Synthesis, molecular modelling and biological evaluation of new 4-aminothiophene and thienopyrimidine compounds

A series of 4-amino-5-substituted-thiophene derivatives 3, 5 and 7, along with their corresponding thieno[3,2-d]pyrimidine compounds 4, 6 and 8, were synthesized and characterized. DFT calculations were utilized to determine frontier orbitals energies. The data showed that the compounds have low HOMO and LUMO energies, where 4-methyl thienopyrimidine 4 and 5 have the lowest values. According to results of antibacterial activity against two distinct strains of Gram positive and negative bacteria through two different concentrations (0.5 and 1.0 mg/mL). Compounds 6 and 8 show (IC50 = 43.53±3.26, 45.64±3.42 and 39.72±2.98, 42.57±3.19 mg/mL) good action toward S. aureus compared to chloramphenicol. Meanwhile, cytotoxic activity for inspected derivatives was evaluated toward three cancer cell lines. The investigated thiophene derivatives 4, 6 and 7 were displayed potent cytotoxic effectiveness (14.53±0.54, 11.17±0.42, 16.76±0.63 μM) against MCF-7 versus 5-fluorouracil as standard drug. In addition, the theoretical study such as molecular docking was stimulated.

However, thiophenes' biological activity was greatly influenced by the type and location of the substitutions [38].Structure and activity correlation for the thiophene analogues demonstrated an outstanding comprehension of the thiophene ring in the cancer treatment of various cancer cells [39].The thiophene analogues play a significant role in the development of agents that efficiently capture the carcinogenic lead compounds inside living cells [40,41].Thiophene's role in inhibiting "topoisomerase, tyrosine kinase, tubulin interaction, and inducing apoptosis" by activating reactive oxygen species is among the key anticancer processes discovered [42].Furthermore, numerous fused pyrimidine skeletons have been created and prepared to serve as drug-like nominees for various pharmacological purposes [43].Many studies in medicinal chemistry have exploited pyrimidines and purine-like structures as curative agents meanwhile it was discovered that they are present as a main component in some nucleic acids [22].Purines, pyrimidines and other fused cyclic molecules like pteridines are displayed interesting biological effectiveness owing to their chemical structures.Amongst the pyrimidine hybrids, thieno [2,3-d]pyrimidine derivatives reveal distinguished and multilevel biological effects such as antimicrobial, antioxidant, anti-inflammatory, antitumour, antiplatelet and antidepressant (Figure 2) [44].We describe here the synthesis of a novel 4-amino-thiophene and thieno [3,2-d]pyrimidine derivatives using straightforward methods.The aminothiophene derivatives are prepared by the cyclizing of N-(4-chlorophenyl)-2-cyano-3-mercapto-3-(phenylamino)-acrylamide with α-halogenated reagents and then converted into their corresponding thieno [3,2-d]pyrimidine compounds by the treatment with phenyl isothiocyanate.The synthesized derivatives were investigated for antibacterial and anticancer activities, in addition, their theoretical studies, such as molecular modeling and docking, were also deliberated.

General remarks
Melting points were measured using a Gallenkamp electric apparatus.The IR spectra (KBr discs) were recorded on a Thermo Scientific Nicolet iS10 FTIR spectrometer.The JEOL's spectrometer was utilized to record the NMR spectra in DMSO-d 6 at frequencies of 125 MHz ( 13 C NMR) and 500 MHz ( 1 H NMR). On a Quadrupole GC/MS Thermo Scientific Focus/DSQII, the mass analyses were carried out with an energy setting of 70 eV.The elemental analyses were carried out using a Perkin-Elmer 2400 analyzer (C, H, and N).

Computational studies
The synthesized compounds were studied using the DFT/B3LYP/6-311 ++ G [45][46][47] incorporated in the Gaussian 09W program [48].The optimized geometries of all derivatives exhibited positive frequencies that cleared their stability.The Fukui indices were determined by the Materials studio package DMol3 module [49] utilizing the GGA and B3LYP functional with DNP (version 3.5) [50].

Antimicrobial assay
At two diverse doses of 0.5 and 1.0 mg/mL, the inspected compounds were solvated in DMSO.To increase the effectiveness of the action, large concentrations of each studied derivative are utilized.It was calculated from the inhibitory zone diameter.For both categories of bacterial strains (+ve Gram) as Staphylococcus aureus "ATCC 25923" and Bacillus subtilis "ATCC 6635" and (−ve Gram) as Salmonella typhimurium "ATCC 14028" and Escherichia coli "ATCC 25922", both chloramphenicol and cephalothin were used as benchmark antibiotics [51,52].The experimental results were carried out in triplicate and the results are presented as mean standard deviation (SD).

Cytotoxic activity
Six 4-amino-thiophene and thienopyrimidine derivatives were investigated via the MTT procedure.On three individual cancer cell lines, including HepG2 (liver carcinoma), HCT-116 (colon carcinoma) and MCF-7 (breast carcinoma) besides WI38 (normal cell) in comparison to 5-Fu (5-flourouracil) as a positive control, the synthesized 4-amino-thiophene and their congruous thienopyrimidine derivatives were examined for in vitro cytotoxicity over the established MTT technique [53].Human cell lines were obtained from National Centre for Cell Sciences, Pune, India.

Molecular docking (MD)
All stable mol file conformers of synthesized molecules were prepared before using MOE 2015.10 software, and the 3D structure of topoisomerase II β was downloaded from the PDB site (PDB entry 3qx3; www.rcsb.org).First, the ligands were prepared in mol file and their energy was minimized to adjust their partial charge, render their atom's charges and assign their potential energy.Then the choice protein was prepared by ignoring the heteroatoms and water, proton was add automatically, fixing the potential energy, selecting the targeted amino-acid sequence, applying the site finder and docking the site of ligand atoms through ten poses.Following preparation and optimization, the ligands were docked.To locate receptor-binding pockets, we employed the MOE ® software's London dG and GBVI/WSA dG tools.The 10 docked poses were scored and then re-scored as directed.The scores "S" (binding efficiency) and values of the root mean square RMS values were used to evaluate and choose the target molecule and receptor configurations.

Chemistry
The synthetic strategy of 4-aminothiophene derivatives is based on the utilization of the versatile precursor, N-
As well, the DFT calculated bond lengths and angles almost coincided with those obtained from single crystal X-rays of similar compounds [55][56][57] where lengths were longer by 1.72 Å maximum with 0.052-0.062Å RMSD, while the angles were deviated by 10.5°maximum with 4.00-5.06°RMSD,which may be attributed to that the quantum chemical calculations carried out for isolated molecule in the gaseous state, no intermolecular columbic interactions, whereas the practical obtained from molecules interacting in solid crystal lattice [58] (Tables S2 and S3).

Frontier molecular orbitals
The HOMO-LUMO structures and energetic values were the main factors affecting the donation or receiving of electrons [59] where reducing the HOMO-LUMO gap will result in the ease of intramolecular charge transfer [60,61] which may affect the molecule's bioactivity [62].The FMO graphs of the examined compounds are presented in Figures 5 and 6.The diagram indicated that compounds 3, 5 and 7 have HOMO spread over the whole molecule and consisted of the π -orbital of the 4amino-2-(phenylamino)thiophene-3-carboxamide moiety along with the heteroatoms lone pair of electrons, while their LUMO was built from the π * -orbitals of the whole molecule.Thus, the HOMO-LUMO charge transfer may be mainly described as π → π * and n → π * transitions (Figure 3).
On the contrary, the HOMO of 4, 6 and 8 derivatives was built mainly of the lone pair of electrons belonging to the thioxo sulphur and nitrogen atoms of the thienopyrimidine-fused ring with a minor contribution of the π -orbital of the phenyl substituent along with moderate involvement of the oxo and imino substituents in 6 and 8.However, the LUMO of compound 4 was constructed from the π * -orbital of the fused thienopyrimidine ring in addition to the phenylamino group, whereas the corresponding 6 and 8 derivatives were made up of thephenylamino and carboxamide substituents mainly beside partial contribution of the thienopyrimidine π * -orbital.Therefore, the HOMO-LUMO charge transfer may be mainly described as n → π * transition (Figure 6).Contrarily, the E L data revealed that the thienopyrimidine derivatives have lower values than those of thiophenes.However, the E H−L gap of the thiophene derivatives was lesser than that of thienopyrimidines except for compound 4 which has the lowest value where the investigated compounds may be ordered due to the energy gap as 4 < 7 < 5 < 3 < 8 < 6 (Table 1).
The data revealed that according to electronegativity (χ), derivatives 5 and 6 exhibited the lowest and highest Lewis's acid character, 4.39 and 4.75 eV, respectively.Whereas, the global softness (δ) and hardness (η) values designated that compound 4 possessed the maximum electrons receiving and charge transfer ability, 1.27 and 0.78 eV, respectively.Hence, consistent with hardness, the studied derivatives may be ordered as 4 < 7 < 5 < 3 < 8 > 6, in agreement with that of the E H−L gap (Table 1).

Atomic Mulliken's charges and Fukui's indices
The charge transfer within a molecule can be correlated to Mulliken's atomic charges [63].In the thiophene derivatives, the thienyl sulphur atom, S 1 (thio) , has a constant negative charge in all derivatives, −0.614 to −0.691, while the thienyl carbon, C 5 (thio) attached to the acetyl (3), carboxylate (5) and cyano (7) groups bears decreasing negative charge, −0.783, −0.275 and −0.145, which may be attributed to the increase of the electron-withdrawing property of the substituents, respectively (Table 2).
In thienopyrimidine derivatives, the thienopyrimidine nitrogen atom N 1 (thyp) and carbon C 4 (thyp) have a positive charge, in the methyl derivative, 4, 0.612 and 0.406, respectively, which reduced to be 0.113 and 0.100; 0.275 and 0.249, in both oxo and imino derivatives, 6 and 8, respectively, which may be attributed to the participation of the lone pair of electrons on the oxo and imino groups in the resonating structure of the fused ring of the latter derivatives.Moreover, the beforementioned behaviour was applied in the case of the thienopyrimidine nitrogen atom N 3 (thyp) which also has a decreasing positive charge, 0.754 to 0.568, whilst the thienopyrimidine carbon atom C 4a (thyp) has an almost constant positive charge, 0.242-0.255,which may be resulted from the electron release effect of the adjacent sulphur atom S 5 (thyp) that has a negative charge.Finally, the data showed that the other heteroatoms had almost the same negative charge in all derivatives (Table 2).The atomic Fukui's reactivity indices, f + k , f − k and f 0 k , towards nucleophilic, electrophilic and radical attacks, respectively, were assessed [64][65][66][67].The nucleophilic attack Fukui's indices (f + k ) of the thiophene derivatives 3, 5 and 7 disclosed that the thienyl sulphur S 1 (thio) was the most labile atom followed by the carboxamide carbon and oxygen atoms, CO (carb) and OC (carb) , respectively.However, the thienopyrimidine data showed that the S 5 (thpy) , S (thox) and C 6 (thpy) were the top three active sites in the derivatives 6 and 8 while in the case of compound 4, they were also the most susceptible atoms but in a different order, S (thox) , S 5 (thpy) and C 4 (thpy) , respectively (Table 3).
However, the electrophilic attack Fukui's indices (f − k ) of the thienyl derivatives 3, 5 and 7 offered different patterns of the highly susceptible atoms wherein the  thienyl sulphur atom S 1 (thio) occupied the first place in 3 and 5, while the cyano nitrogen NC (cyan) was the most active one in 7. The atoms in the second and third place were varied, whereas the amino nitrogen atoms NH 2(thio) and NH (Ph) occupied the fourth and fifth positions, respectively.On the other hand, the thienopyrimidine compounds presented comparable patterns where the thioxo sulphur atom, S (thox) , existed at the top position followed by the S 5 (thpy) atom except in 6 where the O (oxo) atom occupied the second place before the thienopyrimidine sulphur.Eventually, the radical attack (f 0 k ) indices of the synthesized thienyl and thienopyrimidine derivatives exhibited comparable orderliness to those obtained for the electrophilic attack Fukui's indices (f + k ) where the sulphur of thienyl S 1 (thio) and thioxo S (thox) was the most susceptible site, respectively.Moreover, the oxygen of acetyl and carboxamide groups occupied the second place in compounds 3 and 5, respectively, while the cyano nitrogen was in derivative 7.However, the sulphur of the thienopyrimidine ring, S 5 (thpy) , appeared in the second place in 4, 6 and 8 (Table 3).
To overcome the inaccurate prediction of the active sites for the nucleophilic and electrophilic attack via Fukui's indices, the relative electrophilicity and nucleophilicity descriptors, s − k /s + k and s + k /s − k , respectively, were estimated [68][69][70], where and δ is the global softness.The relative nucleophilicity descriptors data, s + k /s − k , of derivative 3 showed that carboxamide CO (carb) and phenylamine C 3 (Ph) were the first and second active atoms, respectively, as well as in 5 and 7.Moreover, the data indicated that the phenylcarboxamide carbon atoms, CO (carb) and C 2 (Phcarb) , were the first two susceptible in compounds 6 and 8, whereas in 4, the thienopyrimidine carbon, C 4 (thpy) , was the first and followed by the thienopyrimidine nitrogen atom, N 1 (thpy) .
Alternatively, the relative electrophilicity descriptors, s − k /s + k , data displayed almost coincided patterns for the thiophene and thienopyrimidine derivatives, wherein in 3 and 5, the thienyl carbons C 5 (thio) and C 4 (thio) were on the top positions, respectively, whilst in 7, they were in a reversed order.Likewise, the thienopyrimidine carbon C 2 (thpy) was the most susceptible site in the 4 and 6, whilst, in 8, the N 1 (thpy) was on the top and followed by the C 2 (thpy) (Table 3).

Antimicrobial activity
Using the disc-agar diffusion technique, the newly prepared thiophene and thienopyrimidine derivatives were investigated for antibacterial effectiveness across Gram +ve and Gram −ve spectra.Figures 7 and 8 demonstrated the antimicrobial results for the 4-aminothiophene and thienopyrimidine derivatives towards the antibacterial action because they could block the creation of folic acid on both types of bacterial strains [71].The checked derivatives, in general, are more susceptible to Gram-positive and (−ve) Gramnegative bacterial strains.Where, compounds 3, 4, 6 and 8 designated higher activity towards Gram-positive bacterial strains rather than Gram-negative strains relative to the results obtained by Chloramphenicol and Cephalothin.Meanwhile, compounds 4, 6 and 8 (characterized by the presence of thienopyrimidine moiety) exhibited the highest activity towards S. aureus and B. subtilis as Gram-positive bacterial strains across two concentrations of 0.5 and 1.0 mg/mL, respectively.
Viewing subtilis.Furthermore, the presence of electron-attracting groups such as (acetyl, cyano and ester) in compounds 3, 5 and 7 containing amino-thiophene moiety represented acceptable improvement in their antibacterial activities against S. typhimurium and E. coli as Gram-negative bacterial strains in contrast to the standard Cephalothin (Table S4).   the growth of (MCF-7) and (HepG2).[72].The results of the studied amino-thiophene and thienopyrimidine compounds exhibit inhibitions toward MCF-7, HepG2, and HCT-116, respectively in consistence to structural activity relationship.Primarily, the 5-acetyl-

Structural activity relationship (SAR)
As a purine base contained in DNA and RNAbioisosteres, adenine, can be structurally compared to the thieno-pyrimidine scaffold, which is a fused heterocyclic ring structure.We are investigating the relationship between its structure and activity because of its wide spectrum of therapeutic uses, such as anticancer preventatives (SAR).Inspired by the previous anticancer results and recent survey, the thienopyrimidine compounds, especially 6 and 8, showed potential activities against MCF-7 and HepG2 [73].Derivative 6 contains a pyrimidinone ring that recorded a superior activity than derivative 4 the methyl-pyrimidine moiety rather and analogue 7 has amino and cyano groups on the thiophene ring which showed reasonable antiproliferative activity between thienopyrimidine derivatives which agree with the earlier literature [74].Meanwhile, derivative 8 with the imino group displayed a lower activity rather than derivatives 6,4, and 7 which may be attributed to the carbonyl group being more attractive electrons than that of the imino group.Temporarily, in analogue 3 substituted with the 5-acetyl and 2-amino group on the thiophene presented poor selective antitumour activity [75].Finally, the order of reactivity can be recorded as 6 > 4 > 7 > 8 > 3,5.

Molecular docking
To investigate the interaction mechanism and binding mode of the synthesized 4-amino-thiophene and thienopyrimidine derivatives and the FDA drug 5fluorouracil at the active site of the (ID: 3qx3)protein, molecular docking studies are carried out using MOE (MOE Version 2015.10).By turning on the MOE software's reduction algorithm, protein energy is reduced to the absolute minimum.By employing the force field topoisomerase II β and a technique known as the conjugate gradient algorithm, the MOE software determines the final and beginning energy of the proteins in kcal/mol.The peptidomimetic ligand was released from Human topoisomerase II beta in a complex with DNA and etoposide after all water molecules were removed [76].The essential residues involved in the substrate's interaction, such as (Phe 738, Arg 743, Gly 1023, Glu 855 and Lys 843), have an adequate level of alignment (RMSD < 1.5).It is thought that they enable the substrate's grid to open to the active state.Inspired by the docking stimulation it was found that the synthesized 4-aminothiophene and thienopyrimidine derivatives revealed nearly acceptable binding scores ranging from −6.6321 to −7.6448 Kcal/mol (Table 4).Meanwhile, 4-amino-5-cyano-2-thiophene-3-carboxamide analogue 7 presented five intermolecular Hbonds (three H-donors and two H-acceptors), N 3 of amino-thiophene moiety with Glu 855, N 10 of N-phenyl moiety with Gly 1023, Cl 17 atom on the phenyl ring with Phe 738 and N 25 of cyano group with Arg 692 and Lys 843.All these interactions were formed over intermolecular distances 3.01, 3.27, 3.22, 3.16 and 3.30 Å, respectively (Figure S3).Moreover, 4-imino-2-thioxo-1,2,3,4tetrahydrothieno [3,2-d]pyrimidine analogue 8 exposed the highest binding score S = −7.6448Kcal/mol, Hdonor among N 28 of aniline moiety with His 1021, H-acceptor between Cl 21 of chlorine atom with Arg 743 and π -H between the pyrimidine ring and Gly 1023 over Rmsd = 1.1378 (Figure 12).Furthermore, 5-fluorouracil (reference) offered an Hacceptor between O3 of one of two carbonyl groups of the pyrimidine ring with Arg 688 and π -cation interaction between the pyrimidine ring with Arg 689 over 2.97 and 3.87 Å (Figure S4).
Lastly, a docking experiment was applied to confirm how much the prepared derivatives interactions alter as they cooperate with many 3QX3 amino acids.Each of the six synthesized amino-thiophene and thienopyrimidine derivative was reinforced by a network of H bonds as the chemical aromatic structures of aminothiophenes and thienopyrimidines interact directly with the amino acids of 3QX3.Most of the prepared derivatives had the 3QX3 pocket, which may be seen as a fork encompassed by polar and nonpolar residues of amino acids (Phe 738, Arg 743, Gly 1023, Glu 855 and Lys 843).Additionally, the fact that the majority of derivatives belong to the same amino acids is seen as significant support for the efficacy of the docking technique.

Conclusion
Three functionalized 4-aminothiophenes are synthesized based on cyclizing the N-(4-chlorophenyl)-2cyano-3-mercapto-3-(phenylamino)-acrylamide with chloroacetone, ethyl bromoacetate and/or chloroacetonitrile.Treatment of these 4-aminothiophenes with phenyl isothiocyanate was the synthetic strategy to obtain their congruous thieno [3,2-d]pyrimidine compounds.The investigated compounds' DFT calculated structure designated that all deviated from planar conformation.The HOMO-LUMO energy gaps of thiophene derivatives have higher values than the thienopyrimidines, as shown in the following order 4 < 8 < 6 < 7 < 5 < 3. The synthesized derivatives were assessed for their antimicrobial activity towards S. aureus, B. subtilis (+ve Gram) and S. typhimurium, E. coli (−ve Gram).From antimicrobial screening results, it was observed that derivatives 3, 4, 6 and 8 designated acceptable activity towards (+ve and −ve) Gram strains compared to standard drugs, Chloramphenicol and Cephalothin.Meanwhile, the examined derivatives' cytotoxic effects on the cancer cell lines "HCT-116, MCF-7, and HepG2" were assessed.In contrast to "5-fluorouracil" the usual reference, the examined derivatives 4, 6, and 7 showed strong effectiveness towards MCF-7 and HepG2 cell lines.Additionally, theoretical molecular docking results displayed acceptable binding scores and Rmsd values for the investigated derivatives alongside the amino acids of 3QX3.

Figure 1 .
Figure 1.Some biological activities of thiophene nucleus.
The HOMO-LUMO energy, E H and E L were affected by the abovementioned facts.Although the data displayed that all the derivatives have close values of the E H and E L , in the range of −5.43 to −6.01 and −3.23 to −3.86 eV, respectively, the thiophene derivatives 3, 5 and 7, generally, exhibited lower E H than the 4, 6 and 8.

Figure 9 Figure 7 .
Figure9displays the cytotoxic results as IC 50 values and compared them to the widely-used anti-cancer standard (5-Fu).Except for HCT-116, several synthesized thienopyrimidines showed good efficacy in preventing

Table 1 .
The HOMO-LUMO energies and chemical reactivity descriptors (eV) of investigated compounds.

Table 2 .
Selected Mulliken's atomic charges of investigated compounds.

Table 3 .
Selected top five atomic Fukui's indices, the local relative electrophilicity and nucleophilicity descriptors' data of the investigated compounds.

Table 4 .
In silico docking results of the prepared thiophene and thienopyrimidine analogues.