WO1991018898A1

WO1991018898A1 - Nucleic acid homolog

Info

Publication number: WO1991018898A1
Application number: PCT/JP1991/000730
Authority: WO
Inventors: Junichi Yano; Tadaaki Ohgi; Kouichi Ishiyama; Hironori Tomi
Original assignee: Nippon Shinyaku Co., Ltd.
Priority date: 1990-06-01
Filing date: 1991-05-31
Publication date: 1991-12-12

Abstract

A novel antisense molecule comprising a nucleic acid homolog represented by general formula (I), available as a drug for suppressing infection with herpes virus, influenza virus, human immunodeficiency virus or the like and the activity of oncogene, wherein R?1 and R2¿ may be the same or different from each other and each represents hydrogen, inorganic acid residue, organic acid residue, etc.; B?1, B2 and B3¿ may be the same or different from one another and each represents adenine, guanine, etc.; and n represents an integer of 0 or above.

Description

Harm

Nuclear acid homolog technology

The present invention relates to nucleic acid homologs having a so-called antisense function. More specifically, by specifically binding to a nucleic acid of DNA or RNA to form a complex, the genetic information is selectively transmitted to a nucleic acid homolog that is inactivated, activated or controlled. o

Background technology

The gene is a double-stranded DNA consisting of a complementary base sequence, and a significant coding sequence (sense sequence) is encoded on one of the strands. Oligonucleotides complementary to this sequence can specifically bind, thereby blocking the telegene function by covering a certain region of interest. A compound having such a function is called an antisense molecule. Antisense molecules act not only on such binding to DNA but also on RNA and can selectively inhibit the process of protein translation from messenger RNA (Harold M. Weintraub., Scientific American, January 1990,

P3-40).

Based on the above principle, antisense molecules can be useful for understanding and controlling biological functions. To date, the following ^ GTM acid homologues have been synthesized (Murakami et al., Organic Synthesis Chemistry, Vol. 48, No. 3, Pl80-193 (1990)). (1) DN A-type oligonucleotide (conductor) (compound)

(2) RNA type oligonucleotide derivative (Compound II) (3) Triester phosphate type oligonucleotide derivative (Compound ③)

(4) Methylphosphonate-type oligonucleotide (conductor) (compound)

(5) Phosphoroamid type oligonucleotide derivative (Compound II)

(6) phosphothioate-type oligonucleotide derivative (compound II)

(7) Phosphorodithioate-type oligonucleotide derivative (Compound II)

(8) Carpalmate-type oligonucleotide derivatives (compounds I and II)

The structural characteristics of the above compound are shown below. Both aim to synthesize antisense molecules that function in vivo by modifying the natural nucleic acid structure (Compound II).

(The following page)

o 0

0 B .0 B

① ②

0 0 0 CHs

0 = P -0 0 = P -R

O

0

(In the formula, B represents a nucleobase, and R, R and R represent hydrogen, alkyl or other substituents.)

Disclosure of the invention

Although many other nucleic acid homologues including the above compounds have been synthesized, satisfactory effects have not always been obtained, and development of practical antisense molecules that sufficiently satisfy the following conditions: Has not been reached.

(a) Binding stability to DNA and RNA (complex forming ability)

(b) Resistance to nucleases

(c) Physicochemical stability exposed to acids, acids, temperature and humidity

(d) Cell membrane permeability

(e) Safety for nucleic acid genes

(f) Simple synthesis and low cost

Thus, the present inventors conduct studies with the aim of finding a new and useful synthetic antisense molecule that solves the above problems. PJ.

The present inventors have conducted intensive studies and found that the following general formula [I]

C I D

R

(Wherein R ¹ and R ² are the same or different and represent hydrogen, an inorganic acid residue, an organic acid residue, alkyl or acyl. B ¹ , B ² and B ³ are the same or different and are adenine and guanine , Hypoxanthine, cytosine, thymine, or parasyl n is an integer of 0 or more, provided that when n is 2 or more, B ² is not limited to the same base The present inventors have found that the nucleic acid homologue represented by the formula has excellent properties as an antisense molecule, and finally completed the present invention. Examples of the inorganic acid residue include, for example, a phosphoric acid residue, a diphosphoric acid residue, a triphosphoric acid residue, a thiol) acid residue or a sulfuric acid residue, and an organic acid residue. Is, for example, a carboxylic acid residue, an alkylphosphonate group or And a honey group.

The antisense molecule of the present invention has extremely excellent properties under the above-mentioned conditions (a) to (f) as compared with conventional antisense molecules. In particular, the ability of (a) to form a complex is most important as the activity of an antisense molecule. It has been reported that all conventional antisense molecules have a lower cleavage temperature (T m) than natural oligonucleotides. However, the thiocarbamate-type antisense molecule of the present invention has a significantly higher melting temperature than natural oligonucleotide, and is superior to the carbamate-type (compound I). In addition, the thiocarbamate-type antisense molecule of the present invention can easily form a triple-stranded complex with DNA or RNA. This property is much stronger than the natural oligonucleotide-carpalmate type, and can be said to be a structural feature of the thiocarpalmate type. Therefore, the inventive molecule of the present invention can extend not only to the normal messenger RNA but also to DNA as its target.

Another excellent property is that the antisense molecule of the present invention has a structure different from that of natural nucleic acids, and thus is resistant to nucleases and, at the same time, is physically stable. In addition, it is highly neutral and rich in fat-solubility, so that it has excellent cell membrane permeability. Furthermore, the synthesis is easy, the reaction yield is good, and mass production is possible. In addition, the antisense molecule of the present invention does not have to worry about the occurrence of a recombinant substitution with DNA in the nucleus unlike the natural oligonucleotide. From the above, the gist of the present invention is that the nucleic acid homolog itself represented by the general formula [I] is itself. Can be.

Uses of the nucleic acid homologs according to the present invention include, as the nucleic acid homologs of the present invention inactivate genetic information, a foreign substance that has invaded the living body, for example, Herpes virus, Inflileenza virus, and human. It can be used as a drug to suppress infections such as immunodeficiency virus (HIV, AIDS virus, etc.) and the function of pain genes. In addition, it can be applied to technologies such as breed improvement by actively controlling the gene transfer of animals and plants. In addition, it has the potential to be used as a DNA probe for analysis of gene function and to check for infectious diseases, bacterial and viral infections, etc. Can be considered.

When the nucleic acid homolog of the present invention is used as a medicament, the compound of the present invention is administered as it is or in a pharmaceutically acceptable nontoxic and inert carrier.

As the carrier, one or more liquid, planar, or semisolid diluents, fillers, and other prescription auxiliaries are used. The pharmaceutical compositions are preferably administered in unit dosage forms. The nucleic acid homologs of the present invention can be administered orally, intraosseously, topically, or rectally. Needless to say, it is administered in dosage forms suitable for these administration methods, for example, various pills, injections, inhalants, eye drops, soft suppositories, suppositories and the like. In particular, intra-tissue administration and local administration are preferred.

(Synthesis method)

Next, a general method for synthesizing the nucleic acid homolog of the present invention will be described. (Synthetic route)

HO B

B HO 1 BR ^l 0 BN

-0 ヽ O H No no

O OH

I

H

® ⑪

(In the formula, B ¹ and B ² are the same or different, and are peracyl, thymine, hypoxanthine or N-benzoyl-denine, N-benzoylguanine, N-penzylcytosine or N-benzoylguanine in which the base moiety is protected. —Indicates dimethytritylated nucleobases, etc. B ³ , B * and B ⁵ may be the same or different, 18 g PCN, guanine, cytosine, thymine or hypoxanthine. n represents an integer of 0 or more. However, when n is 2 or more, B * is not limited to the same base. R ¹ represents hydrogen or imidazolylthiocarbonyl. R ² is a protecting group that can be easily removed with hydrogen or an acid, such as trityl, monomethoxytrityl, dimethoxytrityl or tert-butoxycarbonyl or fluorenylmethylol that can be easily removed with a base. Xycarbonyl (Frooc) and the like. )

Procedure 1 First, the 2,3′-cisdiol portion of a nucleoside or a nucleotide derivative 塩基 whose base moiety is protected is prepared by a known method (for example, BP Stirchak et al., Nucleic Acids Research, 17, 6129-6141). (1989)) to convert to the morpholine derivative ⑪.

Step 2 Next, by a known method, a protecting group capable of easily removing the nitrogen of the morpholine derivative 酸 with an acid, for example, trityl, monomethoxytrityl, dimethoxytrityl or tert-butoxy. Protected with carbonyl and the like, and subsequently, for example, Ι, Γ-carbondiylmidazole (HA Staab et al., Ann, 657,98-103 (1962)) and Ν, Ν-dimethylformolamide (hereinafter “DMFj In a solution 50: heated under neutral conditions to obtain the activated morpholine derivative た in which the hydroxyl group of the morpholine derivative ⑪ has been substituted. When the appropriate morpholine derivative obtained in Step 1 is mixed in a DMF solution at room temperature, it is quantitatively condensed to form a dimer with a terminal nitrogen protected, and when the terminal nitrogen protecting group is removed with a suitable acid, n Dimer with 0 Get ©.

Action 4 Activate the hydroxyl group at the other end of the dimer in which the nitrogen at the terminal is protected with 1-dithiocarberdiimidazole in the same manner as in Step 2, and use the appropriate Condensation with the morpholine derivative in the same manner as in Step 3 and removal of the terminal nitrogen protecting group with an appropriate acid yields a n-trimer.

Step 5 Similarly, activate the hydroxyl group at the other end of the dimer, trimer, etc., whose terminal nitrogen is protected, with 1, dithiocarboerdiimidazole, and use this together with the appropriate moles obtained in Action 1. Condensation of a phosphine derivative or an appropriate dimer or trimer, whose terminal nitrogen is not protected with a protecting group, in the same manner as in step 3 and removal of the terminal nitrogen protecting group with an appropriate acid, any arbitrary An oligomer having a chain length can be prepared.

Action 6 In addition, as the chain length of Oligomer® increases, the solubility in ice decreases. Therefore, the hydroxyl group at the terminal is determined by a known method (PT Gilhara et al., Chem, Ind. (London) , 1959, 542), can be substituted with a phosphoric acid group, an alkylphosphonate group or other substituents. In this way, the solubility in ice is increased about 10 times.

Operation 7 In addition, a nucleoside dinucleoside derivative can be bound to the nitrogen at the terminal of the oligomer of the nucleic acid homolog of the present invention in the same manner as in Steps 2 to 5.

Action 8 Further, in order to sufficiently exert the function of the oligomer of the nucleic acid homologue of the present invention in an organization, a so-called nuclear transport peptide (al deron D. et al., Cell, 39, 499) -509 (1984)) It is also possible to bind to the nitrogen at the terminal of oligomer II by the method described above.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, Test Examples, and Comparative Examples, but the present invention is not limited thereto.

Reference example 1

Synthesis of 6- (ゥ -la-silyl-11-yl) -1-2-hydroxymethylmorpholine [1]

C 1

U: Peracil (the same applies hereinafter)

6.65 g of resin was suspended in 470 ml of methanol, 8.30 g of ammonium tetraborate and 5.90 g of sodium periodate were added, and the mixture was stirred at room temperature for 3 hours. Thereafter, sodium cyanoborohydride (3.00 s) was added to the reaction solution, and a further 30 minutes were submitted (B.P.

Stirchak et al., Ucleic Acids Research, 17, 6129-6141 (1989)). Next, after confirming the end of the reaction by thin-layer chromatography (manufactured by Merck, normal-phase thin-layer plate; hereafter referred to as “TLC”) (10% methanol Z-dichloromethane), the product Rf = 0.15) ー -toluenesulfonic acid 丄

After decomposing the reducing agent, the reaction solution was concentrated to dryness. The residue was dissolved in water and applied to Dowex 50WX8 (Pyridinium type) (registered trademark), and eluted first at 500. Then, 400ml of 2.8% ammonia water was used to elute 160ml of the target substance. After concentration and drying with an aspirator and a vacuum pump, 3.50 g of the present compound [1] was obtained as a white solid (yield: 63%).

Whisper point 227-230t (disassembled)

nnriCDCls, 60MHz) δ: 2.2 to 3.4 (ra, 4H), 3.7 to 4.2 (m, 3H), 5.6 to 5.8 (m, 1H), 5.96 (d.lH, J = 7.6Hz), 7.91 (d, (1H, J = 7.6Hz) Reference example 2

6— (Peryl-Silyl) 1—2—Hydroxymethyl-1 41 Composition of tritylmorpholine [2]

HO U

0

[2]

, Ν '

I

Tr

Tr: Trityl (the same applies hereinafter)

To a solution of 1.14 g of the compound [1] in 25 to π was added 1.00 g of tetraethylamine and 1.68 g of tritrichamine, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was partitioned between dichloromethane and a saturated sodium hydrogen carbonate solution, and the organic layer was dried and concentrated to dryness. Then, the residue was re-dim force Ramuku π Conclusions _{(Si0 2 60H 50g, 0 →} 5% meta The product was purified by Nord Z-pi π-rometan (containing 0.1% viridin)) to obtain 2.40 g of this compound [2] as a white solid (quantitative).

165-168t melting point:

δ: 1.2 to 1.8 (ro, 2H), 2.8 to 3.8 (ra, 5Η), 6.2 to 6.4 (ro, 2H), 6.6 to 7.8 (m, 15Η), 8.4 to 8.8 (ra, 1Η) Reference Example 3 Synthesis of the following compound [3]

HO U

Q

, N

u (3)

S 0

Q

, ΝΓ

I

Tr

Compound [2] Dissolve l.OOg in 20 ml of DMF, add 0.45 g of 1,1'-carbanoldimidazole (manufactured by Wako Pure Chemical Industries, Ltd.) (H. Staab et al., Ann, 657, 98- 103 (1962)) and heated at 50 for 5 hours. After heating, the reaction mixture was concentrated to dryness and concentrated. After partitioning with rometane-ice, the organic layer was dried and then concentrated to dryness. Then, the residue was purified by silica gel column chroma preparative (Si0 ₂ 50 g, main Tano Lumpur / Axis port π meth emissions) to obtain the compounds substituted by Lee Mida sled thiocarbonyl (melting point: 253-255 (Decomposition), MS (PAB) H ⁺ : 580) was obtained (quantitative).

The DMF solution 14 »£ the compound of Compound 0.48 [1] 0.22g was added and after standing for 30 minutes easy at room temperature,«綰乾solidified, the residue silica force Gerukaramuku Roma preparative _{(Si0 3 30g, 2% →} 5X Purification with methanol (dichloromethane, π-methane) yielded 0.63 g of compound [3] as a white solid (quantitative).

230-232t melting point

δ: 2.1 to 2.3 (m, lH), 2.62 (t, 1Η, J = 10 Hz), 2.3 to 3.2 (m, 2H), 3.3 to 3.5 (m, 2H)

Melting point 230-232

δ: 2.1 to 2.3 (ro, 1H), 2.62 (t, 1H, J = 10Hz), 2.3 to 3.2 (m, 2H), 3.3 to 3.5 (ro, 2H), 3.6 to 4.0 (m, 3H), 4.2 ~ 4.6 (ra, 4H), 5.04 (d, 1H, J = 8Hz 5.60 (broad d, 1H, J = 8Hz),

5.76 (t, 2H, J = 8Hz), 6, 15 (broad d, 1H, J = 8Hz), 6.78 (broad d, 1H, J = 8Hz), 7.0-7.6 (m, 15H), 8.74 (broad, 1H),

9.60 (broad, 1H), 9.90 (broad, 1H)

(The following page) Fuse example 1

Synthesis of the following nucleic acid homolog [4] of the present invention:

C 4)

0.20 g of the compound [3] was treated with 7.0 ml of 80% acetic acid at room temperature for 2.5 hours. Then, the reaction solution was concentrated to dryness, after residue partitioned di click _π Rometan monohydrate, facing澳綰dry again the aqueous layer to obtain the present invention the nucleic acid homolog in white surface body [4] (quantitative ).

Furthermore, this was powdered with ethanol-aceton. With a melting point of 167-169

^ nmr (D ₂ G, 200MHz)

δ: ■ 2.8-3.4 (ra, 7Η), 3.6-4.1 (m, 3Η), 4.2-5.l (ro,

4Η), 5.73 (broad d, 1H, J = 8Hz), 5.90 (d, 3H, J = 8Hz),

7.72 (d, 1H, J = 8Hz), 7.82 (d, 1H, J = 8Hz)

MS (FAB) MH ⁺ 497

Reference example 4

One ^6- (Ν ε - benzo I le adenine one 1 Ichii Le) synthesis of single 2-arsenide mud Kishimechirumoruho Li down [5] 〔 Five〕

bzA: N-Penzoyl adenine (the same applies hereinafter)

Using 3.00 g of N-benzoyladenosine, 2.10 g of the present compound [5] was obtained in the same manner as in Reference Example 1.

Melting point 110-115

¹ H _¾Rr (CDsOD, 60MHz)

δ: 2.7-4.l (m, 7Η) 5.9-6.l (m, 1H) 7.2-8.2 (m,

5H), 8.61 (s, 1H, H-2) 8.73 (s, 1H, H-8)

Reference example 5

6— (N ^6— one pen-zil-one-one) Synthesis of 4-dihydroxymethyl-4- (4,1-monomethoxytrityl) morpholine [6]

HO

[6]

MMTr: Mono methoxy trityl (the same applies hereinafter)

Compound (5) 1.50 g and monomethoxytrityl chloride 1. Using 63 g, this compound [6] was obtained in the same manner as in Reference Example 2.

(quantitative) .

Break 180-190: (Disassembly)

'Hn.r (CDaOD, 60MHz)

δ: 2.8 to 4.2 (m, 7H), 3.79 (s, 3H) 6.3 to 6.6. (οι, 1Η),

6.7 to 8.2 (m, 19Η), 8.81 (s.1Η, H-2), 9.05 (s, 1H, Η-8) MS (FAB) H ⁺ 627

Reference example 6

Synthesis of the following compound [7]

[7]

M Tr compound [6] To 2 ml of a 100 mg DOO solution of lOOrag was added 34 rag of Ι, biocarbyldimidazole, and the mixture was reacted at 50 t for 2 hours. Then, the residue was purified by silica gel chromatography to obtain 80 rag of a white powder compound in which the 5'-hydroxyl group was imidazolylthiocarbonylated.

This was redissolved in 2 mL of the DMF solution, compound [5] 46rog was added, and the mixture was allowed to stand at room temperature for 1 hour. After standing, 'concentrate and dry, TLC (5% IB

The compound [7] was obtained from lOOrog.

Melting point 210-215 (decomposition)

δ: 0.89 (t, 2H, H-5ax, J-6Hz), 1.28 (s, 2H, H-3ax), 2.8 to 4.0 (m, 8H), 3.80 (s, 3H), "to 4.8 (m ' 3H), 5. (! To 5.5 (ra, 1H), 5.8 to 6.0 (ra, 1H), 6.3 to 6.5 (ra, 1H), 6.8 to 7.8 (in, 20H), 7.9 to 8,8 (m, 8H)

MS (PAB) MH ⁺ 1024

Reference Example 7

Synthesis of the following compound [8]

HO

[8]

Α: Adenine (same under J¾)

Compound [7] 4Dmg was dissolved in 1.0 ml of finished ice cubes and 1 xd of DMF, sealed, and allowed to stand at room temperature overnight. After standing, the solvent is removed under reduced pressure, and purified by silica gel gel chromatography (5-> 8¾methanol / dichloromouth methane (containing 0.1% pyridine)) to give white 30oig of the present compound [8] was obtained in the form of a facet.

Melting point 235-245t: (decomposition)

MS (PAB) MH ⁺ : 815

Example 2

Synthesis of the following nucleic acid homolog [93 of the present invention (^ below

HO

[9]

After dissolving 30 mg of compound [8] in 1 mL of 80% acetic acid, the mixture was allowed to stand at room temperature for 2 hours, and the solvent was distilled off under reduced pressure using a vacuum pump. Next, the residue was dissolved in 10 mL of water, extracted with dichloromethane, and the aqueous layer was concentrated under reduced pressure and dried to obtain 20 mg of the nucleic acid homolog [9] of the present invention as a white solid. This was recrystallized from n-propanol.

Elemental _{_{_{analysis: C 21 H 26 N 12 [}}} hS 'C 3 H B 0 - as (n Bed opening Pano Lumpur)

C H N

Total value (%) 47.83 5.69 27.89

Actual value (%) 47.54 5.65 27.75

Melting point 235-240 (decomposition)

δ: 3.0-4.2 (m.14H), 5.67 (d, 1H, J = 8 Hz), 6.00 (t, 1H, J = 6 Hz), 7.71 (s, 1H), 7.86 (s, 1H), 8.07 (s , 1H), 8.12 (s, 1H)

U V: ^ SaS 256nm (ε: 39, 600)

MS (FAB) H ⁺ 543

Reference Example 8

Synthesis of the following compound [10] (referred to below as "A o Aj")

〔Ten〕

This compound [10] was synthesized according to the literature (Eugene P. Stirchak et al., Nucleic Acids Res., 17, 6129-6141 (1989)).

114 mg of the compound [6] was dissolved in 1.5 mg of a DMF solution, and 98 mg of bis (p-nitrophenyl) carbonate and 120 z £ of triethylamine were added at room temperature, followed by stirring for 1 hour. After the reaction, the solution was concentrated and dried, and purified by TLC (3% methanol Z-dichloromethane (containing 0.1% pyridine)) to obtain 136 mg of a white solid. this The white solid and the compound [5] were dissolved in 1.5 mL of DMF, and triethylamine 50 was added at room temperature, and the mixture was allowed to stand for 30 minutes. After standing, the mixture was partitioned with dichloromethane and water, and the ice layer was dried and concentrated to dryness. The residue was purified by TLC (4 methanol-dichloromethane) to obtain 117 mg of a white solid.

Dissolve this white solid lOOrag in DMF 1.0m £ and add methanol

2.0m £ and 1.0m 濃 of concentrated ammonia were added and left overnight at room temperature. Then, the reaction solution was concentrated and dried, and then purified by TLC (8J tanol dichloromethan (containing 0.1% pyridine)) to obtain 66 rag of a white solid.

This white solid was treated with 2.0 mL of 80% acetic acid at room temperature and concentrated to dryness. Next, water was added to the mixture, and extraction was performed using a jig-mouth method, and the ice layer was allowed to dry. The residue was recrystallized from isopropanol to obtain 56 mg of the present compound [10] as a white powder.

Elemental _{_{_{析值: C ai H 26 N l 2}}} 0 5 · C 3 H e 0 ( i Sopuropano Lumpur) '3/2

As the H ₃ D

C H N

Calculation 值 (%) 46.98 6.08 27.39

Actual value (%) 46.23 5.94 27.77

Melting point 215-220 (decomposition)

XJV: λΜ258ηπι (e: 27, 800)

MS (FAB) MH + 527

(The following page) Example 3

Synthesis of the following nucleic acid homolog [11] of the present invention:

[11]

The nucleic acid homolog [12] of the present invention was prepared in the same manner as in Reference Examples 4 to 7 and Example 2 except that the compound [6] in Reference Example 6 was changed to the compound [1]. Obtained (quantitative). This was recrystallized from ethanol.

Elemental _{_{_{析值: C3 0 H 35 N s O}}} e S -1 / 2C 2 H 5 0H ( ethanol) · Η ₂ 0 and to

C Η Ν

Calculation 值 (%) 44.49 5.10 22.68

Actual planting (%) 44.99 5.39 22.49

Melting point 240-245:

UV: λίΐΐχ 258nm C ε: 37, 500)

MS (FAB) H ⁺ 520

(Synthesis of tetramer)

Reference Example 9 Synthesis of the following compound [12]

HO

[12]

Compound [3] To 1.5 mg of DOOrog's DMF solution was added 29 mg of 1, dithiocarbonyldimidazole, and the mixture was heated at 50 for 5 hours. After warming, purified with concentrated綰乾solidified reaction solution, the residue was Rikageruka Ramukuro Conclusions (Si0 including ₂ 5g, 0 → 4% main Tano Lumpur Z Axis B Lome motor emissions (0.1% Billiton Gin)) Then, a white-faced llOrag was obtained.

This was dissolved in DMF 2.5 m £, added acetate 82mg and preparative Ryechiru Ryo Mi emissions 50 x the compound [4] at room temperature and left for the _£ left for 30 minutes, solidified the reaction mixture concentrated to dryness, shea Rikageruka Ramukuroma DOO (SiG including ₂ 5 g, 2 → methanol Z Axis Rorometa emissions (G.1% Billiton Gin)) to give the present compound as a white solid [12]

136 rags were obtained. Cut at 254-258 (disassembled)

Example 4

Synthesis of the following nucleic acid homolog [13] of the present invention:

〔13〕

Compound [12] 55rog was simply treated with 1.5 ml of 80% aqueous acid at room temperature for 2 hours, and the reaction solution was slightly dried. After the residue was partitioned with dichloromethane, water and a small amount of methanol, the ice layer was exposed to the surface, and the nucleic acid homologue of the present invention [13] was obtained as a white solid (quantitative). Analytical plant: C ₃₃ H _<6 N ₁₂ 0 _l6 S ₃ '4H ₂ Q

C H N

Calculation 楦 (%) 42.31 4.92 15.18

Actual measurement () 42.32 5.27 15.86

Melting point 250-2601: (decomposition)

UV: ΐΐϋ 255 nm (ε: 62, 500) MS (FAB) MH ⁺ 1036

Reference Example 10

Synthesis of the following compound [14]

14〕

102 mg of the compound [7] was dissolved in 1.O mg of DMF, 23 rag of 1,3-carbocarbonyldimidazole was added, and the mixture was heated at 50 t for 5 hours. After heating, the solvent was removed by filtration and purified by TLC (6 methanol Z dichloromethan (containing 0.1% viridine)) to obtain 78 oig of a white solid compound.

Separately, 78nig of compound [7] was treated with 80% acetic acid at room temperature for 1 hour, concentrated to dryness, and azeotroped with toluene and pyridine.

To this residue, 77 rag of the above-mentioned white solid, 1.3 mL of DMF, and triethylamine 20 were added, and the mixture was left at room temperature for 50 minutes. After standing. The residue that has been concentrated and dried is distributed with dichloromethane, and the organic layer is separated. It was dried and dried again. This was purified by TLC (7-ethanol π-methan (containing 0.1% pyridine)) to obtain 10 mg of the present compound [14] as a white face.

Melting point 240-250: (decomposition)

Example 5

Synthesis of the following nucleic acid homolog [15] of the present invention:

[15]

50 mg of the compound [14] was dissolved in 0.5 mg of DMF, and 3.0 ml of 80% sulfuric acid was added, followed by a reaction at room temperature for 2 hours. Then, after the solvent was concentrated to dryness, the surface was azeotropically dried with a small amount of ethanol.To 2.0 m2 of this DMF solution was added 4.0 ml of methanol and 2.0 ml of concentrated ammonia ice. I left it. After standing, the solvent was concentrated to dryness, and the residue was recrystallized from ethanol-lue ice to obtain 16 mg of the nucleic acid homolog [15] of the present invention as a white powder. Melting point 240-2451: (decomposition)

MS (PAB) MH + 1128

(Synthesis of octamer)

Example 6

Union of the following nucleic acid homologs of the present invention [16]

[16]

H Compound [12] 1,0-thiocarboerdiimidazole lOrag was added to a 0.5 mg solution of 60 mg of DMF, and the mixture was heated at 50 for 5 hours. After heating, the mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (SiO ₂ 4 g, 2 → 4% methanol / dichloromethane) to obtain a white-faced 65 nig.

To 0.6 ml of a DMF solution of 38 rag of this white solid, 33 rag of the acetate salt of compound [13] and 9 juv of triethylamine were added, and the mixture was briefly stirred at room temperature for 1 hour. After the reaction, the residue is concentrated to dryness, and the residue is silica gel column. P

28

Kumato (Si0 ₂ 3.5g, 2 ~> 6% methanol Z-Jig

Purified with H.sub.2 (containing 0.1% pyridine), 40. ling of the present nucleic acid homolog [16] was obtained as a white solid.

Cut point 26G-267: (Disassembly)

S (FAB) H ⁺ 2113

(Synthesis of methyl phosphonate derivative of tetramer)

Reference Example 11

Synthesis of the following compound [17]

[17]

Compound azeotropically pyridine just before reaction [12] Dioxane solution of phosphorylating agent methyl 0,0-bis (1-benzotriazolyl) phosphonate (0.11M) in 20oig in dioxane 0.5ffl £ (JH Van Boom et al., Nucleic Acids Research, 14, 2171-2185 (1986)), and the mixture was stirred at room temperature for 40 minutes. And reversed-phase TLC (Merck Manufactured by DC-Fertigplatten Kiesergel 60Pas si lanisiert, developing solvent: acetone / water = 1/1) After confirming that the raw material has completely disappeared (raw material Rf: 0.25, product Rf: 0.80) , 50% pyridinone 1 was added and the surface was dried. This residue was subjected to reverse phase open chromatography (carrier: Capcell ak C18 AG120, S-20 / 40 μτα (5 id), manufactured by Shiseido Co., Ltd., elution solvent system; 0 to> 35% acetonitrile 10 ιηίί The concentration of this compound [17] was 18.3 rag as a white solid by performing linear concentration gradient with raetyl ammonium acetate buffer (ρΗ7.0)).

244-247t melting point (decomposition)

Example 6

Synthesis of the following nucleic acid homolog [18] of the present invention:

[18]

18 mg of compound (17) in 1.0 mL of 80% acetic acid at room temperature for 1 hour The mixture was concentrated and dried by TLC to confirm that there was no raw material.The residue was applied to the Okayama Oven Chromatography in the same manner as in Reference Example 11, and the nucleic acid of the present invention was used without white powder. 10.3 to homolog [18]

Dig got.

Elemental analysis II: C * ₀ H _4B N ₁₂ O _ie PS ₃ 'C _e H ₁₅ N (triethyl end) • As 7H _a 0

C H N

Calculated value () 1.22 5.87 13.58

Actual measurement () 41.7 5.76 13.52

V: λίύ 252nra (ε: 75,500)

MS (FAB) MH ⁺ 1114

(The following page)

(Synthesis of tetramer derivative of tetramer)

Reference Example 12

Synthesis of the following compound [19]

[19]

0 0

2 ', 3, -0-Isopropylidene peridine (commercial product) 142 mg of DMF solution 5.0 mL of DMF was added with 125 mg of 1,3-carboerdiimidazole and heated at 50 for 6 hours. After warming, the mixture was evaporated to dryness, purified by TLC (5% methanol-Z-dichloromethane), and 180 mg of a white powder compound in which the 5'-hydroxyl group was imidazolyl thiocarbellated was dissolved in llmg of this substance in DMF. Then, 35 rog of the compound [13] and 35 X of triethylamine were added, and the mixture was allowed to stand at room temperature for 30 minutes. After concentrating to dryness, the residue was purified by TLC (10% methanol sigma rometan) to obtain 32 rog of this compound [19] as a white solid. Cut at 251-253 (disassembled)

Example 7

Synthesis of the following nucleic acid homolog [20] of the present invention:

[20]

20 rcg of the HOOH compound [19] was dissolved in 3.0 mL of 80% acetic acid and heated at 80 * C for 5 hours. After heating, the solution was concentrated and dried, and crystallized from ethanol single ice to obtain 14. Orog of the nucleic acid homologue of the present invention [20] as a white powder. Elemental analysis: C HwUwS * · 3H _a 0

C H N

Calculated value (%) 42.79 4.54 14.26

Actual value (%) 43.04 4.74 14.02

Melting point 265-270 (decomposition)

MS (FAB) MH + 1322

(Complex formation ability) P

33 Test Example 1

Measurement of e 值 of As A and AoA lOraM phosphate buffer solution (e 值 of As A and AoA in 0.1 M NaCDpH 7.0 was measured, and the results shown in Table 1 were obtained. Table 1 e 值 compounds of each compound λ.ax (nm) e value (cm ^_1 · mol ' ¹ · i)

A s A 256 39, 600

A o A 258 27, 800

A p A ⁿ 257 27, 200 "d (A p A) ³ ] 257 27, 200"

Poly D 260 8, 880 "

Poly T 260 7,020 ^5} ^l) Adenylyl (3 '→ 5') adenosine (the same applies hereinafter)

^2} J. Brahms et al., J. Mol. Biol., 15, 467-488 (1966),

³ ) 2, -Doxy-endenyl (3, → 5 ') 1 2'-Doxyadenosin (the same applies hereinafter)

^{45 From} package insert (ε value (ερ value) per residue of phosphoric acid, manufactured by Yamasa Oil Co., Ltd.)

^{55 From the} damages attached (e.g., Pharmacia Co., Ltd., ε billion (ερ 值) per residue of phosphoric acid)

Mixing curve ratio example 1

Using A s Α and A 0 Α, the ability to form a complex with Poly II was examined by measuring a mixing curve. Prepare a 260 uM solution of each compound using ε 值 in Table 1, Add Poly U (2-x) and measurement buffer (20ralHJ acid buffer solution (0.2M NaCDpH 7.0, 20mH D acid acid bite solution (1. OM NaCDpH 7.0 or 20raMU)) to each solution X mi (within 2 ^) Acid solution (2. OM NaCDpH 7.0) 2 «2 were mixed with each other, and the absorbance was measured with a Hitachi spectrophotometer U-3200 while stirring at a constant temperature of 4 or 24 t /" 0

As a result, as shown in FIGS. 1 and 2, under the conditions of 0.5 M sodium chloride and 4 tl, As A had a bending point (forming a triple chain with Poly II) only at about 33%. On the other hand, in A 0 Α, the inflection point (Poly I and double strand formation) was observed only at 50%.

Comparative Example 2

Under the conditions of 4 :, the ability to form a complex with PDly T was measured using ApA, d (ApA), AsA, and AoA in the same manner as in Comparative Example 1. investigated.

Table 2 shows the results.

Table 2 Complex formation ability with Poly T based on measurement of mixing curve

N a C 1 degree

Dimer 0.1 M 1.0 M

A p A Do not form complex Do not form complex

d (A p A) does not form complex forms double and triple chains

A o A Form double and triple chains Form double and triple spears

A s A Form double and triple chains Form triple chains

4 in Sectioning curve (measurement of Tm plant)

When nucleic acids form a complex, the absorbance sharply increases around a certain temperature. This is the so-called light-color effect of nucleic acids ( da

This is due to the decrease in the absorbance of the nucleic acid, and the temperature at which the absorbance increased to one-half of the absorbance difference before and after this (called “Tm” under £ 1) was measured to form a complex with the nucleic acid. Noh is known and is commonly used. A compound having a higher Tm 值 value can form a stronger complex with sutonic acid, which is advantageous as a drug or a direct regulator of a gene. Thus, using the dimer of A p A, d (A p A), A o A and As A, a complex with Poly ϋ and Poly T is formed, respectively, and the Tm value of these is measured. Comparative test of complex formation ability Comparative Example 3

Place each dimer and Poly I in a 1: 1 ratio in a stirring cell for a spectrophotometer, and use a Hitachi spectrophotometer U-3200, from 0 t to 30: at a rate of 0.5 per minute. As the temperature rose, the change in absorbance at 260 nm was measured.

As a result, a melting curve as shown in FIG. 3 was obtained, and a Tm value as shown in Table 3 was obtained.

Table 3 Tm 值 of each dimer for Poly U

Tm value

Salt concentration * Poly II: dimer AsA AoA ApA d (ApA)

0.1M 1: 1 11.7 7.7 <0 <0

1.0M 2: 1 16.9 13.6 11.5 12.0 Sodium chloride concentration in Ora iJ phosphate buffer (PH 7.0) As is clear from Table 3 above, the Tmil of As A It is clearly higher than the dimer, indicating that As A has a strong ability to form a complex.

Comparative Example 4

TmiS of each dimer with respect to Poly T was measured in the same manner as in Comparative Example 2, and the results shown in Table 4 were obtained. Table 4 Tm bales of each dimer for Poly T

Ί m £

Salt concentration * Poly T: dimer AsA AoA APA d (ApA)

0.1M2: 1 13.8 11.7 <0 <0

1.0M2: 1 17.9 14.2 <0 15.5

* Concentration of sodium chloride in 10 mM phosphate buffer (PH 7.0) As shown in Table 4 above, As A has the strongest ability to form a complex with Poly T, followed by d (A p A) , AoA, and ApA in this order.

Combining the results of Comparative Example 3 and Comparative Example 4, the strength of the complex-forming ability (from Tm）) of each dimer and nucleic acid under the condition of 1.0 M sodium chloride II is listed as follows. become.

(AsA: Poly T)> ( AsA: Poly ϋ)> (d (A P A): Poly T)> (AoA: Poly T)> (AoA: Poly U)> (d (ApA): Poly ϋ)> (APA: Poly U)> (ApA: Poly T) Thus, As A is stronger with nucleic acids than any other dimer Obviously, it has the ability to form a complex.

Comparative Example 5

The Tm 值 of the tetramer of Example 5 against Poly T and Polyϋ was measured in the same manner as in Comparative Example 3, and the results shown in Table 5 were obtained. Table 5 Tetramer of Example 5 for Poly T and Poly ϋ

Tm plant

Tm plant)

Poly T: Tetramer Poly II: Tetramer

Salt concentration * 1: 1 1: 1

0.15 32.0 32.0

* Natrium chloride concentration in 10fflM phosphate buffer (PH 7.0) As is clear from Table 5 above, the nucleic acid homologue of the present invention is sufficiently stable under physiological conditions in tetramers. It has enough bond stability.

As described above, the thiocarbamate-type nucleic acid homolog that is the antisense molecule of the present invention binds not only to the natural type but also to DNA or RNA more strongly than any known nucleic acid homolog, and forms a stable complex. Can be. Such properties are evidently advantageous as pharmaceuticals or as various gene regulators.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 show the mixing curves of As A and A0A dimers for Poly II in 0.5M sodium chloride at 4%. The abscissa represents the ratio (mol) of each dimer in the solution in each figure, and the ordinate represents the absorbance. FIG. 3 shows the melting curve of each dimer for Poly U in 0.1M sodium chloride at 4 :. The horizontal axis represents temperature (t), and the vertical axis represents the ratio of absorbance (change rate) at a UV wavelength of 260 IHD.

Claims

The scope of the claims

1. The following general formula [: I]

ί I

(Wherein R ¹ and R ² are the same or different and represent hydrogen, an inorganic acid residue, an organic acid residue, alkyl or acyl. B ¹ , B ² , B ³ are the same or different and are adenine, Represents guanine, hypoxanthine, cytosine, thymine or peracil, II represents an integer of 0 or more, provided that when n is 2 or more, B ² is not limited to the same base;

A nucleic acid homolog represented by: