- Corresponding Author:
- P. Selvam
Department of Pharmaceutical Chemistry, Devaki Amma Memorial College of Pharmacy, Chelembra, Malapuram - 673 436
E-mail: periyasamy_selvam@yahoo.co.in
| Date of Submission | 14 November 2009 |
| Date of Revision | 27 August 2010 |
| Date of Acceptance | 16 November 2010 |
| Indian J Pharm Sci, 2010, 72 (6): 806-809 |
Abstract
A series of novel 2,3-disubstitutedquinazolin-4(3H)-ones have been synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and anthranilic acid. Synthesized compounds were evaluated for in vitro antiviral activity against HIV, HSV and vaccinia viruses. 5-Bromo-2-(6-bromo-4-oxo-2-phenyl-4H-quinazolin-3-yl)-benzoic acid (MBR2) exhibited distinct antiviral activity against Herpes simplex and vaccinia viruses.
Keywords
Quinazoline, anthranilic acid, HIV, MT-4 Cells, MTT assay, vaccinia virus
Quinazolin-4-(3H)-one is a versatile lead molecule for designing potential bioactive agents and its derivatives were reported to possess broad spectrum activities. 2-Phenyl-3-substitutedquinazolin-4-(3H)-ones were reported to have antiHIV activity [1-3] and anticancer activity [4-6]. Quinazolinones were screened for their wide spectrum antiviral activity and they have rich potential for further studies [7-9]. A series of some novel 2,3-disubstituted quinazolin-4(3H)-one derivatives has been synthesized by condensation of primary aromatic amino group of anthranilic acid with 2-substituted- 1,3-benzoxazine-4-one [8] to afford 2,3-disubstituted quinazolin-4(3H)-ones (Scheme 1).
Scheme 1: Synthesis of 2-phenyl-3-substituted quinazolin-4(3H)-ones For QAA1, R1 is H, R2 is H, Ar is -phenyl carboxylic acid; for QAA2, R1 is Br, R2 is H, Ar is -4-bromo-2-phenyl carboxylic acid; for QAA3, R1 is Br, R2 is Br, Ar is -4,6-dibromo-2-phenyl carboxylic acid; for MBR1, R1 is Br, R2 is H, Ar is -2-phenyl carboxylic acid; for MBR2, R1 is Br, R2 is H, Ar is -4-bromo-2-phenyl carboxylic acid; for DBR1, R1 is Br, R2 is Br, Ar is -2-phenyl carboxylic acid; for DBR2, R1 is Br, R2 is Br, Ar is -4-bromo-2 phenyl carboxylic acid; for DBR3, R1 is Br, R2 is Br, Ar is -4,6-dibromo-2-phenyl carboxylic acid.
Melting points were determined using open ended capillary tube method and are uncorrected. IR spectrum were recorded In KBr on a Perkin Elmer-1605 grating spectrometer (cm-1). 1H NMR Spectra were recorded at 400 MHz on Bruker FTNMR spectrophotometer using TMS as internal standard. Mass spectra were recorded on a Varian Atlas CH-7 Mass Spectrophotometer at 70 eV by adopting electron impact ionization.
An equimolar mixture (0.01 mol) of 2-phenylsubstituted benzo [1,3]oxazin-4-one and compounds with primary aromatic amino functional group (anthranilic acid, 6-bromo- or 3,5-dibromo anthranilic acid) was mixed and the mixture was refluxed for 6 h in 10 ml of pyridine. Upon cooling, the mixture was poured onto crushed ice. The precipitated solid was collected and recrystalized from ethanol to give the desired title compounds.
The yields and the melting points of the compounds are given in (Table 1) Spectral data as follows 2-(4-oxo-2-phenyl-4H-quinazolin-3-yl)-benzoic acid (QAA1) IR (KBr) cm-1: 1537 (C=C), 1661 (C=N), 1697 (C=O), 3128 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 13H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):342. 5-Bromo-2-(4-oxo-2-phenyl-4H-quinazolin- 3-yl)-benzoic acid (QAA2) IR (KBr) cm-1: 1530 (C=C), 1665 (C=N), 1692 (C=O), 3130 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 12H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):432. 3,5-dibromo-2-(4-oxo- 2-phenyl-4H-quinazolin-3-yl)-benzoic acid (QAA3) IR (KBr) cm-1: 1532 (C=C), 1665 (C=N), 1691 (C=O), 3125 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 11H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):522. 2-(6-Bromo-4-oxo-2-phenyl-4H-quinazolin-3-yl)- benzoic acid (MBR1) IR (KBr) cm-1: 1529 (C=C), 1658 (C=N), 1685 (C=O), 3129 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 12H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):432. 5-Bromo-2-(6-bromo-4- oxo-2-phenyl-4H-quinazolin-3-yl)-benzoic acid (MBR2) IR (KBr) cm-1: 1531 (C=C), 1659 (C=N), 1687 (C=O), 3125 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 11H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):522. 2-(6,8-Dibromo-4-oxo-2-phenyl-4H-quinazolin-3-yl)- benzoic acid (DBR1): IR (KBr) cm-1: 1537 (C=C), 1661 (C=N), 1697 (C=O), 3122 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 12H, Ar-H), 11.6 (s, 1H, COOH); EI-MS(m/z):522. 5-Bromo-2-(6,8-dibromo- 4-oxo-2-phenyl-4H-quinazolin-3-yl)-benzoic acid (DBR2) IR (KBr) cm-1: 1537 (C=C), 1661 (C=N), 1697 (C=O), 3125 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 11H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):612. 3,5-Dibromo-2-(6,8-dibromo-4-oxo-2- phenyl-4H-quinazolin-3-yl)-benzoic acid (DBR3) IR (KBr) cm-1: 1537 (C=C), 1661 (C=N), 1697 (C=O), 3126 (OH): 1H NMR (DMSO-d6): 8.1-7.1 (m, 10H, Ar-H), 11.6 (s, 1H, COOH); EI-MS (m/z):702.
| Compound code | Mol. formula | Yield (%) | M.P(˚) | Rf Value# |
|---|---|---|---|---|
| QAA1 | C21H14N2O3 | 72.7 | 148 | 0.118 |
| QAA2 | C21H13N2O3Br | 66.3 | 165 | 0.51 |
| QAA3 | C21H12N2O3Br2 | 92.6 | 70 | 0.512 |
| MBR1 | C21H14N2O3Br | 72.7 | 154 | 0.118 |
| MBR2 | C21H13N2O3Br2 | 51.6 | 135 | 0.384 |
| DBR1 | C21H12N2O3Br2 | 62.8 | 140 | 0.213 |
| DBR2 | C21H11N2O3Br3 | 55.1 | 150 | 0.407 |
| DBR3 | C21H10N2O3Br4 | 68.1 | 72 | 0.424 |
#Purity of the compounds was checked by TLC using solvent system CHCl3:CH3OH (9:1) and spot is visualized by iodine vapour
Table 1: Physical data for synthesized compounds
The compounds were tested for antiHIV activity against the replication of HIV-1(IIIB) and HIV- 2(ROD) in MT-4 cells [8]. The cells were grown and maintained in RPMI 1640 medium supplemented with 10% heat–inactivated Fetal Calf Serum (FCS), 2 mM- glutamine, 0.1% sodium bicarbonate and 20 μg/ml gentamicin (culture medium). Human immunodeficiency virus-1 (HTLV-IIIB/LAI) strain and HIV-2 (LAV-2ROD) strain were used in the experiment. The virus strains were propagated in MT-4 cells. Titer of virus stock was determined in MT-4 cells and the virus stock was stored at -70° until used. Inhibitory effects of the compounds on HIV-1 and HIV-2 replication were monitored by inhibition of virus-induced cytopathic effect in MT-4 cells and were estimated by MTT assay. Briefly, 50 μl of HIV-1 and HIV-2 (100-300 CCID50) was added to a flat-bottomed MT–4 cells (6×105 cells/ml). After 5 d of incubation, at 37° the number of viable cells were determined by the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cytotoxicity of the compounds for mock-infected MT-4 cells was assessed by the MTT method. AntiHIV activity and cytotoxicity of standard AZT were also performed by a similar method in MT-4 cells. The antiHIV activity and cytotoxicity data are presented in (Table 2).
| Compound code | Strain | IC50a(μg/ml) | IC50b(μg/ml) |
|---|---|---|---|
| QAA1 | IIIB | >125 | >125 |
| ROD | >125 | >125 | |
| QAA2 | IIIB | >88.20 | >88.20 |
| ROD | >88.20 | >88.20 | |
| QAA3 | IIIB | 101 | >125 |
| ROD | >125 | >125 | |
| MBR1 | IIIB | 74.8 | >125 |
| ROD | >125 | >125 | |
| MBR2 | IIIB | >60.40 | >60.40 |
| ROD | >60.40 | >60.40 | |
| DBR1 | IIIB | >83.15 | >83.15 |
| ROD | >83.15 | >83.15 | |
| DBR2 | IIIB | >64.18 | >64.18 |
| ROD | >64.18 | >64.18 | |
| DBR3 | IIIB | >125 | >125 |
| ROD | >125 | >125 | |
| AZT | IIIB | 0.0012 | 65.90 |
| ROD | 0.00062 | 65.90 |
a50% Effective concentration of compound, achieving 50% protection of MT-4 cells against the cytopathic effect of HIV. b50% Cytotoxic concentration of compound, required to reduce the viability of mock-infected MT-4 cells by 50%.
Table 2: Anti-hiv activity of quinazolin-4(3h)-ones
Antiviral activity and cytotoxicity of the synthesized compounds were determined by in vitro cell culture techniques [8]. The antiviral assays were based on inhibition of virus-induced cytopathicity in HEL (HSV-1 and HSV-2, VV, VSV) cultures. Briefly, confluent cell culture in 96-well microtiter plates were inoculated with 100 CCID50 of virus, 1 CCID50 being the virus dose required to infect 50% of the cell cultures. After a 1 h virus adsorption period, residual virus was removed and the cell cultures were incubated in the presence of varying concentrations (400, 200 and 100 μg/ml) of the test compounds. Viral cytopathicity was recorded as soon as it reached as completion in the control virus-infected cell cultures exposed to the test compounds. The antiviral activity and cytotoxicity data are presented in (Table 3).
| Compound | Minimum cytotoxic concentrationa (µg/ml) | EC50B(μg/ml) | |||
|---|---|---|---|---|---|
| Herpes simplex virus-1 (KOS) |
Herpes simplex virus-2 (G) |
Vaccinia virus | Herpes simplex virus-1 TK- KOS ACVr | ||
| AA1 | >100 | 100 | 58 | >100 | 100 |
| AA2 | >100 | 50 | 58 | 100 | 58 |
| AA3 | >100 | 50 | 100 | 100 | 100 |
| DBR1 | >100 | 58 | 50 | 100 | 100 |
| DBR2 | 100 | >20 | >20 | >20 | >20 |
| DBR3 | >100 | >100 | >100 | >100 | >100 |
| MBR1 | 100 | >20 | >20 | >20 | >20 |
| MBR2 | 100 | 12 | 12 | 12 | 12 |
| Brivudin | >250 | 0.04 | 22 | 6 | 10 |
| Ribavirin | >250 | >250 | >250 | >250 | >250 |
| Cidofovir | >250 | 0.8 | 0.8 | 10 | 1 |
aRequired to cause a microscopically detectable alteration of normal cell morphology. bRequired to reduce virus-induced cytopathogenicity by 50 %.
Table 3: Antiviral activity of quinazolin-4(3h)-ones derivatives
From the results the antiviral activity and cytotoxicity of the compound are arrived at the follows compound MBR2 was active against the replication of HSV-1, -2 and vaccinia virus with an IC50 of 12 μg/ml and cytotoxicity at 100 μg/ml. Those compounds displayed cytotoxicity in MT-4 cells were inactive against HIV-1 and -2 replication at non-cytotoxic concentrations. We have previously reported the antiviral activity of novel quinazolinones against vaccinia virus and many of these compounds also exhibited marked cytostatic properties in lymphocytes [8,10]. In this study we evaluated eight new derivatives of quinazolinones synthesized by us for their antiviral activity. This lead molecule is highly suitable for designing newer derivatives and molecular modification in them and may help in optimizing antiviral activity.
Acknowledgements
The author is grateful to the NMR Research centre, Indian Institute of Science, Bangalore for providing the NMR facility for this research work.
References
- Shah BR, Bhatt JJ, Patel HH, Undavia NK,Trivedi PB, Desai NC. Synthesis of 2,3-disubstituted-3,1-quinazolin-4(3H)-ones as potential anticancer and anti-HIV agents. Indian J Chem 1995;34:201-8 .
- Desai NC, Undavia NK, Trivedi PB, Dave D, Vyas GD. Synthesized and screened anti-HIV activity of some non-nucleoside 2,3-disubstitutedquinazoline derivatives. Indian J ExpBiol1998;36:1280-3.
- Alagarsamy V, Pathak US, Pandaya SN, Sriram D, De Clercq E. Anti- HIV and anti bacterial activities of some disubstitutedquinazolonesand their bio-isosterdisubstitutedthienopyrimidones. Indian J Pharm Sci 2000;66:433-7.
- Raffa D, Daidone G, Maggio B, Schillaci D, Plescia F. Synthesis and antiproliferative activity of novel 3-(indazol-3-yl)-quinazolin-4(3H) one and 3-(indazol-3-yl)-benzotriazin-4(3H)one derivatives. Pharmazie1999;332:317-20.
- Murugan V, Padmavathy NP, Ramasarma GV, Sharma SV, Suresh B.Synthesis of some quinazolinone derivatives as possible anticancer agent. Indian J HeterocyclChem 2003;13:143-6.
- Girija K, Selvam P, Nagarajan R. Synthesis anticancer activity of 3- [5-Amino-6-(2,3-dichlorophenyl)- [1,2,4]triazin-3-yl]-6,8-dibromo-2- substituted-3H-quina-zolin-4-one. Asian J Chem 2005;17:1111-5.
- Manoj K, Srivastava S, Bharati M, Nizamuddin N. Pharmacological studies of some 2-methyl-3-(arylthiocarbamido)quinazol-4(3H)-ones and antibacterial activity against Bacillus cereus, S. aureus, S. Lutae and antiviral activity against Gomphrena mosaic. Indian J Chem2001;40:342-4.
- Selvam P, Dinakaran M, De Clercq E, Sridhar SK. Synthesis, antiviral and cytotoxic activity of 6-bromo-2,3-disubstituted-4(3H)- quinazolinones. Biol Pharm Bull 2003;26:1278-82.
- Pandey VK, Synthesized 7-(2’-phenyl-3’-ethyl-4’-oxoquinazolinyl)-3,4-diphenyl isoquinolines and screened for antiviral activity against vaccinia virus. Indian Drugs 1996;26:168-71.
- Selvam P, Chennama B, De Clercq E. Synthesis and antiviral activity of some novel 2-substituted 3-(6-ethyl, 4-amino,5-(4-chlorophenyl)-pyrimidin-2-yl) quinazolin-4(3H)ones. Int J ChemSci 2004;2:627-31.