WO2004048376A1 - Bicyclic naphthylidine nucleosides - Google Patents

Abstract

A nucleoside analog having 4 hydrogen-bond functional groups capable of participating in base pair formation and having a structure selected from the group consisting of the following groups, and an oligonucleotide containing this nucleoside analog. The above nucleoside analog is useful in forming a base pair motif which can further stabilize the higher-order structure of a nucleic acid.

Description

 Specification

Technical field

 The present invention relates to a novel nucleoside derivative and an oligonucleotide incorporating the nucleoside derivative. Background art

DNA stores and transmits genetic information as a carrier. DNA double-stranded structure mainly consists of 1) formation of hydrogen bond by two pairs of complementary base pairs (A: T, G: C), 2) scanning by adjacent base pairs due to stacking of these base pairs. Interaction, and 3) Interaction between hydrophilic and hydrophobic functional groups and the surrounding environment forms a double helical structure and maintains its thermal stability. Interactions such as those found in DNA duplexes are also involved in molecular recognition between DNA and RNA and between nucleic acids and proteins, and are deeply involved in the regulation and expression of various functions in vivo. In recent years, advances in nucleic acid synthesis technology have made it possible to easily synthesize DNA and RNA having various sequences, and have brought dramatic advances in molecular biology. Furthermore, many non-natural artificial nucleic acids have been developed, and their application to nucleic acid drugs such as the antisense method, the antigene method, and the decoy method is being studied. To use in such applications, many properties have been reported so far when many base-modified nucleoside derivatives are chemically synthesized and incorporated into DNA chains. For example, Matteucci et al. Synthesized various nucleosides as shown below and found that they stabilize a DNA duplex by base pairing with the guanine base of a complementary strand in a DNA strand (Lin Jones, RJ; Matteucci, MJ Am. Chem. Soc, 1995, 117, 3873 · 3874, Lin, KY; Matteucci, MJ Am. Chem. Soc, 1998, 120, 3531-3532).

 G-clamp

 It has been reported that the G-clamp forms four hydrogen bonds with guanine bases, and that the introduction of one residue increases the Tm value by as much as 18 degrees, and its application to the antisense method is currently being studied. ing.

 G-clamp

In addition, as an attempt to devise an unnatural base pair motif, Benner et al. Devised a base pair motif such as π: κ, and attempted to extend the dienetic alphabet. (Piccirilli, JA; Krauch, T .; Moroney, SE; Bennner, SA Nature, 1990, 343, 33-37, Leach, ARl Kollman, PAJ Am. Chem. Soc, 1992, 114, 3675-3683).

 7Γ: 1 pair

On the other hand, the present inventors have previously synthesized four types of tricyclic imidazopyridopyrimidine nucleosides (Ira-NN, Im-OO, Im-NO, InrON) having four hydrogen bonding ability. (Kojima, NJ Ueno, Y .; Minakawa, NJ N. Matsuda, A "Nucleic Acis Symp. Ser., 1997, 37, 23-24).

S86M0 / C00Zdf / X3d Contract 00Z OAV 'If these nucleosides are incorporated into DNA oligomers, Im-NN: Im-00 and between the Im-NO: forming a base pair by four hydrogen bonds respectively between I _m -ON stabilize a double-stranded structure I do.

Im-N ^N : in-O ⁰ Im-N ⁰ : Im-0 ^{N When} the thermal stability of the DNA duplex incorporating these base pair motifs was actually evaluated, DNA It was found that the duplex was destabilized. The instability increased when the introduction site increased with discontinuity. However, when three base pairs were successively introduced, the DNA double strand became extremely stable. For example, it was found that the introduction of base pairs of Im-ON: Im-NO stabilized at about 6 degrees per base pair. From the above results, the tricyclic imidazopyridopyrimidine nucleoside thermally stabilizes the DNA double strand by the four hydrogen bonds and the stacking effect by spreading of the aromatic ring, but at the same time, around the base pair introduction site. It was thought that it also had the effect of destabilizing the DNA duplex to prevent base pairing. When only one base pair is introduced, the destabilizing effect exceeds the stabilizing effect, and as a result, the thermal stability of the DNA duplex decreases (Fig. 1A). The effect of instability increases when the introduction points increase further discontinuously (Fig. 1B). On the other hand, when three residues were successively introduced, the stabilizing effect was relatively greater than the instability occurring before and after the introduction, and the thermal stability of the DNA duplex was considered to be increased as a whole. (Figure 1C).

 Therefore, there is a need in the art for a new base pair motif that can further stabilize the higher-order structure of nucleic acids.

An object of the present invention is to provide a novel nucleoside derivative for forming a base pair motif capable of further stabilizing the higher-order structure of a nucleic acid, and an oligonucleotide containing such a nucleoside analog. Disclosure of the invention

 The present invention relates to nucleoside analogs having four hydrogen bonding functional groups that can participate in base pairing, comprising a compound of formula I:

Na-N ^N formula II:

 Na-0 °

Formula I I I:

Na-N ° and Formula IV

からなる群より選択される構造を有するヌクレオシド類似体を提供する。

A nucleoside analog having a structure selected from the group consisting of:

 Here, the hydrogen-bonding functional group means a functional group and an atom that can participate in a hydrogen bond, for example, hydrogen, N of primary, secondary and tertiary amines or amides, aromatic N, carbonyl Or カ ル ボ ン of carboxylic acid, H and の of water molecule, but are not limited thereto.

 The present invention also provides an oligonucleotide comprising at least one of the above-described nucleoside analogs of the present invention.

 The present invention also provides a compound of formula I I I:

で表されるヌクレオシド類似体を製造する方法を提供する。 該方法は、 式：

A method for producing a nucleoside analog represented by the formula: The method has the formula:

[Wherein is a protecting group for a hydroxyl group] In the presence of palladium, a compound represented by the formula:

[式中、 R ₂はアミン基の保護基であり、 Lは脱離基である]

[Wherein, R ₂ is a protecting group for an amine group, and L is a leaving group]

And then deprotecting. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows a schematic diagram of a DNA duplex containing a tricyclic imidazopyridopyrimidine nucleoside.

 FIG. 2 shows stabilization of a DNA double strand by the bicyclic naphthyridine nucleosides of the present invention.

 FIG. 3 shows the thermal stability of the duplex of DNA containing the bicyclic naphthyridine nucleoside of the present invention and DNA containing the tricyclic imidazopyridopyrimidine nucleoside. DETAILED DESCRIPTION OF THE INVENTION

 The novel bicyclic naphthyridine nucleosides (Na-N, Na-OO, Na-NO, Na-ON) of the present invention are novel base pair motifs that can further stabilize the higher-order structure of nucleic acids. These compounds form four hydrogen bonds without distorting the DNA duplex like the base pairs between the tricyclic imidazopyridopyrimidine nucleosides, further stabilizing the DNA duplex. (Figure 2).

The nucleoside analog of the present invention is a novel base pair motif that forms a base pair by four hydrogen bonds, and by incorporating this into a DNA, a very useful functional artificial nucleic acid can be created. Therefore, the present invention is useful for application to nucleic acid pharmaceuticals for controlling and stabilizing the higher-order structure of nucleic acids such as double helical structures. For example, by combining with the above-mentioned tricyclic nucleoside, it is possible to obtain a thermally stable DNA double strand, which is expected to be usable as a decoy molecule. I can wait. In addition, this naphthyridine base can be expected to form a base pair with a natural nucleic acid base, and is considered to be usable as an antisense molecule.

 Further, this compound is a fluorescent nucleoside, similar to the above tricyclic nucleoside. If these compounds show different responses depending on the nucleobases on the complementary strand side, they can be used for the detection of SNPs. Such base-responsive fluorescent nucleosides, such as BPP and BDA, have been reported by the group of Saito et al. (Okamoto, A .; Tainaka, K; Saito, I. 17th Symposium on Biological Function-Related Studies) , 90-91, Okamoto, A .; Tanaka, K; Fukuta, 生 体.; Saito, I. The 17th Symposium on Biofunctional Chemistry, 274-275).

しかし、 本発明では先の三環性ヌクレオシドとあわせて計 8種類の蛍光ヌクレ オシドが得られることから、 より汎用性の高い SNPs検出やハイプリダイゼ一 シヨン検出の蛍光プローブとして用いることができる。

However, in the present invention, a total of eight types of fluorescent nucleosides can be obtained in combination with the above-mentioned tricyclic nucleoside, so that it can be used as a more general-purpose fluorescent probe for SNPs detection or hybridization detection.

 The nucleoside derivative of the present invention can be synthesized as follows. Naphthyridine nucleosides, which are the target compounds, are C-nucleosides. After constructing the base moiety and the sugar moiety, respectively, they are synthesized by performing 'C-glycosylation using a palladium catalyst.

First, the sugar moiety is protected with a TBS group at the sugar moiety of thymidine according to the method described in the literature, and then treated with HMDS and ammonium sulfate to obtain a glycal form 2 (Coleman, RS; Madams, MLJ Org. Chem., 1998, 63, 5700-5703). Then, it is treated with TBAF, and the TBS group at the 5-position is selectively deprotected to give compound 3 (Scheme 1).

The base moiety is reacted with 2,6-diaminopyrimidine and malic acid in sulfuric acid according to the method described in the literature, and neutralized with aqueous ammonia to obtain naphthyridine derivative 4 (Newkome, G.R; Garbis, SJ; Majestic, V. K; Fronczek, FR; Chiari, GJ Org. Chem., 1981, 46, 833-839). Thereafter, the compound 4 is treated with an equivalent amount of NIS, and the amino group is protected with a dimethylamidine group to give the 6-position 5 (Scheme 2).

2,6-diaminopyridine 5

Subsequently, the compound 6 is obtained by the Heck reaction of the glycal derivative 3 and the naphthyridine derivative 5 and then treated with TBAF to obtain a 3′-position ketone 7. Next, this is reduced, and after deprotection of the dimethylamidine group, bicyclic naphthyridine nucleoside (Na-NO) can be synthesized (Scheme 3) (Zhang, HC; Daves, GD, Jr. J. Org. Chem., 1992, 57, 4690-4696).

That is, the present invention also provides a compound of the formula II I I:

で表されるヌクレオシド類似体

A nucleoside analog represented by

ぼ中、 は水酸基の保護基である]

Is a hydroxyl-protecting group.

In the presence of palladium, a compound represented by the formula

Enter N eight.

[Wherein R ₂ is a protecting group for an amine group and L is a leaving group], followed by deprotection. formula:

の化合物は、 天然のヌクレオシド、 例えばチミジンの 3 ' —OHを適当な保護基 R iで保護した後に、 常法により塩基を除去することにより製造することができ る。 た、 式：

Can be produced by protecting a natural nucleoside, for example, 3′-OH of thymidine with a suitable protecting group Ri, and then removing the base by a conventional method. The formula:

の化合物は、 2 , 6—ジァミノピリジンをリンゴ酸と反応させてナフチリジン環 を形成し、 次に、 6位に適当な脱離基 Lを導入するとともに、 アミノ基を適当な 保護基 R ₂で保護することにより製造することができる。

Reacts 2,6-diaminopyridine with malic acid to form a naphthyridine ring, then introduces an appropriate leaving group L at the 6-position and protects the amino group with an appropriate protecting group R ₂ It can be manufactured by doing.

Other bicyclic naphthyridine nucleosides (Na-NN, Na-O ⁰ , Na-ON) can be synthesized by the same method.

The nucleoside analogs of the present invention can be converted to amidites by methods known in the art and incorporated into oligonucleosides. Specifically, the obtained Na-NO form was protected with a dibutylamidine group at the amino group of the base to give compound 9, and then the 5'-7K acid group was subjected to dimethoxytrityl iridyl according to a conventional method. Subsequently, the hydroxyl group at the 3'-position is converted to an amidite 11 by phosphoramidation (Scheme 4). The obtained amidite body 11 can be introduced into a DNA oligomer according to a solid phase phosphoramidite method.

S86M0 / C00Zdf / X3d Contract OOZ OAV That is, in another aspect, the present invention provides an oligonucleotide comprising the above-described bicyclic naphthyridine nucleoside. The thus obtained oligonucleotides of the present invention can be used as diagnostic, therapeutic and research reagents as antisense oligonucleotides, lipozymes, primers, abutamas, antigenes, probes and the like. Preferably, the oligonucleotides of the invention are from about 6 to about 100 nucleotides in length. In a more preferred embodiment of the invention, the oligonucleotide is from about 12 to about 20 nucleotides in length. Oligonucleotides may include modified sugars, such as those having a substituent at the 2 'position, and nucleic acid bases other than adenine, guanine, cytosine, thymine, peracil, such as hypoxanthine, 5-alkylcytosine, It may contain 5-alkylperacyl, 5-haloperacyl, 6-azapyrimidine, 6-alkylpyrimidine and the like. Further, it may contain an internucleoside bond other than the phosphodiester, for example, a phosphorothioate bond.

 The contents of all patents and references explicitly cited herein are hereby incorporated by reference. In addition, the entire contents of the description and drawings of Japanese Patent Application No. 200-232-428, which is the application on which the priority claim of the present application is based, are incorporated herein by reference. To quote. Example

 Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention. Example 1

 3 ', 5, -0- (tert-butyldimethylsilyl) thymidine (1)

Under Ar atmosphere, thymidine (9.7 g, 40.0 mmol) was dissolved in DMF (150 mL), and imidazole (13.6 g, 200.0 mmoL) and TBSC1 (15.0 g, 100.0 bandol) were added, followed by stirring at room temperature for 2 hours. did. The solvent was distilled off, and the residue was dissolved in ethyl acetate (600 mL), washed twice with water (200 mL) and once with brine (200 mL), and dried over anhydrous sodium sulfate. The solvent is distilled off, and the residue is dissolved in a small amount of ethyl acetate. After drying under reduced pressure and adsorbing to silica gel, the mixture was separated and purified by silica gel column chromatography (Φ 7.8 X (15.0 + 3.0) cm, hexane: ethyl acetate = 3: 1 to 1: 1). Thus, compound 1 (18.6 g, 39.6 mmol, 99%) was obtained as a white solid.

44-NMR (CDCla) δ; 8.44 (br s, 1 H, NH), 7.45 (d, 1 H, H'6, J ₆ , _{5. Me} = 1.3 Hz), 6.31 (dd, 1 H, Η- Γ, Ji ', ₂ ' _a = 5.9, Jr _{) 2} ¾ = 7.9 Hz), 4.38 (ddd, 1 H, H'3, J ₃ ', 2' _a = 2.6, J ₃ ', ₂ ' b = 5.9, J ₃ ', 4' = 5.2 Hz), 3.90 (ddd, 1 H, H_4 ,, J ₄ ', ₃ ' = 5.2, J ₄ ', ₅ ' _a = 2.6, J ₄ ', ₅ ' b = 2.0 Hz), 3.85 (dd, 1 H, Η-5'a, J ₅ ' _a ' 4, = 2.6, J ₅ ' _a .5'b = 11.2 Hz), 3.73 (dd, 1 H, Η-5 'b, J ₅ ' b, ₄ '= 2.0, Js-b.s'a = 11.2 Hz), 2.22 (ddd, 1 H, Η-2'a, J ₂ ' _a , i '= 5.9, J ₂ 'a, 2'b = 13.2, J ₂ ' a, 3 '= 2.6 Hz), 1.97 (ddd, 1 H, Η-2'b, J ₂ ' b, i '= 7.9, J ₂ ' b, 2 ' _a = 11.2, J ₂ ' b, 3, = 5.9 Hz), 1.89 (d, 3 H, 5-Me, J ₅ -Me, 6 = 1.3 Hz), 0.91 and 0.87 (each s, each 9 H, tert-Bu), 0.09 (s, 6 H, Mex2), 0.06 and 0.05 (each s, each 3 H, Me) Example 2

 1,4-anhydride-3,5-bis-0- (tert-butyldimethylsilyl) -2-deoxy-D-elis-vent-vent-1-enyl (2)

 Compound 1 (10.3 g, 21.0 mmol) was dissolved in HMDS (100 mL), ammonium sulfate (5.5 g, 42.0 mmol) was added, and the mixture was heated and stirred at 135 ° C for 2 hours. The solvent was distilled off, the residue was dissolved in hexane (300 mL), washed twice with water (150 mL) and once with saturated saline (150 mL), and dried over anhydrous sodium sulfate. The solvent was distilled off, and the residue was separated and purified by silica gel column chromatography (Φ4 × 8 cm, hexane: ether = 7: 1) to give Compound 2 (5.3 g, 15.0 mmol, 72%) Was obtained as a colorless liquid substance.

Ή-NMR (CDCls) δ; 6.45 (d, 1 H, H_l, Ji) 2 = 2.6 Hz), 4.99 (dd, 1 H, H-2, J¾i = 2.6, J 2, 3 = 5.3 Hz), 4.84 (d, 1 H, H-3, J _{3) 2} = 5.3, J ₃ , 4 = 2.6 Hz), 4.27 (dt, 1 H, H'4, J _{4> 3} = 2.6, J ₄ , 5a = J _{4> 5} b = 5.9 Hz), 3.68 (dd, 1 H, H_5a, J _5a , 4 = 5.9, J _5a , 5b = 10.6 Hz), 3.48 (dd, 1 H, H-5b, J ₅ b, 4 = 5.9, J _5b> 5a = 10.6 Hz), 0.88 and 0.87 (each s, each 9 H, tert-Bu), 0.07 and 0.05 (each s, each 6 H, each Mex2) Example 3

 1,4-Anhydro-3-0- (tert-butyldimethylsilyl) -2, -doxy-5-hydroxy-D-erythrox-pent-1-enitol (3)

 Under an Ar atmosphere, compound 2 (5.3 g, 15.0 mmol) was dissolved in THF (40 mL), TBAF (15.7 mL, 15.7 mmol) was added dropwise under ice cooling, and the mixture was stirred at the same temperature for 1 hour. The solvent was distilled off, the residue was dissolved in a small amount of ether, silica gel was added, and the mixture was evaporated to dryness under reduced pressure and adsorbed on silica gel. Separation and purification with ether = 5: 1 to 3: 1) yielded compound 3 (2.2 g, 9.3 mmol, 62%) as a colorless liquid. .

! H-NMR (CDCls) δ; 6.47 (d, 1 Η, Η · 1, J _{1> 2} = 1.9 Hz), 5.04 (dd, 1 H, H-2, J ₂₎ i =

I.9, J _{2) 3} = 5.3 Hz), 4.79 (dd, 1 H, H-3, J ₃ , ₂ = 5.3, J _{3> 4} = 3.3 Hz), 4.33 (ddd, 1 H, H-4 _{, J43 = 3.3, J 4,} 5a = 6.6, J 4, 5b = 7.3 Hz), 3.68 (dd, 1 H, H_5a, J 5a, 4 = 6,6, J 5a, 5b =

II.2 Hz), 3.60 (dd, 1 H, H-5b, J ₅ b, 4 = 7.3, J ₅ b, 5a = 11.2 Hz), 0.88 (s, 9 H, tert-Bu): 0.07 (s , 6 H, Mex2) Example 4

 2-amino-7-hydroxy-1,8-naphthyridine (4)

 Sulfuric acid (50 mL) was added to 2,6-diaminopyrimidine (ll.Og, 0.10 mmol) and malic acid (15.0 g, 0.11 mmol) under ice-cooling, and the mixture was heated and stirred at 110 ° C for 2 hours. . Ice was added to the reaction solution and neutralized with aqueous ammonia. The precipitated precipitate was collected by filtration, washed with water, ethanol and acetone to obtain Compound 4 (14.5 g, 0.09 mmol, 90%) as a tan solid.

86-NMR (DMSO-de) δ; 11.86 (br s, 1 H, Hl), 7.63 (d, 1 H, H-4, J _{4> 3} = 9.2 Hz), 7.62 (d, 1 H, H- 5, J ₅ , 6 = 8.6 Hz), 7.03 (s, 2 H, H-4, NH2), 6.33 (d, 1 H, H.6, J _{6> 5} = 8.6 Hz), 6.10 (d, 1 (H, H "3, J = 9.2 Hz) Example 5

2- (N, N-dimethylformamidino) amino-7-hydroxy-6-odo-1,8-naphthyridin (5) Under Ar atmosphere, compound 4 (5.0 g, 31 mmol) was suspended in DMF (30 mL), NIS (8.4 g, 37 mmol) was added, and the mixture was stirred for 24 hours. The precipitate was collected by filtration and washed with DMF, ethanol and acetone to give a mixture of the 6-odo compound and compound 4 (1.3 g, 6-odo compound = 3.5 mmol, 4 = 1.4 mmol) as a tan solid.

Under an Ar atmosphere, a mixture of the 6-odo compound and the compound 4 (500 mg, 6-odo compound = 1.30 mmol, 4 = 0.60 mmol) was suspended in DMF (10 mL), and 1,1-dimethoxytrimethylamine was suspended. (0.28 mL, 2.1 mmol), and the mixture was heated and stirred at 80 ° C. After 12 hours, 1,1-dimethoxytrimethylamine (0.12 mL, 0.90 mmol) was added, and the mixture was further heated and stirred for 12 hours. After allowing the reaction solution to cool, the precipitate was collected by filtration, and the obtained solid was dissolved in a small amount of a mixed solvent of chloroform: methanol = 1: 1, silica gel was added, and the mixture was dried under reduced pressure and adsorbed on silica gel. after silica gel column chromatography (Φ 1.8 X (11.0 + 3.0 ) cm, 0-5% EtOH in CHC1 3) separated by and purified to give compound 5 (327 mg, 0.95 mmol, 73%) as a yellow solid Was. ·

mp 244-246 ° C

EI-MS (LR): m / z 342 (M +)

01-NMR (DMSO-de) δ; 12.01 (br s, 1 H, NH), 8.50 (s, 1 H, CH), 8.49 (s, 1 H, H-5), 7.79 (d, 1 H, H-3, J _{3> 4} = 8.6 Hz), 6.67 (d, 1 H, H-4, J ₄ , ₃ = 8.6 Hz), 3.10 (s, 3 H, Me) 3.02 (s, 3 H, Me )

 13C-NMR (DMSO-de) δ; 162.95, 159.72, 156.11, 149.63, 147.16, 136.42, 113.37, 110.57, 90.30, 34.49

Calculated: CUHUINAO: C, 38.62; H, 3.24; N, 16.38; I, 37.09.

Found: C, 38.42; H, 3.32; N, 15.95; I, 36.72.

6-(/ 3-D-glycerose-pentofuran-3'- ゥ ros --- yl) -2- (Ν, Ν-dimethylformamidino) amino-7-7-doxy-1,8-naphthyridine (7)

Under Ar atmosphere, palladium acetate (0.09 g, 0.4 mmol) and triphenylenylarsine (0.25 g, 0.8 mmol) were dissolved in DMF (10 mL), and the mixture was stirred at room temperature for 20 minutes. Under an Ar atmosphere, compound 5 (1.37 g, 4.0 mmol), compound 3 (1.01 g, 4.4 mmol), tri Butylamine (1.1 mL, 4.5 mmol) was dissolved in DMF (10 mL), the previously adjusted palladium solution was added thereto, and the mixture was heated and stirred at 60 ° C for 30 hours. After confirming the formation of compound 6 by TLC, the reaction solution was ice-cooled, acetic acid (1.0 mL) was added, followed by TBAF (8.0 mL, 8.0 mmol), and the mixture was stirred at the same temperature for 45 minutes. The solvent was distilled off, the residue was dissolved in a small amount of methanol, silica gel was added, and the mixture was evaporated to dryness under reduced pressure and adsorbed on silica gel. Then, silica gel ram chromatography (Φ3.0χ (13.0 + 3.0) cm, 0-10 % MeOH in CHCls) to give Compound 7 (1.09 g, 3.3 mmol, 82%) as a yellow solid.

FAB-MS (LR): m / z 330 (M +)

iH-NMR (DMSO-de) δ; 8.49 (s, 1 H, CH), 8.00 (s, 1 H, H.5), 7.84 (d, 1 H, H-4, J ₄ , 3 8.6 Hz ), 6.70 (d, 1 H , H-3, J 3) 4 = 8.6 Hz), 5.21 (dd, 1 H, Η-Γ, Jr, 2 'a = 6.0, Jr, 2' b = 10.6 Hz) , 4.96 (s, 1 H, OH), 4.02 (m, 1 H, Η4 '), 3.70 (m, 2 H, Η-5'a and Η-5'b), 2.85 (dd, 1 H , Η-2'a, J ₂ v = 6.0, J ₂ 'a, 2'b = 18.5 Hz), 2.30 (dd, 1 H, Η-2'b, Jzb, v = 10.6, J ₂ ' b, (2 ' _a = 18.5 Hz)

 2-Amino-6- (2, -deoxy- / 3-D-lipofuranosyl) -7-hydroxy-1,8-naphthyridine (Na-NO)

Compound 7 (1.08 g, 3.26 mmol) was dissolved in acetic acid (50 mL) and acetonitrile (50 mL) under an Ar atmosphere, and NaBH (OAc) ₃ (1.04 g, 4.9 mmol) was added and the mixture was heated at the same temperature. The mixture was stirred at room temperature for 1 hour. After distilling off the solvent and azeotroping with methanol, the residue was dissolved in a small amount of methanol, silica gel was added, and the mixture was evaporated to dryness under reduced pressure and adsorbed on silica gel. +4.0) cm, separated on 5-25% MeOH in CHC1 _3), to give compound 8 (1.16 g, 3.4 mmol, the quant.) as a yellow solid.

Compound 8 (1.16 g, 3.4 mmol) was suspended in a small amount of methanol, placed in a steel sealed tube, added with methanolic ammonia (100 mL), and heated and stirred at 80 ° C for 24 hours. The solvent was distilled off, the residue was dissolved in a small amount of methanol, silica gel was added, and the mixture was evaporated to dryness under reduced pressure and adsorbed on silica gel. (10.0 + 2.0) cm, separated on 10-25% MeOH in CHC1 _3), to give, compounds Na_NO

(0.67 g, 2.4 mmol, 72%) was obtained as a yellow solid.

mp> 280 ° C

 FAB-MS (LR): m / z 278 (M +)

Ή-NMR (DMSO-de) δ; 11.72 (bs s, 1 H, NH), 7.69 (s, 1 H, H-5), 7.84 (d, 1 H, H-4, J _{4) 3} = 8.6 Hz), 6.77 (s, 2 H, NH ₂ ), 6.33 (d, 1 H, H-3, J _{3; 4} = 8.6 Hz), 4.98 (m, 2 H, Η-Γ and OH), 4.77 ( m, 1 H, OH), 4.12 (m, 1 H, H-3 '), 3.75 (m, 1 H, H-4'), 3.45 (m, 2 H, Η-5'a and Η-5 'b), 2.31 (m, 1 H, Η-2'a), 1.65 (m, 1 H, H "2'b) ¹³ C-NMR (DMSO-de) <5; 162.15, 159.95, 149.21, 137.00 , 133.73, 127.51, 105.12, 104.68, 87.12, 74.68, 72.32, 62.42, 41.22

_{Calculated: Ci 3 H 15 N 3 O} 4 · 0.72H 2 O: C, 53.80; H, 5.71; N, 14.48.

Found: C, 53.66; H, 5.35; N, 14.36.

Measurement of thermal stability of DNA duplex

2-amino-6- (2, -deoxy-; 6-D-lipofuranosyl) -7-hydroxy-1,8-naphthyridine (Na-NO) obtained in Example 7 was replaced with an amino group in the base moiety. After protecting with a dibutylamidine group to give Compound 9, the 5'-hydroxyl group was dimethoxytritylized according to a conventional method, and then the 3'-hydroxyl group was converted to an amidite body 11 by phosphoramidation (Scheme 4). .

The resulting amidite 11 is introduced into a DNA oligomer according to the solid phase phosphoramidite method, and the DNA oligomer incorporating the tricyclic imidazopyridopyrimidine nucleoside Im-ON is used as a complementary strand to form a DNA double strand The thermal stability was evaluated. As a result, even when one base pair of Im-ON: Na-NO base pair is introduced, the DNA duplex is extremely stable, unlike the base pair of tricyclic imidazopyridopyrimidines, and G: C It was found to be 8.6 degrees more stable than the base pair (Fig. 3A). In addition, when 3 base pairs were introduced successively, the Tm value was 95.7 ° C, which revealed that the DNA duplex was stabilized at 26.7 ° C. This is stabilization of 8.9 degrees per base pair, and the DNA double strand was able to be stabilized when either one or three base pairs were introduced (Fig. 3B).

 Based on the above, the novel bicyclic naphthyridine nucleoside functions as a complementary base to the tricyclic imidazopyridopyrimidine, forms four hydrogen bonds without causing distortion in the DNA duplex, and forms the DNA double strand. It has become clear that this will further stabilize.

Claims

The scope of the claims 1. Nucleoside analogs having four hydrogen-bonding functional groups that can participate in base pairing, comprising a compound of formula I:

Equation II

Formula I I I

And formula IV

A nucleoside analog having a structure selected from the group consisting of: 2. Oligonucleotides comprising at least one nucleoside analogue according to claim 1. 3. Formula I I I:

A method for producing a nucleoside analog represented by the formula:

 [Wherein is a protecting group for a hydroxyl group]  In the presence of palladium, a compound represented by the formula:

[Wherein, R ₂ is a protecting group for an amino group, and L is a leaving group]  And then deprotecting.