Synthesis, spectroscopic characterization and antibacterial screening of novel Mannich bases of Ganciclovir

Sheela Joshi; Purti Bilgaiyan; Anju Pathak

doi:10.1016/j.arabjc.2013.02.013

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Original article

10 (

1_suppl

); S1180-S1187

doi:

10.1016/j.arabjc.2013.02.013

Synthesis, spectroscopic characterization and antibacterial screening of novel Mannich bases of Ganciclovir

Sheela Joshi^⁎, Purti Bilgaiyan, Anju Pathak

School of Chemical Sciences, Devi Ahilya University, Takshshila Campus, Khandwa Road, Indore 452001, Madhya Pradesh, India

⁎Corresponding author. Tel.: +91 9826085169; fax: +91 0731 2365782. spjoshi11@rediffmail.com (Sheela Joshi), http://www.chemical.dauniv.ac.in (Sheela Joshi),

Received: 2011-12-23, Accepted: 2013-2-13, Published: 2013-02-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Biologically active Mannich bases with heteroaromatic ring system of substituted guanine derivative (2-amino-9 [{(1,3 di hydroxy propane-2yl) oxy} methyl] 6-9 dihydro-3H-purine-6-one) (ganciclovir), have been synthesized via Mannich reaction. The aminomethylation of ganciclovir with various biologically potent sulphonamides was carried out and then characterized by elemental analysis and spectral studies – UV, IR, ¹H NMR, powder X-ray diffraction and Scanning Electron Microscopy. The compounds were screened for their antibacterial activity against various pathogenic bacteria at varying concentrations. The antibacterial activity of derived Mannich bases was compared with parent sulphonamides. The toxicity of synthesized Mannich bases was ascertained by LD₅₀ test.

Keywords

2-Amino-9[{(1,3 di hydroxy propane-2yl) oxy} methyl] 6-9 dihydro-3H-purine-6-one (ganciclovir)

Sulphonamides

Mannich reaction

Mannich bases

Antibacterial activity

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1 Introduction

The chemistry of the aminoalkylation of aromatic substrates by the Mannich reaction is of great interest for the synthesis and modification of biologically active compound having physical (Tramontini and Angiolini, 1990) and chemical importance (Tramontini et al., 1998) as well as physiological properties (Sriram et al., 2006; Mandloi et al., 2005; Ali and Shaharynar, 2007 ) because the amino group can be easily converted into a variety of other functionalities (Tramontini and Angiolini, 1994). Mannich reaction offers a judicious method for introduction of basic aminoalkyl chain in various drugs/compounds. Further a considerable amount of work has been reported on synthesis and pharmacological activity of various Mannich bases for analgesic, antiinflammatory, anaesthetic and antimicrobial activity as well as intermediates in drug synthesis (Blanton and Nobles, 1962; Jesudason et al., 2009). The heterocyclic system is reported to have tremendous pharmaceutical importance (Katritzky, 2004). In this context, literature survey has revealed a number of reports on antimicrobial activity of N-Mannich bases. 2-amino-9[{(1,3 dihydroxy propane-2yl) oxy} methyl] 6-9 dihydro-3H-purine-6-one, a guanine derivative is an antiviral drug, used in prevention and treatment of cytomegalovirus (CMV) infection in HIV and transplant patients. It does not kill the virus but keeps CMV infection under control (Sullivan et al., 1996). Ganciclovir is indicated for prevention of CMV disease in bone marrow and solid organ transplant recipients and it is also used for acute CMV colitis in HIV/AIDS and CMV pneumonitis in immunosuppressed patients (Rossi, 2006). In addition to this the sulphonamide is well-known antibacterial (Joshi et al., 2003; Almajan et al., 2009; Bayrak et al., 2009 ), antitubercular (Nishihara, 1953), anti-inflammatory (Li et al., 1995), carbonic inhibitory (Supuran et al., 1998), insecticidal (Singh, 1970).

The Mannich bases incorporated with sulphonamides are reported to be potent antibacterial agents and less toxic than parent sulphonamide (Joshi et al., 2006). Keeping in view the unique features of these compounds 2-amino-9[{(1,3 di hydroxy propane-2yl) oxy} methyl] 6-9 dihydro-3H-purine-6-one as a substrate and sulphonamide as amine component were condensed via Mannich reaction. A series of Mannich bases were synthesized with different sulphonamides/secondary amines (Scheme 1). The synthesized Mannich bases were characterized by elemental analysis and spectral studies-UV, IR, ¹H NMR, powder X-ray diffraction, SEM and screened for in vitro antibacterial activity of gram-positive and gram-negative bacteria at arbitrarily chosen concentrations.

Synthesis of Mannich bases from primary amines and secondary amines.

2 Experimental

All the melting points were determined in open capillary tubes and are uncorrected. Thin layer chromatography was used for monitoring the reaction and to check purity. UV spectra were studied on Schimadzu UV-160A, UV–visible spectrophotometer; IR spectra (KBr) were recorded as potassium bromide pellets on Schimadzu 820 IPC FTIR spectrometer and ¹H NMR spectra on Bruker DRX-300 FT NMR Spectrometer and chemical shifts were expressed as (ppm) values against tetramethylsilane (TMS) as internal reference. The XRD measurements were carried out on Bruker D8 Advance X-ray diffractometer using CuKα at a wavelength of 1.54 Å. SEM studies were performed with a Jeol JSM 5600 instrument having a magnification range of ×18 to ×300,000 and at an accelerating voltage of 0.5–30 kV. The chemical reagents used in the synthesis were purchased from E. Merck and Aldrich.

2.1

2.1 Chemistry

The reaction routes for the synthesis of the title compound were described as shown in Scheme 1. The synthesized Mannich bases (3a–3j) were obtained thus in (⩾85%) yield.

2.1.1

2.1.1 Synthesis of Mannich bases (3a–3e)

In ethanolic solution of 0.01 mol of substrate (Comp.-1), 0.01 mol of sulphonamide and 2.5 mL of formaldehyde solution (37% v/v) were added. The pH of mixture was adjusted to 3.5 by adding 0.5 ml of 1 mol⁻¹HCl. The mixture was kept in an efficient ice cooler for half an hour and then refluxed on a water bath. The reflux time is varied with the sulphonamide used. Refluxed mixture was kept at 0 °C for four days when crystalline product was obtained. The obtained product was recrystallized with dry distilled ethanol and DMF (1:1).

2.1.2

2.1.2 Synthesis of Mannich bases (3f–3j)

Secondary amine 0.01 mol was added in an ethanolic solution of 50 mL of substrate (Comp. -1) 0.01 mol in a flat bottom flask. Amount of 0.4 mL of formaldehyde solution (37%) was added slowly with constant stirring. The reaction mixture was stirred at 70–75 °C for 3.0–8.5 h, depending upon the secondary amine. The remaining portion of formaldehyde solution was added in two instalments after 1 and 2 h, respectively. The reaction mixture was kept overnight in the refrigerator. Next day, the excess of solvent was distilled off from the reaction mixture under reduced pressure. It was again kept for crystallization in the refrigerator. The product obtained was purified by recrystallization from dry distilled ethanol and DMF (1:1).

2.2

2.2 Spectral Studies

Compound 3a: C₂₀H₂₃N₉O₆S; yield 80%, m.p. 160–163 °C. Anal. Calcd C, 46.40; H, 4.58; N, 24.36 Found C, 46.16; H, 4.42; N, 24.23. UV (λ max) nm: 208 (C⚌O), 190 (C⚌N), 205 (sulphonamide group), 186, 207, 251 (for benzene chromophore), 254 (sulphonamide moiety). IR (KBr) v max in cm⁻¹: 3442 υ_s N–H, 3398 υ_as N–H in SO₂NH, 3302 υ_s O–H, 2940 υ_as CH₂, 1688 υ_s C⚌O, 1345 υ_s S⚌O, 1130 C–H in plane bending vibration of 1:4 disubstituted benzene. ¹H NMR (DMSO) δ ppm: 5.04 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 6.36 (S, 1H of sulphonamide), 7.93 (s, 1H, CH of purine ring), 7.84 (s, 1H, free OH of Comp. 1), 9.03 (s, 1H of SO₂NH), 6.6–7.2 (m, ring proton of sulphonamide), 7.48–8.47 (m, 3H of sulphadiazine ring).

Compound 3b: C₂₀H₂₂N₉O₈S; yield 72%, m.p. 105–107 °C. Anal. Calcd. C, 45.74; H, 4.71; N, 21.82; Found C, 45.61; H, 4.46; N, 21.64. UV (λ max) nm: 210 (C⚌O), 197 (C⚌N), 208 S⚌O, 182, 205, 250 for benzene chromophore; 258 sulphonamide moiety. IR (KBr) v max in cm⁻¹: 3440 υ_s N–H, 3380 υ_as N–H in SO₂NH, 3302 υ_s O–H, 2910 υ_as C–H in CH₂, 1653 υ_s C⚌O, 1380 υ_s S⚌O, 1135 C–H in plane bending vibration of 1:4 disubstituted benzene; ¹H NMR (DMSO) δ ppm: 3.43 (s, 3H of OCH₃) 5.04 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 6.4 (s, 1H of sulphonamide), 7.9 (s, 1H, CH of purine ring), 7.4 (s, 1H, free OH of Comp. 1), 9.2 (s, 1H of SO₂NH), 6.6–7.07 (m, ring proton of sulphonamide).

Compound 3c: C₂₀H₂₄N₈O₇S; yield 84%, m.p. 180–181 °C, Anal. Calcd. C, 46.13; H, 4.65; N, 21.53; Found C, 46.13; H, 4.25; N, 21.33. UV (λ max) nm: 201 (C⚌O), 192 (C⚌N), 208 (S⚌O), 186, 207, 250 for benzene chromophore; 255 (sulphonamide moiety). IR (KBr) v max in cm⁻¹: 3475 υ_s N–H, 3350 υ_as N–H in SO₂NH, 3318 υ_s O–H, 2915 υ_as C–H in CH₂, 1685 υ_s C⚌O, 1349 υ_s S⚌O, 1110 C–H in plane bending vibration of 1:4 disubstituted benzene. ¹H NMR (DMSO) δ ppm: 2.71(s, 3H of CH₃) 5.04 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 6.3 (s, 1H of sulphonamide).

Compound 3d: C₁₈H₂₂N₇O₇NaS; yield 78%, m.p. 90 °C, Anal. Calcd. C, 42.93; H, 4.41; N, 19.48 Found C, 42.52; H, 4.21; N, 19.22. UV (λ max) nm: 208 (C⚌O), 190 (C⚌N), 205 S⚌O, 186, 207, 251 for benzene chromophore; 254 sulphonamide moiety. IR (KBr) v max in cm⁻¹: 3482 υ_s N–H, 3351 υ_as N–H in SO₂NH, 3295 υ_s O–H, 2950 υ_as C–H in CH₂, 1647 υ_s C⚌O, 1385 υ_s S⚌O, 1109 C–H in plane bending vibration of 1:4 disubstituted benzene. ¹H NMR (DMSO) δ ppm: 2.16 (s, 3H acetamide CH₃) 5.04 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 6.3 (s, 1H, NH of sulphonamide), 7.9 (s, 1H, CH of purine ring), 6.7 (s, 1H, OH of Comp. 1), 9.42 (s, 1H of SO₂NH), 6.6–7.07 (m, ring proton of sulphonamide).

Compound 3e: C₂₀H₂₂N₉O₆SAg; yield 85%, m.p. 222 °C. Anal. Calcd. C, 38.46; H, 3.55; N, 20.19 Found C, 38.32; H, 3.51; N, 20.24. UV (λ max) nm: 203 (C⚌O), 194 (C⚌N), 208 S⚌O, 180, 203, 250 for benzene chromophore; 257 sulphonamide moiety. IR (KBr) v max in cm⁻¹: 3496 υ_s N–H, 3348 υ_as N–H in SO₂NH, 3300 υ_s O–H, 2952 υ_as C–H in CH₂, 1638 υ_s C⚌O, 1355 υ_s S⚌O, 1100 C–H in plane bending vibration of 1:4 disubstituted benzene. ¹H NMR (DMSO) δ ppm: 5.04 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 6.1 (s, 1H, NH of sulphonamide), 7.9 (s, 1H, CH of purine ring), 6.72 (s, 1H, OH of Comp. 1), 6.6–7.2 (m, ring proton of sulphonamide).

Compound 3f: C₁₄H₂₄N₆O₆; yield 80%, m.p. 160–162 °C. Anal. Calcd. C, 45.14; H, 6.49; N, 22.56 Found C, 45.13; H, 6.51; N, 22.50. UV (λ max) nm: 208 (C⚌O), 191 (C⚌N), 185 205, 250 for benzene chromophore. IR (KBr) v max in cm⁻¹: 3482 υ_s N–H, 3352 υ_s O–H, 2944 υ_as C–H in CH₂, 1680 υ_s C⚌O. ¹H NMR (DMSO) δ ppm: 4.51 (s, 2H,CH2), 5.60 (s, 2H, CH₂ attached to purine ring), 7.9 (s, 1H, CH of purine ring), 4.92 (s, 1H, OH of Comp. 1).

Compound 3g: C₁₂H₂₀N₆O₄; yield 71%, m.p. 225–227 °C, Anal. Calcd. C, 46.13; H, 6.46; N, 26.90 Found C, 46.4; H, 6.51; N, 26.82. UV (λ max) nm: 210 (C⚌O), 195 (C⚌N), 185, 205, and 255 for benzene chromophore. IR (KBr) v max in cm⁻¹: 3412 υ_s N–H, 3310 υ_s O–H, 2909 υ_as C–H in CH₂, 1655 υ_s C⚌O. ¹H NMR (DMSO) δ ppm: 4.48 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 7.9 (s, 1H, CH of purine ring), 6.12 (s, 1H, OH of Comp. 1).

Compound 3h: C₁₄H₂₂N₆O₅; yield 79%, m.p. 215–218 °C, Anal. Calcd. C, 47.43; H, 6.26; N, 23.71 Found C, 47.32; H, 6.30; N, 23.52. UV (λ max) nm: 204 (C⚌O), 190 (C⚌N), 184, 209, and 254 for benzene chromophore. IR (KBr) v max in cm⁻¹: 3477 υ_s N–H, 3361 υ_s O–H, 2947 υ_as C–H in CH₂, 1630 υ_s C⚌O, 1244 υ_s C–O. ¹H NMR (DMSO) δ ppm: 4.4 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purin ring), 7.93 (s, 1H, NH of purine ring), 6.21 (s, 1H, OH of Comp. 1).

Compound 3i: C₂₂H₂₄N₆O_4; yield 82%, m.p. 235–236 °C, Anal. Calcd. C, 60.52; H, 5.55; N, 19.25 Found C, 60.61; H, 5.32; N, 19.10. UV (λ max) nm: 209 (C⚌O), 190 (C⚌N), 184, 206, and 260 for benzene chromophore. IR (KBr) v max in cm⁻¹: 3406 υ_s N–H, 3318 υ_s O–H, 2932 υ_as C–H in CH₂, 1643 υ_s C⚌O. ¹H NMR (DMSO) δ ppm: 4.5 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 7.9 (s, 1H, CH of purine ring), 6.17 (s, 1H, OH of Comp. 1).

Compound 3j: C₁₄H₂₃N₇O₄; yield 80%, m.p. 220 °C. Anal. Calcd C, 47.57; H, 6.56; N, 27.74 Found C, 47.80; H, 6.32; N, 27.62. UV (λ max) nm: 205 (C⚌O), 190 (C⚌N), 184, 206, and 260 for benzene chromophore. IR (KBr) v max in cm⁻¹: 3458 υ_s N–H, 3342 υ_s O–H, 2937 υ_as C–H in CH₂, 1639 υ_s C⚌O. ¹H NMR (DMSO) δ ppm: 4.5 (s, 2H, CH₂), 5.5 (s, 2H, CH₂ attached to purine ring), 7.9 (s, 1H, CH of purine ring), 5.5 (s, 1H, OH of Comp. 1).

2.2.1

2.2.1 Powder X-ray diffraction studies

Powder X-ray diffraction patterns of three of the synthesized compounds namely ganciclovir methyl silver sulphadiazine (3e), ganciclovir methyl diphenylamine (3i) and ganciclovir methyl Piperazine (3j) (Figs. 1–3 , respectively) have been reported and important structural information has been shown in Tables 1–3 , respectively.

Powder X-ray diffraction pattern of ganciclovir methyl silver sulphadiazine.

Powder X-ray diffraction pattern of ganciclovir methyl diphenylamine.

Powder X-ray diffraction pattern of ganciclovir methyl piperazine.

Table 1 The experimental data and the calculated results for powder X-ray pattern of the ganciclovir methyl silver sulphadiazine.

2θ	d Spacing (Å) observed	d Spacing (Å) calculated	Relative intensity (%)	hkl
19.79	4.48	4.46	98.96	210
20.58	4.31	4.14	73.17	023
22.37	3.96	4.00	43.61	220
24.19	3.67	3.67	84.83	004
25.53	3.48	3.48	37.33	141
26.59	3.34	3.04	50.69	043
27.79	3.20	3.32	100	133
29.17	3.05	3.31	19.97	114
30.81	2.89	2.73	22.32	152
31.98	2.79	2.85	46.99	134
33.09	2.70	2.46	55.81	134
34.50	2.59	2.59	25.63	060
35.94	2.49	2.40	27.90	045
37.08	2.42	2.42	32.71	340
38.43	2.33	2.33	42.30	400
39.45	2.28	2.26	15.31	260
40.89	2.20	2.36	29.36	106
43.17	2.09	2.46	24.98	161
45.99	1.97	1.98	31.87	360
47.78	1.90	2.20	19.51	421
48.89	1.86	2.13	15.74	171
49.21	1.84	1.81	17.32	520

Lattice parameters and angle calculated are: a = 9.32 Å or 0.93 nm, b = 15.54 Å or 1.55 nm, C = 14.68 Å or 1.46 nm, β = 94.18°. Unit cell volume of the complex: 2,126.14 × 10⁻⁸ cm.

Table 2 The experimental data and the calculated results for powder X-ray pattern of the ganciclovir methyl biphenyl amine.

2θ	d spacing (Å) Observed	d spacing (Å) Calculated	Relative intensity (%)	h k l
10.97	8.05	8.00	27.38	110
14.87	5.94	5.94	10.07	120
20.76	4.27	4.24	22.82	102
21.99	4.03	4.00	31.25	220
23.75	3.74	4.20	100	201
24.74	3.59	3.56	15.36	140
27.97	3.18	3.18	10.12	003
29.05	3.06	3.34	10.54	202
30.46	2.14	3.01	8.14	103
32.30	2.76	3.28	17.48	212
36.46	2.46	2.61	7.52	302
37.61	2.38	2.96	7.94	301
38.40	2.34	2.34	7.63	400
40.04	2.24	2.25	7.28	260
44.00	2.05	2.40	7.03	161
45.02	2.01	1.98	6.48	360
46.81	1.93	1.93	7.01	080
47.04	1.92	2.18	6.98	421
48.54	1.87	2.10	7.49	171
49.15	1.85	1.81	6.18	520

Lattice parameters and angle calculated are: a = 9.36 Å or 0.93 nm, b = 15.44 Å or 1.54 nm, C = 9.54 Å or 0.954 nm, β = 32.03°. Unit cell volume of the complex: 1,378.70 × 10⁻⁸ cm.

Table 3 The experimental data and the calculated results for powder X-ray pattern of the ganciclovir methyl piperazine.

2θ	d spacing(Å) Observed	d spacing(Å) Calculated	Relative intensity (%)	h k l
19.85	4.53	4.53	10.43	200
22.25	4.06	4.12	46.49	113
23.78	3.81	3.81	100	040
25.71	3.54	3.51	21.29	140
27.97	3.28	3.38	6.57	042
28.67	3.20	3.27	55.90	133
30.08	3.07	3.51	4.90	140
31.98	2.90	2.80	7.92	320
34.76	2.70	2.72	14.79	025
39.16	2.43	2.43	4.59	006
40.16	2.38	2.95	4.99	301
41.68	2.31	2.83	6.86	151
43.06	2.25	2.21	4.76	260
44.26	2.20	2.52	4.55	144
46.81	2.11	2.14	4.96	206
47.89	2.07	2.41	6.18	161
49.39	2.02	1.94	4.30	360

Lattice parameters and angle calculated are: a = 9.06 Å or 0.90 nm, b = 15.24 Å or 1.52 nm, C = 14.58 Å or 1.45 nm, β = 64.13°. Unit cell volume of the complex: 2,013.12 × 10⁻⁸ cm.

Average particle size of the synthesized compound was determined with the help of the Scherrer formula, in which particle size D is defined as 0.9λ/B cos θ,

Where 0.9 = constant, λ = wavelength, B = angular width and θ = diffraction angle average particle size of the compounds determined was 94.18, 32.03 and 64.13 nm respectively. Diffraction data of the compounds are listed in Tables 1–3 . All of our synthesized compounds had an orthorhombic crystal system and XRD patterns showed that all are polycrystalline in nature. The largest relative deviation between the calculated and experimental d value was nearly equal to one, which indicates that all the synthesized compounds are multiphase compounds. Interplaner d spacing and unit cell volume of the synthesized compound were calculated by the formulae: $\begin{matrix} 1 / d^{2} = h^{2} / a^{2} + k^{2} / b^{2} + l^{2} / c^{2} \\ V = abc \end{matrix}$ Peaks having important characteristic information have been identified with the help of standard diffraction card JCPDS 31–1859, 38–1709, 49–2499 and 35–1687 and indicate the formation of nanosized, polycrystalline, two phase and low symmetry compounds.

2.2.2

2.2.2 Scanning Electron Microscopy (SEM) studies

Scanning Electron Microscopy studies of two of the synthesized compounds namely ganciclovir methyl silver sulphadiazine (3e), and ganciclovir methyl piperazine (3j) have been carried out at a magnification of ×1000 − 10 μm and ×600 − 2 μm. SEM uses a focused beam of high energy electrons to generate a variety of signals from the surface of signals revealing information about the sample, including external morphology, topography, chemical composition crystalline structure, and orientation of materials making up the sample.

We recorded different magnifications, as described above. Compound ganciclovir methyl silver sulphdiazine (3e) (Figs. 4 and 5) has a different topography and morphology at the two different magnifications. In the images, the particles exhibit different sizes and shapes, and are present in the form of closed polygonal structure hand, the compound ganciclovir methyl piperazine (3j) (Figs. 6 and 7) shows at different magnifications a finer morphology as compared to the other compound, appears in the form of simple polygonal structures.

SEM image of ganciclovir methyl silver sulphadiazine (magnification of ×1,000 − 10 μm).

SEM image of ganciclovir methyl silver sulphadiazine (magnification of ×600 − 2 μm).

SEM image ganciclovir methyl piperazine (magnification of ×1000 − 10 μm).

SEM image ganciclovir methyl piperazine (magnification of ×600 − 2 μm).

SEM studies show microstructure of the compounds, which mainly include surface morphology.

2.3

2.3 Antimicrobial activity and LD₅₀ test

The newly synthesized Mannich bases 3a–3j were screened for their antibacterial activity against pathogenic strains of Escherichia coli, Staphylococcus aureus and Bacillus subtilis at varying concentrations-80 μg/ml, 160 μg/ml and 320 μg/ml using corresponding sulphonamides as their standards by the cup plate method. Nutrient agar media were prepared for bacterial growth. The media were autoclaved at 15 lbs pressure (121.6 C) for 30 min. The culture of bacterium was mixed with autoclaved media and poured in plates and bored. The solution of Mannich bases was poured in these cups in triplicate and incubated at 37 °C for 24 h. Antibacterial activity was ascertained by the zone of inhibition measured in mm as shown in Table 4. The similar procedure was followed for the parent sulphonamides.

Table 4 Antibacterial screening of synthesized Mannich bases and sulphonamides against E. coli, S. aureus and B. subtilis (Zone of inhibition in mm).

Comp. No.	E.coli Concentration in μg/ml				S.aureus Concentration in μg/ml				B.subtilis Concentration in μg/ml
	80	160	320	Avg	80	160	320	Avg	80	160	320	Avg
3a	8.6	9.6	11.6	9.9	8.6	10.3	12.3	10.4	–	–	–	–
3b	8.6	9.6	12.6	10.2	9.3	10.6	13.3	11.0	7.6	8.0	12.3	9.3
3c	7.6	8.3	11.6	9.1	7.3	8.0	11.0	8.7	8.3	10.0	12.6	10.3
3d	9.6	11.3	13.3	11.4	9.6	11.0	14.3	11.6	–	7.3	15.6	7.6
3e	13.3	14.0	17.6	15.1	7.3	10.6	12.3	10.1	8.6	10.6	11.6	10.2
3f	–	–	11.6	3.8	–	–	7.3	2.4	–	–	–	–
3g	–	–	9.0	3.0	–	–	8.3	2.7	7.3	8.3	10.0	8.5
3h	–	–	14.6	4.8	–	9.0	11.6	6.8	–	–	–	–
3i	–	–	10.3	4.8	–	8.0	11.6	6.8	7.3	8.3	10.0	8.5
3j	–	–	15.3	5.1	–	–	11.6	3.8	7.6	9.6	10.6	7.2
2a	12.0	16.6	20.0	16.0	15.6	18.6	21.3	18.5	17.0	19.6	27.6	21.4
2b	14.6	17.0	19.0	16.8	15.3	19.0	20.0	18.0	17.3	21.6	27.3	22.1
2c	14.0	16.3	20.0	16.7	10.6	16.0	19.3	15.3	15.6	18.0	19.6	17.7
2d	9.6	11.6	17.6	12.9	9.0	11.0	14.6	11.5	9.6	17.6	21.6	16.2
2e	9.6	10.3	16.0	11.7	7.3	8.3	11.0	8.8	8.6	10.3	12.6	10.5

^∗Avg: average value of antibacterial activity (for 80, 160 and 320 μg).

The toxicity of synthesized Mannich bases was ascertained by LD₅₀ test. The test performed on white mice weighing 25 g. Doses were given orally as well as intraperitoneally and mice were kept under observation for 72 h for each trial. The Mannich bases showed no adverse toxic effect even at an oral dose of 1400 mg/kg of the body weight of mice. However when the dose was administered intraperitoneally they proved be lethal at the dose level of 800 mg/kg of the body weight of mice.

3 Results and discussion

The Mannich bases synthesized by Mannich reaction were obtained in good yield (⩾85%). They were analysed for elemental analysis and results were found to be in full agreement with the calculated values. The anticipated structure was in agreement with the spectral data – UV, IR and ¹H NMR. The purity of synthesized compounds was assured with the aid of the chromatographic technique. The stationary phase was silica gel-G. It was of chromatographic grade. The solvents used for the mobile phase were methanol and chloroform. They were distilled before use. The spectral studies have shown a characteristic band due to the methylene group incorporated between active hydrogen substrate and the amine component as a result of Mannich reaction at (2940–2950) and (1442–1450). This shows the presence of amino methyl linkage in the synthesized Mannich bases. The ¹H NMR also confirms aminomethyl linkage (–CH₂) between amine and active hydrogen (5.04–5.20). Some of the synthesized compounds are of nano size. The use of nano particle in medicine offers some exciting possibilities. Some techniques are only imagined, while others are at various stages of testing, or actually being used today. The Mannich bases were screened for their biological significance. They were evaluated for antibacterial activity against pathogenic strains of E. coli, S. aureus and B. subtilis at varying concentrations – 80, 160 and 320 μg/ml. These pathogens were subcultured on specific media. The Mannich bases and the standard compound (sulphonamide and secondary amines) were dissolved in DMF. The activities reported were mean of zone of inhibition in millimetre (in triplicate). All the reported compounds exhibit remarkable in vitro activity against these pathogens. Their activity was also compared with their parent sulphonamides.

Table 4 reflects that most of the compounds had shown remarkable activity only at 320 μg/ml. Derived Mannich bases show activity against this pathogen but 3e was superior to others followed by 3d and 3b in inhibiting the growth of E. coli. A comparison with parent sulphonamides shows that Mannich bases 3e were superior to the corresponding sulphonamides and the other mannich bases are equally potent than the corresponding sulphonamides.

The Mannich bases 3b, 3d, 3e were significantly superior to other compounds in exhibiting antibacterial activity against S. aureus. Comparative study with sulphonamides indicates that Mannich base 3d and 3e are superior to the corresponding sulphacetamide sodium and silver sulphadiazine. Moreover, concentration of 320 μg/ml was superior for inhibiting the growth of the bacterium.

Study against B. subtilis revealed that 3c was best among all Mannich bases, which were moderately active, followed by 3e and 3b, in checking the growth of the pathogen. Moreover, concentration of 320 μg/ml was most effective for showing antibacterial activity.

Comparison with sulphonamides shows that, Mannich bases and sulphonamides show antibacterial activity, but the former is less toxic than the latter as revealed by LD₅₀ test on white mice of weight 25 g. The newly synthesized compounds seem to be really promising compounds for their antibacterial activity. In the light of those findings we will undertake further synthetic studies on the new compounds in the future.

4 Conclusion

Conclusions suggest that the newly synthesized Mannich bases of ganciclovir have a very noticeable and prolonged antibacterial activity. These derived Mannich bases are less toxic in comparison with their parent sulphonamides. This work shows that Mannich bases are a potential source of compounds for inhibition of bacteria and could be used as efficient drugs with minimum side effects.

Acknowledgements

Our sincere thanks are due to CSMCRI, Bhavnagar for elemental analysis, SAIF Chandigarh and UGC-DAE CSR Indore for spectral studies. We also extend our sincere thanks to Dr. Tushar Banerjee, School of Life Sciences, DAVV, Indore for providing facilities to conduct antibacterial studies.

References

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Almajan G.L., Barbusceanu S.P., Almajan E.R., Draghici C., Saramet G., . Eur. J. Med. Chem.. 2009;44:3083-3089.
Bayrak H., Demirbas A., Karaoglu S.A., Demirbas N., . Eur. J. Med. Chem.. 2009;44:1057-1066.
Blanton C.D., Nobles W.L., . J. Pharm. Sci.. 1962;51:878.
Jesudason P.E., Inayatullah M., Selvaraj D., Jayakumar R., . Eur. J. Med. Chem.. 2009;44(5):2307-2312.
Joshi S., Khosla N., Tiwari P., . Bioorg. Med. Chem.. 2003;12:571.
Joshi S., Manikpuri A., Tiwari P., . Bioorg. Med. Chem. Lett.. 2006;17:645.
Katritzky A.R., . Chem. Rev.. 2004;104:2125.
Li J.J., Anderson Q.D., Burton E.G., Cogburn J.N., Collins J.T., Garland D.J., Gregory S.A., Huang H.C., Isakson P.C., Koboldt C.M., Logush E.W., Norton M.B., Perkns W.E., Reinherd E.J., Seibert K., Veenhuizen A.W., Zang Y., Reitz D.B., . J. Med. Chem.. 1995;38:4570.
Mandloi D., Joshi S., Khadikar P.V., Khosla N., . Bioorg. Med. Chem. Lett.. 2005;15(2):405-411.
Nishihara H., . J. Biol. Chem.. 1953;40:579.
Rossi S, editor. Austrlian Medicines Handbook 2006. Adelaid, ISBN 0-9757919-2-3.
Singh B.J., . J. Indian Chem. Soc.. 1970;56:720.
Sriram D., Yogeeswari P., Reddy P., . Bioorg. Med. Chem. Lett.. 2006;16:2113-2116.
Sullivan S.D., Mozaffari E., Johnson E.S., Wolitz R., Follansbee S.E., . Clin. Ther.. 1996;18:546.
Supuran C.T., Scozzafava A., Jurca B.C., Ilies M.A., . Eur. J. Med. Chem.. 1998;33:83.
Tramontini M., Angiolini L., . Tetrahedron. 1990;46:1791.
Tramontini M., Angiolini L., . Mannich Base: Chemistry and Uses. Boca Raton: CRC Press; 1994. pp. 7–20
Tramontini M., Angiolini L., Ghedini N., . Polymer. 1998;29:771.

Introduction

Experimental

Chemistry
Spectral Studies
Antimicrobial activity and LD50 test

Results and discussion

Conclusion

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[1] Ali A.M., Shaharynar M., . Bioorg. Med. Chem. Lett.. 2007;17:3314-3316.

[2] Almajan G.L., Barbusceanu S.P., Almajan E.R., Draghici C., Saramet G., . Eur. J. Med. Chem.. 2009;44:3083-3089.

[3] Bayrak H., Demirbas A., Karaoglu S.A., Demirbas N., . Eur. J. Med. Chem.. 2009;44:1057-1066.

[4] Blanton C.D., Nobles W.L., . J. Pharm. Sci.. 1962;51:878.

[5] Jesudason P.E., Inayatullah M., Selvaraj D., Jayakumar R., . Eur. J. Med. Chem.. 2009;44(5):2307-2312.

[6] Joshi S., Khosla N., Tiwari P., . Bioorg. Med. Chem.. 2003;12:571.

[7] Joshi S., Manikpuri A., Tiwari P., . Bioorg. Med. Chem. Lett.. 2006;17:645.

[8] Katritzky A.R., . Chem. Rev.. 2004;104:2125.

[9] Li J.J., Anderson Q.D., Burton E.G., Cogburn J.N., Collins J.T., Garland D.J., Gregory S.A., Huang H.C., Isakson P.C., Koboldt C.M., Logush E.W., Norton M.B., Perkns W.E., Reinherd E.J., Seibert K., Veenhuizen A.W., Zang Y., Reitz D.B., . J. Med. Chem.. 1995;38:4570.

[10] Mandloi D., Joshi S., Khadikar P.V., Khosla N., . Bioorg. Med. Chem. Lett.. 2005;15(2):405-411.

[11] Nishihara H., . J. Biol. Chem.. 1953;40:579.

[12] Rossi S, editor. Austrlian Medicines Handbook 2006. Adelaid, ISBN 0-9757919-2-3.

[13] Singh B.J., . J. Indian Chem. Soc.. 1970;56:720.

[14] Sriram D., Yogeeswari P., Reddy P., . Bioorg. Med. Chem. Lett.. 2006;16:2113-2116.

[15] Sullivan S.D., Mozaffari E., Johnson E.S., Wolitz R., Follansbee S.E., . Clin. Ther.. 1996;18:546.

[16] Supuran C.T., Scozzafava A., Jurca B.C., Ilies M.A., . Eur. J. Med. Chem.. 1998;33:83.

[17] Tramontini M., Angiolini L., . Tetrahedron. 1990;46:1791.

[18] Tramontini M., Angiolini L., . Mannich Base: Chemistry and Uses. Boca Raton: CRC Press; 1994. pp. 7–20

[19] Tramontini M., Angiolini L., Ghedini N., . Polymer. 1998;29:771.

Synthesis, spectroscopic characterization and antibacterial screening of novel Mannich bases of Ganciclovir