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Current Pharmaceutical Design, 2013, 19, 1343-1355 1343

Antimycobacterial Activity of Quaternary Pyridinium Salts and Pyridinium N-oxides

- Review

Martin Krátka,b and Jarmila Vinováb,*

a
Department of Chemistry, Faculty of Science, University of Hradec Králové, Rokitanského 62, 500 03 Hradec Králové, Czech Re-
public; bDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05
Hradec Králové, Czech Republic

Abstract: The treatment of tuberculosis and other mycobacterioses is still a major world health problem and new antimycobacterial
compounds unrelated to approved drugs are in demand. Quaternary ammonium salts have revealed many usable properties especially as
antimicrobials; they are widely used as disinfection and antiseptic agents. Some of these compounds, including pyridinium salts, have re-
vealed substantial antimycobacterial action, although the presence of the cationic nitrogen itself is not sufficient for activity. A long N-
alkyl chain is also not necessary for antimycobacterial activity, although it is associated with improved activity.
Compounds that have shown significant in vitro activity, e.g., cetylpyridinium, N-(substituted alkyl)pyridinium salts, 3-[(5-
cyclopentylpentyl)(substituted phenyl)amino]-1-methylpyridinium iodides or pyridinium alkyl ethers of steroids (good activity with
minimum inhibitory concentrations – MIC from 0.4 g/mL). However, most pyridinium salts have mild or moderate activity against fast-
and/or slow-growing mycobacteria, including N-methylated isoniazids or pyridinium-based oximes. Moreover, a pyridinium ring is pre-
sent in some cefalosporines (e.g., cefaloridine, ceftazidime and cefsulodine) with antimycobacterial properties. The N-oxidation of pyri-
dine mostly resulted in retained or increased minimum inhibitory concentrations. Additionally, the action of pyridinium N-oxides against
mycobacteria is not especially robust.
The mechanism of action of pyridinium compounds remains elusive, but the inhibition of some mycobacterial enzymes has been de-
scribed for a few derivatives.

Keywords: Antimicrobial activity, mycobacteria, pyridinium N-oxides, pyridinium salts, quaternary ammonium salts, tuberculosis.

1. INTRODUCTION fluoroquinolones, thioamides, serine analogues and salicylic acid

Tuberculosis (TB), a contagious infectious disease caused by
Mycobacterium tuberculosis complex, has been responsible for
millions of human deaths. World Health Organization declared TB
a global public health problem in 1993 and it still represents a
worldwide threat to public health. About 1.4 million people died
from TB in 2010 [1]. Patients who have no history of prior TB
treatment are currently presumed to have drug-susceptible TB.
They typically receive a six-month regimen of two months daily
isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and etham-
butol (EMB), and then four months of INH and RIF combination
[2]. Resistance to any of these antimicrobial drugs creates many
possible difficulties.
Drug-resistant tuberculosis is not a recent phenomenon. M.
tuberculosis strains that were resistant to streptomycin, the first
effective anti-TB drug, appeared soon after its introduction into the
treatment of TB. The currently prevailing scenarios of drug-
resistant TB are particularly alarming and evoke a serious challenge
for the effective control of tuberculosis [3]. Multidrug-resistant
tuberculosis (MDR-TB) is defined as an infection that is resistant to
at least isoniazid and rifampicin, the most effective first-line oral
agents [4]. With the increasing rate of tuberculosis insusceptibility
to existing drugs, extensive drug-resistance (XDR) has emerged.
XDR-TB is MDR-TB in combination with both resistance to any
fluoroquinolone and at least one second-line injectable drug (e.g.,
kanamycin, amikacin or capreomycin) [5]. The situation has re-
cently become more serious with the development of new forms of
drug-resistant M. tuberculosis called totally drug-resistant (TDR)
tuberculosis. TDR-TB is defined as MDR strains that are resistant
to all second-line drug classes (i.e., aminoglycosides, polypeptides,
*Address correspondence to this author at the Heyrovského 1203, 500 05
Hradec Králové, the Czech Republic; Tel: +420-495067343;
Fax: +420-495067166; E-mail: vinsova@faf.cuni.cz
derivatives) [6]. Clearly there exists a strong need for new drugs to
treat and prevent mycobacterial infections [7-9]. The principal rea-
sons are their epidemiology, health and socio-economic seriousness
of drug-sensitive TB and an increasing emergence of drug-resistant
forms of tuberculosis and other mycobacterial infections, as well as
co-incidence of TB with human immunodeficiency virus (HIV)
infection.
Quaternary ammonium compounds (QAC; general formula
R4N+X-) belong to the large group of surface-active agents of cati-
onic compounds. They have two structural regions: a hydrophobic
hydrocarbon (aryl or longer alkyl chain) region, and a hydrophilic
or polar group, with QAC represented by a positively charged ni-
trogen [10].
QAC have been useful as antiseptics, disinfectants, detergents,
preservatives, surface cleaning and deodorization agents. They have
been purposed for a variety of medicinal and pharmaceutical, cos-
metic, food and agriculture, industrial and technical purposes. The
first evidence of antimicrobial activity in QAC was published in the
1910s, but their potential was not been exploited until the 1930s. In
1935, Domagk disclosed the significant antibacterial activity of at
least one long-chain containing QAC and then their synthesis and
evaluation boomed [10-12]. However, their widespread use brought
some risks, especially environmental load and resistance develop-
ment [12].
Pyridinium compounds, where three alkyls and/or hydrogens
are replaced by a six-membered aromatic ring with incorporated
nitrogen, represent an important class of organic compounds used
as biocides, cationic surfactants, drugs and herbicides. The formal
positive charge on the nitrogen atom of these compounds possesses
many unique properties that have been utilized in numerous appli-
cations [11] including medicine, the inhibition of corrosion, clean-
ing, emulsification, extractions, phase transfer catalysis, polymeri-
zation, purifications and flotation. A concise review of the synthesis

1873-4286/13 $58.00+.00 © 2013 Bentham Science Publishers

1344 Current Pharmaceutical Design, 2013, Vol. 19, No. 7
of various pyridinium compounds has been published [13]. Addi-
tionally, some antimycobacterial compounds contain a pyridine
ring, including the clinically relevant isoniazid and thioamides (e.g.,
ethionamide and prothionamide) but also some novel scaffolds [14-
16].
2. ANTIMYCOBACTERIAL ACTIVITY OF QAC – AN IN-
SIGHT
QAC have antimicrobial effects against a broad range of organ-
isms. At high concentrations, QACs are lethal to vegetative bacte-
ria, yeasts, molds, algae and lipophilic viruses, but not to bacterial
spores or hydrophilic viruses. A high microbial density and the
presence of organic materials reduce their efficacy, as antimicrobial
activity is primarily related to their cationic surfactant properties
[12]. However, their activity against mycobacteria is still a topic of
heated debate.
Mycobacteria are known to possess an intrinsic resistance to
many antimicrobial agents. The most likely and believed mecha-
nism of resistance is associated with their highly hydrophobic and
complex cell walls containing mycolic acids. QAC are mycobacte-
riostatic even when used at high concentrations, and their activity
can be substantially increased by formulation [10,12]. The possible
mechanisms of acquired resistance to quaternary ammonium com-
pounds may be found in decreased cell wall permeability or their
excretion by efflux pumps [17,18].
Some “non-pyridinium” quaternary ammonium salts have been
reported to inhibit mycobacterial strains including drug-resistant
ones (e.g., benzyl(dimethyl)tetradecylammonium fluoride [19],
benzyl(hexadecyl)dimethylammonium chloride, hexadimethrine
bromide, methylbenzethonium chloride, pyrvinium pamoate, thon-
zonium bromide and demecarium bromide [20]). On the other hand,
benzalkonium chloride had no activity against M. tuberculosis
H37Rv, M. avium and M. kansasii [21], as did other quaternary am-
monium salts against various mycobacterial species [22-25]. How-
ever, later study found activity for benzalkonium against M. avium
complex with minimum inhibitory concentrations (MIC) between 2
and 8 g/mL [26].
It has been known for many years that QAC are membrane
active drugs which target the cytoplasmic (inner) membrane
through (i) adsorption and penetration of the agent through the cell
wall; (ii) binding to the cytoplasmic membrane components result-
ing in membrane disintegration; (iii) leakage of intracellular small
molecules; (iv) degradation of proteins, enzymes and nucleic acids;
and finally (v) cell wall lysis due to the activity of released autolytic
enzymes. Additionally, QAC react with phospholipids to cause
general rather than specific membrane damage. This action involves
association of the positively charged quaternary nitrogen with the
head group of acidic phospholipids before integration of the hydro-
phobic tail into the hydrophobic membrane core. At high concentra-
tions, QAC release and solubilize hydrophobic cell membrane
components by forming mixed micellar aggregates. Additionally,
they were found to influence proton-motive force necessary for
ATP synthesis, resulting in the inhibition of membrane ATPase. For
Gram-negative bacteria, QAC are also believed to damage the outer
membrane, thus promoting their own uptake [10,12,27]. Based on
the analogy between cationic antimicrobial peptides and QAC, they
may also interact with lipid II, but this is still only a hypothesis
[27].
As noted previously, some QAC showed antimycobacterial
activity. The bisquaternary ammonium compound dequalinium
chloride (1,1’-(decane-1,10-diyl)bis(4-amino-2-methylquinolinium)
dichloride; (Fig. 1)) showed antimicrobial activity against Myco-
bacterium phlei (MIC of 1.66 g/mL) and M. tuberculosis growing
both aerobically and anaerobically with MICs of 1.2 and 0.3
g/mL, respectively, among others. In contrast to some QAC, the
mode of action of dequalinium seems to be more specific, despite
Krátk and Vinová
H2N
N+ N+
2 Cl- H3C NH2
CH3
Fig. (1). The structure of dequalinium chloride.
its seemingly non-specific antimicrobial properties. This drug is
taken up by the cells, but the cell wall is not the primary site of
absorption; the morphological abnormalities are mainly intracellu-
lar [27].
Dequalinium has been indicated to inhibit one mycobacterial
enzyme in particular, mycothiol ligase, which is a key enzyme in
the biosynthesis of mycothiol, whose primary role is to maintain
intracellular redox balance and to remove toxins. This enzyme is
essential for M. tuberculosis, thus representing an attractive target.
An IC50 value of 24 M with a Kd of 0.22 M was found for myco-
bacterial mycothiol ligase; the mode of action was ATP-
competitive in manner. Other quinolinium salts were also potent
inhibitors, but at higher concentrations. The inhibition zone for
M. smegmatis was 18 mm (at the presence of 50 g of de-
qualinium), slightly higher than the 15 g of RIF [28].
3. ANTIMYCOBACTERIAL ACTIVITY OF PYRIDINIUM
COMPOUNDS
3.1. Cetylpyridinium Salts
In 1953, 1-hexadecylpyridinium chloride (cetylpyridinium chlo-
ride, CPC; (Fig. 2a)) was reported to completely inhibit the growth
of M. tuberculosis at a dilution of 1:105 [29].
The cetylpyridinium salt of sulfathiazole (Fig. 2b), a well-
known and widely used antibacterial agent with antimycobacterial
properties [20,30], did not affect mycobacteria at dilutions greater
than 10-4. When the anion was derived from sulfapyridine (Fig. 2c),
complete cessation of mycobacterial viability was only observed at
a dilution of 1:105. Unfortunately, the combination of a pyridinium
ring with a sulfonamide avoided any additive or synergistic effect.
Rather, the results indicated decreased activity with respect to ce-
tylpyridinium chloride. High surface tension contributed to better
activity [31].
Furthermore, cetylpyridinium chloride diluted in the ratio 1:103
with 50% aqueous ethanol has been shown to be an effective ger-
micidal agent for the sterilization of objects contaminated with
virulent M. tuberculosis [32].
The activity of cetylpyridinium chloride (as well as chlorhexi-
dine) against M. avium and M. tuberculosis could be potentiated by
the presence of ethambutol, most likely due to increased cellular
penetration, which normally limits their efficacy. The MICs of
EMB and CPC were determined to be 5 and 25 – 50 g/mL for M.
avium, respectively. When 0.5 g/mL (but not 0.1 g/mL) EMB
(i.e., sub-inhibitory concentration) was added to the CPC, the MIC
of this salt fell to 10 g/mL. Growth was completely inhibited by
ethambutol (0.5 g/mL) with cetylpyridinium (10 g/mL) or by
ethambutol (0.1 g/mL) with cetylpyridinium at 50 g/mL, al-
though CPC alone was inhibitory at 50 g/mL. The MICs of CPC
towards Mycobacterium bovis BCG, Mycobacterium fortuitum and
M. phlei were 10 – 25, 25 – 50 and 5 – 10 g/mL, respectively.
These results suggested that arabinogalactan, whose biosynthesis is
targeted by ethambutol, is a cell wall component acting as a perme-
ability barrier for QAC [10,33]. A composition containing 1% 1-
hexadecylpyridinium chloride decreased the viability of M. fortui-
tum [34,35].
Antimycobacterial Activity of Quaternary Pyridinium Salts Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1345

2a: X = Cl

O
N- S
S
2b: X = O N

H3C N+ H2N

X- O
N- N
S
2c: X =
O
H2N

Fig. (2). The general structure of cetylpyridinium salts.

Unfortunately, increasing outbreaks of soft tissue infections

caused by non-tuberculous mycobacteria appear to be related to the
use of quaternary ammonium compounds. The frequency of sur-
vival after treatment with five disinfectants including CPC was < 1
in 105 bacteria for Mycobacterium smegmatis, but > 1 in 100 for M.
bovis BCG, M. massiliense, M. abscessus, M. fortuitum, M. che-
lonae and M. terrae. Although quaternary ammonium salts kill
most mycobacteria, survivors appear at high frequencies in M. che-
lonae and M. abscessus. Thus, this group of compounds selects a
non-genetically determined reversibly resistant phenotype within
several rapidly growing mycobacteria. In this assay, CPC reduced
mycobacterial growth starting at 0.0015 % [18].
Transportation of sputum samples to laboratories for the diag-
nosis of mycobacterial infections may take several days, which can
lead to overgrowth of non-mycobacterial bacterial and fungal flora
and to the possible loss of viable mycobacteria [36]. Cetylpyridi-
nium chloride has been proved to be a good preservative of M. tu-
berculosis during sputum collection, transport and storage with 71 –
98% preservation of viability [37]. The recovery of M. tuberculosis
from sputa treated with CPC and stored an average of 20 days was
significantly higher than that from sputa that were untreated. The
addition of CPC is therefore useful for isolation of M. tuberculosis
from sputa subjected to long-term storage. CPC increases the cul-
ture positivity and reduces contamination. However, some myco-
bacterial strains and species may be nonviable in the presence of
CPC or even CPC-susceptible [38]. A recent report suggested a
negative effect for CPC on mycobacterial detection rate and in-
creased time for mycobacterial growth readings [36], but conclu-
sions are still difficult to draw.
3.2. Other N-(Substituted alkyl)pyridinium Salts
The first reports of antimycobacterial activity in QAC indicated
that they were effective bacteriostatic agents against mycobacteria,
but with poor bactericidal properties. Bacteriostatic quaternary
ammonium compounds are generally effective at dilutions of 1:104
and 1:105, and only a few were found to be effective at 1:106 [29].
R1
N+
H3C R
N+ 2 X-
Fig. (3). The general formula for nicotinium salts.
1:105 in contrast to almost no activity for four related compounds
((Fig. 3), where R = R1 = methyl or benzyl, X = Br or R = H, R1 =
methyl or 4-chlorobenzyl, X = I and Cl, or R = R1 = 4-chloro-
benzyl, X = Cl). Interestingly, when benzyl derivatives were not
active, the introduction of chlorine into the p-position of the diam-
monium compound increased activity [29].
In the pyridinium salt series, 1-tetradecyl, 1-hexadecyl and 1-
[2-oxo-2-(tetradecyloxy)ethyl]pyridinium chlorides completely
abolished mycobacterial growth at a dilution of 1:105. Two ammo-
nium salts with substituted carboxamide moieties, 3-(diethyl-
carbamoyl)-N-hexadecylpyridinium chloride (Fig. 4) and 1-benzyl-
4-(dodecylcarbamoyl)pyridinium chloride (Fig. 5), were both found
to be completely inhibitory at 1:105, as was N-allyl-4-tridecyl-
pyridinium chloride (Fig. 6). Quinolinium and isoquinolinium hal-
ides showed lower activity, with only 2-dodecylisoquinolinium p-
toluenesulfonate completely inhibiting growth at 1:105 [29].
CH3
H3C N+ N CH3
Cl-
O
Fig. (4). 3-(Diethylcarbamoyl)-N-hexadecylpyridinium chloride.
O

A series of pyridinium, nicotinium, quinolinium and isoquino- H3C N Cl-

linium salts were investigated in terms of inhibition index in the H
N+
range from 0 to 10 with 10 indicating complete growth inhibition
and 0 indicating no inhibition. Many of the compounds tested gave
complete growth inhibition at a dilution of 1:105, but none were Fig. (5). 1-Benzyl-4-(dodecylcarbamoyl)pyridinium chloride.
completely inhibitory at 1:106. Clearly, this method for determining
antimycobacterial activity is not fully comparable with contempo- H3C
rary assays based on the exact MICs in mol/L or g/mL, although Cl-
it could quickly indicate activity. N+
In general, the inhibition of bacterial growth by quaternary CH2
ammonium compounds is enhanced by increasing the chain length
from C-6, reaching a maximum at C-16. For example, 1-dodecyl-3- Fig. (6). N-Allyl-4-tridecylpyridinium chloride.
(1-dodecyl-1-methylpyrrolidinium-2-yl)pyridinium dichloride
((Fig. 3); R = R1 = dodecyl, X = Cl) completely inhibited growth at In addition, 1-(2-oxo-2-{[2-(acyloxy)ethyl]amino}ethyl)pyridi-
nium chloride ((Fig. 7); R = acyl) showed almost complete inhibi-
1346 Current Pharmaceutical Design, 2013, Vol. 19, No. 7
tion of M. tuberculosis growth at 1:105 dilution, but not at 1:106
[31].
O 1a; R = H) displayed an IC50 of 0.55 g/mL and a MIC of 1.3
R O N+
N the phenyl ring retained or increased the antimycobacterial activity
H Cl-
O
Fig. (7). 1-(2-Oxo-2-{[2-(acyloxy)ethyl]amino}ethyl)pyridinium chloride.
Twenty-four 1-alkoxymethyl-3-(1-benzimidazolmethylamino)
pyridinium chlorides, N,N´-bis[3-(1-alkoxymethyl)pyridinium chlo-
ride]methylenediamines and 1-alkoxymethyl-3-[1-(benzotriazol-1-
yl)methylamino]pyridinium chlorides were assayed for antimicro-
bial activity against various strains of bacteria and fungi. They were
found to affect a wide spectrum of pathogens and that their activity
was greatly affected by alkyl chain length in the alkoxymethyl sub-
stituent and the type of quaternary ammonium in the molecule. The
preferred alkyl group contained from 9 to 12 carbon atoms. Pyridi-
nium salts exceeded the efficacy of benzimidazolium chlorides and
were comparable to benzalkonium chloride [11].
The three most active pyridinium salts against fungi, Gram-
positive and negative bacteria (3-[(1H-benzimidazol-1-ylmethyl)
amino]-1-(undecyloxymethyl)pyridinium chloride ((Fig. 8); X =
CH), 3-[(1H-benzotriazol-1-ylmethyl)amino]-1-(undecyloxy-
methyl)pyridinium chloride ((Fig. 8); X = N) and 3,3'-methyl-
enebis(azanediyl)bis[1-(decyloxymethyl)pyridinium] chloride (Fig.
9) were evaluated against M. tuberculosis H37Rv in the Tuberculo-
sis Antimicrobial Acquisition and Coordinating Facility (TAACF)
program. They revealed significant antimycobacterial activity with
MIC < 6.25 g/mL with more than 92% inhibition at this concen-
tration [11]. In another study of antimycobacterial activity reported
by De Souza et al. [39], these three molecules expressed MICs of
14 mol/L. Therefore, they have some potential for combating my-
cobacterial infections.
N trone blocked the growth of M. smegmatis, M. vaccae, M. aurum
X and M. fortuitum at concentrations of 0.4 – 25 g/mL. The most
N active derivatives in vitro contained an eight carbon bridge (0.4 –
HN
Cl-
N+
O terial agents than those derived from estrone, especially against M.
C11H23
Fig. (8). The structure of benzimidazole- and benzotriazole-pyridinium
chlorides.
2 Cl-
O N+ N+ O
C10H21 N N C10H21
H H
Fig. (9). 3,3'-Methylenebis(azanediyl)bis[1-(decyloxymethyl)
pyridinium] chloride.
It was reported that
-carbolines represent a pharmacophore for
anti-infective activity. Corresponding pyridinium salts as their ring-
opened analogues (Scheme 1) were tested along with
-carbolinium
compounds and exhibited almost equally high antibacterial activity,
especially against Gram-positive strains [40]. Carbolines (Scheme
1, general structure a) displayed a high activity against both fungi
Krátk and Vinová
(Candida sp., Aspergillus fumigatus) and bacteria including methi-
cillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium
intracellulare; however, this series was cytotoxic at concentrations
below 10 g/mL (Vero cells). Non-substituted carboline (Scheme
g/mL against M. intracellulare. The introduction of chlorine into
(for 6-Cl – MIC 0.6 g/mL). The presence of other lipophilic moie-
ties on position 8 did not improve antimycobacterial activity. The
potency of substituents decreased in the order: SCH3  CF3 > F >
CH3  OCH3 > CN. In general, IC50 values ranged from 0.5 to 2.1
g/mL and MICs from 0.6 to 2.5 g/mL. All substituted derivatives
showed lower cytotoxicity than the parent molecule [40].
Ring-opened analogues of
-carbolinium compounds, i.e., de-
rivatives of 3-[(5-cyclopentylpentyl)(phenyl)amino]-1-methyl-
pyridinium iodide (Scheme 1, general structure b), shared satisfac-
torily low cytotoxicity. Nevertheless, these pyridinium salts showed
less potent antimycobacterial activity with IC50 values of 0.6 – 3.3
g/mL and MICs of 1.3 – 25 g/mL. The presence of a substituent
on the p-position of the phenyl ring enhances the antimycobacterial
activity when compared with the m- and o-positions. The most ac-
tive was p-ethyl (IC50 of 0.5 g/mL, MIC of 1.3 g/mL), which was
closely followed by p-Cl, p-SCH3, p-CF3 and p-OCH3. Similarly to

-carbolines, the presence of a cyano group resulted in very poor
activity [40].
An interesting approach in the search for new antimicrobial
agents is the combination of a pyridinium ring with steroid frag-
ments. The advantage of employing these large hydrophobic units
is their ability to interact with cell membranes and thus pave the
way for biological activity of such hybrid molecules [41]. The en-
hanced membrane permeability may be particularly beneficial for
treating mycobacteria [42].
Water soluble -pyridinium alkyl ethers of estrone ((Fig. 10); n
= 4, 6, 8, 10) and 1-hydroxy-4-methylestra-1,3,5(10)-triene-17-one
((Fig. 11); n = 4, 6, 8, 10) were synthesized; the most active deriva-
tives exhibited low MICs towards MRSA, and vancomycin-
resistant Enterococcus faecalis. Pyridinium salts derived from es-
3.12 g/mL), followed by ten-, six- and then four- (12.5 – 25
g/mL) membered chains. Estrone alone showed weak antimyco-
bacterial activity of
100 g/mL. 1-Hydroxy-4-methylestra-
1,3,5(10)-triene-17-one, a synthetic steroid without any estrogenic
activity, displayed a weak activity against mycobacteria (
50
g/mL). Hybrids of this molecule were slightly better antimycobac-
smegmatis. An eight carbon chain was again the best for antimyco-
bacterial activity in this study with MICs ranging from 0.4 to 3.12
g/mL.
In general, M. aurum was the least sensitive strain and M. vac-
cae the most susceptible one. For comparison, cetylpyridinium
chloride and bromide inhibited the growth of mycobacteria at 0.8 –
6.25 g/mL. It was confirmed that the antimicrobial activity de-
CH3 O
Br-
O
N+
n
Fig. (10). Pyridinium-estrone conjugates.
Antimycobacterial Activity of Quaternary Pyridinium Salts
H3C I-
N+
8
R R
N N
7
6
a b
Scheme 1. The modification of
-carbolines (a) leading to the ring-opened analogues (b).
N+ Br-
CH3 O
n(H2C)
O
CH3
Fig. (11). Pyridinium and 1-hydroxy-4-methylestra-1,3,5(10)-triene-17-one
conjugates.
pended on the length of the alkyl linking chain; the activity of hy-
brid steroid pyridinium salts was significantly higher for eight- and
ten-membered alkyl chains, while the four-membered rings were
less active. Ciprofloxacin, a fluoroquinolone antibacterial agent,
was superior against M. aurum, four-times more efficient towards
M. vaccae and M. fortuitum and comparable for M. smegmatis
when compared with the most active hybrids.
The antiproliferative (on mouse fibroblast L-929 and human
leukemia K-562 cells) and cytotoxic (HeLa) effects of steroid hy-
brids also depended on the length of the alkyl group, but a higher
concentration was generally necessary. This was in contrast to ce-
tylpyridium salts, where the antimicrobial and cytotoxic concentra-
tions were similar [41].
Based on these results, additional hybrid estrone molecules
were synthesized. Various heterocycles were connected to estrone
by an n-heptyloxy bridge, with some containing a pyridinium frag-
ment. The presence of a charge on the molecule was found to be
important for all the studied biological properties (antibacterial
including MRSA and vancomycin-resistant E. faecalis, antimyco-
bacterial, antifungal, cytotoxic and antiproliferative). The condensa-
tion of the steroid structure with the heterocycles resulted in in-
creased activity, most likely due to the better permeability of the
heterocyclic core through biological barriers [43].
Estrone pyridinium and benzylsulfanyl pyridinium salts were
evaluated against four fast growing mycobacterial strains (M.
smegmatis, M. aurum, M. vaccae and M. fortuitum), as well as
against drug-sensitive M. tuberculosis and non-tuberculous myco-
bacteria (M. avium, two strains of M. kansasii including one clinical
isolate). Antibacterial screening revealed that these hybrid deriva-
tives were only moderately active. The estrone alone suppressed the
growth of the last four mycobacteria at 16 – 62.5 mol/L. The N-
pyridinium salt (Fig. 10; n = 7) affected the four fast growing my-
cobacteria with MICs from 3.12 to 6.25 g/mL and was also active
Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1347
H3C I-
N+
against M. tuberculosis at 16 – 32 mol/L and both strains of M.
kansasii. For comparison, MICs for M. avium reached 32 mol/L.
4-Benzylsulfanylpyridinium salts (Fig. 12; R = H, Cl, CH3)
exhibited uniform inhibition of M. vaccae, M. aurum, M. fortuitum
and M. smegmatis with MICs of 1.56, 3.12, 6.25 and 6.25 g/mL,
respectively. However, only one benzylsulfanyl molecule (Fig. 12;
R = H) blocked the growth of M. smegmatis at 1.56 g/mL. Its
MICs against M. tuberculosis ranged from 4 to 16 mol/L, while
M. avium was the most susceptible strain affected at 2 – 8 mol/L.
Meanwhile, the concentrations inhibiting M. kansasii ranged from 8
to 16 mol/L. This unsubstituted 4-benzylsulfanylpyridinium de-
rivative (Fig. 12; R = H) exhibited superior antimycobacterial activ-
ity with an MIC for M. tuberculosis of 4/8 mol/L after 14/21 days
of incubation, while those for M. avium and M. kansasii were 2
mol/L and 8/16 mol/L.
CH3 O
Br-
N+ O
S
R
Fig. (12). The general structure of estrone-benzylsulfanylpyridinium conju-
gates.
Cytotoxicity and antiproliferative assays were conducted
against HeLa cells and cell lines K-562 and L-929, respectively.
The antiproliferative activity of pyridinium salts ranged from 1.0 to
16.7 g/mL and the cytotoxicity ranged from 1.9 to 17.5 g/mL
(IC50) [43].
Isoniazid, pyridine-4-carboxylic acid hydrazide, represents a
key first-line oral antimycobacterial agent. Currently, it is widely
used in the treatment and chemoprophylaxis of mycobacterial infec-
tions. INH is bacteriostatic for resting mycobacteria and bacteri-
cidal for rapidly growing and dividing cells. The mode of action is
complex and not fully elucidated, but inhibition of mycolic acid
synthesis is considered to be a major target [44].
The pharmacophore moiety of isoniazid has been introduced
into a number of molecules to improve their activity against Myco-
bacterium species, as well as their drug-resistant strains. Many
Schiff bases, hydrazones, hydrazides, heterocycles, metal com-
plexes derived from INH and substituted and modified molecules
have been designed and evaluated. N-Quarternization may be a
simple such modification of INH. However, it was shown that this
modification was disadvantageous and decreased activity [44].
Additionally, a critical review was published in 2006 with the con-
1348 Current Pharmaceutical Design, 2013, Vol. 19, No. 7 Krátk and Vinová

H
HN X N
H H
13a: R = N 13d: R = N N
N N R1 N CH3
H
O O
H3C CH3
N+ S R2
H
I- 13b: R = N CH3 N R1
13e: R =
R N S
O N N
CH3
H S R1
O
N R1
13c: R = N 13f: R =
N
O H N N

Fig. (13). N-Methylated isoniazid analogues.

clusions that many so-called “novel leads” are only precursors of an

INH-based scaffold, and INH ring-substitution or analogous back-
R1 X-
bones never achieve the efficacy of INH [45].
N+ R
Foks et al. [46] synthesized among other compounds N-
methylpyridinium iodides of various INH derivatives (13a-f). When
examined against M. tuberculosis H37Rv and two wild strains (one Fig. (15). The general structure of monoquaternary pyridinium salts.
fully susceptible, another one resistant to INH, RIF, EMB and p-
aminosalicylic acid), the MICs for most of the compounds were
unsatisfactory (250 – 1000 g/mL). As mentioned above, this INH 2 X-
transformation led to almost complete loss of antituberculous activ- R1 R2
N+ A N+
ity.
The “doubled” molecule of thioisonicotinamide (pyridine-4-
carbothioamide) (Fig. 14) via a methylene bridge was found to have Fig. (16). Bisquaternary pyridinium salts.
weaker antituberculosis activity than the parent molecule [47].
S
HO N
S S
2 X-
H2N NH2
N+
N+ N+ Cl-

Fig. (14). 1,1'-Methylenebis(4-carbamothioylpyridin-1-ium).

Pyridinium-based oximes have been utilized as antidotes to
acetylcholinesterase inhibitors. Some of these structures were
evaluated in vitro for potential antimicrobial and antiprotozoal ac-
tivity. However, this compound panel had little or no antimicrobial
action except for thiophene and benzothiophene-substituted mono-
quaternary pyridinium compounds (Fig. 15; R = -bromoalkyl,
thiophen-2/3-yl, 5-bromothiophen-2-yl, benzothiophen-3-yl, 5-
chloromethylthiophen-2-yl, furan-2/3-yl, 3-methylisoxazol-5-yl; R 1
= 2/3/4-CHNOH, 4-CONH2; X = Cl, Br), which showed moderate
antibacterial activity against S. aureus and MRSA, as well as an-
tileishmanial activity. None of the bisquaternary pyridinium salts
((Fig. 16); R1 = R2 = 2/3/4-CHNOH, 4-CONH2; A = O, CH2,
CH2CH2, thiophen-2,5-diyl, furan-2,5-diyl, isoxazol-3,5-diyl; X =
Cl, Br, methanesulfonate) showed antimicrobial or antiprotozoal
properties. Only one of the tested derivatives (Fig. 17) was active
against M. intracellulare with IC50 > 20 g/mL and without signifi-
cant cytotoxicity. These results supported the aliphatic chain at-
tached to the pyridinium skeleton as being important for antimicro-
bial activity (i.e., the presence of only a pyridinium ring is not suf-
ficient to inhibit pathogens) [48].
3/4-(N'-Hydroxycarbamimidoyl)-1-methylpyridinium iodides
(Fig. 18) showed mild antimycobacterial activity towards M. tuber-
culosis H37Rv, as well as two clinically isolated strains with MICs
above 25 g/mL [49].
Fig. (17). (Z)-1-(Benzo[b]thiophen-3-ylmethyl)-2-[(hydroxyimino)methyl]
pyridin-1-ium chloride.
CH3
N+ I-
N
OH
NH2
Fig. (18). 3/4-(N'-Hydroxycarbamimidoyl)-1-methylpyridinium iodides.
Eight chiral pyridinium salts ((Fig. 19a); R1 = H, CH3,
CH2CH3; R2 = CH3, CH2OH; Ar = phenyl, 2-chlorophenyl; X = Cl,
dodecyl sulfate), including three Zincke’s salts ((Fig. 19b); R1 = H,
CH3, CH2CH3; X = Cl) as their synthetic precursors, were synthe-
sized and assayed against M. tuberculosis H37Rv. 3-Methylated and
3-ethylated Zincke´s salts exhibited very mild activity with MICs of
807 and 845 mol/L, respectively. Unfortunately, 1-[(1R)-2-
hydroxy-1-phenylethyl]pyridinium chloride ((Fig. 19a); R1 = H, R 2
= CH2OH, Ar = phenyl, X = Cl) had an MIC of 1000 mol/L, while
the other tested compounds were even higher; their antimycobacte-
rial activity is trifling [39].
Antimycobacterial Activity of Quaternary Pyridinium Salts Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1349

One first-generation cefalosporine drug, cefaloridine, contains a

R2
19a: R =
Ar
H three of them with IC50 values in the range 0.4 – 1.2 g/mL, making
R1
N+
19b: R =
R X-
O2N
Fig. (19). Zinckes (b) and chiral (a) pyridinium salts.
Similarly to alkylpyridinium derivatives, 1-alkoxymethyl-3-
carbamoylpyridinium salts possess antimicrobial activity against
cocci, rods, fungi and bacillus. As observed for other quaternary
ammonium halides, the activity depends on the length of the sub-
stituent; the most favorable alkoxymethyl group in 1-alkoxy-
methyl(carbamoyl)pyridinium compounds contained 10 – 12 car-
bon atoms. Additionally, the antimicrobial potency depended on the
anionic moiety, as acetate and complex metal counteranions like
MgCl42-, CoCl42-, ZnCl42- or FeCl4- enhanced the activity of the
investigated compounds. Twenty-four derivatives from this study
were screened against M. tuberculosis H37Rv, but only three salts –
(Fig. 20a) (n = 1; X- = CH3COO-; R = C12H25), 20b (n = 2; X- =
MgCl42-; R = C10H21) and 21 – were active with MICs > 6.25
g/mL and 40% inhibition at this dilution [50].
n H2N N+ O inhibited M. smegmatis. In the same assay, its CuCl2 hydrate com-
R
X-
O idly growing mycobacteria (M. abscessus, M. smegmatis, M. che-
Fig. (20). 3-Carbamoyl-1-[(alkyloxy)methyl]pyridin-1-ium salts.
H 30 g/mL, while the resistance rates for other cefalosporines were
O O N N+
C9H19
Cl-
O
Fig. (21). 3-({[(Nonyloxy)methoxy]methyl}carbamoyl)-1-[(nonyloxy)
methyl]pyridin-1-ium chloride.
3.3. Cefalosporines Containing a Pyridinium Ring
The role of beta-lactam antibiotics in the treatment of infections
caused by both drug-sensitive and drug-resistant strains of M. tu-
berculosis still remains controversial. This group of compounds has
traditionally been regarded as being ineffective against mycobacte-
ria due to the presence of beta-lactamases and a highly lipophilic
cell wall, although some positive clinical and in vitro results have
been reported [7,8].
pyridinium ring. Four major penicillin-binding proteins in M. tu-
berculosis H37Ra (similarly as in M. avium-intracellulare and M.
fortuitum) have been described. Cefaloridine (Fig. 22a) inhibited
it more active than cefalothin and worse than ceftriaxone. Cefalo-
ridine penetrates the mycobacterial cell wall very well despite its
NO2
relatively high hydrophobicity. Thus, Mycobacterium should be
affected at clinically achievable concentrations of this compound.
Cefaloridine expressed variable activities with MICs
8 g/ml
against M. tuberculosis H37Ra, H37Rv and five clinical isolates,
which were less susceptible. Additionally, this antibiotic is rapidly
hydrolyzed by M. tuberculosis beta-lactamases [51].
Cefaloridine was reported to affect the growth of M. smegmatis
at 16 g/mL. Meanwhile, the strain lacking the major porin MspA
was affected at 128 g/mL, indicating its role for the permeability
and antimycobacterial action of hydrophilic compounds [52]. In
another assay, MICs against four M. avium strains were
64
mol/L. In the same assay, the MIC of ceftazidime was higher than
800 g/mL [53].
A study from 1984 did not find any activity for the cefalospori-
nes cefaloridine and cefsulodine (Fig. 22b) towards M. fortuitum at
100 g/mL [54]. However, the third generation drug cefsulodine
did cause 45% inhibition of colony formation of Mycobacterium
terrae [55].
Ceftazidime (Fig. 22c) represents another cefalosporine con-
taining a pyridinium ring. It is a third-generation cefalosporine
antibiotic with broad-spectrum activity against bacteria including
Pseudomonas aeruginosa. In the disc diffusion assay, ceftazidime
plex was less active but still significant [56]. A wide range of rap-
lonae, M. immunogenum, M. farcinogenes, and M. fortuitum bio-
variant complex III) was evaluated for sensitivity to clinically used
antimicrobial drugs including ceftazidime. Nevertheless, approxi-
mately 76 % of these isolates were resistant to ceftazidime at
O
C9H19 75 – 87 % [57]. Ceftazidime avoids inactivation by chromosomal A
beta-lactamase in M. fortuitum [58]; the tolerance to this drug can-
not be explained on the basis of its rapid enzymatic hydrolysis at
least for a part of mycobacterial strains.
Ceftazidime was previously reported to not affect the growth of
three strains of M. chelonae and eight strains of M. fortuitum [59].
In another assay, ceftazidime inhibited M. smegmatis with an MIC
of 0.015625 g/mL, while in combination with sulbactame, a clini-
cally used beta-lactamase inhibitor, the MIC decreased to
0.0078125 g/mL [60]. The MIC for M. tuberculosis H37Ra was
128 g/mL, while it decreased to 8 g/mL when coadministered
with clavulanic acid (32 g/mL) [61].
As observed, the antimycobacterial activity of cefalosporines is
inconsistent. Additionally, there is no sharp correlation between an
affinity to penicillin-binding proteins and antimycobacterial activ-

S
22a (cefaloridine): R1 = R2 = H; Ar =

R2 H H H
S R1
N 22b (cefsulodine): R1 = CONH2; R2 = SO3Na; Ar = phenyl
O
Ar N N+ N NH2
O O
O 22b (ceftazidime): R1 = H; R2 = N OH; Ar =
S
O O- H3C CH3

Fig. (22). Pyridinium-scaffold based antimycobacterial cefalosporines.

1350 Current Pharmaceutical Design, 2013, Vol. 19, No. 7 Krátk and Vinová

ity. Nonetheless, the inhibition of mycobacteria does not represent a a pyridine moiety in the 2-position were synthesized. Three deriva-
unique property of pyridinium ring-containing cefalosporines and tives contained pyridinium N-oxide functionality (Fig. 25a-c). All
these agents are not the most promising drugs. compounds were examined for their activity against M. tuberculosis
H37Rv and two wild strains isolated from patients: strain 210 was
3.4. Pyridinium N-Oxides resistant to p-aminosalicylic acid, INH, EMB and RIF, whereas
Oxidation of pyridine nitrogen to form N-oxides (pyridine 1- strain 192 was fully susceptible. MICs for all tested compounds
oxides), where the nitrogen possess a formal positive charge and the were within 50 – 100
g/mL, which indicated low antituberculosis
oxygen a negative, may provide derivatives with interesting bio- activity despite the presence of an N-oxide [66].
logical activity, as in the case of quinoxaline-1,4-dioxides, highly
efficient antituberculosis agents [62,63]. However, this is not a S
25a: R = CH3
general rule; in some cases this modification has not improved or
even hampered the pharmacological properties of the parent com- -O O
pounds. N+ N N
25b: R = S
For antimycobacterial agents, N-oxidation reduced the activity R CH3
N S
of pyridine-2-carboxamides, pyridine-2-carbothioamides, nicotina- H
mides and isonicotinamides, as well as 4-pyridylthioureas [47].
However, one pyridinium N-oxide (pyridine 1-oxide) (Fig. 23) was 25c: R = N N
described to have antimycobacterial properties with MIC95 values
against M. bovis and M. tuberculosis of 12.5 and 5 g/mL, respec-
tively. However, this molecule exhibited significant cytotoxicity
(IC50 for Vero cells 5.6 g/mL) and did not improve the activity of Fig. (25). Derivatives of 4-[(1,3,4-thiadiazol-2-yl)amino]pyridine 1-oxide.
the parent pyridine compound [64].
Similar compounds (Fig. 26a-c) expressed low antimycobacte-
CH3 rial activity against the same three strains with MICs in the range
CH3 50 – 500 g/mL [67].

O
S S
26a: R = CH3
N+ O CH3 -O N N
-O
CH3 N+ H
N
S
R 26b: R =
N NH
Fig. (23). 3-(4,5,7,7-Tetramethyl-8,9-dihydro-7H-furo[3,2-f]chromen-2-
yl)pyridine 1-oxide. S
N CH3
2-[(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio]pyridine 1-oxide 26c: R = N S
H
(Fig. 24), also an N-oxide, was reported to disrupt the function of NH2
adenosine 5-phosphosulfate reductase, an enzyme essential for the
bacterial reduction of sulfate. Moreover, this biomolecule is re-
Fig. (26). 4-Substituted pyridinium N-oxides.
quired for survival in latent TB infection and not present in humans;
thus representing a promising target. This lead compound was iden-
tified by docking and assaying compounds from similarity searches A study was recently published where the N-oxidation of pyri-
of the first-generation inhibitors. It blocked the enzyme with a dis- dine nitrogen led to derivative with no in vitro activity [68].
sociation constant of 19.51 M, but any whole cell assays have not A cyclic nitrogenous isostere of INH, 2-[5-(pyridin-4-yl)-1H-1,2,4-
yet been published [65]. triazol-3-yl]pyridine, exhibited moderate MICs for M. tuberculosis
H37Rv and M. intracellulare (4 and 8 g/mL, respectively), but its
N-oxide (Fig. 27) lacked activity up to 32 g/mL. MICs for both
NO2 derivatives against M. avium and Mycobacterium lufu also ex-
ceeded 32 g/mL.
N
O
N N
O-
S N+ N

N+ NH
N
O-

Fig. (24). Pyridinium N-oxide inhibitor of adenosine 5’-phosphosulfate
reductase
Many 1,3,4-thiadiazole derivatives show various pharmacologi-
cal activities including antimycobacterial, and some agents possess-
ing a pyrazine or pyridine ring are established antituberculosis
drugs, so a series of 1,3,4-thiadiazoles substituted with pyrazine or
Fig. (27). 2-[5-(Pyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine 1-oxide.
4-[Chloro(hydroxyimino)methyl]pyridine 1-oxide (Fig. 28a), 4-
(N'-hydroxycarbamimidoyl)pyridine 1-oxide (Fig. 28b), and 4-
{[hydroxyimino][4-(substituted)piperazin-1-yl]methyl}pyridine 1-
oxides (Fig. 28c; R1 = phenyl, 4-fluorophenyl, benzyl or piperonyl)
were evaluated as weak inhibitors of three mycobacterial strains
with MICs > 25 g/mL [49].
Antimycobacterial Activity of Quaternary Pyridinium Salts Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1351

-O conjugate amidase activity in M. tuberculosis and M. smegmatis

N+ 28a: R = Cl with excellent IC50 values of 0.1 M for both [70].
N 28b: R = NH2 Two pyridinium N-oxide analogues of ircinol showed mild
OH antimycobacterial potency. The aliphatic portion of this alkaloid
R 28c: R = N N R1 appeared to be the key pharmacophore for antimycobacterial and
antimalarial activity in vitro with the -carboline also contributing
to the activity in vivo. One derivative (Fig. 30) had a measured MIC
against M. tuberculosis H37Rv of 27.6 g/mL, whereas its isomer
Fig. (28). Functional derivatives of 4-[hydroxy(hydroxyimino)methyl]
derived from 3-carboxypyridine 1-oxide had an MIC of only
pyridine 1-oxide.
128.0 g/mL. These compounds were also cytotoxic and displayed
3.5. Natural Pyridinium and Pyridinium N-oxide Compounds anti-HIV and antiprotozoal properties [71].
and Their Derivatives
Novel biologically active structures may also be isolated from
natural sources. Cyclic pyridinium-containing cyclostellettamines A
– F were isolated from the marine sponge Pachychalina sp. Cyclos- N+
tellettamines G – I, K and L were obtained by total synthesis (Table Cl-
1). All of these were evaluated for antimicrobial activity against
susceptible and drug-resistant Staphylococci and P. aeruginosa, as
8
well as Escherichia coli, Candida albicans and M. tuberculosis
H37Rv (Table 1). Cyclostellettamines displayed multiple antimicro- N+
bial activities that varied with alkyl chain size, suggesting depend- Cl-
ence of activity on the distance between pyridinium moieties. Cy-
clostellettamines B and C (in general the most active derivatives), Fig. (29). The structural motif of 1,3-pyridinium inhibitors of mycothiol-S-
were significantly antimycobacterial active with MICs of conjugate amidase.
4.0 g/mL. Other derivatives reached MICs from 4.6 up to 32
g/mL. For M. tuberculosis, cyclostellettamines with longer alkyl
linkers connecting two pyridinium rings chains were suggested to O-
be less active than compounds with shorter alkyl chains; cyclostel- N+
lettamine A was an exception [69]. O
Table 1. Cyclostellettamines A – L and their Antimycobacte-
rial Properties. O
H

y OH
N+ N
N+
N H
H
x

x y MIC for M. tuberculosis H37Rv [g/mL]

A 3 3 32 Fig. (30). Antimycobacterial ircinol derivative.

B 3 4 4.0 Three groups of pyridinium N-oxide alkaloids possessing a

C 4 4 4.0 disulfide moiety were isolated from Allium stipitatum (2-(methyl-
disulfanyl)pyridinium 1-oxide (Fig. 31a), 2-{[(methylsulfanyl)
D 3 5 8.0 methyl]disulfanyl}pyridine 1-oxide (Fig. 31b) and 2,2-disulfane-
E 4 5 11.0 diylbis(pyridine 1-oxide) (Fig. 31c)). They were evaluated against
Staphylococci including drug-resistant strains with MICs
2
F 5 5 8.0 g/mL. These compounds were also evaluated against fast-growing
G 1 3 4.6 mycobacterial species M. fortuitum, M. smegmatis and M. phlei, as
well as M. tuberculosis H37Rv and M. bovis BCG. MICs for non-
I 2 3 9.3 tuberculous species ranged from 2 to 16 g/mL with 31b being the
most potent (2 – 8 g/mL). M. phlei was the most susceptible
J 1 4 6.6
strain. It seemed that the disulfide bridge was not the only factor
K 1 5 5.3 responsible for activity, but it should be “activated” by the presence
of electron-withdrawing functional groups such as pyridine, 1-
L 2 5 4.6
oxidopyridin-2-yl, pyrimidine and quinoline, but not by phenyl or
thiophene [72].
Mixture of 1,3-pyridinium polymers from the marine sponge Compound 31a was bactericidal against non-replicating M.
Amphimedon sp. represented the most potent inhibitors of my- tuberculosis cells at a concentration of 1.25
g/mL and the growth
cothiol-S-conjugate amidase from thirteen investigated natural was stopped at 0.1 g/mL. This molecule did not affect protein
products. In mycobacteria, this enzyme plays an important role in biosynthesis, but when replicating M. smegmatis were treated with
the detoxification of some xenobiotics; thus indicating a promising 30
g/mL of this compound, complete inhibition of acetate incorpo-
drug target. It was reported that these compounds with a 1,3- ration into soluble lipids was reported. This result indicated inhibi-
pyridinium structural fragment (Fig. 29) affected mycothiol-S-
1352 Current Pharmaceutical Design, 2013, Vol. 19, No. 7
a: R = CH3
O-
CH3
b: R = S
N+ S R
S lipophilicity resulting from the presence of the cyanoborane moiety.
c: R =
N+
-O
Fig. (31). N-Oxide moiety containing disulfides from Allium stipitatum.
tion of fatty acid biosynthesis, likely through FAS I. Additionally,
the molecule possibly generated damaged proteins and other oxida-
tive stress signals as part of its mechanism of action. This com-
pound was also evaluated in vivo, including for efficacy in a mouse
model of TB infection. Acute adverse effects were observed at
doses
100 mg/kg. Regardless, derivative 31a was inactive in mice
models at 30 mg/kg by oral administration by not effectively reduc-
ing bacterial numbers in the lungs and spleens. It is possible that
compound 31a, as an N-oxide, is rapidly excreted.
Both compounds 31a and 31b showed significant activity in
antiproliferative assays using breast carcinoma and lung carcinoma
cancer cells, as well as human colon adenocarcinoma cells [72].
3.6. Other Pyridinium Derivatives
The adducts of isoniazid derivatives with cyanoborane (Fig. 32;
R = H, CH3; R1 = phenyl, 2/3/4-fluorophenyl, 3-(trifluoromethyl)
phenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 3,4-dimethoxy-
phenyl or trifluoromethyl) constituting a quaternary pyridine nitro-
gen displayed moderate or mild activity against M. tuberculosis
H37Rv. The coordination of INH derivatives to a cyanoborane gen-
erally brought remarkably decreased antimycobacterial activity,
while cytotoxicity increased. However, it was expected that this
lipophilic group could potentially facilitate membrane permeability
or prolong biological activity. Six cyanoboranes ((Fig. 32); R =
CH3; R1 = 2/3/4-fluorophenyl, 3-(trifluoromethyl)phenyl, 2,4-
dichlorophenyl and 3,4-dimethoxyphenyl) from a total of fourteen
were inactive with MICs > 12.5 g/mL. At 12.5 g/mL, other ad-
ducts showed 93 – 98% inhibition with MIC90 ranged from 0.39 to
12.5 g/mL, and selectivity indexes of 0.8 to 122. The presence of
the BH2CN moiety appeared to be detrimental, although useful
when searching for antiproliferative agents [73].
N rivatives with 2/3/4-chloro- or 2-bromophenyl were the least effec-
H tive, while methylated, methoxylated or pentafluorinated ones
B-
H showed the best IC50 values (for R = 3-OCH3 had the lowest IC50 =
N+
H pyridinium motif (seven-fold increased activity for the basal sul-
N R
O N
H
R1
Fig. (32). The general structure of cyanoborane - isoniazid derivatives
adducts
In general, when R = H for this group of compounds, the activ-
ity was higher than for R = methyl. Only one compound ((Fig. 32);
R = H, R1 = CF3) showed a good activity profile with MIC and IC50
(Vero cells) of 0.78 and 95 g/mL, respectively, followed by an-
other derivative ((Fig. 32); R = H, R1 = phenyl) with MIC of 0.8
g/mL and cytotoxicity > 10 g/mL [73]. These two adducts/salts
were selected for further tests, including killing of M. tuberculosis
Erdman in monolayers of mouse bone marrow macrophages. Inter-
estingly, the second derivative was superior with an EC90 of 0.116
g/mL, much lower than the corresponding MIC against the H37Rv
Krátk and Vinová
strain and the Erdman strain in culture medium (12.5 g/mL). It
was thus clear this compound was active against both intracellular
and extracellular M. tuberculosis and even more efficient for bacilli
growing within macrophages. This activity might be related to its
From this point of view, this modification appeared to be beneficial
for antimycobacterial activity in infected cells over culture medium.
When evaluated against single drug-resistant (INH, RIF, EMB,
ethionamide, thioacetazone) mycobacterial strains, EMB- and RIF-
resistant strains were affected at concentrations from 6.25 to
12.5 g/mL, while the others were insensitive at 25 g/L. These
results indicated that the mechanism of action was bactericidal in a
similar manner to INH. M. avium was completely resistant to men-
tioned cyanoborane adducts ((Fig. 32); R = H, R1 = phenyl and
CF3) at the concentration of 6.25 g/mL [73,74].
A series of 2-(hydrazinecarbonyl)-3-phenyl-1H-indole-5-sulfo-
namides were modified to generate pyridinium derivatives ((Fig.
33); R = H, 2/3/4-methyl/halogen/methoxy-, 2,3,4,5,6-penta-fluoro)
and evaluated against two -carbonic anhydrases Rv1284 and
Rv3273 from M. tuberculosis. The whole series showed excellent
nanomolar inhibitory activity, with several sub-nanomolar inhibi-
tors being detected [75].
H3C
H
N HN N+ CH3
H2NO2S O H3C
ClO4-
R
Fig. (33). Pyridinium-based sulfonamide inhibitors of
-carbonic anhy-
drases.
Rv1284 and Rv3273 have potential for screening for antimyco-
bacterial agents with an alternative mechanism of action. These
enzymes catalyze CO2 hydration to bicarbonate and a proton and
are essential for the growth and virulence of M. tuberculosis. Inhi-
bition constants of pyridinium compounds for these two enzymes
ranged from 0.88 up to 35.3 nM, while two human isoforms were
less sensitive (0.93 – 3389 nM). Against the Rv1284 isoform, de-
0.92 nM). This excellent activity was not only the function of the
sulfonamide core (IC50 of the 2-(hydrazinylcarbonyl)-3-phenyl-1H-
indole-5-sulfonamide was 48 nM) but also the 2,4,6-trimethyl-
fonamide, and more than fifty-fold for the most active derivatives).
The nature of the substitution on the 3-phenyl ring also strongly
influenced inhibitory activity. Similar positive influences of the
pyridinium ring were observed for the isoenzyme Rv3273 with K i
values in the range of 0.88 – 31 nM; 4-chlorine, 2/3/4-bromine and
five (2,3,4,5,6-) fluorines were optimal substitution patterns for the
phenyl ring (R in the Fig. 33) [75].
CONCLUSION
The search for new antimycobacterial drugs is critical due to
deficiencies in current antituberculosis therapy, the need for con-
comitant therapy of TB-HIV infections and the growing prevalence
of drug-resistance. Compounds with no structure similarity and no
cross-resistance with conventionally used drugs are particularly
needed.
Antimycobacterial Activity of Quaternary Pyridinium Salts
Quaternary ammonium compounds, including pyridinium salts,
have demonstrated a wide range of interesting biological activities
such as antimicrobial activity against various fast- and slow-
growing mycobacterial species. However, quaternary ammonium
salts are considered to be largely inactive against mycobacteria.
Their mechanism of action is likely through mainly non-specific
interactions with cytoplasmic membranes, but some specific effects
have been described. For example, heterocyclic QAC has been
Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1353
culosis: Causes, Diagnosis and Treatments. New York: Nova Sci-
ence Publishers 2009; pp. 59-141.
[8] Koul A, Arnoult E, Lounis N, Guillemont J, Andries K. The chal-
lenge of new drug discovery for tuberculosis. Nature 2011; 469:
483-90.
[9] Speck-Planche A, Scotti MT, de Paulo-Emerenciano V. Current
Pharmaceutical Design of Antituberculosis Drugs: Future Perspec-
tives. Curr Pharm Des 2010; 16: 2656-65.
[10] McDonnell G, Russell, AD. Antiseptics and Disinfectants: Activ-
reported to affect mycobacterial mycothiol ligase, -carbonic anhy-
drases and adenosine 5-phosphosulfate reductase.
Some pyridinium salts and related compounds have revealed
promising antimycobacterial properties, but the presence of a cati-
onic heterocyclic nitrogen is not solely sufficient for activity. In
some cases, generation of the quaternary nitrogen is detrimental
(e.g., the introduction of a small alkyl onto isoniazid derivatives
significantly attenuated antimycobacterial activity). Additionally, a
range of pyridinium compounds was evaluated against mycobacte-
ria in the 1950s, but these compounds should be re-evaluated to
make sure no important compounds were missed. Similarly, a wide
range of newly synthesized quaternary ammonium compounds
designed as potential antibacterials have not been evaluated for
antimycobacterial action. This is most likely the result of the mis-
conception that mycobacteria are universally resistant to quaternary
ammonium compounds.
Nonetheless, the design, synthesis and evaluation of new com-
pounds based on quaternary ammonium salts including pyridinium
can generate derivatives with promising in vitro antimycobacterial
activity at least, and may be potentially useful for suppressing my-
cobacterial spread.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
The work on this thesis was financially supported by IGA NT
13346 (2012).
This publication is a result of the project implementation:
“Support of establishment, development, and mobility of quality
research teams at the Charles University“, project number CZ.1.07/
2.3.00/30.0022, supported by The Education for Competitiveness
Operational Programme (ECOP) and co-financed by the European
Social Fund and the state budget of the Czech Republic.
The assistance of Jaroslava Urbanová for language help is
gratefully acknowledged.
REFERENCES
[1] 2011/2012 TUBERCULOSIS GLOBAL FACTS [homepage on the
Internet]. World Health Organization (WHO) [cited 2012 Jun 20].
Available from: http://www.who.int/tb/publications/2011/fact-
sheet_tb_2011.pdf
[2] Treatment of tuberculosis Guidelines Fourth edition [homepage on
the Internet]. World Health Organization (WHO) [cited 2012 Jun
20]. Available from: http://whqlibdoc.who.int/publications/
2010/9789241547833_eng.pdf
[3] Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacte-
rium tuberculosis. Int J Tuberc Lung Dis 2009; 13: 1320-30.
[4] Laurenzo D, Mousa SA. Mechanisms of drug resistance in Myco-
bacterium tuberculosis and current status of rapid molecular diag-
nostic testing. Acta Trop 2011; 119: 5-10.
[5] Caminero JA. Extensively drug-resistant tuberculosis: is its defini-
tion correct? Eur Respir J 2008; 32: 1413-5.
[6] Velayati AA, Masjedi MR, Farnia P, et al. Emergence of New
Forms of Totally Drug-Resistant Tuberculosis Bacilli: Super Ex-
tensively Drug-Resistant Tuberculosis or Totally Drug-Resistant
Strains in Iran. Chest 2009; 136: 420-5.
[7] Vin
ová J, Krátk M. Tuberculosis — The Development of New [30] Kratky M, Vinsova J, Volkova M, Buchta V, Trejtnar F, Sto-
MDR-TB Drugs. In: Nguy S, Kung Z. Eds. Drug-Resistant Tuber-
ity, Action, and Resistance. Clin Microbiol Rev 1999; 12: 147-79.
[11] Pernak J, Rogoza J, Mirska I. Synthesis and antimicrobial activities
of new pyridinium and benzimidazolium chlorides. Eur J Med
Chem 2001; 36: 313-20.
[12] Hegstad K, Langsrud S, Lunestad BT, Scheie AA, Sunde M,
Yazdankhah SP. Does the Wide Use of Quaternary Ammonium
Compounds Enhance the Selection and Spread of Antimicrobial
Resistance and Thus Threaten Our Health? Microb Drug Resist
2010; 16: 91-104.
[13] Madaan P, Tyagi VK. Quaternary Pyridinium Salts: A Review. J
Oleo Sci 2008; 57: 197-215.
[14] Janin YL. Antituberculosis drugs: Ten years of research. Bioorg
Med Chem 2007; 15: 2479-513.
[15] Chauhan PMS, Sunduru N, Sharma M. Recent advances in the
design and synthesis of heterocycles as anti-tubercular agents. Fu-
ture Med Chem 2010; 2: 1469-500.
[16] Sharma S, Sharma PK, Kumar N, Dudhe R. A review on various
heterocyclic moieties and their antitubercular activity. Biomed
Pharmacother 2011; 65: 244-51.
[17] Takiff HE, Cimino M, Musso MC et al. Efflux pump of the proton
antiporter family confers low-level fluoroquinolone resistance in
Mycobacterium smegmatis. Proc Natl Acad Sci USA 1996; 93:
362-6.
[18] Cortesia C, Lopez GJ, de Waard JH, Takiff HE. The use of quater-
nary ammonium disinfectants selects for persisters at high fre-
quency from some species of non-tuberculous mycobacteria and
may be associated with outbreaks of soft tissue infections. J Antim-
icrob Chemother 2010; 65: 2574-81.
[19] Byrne C, Healy TM. Efficacy of a New Quarternary Ammonium
Compound Against TB. Ir J Med Sci 1999; 168: 45-6.
[20] Lougheed KEA, Taylor DL, Osborne SA, Bryans JS, Buxton RS.
New anti-tuberculosis agents amongst known drugs. Tuberculosis
2009; 89: 364-70.
[21] Rikimaru T, Kondo M, Kondo S, Oizumi K. Efficacy of common
antiseptics against mycobacteria. Int J Tuberc Lung Dis 2000; 4:
570-6.
[22] Best M, Sattar SA, Springthorpe VS, Kennedy ME. Efficacies of
Selected Disinfectants against Mycobacterium tuberculosis. J Clin
Microbiol 1990; 28: 2234-9.
[23] Mainous ME, Smith SA. Efficacy of common disinfectants against
Mycobacterium marinum. J Aquat Anim Health 2005; 17: 284-8.
[24] Rutala WA, Cole EC, Wannamaker NS, Weber DJ. Inactivation of
Mycobacterium tuberculosis and Mycobacterium bovis by 14 hos-
pital disinfectants. Am J Med 1991; 91(Suppl 3B): 267-71S.
[25] Best M, Sattar SA, Springthorpe VS, Kennedy ME. Comparative
Mycobactericidal Efficacy of Chemical Disinfectants in Suspension
and Carrier Tests. Appl Environ Microb 1988; 54: 2856-8.
[26] Garcia-Garcia A, Galvez J, de Julian-Ortiz JV et al. New agents
active against Mycobacterium avium complex selected by molecu-
lar topology: a virtual screening method. J Antimicrob Chemother
2004; 53: 65-73.
[27] Tischer M, Pradel G, Ohlsen K, Holzgrabe U. Quaternary Ammo-
nium Salts and Their Antimicrobial Potential: Targets or Nonspe-
cific Interactions? ChemMedChem 2012; 7: 22-31.
[28] Gutierrez-Lugo MT, Baker H, Shiloach J, Boshoff H, Bewley CA.
Dequalinium, a New Inhibitor of Mycobacterium tuberculosis My-
cothiol Ligase Identified by High-Throughput Screening. J Biomol
Screen 2009; 14: 643-52.
[29] Nishihara H. Bacteriostatic activity of some pyridinium, nicotin-
ium, quinolinium, and isoquinolinium quaternary salts on Myco-
bacterium tuberculosis H37Rv. J Biochem-Tokyo 1953; 40: 579-
87.
larikova J. Antimicrobial activity of sulfonamides containing 5-
1354 Current Pharmaceutical Design, 2013, Vol. 19, No. 7
chloro-2-hydroxybenzaldehyde and 5-chloro-2-hydroxybenzoic
acid scaffold. Eur J Med Chem 2012; 50: 433-40.
[31] Nishihara H. Bacteriostatic effect of cationic surface active agents
on Mycobacterium tuberculosis H37Rv. J Biochem-Tokyo 1953;
40: 589-97.
[32] Ritter HW. The Germicidal Effect of a Quaternary Ammonium
Compound (Cetylpyridinium Chloride) on Mycobacterium tubercu-
losis. Appl Microbiol 1956; 4: 114-6.
[33] Broadley SJ, Jenkins PA, Furr JR, Russell AD. Potentiation of the
effects of chlorhexidine diacetate and cetylpyridinium chloride on
mycobacteria by ethambutol. J Med Microbiol 1995; 43: 458-60.
[34] Annis TC, inventor; NanoBio Corporation, assignee. Compositions
for inactivating pathogenic microorganisms, methods of making the
compositions, and methods of use thereof. United States patent US
2005/0208083 A1. 2005 Sept.
[35] Baker JR, Annis TC, Hamouda T, Vengroff L. inventors; NanoBio
Corporation, assignee. Compositions for inactivating pathogenic
microorganisms, methods of making the compositions, and meth-
ods of use thereof. Patent WO 2005/027872 A2. 2005 Mar.
[36] Sankar MM, Kumar P, Munawwar A, Singh J, Parashar D, Singh
S. Recovery of Mycobacterium tuberculosis from Sputum Treated
with Cetyl Pyridinium Chloride. J Clin Microbiol 2009; 47: 4189-
90.
[37] Bobadilla-del-Valle M, Ponce-de-Leon A, Kato-Maeda M, et al.
Comparison of Sodium Carbonate, Cetyl-Pyridinium Chloride, and
Sodium Borate for Preservation of Sputa for Culture of Mycobacte-
rium tuberculosis. J Clin Microbiol 2003; 41: 4487-8.
[38] Pardini M, Varaine F, Iona E, et al. Cetyl-Pyridinium Chloride Is
Useful for Isolation of Mycobacterium tuberculosis from Sputa
Subjected to Long-Term Storage. J Clin Microbiol 2005; 43: 442-4.
[39] De Souza AO, Galetti FCS, Silva CL, et al. Antimycobacterial and
cytotoxicity activity of synthetic and natural compounds. Quim
Nova 2007; 30: 1563-6.
[40] Mazu TK, Etukala JR, Jacob MR, Khan SI, Walker LA, Ablordep-
pey SY.
-Carbolines and their ring-opened analogs: Synthesis and
evaluation against fungal and bacterial opportunistic pathogens.
Eur J Med Chem 2011; 46: 2378-85.
[41] Lange C, Holzhey N, Schonecker B, Beckert R, Mollmann U,
Dahse HM. -Pyridiniumalkylethers of steroidal phenols: new
compounds with potent antibacterial and antiproliferative activities.
Bioorg Med Chem 2004; 12: 3357-62.
[42] Beran V, Havelkova M, Kaustova J, Dvorska L, Pavlik I. Cell wall
deficient forms of mycobacteria: a review. Vet Med-Czech 2006;
51: 365-89.
[43] Adamec J, Beckert R, Weiss D, et al. Hybrid molecules of estrone:
New compounds with potential antibacterial, antifungal, and anti-
proliferative activities. Bioorg Med Chem 2007; 15: 2898-906.
[44] Vinsova J, Imramovsky A, Jampilek J, Ferriz JM, Dolezal M. Re-
cent Advances on Isoniazide Derivatives. Anti-Infect Agents Med
Chem 2008; 7: 12-31.
[45] Scior T, Garcés-Eisele SJ. Isoniazid is Not a Lead Compound for
its Pyridyl Ring Derivatives, Isonicotinoyl Amides, Hydrazides,
and Hydrazones: A Critical Review. Curr Med Chem 2006; 13:
2205-19.
[46] Foks H, Mieczkowska J, Janowiec M, Zwolska Z, Andrzejczyk Z.
Synthesis and tuberculostatic activity of methyl 3-
isonicotinoyldithiocarbazate and S,S'-dimethyl dithiocarbonate
isonicotinoylhydrazone, and their reactions with amines and hydra-
zines. Chem Heterocycl Comp 2002; 38: 810-6.
[47] Gardner TS, Wenis E, Lee J. Synthesis of Compounds for Chemo-
therapy of Tuberculosis. VII. Pyridine N-Oxides with Sulfur-
Containing Groups. J Org Chem 1957; 22: 984-6.
[48] Bharate SB, Thompson CM. Antimicrobial, Antimalarial, and
Antileishmanial Activities of Mono- and Bis-quaternary Pyridi-
nium Compounds. Chem Biol Drug Des 2010; 76: 546-51.
[49] Gobis K, Foks H, Kedzia A, Wierzbowska M, Zwolska Z. Synthe-
sis and Antibacterial Activity of Novel Pyridine and Pyrazine De-
rivatives Obtained from Amidoximes. J Heterocycl Chem 2009;
46: 1271-9.
[50] Pernak J, Kalewska J, Ksycinska H, Cybulski J. Synthesis and anti-
microbial activities of some pyridinium salts with alkoxymethyl
hydrophobic group. Eur J Med Chem 2001; 36: 899-907.
Krátk and Vinová
[51] Chambers HF, Moreau D, Yajko D, et al. Can Penicillins and Other
Beta-Lactam Antibiotics Be Used To Treat Tuberculosis? Antimi-
crob Agents Chemother 1995; 39: 2620-4.
[52] Stephan J, Mailaender C, Etienne G, Daffe M, Niederweis M.
Multidrug Resistance of a Porin Deletion Mutant of Mycobacte-
rium smegmatis. Antimicrob Agents Chemother 2004; 48: 4163-70.
[53] Danilchanka O, Pavlenok M, Niederweis M. Role of Porins for
Uptake of Antibiotics by Mycobacterium smegmatis. Antimicrob
Agents Chemother 2008; 52: 3127-34.
[54] Saito H, Sato K, Jin BW. Activities of Cefoxitin and Cefotetan
Against Mycobacterium fortuitum Infections in Mice. Antimicrob
Agents Chemother 1984; 26: 270-1.
[55] Chingwaru W, Duodu G, van Zyl Y, et al. Antibacterial and anti-
candidal activity of Tylosema esculentum (marama) extracts. S Afr
J Sci 2011; 107: 79-89.
[56] Muslu H, Golcu A, Ozkan SA. Electrochemical Study of Ceftaz-
idime-Copper (II) Complex: Synthesis, Characterization, Biologi-
cal Activity and Analytical Application to Pharmaceutical Dosage
Forms. Curr Anal Chem 2010; 6: 299-309.
[57] Gayathri R, Therese KL, Deepa P, Mangai S, Madhavan HN. Anti-
biotic susceptibility pattern of rapidly growing mycobacteria. J
Postgrad Med 2010; 56: 19-21.
[58] Galleni M, Franceschini N, Quinting B, et al. Use of the Chromo-
somal Class-A Beta-Lactamase of Mycobacterium fortuitum D316
To Study Potentially Poor Substrates and Inhibitory Beta-Lactam
Compounds. Antimicrob Agents Chemother 1994; 38: 1608-14.
[59] Hoffner SE, Klintz L, Olsson-Liljequist B, Bolmstrom A. Evalua-
tion of Etest for Rapid Susceptibility Testing of Mycobacterium
chelonae and M. fortuitum. J Clin Microbiol 1994; 32: 1846-9.
[60] Shrivastava SM, Shukla SK, Chaudhary M. Comparison of Antim-
icrobial Efficacy of a Fixed Dose Combination of Ceftazidime +
Sulbactam with Ceftazidime and Sulbactam Alone against Five
Bacteria. Folia Microbiol 2009; 54: 391-4.
[61] Dincer I, Ergin A, Kocagoz T. The vitro efficacy of beta-lactam
and beta-lactamase inhibitors against multidrug resistant clinical
strains of Mycobacterium tuberculosis. Int J Antimicrob Agents
2004; 23: 408-11.
[62] Carta A, Corona P, Loriga M. Quinoxaline 1,4-Dioxide: A Versa-
tile Scaffold Endowed With Manifold Activities. Curr Med Chem
2005; 12: 2259-72.
[63] Torres E, Moreno E, Ancizu S, et al. New 1,4-di-N-oxide-
quinoxaline-2-ylmethylene isonicotinic acid hydrazide derivatives
as anti-Mycobacterium tuberculosis agents. Bioorg Med Chem Lett
2011; 21: 3699-703.
[64] Alvey L, Prado S, Saint-Joanis B, et al. Diversity-oriented synthe-
sis of furo[3,2-f]chromanes with antimycobacterial activity. Eur J
Med Chem 2009; 44: 2497-505.
[65] Cosconati S, Hong JA, Novellino E, Carroll KS, Goodsell DS,
Olson AJ. Structure-Based Virtual Screening and Biological
Evaluation of Mycobacterium tuberculosis Adenosine 5
-
Phosphosulfate Reductase Inhibitors. J Med Chem 2008; 51: 6627-
30.
[66] Gobis K, Foks H, Zwolska Z, Augustynowicz-Kopec E. Synthesis
of 2-Aminoaryl-5-Substituted-1,3,4-Thiadiazoles in a Thermal 1,3-
Dipolar Cycloaddition Reaction. Phosphorus, Sulfur, and Silicon
2005; 180: 2653-66.
[67] Orlewska C, Pancechowska-Ksepko D, Foks H, Zwolska Z,
Augustynowicz-Kopec E. Reactivity of N1-Dithioester Substituted
Pyridin- and Pyrazincarboxamidrazones. Phosphorus, Sulfur, and
Silicon 2006; 181: 737-44.
[68] Ranft D, Lehwark-Yvetot G, Schaper KJ, Buege A. N1-
Hetarylcarbonylsubstituierte Amidrazone und 3,4-disubstituierte
1,2,4-Triazole als potentielle antimycobacterial Wirkstoffe.
Pharmazie 2001; 56: 266.
[69] de Oliveira JHHL, Seleghim MHR, Timm C, et al. Antimicrobial
and Antimycobacterial Activity of Cyclostellettamine Alkaloids
from Sponge Pachychalina sp. Mar Drugs 2006; 4: 1-8.
[70] Nicholas GM, Eckman LL, Newton GL, Fahey RC, Ray S, Bewley
CA. Inhibition and Kinetics of Mycobacterium tuberculosis and
Mycobacterium smegmatis Mycothiol-S-conjugate Amidase by
Natural Product Inhibitors. Bioorg Med Chem 2003; 11: 601-8.
Antimycobacterial Activity of Quaternary Pyridinium Salts
[71] Hamann MT, Rao KV, Peng J, inventors. Methods of treating dis-
ease through the administration of a manzamine analog or deriva-
tive. United States patent US 2005/0085554 A1. 2005 Apr.
[72] O’Donnell G, Poeschl R, Zimhony O, et al. Bioactive Pyridine-N-
oxide Disulfides from Allium stipitatum. J Nat Prod 2009; 72: 360-
5.
[73] Maccari R, Ottana R, Monforte F, Vigorita MG. In vitro Antimy-
cobacterial Activities of 2´-Monosubstituted Isonicotinohydrazides
Received: August 10, 2012 Accepted: October 22, 2012
Current Pharmaceutical Design, 2013, Vol. 19, No. 7 1355
and Their Cyanoborane Adducts. Antimicrob Agents Chemother
2002; 46: 294-9.
[74] Maccari R, Ottana R, Vigorita MG. In vitro advanced antimycobac-
terial screening of isoniazid-related hydrazones, hydrazides and
cyanoboranes: Part 14. Bioorg Med Chem Lett 2005; 15: 2509-13.
[75] Guzel O, Maresca A, Scozzafava A, Salman A, Balaban AT,
Supuran CT. Discovery of Low Nanomolar and Subnanomolar In-
hibitors of the Mycobacterial beta-Carbonic Anhydrases Rv1284
and Rv3273. J Med Chem 2009; 52: 4063-7.