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

Abstract

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.

KEYWORDS:

• 4-Aminothiophene
• thienopyrimidine
• Mulliken’s charges
• antimicrobial
• HCT-116
• molecular docking

1. Introduction

Population growth has been negatively impacted by infection control that can be fatal and brought on by microbes decades ago [1]. Besides, contributing to the rise in infection rates and death, microbial fighting too increased the cost of health care associated with treating illnesses that are reluctant to antibiotics [2]. The second-leading cause of death in human beings and one of the major worldwide health issues is cancer [3,4]. The dispersal and death rate of cancer continue to be high regardless of significant breakthroughs in cancer therapy. Recently, several researchers focus on finding cancer treatments that are more efficient and less harmful [5–14]. Meanwhile, the discovery of new drugs for apoptosis, thymidylate synthase and NEK7 protein inhibitors, is heavily relying on various heterocyclic molecules, such as benzimidazole, 1,3,4-oxadiazole, benzo[f]chromene-2-carbonitrile, 1,2,3-Triazole, pyridine and pyrimidine [15–27]. Due to its notable variation in biological effectiveness, thiophene and its products have emerged as prominent scaffolding that has attracted the attention of scientists in the domain of pharmacological applications (Figure 1). The advantages of the thiophene ring have been demonstrated by the successful development of its analogues, which include antioxidant [28–30], anti-inflammatory [31], antibacterial [32,33] and anticancer effectiveness [34–37].

Figure 1. Some biological activities of thiophene nucleus.

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.

Figure 2. Some biological activities of thieno[2,3-d]pyrimidine nucleus.

2. Experimental

2.1. Synthesis of 4-aminothiophenes and their congruous thienopyrimidines

2.1.1. 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-d₆ at frequencies of 125 MHz (¹³C NMR) and 500 MHz (¹H 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).

2.1.2. Synthesis of 4-amino-N-(4-chlorophenyl)-2-(phenylamino)thiophene-3-carboxamide derivatives 3, 5 and 7

In a 100 mL round-bottom flask (RBF), N-(4-chlorophenyl)-2-cyano-3-mercapto-3-(phenylamino)acrylamide (2) (1.65 g, 5 mmol) was dissolved in 35 mL dioxane. Then, 5 mmol of each of the appropriate α-halogenated reagents (namely, chloroacetone, ethyl bromoacetate and/or chloroacetonitrile) and 0.5 mL triethylamine were added. The mixture was refluxed for 6 hours and then allowed to cool. The solid that formed in each case was collected to furnish the targeting 4-amino-5-substituted-thiophene compounds 3, 5 and 7, respectively.

5-Acetyl-4-amino-N-(4-chlorophenyl)-2-(phenylamino)thiophene-3-carboxamide (3): Yield = 83%, m.p. = 211–212°C. IR (ν/cm⁻¹): 3318, 3268 (NH₂ and N-H), 1642 (C=O, amide), 1597 (C=O, acetyl). ¹H NMR (δ/ppm): 2.09 (s, 3H, COCH₃), 7.09–7.66 (m, 9H, Ar-H), 7.47 (s, 2H, NH₂), 9.76 (s, 1H, N-H), 9.96 (s, 1H, N-H). ¹³C NMR (δ/ppm): 28.04, 105.70, 120.31 (2C), 121.89 (2C), 124.01, 126.97, 128.29 (2C), 129.39 (3C), 137.90, 140.85, 154.36, 157.51, 162.46, 186.67. MS m/z (%): 387 (M⁺ + 2, 21.44), 385 (M⁺, 60.87). Anal. Calcd. for C₁₉H₁₆ClN₃O₂S (385.07): C, 59.14; H, 4.18; N, 10.89%. Found: C, 59.23; H, 4.14; N, 10.81%.

4-Amino-N-(4-chlorophenyl)-5-ethoxycarbonyl-2-(phenylamino)thiophene-3-carboxamide (5): Yield = 78%, m.p. = 227–228°C. IR (ν/cm⁻¹): 3362, 3324, 3262 (NH₂ and N-H), 1663 (C=O, amide), 1636 (C=O, ester). ¹H NMR (δ/ppm): 1.20 (s, 3H, OCH₂CH₃), 4.12 (s, 2H, OCH₂CH₃), 6.67 (s, 2H, NH₂), 7.08–7.65 (m, 9H, Ar-H), 9.68 (s, 1H, NH), 9.90 (s, 1H, NH). ¹³C NMR (δ/ppm): 14.57, 59.04, 106.09, 119.91 (2C), 121.85 (2C), 123.68, 126.94, 128.30 (2C), 129.38 (3C), 137.88, 141.01, 154.03, 157.14, 162.58, 163.49. MS m/z (%): 415 (M⁺, 15.63). Anal. Calcd. for C₂₀H₁₈ClN₃O₃S (415.08): C, 57.76; H, 4.36; N, 10.10%. Found: C, 57.90; H, 4.42; N, 10.19%.

4-Amino-N-(4-chlorophenyl)-5-cyano-2-(phenylamino)thiophene-3-carboxamide (7): Yield = 70%, m.p. = 236–237°C. IR (ν/cm⁻¹): 3309, 3256 (NH₂ and N-H), 2207 (C≡N), 1657 (C=O, amide). ¹H NMR (δ/ppm): 6.06 (s, 2H, NH₂), 6.76–7.54 (m, 9H, Ar-H), 9.71 (s, 1H, N-H), 10.04 (s, 1H, N-H). ¹³C NMR (δ/ppm): 87.29, 106.35, 114.33, 120.21 (2C), 121.86 (2C), 124.11, 128.37 (2C), 129.54 (2C), 130.13, 133.64, 137.80, 140.93, 158.07, 162.67. MS m/z (%): 370 (M⁺ + 2, 13.94), 368 (M⁺, 40.78). Anal. Calcd. for C₁₈H₁₃ClN₄OS (368.05): C, 58.62; H, 3.55; N, 15.19%. Found: C, 58.50; H, 3.61; N, 15.28%.

2.1.3. Synthesis of N-(4-chlorophenyl)-3-phenyl-6-phenylamino-thieno [3,2-d]pyrimidine-7-carboxamide analogues 4, 6 and 8

In a 100 mL RBF, a mixture of each 4-amino-1-(4-chlorophenyl)-thiophene derivative 3, 5 or 7 (2 mmol) and phenyl isothiocyanate (0.24 mL, 2 mmol) was refluxed for 4 hours in 25 mL pyridine (in compounds 3 and 5) or 25 mL DMF (in compound 7). The mixture was cooled and then poured into ice water (25 g). The solid produced in each case was collected and recrystallized from the EtOH-DMF mixture (4:1) to provide the targeting thieno[3,2-d]pyrimidine analogues 4, 6 and 8, respectively.

N-(4-Chlorophenyl)-4-methyl-3-phenyl-6-(phenylamino)-2-thioxo-2,3-dihydrothieno[3,2-d]pyrimidine-7-carboxamide (4): Yield = 58%, m.p. > 300°C. IR (ν/cm⁻¹): 3233, 3171 (N-H), 1648 (C=O, amide). ¹H NMR (δ/ppm): 2.35 (s, 3H, CH₃), 7.03–7.74 (m, 14H, Ar-H), 9.91 (s, 1H, N-H), 10.13 (s, 1H, N-H). MS m/z (%): 504 (M⁺ + 2, 12.06), 502 (M⁺, 34.94). Anal. Calcd. for C₂₆H₁₉ClN₄OS₂ (502.07): C, 62.08; H, 3.81; N, 11.14%. Found: C, 61.91; H, 3.88; N, 11.27%.

N-(4-Chlorophenyl)-4-oxo-3-phenyl-6-(phenylamino)-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine-7-carboxamide (6): Yield = 63%, m.p. > 300°C. IR (ν/cm⁻¹): 3241, 3180 (N-H), 1671 (C=O, ring), 1656 (C=O, amide). ¹H NMR (δ/ppm): 7.05–7.78 (m, 14H, Ar-H), 9.80 (s, 1H, N-H), 10.07 (s, 1H, N-H), 12.91 (s, 1H, N-H). MS m/z (%): 504 (M⁺, 21.37). Anal. Calcd. for C₂₅H₁₇ClN₄O₂S₂ (504.05): C, 59.46; H, 3.39; N, 11.09%. Found: C, 59.21; H, 3.30; N, 11.00%.

N-(4-Chlorophenyl)-4-imino-3-phenyl-6-(phenylamino)-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine-7-carboxamide (8): Yield = 55%, m.p. = 282–283°C. IR (ν/cm⁻¹): 3351, 3243, 3188 (N-H), 1657 (C=O, amide), 1639 (C=N). ¹H NMR (δ/ppm): 7.08–7.65 (m, 14H, Ar-H), 9.88 (s, 1H, N-H), 10.31 (s, 1H, N-H), 11.22 (s, 1H, N-H), 12.98 (s, 1H, N-H). MS m/z (%): 503 (M⁺, 15.48). Anal. Calcd. for C₂₅H₁₈ClN₅OS₂ (503.06): C, 59.58; H, 3.60; N, 13.90%. Found: C, 59.81; H, 3.68; N, 13.77%.

2.2. Computational studies

The synthesized compounds were studied using the DFT/B3LYP/6-311⁺⁺G [45–47] 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].

2.3. 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).

2.4. 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.

2.5. 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.

3. Results and discussion

3.1. Chemistry

The synthetic strategy of 4-aminothiophene derivatives is based on the utilization of the versatile precursor, N-(4-chlorophenyl)-2-cyano-3-mercapto-3-(phenylamino) acrylamide (2) [54], by its cyclizing with various halogenated reagents. Thus, the production of 5-acetyl-4-amino-N-(4-chlorophenyl)-2-(phenylamino)thiophene-3-carboxamide (3) has been achieved by heating the thiocarbamoyl precursor 2 with chloroacetone in dioxane and triethylamine (Scheme 1). The reaction of 5-acetyl-4-aminothiophene derivative 3 with phenyl isothiocyanate has proceeded in boiling pyridine to produce the corresponding 4-methyl-thieno[3,2-d]pyrimidine compound 4. The compatible spectral and elemental analyses support the structures of the 5-acetyl-4-aminothiophene derivative 3 and its congruous thieno[3,2-d]pyrimidine analogue 4.

Scheme 1. Synthesis of 5-acetyl-4-aminothiophene analogue 3 and its congruous theino[3,2-d]pyrimidine derivative 4.

In addition, the treatment of N-(4-chlorophenyl)-2-cyano-3-mercapto-3-(phenylamino)-acrylamide (2) with ethyl bromoacetate was carried out in refluxing dioxane and triethylamine to afford the corresponding 5-ethoxycarbonyl-4-amino-thiophene analogue 5. The reaction mechanism is initiated by the ability of acidic thiol function (–SH) to replace the bromine from ethyl bromoacetate. The produced intermediate (A) undergoes intramolecular cyclization through the nucleophilic addition of a methylene group on the nitrile function to produce the corresponding 4-amino-5-ethoxycarbonyl-thiophene derivative 5 (Scheme 2). The cyclization of this 4-aminothiophene derivative 5 with phenyl isothiocyanate in boiling pyridine yielded the bicyclic ring system, 4-oxo-thieno[3,2-d]pyrimidine compound 6. The structures of 4-aminothiophene analogue 5 and its congruous thieno[3,2-d]pyrimidine compound 6 were secured by compatible spectral analyses.

Scheme 2. Synthesis of 4-amino-5-ethoxycarbonyl-thiophene analogue 5 and its congruous theino[3,2-d]pyrimidine derivative 6.

Furthermore, the reaction of N-(4-chlorophenyl)-2-cyano-3-mercapto-3-(phenylamino)-acrylamide (2) with chloroacetonitrile in boiling dioxane and triethylamine furnished the corresponding 4-amino-5-cyano-thiophene derivative 7. The nucleophilic substitution of the chlorine from chloroacetonitrile by acidic thiol function (–SH) of thiocarbamoyl precursor 2 furnished the intermediate (B). This undergoes intramolecular cyclization through the nucleophilic addition of a methylene group on the nitrile function to produce the corresponding 4-amino-5-cyano-thiophene compound 7 (Scheme 3). The production of 4-imino-thieno[3,2-d]pyrimidine analogue 8 has been achieved upon heating 4-amino-5-cyanothiophene derivative 7 with phenyl isothiocyanate in boiling dimethylformamide (DMF) for four hours. The spectral analyses are utilized to secure the structures of 4-amino-5-cyanothiophene derivative 7 and its congruous 4-imino-thieno[3,2-d]pyrimidine analogue 8.

Scheme 3. Synthesis of 4-amino-5-cyanothiophene analogue 7 and its congruous theino[3,2-d]pyrimidine derivative 8.

3.2. DFT structural and electronic features

The dihedral data of 4-amino-N-(4-chlorophenyl)-2-(phenylamino)thiophene-3-carboxamide derivatives 3, 5 and 7 have a non-planar configuration in which the phenylamino nitrogen and phenyl carboxamide carbon and oxygen atoms were situated out of the thiophene ring plane, e.g. the average values of C⁴_(thio)-C³_(thio)-C²_(thio)-NH_(Ph), CO_(carb)-C³_(thio)-C²_(thio)-S¹_(thio) and C²_(thio)-C³_(thio)-CO_(carb)-OC_(carb) were 171.5, 175.6 and 19.3°, respectively. Furthermore, the carboxamide phenyl ring was tilted more than the other phenyl ring as the C³_(thio)-CO_(carb)-NH_(carb)-C¹_(Phcarb) = 39.7° while S¹_(thio)-C²_(thio)-NH_(Ph)-C¹_(Ph) = −7.1°. The 4-amino substituent in all derivatives was slightly shifted above the thiophene plane, e.g. the NH_2(thio)-C⁴_(thio)-C⁵_(thio)-S¹_(thio) = 174.5°, and so the 5-substituents, acetyl, carboxylate and cyano (Figure 3) (Table S1).

Figure 3. DFT Optimized structures of the thiophene derivatives 3, 5 and 7.

Alternatively, the N-(4-chlorophenyl)-3-phenyl-6-(phenylamino)-2-thioxo-2,3-dihydrothieno [3,2-d]pyrimidine-7-carboxamide derivatives, 4, 6 and 8, have also a non-planar configuration. The dihedral angle data showed that the thienopyrimidine-fused ring slightly deviated from planarity, 0.1–3.5°. Also, the methyl and oxo substituents in 4 and 6 were marginally out of the fused ring plane, i.e. S⁵_(thpy)-C^4a_(thpy)-C⁴_(thpy)-Me_(thpy) = -1.3° and S⁵_(thpy)-C^4a_(thpy)-C⁴_(thpy)-O_(Oxo) = 1.1°, respectively, while both imino groups were almost coplanar, S⁵_(thpy)-C^4a_(thpy)-C⁴_(thpy)-NH_(imn) = 0.3°. The thioxo substituent showed perfect planarity with the thienopyrimidine in 4 as C⁴_(thpy)-N³_(thpy)-C²_(thpy)-S_(thoxo) = 180.0°, whereas the corresponding angle in 6 and 8 was 175.4°, which is clearly shifting out. However, the data presented that the phenyl substituent was almost perpendicular on the fused ring plane, e.g. C²_(thpy)-N³_(thpy)-C¹_(Ph)-C²_(Ph) = 89.4–95.0°. Despite the phenylamino nitrogen atom exhibited a little non-planarity with respect to the thienopyrimidine moiety where NH_(Phamn)-C⁶_(thpy)-S⁵_(thpy)-C^4a_(thpy) = 175.6–177.7°, the corresponding phenyl ring was strongly tilted on the fused ring plane, e.g. the C⁶_(thpy)-NH_(Phamn)-C¹_(Phamn)-C²_(Phamn) ranged from 160.3°, in 4, to 171.3°, in 8. Likewise, the carboxamide carbon atom was placed out of the thienopyrimidine ring plane in all derivatives by 8.5–9.9°, while its oxygen and nitrogen atoms were strongly shifted out of the plane, ∼17.5° and 40.0°, respectively, in 6 and 8 (Figure 4) (Table S1).

Figure 4. DFT Optimized structures of the thienopyrimidine derivatives 4, 6 and 8.

As well, the DFT calculated bond lengths and angles almost coincided with those obtained from single crystal X-rays of similar compounds [55–57] where lengths were longer by 1.72 Å maximum with 0.052–0.062 Å 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).

3.2.1. 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 4-amino-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).

Figure 5. The frontier molecular orbital of the thiophene compounds 3, 5 and 7.

Figure 6. The frontier molecular orbital of the thienopyrimidine compounds 4, 6 and 8.

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).

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. Contrarily, the E_L data revealed that the thienopyrimidine derivatives have lower values than those of thiophenes. However, the ΔE_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).

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

Compound	EH	EL	ΔEH-L	χ	η	δ	ω
3	−5.68	−3.31	2.37	4.50	1.18	0.85	8.55
4	−5.66	−4.32	1.33	4.99	0.67	1.50	18.69
5	−5.56	−3.23	2.32	4.39	1.16	0.86	8.31
6	−6.17	−3.97	2.20	5.07	1.10	0.91	11.71
7	−5.68	−3.43	2.25	4.56	1.13	0.89	9.21
8	−5.97	−3.84	2.13	4.91	1.07	0.94	11.29

Correspondingly, chemical reactivity descriptors, electronegativity (χ), global hardness (η), softness (δ) and electrophilicity (ω) were evaluated using the E_H and E_L values by the following formula [60]. $\begin{array}{rl}\chi & =-\frac{1}{2}(E_{\text{HOMO}}+E_{\text{LUMO}})\eta =-\frac{1}{2}(E_{\text{HOMO}}-E_{\text{LUMO}})\\ \delta & =\frac{1}{\eta }\omega =\frac{{\chi }^2}{2\eta }\end{array}$ 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 ΔE_H-L gap (Table 1).

3.2.2. 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, S¹_(thio), has a constant negative charge in all derivatives, −0.614 to −0.691, while the thienyl carbon, C⁵(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).

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

Atom	3	5	7	Atom	4	6	8
S^1(thio)	−0.648	−0.691	−0.614	N^1(thpy)	0.612	0.113	0.100
C^2(thio)	0.532	0.712	0.598	C^2(thpy)	−0.325	−0.485	−0.404
C^3(thio)	−0.335	−0.410	−0.485	N^3(thpy)	0.754	0.568	0.609
C^4(thio)	−0.484	−0.396	−0.983	C^4(thpy)	0.406	0.275	0.249
C^5(thio)	−0.783	−0.275	−0.145	C^4a(thpy)	0.252	0.242	0.255
NH(Ph)	−0.166	−0.172	−0.136	S^5(thpy)	−0.924	−0.639	−0.672
C^1(Ph)	−0.955	−1.102	−1.101	C^6(thpy)	−0.113	0.256	0.134
C^4(Ph)	−0.232	−0.215	−0.276	C^7(thpy)	0.700	0.074	0.011
CO(carb)	−0.139	−0.363	−0.498	C^7a(thpy)	0.430	−0.228	−0.102
OC(carb)	−0.348	−0.363	−0.358	S(thox)	−0.299	−0.473	−0.534
NH(carb)	−0.659	−0.669	−0.664	C^1(Phthpy)	−0.211	−0.203	−0.042
C^1(Phcarb)	−0.517	−0.323	−0.467	C^4(Phthpy)	−0.262	−0.309	−0.285
C^4(Phcarb)	0.604	0.589	0.669	NH(Phamn)	−0.323	−0.161	−0.181
Cl(Phcarb)	0.812	0.832	0.778	C^1(Phamn)	−0.273	−0.505	−0.404
NH2(thio)	−0.637	−0.687	−0.497	C^4(Phamn)	−0.492	−0.326	−0.344
CO(act)	0.750			CO(carb)	0.441	0.129	0.147
OC(act)	−0.458			OC(carb)	−0.205	−0.320	−0.317
CO(Est)		0.416		NH(carb)	−0.179	−0.269	−0.277
O^1(Est)		−0.374		C^1(Phcarb)	−0.701	−0.983	−0.990
O^2(Est)		0.024		C^4(Phcarb)	0.836	0.805	0.823
CN(cyan)			0.637	Cl(Phcarb)	0.806	0.822	0.819
NC(cyan)			−0.237	Me(Phthpy)	−0.520
				O(Oxo)		−0.236
				NH(imn)			−0.151

In thienopyrimidine derivatives, the thienopyrimidine nitrogen atom N¹_(thyp) and carbon C⁴_(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 N³_(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 S⁵_(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 $f_k^0$, towards nucleophilic, electrophilic and radical attacks, respectively, were assessed [64–67]. The nucleophilic attack Fukui’s indices $(f_k^{+})$ of the thiophene derivatives 3, 5 and 7 disclosed that the thienyl sulphur S¹_(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 S⁵_(thpy), S_(thox) and C⁶_(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), S⁵_(thpy) and C⁴_(thpy), respectively (Table 3).

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

3		5		7		4		6		8
Atom	${\mathit{f}}_{\mathit{k}}^{-}$	Atom	${\mathit{f}}_{\mathit{k}}^{-}$	Atom	${\mathit{f}}_{\mathit{k}}^{-}$	Atom	${\mathit{f}}_{\mathit{k}}^{-}$	Atom	${\mathit{f}}_{\mathit{k}}^{-}$	Atom	${\mathit{f}}_{\mathit{k}}^{-}$
S^1(thio)	0.09	S^1(thio)	0.10	NC(cyan)	0.13	S(thox)	0.42	S(thox)	0.314	S(thox)	0.307
OC(act)	0.09	C^5(thio)	0.09	S^1(thio)	0.10	S^5(thpy)	0.061	O(Oxo)	0.052	S^5(thpy)	0.048
C^5(thio)	0.08	O^1(Est)	0.07	C^5(thio)	0.09	C^2(thpy)	0.042	S^5(thpy)	0.052	NH(imn)	0.047
NH2(thio)	0.05	NH2(thio)	0.06	NH2(thio)	0.06	C^4(Phthpy)	0.031	Cl(Phcarb)	0.035	Cl(Phcarb)	0.033
NH(Ph)	0.05	NH(Ph)	0.05	NH(Ph)	0.05	Cl(Phcarb)	0.031	C^2(thpy)	0.032	C^2(thpy)	0.031
Atom	${\mathit{f}}_{\mathit{k}}^{+}$	Atom	${\mathit{f}}_{\mathit{k}}^{+}$	Atom	${\mathit{f}}_{\mathit{k}}^{+}$	Atom	${\mathit{f}}_{\mathit{k}}^{+}$	Atom	${\mathit{f}}_{\mathit{k}}^{+}$	Atom	${\mathit{f}}_{\mathit{k}}^{+}$
S^1(thio)	0.08	S^1(thio)	0.09	S^1(thio)	0.10	S(thox)	0.108	S(thox)	0.088	S(thox)	0.088
CO(carb)	0.08	OC(carb)	0.07	CO(carb)	0.08	S^5(thpy)	0.072	S(thox)	0.072	S(thox)	0.065
OC(carb)	0.08	CO(carb)	0.07	OC(carb)	0.08	C^4(thpy)	0.068	C^6(thpy)	0.062	C^6(thpy)	0.064
Cl(Phcarb)	0.06	Cl(Phcarb)	0.06	NC(cyan)	0.06	C^6(thpy)	0.059	Cl(Phcarb)	0.05	CO(carb)	0.053
C^2(thio)	0.05	C^2(thio)	0.05	Cl(Phcarb)	0.06	C^7a(thpy)	0.053	CO(carb)	0.048	Cl(Phcarb)	0.052
Atom	${\mathit{f}}_{\mathit{k}}^0$	Atom	${\mathit{f}}_{\mathit{k}}^0$	Atom	${\mathit{f}}_{\mathit{k}}^0$	Atom	${\mathit{f}}_{\mathit{k}}^0$	Atom	${\mathit{f}}_{\mathit{k}}^0$	Atom	${\mathit{f}}_{\mathit{k}}^0$
S^1(thio)	0.09	S^1(thio)	0.10	S^1(thio)	0.10	S(thox)	0.264	S(thox)	0.193	S(thox)	0.186
OC(act)	0.07	OC(carb)	0.05	NC(cyan)	0.10	S^5(thpy)	0.067	S^5(thpy)	0.07	S^5(thpy)	0.068
OC(carb)	0.06	O^1(Est)	0.05	OC(carb)	0.05	C^4(thpy)	0.04	O(Oxo)	0.047	NH(imn)	0.044
Cl(Phcarb)	0.05	Cl(Phcarb)	0.05	C^5(thio)	0.05	C^6(thpy)	0.037	Cl(Phcarb)	0.043	Cl(Phcarb)	0.043
CO(carb)	0.05	C^5(thio)	0.05	Cl(Phcarb)	0.05	C^7a(thpy)	0.034	C^6(thpy)	0.037	C^6(thpy)	0.038
Atom	S^+/S^−	Atom	S^+/S^−	Atom	S^+/S^−	Atom	S^+/S^−	Atom	S^+/S^−	Atom	S^+/S^−
CO(carb)	5.27	CO(carb)	5.69	CO(carb)	5.85	C^4(thpy)	6.18	CO(carb)	16.00	C^2(Phcarb)	18.00
C^3(Ph)	3.17	C^3(Ph)	4.00	C^3(Ph)	3.17	N^1(thpy)	5.00	C^2(Phcarb)	16.00	CO(carb)	17.67
C^2(Ph)	3.00	C^2(Ph)	2.80	C^2(thio)	2.67	C^2(Phamn)	5.00	NH(carb)	5.25	C^1(Phamn)	6.00
C^2(thio)	2.78	C^2(thio)	2.79	OC(carb)	2.34	C^6(thpy)	3.93	C^6(thpy)	5.17	NH(carb)	5.75
OC(carb)	2.36	OC(carb)	2.39	C^6(Ph)	2.18	NH(carb)	3.50	C^1(Phamn)	3.67	C^6(thpy)	5.33
Atom	S^−/S^+	Atom	S^−/S^+	Atom	S^−/S^+	4	S^−/S+	6	S^−/S+	8	S^−/S+
C^5(thio)	6.15	C^5(thio)	6.77	C^4(thio)	6.00	C^2(thpy)	4.20	C^2(thpy)	6.40	N^1(thpy)	22.00
C^4(thio)	4.57	C^4(thio)	6.20	C^5(thio)	5.12	S(thox)	3.89	N^1(thpy)	5.25	C^2(thpy)	7.75
NH2(thio)	3.92	NH2(thio)	4.54	NH2(thio)	4.43	C^2(Phthyp)	3.00	C^2(Phthyp)	5.00	C^2(Phthyp)	6.00
OC(act)	2.17	O^2(Est)	2.43	CN(cyan)	3.50	C^6(Phthyp)	3.00	S(thox)	4.36	C^6(Phthyp)	5.00
CO(act)	2.06	CO(Est)	2.42	NC(cyan)	2.02	C^2(Phcarb)	2.33	C^6(Phthyp)	4.00	S(thox)	4.72

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 S¹_(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 S⁵_(thpy) atom except in 6 where the O_(oxo) atom occupied the second place before the thienopyrimidine sulphur.

Eventually, the radical attack $(f_k^0)$ 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 S¹_(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, S⁵_(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 [68–70], where $s_k^{+}=f_k^{+}\times \delta ,s_k^{-}=f_k^{-}\times \delta$ 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 C³_(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 C²_(Phcarb), were the first two susceptible in compounds 6 and 8, whereas in 4, the thienopyrimidine carbon, C⁴_(thpy), was the first and followed by the thienopyrimidine nitrogen atom, N¹_(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 C⁵_(thio) and C⁴_(thio) were on the top positions, respectively, whilst in 7, they were in a reversed order. Likewise, the thienopyrimidine carbon C²_(thpy) was the most susceptible site in the 4 and 6, whilst, in 8, the N¹_(thpy) was on the top and followed by the C²_(thpy) (Table 3).

3.3. Biological evaluation

3.3.1. 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-amino-thiophene 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) Gram-negative 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.

Figure 7. Antibacterial activity of the examined derivatives towards Gram’s positive bacteria.

Figure 8. Antibacterial activity of the tested compounds against Gram’s negative bacteria.

Viewing the results, it became clear that compound 8 displayed (39.72 ± 2.98 and 42.57 ± 3.19) against S. aureus and (33.47 ± 2.51 and 35.91 ± 2.69) against B. subtilis, hybrid 6 revealed (43.53 ± 3.26 and 45.64 ± 3.42) against S. aureus and (38.22 ± 2.87 and 39.83 ± 2.99) against B. subtilis and hybrid 4 revealed (34.34 ± 2.58 and 35.16 ± 2.64) against S. aureus and (21.15 ± 1.59 and 23.41 ± 1.76) against B. subtilis. Moreover, derivative 3 having the 5-acetyl-4-amino-thiophene moiety offered better activity (37.22± 2.79 and 38.56 ± 2.89) against S. aureus and (31.82 ±2.39 and 32.40 ± 2.43) against B. 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).

3.3.2. Anti-tumour activity

Figure 9 displays the cytotoxic results as IC₅₀ 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 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-4-amino-thiophene-3-carboxamide thiophene analogue 3 has a reputable weak activity with IC₅₀ = 29.51 ± 0.47, 47.16 ± 0.12 and 37.63 ± 0.32 μM. While analogue 4 with 4-methyl-2-thioxo-2,3-dihydrothieno[3,2-d]pyrimidine moiety offered good activities with IC₅₀ =14.53 ± 0.16, 12.27 ± 0.12 and 15.75 ± 0.42μM. Meanwhile, 2-carboethoxy-3-amino-thiophene analogue 5 displayed poor effectiveness with IC₅₀ =38.44 ± 0.19, 36.35 ± 0.27 and 49.82 ± 0.38 μM. However, 4-oxo-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine analogue 6 showed excellent activities with IC₅₀ = 11.17 ± 0.06, 9.33 ± 0.27 and 10.63 ± 0.40 μM against MCF-7, HepG2 and HCT-116, respectively. Moreover, 4-amino-5-cyano-2-thiophene-3-carboxamide analogue 7 showed reasonable cytotoxic activities with IC₅₀ = 16.76 ± 0.36, 15.80 ± 0.23 and 18.42 ± 0.46 μM. Furthermore, 4-imino-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine analogue 8 with revealed moderate anti-proliferative activities with IC₅₀ = 19.33 ± 0.18, 18.02 ± 0.39 and 23.81 ± 0.11 μM.

Figure 9. In vitro cytotoxicity of amino-thiophene and thienopyrimidine analogues.

3.3.3. Structural activity relationship (SAR)

As a purine base contained in DNA and RNA-bioisosteres, 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 with 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 anti-tumour activity [75]. Finally, the order of reactivity can be recorded as 6 > 4 > 7 > 8 > 3,5.

3.4. Molecular docking

To investigate the interaction mechanism and binding mode of the synthesized 4-amino-thiophene and thienopyrimidine derivatives and the FDA drug 5-fluorouracil 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-amino-thiophene and thienopyrimidine derivatives revealed nearly acceptable binding scores ranging from −6.6321 to −7.6448 Kcal/mol (Table 4). The 5-acetyl-4-amino-thiophene-3-carboxamide derivative 3 displayed two interactions, Cl 20 on the phenyl ring with Phe 738 and Arg 743 amino acids over H-donor through (3.34 Å) and H-acceptor through (3.51 Å) (Figure S1). Similarly, 4-methyl-2-thioxo-2,3-dihydrothieno[3,2-d]pyrimidine 4 exhibited H-acceptor interaction between S 7 of the thioxo group with Arg 581, two π- H attractions between the phenyl ring of aniline moiety with Lys 1107 and a pyrimidine ring with Asn 840 over intermolecular distances 4.20, 4.27 and 4.02 Å (Figure 10).

Figure 10. Docking interactions over analogue 4 with 3QX3.

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

Code	S (energy score) (Kcal/mol)	Rmsd (Refine unit)	Interaction with ligand	Types of Interactions	Distance ($\text{Å}$)
3	−6.6321	0.7603	Cl 20 with Phe 738	H-donor	3.34
			Cl 20 with Arg 743	H-acceptor	3.51
4	−7.2571	1.3348	7 with Arg 581	H-acceptor	4.20
			6-ring with Asn 840	π-H	4.27
			6-ring with Lys 1107	π-H	4.02
5	−7.0238	0.9526	Cl 21 with Phe 738	H-donor	3.32
			Cl 21 with Arg 743	H-acceptor	3.69
6	−7.6356	0.9029	11 with Glu855	H-donor	3.52
			28 with Glu855	H-donor	3.04
			S7 with Arg 689	H-acceptor	3.22
7	−7.0014	1.2398	3 with Glu 855	H-donor	3.01
			10 with Gly 1023	H-donor	3.27
			Cl 17 with Phe 738	H-donor	3.22
			25 with Arg 692	H-acceptor	3.16
			25 with Lys 843	H-acceptor	3.30
8	−7.6448	1.3112	28 with His 1021	H-donor	3.02
		1.1378	Cl 21 with Arg 743	H-acceptor	3.46
			6-ring with Gly 1023	π-H	3.98
5-Fu	−5.9285	0.9449	3 with Arg 688	H-acceptor	2.97
			6-ring with Arg 689	π-cation	3.87

Likewise, 2-ethoxycarbonyl-3-amino-thiophene analogue 5 demonstrated the identical interactions between Cl 21 with Phe 738 and Arg 743 amino acids with the same H-bonds (donor and acceptors) and intermolecular distances 3.32 and 3.69 $\text{Å}$ (Figure S2). Temporarily, 4-oxo-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine analogue 6 displayed two H-donors between Glu855 amino acid through S11 of thiophene ring and N 28 of aniline moiety in addition to one H-acceptor between S7 thioxo moiety with Arg 689 with prefect Rmsd = 0.9029 (Figure 11).

Figure 11. Docking interactions over analogue 6 with 3QX3.

Meanwhile, 4-amino-5-cyano-2-thiophene-3-carboxamide analogue 7 presented five intermolecular H-bonds (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 $\text{Å}$, respectively (Figure S3). Moreover, 4-imino-2-thioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine analogue 8 exposed the highest binding score S = −7.6448 Kcal/mol, H-donor 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).

Figure 12. Docking interactions over analogue 8 with 3QX3.

Furthermore, 5-fluorouracil (reference) offered an H-acceptor 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 $\text{Å}$ (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 amino-thiophenes 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.

4. Conclusion

Three functionalized 4-aminothiophenes are synthesized based on cyclizing the N-(4-chlorophenyl)-2-cyano-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.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the Deanship of Scientific Research at Umm Al-Qura University [grant number 22UQU4350527DSR02].