AU5334900A - Novel bicyclonucleoside and oligonucleotide analogue - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Takeshi Imanishi Actual Inventor(s): TAKESHI IMANISHI Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL BICYCLONUCLEOSIDE AND OLIGONUCLEOTIDE ANALOGUE Our Ref: POF Code: 623433 1594/370752 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1 6006q -1a-

SPECIFICATION

NOVEL BICYCLONUCLEOSIDE AND OLIGONUCLEOTIDE

ANALOGUE

TECHNICAL FIELD This application is a divisional application of Australian Patent Application 61209/98 (720742) the entire content of which is hereby incorporated by reference.

This invention relates to a novel nucleoside analogue and a novel nucleotide analogue, and more particularly, to a nucleotide analogue suitable as an antisense molecule.

BACKGROUND ART In 1978, it was reported for the first time that an antisense molecule inhibited influenza virus infection. Since then, reports have been issued that antisense molecules inhibited the expression of oncogenes and AIDS infection. In recent years, antisense oligonucleotides have become one of the most promising S. pharmaceuticals, because they specifically control the expression of undesirable genes.

The antisense method is based on the idea of controlling a unidirectional flow called the central dogma, DNA->RNA->protein, by use of an antisense "oligonucleotide.

When a naturally occurring oligonucleotide was applied to this method as an antisense molecule, however, it was decomposed with various nucleases in vivo, or its permeation through the cell membrane was not high. To solve these problems, numerous nucleic acid derivatives and analogues have been synthesized, and their studies have been conducted. Examples of the synthesized products include a phosphorothioate having a sulfur atom substituting for an oxygen atom on the phosphorus atom, and a methylphosphonate W:\floa\SpecieslDiv of720472.doc having a substituting methyl group. Recently, products have been synthesized in which the phosphorus atom has also been substituted by a carbon atom, or the structure of the sugar portion has been changed, or the nucleic acid base has been modified. Any resulting derivatives or analogues, however, have not been fully satisfactory in terms of in vivo stability, ease of synthesis, and sequence specificity (the property of selectively controlling the expression of a particular gene alone).

Under these circumstances, there has been a demand for the creation of an antisense molecule which is minimally decomposed with a nuclease in vivo, binds to target messenger RNA with high affinity, has high specificity, and e can thus efficiently control the expression of a particular 15 gene.

DISCLOSURE OF THE INVENTION The inventors of the present invention designed a nucleic acid analogue with immobilized conformation of the *"sugar portion in a nucleic acid, which would be useful in the antisense method. They synthesized a nucleoside analogue which will be a unit structure therefor, and confirmed that an oligonucleotide analogue prepared using it was very useful as an antisense molecule.

2 2a In one aspect the present invention provides a method of treatment of a medical condition comprising administering a therapeutically effective amount of an oligonucleotide or polynucleotide analogue having one or more structures of the general formula (la)

-O

B

(la) vO where B is a pyrimidine or purine nucleic acid base, or an analogue thereof.

In a further aspect the present invention provides a method of treatment of a medical condition comprising administration of a therapeutically effective amount of an oligonucleotide or polynucleotide analogue of the general formula

(II)

1 w 0 B

O

O

B

*O

SO II o R 0 O -P -OW 2 3 where B and B are identical or different, and each represent a pyrimidine or purine nucleic acid base, or an analogue thereof, R is a hydrogen atom,

S

a hydroxyl group, a halogen atom, or an alkoxy group, W and W 2 are identical or different, and each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkinyl group, a cyloalky g aroup, an aralkyl group, an aryl group, an acyl group, a silyl group, a phosphoric acid residue, a naturally occurring nucleoside or a synthetic nucleoside bound via a phosphodiester bond, or an oligonucleotide or polynucleotide containing the nucleoside, n's or n 2 's are identical or different, and each denote an integer of 0 to 50, provided that n's or n 2 's are not zero at the same time, and that not all of n 2 's are zero at the same time, n 3 denotes an integer of 1 W:\fiona\Species\Div of 720472.doc 2b to 50, provided that n' and/or n 2 are or is 2 or more, 13 1 and B need not be identical, and R's need not be identical.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a chart showing the time course of the ultraviolet absorption (260 nm) of a naturally occurring oligonucleotide decomposed with an exonuclease; and Fig. 2 is a chart showing the time course of the W:\flona\Sp~disSfliV of 7 20472.doc ultraviolet absorption (260 nm) of an oligonucleotide of the present invention (X2) decomposed with an exonuclease.

Details of the present invention will now be described.

The structure of a nucleoside analogue according to the present invention is a nucleoside analogue of the following general formula (I)

B

Y

S

15 where B is a pyrimidine or purine nucleic acid base, or an analogue thereof, and X and Y are identical or different, and each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkinyl group, a cycloalkyl group, an aralkyl group, an aryl group, an acyl group, or a silyl group, or an amidite derivative thereof.

The alkyl group represents a straight chain or branched chain alkyl group with 1 to 20 carbon atoms. Its examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.

The alkenyl group represents a straight chain or branched chain alkenyl group with 2 to 20 carbon atoms. Its examples include vinyl, allyl, butenyl, pentenyl, geranyl, 3 and farnesyl.

The alkinyl group represents a straight chain or branched chain alkinyl group with 2 to 20 carbon atoms. Its examples include ethynyl, propynyl, and butynyl.

The cycloalkyl group represents a cycloalkyl group with 3 to 8 carbon atoms, and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Another example is a heterocyclic group in which one or more arbitrary methylene groups on the ring of the cycloalkyl group have been substituted by an oxygen atom, a sulfur atom, or an alkyl-substituted nitrogen atom. It is, for instance, a tetrahydropyranyl group.

The aryl group refers to a monovalent substituent formed by removing one hydrogen atom from an aromatic heterocyclic group or an aromatic hydrocarbon group. Preferably, it represents a monovalent substituent formed by removing one hydrogen atom from an aromatic hydrocarbon group, and includes, for example, phenyl, tolyl, xylyl, biphenyl, naphthyl, anthryl, and phenanthryl. The carbon atom on the ring of the aryl group may be substituted by one or more of a halogen atom, a lower alkyl group, a hydroxyl group, an alkoxy group, an amino group, a nitro group, a trifluoromethyl group and an aryloxy group.

The aralkyl group refers to an alkyl group bonded to an aryl group,'and may be substituted. The aralkyl group be substituted. The aralkyl group \\MELS\DATA\apps\winword\FIONAflonaSPECl 1209 doc that may be substituted represents an alkyl group bonded to an aryl group, with one or more arbitrary hydrogen atoms of the aryl group and the alkyl group being optionally substituted by the following substituents: Examples of the substituents are acyl, amino, aryl, alkyl, cycloalkyl, alkoxy, hydroxyl, nitro, and halogen.

The amino group need not be substituted, but the amino group when substituted includes, for example, alkylamino, arylamino, and acylamino. Examples of the alkoxy group are methoxy, ethoxy, n-propoxy, i-propoxy, nbutoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, hexyloxy, and phenoxy. Examples of the halogen atom are fluorine, chlorine, bromine, and iodine.

S The preferred examples of the aralkyl group are 15 trityl, benzyl, phenethyl, tritylmethyl, diphenylmethyl, naphthylmethyl, and 4,4'-dimethoxytrityl (DMTr).

*g Particularly preferred is a DMTr group.

As the acyl group, acetyl, formyl, propionyl, benzoyl, and benzyloxycarbonyl can be exemplified. An example of the silyl group is a trialkylsilyl group, preferably trimethylsilyl, triethylsilyl, triisopropylsilyl, tbutyldimethylsilyl or t-butyldiphenylsilyl, and more preferably trimethylsilyl.

The nucleotide analogue of the present invention is an oligonucleotide or polynucleotide analogue having one or more structures of the general formula (Ia) 5 (Ia) where B is a pyrimidine or purine nucleic acid base, or an analogue thereof, or an oligonucleotide or polynucleotide analogue of the general formula (II) S1 00 'O-P OW S15 0-P

II

II 0

O

In 0 represent a pyrimidine or purine nucleic acid base, 1 n3 or an analogue thereof, R is a hydrogen atom, a hydroxyl group, a halogen atom, or an alkoxy group, where B and B are identical or different, and each represent a pyrimidine or purine nucleic acid base, or an analogue thereof, R is a hydrogen atom, a hydroxyl group, a halogen atom, or an alkoxy group,

W

1 and W 2 are identical or different, and each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkinyl group, a cycloalkyl group, an aralkyl group, an aryl group, an acyl group, a silyl group, a phosphoric acid residue, a naturally occurring nucleoside or a synthetic nucleoside bound 6 via a phosphodiester bond, or an oligonucleotide or polynucleotide containing the nucleoside, n 1 's or n 2 's are identical or different, and each denote an integer of 0 to 50, provided that n 1 's or n 2 's are not zero at the same time, and that not all of n 2 's are zero at the same time, n 3 denotes an integer of 1 to provided that when n i and/or n 2 are or is 2 or more, B 1 and B need not be identical, and R's need not be identical.

The pyrimidine or purine nucleic acid base in the present invention refers to thymin, uracil, cytosine, adenine, guanine, or a derivative thereof.

The nucleoside analogue and nucleotide analogue of the present invention can be synthesized in the manner 15 described below.

Synthesis of nucleoside analogue SHO HO U H TaCI PCTFAH CO too H _0 TFA-H ZC 0_ H O pyrdlne SO 0 (94) TsO ZnII OH OH 20 201 2 3 oo. HO 0 NeSHCN HO HO C NaHMoS 0 H,/Pd-C TsO

M

eCN T HF MeOH OB n OH O (quan.) Ph 4 5 6 _U DMTrCI 0i pyrdhaa

OMAP

0(66%) O 7 8 7 Compound 1, synthesized from uridine in accordance with the literature J.A. Secrist et al., J. Am. Chem.

Soc., 101, 1554 (1979); 2) G.H. Jones et al., J. Org. Chem., 44, 1309 (1979)], was treated with tosyl chloride (TsC1) to tosylate only one of the two primary alcohols, leading to Compound 2. Compound 2 was acid hydrolyzed into a triol compound 3. Compound 3 was condensed with benzaldehyde in the presence of an acid catalyst to form a benzylidene compound 4. Compound 4 was reduced with sodium cyanoborohydride (NaBH 3 CN) in the presence of titanium tetrachloride (TiCl 4 to obtain Compound 5. This compound was reacted with sodium hexamethyldisilazide (NaHMDS) in tetrahydrofuran (THF) to obtain a bicyclo compound 6 "(Compound I: B uracil X H, Y benzyl). When 15 Compound 6 was catalytically reduced in the presence of a palladium carbon catalyst, a diol compound 7 (Compound B U, X Y H) was obtained. Further treatment of Compound 7 with 4,4'-dimethoxytrityl chloride (DMTrCl) gave a trityl compound 8 (Compound I: B U, X DMTr, Y H).

S 20 Compounds 6, 7 and 8 can be used as starting materials for various compounds I.

Compounds having various nucleic acid bases, whether natural or nonnatural, other than uridine, can be synthesized by any of the following three methods: The first method is conversion from Compound 8. That is, Compound 8 is acetylated into Compound 9, and then reacted with 1,2,4-triazole to form Compound 10. Hydrolysis of this compound gave Compound 11 (Compound B 8 cytosine X DMTr, Y Compound 12 (Compound B benzoylcytosine X DMTr, Y which will become a starting material for oligonucleotide synthesis, can be easily obtained by benzoylation of Compound 11.

S1 0

N.

N

OAc DMT N O 8 0 DMTO DMT OAc S- OH OH Io* with the literature A.G.M. Barrett et al., J. Org. Chem., 3853 (1990); 4) G.H. Jones et al., ibid., 44, 1309 12 11 (1979) 15 The second method is a metholed perfo Compound 16 by three steps, and cyclized under basic conditions to obtain a desired methylglycosyl compound 17. The OMe group at the 1- 13 whichtion can be easiy obtained from D-ribose in accordance natural nucleic acid bases or nonnatural nucleic acid base analoguwith the literature A.G.M. Barrett et already., J. Org. Chem.,been 3853 (1990); 4) G.H. Jones eth al., ibid., 44, 1309scheme ranging(1979)]. That is Compound 13 was to Compound 20 can be employed.

three steps, and cyclized under basic conditions to obtain a desired methylglycosyl compound 17. The OMe group at the 1position of this compound can be substituted by different natural nucleic acid bases or nonnatural nucleic acid base analogues by various methods which have already been developed. For example, a method as shown by a scheme ranging from Compound 17 to Compound 20 can be employed.

9 HO7 O OH OH D-ribose MeOH, aceo ne cHCI reflux, 19 hr so HO O Me CrO%, Py

CH

2

CI

2 rt, 20 min Hly

M

e) HCHO aq., NaOH aq.

p-dioxane rt, 15 hr (37 2 steps) HO OMa 2) 0 O TB DP C TBPSO 0 OCHz TsCV DMAPTBPSO.0 OcHa E13N I CHZCI 2 z E1 3 N I CH 2

ZCI

2 rt,13 hr HO0x0rt 7 hi 1TsO 0>< 09*** **o TFA -H 2 0 TBPSOj 1 OCH3 NaHDS1< 0 CH ~HF TSO OOH THF rt, 20min rt. 1 hr TBDPSO OH 0 OH (43%) (77%) 16

TBDF

DMAP/ Py rt, 3 hr (86%) k va)

OTIMS

Me- NOT7MS TMSOTf/ 1, 1 -dichloroethane CH3CN rt, 18 hr (ca. 70 IW-

TBOP

10 The third method starts with diacetone D-glucose.

which is obtained from D-glucose by one step and is commercially available. Compound 31 was prepared in accordance with a reference 5) R.D. Youssefyeh, J.P.H.

Verheyden and-J.G. Moffatt., J. Org. Chem.,, 44, 1301-1309 (1979). Then, Compound 31 was treated as shown by the following scheme to protect the two primary hydroxyl groups with a t-butyldiphenylsilyl group and a p-toluenesulfonyl group progressively. The protected compound was acetylated into Compound 34.

:i~0 reference 5) F1 B TBDPSr? Et 3

N

H CHCI'2 *Diacetone D-glucose 67% S. S ova TBDPS TsCI /DMAP IEtN TBDPS

CH

2 C1 2 H* en r t. Ts) Bn 97% -32 3 ACO .C0 coc SAA

TBDPSO"'

5.55 t TsO>rB 7 nAc 86% S4 Compound 34 was condensed, separately, with thymine, benzoyladenine, and isobutyrylguanine activated upon trimethylsilylation (referred to as 2TMS-T, 2TMS-A, and 3TMS-GB, respectively), to obtain Compounds 35, 40 and 44 in high yields, as indicated by 'the scheme offered below.

Then, these condensates were subjected to deacetylation 11 (Compounds 36, 41, 45), five-membered ring formation (Compounds 37, 42, 46), desilylation (Compounds 38, 43, 47), and further debenzylation to form desired compounds 39.

2TMS -T or TBDPS C 2TMS or Ac 3TMS

-GB

TsO Bn Ac 'TMSOTf/CICH 2

CH

2

CI

34 r t.

(70-97%)

TBDO

3 5: B=T E=Aaz .44: B=GU aq. K 2 00 3 MeCH r t.

(64-92%) TBDPS

B

Tsa 4nH S6: B=T 41: B=ABZ 45: B=G"uu NaHMDS/THFTBDPSO oP TBAFITHF r t. r t (44-100U%) (83-100%) 37: B=T 42: B=ABZ 4.6: S=Ge'u

B=T

43: B=ABZ 47: B=GiBU

H

2 20%Pd(OH) 2

-C

MeOH r t.

96%

HOW

3 9a: B=T 39b: B=A"Z 39c: B=GBEu 12 Synthesis of oligonucleotide analogue Compound 8 is reacted with 2-cyanoethyl-N,N,N',N'tetraisopropylphosphoramidite to obtain an amidite compound 21. This compound is combined with a naturally occurring nucleoside amidite, and subjected to a DNA synthesizer to synthesize various oligonucleotide analogues. The synthesized crude products are purified using a reversed phase chromatographic column (Oligo-Pak). The purity of the purified product is analyzed by HPLC, whereby the formation of a purified oligonucleotide analogue can be confirmed.

Natural nucleoside amidite DMTrO- B DMTrO DNA synthesizer Various oligonucleotide .o analogues a IPr2N' 27 27 At least one monomer unit as compound 8 can be contained in the oligonucleotide analogue. Alternatively, the monomer units may be present at two or more locations in the oligonucleotide analogue in such a manner as to be separated from each other.via one or more naturally occurring nucleotides. The present invention makes it possible to synthesize an antisense molecule incorporating a necessary number of the nucleotide analogues (nucleoside analogues) of the invention (a necessary length of the nucleotide or nucleoside analogue) at a necessary location.

The length of the entire oligonucleotide analogue is 2 to preferably 10 to 30, nucleoside units.

13 Such an oligonucleotide analogue (antisense molecule) is minimally degradable by various nucleases, and can be existent in vivo for a long time after administration. This antisense molecule functions, for example, to form a stable double helix together with a messenger RNA, thereby inhibiting the biosynthesis of a potentially pathogenic protein; or form a triple helix in combination with doublestranded DNA in a genome to inhibit transcription to messenger RNA. The oligonucleotide analogue can also suppress the proliferation of a virus which has infected.

In light of these findings, an oligonucleotide analogue (antisense molecule) using the nucleoside analogue of the present invention is expected to be useful as drugs, including antineoplastics and antivirals, for treatment of diseases by inhibiting the actions of particular genes.

The antisense molecule using the nucleotide 0 (nucleoside) analogue of the present invention can be formulated into parenteral preparations or liposome preparations by incorporating customary auxiliaries such as buffers and/or stabilizers. As preparations for topical application, it may be blended with pharmaceutical carriers in common use to prepare ointments, creams, liquids or plasters.

Synthesis of the nucleoside analogue and nucleotide analogue of the present invention will be described in more detail by way of the following Examples and Production Examples. In these Examples, uracil is mainly used as a base, but other purine nucleic acid bases can also be used 14 similarly.

[Example 1] Synthesis of nucleoside analogue Synthesis of 2',3'-O-cyclohexylidene-4'-(ptoluenesulfonyloxymethyl)uridine (Compound 2) To an anhydrous pyridine solution (13.5 ml) of Compound 1 (956 mg, 2.70 mmols) known in the literature, ptoluenesulfonyl chloride (771 mg, 4.05 mmols) was added at room temperature in a stream of nitrogen, and the mixture was stirred for 5 hours at 60 0

C.

To the reaction mixture, a saturated sodium bicarbonate solution was added, whereafter the reaction system was extracted with benzene 3 times. The organic phase was washed once with a saturated sodium chloride Ssolution, and dried over anhydrous MgSO 4 The solvents were distilled off under reduced pressure, and the residue was subjected to azeotropy with benzene 3 times. The resulting crude product was purified by silica gel column chromatography (CHC1 3 :MeOH 15:1), and then reprecipitated from benzene-hexane to obtain a white powder (Compound 2) (808 mg, 1.59 mmols, 59%).

Compound 2: White powder, m.p. 104-106 0 C (benzenehexane). IR v (KBr): 3326, 2929, 2850, 1628, 1577, 1544, 1437, 1311, 1244 cm 'H-NMR (d 6 -acetone): 6 1.45-1.67 2.45 (3H, 3.71 (2H, ABq, J 12 Hz), 4.20 (2H, ABq, J 11 Hz), 4.92 (1H, d, J' 6 Hz), 5.05, 5.06 (1H, dd, J 4.6 Hz), 5.60 (1H, d, J 7 Hz), 5.75 (1H, d, J 4 Hz), 7.48 (2H, d, J 8 Hz), 7.77 (1H, d, J 8 Hz), 7.81 (2H, d, J 8 Hz), 10.10 (1H, 3 C-NMR (d 6 -acetone): 6 21.5, 24.1, 15 24.5, 25.5, 34.8, 36.9, 63.5, 69.7, 82.5, 84.7, 87.8, 92.9, 102.9, 115.4, 128.8, 130.8, 133.9, 142.7, 145.9, 151.3, 163.5. Mass m/z 481 (M'-H 2

O).

Anal. Calcd. for C 23

H

28

N

2 0,S-1/3 H 2 0: C, 53.69; H, 5.61; N, 5.44; S, 6.22. Found: C, 53.99;H, 5.48;N, 5.42;S, 6.10.

Synthesis of 4'-(p-toluenesulfonyloxymethyl)uridine (Compound 3) The above compound 2 (107 mg, 0.21 mmol) was stirred in TFA-H 2 0 (98:2, 1 ml) for 10 minutes at room temperature.

The reaction mixture was distilled off under reduced pressure, and EtOH was added to the residue, followed by performing azeotropy 3 times. The resulting crude product was purified by silica gel column chromatography (CHCl 3 :MeOH 10:1) to obtain Compound 3 (85.0 mg, 0.20 mmol, 94%).

Compound 3: White powder, m.p. 119-120 0 C. IR v (KBr): 3227, 3060, 2932, 2837, 1709, 1508, 1464, 1252, 978, 835, 763, 556 cm-. H-NMR (d 6 -acetone): 6 2.31 (3H, 2.84 (3H, 3.71 (2H, 4.13, 4.20 (2H, ABq, J 11 Hz), 4.28, 4.31 (1H, dd, J' 9.6 Hz), 4.36 (1H, d, J' 6 Hz), 5.54 (1H, d, J' 8 Hz), 5.75 (1H, d, J 7 Hz), 7.32 (2H, d, J i 8 Hz), 7.67 (2H, d, J 8 Hz), 7.70 (1H, d, J' 8 Hz), 10.14 (1H, 1 3 C-NMR (d 6 -acetone): 6 21.5, 63.7, 70.8, 72.7, 74.6, 86.8, 88.8, 103.1, 128.8, 130.7, 133.9, 141.7, 145.8, 151.8, 163.9. Mass m/z 256 (M'-OTs).

Synthesis of 2',3'-0-benzylidene-4'-(ptoluenesulfonyloxymethyl)uridine (Compound 4) In a stream of nitrogen, benzaldehyde (2.4 ml, excess) and zinc chloride (670 mg, 5.0 mmols) were added to 16 the above compound 3 (400 mg, 0.93 mmols), and the mixture was stirred for 5 hours at room temperature. After the reaction was stopped by addition of a saturated sodium bicarbonate solution, the reaction mixture was extracted with chloroform, and washed with a saturated sodium bicarbonate solution, water, and a saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate. The solvents were distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (CHCl 3 :MeOH 40:1) to obtain Compound 4 (380 mg, 0.74 mmol, Compound 4: White powder, m.p. 99-102 0 C (CH 2 C1 2 hexane). [a]D 2 3 -26.7 0 (c 1.0, CHC1 3 IR v (KBr): 3059, 1691, 1460, 1362, 1269, 1218, 1177 'H-NMR (CDC1 3 6 2.41 (3H, 3.25 (1H, br), 3.79 (2H, 4.19 (2H, s), 5.09 (1H, d, J 7 Hz), 5.28 (1H, dd, J 3.7 Hz), 5.60 (1H, d, J 4 Hz), 5.73 (1H, d, J 8 Hz), 5.94 (1H, 7.24 (1H, d, J 8 Hz), 7.38 (2H, d, J 9 Hz), 7.42 (5H, br), 7.69 (2H, d, J 9 Hz), 9.11 (1H, br). 13 C-NMR (CDCL 3 6 21.6, 63.5, 68.3, 77.2, 82.8, 84.2, 87.7, 94.9, 102.6, 107.5, 126.5, 127.9, 128.5, 129.7, 132.2, 135.0, 143.0, 145.0, 150.4, 163.5.

Anal. Calcd. for C 24

H

24

N

2 0OS1/3 H 2 0: C, 55.17; H, 4.76; N, 5.36; S, 6.14. Found: C, 55.19;H, 4.66;N, 5.29;S, 5.98.

Synthesis of 3'-O-benzyl-4'-(ptoluenesulfonyloxymethyl)uridine (Compound To an acetonitrile solution (3 ml) of Compound 4 (150 mg, 0.29 mmol), sodium borocyanohydride (92 mg, 1.5 mmols) 17 was added at room temperature in a stream of nitrogen. Then, titanium tetrachloride (0.16 ml, 1.5 mmols) was added dropwise under cooling with ice, and the mixture was stirred for 15 hours at room temperature. The reaction mixture was diluted with chloroform, and washed with a saturated sodium bicarbonate solution, water, and a saturated sodium chloride solution. Then, the organic phase was dried over anhydrous sodium sulfate. After the solvents were distilled off, the residue was purified by silica gel column chromatography (CHCl 3 :MeOH 25:1) to obtain Compound 5 (112 mg, 0.22 mmol, 0* Compound 5: Colorless crystals. m.p. 195-197 0

C

(AcOEt-hexane). [a]D 23 -14.60 (c 1.0, CHCl 3 IR v (KBr): 3033, 2885, 2820, 1726, 1470, 1361, 1274, 1175, 1119 cm-.

1 S: 15 'H-NMR (CDC3) 6: 2.40 (3H, 3.59-3.77 (3H, 4.10, 4.24 (2H, AB, J 11 Hz), 4.32 (1H, d, J 6 Hz), 4.56 (2H, 4.69 (1H, d, J 11 Hz), 5.52 (1H, d, J 6 Hz), 5.67 (1H, d, J 8 Hz), 7.24-7.29 (7H, 7.48 (1H, d, J 8 Hz),

C

7.70 (2H, d, J 9 Hz), 9.91 (1H, 3 C-NMR (CDCl 3 6 21.6, 63.2, 69.2, 73.6, 74.6, 78.1, 86.6, 92.9, 102.5, 127.9, 128.2, 128.3, 128.6, 129.9, 132.3, 136.9, 142.4, 145.2, 0 150.7, 163.8.

Anal. Calcd. for C 24

H

26

N

2 0,S: C, 55.59; H, 5.05; N, 5.40; S, 6.18. Found: C, 55.41;H, 5.02;N, 5.32;S, 6.15.

Synthesis of 3'-O-benzyl-2'-O, 4'-C-methyleneuridine (Compound 6) To an anhydrous THF solution (1.5 ml) of Compound mg, 0.16 mmol), an anhydrous benzene suspension (0.7 ml) 18 of NaHMDS (3.2 mmols) was added at room temperature in a stream of nitrogen, and the mixture was stirred for 20 hours at room temperature. A saturated sodium bicarbonate solution was added to the reaction mixture, followed by extracting the mixture with CHC13. The organic phase was washed with a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. After the solvents were distilled off under reduced pressure, the resulting crude product was purified by silica gel column chromatography (CHC1 3 :MeOH 10:1), and then recrystallized from MeOH to obtain Compound 6 (41 mg, 0.10 mmol, 61%).

Compound 6: Colorless crystals. m.p. 217-219 0

C

(MeOH). []D 23 +108.4 0 (c 0.3, MeOH). IR v (KBr): 3059, 2951, 1688, 1459, 1271, 1053 cm- 1 'H-NMR (d 6 -DMSO) 6: 3.75, 3.85 (2H, AB, J 8 Hz), 3.77 (2H, d, J 5 Hz), 3.92 (1H, 4.44 (1H, 4.60 (2H, 5.39 (1H, t, J 5 Hz), 5.48 (1H, 7.31 (5H, 7.72 (1H, d, J 8 Hz), 11.37 (1H, s).

13 C-NMR (d 6 -DMSO) 6: 56.0, 71.1, 71.6, 75.8, 76.5, 86.5, 88.3, 100.9, 127.4, 127.6, 128.2, 137.9, 139.0, 150.0, 163.3.

Mass m/z 346 1.1).

Anal. Calcd. for C 1

H,,N

2 0 6 C, 58.96; H, 5.24; N, 8.09.

Found: C, 58.67;H, 5.23;N, 8.05.

Synthesis of 2'-O,4'-C-methyleneuridine (Compound 7) To a methanol solution (2.5 ml) of Compound 6 (25 mg, 0.072 mmol), 10% Pd-C (25 mg) was added, and the mixture was stirred for 15 hours at atmospheric pressure in a stream of hydrogen. The reaction mixture was filtered, and the solvent was distilled off. Then, the residue was purified 19 by silica gel column chromatography (CHCl 3 :MeOH 10:1, then 5:1) to obtain Compound 7 (18.3 mg, quant.).

Compound 7: Colorless crystals. m.p. 239-243 0

C

(MeOH). [n]D 23 +92.2 0 (c 0.3, MeOH). IR v (KBr): 3331, 3091, 3059, 2961, 1689, 1463, 1272, 1049 cm" 1 H-NMR (CD 3

OD)

6: 3.76, 3.96 (2H, AB, J 8 Hz), 3.90 (2H, 4.04 (1H, 4.28 (1H, 5.55 (1H, 5.69 (1H, d, J 8 Hz), 7.88 (1H, d, J 8 Hz).

Anal. Calcd. for C, 1 0

H

2

N

2 0 6 C, 46.88; H, 4.72; N, 10.93. Found: C, 46.74;H, 4.70;N, 10.84.

5'-O-(4,4'-dimethoxytrityl)-2'-0,4'-Cmethyleneuridine (Compound 8) To Compound 7 (140 mg, 0.53 mmol), anhydrous pyridine was added, followed by performing azeotropy of the mixture 3 15 times. Then, the product was converted into an anhydrous pyridine solution (1.5 ml), and 4,4'-dimethoxytrityl *999 *chloride (210 mg, 0.63 mmol) and DMAP (6.5 mg, 0.053 mmol) were added at room temperature in a stream of nitrogen. The mixture was stirred for 5 hours at room temperature. To the reaction mixture, a saturated sodium bicarbonate solution sees was added, followed by extraction with CH 2 Cl,. The organic phase was washed with water and a,saturated sodium chloride solution, and then dried over anhydrous sodium sulfate.

After the solvents were distilled off under reduced pressure, the resulting crude product was purified by silica gel column chromatography (CHCl 3 :MeOH 40:1) to obtain Compound 8 (230 mg, 0.34 mmol, 66%).

Compound 8: White powder. m.p. 117-120°C (CHC1,).

20 [a]D 23 +17.20 (c 1.0, CHCl 3 IR v (KBr): 3393, 3101, 2885, 1689, 1464, 1272, 1047 cm- 1 'H-NMR (CDC1 3 6: 2.59 (1H, br), 3.56 (2H, q, J 7, 11 Hz), 3.87 (1H, d, J 7 Hz), 4.26 (1H, 4.47 (1H, 5.60 (1H, d, J 9 Hz), 5.63 (1H, 5.84 (4H, d, J 9 Hz), 7.22-7.45 (9H, 7.93 (1H, d, J 9 Hz).

[Example 2] Synthesis of nucleoside analogue Synthesis of methyl=5-O-(t-butyldiphenylsilyl)-4hydroxymethyl-2,3-O-isopropylidene-3-D-ribofuranoside (Compound 14) In a stream of nitrogen, Et 3 N (2.62 ml, 18.8 mmols) and t-butyldiphenylsilyl chloride (4.88 ml, 18.8 mmols) were added to an anhydrous CH 2 C1 2 solution (40 ml) of Compound 13 (2.00 g, 8.54 mmols) known in the literature under cooling with ice, and the mixture was stirred for 13 hours at room S 15 temperature. To the reaction mixture, a saturated sodium bicarbonate solution was added, whereafter the reaction S* system was extracted with AcOEt 3 times. The organic phase was washed once with a saturated sodium chloride solution, and then dried over anhydrous Na 2

SO

4 The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane:AcOEt 5:1) to obtain colorless oily matter (Compound 14) (2.82 g, 5.98 mmols, [aC]D"-16.20 (c 0.52, CHC13). IR v (KBr): 3510, 3061, 2938, 2852, 1465, 1103 cm 1 'H-NMR (CDC1 3 6: 1.09 (9H, 1.28 (3H, 1.49 (3H, s), 3.22 (3H, 3.67, 3.76 (2H, AB, J 11 Hz), 3.88, 3.93 (2H, AB, J 11 Hz), 4.49 (1H, d, J 6 Hz), 4.57 (1H, d, J 6 21 Hz), 4.93 (1H, 7.38 7.43 (6H, 7.67 (4H, d, J 7 Hz).

13 C-NMR (CDC13) 60: 19.2, 24.4, 25.9, 26.9, 55.0, 62.9, 64.8, 82.2, 85.9, 88.7, 108.6, 112.6, 127.8, 129.9, 133.0, 135.7.

Anal. Calcd. for C 26

H

3 6 OSi'1/4 H20: C, 65.45; H, 7.71. Found: C, 65.43; H, 7.59.

Synthesis of methyl=5-O-(t-butyldiphenylsilyl)-2,3-Oisopropylidene-4-(p-toluenesulfonyloxymethyl)-|ribofuranoside (Compound In a stream of nitrogen, Et3N (3.92 g, 28.0 mmols), p-toluenesulfonyl chloride (1.34 g, 7.22 mmols), and 4dimethylaminopyridine (90 mg, 0.72 mmol) were added to an anhydrous CH 2 Cl 2 solution (15 ml) of Compound 14 (2.13 g, 4.51 mmols), and the mixture was stirred for 17 hours at 15 room temperature. To the reaction mixture, a saturated sodium bicarbonate solution was added, whereafter the reaction system was extracted with AcOEt 3 times. The organic phase was washed once with a saturated sodium chloride solution, and then dried over anhydrous Na 2

SO

4 The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane:AcOEt 10:1) to obtain colorless oily matter, Compound 15 (2.76 g, 4.42 mmols, [a]D 17 3.820 (c 0.56, CHCl 3 IR v (KBr): 2934, 2852, 1369, 1104 cm 1 'H-NMR (CDC1 3 8: 1.02 (9H, 1.20 (3H, 1.32 (3H, s), 2.41 (3H, 3.09 (3H, 3.51, 3.77 (2H, AB, J 10 Hz), 4.34 (1H, d, J 6 Hz), 4.25, 4.39 (2H, AB, J 9 Hz), 4.47 22 (1H, d, J =6 Hz), 4.77 (1H, 7.28, 7.81 (4H, AB, J 9 Hz), 7.39 -7.44 (6H, in), 7.62 7.65 (4H, mn), 7.81 (2H, d, J 9 Hz).

1 3 C -NMR (CDCl 3 8c 19.2, 21.6, 24.5, 25.8, 26.8, 54.9, 62.7, 68.8, 81.9, 85.6, 87.5, 108.7, 112.8, 127.7, 127.8, 128.2, 129.6, 129.9, 132.9, 135.6, 144.4.

Anal. Calod. for C 33

H

42 0,SS1: C, 63.23; H, 6.75; S, 5.11.

Found: C, 62.99; H, 6.53; S, 5.13.

Synthesis of methyl=5-O-(t-butyldiphenylsilyl)- 4 toluenesulfonyloxymfethyl) -Ip-D-ribofuranoside (Compound 16) Trifluoroacetic acid (14 ml) was added to a THF-H 2 0 ml, 8:3 (vlv)] solution of Compound 15 (645 mg, 1.03 inmol at room temperature, and the mixture was stirred f or 20 min Le: 15 utes at room temperature. The solvents were distilled off un der reduced pressure, and the resulting crude product was pur if ied by silica gel column chromatography (hexane:AcOEt 1) to obtain colorless oily matter, Compound 16 (464 mg, 0.79 inmol, [cj'-35. 8 0 90, CHCl 3 IR v (KBr) :3499, 3051, 2931, 2840, 1594, 1468,1362, llO9cm- 1 'H-NMR (CDCl 3 )8 1.02(9H,s) 2.42(3H,s) 3.16(3H,s) 3.54, 3.70(2H,AB,J=1OHz), 3.97(1H,d,J=5Hz), 4.18(1H,d,J=5Hz), 4.26, 4.39(2H,AB,J=1OHz) 4.73(1H,s), 7.30(2H,d,J=8Hz), 7.36-7.44 (6H,in), 7 .5 9 -7.66(4H,m),7.78(2H,d,J=8Hz). 13 C-NMR (CDC1 3 k: 19.2, 21.6, 26.7, 55.2, 66.5, 69.6, 74.0, 75.2, 76.5, 84.8, 107.5, 127.7, 128.0, 129.8, 132.6, 132.7, 132.8, 135.5, 135.6, 144.9.

Anal. Calcd for C 3 0

H

3 SSiO1-1/4

H

2 0:C,60.94; H,6.56.Fould:C, 23 60.94; H,6.43.

Synthesis of methyl=5-O-(t-butyldiphenylsilyl)-2-0, 4 C-methylene-P-D-ribofuranoside (Compound 17) and methyl=5-O-(t-butyldiphenylsilyl)-3-0,4-C-methylene- (-D-ribofuranoside (Compound 18) In a stream of nitrogen, a benzene suspension (1.6 ml) of NaHMDS (3.30 mmols) was added to an anhydrous THF solution (4 ml) of Compound 16 (194 mg, 0.33 mmol) at room temperature, and the mixture was stirred for 1 hour at room temperature. After a saturated sodium bicarbonate solution was added to the reaction mixture, the reaction solvents were distilled off, and the residue was extracted with AcOEt 3 times. The organic phase was washed once with a saturated sodium chloride solution, and then dried over anhydrous 9 15 Na 2

SO

4 The solvent was distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane:AcOEt 5:1) to obtain colorless oily matter, Compound 17 (48 mg, 0.116 mmol, S. and colorless oily matter, Compound 18 (59 mg, 0.142 mmol, 43%).

Compound 17: IR v (KBr):3438, 3064, 1103, 1036cm 1 1 H-NMR (CDCl 3 1.08(9H,s), 2.04(1H,br 3.39(3H,s), 3.65, 3.98(2H,AB,J=8Hz), 3.95,4.02(2H,AB,J=12Hz), 4.02(1H,s), 4.30 4.79(1H,s), 7.38-7.46(6H,m), 7.65-7.69(4H,m).

3 C-NMR (CDC13) 68: 19.2, 26.7, 55.0, 60.7, 71.2, 73.1, 79.9, 8 104.3, 127.8, 129.9, 130.0, 132.9, 135.6, 135.7.

Anal.Calcd for C 23

H

30 0 5 Si'1/4 H 2 0:C,65.68; H,7.34.Found:C,65.9 8; H,7.23.

24 Compound 18: IR v (KBr):3456, 3058, 2938, 2852, 1467, 1108cm 1 1 H-NMR (CDC1 3 )6 :1.10(9H,s), 3.26(3H,s), 3.71(2H,s), 4.02(1H, d,J=6Hz), 4.35,4.95(2H,d,J=7Hz), 5.01(1H,s), 5.11(1H,d,J=6H 7.38-7.44(6H,m), 7.66(4H,d,J=7Hz).

1 3 C-NMR(CDC1 3 19.3, 26.8, 55.4, 63.7, 75.1, 77.9, 84.5, 86.3, 111.9, 127.8, 128.0, 129.9, 132.9, 133.0, 135.6, 135.8, 135.9.

Anal.Calcd for C 23

H

0 0oSi'1/4 H 2 0:C,65.91; H,7.34.Found:C, 66.07; H,7.14.

Synthesis of methyl=3-0-acetyl-5-O-(tbutyldiphenylsilyl)-2-0,4-C-methylene-p-Dribofuranoside (Compound 19) In a stream of nitrogen, acetic anhydride (0.38 ml, 15 4.08 mmols) and 4-dimethylaminopyridine (21 mg, 0.170 mmols) were added to an anhydrous pyridine solution (10 ml) of Compound 17 (704 mg, 1.70 mmols) at room temperature, and the mixture was stirred for 3 hours at room temperature.

After a saturated sodium bicarbonate solution was added to the reaction mixture, the system was extracted with AcOEt 3 times. The organic phase was washed once with a saturated sodium chloride solution, and then dried over anhydrous Na 2

SO

4 The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane:AcOEt 7:1) to obtain colorless oily matter, Compound 19 (665 mg, 1.46 mmols, 86%).

[a]D 17 -34.3 0 (c=0.93,CHC1 3 IR v (KBr):3438, 3064, 2934, 1749, 25 1468, 1103, 1036cm 1 1 H-NMR (CDCl 3 0.99(9H,s), 1.97(3H,s), 3.34(3H,s), 3.69, 3.86(2H,AB,J=8Hz), 3.86(2H,s), 4.17(1H,s), 4.77(1H,s), 5.06 7.28-7.39(6H,m), 7.58-7.63(4H,m).

13C-NMR(CDC13)8 19.3, 20.9, 26.7, 55.0, 60.3, 72.0, 73.6, 78.3, 85.3, 104.4, 127.7, 129.8, 133.0, 135.6, 169.8.

Anal.Calcd for C 25

H

32 OSi'1/4 H 2 0:C,65.12; H,7.10. Found:C, 65.27;H,7.00.

Synthesis of 5'-O-(t-butyldiphenylsilyl)-2'-O,4'-Cmethylene-5-methyluridine (Compound In a stream of nitrogen, 0,0'bistrimethylsilylthymine (154 mg, 0.598 mmols) was added to an anhydrous CH 3 CN solution (2 ml) of Compound 19 (109.2 g, 0.239 mmol) at room temperature. Then, a 1,1-dichloroethane (0.31 ml) solution of trimethylsilyltrifluoromethane sulfonate (0.82 ml, 8.74 mmols) was added under cooling with ice, and the mixture was stirred for 18 hours at room temperature. The reaction mixture was diluted with CHCl1, and a saturated sodium bicarbonate solution was added, followed by extracting the system with AcOEt 3 times. The organic phase was washed once with a saturated sodium chloride solution, and then dried over anhydrous Na 2

SO

4 The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane:AcOEt 3:1) to obtain colorless oily matter, Compound 20 (87.7 mg, 0.173 mmol, IR v (KBr):3048, 2935, 2852, 1749, 1466, 1369, 1234, 1108, 1040 cm 26 1 H-NMR (CDC1 3 1.06(9H,s), 1.94(3H,s), 2.98(1H,br 3.63, 4.00(2H,AB,J=10Hz), 3.72(1H,d,J=7Hz), 3.82-3.84(2H,m), 4.30 5.25(1H,s), 7.40-7.46(6H,m), 7.60(4H,d,J=6Hz), 7.66 9.68(1H,br s).

[Example 3] Synthesis of nucleoside analogue (different method) Synthesis of 3-O-benzyl-5-O-t-butyldiphenylsilyl- 4 (hydroxymethyl)-1,2-O-isopropylidene-a-Derythropentofuranose (Compound 32) In a stream of nitrogen, triethylamine (3.71 ml, 26.6 mmols) and t-butyldiphenylsilyl chloride (6.94 ml, 26.7 mmols) were added, under cooling with ice, to a methylene chloride solution (50 ml) of Compound 31 (2.50 g, 8.08 mmols) prepared in accordance with the aforementioned reference The mixture was stirred for 10.5 hours at S. room temperature. After a saturated sodium bicarbonate solution was added to the reaction mixture, the system was extracted with ethyl acetate. The organic phase was washed with a saturated sodium chloride solution, and then dried 20 over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane: 1:4 1:3) to obtain a white solid, Compound 32 (2.97 g, 5.41 mmols, 67%).

m.p. 98-990C (hexane). [C]D 2 0 +54.8 0 (c 1.12, acetone).

IR v max (KBr) 3553, 2936, 1463, 1379, 1107 cm- 1 'H-NMR(CDC1 3 1.13 (9H, 1.50 (3H, 1.78 (3H, 2.56 (1H, t, J 7 Hz), 3.82, 3.92 (2H, AB, J 11 Hz), 3.94 (2H, t, 27 J 6 Hz), 4.57 (1H, d, J 5 Hz), 4.64, 4.95 (2H, AB, J 12 Hz), 4.83 (1H, dd, J 4, 5 Hz), 5.95 (1H, d, J 4 Hz), 7.44- 7.55 (11H, 7.72-7.78 (4H, 1 C-NMR(CDCl 3 )5 0 19.2, 26.2, 26.5, 26.8, 63.2, 65.4, 72.5, 77.9, 79.1, 87.4, 104.4, 113.7, 127.6, 127.7, 128.0, 128.5, 129.5, 129.7, 132.9, 133.1, 134.7, 135.5, 137.2.

Anal. Calcd for C 32

H

40 06Si C, 70.04; H, 7.38. Found C, 70.19; H, 7.35.

Synthesis of 3-O-benzyl-5-O-(t-butyldiphenylsilyl)-4- (p-toluenesulfonyloxymethyl)-1,2-a-Derythropentofuranose (Compound 33) In a stream of nitrogen, triethylamine (395 p1, 2.83 mmols), p-toluenesulfonyl chloride (139.2 mg, 0.730 mmol), 'and 4-dimethylaminopyridine (8.92 mg, 0.0730 mmols) were added, under cooling with ice, to a methylene chloride solution of Compound 32 (250 mg, 0.456 mmol). The mixture was stirred for 15.5 hours at room temperature. After a saturated sodium bicarbonate solution was added to the reaction mixture, the system was extracted with ethyl acetate. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate.

S

The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane: 1:6) to obtain light yellow oily matter, Compound 33 (310.6 mg, 0.442 mmol, 97%).

20+16.00 (c 0.44, acetone). IR v max (KBr) 2935, 1595, 1462, 1363, 1174, 1106 cm-.

'H-NMR(CDC13)8: 1.08 (9H, 1.40 (3H, 1.46 (3H, 2.48 28 (3H, 3.68, 3.83 (2H, AB, J 11 Hz), 4.45 (2H, dd, J 4, Hz), 4.64, 4.81 (2H, AB, J 12 Hz), 4.68 (1H, dd, J 4, Hz), 5.81 (1H, d, J 4 Hz), 7.32 (2H, d, J 8 Hz), 7.42-7.72 7.82, (2H, d, J 8 Hz), 7.66 (4H, 7.72 (2H, d, J 8 Hz).

3 C-NMR(CDC13)68: 19.1, 21.5, 26.1, 26.4, 26.7, 64.4,70.0, 72.5, 78.1, 78.9, 85.4, 104.2, 113.6, 127.3, 127.7,127.9, 128.0, 128.4, 129.6, 129.7, 129.8, 132.7, 132.8, 135.5, 137.2, 144.4. MS(EI) m/z 646 High-MS(EI): Calcd for C3sH 37 O0SSi 645.1978, Found 645.1969.

Synthesis of 1,2-di-O-acetyl-3-O-benzyl-5-O-t- S butyldiphenylsilyl-4-(p-toluenesulfonyloxymethyl)-aand -p-D-ribofuranose (Compound 34) S"In a stream of nitrogen, acetic anhydride (6.0 ml, 63.6 mmols) and concentrated sulfuric acid (56 1.10 mol) were added to an acetic acid solution (56 ml) of Compound 34 (3.70 g, 5.27 mmols). The mixture was stirred for 2 hours at room temperature. The reaction mixture was emptied into iced water (300 ml), and stirred for 30 minutes.

After a saturated sodium chloride solution was added, the mixture was extracted with ethyl acetate. Then, the organic phase was dried over magnesium sulfate. The solvents were distilled off, and the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane, 2:1) to obtain yellow oily matter, Compound 34 (3.36 g, 4.53 mmols, as an a-3 mixture.

IR v max (KBr) 2934, 2863, 1751, 1365, 1217, 1106 cm 1 H-NMR (CDC13) [P-configuration] 6: 1.02 (9H, 1.77 (3H, s), 29 1.98 (3H, 2.39 (3H, 3.61, 3.76 (2H, AB, J 11 Hz), 4.21-4.58 (5H, 5.26 (1H, d, J 5 Hz), 5.94 (1H, 7.15- 7.59 (13H, 7.58-7.66 (4H, 7.72 (2H, d, J 8 Hz). [aconfiguration] d: 1.02 (9H, 1.98 (3H, 2.36 (3H, s), 3.48, 3.58 (2H, AB, J 11 Hz), 4.21-4.58 (5H, 5.12 (1H, dd, J 5, 6 Hz), 6.33 (1H, d, J 5 Hz), 7.15-7.59 (13H, m), 7.58-7.66 (4H, 7.72 (2H, d, J 8 Hz).

13 C-NMR (CDCl 3 6c: 14.2, 19.3, 20.5, 20.8, 21.6, 26.7, 26.8, 60.3, 64.8, 69.1, 73.6, 74.1, 78.6, 85.3, 97.4, 127.4, 127.6, 127.7, 127.8, 127.9, 128.0, 128.2, 128.3, 128.4, 129.5, 129.6, 1289.8, 129.9, 132.4, 132.8, 132.9, 135.4, 135.5, 135.6, 136.9, 144.5, 168.7, 169.4. High-MS(FAB) Calcd for C 40

H

46

N

2

,O

1 SSiNa 769.2479, Found 769.2484.

Synthesis of 2'-O-acetyl-3'-O-benzyl-5'-O-tmethyluridine (Compound In a stream of nitrogen, 2TMS-T (1.04 g, 4.03 mmols) and trimethylsilyltrifluoromethane sulfonate (730 pl, 4.03 immols) were added, under cooling with ice, to a 1,2dichloroethane solution (26 ml) of Compound 34 (1.88 g, 2.52 mmols), and the mixture was stirred for 17 hours at room temperature. A saturated sodium bicarbonate solution was added to the reaction mixture, and the system was filtered through Celite, followed by extracting the mother liquor with chloroform. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was 30 purified by silica gel column chromatography (AcOEt-hexane, 2:3) to obtain a white powder, Compound 35 (2.00 g, 2.44 mmols, 97%).

m.p. 70-71.5 0 C. [a]D 2 4 +4.58 0 (c 1.25, acetone).

IR v max (KBr) 3059, 2934, 1694, 1465, 1368, 704 cm- 1 1 H-NMR(CDC13)8: 1.18 (9H, 1.63 (3H, d, J 1 Hz), 2.10 (3H, 2.42 (3H, 3.73, 3.86 (2H, AB, J 11 Hz), 4.12, 4.20 (2H, AB, J 11 Hz), 4.44, 4.57 (2H, AB, J 11 Hz), 4.45 (1H, d, J 6 Hz), 5.38 (1H, t, J 6 Hz), 6.02 (1H, d, J 6 Hz), 7.21-7.60 (13H, 7.62-7.69 (7H, 8.91 (1H, br s).

13 C-NMR(CDC1 3 11.9, 19.3, 20.6, 21.6, 27.0, 65.3, 68.6, 74.1, 74.8, 77.2, 77.3, 86.0, 86.4, 111.6, 127.9, 128.0, *ft 128.2, 128.5, 129.7, 130.1, 130.2, 131.8, 132.3, 132.5, 135.3, 135.5, 135.6, 136.8, 144.9, 150.2, 163.4, 170.2. MS (FAB) m/z 813 Anal. Calcd for C 43

H

48

N

2 O,SSi'2HO2: C, 60.83; H, 6.17; N, 3.30.

Found C, 60.55; H, 5.78; N, 3.22.

Synthesis of (Compound 36) Potassium carbonate (12.75 mg, 0.0923 mmol) and water ml) were added, under cooling with ice, to a methyl alcohol solution (4 ml) of Compound 35 (250 mg, 0.308 mmol), and the mixture was stirred for 22 hours at room temperature.

Under cooling with ice, acetic acid was added to the reaction mixture to neutralize it, whereafter the solvent was distilled off under reduced pressure. After water was added to the residue, the mixture was extracted with ethyl 31 acetate. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate.

The solvent was distilled off under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane, 3:2) to obtain a white powder, Compound 36 (216.7 mg, 0.283 mmol, 92%).

mp. 74 77 0 C. [a]D 23 5.150 (c 1.23, CHCl 3 IR v max (KBr) 3048, 2934, 1695, 1363, 1181, 1108, 977, 819, 704 cm 1 'H-NMR (CDCl 3 d: 1.05 (9H, 1.65 (3H, d, J 1 Hz), 2.39 (3H, 3.04 (1H, br d, J 9 Hz), 3.72 (2H, 4.17 (2H, s), 4.18 (1H, d, J 5 Hz), 4.24-4.32 (1H, 4.54, 4.62 (2H, AB, J 11 Hz), 5.62 (1H, d, J 6 Hz), 7.19-7.69 (20H, 8.46 (1H, br s).

13 C-NMR (CDC13) 6c: 12.1,19.4, 26.9, 58.8, 72.0, 72.2, 75.8, S 15 76.7, 87.4, 88.8, 110.4, 127.7, 12.79, 128.1, 128.2, 128.5, 128.7, 129.8, 130.0, 130.1, 132.2, 134.3, 135.3, 135.5, 136.8, 149.8, 163.9. MS(FAB) m/z 771 Anal. Calcd for C 41

H

4

,N

2 OSSi: C, 63.41; H, 6.16; N, 3.51; S, 3.95.

Found C, 63.87; H, 6.01; N, 3.63; S, 4.16.

Synthesis of 2'-O,4'-C-methylene-5-methyluridine (Compound 37) In a stream of nitrogen, sodium bis(trimethylsilyl)amide (1.0 M in THF, 8.47 ml, 8.47 mmols) was added, under cooling with ice, to a tetrahydrofuran solution (30 ml) of Compound 36 (1.86 g, 2.42 mmols), and the mixture was stirred for 1 hour at room temperature. A saturated sodium bicarbonate solution (14 ml) was added to 32 the reaction mixture, and then the solvent was distilled off under reduced pressure. After water was added to the residue, the mixture was extracted with chloroform. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane, 2:3) to obtain a white powder, Compound 37 (1.42 g, 2.37 mmols, 98%).

m.p. 70.5-72 0 C. 22 +52.47 0 (c 1.025, acetone). IR v max (KB r) 2936, 1694, 1465, 1275, 1106, 1055, 809, 704cm- 1 'H-NMR(CDCl 3 1.21 (9H, 1.76 (3H, 3.88, 4.07(2H, AB, J 8 Hz), 4.07, 4.15 (2H, AB, J 11 Hz), 4.16 (1H, 4.66, 4.80 (2H, AB, J 11 Hz), 4.76 (1H, 7.34-7.79 (16H, m), 10.0 (1H, br MS (FAB) m/z 599 Anal. Calcd for C 34

H

3

,N

2 OSi'2H20: C, 64.33; H, 6.03; N, 4.41.

Found C, 64.58; H, 6.15; N, 4.28.

Synthesis of 3'-O-benzyl-2'-O,4'-C-methylene-5methyluridine (Compound 38) In a stream of nitrogen, tetrabutylammonium fluoride M in THF, 379 0.379 Vmol) was added to a tetrahydrofuran solution (1 ml) of Compound 37 (188.7 mg, 0.316 mmol), and the mixture was stirred for 2.5 hours at room temperature. The reaction mixture was distilled under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (AcOEt-hexane, to obtain a white powder, Compound 38 (94.6 mg, 0.262 mmol, 83%).

33 IR v max :3424, 3183, 3063, 2950, 1691, 1463, 1273, 1057, 734cif 1 'H-NMR (CDCl 3 1.90(3H, d, J 1 Hz), 3.83, 4.05(2H, AB, J 8 Hz), 3.93, 4.02(2H, AB, J 12 Hz), 3.94(1H, 4.53(1H, s) 4. 56, 4. 58 (2H, AB, J 12 Hz) 5. 65 (1H, s) 7. 32 (5H, s), 7.44 (1H, d, J 1 Hz) High-MS (El) Calcd for C,,H,,N0 6 :360.1321, Found :360.1312.

Synthesis of (Compound 39a) To a methyl alcohol solution (4 ml) of Compound 38 (86.5 mg, 0.240 inmol), 20% Pd(OH) 2 -C (86.5 mg) was added, and the mixture was stirred for 14.5 hours at atmospheric pressure in a stream of hydrogen. The reaction mixture was filtered, and then the solvent was distilled off under reduced pressure to obtain colorless crystals, Compound 39 (62.5 mg, 0.230 mmol, 96%).

mp. 194-195 0 C. [oj],2o 53.70 (c =1.02, EtOH). IR v max 999 (KBr) 3323, 3163, 3027, 2889, 2826, 1689, 1471, 1276, 1057 1 H-NMR (CD 3 OD) 89 (3H, q, J 1 Hz) 3.74, 3.95 (2H, AB, J 8 Hz), 3.90 (1H, 4.07 (1H, 4.26 (1H, s), 5.53 (1H, s) 7.74 (1H, d, J 1 Hz).

1 3 C -NMR (CD 3 OD) 6c: 12.6, 57.6, 70.3, 72.4, 80.8, 88.3, 90.4, 110.7, 136.8, 151.8, 166.5.

[Example 4] Synthesis of 2'-O-acetyl-3'-O-benzyl-5'-O-tbutyldiphenylsilyl-4 '-p-toluenesulfonyloxymethyl-N 6 benzoyladenosine (Compound 34 In a stream of nitrogen, a 1,2-dichloroethane solution (5.0 ml) of Compound 34 (250 mg, 0.336 mmol) and trimethylsilyltrifluoromethane sulfonate (6.7 Al, 0.0336 mmols) were added, at room temperature, to 2TMS*ABZ (128.7 mg, 0.336 mmol) prepared in accordance with a reference 6) (H.

Vorbrggen, K. Krolikiewicz and B. Bennua, Chem., Ber., 114, 1234-1255 (1981)). The mixture was heated under reflux for 26 hours. After a saturated sodium bicarbonate solution was added to the reaction mixture, the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHC1,-MeOH, 1:3) to obtain a white powder, Compound 40 (234.5 mg, 0.253 mmol, m.p. 77-78 0 C (AcOEt/hexane). [a]D 24 13.2 (c 1.00, CHC 3 1).

IR v max (KBr): 3058, 2934, 1749, 1703, 1606, 1105 cm 1 'H-NMR (CDC1 3 0.99 (9H, 2.04 (3H, 2.38 (3H, 3.7 20 4, 3.85 (2H, AB, J 11 Hz), 4.31, 4.43 (2H, AB, J 11 Hz), 4.

52, 4.58 (2H, AB, J 11 Hz), 4.81 (1H, d, J 6 Hz), 5.94 (1H, d, J 6 Hz), 6.04 (1H, d, J 5 Hz), 7.18 7.61 (20H, m), 7.69 (2H, d, J 8 Hz), 7.99 (1H, 8.01 (2H, d, J 7 Hz), 8.56 (1H, 8.99 (1H, br "C-NMR (CDC13) 6c: 19.1, 20.5, 21.5, 26.7, 64.1, 68.4, 74.0, 74.6, 77.9, 86.57, 86.64, 123.4, 127.7, 127.8, 127.9, 128.1, 128.5, 128.8, 129.6, 129.9, 132.0, 132.3, 132.6, 132.7, 133.5, 135.4, 135.5, 136.8, 142.0, 144.7, 149.6, 151.2, 152.6, 164.5, 169.8. MS(FAB) m/z 926 35 Synthesis of 4'-p-toluenesulfonyloxymethyl-N6-benzoyladenosne (Compound 41) To a methyl alcohol solution (3.0 ml) of Compound (167.9 mg, 0.182 mmol), potassium carbonate (15.0 mg, 0.109 mmol) was added at room temperature, and the mixture was stirred for 15 minute at room temperature. Concentrated hydrochloric acid was added to the reaction mixture to neutralize it, whereafter the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHC 3 1-MeOH, 30:1) to obtain a white powder, Compound 41 (140.5 mg, 0.160 .mmol, 88%).

m.p. 82-83 0 C (AcOEt-hexane). [a]D 2 5 6.020 (c 0.96, CHCl 3 IR v max (KBr) 3306, 3066, 2935, 2859, 1701, 1611 cm- 1 1H-NMR (CDC1 3 6: 0.98 (9H, 2.37 (3H, 3.76 (2H, 4.

20 39, 4.45 (1H, AB, J 11 Hz), 4.54 (1H, d, J 6 Hz), 4.67, 4.

•76 (2H, AB, J 11 Hz), 4.85 (1H, dd, J 5, 6 Hz), 5.79 (1H, d, J 5 Hz), 7.20 7.58 (21H, 7.73 (2H, d, J 8 Hz), 7.80 (1H, 7.96 (2H, d, J 8 Hz), 8.49 (1H, 9.18 (1H, br s).

13C-NMR (CDCl 3 6c: 19.1, 21.6, 26.8, 64.4, 68.9, 74.1, 74.6, 79.2, 86.8, 89.8, 123.1, 127.7, 127.8, 128.0, 128.2, 128.4, 1 28.6, 128.8, 129.7, 130.0, 132.1, 132.5, 132.6, 132.8, 133.4, 135.4, 135.5, 136.8, 142.1, 144.8, 149.4, 152.3, 164.5.

Synthesis of 36 2'-0,4'-C-methylene-N 6 -benzyladenosine (Compound 42) In a stream of nitrogen, sodium bis(trimethylsilyl)amide (1.0 M in THF, 0.58 ml, 0.572 mmol) was added to a tetrahydrofuran solution (8.0 ml) of Compound 41 (210.5 mg, 0.238 mmol) at room temperature, and the mixture was stirred for 3 hours at room temperature. A saturated sodium bicarbonate solution was added to the reaction mixture, and then the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHCl 3 -MeOH, 30:1) to obtain a white powder, Compound 42 (169.5 mg, 0.238 mmol, quant.).

mp. 80- 81 0 C. IR v max (KBr): 3259, 3064, 2932, 2858, 1703, 1607 cm- 1 'H-NMR(CDCl 3 1.07 (9H, 3.95, 4.10 (2H, AB, J 8 Hz), 4.

02 (2H, d, J 8 Hz), 4.56, 4.64 (2H, AB, J 12 Hz), 4.26 (1H, 20 4.86 (1H, 6.14 (1H, 7.26 7.70 (18H, 8.04 (2 H, d, J 7 Hz), 8.22 (1H, 8.78 (1H, 9.18 (1H, brs).

13 C-NMR(CDCl 3 6c: 19.2, 26.5, 26.8, 29.7, 59.2, 72.4, 72.6, 7 76.8, 86.7, 88.6, 123.4, 127.7, 127.8, 127.9, 128.1, 128.4, 128.8, 129.5, 130.0, 132.4, 132.5, 132.8, 133.5, 134.8, 135.2, 135.5, 135.6, 136.8, 140.4, 152.7.

Synthesis of 3'-O-benzyl-2'-0,4'-C-methylene-N 6 benzoyladenosine (Compound 43) Tetrabutylammonium fluoride (1.0 M in THF, 1.0 ml, 37 mmol) was added, at room temperature, to a tetrahydrofuran solution (7.0 ml) of Compound 42 (173.6 mg, 0.244 mmol), and the mixture was stirred for 25 minutes at room temperature. The reaction mixture was distilled under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHC1 3 -MeOH, 15:1) to obtain a white powder, Compound 43 (115.4 mg, 0.244 mmol, quant.).

mp. 154 155 0 C (Et20). IR v max(KBr): 3339, 2944, 1701, 1611 cm 1 H-NMR(CDC1 3 3.91, 4.13 (2H, AB, J 8 Hz), 3.93, 4.01 (2H, i" AB, J 12 Hz), 4.38 (1H, 4.64 (1H, 4.85 (1H, s), 6.08 (1H, 7.29 (1H, 7.51 (2H, d, J 8 Hz), 7.58 (1H, d, J 7 Hz), 8.05 (2H, d, J 7 Hz), 8.14 (1H, 8.75 (1H, 9.50 (1H, br s).

1 3 C-NMR(CDCl 3 )6c: 57.1, 72.4, 77.0, 77.1, 86.9, 88.6, 122.9, 127.6, 128.0, 128.1, 128.4, 128.7, 132.8, 133.5, 136.9, 140.5, 149.8, 150.5, 152.8, 165.0.

[Example 20 Synthesis of 2'-O-acetyl-3'-O-benzyl-5'-O-tbutyldiphenylsilyl-4'-p-toluenesulfonyloxymethyl-N 2 isobutyrylguanosine (Compound 44) In a stream of nitrogen, a 1,2-dichloroethane solution (5.0 ml) of Compound 4 (250 mg, 0.336 mmol) and trimethylsilyltrifluoromethane sulfonate (6.7 tl, 0.0336 mmol) were added, at room temperature, to 3TMS.G iBu (146.8 mg, 0.336 mmol) prepared in accordance with the aforementioned reference The mixture was heated under reflux for 38 hours. After a saturated sodium bicarbonate solution was added to the reaction mixture, the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHCl 3 -MeOH, 30:1) to obtain a white powder, Compound 44 (213.6 mg, 0.235 mmol, m.p. 96-97 0 C (AcOEt-hexane). 2 4 -11.090 (c 0.97, CHCl 3 IR v max (KBr): 3152, 3065, 2934, 1746, 1681, 1606 cm 1 'H-NMR (CDCl 3 d: 0.96 (9H, 1.10 (3H, d, J 9 Hz), 1.13 (3H, d, J 9 Hz), 1.98 (3H, 2.36 (3H, 2.48 (1H, m), 3.65, 3.72 (2H, AB, J 11 Hz), 4.23, 4.43 (2H, AB, J 11 Hz), 4.47 (2H, 4.63 (1H, d, J 6 Hz), 5.74 (1H, t, J 6 Hz), 5.96 1 H d, J 6 Hz), 7.14 7.68 (20H, 9.15 (1H, 12.

(1H, s).

S" 13 C-NMR(CDC1 3 )6c: 19.1, 19.3, 19.4, 20.8, 21.9, 27.0, 27.2, 36.

64.5, 68.9, 74.4, 74.9, 76.7, 86.1, 86.7, 122.0, 127.6, 12 20 7.7, 127.9, 128.1, 128.3, 128.4, 128.8, 130.1, 130.4, 132.3, 132.7, 132.9, 135.7, 135.8, 137.3, 137.8, 145.2, 147.8, 148.5, 156.2, 170.2, 178.8.

Synthesis of 3 4'-p-toluenesulfonyloxymethyl-N 2 -isobutyrylguanosine (Compound To a methyl alcohol solution (3.0 ml) of Compound 44 (137.0 mg, 0.151 mmol), potassium carbonate (15.8 mg, 0.113 mmol) was added at room temperature, and the mixture was 39 stirred for 45 minutes at room temperature. Concentrated hydrochloric acid was added to the reaction mixture to neutralize it, whereafter the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHC1,-MeOH, 30:1) to obtain a white powder, Compound 45 (83.4 mg, 0.097 mmol, 64%).

.mp. 102-103 0 C (AcOEt-hexane). [a]D 25 2.00 (c 0.40, CHCl 3 IR v max(KBr): 3166, 2932, 1684, 1607 cm 1 H-NMR (CDCl 3 6: 0.90 (9H, 1.09 (3H, d, J 7 Hz), 1.13 (3H, d, J 7 Hz), 2.30 (1H, 2.37 (3H, 3.71, 3.76 (2H, 15 AB, J 11 Hz), 4.32, 4.48 (2H, AB, J 11 Hz), 4.35 (1H, d, J 6 Hz), 4.63, 4.90 (2H, AB, J 12 Hz), 4.96 (1H, t, J 6 H 5.67 (1H, d, J 7 Hz), 7.17 7.71 (20H, 8.82 (1H, s), 12.05 (1H, br s).

13 C-NMR(CDCl 3 )6c: 18.7, 19.0, 21.6, 26.5, 36.2, 63.5, 69.1, 20 73.7, 74.3, 78.8, 86.2, 89.5, 127.7, 127.8, 128.0, 128.1, S128.5, 129.7, 130.0, 132.0, 132.6, 132.7, 135.3, 135.4, 137.4, 138.2, 144.8, 146.9, 155.5, 178.5.

Synthesis of 2'-0,4'-C-methylene-N 2 -isobutyrylguanosine (Compound 46) In a stream of nitrogen, sodium bis(trimethylsilyl)amide (1.0 M in THF, 0.31 ml, 0.315 mmol) 40 was added to a tetrahydrofuran solution (3.0 ml) of Compound (92.1 mg, 0.102 mmol) at room temperature, and the mixture was stirred for 3 hours at room temperature. A saturated sodium bicarbonate solution was added to the reaction mixture, and then the system was extracted 3 times with methylene chloride. The organic phase was washed with a saturated sodium chloride solution, and then dried over sodium sulfate. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (CHCl 3 -MeOH, 25:1) to obtain a white powder, Compound 46 (31.4 mg, 0.160 mmol, 44%).

mp. 99-100 0 C. IR v max(KBr): 3162, 3068, 2932, 1683, 1610 cm- 1 IH-NMR(CDCl 3 1.06 (9H, 1.25 (3H, d, J 7 Hz), 1.27 (3H, d, J 7 Hz), 2.64 (1H, 3.83, 4.01 (2H, AB, J 8 Hz), 3.97 (2H, d, J 7 Hz), 4.18 (1H, 4.51 (1H, 4.54 (2H, 9 d, J 2 Hz), 5.77 (1H, 7.17-7.42 (5H, 7.64 7.72 (10H, 7.84 (1H, 9.03 (1H, 12.08 (1H, br s).

13 C-NMR(CDC13)8c: 18.9, 19.0, 19.1, 26.5, 26.7, 36.4, 59.1, 20 72.4, 72.5, 76.8, 77.5, 86.3, 88.3, 121.7, 127.6, 127.7, 127.

8, 127.9, 128.1, 128.4, 129.6, 130.0, 132.36, 132.42, 134.8, 135.45, 135.54, 135.8, 136.8, 146.8, 147.7, 155.4, 178.6.

Synthesis of 3'-O-benzyl-2'-O,4'-C-methylene-N 2 isobutyrylguanosine (Compound 47) Tetrabutylammonium fluoride (1.0 M in THF, 0.90 ml, 0.90 mmol) was added, at room temperature, to a tetrahydrofuran solution (3.0 ml) of Compound 46 (41.3 mg, 0.060 mmol), and the mixture was stirred for 1 hour at room 41 temperature. The reaction mixture was distilled under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (AcOH-EtOH, 20:1) to obtain a white powder, Compound 47 (27.1 mg, 0.060 minol, quant.).

mp. 228 229 0 C(Et2O). 2 5 32.900 (c 0.875, CHCl 3 IR v max (KBr) :3162, 2934, 1683, 1608 cm-1.

1 H-NMR (CDCl 3 6: 1.24 (3H, d, J =7 Hz), 1.26 (3H1, d, J 7 Hz) 2.76 (1H, in), 3.83, 4.03 (2H, AB, J 8 Hz) 3. 92, 4.02 (2H, AB, J 13 Hz) 4.33 (1H, s) 4.55 (1H1, s) 4. 62 (211, s) 00 5.80 (1H, 7.25 7.91 (111, 9.85 (1H, 12.05 (111, s).

13 C-NMR (CDCl 3 8C: 19.19, 19.25, 36.4, 57.4, 72.5, 77.0, 77.5, 86.5, 88.8, 121.0, 127.8, 128.1, 128.2, 128.3, 128.4, 128.6, 137.1, 137.5, 147.5, 148.2, 155.7, 179.9.

(Example 61 Synthesis of oligonucleotide analogue Natural nucleoside ainidite DINA snhesizer -10 L synVarious oligonucleotide 0.o~ analogues 5'-GCGXTT1TGCT-3' (XTB) (T2XT3) C U 5'.GCGTT7XFGCT3* fT37) X0 0 256'GCGTTTXGCT-3- J5SX) X 6'-GCGXXTTTGCT4 (X2T4) S'-GCGTTXXTTGCT-3- (T2X2T2) S'.0CGXTFXXXGCT4v (X6)) 5SG7 r ,,ca 42 3'-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5'-O- (4,4'-dimethoxytrityl)-2'-0,4'-methanouridine (Compound 21) Compound 8 (200 mg, 0.31 mmol) and diisopropylammonium tetrazolide (39.6 mg, 0.23 mmol) were subjected to azeotropy with anhydrous CH 3 CN three times, and then the system was converted into an anhydrous CH 3

CN-

anhydrous THF solution 4 ml). In a stream of nitrogen, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.12 ml, 0.37 mmol) was added, and the mixture was stirred for 90 minutes at room temperature. The solvents were distilled off under reduced pressure, and the resulting crude product was purified by silica gel column

S

chromatography (AcOEt:hexane:Et 3 N 75:25:1). Then, the purified product was reprecipitated from AcOEt-hexane to 6,Q obtain an amidite compound 21 (181 mg, 0.25 mmol, 81%).

m.p. 71-74 0 C (AcOEt-hexane).

31P-NMR (CDCl 3 6 149.6, 149.5, 149.4, 149.3, 149.2.

General synthesis of oligonucleotide analogues 20 The synthesis of an oligomer was performed by means of Pharmacia's DNA synthesizer, Gene Assembler Plus, on a 0.2 pmol scale. The concentrations of solvents, reagents, and phosphoramidite were the same as for the synthesis of natural DNA. A DMTr group of 5'-O-DMTr-thymidine (0.2 imol) having a 3'-hydroxyl group bound to a CPG support was deprotected with trichloroacetic acid. On its group, condensation reaction was repeated using an amidite comprising four nucleic acid bases for natural DNA synthesis 43 and Compound 21 to synthesize oligonucleotide analogues of respective sequences. The synthetic cycle was as follows: Synthetic cycle (0.2 pmol scale) 1) Detritylation 1% CC1 3 COOH in CH 2

CICH

2 C1, 6 sec 2) Coupling 0.1 M phosphoramidite (25 equiv.), M 1H-tetrazole (500 equiv.) in MeCN, 2 min 3) Capping 3% 4-(dimethylamino)pyridine, 10% Ac 2

O,

in MeCN, 18 sec 10 4) Oxidation 0.01 M 12 in 2,4,6-collidine/H 2 0/MeCN 6 sec The synthesized oligomer was cleaved from the support by treatment with concentrated aqueous ammonia in the customary manner. At the same time, the protective 15 cyanoethyl group was detached from the phosphorus atom, and the protective groups for the adenine, guanine and cytosine were also removed.

The resulting oligonucleotide analogue was rid of the DMTr group by use of 20 5 ml trifluoroacetic acid on a reversed phase chromatographic column (Millipore, Oligo-Pak T M SP), and further purified to obtain the desired oligonucleotide analogue.

In accordance with the foregoing method for general synthesis, the following oligonucleotide analogues were synthesized: 5'-GCGXTTTTTGCT-3' Yield 0.06 imol (30% yield) 44 5'-GCGTTXTTTGCT-3' (T2XT3) Yield 0.05 [mol (25% yield) 5'-GCGTTTXTTGCT-3' (T3XT2) Yield 0.03 [mol (15% yield) 5'-GCGTTTTTXGCT-3' Yield 0.06 imol (30% yield) 5'-GCGXXTTTTGCT-3' (X2T4) Yield 0.06 Rmol (30% yield) 5'-GCGTTXXTTGCT-3' (T2X2T2) Yield 0.05 atmol (25% yield) 5'-GCGTTTTXXGCT-3' (T4X2) Yield 0.06 tmol (30% yield) 5'-GCGXXXXXXGCT-3' (X6) Yield 0.06 lmol (30% yield) (10) 5'-GTTTTTTTTTXXC-3' (X2) Yield 0.07 !Amol (35% yield) [Experimental Example 1] Measurement of melting temperature (Tm) 20 The melting temperatures (Tm's) of annealing products between antisense strands, which were the various oligonucleotide analogues synthesized in Example 2, and natural DNA- or RNA-based sense strands were measured to investigate the hybridizing ability of the oligonucleotide analogues of the present invention for complementary DNA and

RNA.

Each sample solution (500 iL) with end concentrations of 100 mM NaCl, 10 mM sodium phosphate buffer (pH 4 [M 45 antisense strand, and 4 [M sense strand, respectively, was bathed in boiling water, and slowly cooled to room temperature over the course of 10 hours. The sample solution was gradually cooled to 5 0 C, kept at 5 0 C for a further period of 20 minutes, and then started to be measured, with a stream of nitrogen being passed through a cell chamber of a spectrophotometer (UV-2100PC, Shimadzu) for prevention of moisture condensation. The sample temperature was raised at a rate of 0.2 0 C/minute until 90 0

C,

10 and the ultraviolet absorption at 260 nm was measured at intervals of 0.1 0 C. To prevent changes in the sample concentration with increases in the temperature, the cell was provided with a closure, and a drop of a mineral oil was applied onto the surface of the sample solution during 15 measurement.

The results are shown in the following table.

46 Tabe I Melting Temperatures (Tm's) of Antisense Oligonucleotide Analogues for Complementary DNA and RNA S. S. C

S.

*SS C

S

S S

S*

S.

Tm for Tm for Antisense complementary DNA a complementary RNAb) molecule (ATm/mod.) (ATm/mod.) '-GCGTTTTTTGCT-47C5C 3' (natural)47C5C -GCGXTTTTTGCT-50C(3 )49C +4 3' (XT5) 0C(3C49C +4) -GCGTTXTTTGCT-49C(2 )49C +4 3' (T2XT3)49C(2C49C +4) 5'-GCGTTTXTTGCT- 49 0 C (+2 0 C) 50 0 C (+5 0

C)

3' (T3XT2) 52 0 C (+4 0 C) 51 0 C (+6 0

C)

3' -GCGXXTTTTGCT-51C(2 )53C(4C 3' (X2T4)51C(2C 3C +4) 5'-GCGTTXXTTGCT- 49 0 C (+1 0 C) 53 0 C (+4 0

C)

3' (T2X2T2) 54 0 C 5 0 C) 55 0 C (+5 0

C)

3'.(T4X2) -GCGXXXXXXGCT-58C(18C71C(43 3' (X6)58C(18C71C(43) 3'-CGCAAAAAACGA-5'.

b) -r(GGCAAAAAACGA).

As shown in the table, in the case of the oligomer having one or two units of the nucleoside analogue of the present invention (general formula introduced into a natural DNA strand, the ability to hybridize with the 47 complementary DNA oligomer, evaluated by the Tm, rose by 2 to 7 degrees (about 2 degrees per modified residue) as compared with the natural strand. With the oligomer having all T's substituted by X's the increase in the ability was as high as 11 degrees. When the ability to hybridize with complementary RNA was evaluated, the oligomer incorporating one or two X's had an increase in Tm of 4 degrees (4 to 6 degrees per modified residue) over the natural strand. In the case of X6, the ability to hybridize 10 with complementary RNA was further enhanced, showing an increase in Tm of more than 25 degrees (4 degrees per modified residue). There have been no examples of analogues undergoing such increases in Tm as compared with natural strands, and the affinity of the claimed oligomer was higher 15 for RNA than for DNA. These facts mean that the oligonucleotide analogue composed of the bicyclooligonucleoside analogue of the present invention has extremely high performance as an antisense molecule, and is useful as a material for pharmaceuticals.

[Experimental Example 2] Measurement of nuclease resistance A buffer solution (0.003 U/ml, 400 Rl) of a snake venom phosphodiesterase was mixed with a buffer solution tM, 400 [tl) of the oligonucleotide held at 37 0 C for minutes. The mixed solution was placed in a quartz cell (800 tl) kept at 37 0 C, and increases in the ultraviolet absorption (260 nm) due to the decomposition of the oligonucleotide were measured over time by means of SHIMADZU 48 UV-2100PC. The buffer used comprised 0.1 M Tris-HCl (pH 0.1 M NaCI, and 14 mM MgCl 2 and was sufficiently degassed before measurement.

Measurement of half-life A calculation was made of the average of the values of the UV absorption measured at the start of measurement and that measured at the time when no increase in this parameter was noted. The time corresponding to this average was designated as the half-life (ti/ 2 Oligonucleotide sequence tl/2 (seconds) 5'-GTTTTTTTTTTTC-3' (natural type) 260 5'-GTTTTTTTTT-XX-C-3' (X2) 850 Charts showing the time course of the ultraviolet absorption are presented as Fig. 1 (natural strand) and Fig.

2 The ultraviolet absorption reached a plateau in about 30 minutes for the natural strand, and about 20 minutes for X2, after initiation of the enzyme reaction.

INDUSTRIAL APPLICABILITY The use of this analogue provides an oligonucleotide analogue antisense molecule, which is minimally hydrolyzable with an enzyme in vivo, has a high sense strand binding ability, and is easily synthesized.

49 Sequence Listing Name of the applicant: Takeshi Imanishi Title of the invention: Novel bicyclonucleoside and oligonucleotide analogue Reference No.: Application No.: Date of application: March 1998 Priority No.: JPA 53409/97 Priority date: March 7, 1997 10 Number of sequences: 11 Seq. ID No.: 1 Length of sequence: 12 Type of sequence: Nucleotide C g Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGTTTTTTGCT-3' Seq. ID No.: 2 20 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGXTTTTTGCT-3' Seq. ID No.: 3 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue 50 Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGTTXTTTGCT-3' Seq. ID No.: 4 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue Number of strands: Single stranded Topology: Linear 0 Sequence: 5'-GCGTTTXTTGCT-3' Seq. ID No.: 9 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue 15 Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGTTTTTXGCT-3' Seq. ID No.: 6 S• 20 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGXXTTTTGCT-3' Seq. ID No.: 7 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue 51 Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGTTXXTTGCT-3' Seq. ID No.: 8 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue Number of strands: Single stranded Topology: Linear 0 Sequence: 5'-GCGTTTTXXGCT-3' Seq. ID No.: 9 Length of sequence: 12 Type of sequence: Nucleotide, nucleotide analogue 15 Number of strands: Single stranded Topology: Linear Sequence: 5'-GCGXXXXXXGCT-3' Seq. ID No.: S 20 Length of sequence: 13 Type of sequence: Nucleotide, nucleotide analogue Number of strands: Single stranded Topology: Linear Sequence: 5'-GTTTTTTTTTTTC-3' Seq. ID No.: 11 Length of sequence: 13 Type of sequence: Nucleotide, nucleotide analogue 52

U

Number of strands: Single stranded Topology: Linear Sequence: 5' -GTTTTTTTTXXTC-3'

S*

S. S 5555 S. 9

S

S. *S S S

S

S.

S S 555 5 9 *5*e

S

*SSS

S 9

SS

53

Claims (9)

1. A method of treatment of a medical condition comprising administering a therapeutically effective amount of an oligonucleotide or polynucleotide analogue having one or more structures of the general formula (la) -0 B (1a) 0 O where B is a pyrimidine or purine nucleic acid base, or an analogue thereof.

2. A method according to claim 1 wherein said condition would benefit from 10 administration of an antineoplastic or antiviral agent.

3. A method according to claim 1 or 2 wherein said condition is a viral infection 15

4. A method according to claim 1 or 2 wherein said condition is cancer. 0*

5. A method according to claim 1 or 2 wherein said condition is influenza.

6. A method of treatment of a medical condition comprising administration of 2o.. a therapeutically effective amount of an oligonucleotide or polynucleotide analogue of the general formula (II) 4 W 0 o oB 0 o0 SII O O 1 2 n 3 n W:\fiona\Species\Div of 720472.doc where B 1 and B are identical or different, and each represent a pyrimidine or purine nucleic acid base, or an analogue thereof, R is a hydrogen atom, a hydroxyl group, a halogen atom, or an alkoxy group, W 1 and W 2 are identical or different, and each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkinyl group, a cycloalkyl group, an aralkyl group, an aryl group, an acyl group, a silyl group, a phosphoric acid residue, a naturally occurring nucleoside or a synthetic nucleoside bound via a phosphodiester bond, or an oligonucleotide or polynucleotide containing the nucleoside, n's or n 2 's are identical or different, and each denote an integer of 0 to 50, provided that n"s or n 2 's are not zero at the same time, and that not all of n 2 's are zero at the same time, n 3 denotes an integer of 1 to 50, provided that n' and/or n 2 are or is 2 or more, B 1 and B need not be identical, and R's need not be identical. .15

7. A method according to claim 6 wherein said condition would benefit from S administration of an antineoplastic or antiviral agent. S S

8. A method according to claim 6 or 7 wherein said condition is a viral infection 0

9. A method according to claim 6 or 7 wherein said condition is cancer. *i S A method according to claim 6 or 7 wherein said condition is influenza. DATED: 11 August, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for: TAKESHI IMANISHI W:\fiona\Species\Div of 720472.doc