The subject of the invention includes derivatives of nucleoside-5′-O-hypophosphates and their mono- and dithiohypophosphate analogues, in particular 5′-O-[β,β-dialkyl-(α-thiohypophosphate)]- and 5′-O-[β,β-dialkyl-(α,α-dithiohypophosphate)]-and 5′-O-[β,β-dialkyl-(α,β-dithiohypophosphate)]- and 5′-O-[β-alkyl-(α-thiohypophosphate)]- and 5′-[β-alkyl-(α,α-dithiohypophosphate)]- and 5′-O-(α-thiohypophosphate)]- and 5′-O-(α,α-dithiohypophosphate-nucleosides of general formula 1,wherein A1 is a fluorine atom, azide or hydroxyl group, A2 is a hydrogen atom, B1 is adenine, 2-chloroadenine, 2-bromoadenine, 2-fluoroadenine, 2-iodoadenine, hypoxantine, guanine, cytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, 5-chlorocytosine, azacytosine, thymine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 5-chlorouracil, 5-(2-bromovinyl)uracil, 2-pyrimidione residue, W1 is an oxygen or carbon atom or a methylidene group, W2 is a carbon atom or W2 with A1 and A2 represent a sulfur atom or an oxygen atom, Z1 is a hydrogen or fluorine atom or a hydroxyl group or an alkoxyl group, Z2 is a hydrogen or fluorine atom or a hydroxyl or methyl group or Z1 and Z2 jointly represent a fluoromethylene group or A1, A2, Z1 and Z2 jointly represent a carbon-carbon double bond, X1, X2 and Y represent an oxygen atom or a sulfur atom, R1 and R2 represent an alkyl, aryl or a hydrogen atom associated with amine, and the process for the manufacture of derivatives of nucleoside-5′-O-hypophosphates and their mono- and dithiohypophosphate analogues of general formula 1,wherein A1, A2, B1, W1, W2, Z1, Z2, R1, R2, X1, X2 and Y are as above.
Nucleoside polyphosphates whose structures contain a phosphorus-phosphorus bond between the phosphorus atoms at the alpha and beta positions of the polyphosphate chain may reveal inhibiting activity with respect to polymerases.
The derivatives of nucleoside-5′-O-hypophosphates and their mono- and dithiohypophosphate analogues, in particular 5′-O-[β,β-dialkyl-(α-thiohypophosphate)]- and 5′-O-[β,β-dialkyl-(α,α-dithiohypophosphate)]- and 5′-O-[β,β-dialkyl-(α,β-dithiohypophosphate)- 5′-O-[β-alkyl-(α-thiohypophosphate)]- and 5′-O-[β-alkyl-(β,β-dithiohypophosphate)]- and 5′-O-(α-thiohypophosphate)]- and 5′-O-(α,α-dithiohypophosphate)-nucleosides of the present invention are of general formula 1,wherein A1 is a fluorine atom, azide or hydroxyl group, A2 is a hydrogen atom, B1 is adenine, 2-chloroadenine, 2-bromoadenine, 2-fluoroadenine, 2-iodoadenine, hypoxantine, guanine, cytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, 5-chlorocytosine, azacytosine, thymine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 5-chlorouracil, 5-(2-bromovinyl)uracil, 2-pyrimidione residue, W1 is an oxygen or carbon atom or a methylidene group, W2 is a carbon atom or W2, A1 and A2 jointly represent a sulfur atom or an oxygen atom, Z1 is a hydrogen or fluorine atom or a hydroxyl group or an alkoxyl group, Z2 is a hydrogen or fluorine atom or a hydroxyl or methyl group or Z1 and Z2 jointly represent a fluoromethylene group or A1, A2, Z1 and Z2 jointly represent a carbon-carbon double bond, X1, X2 and Y represent an oxygen atom or a sulfur atom, R1 and R2 represent an alkyl, aryl or a hydrogen atom associated with amine.
The process for the manufacture of derivatives of nucleoside-5′-O-hypophosphates and their mono- and dithiohypophosphate analogues of general formula 1, wherein A1, A2, B1, R1, R2, W1, W2, Z1, Z2, X1, X2 and Y are as above according to the present invention consists in that the nucleoside derivatives of general formula 2, wherein R3, R4, R5 and R6 represent a hydrogen atom, simple alkyl or aryl with 1 to 6 carbon atoms, wherein A2, W1 are as above, A3 is a fluorine atom, azide group or a protected hydroxyl group, W2 is a carbon atom or A2, A3, W2 jointly represent a sulfur atom or oxygen atom, B2 is adenine, 2-chloroadenine, 2-bromoadenine, 2-fluoroadenine, 2-iodoadenine, hypoxantine, guanine or cytosine residue of formulae 3, 4, 5 wherein Z5 is a hydrogen atom or a known exoamine protecting group, Z6 is a hydrogen atom or a chlorine, fluorine, bromine or iodine atom, Z7 is a hydrogen atom or a chlorine, fluorine, bromine or iodine atom or B2 is a thymine residue or azacytosine residue or 5-fluorouracil, 5-bromouracil, 5-iodouracil, 5-chlorouracil, 5-(2-bromovinyl)uracil residue or 2-pyrimidione residue and Z3 is a hydrogen, fluorine atom or a protected hydroxyl group, Z4 is a hydrogen, fluorine atom or a protected hydroxyl group or a methyl group or Z3 and Z4 jointly represent a fluoromethyl group or A2, A3, Z3, Z4 jointly represent a carbon-carbon double bond undergo a condensation reaction with phosphorous acid diesters of general formula (R7O)(R8O)POH or thiophosphorous acid diesters of general formula (R7O)(R8O)PSH, wherein R7 and R8 represent an alkyl or aryl, and the condensation is carried out in anhydrous organic solvents in the presence of condensation activators and after reaction completion the groups which protect 2′- and 3′-hydroxyl groups and the groups which protect nucleoside exoamine groups are removed according to known prior art.
The protective groups for the 2′- and 3′-hydroxyl groups preferably include known protecting groups selected from a group consisting of the acyl, benzoyl, 4,4′-dimethoxytriphenylmethyl, benzyl, trialkylsilyl, in particular a trimethylsilyl group.
The protective groups used for the exoamine groups include known protecting groups preferably selected from a group consisting of the phenoxyacetyl, isopropoxyacetyl, isobutyryl, benzoyl, (dialkylamino)methylidene and (dialkylamino)ethylidene group.
The condensation activators used include non-nucleophilic alcoholates, such as potassium tert-butanolate, or amines, such as imidazole, 1-methylimidazole, 4-dimethylaminopyridine, triethylamine and in particular 1,8-diazabicyclo[5.4]undec-7-ene (DBU).
The condensation reaction is preferably carried out in an anhydrous organic solvent selected from a group consisting of acetonitrile, methylene chloride, N,N-dimethylformamide, pyridine, dioxane and tetrahydrofuran.
In the process according to the present invention, compounds of formula 1,wherein X1, X2 and Y represent an oxygen atom, are preferably obtained from previously prepared compounds of formula 1 wherein X1=S or X1=O, X2=S, Y=S or Y=O in the oxidation reaction using oxidation reagents known in the art, particularly iodosobenzene and iodoxobenzene. The process according to the present invention is general and may be used in the direct synthesis of nucleoside-5′-O-hypophosphates of general formula 1.
In the process according to the present invention, compounds of formula 1,wherein R1 represents a hydrogen atom associated with amine, are preferably obtained from previously prepared compounds of formula 1,wherein R1 is a methyl group and R2 is an alkyl or aryl in the reaction with primary amines or ammonia, particularly with tert-butylamine. The process according to the present invention is general and may be used in the direct synthesis of 5′-O-[β-alkyl(α-thiohypophosphate)]- and 5-O-[β-alkyl-(α,α-dithiohypophosphate)]nucleosides of general formula 1.
In the process according to the present invention, compounds of formula 1,wherein R1 and R2 represent a hydrogen atom associated with amine, are preferably obtained from previously prepared compounds of formula 1, wherein R1 and R2 represent an alkyl or R1 is a hydrogen atom associated with amine and R2 is an alkyl in the reaction with trimethylsilyl halide, particularly with bromotrimethylsilane. The process according to the present invention is general and may be used in the direct synthesis of 5′-O-(α-thiohypophosphate)- and 5′-O-(α, α-dithiohypophosphate)-nucleosides of general formula 1.
The process of the invention may be utilised to manufacture 5′-O-[β,β-dialkyl-(α-thiohypophosphate)]- and 5′-O-[β,β-dialkyl-(α,α-dithiohypophosphate)]- and 5′-O-[β,β-dialkyl-(α,β-dithiohypophosphate)- and 5′-O-[β-alkyl-(α-thiohypophosphate)]- and 5′-O-[β-alkyl-(α,α-dithiohypophosphate)]- and 5′-O-(α-thiohypophosphate)]- and 5′-O-(α,α-dithiohypophosphate)-nucleosides of general formula 1,wherein A1 is a fluorine atom, azide or hydroxyl group, A2 is a hydrogen atom, B1 is adenine, 2-chioroadenine, 2-bromoadenine, 2-fluoroadenine, 2-iodoadenine, hypoxantine, guanine, cytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, 5-chlorocytosine, azacytosine, thymine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 5-chlorouracil, 5-(2-bromovinyl)uracil, 2-pyrim idione residue, W1 is an oxygen or carbon atom or a methylidene group, W2 is a carbon atom or A1, A2, W2 jointly represent a sulfur atom or an oxygen atom, Z1 is a hydrogen or fluorine atom or a hydroxyl group or an alkoxyl group , Z2 is a hydrogen or fluorine atom or a hydroxyl or methyl group or Z1 and Z2 jointly represent a fluoromethylene group or A1, A2, Z1 and Z2 jointly represent a carbon-carbon double bond, X1, X2 and Y represent an oxygen atom or a sulfur atom, and X1, X2 and Y may independently represent an oxygen or sulfur atom, R1 and R2 represent an alkyl, aryl or a hydrogen atom associated with amine.
The process according to the present invention is illustrated in the examples which follow.
5′-O-[β,β-diethyl-(α-thiohypophosphate)]-uridine
To a solution of 0.05 mmol of 5′-(2-thio-[1,3,2]-oxathiaphospholanyl)-O2′, O3′-diisopropoxyacetyluridine in 0.5 mL of acetonitrile 0.05 mmol of diethyl phosphite was added and subsequently 0.055 mmol of DBU was added dropwise. The reaction was carried out at ambient temperature for 2.5 hours (TLC and 31P NMR analyses). The reaction mixture was then concentrated under reduced pressure and aqueous saturated ammonia (3 mL) was added to the residue (ambient temperature, 1 hour). The ammonia was subsequently distilled off under reduced pressure. The product was isolated in a 19% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.10-0.80 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:55.790, 13.225 ppm, 1Jp−p=501 Hz, MALDI-TOF m/z:(M-1) 459.2.
5′-O-[β,β-dimethyl-(α-thiohypophosphate)]-uridine
To a solution of 0.05 mmol of 5′-(2-thio-[1,3,2]-oxathiaphospholanyl)-O2′, O3′-diisopropoxyacetyluridine in 0.5 mL of acetonitrile 0.05 mmol of dimethyl phosphite was added and subsequently 0.055 mmol of DBU was added dropwise. The reaction was carried out at ambient temperature for 2.5 hours (TLC and 31P NMR analyses). The reaction mixture was then concentrated under reduced pressure and aqueous saturated ammonia (3 mL) was added to the residue (ambient temperature, 1 hour). The ammonia was subsequently distilled off under reduced pressure. The product was isolated in a 26% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.10-0.60 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:55.177, 15.653 ppm, 1Jp−p=501 Hz, MALDI-TOF m/z:(M-1) 431.0.
5′-O-[β-methyl-(α-thiohypophosphate)]-uridine
To 6 μmol of 5′-O-[β,β-dimethyl-(α-thiohypophosphate)]-uridine 0.5 ml of t-butylamine was added. The reaction was carried out at ambient temperature for 4 days (HPLC and 31P NMR analyses) until the complete conversion of the substrate into the product. The reaction mixture was subsequently concentrated under reduced pressure with the final yield of 100%. 31P NMR(D2O)δ:65.107, 9.813 ppm, 1Jp−p=531 Hz, MALDI-TOF m/z:(M-2) 416.9.
5′-O-α-thiohypophosphate)-uridine
To a solution of 0.05 mmol of 5′-(2-thio-[1,3,2]-oxathiaphospholanyl)-O2, O3-diisopropoxyacetyluridine in 0.5 mL of acetonitrile 0.05 mmol of dimethyl phosphite was added and subsequently 0.055 mmol of DBU was added dropwise. The reaction was carried out at ambient temperature for 2.5 hours (TLC and 31P NMR analyses). The reaction mixture was subsequently cooled to −40° C. and 0.2 mmol of bromotrimethylsilane was added dropwise. The mixture was heated at a rate of 10° C. per 0.5 hour. Once the mixture was heated to ambient temperature, the reaction was carried out for 12 hours. The reaction mixture was then concentrated under reduced pressure and aqueous saturated ammonia (3 mL) was added to the residue (ambient temperature, 1 hour). The ammonia was subsequently distilled off under reduced pressure. The product was isolated in a 18% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.10-0.60 M; pH=7.5) as the eluent and gel filtration on Sephadex LH-20 using water as the eluent. 31P NMR(D2O)δ:66.366, 7.643 ppm, 1Jp−p=543 Hz, MALDI-TOF m/z:(M-1) 403.0.
5′-O-[β,β-diethyl-(α,β-dithiohypophosphate)]-uridine
To a solution of 0.05 mmol of 5′-(2-thio-[1,3,2]-oxathiaphospholanyl)-O2′, O3′-diisopropoxyacetyluridine in 0.5 mL of acetonitrile 0.05 mmol of diethyl thiophosphite was added and subsequently 0.055 mmol of DBU was added dropwise. The reaction was carried out at ambient temperature for 16 hours (TLC and 31P NMR analyses). The reaction mixture was then concentrated under reduced pressure and aqueous saturated ammonia (3 mL) was added to the residue (ambient temperature, 1 hour). The ammonia was subsequently distilled off under reduced pressure. The product was isolated in a 23% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.10-0.60 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:83.995, 83.804, 60.632, 60.467 ppm, 1Jp−p=384 Hz, MALDI-TOF m/z:(M-1) 475.1.
5′-O-β-methylhypophosphatecytidine
To a solution of 16 μmol of 5′-O-[β-methyl-(α-thiohypophosphate)]-cytidine in 2 ml of methanol 16 μmol of iodoxobenzene was added. The reaction was carried out at ambient temperature for 12 hours (HPLC and 31P NMR analyses). The reaction mixture was subsequently concentrated under reduced pressure. The product was isolated in an 82% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.0-0.3 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:9.725 ppm, MALDI-TOF m/z:(M-1) 412.0.
5′-O-β,β-dimethylhypophosphateuridine
To a solution of 15 μmol of 5′-O-[β,β-dimethyl-(α-thiohypophosphate)]-uridine in 0.5 ml of methanol 15 μmol of iodoxobenzene was added. The reaction was carried out at ambient temperature for 12 hours (HPLC and 31P NMR analyses). The reaction mixture was subsequently concentrated under reduced pressure. The product was isolated in a 79% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.0-0.3 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:19.488, −0.450 ppm, 1Jp−p=657 Hz, MALDI-TOF m/z:(M-1) 415.0.
5′-O-[β-methyl-(α-thiohypophosphate)]-2′-O-methyl-guanosine
To a solution of 0.20 mmol of 5′-(2-thio-[1,3,2]-oxathiaphospholanyl)-O-3′-acety-2′-O-methyl-N-isobutyryl-guanosine in 1 mL of acetonitrile 0.20 mmol of dimethyl phosphite was added and subsequently 0.23 mmol of DBU was added dropwise. The reaction was carried out at ambient temperature for 2.5 hours (TLC and 31P NMR analyses). The reaction mixture was then concentrated under reduced pressure and aqueous saturated ammonia (3 mL) was added to the residue (temperature 45° C., 4 hour). The ammonia was subsequently distilled off under reduced pressure. The product was isolated in a 16% yield using ion-exchange chromatography (DEAE-Sephadex A-25) with TEAB (0.10-0.60 M; pH=7.5) as the eluent. 31P NMR(D2O)δ:60.79, 6.28 ppm, 1Jp−p=450 Hz, MALDI-TOF m/z:(M-1) 470.1.
Number | Date | Country | Kind |
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PL382824 | Jul 2007 | PL | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/PL08/00049 | 7/1/2008 | WO | 00 | 12/31/2009 |