WO2009145839A1

WO2009145839A1 - Polyamine compounds that bind tar rna of hiv and methods of treating viral disorders

Info

Publication number: WO2009145839A1
Application number: PCT/US2009/002116
Authority: WO
Inventors: Daniel Appella; Jaclyn Iera; Deyun Wang; Jonathan Pokorski
Original assignee: Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2008-04-04
Filing date: 2009-04-03
Publication date: 2009-12-03

Abstract

The invention provides for novel polyamine compounds, substituted at the side chains, which can treat various disease states including viral infection, e.g. HIV infection, at low cytotoxicity values.

Description

POLYAMINE COMPOUNDS THAT BIND TAR RNA OF HIV AND METHODS OF

TREATING VIRAL DISORDERS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/123,076, filed April 4, 2008, the teachings of which are incorporated herein by reference.

FIELD OF THE INVENTION

In one aspect, the invention provides for novel polyamine compounds. Preferred polyamine compounds of the invention can target an RNA structure associated with a disease state, such as a variety of viruses (e.g. HIV, SARS), bacterial infections and cancers. In a particular aspect, polymaine compounds are provided which selectively bind to TAR RNA of HIV, which are useful to treat HIV infection.

BACKGROUND

The human immunodeficiency virus (HIV) and various mutants including type 1 (HIV-I), is the etiological agent of acquired immune deficiency syndrome (AIDS) and related disorders. The screening and investigation of novel drugs against HIV remains critical because of the ongoing AIDS epidemics and of the fast emergence of virus variants resistant to present antiviral therapy.

In eukaryotic cells, naturally occurring polyamines have a role in regulating specific gene expression. Binding of different classes of polyamines to RNA has been studied, and the usefulness of RNA binding in controlling gene expression is recognized. In the HIV setting, polyamine binding to specific viral RNA offers a way to differentiate HIV targets from the host cell targets.

Two primary HIV RNA elements are viable targets for inhibition: TAR and RRE. TAR and RRE are binding sites for HIV proteins Tat and Rev, respectively. Both Tat and Rev are required for viral replication and work through interaction with the RNA elements. Therefore, the disruption of modest inhibition of Tat or Rev with its corresponding RNA element is desirable. A few studies have shown very modest inhibition of Tat-TAR interaction by polyamines. Polyamines were first observed in crystalline form in the late 1600s. Polyamines are amino acid-derived molecules that are produced naturally by all cells, both eukaryotic and prokaryotic. Polyamines typically refer to several related classes of synthetic compounds having various structural variation.

While chemists have excelled at the design and synthesis of organic molecules that inhibit protein functions by binding to active sites, there exists a lack of basic knowledge about how one should design a molecule to target a folded RNA. The fact that proteins adopt folded, three-dimensional structures with unique binding pockets allows chemists to develop small organic molecules that bind with high affinity and specificity to a target protein. Because RNA can also possess folded, three-dimensional structures, it should be possible for chemists to design new molecules that bind a target RNA with high affinity and specificity. In recent years, a wealth of structural information on RNA has demonstrated that this biopolymer can adopt a multitude of folded structures. In the cell, RNA often adopts folded structures to create protein or small molecule binding sites or to perform catalytic functions. In many cases, the folded RNA structures approach the complexity of folded protein structures. Despite the emerging amount of structural information, RNA continues to be underutilized as a target for drug development because there is a lack of RNA-binding molecules with well-defined molecular recognition properties combined with biological activity.

The most common types of molecules that have been previously investigated for RNA binding include aminoglycosides, polypeptides, and polycyclic aromatic molecules. By incorporating a significant amount of cationic charge or aromatic density in each of these molecular types, excellent binding affinity to a target RNA can be achieved; however, affinity is usually attained at the expense of specificity for the target. Other approaches to identify RNA-binding molecules have explored high-throughput screening of chemical libraries (either in vitro or in silico). While a few interesting leads from such studies have been identified, most results contribute more to the techniques of screening rather than identifying new chemical scaffolds for development into RNA-binding drugs. The emerging picture from these pioneering studies indicates that new types of RNA-specific chemical scaffolds must be developed.

The instant invention seeks to provide novel polyamine compounds that are useful to treat HIV, wherein such compounds affect the Tat-TAR interaction.

BRIEF SUMMARY OF THE INVENTION In one aspect, the invention provides a compound represented by formula I:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof, wherein: each R and R' are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1, 2, or 3 heteroatoms consisting of O, N, and S; each Ri and R₂ are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted haloalkyl, halogen, hydroxy, amino, CN, N₃, NO₂, OR_x, SR_x, SO₂R_x, -NHS(O)₂-R_x, -NH(SO₂)NR_xR_x, NR_xRx₄, CO₂R_x, COR_x, CONR_xR_x, and N(R₃)COR_x; or each Ri and R₂ together form =0 or =S; each R_x is independently selected from an optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, optionally substituted aralkyl, an optionally substituted heteroaralkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylcycloalkyl, optionally substituted cycloalkenyl, and optionally substituted alkylcycloalkenyl;

R₃, R₄, R₅ and R₆, are each independently, H, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(O)R_x, -C(S)R_x, -C(NR)R_x, -S(O)R_x, or -S(O)₂R_x; or R₃ and R₄ taken together, or R₅ and R₆ taken together, with the N to which they are attached is an optionally substituted heterocycloalkyl, or an optionally substituted heteroaryl; m is O, 1, or 2; and n is an integer from 0-20. In another aspect, the invention provides for a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester, or prodrug thereof, in combination with a pharmaceutically acceptable carrier or excipient.

In another aspect, the invention provides a kit comprising an effective amount of a compound of formula I in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a viral infection.

In certain embodiments, the compounds of the invention effectively inhibit the Tat- TAR interaction with an IC50 in the low micromolar range. Additionally, the inhibition is specific for Tat-TAR, unlike the non-specific interactions typically observed. In certain aspects, the toxicity values are at greater than 4-fold above the IC50 values. The low concentration required for activity in whole cells is orders of magnitude less than any previously reported for any class of polyamine used for the same types of HIV assays.

In one aspect, the invention provides a method of treating a subject suffering from or susceptible to a viral infection comprising administering to the subject an effective amount of a compound of formula I.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibition of HIV-I TAT function in DLTAT cells by 4.

Figure 2 shows the inhibition of HIV-I TAT function in DLTAT cells by 7.

Figure 3 shows the inhibition of HIV-I TAT function in DLTAT cells by 16.

Figure 4 shows the inhibition of HIV-I TAT function in DLTAT cells by Temacrazine.

Figure 5 shows the inhibition of HIV-I Clade A (UG/92/029) replication in human PBMC by 4.

Figure 6 shows the inhibition of HIV-I Clade A (UG/92/029) replication in human PBMC by 7.

Figure 7 shows the inhibition of HIV-I Clade A (UG/92/029) replication in human PBMC by 16.

Figure 8 shows the inhibition of HIV-I Clade A (UG/92/029) replication in human PBMC by AZT.

Figure 9 shows the inhibition of HIV-I Clade D (UG/92/001) replication in human PBMC by 4. Figure 10 shows the inhibition of HIV-I Clade D (UG/92/001) replication in human PBMC by 7.

Figure 11 shows the inhibition of HIV-I Clade D (UG/92/001) replication in human PBMC by 16.

Figure 12 shows the inhibition of HIV-I Clade D (UG/92/001) replication in human PBMC by AZT.

Figure 13 shows the inhibition of HIV-I Clade E (CMU06) replication in human PBMC by 4.

Figure 14 shows the inhibition of HIV-I Clade E (CMU06) replication in human PBMC by 7.

Figure 15 shows the inhibition of HIV-I Clade E (CMU06) replication in human PBMC by 16.

Figure 16 shows the inhibition of HIV-I Clade E (CMU06) replication in human PBMC by AZT.

Figure 17 shows the inhibition of HIV-I Clade F (BR/93/020) replication in human PBMC by 4.

Figure 18 shows the inhibition of HIV-I Clade F (BR/93/020) replication in human PBMC by 7.

Figure 19 shows the inhibition of HIV-I Clade F (BR/93/020) replication in human PBMC by 16.

Figure 20 shows the inhibition of HIV-I Clade F (BR/93/020) replication in human PBMC by AZT.

Figure 21 shows the inhibition of HIV-I Clade G (JVl 083) replication in human PBMC by 4.

Figure 22 shows the inhibition of HIV-I Clade G (JV1083) replication in human PBMC by 7.

Figure 23 shows the inhibition of HIV-I Clade G (JVl 083) replication in human PBMC by 16.

Figure 24 shows the inhibition of HIV-I Clade G (JV1O83) replication in human PBMC by AZT.

Figure 25 shows the inhibition of HIV-I Clade O (BCF02) replication in human PBMC by 4.

Figure 26 shows the inhibition of HIV-I Clade O (BCF02) replication in human PBMC by 7. Figure 27 shows the inhibition of HIV-I Clade O (BCF02) replication in human PBMC by 16.

Figure 28 shows the inhibition of HIV-I Clade O (BCF02) replication in human PBMC by AZT.

Figure 29 shows the visualization of positive beads as red in color where beads containing non-binding polyamines were seen as green under the microscope.

Figure 30 shows the results of experiments that identified the sequence XFF as a binding motif specific for the bulge region of TAR.

Figure 31 shows: A. Sequence and secondary structure of TAR, initial lead MBO (YYY) with Kd ~ 5 μM for binding to the bulge of TAR and the fluorescein-labeled derivative (YYY-Fl). B. Cellular uptake of YYY-Fl in HeLa cells (top three rows show 2 μM of YYY-Fl incubated for 30 min. at 37 ⁰C followed by washing, fixing, permeabilization and staining; bottom row shows results with fluorescein alone).

Figure 32 shows a Fluorescence-based competition assay to determine IC₅₀ values for MBO inhibition of tat-TAR association in vitro (for all experiments, TAR was at X nM and labeled tat-peptide was at X nM, A(lambda) was recorded at different concentrations of MBO (include reference). Similar system for rev-RRE binding.

DETAILED DESCRIPTION

Compounds of the Invention

The invention is directed towards the RNA-binding properties of a novel class of molecules termed multivalent binding oligomers (MBOs). These molecules are derived from amino acids and are synthesized similarly to polypeptides. MBOs are oligomeric, like polypeptides, but an amine linkage holds the adjacent amino acids together instead of an amide bond. MBOs were designed to bind to RNA targets while avoiding the disadvantages of other classes of RNA-binding molecules. Specifically, MBOs contain sidechains with non-ionic functional groups to direct the specificity of binding through hydrogen bonding or aromatic-aromatic interactions while the amines in the backbone contribute ionic interactions to facilitate binding to the anionic RNA backbone. At the same time, the presence of the sidechains is hypothesized to reduce the amount of non-specific binding to non-target RNA when interactions between the sidechains and RNA are unfavorable.

In one aspect, the invention provides the development and characterization of an MBO to target TAR, an HIV RNA that forms a hairpin-loop with a unique bulge. The HIV protein tat must bind to TAR for viral transcription to proceed efficiently, and molecules that inhibit the association of tat with TAR can shut down replication of the virus. TAR is present in all HIV-I transcripts, is highly conserved, and must fold into a stem-bulge-loop structure to be recognized by tat. Therefore, molecules that can bind with high affinity and selectivity to the bulge of TAR could evolve into new treatments for HIV infection. Previously developed TAR-binding molecules have demonstrated that this is a good target to attack HIV, but most of the currently available TAR binders are not suitable for further drug development due to non-specific binding, toxicity, poor biological activity (sometimes due to poor cellular uptake), or a combination of these problems. The results demonstrate that proper selection of sidechains and oligomer length lead to MBOs that inhibit tat-TAR association in vitro, are cell-permeable, maintain activity in a cell-based model system, and exert anti-HIV activity in infected white blood cells across a range of different clinically-derived strains of HIV-I.

The invention provides various polyamines that bind to HIV RNA and have antiviral activity. The polyamines of the invention have been functionalized at a side chain, which permits the polyamine to more effectively and more specifically bind to RNA. In certain instances, the polyamines of the invention bind selectively to the HIV RNA target sequence TAR (transactivating response element), when binding is compared to the HIV RNA target RRE (Rev response element).

In one aspect, the invention provides a compound represented by formula I:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof, wherein: each R and R' are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1, 2, or 3 heteroatoms consisting of O, N, and S; each Ri and R₂ are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted haloalkyl, halogen, hydroxy, amino, CN, N₃, NO₂, OR_x, SR_x, SO₂R_x, -NHS(O)₂-R_x, -NH(SO₂)NR_xR_x, NR_xRx₄, CO₂R_x, COR_x, CONR_xR_x, and N(R₃)COR_x; or each Ri and R₂ together form =O or =S; each R_x is independently selected from an optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, optionally substituted aralkyl, an optionally substituted heteroaralkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylcycloalkyl, optionally substituted cycloalkenyl, and optionally substituted alkylcycloalkenyl;

R₃, R₄, R₅ and R₆, are each independently, H, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(O)R_x, -C(S)R_x, -C(NR)R_x, -S(O)R_x, or -S(O)₂R_x; or R₃ and R₄ taken together, or R5 and R₆ taken together, with the N to which they are attached is an optionally substituted heterocycloalkyl, or an optionally substituted heteroaryl; m is O, 1, or 2; and n is an integer from 0-20.

In one embodiment, the invention provides a compound of formula II:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

In certain embodiments, each R and R' are independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by O, 1, 2, or 3 heteroatoms consisting of O, N, and S.

In other embodiments, each Ri and R₂ are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, or each Ri and R₂ together form =O.

In one embodiment, R₃ and R₅ are each independently, H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(O)R_x, -C(S)R_x, -C(NR)R_x, -S(O)R_x, or -S(O)₂R_x.

In yet another embodiment, m is O or 1. In still another embodiment, n is 4-10.

In certain embodiment, the invention provides a compound formula III:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

In a further embodiment, each Ri and R₂ are independently selected from H, an optionally substituted alkyl, or each Ri and R₂ together form =0.

In further embodiment, each Ri and R₂ are independently H or each Ri and R₂ together form =O.

In another embodiment, each R and R'are independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by O, 1, 2, or 3 heteroatoms consisting of O, N, and S.

In a further embodiment, each R and R' are independently selected from methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, pentyl, i-pentyl, neo-pentyl, hexyl, heptyl, benzyl, phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, pyridyl, furanyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, indazolyl, imidazopyridyl, quinazolinyl, or purinyl, each of which may be optionally substituted; or a groups selected from the following: CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

In still another further embodiment, each R is independently selected from the following:

In other embodiments, n is 4-10.

In certain embodiments, the invention provides a compound of formula IV:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

In one embodiment, each R and R' are independently selected from a natural or unnatural amino acid side chain.

In another embodiment, each R and R' are is independently selected from methyl, ethyl, propyl, i-propyl, CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

In a further embodiment, each R is independently selected from the following:

In still a further embodiment, each R is

In one embodiment, n is 4-10.

Compounds of the invention include the following:

(8) YYYYAY

(9) YYYYA:

(1O)YYYKY

(H)YYYKW

(12) YKYKY

(13) YKYKW

In certain embodiments, the invention provides a compound of formula V:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof. In one embodiment, each R₁ and R₂ are independently H or each Ri and R₂ together form =0.

In another embodiment, each R is independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1, 2, or 3 heteroatoms consisting of O, N, and S.

In a further embodiment, each R is independently selected from H, methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, pentyl, i-pentyl, neo-pentyl, hexyl, heptyl, benzyl, phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, pyridyl, furanyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, indazolyl, imidazopyridyl, quinazolinyl, or purinyl, each of which may be optionally substituted; or a groups selected from the following: CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

In one embodiment, n is 4-10.

In certain embodiments, the compound is of formula VI:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

In one embodiment each R is independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by O, 1, 2, or 3 heteroatoms consisting of O, N, and S.

In certain embodiments, the invention provides a compound of formula VII: O ^π R O R O ^π R (VII) as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

In another aspect, the invention provides for a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester, or prodrug thereof, in combination with a pharmaceutically acceptable carrier or excipient.

In one embodiment, the invention provides a composition further comprising an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an antiviral agent.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to HIV infection comprising administering to the subject an effective amount of a compound of formula I.

Synthesis of Compounds of the Invention

The compounds of the invention were synthesized according to the steps found in reaction schemes 1-7.

Scheme 1 shows that polyamines were prepared using a series of reductive alkylations between primary amines on solid support and protected α-amino aldehydes derived from amino acid side chains. A reducing agent was subsequently utilized to reduce the imine to a secondary amine, which was then protected. The steps were repeated until the polyamine of the desired length and substitution was obtained. Cleavage was carried out under acidic conditions and the polyamine was purified to provide the polyamine of the invention.

Scheme 1. Solid Phase Synthesis of Polyamines

2. Reducing agent

3. Reducing agent

3. Reducing agent

3. Reducing agent

Scheme 2 provides for the synthesis of various polyamines which incorporate any number of amino acids along the backbone of the compound. The reductive alkylations provide for the reduced amines, as described in Scheme 1. For the incorporation of amino acid residues, an alkylation reaction occurs to form an intermediate imine, which is oxidized back to the amine in situ, providing an amino acid derivative. The process can be repeated to synthesize a polyamine having any number of reduced amine moieties and amino acid moieties embedded into the desired final polyamine.

Scheme 2. Synthesis of Amino Acid Based Polyamine

2. Reducing agent

3. Oxidizing agent

3. Reducing agent

The polyamines were synthesized on Rink Amide resin through a series of reductive aminations and using standard Fmoc-chemistry. Initially, Fmoc-amino acids were converted to Weinreb amides followed by reduction to aldehydes using lithium aluminum hydride (LAH) (Wen, J. J.; Crews, C. M. Tetrahedron-Asymmetry 1998, 9, 1855-1858; Wang, G.; Mahesh, U.; Chen, G. Y. J.; Yao, S. Q. Organic Letters 2003, 5, 737-740) and the Fmoc- amino aldehydes can be stored in a -20 ⁰C freezer for at least a month with no degradation. Fmoc-β-alanine linker was coupled to the Rink Amide resin using a solution of HATU/HOBt/DIEA in DMF (Scheme 3).

Scheme 3. Fmoc Amino Acid Aldehyde Synthesis and Linker Coupling to Rink Resin

"

/-^. 1 ) 20% Piperidine/DMF -^ O

LjB — NHFmoc ► ^»L U

W 2) Fmoc-β-alanine ^^^N^^^^NHFmoc

Rink amide resin _{HATUi H0Bt D},_{EA| DMF} H

Once the linker was on the resin, the β-alanine was Fmoc-deprotected with 20% piperidine in DMF. An amino aldehyde was added to the resin in DCM and allowed to shake for 10 minutes before it was drained and followed by a solution OfNaBH(OAc)₃ in DCM for 45 minutes. After the completeion of the reductive animation, the secondary amine was Boc- protected to prevent branching. The series of Fmoc-deprotection, reductive animation, and Boc-protection was repeated two more times to synthesize a polyamine trimer. One final Fmoc-deprotection took place before the molecule was cleaved from the resin with a 10% TFA/DCM. All of the protecting groups were successfully removed with a solution of 95% TF A/1 % Triethylsilane/DCM. The final product was precipitated from ether and purified by HPLC to produce a white solid, which is stored in water at 4 ⁰C or colder (Scheme 4).

Scheme 4. Solid Phase Synthesis of Polyamines

Linker Studies

One difficulty with the synthesis was that despite Boc-protection of the secondary amines, branching was still occurring at the first amine due to over-alkylation (Scheme 3). Over-alkylation did not appear to be a problem at the subsequent amines because of the steric bulk the amino acid residues provided. It was reasoned that introducing some bulk in the linker molecule would circumvent this dilemma. Two other linkers, Fmoc-β-homoalanine and Fmoc-β-homophenylalanine, were tested to see if they would hinder branching (Scheme 5).

Scheme 5. Over-alkylation at the first secondary amine 1) 20% Piperidine/DMF

NHFmoc 2) NHFmoc

T NHFmoc ^{H R}'-J I

R² NHFmoc

NaBH(OAc)₃, CH₂CI₂

Three batches of Rink Amide resin were synthesized with each β-amino acid, and the reductive amination was performed with Fmoc-alanine aldehyde (Scheme 6). Alanine is not a bulky amino acid and should branch easily; the Fmoc-protecting group was left intact on the alanine to aid HPLC visualization.

Scheme 6. Derivatization of Rink Amide resin with A) β - alanine, B) β-homoalanine, or C) β-homophenylalanine

X-*. 1 ) 20% Piperidine/DMF U H

LJP — NHFmoc ••

^"^ 2) Fmoc-β-Amino Acid NHFmoc

Rink Amide Resin _{HATU H0Bt D},_{EA DMF}

When the HPLC of the β-alanine-alanine molecule was run, three distinct peaks were found and each peak was collected and its mass determined. The first and major peak was the desired product and the third peak, which was also strong, contained the branched molecule. The second HPLC peak contained a mass that was 28 units greater than our desired product (M+28 peak). Three peaks were also present the HPLC trace of the β- homoalanine-alanine molecule, although both the M+28 and branching peaks were greatly reduced. These two peaks were virtually non-existent in the chromatograph of the β- homophenylalanine-alanine molecule. As we had hypothesized, introducing some steric bulk into the linker of the molecule did inhibit branching, but we had to determine where the extra 28 mass units were originating.

In the synthesis, sodium triacetoxyboroydride is used as the reducing agent to avoid the toxicity associated with sodium cyanoborohydride. However, sodium triacetoxyborohydride will undergo a self-reduction producing acetaldehyde from the acetate groups. It was suspected that the M+28 peak is the result of a reductive amination of acetaldehyde with the amine to produce and ethyl group branching from the amine (Scheme 7). Since this occurrence was not originally considered as a possible by-product and branched and truncated molecules were more of a concern, it was not noticed in the original syntheses.

Scheme 7. Self reduction Of NaBH(OAc)₃

NaBH(OAc)₃, CH₂CI₂

Fortunately the addition of bulkier linkers alleviated this difficulty along with the branching. We proceeded to use Fmoc-β-homoalanine as the linker for future syntheses even though small amounts of branching still occurred. The Fmoc-β-homophenylalanine may have proved to be too bulky thus hindering reductive amination with larger amino acids. In addition, we believed that addition of a phenyl group may affect the binding affinity of the molecule while a methyl group would be relatively benign.

Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.

Nucleophilic agents are known in the art and are described in the chemical texts and treatises referred to herein. The chemicals used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents and the like. The methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compound of the formulae described herein. The methods delineated herein contemplate converting compounds of one formula to compounds of another formula. The process of converting refers to one or more chemical transformations, which can be performed in situ, or with isolation of intermediate compounds. The transformations can include reacting the starting compounds or intermediates with additional reagents using techniques and protocols known in the art, including those in the references cited herein. Intermediates can be used with or without purification (e.g., filtration, distillation, crystallization, chromatography). Other embodiments relate to the intermediate compounds delineated herein, and their use in the methods (e.g., treatment, synthesis) delineated herein.

The compounds of the invention are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

In addition, some of the compounds of this invention may have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double isomeric forms. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treatment or prevention of disorders disclosed herein). The compounds produced by the methods herein can be incorporated into compositions, including solutions, capsules, cremes, or ointments for administration to a subject (e.g., human, animal).

Methods of Treatment In one aspect, the invention provides a method of treating a subject suffering from or susceptible to a viral infection comprising administering to the subject an effective amount of a compound of formula I.

In one embodiment, the subject is infected with a retrovirus.

In a further embodiment, the viral infection is HIV infection.

In another further embodiment, the invention provides a method wherein the compound treats HIV by binding to HIV RNA.

In another embodiment, the RNA is TAR RNA.

In another embodiment, the subject is identified as having a viral infection and the compound of formula I is administered to the identified subject.

In one embodiment, the compound treats HIV by binding to HIV RNA.

In another embodiment, the RNA is TAR RNA.

In certain embodiments, the HIV is wild type or drug resistant.

In other embodiments, the invention provides a method further comprising administration of one or more additional anti-HIV therapeutic agents.

In a further embodiment, the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor, or combination thereof.

In another further embodiment, the other agent or agents are nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, HIV entry inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

In another embodiment, the therapeutically effective amount is from about 0.01 mg to about 5,000 mg per day.

In certain embodiments, the subject is a human.

Treatment of Diseases

The invention provides methods of treating or preventing a viral or retroviral infection comprising the administration of a compound of formula I to a subject infected with or susceptible to infection by a virus or retrovirus, such as HIV. Therapeutic methods of the invention can also include the step of identifying that the subject is in need of treatment of diseases or disorders described herein, e.g., identifying that the subject is in need of treatment for a viral infection. The identification can be in the judgment of a subject or a health professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or a diagnostic method). Tests for retroviral infection such as HIV infection are known in the art and include polymerase chain reaction-based (PCR-based) amplification and detection of viral RNA; Western blot detection of anti-HIV antibodies; agglutination assays for anti-HIV antibodies; ELISA-based detection of HIV- specific antigens; and line immunoassay (LIA). In each of these methods, a sample of biological material, such as blood, plasma, semen, or saliva, is obtained from the subject to be tested. Thus, the methods of the invention can include the step of obtaining a sample of biological material (such as a bodily fluid) from a subject; testing the sample to determine the presence or absence of retroviral infection such as HIV infection, HIV particles, or HIV nucleic acids; and determining whether the subject is in need of treatment according to the invention.

The methods delineated herein can further include the step of assessing or identifying the effectiveness of the treatment or prevention regimen in the subject by assessing the presence, absence, increase, or decrease of a marker, including a marker or diagnostic measure of a retroviral infection such as HIV infection, HIV replication, viral load, or expression of an HIV infection marker; preferably this assessment is made relative to a measurement made prior to beginning the therapy. Such assessment methodologies are known in the art and can be performed by commercial diagnostic or medical organizations, laboratories, clinics, hospitals and the like. As described above, the methods can further include the step of taking a sample from the subject and analyzing that sample. The sample can be a sampling of cells, genetic material, tissue, or fluid (e.g., blood, plasma, sputum, etc.) sample. The methods can further include the step of reporting the results of such analyzing to the subject or other health care professional. The method can further include additional steps wherein (such that) the subject is treated for the indicated disease or disease symptom.

Compounds of the invention may be administered singularly (i.e., sole therapeutic agent of a regime) to treat or prevent diseases and conditions such as viral infection as disclosed herein.

Compounds of the invention also may be administered as a "cocktail" formulation, i.e., coordinated administration of one or more compounds of the invention together with one or more other active therapeutics. For an antiviral therapy, one or more compounds of the invention including those of Formula I may be administered in coordination with a regime of one or more other antiviral agents such as reverse transcriptase inhibitors such as dideoxynucleosides, e.g. zidovudine (AZT), 2',3'-dideoxyinosine (ddl) and 2^l,3'-dideoxycytidine (ddC), lamivudine (3TC), stavudine (d4T), and TRIZIVIR (abacavir + zidovudine + lamivudine), nonnucleosides, e.g., efavirenz (DMP-266, DuPont Pharmaceuticals/Bristol Myers Squibb), nevirapine (Boehringer Ingleheim), and delaviridine (Pharmacia-Upjohn), TAT antagonists such as Ro 3-3335 and Ro 24-7429, protease inhibitors, e.g., indinavir (Merck), ritonavir (Abbott), saquinavir (Hoffmann LaRoche), nelfϊnavir (Agouron Pharmaceuticals), 141 W94 (Glaxo- Wellcome), atazanavir (Bristol Myers Squibb), amprenavir (GlaxoSmithKline), fosamprenavir (GlaxoSmithKline), tipranavir (Boehringer Ingleheim), KALETRA (lopinavir + ritonavir, Abbott), and other agents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), interferon, e.g., alpha-interferon, interleukin II, and phosphonoformate (Foscamet), or entry inhibitors, e.g., T20 (enfuvirtide, Roche/Trimeris) or UK-427,857 (maraviroc, Pfizer). Because many of these drugs are directed to different targets, e.g., viral integration, a synergistic may result with this combination.

In one embodiment, one or more compounds of the invention including those of the formulae herein are used in conjunction with one or more therapeutic agents useful for treatment or prevention of HIV, a symptom associated with HIV infection, or other disease or disease symptom such as a secondary infection or unusual tumor such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus-related lymphomas among others, that can result in HIV immuno-compromised subjects.

In certain embodiments of the invention, one or more compounds of the invention including those of Formula I are used in conjunction with a standard HIV antiviral treatment regimens. In another aspect, the treatment methods herein include administration of a so- called HIV-drug "cocktail" or combination therapy, wherein a combination of reverse transcriptase inhibitor(s) and HIV protease inhibitor(s) is co-administered.

For antiviral therapies, in a particular aspect, the compounds of the invention can be administered to HIV infected individuals or to individuals at high risk for HIV infection, for example, those having sexual relations with an HIV infected partner, intravenous drug users, etc.

Since the initial discovery of tat-TAR association as an important part of HIV replication, there have been numerous attempts to block formation of this protein-RNA complex with synthetic molecules. To date, very few of the currently available inhibitors have the correct features for further development into a drug. While research into new types of inhibitors for tat-TAR association is ongoing, it seems that many initial strategies have since been abandoned. Apparently, the lack of good drug candidates is part of a larger problem in which there exists a lack of molecular scaffolds designed for specific targeting of RNA.

The data presented herein provides a method to develop RNA-targeting molecules for TAR as well as other RNA structures. The important feature of the MBOs described herein is that they behave as multivalent binders of RNA that rely on a combination of sidechain and backbone to achieve their binding and specificity to TAR. The alanine scan indicates that there is not one sidechain that provides the main driving force for tat-TAR inhibition, but rather the combination of all sidechains is essential. The results show the delicate balance of charge that must be achieved to attain specific TAR binding in a cell-based system. While cationic sidechains can be incorporated into an MBO to gain increased activity in vitro, the cell-based model system shows very rapid loses in activity or specificity with modest increases in MBO charge. The best inhibitors maintained consistent activity across in vitro tests, model cell-based systems, and also displayed similar anti-HIV activity.

While the specificity of the MBOs from these studies does not approach the levels seen for small molecule drugs that target proteins, there is currently no well-defined level of specificity for targeting HIV RNA. Since HIV is a highly variable and heterogeneous disease, molecules that are too specific may also be ineffective. While the various TAR sequences appear in some HIV strains, there is variability in the sequence of the bulge from one clade to another. The fact that MBOs of the invention display antiviral activity against a number of different HIV clades indicates the MBOs retain their inhibitory activity despite the natural variance among different TAR sequences. At the same time, toxicity of these MBOs to PBMCs did not appear problematic. While specificity is an important feature to be engineered into RNA-binding molecules, complete specificity may not necessarily be required or desired when targeting an RNA in HIV. In particular, the propensity of HIV to mutate indicates that a highly specific TAR-binding molecule may become rapidly ineffective due to the development of resistance.

Pharmaceutical Compositions

Pharmaceutical compositions and dosage forms of the invention comprise one or more of the active ingredients disclosed herein. Pharmaceutical compositions and dosage forms of the invention typically also comprise one or more pharmaceutically acceptable excipients or diluents.

The term "pharmaceutically acceptable," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.

The invention also provides compositions comprising an effective amount of a composition containing a compound of the invention and an acceptable carrier. Preferably, a composition of this invention is formulated for pharmaceutical use ("a pharmaceutical composition"), wherein the carrier is a pharmaceutically acceptable carrier. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in amounts typically used in medicaments.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

Single unit dosage forms of the invention are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, intraarterial, intracutaneous, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.), by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention will typically vary depending on their use. For example, a dosage form used in the acute treatment of a disorder may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed by this invention will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

A composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. A nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A compound of this invention can also be administered in the form of suppositories for rectal administration.

Because of their ease of administration, tablets and capsules represent an advantageous oral dosage unit form, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

A sterile injectable composition, for example, a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

The carrier in the pharmaceutical composition must be "acceptable" in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the compounds of this invention, or one or more solubilizing agents, can be utilized as pharmaceutical excipients for delivery of the compounds of the invention. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a compound of this invention can range from about 0.001 mg/kg to about 1000 mg/kg, more preferably 0.01 mg/kg to about 100 mg/kg, more preferably 0.1 mg/kg to about 10 mg/kg; or any range in which the low end of the range is any amount between 0.001 mg/kg and 900 mg/kg and the upper end of the range is any amount between 0.1 mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg and 20 mg/kg). Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other agents.

A compound of the invention can, for example, be administered with a dosage ranging from about 0.5 to about 100 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Definitions

As used herein, the term "alkyl" refers to a straight-chained or branched hydrocarbon group containing 1 to 12 carbon atoms. The term "lower alkyl" refers to a C1-C6 alkyl chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-buty\, and n-pentyl. Alkyl groups may be optionally substituted with one or more substituents.

The term "alkenyl" refers to an unsaturated hydrocarbon chain that may be a straight chain or branched chain, containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may be optionally substituted with one or more substituents.

The term "alkynyl" refers to an unsaturated hydrocarbon chain that may be a straight chain or branched chain, containing the 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may be optionally substituted with one or more substituents.

The sp² or sp carbons of an alkenyl group and an alkynyl group, respectively, may optionally be the point of attachment of the alkenyl or alkynyl groups.

The term "alkoxy" refers to an -O-alkyl radical.

As used herein, the term "halogen," "halo," or "hal" means -F, -Cl, -Br or -I.

As used herein, the term "haloalkyl" means an alkyl group in which one or more (including all) of the hydrogen radicals are replaced by a halo group, wherein each halo group is independently selected from -F, -Cl, -Br, and -I.

The term "cycloalkyl" refers to a hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring system having at least one saturated ring, or hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring system having at least one non- aromatic ring, wherein the non-aromatic ring has some degree of unsaturation. Cycloalkyl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a cycloalkyl group may be substituted by a substituent. Representative examples of cycloalkyl group include cyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclohexenyl, bicyclo[2.2.1]hept-2-enyl, dihydronaphthalenyl, benzocyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, cyclooctatetraenyl, cyclononenyl, cyclononadienyl, cyclodecenyl, cyclodecadienyl and the like.

The term "aryl" refers to a hydrocarbon monocyclic, bicyclic or tricyclic aromatic ring system. Aryl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, 4, 5 or 6 atoms of each ring of an aryl group may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

As used herein, the term "aralkyl" means an aryl group that is attached to another group by a (Ci-C₆)alkylene group. Aralkyl groups may be optionally substituted, either on the aryl portion of the aralkyl group or on the alkylene portion of the aralkyl group, with one or more substituents. Representative aralkyl groups include benzyl, 2-phenyl-ethyl, naphth- 3-yl-methyl and the like.

The term "heteroaryl" refers to a 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon (with appropriate hydrogen atoms unless otherwise indicated), wherein at least one ring in the ring system is aromatic. Heteroaryl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a heteroaryl group may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, 1-oxo-pyridyl, furanyl, benzo[l,3]dioxolyl, benzo[l,4]dioxinyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl, and benzo(b)thienyl, 3H-thiazolo[2,3-c][l,2,4]thiadiazolyl, imidazo[l,2-d]-l,2,4-thiadiazolyl, imidazo[2,l-b]-l,3,4-thiadiazolyl, lH,2H-furo[3,4-d]- 1,2,3-thiadiazolyl, lH-pyrazolo[5,l-c]-l,2,4-triazolyl, pyrrolo[3,4-d]-l,2,3-triazolyl, cyclopentatriazolyl, 3H-pyrrolo[3,4-c]isoxazolyl, lH,3H-pyrrolo[l ,2-c]oxazolyl, pyrrolo[2,lb]oxazolyl, and the like.

As used herein, the term "heteroaralkyl" means a heteroaryl group that is attached to another group by a (Ci-C₆)alkylene. Heteroaralkyl groups may be optionally substituted, either on the heteroaryl portion of the heteroaralkyl group or on the alkylene portion of the heteroaralkyl group, with one or more substituent. Representative heteroaralkyl groupss include 2-(pyridin-4-yl)-propyl, 2-(thien-3-yl)-ethyl, imidazol-4-yl-methyl and the like.

The term "heterocycloalkyl" or "heterocyclic" refers to a nonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic, or 10-14 membered tricyclic ring system comprising 1- 3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, B, P and Si, preferably O, N, and S, wherein the nonaromatic ring system is completely saturated. The term "heterocycloalkyl" or "heterocyclic" also refers to nonaromatic 5-8 membered monocyclic, 7-12 membered bicyclic, or 10-14 membered tricyclic ring system comprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, B, P or Si, wherein the nonaromatic ring system has some degree of unsaturation. Bicyclic and tricyclic ring systems may be fused ring systems or spiro ring systems. Heterocycloalkyl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a heterocycloalkyl group may be substituted by a substituent. Representative heterocycloalkyl groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 4-piperidonyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane, 1,4- dioxa-8-aza-spiro[4.5]dec-8-yl, tetrahydrofuranyl, tetrahydrothienyl, thiirene, thiirenyl, thiadiazirinyl, dioxazolyl, 1,3-oxathiolyl, 1,3-dioxolyl, 1,3-dithiolyl, oxathiazinyl, dioxazinyl, dithiazinyl, oxadiazinyl, thiadiazinyl, oxazinyl, thiazinyl, l,4-oxathiin,l,4-dioxin, 1,4-dithiin, lH-pyranyl, oxathiepinyl, 5H-l,4-dioxepinyl, 5H- 1 ,4-dithiepinyl, 6H-isoxazolo[2,3-d] 1,2,4- oxadiazolyl, 7aH-oxazolo[3,2-d]l,2,4-oxadiazolyl, and the like.

The term "hydroxyalkyl" or "hydroxylalkyl" refers to an alkyl substituent which is further substituted with one or more hydroxyl groups.

As used herein the term "substituent" or "substituted" means that a hydrogen radical on a compound or group (such as, for example, alkyl, alkenyl, alkynyl, alkylene, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, or heterocycloalkyl group) is substituted or optionally substituted with any desired group that do not substantially adversely affect the stability of the compound. In one embodiment, desired substituents are those which do not adversely affect the activity of a compound. The term "substituted" refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of substituents include, but are not limited to, halogen (F, Cl, Br, or I), hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, alkylarylamino, cyano, nitro, mercapto, thio, imino, formyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfoamido, sulfonylalkyl, sulfonylaryl, alkyl, alkenyl, alkoxy, mercaptoalkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, wherein alkyl, alkenyl, alkyloxy, alkoxyalkyl, aryl, heteroaryl, cycloalkyl, are heterocycloalkyl are optionally substituted with alkyl, aryl, heteroaryl, halogen, hydroxyl, amino, mercapto, cyano, nitro, oxo (=0), thioxo (=S), imino (=NR), C(=N-NR)R, or C(=N-OR)R.

In other embodiments, substituents on any group (such as, for example, alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, and heterocycloalkyl) can be at any atom of that group, wherein any group that can be substituted (such as, for example, alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocycloalkyl) can be optionally substituted with one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of suitable substituents include, but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halogen, haloalkyl, cyano, nitro, alkoxy, aryloxy, hydroxyl, hydroxylalkyl, oxo (i.e., carbonyl), carboxyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, aryloxycarbonyl, heteroaryloxy, heteroaryloxycarbonyl, thio, mercapto, mercaptoalkyl, arylsulfonyl, amino, aminoalkyl, dialkylamino, alkylcarbonylamino, alkylaminocarbonyl, or alkoxycarbonylamino; alkylamino, arylamino, diarylamino, alkylcarbonyl, or arylamino-substituted aryl; arylalkylamino, aralkylaminocarbonyl, amido, alkylaminosulfonyl, arylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, imino, carbamido, carbamyl, thioureido, thiocyanato, sulfoamido, sulfonylalkyl, sulfonylaryl, or mercaptoalkoxy.

Additional suitable substituents for an alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, and heterocycloalkyl include, without limitation halogen, CN, NO₂, OR¹⁵, SR¹⁵, S(O)₂OR¹⁵, NR¹⁵R¹⁶, Ci-C₂ perfluoroalkyl, Ci-C₂ perfluoroalkoxy, 1,2- methylenedioxy, (=0), C=S), (=NR¹⁵), C(O)OR¹⁵, C(O)NR¹⁵R¹⁶, OC(O)NR¹⁵R¹⁶, NR¹⁵C(O)NR¹⁵R¹⁶, C(NR¹⁶)NR¹⁵R¹⁶, NR¹⁵C(NR¹⁶)NR¹⁵R¹⁶, S(O)₂NR¹⁵R¹⁶, R¹⁷, C(O)H, C(O)R¹⁷, NR¹⁵C(O)R¹⁷, Si(R¹⁵)₃, OSi(R¹⁵)₃, Si(OH)₂R¹⁵, B(OH)₂, P(O)(OR¹⁵)₂, S(O)R¹⁷, or S(O)₂R¹⁷. Each R¹⁵ is independently hydrogen, CpC₆ alkyl optionally substituted with cycloalkyl, aryl, heterocycloalkyl, or heteroaryl. Each R¹⁶ is independently hydrogen, C₃-C₆ cycloalkyl, aryl, heterocycloalkyl, heteroaryl, Ci-C₄ alkyl or Ci-C₄ alkyl substituted with C₃- C_O cycloalkyl, aryl, heterocycloalkyl or heteroaryl. Each R¹⁷ is independently C₃-C₆ cycloalkyl, aryl, heterocycloalkyl, heteroaryl, Ci-C₄ alkyl or Ci-C₄ alkyl substituted with C₃- Ce cycloalkyl, aryl, heterocycloalkyl or heteroaryl. Each C₃-C₆ cycloalkyl, aryl, heterocycloalkyl, heteroaryl and Ci-C₄ alkyl in each R¹⁵, R¹⁶ and R¹⁷ can optionally be substituted with halogen, CN, Ci-C₄ alkyl, OH, Ci-C₄ alkoxy, COOH, C(O)OCi-C₄ alkyl, NH₂, Ci-C₄ alkylamino, or Ci-C₄ dialkylamino.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups.

The compounds of this invention include the compounds themselves, as well as their salts, solvate, clathrate, hydrate, polymorph, or prodrugs, if applicable.

As used herein, the term "polymorph" means solid crystalline forms of a compound of the present invention or complex thereof. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability {e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g. , differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics {e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both {e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing. For example, one polymorph might be more likely to form solvates or might be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution of particles of it.

As used herein, the term "hydrate" means a compound of the present invention or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

As used herein, the term "clathrate" means a compound of the present invention or a salt thereof in the form of a crystal lattice that contains spaces {e.g., channels) that have a guest molecule {e.g. , a solvent or water) trapped within.

As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions {in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds of any one of the formulae disclosed herein that comprise -NO, -NO₂, -ONO, or - ONO₂ moieties. Prodrugs can typically be prepared using well-known methods, such as those described by 1 BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995) 172- 178, 949-982 (Manfred E. Wolff ed., 5^th ed).

The term "effective amount" is used throughout the specification to describe concentrations or amounts of compounds according to the present invention which may be used to produce a favorable change in the disease or condition treated, whether that change is a remission, a favorable physiological result, or the like, depending upon the disease or condition treated.

As used herein, the terms "prevent," "preventing," "prevention," and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The term "subject" is used throughout the specification to describe an animal, preferably a mammal, preferably a human, to whom treatment, including prophylactic treatment, with the compounds according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the term patient refers to a human patient.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "animal", "subject" and "patient", include, but are not limited to, a cow, monkey, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig and human (preferably, a human).

Examples The invention is further illustrated by the following examples which in no way should be construed as being further limiting. The compounds of the invention were synthesized according to the examples provided herein and according to any reaction schemes provided supra.

Example 1: Synthesis of Compounds General Methods

Proton nuclear magnetic resonances (¹H NMR) were recorded in deuterated solvents on a Gemini 300 (300 MHz) relative to tetramethylsilane (δ 0.00). Proton-decoupled carbon (¹³C-NMR) spectra were recorded on a Gemini 300 (75 MHz) and are reported in ppm using the solvent as an internal standard (CDC13, δ 77.23; DMSO, δ 39.52). Electrospray mass spectra (ESI-MS) were obtained using an Agilent 6100 series LC-MS. Nitrogen was bubbled through dimethylformamide (DMF) for 16 hours prior to use. All solution phase reactions were performed in oven dry glassware under a positive pressure of nitrogen. Silanization of glassware was performed using Sigmacote, in accordance with the manufacturer's instructions. All protected amino acids, Rink resin, and HOBt hydrate were purchased from Advanced ChemTech. HATU was purchased from Applied Biosystems. Kaiser test reagents, Fmoc-β-alanine, Fmoc-β-homoalanine, and Fmoc-β-phenylalanine were purchased from Fluka. All other chemicals were purchased from Sigma-Aldrich. All HPLC purification was done via reverse phase on an Agilent 1100 series semi-prep system with UV detection at 254 nm. A Vydac Cl 8 was utilized. The column was kept at room temperature. Solution A was 0.05% TFA in water and solution B was 0.05% TFA in acetonitrile. A typical elution was a gradient of 100% A to 100% B over 40 minutes at a flow rate of 5.0 mL/min.

Abbreviations

(Fmoc), N-(9-fluorenylmethoxycarbonyl); (DIEA), N-diisopropylethylamine; (HATU), 6>-(7-Azabenzotriazol-l-yl)-N_vΛ^_v/V'_:(N'-tetramethyluronium hexafluorophosphate); (HOBt), 1-hydroxybenzotriazole; (EDC), l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (Boc), f-butoxycarbonyl); (DMF), dimethylformamide; (THF), tetrahydrofuran; (DCM), dichloromethane; (ΝMP), N-methylpyrrolidinone; (TFA), trifloroacetic acid; (TES), triethylsilane

Example 2: Synthesis of Fmoc-Tyr(*Bu)-Weinreb Amide

Fmoc-Tyr-OH (5.0 g, 10.9 mmol) was dissolved in DCM with DIEA (1.9 mL, 10.9 mmol) and the reaction was allowed to cool to 0 ⁰C. Once cool, HOBt hydrate (1.98 g, 13.1 mmol) and EDC (2.5 g, 13.1 mmol) were added to the reaction, which was allowed to stir for 10 minutes at 0 ⁰C. N,0-dimethylamine hydrochloride (1.3 g, 13.1 mmol) and second portion of DIEA (2.3 mL, 13.1 mmol) were added to the flask. The mixture was allowed to stir for an hour at 0 ⁰C and then warmed to room temperture and allowed to stir overnight. Upon completion, the reaction was transferred to a separatory funnel with DCM and washed with 1 M HCl (3 x 40 mL), sat. NaHCO₃ (2 x 40 mL), and sat. NaCl (2 x 40 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo to yield 5.15 g (94%) of Weinreb amide as a white solid. ¹H-NMR (CDCl₃-J, 300 MHz): δ 7.73 (d, J = 7.41 Hz, 2H, Fmoc aromatic CH), 7.56 (t, J= 6.85 Hz, 2 H, Fmoc aromatic CH), 7.37 (t, J= 7.41 Hz, 2H, Fmoc aromatic CH), 7.28 (t, J= 7.41 Hz, 2H, Fmoc aromatic CH), 7.08 (d, J= 8.17 Hz, 2H, Tyr aromatic CH), 6.89 (d, J= 8.24 Hz, 2H, Tyr aromatic CH), 5.72 (d, J= 8.88 Hz, IH, carbamate NH), 4.99 (m, IH, CH), 4.29 (m, 2H, Fmoc CH₂), 4.16 (t, J= 7.12, IH, Fmoc CH), 3.60 (s, 3H, -OCH₃), 3.14 (s, 3H, -NCH₃), 3.00 (m, 2H, CH₂), 1.28 (s, 9H, C(CH₃)₃; ¹³C-NMR (CDCl₃-C?, 75 MHz): δ 172.2, 155.9, 154.3, 144.0, 143.9, 141.3, 131.4, 130.0, 127.8, 127.1, 125.3, 124.2, 120.0, 78.5, 67.1, 61.6, 52.2, 47.2, 38.3, 32.1, 29.1, 28.9; ESI-MS m/z = 503

Example 3: Synthesis of Fmoc-Tyrosine Aldehyde

Fmoc-Tyr-Weinreb amide (2.26 g, 4.5 mmol) was dissolved in dry THF and cooled to 0 ⁰C. Lithium aluminum hydride (212 mg, 5.6 mmol) was added slowly to the reaction. The mixture was allowed to stir for 1 hour at 0 ⁰C. The reaction was quenched with 0.1 M NaHSO₄ (2.6 g, 18.9mmol), which was added dropwise. The mixture was allowed to stir an additional 10 minutes at 0 ⁰C before being transferred to a separatory funnel with EtOAc and sat. NaCl. The aqueous layer was extracted with EtOAc and combined organic layers were washed with 1 M HCl (3 x 40 mL), sat. NaHCO₃ (2 x 40 mL), and sat. NaCl (2 x 40 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo to yield a yellow oil. Re-dissolving the product in ether and removing the solvent under vacuum produced 1.77 g (89%) of aldehyde as a yellow solid. The aldehyde was found to be stable for 1 month if stored at -20 ⁰C. ¹H-NMR (CDCl₃-*/, 300 MHz): δ 9.59 (s, IH, CHO), 7.75 (d, J = 7.47 Hz, 2H, Fmoc aromatic CH), 7.55 (d, J= 7.10, 2H, Fmoc aromatic CH), 7.39 (t, J = 7.44, 2H, Fmoc aromatic CH), 7.30 (t, J= 7.38 Hz, 2H Fmoc aromatic CH), 7.00 (d, J= 8.03 Hz, 2H, Tyr aromatic CH), 6.89 (d, J= 8.30 Hz, 2H, Tyr aromatic CH), 5.36 (d, J= 6.88 Hz, IH, carbamate NH), 4.42 (m, 3H, CH, Fmoc CH₂), 4.19 (t, J= 6.68, IH, Fmoc CH), 3.07 (d, J= 6.47, 2H, CH₂), 1.32 (s, 9H, C(CH₃)₃; ¹³C-NMR (CDCl₃-J, 75 MHz): δ 198.97, 155.94, 154.60, 143.78, 141.42, 127.85, 127.00, 125.10, 124.41, 124.09, 120.10, 119.92, 100.33, 67.03, 61.24, 47.28, 34.80, 28.92; ESI-MS m/z = 444

Example 4: Synthesis of Rink Amide Resin Linkers

Rink amide resin (1.0 g, 0.75 mmol) was swelled in a silanized filter vessel with DMF. The Fmoc protecting group was cleaved with 20% piperidine in DMF (5 min., DMF wash, 20 min.). The resin was washed with DMF (3x) and DCM (3x). A blue Kaiser test indicated the presence of a primary amine. Fmoc-amino acid 2.25 mmol) was dissolved in DMF and added to the resin. HATU (843 mg, 2.25 mmol), HOBt (340 mg, 2.25 mmol), and DIEA (888 μL, 5.1 mmol) were dissolved in DMF and added to the resin. The mixture was shaken for 1.5 hours. The reagents were drained and the resin was washed with MeOH, DMF, and DCM. A negative chloranil test indicated the absence of free amine. The resin was dried under vacuum and transferred to a silanized vial for storage.

Example 5: Synthesis of polyamine

Fmoc-β-homoalanine rink resin (500 mg, 0.375 mmol) was swelled in a silanized filter vessel with DMF. The Fmoc group was deprotected with 20% piperidine in DMF (3 x 5 min) and the resin was washed with DMF and DCM. This method was used for all subsequent Fmoc deprotections. A blue Kaiser test indicated a primary amine. Fmoc- Tyrosine aldehyde (832 mg, 1.875 mmol) was dissolved in DCM and added to the resin and shaken for 10 min. The aldehyde was drained and the resin washed with DCM. A mixture of NaBH(OAc)₃ (238 mg, 1.125 mmol) in DCM was added to the resin and shaken for 45 min. The solution was drained and the resin was washed with MeOH, DMF, and DCM. A positive chloranil test indicated the presence of a secondary amine. A solution of BoC₂O (818 mg, 3.75 mmol) and DIEA (261 μL, 1.5 mmol) in DCM was added to the resin and shaken for 2 hours. The solution was drained and the resin was washed with MeOH, DMF, and DCM. A negative chloranil test indicated the absence of any free amine. This procedure, starting with the Fmoc deprotection, was repeated two more times, followed by a final Fmoc deprotection. The resin was cleaved with 10% TFA in DCM for 10 min. The solution was evaporated under nitrogen and the product was re-dissolved in 95% TF A/1% TES in DCM and stirred for 30 min. to 1 hr. to deprotect the Boc groups. The solution was evaporated under nitrogen. Product was re-dissolved in a minimum amount of DCM and crashed out OfEt₂O. The mixture was centrifuged and the Et₂O layer was removed. The pellet was dissolved in H₂O and purified via HPLC.

Example 6: Synthesis of additional polyamines

The other polyamine oligomers of the invention were synthesized following the procedure similar to that described above for Example 5. The following examples are not limiting:

Polyamine 20:

Polyamine 1 :

Polyamine 2:

Polyamine 3 :

Polyamine 4:

Polyamine 8:

Polyamine 15:

Polyamine 17:

Table 1. Mass spectra results of polyamines polyamine Sequence of Polyamine Expected [M+H]⁺ Found [MH-H]⁺

20 β-homoalanine- YYY-NH₂ 550.3 550.3

1 β-alanine-YYY-NH₂ 536.3 536.3

2 β-alanine-YYYY-NH₂ 685.4 685.5

3 β-alanine-YYYYY-NH₂ 834.5 834.5

4 β-alanine-YYYYYY-NH₂ 983.6 983.5

8 β-alanine-YYYYAY-NH₂ 891.5 891.5

15 β-alanine-YYYYYYY-NH₂ 1 132.7 1 132.6

17 β-alanine-YYYYYYYY-NH₂ 1281.7 1282.7 Example 7: Synthesis of Polyamine-Fluorescein

The polyamine was synthesized using the described procedure (200 mg, 0.15 mmol). After the final Fmoc deprotection, the resin was washed with NMP. NHS-fluorescein (78 mg, 0.165 mmol) and DIEA (29 μL, 0.165 mmol) were added to the resin in NMP and shaken for 24 hours (peptide vessel was wrapped in foil to protect from light). The solution was drained and the resin was washed with NMP, DMF, and DCM. Cleavage and purification procedures proceeded as described for the unlabeled polyamine.

Example 8: HIV-I TAR RNA Binding Assay

Before used, HIV-I TAR RNA (5'-GGC AGA UCU GAG CCU GGG AGC UCU CUG CC -3', Thermo Scientific) in 10 μM batch in sterile water was heated to 95 ⁰C for 4.5 min, then cooled rapidly in ice bath for 5 min. This snap-cooling causes the RNA to adopt the kinetically favored hairpin rather than thermodynamically favored duplexes.

21

The relative affinity of each polyamine for HIV-I TAR RNA was determined using Fluorescence Resonance Transfer (FRET)-based competitive binding assay with fluorescein- labeled HIV-I TAT peptide 21 as described in the literature (Tor, Y. Angewandte Chemie- International Edition 1999, 38, 1579-1582). The fluorescence experiments were performed with a Spectra Max Fluorimeter (Molecular Devices) at 25°C, with excitation and emission wavelengths of 495 and 570 nm, respectively. All samples were prepared in 96 well plates in Ix TK buffer (50 mM Tris, 20 mM KCl, pH = 7.4) with 0.1% Trixton-100 (Sigma). The binding affinity (K_D) values reported for each polyamine are the averages of 3~5 individual measurements, and were determined by fitting the experimental data to a sigmoidal dose- response nonlinear regression model on GraphPad Prism 4.0. Prior to the competition experiments, the affinity of fluorescein-labeled peptide 21 for HIV-I TAR RNA was determined by monitoring fluorescence intensity changes of the fluorescent probe upon addition of HIV-I TAR RNA. Addition of an increasing concentration (0 nM to 1000 μM) of HIV-I TAR RNA to a 100 nM solution of fluorescein- labeled peptide 21 in TK buffer at 25 ⁰C afforded a saturation binding curve. The IC₅₀ value obtained from this binding curve was used with equation (1) to obtain the dissociation constant (K_DI) for the fluorescein-labeled peptide 21 and HIV-I TAR RNA complex (Batey, R. T.; Rambo, R. P.; Doudna, J. A. Angew Chem Int Ed Engl 1999, 38, 2326-2343):

^KD\ - τ, ^Lsτ κ^ι)

^BS

KDI: KD of fluorescein-labeled HIV-I TAT peptide 21;

K_D2: K_D of polyamine 20, 1, 2, 3, 4, 8, 15, 17;

R_T: Total concentration of HIV-I TAR RNA;

Lsf- Total concentration of fluorescein-labeled HIV-I TAT peptide 21;

F_SB- Fraction of bound fluorescein-labeled HIV-I TAT peptide 21;

LT: Total concentration of polyamine 20, 1, 2, 3, 4, 8, 15, 17.

Example 9: Competition FRET Assay

To a solution of 100 nM HIV-I TAR RNA and 100 nM fluorescein-labeled HIV-I TAT peptide 21, appropriate concentrations (0 nM to 500 μM) of the polyamine antagonists (20, 1, 2, 3, 4, 8, 15, 17) were added at 25 ⁰C; total volume of the incubation solution was 80 μL. After 60 min, fluorescence changes of the sample solution were determined by the Spectra Max Fluorimeter Detector. The experimental dose-response data for a given polyamine were fit to a sigmoidal dose-response nonlinear regression model on GraphPad Prism 4.0. The IC₅₀ value obtained from this analysis was used in equation (2) to calculate the Kp₂ value for the polyamine (Table 2). Table 2. Affinities of polyamine 1-8 for HIV-I TAR RNA polyamine Sequence of Polyamine IC₅₀ (μM) K_D (μM)

20 β-homoalanine- YYY-NH₂ 82 8 18 1

1 β-alanine-YYY-NH₂ 83 8 18 3

2 β-alanine-YYYY-NH₂ 8 90 1 94

3 β-alanine-YYYYY-NH₂ 2 96 0 64

4 β-alanine-YYYYYY-NH₂ 1 70 0 36

8 β-alanine-YYYYA Y-NH₂ ~ -

15 β-alanine- YYYYYYY-NH₂ 0 47 0 09

17 β-alanine-YYYYYYYY-NH₂ 0 18 0 03

Example 10: Biological Assays

Test Material Handling and Storage ^■

Compounds were solubilized in 100% DMSO and stored at -80 ⁰C until tested, unless alternative solvents and storage conditions are specified. Frozen stocks were thawed at room temperature, pre-warmed for 15 min at 37 ⁰C and vortexed prior to preparation of working solutions in tissue culture medium. During all stages of compound dilution and handling, compounds were protected from incidental light by opaque coverings and by storage and dilution in opaque or amber-colored tissue culture plastics. Additionally, incidental room and laminar flow tissue culture hood light exposure was controlled by reducing total fluorescent lighting in the laboratory by 50%. The final DMSO concentration was 0.25% at the highest test concentration, which had no effect on assay performance.

Cell-based assay for HIV-I Tat.

Inhibition of HIV-I Tat function was determined using a standard Tat/LTR-reporter gene system similar to that described by Jeeninga et al. (J. Virol. 74: 3740-3751, 2000). HeLa cells engineered to express HIV-I Tat and Firefly Luciferase on a single bicistronic mRNA from a tetracycline-controllable promoter were transfected with an HIV-I LTR-Renilla Luciferase reporter construct to generate a stable cell line (DLTat cells) in which Renilla Luciferase expression was dependent upon Tat function. In contrast, Firefly Luciferase expression in DLTat cells was independent of Tat function and was used to assay for non-specific or toxic compounds. Using this system, compounds that inhibit Tat function were identified by their ability to reduce the expression of Renilla Luciferase with no effect on the expression of Firefly Luciferase. Assays were performed in 96 well format by plating DLTat cells (2xlO⁴/well) in the presence of test compound (triplicate wells) and incubating at 37 ⁰C for 48 hours. Luciferase expression levels were subsequently determined using Dual- Luciferase assay reagents (Promega) following the manufacturer's instructions. Temacrazine, an inhibitor of HIV-I LTR transcription initiation, and Ro5-3335, a reported HIV-I Tat inhibitor, was used as positive control compounds for the Tat assay. Although both of these compounds exhibited some non-specific effects in the assay, they were currently the best available controls for this assay.

Cell-based assay for HIV-I Rev

Inhibition of HIV-I Rev function was determined using the pDM128 Rev reporter plasmid previously described by Hope et al. (Proc. Natl. Acad. Sci. USA 87:7787-7791, 1990). The pDM128 plasmid was modified by replacing the chloramphenicol acetyltransferase coding sequence with that of Renilla Luciferase. HeLa cells engineered to express HIV-I Rev and Firefly Luciferase on a single bicistronic mRNA from a tetracycline- controllable promoter were subsequently transfected with the modified pDM128 plasmid to generate a stable cell line (DLRev cells) in which Renilla Luciferase expression was dependent upon Rev function. In contrast, Firefly Luciferase expression in DLRev cells was independent of Rev function and was used to assay for non-specific or toxic compounds. Using this system, compounds that inhibited Rev function were identified by their ability to reduce the expression of Renilla Luciferase with no effect on the expression of Firefly Luciferase. Assays were performed in 96 well format by plating DLRev cells (2xlO⁴/well) in the presence of test compound (triplicate wells) and incubating at 37 ⁰C for 24 hours. Luciferase expression levels were subsequently determined using Dual-Luciferase assay reagents (Promega) following the manufacturer's instructions. Leptomycin B, an inhibitor of hCRMl mediated Rev nuclear export, was used as a positive control compound for the Rev assay. Although quite toxic (therapeutic index in the range of 1.5-5), this compound was currently the best available control for this assay. Efficacy Evaluation in Human Peripheral Blood Mononuclear Cells (PBMCs) a. Materials

Fresh human blood was obtained commercially from Biological Specialty Corporation (Colmar, PA). The virus isolate HIV-l_Ba-_L (Subtype B, R5-tropic, laboratory adapted strain) was obtained from the NIAID AIDS Research and Reference Reagent Program. Pre-titered aliquots of virus were removed from the freezer (LN₂ or -80 °C) and thawed rapidly to room temperature in a biological safety cabinet immediately before use. Phytohemagglutinin (PHA) was obtained from Sigma (St. Louis, MO; catalog # Ll 668) and recombinant IL-2 was obtained from R&D Systems Inc. (Minneapolis, MN; catalog # 202IL).

b. Anti-HIV Efficacy Evaluation in Fresh Human PBMCs

Fresh human PBMCs were isolated from screened donors, seronegative for HIV and HBV. 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, 50 LVmL penicillin, 50 μg/mL streptomycin, and 2 μg/mL PHA. 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. 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 μL/well (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 was used to minimize the variability observed between individual donors, which resulted from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations. Each plate contained 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 μL of each concentration was placed in appropriate wells using the standard format. 50 μL 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 ⁰C, 5% CO₂. 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. AZT was included as a positive antiviral control.

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 dH₂O:Ethanol at 1 mCi/ml. Poly rA:oligo dT template: primer (Pharmacia) was prepared as a stock solution by combining 150 μl poly rA (20 mg/ml) with 0.5 ml oligo dT (20 units/ml) and 5.35 ml sterile dH₂O followed by aliquoting (1.0 ml) and storage at -20 ⁰C. The RT reaction buffer was prepared fresh on a daily basis and consisted of 125 μl 1.0 M EGTA, 125 μl dH₂O, 125 μl 20% Triton XlOO, 50 μl 1.0 M Tris (pH 7.4), 50 μl 1.0 M DTT, and 40 μl 1.0 M MgCl₂. The final reaction mixture was prepared by combining 1 part ³H-TTP, 4 parts dH₂O, 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 μl of virus-containing supernatant was added and mixed. The plate was incubated at 37 ⁰C 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 was 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 was a stable, single solution that does not require preparation before use. At termination of the assay, 20-25 μL of MTS reagent was added per well and the microtiter plates were then incubated for 4-6 hrs at 37 ⁰C, 5% CO₂ 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.

Example 11 : Amino acid-based polyamines

General Procedure A: Weinreb Amide Synthesis

An Fmoc-protected amino acid (10 mmol) and DIEA (1.6 mL) were dissolved in dry CH₂Cl₂ (50 mL) and cooled to 0 ⁰C. EDC (2.4 g, 12 mmol) and HOBt (1.8 g, 12 mmol) were added to the reaction. The reaction mixture continued to stir at 0 ⁰C for 10 minutes to pre-activate the carboxylic acid. After 10 minutes, N,O-dimethylhydroxylamine hydrochloride (1.2 g, 12 mmol) and DIEA (2.0 mL) were added to the reaction mixture. The reaction was allowed to slowly warm to room temperature and was stirred for a total of 16 hours. The reaction was then transferred to a separatory funnel with CH₂Cl₂ (100 mL) and washed with 2 M HCl (3x- 50 mL), saturated aqueous NaHCO₃ (2x - 50 mL), and brine (2x - 50 mL). The organic layer was dried over sodium sulfate and solvent was removed under reduced pressure to yield pure Weinreb amides as solid white foams.

General Procedure B: Reduction of Weinreb Amides to α-amino Aldehydes Dry THF (50 mL) and Weinreb amide (5 mmol) were added to an oven dried 250 mL round bottom flask, placed under N₂, and cooled to 0 ⁰C in an ice bath. Lithium aluminum hydride (250 mg, 6.25 mmol) was slowly added to the solution over a period of approximately 30 seconds. The reaction was stirred vigorously at 0 ⁰C for 60 minutes and then slowly quenched with a IM solution Of NaHSO₄ (50 mL). The biphasic mixture continued to stir at 0 ⁰C for 10 minutes at which point it was transferred to a separatory funnel using EtOAc (50 mL) and brine (50 mL). The aqueous layer was extracted with EtOAc (75 mL) and the combined organics were washed with 1.5 M HCl (2x - 50 mL), saturated aqueous NaHCO₃ (2x - 50 mL), brine (2x - 50 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting amino aldehydes were taken forward crude.

General Procedure C: Reductive Animation to form the PAA Backbone

An Fmoc-protected α-amino aldehyde (4.5 mmol) was dissolved in dry CH₂Cl₂ (30 mL) at room temperature. To the stirring solution was added a benzyl protected amino acid hydrochloride (4.9 mmol) and DIEA (0.85 mL). NaBH(OAc)₃ was immediately added to the reaction mixture and stirred vigorously for 75 minutes. The reaction was quenched with a mixture of saturated aqueous K₂CO₃ (10 mL) and saturated aqueous NaHCO₃ (30 mL) and allowed to stir for an additional 10 minutes. The biphasic mixture was transferred to a separatory funnel and extracted with CH₂Cl₂ (3x - 30 mL). The organic layers were combined and dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified using a biotage flash chromatography system (40+M column, 60:40 Hexanes: EtOAc).

General Procedure D: Boc Protection of Secondary Amine in the PAA Backbone

The PAA secondary amine backbone (2.0 mmol) was dissolved in dry CH₂Cl₂ (40 mL) and stirred at room temperature. To the solution was added di-tert-butyl dicarbonate (0.9 g, 4 mmol) and DIEA (0.6 mL, 2 mmol). The reaction was allowed to stir for 48 hours and was then transferred to a separatory funnel with CH₂Cl₂ (50 mL). The reaction mixture was washed with 1 M HCl (2x - 50 mL), saturated aqueous NaHCO₃ (2x - 50 mL), and brine (2x - 50 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified using a biotage flash chromatography system (40+M column, 80:20 Hexanes: EtOAc). General Procedure E: Hydrogenolysis of Benzyl Ester to Yield PAA Monomers

A Parr flask was purged with N₂ and then charged with 10% palladium on carbon. The palladium catalyst was wetted with a minimum amount of methanol (~5 mL) while under an N₂ atmosphere. The PAA-monomer ester (1.5 mmol) was dissolved in a minimal amount of methanol (typically -20 mL) and added to the Parr flask. The flask was placed under an H₂ atmosphere (40 psi) and shaken on a Parr shaker for 2 hours. The reaction mixture was then filtered through a bed of celite to remove the palladium from the mixture. The resulting solution was concentrated under reduced pressure to yield an off-white solid as the crude product. The crude product was purified using a biotage flash chromatography system (40+M column, 0%-5% MeOH gradient in CH₂Cl₂).

Screen for TAR RNA Binding to PAA Library Members

Several beads from each well of the PAA library were transferred to a 384-well filter plate, keeping their spatial separation and orientation intact. The beads were first washed with water (5x - 50 μL), then Ix TK buffer (50 mM Tris, 20 mM KCl, 0.1% Triton X-100, pH 7.4; 4x - 50 μL). To each well, BSA (0.1 mg/mL) was added in Ix TK buffer (20 μL) and agitated with mechanical shaking for 60 minutes at room temperature. The microplate was drained under vacuum and washed with Ix TK buffer (3x - 50 μL). Following BSA blocking, bulge mutant TAR in Ix TK buffer (2.5 μM, 20 μL/well) was added to each individual well. The library was incubated with bulge mutant TAR for 24 hours at 4 ⁰C before being drained under vacuum. Immediately following solvent removal, a mixture of bulge mutant TAR (2.5 μM) and 5'-biotin labeled TAR (Dharmacon, 250 nM) in Ix TK buffer (20 μL/ well) were introduced to the library. (Note: RNA was snap-cooled by heating at 95 ⁰C for 5 minutes followed by an immediate transfer to dry ice for 5 minutes to promote hairpin formation) The library was incubated with this solution for 2.5 days at 4 ⁰C, drained, and washed with water. To each well a solution of Qdot605 (50 nM, 15 μL/well) in Ix TK buffer was added and agitated at room temperature for 3 hours. The solution was drained and each well was washed with Ix TK (3x - 50 μL), followed by a 2 hour wash with Ix TK buffer and drainage under vacuum. The library was then visualized using a fluorescent microscope equipped with a triple bandpass filter. Beads that appeared red or orange under the microscope were selected for further characterization while those that were green were disregarded.

Results and Discussions Synthesis of Polyamines. Polyamine monomers were synthesized starting from commercially available Fmoc protected amino acids utilizing a solution-phase reductive amination strategy. Initially we sought to synthesize five polyamine monomers starting from orthogonally protected serine, tryptophan, tyrosine, 4-amino-phenylalanine, and phenylalanine (Scheme 1). The monomers were selected based on their likelihood to interact with folded RNA, thus incorporating amino acids capable of π-stacking, hydrophobic burial, and hydrogen bonding. The Fmoc protected amino acid was first converted to the Weinreb amide intermediate (1) in high yield under EDC mediated amide bond forming conditions. Subsequently, intermediate 1 was reduced to the corresponding aldehyde intermediate (2) using lithium aluminum hydride. The aldehyde product was taken forward crude to the key reductive amination step where intermediate 2 was condensed with the hydrochloride salt of benzyl glycinate. The resulting imine was reduced using sodium triacetoxy borohydride to install the secondary amine in the backbone, yielding intermediate 3. Following chromatographic purification, the secondary amine was Boc protected in high yield to afford the PAA monomer ester. Hydrogenolysis of the benzyl ester yielded the PAA monomer intermediate (5).

Scheme 8. Synthesis of PAA monomers

n H

From these five monomers, a library of 125 polyamine trimers was synthesized on solid support. The synthesis was designed to be compatible with the development of an on- bead screen for RNA binding. In order to create a library suited for aqueous screening conditions, Tentagel-NH₂ resin was chosen as a synthetic platform due to its unique ability to swell in both aqueous and organic solvents. In addition, the library synthesis was performed in 96-well filter plates to provide a physical separation between distinct polyamines. The strategy outlined provided an accessible synthetic platform that negated the need for molecular deconvolution during the screening process. The synthesis began by functionalizing the resin with an Fmoc-β-alanine spacer. The spacer was deprotected and PAA trimers were synthesized through HATU-mediated solid phase peptide synthesis (Supporting Information). Upon completion of the trimers, a global deprotection of the backbone and sidechain protecting groups afforded a 125-member library of resin-bound polyamine trimers.

On Bead Screen for TAR Binding. Initially we aimed to develop a simple fluorescent screening procedure for assessing TAR binding to our resin-bound polyamines. To this end, we adapted protocols developed by the Rana and Kodadek laboratories to our system. The polyamine containing beads were first incubated with a solution of BSA to block any nonspecific binding to the bead surface. Subsequently, the library was incubated with a bulge mutant TAR construct containing a single base bulge, rather than the wild-type trinucleotide bulge. The bulge mutant acted as a competitive inhibitor to exclude compounds that were not specific for the bulge region of TAR. Next, 5'-biotinylated wild-type TAR was added to the resin in the presence of an excess of bulge mutant TAR. Binding events were visualized using a fluorescent microscope equipped with a triple bandpass filter after the addition of streptavidin-coated quantum dots (Qdot605). This method allowed for the visualization of positive beads as red in color where beads containing non-binding polyamines were seen as green under the microscope (Figure 15). From this initial screen the six brightest beads were selected, as determined by visual inspection, and re-synthesized on a larger scale to allow determination of binding constants, SFF, YFF, FFF, SYS, YSF, and FYY.

Terbium footprinting experiments. Previous work showed that RNA footprinting studies using terbium (III) ions as an RNA cleavage agent to be a reliable method for quantification of polyamine binding to TAR RNA (Krebs, A.; Ludwig, V.; Boden, O.; Gobel, M. W. Chembiochem 2003, 4, 972-8; Hwang, S.; Tamilarasu, N.; Ryan, K.; Huq, I.; Richter, S.; Still, W. C; Rana, T. M. Proc Natl Acad Sci USA 1999, 96, 12997-3002). The six selected polyamines were synthesized, purified by reversed phase HPLC, and quantified by UV absorbance. The polyamines were then titrated into buffered solutions containing TAR up to 1 niM concentrations and effects on RNA cleavage patterns were assessed as a function of polyamine concentration via denaturing gel electrophoresis. The results of these experiments identified the sequence XFF as a binding motif specific for the bulge region of TAR. Three of the six ligands selected showed appreciable binding affinity for the bulge region, with SFF, YFF, and FFF exhibiting binding constants in the low micromolar range (Figure 16). The best ligand derived from our initial screen was SFF, exhibiting a KD of 14 μM for the bulge region.

Alanine Scan. We next sought to probe the importance of sidechain interactions in an effort to define a minimal binding motif for the bulge region. The most direct way for us to probe this question was to substitute each sidechain in our best ligand (SFF) with a moiety deemed to be unlikely to interact with the RNA. In analogy to alanine scans in peptides, we synthesized the alanine-derived PAA monomer and iteratively replaced each monomer in the SFF polyamine with that bearing a methyl sidechain. The three polyamines were synthesized and subjected to terbium footprinting assays. We found that all three sidechain replacements yielded compounds with very low affinity for the TAR target, thus leading us to conclude that all three sidechains were critical to maintain binding affinity.

Secondary Library Synthesis and Screen. The polyamine scaffold was intentionally designed such that modifications to the backbone could be easily installed in order to determine structural activity relationships (SARs). We posited that modification to the backbone of the SFF core motif could lead to enhanced binding affinity for TAR. In this vein, we aimed to synthesize a library of compounds based on the SFF core but bearing sidechains in place of the glycine subunit of the PAA monomers. Six new monomers were synthesized, where the key step involved condensation between serine or phenylalanine aldehydes and the hydrochloride salts of either tryptophan, tyrosine, or lysine. The new monomers and the original serine and phenylalanine monomers were incorporated into a 64-member polyamine library, where all library members contained the previously identified SFF core. The library was again subjected to the Qdot based screen and the six brightest hits were chosen for further characterization. Unfortunately, all polyamines derived from the second screen showed either very weak affinity for TAR or no affinity at all in the footprinting assays. The synthesis of different types of secondary library monomers is found below.

^✓'"v^,

R= Ser or Phe R¹ = Lys, Trp, Tyr

Example 12: Trimer and cell-permeability

The initial point of development of MBO inhibitors of tat-TAR association was the molecule YYY, which is derived from tyrosines and binds to the bulge of TAR with moderate affinity (K_d ~ 5μM). Previously, we had developed the chemistry to make such molecules and provided initial characterization of the binding properties to TAR. Before developing more potent derivatives, the ability of YYY to enter cells was investigated. Therefore, a fluorescein-labeled version of this molecule was made (19: YYY-Fl), and cell- uptake studies were performed using HeLa cells. Fluorescence imaging showed reasonable cellular penetration, with concentration of the molecule in the nucleus and nucleolus (Figure 31). Based on these encouraging results, we felt that other MBO derivatives would similarly be able to permeate cells.

Example 13: In vitro Inhibition Assays and Optimization

Binding between TAR and tat is an important part of HIV replication. To be an effective antiviral agent, an inhibitor must be able to prevent this association in vitro before additional cell-based studies can be performed. Using an established competition assay, a series of MBO derivatives were investigated for their ability to inhibit association between TAR and a fluorescently-labeled peptide derived from tat (Figure 32). Each inhibition curve was fit to a single-site binding model to provide an IC₅₀ value. This system was used as the principle method to quickly evaluate an MBO' s inhibitory.

As shown in the following table, the length of an MBO is a significant component of inhibitory activity. For instance, the IC₅₀ improves by two orders of magnitude as the length of the polymer chain increases. Next, the importance of sidechains for inhibition of tat binding were investigated using a series of MBO hexamers. Systematic replacement of a tyrosine sidechain with alanine shows only modest decreases in activity, and there is no change in activity when this modification is introduced at positions 2 and 4 within the sequence. A derivative that consists of one tyrosine and five alanine sidechains displays considerably weaker activity compared to hexamers composed mostly of tyrosines. To test whether MBOs can selectively inhibit tat-TAR over another protein-RNA complex, an established competition assay that monitors rev-RRE binding was used. RRE is an HIV- derived RNA that has a hairpin-loop structure with an internal bulge that is the biding site for the rev protein. Using a fluorescence-based competition assay, the MBOs YYYYYY and YYYAYY displayed no inhibition of rev binding to RRE up to an MBO concentration of 20 μM. Therefore, these two MBOs are at least 20 times more selective for inhibition of tat- TAR over rev-RRE.

Length Variation Alanine Scan

MBO IC₅₀ (μM) MBO IC₅₀ (μM)

Sequence in vitro Sequence in vitro

YYY 83.8 ± 3.7 YAYYYY 1.9 ± 0.3

YYYY 8.9 ± 1.0 YYAYYY 3.2 ± 0.4

YYYYY 3.0 ± 0.7 YYYAYY 1.6 ± 0.3

YYYYYY 1.7 ± 0.4 YYYYAY 3.9 ± 0.5

YYYYYYY 0.5 ± 0.2 YYYYYA 3.3 ± 0.5

YYYYYYYY 0.2 ± 0.1 YAAAAA 14 ± 2

IC₅₀ values for inhibition of tat-peptide binding to TAR in vitro. One letter amino acid codes are used to represent the sidechain.

Other amino-acid derived sidechains were incorporated into hexameric MBOs to determine the effects on inhibition of tat-TAR binding. Lysine and tryptophan have commonly been used in peptides to improve RNA binding by increasing the amount of cationic charge or pi-stacking between the peptide and the RNA. Introducing one lysine sidechain in position 4 (YYYKYY) improved the activity 3 times compared to the analog made entirely from tyrosine, but incorporation of an additional lysine or a tryptophan sidechain did not further benefit the activity. Since position 4 was amenable to sidechain variation in the MBO hexamer, the same position was changed to an alanine sidechain in the corresponding heptamer and octamer (YYYAYYY and YYYAYYYY). Similarly, no detectable change in inhibition of tat-TAR association was observed.

Sidechain Variation

MBO IC₅₀ (μM) MBO IC₅₀ (μM)

Sequence in vitro Sequence in vitro

YYYKYY 0.5 ± 0.1 YKYKWY 0.2 ± 0.1

YYYKWY 0.3 ± 0.1 YYYAYYY 0.5 ± 0.1

YKYKYY 0.4 ± 0.1 YYYAYYYY 0.20 ± 0.02 IC₅₀ values for inhibition of tat-peptide binding to TAR in vitro. One letter amino acid codes are used to represent the sidechain.

The MBOs of the current study all contain a polyamine backbone that may be protonated under the conditions of the binding assays and could cause non-specific RNA aggregation similar to other types of highly charged molecules. Using gel electrophoresis, the point at which several MBOs induce TAR to aggregate was determined. Studies with ³²P- labeled TAR (residues 17-45) showed MBO-induced aggregation of the RNA at ECso's of 40 μM for YYYYYY, 224 μM for YYYYY, and 451 μM for YYYY). Since the IC₅₀ values reported in the above tables are significantly lower than the ECso's where the MBOs induce TAR aggregation, the fluorescence-based competition assays correctly report specific inhibition of the tat-TAR complex by MBOs.

Additional methods were employed to further characterize the association of the most active MBOs with TAR. Gel shift studies have been used to investigate the ability of TAR- binding molecules to inhibit binding of tat. While the aggregation of RNA was readily observed by gel electrophoresis (as described above), a gel shift of the tat-TAR complex in the presence of an MBO was not observable under a number of different conditions. The lack of a gel shift does not negate the previously observed inhibition results, but it did drive further investigation into the binding mode between MBOs and TAR. Multidimensional NMR techniques have been previously used to examine the structure of TAR in both ligand- bound and free states. Therefore, following the changes in chemical shifts of TAR as YYYAYY was titrated into solution provided an indication of how the MBO interacts with the RNA. The results from this study showed specific chemical shift changes in the bulge and loop area of the RNA, confirming that this MBO specifically targets the tat-binding area of TAR.

Example 14: Characterization of tat-TAR inhibition in a cell-based model

An established method was used to investigate whether the in vitro inhibition of tat- TAR formation by MBOs could be similarly observed in a cell-based assay. The assay directly probed for inhibition of tat-TAR relative to non-specific binding, and the results guided which MBOs would be suitable for antiviral tests. In this assay, HeLa cells were modified such that a fused tat-firefly luciferase gene and a fused TAR-renilla luciferase gene were both integrated into a HeLa cell's DNA. The two different luciferase proteins generated luminescence at different wavelengths, and comparison of these differences indicated a molecule's specificity for tat-TAR inhibition. For instance, dose-dependent decreases in luminescence from renilla luciferase indicated that inhibition of tat-TAR binding occured, while decreases in luminescence from firefly luciferase indicated that expression of the tat protein was affected (presumably due to non-specific binding). The ideal inhibitor should decrease luminescence from renilla luciferase without affecting firefly luciferase. In these experiments, the IC₅₀ was derived from the dose-dependent decrease in renilla luciferase while the TC₅₀ comes from changes in firefly luciferase. An analogous system has also been established to monitor rev-RRE interactions. Performing the same experiments with the rev- RRE system provided an additional measure of specificity for inhibition of tat-TAR over rev- RRE.

Results from the tests of several MBOs on the HeLa-derived model cell systems are shown in the table below and in the figures. The results demonstrate that the activity of the MBOs is ideal at the hexamer and heptamer lengths. In particular, YYYYYY and YYYAYY have IC₅o's against tat function in the low micromolar range with TCso's above 100 μM in the same assay. In the tests for rev function, YYYYYY showed activity at 34 μM while YYYAYY did not display any activity up to 100 μM. Therefore, YYYAYY appears to be highly selective. It is interesting to note that MBO hexamer variants with additional lysine sidechains or a tryptophan sidechain actually lost potency or selectivity despite observed improvements in the in vitro assay previously mentioned. For instance, YYYKYY is about 3 times more effective at inhibiting tat-TAR binding than YYYYYY according to the in vitro fluorescence competition assay, but it's ICs₀ value is twice as weak in the tat function assay. Incorporation of an additional lysine (as in YKYKYY) eliminated specificity for inhibition of tat-TAR binding compared to non-specific inhibition (IC₅₀ and TC₅₀ values were about the same). The heptamer YYYAYY also has good activity, but it was less specific for tat-TAR inhibition over rev-RRE. An octamer displayed considerable non-specific activity in the tat function assay.

MBO tat function rev function

Sequence IC₅₀ (μM) TC₅₀ (μM) IC₅₀ (μM) TC₅₀ (μM)

YYYY 19 >100 >100 >100

YYYYY 30 >100 >100 >100

YYYYYY 7 >100 34 >100

YYYAYY 6 >100 >100 >100

YYYKYY 12 >100 >100 >100

YKYKYY 35 38 - -

YKYKWY 10 12 - -

YYYAYYY 16 >100 60 >100

YYYAYYYY O.J 5 2.4 - -

Activities of MBOs against tat and rev function in HeLa model cell systems.

Example 15: Antiviral Activity

Based on the results of the model cell system, three MBOs (two hexamers and a heptamer) were advanced into tests for antiviral activity against HIV-I infection of peripheral blood mononuclear cells (PBMCs, collections of white blood cells that contain the CD4 cells that HIV infects). Initial tests used a laboratory-adapted strain of HIV (Ba-L), and all three MBOs showed activity in the low-micromolar range. Based on these results, the study was expanded to include tests against clinical HIV isolates (Clades A-O). All the MBOs displayed some activity against each HIV clade that was tested. While some variation is present between clades, the variance is typical due to the heterogeneous nature of HIV. Toxicity levels of PBMCs to the MBOs were all significantly higher than the concentrations at which the molecules show activity against HIV.

MBO HIV-1 A B C D E F G O

Sequence Ba-L UG/92/029 HT/92/599 97ZA003 UG/92/001 CMU06 BR/93/020 JV1083 BCF02

YYYYYY 2 6.7 8.8 27 6.0 19.0 4.7 13.1 18.9

YYYAYY 5 4.5 12.7 32 5.1 7.7 7.5 17.8 20.1

YYYAYYY 4 2.3 4.5 25 2.3 18.2 5.1 11.9 16.3

Anti-HIV activity of MBOs against infection in PBMC (All values are ICso's in μM). For Ba-L, the TC₅₀ is 76, 56, 55 μM, respectively. For Clades A-O, the TC_5O is > 100 μM for the hexamer MBOs and 75 μM for the heptamer MBO.

With antiviral activity, there is always a concern that the molecules inhibit the virus by preventing entry. To test for viral entry inhibition, a TZMBL assay was performed and showed that there is no viral entry inhibition of the three MBOs in the above table up to concentrations of 300 μM. Incorporation by Reference

All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, technical data sheets, internet web sites, databases, patents, patent applications, and patent publications.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A compound represented by formula I:

R₃, R₄, R₅ and R₆, are each independently, H, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(O)R_x, -C(S)R_x, -C(NR)R_x, -S(O)R_x, or -S(O)₂R_x; or R₃ and R₄ taken together, or R₅ and R* taken together, with the N to which they are attached is an optionally substituted heterocycloalkyl, or an optionally substituted heteroaryl; m is O, I, or 2; and n is an integer from 0-20.

2. The compound of claim 1 , of formula II:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

3. The compound of claim 2, wherein each R and R' are independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1, 2, or 3 heteroatoms consisting of O, N, and S.

4. The compound of claim 2, wherein each Ri and R₂ are independently selected from H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, or each Ri and R₂ together form =O.

5. The compound of claim 2, wherein R₃ and R₅ are each independently, H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(O)R_x, -C(S)R_x, -C(NR)R_x, -S(O)R_x, or -S(O)₂R_x.

6. The compound of claim 2, wherein m is 0 or 1.

7. The compound of claim 2, wherein n is 4-10.

8. The compound of claim 2, of formula III:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

9. The compound of claim 8, wherein each Rj and R₂ are independently selected from H, an optionally substituted alkyl, or each Ri and R₂ together form =O.

10. The compound of claim 9, wherein each Ri and R₂ are independently H or each R) and R₂ together form =0.

11. The compound of claim 10, wherein each R and R'are independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1, 2, or 3 heteroatoms consisting of O, N, and S.

12. The compound of claim 11, wherein each R and R' are independently selected from methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, pentyl, i-pentyl, neo-pentyl, hexyl, heptyl, benzyl, phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, pyridyl, furanyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, indazolyl, imidazopyridyl, quinazolinyl, or purinyl, each of which may be optionally substituted; or a groups selected from the following: CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

13. The compound of claim 12, wherein each R is independently selected from the following:

14. The compound of claim 8, wherein n is 4-10.

15. The compound of claim 2, of formula IV:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

16. The compound of claim 15, wherein each R and R' are independently selected from a natural or unnatural amino acid side chain.

17. The compound of claim 16, wherein each R and R' are is independently selected from methyl, ethyl, propyl, i-propyl, CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

18. The compound of claim 17, wherein each R is independently selected from the following:

19. The compound of claim 18, wherein each R is

20. The compound of claim 19, wherein n is 4-10.

21. A compound of claim 1, selected from the following:

^■

(4) YYYYYY:

(5) YAYYYY:

(13) YKYKWY:

22. The compound of claim 2, of formula V:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

23. The compound of claim 22, wherein each Ri and R₂ are independently H or each Ri and R₂ together form =0.

24. The compound of claim 23, wherein each R is independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1 , 2, or 3 heteroatoms consisting of O, N, and S.

25. The compound of claim 24, wherein each R is independently selected from H, methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, pentyl, i-pentyl, neo-pentyl, hexyl, heptyl, benzyl, phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, pyridyl, furanyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, indazolyl, imidazopyridyl, quinazolinyl, or purinyl, each of which may be optionally substituted; or a groups selected from the following: CH₂CONH₂; CH₂CH₂CONH₂; CH₂OH; CH(OH)CH₃; CH₂SH; CH₂CH₂SCH₃; CH₂CO₂H; CH₂CH₂CO₂H; CH₂CH₂CH₂CH₂NH₂;

26. The compound of claim 22, wherein n is 4-10.

27. The compound of claim 22 of formula VI:

as well as pharmaceutically acceptable salts, esters and prodrugs thereof.

28. The compound of claim 27, wherein each R is independently selected from H, an optionally substituted alkyl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, an optionally substituted heterocyclic, an optionally substituted hydroxyalkyl, an optionally substituted thiolalkyl, or a natural or unnatural amino acid side chain; each of which may be substituted by 0, 1 , 2, or 3 heteroatoms consisting of O, N, and S.

29. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt, ester, or prodrug thereof, in combination with a pharmaceutically acceptable carrier or excipient.

30. The composition of claim 29 further comprising an additional therapeutic agent.

31. The composition of claim 30 wherein the additional therapeutic agent is an antiviral agent.

32. A kit comprising an effective amount of a compound of claim 1 in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a viral infection.

33. A method of treating a subject suffering from or susceptible to a viral infection comprising administering to the subject an effective amount of a compound of claim 1.

34. The method of claim 33 wherein the subject is infected with a retrovirus.

35. The method of claim 33 wherein the viral infection is HIV infection.

36. The method of claim 35, wherein the compound treats HIV by binding to HIV RNA.

37. The method of claim 36, wherein the RNA is TAR RNA.

38. The method of claim 33 wherein the subject is identified as having a viral infection and the compound of claim 1 is administered to the identified subject.

39. A method of treating a subject suffering from or susceptible to HIV infection comprising administering to the subject an effective amount of a compound of claim 1.

40. The method of claim 39, wherein the compound treats HIV by binding to HIV RNA.

41. The method of claim 40, wherein the RNA is TAR RNA.

42. The method of claim 39, wherein the HIV is wild type or drug resistant.

43. The method of claim 33 or 39, further comprising administration of one or more additional anti-HIV therapeutic agents.

44. The method of claim 43 wherein the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor, or combination thereof.

45. The method of claim 43, wherein the other agent or agents are nucleoside HTV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, HIV entry inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, or HIV integrase inhibitors or combinations thereof.

46. The method of claim 33 or 39, wherein the therapeutically effective amount is from about 0.01 mg to about 5,000 mg per day.

47. The method of claim 33 or 39, wherein the subject is a human.