POLY(ALKYL AND ALKENYL PHOSPHATE)S AND THEIR THIOPHOSPHATE AND SELENOPHOSPHATE DERIVATIVES AS ANTIVIRAL AGENTS

Abstract: Methods and compositions employing poly(alkyl and alkenyl phosphate)s and/or their thiophosphate derivatives are provided for treating viral infections. Preferably, polymers of the invention 20-30 monomers in length are used alone or in combination to treat HIV infections.

Description: POLY(ALKYL AND ALKENYL PHOSPHATE)S AND THEIR THIOPHOSPHATE AND SELENOPHOSPHATE DERIVATIVES AS ANTIVIRAL AGENTS Field of the Invention The invention concerns the use of certain 0 phosphate- and thiophosphate-based oligomers to treat viral infections. BACKGROUND The development of compounds useful for the 5 prophylaxis and therapy of viral disease has presented more difficult problems than those encountered in the search for 'drugs effective in disorders produced by other microorganisms. In contrast to most other infectious agents, viruses are obligate intracellular 0 parasites that require the active participation of the metabolic processes of the invaded cell. Thus, it is very difficult to find drugs that will inhibit viral functions without simultaneously damaging the closely related host cell mechanisms. At present, only a 5 handful of drugs are available for treating systemic viral infections, e.g. Galasso, pgs. 382-388 in Notkins et al, eds. Concepts in Viral Pathogenesis (Springer- Verlag, New York, 1984); Mandell et al, eds. Principles and Practice of Infectious Diseases, 2nd Ed. (John 0 Wiley & Sons, New York, 1985); and Dolin, Science, Vol. 227, pgs. 1296-1303 (1985). With the development of efficient methods of synthesis, interest has arisen in the use of anti-sense oligonucleotides as antiviral agents, especially in the case of the human inmmunodeficiency virus (HIV), e.g. Matsukura et al, Proc. Natl. Acad. Sci., Vol. 84, pgs. 7706-7710 (1987). An anti-sense oligonucleotide is a synthetic oligonucleotide of varying length, usually in the range of about 12 to 30 nucleotides, or nucleotide analogs, whose sequence is complementary to a predetermined segment of the RNA, either freshly transcribed or the messenger (mRNA), critical to some viral function. It is believed that when an anti-sense oligonucleotide hybridizes to its target RNA, it either blocks translation or processing of the RNA or makes it susceptible to enzymatic degradation. In view of the number of viral infections that remain refractory to prevention and therapy, and the dearth of effective antiviral agents, the discovery of new classes of compounds with .antiviral effects could be of great ' benefit to clinical medicine. SUMMARY OF THE INVENTION The invention relates to a method of treating viral diseases by administering with a pharmaceutical carrier an effective amount of a poly(alkyl or alkenyl phosphate) or a thiophosphate derivative thereof defined by the following formula: . ~ Fbrmn.la-X ~* K wherein: X and Y are separately selected from the group consisting of selenium, oxygen and sulfur; more preferably, X and Y are separately selected from the group consisting of oxygen and sulfur; and most preferably, at least one of X and Y are sulfur; n is in the range of about 16 to 60 inclusive; and more preferably, n is in the range of about 20 to 30; R is alkyl or alkenyl having from 1 to 8 carbon atoms, alkyloxy or alkylthio having from 1 to 8 carbon atoms and having from 0 to 2 heteroatomε; more preferably R is alkyl or alkenyl having from 3 to 6 carbon atoms, or alkoxy having from 3 to 6 carbon atoms and one oxygen atom; most preferably, R is alkyl having from 3 to 6 carbon atoms or a cyclic alkoxy having from 4 to 5 carbon atoms and one oxygen; and Q and Q' are separately hydrogen, R-OH, or a nucleoside selected from the group consisting of adenoεine, cytidine, guanosine, and thymidine. Brief Description of the Drawings Figures 1A and B illustrate data on the anti-HIV effect of two 28-mer compounds of the invention in a cytopathic effect inhibition assay. Figure 1C illustrates data on the anti-HIV effect of the control compound, 28-mer polydeoxycytidine, in a cytopathic effect inhibition assay. Figures 2 and 3 illustrate data on the anti-HIV effect of a 14-mer and a 28-mer of the invention, respectively, in a cytopathic effect inhibition assay. DETAILED DESCRIPTION OF THE INVENTION A. Preparation of Poly(alkyl and alkenyl phosphate)s and Their Selenophosphate and Thiophosphate Derivatives Compounds of the invention can be synthesized by several different routes. Preferably, they are synthesized by solid phase procedures using hydrogen phosphonate, phosphoramidite, or thiophosphoramidite precursors. Preferably, phosphates and monothiophosphates of the invention are synthesized by hydrogen phosphonate chemistry and polyphosphorodithioates of the invention are synthesized by thiophosphoramidite chemistry. Polyphosphates and polyphosphoromonothioates of the invention may be synthesized by the following steps using the hydrogen phosphonate approach. Precusor 1_ defined by the following formula: RT- - C - O - R - ° H lit R is reacted with a tris-phosphoramidite, or alternative synthon according to reported procedures, e.g. Garreg et al, Chem. Scripta, Vol. 25, pg. 280 (1985), or Marugg et al, Tetrahedron Lett., Vol. 27, pg. 227 (1986), to obtain the corresponding alkane hydrogen phosphonate 2 ^ defined by the formula: - o * H wherein R is as defined above and the moiety (R 1 )(R ) (R'")C is an acid labile protection group as commonly used to protect 5 1 hydroxylε in solid phase synthesis of polynucleotides. Preferably, (R , )(R")(R"')C is trityl, or a derivative thereof. The unspecified cation associated with 2, is usually triethylammonium. _2 is employed as a reagent in n cycles of automated solid phase synthesis using conventional procedures, e.g. Andrus et al, U.S. patent 4,816,571, to obtain a hydrogen phosphonate-linked oligomer .3 attached to a solid phase support: where B^ iε a conventionally protected purine or pyrimidine base, or a derivative thereof, k iε an integer between 1 and n, inclusive, and Z=H for 2'- deoxynucleoεideε, or Z=0-E for ribonucleosideε, E being a εuitable protection group for the 2' hydroxyl. W=0- D-G, where G iε a standard support material, e.g. controlled pore glasε or polystyrene, and D iε a linking group, usually cleavable under basic conditions. ,3 is preferably syntheεized in the form in which the nucleoside is between the growing chain and • the solid phase support. Commercially available solid phase supportε that have been derivatized with protected nucleosides are a convenient starting material. The presence of the nucleoside also provides a convenient method of identifying and quantifying the final product by means employing the spectrophotometric propertieε of the nucleoside. The final step of the chain elongation can be to add either a terminal nucleoside or terminal monomer .1, to achieve the following intermediate A i j4 can be sulfurized by standard techniques, e.g.Matsukura et al. Gene, Vol. 72, pg. 343 (1988). After cleavage from the solid phase support and deprotection of the bases the following material j> is obtained: S " wherein B iε the de-protected base βP. Material J5 is readily purified and isolated by meanε of conventional reverse-phase HPLC, and other purification procedures common in polynucleotide syntheεiε e.g. Zon et al, BioChromatography, Vol. 1, pg. 22 (1986). The purified material !> iε then treated with an aqueouε acetic acid to remove the (R 1 )(R")(R MI )C moiety, which iε uεually εeparated from ϊ> by extraction with ethyl acetate, ether, or the like, to give the final product. Preferably, dithiophosphates of the invention may be synthesized using thiophosphoramidite chemiεtry. Brill et al, J. Am. Chem. Soc, Vol. Ill, pg. 2321 (1989), which iε incorporated herein by reference, provideε instructionε to convert precursor .1 into intermediate of the following formula: Ac wherein R and (R 1 ) (R ) (R" ' )C are as defined above, Ar is halo-substituted phenyl (such as, 2,4- dichlorophenyl) , and R2 is dimethyl or pyrrolidinyl. j6 is employed as a reagent in n cyc les of automated so lid phase synthesis , as taught by Bril l et al (cited above) , to obtain dithiophosphates 7_: Preferably, 1_ i- s treated with thiophenol:triethylamine:dioxane (1:1:2 vol:vol:vol) for 24 hours, and then with concentrated NH 4 0H at 55°C for 12-18 hours to give jJ (provided that the R group selected remains stable under these deprotection conditions) : 8 jB_ iε readily purified and isolated as described above for 5. The purified material JB is treated with aqueous acetic acid to remove the (R')(R )(R'")C moiety, which is separated from 8 ^ by extraction with ethyl acetate, ether, or the like, to give the final product. Preferably, the size of the poly(alkyl or alkenyl phosphate)s and/or their thio analogs ranges between about 20 and 30 monomeric units. Compounds larger than 30-mers may be readily employed for their biological effect, but longer polymers are increaεingly difficult to εyntheεize and purify. Selenophoεphateε for uεe with the invention are εyntheεized in accordance with Mori et al. Nucleic Acidε Research, Vol. 17, pgs. 8207-8219 (1989), which iε incorporated herein by reference. B. Cytopathic Effect Inhibition Assay for Assesεing Anti-HIV Activity In this iri vitro assay syεtem, ATH8 cells, an immortalized T4+ T cell line, are used aε target cellε becauεe of their sensitivity to the cytopathic effect of HIV, Mitsuya et al, Proc. Natl. Acad. Sci., Vol. 83, pgε. 1911-1915 (1986). ATH8 cells are available under catalog number 307 from the AIDS Research and Reference Reagent Program Repository establiεhed by the National Inεtitute of Allergy and Infectiouε Disease and operated by ERC BioServices Corporation (Rockville, MD). 2xl0 5 ATH8 cellε incubated with HTLV-III B for 30 minuteε (about 500 viruε particleε per cell). The HTLV-III B viruε iε alεo available from the AIDS Reεearch and Reference Reagents Program Repository (under catalog number 398). This dose is about 100- 1000 times higher than the minimum cytopathic dose in the assay. Complete medium (2 ml of RPMI 1640) supplemented with L-glutamine (4 mM), 2-mercaptoethanol (50 nM), penicillin (50 units/ml), and streptomycin (50 ug/ l) and containing 15% fetal calf serum and interleukin-2 is uεed with addition of the indicated concentrations of the compounds of the invention added. The number of viable cells is counted in a hemocytometer using the trypan blue dye-exclusion method on day 7 following exposure to the virus. A mock sample without virus can be used at each concentration tested to evaluate toxicity. The highly active phosphorothioate oligodeoxycytidine 28-mer (S- dC2 R in figure 1C) can be used as a poεitive control for antiviral activity with the particular batches of ATH8 cells and HTLV-III B viruε particleε uεed for the preεently deεcribed de novo infection assay. C. Pharmaceutical Carriers Compounds of the invention are administered as a pharmaceutical compoεition. Such compositions contain a therapeutic amount of at least one of the poly(alkyl or alkenyl phosphate)s of the invention and/or at least one of their thiophosphate analogε in a pharmaceutically effective carrier. The poly(alkyl or alkenyl phoεphate)s and their thio analogs may be administered either as a εingle chainlength (i.e. one value of n), or aε a defined mixture containing polymerε of more than one chainlength, e.g. a composition can contain a mixture of the compound of Formula I wherein X, Y, R, B, and Q are the εame for each component of the mixture, but wherein n may have more than one value or be diεtributed around some average value. A pharmaceutical carrier can be any compatible, non-toxic εubεtance suitable for delivering the compositions of the invention to a patient. Sterile water, alcohol, fatε, waxes, neutral lipids, cationic lipidε, and inert εolidε may be included in a carrier. Pharaceutically acceptible adjuvants, e.g. buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical composition. Generally, compositions useful for parenteral adminiεtration of drugε are well known, e.g. Remington's Pharmaceutical Science, 15th ED. (Mack Publishing Company, Easton, PA, 1980). Alternatively, compositions of the invention may be administered by way of an implantable or injectable drug delivery system, e.g. ϋrquhart et al, Ann. Rev. Pharmacol. Toxicol., Vol.24, pgs.199-236 (1984); Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981); U.S. patent 3,773,919; U.S. patent' 3,270,960; or the like. Preferably, compositions of the invention are administered parenterally, and more preferably, intravenously. In such cases, pharmaceutical carriers include saline solutionε, dextrose solutionε, combinations of the two, nonaqueous solutions such as ethyl oleate, and the like. Selecting an administration regimen for a co poεition of the invention depends on several factors, including the rate of degradation of the particular compounds in serum, the acceεεibility of the target tissues and cells, pharmacokinetics, toxicity, and the like. Preferably, an adminiεtration regimen maximizeε the amount of compound delivered to a patient conεiεtent with an acceptable level of side effects. Accordingly, the amount of compound delivered may depend on the particular compound and the severity of the viral infection being treated. Preferably, a daily doεe of the compoundε of the invention iε in the range of about 1-2 ug/kg to about 10-20 mg/kg. EXAMPLES Example 1. Syntheεiε of a 1-0-4,4'-dimethoxytrityl- butane-3-ol ( la ) 4,4'-dimethoxytrityl chloride (2g, 5.9 mmol) iε disolved in anhydrous methylene chloride (5 ml) and added dropwise, with εtirring, to the solution of racemic 1,3-butane diol (530 mg, 5.9 mmol) in anhydrous pyridine (2.5 ml) at -20°C over 0.5 h. The reaction mixture is allowed to warm to 25°C. After 2 h, the solvents are evaporated in vacuo and the residue is dissolved in diethyl ether-hexane (3:1) and purified on a silica gel column (3x10 cm) using diethyl ether- hexane (4:1) as the eluent. The product la iε isolated as colorleεε resinous material (yield 2g, 90%); tic 2H, Hz ' .18, , Example 2. For ation of triethylammonium 1-0-4,4' ■ dimethoxytrityl-butane-3-0-hydrogen phoεphonate (Ila) The trityl alcohol precursor la (388 mg, 1 mmol) iε converted into corresponding hydrogen phoεphonate Ila with tris(imidazoyl)phosphoramidite according to known procedures, e.g. Garreg et al (cited above). The product Ila is isolated by chromatography on a silica column (5x3 cm) as a colorlesε semisolid (470 mg, yield 85%); tic (chloroform-methanol)(9:l) R f =0.1, (acetonitrile-water)(85:15) 31p_ P-NMR 0.5 ppm, jp_ H =605.55 Hz. Example 3. Synthesiε of poly(1 r 3-butanediyl) phos horothioate (Ilia) ___ . Synthesiε of oligophosphorothioate Ilia is performed using triethylammoniu 1-0-4,4'- dimethoxytrityl-butane-3-0-hydrogen phosphonate Ila on a Cyclone 6000-Biosearch DNA synthesizer, or like instrument. B of Ilia is as defined above. The starting solid phase support IVa is controlled pore glass (CPG) with 1 umol nucleoside loaded at 40 umol/g. Pivaloyl chloride is used as the H-phoεphonate activator. βP of IVa iε aε defined above. Average coupling efficiency iε 98% (dimethoxytrityl cation aεsay). The final sulfurization of internucleotide H-phosphonate linkages is performed manually using a saturated solution of sulfur (Sg) in N,N-diisopropylethylamine (25°C, 3h). After cleavage from the solid support with concentrated NH 4 0H, the resultant 5'-0-4,4'- dimethoxytrityl (5'-DMT) derivative of Ilia is purified by HPLC on a uBondapak C 18 column (Waters), elution time 17 minutes, gradient 5-40% (or more) CH3CN versus 0.1 M triethylammonium acetate, pH 7.4, for about 20 minuteε, followed by iεocratic flow, all at about 1.5 ml/min. The 5'DMT IVa iε collected, concentrated in vacuo and then detritylated with 80% acetic acid in water prior to HPLC collection of final product Ilia, elution time about 15 minutes using the same column and flow rate but with a gradient of 5-30% (or more) CH3CN versuε 0.1 M triethylammonium acetate, pH 7.4, at 1.25 ml/min. -^P-NMR of Ilia: about 55 ppm. The final material as a triethylammonium salt iε obtained by evaporation in vacuo. Example 4. The Anti-HIV Effect of CV 26 C A compound of the following formula, designated cv 26 c w s s Y nt hesized using the methods described above: wherein B iε cytosine. CV2gC waε used in the cytopathic effect inhibition asεay deεcribed above in the concentrationε indicated in Figure 1A. Controlε were employed aε deεcribed. A clear inhibition of the cytopathic effectε of the HIV viruε iε εeen at a concentration of 2.5 uM. Example 5. The Anti-HIV Effect of CE 26 C A compound of the following formula, designated C _-26 C ' was synthesized using the methods described above: wherein B is cytoεine. CE2gC waε uεed in the cytopathic effect inhibition assay described above in the concentrations indicated in Figure IB. Controlε were employed as described. A clear doεe-reεponεe relationεhip iε εhown between inhibition of cytopathic effect and concentration of CE25C at 2.5 uM. Example 6. Lack of cytopathic inhibition of a_ 14-mer compound A 14-mer analog of the compound of Example 4, deεignated CV-^C, was syntheεized uεing the methodε described above. As shown in Figure 2, no discernable cytopathic inhibition was observed over the indicated range of concentrations. Example 7. Cytopathic inhibition of a_ 28-mer compound A 28-mer analog of the compound of Example 4, designated V^gC waε re-tested in parallel with cV- j ^C using the methods described above. As shown in Figure 3, a clear inhibition of the cytopathic effectε of HIV iε εeen.

Claims: WE CLAIM: 1. A pharmaceutical composition consiεting essentially of an effective antiviral amount of a poly(alkyl or alkenyl phosphate) or a thio- or εeleno- phoεphate derivative selected from the group defined by 10 the following formula: wherei : X and Y are separately selected from the group consisting of selenium, oxygen and sulfur; n is in the range of 16 to 60 inclusive ; R is a lkyl or a lkeny l having from 1 to 8 carbon atoms , alkyloxy or alkylthio having from 1 to 8 carbon atoms and having from 0 to 2 heteroatoms ; and Q and Q' are separately hydrogen, R-OH, or a nucleoside selected from the group consiεting of adenoεine, cytidine, guanoεine, and thy idine. 2. The pharmaceutical composition of claim 1 wherein n is in the range of 20 to 30 , wherein R iε alkyl or alkenyl having from 3 to 6 carbon atomε, or alkoxy having from 3 to 6 carbon atomε and one oxygen atom, and wherein X and Y are εeparately selected from the group consiεting of oxygen and εulfur. 3. The pharmaceutical compoεition of claim 2 wherein R is alkyl having from 3 to 6 carbon atoms or cyclic alkoxy having 4 to 5 carbon atomε and one oxygen atom. 4. The pharmaceutical compoεition of claim 3 wherein at least one of X or Y iε εulfur. 5. The pharmaceutical compoεition of claim 4 wherein εaid poly(alkyl or alkenyl phoεphate) or thio- or selenophospliate derivative is defined by the following formula: 6. The pharmaceutical composition of claim 4 wherein said poly(alkyl or alkenyl phosphate) or thiophosphate derivative is defined by the following formula: 7. A method of treating viral infections comprising the εtep of adminiεtering an effective amount of a poly(alkyl or alkenyl phoεphate) or a thiophosphate derivative thereof selected from the group defined by the following formula: wherein: X and Y are εeparately εelected from the group conεisting of selenium, oxygen and sulfur; n is in the range of 16 to 60 inclusive; R is alkyl or alkenyl having from 1 to 8 carbon atomε, alkyloxy or alkylthio having from 1 to 8 carbon atoms and having from 0 to 2 heteroatoms; and Q and Q 1 are εeparately hydrogen, R-OH, or a nucleoεide εelected from the group consiεting of adenoεine, cytidine, guanoεine, and thymidine. 8. The method of claim 7 wherein n iε in the range of 20 to 30, wherein R iε alkyl or alkenyl having from 3 to 6 carbon atomε, or alkoxy having from 3 to 6 carbon atomε and one oxygen atom, and wherein X and Y are εeparately εelected from the group conεiεting of oxygen and εulfur. 9. The method of claim 8 wherein R iε alkyl having from 3 to 6 carbon atomε or cyclic alkoxy having 4 to 5 carbon atoms and one oxygen atom. 10. The method of claim 9 wherein at least one of X or Y is sulfur. 11. The method of claim 10 wherein εaid poly(alkyl or alkenyl phosphate) or thio- or selenophoεphate derivative iε defined by the fol lowing formula: 12. The method of claim 10 wherein said poly(alkyl or alkenyl phosphate) or thiophosphate derivative iε defined by the fol lowing formula: 13. The method of claim 7 wherein εaid effective amount iε between about 1 ug/kg of body weight to about 20 mg/kg of body weight.

Inventors: MATSUKURA MAKOTO, STEC WOJCIECH J, WILK ANDRZEJ, ZON GERALD, MATSUKURA, MAKOTO, STEC, WOJCIECH, J., WILK, ANDRZEJ, ZON, GERALD
Applicants: APPLIED BIOSYSTEMS, APPLIED BIOSYSTEMS, INC.
Application year: 1990
Main classification: A01N43
IPC codes: A61K31, C07F9, C07H21