Novel Pyridothienopyrimidine Derivatives: Design, Synthesis and Biological Evaluation as Antimicrobial and Anticancer Agents

Abstract

The growing risk of antimicrobial resistance besides the continuous increase in the number of cancer patients represents a great threat to global health, which requires intensified efforts to discover new bioactive compounds to use as antimicrobial and anticancer agents. Thus, a new set of pyridothienopyrimidine derivatives 2a,b–9a,b was synthesized via cyclization reactions of 3-amino-thieno[2,3-b]pyridine-2-carboxamides 1a,b with different reagents. All new compounds were evaluated against five bacterial and five fungal strains. Many of the target compounds showed significant antimicrobial activity. In addition, the new derivatives were further subjected to cytotoxicity evaluation against HepG-2 and MCF-7 cancer cell lines. The most potent cytotoxic candidates (3a, 4a, 5a, 6b, 8b and 9b) were examined as EGFR kinase inhibitors. Molecular docking study was also performed to explore the binding modes of these derivatives at the active site of EGFR-PK. Compounds 3a, 5a and 9b displayed broad spectrum antimicrobial activity with MIC ranges of 4–16 µg/mL and potent cytotoxic activity with IC₅₀ ranges of 1.17–2.79 µM. In addition, they provided suppressing activity against EGFR with IC₅₀ ranges of 7.27–17.29 nM, higher than that of erlotinib, IC₅₀ = 27.01 nM.

1. Introduction

In the past twenty years, the incidence of microbial infections associated with increased antimicrobial resistance has increased by alarming levels worldwide, endangering the possibility of curing many infectious diseases, and representing a global threat to population health [1,2,3]. One of the potential approaches to combat this resistance problem is based on designing innovative new molecules with different modes of action to overcome cross-resistance to present therapeutics [4,5]. Efforts in developing broad-spectrum as well as specific and targeted drugs against various microbes are continuous in all directions [5]. One of the recent strategies used for developing new antimicrobial agents is hybridization of different pharmacophores binding diverse biomolecular targets to exert synergistic effects against drug-resistant infectious diseases [6].

On the other hand, despite large advances in the diagnosis and treatment of various types of cancers, the survival of cancer patients remains poor because of the widespread side effects of anticancer therapeutics as well as the acquisition of multiple drug resistance by the cancer cells [7]. Breast and liver cancers are among the most medically significant cancers due to their high incidence and morbidity [8]. Therefore, obtaining new, effective, more selective, and less toxic anticancer agents remains one of the most urgent demands [9]. In addition, cancer patients are at higher risk of drug-resistant infectious diseases because of the weakness and immunosuppression caused by anticancer drug therapy [10], which necessitates the development of new drugs that have potent dual activity against cancer and pathogenic microbes [11].

Heterocyclic ring systems are considered key molecular structures in medicinal chemistry. In addition to their presence in the skeleton of many biological molecules such as hemoglobin, DNA, RNA, vitamins, hormones, and heterocyclic compounds [12,13,14], they produce a wide range of biological activities [15,16]. Accordingly, the structural diversity and biological prominence of heterocyclic compounds have made them attractive synthetic targets in drug design and discovery for many years [12,13]. Thieno[2,3-b]pyridines constitute a set of heterocyclic compounds that have beneficial effects in the treatment of many diseases. Recently, many studies have described various substituted thienopyridine compounds as significant antiproliferative agents against a wide range of human cancer cell lines [17,18,19] and as antimicrobial [20,21,22], anti-Alzheimer’s [23], anti-platelet [24], antiviral [25,26] and anti-inflammatory [27] candidates.

Furthermore, the pyrimidine structure is closely related to the nucleobases—uracil, thymine, and cytosine—which makes pyrimidine molecules important building blocks in all living cells [28,29]. Pyrimidine-based compounds exhibit a broad spectrum of pharmacological activity, such as antimalarial [30], antidiabetic [31], antimicrobial [32,33], and anticancer activities [34,35,36]. Therefore, combining thienopyridine and pyrimidine cores in the same molecular architecture, forming a pyridothienopyrimidine nucleus, serves as an attractive strategy for designing a novel scaffold with more favorable pharmacological effects [37]. Recently, various pyridothienopyrimidine derivatives were reported to produce significant antimicrobial [38,39] and anticancer activities [40,41], as well as to suppress protein kinases such as serine/threonine kinase and vascular endothelial growth factor receptor (VEGFR-2) [42,43].

Prompted by the abovementioned issues, the strategy of this work was focused on the design of new tricyclic pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine compounds with structures that hybridize features of the reported thienopyridine and pyrimidine antimicrobial and/or anticancer agents [21,22,33,34] Figure 1. The resulting new pyridothienopyrimidines 2a,b–9a,b were synthesized via different cyclization reactions of the key starting compounds, 3-aminothieno[2,3-b]pyridine-2-carboxamides 1a,b. The tricyclic ring system of the target pyridothienopyridine compounds was linked at position-2 and/or position-4 to different privileged structural motifs such as morpholine, piprazine, spiro cycloalkane, oxirane, and acetamide, which are renowned for their valuable and diverse pharmacological activity [44,45,46,47,48]. All synthesized derivatives were evaluated as antimicrobial agents against a panel of Gram-positive and Gram-negative bacterial and fungal pathogens. Then, they were evaluated as cytotoxic agents against human liver carcinoma cells (HepG2) and human breast cancer cells (MCF-7) in an effort to gain new compounds possessing dual antimicrobial and anticancer activities. Since epidermal growth factor receptor (EGFR) is a key mediator in the regulation of different important cellular processes [49,50] and its overexpression displays a significant role in the development of many human cancers, including hepatic cancer and breast cancer [51,52], it was of interest to examine the EGFR inhibition activities of the compounds that revealed the most potent cytotoxic activities, as one of their cytotoxic mechanisms of action. Furthermore, molecular docking study was carried out to detect the compounds’ binding modes at the active site of EGFR-TK.

2. Results and Discussion

2.1. Chemistry

The synthetic pathways utilized for the synthesis of the target pyridothienopyrimidine compounds 2a,b–9a,b are depicted in Scheme 1 and Scheme 2. The structures of all new compounds were confirmed using ¹H-NMR, ¹³C-NMR (Supplementary Materials), IR, and mass spectral data alongside the elemental microanalyses. Heating 3-amino-4,6-dimethyl/6-phenyl-4-(p-tolyl)thieno[2,3-b]pyridine-2-carboxamides 1a,b, the starting compounds, with urea at 180 °C for 1 h led to the formation of the corresponding pyridothienopyrimidine-2,4-diones 2a,b, which were further refluxed with a mixture of phosphorus oxychloride and phosphorus pentachloride to give the corresponding 2,4-dichloro derivatives 3a,b, respectively. On the other hand, the treatment of the starting compounds 1a,b with 2-chloroacetyl chloride in cold acetonitrile resulted in cyclization via substitution reaction followed by intramolecular elimination to give the 2-(chloromethyl)-pyridothienopyrimidin-4(3H)-ones 4a,b, respectively. The subsequent treatment of 4b with different amines, namely, morpholine and 1-methylpiperazine, resulted in the corresponding 2-(morpholinomethyl)-/2-((4-methylpiperazin-1-yl)methyl)-pyridothieopyrimidin-4(3H)-ones 5a,b (Scheme 1). IR spectra of compounds 2a,b found two bands in the region 3394–3112 cm⁻¹ ascribed to 2NH and two bands in the region 1728–1640 cm⁻¹ related to the 2C=O groups of the pyrimidine-2,4(1H,3H)-dione ring. By comparison, IR spectra of the 2,4-dichloro derivatives 3a,b revealed the absence of the previous bands; instead, they showed two bands in the region 825–768 cm⁻¹ referring to the newly formed C-Cl groups. In addition, ¹H-NMR spectra of compounds 2a,b revealed the two 2NH groups to be two D₂O exchangeable singlets at δ 10.42–11.71 ppm, which vanished in the ¹H-NMR spectra of 2,4-dichloro derivatives 3a,b. Furthermore, the 2CH₃ of 2a, 3a and CH₃ of the p-tolyl moiety of 2b, 3b exhibited as singlet signals in the range 2.43–2.79 ppm along with aromatic protons in the range δ 7.21–8.24 ppm. The ¹³C NMR spectra of compounds 2a,b and 3a,b revealed CH₃ moieties at δ 20.40–24.36 ppm in addition to the aromatic carbons.

IR spectra of compounds 4a,b, 5a,b represented NH and C=O groups as absorption bands in the regions 3440–3387 cm⁻¹ and 1669–1655 cm⁻¹, respectively, while the C-Cl band of 4a appeared at 772 cm⁻¹ and that of 4b was at 776 cm⁻¹. Furthermore, ¹H-NMR spectra of 4a,b and 5a,b exhibited, in addition to the signals of the parent protons, a singlet signal in the region δ 3.29–4.66, referring to the methylene protons of the -NCH₂ moiety. Additionally, the eight protons of the morpholine moiety of 5a appeared as two singlets at δ 2.38 and 3.54 ppm, while those of the piperazine ring of 5b appeared as multiplet signals in the range δ 2.81–3.01 ppm alongside a singlet signal at δ 2.54 ppm due to its -N-CH₃ group. The ¹³C-NMR spectra of 5a exhibited the 2CH₂N- and 2CH₂O of the morpholine moiety as two signals at δ 53.37 and 60.65 ppm, respectively.

The synthesis of spiro cycloalkane-pyridothienopyrimidin-4′-ones 6a–d in good yields was achieved via cyclocondensation reactions of the starting 1a,b with the appropriate cyclic ketones, cyclopentanone and/or cyclohexanone, in DMF containing zinc chloride anhydrous. Treating the latter compounds (6a–d) with phosphorus pentasulfide in refluxing pyridine yielded the corresponding 1′H-spiro[cycloalkane-1,2′-pyridothienopyrimidine]-4′(3′H)-thiones 7a–d (Scheme 2). IR spectra of 6a–d and 7a–d revealed the presence of different absorption bands in the regions 3427–3157 cm⁻¹ related to 2NH groups, 1660–1644 cm⁻¹ related to the C=O groups of 6a-d and 1247–1232 cm⁻¹ related to the C=S groups of compounds 7a–d. ¹H-NMR spectra of 6a–d and 7a–d represented the eight protons of the spiro-cyclopentane and the ten protons of the spiro-cyclohexane substituents as multiplet signals in the range δ 1.21–2.11 ppm alongside one D₂O exchangeable singlet in the range δ 4.59–6.49 ppm, corresponding to the NH at position-1, and another D₂O exchangeable singlet appeared downfield in the range δ 7.81–9.85 ppm, related to the NH group at position-3. ¹³C-NMR spectra of 6a–d and 7a–d revealed various singlets in the ranges δ 19.26–38.33 ppm assignable to the cyclopentane and cyclohexane carbons, δ 69.49–70.81 ppm related to the spiro carbons, δ 164.40–167.80 ppm assigned to the C=O groups of 6a–d and δ 181.33–183.35 ppm ascribed to C=S moieties of compounds 7a–d, as well as the signals of the parent carbons, which appeared in their correct regions.

Compounds 7c,b were treated with 2-chloroacetamide in refluxing DMF containing sodium carbonate anhydrous to obtain the corresponding 4-sulfanyl acetamide derivatives 8a,b, while the treatment of 7c,b with epichlorohydrin in refluxing acetone containing a catalytic amount of triethyl amine resulted in the formation of the corresponding 4-(oxiran-2-ylmethy)sulfanyl derivatives 9a,b (Scheme 2). The S-alkylation at position-4 was supported by ¹H NMR and ¹³C-NMR spectra of 8a,b and 9a,b due to the vanishing of both the signal related to one of the two NH protons and that of the C=S carbon. Moreover, the ¹H NMR spectra of compounds 8a,b exhibited singlet signals at δ 4.05 and 4.19 ppm due to SCH₂ protons of the newly formed acetamide side chain. ¹H NMR spectra of compounds 9a,b represented the protons of the oxirane ring as two multiplets in the range δ 2.86–2.94 ppm, related to the methylene protons, and a third multiplet at δ 3.72–3.95 ppm, due to the methine proton, while the methylene protons of SCH₂ appeared as a doublet signal at δ 3.51 and 3.67 ppm. Additionally, the ¹³C-NMR spectra 9a,b showed three signals at the range δ 31.88–53.45 ppm referred to SCH₂, OCH₂, and OCH moieties.

Confirmation of the molecular structures of the new compounds was also supported by their mass spectra, which represented the correct molecular ion peaks.

2.2. Biological Activity

2.2.1. Antimicrobial Activity

The newly synthesized pyridothienopyrimidine componds 2a,b–9a,b were investigated for their antimicrobial activities against a panel of microbial strains, three Gram-positive bacteria viz. Staphylococcus aureus 25923, Bacillus subtilis 6633, Bacillus cereus 33018, two Gram-negative bacteria viz. Escherichia coli 8739, Salmonella typhimurium 14028, three yeasts viz. Candida albicans 10231, Candida tropicals 750, Saccharomycese cerevisiae and two fungi viz. Aspergillus flavus, Aspergillus niger EM77 (KF774181). Their activities were expressed in terms of minimal inhibitory concentration (MIC) (μg/mL). Additionally, amoxicillin trihydrate and clotrimazole were utilized as positive antibiotic and antifungal controls. The obtained MIC results are represented in

Table 1. In vitro antibacterial activities of the synthesized compounds expressed as MICs (μg/mL) against the tested pathogenic bacteria.

Compd. No.	Gram +ve Bacteria			Gram −ve Bacteria
	S. aureus	B. subtilis	B. cereus	E. coli	S. typhimurium
2a	NA	NA	64	NA	NA
2b	64	128	64	128	64
3a	4	4	8	8	16
3b	32	8	32	16	32
4a	4	8	4	8	16
4b	8	128	16	128	128
5a	8	8	16	8	16
5b	8	8	16	32	32
6a	32	16	8	32	16
6b	4	8	16	8	16
6c	4	4	8	8	16
6d	8	32	8	32	32
7a	4	NA	8	8	16
7b	64	64	64	64	128
7c	32	32	8	32	32
7d	4	64	64	32	32
8a	8	16	8	32	32
8b	4	8	16	8	16
9a	16	32	8	32	16
9b	4	4	8	4	16
Amoxicillin	4	8	16	8	16

NA = No Activity (MIC ˃ 128 µg/mL).

and

Table 2. In vitro antifungal activities of the synthesized compounds expressed as MICs (μg/mL) against the tested pathogenic yeasts and fungi.

Compd. No.	Yeasts			Fungi
	C. albicans	C. tropicals	S. cerevisiae	A. flavus	A. niger
2a	128	128	16	NA	NA
2b	64	32	32	32	32
3a	16	8	8	8	8
3b	32	16	64	32	16
4a	16	16	8	8	8
4b	8	16	8	16	16
5a	16	8	8	8	16
5b	64	16	16	32	64
6a	64	16	32	64	32
6b	8	8	8	16	8
6c	16	4	16	4	8
6d	64	64	32	32	16
7a	16	16	8	16	16
7b	64	64	32	64	128
7c	16	8	8	4	16
7d	32	64	32	64	64
8a	64	16	16	16	16
8b	8	16	16	8	8
9a	16	32	32	16	64
9b	4	4	8	8	8
Clotrimazole	16	8	8	8	8

NA = No Activity (MIC ˃ 128 µg/mL).

and Figure 2 and Figure 3.

Based on the MIC values in Table 1, the antibacterial activity of the target compounds 2a,b–9a,b was compared with that of amoxicillin, whose MICs ranged between 4–16 μg/mL against the five bacterial strains. It is evident that pyridothienopyrimidine-2,4(1H,3H)-dione derivatives 2a,b showed antibacterial activity varying from weak to inactive against the tested strains, with MICs ranging from 64 to >128 μg/mL. However, the conversion of 2a,b to 2,4-dichloro derivatives 3a,b led to a significant increase in antibacterial activity, especially for 7,9-dimethyl derivative 3a, which exhibited more potent activity than amoxicillin against B. subtilis and B. cereus and equalized with it against the other strains. In addition, 2-chloromethyl derivatives 4a,b revealed significant antibacterial activity, particularly 7,9-dimethyl derivative 4a, which showed the most potent activity against B. cereus with MIC = 4 μg/mL and had equipotent activity to amoxicillin against the other strains. While 7-Phenyl-9-(p-tolyl) analogue 4b revealed potent activity against S. aureus and B. cereus, it showed weak activity against other bacterial strains with MIC = 128 μg/mL. Further reaction of 2-chloromethyl derivative 4b with amines led to enhanced antibacterial activity, where 2-morpholinomethyl derivative 5a showed potent activity equal to amoxicillin against all the tested bacterial strains except S. aureus, with MICs ranging from 8 to 16 μg/mL. In addition, 2-(4-methylpiperazin-1-yl) derivative 5b revealed increasing activity against B. subtilis, E. coli and S. typhimurium.

Spiro cycloalkane-1,2′-pyridothienopyrimidin]-4′(3′H)-ones 6a–d exhibited antibacterial activity varying from potent to moderate against the tested strains, with MICs ranging from 4 to 32 μg/mL. Compound 6b showed an activity equal to that of the reference drug against the five bacterial strains, while 6c exceeded the potency of amoxicillin against B. subtilis and B. cereus and gave equipotent activity against the other strains. The other derivatives, 6a and 6d, showed activity varying from potent to moderate with MICs ranging 8–32 μg/mL. The conversion of 6a–d to 4′(3′H)-thiones analogues 7a–d resulted in an obvious weakening in the activity against the most of the tested strains, particularly derivative 7b, which showed weak activity against the five strains with MICs in the range 64–128 μg/mL. However, the S-alkylation of 7b and 7c at position-4 afforded increases in the antibacterial activity. The spiro cyclopentane 4-sulfanylacetamide derivative 8b revealed potent activity similar to that of amoxicillin, and the spiro cyclohexane analogue 8a showed potent to moderate activity with MICs ranging from 8 to 32 μg/mL. Spiro cyclopentane 4-(oxiran-2-ylmethyl)sulfanyl derivative 9b showed the most potent antibacterial activity against all tested strains, especially against E. coli, while spiro cyclohexane analogue 9a showed antibacterial activity ranging from potent to moderate with MICs in the range 8–32 μg/mL.

The antifungal activity of the new compounds 2a,b–9a,b was evaluated according to their MIC values in comparison to the MIC values of the reference antifungal drug clotrimazole, listed in Table 2. The target compounds (3a, 4a, 4b, 5a, 6b, 6c, 7a, 7c, 8b and 9b) appeared to be potent antifungal candidates, representing MIC values ranging from 4 to 16 μg/mL against all the tested yeasts and fungi strains, similar to the range of clotrimazole (MICs; 4–16 μg/mL). Furthermore, 4-(oxiran-2-ylmethyl)sulfanyl derivative 9b represented more potent antifungal activity than that of clotrimazole against the yeast pathogens C. albicans and C. tropicals, with MICs of 4 and 4 μg/mL, respectively. The rest of the target compounds displayed moderate to weak activity against the tested yeasts and fungi strains. The obtained results suggested that the conjugation of chloroine, chloromethyl, morpholinomethyl and spiro cyclopentane/cyclohexane at position-2 of the parent pyridothienopyrimid-4(3H)-one (3a, 4a,b, 5a, 6b,c, 7c) as well as the attachment of (oxiran-2-ylmethyl)sulfanyl and/or sulfanylacetamide side chains at position-4 of the pyridothienopyrimidine scaffold (8b, 9b) produced a beneficial impact for antimicrobial activity, resulting in promising broad-spectrum growth inhibition activity against the examined Gram-positive and Gram-negative microbes as well as fungal pathogens.

2.2.2. Cytotoxic Activity

The newly synthesized pyridothienopyrimidine derivatives 2a,b–9a,b were subjected to antiproliferative activity evaluation against two cancer cell lines, hepatocellular carcinoma (HepG2) and breast cancer (MCF-7), by using the MTT colorimetric assay[53]. The cytotoxic activity of the target compounds was compared with that of doxorubicin, utilized as a positive control. The concentrations of the examined derivatives that induced 50% inhibition of cell viability (IC₅₀, μM) were detected and are listed in

Table 3. Cytotoxic activities (IC₅₀ µM) of the new compounds and doxorubicin against HepG2, MCF7 and WISH cells.

Compd. No.	HepG2 (µM)	MCF7 (µM)	WISH
2a	56.57 ± 3.64	64.34 ± 2.91
2b	33.21 ± 1.79	42.39 ± 2.24
3a	2.31 ± 0.35	7.24 ± 0.64	416.83 ± 15.17
3b	11.34 ± 0.65	24.72 ± 1.32
4a	2.99 ± 0.15	15.42 ± 0.45	460.23 ± 11.08
4b	36.52 ± 1.82	43.27 ± 2.28
5a	1.99 ± 0.09	2.79 ± 0.18	408.48 ± 15.93
5b	10.16 ± 0.29	21.06 ± 1.16
6a	52.18 ± 1.45	77.41 ± 3.62
6b	2.75 ± 0.13	9.89 ± 0.55	394.98 ± 10.20
6c	12.11 ± 0.33	22.24 ± 0.67
6d	4.45 ± 0.22	21.67 ± 0.70
7a	26.05 ± 1.89	28.11 ± 0.92
7b	39.74 ± 1.89	41.62 ± 2.20
7c	10.35 ± 0.31	20.9 ± 0.93
7d	23.25 ± 0.35	26.55 ± 1.62
8a	6.78 ± 0.73	20.88 ± 1.46
8b	2.79 ± 0.08	13.54 ± 0.76	401.37± 17.32
9a	4.88 ± 0.65	23.56 ± 1.24
9b	1.17 ± 0.09	1.52 ± 0.08	417.55 ± 14.1
Doxorubicin	2.85 ± 0.21	3.58 ± 0.33	432.10 ± 19.30

.

Based on IC₅₀ values from Table 3, the examined compounds displayed versatile anti-proliferative activities against the tested cell lines, producing more potent growth inhibitory activity against HepG2 cells than MCF-7 cells with IC₅₀ ranges of 1.17–56.18 µM and 1.52–77.41μM, respectively, compared to doxorubicin’s IC₅₀ values of 2.85 and 3.58 µM, respectively. The most active cytotoxic activity was exhibited by the target compounds (9b, 5a, 3a, 6b, 8b and 4a), listed in descending order according to their activity against the two cell lines, as represented in Figure 4. Interestingly, the attachment of the sulfanylmethyl-oxirane side chain at position-4 of the spiro cyclopenane-1,2′-pyridothienopyrimidine nucleus in compound 9b produced the most potent growth inhibition activity against both HepG2 and MCF-7 cancer cells, approximately 2–3 fold higher than doxorubicin, representing IC₅₀ values of 1.17 and 1.52 μM, respectively. However, the activity slightly decreased against HepG2 cells and detectably decreased against MCF-7 for the spiro cyclohexane sulfanylmethyl oxirane analogue 9a compared to doxorubicin, with IC₅₀ values of 4.88 and 23.56 µM, respectively. Furthermore, the incorporation of the morpholine nucleus into the pyridothienopyrimidine scaffold at position-2 in compound 5a led to a significant cytotoxic potency against both HepG2 and MCF-7, greater than that obtained from the reference drug, at IC₅₀ values of 1.99 and 2.79 µM, respectively. However, the 4-methylpiperazinyl analogue 5b exhibited an observable reduction in growth inhibition activity towards both tested cancer cell lines with IC₅₀ values of 10.16 and 21.06 µM, respectively. A comparable growth inhibitory activity to doxorubicin was displayed against HepG2 cancer cells by the 2,4-dichloro-pyridothienopyrimidine derivative 3a at an IC₅₀ value of 2.31 μM, but its activity was less against MCF-7 with an IC₅₀ value of 7.24 µM, while its 7-phenyl-9-p-tolyl analogue 3b represented a detectable drop in potency, with IC₅₀ values of 11.34 and 24.72 µM against HepG2 and MCF-7, respectively. The spirocyclopentane-pyridothienopyrimidin-4-one derivative 6b was nearly equipotent to doxorubicin in inhibiting the growth of HepG2 cancer cells, with an IC₅₀ value of 2.75 µM, but produced a mild decrease in activity against MCF-7 with IC₅₀ of 9.89 µM; however, the displacement of the spiro pentane moiety with spirocyclohexane in the analogue 6d led to a twofold decrease in cytotoxic activity against HepG2 cells and a significant drop in potency against MCF-7 cancer cells with IC₅₀ values of 4.45 and 21.67 µM, respectively. The other members in this series, 6a,c, appeared to be significantly less potent candidates than the reference drug, with IC₅₀ ranges of 12.11–77.41 µM.

Additionally, the spiro cyclopentane sulfanylacetamide 8b produced an equivalent cytotoxic potency to that obtained by doxorubicin against HepG2 cells, with an IC₅₀ value of 2.79 µM, but less potency than doxorubicin against MCF7, with an IC₅₀ value of 13.54 µM. The spiro cyclohexane sulfanylacetamide analogue 8a appeared to be a less potent growth inhibitor towards both HepG2 and MCF-7 cancer cells, with IC₅₀ values of 6.78 and 20.88 µM, respectively. Finally, the 2-chloromethyl-7,9-dimethyl derivative 4a, the last among the most potent compounds, showed significant growth inhibitory activity against HepG2 cancer cells with IC₅₀ = 2.99 µM, but its potency decreased against MCF-7 cells with an IC₅₀ value of 15.42 µM, whereas a high drop in activity was shown by the 7-phenyl-9-p-tolyl analogue 4b towards the two cancer cell lines with IC₅₀ values of 36.52 and 43.27 µM, respectively. The rest of the target compounds, the spiro[cycloalkane-1,2′-pyridothienopyrimidine-4′(3′H)-thiones 7a–d and the pyridothienopyrimidine-2,4(1H,3H)-diones 2a,b, showed moderate to weak cytotoxic activities in the IC₅₀ range of 10.35–41.62 µM, compared to doxorubicin.

The frequency and severity of undesirable effects on normal cells at therapeutic doses are considered among the most important characteristics that differentiate anticancer drugs from each other. Accordingly, the cytotoxic activity of the most active compounds (3a, 4a, 5a, 6b, 8b, 9b) was evaluated against the normal WISH cell line (Human amnion normal Liver cells) via MTT assay as IC₅₀ values in μM, listed in Table 3. The obtained results revealed that the tested compounds had IC₅₀ values in the range 394.98–460.23 µM, nearly equal to that obtained by the reference drug doxorubicin, IC_{50 doxorubicin} 432.10 µM, which confirmed the high safety of the most potent compounds towards normal cells.

2.2.3. In Vitro EGFR Enzyme Inhibition Assay

The most potent cytotoxic compounds (3a, 4a, 5a, 6b, 8b, 9b) were subjected to further investigations of their inhibiting profiles against EGFR, in order to study one of their proposed modes of action as potent cytotoxic agents [54]. Accordingly, these derivatives were assessed as EGFR kinase inhibitors, using erlotinib as a reference drug as it is one of the most potent EGFR inhibitors [55]. The obtained results were expressed as IC₅₀ values (nM) (

Table 4. In vitro enzymatic inhibitory activity against EGFR kinase.

Compound No.	EGFR IC50 (nM)
3a	17.29 ± 0.24
4a	53.57 ± 0.41
5a	9.66 ± 0.08
6b	53.19 ± 0.46
8b	38.44 ± 0.25
9b	7.27 ± 0.11
Erlotinib	27.01 ± 0.16

). Interestingly, the most cytotoxic derivatives, spiro cyclopentane-1,2′ pyridothienopyrimidine–oxirane 9b and pyridothienopyrimidine–morpholine 5a, appeared to be 3–2 times more potent than erlotinib, representing IC₅₀ values of 7.27, 9.66 and 27.01 nM, respectively. The 2-chloromethyl-pyridothienopyrimidin-4-one derivative 4a displayed a slight decrease in inhibition activity, but remained 1.5-fold more potent than erlotinib. An evident drop in EGFR inhibition activity was observed for the rest of the examined derivatives (3a, 6b, 8b) representing an IC₅₀ range of 38.44–53.57 nM.

2.3. Molecular Docking Studies

Molecular docking studies were performed to study the binding modes of the most potent cytotoxic compounds (3a, 4a, 5a, 6b, 8b, 9b) to the active sites of the target EGFR compared with erlotinib (ERL) as EGFR inhibitor. Docking setup was first validated through self-docking of the co-crystallized ligand (ERL) in the vicinity of the binding site of the enzyme. The docking score (S) was –10.48 kcal/mol. and root mean square deviation (RMSD) was 1.03 Å (Figure 5). The calculated RMSD value confirms the validity of the docking procedure.

Examination of the binding interactions of erlotinib to the active site of the EGFR kinase domain showed several conventional hydrogen bond interactions with the Met769, Leu768, Val702 and Leu820 amino acids (Figure 6).

From the docking results of the examined compounds (

Table 5. Molecular docking results of the most active pyridothienopyrimidine compounds.

Compound	S (kcal/mol)	Amino Acids	Interacting Groups	Type of Bond	Length (Å)
3a	–11.42	Val702	N (Pyrimidine)	H-bond acceptor	4.12
		Lys721	N (Pyridine)	H-bond acceptor	3.25
		Met769	Cl (Pyrimidine)	Halogen bond	3.47
		Leu768	Cl (Pyrimidine)	Halogen bond	4.16
		Thr830	Cl (Pyridine)	Halogen bond	3.26
		Met742	Cl (Pyridine)	Halogen bond	4.13
		Thr766	Cl (Pyridine)	Halogen bond	3.03
		Thr830	S	σ-hole bond	3.79
4a	–8.94	Lys721	Cl	Halogen bond	3.69
		Cys751	O (C=O)	σ-hole bond	3.78
		Thr766	S	σ-hole bond	4.21
		Met769	S and N (Pyridine)	H-bond acceptor	3.41/3.55
		Leu768	N (Pyridine)	H-bond acceptor	3.84
5a	–11.48	Asp831	NH+	Ionic interaction	3.71
		Lys721	O (Morpholine)	H-bond acceptor	3.51
		Cys773	O (C=O)	σ-hole bond	3.46
		Asp776	S	σ-hole bond	4.18
6b	–10.06	Leu820	NH	H-bond acceptor	3.78
		Cys751	O (C=O)	H-bond acceptor	3.49
		Gln767	S	σ-hole bond	3.12
		Thr766	S	σ-hole bond	3.87
		Met769	S	H-bond acceptor	3.83
8b	–9.11	Leu694	S (Thiophene)	H-bond acceptor	4.38
		Leu694	S (Side chain)	σ-hole bond	3.79
		Thr766	N (Pyridine)	H-bond acceptor	3.71
9b	–12.01	Met769	S (thiophene)	H-bond acceptor	4.09
		Leu768	S (thiophene)	H-bond acceptor	4.37
		Leu820	S (thiophene)	H-bond acceptor	4.47
		Thr766	S (Side chain)	σ-hole bond	4.20
		Leu820	N and NH (Pyrimidine)	H-bond acceptor	3.73/3.74
		Val702	O (Oxirane)	H-bond acceptor	3.64
erlotinib	–10.48	Leu768	N (Pyrimidine)	H-bond acceptor	3.64
		Met769	N (Pyrimidine)	H-bond acceptor	2.70

), it was noticed that all compounds showed binding interactions within the active site of EGFR kinase domain with binding scores ranging from –12.01 to –8.94 kcal/mol. The spiro cyclopentane 4-((oxiran-2-ylmethyl)sulfanyl derivative 9b, which showed the highest biological activity, also showed the highest binding score of –12.01 kcal/mol, through binding with the key amino acids, Met769, Leu768, and Leu820, via the S atom of thiophene ring using a hydrogen bond acceptor, with further interactions with Thr766 via σ-hole bonding with the S atom of the S-methyl side chain. In addition, it bound to the amino acid Val702 through a hydrogen bond acceptor with the O atom of the oxirane moiety and showed high fitting in the vicinity of the active site, contributing to its high biological activity (Figure 7).

2-morpholinomethyl derivative 5a and 2,4-dichloro derivative 3a also showed higher binding scores than the co-crystallized ligand, erlotinib, at –11.48 and –11.42 kcal/mol, respectively; they showed a good binding pattern with the key amino acids, and compound 5a showed an ionic interaction with Asp831 revealing its high biological activity (Figure 8 and Figure 9). Other tested derivatives showed a lower binding score than erlotinib, with less binding interaction with the key amino acids.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

The melting points were obtained in open capillary tubes using an Electrothermal IA9100 digital melting point apparatus. Elemental microanalyses were carried out at the Micro Analytical Unit at Cairo University. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker High Performance Digital FT-NMR Spectrometer Advance III (400/100 MHz) in the presence of TMS as the internal standard at Ain Shams University, Cairo, Egypt. Infrared spectra were measured using the KBr disc technique on a Jasco FT/IR-6100 Fourier transform IR spectrometer (Japan) at the scale of 400–4000 cm⁻¹. ESI-mass spectra were determined using an Advion Compact Mass Spectrometer (CMS), NY, USA. TLC on silica gel-precoated aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) was used for following up the reactions and checking the purity of the chemical compounds using chloroform/methanol (3:1, v/v), and spots were detected with iodine vapor or through exposure to a UV lamp at δ 254 nm for several seconds. The nomenclature of the compounds is according to the IUPAC system. The starting compounds, 3-amino-thieno[2,3-b]pyridine-2-carboxamides (1a,b), were prepared using the reported method [56].

3.1.2. Synthesis of Pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-diones 2a,b

A mixture of compounds 1a,b (5 mmol) and urea (0.42 g, 7 mmol) was heated at 180°C for 1 h. The solidified residue was treated with hot water and the obtained solid was separated by filtration. The solid was washed with hot water several times and recrystallized from acetone to yield compounds 2a,b.

7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (2a) was obtained from 1a (1.11 g, 5 mmol) in 85% yield (1.05 g) as a brown solid, m.p. 325 °C. IR (KBr, υ_max/cm⁻¹): 3394, 3112 (2NH), 3010 (CH-aromatic), 2828 (CH-aliphatic), 1728, 1662 (2C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.55 (s, 3H, CH₃), 2.79 (s, 3H, CH₃), 7.21 (s, 1H, Ar-H), 10.59, 11.65 (2s, 2H, 2NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 20.40 (CH₃), 24.36 (CH₃), 108.64, 120.72, 122.85, 140.22, 145.63, 152.01, 160.07, 160.41, 161.48 (Ar-C, 2 C=O); ESI-MS: m/z = 247.33 [M − H⁺]. Anal. Calcd. for C₁₁H₉N₃O₂S (247.27): C, 53.43; H, 3.67; N, 16.99; S, 12.97% Found: C, 53.12, H, 3.91; N, 16.62; S, 13.31%.

7-Phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (2b) was obtained from 1b (1.79 g, 5 mmol) in 83% yield (1.60 g) as an orange solid, m.p. 316–317 °C. IR (KBr, υ_max/cm⁻¹): 3370, 3154 (2NH), 3024 (CH-aromatic), 2925, 2855 (CH-aliphatic), 1702, 1640 (2C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.43 (s, 3H, CH₃), 7.46–7.52 (m, 5H, Ar-H), 7.60 (d, 2H, J = 8 Hz, Ar-H), 7.96 (s, 1H, Ar-H), 8.24 (d, 2H, J = 5.2 Hz, Ar-H), 10.42, 11.71 (2s, 2H, 2NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.42 (CH₃), 112.45, 115.62, 118.27, 121.46, 127.21, 127.87, 129.30, 129.69, 130.23, 133.99, 138.39, 139.64, 144.46, 149.89, 151.96, 156.07, 157.40, 160.46 (Ar-C, 2 C=O); ESI-MS: m/z = 384.39 [M − H⁺]. Anal. Calcd. for C₂₂H₁₅N₃O₂S (385.44): C, 68.56; H, 3.92; N, 10.90; S, 8.32% Found: C, 68.74, H, 4.21; N, 10.68; S, 8.59%.

3.1.3. Synthesis of 2,4-Dichloropyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines 3a,b

To a solution of compounds 2a,b (1 mmol) in phosphorus oxychloride (20 mL), phosphorus pentachloride (0.41 g, 2 mmol) was added. The reaction mixture was refluxed for 15 h, then left to cool. The mixture was poured slowly onto crushed ice and the formed solid was separated by filtration, washed with water several times, and recrystallized from ethanol to yield the 2,4-dichloro compounds 3a,b.

2,4-Dichloro-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (3a) was obtained from 2a (0.25 g, 1 mmol) in 67% (0.19 g) yield as a buff solid, m.p. 250–251 °C. IR (KBr, υ_max/cm⁻¹): 3015 (CH-aromatic), 2920, 2853 (CH-aliphatic), 825, 768 (C-Cl); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.59 (s, 3H, CH₃), 2.76 (s, 3H, CH₃), 7.34 (s, 1H, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 20.43 (CH₃), 24.29 (CH₃), 116.33, 120.63, 123.01, 140.22, 145.69, 152.10, 159.19, 161.22, 163.17 (Ar-C); ESI-MS: m/z = 283.13 [M − H⁺]. Anal. Calcd. for C₁₁H₇Cl₂N₃S (284.16): C, 46.50; H, 2.48; N, 14.79; S, 11.28% Found: C, 46.32, H, 2.59; N, 14.63; S, 11.09%.

2,4-Dichloro-7-phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (3b) was obtained from 2b (0.39 g, 1 mmol) in 73% yield (0.31 g) as a brown solid, m.p. 220 °C. IR (KBr, υ_max/cm⁻¹): 3094 (CH-aromatic), 2922 (CH-aliphatic), 1621 (C=N), 819, 768 (C-Cl); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.47 (s, 3H, CH₃), 7.34 (d, 2H, J = 8.4 Hz, Ar-H), 7.49–7.61 (m, 3H, Ar-H), 7.77 (d, 2H, J = 8.4 Hz, Ar-H), 8.14 (s, 1H, Ar-H), 8.24 (d, 2H, J = 3.6 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.38 (CH₃), 113.52, 118.73, 119.18, 127.79, 129.21, 129.46, 130.38, 132.69, 133.23, 137.29, 138.95, 140.27, 150.70, 155.97, 157.27, 160.01, 161.26 (Ar-C); ESI-MS: m/z = 421.31 [M − H⁺]. Anal. Calcd. for C₂₂H₁₃Cl₂N₃S (422.33): C, 62.57; H, 3.10; N, 9.95; S, 7.59% Found: C, 62.76, H, 3.35; N, 9.73; S, 7.78 %.

3.1.4. Synthesis of 2-(Chloromethyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-ones 4a,b

To a cold solution of 1a,b (5 mmol) in acetonitrile (30 mL) at 0–5 °C, 2-chloroacetyl chloride (0.56 g, 5 mmol) was added dropwise while stirring. After addition, the reaction mixture was stirred for 1 h at room temperature and the solution was evaporated until dryness under reduced pressure, and then the oily residue was treated with hot petroleum ether 40–60. Then the formed solid was collected by filtration and recrystallized from ethanol to yield 4a,b.

2-(Chloromethyl)-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (4a) was obtained from 1a (1.11 g, 5 mmol) in 78% yield (1.09 g) as a yellow solid, m.p. 330–331 °C. IR (KBr, υ_max/cm⁻¹): 3387 (NH), 3097 (CH-aromatic), 2821 (CH-aliphatic), 1669 (C=O), 772 (C-Cl); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.59 (s, 3H, CH₃), 2.88 (s, 3H, CH₃), 4.66 (s, 2H, CH₂Cl), 7.28 (s, 1H, Ar-H), 13.20 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.17, 24.37 (2CH₃), 52.80 (CH₂-Cl), 123.27, 124.88, 144.56, 147.42, 152.87, 158.65, 159.42, 160.22 (Ar-C), 161.93 (C=O); ESI-MS: m/z = 278.69 [M − H⁺]. Anal. Calcd. for C₁₂H₁₀ClN₃OS (279.74): C, 51.52; H, 3.60; N, 15.02; S, 11.46% Found: C, 51.28; H, 3.35; N, 14.82; S, 11.20%.

2-(Chloromethyl)-7-phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (4b) was obtained from 1b (1.79 g, 5 mmol) in 82% yield (1.71 g) as a yellow solid, m.p. 310 °C. IR (KBr, υ_max/cm⁻¹): 3437 (NH), 3119 (CH-aromatic), 2920 (CH-aliphatic), 1669 (C=O), 776 (C-Cl); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.43 (s, 3H, CH₃), 4.38 (s, 2H, CH₂Cl), 7.32 (d, 2H, J = 6.4 Hz, Ar-H), 7.45–7.57 (m, 3H, Ar-H), 7.66 (d, 2H, J = 6.4 Hz, Ar-H), 7.99 (s, 1H, Ar-H), 8.21 (d, 2H, J = 8.4 Hz, Ar-H), 9.79 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.32 (CH₃), 53.71 (CH₂-Cl), 119.37, 126.63, 127.15, 127.94, 129.16, 129.51, 130.08, 132.64, 139.94, 140.57, 147.84, 148.12, 149.61,154.64, 155.69, 159.02, 160.33 (Ar-C, C=O); ESI-MS: m/z = 416.87 [M − H⁺]. Anal. Calcd. for C₂₃H₁₆ClN₃OS (417.91): C, 66.10; H, 3.86; N, 10.06; S, 7.67% Found: C, 66.41, H, 4.06; N, 9.86; S, 7.88%.

3.1.5. Synthesis of 2-Substituted-7-phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-ones 5a,b

A mixture of 4b (0.42 g, 1 mmol) and the appropriate amine (1 mmol) in DMF (15 mL) was refluxed for 1 hr, and then the reaction mixture was poured onto cold water. The obtained precipitate was separated by filtration, washed with water, and recrystallized from ethanol to yield 5a,b.

2-(Morpholinomethyl)-7-phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (5a) was obtained by reaction of 4b with morpholine (0.087 g, 1mmol) in 79% yield (0.37 g) as a pale yellow solid, m.p. 245 °C. IR (KBr, υ_max/cm⁻¹): 3440 (NH), 3094 (CH-aromatic), 2919, 2854 (CH-aliphatic), 1662 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 2.38 (s, 4H, 2CH₂N-morpholine), 2.39 (s, 3H, CH₃), 3.29 (s, 2H, CH₂N), 3.54 (s, 4H, 2CH₂O), 7.28 (d, 2H, J = 10.4 Hz, Ar-H), 7.49–7.60 (m, 3H, Ar-H), 7.63 (d, 2H, J =10.4 Hz, Ar-H), 7.97 (s, 1H, Ar-H), 8.27 (d, 2H, J = 8.4 Hz, Ar-H), 12.66 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.44 (CH₃), 53.37 (2CH₂N-morpholine), 60.65 (CH₂N), 66.56 (2CH₂O), 119.80, 127.49, 127.82, 128.47, 129.49, 130.45, 130.83, 132.14, 139.71, 140.50, 150.02, 151.48, 155.65, 157.06, 158.75 (Ar-C), 163.50 (C=O); ESI-MS: m/z = 467.50 [M − H⁺]. Anal. Calcd. for C₂₇H₂₄N₄O₂S (468.58): C, 69.21; H, 5.16; N, 11.96; S, 6.84 % Found: C, 69.48; H, 5.39; N, 11.72; S, 6.51%.

2-((4-Methylpiperazin-1-yl)methyl)-7-phenyl-9-(p-tolyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (5b) was obtained by reaction of 4b with 4-methylpiperazine (0.100 g, 1mmol) in 73% yield (0.35 g) as a yellowish white solid, m.p. 260–261 °C. IR (KBr, υ_max/cm⁻¹): 3433 (NH), 3032 (CH-aromatic), 2920 (CH-aliphatic), 1655 (C=O); ¹H-NMR (CDCl₃, 400 MHz): δ = 2.48 (s, 3H, CH₃), 2.54 (s, 3H, NCH₃), 2.81–3.01 (m, 8H, 4CH₂N piperazine), 3.57 (s, 2H, NCH₂), 7.32 (d, 2H, J = 8 Hz, Ar-H), 7.48–7.54 (m, 3H, Ar-H), 7.64 (d, 2H, J = 8.4 Hz, Ar-H), 7.81 (s, 1H, Ar-H), 8.17 (d, 2H, J = 6.8 Hz, Ar-H), 11.32 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.34 (CH₃), 48.22 (CH₃N), 56.13, 57.56 (4CH₂N-piperazine), 61.49 (CH₂N), 119.11, 126.96, 127.21, 127.79, 128.90, 129.01, 129.44, 130.08, 132.56, 139.95, 140.21, 145.01, 149.47, 150.17, 155.56, 157.15, 158.44 (Ar-C), 163.25 (C=O); ESI-MS: m/z = 480.64 [M − H⁺]. Anal. Calcd. for C₂₈H₂₇N₅OS (481.62): C, 69.83; H, 5.65; N, 14.54; S, 6.66% Found: C, 69.62, H, 5.90; N, 14.21; S, 6.79%.

3.1.6. Synthesis of 1′H-Spiro[cycloalkane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′(3′H)-ones 6a–d

A mixture of 1a,b (0.02 mol)) and the appropriate cyclic ketone (0.03 mol) in DMF (50 mL) containing zinc chloride anhydrous (2.72 g, 0.02 mol) was heated under reflux for 6 h, and then the reaction mixture was poured onto cold water. The obtained precipitate was separated by filtration, washed with water, and recrystallized from DMF/H₂O to yield 6a–d.

7′,9′-Dimethyl-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′(3′H)-one (6a) was obtained by reaction of 1a (4.42 g, 0.02 mol) with cyclopentanone (2.52 g, 0.03 mol) in 75% yield (4.31 g) as a brown solid, m.p. 245–246 °C. IR (KBr, υmax/cm−1): 3319, 3210 (2NH), 3070 (CH-aromatic), 2921, 2828 (CH-aliphatic), 1650 (C=O); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.72–2.07 (m, 8H, 4CH2), 2.51 (s, 3H, CH3), 2.66)s, 3H, CH3), 6.48 (s, 1H, NH, D2O exchangeable), 7.04 (s,1H, Ar-H), 9.82 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6, 100 MHz): δ = 20.41 (CH3), 22.65, (2CH2), 24.33 (CH3), 37.55 (2CH2), 70.15 (spiro C), 122.54, 123.29, 135.88, 145.63, 148.71, 159.30, 161.57 (Ar-C), 167.65 (C=O); ESI-MS: m/z = 286.41 [M − H⁺]. Anal. Calcd. for C15H17N3OS (287.38): C, 62.69; H, 5.96; N, 14.62; S, 11.16% Found: C, 62.52, H, 5.74; N, 14.41; S, 10.98%.

7′-Phenyl-9′-(p-tolyl)-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′(3′H)-one (6b) was obtained by reaction of 1b (7.19 g, 0.02 mol) with cyclopentanone (2.52 g, 0.03 mol) in 71% yield (6.04 g) as a yellow solid, m.p. 210 °C. IR (KBr, υ_max/cm⁻¹): 3410, 3200 (2NH), 3055 (CH-aromatic), 2919 (CH-aliphatic), 1644 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.21–1.85 (m, 8H, 4CH₂), 2.43 (s, 3H, CH₃), 5.94 (s, 1H, NH, D₂O exchangeable), 7.29 (d, 2H, J = 7.4 Hz, Ar-H), 7.41–7.62 (m, 5H, Ar-H), 7.81(s, 1H, NH, D₂O exchangeable), 7.96 (s, 1H, Ar-H), 8.22 (d, 2H, J = 10.2 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.36, 21.54 (2CH₂, CH₃), 38.33 (2CH₂), 70.81 (spiro C), 118.26, 121.25, 127.54, 127.65, 129.39, 129.79, 130.32, 133.82, 139.15, 139.31, 148.17, 150.62, 155.15, 163.25 (Ar-C), 167.38 (C=O); ESI-MS: m/z = 424.50 [M − H⁺]. Anal. Calcd. for C₂₆H₂₃N₃OS (425.55): C, 73.38; H, 5.45; N, 9.87; S, 7.53% Found: C, 73.64, H, 5.21; N, 10.08; S, 7.38%.

7′,9′-Dimethyl-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′(3′H)-one (6c) was obtained by reaction of 1a (4.42 g, 0.02 mol) with cyclohexanone (2.94 g, 0.03 mol) in 79% yield (4.76 g) as a brown solid, m.p. 290–291 °C. IR (KBr, υ_max/cm⁻¹): 3317, 3168 (2NH), 3043 (CH-aromatic), 2932 (CH-aliphatic), 1649 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.23–2.06 (m, 10H, 5CH₂), 2.51 (s, 3H, CH₃), 2.71 (s, 3H, CH₃), 5.91 (s, 1H, NH, D₂O exchangeable), 7. 07 (s, 1H, Ar-H), 7.86 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.36, 20.16, 22.27, 24.32 (3CH₂, 2CH₃), 36.24 (2CH₂), 69.49 (spiro C), 122.11, 123.58, 134.96, 145.31, 148.44, 159.14, 161.51 (Ar-C), 167.80 (C=O); ESI-MS: m/z = 300.46 [M − H⁺]. Anal. Calcd. for C₁₆H₁₉N₃OS (301.41): C, 63.76; H, 6.35; N, 13.94; S, 10.64% Found: C, 63.49, H, 6.17; N, 13.69; S, 10.41%.

7′-Phenyl-9′-(p-tolyl)-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′(3′H)-one (6d) was obtained by reaction of 1b (7.19 g, 0.02 mol) with cyclohexanone (2.94 g, 0.03 mol) in 77% yield (6.77 g) as a yellow solid, m.p. 263 °C. IR (KBr, υ_max/cm⁻¹): 3415, 3166 (2NH), 3053 (CH-aromatic), 2924, 2854 (CH-aliphatic), 1660 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.34–1.87 (m, 10H, 5CH₂), 2.42 (s, 3H, CH₃), 4.59 (s, 1H, NH, D₂O exchangeable), 7.38 (d, 2H, J = 6.8 Hz, Ar-H), 7.49–7.54 (m, 5H, Ar-H), 7.83 (s, 1H, Ar-H), 8.06 (s, 1H, NH, D₂O exchangeable), 8.21 (d, 2H, J = 6 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.96, 21.38, 22.54 (3CH₂, CH₃), 35.86 (2CH₂), 70.67 (spiro C), 118.52, 121.27, 127.48, 127.84, 129.24, 129.67, 129.96, 130.86, 133.48, 137.29, 139.20, 139.50, 148.67, 155.50, 156.14 (Ar-C), 167.73 (C=O); ESI-MS: m/z = 438.52 [M − H⁺]. Anal. Calcd. for C₂₇H₂₅N₃OS (439.58): C, 73.77; H, 5.73; N, 9.56; S, 7.29% Found: C, 73.54, H, 5.49; N, 9.69; S, 7.59%.

3.1.7. Synthesis of 1′H-Spiro[cycloalkane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine]-4′(3′H)-thiones 7a–d

A mixture of 6a–d (5 mmol) and phosphorus pentasulfide (1.11 g, 5 mmol) in pyridine (40 mL) was heated under reflux for 12 h. The reaction mixture was poured onto cold water and left overnight. The formed solid was separated by filtration, washed with water, and recrystallized from 1,4-dioxane to yield compounds 7a–d.

7′,9′-Dimethyl-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine]-4′(3′H)-thione (7a) was obtained from 6a (1.44 g, 5 mmol) in 63% yield (0.95 g) as a burnt orange solid, m.p. 220–221 °C. IR (KBr, υ_max/cm⁻¹): 3372, 3157 (2NH), 3063 (CH-aromatic), 2922 (CH-aliphatic), 1247 (C=S); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.36–2.03 (m, 8H, 4CH₂), 2.57 (s, 3H, CH₃), 2.73 (s, 3H, CH3), 6.49 (s, 1H, NH,D₂O exchangeable), 7.03 (s,1H, Ar-H), 9.85 (s, 1H, NH,D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ =19.56, 22.53 (CH₃, 2CH₂), 24.43 (CH₃), 37.67 (2CH₂), 78.59 (spiro C), 117.93, 122.11, 123.15, 139.87, 146.17, 159.21, 162.86 (Ar-C),182.25 (C=S); ESI-MS: m/z = 302.40 [M − H⁺]. Anal. Calcd. for C₁₅H₁₇N₃S₂ (303.44): C, 59.37; H, 5.65; N, 13.85; S, 21.13% Found: C, 59.09, H, 5.47; N, 13.56; S, 20.89%.

7′-Phenyl-9′-(p-tolyl)-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine]-4′(3′H)-thione (7b) was obtained from 6b (2.13 g, 5 mmol) in 68% yield (1.50 g) as a brown solid, m.p. 180 °C. IR (KBr, υ_max/cm⁻¹): 3402, 3186 (2NH), 3055 (CH-aromatic), 2923, 2852 (CH-aliphatic), 1232 (C=S); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.34–2.01 (m, 8H, 4CH₂), 2.42 (s, 3H, CH₃), 4.67 (s, 1H, NH, D₂O exchangeable), 7.42 (d, 2H, J = 7.2 Hz, Ar-H), 7.48–7.53 (m, 5H, Ar-H), 7.83 (s, 1H, Ar-H), 8.22 (d, 2H, J = 7.2 Hz, Ar-H), 9.68 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.47, 21.96 (2CH₂, CH₃), 36.50 (2CH₂), 77.79 (spiro C), 118.42, 121.20, 124.67, 127.47, 127.71, 129.11, 129.74, 130.20, 133.56, 139.21, 139.34, 148.17, 149.62, 155.15, 163.25 (Ar-C), 183.35 (C=S); ESI-MS: m/z = 440.69 [M − H⁺]. Anal. Calcd. for C₂₆H₂₃N₃S₂ (441.61): C, 70.72; H, 5.25; N, 9.52; S, 14.52% Found: C, 70.53, H, 5.01; N, 9.37; S, 14.38%.

7′,9′-Dimethyl-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine]-4′(3′H)-thione (7c) was obtained from 6c (1.51 g, 5 mmol) in 71% yield (1.13 g) as a greenish grey solid, m.p. 201 °C. IR (KBr, υ_max/cm⁻¹): 3427, 3190 (2NH), 3048 (CH-aromatic), 2929, 2853 (CH-aliphatic), 1234 (C=S); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.21–2.11 (m, 10H, 5CH₂), 2.51 (s, 3H, CH₃), 2.71 (s, 3H, CH₃), 6.22 (s, 1H, NH, D₂O exchangeable), 7. 07 (s, 1H, Ar-H), 9.68 (s, 1H, NH, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.26, 22.02, 24.32, 25.15 (3CH₂, 2CH₃), 34.46 (2CH₂), 70.16 (spiro C), 118.38,122.29, 123.61, 138.66, 146.42, 159.31, 162.71 (Ar-C), 181.33 (C=S); ESI-MS: m/z = 316.40 [M − H⁺]. Anal. Calcd. for C₁₆H₁₉N₃S₂ (317.47): C, 60.53; H, 6.03; N, 13.24; S, 20.20% Found: C, 60.79, H, 6.17; N, 13.49; S, 20.44%.

7′-Phenyl-9′-(p-tolyl)-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine]-4′(3′H)-thione (7d) was obtained from 6d (2.19 g, 5 mmol) in 66% yield (1.50 g) as a yellow solid, m.p. 178–179 °C. IR (KBr, υ_max/cm⁻¹): 3402, 3188 (NH), 3068 (CH-aromatic), 2923, 2852 (CH-aliphatic), 1232 (C=S); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.2 4–2.07 (m, 10H, 5CH₂), 2.43 (s, 3H, CH₃), 4.79 (s, 1H, NH, D₂O exchangeable), 7.39 (d, 2H, J = 8.4 Hz, Ar-H), 7.48–7.57 (m, 5H, Ar-H), 7.87 (s, 1H, Ar-H), 9.66 (s, 1H, NH, D₂O exchangeable), 8.22 (d, 2H, J = 6.4 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.22, 21.47, 24.46 (3CH₂, CH₃), 34.87 (2CH₂), 70.46 (spiro C), 118.21, 121.45, 124.25, 127.39, 127.73, 129.39, 129.66, 130.42, 133.92, 137.90, 139.96, 140.00, 148.82, 156.27, 163.83 (Ar-C), 182.12 (C=S); ESI-MS: m/z = 454.60 [M − H⁺]. Anal. Calcd. for C₂₇H₂₅N₃S₂ (455.64): C, 71.17; H, 5.53; N, 9.22; S, 14.07% Found: C, 71.36, H, 5.19; N, 9.46; S, 13.89%.

3.1.8. Synthesis of 2-((1′H-Spiro[cycloalkane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′-yl)sulfanyl)acetamides 8a,b

A mixture of compounds 7c,b (2 mmol) and 2-chloroacetamide (0.187 g, 2 mmol) in DMF (30 mL) containing sodium carbonate anhydrous (0.5 g) was refluxed for 6 h. The reaction mixture was poured onto iced water and the medium was neutralized with 1N HCl to pH = 7. The formed precipitate was separated by filtration, washed with water, and recrystallized from chloroform to yield the acetamide derivatives 8a,b.

2-((7′,9′-Dimethyl-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′-yl)sulfanyl)acetamide (8a) was obtained from 7c (0.63 g, 2 mmol) in 73% yield (0.55 g) as a brown solid, m.p. 160–161 °C. IR (KBr, υ_max/cm⁻¹): 3403, 3233 (NH), 3048 (CH-aromatic), 2926, 2854 (CH-aliphatic), 1674 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.22–2.09 (m, 10H, 5CH₂), 2.68 (s, 3H, CH₃), 2.76 (s, 3H, CH₃), 4.05 (s, 2H, SCH₂), 6.24 (s, 1H, NH, D₂O exchangeable), 7. 09 (s, 1H, Ar-H), 7.35 (s, 2H, NH₂, D₂O exchangeable); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.21, 22.71, 24.73, 25.41 (3CH₂, 2CH₃), 36.82 (2CH₂), 41.93 (SCH₂), 72.43 (spiro C), 118.78, 122.54, 123.01, 124.67, 144.63, 147.53, 159.35, 163.15 (Ar-C), 168.23 (C=O); ESI-MS: m/z = 373.50 [M − H⁺]. Anal. Calcd. for C₁₈H₂₂N₄OS₂ (374.52): C, 57.73; H, 5.92; N, 14.96; S, 17.12% Found: C, 57.49, H, 5.18; N, 14.58; S, 17.43%.

2-((7′-Phenyl-9′-(p-tolyl)-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin]-4′-yl)sulfanyl)acetamide (8b) was obtained from 7b (0.88 g, 2 mmol) in 69% yield (0.69 g) as a brown solid, m.p. 148–149 °C. IR (KBr, υ_max/cm⁻¹): 3400, 3200 (NH), 3054 (CH-aromatic), 2918, 2853 (CH-aliphatic), 1667 (C=O); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.21–2.03 (m, 8H, 4CH₂), 2.43 (s, 3H, CH₃), 4.19 (s, 2H, SCH₂), 6.86 (s, 1H, NH, D₂O exchangeable), 7.34 (d, 2H, J = 5.6 Hz, Ar-H), 7.44–7.63 (m, 5H, Ar-H), 7.72 (s, 2H, NH₂, D₂O exchangeable), 7.83 (s, 1H, Ar-H), 8.24 (d, 2H, J = 6.8 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.43 (CH₃), 29.61(2CH₂), 33.86 (2CH₂), 41.41(SCH₂), 74.27 (spiro C), 118.55, 122.05, 126.94, 127.85, 127.96, 128.90, 129.50, 130.44, 133.49, 139.05, 140.57, 147.93, 150.02,155.45, 157.40, 163.26 (Ar-C), 169.38 (C=O); ESI-MS: m/z = 497.68 [M − H⁺]. Anal. Calcd. for C₂₈H₂₆N₄OS₂ (498.66): C, 67.44; H, 5.26; N, 11.24; S, 12.86% Found: C, 67.63, H, 5.44; N, 11.50; S, 13.05%.

3.1.9. Synthesis of 4′-((oxiran-2-ylmethyl)sulfanyl)-1′H-spiro[cycloalkane-1,2′-pyrido[3′,2′:4,5]thieno-[3,2-d]pyrimidine] 9a,b

A mixture of compounds 7c,b (1mmol)) and epichlorohydrin (0.092 g, 1mmol) in acetone (20 mL) containing triethyl amine (0.1 mL) was refluxed for 5 h. The solvent was evaporated until dryness and the oily residue was treated with hot petroleum ether (30 mL). The formed solid was separated by filtration and recrystallized from ethanol to yield 9a,b.

7′,9′-Dimethyl-4′-((oxiran-2-ylmethyl)sulfanyl)-1′H-spiro[cyclohexane-1,2′-pyrido[3′,2′:4,5] thieno[3,2 -d]pyrimidine] (9a), was obtained from 7c (0.32 g, 1 mmol) in 78% yield (0.29 g) as a grey solid, m.p. 155 °C. IR (KBr, υ_max/cm⁻¹): 3430 (NH), 3040 (CH-aromatic), 2928, 2856 (CH-aliphatic), 1623 (C=N); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.24–1.89 (m, 10H, 5CH₂), 2.60 (s, 3H, CH₃), 2.74 (s, 3H, CH₃), 2.86–2.94 (2m, 2H, OCH₂- oxirane), 3.67 (d, 2H, J = 10.8 Hz, SCH₂), 3.72–3.77 (m, 1H, OCH-oxirane), 6.12 (s, 1H, NH, D₂O exchangeable), 7. 06 (s, 1H, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 19.27, 22.91, 24.70, 25.33 (3CH₂, 2CH₃), 32.19 (SCH₂), 37.02 (2CH₂), 44.76, 52.45 (OCH₂, OCH, oxirane), 70.03 (spiro C), 118.74, 122.33, 123.12, 124.67, 145.00, 147.21, 159.19, 163.66 (Ar-C); ESI-MS: m/z = 372.60 [M − H⁺]. Anal. Calcd. for C₁₉H₂₃N₃OS₂ (373.53): C, 61.09; H, 6.21; N, 11.25; S, 17.17% Found: C, 61.23, H, 6.44; N, 11.51; S, 17.01%.

4′-((Oxiran-2-ylmethyl)sulfanyl)-7′-phenyl-9′-(p-tolyl)-1′H-spiro[cyclopentane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine] (9b) was obtained from 7b (0.44 g, 1 mmol) in 74% yield (0.37 g) as a pale yellow solid, m.p. 120 °C. IR (KBr, υ_max/cm⁻¹): 3422 (NH), 3094 (CH-aromatic), 2919 (CH-aliphatic), (C=N); ¹H-NMR (DMSO-d6, 400 MHz): δ = 1.35–1.94 (m, 8H, 4CH₂), 2.43 (s, 3H, CH₃), 2.94–3.13 (2m, 2H, OCH₂- oxirane), 3.51 (d, 2H, J = 6.0 Hz, SCH₂), 3.91–3.95 (m, 1H, OCH-oxirane), 6.87 (s, 1H, NH, D₂O exchangeable), 7.32 (d, 2H, J = 7.2 Hz, Ar-H), 7.48–7.69 (m, 5H, Ar-H), 7.87 (s, 1H, Ar-H), 8.26 (d, 2H, J = 7.6 Hz, Ar-H); ¹³C-NMR (DMSO-d6, 100 MHz): δ = 21.40 (CH₃), 29.15 (2CH₂), 31.88 (SCH₂), 36.33 (2CH₂), 46.33, 53.45 (OCH₂, OCH, oxirane), 69.33 (spiro C), 118.49, 123.62, 127.81, 127.93, 128.78, 128.94, 129.43, 129.50, 130.53, 133.77, 139.17, 140.27, 148.73, 152.73,156.17, 163.83 (Ar-C); ESI-MS: m/z = 496.70 [M − H⁺]. Anal. Calcd. for C₂₉H₂₇N₃OS₂ (497.68): C, 69.99; H, 5.47; N, 8.44; S, 12.88% Found: C, 69.81, H, 5.31; N, 8.20; S, 12.63 %.

3.2. Antimicrobial Assay

All synthesized compounds (2a,b–9a,b) were screened for their in vitro antimicrobial activity against five bacterial strains (S. aureus 25923, B. subtilis 6633, B. cereus 33018, E. coli 8739, S. typhimurium 14028), three yeasts (C. albicans 10231, C. tropicals 750, S. cerevisiae) and two fungi (A. flavus, A. niger EM77). The MIC values (in μg/mL) of the tested compounds were determined using the broth dilution method and are listed in Table 1 and Table 2 [57]. (More details are provided in the Supplementary Materials).

3.3. In Vitro Cytotoxicity Screening

The in vitro cytotoxic activity of the target compounds 2a,b–9a,b was screened against HepG-2 and MCF-7 cancer cell lines, as well as the WISH normal cell line for the most active compounds, using the MTT assay [53]. The cells used in the cytotoxicity assays were cultured in a RPMI 1640 medium supplemented with 10% fetal calf serum. The cytotoxicity was estimated as IC₅₀ in μM for the tested compounds and the reference drug doxorubicin, and are listed in Table 3. (More details are provided in Supplementary Materials).

3.4. EGFR Kinase Inhibitory Assay

EGFR kinase inhibitory assays were performed for target compounds 3a, 4a, 5a, 6b, 8b and 9b with erlotinib as a reference inhibitor using the EGFR kinase assay kit (Cat. # 40321). The assay kit is designed to measure EGFR Kinase activity for screening applications using Kinase-Glo^® MAX as a detection reagent. The luminescence was measured using a microplate reader (Infinite M200 microplate reader, Tecan, Männedorf, Switzerland) [54]. All assays were performed in triplicate and the relative inhibition (%) of inhibitors was then calculated via comparison with the control with no inhibitor. Then, the IC₅₀ values (the concentration that provides 50% enzyme inhibition) and their standard deviation (SD) for the tested compounds and the reference drug were determined in μM and are listed in Table 4. (More details are provided in the Supplementary Materials).

3.5. Molecular Modeling Studies

To investigate molecular interactions between the most potent compounds and the active site of the epidermal growth factor receptor (EGFR), molecular docking study was performed using molecular operating environment software (MOE 2019.0102). Energy minimization was carried out until a RMSD gradient of 0.1 kcal∙mol⁻¹Å⁻¹ was achieved using a MMFF94x force field. The co-crystalized ligand (Erlotinib) was used to define the binding site for docking [58,59]. (More details are provided in the Supplementary Materials).

4. Conclusions

In conclusion, two novel series of pyridothienopyrimidine and spiro[cyclopentane/hexane-1,2′-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine] derivatives 2a,b–5a,b and 6a,b–9a,b, were synthesized and structurally elucidated. The new derivatives were subjected to in vitro antimicrobial screening against a panel of bacterial and fungal pathogens. According to the MIC values, derivatives 3a, 4a,b, 5a, 6b,c, 7c, 8b, and 9b exhibited significant antibacterial and antifungal activity with MIC ranges of 4–16 μg/mL, compared to amoxicillin trihydrate and clotrimazole as reference drugs with MIC ranges of 4–16 µM.

Furthermore, all new derivatives were evaluated as cytotoxic agents against HepG2 and MCF7 cancer cell lines. Compounds 9b, 5a, 3a, 6b, 8b, 4a produced the most potent antiproliferative activity with IC₅₀ values ranging between 1.17–2.99 µM against HepG2 cells and IC₅₀s ranging between 1.52–15.42 µM against MCF-7 cells, compared to doxorubicin as reference drug with IC₅₀s of 2.85 and 3.58 µM, respectively. In addition, these derivatives exhibited a promising safety profile when evaluated against the human normal WISH cell line.

Compounds 3a, 4a, 5a, 6b, 8b, 9b presented promising dual antimicrobial and antiproliferative activities, achieving the desired goal of this study.

The suppressing effect of compounds 3a, 4a, 5a, 6b, 8b, 9b against EGFR TK was also evaluated. It was detected that compounds 9b, 5a, 4a showed higher suppressing activity than erlotinib with IC₅₀ values of 7.27, 9.66 and 27.01 nM, respectively. In addition, molecular docking study was performed to determine the modes of interaction of the examined derivatives with amino acid residues at the active site of EGFR-PK. The docking results revealed that compounds 9a and 5a showed higher binding scores (–12.01 and –11.48 kcal/mol) than that of erlotinib (–10.48 kcal/mol).