Cross-Reference to Related Applications

This application is a continuation-in-part of pending United States patent application serial no. 08/234,121, filed April 18, 1994 which is a continuation-in- part of pending United States patent application serial no. 08/153,981, filed on November 17, 1993.

Field of the Invention

The present invention relates to anti-viral guanidino-substituted compounds. More particularly, the invention relates to the use of guanidino-substituted compounds as anti-HTV agents.

Background to the Invention

Typically, anti-viral agents are modelled to inhibit viral replication within an infected cell. Viral replication may be prevented by down-regulation or inhibition of a protein required in the viral replication pathway, or by interference with the translocation of proteins and viral nucleic acid within the cell. Effective anti-viral agents specifically target steps within the viral replication pathway thereby inhibiting or hindering viral replication within infected host cells while having a minimal cytotoxic effect on the host. Thus, many anti-viral agents are specific inhibitors to virus-specified enzymes and proteins, such as viral DNA and RNA polymerases, virus-specific thymidine kinase and cleavage enzymes for viral capsid protein. Nucleoside analogues, for example, have been developed which target particular enzymes in the viral replication pathway by mimicking a natural substrate of the enzyme.

Cytotoxicity is a common obstacle in developing anti-viral agents, particularly at the concentrations required to attain effective antiviral activity, due to a lack of viral specificity. Presently, there are very few anti-viral agents which are considered to be efficacious, i.e. agents having a high level of viral toxicity

and a low level of cytotoxicity. Accordingly, there is a need to develop anti¬ viral agents having an effective level of anti-viral activity while exhibiting minimal cytotoxicity.

It is a general object of the present invention to provide novel guanidino- substituted compounds having anti-viral activity, and compositions containing these compounds.

SUMMARY OF THE INVENTION

It has been discovered that certain guanidino-substituted compounds are effective inhibitors of HIV replication. The present invention, thus, provides, in one of its aspects, a compound of the general formula (I):

or a salt, hydrate or solvate thereof, wherein X is selected from C, O, N and S; Rj and R ₃ are independently -(W)--(R ₆ ) _b -Z, wherein a and b are independently 0 or 1; W is NH, O or S;

R^ is C _1-8 alkylene in which one or two non-adjacent, intervening carbon atoms thereof is optionally replaced with nitrogen or oxygen; and Z is H, OH or -NH-C(NH)-NH ₂ ; provided that when b is 0 then Z is H; R ₂ is selected from H and OH; and ₄ and R ₅ are independently C _1-8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen.

In another aspect, the present invention provides an anti-HIV composition which includes a compound as defined by formula (I), or a salt, hydrate or solvate thereof, in admixture with an acceptable carrier.

In another aspect, the present invention provides a method of inhibiting

HIV replication in a mammal, in which a composition comprising a therapeutically effective amount of a compound as defined by formula (I), is administered to a mammal.

Embodiments of the present invention are described in greater detail with reference to the accompanying drawings in which:

Brief Reference to the Drawings

FIGURE 1 illustrates the chemical process for preparing a guanidino-substituted compound of the present invention;

FIGURE 2 graphically represents the Tar-binding affinity of compounds in accordance with the present invention;

FIGURES 3-6 illustrate the chemical processes for preparing specific guanidino- substituted compounds of the present invention.

Detailed Description of the Invention and Its Preferred Embodiments

The present invention provides novel guanidino-substituted compounds having anti-HIV activity. The term "anti-HIV ¹¹ as it is used herein refers to those compounds which inhibit replication of a member of the HIV family of retroviruses as determined using conventional cell culture assays, such as the well- established P24 antigen assay in which the anti-HIV nature of a compound is indicated by inhibition of viral P24 synthesis following treatment of virally infected cells with the given compound, relative to a virally infected, untreated control. Alternatively, a binding assay model, indicative of HIV inhibition in cell culture, can be used to determine the anti-HIV nature of a compound.

The term "HIV" is meant to encompass members of the HIV retroviral family such as HIV-1, HIV-2, SIV-1 and HTLV-1, the various strains thereof, such as the LAV, Ada, Ada-M, NL4-3, JR-FL, HXB2 and IIIB strains of HIV-1, and other viruses related thereto.

In one of its aspects, the present invention provides a guanidino-substituted compound, or a salt, hydrate or solvate thereof, which is generally defined by formula (I):

wherein

X is selected from C, O, N and S;

Ri and R ₃ are independently -(W)--(Rή) _b -Z, wherein a and b are independently 0 or 1;

W is NH, O or S; ή is C _1-8 alkylene in which one or two non-adjacent, intervening carbon atoms thereof is optionally replaced with nitrogen or oxygen; and

Z is H, OH or -NH-C(NH)-NH ₂ ; provided that when b is 0 then Z is H; R ₂ is selected from H and OH; and R, and R ₅ are independently Cι _-8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen.

The terms alkyl, alkylene, alkoxy, alkyleneoxy, alkoxyalkyl, alkylamino and aminoalkyl used herein, refer to those groups comprising linear or branched chains having from 1-8 atoms in length.

R, and R ₃ are independently the group -(W)--(R ₆ ) _b -Z, wherein a and b are

independently 0 or 1; W is NH, O or S; Re is C,. _g alkylene in which one or two non-adjacent, intervening carbon atoms thereof is optionally replaced with nitrogen or oxygen; and Z is H, OH or -NH-C(NH)-NH ₂ ; provided that when b is 0 then Z is H. In one embodiment, W, Rή, Z, a and b are selected such that R, and R ₃ independently represent a group selected from H, OH, NH ₂ , and linear or branched alkyl, alkoxy, alkoxyalkyl, alkylamino and aminoalkyl. The term "intervening" with respect to the alkylene chain of ^ means that the first and last carbon atoms of the chain are not to be replaced with nitrogen or oxygen.

The terms alkyl, alkylene, alkoxy, alkyleneoxy, alkoxyalkyl, alkylamino and aminoalkyl used herein, refer to those groups comprising linear or branched chains having from 1-8 atoms in length. Branched groups preferably comprise branched moieties having from 1-3 carbon atoms, e.g. methyl, ethyl or propyl alkyl branches. More preferably, however, Rj and R ₃ are linear containing from 1-6 atoms, i.e. an alkyl group such as methyl, ethyl, propyl, butyl, pentyl or hexyl; an alkoxy group such as methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, or hexyloxy; an alkoxyalkyl group such as methoxy methyl, ethoxy methyl, methoxyethyl, propyloxy methyl, or butyloxymethyl; an alkylamino group such as methylamino, ethylamino, propylamino, butylamino or pentylamino; or an aminoalkyl group such as aminomethyl, aminoethyl, aminopropyl, aminobutyl or aminopentyl.

In a preferred embodiment, W, Re, Z, a and b are selected such that R, is the group -O-R^-H or -O-- NH-C(NH)-NH ₂ ^herein ^ s C-. ₈ alkylene in which one or two non-adjacent, intervening carbon atoms are optionally replaced with nitrogen or oxygen. In specific embodiments R _t is methoxy, ethoxy, -O-ethylene- O-ethylene-O-ethyl, or -O-propylene-NH-C(NH)-NH ₂ and most preferably methoxy or ethoxy.

In another preferred embodiment, W, R^, Z, a and b are selected such that

R ₃ represents H, OH or the group -O-R ₆ -NH-C(NH)-NH ₂ wherein Ε^ is as previously defined. In specific embodiments, R ₃ is H, OH or -O-propylene-NH-

C(NH)-NH ₂ and most preferably R ₃ is H.

R ₄ and R ₅ are divalent groups independently selected from linear or branched C _1-8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen. In a preferred embodiment, R ₄ and R ₅ are linear Cι. ₈ alkylene in which a carbon atom is replaced with oxygen. In a more preferred embodiment, R ₄ is C,. _g alkylene in which the carbon atom adjacent to the ring is replaced with oxygen to form an alkyleneoxy group such as methyleneoxy, ethyleneoxy, propyleneoxy, butyleneoxy or pentyleneoxy. In another more preferred embodiment R ₅ is Cι _-8 alkylene in which the carbon atom that is beta to the ring (i.e. the second carbon from the ring) is replaced with oxygen. For example, R ₅ is preferably one of methyleneoxy-methylene, ethyleneoxy-methylene, propyleneoxy-methylene, butyleneoxy-methylene, pentyleneoxy-methylene or heptyleneoxy-methylene. In specific embodiments, R, is propyleneoxy and R ₅ is propyleneoxy-methylene.

As defined above, X may be any one of a carbon, oxygen, sulfur or nitrogen atom. In the most preferred embodiment, X is an oxygen atom, and the substituents R ₁ -R ₅ represent groups that, combined with the oxygen-containing ring, form a substituted sugar, e.g. a substituted pyranose such as a pyranoside.

In this regard, particular compounds in accordance with formula (I) include: methyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-D-erythro-he xoside;

1,4, 6-tri-O- [n-propylguanidine]-2 , 3-dideoxy-α-D-ery thro-hexoside;

1,4, 6-tri-O-[n-propylguanidine]-2 ,3-dideoxy-β-D-erythro-hexoside; 3,4,6-tri-O-[n-propylguanidine]-dihydro-D-glucal;

4 , 6-di-O-[n-propylguanidine]-methyl-Q!-D-glucopyranoside;

4,6-di-O-[n-propylguanidine]-dihydro-D-glucal;

4 , 6-di-O-[n-propylguanidine]-3-O-methyl-dihydro-D-glucal; ethyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-o!-D-erythro- hexoside; ethoxyethoxyethyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-c .-D-erythro-hexoside; and

3,4,6-tri-O-[n-propylguanidine]-dihydro-D-galactal.

Such guanidino-substituted sugar compounds may be made using suitable sugar-based starting materials, such as, for example, a protected glucal. To provide an intermediate which is substituted at position 1, the glucal is reacted with the appropriate alcohol to yield an enopyranoside substituted at position 1 by the deprotonated alcohol. The enopyranoside is then prepared for substitution at positions 4 and 6 by alkylation at these positions, followed by a series of reactions which introduce the desired guanidino-substituted groups at these positions. Included within these process steps is the hydrogenation of the double-bond between C ₂ and C ₃ of the enopyranoside to yield a fully saturated hexoside. Figure 1 illustrates the process steps for the synthesis of the compound, methyl-

4,6-di-O-N-propylguanidine-2,3-dideoxy-hexoside, which is described in detail in the specific examples. One of skill in the art will appreciate that similar process steps can be applied to appropriate starting materials to yield other sugar-based guanidino-substituted compounds, such as l,4,6-tri-O-N-propylguanidine-2,3- dideoxy-hexoside, or 3,4,6-tri-O-N-propylguanidine-2-deoxy-hexoside, the processes for making which are described herein in Examples 2 and 3, respectively.

Compounds in which W is NH, may be synthesized from a protected glucal such as tri-O-acetyl-D-glucal. As shown in the scheme below, a protected glucal is reacted with an N-protected alkyl-amine (Y-NH-Pr ¹ ) to give the corresponding N-glucosylated compound wherein Y is alkyl, guanidino substituted alkyl, hydroxy-alkyl or alkoxy-alkyl; Pr ¹ is a suitable amino protecting group such as Boc or Cbz and Pr ² is a suitable hydroxyl protecting group such as acetyl. The reaction is carried out in an aprotic solvent such as benzene, toluene or dichloromethane in the presence of a Lewis acid such as SnCl ₄ , A1C1 ₃ , ZnCl or BF ₃ in a temperature range of approximately -40 °C and +20 °C. The amino protecting group Pr ¹ is removed with a suitable reagent allowing the N- glucosylated compound then to be substituted at the 4 and 6 positions, as previously described, ie. alkylation followed by a series of reactions which introduce the desired guanidino-substituted groups at these positions.

Moreover, in accordance with further embodiments of the present invention, appropriate heterocyclic starting materials can be used to prepare corresponding substituted piperidine-based compounds in which X is nitrogen, cyclohexane-based compounds in which X is carbon and tetiahydrothiopyran-based compounds in which X is sulfur. For example, when X is sulfur, 5-thio-D- glucose (Aldrich catalogue, item no. 85,986-9) may be used as a starting material and when X is nitrogen, 1-deoxynojirimycin may be used, the synthesis of which is described in Shankar et al, Tetrahedron Letters, 1993, 34(45):7171. N and S- containing homologues of the oxygen-containing sugar compounds listed above are prepared from these starting compounds using the following steps: 1) protection at the 4 and 6 positions; 2) deoxygenation followed by reduction at positions 2 and 3; 3) oxidation followed by alkylation at position 1 to yield the desired R _j substituent; 4) deprotection at positions 4 and 6 followed by alkylation with N— Boc-aminoalkylbromide, hydrolysis of the N-Boc group to obtain free amino, transformation of the free amino to bis-Boc-guanidino and hydrolysis to yield free guanidino. Steps 1) to 4) may also be used to obtain homologues wherein X is carbon from a suitable starting compound such as quebrachitol (Aldrich catalogue, item no. 36,060-0) or inositol. In this case, a carbon-carbon double bond is introduced at position 5 prior to the protection step 1) by established techniques such as the classical Wittig reaction or organometallic chemistry.

As will be appreciated by one of skill in the art, diastereomers of compounds of the present invention may be separated using techniques well- established in the art, for example, silica gel column chromatography, and utilized equally in anti-HIV compositions according to the present invention. Example 2 describes the separation of diastereomers of the compound 1,4,6-tri-O- propylguanidine-2,3-dideoxy-hexoside to its and β anomers. Moreover, each of

these anomers exhibits similar anti-HIV activity as set out in Example 11.

Functionally equivalent forms of compounds according to formula (I), such as salts, solvates and hydrates, are also included within the scope of the present invention. Acid addition salts of the present compounds, for example, are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. The preparation of an acid addition salt comprises admixing a compound of formula (I) with the requisite organic or inorganic acid. To convert an acid addition salt of a compound to an alternative salt, an aqueous solution of the salt is treated with base, e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted using an appropriate solvent such as ether. The free base is then separated from the aqueous portion, dried and treated with the appropriate acid to yield the desired alternative salt compound.

Once a compound according to the present invention has been synthesized, it is desirable to analyze the compound further to ensure its chemical authenticity and purity. Typically, methods of specttoscopy are used to confirm the identity of a compound including, for example, infra-red, ultra-violet, nuclear magnetic resonance (NMR), electron spin resonance (ESR), and mass specttoscopy (MS).

Having confirmed the identity of the compound, prior to its use as an anti¬ viral agent, it must be purified so as to remove contaminants which may adversely affect its anti-HIV activity. In this regard, it will be appreciated that strict standards of purity, such as those required for pharmaceutical compounds, may not be required for use of the present compounds in vitro. On the other hand, if a compound according to the present invention is to be used in a pharmaceutical sense, it must be purified so as to meet the standards set out by the appropriate

regulatory agencies. Any one of a number of conventional purification procedures may be used to attain the required purity. Reverse phase high-pressure liquid chromatography (RP-HPLC), for example, is one method that is commonly used to purify end products. Fractionation of components in the product mixture using liquid chromatography is generally accomplished by running a linear gradient, e.g. a mobile phase comprising an increasing percentage of organic solvent such as acetonitrile, in an aqueous buffer usually containing a small amount of an ion- pairing agent such as 0.1 % trifluoroacetic acid (TFA), through alkylated silica columns, e.g. Q-, C ₈ -, or C ₁₈ - silica. Alternatively, methods such as ion- exchange chromatography may be used to purify the product.

The guanidino-substituted compounds of formula (I), or salts, solvates or hydrates thereof, are useful to prepare anti-HIV compositions, according to an aspect of the invention. These compositions comprise an effective amount of a guanidino-substituted compound in admixture with an acceptable carrier. The expression "an effective amount" is meant to encompass amounts of the anti-HIV compound sufficient to prevent or cause a reduction in HIV replication. Such compositions have use in preventing growth of HIV, i.e. by inhibiting viral replication, both in vitro as well as in vivo.

For in vitro applications, an anti-HIV compound in accordance with the invention is combined with a carrier suitable for the in vitro application for which it is to be used. For example, for use in preventing HIV replication in cell culture, the selected anti-HIV compound will preferably be combined with a carrier such as an aqueous solvent. In some cases, the compound will require combination with an organic carrier such as DMSO. Once combined with a suitable carrier, the compound may be used as a media supplement and is subsequently combined with a growth media appropriate for the cells to be cultured. For these purposes, the concentration of anti-HIV compound will vary depending on the compound itself and on the cell culture to be treated. Generally, addition of the anti-HIV compound to the cell culture in a concentration of from 0.1 - 10 μM will be suitable for its use as an anti-HIV media supplement.

Anti-HIV compositions for in vivo administtation, i.e. for treating infected individuals, are also contemplated. Such compositions comprise a therapeutically effective amount of an anti-HIV compound together with a pharmaceutically acceptable carrier. In this context, the term "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. non-toxic and not adversely affecting the anti-viral activity of the compounds of the present invention. The term "therapeutically effective amount" means an amount of the compound sufficient to cause a reduction in the replication of EfTV in the infected individual without causing adverse effects. Such reduction is most properly revealed by assaying virus titer in serum samples derived from the individual before and after treatment.

Pharmaceutically acceptable carriers useful to prepare compositions for in vivo administtation include conventional carriers generally selected for combination with sugar-based drugs such as diluents, excipients and the like.

Reference may be made to "Remington's Pharmaceutical Sciences", 17th Ed., Mack Publishing Company, Easton,

Penn., 1985, for guidance on drug formulations generally. As will be appreciated, the pharmaceutical carriers used to prepare compositions in accordance with the present invention will depend on the administrable form required to treat the infection.

According to one embodiment of the invention, the compounds are formulated for administration by injection, either sub-cutaneously or intravenously, and are accordingly provided as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in sterile water or, more desirably, in saline or 5% dextrose solution. The compounds herein designated as preferred compounds are substantially water- soluble. Water solubility of these and other compounds of the invention may be enhanced, if desired, by incorporating a solubility enhancer, such as

cetyltrimethylammonium bromide or chloride. Lyoprotectants, such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.

Alternatively, the compounds of the present invention may be formulated for administtation by routes other than injection. Oral dosage forms, such as tablets, capsules and the like, formulated in accordance with standard pharmaceutical practise, may be employed, as well as inhalable aerosol formulations.

For use in treating individuals infected with HIV, precise dosage sizes of a pharmaceutical composition appropriate for treatment can readily be established in appropriately controlled trials, and will correspond to an amount of anti-viral compound that affords effective results against HIV replication without causing any harmful or deleterious side effects to the host being treated. In such trials, inhibition of HIV replication can be monitored using the well-established P24 antigen assay. It is anticipated that an effective treatment regimen for HTV- infected patients will involve the systemic administtation of dosage sizes in the range of from 1 μg to about 10 mg per kg, e.g., between about 0.01 mg/kg to about 5 mg/kg. It will be appreciated, however, that effective dosage sizes will vary according to the route of administtation, the frequency of administtation, and, of course, with the particular individual to be treated.

In another aspect of the invention, the present anti-HIV compounds may be used as a diagnostic tool. Thus, a biological sample, including for example, blood, urine or saliva, may be analyzed for the presence of HTV. Specifically, an aliquot of the biological sample to be analyzed is combined with a culture of cells normally killed by the virus, e.g. HIV-sensitive cells such as HeLa-CD ₄ , MT4, CEM-T4, Hut-78, PBMC or PBL cells, or differentiated monocytes/macrophages, to provide a conttol. Another aliquot of the biological sample is combined with a culture of the HIV-sensitive cells along with an anti-HIV compound to provide a test sample. The conttol and test samples are incubated under conditions suitable

for cell growth, and are subsequently analyzed for growth of the cells using methods well-established in the art. If the biological sample is HIV-infected, the cells of the conttol will be killed by the virus. In contrast, the cells in the test sample will continue to grow, or will at least grow to a greater extent than those of the conttol. The presence of the anti-HIV compound will inhibit, or at least hinder, HIV replication in the test sample thereby allowing at least some growth of the HIV-sensitive cells to continue.

Specific embodiments of the present invention are described in more detail in the following examples which are not to be construed as limiting.

Example 1 - Synthesis of methyl-4.6-di-O-fn-propylguanidine1-2.3-dideoxy-D- erythro-hexoside

The compound, methyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-D- erythro-hexoside was prepared as generally outlined in Figure 1, and as set out in detail in the following:

A) In a first step to the desired compound, methyl-4,6-di-O-acetyl-2,3- dideoxy-o;,β-hex-2-enopyranoside was prepared as follows. To a stirred solution of tri-O-acetyl-D-glucal (obtained from Aldrich) (6 g, 22.038 mmol) in dry benzene (20 ml) and 2.2 mL of dry methanol, 1.5 ml of boron trifluoride-ether was added dropwise. The reaction mixture was kept under nitrogen for 45 min., and was then diluted with ethyl acetate, washed twice with saturated aqueous sodium bicarbonate and water, and dried over MgSO ₄ . Removal of the solvent yielded methyl-4,6-di-O-acetyl-2,3-dideoxy-α,β-D-erythro-hex-2-eno pyranoside. The crude product was purified by flash chromatography [the eluant was ethyl acetate: hexane (30:70)] to yield the title compound as a colorless oil (80% yield).

B) The methyl-2,3-dideoxy-α,β-D-erythro-hex-2-enopyranoside intermediate was prepared next. The methyl-4,6-di-O-acetyl-2,3-dideoxy-α,β-D-erythro-hex-2- enopyranoside product of A) (2 g, 8.926 mmol) was stirred in 45 ml of dry

methanol. A catalytic amount of sodium methylate (240.9 mg, 4.463 mmol) was added to the stirred solution and stirring at room temperature was continued for 1 hour. The solvent was evaporated and the remaining residue was diluted with ethyl acetate. This mixture was washed twice with brine, dried over MgSO ₄ and the remaining solvent was evaporated. The resulting syrup was purified on silica gel using a 60% ethyl acetate/hexane eluant to yield, as a colorless oil, the methyl- 2,3-dideoxy-o.,β-D-erythro-hex-2-enopyranoside intermediate (yield was >95%).

C) Methyl-4,6-di-O-[ -N-Boc-propylamine]-2,3-dideoxy-c.,β-D-erythro-hex-2- enopyranoside, the third intermediate in the pathway, was prepared by stirring methyl-2,3-dideoxy-o;,β-D-erythro-hex-2-enopyranoside from B) (lg, 6.248 mmol) in 31 ml of dry tettahydrofuran, and then adding thereto freshly powdered potassium hydroxide (1.4 g, 24.992 mmol), 18-Crown-6 catalyst (660 mg, 2.499 mmol) and 1-N-Boc-amino propylbromide (3.7 g, 13.745 mmol). The reaction mixture was stirred at room temperature until all starting material was consumed as indicated by TLC (approx. 2 hrs.). The mixture was then diluted with ethyl acetate and washed several times with water. The desired dialkylated intermediate (crude) was obtained following drying of the organic phase over MgSO ₄ , and evaporation of remaining solvent. Purification of the product was conducted on silica gel using ethyl acetate: hexane (50:50) as the eluant to provide the title compound (60-70% yield).

D) The next intermediate in the pathway, methyl-4,6-di-O-[ -N-Boc- propylamine]-2,3-dideoxy-o!,β-D-erythro-hexoside, was prepared as follows. The dialkylated product from C) (750 mg, 1.581 mmol) was dissolved in 10 mL of dry ethyl acetate. To the solution was added 50 mg of 10% Pd/c suspended in 5 ml of ethyl acetate. The reaction mixture was stirred at room temperature under a balloon of hydrogen for 2 hours, and then filtered through celite. The filtrate was concentrated and passed through a short column (approx. 2-6 cm in length) of silica gel using an ethyl acetate: hexane (50:50) eluant. The desired hydrogenated product was obtained as a colorless oil in quantitative yield.

E) Methyl-4 , 6-di-O-[n-propylamine]-2 , 3-dideoxy-α ,β-D-erythro-hexoside was prepared from the hydrogenated product of D) which was dissolved (170 mg, 0.357 mmol) in 0.5 mL of 3N HC1 in ethyl acetate and stirred at room temperature for 2 hours. The solution was removed under vacuum and the resulting oil was triturated with dry ether followed by evaporation to dryness. The crude salt was converted to the corresponding free base by passage through an ion exchange resin column (Amberlite IRA-400 OH) using pure methanol as the solvent. The free base was obtained as a white foam in quantitative yield.

F) Methyl-4,6-di-O-[bis-Boc-n-propylguanidine]-2,3-dideoxy-o;,Î ²-D-erythro- hexoside was then prepared from the product of E) as follows. To a stirred solution of methyl-4,6-di-O-propylamine-2,3-dideoxy-o.,β-D-erythro-hexo side (100 mg, 0.359 mmol) in dry DMF (1.5 ml) was added 1.5 ml of dry triethylamine. Following 15 minutes of stirring at room temperature, a solution of bis-Boc- thiourea (198.5 mg, 0.719 mmol) in 0.5 mL of dry DMF was added dropwise.

The reaction mixture was stirred at room temperature under argon atmosphere for 20 hr. The mixture was then diluted with ethyl acetate, quenched with water and washed twice with brine. The organic phase was dried over MgSO ₄ and the solvent was evaporated. The crude product was purified on silica gel using ethyl acetate:hexane (40:60) as the eluant to render the product as a white foam (60% yield).

G) The methyl-4,6-di-O-[bis-Boc-n-propylguanidine]-2,3-dideoxy-α,Î ²-D- erythro-hexoside product of F) (200 mg, 0.263 mmol) was dissolved in 6 ml of 3N-HC1 in ethyl acetate, and the reaction mixture was stirred at room temperature for 2 hours. The solution was removed under vacuum and the resulting oil was triturated with dry ether. The solvent was evaporated from the product to yield the title di-guanidino sugar, in quantitative yield, in the form of a white solid. The melting point of the product was determined to be 135-139°C.

Example 2 - Synthesis of 1.4.6-tri-O-[n-propylguanidine1-2.3-dideoxy-D-erythro- hexoside

The synthesis of the compound, l,4,6-tri-O-[n-propylguanidine]-2,3- dideoxy-D-erythro-hexoside ( and β anomer), was essentially the same as the synthesis outlined for the compound of Example 1 with the following exception;

A) To a stirred solution of tri-O-acetyl-D-glucal (5 g, 18.4 mmol) in dry benzene (20 ml) was added 4.17 g of 1-N-Boc-propanol. Then 1.5 ml of boron trifluoride-ether was added dropwise. The reaction mixture was kept under nitrogen at room temperature for 45 min., and was then diluted with ethyl acetate, washed twice with saturated aqueous sodium bicarbonate and water, and dried over MgSO ₄ . Removal of the solvent yielded crude N-Boc-n-propylamine-4,6-di- O-acetyl-2,3-dideoxy-α,β-D-erythro-hex-2-enopyranoside in the form of a colorless oil. The crude product was purified by flash chromatography to an and β anomeric mixture (yield was 66%).

To separate the - and β- anomers, the anomeric N-Boc-n-propyl-4,6-di-O- acetyl-2,3-dideoxy-α,β-D-erythro-hex-2-enopyranoside mixture (3 g, 7.74 mmol) was stirred with dry methanol (100 ml). A catalytic amount of sodium methylate

(208 mg, 3.87 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. The solvent was evaporated, the remaining residue diluted with ethyl acetate (500 ml) and then washed several times with brine. The organic phase was separated and dried over MgSO ₄ . A syrup was resulted, following removal of the solvent, which was purified on silica gel using pure ethyl acetate as eluant to yield the pure a anomer (1.3 g) and the pure β anomer (400 mg).

The remaining steps B)-G) were conducted separately as described in Example 1 to yield the title c. and β anomers.

Example 3 - Synthesis of 3.4.6-tti-O-[n-propylguanidine ^" |-dihydro-D-glucal

The compound, 3,4,6-tri-O-[n-propylguanidine]-dihydro-D-glucal, was synthesized as follows:

A) D-glucal (500 mg, 3.424 mmol) was stirred in dry ethyl acetate (17 ml) with 10% palladium catalyst on carbon (100 mg) under an atmosphere of hydrogen (balloon) for 1.5 hrs. The catalyst was removed by filtration on celite, the solvant was evaporated off, and the remaining residue was filtered on silica gel using an ethyl acetate: methanol (95:5), eluant to yield dihydro-D-glucal as a colorless liquid

(yield was 100%).

B) To a stirred solution of dihydro-D-glucal (200 mg) in 7 ml dry tettahydrofuran, freshly powdered potassium hydroxide (454 mg, 8.1 mmol), 18-Crown-6 catalyst (222 mg, 0.81 mmol) and 1-N-Boc-aminopropylbromide

(1.1 g, 4.86 mmol) were added. The reaction mixture was stirred at room temperature until all starting material was consumed as indicated by TLC (approx. 3 hrs.). The residue was then diluted with ethyl acetate and washed several times with brine. The organic phase was extracted with ethyl acetate (3X), dried over MgSO ₄ , evaporated and following flash chromatography, using an ethyl acetate:hexane (50:50) eluant, the product, 3,4,6-tri-O-[N-Boc-n-propylamine]- dihydro-D-glucal, was obtained as a colorless oil (yield was 52%).

C) To obtain the title compound, the steps E) - G) in Example 1 were followed.

Example 4 - Synthesis of 4.6-di-O-| ^" n-propylguanidine1-methyl-o.-D- glucopyranoside

The synthesis of the compound, 4,6-di-O-[n-propylguanidine]-methyl-α-D- glucopyranoside was prepared as follows:

A) To a stirred solution of methyl-αr-D-glucopyranoside (5 g, 25.75 mmol) in dry DMF (60 mL) and benzaldehyd dimethyl acetal (15 mL), was added a catalytic amount of p-toluene-sulfonic acid (489.6 mg). The reaction mixture was stirred at room temperature for 24 hrs. The solution was quenched with water and diluted with ethyl acetate (200 ml). The aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were washed with brine (2X), dried over magnesium sulfate, filtered and evaporated to dryness to give a yellow oil. The oil was purified by flash chromatography (eluant was ethyl acetate: hexane, 10%, followed by ethyl acetate: hexane, 80%) to yield 4,6-0- benzylidene-X-methyl-D-glucopyranoside as a white solid (mp was 163-164°C; yield was 75%).

B) To a stirred solution of 4,6-O-benzylidene-methyl-α-D-glucopyranoside (2 g, 7.085 mmol) in dry THF (17.7 ml), powdered potassium hydroxide (1.59 g, 28.3 mmol) 18-Crown-6 (749 mg, 2.834 mmol) and benzylbromide (1.85 ml, 15.6 mmol) were added successively. The reaction mixture was stirred at room temperature until all starting material was consumed (approx. 45 minutes as determined by TLC). The mixture was quenched with water and diluted with ethyl acetate. The aqueous layer was then extracted with ethyl acetate (3X), and the combined organic layers were washed with brine (2X), dried over magnesium sulfate, filtered and evaporated to dryness to yield a yellow oil. Purification by flash chromatography (eluant was ethyl acetate: hexane, 10%) yielded a 4,6-0- benzylidene-2,3-di-O-benzyl-memyl-α-D-glucopyranoside as a white solid (mp=182-184°C; yield was 92%).

C) Aqueous 1 N HCL (7 ml) was added dropwise to a solution of 4,6-0- benzylidene-2,3-di-O-benxyl-methyl-o!-D-glucopyranoside (1.2 g, 2.6 mmol) in THF (7 ml). The reaction mixture was stirred overnight at room temperature, diluted with ethyl acetate, and then solid NaCl (5 g) was added. The aqueous layer was extracted with ethyl acetate (2X), and the combined organic layer extracts were washed with brine (2X), dired over magnesium sulfate, filtered and evaporated to dryness. A yellow oil resulted which was purified by flash

chromatography (eluant was pure ethyl acetate) to yield 2,3-di-O-benxyl-methyl-α- D-glucopyranoside (91 % yield) as a colorless oil.

D) Step (C) of Example 1 was followed using the 2,3-di-O-benzyl-methyl-o.-D- glucopyranoside product of step (C) above to yield 4,6-di-O-[l'-N-Boc- propylamine]-2,3-di-O-benzyl-methyl-Q!-D-glucopyranoside (55% yield) as a colorless oil.

E) Step (E) of Example 1 was followed using the 4,6-di-O-[l'-N-Boc- propylamine]-2,3-di-O-benzyl-methyl-α-D-glucopyranoside product of step (D) above to yield 4,6-di-O-[n-propylamine]-2,3-di-O-benzyl-methyl-α;-D- glucopyranoside as a viscous oil (85% yield).

F) Step (F) of Example 1 was followed using the free base product of step (E) above to yield 4,6-di-O-[bis-Boc-propylguanidine]-2,3-di-O-benzyl-methyl-α -D- glucopyranoside (yield was 64%).

G) Step (D) of Example 1 was followed using the product of step (F) above to yield 4,6-di-O-[Bis-Boc-propylguanidine]-methyl-o;-D-glucopyranosi de as a white foam (55% yield).

H) Step (G) of Example 1 was followed using the product of step (G) above to yield the title compound.

Example 5 - Synthesis of 4.6-di-O-rn-propylguanidinel-dihydro-D-glucal

The compound, 4,6-di-O-[n-propylguanidine]-dihydro-D-glucal, was synthesized as follows:

A) A mixture of D-glucal (5 g, 34.24 mmol) was stirred in dry ethanol (170 mL) with Pd/c (10%) (400 mg) under an atmosphere of hydrogen for 2 hours at room temperature. The catalyst was removed by filtration on celite. The solvent

was evaporated off, and the residue was filtered on silica (eluant was methanol:ethyl acetate, 2%) to yield a colorless liquid (100% yield).

B) To a mixture containing the liquid product (3g, 20.3 mmol) of step (A), N,N-dimethylformamide (50 mL) and benzaldehyde dimethyl acetal (15 mL, 101.3 mmol) was added a catalytic amount of p-toluenesulfonic acid (771 mg, 4.05 mmol). The reaction mixute was stirred at room temperature for 24 hours, diluted with ethyl acetate, and quenched with water (100 mL). The aqueous layer was extracted with ethyl acetate (3X). The combined organic layer was washed successively with saturated aqueous NaHNO ₃ and brine, and dried over magnesium sulfate. The solvent was evaporated off, and the remaining was purified by chromatography on silica gel (eluant was ethyl acetate: hexane, 50%) to yield a colorless liquid (yield was 75%).

C) This step corresponded to step (C) as described in Example 1.

D) To a solution of the product (1 g, 3.06 mmol) of step (C) in THF was added 15 mL of aqueous 1 N HCL dropwise. The reaction mixture was stirred overnight at room temperature and then diluted with ethyl acetate. Solid NaCl (5 g) was added to the mixture. The aqueous layer was extracted with ethyl acetate

(2X), and the combined organic extracts were washed with brine (2X), dried over magnesium sulfate, filtered and evaporated to dryness to yield a yellow oil. Purification by flash chromatography (eluant was ethyl acetate:hexane, 50%) yielded an oil (yield was 80%).

E) The title compound was prepared by using the intermediate product of step D) and following respectively steps C), E), F) and D) as described in Example 1.

Example 6 - Synthesis of 4.6-di-O-rn-propylguanidine1-3-O-methyl-dihydro-D- glucal:

The compound 4,6-di-O-[n-propylguanidine]-3-O-methyl-dihydro-D-glucal

was prepared by the steps outlined in Figure 3 and described in detail below.

A: 4,6-O-isopropylidene-3-O-methyl dihydro-D-glucal 2

To a stirred solution of 4,6-O-isopropylidene dihydro-D-glucal I (lg, 5,313mmol) in dry THF (27mL) were added successively freshly powdered potassium hydroxide (596mg, 10.626mmol), 18-crown-6 catalyst (589mg, 2.330mmol) and methyl iodide (829.5mg, 5,844mmol). The reaction mixture was stirred at room temperature under argon atmosphere for one hour. The mixture was then diluted with ethyl acetate (5mL) and quenced with water (lOmL). The aqueous phase was extracted twice with ethyl acetate. The organic layer was then dried over MgSO ₄ and evaporated to give the crude product which was purified on silica gel column using ethyl acetate: hexane (10:90) as eluant, giving 90% yield of 4,6-O-isopropylidene-3-O-methyl dihydro-D-glucal 2 as a colorless oil.

B: 3-O-methyl dihydro-D-glucal 3

To a solution of intermediate 2 (l.lg, 4.903mmol) in THF (12mL), was added dropwise IN aqueous HC1 solution (12mL). The reaction mixture was then stirred for 2hrs. The solution was diluted with ethyl acetate (lOOmL) and solid sodium chloride was added with stirring. The aqueous layer was separated and extracted twice with ethyl acetate (2X, lOOmL). The combined organic layers were washed successively with saturated sodium bicarbonate solution and brine, dried over MgSO ₄ and evaporated to give 3-O-methyl dihydro-D-glucal 3 which was purified by flash chromatography (EtOAc:Hex, 50:50) to 85% yield.

The remaining steps C-F in the preparation of the title compound are the same as steps C and E-G described in example 1.

Example 7 - Synthesis of ethyl-4.6-di-O-fn-propylguanidinel-2.3-dideoxy-o.-D- erythro-hexoside

The compound ethyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-o.-D- erythro-hexoside was prepared as outlined in Figure 4.

A: Preparation of intermediate (ethyl-2,3-dideoxy-D-erythro-hex-2- enopyranoside) 2 To a stirred solution of ethyl-4,6-di-O-acetyl dideoxy-erythro-2-enopyranoside 1 (2g, 7.744mmol) in dry methanol (40ml), was added a catalytic amount of sodium methylate (168mg, 3.097mmol). The reaction mixture was stirred at room temperature for 2hrs. The solvent was removed and the remaining residue was diluted with ethyl acetate (200ml). This mixture was washed twice with brine. The organic layer dried over MgSO and the remaining solvent was evaporated. The resulting syrup was purified on silica gel column using an ethyl acetate/hexane

(60%) as eluant to yield as a white solid (mp 65-67°C) ethyl-2,3-dideoxy-D- erythro-hex-2-enopyranoside 2 (82%).

B: Preparation of intermediate ethyl-4,6-di-O-[l'-N-Boc-propylamine]-2,3- dideoxy-D-erythro-hex-2-enopyranoside 3

To a stirred solution of ethyl-2,3-dideoxy-D-erythro-D-hex-2-enopyranoside 2 (770mg, 4,370mmol) in dry THF (22ml), were added successively freshly powdered potassium hydroxide (980.8mg, 17.480mmol), 18-crown-6 catalyst (577.5mg, 2.185mmol) and 1-N-Boc-aminopropylbromide (2.63g, 9.614mmol).

The reaction mixture was stirred at room temperature until all starting material was consumed as indicated by TLC (2hrs). The mixture was then diluted with ethyl acetate and washed several times with water. The organic layer was dried over MgSO ₄ . After evaporation of the solvent, the crude syrup product was purified on silica gel column using 50% ethyl acetate-hexane as eluant to give as a colorless syrup the ethyl-4,6-di-O-[r-N-Boc-propylamine]-2,3-dideoxy-D-erythro- hex-2-enopyranoside 3 (65%).

C: Preparation of intermediate ethyl-4,6-di-O-[l'-N-Boc-propylamine]-2,3- dideoxy-D-erythro-hexoside 4

Ethyl-4,6-di-O-[l'-N-Boc-propylamine]-2,3-dideoxy-D-eryth ro-hex-2- enopyranoside 3 (lg, 2.046mmol) was dissolved in 15ml of ethyl acetate. To the

solution was added 80mg of 10% Pd/C suspended in 5ml of ethyl acetate. The reaction mixture was stirred at room temperature under a balloon of hydrogen for 2hrs, and then filtered through celite. The filtrate was concentrated and passed through a short silica gel column using an ethyl acetate hexane (50:50) eluant. The desired hydrogenated compound was obtained as a colorless syrup (approx. 100%).

D: Preparation of intermediate ethyl-4,6-di-O-propylamine-2,3-dideoxy-D-erythro- hexoside 5

Ethyl-4,6-di-O-[l '-N-Boc-propylamine]-2,3-dideoxy-D-erythro-hexoside 4 (950mg, 1.936mmol) was dissolved in 9.6ml of 3N HCl in ethyl acetate and stirred at room temperature for 2hrs. The solution was removed under vacuum and the resulting oil was triturated with dry ether followed by evaporation. The crude salt obtained was converted to the corresponding free base by passage through an ion exchange resin column (Amberlite IRA-400 OH) using pure methanol as solvent to leave the free base 5 as a colorless syrup (83.8%).

E: Preparation of intermediate ethyl-4,6-di-O-[bis-Boc-propylguanidine]-2,3- dideoxy-D-erythro-hexoside 6

To stirred solution of ethyl-4,6-di-O-propylamine-2,3-dideoxy-D-erythro-hexoside 5 (460mg, 1.504 mmol) in dry DMF (7ml) was added 2.8ml of dry triethylamine. After 15 minutes of stirring at room temperature, a solution of bis-Boc-thiourea (962.2mg, 3.484mmol) in 2ml of dry DMF was added dropwise. The reaction mixture was then stirred at room temperature under argon atmosphere for 20 hrs.

The mixture was then diluted with ethyl acetate quenched with water and washed twice with brine. The organic layer was dried over MgSO ₄ and the solvent was evaporated, the crude product was purified on silica gel using ethyl acetate: hexane (40:60) as the eluant to yield a white foam (75%).

F: Preparation of final product ethyl-4,6-di-O-propylguanidine-2,3-dideoxy- hexoside The ethyl-4,6-di-O-[bis-Boc-propylguanidine]-2,3-dideoxy-D_eryth ro-hexoside 6 (HOmg, 0.141mmol) was dissolved in 1.5ml of 3N.HC1 in ethyl acetate, and the reaction mixture was stirred at room temperature under argon atmosphere for 2 hrs. The solution was then removed under vacuum and the resulting oil was triturated with dry ether. The solvent was evaporated from the product to yield the final product ethyl-4,6-di-O-propylguanidine-2,3-dideoxy-hexoside as a white foam (98%).

Example 8 - Synthesis of ethoxyethoxyethyl-4.6-di-O-fn-propylguanidine ^~ l-2.3- dideoxy-α-D-erythro-hexoside:

The title compound was prepared as outlined in Figure 5. The synthesis was essentially the same as that described for the compound of example 1 with the exception that in the step A, 2-(2-ethoxy-ethoxy)ethanol was used in place of MeOH to give the intermediate (ethoxy-ethoxy-ethyl)-4,6-di-O-acetyl-2,3-dideoxy- a , β-D-erythro-hex-2-enopyranoside.

Example 9 - Synthesis of 3.4.6-tti-O-fn-propylguanidinel-dihydro-D-galactal:

The compound 3,4,6-tri-O-[n-propylguanidine]-dihydro-D-galactal was prepared as outlined in Figure 6 and described in detail below.

A: To a stirred solution of D-galactal (200mg,1.36mmol) in 7 ml dry tettahydrofuran, freshly powdered potassium hydroxide (454 mg, 8.16 mmol), 18-Crown-6 catalyst (222 mg, 0.81 mmol) and 1-N-Boc-aminopropylbromide (1.1 g, 4.86 mmol) were added. The reaction mixture was stirred at room temperature until all starting material was consumed as indicated by TLC (approx. 12hrs). The residue was then diluted with ethyl acetate and washed several times with brine. The organic phase was extracted with ethyl acetate (3X), dried over MgSO ₄ , evaporated and following flash chromatography, using an ethyl

acetate: hexane (50:50) eluant, the product, 3,4,6-tri-O-[N-Boc-n-propylamine]-D- galactal, was obtained as a colorless oil (yield was 58%).

B: 3,4,6-tri-O-[N-Boc-n-propylamine]-D-galactal (500 mg, 3.424 mmol) was stirred in dry ethyl acetate (17 ml) with 10% palladium catalyst on carbon (100 mg) under an atmosphere of hydrogen (balloon) for 1.5 hrs. The catalyst was removed by filtration on celite, the solvent was evaporated off, and the remaining residue was filtered on silica gel using an ethyl acetate: methanol (95:5), eluant to yield (87%) 3,4,6-tri-O-[N-Boc-n-propylamine] dihydro-D-galactal as a colorless liquid.

Remaining steps C-E were the same as steps E-G described in example 1.

Example 10 - Tat-TAR Competition Binding of methyl-4.6-di-O-rn- propylguanidine ^" |-2.3-dideoxy-D-erythro-hexoside

The ability of a compound to inhibit formation of the HIV Tat-TAR binding interaction provides evidence of the anti-HIV properties of a compound. This inhibition can be assessed using an electtophoretic gel mobility shift assay. Inhibition of Tat-TAR binding by compounds in accordance with the present invention was examined by conducting a series of binding reactions in which the concentrations of Tat and ³² P-labelled TAR remained constant, while the concenttation of the anti-HIV compound being tested was varied. The products of the binding reaction were then separated using non-denaturing polyacrylamide gel electtophoresis. Bands corresponding to free TAR and the Tat-TAR complex were identified by autoradiography and cut from the gel. The amount of RNA in each band was determined by liquid scintillation counting and data were analyzed to determine the IC ₅₀ value for the anti-HIV compound.

Preparation of the TAR substrate:

The TAR substrate for the Tat-TAR binding experiments, having the nucleotide sequence, 5'-GGAGAUCUGAGCCUGGGAGCUCUCUCC-3' (SEQ ID

NO:l), was synthesized by the method described in Ogilvie et al , 1988, Proc. Natl. Acad. Sci. USA, 85:5764, the contents of which are incorporated herein by reference.

Binding Assay:

The inhibition assays were carried out in a reaction volume of 20 μl that contained 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 U/ml RNAsin (Promega), 0.09 mg/ml BSA, 5% (v/v) glycerol, 50 nM TAT (obtained from ABT) and 0.1 nM RNA (2000-5000 cpm). The compound described in Example 1 above, was added at concentrations ranging from 0.1 -

1000 nM. The samples were incubated at 23 °C for 25 min, chilled to 4°C on ice, then loaded onto 5% polyacrylamide gels (30 : 0.8 acrylamide:bis-acrylamide) that contained 5% glycerol. The gels were run in 0.5X TBE buffer at a constant current of 30 mA for 2.5 hr at 4°C, and were pre-run for 15 min prior to loading. The gels were dried onto DEAE paper (Whatman DE81) and exposed to Kodak X-

Omat X-ray film overnight with intensifying screens at -70 °C. Bands corresponding to free TAR and Tat-TAR complexes were cut from the dried gels and radioactivity was measured by liquid scintillation counting in 5 ml of Ecolume (ICN). The following data was obtained:

Conc'n of ex. 1 compound fnM ^* ) Fraction of RNA Bound

0.1 100

1 107.27

5 98.18

10 83.64

50 74.55

100 52.73

500 32.73

1000 27.27

The IC ₅₀ value was determined using the program Grafit (Leatherbarrow, 1990), and the results of this determination are graphically illustrated in Fig. l.

The IC ₅₀ of the compound of example 1 was determined to be 0.051 μM.

Example 11 - Tat-TAR Competition Binding of anomers of 1.4.6-tri-O-fn- propylguanidine ^" |-2.3-dideoxy-D-erythro-hexoside (a and β anomers ^* )

The binding assay described in detail in Example 10 was also used to determine the affinity of the stereoisomers of example 2 for the TAR substrate. The following data was obtained:

Conc'n of ex. 2 (a) (nM) Fraction of RNA Bound

5 100

10 105.56

50 94.44

100 75.93

500 42.59

1000 35.19

5000 25.93

10000 27.78

Conc'n of ex. 2 (R) (nM ^~ Fraction of RNA Bound 5 100

50 82.86

100 80

500 42.86

1000 31.43

5000 25.71

10000 20

The anomer was calculated to have an IC ₅₀ of 0.17 μM, while the β anomer exhibited an IC ₅₀ of 0.22 μM. These results are also graphically illustrated in Fig. 1.

Example 12 - Tat-TAR Competition Binding

The binding assay described in detail in example 10 was used to determine the affinity of the compounds of examples 3-9 for the TAR substtate. The IC ₅₀ value calculated for each compound tested is shown in the following table.

TAR AFFINITY