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International Journal of Biological Chemistry

Year: 2011  |  Volume: 5  |  Issue: 3  |  Page No.: 193 - 199

Synthesis and Antibacterial Activity of Benzotriazole Substituted Acridines

Narinder Pal Singh, Rajesh Kumar, Deo Nandan Prasad, Sarita Sharma and Om Silakari

Abstract

The aim of present study was to carry out the synthesis of benzotriazole substituted acridine derivatives and to evaluate their antibacterial activity. The 9-chloroacridine derivatives 1a-b was treated with o-phenylenediamine in presence of methanol to obtain 9-substituted acridine derivatives 2a-b. These were finally cyclized to get benzotriazole substituted acridine derivatives 3a-b. The synthesized compounds were then established on the basis of IR and 1H NMR spectra data and screened for antibacterial activity. Successfully synthesized four acridine derivatives. Out of these, two compounds 2a and 2b showed moderate activity while two compounds 3a and 3b showed good antibacterial activity. The derivatives with free amine group 2a-b were less prone towards antibacterial activity as compared to derivatives lacking the free amine group 3a-b.

Wainwright, 2001). In the 1920s, their potential in the fight against cancer was first noted. Amsacrine which is an acridine derivative is used clinically for the treatment of cancer (Demeunynck, 2004). Acridine derivatives display a large diversity of physical, physiochemical and biological properties but they share the common feature of interacting with DNA by intercalation (Spicer et al., 1997; Goodell et al., 2008). The length and planar shape of the polyaromatic rings make the overlap between the DNA base pair and the aromatic rings optimal for ð-stacking interaction. As a result, intercalation within DNA double strands interferes with the cell machinery, leading to cell death. Acridine derivatives exhibit antimalarial, antibacterial, anticancer, antiviral, anti-inflammatory, analgesic and other significant activities as shown in Fig. 1 (Gamage et al., 1999; Sondhi et al., 2005; Kumar et al., 2009).

Fig. 1: Multiple activities exhibited by acridine derivatives

The majority of acridine antimicrobial work has employed amino alkyl amino or aryl amino functionality at C-9. The use of cyclic amino groups (i.e., saturated heterocycles containing an N-H moiety) and lower alkyl groups to produce novel 9-substituted acridines have been mentioned briefly by several authors over the last 80 years (Desbois et al., 2008; Kapuriya et al., 2008; Ma et al., 2009).

The objective of study was the synthesis of benzotriazole substituted acridine derivatives and their screening for in vitro antibacterial activity.

MATERIALS AND METHODS

All the chemicals used were of analytical grade and purified as per need of the reaction. Progress of the reaction was monitored by TLC using chloroform: methanol (9:1) ratio and spots were visualized by iodine vapors. Melting points were determined using Veego microprocessor based programmable melting point apparatus, in open capillaries and are uncorrected. IR spectra’s were recorded in cm-1 using KBr pellets on PERKIN-ELMER BX series FT-IR spectrometer. 1H NMR spectra on BRUKER AVANCE II 400 NMR Spectrometer using DMSO-d6 or CDCl3 solvent and TMS as internal standard (chemical shift values expressed in δ ppm).

This study was carried out from 1st August 2009 to 30th June 2010 at Department of Pharmaceutical Chemistry, Shivalik College of Pharmacy, Nangal (Punjab)-140126 India.

Tested microorganism: Laboratory isolates of pure cultures of gram positive (S. aureus and B. subtilis) and gram negative (E. coli) bacteria. These bacterial stains were obtained from IMTEC, Chandigarh, India.

Synthesis of 9-chloro acridine derivatives 1a-b: 1a. 2-methoxy-9-chloroacridine: m.p.146°C, yield 85%; 1H NMR (CDCl3): δ 3.97 (s, 3H, OCH3), 7.40-7.44 (m, 2H, Ar-H), 7.55-7.59 (m, 1H, Ar-H), 7.69-7.73 (m, 1H, Ar-H), 8.04-8.07 (m, 1H, Ar-H), 8.14-8.16 (d, 1H, Ar-H), 8.29-8.32 (t, 1H, Ar-H).

1b. 2-methyl-9-chloroacridine: m.p.142°C, yield 82%; 1H NMR (CDCl3): δ 2.49 (s, 3H, CH3), 7.49-7.54 (m, 2H, Ar-H), 7.66-7.70 (m, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.06-8.08 (d, 1H, Ar-H), 8.15-8.18 (d, 1H, Ar-H), 8.26-8.28 (d, 1H, Ar-H).

Synthesis of 9-substituted acridine derivatives: The o-phenylenediamine (1.2 g, 0.011 moles) was dissolved in dry methanol (50 mL) and heated to reflux, 9-chloroacridine derivatives (0.005 moles) were slowly added and the mixture refluxed for 1-2 h. After cooling the mixture was poured into 50 mL diethyl ether, the hydrochloride salt precipitated and filtered off. The salt was recrystallized from ethanol-ether to give an orange powder 2a-b.

2a: 9-(2’-Aminoanilino)-2-methoxy acridine hydrochloride: m.p. 278°C, yield-80%; IR (KBr) cm-1: 3749, 3424, 3315, 3153, 2952, 2364, 1628, 1587, 1551, 1507, 1454, 1375, 1310, 1249, 1156, 1109, 1028.8; 1H NMR (DMSO-d6): δ 3.66 (s, 3H, OCH3), 5.10 (s, 2H, NH2), 6.70-6.74 (t, 1H, Ar-H), 6.96-7.01 (m, 2H, Ar-H), 7.18-7.22 (m, 1H, Ar-H), 7.27-7.31 (t, 1H, Ar-H), 7.39-7.42 (m, 1H, Ar-H), 7.56-7.59 (t, 1H, Ar-H), 7.70-7.74 (t, 1H, Ar-H), 8.22-8.28 (m, 3H, Ar-H), 10.59 (s, 1H, NH), 14.85 (s, 1H, NH+).

2b: 9-(2’-Aminoanilino)-2-methyl acridine hydrochloride: m.p. 270°C, yield-76%; IR (KBr) cm-1: 3749, 3420, 3305, 2865, 2364, 1628, 1582, 1548, 1509, 1453, 1367, 1325, 1264, 1158, 1037.1; 1H NMR (DMSO-d6): δ 2.40 (s, 3H, CH3), 3.44 (s, 2H, NH2), 6.87-6.91 (t, 1H, Ar-H), 6.98-6.99 (d, 1H, Ar-H), 7.25-7.31 (m, 3H, Ar-H), 7.68-7.70 (d, 1H, Ar-H), 7.78-7.82 (t, 1H, Ar-H), 8.11-8.13 (d, 1H, Ar-H), 8.17-8.24 (m, 3H, Ar-H), 10.91 (s, 1H, NH), 14.68 (s, 1H, NH+).

Synthesis of 9-(Benzotriazol-1-yl)-2-substituted acridines: The aminoanilino acridine derivatives (0.0025 moles) was suspended in 2 M HCl (25 mL) and chilled in an ice bath. A solution of NaNO2 (0.35 mL, 0.005 moles) was added dropwise and the mixture stirred for 1-2 h. The suspension was cooled with ice and made basic with liquid NH3. At neutralization it became notably easier to stir. A brown powder was collected by filtration. The wet solid was dissolved in hot DMF and crystallization occurred upon cooling. A yellow powder 3a-b was collected by filtration. The compounds synthesized by above procedure are listed in Table 1.

Table 1: Physical data of the synthesized compounds

3a: 9-(Benzotriazol-1-yl)-2-methoxy acridine: m.p. 232°C, yield-62%; IR (KBr) cm-1: 3089, 3045, 3013, 2952, 2828, 2288, 1946, 1825, 1629, 1562,1522, 1477, 1447, 1429, 1371, 1351, 1318, 1287, 1221, 1183, 1143, 1104, 1051.9. 1H NMR (CDCl3): δ 3.55 (s, 3H, OCH3), 6.32-6.33 (d, 1H, Ar-H), 7.00-7.02 (m, 1H, Ar-H), 7.14-7.18 (t, 1H, Ar-H), 7.37-7.46 (m, 4H, Ar-H), 7.67-7.71 (m, 1H, Ar-H), 8.16-8.18 (d, 1H, Ar-H), 8.22-8.27 (m, 2H, Ar-H).

3b: 9-(Benzotriazole-1-yl)-2-methyl acridine: m.p. 228°C, yield-60%; IR (KBr) cm-1: 3049, 1631, 1607, 1552, 1515, 1491, 1444, 1426, 1372, 1276, 1234, 1186, 1143, 1056, 1020.6; 1H NMR (CDCl3): δ 2.43 (s, 3H, CH3), 7.08-7.10 (m, 2H, Ar-H), 7.26-7.29 (d, 1H, Ar-H), 7.47-7.56 (m, 3H, Ar-H), 7.69-7.72 (m, 1H, Ar-H), 7.81-7.86 (m, 1H, Ar-H), 8.30-8.34 (m, 2H, Ar-H), 8.39-8.41 (d, 1H, Ar-H).

Antibacterial activity: All the synthesized acridine derivatives were screened for their antibacterial activity. The in vitro antibacterial activity of the synthesized compounds were assessed against S. aureus, B. subtilis and E. coli by cup-plate method on nutrient agar medium at concentration level of 100, 500, 1000, 5000 and 10000 μg mL-1 (Cremieux et al., 1995). The most active concentration was found to be 10000 μg mL-1. Ampicillin (100 μg mL-1) were used as standards for antibacterial activity. The antibacterial activity was determined by measuring the diameter of the inhibition zone in mm. Inoculate a previously liquefied medium appropriate to the assay with the requisite quantity of suspension of the micro-organism, add the suspension to the medium at a temperature between 40 and 50°C and immediately pour the inoculated medium into the petri dishes or large rectangular plates to give a depth of 3 to 4 mm. Ensure that the layers of medium are uniform in thickness, by placing the dishes or plates on a level surface. Prepare solutions of known concentration of the standard preparation and solutions of the corresponding assumed concentrations of the antibiotic to be examined. Apply the solutions to the surface of the solid medium in cavities prepared in the agar. The volume of solution added to each cavity must be uniform and sufficient almost to fill the holes when these are used. When Petri dishes are used, arrange the solutions of standard preparation and the antibiotic to be examined on each dish so that they alternate around the dish and so that the highest concentrations of standard and test preparations are not adjacent. Leave the dishes standing for 1 to 4 h at room temperature or at 40°C, as appropriate, as a period of pre-incubation diffusion to minimize the effects of variation in time between the applications of different solutions. Incubate them for about 18 h at 32-35°C temperature. Accurately measure the diameters or areas of the circular inhibition zones and calculate the results.

RESULTS AND DISCUSSION

To prepare the targeted compounds, the corresponding o-chloro benzoic acid was treated with substituted aniline to give N-phenyl anthranilic acid which was cyclized with phosphorous oxychloride to give 9-chloro acridine derivatives 1a-b (Albert and Ritchie, 1955). 9-chloro acridine derivatives 1a-b were dissolved in dry methanol in the presence of o-phenylenediamine and refluxed for different hours to give corresponding 9-substituted acridine derivatives 2a-b. It was further suspended in hydrochloric acid and sodium nitrite solution was added dropwise and stirred at low temperature for different hours to give corresponding final benzotriazole substituted acridine derivatives 3a-b as shown in the scheme1. The structures of the synthesized compounds were confirmed by spectroscopic techniques. The characterization data supported the proposed structures of the compounds. Thus overall four derivatives were synthesized in sufficient yields. Generally in 1H NMR spectrum the peaks for aromatic proton appear at about δ 6-8. The 1H NMR spectrum of compound 1a showed singlet at δ 3.97 due to -OCH3 protons and 1b showed singlet at δ 2.49 due to -CH3 protons (Parajuli and Medhi, 2004; Cope et al., 2006). Similarly, the 1H NMR spectrum of compound 2a showed singlets at δ 10.59 and δ 14.85 while compound 2b showed singlets at δ 10.91 and δ 14.68 due to -NH and NH+ protons (Rastogi et al., 2002; Su et al., 2006). Absorption in the infrared region is confined to molecular changes for which small energy differences exist between the vibrational and rotational states. Majority of applications of IR measurements have been confined to the region between 4000 and 400 cm-1. The IR spectrum of compound 2a and 2b showed -NH stretching frequency at 3424 cm-1 and 3420 and C-Cl peaks at about 540-785 cm-1, respectively. The synthesized compounds were tested for their antibacterial activity. Two compounds 2a and 2b showed moderate activity while two compounds 3a and 3b showed good antibacterial activity as shown in Table 2 and 3. Synthesized compounds were screened for their antibacterial activity. It has been observed that the substitution at C-2 position of acridine ring either with -OCH3 or -CH3 increases the antibacterial activity. The derivatives with free amine group 2a-b were less prone towards antibacterial activity as compared to benzotriazole substituted acridines 3a-b as the later itself shows antibacterial activity (Swamy et al., 2006; Dubey et al., 2011).

Scheme 1: (I) o-phenylenediamine and dry methanol refluxed for 1-2 h (ii) cyclization by 2M HCl/NaNO2 and liquid ammonia

Table 2: Antibacterial activity of acridine derivatives
10-12 mm poor activity, 13-16 mm moderate, 17-20 mm and above good activity. Concentration of standard compound is 100 μg mL-1

Table 3: Antibacterial activity of acridine derivatives at different concentrations
10-12 mm poor activity, 13-16 mm moderate, 17-20 mm and above good activity

The antibacterial activities of the synthesized compounds were assessed against S. aureus, B. subtilis and E. coli by cup-plate method (Venugopala et al., 2007). Ampicillin was used as standard drug. The activities of synthesized compounds were found to be less than that of the standard drug at 10000μg mL-1 concentration.

In conclusion, the results of tested compounds revealed significant antibacterial activity as compared to the standard drug against these three stains. Further, studies are in progress to design new compounds with better antibacterial activity.

ACKNOWLEDGMENTS

The authors thank the Sophisticated Analytical Instruments Facility (SAIF), Punjab University (Chandigarh) for 1H NMR and IR spectral Data.

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