Evaluation of antitumor potential of synthesized novel 2-substituted 4-anilinoquinazolines as quinazoline-pyrrole hybrids in MCF-7 human breast cancer cell line and A-549 human lung adenocarcinoma cell lines

A series of novel 2 substituted 4-anilinoquinazolines-pyrrole hybrids were synthesized, and cytotoxic activity were evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell line used for the activity was MCF-7 breast cancer cell line and A459 human lung adenocarcinoma cell line. The newly quinazoline-pyrrole hybrid compounds have been synthesized from the 4-chloro-7-(3-chloropropoxy)-6-methoxy-2-phenylquinazoline derivatives. The chemical structure of the synthesized compounds has been confirmed by FTIR, 1HNMR, 13C NMR, and mass spectral data. The cytotoxic study was conducted using morphological study and MTT assay against adenocarcinoma and human breast cancer cell lines. The results of cytotoxic evaluation revealed that few compounds show moderate to promising activity when compared with standard doxorubicin (IC50 value 41.05 μM at 72 h). The synthesized compounds 7d and 7f were found effective in breast cancer cell line with IC50 values 40.64 μM and 44.98 μM at 72 h, respectively. The synthesized compounds 7d, 7f, 7g, and 7h were found effective in adenocarcinoma cell line with IC50 values of 41.05 μM, 45.54 μM, 46.93 μM, and 48.62 μM, respectively. Based on the experimental evidences, we proposed structure activity relationship to provide significant information for the design and development of further potent anticancer agents.


Background
The heterocyclic molecule combined with quinazoline has drawn a colossal thought attributable to their extended applications in pharmaceutical science. Quinazolines are accounted for their enhanced natural exercises and mixes with different substitutions unite to provide information on an objective with comprehension of the molecule types that may collaborate with the objective receptors. Quinazolines are considered as a significant compound for the union of different physiological noteworthiness and pharmacologically used molecule. Quinazolines are the classes of combined heterocycles that are of extensive intrigue as a result of the various scopes of their natural properties [1]. Many substituted quinazoline subsidiaries have a wide scope of bioactivities, for example, antimalarials [2], anticancer [3][4][5], antimicrobial [6,7], antiviral [8], antipsychotic [9], anti-obesity [10], antitubercular [11], anticonvulsant [12], antidiabetic [13], and numerous other natural exercises. Quinazoline and quinazolinone mixes are likewise utilized in planning of different useful materials for engineered science and furthermore present in different medication particles. Pyrroles and their derivatives were found most important classes of heterocyclic compounds. They prove extensive pharmacological properties such as anti-inflammatory [14], antioxidant [15], antitumor [16], antifungal [17], antibacterial [18], and immune suppressant activities [19].
The extensive literature review which reveals the effect of pyrrole derivatives in the inhibition of cancer cell proliferation has been mentioned. The quinazoline derivatives were also approved standard cytotoxic agents as previously cited. Therefore efforts have been taken to synthesised hybrid of quinazoline-pyrrole to expect a promising synergistic effect on the inhibition of cancer cell proliferation. However, there are a no literature reports on 4 substituted quinazoline-pyrrole hybrid synthesis and its anticancer activity on human breast cancer cell line and adenocarcinoma cell line. Owing to current interest on quinazoline and pyrrole as antitumor agents, our ongoing work has been continued to synthesize the quinazoline-pyrrole hybrids and to evaluate them for anticancer activity.
To each of the reaction, 0.1 mol of substituted pyrrole derivatives (X = Cl, Br, and CF 3 ) which was dissolved in glacial acetic acid (0.01 mol) was added and refluxed for 2 h using microwave irradiation. The reaction mixtures were cooled and were added into ice water. The solids so obtained were filtered, washed with water, dried, and recrystallized from methanol: ethyl acetate (2:2) mixture. Uncorrected melting points were determined for all the compounds (7a-7i) cited in Table 2 and were structurally confirmed using spectroscopic studies. The characterization of the studied compounds (7a-i) was given as follows.  13    Trials were performed 24 h present seeding on counteract cell separation. All the molecules used were of 95-97% pure and were gaged by HPLC and verified by mass spectrometry. For subculture, 75-cm 2 flask was used. Cell layer was briefly rinsed with 0.25% (w/v) trypsin-0.53 mM EDTA answer to expel all hints of serum that contains trypsin inhibitor. Accurately 2.0 to 3.0 mL of trypsin-EDTA arrangement was added to cup and watched cells under a rearranged magnifying instrument until cell layer is scattered (for the most part inside 5 to 15 min). Six to 8.0 mL of complete development medium was included and suctioned the cells by tenderly pipetting. Proper aliquots of the cell suspension were added to new culture vessels. Societies can be built up between 2 × 103 and 1 × 104 viable cells/cm 2 . Try not to surpass 7 × 104 cells/cm 2 . Societies were brooded at 37°C. Cultures are maintained at a cell concentration between 6 × 10 3 and 6 × 10 4 cell/cm 2 . A sub cultivation ratio of 1:3 to 1:8 is maintained. Medium renewal was performed at 2 to 3 times per week.

Screening of novel 2-substituted-4-anilinoquinazolines against morphology of MCF-7 and A-549
Morphological observations of MCF-7 and A-549 cells treated with different compounds for cytotoxicity were done to determine the changes induced by the standard and the test compounds. MCF-7 and A-549 cell were treated with 100 μM concentration of novel 2substituted 4-anilinoquinazolines (7a-7i) which were characterized using NMR, mass, and IR spectral techniques prior to use for morphological study. As a part of anti-cancer evaluation, we have screened all 9 newly synthesized molecules of quinazolines against morphological behavior of MCF-7 and A-549. Cells were observed for 24, 48, and 72 h, after treatment of all test molecules. Images were taken by Axiovert 200M phase contrast microscope at the magnification of ×10. Axiovision Rel.4.2 programming was utilized to get the pictures.
Determination of cytotoxic concentration of 7d, 7f, 7 g, and 7 h, against MCF-7 and A-549 using MTT Assay Plate A-549 and MCF-7 cells (100 μL per well) in a 96-well tissue culture plate. (The quantity of cells can differ from 1000 to 80,000 for each well. The volume can differ from 50 to 150 μL, albeit 100 μL is utilized in this trial.) The test molecules (7d, 7f, 7 g, and 7 h) were included and controlled and brooded the cells for the 24 h timeframe. A volume of 20 μL in phosphate-buffered saline (PBS) or culture medium is suggested for the test mixes and controls. The Control Reagent can be helpfully reconstituted with 5 mL PBS. Fifteen microliter (per 100 μL cell culture) of reagent per well was included and brooded for 4 h at 37°C. The volume of the reagent ought to be balanced relying upon the volume of cell culture. One hundred microliter of the solubilizer was included to each well and blended delicately on an orbital shaker for 1 h at room temperature. The volume of the solubilizer ought to be balanced relying upon the volume of cell culture. (On the off chance that precipitation happens in the solubilizer, place the jug in a warm water shower or at 37°C and shake to disintegrate accelerates.) The absorbance was measured at OD 570 nm for each well on an absorbance plate reader. The study [27] was performed in triplicate. Percent proliferation inhibition was calculated using the formula At = Absorbance of the test compound, Ab = Absorbance of the blank, Ac = Absorption of control.
IC 50 values were calculated by analyzing the relationship between concentrations and percent (%) inhibitions using the GraphPad Prism 7 version 7.00 for Windows, GraphPad Software, La Jolla, CA.

Results
In the present study, a series of 2 substituted 4 anilinoquinazolines (7a-i) has been synthesized from the corresponding 4 chloro 7-(3 chloropropoxy) 6-methyl-2 phenyl quinazoline 4 (3H) amine derivatives (6a-c) condensing with substituted pyrrole derivatives. The newly synthesized nine quinazoline-pyrrole hybrids have been characterized and confirmed using FT-IR (Fourier transform infrared spectroscopy) for the functional groups, 1 H NMR (nuclear magnetic resonance) for the equivalent protons, 13 C NMR for the presence of number of equivalent 13 C atoms, and mass spectroscopy for the determination of molecular weight. The details of the reaction scheme are shown in Fig. 1. The melting points and percentage of yield are summarized in Table 2. The obtained percentage of yield was satisfactory. The characterization of the synthesized compounds has been explained and shown in the "Experimental" section.

Result of biological evaluation
The result of the screening of novel 2-substituted 4-anilinoquinazolines against morphology of MCF-7 and A-549 shows that few synthesized molecules have shown significant cytotoxic effect on both MCF-7 and A-549 cells in a time-dependent manner.
The compounds 7d, 7f, 7 g, and 7 h moderately inhibit the proliferation of A-549 cells and therefore exhibited moderate activity against A-549 cells. The compounds 7d and 7f were also found effective in MCF-7 cells as the morphological figures show that there is a prominent inhibition of the MCF-7 breast cancer cell proliferation.
Other molecules have not shown any inhibition of cell proliferation at any time point of observation as shown in Fig. 2. So further anticancer studies that were carried out using the molecules show better results in morphological study.
MTT assay was conducted for the compounds 7d, 7f, 7 g, and 7 h; IC 50 values were calculated for the tested compound and also standard doxorubicin. In A

Discussion
Quinazoline derivatives previously have been demonstrated as potent anticancer agents [28]. A series of 2 substituted 4 anilinoquinazolines (7a-i) has been synthesized from the corresponding 4 chloro 7-(3 chloropropoxy) 6-methyl-2 phenyl quinazoline 4 (3H) amine Morphological studies clearly shows about the moderate inhibition of A-549 cell proliferation due to the effect of 7d, 7f, 7g, and 7h compounds and only two compounds 7d and 7f were able to inhibit MCF-7 cell proliferation. After morphological study, the results of the cytotoxic activity of the tested compounds using MTT assay confirmed that the compounds 7d, 7f, 7g, and 7h exhibited satisfactory cytotoxic activity in A-549 lung adenocarcinoma cancer cells. It was indicated that the presence of para substituted phenyl group at 2nd position of the quinazoline ring is essential for the anticancer activity because the newly synthesized compounds with only  phenyl substitution at 2nd position of the quinazoline ring were found inactive against both A-549 adenocarcinoma cancer cells and MCF-7 human breast cancer cell lines. Only two compounds 7d and 7f were found active against MCF-7 human breast cancer cell lines. The presence of significant electronegative group, trifluoromethyl (-CF 3 ) at the ortho position of the substituted pyrrole ring in the compound 7f, was found to be a major determinant of the cytotoxic activity, specifically in the MCF-7 human breast cancer cell lines. In the compound 7d, the presence of electronegative as well as inductively electron withdrawing chlorine group (-Cl), at the ortho position of the pyrrole ring which is substituted in the 4th position of the quinazoline ring, and the presence of electron-donating amino group (-NH 2 ) reduced the hydrophobicity [29] of the 2 substituted phenyl ring in the quinazoline moiety. Therefore, the compound 7d was found as a determinant for the cytotoxic potential on MCF-7 human breast cancer cell lines. Out of nine synthesized compounds, only four compounds (7d, 7f, 7g, and 7h) were found to have better cytotoxic potential in comparison to standard doxorubicin.

Conclusion
The empirical evidences of the present study revealed that the newly synthesized quinazoline-pyrrole hybrid compounds 7d, 7f, 7g, and 7h showed good activity against the A-459 human adenocarcinoma cell lines. Among the compounds, 7d which had lowest IC 50 value in relation to doxorubicin was identified as most potent.
In MCF-7 breast cancer cell lines, the compounds 7d and 7f showed good activity. The obtained significant and remarkable cytotoxic effects using substituted pyrrole and 2-substituted quinazolines could be consider as a useful template for further derivatization and designed of modification to achieve more compounds with potent cytotoxic effects.