Synthesis, In-silico Studies and Evaluation of New Quinazoline Derivatives as an Anthelmintic Agents

Table no.1 Ligand with their IUPAC names

2. MATERIALS AND METHODOLOGY 

2.1 Materials 

Chemicals used   for the synthetic work were 2- amino benzamide Acetophenone , dimethyl sulphoxide dimethyl  formamide   ,phosphorous  oxychloride iodine, potassium carbonate aniline derivatives ethanol .  All the reactions were performed in   dried borosilicate glass   beakers round bottomed flasks conical flasks. Precoated silica gels plates were   used for TLC to monitor   progress of the  reaction . compounds melting point were deterimend by   capillary method and are uncorrected.  UV chamber was used for detection of spots in TLC. IR spectra   were recorded on BRUKER FTIR spectrometer. 1H NMR spectra were recorded on BRUKER -400MHZ Spectrometer using DMSO as solvent. The  chemical shift data  were expressed as values relatives   to TMS in ppm.  Mass spectra  were recorded 

3. EXPERIMENTAL SECTION  :

3.1 In Silico Screening

Lipinski’s rule of 5 filtration

The files were inserted in *.pdb, *.mol, *.mol2, *.xyz, *.sdf, or .smile formats. Care was taken to avoid whitespace(s) in the input file name. The window opened and the files were uploaded in the above mentioned formats. pH was adjusted from 0-14 as required. Upon submission, results were obtained [Lipinski 2004].

3.2 Prediction of activity spectra for substances (PASS)

Molecules which have been filtered through Lipinski rule were subjected to online PASS software to predict their biological activities. This software may give the Pa and Pi values for respective activity. The Pa and Pi values should be between 0.000 to 1.000. The software may help in selecting type of activity to be predicted.

3.3 OSIRIS property explorer (version 2)

OSIRIS property explores version 2 (which requires JAVA platform to run) was used in the present study. The smiles of the compound were pasted in search bar, and it will show the results at right side with colour coding. A green colour indicates non-toxic and red indicates toxicity. It also gives the compound solubility profile, druglikeness and drug score.

3.4 Molsoft property explorer (version v.3.7-2)

In this present study Molsoft property explorer version v.3.7-2 was used for molecular exposure of compounds. When the structure of compounds drawn directly on the window or when inserted in mol, Inch, smiles formats will calculate properties like MlogP, MlogS.[10]

3.5 Docking (version 4.0)

AutoDock is a computational molecule modelling simulation software. It is especially effective for protein ligand docking. It has two versions Auto Dock 4.0, vina. Vina is a advanced version [11].

3.6 Ligand Modelling

These ligands are selected based Novelty. The structures of ligands were drawn in Chemsketch, generated their SMILES and saved as MDL mole file. By using generated SMILES, we can check ADME properties, toxicity profile and predict the activity of compounds. The selected ligands were converted into PDB format from mole file by using open babel software. Now the ligands are ready for molecular docking.[12]

SCHEME.1

R1=CN 

R2 =OCH3

R3  =  CH3

R4=F

R5=C2H5

Compound  R1                                     Compound R2

Compound R3                                       compound R4

Compound  R5

4.0 SYNTHESIS OF  QUINAZOLINE AND ITS DERIVATIVES

Step 1:  synthesis of 2- phenyl quinazolin -4(3H) -one (compound 1)

To the pre-heated mixture of 2-amino benzamide (1 mol) and acetophenone (1 mol) in DMSO in a round-bottom flask, iodine (10 mol%) was added as oxidising agent with   addition of 20 ml of dimethyl sulphoxide as solvent. The reaction mixture was heated at 110oC for 16 hrs. The product was extracted three times with ethyl acetate (3×10 ml) to get pure quinazolinone.

Step 2:  synthesis of 4- chloro -2 –(3- chlorophenyl ) quinazoline  

 To this Add the quinazolinone derivative to excess POCl₃.Reflux the mixture for 4–6 h. Monitor by TLC Remove excess POCl₃ under reduced pressure. Pour the residue onto crushed ice. Neutralize with sodium bicarbonate solution Filter the precipitate and recrystallize from ethanol

Step 3.  Synthesis of   final quinazoline derivatives

Dissolve 1 mmol of 4-chloro-2-(3-chlorophenyl)quinazoline in 20 mL ethanol. Add 1.2 mmol substituted aniline (R = CH₃, NO₂, OCH₃, F, C₂H₅).Add 2 mmol K₂CO₃. Reflux for 6–8 h with stirring. Monitor the reaction by TLC. Cool the mixture and pour into ice water. Filter the precipitated solid. Wash with water and recrystallize from ethanol.

5.0 EVALUATION OF ANTIHELMINTIC ACTIVITY

The synthesized quinazoline derivatives were evaluated for in vitro antihelminthic activity using adult earthworms (Pheretima posthuma), which are commonly used due to their physiological similarity to intestinal helminths.

 Healthy earthworms of approximately 8–10 cm in length and 0.2–0.3 cm in width were collected and washed with normal saline to remove adhering soil. Test solutions of the synthesized compounds were prepared at the required concentrations using 1% DMSO as the solvent. Albendazole was used as the standard drug, while 1% DMSO served as the negative control.

The earthworms were divided into different groups (six worms per group) and placed in Petri dishes containing the test solutions, standard drug, or control solution. The worms were observed continuously, and the time required for paralysis (P) and time required for death (D) were recorded. Paralysis was confirmed when the worms failed to move even after gentle shaking, while death was confirmed by the absence of movement even after exposure to warm water (50–55°C) and fading of body color.

The antihelminthic activity of the synthesized compounds was compared with that of the standard drug based on the recorded paralysis and death times. Lower paralysis and death times indicated better antihelminthic activity.  

6.0 RESULTS AND DISCUSSION

6.1 Docking Analysis

Docking is used to find the exact binding conformation and orientation of the ligand molecule into the active site of the protein. The synthesized five quinazoline compounds and standard (Albendazole] were docked againstβ-tubulin (PDB ID: 4O2B  using Auto-Dock Tool 4.0, an automated docking tool.

The docking process involves four main steps,

(i) Protein preparation

(ii) Ligand preparation

(iii) Grid preparation and

(iv) Docking.

The Lamarckian genetic algorithm has been used as the search algorithm to search for the best conformers. The initial population size was set randomly as 150 individuals and ten generations was set for each genetic algorithm run and the maximum number of energy evaluations was set to 2,500,000. The grid box size was set as to include all the active site residues present in rigid macromolecules. The grid box was centered at 8.671 Å x -8.036 Å x 0.67 Å and the dimensions of the grid box have been set as 40, 40, 40 (X,Y,Z co-ordinates) so as to include all the active site residues.

Docking studies showed that all ligands chosen for analysis possessed a least binding affinity with the target protein  β-tubulin (PDB ID: 4O2B The protein ligand interactions were studied in terms of minimum binding energy (Kcal/mol) and the number of hydrogen bonds formed with active site residues

Molecular docking studies were carried out against β-tubulin (PDB ID: 4O2B) to evaluate the antihelminthic potential of the synthesized quinazoline derivatives.  All derivatives exhibited favorable binding within the active site of β-tubulin and showed better binding energies than the standard drug Albendazole(-6.09kcal/mol). Among all synthesized compounds, the cyano-substituted derivative (Q1) exhibited the highest binding affinity with a docking score of -7.32 kcal/mol, indicating the strongest interaction with the target protein. The enhanced activity may be attributed to the electron-withdrawing nature of the cyano group, which improves protein–ligand interactions within the binding pocket The methoxy-substituted derivative (Q2) also showed excellent binding affinity with a docking score of -7.22 kcal/mol, suggesting that the methoxy group contributes favorably to receptor binding through electronic and hydrophobic interactions. 

Table No.2

Figure. 2[4-{(Z)-[2-(3-chlorophenyl)quinazolin-4(3H)-ylidene]amino}benzonitrile}

Figure .3[(4Z)-2-(3-chlorophenyl)-N-(4-methoxyphenyl)quinazolin-4(3H)-imine]

Figure .4[4Z)-2-(3-chlorophenyl)-N-(4-methylphenyl)quinazolin-4(3H)-imine]

Figure. 5[(4Z)-2-(3-chlorophenyl)-N-(4-fluorophenyl)quinazolin-4(3H)-imine]

Figure. 6 [(4Z)-2-(3-chlorophenyl)-N-(4-ethylphenyl)quinazolin-4(3H)-imine]

Figure. 7[Albendazole  standard]

Table No.3  Lipinski rule evaluation of ligands

Result of OSIRIS  molecular property explorer

Table N0.4  Results of OSIRIS  molecular  property explorer

Molsoft property explorer

Table No.05 Results of Molsoft   molecular property  explorer

6.2 Pass Studies  

PASS (Prediction of Activity Spectra for Substances) is a computational tool used to predict the probable biological activities of chemical compounds based on their molecular structure. It estimates the probability of activity (Pa) and inactivity (Pi), helping to identify potential pharmacological effects before experimental testing.

Table no.6

Table No.7  Physical Data of synthesized compounds

All the newer quinazoline   derivatives  were synthesized  by different chemical reagents compound  [R=CN]  &[R=ch3]Were obtained high yields . all the synthesized molecules were recrystallized  from the methanol or ethanol   compound   are  purified by column chromatography  and characterized by FTIR ,1H NMR &Mass spectroscopic  techniques

7.0  SPECTRAL INTERPRETATION

4. {(Z)-[2-(3-chlorophenyl)quinazolin-4(3H)-ylidene]amino}benzonitrile:  FTIR: [KBr cm-1]  3420 N-H [amine] 3065 C-H [ aromatic] 2228  C=N [nitrile]  1621 C=N[quinazoline] 1583c=c[ aromatic ] 1298C-N[aromatic  amine]   1H NMR(δ ppm, DMSO) 12.68[s-1H -NH  ]8.73[D-1H-H-8]8.35d[8.4]2H Ar-H[o to CN] 8.11[d8.2-1H -H5] 8.02 t[7.8] 1H-Ar-H[m to CN] EI-MS :m/z354.09(M+1)

(4Z)-2-(3-chlorophenyl)-N-(4-methoxyphenyl)quinazolin-4(3H)-imine:

FTIR :[KBr cm-1]3344 N-H[secondary amine] 3058 C-H[Aromatic] 2955 och3 [methoxyC-H] 1621 C=N[Quinazoline] 1578 C=C[Aromatic] 1170 [Ar-N] 1031 C-O [Methoxy]  1H NMR[δ ppm, DMSO) 10.21 [s-1H-NH] 8.63 [d-1H -H5] 8.18 d[1H-H8] 7.92 [t-1H-H-7] 7.86 [d-2H-H-2,6] 7.50 [d-2H-H-3,5] 6.96 [d-2H-H-3,5]  EI-MS:m/z359.1 [m+1]

(4Z)-2-(3-chlorophenyl)-N-(4-methylphenyl)quinazolin-4(3H)-imine:

FTIR :[KBr cm-1] 3354 N-H 3056 C-H[Aromatic] 2923 C-H[CH3][Aliphatic] 1622 C=N 1587 C=C[Aromatic] 1495 C=N  761 C-Cl[aryl chloride]  1HNMR 10.25 [s-1H-NH] 8.62 [d-1H-H-5] 8.18 [d-1H-H-5] 8.18 [1H-H-8] 7.92 [t-1H-H-7] 7.76 [1H-H-6] 7.82 [d-2H-H2,6] 7.48 [d-2H-H-3,5] 7.58 [d-2H-H-2,6] EI-MS:m/z 343.1[m+1]

(4Z)-2-(3-chlorophenyl)-N-(4-fluorophenyl)quinazolin-4(3H)-imine: 

FTIR :[KBr cm-1]3380 N-H [Secondary amine] 3060 C-H [Aromatic] 2922 C-H[Aliphatic] 1620 C=N  1590 C=C[Aromatic] 1520 C=N  1245 [Ar-N] 840 C-H[aromatic] 760 C-Cl [arylchloride] 1HNMR 10.22 [ s-1H-NH] 8.66 [d-1H -H-5] 8.21[d-1H- H-8] 7.97[t-1H-H-7] 7.88[dd-2H-H-2,6] 7.25[dd-2H- H-3,5] 7.63[d-2H- H-2,6] 7.50[d-2H-H-3,5]  EI-MS:m/z 337.1[m+1]

4Z)-2-(3-chlorophenyl)-N-(4-ethylphenyl)quinazolin-4(3H)-imine:

FTIR  :[KBr cm-1]   3342 N-H [Amine]  3056 C-H [Aromatic] 2928 C-H ] [Aliphatic] 2856 C-H Aliphatic 1618  C=N  1573   C=C  [Aromatic]  1294 C-N ]  1HNMR  12.28 [s-1H-NH] 8.78 [s-1H-H-2]  8.34 dd[1H-H-4] 7.92 td[1H-H-3] 7.79 d[1H-H-5]  7.63 dd[1H-H-7] EI-MS:m/z 358.1 [m+1]

8.0 EVALUATION OF ANTHELMINTIC ACTIVITY

The quinazoline derivatives were screened for their  in-vitro anthelmintic activity by using standard protocol against Indian adult earthworm [pherituma postuma] and compared  with the standard .The indian adult earthworm [pherituma postuma] resemble both anatomically biologically and physiologically to the intestinal round worm  parasites of humanbeing 

All the synthesized compound displayed siginificant Anthelmintic activityTable no.6 Compounds like R=CN, could paralyze and kill the worms in less time . hence this compound found to be more potent than the standard drug albendazole

Table no.8  Biological activity evaluation