WO1990012022A1

WO1990012022A1 - Polynucleotide phosphorodithioates as therapeutic agents for retroviral infections

Info

Publication number: WO1990012022A1
Application number: PCT/US1990/001604
Authority: WO
Inventors: Marvin H. Caruthers; William S. Marshall
Original assignee: University Patents, Inc.
Priority date: 1989-03-31
Filing date: 1990-03-28
Publication date: 1990-10-18
Also published as: AU5418290A

Abstract

The present invention relates to novel oligonucleotides having phosphorodithioate internucleotide linkages which may be used for the treatment of diseases caused by viruses.

Description

Polynucleotide Phosphorodithioates as Therapeutic

Agents for Retroviral Infections

This is a continuation-in-part application of our earlier filed United States Patent Application 198,886, filed May 26, 1988.

Research leading to the making of the invention described herein was supported, in part, with federal funds. Accordingly, the United States Government has certain statutory rights to the invention described herein.

For the past several years, various nucleoside and nucleotide analogs have been screened for antiviral activity and, in some cases, observed to be effective. This approach has now been extended to the retroviruses where it has been found that certain analogs such as 3'-azido-2',2'- dideoxythymidine (Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff Lehrman, S., Gallo, R. C, Bolognesi, D., Barry, D. W., and Broder, S., Proc. Natl. Acad. Sci. USA 82, 7096-7100, 1985) and the 2',3'- dideoxynucleosides (Mitsuya, H. and Broder, S., Proc. Natl. Acad. Sci. USA 83., 1911-1915, 1986) are effective antivirals, the reason being that they inhibit retroviral replication and reverse transcriptase activity. An alternative approach by other investigators has been to use oligonucleotides or their analogs as antivirals. For this purpose several oligonucleotides and oligonucleotide analogs having methylphosphonate, phosphorothioate, and phosphoroamidate intemucleotide linkages have been tested and shown to be effective antivirals (Stein, C. H. and Cohen, J. S., Cancer Research 48, 2659-2668, 1988).

Oligonucleotide therapy is being investigated aggressively because, as antivirals, these compounds have known activities in inhibiting primer binding of reverse transcriptase; they activate reverse transcriptase RNase H activity; they block translation of viral RNA genes through hybridization arrest; or they inhibit RNA splicing reactions. The mechanism of inhibition depends upon the choice of oligonucleotide analog and its nucleotide sequence (Stein, C. A. and Cohen, J. S., Cancer Research .48., 2659- 2668, 1988).

High yielding methodologies are currently available for the rapid synthesis of sequence defined polynucleotides having the natural intemucleotide linkage (Caruthers, M. H., Science 230, 281-285, 1985; Caruthers, M. H. and Beaucage, S. L., U.S. Patent 4,425,732; Caruthers, M. H. and Matteucci, M. D., U.S. Patent 4,458,066). An important step in these methodologies is the oxidation of the intermediate phosphite triester to the naturally occurring phosphate triester with aqueous iodine. These phosphite triesters can also be oxidized, under anhydrous conditions with amines or ammonia and iodine, to yield variable reported amounts of oligonucleotide phosphoramidates, or with sulfur to yield oligonucleotide phosphorothioates (Uznanski, B., Koziolkiewicz, M., Stec, W. J., Zon, G., Shinozuka, K. and Marzili, L. , Chemica Scripta 26, 221-224, 1986; Nemer, M. H. and Ogilvie, K. K., Tetrahedron Letters 21 , 4149-4152, 1980). Other methods employing H-phosphonate intemucleotide linkages can also be used to synthesize oligonucleotide phosphoramidates and oligonucleotide phosphorothioates (Froehler, B. C, Tetrahedron Letters 27, 5575-5578, 1986). Oligonucleotide methylphosphonates are synthesized from nucleoside methylphosphonamidites (Dorman, M. A., Noble, S. A., McBride, L. J. and Caruthers, M. H., Tetrahedron 40, 95-102; Jager, A. and Engels, J., Tetrahedron Letters 25, 1437-1440).

Recently, methods were developed for synthesizing oligonucleotides containing phosphorodithioate intemucleotide linkages (United States Patent Application 314,011, filed 22 February 1989, the disclosure of which is incorporated in toto herein). These developments have now led to the discovery of the present invention that phosphorodithioate-containing oligonucleotides, a new class of antiviral chemotherapeutic agents, are inhibitors of viral reverse transcriptases.

In general, the oligonucleotide phosphorodithioates according to the present invention, can be represented by the formulae I and II:

wherein R is H or a blocking group; A is H, OH, halogen, SH, NH₂ or azide; B is a nucleoside or deoxynucleoside base (including purines, e.g. adenine, hypoxanthine, guanine, or their derivatives, and pyrimidines, e.g., cytosine, uracil, thymine, or their derivatives); n is an integer from zero to thirty; and m is an integer from one to thirty. If the repeat units represented by m and n are within the same oligonucleotide phosphorodithioate, it is understood that the repeat units contained within m and n can be positioned in any sequence and that the sum of m and n usually would not exceed thirty.

The new class of chemotherapeutic compounds according to the present invention and represented by formulae I and II are oligonucleotides having a 3'-5' phosphate diester linkage, ribose and deoxyribose sugars, purine and pyrimidine bases, and the nucleosides and deoxynucleosides linked to phosphorus through oxygen covalently joined at the 3' and 5'-carbons of the sugars. Compound I has two sulfur atoms bonded to each phosphorus whereas compound II has at least one phosphorus bonded to two sulfur atoms and one phosphorus bonded to two oxygens while each remaining phosphorus is bonded to either two fulfurs or two oxygens. Thus it can be seen that compound I is an oligonucleotide having exclusively phosphorodithioate intemucleotide linkages whereas compound II has at least one phosphorodithioate intemucleotide linkage and one phosphate intemucleotide linkage with the remainder being either phosphorodithioate or natural phosphate diester linkages.

The chemical synthesis of compound I is completed using appropriately protected deoxynucleoside or nucleoside phosphorothioamidites as synthons, preferably a deoxynucleoside or nucleoside joined covalently to a silica support. Activation of the synthon is most easily accomplished with tetrazole. The reaction sequence is then completed by oxidation with sulfur, acylation of unreacted, silica bonded deoxynucleoside or nucleoside, and selective removal of appropriate protecting groups. This cycle can then be used repetitively in order to extend the oligonucleotide so that it contains as many as 32 nucleosides (n = 30).

A similar sequence may be used to prepare compound II. In this sequence, two synthons, a deoxynucleoside or nucleoside phosphoramidite and a deoxynucleoside or nucleoside phosphorothioamidite, are used to prepare an oligonucleotide having the phosphate and phosphorodithioate intemucleotide linkages with m + n equal to thirty.

In order to provide a more detailed understanding of the present invention, the following examples and procedures are provided. These depict the formation of compounds I and II, demonstrate how these compounds inhibit viral reverse transcriptases, and provide a more complete understanding and illustration of the present invention. They are, however, examples, and as such are not intended in any manner to limit the scope of the present invention.

Example I

The synthesis of compound la where n = 12, R = H, B = cytosine, and A = H is described. Compound la therefore has the following structure where C represents deoxycytidine and x the phosphorodithioate intemucleotide linkage:

d(CxCxCxCxCxCxCxCxCxCxCxCxCxC)

Bis(dimethylamino)chlorophosphine was prepared by adding tris(dimethylamino)-phosphine (36.3 ml, 32.6 g, 0.2 mole) and trichlorophosphine (8.7 ml, 13.7 g, 0.1 mcle) to anhydrous ether (100 mol) . After stirring for 3 hours at room temperature, solvent was removed by concentration in vacuo at room temperature. The product was then distilled (b.p. 72-75ºC) at reduced pressure (approx. 16 mm Hg) using a water aspirator to yield 30 g. of product.

This procedure is also used to produce dipyrrolidinylchlorophosphine. Preparation of thiophosphoramidites of the formula

wherein:

B = 1-Thyminyl ;

B = 1-(N-4-benzocylcytosinyl);

B = 9-(N-6-benzoyladeninyl);

B = 9-(N-2-isobutyrylguιninyl);

DMT = di-p-anisylphenylmechyl (dimethoxytrityl); M = 4-chlorobenzyl or 2,4-chlorobenzyl; and X = N,N-dimethylamino or pyrrolidinyl and the further use of these compounds to prepare oligonucleotides having phosphorodithioate intemucleotide linkages are presented in this example.

The following example describes the synthesis of 5'- O-dimethoxytrityl-N4-benzoyldeoxycytidylyl-3'-S (4- chlorobenzyl) phosphorthiopyrrolidinite. The same procedure can be used for the other suitably protected deoxynucleosides. Similarly the same procedure is useful for the 2.4-dichlorobenzyl and 4-chlorobenzyl protected sulfur derivatives and for the N,N-dimethylamino and pyrrolidinyl amidites. Table I summarized the ³¹P-NMR data for all these amidites.

³¹P-NMR were recorded in CDCl₃ on a Brucker WM-250 with 85% aqueous H₃PO₄ as external standard. T, C^Bz, A^Bz, and G^iB refer to thymine, N-benzoylcytosine, N-benzoyladenine, and N- isobutyrylguanine respectively; R, is dimethoxytrityl; A is hydrogen. 5'-O-Dimethoxytrityl-N4-benzoyldeoxycytidine (317 mg, 0.5 mmol) was dissolved in a mixture of acetonitrile (2 ml) and triethylamine (1 ml) under argon. Bispyrrolidinylchlorophosphine (124 mg, 0.6 mmol) was added which was followed by the immediate formation of a precipitate. After 5 minutes stirring at room temperature, 4-chlorobenzylmercaptan (159 mg, 1mmol) was added to the reaction mixture and the solution, including the precipitate, was concentrated to a glass In vacuo at room temperature. The glass was resuspended in acetonitrile (2 ml). The ³¹P-NMR spectrum of the reaction mixture indicated that the major phosphorus containing product was the diastereoisomers of the thioamidite (161.5, 159.7 ppm). Minor impurities were an adduct of bispyrrolidinylchlorophosphine and 4-chlorobenzylmercaptan (107.0 ppm) and hydrolysis products (12.4 ppm). Triethylamine was next added to the reaction mixture. The solution was diluted with deacidified ethylacetate (50 ml) and extracted with aqueous saturated sodium bicarbonate (50 ml x 2) and brine. The combined aqueous solutions were back-extracted with deacidified ethylacetate (10 ml). The organic solutions were combined, dried for 1 hour over sodium sulfate in the presence of 10% (volume) triethylamine, filtered, and the filtercake washed with 5 ml deacidified ethylacetate. The organic solution was then concentrated in vacuo to a white foam. This foam was dissolved in toluene (10 ml) containing 1% triethylamine and the product isolated by precipitation into n-pentane: triethylamine (999:1, v/v). After filtration, the product was dried in vacuo over phosphorus pentoxide and potassium hydroxide and isolated in 83.1% yield (741 mg).

Using a deoxynucleoside attached covalently to a silica based polymer support through the 3'-hydroxyl (U.S. Patent 4,458,066), synthesis of deoxyoligonucleotides containing phosphorodithioate linkages proceeded according to the reaction sequence outlined below.

©

Synthesis began by reacting a dry acetonitrile solution of any thiophosphoramidite according to Example I (10 equivalents) and tetrazole (50 equivalents) with 1 μmole of deoxynucleoside on silica for 30 sec followed by a 400 sec oxidation with 5% sulfur in pyridine:carbon disulfide (1:1, v/v). Coupling was performed twice to ensure high yields (greater than 98%. Acylation of unreactive deoxynucleoside, detritylation and various washes were the same as those described previously for synthesizing natural DNA from deoxynucleoside phosphoramidites (U.S. Patent 4,415,732 and Science 230, 281-285, 1985). Repetitions of this cycle an additional twelve times led to the synthesis of compound la. Deoxyoligonucleotides such as compound II having both phosphorodithioate and phosphate intemucleotide bonds may be synthesized when both deoxynucleoside phosphorothioamidites and deoxynucleoside phosphoramidites are used during synthesis.

Synthetic deoxyoligonucleotides were isolated free of protecting groups using a two-step protocol (thiophenol:triethylamine:dioxane, 1:1:2, v/v/v for 24 h followed by cone, ammonium hydroxide for 15 h), and then purified to homogeneity by standard procedures (polyacrylamide gel electrophoresj.s and reverse phase hplc). 3¹P-NMR spectra of phosphorodithioate DNA indicated that this synthesis protocol yielded DNA containing exclusively phosphorodithioate intemucleotide linkages. No hydrolysis of these dithioates to phosphorothioates (³¹P-NMR 56) or phosphate was observed.

Example II

Syntheses are described for lIa, llb and llc where m an n are variable for Ila, llb, and llc, R = H, B = cytosine, and A = H. Compounds Ila, llb and llc have the following structures where C represents deoxycytidine, x represents the phosphorodithioate intemucleotide linkage, and p the natural phosphate intemucleotide linkage.

Ila: d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC)

llb: d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC)

llc: d(CxCpCxCpCxCpCxCpCxCpCxCxCpCpC)

Synthesis of Dinucleoside Phosphorodithioate Triesters of the formula:

wherein

B = 1-thyminyl;

B = 1-(N-4-toluoylcytosinyl);

B = 9-(N-6-benzoyladeninyl);

B = 9-(N2-isobutyrylguaninyl);

DMT = dimethoxytrityl; and

Ac = acetyl

and the further conversions of the deoxydicytidine derivative to deoxycytidine oligodeoxynucleotides having phosphorodithioate intemucleotidd linkages at various positions all presented in this example. A. Synthesis of a Thymidine Dinucleotide Having a Phosp.iorodithioate Intemucleotide Linkage.

5'-O-dimethoxytritylthymidine (1.2 g, 2.21 mmol) was dried by co-evaporation with anhydrous THF and then dissolved in THF (10 ml) and triethylamine (0.46 ml, 3.3 mmol). Bis (diisopropylamino) chlorophosphine (650 mg, 2.44 mmol) was added and the solution stirred at room temperature. After 35 minutes, the precipitate was removed by filtration and washed with THF (1 ml). The combined filtrates containing the deoxynucleoside phosphorodiamidite were pooled, concentrated in vacuo, and redissolved in acetonitrile (5 ml). 3'-O-acetylthymidine (639 mg, 2.25 mmol) and tetrazole (142 mg, 2.0 mmol) were dried by coevaporation with THF (10 ml), redissolved in acetonitrile (5 ml), and added to the acetonitrile solution of the deoxynucleoside phosphorodiamidite. After stirring for 45 minutes at room temperature, the reaction mixture was diluted with dichloromethane (75 ml), extracted with an aqueous sodium bicarbonate solution (5% w/v), dried over sodium sulfate, filtered, and concentrated in vacuo to a gum. The product was then purified by column chromatography (100ml silica, ethylacetate:dichloromethane:-triethylamine; v/v/v) to yield 1.59 g of the doxydinucleoside phosphoramidite (1.66 mmol, 75%).

The deoxydinucleoside phosphoramidite was then converted to the deoxydinucleoside phosphorodithioate triester. The deoxydinucleoside phosphoramidite (1.59 g, 1.66 mmol) was dissolved in acetonitrile (7 ml). 4- Chlorobenzylmercaptan (1.0 ml, 1.20 g, 7.6 mmol) and tetrazole (281 mg, 4.01 mmol) were then added and the reaction mixture stirred at room temperature for 30 minutes. A solution of sulfur in toluene: 2.6-lutidine (19:1, v/v, 10 ml containing 4 mmol atomic sulfur) was added and the resulting solution stirred for 30 minutes. The reaction mixture was diluted with ethylacetate (75 ml), extracted with an aqueous sodium bicarbonate solution (5%, w/v), dried over sodium sulfate, filtered and concentrated in vacuo to an oil. The oil was dissolved in ethylacetate (40 ml) and triturated with hexanes (200 ml) to give a crude product as a white powder. Purification by silica column chromatography (100 ml silica, 2-12% methanol in dichloromethane as % eluant) yields the deoxydinucleoside phosphorodithioate triester (1.59 g, 1.52 mmol, 91%).

Removal of the 3'-O-acetyl group (0.15 M tert- butylamine in methanol, 0ºC, 10 h) yields a deoxydinucleoside phosphorodithioate that can be used for

DNA synthesis (1.26 g, 1.28 mmol, 84%). The deoxydinucleoside phosphorodithioate is converted to the 3'- phosphoramidite and then used to synthesize DNA on a polymer support.

B. Synthesis of Deoxycytidine Oligomers Containing Phosphorodithioates

5'-O-Dimethoxytrityl-N-toluoyldeoxycytidine was prepared by minor modification of a published procedure (H. Koster, K. Kulinowski, T. Liese, W. Heikens, and V. Kohli, Tetrahedron 37, 363, 1981). Deoxycytidine hydrochloride (10 mmol, 2.64 g) was co-evaporated twice with anhydrous pyridine and resuspended in pyridine (50 ml). Trimethylchlorosilane (7.5 ml, 59 mmol) was added and the mixture stirred for 45 minutes at room temperature. o- Toluoyl chloride (1.44 ml, 11 mmol) was added and the reaction stirred for two additional hours. The reaction mixture was chilled in an ice bath, treated with methanol (10 ml) and 25% ammonium hydroxide (20 ml) for 30 min, and the suspension removed by filtration. The resulting solution was concentrated to dryness in vacuo. The resulting solid was suspended in 40 ml dichloromethane:methanol (8:2) and the insoluble salts removed by filtration. The filtrate was concentrated in vacuo to an oil, reconcentrated twice in vacuo after addition of pyridine and redissolved in pyridine (50 ml). After addition of 0.9 equivalents of dimethoxytrityl chloride (3.05 g), the reaction mixture was stirred for 30 min at OºC and 30 min at room temperature. Dimethoxytritylchloride (0.3 equivalents) was added and stirring was continued for 30 minutes. The reaction was quenched by addition of methanol (1 ml) and the solution concentrated in vacuo. The resulting oil was dissolved in dichloromethane (75 ml) and extracted sequentially with aqueous 5% sodium bicarbonate (w/v) and brine. The combined organic phase was dried over sodium sulfate, filtered, concentrated to dryness in vacuo, dissolved in dichloromethane:pyridine (99.5:0.5, v/v) and the product purified by column chromatography (50 g silica, dichloromethane:methanol:pyridine gradient from 0 to 3% methanol; 400 ml each). Fractions containing 5'-O- dimethoxytrityl-N-toluoyldeoxycytidine were pooled, concentrated in vacuo, redissolved in ethylacetate and precipitated into pentane (5.01 g, 7.7 mmol, 77%).

3'-O-Phenoxyacetyl-N-toluoyldeoxycytidine was prepared by minor modification of a published procedure (C. B. Reese and J. C. M. Stewart, Tetrahedron Letters 4273, 1968). 5'- O-Dimethoxytrityl-N-toluoyldeoxycytidine (1.94 g, 3 mmol) and phenoxyacetic anhydride (1.72 g, 6 mmol) was dissolved in tetrahydrofuran (50 ml). After addition of pyridine

(0173 ml, 9 mmol), the solution was stirred for 14 hours at room temperature and then concentrated in vacuo. The resulting oil was dissolved in dichloromethane (75 ml), extracted twice with 5% aqueous sodium bicarbonate (100 ml, w/v) and the combined aqueous phases extracted with dichloromethane (50 ml). The product in the combined organic phase was dried over sodium sulfate, filtered. concentrated to dryness in vacuo, redissolved in ethylacetate and precipitated in pentane. The solid corresponding to totally protected deoxycytidine was dissolved in dichloromethane:methanol (8:2, v/v) and chilled in an ice bath. A solution of p-toluenesulfonic acid (2128 g, 12 mmol) in dichloromethane:methanol (50 ml, 8:2, v/v) was added and the solution stirred for one hour in an ice bath. The reaction was then quenched by addition of 5% aqueous sodium bicarbonate. The organic layer was extracted with brine and the aqueous phase re-extracted with dichloromethane (60 ml). The combined organic phase was dried over sodium sulfate, filtered and concentrated to dryness in vacuo. The resulting oil was dissolved in dichloromethane and the product purified by silica gel column chromatography (20 g of silica, elution with dichloromethane and dichloromethane:methanol (1 to 3% methanol). Fractions containing 3'-O-phenoxyacetyl-N- toluoyldeoxycytidine were pooled, concentrated to an oil, and the product isolated as a precipitate by addition of ethylacetate (1.20 g, 83%).

Deoxydicytidine phosphoroamidite in protected form was prepared using the following procedure. 5'-O- Dimethoxytrityl-N-toluoyldeoxycytidine (647 mg, 1 mmol) was co-evaporated three times with THF, dissolved in THF (5 ml) and triethylamine (0.21 ml, 1.5 mmol) and reacted with bis (N,N-diisopropylamino) chlorosphosphine (320 mg, 1.2 mmol). After 90 minutes under argon, the reaction mixture was filtered under argon pressure to remove insoluble salts. The salts were washed with THF (2 ml). The filtrate was concentrated to dryness and the product redissolved in acetonitrile (2 ml). 3'-O-Phenoxyacetyl-N- toluoyldeoxycytidine (527 mg, 1.1 mmol) and tetrazole (70 mg, 1 mmol) were suspended in acetonitrile (4 ml) and the above solution, including 1.5 ml acetonitrile used to wash the flask, was added. The reaction mixture was stirred under argon for 105 min. and then poured into ethylacetate:triethylamine (99:1, v/v, 50 ml). After two extractions with 2M triethylammonium bicarbonate (20 ml each) and back extraction of the aqueous phase with ethylacetate:triethylamine (99:1, v/v, 25 ml), the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. Purification was achieved by silica gel column chromatography (25 g silica, elution with hexanes:dichloromethane:triethylamine; 50:50:0.5, 400 ml;

45:55:0.5, 200 ml; 40:60:0.5, 200 ml; and 35:65:0.5, 100 ml). Product fractions were pooled, concentrated in vacuo, and precipitated into pentane (67%).

Deoxydicytidine phosphorodithioate was prepared using the following procedure. The deoxydicytidine phosphoramidite as prepared in the previous procedure (1.40 g, 1.12 mmol) was dissolved in acetonitrile (5 ml) (previously flushed with helium to avoid oxygen oxidation of thiophosphite) and 4-chlorobenzyl-mercaptan (0.5 ml, 3.7 mmol) and tetrazole (190 mg, 2.7 mmol) were added. The solution was stirred under argon for 30 min and, without isolation, the resulting thiophosphite was oxidized to the phosphorodithioate triester by addition of 5 ml of a 0.4 M solution of sulfur in toluene:lutidine (19.1, v/v). Based on ³¹P-NMR analysis, oxidation was complete after 10 minutes. The reaction mixture was diluted with ethylacetate (75 ml), extracted twice with 5% aqueous sodium bicarbonate (75 ml each), and the combined aqueous phases back extracted with ethylacetate (50 ml). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo to an oil. The oil was dissolved in a minimal amount of dichloromethane, diluted with ethylacetate to approximately 40 ml, and the product precipitated by addition of 200 ml hexanes. The white precipitate was filtered, redissolved in dichloromethane, and the solution concentrated to dryness. The product was purified by silica gel column chromatography (40 g silica gel, elution with dichloromethane:hexanes:triethylamine, 66:33:0.03, 400 ml and dichloromethane:triethylamine, 100:0.03, 200 ml). Fractions containing the completely protected product were pooled, concentrated in vacuo, redissolved in dichloromethane, and precipitated into pentane (60%).

The 3'-O-phenoxyacetyl protecting group was removed using the following procedure. The completely protected deoxydicytidine phosphorodithioate triester (355 mg, .264 mmol) was dissolved in acetonitrile (3 ml) and diluted with methanol (9 ml). After chilling the solution in an ice bath, tert-butylamine in methanol (0.3 M, 12 ml) was added and the reaction mixture stirred for 90 min in an ice bath.

The reaction solution was concentrated to dryness and the product purified by silica gel column chromatography (30 g silica, elution with dichloromethane:triethylamine,

100:0.03, 100 ml, followed by 200 ml each of dichloromethane:methanol:triethylamine, 99:1:0.03, 98:2:0.03 and 97:3:0.03). Product fractions were concentrated to dryness, redissolved in dichloromethane, and precipitated into pentane (95% yield).

The deoxydicytidine phosphorodithioate was next converted to the 3'-phosphoramidite which is useful as a synthon for synthesizing DNA containing dithioate intemucleotide linkages. The deoxydicytidine phosphorodithioate having a free 3'-hydroxyl (304 mg, 0.251 mmol) was dissolved in acetonitrile (5 ml). Bis (diisopropylamino)-β-cyanoethoxyphosphine (121 mg, 0.402 mmol) and tetrazole (20 mg, 0.236 mmol) were added under argon and the solution stirred for 2 hours. After quenching with ethylacetate:triethylamine (19.5:0.5) and diluting further with ethylacetate (20 ml), the reaction mixture was extracted twice with 2 M triethylammonium bicarbonate (13 ml each) and the aqueous phase back extracted with ethylacetate: triethylamine (19.5:0.5). The organic layer was dried over sodium sulfate, filtered, and concentrated to an oil in vacuo. The resulting oil was redissolved in dry ethylacetate and precipitated into pentane (87% yield).

Deoxycytidine pentadecaroers containing phosphorodithioate intemucleotide linkages at selected sites were synthesized using the deoxydicytidine phosphorodithioate synthons having a 3'-O-(β -cyanoethyl)- N,N-diisopropylphosphoramidite moiety as described above and 5'-O-dimethoxytrityl-N-benzoyldeoxycytidine -3'-O-(β cyanoethyl)-N,N-diisopropylphosphoramidite. The standard phosphoramidite synthesis methodology was used (M. H. Caruthers and S. L. Beaucage, U.S. Patent 4,415,732 and M. H. Caruthers and M. D. Matteucci, U.S. Patent 4,458,066). The average coupling efficiency was 99% (3 minute coupling time, 0.2 mol deoxycytidine on controlled pore glass as a support). The products were freed of protecting groups by t r e a t m e n t w i th a s o l u t i o n o f thiophenol:triethylamine:dioxane (1:1:2, v/v/v) at room temperature for 6 hours (some product remains as the S- protected dithioate (5-10%) when analyzed by gel electrophoresis and concentrated ammonium hydroxide at 55 C (15 hours). Purification of the final product was by either polyaσrylamide gel electrophoresis or high performance liquid chromatography. Three pentadecamers having phosphorodithioate linkages at specific positions were synthesized and have the following sequence:

d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC)

d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC)

d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC)

where x represents a dithioate linkage and p represents the natural intemucleotide linkage. Example III

The ability of deoxyoligonucleotide homopolymers to inhibit viral reverse transcriptases was tested using an assay whereby a deoxyoligonucleotide primer (P) was extended enzymatically using a reverse transcriptase enzyme, deoxynucleotide triphosphates (dNTP), and a deoxyoligonucleotide as template (T). The system is as follows:

P: 5' -GpApTpTpCpApGpCpTpApGpTpCpCpA

T: 3' -CpTpApApGpTpCpGpApTpCpApGpGpTpApGpCpApTpApGpTpGp TpCpApApApC

dATP, dTTP, dCTP, dGTP

Reverse transcriptase

± deoxyoligonucleotide inhibitor

P: 5' -GpApTpTpCpApGpCpTpApGpTpCpCpApTpCpGpTpApTpCpApCp ApGpTpTpTpG

T: 3' -CpTpApApGpTpCpGpApTpCpApGpGpTpApGpCpApTpApGpTpGp TpCpApApApC

Thus it can be seen that the assay involves DNA repair synthesis. Deoxynucleotide triphosphates are incorporated into the primer strand using reverse transcriptase as the DNA polymerizing enzyme. Two reverse transcriptases, the human immunodeficiency virus type I reverse transcriptase (HIV-I reverse transcriptase) and avian myeloblastosis virus reverse transcriptase (AMV reverse transcriptase), and a normal cellular polymerase, the large fragment of E. coli DNA polymerase I (Klenow polymerase), were used in this assay. Several deoxyoligonucleotide homopolymers having phosphorodithioate intemucleotide linkages (la, Ila, llb, llc), one deoxyoligonucleotide having phosphorothioate intemucleotide linkages (Illa), and one having natural phosphate diester linkages (IVa) were tested as inhibitors of the reverse transcriptases. Compounds llla and IVa were prepared using published procedures (Caruthers, M. H. and Beaucage, S. L., U. S. Patent 4,415,731; Caruthers, M. H. and Matteucci, M. D. , U. S. Patent 4,458,066; Stec, W. J., Zon, G., Egan, W. and Stec, B., J. Am. Chem. Soc. 106, 6077- 6079, 1984; Connally, B. A., Potter, V. L., Eckstein, F., Pingond, A. and Grotjahn, L., Biochemistry 23, 3443-3453, 1984). These compounds have the following sequences where intemucleotide linkages are represented by x for phosphorodithioate, p for the natural phosphate, and - for the phosphorothioate:

la: d(CxCxCxCxCxCxCxCxCxCxCxCxCxC) Ila: d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC) lib: d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC) Ilia. d(C-C-C-C-C-C-C-C-C-C-C-C-C-C-C) IVa: d(CpCpCpCpCpCpCpCpCpCpCpCpCpC)

The primer (P) and template (T) deoxyoligonucleotides were also synthesized using published procedures (Caruthers, M. H. and Beaucage, S. L., U. S. Patent 4,415,732; Caruthers, M. H. and Matteucci, M. D., U. S. Patent 4,458,066).

Assays for measuring the inhibition of DNA repair synthesis with phosphorodithyioate containing DNA were completed using the following procedure:

Primer (12 uM) and template (10 uM) in a solution of tris hydrochloride (tris HCl, 50mM, pH 8.3), MgCl₂ (10mM), and dithiothreitol (DTT, 5 mM) were warmed at 90°C for five minutes and then cooled on ice to 0°C. 5'-³²P labeled primer was approximately 0.5% of total primer. Aliquots of primer- template were then mixed with other components to yield assay solutions (20 ul) having the following composition: template (1 uM), primer (1.2 uM), tris HCl (50mM, pH 8.3), MgCl₂ (10 mM), KCl (50 mM), DTT (5 mM), dTTP (250 uM), dCTP (250 uM), dATP (250 uM), dGTP (250 uM), inhibitor oligonucleotide at variable concentrations from zero to 70 uM. Reactions were stated by adding AMV reverse transcriptase (7.2 nM), HIV-I reverse transcriptase (10 nM or 50 nM) or Klenow fragment (200 nM). Assays were incubated at 37°C for 15 minutes, quenched by adding formamide to 50%, and analyzed by electrophoresis on a 15% denaturing polyacrylamide gel. Radioactive bands containing polymerized primer and unextended primer were cut from the gels, dried and analyzed in a scintillation counter. The results are presented in Table 2.

ND = not determined; ID₅₀ = the concentration of inhibitor where the reaction proceeds to 50% of the uninhibited reaction.

*No inhibition was observed at these concentrations whereas HIV-I reverse transcriptase was completely inhibited.

**Only 7% inhibition at 36 uM of IVa.

The results listed in Table 2 can be summarized as follows. Compound la, a phosphorodithioate containing deoxyoligocytidine, is a very potent inhibitor of HIV-I reverse transcriptase (ID₅₀ = 60 nM) and is about 33 fold more inhibitory than Illa, a phosphorothioate linked deoxyoligocytidine of about the same length. Similarly la inhibits a second reverse transcriptase, AMV reverse transcriptase, approximately 168 fold more effectively than Illa. Also of considerable interest was the discovery that la did not inhibit a normal cellular polymerase, the large fragment of E. coli DNA polymerase I or Klenow fragment, even at 800 nM. At this concentration, HIV-I reverse transcriptase is completely inhibited. Two other discoveries merit comment. First normal deoxyoligocytidine, compound IVa, is noninhibitory at concentrations where both HIV-I reverse transcriptase and AMV reverse transcriptase are completely inhibited by la. Also a comparison of the results with HIV-I reverse transcriptase and la, Ila, llb, and lie shows that the extent of inhibition correlates directly with the number of phosphorodithioate linkages present in the deoxyoligonucleotide.

These results demonstrate that we have discovered a new class of potent chemotherapeutic agents for the treatment of viruses. These reagents are the phosphorodithioate containing oligonucleotides which are strongly inhibitory toward reverse transcriptases with ID₅₀ values in the 60 nM range. This means that these reagents are at least 33 fold more inhibitory than the phosphorothioate class of oligonucleotides. As was the case with phosphorothioate oligonucleotides which are also inhibitory against HIV-I reverse transcriptase (Stein, C. A. and Cohen, J. S., Cancer Research 48, 2659-2668, 1988; Majundar, C. Stein, C. A., Cohen, J. S. Broder, S. and Wilson S. H., Biochemistry 28, 1340-1346, 1989), it is to be expected this new class of chemotherapeutic agents, the phosphorodithioate oligonucleotides, to be inhibitory towards viruses containing reverse transcriptases such as HIV-I.

The compounds according to the present invention may be administered transdermally to mammalian host species having pathological conditions brought about by viruses, and other causative agents having a reverse transcriptase requirement for their transportation into the mammalian cell (infection), replication, or genetic expression. In these instances, the compounds may be formulated in suitable compositions determined by the intended means of administration, according to methods and procedures well- known to those skilled in the art. As used herein, the term "transdermal" is to be considered in its broadest meaning, that is administration across an epithelial layer of cells. As such, the term is appropriately used to designate topical, oral, pemasal, intravenous, intramuscular, and other methods of administration. For example, the compounds suitable for use in this invention may be formulated either individually or with other "active agents" or compounded with various conventional bases into preparations such as creams, ointments, gels, lotions, tablets, or pharmaceutical solutions for injection or sprays depending upon the desired mode of administration to the individual. In manufacturing these preparations, the composition may also be mixed with conventional thickening agents, emollients, surfactants, pigments, perfumes, preservatives, fillers, and emulsifiers, all of which are well known and conventionally used in the formulation of transdermal preparations. Typically, these nonactive ingredients will make up the greater part of the final preparation. Preferably, the compositions would be manufactured to allow for slow-release or timed-release delivery. Dosage to be given would, of course, depend upon the route of administration, the administration vehicle, and the degree and severity of the condition to be treated. In each instance, a minimal amount sufficient for bringing about the inhibition of the desired reverse transcriptase enzyme would be administered.

Thus while we. have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification and we therefore do not wish to be limited to the precise terms set forth, but desire to avail ourselves of such changes and alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

Having thus described our invention and the manner and process of making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same;

Claims

We claim

1. A compound according to the formula:

wherein R is H or a blocking group; A is H, OH, halogen, SH, NH₂or azide; B is a nucleoside or deoxynucleoside base; and n is an integer from zero to thirty.

2. A compound according to the formula:

wherein R is H or a blocking group; B is a deoxynucleoside base; and n is an integer from zero to thirty.

3. A compound according to the formula:

wherein R is H or a blocking group; A is H, OH, halogen, SH, NH₂ or azide; B is a nucleoside or deoxynucleoside base; and n and m are integers from one to thirty.

4. A compound according to the formula :

wherein R is H or a blocking group; B is a deoxynucleoside base; and n and m are integers from one to thirty.

5. A method for inhibiting a reverse transcriptase enzyme within a mammalian cell which comprises providing said cell with an amount sufficient to inhibit said enzyme of a compound according to Claims 1, 2 , 3, or 4.

6. A method for the treatment of a pathological condition in a mammalian host, which condition may be altered by inhibiting a reverse transcriptase enzyme activity within a mammalian host cell, which comprises transdermally administering to said host at least one compound selected from the compounds according to Claims 1, 2, 3, or 4 in an amount sufficient to inhibit said enzyme activity.