WO1999036410A1

WO1999036410A1 - Triazine antiviral compounds

Info

Publication number: WO1999036410A1
Application number: PCT/US1999/000945
Authority: WO
Inventors: Jaime E. Arenas; Sharon T. Cload; Elizabeth S. Fleming; Yi Bin Xiang
Original assignee: Scriptgen Pharmaceuticals, Inc.
Priority date: 1998-01-13
Filing date: 1999-01-13
Publication date: 1999-07-22
Also published as: CA2318362A1; WO1999036410A9; EP1053230A1; JP2002509140A

Abstract

The present invention provides pharmaceutical formulations comprising 1,3,5-triazine derivatives. The compounds and formulations of the present invention exhibit a range of activities, including antiviral and antibiotic activities, and the formulations may be used, alone or in combination, as a method of treating a patient in need of antiviral and/or antibiotic therapy. The triazine derivatives of the present invention bind to and inhibit functional nucleic acids, and hence, have broad applicability in the treatment of conditions associated with DNA and RNA viruses.

Description

TRIAZINE ANTIVIRAL COMPOUNDS

Field of the Invention This invention pertains to the interaction between nucleic acids and

1,3,5-triazine derivatives, their use as inhibitors of the replication of the Hepatitis B virus (HBV), and their use in the treatment of viral hepatitis caused by HBN.

Background of the Invention There is a great medical need for novel therapeutic drugs to treat viral infections. Approximately 300,000 Americans become infected annually with hepatitis B virus (HBN). Currently available drugs for the treatment of HBV have limited efficacy and have not exhibited lasting effects, such that virus titres rapidly increase following the termination of drug treatment. In addition, some classes of drugs, such as nucleoside analogs that inhibit the viral polymerase, often become ineffective due to the rapid appearance of resistant viral strains. For these reasons, it has become apparent that combination therapies utilizing multiple drugs that function by different mechanisms are likely to be more successful in the treatment of viral infections. Small molecules can bind RΝA with high affinity and specificity and can block essential functions of the bound RΝA. Examples of such molecules are antibiotics such as erythromycin and aminoglycosides. The first suggestion that some antibiotic translation inhibitors interact specifically with RNA came from the genetic mapping of resistance to kanamycin and gentamicin to the methylation of 16S RNA (Thompson et al., Mol. Gen. Genet. 201:168, 1985). Erythromycin binds to bacterial RNA .and releases peptidyl-tRNA and mRNA (Menninger et al., Mol. Gen. Genet. 243:225, 1994). 2-DOS-containing aminoglycosides bind specifically to the structures of HIV RNA known as the RRE, block binding of the HIV Rev protein to this RNA, and thereby inhibit HIV replication in tissue culture cells (Zapp et al., Cell 74:969, 1993). In addition, although aminoglycosides have long been developed as translation inhibitors, they were only recently shown to bind to rRNA in the absence of proteins (Purohit and Stern, Nature 370:659, 1994).

Hygromycin B inhibits coronaviral RNA synthesis and is thought to do so by binding to the viral RNA and blocking specifically the translation of viral RNA (Macintyre et al., Antimicrob. Agents Chemother. 35:2630, 1991). Therefore, compounds that bind to functionally important regions of nucleic acids of viruses and microorganisms may be useful as inhibitors of replication or other functions, i.e. , as antiviral agents and antibiotics.

The present invention pertains specifically to a novel class of drugs comprising RNA ligands that alter the function(s) of their target RNAs. This class of compounds comprises substituted 1,3,5-triazine derivatives that specifically recognize an essential and multifunctional RNA structure of the HBV pregenomic

RNA known as the encapsidation signal (εRNA). It has been unexpectedly found that this class of compounds can function as inhibitors of HBV replication. εRNA, shown in Figure 2A, consists of a short sequence that folds into a stem-loop structure interrupted by a 6 nucleotide bulge. εRNA, which is contained within the open reading frame encoding the HBV precore protein, is also required for various steps of the HBV viral replication cycle, including encapsidation of the pregenome into viral particles and initiation of minus strand DNA synthesis. εRNA may also play a role in folding and activation of the HBV-encoded polymerase. Derivatives of melamine, l,3,5-triazine-2,4,6-triamine, have been reported in the literature as suitable for various uses. For example, 2,4,6- tris(dimethylamino)-l,3,5-triazine is an antitumor agent known as Altretamine^®, used in the treatment of ovarian cancer (Cancer 71; 4 Suppl.: 1559, 1993). Similarly, Larvadex (N-cyclopropyl-l,3,5-triazine-2,4,6-triamine) has been used as an additive to animal feed stock to control house fly infestation in poultry houses

(Poult. Sci. 62(12): 2371, 1983).

Further, Patel et al. (J. Inst. Chemists (India), 57, 1985) report a number of derivatives of 2-aryl amino-4-(4-methoxy anilino)-6-(4- chlorophenyl/phenyl hydrazido)-l,3,5-triazine having anti-bacterial activity, without data in support of this conclusion and absent any suggestion that the compounds could be used as antiviral agents.

U.S. Patent No. 5,225,405 to Paramelle et al. refers to 4,6-bt.s- allylamino-l,3,5-triazin-2-yl derivatives which reverse acquired resistance to anti- cancer and anti-malarial agents. Paramelle et al. state that the disclosed triazine derivatives, when administered at the same time with a cytotoxic agent, reduce or completely suppress multidrug resistance. The triazine compounds presumably act by inhibiting the action of an inducible membrane protein that normally functions to increase the efflux of the cytotoxic agent, thereby reducing its intracellular concentration. Paramelle et al. are silent as to the use of the triazine derivatives as antiviral agents.

U.S. Patent No. 4,508,898 to Ogilvie relates to nucleoside analogs that have a 1,3,5-triazine moiety, wherein the analog compounds exhibited antiviral activity. The compounds of Ogilvie, however, do not comprise 2,4,6-triamino- 1,3,5-triazine derivatives, but rather, are N-substituted purine and pyrimidine compounds.

European Patent Application No. 172 608 to Kim et al. relates to 1,3,5-triazine derivatives that exhibit anti-ulcer, anti-inflammatory and anti- depressant activities. However, Kim et al. fail to suggest that the disclosed triazine derivatives can be used as antiviral agents.

Golankiewicz, et al. (J. Med. Chem. 38: 3558, 1995) report the isolation of several 1,3,5-triazine derivatives having antiviral activity. However, the derivatives were limited to imidazo-[l,5-α]-l,3,5-triazine derivatives, with special emphasis on thio- and benzyl-substituted derivatives.

Kreutzberger et al. (Arzneim.-Forsch.lDrug. Res. 36 (I)(4): 626, 1986) relates to ahphatically substituted chlorodihexylamino-l,3,5-triazines having antiviral activity. However, these compounds are structurally different from the compounds of the present invention. WO 97/20825 discloses the isolation of various 1,3,5-triazine derivatives structurally distinct from those of the present invention. There is no suggestion that the triazine derivatives can be used as antiviral agents, but rather, that the compounds have utility as herbicides, insecticides, miticides, and bactericides. U.S. Patent No. 4,254,122 to Brown relates to 6- acylaminotetrahydro-l,3,5-triazine-2,4-dione derivatives that exhibit analgesic activities. The disclosed uses of the compound include their use as antiinflammatory agents and as inhibitors of prostaglandin synthetase.

Further, European Patent Application No. 795 549 to Gluzman et al. refers to bis-aryloxy(amino)-triazinyl-oxy(amino)aryl derivatives as antiviral agents.

However, unlike the compounds of the present invention, the compounds of Gluzman et al. are dimers, linked by bicyclic or heterocyclic substituted moieties, and Gluzman et al. fails to suggest the use of the monomers as therapeutic compounds and/or compositions. Thus, there is a need in the art for the identification of compounds that bind to functional viral nucleic acids, thereby inhibiting viral replication, and for specific antiviral agents that inhibit the replication of Hepatitis B virus. Such antiviral agents would be useful in the treatment of viral hepatitis caused by HBV. Summary of the Invention

The present invention provides methods for inhibiting viral and/or microbial replication, preventing or treating viral and/or microbial infection, and pharmaceutical formulations for use in such methods comprising a compound of the formulae IA

or

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non- aromatic heterocyclic, fused or poly cyclic ring, and aryloxy; wherein said alkyl, alkenyl or alkynyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkenyl; and wherein said aryl, aryloxy, heteroaryl, non-aromatic heterocyclic, or fused or poly cyclic ring is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkynyl; or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶ together, optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or poly cyclic ring, said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl; or wherein R⁷ and R⁸ together optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring wherein said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic and fused or polycyclic ring are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl, with the proviso that when R⁷ and R⁸ together form a fused or polycyclic ring, the moiety of the fused or polycyclic ring that binds with N is non-aromatic; and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier or diluent. Further, it is an object of the present invention to provide antiviral and antibiotic formulations comprising one or more compounds represented by the formulae set forth above and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or diluent, and methods of administering such formulations to a patient in need of antiviral and/or antibacterial therapy. It is also an object of the present invention to provide a method of detecting a target nucleic acid by contacting the target nucleic acid with at least one compound of the formulae set forth above and pharmaceutically acceptable salts thereof, .and monitoring an interaction between the target nucleic acid and the at least one compound of the formulae set forth above.

Brief Description of the Drawings Figures 1A-1K are graphic illustrations of preferred 1,3,5-triazine compounds of the present invention.

Figure 2A is a schematic illustration of the HBV pregenomic sequence that corresponds to the encapsidation signal (εRNA).

Figure 2B is a schematic illustration of the target RNAs used in the method of the present invention.

Figure 3 is a graphical illustration of the reduction in virus production when compound 5 and the antiviral drug 2'-deoxy-3'-thiacytidine were tested at various concentrations alone and in combinations in an antiviral assay using 2.2.15 cells producing HBV. Figures 4A, 4B, and 4C are graphical illustrations of the percentage viral reduction when compounds 5 and antiviral drug 2'-deoxy-3'-thiacytidine were tested in combination, with molar ratios of 1:15, 1:5, and 1:1.5, respectively.

Figures 5 A and 5B are photographic and schematic representations, respectively, of the results of the reaction of rRNA, εRNA, RNase, and compound 5 at concentrations ranging from 0 and 200 μM.

Figures 6 A and 6B are graphical and schematic illustrations, respectively, of the change in the melting temperature (tm) of εRNA in the presence and absence of compound 6. Detailed Description of the Invention

All patent applications, patents, and literature references cited in this specification are hereby incoφorated by reference in their entirety. In case of conflict, the present description, including definitions, will prevail.

Definitions

As used herein, the term "ligand" refers to an agent that binds a target RNA. The agent may bind the target RNA when the target RNA is in a native or alternative conformation, or when it is partially or totally unfolded or denatured. According to the present invention, a ligand can be an agent that binds anywhere on the target RNA. Therefore, the ligands of the present invention encompass agents that in and of themselves may have no apparent biological function beyond their ability to bind to the target RNA.

As used herein, the term "test ligand" refers to an agent, comprising a compound, molecule, or complex, which is being tested for its ability to bind to a target RNA.

As used herein, the term "target RNA" refers to a RNA sequence for which identification of a ligand or binding partner is desired. Target RNAs include without limitation sequences known or believed to be involved in the etiology of a given disease, condition or pathophysiological state, or in the regulation of physiological function. Target RNAs may be derived from any living organism, such as a vertebrate, particularly a mammal and even more particularly a human, or from a virus, bacterium, fungus, protozoan, parasite or bacteriophage. Target RNAs may comprise wild type sequences, or, alternatively, mutant or variant sequences, including those with altered stability, activity, or other variant properties, or hybrid sequences to which heterologous sequences have been added. Furthermore, target RNA as used herein includes RNA that has been chemically modified, such as, for example, by conjugation of biotin, peptides, modified bases, fluorescent molecules, and the like.

Target RNA sequences for use in the present invention are typically between about 5 and about 500 nt, preferably between about 30 and about 100 nt, and most preferably about 50 nt. Target RNAs may be isolated from native sources, or, more preferably, are synthesized in vitro using conventional polymerase-directed cell-free systems such as those employing T7 RNA polymerase. In a preferred embodiment, the target RNA is HBV εRNA. Figure 2A shows the actual portion of the HBV pregenomic sequence that corresponds to the encapsidation signal (εRNA). Figure 2B shows the εRNA sequence used as target RNA in the method of the present invention, and the RRE RNA target used for specificity tests. The target RNA was prepared by in vitro transcription of a linearized plasmid containing the cloned target sequence, with bacteriophage SP6 RNA polymerase in the presence of αP³²-UTP to obtain radiolabeled target RNA using methods known to those of ordinary skill in the art. As used herein, the term "treatment" with regard to a viral or microbial infection includes preventing, retarding, and/or reducing a disease, pathological condition or one or more symptoms thereof, in vertebrates, e.g. , birds, and mammals, particularly humans. In the case of HBV, the altering or inhibiting any of the following processes can be considered very useful for the treatment of infection mediated by HBV. They are:

1. Symptoms associated with acute hepatitis, including the onset of the prodromal phase, which is accompanied by anorexia, malaise, nausea and vomiting, and often fever, and which is followed in the icteric phase by the occurrence of urticarial eruptions, arthralgias and jaundice; 2. Elevations in serum aminotransferase and urinary bile levels prior to and during the onset of maximal jaundice;

3. Low-normal white blood cell counts, and appearance on blood smears of atypical lymphocytes; or 4. Symptoms associated with chronic hepatitis infection, including viremia, seroconversion, liver cancer, and cirrhosis.

According to the present invention, treatment constitutes any improvement in one or more clinical or histological symptoms or diagnostic markers observed by the attending physician or determined by quantitative or semiquantitative techniques. Non-limiting examples of appropriate techniques include analysis of blood and urine.

As used herein, "inhibition of replication" refers to any detectable reduction in replication or growth of virus, bacteria or fungi, e.g. between about

1 % and about 100% reduction, preferably between about 5% and about 100% reduction, and more preferably between about 10% and about 100% reduction.

The skilled artisan will appreciate that any reduction in viral replication, bacterial or fungal growth is significant where it is approximately equal to or greater than that which is observed for known inhibitors of viral replication, bacterial or fungal growth. As used herein, the terms "antibiotic", "antibacterial" and

"antifungal" refer to any compound that inhibits growth of or destroys microorganisms, bacteria, or fungi.

As used herein, the term "aryl" means an aromatic carbocyclic ring system having a single radical containing about 6 to about 10 carbon atoms. An aryl group may be a fused or polycyclic ring system. Exemplary aryl groups include phenyl or napthyl.

As used herein, the term "aryloxy" means an O-aryl group. An aryloxy group is optionally substituted on the aryl moiety of the aryloxy. Suitable substituents include halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl. As used herein, the term "alkyl" means a straight or branched saturated hydrocarbon group. Preferred alkyl groups include those having from 1-

12 carbon atoms.

As used herein, the term "cycloalkyl" means a non-aromatic monocyclic or fused or polycyclic ring system of about 3 to about 10 ring carbon atoms. Optionally one or more of the ring carbon atoms of the cycloalkyl may be replaced by a heteroatom, such as nitrogen, oxygen or sulfur. Exemplary cycloalkyl groups include cyclohexyl.

As used herein, the term "cycloalkenyl" means a non-aromatic monocyclic or fused or polycyclic ring system containing a carbon-carbon double bond and having about 3 to about 10 ring carbon atoms. Optionally one or more of the ring carbon atoms of the cycloalkyl may be replaced by a heteroatom, such as nitrogen, oxygen or sulfur.

As used herein, the term "carbonyl" or "carbonyl moiety" refers to any chemical moiety comprising a carbonyl functional group, e.g. , a ketone, aldehyde, carboxylic acid, acid halide, amide, peptide, anhydride and ester. As used herein, when a ring structure is described as substituted with a carbonyl group, the ring carbon atom is replaced by the group

As used herein, the term "heteroatom" includes nitrogen, oxygen and sulfur, as well as any atom other than a carbon.

As used herein, the term "ring system" refers to an aromatic or non- aromatic carbocyclic compound, in which one or more of the ring carbon atoms may be replaced by a heteroatom, such as nitrogen, oxygen or sulfur. The ring system may be optionally substituted by one or more halogens, C₁ to C₁₂ alkyl, aryl, vinyl, alkyl(aryl), vinyl(aryl) and nitro groups.

As used herein, the term "fused ring system" refers to ring systems wherein at least two adjacent carbon centers join one or more cyclic structures. A fused ring system as used herein may be aromatic or non-aromatic, or may be composed of separate aromatic and non-aromatic moieties. Exemplary carbocyclic fused ring systems are represented by the formulas:

Exemplary fused ring systems in which one or more of the ring carbon atoms is replaced by a heteroatom include the following:

As used herein, the term "polycyclic ring system" refers to ring systems having two or more cyclic compounds bonded in tandem. A polycyclic ring system as used herein may be aromatic or non-aromatic, or may be composed of separate aromatic and non-aromatic moieties. An exemplary carbocyclic polycyclic ring system is represented by the formula

An exemplary polycyclic ring system in which one or more of the ring carbon atoms is replaced by a heteroatom include the following:

Additionally, fused or polycyclic ring systems may optionally be substituted by one or more halogens, C, to C₁₂ alkyl, aryl, vinyl, alkyl(aryl), vinyl(aryl) and nitro groups.

As used herein, the term "heteroaryl" means an about 5 to 10- membered aromatic monocyclic or fused or polycyclic ring system having a single radical in which one or more of the carbon atoms in the ring system is other than carbon, for example, nitrogen, oxygen or sulfur. An exemplary heteroaryl group is pyridine. An exemplary fused or polycyclic heteroaryl group is indole. As used herein, the term "heterocyclyl" or "heterocyclic" means an aromatic or non-aromatic about 5 to about 10-membered monocyclic or fused or polycyclic ring system in which one or more of the carbon atoms in the ring system is other than carbon, for example, nitrogen, oxygen or sulfur. A heterocyclyl group may be a fused or polycyclic ring system. Exemplary heterocyclyl groups include piperidine, morpholino, and azepanyl.

As used herein, the term "primary, secondary, or tertiary amine" refers to amine compounds having one, two, or three functional groups, respectively. Suitable functional groups include halogens, amines, to C₁₂ alkyl groups, aryl, vinyl, alkyl(aryl), vinyl(aryl) and nitro groups. As used herein, the term "amide" refers to groups having the amide functional group -C(0)NH-, wherein alkyl, alkenyl or alkynyl groups may be bonded to the C or N atom of the amide group.

As used herein, the term "high affinity" refers to a compound that binds tightly to its target. Preferred compounds of the present invention will exhibit an IC₅₀ at or below 50 μM, preferably at or below 10 μM, and more preferably at or below 1 μM.

As used herein, the term "specificity" refers to a compound having either high or low affinity for its target. A highly specific compound will be unaffected by competitor RNA and will not have an effect on a heterologous assay using a different target, independent of the compound's affinity. In a preferred embodiment, compounds of the present invention will exhibit no activity or at least a 5-fold higher IC₅₀ value in a heterologous assay than in a specific assay.

The present invention provides methods for inhibiting the replication of viruses and microorganisms and for preventing or treating viral or microbial infection, which comprise administering 1,3,5-triazine compounds and pharmaceutically acceptable salts thereof. The compounds bind Hepatitis B virus (HBV) εRNA with high affinity and specificity, and thereby alter its function. The formulations of the invention comprise triazine derivatives represented by the formulae IA

or IB

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non- aromatic heterocyclic, fused or polycyclic ring and aryloxy; wherein said alkyl, alkenyl or alkynyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkynyl; and wherein said aryl, aryloxy, heteroaryl, non-aromatic heterocyclic or fused or polycyclic ring is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkynyl; or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶ together, optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring, said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl; or wherein R⁷ and R⁸ together optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic or fused or polycyclic ring, wherein said cycloalkyl, cycloalkenyl and non-aromatic heterocyclic or fused or polycyclic ring are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl, with the proviso that when R⁷ and R⁸ together form a fused or polycyclic ring, the moiety of the fused or polycyclic ring that binds with N is non-aromatic; and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention is directed to compounds of formula IB wherein one or R¹ and R² is an optionally substituted aryl. In another embodiment, the invention is directed to compounds of formula IB wherein one of R⁷ or R⁸ is an optionally substituted aryl Non-limiting examples of the compounds of the invention include:

IV

The following is a chemical process for the efficient production of triazines of the formula IA:

Commercially

available cyanuric chloride (IX) is first reacted with two equivalents of a reagent chosen from a group consisting of a primary amine and a secondary amine, to afford a singly substituted triazine of the formula (X).

Fig. IX Fig. X

The 2-amino 4,6 di chloro-1,3,5 triazine of the formula (X) is then reacted with two equivalents of a reagent chosen from a group consisting of a primary amine and secondary amine to afford a disubstituted triazine of the formula (XI)

Fig. XI

Fig. X

The 2,4-diamino-6-chloro-l,3,5 triazine of the formula (XI) can then be reacted with two equivalents of a reagent chosen from a group consisting of a primary amine, secondary amine, and hydrazine. In the case of a primary and secondary amine, the reaction affords a trisubstituted triazine of the formula (XII).

Fig. XII

Fig. XI

In the case of hydrazine, condensation reaction forms compound (XIII).

Fig. XI Fig. XIII

Compound (XII) can then be further reacted with a reagent chosen from a group consisting of aldehydes and ketones to afford 2,4,6- substituted-l,3,5-triazines of the formula (XIV).

Fig. XIII Fig. XIV

DDetailed Description of the Preferred Embodiments

The following is a chemical process for the efficient production of triazine derivatives:

Example 1: 2-fluorobenzaldehyde N-[4-(benzylamino)-6-(tert-butylamino)-l ,3,5- triazin-2-yl]hydrazone (compound 93).

20

Step 1:

To a stirred solution of cyanuric chloride in DME at -30 °C was

added a solution of 2 equivalents of benzylamine in water in dropwise fashion. The

mixture was then stirred for 3 hrs. at the reduced temperature, after which time the

mixture was warmed to room temperature and washed sequentially with saturated

sodium bicarbonate and water, dried over magnesium sulfate, and reduced in vacuo.

The product was used in the next step without purification.

Step 2:

To a stirred solution of the product of step 1 in dichloromethane at

room temperature was added 2 equivalents of tert-butyl amine in dropwise fashion.

The solution was stirred for 12 hrs., after which time the mixture was then washed

sequentially with 0.1 M hydrochloric acid, water, and saturated brine, dried over sodium sulfate, reduced in vacuo and recrystallized from hexane:ethyl acetate (4:1) to afford the starting material of step 2.

Step 3:

To a solution of the product of step 2 in dichloromethane was added

2 equivalents of hydrazine in water in dropwise fashion. The solution was then

heated to 60 °C for a period of 12 hrs., after which time the mixture was cooled to

room temperature, washed sequentially with brine and water, dried over sodium

sulfate and reduced in vacuo to a residue which was then recrystallized from

hexane: ethyl acetate (4:1) to afford the starting material of step 4.

Step 4:

To a stirred solution of the product of step 3 in toluene in a round

bottom flask was added one equivalent of 2-fluorobenzaldehyde. The flask was

then fitted with a Dean Stark trap and refluxed to azeotropically remove water. After which time the removal of water was complete, the solution was cooled to

room temperature and the solvent was removed in vacuo. The residue was then

recrystallized from hexane: ethyl acetate (5:1) to afford compound 93.

Example 2: 2-hydroxybenzaldehyde N-[4-(benzylamino)-6-(diethylamino)-l ,3,5- triazin-2-yl]hydrazone (compound 155).

Step 1:

To a stirred solution of cyanuric chloride in DME at -30 °C was

mixture was warmed to room temperature and washed sequentially with saturated

sodium bicarbonate and water, dried over magnesium sulfate and reduced in vacuo.

The product was used in the next step without purification.

Step 2:

To a stirred solution of the product of step 1 in dichloromethane at

room temperature was added 2 equivalents of diethyl amine in dropwise fashion.

The solution was stirred for 12 hrs. , after which time the mixture was then washed sequentially with 0.1 M hydrochloric acid, water and saturated brine, dried over sodium sulfate, reduced in vacuo and recrystallized from hexane:ethyl acetate (4:1) to afford the starting material of step 3.

Step 3:

To a solution of the product of step 2 in dichloromethane was added 2 equivalents of hydrazine in water in dropwise fashion. The solution was then

room temperature, washed sequentially with brine and water, dried over sodium

sulfate and reduced in vacuo to a residue which was then recrystallized from hexane: ethyl acetate (4: 1) to afford the starting material of Step 4.

Step 4:

To a stirred solution of the product of step 3 in toluene in a round

bottom flask was added one equivalent of salicylaldehyde. The flask was then

fitted with a Dean Stark trap and refluxed to azeotropically remove water. After which time the removal of water was complete, the solution was cooled to room

temperature and the solvent was removed in vacuo.

The residue was then recrystallized from hexane: ethyl acetate (5:1) to afford

compound 155:

Determination of Antiviral or Antimicrobial Activity

In Vitro Assay

One assay used in determining the activity of the compounds of the

present invention is disclosed in copending U.S. Patent Application Serial No.

08/709,342, filed September 6, 1996, the disclosure of which is hereby incorporated herein by reference in its entirety. The assay detects the interaction

between target RNA molecule and a test ligand by measuring the ligand 's ability to

inhibit hybridization between the RNA and a specific complementary

ohgonucleotide. In one embodiment, the target RNA is radiolabelled and the

ohgonucleotide is labeled with biotin; in this case, the extent of hybridization is

determined by measuring the amount of radiolabeled RNA detected with a streptavidin/biotin-based capture system. Ligands that exhibit inhibitory activity in a primary in vitro assay are

then titrated and tested in secondary assays to eliminate false positives.

Concentrations of test compounds of between about 0.1 μM and about 200 μM are

assayed for inhibition using radiolabeled εRNA in the absence or presence of a

large excess of an unlabeled non-specific RNA competitor (such as, e.g. , ribosomal

RNA, rRNA). Compounds that bind εRNA non-specifically are competitively

displaced by the rRNA, resulting in reduced or no inhibition of hybridization

between the radiolabeled εRNA and the biotinylated ohgonucleotide. Conversely, the inhibitory activity of compounds that specifically bind εRNA is not affected by

the presence of the competitor rRNA. Specificity is further tested in a third assay in which the radiolabeled

RNA is the HIV derived RRE RNA and the biotinylated ohgonucleotide is

complementary to RRE. Compounds with high specific activity for εRNA are not

expected to be inhibitory in the RRE RNA based assay. The specificity of the

ligand for εRNA is expressed as the ratio between the IC₅₀ value obtained with the

specific target RNA and the IC₅₀ value obtamed with a heterologous target (RRE

RNA), both in the presence of competitor RNA.

The substituted 2,4,6-triamino-l,3,5-triazine derivatives of the

present invention preferably exhibit IC₅₀ values for their interaction with εRNA at

or below 300 μM, more preferably at or below 50 μM, and most preferably at or

below 5 μM, in the presence of an excess of non-specific competitor rRNA. Bioassay

Ligands that exhibit high affinity and specificity for εRNA as

determined above are tested for their ability to inhibit HBV replication. Several cell

lines and cell culture assays have been developed to identify potential therapeutics

effective against chronic HBV infection. One of these cell lines, 2.2.15, has been

used in a standardized assay that has repeatedly proven to be an accurate model of

chronic cellular HBV replication and a predictive model of antiviral response for

chronic hepadnaviral infection in vivo. 2.2.15 cells contain copies of the complete HBV genome integrated into the cell's genome, and 2.2.15 cells are not susceptible

to infection by HBV. (Sells, M. A. et al , J. Virol. 62 (8): 2836-2844 (1988)). These cells express HBV genes producing complete HBV particles capable of infecting chimpanzees (Acs, G. et al , Proc. Natl. Acad. Sci. USA 84 (13): 4641-

4644 (1987)). Briefly, 2.2.15 cells are cultured and pretreated with a test

compound for about 9 days, at which point the media are harvested and subjected to

dot-blot hybridization to detect HBV virion DNA.

Further, the antiviral effect of any compound must be measured

against its toxicity. Cytotoxicity is measured by the neutral red uptake method

using the same cells used for antiviral activity.

It will be understood that the skilled artisan can employ any assay

suitable for assessing the inhibitory potency of a compound to practice the present

invention without undue experimentation. See, e.g. , ]. Sambrook and T. Maniatis,

"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratories Press, Cold Spring Harbor, NY (1989), "Current Protocols in Molecular Biology",

Ed. F.M Ausubel, et al, J. Wiley & Sons, Inc. 1997, D. Leland, "Clinical

Virology", W.B. Saunders Co., 1996, and "Cells: A Laboratory Manual", Ed.

D.L. Spector, et al. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

New York, 1997. The compounds of the present invention are believed to inhibit viral

or microbial replication by binding to functionally important viral or microbial

nucleic acids. The nucleic acids may be RNA or DNA, may form part of the

genome of the virus or microorganism or an intermediate thereof, or may represent expressed mRNA species or any other functional nucleic acid unique to the

replication or function of the virus or microorganism.

Compounds of the present invention inhibit hybridization of the

ohgonucleotide probe in the assay described in copending application U.S. Patent

Application Serial No. 08/709,342, filed September 6, 1996. This indicates that the

compounds interact with the RNA target by stabilizing the RNA structure, thereby

inhibiting the formation of hybrids between the RNA target and the complementary

ohgonucleotide present in the assay.

Supplementary evidence in support of the contention that triazine

compounds interact with the RNA target came from the observation that compound

5 protects specific positions of RNA from digestion with RNases. RNase protection

was assessed by incubating an end-labeled RNA with the amount of RNase needed to yield a ladder representing single cut events at each possible cleavage site in the RNA structure (based upon a gel electrophoresis analysis). The presence of a

ligand bound to the RNA renders the binding site inaccessible to the RNases and

therefore, the binding site will be shown by the disappearance or weakening of

specific bands in the digestion pattern (Figure 5).

Further, the RNA binding ability of the triazine compounds was

demonstrated by preparing an RNA target containing only the bulge region of

εRNA. The melting temperature of the RNA was determined by monitoring the

change in optical density at 260 nm with increasing temperature. Upon addition of

10 μM of triazine compound, the melting temperature was shifted, which indicates

that the RNA structure is stabilized by the ligand (Figure 6). Thus, these experiments demonstrate that the triazine compounds

interact with the bulge region of εRNA by nucleic acid binding.

The methods and formulations of the invention can be used to inhibit

replication and/or prevent or treat infection caused by: hepatitis B virus, hepatitis

C virus, herpes simplex virus, types 1 and 2, varicella-zoster, cytomegalo virus,

Epstein-Barr virus, polyomavirus, papillomavirus, parvovirus, vaccinia virus,

molluscum contagiosum, Marburg and Ebola viruses, influenza A and B, measles,

mumps, respiratory syncytial virus, poliovirus, coxsackie virus A and B,

rhinovirus, rotavirus, human immunodeficiency virus, types 1 and 2, rabies,

rubella, and equine encephalitis. DNA and RNA viruses encompassed by the

invention, include, without limitation, viruses belonging to the viral families adenoviridae, hepadnaviridae, herpesviridae, papovaviridae, parvoviridae, poxviridae, arenaviridae, bunyaviridae, flaviviridae, orthomyxoviridae,

paramyxoviridae, picornaviridae, reoviridae, retroviridae and rhabdoviridae.

Accordingly, compounds and formulations of the present invention

can be used in the prevention and treatment of viral hepatitis caused by HBV, as

well as in the prevention and treatment of disease conditions associated with other

DNA and RNA viruses. Such conditions include, but are not limited to: upper and

lower respiratory tract infections, ocular infections, gastroenteritis, cystidis, and

complications arising in transplant recipients, each of which is associated with

adenoviruses; treatment of immunocompromised individuals, wherein a compromised immune system is associated with cytomegalovirus; infectious

mononucleosis, associated with Epstein-Barr virus; chickenpox and shingles, which

are associated with varicella-zoster; oral, genital and skin lesions, as well as various dermatological anomalies associated with herpesvirus, papillomavirus and

polyomavirus; smallpox, as well as complications arising from smallpox

vaccinations, including allergic rash, progressive vaccinia and postvaccinial

encephalitis, each of which being associated with variola, molluscum and

contagiosum; hemorrhagic fever and aseptic meningitis, which are associated with

lassa fever virus, lymphocytic choriomeningitis virus and other arenaviruses; upper

and lower respiratory infections, serious acute respiratory tract illness and

pneumonia associated with influenza A, B, and C; measles, mumps, and

parainfluenza, associated with paramyxoviruses; enteric, neuromuscular and central

nervous system infections associated with picornaviruses; acquired immune deficiency syndrome (AIDS) and associated infections and clinical syndromes

associated with HIV infection; and encephalitis and measles associated with

togaviruses.

Moreover, given the functional similarities of the compounds of the

present invention and other RNA binding molecules, e.g. , neomycin, erythromycin

and aminoglycosides, the compounds and compositions of the present invention can

be used as antibiotic therapies. Antibiotics are used in the treatment of infectious diseases in plants, animals and man, and may kill and/or inhibit the growth of

microorganisms. Therefore, the compounds and compositions of the present invention may be used as bacteriocidal, bacteriostatic and broad-spectrum antibiotic

agents.

Pharmaceutical Formulations

The compositions of the present invention can be administered in

dosages and by techniques well known to those skilled in the medical, veterinary,

and agricultural arts taking into consideration such factors as the age, sex, weight,

species and condition of the particular patient, and the route of administration. The

compositions of the present invention can be administered alone, or can be co-

administered or sequentially administered with additional, non-triazine based

antiviral agents, such as, e.g. , Acyclovir, α-interferon, ribavirin, and various

protease inhibitors, e.g. , ritonavir, indinavir, saquinavir, and/or additional non- triazine based antibiotics, such as, e.g. , erythromycin, gentamycin, nanamycin, and

streptomycin.

The effect of simultaneous administration of triazine compound 5 and

2'-deoxy-3'-thiacytidine, a well known nucleoside analog having antiviral activity,

was examined in order to assess the interaction between the two therapies. The

results show a strong synergistic interaction when the two drugs are administered

together at a 1:15 molar ratio (3TC: triazine; Figure 4A). The antiviral activity observed with the combination drug treatment is larger than the additive activity

expected based on the activities of each compound administered alone. A

synergistic response allows the use of lower amounts of each drug in combination

therapies, which reduces the toxicity and secondary risks. Moreover, combination therapies with drugs acting on different targets should reduce the probability that

drug resistant viral strains will develop.

Moreover, the formulations of the present invention can be

administered in a formulation suitable for the manner of administration, including

but not limited to liquid preparations for mucosal administration, e.g. , oral, nasal,

anal, vaginal, peroral, intragastric administration and the like, such as solutions,

suspensions, syrups, elixirs. Further, liquid preparations for administration of the

compositions of the present invention for parenteral, subcutaneous, intradermal,

intramuscular, intravenous administrations, and the like, such as sterile solutions,

suspensions or emulsions, e.g, for administration by injection, can be formulated

without undue experimentation. Oral administration is presently preferred. In order for a composition to be administered to an animal or

human, and for any particular method of administration, it is preferred to determine

the toxicity, such as by determining the lethal dose (LD) and LD₅₀ in a suitable

animal model, e.g. , mouse; the dosage of the composition(s), and the concentration

of components in the composition; and the timing of administration in order to

maximize the antiviral and/or antimicrobial response. Such factors can be

determined without undue experimentation by such methods as titrations and

analysis of sera for antibodies or antigens, e.g. , by ELISA and/or EFFIT analysis.

Such determinations do not require undue experimentation from the knowledge of the skilled artisan, the present disclosure and the documents cited herein.

The formulations can be administered in a pharmaceutically effective amount, an antiviral effective amount and/or in an antimicrobial effective amount,

taking into account such factors as the relative activity and toxicity for the target

indication, e.g. , antiviral activity and/or antimicrobial activity, as well as the route

of administration, and the age, sex, weight, species and condition of the particular

patient.

The compositions of the present invention can be solutions,

suspensions, emulsions, syrups, elixirs, capsules, tablets, and the like. The

compositions may contain a suitable carrier, diluent, or excipient, such as sterile

water, physiological saline, glucose, or the like. Moreover, the compositions can

also be lyophilized, and/or may contain auxiliary substances, such as wetting or

emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the

route of administration and the preparation desired. Standard texts, such as

"Remington's Pharmaceutical Science", 17th Ed., 1985, incorporated herein by

reference, may be consulted to prepare suitable preparations, without undue

experimentation.

Suitable dosages for compositions of the present invention can be

determined without undue experimentation based upon the Examples provided

below, and the documents cited herein. For instance, suitable dosages of antiviral

and/or antibiotic agent in a composition can be 0.1 to 250 mg/kg/day, preferably below 100 mg/kg/day, and most preferably below 50 mg/kg/day.

In a further embodiment, the compounds of the present invention can

be used as lead compounds to improve the antiviral and/or antibiotic activities of the compounds. This can be done by modifying certain functional groups of the

compounds of the present invention based upon a recognition of the

structure/activity relationship between a particular functional group in a compound

and its biological activity. Such modifications include synthetic manipulation of the

size, hydrophilicity, hydrophobicity, acidity and basicity of a functional group,

which may inhibit or enhance the activity of a compound.

Moreover, based upon the observed binding interaction between the

compounds of the present invention and nucleic acids, the compounds of the present

invention can be used in a method of detecting and/or purification of nucleic acids,

e.g. binding assays and affinity chromatography. For example, the compounds of the invention can be covalently attached to a chromatographic support and a nucleic

acid-containing solution can be eluted over the solid support, which will bind the

target nucleic acid as the other components of the solution elute from the column in

the flow-through. With regard to binding assays and affinity chromatography,

reference is made to A. Fersht, Enzyme Structure and Mechanism. 2d Ed.. W.H.

Freeman & Co., New York, 1985, and R.K. Scopes, Protein Purification.

Principles and Practice. 2d Ed.. Springer- Ver lag, New York, 1987, the disclosures

of which are hereby incoφorated herein by reference.

Additionally, the compounds of the present invention could be used as inhibitors of specific steps of the viral replication cycle in order to study the

process in vivo or in vitro. In a diagnostic application, the compound could be used for detection of specific RNA species in samples by using a triazine derivative

labeled with a fluorescent, immunochemical, or radioactive moiety. Further, such

labeled triazine compounds could be used to study intracellular localization of the

RNA target by electron-microscopy or light microscopy using tissue culture preparations.

The following examples are intended to further illustrate the

invention without imposing any undue limitations thereon.

Example 1: HIGH-THROUGHPUT IDENTIFICATION OF εRNA LIGANDS

Compounds of the invention were obtained from commercial sources. Compound 32 is available from MicroSource Discovery Systems, Inc., Gaylordsville, Connecticut; compounds 64, 136, and 148 are available from

ComGenex, Inc., Budapest, Hungary; compounds 103 and 123 are available from

ChemBridge Corp., San Diego, California; and the remaining compounds are

available from Specs and Biospecs B.V., Rijswljk, The Netherlands.

The high throughput assays were performed in 96-well plates

containing eighty drugs (5 μl, 60 μg/ml in DMSO) distributed one per well in

columns 2-6 and 8-12 of the 8 x 12 96-well array. The remaining 16 wells in

columns 1 and 7 were filled with 5 μl DMSO to serve as the control reactions. 45

μl of reaction mixture containing buffer, salts and radiolabeled target RNA were

delivered on each well of the compound-containing plates to yield a 50 μl mixture containing 50 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl₂, 5 mM DTT, 6

μg/ml test compound, and 25 nM [³² P] -labeled εRNA. The reaction was started by

addition of the biotinylated ohgonucleotide, 262.104 A (5 μl) to a final

concentration of 100 nM. The reaction was incubated at 25 °C for 60 minutes, and

then 5 μl of 0.3 mg/ml SAAP (streptavidin alkaline phosphatase conjugate, Pierce, Rockford, 111.) was added. The reaction was incubated for an additional 30 minutes

at 25 °C and filtered through 96-well format HATF nitrocellulose filters (Millipore,

Bedford, MA) using a multiscreen vacuum manifold (Millipore, Bedford, MA).

Subsequently, 300 μl of wash buffer (50 mM Tris-HCl, pH 7.5, 200 mM KCl) was

filtered through to wash the filters. The filters were dried and supplemented with

20 μl scintillation fluid (Super Mix, Wallac Oy, Turku, Finland). The amount of hybrids formed was determined by scintillation counting of the radiolabeled εRNA

retained on the filters.

The inhibitory effect of a test compound (expressed as percentage of

inhibition, % Inh.) was calculated using the following formula:

% Inh. = (1 - (Rx/Ro)) x 100

where Ro is the average retention of hybrids in the 16 identical control reactions in

columns 1 and 7 and Rx is the hybrids retained in the presence of a test drug, x.

Biotinylated oligonucleotides 262.104 A (5'-biotin-TTA GGC AC A

GCT TGG AGG CTT GAA CAG TG-3') and 208.92 A (5^*-biotin-CGT CAT TGA

CGC TGC GCC CA-3') were synthetically prepared (Oligos Etc., Willsonville,

OR). These oligonucleotides are complementary to regions of the εRNA and RRE

RNA targets, respectively.

A plasmid containing the target sequence following the bacteriophage

SP6 promoter was used as a template for the synthesis of radiolabeled εRNA using

[α³² P]-UTP and SP6 RNA polymerase as described by the suppliers, e.g. , Ambion,

Austin, TX, and by methods well known to those of ordinary skill in the art.

Compounds that inhibited the primary screening assay described

above were individually titrated in the same assay at concentrations between 0.1 μM

and 200 μM in the absence and presence of 60-fold excess rRNA as a non-specific

competitor. A third titration was performed in a heterologous assay using

radiolabeled RRE RNA as the target and the complementary biotinylated oligonucleotide 208.92 A. From these three assays, the IC₅₀ values and specificity

for each compound was calculated.

Results:

IC₅₀ values using rRNA competitor (from Example 1) and

radiolabeled RRE RNA are shown in Table 1 for the following ligands. A

compound is considered inactive if the titration course does not result in sufficient

inhibition to estimate an IC₅₀ value. Specificity is calculated as the ratio of RRE +

rRNA IC₅₀/εRNA + rRNA IC₅₀. When an εRNA ligand was inactive in the RRE

assay the specificity of the ligand is defined as maximal (max) since a value cannot

be calculated.

Table 1. IC₅₀ Values for 1,3.,5-Triazine Compounds

Example 2: DETERMINATION OF ANTIVIRAL ACTIVITY

i. Materials and Methods

The antiviral activity of the compounds of the present invention is

measured by the methods described in Korba and Gering, Antiviral Res. 19: 55-70

(1992).

Whenever possible, all materials were prepared sterile and solutions

passed through 0.22 micron filters. Materials

1. HBV hybridization probe: A 3.2kb full genome length HBV fragment was

retrieved from a restriction digest of a plasmid clone (e.g. , pAM6)

electrophoresed in a 1 % agarose gel, isolated, and stored at -20 °C.

2. HBV gel standard: l.Oμl HBV standards (100 ng/ml) plus 5μl tracking dye

and 14 μl TE (per lane). HBV standards were made by performing separate

Bam HI and Eco Rl digests of cloned HBV DNA. The digests were combined in equimolar amounts and stored at -20 °C. This produced several HBV fragment (positive hybridization controls) as well as non-HBV

plasmid DNA fragments (negative hybridization control). For example,

pAM6 produces HBV DNA fragments of 3.2, 1.85, and 1.35kb, as well as a plasmid fragment of 4.3kb. This mixture served as a positive and a negative

hybridization control, a size standard, and a quantitation standard that

occupied only one lane of a gel.

3. HBV media standards: Culture medium (RP2) was collected from confluent

non-G418 treated 2.2.15 cells and stored frozen at -70 °C. Aliquots were

pooled as necessary (typically 1-2 liters) and centrifuged at 7000xg, 10

minutes to remove cellular debris. The supernatant was added to an Amicon

Inc. "stirred cell" (8400 series) fitted with a YM100 membrane (cat. No.

13642) 400 ml, and ultrafiltered at approximately 20psi nitrogen with constant stirring, at 4°C (approximately 3-5 hours), to insure that the

membrane was not allowed to dry. This resulted in approximately a 40-fold

concentration by volume. HBV DNA content in the concentrated sample

was quantitated by blot hybridization against a known standard quantity of

HBV DNA. Approximately a 2-fold loss of HBV DNA (as compared to the

theoretical starting concentration) frequently occurred during this process.

The final product was diluted to a standard concentration of lOng HBV

DNA/ml with RPMI 1640 (without FBS). The concentration was then

rechecked by blot hybridization. The adjusted standard pool was again rechecked by blot hybridization. The adjusted standard pool was stored at - 70 °C in 5ml aliquots in screw-capped tubes and stable for at least 5 years.

ii. Guidelines for the Culture of 2.2.15 CeUs

Cell cultures were handled aseptically, without antibiotics, and in

contained facilities (BS level II). The basal culture medium used for the culture of

2.2.15 cells was RPMI 1640. Fetal Bovine Serum (FBS) was added at either 2% or

4% final concentration, and was not heat inactivated or lot tested. L-Glutamine was

added to a final concentration of 4 mM. Complete culture medium was stored at 4

°C for up to 4 weeks. Cells were grown and maintained at 37 °C in a 5% C0₂

humidified atmosphere.

Flasks and plates were routinely seeded at a density of 3-5 x IO⁴

cells/cm². This seeding density produced confluent cultures in 3-5 days. Seeding densities of approximately lxlO⁴ cells/cm² were permissible and prolonged time to

confluence. At confluence, cells were at a density of 3-5 x 10⁵ cells/cm² and

produced their maximal and relatively stable levels of HBV. A confluent, "healthy"

T-75 flask, grown in medium with 4% FBS was subcultured in up to 6, 96-well or

24-well flat bottom plates.

iii. "Primary" or "Screening" Assay (96-well plate format)

This assay format is well suited to the screening of test compounds for potential antiviral activity. The assay provides a threshold assessment of

antiviral activity by measuring (i) the levels of HBV virion release from the cells and (ii) cytotoxicity. Two rows of cells were used for each compound, and 4 rows for the assay controls (two for untreated, and two for positive antiviral control,

e.g. , 3TC). After incubating in the presence of test compound for 9 days, the

media were harvested, transferred to 96-well U-bottom plates, and centrifuged. The

supernatants were transferred to tubes for dot blot hybridization analysis of HBV

virion DNA. The medium was aspirated off of the toxicity plates and discarded.

Toxicity plates were then incubated with neutral red dye (MTT can also be used if

preferred), washed with DPBS, developed with an acetic acid-ethanol solution, and

assayed in a plate reader.

There are many options as to what concentration ranges to use for

these assays. The compounds of the present invention were tested for toxicity at as

high a concentration as the compound's solubility and the toxicity of the diluent (frequently DMSO) allowed. The 2.2.15 cells will tolerate 2-3% DMSO for up to

10 days with little loss of viability.

1. 2.2.15 cells were seeded into 96-well, flat bottom tissue culture

plates as described above. Duplicate plates were used for the

antiviral treatments for each test compound (up to 8 compounds per pair of plates); after three to four days, cells were confluent and

medium was yellow in color; the medium was removed and replaced

with lOOμl RP2 24 hours before the beginning of drug treatment. 2a. Compounds were prepared for antiviral treatment as follows: i) For each compound, a total of 4 concentrations were

examined; this required 9 sets of 4 sterile, 1.1 ml minitubes

(36 tubes per compound). Into the first tube of each set,

sufficient compound was aliquoted to make up 700 μl (for 10-

fold dilution series) or 980μl (for 3.3-fold dilution series) of

the highest test concentration; the other tubes were left empty.

ii) The sample aliquots for the last day of treatment were set up

at a 3 fold higher concentration relative to the other aliquots;

this was done to provide sufficient material for DNA analysis

(see below),

iii) The tubes were covered with the rack lids, labelled

appropriately, and stored at -20 °C or the appropriate

temperature for the test compounds; this procedure prevents multiple freeze-thaw cycles of stock solutions of the test

compound, and ensures that all 9 daily test aliquots are treated

in an identical manner with respect to temperature variations.

If compounds were stored at 4 °C, the tops of the tubes were

covered with a sheet of parafilm to prevent evaporation.

b. Compounds were prepared for toxicity treatment as follows:

For each compound, a total of 4 concentrations were examined. This

required 9 sets of 4 sterile, 1.1 ml minitubes (36 tubes per compound). Into the first tube of each set, sufficient compound was

aliquoted to make up 465 μl of the highest test concentration. The other tubes were left empty. The tubes were covered with the rack lids, and stored at -20 °C or at an appropriate temperature. Tubes

for the last day of treatment for toxicity testing contain the same

amount of compound as used the previous days (not 3xas for the

antiviral assays).

a. The compound dilution series was prepared for antiviral treatment as

follows:

i) For a 10-fold dilution series: to the first (compound

containing) tube was added 700μl (175μl x 4). 630μl (210μl

x 3) RP2 culture medium was added to the remaining 3 tubes.

The first tube was mixed by pipeting up and down with a

pipetman (a multichannel pipetman permits the simultaneous processing of multiple compounds). 70μl of test compound-

containing medium were serially transferred from the first

tube to the other 3 tubes, taking care to thoroughly mix each

tube before transferring medium;

ii) For a 3.3-fold dilution series: 960μl (160μl x 6) of RP2 was

added to the first tube (containing the aliquot of compound)

and 640μl (160μl x 4) RP2 was added to each of the empty tubes. 280μl of test compound-containing medium were

serially transferred from the first tube to the other 3 tubes. 3b. The cytotoxicity of test compounds was analyzed in a 3.3-fold

dilution series as follows. 485μl (155μl x 3) of RP2 were added to the first tube (containing the aliquot of compound) and 310μL (155μl x 2) RP2 to each of the 3 remaining tubes. 150μl of test compound-

containing medium were serially transferred from the first tube to the

other 3 tubes.

4. Treatments were initiated by the following procedure:

a) The culture medium was removed with care to minimize the

time that the cell monolayers are without medium;

b) lOOμl of each dilution of every compound was added to each

of 6 wells (3 wells per plate) using the configuration listed

below as an example. The lowest concentration was added first, and the same tips were used to add the higher

concentrations. The untreated cells received lOOμl RP2 per

well (untreated cells have to be carried on only one pair of the

antiviral assay plates).

Col. 1 -3 Col.4-6 Col.7-9 Col. 1 0-1 2

untreated cells

g 1 @ IX drug 1 @ 1/lOX drug 1 @ 1/lOOX drug 1 @

g 2 @ IX

•

• drug 7 @1/1000

c) Treatments were repeated daily for 9 days. For the last day of

treatment, and additional 200μl RP2 were added to each well

of the antiviral assay plates after the wells are treated with the

test compounds (a total of 300μl medium per well). 5. A single plate was used for the toxicity treatments for each test

compound (up to 7 compounds per plates since the top row were

reserved for untreated cells on every toxicity plate). To initiate the

treatments, the culture medium was removed, and lOOμl of each

dilution of every compound were added to each of 3 wells using a

configuration similar to that shown above for the antiviral treatments.

The untreated cells received lOOμl RP2 per well. Treatment was

repeated daily for 9 days.

6. The assay was terminated and samples harvested for quantitative analysis of HBV virion DNA, by removing the culture medium 24 hours following the 9th day of treatment and storing the culture medium in 96-well U-bottom culture plates. These samples were

stored at 4 °C until blotting was performed. Samples were

eventually be transferred to -20 °C for long term storage.

iv. Assay for Effects on Intracellular HBV Replication (24-well plate format)

This assay format serves to further define the action of potential

antiviral agents by permitting an assessment of the levels of intracellular HBV DNA

replicative forms. This type of assay is usually performed on compounds identified

as active in the 96-well plate format since the effective antiviral concentrations

observed in those experiments can be used as a guide for this type of assay (which

is considerably more labor intensive and costly). In general, a 3- to 5-fold higher concentration of compound will be needed than that observed in the antiviral assay

to produce similar levels of effects on intracellular HBV DNA replication. 2.2.15

cells are seeded in 24-well plates for this assay and treated with test compounds for

9 days. The medium is collected at the end of the treatment period for analysis of

HBV virion DNA. For analysis of intracellular HBV DNA, the monolayers are

lysed with guanidine thiocyanate/sarcosyl/βME, dialyzed, digested with

SDS/Proteinase K, extracted with phenol and chloroform, and precipitated with sodium acetate/isopropanol. The intracellular DNAs are then resuspended, digested

with Hind III, subjected to gel electrophoresis, and transferred to nitrocellulose for

hybridization analysis. 1. 24-well culture plates are seeded as described above. The day before the addition of compounds, the medium is changed to RP2 (0.5 ml

per well). As in the 96-well plate assay, duplicate plates are used. A

total of 2 wells on each plate are treated with each dilution of

compound (4 wells per dilution).

2. Compounds are prepared for antiviral treatment as follows:

For each compound, a total of 4 concentrations are examined. This

requires 9 sets of 4 sterile, 1.1 ml minitubes (36 tubes per

compound). Into the first tube of each set, sufficient compound is

aliquoted to make up 2.2ml of drug-containing medium (additional

medium to make up the proper total volume was added at the time of cell treatment (see step 4 below). Tubes for the last day of treatment for toxicity testing contain the same amount of compound as used the

previous days (not 3X as for the 96-well plate assay). The tubes are

covered with the rack lids, and stored at -20 °C or at an appropriate

temperature.

3. Compounds in this assay are tested in a 3.3-fold dilution series. To

make this dilution series, 720μl (240μl x 3) are added to the first

tube. 460μl (230μl x 2) of medium are added to the remaining 3

tubes. 200μl are serially transferred from the first tube to the remaining 3 tubes.

4. Wells are treated from the lowest concentration of drug to the highest, adding lOOμl to each of the 4 wells. An additional 400μl of

RP2 (without drugs) are added to each well. Culture medium is continually changed and test compounds are added each day for a

total of 9 days.

5. 24-hours following the final addition of compound, the culture

medium is collected and stored in new 24-well plates. A 250μl

aliquot of each stored culture medium sample is transferred to a 96-

well U-bottomed culture plates (one plate can hold samples from up

to 4, 24-well plates), and stored at 4 °C until dot blotting is

performed.

6. The cell monolayers are then lysed for analysis of intracellular HBV

DNA as described above. vi. Neutral Red Dye Determination of Drug Toxicity

The antiviral effect of any compound was evaluated relative to its

toxicity. Cells were seeded and treated following the guidelines above, after which

a neutral red dye uptake assay was performed to assess toxicity as described below.

Other assays of cytotoxicity (e.g. , MTT) can be substituted for the procedure

described below. Note that this procedure assesses toxicity under culture and

treatment conditions which are identical to those used for the antiviral analyses,

thereby permitting a determination as to whether the reduction in virus production

due to a specific antiviral effect or due to a cytotoxic effect on the host cell. Since the cultures, by necessity, are at confluence, the cytotoxic effects of the test

compounds will probably be reduced relative to the cytotoxic effects that would be expected for actively dividing cells.

1. Cultures were treated with test compounds on the designated toxicity

plates as described above.

2. The culture medium was carefully removed 24 hours following the

9th day of treatment. The monolayer was fully removed in the top

left three wells (row A, col. 1-3). These wells were used as "blanks"

for the plate reader.

3. lOOμl DPBS (containing 0.01 % neutral red dye) were added to each

well, including the empty wells, and Incubated in the tissue culture

incubator for 30 minutes. 4. The dye was removed carefully and gently, using a multichannel

pipetman since the monolayers can become fragile after incubation

with neutral red dye. 200μl DPBS were added to all the wells taking

care not to displace the monolayers. The DPBS was removed and

lOOμl 50% EtOH/1 % glacial acetic acid (in H₂0) were added to each

well. The plates were mixed for 15 minutes on an orbital platform

shaker (120-150 RPM) to allow for full extraction of the dye from

the cells. 5. Optical absorbance at 510nm was read in an ELISA type plate

reader, using the 3 empty wells to set the background. An average absorbance value was calculated for the 9 untreated cultures on a plate, and the absorbance of dye for the treated cultures on the same

plate was expressed as a percentage of that value. This procedure

was repeated for each individual plate.

Table 2 shows the antiviral activity of a group of εRNA ligands. The

micromolar concentration at which the production of extra-cellular virus was

reduced by 50% (EC₅₀) and was reduced by 90% (EC^) is reported. Because HBV

does not cause cell death, the cytotoxic potential of the compounds can also be

estimated in the same cultures by measuring cell death by the neutral red uptake

method, as described above. This value is expressed by 50% cell death (CC₅₀ μM). Table 2. Antiviral Activity of Selected 1,3,5-Triazine Compounds

The compounds show a potent antiviral activity. In particular,

compounds 5 and 6 show antiviral activities with ECc*, values within 3 to 5-fold of

that observed for 3TC (2'deoxy-3-thiacytidine), a well known antiviral drug, in the

same assay. The figure " > " represents that no effect was observed at the highest

concentration tested.

It is noted that the compounds tested above evidenced a wide range

of antiviral activity, with some compounds demonstrating more activity than other compounds. It is believed that all structures 1-254 may have activity against the

HBV. However, as is understood by persons of ordinary skill in the art, assay

results demonstrating inactivity may be due to a number of factors, such as cell

permeability and metabolic stability.

Example 3: Antiviral Combination Therapies

Compound 5 and 3TC were tested at varying concentrations, alone

and in combination, in the antiviral assay outlined in Example 2, above. The reduction in virus production when these drugs were tested independently is shown

in Figure 3. 3TC alone causes 90% viral reduction (ECgo) at 216 nM, while

compound 5 alone exhibits an EC^ at 1300 nM.

When compounds 5 and 3TC were tested in combination with molar

ratios of 1:15, 1:5, and 1:1.5, the results are shown in Figures 4 A, 4B, and 4C.

The front row column of each plot shows the expected % viral reduction if the

effects of each drug are additive. The back row in each plot shows the observed

antiviral activity. The results show a strong synergistic effect when the drugs are

mixed at a 1:15 (3TC:compound 5) molar ratio. For example, approximately 7%

viral reduction is the expected additive effect of 20 nM 3TC and 300 nM compound

5, but the observed viral reduction reached 92%, which is indicative of a synergistic

interaction. These results suggest that when combined in a 1:15 ratio these two

drugs could be used at approximately 4 to 10-fold lower concentrations than those

needed when each drug is administered alone.

Example 4: Nucleic Acid Binding of Triazine Compounds

Reaction mixtures (10 μl) containing 50 mM Tris-HCl, pH 7.5, 50

mM KCl, 0.5 mg/ml rRNA (as a carrier), 0.5 pmol of 3 '-end ³²P-labeled εRNA,

0.05 units of 5. Cereus RNase, and compound 5 at concentrations between 0 and 200 μM were incubated at 25 °C for 30 minutes. The reaction products were

resolved by gel electrophoresis in polyacrylamide gels. B. Cereus nuclease is a single strand specific RNase. The results are shown in Figure 5 A, wherein the

sections of the ladder of digestion products corresponding to cleavage events

occurring in the bulge and loop sections of the εRNA structure. It is evident that

increasing concentrations (indicated at top) of compound 5 in the reaction results in

a decrease in cleavage at specific positions of the bulge indicated by stars.

Conversely, the loop and other regions of the structure where not affected. Similar

results were obtained using compounds 6, 15, 28, 33, and 61.

These results indicate that the triazine compound bind RNA and that

it does so at a site overlapping the bulge region. Moreover, an RNA containing a

structural element similar to the bulge of εRNA is likely to be bound by a triazine

compound increasing the stability of the RNA structure. Further, a smaller RNA target containing only the bulge region of

εRNA was prepared, and the melting temperature (tm) of this RNA structure was

determined by monitoring the change in optical density (OD) at 260 nm while

increasing the temperature. A 1 ml aliquot of reaction mixture contained 50 mM

NaCl, 10 mM sodium cacodylate, pH 7.0, 10% DMSO, and 2 μM bulge RNA.

The results are shown in Figure 6. The y-axis shows the first

derivative of the change in OD at 260 nm in OD units/degree C. The reaction containing 2 μM RNA and no drug shows that the RNA has a tm of 65 °C, and

upon addition of 10 μM compound 6, the tm is shifted to 83 °C. These results

suggest that the RNA structure is stabilized by the ligand.

All patents, applications, test methods, and publications mentioned herein are hereby incoφorated by reference.

Many variations of the present invention will suggest themselves to

those skilled in the art in light of the above detailed disclosure. All such

modifications are within the full extended scope of the appended claims.

Claims

What is claimed is:

1. A pharmaceutical formulation comprising a compound of formula IA:

or IB:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently

selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,

heteroaryl, non-aromatic heterocyclic, fused or polycyclic ring and aryloxy;

wherein said alkyl, alkenyl or alkynyl is optionally substituted

with one or more substituents selected from the group consisting of halogen,

hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl,

carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or alkynyl, and

wherein said aryl, aryloxy, heteroaryl, non-aromatic heterocyclic or fused or polycyclic ring is optionally substituted by one or more

substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro,

trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amides, cyano, cycloalkyl, alkenyl, cycloalkenyl and

alkynyl;

or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶

together, optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic,

heteroaryl, or fused or polycylic ring, said cycloalkyl, cycloalkenyl, non-aromatic

heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one

or more substituents selected from the group consisting of halogen, hydroxy, alkyl,

nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl,

cycloalkenyl and alkynyl;

or wherein R⁷ and R⁸ together optionally form a cycloalkyl,

cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring wherein said

cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring are

optionally substituted with one or more substituents selected from the group

consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy,

amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl and alkynyl, with the proviso that when R⁷ and R⁸

together form a fused or polycyclic ring, the moiety of the fused or polycyclic ring

that binds with N is non-aromatic; and pharmaceutically acceptable salts thereof;

and a pharmaceutically acceptable carrier or diluent.

2. The formulation of claim 1 comprising a compound of

formula IB wherein one R¹ or R² is an optionally substituted aryl.

3. For formulation of claim 1 comprising a compound of

formula IB wherein one of R⁷ or R⁸ is an optionally substituted aryl.

4. The formulation of claim 1 , said formulation further

comprising at least one component selected from the group consisting of a diluent, excipient, adjuvant, one or more non-triazine based antiviral or antibiotic agents,

and mixtures thereof.

The formulation of claim 1 , wherein said compound binds one

or more nucleic acids.

The formulation of claim 5, wherein said one or more nucleic

acids are RNA.

7. A method of preventing or treating hepatitis B virus infection

in a patient in need of such treatment, said method comprising administering a

compound of the formula I A:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently selected from the

group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non-

aromatic heterocyclic, fused or polycyclic ring and aryloxy;

wherein said alkyl, alkenyl or alkynyl is optionally substituted with one or more substituents selected from the group consisting of halogen,

hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl,

alkenyl, cycloalkenyl or alkynyl, and

wherein said aryl, aryloxy, heteroaryl, non-aromatic

heterocyclic, or fused or polycyclic ring is optionally substituted by one or more

trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide,

primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl and

alkynyl;

or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶

heteroaryl, or fused or polycyclic ring, said cycloalkyl, cycloalkenyl, non-aromatic

heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl,

nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester,

amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl,

cycloalkenyl and alkynyl;

or wherein R⁷ and R⁸ together optionally form a cycloalkyl,

optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy,

that binds with N is non-aromatic;

and pharmaceutically acceptable salts thereof;

and a pharmaceutically acceptable carrier or diluent, wherein said compound is administered in an amount and for a time sufficient to inhibit viral

replication.

8. The method of claim 7, wherein said method comprises

administering a compound of formula IB wherein one of R¹ or R² is an optionally

substituted aryl.

9. The method of claim 7, wherein said method comprises

administering a compound of formula IB wherein one of R⁷ or R⁸ is an optionally

substituted aryl.

10. The method of claim 7, further comprising administering at

least one component selected from the group consisting of one or more additional

nontriazine based antiviral agents, and mixtures thereof.

11. The method of claim 7, wherein said antiviral formulation is administered orally.

12. The method of claim 7, wherein said compound is

administered at a dosage of 0.1 to 250 mg/Kg/day.

13. A method of preventing or treating microbial infection in a patient in

need of such treatment, said method comprising administering a compound of the formula IA:

or IB:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently selected

from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non- aromatic heterocyclic, fused or polycyclic ring and aryloxy;

wherein said alkyl, alkenyl or alkynyl is optionally substituted with

one or more substituents selected from the group consisting of halogen, hydroxy, alkyl,

nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide,

primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or

alkynyl, and

wherein said aryl, heteroaryl, non-aromatic heterocyclic or fused or

polycyclic ring is optionally substituted by one or more substituents selected from the

group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy,

amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano,

cycloalkyl, alkenyl, cycloalkenyl and alkynyl; or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶ together,

optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused

or polycyclic ring, said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl or

fused or polycyclic ring optionally substituted with one or more substituents selected from

the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy,

alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines,

cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl;

or wherein R⁷ and R⁸ together optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring wherein said cycloalkyl,

cycloalkenyl, non-aromatic heterocyclic, or fused or polycyclic ring are optionally

substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl,

ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl and

alkynyl, with the proviso that when R⁷ and R⁸ together form a fused or polycyclic ring,

the moiety of the fused or polycyclic ring that binds with N is non-aromatic;

and pharmaceutically acceptable salts thereof;

and a pharmaceutically acceptable carrier or diluent wherein said compound

is administered in an amount and for a time sufficient to inhibit viral replication.

14. The method of claim 13, wherein said method comprises

substituted aryl.

15. The method of claim 13, wherein said method comprises

substituted aryl.

16. The method of claim 13, wherein said compound is co-administered

with at least one component selected from the group consisting of one or more additional

non-triazine based antibiotic agents and mixtures thereof.

17. The method of claim 13 wherein said compound is administered

orally.

18. The method of claim 13 wherein said compound is administered at a

dosage of 0.1 to 250 mg/Kg/day.

19. A method of detecting a target nucleic acid comprising

(a) contacting the target nucleic acid with a compound of the formula IA:

or IB:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, non-

aromatic heterocyclic, fused or polycyclic ring and aryloxy;

wherein said alkyl, alkenyl or alkynyl is optionally substituted with

one or more substituents selected from the group consisting of halogen, hydroxy, alkyl, nitro, trihalomethyl, aryl, aryloxy, alkoxy, amino, carbonyl, carboxyl, ester, amide, primary, secondary or tertiary amines, cyano, cycloalkyl, alkenyl, cycloalkenyl or

alkynyl, and

wherein said aryl, aryloxy, heteroaryl, non-aromatic heterocyclic, or

fused or polycyclic ring is optionally substituted by one or more substituents selected from

cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl;

or wherein R¹ and R² together, R³ and R⁴ together, or R⁵ and R⁶ together,

optionally form a cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or poly cy lie ring, said cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, heteroaryl, or fused or polycyclic ring optionally substituted with one or more substituents selected from

cyano, cycloalkyl, alkenyl, cycloalkenyl and alkynyl;

and pharmaceutically acceptable salts thereof;

and a pharmaceutically acceptable carrier or diluent; and

(b) monitoring an interaction between the target nucleic acid and at least

one compound.

20. The method of claim 19, wherein said compound is labeled with a

moiety selected from the group consisting of a fluorescent compound, an antibody for the

target nucleic acid, and a radioactive label.