Synthesis of Some Aryl Ketoxime Derivatives with their in vitro Anti-microbial and Cytotoxic Activity

Benzofuwran derivatives found in several natural compounds and synthesized for various purposes. Due to their molecular structure’s electron behaviors they have several biological activities such as antitumor, cytotoxic, anticancer, antimicrobial, antifungal, ant proliferative etc. We synthesized (3-methylbenzofuran-2-yl) ketoxime derivatives (one of them are new compound) and structure elucidation of the compounds was performed using IR, 1H-NMR, MASS spectroscopy and elemental analysis. X-Ray analysis of H2 compound was elucidated for the fi rst time with this study in the literature. Cytotoxic activity against F2408 and HepG2 cell lines was also evaluated for the fi rst time by MTT and NR uptake methods. Antimicrobial activity of H1, H2 and H3 was also investigated with broth micro dilution test. The results show that these ketoximes have cytotoxic and anti-microbial activity on different higher doses.


Introduction
Benzofuran is a heterocyclic compound which formed by a fused benzene and furan ring. Like other heterocyclic structures, benzofurans have several pharmacological effects due to their scaffolds. Their derivatives have attracted attention in last years. They are found in various natural sources or synthesized for different purposes. Moreover compounds that contains benzofuran heterocyclic earned some features such as solubility, salt formation, absorption and bioavailability [1]. They play key role in design and synthesis of new pharmacologically active compounds. Even some medicinal plants earned pharmacological effect due to benzofuran cores. Primarily they have several biological activities such as antitumor, cytotoxic, anticancer [2], antimicrobial [3], antifungal [4], antiproliferative [5], inhibition of angiogenesis [6].
Cancer is the most dangerous life-threatening disease that cause mortality with a big proportion in all over the world [7]. Previous studies claimed that number of cancer cases will increase by 2050 and reach a peak with 16 million, so that it is very important to understand the mechanism of cancer types. They have several complex mechanisms Activity. Glob J Cancer Ther 5(1): 001-006. DOI: http://dx.doi.org/10.17352/2581-5407.000023 [8]. For example, in a study two different derivative series of benzofurans were synthesized. They tried to understand relationship between the benzene, hydroxy and methhoxy fragments on 4-and 5-positions and antiproliferative activity.
They wanted to search the effects of electron donating groups on antiproliferative activity. They discovered the best activity provides by the methoxy group on para position of the benzoyl moiety. Meta position was not ideal for the tubulin polimerization. In addition the best antiproliferative activity was showed by (5-hydroxy-4-phenylbenzofuran-2yl) (4-methoxyphenyl) methanone derivative (3d), which was activate sub-micromolar concentrations against Molt/4, CEM and HeLa cancer cell lines [9].
In addition to side effects of the cancer drugs is drug resistance to cancer therapy transience [10]. Unconscious usage of antitumors and antibiotics cause to suppression of the immune system. Infection diseases are increasing with the improvement of mutagenicity due to bacteria's resistant to drugs also [11]. This leads to the need for new antimicrobial agents that antibiotics do not resist. It is necessary to discover/ design new antimicrobial agents and fi nd practical/economical ways to synthesis bioactive heterocyclic moieties such as benzofurans. There is many research on antimicrobial potential on benzofuran derivatives which show promising results. For example in a study, researchers studied on a series of different bacteria and showed benzofuran pyrazol derivatives have high antimicrobial activity against nearly all tested organisms [12]. Antifungal studies on benzofurans also gave satisfactory results to different pathogenic fungi. A series of benzofurantriazoles derivatives were studied against fluconazoleresistant Trichophyton rubrum and Cryptococcus neoformans and found as having in vitro antifungal activity [13]. Benzofuran ketoxime analogues were also studied with docking studies as antifungal potency. They found ketoxime moiety and at least one hydrogen bound between enzyme and molecule directly increases the activity [4].
In this study new aryl (3-methyl-benzofuran-2yl) ketones were synthesized and identifi ed with nuclear magnetic resonance (NMR), infrared spectroscopy (IR), mass spectroscopy (MS) and X-ray analysis. Cytotoxicity and anti-microbial potential of this benzofuran derivatives were investigated. Synthesis: 2′-hydroxyacetophenone (5 mmol), 2-bromoacetophenone (5 mmol) and potassium carbonate (6 mmol) used as starting materials to synthesized Aryl (3-methyl-benzofuran-2-yl) ketones at shown as Scheme 2. They were refl uxed in acetonitrile for 4 hours. The reaction was controlled with thin layer chromatography. When the starter materials were run out and the product occurred, the reaction mixture was cooled. The solvent was evaporated and the raw product was fi ltered. The residue was washed with water and crystallized from ethanol [14][15][16]. was treated with any test agent and a negative control in which F2408 cells treated with 10 μM and HepG2 cells was treated with 100 μM cisplatin was maintained. The neutral red uptake (NRU) assay was performed as previously described [17]. The Three independent experiments was done by that way. % viability = (test-blank) / (negative control-blank)*100.

The MTT assay
Three independent experiments were done by that way.

Anti-microbial activity
Anti-microbial activity of the compounds was further determined by Broth Micro dilution (BM) method as previously described [19]. Briefl y, stock solutions of H1, H2 and H3 were diluted to achieve serial decreasing dilutions ranging from 500 to 0.8 μg/ml and transferred to 96-well microtitre plates.

Chemistry
Melting points were determined by using an Electrothermal 9100 digital melting point apparatus and were uncorrected. Spectroscopic data were recorded on the following instrument, IR: Schimadzu 435 IR spectrophotometer. 1 H-NMR: Bruker DPX 400 NMR spectrometer in DMSO-d 6 using TMS as internal standard. MS: VG Platform Mass spectrometer. Analysis for C, H, N were within 0.4% of the theoretical values.

Structure elucidation
As expected, the presence of the derivatives was confi rmed by a thin layer chromatography and NMR spectral data. In the IR spectra C=C and C=N stretching bands, characteristic for all the compounds were obtained at 1510-1616 cm −1 region. Ketone's C=O bands were observed at 1638-1647 regions. All the protons resonated as expected in the NMR spectra. Aliphatic protons resonated in two groups for methyl 2.12 and 2.15, methoxy 3.77 and 3.80 and methylene 5.28 and 5.42 ppm regions, respectively.

X-ray crystallography
The red coloured crystals of the title compound was crystallized from chloroform at room temperature. The crystallographic data are given in table 1 and the selected bond lengths and angles are listed in table 2. Crystallographic data were recorded on a Bruker Kappa APEXII CCD area-detector diffractometer using Mo K  radiation (=0.71073 Å) at T=108 (2) K. Absorption correction by multi-scan was applied. Structure was solved by direct methods and refi ned by full-matrix least squares against F 2 using all data [20].

Biology
A serial concentration of H1, H2 and H3 compounds was tested against seven standard microorganisms and they were not exhibited antimicrobial activities up to 500 μg/ml concentration whereas chloramphenicol as a positive control inhibited the microbial growth. MTT and NRU cytotoxicity assays showed that H1, H2 and H3 compounds were not cytotoxic on both cell lines as normal fi broblast F2408 cells (Scheme 4) and hepatocarcinoma HepG2 cells (Scheme 5) up to 1000 μM tested. On the other hand, cisplatin [22], which is an anticancer drug used as a positive control [23], showed very strong cytotoxicity on the both cell types (Scheme 6) at 100 μM concentration and for all periods of time tested.

Discussion
In this work, we have synthesized molecules named as H1, H2 and H3, completely purifi ed, elucidated and subjected to X-ray analysis. Further the molecules were tested for their bioactivity using anti-microbial and cytotoxicity assays. As a results of activity tests, H1, H2 and H3 compounds was not able to inhibit the microbial growth even at 500 ug/ml concentration whereas chloramphenicol inhibited the microbial growth.
Consistent with the anti-microbial activity, H1, H2 and H3 did