DUAL ACTION 2',5'-OLIGOADENYLATE ANTIVIRAL DERIVATIVES AND USES THEREOF

Abstract: Viral infection is inhibited in mammals by administration of metabolically stable, non-toxic 2',5'-oligoadenylate (2-5A) derivatives that have a dual therapeutic effect. The compounds activate the intracellular latent 2-5A dependent endoribonuclease RNase L and also inhibit the action of viral DNA polymerases. Conjugates of the 2-5A derivatives for therapeutic delivery are also described.

Description: DUAL ACTION 2',5'- OLIGOADENYLATE ANTIVIRAL DERIVATIVES AND USES THEREOF Field of the Invention The invention relates to certain therapeutic 2 ' ,5'-oligoadenylate analogs, pharmaceutical conjugates and compositions of such analogs, and uses thereof. Background of the Invention The full nomenclature of the subject matter of the present invention involves extremely long terms. It is customary for those skilled in the art to abbrevi¬ ate these terms in a manner well known to them. These general and customary abbreviations are set forth herein below and may be utilized in the text of this specifica¬ tion. Abbreviations: RT, reverse transcriptase A, adenosine or adenylate or adenylyl cordycepin or C or 3 , -dA, 3'-deoxyadenosine- (3'-deoxyadenylate) ara-A, 9-β-D-arabinofuranosyladenine EHNA, erthyro-9- (2-hydroxv-3-nonvl)adenine A-3'-amino, 3'-amino-3'-deoxyadenosine tubercidin, 4-amino-7(β-D-ribofuranosyl)- pyrrolo-[2,3-d]pyrimidine 3'-dATP, 3'-deoxyadenosine triphosphate ATP, adenosine triphosphate I, inosine or inosinate or inosinylyl Xylo-A or xyloadenosine, 9-β-D-xylofuranosyl- adenine dCF or 2'-deoxycoformycin, (R)-3-(2-deoxy-β-D- erythropentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5- d] [l,3]diazepine-8-ol 2-5A or 2 / ,5 / -oligo(A) or 2',5'- oligoadenylate, oligomer of adenylic acid with 2',5'- phosphodiester linkages and a triphosphate at the 5'-end C, cordecypin 2' ,5'-cordycepin analog or 2' ,5'-oligocordy- cepin, oligomer of 3'-deoxyadenylic acid with 2',5'- phosphodiester linkages and a triphosphate at the 5'-end 2' , 5 ' -h- or coreoligomer, oligomer of adenylic acid with 2',5'-phosphodiester linkages 2',5'-A 3 or 2',5'-adenylate trimer core, adenylyl(2',5')adenylyl(2',5')adenosine 2',5'-A 4 or 2' ,5'-adenylate tetramer core, adenylyl(2',5')adenylyl(2',5')adenylyl(2' ,5')adenosine 2' ,5'-3'dA 3 or 2',5'-C-C-C or 2' ,5'-cordycepin trimer core, 3'-deoxyadenylyl(2',5')3'-deoxyadenylyl- (2',5')3'-deoxyadenosine 2',5'-C-C-C-C or 2' ,5'-cordycepin tetramer core,3'-deoxyadenyly1(2',5')3'-deoxyadenyly1(2' ,5')3'- deoxyadenylyl(2' ,5')3'-deoxyadenosine 3',5'-A 3 , adenylyl(3',5')adenylyl(3',5')aden¬ osine 2',5'-I 3 or 2',5'-inosine trimer core, inosi- nylyl(2',5')inosinylyl(2',5')inosine dd benz, benzimidazyly1(2',5')5,6-dichlor- obenzimidazole riboside, EBV, Epstein-Barr virus EBNA, Epstein-Barr virus associated early nuclear antigen HBV, hepatitis B virus HIV, human immunodeficiency virus, including HIV-1, HIV-2, and all other HIV subtypes HBLV, human B-cell lymphotropic virus HTLV, human T-cell leukemia virus, including HTLV-I, HTLV-II and HTLV-III, and all other HTLV sub¬ types IFNα: α-interferon rIFN-αA: recombinant α-interferon dsRNA: double-strand ribonucleic acid 2' ,5'-A-A-Tu, adenylyl(2',5')adenylyl(2' ,5')- tubercidin 2' ,5'-Tu-Tu-Tu, 2' ,5'-tubercidylyl(2' ,5') - tubercidylyl(2' ,5')tubercidin 2' ,5'-A-A-ara-A, adenylyl (2' ,5') adenylyl- (2',5')ara-A 2' ,5'-C-C-A, 3'-deoxyadenylyl(2' ,5') 3'-deoxy¬ adenylyl (2' ,5')adenosine 2',5'-A-C-C, adenylyl(2',5') 3'-deoxyadenylyl- (2' ,5') 3'-deoxyadenosine 2' ,5'-A-A-C, adenylyl(2' ,5')adenylyl(2' ,5')3'- deoxyadenosine 2' ,5'-C-A-C, 3'-deoxyadenylyl(2',5')adenylyl- (2' ,5') 3'-deoxyadenosine 2' ,5'-C-C-A, 3'-deoxyadenylyl(2' ,5') 3'-deoxy¬ adenylyl (2' ,5')adenosine 2' ,5'-A-C-A, adenylyl(2' ,5') 3'-deoxyadenylyl- (2' ,5' )adenosine 2' ,5'-xylo-A 3 , xyloadenylyl(2',5')xyloadenylyl- (2' ,5')xyloadenosine 2' ,5'-xylo-A 4 , xyloadenylyl(2',5')xyloadenylyl- (2' ,5' )xyloadenylyl(2' ,5')xyloadenosine Ac, acetyl Bz, benzyl MMTr, 5'-O-p-methoxytrityl 2' ,5'-trityl-C 3 , 5'-O-β-methoxytrityl-3'-deoxy¬ adenylyl(2' ,5') 3'-deoxyadenylyl(2' ,5') 3'-deoxyadenosine 2' ,5'-trityl-A 3 , 5'-O-p-methoxytrityladenylyl- (2' ,5')adenylyl(2' ,5')adenosine 2' ,5'-C-C-dCF, 3'-deoxyadenylyl(2',5') 3'-deo¬ xyadenylyl(2' ,5') 2'-deoxycoformycin 2',5'-A-A-A-3'-amino, adenylyl(2',5')adenylyl- (2' ,5')3'-amino-3'-deoxyadenosine SiTBD, t-butyldimethylsilyl or -Si(CH 3 ) 2 C(CH 3 ) 3 2',5'-A (Si) -A (Si) -A, 3'-O-t-butyldimethylsilyl- adenylyl(2',5')3'-O-t-butyldimethylsilyladenylyl(2',5')- adenosine 2',5'-A-A-A-3'-O-methyl, adenylyl(2',5')adeny¬ lyl(2' ,5')3'-O-methyladenosine 2',5'-A-A-A-3'-O-pentyl, adenylyl(2',5')adeny- lyl(2',5')3'-O-pentyladenosine 2',5'-A-A-A-3'-0-hexyl , adenylyl(2',5')adeny¬ lyl(2',5')3'-0-hexyladenosine 2',5'-A-A-A-3'-0-heptyl, adenylyl(2',5')adeny¬ lyl(2',5')3'-O-heptyladenosine 2',5'-EHNA-A-A, erythro-9-(2-hydroxy-3-nonyl)- adenylyl(2',5')adenylyl(2',5')adenosine The abbreviation for the "tetramer" compounds comprising the adenylyl (A) and 3'deoxyadenylyl (C) moieties is illustrated by the following: 2',5'-A-A-C-C, adenylyl(2',5')adenylyl(2',5')- 3'-deoxyadenylyl(2',5')3'-deoxyadenosine The above compounds are also abbreviated with¬ out the 2'-5' prefix, without hyphens and without the - 3' suffix; hence 2'-5'-C-C-C-3' is also abbreviated CCC. With the expansion of the knowledge of the antiviral state induced by interferon, attention has been focused on the chemical and enzymatic synthesis and biological properties of the 2',5'-oligoadenylates as mediators of the antiretroviral response. 2',5'-01igo- (A) is a component of a natural, broad-spectrum antivi¬ ral defense mechanism in plants and animals. The 2-5A pathway, also known as the 2-5A/RNase L pathway or anti¬ viral system, is widely accepted to be involved in the antiviral mechanism of interferon, and is also involved in the regulation of cell growth and differentiation. The pathway involves the activation by 2-5A of the latent endoribonuclease, RNase L (EC 3.1.27). According to that pathway shown in Fig. 1, 2-5A is syn¬ thesized from ATP by 2',5'-oligoadenylate synthetase [ATP: (2'-5')oligo(A)-adenyltransferase (EC 2.7.7.19) ] , hereinafter "2-5A synthetase". When activated by dsRNA, 2-5A synthetase converts ATP into 2-5A, i.e., a series of 2',5'-linked oligoadenylates characterized by a 5'- terminal triphosphate. 2-5A Synthetase exists in dif- ferent isoenzyme forms, is induced by interferon, but is also detectable at lower levels in the absence of interferon. 2-5A exerts its biological effects by bind¬ ing to and activating its only known target enzyme, the unique 2-5A dependent endoribonuclease RNase L. The latter cleaves viral and cellular mRNA or rRNA, thereby inhibiting protein synthesis. Hovanessian et al., Eur. J. Biochem. 91:515-526 (1979) ; Kerr et al., Proc. Natl. Acad. Sci. USA 75:256-260 (1978). The short half-life of the authentic 2-5A molecule in biological systems is an acknowledged disadvantage in the control of viral replication. Moreover, bioactive 2-5A is inactivated by three enzymes: a relatively unspecific 2'-phosphodi- esterase, a 5'-phosphatase, and a relatively specific 2' ,3'-exonuclease. Some cytokines, e.g. IL-6, activate 2-5A synthetase in such a way as to cause the enzyme or particular forms of the enzyme to produce bioinactive forms of 2-5A (Bickel, M. , Dveksler, G, Dieffenbach, C. , W. , Ruhl, S., Midura, S., B. and Pluznik, D.H., Cvtokine 2:238-246 (1990) and Cohen, B. , Gothelf, Y., Vaiman, D. , Revel, M. and Chebath, J., Cvtokine 3:83-91 (1991)). The 2-5A synthetase/RNase L pathway is acti¬ vated following viral infection by many viruses includ¬ ing HIV-1 (Schroder, H.C., Wenger, R. , Kuchino, Y., and Mύller, W.E.G., J. Biol. Chem. 264. 5669 (1989); Schrό- der, H.C., Wenger, R. , Rottman, M. , and Mύller, W.E.G., Biol. Chem. Hoppe-Seyler 369. 985 (1988) ) . The activa¬ tion of this pathway delays the HIV infection process. To activate RNase L, the naturally occurring 2-5A mole¬ cule requires a 5'-triphosphate, which is unstable. 2- 5A molecules with 5'-monophosphates or no 5'-phosphate (core) do not activate RNase L at physiological concen- trations. "Human B-lymphotropic virus", also known as "human B-cell lymphotropic virus" (HBLV) , now called HHV-6, which is characterized by a large molecular weight double-stranded DNA genome is morphologically similar to viruses of the herpes virus family, but is readily distinguishable from the known human and non- human primate herpes viruses by host range, in vitro biological effects, antigenic features and genome. Salahuddin et al., Science 234:596-601 (1986); Josephs et al., Science 234:601-602 (1986). The virus has been observed to selectively infect freshly isolated human B-cells, which are converted into large, refractile mono- or binucleated cells with nuclear and cytoplasmic inclusion bodies. HBLV is suspected to be the cause of a chronic mononucleosis-like syndrome characterized by chronic fatigue lasting more than a year. Human immunodeficiency virus ("HIV"), also known as human T-cell leukemia virus III ("HTLV-III") , the etiologic agent of acquired immune deficiency syn- drome, is a type D retrovirus. As in all retroviruses, an essential feature of HIV replication is reverse tran¬ scription of the plus-strand RNA genome into DNA, a process which requires an RNA dependent DNA polymerase, reverse transcriptase. This enzyme is viral-encoded and is found associated with genomic RNA in mature HIV vir- ions. The exclusiveness of reverse transcriptase to retroviruses and viruses requiring a short reverse tran¬ scription step makes reverse transcriptase a major tar¬ get for antiviral, and particularly for antiretroviral, therapeutic intervention. The 2-5A synthetase/RNase L system as an anti¬ viral cellular defense mechanism has been shown to be ■ 1 a promising target for antiviral chemotherapy, particu¬ larly due to its interaction with double-stranded seg¬ ments within viral genomes or transcripts such as the HIV-1 RNA genome (Lengyel, P., Annu. Rev. Biochem. 51. 251 (1982); Pestka, S., ed. , Methods Enzvmol. 118. 119 (1986); Lengyel, P., J. Interferon Res. 7. 511 (1987); Sen, G.C., Pro . Nucleic Acid Res. Molec. Biol. 27. 105 (1982)). However, what is needed are derivatives of 2- 5A which will override degradation by enzymes which inactivate authentic 2-5A. What is needed is a method for controlling HIV, chronic fatigue caused by HBLV, and other viral or cytokine-induced disease states charac¬ terized by a 2-5A pathway defect using compounds that are more metabolically stable and active than authentic 2-5A. What is needed is a method for treatment of viral infection which utilizes compounds which have broad spectru , dual action, that is, compounds which both activate the 2-5A pathway and inhibit the activity of viral DNA polymerase. fimnmary of the Invention According to one embodiment, the invention comprises the antiviral use of a novel compound of For¬ mula I Ϋ wherein n is a whole positive integer from 1 to 8, m is 0, 1, 2 or 3, R is independently hydrogen or hydroxyl, X is selected from the group consisting of Cl to C6 alkyl and Cl to C6 alkoxy, or a pharmaceutically acceptable salt thereof. Preferably n is 1 to 3, most preferably 1 or 2. X is preferably selected from Cl to C3 alkyl and Cl to C3 alkoxy. Accordingto another embodiment, the invention is a method of antiviral treatment comprising adminis¬ tering to a mammal an effective amount of one or more compounds according to Formula I, wherein n, m, R and X are defined as above, or according to Formula II II wherein X is NH 2 or CH 2 ; m is zero, 1, 2 or 3; n is an integer from 1 to 8; each R_ is independently selected from the group consisting of oxygen, sulfur, sulfate, selen¬ ium, C x to C 8 alkyl and C_ to C 8 alkoxy; each R 2 is independently selected from the group consisting of oxygen, sulfur, sulfate, selenium, C_ to C 8 alkyl and C_ to C 8 alkoxy; each R 3 is independently selected from hydro¬ gen, hydroxyl, amino and -OSi(CH 3 ) 2 -C(CH 3 ) 3 ; R 4 and R 5 are independently selected from the group consisting of hydrogen; hydroxyl; amino; C λ to C 8 alkyl; C_ to C 8 alkoxy; C α to C 8 alkylamino, alkylcarbonyl, alkyl- carboxyl and alkylhalide; and C_ to C 8 alkoxyamino, alkoxycarbonyl, alkoxycarboxyl and alkoxyhalide; or pharmaceutically acceptable salts thereof, excluding authentic 2' ,5'-oligoadenylate and salts ther¬ eof, to simultaneously cause activation of the 2-5A synthetase/RNase L antiviral pathway of said mammal and inhibition of viral DNA polymerase. Preferably, n is 1 to 3, most preferably 1 or 2. According to yet another preference, the alkyl, alkoxy, substituted alkyl or substituted alkoxy groups, which may contain from one to 8 carbon atoms, preferably contain from 1 to 4 carbon atoms, most preferably from 1 to 3 carbon atoms. For halide-substituted alkyl and (0 alkoxy groups, the halogen atom is preferably chlorine, bromine or fluorine, with chlorine being most preferred. According to a preferred subgenus of the in¬ vention, the group A is wherein m is defined above; and more preferably, at least one of R x are sulfur, where each other R x is oxy¬ gen. An additional preferred embodiment provides that at least one of R 3 or R 4 is hydrogen, the remainder being hydroxyl. According to another embodiment, the invention is a conjugate comprising a compound according to Formu¬ la II covalently linked to an adduct which results in enhanced penetration into intact cells. The adduct is most advantageously coupled to the oligomer through a hydroxyl oxygen at the 2' or 3' position of the 2'-terminal nucleotide, with preference being for the 2' position. The adduct may comprise in one embodiment a vitamin selected from those vitamins which have corre¬ sponding cell receptors on targeted mammalian cells for a receptor-mediated endocytosis of the vitamin. Such vitamins useful as adducts according to the present invention include, for example, vitamin B 12 , biotin, riboflavin and folic acid. Alternatively, the adduct may comprise a lipo- philic molecule or radical, such as an acyl group of the formula C(CH 2 ) X CH 3 // wherein x is an integer from 1 to 20, prefera¬ bly from 2 to 14. Another preferred lipophilic radical is cholesteryl. According to one preferred subgenus of conju- gate, the compound to which the adduct is conjugated has the structure of Formula II wherein m is zero, 1, 2 or 3, n is an integer from 1 to 8, preferably 1, 2 or 3, each R 3 and R 4 is independently selected from hydrogen and hydroxyl, each R 2 is independently selected from sulfur and oxygen, R 5 is hydroxy, or a pharmaceutically acceptable salt thereof; which compound is covalently linked through the 2'-position of the 2'-terminal nucleotide thereof to the adduct. Description of the Figures Fig. 1 is a diagram illustrating the 2-5A syn¬ thetase/RNase L intracellular enzyme pathway. Fig. 2A is a phase-contrast micrograph of H9 cells incubated for 5 hours with fluorescein-bovine serum albumin (BSA) covalently conjugated to folate (25 μg/ l) in folate-deficient medium. Fig. 2B is a fluorescent micrograph of the same H9 cells shown in Fig. 2A. Fig. 2C is a phase-contrast micrograph of H9 cells incubated for 5 hours with fluorescein-BSA not conjugated to folate. Fig. 2D is a fluorescent micrograph of the same H9 cells shown in Fig. 2C. Fig. 2D is a fluorescent micrograph of the same H9 cells shown in Fig. 2C. Detailed Description of the Invention Therapeutic agents that activate the 2-5A synthetase/RNase L antiviral defense pathway and also inhibit the activity of virally-derived DNA polymers (particularly reverse transcriptase) are used for the treatment of viral disease states. By "treatment" is meant to include also prophylaxis. Such dual effect therapeutic agents are selective, broad spectrum inhibi¬ tors of viral DNA polymerases that are produced during viral infection. The polymerases are critical for viral replication. The compounds utilized in the practice of the invention may be characterized as derivatives of ■authentic 2-5A with modifications in the 5'-terminal phosphate group, ribosyl moiety and/or the internucle- otide bonds. These derivatives are metabolically sta¬ ble, non-toxic, and have dual antiviral effects, e.g., they activate the 2-5A synthetase/RNase L antiviral pathway and also inhibit virally-derived DNA polymerase. We have thus approached antiviral therapy by dually activating inherent antiviral mechanisms present in mammalian cells and inhibiting enzymes critical for viral information transfer. According to one class of compounds useful in the practice of the invention, one or more of the 3'- hydroxyl groups was replaced by hydrogen atoms to form the core 2' ,5'-cordycepin derivatives. This modifica- tion resulted in derivatives with increased resistance to degradation by phosphodiesterase. The preparation of such compounds is disclosed in U.S. Patent 4,464,359 (2',5'-oligocordycepin) , and in U.S. Patent 4,859,768 (2' ,5'-oligocordycepin and mixed 2' ,5'-oligo(cordycepin- /adenosine) ) . The core 2' ,5'-cordycepin derivatives are also non-toxic and have broad spectrum antiretroviral activity in infected cells (Montefiori, D.C. Sobol, IS R.W. , Li, S.W., Reichenbach, N.L. , Suhadolnik, R.J., Charubala, R. , Pfleiderer, W. , Modliszewski, A., Robin¬ son, W.E., Jr., and Mitchell, W.M. , Proc. Natl. Acad. Sci. USA 86, 7191 (1989); Suhadolnik, R.J. , Lebleu, B. , Pfleiderer, W. , Charubala, R. , Montefiori, D.C., Mitch¬ ell, W.M. , Sobol, R.W., Li, S.W., Kariko, K. , and Reich¬ enbach, N.L. , Nucleosides & Nucleotides 8, 987 (1989); Mύller, W.E.G., Weiler, B.E., Charubala, R. , Pfleiderer, W. , Leserman, L. , Sobol, R.W. , Suhadolnik, R.J. , and Schroder, H.C., Biochemistry 30. 2027 (1991)). Other 2-5A derivatives have been synthesized with other modi¬ fications of the 3'-hydroxyl groups, such as the substi¬ tution of amino and OSi(CH 3 ) 2 -C(CH 3 ) 3 , as described in Patent 4,859,768. Other 2-5A derivatives contain, in addition to or in lieu of 3'-hydroxyl modifications on one or more nucleotides, modifications in the 5'-termi¬ nal phosphate group. These 2-5A derivatives retain the ability to inhibit viral DNA polymerase and retain meta¬ bolic stability but, in addition, are able to activate RNase L. A variety of 2'-5'-oligoadenylate derivatives in addition to those already synthesized will exhibit enhanced antiviral activity. According to yet another embodiment, the in- ternucleotide phosphodiester linkage is modified. Ac- cording to a preferred embodiment, sulfur is substituted for an oxygen atom to form the 2',5'-phosphorothioate oligoadenylates. The preparation of such compounds, including optical isomers, is described in U.S.Patent 4,924,624. The substitution of sulfur for oxygen in the 2',5'-phosphodiester backbone introduceschirality into the molecules and introduces a new chemistry of the backbone. The core 2',5'-phosphorothioates exhibit increased resistance to phosphodiesterase and phos- phatases and new biological activities compared to au- thentic 2-5A cores. These stereochemically modified molecules are the first 2',5'-linked core molecules able to activate RNase L. The 2',5'-phosphorothioates act a through both the activation of the 2-5A synthetase/RNase L antiviral system and the inhibition of viral DNA poly- merase. The 2',5'-phosphorothioate trimer 5'-monophos- phates activate RNase L at nanomolar concentrations, similar to naturally occurring p 3 A 3 . Other molecular modifications of the internucleotide linkage provided for herein include the substitution of an oxygen atom by sulfate, selenium, C1-C8 alkyl and C1-C8 alkoxy. In yet another embodiment, dual action, anti- viral 2-5A derivatives having increased penetration into intact cells are prepared by conjugating the oligomer to an adduct. One preferred group of adducts comprise the water-soluble vitamins (including but not limited to biotin, folic acid, vitamin B 12 , or riboflavin) . Other preferred adducts include lipophilic molecular and chemical groups such as, for example, acyl or choles- teryl groups. Such conjugates are internalized by in¬ tact cells by exploiting receptor-mediated endocytosis. Administration of exogenous, metabolically stable, dual action analogs of 2-5A will render in¬ creased protection against disorders characterized by a 2-5A defect, particularly protection against viral infection in animals and humans. By "2-5A defect" as used herein is meant any manifestation, condition or interruption of the 2-5A pathway which results in a physiologically consequential change in the production of authentic 2-5A, and/or the interruption of 2-5A-de- pendent activation of RNase L. Afflictions character- ized by a 2-5A defect include, for example, viral infec¬ tions, particularly HTLV infection, most particularly HIV infection, chronic fatigue and other HHV-6 related disorders, hepatitis B and other infections of the hepa¬ titis virus family, cutaneous T-cell lymphoma and other HTLV-1 related disorders, etc. Other diseases that are relevant include chronic myelogenous leukemia; acute leukemia; cancer; T-cell leukemia; Alzheimer's disease; a' Parkinson's disease; multiple sclerosis; autoimmune disease; and surgery- and other trauma-induced immune dysfunction. 2-5A pathway defects are particularly manifested in diseases characterized by both chronic viral infection and immune cell defects. Structural modification of the 2-5A molecule at the 3'-hydroxyl groups and elsewhere provides 2-5A analogues with remarkably increased metabolic stability to 2'-phosphodiesterases and cellular nucleuses, while maintaining the ability to activate RNase L and inhibit retroviral reverse transcriptase. Likewise modification of native 2-5A by substitution of the 3'-terminal nucle¬ otide results in a more stable molecule. Persistent, high intracellular concentration of the metabolically stable 2-5A analogs are a consequence of their increased stability. Additionally, modifications of the 5'-termi¬ nal enhance the ability of the 2-5A molecule to activate RNase L. The longer-lasting pharmacological activity of the 2-5A analogs offer a more favorable therapeutic ratio. This allows a decreased frequency of administra¬ tion relative to 2-5A, which is metabolically unstable. Decreased frequency of administration is important due to the chronic nature of many afflictions characterized by 2-5A pathway defects. In addition, certain modi¬ fications of 2-5A derivatives that facilitate penetra¬ tion into the cell further increase the therapeutic ratio by reducing the amount of the derivative that must be administered to give a therapeutic effect. The 2-5A analogs are particularly useful in the treatment of infections caused by viruses. The 2-5A pathway defect associated with viral infection comprises the inactivation of the pathway caused by the virus' interference with the activation of 2-5A synthetase by dsRNA. In the absence of 2-5A synthetase activation, 2-5A production, and hence activation of RNase L, is reduced. According to the present invention, exogenous, metabolically stable 2-5A analog is administered to counteract this virally-caused defect in the 2-5A path¬ way. The 2-5A analogs, like authentic 2-5A, are capable of activating RNase L, which cleaves viral RNA. The 2-5A analogs are particularly useful in protecting against infection by the various human T-cell leukemia viruses (collectively "HTLV") , such as HTLV-I, which cause cutaneous T-cell lymphoma; HTLV-II, which causes Sezany lymphoma: HTLV-III; and HTLV-IV, which is presently believed to be the etiologic agent of multiple sclerosis. Each of the HTLV viruses is a retrovirus. Also known as "HIV-1", HTLV-III is responsible for caus¬ ing acquired immune deficiency syndrome ("AIDS") . The compounds are further believed useful in treating HIV-2, a second serologically distinct HIV subtype. Herein¬ after (HIV) shall mean either HIV-1 or HIV-2, and any other HIV subtypes now or hereinafter known. The 2-5A analogues are also particularly use¬ ful in protecting against infection by the various hepa- titis viruses which cause viral hepatitis. The hepati¬ tis B virus, in particular, is thought to be a composite of a retrovirus and a virus that employs replication of a DNA genome by DNA dependent DNA polymerase. The 2-5A analogues are also particularly useful in protecting against infection by viruses with a DNA genome, such as herpes viruses. HTLV-infected patients, in particular HIV-1- infected patients, have been shown to demonstrate unusu¬ ally low levels of 2-5A and/or RNase L activity in blood mononuclear cells. Blood ononuclear cells from healthy individuals, by contrast, display higher 2-5A levels, on average, and RNase L activity is readily detectable. Likewise blood mononuclear cells of chronic fatigue-in¬ flicted individuals exhibit low 2-5A levels, and evi- dence the appearance of novel RNA cleavage products, distinct from the specific cleavage products observed in blood mononuclear cells from normal individuals. /I While the practice of the invention is illus¬ trated herein with regard to the treatment of HIV-1 infection, which is generally regarded as a prototypical retrovirus and HbV infection, which has properties both of retroviruses and DNA genome viruses, the method of the invention has application to the treatment of any diseases wherein the etiologic agent comprises a virus. In addition, chronic virus infections are commonly associated with cytokine imbalances that pro- duce additional pathogenic effects, including the accu¬ mulation of bioinactive 2-5A. The present invention operates to correct the effects of this imbalance by supplying bioactive 2-5A. The preparation of certain of the compounds utilized in the practice of the present invention are described in U.S. Patents 4,859,768 and 4,924,624. The other compounds used in the practice of the invention may be prepared by following the general synthetic tech¬ niques described in those patents. Examples of compounds for use in the method of the invention include the following core compounds, their corresponding 5' mono-, di-, and triphosphates, and the pharmaceutically acceptable salts of any of them: Mixed cordycepin/adenosine oligomers: 2',5'-C-C-C, 2',5'-A-A-C, 2',5'-A-C-C, 2',5'-C-C-A, 2',5'-C-A-C, 2',5'-C-C-A, and 2',5'-A-C-A, in addition to the various "tetramer" combinations of A and C, including but not limited to, 2',5'-C-C-C-C, 2',5'-A-A-A-C, 2',5'-A-A-C-C, 2',5'-A-A-C-A, and the like. Alkoxy compounds: 2' ,5'-A-A-A-3'-O-methyl 2' ,5'-A-A-A-3'-0-pentyl 2' ,5'-A-A-A-3'-O-hexyl 2' ,5'-A-A-A-3'-O-heptyl Amino compounds: 2' ,5'-A-A-A-3'-amino Miscellaneous compounds: 7 ' ~\ '-A . A . A 2',5'-A-A-ara-A, 2',5'-A-A-Tu, 2' ,5'-Tu-Tu-Tu, 2',5'-I 3 , 2' ,5'-xylo-A 3 , 2' ,5'-xylo-A 4 , 2',5'-C-C-dCF, 2' ,5-EHNA-A-A, and 5 ,6-di hlorobenzimidazylyl(2,'5')5 ,6-dichloro- benzimidazylyl(2' ,5')5,6-dichlorobenzimidazole ribo- side, 3'-deoxyadenylyl-(2'-5' ) -3'-deoxyaden¬ ylyl-(2'-4')-9-(4'hydroxybutyl)adenine, and 3 ' -deoxyadenylyl- (2 ' -5 ' ) -3 '-deoxy¬ adenylyl-(2'-4') -9-(4'hydroxyethoxy)adenine. The two last mentioned compounds, charac¬ terized by an ether linkage between a cordycepin nucleo¬ tide and adenine, are from a group collectively referred to as "C-C-ether-A". Correspondingly, when the cordey- cepin residues are substituted by adenosine, the com¬ pounds are referred to collectively as "A-A-ether-A". Inhibition of HBV Replication by 2-5A Derivatives The antiviral activity of several 2-5A deriva¬ tives against hepatitis B virus was tested. Human liver /? cells chronically producing HBV (Acs et al. , Proceedings of the National Academy of Sciences. USA 84:4641-4645 (1987)) were seeded into 24 well tissue culture plates and grown to confluence. 2-5A derivatives were then 5 added at 20 μM for a continuous 9 day period. Culture medium was changed daily. Spent culture medium was analyzed for extracellular HBV DNA after 0, 3, 6 and 9 days. Treated cells were lysed 24 hours following the 9 day treatment and analyzed for intracellular genomic 10 DNA forms. The protocol has been published by Korba and Milman (Antiviral Research 217:217 (1991)). The assay parameters are described in more detail below. Both intracellular and extracellular HBV DNA were analyzed by procedures that are routine in the art 15 in order to (1) allow for verification of compound effi¬ cacy, and (2) provide possible data on the target site in the HBV replication pathway for the compound from the examination of the pattern of viral replicative forms. The culture medium was changed daily during the treat- 20 ment period to (1) prevent the buildup of potentially toxic metabolites from the test compounds, and (2) pro¬ vide an analysis of HBV virion production during dis¬ crete 24-hour intervals which enables a quantitative comparison of any effect on virion production. 25 The analysis of HBV DNA was performed using blot hybridization techniques (Southern and slot blot) and [ 32 P]-labeled HBV-specific probes. HBV DNA levels were measure by comparison to known amounts of HBV DNA standards applied to every nitrocellulose membrane (gel 30 or slot blot) . An AMBIS beta scanner, which measures the radioactive decay of the hybridized probes directly from the nitrocellulose membranes, was used for the quantitative analysis. Standard curves, generated by multiple analyses, were used to correlate cpm measure- 35. ments made by the beta scanner with relative levels of target DNA. The levels of HBV virion DNA released into the culture medium were analyzed by a slot blot hybrid- ization procedure. HBV DNA levels were then compared to those at day 0 to determine the effect of drug treat¬ ment. The levels of replicate intermediate and epi- so al monomers were used as an indicator of the relative levels of HBV replication. Integrated HBV DNA was used to normalize the relative amounts of DNA in each lane because the levels of this class of HBV DNA remain con¬ stant-on a per cell basis. Inhibition of HBV DNA repli- cation is indicated by the loss of replicative interme¬ diates without changes in the level of integrated HBV DNA. In this assay, the following 2-5A derivatives showed anti-HBV activity: xyloA 3 , A-A-ether-A, EHNA-AA, CAC, ACCA and dd benz. To study these inhibitions further, 5 of those 2-5A derivatives were diluted and tested at 2 μM and 6 μM, in addition to 20 μM. The five 2-5A derivatives displayed marked anti-HBV inhibitory power at 20 μM and 3 of the 5 (A-A-ether-A, dd benz and CAC) were also very active at 2 μM. These results demonstrate that a vari¬ ety of 2-5A derivatives are active against HBV. In accordance with the invention, it maybe concluded that anti-HBV of these and other 2-5A derivatives will be enhanced by adding 5' terminal modifications that aug- ment the ability of the 2-5A derivatives to activate RNase L. Also in accordance with the invention, it may be concluded that anti-HBV activity will be augmented further by conjugating said 5' modified and 5' unmodi¬ fied 2-5A derivatives with lipophyllic adducts such as cholesterol, palmitate, folate, etc. To determine the therapeutic ratio of the 5 2-5A derivatives that were titrated for anti-HBV activi¬ ty, their toxicity was measured with a conventional neutral red assay. The protocol for determining the toxicity of these compounds in culture is routine in the art and can be summarized as follows. 2.2.15 cells were grown to confluence in 96 well flat-bottom tissue cul- ture plates and treated with compounds in 0.2 ml of culture medium per well. Four concentrations of each compound were assayed, each in triplicate cultures. Untreated control cultures were maintained on each 96 well plate. On each 96 well plate, wells containing no cells were used to correct for light scattering. Toxic¬ ity was determined by the inhibition of uptake of neu¬ tral red dye, as determined by the absorbance at 510 nanometers relative to untreated cells (Finter et al., J. Med. Chem 5:419 (1969)), 24 hours following day 9 of treatment. No toxicity was observed for the aforesaid 2- 5A derivatives at the highest concentration tested for antiviral activity (20 μM) . Of the five 2-5A deriva- tives tested, four showed no toxicity at 9 times the effective antiviral dose (180 μM) . The fifth, dd benz, showed low toxicity at 180 μM. To study further the specificity of the HBV inhibition by the said 2-5A derivatives, their breakdown products were tested for possible anti-HBV activity. No anti-HBV activity was found with benzimidazole, di- benzimidazole 2 , adenosine, cordecypin or 9-β-D-xylo- furanosyladenine. These results show that the 2-5A derivatives specifically inhibit the replication of HBV at concentrations that are not toxic to uninfected cells and that the inhibition is due to the 2-5A derivatives and not to their metabolites. Viral Inhibition bv Phosphorothioate Oligomers. RNase L is a key functional enzyme of the 2-5A synthetase/RNase L antiviral defense pathway. The acti¬ vation of this unique 2-5A-dependent endoribonuclease by 2-5A derivatives and the subsequent hydrolysis of viral RNA is critical in the inhibition of virus repli- cation and regulation of cell growth. Some of the anti- retroviral properties of interferon are mediated by activation of the 2-5A synthetase/RNase L pathway. *- Direct activation of RNase L by 2-5A derivatives bypass¬ es the requirement for interferon in establishing an antiretroviral state. Modification of the 2-5A molecule at the 3'-hydroxyl groups and the 2',5'-internucleotide linkages has resulted in 2-5A derivatives that are meta¬ bolically stable (and therefore resistant to degradation byphosphodiesterases andphosphatases) andbiologically active (capable of activating RNase L) . Manipulation of synthetic procedures permits the design and synthesis of 2-5A derivatives which (i) display increased metabol¬ ic stability against nucleolytic degradation, (ii) are phosphatase- resistant, (iii) do not require 5'-phos- phorylation for activation of RNase-L, and (iv) can be transported into intact cells. Chirality was introduced into the 2',5'-phosphodiester bond of the 2-5A molecule as an approach to differentiate between RNase L activa¬ tion and inhibition at the molecular level. The pure dimers and trimers of the diastereomeric phosphoro- thioate derivatives of 2-5Awere first synthesized enzy- atically. More recently, chemical synthesis, purifica¬ tion and identification of the two dimer, four trimer and eight tetramer diastereomers have been achieved via the phosphotriester and phosphoramidite approach. These chiral molecules exhibit striking biological activities which altered the dogma that three adenylate residues and two 5'-terminal phosphates were required to activate RNase L. In contrast to authentic 2-5A core molecules which are unable to activate RNase L, three of the four 2',5'-phosphorothioate trimer cores (RpRp, SpRp and RpSp) bind to and activate RNase L (at 10 "5 M) , as do the 2 ' ,5'-phosphorothioate 5'-monophosphates (pRpRp, pRpSp, and pSpRp) (at 10 -8 M) . To determine whether these in vitro studies could be extended to an in vivo model with virus-infect- ed cells, the 2',5'-phosphorothioate tetramer 5'— monophosphates were microinjected into the cytoplasm of HeLa cells and the cells were then challenged with VSV. The 2' ,5'-phosphorothioate tetramer 5'-monophosphates (pRpRpRp, pSpRpRp and pRpSpSp) all activated RNase L. However, the pSpSpSp isomer (at concentrations as high as 10 "6 M) did not. Microinjection of the pSpSpSp inhib- itor simultaneously with 2-5A eliminated protection against VSV replication. The R p - and S p -2',5'-phosph¬ orothioates inhibit HIV-1 replication in intact cells in culture. The 2',5'-phosphorothioates protect target cells from HIV-1 infection by inhibition of HIV-1 RT (measured in Triton X-100-activated lysates of HIV-1 virions from H9/HTLV-IIIB culture supernatants) . HIV-1 RT was not inhibited by A 3 or p 3 A 3 at 0.25-256 μM. In contrast to 2-5A, the 2',5'-phosphorothioate tetramer 5'-monophosphate derivatives are very effective inhibi- tors of HIV-1 RT activity. The most effective inhibitor of HIV-1 RT in HIV-1-infected cell lysates is p 3 A 3 αS with 50% inhibition observed at 0.5 μM. AMPS did not inhibit HIV-1 RT up to 200 μM, suggesting that degradation prod¬ ucts are not responsible for the inhibition observed with 2',5'-phosphorothioates. d(T) 16 and tRNA ys - 3 bind specifically to the kinetically significant primer binding site of homoge¬ neously pure recombinant p66 HIV-1 RT as demonstrated in UV crosslinking studies. Binding of oligo d(T) n to p66 RT is not affected by dNTPs; in competition assays, the primer analogs, Sd(C) 28 and d(C) 19 _ 24 , the natural primer tRNA Lya-3 and p 3 A 3 αS inhibit oligo d(T) n -p66/RT complex formation in a first-order exponential manner.. The inhibition of HIV-1 RT by 2-5A and 2-5A derivatives occurs at the primer binding site. Under equilibrium binding conditions, 2',5'-p 3 A 4 exhibits first order binding kinetics with one half competition at 55 μM (K D = 31 x 10" 6 M) . 2',5'-p 3 A 4 αS exhibits first order binding kinetics with one half competition at 5.1 x 10 "6 M (K D =2- .9 x 10" 6 M) . 2',5'-A 3 core, 2',5'-pA 3 , 3',5'-A 3 core, 3',5'-pA 3 , 3',5'-p 3 A 3 and ATP do not inhibit binding of HIV-1 RT to d(T) 16 . The 2',5'-cordycepin trimer core and 5'-mono- phosphate (1 μM) (when incorporated into antibody- targeted liposomes specific for the T cell receptor molecule CD3) inhibited 90% of HIV-1 replication. See Table 1 of Mύller, W.E.G., Weiler, B.E., Charubala, R. , Pfleiderer, W. , Leserman, L. , Sobol, R.W. , Suhadolnik, R.J., and Schroder, H.C., Biochemistry 30. 2027 (1991). Dot-blot and gel-retardation assays showed that 2',5'- cordycepin trimer core and 5'-monophosphate interfere with the binding of tRNA Lys ' 3 to HIV-1 RT. See Figure 3 of Mύller et al. The aforesaid publication is incorpo¬ rated herein by reference. pC 3 did not stimulate RNase L activity and displayed no effect on the amount of cellular RNA or protein. At a concentration of 10 μM, the cellular DNA polymerases α, β and γ were almost insensitive to C 3 or pC 3 [C = cordycepin] . We have available for the first time, anti- retroviral molecules (2-5A derivatives) which possess dual effects on HIV-1-infected cells: (i) they activate the 2-5A synthetase/RNase L antiretroviral system and (ii) inhibit HIV-1 RT. With the 2',5'-phosphorothioate cores and their 5'-monophosphates, it is possible to prevent acute infection (by inhibition of HIV-1 RT) and simultaneously degrade HIV-1 mRNA by activation of RNase L. Conjugates of 2,5'-01igoadenylate Derivatives Having Increased Cellular Uptake To facilitate internalization of 2-5A deriva- tives into intact cells, bioactive 2-5A derivatives are conjugated to water-soluble vitamins (folic acid, bio- tin, riboflavin, or vitamin B 12 ) or to lipophilic chemi¬ cal groups. By exploiting this technique, 2-5A deriva¬ tives may be delivered to intact cells without membrane damage. This approach involves (i) the synthesis of bioactive, metabolically stable derivatives, e.g., 2' ,5'-C n , 2' ,5'-phosphorothioates (prepared accordingto U.S. Patent 4,924,624), and other metabolically stable derivatives such as those disclosed in U.S. Patent 4,- 859,768. 2-5A derivatives according to the disclosure of the above-mentioned documents, have been designed, synthesized and tested for their antiretroviral effect at the level of RNase L and inhibition of RT. 2',5'- Oligonucleotides may be prepared enzymatically in multi- step chemical syntheses via the phosphotriester and phosphoramidite approach, in milligram and gram amounts, respectively. The correct choice of appropriate block¬ ing groups for sugar, base and phosphate protection determines yields, ease of purification and chromato- graphic behavior. The successful facilitated delivery of biolog- ically active, metabolically stable 2-5A derivatives is key to continuation of the mechanistic studies of inhi¬ bition of HIV-1 replication, and effective drug deliv¬ ery. Several strategies for the assisted delivery of 2-5A derivatives into intact cells may be used. 2'.5 ,-Oligoadenγlate Lipophilic Conjugates A first method of assisted delivery involves covalent conjugation of lipophilic groups (e.g., acyl or cholesteryl groups) to the 2',3'-terminus of the molecule. For example, the 2',5'-cordycepin trimer core may be synthesized and purified with palmitoyl (Formula III) or cholesterylformyl (Formula IV) ester linkages at the 2'-terminal hydroxyl group: C-- These esterified compounds have been found to inhibit HIV-1 replication in HIV-1 infected H9 cells in culture. 2-5A-analog cholesterol conjugates may be synthesized by reacting a conjugating compound of Formu¬ 7 wherein m is 0, 1, 2 or 3 n is an integer from 1 to 8, R is independently selected from the group consisting of hydrogen and hydroxyl, R 2 is independently sulfur or oxygen R 3 is independently sulfur or oxygen, and x is an integer from 1 to 17 with chlotesterylchloroformate in the presence of di- methylaminopyridine (DMAP) and dichloromethane. Purifi¬ cation is accomplished by column chromatography. Preferably, not all R groups are hydroxyl, that is, at least one R is hydrogen. Preferably, n is 1 to 3, most preferably 1 or 2. The R 2 of the 5'-termi¬ nal phosphonyl group is preferably sulfur. Finally, the value of X is preferably from 1 to 8, most preferably 1 to 4. The conjugating 2-5A derivative according to Formula V may be prepared by functionalizing an appro¬ priate 5'-phosphorylated 2',5'-oligoadenylate, 2',5'- oligocordycepin or mixed 2' ,5'-oligo(adenylate/cordy- cepin) oligomer, such as may be prepared from either U.S. Patent 4,464,359 or 4,859,768. The oligomer is functionalized by coupling the 2'-terminal nucleotide thereof to an alkyl linker -(CH^ x -R j wherein X and R x are defined above. It should be appreciated that, in addition to oligomers containing phosphodiester internucleotide linkages, the conjugating 2-5A derivative of Formula V may be prepared by attaching the alkyl linker to the 2'- terminal nucleotide of a 2',5'-phosphorothioate oligonu- cleotide. The preparation of the 2',5'-phosphoro¬ thioates, including fully resolved enantiomers thereof (the phosphorothioates are optically active) , is dis¬ closed in U.S. Patent 4,924,624. It may be appreciated that by following the combined synthetic techniques of Patents 4,924,624 and 4,859,768, that a mixed 2',5'- (phosphodiester/phosphorothioate)-oligoadenylatemaybe prepared. In the case where the R 2 group of all inter- nucleotide bonds of the molecule comprise oxygen, i.e., the linkages comprise phosphodiester bonds, the 5'-mono¬ phosphates are readily prepared by reacting the corre¬ sponding unphosphorylated core compound with P0C1 3 . In the case wherein at least one internucleotide linkage comprises a phosphorothioate bond, i.e., R 2 =sulfur, such treatment would result in the elimination of sulfur from the phosphorothioate internucleotide linkage, and the formation of a 2',5'-oligoadenylate. Thus, the 5'-mono¬ phosphates of phosphorothioate bond-containing core oligomers must be prepared from the corresponding fully protected core oligomer fromwhich the monomethoxytrityl blocking groups on the 5'-terminal nucleotide has been removed. The procedure is described in detail in U.S. Patent 4,924,624, columns 26 through 31 thereof. 2-5-derivative/acyl conjugates may be synthe¬ sized by reacting the conjugating compound with acyl chloride, according to standard techniques. Alterna¬ tively, the 2',5'-oligomer may be synthesized directly with an acyl group or cholesteryl formyl group esteri- fied to the 2'-terminal hydroxyl group. One such method is illustrated by but not limited to the preparation of the cholesterol conjugated and acylated cordycepin trimer compounds comprising compounds 12 . and 13 of Scheme 1. SCHEME 1 β*> SCHEME 1 (continued) SCHEME 1 (continued) 35 3- SCHEME l (continued) According to Scheme 1, a multistep chemical synthesis starts from 5'-0-dimethoxytrityl-N 6 -p-nitro- phenylethoxycarbonyl-cordycepin (l) and the correspond¬ ing 2'-O-succinate derivative (2.) . Compound l. is react- ed with colesteryl chloroformate to the carbonate 3 . which is used as the 2'-terminal building block after detritylation to 4.. The succinate 2 is condensed with cholesterol to the ester S_, and detritylation leads to compound 6.. The synthetic route to the trimer conjugates is illustrated in Scheme 2 by one example starting from compound j6. This cordycepin derivative is first reacted with the fully protected phosphoramidite 7_ to give in excellent yield with the dimer 8. Detritylation at the 5'- end forms and subsequent condensation with a sec¬ ond molecule of 7. gives the fully protected trimer 10. The latter is sequentially treated with acid to cleave the dimethoxytrityl group and by DBU to cleave the p- nitrophenylethoxycarbonyl groups simultaneously, to afford the free cordycepin trimer conjugate .11. The advantage of this approach can be seen from the fact that the ester function between cholesterol and the succinate spacer does survive all the chemical manipula¬ tions. In an entirely analogous manner the choles- terylcarbonate 4. is reacted according to the same se¬ quence of steps to prepare the cordycepine trimer conju¬ gate 12. Also in an entirely analogous manner, the cordycepin trimer conjugate .13 . , which carries an acyl residue at the 2'-terminal end of the oligonucleotide, is prepared. Lipophilic conjugates according to the present invention have the potential to enter the cell by diffu- sion, owing to their lipophilic nature. They may be introduced into intact cells with substantially the same biological activity as authentic 2-5A. Once inside the cell, the conjugates are hydrolyzed and release the 2-5A derivative. 2 >,5 -01igoadenylate Vitamin Conjugates A second method of assisted oligonucleotide delivery involves covalent conjugation of 2-5A deriva¬ tives to water-soluble vitamins such as biotin, folic acid, riboflavin or vitamin B 12 . Covalent conjugation of 2-5A derivatives to water-soluble vitamins provides a method for co-delivery of active molecules into intact cells by exploiting receptor-mediated endocytosis with retention of essentially full biological activity. As a mode for demonstrating this principle, we have undertaken internalization of fluorescein-bovine serum albumin (BSA) covalently conjugated to folate in H9 cells in culture (Figure 2A and 2B) . In control experiments, H9 cells incubated for 5 hours with fluo- rescein-BSA, but not conjugated to folate, were not taken up (Figures 2C and 2D) . These results demonstrate the presence of folate receptors on H9 cells, which receptors may be used for targeted delivery of 2-5A derivatives. The folate receptors are able to bind to the folate-conjugated molecule and undergo uptake into 80% of the cells in 5 hours. 2-5A derivatives can be covalently conjugated to water-soluble vitamins to facilitate delivery of the polar 2-5A molecule into intact cells without membrane damage. The water-soluble vitamins are covalently con¬ jugated to the 2',3'-terminus of the 2-5A molecule to exploit receptor-mediated endocytosis for uptake into intact cells. Nondestructive delivery of polar mole¬ cules covalently conjugated to biotin, folic acid, ribo¬ flavin and vitamin B 12 into the cytoplasm and subsequent efficient uptake via receptor-mediated endocytosis has been well demonstrated. In this way, membrane damage arising from other established methods (such as micro- injection, electroporation, sonication, detergentperme- abilization) can be avoided by employing the natural vitamin endocytosis pathway to accomplish uptake. Vita¬ min uptake occurs in all dividing cells at reasonable rates. Based on the known efficiency of uptake of vita- min conjugates, it is believed that an intracellular concentration of a 2-5A/folic acid conjugate of about 10 "6 M can be achieved; these concentrations are suffi¬ cient to activate RNase L and inhibit HIV-1 RT. Similar intracellular concentrations are expected with 2-5A/bio- tin and the 2-5A/vitamin B 12 conjugates on the basis of the similar numbers of folate, biotin and vitamin B 12 receptors. 2-5A Vitamin B 17 Conjugate A vitamin B 12 covalent conjugation of 2-5A or a bioactive 2-5A derivative is prepared by first con¬ verting vitamin B 12 to the mono-acid derivative thereof by mild acid hydrolysis (0.4 M HCl, 72 hours at room temperature) . A 2-5A conjugating compound, such as a compound according to Formula V (R α = NH 2 ) , is reacted with the carboxyl group of vitamin B 12 using an appropri¬ ate coupling agent such as l-ethyl-3-(dimethyl-amino- propyl)-carbodiimide HCl (EDAC) . The 2-5A/vitamin B 12 conjugate is purified by either chromatography on Seph- adex G-25 or reverse phase HPLC. Characterization is based on the reported molar extinction coefficients for 2-5A and vitamin B 12 . 2-5A/Folic Acid Conjugate The 2-5A/folic acid conjugate is synthesized by reacting a conjugating compound, such as a compound according to Formula V (R λ = NH 2 ) , with a suitable cou¬ pling reagent such as EDAC, followed by purification using either column chromatography, thin layer chroma- tography or HPLC. >££ 2-5A/Biotin Conjugate Biotinylated 2-5A conjugatemay be synthesized byreactingcommerciallyavailableN-hydroxysuccidimidyl biotin with a Formula V compound (R x = NH 2 ) , followed by purification as described for the 2-5A/folic acid conju¬ gate. Preparation of 2 ',5'-Cordycepin Analogs Containing 3 / -Terminal Acyclic Nucleoside Trimer "core" cordycepin analogs according to Formula I, i.e., compounds wherein m is O and n is 1 may be prepared according to Scheme 2, wherein "bz" is benzoyl, "MeOTr" is monomethoxytrityl, "npe" is 2-(4- nitrophenyl)ethyl and "npeoc" is [2-(4-nitrophenyl)- ethoxy]carbonyl. For illustration purposes, the com¬ pounds are prepared as ammonium salts, it being under¬ stood that the invention is not so limited. The Scheme is exemplified by the preparation of four compounds, 18, 19. 20 and 21. Scheme 2 Me0TrO 14 bz MeOTr npβ npeoc CH, 15 bz MeOTr npβ npβoc CH 2 CH 2 16 bz MeOTr npe npeoc CH 2 CH,C 17 bz MβOTr npe npβoc CH j CHjO bz " benzoy! 18 H H NH, H CHj MβOTr » mono ethoxytrityl - 19 H H NH, H CH 2 CH 2 , H CH j CH j C np β »2-(4-nhrophβnyl)ethyl 20 H H NH CH j CHjO np β oc - [2-(4-nitrophenyl)βthoxy]carboπyl 21 H H NH, H S The dinucleotide monophosphodiester 9. was prepared from3'-deoxy-5'-0-(monomethoxytrityl)-N 6 -[2-(4- nitrophenyl)ethoxycarbonyl]adenosine (4 . ) according to the method of Charubala et al. , Helv. Chim. Acta 70, 2028 (1987). Compound 4. which may be prepared according to Charubala et al. , was taken and converted into the 2'-phosphotriester 5 . with a 2,5-dichlorophenyl and a 2- (4-nitrophenyl)ethyl group at the phosphate function (Scheme 1) according to the method of said Charubala et a . reference. From 5., the 2,5-dichlorophenyl group was cleaved off with 4-nitrobenzaldehyde oxime to give dies- ter 6.. On the other hand, the 5 '-O-monomethoxytrityl group was removed by acid to give the 5'-hydroxytriester 7_. These two building blocks 6. and 7_ were condensed in presence of 2,4,6-triisopropylbenzenesulfonyl chloride and 1-methyl-lH-imidazole to yield in 88%, after purifi¬ cation and drying, the dimeric phosphotriester 8.. Again, the 2,5-dichlorophenyl group from this dimer block was cleaved off by the oximate method to give the corresponding dinucleotide monophosphodiester 9.. For the syntheses of trimers 14-17, the above dimeric phosphodiester 9^ was condensed with the suitably blocked appropriate acyclic nucleosides 10-13 in pres¬ ence of 2,4,6-triisopropylbenzenesulfonyl chloride and 1-methyl-lH-imidazole in yields of 70-75%. Acyclic nucleosides 10-13 may be prepared according to the pro¬ cedure of Tychinskaya et al. , Bioorg. Khim (USSP) 5 , 1059 (1979) and Mikhailov et al. , Izv. Akad. Nauk. Ser. Khim. 2582 (1974) . Dimer diester 9. (lmmol) and the ap- propriate acyclic nucleoside analog 10-13 (0.75 mmol) were coevaporated with dry pyridine (5 x 10 ml) , and finally dissolved in pyridine (10 ml). Then 0.46 ml (6 mmol) of 1-methyl-lH-imidazole and 0.606 g (2 mmol) of 2,4,6-triisopropylbenzenesulfonyl chloride were added successively. The mixture was stirred at room tempera¬ tures for 20 hours, then diluted with CHC1 3 (100 ml) , and washed with H 2 0(2 x 50 ml) . The organic phase was dried and evaporated, and after coevaporation with toluene (3 x 20 ml) , the residue was purified by CC (silica gel, 10 x 2.5 cm, CHCl 3 /MeOH 100:1) : 14-17 as colorless foams in 85-90% yield. Accordingly, to a solution of protect- ed trimer 15-18 (0.028 g; 15 μmol) was added 0.5M DBU in dry pyridine (5 ml) . The mixture was stirred at room temperature for 20 hours, neutralized with IM AcOH in pyridine (2.5 ml), and evaporated. The residue was stirred with concentrated NH 3 (15 ml) and after stirring for 48 hours, the solvent was removed in vacuo and the residue detritylated by treatment with 80% AcOH/H 2 0 (4 ml) at room temperature for 24 hours. After evapora¬ tion, the residue was coevaporated several times with H 2 0, taken up in H 2 0 and applied onto a DEAE A-25 Seph- adex ® column (60 x 1 cm) and eluted with a linear gradi¬ ent 0.001-0.3M (Et 3 NH) and HC0 3 (TBK) buffer (pH 7.5). The product fractions were evaporated and coevaporated several times with H 2 0 and further purified by paper chromatography (i-PrOH/conc. NH 3 /H 2 06:1:3) . The product band was cut out and eluted with H 2 0 and lyophilized to give3'-deoxyadenylyl-(2'-5' )-3'-deoxyadenylyl-(2'-2')- 9-(2'-hydroxyethy1)adenine (diammonium salt; ljjϊ) , 3'- deoxyadenylyl-(2 '-5')-3'-deoxyadenylyl-(2'-3')-9-(3'- hydroxypropyl)adenine (diammonium salt; .19.), 3'-deoxy- adenylyl-(2'-5')-3'-deoxyadenylyl-(2'-4' )-9-(4'-hydro- xybutyl)adenine (diammonium salt; .20.), and 3'-deoxy¬ adenylyl-(2'-5')-3-deoxyadenylyl-(2'-2")-9-[(2"-hydro- xyethoxy)methyl]adeninediammoniumsalt; 2JL, respective¬ ly, as colorless powders in 75-85% yield. The purity of all products was checked by TLC, UV, and -NMR spec¬ tra. All trimers were deblocked sequentially, using 0.5M DBU (l,8-diazabicyclo[5.4.0]undec-7-ene) in dry pyridine for 20 hours at room temperature to cleave the 2-(4-nitrophenyl)ethyl groups, NH 3 in dioxane to remove the benzoyl groups, and finally acid treatment to de¬ block the 5'-O-monomethoxytrityl group yielding the corresponding fully deblocked trimers 18-21, which were purified by DEAE sephadex column chromatography and paper chromatography. The trimers were characterized by TLC, UV, HPLC, and 1 H- MR spectra (data not shown) . Tetramers (C-C-C-ether-A) may be prepared by substituting for the dinucleotide monophosphodiester 9. in Scheme 2 the corresponding protected trinucleotide 9a which is condensed with the suitably blocked appro¬ priate acyclic nucleoside, as above. Similarly, it should be clear that higher oligomers of the present 3'- acyclic cordycepin oligomers may be prepared by select¬ ing the appropriate higher oligonucleotide for condensa¬ tion with the acyclic nucleoside. The 5'-monophosphates of the unphosphorylated core molecules may be prepared from the fully blocked trimers, e.g., 14-17 by detritylation with acid treat¬ ment, followed by reaction with di-p-nitroph- enylethylphosphoryl chloride. Further deblocking and chromatography results in isolation of the 5'-monophos- phate oligomers. The 5'-monophosphorylation procedure may be carried out according to the method of Example 6 of U.S. Patent 4,859,768. The 5'-diphosphates and 5'-triphosphates of the core molecules may be prepared according to the procedure of the aforesaid patent. Briefly, they may be prepared by adding 0.5 mM of tributylammonium pyro- phosphate dissolved in 5 ml of dimethylformamide to O.lmM of monophosphorylated core as the anhydrous tri¬ butylammonium salt in 1 ml of dimethylformamide and 0.5 mM of l,l'-carbonyldiimidazole. After 20 hours at room temperature, the reactants are treated with 5ml of meth- anol, evaporated to dryness and chromatographed on a 2x20 cm DEAE cellulose column. The 5'- di- and tri¬ phosphates are isolated following a linear gradient (0- 0.4M in 3 1 at pH 7.5) of triethylammoniumbicarbonate, according to the method of Hoard, et al. , J. Amer. Chem. Soc. 87, 1785-1788 (1965). The 5'-diphosphates and 5'- ft triphosphates may then be purified by DEAE-Sephadex ® A25 and Sephadex ® . The 2-5A analogs and conjugates may be admin¬ istered according to the methods described in Patents 4,924,624, 4,859,768 and International Patent Applica¬ tion No. PCT/US85/03634 (International Publication No. WO 89/03683) in the dosages recommended therein. Thus, for pharmaceutical use, the 2 ',5'-oligoadenylate analogs may be taken up in pharmaceutically acceptable carriers. Such carriers for preparation of pharmaceutical composi¬ tions may be either organic or inorganic, solid or liq¬ uid in nature. Suitable liquid carriers include water and alcohols such as ethanol, benzyl alcohol and poly(ethylene glycols) . The preferred liquid carriers for injectable preparations include water, saline solu¬ tion, dextrose solution and glycol solutions such as aqueous propylene glycol or aqueous poly(ethylene gly¬ col) . The properties of the formulations may be en¬ hanced by the addition of one or more adjuvants possess- ing properties as viscosity enhancers, surfactants, pH modifiers, preservatives, sweeteners, stability en¬ hancers, coloring agents, suspending agents, granulating agents, coating agents, disintegration modifiers, pro- pellants, emulsifying agents and hymectants. The re- suiting compositions may be in the form of solutions, suspensions, tablets, capsules, ointments, elixirs, injectable compositions and the like. The dosage administered depends upon the se¬ verity of the infection or affliction and the size and weight of the subject. The dosage may vary over a wide range depending on the nature of the affliction, and the size and weight of the subject. According to one em¬ bodiment, the compounds are prepared as a solution of 0.1-100 mg/ml in water, phosphate-buffered saline or other appropriate fluid, or may be prepared as a tablet containing 0.01-1 gram active compound. The compounds may be administered in the form of water-soluble salts. Pharmaceutically acceptable water-soluble salts include, for example, the sodium, potassium and ammonium salts of the active compounds. They are readily dissolved in water or saline solution, the formulation may contain additional agents, such as a sugar or protein, to maintain the osmotic balance. The 2',5'-oligoadenylate analogs maybe admin¬ istered in doses of about 0.1 g to about 1 gram to animals or humans afflicted by, or suspected of afflic¬ tion by, or at risk or affliction by, any of the various conditions characterized by a 2-5A pathway defect. The total daily dosage may vary, for example, from about 0.001 gram to about 1 gram, although lower or higher amounts may be administered. A preferred daily dose is from about 0.01 gram to about 0.1 g of active ingredi¬ ent. The compounds may be administered by any of sever¬ al routes, including, but not limited to, intravenous injection, intraperitoneal or intramuscular injection, and oral administration. Techniques for accomplishing such administration are routine and known in the medical art. Administration may be as frequent as several time a day or as infrequent as weekly. For intravenous in¬ jection, particularly for treatment of HIV, a solution containing about 0.1 to about 1.0 milligram per ml of active ingredient is preferred. It is also contemplated that the 2' ,5'- oligoadenylate analogs may be administered topically to treat skin lesions associated with any of the disease states characterized 2-5A pathway defect. A sufficient amount of a preparation containing one or more of the 2' ,5'-oligoadenylate analogs may be applied to cover the lesion or affected area. An effective concentration of active agent is from about 10 "3 M to about 10 "5 M, with about 10 ™1 M being preferred. For conjugates if 2-5A analog and cell pene¬ tration-enhancing adducts, the dosage is based upon the weight of the 2-5A analog moiety. In addition to admin¬ istration with conventional carriers, the 2-5A analogs may be administered by a variety of specialized oligonu- cleotide or nucleic acid delivery techniques, such as by encapsulation in unilameller liposomes or reconsti¬ tuted sendai virus envelopes, or by conjugation to car¬ rier molecules such as poly(L-lysine) . Such methods are disclosed in International Patent Application No. PCT/- US85/03634, the entire disclosure of which is incorpo- rated herein by reference. In addition, 2-5A derivatives and conjugates may be administered via microscopic nanometer-size poly¬ styrene carriers which dissolve in the bloodstream to release the active drug. Other suitable carriers are discussed in the aforementioned patent documents, the entire disclosures of which are incorporated herein by reference. All references with respect to synthetic, preparative and analytic procedures are incorporated herein by reference. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims: CLAIMS A compound according to the formula wherein n is a whole positive integer from 1 to 8, m is 0, 1, 2 or 3, R is hydrogen, X is selected from the group consisting of Cl to C6 alkyl and Cl to C6 alkoxy, or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1 wherein X is selected from the group consisting of Cl to C3 alkyl and Cl to C3 alkoxy. 3. A compound according to claim 1 wherein the compound is selected from the group consisting of 3'-deoxyadenylyl-(2'-5')-3'-deoxyadenylyl(2'-2')-9-(2'- hydroxyethyl)adenine, the 5' mono-, di- and tri- phosphates thereof, and any pharmaceutically acceptable salts of any of them. 4. A compound according to claim 1 wherein the compound is selected from the group consisting of 3'-deoxyadenylyl-(2',5')-3'-deoxyadenylyl-(2' ,3')-9-(3'- hydroxypropyl)adenine, the 5' mono-, di- and tri- phosphates thereof, and any pharmaceutically acceptable salts of any of them. 5. A compound according to claim 1 wherein the compound is selected from the group consisting of 3'-deoxyadenylyl-(2',5')-3'-deoxyadenylyl-(2' ,4')-9-(4'- hydroxybutyl)adenine, the 5' mono-, di- and tri- phosphates thereof, and any pharmaceutically acceptable salts of any of them. 6. A compound according to claim 1 wherein the compound is selected from the group consisting of 3'-deoxyadenylyl-(2' ,5')-3'-deoxyadenylyl-(2' ,2')-9- [ (2"-hydroxethoxy)methyl]adenine, the 5' mono-, di- and triphosphates thereof, and any pharmaceutically accept- able salts of any of them. 7. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutical carrier. ■ Yl > 8. Aconjugate comprising a compound accord¬ ing to the formula wherein OH OH OH HO P X P X is NH 2 or CH 2 ; m is zero, 1 2 or 3; n is an integer from 1 to 8; each R 2 is independently selected from the group consisting of oxygen, sulfur, sulfate, sele¬ nium, C 2 to C 8 alkyl and C x to C 8 alkoxy; each R 2 is independently selected from the group consisting of oxygen, sulfur, sulfate, selenium, C x to C 8 alkyl and C x to C B alkoxy; each R 3 is independently selected from hydro¬ gen, hydroxyl, amino and -OS; (CH 3 ) 2 C(CH 3 ) 2 ; R 4 and R 5 are independently selected from the group sonsisting of hydrogen; hydroxyl; amino; C 1 to C 8 alkyl; C to C 8 alkoxy; C x to C 8 alkylamino, alkylcarbonyl, alkyl- carboxyl and alkylhalide; and C 2 to C 8 alkoxyamino, alkoxycarbonyl, alkoxycarboxyl and alkoxyhalide; or pharmaceutically acceptable salts thereof, excluding authentic 2',5'-oligoadenylate and salts thereof, said compound being covalently linked to an adduct which results in enhanced penetration into intact cells. 9. A conjugate according to claim 8 wherein the adduct comprises a vitamin selected from the group of vitamins which have corresponding cell surface recep¬ tors for receptor-mediated endocytosis of the vitamin. 10. A conjugate according to claim 9 wherein the adduct is vitamin B 12 . 11. A conjugate according to claim 9 wherein the adduct is biotin. 12. A conjugate according to claim 9 wherein the adduct is folic acid. 13. A conjugate according to claim 9 wherein the adduct is lipophilic. 14. A conjugate according to claim 13 wherein the adduct is an acyl group of the formula O » -C(CH 2 ) X CH 3 wherein x is an integer from 1 to 20. 15. A conjugate according to claim 13 wherein the adduct comprises the colesteryl group. 16. A conjugate according to claim 8 wherein m is zero, 1, 2 or 3, n is an integer from 1 to 8, each R 3 and R 4 is independently selected from hydrogen and hydroxyl, each R 2 is independently selected from sulfur and oxygen, R 5 is hydroxy, or a pharmaceutically acceptable salt thereof, which compound is covalently linked through the 2'-position of the 2'-terminal nucleotide thereof to said adduct. 17. A conjugate according to claim 16 wherein the adduct is a vitamin. 18. A conjugate according to claim 17 wherein the vitamin is selected from the group consisting of B 12 , folic acid and biotin. 19. A conjugate according to claim 16 wherein the adduct is lipophilic. 20. A conjugate according to claim 16 wherein the adduct is cholesteryl. 21. A conjugate according to claim 19 wherein the adduct is an acyl group of the formula 0 II -C(CH 2 ) X CH 3 wherein X is an integer from 1 to 20. 22. A conjugate according to claim 21 wherein the adduct is palmitoyl. 23. A method for inhibiting viral infection comprising administering to a mammal in need of such treatment an effective amount of a compound according to claim 1. 24. A method for inhibiting viral infection comprising administering to a mammal in need of such treatment an effective amount of a compound according to claim 2. 25. A method for inhibiting viral infection comprising administering to a mammal in need of such treatment an effective amount of a conjugate according to claim 8. 26. A method for inhibiting viral infection comprising administering to a mammal in need of such treatment an effective amount of a conjugate according to claim 9. cτ> 27. A method of antiviral treatment compris¬ ing administering to a mammal an effective amount of one or more compounds according to the formula wherein X is NH 2 or CH 2 ; m is zero, 1 2 or 3; n is an integer from 1 to 8; each R x is independently selected from the group consisting of oxygen, sulfur, sulfate, selenium, C x to C 8 alkyl and C x to C 8 alkoxy; each R 2 is independently selected from the group consisting of oxygen, sulfur, sulfate, selenium, Cj to C 8 alkyl and C to C 8 alkoxy; each R 3 is independently selected from hydro- gen, hydroxyl, amino and -OS; (CH 3 ) 2 C(CH 3 ) 2 ; R 4 and R 5 are independently selected from the group consisting of hydrogen; hydroxyl; amino; C γ to C 8 alkyl; C__ to C 8 alkoxy; Cj to C 8 alkylamino, alkylcarbonyl, alkyl- carboxyl and alkylhalide; and C to C 8 alkoxyamino, alkoxycarbonyl, alkoxycarboxyl and alkoxyhalide; or pharmaceutically acceptable salts thereof, excluding authentic 2',5'-oligoadenylate and salts thereof, to simultaneously cause activation of the 2-5A synthetase/RNase L antiviral pathway of said mammal and inhibition of viral DNA polymerase. 28. A method according to claim 27 wherein R 4 or at least one R 3 is hydrogen. 29. A method according to claim 27 wherein 30. A method according to claim 29 wherein at least one of R x is sulfur. 31. A method according to claim 30 wherein at least two of R 2 is sulfur. 32. A method according to claim 30 wherein each R λ is oxygen. <JC<5^ 33. A method according to claim 27 wherein R 4 and each R 3 are hydrogen. 34. A method according to claim 27 wherein at least one of R 2 is sulfur. 35. A method according to claim 29 wherein n is -1 or 2, R x is independently selected from oxygen and sulfur, and R 2 is independently selected from oxygen and sulfur. 36. A method according to claim 35 wherein each R 3 and R 4 is independently selected from hydrogen and hydroxyl. 37. A method according to claim 36 wherein s is hydroxyl. 38. A method according to claim 37 wherein each R 3 is hydrogen.

Inventors: PFLEIDERER WOLFGANG, SUHADOLNIK ROBERT J, PFLEIDERER, WOLFGANG, SUHADOLNIK, ROBERT, J.
Applicants: UNIV TEMPLE, TEMPLE UNIVERSITY - OF THE COMMONWEALTH SYSTEM OF
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61P31, C07H21, C07J43, C07J51, C08B37