Synthesis, Characterization, and Anticancer Activity of Benzothiazole Aniline Derivatives and Their Platinum (II) Complexes as New Chemotherapy Agents

We describe the synthesis, characterization, molecular modeling, and in vitro anticancer activity of three benzothiazole aniline (BTA) ligands and their corresponding platinum (II) complexes. We designed the compounds based on the selective antitumor properties of BTA, along with three types of metallic centers, aiming to take advantage of the distinctive and synergistic activity of the complexes to develop anticancer agents. The compounds were characterized using nuclear magnetic resonance spectrometry, Fourier transform infrared spectroscopy, mass spectrometry, elemental analysis, and tested for antiproliferative activity against multiple normal and cancerous cell lines. L1, L2, and L1Pt had better cytotoxicity in the liver, breast, lung, prostate, kidney, and brain cells than clinically used cisplatin. Especially, L1 and L1Pt demonstrated selective inhibitory activities against liver cancer cells. Therefore, these compounds can be a promising alternative to the present chemotherapy drugs.


Introduction
The success of cisplatin [cis-diamminedichloroplatinum-(II)] flagged the way for second-and third-generation cancer drugs: carboplatin, oxaliplatin, nedaplatin, and lobaplatin ( Figure 1) [1][2][3]. The efficacy of cisplatin is governed by its ability to covalently bind with DNA and change its helical structure, resulting in cell death [4]. However, new anticancer agents have drawn attention for the reason that current platinum agents present drug resistance and have several limitations, such as a lack of selectivity, poor pharmacokinetic profiles and inadequate water solubility [5][6][7][8]. Moreover, existing platinum-based chemotherapy drugs are mostly correlated with general toxicity, which causes adverse Benzothiazole aniline (BTA), which is chemically known as 2-(4-aminophenyl)-benzothiazole, is a fused heterocyclic pharmacophore that exhibits antitumor activity [20]. Substituent exchange of the 3′-position with methyl or halogens (for instance Cl) contributes to the increased antitumor activity against various cancer cell lines (ovarian, colon, renal, etc.) [21,22]. Moreover, BTA showed selective anticancer activity by demonstrating a distinct cytotoxic response against some tumor cell lines, while no hormonal dependency was recognized [23,24]. Therefore, several studies have been performed in the past years to develop novel BTA derivatives as anticancer agents [25][26][27][28]. For example, the ring-substituted BTA derivative 2-(4-amino-3-methylphenyl) benzothiazole-DF 203was developed as an antitumor agent, and phortress NSC 710,305 underwent phase 1 clinical trials as a prodrug [24,25]. In addition, technium ( 99m Tc) and rhenium ( 186 Re, 188 Re) complexes of BTA conjugate were developed as radiopharmaceuticals for targeted therapy and imaging of breast cancer [29,30]. In our previous study, we developed a bifunctional Gd-DO3A-BTA chelate and evaluated its antiproliferative activity, both in vivo and in vitro, as a theranostic agent [31]. Recently, Mavroidi et al. synthesized Pd (II) and Pt (II) chelates of BTA derivatives to target cancer cells, however, the studied compounds proved less cytotoxic than clinically permitted cisplatin [32]. The approach using different types of biologically active moieties is frequently used in the design of novel metal-based drugs [33][34][35]. For example, a series of biologically active organometallic compounds were synthesized bearing an acetylsalicylic acid (ASA) substructure to inhibit cyclooxygenase (COX) enzymes [36]. Also, the nonsteroidal anti-inflammatory drug was conjugated to Pt (II) with various intracellularly cleavable linkers, which disclosed potent cytotoxic activity against different cancer cell lines [37]. Similarly, few novel Pt (II) complexes were synthesized bearing aliphatic amines and 1,8-naphthalimide moieties with DNA-targeting properties to achieve more potent and selective metallodrugs [38].
Based on the concepts containing biologically active molecules and the cisplatin-like composite, we designed and synthesized novel benzothiazole aniline derivatives (L1, L2, and L3) and their corresponding Pt (II) complexes as selective agents for treating cancer. We have hypothesized that the conjugation of new ligands using BTA will result in compounds that have distinct cytotoxic properties as BTA is well known for its antitumor activity. In vitro screening was performed in different cancer and normal cell lines, and Benzothiazole aniline (BTA), which is chemically known as 2-(4-aminophenyl)-benzothiazole, is a fused heterocyclic pharmacophore that exhibits antitumor activity [20]. Substituent exchange of the 3 -position with methyl or halogens (for instance Cl) contributes to the increased antitumor activity against various cancer cell lines (ovarian, colon, renal, etc.) [21,22]. Moreover, BTA showed selective anticancer activity by demonstrating a distinct cytotoxic response against some tumor cell lines, while no hormonal dependency was recognized [23,24]. Therefore, several studies have been performed in the past years to develop novel BTA derivatives as anticancer agents [25][26][27][28]. For example, the ring-substituted BTA derivative 2-(4-amino-3-methylphenyl) benzothiazole-DF 203-was developed as an antitumor agent, and phortress NSC 710,305 underwent phase 1 clinical trials as a prodrug [24,25]. In addition, technium ( 99m Tc) and rhenium ( 186 Re, 188 Re) complexes of BTA conjugate were developed as radiopharmaceuticals for targeted therapy and imaging of breast cancer [29,30]. In our previous study, we developed a bifunctional Gd-DO3A-BTA chelate and evaluated its antiproliferative activity, both in vivo and in vitro, as a theranostic agent [31]. Recently, Mavroidi et al. synthesized Pd (II) and Pt (II) chelates of BTA derivatives to target cancer cells, however, the studied compounds proved less cytotoxic than clinically permitted cisplatin [32]. The approach using different types of biologically active moieties is frequently used in the design of novel metal-based drugs [33][34][35]. For example, a series of biologically active organometallic compounds were synthesized bearing an acetylsalicylic acid (ASA) substructure to inhibit cyclooxygenase (COX) enzymes [36]. Also, the nonsteroidal anti-inflammatory drug was conjugated to Pt (II) with various intracellularly cleavable linkers, which disclosed potent cytotoxic activity against different cancer cell lines [37]. Similarly, few novel Pt (II) complexes were synthesized bearing aliphatic amines and 1,8-naphthalimide moieties with DNA-targeting properties to achieve more potent and selective metallodrugs [38].
Based on the concepts containing biologically active molecules and the cisplatin-like composite, we designed and synthesized novel benzothiazole aniline derivatives (L1, L2, and L3) and their corresponding Pt (II) complexes as selective agents for treating cancer. We have hypothesized that the conjugation of new ligands using BTA will result in compounds that have distinct cytotoxic properties as BTA is well known for its antitumor activity. In vitro screening was performed in different cancer and normal cell lines, and stability of the lead compounds was measured in buffered aqueous solution. In addition, we conducted molecular modeling studies to predict the best binding poses of ligands in the active site of DNA. We hypothesized that L1 and L1Pt can be promising candidates for treating liver, colon, breast, prostate, cervical, and brain cancers.

Synthesis and Characterization
The syntheses of the BTA derivatives L1, L2, and L3 and their Pt (II) complexes L1Pt, L2Pt, and L3Pt are depicted in Scheme 1. Compound L1 was derived based on our previously published procedure [39], which was then condensed with two salicylaldehyde equivalents to give the L2 ligand (white solid). The reaction of L1 with 2-pyridinecarboxaldehyde at room temperature in the presence of N-bromosuccinamide resulted in L3 (as a yellow solid). Platinum complexes L1Pt, L2Pt, and L3Pt were prepared by reacting the respective ligands L1, L2, and L3 with an equivalent of K 2 PtCl 4 under inert conditions. Potassium carbonate (K 2 CO 3 ) was used to increase the reaction rate of complex L2Pt. The pure Pt (II) chelates were isolated in moderate yield after washing repeatedly with water, methanol and ether. The formation of the ligands and their Pt (II) complexes were confirmed using various spectroscopic methods such as 1 H nuclear magnetic resonance (NMR), 195 Pt-NMR, Fourier transform infrared spectroscopy (FTIR), high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS), matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS), and elemental analysis (EA). stability of the lead compounds was measured in buffered aqueous solution. In additi we conducted molecular modeling studies to predict the best binding poses of ligands the active site of DNA. We hypothesized that L1 and L1Pt can be promising candida for treating liver, colon, breast, prostate, cervical, and brain cancers.

Synthesis and Characterization
The syntheses of the BTA derivatives L1, L2, and L3 and their Pt (II) complexes L1 L2Pt, and L3Pt are depicted in Scheme 1. Compound L1 was derived based on our pre ously published procedure [39], which was then condensed with two salicylaldehy equivalents to give the L2 ligand (white solid). The reaction of L1 with 2-pyridinecarb aldehyde at room temperature in the presence of N-bromosuccinamide resulted in L3 a yellow solid). Platinum complexes L1Pt, L2Pt, and L3Pt were prepared by reacting respective ligands L1, L2, and L3 with an equivalent of K2PtCl4 under inert conditio Potassium carbonate (K2CO3) was used to increase the reaction rate of complex L2Pt. T pure Pt (II) chelates were isolated in moderate yield after washing repeatedly with wat methanol and ether. The formation of the ligands and their Pt (II) complexes were co firmed using various spectroscopic methods such as 1 H nuclear magnetic resonan (NMR), 195 Pt-NMR, Fourier transform infrared spectroscopy (FTIR), high-resolution f atom bombardment mass spectrometry (HR-FAB-MS), matrix-assisted laser deso tion/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and elemental an ysis (EA).  1 H-NMR spectra of the newly synthesized ligands L1, L2, and L3 and their Pt complexes L1Pt, L2Pt, and L3Pt were recorded in different solvents, such as MeOH-d 4 , CHCl 3 , DMSO-d 6 , and the details are presented in the experimental section. The 1 H-NMR spectral data are consistent with the expected structure of the compounds (Supporting Information Figures S1, S4, S6, S9 and S12). In addition, complex L1Pt was characterized using 195 Pt-NMR and the chemical shifts (δ = −2955 ppm, DMSO-d 6 ), indicated no inter-or intramolecular contacts between the Pt (II) center and BTA moieties (Supporting Information Figure S7) [40]. The formation of L2 was confirmed by the presence of a C=N stretching vibration at 1634 cm −1 and the coordination of the metal center was confirmed by the downshift of C=N at 1600 cm −1 due to the development of Ar-O-Pt-N=CH-chelating ring and loss of salicylic effect [41]. The FTIR spectrum of L2 compared well to that of the corresponding L2Pt complex (Supporting Information Figures S3 and S10) Therefore, the 1 H-NMR, 195 Pt-NMR, and FTIR analysis data showed that the complexation of the ligand L1 and L2 with Pt was accomplished by coordination using amine and/or Schiff-bases nitrogen. In addition, high-resolution mass spectrometry in positive-ion mode was performed for all synthesized compounds. Parent peaks were found at (m/z) 578, (m/z) 714, and (m/z) 687 for complexes L1Pt, L2Pt, and L3Pt (Supporting Information Figures S8, S11 and S13). Ion peaks corresponding to the ligands have been observed at (m/z) 313, (m/z) 521, and (m/z) 400 for L1, L2, and L3 (Supporting Information Figures S2 and S5) [39]. The purity of the newly synthesized compounds was confirmed by EA. L1 was soluble in water and all other compounds when dissolved in DMSO.

Anticancer Effects and Cytotoxicity
We have tested the effects of different concentrations of ligands L1, L2, and L3 and their platinum complexes L1Pt, L2Pt, and L3Pt on the viability of various cancerous and normal cell lines (Tables 1 and 2, Figure 2). The half-inhibitory concentration (IC 50 ) values were calculated based on these data. The data showed that each component inhibits the growth of different cell lines at varying degrees. L1, L1Pt, and L2 exhibit similar cytotoxicity to that of cisplatin and BTA used as a control group in HeLa cells (Table 1 and Figure 2A). However, L1, L1Pt, and L2 exhibited excellent toxicity to most of the cancer cells, except for HeLa cells. The L1 and L1Pt exerted the best efficacy in liver and colon cancer cells. In addition, L2Pt revealed the anticancer effect only in liver cancer cells, and L3 had the possibility of the anticancer effect in glioma and prostate cancer cells. Although L3 induced a better anticancer effect than BTA in colon cancer cells, it did not reach the effect of cisplatin. L3Pt did not show anticancer effects in the tested cell lines, and the reason needs clarification. The structure-activity reveals that the conjugation of the 1,2-ethylenediamine with BTA (L1 and L1Pt) exhibited excellent inhibitory activity against numerous cancer cells. Moreover, the exchange of the ethylene bridge with electron-donating hydroxyl group-containing phenol rings (i.e., compounds L2 and L2Pt) displayed better anticancer activity than those of pyridine-containing derivatives L3 and L2Pt. From toxicity comparison in normal cells (Table 2 and Figure 2B), we confirmed that the toxicity of L1, L1Pt, and L2 was improved more than the cisplatin in the liver and brain cells. Although L2Pt, L3, and L3Pt did not exhibit toxicity in most of the normal cell lines, they did not display any anticancer effects, therefore, these compounds are not considered for further study.

Selective Antitumor Activity
A non-malignant mouse liver cell line (AML-12), human colon epithelial cells (FHC), human embryonic kidney cells (HEK-293), human breast epithelial cells (MCF-10A), and mouse brain neural stem-cell line (NE-4C) were used as models of the healthy cells to estimate the selectivity of these compounds in cancer cells regarding normal cells (Table 2, Figure 2B). To correctly term cytotoxicity as "selective" actually, there should be a great difference between tumor and non-tumor cells and it is challenging to compare cytotoxicity from different works. Interestingly, no cytotoxicity was observed for L1, L2, L1Pt, and L2Pt in normal mouse liver hepatocyte cells (AML12) up to 137 μM (Table 2). Moreover,

Selective Antitumor Activity
A non-malignant mouse liver cell line (AML-12), human colon epithelial cells (FHC), human embryonic kidney cells (HEK-293), human breast epithelial cells (MCF-10A), and mouse brain neural stem-cell line (NE-4C) were used as models of the healthy cells to estimate the selectivity of these compounds in cancer cells regarding normal cells (Table 2, Figure 2B). To correctly term cytotoxicity as "selective" actually, there should be a great difference between tumor and non-tumor cells and it is challenging to compare cytotoxicity from different works. Interestingly, no cytotoxicity was observed for L1, L2, L1Pt, and L2Pt in normal mouse liver hepatocyte cells (AML12) up to 137 µM (Table 2). Moreover, compounds L1, L2, L1Pt, and L2Pt displayed an IC 50 in liver cancer cells (HepG2) that are 23, 35, 18.5, and 15.8 folds higher in comparison with its IC 50 value in normal liver cells (AML12) respectively [42]. In contrast, clinically used cisplatin exhibited an IC 50 value in normal liver cells (AML12) 32 µM. In the case of liver cancer cells, L1, L2, L1Pt, and L2Pt showed strong toxicity, while revealed deteriorated toxicity in normal liver cells, demonstrate there is specificity for the liver cancer cells. In addition to liver cancer, L1, L2, L3, and L1Pt showed a more significant anticancer effect than cisplatin or BTA in brain glioma (Table 1). In the toxicity in normal cells, they have significantly improved toxicity than cisplatin in NE-4C cells ( Table 2). We observed that compounds L1, L2, L1Pt, and L2Pt exhibited anticancer activity against colon cancer cells. In addition, compound L3 displayed preferential anticancer activity against colon and breast cancer cells. This characteristic anticancer outcome may be attributed to the presence of the BTA moiety, which is well known for its tumor selectivity [23,24]. The superior selective toxicity toward cancer cells over noncancer cells proposes a strong potential of these compounds toward their antitumor application in liver cancer.

Stability of the Compounds in an Aqueous Solution
An indispensable feature of any promising anticancer agent is its thermodynamic stability in aqueous media. All prospective agents should be able to reach their target under the conditions encountered in living organisms. Therefore, the stability of the compounds was measured at 1 mM in phosphate-buffered saline (PBS) solution using UV-Vis spectroscopy. Due to poor solubility in aqueous media, compound L1Pt was dissolved in 2% DMSO in PBS solution. The stability of the metal complexes is often dependent on the pH value and the pH 4.5-5.0 is crucial since endosomal uptake of the complexes by lysosomes can occur [43]. Therefore, the UV-Vis spectrum was recorded in the range of 200-450 nm at t = 0, 7, and 24 h in different pH-values [(e.g., strong acidic (pH = 2), weakly acidic (pH = 5), neutral (pH = 7.4), alkaline (pH = 12)]. Only the most promising compounds were studied, and Figure 3 represents the time-dependent UV-Vis spectrum of the compound L1 and L1Pt in physiological pH (7.4). The compounds expressed distinct peaks in the 200-450 nm region and did not display any significant changes throughout 24 h. Lack of substantial interactions in the absorptions peak and spectral characteristics for testing compounds over time propose that no structural alterations occurred in the aqueous solution [44,45]. Also, there were no obvious changes in the absorptions peak in other pH-values (such as acidic, weakly acidic, and alkaline) for complexes L1 and L1Pt over time are shown in the Supporting Information ( Figure S14).

Protein-Ligand Docking Simulation
The anticancer mechanism of platinum drugs involves their intercalation with DNA base pairs [46,47]. Thus, molecular docking studies were conducted to predict the binding poses of ligands in the active site of the DNA. Table 3 shows the docking binding energies of L1, L1Pt, and BTA with various DNA structures. The top-ranked binding energies (kcal/mol) in the Auto Dock output file were considered a response in every single run. The best docking result was considered with the lowest binding energy of the conformation. The predicted binding energies of compound L1 and L1Pt were −6.697 and −7.150 kcal/mol, respectively, for binding to the 1BNA, whereas it was −6.658 kcal/mol for the parent compound BTA. Therefore, this negative binding energy suggests that those compounds rationally bind to DNA as their anticancer target. In addition, compounds L1 and L1Pt with high cytotoxic activity also showed elevated binding energies, −5.839 and −5.695 kcal/mol, in binding to 3CO3, respectively. Compounds L1 and L1Pt showed lower docking binding energy to DNA in binding to 1LU5 compared with parent BTA. somes can occur [43]. Therefore, the UV-Vis spectrum was recorded in the range of 200-450 nm at t = 0, 7, and 24 h in different pH-values [(e.g., strong acidic (pH = 2), weakly acidic (pH = 5), neutral (pH = 7.4), alkaline (pH = 12)]. Only the most promising compounds were studied, and Figure 3 represents the time-dependent UV-Vis spectrum of the compound L1 and L1Pt in physiological pH (7.4). The compounds expressed distinct peaks in the 200-450 nm region and did not display any significant changes throughout 24 h. Lack of substantial interactions in the absorptions peak and spectral characteristics for testing compounds over time propose that no structural alterations occurred in the aqueous solution [44,45]. Also, there were no obvious changes in the absorptions peak in other pH-values (such as acidic, weakly acidic, and alkaline) for complexes L1 and L1Pt over time are shown in the Supporting Information ( Figure S14).

Protein-Ligand Docking Simulation
The anticancer mechanism of platinum drugs involves their intercalation with DNA base pairs [46,47]. Thus, molecular docking studies were conducted to predict the binding poses of ligands in the active site of the DNA. Table 3 shows the docking binding energies of L1, L1Pt, and BTA with various DNA structures. The top-ranked binding energies (kcal/mol) in the Auto Dock output file were considered a response in every single run. The best docking result was considered with the lowest binding energy of the conformation. The predicted binding energies of compound L1 and L1Pt were −6.697 and −7.150 kcal/mol, respectively, for binding to the 1BNA, whereas it was −6.658 kcal/mol for the parent compound BTA. Therefore, this negative binding energy suggests that those compounds rationally bind to DNA as their anticancer target. In addition, compounds L1 and L1Pt with high cytotoxic activity also showed elevated binding energies, −5.839 and −5.695 kcal/mol, in binding to 3CO3, respectively. Compounds L1 and L1Pt showed lower docking binding energy to DNA in binding to 1LU5 compared with parent BTA.  DNA intercalation and major or minor groove binding with DNA are the most frequently observed modes of interaction for small molecule drugs [48]. The molecular modeling results suggest that compounds L1 and L1Pt interacted with the minor groove of the DNA (PDB ID: IBNA) (Figure 4). The BTA fragment of these compounds fits into the minor groove of the DNA and interacts through its sulfur group by forming hydrogen bonds with the base pairs. In addition, the -NH group showed a hydrogen bond interaction with the DNA. The docked poses of the compound L1 and L1Pt (Figure 4) revealed that it binds to the minor groove of the DNA (PDB ID: 1BNA) using −6.697 and −7.150 kcal/mol binding energy. Cisplatin requires hydration to form diaqua species, which are considered active agents. However, in this study, L1Pt remains stable over 24 h and probably does not palatinate the DNA (Figure 3), nevertheless it acts by intercalation. In contrast, ligand L1 showed groove binding intercalation with DNA and this can be one of the possible reasons for the higher cytotoxic effects than their corresponding Pt (II) complexes. The interactions of compound L1 and L1Pt with the 3CO3 and 1LU5 structures of the DNA are shown in the Supporting Information [ Figures S15 and S16]. However, we must mention that the docking study alone is not sufficient to probe mechanism of action of compounds. To probe it, at least in vitro binding assays such as ITC and SPR should be carried out on DNA-model systems, as well as on protein models. Therefore, further study using in vitro binding assays is warranted to probe mechanism of action of compounds. that the docking study alone is not sufficient to probe mechanism of action of compounds. To probe it, at least in vitro binding assays such as ITC and SPR should be carried out on DNA-model systems, as well as on protein models. Therefore, further study using in vitro binding assays is warranted to probe mechanism of action of compounds.

Reagents and Instruments
Solvents were dried using standard methods. 2,3-Diaminopropionic acid, di-tert-butyl dicarbonate (Boc 2 O), and triphenylphosphite [P(OC 6 H 5 ) 3 ] were purchased from Tokyo Chemical Industry (Tokyo, Japan). Sodium bicarbonate (NaHCO 3 ) and potassium carbonate (K 2 CO 3 ) were purchased from Daejung Chem. (Siheung-si, Korea). Magnesium sulfate anhydrous (MgSO 4 ) and sodium sulfate anhydrous (Na 2 SO 4 ) were obtained from Duksan Scientific Corp. (Ansan-si, Korea). Salicylaldehyde was acquired from Junsei Chemical Co. Ltd. (Tokyo, Japan). 2-Pyridinecarboxaldehyde, 2-(4-aminophenyl)-benzothiazole, potassium tetra chloroplatinate (II) (K 2 PtCl 4 ), and other commercial-grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), and used as received, unless otherwise stated. Deionized water (DI water) was used for the experiments. Progress of chemical reactions was observed using TLC (silica gel plates 60 F254) and visualized using a UV-Vis spectrometer. 1 H-NMR experiments were performed using an Advance 500 spectrometer (Bruker, Billerica, MA, USA) at the instrumental analysis center of Kyungpook National University (KNU, Daegu, Korea). Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. FTIR spectra were recorded using KBr pellets on a model 883 double beam infrared spectrophotometer (PerkinElmer, Waltham, MA, USA) in 200-4000 cm −1 . Microanalysis was performed using a CHNS elemental analyzer (Thermo Fisher Scientific, Waltham, MA, USA) at the KNU instrumental analysis center. HR-FAB-MS spectra were recorded using a model JMS-700 spectrophotometer (JEOL, Tokyo, Japan) at the Korea Basic Science Institute. Complexation reactions were conducted under an inert atmosphere using standard Schlenk techniques. The purity of the synthesized compounds was confirmed by EA and the tested compounds had at least 95% purity.

Synthesis of Complex L1Pt
A solution of K 2 PtCl 4 (0.14 g, 0.35 mmol) in distilled water (10 mL) was prepared under an inert atmosphere and added to compound L1 (0.11 g, 0.35 mmol). The resultant mixture was stirred overnight in the dark at RT. The precipitate obtained was filtered and rinsed using water, methanol, and ethyl ether. Vacuum drying produced a pale-yellow solid. A characteristic peak was observed at −2754 ppm corresponding to the Pt (II) species. Yield: 0.10 g (53%). 1

Synthesis of Complex L2Pt
Potassium carbonate (0.19 g, 1.35 mmol) was added to a solution of ligand L2 (0.35 g, 0.67 mmol) in DMF (10 mL). Next a solution of potassium tetrachloroplatinate (0.28 g, 0.67 mmol) in water (10 mL) was prepared under a stream of nitrogen and added to the mixture, which was then stirred overnight at a reaction temperature lower than 60 • C. The reaction mixture was left to cool at RT and the solid was filtered. Pure compound was gathered as a pale-yellow solid following repeated flushing with water, methanol, and ethyl ether. Yield: 0.24 g (49%). 1

Synthesis of Complex L3Pt
A solution of K 2 PtCl 4 (0.45 g, 1.10 mmol) in distilled water (15 mL) was prepared under a stream of nitrogen and added to a solution of ligand L3 (0.4 g, 1.10 mmol) in ethanol (15 mL). The resultant mixture was stirred in the dark under a nitrogen atmosphere at 50 • C, overnight. Solid that appeared was filtered and the pale-yellow product harvested after repeated washing using water, ethanol, and Et 2 O. Yield: 0.27 g (39%). 1

Stability of the Compounds in Aqueous Solution
The stability of the most active compounds, L1 and L1Pt, was studied. The compounds, L1 and L1Pt, at a concentration of 1 mM in phosphate-buffered saline (PBS) solution at different pH-values [(e.g., strong acidic (pH = 2), weakly acidic (pH = 5), neutral (pH = 7.4), alkaline (pH = 12)] were evaluated using UV-Vis spectroscopy. In the case of L1Pt, 2% DMSO in PBS was used as solvent due to the poor water solubility. The spectra were recorded in the range of 200-450 nm at t = 0, 7, and 24 h, and compared to each other.

Molecular Docking
Binding poses and energies of L1, L1Pt, and BTA for DNA structures were predicted using a protein-ligand docking simulation application called Glide [49]. Glide searches for possible binding poses of given ligands on DNA structure surface and finds the best binding poses and energy using the empirical scoring function called GlideScore [49]. For docking simulation, the OPLS3 force field was used to describe the atomic forces of ligand and DNA molecules. Flexible ligand sampling of ligand was allowed and the standard precision mode of Glide was used.

Statistical Analysis
Data were evaluated using a one-way analysis of variance with Tukey's multiple comparison tests. Analyses were performed using GraphPad Prism (GraphPad Prism Software Inc., version 5.02). Data are expressed as mean ± SD (standard deviation) or standard error of the mean values, and p < 0.05 was considered significant, statistically.

Conclusions
In this study, three novel benzothiazole aniline derivatives L1, L2, and L3 and their corresponding Pt (II) complexes L1Pt, L2Pt, and L3Pt have been designed and synthesized. The targeted compounds were investigated for their in vitro cytotoxic activity using the CCK-8 assay against various cancer and normal cells lines. Compared to the parental BTA and clinically used cisplatin, compounds L1, and L1Pt demonstrated selective inhibitory activities against liver cancer cells. In addition, docking results indicate that compounds L1 and L1Pt interact with the minor groove of the DNA, and remain stable in aqueous media. Therefore, these compounds may be considered as prospective alternatives to the present chemotherapy drugs.

Conflicts of Interest:
The authors declare no conflict of interest.