*Corresponding Author:
Selvam P
Department of Pharmaceutical Chemistry, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil−636 290, India
E-mail: [email protected]
Date of Submission November 10, 2010
Date of Revision June 24, 2012
Date of Acceptance June 28, 2012
Indian J Pharm Sci, 2012, 74 (3): 275-278  

Abstract

A series of novel N-substituted indophenazine derivatives were synthesised and screened for antiviral activity against a panel of human pathogenic viruses. New compounds were synthesised through modifying the N-hydrogen of indophenazine moiety with different substitution and formaldehyde by Mannich reaction. The structure of the synthetic compounds was characterised by means of infra red and nuclear magnetic resonance spectral data. The compound 10H-indolo-2-Amino pyridine [3,2-b] quinoxalines inhibits Herpes simplex virus-1 and vaccinia virus at a concentration of 12 μg/ml, and the cytotoxicy was found to be 100 μg/ml. 4-Aminobenzene sulfonamide-10H-indolo [3,2-b] quinoxalines inhibit vaccinia virus at a concentration of 12 μg/ml and cytotoxicy was found to be 100 μg/ml. The anti-HIV activities of the new compounds were also screened for in vitro antiviral activity against replication of HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells using zidovudine (AZT) as standard. Pthalimide derivative inhibited the replication of HIV-2 (EC 50 =11.60 μg/ml and CC 50 =61.63 μg/ml) in MT-4 cells.

Keywords

HIV, Herps simplex virus, indophenazine, Mannich base, MT−4 cell, vaccinia virus

Indophenazine (10H−indolo [3,2−b] quinoxaline) is a versatile lead molecule for designing of potential bioactive agents. Indophenazine derivatives were reported to possess anticancer [1], antiviral activity against HSV [2] and inhibition of viral RNA and DNA synthesis [3], and biological activities of these compounds depended upon the functional group attached to the NH group of indophenazine moiety. 10H−indolo [3,2−b] quinoxaline is synthesised by condensing the appropriate isatin and o−phenylenediamine. We have previously reported the antiviral activity of novel heterocyclic compounds against vaccinia virus, and many of those compounds also exhibited marked cytostatic properties in lymphocytes [46]. Though a variety of heterocyclic compounds had been synthesised and studied for wide range of antiviral activity, the antiviral activities of indophenazine against panel of pathogenic viruses have not been extensively explored. Present work is to synthesis novel Mannich bases of indophenazine (Scheme 1) and tested for antiviral activity against HSV, vaccinia and HIV viruses. Cytotoxicity was also tested in mock−infected MT−4 and Human Foreskin cells (HEL).

Scheme 1: Synthetic protocol of heterocyclic compounds. Where IP−SN R1−sulphanilamide, IP−SD R1−sulphadimidine, IP−SMZ R1−sulphamethoxazole, IP−SC R1 sulphacetamide, IP−BA−R1benzamide, IP−AN R1 anthranilic acid, IP−BI−R 1 benzimidazole, IP−MBI R1mercaptobenzimidazole, IP−2AMP R1 2−aminopyridine, IP−PTH R1−pthalimide.

Melting points were determined in open capillary tubes on a Thomas−Hoover melting point apparatus and are uncorrected. IR spectra were recorded for KBr pellets on a (Shimadzu−800) infrared spectrophotometer, PMR spectra were determined Bruker AM, 400 MHZ with tetramethylsilane as an internal standard. The sample was dissolved in DMSO−d6 and the valves were measured in d ppm.

Synthesis of indophenazine derivatives was achieved by refluxing an equimolar (0.01 mol) mixture of formaldehyde, active hydrogen compounds (sulphanilamide, sulphadimidine, sulphamethoxazole, 2−aminopyridine, pthalamide, benzamide, nicotinamide, anthranilic acid and 2−marcapto−benzimidazole) and Indophenazine with methanol for 3 h (Scheme 1). The reaction mixture was allowed to cool overnight in refrigerator. The solid thus obtained was recrystallised from DMF. Physical data of synthesised compounds is presented in the Table 1.

Compound Molecular Yield % M.P. (º) Rf* Log P**
code formula        
IP2−SN C21H17N5O2S 63 2722−280 0.6 3.72
IP2−SD C27H23N7O2S 76 1902−202 0.4 5.37
IP2−SMZ C25H20N6O3S 57 1752−182 0.53 4.61
IP2−SC C23H19N5O3SNa 84 2252−232 0.8 3.5
IP2−2AMP C20H15N5 93 2452−253 0.38 4.14
IP2−BA C21H15N4Br 78 2682−271 0.6 5.59
IP2−PTH C23H14N4O2 58 2632−270 0.7 4.36
IP2−ANTH C22H16N4O2 86 1952−205 0.4 2.78
IP2−NM C21H15N5O 67 1252−132 0.6 3.9
IP2−BI C22H15N5 82 1752−195 0.8 4.11
IP2−2MBI C22H15N5S 59 1632−178 0.7 3.24

Table 1: Physical Data Of Synthesised Compounds.

Indophenazine (10H−indolo [3,2−b] quinoxaline) (IP): IR (KBr): 3420 (NH), 1640 (C=N), 1458 (C=C); 1H NMR (DMSO−d6): 7.0-8.1 (m, 8H, Ar−H), 10.1 (b,1H, NH). 4−[(Indolo [2,3−b] quinoxalin- 6-ylmethyl)-amino]-benzenesulfonamide (IP−SN): IR (KBr): 3502 (NH), 1656 (C=N), 1451 (C=C), 1083 (>N−), 1332 (SO2), 760 (Ar−H); 1H NMR (DMSO−d6): 5.62 (s, 2H, −CH2−), 6.7-8.1 (m, 12H, Ar−H), 4.0 (b, 1H, NH), 2.0 (b, 1H, −SO2NH−). N−(4,6−Dimethyl−pyrimidin−2−yl)−4−[(indolo [2,3−b] quinoxalin−6−ylmethyl)−amino] benzenesulfonamide (IP−SD): IR (KBr): 3378 (NH), 1620 (C=N), 1424 (C=C), 1300 (SO2), 709 (Ar−H); 1H NMR (DMSO−d6): 2.35 (s, 6H, 2×CH3) 5.62 (s, 2H, −CH2−) 6.28 (d, 1H, pyrimidine), 7.0-8.1 (m, 8H, Ar−H), 10.1 (b, 1H, NH). N−Acetyl−4−[(indolo [2,3−b] quinoxalin- 6-ylmethyl)-amino]-benzene sulfonamide (IP−ISC): IR (KBr): 3383 (NH), 1663 (C=N), 1458 (C=C), 1386 (CH3), 1330 (SO2), 1093 (>N−), 762 (Ar−H); 1H NMR (DMSO−d6): 2.03 (s, 3H, −CH3) 5.62 (s, 2H, −CH2−), 6.7-7.8 (m, 12H, Ar−H), 8.2 (b, 1H, NH). 4− [(Indolo [2,3−b] quinoxalin−6−yl methyl)-amino]- N−(5-methyl-isoxazol-3−yl)-benzenesulfonamide (IP−SMZ): IR (KBr): 3383 (NH), 1663 (C=N), 1458 (C=C), 1386 (CH3), 1330 (SO2), 1093 (>N−), 762 (Ar−H); 1H NMR (DMSO−d6): 6.7-8.1 (m, 12H, Ar−H), 10.1 (b, 1H, NH), 2.03 (s, 3H, −CH3) 5.62 (s, 2H, −CH2−), 8.2 (b, 1H, SO2NH).2−Indolo [2,3−b] quinoxalin−6−ylmethyl−isoindole−1,3−dione (IP−PTH): IR (KBr): 1730(C=O), 1663 (C=N), 1458 (C=C), 1093 (>N−), 762 (Ar−H); 1H NMR (DMSO−d6): 6.2 (s, 2H, −N−CH2−N−),7.2-8.1 (m, 12H, Ar−H). Indolo [2,3−b] quinoxalin−6−ylmethyl−pyridin−2−yl−amine (IP−2AMP): IR (KBr): 3383 (NH), 1663 (C=N), 1458 (C=C), 1093 (>N−), 762 (Ar−H); 1H NMR (DMSO−d6): 4.0 (b,1H, NH), 5.6. (s, 2H, −N−CH2−N−), 6.6−8.1 (m, 12H, Ar−H). 6−Benzoimidazol−1−ylmethyl−6H−indolo [2,3−b] quinoxaline (IP−BI): IR (KBr): 1672 (C=N), 1560 (C=C), 1090 (>N−), 770 (Ar−H); 1H NMR (DMSO−d6): 6.2 (s, 2H, −N−CH2−N−), 7.1-8.0 (m,12H, Ar−H), 8.2 (b,1H, CH−Benzimidazole).

Anti−HIV Assay compounds were tested for their inhibitory effects against replication of HIV−1 (IIIB) and HIV−2 (ROD) in MT−4 cells [4]. The MT−4 cells were grown and maintained in RPMI 1640 DM Medium supplemented with 10% (v/v) heat−inactivated fetal calf serum (FCS), 2 mM glutamine, 0.1% sodium bicarbonate and 20 μg/ml gentamicin. Inhibitory effect of test compounds on HIV−1 and HIV−2 replications was monitored by inhibition of virus−induced cytopathic effect in MT−4 cells and was estimated by 3−(4,5−dimethylthiazol−2−yl)−2,5−diphenyl tetrazolium bromide (MTT) method. Briefly, 50 μl of HIV−1 and HIV−2 (100−300 CCID50) were added to a flat−bottomed microtiter tray with 50 μl of medium containing various concentrations of compounds. MT−4 cells were added at a final concentration of 6×105 cells/mL. After 5 d of incubation at 37°, the number of viable cells were determined by the MTT method. Cytotoxicity of test compounds against mock−infected MT−4 cells was also assessed by the MTT method. AntiHIV activity and cytotoxicity of compounds are presented in Table 2.

Compound code Strain EC50a (µg/ml) CC50b (µg/ml)
IP−SN IIIB >14.03 14.03 ± 2.87
  ROD >14.03 14.03 ± 2.87
IP−SD IIIB >90.48 90.48 ± 3.48
  ROD >90.48 90.48 ± 3.48
IP−SMZ IIIB >64.98 64.98 ± 5.92
  ROD >64.98 64.98 ± 5.92
IP−SC IIIB >12.90 12.90 ± 0.95
  ROD >12.90 12.90 ± 0.95
IP−2AMP IIIB >71.80 71.80 ± 8.91
  ROD >71.80 71.80 ± 8.91
IP−BA IIIB >43.03 43.03 ± 4.58
  ROD >43.03 43.03 ± 4.58
IP−PTH IIIB >61.63 61.63 ± 13.97
  ROD 11.60 61.63 ± 13.97
IP IIIB >98.40 98.40
  ROD >98.40 98.40
IP−BI IIIB >12.37 12.37 ± 0.25
  ROD >12.37 12.37 ± 0.25
AZT (STD) IIIB 0.0012 ± 0.003 65.40
  ROD 0.00016 ± 0.00027 65.40

Table 2: Anti−hiv activity and cytotoxicity of Synthesised compounds

Antiviral activity and cytotoxicity of the synthesised compounds were determined by in vitro cell culture techniques [7]. The antiviral assays were based on inhibition of virus−induced cytopathicity in HEL (HSV−1 and HSV−2, Vaccinia) 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 1 h virus adsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity was recorded as soon as it reached 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 Vesicular
stomatitis virus
Herpes simplex
virus−1 TK− KOS ACVr
IP 100 >20 >20 >20 >20 >20
IP−BI 100 >20 >20 >20 >20 >20
IP−SC ≥20 >20 >20 >20 >20 >20
IP−SD ≥20 >20 >20 >20 >20 >20
IP−SMZ 100 >20 >20 >20 >20 >20
IP−SN 100 20 >20 12 >20 20
IP−PTH 20 >4 >4 >4 >4 >4
IP−2AMP 100 12 20 12 >20 20
IP−BA 100 >20 >20 >20 >20 >20
Brivudin (µM) >250 0.04 50 10 >250 >250
Ribavirin (µM) >250 >250 >250 >250 >250 >250
Acyclovir (µM) >250 2 2 7 >250 2
Ganciclovir (µM) >100 0.06 0.1 >100 >100 12

Table 3:Cytotoxicity And Antiviral Activity Of Indophenazine Derivatives In Hel Cell.

We report our results from a study of replacing the NH (hydrogen) of indophenazine moiety with different types of substitutions like sulphanilamide, sulphadimidine, sulphamethoxazole, 2−aminopyridine, pthalamide, benzamide, anthranilic acid, nicotinamide, 2−marcaptobenzimidazole to form N−methyl substituted indophenazine derivatives by Mannich reaction. Synthesised compounds were screened for antiviral activity against HIV−1 and HIV−2 in MT−4 cells. Compound IP−PTH inhibits the replication of HIV−2 in MT−4 cells at a concentration of 11.60 μg/ml, and the cytotoxicy was found to be 61.63 μg/ml. All the compounds except PTH displayed cytotoxic properties in MT−4 cells. Synthesised compounds also tested for antiviral activity against HSV−1, 2 and vaccinia viruses in HEL cells. The compound 10H−indolo−2−Amino pyridine [3,2−b] quinoxalines (IP−2AMP), inhibits Herpes simplex virus−1 and vaccinia virus at a concentration of 12 μg/ml, and the cytotoxicy was found to be 100 μg/ml. incidentally the compound 4−Aminobenzene sulfonamide−10H−indolo [3,2−b] quinoxalines (IP−SN) also inhibits vaccinia virus at a concentration of 12 μg/ml, and the cytotoxicy was found to be 100 μg/ml.

Indophenazine derivatives (IP−2AMP and IP−SN) inhibited the replication of vaccinia and HSV−1 viruses below the cytotoxic concentration. Newly synthesised indophenazine derivative indolo [2,3−b] quinoxalin−6−ylmethyl−isoindole−1,3−dione (PTH) inhibited the replication of the HIV−2 in MT−4 cells. Recently, we reported the activities of certain quinazolinone derivatives with sulphanamide against biodefense viruses in cell culture [8]. The potencies of some of them exceeded those of the present indophenazine−sulphonamide series. The compounds were found to inhibit virus replication as a result of interfering with virus adsorption [9]. There is a need to discover new compounds that are inhibitory to HIV and vaccinia viruses due to the emergence of potentially pandemic virus strains and viral resistance against approved drugs.

Acknowledgement

The author is grateful to the NMR Research centre, Indian Institute of Science, Bangalore for providing the NMR facility for this research work.

References