2-Alkyl-Substituted-4-Amino-Thieno[2,3-d]Pyrimidines: Anti-Proliferative Properties to In Vitro Breast Cancer Models

Thienopyrimidines are structural analogs of quinazolines, and the creation of new 2-alkyl derivatives of ethyl 4-aminothienopyrimidine-6-carboxylates for the study of their anti-proliferative properties is of great pharmacological interest. Some 2-alkyl-4-amino-thieno[2,3-d]pyrimidines 2–5 were synthesized, and their cyto- and phototoxicity against BALB 3T3 cells were established by an in vitro 3T3 NRU test. The obtained results indicate that the tested compounds are not cytotoxic or phototoxic, and that they are appropriate to be studied for their anti-proliferative and anti-tumor properties. The anti-proliferative potential of the compounds was investigated on MCF-7 and MDA-MB-231 cancer cells, as well as a MCF-10A cell line (normal human mammary epithelial cells). The most toxic to MCF-7 was thienopyrimidine 3 with IC50 13.42 μg/mL (IC50 0.045 μM), followed by compound 4 (IC50 28.89 μg/mL or IC50 0.11 μM). The thienopyrimidine 4 revealed higher selectivity to MCF-7 and lower activity (IC50 367 μg/mL i.e., 1.4 μM) than compound 3 with MCF-10A cells. With respect to MDA-MB-231 cells, ester 2 manifested the highest effect with IC50 52.56 μg/mL (IC50 0.16 μM), and 2-ethyl derivative 4 revealed IC50 62.86 μg/mL (IC50 0.24 μM). It was estimated that the effect of the substances on the cell cycle progression was due to cell cycle arrest in the G2 stage for MDA-MB-231, while arrest in G1 was detected for the estrogen (ER)-positive MCF-7 cell line. The tested compound’s effects on the change of the zeta potential in the tumorigenic cells utilized in this study were determined. The calculation which we performed of the physicochemical properties and pharmacokinetic parameters influencing the biological activity suggested high intestinal absorption, as well as drug-likeness.


Introduction
Cancer is a disease caused by mutations in certain genes, and, according to the WHO, it is the second leading cause of death worldwide.The number of cancer-related deaths is expected to reach 11.5 million in 2030.Although significant advances have been made in the treatment of cancer, side effects and drug resistance remain unsolvable problems in the fight against cancer.Therefore, selective and effective chemotherapeutic agents for cancer treatment are still being sought [1][2][3].
Breast cancer, in particular, is the most prevalent cause of cancer death in women, accounting for 23% of all cancers and 14% of cancer deaths.Based on these concerning data, it should be noted that research in this area is important not only for the discovery of new drugs and methods of treatment, but also to overcome the psychological load.The isolation of MCF-7 cells (Michigan Cancer Fondation-7) from pleural effusion in the 1970s led to the formation of the MCF-7 cell line, commonly used in many studies [4,5].The MDA-MB-231 (M.D. Anderson-Metastatic Breast 231) cell line is a highly aggressive epithelial human breast cancer cell line, which is also extracted from pleural effusion of a female with mammary adenocarcinoma.This cell line has served as a basis for many investigations dedicated to the creation of new anti-cancer agents [6].Depending on the breast cancer type, variable therapy treatments can be used, such as immunotherapy, targeted drug therapy, and hormone receptor-positive breast cancer therapy.The list of the utilized clinic drugs was significantly expanded in the last decade.Nevertheless, active research to discover additional specific targets, as well as new chemotherapeutics, is going on [7][8][9].
The significance of the rational drug design is the basis of many studies aiming to generate novel, small, drug-like molecules.After the development of 4-aminoquinazolines lapatinib, gefitinib, and trastuzumab as anti-breast-cancer agents, a number of thienopyrimidines were synthesized as bioisosteres in order to study their anti-proliferative properties [10,11].Structural combinations of chalcone with thieno [2,3-d]pyrimidinone scaffolds were generated, and their inhibitory activity against MCF-7 cells was proven.Some of the compounds showed anti-cancer and anti-proliferative activities at the G1 phase against MCF-7 cells, which were comparable to those of uracil [12].2,3-Disubstituted thieno [2,3-d]pyrimidin-4(3H)-ones prepared through one-pot synthesis were reported to inhibit the proliferation of A549 and MCF-7 cells [13].A series of 2-trifluoromethylthieno [2,3d]pyrimidine derivatives were designed and screened for their anti-tumor effects on MCF-7 and HepG2.Within the series, some derivatives exhibited higher activity than Gefitinib, a selective inhibitor of epidermal growth factor receptor's (EGFR) tyrosine kinase [14,15].
Despite the results which have been achieved, no medicaments effective enough for the treatment of breast cancer have been developed yet.That is why the creation of new small molecules with good efficiency is of great importance.As can be seen from Figure 1, the thienopyrimidines can be considered as bioisosteres of the biological nitrogenous bases uracil and adenosine.
Despite the results which have been achieved, no medicaments effective enough for the treatment of breast cancer have been developed yet.That is why the creation of new small molecules with good efficiency is of great importance.As can be seen from Figure 1, the thienopyrimidines can be considered as bioisosteres of the biological nitrogenous bases uracil and adenosine.Based on the aforementioned properties of thienopyrimidines, we focused our study on the synthesis of some ethyl 2-alkyl-substituted-thienopyrimidine carboxylates through structure modification of derivatives A and B (Figure 2) in order to evaluate their effects against MCF-7 and MDA-MB-231 cancer cells.The choice of these structures is of pharmacological interest, because thienopyrimidines containing an amino group in the 4th and an alkyl fragment in the 2nd position (C) are less studied than the relevant thieno [2,3-d]pyrimidines A and B. Furthermore, these substituents occur in some target breast cancer inhibitors.The current study is a continuation of our previously reported investigations [30][31][32].Herein, we also report the cytotoxicity and phototoxicity of the tested compounds against BAL3 cells, as well as the substance effects on the cell cycle progression of ER-negative MDA-MB-231 and ER-positive MCF-7 cell lines.Based on the aforementioned properties of thienopyrimidines, we focused our study on the synthesis of some ethyl 2-alkyl-substituted-thienopyrimidine carboxylates through structure modification of derivatives A and B (Figure 2) in order to evaluate their effects against MCF-7 and MDA-MB-231 cancer cells.The choice of these structures is of pharmacological interest, because thienopyrimidines containing an amino group in the 4th and an alkyl fragment in the 2nd position (C) are less studied than the relevant thieno [2,3-d]pyrimidines A and B. Furthermore, these substituents occur in some target breast cancer inhibitors.The current study is a continuation of our previously reported investigations [30][31][32].Herein, we also report the cytotoxicity and phototoxicity of the tested compounds against BAL3 cells, as well as the substance effects on the cell cycle progression of ER-negative MDA-MB-231 and ER-positive MCF-7 cell lines.

Chemistry
As mentioned above, the aim of the study was to generate hybrid molecules introducing an amino group in the 4th position of the thieno [2,4-d] Ethyl 2-amino-3-cyano-4-methyl-thiophene-5-carbioxilate 1 was synthesiz according to the Gewald reaction by means of ethyl acetoacetate, malononitrile, and sul in ethanol medium.Diethyl amine was added drop-wise as a catalyst by stirring at ambient temperature [33][34][35].The obtained tetra-substituted thiophene served a precursor for cyclocondensation with different nitriles.The process leading to the tar 4-amino-thieno [2,3-d]pyrimidines was conducted under acidic conditions, accomplish

Chemistry
As mentioned above, the aim of the study was to generate hybrid molecules by introducing an amino group in the 4th position of the thieno [2,4-d]pyrimidine nucleus, as well as to induce ethoxycarbonyl and methyl groups in the thiophene ring instead of the fused tetrahydrobenzene fragment.The aryl substituents at the 2nd position were replaced with 2-ethoxy-2-oxoethylene, 2-chloroethyl, ethyl, and methyl groups.

Chemistry
As mentioned above, the aim of the study was to generate hybrid molecules by introducing an amino group in the 4th position of the thieno [2,4-d]pyrimidine nucleus, as well as to induce ethoxycarbonyl and methyl groups in the thiophene ring instead of the fused tetrahydrobenzene fragment.The aryl substituents at the 2nd position were replaced with 2-ethoxy-2-oxoethylene, 2-chloroethyl, ethyl, and methyl groups.2-Alkylsubstituted-4-amino-thieno [2,3-d]pyrimidines were synthesized as shown in Scheme 1. Ethyl 2-amino-3-cyano-4-methyl-thiophene-5-carbioxilate 1 was synthesized according to the Gewald reaction by means of ethyl acetoacetate, malononitrile, and sulfur in ethanol medium.Diethyl amine was added drop-wise as a catalyst by stirring at an ambient temperature [33][34][35].The obtained tetra-substituted thiophene served as a precursor for cyclocondensation with different nitriles.The process leading to the target 4-amino-thieno [2,3-d]pyrimidines was conducted under acidic conditions, accomplished Ethyl 2-amino-3-cyano-4-methyl-thiophene-5-carbioxilate 1 was synthesized according to the Gewald reaction by means of ethyl acetoacetate, malononitrile, and sulfur in ethanol medium.Diethyl amine was added drop-wise as a catalyst by stirring at an ambient temperature [33][34][35].The obtained tetra-substituted thiophene served as a precursor for cyclocondensation with different nitriles.The process leading to the target 4-aminothieno [2,3-d]pyrimidines was conducted under acidic conditions, accomplished by passing dry hydrogen chloride gas through the reaction mixture.The structures of thienopyrimidines 2-5 were identified through IR, 1 H-NMR spectra, and elemental analysis.The stretching vibration of the ester groups in the thiophene ring gave rise to IR bands within the range of 1722-1711 cm −1 .In the 1 H NMR spectra, the signals of the thiophen ester group -OCH 2 CH 3 appeared in the form of a triplet at 1.3-1.4ppm for the -CH 3 group and a quartet at 4.0-4.4ppm for the -CH 2 -group.The signals of the -CH 3 group attached to the thiophen ring were detected as singlets at 2.85-2.87ppm.In the 13 C NMR spectra, the thiophen ester group -OCH 2 CH 3 was characterized by signals at 61-62 ppm (for the -CH 2 -) and ca.14 ppm (for the -CH 3 ), while the signals for the methyl group at 5-position were observed at ca. 15 ppm.The differentiation of the methylene and methyl signals was clearly visible from the DEPT (distortions enhancement by polarization transfer) NMR spectra (Figures S5d-S8d in the Supplementary Material).The different combinations of methyl/methylene groups of the substituents in the pyrimidine rings also had very characteristic patterns: the methylene groups resonated within the range of 32-42 ppm, and the methyl groups of compounds 4 and 5 within a broader range, i.e., 12-22 ppm, respectively.The assignment of the H and C signals was confirmed by heteronuclear single quantum correlation (HSQC) spectra (Figures S5e-S8e in the Supplementary Material).The assignment of the NMR signals was also supported by the prediction of NMR isotopic shielding by B3LYP/6-311++G** calculations in DMSO solvent (GIAO method) for all compounds (Tables S1-S4 in the Supplementary Material).The comparison between the calculated and experimental data demonstrated that C5 in the thiophen ring (bearing the methyl group) was characterized by a signal at ca. 140 ppm; C6 and βC by signals in the interval of 114-120 ppm; and C2, C4, αC, and the C-atom from the carbonyl group by signals in the interval of 160-169 ppm.The signals for the amino group protons were detected at ca. 7.3-7.4ppm only in the spectra of compounds 2 and 4, while in the spectra of 3 and 4, they were lacking, most probably due to an exchange of protons with the water present in the solvent.Nevertheless, the presence of the amino group in all four compounds was unambiguously confirmed by the two IR absorption bands for N-H stretching vibrations in the ranges of 3490-3400 cm −1 and 3375-3280 cm −1 , as well as the band for scissoring vibration of the amino group at ca. 1640-1660 cm −1 , which was found in the IR spectra.

Biological Study 2.2.1. In Vitro Safety Test
Compounds 2-5 were studied for cytotoxicity/phototoxicity by an in vitro 3T3 NRUtest.The cell line BALB 3T3, clone A31 (mouse embryo fibroblasts), was incubated with the test compounds at a concentration from 15 to 4000 µg/mL for 24 h at 37 • C, 5% CO 2 , and 95% humidity.The cytotoxicity and phototoxicity were expressed in percentages relative to the negative control determined.Dose-response dependence was observed for all of the tested thienopyrimidines.The obtained results are shown in Figure 3. Based on the dose-response curves, the CC 50 /PC 50 values (50% cytotoxic/phototoxic concentration) were calculated by nonlinear regression analysis (Table 1).The CC 50 values can be used to calculate the PIF (photo-irritancy factor) for each test compound according to the following formula: PIF = CC 50 /PC 50 .The PIF demonstrates the probability that the test compound may cause a phototoxic effect (PIF < 2: not phototoxic; PIF ≥ 2 < 5: probable phototoxicity; PIF ≥ 5: phototoxic).The PIF < 2 obtained for all tested compounds shows a high level of photo safety.
In our study, as a positive control for phototoxicity, acridine orange (AO) was used.These results show that compounds 2-5 are safe for use in pharmaceuticals.BALB 3T3 cells have the ability to divide indefinitely, but they are extremely sensitive to inhibition of cell division after fusion and highly susceptible to transformation.Therefore, they are widely used in carcinogenesis studies.BALB 3T3 cells have the ability to divide indefinitely, but they are extremely sensitive to inhibition of cell division after fusion and highly susceptible to transformation.Therefore, they are widely used in carcinogenesis studies.

Anti-Proliferative Effects
As can be seen from the data in Table 1, compounds 2-5 revealed neither cytotoxicity nor phototoxicity; thus, they were suitable to be screened for their anti-proliferative and anti-tumor properties using an MTT-test.The MCF-10A cell line (normal human mammary epithelial cells) served as a reference control.
All studied compounds showed lower anti-proliferative activity against MCF-10A in comparison to the manifested cytotoxicity of MCF-7 and MDA-MB-231, see Table 2 below.As the selectivity of anti-cancer agents plays a significant role in the treatment of cancer diseases, the selectivity index (SI) of the compounds was calculated and results are shown in Table 3.The MTT test results showed that the most toxic to MCF-7 was thienopyrimidine 3, with an IC50 of 13.42 µg/mL (IC50 0.045 µM, respectively), followed by compound 4 (IC50 28.89 µg/mL or IC50 0.11 µM).At the same time, with regard to MCF-

Anti-Proliferative Effects
As can be seen from the data in Table 1, compounds 2-5 revealed neither cytotoxicity nor phototoxicity; thus, they were suitable to be screened for their anti-proliferative and anti-tumor properties using an MTT-test.The MCF-10A cell line (normal human mammary epithelial cells) served as a reference control.
All studied compounds showed lower anti-proliferative activity against MCF-10A in comparison to the manifested cytotoxicity of MCF-7 and MDA-MB-231, see Table 2 below.As the selectivity of anti-cancer agents plays a significant role in the treatment of cancer diseases, the selectivity index (SI) of the compounds was calculated and results are shown in Table 3.The MTT test results showed that the most toxic to MCF-7 was thienopyrimidine 3, with an IC 50 of 13.42 µg/mL (IC 50 0.045 µM, respectively), followed by compound 4 (IC 50 28.89µg/mL or IC 50 0.11 µM).At the same time, with regard to MCF-10A, compound 4 demonstrated a cytotoxicity level eight times lower than compound 3.Moreover, the ethyl substituted thienopyrimidine 4 had a selectivity index (SI = 12.7) greater than that of the 2-chloroethyl-substituted compound 3, confirming compound 4 as a lead structure.Compound 5 demonstrated similar properties, which resulted in the highest SI.The calculated selective index in the positive control (Cisplatin) was SI = 25.10A, compound 4 demonstrated a cytotoxicity level eight times lower than compound 3.Moreover, the ethyl substituted thienopyrimidine 4 had a selectivity index (SI = 12.7) greater than that of the 2-chloroethyl-substituted compound 3, confirming compound 4 as a lead structure.Compound 5 demonstrated similar properties, which resulted in the highest SI.The calculated selective index in the positive control (Cisplatin) was SI = 25.

Cell Cycle Arrest
Our study continued with the examination of the effect of the newly synthesized compounds' IC 50 values on cell cycle progression.The cell aliquots were collected, and FACS analyses were used to detect the effects of cell treatments with the selected compounds (2, 4, and 5).A propidium iodide staining assay was performed.The results are presented in Figure 5.

Cell Cycle Arrest
Our study continued with the examination of the effect of the newly synthesized compounds' IC50 values on cell cycle progression.The cell aliquots were collected, and FACS analyses were used to detect the effects of cell treatments with the selected compounds (2, 4, and 5).A propidium iodide staining assay was performed.The results are presented in Figure 5.The changes in the cell cycle distribution in % (G1, S, and G2 phases), based on the graphical figures A-C, were calculated and are given in Table 4.The changes in the cell cycle distribution in % (G1, S, and G2 phases), based on the graphical figures A-C, were calculated and are given in Table 4.As shown in Table 4, 40.41% of the untreated MCF-7 cells were in the G1 phase of the cell cycle.The G1 phase occurred between the M and S phases, i.e., the stage in which cells are preparing to divide.After treatments, increases of 11%, 20%, and 20.5%, respectively, compared to the control were detected in this phase.Regarding the highly metastatic breast cancer cell line MDA-MB-231, 4.38% of the cells of the untreated control were in phase G2, a period of rapid cell growth and protein synthesis during which the cell prepares itself for mitosis.Dramatic increases in the percentage of cells in this phase were observed after the treatments, i.e., 53.65%, 44.9%, and 45.9%, which suggested the existence of a block at the G2/M phase of the cell cycle.The data presented in Table 4 demonstrate that the tested compounds have different effects on estrogen (ER)-positive and -negative breast cancer lines.The inhibitory effect on cell proliferation occurred due to cell cycle arrest in the G2 stage for the ER-negative MDA-MB-231, while arrest in G1 was detected for the ER-positive MCF-7 cell line.

ζ-Potential Measurement
Surface electrical charge is specific for each cell type, and is especially important in malignant cells because it determines their state of aggregation and adhesion in the organs.Nearly all metabolically active cancer cells are known to secrete a large amount of lactate as mobile anions.The increased levels of glucose uptake and lactate secretion could be up to 30 times those of normal cells, which is the basis for cancer-detection technologies such as PET (positron emission tomography) scan diagnostics.Cancer cells may have slightly elevated surface contents of negatively-charged immobilized molecules (e.g., sialic acid), i.e., 30-50% more than normal cells [33][34][35][36][37][38][39].
The treatment of tumorigenic (MCF-7, MDA-MB-231) and non-tumorigenic (MCF-10A) cell lines with compounds 2, 4, and 5 demonstrated that these compounds were able to affect their ζ-potential, as shown in Figure 6.First, the ζ-potential of each cell line was measured, and all obtained values were negative (Figure 6A).The Student t-test statistical analysis which was applied distinguished the ζ-potentials of the three studied cell lines with p < 0.05, and the values followed the order of MCF-7 > MDA-MB-231 > MCF-10A.The cells treated with compounds 2, 4, and 5 exhibited more negative ζpotential values.Figure 5B  The ζ-potential of all studied cell lines was negative: MCF-7 > MDA-MB-231 > MCF-10A (Figure 6), which is consistent with the data obtained for anti-proliferative activity (Tables 3-5).The investigated compounds 2, 3, and 5 showed lower anti-proliferative activity against MCF-10A in comparison to the manifested cytotoxicity against the tumorigenic MCF-7 and MDA-MB-231 cell lines.As mentioned above, cancer cells have a higher zeta potential due to the negatively charged mobile lactate products in elevated glycolysis.After treatment with compounds 2, 4, and 5, the changes in the zeta potential were not of the same rank.MCF-10A showed higher electrical potential compared to the metastatic cells.One reason for this surprising effect could be the higher percentage of mitotic cells, as can be seen in Table 4, where the surface area is nearly double.The dielectric properties of a cell are determined by the distribution of surface charges, cell size, and morphology, as well as the conductivity and permittivity of their membranes, and could also affect the zeta potential.Henslee et al. have shown that the average radii (µm) are MCF-10A 9.25, MCF-7 9.1, and MDA-MB-231 8.93 with membrane capacitance (F/m 2 ) of MCF-10A 0.0194, MCF-7 0.0186, and MDA-MB-231 0.0163.This describes the specificity of the dipolophoretic electric properties of breast cancer models [4,39].On the other hand, cell membranes' electrical charges are also affected by the sialic acid present in glycolipids and glycoproteins [38,40], as well as the different number of microvili on the cell surface [39,41].Therefore, it could be speculated that the changes occurring after treatment with the three thienopyrimidines are related to the altered sialic acid, the synthesis of more microvilli rich in lipid rafts, and the externalization of a higher content of negatively charged phosphatidyl serine from the inner to the outer part of the bilayer membrane.The fact that surface electrical properties also depend on phospholipid metabolism should not be overlooked.The membrane of MCF-10A is more ordered in comparison to the tumorigenic MCF-7 and MDA-MB-231, which has been confirmed by reports in the literature that the membranes of malignant cells are more fluid compared to the membranes of non-cancerous cells.The highly metastatic cell line MDA-MB-231, however, showed a higher order compared to MCF-7, possibly due to the higher cholesterol content of the highly metastatic cell line [40,42,43].
treatment with the three thienopyrimidines are related to the altered sialic acid, the synthesis of more microvilli rich in lipid rafts, and the externalization of a higher content of negatively charged phosphatidyl serine from the inner to the outer part of the bilayer membrane.The fact that surface electrical properties also depend on phospholipid metabolism should not be overlooked.The membrane of MCF-10A is more ordered in comparison to the tumorigenic MCF-7 and MDA-MB-231, which has been confirmed by reports in the literature that the membranes of malignant cells are more fluid compared to the membranes of non-cancerous cells.The highly metastatic cell line MDA-MB-231, however, showed a higher order compared to MCF-7, possibly due to the higher cholesterol content of the highly metastatic cell line [40,42,43].As a conclusion, regarding the changes in the zeta potential when treated with 2alkyl-substituted-4-amino-thieno[2,3-d]pyrimidines 2, 4, and 5, it can be suggested that the observed changes are not only related to the effects caused by the anti-proliferative activity of the aforementioned compounds.It could be further assumed that the specific physico-chemical features of the three types of cells with different metastatic potential,  As a conclusion, regarding the changes in the zeta potential when treated with 2alkyl-substituted-4-amino-thieno[2,3-d]pyrimidines 2, 4, and 5, it can be suggested that the observed changes are not only related to the effects caused by the anti-proliferative activity of the aforementioned compounds.It could be further assumed that the specific physico-chemical features of the three types of cells with different metastatic potential, such as different dielectrophoretic potential, phospholipid composition, microvilli, or cell cycle distribution, play an additional role in the surface electrical properties of cells after compound treatment (Figure 6).

Physico-Chemical Properties and Drug-Likeness Analysis
An important goal of drug research is to gain sufficient understanding of the molecular properties that limit oral bioavailability in order to facilitate the design of viable new drug candidates.Lipophilicity and the number of hydrogen bond donors appear to be key properties, as they remain essentially constant in oral medications over time.The number of H-donors and H-acceptors affects the polar surface of the molecule and reduces the rate of its membrane penetration, which correlates with the bioavailability.Therefore, it is important to calculate the lipophilicity and the physico-chemical parameters of the studied compounds which affect the absorption, distribution, metabolism, excretion, and toxicological properties.The physico-chemical properties of the compounds given in Table 5 were generated using the Molinspiration software, v2013.09[41,44].The descriptors of the synthesized ligands (Table 5) are consistent with Lipinski's rule of five, without any violations [42,45].Equally important to the characterization of a ligand as a drug candidate is the determination of ligand efficiency (LE).LE is related to the Gibbs free energy of binding for a heavy atom, and may serve to establish the preferred pharmacophores of the leads.The calculation of LE can be performed using the equation given below (1) [43][44][45][46][47][48][49]: Ligand lipophilicity efficiency (LLE), also known as lipophilic efficacy (LipE), is related to the possibility of transferring the ligand from 1-octanol to the active site of binding.It has been proposed as a better alternative to LE to capture the enthalpic component of ligand binding [47,48,50,51]: Since lipophilicity and effectiveness play important roles in the development of new drug candidates, it is essential that their values be greater than 0.3 and 3, respectively [49,52].As can be seen, the LE and LLE values of compounds 2-5, given in Table 5, are higher than 0.3 and 3.
The absorption of a drug intended for oral intake depends on its dissolution, i.e., on the solubility of the chemical compound.For this reason, solubility is another important parameter that should be considered when describing biological properties and bioavailability, in particular.SwissADME [50,53] was used to calculate the possible solubility [51][52][53][54][55][56] and to determine the drug-likeness of the studied compounds.The obtained results regarding the solubility of compounds 2-5 are given in Table 6.Log S (ESOL−Estimated SOLubility) is a method for the estimating the aqueous solubility of a compound directly from its structure [51,54].Log S (Ali) solubility is calculated by an alternative model for predicting aqueous solubility by incorporating TPSA [52,55].Both methods differ from the basic solubility equation, avoiding the melting point parameter.
The third method for the establishment of solubility was developed by SILICOS-IT.The linear correlation coefficient was corrected by molecular weight, and its value was R2 = 0.75 [54,57].The results obtained by the use of the Ali method revealed that all compounds exhibited moderate solubility.The calculations by the other two methods determined the compounds as soluble, except for compound 3.These results are a sign that the compounds can be considered as orally applicable candidates.
The pharmacokinetic properties of a drug include the relationship between its absorption, distribution, and inactivation.The passage of drugs across cell membranes is a key part of most pharmacokinetic processes.The most important route by which a drug crosses cell membranes is passive diffusion, the rate of which is determined by molecular size, concentration gradient, lipid solubility, degree of drug ionization, and protein binding.The calculated results, given in Table 7, indicate that thienopyrimidines 2-5 could exhibit high gastrointestinal absorption.
According to the calculation, a high gastrointestinal absorption (GI) was characteristic of all compounds, although they may not pass the blood-brain barrier (BBB).On the other hand, the compounds were predicted not to act as substrates of Pg (protein that pumps substances out of cells), which is beneficial, as if they were to enter the central nervous system, they would not be eliminated by the Pg protein.The studied thienopyrimidines might behave as CYP (cytochrome P450 superfamily) substrates/inhibitors towards 1A2, 2C19, and 2C9, but not to members of the 2C6 and 2C4 CYP families.The data related to drug-likeness correspond to five rules, bearing the names of their authors.For Ghose et al. [54,57], the calculated logP should be between −0.4 and 5.6, with an average value of 2.52.For molecular weight, the qualifying range is between 160 and 480, with an average value of 357.For molar refractivity, the qualifying range is between 40 and 130, with an average value of 97.According to Egan's rule, a compound demonstrates good bioavailability when its TPSA value is within the range of 0 ≥ tPSA ≤ 132 Å2 and −1 ≥ logP ≤ 6 [55,58].In researching the molecular properties affecting oral bioavailability, Veber et al. concluded that the polar surface area (PSA) should be ≤140 Å2, while a number of rotatable bonds ≤ 10 or a sum of H-bond donors and acceptors ≤ 12 are sufficient and selective criteria for determining a drug candidate [56,59].The results in Table 5 show that the physico-chemical characteristics of the compounds were in accordance with the aforementioned rules.Muegge's rule is based on the so-called pharmacophore point filter, the role of which is to distinguish substances that are drug-like and those that are not, which is based on simple structural rules.To pass filtering, a candidate drug must receive two to seven pharmacophore points [57,60].For compounds with a carboxylic acid, amine, amidine, or guanidine pharmacophore point, the survival threshold is lowered to one.
The compounds which pass the PAINS (pan-assay interference compounds) filter or pan-assay interference compounds are prominent sources of false positives in the drugdiscovery process [61].
The bioavailability radar in Figure 7 is a graphical depiction of the drug-likenesses of compounds 2-5.
As can be seen from Figure 7, the values of the lipophilicity, size, polarity, solubility, saturation, and flexibility of compounds 2-5, summarized in the SwissADME radar of bioavailability, support that the compounds meet the requirements for drug likeness.As can be seen from Figure 7, the values of the lipophilicity, size, polarity, solubility, saturation, and flexibility of compounds 2-5, summarized in the SwissADME radar of bioavailability, support that the compounds meet the requirements for drug likeness.
Bioactive molecules exert their action by interacting with specific target proteins or other macromolecules.The prediction of these targets restricts their numbers, thus contributing to the faster and more relevant study of the biological properties of a molecule [59,62].It would be expected that thienopyrimidine 4 would reveal its action by interacting with kinase enzymes (Figure 8).A kinase is a type of enzyme that is involved Bioactive molecules exert their action by interacting with specific target proteins or other macromolecules.The prediction of these targets restricts their numbers, thus contributing to the faster and more relevant study of the biological properties of a molecule [59,62].It would be expected that thienopyrimidine 4 would reveal its action by interacting with kinase enzymes (Figure 8).A kinase is a type of enzyme that is involved in a number of cellular processes.Some kinases are thought to cause cancer.The obtained results regarding the anti-proliferative activity of compound 4 against MCF-7 and MDA-MB-231 showed lower cytotoxicity compared to the most active compound of series B (Ar = Phen, Figure 2), but thienopyrimidine 4 had lower cytotoxicity compared to BALB 3T3.On the other hand, this compound showed a higher selective action index towards the investigated cancer cells.By performing SwissАdme calculations, it was found that the parent compound exhibited poor solubility and a XLOGP3 > 3.5, which was a violation of the criterion for lead-likeness.

Materials and Methods
For the synthesis of the target compounds, the following commercial products were used without further purification: ethyl acetoacetate, chloropropionitrile (Merck, Rahway, NJ, USA), ethyl cyanoacetate, acetonitrile, propanonitrile, and malononitrile (Alpha Aesar, Karlsruhe, Germany).All inorganic substances and organic solvents utilized were pure for synthesis or analysis (Macron, Merck).
Melting points were determined as the phase transition, from solid to liquid, at atmospheric pressure on a Boetius PHMK 5 microscope heating table, in degrees Celsius ±1.0 °C.The purity levels of all the obtained compounds, as well as the retention coefficients (Rf), were estimated on F254 or Al2O3 60 silica gel plates (Merck, 0.2 mm).IR spectra were measured using KBr tablets on a Varian Scimitar 1000 spectrophotometer, or by the ATR technique on a Bruker Equinox 55 spectrophotometer.All 1 H-NMR spectra were recorded in DMSO-d6 solvent on a Bruker NEO 400 spectrometer (Bruker, Faelanden, Switzerland).Chemical shifts were expressed to tetramethylsilane (TMS) and were represented in δ (ppm).To a solution of 5.5 mL (6.6 g, 0.1 mol) malonodinitrile and 12.7 mL (13.01 g, 0.1 mol) ethyl acetoacetate in 30 mL ethanol, 3.2 g sulfur was added.Via cooling and vigorous stirring, an equimolar amount of the catalyst (diethylamine, 0.1 mol) was dripped for 30 min.The reaction mixture was stirred for a further 75 min, the solution was cooled, and the amino-thiophene was crystallized.The precipitate was filtered off, washed thoroughly The obtained results regarding the anti-proliferative activity of compound 4 against MCF-7 and MDA-MB-231 showed lower cytotoxicity compared to the most active compound of series B (Ar = Phen, Figure 2), but thienopyrimidine 4 had lower cytotoxicity compared to BALB 3T3.On the other hand, this compound showed a higher selective action index towards the investigated cancer cells.By performing SwissAdme calculations, it was found that the parent compound exhibited poor solubility and a XLOGP3 > 3.5, which was a violation of the criterion for lead-likeness.

Materials and Methods
For the synthesis of the target compounds, the following commercial products were used without further purification: ethyl acetoacetate, chloropropionitrile (Merck, Rahway, NJ, USA), ethyl cyanoacetate, acetonitrile, propanonitrile, and malononitrile (Alpha Aesar, Karlsruhe, Germany).All inorganic substances and organic solvents utilized were pure for synthesis or analysis (Macron, Merck).
Melting points were determined as the phase transition, from solid to liquid, at atmospheric pressure on a Boetius PHMK 5 microscope heating table, in degrees Celsius ±1.0 • C. The purity levels of all the obtained compounds, as well as the retention coefficients (Rf), were estimated on F254 or Al 2 O 3 60 silica gel plates (Merck, 0.2 mm).IR spectra were measured using KBr tablets on a Varian Scimitar 1000 spectrophotometer, or by the ATR technique on a Bruker Equinox 55 spectrophotometer.All 1 H-NMR spectra were recorded in DMSO-d6 solvent on a Bruker NEO 400 spectrometer (Bruker, Faelanden, Switzerland).Chemical shifts were expressed to tetramethylsilane (TMS) and were represented in δ (ppm).To a solution of 5.5 mL (6.6 g, 0.1 mol) malonodinitrile and 12.7 mL (13.01 g, 0.1 mol) ethyl acetoacetate in 30 mL ethanol, 3.2 g sulfur was added.Via cooling and vigorous stirring, an equimolar amount of the catalyst (diethylamine, 0.1 mol) was dripped for 30 min.

Cytotoxicity and Phototoxicity Testing
BALB/3T3 cells were cultured in 75 cm 2 tissue culture flasks in DMEM, 10% FBS, 2 mM glutamine, and antibiotics (penicillin 100 U/mL and streptomycin-100 µg/mL) at 37 • C, 5% CO 2 , and 90% relative humidity.Cytotoxicity/phototoxicity was assessed by a validated BALB/3T3 clone A31 neutral red uptake assay (3T3 NRU test) [60,63].Briefly, cells were plated in a 96-well microtiter plate at a density of 1 × 10 4 cells/100 µL/well and incubated for 24 h.A wide concentration range was applied for the test compounds.For the phototoxicity tests, 96-well plates were irradiated with a dose of 2.4 J/cm 2 , and the cells were incubated for an additional 24 h.After treatment with neutral red medium, washing, and treatment with the ethanol/acetic acid solution, the absorption was measured on a TECAN microplate reader (TECAN, Grödig, Austria) at a wavelength of 540 nm.The light source which we used was a light-emitting diode (LED) matrix-an artificial solar light simulator Helios-iO, model LE-9ND55-H-5500K (SERIC Ltd., Tokyo, Japan).
selective index (ratio between IC50 values of MCF-10A and the corresponding tumor cell line for each of the tested compounds).SI = IC50 MCF-10A/IC50 tumor cell line.The cytotoxicity levels of compounds 2-5 against MCF-10A, MCF-7, and MDA-MB-231 cells are presented graphically in Figure 4.

Figure 6 .
Figure 6.ζ-potential of the investigated cell lines, MCF-10A, MCF-7, and MDA-MB-231 (A).Alterations in the ζ-potential after treatment with compounds 2 (pink), 4 (blue), and 5 (green) (B).All zeta potential values were measured in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4; pH 7.4) at 37 °C.Two independent experiments were carried out for each cell line, and each sample was measured 6 times.(*; ***) denote p < 0.05 and p < 0.001 as follows.Student t-test was applied to derive the statistical significance between the cancer cell lines (MCF-7 and MDA-MB-231) and the control (MCF-10A) (A) as well as the treated cancer cell lines with compounds 2, 4 and 5 compared to treated control cell line (B).

Figure 6 .
Figure 6.ζ-potential of the investigated cell lines, MCF-10A, MCF-7, and MDA-MB-231 (A).Alterations in the ζ-potential after treatment with compounds 2 (pink), 4 (blue), and 5 (green) (B).All zeta potential values were measured in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ; pH 7.4) at 37 • C. Two independent experiments were carried out for each cell line, and each sample was measured 6 times.(*; ***) denote p < 0.05 and p < 0.001 as follows.Student t-test was applied to derive the statistical significance between the cancer cell lines (MCF-7 and MDA-MB-231) and the control (MCF-10A) (A) as well as the treated cancer cell lines with compounds 2, 4 and 5 compared to treated control cell line (B).

5 Figure 7 .
Figure 7. SwissADME radar of bioavailability of compounds 2-5.The pink area represents the optimal range for each property (lipophilicity: XLOGP3 between −0.7 and +5.0; size: MW between 150 and 500 g/mol; polarity: TPSA between 20 and 130 Å2; solubility: log S not higher than 6; saturation: fraction of carbons in the sp3 hybridization not less than 0.25; and flexibility: no more than 9 rotatable bonds).

Figure 7 .
Figure 7. SwissADME radar of bioavailability of compounds 2-5.The pink area represents the optimal range for each property (lipophilicity: XLOGP3 between −0.7 and +5.0; size: MW between 150 and 500 g/mol; polarity: TPSA between 20 and 130 Å2; solubility: log S not higher than 6; saturation: fraction of carbons in the sp3 hybridization not less than 0.25; and flexibility: no more than 9 rotatable bonds).

Table 2 .
Anti-proliferative and anti-tumor activity of compounds 2

Table 3 .
Selective effects of compounds 2-5 on the studied cells.
SI*: selective index (ratio between IC 50 values of MCF-10A and the corresponding tumor cell line for each of the tested compounds).SI = IC 50 MCF-10A/IC 50 tumor cell line.

Table 3 .
Selective effects of compounds 2-5 on the studied cells.

Table 4 .
Effect of the treatment with substances 2

Table 5 .
Physico-chemical properties and LE and LLE of 2-5, as revealed in the MCF-7 and MDA-MB-231 test.
* TPSA values calculated by Molinspiration differ from those calculated with SwissAdme.