AU644412C - 6-halo- and 2-amino-6-halo-purine 2',3'-dideoxy nucleosides and their use as antiviral agents - Google Patents

Description

-Halo- and 2-amino-6-halo-purtne 2', 3'-dideoxy nucleosides and their se as antiviral agents.

BACKGROUND OF THE INVENTION Acquired immune deficiency syndrome (AIDS) is a contagious disease which directly or indirectly kills a number of people with the number of cases appearing to continuously increase in spite of various education campaigns, testing, and drug therapy. AIDS is believed to be caused by the Human Immunode iciency Virus (HIV) . This virus is one member of a retrovirus family which includes viruses which cause numerous neoplasms and/or immune system abnormalities in humans and animals. Because the virus can destroy cells critical to a person's immune system the person becomes more susceptible to infection by a large number of microorganisms which would otherwise be easily handled by a fully operable immune system. These opportunistic diseases are frequently the cause of death for people with their immune systems compromised by HIV infection. HIV also causes much morbidity and even some mortality by lesser disease states such as by wasting away with ARC.

While numerous compounds have been tested for activity against HIV in vitro and in vivo, currently only one drug, azidothymidine (AZT) , has been approved for use in preventing the action of HIV. This drug, however, can cause severe toxic side effects. In particular bone marrow suppression is the most serious dose limiting side effect. Moreover some HIV strains isolated from patients, especially those who had been under AZT treatment for more than 6 months, have been found to be resistant to AZT in vitro, which may have clinical relevance. Accordingly, the current need for additional anti-HIV therapeutics is great. This is particularly true for long term therapy. A number of compounds which were shown to be active against HIV in vitro are undergoing clinical trials. See for example Mitsuya et al, Science 226, 172-4 (1984) ; Broder, AIDS, Modern Concepts and Therapeutic Challenges, Marcel Dekker, Inc., NY (1987); Ho et al, Lancet, i, 602-4 (1985); Sandstrom et al. Lancet, i, 1480-82 (1985), Mitsuya et al. Nature 325, p. 773-8 (1987); and Yarchoan et al, New England Journal of Medicine 321, p. 726-38 (1989) .

Recently, certain dideoxynucleosides have been shown to have anti-HIV activity such as ddC, ddl, ddA and ddG. See for example previous U.S. Patent 4,861,759 by Mitsuya et al.

SUMMARY OF THE INVENTION It is an object of the present invention to provide compositions useful for inhibiting viral growth in cells. This is done by administering any of the eight novel dideoxynucleoside purine derivatives described below. All of these compounds have demonstrated effec¬ tiveness at blocking the infectivity, the cytotoxic effect of a virus against cells, and the synthesis of gag protein in T-cells. Furthermore, these compounds have been shown to inhibit infection of HIV in macrophages.

The exact mechanism of how the novel compounds operate is unknown but it is believed that once they enter the cell, the natural cell metabolism converts the novel compounds into a triphosphate form which are recognized by reverse transcriptase and substituted for natural nucleo- tides in the synthesis of viral DNA. These compounds however, do not function in the same manner as a normal nucleoside and therefore are believed to cause the viral DNA strand to terminate its chain elongation as they are incapable of forming a proper linkage, or to compete with normal nucleotides for binding to reverse transcriptase. By terminating the viral DNA chain prematurely, the compounds perhaps block viral DNA synthesis and thereby block HIV replication.

Because these compounds are recognized and used by reverse transcriptase, the invention encompasses their use to block any virus which utilizes this enzyme in its life cycle. For example, these compounds are also used to inhibit the replication of hepatitis B virus which has reverse transcriptase and can cause hepatitis, cirrhosis and hepatic cancer in humans. Viruses of particular concern are in the human retrovirus group, particularly HIV of any type.

These compounds are more lipophilic than other anti-HIV dideoxynucleoside analogues such as AZT, ddC, ddG, ddl or ddA. Accordingly, these compounds are expected to cross the blood-brain barrier better and therefore may be more effective at blocking viral infection in the central nervous system.

These compounds are substrates for adenosine deaminase and convert to ddl or ddG in vivo.

It is a further object of this invention that these compounds can be used to prevent an infection from becoming established as well as treatment once cells become infected. Examples of such preventative use may be for people potentially exposed to a virus but are uncer¬ tain whether or not they are actually infected such as when one has been injured with contaminated equipment or contacted contaminated fluids.

BRIEF DESCRIPTION OF THE DRAWINGS Figures la-d and 2a-d show the number of ATH-8 cells remaining upon exposure to various concentrations of various suspected anti-rviral compounds. For each test 2X10⁵ ATH-8 cells are exposed to 1,000 HIV virus parti¬ cles/cell and cultured in the presence of various concen- trations of a compound. The dark bars represent the cell number after six days of growth following exposure to HIV and the white bars represent the cell number after six days of growth without virus.

Figure la shows the data when 6-fluoro, dideoxy- purine is used as the suspected anti-viral compound.

Figure lb shows the data when 6-chloro, dideoxy- purine is used as the suspected anti-viral compound.

Figure lc shows the data when 6-bromo, dideoxy- purine is used as the suspected anti-viral compound. Figure Id shows the data when 6-iodo, dideoxy- purine is used as the suspected anti-viral compound. Figure 2a shows the data when 2-amiho, 6-fluoro, dideoxypurine is used as the suspected anti-viral com¬ pound.

Figure 2b shows the data when 2-amino, 6-chloro, dideoxypurine is used as the suspected anti-viral com¬ pound.

Figure 2c shows the data when 2-amino, 6-bromo, dideoxypurine is used as the suspected anti-viral com¬ pound. Figure 2d shows the data when 2-amino, 6-iodo, dideoxypurine is used as the suspected anti-viral com¬ pound.

PREFERRED EMBODIMENT The particular compounds discovered to have antiviral compounds tructure illustrated below:

Wherein X is any hal fluorine, chlorine, bromine or iodine, and wherein Y is hydrogen or amino, and wherein Z is hydroxyl, monophosphate, diphosphate or triphosphate. Each of these compounds has demonstrated anti-viral activity, especially anti-retroviral activity.

The HIV infectivity assay using ATH-8 cells is now a recognized in vitro test which reasonably accurately predicts biological activity in vivo. See the previous U.S. Patent 4,704,357 by Mitsuya et al. This has been demonstrated with AZT, ddl, ddC and several promising drugs now undergoing the later stages of clinical trials. The compounds of the invention used alone or in combina¬ tion with each other or other anti-viral compounds not of this invention would typically be used with a pharmaceuti¬ cally acceptable carrier to facilitate administration of the active ingredient. Also esters of the described compounds may also be used. Preferred esters of the anti-viral compounds may include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is straight or branched chain alkyl, alkoxyalkyl (e.g. methoxymethyl) , aralkyl (e.g benzyl) , aryloxyalkyl (phenoxymethyl) , aryl (e.g. phenyl) substituted aryl where a halogen, C_1-4 alkyl or alkoxy sulphonate esters such as alkyl or aralkylsulphonyl (e.g methanesulphonyl) , and mono-, di- or tri-phosphate esters Another embodiment of the present invention invol- ves direct delivery of the triphosphate derivative directly to the host cell. As triphosphate derivatives generally do not penetrate cell membranes well, it needs to be "packaged" in order to cross the cell membrane. One way is by encapsulating the drug in a small liposome (about lμ to 25μ in diameter) which permits normally non- absorbable drugs to cross the cell membrane. The use of liposomes for drug delivery is well known in the art and is based on a phospholipid's ability to spontaneously form lipid bilayers in aqueous environments. One method of forming liposomes is by agitating phospholipids in aqueous suspensions at high frequencies; this results in the closed vesicles characteristic of liposomes. Once a liposome contacts a cell membrane it fuses and thereby "dumps" its contents into the cell. This technique will permit the triphosphate form of the dideoxynucleoside purine derivatives to enter the cell and thereby act in the same manner as the same triphosphate compound formed naturally inside the cell from the dideoxynuceloside purine derivatives. In addition, each time a compound or its deriva¬ tives are mentioned its pharmaceutically acceptable salts thereof is also intended. Unless otherwise specified any alkyl moiety present preferably contains 1-18 carbon atoms, more preferably 1-4 carbon atoms. The aryl moiety preferably comprises a phenyl or substituted phenyl group.

Examples of physiologically or pharmaceutically acceptable salts of the compounds or acceptable deriva¬ tives thereof include base salts, e.g. derived from an appropriate base, such as alkali metal, alkaline earth metal salts, ammonium, and NX₄ wherein each X is hydrogen or an alkyl with 1-4 carbon atoms. Acceptable salts containing a hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, succinic: organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulphonic; and inorganic acids such as hydrochloric, sulfuric, phosphoric, sulfamic acids. Acceptable salts of a compound containing any hydroxy group include the anion of said compound in combination with a suitable cation such as sodium, NX₄, NHX₃, NH s₂, NH₃X wherein X is alkyl having 1-4 carbon atoms.

Specific examples of pharmaceutically acceptable derivatives of the anti-viral compound that may be used in accordance with the invention include sodium salts of 5' esters including mono-, di- and tri- phosphates, acetates, 3-methyl butyrate, octanoate, palmitate, 3-chloro benzo- ate, 4-methyl benzoate, hydrogen succinate, pivalate and mesylate.

Also included within the scope of this invention are the pharmaceutically acceptable salts, esters, salts of such esters, nitrile oxides, or any other covalent linked or non-linked compounds which upon administration to the cells or individual, is capable of providing (directly or indirectly) the substituted dideoxynucleoside anti-viral compound described in the invention or a biologically active metabolite thereof. All of these compounds are active and relatively non-toxic at concen- trations sufficient for effective inhibition of viral cytotoxicity.

It is possible for the compounds of the present invention to be administered alone in solution. However, in the preferred embodiment, the active ingredient(s) may be used or administered in a pharmaceutical formulation. These formulations comprise at least one active ingredi¬ ent, together with one or more pharmaceutically acceptable carriers and possibly other active or inactive therapeutic ingredients. As included within the scope of the inven¬ tion, "acceptable" is defined as being compatible with other ingredients of the formulation and relatively non- injurious to the patient or host cell. These carriers include those well known to practitioners in the art as suitable for oral, rectal, nasal, topical, buccal, sublin- gual, vaginal, transdermal, subcutaneous, intradermal, intramuscular, intravenous or other parenteral administra¬ tion. Specific carriers suitable for use in the invention are further defined below.

In general, a suitable dose in the range of 0.1 to 120 mg per kilogram body weight per day and more preferably in the range of 10-60 mg per kilogram body weight per day. The desired dose is preferably provided in several increments at regular intervals throughout the day or by continuous infusion or sustained release formu¬ lations. The doses will need to be modified according to the type of cells being treated, the species of the cells or patient, the particular virus infection one wishes to treat or prevent, the condition of the patient particularly in regard to the hepatic, renal, and bone marrow functions, and the nature of whatever other treatment is being employed,

Ideally the active ingredient is administered to achieve peak plasma concentrations of the active compound from about 0.5μM to about 200μM and preferably from about lμM to lOOμM. This may be achieved, for example, by intravenous injection of 0.1% to 50% concentration in solution of the active ingredient or may be administered orally in doses of about 0.1-120 mg/kg of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg/kg of the active ingredient. Ideally, concentrations in the cerebrospinal fluid should reach 10- 100% of the circulating plasma concentration. To attain such levels, the ingredient may be administered intra- thecally or systemically. The antiviral compounds may be administered orally in liquid or in solid form and may include any of the following: antacids, lactose (hydrous, fast flow etc.), microcrystalline cellulose, colloidal silicon dioxide, magnesium stearate, stearic acid and other excipients, binders, colorants and other pharmacologically compatible carriers. Compositions for oral use may be administered to patients in fasting or non-fasting states.

Formulations of the present invention suitable for oral administration include sustained release formulations and may be presented in discrete units such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient(s) . The shape and form of the solid are immaterial and it may be composed of smaller solids such as powders or granules. The formulation may be in liquid form such as a solution, suspension, oil-in- water or water-in-oil emulsion. Other acceptable formula¬ tions include a bolus, electuary or paste.

The oral dose may optionally be provided with an enteric coating to provide release in any part of the digestive track so desired.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient with an acceptable flavorant such as sucrose and acacia or tragacanth; with an inert ingredient(s) such as gelatin or glycerin; or a combination of both. Mouth- wash comprising the active ingredient and a liquid carrier are also acceptable in accordance with the invention.

Formulations for topical and transdermal adminis- tration include a suitable carrier such as a cream or base of other material to facilitate contact with the skin or mucus membranes. The active ingredient(s) contained therein may be charged, or converted into a salt in order to permit crossing the surface under the influence of an electrical field. Alternatively, the active ingredient may be derivatized in order to enhance absorption or transport across the cell layer. Formulations for rectal administration may be presented as a suppository with a suitable base, for exam¬ ple, comprising cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulas containing such carriers as are known in the art to be appropriate in addition to the active ingredient(s) .

Formulations suitable for parenteral administra- tion include aqueous and non-aqueous, isotonic and isos- motic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the body fluids of the intended recipient and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and may be stored in a freeze-dried (lyophi- lized) condition requiring only the addition of the sterile liquid carrier (e.g. water, saline) for injection immediately prior to use. Extemporaneous injection solu¬ tions and suspensions may be prepared from powders, granules and tablets of the kind previously described. In all cases, the final product is preferably free of pyrogens.

For long term therapy, oral administration is highly desirable. Since the compounds of the invention may not be stable in the acid range it may be necessary to buffer or otherwise protect the composition in the neutral range to provide adequate bioavailability.

The anti-viral compounds of the invention may be used in conjunction with other anti-viral drugs, antibiotics or immuno odulating chemicals. Other forms of therapy such as cell transplants may also be concurrently used.

EXAMPLES 2• ,3•-dideoxyuridine (DDU) was chemically synthe¬ sized from uridine by a method previously described by Furukawa et al, Chem. Pharm. Bull. 18(3), p. 554-60 (1970) . 6-chloropurine, 6-iodopurine, 2-amino-6-chloro- purine and 2-amino-6-iodopurine were purchased from Sigma Chemical Company. 6-Fluoropurine and 2-amino-6-fluoro- purine were synthesized by the method of Lister et al, J. Chem. Soc. (C) p. 3942-7 (1971) . 6-bromopurine and 2- amino-6-bromopurine were synthesized from the corresponding purine thiols and bromine in the presence of aqueous hydrobromic acid and methanol according to Beamar et al, J. Org. Chem. 27 p. 986 (1962).

Escherichia coli JA-300 (Gene 10. p. 157 (1980) was selected as the best strain for the production of the various 2' ,3*-dideoxynucleosides. This is done by a base exchange reaction catalyzed by the microorganism. The cells of E. coli JA-300 were inoculated into five flasks of 100 ml of medium in a 500 ml flask. The medium used consisted of 0.5 g of yeast extract (Oriental Yeast, Tokyo, Japan), 1.0 g of Polypepton (Nippon-Seiyaku, Tokyo, Japan), and 0.5 g NaCl in 100 ml deionized water. The culture was aerobically grown at 37°C for 24 hours. 500 ml of the culture was added to a 30 1 jar-fermenter and 20 1 of fresh medium added followed by 24 hours aerobic cultivation at 37°C. The cells of E. coli JA-300 were then harvested by centrifugation for 10 minutes at 8000 rpm in 10°C. About 150 g of wet cells were recovered and used as the enzyme source below.

Confirmation of the chemical structure was made using the following equipment. Melting points were determined with a Yanaco melting point apparatus. The ¹ - NMR and ¹³C NMR spectra were recorded on a JEOL FX60Q instrument. Proton chemical shifts are expressed as a values with reference to TMS (tetramethylsilane) . UV spectra were recorded with a Hitachi instrument model 150- 20 spectrophotometer. Positive-ion fast atom bombardment (FAB) mass spectra were obtained on a JEOL JMS-AX505H mass spectrometer. Thin-layer chromatography was carried out on E. Merck 60F 254 precoated silica gel plates. SYNTHESIS OF 6-F, DDP 456 g (3.3 mmol) of 6-fluoropurine and 700 mg (3.3 mmol) of DDU were added to 165 ml of 50 mM potassium phosphate buffer with pH of 6.5. To the mixture 16 g of E. coli JA-300 (wet cells) are added and the combined reaction mixture was incubated for 4 fours at 50°C. At the end of the reaction mixture was centrifuged at 8000 rpm for 20 min at 10°C in order to separate cells from supernatant. The supernatant was recovered and chromato- graphed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% methanol and 100% methanol. The fraction of methanol was concentrated and purified by silica gel column chromatography with ethyl acetate used to elute the column, and treated with activated charcoal to yield 180 mg (0.76 mmol) of 6-fluoro-9-(2,3-dideoxy-_/3- D-glycero-pentofuranosyl)-9H-purine (6-F, ddP) a 23% yield. The structure was confirmed by the following analysis: mp (AcOEt) 109-112°C; TLC Rf (CHCl₃/MeOH, 95/5) 0.40; λ max (water) 250 n (e 6720); ^XH NMR (DMSO-d₆) σ 1.80-2.76 (m,4H,H-2" and 3 • ) , 3,44-3.76 (m,2H,H-5'), 3.99- 4.40 (m,lH,H-4»), 4.99 (t,lH,0H), 6.41 (t,lH,H-l'), 8,71 (s,lH,H-8), 8.92 (s,lH,H-2); FAB-HRMS (High Resolution Mass Spectrum) (m/z) calculated for C₁₀H_l:Lθ₂N₄F+H 239.0944, found 239.0987. SYNTHESIS OF 6-C1, ddP

1.78 g (11.5 mmol) of 6-chloropurine and 2.44 g (11.5 mmol) of DDU were added to 230 ml of 50 mM of potassium phosphate buffer with a pH of 6.5. To the mixture 50 g of E. coli JA-300 (wet cells) were added and reacted for 2 hours at 50°C. The cells were removed by centrifugation at 8000 rpm for 20 min at 10°C and the supernatant was recovered. The supernatant was chromato- graphed on a DIAION HP^20 column (Mitsubishi Kasei Co.) and eluted with water, 10% methanol and 50% methanol. The 50% methanol fraction was treated with the activated charcoal and concentrated by evaporation to give 6-chloro- 9-(2,3-dideoxy-_/3-D-glycero-pentofuranosyl)-9H-purine (6- Cl, ddP) . Recrystallization from ethyl acetate gave 955 mg (3.7 mmol) of a white crystal to yield 32.6%. The chemical structure was confirmed by the following data: mp (AcOEt) 104-105°C; TLC Rf (CHCl₃/MeOH, 95/5) 0.45; λ max (water) 265 nm (e 9810); --H NMR (DMSO-d₆) σ 1.82-2.76 (m,4H,H-2' and 3^«), 3.41-3.78 (m,2h,h-5'), 3.91-4.40 (m,lH,H-4'), 5.00 (t,lH,OH), 6.39 (t,lH,H-l^»), 8.79 (s,lH,H-8), 8.94 (S,lH,H-2), FAB-MS (m/z) 255 (MH+) .

SYNTHESIS OF 6-Br, ddP 1.79 g (9.0 mmol) of 6-bromopurine and 1.91 g (9.0 mmol) of DDU were mixed with 450 ml of 50 mM potassium phosphate buffer with a pH of 6.5. 40 g of E. coli JA-300 (wet cells) and this reaction mixture was incubated for three hours at 50°C. The cells were separated by centrif¬ ugation at 8000 rpm for 20 min at 10°C. The supernatant was recovered and chromatographed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% metha¬ nol, and 40% methanol. The fraction of 40% methanol was treated with activated charcoal and concentrated by evaporation to yield 6-bromo-9-(2,3-dideoxy-β-D-glycero- pentofuranosyl)-9H-purine (6-Br,ddP) . The compound was recrystallized from AcOEt yielding 572 mg (1.9 mmol) of white crystal 6-Br, ddP, a 21.2% yield. Structural identity of the 6-Br, ddP was confirmed by the following data: mp (AcOEt) 106-108°C; TLC Rf (CHCl₃/MeOH, 95/5) 0.46; λ max (water) 267 nm (e 9920); -H NMR (DMSO-d₆) σ 1.80-2.76 m,4H,H-2' and 3 • ) , 3.42-3.75 (m, 2H,H-5'), 3.90- 4.40 (m,lH,H-4), 4.98 (t,lH,0H), 6,38 (t,lH,H-l')_/ 8,73 (s,lH,H-8), 8.93 (s,lH,H-2); FAB-HRMS (m/z) calculated for ^C ₁₀ ^H ₁₁°2 N₄Br+H 299.0144, found 299.0108. SYNTHESIS OF 6-1, ddP

1.50 g (6.1 mmol) of 6-iodopurine and 1.3 g (6.1 mmol) DDU were mixed with 300 ml of 50mM potassium phos¬ phate buffer having a 6.5 pH. To the mixture 28.5 g of E. coli JA-300 (wet cells) and the reaction mixture was incubated for three hours at 50°C. The cells were then separated by centrifugation at 8000 rpm for 20 min at 10°C. The supernatant was collected and chromatographed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 30% methanol, 60% methanol, and 80% methanol. The 60% and 80% methanol fractions were combined and evaporated to yield 6-iodo-9-(2,3-dideoxy-_/3-D-glycero- pentofuranosyl)-9H-purine (6-1, ddP) . The compound was recrystallized from ethanol to yield 458 mg (1.3 mmol) of white needles, a 21.6% yield. The structural identity of the compound was confirmed by the following data: mp (EtOH) 108-111°C, λ max (water 276 nm (e 11650), ^~H NMR (DMSO-d₆) σ 1.73-2.74 (m,4H,H-2^« and H-3•) , 3.43-3.75 (m,2H,H-5'), 3.92-4.38 (m,lH,H-4'), 4.98 (t,lH,0H), 6.35 (t,lH,H-l'), 8.63 (s,lH,H-8), 8.89 (s,lH,-2, FAB-MS (m/z) 347 (MH+) .

SYNTHESIS OF 2-AMINO, 6-F, ddP 457 mg (3.0 mmol) of 2-amino-6-fluoropurine and 637 mg (3.0 mmol) of DDU were mixed with 150 ml of 50 mM potassium phosphate buffer having a pH of 6.5. 13.2 g of E. coli JA-300 (wet cells) were added to make a reaction mixture which was incubated for three hours at 50°C and shaking at 100 strokes per minute. The cells were sepa- rated by centrifugation at 8000 rpm for 20 min at 10°C and the supernatant collected. The supernatant was chromato¬ graphed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 10% methanol and 50% methanol. The 50% methanol fraction was evaporated to yield 164 mg 2- amino-6-fluoro-9-(2,3-dideoxy-3-D-glycero-pentofuranosyl)- 9H-purine (2-amino, 6F, ddP) a 21.7% yield. The struc¬ tural identity of the compound was confirmed by the following data: mp 138-140°C, TLC Rf (CHCl₃/MeOH, 9/1) 0.58, λ max (0.01N NaOH) 245 nm (e 7970), 284 nm (e 6530), ~H NMR (DMSO-d₆) σ 1.74-2.70 (m,4H,H-2' and H-3 • ) , 3.43- 3.79 (m,2H,H-5'), 3.79-4.35 (m,lH,H-4^»), 4.96 (t,lH,0H), 6.14 (t,lH,H-l), 6.93 (bs,2H,NH₂), 8,36 (s,lH,H-8), FAB- HRMS (m/z) calculated for C₁₀H₁₂O₂N₅F+H 254.1053, found 254.1057. SYNTHESIS OF 2-AMINO,6-C1, ddP

3.39 g (20.0 mmol) of 2-amino-6-chloropurine and 4.24 g (20.0 mmol) of DDU were added to 400 ml of 50 mM potassium phosphate buffer with a pH of 6.5. 85 g of E. coli JA-300 (wet cells) were added to form a reaction mixture and incubated for three hours at 50°C and agitated at 100 strokes per minute. The cells were separated by centrifugation at 8000 rpm for 20 minutes at 10°C and the supernatant recovered. The supernatant was chromato¬ graphed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% methanol and 50% menthol. The 50% methanol traction was treated with activated charcoal and evaporated and 2-amino-6-chloro-9-(2,3-dideoxy-_/3-D- glycero-pentofuranosyl)-9H-purine was crystallized as a white crystalline solid from water. The compound was recrystallized from water to give 0.97 g (3.6 mmol) of white crystal, an 18.0% yield. The structural identifica¬ tion of the compound was confirmed by the following data: mp (water) 138-140°C, TLC Rf (CHCl₃/MeOH 9/1) 0.58, λ max (0.001 NaOH) 222 nm (e 27310), 248 nm (e 8800), 307 n (e 9460), ^-NMR (DMSO-d₆) σ 1.74-2.67 (m,4H,H-2' and 3'), 3.44-3.73 (m,2H,H-5'), 3.86-4.34 (m,lH,H-4'), 4.97 (t,lH,OH), 6.13 (t,lH,H-l'), 6.95 (bs, 2H,NH₂) , 8.39 (s,lH,H-8), FAB-HRMS (m/z) Calculated for C₁₀H₁₂0₂N₅C1+H 270.0758, found 270.0718.

SYNTHESIS OF 2-AMINO, 6-Br, ddP 3.98 g (18.6 mmol) of 2-amino-6-bromopurine and 3.95 g (18.6 mmol) of DDU were mixed with 930 ml of 50 mM potassium phosphate buffer having a pH of 6.5. 93g of E. coli JA-300 (wet cells) were added to form a reaction mixture and incubated for three hours at 50°C and shaking at 100 strokes per minute. The cells were separated by centrifugation at 8000 rpm for 20 minutes at 10°C and the supernatant recovered. The supernatant was chromato¬ graphed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% methanol and 50% methanol. The 50% methanol fraction was treated with activated charcoal and evaporated to give 2-amino-6-bromo-9-(2,3-dideoxy-j8-D- glycero-pentofuranosyl)-9H-purine. The compound was recrystallized from water to yield 1.25 g (3.98 mmol) of pale yellow crystals, a 21.3% yield. Chemical structure was confirmed by the following data: mp (water) 137- 142°C, TLC Rf (CHCl₃/MeOH, 9/1) 0.62, λ max^' (0.001 NaOH) 221 nm (e 29740), 249 nm (e 9940), 319 nm (e 10330), ^lE- NMR (DMSO-d₆) a 1.73-2.67 (m,4H,H-2' and 3'), 3.43-3.79 (m,2H,H-5')_f 3.81-4.34 (m,2H,H-4'), 4.95 (t,lH,OH), 6.11 (t,lH,H-l'), 6.94 (bs,2H,NH2). 8.38 (s,lH,H-8), FAB-HRMS (m/z) calculated for C₁₀H₁₂O₂N₅Br+H 314.0253, found 314.0193.

SYNTHESIS OF 2-AMINO, 6-1, ddP 4.43 g (17.0 mmol) of 2-amino-6-iodopurine and 3.61 g (17.0 mmol) of DDU were mixed with 860 ml of 50 mM potassium phosphate buffer with a pH of 6.5. 75.7 g of E. coli JA-300 (wet cells) were added to form a reaction mixture which was incubated for three hours at 50°C with shaking at 100 strokes per minute. The cells were sepa- rated from the reaction mixture by centrifugation at 8000 rpm for 20 minutes at 10°C and the supernatant recovered. The supernatant was chromatographed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% methanol, and 50% methanol. The 50% methanol fraction was evaporated to yield 2-amino-6-iodo-9-(2,3-dideoxy-β-D- glycero-pentofuranosyl-9H-purine (2-amino, 6-1, ddP) . The compound was recrystallized from water to give 2.0 g (5.5 mmol) of white crystal for a yield of 32.3%. The structural identification for the compound was confirmed by the following data: mp (water) 143-146°C, TLC Rf (CHCl₃/MeOH, 9/1) 0.65, λ max 0.001N NaOH) 223 nm (e 28110), 249 nm (e 13860), 312 nm (e 10950), ^-NMR (DMSO- d₆) σ 1.72-2.63 (m,4H,H-2' and 3'), 3.38-3.72 (m,2H,H-5'), 3.79-4.30 (m,lH,H-4'), 4.94 (t,lH,OH), 6.01 (t,lH,H-l'), 6.84 (bs,2H,NH₂), 8.33 (s,lH,H-8), FAB-HRMS (m/z) calculated for C₁₀H₁₂O₂N₅I+H 362.0114, found 362.0065. DETAILS OF THE ASSAY AND CELL LINE A human tetanus toxoid-specific T-cell line was established by repeated cycles of stimulation with antigen as described by Mitsuya et al, Science, 225, p. 1484-6 (1984), and cloned in the presence of lethally irradiated (12,000 rad) human T-lymphotrophic virus type I (HTLV-I) producing MJ tumor cells in 96-well microtiter culture plates (Costar, Cambridge, MA) . Clone ATH-8 was isolated by limiting dilution when plated at 0.5 cells per well. This clone was selected for drug screening on the basis of its rapid growth in the presences of interleukin-2 (IL-2), exquisite sensitivity in vitro to the cytopathic effect of HIV. See previous patent 4,704,357 by Mitsuya et al. For example after ten days in culture, HIV will kill greater than 98% of the ATH-8 cells and profound cytopathic is easily seen after four to six days.

ATH-8 cells bear several distinct copies of HTLV-I in its genome when assessed by Southern Blot Hybridization using a radiolabelled HTLV-I cDNA probe but does not produce detectable amounts of HTLV-I p24 gag protein. 2 X 10⁵ ATH-8 cells were pelleted and exposed to 1,000 HIV viral particles/cell for 40 minutes and resuspended in 2 ml of RPMI medium supplemented with 15% undialysed heat- inactivated fetal calf serum, 4mM L-glutamine, 5X10^"5M 2- mercaptoethanol, 50 U/ml penicillin, and 50 μg/ l strepto- ycin, further containing 15% (vol./vol.) IL-2 (lectin- depleted; Cellular Products, Inc. Buffalo, NY). The cells were cultured in culture tubes (3033, Falcon, Oxnard, CA) at 37 C in 5% carbon dioxide humidified air. On day 6 in culture the total viable cells were counted by the trypan blue dye exclusion method. The number of viable cells cultured at concentrations ranging from 0 μm to 200 μm with each of 6-F-ddP, 6-Cl-ddP, 6-Br-ddP, 6-I-ddP, 2- amino-6-F-ddP, 2-amino-6-Cl-ddP, 2-amino-6-Br-ddP and 2- amino-6-I-ddP are shown in Figs, la-d and Figs. 2a-d. While this invention has been described in detail, it is evident that modifications and variations would be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth herein are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula:

wherein x is either F, Cl, Br or I, and y is either H or NH₂ and z is OH, phosphate, diphosphate or triphosphate.

2. The compound according to claim 1 wherein the compound is 6-fluoro-9-(2,3-dideoxy-3-D-glycero-pento- furanosyl)-9H-purine.

3. The compound according to claim 1 wherein the compound is 6-chloro-9-(2,3-dideoxy-_/9-D-glycero-pento- furanosyl)-9H-purine.

4. The compound according to claim 1 wherein the compound is 6-bromo-9-(2,3-dideoxy-3-D-glycero-pento- furanosyl)-9H-purine.

5. The compound according to claim 1 wherein the compound is 6-iodo-9-(2,3-dideoxy-/3-D-glycero-pentof.urano- syl)-9H-purine.

6. The compound according to claim 1 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-3-D-glycero- pentofuranosyl)-9H-purine.

7. The compound according to claim 1 wherein the compound is 2-amino-6-chloro-9-(2,3-dideoxy-9-D-glycero- pentofuranosyl)-9H-purine.

8. The compound according to claim 1 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-/9-D-glycero- pentofuranosyl)-9H-purine.

9. The compound according to claim 1 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-_/3-D-glycero- pentofuranosyl)-9H-purine.

10. A method for preventing or treating the viral infection of cells comprising; exposing cells to the compound of the formula:

wherein x is either F, CL, Br or I, and y is either H or NH₂ and Z is OH, phosphate, diphosphate or triphosphate.

11. The method of claim 10 wherein the compound is 6-fluoro-9-(2,3-dideoxy-J-D-glycero-pentofuranosyl)-9H- purine.

12. The method of claim 10 wherein the compound is 6-chloro-9-(2,3-dideoxy-J-D-glycero-pentofuranosyl)-9H- purine.

13. The method of claim 10 wherein the compound is 6-bromo-9-(2,3-dideoxy-J-D-glycero-pentofuranosyl)-9H- purine.

14. The method of claim 10 wherein the compound is 6-iodo-9-(2,3-dideoxy-9-D-glycero-pentofuranosyl)-9H- purine.

15. The method of claim 10 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-3-D-glycero-pento- furanosyl)-9H-purine.

16. The method of claim 10 wherein the compound is 2-amino-6-chloro-9-(2,3-dideoxy-_/9-D-glycero-pento- furanosyl)-9H-purine.

SUBSTITUTESHEET

17. The method of claim 10 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-3-D-glycero-pentofurano¬ syl)-9H-p rine.

18. The method of claim 10 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-y3-D-glycero-pentofurano¬ syl)-9H-purine.

19. A method of preventing or treating viral disease comprising administering to an animal a composi¬ tion comprising a compound of the formula:

wherein x is either F, Cl, Br or I, and y is H or NH₂, and Z is OH, phosphate, diphosphate or triphosphate in an amount sufficient to alter the viral disease.

20. The method of claim 19 wherein the virus contains reverse transcriptase.

21. The method of claim 20 wherein the compound is 6-fluoro-9-(2,3-dideoxy-/9-D-glycero-pentofuranosyl)-9H- purine.

22. The method of claim 20 wherein the compound is 6-chloro-9-(2,3-dideoxy-7-D-glycero-pentofuranosyl)-9H- purine.

23. The method of claim 20 wherein the compound is 6-bromo-9-(2,3-dideoxy-/3-D-glycero-pentofuranosyl)-9H- purine.

SUBSTITUTESHEET

24. The method of claim 20 wherein the compound is 6-iodo-9-(2,3-dideoxy-/3-D-glycero-pentofuranosyl)-9H- purine.

25. The method of claim 20 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-,9-D-glycero-pento- furanosyl)-9H-purine.

26. The method of claim 20 wherein the compound is 2-amino-6-chloro-9-(2,3-dideoxy-9-D-glycero-pento- furanosyl)-9H-purine.

27. The method of claim 20 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-_/3-D-glycero-pentofurano¬ syl)-9H-purine.

28. The method of claim 20 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-?-D-glycero-pentofurano- syl)-9H-purine.

29. The method of claim 20 wherein the animal is a human.

30. The method of claim 29 wherein the virus is one which replicates by reverse transcriptase.

31. A pharmaceutical composition comprising a compound having the chemical formula:

wherein x is either F, Cl, Br or I, and y is either H or NH₂, and Z is either OH, phosphate, diphosphate or tri¬ phosphate and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31 wherein the compound is 6-fluoro-9-(2,3-dideoxy-J-D- glycero-pentofuranosyl)-9H-purine.

TESHEET

33. The pharmaceutical composition of claim 31 wherein the compound is 6-chloro-9-(2,3-dideoxy-7-D- glycero-pentofuranosyl)-9H-purine.

34. The pharmaceutical composition of claim 31 wherein the compound is 6-bromo-9-(2,3-dideoxy-_/3-D-gly- cero-pentofuranosyl)-9H-purine.

35. The pharmaceutical composition of claim 31 wherein the compound is 6-iodo-9-(2,3-dideoxy-/3-D-glycero- pentofuranosyl)-9H-purine.

36. The pharmaceutical composition of claim 31 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-?- D-glycero-pentofuranosyl)-9H-purine.

37. The pharmaceutical composition of claim 31 wherein the compound is 2-amino-6-chloro-9-(2,3-dideoxy-_/3- D-glycero-pentofuranosyl)-9H-purine.

38. The pharmaceutical composition of claim 31 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-_/3- D-glycero-pentofuranosyl)-9H-purine.

39. The pharmaceutical composition of claim 31 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-_/9-D- glycero-pentofuranosyl)-9H-purine.

40. The use of a composition in a method of preventing or treating viral disease in an animal, said composition comprising a compound of the following formu- la:

41. The use of claim 40 wherein the virus con¬ tains reverse transcriptase.