Synthesis and Crystal Structure of(Z)-2-Methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one O-Prop-2-yn-1-yl Oxime Derivatives①

CAO Cheng-Qio YAN Xi-Ming YANG Qun-Li LUO Hu-Jun HUANG Nin-Yu②

a (Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, China)

1 INTRODUCTION

Gastric cancer remains the fourth most commonly diagnosed cancer and is the second leading cause of cancer-related mortality worldwide[1]. Regular therapies including chemotherapy, bio- therapy and radiotherapy against gastric cancer have been widely applied, however, they have unavoi- dable side effects[2,3]. Therefore, more effective alternative approaches for the prevention and therapies against gastric cancer without undesirable side-effects have emerged as one of the most intensive areas of investigation in drug discovery.

Recent studies have demonstrated many natural or synthesized compounds are designed as potential chemotherapeutic agents for the treatment of gastric cancer[4,5]. Heterocycles bearing nitrogen, sulphur and thiazole moieties constitute the core structure of many interestingly biological compounds with significant therapeutic applications[6,7]including anti-tumor[8], antimicrobial[9], anti-inflammatory[10],antiviral[11], antioxidant[12]and other activities. In order to present the anti-gastric cancer activities of benzothiazoles, the target 5,6-dihydrobenzo[d]thiazole derivatives were synthesized and characterized in this work (Scheme 1).

2 EXPERIMENTAL

2.1 Reagents and physical measurements

All chemicals were of analytical reagent grade and purchased from commercial sources, which were used directly without further purification.Melting points were determined with an uncorrected X-4 digital melting point apparatus.1H NMR and13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz Plus NMR spectrometer.The single-crystal X-ray diffraction analysis was performed on a Rigaku Mecury CCD diffractometer.IR spectra were recorded on a PE-983 infrared spectrometer as KBr pellets with absorption in cm-1.

2.2 Synthesis and spectroscopy characterization

A mixture of 2-bromocyclohexane-1,3-dione (I,3.80 g, 20 mmol) and thioacetamide (1.50 g, 20 mmol) in pyridine (50 mL) was heated at 60 ℃ for 6 hours under nitrogen atmosphere. After completion of the reaction, the solvent was removed under vacuum, and the residue was washed with saturated brine (50 mL). The product was extracted with dichloromethane (3 × 30 mL). The combined organic extracts were dried and concentrated under vacuum to give the dark viscous oil, which was purified by flash column chromatography (eluent:ethyl acetate/petroleum ether = 1:4, v/v) to yield 2-methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one (II)as a yellow oil[13](2.40 g, 71%).

The 2-methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one (II, 1.67 g, 10.0 mmol) was dissolved in pyridine (40 mL), and hydroxylamine hydrochlororide was added (0.83 g, 12.0 mmol) under nitrogen atmosphere. The mixture was stirred for 4 h at 80 ℃, then cooled and poured into ice-water(250 mL) to give white precipitates. Filtrating the precipitates and drying give the 2-methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one oxime mixtures[14]as white solids (1.80 g), and the Z-oxime isomer (III, 0.88 g) was isolated by column chromatography (n-hexane: EtOAc = 5:1, v/v).

(Z)-2-Methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one oxime (III): Yield 93%, m.p.: 199～201 ℃.1H NMR (400 MHz, CDCl3) δ (ppm): 10.23 (s, 1H,OH), 2.99 (t, J = 4.8 Hz, 2H, CH2), 2.72 (s 3H,CH3), 2.65 (d, J = 6.0 Hz, 2H, CH2), 2.07 (d, J = 4.0 Hz, 2H, CH2).13C NMR (100 MHz, CDCl3) δ(ppm): 170.4, 158.9, 148.8, 118.4, 29.3, 28.0, 22.7,18.7.

The Z-oxime isomer (III, 0.36 g, 2.0 mmol) was dissolved in CH3CN (10 mL), and anhydrous K2CO3(0.41 g, 3.0 mmol) was added. The mixture was stirred for 0.5 h at room temperature, and 3-bromopropyne (0.24 g, 2.0 mmol) was added and stirred for 2 h at 80 ℃. The solvent was removed and the product was extracted with ethyl acetate (3× 20 mL). The combined organic extracts were dried and concentrated under vacuum to leave a yellow crude product. Purification by flash column chromatography (n-hexane:ethyl acetate = 3:1, v/v)afforded pure (Z)-2-methyl-5,6-dihydrobenzo[d]-thiazol-7(4H)-one O-prop-2-yn-1-yl oxime (IV) as white solids (0.41 g). X-ray quality crystals were obtained by the vapor diffusion of petroleum ether into a solution of IV in chloroform at room temperature.

(Z)-2-Methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one O-prop-2-yn-1-yl oxime (IV): Yield 93%, m.p.:81～83 ℃.1H NMR (400 MHz, CDCl3) δ (ppm):4.77 (d, J = 2.4 Hz, 2H, O-CH2), 2.95 (t, J = 6.4 Hz,2H, CH2), 2.69 (s, 3H, CH3), 2.66～2.62 (m, 2H,CH2), 2.51 (t, J = 2.4 Hz, 1H, ≡C-H), 2.09～2.03(m, 2H, CH2).13C NMR (100 MHz, CDCl3) δ(ppm): 170.2, 159.5, 149.5, 118.5, 79.4, 74.5, 61.2,29.2, 28.0, 22.6, 18.7. IR (KBr) ν (cm-1): 2961,2928, 2869, 2116, 1607, 1503, 1450, 1429, 1372,1358, 1310, 1271, 1185, 1099, 1048.

The propargyl oxime ether (IV, 220.1 mg, 1.0 mmol) and 2-azido-1-(3-hydroxyphenyl)ethanone(177.1 mg, 1.0 mmol) were suspended in a 1:1 mixture of water and tert-butyl alcohol (10 mL).Sodium ascorbate (0.1 mmol) was added, followed by copper(II) sulfate pentahydrate (2.5 mg, 0.01 mmol). The heterogeneous mixture was stirred vigorously at 80 ℃ overnight until complete consumption of the reactants. The reaction mixture was diluted with cooled water (50 mL) to give white precipitates. Filtration and drying under vacuum afforded the pure product of V as white solids.

(Z)-1-(3-Hydroxyphenyl)-2-(4-((((2-methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-ylidene)amino)oxy)-methyl)-1H-1,2,3-triazol-1-yl)ethanone (V): Yield 95%, m.p.: 208～209 ℃.1H NMR (400 MHz,DMSO-d6) δ (ppm): 9.90 (s, 1H, OH), 8.09 (s, 1H,triazole-H), 7.53 (d, J = 7.6 Hz, 1H, Ar-H), 7.43～7.38 (m, 2H, Ar-H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H),6.11 (s, 2H, N-CH2), 5.25 (s, 2H, O-CH2), 2.86 (t, J= 5.6 Hz, 2H, CH2), 2.62 (s, 3H, CH3), 2.58～2.50(m, 2H, CH2), 1.94 (t, J = 5.6 Hz, 2H, CH2).13C NMR (100 MHz, DMSO-d6) δ (ppm): 191.9, 169.5,159.3, 157.7, 148.2, 143.0, 135.4, 130.0, 125.9,121.2, 119.0, 117.5, 114.1, 66.9, 55.7, 28.6, 27.5,22.3, 18.3. IR (KBr) ν (cm-1): 3431, 2967, 2925,1697, 1599, 1584, 1453, 1372, 1268, 1179, 1057,1003, 935, 887, 774, 676.

2.3 Single-crystal structure determination

A colorless prism crystal with dimensions of 0.21mm × 0.19mm × 0.18mm was selected for measurement. Diffraction data of the single crystal were collected at 296(2) K on a Rigaku Mecury CCD diffractometer equipped with a graphitemonochromatic MoKα radiation (λ = 0.71073 ?) by Crystal clear software. A total of 11511 reflections were collected in the range of 2.85≤θ≤27.57o by using an ω-scan mode, of which 2591 were unique with Rint= 0.0709 and 2289 were observed with I >2σ(I). Empirical absorption corrections were applied.The structure was solved by direct methods using SHELXS-97 programs[15]. All of the non-hydrogen atoms were located from difference Fourier maps,and then refined anisotropically with SHELXL-97 via a full-matrix least-squares procedure[16]. The hydrogen atoms were added according to the theoretical model. The final R = 0.0457, wR =0.1298, (Δ/σ)max= 0.000, S = 1.017, (Δρ)max= 0.264 and (Δρ)min= –0.291 e/?3.

2.4 3-(4,5)-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide(MTT) assay cell viability assay

Human gastric cancer HGC-27 cells and human gastric mucosa epithelial GES-1 cells were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cell viability was assessed by MTT cell staining as previously described[17]. The cells (1.0 × 104cells/well) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS)and 100 units/mL penicillin at humidified 5% CO2atmosphere in a 96-well plate. Twelve hours later,the cells were exposed to the drugs with different concentrations (100.0, 50.0 and 10.0 μg/mL) for 48 h, and set Paclitaxel (10.0 μg/mL) as the positive control. MTT (50 μL of a 5 mg/mL in PBS;Sigma-Aldrich) was added to each well and the cells were incubated in a CO2incubator at 37 ℃ for 5 h. Following media removal, the MTT-formazan formed by metabolically viable cells was dissolved in 200 μL of DMSO (Sigma-Aldrich) and the absorbance was measured in a plate reader at 492 nm. The survival (%) was calculated by the following formula: No. of viable cells (dye excludeed cells)/No. of the total.

3 RESULTS AND DISCUSSION

3.1 Spectroscopy analysis

The oxime intermediate (III) and oxime ether(IV) were synthesized and confirmed by 1D and 2D NMR spectroscopy. The Z-configuration for the oxime (III) was determined by the NOE correlation between the hydroxyl proton (δ = 10.23 ppm) and 2-methyl proton (δ = 2.69 ppm). In compound IV,the long-range coupling constant (4J) between the terminal alkyne proton and methylene proton in the propargyl group was measured to be 2.4 Hz in its1H NMR spectroscopy, and the corresponding carbon signals in the13C NMR spectra were also found at 79.4, 74.5 and 61.2 ppm, respectively.

The triazole proton of the target compound V could be clearly observed at 8.09 ppm as singlet,and the proton in hydroxyl group is located at 9.90 ppm as singlet. The protons in the methylene groups of N-CH2and O-CH2were accordingly assigned to the two single peaks of 6.11 and 5.25 ppm in the1H NMR spectroscopy, and their corresponding carbon signals are located at 55.7 and 66.9 ppm, respectively. The thirteen resonance peaks in the range from 114 to 192 ppm were attributed to sp2-carbons in the13C NMR spectra, which indicated the dihydrobenzo[d]thiazole ring had been successfully linked to the phenyl group by the triazole moiety.The absorption peaks of hydroxyl and metadisubstituted phenyl groups could be clearly found at 3431 and 887～676 cm-1in the IR spectra.

3.2 Crystal structure description

ORTEP drawing of compound IV with common atom numbering scheme is shown in Fig. 1, and the selected bond lengths, bond angles and torsion angles are listed in Table 1. The imine of C(4)–N(2)in the oxime was purely a double bond[18]in 1.278(2)?. The angles containing unsaturated bonds of N(1)–C(1)–C(8), C(4)–N(2)–O(1) and C(11)–C(10)–C(9) correspond to 123.95(17), 110.35(14) and 177.3(2)°, respectively. The torsion angles of C(7)–C(2)–C(3)–S(1) and C(4)–C(5)–C(6)–C(7) are equal to 179.05(12) and 57.2(2)°, respectively. The oxime ether showed Z-configuration with the C(3)–C(4)–N(2)–O(1) torsion angle of –1.1(2)° (see Table 1).All ring atoms in the thiazole system of S(1)–C(1)–N(1)–C(2)–C(3) were essentially coplanar with a maximum deviation of –0.0022(8) ? from the plane for S(1). The 6-membered ring C(2)–C(3)–C(4)–C(5)–C(6)–C(7) in Fig. 1 shows a distorted chair conformation (Φ = 224.7(3)°, θ = 53.4(2)°, puckering amplitude (Q) = 0.461(2)°).

Fig. 1. Structure of compound IV

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

3.3 Evaluation of the bioactivity

As our continuous interest in search of biological nitrogen-heterocycles as anti-tumor agents[19-21], the target compounds IV and V were also evaluated for in vitro anti-proliferative activities against human gastric carcinoma cell lines (HGC-27) for 48 h at 37 ℃ by MTT assays. All data were presented as mean ± standard deviation[22]and analyzed by SPSS[23]. Paclitaxel was chosen as a positive control with the IC50of 4.16 μmol/L. The results indicated that the synthetic intermediate IV exhibited poor anti-pro- liferative effect against HGC-27 cells,however, the target compound V showed better cytotoxic effect with the IC50value of 59.20 μmol/L(Table 2). Both compounds exhibited low toxicities against the normal human gastric mucosa epithelial GES-1 cells (IC50> 100 μmol/L). Further structure optimi- zation and anti-ulcer mechanism were under way in our research group.

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