Synthesis,Crystal Structure and Antitumor Activity of(E)-N-(4-Fluorobenzylidene)-3-(methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-amine①

LIN Wei-Yuan YANG Fang DUANAn-Na YOU Wen-Wei ZHAO Pei-Liang

(Guangdong Provincial Key Laboratory of New Drug Screening,School of Pharmaceutical Science,Southern Medical University,Guangzhou 510515,China)

The title compound (E)-N-(4-fluorobenzylidene)-3-(methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-amine was synthesized, and its structure was characterized by1H-NMR,13C-NMR, ESI-MS and single-crystal X-ray diffraction.The preliminary bioassay indicated that the title compound exhibited better inhibitory activity against Hela than 5-fluorouracil.

1INTRODUCTION

Recently,increasing considerable attention interest has been focused on the 1,2,4-triazoles due to their broad spectrum of biological activities[1-4],such as antitumor[5],antifungal[6],antileishmanial[7],antimycobacterial[8],and antimicrobial[9]properties.In addition,it is well-known that the derivatives of Schiff bases always exhibit a wide range of biologicalactivities including antitumor[10],antimicrobial[11],antioxidant[12],antifungal[13]and antibacterial[14]activities.

In view of these facts mentioned above,and in a continuation of our interest in the synthesis of nitrogen-containing heterocycles with anticancer activity[15-21],we developed an idea that incorporating 1,2,4-triazole ring with imine moiety might result in new compounds with high biological activities,and the title compound was designed as a novel derivative of triazole-Schiff bases hybrids.In this paper,we describe the synthesis of this compound by using ethyl 3,4,5-trimethoxybenzoate as the starting material(Scheme 1),and also focus on its crystal structure and antitumor activity.

Scheme 1.Synthesis route of the target compound 5

2EXPERIMENTAL

2.1 Instruments and reagents

Melting point was determined with an uncorrected Buchi B-545 melting point apparatus.1H NMR and13C NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3with TMS as the internal reference.MS spectra were determined using a MicromassZQ4000organicmassspectrometry.Elementary analyses were performed on a Vario EL IIIelementary analysisinstrument.Allofthe solvents and materials were of reagent grade and purified as required.

2.2 Synthesis

General synthetic procedure for the title compound5is depicted in Scheme 1.The key intermediate,4-amino-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione(3),was prepared in good yield according to our previously reported method[22].Preparation of (E)-4-(4-fluorobenzylideneamino)-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-5(4H)-thi one(4):A solution of intermediate3(2.82 g,10 mmol)and 4-fluorobenzaldehyde(1.24 g,10 mmol)in glacial acetic acid(20 mL)was refluxed for about 2 h.The resulted solution was then slowly poured into water,filtered and recrystallized from ethanol to give pure product4as a white crystal.Yield,93%;m.p.191～193 ℃.1H NMR(400 MHz,CDCl3),d:3.84(s,6H,2×OCH3),3.89(s,3H,OCH3),7.15～7.24(m,4H,ArH),7.91(s,2H,ArH),9.96(s,1H,CH=N),11.51(s,1H,NH);ESI-MS,m/z:411.7[M+Na]+,389.7[M+1]+;IR(KBr),ν/cm-1:3270,2360,1558,1508,1468,1240,1153,837.

Preparation of(E)-N-(4-fluorobenzylidene)-3-(methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-4-amine(5):A mixture of(E)-4-(4-fluorobenzylideneamino)-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione4(0.43 g,1.1 mmol)and anhydrous K2CO3(0.16 g,1.2 mmol)in dry ethanol(8 mL)was stirred for 0.5 h,followed by the addition of methyl iodide(0.14 g,1.0 mmol).The reaction was continued for 3 h at room temperature,and then filtered off by suction.The solvent was evaporated to give the crude product followed by recrystallization from ethanol to give the target compound5in the yield of 71%.m.p.:168～169℃.1H NMR(400 MHz,CDCl3),d:2.76(s,3H,SCH3),3.80(s,6H,2×OCH3),3.87(s,3H,OCH3),7.20(t,J=6.8 Hz,4H,ArH),7.87(dd,J=5.6,8.4 Hz,2H,ArH),8.49(s,1H,CH=N);ESI-MS,m/z:425.7[M+Na]+,403.8[M+1]+;IR(KBr),ν/cm-1:2939,1614,1522,1487,1234,1158,1122,850,735;.Anal.Calcd.(%)for C19H19FN4O3S:C,56.70;H,4.76;N,13.92;S,7.97.Found(%):C,56.99;H,4.65;N,14.07;S,7.68.

2.3 Crystal data and structure determination

A colorless crystal cultured from acetone with dimensions of 0.20mm×0.20mm×0.20mm was mounted on a glass fiber in a random orientation.The unit cell determination and data collection were performed with a MoKα radiation(λ=0.71073 ?)on a Bruker Smart APEX-CCD diffractometer equipped with a φ-ω scan mode in the range of 1.98≤θ≤26.37°at 296(2)K.A total of 6158 reflections were collected with 3943 unique ones(Rint=0.0645),of which 3181 observed reflections with I>2s(I)were used in the succeeding refinements.The structure was solved by direct methods with SHELXS-97[23],and refined on F2by full-matrix least-squares techniqueswith SHELXL-97 programs[24].The hydrogen atoms were determined with theoretical calculations.A full-matrix least-squares refinement gave the final R=0.0484 and wR=0.1474(w=1/[σ2(Fo2)+(0.1481P)2+0.0629P],where P=(Fo2+2Fc2)/3),(Δ/σ)max=0.000,S=1.029,(Δρ)max=0.324 and(Δρ)min= –0.399 e/?3.

3RESULTSAND DISCUSSION

The structure of the title compound5was confirmed by the1H NMR,13C NMR,EI-MS and elemental analysis.These spectroscopic data are in good agreement with the assumed structure,and its structure was further determined by X-ray singlecrystaldiffraction analysis.The selected bond lengths and bond angles are listed in Table 1,and hydrogen bond details in Table 2.The C–H··π stacking interactions in the crystal are given in Table 3.The molecular structure of the title compound with atomic numbering scheme is shown in Fig.1 and Fig.2 depicts the molecular packing in a unit cell.

Fig.1.Molecular structure of the title compound

Fig.2. Packing diagram of the title compound in a unit cell

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

Table 2.Hydrogen Bonds for the Title Compound(?,°)

T

able 3.C–H××π Hydrogen Bonds for the Title Compound(?,°)

Generally,the average bond lengths and bond angles of triazole and phenyl rings are in normal ranges.The N(1)–C(7)(1.319(3) ?),N(3)–C(8)(1.368(3)?),N(3)–C(7)(1.378(2)?)and N(2)–C(8)(1.310(3)?)bonds are significantly shorter than a normal single C–N bond(1.47 ?)and longer than a C=N bond(1.28 ?),indicating significant electron delocalization in the 1,2,4-triazole ring system.The torsion angle of C(19)–S(1)–C(8)–N(3)is–175.62(2)°.

As shown in Fig.2,the triazole ring is nearly parallel with the 3,4,5-trimethoxyphenyl and 4-fluorophenyl rings with dihedral angles(θ)of 42.6oand 15.2o,respectively.And these two phenyl planes are not coplanar with a dihedral angle(θ)of 36.1o.Furthermore,the intramolecular C(4)–H(4B)×××N(1)and C(17)–H(17)×××O(3)as well as intermolecular C(11)–H(11A)××O(2)and C(19)–H(19A)××O(1)hydrogen bonds found in the title compound play a major role in stabilizing the molecule.Additionally,the intramolecular hydrogen bonds C(9)–H(9A)×××S(1)and C(6)–H(6A)×××N(4)make the E-configuration of the triazole ring with the 4-fluorophenyl ring more stable.It is worth noting that the crystal packing is further stabilized by one C–H×××π hydrogen bond.It has been known that methyl groups can function as hydrogen-bond donors towards aromatic π systems.The C(9)methenyl group is involved in C–H ×××π interactions with the neighboring 3,4,5-trimethoxyphenyl ring(centroid Cg2,C ×××Cg2=3.659 ?,H××Cg2=2.94 ?,C–H××Cg2=135o,symmetry code:1–x,2–y, –z).These interactions together with the weak π-π interactions result in the formation of a three-dimensional framework.

4ANTITUMOR ACTIVITY

The in vitro antitumor activity of the title compound5against three human tumor cells including Hela,MKN45,and HepG2 was evaluated by the MTT assay.Meanwhile,the cytotoxic activity of5was evaluated against the normal human embryonic kidney(HEK-293)cell lines.5-Fluorouracil(5-FU)was used as a positive control.As described in Table 3,the result of preliminary bioassay shows that compound5displays different degree of inhibition against Hela,MKN45,and HepG2 cell lines with the IC50values of 15.10±6.08,61.10±2.17,and 54.65±12.96 μmol·L-1.These encouraging results indicated that5exhibited better inhibitory activity against Hela than 5-fluorouracil.It was worth noting that the compound did not affect the normal human embryonic kidney cells,HEK-293.Hence,the target derivative may have potential to be developed as a promising lead for antitumor agents,and further structural optimization is undergoing.

Table 3.In VitroAntitumorActivity for the Title Compound 5

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