WO2012079032A2

WO2012079032A2 - Compositions and methods of treating drug-resistant retroviral infections

Info

Publication number: WO2012079032A2
Application number: PCT/US2011/064258
Authority: WO
Inventors: Steve Peterson; Richard H. Guenther; Dan Mitchell
Original assignee: Trana Discovery, Inc.
Priority date: 2010-12-09
Filing date: 2011-12-09
Publication date: 2012-06-14
Also published as: WO2012079032A3

Abstract

Methods of treating drug-resistant retroviral infections, such as those caused by drug- resistant HIV-I, HIV-II, and HIV-III, are disclosed. The methods involve administering a compound that is an inhibitor of retroviral propagation. The inhibitors inhibits the ability of any portion of the HIV genome involved in reverse transcription to bind to or associate with a host cell tRNA, or the inhibitor disrupts the RNA/RNA complex formed between the viral genome and the human tRNA primer, or the binding or association of the host cell tRNA to a retroviral genome initiates, primes, or facilitates reverse transcription of the retroviral genome in the absence of the administered compound.

Description

COMPOSITIONS AND METHODS OF TREATING DRUG-RESISTANT RETROVIRAL INFECTIONS

FIELD

The invention generally relates to methods of treating drug-resistant retroviral infections using inhibitors of retroviral propagation.

BACKGROUND

The primate lentiviruses include the human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIVs) (Barre-Sinoussi, F., et al. (1983) Science 220:868-871; Clavel, F. (1987) AIDS 1:135-140; Daniel, M. D., et al. (1985) Science 228: 1201-1204; Desrosiers, R. C. (1990) Ann. Rev. Immunol. 8: 557-578; Gallo, R. C, et al. (1984) Science 224:500-503). HIV-1 and HIV-2 infect humans, HIV-l-like viruses infect chimpanzees, and SIV variants infect African monkeys. Humans infected by HIV-1 and HIV-2 and Asian macaques infected by certain SIV strains often develop life-threatening immunodeficiency due to depletion of CD4-positive T lymphocytes (Fauci, A., et al. (1984) Ann. Int. Med. 100:91-106; Letvin, N. L., et al. (1985) Science 230:71-739,19).

In humans, HIV infection causes Acquired Immunodeficiency Syndrome (AIDS), an incurable disease in which the body's immune system breaks down leaving the victim vulnerable to opportunistic infections, e.g., pneumonia and certain cancers, e.g., Kaposi's Sarcoma. AIDS is a major global health problem. The Joint United Nations Programme on HIV/ AIDS (UNAIDS) estimates that there are now over 34 million people living with HIV or AIDS worldwide; some 28.1 million of those infected individuals reside in impoverished subSaharan Africa. In the United States, approximately one out of every 500 people is infected with HIV or has AIDS. Since the beginning of the epidemic, AIDS has killed nearly 19 million people worldwide, including some 425,000 Americans. AIDS has replaced malaria and tuberculosis as the world's deadliest infectious disease among adults and is the fourth leading cause of death worldwide.

There are a number of agents currently used to treat HIV infection, which block replication of the HIV virus by blocking HIV reverse transcriptase or by blocking HIV protease. Three categories of anti-retroviral agents in clinical use are nucleoside analogs (such as AZT), protease inhibitors (such as nelfinavir), and non-nucleoside reverse transcriptase inhibitors (NNRTI), such as nevirapine.

In general, to exhibit antiviral activity, NRTI must be metabolically converted by host-cell kinases to their corresponding triphosphate forms (NRTI-TP). The NRTI-TP inhibit HIV-1 RT DNA synthesis by acting as chain- terminators of DNA synthesis (see Goody RS, Muller B, Restle T. Factors contributing to the inhibition of HIV reverse transcriptase by chain terminating nucleotides in vitro and in vivo. FEBS Lett. 1991, 291, 1-5). Although combination therapies that contain one or more NRTI have profoundly reduced morbidity and mortality associated with AIDS, the approved NRTI can have significant limitations. These include acute and chronic toxicity, pharmacokinetic interactions with other antiretrovirals, and the selection of drug-resistant variants of HIV-1 that exhibit cross-resistance to other NRTI.

HIV-1 drug resistance within an individual arises from the genetic variability of the virus population and selection of resistant variants with therapy (see Chen R, Quinones- Mateu ME, Mansky LM. Drug resistance, virus fitness and HIV-1 mutagenesis. Curr. Pharm. Des. 2004, 10, 4065-70). HIV-1 genetic variability is due to the inability of HIV-1 RT to proofread nucleotide sequences during replication. This variability is increased by the high rate of HIV-1 replication, the accumulation of pro viral variants during the course of HIV-1 infection, and genetic recombination when viruses of different sequence infect the same cell. As a result, innumerable genetically distinct variants (termed quasi-species) evolve within an individual in the years following initial infection. The development of drug resistance depends on the extent to which virus replication continues during drug therapy, the ease of acquisition of a particular mutation (or set of mutations), and the effect of drug resistance mutations on drug susceptibility and viral fitness. In general, NRTI therapy selects for viruses that have mutations in RT. Depending on the NRTI resistance mutation(s) selected, the mutant viruses typically exhibit decreased susceptibility to some or, in certain instances, all NRTI. From a clinical perspective, the development of drug resistant HIV-1 limits future treatment options by effectively decreasing the number of available drugs that retain potency against the resistant virus. This often requires more complicated drug regimens that involve intense dosing schedules and a greater risk of severe side effects due to drug toxicity. These factors often contribute to incomplete adherence to the drug regimen.

One way that retroviruses develop resistance to NRTI is via an excision mechanism. NRTI resistance mutations associated with the excision mechanism include thymidine analog mutations (TAMS) and T69S insertion mutations. Drugs such as DAPD, tenofovir, and tenofovir DF tend to select for the K65R mutation.

There are a number of mutations associated with the HIV-1 protease, which occur following administration of protease inhibitors. The mutations include L10I, L10F, L10V, L10C, L10R, VI II, I13V, G16E, K20M, K20R, K20T, K10T, K20V, L24I, D30N, V32I, L33F, L33I, L33V, E34Q, E35G, M36I, M36L, M36V, K43T, M46I, M46L, I47A, I47V, I47A, G48V, I50V, I50L, F53L, F53Y, I54V, I54L, I54M, I54A, I54T, I54S, 058E, D60E, I62V, L63P, I64M, I64L, I64V, H69K, A71V, A71T, A71I, A71L, G73C, G73S, G73T, G73A, T74P, V82A, V82F, L76V, V77I, G82T, V82S, V82F, V82T, V82I, G82M, I84V, I84A, I85V, I84C, N83D , N88D, N88S, L89V, and L90M. The G48V mutation is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy.

There are a number of mutations associated with the HIV-1 reverse transcriptase, which occur following administration of nucleoside reverse transcriptase inhibitors. Representative HIV-1 RT mutations include M41L, K65R, D67N, D67G, D67Del, T69D, T69ins, K70R, L74V, V75A, V75M, V75T, V75S, F77L, Y115F, F116Y, M184V, M184I, L210W, T215Y, T215F, T215C, T215D, T215E, T215E, T215S, T215I, T215V, K219Q, K219E, and K219R.

There are also a number of mutations associated with the HIV-1 reverse transcriptase, which occur following administration of non- nucleoside reverse transcriptase inhibitors. Representative NNRTI mutations include L100I, K101E, K101P, K101H, K103N, K103S, V106A, V106M, Y181C, Y181I, Y188L, Y188H, Y188C, G190A, G190S, G190E, G190Q, P225H, M230L, and P236L.

There are also a number of mutations associated with the HIV envelope gene, including G36D, G36S, I37V, V38A, V38M, V38E, Q39R, Q40H, N42T, and N43D.

There are further a number of mutations associated with the HIV integrase gene selected from the group consisting of Y143R, Y143H, Y143C, Q148H, Q148K, Q148R, and N155H.

Some nucleoside (or nucleotide) analogue reverse transcriptase inhibitor (nRTI) mutations, like T215Y and H208Y,1 may lead to viral hyper- susceptibility to the non- nucleoside analogue reverse transcriptase inhibitors (NNRTIs), including etravirine,2 in nRTI-treated individuals. The presence of these mutations may improve subsequent Virologic response to NNRTI-containing regimens (nevirapine or efavirenz) in NNRTI-naive individuals, 3 -7 although no clinical data exist for improved response to etravirine in NNRTI- experienced individuals.

The 69 insertion complex consists of a substitution at codon 69 (typically T69S) and an insertion of 2 or more amino acids (S-S, S-A, S-G, or others). The 69 insertion complex is associated with resistance to all nRTIs currently approved by the US FDA when present with 1 or more thymidine analogue-associated mutations (TAMs) at codons 41, 210, or 215. Some other amino acid changes from the wild-type T at codon 69 without the insertion may be associated with broad nRTI resistance.

Mutations known to be selected by thymidine analogues (M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E, termed TAMs) also confer reduced susceptibility to all approved nRTIs. The degree to which cross-resistance is observed depends on the specific mutations and number of mutations involved.

As with tenofovir, the K65R mutation may be selected by DAPD, didanosine, abacavir, or stavudine (particularly in patients with nonsubtype-B clades) and is associated with decreased viral susceptibility to these drugs. K65R is selected frequently (4 -l l ) in patients with nonsubtype-B clades for whom stavudine-containing regimens are failing in the absence of tenofovir.

The presence of 3 of the following mutations— M41L, D67N, L210W, T215Y/F, K219Q/E— is associated with resistance to didanosine.

The T215A/C/D/E/G/H/I/L/N/S/V substitutions are revertant mutations at codon 215 that confer increased risk of virologic failure of zidovudine or stavudine in antiretroviral- naive patients. The T215Y mutant may emerge quickly from one of these mutations in the presence of zidovudine or stavudine.

Resistance to etravirine has been extensively studied only in the context of coadministration with darunavir/ritonavir. In this context, mutations associated with virologic outcome have been assessed and their relative weights (or magnitudes of impact) assigned. In addition, phenotypic cutoff values have been calculated, and assessment of genotype- phenotype correlations from a large clinical database have determined relative importance of the various mutations. These two approaches are in agreement for many, but not all, mutations and weights. The single mutations Y181C/I/V, K101P, and L100I reduce but do not preclude clinical utility. The presence of K103N alone does not affect etravirine response. Accumulation of several mutations results in greater reductions in susceptibility and virologic response than do single mutations.

Resistance mutations in the protease gene are classified as "major" or "minor." Major mutations in the protease gene are defined as those selected first in the presence of the drug or those substantially reducing drug susceptibility. These mutations tend to be the primary contact residues for drug binding. Minor mutations generally emerge later than major mutations and by themselves do not have a substantial effect on phenotype. They may improve replication of viruses containing major mutations. Some minor mutations are present as common polymorphic changes in HIV-1 nonsubtype-B clades. Many mutations are associated with atazanavir resistance. Their impacts differ, with I50L, I84V, and N88S having the greatest effect. Higher atazanavir levels obtained with ritonavir boosting increase the number of mutations required for loss of activity. The presence of M46I plus L76V might increase susceptibility to atazanavir.

In Pi-experienced patients, the accumulation of six or more mutations is associated with a reduced virologic response to lopinavir/ritonavir. The product information states that accumulation of seven or eight mutations confers resistance to the drug. However, there is emerging evidence that specific mutations, most notably I47A (and possibly I47V) and V32I, are associated with high-level resistance. The addition of L76V to three PI resistance- associated mutations substantially increases resistance to lopinavir/ritonavir.

Clinical correlates of resistance to tipranavir are limited by the paucity of clinical trials and observational studies of the drug. Lists of mutations associated with accumulating resistance have been presented, with some conflicting results. In vitro studies and initial analysis of clinical data show mutations L33F, V82L/T, and I84V as having substantial contributions. Confirmatory studies are pending. A number of mutations (L24I, I50L/V, I54L, and L76V) are associated with decreased resistance in vitro and improved short-term virologic response if two or more are present.

Resistance to enfuvirtide is associated primarily with mutations in the first heptad repeat (HR1) region of the gp41 envelope gene. However, mutations or polymorphisms in other regions of the envelope (eg, the HR2 region or those yet to be identified) as well as coreceptor usage and density may affect susceptibility to enfuvirtide.

The activity of CC chemokine receptor 5 (CCR5) antagonists is limited to patients with virus that uses only CCR5 for entry (R5 virus). Viruses that use both CCR5 and CXC chemokine receptor 4 (CXCR4; termed dual/ mixed [D/M]) or only CXCR4 (X4 virus) do not respond to treatment with CCR5 antagonists.

Virologic failure of these drugs frequently is associated with outgrowth of D/M or X4 virus from a preexisting minority population present at levels below the limit of assay detection. Mutations in HIV-1 gpl20 that allow the virus to bind to the drug -bound form of CCR5 have been described in viruses from some patients whose virus remained R5 after virologic failure of a CCR5 antagonist. Most of these mutations are found in the V3 loop, the major determinant of viral tropism. There is as yet no consensus on specific signature mutations for CCR5 antagonist resistance. Some CCR5 antagonist-resistant viruses selected in vitro have shown mutations in gp41 without mutations in V3; the clinical significance of such mutations is not yet known. Raltegravir failure is associated with integrase mutations in at least 3 distinct genetic pathways defined by 2 or more mutations including (1) a signature (major) mutation at Q148H/K/R, N155H, or Y143R/H/C; and (2) 1 or more additional minor mutations. Minor mutations described in the Q148H/K/R pathway include L74M plus E138A, E138K, or G140S. The most common mutational pattern in this pathway is Q148H plus G140S, which also confers the greatest loss of drug susceptibility. Mutations described in the N155H pathway include this major mutation plus either L74M, E92Q, T97A, E92Q plus T97A, Y143H, G163K/R, V151I, or D232N.60 The Y143R/H/C mutation is uncommon.

Examples of known HIV strains that include one or more of these mutations include HIV-1K65R, HIV- IKTOE, HIV-1L74V, HIV-1MI84V, HIV-1AZT2, HIV-1AZT3 , HIV- IAZTT, HIV-

1AZT9> HIV-1QI51M and HIV- l69Insertion-

There remains a need for the identification of inhibitors of HIV infection, particularly with respect to drug resistant HIV variants, and more particularly with respect to multi-drug resistant HIV variants. The present invention provides such inhibitors, and methods of treating drug resistant HIV infection.

SUMMARY

Compounds which are inhibitors of retroviral propagation, and which are effective against drug resistant HIV variants, including multi-drug resistant HIV variants, are disclosed. The compounds function via a different pathway than conventional anti-retroviral compounds, such as nucleoside reverse transcriptase inhibitors ("NRTI"), non-nucleoside reverse transcriptase inhibitors ("NNRTI"), protease inhibitors ("PI"), entry inhibitors, integrase inhibitors, and other known anti-retroviral compounds. Accordingly, the compounds are effective against drug-resistant HIV, including multi-drug resistant HIV.

Because the inhibitors described herein function via a different mechanism than conventional anti-retroviral agents, they can be administered in combination or alternation with such additional anti-retroviral agents. Indeed, because the inhibitors described herein function via a totally different mechanism, when they are co-administered with such other anti-retroviral agents, they can delay the onset of the development of mutations typically resulting from these agents. Methods of treating and/or preventing retroviral infection using the inhibitors described herein are also disclosed.

The compounds inhibit retroviral propagation by inhibiting retroviral reverse transcription, viral recruitment of the retroviral primer used in translation, human tRNA^Lys3, inhibiting the final packaging and assembly of new virions, and/or inhibiting the binding of a host cell tRNA to a target nucleic acid molecule.

The inhibitory activity of the compounds can be evaluated and/or verified using methods for screening inhibitors of retroviral propagation as described herein. Such methods may involve forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment, a nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound. The mixture is incubated under conditions that allow binding of the tRNA anticodon stem loop fragment and the nucleic acid molecule in the absence of the test compound. One can then determine whether or not a test compound inhibits the propagation of a retrovirus. Inhibition of binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of retroviral propagation.

DETAILED DESCRIPTION

The present invention relates to methods for treating drug-resistant HIV infection, including multi-drug resistant HIV infection, using compounds which inhibit retroviral propagation. Compositions including the compounds, and, optionally, additional anti- retro viral compounds, are also disclosed. Viral propagation can be inhibited by inhibiting reverse transcription, viral replication, translation of viral RNA into proteins, recruitment of human tRNA^Lys3, packaging and assembly of new virions, and/or inhibiting the binding of a host cell tRNA to a target nucleic acid molecule.

Prior to describing this invention in further detail, however, the following terms will first be defined.

Definitions:

As used herein, an "inhibitor" refers to any compound capable of preventing, reducing, or restricting retroviral propagation. An inhibitor may inhibit retroviral propagation, for example, by preventing, reducing or restricting retroviral reverse transcription. In some embodiments, the inhibition is at least 20% (e.g., at least 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%) of the retroviral propagation as compared to the propagation in the absence of the inhibitor. In one aspect, an inhibitor prevents, reduces, or restricts the binding of a tRNA, or fragment thereof, to a target nucleic acid molecule. Inhibitors can also affect recruitment of human tRNA^Lys3, translation of viral RNA into proteins, and/or final packaging and assembly of virions. Assays for analyzing inhibition are described herein and are known in the art. An "RNA-dependent DNA polymerase" or "reverse transcriptase" is an enzyme that can synthesize a complementary DNA copy ("cDNA") from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template (target nucleic acid); thus, they are both RNA- and DNA-dependent DNA polymerases.

As used herein, a "label" or "detectable label" is any composition that is detectable, either directly or indirectly, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include, but are not limited to, radioactive isotopes (for example, ³²P, ³⁵S, and ³H), dyes, fluorescent dyes (for example, Cy5 and Cy3), fluorophores (for example, fluorescein), electron-dense reagents, enzymes and their substrates (for example, as commonly used in enzyme-linked immunoassays, such as, alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or hapten; and proteins for which antisera or monoclonal antibodies are available. Moreover, a label or detectable moiety can include an "affinity tag" that, when coupled with the target nucleic acid and incubated with a test compound or compound library, allows for the affinity capture of the target nucleic acid along with molecules bound to the target nucleic acid. One skilled in the art will appreciate that an affinity tag bound to the target nucleic acid has, by definition, a complimentary ligand coupled to a solid support that allows for its capture. For example, useful affinity tags and complimentary partners include, but are not limited to, biotin-streptavidin, complimentary nucleic acid fragments (for example, oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, oligo G-oligo C), aptamers, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.

The terms "alkyl", "aryl" and other groups refer generally to both unsubstituted and substituted groups unless specified to the contrary.

Unless specified otherwise, alkyl groups are hydrocarbon groups and are preferably C1-C15 (that is, having 1 to 15 carbon atoms) alkyl groups, which can be branched or unbranched, acyclic or cyclic. The above definition of an alkyl group and other definitions would apply also when the group is a substituent on another group (for example, an alkyl group as a substituent of an alkylamino group or a dialkylamino group).

The term "aryl" refers to any functional group or substituent derived from a simple aromatic ring, such as phenyl, thiophenyl, indoyl, etc. The term "alkenyl" refers to a straight or branched chain hydrocarbon group with at least one double bond, preferably with 2-15 carbon atoms.

The term "alkynyl" refers to a straight or branched chain hydrocarbon group with at least one triple bond, preferably with 2-15 carbon atoms.

The terms "alkylene," "alkenylene" and "alkynyllene" refer to bivalent forms of alkyl, alkenyl, and alkynyl groups, respectively.

The terms "halogen" or "halo" refer to fluoro, chloro, bromo, or iodo.

Substituent groups building off of the hydrocarbon groups include alkoxy, aryloxy, acyloxy, haloalkyl, perfluoroalkyl, fluorine, chlorine, bromine, carbamoyloxy, hydroxyl, nitro, cyano, cyanoalkyl, azido, azidoalkyl, formyl, hydrazine, hydroxyalkyl, alkoxyalkyl, and the like.

I. Antiviral Compounds

wherein Single ΟΓ double bond _{^ with the prov}j_{so t}h_at no allenes are intended to be within the scope of the invention.

M and N = 1, 2 or 3 atoms from C, N, O

X and Y = NR¹, O or S

R¹ = H, alkyl, aryl, aralkyl, alkaryl, heterocyclyl, heteroaryl, substituted analogs thereof, wherein the substituents are selected from the list of substituents, Z, defined herein.

Substituents Z as defined herein include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R', -SR', -N₃, -C(=0)NRR", -NRC(=0) R", -C(=0)R, -C(=0)OR, -OC(=0)R, - OC(=0)NRR", -NRC(=0)0 R", -S0₂R, -S0₂NRR", and -NR'S0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl).

More specifically, within these broad formulas are the following representative narrower formulas:

Ar-i (CH₂)_n NR., Ar₂

Formula A

wherein Ari and Ar₂ are, independently, six membered aryl rings, five or six membered ring heteroaryl rings, or analogs thereof in which a five membered heteroaryl or six membered aryl or heteroaryl ring is fused to the six membered aryl rings, five or six membered ring heteroaryl rings,

n is 0 or 1, and

Ri is H or a moiety cleaved in vivo to form H,

and each of the aryl/heteroaryl rings can be substituted with one to three substituents,

Z.

Substituents Z as defined herein include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R, -SR, -N₃, -C(=0)NR'R", -NR'C(=0) R", -C(=0)R, -C(=0)OR, -OC(=0)R, - OC(=0)NR'R", -NRC(=0)0 R", -S0₂R, -S0₂NRR", and -NR'S0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl).

In one embodiment, where one or more aryl rings are present, at least one of the rings is an aniline or substituted aniline (i.e., an aryl ring with an -NH₂, primary amine, or secondary amine substituent).

Representative compounds from the table provided above, in which n is 0, Ari is phenyl, and Ar₂ is pyridinyl or pyrimidinyl, include Compounds 66, 73, and 111.

Compounds 16, 20, and 27 are compounds in which n is 0, Ari and Ar₂ are pyrimidinyl, and one of the pyrimidinyl rings is fused to an aryl ring.

Compound 62 is a compound in which n is 0, Ari is phenyl, and Ar₂ is pyridinyl, and the pyridinyl ring is fused to an aryl ring.

Compounds 6, 11, 12, and 90 are compounds in which n is 0, Ari is phenyl, and Ar₂ is oxathiazole.

Compound 29, 39, and 61 are compounds in which n is 0, and Ari and Ar₂ are phenyl.

Compounds 25, 33, 37, 42, 51, 52, 53, 69, 79, 80, 87, 99, 100, 107, 112 are compounds in which n is 1 , and Ari and Ar₂ are phenyl.

Compound 58 is a compound in which n is 1, and Ari and Ar₂ are both phenyl-fused heteroaryl rings.

Compounds 81 and 91 are compounds in which n is 1, Ari is phenyl, and Ar₂ is a phenyl-fused heteroaryl rings.

Compounds 4, 5, 13, and 15 have a core structure where n is 0, Ari is lH-pyridin-4- one, and g fused to a benzene ring, as shown below:

An (CH₂)_m NR., (CH₂)_{m Af2}

Formula B

where Ari, Ar₂, and Ri are as defined above, m is 0, 1, 2 or 3, and the aryl/heteroaryl rings can be substituted with from 1 to 3 substituents, Z, as described above, with the proviso that at least one m is 2. Specific embodiments are those in which one of m is 1 and the other m is 2, both of m are 2, one of m is 0 and the other m is 2, and one of m is 1 and the other m is 3. Compound 21 is an example of a compound where one of m is 0 and the other m is 2. Compounds 19 and 36 are examples of compounds where one m is 1 and the other m is 2. Compounds 50 and 98 are examples of compounds where one m is 1 and the other m is 3. Compounds 50 and 98 both also include a benzofuran ring, and a phenyl ring substituted with a dimethylamine group at a position para to the linkage to the remainder of the molecule.

Formula C wherein m is 0, 1, or 2, X is NRi, O, or S, and halo is F, CI, Br, I. In one embodiment of Formula B, X is S and halo is CI. Representative azacyclic rings include morpholine, azacyclopentane, and piperidine.

and 88 are examples of compounds of Formula C.

Formula D

where Z, j and Ri are as defined above, with the proviso that two Ri groups can link together to form a 5-7 membered ring azacyclic moiety.

Compounds 9 and 28 are examples of compounds of Formula D.

Formula E

where Z and j are as defined above.

Compounds 45 and 49 are examples of compounds of Formula E.

Formula F

wherein Ari, Ri, Z and j are as defined above, and Ari can include from one to three Z substituents.

64, and 77 are representative compounds of Formula F.

Formula G

wherein X, Ri, Z, j, and n are as defined above, and the =X moiety can be present or not present (i.e., n is 0 or 1).

Compounds 17, 60, 74, and 96 are representative compounds within the scope of Formula G.

Formula H

Alternatively, the compounds of Formula H can have the formula shown below, where the cyclohexadienone double bond is optional (as indicated by a dashed line), as follows:

wherein the dashed line indicates the presence of an optional double bond.

The analogs can have substantially any organic substituent or functional group substituted in place of one or more of the hydrogen atoms on the ring skeleton, for example, a substituent J as defined herein.

In one embodiment, R4, R5, R6, R7, R15, R16, and R17 are, independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (Ci-Ce), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C2-C₆), lower alkynyl (C2-C₆), lower cycloalkyl (C3-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above).

Alternatively, one or more of R4 and R5, R5 and R6, R6 and R7, R15 and R16, and R16 and R17 together form a five, six, or seven-member ring, which ring can include one or more heteroatoms, such as O, S, and N (wherein N can be substituted with H or R').

The compounds of Formula H use the following numbering scheme for the various positions: 1

In one aspect of this embodiment, one or both of the nitrogens at positions 13 and 19 as listed above can be replaced with a CR' moiety.

Naphtho[2',3':4,5]imidazo[l,2-a]pyridine-6,ll-dione is a representative compound of

Formula H.

Formula I

The analogs can have substantially any organic or inorganic substituent or functional group substituted in place of one or more of the hydrogen atoms on the ring skeleton (i.e., at positions 2, 3, 6, 7, and 8), for example, a substituent J as defined herein.

In one embodiment, these substituents are, independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (Ci-Ce), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above).

Rl, R2, R3, and R4, are independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, -COR', and -COOR', wherein R' is, independently H, a lower alkyl (Ci-C₆), lower haloalkyl (Ci-C₆), lower alkoxy (Ci-C₆), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above). In one embodiment, one or both of RI and R2, and R3 and R4, together with the nitrogens to which they are attached, form a 5-7 membered ring, which can include one or more additional heteroatoms such as O, S, or N, wherein the N can be bonded to a substituent R', as defined above).

4,5-Bis(dimethylamino)-l-naphthaldehyde (Compound 10) is a representative

wherein, for compounds of Formulas J, K, and L, Ri, Z and j are as defined above. In a specific embodiment of Formulas J and K, both of Ri adjacent the ring nitrogen are methyl. In another embodiment, both of these Ri moieties link together to form a five, six, or seven- membered ring, which can optionally include a heteroatom such as O, S, or N. Compounds 14, 18, 22, and 24 are specific examples of Formula J. Compounds 23, 34, 35, 68, 82, and 85 are specific examples of Formula K. Compounds 30, 67, 78, and 103 are specific examples of Formula L.

wherein X, Z, j, m and Ri are as defined above.

In a specific embodiment, X in the heteroaryl ring is O and/or X in the bridge between the aryl and heteroaryl ring is O. In one embodiment, n is 0, and in another embodiment, n is 1. Compounds 84 and 101 are specific examples of Formula M, where n is 1. Compounds 63, 89, and 113 are specific examples of Formula M, where n is 0. Examples of heterocyclic rings that can be attached to the heteroaryl ring include piperidine, piperazine, azacyclohept pentane, and morpholine.

Formula N

wherein X,Z, j, are as defined above.

nds of Formula N have the following formula:

That is, these compounds fall within the definition of Formula N, where Ri is alkaryl or aryl. In a specific embodiment, the X variables are selected so as to form a thiourea moiety. In another specific embodiment, at least one Z substituent is present on at least one aryl ring, and the substituent is an -NH₂, primary or secondary amine group. Compounds 76, 94, and 110 are specific examples of Formula M where n is 0. Compounds 41 and 55 are specific examples of Formula N where n is 1.

Other compounds falling generally into the definition of Formula N, where Ri is alkyl iety), include compounds 71 and 109.

Formula O

wherein X, Z, j , and m are as defined above. In a specific embodiment, at least one aryl ring includes a nitro group. Compounds 54 and 93 are specific examples of Formula M.

Formula P

wherein Z, j, n, and Ri are as defined above, and a) K is NRi, or b) K is N(Ri)₂, and the link to the other ring nitrogen is absent, in which case the other NRi moiety is an N(Ri)₂ moiety rather than an NRi moiety.

Specific compounds within the scope of Formula P include compounds 1 and 10.

wherein Z, j, n, and X are as defined above, and R₂ is absent (i.e., a direct link between the aryl ring and the C=X moiety), or is an alkyl or cycloalkyl moiety linking the aryl ring and the C=X moiety. Compounds 97, 104, 106, and 108 are examples of specific compounds falling within Formula Q.

Formula R

wherein X and R] are as defined elsewhere herein, o is an integer from 4 to 8 (in compounds 2 and 3, the number is 5), R₂ is d_₆ alkyl, and R₅ is -C(=X)OR_l5 -C(=X)SR_l5 -C(=X)NHR_l5 - X-C(=X)ORi, -X-C(=X)SRi, -X-C(=X)NHR_l5 -O-Ri, -SRi, or -NHR_L In one embodiment, where the ring nitrogen is bound to Ri, the NRi is an amide moiety, and the amide moiety functions as a prodrug form of compounds in which the NRI is an amine.

Compounds 8, 32, 40, 57, 75, 83, 95, and 114 do not fall within the scope of the various Formulas A through R. These compounds were also active, and compounds of these formulas, where the aryl or heteroaryl rings can be substituted with from one to three Z substituents are also within the scope of the invention. A number of compounds appear active in the assays described herein, and are all highly conjugated molecules. These compounds include Compounds 7, 26, 38, 46, 47, 59, 65, 72, 86, 92, and 102. These compounds are generally types of compounds known to be highly conjugated, including benzophenones, triarylmethanes, fulvenes, and the like.

In each of these compounds, particularly those of Formula Q, an ester group can be replaced with an amide or thioamide moiety to increase the in vivo stability. Carbamate, thiocarbamate, urea, thiourea, ether, and thioether moieties can also be substituted for ester moieties. Aryl rings can be replaced with heteroaryl rings, such as thiophene rings in any of these compounds. For example, Compound 143 is an example of a compound that would fall within Formula Q if one of the aryl rings is replaced with a thiophene ring, n is 0, and R₂ is a direct link between the aryl ring and the C=X moiety. All of the compounds can optionally be substituted with a morpholinyl or piperidinyl moiety, which can be desirable to increase hydrophilicity.

In one embodiment, compounds of Formulas H and I are intended to be specifically excluded.

Novel compounds may also be formed in a combination of substituents which creates a chiral center or another form of an isomeric center. In this embodiment, the compound may exist as a racemic mixture, a pure enantiomer, and any enantiomerically enriched mixture.

The compounds can occur in varying degrees of enantiomeric excess, and racemic mixtures can be purified using known chiral separation techniques.

The compounds can be in a free base form or in a salt form (e.g., as pharmaceutically acceptable salts). Examples of suitable pharmaceutically acceptable salts include inorganic acid addition salts such as sulfate, phosphate, and nitrate; organic acid addition salts such as acetate, dichloroacetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate; salts with an acidic amino acid such as aspartate and glutamate; alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium and calcium; ammonium salt; organic basic salts such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, and Ν,Ν'-dibenzylethylenediamine; and salts with a basic amino acid such as lysine and arginine. The salts can be in some cases hydrates or ethanol solvates. The stoichiometry of the salt will vary with the nature of the components.

Representative compounds include the following:

Compound 1 3-ethyl-6-methoxy-lH-benzo[de]cinnoline Compound 2 methyl 6-(5-methyl-2-oxo-2,3-dihydro-lH-imidazol-4-yl)-6-oxohexanoate Compound 3 ethyl 6-(l-benzoyl-5-methyl-2-oxo-2,3-dihydro-lH-imidazol-4-yl)-6- oxohexanoate

Compound 4 2-[(8-ethoxy-4-methyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(lH)- quinazolinone

Compound 5 2-[(6-methoxy-4-methyl-2-quinazolinyl)amino]-5,6-dimethyl-4(lH)- pyrimidinone

Compound 6 2-[(4,7-dimethyl-2-quinazolinyl)amino]-6-propyl-4(3H)-pyrimidinone Compound 7 tris [4- (dimethylamino)phenyl] methanol

Compound 8 5-(2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoic acid

Compound 9 N-ethyl-5-nitro-N-phenyl-2,l,3-benzoxadiazol-4-amine

Compound 10 4,5-bis(dimethylamino)-l-naphthaldehyde

Compound 11 N,N-dimethyl-N'-[4-(2-pyridinyl)-l,3-thiazol-2-yl]-l,4-benzenediamine hydrobromide

Compound 12 N~2~-(4,6-dimethyl-2-pyrimidinyl)-2,4-quinazolinediamine hydrochloride Compound 13 2-[(4,6,7-trimethyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(lH)- quinazolinone

Compound 14 9-(2-methoxyphenyl)-2,3,7,7-tetramethyl-10-thioxo-9,10-dihydro-7H- isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5-dione

Compound 15 2-[(4,8-dimethyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(lH)- quinazolinone

Compound 16 2- [(4-methyl-2-quinazolinyl)amino] -6-propyl-4( 1 H)-pyrimidinone

Compound 17 2- [(4-acetylphenyl)amino] -3 -( 1 -pyrrolidinyl)naphthoquinone

Compound 18 9-(2,5-dimethoxyphenyl)-2-methoxy-7,7-dimethyl-10-thioxo-9,10-dihydro- 7H-isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5-dione

Compound 19 [3 -( 1 , 3 -benzodioxol- 5 -yl)- 3 -phenylpropyl] [4- (dimethylamino)benzyl] amine hydrochloride

Compound 20 2-[(4,7-dimethyl-2-quinazolinyl)amino]-5-ethyl-6-methyl-4(3H)-pyrimidinone Compound 21 N-[2-(l-cyclohexen-l-yl)ethyl]-2-(3-methylphenyl)-5-nitro-2H-l,2,3-triazol- 4-amine 3 -oxide

Compound 22 2-methoxy-7,7-dimethyl-9-(2-propoxyphenyl)- 10-thioxo-9, 10-dihydro-7H- isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5-dione

Compound 23 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2- methylbenzoate Compound 24 9-(2,3-dimethylphenyl)-2-methoxy-7,7-dimethyl-10-thioxo-9,10-dihydro-7H- isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5-dione

Compound 25 N'-(2-methoxybenzyl)-N,N-dimethyl-l,4-benzenediamine

Compound 26 2-(4-ethylphenyl)naphthoquinone

Compound 27 2-[(4,6-dimethyl-2-quinazolinyl)amino]-l,5,6,7-tetrahydro-4H- cyclopenta[d]pyrimidin-4-one

Compound 28 5-nitro-4-(l-piperidinyl)-2,l,3-benzoxadiazole

Compound 29 N,N-dimethyl-N'-[2-nitro-4-(trifluoromethyl)phenyl]-l,3-benzenediamine

Compound 30 l-[2-(benzyloxy)benzyl]-5-methyl-lH-indole-2,3-dione

Compound 31 l-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]pyrrolidine

Compound 32 2-(4-ethylphenyl)-5-methyl-4-[4-(4-morpholinyl)benzylidene]-2,4-dihydro-

3 H-pyrazol- 3 -one

Compound 33 (2-methoxybenzyl)[4-(l-piperidinyl)phenyl]amine

Compound 34 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2- methoxybenzoate

Compound 35 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2- chlorobenzoate

Compound 36 4-({[3-(2-furyl)-3-(2-methoxyphenyl)propyl]amino}methyl)-N,N- dimethylaniline

Compound 37 N'-[4-(allyloxy)-3-chloro-5-methoxybenzyl]-N,N-dimethyl-l,4- benzenediamine

Compound 38 2,6,7-trihydroxy-9-(5-nitro-2-furyl)-3H-xanthen-3-one

Compound 39 2-[ethyl(4-{ [2-nitro-4-(trifluoromethyl)phenyl]amino}phenyl)amino]ethanol Compound 40 5 - [4-(dimethylamino)phenyl] -3 -(4-methoxyphenyl)-N-methyl-4,5 -dihydro- 1 H-pyrazole- 1 -carbothioamide

Compound 41 N-[4-(dimethylamino)phenyl]-N'-(4-methylbenzyl)thiourea

Compound 42 N-[4-(allyloxy)-3-methoxybenzyl]-4-(l-pyrrolidinyl)aniline

Compound 43 5-methyl-N-[7-(4-morpholinyl)-2,l,3-benzoxadiazol-4-yl]-4-phenyl-3- thiophenecarboxamide

Compound 44 4-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]morpholine

Compound 45 l-(4-fluorophenyl)-2-(2-nitrovinyl)-lH-pyrrole

Compound 46 l,l'-(2,4-cyclopentadien-l-ylidenemethylene)bis(4-methoxybenzene)

Compound 47 3-(2-chlorophenyl)-6-ethyl-7-methoxy-4H-chromene-4-thione

Compound 48 N-[4-hydroxy-3-(phenylthio)-l-naphthyl]-4-methoxybenzenesulfonamide Compound 49 l-(4-chlorophenyl)-2-(2-nitrovinyl)-lH-pyrrole

Compound 50 4-({ [3-(2-furyl)-4-phenylbutyl]amino}methyl)-N,N-dimethylaniline Compound 51 4-(4-benzyl-l-piperazinyl)-N-(4-fluorobenzyl)aniline

Compound 52 (2-ethoxy-3-methoxybenzyl)[4-(l-pyrrolidinyl)phenyl]amine

Compound 53 (4-fluorobenzyl)[4-(l-pyrrolidinyl)phenyl] amine

Compound 54 6-(dimethylamino)-2-(2-methylphenyl)-5-nitro- lH-benzo[de]isoquinoline- l,3(2H)-dione

Compound 55 N-(4-chlorobenzyl)-N'-[4-(diethylamino)phenyl]thiourea

Compound 56 4-fluoro-N-[4-hydroxy-3-(phenylthio)-l-naphthyl]benzenesulfonamide

Compound 57 2-bicyclo[2.2.1]hept-2-yl-5-nitro-lH-isoindole-l,3(2H)-dione

Compound 58 N-[2-(lH-indol-3-yl)ethyl]-9-acridinamine

Compound 59 6-bromo-2-(3-methoxy-4-propoxyphenyl)-3-nitro-2H-chromene

Compound 60 ethyl 3-(4-methoxyphenyl)-l,4-dioxo-l,4-dihydro-2-naphthalenecarboxylate

Compound 61 (2-methoxyphenyl)[2-nitro-4-(trifluoromethyl)phenyl]amine

Compound 62 N'-(2,8-dimethyl-4-quinolinyl)-N,N-dimethyl-l,4-benzenediamine hydrochloride

Compound 63 2-(2-methylphenyl)-5-(l-piperidinyl)-l,3-oxazole-4-carbonitrile

Compound 64 N-[4-hydroxy-3-(phenylthio)-l-naphthyl]benzenesulfonamide

Compound 65 l-(4-chlorophenyl)-5-[(5-nitro-2-furyl)methylene]-2,4,6(lH,3H,5H)- pyrimidinetrione

Compound 66 N'-(4,6-dimethyl-2-pyrimidinyl)-N,N-dimethyl-l,4-benzenediamine Compound 67 5-bromo-l-(2-chlorobenzyl)-lH-indole-2,3-dione

Compound 68 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2- thiophenecarboxylate

Compound 69 2-({[4-(dimethylamino)phenyl]amino}methyl)phenol

Compound 70 2-(2-fluorophenyl)-3-(l-methyl-lH-pyrrol-2-yl)acrylonitrile

Compound 71 N-[4-(diethylamino)phenyl]-N'-isobutylthiourea

Compound 72 2-(4-ethylphenyl)-4-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methyl-2,4- dihydro-3H-pyrazol-3-one

Compound 73 N-(2-methoxyphenyl)-3-nitro-2-pyridinamine

Compound 74 ethyl 3-(4-methylphenyl)-l,4-dioxo-l,4-dihydro-2-naphthalenecarboxylate Compound 75 5-{ [(2-nitrophenyl)thio]amino}-l,3-benzodioxole

Compound 76 N-[4-(diethylamino)phenyl]-N'-(4-ethoxyphenyl)thiourea

Compound 77 N-[4-hydroxy-3-(phenylthio)-l-naphthyl]-2-thiophenesulfonamide Compound 78 l-benzyl-5-bromo-7-methyl-lH-indole-2,3-dione

Compound 79 N-[4-(dimethylamino)benzyl]-6-methyl-2-pyridinamine

Compound 80 2-[2-bromo-4-({ [4-(dimethylamino)phenyl]amino}methyl)-6-ethoxyphenoxy]-

N-(tert-butyl)acetamide

Compound 81 N-[4-(dimethylamino)benzyl]-l-pentyl-lH-benzimidazol-2-amine

Compound 82 4,6-diethyl-4,8-dimethyl-4H-pyrrolo[3,2,l-ij]quinoline-l,2-dione

Compound 83 N-[(l-methyl-lH-pyrrol-2-yl)methylene]-4-(l-naphthylmethyl)-l- piperazinamine

Compound 84 5 -( 1 -azepanyl)-2- [(3 -chlorophenoxy)methyl] - 1 ,3 -oxazole-4-carbonitrile Compound 85 6'-methyl-5',6'-dihydrospiro[cyclohexane-l,4'-pyrrolo[3,2,l-ij]quinoline]- ,2'- dione

Compound 86 4-(di-lH-indol-3-ylmethyl)-l,2-benzenediol

Compound 87 (5-bromo-2-methoxybenzyl)[4-(4-morpholinyl)phenyl]amine

Compound 88 l-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]piperidine

Compound 89 5-(l-azepanyl)-2-(2-fluorophenyl)-l,3-oxazole-4-carbonitrile

Compound 90 4-(2-{ [4-(diethylamino)phenyl]amino}-l,3-thiazol-4-yl)-l,2-benzenediol Compound 91 l-butyl-N-[4-(diethylamino)benzyl]-lH-benzimidazol-2-amine

Compound 92 (4-bromophenyl) [3 -nitro-4-( 1 -piperidinyl)phenyl] methanone

Compound 93 2-(2,5-dimethylphenyl)-6-nitro-lH-benzo[de]isoquinoline-l,3(2H)-dione Compound 94 N-[4-(diethylamino)phenyl]-N'-(2,4-dimethoxyphenyl)thiourea

Compound 95 5-(dimethylamino)-l,3-benzothiazole-2-thiol

Compound 96 ethyl 3-(4-ethylbenzyl)-l,4-dioxo-l,4-dihydro-2-naphthalenecarboxylate Compound 97 4-propylphenyl 4-nitrobenzoate

Compound 98 4-({ [3-(2-furyl)-3-(4-methylphenyl)propyl]amino}methyl)-N,N- dimethylaniline

Compound 99 N-benzyl-5-(4-benzyl-l-piperazinyl)-2-nitroaniline

Compound 100 N-benzyl-4-chloro-2-nitroaniline

Compound 101 5-( 1 -azepanyl)-2- [(4-chlorophenoxy)methyl] - 1 ,3 -oxazole-4- carbonitrile

Compound 102 [4-(2,6-dimethyl-4-morpholinyl)-3-nitrophenyl](4- ethoxyphenyl)methanone

Compound 103 5-chloro-l-(2-chlorobenzyl)-lH-indole-2,3-dione

Compound 104 2-fluorobenzyl 2-chloro-4-nitrobenzoate

Compound 105 3-[(3,4-dimethylphenyl)amino]-l-(4-nitrophenyl)-l-propanone Compound 106 N- [4-(diethylamino)phenyl] -2-phenylcyclopropanecarboxamide Compound 107 2-[2-bromo-6-methoxy-4-({[4-(l- pyrrolidinyl)phenyl] amino } methyl)phenoxy ] acetamide

Compound 108 N- [4-( 1 -azepanyl)phenyl] -2-methylbenzamide

Compound 109 N-cyclohexyl-N'-[4-(dimethylamino)phenyl]thiourea

Compound 110 N-(3-chloro-4-fluorophenyl)-N'-[4-(diethylamino)phenyl]thiourea Compound 111 4,6-dimethyl-N-[4-(l-pyrrolidinyl)phenyl]-2-pyrimidinamine

Compound 112 (2,6-dichlorobenzyl)[4-(l-pyrrolidinyl)phenyl]amine

Compound 113 2-(2-fluorophenyl)-5-(4-phenyl- 1-piperazinyl)- 1 ,3-oxazole-4- carbonitrile

Compound 114 1 - [4-nitro-3 -( 1 -pyrrolidinyl)phenyl] -4-(2-thienylcarbonyl)piperazine

The above-listed compounds were obtained from a list of active compounds determined in an initial screening assay, and were the compounds which appeared to show the highest efficacy.

Potential R-group substitutions:

Novel compounds may also be formed in the event that some combination of substituents creates a chiral center or another form of an isomeric center in any compound of the present list. The list would include any or all of the racemic mixture, pure enantiomers, and any enantiomerically enriched mixture.

For example, in one embodiment, the amino groups at the 4 and/or 5 position on the naphthalene ring in the compounds of formula I can be replaced with -C(Ri)3, -OR], or -SRi.

Representative compounds include the following:

4,5 -bis(dimethylamino)- 1 -naphthaldehyde

4,5-bis(diethylamino)- 1 -naphthaldehyde

4,5-bis(dipropylamino)- 1 -naphthaldehyde

4,5-bis(dibutylamino)-l-naphthaldehyde

4,5-bis(methylethylamino)-l-naphthaldehyde

4,5-bis(methylpropylamino)-l-naphthaldehyde

4,5-bis(methylbutylamino)-l-naphthaldehyde

4,5 -bis(ethylpropylamino)- 1 -naphthaldehyde 4,5-bis(ethylbutylamino)-l-naphthaldehyde

4-dimethylamino-5 -diethylamino- 1 -naphthaldehyde

4-dimethylamino-5 -dipropylamino- 1 -naphthaldehyde

4-dimethylamino-5 -dibutylamino- 1 -naphthaldehyde

4-diethylamino-5-dimethylamino- 1 -naphthaldehyde

4-diethylamino-5-dipropylamino-l-naphthaldehyde

4-diethylamino-5-dibutylamino-l-naphthaldehyde

4-dipropylamino-5-dimethylamino- 1 -naphthaldehyde

4-dipropylamino-5-diethylamino-l-naphthaldehyde

4-dipropylamino-5-dibutylamino- 1 -naphthaldehyde.

II. Synthetic Methods

The following synthetic information is representative only, and not intended to be limiting. The compounds described herein all include at least one aryl or heteroaryl ring, and all of these rings can be further substituted with one or more substituents, as defined herein. Those skilled in the art will readily understand that incorporation of other substituents onto an aryl or heteroaryl ring used as a starting material to prepare the compounds described herein, and other positions in the compound framework, can be readily realized. Such substituents can provide useful properties in and of themselves or serve as a handle for further synthetic elaboration.

Benzene rings (and pyridine, pyrimidine, pyrazine, and other heteroaryl rings) can be substituted using known chemistry, including the reactions discussed below. For example, the nitro group on nitrobenzene can be reacted with sodium nitrite to form the diazonium salt, and the diazonium salt manipulated as discussed above to form the various substituents on a benzene ring.

Diazonium salts can be halogenated using various known procedures, which vary depending on the particular halogen. Examples of suitable reagents include bromine/water in concentrated HBr, thionyl chloride, pyr-ICl, fluorine and Amberlyst-A

A number of other analogs, bearing substituents in the diazotized position, can be synthesized from the corresponding amino compounds, via the diazocyclopentadiene intermediate. The diazo compounds can be prepared using known chemistry, for example, as described above.

The nitro derivatives can be reduced to the amine compound by reaction with a nitrite salt, typically in the presence of an acid. Other substituted analogs can be produced from diazonium salt intermediates, including, but are not limited to, hydroxy, alkoxy, fluoro, chloro, iodo, cyano, and mercapto, using general techniques known to those of skill in the art.

For example, hydroxy-aromatic/heteroaromatic analogues can be prepared by reacting the diazonium salt intermediate with water. Halogens on an aryl or heteroaryl ring can be converted to Grignard or organolithium reagents, which in turn can be reacted with suitable aldehyde or ketone to form alcohol-containing side chains. Likewise, alkoxy analogues can be made by reacting the diazo compounds with alcohols. The diazo compounds can also be used to synthesize cyano or halo compounds, as will be known to those skilled in the art. Mercapto substitutions can be obtained using techniques described in Hoffman et al., /. Med. Chem. 36: 953 (1993). The mercaptan so generated can, in turn, be converted to an alkylthio substitutuent by reaction with sodium hydride and an appropriate alkyl bromide. Subsequent oxidation would then provide a sulfone. Acylamido analogs of the aforementioned compounds can be prepared by reacting the corresponding amino compounds with an appropriate acid anhydride or acid chloride using techniques known to those skilled in the art of organic synthesis.

Hydroxy- substituted analogs can be used to prepare corresponding alkanoyloxy- substituted compounds by reaction with the appropriate acid, acid chloride, or acid anhydride. Likewise, the hydroxy compounds are precursors of both the aryloxy and heteroaryloxy via nucleophilic aromatic substitution at electron deficient aromatic rings. Such chemistry is well known to those skilled in the art of organic synthesis. Ether derivatives can also be prepared from the hydroxy compounds by alkylation with alkyl halides and a suitable base or via Mitsunobu chemistry, in which a trialkyl- or triarylphosphine and diethyl azodicarboxylate are typically used. See Hughes, Org. React. (N. Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996) for typical Mitsunobu conditions.

Cyano-substituted analogs can be hydrolyzed to afford the corresponding carboxamido-substituted compounds. Further hydrolysis results in formation of the corresponding carboxylic acid-substituted analogs. Reduction of the cyano-substituted analogs with lithium aluminum hydride yields the corresponding aminomethyl analogs. Acyl- substituted analogs can be prepared from corresponding carboxylic acid- substituted analogs by reaction with an appropriate alkyllithium using techniques known to those skilled in the art of organic synthesis.

Carboxylic acid-substituted analogs can be converted to the corresponding esters by reaction with an appropriate alcohol and acid catalyst. Compounds with an ester group can be reduced with sodium borohydride or lithium aluminum hydride to produce the corresponding hydroxymethyl-substituted analogs. These analogs in turn can be converted to compounds bearing an ether moiety by reaction with sodium hydride and an appropriate alkyl halide, using conventional techniques. Alternatively, the hydroxymethyl-substituted analogs can be reacted with tosyl chloride to provide the corresponding tosyloxymethyl analogs, which can be converted to the corresponding alkylaminoacyl analogs by sequential treatment with thionyl chloride and an appropriate alkylamine. Certain of these amides are known to readily undergo nucleophilic acyl substitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- or N-arylcarbamoyloxy- substituted compounds by reaction with N-alkyl- or N-arylisocyanates. Amino-substituted analogs can be used to prepare alkoxycarboxamido-substituted compounds and urea derivatives by reaction with alkyl chloroformate esters and N-alkyl- or N-arylisocyanates, respectively, using techniques known to those skilled in the art of organic synthesis.

Synthesis of Compounds of Formula A

The compounds of Formula A include an aryl or heteroaryl ring linked to another aryl or heteroaryl ring to form an amine. Where Ri is H, the compounds are secondary amines. Where Ri is other than H, the compounds are tertiary amines. Where n is 0, the amine nitrogen is linked directly to the Ari ring, and where n is 1, a methylene bridge exists between the amine nitrogen and the Ari ring (i.e., a benzylamine when Ari is a benzene ring). The formation of aniline moieties is well known, as discussed above with respect to forming amine substituents on aryl/heteroaryl rings. The formation of a benzylamine can take place by reacting a benzyl halide with an amine using standard nucleophilic displacement chemistry.

Synthesis of Compounds of Formula B

The compounds of Formula B include an aryl or heteroaryl ring linked to another aryl or heteroaryl ring to form an amine. Where Ri is H, the compounds are secondary amines. Where Ri is other than H, the compounds are tertiary amines. Where n is 0, the amine nitrogen is linked directly to the Ari ring, and where n is 1, 2, or 3, an alkylene bridge exists between the amine nitrogen and the Ari ring (i.e., a benzylamine, benzethylamine, and the like, when Ari is a benzene ring). The formation of aniline moieties is well known, as discussed above with respect to forming amine substituents on aryl/heteroaryl rings. The formation of a benzylamine, benzethylamine, and the like can take place by reacting a arylalkyl halide with an amine using standard nucleophilic displacement chemistry. Synthesis of Compounds of Formula C

The compounds of Formula C are 5-membered ring heteroaryl compounds which include various substituents at various positions on the rings. The nitro group at position 3 can be difficult to attach to an unsubstituted 5-membered ring heteroaryl, since the preference for nitration can be at the 2-position. However, once desired halo substituents are placed on 2 and 5 position on the ring, for example, by reacting the heteroaryl ring with an elemental halogen in the presence of acetic acid, the ring can be nitrated at the 3-position. The halogen at the 5 -position can be reacted with vinyl sulfide in a nucleophilic displacement reaction to form the S-vinyl ether. A halogenation reaction will place a halogen at the 3-position, which can be displaced using a suitable amine to form the heterocyclic ring attached to the heteroaryl ring.

Synthesis of Compounds of Formula D

The compounds of Formula D can be formed by starting with the unsubstituted ring structure, and performing a nitration reaction. Then, halogenation can be used to place a halogen at a position adjacent to the nitro group, and the halogen can be displaced with an amine to form the compounds of Formula D.

Synthesis of Compounds of Formula E

The compounds of Formula E include a haloaryl ring with a pyrrole ring attached para to the halogen. The pyrrole ring includes a CH₂=CHNC>₂ moiety at the 2-position. This moiety can be provided, for example, by starting with a pyrrole with a CHO group at the 2- position, and using Wittig chemistry to attach the CH₂=CHN0₂ moiety. An aryl (i.e., phenyl) ring with a halogen at the 1 -position and a diazonium salt at the 4-position can be reacted with the pyrrole to form a linkage between the pyrrole ring and the aryl ring.

Synthesis of Compounds of Formula F

The compounds of Formula F include a naphthyl ring that further includes an aryl sulfonamide moiety, an aryl thioether moiety, and an -OH or ether moiety. Starting from a naphthyl ring with appropriately positioned amine, thiol, and hydroxy groups, one can selectively protect two of the three groups. A thiol group can be reacted with a diazonium group on a benzene ring to form the aryl thioether. The amine group can be reacted with an aryl sulfonyl halide to form the sulfonamide moiety. The protected hydroxy group can be deprotected to form an OH group, which can then be converted to ethers or esters if desired, using known chemistry. Since an amine group is more nucleophilic than a hydroxy group, the sulfonamide can likely be prepared even in the presence of an unprotected hydroxy group.

Synthesis of Compounds of Formula G

The compounds of Formula G are napthoquinones. They can typically be prepared from appropriately substituted 1 ,4-quinones and dienes using Diels Alder chemistry (see, for example, Witayakron et al., Tetrahedron Letters, Volume 48, Issue 17, 23 April 2007, Pages 2983-2987, the contents of which are hereby incorporated by reference).

Synthesis of Compounds of Formula H

The compounds of Formula H are also substituted napthoquinones. They can similarly be prepared from suitably appropriately substituted 1,4-quinones and dienes using Diels Alder chemistry. The quinones are prepared from 1,4-bisphenols, and the additional ring functionality can be incorporated by starting with 1 ,4-bisphenols with an amine group at the 2 position, and a pyridine carboxamide at the 3-position, where an imine linkage is formed between the carboxy group on the carboxamide and the amine at the 2-position.

Synthesis of Compounds of Formula I

The compounds of Formula I are naphthalenes with amines at the 4 and 5 position, and an aldehydes at the 1-position. Ideally, the amines are dialkylamines, so that they do not react with the aldehyde moiety to form an intramolecular imine group. Amine groups are typically formed on aromatic rings by a combination of nitration with nitric acid, and reduction of the nitro group to an amine group. Alkylation of the amine groups involves routine nucleophilic displacement chemistry with appropriate alkylamines, whereas arylation can involve reaction of an amine with a diazonium salt. The aldehydes moiety can be introduced by reacting an organolithium reagent (a naphthyl-lithium) with isonitriles to the corresponding lithium aldimine. Subsequent hydrolysis effectively converts the organolithium compound to its aldehydes (see, for example, G. E. Niznik, W. H. Morrison, III, and H. M. Walborsky (1988), "1-d- Aldehydes from Organometallic Reagents: 2- Methylbutanal-l-d", Org. Synth., Coll. Vol. 6: 751, the contents of which are hereby incorporated by reference).

Synthesis of Compounds of Formula J The compounds of Formula J can be formed from appropriately functionalized dihydroquinolines. The amine in the dihydroquinoline can be reacted with oxaloyl chloride in a stepwise fashion, and the remaining acid chloride can be reacted with the aromatic ring via Friedel- Crafts acylation conditions to form the cyclic structure. From there, routine chemistry, for example, 3,2 Diels Alder chemistry heterocyclic ring structure, for example, by stepwise reaction of an aniline with appropriately functionalized groups on the dihydroquinoline framework.

Synthesis of Compounds of Formula K

The compounds of Formula K can be formed from appropriately functionalized dihydroquinolines. The amine in the dihydroquinoline can be reacted with oxaloyl chloride in a stepwise fashion, and the remaining acid chloride can be reacted with the aromatic ring via Friedel-Crafts acylation conditions to form the cyclic structure.

Synthesis of Compounds of Formula L

The compounds of Formula L can be formed from appropriately functionalized anilines. The amine in the aniline can be reacted with oxaloyl chloride in a stepwise fashion, and the remaining acid chloride can be reacted with the aromatic ring via Friedel-Crafts acylation conditions to form the cyclic structure. From there, the amine can further react with an appropriately functionalized benzyl halide to form the benzylamine moiety.

Synthesis of Compounds of Formula M

The compounds of Formula M can be formed from appropriately functionalized 5- membered ring heteroaryls. The amine moiety can be formed by initial nitration, which tends to form nitro groups in the 2-position, and subsequent reduction to an amine group (which may be postponed until the other moieties are present). Halogenation occurring after the nitration step can place a halo group at the 3 -position, which can then be nucleophilically displaced by a cyanide ion to form the nitrile, or converted to an organolithium reagent and reacted, for example, with cyanogen bromide to form the nitrile moiety. The side chain (alkylaryl, ether, and the like) can be incorporated using standard chemistry, such as nucleophilic substitution using an organolithium reagent.

Synthesis of Compounds of Formula N The compounds of Formula N include urea, thiourea, and other similar moieties. At least one of these moieties includes an O, S, or N linked to an aryl ring, so the compounds can be synthesized from an appropriately functionalized phenyl isocyanate, thioisocyanate, and the like by nucleophilic reaction with an appropriately functionalized amine, thiol, or hydroxy-containing material (i.e., Ri-XH).

Synthesis of Compounds of Formula O

The compounds of Formula O include a naphthalene ring, and a cyclic ring structure including an imide moiety. The compounds can be prepared from naphthalene dicarboxylic acids and a suitably functionalized aniline in much the same way as phthalimide is formed (i.e., ring cyclization as the amine reacts with the acids, or activated forms thereof). Alternatively, the acids or activated forms thereof, such as anhydrides, acid chlorides, and the like, can be reacted with ammonia, which is then reacted with an aryl-diazonium salt to form the aniline.

Synthesis of Compounds of Formula P

The compounds of Formula P include a naphthalene ring, and a) a cyclic ring structure including two ring nitrogens originating at positions 1 and 8 on the naphthalene ring, b) a cyclic ring structure including one ring nitrogen originating at position 1 or 8 on the naphthalene ring, and a methylamine moiety at the other of these positions, or c) two amines, at positions 1 and 8 on the naphthalene ring. Naphthalene 1,8 diamine is a commercially available compound whose synthesis need not be discussed herein. Alkylamines can be formed by reacting an amine (or ammonia) with a -CH₂Br moiety at the 1 or 8 position, or with another naphthyl halide at this position. Rings with adjacent ring nitrogens can be formed, for example, by step-wise reaction of suitably functionalized hydrazines with diazonium salts (to form a linkage directly on an aromatic ring) or a -CH₂Br moiety on the naphthalene ring (or other suitable leaving group other than bromide on such moiety).

Synthesis of Compounds of Formula Q

The compounds of Formula Q include an amide, ester, thioester, or similar linkage, where to the left and right of the carbonyl/thiocarbonyl moiety lie an aryl or arylalkyl moiety. These compounds can be prepared from suitably functionalized benzoic acid or phenyl- alkanoic acid by forming an acid halide or anhydride (or versions thereof where the carbonyl is replaced by a C(=S) or C(=NRi) moiety), and reacting with a suitably functionalized phenol, thiophenol, aniline, or aryl-substituted hydroxyalkane, thioalkane, or amine.

Synthesis of Compounds of Formula R

The compounds of Formula R are functionalized cyclic ureas. They can be formed from suitably functionalized diamines (with amine moieties on adjacent carbon atoms) by reaction with phosgene, diphosgene, triphosgene, and the like. The carbonyl side chain can be formed, for example, by converting a carboxylic acid to an acid halide, and reacting the acid halide with a suitable Grignard or organolithium reagent.

Additional synthetic details are provided below:

Preparing the 4,5-Bis(Diamino)-l-Naphthaldehyde Framework

1,8-diamino naphthalene is commercially available, and is used as a starting material for other commercially available analogs, such as proton sponge (1,8-bis-dimethylamino naphthalene).

To prepare the simplest analog, where R1-R4 are H, one can react 1,8-diamino naphthalene with carbon monoxide in a Friedel Craft reaction can produce the formyl group at a para-position to one of the amino groups. See, for example, "Aldehyde Syntheses" G.A. Olah, et al., Friedel-Crafts and Related Reactions, Wiley-Interscience, vol. Ill, Chapter XXXVIII, pp. 1153-1256, 1964.

"Superacid-Catalyzed Formylation of Aromatics with Carbon Monoxide," G.A. Olah et al., J. Org. Chem., vol. 50, pp. 1483-1486, 1985. Note that the numbering on the naphthalene ring changes as the formyl substituent is added (i.e., formyl becomes the 1-position, and the amino groups are numbered accordingly, going from 1,8-diamino to 4,5-diamino). This chemistry is shown below in Scheme 1.

N-Alkylation/Arylation

Either before or after the Friedel Crafts reaction to put the formyl substituent on the naphthalene ring, one can react one or both of the amino groups (-NH₂) with an alkylating reagent to alkylate one or both of the amine groups, depending on stoichiometry.

For example, proton sponge (the bis-dimethylamino analogue of 1,8- diaminonaphthalene) is prepared by reacting 1,8-diaminonaphthalene with dimethyl sulfate.

Suitably functionalized aryl groups (i.e., aryl rings with any desired substitution) can be prepared that include a diazonium moiety at the position in which it is desired to attach the aryl group to the amine moiety(ies) on the 1,8-diaminonaphthalene. The amine moiety(ies) can then displaced the diazonium moiety to provide aryl amines.

Protecting groups can be used when it is desirable to alkylate/arylate one amino group in preference to the other. For example, one can selectively protect either the 1 -amine or the 8-amine in the 1,8-diaminonaphthalene starting material, for example, using a t-boc or other protecting groups, such as those described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, June 1999, John Wiley & Sons Inc., the contents of which are hereby incorporated by reference. Then, following the alkylation/arylation reaction(s), the protective groups can be removed. The aldehyde group can be protected, for example, as an acetal group, which can be deprotected at a later time by simply reacting the acetal with water in the presence of an acid catalyst.

Substitution at Positions 2, 3, 6, 7, and 8

Where it is desirable to provide substitution at positions 2, 3, 6, 7, and 8 on the naphthalene ring, electrophilic aromatic substitution can be used to provide other desired functionality. For example, alkyl, aryl, heteroaryl, alkaryl, arylalkyl, alkenyl, alkynyl, and acyl groups can be added using Friedel-Crafts alkylation/arylation/acylation reactions. Other electrophilic aromatic substitution reactions can be used, for example, to provide halogens, such as by forming chloronium or bromonium ions in situ and reacting them with the aromatic ring, or by forming sulfonium or nitronium ions to provide sulfonyl or nitro groups.

Friedel Crafts alkylation is conducted using an appropriate halo-alkyl moiety, and a Lewis acid. The alkyl moiety forms a carbocation, and electrons from the aryl ring form a bond with the carbocation, placing a positive charge on the aryl ring. The aryl ring then loses a proton. Alkyl and alkaryl moieties (such as benzyl moieties) can be added in this fashion.

Friedel Crafts acylation is similar, but uses an acid halide, such as an acid chloride, to place a ketone moiety on the ring. The acid halide can be an alkyl acid, such as acetic acid, propionic acid, butyric acid, and the like, or can be an aromatic acid, such as benzoic acid, p- toluic acid, and the like.

Friedel Crafts arylation (also known as the Scholl reaction) is a coupling reaction with two aryl rings, catalyzed by a Lewis acid. The proton lost during the coupling reaction serves as an additional catalyst. Typical Reagents are iron(III) chloride in dichloromethane, copper(II) chloride, PIFA and boron trifluoride etherate in dichloromethane, Molybdenum(V) chloride and lead tetraacetate with BF₃ in acetonitrile.

Substitution typically occurs at a position ortho or para to the amine groups. So, positions 3, 6, and 8 are typically functionalized using this chemistry. Substitution of the naphthalene ring at a meta position to the amine groups (i.e., positions 2 and 7) can be performed by oxidizing the amine group(s) to nitro groups, which leads to meta substitution. The nitro groups can then be reduced back to the amine groups.

Formation of Heterocyclic Rings Incorporating the Amino Groups

Either or both of the amino groups in the 1,8-diamino naphthalene starting material, or in 4,5-bis(amino)-l-naphthaldehyde, can be cyclized using a di-halo compound. For example, a five membered ring can be formed using nucleophilic subsituition. The amine is reacted with a 1,4-di-halobutane, such as 1,4-dibromobutane, and a six membered ring can be formed using a 1,5-dihalopentane, such as 1,5-dibromopentane. The reaction typically takes place in the presence of a tertiary amine, which reacts with the in situ-formed hydrogen halide, such as hydrogen bromide.

When it is desirable to incorporate an additional heteroatom into the cyclic group, one or more of the carbons in the dihaloalkane can be replaced with a heteroatom, such as O, S, or N (where the N can be substituted with an alkyl, aryl, alkaryl, aralkyl, or other such substituent). Preparation of the Naphtho[2',3':4,5]imidazo[l,2-a]pyridine-6,ll-dione Framework

The compound 2,3-dichloro-l,4-naphthoquinone is commercially available, and is described, for example, in Honda, Nakanishi and Tabe, Bull. Chem Soc. Japan, 56(8):2338- 2340 (1983), the contents of which are hereby incorporated by reference.

The carbonyl moieties on this compound can be protected, for example, as an ethylene ketal, and then reacted with 2-hydroxypyridine, the tautomer of which has the formula:

to provide an intermediate in which the pyridine nitrogen reacts with one of the ring halogens. Nucleophilic displacement of the halogen with ammonia (or an amine, if an N-alkyl, N-aryl, or other such derivative is desired) affords an amine group, which cyclizes with the carbonyl moiety in the pyridine to form an imine linkage in a ring-closure step. Deprotection of the ketone moieties, and catalytic dehydrogenation, afford the final product. This chemistry is shown below in Scheme 2.

Scheme 2

As described above with respect to formation of the 4,5-bis(diamino)-l- naphthaldehyde compounds, substitution on the aromatic ring (substituents R4-R7) can be performed using Friedel Crafts alkylation, acylation, or arylation, or other known electrophilic aromatic substitution. Substitution of the pyridine ring (i.e., R15-17) can be performed using well-known substitution reactions for producing pyridine analogs. These substitution reactions include electrophilic aromatic substitution, and nucleophilic aromatic substitution reactions.

Electrophilic Aromatic Substitution on Pyridine

Electrophiles react preferentially with the lone pair of the nitrogen to generate the pyridinium ion which, being positively charged, is unreactive towards electrophilic substitution. Neutral pyridine, which can react with electrophiles, is present only in a very low equilibrium concentration, so the rate of electrophilic aromatic substitution reactions is slow relative to aromatic rings.

The ring nitrogen polarizes the p-electron system, resulting in decreased electron density on the carbons, and as a result, electrophilic substitution typically forms 3-substituted products (the 3-position is the least disfavored position). This is analogous to how a nitro- substituent directs electrophilic substitution of benzene to the meta position.

Nucleophilic Aromatic Substitution on Pyridine

Pyridines are susceptible to nucleophilic attack at C-2 and C-4. By analogy with nitrobenzene, 2- or 4-halopyridines will undergo preferential substitution of the halide, compared to 3-halopyridines. Strongly basic nucleophiles, such as NH₂ , and alkyllithium and aryllithium or comparable Grignard reagents, will add at C-2 to form the 2-substituted pyridine, even without a leaving group. Where the nucleophile is NH₂ , the reaction is known as the Chichibabin reaction.

Enantiomeric Purification

As used herein, the term "enantiomerically pure" refers to a nucleotide composition that comprises at least approximately 95%, and, preferably, approximately 97%, 98%, 99% or 100% of a single enantiomer of that nucleotide.

As used herein, the term "substantially free of or "substantially in the absence of refers to a nucleotide composition that includes at least 85 to 90% by weight, preferably 95% to 98 % by weight, and, even more preferably, 99% to 100% by weight, of the designated enantiomer of that nucleotide. In a preferred embodiment, the compounds described herein are substantially free of enantiomers.

Similarly, the term "isolated" refers to a nucleotide composition that includes at least 85 to 90% by weight, preferably 95% to 98 % by weight, and, even more preferably, 99% to 100% by weight, of the nucleotide, the remainder comprising other chemical species or enantiomers.

The compounds described herein may have asymmetric centers and occur as racemates, racemic mixtures, individual diastereomers or enantiomers, with all isomeric forms being included in the present invention. Compounds of the present invention having a chiral center can exist in and be isolated in optically active and racemic forms. Some compounds can exhibit polymorphism. The present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric forms, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. The optically active forms can be prepared by, for example, resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution. One can either purify the respective nucleoside, then derivatize the nucleoside to form the compounds described herein, or purify the nucleotides themselves.

Optically active forms of the compounds can be prepared using any method known in the art, including but not limited to by resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

Examples of methods to obtain optically active materials include at least the following. i) physical separation of crystals: a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e. , the material is a conglomerate, and the crystals are visually distinct;

ii) simultaneous crystallization: a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state;

iii) enzymatic resolutions: a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis: a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer;

v) chemical asymmetric synthesis: a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e. , chirality) in the product, which can be achieved using chiral catalysts or chiral auxiliaries;

vi) diastereomer separations: a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer;

vii) first- and second-order asymmetric transformations: a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer;

viii) kinetic resolutions: this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions;

ix) enantiospecific synthesis from non-racemic precursors: a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis;

x) chiral liquid chromatography: a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase (including but not limited to via chiral HPLC). The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography: a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;

xii) extraction with chiral solvents: a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent;

xiii) transport across chiral membranes: a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.

Chiral chromatography, including but not limited to simulated moving bed chromatography, is used in one embodiment. A wide variety of chiral stationary phases are commercially available.

III. Methods of Treatment

The compounds described herein are capable of inhibiting viral propagation, and are therefore able to treat HIV infection, particularly infection by drug-resistant HIV, and, more particularly, infection by multi-drug resistant HIV. The HIV can be HIV-I, HIV-II, or HIV- III. The HIV can include any mutations associated with treatment with NNRTI, NRTI, PI, integrase inhibitors, entry inhibitors, and other conventional anti-HIV therapies. The HIV can even include mutations associated with long-term treatment with HART (highly active retroviral therapies), which involve the simultaneous administration of multiple drugs.

The retroviral propagation can be inhibited by inhibiting retroviral reverse transcription, viral recruitment of the retroviral primer used in translation, human tRNA^Lys3, inhibiting the final packaging and assembly of new virions, and/or inhibiting the binding of a host cell tRNA to a target nucleic acid molecule. The inhibition occurs through the inhibition of a complex formed between retroviral RNA and host cell tRNA, particularly, human tRNA^Lys3. Accordingly, these compounds can be used in methods to treat patients suffering from retroviral infections. That is, a retroviral viral infection can be treated or prevented by administering one or more inhibitors of retroviral propagation, for example, inhibitors of retroviral reverse transcription, binding to host cell tRNA and a target nucleic acid molecule, recruitment of the retroviral primer, human tRNA^Lys3, viral RNA translation into viral proteins, and final viral packaging and assembly of virions. Treatment of viral disease has not been heretofore accomplished by using such inhibitors.

The compounds can be used to treat or prevent viral infections, including infections by retroviruses, and/or to inhibit viral replication, propagation, reverse transcription, mRNA translation, and/or final viral packaging and assembly. The HIV can be any strain, form, subtype or variation in the HIV family. HIV viruses include, but are not limited to, HIV-I, HIV-II, HIV-III (also known as HTLV-II, LAV-I, LAV-2), and the like.

The compounds can also be used as adjunct therapy in combination with existing therapies in the management of the aforementioned types of viral infections. In such situations, it is preferably to administer the active ingredients to a patient in a manner that optimizes effects upon viruses, including mutated, multi-drug resistant viruses, while minimizing effects upon normal cell types. While this is primarily accomplished by virtue of the behavior of the compounds themselves, this can also be accomplished by targeted drug delivery and/or by adjusting the dosage such that a desired effect is obtained without meeting the threshold dosage required to achieve significant side effects.

IV. Pharmaceutical Compositions

The inhibitory compounds as described herein can be incorporated into pharmaceutical compositions and used to treat or prevent a viral infection, such as a retroviral infection. The pharmaceutical compositions described herein include the inhibitory compounds as described herein, and a pharmaceutically acceptable carrier and/or excipient.

The manner in which the compounds are administered can vary. The compositions are preferably administered orally (e.g., in liquid form within a solvent such as an aqueous or non-aqueous liquid, or within a solid carrier). Preferred compositions for oral administration include pills, tablets, capsules, caplets, syrups, and solutions, including hard gelatin capsules and time -release capsules. Compositions may be formulated in unit dose form, or in multiple or subunit doses. Preferred compositions are in liquid or semisolid form. Compositions including a liquid pharmaceutically inert carrier such as water or other pharmaceutically compatible liquids or semisolids may be used. The use of such liquids and semisolids is well known to those of skill in the art.

The compositions can also be administered via injection, i.e., intraveneously, intramuscularly, subcutaneously, intraperitoneally, intraarterially, intrathecally; and intracerebroventricularly. Intravenous administration is a preferred method of injection. Suitable carriers for injection are well known to those of skill in the art, and include 5% dextrose solutions, saline, and phosphate buffered saline. The compounds can also be administered as an infusion or injection (e.g., as a suspension or as an emulsion in a pharmaceutically acceptable liquid or mixture of liquids).

The formulations may also be administered using other means, for example, rectal administration. Formulations useful for rectal administration, such as suppositories, are well known to those of skill in the art. The compounds can also be administered by inhalation (e.g., in the form of an aerosol either nasally or using delivery articles of the type set forth in U.S. Patent No. 4,922,901 to Brooks et al., the disclosure of which is incorporated herein in its entirety); topically (e.g., in lotion form); or transdermally (e.g., using a transdermal patch, using technology that is commercially available from Novartis and Alza Corporation). Although it is possible to administer the compounds in the form of a bulk active chemical, it is preferred to present each compound in the form of a pharmaceutical composition or formulation for efficient and effective administration.

Exemplary methods for administering such compounds will be apparent to the skilled artisan. The usefulness of these formulations may depend on the particular composition used and the particular subject receiving the treatment. These formulations may contain a liquid carrier that may be oily, aqueous, emulsified or contain certain solvents suitable to the mode of administration.

The compositions can be administered intermittently or at a gradual, continuous, constant or controlled rate to a warm-blooded animal (e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey), but advantageously are administered to a human being. In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered can vary.

Preferably, the compositions are administered such that active ingredients interact with regions where viral infections are located. The compounds described herein are very potent at treating these viral infections.

In certain circumstances, the compounds described herein can be employed as part of a pharmaceutical composition with other compounds intended to prevent or treat a particular viral infection, i.e., combination therapy. In addition to effective amounts of the compounds described herein, the pharmaceutical compositions can also include various other components as additives or adjuncts.

Combination or Alternation Therapy

In one embodiment, the compounds of the invention can be employed together with at least one other antiviral agent, chosen from entry inhibitors, reverse transcriptase inhibitors, protease inhibitors, and immune-based therapeutic agents.

For example, when used to treat or prevent HIV infection, the active compound or its prodrug or pharmaceutically acceptable salt can be administered in combination or alternation with another anti-HIV agent. In general, in combination therapy, effective dosages of two or more agents are administered together, whereas during alternation therapy, an effective dosage of each agent is administered serially. The dosage will depend on absorption, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

Combination therapy may be administered as (a) a single pharmaceutical composition which comprises an inhibitory compound as described herein, at least one additional pharmaceutical agent described herein, and a pharmaceutically acceptable excipient, diluent, or carrier; or (b) two separate pharmaceutical compositions comprising (i) a first composition comprising an inhibitory compound as described herein and a pharmaceutically acceptable excipient, diluent, or carrier, and (ii) a second composition comprising at least one additional pharmaceutical agent described herein and a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical compositions can be administered simultaneously or sequentially and in any order.

In use in treating or preventing viral disease, the inhibitory compound(s) can be administered together with at least one other antiviral agent as part of a unitary pharmaceutical composition. Alternatively, it can be administered apart from the other antiviral agents. In this embodiment, the inhibitory compound and the at least one other antiviral agent are administered substantially simultaneously, i.e. the compounds are administered at the same time or one after the other, so long as the compounds reach therapeutic levels for a period of time in the blood. Combination therapy involves administering the inhibitory compound, as described herein, or a pharmaceutically acceptable salt or prodrug of the inhibitory compound, in combination with at least one anti-viral agent, ideally one which functions by a different mechanism than the inhibitors of viral propagation described herein.

Representative Antiviral A2ents

Some antiviral agents which can be used for combination therapy include agents that interfere with the ability of a virus to infiltrate a target cell. The virus must go through a sequence of steps to do this, beginning with binding to a specific "receptor" molecule on the surface of the host cell and ending with the virus "uncoating" inside the cell and releasing its contents. Viruses that have a lipid envelope must also fuse their envelope with the target cell, or with a vesicle that transports them into the cell, before they can uncoat.

There are two types of active agents which inhibit this stage of viral replication. One type includes agents which mimic the virus-associated protein (VAP) and bind to the cellular receptors, including VAP anti-idiotypic antibodies, natural ligands of the receptor and anti- receptor antibodies, receptor anti-idiotypic antibodies, extraneous receptor and synthetic receptor mimics. The other type includes agents which inhibit viral entry, for example, when the virus attaches to and enters the host cell. For example, a number of "entry-inhibiting" or "entry-blocking" drugs are being developed to fight HIV, which targets the immune system white blood cells known as "helper T cells", and identifies these target cells through T-cell surface receptors designated "CRX4" and "CCR5". Thus, CRX4 and CCR5 receptor inhibitors such as amantadine and rimantadine, can be used to inhibit viral infection, such as HIV, influenza, and hepatitis B and C viral infections. Another entry-blocker is pleconaril, which works against rhinoviruses, which cause the common cold, by blocking a pocket on the surface of the virus that controls the uncoating process.

Further antiviral agents that can be used in combination with the inhibitory compounds described herein include agents which interfere with viral processes that synthesize virus components after a virus invades a cell. Representative agents include nucleotide and nucleoside analogues that look like the building blocks of RNA or DNA, but deactivate the enzymes that synthesize the RNA or DNA once the analogue is incorporated. Acyclovir is a nucleoside analogue, and is effective against herpes virus infections. Zidovudine (AZT), 3TC, FTC, and other nucleoside reverse transcriptase inhibitors (NRTI), as well as non-nucleoside reverse transcriptase inhibitors (NNRTI), can also be used. Integrase inhibitors can also be used. Once a virus genome becomes operational in a host cell, it then generates messenger RNA (mRNA) molecules that direct the synthesis of viral proteins. Production of mRNA is initiated by proteins known as transcription factors, and certain active agents block attachment of transcription factors to viral DNA.

Other active agents include antisense oligonucleotides and ribozymes (enzymes which cut apart viral RNA or DNA at selected sites).

Some viruses, such as HIV, include protease enzymes, which cut viral protein chains apart so they can be assembled into their final configuration. Protease inhibitors are another type of antiviral agent that can be used in combination with the inhibitory compounds described herein.

The final stage in the life cycle of a virus is the release of completed viruses from the host cell. Some active agents, such as zanamivir (Relenza) and oseltamivir (Tamiflu) treat influenza by preventing the release of viral particles by blocking a molecule named neuraminidase that is found on the surface of flu viruses.

Still other active agents function by stimulating the patient's immune system. Interferons, including pegylated interferons, are representative compounds of this class. Interferon alpha is used, for example, to treat hepatitis B and C. Various antibodies, including monoclonal antibodies, can also be used to target viruses.

Any of the above-mentioned compounds can be used in combination therapy with the inhibitors described herein.

The appropriate dose of the compound is that amount effective to prevent occurrence of the symptoms of the disorder or to treat some symptoms of the disorder from which the patient suffers. By "effective amount", "therapeutic amount" or "effective dose" is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder.

When treating viral infections, an effective amount of the inhibitory compound is an amount sufficient to suppress the growth and proliferation of the virus. Viral infections can be prevented, either initially, or from re-occurring, by administering the compounds described herein in a prophylactic manner. Preferably, the effective amount is sufficient to obtain the desired result, but insufficient to cause appreciable side effects.

The effective dose can vary, depending upon factors such as the condition of the patient, the severity of the viral infection, and the manner in which the pharmaceutical composition is administered. The effective dose of compounds will of course differ from patient to patient, but in general includes amounts starting where desired therapeutic effects occur but below the amount where significant side effects are observed.

The compounds, when employed in effective amounts in accordance with the method described herein, are effective at inhibiting the proliferation of certain viruses, but do not significantly effect normal cells.

For human patients, the effective dose of typical compounds generally requires administering the compound in an amount of at least about 1, often at least about 10, and frequently at least about 25 μg/ 24 hr/ patient. The effective dose generally does not exceed about 500, often does not exceed about 400, and frequently does not exceed about 300 μg/ 24 hr/ patient. In addition, administration of the effective dose is such that the concentration of the compound within the plasma of the patient normally does not exceed 500 ng/mL and frequently does not exceed 100 ng/mL.

V. Methods for Identifyin2 an Inhibitor of Retroviral Propo2ation

The compounds described herein can be evaluated for their ability to inhibit viral propagation, for example, retroviral propagation, using the methods described herein. The retroviral propagation can be inhibited, for example, by

a) inhibiting retroviral reverse transcription,

b) inhibiting the binding of a host cell tRNA and a target nucleic acid molecule, c) inhibiting the viruses recruitment of the retroviral primer, human tRNA^Lys3, d) inhibiting HIV translation of viral RNA to precursor proteins, and/or

e) inhibiting HIV's final packaging and assembly.

These individual methods for identifying inhibitors of retroviral propagation are discussed below.

Identifying Inhibitors of Retroviral Reverse Transcription

In one aspect, putative inhibitors of retroviral reverse transcription can be identified. In another aspect, putative inhibitors of tRNA's ability to bind to a target nucleic acid molecule can be identified. The identification can be done in in a high through-put manner. Transfer RNA (tRNA) is involved in reverse transcription through the recognition of a corresponding site on the retroviral genome priming reverse transcription. Identifying inhibitors of reverse transcription may lead to the identification of therapeutic compounds for use in treating retroviral infection in a host cell.

The screening methods involve forming a mixture having a tRNA anticodon stem- loop (ASL) fragment, a target nucleic acid molecule that is capable of binding to the tRNA fragment, and a test compound. In one aspect, the target nucleic acid molecule corresponds to a fragment of the retroviral genome involved in reverse transcription. The resulting mixture is incubated under conditions that allow binding of the tRNA fragment and the target nucleic acid in the absence of the test compound. The method further involves detecting whether the test compound inhibits the binding of the tRNA fragment to the target nucleic acid, where the absence of binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of retroviral reverse transcription. In one aspect, the detection involves the use of labels to detect the inhibition of binding of the tRNA fragment to the target nucleic acid molecule.

Methods for Identifying Inhibitors of Binding of a Host Cell tRNA to a Target Nucleic Acid Molecule

In another aspect, the ability of a putative inhibitor to bind a tRNA to a target nucleic acid molecule can be assayed. The assay involves forming a mixture containing a host cell tRNA ASL fragment, a target nucleic acid molecule that is capable of binding to the tRNA fragment, and a test compound. The resulting mixture is incubated under conditions that allow binding of the tRNA fragment and the target nucleic acid in the absence of the test compound. The method further involves detecting whether the test compound inhibits the binding of the tRNA fragment to the target nucleic acid, where binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of binding of a tRNA to a target nucleic acid molecule. In one aspect, the detection involves the use of labels to detect the inhibition of binding of the tRNA fragment to the target nucleic acid molecule.

Methods for Identifying Inhibitors of HIV Reverse Transcription (RT) Complex Formation

In another aspect, the ability of a compound to function as an inhibitor of HIV reverse transcriptase (RT) complex formation can be assayed. The assay involves forming a mixture containing a tRNA ASL fragment, a target nucleic acid molecule capable of binding to the tRNA fragment, and a test compound. The resulting mixture is incubated under conditions that allow binding of the tRNA fragment and the target nucleic acid in the absence of the test compound. The method further involves detecting whether the test compound inhibits the binding of the tRNA fragment to the target nucleic acid. In one aspect, the detection involves the use of labels to detect the inhibition of binding of the tRNA fragment to the target nucleic acid molecule, where the inhibition indicates that the test compound is capable of inhibiting the formation of the RT complex.

In another aspect, the assay may involve detecting the binding of the putative inhibitor to either the tRNA fragment, the target nucleic acid, or both the tRNA fragment and the target nucleic acid. In one aspect, the binding of the putative inhibitor is indicative of the test compound being an inhibitor of retroviral propagation, retroviral infection, reverse transcription, or tRNA binding.

Methods for Identifvin2 Inhibitors of Viral Recruitment of Human tRNA^Lys3.

In yet another aspect, the ability of a putative inhibitor to inhibit HIV's recruitment of the retroviral primer, human tRNA^Lys3 can be assayed. The assay involves forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment that is not capable of forming a stem-loop, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound, wherein the target nucleic acid molecule corresponds to a portion of a retroviral genome involved in recruitment of retroviral primer recruitment. The mixture is incubated under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound. One can then detect whether or not the test compound inhibits the binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule. The absence of binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor retroviral primer recruitment.

Methods for Identifvin2 Inhibitors of Viral RNA Translation

In still another aspect, a ability of a putative inhibitor of viral RNA translation to viral precursor proteins can be assayed. The assay involves forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment that is not capable of forming a stem-loop, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound; incubating the mixture under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound; and detecting whether or not the test compound inhibits the binding of the tRNA fragment and the target nucleic acid molecule where binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of tRNA recruitment during viral RNA translation to viral precursor proteins. The inhibitors can inhibit the retroviral infection by inhibiting any step of a virus lifecycle, including, but not limited to, reverse transcription, viral assembly, RT complex formation, recruitment of the retroviral primer, human tRNA^Lys3, translation of viral RNA to precursor proteins, and the final packaging and assembly. Moreover, the inhibitors may inhibit retroviral infection, delay the infection, or slow the progression of the infection.

VI. tRNA Fra2ments Useful in the Methods Described Herein

The tRNA fragments (or "tool tRNA fragments") for use in the screening methods described herein can be a fragment from any tRNA. Specific tRNA fragments described in the formulas below are another aspect of the invention, and these fragments can be included in the kits described herein.

In a preferred aspect, the fragment tRNA contains modified nucleic acids corresponding to positions 34, 37, and 39 in the anticodon stem loop of a tRNA. The position numbers used herein refer to the nucleotide position numbering of the conventional tRNA numbering as disclosed in Sprinzl, et al. Nucl. Acids. Res., 26, 148-153 (1998). In one aspect, the tRNA fragment comprises, or consists of, a molecule having the sequence 5'- GCUXUUAYZCUG, in which the X, Y, and Z refer to modified or unmodified nucleosides. In one aspect, the X, Y, and Z refer to modified nucleosides, such as mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, and t6A. In another aspect, the tRNA fragment has the nucleic acid sequence 5'-CU(mnm5s2U)UU(ms2t6A)A(\|/)CUGC. In another aspect, the tRNA fragment has the nucleic acid sequence 5'-GCU(mnm5s2U)UU(ms2t6A)A(\|/)CUG.

The tRNA fragment may correspond to any portion of the tRNA involved in propagation of the retrovirus through binding, directly or indirectly, to the retroviral genome. In a preferred aspect, the tRNA fragment corresponds to the anticodon stem loop (ASL) of the tRNA.

The tRNA fragment may correspond to any portion of the host cell's tRNA involved in nucleotide binding, such as involvement in the reverse transcription (RT) complex formation. For example, the tRNA may be involved in binding to a retroviral genome to initiate, prime, or facilitate reverse transcription of the retroviral genome. In one aspect, the fragment tRNA corresponds to a fragment of the anticodon stem loop of any tRNA. In one aspect, the fragment corresponds to a fragment from the anticodon stem loop of tRNA^"Lys. In another aspect, the tRNA fragment corresponds to a fragment from the anticodon stem loop of human tRNA^"Lys. In another aspect, the tRNA fragment corresponds to a fragment from nucleotides 32-43 of human tRNA^Lys3. In another aspect, the target nucleic acid molecule corresponds to a nucleic acid molecule from a Human Immunodeficiency Virus (HIV), such as HIV-1 or HIV-2. In another aspect, the target molecule corresponds to HIV-1. In another aspect, the target nucleic acid molecule corresponds to a nucleic acid molecule involved in priming HIV reverse transcription.

Such target nucleic acid molecules can be derived from or correspond to any portion of the HIV genome involved in reverse transcription through the binding or association with a host cell tRNA. In one aspect, the target nucleic acid molecule is derived from or corresponds to the 5' untranslated region of the HIV genome. In another aspect, the target nucleic acid molecule corresponds to a fragment including residues 157 to 169 of the 5' untranslated region of HIV-1. The target nucleic acid sequence may be complementary to the tRNA fragment. In a one aspect, the target nucleic acid molecule comprises the nucleic acid sequence 5'-GCGGUGUAAAAG.

Specific Isolated tRNA Fragments

In one aspect, the isolated tRNA fragment comprises the sequence 5'- GCUXUUAYZCUG, in which the X, Y, and Z refer to modified nucleosides.

Representative modified nucleosides include unknown modified adenosine (?A), 1- methyladenosine (mlA), 2-methyladenosine (m2A), N⁶-isopentenyladenosine (i6A), 2- methylthio-N⁶-isopentenyladenosine (ms2i6A), N⁶-methyladenosine (m6A), N⁶- threonylcarbamoyladenosine (t6A), N⁶-methyl-N⁶ threonylcarbomoyladenosine (m6t6A), 2- methylthio-N⁶-threonylcarbamoyladenosine (ms2t6A), 2'-0-methyladenosine I Inosine (Am), 1-methylinosine Ar(p) 2'-0-(5-phospho)ribosyladenosine (mil), N⁶-(cis- hydroxyisopentenyl)adenosine (io6A), Unknown modified cytidine (?C), 2-thiocytidine (s2C), 2'-0-methylcytidine (Cm), N⁴-acetyl cytidine (ac4C), 5-methylcytidine (m5C), 3- methyl cytidine (m3C), lysidine (k2C), 5-formylcytidin (f5C), 2'-0-methyl-5-formylcytidin (f5Cm), unknown modified guanosine (?G), 2'-0-(5phospho) ribosylguanosine (Gr(p)), 1- methylguanosine (mlG), N²-methyl guanosine (m2G), 2'-0-methylguanosine (Gm), N²N²- dimethylguanosine (m22G), N²,N²,2'-0-trimethyl guanosine (m22Gm), 7-methylguanosine (m7G), archaeosine (fa7d7G), queuosine (Q), mannosyl-queuosine (manQ), galactosyl- queuosine (galQ), wybutosine (yW), peroxywybutosine (02yW), unknown modified uridine (?U), 5-methylaminomethyluridine (mnm5U), 2-thiouridine (s2U), 2'-0-methyluridine (Um), 4-thiouridine (s4U), 5carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5methylaminomethyl-2-thiouridine (mnm5s2U), 5-methoxycarbonylmethyl-2- thiouridine (mcm5s2U), uridine 5-oxyacetic acid (cmo5U), 5-methoxyuridine (mo5U), 5carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 5-

(carboxyhydroxymethyl)uridinemethyl ester (mchm5U), 5-carboxymethylaminomethyl-2'-0- methyluridine (cmnm5Um), 5-carbamoylmethyl-2'-0-methyluridine (ncm5Um), Dihydrouridine (D), pseudouridine (ψ), 1-methylpseudouridine (ηιΐψ), 2'-0- methylpseudouridine (ψηι), ribosylthymine (m5U), 5-methyl-2-thiouridine (m5s2U), and5,2'- O-dimethyluridine (m5Um).

In one embodiment, the modified nucleosides are mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, or t6A.

One specific tRNA fragment comprises the nucleic acid sequence 5'- CU(mnm5s2U)UU(ms2t6A)A(T)CUGC.

Another specific tRNA fragment comprises the nucleic acid sequence 5'- GCU(mnm5s2U)UU(ms2t6A)A(T)CUG.

Any of these tRNA fragments can further comprise a label. The label can be detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Representative labels include radioactive isotopes (for example, ³²P, ³⁵S, and ³H), dyes, fluorescent dyes (for example, Cy5 and Cy3), fluorophores (for example, fluorescein), electron-dense reagents, enzymes and their substrates (for example, as commonly used in enzyme-linked immunoassays, such as, alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or hapten; and proteins for which antisera or monoclonal antibodies are available. The label can also be an "affinity tag."

Where the label comprises an affinity tag, the isolated tRNA fragments can be captured with a complimentary ligand coupled to a solid support that allows for the capture of the affinity tag-labeled tRNA fragment. Representative affinity tags and complimentary partners include biotin-streptavidin, complimentary nucleic acid fragments (for example, oligo dT-oligo dA, oligo T-oligo A, oligo dO-oligo dC, oligo O-oligo C), aptamers, or haptens and proteins for which antisera or monoclonal antibodies are available.

When a biological interaction brings the beads together, a cascade of chemical reactions acts to produce a greatly amplified signal. On laser excitation, a photosensitizer in the "Donor" bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse across to react with a thioxene derivative in the Acceptor bead generating chemiluminescence at 370 nm that further activates fluorophores contained in the same bead. The fluorophores subsequently emit light at 520-620 nm.

In one example of a commercially-available alpha bead, the Donor beads comprise biotin or are directly bound to RNA. The Acceptor beads include a His6 tag, hemagglutinin (HA), digoxin/digoxigenin (DIG), or fluorescein (FITC).

VI. Methods for Detecting Binding (or Inhibition Thereof) of Target RNA to tRNA

The methods for detecting binding of the target RNA to the tRNA or the inhibition of such binding may be performed using any method for such detection. For example, the AlphaScreen® assay (Packard Instrument Company, Meriden, Conn.). AlphaScreen® technology is an "Amplified Luminescent Proximity Homogeneous Assay" method utilizing latex microbeads (250 nm diameter) containing a photosensitizer (donor beads), or chemiluminescent groups and fluorescent acceptor molecules (acceptor beads). Upon illumination with laser light at 680 nm, the photosensitizer in the donor bead converts ambient oxygen to singlet-state oxygen. The excited singlet-state oxygen molecules diffuse approximately 250 nm (one bead diameter) before rapidly decaying. If the acceptor bead is in close proximity to the donor bead (i.e., by virtue of the interaction of the target RNA and tRNA fragment), the singlet-state oxygen molecules reacts with chemiluminescent groups in the acceptor beads, which immediately transfer energy to fluorescent acceptors in the same bead. These fluorescent acceptors shift the emission wavelength to 520-620 nm, resulting in a detectable signal. Antagonists of the interaction of the target RNA with the tRNA fragment will thus inhibit the shift in emission wavelength, whereas agonists of this interaction would enhance it.

The disclosed methods may be performed by mixing the component nucleotide (e.g. the tool tRNA and the target RNA) and the test compound in any order, or simultaneously. For example, a target RNA may be first combined with a test compound to form a first mixture, and then a tool tRNA fragment may be added to form a second mixture. In another example, a target RNA, a tool tRNA and the test compound may all be mixed at the same time before incubation. In one aspect, the mixture is incubated under conditions that allow binding of the tRNA fragment and the target nucleic acid in the absence of the test compound.

The inhibition of binding of the tRNA fragment and the target nucleic acid molecule by the test compound may be detected using any method available for the detection of inhibition. In one aspect, the determining step may be performed using methods including, but not limited to, gel shift assays, chemical and enzymatic footprinting, circular dichroism and NMR spectroscopy, equilibrium dialysis, or in any of the binding detection mechanisms commonly employed with combinatorial libraries of probes or test compounds. The inhibition of binding indicates that the test compound may be useful for inhibiting propagation of the virus in the host.

The invention will be further explained by the following illustrative examples, which are intended to be non-limiting.

EXAMPLES

Example 1: Inhibitor screening assay

Two assays were developed using tool and target RNAs, the immobilized assay and the Alphascreen assay. Both assays use the same two RNA components (the target RNA and the tRNA fragment). In the example, the HIV viral RNA target is a 12mer with a 3' Biotin, while the Human tRNA mimic is a synthetic 12mer containing the native modified nucleotides and 3' fluorescein. These two RNAs mimic an essential complex of the HIV replication complex.

As set forth more fully below, the immobilization assay uses a three step process that first involves the binding of the target RNA to an avidin coated microtiter plate. Then, the test compound (drug/small molecule), denoted as a star, is incubated with the target sequence for 30 min. Then, the tRNA mimic was added to determine if the complex was formed or inhibited. In this assay a phosphate buffer may be used with 1M NaCl to improve the affinity for the two RNA. The stability of the complex is concentration dependent so that μΜ concentrations are used and the assay is run at 4 degrees C.

The 5' labeled target RNA sequence (5'- CGGUGUAAAAGC) is bound to a avidin microtiter plate (Roche High Load plates, 96-well avidin microtiter plates) by adding 150μ1 of target solution to each well. The plates can be covered and incubated at 37° C for 1 hour. The plates are then rinsed twice with binding buffer, the second rinse is incubated at 37° C for 5 minutes. The plates are then rinsed two additional times with binding buffer, covered, and ready for use.

The test compounds can be prepared by thawing solutions of the compounds to room temperature. Dilutions of the test compounds (1:10 and 1:500) can be prepared by dilution in DMSO and shaking for 1 hour.

The assays were performed by adding 98.5μ1 of loading buffer (100 mM Tris HC1, pH 7.5, 150 mM NaCl and 0.1% Tween 20, pH adjusted from around 4.5 to 7.5 with 10 M NaOH) to each well of the plate. Test compounds can be added individually to each well (1.5 μΐ each), and the plates were mixed for 1 hour.

Fifty microliters of solution containing the tool tRNA (5' - GCUXUUAYZCUG; where the X, Y, and Z are independently selected from modified nucleosides mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, and t6A) can then be added to each well and the plates can be incubated at 4° C for 1 hour with shaking. The reaction mixture can then be removed, while the mixture was still cold, and the remaining compound solution can also be removed.

After removing the remaining solution, reading buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, PEG (40mg/200ml)) can then be added to each well and the results were read using a plate reader.

A positive (+) reaction indicates that the test compound inhibits binding of the tool tRNA to the target nucleic acid (e.g. the test compound binds to either the tool tRNA, the target nucleic acid molecule or both the tool tRNA and the target nucleic acid molecule). A negative (-) reaction indicates that the test compound does not inhibit the binding of the tool tRNA to the target nucleic acid (e.g. the test compound does not bind to either the tool tRNA or the target nucleic acid).

In the AlphaScreen configuration, the assay is done in solution using the same RNA as the immobilization assay. The donor and acceptor beads are bound to their respective RNA's. During the screening the RNAs and test drugs/small molecules are incubated together and formation of the complex is measured using the AlphaScreen detection conditions. Utilization of the AlphaScreen assay may allow for the assay to be run at a lower RNA concentration at room temperature, and increase the stability of the complex.

Example II. Validation of HIV screening assay

The HIV screening assay was validated to confirm that positive and negative controls would function as expected and to test a small compound library to verify that differential inhibition could be detected. Two validation runs were completed with 4,275 and 4,616 compounds, respectively, using 17 plates in each run. There were 3,961 compounds in common between the two assays and the statistical analysis was completed using only these compounds and the positive and negative controls. Each plate contained approximately 30 positive and 30 negative controls and these controls performed as expected. Differences were observed between validation runs when analyzing the luminescence; however, these differences were minimized or eliminated when evaluating the percent inhibition by compounds (hits) that were active in both runs. This assay met the functional requirements based on the results of the positive and negative controls.

To evaluate the inhibition exhibited in the screening of this small compound library, a cutoff was set at 42.96% inhibition, the average plus three times standard deviation of compound percent (%) inhibition. Using this cutoff, 34 repeated compounds (hits) were identified. By using 99.75% inhibition as the cutoff, the average minus three times standard deviation of positive control (Tool + Target) percent (%) inhibition, there is 1 repeated hit. If 29.02%, which is the average plus three times standard deviation of negative control (Tool) percent (%) Inhibition, is defined as the cutoff, there are 51 repeated hits, out of 3961 compounds analyzed. These results are in line with expectations when evaluating a small random compound library.

To select compounds for use in a secondary HIV assay to verify that this assay was capable of identifying HIV specific compounds with biological activity a cutoff was set at greater than 60% inhibition in at least one of the two validations runs. This resulted in the selection of29 compounds. These compounds were analyzed for anti-HIV activity in freshly harvested PBMC cells. Of the 30 tested compounds, 15 were active at a concentration of less than 100 ~M (the highest tested concentration). Of these 15 compounds, 9 were not toxic to the PBMC cells at the 100 ~M concentration; thus, an absolute conclusion regarding the differential toxicity to HIV and PBMC cells cannot be drawn with these 9 compounds. Two other compounds had an antiviral index (inhibited HIV cells and not PBMC cells) greater than 25 which is acceptable. The two compounds identified were:

0 naphtho[2',3',4,5]imidazo[l,2-a]pyridine-6, 11-dione; and

4,5-bis(dimethylamino-l-naphthaldehyde.

The two compounds demonstrated anti-HIV activity at 0.63 and 0.022 μΜ and had an antiviral index greater than 25. One compound was inactive in the reverse transcriptase assay indicating that the compound does not inhibit this enzyme and indicating that the compound inhibits the RNA:RNA interaction that the assay is designed to mimic.

In one embodiment, the compounds as described herein include all analogs other than the two compounds identified above. In another embodiment, the compounds described herein include the two compounds identified above.

Example III: High Throughput Assay on a Large Compound Library Using HIV That Does Not Include Mutations Associated With Drug-Resistance

Prior to testing compounds for their ability to treat drug-resistant HIV, it was appropriate to verify that the compounds were useful for treating HIV that was not associated with drug resistance, so that the effectiveness of the compounds against non-resistant strains could be compared to the effectiveness of resistant strains. The manner in which the non- resistant strains were tested is described below.

TRANA Discovery Biochemical HIV-1 tRNA Inhibition Assay: HIV-1 has evolved to use Human tRNA^Lys3 as a primer for initiation of reverse transcription. Therefore, the interaction between tRNA^Lys3 and viral genomic RNA represents a potential novel target for HIV- 1 drug development. Based on this hypothesis, a biochemical assay to identify inhibitors of the interaction between Human tRNA^Lys3 and HIV-1 genomic RNA was developed by TRANA Discovery and transferred to Southern Research Institute for high-throughput screening. This assay was developed as a homogeneous amplified luminescent proximity assay using AlphaScreen reagents from PerkinElmer. Based on this assay technology, the AlphaScreen™ luminescent signal serves as a mechanism for detecting the interaction between RNA molecules that represent Human tRNA^Lys3 and HIV-1 genomic RNA. The inhibition of the interaction between these RNA molecules by test compounds is detected as a decrease in the AlphaScreen™ luminescent signal.

Compounds Screened: For the results described herein, a 101,000 compound library that was purchased from ChemBridge for the NIAID TAACF program was used. The compounds were screened at a concentration of 12.5 μg/mL (first 3 batches) or 25 μg/mL (fourth and final batch).

RESULTS AND CONCLUSIONS

Screening Results: The median Z-value for the 78 assay plates used in the screen was 0.76, with a range from 0.64 to 0.86. Following analysis of the data, 99,303 valid screening results were obtained from all four compound batches screened. Statistical analysis identified 38.59% inhibition (batches 1-3 screened at 12.5 μg/mL) and 44.89% inhibition (batch 4 screened at 25 μg/mL) as the cutoffs between inactive and hit compounds. Based on these statistical criteria, a total of 315 compounds were identified as hits. There were 202 hits identified from the 75,144 compounds screened in compound batches 1-3 (hit rate of 0.27%). Similarly, there were 113 hits identified from the 24,159 compounds screened in compound batch 4 (hit rate of 0.47%). The resulting overall combined hit rate for the screen was 0.32%. The range of the percent inhibition observed for the 315 hits was from 38.59% to 99.66%. Overall, the statistical cutoffs and hit rate observed for this screen are somewhat lower than the values previously observed when screening the NINDS Diversity Set (72.31% and 1.09% for hit cutoff and hit rate, respectively). However, these general differences can be explained by the lower test concentrations and plate format used for this screen (12.5 and 25 μg/mL; 1536-well plates) compared to the NINDS Diversity Set (40 μg/mL; 384-well plates).

Dose-response Testing Results: For initial follow-up testing of the compounds, resupplies of the hits were purchased from ChemBridge for dose-response testing in the TRANA Discovery biochemical HIV-1 tRNA inhibition assay. Of the 315 hits identified in the screen, 309 were available for resupply and were evaluated in dose-response, in duplicate. 183 hits (59.2%) were confirmed to be active in the assay. In addition, 125 hits (40.5%) reached an IC₅₀ value within the concentration range evaluated (0.049-25 μg/mL). The observed IC₅₀ values ranged from 0.205 to 24.97 μg/mL.

Based on dose-response testing of hits, 125 compounds that achieved an IC₅₀ value of less than 25 μg/mL have been identified for additional follow-up testing. These compounds have been prioritized based on their IC₅₀ values and are being scheduled for dose-response testing against HlV-lnm replication in a CEM-SS cytoprotection assay. Based on results, compounds found to inhibit virus replication in this cell-based assay will be considered for additional testing (e.g., in PBMCs) following discussion of the results with the Project Officer and the Sponsor, TRANA Discovery.

OVERALL SCREENING SUMMARY

Example IV: Antiviral Activity and Cytotoxicity of Bioactive Hits Tested Against NNRTI-Resistant HIV-1 Isolates in Fresh Human PBMCs

INTRODUCTION

This assay is based on the premise that HIV-1 has evolved to use Human tRNA^Lys3 as a primer for initiation of reverse transcription. Therefore, the interaction between tRNA^Lys3 and viral genomic RNA represents a potential novel target for HIV-1 drug development. A biochemical assay to identify inhibitors of the interaction between Human tRNA^Lys3 and HIV-1 genomic RNA was developed for high-throughput screening. This assay was developed as a homogeneous amplified luminescent proximity assay using AlphaScreen™ reagents from PerkinElmer. Based on this assay technology, the AlphaScreen™ luminescent signal serves as a mechanism for detecting the interaction between RNA molecules that represent Human tRNA^Lys3 and HIV-1 genomic RNA. The inhibition of the interaction between these RNA molecules by test compounds is detected as a decrease in the AlphaScreen™ luminescent signal. Five hit compounds identified during the validation of the assay and from the screening of the NIAID/TAACF ChemBridge and NINDS Diversity Set libraries were selected for additional follow-up testing in dose-response against NNRTI-resistant HIV-1 isolates in PBMCs. These five compounds were previously found to have modest antiviral activity against HIV-1 in a cell-based assay (e.g., HIV-1 cytoprotection assay in CEM-SS cells). Testing against NNRTI-resistant viruses was requested by TRANA Discovery as a mechanism to study whether or not the compounds are acting as NNRTIs instead of inhibiting the intended target of the interaction between tRNA^Lys3 with viral genomic RNA.

RESULTS AND CONCLUSIONS

Although there were some differences, the overall results from this study were consistent with previous results obtained for these compounds:

The Validation Study Hit #1 was determined to be less potent in this experiment (average IC₅₀ = 1.23 μΜ) compared to the previous experiment in which it was tested against HIV-l_BA-_L in PBMCs (IC₅₀ = 0.02 μΜ). In the current experiment, the antiviral activity closely paralleled the cytotoxicity of the compound (average Therapeutic Index = 1.45); however, the cytotoxicity observed was consistent with the previous data.

The Validation Study Hit #2 demonstrated modest antiviral activity (average IC₅₀ = 7.28 μΜ) that was not well separated from the cytotoxicity of the compound (average Therapeutic Index = 2.23). The observed activity was approximately 5- to 10-fold less than the activity previously observed against HIV-I_BB-_L in PBMCs (IC₅₀ = 0.63 μΜ).

The 15K NINDS Diversity Set Hit #12 exhibited modest antiviral activity (average IC₅₀ = 28.8 μΜ) that was similar to the antiviral activity previously observed in the CEM SS/HIV-lum cytoprotection assay (IC₅₀ = 23.8 μΜ).

The 15K NINDS Diversity Set Hit #84 also exhibited modest antiviral activity (average IC₅₀ = 3.97 μΜ) that was similar to the antiviral activity previously observed in the CEM SS/HIV-lum cytoprotection assay (IC₅₀ = 1.49 μΜ).

The 15K NINDS Diversity Set Hit #98 showed low-level antiviral activity (IC5₀S ranging from 61.8 μΜ to > 100 μΜ) and no cytotoxicity, which is consistent with previous results in the CEM SS/HIV-lum cytoprotection assay (IC5₀ = 69.8 μΜ).

Importantly, there was no apparent difference in antiviral activity based on the presence of NNRTI-resistance engendering mutations in the viruses, which suggests the compounds are likely not acting as NNRTIs. Although the antiviral activity of these five compounds is not high enough to pursue additional development, these results help to demonstrate that the TRANA Discovery Biochemical HIV-1 tRNA Inhibition Assay is not identifying compounds that act off-target as NNRTIs.

INTRODUCTION

HIV-1 has evolved to use Human tRNA^Lys3 as a primer for initiation of reverse transcription. Therefore, the interaction between tRNA^Lys3 and viral genomic RNA represents a potential novel target for HIV-1 drug development. Based on this hypothesis, a biochemical assay to identify inhibitors of the interaction between Human tRNA^Lys3 and HIV-1 genomic RNA was developed for high-throughput screening (HTS). A preliminary 15,000 compound high-throughput screen was performed using the biochemical HIV-1 tRNA inhibition assay described herein. One hundred thirty- six (136) hit compounds were identified to have the greatest activity against HIV-1 tRNA in this screen, and were subsequently tested in dose-response against HIV-lnm replication in a CEM-SS cytoprotection assay

Based on the results from this preliminary 15,000 compound screen and related follow-up testing, a larger campaign for the screening of an additional 300,000 compounds was initiated. Screening of the first 100,000 compounds was completed, and one hundred twenty-five (125) hit compounds identified to have the greatest activity against HIV-1 tRNA in this screen were subsequently tested in dose-response against HIV-lnm replication in a CEM-SS cytoprotection assay.

This report summarizes the results from the additional follow-up testing of 7 hit compounds identified during the validation of the assay and from the screening of the NIAID/TAACF ChemBridge and NINDS Diversity Set libraries. These compounds were selected for additional follow-up testing in dose-response against NNRTI-resistant HIV-1 isolates in PBMCs based on previous results indicating these compounds have modest antiviral activity against HIV-1 in a cell-based assay (e.g., HIV-1 cytoprotection assay in CEM-SS cells). Testing against NNRTI-resistant viruses was requested by TRANA Discovery as a mechanism to study whether or not the compounds are acting as NNRTIs instead of inhibiting the intended target of the interaction between tRNALys3 with viral genomic RNA.

GOALS & OBJECTIVES

The goal of this study was to determine the antiviral activity of 7 compounds against NNRTI-resistant HIV-1 isolates in PBMCs. Identification of hit compounds from the Biochemical HIV-1 tRNA Inhibition Assay described herein that inhibit HIV-1 replication serves to identify compounds with potential for development as a novel class of antiviral drug. Confirming that NNRTI-resistant virus isolates are not resistant to these hit compounds serves to verify that the compounds are not acting off target as NNRTIs inhibiting reverse transcriptase and provides further data to support the hypothesis that the compounds are targeting the interaction between tRNA^Lys3 with viral genomic RNA.

METHODS

1. Compound Information

Five hit compounds were selected for evaluation in this study. Two of these hit compounds were originally identified during the assay validation study performed at Southern Research in collaboration with TRANA Discovery. In addition, three compounds were identified from the NINDS Diversity Set screen and two compounds were identified from the NIAID/TAACF ChemBridge library screen. These five compounds were found to have the greatest level of bioactivity in follow-up testing against HIV-1 in cell-based assays. Table 1 summarizes the antiviral activity previously observed for these compounds.

Table 1. Hit Compound Background Data

15K NINDS HIV- liiiB

Diversity Set 69.8 μΜ > 100 μΜ > 1.43 Cytoprotection

Hit #98 Assay

Antiviral Index = TC5₀/IC5₀

The compounds have the following chemical formulas:

All five compounds were obtained from ChemBridge as part of previous testing efforts performed under the contract and solubilized soon after receipt in DMSO as 40 mM stocks (Validation Study and 15K NINDS Diversity Set hits) or 40 mg/mL stocks (100K NIAID/TAACF ChemBridge hits). The solubilized stocks were stored at -80°C until the day of the assay. Stocks were thawed at room temperature on each day of assay setup (dates are listed on the individual graph pages) and were used to generate working drug dilutions used in the assays. Working dilutions were made fresh for each experiment and were not stored for re-use in subsequent experiments performed on different days. The compounds were tested at a high-test concentration of 10 μΜ (10,000 nM), 100 μΜ, or 100 μg/mL with 8 additional serial half-log dilutions in the PBMC assays. The high-test concentration for each compound was selected based on individual compound stock concentrations and based on the antiviral/cytotoxicity data from the previous testing of each compound. AZT (NRTI), nevirapine (NNRTI), and efavirenz (NNRTI) were included in the PBMC assays as control antiviral compounds using 1.0 μΜ (1,000 nM), 10 μΜ (10,000 nM), and 1.0 μΜ (1,000 nM) high-test concentrations, respectively.

2. Efficacy Evaluation in Human Peripheral Blood Mononuclear Cells (PBMCs)

Virus Strains

Five HIV-1 isolates were selected for use in these experiments. The virus isolates HIV- I_BB-_L (lab-adapted, Group M, Subtype B, CCR5-tropic) and HIV-1_NL4-3 (Subtype B, CXCR4-tropic, molecular clone) were selected as representative "wild- type", drug- sensitive viruses. The remaining three viruses were chosen based on mutations associated with NNRTI resistance. Based on experiments performed at Southern Research, HIV- I92BR014 (Subtype B, Dual-tropic clinical isolate) has been found to be resistant to all NNRTIs tested (unpublished data). Sequencing of the reverse transcriptase from this virus indicates it contains the following amino acid changes compared to the HIV-1 consensus B sequence: D177E, G359G/S, K390R, T403V, and L491S. None of these changes are known to be associated with NNRTI resistance. All of these changes except T403 V are common polymorphisms in HIV- 1 RT. Therefore, it is hypothesized that T403V may be the mutation associated with NNRTI resistance (studies are ongoing to test this hypothesis). The NNRTI resistant isolate HIV- -17 is a K103N/Y181C dual RT mutant in the HlV-lum background. HIV-IBB-L, HIV-1NL4-3, HIV-I92BR014, and HIV-1A I7 were all obtained from the NIH AIDS Research and Reference Reagent Program. The clinical isolate HIV-1_MDR769 (presumed Group M, Subtype B) was obtained from Dr. Thomas C. Merigan (Stanford University) and is described in the following reference: Palmer S, RW Shafer and TC Merigan. Highly drug -resistant HIV-1 clinical isolates are cross-resistant to many anti-retroviral compounds in current clinical development. AIDS 1999, 13:661-667.

Table 2 lists the mutations found in HIV-1MDR769 (based on sequencing of current stocks) compared to the HIV- 1 consensus B sequence.

Low passage stocks of each virus were prepared using fresh human PBMCs and stored in liquid nitrogen. Pre-titered aliquots of each virus were removed from the freezer and thawed rapidly to room temperature in a biological safety cabinet immediately before use.

K122E, NFV, RTV, SQV Q151M,

1167 V,

Y181I. E248E/K,

A272P,

K277R,

D324E,

G359S,

A376V,

T386A,

A400T,

D460N,

T470P,

L491S,

K512R,

S519N

L10I,

M36M/V,

M46L,

I54V,

D60E,

PR I62V,

L63P,

A71V,

V82A,

I84V,

L90M

Bold figures represent the signature mutations of the gene. Italics figures represent key NNRTI mutations

*Predicted resistance from the Stanford HIV Drug Resistance Database (http ://hivdb. stanford.edu/) b. Anti-HIV Efficacy Evaluation in Fresh Human PBMCs

Fresh human PBMCs were isolated from screened donors, seronegative for HIV and HBV (Biological Specialty Corporation, Colmar, PA). Cells were pelleted/washed 2-3 times by low speed centrifugation and resuspension in Dulbecco's phosphate buffered saline (PBS) to remove contaminating platelets. The leukophoresed blood was then diluted 1 : 1 with PBS and layered over 14 mL of Ficoll- Hypaque density gradient (Lymphocyte Separation Medium, Cell Grow #85-072-CL, density 1.078+/-0.002 gm/ml) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs were gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation. After the final wash, cells were enumerated by trypan blue exclusion and re-suspended at 1 x 10⁶ cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 μg/mL Phytohemagglutinin (PHA; Sigma, St. Louis, MO; catalog #L1668). The cells were allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15% FBS, L-glutamine, penicillin, streptomycin, non-essential amino acids (MEM/NEAA; Hyclone; catalog # SH30238.01), and 20 U/mL recombinant human IL-2 (R&D Systems Inc., Minneapolis, MN; catalog #202IL). PBMCs were maintained in this medium at a concentration of 1-2 x 10⁶ cells/mL, with twice-weekly medium changes until they were used in the assay protocol. Monocytes-derived-macrophages were depleted from the culture as the result of adherence to the tissue culture flask.

For the standard PBMC assay, PHA stimulated cells from at least two normal donors were pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 10⁶ cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 μίΛ βΙΙ (5 x 10⁴ cells/well) in a standard format developed by the Infectious Disease Research department of Southern Research Institute. Pooling (mixing) of mononuclear cells from more than one donor is used to minimize the variability observed between individual donors, which results from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations. Each plate contains virus control wells (cells plus virus) and experimental wells (drug plus cells plus virus). Test drug dilutions were prepared at a 2X concentration in microtiter tubes and 100 \lL of each concentration was placed in appropriate wells using the standard format. 50 lL of a predetermined dilution of virus stock was placed in each test well (final MOI≡ 0.1). Separate plates were prepared identically without virus for drug cytotoxicity studies using an MTS assay system (described below; cytotoxicity plates also include compound control wells containing drug plus media without cells to control for colored compounds that affect the MTS assay). The PBMC cultures were maintained for seven days following infection at 37 °C, 5% C0₂. After this period, cell-free supernatant samples were collected for analysis of reverse transcriptase activity, and compound cytotoxicity was measured by addition of MTS to the separate cytotoxicity plates for determination of cell viability. Wells were also examined microscopically and any abnormalities were noted.

c. Reverse Transcriptase Activity Assay

A microtiter plate-based reverse transcriptase (RT) reaction was utilized (Buckheit et al., AIDS Research and Human Retroviruses 7:295-302, 1991). Tritiated thymidine triphosphate (³H-TTP, 80 Ci/mmol, NEN) was received in 1 : 1 dH₂0:Ethanol at 1 mCi/ml. Poly rA:oligo dT template:primer (Pharmacia) was prepared as a stock solution by combining 150 1 poly rA (20 mg/ml) with 0.5 ml oligo dT (20 units/ml) and 5.35 ml sterile dH₂0 followed by aliquoting (1.0 ml) and storage at -20°C. The RT reaction buffer was prepared fresh on a daily basis and consisted of 125 μΐ 1.0 M EGTA, 125 μΐ dH₂0, 125 μΐ 20% Triton X100, 50 μΐ 1.0 M Tris (pH 7.4), 50 μΐ 1.0 M DTT, and 40 μΐ 1.0 M MgCl₂. The final reaction mixture was prepared by combining 1 part ³H-TTP, 4 parts dH₂0, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 1 of virus-containing supernatant was added and mixed. The plate was incubated at 37°C for 60 minutes. Following incubation, the reaction volume was spotted onto DE81 filter-mats (Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies), 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Incorporated radioactivity (counts per minute, CPM) was quantified using standard liquid scintillation techniques.

d. MTS Staining for PBMC Viability to Measure Cytotoxicity

At assay termination, the uninfected assay plates were stained with the soluble tetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondria enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 20-25 L of MTS reagent is added per well and the microtiter plates are then incubated for 4-6 hrs at 37 °C, 5% C0₂ to assess cell viability. Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 490/650 nm with a Molecular Devices SPECTRAmax plate reader.

e. Data Analysis

Using an in-house computer program, the PBMC data analysis includes the calculation of ICso (50% inhibition of virus replication), IC9₀ (90% inhibition of virus replication), IC95 (95% inhibition of virus replication), TCso (50% cytotoxicity), TC9₀ (90% cytotoxicity), TC95 (95% cytotoxicity) and therapeutic index values (TI =TC/IC; also referred to as Antiviral Index or AI). Raw data for both antiviral activity and toxicity with a graphical representation of the data are provided in a printout summarizing the individual compound activity.

RESULTS & CONCLUSIONS

The results from the testing of the 5 hit compounds against the 5 HIV-1 isolates in PBMCs are summarized below in Table 3 with data and graphs provided in Appendix I.

Table 3. Antiviral Efficacy of Bioactive Hit Compounds Against HIV-1 in PBMCs

Five bioactive hit compounds from the screening initiative using the TRANA Discovery Biochemical HIV- 1 tRNA Inhibition Assay were evaluated for cytotoxicity and antiviral efficacy against five HIV-1 isolates in PBMCs. The results of the testing are summarized above in Table 3. Although there were some differences, the overall results from this study were consistent with previous results obtained for these compounds:

The Validation Study Hit #1 was determined to be less potent in this experiment (average IC₅₀ = 1.23 μΜ) compared to the previous experiment in which it was tested against HIV- l_BA-L in PBMCs (IC50 = 0.02 μΜ; see Table 1). In the current experiment, the antiviral activity closely paralleled the cytotoxicity of the compound (average Therapeutic Index = 1.45); however, the cytotoxicity observed was consistent with the previous data. The lower level of antiviral activity observed in the current experiment is possibly due to the use of PBMCs from a different donor.

The Validation Study Hit #2 demonstrated modest antiviral activity (average IC₅₀ = 7.28 μΜ) that was not well separated from the cytotoxicity of the compound (average Therapeutic Index = 2.23). The observed activity was approximately 5- to 10-fold less than the activity previously observed against HIV- IB_B-L in PBMCs (IC₅₀ = 0.63 μΜ; see Table 1). Again, the lower level of antiviral activity and slightly greater cytotoxicity observed in the current experiment compared to previous testing is possibly due to the use of PBMCs from a different donor.

The 15K NINDS Diversity Set Hit #12 exhibited modest antiviral activity (average IC₅₀ = 28.8 μΜ) that was similar to the antiviral activity previously observed in the CEM-SS/HIV- lum cytoprotection assay (IC50 = 23.8 μΜ; see Table 1). Slightly greater cytotoxicity was observed in the current experiment, resulting in an average Therapeutic Index of 2.29. The 15K NINDS Diversity Set Hit #84 also exhibited modest antiviral activity (average IC₅₀ = 3.97 μΜ) that was similar to the antiviral activity previously observed in the CEM-SS/HIV-l_ni_B cytoprotection assay (IC50 = 1-49 μΜ; see Table 1). The cytotoxicity of the compound was also similar to previous results, resulting in an average Therapeutic Index of 5.51.

The 15K NINDS Diversity Set Hit #98 only showed low-level antiviral activity (IC₅₀S ranging from 61.8 μΜ to > 100 μΜ) and no cytotoxicity, which is consistent with previous results in the CEM-SS/HIV- I_HIB cytoprotection assay (IC₅₀ = 69.8 μΜ; see Table 1).

The overall assay performance was validated by the control compounds, nevirapine, efavirenz, and AZT, which exhibited the expected levels of antiviral activity and resistance profiles in the assays. Macroscopic observation of the cells in each well of the microtiter plates confirmed the cytotoxicity results obtained following staining of the cells with the MTS metabolic dye.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A method of treating a drug-resistant HIV infection in a patient, comprising:

administering to a patient suffering from a drug-resistant HIV infection an effective amount of an inhibitor of retroviral propagation, wherein:

i) the inhibitor inhibits the ability of any portion of the HIV genome involved in reverse transcription to bind to or associate with a host cell tRNA, or the inhibitor disrupts the RNA/RNA complex formed between the viral genome and the human tRNA primer, or

ii) the binding or association of the host cell tRNA to a retroviral genome initiates, primes, or facilitates reverse transcription of the retroviral genome in the absence of the administered compound.

2. The method of Claim 1, wherein the portion of the HIV genome involved in reverse transcription is the 5' un-translated region of the HIV genome.

3. The method of Claim 2, wherein the region of the HIV genome comprises residues 157 to 169 of the 5' un-translated region of HIV-1.

4. The method of Claim 1, or wherein the portion of the host cell tRNA comprises human tRNA^Lys3.

5. The method of Claim 4, wherein the portion of the host cell tRNA comprises nucleotides 32-43 of human tRNA^Lys3.

6. The method of Claim 1, wherein the inhibitor inhibits retroviral reverse transcription, inhibits viral recruitment of the retroviral primer used in translation, human tRNA^Lys3, inhibits the final packaging and assembly of new virions, or inhibits the binding of a host cell tRNA to a target nucleic acid molecule.

7. The method of Claim 1, wherein the HIV is selected from the group consisting of drug-resistant HIV-I, HIV-II, and HIV-III.

8. The method of Claim 1, wherein the inhibitor directly interacts with nucleotides 32-43 of the host tRNA^Lys3 (the HIV human primer) such that binding of the retroviral RNA to those nucleotides is inhibited, or with nucleotides 157-169 of the retroviral RNA, such that binding of the host tRNA^Lys3 (the HIV human primer) to those nucleotides is inhibited.

9. The method of Claim 1, wherein the HIV is resistant to more than one anti- retroviral drug.

10. The method of Claim 1, wherein the HIV comprises one or more HIV-1 protease mutations selected from the group consisting of L10I, L10F, L10V, L10C, L10R, VI II, I13V, G16E, K20M, K20R, K20T, K10T, K20V, L24I, D30N, V32I, L33F, L33I, L33V, E34Q, E35G, M36I, M36L, M36V, K43T, M46I, M46L, 147 A, I47V, 147 A, G48V, I50V, I50L, F53L, F53Y, I54V, I54L, I54M, I54A, I54T, I54S, 058E, D60E, I62V, L63P, I64M, I64L, I64V, H69K, A71V, A71T, A71I, A71L, G73C, G73S, G73T, G73A, T74P, V82A, V82F, L76V, V77I, G82T, V82S, V82F, V82T, V82I, G82M, I84V, I84A, I85V, I84C, N83D , N88D, N88S, L89V, and L90M.

11. The method of Claim 1, wherein the HIV comprises one or more HIV-1 RT mutations selected from the group consisting of M41L, K65R, D67N, D67G, D67Del, T69D, T69ins, K70R, L74V, V75A, V75M, V75T, V75S, F77L, Y115F, F116Y, M184V, M184I, L210W, T215Y, T215F, T215C, T215D, T215E, T215E, T215S, T215I, T215V, K219Q, K219E, and K219R.

12. The method of Claim 1, wherein the HIV comprises one or more HIV-1 RT mutations selected from the group consisting of V90I, A98G, L100I, K101E, K101P, K101H, K103N, K103S, V106I, V106A, V106M, V108I, E138A, V179D, V179F, V179T, Y181C, Y181I, Y181V, Y188L, Y188H, Y188C, G190A, G190S, G190E, G190Q, P225H, M230L, and P236L.

13. The method of Claim 1, wherein the HIV comprises one or more mutations in the envelope gene selected from the group consisting of G36D, G36S, I37V, V38A, V38M, V38E, Q39R, Q40H, N42T, and N43D.

14. The method of Claim 1, wherein the HIV comprises one or more mutations in the integrase gene selected from the group consisting of Y143R, Y143H, Y143C, Q148H, Q148K, Q148R, and N155H.

15. The method of Claim 1, wherein the HIV comprises two or mutations, where the mutations are present in a gene selected from the HIV envelope gene, integrase gene protease gene, and reverse transcriptase gene.

16. The method of any of Claims 1-15, wherein the compound has one of the following formulas:

A. B.

wherein

Single or double bond

M and N = 1, 2 or 3 atoms from C, N, O

X and Y = NR¹, O or S

Ri and R₂ are, individually, H, alkyl, aryl, aralkyl, alkaryl, heterocyclyl, heteroaryl, substituted analogs thereof, wherein the substituents are selected from the list of substituents, Z,

wherein substituents Z include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R, -SR', -N₃, -C(=0)NR'R", -NR'C(=0) R", -C(=0)R', -C(=0)OR, -OC(=0)R', - OC(=0)NR'R", -NRC(=0)0 R", -S0₂R, -S0₂NRR", and -NR'S0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl).

17. The method of any of Claims 1-15, wherein the compound has one of the following formulas:

Ar-i (CH₂)_n NR., Ar₂

Formula A wherein Ari and Ar₂ are, independently, six membered aryl rings, five or six membered ring heteroaryl rings, or analogs thereof in which a five membered heteroaryl or six membered aryl or heteroaryl ring is fused to the six membered aryl rings, five or six membered ring heteroaryl rings,

n is 0 or 1, and

Ri is H or a moiety cleaved in vivo to form H,

Z, and

substituents Z as defined herein include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R, -SR', -N₃, -C(=0)NR'R", -NR'C(=0) R", -C(=0)R', -C(=0)OR, -OC(=0)R', - OC(=0)NR'R", -NR'C(=0)0 R", -S0₂R, -S0₂NRR", and -NR'S0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl),

An (CH₂)_m NR-i (CH₂)_{m Ar2}

Formula B where Ari, Ar₂, and Ri are as defined above, m is 0, 1, 2 or 3, and the aryl/heteroaryl rings can be substituted with from 1 to 3 substituents, Z, as described above, with the proviso that at least one m is 2,

a o

Formula C

wherein m is 0, 1, or 2, X is NRi, O, or S, and halo is F, CI, Br, I. In one embodiment of Formula B, X is S and halo is CI,

Formula D

where Z, j and Ri are as defined above, with the proviso that two Ri groups can link together to form a 5-7 membered ring azacyclic moiety, halo

Formula E where Z and j are as defined above,

wherein Ari, Ri, Z and j are as defined above, and Ari can include from one to three Z substituents, Z,

wherein X, Ri, Z, j, and n are as defined above, and the =X moiety can be present or not present (i.e., n is 0 or 1),

Formula H

where the compounds of Formula H can alternatively have the formula shown below, where the cyclohexadienone double bond is optional (as indicated by a dashed line), as follows:

wherein the dashed line indicates the presence of an optional double bond, wherein

R4, R5, R6, R7, R15, R16, and R17 are, independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (C C₆), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-C₆), lower alkynyl (C₂- C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above), or, alternatively, one or more of R4 and R5, R5 and R6, R6 and R7, R15 and R16, and R16 and R17 together form a five, six, or seven-member ring, which ring can include one or more heteroatoms, such as O, S, and N (wherein N can be substituted with H or R'),

Formula I

wherein positions 2, 3, 6, 7, and 8 can include a substituent Z as defined herein, and Rl, R2, R3, and R4, are independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, -COR', and -COOR', wherein R' is, independently H, a lower alkyl (Ci-Ce), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-C6), lower alkynyl (C₂-C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above), and

in one embodiment, one or both of Rl and R2, and R3 and R4, together with the nitrogens to which they are attached, form a 5-7 membered ring, which can include one or more additional heteroatoms such as O, S, or N, wherein the N can be bonded to a substituent R', as defined above),

Formula

wherei , Z and j are as defined above,

wherein X, Z, j, m and Ri are as defined above,

Formula N

wherein X,Z, j, are as defined above,

wherein Χ,Ζ, j , are as defined above,

Formula O wherein X, Z, j , and m are as defined above,

Formula P

wherein Z, j, n, and R] are as defined above, and a) K is NR], or b) K is N(R])₂, and the link to the other ring nitrogen is absent, in which case the other NRi moiety is an N(Ri)₂ moiety rather than an NRi moiety,

wherein Z, j, n, and X are as defined above, and R₂ is absent (i.e., a direct link between the aryl ring and the C=X moiety), or is an alkyl or cycloalkyl moiety linking the aryl ring and the C=X moiety, or

Formula R

wherein X and Ri are as defined elsewhere herein, o is an integer from 4 to 8 (in compounds 2 and 3, the number is 5), R₂ is Ci_₆ alkyl, and R₅ is -C(=X)ORi, -C(=X)SRi, -C(=X)NHR_l5 - X-C(=X)ORi, -X-C(=X)SRi, -X-C(=X)NHRi, -O-Ri, -SRi, or -NHR_L

18. The method of any of Claims 1-15, wherein the compound has one of the following formulas:

82

19. The method of any of Claims 1-15, wherein the compound has the following formula:

wherein:

the dashed line indicates the presence of an optional double bond,

R4, R5, R6, R7, R15, R16, and R17 are, independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (C C₆), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-C₆), lower alkynyl (C₂- C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above),

alternatively, one or more of R4 and R5, R5 and R6, R6 and R7, R15 and R16, and R16 and R17 together form a five, six, or seven-member ring, which ring can include one or more heteroatoms, such as O, S, and N (wherein N can be substituted with H or R'), and

one or both of the ring nitrogens can be replaced with a CR' moiety, and pharmaceutically-acceptable salts thereof,

and a pharmaceutically-acceptable carrier.

20. The method of any of Claims 1-15, wherein the compound has the following formula:

wherein one or more positions on the ring skeleton (i.e., at positions 2, 3, 6, 7, and 8) can, individually, be replaced with a substituent selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (C C₆), lower haloalkyl (Ci-C₆), lower alkoxy (Ci-C₆), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆), lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above),

Rl, R2, R3, and R4, are independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, -COR', and -COOR', wherein R' is, independently H, a lower alkyl (Ci-Ce), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆), lower cycloalkyl (C3-C6) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above), and

one or both of Rl and R2, and R3 and R4, together with the nitrogens to which they are attached, can form a 5-7 membered ring, which can include one or more additional heteroatoms such as O, S, or N, wherein the N can be bonded to a substituent R', as defined above, and pharmaceutically-acceptable salts thereof, and a pharmaceutically-acceptable carrier.

21. The method of Claim 19, wherein at least one of R4, R5, R6, R7, R15, R16, and R17 is other than H.

22. The method of Claim 21, wherein at least one of Rl, R2, R3, and R4 is other than H, when the aryl rings are otherwise unsubstituted (i.e., where all other positions on the aryl rings are H).

23. The method of Claim 1, wherein the compound is selected from the group consisting of 3-ethyl-6-methoxy-lH-benzo[de]cinnoline, methyl 6-(5-methyl-2-oxo-2,3- dihydro-lH-imidazol-4-yl)-6-oxohexanoate, ethyl 6-(l-benzoyl-5-methyl-2-oxo-2,3-dihydro- lH-imidazol-4-yl)-6-oxohexanoate, 2-[(8-ethoxy-4-methyl-2-quinazolinyl)amino]-5, 6,7,8- tetrahydro-4(lH)-quinazolinone, 2-[(6-methoxy-4-methyl-2-quinazolinyl)amino]-5,6- dimethyl-4(lH)-pyrimidinone, 2-[(4,7-dimethyl-2-quinazolinyl)amino]-6-propyl- 4(3H)-pyrimidinone, tris[4-(dimethylamino)phenyl]methanol, 5-(2-oxohexahydro- 1H- thieno[3,4-d]imidazol-4-yl)pentanoic acid, N-ethyl-5-nitro-N-phenyl-2,l,3-benzoxadiazol-

4- amine, 4,5-bis(dimethylamino)-l-naphthaldehyde, N,N-dimethyl-N'-[4-(2- pyridinyl)-l,3-thiazol-2-yl]-l,4-benzenediamine hydrobromide, N-2-(4,6-dimethyl-2- pyrimidinyl)-2,4-quinazolinediamine hydrochloride,2-[(4,6,7-trimethyl-2- quinazolinyl)amino]-5,6,7,8-tetrahydro-4(lH)-quinazolinone, 9-(2-methoxyphenyl)- 2,3,7,7-tetramethyl-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline- 4,5-dione, 2-[(4,8-dimethyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(lH)-quinazolinone, 2-[(4-methyl-2-quinazolinyl)amino]-6-propyl-4(lH)-pyrimidinone, 2-[(4- acetylphenyl)amino]-3-(l-pyrrolidinyl)naphthoquinone, 9-(2,5-dimethoxyphenyl)-2- methoxy-7,7-dimethyl-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,l- ij]quinoline-4,5-dione, [3-(l,3-benzodioxol-5-yl)-3-phenylpropyl][4- (dimethylamino)benzyl] amine hydrochloride, 2-[(4,7-dimethyl-2-quinazolinyl)amino]-

5- ethyl-6-methyl-4(3H)-pyrimidinone, N-[2-(l-cyclohexen-l-yl)ethyl]-2-(3- methylphenyl)-5-nitro-2H-l,2,3-triazol-4-amine 3-oxide, 2-methoxy-7,7-dimethyl-9-(2- propoxyphenyl)-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5- dione, 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2- methylbenzoate, 9-(2,3-dimethylphenyl)-2-methoxy-7,7-dimethyl- 10-thioxo-9, 10- dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,l-ij]quinoline-4,5-dione, N'-(2-methoxybenzyl)- Ν,Ν-dimethyl- 1 ,4-benzenediamine, 2-(4-ethylphenyl)naphthoquinone, 2-[(4,6-dimethyl- 2-quinazolinyl)amino]-l,5,6,7-tetrahydro-4H-cyclopenta[d]pyrimidin-4-one, 5-nitro-4- (l-piperidinyl)-2,l,3-benzoxadiazole, N,N-dimethyl-N'-[2-nitro-4- (trifluoromethyl)phenyl] - 1 ,3 -benzenediamine, 1 - [2- (benzyloxy)benzyl] - 5 -methyl- 1 H- indole-2,3-dione, l-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]pyrrolidine, 2-(4- ethylphenyl)-5-methyl-4-[4-(4-morpholinyl)benzylidene]-2,4-dihydro-3H-pyrazol-3-one, (2- methoxybenzyl)[4-(l-piperidinyl)phenyl] amine, 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro- 4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2-methoxybenzoate, 4,4,6-trimethyl-l,2-dioxo-l,2- dihydro-4H-pyrrolo[3,2,l-ij]quinolin-8-yl 2-chlorobenzoate, 4-({[3-(2-furyl)-3-(2- methoxyphenyl)propyl]amino}methyl)-N,N-dimethylaniline, N'-[4-(allyloxy)-3-chloro- 5-methoxybenzyl]-N,N-dimethyl-l,4-benzenediamine, 2,6,7-trihydroxy-9-(5-nitro-2- furyl)-3H-xanthen-3-one, 2-[ethyl(4-{[2-nitro-4- (trifluoromethyl)phenyl]amino}phenyl)amino]ethanol,

5 - [4-(dimethylamino)phenyl] -3 -(4-methoxyphenyl)-N-methyl-4,5 -dihydro- lH-pyrazole- 1 - carbothioamide, N-[4-(dimethylamino)phenyl]-N'-(4-methylbenzyl)thiourea, N- [4-(allyloxy)-3-methoxybenzyl]-4-(l-pyrrolidinyl)aniline, 5-methyl-N-[7-(4-morpholinyl)- 2,l,3-benzoxadiazol-4-yl]-4-phenyl-3-thiophenecarboxamide, 4-[2-chloro-4-nitro-5- (vinylthio)-3-thienyl]morpholine, l-(4-fluorophenyl)-2-(2-nitrovinyl)-lH-pyrrole,

1 , 1 '-(2,4-cyclopentadien- 1 -ylidenemethylene)bis(4-methoxybenzene), 3-(2- chlorophenyl)-6-ethyl-7-methoxy-4H-chromene-4-thione, N-[4-hydroxy-3-(phenylthio)-l- naphthyl]-4-methoxybenzenesulfonamide, l-(4-chlorophenyl)-2-(2-nitrovinyl)-lH-pyrrole,

4-({[3-(2-furyl)-4-phenylbutyl]amino}methyl)-N,N-dimethylaniline, 4-(4- benzyl-l-piperazinyl)-N-(4-fluorobenzyl)aniline, (2-ethoxy-3-methoxybenzyl)[4-(l- pyrrolidinyl)phenyl] amine, (4-fluorobenzyl) [4-( 1 -pyrrolidinyl)phenyl] amine, 6- (dimethylamino)-2-(2-methylphenyl)-5-nitro-lH-benzo[de]isoquinoline-l,3(2H)-dione, N- (4-chlorobenzyl)-N'-[4-(diethylamino)phenyl]thiourea, 4-fluoro-N-[4-hydroxy-3- (phenylthio)- l-naphthyl]benzenesulfonamide, 2-bicyclo[2.2. l]hept-2-yl-5-nitro- 1H- isoindole-l,3(2H)-dione, N-[2-(lH-indol-3-yl)ethyl]-9-acridinamine, 6-bromo-2-(3- methoxy-4-propoxyphenyl)-3-nitro-2H-chromene, ethyl 3-(4-methoxyphenyl)-l,4-dioxo- l,4-dihydro-2-naphthalenecarboxylate, (2-methoxyphenyl)[2-nitro-4- (trifluoromethyl)phenyl] amine, N'-(2,8-dimethyl-4-quinolinyl)-N,N-dimethyl-l,4- benzenediamine hydrochloride, 2-(2-methylphenyl)-5-(l-piperidinyl)-l,3-oxazole-4- carbonitrile, N-[4-hydroxy-3-(phenylthio)-l-naphthyl]benzenesulfonamide, l-(4- chlorophenyl)-5-[(5-nitro-2-furyl)methylene]-2,4,6(lH,3H,5H)-pyrimidinetrione, N'-(4,6- dimethyl-2-pyrimidinyl)-N,N-dimethyl-l,4-benzenediamine, 5-bromo-l-(2- chlorobenzyl)-lH-indole-2,3-dione, 4,4,6-trimethyl-l,2-dioxo-l,2-dihydro-4H- pyrrolo[3,2,l-ij]quinolin-8-yl 2-thiophenecarboxylate, 2-({ [4- (dimethylamino)pheny 1] amino } methyl)phenol , 2- (2-fluorophenyl)- 3 - ( 1 -methyl- 1 H- pyrrol-2-yl)acrylonitrile, N-[4-(diethylamino)phenyl]-N'-isobutylthiourea, 2-(4- ethylphenyl)-4-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methyl-2,4-dihydro-3H-pyrazol-3- one, N-(2-methoxyphenyl)-3-nitro-2-pyridinamine, ethyl 3-(4-methylphenyl)-l,4- dioxo- 1 ,4-dihydro-2-naphthalenecarboxylate, 5- { [(2-nitrophenyl)thio] amino } - 1 ,3- benzodioxole, N-[4-(diethylamino)phenyl]-N'-(4-ethoxyphenyl)thiourea, N-[4-hydroxy-3- (phenylthio)- l-naphthyl]-2-thiophenesulfonamide, l-benzyl-5-bromo-7-methyl- lH-indole- 2,3-dione, N-[4-(dimethylamino)benzyl]-6-methyl-2-pyridinamine, 2-[2-bromo-4- ({[4-(dimethylamino)phenyl]amino}methyl)-6-ethoxyphenoxy]-N-(tert-butyl)acetamide, N- [4-(dimethylamino)benzyl]-l-pentyl-lH-benzimidazol-2-amine, 4,6-diethyl-4,8-dimethyl- 4H-pyrrolo[3 ,2, 1 -ij Jquinoline- 1 ,2-dione, N- [( 1 -methyl- lH-pyrrol-2-yl)methylene] -4-( 1 - naphthylmethyl) - 1 -piperazinamine , 5 -( 1 - azepanyl) -2- [(3 -chlorophenoxy)methyl] -1,3- oxazole-4-carbonitrile, 6'-methyl-5',6'-dihydrospiro[cyclohexane-l,4'-pyrrolo[3,2,l- ij]quinoline]-l',2'-dione, 4-(di-lH-indol-3-ylmethyl)-l,2-benzenediol,(5-bromo-2- methoxybenzyl) [4-(4-morpholinyl)phenyl] amine, 1 - [2-chloro-4-nitro-5 -(vinylthio)-3 - thienyljpiperidine, 5-(l-azepanyl)-2-(2-fluorophenyl)-l,3-oxazole-4-carbonitrile, 4- (2- { [4-(diethylamino)phenyl] amino } - 1 ,3 -thiazol-4-yl)- 1 ,2-benzenediol, 1 -butyl-N- [4- (diethylamino)benzyl]-lH-benzimidazol-2-amine, (4-bromophenyl)[3-nitro-4-(l- piperidinyl)phenyl]methanone, 2-(2,5-dimethylphenyl)-6-nitro-lH- benzo[de]isoquinoline-l,3(2H)-dione, N-[4-(diethylamino)phenyl]-N'-(2,4- dimethoxyphenyl)thiourea, 5-(dimethylamino)-l,3-benzothiazole-2-thiol, ethyl 3-(4- ethylbenzyl)- 1 ,4-dioxo- 1 ,4-dihydro-2-naphthalenecarboxylate, 4-propylphenyl 4- nitrobenzoate, 4-({[3-(2-furyl)-3-(4-methylphenyl)propyl]amino}methyl)-N,N- dimethylaniline, N-benzyl-5-(4-benzyl-l-piperazinyl)-2-nitroaniline, N-benzyl-4- chloro-2-nitroaniline, 5-(l-azepanyl)-2-[(4-chlorophenoxy)methyl]-l,3-oxazole-4- carbonitrile, [4-(2,6-dimethyl-4-morpholinyl)-3-nitrophenyl](4-ethoxyphenyl)methanone,

5-chloro-l-(2-chlorobenzyl)-lH-indole-2,3-dione, 2-fluorobenzyl 2-chloro-4- nitrobenzoate, 3-[(3,4-dimethylphenyl)amino]-l-(4-nitrophenyl)-l-propanone, N-[4- (diethylamino)phenyl]-2-phenylcyclopropanecarboxamide, 2-[2-bromo-6-methoxy-4-({[4-(l- pyrrolidinyl)phenyl] amino } methyl)phenoxy] acetamide, N- [4-( 1 -azepanyl)phenyl] -2- methylbenzamide, N-cyclohexyl-N'-[4-(dimethylamino)phenyl]thiourea, N-(3- chloro-4-fluorophenyl)-N'-[4-(diethylamino)phenyl]thiourea, 4,6-dimethyl-N-[4-(l- pyrrolidinyl)phenyl] -2-pyrimidinamine, (2,6-dichlorobenzyl) [4-( 1 - pyrrolidinyl)phenyl] amine, 2-(2-fluorophenyl)-5-(4-phenyl-l-piperazinyl)-l,3-oxazole-4- carbonitrile, and l-[4-nitro-3-(l-pyrrolidinyl)phenyl]-4-(2-thienylcarbonyl)piperazine

24. The method of any of Claims 1-15, wherein the compound is co-administered with one or more additional antiviral agents.

25. The method of Claim 24, wherein the one or more additional antiviral agents are selected from the group consisting of entry inhibitors, integrase inhibitors, reverse transcriptase inhibitors, protease inhibitors, and immune-based therapeutic agents.

26. A pharmaceutical composition comprising an inhibitor as described in any of Claims 1-23, along with a pharmaceutically-acceptable carrier.

27. The composition of Claim 26, wherein the inhibitor inhibits retroviral reverse transcription.

28. The composition of Claim 26, wherein the inhibitor inhibits viral recruitment of the retroviral primer used in translation, human tRNA^Lys3.

29. The composition of Claim 26, wherein the inhibitor inhibits the final packaging and assembly of new virions.

30. The composition of Claim 26, wherein the inhibitor inhibits the binding of a host cell tRNA to a target nucleic acid molecule.

31. The composition of Claim 26, further comprising a second antiretro viral compound.

32. The composition of Claim 31, wherein the second antiretroviral agent is selected from the group consisting of NRTIs, NNRTIs, VAP anti-idiotypic antibodies, CD4 and CCR5 receptor inhibitors, entry inhibitors, antisense oligonucleotides, ribozymes, protease inhibitors, neuraminidase inhibitors, tyrosine kinase inhibitors, PI-3 kinase inhibitors, and Interferons.

33 The use of an inhibitor of retroviral propagation in the preparation of a medicament for treating or preventing a drug-resistant HIV infection in a patient, wherein: i) the inhibitor inhibits the ability of any portion of the HIV genome involved in reverse transcription to bind to or associate with a host cell tRNA, or the inhibitor disrupts the RNA/RNA complex formed between the viral genome and the human tRNA primer, or

34. The use of Claim 33, wherein the portion of the HIV genome involved in reverse transcription is the 5' un- translated region of the HIV genome.

35. The use of Claim 2, wherein the region of the HIV genome comprises residues 157 to 169 of the 5' un-translated region of HIV-1.

36. The use of Claim 33, or wherein the portion of the host cell tRNA comprises human tRNA^Lys3.

37. The use of Claim 36, wherein the portion of the host cell tRNA comprises nucleotides 32-43 of human tRNA^Lys3.

38. The use of Claim 33, wherein the inhibitor inhibits retroviral reverse transcription, inhibits viral recruitment of the retroviral primer used in translation, human tRNA^Lys3, inhibits the final packaging and assembly of new virions, or inhibits the binding of a host cell tRNA to a target nucleic acid molecule.

39. The use of Claim 33, wherein the HIV is selected from the group consisting of drug-resistant HIV-I, HIV-II, and HIV-III.

40. The use of Claim 33, wherein the inhibitor directly interacts with nucleotides 32- 43 of the host tRNA^Lys3 (the HIV human primer) such that binding of the retroviral RNA to those nucleotides is inhibited, or with nucleotides 157-169 of the retroviral RNA, such that binding of the host tRNA^Lys3 (the HIV human primer) to those nucleotides is inhibited.

41. The use of Claim 33, wherein the HIV is resistant to more than one anti-retroviral drug.

42. The use of Claim 33, wherein the HIV comprises one or more HIV-1 protease mutations selected from the group consisting of L10I, L10F, L10V, L10C, L10R, VI II, I13V, G16E, K20M, K20R, K20T, K10T, K20V, L24I, D30N, V32I, L33F, L33I, L33V, E34Q, E35G, M36I, M36L, M36V, K43T, M46I, M46L, 147 A, I47V, 147 A, G48V, I50V, I50L, F53L, F53Y, I54V, I54L, I54M, I54A, I54T, I54S, 058E, D60E, I62V, L63P, I64M, I64L, I64V, H69K, A71V, A71T, A71I, A71L, G73C, G73S, G73T, G73A, T74P, V82A, V82F, L76V, V77I, G82T, V82S, V82F, V82T, V82I, G82M, I84V, I84A, I85V, I84C, N83D , N88D, N88S, L89V, and L90M.

43. The use of Claim 33, wherein the HIV comprises one or more HIV-1 RT mutations selected from the group consisting of M41L, K65R, D67N, D67G, D67Del, T69D, T69ins, K70R, L74V, V75A, V75M, V75T, V75S, F77L, Y115F, F116Y, M184V, M184I, L210W, T215Y, T215F, T215C, T215D, T215E, T215E, T215S, T215I, T215V, K219Q, K219E, and K219R.

44. The use of Claim 33, wherein the HIV comprises one or more HIV-1 RT mutations selected from the group consisting of V90I, A98G, L100I, K101E, K101P, K101H, K103N, K103S, V106I, V106A, V106M, V108I, E138A, V179D, V179F, V179T, Y181C, Y181I, Y181V, Y188L, Y188H, Y188C, G190A, G190S, G190E, G190Q, P225H, M230L, and P236L.

45. The use of Claim 33, wherein the HIV comprises one or more mutations in the envelope gene selected from the group consisting of G36D, G36S, I37V, V38A, V38M, V38E, Q39R, Q40H, N42T, and N43D.

46. The use of Claim 33, wherein the HIV comprises one or more mutations in the integrase gene selected from the group consisting of Y143R, Y143H, Y143C, Q148H, Q148K, Q148R, and N155H.

47. The use of Claim 33, wherein the HIV comprises two or mutations, where the mutations are present in a gene selected from the HIV envelope gene, integrase gene protease gene, and reverse transcriptase gene.

48. The use of any of Claims 33-47, wherein the compound has one of the following formulas:

A. B.

G.

wherein

Single or double bond

M and N = 1, 2 or 3 atoms from C, N, O

X and Y = NR¹, O or S

wherein substituents Z include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R', -SR', -N₃, -C(=0)NRR", -NRC(=0) R", -C(=0)R, -C(=0)OR, -OC(=0)R, - OC(=0)NR'R", -NRC(=0)0 R", -S0₂R, -S0₂NRR", and -NRS0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl).

49. The use of any of Claims 33-47, wherein the compound has one of the following formulas: Ar-ι (CH₂)_n NR., Ar₂

n is 0 or 1, and

Ri is H or a moiety cleaved in vivo to form H,

Z, and

substituents Z as defined herein include Ci_6 alkyl (including cycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, CI, Br, or I), -OR', -NR'R", -CF₃, -CN, -N0₂, - C₂R, -SR', -N₃, -C(=0)NR'R", -NR'C(=0) R", -C(=0)R', -C(=0)OR, -OC(=0)R', - OC(=0)NR'R", -NRC(=0)0 R", -S0₂R, -S0₂NRR", and -NR'S0₂R", where R and R" are individually hydrogen, Ci_6 alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (such as benzyl),

An (CH₂)_m NR-i (CH₂)_m Δ

Formula C

Fonnula D

where Z and j are as defined above,

Formula F wherein Ari, Ri, Z and j are as defined above, and Ari can include from one to three Z substituents, Z,

wherein X, R], Z, j, and n are as defined above, and the =X moiety can be present or not present (i.e., n is 0 or 1),

Formula H

R4, R5, R6, R7, R15, R16, and R17 are, independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo (F, CI, Br, I), OR', N(R')₂, SR', OCOR', NHCOR', N(COR')COR', SCOR', OCOOR', and NHCOOR', wherein each R' is independently H, a lower alkyl (C C₆), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C2-C₆), lower alkynyl (C₂- C₆), lower cycloalkyl (C3-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above), or, alternatively, one or more of R4 and R5, R5 and R6, R6 and R7, R15 and R16, and R16 and R17 together form a five, six, or seven-member ring, which ring can include one or more heteroatoms, such as O, S, and N (wherein N can be substituted with H or R'),

Formula I

wherein positions 2, 3, 6, 7, and 8 can include a substituent Z as defined herein, and Rl, R2, R3, and R4, are independently, the same or different, and are selected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, -COR' , and -COOR' , wherein R' is, independently H, a lower alkyl (Ci-Ce), lower haloalkyl (Ci-Ce), lower alkoxy (Ci-Ce), lower alkenyl (C₂-Ce), lower alkynyl (C₂-Ce), lower cycloalkyl (C3-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, wherein the groups can be substituted with one or more substituents as defined above), and

in one embodiment, one or both of Rl and R2, and R3 and R4, together with the nitrogens to which they are attached, form a 5-7 membered ring, which can include one or more additional heteroatoms such as O, S, or N, wherein the N can be bonded to a substituent R' , as defined above),

Formula K wherein, for compounds of Formulas J, K, and L, R_l5 Z and j are as defined above,

wherein X, Z, j, m and R] are as defined above,

Formula N

wherein X,Z, j, are as defined above,

wherein X,Z, j, are as defined above,

Formula O

wherein X, Z, j, and m are as defined above,

Formula P

wherein Z, j, n, and Ri are as defined above, and a) K is NRi, or b) K is N(Ri)₂, and the link to the other ring nitrogen is absent, in which case the other NRi moiety is an N(Ri)₂ moiety rather than an NRi moiety,

Formula R

50. The use of any of Claims 33-47, wherein the compound has one of the following formulas:

98

51. The use of any of Claims 33-47, wherein the compound has the following formula:

wherein:

the dashed line indicates the presence of an optional double bond,

and a pharmaceutically-acceptable carrier.

52. The use of any of Claims 33-47, wherein the compound has the following formula:

53. The use of Claim 51, wherein at least one of R4, R5, R6, R7, R15, R16, and R17 is other than H.

54. The use of Claim 53, wherein at least one of Rl, R2, R3, and R4 is other than H, when the aryl rings are otherwise unsubstituted (i.e., where all other positions on the aryl rings are H).

55. The use of Claim 1, wherein the compound is selected from the group consisting of 3-ethyl-6-methoxy-lH-benzo[de]cinnoline, methyl 6-(5-methyl-2-oxo-2,3-dihydro-lH- imidazol-4-yl)-6-oxohexanoate, ethyl 6-(l-benzoyl-5-methyl-2-oxo-2,3-dihydro-lH- imidazol-4-yl)-6-oxohexanoate, 2-[(8-ethoxy-4-methyl-2-quinazolinyl)amino]-5, 6,7,8- tetrahydro-4(lH)-quinazolinone, 2-[(6-methoxy-4-methyl-2-quinazolinyl)amino]-5,6- dimethyl-4(lH)-pyrimidinone, 2-[(4,7-dimethyl-2-quinazolinyl)amino]-6-propyl- 4(3H)-pyrimidinone, tris[4-(dimethylamino)phenyl]methanol, 5-(2-oxohexahydro- 1H- thieno[3,4-d]imidazol-4-yl)pentanoic acid, N-ethyl-5-nitro-N-phenyl-2,l,3-benzoxadiazol-

5-[4-(dimethylamino)phenyl]-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-lH-pyrazole-l- carbothioamide, N-[4-(dimethylamino)phenyl]-N'-(4-methylbenzyl)thiourea, N- [4-(allyloxy)-3-methoxybenzyl]-4-(l-pyrrolidinyl)aniline, 5-methyl-N-[7-(4-morpholinyl)- 2,l,3-benzoxadiazol-4-yl]-4-phenyl-3-thiophenecarboxamide, 4-[2-chloro-4-nitro-5- (vinylthio)-3-thienyl]morpholine, l-(4-fluorophenyl)-2-(2-nitrovinyl)-lH-pyrrole,

56. The use of any of Claims 33-47, wherein the compound is co-administered with one or more additional antiviral agents.

57. The use of Claim 56, wherein the one or more additional antiviral agents are selected from the group consisting of entry inhibitors, integrase inhibitors, reverse transcriptase inhibitors, protease inhibitors, and immune-based therapeutic agents.