H2O2:HCl Catalyzed Synthesis of 5-(3-Substituted-Thiophene) Pyrimidine Derivatives and Evaluation for Their Pharmacological Effects

A series of 5-(3-substituted-thiophene) pyrimidine derivatives ( 3a-d ) were synthesized via Knoevenagel condensation reaction in aqueous ethanol using H 2 O 2 :HCl as a green halogenating catalyst and evaluated for their pharmacological effects. The structures of the targets were confirmed by analytical and spectroscopic methods . From antibacterial activity result reveals that, all the four compounds showed appreciable activity with varied zone of inhibition, in that the compounds 3b & 3d exhibited most effective zone of inhibition against bacterial strains E. coli & S. aureus respectively. In-vitro cytotoxicity was carried by MTT assay method against MCF-7 (Breast cancer) cell line and results of all the four compounds showed excellent selectivity, in that compound 3a exhibited excellent cytotoxicity with minimum cell viability range of 23.68 to 44.16 %. The interaction of compounds with CT-DNA was determined by using UV-absorption spectroscopy and results were confirmed that, all the synthesized compounds interacted strongly with CT-DNA through electrostatic or groove binding. In-silico ADME-toxicology results indicated that, all the targets are non-toxic, good oral bioavailability and druglikeness score indicated that they are suitable as drug-leads. From In-silico molecular docking results showed compound 3b was bound with GlcN-6-P


Introduction
Heterocyclic compounds play a predominant role in medicinal chemistry and synthetic organic chemistry due to their massive biological importance. Sulphur and nitrogen containing heterocyclic compounds are always attempting to attract the attention of medicinal chemists and researchers due to their multiform pharmacological and biological activities [1][2][3]. Cancer is one of the leading diseases around the world and it caused by mutations in genes that regulate cell growth. The mutations led to abnormal cells division and multiply in an uncontrolled way. Breast cancer is the most common cancer in women worldwide, with more than 1.5 million new cases recorded every year. It is also the fifth highest cause of cancer death in world [4]. Current chemotherapies suffers major limitation of side effect and drug resistance, therefore continued search for novel and safer anticancer drugs remains as important [5]. Pyrimidine and thiophene have been recognized as key scaffolds due to its biological significances such as antiviral, antibacterial, antifungal, anticancer, antiinflammatory, anti-diabetic, anti-tubercular, antioxidant, anti-parasitic, anti-convulsants, antidepressant, analgesic, antitumor, gastro protectors and kinase inhibitors [6,7].
Moreover the heterocyclic compounds increase the strength of the complex by forming hydrogen bonds with DNA. Interactive study of heterocyclic moieties with DNA is essential for estimation of their anticancer activity and elucidates viable mechanism of their action.
Hence, DNA binding is considered to be the most essential experimental step to measure the activity of anticancer drugs, because most of the anticancer drugs target DNA specifically [8].
Pollution is one of the critical problems faced by the synthetic organic chemist in designing the organic reactions for the synthesis of pharmacologically active compounds. Thus, the development of environment-friendly chemical process that induces necessary organic transformation is the important objectives of sustainable development [9]. Therefore, the "green chemistry" is an auspicious approach that meets the requirement of chemical and pharmaceutical industries. The replacement of hazardous solvents with eco-friendly solvents is an acceptable and valuable approach in a chemical reaction and use of a combination of hydrogen peroxide and the respective hydrohalic acid as a green halogenating agent [10].
Hypochlorous acid (HOCl or HClO) is a weak acid that forms when chlorine dissolves in water or H2O2 react with HCl and itself partially dissociates, forming hypochlorite (ClO − ).
HOCl and ClO − are powerful oxidizers and the primary disinfection agents [11,12]. HOCl cannot be isolated from these solutions due to rapid equilibrium with its precursor. Earlier, our research group has reported different derivatives of pyrimidine moieties and other biologically important heterocyclic compounds [13,14]. Some of the drugs containing pyrimidine nucleus available in the market (Fig. 1).

Chemistry
In this report, we developed a simple and convenient method for the synthesis of 5-( Firstly, we studied the effect of catalyst on the reaction. In the previous reports, the same reaction was carried out in presence of different catalysts such as CuO NPs, PVP-Ni NPs, Fe3O4 NPs, L-tyrosine, NH2SO3H, EAN, Bi(NO3)3. 5H2O and also in the absence of catalyst (Table 1), were used to find the influence of catalyst in progress of reaction as well as in the increase of product yield. But our observation of the experiment revealed that, the starting materials remained in the reaction media which was clearly indicated in the TLC even prolonged time of stirring. Therefore we concluded that the best result was obtained in the presence green halogenating catalyst H2O2:HCl, whereas further increase in the quantity of catalyst doesn't have a significant effect on reaction kinetics. -EtOH Reflux 120 89 [22] Consequently, to study the effect of temperature on synthesized compound 3a, we carried a reaction at RT, 50 °C and 80 °C ( Table 2) as a result while increase the reaction temperature decreases the reaction time from 60 to 20 min and 20 to 10 min respectively, but yield of the product does not affected by increase in temperature. The structures of the intended 5-(3-substituted-thiophene) pyrimidine derivatives (3a-d) were confirmed by IR, 1 H NMR, 13 C NMR and HRMS spectral data. IR spectrum of compound 3a showed the absorption band in the region 3373 cm -1 is attributed to the amide stretching vibration and the absorption band at 1654 cm -1 corresponds to stretching vibration of the carbonyl group (C=O). Another stretching vibrational band at 1529 cm -1 corresponds to C=C bond. The 1 H NMR spectrum of compound 3a exhibited two singlet peaks at δ 11.21 and 11.17 ppm which corresponds to two NH protons of pyrimidine nucleus (s, 2H, NH) and another singlet peak at δ 8.53 ppm due to CH proton (s, 1H, CH). A multiplet peak was observed in the range of δ 8.24-8.13 ppm corresponds to two aromatic protons (m, 2H, Ar-H) and a triplet peak at 7.33-7.31 ppm due to one aromatic proton (t, J= 8 Hz, 1H, Ar-H). In addition, 13 C NMR spectrum of the compound 3a exhibited peaks at δ 163.950 and 163.453 ppm which correspond to carbonyl carbons.
The mass spectrum showed molecular ion peak [M+H] + at m/z is 221. 9054 which correspond to the molecular weight of the compound 3a (Supporting information: S1 to S16). The physical and analytical data of synthesized compounds (3a-d) was appended in Table 3.

Fig. 2
Images of antibacterial study of the synthesized compounds (3a-d)

Cytotoxicity study
All the four synthesized compounds were investigated for their in-vitro cytotoxicity against MCF-7 (Breast cancer) cell line (Fig. 3). Plot of compound concentration versus the survival fraction was performed (Fig. 4) and percentages of cell survival of the tested compounds are listed in Table 5.   involves the changes in absorbance and wavelength [23]. The molecules are bind to DNA with two modes (covalent or non-covalent of binding). Covalent bonding led to bathochromism and hyperchromism due to breaking of the DNA structure when a compound interacted with DNA covalently. While in non-covalent binding there are "electrostatic", "groove" and "intercalative" types of interactions. Decreased in absorption (hypochromic shifts) and red shift (bathochromic shift) revealed the intercalative binding of compounds with DNA. Lower hypochromic/hyperchromic effect with no or negligible bathochromic shift led to electrostatic binding. Minor or no effect and rarely, some hyperchromism shows the groove binding [24][25][26].
The DNA binding efficiency of synthesized compounds (3a-d) was monitored by comparing their absorption spectra with and without CT-DNA. The absorption spectra was carried out at    Table 7).
In-silico pharmacodynamics studies revealed that, all the four molecules are non-mutagenic, non-tumorigenic, non-irritant, AMES non-toxic with high reproductive effects and possible hepatotoxicity. The bioactivity assessment indicated that, the molecules do not belong to GPCR group of ligands, do not modulate ion channels, non-kinase inhibitors, non-nuclear receptor ligands, non-protease and non-enzyme inhibitors ( Table 8).  Table 8 In-silico pharmacodynamics and bioactivity assessment of synthesized compounds (3a-d)

In-silico molecular docking studies
The in-silico molecular docking studies were performed to predict the most effective binding among the synthesized molecules to appropriate targets [28,29]. The results revealed that, the compound 3b was bound with GlcN-6-P and P38 MAPk with minimum binding energy of -7.9 and -6.4 kcal/mol while the molecules 3a, 3d and 3c interacted with a binding energy of -7.6 and -6.4, -7.4 and -6.2 and -7.4 and -6.0 with GlcN-6-P and P38 MAPk targets respectively. The interaction of all the molecules with GlcN-6-P and P38 MAPk were compared with antibacterial agent Ciprofloxacin (-7.7 kcal/mol) and anticancer agent 5fluorouracil (-4.7 kcal/mol) and results are appended in Table 9.  In-silico ADME-toxicology results indicated that, all the four compounds are non-toxic and good oral bioavailability and druglikeness score indicated that they are suitable as drug-leads. From In-silico molecular docking results, the compound 3b was bound with GlcN-6-P and P38 MAPk with least binding energy of -7.9 and -6.4 kcal/mol respectively. In future, the obtained compounds can be used as antibiotics, anticancer agents, dyes and pigments, paints and food industries.

Materials and Method
All chemicals and calf thymus DNA were purchased from Aldrich Chemical Company and reaction was performed at refluxed condition and solvents were used without further purification. Analytical TLC was performed with E. Merck silica gel GF254 glass plates.
Visualization of the developed chromatogram was performed by UV light (254 and 356 nm).
The melting points of the products were determined in open capillary tubes and uncorrected.
The ATR-IR spectra were obtained using Bruker FTIR Alpha spectrometer. The 1 H NMR and 13   [DNA] (∈ B −∈ F ) Where, ∈ , ∈ and ∈ corresponds to the apparent, bound and free compound extinction coefficients respectively. A plot of . Hence Kb is the ratio of slope to intercept. The % of hyperchromicity or hypochromicity (% H) for the CT-DNA/[Ligand] was obtained from (εaεf)/ εf × 100.

In-silico oral bioavailability assessment and ADME-toxicology studies
The oral bioavailability of the synthetic molecules (3a-d) can be predicted by considering their structural properties to screen based on the Rule of five or Lipinski rule-of-five (RO5) filter [35]. Rule of five describes the molecular properties necessary to filter candidate drug's pharmacokinetic (PK) and pharmacodynamics (PD) [36][37][38].

In-silico molecular docking studies
The docking of the synthesized compounds to the binding pocket of glucosamine-6-phosphate synthase (GlcN-6-P) and P38 MAP kinase was carried out using Autodock Vina program [43]. The co-crystallized structure of GlcN-6-P (PDB ID: 2VF5) and P38 MAP kinase (PDB ID: 1OUK) were retrieved from protein databank and their substrate binding sites were identified using pdbsum server [42,44,45]. the obtained molecules, all the torsions were allowed to rotate during docking [38]. The grid box was set around the residues forming the active pocket [40]. The binding interactions were visualized using Biovia Discovery Studio Visualizer V.20.1 and Schrodinger-Maestro V.12.7.
The in-silico studies were performed on a local machine equipped with AMD Ryzen 5 sixcore 3.4 GHz processor, 8GB graphics and 16 GB RAM with Microsoft Windows 10 operating system.