WO1993008165A1 - Dna bis-intercalators - Google Patents

Abstract

Compounds having the formula X-A-Y are disclosed where X and Y are groups capable of intercalating DNA and comprise optionally substituted fused bi- or polycyclic, carbocyclic or heterocyclic at least partially aromatic ring systems having a substantially flat conformation. A is a substantially rigid divalent group which is extended such that X and Y are kept apart at a substantially fixed distance from each other such that X and Y are capable of intercalating different DNA duplexes. The compounds have potential applications as anti-tumour and antiviral agents.

Description

DNA BIS-INTERCALATORS

This invention relates to a novel class of DNA bis-intercalators.

DNA intercalators are compounds which bind to DNA duplexes by inserting between base pairs in the DNA chain. DNA bis-intercalators have two intercalating groups in a single molecule and this enables these compounds to bind twice to a double stranded DNA chain. Many DNA bis-intercalators are known and have been discussed by, for example, LePecq et al. (1975) Proc. Natl. Acad. Sci. USA, 72, 2915-2919, Cannellakis et al. (1976) Biochim. Biophys. Acta, 418, 277-289, Gaugain et al. (1978) Biochem., 17, 5078-5088, Welsh et al. (1987) J. Mol. Biol., 198, 63-71. In all of these references, the two intercalating groups of the molecule are joined together by a flexible linking group about which they can rotate freely. Since the linking group is flexible, the free energy of binding is more favourable i if the intercalation occurs intramolecularly rather than intermolecularly. Hence the binding of one intercalating group inevitably leaves the other in close proximity to other' binding sites in the same duplex and hence relative movement of the intercalating groups leads to intramolecular cross-linking. Wakelin, Medicinal Research Reviews, 1986, 6, 275, has shown that in such cases the intercalating groups insert between base pairs which are only a few nucleotides apart in the DNA chain. Previous bis-intercalators have thus achieved intramolecular rather than intermolecular cross-linking.

The present invention relates to DNA bis-intercalators having two intercalating groups linked by a rigid linker unit which has an extended configuration. The requirement that the linker should be rigid and have an extended configuration ensures that the two intercalating groups are kept apart from each other at an essentially fixed distance and angle. The linker should be rigid so that the intercalating groups are held apart and are extended such that they are kept apart to permit one intercalating group to be free to bind to a DNA duplex when the other is already bound to a different DNA duplex. The binding of both intercalators into the same duplex is thus prohibited unless the duplex folds back on itself to cross-link distant parts of the same molecule. The bis- intercalators with rigid linkers of the present invention, therefore, cross-link DNA duplexes to form intermolecular links or cross-link distant parts of the same molecule. The binding of one intercalating group leaves the other group pointing out from the binding site by virtue of the rigid linker and so available for further binding to another DNA duplex. The bis-intercalating molecules of the present invention are of interest since they may be used to probe the organisation of DNA in three-dimensional space, especially near sites of replication, recombination or topoisomerase action where two duplexes must be in close proximity. This could not be achieved by previous flexibly linked DNA bis-intercalators. Moreover, it can be understood that by binding DNA at a replicating fork (e.g., during cell division), the bis-intercalators of the present invention would be expected to show antibacterial and/or anti-tumour and/or anti-viral and/or antifungal activity. These properties would not be expected to be the same as those of flexibly linked DNA intercalators. In addition, they can be used to determine the kind of structure formed during the synthesis (e.g., by ligation) of large DNA structures from smaller pieces of DNA. A further advantage of the compounds of the present invention is that the linking groups are cleavable by a variety of methods without damaging neighbouring bound and unbound DNA providing further assistance in the investigation of DNA organisation.

The invention accordingly provides a compound having the formula X-A-Y where X and Y may be the same or different and are groups capable of intercalating DNA comprising an optionally substituted fused bi- or polycylic, carbocyclic or heterocyclic, at least partially aromatic ring system having a substantially flat conformation and A is a substantially rigid divalent group which is extended such that X and Y are kept apart at a substantially fixed distance and angle from each other so that X and Y are capable of intercalating different DNA molecules or distant parts of the same molecule.

A may consist of two or more of one or any combination of the following groups: -C≡C- ; anti--N=N- ; trans- -N=CH- ; trans- -CH=CH- ; 1,4-phenylene; 1,4- or 2,5- six-membered heterocycles; optionally substituted by a group replacing a hydrogen atom such that the rigidity of A is substantially unaffected. A should consist of enough of these groups to ensure that there is a chain of adequate length between X and Y, such that they are capable of intercalating to different DNA duplexes. X and Y may be neutral or positively charged polycyclic aromatic nitrogen-containing heterocycles such as, for example, optionally substituted 6-phenanthridine or 10-acridine groups, optionally substituted 5-alkylated, 6-phenanthridinium groups (the alkyl group being branched or unbranched C₁ to C₆ ) or 9-alkylated 10-acridinium groups, the alkyl group being branched or unbranched C₁ to C₆.

The intercalating groups of the present invention may be any flat polycyclic aromatic groups which are capable of intercalating DNA. The term "flat" as used herein refers to the basic polycyclic ring system and not to any substituents on the ring system which may be out of plane. This term will be understood by those skilled in the art .to cover fused aromatic rings which show little or no deviation from a common plane. The intercalating groups may be fully or partially aromatic and may be made up of fused carbocyclic or heterocyclic rings. Any flat polycyclic aromatic groups which bind to DNA by intercalation may be used as the intercalating groups of the compounds of the present invention. Accordingly, the ring system may be substituted in any way which enhances the binding ability to DNA or substantially unaffects it. Preferred intercalating groups are substituted or unsubstituted phenanthridine aⁿd acridine groups. Also preferred are the N- alkylated derivatives of these groups, the phenanthridinium and acridinium salts, where the alkyl group is branched or unbranched C₁-C₆ alkyl, particularly methyl. The anion accompanying these salts in the bis-intercalating co.mpounds of the present invention may be any pharmaceutically acceptable anion, especially halide ion such as iodide. The intercalating group may be attached to the linker part of the molecule at any position on the ring at which substitution may be carried out. For synthetic reasons, presently preferred positions for attachment to the linker are the 6- position for phenanthridine or phenanthridinium and the 10-position for acridine or acridinium groups.

The linker group is that part of the molecule which connects the two intercalating groups in such a way that they are kept apart. This spacing of the intercalating groups is necessary to ensure that once one of the groups is bound to DNA, the other is not bound in the same molecule but is held at a distance from the binding site for possible binding to another DNA duplex or distant parts of the same molecule. The linker group must therefore be rigid in the sense that the conformation keeping the two intercalating groups apart at a significant distance to prevent binding to neighbouring sites on the same DNA molecule is significantly energetically favoured over any conformation which brings the binding groups closer together. The linker group must thus be both rigid and extended in order to maintain the intercalating groups spaced apart from each other such that each may bind to a different DNA duplex or distant parts, of the same molecule. The linker group may be made up of a single repeating unit or a combination of two or more units giving rise to a rigid extended chain. Possible groups constituting the linker include -C≡C-, anti- -N=N-, trans -CH=N-, substituted or unsubstituted trans- -CH=CH-, substituted or unsubstituted aromatic rings linked into the chain in a para-arrangement and no more than one substituted or unsubstituted aromatic ring linked into the chain in a meta-arrangement. Possible aromatic groups in the linker include substituted or unsubstituted: 1,3- and 1,4-phenylene, and 1,4- or 2,5- linked six-membered heterocycles such as pyridine; and 1,4-linked five membered heterocycles such as furan, thiophene and pyrrole. Essentially any group which may form a rigid extended linker may be employed. It will be appreciated that the constituent parts of the linker may be substituted i.e., other groups may replace the hydrogen atoms. Substitution on the linker is envisaged for the compounds of the present invention provided that such substitution does not affect the ability of the linker to be rigid and extended (through steric or electronic effects, for example). Preferred groups constituting the linker in the compounds of the present invention are trans-1,2- vinylene, 1,4-phenylene and 1,4-pyridinium ion. These may be linked as alternating trans-1,2-vinylene and

1,4-phenylene or alternating trans-1,2-vinylene and 1,4-pyridinium ion. The counter ion in the case of

1,4-pyridinium ion containing linkers may be any of a variety of pharmaceutically acceptable anions such as halide ions, for example chloride. The water solubility of bis-intercalators may be altered by varying the groups in the linker.

The presence of vinyl groups in the linker allows for cleavage of the DNA bis-intercalating molecules of the present invention. The olefinic bonds are potential sites of cleavage for the bis- intercalators by a variety of methods when DNA cross- linking has been achieved.

The invention includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent and a bis-intercalating compound of the present invention in an amount effective against tumours and/or bacterial, fungal or viral infections. The pharmaceutical composition may be in a form suitable for topical application to the skin (e.g., for the treatment of skin cancer).

It is envisaged that the bis-intercalators of the present, invention may be of use in the field of molecular electronics. The bis-intercalators with rigid and extended linkers could be used to connect small circles of double stranded DNA. Circles of DNA with as few as 40 base pairs can be made synthetically. The size of the circles of DNA could be used to limit the number of links made to other circles of DNA through the bis-intercalator molecules. The circles of DNA could be used to carry molecular shuttles which fit into either the major or minor groove of the DNA. Alternatively, the molecular shuttle could be a molecular ring/bracelet which could move along the double stranded DNA and carry information from linker (bis-intercalator) to linker.

If one strand of the circular DNA were made entirely of purine and the other of pyrimidine nucleotides it is possible to obtain triple stranded DNA through Hoogsteen base pairing. By this means buffers could be incorporated on to the molecular tracks at any predetermined site since the sequence of the track could be made specific and the complementary sequences of the oligonucleotide buffers would seek out specific sites. This could be used to preclude molecular shuttles from connecting some neighbouring linker pairs but allowing others to be connected by molecular shuttles.

Bis-intercalators of the present invention could also determine the architecture of three-dimensional DNA networks which could be used as tracks for molecular and electronic traffic. Networks can be made by- synthesising large DNA molecules from smaller branched oligonucleotides. Bis-intercalators with rigid and extended linkers could be used during synthesis of the network to determine the geometry of the X- and Y-shaped pieces and so the geometry of the resulting junctions. This in turn would determine which branches were connected. They could also be used after synthesis to regulate the passage of traffic at nodes xn the network permitting the traffic to pass or switching it from one track to another. Intercalating groups can be envisaged which permit or prevent the passage of traffic, so .bis-intercalators of the present invention with appropriate intercalating groups bound close to a node might regulate traffic passing through the node. They might also regulate traffic by changing the geometry of the DNA at the node when they bind, for example, by stacking and unstacking bases.

The compounds of the invention may have applications as herbicides, particularly those with at least two 1,4-pyridinium groups in the linker. The invention, thus, includes the use of the compounds or herbicidally acceptable acid addition salts thereof in compositions for combating undesirable plant growth.

The compounds of the present invention may be prepared by a number of synthetic routes. Presently preferred synthetic routes for the formation of substituted or unsubstituted phenanthridine, acridine, phenanthridinium and acridinium based compounds having 1,2-vinylene and, 1,4-phenylene and/or 1,4-pyridinium diyl containing linkers are outlined in the following reaction scheme.

1 Reaction of 6-methylphenanthridine or its derivatives

(a) 6-methylphenanthridine (and its substituted derivatives) may be condensed with an aldehyde preferably in the presence of an acid chloride with or without a solvent, preferably at an elevated temperature for a time sufficient to substantially complete the reaction.

where Q is branched or unbranched alkyl, substituted or unsubstituted phenyl

A¹ and A² are the same or different optionally substituted aromatic ring systems

R¹, R², R³ and R⁴ are substituents which do not affect the rigidity of the linker or the ability of the compound to act as an intercalator. e.g.

The resulting compounds may subsequently be N- alkylated by a number of well known routes to form a mono-intercalating compound. e.g.

(b) 6 - methylphenanthridine or its derivatives may be condensed with a para-dialdehyde preferably in the presence of an acid chloride, optionally in a solvent preferably at elevated temperature for a time sufficient to substantially complete the reaction in one of two ways:

to form a bis-adduct

These compounds may be bis N-alkylated by a number of known routes to give compounds having the following general structure:

The above compound may also be formed from 5,6- dimethylphenanthridine salts by reaction with a para- dialdehyde preferably in the presence of a base such as piperidine.

(ii) to form a mono adduct

e.g.

2 equivalents of the resulting aldehyde may then be reacted with a bis-phosphonium ylid (preferably formed from the phosphonium salt and a base in situ) in a Wittig reaction with or without a solvent preferably at room temperature for a time to substantially complete the reaction.

The resulting bis-phenanthridine compound may then be bis N-alkylated by any of the methods well known to those skilled in the art to give the following:

2 Reaction of acridine derivatives

(a) 9-methylacridine and its substituted derivatives may be reacted in a number of ways to form mono- and bis-intercalating compounds as outlined in the following scheme.

Step (ii) involves N-alkylation which may be carried out by means of any one of a number of routes well known to those skilled in the art.

Steps (iii) and (iv) involve condensation with an aldehyde or dialdehyde, respectively. These reactions are preferably carried out in the presence of an acid chloride (such as benzoyl chloride) with or without a solvent (e.g., DMF) and preferably at an elevated temperature for a time to substantially complete the reaction.

(b) 9-substituted acridines (e.g., 9-chloroacridine) and their substituted derivatives may be used to form compounds of the present invention in accordance with the following reaction schemes:

Where X is a leaving group such as Cl

and n is O or a positive integer

(c) 9-substituted acridines may also be reacted with substituted or unsubstituted 1,4-bis(4- vinylpyridyl)-benzenes to form the compounds of the invention.

1,4-bis(4-vinylpyridyl)-benzenes may be formed by the condensation of a 4-picoline with two equivalents of a terephthaldicarboxaldehyde:

The 1,4-bis(4-vinylpyridyl)-benzenes react with 9- substituted acridines to form compounds of the present invention:

It will be appreciated that the synthetic routes outlined above are equally applicable to substituted analogues.

EXAMPLES

1. Preparation of bis-intercalating compounds

N.m.r. spectra were recorded on either a Varian Gemini 200 MHz, a Bruker WM300 MHz or AM 500 MHz FT spectrometer all with internal reference. Mass spectral data were obtained in either El or positive FAB mode on a VG Micromass 30FD or 16F spectrometer. All solvents were dried by distillation from suitable drying agents prior to use. Melting points are uncorrected. (i) 5, 6-Dimethylphenanthridinium iodide (2) 6-Methyl-phenanthridine (1) (0.89 g, 4.6 mmol), white needles m.p.84°C prepared in 70% yield (Morgan and Walls, 1931), was dissolved in nitrobenzene (1 ml), methyl iodide (1 ml) was added and the mixture heated at 140°C (oil bath temperature) for 5 h. After cooling to room temperature the resulting crystalline product was collected and washed with copious amounts of benzene followed by diethyl ether to give yellow needles of 5,6-dimethyl phenanthridinium iodide (1.3 g, 84%), m.p. 260°C (decomp) (Found: C, 53.5; H, 4.1; N, 4.1. C₁₅H₁₄NI requires: C, 53.7; H, 4.2; N, 4.2%); ^vmax. (KBr disc) 3080 (aromatic C-H stretch), 3040 (aromatic C-H stretch), 1605 (C=C stretch), 1570 (aromatic C=C stretch); δ_H [300 MHz, (CD₃)₂SO] 3.44 (3H, s, Me), 4.57 (3H, s, Me-N+), 8.02-8.11 (3H, m, H-2, H-3, and H-8), 8.34 (1H, m, H-9), 8.61 (1H, d, J 8 Hz, H-7), 8.90 (1H, d, J 8 Hz, H-4), 9.13 (2H, d, J 8 Hz, H-1 and H-10); m/z ( +ve FAB) 208 (M+ cation).

(ii) 6-p-methylbenzylidenephenanthridine (3)

Benzoyl chloride (3.1 ml, 3.75 g, 0.026 mol) was added to a solution of 6-methylphenanthridine (5.0 g, 0.026 mol) in dry DMF (60 ml) and the mixture was stirred at ambient temperature for 0.5 h. -p-Tolualdehyde (3.0 g, 0.026 mol) was added and the mixture refluxed for 4 h.

After cooling to room temperature the DMF was removed under reduced pressure and the resulting residue was dissolved in ethyl acetate and put in the refrigerator to crystallise. The yellow crystals were recrystallised in ethyl acetate to give bright yellow crystals (2.34 g, 31%), m.p. 149-151°C (decomp.) (Found: C, 89.6%; H, 5.8%; N, 4.7%. C₂₂H₁₇N requires: C, 89.5%; H, 5.8%; N, 4.7%); δ_H (300 MHz, CDCI₃), 8.73 (1H, d, H-1), 8.69 (1H, d, H-10), 8.51 (1H, d, H-4), 8.25 (1H, d, H-7), 8.03 (1H, d, J 15Hz, olefinic-H), 8.15 (1H, d, J 15 Hz, olefinic-H), 7.87-7.73 (4H, m, H-2, H-3, H-8 and H-9), 7.64 (2H, d, benzenoid-H), 7.25 (2H, d, benzenoid-H), 2.37 (3H, s. Me).

(iii) 5-Methyl-6-(p-methylbenzylidene) phenanthridinium Iodide (4)

Methyl iodide was added to a solution of 6-p- methylbenzylidenephenanthridine (0.2 g, 0.678 mmol) in nitrobenzene (30 ml), and the mixture was refluxed for 2 h. The reaction mixture was cooled to ambient temperature and poured into diethyl ether and cooled in a dry ice/acetone bath. The bright yellow solid was filtered and washed with copious amounts of ether and recrystallised from ethanol (0.25 g, 85%). m.p. 243-244°C (decomp.) (Found: C, 63.4%; H, 4.5; N, 3.0%. C₂₃H₂₀IN requires: C, -63.2%; H, 4.6%; N, 3.2%); δ_H [300 MHz, (CD₃)₂SO] 9.24 (1H, d, H-1), 9.16 (1H, d, H- 10), 8.65 (1H, ra, H-4), 7.82-8.43 (6H, m, H-7, H-8, H-9, H-2, H-3,- benzenoid-H), 7,64 (1H, d, benzenoid-H) , 7.38 (1H, t, benzenoid-H) , 7.19 (1H, d, benzenoid-H), 6.96-6.89 (2H, q, olefinic-H), 4.62 (3H, s, Me-N+), 2.14 (3H, s, Me). (iv) 1,4-Bis(phenanthridine-6-vinyl)benzene (5)

6-Methyl-phenanthridine (5 g, 0.026 mol) was dissolved in dry DMF (60 ml). Benzoyl chloride (3.1 ml, 3.75 g, 0.026 mol) was added and the mixture stirred at room temperature for 0.5 h. Terephthaldicarboxaldehyde (1.74 g, 0.013 mol) was then added and the mixture heated under reflux for 5 h. After cooling to room temperature the DMF solution was poured into excess hydrochloric acid and steam distilled until 1200 ml of distillate was collected. The acidic residue was made alkaline by the addition of 0.88 ammonia and the yellow precipitate collected and recrystallised from chlorobenzene (200 ml) to give (5) as a mat of fine bright yellow, feathery needles (4.73 g, 75%), m.p. 300°C (Found: C, 89.3; H, 5.0; N, 5.8. C₃₅H₂₄N₂ requires C, 89.2; H, 5.0; N, 5.8%); m/z (El) 484 (M+, 100%); due to lack of solubility of the compound it proved impossible to obtain an N.M.R. spectrum. It was N-methylated without further purification. (v) 1,4-Bis(N-methylphenanthridinium-6-vinyl)benzene di-iodide (6)

a) 1,4-Bis(phenanthridine-6-vinyl)benzene (148 mg, 3.1 mmol) was dissolved in hot nitrobenzene (30 ml). Methyl iodide (2 ml) was cautiously added and the mixture heated at 140°C (oil bath temperature) for 4 h. After cooling to room temperature the resulting crystals were collected and washed with diethyl ether. Proton n.m.r. of this crystalline solid shows it to be the desired product, however it contains one molecule of nitrobenzene as solvent of crystallisation per molecule of (6). Treating the solid with boiling ethanol removes the nitrobenzene to give a pure sample (185 mg, 79%), m.p. >300°C, (Found: C, 59.5; H, 3.9; N, 3.5. C₃₈H₃₀N₂I₂ requires: C, 59.4; H, 3.9; N, 3.6%); δ_H p00 MHz, (CD₃)₂SO] 4,67 (6H, s, Me-N+), 7.5 (2H, d, J 17 Hz, vinylic-H), 8.07-8.21 (12H, m, H-2, H-2', H-3, H-3', H-8, H-8', benzene-H and vinylic-H), 8.41 (2H, m, H-9 and H-9'), 8.68 (4H, m, H-4, H-4', H-7 and H-7'), and 9.22 (4H, d, J 8 Hz, H-1, H-1'. H- 10, and H-10'); m/z ( +ve FAB) 514 (M+. reduced cation). b) 5,6-Dimethyl phenanthridinium iodide (0.113 g, 0.337 mmol) was dissolved in hot ethanol (30 ml) and terephthaldicarboxaldehyde (22 mg, 0.168 mmol) was added followed by piperidine (0.5 ml). The mixture was heated under reflux for 2 h and then half the solvent was removed by distillation. On cooling to room temperature the desired product (6) precipitated as a dark red powder which was collected and dried (50 mg, 39%) . Analysis showed this material to be identical to that synthesised in a).

(vi) Phenanthridine-6-vinyl-1,4-benzenecarboxaldehyde

(7)

6-Methylphenanthridine (1.17 g, 6 mmol) was dissolved in dry DMF (20 ml). Benzoyl chloride (1 ml) was added and the solution stirred at room temperature for 30 min during which time a precipitate formed. Terephthaldicarboxaldehyde (0.811 g, 6 mmol) was then added and the mixture heated under reflux for 5 h. After cooling to room temperature the DMF solution was added dropwise to water (400 ml) and the resulting yellow precipitate collected by filtration and recrystallised from ethanol to give the aldehyde (7) (1.2 g, 65%) m.p. >250°C. δ_H (300 MHz, CDCl₃), 7.68 (1H, m, H-8), 7.74 (1H, m, H-3), 7.76 (1H, m, H-2), 7.87 (1H, m, H-9), 7.87 (4H, AA'BB' , benzene ring-H), 8.09 (1H, -d, J 16 Hz, vinyl-H), 8.17 (1H, d, J 16 Hz, vinyl-H), 8.22 (1H, d, J 8 Hz, H-7), 8.44 (1H, d, J 8 Hz, H-4), 8.65 (1H, d, J 8 Hz, H-10), 8.67 ( 1H , d, J 8 Hz, H-1), 10.04 (1H, s, aldehyde-H); m/z (El) 309 (M+ 100%).

(vii) 1,4-Bis [N-methylphenanthridinium-6-(1,4-vinyl-benzenevinyl] benzene (9)

Sodium ethoxide (0.374 g, 5.5 mmol, as a 21% w/v solution in ethanol) was added slowly to a solution of phenanthridine-6-vinyl-1,4-benzenecarboxaldehyde (7) (1.7 g, 5.5 mmol) and p-xylylenebis-(triphenylphosphonium) bromide (2.17 g, 2.75 mmol, Aldrich Chemical Co. Ltd. ) in dry DMF (50 ml) at room temperature. The red colouration generated on addition of sodium ethoxide solution was allowed to dissipate before addition of a further drop of the ethoxide solution. After addition was complete the mixture was stirred at room temperature for 3 h, poured into water (500 ml) and the resulting solid collected and washed with copious amounts of hot methanol. The resulting solid was recrystallised from chlorobenzene to give (8) (0.62 g, 33%) m.p. >250°C; m/z (El) 688 (M+).

Methyl iodide ( 3 ml) was added cautiously to a solution of (8) (0.3 g, 0.44 mmol) in nitrobenzene (10 ml) at 140°C. The mixture was heated with stirring at 140°C (oil bath temperature) for 5 h. After cooling to room temperature the mixture was added dropwise to diethyl ether ( 500 ml) and the red precipitate collected by filtration. The product was recrystallised from methanol (100 ml) to yield the bis-phenanthridinium salt (9) (0.3 g, 69%) , m.p. >250°C. δ_H(300 MHz, ( CD₃ ) ₂SO ) 4.65 (6H, s, Me-N+).

7.42 (2H, d, J 14 Hz, vinyl-H), 7.47 (4H, s, central benzene ring) , 7.72 (4H, m, vinyl-H) , 7.85 (8H, AA'BB' , benzene ring-H), 8.03-.8.25 (8H, m, H-2, H-2¹ , H-3, H-3¹ , H-8, H-8' , and vinyl-H) , 8.38 (2H, m, H-9, H-9' ), 8.64 (2H, d, J 8 Hz, H-7 and H-7' ), 8.70 ( 2H, d, J 8 Hz, H-4 and H-4' ), 9.19 (4H, d, J 8 Hz, H-1, H-1' , H-10 and H-10' ); m/z (El) 359(M++).

(viii) 9,10-Dιmethylacridiniurn Iodide (11)

9-Methylacridine ( 10) (3.7 g, 19 mmol) a yellow crystalline solid m.p.117-118 °C prepared in 66% yield, (Tsugo et al. , 1963) and methyl iodide (3.7 ml) were heated together under reflux for 48 h. After cooling to room temperature the mixture was added dropwise to diethyl ether (200 ml) and the resulting precipitate collected by filtration and washed with boiling diethyl ether (2 × 400 ml). The solid was recrystallised from water to give the desired product (4.6 g, 72%), m.p. 245°C (decomp.) (Found: C, 53.4; H, 4.2; N, 4.0. Calc. for C₁₅H₁₄NI: C, 53.7; H, 4.2; N, 4.2%); δ_H (200 MHz; (CD₃)₂SO) 3.5 (3H, s. Me), 4.8 (3H, s, Me-N+), 8.05 (2H, m, H-2 and H-7), 8.4 (2H, m, H-3 and H-6), 8.75 (2H, d, J 8.7 Hz, H-4 and H-5), 8.95 (2H, d, J 8.7 Hz, H-1 and H-8); m/z (+ve FAB) 208 (M+).

(ix) 9-p-methylbenzylideneacridine (12)

Benzoyl chloride (1.5 g, 1.2 ml, 10.5 mmol) was added to 9-methylacridine (5.2 g, 10.5 mmol) dissolved in dry DMF (25. ml). The mixture was stirred at ambient temperature for 20 min. p-Tolualdehyde (1.26 g, 10.5 mmol) was added and the reaction mixture refluxed for 5 h. After cooling to room temperature, the solvent was removed under vacuum, dilute ammonia solution was added to neutrality. The organic material was extracted with ethyl acetate and the extract washed with brine, and sodium bicarbonate solution and dried (MgSO₄). The ethyl acetate was removed to give a brown oil which was column chromatographed (silica, 3:lr hexane/ethyl acetate). The second fraction was collected and crystallised from hexane/ethyl acetate to give yellow crystals (1.89 g, 25%) of (12), m.p. 167-169°C (decomp.) Found: C, 89.4%; H, 5.7%; N, 5.0% C₂₂H₁₇N requires: C, 89.5%; H,. 5.7%; N, 4.8%; δ_H (300 MHz, CDCl₃) 8.35 (2H, d, H-1 and H-8), 8.27 (2H, d, H-4 and H-5), 7.81 (2H, m, H-3 and H-6), 7.61 (2H, d, benzenoid-H), 7.55 (2H, m, H-2 and H-7), 7.29 (2H, d, benzenoid-H), 7.05 (1H, d, olefinic-H), 6.99 (1H, d, olefinic-H), 2.45 (3H, s, Me). (x) 10-Methyl-9-(p-methylbenzylidene)acridinium Iodide (13)

9-p-Methylbenzylideneacridine (12) (0.2 g) was dissolved in excess nitrobenzene (20 ml), methyl iodide (10 ml) was added, and the mixture refluxed for 2 h. The reaction mixture was cooled, poured into ether cooled in a dry ice/acetone bath, and the deep maroon solid was filtered and washed with copious amounts of ether. The solid was recrystallised from ethanol (0.24 g, 82%). m.p. 260-263°C. (Found: C, 67.7%; H, 4.7%, N, 2.9%. C₂₃H₂₀IN requires: C, 67.7%; H, 4.6%, N, 3.2%). δ_H (300 MHz; (CD₃)₂SO) 8.80 (2H, d, H-1 and H-8), 8.55 (2H, d, H-4 and H-5), 8.43 (2H, t, H-2 and H-7), 7.89-7.94 (2H, t, H-3 and H-6), 7.43-7.55 (2H, q, olefinic-H), 6.71-6.84 (4H, m, benzenoid-H), 4.88 (3H, s, MeN+), 2.15 (3H, s, Me).

(xi) 1,4-Bis(acridine-9-vinyl)benzene (14)

9-Methylacridine (10) (5.2 g, 10.5 mmol) was dissolved in dry DMF (25 ml), benzoyl chloride (1.5 g, 1.2 ml, 10.5 mmol) was then added and the mixture stirred at room temperature for 20 min. Terephthaldicarboxaldehyde (0.7 g, 5.25 mmol) was added and the reaction mixture heated under reflux for 5 h. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure, hydrochloric acid was added to the residue and the solvent evaporated to dryness. The gum was triturated with dilute ammonia and the resulting solid collected and washed with more dilute ammonia then water. The crude product was recrystallised from chlorobenzene to give (14) as an orange solid (0.7 g, 28%), m.p. >300°C (Found: C, 89.2%; H, 5.2; N, 5.3. C₃₆H₂₄N₂ requires C, 89.2; H, 5.0; N, 5.7%); fo (300 MHz, CDCl₃) 7.12 (2H, d, J 17 Hz, vinylic-H), 7.57 (4H, m, H-2, H-2', H-7 and H-7'), 7.8. (4H, d, J 9 Hz, H-3, H-3', H-6, and H-6'), 8.0 (2H, d, J 17 Hz, vinylic-H), 8.28 (4H, d, J 9 Hz, H-4, H-4', H-5, and H-5'); m/z (DCI NH₃ ) 485 (M +1).

(xii) 1,4-Bis(N-methylacridinium-9-vinyl)benzene diiodide (15)

Methyl iodide (2 ml) was added cautiously to a hot solution of (14) (18 mg) in nitrobenzene (1.5 ml). The solution was heated at 140°C (oil bath temperature) for 6 h. After cooling to room temperature the mixture was added dropwise to dry benzene and the resulting precipitate collected, washed with benzene, diethyl ether and dried (MgSO₄).

The bis-acridinium salt (15) was obtained as a dark red powder (21.2 mg, 74%), m.p. >250°C (Found: C,

59.2; H, 3.9; N, 3.8. C₃₈H₃₀N₂l₂ requires: C, 59.4; H, .

3.9r N, 3.6%)r δ_H (300 MHz, (CD₃)₂SO) 4.84 (6H, s, me-N+), 7.35 (2H, d, J 17 Hz, vinylic-H), 8.03 (4H, m,

H-2, H-2¹, H-7, and H-7'), 8.18 (4H, s, benzene-H),

8.46 (4H, m, H-3, H-3', H-6, and H-6'). 8.75 (2H, d, J

17 Hz, vinylic-H), 8.79 (4H, d, J 8.8 Hz, H-4, H-4' ,

H-5, and H-5' ), 8.89 (4H, d, J 8.8 Hz, H-1, H-1', H-8, and H-8' ); m/z (+ve FAB) 514 M.⁺ reduced cation), 499 (M.⁺-Me).

(xiii) N,N'-Bis(9-acridine)-4,4' -dipyridyliurm dichloride (16)

9-Chloroacridine (0.214 g, 1.0 mmole) and 4,4'-dipyridyl (0.078 g, 0.50 mmole) were intimately mixed before heating in an oil bath at 120°C until the mixture melted and then solidified. The temperature of the oil bath was then raised to 150°C for 20 min.

After the reaction mixture had cooled it was dissolved in hot ethanol and the solution kept at 0°C. The crystals were filtered off and recrystallised from ethanol to give an orange powder (0.22 g, 68%), (Found: C, 66.9; H, 4.7; N, 8,5. C₃₆H₂₄Cl₂N₄. 3.5H₂O requires C, 66.9; H, 4.7; N, 8.8%), δ _H (200MHz, d₆-DMSO) 10.00 (d, 4H), 9.49 (d, 4H), 8.50 (d, 4H), 8.13 (t, 4H), 7.90 (t, 4H), 7.70 (d, 4H).

(xiv) N,N'-Bis(9-acridine)-4,4'-trans-vinylidenedipyridylium dichloride ( 17 )

9-Chloroacridine (0.214 g, 1.0 mmole) and 4,4'-trans-vinylidenedipyridine (0.091 g, 0.50 mmole) were intimately mixed before heating in an oil bath at 120°C until the mixture melted and solidified. The temperature of the oil bath was then raised to 150°C for 30 min. After the reaction mixture had cooled it was dissolved in hot ethanol and the solution kept at 0°C. The crystals were filtered off and recrystallised from ethanol to give a red-brown powder (0.15 g, 46%), (Found: C, 70.4; H, 4.9; N, 8.9. C₃₈H₂₆Cl₂N₄. 2H₂O requires C, 70.7; H, 4.7; N, 8.7%), δ _H (200MHz, de-DMSO) 9.64 (d, 4H), 8.95 (d, 4H), 8.80 (s, 2H), 8.42 (d, 4H), 8.07 (t, 4H), 7.81 (t, 4H), 7.69 (d, 4H). (xv) 1,4-bis(4-vinylpyridyl)benzene (18)

Benzoyl chloride (6g, 43mmol) was added slowly to a solution of 4-methylpyridine (4g, 0.043mol) in dimethylformamide (50ml) at room temperature. After stirring for 0.5h at room temperature the solution was heated at 50°C for 0.5h, a solution of terephthaldi-carboxadehyde (2.88g, 21.5mmol) in DMF (20ml) was then added and the reaction mixture heated under reflux for 12h. The resulting solution was cooled to room temperature and poured into water (300ml) which was rendered basic by addition of 0.88 ammonia. The crude product was collected by filtration and dissolved in hydrochloric acid (2M, 200ml) . Impurities were removed by filtration and the acidic solution neutralised by addition of 0.88 ammonia. The resulting precipitate was collected and recrystallised from toluene to give JL8_^ (3.96g, 65%) m.p. 272-273°C; (Found: C, 8.3.6; H, 5.7; N, 9.4. C₂₀H₁₆N₂ requires C, 84.2; H, 5.6; N, 9.8%); δ_H (d₆ DMSO, 200 MHz) 7.27 (1H, J 16.5Hz, vinylic-H) , 7.52 (1H, J 16.5Hz, vinylic-H) , 7.41 and 8.56 (8H, A₂B₂ J 20.1 Hz, pyridinyl-H), 7.7 (4H, s, benzenoid-H); m/z (El) 284 (M⁺).

(xvi) N,N'-Bis(9-acridine)-4,4'-(vinyl-p-phenylenevinyl)dipyridinium dichloride (19)

9-Chloroacridine (0.230g, 1.1 mmol) was dissolved in nitrobenzene (5ml) and the solution was heated to 130°C. A solution of 1,4-bis(4-vinylpyridyl)benzene (0.153g, 0.53mmol) was added and the mixture heated at 140°C for 3h. After cooling to room temperature the nitrobenzene solution was added dropwise to benzene (100ml) and the solid precipitate collected by filtration. The crude material was purified by recrystallisation from ethanol to give 19 as its tetrahydrate (0.23g, 55%), m.p.>250°C; (Found: C, 70.8; H, 4.7; N, 7.3. C₄₆H₃₂N₄Cl₂.4H₂O requires C, 70.8, H, 5.1; N, 7.2%); δ_H (200 MHz d₆ DMSO) 7.63 (d J 8.5 Hz, 4H, 4-, 5-, 4'-, 5'-H) 7.81 (m, 4H, 3-, 6-, 3'-, 6'-H), 7.69 and 8.16 (AB J 16Hz, 4H, vinylic-H), 8.05 (m, 8H, 2-, 7-, 2'-, 7'- and phenylene-H), 8.44 (d J 9.2Hz, 4H, 1-, 8-, 1'-, 8'-H) , 8.70 and 9.39 (A₂B_2, J 14.0Hz, 4H pyridinyl H).

2. Results of tests of the compounds of the invention for anti-tumour activity (a) Cell-killing in vitro:

Compounds were evaluated using three cell lines: L1210 (mouse leukemia) , ADJ/PC6 (mouse plasmacytoma) and CHI (human ovarian carcinoma).

IC₅₀ (ug/ml) - 2 days

Compound L1210 ADJ/PC6 CHI

11 3 5.6

13 0.27 1.5

15 0.38 1.1

2 1.5 5.1

4 0.13 0.48

6 0.6 4.1

9 0.12 0.08

IC₅₀ (μM)

Compound L1210 ADJ/PC6 CHI

9-acridine> 100 16.0 25.0

pyridinium chloride

16 24.5 > 100 2.5

17 24.5 16.0 1.6

19 14.0 > 50 0.52

(b) Anti-tumour activity in vivo

Compounds were evaluated using the ADJ/PC6 plasmacy toma, an alkylating-sensitive solid mouse tumour. They were administered as a suspension in 10%

DMA/arachis oil given by a single in traper itoneal injection. All compounds gave <90% inhibition of tumour growth.

Compound LD₅₀ % inhibition at max. tolerable dose

13 17.5 48

15 470 44

9 142 28

(c) In vitro cytotoxicity evaluation against six human ovarian carcinoma cell lines

IC₅₀ (μM)

Compound HX/62 SKQV-3 CHI CHlcisR A2780 A2780 cisR

16 22.5 50 5.6 12 4.5 20.7

17 19 40 7.7 12.7 14.1 19.2

19 33 90 6.2 6 15 7.4

9-acridine > 100 >100 11 26 110 83

-pyridinium

chloride

Cisplatin 12.6 4.4 0.1 0.65 0.33 5.2

CHlcisR and A2780cisR are lines with derived resistance to the antitumour agent, cisplatin, from their respective parent lines. The result for cisplatin is included for reference.

The compounds were made up immediately prior to adding to the cells in either water or (for 19) in DMSO.

Compounds were present throughout a 96h incubation of cells. Thereafter, the growth inhibitory properties of the compounds were assessed using a protein stain ( sulforhodamine B).

The data show the potential for the compounds of the invention for use as anti-tumour agents. Compounds 17 and 19 show good retention of potency against the two cell lines with acquired res.istance to cisplatin. 3. Anti-viral activity a) Anti-HSV activity

Herpes Simplex Virus types 1 (HSV 1) and ( HSV 2) were assayed in monolayers of Vero cells in multiwell trays. The virus strains used were SC16 and

186 for HSV-1 and HSV-2 respectively. Activity of compounds was determined in the plaque reduction assay, in which a cell monolayer was infected with a suspension of the appropriate HSV, and then overlaid with nutrient agarose in the form of a gel to ensure that there was no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient agarose overlay. Plaque numbers at each concentration were expressed as percentages of the control and a dose-response curve was drawn. b) Anti-CMV activity

Human cytomogalovirus (HCMV) was assayed in monolayers of either MRC5 cells (human embryonic lung) in multiwell trays. The standard CMV strain AD 169 was used. Activity of compounds is determined in the plaque reduction assay, in which a cell monolayer is infected with a suspension of HCMV, and then overlaid with nutrient agarose in the form of a gel to ensure that there is no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient agarose overlay. c) Anti-VZV activity Clinical isolates of varicella zoster virus

(VZV) were assayed in monolayers of MRC-5 cells. MRC-5 cells are derived from human embyonic lung tissue. A plaque reduction assay was used in which a suspension of the virus stock was used to infect monolayers of the cells in multiwell trays. A range of concentrations of the compound under test of known molarity was added to the wells.

The following table shows the activity of the compounds of the invention:

Herpes simplex Herpes simplex Varicella zoster Human HIV 1 type 1 type 2 Cytomegalovirus

Compound Testing @ Testing @ Testing Testing @ Testing @

10 & 100 μM 10 & 100 μM 40 μM 100 μM 5 & 50 μM

5 Inactive Inactive Slight Toxicity Inactive & Toxic Inactive

11 Inactive @ 10, Inactive @ 10, Toxic Toxic Inactive @ 5

Toxic @ 100 Toxic @ 100 Toxic @ 50

Inactive @ 10, Inactive @ 10, Toxic Toxic Toxic @ 5

Slight Toxicity 77% Inhibition

@ 100 & Slight

Toxicity @ 100

15 Inactive Inactive Slight Toxicity Active Inactive @ 5,

Toxic @ 50

16 Inactive Inactive Slight Toxicity Toxic Active @ 50

but Slight Toxicity @ 5 & 50

AP Inactive @ 10, Inactive @ 10, Inactive Toxic I nact ive @ 5 ,

90% inhibition 100% inhibition Toxic @ 50

(3 100 @ 100

2 Inactive @ 10, Inactive @ 10, Toxic Toxic Tox ic @ 5

83% inhibition 82% inhibition

@ 100 @ 100

17 Inactive @ 10, Inactive (3 10, Inactive Toxic Inactive &

100% inhibition 100% inhibition Slightly Toxic

(a 100 @ 100 @ 5, Toxic @ 50

Evidence for bis-intercalation of DNA by the compounds of the present invention.

In order to study the DNA intercalation and cross-linking properties of the compounds of the present invention, an indirect, but decisive, assay for unwinding (and so intercalation) and knotting (and so cross-linking) has been developed. The test compound is allowed to bind to linear DNA molecules and alter their shape. Then the linear molecules are treated with DNA ligase; some are ligated end-to-end, others into circles, catenanes or knots. (Wasserman and Cozzarelli (1986) have reviewed the structure of knots and catenanes). If the test compound is an intercalator (either mono- or bis-), its removal has little effect on the overall shape of the linear forms but it compacts the circular forms by inducing compensatory supercoiling in them. If the test- compound also cross-links, ligation of entwined linear molecules yields catenanes. Cross-linking distant parts of the same molecule inevitably leads to knotting; as binding sites are helically arranged, cross-linking tends to intertwine the two parts and subsequent circularisation then produces a knot. The various different structures (i.e. supercoils, catenanes, knots, etc. ) are resolved by gel electrophoresis after removal of the test compound (Keller, 1975; Wasserman and Cozzarelli, 1986).

This assay has an important advantage over others. Inter-molecular bis-intercalators can be expected to be only weak cross-linkers as the entropic factor involved in bringing two DNA molecules together is large. The bis-intercalators of the present invention (6), (9) and (15) were indeed relatively weak cross-linking agents and only soluble in aqueous buffers at concentrations up to about 10 μg/ml. Fortunately, intercalation and cross-linking can be demonstrated in the accessible concentration range because a minor fraction of a supercoiled or knotted form can be detected in the presence of an excess of other forms. However, the assay is limited if the agents inhibit the DNA ligase (Montecucco et al., 1990). The ligation assay

pSVtkneo, a 5.3kbp plasmid (Townsend et al., 1984), was linearized with Hind III (2 units/ μg DNA; lh; 37°C), ethanol precipitated and redissolved. Ligations generally contained 250 - 750ng linear DNA in ImM ATP, 10mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 1 unit T4 DNA ligase (Boehringer) and various concentrations of test compound in a final volume of 250μl. Compounds (16) and (17) were dissolved in water and used immediately. The remaining test compounds were dissolved in dimethylsulphoxide and control experiments showed that this solvent had no effect on the mobility of the ligated products at the concentrations used. After incubation on ice overnight, the ligated DNA was ethanol precipitated to remove the test compound, redissolved in a sample buffer containing 1% sodium dodecyl sulphate and then subjected to electrophoresis in 0.8% agarose gels containing 40 mM Tris, 2 mM EDTA and 20 mM sodium acetate ( pH 8.3), and the gel stained with ethidium and photographed (Keller, 1975; Maniatis et al., 1982). In some cases samples were gammairradiated (1180J/kg; Cook et al. 1976) in the sample buffer used for electrophoresis; control experiments showed that this dose nicked >99% supercoiled molecules. The Phenanthridinium Series: intercalation

Fig. 1 illustrates a typical ligation assay. Linear DNA was ligated in the presence of different concentrations of the compounds, the compounds removed and the ligation products resolved electrophoretically before staining and photographing the resulting gel. Lane 1: lambda/Hind III markers. Lane 2: unligated DNA. Lane 3: linear DNA ligated in the absence of any test compound. Lane 20: ligation in 2% dimethylsulphoxide, the maximum concentration of solvent present during ligation. The positions of forms II and III are indicated. Linear DNA runs as a single band (form III, lane 2) and most is ligated in the absence of any test compounds into a complicated set of products (lane 3). At the DNA concentration used, the majority of these are circles. As some linear molecules originally contained nicks, some circles are nicked and run just behind the linear molecules. Such relaxed circles (i.e. form II molecules) constitute a background present whenever the ligase is active. However, about half the circles contain no nicks and run as a number of bands slightly ahead of the nicked circles. These are topoisomers containing positive supercoils generated after ligation by the duplex unwinding that occurs on transfer of DNA from the ligation buffer (containing a high Mg2⁺ concentration) into the electrophoresis buffer (Depew and Wang, 1975). There are additional, but minor, bands at the top of the gel formed by end-to-end ligation which give linear molecules of two or more unit lengths, together with their circular and catenated counterparts (both supercoiled and relaxed). (As these forms are difficult to identify, DNA Is ligated at a low concentration to minimise their formation). If the known intercalating agent, ethidium (Et), is present during ligation, a different pattern of ligation products is obtained. At lower concentrations of ethidium than those shown (i.e. 0.1 μg/ml), there is little intercalation and the double helix is only slightly unwound. When ethidium is removed after ligation and the DNA transferred into electrophoresis buffer, intact circles, already slightly unwound, need to unwind less. As a result, the topoisomers are less positively supercoiled than those in lane 3 and run more slowly. As the concentration of ethidium is increased, its unwinding eventually balances that due to transfer between buffers and the topoisomers are centred around the mobility of the relaxed circle. At higher ethidium concentrations, intercalative unwinding becomes larger than the slight effects due to transfer between buffers. Then, DNA unwound by intercalation, rewinds on transfer to electrophoresis buffer, inducing negative supercoils. These run rapidly as an unresolved group of topoisomers (lane 4). After ligation in lμg/ml ethidium, this group of topoisomers is even more negatively supercoiled (lane 5). At a high concentration of ethidium, 3μg/ml, ligase is inhibited (lane 6; Montecucco et al., 1990). Intercalating agents are known to have these characteristic effects on DNA supercoiling (Wang et al., 1983).

This shift from positively-supercoiled topoisomers, through a relaxed group to negatively- supercoiled forms is shown clearly by the mono- intercalator, (2), as its concentration is increased. In lane 13, most topoisomers are fully relaxed and as the concentration is increased they become more negatively supercoiled, migrating faster (lanes 14- 19). Unlike ethidium, ligase is not inhibited at 10 μg/ml (lane 19) but is inhibited by higher concentrations (results not shown). The bifunctional compound, (9), shows a similar pattern, but begins to inhibit at 1.5 μg/ml (lane 10), with almost complete inhibition occurring at 3 μg/ml (lane 12). Between 1.5 and 3 μg/ml, some DNA has aggregated (presumably as catenanes) and cannot migrate far into the gel (lanes

10-12), providing circumstantial evidence for cross- linking. These results, and others for the remaining members of this series (Fig. 3), show that all unwind like ethidium. Unfortunately, inhibition of the ligase precludes testing at higher concentrations. The Phenanthridinium Series: evidence for knotting

A bis-intercalating cross-linking agent would be expected to promote catenation and knotting by DNA ligase because its binding sites are arranged helically (Fig. 2A). It is clear from Fig. 1 that the pattern produced by the mono-intercalator, (2), is broadly similar to that produced by the possible bis- intercalator, (9); therefore the latter cannot be a powerful bis-intercalator. However, close Inspection of Fig. 1, lane 11 shows there to be an extra faint band just below the linear molecule (i.e. form III), in the position characteristic of a relaxed knot with 3 nodes, the trefoil (Dean et al., 1985; Wasserman and Cozzarelli, 1986). This suggests that (9) might be acting as a weak cross-linking agent, even at these low concentrations.

Trefoils - as well as more complicated knots - are formed as the result of any ligation, but their concentration is usually too low to detect. Even if their concentration is artificially increased, their presence In gels is often obscured by the presence of supercoiled topoisomers that have similar mobilities. Therefore they are usually detected after removing the supercoiled forms by nicking (Wasserman and Cozzarelli, 1986). Fig. 2B illustrates such an experiment where the products formed by ligation have been nicked by gamma-irradiation. Linear DNA was ligated (750ng in 250 μl) in the presence of different concentrations of the compounds, the compounds removed, some samples nicked and the ligation products resolved electrophoretically before staining and photography. Lane 1: lambda/Hind III markers. Lane 2: linear DNA. Lanes 3, 4: linear DNA ligated in the absence of any test compound. The positions of forms II and III are indicated on the left. White lines between lanes 13 and 14 indicate the position of the knots.

Linear DNA was ligated in the presence of ethidium ( Et), (2), or (9) and the products analysed as before. (Note that in this experiment the DNA concentration was increased three-fold to allow visualisation of minor species and this inevitably leads to more intermolecular ligation.) Half of each sample was then irradiated with a dose of gamma rays sufficient to nick >99% of the circles. Ligation of linear DNA (lane 2) in the absence of any compound produced a set of positively-supercoiled topoisomers (lane 3) which were relaxed by irradiation (lane 4). (The faint smear extending below the unit length DNA is due to a continuous range of smaller linear fragments produced when two nicks occur opposite each other.) The presence of ethidium or (2) (odd-numbered lanes 5-11) during ligation led to rapidly-migrating negatively supercoiled topoisomers; these, too, were relaxed by irradiation (even-numbered lanes 6-12). The pattern of the products generated by ligation in the presence of (9) (lanes 13, 15) included bands (marked by white lines) below that of linear DNA that were out of register with the topoisomers in lane 3; this is characteristic of knots lacking supercoils and which have an increasing number of nodes (Wasserman and Cozzarelli, 1986). Ligation of nicked linear molecules produces such relaxed knots; ligation of intact linear molecules forms supercoiled trefoils and other knots with more nodes which migrate faster. Irradiation destroyed most rapidly-migrating material but left these out-of-register bands, again characteristic of relaxed knots containing different numbers of nodes. As these out-of-register bands are undetectable in irradiated samples ligated in the presence of ethidium or (2), we conclude that (9) is able to cross-link DNA to a small extent at the concentrations used here. Note also, that some DNA has been ligated into a form unable to migrate far into the gel (lanes 13-16), again providing circumstantial evidence for cross-linking. Unfortunately, higher concentrations of (9) inhibit the ligase and so cannot be tested using this assay. The concentration of (9) generating knots depends on the DNA concentration (results not shown); this Is to be expected as its molar concentration roughly equals the concentration of base pairs in the assay.

Results for the remaining members of the group are presented in Fig. 3. Linear DNA was ligated (750ng for lanes 1-7 and 1000ng for lanes 8-14) in the presence of different concentrations of test compounds, the compounds removed, some samples nicked and the ligation products resolved electrophoretically before staining and photography. Lanes 1, 4, 7, 8, 11 and 14: markers provided by ligating linear DNA in the absence of any test compound. The positions of forms II and III are indicated on the left. The white lines between lanes 6, 7 and 12, 13 indicate the position of knots. Ligating the linear DNA in the presence of the phenanthridinium salt (4) or the bis-phenanthridinium salt (6) yields rapidly-migrating supercoiled circles (lanes 2, 5). Note that the supercoiled forms generated with (6) (lane 5) migrate slightly faster than the corresponding forms generated with (4), as expected for highly supercoiled knots. However, only the supercoiled forms produced by (6) when nicked give out-of-register bands (lane 6, white lines). These results show that all members of this group (i.e. 2, 4, 6 and 9) unwind, and so intercalate, but only the bis-phenanthridinium salts (i.e. 6 and 9) knot, and so cross-link.

The Acridinium Series: unwinding and knotting

A similar series of experiments showed that the monofunct ional members of the group based on acridine unwind DNA, but only the bis-acridinium salts, (15) (16) (17) and (19) knot. Fig. 4 shows the results of an experiment in which linear DNA was ligated in the presence of various concentrations of test compounds, the compounds removed and the ligation products resolved electrophoretically before staining and photography. Lane 1: lambda/Hind III markers. Lane 2: linear DNA. Lane 3: linear DNA ligated in the absence of any test compound. Lane 20: ligation in 2% dimethylsulphoxide. The positions of forms II and III are indicated. White lines between lanes 15 and 16 indicate the position of knots. Thus, acridine orange (AO) and the mono-acridinium salts, (11) and (13) produced rapidly-migrating supercoils (Fig. 4), whilst the bis-acridinium salt (15) produced a few even more rapidly-migrating forms (Fig. 4, lanes 15 and 16; Fig. 3, lane 12), again characteristic of supercoiled knots. It also produced out-of-register bands (Fig.

4, lane 15, white lines). The concentration range over which (15) yielded even these few supercoiled knots was narrow; ligase was Inhibited at higher concentrations (Fig. 4, lane 17) and, as with other weak intercalators, the precise range critically depended on the DNA concentration (compare Fig. 4, lane 19 with Fig. 3, lane 12). This made the demonstration of intercalation and knotting difficult. Therefore higher concentrations of DNA were used to confirm the presence of knots after nicking (Fig. 3, lanes 8-14). This meant that trefoils obtained with the mono-acridinium salt (13), which were too low in concentration to be detected in Fig. 4, lane 13, became visible after nicking (Fig. 3, lane 10). However no knots with more nodes could be seen. In contrast, bis-acridinium salt (15) yielded a higher concentration of trefoils and knots with more nodes (i.e. the out-of-register bands in Fig. 3, lanes 12 and 13, white lines). Figures 5, 6 and 7 show similar results for bis-acridinium salts (16) and (17).

Fig. 5 shows that the mono-acridinium salt AP (N-(9-acridineIpyridinium chloride), (16) and (17) are all intercalating agents. Linear DNA (500 ng in 250 μl) was ligated in the presence of different concentrations of the compounds, the compounds removed and the ligation products resolved electrophoretically before staining and photographing the resulting gel. Lane 1: lambda/HindIII markers. Lane 2: unligated DNA. Lanes 3 and 19: linear DNA ligated in the absence of any test compound. The positions of forms II and III are Indicated. White lines to the right of lanes 11 and 16 indicate the position of simple relaxed knots with increasing numbers of nodes. Fig. 6 illustrates that (16) and (17) knot DNA. Linear DNA was ligated (1 μg in 250 μl) in the presence of different concentrations of the compounds, the compounds removed, some samples nicked and the ligation products resolved electrophoretically before staining and photography. Lane 1: lambda/Hind III markers. Lane 2: linear DNA. Lanes 3, 4: linear DNA ligated in the absence of any test compound. + and - indicate whether samples are nicked^' or not. The positions of forms II and III are indicated on the left. White lines to the right of lanes 12 and 17 indicate the position of simple relaxed knots with increasing numbers of nodes. Samples in lanes 9, 14 and 19 are identical to those in lane 5 and serve as markers (M).

Fig. 7 shows the effects of ligation volume, nicking and cutting on knots and supercoils generated in the presence of AP and (16). Linear DNA was ligated (500 ng in 25 or 250 μl ) in the presence of AP (30 μg/ml) or (16) (3 μg/ml) and the compounds removed by ethanol precipitation. Some samples (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17) were redissolved directly in sample buffer, others (3, 4, 7, 8, 11, 12, 15, 16) were redissolved in 50 μl 50mM Tris-HCl (pH8.0), 50mM NaCl, 5mM MgCl₂ and incubated with or without 2.5 units Hindlll for 1h at 37°C before ethanol precipitation and dissolving in sample buffer. Some samples were nicked and ligation products were resolved electrophoretically before staining and photography. Lane 17: linear DNA ligated in the absence of any test compound. + and - indicate whether samples are nicked or not, or cut with Hindlll or not. The positions of lambda /HindIII markers and

-forms II and III are indicated on the left.

Both (16) and (17) prove to be powerful cross-linking agents. For example, 10 μg/ml (16) or (17) convert a significant fraction of DNA into unresolved forms that migrate even more rapidly than the highly-supercoiled topoisomers generated by 30 μg/ml AP (Fig. 5, compare lanes 12 and 17 with 8); knotting compacts them even further. This occurs even though the ligase is partially inhibited. Relaxed knots are clearly visible in lanes 11, 12, 16 and 17 (white markers). Nicking has relatively little effect on such rapidly-migrating forms (Fig. 6, compare lane 12 with 13 and lane 17 with 18); it reduces only slightly the intensity of the smear with a corresponding increase in the intensity of the relaxed knots (white markers).

Fig. 8 shows that acridine orange ( AO), AP

(9-acridine-pyridinium chloride) and (19) are intercalating agents. Linear DNA (500ng) was ligated in the presence of different concentrations of the compounds, the compounds removed and the ligation products resolved electrophoretically before staining and photographing the resulting gel. Lane 1: lambda/Hind III markers. Lane 2: unligated DNA. Lane 3: linear DNA ligated in the absence of any test compound. Lane, 19: ligation in 0.1% dimethylsulphoxide, the maximum concentration of organic solvent present during ligation. The positions of forms II and III are indicated. White lines to the right of lane 17 indicate the positions of simple knots lacking supercoils.

Fig. 9 shows that ( 19) but not acridine orange (AO) or 9-acridine-pyridinium chloride (AP) knots DNA. Linear DNA was ligated (750ng In 250 1) in the presence of different concentrations of the compounds, the compounds removed, some samples nicked and the ligation products resolved electrophoretically before staining and photography. Lane 1: lambda/Hind III markers. Lane 2: linear DNA. Lane 3, 4: linear DNA ligated in the absence of any test compound. + and - indicate whether samples are nicked or not. The positions of forms II and III are indicated on the left. White lines to the right of lanes 15 and 17 indicate the position of simple relaxed knots with increasing numbers of nodes. Samples in lanes 3, 9, 14 and 19 are identical and serve as markers. The bifunctional compound, (19), gives a different pattern from AO and AP. After ligation in 3 μg/ml (Fig. 8, line 17), a significant fraction of the DNA runs faster than the most rapidly-migrating forms given by acridine orange. These must be very compact and are probably superhelical forms condensed even further by knotting. At 10 μg/ml, ligase is almost completely inhibited (lane 18), precluding testing at higher concentrations. In lanes 17 and 18, some DNA remains at the top of the gel, providing circumstantial evidence for cross-linking into large catenanes These results, show that AP unwinds DNA like acridine orange and that (19) additionally gives more complicated forms, probably knots.

A close inspection of Fig. 8, line 17 (white markers) shows there to be extra faint bands below the linear form III, in the positions characteristic of simple relaxed knots with 3 or more nodes. Note that these bands are not in register with those given by the topoisomers produced by mono-intercalating agents like acridine orange and AP. This suggests that (19) acts as a cross-linking agent.

The rapidly-migrating molecules produced by (19) (Fig. 9; lanes 15 and 17) continue to migrate faster than form II and form III molecules even after nicking (lanes 16 and 18); they must be knots. Ligation of nicked linear molecules produces relaxed knots, faintly visible as the discrete bands in lanes 15 and 17 marked by the white lines. These are out of register with topoisomers in lane 12 and are characteristic of relaxed knots with increasing numbers of nodes. Ligation of intact linear molecules forms supercoiled trefoils and other knots with more nodes that migrate faster as an unresolved smear. Irradiation nicks some of this rapidly-migrating material, increasing the concentration of the relaxed knots marked with the white lines in lanes 16 and 18. Nicking has little effect on the more complicated knots in the faint and unresolved smear (compare lanes 15 with 16, and 17 with 18); releasing their supercoils has little effect on compaction. We conclude that (19) acts as both an unwinding and weak cross-linking agent to generate these forms.

This confirms that all members of this series (i.e. 11, 13, 15, 16 17 and 19) unwind, and so intercalate, but only the bis-acridinium salts (15), (16), (17) and (19) increase the percentage of knots, and so cross-link.

The assay requires that all test compound is removed; if not, residual bound molecules would alter mobilities, confounding interpretation. However, establishing exactly how much might remain during electrophoresis is technically difficult, given the low concentrations used. Most test compound in the ligation reaction is necessarily diluted 50-fold prior to loading on the gel. Thus, samples are first-ethanol precipitated (all compounds are relatively soluble in ethanol) before redissolving them in sample buffer. This contains sodium dodecyl sulphate, which would be expected to enhance dissociation of any remaining test compound. However, the best evidence that essentially all test compound has been removed is provided by the similarity of the electrophoretic profiles of samples prepared routinely and after redissolving and reprecipitating them (Fig. 7; compare lanes 1 with 3, 5 with 7, 9 with 11 and 13 with 15); any residual compound cannot influence mobilities.

Cross-linking by bis-intercalation

The results show that all the phenanthridinium salts (i.e. 2, 4, 6 and 9) and acridinium salts (i.e. 11, 13, 15, 16, 17 and 19) unwind DNA (Figs. 1-5). Ligating linear molecules in the presence of the bifunctional agents (i.e. 6, 9, 15, 16,17 and 19) - but not the analogous monofunctional compounds - also increases the proportion of complex structures (perhaps catenanes) that do not migrate far into the gel. More significantly, only these molecules also increase the proportion of knots in the ligation mixture (Figs. 2-6).

Evidence of unwinding, though circumstantial, is generally accepted as proof of intercalation (Waring, 1981; Waring and Fox, 1983). Therefore the results provide strong evidence that all these compounds are, at least, mono-intercalators. However, proof of bis-intercalation requires the demonstration that both groups intercalate simultaneously. Such evidence is much more difficult to obtain. Formation of catenanes and knots by the bifunctional agents provides evidence only for cross-linking o.f some sort. It remains possible that they might do this non-intercalatively, for example, in the same way that a spefmine molecule might link two duplexes. However, in view of the above demonstration that, the phenanthridinium and acridinium groups intercalate, it seems probable that the bifunctional molecules (i.e. 6, 9, 15, 16, 17 and 19) do bis-intercalate simultaneously and so cross-link. Although knotting provides circumstantial evidence for cross-linking, formal proof of bis-intercalation must await evidence from X-ray crystallography or n.m.r. spectroscopy.

References

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Claims

1 A compound having the formula X-A-Y where X and Y may be the same or different and are groups capable of intercalating DNA comprising an optionally substituted fused bi- or polycyclic, carbocyclic or heterocyclic, at least partially aromatic ring system having a substantially flat conformation and A is a substantially rigid divalent group which is extended. such that X and Y are kept apart at a substantially fixed distance from each other so that X and Y are capable of intercalating different DNA duplexes, or a pharmaceutically acceptable acid addition salt thereof.

2 A compound as claimed in claim 1, wherein A consists, optionally, of one substituted or unsubstituted 1,3-phenylene group and of two or more groups selected from one or any combination of the following groups: -CΞC- ; anti- -N=N- ; trans- -N=CH- ; trans- -CH=CH- ; 1,4-phenylene; 1,4- or 2,5- six- membered heterocycles; 1,4- five-membered heterocycles; optionally substituted by a group replacing a hydrogen atom such that the ridigity of A is substantially unaffected.

3 A compound as claimed in claim 1 or claim 2, wherein at least one of X and Y is a polycyclic aromatic nitrogen-containing heterocycle or an alkyl quaternary salt thereof.

4 A compound as claimed in claim 3, wherein at least one of X and Y is an optionally substituted 6-phenanthridine or 10-acridine group.

5 A compound as claimed in claim 3, wherein at least one of X and Y is an optionally substituted 5-alkylated 6-phenanthridinium group, the alkyl group being branched or unbranched C₁ to C₆. 6 A compound as claimed in claim 3, wherein at least one of X and Y is an optionally substituted 9- alkylated 10-acridinium group, the alkyl group being branched or unbranched C₁ to C₆.

7 A compound as claimed in any one of claims 1 to 6, wherein A consists of at least three constituent groups, said groups being alternating trans- 1,2-vinylene and para- aromatic groups.

8 A compound as claimed in claim 7, wherein the para- aromatic groups are 1,4-phenylene, 1,4-pyridinium diyl or 2,5-pyridinylene.

9 - A pharmaceutical composition comprising any of the compounds of claims 1 to 8 and a pharmaceutically acceptable carrier or diluent.

10 A pharmaceutical composition as claimed in claim 9 in a form suitable for topical application to the skin.