WO1999030712A1 - Agents - Google Patents

Abstract

Agents for stabilizing natural or backbone modified DNA and RNA triplexes and hybrids thereof by partly intercalative, partly non-intercalative binding wherein the agent is a compound of fomula (I) wherein n = 2 or 3 and x is 1 or 2. Use of the ligands as antigene/antisense enhancing agents and in preparing drugs for treating retroviral infections are also described.

Description

AGENTS

The present invention relates to agents of the general formula I which interact with nucleic acid structures by binding to natural or backbone modified DNA and RNA duplexes and triplexes and hybrides thereof, whereby complexes are formed with said DNA and RNA structures by means of which said structures are stabilized.

With growing insight of the potential biological role of triple helical nucleic acids and the therapeutic potential of oligonucleotide-directed triplex formation in the control of gene expression according to the antigene strategy, research in triple helical structures has been considerably stimulated. Thus, in the antigene approach, oligonucleotides are targeted to the unique gene that specifies a disease-related protein and stall transcription by binding to the major groove of the doublestran- ded DNA target. An article which contains a good review of this is that by Thuong & Helene in Angew.Chem.Int. Ed.Engl. 1993 32, pages 666-690.

A review of the development of the triplex and antisense strategies for designing drugs that will bind to selected sites on the nucleic acids (DNA and RNA) is found in an article by J.S. Cohen and M.E. Hogan in Scientific American, december 1994, pages 50-55 and in the monograph by Soyfer, V.N. & Potaman, VN. (1996). "Triple- helical nucleic acids", Springer- Verlag, New York.

A limiting feature of antigene therapy is that triplexes are, in general, thermodyna- mically less stable than the corresponding duplexes which thus prevents effective use of triplex formation under physiological condition. One approach to circumvent this problem is to design compounds that bind specifically to triplexes and significantly increase the thermal stability of the triplex relative to its corresponding duplex and single strand components. These ligands should then be covalently conjugated to the third strand oligonucleotides to enhance their activity as antige- ne agents, cf. the above article by Thuong & Helene 1993 and the article by Silver et al in J. Amer. Chem. Soc. 1997, 119, pages 263-268.

The two most efficient DNA triplex stabilizers reported to date are the ben- zo[g]pyridoindole derivative BgPI (targeted to a mixed sequence oligonucleotide complex, see the article of Escude et al in J.Amer. Chem. Soc. 1995, 1 17, pages 10212- 10219) and 2-naphtyl quinoline derivative targeted to poly(dA).2poly(dT), cf the article of Wilson et al in Biochemistry 1993, 32, pages 10614- 10621. Each of said derivatives provides increments of the triplex-to-duplex transitions by 31°C and 35,6°C respectively.

It has now surprisingly been found that under ionic conditions virtually identical to those used for BgPI and for the 2-naphtyl quinoline derivative a substituted indolo- [2,3b]-quinoxaline, hydroxylated in any of the postions 7, 8, 9 and 10, preferably in position 9, or containing a methoxy group in any of said positions, induces a larger thermal enhancement of the triple helical state of the synthetic DNA triplex poly(dA).2poly(dT) than either of the above mentioned derivatives. These compounds also display a high discrimination between the triplex and duplex states of poly(dA).2poly(dT). Thus, the compound 2,3-dimethyl-6-(dimethyl aminoethyl)-9- hydroxy-6H-indolo[2,3b]quinoxaline (9-OH B220) binds to poly(dA).2poly(dT) under near physiological conditions with an affinity of 1.10⁵ M^{" 1} at 20°C.

An object according to the present invention is an agent interacting with nucleic acid structures by binding to natural or backbone modified DNA and RNA triplexes and hybrides thereof by partly intercalative, partly non-intercalative binding, whereby complexes are formed with said DNA and RNA structures which are thereby stabilized, characterized in that the agent is a compound of the formula

wherein n = 2 or 3

R^ and R2, which are identical or different, represent lower alkyl with the provisio that the total sum of carbon atoms in R_j and R2 is maximum 8 and R _j and R2 can also form part of a ring, e.g. piperidino, morpholino or pyrrolidino,

R3 and R4, which are identical or different can be hydrogen or lower alkyl with 1 to

4 carbon atoms,

R5 is hydrogen or methyl, x is 1 or 2.

An other object according to the invention is use of the agents as antigene /an tisense enhancing agents when combined with oligonucleotides in form of a covalent or non-covalent complex.

A further object of the invention is the use of the agents either alone together with a host ribonucleic acid or combined with an oligonucleotide in form of a covalent or non-covalent complex in preparing drugs for treating HIV and other retroviral infections.

Substituted indolo-[2,3b]-quinoxalines of the general formula I, which are not hyd- roxylated in any positions 7-10 are described in the European patent 0238459 and are stated to have antiviral and anticancer effect. The Swedish patent 504,289 describes the same compounds for use in preparing a drug for inhibiting the enzyme HIV integrase and the Swedish patent 504,862 describes the same compounds and also substituted in any position 7-10 with a hydroxyl group, for protecting DNA in the initiating phase and/ or promotion phase of carcinogenes and for preventing oxidative stress. The preparation of the hydroxylated compounds are also described in the last mentioned patent.

It has now been found that the substituted indolo-[2,3b]-quinoxalines, especially the hydroxylated and methoxylated derivatives thereof, are excellent agents for stabilizing natural or backbone modified DNA and RNA triplexes and hybrids thereof by partly intercalative, partly non-intercalative binding. This stabilizing effect can be used in gene expression modification and gene therapy such as use as antigene/ an tisense enhancing agents in combination with the oligonucleotides in form of complex and in gene expression modification as pesticides in plants for plant protection purposes. The agents act by helping to block gene expression of specific genes. They strengthen the binding interaction of any synthetic oligonucleotide or oligonucleotide analog tailored to form a base triplex complex with a specific chosen gene or its control region, so that the RNA polymerase cannot transcribe this gene. Therefore the agents are potentially useful in all types of living cells where transcription of a particular gene should be blocked for a particular reason. A typical reason would be to kill the cell, but other reasons are certainly possible, e.g. to immortalize the cell.

From the increasing knowledge of the gene sequences of many organisms one should be able to target the gene expression specifically in a certain organism but not in others, and thus the agents are useful e.g. in killing microorganisms while leaving the host less affected. This means that the agents according to the present invention are useful not only in medicine but also in veterinary medicine and pesticide development.

The interactions of 9-OH-B220 with both duplexes and triplexes of synthetic poly- deoxoribonucleotides (poly(dA).poly(dT) and poly(dA).2poly(dT)] and polyribonucle- otides [poly(rA).poly(rU) and poly(rA).2poly(rU)] were investigated. Optical absorption and linear dichroism (LD) were utilized as spectroscopic tools to elucidate the capacity and mode of binding of the ligand. In addition the thermal melting behavior of these duplexes and triplexes was studied in the absence and presence of the drug. The first objective was to establish the conditions at which duplex and triplex forms of the nucleic acid polymers are present as given by the stoichiometry of the single stranded polymers in the sample, without undergoing disproportionation. Particularly, the drug-triplex interactions under solution environments resembling those of physiological media, i.e. in the presence of both mono- and divalent cations (here

Na and Mg^ ) were studied.

Order-Disorder Transitions

Thermal denaturation of the duplexes and triplexes of poly(dA)/poly(dT) and poly(rA)/poly(rU) was studied at different ionic strengths in the absence and presence of drug molecules at or near saturating conditions, defined such that no change in the optical spectrum of the drug is observed upon addition of more nucleic acids. The drug-free duplex structures melt via a single transition. The melting profiles of the triplex structures, on the other hand, are generally biphasic; the low temperature step corresponds to the triplex to duplex transition (3- 2), whereas the high temperature transition is assigned to the conversion of the duplex into its component single strand (2→T). In Figure 1, some selected first derivatives of the obtained melting curves for the DNA and RNA host structures are depicted. The thermal melting temperatures, for each optically detected transition, are summarized in Table 1.

Effects of Salt Concentration

poly (dA). poly {dT) duplex. The results presented in Table 1 show, as expected, that the poly (dA). poly (dT) duplex structure is thermodynamically stabilized when the ionic strength of the solution medium increases. Further inspection of the data reveals that an MgCl2 concentration of 2 mM is sufficient to induce disproportionation of a stoichiometric poly(dA).poly(dT) duplex. The triplex structure thus formed is cognate with that of poly(dA).2poly(dT) under comparable salt conditions, as judged from the similar values of the first transition temperature (characterized by

poly(dA).2poly(dT) triplex. To hybridize poly(dA) and poly(dT) in a 1:2 molar ratio into a triplex structure, an NaCl concentration of 100 mM or an MgCl2 concentration of 2 mM is adequate, which is apparent from the two melting transitions observed at this salt concentration (Table 1). However, the thermal stability of each transition is strongly dependent on the ionic strength of the solution medium. In the presence of 100 mM NaCl, increasing amounts of MgCl2 give rise to increased thermal stabilities of both the triplex to duplex and the duplex to single strand equilibria. On the contrary, in the presence of 2 mM MgC^, addition of 100 mM

NaCl results in a destabilization of the triplex to duplex equilibrium as well as the duplex to single strand equilibrium, although the latter to less extent. This is ratio- nalized in terms of a competition between Na and Mg^ for available nucleic acid phosphate binding sites, causing the stabilizing capability of Mg²⁺ to be inhibited.

This capacity of Mg ⁺ to stabilize the poly(dA).2poly(dT) triplex structure was examined both in the absence and presence of 1 mM EDTA in the buffered solutions containing 2 mM MgC^; as EDTA was added to the solutions, t_χa 3^→2 decreased from 75°C to 69°C, while t_m ^{2→ 1} shifted from 78°C to 76°C, clearly demonstrating the importance of the effective Mg²⁺ concentration on nucleic acid stability.

poly (r A), poly (rU) duplex. The data presented in Table 1 reveals that , as for the DNA hos duplex, the thermodynamic stability of the poly(rA).poly(rU) duplex is enhanced as the ionic strength of the solvent is increased. From the biphasic melting profile obtained at 280 nm it is evident that even in the absence of any added MgCL^, an

NaCl concentration of 100 mM is sufficient to promote heating induced transformation of the poly(rA).poly(rU) duplex into the poly(rA).2poly(rU) triplex and single stranded poly(rA) (spectra not shown). The same behavior is observable in buffers containing 2 mM MgCl2 alone. Melting of poly (r A), poly (rU) at and above these ionic strengths is accompanied by an initial hypochromic absorbance change at 280 nm, corresponding to the so called 2→3 disproportionation transition followed by a hy- perchromic transition attributed to the melting of the poly (r A).2poly(rU). triplex to its single strand components (i.e. the 3->l transition). Hence, in a stoichiometric mixture of poly(rA) and poly(rU) at high ionic strength, the stability of the thermally induced poly(rA).2poly(rU) triplex is increased relative to that of the poly(rA).poly(rU) duplex. Therefore, in these cases, the first transition temperature characterizes the 2- 3 transition, while the second one represents the 3—> l transition. The 2- 3 transition is not observable at 260 nm, which is the reason why detection of any disproportionation reaction has to be performed near 280 nm.

poly(rA).2poly(rU) triplex. Triplex formation by the association of poly(rA) and poly(rU) in a 1:2 stoichiometry is readily accomplished in a buffer containing 50 mM NaCl, as indicated by two sequential steps in the melting profile at 260 nm (Table 1 , spectra not shown). However, the ionic strength has to be increased to at least 100 mM NaCl + 2 mM MgC^ for the formation to be complete. Inspection of

Table 1 reveals that the influence of ionic strength on each transition is similar to that just described for the poly(dA).2poly(dT) triplexes, with the same type of competition between mono- and divalent cations for appropriate sites of interaction. Note that at 100 mM Nacl + 5 mM MgC^, poly(rA).2poly(rU) exhibits a broad single melting transition (depicted in Figure Id, solid curve), which is attributed to a simultaneous dissociation of all three strands involved in the triplex structure. This assignment was confirmed by monitoring the melting process also at 280 and 283.5 nm (data not shown). At 280 nm, where the duplex-to-single-strand transition is masked while melting of the triplex state is observable, a single hyperchro- mic transition is observed with a melting temperature of 75.5°C, i.e. equal to that obtained at 260 nm. In addition, the melting profile at 283.5 nm, where a direct dissociation of poly(rA).2poly(rU) to the single stranded homopolymers cannot be detected, does not contain any transition at all, well in agreement with a disruption of the three stranded complex without any stable intermediate.

Effects of interactions with 9-OH-B220

Drug binding to deoxyribonucleotide duplex and triplex. The influence of 9-OH-B220 on the thermal denaturation profiles of the deoxyribonucleotide host structures was examined. Figure la and Table 1 shows that binding of the drug to the poly(dA).poly(dT) duplex at 12.5 mM NaCl thermally stabilizes the duplex to single strand equilibrium with a At^^2-* of +26.5°C at a drug to base pair ratio of 0.50.

This observation is consistent with the drug exhibiting a preference for the duplex state over the single stranded coil state. The displayed biphasic melting profiles at certain drug to base pair ratios are typical and in agreement with previous work and with the expectations predicted from theoretical considerations of the effect of intercalating ligands on the nucleic acid melting behavior.

Comparison of the different traces in Figure lc reveals further that addition of 9- OH-B220 to the poly(dA).2poly(dT) triplex structure in the presence of 100 mM NaCl + 5 mM MgCl2 results in stabilization of the triplex state, with an increase in t_m of 20°C at saturation (r = 0.50), suggesting a binding preference of 9-OH-

B220 for the host triplex structure over the duplex state. At this high salt concentration, however, the drug has a much smaller effect on the duplex to single strand equilibrium. At the highest drug to base triplet ratio (r = 0.50) the melting temperatures for the first (tm ) and second (t_ιa ^~^ ) transitions have merged (Table

1 and Figure lc), indicative of equal stabilities of the triple and double helical states in the presence of 9-OH-B220 under these conditions.

Drug binding to ribonucleotide duplex and triplex. As with the poly(dA).poly(dT) host duplex, the poly(rA).poly(rU) duplex experiences a thermal stabilization (Δt T- =

+ 17.5°C at 12.5 mM NaCl) in the presence of 9-OH-B220 at a drug to base pair ratio of 0.50 (Figure lb and Table 1). In parallel, the melting behavior at low and intermediate drug to base pair ratios is similar to that of the ligand-poly(dA).poly(dT) complex, which implies cognate drug modes of binding in the two nucleic acid duplex host structures. The effects of drug binding on the ρoly(rA).2poly(rU) triplex structures are, on the other hand, different from those observed for the DNA triplexes. Inspection of Figure Id discloses that 9-OH-B220 has a much more moderate effect on the poly(rA).2poly(rU) triplex at 100 mM NaCl + 5 mM MgCl₂ than on the DNA triplex under the same ionic strength; the single melting transition of the polymer structure in the absence of drug remains upon drug binding as a single transition but at gradually increasing temperatures (Δt^ +3°C at r = 0.50), indicative of a slight preference of the drug for the triplex state over the duplex state. In order to confirm the stabilizing capability of the drug on the RNA triplex structure, the melting process was monitored at 280 nm as well (not shown) At this wave- length, the denaturation profile is monophasic at all drug binding ratios with t^ values coinciding with those obtained at 260 nm. In addition, the total hyperchro- micity of the transition remains constant over the whole titration range, which strongly suggests that 9-OH-B220 binds to the poly(rA).2poly(rU) triplex without displacing the major groove-bound third strand from the underlying duplex.

Absorption and Linear Dichroism

The upper and lower panels of Figure 2 show isotropic absorbance (A_jso) and reduced linear dichroism (LD^r) spectra, respectively, of the complexes formed between 9-OH-B220 and poly(dA).poly(dT) (a), poly (rA). poly (rU) (b), poly(dA).2poly(dT) (c) and poly(rA).2poly(rU) (d).

Light absorption

As evident from the top panels of Figure 2, the absorption spectrum of free 9-OH- B220 consists of three absorption bands in the near UV/vis regions: (i) the highest- energy band is strong with a maximum at 270 nm and has the tendency of a shoulder around 290 nm, (ii) the strong band in the visible region has a peak at 370 nm and a plateau at the high-energy side of the maximum, while (iii) the lowest- energy band, centered around 430 nm, is broad and very weak. In presence of the nucleic acid polymers, these bands are affected differently, depending on the host nucleic acid structure. In all drug-DNA/RNA complexes, except for that with poly(rA).2poly(rU), the visible band at 370 nm is red-shifted by 8-10 nm (~ 600-700 cm^"') and shows 10-20% hypchromicity. For the highest-energy band at 270 nm, the situation is more complex; although a hypochromic effect is observed upon binding to any of the host nucleic acid structures, the extent of hypochromicity varies from 23% for the poly (rA). poly (rU) complex to 46% for the poly (dA). poly (dT) complex. Moreover, whereas the peak is shifted towards shorter wavelengths by 1-3 nm (~ 130- 140 cm^{" 1}) upon binding of the drug to either poly (dA). poly (dT) or poly (rA). poly (rU), a red-shift of 0.5-10.5 nm (~ 70-1380 cm^{" 1}) is displayed by the drug upon interacting with poly(dA).2poly(dT) and poly(rA).2poly(rU). However, binding of 9-OH-B220 to any of the nucleic acid polymers produces significant chang- es of the spectral envelope so that the broad drug absorption bands become more dispersed with pronounced shoulders in the regions where they are just hinted in the spectrum of the free drug.

Spectral effects of this nature are typical for ligands interacting with nucleic acids. Another very useful method for detection and characterization of ligand binding to nucleic acids is linear dichroism. If the absorbing ligands interact with the nucleic acid polymer, an LD signal appears in the drug absorption region when the host nucleic acid becomes oriented, as a result of indirect orientation of the ligand chromophores. If, on the contrary, no binding occurs, no LD from the ligand molecules can be observed.

Reduced linear dichroism (LD^r)

The LD^r spectrum of 9-OH-B220 with the poly(dA).poly(dT) duplex (bottom panel of Figure 2a) has a larger negative amplitude in the visible drug absorption bands than in the DNA band around 260 nm. This is indicative of the drug transitions around 380 nm and 430-470 nm being oriented more perpendicular to the helix axis than are on average the base pairs, a phenomenon that has been observed earlier for various intercalating ligands. Table 2 lists the LD^r parameters obtained. Like in the drug-poly(dA).poly(dT) system, 9-OH-B220 seems to adopt a nearly perpendicular binding geometry upon interacting with the poly(dA).2poly(dT) triplex, emphasized by the strong negative LD^r amplitudes and the calculated effective drug transition moment angles (Figure 2c, lower panel, and Table 2). Note, however, that binding of 9-OH-B220 to any of the DNA host structures causes a numerical decrease in the LD^r amplitude at 260 nm, implying that the orientability of the nucleic acid polymers is reduced (S decreases, Eqn (2) in Experiments, Materials and Methods), which contrasts the usual behavior of classical intercalators. Nevertheless, similar observations have been made with poly(dA)/poly(dT) poljmiers complexed with several intercalating molecules.

Whereas the LD technique has been widely adopted for studies of DNA and DNA- ligand complexes, there are only a few reports of LD measurements on RNA systems including two investigations of RNA-ligand complexes (Gatti et al., 1975 Bio- chim. Biophys. Acta, pp 407, 308-319; Bailly et al, 1996 J.Mol.Recogn, 5, pp 155- 171). In all but the work by Wada et al (1971 Biopolymers, 10, pp 1153- 1 157), which used flow orientation, the RNA molecules have been oriented by electric fields, which have restricted the measurements to solutions of low ionic strength. To the best of our knowledge, the present work provides the first demonstration of flow-oriented drug-RNA complexes and, in particular, RNA triplex structures.

As with the host DNA structures, binding of 9-OH-B220 to the poly (r A), poly (rU) duplex results in negative LD^r signals at all wavelengths (Figure 2b, lower panel, and Table 2). Upon binding of the drug to the RNA duplex, a behavior similar to that described for the drug-poly(dA).poly(dT) system is observed, with LD^r signals of higher magnitude in the long wavelength end of the spectrum than in the UV region.

An interesting observation is the increasing amplitude of the LD^r signal around 260 nm with increasing ionic strength of the solvent, showing that the RNA duplex polymers become better oriented upon increasing the salt concentration (the orientation factor S increases) . This behavior is diametrically opposed to that of DNA polymers, which are more readily oriented at lower compared to higher ionic strengths, attributed to an increase in persistence length of the DNA chain as the salt concentration is decreased.

Inspection of the lower panel of Figure 2d and Table 2 reveals that the orientability of the poly(rA).2poly(rU) triplex is dependent of the ionic strength in a manner similar to that of the poly (r A), poly (rU) duplex. The orientation function S for the drug-free RNA triplex is increased by a factor of 2.1 upon increasing the salt concentration from 50 mM NaCl to 100 mM NaCl + 5 mM MgC^. It has to be emphasized , however, that at 50 mM NaCl, the poly(rA).2poly(rU) triplex is not completely formed. Therefore, single stranded poly(rU) will be present it the solution and give rise to absorption but not to linear dichroism, since the single stranded polymer is too flexible to be oriented along the flow lines. As a result, the average reduced linear dichroism (LD^r = LD/A_jso) will appear with a lower amplitude than that expected from a completely formed triplex. Nevertheless, at 260 nm, the isotropic ab- sorbance for poly(rA).2poly(rU) at 50 mM NaCl is only 10% higher than that of the same system at 100 mM NaCl + 5 mM MgC^, i.e. under conditions of complete triplex formation. Hence, merely the presence of uncomplexed poly(rU) single strands in the low salt mixture is not sufficient to explain the two-fold increase in orientability observed here when more salt is added to the solution.

Binding of 9-OH-B220 to poly(rA).2poly(rU) in the presence of 100 mM NaCl + 5 mM MgCl2 induces LD^r spectra with amplitudes that are significantly smaller in the wavelength region above 320 nm than in the UV region, and the calculated average angles between the nucleic acid helix axis and the drug transitions responsible for absorption in the visible region indicate a binding geometry that is nonin- tercalative. Such an interpretation is well in agreement with the observed rather small perturbations of the drug light absorption envelope upon binding to poly(rA).2poly(rU) at this ionic strength (Figure 2d, top).

It may be noted that, as with the DNA host structures, the LD^r signal in the predominantly RNA absorption region around 260 nm decreases upon binding of 9- OH-B220 to any of the RNA host structures, indicating an impaired orientability of the RNA structures as a consequence of drug binding.

Conclusions

The purpose of the present experiments was to characterize the nature of interactions between the antiviral quinoxaline derivative 9-OH-B220 and triple helical nucleic acid polymer structures, and to compare with those of their precursor duplexes. In order to obtain the relevant binding data, we optimized the ionic conditions for the duplex and triplex structures, respectively. Emphasis was put on achieving solution compositions where each of the poly (dA). poly (dT), poly(rA) .poly(rU), poly(dA).2poly(dT) and poly(rA).2poly(rU) complexes were completely formed and remained purely in one state at room temperature. In addition, since we wanted to ascertain the possible role of 9-OH-B220 as an antigene enhancer, we found it relevant to study the drug-triplex association in the presence of both mono- and divalent cations at relatively high concentrations (i.e. in the millimolar range). The reasoning was that a characteristic feature of intracellular media, which is where any application of triplex formation to stall transcription has to be accomplished, is that it is composed of a mixture of various cations at tightly regulated levels.

Ionic effects on complex formation and physicochemical properties

The thermal denaturation experiments carried out here demonstrate that, in solutions containing cations of different valence, the influence of a specific counterion on the nucleic acid association equilibria depends on the valence of that ion (cf. Table 1). For example, in case of the poly(dA).2poly(dT) triplex, increasing amounts of NaCl at a constant MgCl2 concentration of 2 mM causes a significant destabili- zation of the triplex to duplex as well as duplex to single strand equilibria. On the other hand, in the presence of a constant NaCl concentration, increasing levels of MgCl2 results in enhanced thermal stabilities of both equilibrium reactions. These findings are in perfect agreement with expectations and previous work (Plum et al, 1995 Annu. Rev. Biophys. Biomol. Struct. 24, pp 319-350 and references therein) and are interpreted as a competitive interplay among counterions of different valence in the ion atmosphere surrounding the polymer structures.

In addition to the equilibrium melting temperatures, a parameter to be considered in the search of optimal ionic conditions for the formation of multistranded nucleic acid complexes is the resulting hypochromicities for each helix forming reaction. When a complex is formed in a mixture of single- stranded polymers, the absorban- ce is reduced as a consequence of base stacking interactions in the resulting structure. Hence, the hypochromicity can be utilized to estimate the extent of complex formation. Then, it is interesting to note that even though the melting temperatures of the poly(dA).2poly(dT) and poly(rA).2poly(rU) triple helical structures in the presence of 2 mM MgCl2 alone are higher relative to the melting temperatures in mixtures containing both 100 mM NaCl and 2 mM MgC^, the hyperch- romicities of the triplex denaturation transitions are larger in the latter case. This observation implies that despite the displayed high thermal stabilities, a MgCl2 concentration of 2 mM is not sufficient for the triple helical complexes to adopt their ideal solution structures. Influence of drug binding on complex stability

Interaction with poly (dA). poly (dT) and poly (rA). poly (rU) duplexes. While ligand interactions with DNA polymers have been thoroughly investigated, relatively few studies have been performed on ligand-RNA systems. However, classical intercalators such as ethidium bromide, ellipticine and quinacrine were recently demonstrated to induce increases in the thermal stabilities of both poly(dA).poly(dT) and poly (r A), poly (rU) (Tanious et al, 1992 Biochemistry, 31, pp 3103-31 12; Wilson et al, 1993 loc.lit).

The strong increments in melting temperatures, observed here for poly (dA). poly (dT) and poly(rA).poly(rU) in presence of 9-OH-B220 (Figure 1, panels a and d, and Table 1), show that the drug effectively stabilizes each of these duplex structures and suggest a specific mode of interaction between the drug and the DNA as well as the RNA helix. Furthermore, the higher Δ^ value observed for poly(dA).poly(dT) compared to poly (r A), poly (rU) indicates a slight preference of the drug for the DNA polymer structure, a feature that it shares with a vast majority of the ligands studied so far.

The described observations are consistent with intercalation of 9-OH-B220 between the base pairs of each duplex structure. This interpretation is strongly supported by the spectroscopic properties of these complexes (vide infra).

Interaction with poly (dA). poly (dT) and poly (rA). poly (rU) triplexes. The results from the triplex denaturation experiments presented here clearly demonstrate that 9-OH- B220 binds to, and stabilizes, the poly(dA).2poly(dT) triplex structure (Figure lc and Table 1). As the drug to base triplet ratio is increased from 0 to 0.2 the coope- rativity of the triplex to duplex transition decreases and the melting temperature is shifted from 65,5°C to 79°C, while dissociation of the remaining duplex occurs att 77.5°C and 80.5°C at the respective r ratio. Hence, 9-OH-B220 displays a preferential stabilization of the Hoogsteen-paired third strand in poly(dA).2poly(dT) . Similar observations have been made with several DNA-interacting agents, e.g. the ben- zo[e]- and benzo[g]pyridoindole derivatives BePI (Pilch et al, 1993 J. Mol. Biol. 232, pp 926-946) and BgPI (Escude et al, 1995 loc.cit) all suggested to bind to DNA tri- plexes by intercalation. Groove binding compounds, on the other hand, normally destabilizes DNA triplex structures.

The two most efficient DNA triplex stabilizers reported to date are the ben- zo[g]pyridoindole derivative BgPI (targeted to a mixed sequence oligonucleotide. Escude et al, 1995 loc.cit) and a 2-naphtyl quinoline derivative (target to poly(dA).2poly(dT), Wilson et al, 1993b loc.cit), each providing melting temperature increments of the triplex-to-duplex transitions by 31°C and 35.6°C. Both these drugs were, however, investigated in buffer solutions containing significantly smaller amounts of salt than those used in the present work. To be able to compare the relative efficacy of 9-OH-B220 as a DNA triplex stabilizing agent, thermal denaturation experiments were thus performed on poly(dA).2poly(dT) under ionic conditions virtually identical to those used for BgPI (Figure 3a) as well as for the 2- naphtyl quinoline derivative (Figure 3b). Remarkably, 9-OH-B220 induces a larger thermal enhancement of the triple helical state of poly(dA).2poly(dT) than either

BgPI or the 2-naphtyl quinoline derivative, the Δt^^3-*² being more than 50°C and almost 40°C at a drug to base triplet ratio of 0.50 in buffers containing 100 and 190 mM NaCl, respectively. In addition, the drug displays a high discrimination between the triplex and duplex states of poly(dA).2poly(dT) at these ionic strengths, exhibiting fractional Δt_m ^{2→ 1}/Δt_m ^3→2 increments of 0.18 and 0.16 at 100 and 190 mM NaCl, respectively.

In the same manner as above the relative efficacy of 8,10-dimethoxy-B220 as a DNA triplex stabilizing agent were investigated by performing thermal denaturation experiments on poly(dA).2poly(dT) under ionic conditions virtually identical to those used for BgPI (Figure 4). 8, 10-dimethoxy-B220 (6-dimethylaminoethyl-2,3- dimethyl-8,10-dimethoxy-6H-indolo[2,3-b]quinoxaline) showed a somewhat lower stability effect (Δt_ιn ^→2 =48°C) but the selectivity, as evident from Figure 4, was better.

The general conclusion to be drawn from this and other reports is that the size and shape of the ligand chromophore ring system, as well as the positioning of charged side chains, are crucial factors to be considered in developments of DNA triplex stabilizing agents. The compound 9-OH-B220 studied here has a ring system, which shows strong stacking interactions with each of the bases in a triad, and a positively charged side chain that may form favorable interactions with proton acceptor groups in one of the grooves of the triple helical host structure. The above comparison and the fact that 9-OH-B220 behaves like a typical intercalator in terms of triplex stabilizing capacity suggests that 9-OH-B220 binds to the poly(dA).2poly(dT) triplex by intercalation, as will be discussed in more detail from a spectroscopic point of view.

The triplex stabilizing effect of 9-OH-B220 on the poly(rA).2poly(rU) triplex is much smaller than that on the corresponding DNA triplex (Figure Id and Table 1). This implies that the drug interactions are weaker with the triple helical RNA structure than with the poly(dA).2poly(dT) triplex. At the same time, the stabilization at all drug to base triplet ratios clearly demonstrates a drug preference for the triple helical state of poly(rA).2poly(rU) over the parent duplex structure.

Drug binding modes

Complexation of 9-OH-B220 with duplex structures of DNA and RNA. The spectroscopic data for 9-OH-B220 in complex with poly(dA).poly(dT) and poly(rA).poly(rU) have many features in common with each other. The large hypochromicities and red shifts of the long wavelength drug absorption bands, observed upon binding to each duplex polymer structure, indicate strong interactions between the drug and the nucleotide bases in both cases (Figures 2a and b, upper panels), as would be expected if the drug was bound intercalatively.

The above results are indicative of intercalation of the drug chromophore to each duplex structure, but they might also be consistent with a groove binding geometry. However, important evidence in contradiction of such an interpretation comes from the performed LD measurements. The LD^r in the long wavelength region is strongly negative, showing that the orientations of the in-plane polarized transition moments, responsible for absorption in this wavelength range, of 9-OH-B220 when bound to either poly(dA).poly(dT) or poly (rA). poly (rU) are essentially perpendicular to the helix axis of the respective nucleic acid duplex (Figures 2a and b, lower pa- nels, and Table 2). This behavior is in excellent agreement with a sandwiching of the drug molecules between the base pairs, but not with a geometry in which the ligand chromophores are positioned edgewise along one of the grooves, since this latter possiblity would have been associated with positive LD^r signals from transitions polarized along the long-axis of the chromophore, which would be directed parallel to the groove, as opposed to the observed negative ones.

Complexation of 9-OH-B220 with triplex structures of DNA and RNA. In contrast to the striking similarities in spectroscopic properties displayed by the drug complexes of the double helical polymers, those of the poly(dA).2poly(dT) and poly(rA).2poly(rU) triplexes exhibit markedly different characteristics. In the first place, the absorption spectra reveal that the perturbations in the excited states of the 9-OH-B220 chromophore are stronger in its complex with poly(dA).2poly(dT) than in that of the corresponding RNA triplex. This is judged from the larger red shifts and hypochromic effects of the drug absorption bands observed in the drug- DNA triplex system (Figures 2c and d, upper panels), and is a first important indication of different interaction modes of 9-OH-B220 in poly(dA).2poly(dT) and poly(rA).2poly(rU). In fact, of all the drug-nucleic acid polymer complexes studied here, the absorption envelope of 9-OH-B220 has suffered the greatest perturbations in the case of poly(dA).2poly(dT), which implies that the interactions between the drug transition moments and those of the nucleic acid bases are strongest in this complex.

The most solid support for a nonintercalative binding mode of 9-OH-B220 in complex with the poly(rA).2poly(rU) RNA triplex is provided by the LD data on this system (Figure 2d, lower panel, and Table 2). The effective average angles of all drug transition moments in the visible wavelength region are found within the range of 60 to 65°, independent of the drug binding ratio between 0.05 and 0.20. If the drug chromophore had been intercalated, one would have expected angles much closer to 77°, which is assumed to be the average angle between the nucleic acid bases and the polymer helix axis in poly(rA).2poly(rU). Moreover, the LD data argues against a nonspecific electrostatic association of the drug molecules to the phosphate groups at the outside of the triple helical structure, since such surface binding would result in very weak LD signals due to poor average orientation of the interacting ligands, which is evidently not the case here.

In the triple helical poly(rA).2poly(rU) structure, the major groove of the underlying duplex is blocked due to the presence of the Hoogsteen-paired third strand. Yet, the minor groove is still accessible to interacting agents. Given that the minor groove is wide and shallow in this A-type helical structure, an arrangement of the drug molecules in, and possibly across, this groove constitute an attractive binding geometry model for 9-OH-B220 in complex with poly(rA).2poly(rU). Hence, all findings on the drug-RNA triplex complex point towards homogeneous binding of the 9-OH- B220 molecules in the minor groove of the poly(rA).2poly(rU) host polymer.

The deviating drug binding mode in the poly(rA).2poly(rU) triplex, as compared to the other drug-nucleic acid complexes, suggests that intercalation into the poly (r A), poly (rU) duplex occurs from the major groove side of the helix structure; when the major groove is blocked by a third strand, such as in the poly(rA).2poly(rU) triplex, the drug is hindered from binding intercalatively and is directed to the minor groove of this polymer structure. When DNA polymers serve as host nucleic acids, on the other hand, intercalation occurs from the minor groove. Therefore, a third strand in the major groove of a DNA duplex structure is not expected to severely affect the intercalation process of 9-OH-B220, in agreement with the observations made here. In the A-form RNA duplex structure, the major groove is less hydrophobic and has a lower electrostatic potential than the minor groove. Hence, the positively charged side chain of 9-OH-B220 is likely to preferentially interact with atoms in the major groove of the poly(rA).poly(rU duplex. We consider this groove selectivity of the side chain to be the most probable explanation to the different intercalation directionalities seen for the drug between the RNA- and DNA-polymers.

Our experiments have demonstrated that the biologically active drug 9-OH-B220 interacts with triple helical host nucleic acid structures under cellular concentrations of cations. Thus, 9-OH-B220 in our experiments has been shown to bind to both DNA and RNA triplexes formed in high salt containing solutions. From a combination of optical spectroscopic measurements, the binding of this drug to poly(dA).2poly(dT) and poly(rA).2poly(rU) as well to their parent duplex structures has been characterized. It was found that the drug intercalates to, and stabilizes, both DNA structures. Also when bound to the RNA polymers 9-OH-B220 exerts a thermally stabilizing effect, being bound intercalatively in the poly(rA).poly(rU) duplex but exhibiting properties characteristic of minor groove binding in the poly(rA).2poly(rU) triplex.

Thus, it has unexpectedly according to the present invention been shown that the antiviral, low-toxic and highly water soluble compound 9-OH-B220 recognizes and stabilizes both ribonucleotide and deoxyribonucleotide containing helices, which makes it suitable to be conjugated to oligonucleotides to enhance their activity as antigene/ an tisense agents. Furthermore, the present results on particularly the 9- OH-B220-RNA interactions in combination with the known effects of its parent compound B220 on HIV integrase, cf. Swedish patent SE 504,289 makes it a suitable compound for preparing an anti-HIV agent.

Experiments, Materials and Methods

The compounds of formula I were prepared in the manner described in SE patent 504.862 in the following manner with the compound 9-OH-B220 2,3-dimethyl-6-(2- dimethylaminoethyl)-9-hydroxy-6H-indolo[2,3b]quinoxaline as an example.

Compound lb was prepared by nitration of 1 eq B220 (la) with 1 eq of potassium nitrate in sulfuric acid followed by reduction of the nitro group to give 9-NH2-B220

(3), which was converted to the corresponding diazonium salt by treatment with sodium nitrite in aqueous sulfuric acid. The diazonium salt was transformed to 9- OH-B220 by treatment with copper (I) oxide and copper (II) nitrate. Omission of the copper (I) oxide resulted in drastic decrease in the yield of 9-OH-B220.

2, 3-dimethyl-6-(2-dimethylaminoethyl)-9-nitro-6H-indolo[2, 3]quinoxaline (2) : Potassium nitrate (5.06 g, 50 mmol) was added to a solution of la (15.9 g, 50 mmol) in H2SO (200 ml) so that the temperature did not exceed 10°C. The solution was stirred for 2 h at 5-10°C and then poured into ice-water. The mixture was made alkaline with 20% KOH-solution. The solid formed was collected by filtration and recrystallized from EtOH to give 13.1 g (72%) of 2 as a yellow solid; m.p. 218.5- 219°C. IR (KBr) v_maχ: 2943, 2757, 1581 , 1479, 1463, 1332,

1316, 1 104 cm-¹. ^JH-NMR (DMSO-d₆) δ: 9.04 (s, 1H), 8.56 (d, 1H), 8.05 (s, 1H), 7.98 (d, 1H), 7.93 (s, 1H), 4.64 (t, 2H, CH₂), 2.77 (t, 2H, CH ), 2.5 (s, 3H, Me), 2.5 (s, 3H, Me),

2.19 (s, 6H, Me) ppm.

9-Amino-2, 3-dimethyl-6-(2-dimethylaminoethyl)-6H-ιndolol2, 3-b]quinoxaline (3) : A suspension of 2 (3,63 g, 10 mmol) and Pd/C (10%, 0.36 g) in N,N- dimethylacetamide (160 ml) was left under hydrogen pressure (2.7 atm) for 24 h. The catalyst was filtered away with celite. The filtrate was poured into water and basified (NaHCO ) and the solid formed was collected by filtration and purified by flash chromatography (MeOH/CH₂Cl_2. 2/8) to give 2.80 g (84%) of 3 as a red solid; m.p. 250-251°C. IR (KBr) v_maχ: 3400, 3322, 2938, 2763,

1580, 1491, 1401, 1213, 806 cm"¹. *H-NMR (DMSO-d₆) δ: 7.9 (s, 1H), 7.8 (s, 1H), 7.5 (s, 1H), 7.4 (d, 1H), 7.1 (d, 1H), 5.1 (br s, 2H, NH₂), 4.4 (t, 2H, CH₂), 2.7 (t, 2H, CH₂), 2.5 (s, 3H, Me), 2.5 (s, 3H, Me), 2.2 (s, 6H, Me) ppm. ¹³C-NMR (DMSO-d₆) δ: 145.0 (s), 143.3 (s), 138.7 (s), 138.6 (s), 137.1 (s), 136.0 (s), 135.0 (s), 128.0 (d), 126.5 (d), 1 19.5 (s), 1 19.0 (d), 1 10.6 (d), 105.4 (d), 56.7 (t), 45.2 (q), 38.9 (t), 19.9 (q), 19.6 (q) ppm.

2, 3-Dimethyl-6-(2-dimethylaminoethyl)-9-hydroxy-6H-indolo[2, 3-b]quinoxaline ( lb) : Compound 3 (660 mg, 2 mmol) was dissolved in a solution of H2SO (30%, 6 ml) at

15°C and thereafter the solution formed was cooled to 5°C. A solution of sodium nitrite (176 mg, 2.6 mmol) in water (2 ml) was added keeping the temperature below 5°C. The reaction mixture was stirred for 30 min at 0°C, thereafter urea was added to consume unreacted nitrous acid. A cold (0°C) solution of copper(II)nitrate trihydrate (4 g) in water (50 ml) was added to the reaction mixture whereupon cop- per(I)oxide (286 mg) was added, which resulted in a vigorous formation of N2~gas.

When the ^-gas evolution had ceased the mixture was left for 30 min and then the pH was increased to 9-10. The mixture was extracted with CH2CI2. The dried

(MgSθ4) extract was evaporated and the residue flash chromatographed

(MeOH/CH₂Cl₂, 1/9) to give 133 mg (20%) of lb as a yellow solid; m.p. >250°C. IR (KBr) ^vmax^:

3420, 3130, 2940, 2765, 1585, 1490, 1425, 1350, 1240, 1210, 1155, 1025, 1000, 865,

855, 725 cm-l. »H-NMR (DMSO-d₆) δ: 9.5 (1H, s, OH), 8.0 (1H, s) 7.8 (1H, s), 7.6 (1H, s),

7.6 (1H, d), 7.2 (1H, d), 4.5 (2H, t, CH₂), 2.7 (2H, t, CH₂), 2.5 (3H, s, Me), 2.2 (6H, s, Me) ppm. J3C-NMR (DMSO-d₆) δ:152.0 (s), 145.0 (s), 138.8 (s), 138.6 (s), 138.3 (s), 137.2 (s),

137.1 (s), 135.2 (s), 127.9 (d), 126.5 (d), 1 19.4 (d), 1 19.3 (s), 1 10.9 (d), 106.6 (d), 56.7 (t), 45.2 (q), 39.2 (t), 19.8 (q), 19.5 (q) ppm. MS: m/z 334.2 (M+, 0.19), 279.2 (1.84), 263.1

(1 1.8), 72.1 (24.06), 28.0 (100).

2, 3-Dimethyl-8, 10-dimethoxy-6H-indolo[2, 3-bJ-quinoxaline. 4 , 5-Dimethyl-o- phenylenediamine (1.36 g, 10 mmol) was added to a refluxing solution of 4,6- dimethoxyisatine (2.07 g, 10 mmol) in acetic acid (30 ml). After a reflux period of 3 h the mixture was allowed to cool and the yellow precipitate of the product obtained collected and dried. Yield: 2.85 g (93%), mp. >260°C.

6-Dimethylaminoethyl-2,3-dimethyl-8, 10-dimethoxy-6H-indolo[2,3-b]-quinoxaline. 2,3- Dimethyl-8, 10-dimethoxy-6H-indolo[2-3-b]-quinoxaline (614 mg, 2 mmol) was dissolved in dry dimethyl sulf oxide (15 ml) whereupon sodium hydride (4.4 mmol) was added at 35°C to the stirred solution under nitrogen. When the evolution of hydrogen had ceased (ca 30 min) 2-chloro-N,N-dimethylethylamine hydrochloride (288 mg, 2 mmol) was added to the stirred, red solution. After 1 h at 35°C the temperature was rised during 1 h to 55°C and kept at this temperature for 2 h. The reaction mixture was allowed to cool, water was added and the product extracted with ether. After concentration and drying the product was collected as a hydrochloride by introduction of HCl gas, 480 mg (53%), mp. >260°C.

The free base (mp. 117-119°C) of this hydrochloride gave the following IR data: 2946, 1623, 1588, 1469, 1393, 1208, 1151 cm^{" 1}.

^-NMR (CDC1₃) δ: 2.38 (s, 6H, NMe₂), 2.48 (s, 3H, CH3), 2.50 (s, 3H, CH3), 2.79 (t, 2H, CH₂), 3.96 (s, 3H, OCH3), 4.13 (s, 3H, OCH3), 4.51 (t, 2H, CH₂), 6.37 (d, 1H, J=3.2), 6.57 (d, 1H, J=3.2), 7.81 (s, 1H), 8.05 (s, 1H). ¹³C-NMR (CDCI3) δ: 20.5 (q, CH3), 20.7 (q, CH3), 40.4 (t, CH₂), 46.3 (q, NCH3),

56.3 (q, OCH3), 56.7 (q, OCH3), 57.5 (t, CH₂), 87.2 (d, CH), 92.0 (d, CH), 127.2 (d,

CH), 129.2 (d, CH), 135.9 (s), 138.1 (s), 138.4 (s), 138.9 (s), 139.3 (s), 141.0 (s), 145.6 (s), 147.2 (s), 158.8 (s), 164.4 (s).

6-Dimethylaminoethyl-9-methoxy-6H-indolo[2,3-b]-quinoxaline. The procedure in the two preceding experiments was used starting with 6-methoxyisatine and o- phenylene diamine which gave 9-methoxy-6H-indolo[2,3-b]-quinoxaline, which was subsequently alkylated following the procedure in the preceding experiment which gave the title compound, as hydrochloride, in 75% yield.

The free base (mp. 125-127°C) of this hydrochloride gave the following proton NMR data: 2.7 (t, 2H, CH₂), 3.4 (s, 3H, OCH3), 3.9 (s, 6H, NCH3), 4.4 (t, 2H, CH₂), 7.5-

8.3 (m, 7H, arom. CH) ppm (in CDCI3).

Chemicals and Solutions

All chemicals used were of analytical grade. DNA and RNA polymers were obtained from Pharmacia Biotech (Sollentuna, Sweden) and used without further purification. The polymers were dissolved in 10 mM sodium cacodylate buffer containing 1 mM EDTA (pH 7.0). The buffer was autoclaved to overcome possible RNase contamination. Concentrations were determined spectrophotometrically using base molar extinction coefficients of: ε₂57 = 8600 for poly(dA), ε₂64 = 8520 for poly(dT), ^ε260 ^{= 9400} f°^r P°ly(^rA) and ε₂60 = 8480 for poly(rU). Duplexes of poly (dA). poly (dT) and poly(rA).poly(rU) were prepared by mixing each strand at a molar ratio of 1: 1. The triplexes were annealed by incubating 1:2 molar ratios of poly (dA)/ poly (dT) and poly(rA)/poly(rU), respectively, at 90°C for 30 min followed by slow cooling to 4°C.

Stock solutions of 9-OH-B220 were prepared in plastic tubes by weighing an amount of the compound and dissolving it in a buffer containing 10 mM sodium cacodylate and 1 mM EDTA at pH 7.0. The solutions were acidified until the drug was completely dissolved, and the concentrations were determined spectrophotometrically using the extincion coefficient of 8370 ⁼ 13400 M^' -^cm^{" 1} previously re- ported for the compound in 10 mM phosphate buffer, pH 7.0 (Behravan et al, 1994). The samples were kept protected from light in order to avoid photodegrada- tion.

The mixing ratio, R, is defined as the total number of added drug molecules per nucleotide base pair/triplet. Under the conditions used in this study, however, the binding constant of 9-OH-B220 to all nucleic acid polymers was estimated from light absorption measurements to be in the order of 10^ M^{" 1} at 20°C. Therefore, R can be approximately regarded as the true binding ratio, r, even at the highest mixing ratio studied here (R = 0.50) and, accordingly, the amounts of free drug in the samples can be neglected.

Absorption Spectroscopy

All absorption experiments were carried out on a Gary 4 spectrophotometer connected to an ethylene glycol-water circulating RM6 Lauda temperature bath. Titra- tion studies were performed at 20°C. At each point of the titration, the nucleic acid polymer concentration was kept constant and the obtained data were normalized to unit drug concentration and a 1 cm pathlength. In addition, certain experiments on the RNA polymers were performed in a medium containing 10 mM sodium cacodylate (pH 7.0) with 1 mM EDTA and 50 mM NaCl.

Thermal denaturation measurements of the polynucleotides in their free and drug- bound states, respectively, were monitored by following the absorption change at 260, 280 or 283.5 nm as a function of temperature. The temperature was increased at a rate of 0.1°C/min; increasing the scan rate to l°C/min had little effect on the melting curves or the derived parameters. Thermal melting temperatures, t_ιn, were determined from maxima in the calculated dA/dT derivative curves. The studies were performed in 10 mM sodium cacodylate buffer (pH 7.0) with 1 mM EDTA and varying concentrations of NaCl and MgC^. Linear Dichroism

LD at a given wavelength is defined as the differential absorption of light polarized with its electric field vector parallel and perpendicular to a macroscopic orientation direction, which equals the flow direction when the sample is oriented by shear gradients. The strength of the LD signal is dependent on the degree of sample orientation, as well as the molar absorptivity and concentration of the absorbing species. To compensate for dependence on the latter two quantities, the LD is usually normalized by dividing with the absorption spectrum of the corresponding unori- ented sample, _so, giving the dimensionless reduced linear dichroism, LD^r:

LD^r(λ)=LD(λ)/A_iso(λ) (1)

DNA and RNA molecules can be assumed to possess an effective cylindrical symmetry about their respecitve helix axis, which makes it possible to express the reduced linear dichroism as a product between an orientation factor S and an optical factor O o r

LD^r(λ) = Sx O =— (3<cos² α>- l) (2)

2

The optical factor depends on the angle α between the absorbing transition moment of the chromophore and the polynucleotide helix axis; the angle brackets indicate an ensemble average over the angular distribution. The orientation function S describes the degree of orientation of the nucleic acid polymers such that S= 1 for perfect alignment along the flow lines and S=0 for random orientation. S is affected by the flexibility and length of the nucleic acids, the flow rate, and the viscosity and temperature of the sample solution.

Under the prevailing solution conditions during the experiments (identical to those in the absorption titration studies), B-like conformations are adopted by poly(dA).poly(dT) and poly(dA).2poly(dT) , while the RNA polymers are in an A-like geometry. Hence, S can be determined from the reduced dichroism obtained for the polynucleotides at 260 nm, where the π-»π* transitions of the nucleobases are in- plane polarized; it has been shown that the effective angle between the plane of the nucleic acid bases and the helix axis can be assumed to be 77° and 86° for the canonical fiber A- and B-conformations, respectively. LD was measured on a Jasco J-500A spectropolarimeter adapted with an Oxley prism to convert the circularly polarized incident light to linearly polarized. Orientation of the nucleic acid polymers was generated by shear flow in a Couette cell with an outer rotating cylinder. Prior to each measurement, the Couette cell was treated for 30 minutes with a solution of 0.1% DEPC (a strong inhibitor of RNase) in ethanol at 60°C. Thereafter, the Couette cell was rinsed with 25 volumes of ethanol at 75°C to remove traces of DEPC. All experiments were performed at 20°C with a shear gradient of 3100 s .

Figure Legends

Figure 1. Thermal denaturation profiles of free and drug-bound poly(dA)-poly(dT) (a), poly(rA)-poly(rU) (b), poly(dA)-2ρoly(dT) (c) and ρoly(rA)-2poly(rU) (d) in 10 mM sodium cacodylate buffer at pH 7.0 with 1 mM EDTA and 12.5 mM NaCl or 100 mM NaCl + 5 mM

MgCl₂ for duplexes and triplexes, respectively. In each figure, ( ) Free polymer, (

— ) r = 0.05, ( ) r - 0.20, ( ) r = 0.50. The nucleic acid concentrations were 80 μM in base pairs/triplets for duplex/triplex polymers, respectively.

Figure 2. Isotropic absorption (A_js0) and reduced linear dichroism (LD^r) of 9-OH-B220 in the presence of poly(dA)-poly(dT) (a), poly(rA)-poly(rU) (b), poly(dA)-2poly(dT) (c) and poly(rA)-2poly(rU) (d). The nucleic acid concentrations were 150 μM and 350 μM in base pairs/triplets for the DNA and RNA polymers, respectively, and the concentration of free 9- OH-B220 was 30 μM (panels a and c) or 70 μM (panels b and d). In panels b and d LD^r spectra of the drug-free RNA structures formed in 50 mM NaCl are displayed with thin dashed lines for comparative reasons.

Figure 3. Thermal denaturation profiles of free ( ) poly(dA)-2poly(dT) and in the presence of 9-OH-B220 at a drug to base triplet ratio of 0.50 ( ). The triplexes were formed from stoichiometric 1:2 ratios of the single strands in 10 mM sodium cacodylate buffer at pH 7.0 with 1 mM EDTA and (a) 100 mM NaCl (b) 190 mM NaCl. The base triplet concentration was 80 μM.

Figure 4. Thermal denaturation profiles of free ( ) poly(dA)-2poly(dT) and in the presence of 8,10-diMeO-B-220 at a drug to base triplet ratio of 0.50 ( ). The ad^¬ ditional conditions were the same as stated in legend 3a.

Table 1

Ionic strength dependence of UV melting temperatures for the free and drug-saturated nucleic acids. The values within parentheses represent the melting temperatures in presence of the drug.^α

Nucleic acid polymer [NaCl] (mM) [MgCl₂] (mM) _m3→2 ₍ ^o _C) t_m2→i (°C)

DNA poly(dA)-poly(dT) 12.5 0 — — 5 555..55 ((8822))

50 0 — — 6 633..55

100 0 — — 7 700

100 2 5 533 7 744

100 5 6 644..55 7 766..55

0 2 6 688..55 7 755..55 poly(dA)-2poly(dT) 100 0 2 211..55 7 700..55

100 2 5 544 7 744..55

100 5 6 644..55 ((8844..55))** 7 777..55 ((8844..55))**

0 2 6 699 7 766

RNA poly(rA) poly(rU) 12.5 0 — — 4 477 ((6644..55))

50 0 — — 5 555..55

100 0 2 233^c* 5 599..55*

100 2 4 411** 7 700..55^CC

100 5 4 499*c . .7755..55**

0 2 4 411** 7 766** poly(rA)-2poly(rU) 50 0 4 499 ((4411..55)) 5 555..55 ((6655))

100 2 7 700..55** 7 700..55**

100 5 7 755..55** ((7788..55))** 7 755..55** ((7788..55))**

0 2 7 766** 7 766**

" The nucleic acid concentrations were 80 μM in base pairs/triplets and the drug to base pair/triplet ratio was 0.50. All samples were in 10 mM sodium cacodylate (pH 7.0), with 1 mM EDTA, and the measurements were performed either at 260, 280 and/or 283.5 nm. ^b When duplex and triplex transitions overlap, t_m ^3→2 is assumed to equal t,,,^2-", and is listed in both columns. Due to heating induced disproportionation of the duplex structures, the low melting temperature characterizes the 2→3 transition while the high melting temperature represents the 3→1 transition (see text for further explanation). Table 2

Analysis of LD^r parameters for the free and drug-saturated nucleic acids. The values within i parantheses represent data for the drug-free nucleic acid polymers.⁰

Reduced linear dichroism* Effective transition moment angle

Nucleic acid polymer [NaCl] (mM) [MgCl₂] (mM) 260 nm 380 nm 430-470 nm Orientation factor (S) ^α380 ^α430-470

DNA poly(dA)-poly(dT) 12.5 0 -0.054 (-0.115) -0.067 -0.055 0.037 (0.078) 90° 90° poly(dA)-2poly(dT) 100 5 -0.143 (-0.165) -0.119 -0.1 12 0.097 (0.1 12) 76° 74°

RNA poly(rA)-poly(rU) 12.5 0 -0.058 (-0.084) -0.066 -0.056 0.046 (0.066) 83° 75°

50 0 (-0.104) (0.082) poly(rA)-2poly(rU) 50 0 (-0.047) (0.037)

100 5 -0.060 (-0.099) -0.033 -0.018 0.047 (0.078) 65° 60°

" The data are obtained from Figure 2 and evaluated according to equation 2, assuming an average angle between the nucleic acid bases and the helix axis of 86° and 77° for the DNA and RNA polymers, respectively (see the Materials and Methods section). The experimental conditions are as stated in the legend of Figure 2. ^b The reduced linear dichroism is given for the DNA/RNA transition around 260 nm and for the the lowest-energy drug transitions around 380 and 430-470 nm, assumed to be polarized i the plane of the chromophore.

la

Hz/Pd-C

DMA

1. HN0₂

lb

Scheme 1. Organic synthetic scheme for 9-OH-B220

Claims

Claims

1. Agent for stabilizing natural or backbone modified DNA and RNA triplexes and hybrids thereof by partly intercalative, partly non-intercalative binding wherein the agent is a compound of the formula

wherein n = 2 or 3

R^ and R2, which are identical or different, represent lower alkyl with the provisio that the total sum of carbon atoms in R_j and R2 is maximum 8 and R^ and R2 can also form part of a ring, e.g. piperidino, morpholino or pyrrolidino,

R3 and R4, which are identical or different can be hydrogen or lower alkyl with 1 to

4 carbon atoms,

R5 is hydrogen or methyl, x is 1 or 2.

2. Agent according to claim 1 wherein R5 is hydrogen.

3. Agent according to claim 1 or 2 wherein R5 is hydrogen and x is 1.

4. Agent according to claim 3 wherein the compound is 2,3-dimethyl-6-(dimethyl aminoethyl)-9-hydroxy-6H-indolo[2,3b]quinoxaline.

5. Agent according to claim 1 wherein R5 is methyl and x is 1 or 2.

6. Agent according to claim 5 which is 6-c methylaminoethyl-2,3-dirnethyl-8, 10- dimethoxy-6H-indolo[2,3-b]-quinoxaline.

7. Agent according to claim 5 which is 6-dimethylaminoethyl-9-methoxy-6H- indolo[2,3-b]-quinoxaline.

8. Agent according to claim 5 which is 2,3-dimethyl-8, 10-dimethoxy-6H- indolo[2,3-b]-quinoxaline.

9. Agent according to any of the preceding claims for thermally stabilizing the DNA triplex poly(dA).2poly(dT).

10. Agent according to any of the claims 1-8 which stabilizes RNA triplex poly(rA).2poly(rU) by minor groove binding.

11. Agent according to any of the preceding claims for use as antigene/antisense enhancing agents when combined with oligonucleotides in form of a covalent or non-covalent complex.

12. Use of an agent according to claim 4, either alone together with a host ribo- nucleic acid or combined with an oligonucleotide in form of a covalent or non- covalent complex for preparing a drug for treating HIV and other retroviral infections.