Acridine—a neglected antibacterial chromophore

Mark Wainwright*

Centre for Forensic Science, University of Central Lancashire, Preston PR1 2HE, UK


    Abstract
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 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
The use of acridines as antimicrobial agents was first proposed by Ehrlich and Benda in 1912, and the first clinical use of these agents occurred in 1917. Many compounds containing the acridine chromophore were synthesized and tested, and the aminoacridines found wide use, both as antibacterial agents and as antimalarials, during World War II. The emergence of the penicillins eclipsed the acridines in antisepsis due to the greater therapeutic efficacies of the former. However, with the current massive increases in drug-resistant bacterial infection, new acridine derivatives may be of use. In addition, the topical utilization of aminoacridines in conjunction with directed low-power light offers bactericidal action at much lower doses.


    Introduction
 Top
 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
Of the many advances in medical science in the twentieth century, few have been as pervasive as those made in the field of antimicrobial chemotherapy. Although the early breakthroughs made by Ehrlich using arsenicals may have been associated with considerable morbidity, they introduced the idea of synthetic chemotherapy. Compounds such as Salvarsan were products of the systematic screening of available series of chemicals, most of which were produced as dyestuffs. However, there was a considerable antibacterial gap between Salvarsan and the widespread availability of penicillins (1944). From the latter part of World War I to the early-mid part of World War II this was filled by mercury salts, therapeutic dyes and dye derivatives such as the sulphonamides (1935). In addition, the considerable progress made in the treatment of protozoal tropical diseases such as trypanosomiasis and malaria was based squarely on acridine, phenothiazine and quinoline derivatives. The genesis of the alkylamino side chain, so common in modern drugs, can be traced to this period.1

The use of heteroaromatic dyes as antibacterial agents evolved directly from the experimentation of Ehrlich in the late nineteenth century. The trypanocidal activity of 10-methyl-3,6-diaminoacridinium chloride (Trypaflavin, acriflavine; TableGo) was reported in 1912 by Ehrlich and Benda, and the antibacterial activity of the same compound and the neutral (non-methylated) acridine proflavine (TableGo) in the following year by Carl Browning, a major figure in the development of acridine-based chemotherapy.2 A student of Ehrlich's, Browning was instrumental in the introduction of proflavine and acriflavine as wound antiseptics in base hospitals serving the Western Front. In the post-war era Browning worked with the then Professor of Organic Chemistry at the University of Leeds, J. B. Cohen, on the early structure–activity relationships pertaining to the antimicrobial action of a variety of dye-derived cationic heterocyclics, both in the antibacterial and trypanocidal fields.3 These cationics included cyanine and styryl derivatives of several classes of heterocycle, such as quinoline and phenazine, as well as the established acridine chromophore.


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Table. Clinically used acridines, 1917–1946
 
Similarly, in Germany, workers at the chemical giant I. G. Farben were following antimicrobial research programmes broadly based on biologically active dyestuffs. At this time the use of the phenothiazinium dye methylene blue as a lead compound resulted in the development of the antimalarials primaquine and pamaquine, both based on the 8-aminoquinoline chromophore, and of chloroquine, based on 4-aminoquinoline. The analogous acridine-based antimalarial Mepacrin (Atebrin, Quinacrine; Figure 1Go) was to find widespread use by the Allies in eastern theatres of World War II, in the absence of quinine from Japanese-held Java.4



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Figure 1. Acridine structures: (a) Mepacrine; (b) Azacrine; (c) m-AMSA; (d) dercetin; (e) heterocyclic styrylacridine; and (f) Nitroakridin 3582.

 
The second key figure in acridine antibacterial development was Adrien Albert. An Australian chemist, Albert was interested in the idea of structure–activity relationships, and it was his work on acridines that ultimately led to the understanding of their mode of action.5

Having synthesized and tested many different acridines, Albert rationalized the following parameters as being necessary for antibacterial activity:

This unifying hypothesis explained the activity against bacteria of many fused aromatic compounds. For example, the presence of the acridine nucleus per se is not required, e.g. isomeric aminobenzoquinolines and phenanthridines are also active. In fact, a heteroaromatic nucleus is not essential—2-guanidinoanthracene is antibacterial.6 Albert concentrated on the aminoacridine derivatives since these had been known since Browning to be active and of low toxicity. Within the series, Albert showed that those aminoacridines having electronic conjugation between the ring nitrogen and the amino group were the most active, due to the high ionization of such compounds. In this respect, the important positions in the acridine chromophore are 3, 6 and 9 (Figure 2Go), and many derivatives based on these structures are effective antibacterial agents.7 In terms of clinically useful materials, the TableGo gives the structures of acridines that were employed as antibacterial agents in the period up to the end of World War II. The introduction of the derivatives Aminacrine and Salacrin into clinical usage should be accredited to Albert himself, although the useful lifetime of these agents was shortened considerably by the advent of the ß-lactam agents that became available in quantity towards the end of World War II.



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Figure 2. Numbering of the acridine chromophore.

 
With the acceptance of the activity of aminoacridines as antibacterials, and the widespread use of the Allies' alternative antimalarial preparation, Mepacrine, research into the medicinal properties of the acridine chromophore reached its zenith in the immediate post-war period. At this time chromophoric analogues such as the pyridoquinolines (‘azacridines', e.g. Azacrine, Figure 1Go) were also examined as antimicrobials, although mainly in the domain of antimalarial research,16,17 reflecting the focus of contemporary antibacterial research (and clinical reliance) on ß-lactam drugs. In the 1950s and early 1960s, the work of Steck on anti-rickettsial acridines,18 based on a nitroaminoacridine system (previously Nitroakridin 3582, Figure 1Go), and of Elslager on acridine N-oxides19 are noteworthy as being the last major research efforts in systemic acridine antibacterials. Tabern's ‘Phenacridane' series20 was intended as a topical anti-infective and this has been the major area of acridine-based antibacterial treatment since that time, mainly utilizing proflavine or acriflavine.

Nucleic acids are the established sites of action of simple aminoacridine derivatives in bacteria,21 the planar area of the tricyclic acridine nucleus being ideally suited to intercalation between nucleotide base pairs in the helix and the positive charge aiding targeting. The inter-calative binding of proflavine to bacterial nucleic acids was first demonstrated by Lerman.21 This site of action also led to the development of acridine derivatives for modern anticancer chemotherapy, e.g. m-AMSA (Figure 1Go), although the actual site of action of such derivatives is now established at the level of DNA-coiling enzymes (topoisomerases) rather than DNA itself, damage being caused by the stabilization of the enzyme–DNA cleavage complex.22 DNA intercalation has also formed the main foundation for opposition to the widespread use of acridines as mainstream antibacterials in modern clinical practice, the nucleic acid site of action resulting in positive mutagenicity testing in vitro.

In terms of modern chromophoric alternatives, a range of naturally occurring ring-fused acridines have been discovered of the pyrido-/thiazolo-fused variety, e.g. dercetin (Figure 1Go). The site of action of these natural products is again reported to be DNA, in agreement with the above.23 However, the most abundant acridine-containing natural products are the acridine alkaloids present in plants of the family Rutaceae.24,25 An observation that might be made concerning acridine-based natural products is that they are usually tested for anticancer properties exclusively. That this is so is probably due to the in vitro mutagenicity found with some aminoacridines, alluded to above. Unfortunately, this may mean that promising antimicrobials have been overlooked. A rare exception to this is the work of Queener concerning the activity of Rutaceae acridones against Pneumocystis carinii.26


    Clinical use
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 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
Systemic administration

Although acriflavine (as Gonoflavin, I. G. Farben, Germany) was given iv for the treatment of gonorrhoea (0.1 g three times per week27 or 10 doses of 40–80 mg at 2–3 day intervals28), the use of simple aminoacridines in the clinical treatment of blood-borne infection, e.g. staphylococcal septicaemia, has never been a realistic option owing to the short half-life of drugs such as acriflavine or proflavine in the bloodstream. For example, iv treatment of septicaemia with Argoflavin (I. G. Farben, Germany), a mixture of euflavine lactate and silver lactate, was not usually successful.29 This is unsurprising, since the concentration in the blood of iv-administered acriflavine (200 mg) was found to have decreased by 90% over 5 min and to be undetectable at 30 min.30 Aminacrine and rivanol exhibited similar pharmacokinetics.13,31

The aqueous solubility of aminoacridine derivatives is obviously an important parameter in the proposed use of such compounds as injectable antibacterials, i.e. for systemic use. In this respect one approach is the use of quaternary salts, i.e. aminoacridines with the ring nitrogen alkylated and thus carrying a permanent positive charge. Euflavine, in the form of acriflavine preparations (q.v.) constitutes the lead compound in this area, but other simple aminoacridines were synthesized as blood antimicrobials, notably by I. G. Farben, e.g. 3-amino-10-methyl-6-haloacridinium species.32

A great amount of research and development was carried out on simple monoaminoacridine derivatives in Germany (I. G. Farben) before the outbreak of World War II. This work does not appear in the mainstream literature, but a high rate of activity may be surmised from contemporary patents. Therapeutic 3-aminoacridine derivatives were originally produced using the reduction of analogous 3-nitro species, as well as further reactions of diamino compounds such as proflavine to provide amino-/halogen-type compounds.33 The quaternization of such compounds led to derivatives that were effective antiprotozoal treatments, in line with the activity of the established quaternary trypanocide acriflavine.34 Although the work on aminoacridines eventually led to the synthesis of Mepacrine,4 it is clear from the patent literature that the antibacterial pharmacy of the simpler (amino) derivatives was being investigated, e.g. their formulation as injectables.

Owing to the low aqueous solubilities of the usual mineral acid salts (chloride, sulphate, etc.) of the aminoacridines, many of these compounds were unsuitable for clinical use in the treatment of systemic microbial disease. Thus, recourse was made to the synthesis of methanesulphonate salts, which proved to have stability equal to that of the earlier materials, but which could be produced in an aqueous injectable form.35

The short bloodstream half-life of simple aminoacridines, such as ethacridine (TableGo), is greatly extended in the antimalarial acridine, Mepacrine.36 Since the main difference between the former and the blood schizonticide lies in the size of the side chain at position 9 of the acridine chromophore (amino in the former compared with aminoalkylamino in the latter), the search for clinically useful iv-administrable acridines logically should use Mepacrine as a lead compound. Although Mepacrine is not highly antibacterial, other derivatives have been reported to exhibit increased antibacterial action. The influence of 9-substitution on activity is discussed below.

The oral administration of acridine drugs has been much more common, e.g. throat pastilles of euflavine [Panflavine (I. G. Farben, Germany) or Planacrine (May & Baker, UK) brands]. Gonorrhoea was also treated via the oral route using acriflavine. Hanschell reported the efficacious treatment of 2500 men using a daily dosage of 90–120 mg po over a period of 2 weeks, and 5 months with reportedly high tolerance and a significant side effect (jaundice) in only one patient.37

Ethacridine (2-ethoxy-6,9-diaminoacridine; TableGo) resulted from the attempted combination of the activities of acriflavine and hydrocupreine in the 1920s.38 Rivanol (Winthrop Chemical Co., USA), the highly soluble lactate salt, is an effective antibacterial and has found long-term use in the oral treatment of enteric disease such as traveller's diarrhoea and shigellosis because of its poor absorption.39 Orally administered rivanol is almost completely (99%) excreted in the faeces.40 Rivanol was also found to be far less toxic for systemic administration than acriflavine.41

Topical administration

From the earliest period of chemotherapy, acridines were recognized as effective topical antibacterials, not least because their action is undiminished in the presence of serum.42 However, their current status, even in topical therapy, appears to have been generally diminished in the absence of wartime exigencies. The fact remains that proflavine was used thoughout World War II as a standard topical treatment for combat wounds. The availability of sulphathiazole did little to alter this.

Proflavine was generally employed, as the neutral sulphate, at a reasonably high (solid) dose. For example, a series of nearly 300 wounds were treated successfully via topical administration of 500 mg of powder per wound at intervals of 4–28 days.43 Importantly for present day considerations, proflavine was also successful in cases of sulphonamide-resistant infection,43 including streptococcal complication.8 Acriflavine and proflavine resistance has been reported in methicillin-resistant strains of Staphylococcus aureus and is due to a penicillinase plasmid, pSK57.44 Resistance is due to an energy-dependent efflux mechanism encoded by the qacA gene.45 However, there are no reported incidences of clinical resistance to Aminacrine derivatives, and laboratory-produced proflavine-resistant strains did not exhibit cross-resistance to Aminacrine and Diflavine.46

Proflavine and sulphathiazole were found to exert a synergistic action and were thus used as a combined therapy for battle casualties.47 A small proportion of proflavine was found to exert a considerable synergistic effect, and this led to the use of preparations containing one part proflavine to 99 parts sulphathiazole, which were effectively antibacterial, either for wound disinfection or as a prophylactic measure administered topically at a dose of 5 mg/cm2.48,49 Owing to the basic and acidic natures, respectively, of proflavine and sulphathiazole, the salt proflavine sulphathiazolate was also employed and subsequently marketed under the brand name Flavazole (Boots Pure Drug Co., UK).50

Topical proflavine is much less damaging to human tissue than is acriflavine51,52 and it was recommended by Garrod53 that the former should be the agent of choice in wound therapy. However, the non-staining properties of the equally effective Aminacrine and Salacrin (TableGo) suggested their use in preference, in order to avoid the jaundiced appearance of some patients. Diflavine (2,6-diaminoacridine; TableGo) was also found to be a clinically effective antibacterial,54 but one that leaves a blood-red stain and, in this respect, was thought disadvantageous in comparison with the Aminacrine derivatives.8,10 Aminacrine has also found current topical use in vaginal suppositories for the treatment of trichomoniasis.55


    Structure–activity relationships in acridines
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 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
As has been mentioned, the site of action of the established aminoacridine antibacterials is bacterial nucleic acid, intercalation by the acridines being facilitated by cationic ionization and sufficient molecular planarity. Initially, this was also the accepted site and mode of action for the acridine-based antimalarial drug Mepacrine, but the weak-base hypothesis, in line with other antimalarial heterocyclic bases such as chloroquine, is equally applicable.56 Since bacterial nucleic acid must be located de facto within the bacterial cell, the aminoacridine molecule must also enter the cell in order to interact. In other cell types, aminoacridine derivatives have been shown to localize in different regions/organelles depending on a combination of lipophilicity and degree of ionization.57 Logically, it should follow that the Aminacrine skeleton (TableGo) can be so functionalized, in terms of hydrophilic/lipophilic balance, as to be excluded from the interior of the bacterial cell. This suggests the possibility that non-intercalative antibacterials exist between these two extremes. The series of (cationic) aminoacridine derivatives that have been published, e.g. by Albert,5 in tables of antibacterial data exhibit a wide range of lipophilicities. Yet it has been accepted that each of these compounds acts at the level of nucleic acid by intercalation.

The following sections deal with the effect on activity of changes in the substitution pattern or identity of different sites in the acridine chromophore (see Figure 2Go for numbering).

9-Arylaminoacridines

9-Arylaminoacridine anticancer drugs act via interference with the mammalian topoisomerase II enzyme.58 The differences between such compounds and the aminoacridine antibacterials lie in the higher lipophilicity, and presumably greater steric bulk of the former. This supports the idea that intercalative activity (and thus potential mutagenicity) might be ‘designed out' of the aminoacridine profile. This has indeed been shown in derivatives of acridine yellow, the chromophoric methyl groups (2,7) being replaced by ethyl, propyl and tertiary butyl groups. Compounds derived from the latter two groups were only weakly or nonintercalating, respectively.59

Little research has been published regarding the correlation between the size and character of the group at C-9 and the type of antimicrobial activity, i.e. antiplasmodial (antiprotozoal) or antibacterial. For example, it has been shown that the Mepacrine chromophore (3-chloro-7-methoxyacridine) can exhibit either type of activity depending on the group at C-9.60 The majority of acridine antimicrobial work has employed amino alkylamino (especially alkylaminoalkylamino) or arylamino functionality at C-9. The use of cyclic amino groups (i.e. saturated heterocycles containing an N–H moiety) and lower alkyl groups to produce novel 9-substituted acridines has been mentioned briefly by several authors over the last 80 years,5,35,37 but no biological correlation has been published. Such work is currently being carried out by the author, giving antibacterial effficacy comparable to that of Aminacrine (M. Wainwright, D. A. Phoenix & T. Kabelo, unpublished results).

The target site of AMSA and its analogues is topoisomerase II. Compounds that have shown activity against this enzyme are also active against Leishmania spp., and this has proved to be true for AMSA analogues. Significantly, several lipophilic derivatives exhibited high therapeutic indices between human cells and Leishmania major.58

Such 9-anilinoacridine compounds offer huge scope for functionalization, both in the acridine and the aryl rings, and thus a range of activities may be expected, including possible activity against bacteria. Although latterly such compounds have been developed as anticancer agents, some activity against Streptococcus pneumoniae type 1 has been reported.61

A series of 9-anilino derivatives based on the 4-methoxy-7-chloroacridine chromophore exhibited marginally higher activities against both S. aureus and Escherichia coli than that of Aminacrine. In common with other acridine substitution patterns, e.g. 2-alkoxy-7-halo-, higher activities were associated with electron-releasing groups (+R) in position 4 (para-) of the aniline substituent.62 This significant structure–activity parameter was also reported in the previously mentioned series of anti-leishmanial acridines.58

Owing to the success of Mepacrine as an antimalarial/ antiprotozoal substance, it was tested, alongside side-chain analogues, for antibacterial properties. Against standard bacteria such as S. aureus, antibacterial efficacies were low (bactericidal range of 600 µM for Mepacrine compared with 250 µM for the derivatives and 60 µM for acriflavine), although the activities increased in the presence of serum and, additionally, no adverse effects were seen in animal tests.63 However, the activity of Mepacrine against Streptococcus spp. and Gram-negative organisms was considerably lower than that of either acriflavine or rivanol.64

Noticeably, shorter, simpler side-chain analogues of Mepacrine (e.g. having a 2-hydroxyethylamino group in position 9) exhibited increased activity compared with the lead compound in tests against enteric pathogens such as Shigella dysenteriae.60 The observed activities of the derivatives were, in fact, very similar to that of rivanol.

Comparable side-chain derivatives, e.g. containing larger alkyl or heterocyclic termini at C-9, were examined against Mycobacterium spp. in vitro, again showing activity at concentrations of c. 100 µM. However, although a wide range of side chains was examined, there was no correlation between the physicochemical characteristics and the concomitant activities of the derivatives.65 Having a piperidyl moiety as the distal nitrogen-containing group (i.e. instead of diethylamino) was reported, in conjunction with a 3-methoxy-7-chloro- couple, to yield a highly antistreptococcal compound, with approximately 10-fold higher activity, under the same conditions, than Aminacrine.66

The substitution of hydrazino (-NHNRR') groups for the distal dialkylamino groups in acridine antimalarial compounds such as Mepacrine and Azacrine was found to decrease the antimalarial activity, but to maintain or increase the antibacterial activity.67 This was particularly noticeable in tests against Mycobacterium tuberculosis where the derivatives were c. 35 times more active.

Styrylacridines

Owing to the efficacy of the styryl and anil derivatives of several heterocycles, particularly as trypanocidal agents, which constituted much of Browning's work, heterocyclic-containing styryl groups—typically pyridyl or quinolinyl—were incorporated into the acridine system at position 9. The resulting derivatives were tested as trypanocides and also for antibacterial activity.

In line with many of the other acridines, the quaternized derivatives (i.e. having the pyridyl or quinolinyl nitrogen and/or the acridine nitrogen methylated; see Figure 1Go) were active antibacterials, having MICs of the order of 5 µM against S. aureus. Notably, there was no perceived difference in activity between mono- and dicationic styrylacridines.68 None of these compounds was trypanocidal, such activity requiring peripheral amino substitution.69 In addition, the activity of these compounds against S. aureus was far higher than that of the more simple precursor compounds based on 9-methylacridine. Although this is perhaps not surprising owing to the low ionization of the methyl derivative compared with the 9-amino compound, for example, quaternization of the 9-methyl derivative did not increase its activity relative to those of the styryl derivatives.70,71

It is interesting to speculate on the reasons for the lack of trypanocidal activity of the styryl compounds coupled with reasonable antibacterial properties, in comparison with the 9-arylamino derivatives covered previously, which are trypanocidal but only weakly antibacterial. The lipophilicities and ionization behaviour of the two types are similar, the main apparent difference being the structure of the group linking the aromatic moiety to the acridine. The ethenyl (-CH=CH-) linker means that the styryl groups are considerably bulkier than the arylamino type. While it is logical to suggest that this extra steric bulk inhibits the interaction of the styryl molecule with the parasitic receptor, it should also abrogate the DNA intercalation mechanism of the simple aminoacridine antibacterials. The extra steric bulk of the styryl group also means that the two chromophoric moieties (acridine and pyridine or quinoline) will not be able to maintain coplanarity. This seems to indicate different sites of antibacterial action for the styrylacridines and traditional aminoacridines.

Aminoacridines

The basis of acridine antibacterial chemotherapy lies, for the most part, in the use of the aminoacridines. The position of the amino function is important, as described above, in endowing sufficient basic character to the molecule so that there is a high degree of positive ionization at physiological pH. Although, principally, this limits amino functionality to positions 3, 6 and 9 of the parent chromophore, there is considerable evidence to support the inclusion of amino groups in addition to these, to the benefit of the activity of the resulting molecule. Diflavine is such an example, having amino groups at positions 2 and 6, being highly antibacterial in vivo and of lower toxicity than other simple aminoacridines.72,73 Notably, in tests in mice, the toxicities of several diaminoacridines (1,6-, 2,6-, 3,9-, 3,5-, 2,5-, and 1,7-) were significantly lower (<50%) than that of proflavine.74

The toxicity of Mepacrine (Russian = acriquine) was decreased considerably by the inclusion of an additional amino group in position 7 of the acridine chromophore.75 Although this also diminished the antimalarial activity, the resulting aminoacriquine analogues may be of interest as antibacterials, particularly those including the Nitroakridin 3582 side chain.

The presence of a primary arylamino group in the putative antibacterial acridine is suggested as a source of mutagenic activity (e.g. in proflavine). Again, it is possible to synthesize compounds with ‘protected' amino functionality (e.g. alkyl- or dialkylamino, nitrogen-containing heterocyclo- groups, etc.) or to use acridines without amino groups altogether. However, the use of amido groups (e.g. acetamido or sulphonamido) generally leads to a decrease in antibacterial potency. Whereas this is to be expected in 3- or 9-aminoacridine derivatives, since there results a concomitant drop in conjugation between the amino group and the ring nitrogen (and thus a decrease in the basicity of the system), the deleterious effect of amidation of non-conjugated amino groups, e.g. at position 2 of the chromophore, is less easy to explain.62 It is possible that there are secondary factors, e.g. hydrogen bonding, involved at the site of action, which are altered by amidation.

The use of amino groups, other than primary -NH2 in the acridine chromophore, has been fairly limited to the 9-(dialkylaminoalkylamino) variety. There are very few instances of simpler alkylamino functionality.76 However, the inclusion of the amino group as part of a nitrogencontaining ring has been shown to yield, for example, Aminacrine analogues with DNA binding properties similar to those of the parent.77 The use of nitrogen-containing rings in antibacterial structures is, of course, well established in the fluoroquinolone series78 and would allow the variation of physicochemical properties such as lipophilicity. Larger rings would have associated steric problems in position 9, which would lead to lower basicities—due to a lack of coplanarity between the ring and the acridine system—and, using Albert's hypothesis, decreased antibacterial activities. However, this may be overcome by optimal functionalization elsewhere in the chromophore. This has been shown in recent work by the author (unpublished observations).

Halogenoacridines

Analogues of Aminacrine containing fluorine in positions 1–4 have been synthesized and tested.79 Although highly electron-withdrawing in nature, fluorine did not decrease the basicity of the analogues significantly, and ionization levels were comparable to those of other substituted Aminacrines. In the short series examined, including trifluoromethyl-substituted compounds, significant increases in activity were shown only with the quaternary (methobromide) salts of 3-fluoro- (and 3-chloro-) Aminacrine derivatives.80 Chloroaminoacridines were found to be less active than Aminacrine, a chlorine atom in position 2 or 3 giving maximum activity.81

Varying the chromophoric position of chlorine and methoxyl substituents was investigated by Singh and coworkers in the immediate post-war period, in research initiated by the moderate antibacterial properties of Mepacrine. Noticeably, the chloro-/methoxy-substituent couple gave rise to compounds that were more effective, in general, against Gram-negative than against Gram-positive bacteria,62 in line with ethacridine-type activity.

The use of bromine and iodine in place of chlorine again yielded improved antibacterials compared with Aminacrine itself, similar to the chlorine-substituted analogues and having a similar activity profile with respect to Gram type.82 The iodinated Aminacrine derivatives were of sufficient promise to warrant animal testing and here they were found to be somewhat less toxic than the parent compound, Aminacrine, usually by a factor of two or three. Given the improvements in activity and toxicity over the lead compound, it is difficult to explain why such compounds did not find their way into contemporary pharmacopoeiae: arguments concerning probable mutagenicity were not established at the time of the study (1955). In addition, substitution patterns have since been reported that lower mutagenic effects.

The planarity of the acridine molecule is important in intercalation: this is the reason for the antibacterial activity of Aminacrine relative to its tetrahydro analogue tacrine. The presence of a chromophoric methyl group (at carbon 2) in the Aminacrine structure is reported to be sufficient to reduce both DNA intercalative ability and frameshift mutagenicity in Salmonella typhimurium TA1537. However, the inclusion of a 2-bromo or 2-iodo substituent caused increased intercalation relative to the 2-chloro analogue but led to decreased mutagenicity.83 Although the substituent Hammett constants do not enable simple correlation, the possibility of more extensive structure–activity relationship studies on substituted Aminacrine derivatives aimed at non-mutagenic acridine antibacterials may be fruitful.

Alkyl or alkoxyl substitution in positions 4 or 5 is reported to lead to lowered antimalarial potency in Mepacrine analogues,84 although 4,5-dialkyl substitution increases activity.85 Again, either substitution pattern increases antibacterial activity.7,82 In addition, the toxicity of Mepacrine analogues was found to be much lower when a 2-(2-hydroxyethoxy) moiety was employed in place of the usual methoxy.86

Nitroacridines

In the literature, the nitroaminoacridines have been associated with high antibacterial activity—3-nitro-9-aminoacridine was reported to be anti-streptococcal7 at a concentration of 1.5 µM—but also with significant toxicity in mammals. While mutagenic effects have been reported for some compounds in vitro,87 the position of the nitro group in the acridine chromophore is important in this respect. For example, the anticancer drug Ledakirin is cytotoxic due to the close proximity and interaction of the 9-amino and 1-nitro groups.88 However, the range of antimicrobial activities associated with the nitroaminoacridines89 is such that they merit re-examination. In particular, the activity of Nitroakridin 3582 [2,3-dimethoxy-6-nitro-9-(3'-diethylamino-2'-hydroxypropylamino)acridine; Figure 1Go] in the treatment of clinical typhus90 is worthy of comment. This and side-chain analogues (e.g. containing the Mepacrine side chain) were found, in addition, to exhibit low toxicity in mice at 1–4 mg/day.91 Reports of drug-resistant Rickettsia spp.92,93 suggest that further work on this group of compounds, including the similarly active simpler nitroaminoacridine analogues,94 might be profitable.

Although Albert's basic structure–activity postulate (above) holds true for the majority of acridine derivatives, there are exceptions. Acridines containing an amidino group [-C(=NH)NH2] in position 2 or 3 were synthesized in order to increase the aqueous solubility of the system. However, these were almost inactive against bacteria.95

Quaternary acridines

Quaternized alkoxyacridines were reported to be highly antibacterial, of low toxicity and also to be non-staining. Sinflavin (TableGo) is illustrative of this type, being an active antibacterial having chromophoric methoxy rather than amino groups, positive ionization being achieved via the quaternization of the ring nitrogen.11 The low activity of this compound against Gram-negative bacteria explains its lack of use.96 The increased basicity of 3,6-dimethoxyacridine97 compared with the dihydroxy species is now understood to endow the highly antibacterial activity of the former, e.g. against streptococcal infection.

In tests against S. aureus, Flavicid (TableGo) was found to be bactericidal at 10 µM, 12 times the activity of the standard acriflavine (120 µM).12 However, the activity of Flavicid against Gram-negative bacteria is very low,13 leading to its early removal from clinical use.

Although there are reported incidences of the activity of acriflavine being greater than that of its precursor, proflavine, quaternization of established acridine molecules is not necessarily a guarantee of increased antibacterial efficacy. However, owing to early evidence of the greater activity against Gram-positive bacteria of acriflavine, compared with that of ethacridine,98 methylation of the ethacridine molecule was carried out. The resulting quaternary methiodide exhibited a two-fold increase in activity against S. aureus and similar results against E. coli.99

The quinoline nucleus has been used widely to generate analogues of biologically active acridine compounds and vice versa. The dequalinium system, based on two 2-methyl-4-aminoquinoline groups linked by a 10-carbon chain at the ring nitrogen positions, forms part of a local antibacterial preparation available over the counter in the UK (e.g. Dequacain, Crookes Healthcare, UK). Logically, the acridine analogues of such compounds should be at least equally effective due to the inherent antibacterial action of the quaternary acridine system. However, there are synthetic difficulties in the linking of, for example, two molecules of 9-aminoacridine via the ring nitrogens. These are thought to result from the steric considerations on the inclusion of the third rings of the acridine nuclei compared with the 2-methyl group in the quinolines.100 In addition, although the bis-acridine system linked via the same type of alkyl chain through the 9-amino position is antibacterial,101 this has also been shown to act as a bis-intercalator in mammalian cell lines.102 Interestingly, bis(styrylquinolinium) compounds, derived from the same 2-methyl-4-aminoquinoline, were reported to be highly bactericidal.103

Acridine N-oxides

This group of compounds, originally intended as amoebicides, was based on the activity of the benz[c]acridine and nitroaminoacridine series by Elslager. The activity of one of the lead compounds, Nitroakridin 3582, was improved upon considerably, e.g. versus Streptococcus C203, the MIC of Nitroakridin 3582 is 5 µM, while the MIC of N-oxide is 20 nM.19 In addition, the N-oxide was much less toxic in mammals, and was effectively antibacterial whether administered orally or subcutaneously. Again, the short side chain of the active derivatives (-NHCH2CHOHCH2NEt2) can be considered to be halfway between the small 9-amino moiety and the large aromatic residues of the amodiaquine analogues. The latter compounds were the least active derivatives tested against each bacterial strain.

Aza analogues

The substitution of nitrogen for an aromatic -CH= is standard biosteric practice in drug development programmes. In the case of acridine, this is less straightforward than for other aromatic systems owing to the synthetic routes employed, although 9-(substituted amino) derivatives are produced via the same route as for standard acridines.104 Inclusion of nitrogen at position 1 of the acridine chromophore allowed the synthesis of Mepacrin analogues with features common to both 4- and 8-aminoquinoline antimalarials. The direct Mepacrin analogue, Azacrin, was successful in field trials against malaria17 and such analogues were also highly active against human schistosomiasis. For example, the 9-(2-(2',3'-dihydroxypropylamino)ethylamino) derivative was effective at 12 mg/kg (55 µM/kg) daily for 5 days. The toxicity of such compounds is also low, in the order of 800 mg/kg (4 mmol/kg) in mice.105

Neither the synthesis nor the antibacterial efficacies of aza-substituted Aminacrine analogues has been studied in depth. 1-Aza analogues of Aminacrine and rivanol were, however, shown to be more active than the parent compounds against a haemolytic streptococcal strain.106

Reduced acridine systems

Dihydro analogues of the acridine nucleus, the acridans, were found to have no activity against a range of bacteria;107 however, there is evidence that the reduced form of acriflavine is strongly bactericidal via an oxidative mechanism, viz. the generation of the superoxide radical anion together with acriflavine. This is reported to lead to a much more rapid bactericidal effect.108

Although the use of tetrahydroacridine derivatives, i.e. the chromophore having two fused, planar aromatic rings and one non-planar alicyclic, still allows the production of antimalarial compounds,109 there is scant evidence for the maintenance of antibacterial activity under similar circumstances. As the tetrahydroacridine nucleus is less toxic in mammalian systems than its completely aromatic counterpart, the screening of putative antibacterials based on the former system is attractive, although, as with the acridans, there is little to support an intercalative mode of action.

The activity of 9-amino-1,2,3,4-tetrahydoacridine (tacrine) in neurological disorders such as Alzheimer's disease is now well established, the drug being an effective inhibitor of acetylcholinesterase. Owing to hepatotoxic considerations,110 a considerable quantity of research into improved analogues is currently underway.111,112 Rather surprisingly, given the range of chromophoric variations produced, there is scant evidence for antibacterial screening. Although Bindra77 reported (expected) zero activity against Bacillus subtilis for both tacrine and the cyclohepto-fused derivative, the pseudo-planarity of the cyclopento analogue was reflected in only slightly lower activity than Aminacrine itself. However, DNA binding data for the active cyclopento and inactive cyclohexo derivatives were similar, and the interactions were weak compared with Aminacrine. Although the 9-hexylamino derivative of tacrine exhibited similar DNA binding to Aminacrine, no antibacterial data have been presented, and the nature of the acridine–DNA interaction is not clear.


    Conclusions and future directions
 Top
 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
It has been established for many years that several of the acridine dyes are photosensitizers. However, use has not been made of this property other than in the examination of the experimental photoinduced mutagenicity,113 either of standard dyes such as acridine orange114 or of azido-labelled analogues.115 Such work is aided by the targeting of DNA, mentioned previously.

Given that dyes such as proflavine and acridine orange can cause increased mutagenicity, and that the phototoxic side effects of systemically administered Quinacrine are due to the production of superoxide and singlet oxygen,116 monoamino analogues including some of the acridine antibacterial agents were also tested.117 Increased mutagenicity here was also indicative of photodynamic action and, due to the previous use of the aminoacridines as antibacterial agents, encouraged the current author to examine this effect against pathogenic bacteria. In a range of organisms (including S. aureus and Pseudomonas aeruginosa), the activity of several of the aminoacridines was increased up to 20-fold,118 being noticeably higher against the Gram-positive strains. Since the difference between typical drug-sensitive and drug-resistant bacteria such as MRSA is based on differences in the expression of penicillin binding proteins,119 with no increase in antioxidant enzymes, it is likely that the aminoacridines could be used in topical photodisinfection in cases of colonization with drugresistant organisms. High photobactericidal activities against methicillin-sensitive and -resistant S. aureus have been reported by the author for the related phenothiazinium photosensitizers.120

Owing to the high levels of morbidity associated with drug-resistant bacterial infections, many different drugs have been used in treatment. Crystal violet (gentian violet) was an early antibacterial—indeed Browning used this in wound disinfection—and this has been employed in the topical treatment of MRSA outbreaks in Japan.121 It is not unexpected, then, that the aminoacridines, which have been used far more extensively than crystal violet, might find a therapeutic niche, if only in topical disinfection.

In terms of systemic treatment there are no established acridine-based drugs suitable for use as intravenously administered antibacterials. Although the anti-rickettsial nitroaminoacridines would be a useful starting point, the toxicity and possible mutagenicity associated with the nitro group remains a problem. However, there is little doubt that position 9 side chains intermediate in size between Mepacrine and Aminacrine (e.g. Nitroakridin) would allow sufficient bloodstream half-life without depleting the antibacterial efficacy. Ethacridine/rivanol, often in conjunction with Nitroakridin, remains in use as an orally administered antibacterial, but usually only for the treatment of shigellosis.

There remains a feeling of mistrust about acridines owing to the fact that several examples intercalate efficiently with DNA; this is after all, the site of action of the simple aminoacridines in bacteria. However, this does not mean that because a drug is based on the acridine chromophore that it will intercalate; indeed, there is sufficient knowledge about the system now to allow the synthesis of non-intercalating compounds. In addition, DNA intercalation in bacteria does not necessarily imply the same in human cells, and there are many new chromophores based on the acridine system that may exhibit modified intercalative behaviour. Also, the number and type of atoms/groups attached to the acridine nucleus (let alone in combination with the newer chromophores) is far from exhaustive, e.g. sulphur-containing groups in acridine compounds seem to be limited to antimalarial/trypanocidal work.122

Useful systemic drugs based on the acridine nucleus will be discovered only as a result of properly organized drug screening programmes. Compounds that have shown considerable promise in the past have not been properly tested in terms of structure–activity, as they would be in modern pharmaceutical research. Particularly in view of today's serious drug-resistance problems, such testing is worthwhile.


    Notes
 
* E-mail: MWainwright{at}UCLan.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Clinical use
 Structure-activity relationships...
 Conclusions and future...
 References
 
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Received 26 July 2000; returned 7 September 2000; revised 18 September 2000; accepted 28 September 2000





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