutions are used for accurately calibrating the glass electrode to NBS standards [37].
Where log10 UH = solvent composition correction factor and the ionic strength read by B. pKw for titrated samples were estimated as previously described [40]. All precautions and procedures comply with literature requirements [41][42][43].

Processing of data
MINIQUAD-75 computer program has been applied to calculate ca. 100-150 readings for each titration [44]. Species distribution diagrams for the studied samples were given by the SPECIES program [45].

Molecular modeling studies
DFT calculations were performed using DMOL 3 program [46,47] in Materials Studio package [48]. Calculations for DFT semi-core pseudopods (dspp) were created with dual numerical base sets and polarization properties (DNP) [49]. The RPBE model is focused on the (GGA) generalized gradient approximation as the best functional approximation [50,51].

In vitro antibacterial activity
Ability of the synthesized thiosemicarbazone compounds to suppress bacterial growth was checked by the disc diffusion process, [52,53]

In vitro antitumor activity
The synthesized thiosemicarbazone compounds were screened for their cytotoxicity against liver cancer (HepG2) and breast cancer (MCF-7) cells by using SRB assay protocol [54].
Potential cytotoxicity of the compounds was tested using the method of Skehan and

UV-Vis spectrum
The strong absorption band detected at 33003-32787 cm -1 were assigned to π→ π * transitions (C=N)azomethine group while the possible assignments for the bands at 26809-27548 cm -1 are attributed to the n → π * thiosemicarbazone compounds transitions, respectively.

Mass spectra
The proposed formulae can be further proven by mass spectroscopy. The electron impact mass spectrum of TSC1 confirms the suggested formula by displaying a peak at 249 equivalents to (C11H15N5S) compound moiety in addition to a series of peaks which attributable to different fragments of TSC1 compound. These data suggest that a ketone PTHP group is condensed with the NH2 group of thiosemicarbazide or its derivatives. The mass spectra of TSC1, TSC2 and TSC3 showed peaks at 249, 277 and 325 confirming the proposed structural formula of the synthesized TSC1, TSC2 and TSC3 compounds respectively.

Crystallography
The structure of the two of the representing thiosemicarbazone compounds (TSC2 and TSC3) was established through X-ray crystallography. Recrystallisation of the compounds from hot ethanol followed by slow evaporation leads to formation of single crystals. Data of TSC2 and TSC3 are summarized in the Table 1, CCDC 2026108 and CCDC 2033322 contain the supplementary crystallographic data for this paper.
The X-ray single-crystal structures of monomeric TSC2 and TSC3 compounds were given in Fig. 2a and 2b, respectively. It suggested that TSC2 was crystallized in monoclinic It is worth to mention that TSC2 molecules are stacked non-covalently altogether via inter-molecular interactions i.e. van der Waals as well as H-bonding interactions. This is can be clearly illustrated in Fig. 3. The packing structures of TSC2 and TSC3 compounds is shown in the Supplementary Fig. S5a and S5b, respectively. In the packing structure of TSC2 and TSC3 molecules, the unit cell includes four and seven molecules stacked to each other's per unit cell, respectively. The inverse of the global hardness is called softness σ [66].
Since the geometric optimization of the prepared compounds can be characterized using theoretical calculations; therefore, the optimized structure for the synthesized compounds could be obtained by calculating theoretical physical parameters like bond lengths and bond angles using DFT calculations.
Quantum parameters of the synthesized TSC1, TSC2 and TSC3 compounds have been calculated using Eqs. 2-11. From data given in Table 2 and Table 3 j) The HOMO level in the thiosemicarbazone compounds are commonly localized on the C=N groups demonstrating they are the favored sites for Nu attack at the central metal ion. k) Negativity of EHOMO and ELUMO indicates thiosemicarbazone compounds stability [71].

Antibacterial and antifungal activities:
Antimicrobial activities of thiosemicarbazone compounds were screened. Results of antimicrobial activity of thiosemicarbazone compounds versus all tested microbes are shown in Table 4

Antitumor activities:
Cytotoxic study of thiosemicarbazones was investigated versus liver cancer cell line

Molecular modeling and biological activity
Theoretical calculations were performed with the purpose of physicochemical properties investigation that possibly correlated to the antimicrobial action of investigated thiosemicarbazones. From the obtained data, we can deduce that: a) TSC1 thiosemicarbazone compound, which offered the lowest value of HOMO energy among the synthesized thiosemicarbazone compounds, showed the highest biological activity among the synthesized compounds.
b) Inverse relation between dipole moment and lipophilicity indicates that as dipole moment decreases, the lipophilic nature of the compound increases, which favors its penetration more powerfully via lipid layer of microorganism [72,73], thus destroying them more violently.
From the results in Table 3, the lipophilicity of the TSC1 is larger than the other thiosemicarbazone compounds, which sequentially deactivates enzymes accountable for respiration process of the investigated microbes more than other compounds and accordingly increase its cellular uptake by bacterial cells.
c) Thiosemicarbazone compounds have antibacterial activity due to existence of toxophorically essential imine groups (-C=N) where the action mode of these compounds could include formation of H-bonds via azomethine group with an active center of cells which may interfere with ordinary cell processes [74].

Equilibrium Studies
The protonation constants of TSC1 ligand are calculated (  (17) We can conclude that the 1 st and 2 nd deprotonation constants correspond to the deprotonation of the two N-imino sites in TSC ligand as given in Scheme 3a. While the 3 rd deprotonation constant correspond to the thiolate group site in TSC ligand as shown in Scheme 3b.
However, a similar conclusion was obtained in literature [75]. and/or where they coexist, as well as their relative formation percentages. Thus, the species distribution graph is a good tool for obtaining complete picture about the concentration of each species present as a function of pH. It enables us to obtain the best conditions for preparation a solid complex as pH, concentration and ligand: metal ratio. At low pH, (TSC1) exists initially in a fully protonated form with maximum percent of 100% as H3L below pH < 2. On addition of base, pH value increases so the (H3L) species loses its first proton from an imino group to form (H2L), which is the major species at pH = 3.3. As conditions become more alkaline, the second proton released from the second imino group begins deprotonation to HL ligand accomplish highest percent of 99.1 % at pH = 5.8. More increase of pH is followed by liberation of the third H + forming the fully deprotonated ligand L with maximum percent species 98.4% at pH = 10.0.

Species distribution curves
The calculation of equilibrium complex concentrations of Cd(II) with TSC1 (Table   7) as a function of pH gives a valuable picture of metal(II) binding in the biological system. As an illustrative example of metal complexes, Fig. 4b showed a concentration distribution diagram for the complex Cd(II)-TSC1. The Cd-TSC1 complex begins to form in acidic pH range reaching a constant concentration of 99.9 % at pH = 5.0, whereas Cd(TSC1)2 complex species reaches a maximum concentration of 45 % at pH 9.8.

Thermodynamics
The data derived for H o , S o and G o related to protonation of TSC1 and Cd(II)complex formation were calculated from the data tabulated in tables 8 and 9. H o for the ligand protonation or complexation was determined from the plot slope ( Fig. 5a-b) through graphical representation of the Van't Hoff equation With the well-known relations (6) and (7), from the values of (G) and enthalpy change (H), one can calculate (S): The main reasons for the protonation constant determination can be explained as follows: (1) The ratio and pH of the various substance forms can be determined using its protonation constants.
(2) Very useful in preparation of newly synthesized compounds. The suggested structure can be reliable where protonation constants are theoretically well calculated according to the experimental values. Additionally, their protonation constants are used for calculating the stability constants of the dynamic formation of bioactive compounds with metal ions [81]. (6) The equilibrium constants of certain compounds must be understood to measure concentration of each ionized species at pH which is fundamental to understand their physiochemical behavior [81]. Tables 8, 9 describes the thermodynamic functions measured and can be interpreted as: 1. The corresponding thermodynamic processes for the protonation reactions are: a) Exothermic processes for neutralization reactions; ii) Endothermic for ions desolvation; iii) Structure alteration and alignment of H-bonds around protonated and free ligands.
2. When the temperature rises the value of log10 K H decreases and its acidity rises as the temperature rises.
3. Negative ∆H o for protonation of TSC1 ligand indicates that this process is exothermic followed by heat release.
4. Positive entropy of TSC1 protonation reaction indicates increased disorder due to desolvation processes and breakdown of H-bonds. These results provide the following findings: 1-Negative ∆H o show that the coordinating process is exothermic suggesting that the metalligand bonds are fairly strong and complexity reactions are preferred at low temperatures.          (4)    # http://www.iucr.org/resources/cif/spec/version1.1/semantics#namespace # A reserved prefix, e.g. foo, must be used in the following way # " If the data file contains items defined in a DDL1 dictionary, the # local data names assigned under the reserved prefix must contain it as # their first component, e.g. _foo_atom_site_my_item. " # However, this seems to say the opposite: # http://www.iucr.org/__data/iucr/cif/standard/cifstd8.html # According to advice from the IUCr, CRYSTALS is correct