EP0541742A1 - Analogues de 2',5'-nucleotides tels que des agents antiviraux - Google Patents

Analogues de 2',5'-nucleotides tels que des agents antiviraux

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Publication number
EP0541742A1
EP0541742A1 EP19920909806 EP92909806A EP0541742A1 EP 0541742 A1 EP0541742 A1 EP 0541742A1 EP 19920909806 EP19920909806 EP 19920909806 EP 92909806 A EP92909806 A EP 92909806A EP 0541742 A1 EP0541742 A1 EP 0541742A1
Authority
EP
European Patent Office
Prior art keywords
group
butyldimethylsilyl
mmol
adenosine
residue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19920909806
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German (de)
English (en)
Inventor
Carlo Battistini
Maria Gabriella Brasca
Antonio Giordani
Silvia Fustinoni
Antonella Ermoli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Italia SRL
Original Assignee
Farmitalia Carlo Erba SRL
Carlo Erba SpA
Pharmacia and Upjohn SpA
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Publication date
Application filed by Farmitalia Carlo Erba SRL, Carlo Erba SpA, Pharmacia and Upjohn SpA filed Critical Farmitalia Carlo Erba SRL
Publication of EP0541742A1 publication Critical patent/EP0541742A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the present invention relates to the use of nucleotide analogs having 2',5' unusual internucleotide linkage as antivirals, antitumors, immunomodulators, enhancers of interferons or interferon inducers, to new diribonucleotide analogs and to a stereoselective route for their preparation.
  • the recent literature describes the "2-5A system” as the main mechanism by which interferon promotes an antiviral state and carries on its activity resulting in the degradation of viral mRNA and therefore in the inhibition of protein synthesis. Interferon seems to provide a membrane-mediated signal to a healthy cell with risk of viral infection. Such a signal leads to a level increase of the enzyme 2-5A Synthetase that is activated after infection by the ds RNA formed during the virus life cycle or introduced by the virus itself.
  • This enzyme generates 2',5'-oligoadenylates (2-5A) from ATP and these unusual oligonucleotides activate a 2-5A-dependent endonuclease, responsible for the degradation of single strand RNA and hence for the inhibition of protein synthesis.
  • 2',5'-Oligoadenylates (2-5A) are oligoribonucleotides where 3 to 15 adenosine units are linked by phosphate moieties unusually between the position 2' and 5', besides they have a triphosphate capping at the 5' end.
  • 2-5A system seems to be involved in cell regulation and differentiation. In any case the level of 2-5A has a fundamental importance and the regulation is obtained through a formation-degradation balance. While the formation is provided by 2-5A Synthetase, 2-5A are degraded by the specific enzyme 2'-Phosphodiesterase (2'-PDE) that starts its action at the 2' end of the oligomer chain and gives 5'-AMP and 5'-ATP (B. LEBLEU AND J. Content, in "Interferon 4" (I. Gresser ed.) pp. 47-94, Academic Press - New York, 1982). Selectivity of action of the 2-5A system for the inhibition of viral translation respect to the cellular one is thought to be due to the localization of the system activity within the cell, the stimulation by ds RNA being restricted to the particular area where it appears.
  • 2'-PDE specific enzyme that starts its action at the 2' end of the oligomer chain and gives 5'-AMP and 5'-ATP
  • the object of the present invention is to provide compounds that increase the level of endogenous 2-5A by acting on the formation-degradation balance through the inhibition of the 2'-phosphodiesterase (2'-PDE).
  • 2'-PDE 2'-phosphodiesterase
  • the alteration will still depend on the activation of 2-5A synthetase by infectious particles keeping the localization of action within particular region of the cell and hence maintaining selectivity against viral agents.
  • the less charged or even uncharged compounds of the present invention render the obstacle of cell penetration easier to be overcome.
  • the present invention relates to the use of nucleotide analogs having 2',5' unusual internucleotide linkage as antivirals, antitumors, immunomodulators, enhancers of interferons or interferon inducers having the formula I
  • R 1 represents (i) a hydrogen atom, (ii) a 2' or 3'-P-thionucleotidyl residue conjugated at its 5' position with an acyl group, (iii) a phoshoryl group, a P-thiophosphoryl group, a PO 2 R 2 H group optionally esterified with a linear or branched, cyclic or acyclic aliphatic hydroxy alkyl group having from 1 to 6 carbon atoms or with a lipophilic moiety or (iv) an acyl group; R 2 represents a C 1 -C 6 alkyl group or hydrogen atom,. and the pharmaceutically acceptable salts thereof.
  • the present invention provides a compound of the formula I'
  • R 2 is as above defined and R 3 has the same meanings of R 1 above defined but not hydrogen atom, P-thiophosphoryl or phosphoryl group.
  • C 1 -C 6 alkyl group encompasses linear or branched, acvlic or cvclic alkyl groups for example methyl, ethyl, propyl, n-butyl, s-butyl, i-propyl, tert-butyl, amyl, cyclichxyl, cyclopentyl.
  • R 3 or R 1 represent a P-thianycleotidyl residue, they are preferably a 2- P -thioedenyl residue.
  • Preferred lipophilic moieties are cholesteryl group or glycerol esterified with saturated on unsaturated long chain fatty acids having from 6 to 20 carbon atoms. Such fatty acids are preferably palmitic or miristic.
  • the C 1 -C 6 alkyl phosphonyl group which R 1 and R 3 may represent is preferably methylphosphonyl group.
  • the acyl group which R 3 or Ra. may represent is preferably the residue of
  • octanoic cyclohexylacetic or palmitic acid
  • a difunctional acid more preferably oxalic, carbonic, phosphoric, thiophosporic, malonic or succinic acid, in which the other acidic moiety is esterified with a C 1 -C 6 hydroxy alkyl group or with a lipophilic moieity as defined above;
  • carboxylic acid endowed with positive charged part at physiological pH and with or without lipophilic group preferably non- ⁇ aminoacids, more preferably ß-aminopropionic and 3- or 6-aminocaproic acids, or ⁇ -aminoacids of basic nature, more preferably lysine, or a short peptide chain, for example of two to six, preferably two to four, amino acid residues, and containing at least one basic residue like lysine.
  • the terminal ⁇ -amino group is optionally protected.
  • Preferred terminal nitrogen atom protecting groups include formyl, acetyl, trifluoroacetyl, propionyl, benzoyl;
  • benzyloxycarbonyl (Z), 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), 3,5-dimethoxy- ⁇ , ⁇ '-dimethylbenzyloxycarbonyl (Ddz) t-butoxycarbonyl, 1-mebhylcyclobutoxycarbonyl, adamtanyloxycarbonyl, isobornyloxycarbonyl; trityl, benzyl, methyl and isopropyl groups.
  • Preferred peptidic chain are Lys-Gly and Lys-Phe, optionally protected by benzyloxycarbonyl group.
  • Salts of compounds according to the invention with pharmacuetically acceptable acids or bases are included.
  • Such acid addition salts can be derived from a variety of inorganic and organic acids as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicyclic, gluconic, ascorbic and related acids.
  • Such base addition salts can be derived from a variety of inorganic and organic bases as sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dicyclohexylamine.
  • the partial or whole negative charge may be located on the oxygen atom or on the sulphur atom or in equilibrium between them.
  • the compounds of the invention may be diastereomer mixture or pure diastereomer having S P or R P configuration.
  • At least one of the phosphorothioate groups is of the S P configuration, more preferably the phosphorotiate group adjacent to the 2'/3' terminal adenylate mojety is of the S P configuration.
  • the invention further provides a process for preparing a compound of the formula I' as above defined which comprises introducing the desired R 3 group at the 5' position of an appropiately protected compound of the formula II
  • Z represents hydrogen or a protective group such as an acyl group, preferably a benzoyl group
  • X represents any of the protective groups generally used for the hydroxyl group like acyl, trityl, tetrahydropyranyl, methoxytetrahydropyranyl, allyloxycarbonyl, p-nitrophenylethylcarbonyl, p-nitrophenylethylsulphonyl, tert.butyldimethylsilyl, 1,3-tetraisopropyldisiloxyl, the last two being the preferred ones;
  • Y represents hydrogen or just a negative charge (that is diester moiety under salt form) or an usual protecting group of the phosphate function like for example O-chloro- phenyl, methyl, trichloroethyl, allyl, p-nitrophenylethyl, ß-cyanoethyl, unprotected and hence diester form being the preferred one, and
  • the preparation of the compounds of the formula I' wherein R 3 is a 2' or 3' P-thionucleotidyl residue conjugated at its 5' position with an acyl group may be carried out from 2', 5' phosphorothioate trimers.
  • the 2', 5'-phosphorothioate trimers are obtained by allowing the 2'-hydrogenphosphonate of a properly protected adenosine like compound 2 to react with a properly protected 2', 5'-diadenqsyl phosphorothioate like for example compound 7 where base amino groups can be conveniently unprotected as well as the phosphorothioate moiety. In this process the only necessary protections are for the hydroxyl groups other than the 5'-hydroxyl group that has to be involved in the esterification.
  • the coupling reaction is performed by adding a hindered acyl chloride as condensing agent, preferably pivaloyl chloride or adamantoyl chloride in a solvent like pyridine.
  • a hindered acyl chloride as condensing agent, preferably pivaloyl chloride or adamantoyl chloride in a solvent like pyridine.
  • the process is completed by thio-oxidation performed with elemental sulphur, better a suspension of suphur in pyridine or other known thiooxidizing agents.
  • the configuration of the starting dimer phosphorothioate is S P
  • the trimer is obtained in high yield (85% in the example) as a mixture of diastereomers R P , S P and S P , S P with a ratio 2:8, that is with a marked prevalence of the last one.
  • diastereoisomers can be separated by silica-gel column chromatography and independently converted to the protected intermediate trimers by conjugation of the 5' position, after detritylation, with an appropriate group, like a phosphoric or carboxylic acid, as described hereinbelow for the dinucleotides.
  • the final compounds are obtained by deprotection with known methodologies like for example treatment with hydrazine in pyridine/acetic acid for debenzoylation, treatment with an acid like trifluoroacetic acid in methylene chloride for detritylation and treatment with tetrabutylammonium fluoride in tetrahydrofuran and pyridine for desilylation.
  • the coupling between the nucleotidic dimer 5 or 7 and cholesterol 3-H-phosphonate 21 can be performed by using a hindered acyl halide like adamantoyl chloride as activacting agent followed by thiooxidation with a proper reagent like for example elemental sulphur.
  • a hindered acyl halide like adamantoyl chloride as activacting agent followed by thiooxidation with a proper reagent like for example elemental sulphur.
  • the base-deprotected dinucleotide 7 directly gives the (3-cholesteryl)thiophosphoryl derivative 23, while the base-protected dinucleotide 5 gives the intermediate 22 that in turn can be deprotected to 23 by basic treatment like ammonia in pyridine.
  • Desilylation of the intermediate 23 for example by treatment with tetra-butylammonium fluoride affords the final compound 24, 5'-O-[(3-cholesteryl)thiophospho
  • the coupling can be performed by adding a condensing agent, for example a hindered acyl chloride preferably pivaloyl chloride or adamantoyl chloride in pyridine, and can be followed by oxidation with a I 2 solution to get the phosphate moiety or by oxidation with a proper sulphur containing reagent, like for example elemental sulphur, to get the phosphorothioate moiety like in the dipalmitoylthiophosphatidyl derivative 27.
  • a condensing agent for example a hindered acyl chloride preferably pivaloyl chloride or adamantoyl chloride in pyridine
  • the last intermediate can be desilylated by known procedures, like using tetrabutylammonium fluoride, to give 5'-O-[ (1,2-dipalmitoylglyceryl)thiophosphoryl]adenosyl(2'—>5') thiophosphoryladenosine (28) as final compound.
  • a proper dinucleotide with available 5'-hydroxyl group. like 7 can also be made to react with an alkyl-phosphonylating mixture like for example a mixture composed by methylphosphonyldichl ⁇ ride, triazole, triethylamine and pyridine to afford the methylphosphonyl derivative 29.
  • This intermediate can undergo a proper desilylating treatment, like with tetrabutylammonium fluoride, to give the final product 30, namely 5,'-O-methylphosphonyladenosyl (2'—>5')thiophosphoryladenosine.
  • cholesterol or other cholestery ⁇ derivatives can be properly functionalized by preesterification with a dicarboxylic linker like succinic acid to intermediate 31 that can be made to react with the P-thio-dinucleotide 7 in the presence of a condensing reagent like dicyclohexylcarbodiimide (DCC) with dimethylaminopyridine and/or hydroxybenzotriazole at least in catalytic amount in pyridine or other proper solvent like methylene chloride at a temperature between -30°C and 50°C preferably at room temperature and during a period lasting at least 30 minutes to 48 hours preferably 24 hours.
  • a condensing reagent like dicyclohexylcarbodiimide (DCC) with dimethylaminopyridine and/or hydroxybenzotriazole
  • P-thiooligonucleotide like 5 obtaining in this case a base-protected intermediate like 35 that successively has to undergo selective deacylation at the base without concomitant lysis of the ester bond just formed.
  • This can be done by reaction with aqueous hydrazine in pyridine-acetic acid obtaining for example intermediate 36 from 35.
  • Aminoacyl and peptidyl derivatives are synthetically analogous cases. Conjugation through esterification of the nucleotidic 5'-hydroxyl group with an aminoacid shows to be a process feasible in the same manner as above explained for lipophilic derivative, as outlined in scheme
  • 2',5'-P-thio oligonucleotide can be accomplished on either base-protected or base-unprotected nucleotidic substrates. like respectively 5 and 7.
  • esterification step is followed by selective base deprotection, effected by hydrazine treatment as above mentioned, to convert compounds of type 41 to compounds of type 42.
  • esterification procedure using aminoprotected aminoacids can be performed as described above by using dicyclohexylcarbodiimide with dimethylaminopyridine and/or hydroxybenzotriazole or other similar activating agents in a dry polar organic solvent like pyridine or methylene chloride at a temperature between -30°C and 50°C preferably at room temperature for a period lasting at least 30 minutes to 96 hours depending on the case testing the reaction mixture by TLC.
  • Replacement of pyridine with a solvent like methylene chloride is useful to allow to filter off most of the formed dicyclohexylurea.
  • usual work-up gives a crude compound that can be either used for the subsequent step or purified by column chromatography.
  • esterification step and the hydrazine treatment for base deprotection can be made either in this order or in the reverse order to give as example the protected ß-aminopropionyl derivative 42a, the protected ⁇ -aminohexanoyl derivative 42b, and the protected N ⁇ -benzyloxycarbonyllysyl derivative 42c.
  • deprotection steps namely the t.butoxycarbonyl group removal for example by trifluoroacetic acid treatment and the desilylation performed for example by tetrabutyl-ammonium fluoride treatment, have necessarily to follow in this order and not in the reverse one to give 5'-O-(ß-aminopropionyl) adenosyl(2'—>5')-S P -thiophosphoryladenosine (43a), 5'-O-( ⁇ -aminohexanoyl)adenosyl(2 , —>5')-S P -thiophosphoryladenosine (43b), and 5'-O-[(N ⁇ -benzyloxy-carbonyl)lysyl]adenosyl(2'—>5')-S P -thiophosphoryladenosine (43c) respectively as final compounds.
  • an oligopeptide chain can also be conjugated to an oligonucleotide as appears in these examples through the formation of an ester bond.
  • ester-type conjugation can be effected either on base-protected or base-unprotected nucleotidic substrates also in this case.
  • Two possible routes are exemplified in scheme 5. Elongation of the peptidic chain can be either done before conjugation with the oligonucleotide (like in route A) or after the coupling of the carboxy-terminal aminoacid (like in route B) and adding sequentially the other aminoacids using the known methodologies for peptide synthesis.
  • the esterification between a nucleotidic 5' hydroxyl group and a peptide can be performed by treatment with dicyclohexylcarbodiimide or other carbodiimides in the presence of catalyst like dimethylaminopyridine or hydroxybenzotriazole as above explained.
  • Such treatment can give for example base-protected peptidyl conjugates like 44 abd 45 (from 5) or directly base-unprotected peptidyl conjugates like 46 and 47 (from 7).
  • base debenzoylation to the last compounds can be effected by treatment with aqueous hydrazine as above described.
  • the same peptidylester intermediates 46 and 47 can be obtained as examples through the route B that involve a first aminoacid conjugation even starting from base-unprotected P-thio-(2',5')oligonucleotide like 7 to give a monoaminoacyl intermediate like 48, as a further example of the case already illustrated in scheme 4.
  • Subsequent butoxycarbonyl removal by trifluoroacetic acid treatment give an intermediate like 49 in which an amino group is available for peptidic chain elongation.
  • So peptidic bond for example with dicyclohexylcarbodiimide in the presence of hydroxybenzotriazole in methylene chloride giving for example a peptidic product like 47 in a selective manner although the presence of unprotected base amino groups and an unprotected phosphorothioate moiety.
  • the final deprotection treatments like t.butoxycarbonyl group removal by trifluoroacetic acid and desylalation by tetrabutylammonium fluoride in the necessary order as above mentioned provide the final compounds, namely 5'-O-[(N ⁇ -benzyloxycarbonyl)-L-lysylglycyl]adenosyl(2'—>5')-S P -thiophosphoryladenosine ( 50) and 5'-O-[(N ⁇ -benzyloxycarbonyl)-L-lysyl-L-phenylalanyl]adenosyl(2'—>5')-S P -thiophosphoryladenosine (51) as representative examples.
  • nucleotides of the formula I wherein R 1 represents hydrogen atom a p-thiophosphoryl or a phosphoryl group and the starting compounds of the formula II are known compounds or may be prepared according to known methods, see for example WO 89/03683. Also the scientific paper "High stereoselectivity in the formation of the inter-ribonucleotide phosphorothioate bond", of C.
  • 6-N-benzoyl-5'-O-dimethoxytrityl-3'- O-t.butyldimethylsilyladenosine 1 is made to react with a P (III) phosphorylating agent, like phosphorus trichloride or better what is obtained by mixing phosphorus trichloride, triazole, N-methylmorpholine in dichloromethane.
  • a P (III) phosphorylating agent like phosphorus trichloride or better what is obtained by mixing phosphorus trichloride, triazole, N-methylmorpholine in dichloromethane.
  • the resulting 2 '-H-phosphonate 2 is coupled with a 2', 3'-0, 0-diprotected nucleoside like 6-N-benzoyl-2', 3'-0-0-bis-t.butyldimethylsilyl adenosine 3 by adding a condensing agent, for example a hindered acyl chloride, preferably pivaloyl chloride or adamantoyl chloride in pyridine.
  • a condensing agent for example a hindered acyl chloride, preferably pivaloyl chloride or adamantoyl chloride in pyridine.
  • This coupling procedure has to be followed by an oxidation stage performed with elemental sulphur (S 8 ) or better a suspension of sulphur in pyridine, or other thiooxidizing agent.
  • S 8 elemental sulphur
  • diastereomers can be separated by silica-gel column chromatography and they can independently be deprotected by known methodologies either completely to the final dimers S P -15 and R P -15, or partially to proper intermediates useful for further conjugation to the 5'-position.
  • compounds S P -4 or Rp-4 can be detritylated by acids at the position 5' of the first ribose to have the corresponding S P or R P form of the benzoylated dimer 5 ready for further reactions at the first 5' position.
  • debenzoylation of 4 can afford dimers of type 6, whose detritylation gives debenzoylated dimers 7 ( S P or R P ), with only the first 5' position available for further synthetic operations.
  • the S p -stereo- selectivity of the coupling can be modulated by the nature of the protecting groups at the first 5' and 3' positions and the second 3' and 2' positions (first and second being referred to the conventional way of reading a molecule of this type, from the 5' and to the 2', 3' end).
  • This is shown by two other examples, one being the coupling of the above mentioned 2'-H-phosphonate 2 with a different 5' component (8) having the tetraisopropyldisiloxane moiety as protecting group for both the positions 2' and 3'.
  • Conveniently intermediate 9 (S P or R P ) has been partially deprotected by acid-promoted detritylation to the corresponding derivative 10 ( S P or R P ) having the 5' position the only free hydroxyl group, as a useful intermediate.
  • Synthetically useful also is the debenzoylated analog 11 (either in S P or R P configuration) easily obtainable from 10. All the partially deprotected dinucleotide analogs, benzoylated or not, mentioned in the examples (5, 7, 10, 11) are key intermediate for further synthetic modifications aiming to 5'-elongation or conjugation, but they can also be conveniently converted to the final 2',5'-phosphorothioate dimers S P -15 and R P -15 by known methodologies.
  • the compounds object of this patent can be useful as antivirals, antitumorals and/or immunomodulators and/or potentiating agent of interferon (IFN) action.
  • IFN interferon
  • the same compound (S P -15) shows a marked potentiating action towards interferon (IFN); in fact it potentiates IFN 4 times on normal cells (Hep2, monolayer) and 8 times on partially trypsinized cells (suspension) against Semliki Forest virus (SFV).
  • IFN interferon
  • the maximum rolered dose of S P -15 for Hep2 cells is 400 ⁇ M.
  • RNA viruses both enveloped and non-enveloped.
  • compound 37 has a MIC of 12.5 ⁇ M against HRS virus (human respiratory syncytial virus), that is a paramixovirus and also 12.5 ⁇ M against SFV (Semliki Forest virus) belonging to the family togaviridae; both being examples of enveloped RNA viruses.
  • HRS virus human respiratory syncytial virus
  • SFV Semliki Forest virus
  • enteroviruses like Coxsackie B, a non-enveloped RNA virus, with a MIC of 25 ⁇ M, evaluating the cytopathic effect.
  • the palmitoyl derivative 40 is an example of wide spectrum antiviral showing a MIC of 12.5 ⁇ M against HSV-1; 10 ⁇ M against HRS; 37 ⁇ M against SFV; 12.5 ⁇ M against Cox B (Coxackie B virus); 7 ⁇ M against Col SK (encephalomyocarditis Columbia SK virus) that is a non-enveloped RNA virus, by evaluation of the cytopathic effect.
  • Compound 40 also showed an activity against retroviruses like MoSV (Moloney sarcoma virus) with ID 50 12.5 ⁇ M for foci formation.
  • Table 2 summarizes the activity data of the example compounds 37 and 40.
  • dipalmitoylphosphatidyl derivative 28 shows a MIC of 6.2 ⁇ M against the cytopathic effect of HRS
  • RNA viruses activity against RNA viruses
  • ⁇ -aminoacyl derivatives are endowed with antiviral activity as exemplifies by the ⁇ -carbobenzoxylysyl derivative with the phosphorothioate moiety having the R configuration (R P -43c) that inhibits the cytopathic effect by HRS virus with a MIC of 50 ⁇ M and by Coxsackie B virus with a MIC of 37 ⁇ M.
  • R P -43c the isomer with S P configuration at the phosphorothioate moiety, namely S P -43c inhibits the cytopathic effect due to Columbia SK virus at 75 ⁇ M concentration.
  • the compounds of the invention are useful in methods of treatment of the human or animal body by therapy. They have antiviral activity and can be used against RNA viruses in humans and other mammals. They show also antitumor and immunomodulating activity and can therefore be used as anticancer agents and immunomodulators. For these purposes, they can be formulated into oral dosage forms such as tablets, capsules and the like.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as active ingredient a compound of formula (I) or a pharmaceutically acceptable base addition salt thereof, together with a pharmaceutically acceptable carrier or diluent.
  • the compounds can be administered alone or by combining them with a conventional carrier or diluent, such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter, and the like.
  • a conventional carrier or diluent such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter, and the like.
  • Flavoring agents solubilizers, lubrificants, suspending agents, binders, tablet-disintegrating agents and the like may be employed.
  • the compounds may be encapsulated with or without other carriers. In all cases the proportion of active ingredients in said compositions both solid and liquid will be at least sufficient to impart antiviral activity thereto on oral administration.
  • the compounds may also be injected parenterally, in which case they are used in the form of a sterile solution containing other solutes, for example, enough saline or glucose to make the solution isotonic. Typically, a dose of 20-2000 mg of a compound of the invention may be administered per day to a human under treatment.
  • NMR data are given indicating with A, B, C, ... respectively the first, second, third .. ribose ring going from the 3' end to the 5' end of the oligonucleotide molecule, i.e. from the right bottom to the left top of the structure as drawn on the schemes.
  • 1,2,4-Triazole (11.5 g, 167.5 mmol) was added to a stirred solution of phosphorus trichloride (4.36 ml, 50 mmol) and N-methyl morpholine (53.7 ml, 500 mmol) in anhydrous methylene chloride (500 ml) at room temperature.
  • reaction mixture was quenched by adding 1M aqueous triethylammonium hydrogen carbonate (400 ml), then shaken and separated.
  • the aqueous phase was washed with methylene chloride and the combined organic layer was dried (Na 2 SO 4 ) and evaporated under reduced pressure.
  • Purification of the crude was performed by silica gel colum chromatography (eluent: ethyl acetate/methanol/triethylamine 10:1:0.2) to obtain 7.5 g (96% yield). of the title compound.
  • reaction mixture was concentrated and the residue, dissolved in methylene chloride (50 ml), was added a solution of trifluoroacetic (0.6 ml) in methylene chloride (10 ml), at 0°C. After 5 minutes the reaction mixture was poured into 1N aqueous triethylammonium hydrogen carbonate 1100 ml). The orgnic phase was separated and the aqueous phase was. extracted 3 times with methylene chloride. The combined extracts were dried (Na 2 SO 4 ) and concentrated.
  • the reaction mixture was kept stirring in a sealed vassel at 50°C for 4 hours then evaporated under reduced pressure.
  • the residue dissolved in pyridine/ dioxane 1:4 (10 ml) was added with tetrabutylammonium fluoride (trihydrate) (435 mg, 1.38 mmol) and stirred overnight.
  • the reaction mixture was quenched with water, and washed with methylene chloride.
  • the aqueous phase was evaporated under reduced pressure.
  • the crude was purified by reverse phase colomn chromatography performing a linear gradient using water/acetonitrile (from pure water to 85:15).
  • the fractions containing the wished product were collected and passed through a column of Dowex 50W-X8, sodium cation strong exchanger.
  • the aqueous solution was lyophilized to obtain 70 mg (80% yield) of title compound as sodium salt.
  • 1,2,4-Triazole (3.04 g, 44 mmol) was added to a stirred solution of phosphorus trichloride (1.1 ml, 12.8 mmol) and N-methylmorpholine (14.1 ml, 128 mmol) in dry methylene chloride (100 ml) at room temperature. After 30 minutes the reaction mixture was cooled to 0°C and cholesterol (20) (1 g, 2.6 mmol, dried by coevaporation from pyridine) in dry methylene chloride (20 ml) was added dropwise during 30 minutes. The mixture was stirred for 45 minutes, poured into 1M aqueous triethylammonium bicarbonate at pH 8.5 (100 ml), shaken and separated.
  • Cholesterol-3-hydrogenphosphonate triethylammonium salt (21) 250 mg, 0.45 mmol
  • N 6 -benzoyl-3'-O-(t.butyldimethylsilyl)-(S P )-P-thioadenylyl-(2' ⁇ >5')-N 6 -benzoyl-2',3'-O-(di-t.butyldimethylsilyl)adenosine (5) (630 mg, 0.49 mmol) were coevaporated twice with dry pyridine and dissolved in the same solvent ( 6 ml) .
  • 1,2,4-Triazole (5.05 g, 73.2 mmol) was added to a stirred solution of phosphorous trichloride (1.9 ml, 21.95 mmol) and N-methyl-morpholine (42 ml, 219.5 mmol) in anhydrous methylene chloride (150 ml), at room temperature.
  • Methylphosphorodichloridate (106 mg, 0.8 mmol) was added to a solution of triazole (110 mg, 1.6 mmol) and triethyl amine (445 ⁇ l, 3.2 mmol) in dry pyridine (5 ml) and the resulting suspension stirred at 0°C for 15 minutes under nitrogen.
  • Succinic anhydride (174 mg, 1.74 mmol) was added to a solution of cholesterol (20) (518 mg, 1.34 mmol) in dry toluene (30 ml)containing benzensulfonic acid as catalyst. The solution was refluxed for 45 minutes, neutralized with 2% aqueous sodium bicarbonate and washed with water. The organic phase was dried (Na 2 SO 4 ) and evaporated under vacuum. The residue was purified by silica gel column chromatography eluting with a gradient of ethyl acetate 0 to 20% in cyclohexane to give the title compound (31) (500 mg, 76.6% yield).
  • Succinic anhydride 255 mg, 2.55 mmol
  • dimethylaminopyridine 90 mg, 0.75 mmol
  • the solution was heated at 90oC for 4 hours, then stirred for 18 hours at room temperature, diluted with dichloromethane (60 ml) and washed with water.
  • the organic phase was dried (Na 2 SO 4 ) and evaporated under vacuum with complete elimination of pyridine.
  • 3-O-Succinylcholesterol (31) (400 mg, 0.82 mmol) and 3'-O-(t.butyldimethylsilyl)-(S P )-P-thioadenylyl-(2' ⁇ >5')-2', 3'-O-(di-t.butyldimethylsilyl)adenosine triethylammonium salt (7) (440 mg, 0.45 mmol) were coevaporated twice with dry pyridine and dissolved in the same solvent (8 ml).
  • N,N'-dicyclohexylcarbodiimide (185 mg, 0.9 mmol) and 4-dimethylaminopyridine (14.6 mg, 0.12 mmol) were added to the stirred solution under nitrogen atmosphere.
  • the reaction mixture was stirred for 24 hours at room temperature and then evaporated to dryness.
  • the residue was redissolved in dichloromethane, the insoluble dicyclohexylurea was filtered off and the organic solution was washed with water, dried (Na 2 SO 4 ) and evaporated under reduced pressure.
  • the residue was purified by silica gel column chromatography using a gradient from dichloromethane to dichloromethane/methanol 70:30, to give the title compound (32) (350 mg, 54% yield).
  • TLC on silica gel R f 0.23 eluting with dichloromethane/methanol 8:2.
  • N',N'-Dicyclohexylcarbodiimide 80 mg, 0.38 mmol
  • 4-dimethylaminopyridine 12 mg, 0.1 mmol
  • octanoic acid 55 mg, 0.38 mmol
  • the mixture was evaporated to dryness, the residue thus obtained was dissolved in a small amount of precooled methylene chloride and insoluble N,N'-dicyclohexylurea was filtered off.
  • Methylene chloride 100 ml was added and the organic solution was washed with water, dried (Na 2 SO 4 ) and evaporated.
  • N,N'-Dicyclohexylcarbodiimide (103 mg, 0.5 mmol), 4-dimethylaminopyridine (15 mg, 0.125 mmol) and cyclohexylacetic acid (71 mg, 0.5 mmol) were added and the mixture was stirred overnight at room temperature. The mixture was evaporated to dryness, the residue thus obtained was dissolved in a small amount of precooled methylene chloride and insoluble N,N'-dicyclohexylurea was filtered off. Methylene chloride (100 ml) was added, the organic solution was washed with water, dried (Na 2 SO 4 ) and the solvent evaporated.
  • the crude was purified by silica gel column chromatography eluting with a linear gradient of methylene chloride/methanol 90 : 10 to 75 : 25 .
  • the expected compound ( 3_4) was obtained as a white foam ( 217 mg, 70% yield) .
  • the pure sodium salt was then obtained by stirring a solution of the above compound in acetone/water 1:1 (60 ml) with Dowex-50W-X8 resin, Na + form (500 mg), for 20 minutes and then by passing the solution through a 2x8 cm column of the same resin. Evaporation of organic solvents and lyophilization afforded the title compoud (40) (315 mg, 67% yield).
  • N,N'-Dicyclohexylcarbodilmide (195 mg, 0.948 mmol) and 4-dimethylaminopyridine (11.6 mg, 0.095 mmol) were added and the resulting mixture was stirred at room temperature for 48 hours.
  • the reaction mixture was concentrated, diluted with methylene chloride and washed with water.
  • the organic phase was dried (Na 2 SO 4 ) and concentrated. Purification by silica gel column chromatography eluting with methylene chloride/methanol/ triethylamine 90:5:0.2, gave the title compound (42a) (522 mg, 90% yield).
  • Trifluoroacetic acid (3 ml) was added to a solution of 5'-O-[N-(t.butoxycarbonyl)- ⁇ -alanyl]-3'-O-(t.butyldimethyl silyl)-(S P )-P-thioadenylyl-(2' ⁇ >5')-2',3'-O-(di-t.butyldimethylsilyl)adenosine, triethylammonium salt (42a) (470 mg, 0.383 mmol) in methylene chloride (3 ml), containing 1,2-ethanedithiol (0.32 ml) as a carbocation scavenger, at 0°C.
  • reaction mixture was diluted with methylene chloride and neutralized with 1N aqueous triethyammonium hydrogen carbonate.
  • the organic phase was dried (Na 2 SO 4 ) and concentrated.
  • a 1N tetrabutyl ammonium fluoride solution in tetrahydrofuran (2.5 ml) was added to the residue dissolved in tetrahydrofuran (10 ml).
  • the reaction mixture was diluted with water and extracted with diethylether.
  • the aqueous phase was concentrated, purified by reverse phase chromatography on RP8 eluting with a stepwise gradient of acetonitrile from 0% to 60% in water and passed through a column of Dowex 50W-X8 resin, Na + form. Evaporation of the solvent gave the title compound 43a (182 mg, 70% yield over the 2 steps).
  • N,N'-Dicyclohexylcarbodiimide (117 mg, 0.570 mmol) and 4-dimethylaminopyridine (6.9 mg, 0.057 mmol) were added and the resulting mixture was stirred at room temperature for 48 hours.
  • the reaction mixture was concentrated, diluted with methylene chloride and washed with water.
  • the organic phase was dried (Na 2 SO 4 ) and concentrated. Purification of the residue by silica gel column chromatography eluting with methylene chloride/ methanol/triethylamine 90:5:0.2 gave the title compound (42b) (325 mg, 90% yield).
  • Trifluoroacetic acid (2 ml) was added to a solution of 5'-O- [N-(t.butoxycarbonyl)-6-aminocaproyl]-3'-O-(t.butyldimethylsilyl)-(S P )-P-thioadenylyl-(2' ⁇ >5')-2',3'-O-(di-t.butyldimethyl silyl)adenosine, triethylammonium salt (42b) (260 mg, 0.205 mmol) in methylene chloride (2 ml) containing 1,2-ethanedithiol (0.17 ml) as a carbocation scavenger, at 0oC.
  • reaction mixture was diluted with methylene chloride and neutralized with 1N aqueous triethylammonium hydrogen carbonate.
  • the organic phase was dried (Na 2 SO 4 ) and concentrated.
  • a 1 N tetrabutylammonium fluoride solution in tetrahydrofuran (1.3 ml) was added to the residue dissolved in tetrahydrofuran (5.1 ml). After 3 hours the reaction mixture was diluted with water and extracted with diethylether.
  • the aqueous phase was concentrated, purified by reverse-phase chromatography on RP8 eluting with a stepwise gradient of acetonitrile from 0% to 60% in water, and passed through a column of Dowex-50W-X8 resin, Na + form. Evaporation of the solvent gave the title compound 43b (.118 mg, 80% yield over the two steps).
  • N,N'-Dicyclohexylcarbodiimide (195 mg, 0.948 mmol) and 4-dimethyl-aminopyridine (11.6 mg, 0.095 mmol) were added and the resulting mixture was stirred at room temperature for 48 hours. The reaction mixture was then concentrated, diluted with methylene chloride and washed with water. The organic phase was dried (Na 2 SO 4 ) and concentrated. Purification of the residue by silica gel column chromatogra eluting with methylene chloride/ methanol/triethylamine (90:5:0.2) gave the title compound (S P -42c) (590 mg, 88% yield).
  • Trifluoroacetic acid (2 ml) was added to a solution of 5'-O-[ -(benzyloxycarbonyl)-N ⁇ -(t.butoxycarbonyl)-L-lysyl]-3'-O-(t.butyldimethylsilyl)-(Sp)-P-thioadenylyl-(2' ⁇ >5')-2',3'-O-(di-t.
  • N 6 -benzoyl-3'-O-(t.butyldimethylsilyl)-(Rp)-P-thioadenylyl-(2' ⁇ >5')-N 6 -benzoyl-2',3'-O-(di-t.-butyldimethylsilyl)adenosine (R P -5) 500 mg, 0.5 mmol
  • N ⁇ -benzyloxycarbonyl-N ⁇ -t.butoxycarbonyl-L- lysine 350 mg, 0.98 mmol
  • 4-dimethylaminopyridine 200 mg, 1.6 mmol
  • the N,N'-dicyclohexylcarbodilmide 200 mg, 1 mmol
  • dry pyridine (10 ml) was slowly added. The resulting solution was stirred overnight at room temperature.
  • N 6 -Benzoyl-3'-O-(t.butyldimethylsilyl)-(S P )-P-thioadenylyl-(2' ⁇ >5') -N 6 -benzoyl-2',3'-O-(di-t.butyldimethylsilyl)adenosine (S P -5) (1.9 g, 1.8 mmol), was dissolved in pyridine (150 ml),and the resulting solution was cooled at 0oC. 4-Dimethylaminopyridine (0.20 g, 1.6 mmol) and N,N'- dicyclohexylcarbodiimide (0.45 g 2.2 mmol) were added.
  • reaction mixture was neutralized with solid sodium bicarbonate and partitioned between water and dichloromethane.
  • organic phase was dried (Na 2 SO 4 ) and evaporated leaving as residue the title compound (49) (1.1 g, 85% yield) as a slight brown foam sufficiently pure to be used for the next step.
  • N,N'-dicyclohexylcarbodiimide (247 mg, 1.2 mmol) was added in one portion, under stirring.
  • the resulting solution was stirred at room temperature for 6 hours; some precipitated dicyclohexylurea was filtered off, and washed with cold dichloro methane. The filtrate and the washing were combined, washed with 5% sodium bicarbonate solution, and then with brine.
  • reaction mixture was stirred at 0°C for two hours, neutralized with solid sodium bicarbonate, and partitioned between water and dichloromethane.
  • the aqueous phase was extracted with dichloromethane (2 ⁇ 20 ml) and the combined organic extracts were washed with water, dried (Na 2 SO 4 ) and evaporated under vacuum to give a slight brown solid (400 mg, 86% yield).
  • This solid was dissolved in dry tetrahydrofuran (60 ml), and the solution was cooled at -10°C. 1 M tetrabutylammonium fluoride in tet.rahydrofuran (1.66 ml, 1.66 mmol) was added under dry nitrogen atmosphere.

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Abstract

L'invention concerne l'utilisation d'un analogue de nucléotide de la formule (I) dans laquelle R1 représente (i) un atome d'hydrogène, (ii) un résidu de 2' ou 3'-P-thionucléotidyle conjugué en sa position 5' à un groupe acyle, (iii) un groupe phosphoryle, un groupe P-thiophosphoryle, un groupe PO2R2H éventuellement estérifié avec un groupe hydroxy alkyle aliphatique linéaire ou ramifié, cyclique ou acyclique, constitué de 1 à 6 atomes de carbone ou avec une fraction lipophile, ou (iv) un groupe acyle; R2 représente un groupe alkyle C1C6 ou un atome d'hydrogène, et les sels pharmaceutiquement acceptables de celui-ci, dans la fabrication d'un médicament pouvant être utilisé comme agent antiviral, antitumeur, immunomodulateur, stimulateur d'interféron ou inducteur d'interféron. Certains composés de la formule (I) sont nouveaux. Un procédé pour leur préparation et des compositions pharmaceutiques les contenant sont également décrits.
EP19920909806 1991-06-04 1992-05-14 Analogues de 2',5'-nucleotides tels que des agents antiviraux Withdrawn EP0541742A1 (fr)

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GB919111967A GB9111967D0 (en) 1991-06-04 1991-06-04 2,5'-nucleotide analogs as antiviral agents

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TW517067B (en) 1996-05-31 2003-01-11 Hoffmann La Roche Interferon conjugates
US5955446A (en) * 1997-01-16 1999-09-21 Pentose Pharmaceuticals, Inc. Method of treating herpes infections with 2',5'-oligoadenylate-2',3'-cyclophosphate compounds
JP2002509715A (ja) * 1998-03-30 2002-04-02 イ・デ・エム・イミュノ−デジネ・モレキュル 刺激された単球に由来する細胞、その調製及び用途

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US5223263A (en) * 1988-07-07 1993-06-29 Vical, Inc. Liponucleotide-containing liposomes
US4924624A (en) * 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof

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See references of WO9221691A1 *

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