Antimicrobial activity studies on some piperidine and pyrrolidine substituted halogenobenzene derivatives

Abstract

The in vitro antibacterial and antifungal activities of the compounds synthesised from some 1,2,3,5-tetrahalogeno benzenes in presence of sodium piperidide and sodium pyrrolidide (2,6-dipiperidino-1,4-dihalogenobenzenes; 2,6-dipyrrolidino-1,4-dibromobenzene; 2,4,6-tripyrrolidino chlorobenzene; and 1,3-dipyrrolidino benzene) were investigated. The in vitro antimicrobial activities were screened against the standard strains: Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633 as Gram positive, Yersinia enterocolitica ATCC 1501, Escherichia coli ATCC 11230 and Klebsiella pneumoniae as Gram negative, and Candida albicans as yeast-like fungus. Compounds (3, 5, 6, 7) inhibited the growth of all the test strains at MIC values of 32–512 μg/ml. None of the four compounds (1, 2, 4, 8) studied showed antimicrobial activity against any of the test strains within the MIC range 0.25–512 μg/ml.

Keywords::

• Antimicrobial activity
• Piperidine derivatives
• Pyrrolidine derivatives
• Halogeno benzenes
• MIC

Introduction

Piperidine, an alicyclic base is used as a raw material for pharmaceuticals, agrochemicals, rubber chemicals, surface active agents and other organic chemicals. Pyrrolidine, a similar base found in tobacco leaves with a strong piperidine-like odor provides derivatives used in pharmaceuticals, especially as modifiers of quinolone antibacterial agents [1,2]. Numerous biological compounds possess pyrrolidine and piperidine rings in their framework, and some of them are pharmaceutically important [3,4]. In the last several decades, halogenated aromatic hydrocarbons have been used extensively as intermediates in the chemical industry and agriculture. Some of these chemicals are carcinogenic and have deleterious effects on organisms [5,6].

Pharmaceuticals have a history of success in controlling morbidity and mortality caused by infectious diseases but as a consequence of frequent use, bacterial resistance to known classes of antibiotics has become a severe global problem in recent years [7,8]. There are serious concerns that untreatable pathogens may develop at an alarming rate in the near future. In this respect, we screened the antimicrobial activities of some piperidine and pyrrolidine substituted halogenobenzene derivatives.

Materials and methods

Synthesis

The tetrasubstituted benzene derivatives were prepared using well-known procedures [9]. Tetrasubstituted benzene derivatives, 1-chloro (or bromo or iodo)-2,4,6-trichloro, 1-chloro (or bromo or iodo)-2,4,6-tribromo, and 1-chloro (or bromo or iodo)-2,4,6-triiodo benzenes were synthesized as reactants. 2,4,6-trichloro, tribromo and triiodo anilines were used as the starting material and the procedures were carried out according to the methods given in literature. In nucleophilic substitution experiments, the sodium salts of piperidine and pyrrolidine were prepared with sodium amide and the reactions of these derivatives were successful [9].

Microbiological assays

Minimum inhibitory concentrations (MICs) were determined by broth microdilution following the procedures recommended by the National Committee for Clinical Laboratory Standards [10,11]. All the compounds were tested for their in vitro growth inhibitory activity against Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633 as Gram positive bacteria, Yersinia enterocolitica ATCC 1501, Escherichia coli ATCC 11230 and Klebsiella pneumoniae as Gram negative bacteria and Candida albicans as a yeast-like fungus. K. pneumoniae and C. albicans were obtained from the Department of Microbiology, Faculty of Medicine, Abant Izzet Baysal University, Turkey.

All tests were performed in Mueller-Hinton Broth (MHB). Bacterial strains were cultured overnight at 37°C in Brain Heart Infusion broth (BHI) and the yeast was cultured overnight at 30°C in Sabouraud Dextrose Broth (SDB). Test strains were suspended in MHB to give a final density of 5 × 10⁵ cfu/ ml. The compounds under test were dissolved in 100% DMSO and the final twofold dilutions gave concentrations from 1–512 μg/ml. Ampicillin and fluconazole were used as reference antibiotics for the bacteria and fungus, respectively 0.25–512 μg/ml. Microtiter plates were incubated for 18–24 h at 37°C for the bacterial tests and for the yeast-like fungus, MICs were determined after incubation for 48 h at 30°C. The lowest concentration of the compounds that prevented visible growth was considered to be the minimal inhibitory concentration (MIC). MIC values for the compounds and the standards are presented in Table IV.

Results and discussion

Synthesis

The compounds synthesised were 2,6-Dipiperidino-4-chlorobromobenzene (1); 2,6-Dipiperidino-4-chloroiodobenzene (2); 2,6-Dipiperidino-4-bromochlorobenzene (3); 2,6-Dipiperidino-1,4-dibromobenzene (4); 2,6-Dipiperidino-4-bromoiodobenzene (5); 2,4,6-Tripyrrolidinochlorobenzene (6); 2,6-Dipyrrolidino-1,4-dibromobenzene (7); 1,3-Dipyrrolidinobenzene (8) [9] (Scheme 1). These compounds were prepared from sodium piperidide and sodium pyrrolidide and 2,4,6-trichloro-, tribromo- and triiodoanilines according to the literature methods [9]. The structures of all the tetrasubstituted benzene derivatives are given in Table II and were confirmed by IR, NMR (100 M Hz spectrometer) and elemental analysis ().

Scheme 1 Synthesis of the compounds.

Table I.  IR spectral data for the compounds.

Bond	Peak (cm^− 1)
Aromatic C-H	3025
Alifatic C-H	2940
Aromatic primary Amine C-N	1340
Halogen C-Cl	790
Halogen C-Br	665
Halogen C-I	490

Table II.  NMR spectral data of the Dipiperidino- and Pyrrolidinobenzenes.

		Group peaks (ppm)
				Aromatic
Compounds	Structures	-CH2-	-CH2-N-	N-CH(1)-N	N-CH(2)-CH	N-CH-CH(3)
1		1.58	3.0		5.9
2		1.53	3.0		5.9
3		1.58	3.0		5.9
4		1.58	3.0		5.9
5		1.58	3.0		5.9
6		1.94	3.24		5.1
7		1.94	3.2		5.0
8		1.90	3.2	(H1) 5.5	(H2) 5.74	(H3) 6.79

Table III.  Physical and chemical data for the compounds.

Comp. No	Empirical formula	Found %C	Calc. %C	Found %H	Calc. %H	Yield (%)	M.p. (°C)
1	C16H22BrClN2	53.37	53.70	6.12	6.15	37.8	179–182
2	C16H22IClN2	47.23	47.46	5.28	5.43	20	170–173
3	C16H22BrClN2	53.55	53.70	5.99	6.15	37.8	170–172
4	C16H22 Br2N2	47.36	47.76	5.33	5.47	35	180–183
5	C16H22IBrN2	42.35	42.76	4.78	4.89	26.6	177
6	C18H26ClN3	67.45	67.60	8.04	8.13	40	173–179
7	C14H18Br2N2	44.78	44.91	4.76	4.81	34	172–175
8	C14H20N2	77.49	77.77	9.23	9.26	92.6	94

Antimicrobial activity

The in vitro antibacterial screening of the compounds against S. aureus, B. subtilis, Y. enterocolitica, E. coli and K. pneumoniae and the antifungal screening against C. albicans was carried out by the minimum inhibitory concentration method [10,11]. The antimicrobial activities (MIC values) are given in Table IV, which also contains those for ampicillin and flucanozole used as reference compounds as comparism and a check on the reliability of the method used.

Table IV.  The MICs (μg/ml) of the tested compounds.

Compounds	S. aureus	B. subtilis	E. coli	Y. enterocolitica	K. pneumoniae	C. albicans
1	na^a	na	na	na	na	na
2	na	na	na	na	na	na
3	64	256	256	512	512	128
4	na	na	na	na	na	na
5	64	256	512	512	512	64
6	32	128	256	256	256	32
7	32	32	64	128	256	32
8	na	na	na	na	na	na
Ampicillin	2	8	4	8	64	nt^b
Fluconazole	nt	nt	nt	nt	nt	128

^a na = not active;

^b nt = not tested.

In this study, eight derivatives were tested against standard strains of gram-positive bacteria, gram-negative bacteria and fungus. Compounds 3, 5, 6 and 7 inhibited the growth of all microorganisms, include fungus, with MICs 32–512 μg/ml (Table IV). Among the tested compounds, 5, 6 and 7 showed good antifungal activity against C. albicans with MICs 32–64 μg/ml, which was comparable with that for fluconazole (128 μg/ml). Four compounds (1, 2, 4, 8) did not show antimicrobial activity against any of the test strains within the range MICs 0.25–512 μg/ml. The inhibitory compounds had the highest antimicrobial activities against S. aureus and C. albicans with MICs of 32–128 μg/ml, but poor activity of the compounds was observed against gram-negative bacteria such as Y. enterocolitica ATCC 1501, E. coli ATCC 11230 and K. pneumoniae, which are highly pathogenic to humans. Indeed, the bacterium K. pneumoniae showed lower microbial susceptibility compared to the other tested microorganisms. The compounds tested here generally show lower antimicrobial activity against gram-negative bacteria. The antimicrobial actitivity against gram-positive bacteria and fungus may depend on the differences between the cell structures of these microorganisms.

The results (Table IV) show that 1,3-dipyrrolidinobenzene (8) had no activity against any of the test microorganisms so we can say that the bioactivities of compounds 6 and 7 (pyrrolidine derivatives) are mainly correlated with the halogen substituents. Here the bromo, chloro and pyrrolidino substituents in combination showed inhibitory activity against the microorganisms (Table IV). However, the results of the antimicrobial tests with compounds 1–5 indicated that the type and the position of the halogen and piperidine tetrasubstituents had a significantly different effects on the growth of microorganisms. In conclusion, we report that some of the compounds (piperidine and pyrrolidine substituted halogenobenzene derivatives) had antimicrobial activity against some hazardous microbes.