Facile synthesis of some 5-(3-substituted-thiophene)-pyrimidine derivatives and their pharmacological and computational studies

A series of 5-(3-substituted-thiophene)-pyrimidine derivatives (3a-d) were synthesized via Knoevenagel condensation reaction in aqueous ethanol using H2O2:HCl as a catalyst. Their pharmacological effects were evaluated. Analytical and spectroscopic methods confirmed the structures of the target molecules. The antibacterial activity studies revealed that compounds 3b and 3d exhibited the most effective zone of inhibition against bacterial strains E. coli and S. aureus, respectively. The in vitro cytotoxicity was carried out by MTT assay against MCF-7 cell line. The results showed the excellent selectivity for all four compounds, among which the compound 3a exhibited remarkable cytotoxicity with a minimum cell viability range of 23.68 to 44.16%. The interaction of compounds with calf thymus DNA was determined using UV-absorption spectroscopy. The results confirmed that all the synthesized compounds interacted strongly with CT DNA through electrostatic or groove binding. In silico ADME-toxicology studies indicated that all the molecules under investigation are nontoxic with good oral bioavailability. The drug-likeness score indicated that they are suitable as drug-leads. In silico molecular docking the specified compound 3b bounds with GlcN-6-P and P38 MAPk with a minimum binding energy of –7.9 and –6.4 kcal/mol, respectively. DFT study demonstrated that the compound 3d was chemically and biologically more reactive due to less energy gap. Keywords

Moreover, the heterocyclic compounds increase the strength of the molecules by forming hydrogen bonds with DNA. Hence, the interactive study of heterocyclic moieties with DNA is essential for estimating their anticancer activity and elucidates the viable mechanism of their action. Therefore, DNA binding is considered as an essential ex-perimental step to measure anticancer drugs activity because most anticancer drugs specifically target the DNA [16]. Heterocyclic compounds appear to be most effective against various cancers. Around 60% of the medicines used for cancer treatment are based on heterocyclic moieties. Among the various heterocyclic moieties, nitrogen and sulphur-based compounds are effective against different types of cancers [17][18][19].
Cancer is a group of diseases in which the abnormal cells grow uncontrollably by disregarding the normal rules of cell division. Cancer is caused by many external (tobacco, chemicals, radiation and infectious organisms) as well as some internal factors (inherited mutations, hormones, immune conditions and random mutations). There are different types of cancer such as breast cancer, bladder cancer, kidney cancer, leukaemia, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer etc. [20]. The breast cancer is one of the most frequently diagnosed cancers and the leading cause of death in females worldwide, with more than 1.5 million new cases recorded every year. It is also the fifthhighest cause of death in the world [21]. The modern methods of chemotherapy suffer from the main disadvantages: side effects and drug resistance. Therefore, continued search for novel and safer anticancer drugs remains essential [22].
Pollution is one of the critical problems that the synthetic organic chemist faces in designing the organic reactions for the synthesis of pharmacologically active compounds. Thus, development of an environmentally-friendly chemical process that induces necessary organic transformation is one of the most important goal of sustainable progression [23]. Therefore, "green chemistry" becomes a promising approach that meets the requirements of the chemical and pharmaceutical industries. The replacement of hazardous solvents with eco-friendly solvents is an acceptable and valuable approach in a chemical reaction. Hence, the use of a hypochlorous acid (HOCl) as a green halogenating agent [24] is highly advantageous over other catalysts, including cost-effectiveness, efficiency and readily availability. Earlier, our research group has reported the synthesis of different pyrimidine derivatives and other biologically important heterocyclic compounds [25][26][27]. Some of the drugs containing pyrimidine nucleus available in the market are depicted below (Fig. 1).
Based on the above findings, we herein report the application of H2O2:HCl as a green halogenating catalyst for the synthesis of 5-(3-substituted-thiophene)-pyrimidine derivatives of considerable pharmacological relevance.

Materials and Method
All chemicals and calf thymus DNA were purchased from Aldrich Chemical Company, and the 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 C NMR spectra were recorded on Bruker 400 MHz and 100 MHz in DMSO-d6 as a solvent. Mass spectra were obtained by Agilent 1200 series LC and Micromass Q spectrometer. The DNA binding studies were carried on Elico SL 159 UV-Visible spectrophotometer in 200-500 nm range equipped with 1.0 cm quartz cell at room temperature. The anticancer activity was carried out in the Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka. The computational studies were carried out by Density functional theory (DFT)/B3LYP method using Gaussian 09 software using 6-31G (d, p) basis set at gaseous phase.
2.2. General procedure for the synthesis of 5-(3substituted-thiophene)-pyrimidine derivatives (3a-d) The synthesis of 5-(3-substituted-thiophene)-pyrimidine derivatives (3a-d) were carried out by the reaction of barbituric/thiobarbituric acid (1, 1mmol) with 3-substitutedthiophene-2-carboxaldehyde (2, 1mmol) in the presence of 15 mL aqueous ethanol using 6% H2O2:HCl (2:1) as a catalyst. The reaction mixture was refluxed at 80 °C for about 10-15 min and the progress of the reaction was monitored by TLC (Ethyl acetate and Petroleum ether). After the completion of the reaction, the reaction mixture was cooled to room temperature and poured into the 100 mL flake ice with vigorous stirring to get solid precipitated out. The crude mixture was filtered, washed and recrystallized with absolute ethanol to afford pure solid products.    Antibacterial activity of the synthesized compounds (3a-d) was carried out by the agar well diffusion method [28] using two bacterial strains (Gram-negative and Grampositive) Escherichia coli (MTCC-1559) and Staphylococcus aureus (MTCC-902). DMSO was used as negative control and Ciprofloxacin as standard drug. The test compounds were dissolved in DMSO at two different concentrations, 20 and 40 µg/mL.

Cytotoxicity
In vitro cytotoxicity was assessed by the MTT assay method [29] against MCF-7 (Breast cancer) cell line. The cells were seeded in a 96-well flat-bottom microplate and maintained at 37 °C in 95% humidity and 5% CO2 overnight. Different concentration (200, 100, 50, 25, 12.5 and 6.25 µg/mL) of samples were treated. The cells were incubated for another 48 h, and the wells were washed twice with PBS. 20 µL of the MTT staining solution was added to each well, and the plate was incubated at 37 °C for 4 h. The medium with MTT was discarded, and 100 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was recorded at 570 nm using a microplate reader. The percentage of cell survival was calculated by using the following formula: (1)

DNA binding study
DNA binding study was assessed by using electronic spectroscopy. A solution of CT DNA in 50 mM Tris-HCl/50 mM NaCl buffer solution was prepared at pH 6.9-7.01. In buffer solution, the ratio of absorption values of CT DNA at 260 and 280 nm is 1.8-1.9, indicating that DNA was free of proteins [30]. Then a concentrated stock solution of DNA was prepared in 50 mM Tris-HCl, 50 mM NaCl in double distilled water at pH 6.9-7.01. The concentration of CT DNA was determined per nucleotide by taking the absorption coefficient (6600 dm 3 mol -1 cm -1 ) at 260 nm [31]. Stock solutions were stored at 4 °C. 2 mL of the solution was taken containing a fixed concentration of the compounds (3a-d) with CT DNA (0 to 350 µL of a 0.5025-6.067010 -7 M stock CT DNA solution). A blank solution containing the same concentration of DNA was used as a reference. Solutions were prepared by mixing the compound and CT DNA in DMSO medium and then recording the UV absorption spectra by adding 25 to 350 µL DNA to the compound. The spectra were recorded against a blank solution containing the same concentration of DNA (4.090910 -6 mol L -1 ). The intrinsic binding constant Kb was obtained by using the following equation [32]: where, ∈ , ∈ and ∈ corresponds to the apparent, bound and free compound extinction coefficients, respectively. A plot of and Y-intercept equal to . Hence Kb is the ratio of slope to intercept. The % of hyperchromicity or hypochromicity (% H) for the CT DNA/[Ligand] was obtained from (∈ A -∈ F )/ ∈ F  100.

In silico oral bioavailability assessment and ADMEtoxicology 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 [33]. Rule of five employs the molecular properties necessary to filter candidate drug's pharmacokinetics (PK) and pharmacodynamics (PD) [34][35][36].
Oral bioavailability assessment was done using Osiris Data warrior V.4.4.3 [37] based on total molecular weight, ClogP, H-acceptors, H-donors, rotatable bonds as part of RO5 filters, along with TPSA (Topological polar surface area) and drug-likeness assessment [38]. Pharmacodynamic properties like mutagenicity, tumorigenicity, reproductive effects, irritancy, Ames toxicity and hepatotoxicity were predicted using the admetSAR server. Bioactivity scores were predicted using the molinspiration server for GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor inhibitor, protease inhibitor, enzyme inhibitor. Pharmacokinetic properties like blood-brain barrier penetration, human intestinal absorption, Caco-2 permeability and CYP450 2D6 substrate were predicted by submitting each molecule individually to the admetSAR server [39].

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 the Autodock-Vina program [40]. 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 [41,42]. The in silico studies were performed on a local machine equipped with AMD Ryzen 5 six-core 3.4 GHz processor, 8 GB graphics and 16 GB RAM with Microsoft Windows 10 operating system.

Computational studies
Computational studies of synthesized compounds (3a-d) were conducted by using the Gaussian 09 software [43] with the help of the density functional theory at Becke-3-Lee-Yang-parr (DFT)/B3LYP level with 6-31G (d,p) basis set [44]. The energy minimization process has been conducted at the same level in the gas phase to obtain a stable structure. The 3D representation of the optimized structure was presented in a molecular visualization program Gauss view 5.0 and the output was processed using the Avogadro software [45].
A possible mechanism for the formation of 5-(3-substituted-thiophene)-pyrimidine derivatives is shown in Scheme 2. Firstly, hypochlorous acid (HOCl) forms by the reaction of H2O2 with HCl, and it acts as a powerful oxidizing agent. Then the reaction is initiated by the generation of carbanion 2 from active methylene compound 1. The carbanion 2 attacks the carbonyl carbon of aldehyde 3 to form intermediate 4, which undergo subsequent dehydration to desired Knoevenagel product 5. HOCl increases the abstraction of acidic proton from active methylene compound and electrophilicity of carbonyl group of aldehydes due to hydrogen bonding.
Firstly, we studied the effect of catalyst on the reaction. In the previous reports, the same reaction was carried out in the 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). We studied the influence of catalyst in the progress of reaction as well as on the increased product yield. The results were not very encouraging. Therefore, we concluded that the best result was obtained in the presence of green halogenating catalyst 6% H2O2:HCl, where a further increase in the quantity of catalyst doesn't significantly affect reaction kinetics.
Consequently, to study the effect of temperature on synthesized compound 3a, we carried out the reaction at room temperature, 50 °C and 80 °C ( Table 2). As a result, an increase in the reaction temperature decreased the reaction time from 60 to 20 and 20 to 10 min, respectively. Still, the yield of the product was not affected by an increase in temperature.
The structures of the intended 5-(3-substitutedthiophene)-pyrimidine derivatives (3a-d) were confirmed by IR, 1 H NMR, 13 C NMR and HRMS spectral data. IR spectrum of compound 3a showed that 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, the 13 C NMR spectrum of 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 corresponds to the molecular weight of compound 3a (Supporting information: S1 to S16). The physical and analytical data of synthesized compounds (3a-d) were appended in Table 3.

Antibacterial activity
The synthesized 5-(3-substituted-thiophene)-pyrimidine derivatives (3a-d) were screened for their in vitro antibacterial activity at two different concentrations (20 and 40 µg/mL). All four compounds showed appreciable antibacterial activity with a varied zone of inhibition in the range of 3.3 to 3.8 and 7.1 to 7.8 mm against E. coli and 3.0 to 3.7 and 7.4 to 7.9 mm against S. aureus, respectively (Table 4).  The results revealed that compounds 3b (3.8 and 7.8 mm) and 3d (3.7 and 7.9 mm) having electrondonating group (methyl) on C-3 of the thiophene ring exhibited the most inhibitory effect against bacterial strains E. coli and S. aureus, respectively, compared to the standard drug Ciprofloxacin.

Cytotoxicity
All the four synthesized compounds were investigated for their in vitro cytotoxicity against MCF-7 (Breast cancer) cell line (Fig. 2). The plot details compound concentration versus the survival fraction (Fig. 3). The percentages of cell survival of the tested compounds are listed in Table 5.
In vitro cytotoxicity results revealed that all four compounds displayed a superior selectivity against the MCF-7 cell line. Among the compounds tested, compound 3a exhibited promising cytotoxicity with a minimum cell survival range of 23.68 to 44.16% at the concentration range of 200 to 6.25 µg/mL. Whereas 3b, 3c and 3d displayed reliable selectivity at all the concentrations with cell sur-vival ranges of 29.00 to 50.93%, 31.31 to 66.82% and 26.95 to 53.12%, respectively.

DNA binding study
DNA binding was assessed using electronic spectroscopy. The UV-absorption spectral studies were employed to examine the binding mode of compounds to CT DNA, which involves the changes in absorbance and wavelength [55]. The molecules are bound to DNA with two modes (covalent or noncovalent) of binding. The covalent binding led to bathochromism and hyperchromism due to breaking 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 absorption (hypochromic shifts) and redshift (bathochromic shift) revealed the intercalative binding of compounds with DNA. Lower hypochromic/hyperchromic effect with no or negligible bathochromic shift led to the electrostatic binding. Minor or no effect and, rarely, some hyperchromism show the groove binding [56][57][58].
The DNA binding efficiency of synthesized compounds (3a-d) was monitored by comparing their absorption spectra with and without CT DNA. The absorption spectra were carried out at a fixed concentration of synthesized compounds and varying with DNA concentrations (25-350 µL of 0.502510 -7 to 6.067010 -7 mol L -1 ) under the physiological condition of pH 7.01. The absorption spectra of all the synthesized compounds (3a-d) exhibited absorption bands at 235 to 240 nm due to π-π* transitions (Fig. 4). The Kb values of compounds (3a-d) are found to be 1.121610 7 , 1.407210 7 , 1.063410 7 and 3.487210 7 respectively are shown in Table 6.
These Kb values confirm that all the synthesized compounds interacted strongly with CT DNA. Among the four compounds, compound 3d showed a prominent binding ability with CT DNA compared to other compounds. The absorption bands of the compounds were affected due to the gradual increase of CT DNA concentration resulting hyperchromism/hypochromism. No/or negligible blue/red shift indicates strong interaction of the compounds with CT DNA mainly through electrostatic or groove binding [59]. The kinetics and thermodynamics of compounds-DNA interaction in terms of binding constant (Kb) and Gibbs free energy change (ΔG) were evaluated using the classical Van't Hoff's equation, ΔG= -2.303RT logKb. The negative ΔG values confirmed spontaneous binding of compounds with CT DNA through the formation of stable complexes.

In silico ADME-toxicology studies
The bioavailability and drug-likeness were estimated for all the synthesized compounds (3a-d) based on the molecular properties. The results indicated that all the four compounds under study could pass through Lipinski's filter without any violation, demonstrating a positive druglikeness score indicating their suitability as drug-leads.
In silico pharmacokinetic studies showed that all the molecules under investigation could penetrate the blood-brain barrier and are readily absorbed by the human intestine while they are impermeable to Caco-2, and non-substrate Cytochromes P450 (CYP450) group of enzymes (Table 7).   In silico pharmacodynamics studies revealed that all the four molecules are non-mutagenic, nontumorigenic, non-irritant, AMES non-toxic with high reproductive effects with possible hepatotoxicity. The bioactivity assessment indicated that the molecules do not belong to the GPCR group of ligands, do not modulate ion channels, non-kinase inhibitors, non-nuclear receptor ligands, non-protease and non-enzyme inhibitors (Table 8).

Structure-activity relationship (SAR) studies
The evaluation of the antibacterial activity of the newly synthesized compounds (3a-d) revealed that the presence of electron-donating methyl group has a significant effect on enhancing their potency. The compounds 3b and 3d have a methyl group at C-3 of the thiophene ring. This could have improved their cell permeability, which improved their activity profile compared to 3a and 3c. The combination of heterocyclic rings like pyrimidine and thiophene is presumed to be the main reason for the profound cytotoxicity of the newly synthesized compounds (3a-d). Furthermore, the presence of the urea group in the pyrimidine ring could have significantly contributed to the superior cytotoxicity of 3a and 3b compared to other synthesized molecules.

In silico molecular docking studies
The results revealed that compound 3b bound with GlcN-6-P and P38 MAPk with a minimum binding energy of -7.9 and -6.4 kcal/mol, respectively. 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 5-fluorouracil (-4.7 kcal/mol) (Table 9, Fig.5). The computational methods in drug discovery have gained enormous importance in modern drug research. They play a critical role in reducing the virtual chemical space in synthesizing, modifying, and screening chemical drugs against a specific disease target. Their effectiveness can be validated using microbial pathogens due to their simpler and clearer cellular understandings. Similarly, cell death modalities can also be studied using in vitro cell culture studies, particularly focusing on necrosis, apoptosis, necroptosis, autophagic cell death, etc. [42]. In the present study, in silico molecular docking studies were performed to predict the most effective binding among the synthesized molecules to appropriate targets [60,61]. The studied molecules showed remarkable binding interactions with the selected target proteins, supporting their remarkable antimicrobial and anticancer effects.
The calculated HOMO-LUMO energies and global reactivity parameters of the synthesized compounds (3a-d) are displayed in Table 10. A smaller HOMO-LUMO energy gap (ΔE) indicates a soft molecule, while a larger gap indicates a hard molecule. Lower energy gap, less ionization potential, electron affinity, chemical hardness, electronegativity, electrophilicity index values and more softness values indicate that a molecule is more chemically and biologically active with low kinetic stability [65].  DFT studies data reveals that the energy gaps (ΔE) of compounds 3a, 3b, 3c and 3d are 0.16038 eV, 0.15988 eV, 0.12799 eV and 0.12636 eV, respectively. 3a, 3b and 3c, 3d moleculs show nearly similar energy gap due to the similar structure. Among them 3d molecule shows less energy gap (0.12636 eV) and more softness value (15.8277 eV); hence, it is chemically more reactive compared to the other molecules. 3a molecule has more electronegative value (0.19923 eV); hence, it has more tendency to attract a bonding electron pairs compared to the other molecules.

Conclusions
We described a mild, easy and green protocol for synthesizing 5-(3-substituted-thiophene)-pyrimidine derivatives (3ad) using H2O2:HCl as a catalyst under reflux condition. This synthetic approach has a short reaction time, excellent yield, and clean reactions make this procedure a magnificent alternative to the existing methods. Furthermore, this method is environmentally greener and safer. The activity results revealed that the compounds 3b and 3d exhibited more potent antibacterial activity against E. coli and S. aureus than the standard drug Ciprofloxacin. In vitro cytotoxicity results disclosed the outstanding selectivity on MCF-7 cell line, mainly compound 3a exhibiting the most effective cytotoxicity with a minimum cell survival range of 23.68 to 44.16%. DNA binding results indicated that all the synthesized compounds interacted strongly with CT DNA, and compound-DNA complexes were stabilized by electrostatic or groove binding. In silico ADME-toxicology results showed that all the four compounds are non-toxic and suitable for oral bioavailability and drug-likeness, indicating their suitability as drug-leads. In silico molecular docking results showed compound 3b bound with GlcN-6-P and P38 MAPk with a least binding energy of -7.9 and -6.4 kcal/mol, respectively. DFT study result revealed that the compound 3d was chemically and biologically more reactive with low kinetic stability due to less energy gap. In future, the obtained compounds can be used as antibiotics, anticancer agents, dyes and pigments in compounds as food industries.