Synthesis, Computational studies, DNA-binding and cytotoxicity of 4-Thiazolidonone-cyclopropyl hybrid

A derivative of 4-Thiazolidinone derivative endowing cyclopropyl ring substituted at 3-nitrogen positioned was synthesized that was further evaluated against cancerous cell lines MCF-7. The structure of synthesized compound (6) was well characterized by different spectral techniques such as FT-IR, UV-Visible, 1 H-NMR, 13 C-NMR and mass spectrophotometer. X-ray single crystal structure and Computational study (DFT) study revealed that compound (6) adopted (2Z, 5Z)-conguration. Preliminary In vitro study suggested that compound (6) displayed moderate activity bearing IC 50 (161.0 μM). The DNA binding studies (Ct-DNA) with compound (6) was performed. The study suggested that bound with DNA exhibiting binding constant K b = 3.3 x 10 4 LMol -1 ). Furthermore, the binding study was complemented by Molecular docking possessing DNA binding studies (Ct-DNA) were performed. Final compound (6) exhibited moderate cytotoxicity effect (IC 50 = 161.0 μM) and DNA binding ability (K b = 3.3 x 10 4 LMol -1 ). The experimental ndings were completed by molecular docking study.


Introduction
Cancer is one of the notorious and gruesome diseases hovering around the globe. According the report published by International agency of Research on cancer (IARC) in 2012, 14.1 million new cases were estimated out of which 8.2 million were deaths. It was also reported that 19.3 cancer million cases would be confronted up to 2025 [1]. However, 1.1 million cancer cases were estimated out of 184 countries contributing 7.8 % global cancer burden [1]. In spite of advancements in modern medical sciences, cancer is of the biggest challenges. There are many anti-cancer agents such as cis-platinum, doxorubicin, vincristine sulphate, etoposide etc but these drugs lack of selectivity and are associated with several effects sides. So, in order to curb these problems, there is an urgent need to develop new anti-cancer chemotherapeutics. For this purpose, heterocyclic compounds are important owing to the manifestation a wide spectrum of biological activities. To date, almost every drug in clinical applications contains one or more heterocyclic molecular scaffolds [2]. Among the heterocyclic compounds, 4-Thaiazolidinone endowing heteroatoms such as nitrogen, Oxygen and sulfur are ve membered heterocyclic compounds which are known to exhibit a diverse range of pharmacological activities [3][4][5][6]. Drugs based on thiazolidinones core such as Pioglitazone, Troglitazone, Rosiglitazone Rivoglitazone, and Balaglitazone as shown in Chart1., have already been approved for the treatment of type diabetic and in ammatory conditions [7].
Considering versatile importance of thiazolidinone molecular scaffold, the scientists and researchers have explored the biological activities. The potent activities of 4-Thiazolidinone derivatives are antimicrobial [8][9][10], anti-cancer [11,12] anti-viral [13], anti-convulsant [14], anti-in ammatory [15][16][17] and antiamoebic [18]. We have a long-standing interest in the design and development of 4-thiazolidinones cores [3,5,18]. Our research group has earlier reported the stereochemistry and biological activity of 4-Thiazolidinone derivatives such as anti-amoebic activity[18] and anti-cancer activity [4][5][6]. In quest of developing a novel chemotherapeutic agent, our research group evaluated the compound against the cancerous cell lines. So, in the view of pharmacological signi cance, we herein report the synthesis, stereochemistry elucidation and pharmacological studies of 4-Thiazolidinone derivative bearing of cyclopropyl ring as a tail.

Chemistry
The synthesis of the lead compound (6) has been outlined in the scheme1, whose methodology has already been reported in our previous papers [5,18]. The presented compound (6) was found to be stable at room temperature. The spectral techniques FT-IR, 1 H-NMR, 13 C-NMR and mass spectroscopy have been exploited to assign the structure of synthesized compounds. The nal compound (6) was identi ed by Xray single crystal structure as shown in Fig. 1.
The structures of the intermediate compounds (3 & 4) have already been reported in our previous work [5].
In this paper, the structure of the lead compound (6) has been explained with the help of spectral techniques. In FT-IR spectrum is used to detect the functional group which helps in identifying the presence of compound. In our investigated compound (6), the main identifying functional groups are (C = O) and (C = C) and the wavelengths of these functional groups were found to be at 1710.14 cm − 1 and 1592.87 cm − 1 . These bands showed the presence of compound (6). 1 H-NMR which is one of the most important techniques used to identify the organic and organometallic compounds. In our reported compound (6), the exocyclic alkenic proton (H-C = C-) resonated at high chemical shift δ 8.045 ppm which con rmed formation of compound (6). In the same way, the presence of characteristic chemical shifts

X-ray single crystal structure
The ORTEP diagram of the compound (6) is shown in Figure (1). Selected bond distances and angles are given in Table (supplementary table (

Theoretical calculation on geometrical isomerism:
It is obvious that compound (6) bearing alkenic and exocylic nitrogen bonds may display four different con gurations such as (2Z, 5Z), (2Z,5E), (2E,5Z) and (2E,5E). Among these most stable would be major product. So, in order to con rm the most stable the geometrical isomers of the compound (6), the DFT calculations were performed with Gaussian 09 software [19]. The structures of the various isomers i.e.
(2Z, 5Z), (2Z, 5E), (2E,5Z) and (2E,5E) were energetically optimized at B3LYP/6-31G(d,p) level of theory and the results have been depicted in Table ( 1). In order to con rm that all the possible isomers have minima (zero point frequency), frequency calculations were performed. The geometry optimization of was performed at 6-311 + + G(d,p) basis set with B3LYP and M06-2X levels of theories. The energy obtained was exploited for further analysis. The optimized geometries have been visualized by UNIVIS software [20].
The results of the study suggested that compound (6) displayed most stable con guration in the form of (2Z, 5Z)-con guration. This computational study is completely in agreement with the experimental X-ray single crystal structure determination.

Cytotoxicity study
In order to know the anti-cancerous property of the compound (6), the cytotoxic study against cancerous cell lines (MCF-7) was evaluated by MTT-assay using vital dye. This dye is a chemical compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. This dye is reduced by the succinate dehydrogenase enzyme of mitochondrial living cells to produce water insoluble purple formazan crystals [21,22] which can be measured spectrophotometrically after solubilization. It is well known fact that the quantity of formazan crystals produced is directly proportional to the number of active cells in the culture. Hence, MTT has long been exploited to measure the cell viability in cell proliferation and cytotoxicity [23,24]. The compound (6) was evaluated against (MCF-7) cancerous cell lines for 48 hours treatment at the concentration range 0-320 µM as shown in Figure (5). The cytotoxic study revealed that compounds (6) displayed moderate activity (IC 50 = 161.0 µM) which is considerable anti-cancer property.

DNA-binding study
In the prevalence of cancer DNA is quite important. Cell is multiply increased through the replication of DNA. So cell proliferation is prevented through DNA damage. In order to develop anti-cancer drug, DNA is regarded as one of the important targets. The evaluation of anti-cancer property of the newly synthesized Organic/organometallic compounds through DNA binding is quite important which bind with DNA in various ways [5,25]. UV-Visible spectroscopy is one of the most commonly exploited techniques for the measurement of DNA-binding among all the available techniques [25]. The effectiveness of DNA-binding of the active compounds is explained by the change in absorption and wavelength. It is well known fact that non-covalent binding and covalent binding are characterized by bathochromism and hypochromism and hyperchromism [25]. The lowering of hypochromicity with no bathochromic shift describes the electrostatic interaction of a compound with DNA [26,27]. From the Fig. 6 (a-b), it is evident that there are three signi cant peaks in the range of 200-405 nm, indicating interaction of compound (6) with Ct-DNA.
It was found that on increment of the concentrations of the compound (6), hyperchromism was observed.
The rst electronic transition was found be at 402 nm due to n →π * and the second transition was at 366 nm due to π→π*.The last electronic transition was observed at 230 nm is π→π* due to benzene ring electrons. These transitions showed the considerable DNA-binding. From DNA-binding calculation, it was found that compound (6) exhibited binding constant (K b ) 3.3 x 10 4 L mol − 1 . The lower value of K b comparison to classical intercalations may be attributed to non-planarity in molecules[28] interestingly, same ndings (grove binding) have been found in the docking studies. The DNA-binding studies have been presented in Fig. 6(a-b).

Molecular docking
Molecular docking is one of the best methods to select the hit compounds virtually. These days, it is very common and popular in the drug design and medicinal chemistry. The designed compounds are made to bind with a particular protein through which hit compound is scrutinized for the further process. With the help of molecular docking, active pocket and interacting residues are determined. In the present study, compound 6 was docked with DNA (PDBID: 1BNA) to complement Ct-DNA-binding outcomes. The DNA docking studies was performed by Auto Dock-vina [29,30]using DNA dodecamers d(CGCGAATTCGCG)2 (PDB ID:1BNA). The study of the compound showed that compound (6) interacted with residues of the both the strands of DNA through polar and non-polar attractions as shown in Fig. 7(a-c). Similar result was obtained from in vitro DNA binding studies. Besides, binding energy, is one of the physical descriptors to compare the docking results, was found to have − 9.30 kcal/mol. The molecular docking as shown in Fig. 7(a-c) made ve polar and non-polar interactions. The oxygen atoms of thiazolidinone ring of compound (6) form two hydrogen bonds with residues DC-10 & DC-9 of Chain A of DNA. However, the exocyclic nitrogen at position-2 formed three hydrogen bonds with the residues DA-17 and DA-18 of Chain B of DNA and with residue DC-9 of Chain A of DNA respectively. The 3D and 2D representations of compound 6 and DNA-interacting residues have been shown in Fig. 7(a-c).

Conclusion
We herein conclude on the basis of experimental and computational studies that the synthesized compound (6) adopted (2Z, 5Z)-con guration. Furthermore, the computational molecular docking study showed that compound 6 formed ve hydrogen bonds bearing docking energy − 9.03 kcal/mol. Besides, the in vitro DNA-binding study revealed that binding constant of compound (6) was found to be K b (3. penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin B, at 37 0 C in a relative humidity 80 %, 5 % CO 2 [31].

MTT assay
The MTT assay is a standard colorimetric assay, in which mitochondrial activity is measured by splitting tetrazolium salts with mitochondrial dehydrogenases in viable cells only [32]. Cytotoxicity of compound (6) was evaluated through MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, M2128 Sigma Aldrich) assay on MCF-7. MTT is a validated assay for the in vitro cytotoxicity of any natural, synthetic compounds and extracts. The cell count 1.2 × 104 cells/well were seeded in 96 well plate (150 µL/well). After the overnight incubation, cells were treated with different concentration of compound (6) for 48 h. After the 48 h of treatment, the medium was remove and incubated with 20 µL of MTT solution (5 mg/mL in Phosphate saline buffer) for 4 hour. The formazan crystals were formed by mitochondrial enzyme reduction, nally solubilized in DMSO (150 µL/well) and absorbance was recorded at 570 nm through the Microplate reader (iMark, BIORAD, S/N 10321). Percent viability was de ned as the relative absorbance of treated versus untreated control cells.

DNA-binding
The stock solution of disodium salt of Ct-DNA was prepared in tris-HCl buffer (pH 7.2-7.3) and stored at 4 0 C temperature. Once prepared, the stock solution was used within 4 days. The concentration of the solution was determined spectrometrically. The ratio of absorbance at 260 and 280 (≥ 1.8) indicated that DNA was su ciently free of protein. The concentration of DNA was measured using its extinction coe cient at 260 nm (6600 M − 1 cm − 1 ) after dilutions. For the titration purpose, DNA stock solution was diluted using tris-HCl buffer. The compounds were dissolved in minimum amount of DMSO (1.6 x 10 − 4 M). UV-Vis absorption spectra were recorded after each addition of different concentrations of DNA.
Absorption titration was conducted by adding varying concentrations (2.8-6.8 x 10 − 5 M) of DNA. The intrinsic binding constant (K b ) was determined by Eq. (1), which was originally known as Benessi-Hilderbrand equation and further modi ed by Wolfe et. al. [33].
Where the apparent absorption coe cients a , f and a correspond to A obs/[compounds], the extinction coe cient for the compounds, and the extinction coe cient for the compounds in the fully bound form. In plots of [DNA]/( a− f ) versus [DNA], K b is given in the ratio of the slope to intercept.

DNA docking studies
Docking studies were performed at Intel(R) Core(TM) i3 CPU (2.3 GHz) with XP-based operating system (Windows 2010). 3D Structures of the compounds (6) was drawn by Marvin sketch and saved in pdb le format. The preparation of the compound was done by assigning Gastegier charges, merging non-polar hydrogens, and saving it in PDBQT le format using Auto-Dock Tools (ADT4.2) [29,34,35]. The X-ray crystal structure of DNA (PDB ID: 1BNA) was obtained from the Protein Data Bank [http://www.rcsb.org/pdb]. Using ADT 4.2, DNA was saved in PDB le format leaving heteroatoms (water). Gastegier charges were assigned to DNA and saved in PDBQT le format. Preparation of parameter les for grid and docking was done using ADT. Docking was performed with Auto Dock Vina 4.2[36] considering all the rotatable bonds of ligand (compound 3 and 4) as rotatable and receptor (DNA) as rigid [37]. A grid box of size 64 × 64 × 118 Å with 0.375Å spacing was used that included the whole DNA. The nal structure of the docked complexes was drawn using PyMol[38] and 2D plot of docked complexes were constructed using Schrödinger visualizer (Maestro 10.5 trial version, Maestro, 2016).

X-Ray crystal structure determination
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at low temperature by the φ-ωscan method. Re ections were measured from a hemisphere of data collected from frames, each of them covering 0.3º in ω.A total of 59758 for 6, re ections measured were corrected for Lorentz and polarization effects and for absorption by multi-scan methods based on symmetryequivalent and repeated re ections. Of the total, 4744 independent re ections exceeded the signi cance level ( F /σ F ) > 4.0. After data collection, an multi-scan absorption correction (SADABS) [39] was applied, and the structure was solved by direct methods and re ned by full matrix least-squares on F 2 data using SHELX suite of programs [40]. Hydrogen atomswerelocated in difference Fourier map and left to re ne freely, except for C (20), which were included in calculation position and re ned in the riding mode. Re nements were done with allowance for thermal anisotropy of all non-hydrogen atoms. A nal difference Fourier map showed no residual density outside: 0.403 and − 0.265 e.Å −3 .A weighting scheme w = 1/[σ 2 (F o 2 ) + (0.052700 P) 2 + 0.584300P] for 6, where P = (|F o | 2 + 2|F c | 2 )/3, was used in the latter stages of re nement. Further details of the crystal structure determination are given in Table (  ORTEP for the compound (6). Hydrogen atoms are omitted by clarity. All the non-hydrogen atoms are presented by their 50% probability ellipsoids.

Supplementary Files
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