Designing Potent Anti-Cancer Agents: Synthesis and Molecular Docking Studies of Thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidine Derivatives

A new series of thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidines was designed and synthesized using readily available starting materials, specifically, β-enaminoester. Their cytotoxicity was screened against three cancer cell lines, namely, MCF-7, HCT-116, and PC-3. 2-(4-bromophenyl)triazole 10b and 2-(anthracen-9-yl)triazole 10e afforded excellent potency against MCF-7 cell lines (IC50 = 19.4 ± 0.22 and 14.5 ± 0.30 μM, respectively) compared with doxorubicin (IC50 = 40.0 ± 3.9 μM). The latter derivatives 10b and 10e were further subjected to in silico ADME and docking simulation studies against EGFR and PI3K and could serve as ideal leads for additional modification in the field of anticancer research.


Introduction
Cancer is a global health crisis marked by increasing incidence and mortality rates.Prompt diagnosis and effective treatment strategies are crucial due to the growing number of individuals affected by cancer.The relentless pursuit of innovative therapeutic strategies for combating cancer has led to an increased emphasis on the design and synthesis of novel compounds with potent anti-cancer properties [1].The thienopyrimidine scaffold has emerged as a frequently utilized chemical framework in drug development.Compounds containing thienopyrimidine exhibit structural and isoelectronic characteristics similar to purines, making them attractive in the production of pharmaceutical drugs [2,3] and have demonstrated significant pharmacological properties, including antibacterial [4][5][6], antiviral [7,8], anti-inflammatory [9,10], antiprotozoal [11], and anticancer activities [12][13][14][15].
Since the epidermal growth factor receptor tyrosine kinase (EGFR) participates in the growth and progression of cancer, it represents a compelling target for cancer treatment [24].EGFR is a desirable target in many illnesses such pancreatic cancer, breast cancer, and nonsmall cell lung cancer as well as those of the lung, ovarian, and breast regions [25][26][27][28].Additionally, the signaling pathway that involves phosphatidylinositol 3-kinase (PI3K) is a key player in regulating cell viability, proliferation, migration, glucose metabolism, and death.It has been extensively investigated over the past few decades to create novel cancer therapies that target the earlier pathways [29,30].
Molecules 2024, 29, x FOR PEER REVIEW 2 of 22 moiety to the thienopyrimidine system is anticipated to significantly influence the cytotoxic activity [20].Conversely, the combination of glycosides with heterocyclic molecules generated essential hybrids with biologically interesting properties, such as antiviral, anticancer, and antibacterial properties [21][22][23].
Since the epidermal growth factor receptor tyrosine kinase (EGFR) participates in the growth and progression of cancer, it represents a compelling target for cancer treatment [24].EGFR is a desirable target in many illnesses such pancreatic cancer, breast cancer, and non-small cell lung cancer as well as those of the lung, ovarian, and breast regions [25][26][27][28].Additionally, the signaling pathway that involves phosphatidylinositol 3-kinase (PI3K) is a key player in regulating cell viability, proliferation, migration, glucose metabolism, and death.It has been extensively investigated over the past few decades to create novel cancer therapies that target the earlier pathways [29,30].
Several examples of diverse thienopyrimidine-containing drugs highlight their broad applications in various therapeutic areas.Pictilisib (GDC-0941) is currently under clinical investigation for its potential in addressing advanced solid tumors by inhibiting PI3K.Also, olmutinib has proven effective as an EGFR inhibitor, applied in the treatment of non-small cell lung cancer (NSCLC) [31].Thieno [2,3-d]pyrimidine I was discovered to exceed the commercially available medicine lapatinib in terms of EGFR inhibition, which piqued a lot of interest [32].Moreover, the derivative II illustrated powerful cytotoxicity against colorectal HCT-116, SW480, ovarian SKOV3, glioblastoma U87 and breast SKBR3 cancer cell lines, with IC50 values ranging from 3.83 to 11.94 μM when compared to erlotinib through EGFR inhibition behavior [33].Thieno [2,3-d]pyrimidine III demonstrated significant antitumor efficacy via PI3K inhibition against NCI 60 cell lines [34].Thieno [2,3-d]pyrimidine-1,2,3-triazole-glycoside IV, 1,2,4-triazole V and 1,2,4-triazole -glycoside VI also exhibit substantial anticancer activity, mainly against MCF-7, through their inhibitory activity against EGFR [35][36][37] (Figure 1).Building on insights from the cited reports and our ongoing research in synthesizing biologically active compounds [22,[38][39][40], this study focuses upon designing derivatives with the thieno [3,2-d]pyrimidin-4(3H)-one cores bearing 1,2,4-triazole and glycoside scaffolds.The effectiveness of these compounds will be assessed against MCF-7, HCT-116, and PC-3 cancer cell lines.The promising derivatives were further evaluated through molecular docking studies against EGFR and PI3K to predict their mechanism of action.Finally, in silico ADME studies were applied to determine the physicochemical and pharmacokinetic properties to facilitate valuable insights in development of more effective anticancer therapies.

Chemistry
The synthetic approaches utilized for creating the intermediate and final compounds are illustrated in Schemes 1-6, respectively.In Scheme 1, ethyl 2-amino-5,6-dihydro-4Hcyclopenta[b]thiophene-3-carboxylate 1 underwent an efficient transformation to yield ethyl 2-(1H-tetrazol-1-yl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate 2. This conversion was achieved by subjecting compound 1 to a reaction with triethyl orthoformate and sodium azide in glacial acetic acid [41,42].Subsequently, treatment of the resulting compound 2 with hydrazine hydrate led to the successful synthesis of cyclized thienotriazolopyrimidine 3, obtained in a good yield.The chemical structure of compound 3 was confirmed through analysis of its analytical and spectral data (see experimental).Building on insights from the cited reports and our ongoing research in synthesizing biologically active compounds [22,[38][39][40], this study focuses upon designing derivatives with the thieno [3,2-d]pyrimidin-4(3H)-one cores bearing 1,2,4-triazole and glycoside scaffolds.The effectiveness of these compounds will be assessed against MCF-7, HCT-116, and PC-3 cancer cell lines.The promising derivatives were further evaluated through molecular docking studies against EGFR and PI3K to predict their mechanism of action.Finally, in silico ADME studies were applied to determine the physicochemical and pharmacokinetic properties to facilitate valuable insights in development of more effective anticancer therapies.

Chemistry
The synthetic approaches utilized for creating the intermediate and final compounds are illustrated in Schemes 1-6, respectively.
This conversion was achieved by subjecting compound 1 to a reaction with triethyl orthoformate and sodium azide in glacial acetic acid [41,42].Subsequently, treatment of the resulting compound 2 with hydrazine hydrate led to the successful synthesis of cyclized thienotriazolopyrimidine 3, obtained in a good yield.The chemical structure of compound 3 was confirmed through analysis of its analytical and spectral data (see experimental).

Scheme 1. Synthesis of compound 3.
The reaction of 2,3-diamino-6,7-dihydro-3H-cyclopenta [4,5]thieno [2,3-d]pyrimidin-4 (5H)-one 3 with triethyl orthoformate and acetic anhydride (Scheme 2) readily provided the cyclized thienotriazolopyrimidins 4 and 5, respectively.The confirmation of the chemical structures of compounds 4 and 5 was achieved through the analysis of both analytical and spectral data.For instance, in the case of compound 4, the IR spectrum revealed absorption bands at 3309 and 1670 cm −1 , corresponding to the NH and C=O groups, respectively.Additionally, in the 1 H NMR spectrum, a singlet signal at 7.54 ppm was observed, which was attributed to the triazolo proton.Moreover, the 13  The reaction of 2,3-diamino-6,7-dihydro-3H-cyclopenta [4,5]thieno [2,3-d]pyrimidin-4(5H)-one 3 with triethyl orthoformate and acetic anhydride (Scheme 2) readily provided the cyclized thienotriazolopyrimidins 4 and 5, respectively.The confirmation of the chemical structures of compounds 4 and 5 was achieved through the analysis of both analytical and spectral data.For instance, in the case of compound 4, the IR spectrum revealed absorption bands at 3309 and 1670 cm −1 , corresponding to the NH and C=O groups, respectively.Additionally, in the 1 H NMR spectrum, a singlet signal at 7.54 ppm was observed, which was attributed to the triazolo proton.Moreover, the 13  Upon heating compound 3 with diethylmalonate, the ethyl acetate ester 6 was formed.The IR spectrum of compound 6 displayed prominent absorption bands at 3395, 1718 and 1684 cm −1 due to NH2, ester, and amidic C=O groups.The 1 H NMR spectrum revealed expected triplet and quartet signals corresponding to the ester group, in addition to a singlet at 3.33 ppm, corresponding to the CH2CO protons.Upon heating compound 3 with diethylmalonate, the ethyl acetate ester 6 was formed.The IR spectrum of compound 6 displayed prominent absorption bands at 3395, 1718 and 1684 cm −1 due to NH 2 , ester, and amidic C=O groups.The 1 H NMR spectrum revealed expected triplet and quartet signals corresponding to the ester group, in addition to a singlet at 3.33 ppm, corresponding to the CH 2 CO protons.
The synthesis of the 3-cyanomethyl derivative 7 was accomplished with a satisfactory yield by subjecting compound 3 to a reaction with ethyl cyanoacetate at 180 • C. In the 1 H NMR spectrum, a singlet appeared at 4.32 ppm, which was ascribed to the CH 2 protons.Additionally, the IR spectrum displayed distinctive signal of the nitrile group at 2265 cm −1 (Scheme 2).
Subjecting compound 3 to heat in the presence of excess acetyl acetone resulted in the formation of the 1-methyl-3-oxobutylideneamino derivative 8 (Scheme 3).In its 1 H-NMR spectrum, the upfield resonance of the N=C-CH 3 protons and the methyl carbon provided confirmation of an E configuration at C1 [43,44].Upon heating compound 3 with diethylmalonate, the ethyl acetate ester 6 was formed.The IR spectrum of compound 6 displayed prominent absorption bands at 3395, 1718 and 1684 cm −1 due to NH2, ester, and amidic C=O groups.The 1 H NMR spectrum revealed expected triplet and quartet signals corresponding to the ester group, in addition to a singlet at 3.33 ppm, corresponding to the CH2CO protons.
The synthesis of the 3-cyanomethyl derivative 7 was accomplished with a satisfactory yield by subjecting compound 3 to a reaction with ethyl cyanoacetate at 180 °C.In the 1 H NMR spectrum, a singlet appeared at 4.32 ppm, which was ascribed to the CH2 protons.Additionally, the IR spectrum displayed distinctive signal of the nitrile group at 2265 cm −1 (Scheme 2).
Subjecting compound 3 to heat in the presence of excess acetyl acetone resulted in the formation of the 1-methyl-3-oxobutylideneamino derivative 8 (Scheme 3).In its 1 H-NMR spectrum, the upfield resonance of the N=C-CH3 protons and the methyl carbon provided confirmation of an E configuration at C1 [43,44].By subjecting compound 3 to heat in the presence of carbon disulfide in ethanol with sodium ethoxide, we successfully synthesized the 2-thioxo analog 9 (Scheme 3).The validation of its structure was established through multiple analyses, including the mass spectrum, where a molecular ion peak at m/z 262 (35.1%) was observed.Additionally, in the 1H NMR spectrum, two singlets were detected at δ 10.57 and 10.90 ppm, which were attributed to the 2NH protons and found to be exchangeable with D2O.
By subjecting compound 3 to heat in the presence of carbon disulfide in ethanol with sodium ethoxide, we successfully synthesized the 2-thioxo analog 9 (Scheme 3).The validation of its structure was established through multiple analyses, including the mass spectrum, where a molecular ion peak at m/z 262 (35.1%) was observed.Additionally, in the 1H NMR spectrum, two singlets were detected at δ 10.57 and 10.90 ppm, which were attributed to the 2NH protons and found to be exchangeable with D 2 O.
The fusion of compound 3 with different aromatic aldehydes, namely 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-nitrobenzaldehyde, 4-methoxybenzaldehyde, and anthracenaldehyde, in an oil bath at 180 • C led to the formation of the desired thienotriazolopyrimidines 10a-e (Scheme 4).The compounds 10a-e displayed strong IR absorption bands at 3396-3308 cm −1 and 1680-1658 cm −1 indicating the presence of NH and C=O groups, respectively.In the analysis of the 1 H NMR spectra of compounds 10a-e, the absence of a singlet corresponding to the N=CH proton, expected for the azomethine proton, confirmed the formation of the cyclized product.
The oxidative condensation of monosaccharides, namely, (D)-glucose, (D)-galactose, (D)-mannose, and (D)-arabinose with 3, occurred readily at room temperature using a catalytic amount of iodine in acetic acid.The reaction was completed within 6-12 h, as monitored by thin-layer chromatography, leading to the formation of 11a-d, which, upon acylation, resulted in the formation of the acylated products 12a-d.The structures of the new deacylated and acylated products were established based on their microanalytical and spectroscopic data (see experimental and Scheme 5).
4-methoxybenzaldehyde, and anthracenaldehyde, in an oil bath at 180 °C led to the formation of the desired thienotriazolopyrimidines 10a-e (Scheme 4).The compounds 10a-e displayed strong IR absorption bands at 3396-3308 cm −1 and 1680-1658 cm −1 indicating the presence of NH and C=O groups, respectively.In the analysis of the 1 H NMR spectra of compounds 10a-e, the absence of a singlet corresponding to the N=CH proton, expected for the azomethine proton, confirmed the formation of the cyclized product.The oxidative condensation of monosaccharides, namely, (D)-glucose, (D)-galactose, (D)-mannose, and (D)-arabinose with 3, occurred readily at room temperature using a catalytic amount of iodine in acetic acid.The reaction was completed within 6-12 h, as monitored by thin-layer chromatography, leading to the formation of 11a-d, which, upon acylation, resulted in the formation of the acylated products 12a-d.The structures of the new deacylated and acylated products were established based on their microanalytical and spectroscopic data (see experimental and Scheme 5).The coupling of compounds 4, 8, and 10d with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide 13 in acetone and potassium carbonate afforded the N-glycosylated nucleosides 14, 15, and 16 in good yields (73, 62, and 65%, respectively (Scheme 6).Thin layer chromatography (chloroform/methanol = 10:1) indicated formation of the pure compounds.The structures of the products 14, 15, and 16 were confirmed by elemental analyses and spectral data (IR, 1 H NMR, 13 C NMR) (see Experimental).For instance, analytical data for compound 16 revealed a molecular formula of C 31 H 32 N 4 O 11 S (M + 668.67).The 1 H NMR spectrum showed the anomeric proton of the glucose moiety as a doublet at δ 5.03-5.25 ppm with a coupling constant J = 10.5 Hz indicating β-configuration of the anomeric center.The other protons of the glucopyranose ring resonated at δ 3.84-6.52ppm, while the four acetoxy groups appeared as four singlets at 1.13-2.21ppm.
Investigating the targets' absorption, distribution, metabolism, and excretion (ADME) is a critical first step in selecting the best possible medication candidate.Swiss-ADME, a free online tool, made this anticipated examination easier [48][49][50].Veber's (molecule with number of rotatable bonds ≤ 10, TPSA ≤ 140 Å 2 ) and Lipinski's (MW ≤ 500, MLogP ≤ 4.15, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5) rules should be taken into consideration while selecting an oral drug.Anthracenyl derivative 10e had one violation with MLogP > 4.15, whereas 4-bromophenyl 10b seemed to be in accordance with the prior rules with no violations (Table 2).The cyclopenta [4,5]thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidinone 10b was identified to be in the optimal range (pink zone) with respect to the critical variables lipophilicity, polarity, size, solubility and flexibility, as illustrated in Figure 3 of the bioavailability radar chart; however, the derivative 10e departed away from solubility and saturation.
Figure 3 and Table 3 represent the investigated pharmacokinetic characteristics of the promising cyclopenta [4,5]thieno [2,3-d] [1,2,4]triazolo [1,5-a]pyrimidinones 10b and 10e.Both derivatives 10b and 10e were anticipated to have a high gastrointestinal absorption with no brain penetration (within the white region and away from the yellow one of the boiled egg chart).The red dot of 10b in Figure 4 suggests that it has a restricted ability to efflux out of the cell, representing its maximal potency as it's not a substrate for p-glycoprotein (P-gp, drug efflux transporter), unlike compound 10e (blue dot, P-gp substrate).These substances also have a satisfactory bioavailability value of 0.55 and no pain alert.4 suggests that it has a restricted ability to efflux out of the cell, representing its maximal potency as it's not a substrate for p-glycoprotein (P-gp, drug efflux transporter), unlike compound 10e (blue dot, P-gp substrate).These substances also have a satisfactory bioavailability value of 0.55 and no pain alert.Based upon the excellent outcomes retrieved from the cytotoxic evaluation of both cyclopenta [4,5]thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-9(6H)-ones 10b and 10e against MCF-7, HCT-116, and PC-3 cell lines and their promising drug-like characteristics, a docking simulation was carried out to predict their mechanism of action.The docking procedure was carried out using MOE-Dock (Molecular Operating Environment) software version 2014.01 [51].First, the process was validated by re-docking the native ligands, erlotinib and quinolone LXX, within the active sites of EGFR and PI3K (PDB codes: 1M17 and 3L54, respectively) [52,53], providing energy score values of −11.75 and −10.60 kcal/mol with relatively minor values of RMSD (1.22 and 0.78 Å, respectively), between the co-crystallized ligands and their docked positions.

General Information
All melting points are uncorrected, were measured by using an electro-thermal IA 9100 apparatus (Shimadzu, Kyoto, Japan).Microanalyses were carried out at the Microanalytical center (Faculty of Science, Cairo University, Egypt).IR spectra were carried out on JASCO FT/IR 6100 Japan spectrometer (National Research Centre, Cairo, Egypt) using KBr discs. 1 H-NMR spectra were measured in DMSO using JEOL EX-270 run for 1 H-NMR at 270 MHz; JEOL ECA-500 run for 1 H-NMR at 500 MHz and run for 13 C-NMR at 125 MHz spectrometers.Chemical shifts were expressed in parts per million (δ ppm) against tetramethylsilane (TMS) as an internal standard.The coupling constant J is expressed in Hz.Mass spectra were recorded on GCMS Finnigan mat SSQ 7000 spectrometer.UV-Vis was recorded using (Shimadzu spectrophotometer).TEM was recorded by using High-Resolution Transmission Electron Microscopy (HRTEM) JEOL (JEM-2100 TEM).All reactions were followed up by thin layer chromatography (TLC).Aluminum sheets were used recoated with UV fluorescent silica gel (Merck Kieselgel 60 F 245 ).It was visualized using a UV lamp and iodine vapor.Fine chemicals were of analytical grade; Selenious acid (H 2 SeO 3 ) (Aldrich, Burlington, MA, USA), ascorbic acid (99%, Aldrich).All solvents were dried before being used.Refer to Supplementary Materials for 1H-NMR and 13C-NMR spectra of sample compounds.
A suspension of 1 (9.95 g, 50 mmol), triethyl orthoformate (38.29 mL, 230 mmol), and sodium azide (3.90 g, 60 mmol) in glacial acetic acid (40 mL) was stirred under reflux for 2 h.The reaction mixture was cooled to room temperature and 7 mL of conc.HCl was added.The separated solid was filtered off and the filtrate was evaporated under reduced pressure.The remaining residue was recrystallized from ethanol to afford brown crystal in 65% yield.

In Vitro Cytotoxic Screening
The cell lines were obtained from Karolinska Center, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.as follows: human breast MCF-7, colorectal HCT-116 and prostate PC-3 cancer cell lines and human skin normal BJ-1 cell line.Exponentially, cells were cultured at a concentration of 10 4 cells/well for 24 h, afterwards fresh medium containing different concentrations of the tested samples was added.Serial two-fold dilution of the tested samples were added using a multichannel pipette.Moreover, all cells were cultivated at 37 • C, 5% CO 2 and 95% humidity.Also, incubation of control cells occurred at 37 • C.However, after incubation for 24 h different concentrations of samples (100, 50, 25 and 12.5 µM) were added and the incubation was continued for 48 h, then crystal violet solution 1% was added to each well for 0.5 h to examine the presence of viable cells.After rinsing the wells using water until stain free, 30% glacial acetic acid was added to all wells with shaking the plates on a Microplate reader (TECAN, Inc.) to measure the absorbance at a wavelength of 490 nm.The cytotoxicity was estimated by IC 50 in µM, which is the concentration that inhibits 50% of growth of cancer cells.

Molecular Docking Study
Molecular docking simulation of the promising in vitro screened cyclopenta [4,5]thieno [2,3d] [1,2,4]triazolo [1,5-a]pyrimidin-9(6H)-ones 10b and 10e against EGFR and PI3K was done using the Molecular Operating Environment software (MOE-Dock) version 2014.01.The co-crystallized structures of EGFR and PI3K kinases complexed with their native ligands, erlotinib and quinolone LXX, were downloaded from the protein data bank (PDB codes: 1M17 and 3L54, respectively).All minimizations were performed using MOE until an RMSD gradient of 0.05 kcal•mol −1 Å −1 with MMFF94x force field and the partial charges were automatically calculated.Preparation of the enzyme structures was done for molecular docking using Protonate 3D protocol with the default options in MOE.London dG scoring function and Triangle Matcher placement method were used in the docking protocol.Initially, the original ligands were re-docked into the active binding site of EGFR and PI3K kinases to assess the root-mean-square deviation values.Thereafter, docking of the newly targeted compounds was performed within the ATP-binding sites of both target kinases after elimination of the co-crystallized ligands.

Molecules 2024 ,
29,  x FOR PEER REVIEW 10 of 22 vailability radar chart; however, the derivative 10e departed away from solubility and saturation.

Figure 3 and
Figure 3 and Table3represent the investigated pharmacokinetic characteristics of the promising cyclopenta[4,5]thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidinones 10b and 10e.Both derivatives 10b and 10e were anticipated to have a high gastrointestinal absorption with no brain penetration (within the white region and away from the yellow one of the boiled egg chart).The red dot of 10b in Figure4suggests that it has a restricted ability to efflux out of the cell, representing its maximal potency as it's not a substrate for p-glycoprotein (P-gp, drug efflux transporter), unlike compound 10e (blue dot, P-gp substrate).These substances also have a satisfactory bioavailability value of 0.55 and no pain alert.

Scheme 2. Synthesis of compounds 4-7.
C NMR spectrum displayed distinct signals at 132.19 and 166.61 ppm, corresponding to the triazole CH and CO, respectively.

Table 1 .
The antitumor activities of the target compounds against MCF-7, HCT-116, PC-3, and BJ-1 cancer cell lines expressed as IC 50 values.