Journal of Antimicrobial Chemotherapy (2001) 47, 1–13 JAC Review

Acridine—a neglected antibacterial chromophore

Mark Wainwright*

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

The use of 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 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 A student of Ehrlich’s, Browning was instrumental in the introduction of proflavine and acriflavine as wound anti- Of the many advances in medical science in the twentieth septics in base hospitals serving the Western Front. In the century, few have been as pervasive as those made in the post-war era Browning worked with the then Professor of field of antimicrobial chemotherapy. Although the early Organic Chemistry at the University of Leeds, J. B. Cohen, breakthroughs made by Ehrlich using arsenicals may have on the early structure–activity relationships pertaining to been associated with considerable morbidity, they intro- the antimicrobial action of a variety of dye-derived cationic duced the idea of synthetic chemotherapy. Compounds heterocyclics, both in the antibacterial and trypanocidal such as Salvarsan were products of the systematic screening fields.3 These cationics included cyanine and styryl deriva- of available series of chemicals, most of which were pro- tives of several classes of heterocycle, such as duced as dyestuffs. However, there was a considerable and phenazine, as well as the established acridine chromo- antibacterial gap between Salvarsan and the widespread phore. availability of penicillins (1944). From the latter part of Similarly, in Germany, workers at the chemical giant I. G. World War I to the early-mid part of World War II this was Farben were following antimicrobial research programmes filled by salts, therapeutic dyes and dye derivatives broadly based on biologically active dyestuffs. At this time such as the sulphonamides (1935). In addition, the consid- the use of the phenothiazinium dye as a erable progress made in the treatment of protozoal tropical lead compound resulted in the development of the anti- diseases such as trypanosomiasis and was based malarials and pamaquine, both based on the squarely on acridine, phenothiazine and quinoline deriva- 8-aminoquinoline chromophore, and of , based tives. The genesis of the alkylamino side chain, so common on 4-aminoquinoline. The analogous acridine-based anti- in modern drugs, can be traced to this period.1 malarial Mepacrin (Atebrin, Quinacrine; Figure 1) was to The use of heteroaromatic dyes as antibacterial agents find widespread use by the Allies in eastern theatres of evolved directly from the experimentation of Ehrlich in World War II, in the absence of from Japanese-held the late nineteenth century. The trypanocidal activity of Java.4 10-methyl-3,6-diaminoacridinium chloride (Trypaflavin, The second key figure in acridine antibacterial develop- acriflavine; Table) was reported in 1912 by Ehrlich and ment was Adrien Albert. An Australian chemist, Albert Benda, and the antibacterial activity of the same compound was interested in the idea of structure–activity relation- and the neutral (non-methylated) acridine proflavine ships, and it was his work on acridines that ultimately led to (Table) in the following year by Carl Browning, a major the understanding of their mode of action.5 figure in the development of acridine-based chemotherapy.2 Having synthesized and tested many different acridines,

*E-mail: [email protected]

1 © 2001 The British Society for Antimicrobial Chemotherapy M. Wainwright

Albert rationalized the following parameters as being This unifying hypothesis explained the activity against necessary for antibacterial activity: bacteria of many fused aromatic compounds. For example, the presence of the acridine nucleus per se is not required, ● cationic ionization; e.g. isomeric aminobenzoquinolines and phenanthridines ● high levels of ionization at neutral pH (i.e. pKa 8); are also active. In fact, a heteroaromatic nucleus is not ● planar molecular surface area 38 Å2. essential—2-guanidinoanthracene is antibacterial.6 Albert

Table. Clinically used acridines, 1917–1946

R2 R3 R4 R6 R9 R10 Reference no.

Proflavine H NH2 HNH2 HH 8 a Euflavine HNH2 HNH2 HCH3 9 Diflavine NH2 HHNH2 HH 10 Sinflavin H CH3OH CH3OH CH3 11 Flavicid CH3 NH2 H (CH3)2NH CH3 12 Ethacridine CH3CH2OH H NH2 NH2 H13 Aminacrine H H H H NH2 H14 Salacrin H H CH3 HNH2 H15

aAs a component of acriflavine, Gonoflavin, Trypaflavin, etc.

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

2 Acridine—a neglected antibacterial chromophore

merases) 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 main- stream antibacterials in modern clinical practice, the nucleic acid site of action resulting in positive mutagenicity Figure 2. Numbering of the acridine chromophore. testing in vitro. In terms of modern chromophoric alternatives, a range concentrated on the aminoacridine derivatives since these of naturally occurring ring-fused acridines have been dis- had been known since Browning to be active and of covered of the pyrido-/thiazolo-fused variety, e.g. dercetin low toxicity. Within the series, Albert showed that those (Figure 1). The site of action of these natural products is aminoacridines having electronic conjugation between the again reported to be DNA, in agreement with the above.23 ring nitrogen and the amino group were the most active, However, the most abundant acridine-containing natural due to the high ionization of such compounds. In this products are the acridine alkaloids present in plants of the respect, the important positions in the acridine chromo- family Rutaceae.24,25 An observation that might be made phore are 3, 6 and 9 (Figure 2), and many derivatives based concerning acridine-based natural products is that they are on these structures are effective antibacterial agents.7 In usually tested for anticancer properties exclusively. That terms of clinically useful materials, the Table gives the this is so is probably due to the in vitro mutagenicity found structures of acridines that were employed as antibacterial with some aminoacridines, alluded to above. Unfortun- agents in the period up to the end of World War II. The ately, this may mean that promising antimicrobials have introduction of the derivatives Aminacrine and Salacrin been overlooked. A rare exception to this is the work of into clinical usage should be accredited to Albert himself, Queener concerning the activity of Rutaceae acridones although the useful lifetime of these agents was shortened against Pneumocystis carinii.26 considerably by the advent of the -lactam agents that became available in quantity towards the end of World War II. With the acceptance of the activity of aminoacridines as Clinical use antibacterials, and the widespread use of the Allies’ altern- Systemic administration ative antimalarial preparation, Mepacrine, research into the medicinal properties of the acridine chromophore Although acriflavine (as Gonoflavin, I. G. Farben, Ger- reached its zenith in the immediate post-war period. At this many) was given iv for the treatment of gonorrhoea (0.1 g time chromophoric analogues such as the pyridoquinolines three times per week27 or 10 doses of 40–80 mg at 2–3 day (‘azacridines’, e.g. Azacrine, Figure 1) were also examined intervals28), the use of simple aminoacridines in the clinical as antimicrobials, although mainly in the domain of anti- treatment of blood-borne infection, e.g. staphylococcal malarial research,16,17 reflecting the focus of contemporary septicaemia, has never been a realistic option owing to the antibacterial research (and clinical reliance) on -lactam short half-life of drugs such as acriflavine or proflavine in drugs. In the 1950s and early 1960s, the work of Steck on the bloodstream. For example, iv treatment of septicaemia anti-rickettsial acridines,18 based on a nitroaminoacridine with Argoflavin (I. G. Farben, Germany), a mixture of system (previously Nitroakridin 3582, Figure 1), and of euflavine lactate and lactate, was not usually success- Elslager on acridine N-oxides19 are noteworthy as being ful.29 This is unsurprising, since the concentration in the the last major research efforts in systemic acridine antibac- blood of iv-administered acriflavine (200 mg) was found to terials. Tabern’s ‘Phenacridane’ series20 was intended as a have decreased by 90% over 5 min and to be undetectable topical anti-infective and this has been the major area of at 30 min.30 Aminacrine and rivanol exhibited similar acridine-based antibacterial treatment since that time, pharmacokinetics.13,31 mainly utilizing proflavine or acriflavine. The aqueous solubility of aminoacridine derivatives is Nucleic acids are the established sites of action of obviously an important parameter in the proposed use of simple aminoacridine derivatives in bacteria,21 the planar such compounds as injectable antibacterials, i.e. for sys- area of the tricyclic acridine nucleus being ideally suited temic use. In this respect one approach is the use of quater- to intercalation between nucleotide base pairs in the helix nary salts, i.e. aminoacridines with the ring nitrogen and the positive charge aiding targeting. The inter-calative alkylated and thus carrying a permanent positive charge. binding of proflavine to bacterial nucleic acids was first Euflavine, in the form of acriflavine preparations (q.v.) demonstrated by Lerman.21 This site of action also led to constitutes the lead compound in this area, but other the development of acridine derivatives for modern anti- simple aminoacridines were synthesized as blood anti- cancer chemotherapy, e.g. m-AMSA (Figure 1), although microbials, notably by I. G. Farben, e.g. 3-amino-10- the actual site of action of such derivatives is now estab- methyl-6-haloacridinium species.32 lished at the level of DNA-coiling enzymes (topoiso- A great amount of research and development was

3 M. Wainwright carried out on simple monoaminoacridine derivatives in Topical administration Germany (I. G. Farben) before the outbreak of World War From the earliest period of chemotherapy, acridines were II. This work does not appear in the mainstream literature, recognized as effective topical antibacterials, not least but a high rate of activity may be surmised from contem- because their action is undiminished in the presence of porary patents. Therapeutic 3-aminoacridine derivatives serum.42 However, their current status, even in topical ther- were originally produced using the reduction of analogous apy, appears to have been generally diminished in the 3-nitro species, as well as further reactions of diamino com- absence of wartime exigencies. The fact remains that pro- pounds such as proflavine to provide amino-/halogen-type flavine was used thoughout World War II as a standard compounds.33 The quaternization of such compounds led topical treatment for combat wounds. The availability of to derivatives that were effective treatments, sulphathiazole did little to alter this. in line with the activity of the established quaternary Proflavine was generally employed, as the neutral sul- trypanocide acriflavine.34 Although the work on amino- phate, at a reasonably high (solid) dose. For example, a acridines eventually led to the synthesis of Mepacrine,4 it is series of nearly 300 wounds were treated successfully via clear from the patent literature that the antibacterial phar- topical administration of 500 mg of powder per wound at macy of the simpler (amino) derivatives was being investi- intervals of 4–28 days.43 Importantly for present day con- gated, e.g. their formulation as injectables. siderations, proflavine was also successful in cases of sul- Owing to the low aqueous solubilities of the usual mineral phonamide-resistant infection,43 including streptococcal acid salts (chloride, sulphate, etc.) of the aminoacridines, complication.8 Acriflavine and proflavine resistance has many of these compounds were unsuitable for clinical use been reported in methicillin-resistant strains of Staphylococ- in the treatment of systemic microbial disease. Thus, cus aureus and is due to a penicillinase plasmid, pSK57.44 recourse was made to the synthesis of methanesulphonate Resistance is due to an energy-dependent efflux mechanism salts, which proved to have stability equal to that of the encoded by the qacA gene.45 However, there are no reported earlier materials, but which could be produced in an aque- incidences of clinical resistance to Aminacrine derivatives, ous injectable form.35 and laboratory-produced proflavine-resistant strains did not The short bloodstream half-life of simple amino- exhibit cross-resistance to Aminacrine and Diflavine.46 acridines, such as ethacridine (Table), is greatly extended Proflavine and sulphathiazole were found to exert a syn- in the antimalarial acridine, Mepacrine.36 Since the main ergistic action and were thus used as a combined therapy difference between the former and the blood schizonticide for battle casualties.47 A small proportion of proflavine was lies in the size of the side chain at position 9 of the acridine found to exert a considerable synergistic effect, and this led chromophore (amino in the former compared with amino- to the use of preparations containing one part proflavine to alkylamino in the latter), the search for clinically useful 99 parts sulphathiazole, which were effectively antibac- iv-administrable acridines logically should use Mepacrine terial, either for wound disinfection or as a prophylactic as a lead compound. Although Mepacrine is not highly measure administered topically at a dose of 5 mg/cm2.48,49 antibacterial, other derivatives have been reported to Owing to the basic and acidic natures, respectively, of pro- exhibit increased antibacterial action. The influence of 9- flavine and sulphathiazole, the salt proflavine sulphathiazo- substitution on activity is discussed below. late was also employed and subsequently marketed under The oral administration of acridine drugs has been much the brand name Flavazole (Boots Pure Drug Co., UK).50 more common, e.g. throat pastilles of euflavine [Panflavine Topical proflavine is much less damaging to human (I. G. Farben, Germany) or Planacrine (May & Baker, UK) tissue than is acriflavine51,52 and it was recommended by brands]. Gonorrhoea was also treated via the oral route Garrod53 that the former should be the agent of choice in using acriflavine. Hanschell reported the efficacious treat- wound therapy. However, the non-staining properties of ment of 2500 men using a daily dosage of 90–120 mg po the equally effective Aminacrine and Salacrin (Table) over a period of 2 weeks, and 5 months with reportedly high suggested their use in preference, in order to avoid the tolerance and a significant side effect (jaundice) in only one jaundiced appearance of some patients. Diflavine (2,6- patient.37 diaminoacridine; Table) was also found to be a clinically Ethacridine (2-ethoxy-6,9-diaminoacridine; Table) resulted effective antibacterial,54 but one that leaves a blood-red from the attempted combination of the activities of stain and, in this respect, was thought disadvantageous in acriflavine and hydrocupreine in the 1920s.38 Rivanol comparison with the Aminacrine derivatives.8,10 Amina- (Winthrop Chemical Co., USA), the highly soluble lactate crine has also found current topical use in vaginal supposit- salt, is an effective antibacterial and has found long-term ories for the treatment of trichomoniasis.55 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 com- Structure–activity relationships in acridines pletely (99%) excreted in the faeces.40 Rivanol was also found to be far less toxic for systemic administration than As has been mentioned, the site of action of the established acriflavine.41 aminoacridine antibacterials is bacterial nucleic acid, inter-

4 Acridine—a neglected antibacterial chromophore calation by the acridines being facilitated by cationic antibacterial effficacy comparable to that of Aminacrine ionization and sufficient molecular planarity. Initially, this (M. Wainwright, D. A. Phoenix & T. Kabelo, unpublished was also the accepted site and mode of action for the results). acridine-based antimalarial drug Mepacrine, but the weak- The target site of AMSA and its analogues is topo- base hypothesis, in line with other antimalarial heterocyclic isomerase II. Compounds that have shown activity against bases such as chloroquine, is equally applicable.56 Since this enzyme are also active against Leishmania spp., and bacterial nucleic acid must be located de facto within the this has proved to be true for AMSA analogues. Signifi- bacterial cell, the aminoacridine molecule must also enter cantly, several lipophilic derivatives exhibited high thera- the cell in order to interact. In other cell types, aminoacri- peutic indices between human cells and Leishmania dine derivatives have been shown to localize in different major.58 regions/organelles depending on a combination of lipo- Such 9-anilinoacridine compounds offer huge scope for philicity and degree of ionization.57 Logically, it should functionalization, both in the acridine and the aryl rings, follow that the Aminacrine skeleton (Table) can be so func- and thus a range of activities may be expected, including tionalized, in terms of hydrophilic/lipophilic balance, as to possible activity against bacteria. Although latterly such be excluded from the interior of the bacterial cell. This compounds have been developed as anticancer agents, suggests the possibility that non-intercalative antibacterials some activity against Streptococcus pneumoniae type 1 has exist between these two extremes. The series of (cationic) been reported.61 aminoacridine derivatives that have been published, e.g. by A series of 9-anilino derivatives based on the 4-methoxy- Albert,5 in tables of antibacterial data exhibit a wide range 7-chloroacridine chromophore exhibited marginally higher of lipophilicities. Yet it has been accepted that each of activities against both S. aureus and Escherichia coli than these compounds acts at the level of nucleic acid by inter- that of Aminacrine. In common with other acridine substi- calation. tution patterns, e.g. 2-alkoxy-7-halo-, higher activities were The following sections deal with the effect on activity of associated with electron-releasing groups (R) in position changes in the substitution pattern or identity of different 4 (para-) of the aniline substituent.62 This significant struc- sites in the acridine chromophore (see Figure 2 for num- ture–activity parameter was also reported in the previously bering). mentioned series of anti-leishmanial acridines.58 Owing to the success of Mepacrine as an antimalarial/ antiprotozoal substance, it was tested, alongside side-chain 9-Arylaminoacridines analogues, for antibacterial properties. Against standard 9-Arylaminoacridine anticancer drugs act via interference bacteria such as S. aureus, antibacterial efficacies were low with the mammalian topoisomerase II enzyme.58 The dif- (bactericidal range of 600 M for Mepacrine compared ferences between such compounds and the aminoacridine with 250 M for the derivatives and 60 M for acriflavine), antibacterials lie in the higher lipophilicity, and presumably although the activities increased in the presence of serum greater steric bulk of the former. This supports the idea and, additionally, no adverse effects were seen in animal that intercalative activity (and thus potential mutagenicity) tests.63 However, the activity of Mepacrine against Strepto- might be ‘designed out’ of the aminoacridine profile. This coccus spp. and Gram-negative organisms was consider- has indeed been shown in derivatives of acridine yellow, ably lower than that of either acriflavine or rivanol.64 the chromophoric methyl groups (2,7) being replaced by Noticeably, shorter, simpler side-chain analogues of ethyl, propyl and tertiary butyl groups. Compounds derived Mepacrine (e.g. having a 2-hydroxyethylamino group in from the latter two groups were only weakly or non- position 9) exhibited increased activity compared with the intercalating, respectively.59 lead compound in tests against enteric pathogens such as Little research has been published regarding the correla- Shigella dysenteriae.60 The observed activities of the deriva- tion between the size and character of the group at C-9 tives were, in fact, very similar to that of rivanol. and the type of antimicrobial activity, i.e. antiplasmodial Comparable side-chain derivatives, e.g. containing larger (antiprotozoal) or antibacterial. For example, it has been alkyl or heterocyclic termini at C-9, were examined against shown that the Mepacrine chromophore (3-chloro-7- Mycobacterium spp. in vitro, again showing activity at con- methoxyacridine) can exhibit either type of activity centrations of c. 100 M. However, although a wide range depending on the group at C-9.60 The majority of acridine of side chains was examined, there was no correlation antimicrobial work has employed amino alkylamino (espec- between the physicochemical characteristics and the con- ially alkylaminoalkylamino) or arylamino functionality at comitant activities of the derivatives.65 Having a piperidyl C-9. The use of cyclic amino groups (i.e. saturated hetero- moiety as the distal nitrogen-containing group (i.e. instead cycles containing an N–H moiety) and lower alkyl groups of diethylamino) was reported, in conjunction with a 3- to produce novel 9-substituted acridines has been men- methoxy-7-chloro- couple, to yield a highly antistrepto- tioned briefly by several authors over the last 80 years,5,35,37 coccal compound, with approximately 10-fold higher but no biological correlation has been published. Such activity, under the same conditions, than Aminacrine.66 work is currently being carried out by the author, giving The substitution of hydrazino (-NHNRR) groups for

5 M. Wainwright the distal dialkylamino groups in acridine antimalarial in endowing sufficient basic character to the molecule so compounds such as Mepacrine and Azacrine was found that there is a high degree of positive ionization at physio- to decrease the antimalarial activity, but to maintain or logical pH. Although, principally, this limits amino func- increase the antibacterial activity.67 This was particularly tionality to positions 3, 6 and 9 of the parent chromophore, noticeable in tests against Mycobacterium tuberculosis there is considerable evidence to support the inclusion of where the derivatives were c. 35 times more active. 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 Styrylacridines highly antibacterial in vivo and of lower toxicity than other Owing to the efficacy of the styryl and anil derivatives of simple aminoacridines.72,73 Notably, in tests in mice, the several heterocycles, particularly as trypanocidal agents, toxicities of several diaminoacridines (1,6-, 2,6-, 3,9-, 3,5-, which constituted much of Browning’s work, heterocyclic- 2,5-, and 1,7-) were significantly lower (50%) than that of containing styryl groups—typically pyridyl or quinolinyl— proflavine.74 were incorporated into the acridine system at position 9. The toxicity of Mepacrine (Russian acriquine) was The resulting derivatives were tested as trypanocides and decreased considerably by the inclusion of an additional also for antibacterial activity. amino group in position 7 of the acridine chromophore.75 In line with many of the other acridines, the quaternized Although this also diminished the antimalarial activity, the derivatives (i.e. having the pyridyl or quinolinyl nitrogen resulting aminoacriquine analogues may be of interest as and/or the acridine nitrogen methylated; see Figure 1) were antibacterials, particularly those including the Nitroakridin active antibacterials, having MICs of the order of 5 M 3582 side chain. against S. aureus. Notably, there was no perceived difference The presence of a primary arylamino group in the puta- in activity between mono- and dicationic styrylacridines.68 tive antibacterial acridine is suggested as a source of muta- None of these compounds was trypanocidal, such activity genic activity (e.g. in proflavine). Again, it is possible to requiring peripheral amino substitution.69 In addition, the synthesize compounds with ‘protected’ amino functionality activity of these compounds against S. aureus was far higher (e.g. alkyl- or dialkylamino, nitrogen-containing hetero- than that of the more simple precursor compounds based on cyclo- groups, etc.) or to use acridines without amino 9-methylacridine. Although this is perhaps not surprising groups altogether. However, the use of amido groups (e.g. owing to the low ionization of the methyl derivative com- acetamido or sulphonamido) generally leads to a decrease pared with the 9-amino compound, for example, quaterniza- in antibacterial potency. Whereas this is to be expected in tion of the 9-methyl derivative did not increase its activity 3- or 9-aminoacridine derivatives, since there results a con- relative to those of the styryl derivatives.70,71 comitant drop in conjugation between the amino group and It is interesting to speculate on the reasons for the lack of the ring nitrogen (and thus a decrease in the basicity of the trypanocidal activity of the styryl compounds coupled with system), the deleterious effect of amidation of non-conjug- reasonable antibacterial properties, in comparison with the ated amino groups, e.g. at position 2 of the chromophore, 9-arylamino derivatives covered previously, which are try- is less easy to explain.62 It is possible that there are panocidal but only weakly antibacterial. The lipophilicities secondary factors, e.g. hydrogen bonding, involved at the and ionization behaviour of the two types are similar, the site of action, which are altered by amidation. main apparent difference being the structure of the group The use of amino groups, other than primary -NH2 in the linking the aromatic moiety to the acridine. The ethenyl acridine chromophore, has been fairly limited to the 9- (-CHCH-) linker means that the styryl groups are consid- (dialkylaminoalkylamino) variety. There are very few erably bulkier than the arylamino type. While it is logical to instances of simpler alkylamino functionality.76 However, suggest that this extra steric bulk inhibits the interaction of the inclusion of the amino group as part of a nitrogen- the styryl molecule with the parasitic receptor, it should containing ring has been shown to yield, for example, also abrogate the DNA intercalation mechanism of the Aminacrine analogues with DNA binding properties simi- simple aminoacridine antibacterials. The extra steric bulk lar to those of the parent.77 The use of nitrogen-containing of the styryl group also means that the two chromophoric rings in antibacterial structures is, of course, well estab- moieties (acridine and pyridine or quinoline) will not be lished in the fluoroquinolone series78 and would allow the able to maintain coplanarity. This seems to indicate differ- variation of physicochemical properties such as lipophil- ent sites of antibacterial action for the styrylacridines and icity. Larger rings would have associated steric problems in traditional aminoacridines. 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 anti- Aminoacridines bacterial activities. However, this may be overcome by The basis of acridine antibacterial chemotherapy lies, for optimal functionalization elsewhere in the chromophore. the most part, in the use of the aminoacridines. The posi- This has been shown in recent work by the author (unpub- tion of the amino function is important, as described above, lished observations).

6 Acridine—a neglected antibacterial chromophore

antibacterial activity.7,82 In addition, the toxicity of Mepa- Halogenoacridines crine analogues was found to be much lower when a Analogues of Aminacrine containing fluorine in positions 2-(2-hydroxyethoxy) moiety was employed in place of the 1–4 have been synthesized and tested.79 Although highly usual methoxy.86 electron-withdrawing in nature, fluorine did not decrease the basicity of the analogues significantly, and ionization levels were comparable to those of other substituted Nitroacridines Aminacrines. In the short series examined, including tri- In the literature, the nitroaminoacridines have been asso- fluoromethyl-substituted compounds, significant increases ciated with high antibacterial activity—3-nitro-9-amino- in activity were shown only with the quaternary (metho- acridine was reported to be anti-streptococcal7 at a concen- bromide) salts of 3-fluoro- (and 3-chloro-) Aminacrine tration of 1.5 M—but also with significant toxicity in derivatives.80 Chloroaminoacridines were found to be less mammals. While mutagenic effects have been reported for active than Aminacrine, a chlorine atom in position 2 or 3 some compounds in vitro,87 the position of the nitro group giving maximum activity.81 in the acridine chromophore is important in this respect. Varying the chromophoric position of chlorine and For example, the anticancer drug Ledakirin is cytotoxic methoxyl substituents was investigated by Singh and due to the close proximity and interaction of the 9-amino coworkers in the immediate post-war period, in research and 1-nitro groups.88 However, the range of antimicrobial initiated by the moderate antibacterial properties of Mep- activities associated with the nitroaminoacridines89 is such acrine. Noticeably, the chloro-/methoxy-substituent couple that they merit re-examination. In particular, the activity gave rise to compounds that were more effective, in gen- of Nitroakridin 3582 [2,3-dimethoxy-6-nitro-9-(3-diethyl- eral, against Gram-negative than against Gram-positive amino-2-hydroxypropylamino)acridine; Figure 1] in the bacteria,62 in line with ethacridine-type activity. treatment of clinical typhus90 is worthy of comment. This The use of bromine and iodine in place of chlorine again and side-chain analogues (e.g. containing the Mepacrine yielded improved antibacterials compared with Aminacrine side chain) were found, in addition, to exhibit low toxicity itself, similar to the chlorine-substituted analogues and in mice at 1–4 mg/day.91 Reports of drug-resistant Rick- having a similar activity profile with respect to Gram type.82 ettsia spp.92,93 suggest that further work on this group of The iodinated Aminacrine derivatives were of sufficient compounds, including the similarly active simpler nitro- promise to warrant animal testing and here they were aminoacridine analogues,94 might be profitable. found to be somewhat less toxic than the parent compound, Although Albert’s basic structure–activity postulate Aminacrine, usually by a factor of two or three. Given the (above) holds true for the majority of acridine derivatives, improvements in activity and toxicity over the lead com- there are exceptions. Acridines containing an amidino pound, it is difficult to explain why such compounds did group [-C(NH)NH ] in position 2 or 3 were synthesized not find their way into contemporary pharmacopoeiae: 2 in order to increase the aqueous solubility of the system. arguments concerning probable mutagenicity were not However, these were almost inactive against bacteria.95 established at the time of the study (1955). In addition, sub- stitution patterns have since been reported that lower mutagenic effects. Quaternary acridines The planarity of the acridine molecule is important in intercalation: this is the reason for the antibacterial activity Quaternized alkoxyacridines were reported to be highly of Aminacrine relative to its tetrahydro analogue tacrine. antibacterial, of low toxicity and also to be non-staining. The presence of a chromophoric methyl group (at carbon Sinflavin (Table) is illustrative of this type, being an active 2) in the Aminacrine structure is reported to be sufficient to antibacterial having chromophoric methoxy rather than reduce both DNA intercalative ability and frameshift amino groups, positive ionization being achieved via the mutagenicity in Salmonella typhimurium TA1537. How- quaternization of the ring nitrogen.11 The low activity of ever, the inclusion of a 2-bromo or 2-iodo substituent this compound against Gram-negative bacteria explains caused increased intercalation relative to the 2-chloro its lack of use.96 The increased basicity of 3,6-dimethoxy- analogue but led to decreased mutagenicity.83 Although the acridine97 compared with the dihydroxy species is now substituent Hammett constants do not enable simple corre- understood to endow the highly antibacterial activity of the lation, the possibility of more extensive structure–activity former, e.g. against streptococcal infection. relationship studies on substituted Aminacrine derivatives In tests against S. aureus, Flavicid (Table) was found aimed at non-mutagenic acridine antibacterials may be to be bactericidal at 10 M, 12 times the activity of the fruitful. standard acriflavine (120 M).12 However, the activity of Alkyl or alkoxyl substitution in positions 4 or 5 is Flavicid against Gram-negative bacteria is very low,13 reported to lead to lowered antimalarial potency in Mepa- leading to its early removal from clinical use. crine analogues,84 although 4,5-dialkyl substitution increases Although there are reported incidences of the activity of activity.85 Again, either substitution pattern increases acriflavine being greater than that of its precursor, pro-

7 M. Wainwright

flavine, quaternization of established acridine molecules is Inclusion of nitrogen at position 1 of the acridine chromo- not necessarily a guarantee of increased antibacterial effi- phore allowed the synthesis of Mepacrin analogues with cacy. However, owing to early evidence of the greater features common to both 4- and 8-aminoquinoline anti- activity against Gram-positive bacteria of acriflavine, com- malarials. The direct Mepacrin analogue, Azacrin, was suc- pared with that of ethacridine,98 methylation of the ethacri- cessful in field trials against malaria17 and such analogues dine molecule was carried out. The resulting quaternary were also highly active against human schistosomiasis. methiodide exhibited a two-fold increase in activity against For example, the 9-(2-(2,3-dihydroxypropylamino)ethyl- S. aureus and similar results against E. coli.99 amino) derivative was effective at 12 mg/kg (55 M/kg) The quinoline nucleus has been used widely to generate daily for 5 days. The toxicity of such compounds is also low, analogues of biologically active acridine compounds and in the order of 800 mg/kg (4 mmol/kg) in mice.105 vice versa. The system, based on two 2- Neither the synthesis nor the antibacterial efficacies of methyl-4-aminoquinoline groups linked by a 10-carbon aza-substituted Aminacrine analogues has been studied in chain at the ring nitrogen positions, forms part of a local depth. 1-Aza analogues of Aminacrine and rivanol were, antibacterial preparation available over the counter in the however, shown to be more active than the parent com- UK (e.g. Dequacain, Crookes Healthcare, UK). Logically, pounds against a haemolytic streptococcal strain.106 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 Reduced acridine systems are synthetic difficulties in the linking of, for example, two molecules of 9-aminoacridine via the ring nitrogens. These Dihydro analogues of the acridine nucleus, the acridans, are thought to result from the steric considerations on the were found to have no activity against a range of bac- inclusion of the third rings of the acridine nuclei compared teria;107 however, there is evidence that the reduced form of with the 2-methyl group in the .100 In addition, acriflavine is strongly bactericidal via an oxidative mech- although the bis-acridine system linked via the same type of anism, viz. the generation of the superoxide radical anion alkyl chain through the 9-amino position is antibacterial,101 together with acriflavine. This is reported to lead to a much this has also been shown to act as a bis-intercalator in more rapid bactericidal effect.108 mammalian cell lines.102 Interestingly, bis(styrylquino- Although the use of tetrahydroacridine derivatives, i.e. linium) compounds, derived from the same 2-methyl-4- the chromophore having two fused, planar aromatic rings aminoquinoline, were reported to be highly bactericidal.103 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 Acridine N-oxides circumstances. As the tetrahydroacridine nucleus is less toxic in mammalian systems than its completely aromatic This group of compounds, originally intended as amoebic- counterpart, the screening of putative antibacterials based ides, was based on the activity of the benz[c]acridine and on the former system is attractive, although, as with the nitroaminoacridine series by Elslager. The activity of one of acridans, there is little to support an intercalative mode of the lead compounds, Nitroakridin 3582, was improved upon action. considerably, e.g. versus Streptococcus C203, the MIC of The activity of 9-amino-1,2,3,4-tetrahydoacridine Nitroakridin 3582 is 5 M, while the MIC of N-oxide is (tacrine) in neurological disorders such as Alzheimer’s dis- 20 nM.19 In addition, the N-oxide was much less toxic in ease is now well established, the drug being an effective mammals, and was effectively antibacterial whether admin- inhibitor of acetylcholinesterase. Owing to hepatotoxic istered orally or subcutaneously. Again, the short side chain considerations,110 a considerable quantity of research into of the active derivatives (-NHCH CHOHCH NEt ) can be 2 2 2 improved analogues is currently underway.111,112 Rather considered to be halfway between the small 9-amino moiety surprisingly, given the range of chromophoric variations and the large aromatic residues of the ana- produced, there is scant evidence for antibacterial screen- logues. The latter compounds were the least active deriva- ing. Although Bindra77 reported (expected) zero activity tives tested against each bacterial strain. against Bacillus subtilis for both tacrine and the cyclo- hepto-fused derivative, the pseudo-planarity of the cyclo- pento analogue was reflected in only slightly lower activity Aza analogues than Aminacrine itself. However, DNA binding data for The substitution of nitrogen for an aromatic -CH is stan- the active cyclopento and inactive cyclohexo derivatives dard biosteric practice in drug development programmes. were similar, and the interactions were weak compared In the case of acridine, this is less straightforward than with Aminacrine. Although the 9-hexylamino derivative of for other aromatic systems owing to the synthetic routes tacrine exhibited similar DNA binding to Aminacrine, no employed, although 9-(substituted amino) derivatives are antibacterial data have been presented, and the nature of produced via the same route as for standard acridines.104 the acridine–DNA interaction is not clear.

8 Acridine—a neglected antibacterial chromophore

Conclusions and future directions DNA; this is after all, the site of action of the simple aminoacridines in bacteria. However, this does not mean It has been established for many years that several of the that because a drug is based on the acridine chromophore acridine dyes are photosensitizers. However, use has not that it will intercalate; indeed, there is sufficient knowledge been made of this property other than in the examination about the system now to allow the synthesis of non-inter- of the experimental photoinduced mutagenicity,113 either calating compounds. In addition, DNA intercalation in of standard dyes such as acridine orange114 or of azido- bacteria does not necessarily imply the same in human labelled analogues.115 Such work is aided by the targeting cells, and there are many new chromophores based on the of DNA, mentioned previously. acridine system that may exhibit modified intercalative Given that dyes such as proflavine and acridine orange behaviour. Also, the number and type of atoms/groups can cause increased mutagenicity, and that the phototoxic attached to the acridine nucleus (let alone in combination side effects of systemically administered Quinacrine are with the newer chromophores) is far from exhaustive, e.g. due to the production of superoxide and singlet oxygen,116 sulphur-containing groups in acridine compounds seem to monoamino analogues including some of the acridine be limited to antimalarial/trypanocidal work.122 antibacterial agents were also tested.117 Increased muta- Useful systemic drugs based on the acridine nucleus will genicity here was also indicative of photodynamic action be discovered only as a result of properly organized drug and, due to the previous use of the aminoacridines as anti- screening programmes. Compounds that have shown con- bacterial agents, encouraged the current author to examine siderable promise in the past have not been properly tested this effect against pathogenic bacteria. In a range of organ- in terms of structure–activity, as they would be in modern isms (including S. aureus and Pseudomonas aeruginosa), pharmaceutical research. Particularly in view of today’s the activity of several of the aminoacridines was increased serious drug-resistance problems, such testing is worth- up to 20-fold,118 being noticeably higher against the Gram- while. 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 References proteins,119 with no increase in antioxidant enzymes, it 1. Schulemann, W. (1932). Synthetic anti-malarial preparations. is likely that the aminoacridines could be used in topical Proceedings of the Royal Society of Medicine 25, 897–905. photodisinfection in cases of colonization with drug- resistant organisms. High photobactericidal activities 2. Browning, C. H., Cohen, J. B., Gaunt, R. & Gulbransen, R. (1922). Relationships between action and chemical con- against methicillin-sensitive and -resistant S. aureus have stitution with special reference to compounds of the pyridine, quino- been reported by the author for the related pheno- line, acridine and phenazine series. Proceedings of the Royal 120 thiazinium photosensitizers. Society 93, 329–66. Owing to the high levels of morbidity associated with 3. Browning, C. H. (1964). Chemotherapy with antibacterial dye- drug-resistant bacterial infections, many different drugs stuffs. In Experimental Chemotherapy, (Schnitzer, R. J. & Hawking, have been used in treatment. Crystal violet (gentian violet) F., Eds), pp. 1–36. Academic Press, New York. was an early antibacterial—indeed Browning used this in 4. Greenwood, D. (1995). Conflicts of interest: the genesis of syn- wound disinfection—and this has been employed in the thetic antimalarial agents in peace and war. Journal of Antimicrobial 121 topical treatment of MRSA outbreaks in Japan. It is not Chemotherapy 36, 857–72. unexpected, then, that the aminoacridines, which have 5. Albert, A. (1966). The Acridines, 2nd edn, p. 403. Arnold, London. been used far more extensively than crystal violet, might 6. Albert, A., Rubbo, S. D. & Burvill, M. I. (1949). The influence of find a therapeutic niche, if only in topical disinfection. chemical constitution on antibacterial activity. IV. A survey of hetero- In terms of systemic treatment there are no established cyclic bases, with special reference to benzoquinolines, phenan- acridine-based drugs suitable for use as intravenously thridines, benzacridines, quinolines and pyridines. British Journal of administered antibacterials. Although the anti-rickettsial Experimental Pathology 30, 159–75. nitroaminoacridines would be a useful starting point, the 7. Albert, A., Rubbo, S. D., Goldacre, R. J., Davey, M. E. & Stone, toxicity and possible mutagenicity associated with the nitro J. D. (1945). The influence of chemical constitution on antibacterial group remains a problem. However, there is little doubt activity. Part II: a general survey of the acridine series. British that position 9 side chains intermediate in size between Journal of Experimental Pathology 26, 160–92. Mepacrine and Aminacrine (e.g. Nitroakridin) would allow 8. Rank, B. K. (1944). Use and abuse of local on sufficient bloodstream half-life without depleting the anti- wounds. Medical Journal of Australia 31, 629–36. bacterial efficacy. Ethacridine/rivanol, often in conjunction 9. Browning, C. H. (1943). The present status of aminoacridine with Nitroakridin, remains in use as an orally administered compounds (flavines) as surface antiseptics. British Medical Journal antibacterial, but usually only for the treatment of shigel- i, 341–3. losis. 10. Turnbull, H. (1944). A rational treatment of gunshot wounds of There remains a feeling of mistrust about acridines owing long bones with established sepsis. Australian and New Zealand to the fact that several examples intercalate efficiently with Journal of Surgery 14, 3–13.

9 M. Wainwright

11. Goldschmidt, R. (1927). Prufung einiger akridinderivate in vitro 31. Keogh, P. P. & Bentley, G. A. (1948). The pharmacology of im tierversuch. Zeitschrift fur Immunitatsforschung 54, 442–5. monacrine. Australian Journal of Science 11, 98–9. 12. Langer, H. (1920). Zur theorie der chemotherapeutischen leis- 32. British Patent no. 367,037—I. G. Farben, Process for the manu- tung. Nach versuchen an akridinium-farbstoffen. Deutsche facture of acridine derivatives (1932). Medezinische Wochenschrift 46, 1015–6. 33. German Patent no. 547,983—Mietzsch, F., Mauss, H. & Klarer, 13. Levrat, M. & Morelon, F. (1933). Contribution à l’étude pharma- J. (1932). codynamique et toxicologique de la trypaflavine, du rivanol et 34. German Patent no. 555,934—Mietzsch, F. & Mauss, H. (1932). d’autres dérivés de l’acridine. Bulletin Science Pharmacologique 40, 582–92. 35. US Patent no. 2,092,131—Mietzsch, F. & Mauss, H., Salts of aminoacridines (1937). 14. Poate, H. G. (1944). Acridines in septic wounds. Use of 5- aminoacridine. Lancet ii, 238–40. 36. Albert, A. (1951). The Acridines, 1st edn, p. 246. Edward Arnold & Co, London. 15. Albert, A. (1951). The Acridines, 1st edn, p. 239. Edward Arnold & Co, London. 37. Hanschell, H. M. (1931). Acute toxic hepatitis after acriflavine— reply. Lancet i, 269–70. 16. Besly, D. M. & Goldberg, A. A. (1954). Antimalarial 2-alkoxy-6- chloro-9-dialkylaminoalkylamino-1:10 diaza-anthracenes. Journal 38. Morgenroth, J., Schnitzer, R. & Rosenberg, E. (1921). Chemo- of the Chemical Society, 2448–55. therapeutic antisepsis. III. A new antiseptic, 2-ethoxy-6,9-diamino- 17. Bruce-Chwatt, L. J. & Archibald, H. M. (1953). Field trials of new acridine hydrochloride (Rivanol). Deutsche Medezinische Wochen- antimalarials in West Africa. British Medical Journal 539–41. schrift 47, 1317–20. 18. Steck, E. A., Buck, J. S. & Fletcher, L. T. (1957). Some 9-amino- 39. Manson-Bahr, P. (1940). In Manson’s Tropical Diseases, 7th 3-nitroacridine derivatives. Journal of the American Chemical Soci- edn, p. 516. Cassell, London. ety 79, 4414–7. 40. Rising, T. J., Fromson, J. M., McEwen, J. & Johnson, P. (1977). 19. Elslager, E. F. & Tendick, F. H. (1962). 9-Amino-2,3-dimethoxy- Absorption studies with the antidiarrhoeal drug in 6-nitroacridine 10-oxides. Journal of Medicinal and Pharmaceutical laboratory animals and man. Arzneimittelforschung 27, 872–8. Chemistry 5, 1149–53. 41. Levrat, M. & Morelon, F. (1933). Toxicité expérimental du 20. US patent nos 2,645,594—Tabern, D. L., Antiseptic acridine rivanol en injection intraveineuse chez le chien et le lapin. Comptes compounds (1953) and 2,684,367—Tabern, D. L., Alkoxyphenyl- Rendus Societé Biologique 114, 643–5. alkoxy-acridines (1954). 42. Albert, A. (1951). The Acridines, 1st edn, p. 234. Edward Arnold 21. Lerman, L. S. (1963). The structure of the deoxyribonucleic acid & Co., London. (DNA)–acridine complex. Proceedings of the National Academy of 43. Mitchell, G. A. G. & Buttle, G. A. H. (1943). Proflavine in closed Sciences, USA 49, 94–102. wounds. Lancet ii, 749. 22. Kreuzer, K. N. (1998). Bacteriophage T4, a model system for 44. Gillespie, M. T., May, J. W. & Skurray, R. A. (1986). Plasmid- understanding the mechanism of type II topoisomerase inhibitors. encoded resistance to acriflavine and quaternary ammonium Biochimica et Biophysica Acta 1400, 339–47. compounds in methicillin-resistant Staphylococcus aureus. FEMS 23. Burres, N. S., Sazesh, S., Gunawardana, G. P. & Clement, J. J. Mcrobiology Letters 34, 47–51. (1989). Antitumor activity and nucleic acid binding properties of 45. Rouch, D. A., Cram, D. S., DiBerardino, D., Littlejohn, T. G. & dercitin, a new acridine alkaloid isolated from a marine Dercitus Skurray, R. A. (1990). Efflux-mediated antiseptic resistance gene species sponge. Cancer Research 49, 5267–74. qacA from Staphylococcus aureus: common ancestry with tetra- 24. Svoboda, G. H., Poore, G. A., Simpson, P. J. & Boder, G. B. cycline- and sugar-transport proteins. Molecular Microbiology 4, (1966). Alkaloids of Acronychia baueri Schott I. Isolation of the alka- 2051–62. loids and a study of the antitumor and other biological properties of 46. McIntosh, J. & Selbie, F. R. (1943). The production of drug- acronycine. Journal of Pharmaceutical Science 55, 758–68. resistant cultures of bacteria in vitro and a study of their inter- 25. Svoboda, G. H. (1966). Alkaloids of Acronychia baueri II. relationships. British Journal of Experimental Pathology 24, 246–52. Lloydia 29, 206–24. 47. McIntosh, J. & Selbie, F. R. (1944). Proflavine-sulphathiazole 26. Queener, S. F., Fujioka, H., Nishiyama, Y., Furukawa, H., mixture. Lancet i, 66. Bartlett, M. S. & Smith, J. W. (1991). In vitro activities of acridone 48. Selbie, F. R. & McIntosh, J. (1943). The action of chemothera- alkaloids against Pneumocystis carinii. Antimicrobial Agents and peutic drugs (including proflavine) and excipients on healthy tissue. Chemotherapy 35, 377–9. Journal of Pathology and Bacteriology 55, 477–81. 27. Jauison, H., Pecker, A. & Medioni, G. (1931). Les accidents de 49. McIntosh, J., Selbie, F. R., Vaughan Hudson, R., Patey, D. H., l’acridinotherapie. Innocuite des doses usuelles. Societe Medicale Parkes, T., McMullen, H. L. et al. (1944). Sulphathiazole–proflavine des Hôpitaux de Paris 47, 397–406. powder in wounds. Lancet i, 591–3. 28. Assinder, E. W. (1936). Acriflavine as a urinary antiseptic. 50. McIntosh, J., Robinson, R. H. M., Selbie, F. R., Reidy, J. P., Lancet i, 304–5. Elliott Blake, H. & Guttmann, L. (1945). Acridine–sulphonamide 29. Sievers, O. & Stein, B. (1936). In Medicine in its Chemical compounds as wound antiseptics. Clinical trials of flavazole. Lancet Aspects, Bayer Leverkusen 3, 60–3. ii, 97–9. 30. Bernstein, F. & Carrié, C. (1933). Zu pharmakologie des 51. Russell, D. S. & Falconer, M. A. (1941). Antiseptics in brain trypaflavins. Dermatologische Zeitschrift 66, 330–5. wounds; experimental study of histological reaction of cerebral

10 Acridine—a neglected antibacterial chromophore tissues to various antiseptic solutions. British Journal of Surgery 28, quinacrine, and 7-{[3-(octylamino)propyl]amino}benz[c]acridine. 472–99. Journal of Medicinal Chemistry 12, 955–7. 52. Rubbo, S. D. (1947). The influence of chemical constitution on 68. Glen, W. L., Sutherland, M. M. J. & Wilson, F. J. (1936). The toxicity. I. A general survey of the acridine series. British Journal of preparation and therapeutic properties of certain acridine deriva- Experimental Pathology 28, 1–11. tives. Part I. Anil and styryl derivatives of 2:8-diaminoacridine and acridine-5-aldehyde respectively. Journal of the Chemical Society, 53. Garrod, L. P. (1940). Action of antiseptics on wounds. Lancet i, 1484–7. 798–9. 69. Glen, W. L., Sutherland, M. M. J. & Wilson, F. J. (1938). Part II. 54. Mitchell, G. A. G. & Buttle, G. A. H. (1943). Diflavine in wound Derivatives of s-(6-amino-2-quinolyl)-5-acridylethenes. Journal of therapy. Lancet i, 287–9. the Chemical Society, 654–7. 55. DuBouchet, L., Spence, M. R., Rein, M. F., Danzig, M. R. & 70. Glen, W. L., Sutherland, M. M. J. & Wilson, F. J. (1943). Part III. McCormack, W. M. (1997). Multicenter comparison of clotrimazole 5-Styrylacridines and their quaternary salts. Journal of the Chemical vaginal tablets, oral and vaginal suppositories Society, 5–7. containing sulfanilamide, aminacrine hydrochloride and allantoin in the treatment of symptomatic trichomoniasis. Sexually Transmitted 71. Glen, W. L., Sutherland, M. M. J. & Wilson, F. J. (1943). Part IV. Disease 24, 156–60. Further 5-styrylacridines and their quaternary salts. Journal of the Chemical Society, 344–7. 56. Egan, T. J., Ross, D. C. & Adams, P. A. (1996). The mechanism of action of quinolines and related antimalarial drugs. South African 72. Albert, A. (1944). Cationic chemotherapy, with special refer- Journal of Science 92, 11–4. ence to the acridines. Medical Journal of Australia 31, 245–8. 57. Rashid, F. & Horobin, R. W. (1990). Interaction of molecular 73. Ungar, J. & Robinson, F. A. (1943). Aminoacridine antiseptics. probes with living cells and tissues. Part 2. A structure-activity Lancet ii, 285–6. analysis of mitochondrial staining by cationic probes, and a discus- 74. Albert, A., Dyer, F. J. & Linnell, W. H. (1937). Chemotherapeu- sion of the synergistic nature of image-based and biochemical tic studies in the acridine series. Part IV. The relations between approaches. Histochemistry 94, 303–8. structure and toxicity. Quarterly Journal of Pharmacology 10, 58. Gamage, S. A., Figgitt, D. P., Wojcik, S. J., Ralph, R. K., Ran- 649–58. sijn, A., Mauel, J. et al. (1997). Structure-activity relationships for the 75. Grigorovsky, A. M. & Veselitskaya, T. A. (1956). Amino- antileishmanial and antitrypanosomal activities of 1 -substituted 9- acriquine and its analogs. Journal of General Chemistry of the anilinoacridines. Journal of Medicinal Chemistry 40, 2634–42. USSR 26, 491–6. 59. Piestrzeniewicz, M. K., Wilmanska, D., Studzian, K., Szemraj, 76. Albert, A. (1966). The Acridines, 2nd edn, p. 438. Arnold, Lon- J., Czyz, M., Denny, W. A. et al. (1998). Inhibition of RNA synthesis don. in vitro by acridines—relation between structure and activity. Zeitschrift für Naturforschung. Section C. Journal of Biosciences 53, 77. Bindra, J. S., Rastogi, S., Patnaik, G. K. & Anand, N. (1987). 359–68. Synthesis, pharmacological activities & physico-chemical properties of 4-(substituted amino/N 4-arylpiperazinyl/aminocarbonyl)-2,3-poly- 60. Lawrence, C. A. (1943). In vitro effects of quinine, atabrine and methylenequinolines. Indian Journal of Chemistry 26B, 318–29. substituted acridine compounds upon Gram negative bacteria. Pro- 78. ceedings of the Society of Experimental Biology and Medicine 52, Domagala, J. (1994). Structure–activity and structure–side- 90–1. effect relationships for the quinolone antibacterials. Journal of Anti- microbial Chemotherapy 33, 685–706. 61. US Patent no. 2,464,171—Britton, E. C. & Coleman, G. H., 79. Wilkinson, J. H. & Finar, I. L. (1947). A study of the properties Derivatives of 9-anilinoacridine (1945). of fluorine-substituted 5-aminoacridines and related compounds. 62. Chaudhri, J. R. & Singh, M. (1948). Antiseptics of the acridine Part I. The monofluoro-5-aminoacridines. Journal of the Chemical series. Part III. Indian Journal of Medical Research 36, 397–404. Society, 759–62. 63. Singh, S. & Chaudri, J. R. (1947). Antiseptics of the acridine 80. Wilkinson, J. H. & Finar, I. L. (1948). A study of the properties series. Part I. Effects of various substituents and loading of the of fluorine-substituted 5-aminoacridines and related compounds. terminal N- in the dialkylaminoalkyl side chain of the type Part II. 5-Amino-2- and -4-trifluoromethylacridines. Journal of the –NH(CH2)4NR2 at position 9 in 3-methoxy-5-chloro-9-aminoacridine. Chemical Society, 32–4. Indian Journal of Medical Research 35, 177–84. 81. Bradbury, F. R. & Linnell, W. H. (1942). Chemotherapeutic 64. Goetchius, G. R. & Lawrence, C. A. (1944). The antibacterial studies in the acridine series. Part VIII. The chloroaminoacridines. effects of various acridine compounds. Journal of Laboratory and Quarterly Journal of Pharmacy and Pharmacology 15, 31–40. Clinical Medicine 29, 134–8. 82. Singh, S. & Singh, M. (1955). Antiseptics of the acridine series. 65. Avery, R. C. & Ward, C. B. (1945). The inhibitory effect of Part IV. In vitro study of antibacterial action of iodoacridines. Indian atabrine and some acridine derivatives upon acid-fast bacilli in vitro. Journal of Medical Research 43, 473–9. Journal of Pharmacology and Experimental Therapy 85, 258–64. 83. Tomosaka, H., Omata, S., Hasegawa, E. & Anzai, K. (1997). 66. Singh, S. & Chaudri, J. R. (1948). Antiseptics of the acridine The effects of substituents introduced into 9-aminoacridine on series. Part II. Effect of changing chlorine atom in N-substituted 3- frameshift mutagenicity and DNA binding affinity. Bioscience, Bio- methoxy-9-amino-acridine from position 5 to 7 and 8. Indian Journal technology and Biochemistry 61, 1121–5. of Medical Research 36, 91–4. 84. Shepard, E. R. & Shonle, H. A. (1948). Nuclear substituted 67. Elslager, E. F. & Worth, D. F. (1969). Antiamebic, antimalarial, 9-(4-diethylamino-1methylbutylamino)-acridines. Journal of the and anthelmintic effects of distal hydrazine analogs of azacrine, American Chemical Society 70, 1979–80.

11 M. Wainwright

85. Hall, D. M. & Turner, E. E. (1945). Structure and antimalarial 103. Glen, W. L., Sutherland, M. M. J. & Wilson, F. J. (1939). The activity. Part I. Some acridine derivatives. Journal of the Chemical preparation and therapeutic properties of certain 4-substituted Society, 694–9. quinoline derivatives. Journal of the Chemical Society, 489–92. 86. Carlson, W. W. & Cretcher, L. H. (1948). The hydroxyethyl 104. US Patent no. 2,650,225—Goldberg, A. A. & Besly, D. M., analog of quinacrine. Journal of the American Chemical Society 70, Manufacture of meso-halogenated aza-acridines (1951). 597–9. 105. US Patent no. 2,775,595—Goldberg, A. A., Substituted 1,10- 87. Ferguson, L. R. & Denny, W. A. (1991). The genetic toxicity of diazaanthracenes (1956); British Patent 769,023—Goldberg, A. A., acridines. Mutation Research 258, 123–60. Improvements in or relating to substituted 1,10-diazaanthracenes 88. Mazerska, Z., Mazerski, J. & Ledochowski, A. (1990). QSAR of (1955). acridines. II. Features of nitracrine analogs for high anti-tumor activ- 106. Besly, D. M. & Goldberg, A. A. (1957). Analogues of amina- ity and selectivity on mice, searched by PCA and MRA methods. crine and rivanol derived from 1:10-diaza-anthracenes. Journal of Anti-Cancer Drug Design 5, 169–87. the Chemical Society, 5085–6. 89. Weston Hurst, E. (1948). Nitroakridin 3582: a compound pos- 107. Bradbury, F. R. & Linnell, W. H. (1938). Chemotherapeutic sessing chemotherapeutic activity against the viruses of psittacosis studies in the acridine series. Part VI. The acridanes. Quarterly and lymphogranuloma venereum. British Journal of Pharmacology Journal of Pharmacy and Pharmacology 11, 240–51. 3, 181–6. 108. Adamus, J., Gebicki, J., Ciebiada, I., Korczak, E. & Denys, A. 90. Miller, C. S. & Wagner, C. A. (1948). 2,3-dimethoxy-6-nitro-9- (1998). 3,6-Diamino-10-methylacridan: uncharged precursor of acri- (-diethylamino--hydroxypropylamino)acridine. Journal of Organic flavine and its unique antimicrobial activity. Journal of Medicinal Chemistry 13, 891–4. Chemistry 41, 2932–3. 91. Greenhalgh, N., Hull, R. & Hurst, E. W. (1956). The antiviral 109. Sargent, L. J. & Small, L. (1947). Studies in the acridine series. activity of acridines in eastern equine encephalomyelitis, Rift Valley IV. Dialkylaminoalkylamines derived from 5,9-, 6,9-, 7,9- and 8(?),9- fever and psittacosis in mice, and lymphogranuloma venereum in dichloro-1,2,3,4-tetrahydroacridines. Journal of Organic Chemistry chick-embryos. British Journal of Pharmacology 11, 220–4. 12, 571–6. 92. Cowan, G. (2000). Rickettsial diseases: the typhus group of 110. Eagger, S. A., Levy, R. & Sahakian, B. J. (1991). Tacrine in fevers—a review. Postgraduate Medical Journal 76, 269–72. Alzheimer’s disease. Lancet 337, 989–92. 93. Drancourt, M. & Raoult, D. (1999). Characterization of muta- 111. Valenti, P., Rampa, R., Bisi, A., Andrisano, V., Cavrini, V., Fin, tions in the rpoB gene in naturally rifampin-resistant Rickettsia L. et al. (1997). Acetylcholinesterase inhibition by tacrine analogues. species. Antimicrobial Agents and Chemotherapy 43, 2400–3. Bioorganic & Medicinal Chemistry Letters 7, 2599–2602. 94. Smadel, J. E., Snyder, J. C., Jackson, E. B., Fox, J. P. & Hamil- 112. McKenna, M. T., Proctor, G. R., Young, L. C. & Harvey, A. L. ton, H. L. (1947). Chemotherapeutic effect of acridine compounds in (1997). Novel tacrine analogues for potential use against Alzheimer’s experimental rickettsial infections in embryonated eggs. Journal of disease: potent and selective acetylcholinesterase inhibitors and Immunology 57, 155–71. 5-HT uptake inhibitors. Journal of Medicinal Chemistry 40, 3516–23. 95. Goldberg, A. A. & Kelly, W. (1947). Synthesis of nuclear 113. Iwamoto, Y., Yoshioka, H. & Yanagihara, Y. (1987). Singlet amidino-derivatives of 5-aminoacridine. Journal of the Chemical oxygen-producing activity and photodynamic biological effects of Society, 637–41. acridine compounds. Chemical and Pharmaceutical Bulletin 35, 96. Albert, A. (1966). The Acridines, 2nd edn, p. 419. Arnold, 2478–83. London. 114. Webb, R. B. & Hass, B. S. (1984). Biological effects of dyes on 97. US Patent no. 1,715,332—Benda, L. & Schmidt, W., Alkoxy- bacteria. VI. Mutation induction by acridine and methylene blue in acridines (1929). the dark with special reference to Escherichia coli WP6(polA1) Mutation Research 137, 1–6. 98. Morgenroth, J. & Schnitzer, R. (1923). Chemotherapeutic antisepsis. V. The action of rivanol on staphylococci. Deutsche 115. Firth, W. J., Messa, A., Reid, R., Wang, R. C., Watkins, C. L. & Medezinische Wochenschrift 49, 745–8. Yielding, L. W. (1984). Identification of an acridine photoaffinity probe for trypanocidal action. Journal of Medicinal Chemistry 27, 99. Weizmann, A. (1947). Quaternary 3-ethoxy-7,9-diaminoacri- 865–70. dinium salts. Journal of the Chemical Society, 1224–5. 116. Molten, A. G., Martinez, L. J., Holt, N., Sik, R. H., Riszka, K., 100. Galanakis, D., Davis, C. A., Del Rey Herrero, B., Ganellin, C. Chignell, C. F. et al. (1999). Photophysical studies on antimalarial R., Dunn, P. M. & Jenkinson, D. H. (1995). Synthesis and quantita- drugs. Photochemistry and Photobiology 69, 282–7. tive structure-activity relationship of dequalinium analogs as K+ channel blockers. Investigations on the role of the charged hetero- 117. Iwamoto, Y., Itoyama, T., Yasuda, K., Morita, T., Shimizu, T., cycle. Journal of Medicinal Chemistry 38, 595–606. Masuzawa, T. et al. (1993). Photodynamic DNA strand breaking activities of acridine compounds. Biological and Pharmaceutical 101. Wainwright, M., Phoenix, D. A., Marland, J., Wareing, D. R. A. Bulletin 16, 1244–7. & Bolton, F. J. (1998). A comparison of the bactericidal and photo- bactericidal activities of aminoacridines and bis(aminoacridines). 118. Wainwright, M., Phoenix, D. A., Marland, J., Wareing, D. R. & Letters in Applied Microbiology 26, 404–6. Bolton, F. J. (1997). In vitro photobactericidal activity of amino- acridines. Journal of Antimicrobial Chemotherapy 40, 587–9. 102. Slama-Schwok, A., Teulade-Fichou, M.-P., Vigneron, J.-P., Taillandier, E. & Lehn, J.-M. (1995). Selective binding of a macro- 119. de Lancestre, H., de Jonge, B. L., Matthews, P. R. & Tomasz, cyclic bisacridine to DNA hairpins. Journal of the American Chemi- A. (1994). Molecular aspects of methicillin resistance in Staphylo- cal Society 117, 6822–30. coccus aureus. Journal of Antimicrobial Chemotherapy 33, 7–24.

12 Acridine—a neglected antibacterial chromophore

120. Wainwright, M., Phoenix, D. A., Laycock, S. L., Wareing, D. R. 122. Obexer, W., Schmid, C., Barbe, J., Galy, J. P. & Brun, R. A. & Wright, P. A. (1998). Photobactericidal activity of pheno- (1995). Activity and structure relationship of acridine derivatives thiazinium dyes against methicillin-resistant strains of Staphylo- against African trypanosomes. Tropical Medicine and Parasitology coccus aureus. FEMS Microbiology Letters 160, 177–81. 46, 49–53. 121. Saji, M., Taguchi, S., Uchiyama, K., Osono, E., Hayama, N. & Ohkuni, H. (1995). Efficacy of Gentian-violet in the eradication of methicillin-resistant Staphylococcus aureus from skin lesions. Jour- Received 26 July 2000; returned 7 September 2000; revised 18 nal of Hospital Infection 31, 225–8. September 2000; accepted 28 September 2000

13