PL160846B1 - Method for the production of new nucleoside derivatives - Google Patents

Abstract

A method for the preparation of new nucleoside derivatives of the general formula 1, wherein Y is a dimethoxytriphenylmethyl group, X is a group of the formula 6, wherein R2 is a 2-cyanoethoxy group and R3 is a diisopropylamino group or X is a solid support covalently bound to the nucleoside, Z is a hydrogen atom or an OH group, and B is a group of the formula 3, 4 or 5, characterized in that the compound of the formula 2, wherein B' is an adenine, guanine or cytosine residue of the formulas 7, 8, 9, respectively, and Z has the meaning given above, is reacted with isopropoxyacetyl chloride or anhydride in the presence of an excess of a trialkylsilyl halide, the silyl groups are selectively hydrolyzed in the presence of water, and then the obtained compound of the formula 1, wherein Z has the meaning given above, X and Y are hydrogen atoms, and B is a group of the formula 3, 4 or 5 is reacted with 4,4'-dimethoxytriphenylmethyl chloride and then reacted in the 3' position either with N,N,N',N'-tetraisopropyldiamidophosphite 0 2-cyanoethyl or acetylated, esterified with 4-nitrophenol and reacted with a solid support with an organic chain containing carbon, nitrogen and oxygen atoms.

Description

The subject of the invention is a process for the preparation of new nucleoside derivatives of the general formula I, in which Y is a dimethoxytrieenomethyl group,

B is a group of formula 3, formula 4 or formula 5, X is a group of formula 6, where Rg is a 2-cyanoethoxy group of formula - OCHgCHgCN and Rj is a diisoprppyoomrnine group or X is a solid support covalently linked to nucleosides, and Z is hydrogen or OH.

The novel nucleoside derivatives of the formula I find use in the production of oligonucleotide gene fragments encoding protein biosynthesis. The synthesis of oligonucleotide gene fragments using the new derivatives is presented in Potential Application No. P 273 723 published in BUP No. 2/90.

The earliest known nucleoside derivatives used for the synthesis of gene fragments are nucleosides in which the amine functions of adenine, guanine and cytosine are blocked with a benzoyl or isobutyryl group. Known nucleoside derivatives are not convenient for the synthesis of oligonucleotides because ueumnl allocating groups require heating of the synthesis product for 16 hours in aqueous ammonia solution at 50-60 ° C or for 48 hours at room temperature.

Surprisingly, it was found that the new nucleoside derivatives obtained by the method according to the invention are very advantageous starting compounds in the synthesis of gene fragments, because they allow to obtain a product with high efficiency, more than 99.6% for a single chain extension reaction, and also the removal of groups blockers are digested for 2 hours in a 25% aqueous ammonia solution at a temperature of 20 ° C, which favorably affects the purity of the product.

160 846

The method according to the invention consists in reacting a compound of formula II, in which B is an adenine, guanine or cytosine residue of the formulas 7, Θ, 9, respectively, and Z is as defined above, is reacted with an isopropoxyacetyl chloride or an anhydrous, in the presence of an excess of a trialkylsilyl halide, followed by selectively hydrolyzing the silLUwe groups in the presence of water to give the compound of Formula I, wherein X and Y are hydrogen.

Z represents a hydrogen atom or an OH group, and B represents a group of formula 3, 4 or 5. The obtained compound is reacted with 4,4'-dimethokaytriphenylmethyl chloride, which is a known 5-hydroxy group protection reaction, followed by a known reaction at the position 3 'or. With Η, Η, Η', H '-teOraioopropnillamL-0-2-cyanoyl-phosphorne to give the compound of formula 1, where B is as defined, Y is d10θtlxytripheneOomstyl and X is the group of formula 6, where Rg is Z-cyanoethoxy and Rj is diisoplopylaam or acetylation followed by esterification with 4-nitloSenoS to give an active ester which is reacted with a solid support with an organic chain containing carbon, nitrogen and oxygen atoms, to give a compound of formula I in which B is as defined above, Y is dl-methocytrifinyl methyl and X is a solid support covalently linked to nucleoside.

The reaction of a compound of formula II with isopropokayacetyl chloride or anhydride may be carried out in a solvent, for example pyridine. In this reaction, a trialkyl8llyl halide is used for the transient protection of hydroxyl groups. Selective hydrolysis of the silyl groups is carried out in the presence of water.

In the acetylation reaction, for example, succinic anhydride can be used before binding the nucleoside to the solid support. An exemplary solid support bound nucleoside has formula 10, where CPG is a controlled pore glass, Y is a dimethoxytriphenyl ethyl group, and B is as defined above.

As already mentioned, the new nucleoside derivatives are very advantageous compounds in the process of obtaining synthetic gene fragments by condensation of appropriately blocked nucleosides. The following examples illustrate the images.

Example I.A, Ng-isopropoxyectyl ~ 2 '"dookoysd" elo-et 50mmoH.

"= D. 13x2xt) dried under high vacuum in an oil bath at 50 ° C for 6 hours, then suspended in 80 µl of anhydrous pyridine and added with stirring 250 mAh of thioethylchloroθilίιn at room temperature. After two hours, 75 mnoH of isopooxic anhydride was added and stirred for 2 hours. The precipitate of pyridine hydrochloride was filtered off, washed with 20 ml of benzene, the filtrates were combined and concentrated to about 40 ml. Then 50 ol of dioxane and 30 ol of water were added. Stirred for 2 hours. The reaction mixture was concentrated to dryness. The oil obtained was purified on a gel column (Merck 60) with the eluent: chloroform / methanol 99: 1 - 90:10. The actual fractions were concentrated to dryness. Crystallilaafano product from chloroform. Mp 134-135 ° C; Rf - 0.52; / CH 2 CN: H 2 O 85: 15/5 yield 76%. Elemental analysis consistent with theoretical.

B. 5 '-dioethoxyStriphenylmethyl-Ng-isopropoxy-acetyl-2'-deoxyadenosine. Obtained in stage I.A. the compound (2) was dried by evaporation of anhydrous pyridine (5 ml) and suspended in 10 ol of anhydrous pyridine. Chloride 4,4''im to the metallo tide (2.2 m) was added. The reaction mixture was stirred at room temperature for 5 hours. 2 L of methanol was added and concentrated to dryness. The crude product was purified on a r-erck 60K gel column as an element

160 846 meazanine: chlornform / methanol / 99: 1 / containing 0.1 I of pyridine, Rf - 0.68, / CHCl3 / C & D < 2 > H 90: 10 / HPTLC, 76% yield Elemental analysis consistent with the theoretical

C. C £ glzopropokeyaaceylo-5'-di-ethokaytriphenylmethyl-2'-deoxysdanosine-3'-0- / 0-2c _C ylβnoethyl / cϊI, 1'-dlysopylamidophosphines.

The compound obtained in step (1 mol) and 1-H-tetrazole (1 mol) was dried by evaporation of anhydrous pyridine (3 ml) and for 2 hours under high vacuum. Dissolved in anhydrous methylene chloride (3 ml) and added with stirring. ', H'-tetraisopropyl diamidophoephorin O 4 -cyanoethyl / 1.1 mm. The mephate was continued for 1 hour at room temperature. The reaction mixture was concentrated to dryness, purified on a Merck 60 gel column, eluting with benzene / meeylene chloride / triethylamine 8: 1: 1. The product was obtained with a yield of 80 Ui R _f - 0.75 and 0.62, [benzene / 0 ^^ / 60:16:16, < ^{ppm 31p} BMB

149.73, 149.46, (cD-OT) 85% H 2 PO 3

Example I ^ A. H2-isopropokβylcetylO22'-dodagulanosinil. Proceeding analogously to Example 1A, except that after the addition of dioxane and water, the mixture was stirred for 12 hours, and the product kryitllZlowanl from the mixture of dioxane-ter-ethyl II, the title compound was obtained with an yield of 81%. Melting point> 000 ^o C / with decomposition, Rf - 0.50, (CH3CB: H2O 85: 15). Elemental analysis consistent with theoretical.

B. 5 * -aethokβytrlf ^> enylooethyl-C2-isoeΓopokayaaltyl-2'-deoxyguanosine. Following the procedure as described in Example IB, the product of wyddjnością 71%, Rf - 0.53 / CHC! ^ ^W ^ H 90 CH 10 / HPTLC. A ^ aUza elmentary consistent with the theoretical.

C * H2-isopropokβyaactylo-5 * -aioethok8ytrifinyloethyll-2'-de-xyguanosine3 * d) - (0-2-oyanoethyl) -Ν, Ν-diisopyll-aolaoflβforin. By proceeding analogously to Example IC, the product was obtained in 82% yield, R f -0.7 benzene / methylene chloride (m / m) 60:16:16, <T ppo NMR 149.86, 149.46) CD3CN / 85% H.jPO ₄ .

Example III.A. N ₄ “izoerlpoksya ¢ θtyio = -2'-deokeycytidine. By proceeding analogously to Example 1A, except that the crystallization was carried out in carbon tetrachloride, the product was obtained with an elution of 87%; Rf - 0.59 (CH3CN2GO 85: 15) Melting point 157-158 ° Co Elentary analysis is consistent with the theoretical.

B. 5'-dimethylk8ytriphenylmethyl-N ₄ -lzlerlpokβyacetyl-2 '-aezlkyycytyaynl. By proceeding analogously to example 1b, the product was obtained in a yield of 80%, Rf - 0.75 [mu] C ^ 3: C ^ O ^ C 90: 10 / HPTLC. Elemental analysis consistent with theoretical.

C. N ₄ -isopropokayacetyl-5'-dimethokaytriphenylmethyl-2'-deoxycytidine-3 '

-O- (O-2-cyanoethyl) -N, N-diisopropylamide, sulfite. By proceeding analogously to the example IC, the product was obtained in a yield of 80%, Rf - 0.62 ⁽ methylene chloride / triethylamine ⁸ 4: l6). ([Ppm KMil 149.66, 14 ^{9.52 / c} Dy ^CN / ⁸ 5% h / by _4. ,

Example IV. 5'-dimethoxytriphenylamphthyl-N'-isopropoxyacetyl-2'-deocayadenosine bound to a solid carrier. 10 mumH of the compound obtained in Example I.B., 10 mmmH of burazine anhydride and 10 mmmH of dimethylaminopyridine were dried under high vacuum for 2 hours, 10 ml of anhydrous pyridine were added and stirred for 12 hours at room temperature. The product is concentrated to dryness, the product is purified on a gel column (Merck 60), eluted with a mixture of chloroorms-methanol 100: 5 26 mmH product, 28 mmH 4-nitrophenol and 31 mmmH dicyclohexyl acetate and dried for 2 hours under high vacuum. Under anhydrous conditions, 3 ol of methylene chloride were added, stirred for 1 hour.

160 846

The product in 1 ml of triethoamine was added to 1g of a controlled porosity glass consisting of carbon, nitrogen and oxygen, stirred for 20 hours, filtered and washed with methanol and pyridine. The product was obtained with a content of 35 µmol per 1 g of solid support.

EXAMPLE 5 5 * -dimethokeytriphenylmethyl-2-ddzxaguaanosine bound to a solid support. Analogously to example 4, a product was obtained with a hardness of 41.3 / mole per 1 g of solid support.

Example VI. 5-dimethokaytΓiphenalomethyl-2 * -dzzkkaycayidine bound to solid support. By analogy with Example 4, the product was obtained with a content of 39.8% Bw per 1 g of solid support.

New nucleoside derivatives were used for the production of gene fragments by the CondenasCi method. Fragments containing 14 to 40 nucleotides were obtained, which were enzymatically combined to obtain, for example, a gene encoding human cachectin, which is a known drug for <»Γoaιroroym

160 846

PATTERN 9

ch ₂

I o = c

NHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ fyH

C®0

ĆH ₂ ch ₂ oEt o = ę

CPG — O-St —ch ₂ ch ₂ ch ₂ nh

MODEL 10

OEt

160 846 ch ₃

II I hnc-ch ₂ -o-ch nA Ćh ₃ pr ₃

No. ₂

PATTERN 5 PATTERN 6

PATTERN?

160 846

Ηθη 0

OH Z

W2ÓR1

PATTERN 2

II

CH,

H-N-C-CH, -O-CH

I 4 I ch ₃

MODEL 3

MODEL A

Department of Publishing of the UP RP. Circulation of 90 copies

Price: PLN 10,000

Claims (1)

Patent claim A method for the preparation of novel nucleoside derivatives of general formula I, wherein Y is a dimethokaytriphenylmethyl group. Z is a group of formula 6, where Rg is 2-cyanoethoxy and Rj is a diisopropylamino group, or Z is a support covalently linked to a nucleoside, Z is a hydrogen atom or an OH group, and B is a group of formula 3, 4 or 5, characterized in that the compound of formula 2 wherein B'oznacza residue of adenine, guanine or cytosine odpcowedido of formulas 7, 8, 9, and Z has the ^desired PE ®j defined AIE treated or reacted with Isopropokayacetyl anhydride, in ob ^ <^^ <^^ <c and excess of the trialkylalkyloylvago halide, selectively hydrolyzes a group of ailU in the presence of water, then the obtained compound of formula I, where Z m and the above meaning, X and Y are hydrogen, and B is a group of formula 3, 4 or 5 reacts with 4,4-dimethokaytriphenylmethyl chloride, followed by reaction at the 3-position or with Ν, Ν, ΝΝ'te traisopropyl diamidophoyl 0-2-cyanoelastic, or acetylation, eetryification and 4- nitrophenol and reacting with a solid support with an organic chain hu containing carbon, nitrogen and oxygen atoms. * ♦ *