PL184377B1 - Novel phosphoro-organic derivatives and method of obtaining them - Google Patents

Abstract

In New Organophosphorus Pochoshos presented general formula 1, where X means oxygen or sulfur Y means - NHPH, F or a group of formula -X'r3 where X is as defined above and R3 is a methyl group, or a group of the formula - CH2C6H4-R4, wherein R6 is a hydrogen atom, cHloro or -NO2, Ri is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-O-enylxanthen-9-yl (Px), R "is hydrogen or alkoxy preferably mets) xyl and B is N-blocked purine or pyrimidine base preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanina.

Description

The subject of the invention is new organophosphorus derivatives of the general formula I in which X is oxygen or sulfur, Y is -NHPh, F or a group of the formula -X'R ^: 3 in which X1 is as defined above for X and R3 is the group methyl, or a group of formula CH ₂ C ₆ H ₄ -R 4, where R 4 is a hydrogen atom, a chlorine atom or a NO ₂ group, R ¹ is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px ), R2 is hydrogen or alkoxy. preferably methoxy, and B is an N-blocked purine or pyrimidine base preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, and a method of their preparation.

The concept of using oligonucleotides as potential antiviral drugs proposed by Zamecnik and Stevenson (ANTISENSE concept) (PCZamecnik, ML Stephenson, 1978. Inhibition of Rous Sarcoma Virus Replication and Celi Transformation by a Specific Oligodeoxynucleotide, Proc.Natl.Acid.Sci. USA, 1975 , 280) has increased interest in the synthesis and properties of oligonucleotides and their modified analogues, especially phosphorothioates and methylphosphonates.

184 377

This concept is based on the use of interactions between oligonucleotides complementary to fragments of pre-mRNA or mRNA molecules, resulting in inhibition of the biosynthesis of undesirable and harmful proteins. It is also possible for antisense DNA to interact with a DNA double helix (formation of a triple-stranded structure), resulting in the same end result, expressed in a specific inhibition of the transcription process. Both of these processes are characterized by high selectivity caused by interactions with precisely defined fragments of dNa or RNA at various stages of the gene expression process in eukaryotic cells. The affinity of the oligonucleotides for the recognition sequence is the result of hybridization interactions (Watson-Crick base pairing and layered interactions in the forming double helix) and increases with the length of the oligonucleotide-recognition sequence complex.

Modifications to the ANTISENSE molecule may change these interactions, resulting from changes in the geometry around the modified bonds (YN Vorobyov, Non-ionic Analogs of Oligonucleotide Duplexes. Molecular Mechanical Calculations of the Influence of Configuration of Asymmetrical Phosphorus Atoms in Methyl-phosphonate d (TPMe) 6 and Phosphotriester Derivatives d (TPEt) 6 on the Structure and Stability of their Complexes with dA6, 1990; Molekulyamaya Biologiya 24, 58), and an increase in their stability in cells as a result of resistance to nucleolytic enzymes (YLPotter, BAConnolly, F. Eckstein, Synthesis and Configurational Analysis of a Dinucleoside Phosphate IsotopicallyChiral at Phosphorus. Stereochemical Course of Penicillium Citrum Nuclease PI Reaction, 1983, Biochemistry, 22, 1369; FR Bryant. SJBenkovic, Stereochemical Course of the Reaction Catalyzed by Nuclesterase from Phosphodake Venom, 1979, Biochemistry, 18, 2825).

Nucleoside oligomethane phosphonates (OligoMe) are non-ionic DNA analogues containing neutral methane phosphonate moieties that replace the phosphodiester bonds in the natural DNA molecule. The methanophosphonate bond is completely resistant to the action of nuclease, and its non-ionic nature allows the OligoMe chains to be introduced intact through the cell membrane into the cytoplasm (S. Agrawal, PSSarin, M. ZamecnikP.C.Zamecnik, Cellular Uptake and Anti-HlV Activity of Oligonucleotides and their Analogs. In Gene Regulation. Biology of Antisense RNA and DNA, 1991, Vol. 1; Robert P. Erickson and Jonathan G. Izant eds., Reven Press, 273-284).

A number of interesting results indicate the effectiveness of the use of complementary (antisense) OligoMe fragments for the specific inhibition of the synthesis of undesirable (harmful) proteins both in vitro and in vivo. Short fragments (7-12 mer) of OligoMe have been successfully used to inhibit the process of Herpes simplex pre-mRNA mRNA (type 1) CCSmith, L. Aurelian, MPReddy, PSMiller, POPTs'o, Antiviral Effect of an O1igo ( nucleoside methylphosphonate) Complementary to the Splice Junction of Herpes simplex virus type 1 Immediate Early pre-mRNAs 4 and 5, 1986, Proc.Natl.Acad. Sci., USA, 83, 2787), as well as multiplication of this virus in frog oocytes ( Xeropus laevis) (C. Casenave, M. Chemer, TTNauyen, C. Helene, Ratę of Degradation of and βOligodeoxynucleotides in Xenopus oocytes: Implications for Anti-messenger Strategies. Nucl.Acids. Res. 1987, 15, 10507). Similarly, it was possible to inhibit the expression of the βglobin gene in rabbit reticulocytes (K. Blake, A. Murakami, P.S. Miller, Inhibition of Rabbit Globin mRNA Translation a Sequence - Specific Oligodeoxyribonucleotides, 1985, Biochemistry, 24, 132). These properties indicate significant potential uses of oligoMe. Hence the great interest in the synthesis and properties of this class of compounds in many research laboratories and biotechnology companies (POPTs'o, L. Aurelian, E.Chang, PSMiller, in Antisense Strategies, Annals of the New York Academy of Sciences, 1993, 660). , 159).

An important aspect, and at the same time hindering research on OligoMe, is the asymmetry center on the phosphorus atom, resulting from the exchange of the oxygen ligand with a non-ionic methyl group. The stereochemical consequence is the presence in the oligomer of n base pairs - 2n-1 isomers formed as a result of consecutive non-stereospecific nucleotide chain growth reactions. It was found that the homochiral chains Rp and Sp,

184 377, i.e. those whose absolute configuration on all intemucleotide phosphorus methanophosphonates is Rp or Sp, and non-stereoregular chains, show different physicochemical properties, as well as different ability to create adducts from oligonucleotides with complementary sequences. Studies on dinucleotide thiophosphate analogs confirmed the differences in the behavior of oligo-Rp and oligo-Sp, including stereoselectivity towards nuclease (BYLPotter, BA Connolly, F. Eckstein, Biochemistry, 22, 1983, 1369; FRBryant, SJBenkovic, Biochemistry, 18, 1979, 2825).

In recent years, it has been shown that octa (thymidylmethane phosphonates), in which six out of the seven methylphosphonate bonds have a well-defined configuration on the phosphorus atom and high diastereomeric purity, obtained by non-stereospecific condensation of the corresponding diastereoisomerically pure tetramers, show dramatic differences in the softening point Tm, indicating different stability of isomeric complexes of compounds with the pentadecadeoxyadenylic acid matrix (ZJLeśnikowski, M. Jaworska, WJStec, Nuci. Acids Res., 18, 1990, 2109).

The OligoMe with a defined configuration on the phosphorus atom used in these studies was obtained as a result of a stereocontrolled reaction between the nucleoside 5'-hydroxyl group activated by Grignard's reagent and the isomerically pure nucleoside p-nitrophenylmethyl phosphonate (J. Leśnikowski, M. Jaworska, WJStec, 1990, Octa (thymidine methanephosphonates) of Partially Defined Stereochemistry: Synthesis and Effect of Chirality at Phosphorus on Binding to Pentadeca-deoxyriboadenylic Acid, Nucleic Acids Res., 18 1990, 2109; ZJLeśnikowski, M.Jaworska-MaślaNka, WJStific, Stereospec Synthesis of -chiral Di (2'-O-Deoxyribonucleoside) methanephosphonates, Nucleosides & Nucleotides, 1991, 10, 733); Z. J. Leśnikowski, M. Jaworska, W.J. Stec, Sfereoselective Synthesis of P-homochiral Thymidine Methylphosphonates. Humming. Acid Res., 16, 11675).

However, this method requires long reaction times and so far has only been proven in the synthesis of tetramer homo-thymidyl fragments and heteromeric hexamer structures.

Attempts to obtain diastereoisomerically pure OligoMe by reactions between methyldichlorophosphine and the corresponding 5 'or 3'-blocked nucleosides at low temperatures (-80 ° C) allowed to obtain predominantly (8: 1) Rp isomers of the corresponding dinucleoside methylphosphonates (T. Loschner, J.Engels, One Pot Rp-Diastereoselective Synthesis of Dinucleoside Methylphosphonates Using Methyldichlorophos-phine, Tetrahedron Lett., 1989, 30 (41), 5587; JWEngels, T.Loschner, A.Frauendorf, Diastereoselective Synthesis of Thymidine Methylphosphonate ., Nucleosides & Nucleotides, 1991, 10, 347).

However, this method cannot be used to obtain longer stereoregular chains, because the 3'-O-nucleosides formed temporarily as a result of successive chloromethylphosphonine condensation reactions are configuration labile even at low temperatures.

The recently described method involving the use of 5'-DMT2 (O-nucleoside-3'-O- (Se-methylselenomethane-phosphonates) separated into diastereoisomers as monomers allows to obtain diastereomerically pure oligoMe in solution (LA Woźniak, J. Pyzowski, M. Wieczorek , WJStec, J. Org. Chemistry, 1994, 59, 5843) Catalysts for this reaction are lithium salts and 1,8-diazabicyclo [5.4.0] -undec-7-ene (DBU).

Oligonucleotides containing n-chiral intemucleotide linkages exist as 2n isomers. Chromatographic methods allow for efficient separation of short fragments, especially dimers, which can be successfully incorporated in a diastereomerically pure form into chimeric chains alternating methanophosphonate bonds with a defined configuration on the phosphorus atom [Rp] and phosphodiester or phosphorothioate bonds. Such a strategy has been shown to be very promising in view of the high degree of inhibition achieved by chimeric oligonucleotides (L. Amold, First International Antisense Conference of Japan, Kyoto, Abs. # 25, 1994).

184 377

The new organophosphorus derivatives according to the invention are represented by the general formula 1, in which X is oxygen or sulfur, Y is -NHPh, F or a group of the formula X'R ³ , in which X ¹ is as defined for X and R ³ is a methyl group, or a group of the formula -CH2C6H4-R ^4, wherein R ⁴ is hydrogen, chlorine or -NO 2, R ¹ is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthene-9-yl (Px ), R2 is hydrogen or alkoxy, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine.

A method for the preparation of new organophosphorus derivatives of the general formula I, in which X is a sulfur or oxygen atom, Y is -NHPh, Ri is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px), R ² is hydrogen or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, according to the invention, the methyldichlorophosphine is reacted in presence of a hydrogen chloride-binding reagent, preferably amines, especially triethylamine with a nucleoside of formula 2, in which R 1, R 2 and B are as defined above, then aniline and elemental sulfur, selenium or an oxidizing reagent are added to the mixture, and after the reaction is completed, the product is isolated in known way.

A method for the preparation of new organophosphorus derivatives of the general formula I, in which X is an oxygen atom, and Y is a group of the formula -X ³ R3, in which Xi is a sulfur atom and R3 is a methyl group, or a group of the formula -CH ₂ C ₆ H4-R 4, wherein R is hydrogen, chlorine or -NO 2 group, R is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthene-9-yl (Px), R ² is hydrogen or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine. amines, especially triethylamine with a nucleoside of formula 2, where R1, R2 and B are as defined above, then aniline and elemental sulfur are added to the mixture, and the resulting mixture of nucleoside diastereoisomers is 3'-O-methanophosphonothi oanilidu of formula 1 wherein X is sulfur, R 1 R ² and B are as defined above and Y is -NHPh, is reacted with sodium hydride or DBU and carbon dioxide, and the resulting acid transient nucleoside 3'-O -methanothiophosphonic acid (sodium or DBU-iodine salt) is alkylated with an alkylating reagent of formula R ³ Z in which R ³ is as defined above and Z is chlorine, bromine or iodine.

A process for preparing novel organophosphorus of formula 1 wherein X is oxygen, Y is -NHPh, R 1 is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthene-9-yl (Px) ² R represents a hydrogen atom or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, according to the invention the reaction of methyldichlorophosphine is carried out in the presence of the reagent hydrogen chloride binding, preferably amines, especially triethylamine with a nucleoside of formula 2, in which R 1, R 2 and B are as defined above, then aniline and elemental sulfur are added to the mixture, and the resulting mixture of nucleoside diastereoisomers of 3'-O-methanophosphonothioanilide of the general formula Wherein R1, R2 and B are as defined above, and X is a sulfur atom, is oxidized with known oxidizing agents, preferably with potassium peroxymonosulfate or hydrogen peroxide.

A process for preparing novel organophosphorus of formula 1 wherein X is oxygen and Y is a group of formula -X'R3, wherein X1 is oxygen or sulfur, and R3 is methyl or -CH ₂ C ₆ H4-R4, where R4 is a hydrogen atom, a chlorine atom or a -NO2 group, R1 is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px), R2 is a hydrogen atom or a group alkoxy, preferably methoxy, and B is an N-blocked purine base

The invention consists in reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent, preferably an amine, especially triethylamine, with a nucleoside of formula 2, according to the invention, in the presence of a pyrimidine, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine. R ^1, R ² and B are as defined above, followed by the addition of aniline and elemental sulfur and the resulting mixture was dlastereoizomerów nucleoside 3'-O-metanofosfonotioanilidu of formula 1 wherein X is sulfur, R ^1, R 2 and B are as defined above and Y is -NHPh, subjected to an oxidation reaction with known oxidizing agents, preferably with potassium peroxymonosulfate or hydrogen peroxide, then the compound obtained is reacted with sodium hydride or DBU and with carbon dlsulfide and the resulting compound is reacted intermittently nucleoside 3'-O-methanothiophosphonic acid (sodium or DBU-nium salt) alkylated with an alkylating reagent of formula R ³ Z, in which R ³ is as defined above and Z is chlorine, bromine or iodine.

A process for the preparation of new organophosphorus derivatives of general formula I, in which X is a sulfur or oxygen atom, Y is F, Ri is a blocking group, preferably 4'-dl-methoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px), r2 is a hydrogen atom or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, according to the invention the reaction of methyldichlorophosphine is carried out in the presence of a hydrogen chloride binding reagent , preferably amines, especially triethylamine with a nucleoside of formula II, in which R1, R2 and B are as defined above, then aniline and elemental sulfur or an oxidizing agent are added to the mixture, the resulting mixture of nucleoside diastereolzomers 3'-O-methanophosphonoanilide of the general formula Is reacted with hydrogen, where X is oxygen or sulfur, R1, R2 and B are as defined above, and Y is -NHPh. rkiem sodium or DBU and carbon disulfide, or carbon, and the resulting mixture of diastereomers nucleoside 3'-Ometanofosfonotloloester of formula 1 wherein X, R ^1, R 2 and B-are as defined above, and Y is S, is reacted with a fluorinating reagent of formula MF, wherein M is Ag, -NEt4, -NET ₃ H, and the product is isolated in a known manner.

The following are non-limiting examples of the invention.

Example 1 General recipe for the preparation of a compound of formula 1 (X = S, Y = -NHPh).

To a solution of methyldichlorophosphine (0.29 g, 2.5 mmol) in THF and triethylamine (0.55 g, 55 mmol) cooled to 40 ° C, a solution of the appropriate nucleoside (1 mmol) in THF (10 ml) was added dropwise. After 30 minutes, the reaction temperature was raised to room temperature. To this mixture was added aniline (0.28 g, 3 mmol) and elemental sulfur. After completion of the reaction, the reaction mixture (diluted with chloroform) was extracted with carbonate buffer and purified by column chromatography on silica gel. The appropriate fractions were combined and concentrated under reduced pressure.

Example II. [Rp, Sp] -3'-O-methanophosphonothioanil 1d 5'-O-DMT-thymidyl formula 1 (B = thym1na, X = S, Y = -NHPh)

Prepared from 5'-O-DMT-thymidine (0.540 g, 1 mmol) as in Example 1. Solid, colorless foam solidified; 93% yield (0.66 g); ³ 'P NMR: 79.44; 79, 58 ppm; FAB-MS [MH]: 712.4.

Example IIL [Rp, Sp] -3'-O-methanophosphonothioanilide 5'-O-DMT-N4-benzoyl-2'-Odeoxycytidyl and (X = S, Y = -NHPh). Prepared from 5'-O-DMT-N4-benzoyl-2'-Odeoxycytidine (0.633 g, 1 mmol) as in Example 1. Diastereoisomeric mixture as colorless glaze. Yield 7θ% (0.56 g). 3'P NMR (CH ₂ Cl ₂ / C ₆ D ₆ ): 79.38; 79.74 ppm.

Example IV. [Rp, Sp] -3'-O-methanophosphonothioanil 1d 5'-O-DMT-N6-Benzoyl-2'-Odeoxyadenyl 1 (X = S, Y = -NHPh). Prepared from 5'-O-DMT-N6-Benzoyl-2'-Odeoxyadenosine (0.657 g, 1 mmol) as in Example 1. Diastereomeric mixture obtained as a colorless solid with a solid foam structure; 75% efficiency; 3'P NMR: 79.21; 79.60 ppm.

184 377

EXAMPLE 5 [Rp, Sp] p3'-O-methanesoonetielide 5'pO-DMT-N2-isobutyryl-2'-Odeexyguanyl 1 (X = S, Y = -NHPH):

Prepared from 5'-O-DMT-N2-isobutyryl-2'-O-deoxyguanosine (0.640 g; 1 mmol) as in Example 1. Isolated as a mixture of diastereoisomers as colorless glaze. Yield 98% (0.71 g). 3'P NMR: 79.65; 79.72 ppm.

Example d VI. [Rp, Sp] -5'pO-DMT 3'-O-methanoloso-thioanilide '-O-methyluridylide 1 (X = S, Y = -NHPH):

Prepared from 5'-OpDMT-2'-O-methyl uridine (0.560 g, 1 mmol) as in Example 1. Isolated as a mixture of diasternoisemers in the form of a colorless glaze. Yield 85% (0.68 g). 3'P NMR: 81.96 (SLOW-formula Ia); 81.38 (FAST formula 1b) ppm.

Example VII. Separation into FAST and SLOW diasteressers was carried out on silica gel, eluting with celoroether containing 10-20% Heptane.

The diastereomeric purity of the resolved FAST and SLOW isomers was checked by the ^3L P NMR method.

Table 1

No example B: Diastereoigomer * 3'PNMR (ppm) II B = thymine FAST 79.44 WORDS 79.58 III B = cytosine FAST 79.38 WORDS 79.74 IV B = adenine FAST 79.21 WORDS 79 60 V B = guanine FAST 79.65 WORDS 79.72 VI B = uracil FAST 81.38 WORDS 81.96

* Mobility on gel (Kieselgel 60, 240-400 mesh) in a CHCl ₃ / MeOH development system (95.5 v / v).

Example VIII. General Method for the Synthesis of 3-O- (Spbenzyl methanothioosoonates) 5'-ODMT- (N-blocking) nuclosides formula 1 (X = O, Y = SCHPH).

NaH (1.2 mmol) was added to a solution of the compound of formula 1 (X = S, Y = -NHPH) (1 mmol) in sucHym DMF (10 ml). The solution was stirred until the evolution of hydrogen gas ceased. Dry CO ₂ gas was passed through the suspension thus obtained. The course of the reaction was monitored by TLC. Then benzyl bromide (5 mmol) was added to the reaction mixture. Upon completion of the alkylation reaction, the solvents and excess benzyl bromide were distilled off under reduced pressure, and the residue was dissolved in a celerorm. The solution was extracted with carbonate buffer and, after concentration and drying of the organic layer, the product was purified by cHromategraii on silica gel.

If the reactant is a mixture of diastereoisomers, the purification on silica also separates into diastereoisomers Ia (FAST) and Ib (SLOW) (X = O, Y = SCH ₂ PH). Eluent: cHlereorm with addition of ethanol (0-5%).

Example IX. 3-O- (Spbenzyl methanethio-sulfonate) 5'-O-DMT-Thymidyl, 1 (B = thymine, X = O, Y = SCH ₂ PH): Prepared from 1 (B = Thymine, X = S, Y = -NHPH ) (0.712 g; 1 mmol) as in Example VII. Total yield: 86% (0.55 g); 3'P NMR: 56.44: 55.94 ppm.

184 377

Example X. 3'-O- (S-benzyl methanothiophosphonate) 5'-O-DMT-N4-Benzoyl-2'-Odeoxycytidyl 1a, (B = cytosine, X = O, Y = SCH ₂ Ph): Prepared from 1 , (B = cytosine, X = S, Y = -NHPh) (1mmol) as in Example VII. Overall yield 82%; ^{3 I} P NMR (CDCl ₃ ): 55.41; 55.21 ppm.

Example XI. 3'-O- (S-benzyl methanothiophosphonate) 5'-O-DMT-N6-Benzoyl-2'-Odeoxyadenyl, Ia (B = adenine, X = O, Y = SCH ₂ Ph): Prepared from 1, (B = adenine, X = S, Y = -NHPh) (1 mmol) as in Example VIII; 75% efficiency; ³¹ P NMR (CH ₂ C ₁₂ / C ₆ D ₆ ): 55.80; 55.53 ppm.

Example XII. 3'-O- (S-benzyl methanothiophosphonate) 5'-O-DMT-N4-Isobutyryio-2'O-deoxyguanidyl 1a, (B = guanine, X = O, Y = SCH ₂ Ph): Prepared from 1, ( B = guanine, X = S, Y = -NHPh) (1 mmol); 75% efficiency; ³¹ P NMR (CDCl ₃ ): 56.01; 55.85 ppm.

Example XIII. Oxidation reaction of 5'-O-DMTthymidyl 3'-O-methanophosphonothioanide 1 (B = thymine, X = S, Y = -NHPh) to 5'-O-DMTthymidyl 3'-O-methanophosphonate 1, (B = thymine, X = -O, Y = -NHPh) using potassium peroxymonosulfate.

FAST compound - formula 1 (B = thymine, X = S, Y = -NHPh) (0.072 g; 1 mmol) was dissolved in a mixture of methanol and tetrahydrofuran and an aqueous solution of potassium peroxymonosulfate (pH 6.7-7) (2 mmol) was added . After 10 minutes, 10% sodium thiosulfate was added and the product was extracted from the reaction mixture with chloroform.

Product 1 a (B = thymine, X = O, Y = -NHPh) was purified by silica gel chromatography. Diastereomerically pure FAST 1 was obtained in a yield of 73% (0.047 g); 3'P NMR (CDC1 _3): 30.1 ppm. Mass Spectroscopy: FAB MS: 696.4.

Example XLV. Analogously as in example XIII, the conversion of SLOW - formula 1b (B = ymin, X = S, Y = -NHPh) to SLOW - 1b (B = thymine, X = O, Y = -NHPh)) was performed with the efficiency of 70%; ³¹ P NMR (CDCl3): 29.89 ppm.

Example XV. Conversion of 5'-O-DMT-thymidyl 3'-O-methanophosphonate formula 1 (B = thymine, X = O, Y = -NHPh) to thymidyl 3'-O- (S-benzylmethanothiophosphonate) thymidyl 1, (B = thymine , X = O, Y = SCH ₂ Ph). Compounds 1 (B = thymine, X = O, Y = -NHPh) (FAST or SLOW) (50 mg) was dried and dissolved in THF (2 ml) with the addition of 1,8-diazabicyclo [5.4.0] undec-7-ene ( DBU) (0.02 ml). Stirring was continued for 45 min. at room temperature, then carbon disulfide (1 mL) was added. After 20 minutes, benzyl bromide (5 eq.) Was added.

The product was purified by extraction (chloroform-water) and silica gel column chromatography. From FAST 1a (B = thymine, X = O, Y = -NHPh), the FAST Ia product (B = thymine, X = O, Y = SCH ₂ Ph) was obtained in 65% yield (0.036 g). ³¹ P NMR (CDCl 3): 56.85 ppm. SLOW compound -1b (B = thymine, X = O, Y = NHPh) was SLOW -1b (B-thymine, X = O, Y = SCH2Ph 70% yield (0.039 g). ^Fl P NMR (CDC13) : 55.38 ppm.

Example XVI. AgF (25% aqueous solution) (1.25 eq.) Was added to a solution of the compound of formula 1 (X = O, Y = XRs) (1 mmol) in acetonitrile (75 ml). After completion of the reaction, product 1 (X = O, Y = F) was extracted from CHCl ₃ -saturated brine. Dried over MgSO4 and the pure product was precipitated from CHCl ₃ -petroleum ether.

The spectroscopic properties of the substrates and products are presented in Table 2.

184 377

Table 2

Substrate (X = O, Y = CH ₂ pH) Product (X = O, Y = F) B 3P nmr * ^3I P NMR J1P-F FAB-MS T. 56.83 30.7 1060 623.4 30.6 1059 C ^B2 56.10 30.3 1060 712.2 30.2 1059 AB2 55.85 30.6 1057 736.2 30.3 1059 G ' ^bu 56.58 31.3 1058 718.2 30.9 1060

* spectra made in C ₆ D ₆

184 377

184 377

Formula 1

Formula la Formula 1b

Formula 2

Publishing Department of the UP RP. Circulation of 70 copies. Price PLN 4.00.

Claims (6)

Patent claims 1. New organophosphorus derivatives represented by the general formula 1, in which X is an oxygen or sulfur atom, Y is -NHPh, F or a group of the formula -X'R ³ , in which X is as defined above and R ³ is a methyl group, or -CH2C6H4 of the formula-R ^4, wherein R 4 is hydrogen, chlorine or -NO 2, R ¹ is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthene-9-yl (Px) ² R is a hydrogen atom or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine. and Guanina. 2. Process for the preparation of new organophosphorus derivatives of general formula I, in which X is a sulfur or oxygen atom, Y is -NHPh, Ri is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px) , R2 is hydrogen or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, characterized by reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent , preferably amines, especially triethylamine with a nucleoside of formula II in which R1, R2 and B are as defined above, then aniline and elemental sulfur are added to the mixture, and after the reaction is completed, the product is isolated in a known manner. 3. A process for preparing novel organophosphorus of the general formula 1, wherein X represents an oxygen atom, Y a group of formula -X'R ^3, wherein X is as defined above and R3 is methyl or -CH ₂ C ₆ H | -R4, where R4 is hydrogen, chlorine or -NO2, is Ri is a blocking group, preferably 4'-dimethoxytryre (DMT) or 9-phenylxanthen-9-yl (Px), R2 is a blocking group hydrogen or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, characterized by reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent, preferably amines, especially triethylamine with a nucleoside of formula 2 in which R 1, R 2 and B are as defined above, then aniline and elemental sulfur are added to the mixture, and the resulting mixture of nucleoside diastereoisomers of the 3'-O-methanophosphonothioanilide of formula general clause 1, in which R 1, R ^{2 and B} are as defined above, and Y is -NHPh, is reacted with sodium hydride or DBU and carbon dioxide, and the transiently obtained nucleoside 3'-O-methanothiophosphonic acid (sodium or DBU sodium salt) ) is alkylated with an alkylating reagent of formula R ³ Z in which R ³ is as defined above and Z is chlorine, bromine or iodine. 4. Process for the preparation of new phosphorus acid derivatives of general formula I, in which X is oxygen, Y is -NHPh, R1 is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px), IU is a hydrogen atom or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, characterized by reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent, preferably amines especially triethylamine nucleoside of formula 2 wherein R1 ^R2 and B are as defined above then added to the mixture of aniline and elemental sulfur and the resulting mixture of diastereomers nucleoside 3'-O-metanofosfonotioanilidu of general formula 1, wherein R ^1, R2 and B are as defined above and X is a sulfur atom and is oxidized with known oxidizing agents, preferably peroxy potassium monosulfate or hydrogen peroxide. 184 377 5. Process for the preparation of new organophosphorus derivatives of general formula I, in which X is oxygen and Y is a group of the formula -X'R ³ , in which X ¹ is oxygen or sulfur, and R3 is a methyl group, or a group of the formula - -CH2CĆH4-R ^4, wherein R ⁴ is hydrogen, chlorine or -NO 2, R? is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthene-9-yl (Px), R ² is hydrogen or alkoxy, preferably methoxy, and B is N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, characterized by reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent, preferably an amine, especially triethylamine, with a nucleoside of formula 2, wherein R 1, R 2 and B are as defined above, then aniline and elemental sulfur are added to the mixture, and the resulting mixture of nucleoside diastereoisomers of the 3'-O-methanophosphonothioanilide of the general formula I, in which X is a sulfur atom, R1, R2 and B are as defined above, and Y is -NHPh, is subjected to the oxidation reaction by known oxidizing agents, preferably with potassium peroxymonosulfate or hydrogen peroxide, then the compound obtained is reacted with a hydride with sodium or DBU and with carbon disulfide, and the transiently obtained nucleoside 3'-O-methanothiophosphonic acid (sodium or DBU-sodium salt) is alkylated with an alkylating agent of formula R ³ Z, where R 3 is as defined above and Z is chlorine, bromine or iodine. 6. Process for the preparation of new organophosphorus derivatives of general formula I, in which X is sulfur or oxygen, Y is F, Ri is a blocking group, preferably 4'-dimethoxytrityl (DMT) or 9-phenylxanthen-9-yl (Px), r2 is a hydrogen atom or an alkoxy group, preferably methoxy, and B is an N-blocked purine or pyrimidine base, preferably selected from the group consisting of Thymine, Uracil, Adenine, Cytosine and Guanine, characterized by reacting methyldichlorophosphine in the presence of a hydrogen chloride binding reagent, preferably amines, especially triethylamine with a nucleoside of formula 2, where R 1, R 2 and B are as defined above, then aniline and elemental sulfur or an oxidizing agent are added to the mixture, the resulting mixture of nucleoside diastereoisomers of the 3'-O-methanophosphonoanilide of the general formula 1, where X is oxygen or sulfur, R1, R2 and B are as defined above, and Y is -NHPh, reacted with sodium hydride 1 or DBU and carbon dioxide or disulfide, and the resulting mixture of nucleoside diastereoisomers of the 3-0-methane phosphonothiolester of the general formula 1, in which X, R1, R2 and B are as defined above, and Y is S or -SeR3, is reacted with a fluorinating reagent of formula MF, wherein M is Ag, -NEt4, -NET ₃ H, and the product is isolated in a known manner.