Antimicrobial, antimalarial and antileishmanial activities of mono- and bisquaternary pyridinium compounds

Abstract

Pyridinium-based oxime compounds have been utilized worldwide as antidotes following exposure to anticholinesterase agents. In the event of combined chemical and biological incident, it is of vital importance to know the ability of antidotes to provide additional protection against biological threats. This paper reports results of in vitro antimicrobial and antiprotozoal activities of a series of quaternary pyridinium oximes against a number of lower pathogenicity BSL-1 and 2 agents. In general, our compound panel had little to no antimicrobial action except for thiophene- and benzothiophene-substituted monoquaternary pyridinium compounds 21 and 24 that showed moderate antibacterial activity against Staphylococus aureus and methicillin resistant S. aureus with IC₅₀ values ranging from 12.2–17.7 µg/mL. Compounds 21 and 24 also exhibited antileishmanial activity against Leishmania donovani with IC₅₀ values of 19 and 18 µg/mL, respectively. Another monoquaternary pyridinium compound with a bromobutyl side chain 17 showed antimalarial activity against both a chloroquine sensitive and resistant strains of Plasmodium falciparum with IC₅₀ values of 3.7 and 4.0 µg/mL, respectively. None of the bisquaternary pyridinium compounds showed antimicrobial, or antiprotozoal activity. None of the compounds showed cytotoxic effects towards mammalian kidney fibroblasts. Results of this study indicate that the pyridinium compounds, some of which are already in use as antidotes, do not have significant antimicrobial and antiprotozoal activities and cannot be relied upon for additional protection in the event of combined chemical-biological incident.

Quaternary pyridinium bearing an oxime functional group are well known for their acetylcholinesterase reactivation activities and are used as antidotes in organophosphorus (OP) poisoning (1, 2). Examples of well known AChE reactivators are pralidoxime, HI-6, trimedoxime and obidoxime. In the event of combined chemical-biological incident, it is of prime importance to provide antidotes against both chemical as well as biological threats. Quaternary pyridinium oximes are widely used as antidotes for chemical (nerve agent) poisoning but the ability of these antidotes to provide additional support against biological threats is unknown. Thus, it is important to know whether pyridinium oxime antidote provides additional protection against biological threats besides their AChE reactivation ability. Therefore, herein our aim was to screen series of quaternary pyridinium oximes with known AChE reactivation ability for their ability to provide protection against biological threats.

Besides this main objective, literature reports on interesting antimicrobial and antiprotozoal activities of several quaternary pyridinium class of compounds (3) prompted us to evaluate pyridinium class of AChE reactivators for these activities. Quaternary salts exhibit antimicrobial properties by adsorption to the cell wall of microorganisms, and bind to other biochemical components causing disruption of cellular processes which leads to cessation of bacterial growth and thus cell death (3). Chemical structures and IC₅₀/ MIC/ MBC values of representative mono- and bis-quaternary pyridinium compounds with antimicrobial 1–9, (4–11) antimalarial 10–14 (12–16) and antileishmanial 15 (17) activities are shown in Figure 1 and Table 1 respectively.

Figure 1.

Structures of antimicrobial (1–6, 9), antimalarial (7, 8, 10–14) and antileishmanial (15) pyridinium salts / dications

Table 1.

Antimicrobial and antiprotozoal activities of reported pyridinium quaternary compounds

Entry	IC50/ EC50/ MIC/ MBC	Reference
1	MIC 9.8 (S. aureus), 39.1 (E. coli), 19.5 (C. albicans), and 9.8 (E. faecalis) µg/mL	(4)
2	MIC 1.4 (S. aureus), 11 (C. albicans), 6 (B. subtilis) and 22 (E. coli) µM	(5)
3	MBC 15.6 (S. aureus), 10.4 (E. coli), 16.3 (E. faecalis) µM	(6)
4 – 5	MBC 62.5 µg/mL (S. aureus, S. albus and MRS)	(7)
6	MIC 7 (S. aureus), 27 (E. coli), 13.5 (C. albicans) and 14 (B. subtilis) µM	(8)
7	IC50 1.5 µM (P. falciparum)	(9)
8	EC50 52 nM (FCR-3 - Chloroquine sensitive strain of P. falciparum)	(10)
9	MIC 1.1 (S. aureus), 1.4 (MRS), 1.4 (B. subtilis), 5.2 (E. coli) and 13.2 (P. aeruginosa) µM	(11)
10	IC50 0.5 nM (P. falciparum)	(12)
11	EC50 8 nM (P. falciparum)	(13)
12	IC50 0.65 nM (P. falciparum)	(14)
13	IC50 19 nM (P. falciparum)	(15, 30)
14	IC50 5.5 nM (P. falciparum)	(16, 30)
15	IC50 0.82 µM (L. donovani)	(17)

S. aureus: Staphylococcus aureus; E. coli: Escherichia coli; C. albicans: Candida albicans; E. faecalis: Enterococcus faecalis; M. catarrhalis: Moraxella catarrhalis; B. subtilis: Bacillus subtilis; P. aeruginosa: Pseudomonas aeruginosa; P. falciparum: Plasmodium falciparum; L. donovani: Leishmania donovani; MRS: Methicillin resistant Staphylococcus

Biosafety level 1 and 2 agents have been widely used as model organisms and recently Utrup and Frey (18) used several model organisms as surrogates of biological threat agents. These include Bacillus subtilis for B. anthracis (anthrax), E. coli for Variola major (Smallpox), Pseudomonas fluorescens for Burkholderia pseudomallei (melioidosis) (18). The present paper reports results of an initial screen of a series of quaternary pyridinium oximes (19, 20) against a number of lower pathogenicity BSL-1 and 2 model organisms. Use of model organisms in the first stage screening saves on the time and expense of BSL-3 studies, and allows for a determination of which compounds and BSL-3 organisms are worth further exploration.

Experimental section

General procedure for synthesis of quaternary pyridinium compounds

Hydroxyiminomethylpyridine (1 mmol) and the alkylating agent (1.5 mmol) in solvent (20 mL) were stirred at 60–80 °C for 1–24 h. The mixture was cooled to rt, the precipitate collected, washed with acetone (3 × 20 mL), dried under vacuum, and characterized by ¹H NMR, ¹³C NMR, ESI-MS and IR (19). Spectral data for representative compounds is as follows: Bromothiophen-2-yl)-methyl-2-hydroxyiminomethylpyridinium chloride (21): Brownish black viscous oil; yield: 74%; ¹H NMR (400 MHz, CD₃OD): δ 8.82 (t, J = 7.2 Hz, 1H), 8.64 (m, 1H), 8.44 (t, J = 12 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.09 (m, 1H), 7.19 (d, J = 4.0 Hz, 1H), 7.06 (d, J = 4.0 Hz, 1H), 6.28 (s, 2H); ¹³C NMR (100 MHz, CD₃OD): δ 147.4, 146.2, 142.0, 141.4, 141.0, 131.0, 128.0, 127.5, 126.9, 125.7, 56.4; ESI-MS: m/z 296.95 [M]⁺ (calcd for [C₁₁H₁₀BrN₂OS]⁺ 297.97); 1-(Benzothiophene-3-yl)-methyl-2-hydroxyiminomethylpyridinium chloride (24): Greenish black viscous oil; yield: 64%; ¹H NMR (400 MHz, CD₃OD): δ 8.96 (s, 1H), 8.67 (s, 1H), 8.51 (m, 2H), 8.04 (m, 1H), 7.92 (m, 1H), 7.85 (m, 1H), 7.51 (s, 1H), 7.44 (m, 2H), 6.30 (s, 2H); ¹³C NMR (100 MHz, CD₃OD): δ 148.6, 146.1, 145.9, 145.7, 143.1, 141.8, 140.9, 127.9, 127.7, 126.4, 125.5, 125.1, 123.1, 121.2, 56.3; ESI-MS: m/z 269.1 [M]⁺ calculated for [C₁₅H₁₃N₂OS]⁺.

Biological testing

Antimicrobial, antimalarial, antileishmanial activities and cytotoxicity testing were performed using the protocols as reported by Samoylenko et al (21).

Results and Discussion

Mono- (16–34) and bisquaternary (35–61) pyridinium compounds used in this study were synthesized using a general synthetic strategy as depicted in Figure 2 and were characterized by ¹H NMR, ¹³C NMR and ESIMS spectral data (19, 22). All synthesized compounds were screened for in vitro antimicrobial, antimalarial, antileishmanial and cytotoxic activities. Susceptibility testing for antifungal and antibacterial activity was done using a modified CLSI (formerly NCCLS) methods (21). Organisms include the fungi Candida albicans, C. glabrata, C. krusei, Aspergillus fumigates, Cryptococcus neoformans and the bacteria Staphylococcus aureus, methicillin resistant S. aureus (MRS), Pseudomonas aerugenosa, Escherichia coli. Ciprofloxacin (0.07 and 0.08 µg/ml for S. aureus and MRS) and Amphotericin B (IC₅₀ 0.76 µg/ml for Cryptococcus neoformans) were used as reference standards. Susceptibility of Mycobacterium intracellulare was done using the modified Alamar Blue procedure of Franzblau et al (23). Of all compounds tested, only monoquaternary pyridinium compounds 21 and 24 showed moderate antibacterial activity against S. aureus and methicillin-resistant S. aureus (MRS): compound 21: IC₅₀ 15.3 and 14.2 µg/mL; compound 24: IC₅₀ 12.2 and 17.7 µg/mL for S. aureus and MRS, respectively (Table 2). Both compounds 21 and 24 were inactive against all other test organisms. All other compounds showed no in vitro antifungal or antibacterial activity. Similar observations in the activity profile has been reported by Sidorchuk et al for quaternary quinolinium salts (7).

Figure 2.

General Scheme for synthesis of mono- (16–34) and bis- (35–61) quaternary pyridinium compounds (R₁ = CHNOH, CONH₂; R₂ = CHNOH; Position of R₁ or R₂: 2, 3 or 4; X = Br, Cl, OMs; R = CH₂CH₂Br, CH₂CH₂CH₂Br, heterocycles – thiophene, furan or isoxazole; A = O, CH₂, CH₂CH₂, heterocycles - thiophene-furan or isoxazole).

Table 2.

Antibacterial and antifungal activities of pyridinium oximes

		IC50 (MIC) µg/mL
Entry		Antibacterial			Antifungal
		S. aureus	MRS	M. intracellulare	C. neoformans	C. glabrata	A. fumigatus
21	R = 5-bromothiophen-2-yl, R1 = 2-CHNOH, X = Cl	15.27	14.19	NA	na	>20	na
24	R = benzothiophen-3-yl, R1 = 2-CHNOH, X = Cl	12.23 (20)	17.72 (20)	>20	na	na	na
25	R = benzothiophen-3-yl, R1 = 3-CHNOH, X = Cl	na	na	na	na	na	>20
33	R = 3-methyl-isoxazol-5-yl, R1 = 3-CHNOH, X = Br	na	na	na	>20	na	na
45	R1 = 4-CONH2, R2 = 2- CHNOH, A = CH2CH2, X = Br	na	na	na	>20	na	na
46	R1 = 4-CONH2, R2 = 3- CHNOH, A = CH2, X = Br	na	na	na	>20	na	na
50	R1 = R2 = 2-CHNOH, A = thiophen-2,5-yl, X = Cl	na	na	na	>20	na	na
51	R1 = R2 = 3-CHNOH, A = thiophen-2,5-yl, X = Cl	na	na	na	>20	na	na
52	R1 = R2 = 4-CHNOH, A = thiophen-2,5-yl, X = Cl	na	na	na	>20	na	na
53	R1 = 4-CONH2, R2 = 4- CHNOH, A = thiophen-2,5- yl, X = Cl	na	na	na	>20	na	na
58	R1 = R2 = 4-CONH2, A = furan-2,5-yl, X = Br	na	na	na	>20	na	na
60	R1 = R2 = 3-CHNOH, A = isoxazol,3,5-yl, X = Br	na	na	na	>20	na	na
61	R1 = R2 = 4-CHNOH, A = isoxazol,3,5-yl, X = Br	na	na	na	>20	na	na
Ciprofloxacin		0.1 (0.5)	0.09 (0.25)	0.39 (0.5)	nt	nt	nt
Amphotericin B		nt	nt	nt	1.28 (2.5)	0.7 (1.25)	0.98 (2.5)

IC₅₀, the concentration that affords 50% inhibition of bacterial/ fungal growth; na, not active; nt, not tested; MIC (Minimum inhibitory concentration) is the lowest concentration (µg/ml) that allows no detectable growth; *None of the compound was active against C. albicans, C. krusei, E. coli and P. aeruginosa (data not shown).

Although compounds 33, 45, 46, 50–53, 58, 60 and 61 showed some antifungal activity against Cryptococcus neoformans with IC₅₀ > 20 µg/mL, none of the compound showed significant antifungal activity against any of the organisms. In vitro antimalarial activity was evaluated against chloroquine sensitive (D6) and chloroquine resistant (W2) clones of P. falciparum. Determination of in vitro antimalarial activity was based on the determination of plasmodial LDH activity (24). Compounds were tested at three concentrations 4.76, 1.59 and 5.29 µg/mL. Chloroquine and artemisinin were used as reference standards. Monoquaternary pyridinium compound 17 showed antimalarial activities against both the clones of Plasmodium falciparum with the IC₅₀ of 3.7 (D6 clone) and 4.0 (W2 clone) µg/mL with selectivity index of more than 1.3 (Table 3). None of the other compounds were active.

Table 3.

Antimalarial and antileishmanial activities of pyridinium oximes

Entry		Antimalarial		Antileishmanial		Cytotoxicity
		P. falciparum		L. donovani		Vero cells
		D6 Clone	W2 Clone
		IC50 µg/mL		IC50 µg/mL	IC90 µg/mL	IC50 ng/mL
17	R = CH2CH2CH2Br, R1 = 4-CONH2, X = Br	3.7 (S.I. > 1.3)	4.0 (S.I. > 1.2)	na	na	nc
21	R = 5-bromothiophen-2-yl, R1 = 2-CHNOH, X = Cl	na	na	19	40	nc
24	R = benzothiophen-3-yl, R1 = 2-CHNOH, X = Cl	na	na	18	40	nc
Chloroquine		0.015	0.12	nt	nt	nt
Artemisinin		0.010	0.0065	nt	nt	nt
Pentamidine		nt	nt	1.2	5	nt
Amphotericin B		nt	nt	0.2	0.4	nt

IC₅₀, the concentration that affords 50% inhibition of plasmodial / leishmanial growth; IC₉₀, the concentration that affords 90% inhibition of leishmanial growth; na, not active; nt, not tested; nc, Not cytotoxic; S.I., selectivity index = IC₅₀ vero cells/IC₅₀ P. falciparum.

Antileishmanial activity against L. donovani promastigotes was determined by the Alamar Blue assay (25, 26). Monoquaternary pyridinium compounds bearing thiophene functionality 21 and 24 showed antileishmanial activity against L. donovani with IC₅₀ of 19 and 18 µg/mL, respectively (Table 3). None of the compounds were found to have any cytotoxic effects towards mammalian kidney fibroblasts (vero cells) (27).

Although a series of pyridinium compounds tested in the present study varied in structural features to allow for a critical study of structure-activity relationships, but this class of compound was almost devoid of any significant antimicrobial, antimalarial or antileishmanial properties. None of the compound demonstrated any activities comparable to standards used in the study. Mainly, bisquaternary pyridinium oximes displayed a complete lack of activity. Therefore, a detailed, meaningful analysis of structure-activity relationships was not possible. The only active compounds were two monoquaternary compounds 21 and 24 possessing a thiophene and banzothiophene linker chains with antibacterial and antileishmanial activities and another monoquaternary compound 17 with antimalarial activity. It is noteworthy to mention that compounds 21 and 24 which were already reported to possess AChE reactivation ability (19) also exhibited moderate antimicrobial and antileishmanial activities. In contrast, commercially used bisquaternary pyridinium antidotes trimedoxime (42) and obidoxime (37) with potent AChE reactivation abilities (19) were completely devoid of any antimicrobial or antiprotozoal activities. The major difference between reported antimicrobial / antimalarial quaternary pyridinium salts and compounds studied in this paper is that most of the compounds have long aliphatic chains (C₄–C₂₀) (Figure 1). This long aliphatic chain appears important for antimicrobial or antiprotozoal activity of quaternary salts. The poor or complete lack of activity of our compounds may be due to this difference. Thus, this work suggests that the aliphatic chain attached to pyridinium skeleton may be important for retaining antimicrobial/ antiprotozoal activity. Merely, the presence of pyridinium skeleton does not suffice requirements to inhibit microbes or parasites.

Trimedoxime, obidoxime, HI-6 and several other oximes are known to be tolerated in high doses in humans and have been used as warfare agents for years (28, 29). It was anticipated that the ability for safely achieving high serum concentrations of these compounds would allow for a significant antimicrobial effect against biological threats and might provide advantage in the event of a combined nerve agent/ biological-agent incident. Unfortunately, based on initial in-vitro studies in model organisms, the compounds appear to be tolerated as well by bacteria and protozoa as seen for mammals. Therefore, the utility of this class of compound does not extend to biological agents.

In conclusion, pyridinium oxime compounds which are widely used as antidotes for nerve gas poisoning, do not have ability to provide protection against microbial and protozoal infections. Results from this work provided important information about antidotes of OP poisoning that they cannot be relied upon for additional protection against biological threats in the event of combined chemical-biological incident.