ONE-POT

. Novel five-membered ring (pyrrole, pyrazole and imidazole)-based pyrimidine and quinazoline derivatives were synthesized by one-pot domino approach. This approach has the advantages of high yield, mild reaction conditions and a simple work-up procedure. The structure of the synthesized compound was elucidated by spectroscopy technique and elemental analysis. The synthesized compound were examined for antimicrobial activity against four bacteria ( E. coli , S. pyogenes , S. aureus


INTRODUCTION
Green methods play a crucial role in synthetic organic chemistry and are thought to be a significant engine of transformation without affecting the environment. The one-pot green approach incorporated distinct features within the "Twelve Principles of Green Chemistry", including simple operation, short reaction time, mild reaction condition and high yield. The one-pot reaction is atom economic, time saving and most notably does not require purification at each step to afford high regio-and stereoselective products. Thus, one-pot strategy has been considered as best method for the preparation of heterocyclic compounds and gaining enormous attention of synthetic organic chemists owing to their eco-friendliness, shorter reaction time, improved selectivity and superior work-up procedures.
Several research articles on the application of one-pot domino approach in chemical synthesis have been published. The one-pot multicomponent reaction is a more pronounced approach for the synthesis of pyrimidine and quinazoline derivatives [1][2][3].
In COVID-19 scenario, the requirement of new antifungal, antibacterial and immunityboosting synthetic molecules has been increased throughout the world. The present paper focuses on the incorporation of five membered nitrogen containing heterocyclic motifs (pyrrole, pyrazole, imidazole) onto the privileged pyrimidine and quinazoline nucleus linked to each other through a Scheme 4. Synthesis of pyrrole, pyrazole, imidazole bearing diphenyl-quinazoline derivatives from N-phenylbenzamide.

Synthesis of N-hydroxy-4-(pyrrol-1-yl)benzimidamide (4)
Potassium hydroxide (0.196 g, 0.0035 mol) was added to hydroxyl amine hydrochloride (0.243 g, 0.0034 mol) in distilled water to break the hydrochloride salt. This solution was added to 4-(1H-pyrrol-1-yl)benzonitrile (1.2 g, 0.0071 mol) (3) in ethanol and the reaction mass was subjected to reflux at 100 o C for 2 h. The completion of reaction mixture was checked by TLC (Hex:EtOH/8:2). The reaction mass was cooled to RT (Room temperature), then extracted with ethylacetate (3 x 10 mL), combined organic layer was dried with anhydrous magnesium sulfate and concentrated under vaccum to obtain crude product which was purified by flash column chromatography (15- Synthesis of methyl 2-4-pyrrol-1-phenyl-5,6-dihydroxy pyrimidine-4-carboxylate (6) Amidoxime (4) (1.1 g, 0.0054 mol) and dimethylacetylenedicarboxylate (0.7 g, 0.0054 mol) was refluxed in chloroform and then in acidic medium for 14 h, the reaction completion was checked by TLC (Hex:EtOH/5:5). The solvent chloroform was completely distilled off under vacuo to obtained brown oil, in which xylene was added dropwise and stirred the reaction mass for 16 h at 100 o C after the reaction completion. Brown colored solid was found in the reaction which were recrystallize with xylene followed by hexane and then dried to obtain pyrrole linked pyrimidine derivatives 6. Molecular
Molecular  (13) 4-Cyano phenylhydrazine (1.6 g, 0.01201 mol) 11 was mixed with acetyl acetone (1.5 g, 0.0149 mol) 12 in ethanol and methane sulfonic acid was added in catalytic amount to the reaction mixture, further it was condensed at 90 °C for 2 h. The reaction completion was checked by thin layer chromatography (Hex:EtOH/7:3). After completion of reaction, ethanol was distilled under attenuated pressure and the crude product was purified by column chromatography (

Synthesis of 4-(3,5-dimethyl-1-pyrazolyl)-hydroxybenzimidamide (14)
Potassium hydroxide (2 g, 0.023 mol) was mixed to hydroxyl amine hydrochloride (1.62 g, 0.023 mol) in distilled water to break the hydrochloride salt. This solution was added to 13 (2 g, 0.010 mol) in ethanol and the reaction was subjected to reflux at 100 °C for 2 h. The reaction completion was monitored by TLC with mobile phase (Hex:EtOH/7:3), After the reaction completion, reaction mass was cooled to RT, extracted with ethyl acetate and chloroform, dried with sodium sulfate. The excess solvent was evaporated by rotatory evaporator and purified by column chromatography (17-20%

Synthesis of methyl 3,5-dimethyl(-1-pyrazolyl)-pyrimidine-4-carboxylate(15)
Amidoxime 14, (0.5 g, 0.00217 mol), dimethylacetylenedicarboxylate (0.77 g, 0.0054 mol) 5 and catalytic amount of DABCO (0.045 g, 0.0004 mol) was mixed in dichloromethane and the reaction mass was refluxed for 2h at 60 o C, the reaction completion was checked by TLC with mobile phase (Hex:EtOH/7:3), on completed dichloromethane was completely distilled off by vacuum to obtain brown oil, which was stirred for 6 h at 105 o C after addition of xylene. Brown colored solid was found after filtration and washing first with xylene followed by hexane to yield crude 15. Finally, crude product was purified by column chromatography (20-

Synthesis of 4-cyanophenyl-3-phenyl-1-pyrazol benzamide(19)
4-Cyano phenyl hydrazine (1 g, 0.0751 mol) 11 and benzoylacetonitrile (1.2 g, 0.0082 mol) 16 was mixed without any solvent and heated under stirring for 3 h at 110-120 o C. The reaction mass was cooled at RT by adding dichloromethane and slowly benzoyl chloride was added 18 (1.86 g, 0.0002 mol) in the reaction mass under N2 atmosphere. The reaction mixture was well shaken in stirrer for 3 h and completion of reaction was determined by TLC. The reaction mass was extracted in methanol. Then, the filtrate was concentrated to yield desired product 19 which was isolated without further purification.

Synthesis of (Z)-N-(1-(4-(N'-hydroxycarbamimidoyl)phenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (20)
Potassium hydroxide (2.53 g, 0.030 mol) and hydroxyl amine hydrochloride (1.9 g, 0.0273 mol) 2.044 were added in 50 mL of distilled water, then this solution was mixed to the solution of nitrile substrate 19 in 50 mL ethanol at RT. The reaction mass was heated to 85 o C for 10 h. The reaction was checked by TLC (Hex:EtOH/7:3). The reaction mass was extracted with DCM and H2O. The compound containing organic layer was separated and dried by sodium sulfate led to obtain crude white crystalline solid. Purification was done by flash chromatography (20-

Synthesis of 4,5-diphenyl-1-imidazol-2-yl)benzonitrile (24)
A reaction mixture of benzil (0.8 g, 0.0038 mol), equimolar quantity of 4-cyano benzaldehyde, ammonium acetate ( 0.99 g, 131.13 mol) and 1 g mixture of P2O5/SiO2, all were mixed in a single neck 100 mL flask and placed in a microwave synthesizer at 50 W, 80-100 o C for 5-8 min. Then, reaction completion was checked by TLC with mobile phase (Hex:EtOH/6:4). Ethylacetate was added to the reaction mixture and stirring was done for 1 h at 50 o C. Further, reaction mass was recrystallized with n-hexane to obtain pale yellow solids of 24.

Synthesis of (Z)-4-(4,5-diphenyl-1H-imidazol-2yl)-N'-hydroxybenzimidamide (25)
Sodium bicarbonate (1.99 g, 0.0238 mol) was mixed to hydroxyl amine hydrochloride (1.61 g, 0.0233 mol) in distilled water to break the hydrochloride salt. This solution was added to 24 (3 g, 0.0093 mol) in ethanol and reaction mass was subjected to reflux at 100 °C for 2 h. The completion of reaction was checked by TLC (Hex:EtOH/7:3). After the reaction completion, the temperature of reaction mixture was lowered to RT and filtration of reaction mass was done to obtained crude compound. Purification was done by silica flash chromatography (18-20%
The synthesized compound (4) was affirmed by the appearance of peak at 3600 cm -1 (O-H group) and 3410 cm -1 (N-H group) in the IR spectrum. Further, the structure of compound (4) was ascertained by the presence of additional peak at δ 9.68 and δ 5.88, which may be attributed to the presence of -OH and -NH2 respectively of amidoxime group. The appearance of peak at 1700 cm -1 and 1646 cm -1 (C-O, stretching) affirmed the formation of (6) from (4). The synthesis of a pyrimidine ring in (6) was assured by one upfield at δ 3.36 for three proton of methyl ester group and sharp singlet at δ 8.01 for one proton of OH group. The presence of 2913 cm -1 peak (C-H Str, aliphatic) along with peak at 1218 cm -1 (C-N bending) ascertained the formation of (8). The IR spectrum of (9) revealed presence of Fermi resonance peak of -NH2 group at 3475-3392 cm -1 and appearance of peak at 1739-1833 cm -1 (-COOMe) due to the coupled vibration confirmed the formation of (10). The 1 H NMR of (8) signifies the presence of six methyl protons which appeared in the form of sharp singlet at δ 2.45 and the formation of (9) was confirmed through the sharp downfield singlet at δ 9.77 which represents the presence of -OH of amidoxime. Finally the formation of (10) was ascertained by the appearance of additional sharp singlet of methyl ester at δ 3.87 in its 1 H NMR spectra. Similarly, the structures of synthesized compounds 15, 21 and 26 was confirmed.
Another one pot pioneering reaction deals with the activation of less active N-aryl amide like benzanilide (27). The amide was activated in two steps, firstly reacted with Tf2O and secondly reacted with 2-chloropyridine. The activated benzanilide (29) was now ready to couple with substituted nitrile 3, 8, 13, 19, 24. The synthesized nitrilium (30)(31)(32)(33)(34) undergo cyclocondensation with the π-electron system of benzene ring to yield the quinazoline nucleus in a single step 35-39 depicted in Scheme 4 and mechanism shown in Scheme 6. Scheme 5. Plausible mechanism for the formation of pyrimidine derivatives. Scheme 6. Plausible mechanism for the formation of quinazoline derivatives.

Antimicrobial activity
The synthesized derivatives were examined for antibacterial and antifungal activity. The antibacterial activity of the synthesized compound was screened against the gram negative bacteria E. coli, P. aeruginosa and gram positive bacteria S. pyogenes and S. aureus. The derivatives were also screened for antifungal activity against C. albicans and A. clavatus. The microbroth dilution method was used for the determination of MIC, MBC and MFC. Among all the ten synthetically derived compounds, compound 39 showed maximum antibacterial potential against all tested bacterial pathogens with a zone of inhibition of 23, 20, 23 and 20 mm against S. pyogenes, S. aureus, E. coli and P. aeruginosa, respectively, at the concentration of 250 µg/mL while derivatives 10 and 21 both showed maximum antifungal potential with zone of inhibition of 23 and 22 mm against C. albicans and A. clavatus, respectively (Table 1). The compound 39 also displayed least MBC against E. coli, P. aeruginosa, S. aureus and S. pyogenes which was comparable to reference drugs ampicillin, ciprofloxacin, norfloxacin and gentamycin revealing its potent antibacterial activity. While each of the synthesized compound exhibited different MFC values against each of the tested fungal pathogens revealing their moderate antifungal activity when compared to standard drugs nystatin and griseofulvin.

Minimum inhibitiory concentration
Compound No.

E. coli P. aeruginosa S. aureus S. pyogenes Ampicillin
One-pot mediated synthesis of pyrimidine and quinazoline annulated derivatives

CONCLUSION
The novel and efficient methodologies were described for the preparation of pyrimidine and quinazoline derivatives using a one-pot domino approach. Compound 39 showed maximum antibacterial potential against the bacterial pathogens with a zone of inhibition of 23 mm, 20 mm, 23 mm and 20 mm against P. aeruginosa, S. pyogenes, E. coli, S. aureus, respectively, at a concentration of 250 µg/mL while compound 10 exhibited potential antifungal activity against C. albicans and A. clavatus. This work exploited a synthesis of various pyrimidine and quinazoline The findings of the present study are promising, however attaining the same extent of response in in vitro and in vivo studies would be a great challenge to the investigators. Thus, further efforts are required to conduct in depth in vitro and in vivo studies to establish the safety and efficacy of these compounds for their future development in clinical practice.