IE83348B1 - Therapeutic uses of 2',5'-oligoadenylate derivatives - Google Patents

Description

THERAPEUTIC USES OF 2' 5'—0LIGOADENYLATE DERIVATIVES Field of the Invention The invention relates to certain 2’,5’—oligoadenylate analogs and their use in the preparation of medicaments and pharmaceutical compositions of such analogs.

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 abbreviate 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: A, adenosine or adenylate or adenylyl cordycepin or C or 3'-dA, 3'-deoxyadenosine(3'- deoxyadenylate) ara-A, 9—fl—Q~arabinofuranosyladenine EHNA, erthyro(2—hydroxy-3—nonyl)adenine A—3'—amino, 3'—amino—3'—deoxyadenosine tubercidin, 4-amino—7(fi—Q—ribofuranosyl)pyrro1o— [2,3—g]pyrimidine ‘ '-dATP, 3'—deoxyadenosine triphosphate ATP, adenosine triphosphate I, inosine or inosinate or inosinylyl Xylo—A or xyloadenosine, 9-fl-Q—xylofuranosy1ade— nine dCF or 2'-deoxycoformycin, (R)-3—(2—deoxy~fi—Q— erythropentofuranosyl)-3,6:7,8-tetrahydroimidazo[4,5~ g][1,3]diazepine—8—ol —5A or 2',5'-oligo(A) or 2',5'-oligoadenylate, oligomer of adenylic acid with 2',5'—phosphodiester link—. ages and a triphosphate at the 5'—end 2',5'—cordycepin analog or 2',5'-oligocordycepin, oligomer of 3'-deoxgadenylic acid with 2',5'-phosphodi— ester linkages and a triphosphate at the 5'-end 2',5'-An or core oligomer, o1igomeI' of adenylic .acid with 2',S'-phosphodiester linkages ',5'-A3 or 2',5'-adenylate trimer core, adenylyle (2',5')adenyly1(2',5')adenosine ',5'-A"or 2',5'-adenylate tetramer core, adenyl- y1(2',5')adenyly1(2',5')adeny1yl(2',5')adenosine ',5’—3'dA3 or 2',5‘-C—CFC or 2',5'—cordycepin trimer core,_ 3'—deoxyadeny1yl(2',5')3'—deoxyadenylyl~ (2’,5')3'—deoxyadenosine ',5'-C-C—C—C or 2',5'—cordycepin tetramer core, 3’—deoxyadenylyl(2',5')3'—deoxyadenylyl(2',5')3'-deoxy- adenylyl(2',5')3'~deoxyadenosine ',5'—A3, adenylyl(3',5f)adeny1yl(3',5')adenosine ',5'-I3 or 2',5'-inosine trimer core, inosinylyl— (2',5')inosinylyl(2',5')inosine EBV, Epstein—Barr virus EBNA, Epstein~Barr virus associated early nuclear antigen HIV, human immunodeficiency virus, including HIV-1, HIV-2, and all other HIV subtypes HBLV, human B—cel1 lymphotropic virus HTLV, human T-Cell leukemia virus, including HTLV- I, HTLV-II and HTLV-III, and all other HTLV sub-types IFNa: adinterferon rIFN-«A: recombinant a—interferon dsRNA: double-strand ribonucleic acid ._3.. ..4_ ',5‘—A-A-A-3'—0-methyl, adenylyl(2',5‘)adenylyl- (2',5')3'—O-methyladenosine i ',5'-A-A-A-3‘-0—pentyl, adeny1yl(2',5')adenylyl— (2',5')3'pentyladenosine ',5'-A—A—A0-hexyl, adeny1y1(2',5')adenylyl- (2',5‘)3'hexyladenosine A 7 ',5'-A—A—A—3'—0-heptyl, adenyly1(2',5')adenylyl— n(2',5')3'—0—heptyladenosine The abbreviation for the "tetramer" compounds comprising the adenylyl (A) and 3'deoxyadeny1yl (C) moie- ties is illustrated by the following: ',5'-A-A—C—C, adenylyl(2',5')adenylyl(2',5')3'~ deoxyadeny1yl(2',5')3'-deoxyadenosine With the expansion of the knowledge of the anti- viral 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 antiviral response. 2Y,5'~0ligo(A) is a component of a natural, broad-spectrum antiviral defense mechanism in plants and animals. The 2—5A pathway, also known as the 2~5A/RNase L pathway or antiviral system, is widely accepted to be involved in the antiviral mechanism of interferon, and may also be involved in the regulation of cell growth and differentiation. According to that pathway, 2—5A is synthesized from AT~P' by 2',5'-oligo-i adenylate synthetase [ATP: (2‘-5')oligo(A)—adenyltrans~ ferase (EC 2.7.7.19)], hereinafter "2—5A synthetase".

The enzyme is activated by dsRNA. 2-SA exerts its biological effects by binding to and activating its only known target enzyme, the unique 2—5A dependent endoribo- nuclease RNase L. The latter cleaves viral and cellular "Human B—lymphotropic virus", also known as "human B-cell lymphotropic virus" (HBLV), 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 immunodeficiency virus ("HIV"), also known as human T—cell leukemia virus III ("HTLV-III"), the etio- logic agent of acquired immune deficiency syndrome, is a type D retrovirus. As in all retroviruses, an essential feature of HIV replication is reverse transcription of the plus—strand RNA genome into DNA, a process which requires an RNA dependent DNA polymerase, reverse tran— scriptase. This enzyme is viral—encoded and is found associated with genomic RNA in mature_HIV virions. The exclusiveness of reverse transcriptase to retroviruses and viruses requiring a short reverse transcription step makes reverse transcriptase a major target for antiviral, and particularly for antiretroviral, therapeutic inter~ vention.

What is needed is a method for controlling HIV, chronic fatigue caused by HBLV, and other disease states characterized by a 2—5A pathway defect using compounds that are more metabolically stable and active than authentic 2—5A.

Summary of the invention In accordance with the invention, ,6—dichlorobenzimidazylyl(2,’5’)5,6— dichlorobenzimidazylyl(2’,5’)5,6-dichlorobenzimidazole riboside riboside, or the 5’ mono—, di— and triphosphate thereof, or a pharmaceutically acceptable salt of any of them (hereinafter "5,6~dichlorobenzimidazyl 2—5A analogs") is provided.

The invention is further directed to pharmaceutical compositions of the compounds, the compounds for use in medicine and their use for the preparation of medicaments for treating retroviral infections.

Detailed description of the invention Administration of the 5,6—dichlorobenzimidazyl 2—5A analogs will render increased protection against disorders characterized by 21 2—5A defect, particularly protection against retroviral infection in animals and By "2-5A defect" manifestation, pathway which results in a decrease in the production of authentic 2—5A, and/or the interruption of 2—SA—dependent .Afflictions characterized by a 2- example, the following: retroviral humans. as used herein is meant any condition or interruption of the 2-SA activation of RNase L.

SA defect include, infection, particularly HTLV infection, most particularly T-cell HIV infection, chronic fatigue, and cutaneous lymphoma; chronic myelogenous leukemia; acute leukemia: cancer; T—cell leukemia; Alzheimer's disease; Parkinson's ._7.... disease; multiple sclerosis autoimmune disease; and surgery— and other trauma—induced immune dysfunction.

The defect is apparent in diseases, such as the above disorders caused by chronic viral infection, immune cell defects or both. 2-5A pathway defects are par- ticularly manifested in diseases characterized" by both chronic viral infection and immune cell defects.

Structural modification of the 2—5A moleculéiat the 3'-hydroxyl groups and elsewhere provides 2~5A analogues with remarkably increased_metabolic stability to 2'-phos- phodiesterases and cellular nucleuses, while maintaining the ability to activate RNase L. Likewise modification of native 2-SA by substitution of the terminal nucleotide results in a more stable molecule. Persistent, high intracellular concentration of the metabolically stable 2—5A analogs are a consequence of their increased stabil— ity.

The longer-lasting pharmacological activity of the, -SA analogs offer a more favorable therapeutic ratio.

This allows a decreased frequency of administration rela- tive to 2-SA, which is metabolicly unstable. Decreased frequency of ‘administration .is important due to the chronic nature of many afflictions characterized by 2-SA pathway defects.

The 2-5A analogs are particularly useful in ‘the treatment of infections caused by retroviruses. The 2—SA pathway defect associated with retroviral infection com- prises the inactivation of the pathway caused iby' the virus’ interference with the activation of 2—5A syn- thetase by dsRNA. .In .-the absence of 2-511 synthetase activation, 2—SA production, and hence activation of RNase L, is reduced. According to the present invention, exogenous, metabolically stable 2~5A analog is admini— stered to counteract this retrovirally~caused defect in the 2~5A pathway. The 2-5A analogs, like authentic 2—5A, ..8_ are capable of activating RNase L, which cleaves viral RNA.

The 2-SA analogs are particularly useful in protec- ting" 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 HTEV viruses is a retrovirus.

Also‘ known as "HIV-1", HTLV-III is responsible for causing acquired immune deficiency syndrome ("AIDS").

The compounds are further‘ believed useful in ‘treating HIV-2, a second serologically distinct HIV. subtype.

Hereinafter (HIV) shall mean either HIV-1 or HIV-2, and any other HIV subtypes now or hereinafter known.

HTLV-infected patients, in particular HIV-1~in— fected patients, have been shown to demonstrate unusually low levels of 2-SA and/or RNase L activity in blood mono- nuclear cells. Blood mononuclear cells from healthy in- dividuals, 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-SA levels, and evidence the appearance of novel RNA cleavage products, distinct from the specific cleavage products observed in lalood mononuclear cells from normal individuals.

While the practice of the invention is illustrated herein with regard to the treatment of HIV-1 infection, which is generally regarded as a prototypical retrovirus, the method_of the invention has application to the treat- ‘ment of any diseases wherein the etiologic agent com-’ prises a retrovirus. Additional retrovirus which infect man include, for example, the various non~HIV HTLV virus, discussed above. .

The various afflictions characterized by a 2—5A pathway defect, in particular retroviral infection, most particularly HIV infection, may therefore be treated by the administration of exogenous, metabolically stable analogs of 2-51; to counteract the 2-5A system defect associated with the disease state.

For pharmaceutical use, the 5,6—dichlorobenzimidazyl 2’,5’— oligoadenylate analogs may be taken up in pharmaceutically acceptable carriers. Such carriers for preparation of pharmaceut- ical compositions of-the invention may be eitherfibrganic or inorganic, solid or liquid in nature. Suitable solid‘ carriers include gelatin, microcrystalline cellulose, . lactose, starches, and magnesium stearate. 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 solution, dextrose solution and glycol solutions such as aqueous propylene glycol or aqueous ‘poly(ethylene glycol). s The properties of the formulations may be enhanced by the addition of one or more adjuvants possessing properties as viscosity enhan- cers, surfactants, {xi modifiers, preservatives, sweet- eners, stability enhancers, coloring agents, suspending agents, granulating agents, coating agents, disintegra- tion modifiers, propellants, emulsifying agents and' hymectants. The resulting compositions may be in the form of solutions, suspensions, tablets, capsules, oint- ments, elixirs, injectable compositions and the like.

The dosage administered depends upon the severity 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 embodiment, 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. ._]_'0..

The compounds may be administered in the form of water-soluble salts. 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 Pharmaceutically acceptable water- formulation may contain additional agents, such as a sugar or protein, to maintain the osmotic balance. - The 5,6—dichlorobenzimidazyl 2',5'~oligoadenylate analogs may be administered in doses of about O.lmg to about 1 gram to animals or humans afflicted by, or suspected of affliction by, or at risk of 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 ingredient. The compounds may be administered by any of several routes, including, but not limited to, toneal or intramuscular injection, intravenous injection, intraperi— and oral administra- tion. ‘Techniques for accomplishing such administration are routine and known in the medical art. Administration may be as frequent as several times a day or as infre- quent as weekly. For intravenous injection, particularly for treatment of HIV, a solution containing about 0.1 to about 1.0 milligram per ml of active ingredient is pre~ ferred.

It is also contemplated that the 5,6—dichlorobenzimidazyl 2’,5’— oligoadenylate analogs may be administered topically to treat skin lesions associated with any of the disease states charac- terized 2~5A pathway defect. A sufficient amount of a preparation containing one or more of the 2',5'—o1igo- adenylate analogs may be applied to cover the lesion or An effective concentration of active M to about 10"‘ M, with about affected area. agent is from about 10'3 "? M being preferred.

In addition to administration with conventional carriers, the ,6~dichlorobenzimidazyl 2—5A analogs may be administered by a Variety of specialized oligonucleotide or nucleic acid delivery techniques, such as by encapsulation in uni—lameller liposomes or reconstituted sendai virus envelopes, or by conjugation to carrier molecules such as poly(L—lysine). Such methods are disclosed in International Patent Application WO 89/03683.

The 5,6—dichlorobenzimidazyl 2',5’—oligoadenylate analogs may be chemically synthesized from 5,6—dichlorobenzimidazyl riboside according to the following general protection, condensation and deprotection methods as appropriate.

The blocked adenosine—2'—phosphodiester is condensed with the blocked nucleoside in the presence of a condensing reagent which causes blocking of the phos- phate functions to form a fully protected dinucleoside~ monophosphotriester.

The resulting fully protected condensate is then detritylated at the terminal 5' position with a detrity1- ating agent and condensed with a further adenosine-2'- phosphodiester, blocked as described above, to form a fully protected 2',5'—trinucleosidediphosphoditriester, or 2',5' trimer core, The fully protected trimer-eore is then treated with appropriate deprotecting reagents to achieve complete deprotection and conversion to 2',5' ,trimer core. , i 5,6—dichlorobenzimidazylyl—(2,’5’)5,6— dichlorobenzimidazylyl—(2’,5’)5,6—dichlorobenzimidazole riboside is prepared by condensing commercially available 5,6- dichlorobenzimidazole riboside (Sigma, Cat. No. D5893) according to methods described herein.

The preparation of 2’,5’—oligoadenylate analogs is illustrated in the following non—limiting examples, although the specific compounds prepared fall outside the scope of the present invention.

Example 1 Preparation of structurally Modified 2',5'—Adenv1ate Trimer Cores The various structurally modified novel trimer core analogs of 2',5’—adenylate may generally be prepared as follows, although the specific compounds prepared in this example fall outside the present invention. One mmole of suitably blocked adenosine—2’— phosphodiester having the general formula of reactant (I) was prepared from adenosine or cordycepin-according to the method of Charubala, R., Uhlmann, E., and Pfleiderer, W., Liebigs Ann. Chem.,-2392 (1981) and Charubala, R., and Pfleiderer, W., Tetrahedron Lett. gl, 4077 (1980). Ex- amples of suitably blocked adenosine—2'-phosphodiesters are represented by compounds ; and ;, listed in Table 1.

NHBz Runaw- \ N ® MM‘nocH 2 N - ‘J O N R 0 O=Pé0* / I HN +\\ OR‘ TABLE 1 . Compohnd butyldimethy1si1y1—5'—0-Q— I OSiTBD 2—chloro— methoxytrityladenosine—2- phenyl (2—ch1oropheny1)~phosphate Pyridium N‘—benzoy1-5'—0~p~ V 2~Chloro— methoxytrity1—3‘—deoxyadenosine~ 2'—(2—chloropheny1)—phosphate phenyl Tab1e.2.

NHBZ " ‘N Reactant HOCH2 R on‘ TABLE 2 Compound adenosinei N°,2'-0—dibenzoyl—3'e_ A H _Bz N deoxyadenosine N‘,2'dibenzoy1-3'- 1 0-Q-C,H15 Bz N —Q—hepty1adenosine ethoxycarbony1amino~ 3'—deoxyadenosine Compounds 3 and Q are prepared treating N‘-benzoy— lated adenosine with monomethoxytrityl chloride in pyridine to yield the 5'-monomethoxytrityl derivative.

The derivative is treated with t-butyldimethylsi1ylch1or- ide in a mixture of pyridine and methylimidazole to form the N‘-benzoy1—2',3'—di-0—t—buty1dimethy1si1y1-5'—mono— methoxytrityladenosine. The trityl group is removed by acetio acid to produce compound ;. Compound 3 is syn- thesized analogously; starting with .N°—benzoy1ated tubercidin. This method of preparation is a described in Charubala, et a1., Liebigs Ann. Chem. 2392 (1981); Compound 3 is prepared by treating N°—benzoy1ated— 3'-deoxyadenosine with monomethoxytritylchloride to yield N°—benzoy1—5'-0—monomethoxytrityl-3'~deoxyadenosine, which is converted to N6—2‘—0—dibenzoyl-3'—deoxyadenosine by benzoylation of the 2'~hydroxyl group with benzoyl- chloride followed by detritylation with 80% acetic acid for 30 minutes. This method of preparation is as de- scribed in Tetrahedron Lett. gl, 4077 (1980).

' Compound :5 is prepared by converting N°—benzoyl- adenosine with jg-butyldiphenylchlorosilane to the 5'- silylated nucleoside in pyridine. The 5'-silylated nucleoside is treated with triethyl orthoacetatefollowed by boron trifluoride/diethyl ether and sodium iodide in CH3‘CN (0°C, '1 hour) to yield the 3'-‘-iodoacetyl deriva- tive. The iodoacetyl derivative is converted to compound Q by treatment with tributyltinhydride in toluene (80'C, l.hour) and desilylation with ammonium tetrabutyl fluor- ide in tetrahydrofuran. This method of preparation is as described/ by Engles, J ., in Tetrahedron Lett. 2;, 4339 (1980)_ Compounds g and 1 are prepared by treating N‘-— benzoyl adenosine with tritylchloride in pyridine and refluxing for 2 hours. N‘-benzoy1—5'—trity1adenosine is isolated by extraction with chloroform. The water from the chloroform phase is removed by drying with sodium sulfate. N‘-benzoyl—2',5'—di-0—trityladenosine and N‘~ benzoyl-3’,5‘-di-0—trityladenosine are isolated by preparative silica gel thin layer chromatography in chloroform/ethanol. Compounds Q and 1 are prepared by treating the isolated compounds with g—penty1chloride and Q-heptylchloride, respectively, under reflux in a suspen- sion of sodium hydroxide in benzene. The solution is neutralized by refluxing in acetic acid followed by the addition of diethyl ether and water. The reaction products are extracted with chloroform followed by thin layer chromatography with chloroform: methanol (4:1) on silica gel plates. The trityl and benzoyl groups are removed by refluxing in acetic acid for one hour, cool- ing, extraction owith diethyl ether, followed by con- and 1, This method of preparation is carried out accord- ing to Blank, H.U., Franne, D., Myles, A. and Pfleiderer, W., Justus Liebigs Ann. Chem. 742, 34 (l970L Compound Q was prepared as follows. 1_mmole of 3'- amino—3'—deoxyadenosine was reacted with 1.2 mmole of 1- methyl-3—nitrophenylethoxy carboxylimidazolium chloride in dimethylformamide, followed by the addition-of hexa- methyldisilazane to (block the 2',5' and 6-amino‘ posi- tions. 1.1 mmole of benzoyl chloride in pyridine was .added at room temperature to produce blocked N°~benzoyl- 2',5'—disilyladenosine. The reaction mixture was poured Reactants (I) and (II) were combined to produce the intermediate having the general formula of dimer (III) as follows. 0.95 Mmole‘ of reactant (II) and condensing reagents 2,4,6—triisopropylbenzenesu1fonyl chloride (2 mmole) and 1—methylimidazole (6 mmole) were combined and stirred for 1 hr at room temperature. The reaction is stopped by adding 30 ml of aqueous phosphate buffer pH 7 and extracted with 150 ml of chloroform. The chloroform layer was washed twice with 50 ml of water, dried over sodium sulfate about 1-2 hrs and filtered. The chloro— form was evaporated to a small volume and then applied to a silica gel column (20 x 2.5 cm) for purification.

Chromatography was performed first with chloroform and then with chloroform/methanol (99/1, v/v) to elute the fully protected dinucleosidemonophosphotriester produce of the general formula of dimer (III). Evaporation gave a solid foam of dimer (III) exemplified by compounds lg- ;1 (Table 3). ‘—17~ RzOR3 TABLE 3 Compound Compound One mmole of the fully protected dimer (III) was stirred at room temperature for 30 minutes in 20 ml of 2% p~toluenesulfonic acid ih dichloromethane/methanol (4/1, v/v) for detritylation. 20 ml of phosphate-buffer pH? was added and'subsequently extracted several times with 200 ml of dichloromethane. The organic phase was washed with water, dried over sodium sulfate, evaporated to a The fully protected 2',5'—trinucleosidediphospho— ditriester, having th_e general formula of trimerc(_2E(V) and exezixplified by compounds ;_6_ - 11 (Table 4, below), was prepared from the 5'—detritylated dimer (III) as follows.

Trimer ( IV ) TABLE 4 .05 Mmole. of the startingt adenosine-2'—phospho— diester (reactant (I)), was condensed with 1 mmole of the '—detritylated dimmer (III) (compounds lg — gg) in 10 ml of absolute pyridine using 3 mmole of 2,4,6-triisopropyl— benzenesulfonyl chloride and 9 mmole of 1~methylimidazole as condensing agents. ‘Work-up was performed after 2 hrs in the manner as described above. Quenching with phos- phate buffer, followed by extraction with dichloromethane and silica gel chromatography in chloroform and chloroform/ methanol (99/1/ to 98/2, v/V) yielded 70-90% of fully protected trimer (IV) (compounds gg — Q3), as a chromatographically pure amorphous powder.

The fully‘ protected 2‘*5'-trinucleosidediph0spho~ ditriester, trimer (IV), was deprotected to trimer core (V) as follows. .01 Mmole of trimer (IV) was treated with a solution of 0.073 g Q-nitrobenzaldoxime and 0.07 g tetra- methylguanidine in 2 ml of dioxane water (1/1, v/v) for 16 hrs at room temperature to deblock the Q-chlorophenyl group. After evaporation to dryness and coevaporation four times with water, 20 1ml of concentrated ammonium hydroxide was added and the solution stirred for 2 days at room temperature to deprotect the acyl groups. The solution was then evaporated again, and the residue was dissolved in 25 ml of water and washed four times with 10 ml of chloroform each time. The water layer was evapor- ated to dryness and coevaporated ten times with 10 ml The residue was then absolute pyridine each time. treated with 2 ml of an 0.5 M .solution of anhydrous tetrabutylammonium fluoride in absolute pyridine 16 t-butyldimethylsilyl groups. treatment of the residue with 5 ml of 80% hrs to remove the evaporation, acetic acid for 6 hrs at room temperature lead to clea- vage of the p—methoxytrityl group. The solution was again evaporated, the residue dissolved in 15 ml of and extracted four times with 5 ml of chloroform The aqueous layer was evaporated and then water, each time. coevaporated several times with water until the smell of acetic acid disappeared. The residue was dissolved in 10 ml of water and applied to a DEAE—Sephadex A—-25 column (60 x 1 cm) for ion——exchange chromatography with a gradient of 0-001 — 0.5 M triethylammonium bicarbonate.

The main fraction was evaporated, then coevaporated several times with water. Trimer core (V), a fully I deprotected 2 ' , 5 ' —trinucleosidediphosphate, was isolated by lyophilization of the aqueous solution to give 70-90% of an amorphous solid. Trimer core (V) is exemplified by After’ R3 ON TABLE 5 Compound Compound '-monophosphate Examp1e'2 Preparation of 3'—0—t—butyldimethylsilyladenylyl ‘,5’)3'—0—t-butvldimethvlsilyladenvlvl(2'.5')adenosine Example 3 Preparation of adenylyl(2',5')adenylyl 13'.5')3'—amino~3'—deoxvadenosine 0.01 Mmole of N‘-benzoyl-3'—0~t—buty1dimethylsilylF '-0—p-methoxytrityladenylyl(2'—g—chlorophenyl-5!)N‘— benzoyl-3't—butyldimethylsilyladenylyl(2‘—g—ch1oro— pheny1-5')N°-benzoyl- 2'—0-t—butyldimethy1si1yl—3'~p— nitrophenylethoxycarbonylamino~3'~deoxyadenosine (com- pound gg, Table 4) was treated according to the deprotec— tion procednre of Example. 1 wherein the Q-nitrophen— ylethoxycarbonyl group was cleaved simultaneously’ with the silyl groups by tetrabutylammonium fluoride in a fi- elimination process. Subsequent decarboxylation yields ',5'—A-A—3'—amino (compound 5;, Table 5). treated according to the deprotection procedure of Example 1 except that the last step of acetic acid treatment was omitted. The product, 2',5'—trityl~A3 (compound gg, Table 5) was isolated, purified by DEAE— Sephadex A—25 chromatography, and lyophilized to form the amorphous pure compound 53 as a powder in 85% yield.

Preparation of 5'Q-nethoxytrityl-3'—deoxyadenyly1 12'.5')3'—deoxvadeny1vl(2'.5')3'—deoxvadenosine ‘ 0.01 Mmole of N‘—benzoyl-5'—0—p-methoxytrityl—3'- deoxyadenylyl(2'~g-chlorophenyl—5')N°ebenzoyl-3'—deoxy- adeny1y1(2'-gechlorophenyl-5')N°,N‘,2'-0—tribenzoy1-3'- deoxyadenosine prepared according to the method of Charu— bala, R., and Pfleiderer, W., Tetrahedron Lett. gl, 4077 (1980) was treated according to the deprotection proce- dure of Example 1 except that the steps of treatment with tetrabutylammonium fluoride and acetic acid were omitted.

The product, 2',5'-trityl-C3 (compound 35, Table 5) was isolated, purified by DEAE—Sephadex A—25 chromatography and lyophilized to form the amorphous pure compound.

The 5'—monophosphates of the trimer core molecules of the present invention may be prepared from the fully blocked 2',5'-trinucleosidediphosphoditriester by detri- tylation as in Example 1 followed by reaction with di—p— nitrophenyl~ethylphosphoryl chloride. Extraction, chromatography and deblocking according to Example 1 results in isolation of the 5'—monophosphate trimers.

The preparation is exemplified in the method of Example Example 6 Preparation of 5'—0—phosphory1—3'~deoxyadenylyl 12'.5’)3'-deoxyadenv1v1(2'.5')3'—deoxvadenosine 0.1 Mmole of N‘-benzoyl-5'—0—Q—methoxytrityl-3'- deoxyadenylyl(2'-Q-chloropheny1—5')N°-benzoy1—3'—deoxya- deny1yl(2'—g~ch1oropheny1—5!)N‘,N°,2'—Otribenzoy1-3'- deoxyadenosine is prepared" from 3'—deoxyadenosine by benzoylation, 5'~tosylation, and 2'—phosphorylation, with formation of the dinucleoside phosphotriester, N"- benzoyl(2—g-chlorophenylphosphoryl-5)3'-deoxyadenosine, by treatment of the reaction products N°—benzoy1(2- triethylammonium:Q-chlorophenylphosphoryl-5)-5'-tosyl-3'- deoxyadenosine_ and_ 2’-cyanoethylphosphoryl—g:qh1oro- phenyl-3'-deoxyadenosine with triisopropy1benzenesu1- fony1—nitro-1,2,4—triazolide. The fully7 blocked. dimer ‘thus formed is condensed with N‘,2'-0—dibenzoyl—3'— deoxyadenosine to form the trimer. 0.01 mM of this trimer prepared according to the method of Charubala, R.} and Pfleiderer, W., Tetrahedron Lett. g;, 4077 (1980) was treated with 2.ml of a solution of 2% Q-toluenesulfonic acid -in dichloromethane/ methanol (7/3, v/v) for 30 minutes at room temperature to remove the p—methoxytrityl group. Purification kn! silica gel chromatography on a preparative plate with chloroform/methanol (95/5, v/v) gave a 90% yield of the 5'-deprotected analog.

This product was dissolved in 1 ml of absolute pyridine and treated with 0.27 mmole of di—;nitropheny1- ethyl—phosphoryl chloride as described by Himmelsbach, F., and Pfleiderer, W., Tetrahedron Lett. gg, 4973 (1982) for 1 hr. at room temperature. After dilution with 15 ml of chloroform, the reaction mixture was extracted three times with phosphate buffer pH 7. The organic layer was dried over sodium sulfate, filtered, evaporated and coevaporated three times with 10 ml of toluene each time.

The residue was purified by silica gel chromatography on preparative plates in chloroform/methanol (9/1, v/v) to yield 81% of 5'-0—di—p-nitrophenylethy1phosphory1—N‘— benzoyl—3'—deoxyadeny1yl-(2'-g—chlorophenyl-5')N°-ben- zoyl—3'—deoxyadeny1yl(2'—9:chlorophenyl—S')N‘,N‘,2'—0— tribenzoy1—3'—deoxyadenosine in the form of an amorphous solid. .01 Mmole of the latter material was treated with Q—nitrobenzaldoximate according to the deprotection pro- cedure cu? Example 3. to remove g—chlorophenyl blocking groups. After evaporation to dryness and several coevap— orations with absolute pyridine, the deprotected product was dissolved in 10 ml of a 0.5 M solution of diazabi— cyclo [4.3.0]undecene in absolute pyridine and stirred for 36 hours at room temperature to cleave the pjnitro— phenylethyl group by fl-elimination. The solution was again evaporated and then treated with 20 ml of con— centrated ammonium hydroxide for 24 hours at room temper- }ature. Purification and isolation of the trimer core 5'- monophosphate (compound gg, Table 5) was achieved by DEAE—Sephadex chromatography’ and lyophilization of the main fraction.

.The tetramer core molecules of the present inven- tion may be prepared by following the method of Examples or 8. rfixamgle 7 Preparation of adenylyl(2',5')adenyly1(2',5')3'— deoxvadenv1Y1(2',5')3'—deoxvadenosine 0.5 Mmole of fu1ly—protected compound 51 having the formula (VI) and 0.4 mmole of N°—benzoyl—3'~deoxyadenylyl (2'—9—chloropheny1-5')N‘,2'—0—dibenzoyl—3'-deoxyadenosine (compound gg, Table 3) were dissolved in 5 ml of absolute pyridine.

Reactant ( VI ) +33 so 9 Q°"?* 9 CI 0" HN +\ Following addition of 1 mmole of 2,4,6-triisopropyl— benzenesulfonyl chloride and 3 mmole of l-methylimidaz— one, the mixture was stirred for 2 his at loom tempera- ture. The solution was diluted with 400 ml of chloro~ form, washed twice with 400 ml of water, then the organic layer was dried over sodium sulfate, filtered and evapor— ated to dryness. The residue was coevaporated twice with 50 ml of toluene. Purification was achieved by chromato- graphy on a silica gel column (20 x 2.5 cm) first with chloroform and then with a gradient of chloroform/methan- 01 of 99/1 to 98/2 (v/V). On evaporation, the main fraction gave compound gg (Table 6) as a solid foam in 80% yield. éompound gg is a fully-blocked 2',5'—tetra- nucleosidetriphosphotritriester according to the general formula of tetramer core (VIi), below. Deprotection of the blocking groups was performed by the procedure of Example 1 to yield 2',5'~A-A—C—C (compound 52, Table 6).

DEAE—Sephadex chromatography, evaporation and lyophiliza— tion resulted in an amorphous so1id.in 80% yield. *27~‘ N \N ’ I <.. N» - Tetramer Z i ‘core 2 . 8° ( VH ) 3 9 NHR R ‘O-P30 N I \N o ( I J in " .

H 9 R_o_pq) NHR I H OR TABLE 6 Compound No. R R1 R2 R3 48 B2 MMTr SiTBD 2—ch1oropheny1 49 H H H H Examgle 8 Tdeoxyadeny1y1(2'-o—ch1oropheny1—5')N°—benzoy1—3'—deoxya— ', 5'—trinuc1eosidediphosphoditriester according to the general formula of reactant (VIII), NH8z I ‘" ROGQ N NJ 0 u Reactant ( VHI ) _ , u_ Nflzz O-P—0 | N \N C was treated with 2 ml of a 2% solution of Q-toluenesu1— fonic acid in dichloromethane/methanol (4/1, v/v) for 30 minutes at room temperature. The reaction was stopped by adding 20 ml of phosphate buffer pH 7. The solution was extracted several timesfwith 200 ml of chloroform. The organic layer was washed with water, dried over sodium sulfate, filtered and evaporated to a small volume for purification on preparative silica gel plates in chloro- form/methanol (95/5, v/v). The main band was eluted by chloroform/methanol (4/1, v/v) to give the 5'~detri- .05 Mmole of compound g; (Table 7) was then condensed with 0.1 mmole of pyridinium N‘-benzoyl—5'~O-p- methoxytrityl—3'—deoxyadenosine—2'—(2-g~chlorophenyl) phosphate (compound g, Table 1) in 0.6 ml of absolute pyridine in the presence of 0.2 mmole of 2,4,6-triisopro— pylbenzene—sulfony1 chloride and 0.6 mmole of‘\.;L;-methyl- imidazole for 2 hrsfat room temperature. The solution was diluted with 100 ml of chloroform, washed twice with water, dried over sodium sulfate and evaporated to a ‘small volume for separation (n1 preparative silica gel plates in chloroform/methanol (95/5, v/v). ,The main band was eluted with chloroform to give the fully-protected 2', 5'-tetranucleosidetriphosphotritriester compound (Q; (Table 8, below) as an amorphous solid upon evaporation in 84% yield.

The 5'-O-monophosphates of the tetramer core molecules of the present invention may be prepared from the fully blocked 2',5'—tetranucleosidetriphosphotritri- ester by 5'—detritylation as _n1 Example 1. followed by reaction with di-p—nitrophenylethylphosphoryl chloride.

Extraction, chromatography and deblocking according to Example 1 results in isolation of the 5'—0—monophosphate tetramers. The preparation is exemplified in Example 6, above.

/N \j 0 / EH20 N Teframer " core — ( IX ) u 'NHR R2.0-P=o \N o 6 l ,. ,4 0 H ? NM R—o—P:0 é H OR TABLE 8 Compound R R‘ R’ No- Q; Bz MTr 2—ch1orophenyl 53 H H H The 5'—diphosphate and 5'—triphosphate of the trimer and tetramer core.mo1ecu1es of the present inven- tion may be prepared by adding 0.5 mM of tributylammonium pyrophosphate qissolved in 5 ml of aimethylformamide to 0.1 mM of monophosfihorylated core as the anhydrous tributylammonium salt ix: 1 ml of dimethylformamide and 0.5 mM of 1,1‘—carbony1diimidazole. After 20 hours at Structural modification of the 2',5'—oligoadenylate [molecule at‘ the 2'-terminal nucleoside, aglycon and/or ribosyl moiety have provided molecules that are potent inhibitors of virus replication, particularly replication of retroviruses, such as HIV. These synthetic molecules are biologically more active and metabolically more stable than the naturally occurring 2',5'-oligoadenylate molecule.

The antiretroviral activity of the compounds of the present invention is demonstrated by the following experimental methods in which any of the core compounds of the invention or their 5' mono, -di—, or triphosphate counterparts may be substituted for any of the analogs in the following experiments.with the efficacy disclosed for such compounds in the specification.

According to the following experiment, a 5,6,- dichlorobenzimidazyl 2’,5’—oligoadenylate analog was observed to inhibit HIV-1 reverse transcriptase activity when compared with other 2—5A analogs.

Example 9 Inhibition Of HIV-1 Reverse Transcriptase Activity In Vitro Reverse» Transcriptase Assays. Virus was concentrated from cell?free (0.45. micromolar—filtered) conditioned H9/HTLV-IIIB culture supernatants by’ centrifugation at 18,000 r.p.m. for 4 hrs at 20°C in a Beckman JA—20 rotor.

A viral pellet obtained from 50 ml of conditioned culture fluid was dissolved in 0.5 mL of a solution-containing 17 mM Tris-HC1 (pH 7.8), 3mM dithiothreitol (MT), 55 mM KC1, 0.32% w/v’ Triton X-100, and 33%’ glycerol. This viral lysate was stored at —20°C and was used as a source of HIV-1 reverse transcriptase. Reverse transcriptase reactions~ were performed in 100 uL reaction volumes containing 40 mM Tris-HC1 (pH 7.8), 4 mM DTT, 50 mM KC1, mM MgCl2, 0.0325% W/V’ Triton X-100, 3 micromolar [33H]dTTP (80 Ci/mmol,NEN) and poly (A).(d‘I‘)15(2.S microgram/mL) template-primer after the addition of 10 microliter enzyme. Inhibitors were added at various concentrations after adjusting water volumes so that reaction volumes remained constant. Reactions were incubated at 37°C for l hr in a humidified environment and terminated by adding 2 mL of 10% cold trichloroacetic acid. Precipitate was collected on 0.45 micron cel- 1ulose—acetate Millipore filters which were then dis- solved in 10 ml. of 3a70B aqueous scintillant and the counts per minute quantitated using ea Beckman LS 6800 liquid scintillation spectrometer{ The effect of a 5,6—dichlorozimidazylyl analog on 2—5A on HIV-1 reverse transcriptase activity is shown in Table 9. The ,6—dichlorobenzimidazole riboside 2’,5’—trimer 2’,5’—cordycepin trimer 5’—triphosphate (pfig) was the most effective inhibitor (73% inhibition at 200 micromolar), followed by 2’,5’—pC4(64% inhibition at 200 micromolar), the 2’-5’~cordycepin trimer 5’~ triphosphate (pfig) (62% inhibition at 200 micromolar), 2’—5’~A—C— A (58% inhibition at 200 micromolar) and 2’,5’—A—A—ara—A (53% inhibition at 200 micromolar)(Table 9).

TABLE 9 Effect of 2—5A Analogues on HIV-1 Reverse Transcriptase Activity —5A or 2',5' Analggge (adenosine) A A3 PA3 Pa 3‘-.3 Acordycepin (C) ‘c PC3 psca C pc.’ A-C-C A-A—C C-A-C A-C-A A-A-C—C A—A—C-A P13 A—A—ara-A Tu-Tu—Tu xylo—A‘ A-A—A—3'—amino EHNA—AfA ,6—dichlorobenzimid— azy1y1(2'—5')~5,6—di— ch1orobenzimidazy1yl~ (2',5')—5,6—dich1oro— Concentration (micromolar) 200 200 400 200 2200 100 200 200 200 200 200 200 200 200 200 200 200 200 200 benzimidazole riboside Percent Inhibitioné a Values ~for control reactions present) were greate reaction (no enzyme 12,000 cpm. (i.e. b Average of two or more experiments; no inhibitor r than 180,000 cpm while blank present) values were less than

Claims (4)

1. 5,6—Dich1orobenzimidazyly1(2’,5’)5,6— 5 dichlorobenzimidazylyl(2’,5’) 5,6—dichlorobenzimidazole riboside, the 5’ mono, di, and triphosphates thereof, and pharmaceutically acceptable salts of any of them.

2. A compound according to claim 1 for use in medicine. 10

3. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of a compound according to claim 1 for the preparation 15 of a medicament for treating ietroviral infections. Tomkins & Co.