Synthetic Approach for the Novel Semicarbazones of Quinazoline ring and its Biological Activity

 

Ponnilavarasan I^1*, Rajasekaran A¹, Sivakumar KK¹, Sundaramoorthi C¹, Swastika Ganguly² and Sivasakthi R³

¹Department of Pharmaceutical Chemistry, College of Pharmacy, Kovai Medical Center Research and Educational Trust, Coimbatore, 641048, India.

² Birla Institute of Technology, Mesra, Ranchi, India.

³Cherrann’s College of Pharmacy, Coimatore, Tamilnadu, India.

*Corresponding Author E-mail: ponns75@gmail.com, ponns75@rediffmail.com

ABSTRACT:

A number of organic compounds obtained by chemical synthesis as model compounds have useful antimicrobial activities. Quinazoline ring is an aromatic benzopyrimidine system; many of its derivatives possess interesting biological activities, such as analgesic, anti-inflammatory, anti-microbial, and anti-tumor. In our study, the biological activity of synthesized quinazoline semicarbazone derivatives were characterized by antimicrobial screening against several gram-positive, gram negative bacteria, and fungus. The purity of the synthesized compounds was characterized by physicochemical properties, such as solubility, melting point, and thin layer chromatography (TLC).Elemental analysis for carbon, nitrogen, hydrogen, and oxygen was performed according to standard procedures. The presence of functional groups was analyzed using FT-IR spectra. Molecular structural information for the compounds was analyzed by 1H-NMR spectroscopy. The wavelengths of maximum absorbance for all the synthesized compounds were measured by UV-Visible spectroscopy. Thereby the chemical structures of the synthesized quinazoline derivatives were confirmed. Antimicrobial screening for all the compounds exhibits characteristic microbial inhibition. A detailed study is in progress to modify the synthetic route, structural activity, and toxicological barriers for the enhanced pharmacological efficiency of synthetic antibiotics.

 

 

 

INTRODUCTION:

The clinical potential of microbial product as therapeutic agents was first investigated by Pasteur and Joubert, who recorded their observation and speculations in 1877. The golden age of antibiotics began with the production of penicillin in 1941. Common usage of antibiotics includes synthetic antibacterial agents, such as sulfonamides and quinolones, which are not products of microbes. Antibiotics differ markedly in physical, chemical, and pharmacological properties, antibacterial spectra, and mechanism of action. Synthetic quinazolines and its derivatives of semicarbazones possess interesting biological activities. Most biologically active quinazolines are having substitutions at C-2 and N-3 positions.

 

Isatin derivatives with 3-amino-2-methyl mercaptoquinazolin-4(3H)-one form Schiff bases and N-Mannich bases of quinazolines¹,quinazolinone derivative was converted into bis [quinazoline-4-thiozo-2-yl]–m-phenylene via different routes² synthesis of quinazoline and naphthalene from 3-methyl-5-phenyl-6-benzylidene-2-cyclohexene-1-one³, thioglycolic acid and pyrazole derivatives of 4(3H) quinazolinone were synthesized⁴. Anti fungal, antibacterial, anti-HIV activity of Schiff, and Mannich bases derived from isatin derivatives and N-[4-(4’- chlorophenyl) thiazol-2-yl] thiosemicarbazide⁵, fluorinated hydroquinazoline derivatives as antifungal agents⁶ antibacterial activity of quinazolines,⁷ 6-chloro-2-morpholino 4-quinazolyl-5-vitro-20-furyl hydrazone as antibacterial agent⁸  also were studied. On this basis, we have synthesized some derivatives of quinazoline semicarbazone by condensation of semicarbazide with different aromatic carbonyl groups. Elemental analysis and spectroscopic techniques, such as UV-Visible, FT-IR, and 1H-NMR, were used to ensure the chemistry of the synthesized quinazoline semicarbazone derivatives. Investigation of antimicrobial activity of compounds was performed by the disk diffusion method using four gram negative and four gram-positive nonpathogenic bacteria and one fungus.

 

MATERIALS AND METHODS:

Structural investigation:

The synthesized quinazoline semicarbazone derivatives were denoted by specific codes for identity. Detailed physicochemical analysis of synthetic organic compounds enables the measurement of extent of purity, structural evidence, solubility variations, melting temperature measurements, etc. Melting points were determined in open capillary tubes with electro thermal melting point apparatus and are uncorrected. The solubility of all the compounds were tested by using water, chloroform, ethanol, DMF, DMSO, benzene, acetic acid, ethyl acetate, and dilute acids. TLC was performed as⁹ dimension of plates: 5 × 20 cm, stationary phase: silica gel-G (E-Merck), mobile phase: ethyl acetate: n-butanol: water (6:3:1), technique involved is ascending and iodine vapor was used as the detecting agent. The synthesized compounds were analyzed and the retardation factor (RF) values were calculated. Elemental analysis of the percentage weights of carbon, hydrogen, nitrogen, and oxygen of different derivatives was performed¹⁰. Infrared analysis was performed by JASCO 4100 FT-IR using KBr pellet disc technique. 1H-NMR spectral study for new quinazoline semicarbazone derivatives were performed by using DMSO as a solvent in ‘‘BRUKER, AV 300 MHz,’’ transform 1H-NMR spectrometer. The maximum absorbance or   kmax of synthesized compounds were determined in the concentration of 0.01%w/w in DMSO by using Shimadzu 1700A UV-Visible spectroscopy.

 

Chemistry:

Synthesis of 2-aryl-3-amino-4(3H)-quinazolinone:

Anthranilic acid (0.01mole) was dissolved in dry pyridine (30ml) by stirring slowly at room temperature. The solution was cooled to 0° and a solution of benzoylchloride (0.02mole) in dry pyridine (30ml) was added slowly with constant stirring. After this addition the reaction mixture was further stirred for half an hour at room temperature and set aside for 1hr. The pasty mass obtained was diluted with water (50ml) and treated with aqueous sodium bicarbonate solution. When the effervescence ceased the precipitate obtained was filtered off and washed with water, dried and recrystallized from diluted ethanol.

 

Synthesis of quinazolinone urea:

2-Aryl-3-amino-4(3H)quinazolinone (0.1mole) was dissolved in 10ml of glacial acetic acid and diluted to 100ml with water. To this equimolar quantity of sodium cyanate in 50ml of warm water was added with stirring then allowed to stand for 30mts, cooled in ice for further 30mts. The precipitate obtained was filtered, washed with water, dried and recrystallized from boiling water.

 

 

Synthesis of quinazoline semicarbazide:

To a solution of quinazolinone urea (0.1mole) in 200ml of water equimolar quantity of hydrazine hydrate was added. The reaction mixture was made alkaline by 4gm of NaOH, and then added required quantity of ethanol to give a clear solution. The reaction mixture was refluxed for 1.5hrs, cooled and filtered the precipitate. The precipitate was recrystallized from ethanol.

 

General procedure for the synthesis of 2-Aryl-3-amino-4(3H) quinazolinone semicarbazone:

2-Aryl-3-amino-4(3H) quinazolinone semicarbazide (0.01mole) was dissolved in ethanol (20ml) and added slowly to an ethonolic solution of aromatic carbonyl compound (0.01mole). The reaction mixture was catalyzed with 5ml of glacial acetic acid and refluxed for half an hour. The precipitate was collected washed with ether and water, dried and recrystallized from ethanol.

 

BIOLOGICAL INVESTIGATION:

The biological evaluation of synthesized compound was performed using the disk diffusion method^11,12. In the present study four gram-positive, four-gram negative, and one fungus were selected. The gram positive strains were Bacillus lentus (NCIM 2018), Bacillus cereus (NCIM 2018), Micrococcus luteus (NCIM 2155), Staphylococcus albus (NCIM 2178); gram negative strains were Escherichia coli (NCIM 2065), Klebsiella aerogenes (NCIM 2075), Salmonella paratyphi (NCIM 2075), Proteus vulgaris (NCIM 2239), and fungus Candida albicans (NCIM 0707). The strain was confirmed for its purity and identity by the gram-staining method and it was further characterized by chemical reaction. The selected strains were preserved by periodical sub culturing on agar slant and storing them under frozen condition; for the study fresh 24 hours broth cultures were used. Each bacterial and fungal pure culture was transferred into 100 ml of Muller Hinton nutrient broth and Sabouraud’s dextrose broth, respectively. The inoculated broths were incubated at 37°C for 24 hours and 27°C for 72 hours for bacteria and fungus respectively. After incubation, inocula were standardized to 108 colony-forming units (CFU)/ml for bacteria and 106 CFU/ml for fungus by colony forming unit method. Muller Hinton agar media was prepared by using Beef infusion 300 g, Casein acid hydrolysation 17.5 g, starch 1.5 g, and agar 17 g. Accurately weighed quantities of these ingredients were suspended in 1,000 ml of distilled water. They were boiled to dissolve completely. The pH was adjusted to 7.3 ± 0.2 at 25°C. It was then sterilized by autoclaving at 15 lbs. pressure (121°C for 15 minutes). The prepared Muller Hinton agar medium was transferred into sterile Petri plates; 200 µl of the standardized bacterial inoculums and fungus inoculum were spread on agar medium using sterile cotton swab. The synthesized product of quinazoline semicarbazone derivatives were dissolved in suitable chloroform solvent to a final concentration of 50 µl of drug solution, assuming that each disk absorbed approximately 10 µl of the drug. The drug was impregnated on disk and placed on the inoculated agar medium.

 

TABLE 1: Physical data of the synthesized compounds

Compound	Melting point  (⁰C)	Rf	Yield (%)	Log P*	Molecular formula	Molecular weight
1.	232	0.60	92	2.07	C22H17N5O3	399.4
2.	234	0.63	90	2.19	C22H17N5O2	383.14
3.	209	0.52	92	1.97	C20H15N5O3	373.36
4.	214	0.46	90	1.88	C22H17N5O3	399.4
5.	241	0.52	90	2.06	C23H15BrN6O3	503.441

*log P was calculated by partition coefficient determination using Octanol and buffer system.

 

TABLE 2: Values obtained by elemental analysis for individual compounds

Compound	Carbon		Hydrogen		Nitrogen		Oxygen		Bromine		Exact mass
	A	B	A	B	A	B	A	B	A	B
1.	69.34	69.45	4.55	4.69	14.06	14.17	12.05	12.20	-	-	398.14
2.	72.24	72.36	4.74	4.90	14.65	14.76	8.37	8.49	-	-	382.14
3.	67.73	67.90	4.33	4.46	15.05	15.17	12.89	13.99	-	-	372.12
4.	69.34	69.46	4.55	4.67	14.06	14.18	12.05	13.18	-	-	398.14
5.	54.89	54.98	3.00	3.11	16.70	16.82	9.54	9.70	15.88	15.98	502.04

A- Calculated    B-Found

 

TABLE 3: SPECTRAL DATA FOR INDIVIDUAL COMPOUNDS

Comp.	IR(cm-1)	NMR(δppm)	UV(λmax) nm
1.	780(Ar-CH),1580(C=N) 1660(C=O),1261(OH),3446(NH)	6.68-7.67 δppm- aromatic H, 7.92 δppm- HC=N, 9.21 δppm- NHCO, 10.06- δppm OH	336
2.	776(Ar-CH),1563(C=N) 1669(C=O), 3443(NH)	7.02-7.79 δppm- aromatic H, 7.89 δppm- HC=N, 9.30 δppm- NHCO	322
3.	790(Ar-CH),1554(C=N) 1650(C=O), 3440(NH)	7.23-7.64 δppm- aromatic H, 7.78 δppm- HC=N, 9.24 δppm- NHCO	380
4.	789(Ar-CH),1589(C=N) 1666(C=O),1263(OH),3446(NH)	6.71-7.62 δppm- aromatic H, 7.83 δppm- HC=N, 9.32 δppm- NHCO, 10.16- δppm OH	390
5.	740(Ar-CH),1600(C=N) 1658(C=O), 3423(NH)	6.4–6.68 δppm- aromatic H, 7.65 δppm- isatin NH, 7.73 δppm- HC=N, 9.11 δppm- NHCO	296

 

 

Ciprofloxacin and clotrimidazole were used as a standard for the antibacterial and antifungal activity, respectively. All the bacterial Petri plates were kept in an incubator and the fungal Petri plate was kept at room temperature for approximately 18 hours. Then the zones of inhibition were measured.

 

Fig. 1 Schematic structure for the synthesized quinazoline semicarbazone derivatives

 

Compound 1 

3-(2-hydroxy-phenylidenesemicarbazone)- 2-phenyl-3H-quinazolin-4-one,

 

 

Compound 2 

3-(phenylidenesemicarbazone)-2-phenyl-3H-quinazolin-4-one,

 

 

Compound 3

3-(2-furyl semicarbazone)-2-phenyl-3H-quinazolin-4-one,

 

 

Compound 4 

3-(4- hydroxy-phenylidenesemicarbazone)- 2-phenyl-3H-quinazolin-4-one,

 

 

Compound 5 

3-(2-oxo-1, 2–dihydro-indolydenesemicarbazone)-2-phenyl-3H-quinazolin-4-one,

 

RESULTS:

The schematic molecular structures of all the compounds are shown in Fig. 1.The scheme of the reaction for the synthesized compounds are shown in Fig.2. The physicochemical properties, RF values of TLC, and the calculated and found elemental composition of all the elements are reported in Tables 1 and 2 consecutively. FT-IR spectral interpretation of newly synthesized compounds for functional groups shows the specific range of stretching and bending frequencies. The entire spectral data’s and NMR spectrum were individually reported in Table 3. Biological investigation of this new series of quinazoline semicarbazone showed an excellent zone of inhibition against several gram-positive and gram-negative bacteria and fungus Candida albicans. For the solvent chloroform, the same was done separately; their inhibition values are included in Table 4.

 

Fig.2.Scheme of the synthesized compounds

 

DISCUSSION:

From the structural investigation, IR spectra showed the stretching frequency range between 1588 and 1629 cm-1, which evinced the presence of imine linkage and also the absence of -NH2 peak for the synthesized quinazoline semicarbazone derivatives. Dependant substitution of double-bonded nitrogen group of imine C=N could be the reason for the characteristic absorption close to the carbonyl C=O of amide (1630–1680 cm-1) or C=C of alkene (1600–1680 cm-1) double bond stretching region¹³. 1H-NMR spectra give a characteristic proton resonance shifts for all the synthesized quinazoline semicarbazone derivatives, which ensured the existence of aromatic, amine, amide, and imine protons. The wavelengths of maximum absorbance (kmax) for all the synthesized compounds were shown the specific absorptivity. The energetically most favorable p-p* excitation occurs from the highest energy bonding pi-electron to the lowest energy antibonding pi-electron systems. Conjugated pi-electron systems (unsaturated carbonyl and aromatic ring compounds) of synthesized compounds act as chromophores and absorbed the light, in the region of 296–390 nm. Estimated elemental compositions were within ±0.4% of the calculated values. The antimicrobial screening of all the compounds showed an excellent zone of inhibition against both gram-positive and gram-negative bacteria than standard ciprofloxacin. Similarly, the zone of inhibition on the fungal strain showed a stronger activity than the standard clotrimidazole. New derivatives of quinazoline semicarbazone series exhibits stronger inhibition on gram-negative Escherichia coli compared with other bacterial strains. On the other hand, Candida albicans zone was highly inhibited by quinazoline semicarbazone derivatives, which proves the efficiency of antifungal activity than antibacterial activity. The zone of inhibition on Staphylococcus albus comparatively smaller than other bacterial species. Discussing the antimicrobial activity against individual organisms, it was clear that all the compounds have significant inhibitions. It was found that the Escherichia coli and Candida albicans were highly susceptible to killing by the synthesized quinazoline semicarbazone. Selective thymidylate synthetase and cell growth inhibition by 3-chloro-N-((3, 4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl)-4-(phenylsulfinyl)-N-(prop-2-ynyl)-aniline was found to be show a potent antitumor activity¹⁴. Some aryl semicarbazone derivatives¹⁵ were reported to be potent antituberculosis agent. 3-Chloro-2-methyl phenyl substituted semicarbazones was characterized as an anticonvulsant, which evinced the anticonvulsant and antitubercular activity of semicarbazones. In our study, we have synthesized the biologically active condensed product of quinazoline semicarbazones. The semicarbazones was substituted at N-3 position of quinazoline, which again proves the novelty of biological efficiency of our new quinazoline series as synthetic antibiotics. In the future, the compounds will be modified further to reduce the molar mass and toxicological barriers. Based on the literature review, the compound will be screened for central nervous system activity, such as sedatives, hypnotics, and psychotics.

TABLE 4: DIAMETER OF ZONE OF INHIBITION BY INDIVIDUAL COMPOUNDS AGAINST GRAM-POSITIVE, GRAM-NEGATIVE BACTERIA, AND FUNGUS

Zone of inhibition in mm	Compounds
Organism	Standard	1	2	3	4	5	Solventb
Gram positive bacteria
Bacillus lentus	09	16	17	17	17	18	6
Micrococcus luteus	08	16	15	14	12	15	3
Bacillus cereus	09	12	14	14	15	16	3
Staphylococcus albus	08	13	13	12	10	15	4
Gram negative bacteria
Escherichia coli	16	21	19	18	21	19	4
Klebsiella aerogenes	10	17	16	16	15	17	5
Salmonella paratyphi	08	16	15	15	16	15	4
Proteus vulgaris	08	17	16	17	16	18	4
Fungus
Candida albicans	14	22	21	20	25	21	3

_a Standard ciprofloxacin for bacteria, clotrimazole for fungal

_bChloroform

 

The present study provides the further broad-spectrum activity of substituted quinazoline semicarbazones that are comparatively higher or equipotent to the antibiotic and antifungal agents in the comparison tests.

 

ACKNOWLEDGEMENT:

This work is supported by the Kovai Medical Centre Hospital, Research and Educational institute. Authors are thankful to the management of Kovai Medical Centre Hospital, Research and Educational institute.

 

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Received on 22.11.2009        Modified on 29.12.2009

Accepted on 27.01.2010        © AJRC All right reserved