Synthesis and Crystal Structure of 4-Chloro-N- (2-(2-nitrophenyl)acetoxy)-N-(m-tolyl)benzamide①

HE Din YANG Zhu-Qing HOU Meng

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Synthesis and Crystal Structure of 4-Chloro-N- (2-(2-nitrophenyl)acetoxy)-N-(-tolyl)benzamide①

HE Diana, b②YANG Zhu-QingaHOU Menga

a(730000)b(730000)

The new title compound 4-chloro-N-(2-(2-nitrophenyl)acetoxy)-N-(-tolyl)benza- mide (C22H17ClN2O5,M= 424.82) has been synthesized via the reaction of 4-chloro-N-hydroxy-N- (-tolyl)benzamide with 2-(2-nitrophenyl)acetyl chloride. The structure of the product was confirmed by1H NMR,13C NMR, IR, HRMS (ESI) and single-crystal X-ray diffraction. The title compound crystallizes in the monoclinic system, space group21/with13.0269(10),6.7251(6),23.9313(16) ?,99.931(6)o,2065.1(3) ?3,4,D= 1.366 g/cm3,(000) = 880,= 0.221 mm-1, the final= 0.0600 and= 0.1754 for 1981 observed reflections (> 2()). X-ray analysis indicates that the chloro-phenyl ring (C(10)～C(15)) and the methyl-substituted benzene ring (C(16)～C(21)) are not coplanar with the nitro-substituted benzene ring (C(1)～C(6)), with the dihedral angle to be 70.78° and 63.72°, respectively. Hydrogen bonds C(2)–H(2)×××O(2) and C(7)–H(7B)×××O(5) are observed.

crystal structure, synthesis,4-chloro-N-(2-(2-nitrophenyl)acetoxy)-N-(-tolyl) benzamide

1 INTRODUCTION

Hydroxamic acid derivatives played an important role in antitumor as an inhibitor of histone deacety- lases (HDAC) due to its special structure and the corresponding physical and chemical properties[1-3]. Therefore, we design novel phenyl hydroxamic acid derivatives using-nitrophenyl acetic acid as the carrier[4]. These derivatives can hardly be reduced in normal cells, but because of the low pH and active hydrolase and reductase ester in the environment of tumor cells[5], these compounds can release the activebenzohydroxamic acid, which is helpful to the antitumor activity. In addition,-nitrophenyl acetic acid also has intensive inhibitory activity against VEGF.

N-arylhydroxamic acid derivatives have been less studied. We divided the synthesis of N-arylhydro- xamic acid derivatives into three parts. At first, the preparation of substituted phenyl hydroxylamine has been reported by the method of chemical reduction and electrochemical reduction whereas the shortco- ming of costliness and uncontrolled reaction rate limit their application[6-9]. We applied catalytic hy- drogenation, in which the hydrazinium hydroxide with low boiling point, obvious different solubility form hydroxylamine, was used as reducing agent under the catalyst of raney nickel[10]. Secondly, there were many methods to prepare the hydroxamic acid such as N-acylation[11], ammonolysis[12]and para- helium oxidation[13]. We obtained the hydroxamic acid though the acylation of hydroxylamine with corresponding chloride. The reaction was easy to operate with low pollution. Finally, N-arylhydro- xamic acid derivatives were synthesized though alcoholysis reaction between the hydroxamic and acylated-nitroaromatic acids.

In this work, we used-nitrotoluene as the raw material, in the presence of raney nickel and hydra- zinium hydroxide, to prepare N-(-tolyl) hydroxyla- mine. Then we can obtain the target compound through two steps of acylation (Scheme 1). The title compound in tumor cell was easy to release active phenylhydroxylamine derivatives owing to the immediate and stable hexatomic or hepatomic cyclization under higher activity of ester hydrolase, lower pH value, and neighboring group assistance of amino translated from the nitryl. It means that their selectivity and safety are both improved. In this paper, we describe the synthesis method, the inhibitory activity against tumor cells and the crystal of 4-chloro-N-(2-(2-nitrophenyl)acetoxy)-N-(-tol- yl)benzamide.

Scheme 1. Synthetic route of the title compound

Reagents and conditions: (a) NH2NH2·H2O, raney nickel, CH3CH2OH/ClCH2CH2Cl (3:2), 0 ?C; (b) THF, NaHCO3, 0 ?C; (c) CH2Cl2, NaHCO3, rt.

2 EXPERIMENTAL

2. 1 Materials and instruments

Starting materials and solvents were obtained from Keheng (China). The melting point determi- nation was performed on an electrothermal PIF YRT-3 apparatus. Infrared spectra (IR) were recor- ded on NEXUS 670 FT-IR (Nicolet).1H NMR and13C NMR (ppm) spectra were recorded on a Varian Mercury (400 MHz) using TMS as the internal standard. Mass spectra were recorded on a VGZAB- HS (70 ev) spectrometer with ESI source as ionization. Reaction progress was monitored through thin layer chromatography (TLC) on precoated glass plates (silica gel 60 F254, 0.25 mm thickness).

2. 2 Preparation of N-(m-tolyl)hydroxylamine (2)

The N-(-tolyl)hydroxylamine was prepared according to the literature[14]. However, we used the method of hydrazinium hydroxide reduction under the catalyst of raney nickel, which makes the product easier to be separated and controls the reac- tion rate. To a solution of 32 mmolhydrazinehy- drate in mixed solution (60% ethanol/40% 1,2-di- chloroethane, 20 mL) was added 8 mmol-nitro- toluene. The solution was stirred at 0 ℃ for 20 min (monitored by TLC) and then filtered to remove the raney nickel. The excess solvent was steamed out by the reduced pressure. Residual liquid was extracted with dichloromethane (20 mL ×3) and water (20 mL). Organic layer was dried and then dichloro- methane was removed under vacuum, giving the light-yellowish oil.

2. 3 Synthesis of 4-chloro-N-hydroxy-N-(m-tolyl)benzamide (4)

To a mixture of compound 2 (15 mmol), sodiumbicarbonate (22.5 mmol) and tetrahydrofuran (40 mL) was slowly added compound 3 (15 mmol) with vigorously stirring for 1.5 h. After filtration, part of the solvent was removed under vacuum and recrys- tallized from hexane to give yellow powder[15]. Yield 91%,m.p.: 133.4～133.6 ℃.1H NMR (400 MHz, DMSO-d): 10.77(s, 1H), 7.66(d,= 7.6 Hz, 2H), 7.62～7.25(m, 5H), 7.03(d,= 7.2 Hz, 1H), 2.38(s, 3H).13C NMR (100 MHz, DMSO-d): 166.8, 141.8, 137.9, 134.9, 134.4, 130.3, 128.4, 128.0, 126.5, 122.5, 119.4, 21.1. IR(KBr) (cm-1): 3437.8(-OH), 1598.6(-C=O), 1513.4(Ar), 1445.3(Ar). H RMS (ESI): (M+H) 262.0629 (calculated 262.0635), error = 1.5 ppm.

2. 4 Synthesis of the title compound (6)

Compound 4 (8 mmol) was dissolved in 30 mL dichloromethane, into which compound 5 (8 mmol) was then slowly poured. The mixture was stirred for 2 h and monitored by TLC. After filtration, the solution was concentrated under reduced pressure and recrystallized from methanol to give the light-yellowish solid. Colorless crystals of the title compound suitable for X-ray structure analysis was obtained from the mixture of dichloromethane and ethanol (, 1:3) by slow evaporation at room tem- perature. Yield 66%, m. p.: 101.2～101.3 ℃.1H- NMR (400 MHz, CDCl3): 8.14(d,= 8Hz, 1H), 7.63(t,1= 7.2 Hz,2= 7.2 Hz, 1H), 7.52～7.43(m, 4H), 7.26～7.03(m, 6H), 4.18(s, 2H), 2.31(s, 3H).13C NMR(100 MHz, CDCl3): 21.2(1C, Ar-CH3), 37.3 (1C, -CH2(C=O)-), 124.1, 125.4, 127.3, 128.1, 128.4, 129.0, 129.1, 129.6, 130.3, 131.5, 133.5, 133.9, 137.3, 139.5, 139.9, 148.3(16C, Ar), 165.7(1C, CH3-C=O), 167.6(1C, Ar-C=O); IR(KBr)(cm-1): 3425(-N-C=O), 1791(-C=O), 1523(-NO2), 1340(-NO2), 1087(N-O), 877(C-N). H RMS (ESI): (M+Na+) 447.0721 (calculated 447.0718), error = 0.7 ppm.

2. 5 Crystal data and structure determination of the target compound

A single crystal suitable for X-ray diffraction study was cultivated from dichloromethane and ethanol (/, 1:3) by a slow evaporation method at room temperature. The clear light colorless crystal of the title compound having approximate dimensions of 0.37mm × 0.34mm × 0.31mm was moun- ted on the top of a glass fiber. All measurements were performed with Moradiation (= 0.71073 ?) on aSuperNova, Dual, Cu at zero, Eos diffractometer[16, 17]. Out of the 8411 total reflections collected in the range of 3.150o≤≤26.021o (–16≤≤14, –6≤≤8, –29≤≤29) by using a phi and omega scan mode, 4055 were independent withint= 0.0485, of which 1981 were observed with> 2() and used in the structure determination and refine- ments. The structure was solved by direct methods with SHELXS-97[18]and refined by SHELXL-97[19]. All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atomswere placed in the calculated positions. The final full-matrix least-squares refinement gave= 0.0600,= 0.1754 (= 1/[2(F2) + (0.0544)2+ 0.00], where= (F2+ 2F2)/3),= 1.026, (Δ)max= 0.000, (Δ)max= 0.212 and (Δ)min= –0.221 e/?3.

2. 6 MTT assay to measure the cell growth inhibition in vitro

The four cell lines containing Fibroblast, A549, BEL-7402 and MGC-803 were chosen for screening cell growth inhibition activity of the target com- pound. All cell lines were maintained at 37 ℃ in 5% CO2in DMEM medium, supplemented with 10% fetal calf serum. The logarithm vegetal period cells were inoculated at the appropriate concentration (100 μL per well) into 96-well plates while the marginal wells should be omitted. Then the cells were preincubated for 24 h. The solutions of the compounds of the corresponding concentration were applied carefully on the monolayers of cells for 48 h. Then 10 μL of MTT stock solution (5 mg/mL in PBS) was added in each well and the plate was incubated for 4 h. The blue-coloured formazan that formed was dissolved in 100 μL of DMSO per well. After being mixed for 10 min, the absorbance was determined at 490 nm to calculate the IC50which was defined as the amount of cytotoxin causing an inhibition of 50% in the viability of the cells compared with the control cell culture.

3 RESULTS AND DISCUSSION

Our title compound was generated from the reaction of 4-chloro-N-hydroxy-N-(-tolyl)benza- mide obtained by the acylation of reductive nitro- benzene and 2-(2-nitrophenyl)acetyl chloride in methanol with fairly good yield. The synthetic route can be summarized in Scheme 1. The structure of the product was confirmed by1H NMR,13C NMR, IR and H RMS (ESI).

The structure of the title compound verified by single-crystal X-ray diffraction is shown in Fig. 1. The selected bond lengths and bond angles are listed inTable 1. The perspective view of the crystal pac- king in a unit cell of the title compound is revealed in Figs. 2 and 3.

Table 1. Selected Bond Lengths (?) and Bond Angles (°) for the Title Compound

Fig. 1. Structure of the title compound

Fig. 2. Crystal packing of the title compound

Fig. 3. Two-dimensional structure of the title compound with hydrogen bonds (green lines)

Table 2. Geometry of Proposed C–H×××O Close Contacts

Symmetry code: (i) 1–, –1/2+, 1/2–

In the crystal, neighboring molecules are parallel and crisscross to each other in the cell. The short intermolecular distance between the adjacent benzene rings of C(1)～C(6) is 3.7152 ? (symmetry code: 1–, –1–, 1–z), indicating the presence of face-to-facestacking (Table 3). Obviously, the hydrogen bonds andinteraction together stabili- ze the compound structure.

Table 3. π–π Stacking Interactions (?, °)

Symmetry codes: [3576] = 1–, –1–, 1–

4 BIOLOGICAL ACTIVITIES

The cytotoxic activities of the title compound were measured in three different tumor cell lines and one normal cell from people by MTT assay, and the results are summarized in Table 4. The inhibitory activity of SAHA against these cells is in agreement with literature[20]andthe National Cancer Institute- Development Theraputic Program (NCI-DTP) data. Our target compound presents good inhibition profile against A549, BEL-7402 and MGC-803, and is equivalent with the positive group SAHA. The values of IC50are 3.25, 4.76 and 4.21 μM, respec- tively for three different tumor cells. Moreover, the inhibitory activity against A549 (IC50= 3.25 μM) is the best, which provides important information for us to conduct the further activity studies. The cyto- toxic experiment presents the probability that the title compound will be a potent antitumor agent. Further study with the title compound is underway to investigate therapeutic efficacy against human cancers.

Table 4. Inhibitory Activity the Title Compound against Human Tumor Cellsa (IC50/μM)

aValues are the mean of three separate experiments.

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24 February 2014;

28 May 2014 (CCDC 975835)

the Fundamental Research Funds for the Central Universities (lzujbky-2010-137) and Lanzhou Science and Technology Bureau Program Funds (2012-2-90)

. Born in 1970. Tel: +86-931-13008738922, Fax: +86-0931-8915685, E-mail: hed@lzu.edu.cn