In Silico Screening and Anticancer-Apoptotic Evaluation of Newly Synthesized Thienopyrimidine/Sulfonamide Hybrids

This work describes the design and synthesis of new hybrids of thienopyrimidine and sulfonamides. The binding affinity of the prepared compounds to FGFR-1 enzyme and caspase-3 was investigated via molecular docking. The cytotoxic effect was estimated for the synthesized compounds against human breast cancer cell lines (MCF-7 and MDA-MB231) using Doxorubicin as a reference. All the tested compounds exhibited moderate to excellent anticancer efficacy against both tested cell lines, among which 3b and 4bi were the best. All the synthesized compounds exhibited distinguishing selectivity index values greater than Doxorubicin. The influence of the new hybrids under inquiry was further examined on both FGFR-1 and Caspase-3. The results revealed that compound 3b showed observed concordance between anti-proliferative activity and Caspase-3 activity. In respect to the compounds’ effect on the apoptosis, compound 3b significantly increased the population of late apoptotic cells and necrotic cells. In silico pharmacokinetic investigation revealed that compound 3b showed the best intestinal absorption, BBB permeability, and, along with 4bi and 4bii, the best CNS penetrability.


Introduction
The fibroblast growth factor (FGF) family and its four receptor tyrosine kinases (FGFR-1-4) are necessary for a variety of physiological processes, such as embryogenesis, tissue repair, tissue homeostasis, wound healing, and inflammation [1][2][3]. Fibroblast growth factor receptor 1 (FGFR-1) is involved in the regulation of cell proliferation and differentiation. FGFR-1 is also involved in other critical physiological processes and in the process of oncogenic angiogenesis; accordingly, it participates in solid tumor development [1][2][3]. FGFR-1 is considered a promising therapeutic target for the development of tumor growth inhibitors as many diseases, including cancer (such as breast, prostate, and ovarian cancers), are linked to its malfunction. Approximately 15% of breast cancers that are hormone receptor-positive and 5% of the more dangerous triple-negative breast cancers have been shown to have FGFR-1 overexpression, which is reported as frequent in breast cancer [1][2][3][4][5].
Several classes of heterocyclic rings were reported as FGFR-1 inhibitors, such as Nphenylnaphthostyril-1-sulfonamides [1], quinazolines [6], oxindoles [7], and N-phenylthieno pyrimidine derivatives [5]. The latter-mentioned ring "thienopyrimidine" is reported as structurally unique, with its wide range of biological activities; it has been widely used as a scaffold in the design of compounds in the chemical sector with a variety of biologically active compounds such as enzyme inhibitors including kinases [5,8], poly-ADP ribose polymerase (PARP) inhibitors [9], and anticancer agents against some tumor cell lines [10][11][12]. In addition, its antioxidant activity [13][14][15] has been shown to be useful not only in the prevention but also in the treatment of complex diseases such as Alzheimer's, cancer, and stroke. Additionally, in an area of anticancer drug discovery, thiourea and sulfonamide derivatives were reported to exhibit their anticancer actions via inhibiting diverse molecular targets [16], which make them promising scaffolds for novel anticancer compounds.
The foregoing facts piqued our interest, so we set out to design and synthesize thiophene/thienopyrimidine derivatives with the goal of obtaining new cytotoxic compounds, keeping in mind that the sulfonamide moiety incorporated in the designed compounds will mostly target the fibroblast growth factor receptor-1 enzyme.
Design strategy: The design technique involved introducing cyclohexylthieno[2,3-d] pyrimidine as a core structure (Figure 1). This was performed based on reported research that related the increased anticancer activity of thieno [2,3-d]pyrimidine with the incorporation of a lipophilic cycloalkyl ring [12,16,17]. Replacing the cyclohexyl side chain with an open ester one (R1) and a methyl group (R2) was carried out to further explore the effect of this replacement on biological activity and compare results between a cyclic side chain and the open one. Furthermore, sulfonamides had been previously incorporated in compounds resulting in promising anticancer activity, especially kinase inhibition profiles [18][19][20]. Accordingly, it prompted our interest to link the coplanar cyclic structure of thienopyrimidine with different substituted sulfonamide groups which were incorporated in the thienopyrimidine core at position 4 to afford two novel series of thienopyrimidine-sulfonamide hybrids "series a from 1a-4a(i-iii)" and "series b from 1b-4b(i-iii)", aiming to explore their biological anticancer and apoptotic activities ( Figure 1).
reported as structurally unique, with its wide range of biological activities; it has been widely used as a scaffold in the design of compounds in the chemical sector with a variety of biologically active compounds such as enzyme inhibitors including kinases [5,8], poly ADP ribose polymerase (PARP) inhibitors [9], and anticancer agents against some tumo cell lines [10][11][12]. In addition, its antioxidant activity [13][14][15] has been shown to be usefu not only in the prevention but also in the treatment of complex diseases such as Alz heimer's, cancer, and stroke. Additionally, in an area of anticancer drug discovery, thiou rea and sulfonamide derivatives were reported to exhibit their anticancer actions via in hibiting diverse molecular targets [16], which make them promising scaffolds for nove anticancer compounds.
The foregoing facts piqued our interest, so we set out to design and synthesize thio phene/thienopyrimidine derivatives with the goal of obtaining new cytotoxic compounds keeping in mind that the sulfonamide moiety incorporated in the designed compound will mostly target the fibroblast growth factor receptor-1 enzyme.
Design strategy: The design technique involved introducing cyclohexylthieno[2,3 d]pyrimidine as a core structure ( Figure 1). This was performed based on reported re search that related the increased anticancer activity of thieno [2,3-d]pyrimidine with the incorporation of a lipophilic cycloalkyl ring [12,16,17]. Replacing the cyclohexyl side chain with an open ester one (R1) and a methyl group (R2) was carried out to further explore the effect of this replacement on biological activity and compare results between a cyclic side chain and the open one. Furthermore, sulfonamides had been previously incorpo rated in compounds resulting in promising anticancer activity, especially kinase inhibi tion profiles [18][19][20]. Accordingly, it prompted our interest to link the coplanar cyclic structure of thienopyrimidine with different substituted sulfonamide groups which were incorporated in the thienopyrimidine core at position 4 to afford two novel series o thienopyrimidine-sulfonamide hybrids "series a from 1a-4a(i-iii)" and "series b from 1b-4b(i-iii)", aiming to explore their biological anticancer and apoptotic activities ( Figure  1).  Design strategy for the synthesis of thienopyrimidine-sulfonamides hybrids [18,19].  [18,19].

In Vitro Anti-Proliferative Activity
The cytotoxic effect of the synthesized compounds was estimated against human breast cancer cell lines (MCF-7 and MDA-MB231). The IC50 values of all compounds compared to Doxorubicin are summarized in Table 1. The results of cytotoxic evaluation revealed that the synthesized compounds exhibited moderate to excellent anticancer efficacy against both tested cell lines. In the estrogen receptor-positive (MCF-7) cells, compounds 3b, 4bi, and 4biii demonstrated the best cytotoxic effect among all the investigated compounds, with IC50 9.74 ± 0.13, 6.17 ± 1.3, and 9.54 ± 3.1 µM, respectively. The comparison of IC50 values against the triple-negative MDA-MB-231 cells revealed that 3b and 4bi had the highest potency, with IC50 4.45 ± 0.31 and 8.68 ± 4.3 µM, respectively.
However, structural differences result in a distinguishing variation in cytotoxicity. The presence of three derivatives, 3b, 4bi, and 4biii, possessing IC50 values less than 10 µM is an indication of how structure belonging to series "b" can prominently inhibit the proliferation of breast cancer cells and angiogenesis. The absence of a cyclohexyl ring and its replacement with carboxylate (open side chain) could be a contributing factor to the anticancer activity of compounds from series b (Table 1).   Table 1. The results of cytotoxic evaluation revealed that the synthesized compounds exhibited moderate to excellent anticancer efficacy against both tested cell lines. In the estrogen receptor-positive (MCF-7) cells, compounds 3b, 4bi, and 4biii demonstrated the best cytotoxic effect among all the investigated compounds, with IC 50 9.74 ± 0.13, 6.17 ± 1.3, and 9.54 ± 3.1 µM, respectively. The comparison of IC 50 values against the triple-negative MDA-MB-231 cells revealed that 3b and 4bi had the highest potency, with IC 50 4.45 ± 0.31 and 8.68 ± 4.3 µM, respectively.
However, structural differences result in a distinguishing variation in cytotoxicity. The presence of three derivatives, 3b, 4bi, and 4biii, possessing IC 50 values less than 10 µM is an indication of how structure belonging to series "b" can prominently inhibit the proliferation of breast cancer cells and angiogenesis. The absence of a cyclohexyl ring and its replacement with carboxylate (open side chain) could be a contributing factor to the anticancer activity of compounds from series b (Table 1).

Selectivity Assessment
Selectivity for cancer cells was investigated among the synthesized compounds to identify the compounds that have high potency towards cancer cells and low cellular toxicity in normal cells. The selectivity index (SI) was calculated using the ratio of the IC 50 value of the selected compounds on non-cancerous cells (MCF10A) to the IC 50 value of the compounds on both cancer cells (MCF-7 and MDA-MB-231). It has been proposed that compounds with an SI > 3 were identified to have potential selectivity towards cancer cells [26,27] (Table 2). These results demonstrate the potential of the synthesized compounds to selectively target cancer cells while minimizing damage to normal cells, which is a desirable characteristic in chemotherapeutic agents ( Table 2).

Target Prediction
Target prediction was performed in silico on the synthesized compounds in an attempt for exploration of the anticancer target. Compounds recorded affinity towards different targets, among them, kinases, proteases, and oxidoreductases, with percentages ranging from >60% to 6%, whereas kinase affinity was expressed in a promising value (>60% to 40%) ( Figure 2).

Modeling
The binding affinity of the prepared compounds to FGFR-1 enzyme and caspase-3 was investigated via molecular docking. The choice of these targets was based upon a target prediction step that showed high probability for FGFR-1, while caspase-3 selection was based on our interest to explore the apoptotic activity of the newly prepared hybrids. FGFR-1 and caspase-3 proteins bounded to their ligand were downloaded from a protein data bank for modeling study with PDB codes for their crystal structures (pdb:5O49 and 7JL7, respectively). Interestingly, it was noticed that among the investigated compounds, those bearing a carboxylate side chain showed better results than those with the cyclohexyl group in both the modeling study and biological evaluations.

Modeling
The binding affinity of the prepared compounds to FGFR-1 enzyme and caspase-3 was investigated via molecular docking. The choice of these targets was based upon a target prediction step that showed high probability for FGFR-1, while caspase-3 selection was based on our interest to explore the apoptotic activity of the newly prepared hybrids. FGFR-1 and caspase-3 proteins bounded to their ligand were downloaded from a protein data bank for modeling study with PDB codes for their crystal structures (pdb:5O49 and 7JL7, respectively). Interestingly, it was noticed that among the investigated compounds, those bearing a carboxylate side chain showed better results than those with the cyclohexyl group in both the modeling study and biological evaluations.
Regarding the FGFR-1 target protein (pdb:5O49): All compounds showed promising binding affinity to the target protein, ranging from −7.123 to −5.239. Amino acids that are commonly involved in interaction were ASP 641, LEU 484, LYS 514, and ALA 564. Compounds 3b, 4aii, 4bi, and 4bii showed interactions with the target phosphate binding pocket at ASP 641, whereas compounds 4aiii and 4biii revealed binding to the hinge region at ALA 564. Compounds recording the best binding affinities are illustrated in Figure  3. It worth mentioning that compound 3b recorded the best anticancer activity, reflected in it IC50 values of 9.74 ± 0. 13    Regarding the FGFR-1 target protein (pdb:5O49): All compounds showed promising binding affinity to the target protein, ranging from −7.123 to −5.239. Amino acids that are commonly involved in interaction were ASP 641, LEU 484, LYS 514, and ALA 564. Compounds 3b, 4aii, 4bi, and 4bii showed interactions with the target phosphate binding pocket at ASP 641, whereas compounds 4aiii and 4biii revealed binding to the hinge region at ALA 564. Compounds recording the best binding affinities are illustrated in Figure 3. It worth mentioning that compound 3b recorded the best anticancer activity, reflected in it IC 50 values of 9.74 ± 0.13 and 4.45 ± 0.31 µM against cancer cell lines MCF-7 and MDA-MB-231, respectively, as well as its remarkable selectivity index values (SI) of 27.20 and 44.80 For MCF-7 and MDA-MB-231 cell lines, respectively. It totally fits the protein pocket ( Figure 3), with hydrophobic interaction with ASP 641 at the protein phosphate binding pocket and a binding affinity of −5.239 at a perfect fit with an RMSD value of 1.0171. The binding energy and root mean square deviation (RMSD) as well as residues of amino acids incorporated in the interactions between the prepared ligands and the active site either hydrogen bonding or hydrophobic interactions are tabulated below in Table 3.  Figure 3. Synthesized chloro thienopyrmidine derivative 3b which has the best biological results in green sticks and thienopyrimidine-sulfamethoxazole hybrid derivative 4biii with the best binding energy docking score in grey sticks interacting with FGFR-1 protein crystal structure 5O49.pdb.
Regarding the caspase-3 target protein (pdb: 7JL7): Binding affinity to the downloaded protein range from −6.476 to −4.059, and RMSD values range between 1.372 to 2.455A. Common amino acid residues involved in interactions are ARG 212, TRP 219, ARG 67, THR 255, and CYS 166; interestingly, these are the same residues involved in the interactions between the protein and the co-crystalized ligand, as shown in Table 4 and the overlay-complex 3D model in Figure 4. Binding energy and root mean square deviation (RMSD), as well as residues of amino acids incorporated in the interactions between the prepared ligands and the active site in addition to the types of bonding (H-bond, Hπ or π-π), are tabulated below, Table 4.  . Synthesized chloro thienopyrmidine derivative 3b which has the best biological results in green sticks and thienopyrimidine-sulfamethoxazole hybrid derivative 4biii with the best binding energy docking score in grey sticks interacting with FGFR-1 protein crystal structure 5O49.pdb.
Regarding the caspase-3 target protein (pdb: 7JL7): Binding affinity to the downloaded protein range from −6.476 to −4.059, and RMSD values range between 1.372 to 2.455A. Common amino acid residues involved in interactions are ARG 212, TRP 219, ARG 67, THR 255, and CYS 166; interestingly, these are the same residues involved in the interactions between the protein and the co-crystalized ligand, as shown in Table 4 and the overlaycomplex 3D model in Figure 4. Binding energy and root mean square deviation (RMSD), as well as residues of amino acids incorporated in the interactions between the prepared ligands and the active site in addition to the types of bonding (H-bond, H-π or π-π), are tabulated below, Table 4. Pi-H Pi-sulfur H-donor π-π π-π π-sulfur π-sulfur

FGFR-1 Inhibitory Activity
After analyzing the target prediction results, it was identified that FGFR-1 had the highest probability among kinases; thus, FGFR-1 enzyme assays were conducted. Considering potency, selectivity, and affinity percentages, the inhibitory activity of all synthesized compounds against FGFR-1 was evaluated, and the results are summarized in Table 5. There was a wide range of inhibitory potencies, ranging from 131.95 ± 4.3 µM to 325.65 ± 7.4 µM. Compounds 4ai and 4aii had the highest FGFR-1-inhibiting activity among those tested, with IC 50 values of 131.95 ± 4.3 and 119.43 ± 5.6 µM, respectively. However, it was challenging to correlate the anticancer effect of the drugs that were examined to the inhibition of FGFR-1 alone owing to the compounds' modest inhibitory action against FGFR-1. This may imply the presence of alternate underlying mechanisms other than interacting with FGFR-1 to boost the anticancer effects of the compounds that were tested, or it might suggest that another kinase enzyme might be involved in the mechanism of action.

Caspase-3 Activity
To further explore the cytotoxicity of the prepared hybrids, their influence on Caspase-3 activity was examined. MDA-MB-231 cells were incubated with the predetermined IC 50 s of tested compounds for a duration of 24 h. The results revealed that all compounds induced Caspase-3 activity in comparison to control cells, with fold change ranging from 1.4 to 2.3 folds ( Figure 5). However, compound 3b showed 2.3-fold concordance between anti-proliferative activity and the Caspase-3 activity, ( Figure 5, p < 0.01).
crystalized ligand of the target protein 7JL7.pdb in cyan. (B) Compound 4bi in green, featuring 2D and 3D interactions with the receptor protein PDB: 7JL7. (C) Compound 4biii in cyan, featuring 2D and 3D interactions with the receptor protein PDB: 7JL7.

FGFR-1 Inhibitory Activity
After analyzing the target prediction results, it was identified that FGFR-1 had th highest probability among kinases; thus, FGFR-1 enzyme assays were conducted. Consid ering potency, selectivity, and affinity percentages, the inhibitory activity of all synthe sized compounds against FGFR-1 was evaluated, and the results are summarized in Tabl 5. There was a wide range of inhibitory potencies, ranging from 131.95 ± 4.3 µM to 325.6 ± 7.4 µM. Compounds 4ai and 4aii had the highest FGFR-1-inhibiting activity amon those tested, with IC50 values of 131.95 ± 4.3 and 119.43 ± 5.6 µM, respectively. However it was challenging to correlate the anticancer effect of the drugs that were examined to th inhibition of FGFR-1 alone owing to the compounds' modest inhibitory action agains FGFR-1. This may imply the presence of alternate underlying mechanisms other than in teracting with FGFR-1 to boost the anticancer effects of the compounds that were tested or it might suggest that another kinase enzyme might be involved in the mechanism o action.

Caspase-3 Activity
To further explore the cytotoxicity of the prepared hybrids, their influence o Caspase-3 activity was examined. MDA-MB-231 cells were incubated with the predeter mined IC50s of tested compounds for a duration of 24 h. The results revealed that all com pounds induced Caspase-3 activity in comparison to control cells, with fold change rang ing from 1.4 to 2.3 folds ( Figure 5). However, compound 3b showed 2.3-fold concordanc between anti-proliferative activity and the Caspase-3 activity, ( Figure 5, p < 0.01).

Annexin V/Propidium Iodide (PI) Flow Cytometric Analysis
Apoptosis and necrosis are two routes that make up the overall division of the cell death pathway carried out by anticancer agents. A double labeling flow cytometry assay using Annexin V-FITC/propidium iodide was performed to identify which mechanism, necrosis, or apoptosis is responsible for cell death [28]. In accordance with the IC 50 values obtained from Table 1, compounds 3b, 4bi, and 4biii were selected to be examined further in respect to their effect on the apoptosis (Figure 6). Among all compounds examined, compound 3b significantly increased the population of late apoptotic cells and necrotic cells 4.8 and 5.5 fold, respectively. In addition to demonstrating that the calculated IC 50 value is indicative of cytotoxic effects with/without moderate antiproliferative activity, this explains the cell killing effects attributed to compound 3b against MDA-MB231 cells.

Annexin V/Propidium Iodide (PI) Flow Cytometric Analysis
Apoptosis and necrosis are two routes that make up the overall division of the cell death pathway carried out by anticancer agents. A double labeling flow cytometry assay using Annexin V-FITC/propidium iodide was performed to identify which mechanism, necrosis, or apoptosis is responsible for cell death [28]. In accordance with the IC50 values obtained from Table 1, compounds 3b, 4bi, and 4biii were selected to be examined further in respect to their effect on the apoptosis (Figure 6). Among all compounds examined, compound 3b significantly increased the population of late apoptotic cells and necrotic cells 4.8 and 5.5 fold, respectively. In addition to demonstrating that the calculated IC50 value is indicative of cytotoxic effects with/without moderate antiproliferative activity, this explains the cell killing effects attributed to compound 3b against MDA-MB231 cells.

Investigation of Drug Likeness and Physicochemical Properties
In silico screening of pharmacokinetic and physicochemical properties was performed on the biologically evaluated thienopyrimidine-sulfonamide hybrids 3b, 4a(i-iii), and 4b(i-iii). All the tested compounds recorded log p < 5, which indicated their high cell membrane tolerability [29]. In addition, the topological polar surface area (TPSA) values range from 146.38 to 196.77. The number of hydrogen bond acceptors (HBAs) < 10 acceptors, while the number of hydrogen bond donors (HBDs) are either two or four, and their molecular weights (MW) are less than 500, which is consistent with Lipinski's rule of five (Table 6).
According to reported literature [30,31], compounds with a positive drug likeness score are considered good drug candidates. As shown in Table 6, all the investigated compounds except 3b expressed positive drug likeness values ranging from 0.73 to 1.58,

Investigation of Drug Likeness and Physicochemical Properties
In silico screening of pharmacokinetic and physicochemical properties was performed on the biologically evaluated thienopyrimidine-sulfonamide hybrids 3b, 4a(i-iii), and 4b(i-iii). All the tested compounds recorded log p < 5, which indicated their high cell membrane tolerability [29]. In addition, the topological polar surface area (TPSA) values range from 146.38 to 196.77. The number of hydrogen bond acceptors (HBAs) < 10 acceptors, while the number of hydrogen bond donors (HBDs) are either two or four, and their molecular weights (MW) are less than 500, which is consistent with Lipinski's rule of five (Table 6).

Pharmacokinetics In Silico Assessment for the Prepared Hybrids
ADMET analysis is especially helpful in simplifying clinical trials, especially in th early stage of drug design. Intestinal absorption, skin sensitization, and oral bioavailabil ity are the absorption parameters considered in drug discovery [33]. An intestinal absorp tion score >30% indicates perfect absorbance. As tabulated in Table 7, all compounds rec orded intestinal absorption of significantly more than 30%, with a minimum of 64% fo compound 4bi and a maximum value of 96% demonstrated by 3b, which reflects an ex cellent absorbance rate. A compound is known to have a relatively low skin permeability if it has log Kp > −2.5 [33]. The prepared compounds revealed good skin permeability with a skin permeability average score around −2.88 cm/h. Compounds are considered to possess high human colon adenocarcinoma (Caco2) permeability when they record Caco values > 0.9 [33]; however, the currently investigated thienopyrimidine sulfonamides hy brids revealed low human colon adenocarcinoma permeability as all compounds showed values < 0.9, except 3b, which showed Caco2 permeability of 1.324.
To investigate the compounds' distribution in silico, volume of distribution (VDss) blood-brain barrier (BBB) membrane permeability, and CNS permeability were assessed With VDss values, a larger distribution volume was observed in compound 4ai, with 0.39 log L/kg (Table 7). Log BB value for compounds will reflect low BBB if <−1 as reported [33]. Among the tested compounds, 3b expressed the best and most promising permea bility of the BBB membrane with a score of 0.184. Log PS values for CNS permeability range from −2.18 to −3.24, and since low CNS permeability is reported if log PS is <−3 [33] 4bi 4ai Figure 7. Illustration of the best drug likeness scores: thienopyrimidine with sulfaguaidineincorporated hybrids (4ai and 4bi).

Pharmacokinetics In Silico Assessment for the Prepared Hybrids
ADMET analysis is especially helpful in simplifying clinical trials, especially in the early stage of drug design. Intestinal absorption, skin sensitization, and oral bioavailability are the absorption parameters considered in drug discovery [33]. An intestinal absorption score >30% indicates perfect absorbance. As tabulated in Table 7, all compounds recorded intestinal absorption of significantly more than 30%, with a minimum of 64% for compound 4bi and a maximum value of 96% demonstrated by 3b, which reflects an excellent absorbance rate. A compound is known to have a relatively low skin permeability if it has log Kp > −2.5 [33]. The prepared compounds revealed good skin permeability, with a skin permeability average score around −2.88 cm/h. Compounds are considered to possess high human colon adenocarcinoma (Caco2) permeability when they record Caco2 values > 0.9 [33]; however, the currently investigated thienopyrimidine sulfonamides hybrids revealed low human colon adenocarcinoma permeability as all compounds showed values < 0.9, except 3b, which showed Caco2 permeability of 1.324.
To investigate the compounds' distribution in silico, volume of distribution (VDss), blood-brain barrier (BBB) membrane permeability, and CNS permeability were assessed. With VDss values, a larger distribution volume was observed in compound 4ai, with 0.397 log L/kg (Table 7). Log BB value for compounds will reflect low BBB if <−1 as reported [33]. Among the tested compounds, 3b expressed the best and most promising permeability of the BBB membrane with a score of 0.184. Log PS values for CNS permeability range from −2.18 to −3.24, and since low CNS permeability is reported if log PS is <−3 [33], compounds 3b, 4bi, and 4bii are considered to have promising CNS permeability, while compounds 4a(i-iii) and 4biii recorded log PS < −3, indicating impenetrability.  Hepatic and renal clearance were used to examine overall drug clearance. Total clearance calculates the drug's concentration in the body utilizing the elimination rate.
Compounds' excretion rate is demonstrated in log(mL/min/kg) in Table 7. The anticipated scores for ADMET analysis are summarized in Table 7.

Chemistry
All NMR analyses were performed with the Bruker magnet system 400 54 Ascend/R (USA) using 400 MHz and 100 MHz for 1 H-NMR and 13 C-NMR. Mass spectrum was performed on Direct Inlet part to mass analyzer in GCMS model with ISQ single quadrupole thermoscientific Electron Impact mode (UK). Melting points (m.p.) were determined using the Stuart scientific melting point apparatus and are uncorrected.
Compounds 1 and 2 were prepared according to the respectively reported procedures in [34] (Scheme 1). Compounds 1a and 1b were prepared following Gewald reaction between sulfur powder, morpholine, ethylcyanoacetate, and either cyclohexanone or ethylacetoacetate to yield 1a and 1b, respectively. Compounds 2a and 2b were obtained upon reacting the aminocarboxylate esters 1a and 1b, respectively, with formamide, which is then chlorinated to the chloride derivatives 3a and 3b using phosphorus oxychloride [21].
General procedures for synthesis of 4a(i-iii) and 4b(i-iii): Equimolar amounts of the chloride derivatives 3a/3b and the appropriate sulfonamide, namely, sulfaguanidine, sulfadiazine, and sulfamethoxazole, were refluxed in glacial acetic acid (15 mL) for 15 h; the reaction mixture was then left to cool at R.T and then poured onto ice water. The formed solid was filtered and crystalized from absolute ethanol to yield 4a(i-iii) and 4b(i-iii) series, respectively.

Selectivity Index (SI)
To determine if the new derivatives have selectivity towards cancer cells, the cytotoxic effect on non-tumorigenic cell line MCF10A was evaluated. The selectivity ratio in noncancerous cells (MCF10A) versus cancer cells (MCF-7 and MDAmb231) was calculated as reported in [26,27] using the following equation: SI = IC 50 of normal cells/IC 50 of cancer cells.

FGFR-1 Inhibitory Activity
To determine basal FGFR-1 activity, a FGFR-1 ELISA Kit was used according to the protocol of the manufacturer (G-Biosciences, Saint Louis, MO, USA). In brief, MDA-MB-231 cell lysates (100 ng/mL) were diluted and added to the Eliza plate. After 90 min of incubation at 37 • C, 100 µL Biotinylated Detection Antibody was added and incubated for 60 min. After washing twice each with washing buffer, 100 µL ELISA Detection Reagent was added to each well and incubated for 30 min. After washing, 90 µL of Detection Substrate (TMB) was added to visualize the enzymatic reaction. TMB was catalyzed to produce a blue color that changed into yellow after adding 50 µL of acidic stop solution. The density of yellow is directly proportional to the concentration of FGFR-1 captured on the plate using a Varioskan TM LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA) [36].

Caspase-3 Activity Assay
Caspase-3 activity was assessed with a colorimetric assay based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by Caspase-3 and releasing p-nitroaniline (pNA). MDA-MB-231 cell lysates (100 ng/mL) were diluted and subjected to analysis via Caspase-3 Colorimetric Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA) based on the protocol of the manufacturer. The reaction for color development was held at 37 • C for 2 h, and the value of OD405 was determined using a Varioskan TM LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA). Each caspase-3 activity was expressed as a value of OD405.

Evaluation of Apoptosis by Annexine V
Apoptosis was detected after 48 h of cell culture using V-FITC apoptosis detection kit (BD Pharmingen TM, Franklin Lakes, NJ, USA). MDA-MB-231 cells were treated with the predetermined IC 50 s of the most potent compounds for 24 h, and the untreated group was included as a control group. After treatment, cells were collected, washed with ice-cold PBS and centrifuged at 200× g for 5 min. The cell pellet was suspended in 200 µL of annexin V-FITC/PI solution for 15 min in the dark. The stained and Annexine V propidium iodide negative cells were evaluated via CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA). Ten thousand cells (gated events) were captured for each sample. The mean fluorescence intensity was analyzed with Cytoexpert (Beckman Coulter).

Target Prediction
Scanning for targets was performed using Swiss Target [37]; smiles were copied to the free available software accessed on 6 September 2022, where calculations were run for target prediction and indications.

Molecular Modeling
Modeling studies were performed with the aid of molecular operating environment MOE [38] and Discovery Studio 2021 software v21.1.0.20298. Two proteins were downloaded for modeling study from the protein data bank (pdb:5O49 and 7JL7). The crystal structure FGFR-1 bound to its co crystallized ligand was downloaded from Protein Data Bank (PDB code:5O49), as was caspase-3 with its co-ligand at chain F [39,40]. All compound structures were built on MOE builder, corrected, energy minimized, and saved as mol2 format. Induced fit was the selected protocol. Ligand was set as site of docking in caspase-3, while dummy atoms were set as a placement guide for FGFR-1 docking. The gradient for energy minimization was set as default at 0.05, and the applied force field was also applied as default MMFF94X.

Statistical Analysis
The data were analyzed using Prism ® for Windows, ver. 9.00 (GraphPad Sofware Inc., La Jolla, CA, USA) and presented as mean ± S.D. The statistical tool used for testing the significance in this study was Analysis of Variance (ANOVA) with the Least Significant Difference (LSD) post hoc test. The software used for the analysis was SPSS ® for Windows, version 17.

Conclusions
This work describes the biological evaluation of new synthesized hybrids of thienopyrimidine and sulfonamides. Docking studies were applied to calculate the binding affinity scores of the synthesized hybrids to FGFR-1 enzymes and caspase-3. All compounds showed promising binding affinity to the FGFR-1 target protein, ranging from −7.123 to −5.239, whereas binding affinity to Caspase-3 ranged from −6.476 to −4.059. The cytotoxic effect of the synthesized compounds was estimated against two human breast cancer cell lines (MCF-7 and MDA-MB231) using Doxorubicin as a reference. All the tested compounds exhibited moderate to excellent anticancer efficacy against both tested cell lines, among which 3b and 4bi were the best. In MCF-7 cells, compound 4bi displayed the highest cytotoxic activity among all the tested compounds, with IC 50 of 6.17 ± 1.3 µM, while compound 3b recorded the lowest IC 50 value against MDA-MB-231 cells with IC50 of 4.45 ± 0.31 µM. For further exploration of the anticancer activity of the tested compounds, their selectivity was examined through calculating their selectivity index using normal MCF-10A cells. All the synthesized compounds exhibited distinguishing selectivity index values greater than the Doxorubicin. To further dissect the cytotoxic effects of the compounds under investigations, the influence of these compounds on both FGFR-1 and Caspase-3 activity was examined. The results revealed that compound 3b showed concordance between anti-proliferative activity and Caspase-3 activity. In respect to compounds' effect on apoptosis, compound 3b significantly increased the population of late apoptotic cells and necrotic cells by 4.8 and 5.5 times, respectively. Structural changes produced a distinct difference in cytotoxicity. Three derivatives from the series "b" structure, 3b, 4bi, and 4biii, all have IC 50 values below 10 µM, which shows how effectively they can block angiogenesis and the proliferation of breast cancer cells. The absence of a cyclohexyl ring and its replacement with a carboxylate (open side chain) may contribute to the anticancer activity of compounds from series b. In silico pharmacokinetic assessment revealed that all compounds had no violations to Lipinski's rule. Drug likeness investigation showed that compounds 4a(i-iii) and 4b(i-iii) are drug-like, in particular compound 4ai, which has a guanidine group engaged with the thienopyrimidine core that recorded the highest positive score of 1.58.