INTRODUCTION

Heterocyclic compounds, and specifically for the current study benzothiazoles, demonstrate a broad range of biological activities including anticancer [1], antimicrobial [2], antiviral [3], analgesic [4], antimalarial [5], fungicidal [6], antihelmintic [7], antitubercular [8], and many more. For example, molecules of compounds NSC-710305 (1, Fig. 1) and violatinctamine (2, Fig. 1) [9] contain benzothiazole moieties in their structures. Compound1 demonstrated anticancer activity and has been processed to phase-1 clinical trial [10].

Fig. 1.
figure 1

Structures of anticancer drugs: (a) NSC-710305, 1, (b) Violatinctamine, 2, and (c) NVP-AUY922, 3.

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Similarly, 1,2-isoxazoles demonstrated antitumor [11], anti-oxidant [12], antifungal [13], and a range of other activities. One of the anticancer drug candidates Luminespib (3, NVP-AUY922, Fig. 1) contains a synthetic 1,2-isoxazole scaffold [14].

In view of the above and in continuation of the ongoing studies, we have designed and synthesized a new series of bis-isoxazole incorporated benzothiazole derivatives (14a–14j) and confirmed their structures by 1H and13C NMR, and mass spectra. The products were screened for their anticancer activity towards four human cancer cell lines.

RESULTS AND DISCUSSION

Synthesis of new series of bis-isoxazole incorporated benzothiazole derivatives14a–14j is outlined in Scheme 1. The 3,4,5-trimethoxyphenyl oxime (4) was reacted with 1-ethynyl-4-nitrobenzene (5) in presence aq NaOCl in anhydrous media of CH2Cl2 at room temperature to give pure isoxazole derivative 6.

Scheme 1.
scheme 1

Synthetic approach to the target bis-isoxazole incorporated benzothiazole derivatives 14a–14j.

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Reduction of the nitro group in the intermediate 6 by Pd/C, H2 in ethanol yielded pure amino intermediate 7. Its reaction with 4-isothiocyanatophenol (8) in ethanol led to pure intermediate 9, cyclization of which in the presence of [bbim][Br3] ionic liquid at 70°C afforded pure benzothiazole intermediate 10. Reaction of compound10 with propargyl bromide 11 in presence of K2CO3 in anhydrous DMF resulted in formation of the intermediate 12, following cyclization of which with different types of aryl oximes (4, 13a–13i) in presence of aq. NaOCl and TEA gave the corresponding pure target compounds 14a–14j.

Biological activity of the products. The newly synthesized bis-isoxazole incorporated benzothiazole derivatives 14a–14j were tested for their in vitro anticancer activity against breast MCF-7 and MDA MB-231, lungs A549, and prostate DU-145 cancer cell lines by using the MTT assay and etoposide as the standard drug (Table 1). All the compounds exhibited low to moderate activity. Among those the products 14b, 14c, 14g–14j were determined to be more potent than etoposide. The structure-activity relationship (SAR) analysis indicated that the compound with 3,4,5-trimethoxyphenyl ring (14b) exhibited high anticancer activity on all cell lines, whereas the one with 2,4-dimethoxyphenyl ring (14c) displayed somewhat lower activity, and the 2,3-dimethoxyphenyl substituted analogue (14d) was characterized by very poor activity. The compounds 14e and 14f with 3-bromophenyl and 4-nitrophenyl rings exhibited moderate activity. The compound 14g containing 4-pyridyl heterocyclic ring demonstrated excellent activity on all cell lines. Replacement of 4-pyridyl ring with 2-pyrrolyl ring resulted in slightly lower activity of compound 14h. Compound 14i containing 2-thiophenyl ring exhibited lower activity than 14h. 5-Bromothiophenyl substituent in derivative 14j caused its slightly decreased activity than that of 14i.

Table 1. In vitro cytotoxicity of the synthesized compounds 14a–14j (IC50, µM)a
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Molecular docking studies. Molecular docking studies were carried out for the synthesized compounds 14a–14j to identify the molecular interactions between anticancer target human topoisomerase II beta in complex with DNA ligands bound with inhibitor etoposide (PDB ID:3QX3) by using AutoDock4.2.4.

All the docked ligands demonstrated interactions with Glu477, Asp479, Arg503, Met555, and Gln778 of Topo-II enzyme and DT9, DC8, DA12, and DG13 of DNA nucleotide residues (Table 2).

Table 2. Molecular docking data for the synthesized compounds against the DNA topoisomerase II beta in complex with DNA (PDB ID: 3QX3)
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Binding affinity of the docked compounds was expressed as negative energy in kcal/mol (dock score). The ligands with more negative value of dock score had higher affinity with Topo-II and DNA binding. As an example, pyridinyl substituted compound14g formed H-bond with Gln778 (2.71 Å) and Arg503 (3.01 Å) residues, as well as hydrophobic and electrostatic interactions with Arg503 residue (Table 2, Fig. 2).

Fig. 2.
figure 2

(a) 2D and (b) 3D interactions of compound 14g with DNA Topoisomerase II beta in complex with DNA (PDB ID: 3QX3).

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Similarly, bromothiophene substituted compound 14j demonstrated H-bond interactions with Glu777(2.73), DG10(2.09), DT9(2.82), DA12(2.41), and electrostatic interactions with Arg503, DC8, DT9, and hydrophobic interactions with DC8, DA12, DG10, Arg503 active residues (Table 2, Fig. 3).

Fig. 3.
figure 3

(a) 2D and (b) 3D interactions of 14j with DNA topoisomerase II beta in complex with DNA (PDB ID: 3QX3).

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All ligands demonstrated the same interactions with Topo-II protein and DNA nucleotides and accordingly the compounds had potential of inhibiting the Topo-II protein and intercalating with the base pairs of DNA nucleotides.

EXPERIMENTAL

All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO, USA) and Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA), and used without further purification. Reactions were monitored by TLC, performed on silica gel plates covered by 60 F-254 and visualized under UV light or by iodine indicator. Melting points were determined with an electro thermal melting point apparatus and are uncorrected.

1H and 13C NMR spectra were measured on a BRUKER NMR 300 MHz and 400 MHz spectrometers using TMS as the internal standard. ESI spectra were measured on a Micro mass, Quattro LC using ESI+ software with capillary voltage 3.98 kV and ESI mode positive ion trap detector.

3-(3,4,5-Trimethoxyphenyl)-5-(4-nitrophenyl)isoxazole (6). The compound 4 (33.7 g, 0.158 mmol) and 1-ethynyl-4-nitrobenzene 5 (15 g, 0.101 mmol) were mixed with dichloromethane (80 mL), and then cooled down to 0°C. Aqueous NaOCl (11 mL, 0.158 mmol) was added drop wise to the above mixture for over 30 min, and then stirred vigorously for 8 h at room temperature. The layers were separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried with Na2SO4 and concentrated. The crude product was purified by column chromatography with ethyl acetate/hexanes (4 : 6) to afford pure compound 6, yield 66%.1H NMR spectrum, δ, ppm: 3.71 s (3H), 3.92 s (6H), 7.23 s (2H), 7.91 s (1H), 8.21 d (2H, J = 8.7 Hz), 8.42 d (2H, J = 8.7 Hz). MS (ESI): 357 [M + H]+. Found, %: C 60.62; H 4.49; N 7.91. C18H16N2O6. Calculated, %: C 60.67; H 4.53; N 7.86.

4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]benzenamine (7). Compound 6 (22 g, 0.0617 mmol) was dissolved in ethanol and mixed with 10% Pd/C (656 mg 0.00617 mmol). The reaction mixture was stirred under the atmosphere of H2 (1 atm) at room temperature for 6 h. After completion of reaction (TLC), the catalyst was filtered off over celite and the filtrate was concentrated in vacuo to provide the product 7 (yield 91%) which was used further without purification.1H NMR spectrum, δ, ppm: 3.71 s (3H), 3.92 s (6H), 7.23 s (2H), 7.31 s (2H), 7.89 s (1H), 8.15 d (2H, J = 8.4 Hz), 8.29 d (2H, J = 8.4 Hz). MS (ESI): 327 [M + H]+. Found, %: C 66.21; H 5.52; N 8.63. C18H18N2O4. Calculated, %: C 66.25; H 5.56; N 8.58.

1-(4-Hydroxyphenyl)-3-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}thiourea (9). To a solution of compound 7 (17 g, 0.0521 mmol) in absolute ethanol (5 mL), 4-isothiocyanatophenol (8) (8.45 mL, 0.0782 mmol) was added at 0oC, and stirring of the mixture was continued for 6 h at room temperature. The formed solid was filtered off and washed thoroughly with ethanol to afford the pure compound 9, yield 87%. 1H NMR spectrum, δ, ppm: 3.71 s (3H), 3.92 s (6H), 7.23 s (2H), 7.34 d (2H, J = 7.56 Hz), 7.65 d (2H, J = 7.56 Hz), 7.93 s (1H), 8.13 d (2H, J = 8.1 Hz), 8.25 d (2H, J = 8.1 Hz), 9.41 s (1H), 10.32 bs (1H), 11.40 s (1H). MS (ESI): 478 [M + H]+. Found, %: C 62.84; H 4.82; N 8.84. C25H23N3O5S. Calculated, %: C 62.88; H 4.85; N 8.80.

2-{4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]phenylamino}benzo[d]thiazol-6-ol (10). Compound 9 (20 g, 0.0419 mol) was added to 1,3-di-n-butylimidazolium bromide ([bbim][Br]) (11 g, 0.0419 mmol), and the reaction mixture was stirred at 70°C for 40 min. After completion of the reaction (TLC), chloroform was added to the mixture and the product was separated as crude residue. The mixture was filtered and the residue was recrystallized from ethanol to afford pure compound 10, yield 87%. 1H NMR spectrum, δ, ppm: 3.72 s (3H), 3.93 s (6H), 7.24 s (2H), 7.41 s (1H), 7.48 d (1H,J = 8.6 Hz), 7.62 d (1H, J = 8.6 Hz), 7.95 s (1H), 8.16 d (2H, J = 8.3 Hz), 8.27 d (2H, J = 8.3 Hz), 10.36 bs (1H), 12.54 s (1H). MS (ESI): 476 [M + H]+. Found, %: C 63.11; H 4.42; N 8.89. C25H21N3O5S. Calculated, %: C 63.15; H 4.45; N 8.84.

N-{4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]phenyl}-6-(prop-2-ynyloxy)benzo[d]thiazol-2-amine (12). Compound 10 (16 g, 0.0336 mmol) was dissolved in dry N,N-dimethyl formamide (50 mL), and K2CO3 (9.3 g, 0.0673 mmol) was added to the mixture which was stirred for 15 min at room temperature. Propargyl bromide 11 (2.6 mL, 0.0336 mmol) was slowly added drop wise to the above mixture over a period of 15 min and stirring was continued for 4 h. The reaction was quenched with water and extracted with ethyl acetate (3×20 mL). The combined extracts were washed with water (3×25 mL) and brine (20 mL). The organic layer was dried over Na2SO4, filtered, evaporated under reduced pressure, and purified by silica gel chromatography with ethyl acetate/hexane (1 : 1) to afford pure intermediate12, yield 80%. 1H NMR spectrum, δ, ppm: 3.10 s (1H), 3.72 s (3H), 3.93 s (6H), 4.90 s (2H), 7.24 s (2H), 7.43 s (1H), 7.50 d (1H, J = 8.6 Hz), 7.63 d (1H, J = 8.6 Hz), 7.95 s (1H), 8.15 d (2H,J = 8.4 Hz), 8.26 d (2H, J = 8.4 Hz), 12.55 s (1H). MS (ESI): 514 [M + H]+. Found, %: C 65.44; H 4.47; N 8.23. C28H23N3O5S. Calculated, %: C 65.48; H 4.51; N 8.18.

6-[(3-Phenylisoxazol-5-yl)methoxy]-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14a). Benzaldoxime (13a) (0.17 mL, 15.5 mmol) and compound 12 (500 mg, 9.7 mmol) were dissolved in dichloromethane (10 mL), and the solution was cooled to 0°C. Aqueous NaOCl (0.1 mL, 15.5 mmol) was added dropwise over 30 min, and the reaction mixture was stirred vigorously for 8 h at room temperature. The layers were separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried with Na2SO4 and concentrated. The crude product was purified by column chromatography with ethyl acetate/hexanes (6 : 4) to afford pure compound 14a. Yield 50%, mp 212–214°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.12 s (2H), 7.23 s (2H), 7.39–7.56 m (5H), 7.64 d (1H, J = 8.7 Hz), 7.71 d (2H, J = 7.65 Hz), 7.81 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.5 Hz), 8.27 d (2H, J = 8.5 Hz), 12.56 s (1H). 13C NMR spectrum, δ, ppm: 57.4, 61.8, 65.3, 96.3, 96.9, 107.3, 108.5, 115.3, 119.6, 120.5, 124.5, 125.6, 126.2, 126.8, 127.5, 128.2, 130.4, 134.5, 137.3, 142.5, 149.6, 154.7, 155.4, 157.6, 160.5, 164.7, 165.8, 171.8. MS (ESI): 633 [M + H]+. Found, %: C 66.40; H 4.41; N 8.90. C35H28N4O6S. Calculated, %: C 66.44; H 4.46; N 8.86.

The compounds 14b–14j were synthesized according to the method developed for 14a using the appropriate benzaldoximes 4, 13b–13i.

6-{[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl) isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14b). Yield 52%, mp 222–224°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.86 s (3H), 3.92 s (6H), 3.96 s (6H), 5.09 s (2H), 7.24 s (2H), 7.43 s (1H), 7.52–7.60 m (3H), 7.64 d (1H, J = 8.5 Hz), 7.80 s (1H), 7.95 s (1H), 8.14 d (2H, J = 8.4 Hz), 8.26 d (2H, J = 8.4 Hz), 12.54 s (1H).13C NMR spectrum, δ, ppm: 57.5, 58.3, 61.7, 62.4, 65.4, 96.4, 96.8, 106.5, 107.4, 107.7, 115.6, 119.6, 120.4, 126.4, 127.6, 128.7, 130.5, 134.2, 137.6, 142.3, 149.6, 154.3, 155.5, 157.3, 158.1, 159.5, 164.6, 165.7, 171.8. MS (ESI): 723 [M + H]+. Found, %: C 63.11; H 4.70; N 7.80. C38H34N4O9S. Calculated, %: C 63.15; H 4.74; N 7.75.

6-{[3-(2,4-Dimethoxyphenyl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14c). Yield 47%, mp 227–229°C NMR spectrum, δ, ppm: 3.70 s (3H), 3.72 s (3H), 3.86 s (3H), 3.92 s (6H), 4.97 s (2H), 6.85 s (1H), 7.09 d (1H,J = 7.34 Hz), 7.24 s (2H), 7.43 s (1H), 7.55 d (1H, J = 8.3 Hz), 7.61–7.68 m (2H), 7.81 s (1H), 7.95 s (1H), 8.15 d (2H, J = 8.5 Hz), 8.26 d (2H, J = 8.5 Hz), 12.55 s (1H).13C NMR spectrum, δ, ppm: 56.4, 57.6, 58.5, 61.7, 65.7, 96.4, 97.6, 101.7, 107.4, 107.8, 109.5, 110.3, 115.4, 119.6, 120.3, 126.4, 126.7, 127.5, 130.5, 134.2, 137.5, 142.3, 149.5, 154.3, 157.6, 158.2, 160.4, 162.4, 164.5, 164.7, 165.6, 171.7. MS (ESI): 693 [M + H]+. Found, %: C 64.10; H 4.62; N 8.14. C37H32N4O8S. Calculated, %: C 64.15; H 4.66; N 8.09.

6-{[3-(2,3-Dimethoxyphenyl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl) isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14d). Yield 51%, mp 225–227°C NMR spectrum, δ, ppm: 3.71 s (3H), 3.73 s (3H), 3.87 s (3H), 3.92 s (6H), 4.97 s (2H), 6.86–6.90 m (1H), 7.12 t (1H), 7.23 s (2H), 7.44 s (1H), 7.56 d (1H, J = 8.2 Hz), 7.60–7.69 m (2H), 7.82 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.3 Hz), 8.27 d (2H, J = 8.3 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 56.4, 57.7, 61.5, 61.8, 65.7, 96.4, 97.5, 107.3, 107.8, 115.4, 118.2, 119.5, 120.2, 120.6, 121.5, 123.4, 126.5, 127.6, 130.4, 134.2, 137.2, 142.4, 147.5, 149.7, 154.3, 154.7, 157.4, 157.8, 162.4, 164.7, 165.3, 171.8. MS (ESI): 693 [M + H]+. Found, %: C 64.11; H 4.63; N 8.14. C37H32N4O8S. Calculated, %: C 64.15; H 4.66; N 8.09.

6-{[3-(3-Bromophenyl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14e). Yield 58%, mp 229–231 NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.08 s (2H), 7.24 s (2H), 7.39–7.51 m (3H), 7.56 d (1H, J = 8.3 Hz), 7.59–7.70 m (2H), 7.84 s (1H), 7.96 s (1H), 8.10 s (1H), 8.15 d (2H, J = 8.5 Hz), 8.27 d (2H, J = 8.5 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.4, 61.7, 65.8, 96.4, 97.2, 107.3, 107.7, 115.3, 119.5, 120.5, 125.4, 126.4, 127.5, 128.2, 130.4, 131.3, 133.4, 133.6, 134.7, 137.4, 142.5, 149.7, 154.6, 155.6, 157.4, 160.5, 164.5, 165.3, 171.8. MS (ESI): 713 [M + H]+. Found, %: C 59.04; H 3.77; N 7.92. C35H27N4O6BrS. Calculated, %: C 59.08; H 3.82; N 7.87.

6-{[3-(4-Nitrophenyl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14f). Yield 60%, mp 230–232°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.13 s (2H), 7.24 s (2H), 7.44 s (1H), 7.56 d (1H, J = 8.4 Hz), 7.66 d (1H, J = 8.4 Hz), 7.86 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.5 Hz), 8.22 d (2H, J = 7.67 Hz), 8.27 d (2H, J = 8.5 Hz), 8.34 d (2H, J = 7.67 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.6, 61.8, 65.6, 96.4, 97.6, 107.6, 108.3, 115.4, 119.5, 120.7, 126.4, 127.3, 127.8, 128.6, 130.5, 131.3, 134.7, 137.6, 142.4, 149.6, 150.4, 154.6, 155.5, 157.4, 160.6, 164.5, 165.8, 171.8. MS (ESI): 678 [M + H]+. Found, %: C 62.99; H 3.97; N 10.37. C35H27N5O8S. Calculated, %: C 62.03; H 4.02; N 10.33.

6-{[3-(Pyridin-4-yl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14g). Yield 56%, mp 255–257°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.15 s (2H), 7.24 s (2H), 7.45 s (1H), 7.56 d (1H, J = 8.5 Hz), 7.65 d (1H, J = 8.5 Hz), 7.87 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.6 Hz), 8.27 d (2H, J = 8.6 Hz), 8.52 d (2H, J = 8.01 Hz), 8.78 d (2H, J = 8.01 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.4, 61.8, 65.7, 96.4, 97.8, 107.5, 108.3, 115.3, 119.6, 120.5, 121.8, 126.5, 127.4, 130.5, 134.6, 137.3, 138.5, 142.6, 149.7, 152.4, 154.6, 155.7, 157.4, 160.5, 164.5, 165.8, 171.8. MS (ESI): 634 [M + H]+. Found, %: C 64.39; H 4.25; N 11.10. C34H27N5O6S. Calculated, %: C 64.44; H 4.29; N 11.05.

6-{[3-(1H-Pyrrol-2-yl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14h). Yield 60%, mp 258–260°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.10 s (2H), 7.24 s (2H), 7.32 d (1H, J = 8.10 Hz), 7.39 t (1H), 7.44 s (1H), 7.52–7.59 m (2H), 7.66 d (1H, J = 8.4 Hz), 7.86 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.6 Hz), 8.27 d (2H, J = 8.6 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.6, 61.8, 65.7, 94.5, 96.7, 107.3, 107.8, 113.4, 115.4, 116.7, 119.5, 120.4, 122.3, 126.5, 127.3, 129.5, 130.4, 134.2, 137.5, 142.4, 149.6, 150.3, 154.5, 157.3, 157.8, 164.5, 165.2, 171.7. MS (ESI): 622 [M + H]+. Found, %: C 63.72; H 4.33; N 11.32. C33H27N5O6S. Calculated, %: C 63.76; H 4.38; N 11.27.

6-{[3-(Thiophen-2-yl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14i). Yield 61%, mp 263–265°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.11 s (2H), 7.24 s (2H), 7.32 t (1H), 7.38 d (1H, J = 8.3 Hz), 7.44 s (1H), 7.54–7.59 m (2H), 7.66 d (1H,J = 8.4 Hz), 7.86 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.6 Hz), 8.27 d (2H, J = 8.6 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.5, 61.8, 65.7, 96.4, 97.3, 107.4, 107.8, 115.6, 119.4, 120.4, 125.2, 126.4, 127.5, 128.2, 130.2, 130.8, 134.5, 137.6, 139.6, 142.4, 149.6, 151.5, 154.3, 157.3, 157.6, 164.5, 165.2, 171.8. MS (ESI): 639 [M + H]+. Found, %: C 62.01; H 4.05; N 8.82. C33H26N4O6S2. Calculated, %: C 62.05; H 4.10; N 8.77.

6-{[3-(5-Bromothiophen-2-yl)isoxazol-5-yl]methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]thiazol-2-amine (14j). Yield 59%, mp 270–272°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.13 s (2H), 7.24 s (2H), 7.42–7.50 m (2H), 7.56 d (1H, J = 7.95 Hz), 7.66 d (1H, J = 8.5 Hz), 7.69 d (1H, J = 8.2 Hz), 7.87 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.7 Hz), 8.27 d (2H, J = 8.7 Hz), 12.56 s (1H).13C NMR spectrum, δ, ppm: 57.6, 61.8, 65.8, 96.5, 97.2, 107.5, 107.9, 115.2, 115.7, 119.5, 120.7, 126.4, 127.4, 130.3, 130.7, 131.5, 134.5, 137.3, 138.6, 141.4, 149.7, 152.4, 154.5, 157.3, 157.8, 164.5, 165.6, 171.9. MS (ESI): 719 [M + H]+. Found, %: C 55.19; H 3.47; N 7.86. C33H25N4O6S2Br. Calculated, %: C 55.23; H 3.51; N 7.81.

MTT assay. Individual wells of a 96-well tissue culture micro titer plate were inoculated with 100 µL of complete medium containing 1×104 cells. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 18 h prior to the experiment. After medium removal, 100 µL of fresh medium containing the test compounds and etoposide at concentrations 0.5, 1 and 2 µM were added to each well and incubated at 37°C for 24 h. Then the medium was discarded and replaced with 10 µL MTT dye. Plates were incubated at 37°C for 2 h. The resulting formazan crystals were solubilized in 100 µL extraction buffer. The optical density was recorded at 570 nm with a micro plate reader (Multi-mode Varioskan Instrument-Themo Scientific). Percentage of DMSO in the medium never exceeded 0.25%.

Molecular docking. The DNA ligand structures were drawn using the build panel and prepared using Maestro. Energy minimization was carried out using OPLS-2005 forcefield. For docking, grid parameter file (.gpf) and docking parameter files (.dpf) were written using MGL Tools-1.5.6. Receptor grids were generated using 86×68×96 Grid points in xyz with grid spacing of 0.441 Å with grid centre, x = 30.911, y = 101.462, and z = 41.536. Grid box was generated by considering active site residues. Map types were generated using Autogrid 4.2. Docking was carried out with a number of runs: 50, population size: 150, number of evaluations: 2,500,000, and number of generations: 27,000, using Autodock 4.2. Analysis of docking results was done using MGL Tools-1.5.6. Top scoring molecule in the largest cluster was analysed for its interactions with the protein. The 2D interactions were generated from Proteinplus online pose viewer. The 3D interactions were obtained with Discovery studio Visualizer. The synthesized compounds were docked into the X-ray crystal structure of human topoisomerase IIbeta domain (PDB ID: 3QX3) for considering the possible target mechanism of action.

CONCLUSIONS

In summary, we have designed and synthesized a novel series of bis-isoxazole incorporated benzothiazole derivatives and tested those in vitro against human cancer cell lines such as breast MCF-7 and MDA MB-231, lungs A549, and prostate DU-145 using the MTT assay and etoposide as a standard drug. Most of the compounds have demonstrated good to moderate activity, and some of those have exhibited more potent activity than etoposide. According to molecular docking, the compounds can be characterized by high free energy of binding interactions with human topoisomerase II beta in complex with DNA.