Investigation of Antimicrobial Activity of 1,3-benzoxazine Derivatives

S. P. Zahorulko, S. A. Varenichenko, O. K. Farat © 2019 S. P. Zahorulko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 547.867.772.1


Introduction
Today, the problem of finding new effective medicines remains topical, despite the fact that branded medicines of various pharmacological groups are widely represented in the modern pharmaceutical market. One of the promising classes of chemical compounds, from the point of view of obtaining new biologically active substances on their basis, is the derivatives of benzoxazines. Professional journals quite often publish new information about the possibilities Bioorganic Chemistry ISSN 1993-6842 (on- of pharmacological application of the derivatives of compounds of this class. Benzoxazines are reported to be used as th potent progesterone receptor agonists, DNA-binding antitumor agents, human leukocyte elastase (HLE) and Clr serine protease inhibitors, as well as the fungicidal, anti-inflammatory and anticonvulsant drugs [1]. The derivatives of 1,4-benzoxazine exhibit the inhibitory activity in cell proliferation, which impedes the endothelial cell migration, and inhibition of angiogenesis in the chorioallantoic membrane assay [2]. Among the derivatives of 1,2-dihydrobenzoxazines, some promising compounds with biological activity have also been identified [3][4][5]. In particular, the study of 6-aryl-1,2dihydro-4H-3,1-benzoxazine and 6-aryl-1,2dihydro-4H-3,1-benzoxazine-2-thione has led to the development of potent and selective nonsteroidal progesterone-receptor agonists. Acridines with 1,2-dihydrobenzoxazines have demonstrated the cytotoxic activity against some lines of human cancer. The benzoxazine fragments are often found in natural compounds. For example, four alkaloids with antimicrobial action have been allocated from the sea sponge Jaspis splendans. One of them contains a substituent of the benzoxazine type [6]. High practical significance has prompted us to investigate the antimicrobial activity of 1,3-benzoxazines derivatives.

Methodology for the research] of the antibacterial activity
All bacteria were cultured in Cation-Adjusted Mueller-Hinton Broth (CAMHB) at 37 °C overnight. Each sample was diluted 40 times in a fresh medium and then, incubated at 37 °C for 1.5-3 hours. The samples of the mean logarithmic phase were diluted (4.5-5 × 10 5 CFU/ ml, measured by 600 nm (OD600)). Then, the compounds containing the plates were added to each well, yielding a cell density of 5 × 10 5 CFU/ml and a total volume of 50 μl. All plates were coated and incubated at 37 °C for 18 hours without shaking. Inhibition of growth of all bacteria was determined measuring absorbance at 600 nm (OD600), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as reference.

Methodology of research of the antifungal activity
The fungi strain was cultivated for 3 days on YPD at 30 °C. A suspension of yeast from 1 × 10 6 to 5 × 10 6 CFU / ml (as defined by OD530) was prepared from five colonies. The suspension was then diluted and added to each well of the plates containing the compound, which gave the final density of fungi cells suspension of 2.5 × 10 3 CFU / ml and a total volume of 50 μl. All plates were coated and incubated at 35 °C for 36 hours without sha king.
The growth inhibition of C. albicans was determined measuring absorbance at 530 nm (OD530). The growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD600-570), after the addition of resazurin (0.001 % final concentration) and incubation at 35 °C for additional 2 hours. The absorbance was measured using Biotek Synergy HTX Microplate Reader. The percentage of growth inhibition was calculated for each well, using negative control (media only) and positive control (fungi without inhibitors) on one plate.
The percentage [of] growth inhibition of an individual sample is calculated based on negative controls (media only) and positive controls (bacterial / fungal media without inhibitors). The negative inhibition values indicate that the growth rate (or OD600) is higher compared to the negative control (bacteria / fungi only set to 0 % inhibition). The growth rate for all bacteria and fungi has a variation of -+ 10 %, which is within the reported normal distribution of bacterial / fungal growth.

Results and Discussion
The methods of synthesis of geminal 1,3-benzoxazines derivatives had been developed in the previous studies authors. The Derivatives of 1,3-benzoxazines (1-4) have been obtained through the interaction of substituted salicylamides with ketones in benzene in the presence of p-TsOH removing water with a Dean-Stark trap (Scheme 1) [11,12].
In cooperation with the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, we sampled the synthesized compounds. These compounds were compared with the compounds whose antimicrobial activity had already been investigated (CHEMBL [13] and DrugBank [14] databases.) For antimicrobial screening, we chose the compounds that differed from the investigated ones by 30 %. Five compounds were selected and their antimicrobial activity against five bacterium and two  Table 2.

The percentage of growth inhibition of whole cells by synthesized compounds
Compound fungus strains was studied by the microbial growth inhibition assay. The data on the tested strains of microorganisms are given in Table 1.

Strain (Sa) (Ec) (Kp) (Pa) (Ab) (Ca) (Cn)
The screening is performed as two replica (n = 2), with both replicas on different assay plates, but from single plating and performed in a single screening experiment (microbial incubation). Each individual value is reported in the Tables  (see 2 and 3). During the analysis of the results obtained, it was found that among the synthesized compounds the most active with respect to Acinetobacter baumannii were the compounds (5) and (1) ( Table 2 and Figure 1).
The repeated experiment confirmed the activity of compounds numbered (5) and (1) with regard to the Acinetobacter baumannii strain, as well as the activity of compound (4) in relation to the Candida albicans fungus (Table 3 and Figure 2).