Full HTML 01 V1 I3

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 4-PYRROLIDINO/ MORPHOLINO -1-ARYL / ALKYLPYRIDINIUM BROMIDES


YASODHA*1, A. SIVAKUMAR, A. PURATCHIKODY2
2. Department of Pharmaceutical Chemistry, Dhanvanthri college of Pharmaceutical Sciences, Mahabubnagar, Telugana, India.
2. Department of Pharmaceutical Technology, Anna University Chennai, BIT Campus, Tiruchirappalli, Tamilnadu, India.

Abstract

A series of bromides (RCH2Br) containing aryl / alkyl group readily reacted with 4-pyrrolidino / morpholinopyridine to give stable pyridinium bromide (1-30). The physical and spectral (IR, 1H & 13C NMR and MS) data for 1-30 were collected to confirm the structure (s) assigned. Invitro antibacterial and antifungal activities of 1-30 against Gram positive / Gram negative and fungal species were evaluated. The 4-pyrrolidino / morpholinopyridine -1-alkylpyridinium bromides (Group III & IV) were found higher antibacterial and antifungal activity than the standard drugs ciprofloxacin / fluconazole respectively.

Key words: Antimicrobial activity, 4- pyrrolidinopyridine, 4-morpholinopyridine, Pyridinium bromides.

INTRODUCTION

Quaternary ammonium salts were reported as good antimicrobial agents1-4. Antimicrobial activity of quat salts was endorsed to their effect on the cell wall resulting in a direct and indirect lethal effect on the cell viability 5. Pyridinium salts are considered as one of the categories of quaternary ammonium salts which are heterocyclic compounds having different functional groups 6. Pyridinium salts have wide range of therapeutic properties like antibacterial7-8, antifungal 9, antimycobacterial 10, antiviral 11, antitumoral 12, antiAChE 13, antimalarial 14 and hepatoprotective activities 15. Several well-known drugs contain pyridinium nucleus such as Cephaloridine, Pralidoxime, Pyridostigmine, Cefalonium, Stilbazium, Fazadinium etc, indicating the fact that pyridinium nucleus possess vast spectrum of biological activities. These activities indicate that pyridinium frame work plays an essential role in drug discovery and represents an interesting template for combinatorial and medicinal chemistry16. It is, therefore, part of interest to prepare some pyridinium salts with different functional groups at 4-and 1-position of pyridine nucleus with a view to knowing their biological activity. The present investigation is one more addition to the pyridine derivatives of biological interest.

MATERIALS AND METHODS

Melting points determined are uncorrected. IR (KBr) spectra of the compounds 1-30 were recorded on Perkin Elmer 1600 FT spectrophotometer. 1H CNMR spectra were recorded in DMSO – d6 on a Bruker AC, 300MHz spectrometer using TMS as standard and Mass Spectra on API 3000 Centroid Turbo Spray Analyzer.

General Procedure for Synthesis of 1-aryl / alkyl-4- pyrrolidino / morpholinopyridinium bromides (1-30): 4- pyrrolidino / morpholinopyridine in dry acetone (30 ml) was added in to the solution containing required amount of aryl or alkyl bromide (0.1mol) in acetone (25 ml). The reaction mixer was stirred at room temperature for 2-12 h. The solid that separated was filtered off, washed with toluene, dried in vaccum and recrystallized from chloroform and acetone (1:1) to give 1-30.

4-Pyrrolidino-1-(phenylmethyl)pyridinium bromide (1): IR (KBr, cm-1): 1355, 1443, 1559, 1643, 2555, 2868 and 3017; 1H NMR (300 MHz, DMSO-d6) δ: 1.98-2.02(m, 4H), 3.48-3.50 (t, 4H), 5.46 (s, 2H), 6.92 (d, 2H), 7.37-7.44 (m, 5H) and 8.47(d, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 24.6, 48.3, 55.2, 108.6, 128.0, 128.6, 128.9, 129.0, 135.9, 142.0 and 153.0; Mass ESI-MS (m/z): 239 (M+). Anal. Cal. (%): C, 80.33; H, 7.94; N, 11.71; Found (%): C, 80.32; H, 7.92; N, 11.70.

4-Pyrrolidino-1-[(4-bromophenyl)methyl]pyridinium bromide (2): IR (KBr, cm-1): 802, 1169, 1413, 1560, 1644, 2868 and 2976; 1H NMR (300 MHz, DMSO-d6) δ: 1.98–2.00 (m, 4H), 3.48-3.49 (m, 4H), 5.43(s, 2H), 6.92 (d, 2H), 7.38 (d, 2H), 7.62 (d, 2H) and 8.43(d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.6, 48.4, 58.5, 108.6, 122.0, 130.2, 131.9, 135.2, 142.0 and 153.0 ppm; Mass ESI-MS (m/z): 317 (M+). Anal. Cal. (%): C, 60.75; H, 5.69; N, 8.86; Found (%): C, 60.73; H, 5.68; N, 8.88.

4-Pyrrolidino-1-[(4-nitrophenyl)methyl]pyridinium bromide (3): IR (KBr, cm-1): 840, 1176, 1344, 1523, 1646, 2873 and 3046; 1H NMR (300 MHz, DMSO-d6) δ: 1.99-2.02 (m, 4H), 3.50-3.52 (m, 4H), 5.65 (s, 2H), 6.96 (d, 2H), 7.66 (d, 2H),  8.26 (d, 2H) and 8.48 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.6, 48.4, 58.2, 108.8, 124.0, 129.1, 142.2, 143.2, 147.5 and 153.1ppm; Mass ESI-MS (m/z): 284 (M+). Anal. Cal. (%): C, 67.60; H, 6.38; N, 14.78; Found (%): C, 67.58; H, 6.37; N, 14.77.

4-Pyrrolidino-1-[(4-methylphenyl)methyl]pyridinium bromide (4): IR (KBr, cm-1): 1172, 1352, 1559, 1643 and 3017; 1H NMR (300 MHz, DMSO-d6) δ: 1.98–2.01 (m, 4H), 2.29 (s, 3H), 3.47-3.49 (m, 4H), 5.39 (s, 2H), 6.91(d, 2H), 7.21 (d, 2H), 7.32 (d, 2H) and 8.43 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 20.7, 24.6, 48.3, 59.1, 108.5, 128.0, 128.2, 129.5, 132.9, 138.1, 141.9 and 153.0 ppm; Mass ESI-MS (m/z): 253 (M+). Anal. Cal. (%): C, 80.63; H, 8.30; N, 11.06; Found (%): C, 80.62; H, 8.28; N, 11.05.

4-Pyrrolidino-1-(2-oxo-2-phenylethyl) pyridinium bromide (5): IR (KBr, cm-1): 1171, 1355, 1443, 1559, 1717, 2858 and 3017; 1H NMR (300 MHz, DMSO-d6) δ: 1.98-2.00(t, 4H), 3.50-3.48 (m, 4H), 5.46 (s, 2H), 6.92 (d, 2H), 7.37-7.44 (m, 5H) and 8.47 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.6, 45.6, 59.2, 108.6, 128.0, 128.6, 129.0, 135.9, 142.0, 153.0 and 192.2 ppm; Mass ESI-MS (m/z): 267 (M+). Anal. Cal. (%): C, 76.40; H, 7.11; N, 10.48; Found (%): C, 76.42; H, 7.10; N, 10.46.

4-morpholino-1-(phenylmethyl)pyridinium bromide (6): IR (KBr, cm-1): 1170, 1262, 1440, 1550, 1643, 2853 and 3026; 1H NMR (300 MHz, DMSO-d6) δ: 3.68-3.66 (t, 4H,), 3.73-3.71 (t, 4H), 5.44 (s, 2H), 7.29-7.27, (d, 2H), 7.44-7.37 (m, 5H) and 8.51-8.49 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ:  59.4, 65.4, 108.4, 128.0, 128.7, 129.0, 135.6, 142.7 and 155.7 ppm; Mass ESI-MS (m/z): 255 (M+). Anal. Cal. (%): C, 75.29; H, 7.45; N, 10.98; Found (%): C, 75.27; H, 7.44; N, 10.96.

4-morpholino-1-[(4-bromophenyl)methyl]pyridinium bromide (7): IR (KBr, cm-1): 787, 1177, 1263, 1416, 1546, 1642, 2854 and 2992; 1H NMR (300 MHz, DMSO-d6) δ: 3.69-3.67 (t, 4H), 3.73-3.72 (t, 4H), 5.43 (s, 2H), 7.29-7.28 (d, 2H), 7.42-7.41 (d, 2H), 7.64-7.63 (d, 2H) and 8.50-8.49 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ:  46.6, 59.1, 65.9, 108.9, 122.6, 130.9, 132.4, 135.5, 143.1 and 156.2 ppm; Mass ESI-MS (m/z): 383 (M+, 100%), 384 (M++1, 3%), 382 (M+). Anal. Cal. (%): C, 50.26; H, 4.71; N, 7.32; Found (%): C, 50.25; H, 4.70; N, 7.30.

4-morpholino-1-[(4-nitrophenyl)methyl]pyridinium bromide (8): IR (KBr, cm-1): 839, 1180, 1263, 1346, 1535, 1643 and 2858; 1H NMR (300 MHz, DMSO-d6) δ: 3.69-3.67 (t, 4H), 3.73-3.72 (t, 4H), 5.60 (s, 2H), 7.23-7.22 (d, 2H), 7.68-7.67 (d, 2H), 8.30-8.29 (d, 2H) and 8.52-8.50 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 32.6, 46.4, 46.7, 58.9, 65.9, 107.9, 109.0, 124.1, 124.3, 124.5, 124.6, 127.5, 129.3, 129.7, 131.0, 131.3, 140.3, 143.4, 148.1 and 156.3 ppm; Mass ESI-MS (m/z): 300 (M+). Anal. Cal. (%): C, 64.00; H, 6.00; N, 14.00; Found (%): C, 63.98; H, 6.01; N, 13.99.

4-morpholino-1-[(4-methylphenyl)methyl]pyridinium bromide (9): IR (KBr, cm-1):1113, 1262, 1424, 1544, 1643, 2855 and 3013; 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 3.54-3.52 (t, 4H), 3.72-3.70 (t, 4H), 5.38 (s, 2H), 7.08-7.07 (d, 2H), 7.34-7.33 (d, 2H), 8.26-8.24 (d, 2H) and 8.48-8.47 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 21.2, 46.3, 46.6, 59.7, 65.9, 66.1, 108.8, 128.6, 130.0, 133.1, 138.7, 143.1 and 156.2 ppm; Mass ESI-MS (m/z): 270 (M+). Anal. Cal. (%): C, 76.11; H, 7.83; N, 10.44; Found (%): C, 76.10; H, 7.81; N, 10.42.

4-morpholino-1-(2-oxo-2-phenylethyl)pyridinium bromide (10): IR (KBr, cm-1): 1170, 1262, 1308, 1440, 1550, 1743, 2853 and 3026 ppm; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.73-3.71 (t, 4H), 5.42 (s, 2H), 6.83-6.82 (d, 2H), 7.43-7.35 (m, 5H) and 8.20-8.18 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 46.1, 66.2, 108.7, 128.6, 150.3, 155.3 and 194.4 ppm; Mass ESI-MS (m/z): 283 (M+). Anal. Cal. (%): C, 72.08; H, 6.71; N, 9.89; Found (%): C, 72.06; H, 6.70; N, 9.87.

4-Pyrrolidino-1-propylpyridinium bromide (11): IR (KBr, cm-1): 1244, 1377, 1547, 1601, 2884, 2880 and 2955; 1H NMR (300 MHz, DMSO-d6) δ: 2.51 (t, 4H), 3.28-3.26 (t, 4H), 3.57 (m, 5H), 3.73-3.71 (t, 2H), 6.84-6.83 (d, 2H) and 8.20-8.19 (d, 2H) ppm; 13C NMR (100 MHz, MSO-d6) δ: 19.6, 21.1, 46.1, 66.2, 108.7, 150.1 and 155.4 ppm; Mass ESI-MS (m/z): 191 (M+). Anal. Cal. (%): C, 75.39; H, 9.94; N, 14.65; Found (%): C, 75.38; H, 9.92; N, 14.64.

4-pyrrolidino-1-butylpyridinium bromide (12): IR (KBr, cm-1): 1243, 1375, 1545, 1601, 2834 and 2955; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.58(m, 9H), 3.72-3.70 (t, 4H), 6.83-6.82 (d, 2H) and 8.19-8.18 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 13.5, 18.6, 33.2, 45.6, 65.7, 108.2, 149.7 and 154.8 ppm; Mass ESI-MS (m/z): 205 (M+). Anal. Cal. (%): C, 76.09; H, 10.24; N, 13.65; Found (%): C, 76.07; H, 10.23; N, 13.66.

4-Pyrrolidino-1-pentylpyridinium bromide (13): IR (KBr, cm-1): 1375, 1446, 1545, 1601, 2683 and 2983; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.50-3.45            (m, 11H), 3.72-3.71 (t, 4H), 6.83 (d, 2H) and 8.2 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 22.7, 23.9, 30.7, 38.6, 46.1, 66.2, 108.7, 150.3 and 155.3 ppm; Mass ESI-MS (m/z): 219 (M+). Anal. Cal. (%): C, 76.61; H, 10.50; N, 12.78; Found (%): C, 76.62; H, 10.48; N, 12.77.

4-Pyrrolidino-1-hexylpyridinium bromide (14): IR (KBr, cm-1): 1381,1444, 1549, 1644, 2872 and 3027; 1H NMR (300 MHz, DMSO-d6) δ: 1.25 (t, 3H), 1.73-1.77 (m, 2H),  2.00-2.02 (m, 6H), 3.48-3.50 (m, 4H), 3.52-3.59(m, 4H), 4.17-4.16 (m, 2H), 6.89 (d, 2H) and 8.33 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.7, 30.1, 31.9, 32.2, 35.0, 48.3, 56.5, 60.4, 108.2, 142.0 and 154.8 ppm; Mass ESI-MS (m/z): 233 (M+). Anal. Cal. (%): C, 77.25; H, 10.72; N, 12.01; Found (%): C, 77.23; H, 10.71; N, 12.02.

4-morpholino-1-propylpyridinium bromide (15): IR (KBr, cm-1): 1114, 1243, 1376, 1546, 1602, 2834 and 2954; 1H NMR (300 MHz, DMSO-d6) δ: 2.51 (t, 3H), 3.28-3.26 (t, 4H), 3.57(m, 4H), 3.73-3.71 (t, 4H), 6.84-6.83 (d, 2H) and 8.20-8.18 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 38.4, 39.2, 46.1, 66.2, 108.7, 150.1 and 155.4 ppm; Mass ESI-MS (m/z): 208 (M+). Anal. Cal. (%): C, 69.56; H, 9.17; N, 13.52; Found (%): C, 69.54; H, 9.15; N, 13.50.

4-morpholino-1-butylpyridinium bromide (16): IR (KBr,        cm-1): 1115, 1244, 1377, 1547, 1601, 2833 and 2956; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.57 (m, 9H), 3.72-3.70 (t, 4H), 6.83-6.82 (d, 2H) and 8.20-8.19 (d, 2H) ppm;13C NMR (100 MHz, DMSO-d6)  δ: 23.75, 29.59, 39.10, 46.09, 66.19, 108.70, 150.28 and 155.30 ppm; Mass ESI-MS (m/z): 221 (M+). Anal. Cal. (%): C, 70.58; H, 9.50; N, 12.66; Found (%): C, 70.57; H, 9.51; N, 12.65.

4-morpholino-1-pentylpyridinium bromide (17): IR (KBr, cm-1): 1116, 1245, 1379, 1550, 1603, 2837 and 2983; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.48 (m, 11), 3.72-3.71 (t, 4H), 6.83-6.82 (d, 2H) and 8.20-8.19 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 21.7, 23.8, 29.0, 39.1, 46.1, 66.2, 108.7, 150.3 and 155.3 ppm; Mass ESI-MS (m/z): 235 (M+). Anal. Cal. (%): C, 71.48; H, 9.78; N, 11.91; Found (%): C, 71.46; H, 9.77; N, 11.90.

4-morpholino-1-hexylpyridinium bromide (18): IR (KBr, cm-1): 1116, 1247, 1381, 1549, 1602, 2862 and 2963; 1H NMR (300 MHz, DMSO-d6) δ: 3.27-3.25 (t, 4H), 3.47 (m, 13H), 3.72-3.71 (t, 4H), 6.83-6.82 (d, 2H) and 8.20-8.19 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.3, 30.1, 32.3, 34.8, 46.1, 55.3, 66.2, 108.7, 150.3 and 155.4 ppm; Mass ESI-MS (m/z): 249 (M+). Anal. Cal. (%): C, 72.28; H, 10.04; N, 11.24; Found (%): C, 72.26; H, 10.03; N, 11.25.

4-Pyrrolidino-1-(3-bromopropyl)pyridinium bromide (19): IR: 805, 1243, 1376, 1546, 1602, 2872 and 2956; 1H NMR (300 MHz, DMSO-d6) δ: 2.5 (t, 4H), 3.28-3.26 (t, 4H), 3.57 (m, 4H), 3.73-3.71 (t, 2H), 6.84 (d, 2H), and 8.20-8.19 (d, 2H) ppm; 13C NMR (100 MHz,     DMSO-d6) δ: 46.1, 66.2, 108.7, 141.9, 142.0 and 153.0 ppm; Mass ESI-MS (m/z): 268 (M+). Anal. Cal. (%): C, 53.93; H, 6.74; N, 10.48; Found (%): C, 53.91; H, 6.73; N, 10.47.

4-Pyrrolidino-1-(4-bromobutyl)pyridinium bromide (20): IR (KBr, cm-1): 835, 1185, 1434, 1558, 1646, 2872 and 3027; 1H NMR (300 MHz, DMSO-d6) δ: 1.79-1.72 (quintet, 2H), 1.92-1.85 (quintet, 2H), 2.02-2.00 (t, 4H), 3.51-3.48 (t, 4H), 3.57-3.55 (t, 2H), 4.26-4.23 (t, 2H), 6.92 (d, 2H) and 8.36-8.35 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.7, 27.0, 27.3, 28.7, 29.1, 34.1, 48.3, 55.9, 56.6, 60.0, 141.9, 108.3, 142.0 and 153.0 ppm; Mass ESI-MS (m/z): 282 (M+). Anal. Cal. (%): C, 55.12; H, 7.06; N, 9.89; Found (%): C, 55.11; H, 7.04; N, 9.88.

4-Pyrrolidino-1-(5-bromopentyl)pyridinium bromide (21): IR (KBr, cm-1): 807, 1245, 1379, 1550, 1603 and  2956; 1H NMR (300 MHz, DMSO-d6) δ: 8.37-8.36 (d, 2H), 6.91-6.89 (d, 2H), 4.21-4.18 (t, 2H), 3.50-3.48 (t, 4H), 3.37-3.35 (t, 2H), 2.51-2.50 (t, 4H), 1.84-1.16 (m, 6H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 22.3, 25.2, 30.1, 48.8, 56.7, 108.7, 142.5, 142.5 and 152.2 ppm; Mass ESI-MS (m/z): 297 (M+). Anal. Cal. (%): C, 56.56; H, 7.40; N, 9.42; Found (%): C, 56.54; H, 7.41; N, 9.40.

4-Pyrrolidino-1-(6-bromohexyl)pyridinium bromide (22 IR (KBr, cm-1): 802, 1247, 1381, 1549, 1602 and 2962; 1H NMR (300 MHz, DMSO-d6) δ: 1.76-1.24 (m, 6H), 2.02-1.99 (t, 4H), 2.51-2.50 (quintet, 2H), 3.50-3.48 (t, 4H), 3.58-3.52 (t, 2H), 4.17-4.16 (t, 2H), 6.90-6.88 (d, 2H) and 8.34-8.33 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 24.7, 30.1, 31.9, 32.2, 35.0,  48.3, 56.5, 60.4, 108.2, 142.0 and 152.9 ppm; Mass ESI-MS (m/z): 311 (M+). Anal. Cal. (%): C, 57.87; H, 7.71; N, 9.00; Found (%): C, 57.85; H, 7.70; N, 8.98.

4-morpholino-1-(3-bromopropyl)pyridinium bromide (23): IR (KBr,  cm-1): 822, 1189, 1262, 1421, 1547, 1646, 2863 and 2910; 1H NMR (300 MHz, DMSO-d6) δ: 2.39 (quintet, 2H), 3.53-3.50 (t, 4H), 4.34-4.30 (t, 2H), 3.71-3.70 (t, 2H), 3.73-3.72 (t, 4H), 7.31-7.29 (d, 2H) and 8.44-8.40 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 30.8, 33.4, 46.6, 46.7, 54.2, 55.9, 66.0, 108.7, 143.2, 143.3 and 156.2 ppm; Mass ESI-MS (m/z): 285 (M+). Anal. Cal. (%): C, 50.70; H, 6.33; N, 9.85; Found (%): C, 50.68 H, 6.32; N, 9.84.

4-morpholino-1-(4-bromobutyl)pyridinium bromide (24): IR (KBr, cm-1):  837, 1180, 1253, 1429, 1545, 1643, 2853 and 3032; 1H NMR (300 MHz, DMSO-d6) δ: 1.92-1.74 (m, 4H), 3.58-3.56 (t, 2H), 3.70-3.68 (t, 4H), 3.74-3.72 (t, 4H), 4.25-4.22 (t, 2H), 7.29-7.27 (d, 2H) and 8.41-8.40 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 27.4, 29.2, 29.5, 34.5, 46.6, 56.3, 56.4, 66.0,108.6, 143.1, 143.2 and 156.2 ppm; Mass ESI-MS (m/z): 299 (M+). Anal. Cal. (%): C, 52.34; H, 6.71; N, 9.39; Found (%): C, 52.32; H, 6.70; N, 9.37.

4-morpholino-1-(5-bromopentyl)pyridinium bromide (25): IR (KBr, cm-1): 827, 1185, 1263, 1424, 1543, 1646, 2859 and 3036; 1H NMR (300 MHz, DMSO-d6) δ:  1.86-1.77 (m, 4H), 3.55-3.52 (t, 2H), 3.67 (t, 4H), 3.72-3.71 (t, 4H), 4.21-4.18 (t, 2H), 7.27-7.28 (d, 2H) and 8.43-8.41 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 22.3, 24.5, 29.8, 30.1, 32.0, 35.4, 46.5, 46.6, 56.8, 57.0, 66.0,  108.6, 143.2 and 156.2 ppm; Mass ESI-MS (m/z): 313 (M+). Anal. Cal. (%): C, 53.67; H, 7.02; N, 8.94; Found (%): C, 53.65; H, 7.01; N, 8.93.

4-morpholino-1-(6-bromohexyl)pyridinium bromide (26): IR (KBr, cm-1): 828, 1180,1262, 1427, 1544, 1644, 2860 and 3049 ; 1H NMR (300 MHz, DMSO-d6) δ:   1.81-1.22 (m, 8H), 3.53-3.51 (t, 2H), 3.68-3.66 (t, 4H), 3.73-3.71 (t, 4H), 4.20-4.18 (t, 2H), 7.27-6.88 (d, 2H) and 8.41-8.39 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 25.0, 25.3, 27.4, 30.5, 32.4, 35.5, 46.5, 61.0, 66.0, 108.6, 143.1 and 156.2 ppm; Mass ESI-MS (m/z): 327 (M+). Anal. Cal. (%): C, 55.04; H, 7.33; N, 8.56; Found (%): C, 55.03; H, 7.32; N, 8.54.

4-Pyrrolidino-1-(2-ethoxy-2oxoethyl)pyridinium bromide (27): IR (KBr, cm-1): 1091, 1210, 1382, 1552, 1654, 1744,  2858 and 3017; 1H NMR (300 MHz, DMSO-d6) δ: 2.00 (s, 7H), 3.47-3.52 (m, 6H),  5.15 (s, 2H), 6.93 (d, 2H) and 8.26 (d, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 25.1, 48.7, 48.9, 57.3, 108.1, 108.4, 139.0, 139.4, 143.5, 153.6, 154.6 and 169.4 ppm; Mass ESI-MS (m/z): 235 (M+). Anal. Cal. (%): C, 66.38; H, 8.08; N, 11.91; Found (%): C, 66.36; H, 8.07; N, 11.90.

4-Pyrrolidino-1-allylpyridinium bromide (28): IR (KBr, cm-1):  1243, 1376, 1508, 1546, 1602, 2834 and 2955;      1H NMR (300 MHz, DMSO-d6) δ:  3.26-3.28 (m, 4H), 3.71-3.73(m, 4H), 5.38 (d, 2H), 6.43 (d, 2H), 6.92 (d, 2H), 7.62 (d, 1H) and 8.43 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 46.1, 65.7, 108.2, 149.7 and 154.8 ppm; Mass ESI-MS (m/z): 189 (M). Anal. Cal. (%): C, 76.19; H, 8.99; N, 14.81; Found (%): C, 76.17; H, 8.98; N, 14.80.

4-morpholino-1-(2-ethoxy-2oxoethyl)pyridinium bromide (29): IR (KBr, cm-1): 1114, 1210, 1242, 1376, 1551, 1655,1747, 2834 and 2954; 1H NMR (300 MHz, DMSO-d6) δ: 3.65-3.63 (t, 3H), 3.73-3.71 (t, 4H), 3.75-3.74 (t, 4H), 4.23-4.19 (q, 2H), 5.22 (s, 2H), 7.33-7.28 (d, 2H) and 8.31-8.29 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 14.5, 46.4, 46.6, 46.7, 57.9, 62.3, 66.0 67.0, 108.8, 108.1, 108.2, 156.3, 141.3, 144.2, 144.2 and 168.0 ppm; Mass ESI-MS (m/z): 251 (M+). Anal. Cal. (%): C, 62.15; H, 7.56; N, 11.15; Found (%): C, 62.14; H, 7.54; N, 11.13.

4-morpholino-1-allylpyridinium bromide (30): IR (KBr,  cm-1): 1114, 1243, 1374, 1508, 1525, 1673, 2834 and 2954; 1H NMR (300 MHz, DMSO-d6) δ: 3.28-3.26 (t, 4H), 3.57 (m, 1H), 3.73-3.71 (t, 4H), 5.39-5.37 (d, 2H), 6.43-6.42 (d, 2H), 6.93-6.91 (d, 2H) and 8.43-8.42 (d, 2H) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 46.1, 66.2, 108.7, 119.4, 135.7, 150.1, and 155.7 ppm; Mass ESI-MS (m/z): 205 (M+). Anal. Cal. (%): C, 70.24; H, 8.29; N, 13.65; Found (%): C, 70.23; H, 8.28; N, 13.63.

Antimicrobial Testing:

The antibacterial activity was performed against Gram positive (Staphylococcus aureus and Streptococcus mutants) and Gram negative (Escherichia coli and Klebesilla pneumoniae) bacteria and antifungal activity was performed against Rhizopus arrhizus and Aspergillus niger by tube dilution method 17. Dilutions of test and standard compounds [ciprofloxacin (antibacterial) and fluconazole (antifungal)] were prepared in double strength nutrient broth – I.P. (bacteria) and Sabouraud dextrose broth I.P. (fungi). The samples were incubated at 37 °C for 24 h (bacteria), at  25 °C for 72 h (Aspergillus niger) and at 37 °C for 48 h (Rhizopus arrhizus), respectively, and the results were recorded in terms of minimum inhibitory concentration (MIC) (the lowest concentration of test substance which inhibited the growth of microorganisms).

RESULTS AND DISCUSSION

Chemistry:

The reaction between aryl / alkyl bromide(s) (RCH2Br) and 4-pyrrolidino / morpholinopyridine reacts to give 4-pyrrolidino / morpholinopyridine -1-aryl / alkylpyridinium bromide (1-30) in dry acetone at room temperature under stirring for 2-12 hours (Scheme 1).

Compd. No 1 2 3 4 5 6 7 8 9 10
Z -N C4H4 -N C4H4 -N C4H4 -N C4H4 -N C4H4 -C4H4ON -C4H4ON -C4H4ON -C4H4ON -C4H4ON
R -C6H5 -C6H4-Br(4) -C6H4-NO2(4) -C6H4-CH3(4) -COC6H5 -C6H5 -C6H4-Br(4) -C6H4-NO2(4) -C6H4-CH3(4) -COC6H5
Compd. No 11 12 13 14 15 16 17 18 19 20
Z -N C4H4 -N C4H4 -N C4H4 -N C4H4 -C4H4ON -C4H4ON -C4H4ON -C4H4ON -N C4H4 -N C4H4
R -CH2CH3 -(CH2)2-CH3 -(CH2)2-CH3 -(CH2)3-CH3 -CH2CH3 -(CH2)2-CH3 -(CH2)3-CH3 -(CH2)4-CH3 -CH2-CH2Br -(CH2)2-CH2Br
Compd. No 21 22 23 24 25 26 27 28 29 30
Z -N C4H4 -N C4H4 -C4H4ON -C4H4ON -C4H4ON -C4H4ON -N C4H4 -N C4H4 -C4H4ON -C4H4ON
R -(CH2)3-CH2Br -(CH2)4-CH2Br -CH2-CH2Br -(CH2)2-CH2Br -(CH2)3-CH2Br -(CH2)4-CH2Br -OCOCH2CH3 -CH=CH2 -OCOCH2CH3 -CH=CH2

Scheme 1: Synthetic route followed for the synthesis of 4-amino / dimethylamino-1-aryl / alkylpyridinium bromides

The physical data of 1-30 were collected and are presented in Table 1. The physical (m.p., yield, etc.) data of some of the 4-pyrrolidinopyridinium bromides and 4-morpholinopyridinium bromides are found comparable with their reported values elsewhere18. Each of 4-pyrrolidino / morpholinopyridine is small organic molecule but is a potential nucleophile 19.

The presence of -NC4H4 and -C4H4ON group at 4-position of pyridine has electron releasing property, whereas, the nitrogen at 1-position has electron accepting tendency. As a result the charge density on the endocyclic nitrogen is more than the exocyclic nitrogen 20. Therefore, electrophile preferably attacks on the endocyclic nitrogen. In the case of aryl bromide, electron withdrawing group (EWG) at 4-position of benzene ring causes for C-Br bond to be more facile to cleave which makes the reaction faster and form more yield more (>80%) (1-10). Whereas,  C-Br cleavage is less facile in the case of alkyl bromide(s) (11-18) which results the less yield (70-80%).  The isolated yield for 19-26 is found to be comparable with the yield of 1-10.

The presence of other bromine atom in the alkyl chain may cause for increasing the electrophilicity of the alkyl group. The reaction between 4-pyrrolidino / morpholinopyridine and dibromoalkane under 1:1 mole ratio prefers to attack either one of the C-Br bonds 21. Increasing the alkyl chain length is also favored for the approach of 4-pyrrolidino / morpholinopyridine nucleophile which in turn increases the yield. The reaction between 4-pyrrolidino / morpholinopyridine and carboethoxymethyl / allyl bromide gave the corresponding pyridinium salt (27-30) with poor yield. This may be due to the formation of carbocation which is to be stabilized by inductive and conjugative effect.

Table 1: Physical data of the synthesized compounds (1-30)

Compd.

No

MF Colour Yield (%) MP (°C)
1. C16H19BrN2 Colourless 78 114-116
2. C16H18Br2N2 Colourless 80 100-102
3. C16H18BrN3O2 Yellow 84 150-152
4. C17H21BrN2 Pale Yellow 76 178-180
5. C17H19BrN2O Colourless 50 124-126
6. C16H19BrN2O Colourless 70 288-290
7. C16H19Br2N2O Colourless 72 295-297
8. C16H19BrN3O3 Yellow 69 240-242
9. C17H21BrN2O Pale Yellow 74 282-284
10. C17H19BrN2O2 Colourless 72 235-237
11. C12H19BrN2 Colourless 82 166-168
12. C13H21BrN2 Pale yellow 70 135-137
13. C14H23BrN2 Yellow 75 154-156
14. C15H25BrN2 Colourless 78 128-130
15. C12H19BrN2O Colourless 70 210-212
16. C13H21BrN2O Colourless 73 250-252
17. C14H23BrN2O Colourless 70 294-296
18. C15H25BrN2O Colourless 78 206-208
19. C12H18Br2N2 Pale yellow 80 120-122
20. C13H20Br2N2 Yellow 86 180-182
21. C14H22Br2N2 Colourless 70 155-157
22. C16H19BrN3O3 Colourless 84 179-180
23. C12H18N2OBr2 Colourless 73 268-270
24. C13H20Br2N2O Colourless 70 258-260
25. C14H22Br2N2O Colourless 74 284-286
26. C15H24Br2N2O Colourless 72 296-298
27. C13H19BrN2O2 Colourless 65 165-167
28. C12H17BrN2 Pale Yellow 55 140-142
29. C13H19BrN2O3 Colourless 68 208-210
30. C12H17BrN2O Pale Yellow 71 215-217

 Antimicrobial activity:

The minimum inhibitory concentration (MIC) assay of the 4-amino / dimethylamino-1-aryl / alkylpyridinium bromides (Group I, II, III & IV) showed a significant activity against all the mentioned pathogenic organisms (Figure 1).

Antibacterial activity:

Pyridinium bromides (Group III) with pyrrolidino moiety and alkyl side chain displayed excellent activity against both Gram positive and Gram negative bacteria at the low range of MIC value (6.25 -0.39 µg/mL). In addition , the same (Group III) compounds with the substitution of bromine atom at the end of the alkyl side chain displayed strong inhibitory action against both of the Gram negative bacteria Escherichia coli and Klebesilla pneumoniae (MIC range 6.25-0.39 µg/mL). The compounds (Group IV) with morpholino moiety bearing substitution of bromine at the end of the aliphatic side chain exerted remarkable activity against Klebesilla pneumoniae (3.12-0.39 µg/mL) and promising activity against Streptococcus mutans (6.25 -0.78 µg/mL).

Antifungal activity:

Investigation on antifungal screening revealed that the synthesized compounds showed variable degree of inhibition against the tested fungi. Pyridinium bromides (Group II & IV) with pyrrolidino/morpholino moiety bearing alkyl/aryl side chain exhibited higher inhibitory effect (3.12 -0.78, 6.25-0.39 µg/mL) than others (Group I & IV) against Rhizopus arrhizus. Pyridinium bromides (Group III) with morpholino moiety containing aliphatic side chain displayed excellent inhibition against Aspergillus niger (6.25-0.39 µg/mL). The synthesized compounds with aryl side chain bearing pyrrolidino and morpholino moiety (Group I & II)  and  4- pyrrolidino-1-alkylpyridinium bromides (Group III) showed substantial activity against Aspergillus niger (12.5 -3.12 µg/mL).

4- pyrrolidino / morpholino -1-alkylpyridinium bromides (Group III & IV) have showed a good deal of antibacterial activity than 4- pyrrolidino / morpholino -1-arylpyridinium bromides (Group I & II). But 4-pyrrolidinyl 1-aryl (Group I) and 4-morpholinyl 1-alkyl (Group IV) have showed a good deal of antifungal activity.  It is worth noting that the presence of a polar amino substituted pyridinium head group and the long liphophilic carbon chain could give rise to surfactant like activity. This surfactant like character may be helpful in achieving successful penetration into the lipid cell membrane and better biological activity for the compounds 6. In addition, it was proved that the factors such as molecular hydrophobicity 22,23 and electron density of the pyridinium nitrogen atom 24,25 control their antimicrobial activity.

Figure 1: Graph showing relation between the mean value of MIC of compounds 1-30 against bacterial (a) & fungal (b) microorganisms.

GI           –               1-5                                                                         

GII         –               6-10                                                        SA: Staphylococcus aureus

GIII        –               11-14, 19-22, 27 & 28                        SM: Streptococcus mutans

GIV        –               15-18, 23-26, 29 & 30                        KP: Klebsiella pneumoniae

STD 1    –               Ciprofloxacin                                     EC: Echerichia coli

STD 2    –               Fluconazole                                         RH: Rhizopus arrhizus


CONCLUSION

This study clearly shows that quaternary 4-pyrrolidino / morpholino-1-alkylpyridinium bromides are not only active against Gram negative, Gram positive bacteria and also active against opportunistic pathogenic fungi ultimately, these compounds have potential to be explored as a broad spectrum antimicrobial agent. These compounds are rather simple, easy to prepare in large scale and show interesting biological activity. Thus, there is a good scope for large-scale preparation of these compounds and application as both bactericide and fungicide.

REFERENCES

  • Thorsteinsson T, Masson M, Kristinsson K.G, Hjalmarsdottir M.A, Hilmarsson H, Loftsson T: Journal of Medicinal Chemistry 2003; 46: 4173-4181.
  • Vnutskikh Z.A, Shklyaev Y.V, Odegova T.F, Chekryshkin Y.S, Tolstikov A.G, Elchishcheva V, Syropyatov, B.Y: Khimiko Farmatsevticheskii Zhurnal 2006; 40: 19-22.
  • Ovchinnikova I.G, Fedorova O.V, Rusinov G.L, Zeuva M.N, Morodvskoi G.G: Khimiko Farmatsevticheskii Zhurnal 2003; 37: 17-19.
  • Burke J.R, Frey P.A: Journal of Organic Chemistry 1996; 61: 530-533.
  • Kourai H, Yabuhara T, Nagamune H: European Journal of Medicinal Chemistry 2006; 41: 437-444.
  • Madaan P, Tyagi V.K: Journal of Oleo Sciences 2008; 57: 197-215.
  • Alptiziin V, Parlar S, Tasli H, Erciyas E: Molecules 2009; 14: 5203-5215.
  • Darwish E.S, Facile: Molecules 2008; 13: 1066-1078.
  • Bull J.A, Balskuls E.P, Horan R.A.J, Langner M, Ley S.V: European Journal of Medicinal Chemistry 2007; 13: 5515-5538.
  • Kumar, P.V., Rao, V.R., Indian J. Chem. 2005; 44B, 2120-2125.
  • Song Y, Goel A, Basrur V, Roberts P.E.A, Mikovits J.A., Inman J.K, Turpin J.A, Rice W.G, Appella E: Bioorganic Medicinal Chemistry 2002; 10: 1263-1273.
  • Shabani F, Ghammamy S, Mehrani K, Teimouri M.B, Soleimani M, Kaviani S, Bioinorganic Chemistry and Applications 2008; Doi:10.1155/2008/501021.
  • Mancini I, Sicurelli A, Guella G, Turk T, Macek P, Sepcic K, Organic Biomedicinal Chemistry 2004; 2: 1368-1375.
  • Fujimot K, Morisaki D, Yoshida M, Namba T, Hye-Sook K, Wataya Y, Kourai H, Kakuta H, Sasaki K: Bioorganic Medicinal Chemistry Letters 2006; 16: 2758-2760.
  • Perez-Alvarez V, Fernandez-Martinez E: Proceedings of the western Pharmacology Society 2003; 46: 136-138.
  • Cantalejo Y.M, Saez B, Monterde M.I, Murillo M.T: European Journal of Medicinal Chemistry 2011; 46: 5662-5667.
  • Cappucino J.G, Sherman N, Microbiology-A Laboratory Manual, Addison Wesley longmanInc, California, 1999;
  • Nallu M, Sathishkumar V, Sangeetha M.K, Sarkunam K: Indian Journal of Chemistry 2001; 40: 295-298.
  • Leslie A, Hull: Journal of Chemical Education 2001; 78: 420.
  • Krishnapillay M, Jeyaraman R, Nallu M, Venuvanalingam P: Indian Journal of Chemistry 1997; 36A: 414-417.
  • Sarkunam K, Nallu M: Journal of Indian Chemical Society 2004; 81: 978-980.
  • Kourai H, Machikawa F, Horie T, Takeichi K, Shibasaki I: Journal of Antibacterial and Antifungal Agents 1983; 11: 553-562.
  • Kourai H, Manabe Y, Matsutani E, Hasegawa Y, Nakagawa K: Journal of Antibacterial and Antifungal Agents 1995; 23: 271-280.
  • Maeda T, Goto S, Manabe Y, Okazaki K, Nagamune H, Kourai H: Biocontrol Science 1996; 1: 41-49.
  • Okazaki K, Maeda T, Nagamune H, Kourai H: Biocontrol Science 1996; 1: 51-59.