CA2075490A1 - 6-halo-and 2-amino-6-halo-purine 2',3'-dideoxy nucleosides and their use as antiviral agents - Google Patents

Abstract

Compositions containing 6 halo, 2',3'-dideoxynucleoside purines and 2 amino, 6 halo, 2',3'-dideoxy-nucleoside purines are useful for the treatment of viral infections. Particularly, these compounds are useful for preventing viral replication and cytotoxic effects of human immunodeficiency virus (HIV) and hepatitis B virus in humans infected by these viruses.

Description

W O 91/1~260 PCT/US91/00886 207~0 6-Ha~o- and 2-~mino-6-halo-purine 2', 3'-dideoxy nucleosides and ~eir us~ as 2nt~viral agen~s.
BACXGROUND_ OF THE I ~ ENTION
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, te ting, and drug therapy. AIDS is believed to be caused by the Human Immunodeficiency 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. ~oreover 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 Yitro~ which may have clinical relevance. Accordingly, the current need for additïonal 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 Challenge~, Marcel Dekker, Inc., NY (1987~; Ho et al, Lancet, i, 602-4 (1985); Sandstrom et al, ~ancet, i, 1480-82 (1985), .- . . . . . .

WO9l/12~60 ~CT/US91/00886 207~ Mitsuya et al, Nature 325, p. 773-8 (1987); and Yarchoan et al, New England Journal of ~edicine 321, p. 726-38 (1989~.
Recently, certain dideoxynucleosides have been shown to have anti-HIV activity such as ddC, ddI, ddA and ddG. See for example previous U.S. Patent 4,861,759 by Nitsuya 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 e~fect of a virus against cells, and the synthesis of gag protein in T~cells. Furthermore, thase 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 substil:uted 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 thereSore are believed to cause the viral DNA strand to terminate its chain elongation as they are in~apable 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 examplz, 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 , : ... .
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3 2~7~a concern are in the human retro~irus group, particularly HIV of any type.
Thes~ compounds are more lipophilic than other anti-HIV dideoxynucleoside analogues such as AZT, ddC, ddG, ddI or ddA. Accordingly, these compounds are expected to cross the blood-brain barrier better and therefore may be ~ore effective at blocking viral infection in the central nervous system.
These compounds are substrates for adenosine deaminase and convert to ddI or ddG in vivo.
It is a further object of this invention that these compounds can be used to prevent an infection from becoming e~tablished 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 aotually 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 ~arious concentrations of various suspected anti-viral compounds. For each test 2Xl05 AT~-8 cells are exposed to l,000 HIV virus parti-cles/cell and cultured in the presence of various concen-trations of a compound. Tha dark bars represent the cell number after six days o~ growth following exposure to HIV
and the white bars represent the ~ell 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 dat~ when 6-chloro, dideoxy-purine is used as the suspected anti-viral compound. -Figure l~ shows the data when 5-bromo, dideoxy-purine is used as the suspected anti-viral compound.
Figure ld shows the data when 6 iodo, dideoxy-purine is used as the suspected anti-viral compound.

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WO91/12260 PCT/U~91/00~86 2 ~ 7 ~ Figure 2a shows the data when 2-amino, 6-fluoro, dideo~ypurine is u~ed as the suspected anti-viral com-pound.
Fiyure 2b shows the data when 2-amino, 5-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 wh~n 2-amino, 6-iodo, dideoxypurine is used as the suspected anti-viral com-pound.
PREFERRED ~MBODIMENT
The particular compounds discovered to have antiviral compounds have the general structure illustrated below~
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Wherein X i5 any halo compound such as fluorine, chlorine, bromine or iodine, and wherein Y :Ls hydrogen or amino, and wherein Z is hydroxyl, monophosphate, diphosphate or triphosphate. Each of these compounds has de~onstrated anti-Yiral activity, especially anti retroviral activity.
The HIV in~ectivity assay using ~TH-8 cells is now a recogni~ed 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 demsnstrated with A~T, ddI, ddC and several promising drugs now undergoing the later stages of clinical trials.
~he compounds of the invention used alone or in combina-tion with each o~her or other anti-viral compounds not of this in~ention would typically be used with a pharmaceuti-cally acceptable carrier to facilitate administration of the active ingrediant. Also esters of the described compounds may also be used.

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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 hain alkyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl ~e.g.
benzyl), aryloxyalkyl (phenoxymethyl), aryl (e.g. phenyl), substituted aryl where a halogen, Cl_4 alkyl or alkoxy, sulphonate esters such as alkyl or aralkylsulphonyl (e.g.
methanesulphonyl), and mono-, di~ or ~ri-phosphate esters.
Another embodiment of the present invention invol-ves direct delivery of the triphosphate derivat~ve 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 1~ to 25~ in diameter) which permits normally non-absorbable drugs to cross the cell membrane. The use of liposomes for drug delivery is w~all 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 ~5 permit the triphosphat~ 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 ': : : ' : - , , : .
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2 ~ 7 ~ 6 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 ~ul~onic 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, NH2s2, NH3X wherein X is alkyl having 1-4 carbon atoms. _ Specific examples of pharmaceutically acceptable lS derivatives of the anti-viral compound that may be used in accordance with the invenkion include sodium salts of 5' esters including mono-, di- and tri- phosphates, acetates, 3-methyl butyrate, octanoate, palmitate, 3~chloro benæo-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 viralcyto~oxicity.
It is possible ~or 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 phaxmaceutical formulation.
These formulations comprise at least one active ingredi-ent, togethar with one or more pharmaceutically acceptable carriers and possibly other active or inactive therapeutic ~ ::

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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 modifiecl according to the type of cells being treated, the species of the cells or pa~ient, the particular viru!3 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 ab~ut 0.5~M to about 200~M and preferably from about l~M to lOO~M. This may be achieved, for example, by intravenous injection of 0.1~ to 50% concentration in solution of the active ingredient or may ~e 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 ~g/~g/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.

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2 ~ ~ 5 ~ he 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.
Foxmulations 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 ~lavorant 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.
For~ulations for topical and transdermal adminis-tration include a suitable c~rrier such as a cream or baseof 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 ingrPdient may be derivatized in order to enhance absorption or transport across the cell layer.

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WO91~12260 PCT/US91/00886 9 2~7~0 Formulations for rec~al 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 steri}e 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.q. watler, saline) for injection immediately prior to use. Extemporaneous injection solu-tions and suspensions may ~e prepared from powders, qranules and tablets of the kind previously described. In all cases, the final product is preferably ~ree 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 immunomodulating chemicals. Other forms of therapy such as cell transplants may also be concurrently used.
EX~MPL~S
2',3'-dideoxyuridine (DDU) was chemically synthe-sized from uridine by a method previously described by , :~

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WO9t/12260 PCT/U~91/00886 2 ~ 9 0 lO
Furukawa et al, Chem. Pharm. Bull. 18(3), p. 554-60 (1970). 6-chloropuxine, 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 o~ Lister et al, J.
Chem. Soc. (C) p. 3942-7 (1971). ~-bromopurine and 2-amino-S-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 lGene 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 rnl deionized water. The culture was aerobically grown at 37C for 24 hours. 500 ml of the culture was added to a 30 1 jar-fermenter and 20 l of fresh medium added followed by 2~ hours aerobic cultivation at 37C. The cells of E. coli JA-300 were then harvested by centrifugation for 10 minu~es at 8000 rpm in 10C~ About 150 g of wet cells were recovered ~nd used as the enzyme source below.
Confirmation of the chemical structure was made using the following equip~ent. Melting points were determined with a Yanaco melting point apparatus. The lH-NMR and 13C NMR spectra were recorded on a JEOL FX60Q
in~trument. Proton chemical shifts are expressed as o values with reference to T~S (tetramethylsilane). W
spectra were recorded with a Hitachi instrument model 150-20 spectrophotometer. Positive-ion fast atom bombardment ~F~B) mass spectra were obtained on a JEOL JMS-AX505H mass spectrometer. Thin-layer chromatoyraphy was carried out on E. Merck 60F 254 precoated silica gel plates.

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WO9l/12260 PCT/~S91/00886 11 2~7~'~90 SYNTHESIS OF 6-F, DDP
456 mg (3.3 mmol~ of 6-fluoropurine and 700 mg (3.3 mmol) of DDU were added to 165 ml of 50 m~ potassium phosphate ~uffer with pH of 6.5. To the mixture 16 g of E. coli JA-300 (wet cells) are added and the combined reaction mixture wa~ incuhated for 4 fours at 50C. At the end o~ the reaction mixture wa~ centrifuged at 8000 rpm for 20 min at 10C in order to separate cells from supernatant. The supernatan~ was recovered and chromato-graphed on a DIAION HP-20 column (Mitsubishi Kasei Co.) and eluted with water, 20% methanol and 100% methanol.
The frartion of methanol was concentrated and purified by si1ica 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-~-D-glycero-pentofuranosyl)-9H-purine (6-F, ddP) a 23%
yield. The structure was confirmed by the ~ollowing analysis: mp (AcOEt) 109-112C; TLC Rf (CHCl3/MeOH, 95/5) 0.40; 1 max (water) 250 nm (~ ~720); lH NMR (DMSO-d6) a 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,OH), 6.41 (t,lH,H-l'), 8,71 (s,lH,H-8), 8.92 (s,lH,H-2); FAB-HRMS (High Resolution Mass Spectr~m) (m/z) calculated i`or CloH1lO7N4F+H 239.0944, found 239.0987.
SYNTHESIS OF 6--Cl, ddP
1.78 ~ (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 o~ E. coli JA-300 (wet cells) were added and reacted for ~ hours at 50C. The cells were ramoved by centrifugation at 8000 rpm for 20 min at 10C 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-~-D-glycero-pentofuranosyl)-9H-purine (6-Cl, ddP). Recrystallization from ethyl acetate gave 955 . . .
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WO~t/12260 PCT/US91/008~6 2 ~ 7 ~ 12 mg (3.7 mmol) of a white crystal to yield 32.6%. The chemical structure was confirmed by the following data:
mp (AcOEt) 104-105C; TLC Rf (CHCl3/MeOH, 95/5) 0.45; A
max (water) 265 nm (~ 9810); lH NMR (DMSO-d6) a 1.82-2.76 (m,4H,H-2' and 3'), 3.41-3.78 (m,~h,h-5'), 3.91-4.40 (m,lH,H-4'), 5.00 (t,lH,OH), 6.39 ~t,lH,H-1'), 8.79 (s,lH,H-8), 8.94 (s,lH,X-2), FAB-~S (m/z) 255 (~H+).
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 ~ potassium phosphate buffer with a pH of 6.5. 40 g of E. coli JA-300 (wet cells) and this reaction mixture was incubatad for _ _ three hours at 50C. The cells were separated by centrif-ugation at 8000 rpm for 20 min at 10C. 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-~-D-glycero-pentofuranosyl)-9H-purine (6-Br,ddP). The compound was recrystallized ~rom 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 ~`ollowing data: mp (AcOEt) 106-108C; ~LC Rf (CHCl3/MeOH, 95/5) 0.46; A max (water) 267 nm (~ 9920); lH NMR (DMSO-d63 o 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,OH), 6,3~ (t,lH,H-1'), 8,73 (s,lH,H-8), 8.93 (s,lH,H-2); ~AB-HRMS (m/z) calculated for CloHllO2 N4Br+H 299.0144, ~ound 299.0108.
SYNTHESIS OF 6-I, 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 haYing a 6.S pH. To the mixture 28.5 g of E.
coli ~A-300 (wet cells) and the reaction mixture was incubated for three hours at 50C. 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 .
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13 2~7~90 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-~-D-glycero-pentofuranosyl)-9H-purine (6-I, 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 con~irmed by the following data: mp (EtOH) 108-111C, A max (water 276 nm (~ 11650), lH NMR
(DMSO-d6) ~ 1.73-2.74 (m,4H,H-2' and H-3'), 3.43-3.75 (m,2H,H-57), 3.92-4.38 (m,lH,H-4'), 4.98 (t,lH,OH), 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 50C and shaking at 100 strokes per minute. The cells were sepa-rated by centrifugation at 8000 rpm for 20 min at 10C 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-~fluoro-9-(2,3-dideoxy-~-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 138-140C, TLC Rf (CHCl3/MeOH, 9/1) 0.58, A max (0.OlN NaOH) 245 nm (~ 7970), 284 nm (E 6530), lH NMR (DMSO-d6) a 1.74-2.70 (m,4H,H-2' and H-3'), 3.43-3.7~ (m,2H,H-5'), 3.79-4.35 (m,lH,H-4'), 4.96 (t,lH,OH), - 6.14 (t,lH,H-1), 6.93 (bs,2H,NH2), 8,36 (s,lH,H-8), FAB-HRMS (mlz) calculated for CloH12O2N5F+H 254.1053, found 254.1057.
SYNTHESIS OF 2-AMINO,6-Cl, 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
potas~ium phosphate buffer with a pK of 6.5. 85 g of E.

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2 a7'a ~ 14 coli JA-300 (wet cells) were added to form a reaction mixture and incubated for three hours at 50OC 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 (~itsubishi 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~-D-glycero-pentofuranosyl)-9H-purine was crystallized as a white crystalline solid from water. The compound was recrystalli~ed 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) 138-140C, TLC Rf (CHCl3/MeOH 9/1) 0.58, 1 max (0.001 NaOH) 222 nm (~ 27310), 248 nm (~ 8800), 307 nm (~
9460), lH-NMR (DMSO-d6) o 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-1'), 6.95 (bs, 2H,NH2), 8.39 (s,lH,H-8), FAB-HRMS (m/z) Calculated for CloH1202N5Cl+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
pota~sium phosphate buffer haviny a pH of 6.5. 93g of E.
coli JA-300 (wet cells) were added to ~orm a reaction mixture and incubated for three hours at 50C and shaking at 100 strokes per minute. The cells were separated by centrifugation at 8000 rpm for 20 minutes at 10C 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 e~aporated to give 2-amino-6 bromo 9-~2,3-dideoxy-~-D-glycero-pentofuranosyl)-9H-purine. The compound was recrystallized from water to yield 1.25 g (3.98 mmol) of pal~ yellow crystals, a Z1.3% yield. Chemical structure was confirmed by the following data: mp (water) 137-?

WO91/12260 PCr/US91/00886 15 207~ 0 142~C, TLC Rf (CHCl3/MeOH, 9~1) 0.62, A max (0.001 NaOH) 221 nm (e 29740), 249 nm (e 9940), 319 nm (~ 10330), 'H-NMR (DMSO-d6) o 1.73-2.67 (m,4H,H-2' and 3'), 3.43-3.79 (m,2H,H-5~), 3.81-4.34 (m,2H,H-4'), 4.95 (t,lH,OH), 6.11 (t,lH,H-l'), 6.94 (bs,2H,NH~). 8.38 (s,lH,H-8), FAB-HRMS
(m~z) calculated for C,0H12O2N5Br+H 314.0253, found 314.0193.
SYNTHESIS OF 2~AMINO, 6-I, 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 50C 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 10C and the supernatan~ 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 ~0 evaporated to yield 2-amino-6-iodo-9-(2,3-dideoxy-~-D-~lycero-pentofuranosyl 9H-purine (2-amino, 6-I, ddP). The compound was recrystallized from water to give 2.0 g (5.5 mmol) of white crystal for a yield of 32.3%. ~he structural identification for the compound was confirmed by the following data: mp (water) 143-146C, TLC Rf (CHCl3/MeOH~ 9~1) 0.65, A max 0.001N NaOH) 223 nm (e 28110), 249 nm (e 138~0), 312 nm ~ 10950), lH-NMR (DMSO-d6) ~ 1.72-2.63 (m,4II,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-1'), 6.84 (bs,2H,NH2), 8.33 (s,lH,H-8), FAB~HRMS (m/z) calculated for Cl~H~2O2N5I~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) . . .
; :.. :. . . . : .
.... ..
:: ~ :, -: :, - . .

W091/l2260 PCT/US91/00886 2~7~l19 0 16 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 cytopa~hic is easily seen after four to six days.
ATH-8 cells bear several dis~inct 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 RP~I medium supplemented with 15% undialysed heat-inactivated fetal calf serum, 41nM L-glutamine, 5Xl0-5M 2-mercaptoethanol, 50 U/ml penicillin, and 50 ~g/ml strepto-mycin, 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 ~m to 200 ~m 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 in~ention 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.

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Claims (37)

WHAT IS CLAIMED IS:

1. A compound of the formula:

wherein x is either F, Br or I, and y is H or NH2 and z is OH, phosphate, diphosphate or triphosphate and wherein said compound is a substrate for adenosine deaminase, with the proviso that it is not 6-iodo-9-(2,3-dideoxy-.beta.-D-glycero-pento-furanosyl)-9H-purine.

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

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

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

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

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

7. 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, Br or I, and y is H or NH2 and z is OH, phosphate, diphosphate or triphosphate.

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

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

10. The method of claim 7 wherein the compound is 6-iodo-9-(2,3-dideoxy-.beta.-D-qlycero-pentofuranosyl)-9H-purine.

11. The method of claim 7 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

12. The method of claim 7 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

13. The method of claim 7 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

14. A method of preventing or treating viral disease comprising administering to an animal a composition comprising a compound of the formula:

wherein x is either F, 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.

15. The method of claim 14 wherein the virus contains reverse transcriptase.

16. The method of claim 14 wherein the compound is 6-fluoro-9-(2,3-dideoxy-.beta.-D-gylycero-pentofuranosyl)-3H-purine.

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

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

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

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

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

22. The method of claim 14 wherein the animal is human.

23. The method of claim 22 wherein the virus is one which replicates by reverse transcriptase.

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

wherein x is either F, Br or I, and y is H or NH2, and z is either OH, phosphate, diphosphate or triphosphate and a pharmaceutically acceptable carrier.

25 . The pharmaceutical composition of claim 24 wherein the compound is 6-fluoro-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

26 . The pharmaceutical composition of claim 24 wherein the compound is 6-bromo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

27 . The pharmaceutical composition of claim 24 wherein the compound is 6-iodo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

28 . The pharmaceutical composition of claim 24 wherein the compound is 2-amino-6-fluoro-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

29 . The pharmaceutical composition of claim 24 wherein the compound is 2-amino-6-bromo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

30 . The pharmaceutical composition of claim 24 wherein the compound is 2-amino-6-iodo-9-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-9H-purine.

31 . 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 formula:

wherein x is either F, Br or I, and y is H or NH2 and z is OH, phosphate, diphosphate or triphosphate, and wherein said compound is a substrate for adenosine deaminase.

32. The use of claim 31 wherein the virus contains reverse transcriptase.

33. The use of claim 31 wherein said virus is a retrovirus.

34. The use of claim 31 wherein said retrovirus is a human immunodeficiency virus.

35. The method of claim 7 wherein said virus is a retrovirus.

36. The method of claim 35 wherein said retrovirus is a human immunodeficiency virus.

37. The method of claim 36 wherein said human immunodeficiency virus is located in the central nervous system.