In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

: Dihydrofolate reductase (DHFR) is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ‘best fit’ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino) quinazolinones (Quinazolinone Shiff’s bases) QSB 1-5 and pyridine-4-carbohydrazide Shiff’s bases ( ISB 1- 5 ) derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1 H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX) was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB 3 (4-( N , N -dimethyl-amino)-phenyl) Schiff’s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute) guidelines.


Introduction
In silico screening methods such as docking have a great advantage as compared to 2D similarity and 3D pharmacophore search methods as it utilizes the 3D receptor structure in a quantitative way 1 .Docking calculations alone or combined with the virtual screening has been carried out to develop the DHFR and tyrosine kinase inhibitors etc [2][3][4][5][6][7][8] .
Dihydrofolate reductase (E.C.1.5.1.3)functions as the catalyst for the reduction of the dihydrofolate to tetrahydrofolate that generates reduced folate carriers of one carbon fragments and it is the important co-factor in the biosynthesis of nucleic acids and amino acids .The inhibition DHFR leads to the partial depletion of intracellular reduced folates with the subsequent limitation of cell growth 9 .MTX is a potent inhibitor of DHFR 10 .The quinazoline moiety has the potential for the DHFR inhibition and several 2, 4-diamino-quinazoline analogues.Isoniazid a pyridine Carbohydrazide derivative is hypothesized to act on M. Tuberculi DHFR 11 .Thus Schiff's bases of quinazolinones and pyridine carbohydrazide were designed and in silico screened, amongst which the best fit molecules selected for synthesis and in vitro anticancer evaluation.

Molecular docking
Molecular docking was performed for quinazolinones and pyridine analogues using the GLIDE ® integrated Maestro ® 7.5 interface on the Linux operation system.The high-resolution (2.10 Å X-ray structure of human DHFR complex with inhibitor (RCSB PDB id code 1BOZ) was imported into GLIDE module and the docking simulations were carried out without cofactor NADPH to explore the binding interactions of quinazolinone Schiff's bases (QSB 1-5 ) and pyridine-4-carbohydrazide-Schiff's bases (ISB 1-5 ) derivatives with amino acid residues of the enzyme.MTX was included in the docking run.The low energy conformations of these ligands were generated by LigPrep module with OPLS-2005 force field.

Scoring function
GLIDE module uses force field based MCSA minimization with free energy scoring.This produces G-Scores and ∆G Energy of binding scores.These scores are mentioned in Table 1.

Validation of the docking protocol
Validation of the docking protocol was carried out by correlation coefficient method 12,13 and by pose regeneration of the same ligand structure as seen in the crystallized PDB.

Synthesis of the molecules (QSB 1-5 and ISB 1-5 )
1 H-NMR spectra were recorded in CDCl 3 for quinazolinones and DMSO-d 6 for pyridines, on a Varian Mercury-300 MHz Tetramethylsilane (TMS) as internal standard (chemical shifts in δ ppm).The IR spectra were recorded on a Spectrum RX-1 Perkin Elmier FT-IR spectrometer in potassium bromide discs.MerckGF 254 pre-coated silica plates were used for TLC.The melting points are uncorrected and taken on digital melting point apparatus.The compounds were synthesized using the Scheme 1 and 2. Samsung domestic microwave oven M-183DN was used for microwave irradiated (MWI) synthesis.

General procedure for synthesis of Schiff's bases of 3-amino-2-phenyl-quinazolin-4-(3H)-one (QSB 1-5 )
Equimolar quantities of 2 and aldehyde were taken in 10 mL of ethanol and one drop of concentrated sulphuric acid was added to the mixture and irradiated at 300 watts at for 1.5 minutes.The reaction was monitored by TLC, the solid obtained was washed with water, ethanol and columned over silica gel 120 column and purified (chloroform : ethanol (9:1) solvent system).Recrystallized from ethanol:water (95:5).The physical data of these compounds is mentioned in Table 2.
Spectral data of selected compounds

General procedure for synthesis of Schiff's bases of pyridine-4-carbohydrazide (ISB 1-5 )
Equivalent quantities of isoniazid and aryl aldehydes were dissolved in ethanol and 3 drops of concentrated sulphuric acid or ZnCl 2 (250 mg /mol) or LaCl 3 (300 mg/mol) was added and mixture was irradiated in Domestic Microwave at 150 watts for 15-20 minutes.The reaction was monitored with TLC using choloroform and ethanol (9:1) as eluent.On completion of the reaction the solvent was removed under reduced pressure on rotary evacuator.The solid obtained washed with Pet.ether:ethyl acetate (1:5), ethanol or passed over silica gel column to obtain pure compounds.Recrystallize from 99% absolute ethanol.Physical data of these compounds is given in Table 2.

In vitro anticancer evaluation
The study was performed at Indian Institute of Integrative Medicine, Jammu.The cancer cell-line cytotoxixicity assay was performed on five human cancer cell-lines for the percent total growth inhibition (TGI 50 ) values.The standards such as MTX, 5-fluorouracil, placlitaxel and adriamycin were used in the assay to give a comparable value of the inhibition of DHFR, thymidylate synthase, DNA topoisomerase and microtubilin inhibition.The data of the compounds is given in Table 3.

Docking of the molecules and synthesis
MTX has seven inter hydrogen bondings with amino acid residues of DHFR and the ∆G energy of binding for MTX being -163.3 kcal/mol.The ligands interacting with at least one of these residues having comparable G-Scores, and ∆ G energy of binding lower than MTX were selected for synthesis and in vitro human cancer cell-line cyto-toxicity assay.The molecules were synthesized by microwave irradiation procedure.The 1 H-NMR of the molecules showed the characteristic -N=CH proton shift at 9.40 ppm and for pyridine Shiff's bases at 8.5 ppm.Other aromatic protons were present in 7.00-9.00ppm region.IR spectrum showed the presence of the amide group of 1745cm -1 for the pyridines and 1680 cm -1 for the quinazolinones and peaks at 1601 cm -1 and 1605 cm -1 1585 cm -1 for C=N were observed for quinazolinones and pyridines.

In vitro anticancer evaluation
The quinazolinone Schiff's bases (QSB 1-5 ) did not show anticancer activity but showed comparable inhibition.Amongst pyridine carbohydrazide Schiff's basses ISB 1-5 , one molecule was found to be equipotent with Methotrexate showing the possibility of inhibition of DHFR.The other results were comparable with paclitaxel and adriamycin as per the NCI guidelines for this molecule.

Table 1 .
G-Scores and ∆G binding energy scores.

Table 2 .
Physical constants of the compounds and yields.

Table 3 .
Cancer cell-line cyto-toxicity assay and G-scores.
*Methotrexate , 3, (CH3) 2 -N-C 6 H 4 Shiff's base of pyridine-4-carbohydrazide with G-Score -8.10, (approximately similar for all the compounds from this series) was found to be active as per NCI guidelines.Thus molecule may not only act by DHFR inhibition but also by other mechanisms of anticancer activity.