Microwave Assisted Synthesis and Its Cytotoxicity Study of 4H-Pyrano[2,3-a]acridine-3-carbonitrile Intermediate: Experiment Design for Optimization Using Response Surface Methodology

Several synthetic routes have been achieved to synthesize pyrane fused systems. As a follow up of earlier work, we hereby report the microwave assisted synthesis of key intermediate, pyrane fused acridine compounds 3a–f. It was obtained by treating α,β-unsaturated ketone 1a–f and malononitrile 2 in presence of piperidine with ethanolic solution at 50 °C under 200 W power. This method also optimized via microwave method using RSM methodology. All the synthesized derivatives and target compounds were evaluated for cytotoxicity effect on human hepatoblastoma (HepG2) cell line and HDAC enzyme activity.


Introduction
Pyrane with amine substitution was one of the major classes of heterocycle [1] because their core fragments. It is constituted by a variety of natural products and biologically active compounds [2]. In the latter case, these kinds of compounds have many pharmacological properties and play a major role in biochemical processes [3]. On the other hand, 2-amino-4H-pyran-3-carbonitriles act as an important key intermediate for the synthesis of enormous new organic molecules such as pyridine-3-carobnitriles, pyrimidine thiones, pyrano pyrimidines, pyrazolo-3-amines, pyran-3-carbonitrile, chromene-3-carbonitrile, chromenoprimidinone, pyranothiazine, pyranoquinoline, dihydropyridine, dihydropyranopyrrole, dihydrofuro pyridine, pyrimidine dithione, chromenopyrimidine, chromeno pyrimidine thion, triazolopyrimidine respectively [4]. Among many diseases, cancer has been considered to be a genetic disease which occurs as a result of genetic alterations such as deletions, amplifications, point mutations, insertions and translocations [5][6][7]. The above described genetic reasons are not only the cause for cancer to occur, but it may also involve epigenetic changes which undergoes methylation of DNA and post-translational histone modifications like acetylation, methylation, phosphyralation, etc. [8,9]. With the help of histone post-translational modifications leads to acetylation which provides a link with early gene transcription and chromatin modification. Histone deacetylases (HDAC) and Histone acetyl transferases (HATs) are the two antagonist enzymes, which controls histone acetylation levels. Lysine acetylation was induced by HATs enzymes that is present in core N-terminal histone domains. The removal of acetyl groups from lysine residues is done by Histone deacetylases [10]. Mammalian HDAC family includes eighteen HDAC enzymes and it is further divided into four groups as follows class I-IV due to their homology sequences of proteins present in the yeast [11]. These classes of HDAC's contain zinc in their catalytic site. Class I HDAC's are predominantly localized within the nucleus [10]. The central role of class I (HDACs) is to control regulation, differentiation and tissue development of cell. These class I HDAC enzymes exert their function by removing histone acetyl groups from tails, which replaced by a non-protein histone complex which regulates the gene expression of cellular process [11]. In class I HDAC, the enzymes 1 and 2 are most probably same this plays a major role from cell growth to apoptosis stage. Cell cycle processes and DNA damage responses are the major roles of HDAC3 enzymes. HDAC8 enzymes are mostly present in cytosol which was expressed by smooth muscle cells for differentiation and plays a major role in tumor cell proliferation.
The RSM (Response Surface Methodology) is an extensively employed mathematical and statistical method for modeling. It is an analysis process where different variables affect the response of interest [12]. The aim of this method is to optimize the response [13]. The parameters influencing the method are called independent variables, while the response is called dependent variables [14]. The RSM explores a suitable relationship of approximation between input and output variables and it also determines the optimum operating conditions for a process under analysis or a factor area region that meets operating requirements [15,16]. Box-Behnken designs (BBD) and central composite design (CCD) are two major experimental designs used in the response surface methodology [13].
Microwave heating has gained attention in the preparation of organic synthesis because of the short treatment time, low energy cost, high heating rate, selective heating, and controllable heating process. Here RSM (Response surface methodology) has been used for microwave optimization to prepare high-performance of pyrane fused acridines 3a-f to develop various analogues of acridines.

Results and Discussion
The current study, the relationship between response (yield of 4H-pyrano[2a]acridine-3-carbonitrile, Table 1) was investigated through RSM. The set of 21 experimental runs via CCD template are showed in Table 2. From the experimental data of Table 2, also, the quadratic model equation the relationship between yield of synthesized compound (response) and the three independent reaction variables were (Table S1)  where, Y-Yield of the product; Ti-reaction time; Te-reaction temperature; W-Wattz used in microwave reactor. Positive sign before the linear term indicates that the response increases linearly (synergistic influence) with an increase in the parameter, while negative sign indicates antagonistic influence on the other side. For the fitting of quadratic second order model (Table S2), statistical analysis based on variance analysis (ANOVA) was used. Table S2 reflects all the terms of the model for all RSM responses. At a confidence level of 95 percent, the model's F-value of 22.51 and a very low probability value (p < 0.005) indicated that the fitted model was very significant.
This also indicated that the regression model used was the accurate one to estimate the final product yield. The model's suitability/fit were evaluated using the regression formula (Equation (1)) and determination coefficient (R 2 ). A strong determination coefficient value (R 2 0.948) indicated an extraordinary relationship between the independent process variables, which also intended the second order model to be accurate. The model can elucidate at least 94.8% of the data variance. Adequate accuracy ratio of 69.76 was an acceptable signal in the current investigation and showed the model's ability to navigate the development space.
In Figure 1, three-dimensional response surface plots ( Figure S1) and two-dimensional contour (interaction) plots are drawn to investigate the individual and interactive effects of system variables on yield. The three-dimensional surfaces are the regression equation's graphical representation. The contour curve (two-dimensional) represented two test variables combined with the other one retained at zero (central value) level. The circular contours have been stated to denote the negligible interaction between the corresponding variables. Theoretical yield is fitted with the yield obtained ( Table 2).

Cytotoxicity
All the synthesized compounds (3a-f) were subjected to identify its cytotoxicity using MTT assay. Among the various concentrations, 25 mg/mL is elaborated in Figure S2. Data are presented as mean ± SD calculated from the three independent experiments. It shows 29% of mortality after 24 h which results in 41% of cytotoxicity at 100 μg/mL. When it treated with 500 μg/mL the authors inferred the 81.5% of mortality rate were clearly illustrated in ( Figure S2).

Morphological Alterations
To identify the alterations in morphology further the researchers focused on cancer cell line named HepG2 which were treated with several concentrations of synthesized compounds (3a-f). With the help of HDAC assay kit morphological alterations were resulted in percentage of HDAC. ( Figure S3) Values were expressed as the percentage of HDAC. The Trichostatin A was used as positive control drug. (Figure S4).

Histone Deacetylase Assay
Inhibition of HDAC activity has been associated with cell-cycle arrest and growth inhibition.

Synthesis of Pyrane Fused Acridines 3a-f
Synthesis of pyrane fused acridines 3a-f was achieved by simple conversion from α,β-unsaturated ketone [17,18] and malanonitrile in the presence of ethanol at 50 °C under 200 W powers in a synthesis/extraction reactor of Sineo-UWave-1000 MW-Uv-Us. During this process the reaction has been monitor through thin layer chromatography. We have observed the consumption of all reactant after 5 min (Table 1). Synthesized derivatives were summarized in Table 3. All the synthesized motifs 3a-f characterization information is denoted in the Table 3.

Conclusions
All the synthesized derivatives 3a-f were subsequently evaluated for cytotoxicity activity by the MTT assay on human hepatocellular carcinoma (HepG2) cell line, after and during a period of about 6 days. The cells were exposed to concentrations of 0% (control), 5%, 10%, 15%, 20% and 25%. The MTT assay resulted in apoptosis of cancer cell line by damaging structure of DNA were observed.