Synthesis, Crystal Structure and Antiproliferative Activity of 2-(((5-(((5,7-Dimethyl-[1,2,4]triazolo-[1,5-a]pyrimidin-2-yl)thio)methyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)methyl)-4H- chromen-4-one Methanol Solvate①

SUN Hi-Yn

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Synthesis, Crystal Structure and Antiproliferative Activity of 2-(((5-(((5,7-Dimethyl-[1,2,4]triazolo-[1,5-a]pyrimidin-2-yl)thio)methyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)methyl)-4H- chromen-4-one Methanol Solvate①

SUN Hai-Yana②

(210048)

The novel crystal of the title compound 2-(((5-(((5,7-dimethyl-[1,2,4]triazolo-[1, 5-a]-pyrimidin-2-yl)thio)methyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)methyl)-4H-chromen-4-one methanol solvate (C27H25N7O3S2,M= 559.66) has been prepared and its structure was determined by single-crystal X-ray diffraction. The crystal belongs to the monocline system, space group21/with= 11.9879(9),= 14.0743(10),= 15.9706(11)?,= 98.509(2)°,= 2664.9(3) ?3, Z = 4,D= 1.395 g/cm3,(000) = 1168,= 0.116 mm-1, Moradiation (= 0.71073),= 0.0652, and= 0.1425 for 5220 observed reflections with> 2(). X-ray diffraction analyses reveal that the molecule adopts a C-shape. The planes of the benzopyrone and triazolopyrimidine were nearly parallel to each other, with a dihedral angle of 1.21(3)°.Intramolecular and intermolecular hydrogen bonds together withinterations are found to exist in the structure. The results of MTT assay indicate that the title compound displays excellent antiproliferative activity against two human cancer cell lines.

flavonoid, 1,2,4-triazolo[1,5-a]pyrimidine, single-crystal X-ray diffraction, crystal structure, synthesis, antiproliferative activity

1 INTRODUCTION

Flavonoid derivatives are kinds of important natural products, which can be applied to protect cardiovascular, anti-inflammatory, antiviral, anti- tumor and so on[1-3].Therefore, the researches on isolation, screening and structural modification of flavonoid derivatives have attracted great attention. Therein, 1,2,4-triazolo[1,5-a]pyrimidine derivatives were widely applied in the fields of pesticides and medicines because of their excellent biological activities. Some derivatives were synthesized as potential anti-infective, anti-inflammatory and antiproliferative agents[4, 5].In this work, we des- cribe the synthesis and X-ray structure of 2-(((5- (((5,7-dimethyl-[1,2,4]triazolo[1,5-a]-pyrimidin-2-yl)-thio)methyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)methyl)-4H-chromen-4-one (compound 6). The syn- thetic route of6 is shown in Fig. 1.In the presence of sodium methoxideas base,-hydroxyaceto- phenone was condensed with diethyl oxalate to produce 1,3-dione ester (compound 1);compound 1 was cyclized to afford ethyl 4-oxo-4H-chromene- 2-carboxylate (compound 2); compound 2 was reduced by NaBH4to obtain 2-(hydroxymethyl)- 4H-chromen-4-one (compound 3); 3 reacted with SOCl2to give 2-(chloromethyl)-4H-chromen-4-one (compound 4); then 4 reacted with 5-(((5,7-dime- thyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)me-thyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (compound 5) at 90 ℃to get the desired title compound (6).The structure of 6 was characterized by1H NMR, MS andelemental analysis, and its crystal structure was further determined by X-ray diffraction method.The anti-proliferative activity of compound 6 against two types of human tumor cell lines was tested by using the MTT method[6].

2 EXPERIMENTAL

2.1 Reagents and instruments

1H NMR spectra were determined by Bruker 400 MHz NMR instrument (TMS as internal standard, Chloroform-d as solvent); MS spectra were recorded by Finnigan Trace Ms mass spectrometer; and Elementar Vario EL III elemental analyzer was used for elemental analysis. The other reagents and solvents were analytically or chemically pure if there is no special instruction. Mammary adenocar- cinoma cells (MDA-MB-231) and human gastric cancer cells (BGC-823) were obtained from CAS Shanghai Cell Bank; 96 well cell culture plates (made by Costar); MTT (Sigma, USA); Microplate Reader (made by Tokyo); the other reagents and solvents were analytically or chemically pure unless otherwise specified.

2.2 Synthesis and characterization

2.2.1 Synthesis of 2-(chloromethyl)-4H-chromen-4-one (compound 4)

According tothe procedures reported in Ref. [7], compound 4 can be obtained easily. ESI-MS: 195.5 [M+H]+.

2.2.2 Synthesis of 5-(((5,7-dimethyl- [1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (compound 5)

According tothe procedures reported in Ref. [4], compound 5 can be also prepared easily. ESI-MS: 370.5 [M+H]+.

2.2.3 Synthesis of 2-(((5-(((5,7-dimethyl- [1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)- methyl)-4-phenyl-4H-1,2,4-triazol-3-yl)-thio)methyl)-4H-chromen-4-one (compound 6)[6]

A solution of 5-(((5,7-dimethyl-[1,2,4]triazolo[1,5- a]pyrimidin-2-yl)thio)methyl)-4-phenyl-4H-1,2,4-tri-azole-3-thiol (5, 0.37 g, 1 mmol) was prepared in 1% aqueous NaOH (5 mL). Then, 1 mL of com- pound 4 (0.20 g, 1 mmol) was added in DMF. The mixture was heated to 90 ℃ in an oil bath. After the reaction was completed, the mixture was diluted with water (40 mL) and the precipitate was filtered. Finally, the precipitate was recrystallized from ethyl alcohol to give compound 6.

m.p: 159～160 ℃.1H NMR (400 MHz, CDCl3):: 2.59 (s, 3 H, CH3), 2.66 (s, 3 H, CH3), 4.37 (s, 2 H, CH2), 4.61 (s, 2 H, CH2), 6.42 (s, 1 H, 3-CH), 6.72 (s, 1 H, Het-ArH), 7.28～7.40 (m, 7 H, ArH), 7.64 (t,= 8.0 Hz,1 H, ArH), 8.14 (d,= 8.0 Hz, 1 H, ArH); Ms (m/z) 528.5 ([M+H]+). Anal. Calcd. (%) for C26H21N7O2S2: C, 59.19; H, 4.01; N, 18.58. Found (%): C, 58.99; H, 4.02; N, 18.28.

2.3 Diffraction and structure analyses

The recrystallized target compound was dissolved by methanol in 50 mL flask and then placed in dark place for natural volatilization. Single crystals were obtained after the solvent was completely vola- tilized.

The single crystal of the title compound with dimensions of 0.3mm × 0.2mm × 0.1mm was taken for X-ray diffraction analysis. The diffraction inten- sity data were collected by an Enraf-Nonius CAD4 EXPRESS diffractometer equipped with a graphite- monochromatized Mo-radiation (= 0.71073 ?) using an-2scan mode at 293±1 K.A total of 5220 independent reflections were collected in the range of 2.4<<27.9°, of which 3022 were con- sidered to be observed. The structure was solved by direct methods. Non-hydrogen atoms were deter- mined with successive difference Fourier syntheses. The hydrogen atoms were located at the calculated positions. The anisotropic thermal parameters for non-hydrogen atoms were refined by full-matrix least-squares of2.All calculations were performed on a computer with SHELXTL program package[8]. The final= 0.0652,= 0.1425 (= 1/[2(F2) + (0.0419)2+ 0.9496],where= (F2+ 2F2)/3),= 1.014,(Δ)max= 0.207 and (Δ)min= –0.246 e/?3.Crystal parameters: monocline system, space group21/,= 11.9879(9),= 14.0743(10),= 15.9706(11) ?,= 98.509(2)°,= 4,= 2664.9(3) ?3,D= 1.395 g/cm3,(000) = 1168 andM= 559.66. The selected bond lengths and bond angles are listed in Table 1, and the hydrogen bond lengths and bond angles are given in Table 2.

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

Table 2. Hydrogen-bonding Interactions (?, °)

Symmetry codes:#1:,,+1; #2:, –+1/2,–1/2

2.4 Biological activity evaluation

Evaluation on the anti-proliferative activity of the title compound against mammary adenocarcinoma cells (MDA-MB-231) and human gastric cancer cells (BGC-823) were carried out in accordance with the methods reported[6].5-Fluorouracil (5-Fu) was selected as the positive control substances. IC50(concentration of the drug required to reduce the cell viability by 50%) was used to evaluate the antiproliferative activity.

3 RESULTS AND DISCUSSION

3.1 Crystal structure

The selected bond lengths, bond angles and torsion angles are listed in Table 1. The molecular structure of the title compound with atomic labeling scheme is shown in Fig. 2. And the molecular packing in the crystalline cells is shown in Fig. 3. In the molecular structure, the C(1)–C(6), C(2)–C(3) and C(23)–C(24) bonds are obviously longer than the nomal C=C (1.34 ?), and the corresponding C(4)–N(2), C(5)–N(1), C(9)–N(4), C(12)–N(4), C(18)–N(6), C(19)–N(6), C(1)–O(1) and C(9)–O(1) bonds are evidently shorter than the normal C(2)– N (1.35 ?) and C(2)–O (1.43 ?), showing a certain degree of delocalization.

a) Diethyl oxalate,sodium methoxide, methanol,reflux, 0.5 h; b)Concentrated sulfuric acid, acetic acid, 80 ℃, 2 h; c) NaBH4, methanol, 25 ℃, 15 h; d) SOCl2, Ether, 25 ℃; e) NaOH, DMF, 90 ℃, 4 h

Fig. 1. Synthetic route of 2-(((5-(((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)- phenyl-4H-1,2,4-triazol-3-yl)thio)methyl)-4H-chromen-4-one (compound 6)

Fig. 2. Molecular structure of (I) showing the atomic labeling scheme

Fig. 3. A packing view of the title compound, showing the hydrogen bonds (dashed lines) andstacking interactions

As we expected, the triazole-pyrimidine ring Cg7 (N(4)/N(5)/C(24)/C(23)/C(22)/N(7)/C(21)/N(6)/C(20))and benzopyrone ring Cg8 (O(1)/C(1)/C(2)/C(3)/C(4)/C(5)/C(6)/C(7)/C(8)C(9)) show good co- planarity, with the max deviations of 0.011(3) ? at the N(5) atom and 0.025(43) ? at the C(7) atom. As shown in Fig. 2, the molecule adopts a C-shape. The planes of benzopyrone and triaz olopyrimidine were nearly parallel to each other, with a dihedral angle of 1.21(3)°. The dihedral angle between the triazole- pyrimidine ring Cg7 and the triazole ring Cg1(N(1)/ C(24)/C(11)/N(2)/N(3)/C(18)), the benzopyrone ring Cg8 and triazole ring Cg1 are 81.29(4) and 80.27(4)°, respectively.

The packing diagram of the title compound (Fig. 3) displays that the molecules are connected by intra- molecular and intermolecular O–H···N, C–H···O and C–H···N hydrogen bonds which make the crystal structure stable. The dihedral angles between the triazole ring Cg2 (N(4)/N(5)/C(21)/N(6)/C(20)) and the pyrimidine ring Cg4 (N(5)/C(21)/ N(7)/C(22)/ C(23)/C(24)), triazole ring Cg2 and the benzene ring Cg5 (C(1)/C(2)/C(3)/C(4)/C(5)/C(6)), the pyrimi- dine ring Cg4 and pyranone Cg3 (O(1)/C(1)/C(6)/ C(7)/C(8)C(9)), the pyrimidine ring Cg4 and the pyrimidine ring Cg4, the pyrimidine ring Cg4 and the benzene ring Cg5 in adjacent molecules (symmetry code: 2–, –, –) are in the range of 3.67(6)～10.77(9)°, and the distances between these ring centroids vary from 3.536(2)to 3.962(1) ? (Fig. 2). These short approaches suggest the existence of-interactions. Intermolecular hydrogen bonds form one-dimensional chains parallel to theaxis, and-stacking interactions and intermolecular hydrogen bonds connect such chains to form a two-dimensional grid, which further stablizes the crystal structure.

3.2 Antiproliferative activity

The MTT screening results show that compound 6 has good anti-proliferative activity against both MDA-MB-231 and BGC-823 carcinoma cell lines, with the half maximal inhibitory concentration (IC50) of 12.9 and 10.4M, respectively. And further structural optimization is undergoing.

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(8), Bruker AXS Inc., 6300 Enterprise Lane, Madison, WI 53719–1173, USA

12 June 2014;

11 July 2014 (CCDC 992485)

the Natural Science Foundation of Jiangsu Province (No. BK2011088)

. Sun Hai-Yan, E-mail: shy198209210@126.com