Although of little current utility in terms of infection control, azo dyes have a considerable pedigree in terms of the development of conventional drugs and antiseptics. It was, after all, the azo dyes KL695 and prontosil that ushered in what is now considered to be the “antibiotic era” in the 1930s, as a result of a considerable chemical and microbiological effort at the Bayer company premises in Elberfeld.10
The discovery that the azoic structure represented a prodrug, breaking down to sulfanilamide in the mammalian colon, gave rise to the sulfonamide class of antibacterial agents, and it is this class—not the penicillins—that furnished the commencement of useful clinical infection control.10
The continued presence, although in diminishing import, of drugs such as sulfadiazine and sulfamethoxazole is entirely due to Domagk’s screening of azoic dyes, but it is less well appreciated that other related drug classes were developed by him during World War II, such as the thiosemicarbazones with useful activity against mycobacterial infection.11
The demonstrable efficacy of the many sulfanilamide derivatives arising from Bayer’s eventual disclosure of prontosil in 1935 belies the fact that several azoic dyes had been previously reported to have antibacterial activity that did not contain the azobenzenesulfonamide moiety (ie, would not produce sulfanilamide on breakdown).
Azo dyes had, in fact, been suggested and trialed in vitro as antiseptic agents as early as 1913. Excellent results had previously been reported for the simple dye chrysoidine by Eisenberg12
and for pyridium and serenium by Ostromislensky.13
The latter two were still in use as urinary antiseptics in the 1930s,14
and pyridium remains in use.15
Although such results were not observed in Bayer’s animal testing,16
the structural similarity to the prontosil rubrum molecule is obvious, but the lack of a sulfonamide residue (Figure 13.1
) suggests that the mode of antibacterial action is different.
It can be argued that the first major impact of dyes as antimicrobial agents was, in fact, the use of the triarylmethane derivative crystal (gentian) violet (Figure 13.2
Hans-Christian Gram’s development of bacterial typing. Although this immensely valuable work was not aimed at bacterial killing, the demonstrated dye-cell interactions and selectivity underpinned the ensuing labors of scientists such as Koch and Ehrlich and led, ultimately, to the development of antimicrobial chemotherapy.1
As noted earlier, “flavine therapy” was introduced by Browning and Gulbransen3
during the second half of World War I, “flavines” referring generally to the dyes employed for wound disinfection, rather than being chemically specific. One of the principal dyes used here was brilliant green, a closely related analogue of crystal violet (see Figure 13.2
). Similar work was carried out at the same time by others.17
FIGURE 13.1 Antiseptic (in vitro) azoic dyes and prontosil rubrum.
FIGURE 13.2 Simple antimicrobial triarylmethane dyes.
Interestingly, Fung and Miller21
examined the effects of a range of dyes—42 in all—from different classes and observed a distinct difference in activity along Gram classification lines, with very little activity shown against gram-negative bacteria but high activity by over half of the examples used against gram-positive bacteria. Little modern work has focused on this class of agents in terms of new and improved derivatives. Crystal violet was also found to be active against fungi and was used until relatively recently against skin infections such as ringworm.22
In a parallel field, the dye was, until recently, added to donated blood by South American agencies to inactivate the parasite responsible for Chagas disease, Trypanosoma cruzi
Additionally, crystal violet has proved to be highly effective in the treatment of patients infected with methicillin-resistant Staphylococcus aureus
The use of acridines as antimicrobial agents was first proposed25
following the clinical introduction of proflavine and acriflavine as wound disinfectants3
in 1917; several other derivatives were introduced during the World War II, mainly due to the work of Albert et al26
and including the antimalarial drug mepacrine. Albert et al’s efforts in this area allowed the development of structure-activity ideas, indicating that the microbial target was DNA and that active acridines intercalated into the structure. However, the interaction of quinolone antibacterials27
and some acridine-based anticancer drugs28
with DNA-managing enzymes such as gyrases and topoisomerases suggests that alternative/additional modes of action are possible. Albert et al’s experimentally derived criteria for antibacterial activity among the acridines, to include their cationic nature, high levels of ionization at physiological pH, and a planar molecular surface area ≥38 Å2
, led to the discovery that the heteroaromatic chromophore could be varied, or that a purely benzenoid structure was equally effective.29
Thus, activity became predictable (or explainable) not only in the related azine dyes (phenazines, phenoxazines, phenothiazines, etc) but also in cationic anthracene derivatives.29
Furthermore, nonlinear ring fusion (as in the phenanthridines) or bridged structures, where the bridge is part of the delocalized pi system of the molecule (eg, styryl and cyanine derivatives), also furnished active examples.
In terms of the clinical use of acridine derivatives, the principal entries are provided in Table 13.1
In antiseptic and disinfection applications, the main acridine derivatives used have been proflavine, acriflavine/euflavine, and aminacrine, although local use of these agents was rapidly superseded by systemic β-lactam administration in the mid-1940s. Local application, for example, in wound disinfection, continued for a considerable time, both alone and in combination with sulfonamide derivatives.31
In addition, acriflavine, the quaternary 10-methyl derivative of proflavine containing varying levels of proflavine hydrochloride, had been used as a systemic therapy in cases of trypanosomiasis (as trypaflavin) and gonorrhoea (as gonaflavin), the latter being administered intravenously at 0.1 g 3 times per week,32
or in 10 doses of 40 to 80 mg at 2- to 3-day intervals.33
The pure quaternary compound is euflavine (see Table 13.1
The aqueous solubility of aminoacridine derivatives is obviously an important parameter in the proposed use of such compounds as injectable antibacterials, that is, for systemic use. The hydrophilic nature of the simple aminoacridines covered here furnishes correspondingly short half-lives in the bloodstream, and their use in the clinical treatment of bloodborne infection, for example, staphylococcal septicemia, has never been a realistic option as a consequence. For example, intravenous treatment of septicemia with argoflavin, a mixture of euflavine lactate and silver lactate, was not usually successful,34
the concentration in the blood of administered acriflavine (200 mg) having decreased by 90% over 5 minutes and being undetectable at 30 minutes.35
Aminacrine and rivanol exhibited similar pharmacokinetics.36
Despite the absence of currently suitable injectables, there is a considerable patent literature, particularly concerning quaternized acridines, which emanated from earlier work on euflavine. However, simple aminoacridines were synthesized as blood antimicrobials, notably by IG Farben, for example, 3-amino-10-methyl-6-haloacridinium species.38
Further forays in this area might well use the antimalarial acridine mepacrine (quinacrine, atebrin) as a lead compound from a pharmacologic standpoint, with alterations to the much longer C-9 side chains, for example,
with Nitroakridin 3582. This compound, [2,3-dimethoxy-6-nitro-9-(3′-diethylamino-2′-hydroxypropylamino) acridine], has been used in the treatment of clinical typhus.41
The works of Steck et al42
on antirickettsial acridines, based on Nitroakridin 3582, and of Elslager and Tendick43
on acridine N
-oxides represent the last major research efforts in systemic acridine antibacterials. Tabern’s44
“Phenacridane” series was intended as a topical anti-infective, and this has indeed been the major area of acridine-based antibacterial treatment since that time, mainly using proflavine or acriflavine.
Clinical antibacterial acridinesa
a From Wainwright M et al.30 Reproduced by permission of British Society for Antimicrobial Chemotherapy.
b Photobactericidal examples.
c Purified acriflavine.
The use of orally administered acridine drugs has been much more common, for example, euflavine pastilles employed for throat disinfection (Panflavine or Planacrine brands). Gonorrhoea was also treated via the oral route using acriflavine.46
Ethacridine (2-ethoxy-6,9-diaminoacridine; see Table 13.1
) has found long-term use in the oral treatment of enteric disease such as shigellosis due to low absorption and lower toxicity than acriflavine.36
Heteroatomic replacement of the C9 carbon of the acridine nucleus with nitrogen furnishes the phenazine analogue, responsible for biological stains such as neutral red and safranin, among others. Although a range of phenazines was included in Browning’s examination of a range of available dyes for antimicrobial activity in the post-World War I period,48
no significant activity was reported. However, clofazimine, a phenazine-containing molecule, has formed part of the antimycobacterial effort since the 1950s. Clofazimine (Figure 13.3
) was originally synthesized in the search for drugs against Hansen disease.49
Clofazimine is a purple- or damson-colored compound. Although, as noted, Barry’s original drug target was Hansen bacillus (Mycobacterium leprae
it is also active against Mycobacterium avium
, one of the opportunistic pathogens associated with human immunodeficiency virus (HIV)-positive status.51
Clofazimine has also found use in the treatment of Crohn disease52
and in drug-resistant tuberculosis.53
FIGURE 13.3 Clofazimine.
Cyanine and Styryl Dyes
Research in this area grew out of the success of flavine therapy during World War I and was due, in the most part, to Carl Browning, a one-time assistant of Paul Ehrlich.54
Browning and his main chemical collaborator, Cohen, deconstructed the acridine chromophore and thus experimented on amino derivatives of pyridines and quinolines with olefinic groups attached in partial replacement of the fused benzene rings in acridine, producing both cyanine- and styryl-type molecules. As Browning was interested both in bacterial and trypanosomal infection, the resulting compounds were tested against both types of pathogen, producing a considerable body of work. In addition, styryl (aryl-CH=CH-aryl) and imino (aryl-CH=N-aryl) derivatives were examined.48
The activity of several of the derivatives was such that a clinical trial was run with compound 48S (Table 13.2
) at the General Infirmary in Leeds, United Kingdom,61
and included more than 2000 subjects. The 48S was employed in most areas requiring antisepsis/antiseptic dressings, local application, wound or infection site irrigation, and even intravenous administration in septicemia. Due to the instability of imino structures in acidic solution where hydrolysis occurs, 48S was not administered by mouth. Generally, the activity of 48S in human subjects was sufficient to give cures in most cases where it was applied directly rather than systemically, although some successful treatments of septicemia were reported. In many ways, 48S appears to be similar in activity to acriflavine and proflavine, although these are not effective in blood-borne disease. That the clinical trial of the iminoquinoline was not extended cannot be due to lack of efficacy, and it was described as being bland and nonirritating to the tissues. As can be seen from Table 13.2
, the variation in amino side chain length produced considerable differences in activity, longer chain derivatives generally being more active, in many cases more so than 48S itself. This may indicate a “lipophilic tail” influence due to interaction with outer/inner membrane structures in the bacterial target.
Antibacterial activity of analogues of clinical compound 48, provided in µmol (10-6
MBC Staphylococcus aureus (µmol)
MBC Escherichia coli (µmol)
Abbreviation: MBC, minimum bactericidal concentration.
a Data from Browning et al.59,60
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