Synthesis and in vitro anticancer activity of 6-chloro-7-methyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one derivatives: molecular docking and interaction with bovine serum albumin

ABSTRACT A novel series of 6-chloro-7-methyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one derivatives were synthesized. Structure of the newly synthesized compounds was established by their analytical and spectroscopic data. The title compounds were evaluated for their anticancer activity against human breast cancer (T-47D) and lung cancer (NCI-H226) cell lines. Effects of compounds on the cell morphology of these cell lines were studied. Among the series of compounds tested, 6-chloro-7-methyl-2-(4-((2-(piperidin-1-yl)ethylamino)methyl) phenyl)-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one [MTDP4(9)] exhibited good anticancer activity against both cell lines. Further, the binding interaction of [MTDP4(9)] with bovine serum albumin has been investigated by UV, fluorescence and molecular docking studies.


Introduction
Cancer is a potentially fatal disease causing 24% of deaths worldwide and stands next to the cardiovascular fatalities in the world [1]. Treatment of cancer remained as a major and challenging therapeutic problem. The search of novel, more potent and less toxic chemotherapeutic agents for its treatment is one of the most important and challenging tasks in medicinal chemistry. Hence, many research projects are aimed towards the search of new drugs for cancer therapy.
Pyrimidine scaffolds are important pharmacophores with a wide range of biological activities [2][3][4][5][6]. Pyrimidine ring is the important constituents of DNA and RNA; hence compounds containing pyrimidine skeleton exhibits diverse biological activities such as antineoplastic, antibacterial, antifungal, antiviral, anthelmintic, etc. [7]. There are numerous reports in the literature on the anticancer activity of pyrimidine derivatives [8][9][10]. Heterocyclic compounds containing 1,3,4-thiadiazoles have received the attention of medicinal chemists due to their broad spectrum of activities. These compounds have been reported to possess anticancer [11], antiviral [12], antimicrobial [13], anti-tubercular [14] and anticonvulsant activities [15]. The anticancer activity studies revealed that the pyrimidine-1,3,4-thiadiazole hybrids have exhibited potent activity [16,17]. In continuation of our search for new and effective anticancer agents, we have incorporated these heterocyclic systems in a CONTACT Imtiyaz Ahmed M. Khazi  single molecular framework to explore their anticancer potential [18].
Serum albumins are the most important transport proteins present in the blood plasma which carries fatty acids, amino acids and other small molecules. Bovine serum albumin (BSA) is the most abundant and frequently used globular protein for the binding interaction studies between drugs and protein because of its structural similarity with human serum albumin, low cost, binding properties and easy availability [19][20][21][22]. In present work, we explore the binding interaction of a compound MTDP4 (9) with BSA by fluorescence quenching and molecular docking studies.

General
General techniques and information about instruments are mentioned in the supplementary data.

5-p-tolyl-1,3,4-thiadiazol-2-amine (MTDP1)
A mixture of 4-methylbenzoic acid (5 g, 36.72 mmol), thiosemicarbazide (3.35 g, 36.75 mmol) and phosphorous oxychoride (50 ml) were taken in a round bottom flask and heated at reflux for about 12 h. Completion of the reaction was monitored by TLC. The reaction mixture was cooled, added dropwise to crushed ice and neutralized by using NaOH (50%) solution. The resulting precipitate was filtered, washed with water and dried over reduced pressure. The crude product obtained above was filtered off and recrystallized from ethanol to get light yellow solid (80%

Sample preparation
BSA was procured from Sigma Aldrich, India. The phosphate buffer was prepared by using sodium dihydrogen phosphate and disodium hydrogen phosphate (Sigma Aldrich). The physiological pH (pH = 7.4) was maintained by using this phosphate buffer. The stock solutions of MTDP4(9) and BSA of concentration 250 µM were prepared in 0.1 M phosphate buffer of pH 7.4. BSA solution was prepared based on molecular weight of 66,500. All the analytical reagent grade chemicals were used and solutions were prepared in Millipore water.

MTDP4(9)-BSA interaction study by fluorescence
The stock solutions of MTDP4(9) and BSA with 250 µM concentration were prepared in phosphate buffer solution of pH 7.4. The concentration of MTDP4(9) solution was varied in the range from 2.5 to 22.5 µM and BSA was fixed at 2.5 µM. Fluorescence spectra were recorded at the room temperature in the range 280-550 nm upon excitation at wavelength of 296 nm in each case.

UV measurements
The UV measurements of BSA in the presence and absence of MTDP4(9) was made in the range of 200-400 nm. BSA concentration was fixed at 2.5 µM while the MTDP4(9) concentration was varied from 2.5 to 22.5 µM in the presence of phosphate buffer as a solvent.

3D fluorescence studies
The 3-D fluorescence spectrum was recorded as excitation wavelength range of 250-350 nm and emission wavelength range of 200-800 nm and an increment of 10 nm with other parameters were just the same as that of fluorescence quenching spectra (CBSA = 2.5 µM and CMTDP4(9) = 2.5-22.5 µM).

Molecular docking
The crystal structure of BSA (PDB ID: 4F5S) used for the docking study was obtained from the Protein Data Bank. The proteins were prepared for docking by adding polar hydrogen atom with Gasteiger-Huckel charges [23] and water molecules were removed. The three-dimensional structure of the ligands was obtained by the SKETCH module implemented in the SYBYL program (Tripos Inc., St. Louis, USA) and its energy-minimized conformation was obtained with the help of the Tripos force field using Gasteiger-Huckel charges and molecular docking was performed with Surflex-Dock program that is interfaced with Sybyl-X 2.0.
[24] and other miscellaneous parameters were assigned with the default values given by the software.

Cell morphology
Effect of compounds on the cell morphology of NCI-H226 and T-47D were studied. Concentration of active compound MTDP4(9) on NCI-H226 and T-47D produced rapid changes in cell shape. These results indicate the cytotoxicity of the compound which induce cell death (Figures 3 and 4). By contrast, no significant changes were obsereved in the cell morphology of inactive compounds.

Characterization of the quenching behaviour of MTDP4(9) on BSA
To unveil the structural interaction between BSA with compound MTDP4(9), UV-visible and fluorescence spectral studies were carried out. The UV-visible spectrum ( Figure 5) of BSA (2.5 µM) showed an intense band at 276 nm in phosphate buffer of pH 7.4, which arises due to the presence of phenyl rings in Trp (tryptophan), Tyr (tyrosine) and Phe (phenylalanine) residues. Successive addition of MTDP4 (9) to BSA solution resulted in gradual increase in absorbance intensity at 276 nm with appearance of new absorption peak at 328 nm. This change in BSA absorbance upon regular introduction of compound MTDP4 (9) indicates the interaction between BSA and compound MTDP4(9) leading to an increase of hydrophobicity in the vicinity of Trp, Tyr and Phe residues. Moreover, the maximum absorption wavelength of BSA remains unchanged, indicating that the interactions between compounds and BSA are noncovalent in nature and likely occur through ππ stacking between aromatic ring of compound and phenyl ring of Trp, Tyr and Phe residues located in the binding cavity of BSA.
The quenching mechanism of BSA by drugs can be obtained by fluorescence methods. The fluorescence spectra of BSA at various concentrations of MTDP4 (9) are shown in Figure 6. The fluorescence intensity of BSA decreased regularly with an increasing concentration of MTDP4(9), which indicated that MTDP4(9) binds to BSA [30]. The Stern-Volmer equation [31] was employed to confirm the mechanism as shown in Equation (1).
where F 0 and F are the fluorescence intensities of BSA and BSA with MTDP4(9) respectively, K SV is the Stern-Volmer quenching constant, [Q] is the concentration of the MTDP4 (9). k q is the quenching rate constant of the bio-molecule and 0 is the average lifetime of bio-molecule without the quencher and the literature value is 10 −8 s [32]. Notes: LC 50 = concentration of drug causing 50% cell kill, GI 50 = concentration of drug causing 50% inhibition of cell growth, TGI = concentration of drug causing total inhibition of cell growth, ADR = adriamycin, positive control compound and NE = not equated. In this work, the fluorescence intensities of all BSA solutions were corrected for the inner-filter effect of fluorescence according to the following equation [33]: where F cor and F obs are the corrected and uncorrected fluorescence intensities and A exc and A em are the absorbance values at the current excitation and emission wavelengths. The Stern-Volmer plot of F 0 /F vs. [MTDP4 (9)] was observed to be linear (Figure 7). From the slope of the Stern-Volmer plot for BSA-MTDP4(9) system at 298 K, the quenching constant (K SV ) was found to be 5.21 × 10 4 L/mol, the value k q for MTDP4(9) -BSA system at 298 K was found to be 5.21 × 10 12 L/ mol/s. Thus, this investigation revealed the presence of strong quenching between BSA and MTDP4(9).

Binding parameters
Binding site is a region on protein to which small molecules or ions may bind via chemical bond. The   number of binding sites (n) and the binding constant (K) can be obtained [34] from the formula.
where n is the number of Q molecules that interact simultaneously with each site and K is the binding constant. The slope of the above equation gave the exact stoichiometry, namely quencher to protein. The values of K and n were obtained from the slope and intercept of the plot of log[(F 0 − F)/F] vs. log[Q.] (Figure 8). The value of K was found to be 8.8 × 10 4 L/mol, indicates that a long-lasting interaction exists between MTDP4(9) and BSA. Even if a low concentration of MTDP4(9) is present in organs, it can interact with BSA easily. The number of binding sites, n at the experimental temperature was 1.05 which indicated that there was one class of binding site for MTDP4(9) on BSA.

Three-dimensional fluorescence spectra
Three-dimensional fluorescence is a well-known computerized technique that provides more detailed  picture of the binding, conformational changes of protein when quenched by drug. Figures 9 and 10 represent the contour one and three-dimensional fluorescence spectra of BSA (A) and BSA-MTDP4(9) (B), respectively. The contour map displayed a bird's eye view of the fluorescence spectra. The contour map of BSA was changed upon introduction of MTDP4 (9) which revealed the changes in secondary structure of BSA ( Figure 9). Further, from Figure 10, peak (a) is the Rayleigh scattering peak (λ ex = λ em ) [35]. With the addition of MTDP4(9), the fluorescence intensities of peak (a) increased. The reason for this is that when the BSA-MTDP4(9) complex was formed, the diameter of the macromolecule is increased which in turn resulted in enhanced scattering effect. As referred to peak (b), it mainly reveals the spectral characteristic of tryptophan and tyrosine residues. The reason is that when serum albumin is excited at 296 nm, it mainly reveals the intrinsic fluorescence of tryptophan and tyrosine residues. The fluorescence intensity of the peak (b) decreased markedly and the maximum emission wavelengths of the peak (b) have obvious blue shift following the addition of BSA-MTDP4(9), indicating that the conformations of the tryptophan and tyrosine residues of BSA were transformed. Hence, we conclude that the binding of BSA-MTDP4(9) induced some micro environmental and conformational changes in BSA and a complex between BSA and MTDP4(9) was formed.

Molecular docking study
Molecular docking study was performed to support the interaction and preferred binding mode of thiadiazolopyrimidinone derivative with BSA. The docked compound MTDP4(9), as depicted in Figure 11, have two hydrogen bonding interaction with amino acid residues, oxygen atom of carbonyl group present at 5th position of pyrimidine ring makes a hydrogen bonding interaction with hydrogen of amino acid residue LYS132 (C = O ---H-LYS132; 2.01 Å) and another Figure 10. The three-dimensional fluorescence spectra of BSA (2.5 μM) (i) and BSA-MTDP4(9) system with MTDP4(9) concentration (ii) 2.5, (iii) 10, (iv) 17.5 and (v) 25 μM. one hydrogen bonding interaction raised from the amine group (-NH) with oxygen of amino acid residue PHE133 (NH ---O-PHE133; 2.06 Å). Hence, compound MTDP4 (9) bound to the hydrophobic pocket of subdomain IB of BSA [36].