New pyrimidine-N-β-D-glucosides: synthesis, biological evaluation, and molecular docking investigations

In this study, syntheses of new pyrimidine-coupled N-β-glucosides and tetra-O-acetyl derivatives were carried out. All glycoconjugates were investigated in comparison with known chemotherapeutic agents in terms of their antimicrobial and anticancer functions and DNA/protein binding affinities. Spectral data showed that all glycoside derivatives were obtained by diastereoselectivity as β-anomers. Both tested groups exhibited strong antiproliferative activity (2.29–66.84 μg/mL), but some of them had sufficiently ideal % cytotoxicity values (10.01%–16.78%). And also all synthetic compounds exhibited remarkable antibacterial activity against human pathogenic bacteria. Binding of these compounds to CT-DNA resulted in significant changes in spectral properties, consistent with groove binding. Molecular docking studies of some compounds revealed that the docking score, complex energy, and MM-GBSA ΔGBind energy values were consistent with the experimental results, which showed that the new compounds were potent chemotherapeutic agents. Overall bioactivity results suggest that these compounds may be candidates as new chemotherapeutic agents and deserve further pharmacological evaluation.

The 1 H NMR spectrum of compounds 1-9 showed 13 proton signals for the aglycone moiety (2-amino-4,6diarylsubstituted pyrimidines) and seven protons at δ 3.7-5.5 ppm for the glycone part of 1-9. The 1 H-NMR spectrum showed one anomeric proton signal assignable to H-1''' of glucosyl moiety at δ ~ 5.4 (d, J = 8.8-9. 2 Hz). The 13 C-NMR spectrum of compounds 1-9 gave one anomeric carbon signal detected at δ ~ 82.5 (C-1''') bound to the anomeric proton of 1-9. The coupling constant (8.8-9.2 Hz) of the anomeric proton indicated a β-glucopyranosyl unit which were in good agreement with the literature [11,12,[25][26]. All the compounds obtained adopted the β-configuration. This is because for pyranoses, the usual energetically preferred form is the equatorial orientation of the substituents [27]. The other H-atoms of the glucopyranosyl (H-2'''-H-5''') formed a coupling system and the multiplet peak arising at 3.4-3.6 ppm was defined as the H-2'''-H-5''' protons. Due to the different chemical environment, the -CH 2 protons (H-6a'''/6b''') exhibited different chemical shifts. The H-6a''' was observed as a doublet (d, J = 11. 8 Hz) or doublet of doublets (dd, J = 11.8/2.2 Hz) at δ 3.9 ppm. The peak belonging to H-6b''' appeared as dd at 3.7 ppm with coupling constant J = 11.8-12.0/4.0-5. 2 Hz. On the other hand, due to the dissolution of almost all N-β-D-glucopyranoside derivatives in methanol and the preparation of NMR samples in deuteromethanol (CD 3 OD), the -NH and -OH protons of the glycopyranosyl ring were exposed to deuterium exchange in the solvent and their peaks could not be observed in the 1 H-NMR spectra. Preliminary assignments of 13 C NMR data were made using Attached Proton Test (APT) experiments. 13 C NMR spectra gave the peaks at δ 166.9-103.0 ppm for aglycone parts and peaks at δ 82.5-62.0 ppm appeared for glycone unit of compounds 1-9, respectively.
In the second part of the study, compounds 1-9 were acetylated then tetra-O-acetyl derivatives (10)(11)(12)(13)(14)(15)(16)(17)(18) were obtained in high yields (78%-93%) as a result of the nucleophilic acyl substitution of 1-9 with acetic anhydride in the basic medium [28]. The FT-IR spectrum of compounds 10-18 showed the characteristic broad absorption bands for the -NH and C=O groups at 3420 cm -1 and 1740 cm -1 , respectively. LC-MS/MS spectra of 10-18 gave a pseudomolecular ion peak at m/z 615.00 [M + Na] + . In 1 H-NMR spectrum, as a result of the deshielding effect of the C=O or O-acetylated glucopyranosyl group by the electron-withdrawing property, the anomeric H-1''' appeared as a doublet in the low field (δ 6.1-5.9 ppm) with a coupling constant between 9.0 and 10 Hz. The coupling constant also confirmed the β-glucopyranosyl linkage. The acetyl groups' H-atoms of compounds 10-18 resonated in the range of 2.0-1.9 ppm in 1 H-NMR spectra, and the 13 C-NMR signals were observed around 21.6-19.2 ppm. In addition, four new quaternary peaks were observed in the range of 171.1-169.6 ppm, originating from the carbonyl groups and confirming the structure. All the spectra of 1-18 were given in the supplementary data.

Biological evaluation 2.2.1. Evaluation of anticancer properties of the compounds
It is widely known that natural or synthetic glycosides play many important roles in living systems, such as apoptotic or arrhythmic effects. As the findings imply that glycosides are cytotoxic, glycosides can also be attributed to promising drug candidates that exhibit significant anticancer activity. Therefore, the synthesis and biological evaluation of novel glycosides are of interest to our research group. Accordingly, the anticancer effects of 18 new compounds synthesized as pyrimidinecoupled N-β-glucosides (1)(2)(3)(4)(5)(6)(7)(8)(9) and their tetra-O-acetyl derivatives (10)(11)(12)(13)(14)(15)(16)(17)(18) were evaluated using the MTT protocol. The half-maximal inhibitory concentration 50 (IC 50 ) inhibition values, generally used for inhibition studies, and growth inhibiting concentration 50 (GI 50 ), total growth inhibition (TGI), and lethal concentration 50 (LC 50 ) were determined using spectrophotometric data obtained from the MTT assay using cisplatin and 5-fluorouracil (5 FU) as anticancer control drugs, as recommended by the NCI. When the synthetic compounds were compared with the control group (Tables 1  and 2), they were not sufficiently antiproliferative in the A549 cancer cell line, even at a high concentration of 500 μg/mL. Among the pyrimidine N-β-D-glucosides (1-9), compounds 1-5, 7, and 8 in C6 cell line (with IC 50 values between 27.78 and 49.71 μg/mL, TGI values between 20.46 and 31.57 μg/mL) and compounds 1, 4, 7, and 8 in the C6 and HeLa cell lines (with IC 50 values between 31.85 and 81.26 μg/mL, TGI values between 21.60 and 50.07 μg/mL) exhibited high antiproliferative properties (Tables 1 and 2). Compounds 5 and 16 in MCF7 and Hep3B cells, and 4 in Hep3B cells showed potent anticancer properties with IC 50 values between 4.12 and 66.84 μg/mL and TGI values between 4.29 and 75.66 μg/mL. In the HT29 cell line, compounds 3-5, 7, and 8 achieved sufficient antiproliferative activity (IC 50 values between 26.69 and 78.38 μg/mL, TGI values between 31.94 and 230.10 μg/mL). When the effect of the pyrimidine-coupled pyrimidine N-β-D-glucosides on cells was investigated according to the screening method recommended by the NCI, compound 5 with low GI 50 and high LC 50 values could be considered a candidate for further pharmacological testing (Tables 1 and 2).
When the IC 50 and TGI data of the test results were examined, the tetra-O-acetyl-N-β-D-glucopyranoside derivatives (10-13, 15-16, 18), with the exception of 14 and 17, showed similar or stronger antiproliferative properties than 5-fluorouracil (5FU) and cisplatin (Table 2). When the GI 50 values presented in Table 1 were examined, the tetra-O-acetyl derivatives showed greater growth inhibition than cisplatin, and all compounds also caused greater inhibition than 5FU in the C6 cell line. Of the tetra-O-acetyl derivatives (10)(11)(12)(13)(14)(15)(16)(17)(18) in Tables 1 and 2, compound 16 showed very successful anticancer properties against the C6 cell line as well as against HeLa, HT29, MCF7, and Hep3B cells (IC 50 values between 3.26 and 9.13 μg/mL and TGI values ranging from 2.80 to 7.33 μg/mL). Among the other antiproliferative compounds, compounds 13 and 18 against HeLa cells and compound 10 against HT29 cell line showed potent anticancer activity. Considering the obtained growth inhibition values (GI 50 and TGI values), it could be said that all compounds except 14 and 17 have the potential to be used for pharmacological studies for the treatment of glioma cancer in the brain. While compounds 4, 7, 13, 15, 16, and 18 showed lower values than cisplatin in terms of lethal concentration (LC 50 ), compounds 1-3, 5, 8, and 10-12 showed higher LC 50 values than both cisplatin and 5FU (Tables 1 and 2). Higher lethal concentration values indicate that the cytotoxic effects of the test substances are lower, which is desirable. Lower GI 50 and TGI concentration values indicate that the cytotoxic effects of the test substances are greater, which is also desirable. When the IC 50 data of the test results were examined, only compounds 13, 16, and 18 were found to be effective for the HeLa cell line. On the other hand, the GI 50 and LC 50 values of these compounds showed that all compounds were more effective than the control compounds 5FU and cisplatin. However, the TGI values showed that these compounds were toxic to the HeLa cell line except for compounds 16 and 18. In this case, these compounds cannot be used for further studies because they do not have the molecular structure that can be used in cervical cancer research. However, the design of these molecules can be redesigned with Lipinski's rules in mind, and the toxic effects can be reduced to reasonable levels without reducing the antiproliferative effects. However, it should be kept in mind that the MTT method only measures mitochondrial activity of living cells and FL cells may have lower mitochondrial activity than cancer cells. According to this principle, the cells of FL could have lower mitochondrial activity, which could lead to an increased antiproliferative effect. To address this issue, we used LDH-based cytotoxicity measurement in addition to MTT. In evaluating the results of LDH activity measurement, which is our second important test to elucidate the cytotoxicity of these compounds, it was found that the above anticancer compounds (1-3, 5, 7, 8, 10-13, 15-16, and 18) caused membrane damage of approximately 10%-20% at effective concentrations (IC 50 values in the range of approximately 25-50 μg/mL) (Tables 3 and 4).
These values were very close to the values of percent cytotoxicity caused by the positive controls used (5FU and cisplatin) ( Table 3 and 4).
However, the membrane damage caused by these compounds with potent anticancer properties should have more pharmacologically reliable values without altering the anticancer properties of these molecules. According to the proliferation measurements performed on the normal cell line (FL), compounds 1, 4, 5, 13, 16, and 18, which had high LC 50 values (>500 μg/mL), were also found to cause membrane damage of 15%-20% in toxicity tests with the same molecules. The results of these pharmacological measurements on the normal cell line FL are largely consistent with NCI criteria.

Compound (µg/mL)
Therefore, they may be candidates for ADME/Tox and advanced phase studies due to the significant in vitro biological activity exhibited by the respective test compounds.
When the qualitative structure-activity relationship of the compounds was discussed, it was seen that the 2'-and 3'-pyridinyl compounds (4, 5, 7, and 8) in which the methyl group was in the meta and para positions were both anticancer and antimicrobial active (Table 5). However, acetylated derivatives of these compounds (13,14,16, and 17) showed inactive behavior against cells (Table 6). In general, the position of methyl and pyridine nitrogen affected the biological activities,    while acetylation resulted in different cytotoxicities. The decrease in hydrogen bonding capacity by acetylation may have caused this behavior. As can be understood from docking studies, the three-dimensional structure of molecules regulates their interactions with biomolecules. Therefore, the movement of the methyl group and nitrogen atom and acetylation caused the bioactivity of the compound to change.

Morphological changes of molecules on cells
At various concentrations, the effects of pyrimidine-coupled N-β-glucosides and tetra-O-acetyl derivatives on the cell morphology of C6, Hela, A549, Hep3B, MCF7, and FL were visualized and examined by phase-contrast microscopy. The control cells shown in Figure 2 exhibited fibroblast-or epithelium-like normal cell morphology and served as the benchmark for our assessments. The first impression we got from the phase-contrast microscopy images was that the cells began to detach from the flask surface in a concentration-dependent manner. During this detachment, the cells lost their fibroblastic or epithelial normal shape and started to change into round shapes. Then the cells underwent some morphological changes, such as cytoplasmic blistering and spiking, abnormal spherical structures, and apoptotic bodies, and finally the cells floated (indicating that the cells were dead). As we found, concentrations of 60 μg/mL and above caused cells to separate, to be smaller, to be seen in smaller numbers, and to have insufficient cell adhesion. Furthermore, these images may indicate a decrease in cell viability leading to poor proliferation, small cell size, and apoptosis. We observed that the cells could maintain their normal fibroblast-like appearance even under the conditions of the test substance at low concentrations (< 40 μg/mL). We also found that apoptosis-like images as well as partial necrotic damage affected the growth of cells treated with high concentrations of the compounds (>82 μg/mL) ( Figure 2).

Evaluation of Antibacterial Effects of the Compounds
Indeed, the development not only of new anticancer drugs but also of antibiotic derivatives is of paramount importance to the community. This is because the bacterium methicillin-resistant Staphylococcus aureus (MRSA) has been reported to cause more deaths than HIV/AIDS annually worldwide, particularly in the United States [29]. In light of this literature information, the antibacterial activity of the newly synthesized compounds on some gram-positive and gram-negative bacteria that cause disease in the human body was investigated using the minimum inhibitory concentration (MIC) method. Among our test compounds, those with MIC values not exceeding 125 μg/mL and below these dose levels were found to have antibacterial activity. This evaluation was based on the MIC values of antimicrobial drugs in use today.
When the MIC values of the newly synthesized compounds were examined, it was found that the antibacterial activity of the compounds was quite high ( these compounds were at least as sensitive as the positive control SCF antibiotic against the tested bacteria (Tables 7 and 8). However, none of the new compounds showed strong antibacterial activity on E. coli ATCC 25922, E. coli ESBL ATCC 35218, and P. aeruginosa AGME ATCC 27853 strains. When the results of the in vitro antibacterial assays of these structures were evaluated as a whole, it was found that the pyrimidine N-β-D-glucosides (1-9) and the tetra-O-acetyl derivatives (10-18) exhibited similar antibacterial properties. Compound 8 showed the best result with a strong antimicrobial effect Although both groups of compounds were more active on gram (+) bacteria, their activities were much lower on gram (-) bacteria. In addition, potent antimicrobial activity against resistant strains such as VRE, MRSA, ESBL, and AGME was not achieved at the desired level and remained at the same level as the control drug, SCF. In general, both functional groups were found to be effective against some bacteria causing diseases in the human body, and we strongly recommend that some of them should enter advanced pharmacological research immediately and others after remodeling according to Lipinski's rules.

Analyzing of DNA/BSA binding properties of the compounds
The vast majority of anticancer agents used in pharmaceutical chemistry act on functional macromolecules such as DNA and proteins. Given this well-known fact, the relationship between newly developed anticancer drug candidates and macromolecules should be studied in detail. The interaction of molecules with DNA leads to significant changes in the helical structure of DNA, which can be observed by spectrophotometric techniques. In general, the changes in DNA caused by the compounds appear as hyperchromic or hypochromic effects in their absorption spectra [30]. The hypochromic effect shows a decrease in absorbance when the DNA concentration is increased, while the hyperchromic effect shows an increase in absorbance when the DNA concentration is increased. While the hypochromic effect causes changes in DNA structure and shrinkage or shortening of DNA along the helical axis, the hyperchromic effect causes the twisting of DNA in the helical configuration of DNA. Also, the red or blue shift in the absorption bands of the compounds can be an indication of the stability of the compound and the DNA structure. The DNA/BSA binding properties of the new chemicals synthesized by our group were determined using a UV-Vis spectrophotometer. The interactions of these compounds with DNA were as follows. In the spectra obtained by UV-Vis studies of the compounds, a single maximum absorption peak was observed and this peak had no clear bathochromic or hypsochromic effect. When increasing amounts of CT-DNA were added to the reaction mixture, the decrease in the absorption intensities of 3 and 9 of pyrimidine-N-β-Dglucopyranosylamines resulted in a hyperchromic effect, whereas the increase in the absorption intensities of compounds 1, 2, and 4-8 resulted in a hypochromic effect. Similarly, the addition of CT-DNA in increasing amounts led to an increase in the absorption intensities of compounds 10-15, 17, and 18 (hyperchromic effect), while the absorption intensity of compound 16 decreased (hypochromic effect). The physical interactions of our newly synthesized compounds with BSA caused the formation of the spectral bands described below. According to the spectrophotometric analysis of the interaction of pyrimidine-coupled N-β-glucosides with increasing amounts of BSA, compounds 1-3, 5, 7, and 8 showed a hypochromic effect, while compounds 4, 6, and 9 from the same group exhibited hyperchromic activity. Similarly, compounds 10-15, 17, and 18 were found to have a hyperchromic appearance, while only compound 16 behaved hypochromically. As a result of the easy-to-perform spectrophotometric studies, the binding constants (Kb) of the new pyrimidine-coupled N-glucosides and tetra-O-acetyl derivatives showing the affinity of the compound for DNA were determined by the following equation When we compared the DNA binding affinities of these anticancer drugs commonly used in the clinic with the DNA binding affinity of the compounds tested, we concluded that the new pyrimidine glycopyranosyl derivatives bind strongly enough to DNA. In particular, compound 1 was found to show greater interest in DNA than the anticancer drugs cisplatin and 5FU.

In silico studies
Molecular docking studies were performed to determine the interactions of compounds 4, 7, 13, and 16 and different crystal structures (PDB IDs: 4QL3, 6MPP, 4E26, 6QGG, 5MU8, 4QUG, 2DBF, 6SL6, 1CGL, 5Z62, 6EB6, 5ITD, 4EKK, 6GU7, 7BG9, 3GUS). [34]. Schrödinger 2021-2 molecular modeling software was used to determine these interactions. Parameters such as MM-GBSA ΔG Bind , docking score, and complex energy values were calculated with Schrödinger 2021-2. According to these values, the strength of the interactions between the ligand and the target was calculated and the values were compared among themselves and the proteins that could have the best interaction were determined. The free binding energy, docking score and complex energy values of the compounds interacting in silico with the proteins obtained from the protein database are presented in Table 10. The values in Table 10 indicated that NRAS, BRAF, PI3K alpha, Cytochrome c oxidase, and Akt1 were more effective than the other targets listed in the table.
The proteins used in in silico approaches were determined based on the DNA/BSA binding properties of the compounds. These proteins are known to be important in the pathway. The values calculated for the in silico approaches showed us this. Although the data for the mentioned proteins are very good, according to Table 10, we can say that values of the binding parameters of PI3K alpha (PDB ID: 5TID) were the best.
When the interaction results of PI3K alpha with molecular docking were examined in detail, the values of ΔG Bind , docking score and complex energy were found -67. When performing molecular docking analysis, the amino acid residues determined in the active binding site are as important as the energy values of the binding parameters shown in Table 10. The 2D interaction diagrams of the compounds in the binding site for PI3K alpha, calculated according to in silico approaches and having the best binding parameter values, are presented in Figure 3. In Figure 3, there is a hydrogen bond with important amino acids Val851, Asn853, Gln859, and a π-π π-π stacking bond with residue Tyr836, Trp780 in the 2-dimensional interaction diagram between compound 4 and PI3K alpha. When this interaction was analyzed for compound 7, it made hydrogen bonds with Val851, Asn853, Gln859 amino acid residues, and π-π stacking interaction bonds with Trp780, Tyr836. In the interaction between compound 13 and PI3K alpha in Figure 3, there is Trp780, π-π stacking interaction of amino acids Tyr836, and hydrogen bond interaction with amino acid residues Ser774 and Ser773. On the other hand, Lys802 in the active binding site compound 16 made hydrogen bonds with Hie917 amino acid residues.
3D diagrams of amino acid residues of compounds interacting with PI3K alpha are presented in Figure 4. It is understood from Figure 4 that the compounds bind and interact to the active binding site of the crystal structure of the target from the same region.
Biological evaluations (antiproliferative, cytotoxic properties and DNA/protein binding affinities) of 1-18 were investigated. Proliferation measurements revealed that compounds 1, 4, 5 of the pyrimidine N-glucosides and 13, 16, and 18 of the tetra-O-acetyl derivatives exhibited high antiproliferative activity. They were also found to cause membrane damage of 15%-20% in toxicity tests with the same molecules. The results of these pharmacological measurements on the cell line were largely consistent with NCI criteria. As a result, when the in vitro antibacterial test results of these complexes were evaluated overall, it was found that there are similar antibacterial properties between the pyrimidine N-β-D-glucosides (1-9) and the tetra-O-acetyl derivatives (10)(11)(12)(13)(14)(15)(16)(17)(18). Moreover, the new synthesized molecules can bind sufficiently strongly to DNA. In particular, Compound 1 has more affinity for DNA than cisplatin. For this reason, they could be candidates for ADME/Tox and advanced phase studies due to the significant in vitro biological activity exhibited by the respective test compounds.
Recently, calculations with in silico approaches have been applied to support the experimental results. By molecular docking calculations, it was determined that the compounds 4, 7, 13, and 16 had the best interaction with the PI3K alpha protein. By showing the effects of these compounds on ligands in NRAS, BRAF, Akt1, receptor/enzymes, it can be a lead drug candidate research study. Overall bioactivity results suggest that these compounds may be candidates as new chemotherapeutic agents and deserve further pharmacological evaluation.

Experimental
The materials and equipment used in this study are presented in the supplementary information.

General procedure for synthesis of compounds 1-9
A mixture of 2,4,6-trisubstituted pyrimidine (15 mmol, 3.93 g), D-glucose monohydrate (15 mmol, 2.97 g), and glacial acetic acid (15 mmol, 0.85 mL) in dimethyl sulfoxide (6 mL) was heated and refluxed at 100 °C for 24 h under progress control by TLC assay [15]. TLCs were carried on silica gel (Kieselgel 60 F 254 , Merck) plates and the spots were visualized by UV lamp or spraying with 10% alcoholic H 2 SO 4 and heating. After the TLC control, reactions were stopped, the mixtures were allowed to cool to room temperature and placed in the separating funnel, shaken by adding chloroform and then distilled water. When the shaken mixture came to rest, phase separation and the formation of a precipitate between the two phases were observed. The precipitate was obtained after separation of the solvents and was washed again with chloroform and water in a separatory funnel to try to remove impurities that might have come from unreacted pyrimidine and glucose. The purity of the precipitate was checked again with TLC and dried with a freeze dryer. The structure was confirmed by spectroscopic methods ( 1 H, 1 H-1 H COSY and 13 C-APT NMR, LC-MS /MS and FT-IR) and elemental analysis.

General procedure for synthesis of compounds 10-18
Pyrimidine N-glycoside (1-9) (3 mmol, 1.3 g each), acetic anhydride (12 mmol, 1.2 g) and Na 2 CO 3 (12 mmol, 1.1 g) were mixed and this mixture was stirred at 100 °C under reflux conditions for 10-20 min, and the progress was monitored by TLC [28]. Water and then chloroform were added to the finished reaction, and the mixture was placed in the separatory funnel, shaken, and allowed to rest. After phase separation, the organic phase was taken and the solvent was removed in vacuo. The product was washed with diethyl ether and dried at room temperature. This class of compounds was found to dissolve in chloroform. Purity control of the compounds was performed by TLC and their structures were elucidated by spectroscopic methods ( 1 H, 1 H-1 H COSY and 13 C-APT NMR, LC-MS /MS and FT-IR) and elemental analysis.

Pharmacology
Pharmacological experiments that include the preparation of cell culture, cell proliferation assay (MTT assay), cytotoxic activity assay, microdilution assay, and DNA binding studies were performed [35] and provided in supplementary information. The calculation of IC 50 and %inhibition was also explained in the supplementary information.

In silico studies
Molecular docking studies were applied to determine the amino acid residues in the active site of compounds 4, 7, 13, and 16, which interact with all crystal structures in in silico approaches, respectively, and to calculate the binding parameters.

Determination and preparation of proteins
The crystal structures of the proteins with which compounds 4, 7, 13, and 16 interact were obtained from the Protein Data Bank (https://www.rcsb.org/). Based on the pathways in the research study, crystal structures with PDB access codes 4QL3

. Materials and equipment
All starting chemical reagents and solvents used in the synthesis, purification,n and biological activity investigations were high-grade commercial products purchased from Aldrich, Fluka, Sigma, Merck, Amresco, Carlo-Erba, Lonza, Roche and used without further purification. Thin-layer chromatography (TLC) and column chromatography were performed on Merck precoated 60 Kieselgel F 254 analytical aluminum acidic plates and silica gel 60 (0.040{0.063 mm), respectively. All reactions were monitored using TLC. 1 H and 13 C NMR spectra were recorded on a Bruker 400 MHz NMR in CDCl 3 , CDCl 3 /CD 3 OD, CD 3 OD, DMSO-d 6 with tetramethyl-silane (TMS) as an internal standard. The elemental analyses were performed by using a Costech ECS 4010 instrument. Mass spectral analyses were performed on a Micromass Quattro LC-MS/MS spectrophotometer. Infrared spectra were obtained using a PerkinElmer 1600FT-IR (4000-400 cm -1 ) spectrometer. Melting points were determined using a Stuart SMP10 apparatus.                                                              (12) Yield           (14) Yield                          Preparation of cell cultureThe procedure of the pharmacological experiments that include the preparation of cell culture, cell proliferation assay (MTT assay), cytotoxic activity assay, microdilution assay, and DNA binding studies are provided in supplementary information. The calculation of IC 50 and three dose response parameters were explained in the supplementary information. The anticancer potential of the compounds was investigated on cancerous HeLa (ATCC ® CCL2™), HT29 (ATCC ® HTB38™), MCF7 (ATCC ® HTB22™), A549 (ATCC ® CCL185™), C6 (Rat brain glioma, ATCC ® CCL-107™), and Hep3B (ATCC ® HB8064™) and normal FL cells (ATCC ® CCL62™). The cell lines were cultured in a cell medium (Dulbecco's modified eagle's or RPMI 1640) enriched with 10% (v/v) fetal bovine serum and 2% (v/v) Penicillin-Streptomycin (10,000 U/mL). First, old medium was removed out of the flask when the cells reached approximately 80% confluence. Next, cells were taken from the flasks surface using trypsin-EDTA solution and then subjected to centrifugation. Following, the cell pellet was suspended with fresh media and was inoculated into wells.

Cell proliferation assay (MTT assay)
A cell suspension containing approximately 1 × 10 4 cells in 100 µL was seeded into the wells of 96-well culture plates. The cells were treated with the compounds and control drug, cis-platin and 5 fluorouracil (5FU), dissolved in sterile DMSO (max 0.5% of DMSO) at final concentrations of 1.96, 3.91, 7.81, 15.625, 31.25, 62.5, 125, and 250 µg/mL at 37 °C with 5% CO 2 for overnight. The final volume of the wells was set to 200 µL by medium. Cell proliferation assay was evaluated by MTT (yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) methods. Briefly, an MTT stock solution (5 mg of MTT/mL of distilled water) was filtered and kept at -20 °C until use. The cells were exposed to an MTT reagent (consisting of one parts of MTT stock solutions and nine parts of fresh RPMI 1640 without phenol red) for 4 h to form MTT formazan dye followed by the dye dissolved in DMSO with Sorenson's buffer for 30 min at room temperature and then the plate was measured at 560 nm, with 690 nm as a reference interval, using a microplate reader. Each experiment was repeated at least three times for each cell line.

Cytotoxic activity assay
The cytotoxicity of the compounds, cisplatin and 5 fluorouracil on cells was determined through a Lactate Dehydrogenase Assay Kit according to the manufacturer's instructions. Approximately 5 × 10 3 cells in 100 µL were placed into 96-well plates as triplicates and treated with 25, 50, 75, and 100 µg/mL concentrations of test compounds at 37 °C with 5% CO 2 for 24 h. LDH activity was obtained by determining absorbance at 492-630 nm using a microplate reader. The cytotoxicity assay results were noted as the percent cytotoxicity according to the following formula: % Cytotoxicity = [(Experimental Value -Low Control / High Control -Low Control) × 100].

Microdilution assay
MIC values of the compounds against bacterial strains were determined on the basis of a microwell dilution method. To determine the minimal inhibitory concentration (MIC) values, inocula of bacteria were prepared using 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Each substance dissolved in dimethyl sulfoxide (DMSO) and serial twofold dilutions were made in a concentration range from 7.81-1000 µg/mL in microplate wells containing nutrient broth. Growth of microorganisms was determined visually after incubation for 24 h at 35 °C. The lowest concentration at which there was no visible growth (turbidity) was taken as the MIC.

1.3.5.
DNA binding studies To find the interaction of the compounds with calf thymus DNA and to calculate the binding constants ( ) UV-Visible absorption spectroscopy technique was used. A 2.5-mg calf thymus DNA was dissolved in 10.0 mL Tris-HCl buffer (20 mM Tris-HCl, 20 mM NaCl, pH 7.0) and stabile during one week in the refrigerator. The concentration of calf thymus DNA was obtained spectrophotometrically with help of ε value of 6600 M −1 cm −1 at 260 nm. After dissolving the calf thymus DNA fibers in Tris-HCl buffer, the purity of this solution was checked from the absorbance ratio A260/ A280. The calf thymus DNA solution at A260/A280 ratio was equal to 1.87, implying that the DNA was pure enough. These compounds were diluted with Tris-HCl buffer to obtain 25 µM concentrations. Test compounds in the solutions were incubated at room temperature for nearly 30 min before the process. The UV-visible spectral studies were performed in mixed solvent system (1/9 DMSO/Tris-HCl buffer) using eight points that the fine structure is observed for these compounds in this system by UV-visible absorption. The UV absorption titrations were conducted by keeping the concentration of these compounds fixed while varying the CT-DNA concentrations (6.5-800 μM). Absorption spectra were recorded by using 1-cm-path quartz cuvettes at room temperature. To evaluate the interaction of the compounds with BSA, UV-Visible absorption spectroscopy technique was also used. A 2.5 mg BSA was dissolved in 10.0 mL of Tris-HCl buffer (5 mM Tris-HCl, 10 mM NaCl at pH 7.4) and stored in the refrigerator. The UV-Visible absorption spectra of the BSA solutions (6.5-800 μM) in the presence of a conserved concentration of the compounds (25 μM) were scanned in the wavelength range from 300 to 550 nm.

Calculation of IC 50 and % inhibition
IC 50 value is a concentration that inhibits half of the cells in vitro. The half maximal inhibitory concentration (IC 50 ) of the test and control compounds was calculated using XLfit5 or excel spreadsheet and represent in µM at 95% confidence intervals. The proliferation assay results were expressed as the percent inhibition according to the following formula: % Inhibition = [1 -(Absorbance of Treatments / Absorbance of DMSO) × 100]. Three dose response parameters (GI 50 , TGI, LC 50 ) were calculated according to the following formulas using the absorbance measurements of time zero (Tz), control growth (C), and test growth in the presence of drug (Ti). Growth inhibition of 50% (GI 50 ) was calculated from [(Ti-Tz)/ (C-Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net growth increase in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC 50 indicating a net loss of cells following treatment was calculated from [(Ti-Tz)/Tz] × 100 = -50.