Synthesis, Crystal Structure and Cytotoxic Activities of Oxazolidin-2-one Derivatives①

YANG Quan-Li WANG Ai-Ling WANG Jun-Zhi WANG Long LIU Ming-Guo

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Synthesis, Crystal Structure and Cytotoxic Activities of Oxazolidin-2-one Derivatives①

YANG Quan-Li WANG Ai-Ling WANG Jun-Zhi WANG Long LIU Ming-Guo②

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

Four cytotoxic oxazolidin-2-one derivatives were prepared from alkynyl alcohol and isocynate with high yields of 83～95%, and their structures were characterized by IR, H-RESI-MS and NMR analysis. Meanwhile, the crystal of ()-4-benzylidene-3-ethyl-1-oxa-3-azaspiro[4.4] nonan-2-one (5a) was obtained and determined by X-ray single-crystal diffraction. Crystal data: monoclinic system, space group121/1,= 10.9284(2),= 9.47510(10),= 14.2510(2) ?,= 111.917(2)o,= 1369.01(3) ?3,= 4,(000) = 552.0,D= 1.248 Mg/m3,= 0.652 mm-1,= 0.0473 and= 0.1207 for 2699 independent reflections (int= 0.0206) and 2581 observed ones (> 2()).

oxazolidin-2-one, synthesis, crystal structure, cytotoxic activity,X-ray crystallography;

1 INTRODUCTION

At present, the tumor has become a major threat to human health. The world's annual 13% of the number of death is from malignant tumors, and China also has more than 60% of cancer mortality[1].Abnormal lesions of malignant tumors can occur in the cell signal transduction, cell cycle regulation, induction of apoptosis, important proteins of angiogenesis and the interaction between cells and extracellular matrix and other factors[2]. Furthermore, tumor cells that survived chemotherapy are often resistant to a broad range of drugs[3], thus making the continual search for novel types of drugs an important and relevant task.

Nitrogen-containing heterocyclic compounds usually exhibit good biological activity and play an important role in medicine and agricultural pro- duction[4]. Many nitrogen-containing heterocyclic compounds have been developed into new varieties of medicine and pesticides. Among them, oxazo- linone and its derivatives are a kind of heterocyclic compounds with broad biological activity[5]and important organic synthesis intermediates[6].In the course of our research towards the development of new anti-cancer agents, we have previously reported the synthesis of cytotoxic heterocycles bearing nitrogen[7, 8], sulphur[9]and triazole[10]moieties. To further explore the activity of these agents, the target ()-4-benzylidene-3-ethyl-1-oxa- 3-azaspiro[4.4]non an-2-one derivatives were synthesized and charac- terized in this work (Scheme 1).

Scheme 1. Synthetic routes for the title compound 5

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. Thin layer chromatography (TLC) was performed on silica gel GF2540.2 mm precoated plates (Yantai Chemical Industry Research Institute, Yantai, China). Product spots were visualized under UV light at 254 and 365 nm. Melting points were determined with an uncorrected X-4 digital melting point apparatus. The single-crystal X-ray diffraction analysis was per- formed on a Rigaku Mecury CCD diffractometer.1H NMR and13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz plus NMR spectro- meter. IR spectra were recorded on a PE-983 infrared spectrometer as KBr pellets with absorption in cm-1. MS data were measured on the MALDI TOF/TOF 5800 system.

2. 2 Structure determination synthesis and spectroscopy characterization

-Butyllithium (2.0 M in cyclohexane, 3.0 mL) was added dropwise to a stirred solution of alkyne (1, 5.0 mmol) in dry THF (20.0 mL) at –78 ℃ under an argon atmosphere. After 30 min, aldehyde or ketone (2, 5.0 mmol) was added and the mixture was allowed to warm to room temperature and stirred for two hours. The reaction was quenched by the addition of saturated aqueous NaHCO3and the organic phase was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried with MgSO4and concentrated. The residue was purified by column chromatography to give the propargylic alcohol (3)[11, 12].

The mixture of propargylic alcohol (3, 1.0 mmol) and isocyanate or isothiocyanate (4, 1.0 mmol) in anhydrous acetonitrile (5.0 mL) was stirred for 10 minutes under N2, then1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 30.5 μL, 0.2 mmol) was added dro- pwise and stirred for overnight at 30 ℃for 24 h. The reaction was then quenched with water (15.0 mL) and extracted with EtOAc (3 × 10.0 mL). The combined organic layers were washed with brine and dried with Na2SO4. After the removal of solvent, the residue was analyzed/selectivity by1H NMR and further purified by column chromatography (eluent: hexane/EtOAc = 50:1, v/v) to give the corresponding oxazolidin-2-one derivatives (5).

()-4-Benzylidene-3-ethyl-1-oxa-3-azaspiro[4.4]nonan-2-one (5a). White solid, yield 85%. m.p.: 135～137 ℃.1H NMR (CDCl3, 400 MHz)(ppm): 7.34～7.29 (m, 2H, Ar-H), 7.25～7.18 (m, 3H, Ar-H), 5.53 (s, 1H, =CH), 3.38 (dd,= 7.0 Hz, 2H, -CH2), 2.20～2.16 (m, 2H, -CH2), 1.96～1.83 (m, 6H, 3×-CH2), 0.74 (t,= 7.0 Hz, 3H, -CH3).13C NMR (CDCl3, 100 MHz)(ppm): 156.6, 141.5, 134.8, 129.4, 127.8, 126.7, 97.6, 92.4, 41.4, 38.0, 24.4, 12.4. IR (KBr)(cm-1): 3452, 2971, 1748, 1674, 1456, 1364, 1267, 1136, 1040, 963, 707. H RMS (ESI): calcd. for [C16H19NO2+H]+258.1494. Found: 258.1492.

()-4-Benzylidene-5,5-diethyl-3-(4-fluorophenyl)oxazolidin-2-one (5b). White solid, yield 95%. m.p.: 52～54 ℃.1H NMR (CDCl3, 400 MHz)(ppm): 6.91～6.88 (m, 5H, Ar-H), 6.74～6.66 (m, 4H, Ar-H), 5.55 (s, 1H, =CH), 2.11～2.01 (m, 2H, -CH2), 1.91～1.80 (m, 2H, -CH2), 1.57 (s, 1H, -CH), 1.09 (t,= 7.4 Hz, 6H, 2×-CH3).13C NMR (CDCl3, 100 MHz)(ppm): 162.4, 160.0, 156.0, 139.3, 133.3, 131.0, 128.5, 127.7, 127.2, 126.0, 115.2, 115.0, 99.6, 88.5, 33.1, 7.2. IR (KBr)(cm-1): 3449, 2975, 1770, 1661, 1514, 1405, 1332, 1232, 1133, 822, 701, 624. HRMS (ESI): calcd. for [C20H20FNO2+H]+348.1376. Found: 348.1378.

()-4-Benzylidene-3,5-diisopropyl-5-methyloxazo- lidin-2-one (5c). White solid, yield 86%. m.p.: 107～109 ℃.1H NMR (CDCl3, 400 MHz)(ppm): 7.34～7.30 (m, 2H, Ar-H), 7.26～7.18 (m, 3H, Ar-H), 5.37 (s, 1H, =CH), 3.71～3.62 (m, 1H, -CH), 1.94～1.86 (m, 1H, -CH), 1.52 (s, 3H, -CH3), 1.24 (d,= 6.8 Hz, 6H, 2×-CH3), 1.07 (d,= 6.8 Hz, 3H, -CH3), 1.00 (d,= 6.8 Hz, 3H, -CH3).13C NMR (CDCl3, 100 MHz)(ppm): 155.9, 143.4, 135.4, 128.8, 128.1, 126.7, 97.2, 86.2, 47.0, 37.8, 25.3, 18.8, 18.7, 16.4, 16.2. IR (KBr)(cm-1): 3455, 2968, 1748, 1677, 1405, 1380, 1290, 1024, 726, 637. HRMS (ESI): calcd. for [C17H23NO2+Na]+296.1626. Found: 296.1618.

()-4-Benzylidene-3-(4-fluorophenyl)-1-oxa-3-aza-spiro[4.5]decan-2-one (5d). White solid, yield 83%. m.p.: 157～159 ℃.1H NMR (CDCl3, 400 MHz)(ppm): 6.97～6.87 (m, 5H, Ar-H), 6.74～6.64 (m, 4H, Ar-H), 5.60 (s, 1H, =CH), 2.16～2.12 (m, 2H, -CH2), 1.86～1.70 (m, 7H, -CH, 3×-CH2), 1.40～1.32 (m, 1H, -CH).13C NMR (CDCl3, 100 MHz)(ppm): 162.3, 159.8, 155.4, 142.5, 128.4, 127.6, 127.6, 127.1, 125.9, 115.1, 114.9, 99.9, 84.8, 37.2, 24.6, 21.7. IR (KBr)(cm-1): 3442, 2933, 1751, 1658, 1508, 1232, 1111, 954, 835. HRMS (ESI): calcd. for [C21H20FNO2+Na]+360.1376. Found: 360.1377.

2. 3 Single-crystal structure determination

A colorless prism crystal with dimensions of 0.20mm × 0.16mm × 0.14mm was selected for measurement. Diffraction data of the single crystal were collected at 100.00(10) K on a Rigaku Mecury CCD diffractometer equipped with a graphite-mono- chromatic Curadiation (= 1.54184 ?) by Crystal clear software. A total of 10428 reflections were collected in the range of 4.36≤≤73.43o by using an-scan mode, of which 2699 were unique withint= 0.0206 and 2581 were observed with> 2(). Empirical absorption corrections were applied. The structure was solved by direct methods using SHELXS-97 programs[20]. 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[21]. The hydrogen atoms were added according to the theoretical model. The final= 0.0473,= 0.1207, (Δ/)max= 0.000,= 1.066, (Δ)max= 0.32 and (Δ)min= –0.449 e×?-3.

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

Human epidermoid cervical carcinoma (CaSki) cell lines and human hepatoblastoma (HepG2.215) cell lines 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[13]. 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, 50, 25, 12.5 and 6.25 μg/mL) for 48 hours, 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 hours. After the removal of media, the MTT-for- mazan 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 excluded cells)/No. of the total. The growth inhibition (%) was determined in triplicate at concentrations 5, 10, 20, 40, 80 and 160 μM. All data were presented as mean ± standard deviationand analyzed by SPSS 30[14, 15]. The half-inhibitory concentration (50value) was calculated and listed in Table 1 based on the percentage inhibition of cell growth.

Table 1. Selected Bond Lengths (?), Bond Angles (°) and Torsion Angles (°) for Compound 5a

3 RESULTS AND DISCUSSION

3. 1 Synthesis, spectroscopy analysis and cyclization mechanism

All the intermediates and oxazolidin-2-one deriva- tives were characterized by NMR, IR and HR-ESI- MS. In1H NMR spectrum, the olefinic protons of exocyclic double bond of 2-oxazolidinone skeleton are located at 5.37～5.60 ppm as singlet. The strong infrared absorption peak at 1748～1770 cm-1was assigned to the carbonyl group in all the target compounds. Their carbon signs of carbamate group for 5a～5d were found at 155.9～162.4 ppm in13C NMR. In the HR-ESI-MS spectrum, the ion adducts of (M + H)+or (M + Na)+were usually observed as the base peak ion for all the target compounds.

According to some related literatures[16, 17], a pos- sible mechanism of the DBU-promoted nucleophilic addition/cyclization reaction could be tentatively explained as shown in Fig. 2. Firstly, propargylic alcohol intermediate (3) was deprotonated by DBU to give alkoxyl anion (A), which nucleophilically attacked isocyanate (4) to generate carbamate ester (B). Then, a 5intramolecular nucleophilic N–H in carbamate ester (B) attacked to the conjugate base of DBU-activated carbon-carbon triple bond to give the DBU complex (C). Finally, the correspond- ding product (5) was generated through the release of proton from DBU.

Fig. 1. Crystal structure of compound 5a with numbered atoms

Fig. 2. Possible reaction mechanism

3. 2 Crystal structure description

ORTEP drawing of compound 5a 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 O(1)-C(3) and N(1)-C(2) exhibit typical single bonds in 1.4699(15) and 1.4034(16) ?, respectively. The bond length for the carbonyl group of O(2)-C(1) is 1.2125(17) ?. The bond length of 1.3350(18) ? confirmed the signi- ficant double-bond character for C(2)-C(6), whose substituents adopted a (Z)-conformation. The angles containing unsaturated bonds of O(2)-C(1)-O(1) and O(2)-C(1)-N(1) correspond to 122.72(12)° and 127.05(13)°, respectively. The N(1)-C(2)-C(3)-C(16) torsion angle is 125.92(12)°. The oxazolidin- 2-one ring of O(1)-C(1)-N(1)-C(2)-C(3) in Fig. 1 formed almost a plane with a maximum deviation of 0.0423 ? from the plane for C(2), but the cyclo- pentane ring of C(3)-C(13)-C(14)-C(15)-C(16) showed a similar envelope conformation with the puckering parameters of= 0.3616 ? and= 199.5308°.

3. 3 Evaluation of the bioactivity

All the target compounds were evaluated for their cytotoxic activity against a panel of human cancer cell lines including CaSki and HepG2.215 cell lines for 48 hours at 37℃ by MTT assays (Table 2). Compared with the positive control paclitaxel[18, 19], compound 5c showed better anti-proliferative effect against the two selected cell lines with corresponding50values of 26.51 and 4.89 μmol/L, respectively. These results indicated that the N-isopropyl and acyclic substituents on the oxazolidin-2-one ring might be beneficial to increase the cytotoxicities.

Table 2. Bioactivity Evaluation for the Target Compounds

a Positive control

4 CONCLUSION

A series of oxazolidin-2-one derivatives were synthesized by DBU promoted nucleophilic addi- tion/cyclization from alkynyl alcohol and isocynate or thiocynate in high yields of 83～95%. After the structural characterization and biological evaluation, the compound 5csubstituted byN-isopropyl was found to possess most potent cytotoxicities against the test tumor cell lines.

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30 July 2017;

12 December 2017 (CCDC 1514794)

10.14102/j.cnki.0254-5861.2011-1798

① Financially supported by the National Natural Science Foundation of China (21602123), and Scientific Foundation from graduate school of China Three Gorges University (SDYC2016121)

②. Prof. Liu Ming-Guo (1966-), majoring in synthetic medicinal chemistry. E-mail: mgliu1966@163.com