Synthesis, and Antitumor Activity of Some N1-(Coumarin-7-yl) Amidrazones and Related Congeners

A series of new N1-(coumarin-7-yl)amidrazones incorporating N-piperazines and related congeners were synthesized by reacting the hydrazonoyl chloride derived from 7-amino-4-methylcoumarin with the appropriate piperazines. The chemical structures of the newly prepared compounds were supported by elemental analyses, 1H-NMR, 13C-NMR, and ESI-HRMS spectral data. The antitumor activity of the newly synthesized compounds was evaluated. Among all the compounds tested, 7-{2-[1-(4-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl)piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2H-chromen-2-one (3n) was the most potent against MCF-7 and K562 cells, with IC50 values of 20.2 and 9.3 μM, respectively.


Introduction
Due to their structural and therapeutic diversity as pharmaceutical agents, along with their commercial availability, piperazine derivatives continue to capture the attention of synthetic and medicinal chemists. Piperazine-based compounds have been employed as antibacterial, antidepressant, and antitumor drugs, and as α-adrenoceptor antagonists, CCR5 receptor antagonists, 5-HT7 receptor antagonists, and adenosine A2a receptor antagonists [1]. Several piperazine derivatives have reached the stage of clinical application; among the known drugs that are used to treat anxiety is a pyrimidinyl piperazinyl compound (buspirone, BuSpar ® ) [2], while a 3-chlorophenyl piperazinyl drug (trazodone, Desyrel ® ) is used as an antidepressant [3]. Besides, several publications have dealt with the synthesis and evaluation of thrombin inhibitors that incorporate an amidrazone functionality as a structural motif [4][5][6], and there is a report pertaining to the inactivation of lipoxygenase-1 from soybeans by open-chain and cyclic amidrazones [7].
On the other hand, coumarin derivatives have drawn considerable attention from researchers due to their role in natural and synthetic organic chemistry, and their interesting biological activities. Compounds which contain a coumarin nucleus were found to exhibit various biological activities such as anticoagulant and antithrombotic properties [8]. Some derivatives have shown molluscicidal, anthelmintic [9], hypnotic, and insecticidal [10] activity, while others have served as antifungal [11], anti-inflammatory [12] and antiviral agents, including against human immunodeficiency virus [13], and anticoagulant properties [14]. In addition, coumarins have also been used as additives in food and cosmetics [15], and in the preparation of optical brighteners, dispersed fluorescent and laser dyes [16].
In view of the widespread interest in the activity spectrum and profile of coumarins [17][18][19][20], and in continuation of our work on the synthesis of new compounds of pharmacological and biological interest [1,[21][22][23][24], we describe herein the preparation and spectroscopic characterization of some new piperazinyl amidrazones containing coumarin moieties and evaluation of their antitumor activity.

Scheme 2. Synthesis of 4-methyl
The newly synthesized compounds 3a-n were characterized by elemental analyses, MS and NMR spectral data. These data, detailed in the experimental part, are consistent with the suggested structures. Thus, the mass spectra display the correct molecular ion peaks for which the measured high resolution (HRMS) data are in good agreement with the calculated values. DEPT and 2D (COSY, HMQC, HMBC) experiments showed correlations that helped in the 1 H-and 13 C-signal assignments to the different carbons and their attached, and/or neighboring hydrogens.

Antitumor Activity
The antitumor activities of compounds 3c-n were characterized by conducting cell viability assays using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cultures of MCF7 breast cancer cells and K562 human leukemia cells were treated first at one concentration of 50 μg/mL and the results are shown in Table 1. Compounds 3i, 3m and 3n showed potential anti-MCF-7activity. These compounds were able to reduce the viability after 72 hours to less than 50%. With respect to K562 cells, only compound 3n showed considerable inhibition of cell proliferation. Furthermore, we explored the anti-tumor activity for 3i, 3m and 3n (compounds that showed potential activities) in two more cancer cells: breast cancer cell line ZR-75-1 and leukemia cell line HL60. We determined the IC 50 values for compounds 3i, 3m and 3n on ZR-75-1 and HL60 additional cell lines, and on the MCF-7 and K526 cell lines as well (Table 2). Notably, compound 3n was the most potent against MCF-7 cells scoring an IC 50 value of 20.2 μM and showing a very promising activity against the K562 cells with an IC 50 of 9.3 μM ( Table 2). From the structure-activity relationships point of view, the nature of substituents on the X position seems to play a critical role for the cytotoxic activity. For example, in case of MCF-7 breast cancer cells, compounds 3i, 3m, and 3n with the substituent N-CO 2 Et, N-(2-pyrimidyl), and N-(1-benzyl-2-ethyl-4-nitroimidazol-5-yl) appendages, respectively, exhibited cytotoxic IC 50 values of 49, 38, and 20 μM. The shared characteristic feature between these three substituents, which are absent in all others, is the presence of the nucleophilic component next to position X. However, this belief cannot be generalized on the in anti-K562 cell activity, where compound 3n was the only active compound and no activity could be observed with compounds 3i and 3m. What make compound 3n active against K562 cells, giving a relatively low IC 50 of 9 μM, may be linked with the bulky substituent that characterized this compound from all others tested. This bulky substituent on compound 3n may also be associated with the lowest IC 50 value as exhibited by this compound against the MCF-7 cells.

General
The following chemicals, used in this study, were purchased from Acros and were used as received: piperidine, morpholine, thiomorpholine, piperazine, N-alkylpiperazines, N-arylpiperazines, ethyl N-piperazinecarboxylate. 1-(1-Benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl)piperazine was prepared according to a literature procedure [39]. Silica-gel for column chromatography was purchased from Macherey-Nagel GmbH & Co (Germany). Melting points (uncorrected) were determined on a Stuart scientific melting point apparatus in open capillary tubes. 1 H-and 13 C-NMR spectra were recorded on a 300 MHz spectrometer (Bruker DPX-300) with TMS as the internal standard. Chemical shifts are expressed in δ units; J-values for 1 H-1 H, 1 H-F and 13 C-F coupling constants are given in Hertz. High resolution mass spectra (HRMS) were acquired (in positive or negative mode) using electrospray ion trap (ESI) technique by collision-induced dissociation on a Bruker APEX-4 (7-Tesla) instrument. The samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic acid) and infused using a syringe pump with a flow rate of 2 µL/min. External calibration was conducted using arginine cluster in a mass range m/z 175-871. (1) This synthon, required in the present study, was prepared according to a literature procedure [28][29] which involves reaction of m-aminophenol with methoxycarbonyl chloride as the initial step; the resulting N-protected m-aminophenol underwent cyclocondensation upon reaction with ethyl acetoacetate and conc. sulpuric acid, followed by removal of the N-protecting group (via treatment with sodium hydroxide) to deliver the title compound 1; mp = 224-226 °C (lit. mp = 226-227 °C) [28][29]. (2) Compound 1 (17.5 g, 0.10 mol) was dissolved in 17% aqueous hydrochloric acid (160 mL). To this solution was added drop-wise a solution of sodium nitrite (7.6 g, 0.11 mol) in water (15 mL) with efficient stirring at 0-5 °C. Stirring was continued for 20-30 min., and the resulting fresh cold 4-methyl-2-oxo-2H-chromene-7-diazonium chloride [also named as 7-(chlorodiazenyl)-4-methylcoumarin] solution was poured onto cold solution (0 to -10 °C, ice-salt bath) of 3-chloropentan-2,4dione (13.5 g, 0.1 mol) in ethanol/water (160 mL, 1:1 v/v) containing 30.0 g of sodium acetate with vigorous stirring. The resulting yellowish-colored mixture was further stirred until a solid precipitate was formed (5-10 min). The reaction mixture was then diluted with cold water (200 mL), the solid product was collected by suction filtration, washed several times with cold water, dried, and recrystallized from acetonitrile. Yield