KR20030076805A - Novel PNA Monomer Having Photo-labile Protecting Group - Google Patents

Abstract

The present invention relates to a compound of formula (I) having a photo-labile protecting group as a monomer for preparing a peptide nucleic acid (PNA) oligomer and a method for preparing the same.

Formula (1)

In the above formula,

R is

Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1;

B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally. The present invention also relates to a method for producing a PNA oligomer from the novel monomers and a PNA chip which can be produced from the monomers.

Substituents having a sulfonyl group in the compound of formula (1) serve as a protecting group of the PNA backbone, activation of amide bond formation in PNA oligomer synthesis, and three roles applied to photolithography. The monomers of the present invention having these characteristics are PNA oligomers, PNA / DNA chimera, PNA / peptide conjugates and various labelable basic compounds using various combinatorial chemistry techniques. Can be used as In particular, the monomer of the present invention can be used to manufacture PNA chips that can replace DNA chips through exposure techniques.

Description

Novel PNA Monomer Having Photo-labile Protecting Group

The present invention relates to a PNA monomer having a photolysis protecting group and a method for producing the same. The present invention also relates to a method for producing a PNA oligomer from the novel monomers and a PNA chip which can be produced from the monomers.

Photodegradation protecting groups are widely used for the immobilization of organic synthetic chemical and physiologically activated molecules, for the production of light-controlled biopolymers, in particular for the preparation of high-density DNA chips. Monomers of nucleotides or nucleosides with photodegradation protecting groups have been applied to parallel synthesis of oligonucleotides by light control on a substrate, which is an important technique in the manufacture of DNA chips. The oligo DNA chip is divided into two types according to the manufacturing method. The off-chip manufacturing method in which the oligo DNA chip is prepared and purified by a DNA synthesizer in advance and then integrated on a solid surface by a mechanical method, an ink jet method, or an electrochemical method. And on-chip preparations that directly synthesize oligomeric DNA on a chip (E. Crapez, Nature Genet. 21, 1, 1999). The off-chip method has the advantage of controlling the purity of the oligo DNA probe. The purity and amount of oligo DNA can be controlled and the variety of solid surfaces available can be selected to increase the reliability of experimental results using chips. In addition, the type of oligomeric DNA can be easily changed and can be produced at a low price. However, mechanically integrated off-chip has a relatively low degree of integration and has a disadvantage in that the accuracy of the experimental results such as unevenness of spots or contamination between spots in the process of integration on solid surfaces is reduced. The on-chip method is a patented technology developed by Affymetrix, USA (US 5,800,992; US 5,445,934; US 5,744,305) and is the most common DNA chip currently commercially available. This technique uses photolithography, which is used in semiconductor manufacturing, to synthesize oligo DNA on the glass surface step by step and to integrate 65000-400,000 oligo DNA in 1.6cm2. Exposure technology is a technique that has been used in the manufacturing process of a semiconductor chip is a method of passing a light through a photo mask to a surface coated with a photodegradable material to create a uniform shape. This technique has been used to manufacture microlenses used in electronic detectors (P. Nussbaum et al., Pure Appl . Opt . 1977 , 6, 617-636) or to increase the sensitivity of urea detectors (A Steinschaden et al., Sens. Actua B-Chem . 1977 , 44, 365-369). Affymetrix developed the technology to integrate the largest number of oligomeric DNA available today. When a photomask is used to remove a photoprotective group at a specific position on the glass surface coated with a photodegradable material, the newly supplied monomer reacts and attaches only at the position where the protective group is removed. After one monomer is attached to all the positions where the light protecting group is removed, another photo mask is applied to attach the next monomer, where a monomer having a photodegradable protecting group is essential. Repeat these steps to attach 1525 monomers in sequence (SP Fordor et al., Science 1991 , 251, 767-773; L. Wodicka et al., Nat. Biotechnol . 1997 , 15, 1359-1367). The main advantage of this technique is that it can be synthesized directly from the nucleic acid sequence database using four photodegradable protecting groups, A, T, G and C, without the need for direct handling of oligo DNA samples, unlike the off-chip method. In addition, the use of established semiconductor manufacturing process technology can maintain the uniformity of each synthesis step has the advantage of minimizing the quality difference between each chip. On the other hand, there is a disadvantage of the manufacturing technology of the oligo DNA chip using the exposure technique. In order to synthesize oligo DNA consisting of 20 bases, various types of photomasks have to be manufactured, which is expensive and does not respond quickly to synthesis of changed nucleotide sequences. In addition, it is difficult to control the quality of the synthesized oligo DNA due to the characteristics of the disposable DNA chip. Affymetrix manufactures 10 complementary oligomeric DNAs for one gene and a control group with different nucleic acid sequences for each gene to increase chip sensitivity. In the end, 20 kinds of oligo DNA are produced for one gene. In addition, for quality control, a large number of homogeneous chips are manufactured on a large glass plate, and a part of the sample is used to check the quality of the synthesized oligo DNA.

The basic requirement for photodegradation protecting groups used to make DNA chips is that the light must be decomposed at wavelengths longer than 340 nm. This is because damage to nucleic acid bases is more likely to occur at wavelengths shorter than 340 nm (MC Pirrung and L. Fallon, J. Org. Chem . 1998 , 63 , 241). In addition, heat stability should be good and if possible, photolysis should occur in a short time.

Since the discovery of photoreactions in 1901, 2-nitrobenzyl-type photoprotectors have been used the most (G. Ciamician and P. Silber, Chem. Ber . 1901 , 34 , 2040). Understandably (JA McCray and DR Trentham, Annu. Rev. Biophys. Chem . 1989 , 18 , 239), studies have been actively conducted on derivatives. The most frequently used derivative is 1- (3,4- (methylenedioxy) -6-nitrophenyl) ethyloxycarbonyl (MeNPOC). As shown in the following scheme, the derivative increases the photolysis rate, and the half-life (t _1/2 ) changes depending on the solvent, and the photolysis occurs very quickly from 10 seconds to 30 seconds (GH McGall, et al. J. Am) . Chem. Soc . 1997 , 119 , 5081).

The photo-degradation mechanism of the o-nitrobenzyloxy form traps benzylic hydrogen atoms by light-activated nitro groups, causing oxygen atoms to move to the benzylic position, whereby decomposition occurs, together with o-nitroso acetphenone, carbon dioxide and alcohol. (H. Morrison and BH Migdalof, J. Org. Chem . 1965 , 30 , 3996).

Papers and patents have been published by Affymetrix, which published a study on DNA chips using 1- (3,4- (methylene dioxy) -6-nitrophenyl) ethyloxycarbonyl (MeNPOC). 1- (3,4- (methylenedioxy) -6-nitrophenyl) ethyloxycarbonyl (MeNPOC) was used here in combination with 5'-OH of deoxynucleosides (AC Pease, et al. Proc) . Natl.Acad . Sci. USA 1994 , 91 , 5022; USP 5,489,678 to Fodor et al., And US 5,889,165 to Fodor et al.). In addition, 3, 4- dimethoxy may be used instead of a 3, 4- methylenedioxy substituent.

Another class of photolytic protecting groups is benzoin derivatives. The first report of this compound was in 1971 (JC Sheehan et al . J. Am. Chem. Soc . 1971 , 93 , 7222). Research results on the effective synthesis of benzoin have been published (HB Michael et al. Tetrahedron Lett. 1996 , 307; and G. Papageorgiou, et al. Tetrahedron 1997, 3917). It was also used for oligonucleotide synthesis and DNA chips using this photodegrader (MC Pirrung and L. Fallon, J. Org. Chem . 1998 , 63 , 241). According to this report, the half-life is about 10 seconds. In addition, it can be used as a linker capable of photolysis in combinatorial chemistry (HB Lee et al. J. Org. Chem . 1999 , 64 , 3454).

Another widely used compound is a compound that uses a 2-methyl-2- (2-nitrophenyl) propyloxycarbonyl group, but the o-nitro group activates the photoreaction, but 1- (3,4- (methylenedioxy)- It has a different reaction mechanism than 6-nitrophenyl) ethyloxycarbonyl (MeNPOC). That is, it follows the reaction mechanism of the beta-elimination reaction rather than the movement of oxygen atoms. Many studies on the synthesis and physical properties of this compound and its derivatives have been reported (A. Hasan, et al. Tetrahedron 1997 , 53 , 4247).

This photolytic protecting group study was also attempted for use in DNA oligomer synthesis and DNA chip fabrication (S. Walbert, et al. Helv. Chim. Acta 2001 , 84 , 1601; review CHEMTRACKS-ORGANIC CHEMISTRY 2000 , 13 , 487 )

Introduction of photolytic protecting groups corresponding to most oligomeric synthesis and DNA chip manufacturing methods was performed using the 5'-OH position, and a method of introducing a photolytic protecting group at the 3'-OH position was also reported (M. Beier, et al. Helv) . Chim Acta 2001 , 84 , 2089).

On the other hand, there was a report about the oxy remove the amine protecting group introduced directly (M. Rinnova, et al. J. Peptide Sci. 2000, 6 .355).

A method for preparing oligomeric DNA chips by introducing sulfonyl group derivatives as photolysis protecting groups instead of oxycarbonyl groups has also been studied (W. Pflederer et al. USP 6,153,744).

WO 00/61594 describes a process for preparing oligomeric DNA chips by introducing sulfonyl group derivatives as photolysis protecting groups on 3′-OH groups.

In addition, there have been reports of a method for protecting amine groups by directly sulfonating. In this case the photolytic reactivity is determined by the form of the sulfonamide substituents (JET Corrie and G. Papageorgiou, J. Chem. Soc., Perkin Trans . 1 1996 , 1583 ).

Over the last few decades, a large number of chemicals have been reported that modify oligonucleotides to have new properties (biochemical or physical), and many studies have been conducted to optimize the properties by changing the skeleton of nucleobases, phosphates and ribose in parts. It became. One of the biggest shifts has been to develop PNAs that replace ribose rings and phosphates with 2-aminoethylglycine, reported by Nielsen, Denmark (PE Nielsen). , et al. Science , 1991 , 254 , 1497).

PNAs can pair DNA or RNA with Watson-Crick bases and are electrically neutral so PNA / DNA and PNA / RNA complementarity is greater than the affinity of DNA / DNA or DNA / RNA complementarity (M. Egholm, et al. Nature , 1993 , 365 , 566). Moreover, PNA can be parallel or antiparallel complementary to DNA or RNA (E. Uhlmann, et al. Angew. Chem. Int. Ed. Engl . 1996 , 35 , 2632). Irrespective of their complementary properties (S. Tomac, et al. J. Am. Chem. Soc . 1996 , 118 , 5544). On the other hand, PNA / DNA or PNA / RNA complementary bonds are more sensitive to mismatches as they have a high affinity with DNA or RNA. This may mean that PNA can have a big advantage over DNA as a biosensor. In addition, PNA is stable against nucleases or peptidase (V. Demidov, et al. Biochem. Pharmacol. 1994, 48, 1310) DNA or RNA in the presence of strong acids or bases While reactions such as depurination and hydrolysis occur, PNA is very stable in acid and fairly stable under basic conditions.

Due to the above characteristics, PNA chips have many advantages over DNA chips and can be widely applied. Therefore, there is an urgent need for the development of basic technologies for making chips. In such a situation, the inventors of the present invention can not only be used to synthesize oligomers, but also manufacture PNA chips through parallel synthesis of PNA oligomers having various kinds of sequences through lithography by light control on a substrate. A monomer having a photolysis protecting group can be used as a base material that can be used in combination with a DNA chip or a PNA chip that can be substituted.

In one aspect, the present invention provides a PNA monomer represented by formula (1) having a photolytic protecting group:

Formula (1)

In the above formula,

R is

Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1;

B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.

As another aspect, the present invention provides a method for producing the PNA monomer represented by the formula (1).

As another aspect, the present invention provides a PNA monomer represented by the formula (1) to produce a PNA chip.

In another aspect, the present invention provides a compound of formula (5):

Formula (5)

In the above formula,

R is

Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; HX is an inorganic acid or an organic acid.

In another aspect, the present invention provides a method for preparing the compound of formula (5).

Figure 2 is a chart showing the chemical formula of naturally occurring or non-naturally occurring nucleic acid bases useful in the present invention.

The monomer compound of the formula (1) according to the present invention may be prepared by using various combinatorial chemistry techniques, such as a peptide nucleic acid (PNA) oligomer, a PNA / DNA chimera, a labeling compound of a peptide and a PNA (PNA / peptide conjugate), and It can be used as a basic labelable compound. In particular, PNA chips, which can replace DNA chips, can be used to produce photolithography. The sulfonyl derivative as a backbone protecting group of the monomers shown in the present invention may have three roles that may be simultaneously used as protecting groups of the PNA monomer skeleton for PNA oligomer synthesis, activation of amide bond formation in oligomer synthesis, and exposure techniques. You can show the advantages. In particular, when the photolytic protecting group is used, the range to be selected as the protecting group of the nucleic acid base has an advantage.

Preferred R in the monomeric compound of formula (1) according to the invention is a compound represented by the formula:

Wherein R 1, R 4, R 5 and n are as defined above.

Preparation of PNA monomer skeleton having photolysis protecting group

In the present invention, a sulfonyl derivative capable of photolysis is used as a protecting group for the amino group of 2-aminoethylglycine, which is a PNA basic skeleton. In Scheme 1, the compound of formula (5) is an important intermediate for synthesizing the monomer of the present invention.

The first reaction (conversion from compound of formula (1) to compound of formula (3)) is a known method (DW Will et al. Tetrahedron, 1995 , 51 , 12069.). Sulfonylation was carried out using the compound of formula (2) in the presence of a base. The base used may be a tertiary amine organic base such as triethylamine, diisopropylethylamine, N-methylmorpholine, DBU and inorganic bases such as potassium carbonate and cesium carbonate, but a tertiary amine organic base is preferable. Do. The reaction temperature may be from 10 ° C to 50 ° C, but a suitable temperature is from 0 ° C to 30 ° C. Solvents used therein include aromatic hydrocarbons such as water, benzene and toluene, esters such as ethyl acetate, haloalkanes such as dichloromethane and chloroform, ethers such as tetrahydrofuran, diethyl ether and isopropyl ether, Ketone species such as acetone, methyl ethyl ketone, nitrile species such as acetonitrile, propionitrile, amide species such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone and mixed solvents of the above solvents can be used. However, preferred solvents include haloalkane species, nitrile species and amide species.

The conversion of a compound of formula (3) to a compound of formula (4) is a series of two reactions that undergo hydrolysis under basic conditions and then react with di-t-butyl dicarbonate ((t-BuOCO) ₂ O). It can be prepared through the sequential reaction in one reactor. It is most preferable to use R6 as a C1-C4 alkyl group which can be hydrolyzed under basic conditions, or a cyanoethyl group which can be converted into a carboxylic acid in an organic base compound. When hydrolyzing with alkali metal hydroxides such as LiOH, NaOH, KOH, etc., it uses an organic solvent that can be dissolved in water as a solvent, and can be mixed with water, such as tetrahydrofuran, dioxane, dimethoxyethane, ethanol, etc. This is suitable. The reaction temperature range may be -30 ^o C to 70 ^o C, but room temperature is preferred.

The cyclization reaction (conversion of the compound of formula (4) to the compound of formula (5)) is carried out in peptide synthesis such as HATU, HBTU, HOObt, DCC, EDC, BOP, PyBOP, PyProP, TBTU, TSTU, TNTU, TOTU Coupling reagents used to carry out amidation reactions using carboxylic acids and amine groups to form amides can be used. At this time, solvents include dichloromethane, dimethylformamide, dimethylacetamide, amides such as N-methylpyrrolidone, tetrahydrofuran and the like, and amides such as dimethyl formamide, dimethylacetamide and N-methylpyrrolidone. Is preferred. Alternatively, chloroisobutylformate can be reacted with N-methylmorpholine. Dichloromethane, dimethylformamide, tetrahydrofuran, etc. can be used as a solvent, but tetrahydrofuran is preferable. The reaction temperature is -30 ^o C ~ 50 ^o C when using coupling reagents such as HATU, HBTU, HOObt, DCC, EDC, BOP, PyBOP, PyProP, TBTU, TSTU, TNTU, TOTU. When reacting with N-methylmorpholine, -20 ^o C ~ 0 ^o C is suitable. To remove the t-butyloxycarbonyl protecting group, a halogen acid such as HCl / ethanol and HCl / ethyl acetate, an organic acid / organic solvent such as sulfuric acid, nitric acid / organic solvent, and trifluoroacetic acid / dichloromethane can be used. Obtain the compound of 5). At this time, the reaction temperature is performed at room temperature. Room temperature is 25 ^o C unless otherwise specified.

Scheme 1

In the above scheme,

R is

Wherein R1, R2, R3 and R4 are the same or different and each independently hydrogen, nitro group, nitrile group, halogen atom, (C1-C4) alkyl group, (C1-C4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; HX represents halogen acid, sulfuric acid, nitric acid, alkanesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid and the like; R 6 is a (C ₁ -C ₄ ) alkyl or cyanoethyl group.

Preparation of Monomer of Formula (1)

Monomers of formula (1) for preparing oligomers are prepared as shown in Scheme 2 below.

Scheme 2

Wherein R and B are as defined above.

The method for preparing the compound of formula (1) uses methods well known in peptide synthesis chemistry. The amidation reaction (conversion of the compound of formula (5) to the compound of formula (1)) is carried out in peptide synthesis such as HATU, HBTU, HOObt, DCC, EDC, BOP, PyBOP, PyProP, TBTU, TSTU, TNTU, TOTU, etc. Coupling reagents used for carrying out the amidation reaction to form amides using carboxylic acid and amine groups can be used. At this time, solvents include dichloromethane, dimethyl formamide, dimethyl acetamide, amides such as N-methylpyrrolidone and tetrahydrofuran, and amides such as dimethylformamide, dimethylacetamide and N-methylpyrrolidone. Is preferred. In this process, the reaction temperature is -20 ^o C ~ 30 ^o C.

Detailed description by nucleic acid base

Nucleic acid base

Nucleic acid bases that can be usefully used in the present invention are specifically illustrated in FIG. In the present invention, these nucleic acid bases may or may not be protected for the production of PNA oligomers. Protecting groups that can be used in the present invention for nucleic acid bases include, but are not limited to, (4-methoxy) phenyldiphenylmethyl (Mmt) (G. Breipohl et al., Bioorg. Med. Chem. Lett., 1996 , 6, 665), benzhydryloxycarbonyl (Bhoc) (US 6172226), An (il) (DW Will et al., Tetrahedron, 1995, 51, 12069, 2-methylthioethoxycarbonyl (US) 6063569), benzyloxycarbonyl (PE Nielsen et al., Science, 1991, 254, 1497; M. Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677; M. Egholm et al. J. Am. Chem. Soc., 1992, 114, 1895; M. Egholm et al., J. Chem. Soc. Chem. Commun., 1993, 800; KL Dueholm et al., J. Org.Chem. , 1994, 59, 5767; and WO92 / 20702), and a number of protecting groups that can be used as a protecting group of a stable nucleotide amines in the base (TW Greene and PGM Wuts, Protective Group in Organic Synthesis, 3 ^rd Edition, pp 494 653) and 4-methoxy-benzoyl, 4-t-butyl-benzoyl, isobutan oil [Z. Timar, J. Chem. Soc., Perkin Trans. 1, 2 000, 19; DW Will et al. Tetrahedron, 1995, 51, 12069.], such as TW Greene and PGM Wuts, Protective Group in Organic Synthesis, 3 ^rd Edition, pp. 494 to 653. As a preferred embodiment, the nucleic acid base of the present invention is specifically illustrated as thymine, guanine, cytosine and adenine.

Preparation of Thymine (T) Monomer

Thimine-1-ylacetic acid represented by the following formula is synthesized by known methods (KL Dueholm, et. Al. J. Org. Chem. 1994 , 59 , 5767; and O. Buchardt et al. WO 92/01219). Was used:

Through the amidation reaction to make an amide of piperazinone and thymine-1-ylacetic acid represented by the formula (5) protected by a sulfonyl group substituted with R as shown in Scheme 3 below, As a method for synthesizing the monomer represented by the formula (1T), the monomer can be obtained by one step reaction of the secondary amine portion of the skeleton and thymine-1-ylacetic acid. The conversion of the compound of formula (5) to the compound of formula (1T) was obtained by the amidation reaction described above.

Scheme 3

Wherein R and HX are as defined above.

Preparation of cytosine (C), adenine (A), guanine (G) monomers protected with protecting groups

Cytosine, adenine, guanine monomers protected with protecting groups were synthesized from derivatives of nucleic acid bases having respective protecting groups.

1) Preparation of nucleic acid base cytosine having a protecting group and monomer thereof

A) [4-N- (benzyloxycarbonyl) cytosine-1-yl] -acetic acid having a protecting group of benzyloxy carbonyl group on the 4-NH ₂ group of cytosine is described in Dueholm et al. J. Org. Chem . 1994 , 59 , 5767; And in O. Buchardt et al. Synthesized and used in the manner reported in WO 92/21702.

Scheme 4

B) [4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -acetic acid having a protecting group of a benzhydryloxycarbonyl group on the 4-NH ₂ group of cytosine is described in Scheme 5 below. JM Colull, et al. US 6,172,226: and BD Gildea et al. Synthesized by the method reported in US Pat. No. 6,265,559.

Scheme 5

C) {4-N- [2- (methylthio) ethoxycarbonyl] cytocin-1-yl} -acetic acid having a protecting group of 2- (methylthio) ethoxycarbonyl group on 4-NH ₂ group of cytosine is represented by the following scheme As shown in 6, JM Colull, et al. US 6,172,226B] was used synthesized by the method reported.

Scheme 6

D) {4-N- [4- (methoxy) benzoyl] cytosine-1- having a protecting group of 4- (methoxy) benzoyl, 4- (t-butyl) benzoyl isobutyryl group in the 4-NH ₂ group of cytosine Il} -acetic acid, {4-N- [4- (t-butyl) benzoyl] cytosine-1-yl} -acetic acid and [4-N- (isobutyryl) cytosin-1-yl] -acetic acid As shown in 7, 8 and 9, Z. Timar, J. Chem. Soc, Perkin Trans. 1, 2000, 19.] was synthesized by using the method reported in the application.

Scheme 7

Scheme 8

Scheme 9

Preparation of the monomer from the nucleic acid base cytosine having the protective group prepared above was prepared using the method for preparing the thymine monomer. Scheme 10 shows a method of preparing a cytosine monomer (1C) by reacting a compound of formula (5) with cytosine acetic acid having a protecting group.

Scheme 10

In the above formula,

R and HX are as defined above;

Pg represents benzyloxy carbonyl, benzhydryloxy carbonyl, 2- (methylthio) ethoxy carbonyl, 4- (methoxy) benzoyl, 4- (t-butyl) benzoyl or isobutyryl.

2) Preparation of Nucleic Acid Base Adenine and its Monomer Having Protective Group

A) [6-N- (benzyloxycarbonyl) adenin-9-yl] acetic acid having a protecting group of benzyloxy carbonyl group on 6-NH ₂ group of adenine is known as shown in Scheme 11 (SA Thomson et al. Tetrahedron 1995 , 6179.) was used to prepare a monomer.

Scheme 11

B) [6-N- (benzhydryloxycarbonyl) adenin-9-yl] -acetic acid shown in Scheme 12 below was synthesized by a known method (JM Coull, et al. US 6,172,226.) And used to prepare monomers. It was.

Scheme 12

C) {6-N- [2- (methylthio) ethoxycarbonyl] adenin-9-yl} acetic acid shown in Scheme 13 below shows [6-N- (benzhydryloxycarbonyl) adenine-9- Synthesis was carried out by the same method as for acetic acid, and used for preparing a monomer.

Scheme 13

D) {6-N- [4- (methoxy) benzoyl] adenine-9- having a protecting group of 4- (methoxy) benzoyl, 4- (t-butyl) benzoyl isobutyryl group in the 6-NH ₂ group of adenine Il} -acetic acid, {6-N- [4- (t-butyl) benzoyl] adenin-9-yl} -acetic acid and [6-N- (isobutyryl) cytocin-9-yl] -acetic acid As reported in 14, 15 and 16, the method reported in Z. Timar, J. Chem. Soc., Perkin Trans . 1 , 2000 , 19. was used or applied synthetically.

Scheme 14

Scheme 15

Scheme 16

Preparation of the monomer from the nucleic acid base adenine having a protective group prepared above was prepared using the method for preparing the thymine monomer. Scheme 17 below shows a method for preparing adenine monomer (1A) by reacting a compound of formula (5) with adenine acetic acid having a protecting group.

Scheme 17

In the above formula,

R and HX are as defined above;

Pg represents benzyloxycarbonyl, benzhydryloxycarbonyl, 2- (methylthio) ethoxycarbonyl, 4- (methoxy) benzoyl, 4- (t-butyl) benzoyl or isobutyryl.

3) Preparation of nucleic acid base guanine having a protecting group and monomer thereof

A) [2-N- (benzyloxy carbonyl) guanin-9-yl] acetic acid shown in Scheme 18 is described in J. M. Coull, et al. A method known in US Pat. No. 6,172,226 was modified and synthesized. Hydrolysis reaction was carried out with LiOH using the more reactive iodine group instead of chloro at 6 position, ethyl ester instead of benzyl group, ethyl group instead of benzyl group, and iodine group using cyano ethanol. -(Benzyloxy carbonyl) guanine-9-yl) acetic acid was synthesized.

Scheme 18

B) (2-N- (benzhydryloxycarbonyl) guanin-9-yl) acetic acid shown in Scheme 19 is described in J. M. Coull, et al. A method known in US Pat. No. 6,172,226 was modified and synthesized. In the 6th position, hydroxy group is used instead of chloro for more reactivity, ester of acetic acid is used for ethyl group instead of benzyl group for hydrolysis reaction to LiOH, and then iodine group is used for cyanoethanol to give the desired monomer precursor (N2- ( Benzhydryloxy carbonyl) guanine-9-yl) acetic acid was synthesized.

Scheme 19

C) {2-N- [2- (methylthio) ethoxycarbonyl] guanine-9-yl} acetic acid shown in Scheme 20 is described in J. M. Coull, et al. A method known in US Pat. No. 6,172,226 was modified and synthesized. It is a nucleic acid base derivative for synthesizing a desired monomer by using a more reactive iodine group in place of chloro and a hydrolysis reaction with LiOH using ethyl group instead of benzyl group in 6-position, and then using cyanoethanol for iodine group. {2-N- [2- (methylthio) ethoxycarbonyl] guanine-9-yl} acetic acid was synthesized.

Scheme 20

D) Guanine having a protecting group of isobutyryl is described in DW Will et al. Tetrahedron , 1995 , 51 , 12069.] were prepared by the reported method.

Scheme 21

Monomer preparation from the nucleic acid base guanine having a protective group prepared above was prepared using the method for preparing the thymine monomer. Scheme 22 below shows a method for preparing a guanine monomer (1G) by reacting a compound of formula (5) with guanine acetic acid having a protecting group.

Scheme 22

In the above formula,

R and HX are as defined above;

Pg represents benzyloxy carbonyl, benzhydryloxy carbonyl, 2- (methylthio) ethoxy carbonyl or isobutyryl.

Preparation of oligomer

In the present invention, a PNA oligomer may be prepared through a series of reactions as shown in Schemes 23 and 24. By applying a conventional technique using a nitro benzene ring as a photodegradation protecting group to a new monomer of the present invention, oligomers can be prepared on a solid support in a new way, and PNA oligomers having various kinds of sequences can be prepared using exposure techniques. It suggests what can be prepared in solids.

Scheme 23

In the above formula,

R is

Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1;

B ₁ and B ₂ are nucleic acid bases identical or different from one another, protected or unprotected, and either naturally or non-naturally present.

Schematic representation of the reaction scheme is as follows (Figure 1).

1

The invention will be illustrated more specifically in the following examples. However, these embodiments are merely embodiments of the present invention and do not limit the scope of the present invention.

Example

1. Preparation of Sulfonyl Chloride

2- (2-nitro-phenyl) -ethanesulfonyl chloride was prepared by a known method (W. Pfleiderer et al US 6153744) and 1- (6-nitro-benzo [1,3] dioxol-5-yl) Ethanesulfonyl chloride was prepared by the following method.

Example 1

5- (1-chloro-ethyl) -6-nitro-benzo [1,3] dioxol (5- (1-Chloro-ethyl) -6-nitro-benzo [1,3] dioxole

1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanol (21.1 g) prepared by known method (GH McGall, J. Am. Chem. Soc . 1997 , 119 , 5081.). , 0.1 mole) was dissolved in dichloromethane (100 mL), and thionyl chloride (35.7 g, 0.3 mole) was slowly added at room temperature, followed by stirring at room temperature for 12 hours. The reaction solution was removed under reduced pressure and the excess thionyl chloride was dissolved in ethyl acetate (150 mL), washed with water (100 mL) and brine, the organic layer was dried over anhydrous forget-me-not, the solvent was removed, and the residue was subjected to column chromatography. Purification via gave the title compound (15.4 g, 67%).

¹ H NMR (DMSO-d6)

7.37 (s, 1 H), 7.27 (s, 1 H), 6.12 (s, 1 H), 5.78 (q, 1 H), 1.84 (d, 3 H).

Example 2

Thioacetic acid S- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethyl] ester (Thioacetic acid S- [1- (6-nitro-benzo [1,3] dioxol- 5-yl) -ethyl] ester)

5- (1-chloro-ethyl) -6-nitro-benzo [1,3] dioxol (11.48 g, 0.05 mole) was dissolved in dimethyl formamide (100 mL) and then potassium thioacetate (8.59 g, 0.075 mole) ) Is added and stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed under reduced pressure, the residue was dissolved in ethyl acetate (100 mL), washed twice with water (100 mL), the organic layer was dried over anhydrous forget-me-not, the solvent was removed, and the residue was purified by column chromatography. Purification through gave the title compound (12.25 g, 91%).

¹ H NMR (DMSO-d6)

7.46 (s, 1 H), 7.26 (s, 1 H), 6.30 (s, 2 H), 5.08 (q, 1 H), 1.60 (d, 3 H).

Example 3

1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl acid sodium salt (1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonic acid sodium salt)

Thioacetic acid S- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethyl] ester (5.38 g, 0.02 mole) was dissolved in acetic acid (80 mL) and 30% hydrogen peroxide ( 22 mL) is added at room temperature and then stirred at room temperature for 15 hours. After the reaction was completed, sodium acetate (1.64 g, 0.02 mole) was added, followed by stirring for 1 hour, and then 200 mg of Pd / C (10%) was added to remove excess hydrogen peroxide, and the bubble was stopped. (2-3 hours) Stir and filter to remove Pd / C. The solvent in the reaction solution was completely removed under reduced pressure and the residue was solidified with ethyl ether to obtain the title compound (5.88 g, 99%).

¹ H NMR (DMSO-d6)

7.40 (s, 1 H), 7.29 (s, 1 H), 6.17 (d, 2 H), 4.37 (q, 1 H), 1.47 (d, 3 H).

Example 4

1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl chloride (1- (6-Nitro-benzo [1,3] dioxol-yl) -ethanesulfonyl chloride)

1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulphonic acid sodium salt (5.2 g, 0.0175 mole) was added to dichloromethane (60 mL) to form a turbid solution, followed by phosgene (under nitrogen). 18 mL of 20% toluene solution) is added followed by dimethyl formamide (2 mL). The reaction solution was stirred at room temperature for 3 hours, washed twice with water (30 mL), the organic layer was dried over anhydrous forget-me-not, the solvent was removed under reduced pressure, the residue was purified by short column chromatography, and the solvent was removed. Used for the next reaction without purification.

¹ H NMR (DMSO-d6)

7.38 (s, 1 H), 7.27 (s, 1 H), 6.12 (s, 2 H), 5.79 (q, 1 H), 1.85 (d, 3 H).

2. Preparation of Sulfonyl-piperazin-2-one

Example 5

{2- [2- (2-Nitro-phenyl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester ({2- [2- (2-Nitro-phenyl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester.)

Dichloromethane (50 mL) was prepared by divalent methane hydrochloride (2.19 g, 10 mmole) of 2- (amino-ethylamino) -acetic acid ethyl ester prepared by a known method (DW Will et al. Tetrahedron 1995 , 51 , 12069.). Was suspended and triethyl amine (4.04 g, 40 mmole) was added. The suspended reaction solution was stirred at room temperature with 2- (2-nitro-phenyl) -ethanesulfonyl chloride (2.5 g 10 mmole) added dropwise for 5 minutes and allowed to react for 2 hours. When the reaction was completed, 30 ml of water was added to the reaction solution, the organic layer was dehydrated with anhydrous forget-me-not and concentrated under reduced pressure to obtain 3.36 g (93%) of the title compound.

¹ H NMR (DMSO-d6)

7.99 (d, 1H), 7.69 (t, 1H), 7.60 (d, 1H), 7.52 (t, 1H), 7.21 (s, 1H), 4.07 (q, 2H), 3.36 (m, 4H), 3.20 (m, 2H), 3.03 (br. s, 2H), 2.63 (t, 2H), 1.17 (t, 3H).

Example 6

{2- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester ({2- [1- (6-Nitro- benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester.)

In the same manner as in Example 5 above 2 equivalents hydrochloride (3.28 g, 15 mmole) of 2- (amino-ethylamino) -acetic acid ethyl ester The sulfonylchloride prepared in Example 4, above, afforded 5.45 g (90% yield based on 2 equivalents hydrochloride of 2- (amino-ethylamino) -acetic acid ethyl ester) of the desired title compound.

¹ H NMR (DMSO-d6)

7.59 (s, 1H), 7.33 (br. 1H), 6.24 (s, 2H), 5.17 (q, 1H), 4.08 (q, 2H), 3.28 (s, 2H), 2.86 (m, 2H), 2.53 (m, 2H), 1.64 (d, 3H), 1.18 (t, 3H).

Example 7

(t-butoxycarbonyl- {2- [2- (2-nitro-phenyl) -ethanesulfonylamino] -ethyl} -amino) -acetic acid ((t-Butoxycarbonyl- {2- [2- (2- nitro-phenyl) -ethanesulfonylamino] -ethyl} -amino) -acetic acid)

{2- [2- (2-Nitro-phenyl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester To a solution of (2.53 g, 7 mmole) in tetrahydrofuran (15 mL) was added a hydrate of lithium hydride (575 mg, 14 mmole) in water (15 mL), followed by 1 h at room temperature. Stirred. After completion of the hydrolysis, di-t-butyl dicarbonate (2.18 g, 10 mmole) was added to the reaction solution, stirred for 30 minutes, and then lithium hydroxide (290 mg, 7 mmole) dissolved in water (15 mL). Hydrate was added dropwise for 30 minutes. After the reaction was completed, the insoluble substance was filtered off, and the filtrate was washed with diethyl ether (30 mL) and the aqueous layer was separated. The separated aqueous layer was adjusted to pH = 3 by adding 2N aqueous hydrochloric acid, and then extracted by adding dichloromethane (50 mL). The separated organic layer was dried filtered over anhydrous forget-me-not and concentrated under reduced pressure to give 2.94 g (97%) of the title compound.

¹ H NMR (DMSO-d6)

8.00 (d, 1H), 7.69 (t, 1H), 7.60 (dd, 1H), 7.52 (t, 1H), 7.28 (m, 1H), 3.89 (s, 0.9H), 3.86 (s, 1.1H) , 3.30 (m, 4H), 3.19 (m, 2H), 3.11 (m, 2H), 1.38 (s, 4.1H), 1.32 (s, 4.9H).

Example 8

(t-butoxycarbonyl- {2- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethyl} -amino) -acetic acid ((t- Butoxycarbonyl- {2- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethyl} -amino) -acetic acid)

In the same reaction as in Example 7, {2- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethylamino} -acetic acid ethyl ester (4.03 g , 10 mmole) gave the title compound (4.51 g, 95%).

¹ H NMR (DMSO-d6)

7.59 (s, 1H), 7.36 (m, 1H), 7.24 (s, 1H), 6.24 (d, 2H), 5.14 (q, 1H), 3.83 (s, 1H), 3.80 (s, 1H), 3.58 (m, 2H), 3.18 (m, 2H), 2.91 (m, 2H).

Example 9

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride (1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one, HCl salt)

(t-butoxycarbonyl- {2- [2- (2-nitro-phenyl) -ethanesulfonylamino] -ethyl} -amino) -acetic acid (2.17 g, 5 mmole) in dichloromethane (30 mL) After dissolving, dicyclohexyl carbodiimide (1.24 g, 6 mmole) was added at room temperature, followed by stirring for 2 hours. Upon completion of the reaction, the insoluble dicyclohexyl urea was filtered off and the solution removed was concentrated under reduced pressure. Ethyl acetate (30 mL) was added to the concentrated residue, and the insoluble dicyclohexyl urea was filtered again, and the removed solution was cooled to 0-5 ° C., and then 2N hydrochloric acid ethyl acetate solution (30 mL) was added thereto. After stirring the reaction solution at room temperature for 10 hours, the resulting solid was filtered, washed with ethyl acetate (100 mL) and dried to give 1.50 g (85%) of the title compound as a white solid.

¹ H NMR (DMSO-d6)

10.05 (br.s, 1H), 8.04 (d, 1H), 7.72 (t, 1H), 7.64 (d, 1H), 7.55 (t, 1H), 3.96 (m, 4H), 3.49 (m, 4H) , 3.35 (m, 2 H).

Example 10

1- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride (1- [1- (6-Nitro-benzo [ 1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one, HCl salt

In the same reaction as in Example 9, (t-butoxycarbonyl- {2- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonylamino] -ethyl} -Amino) -acetic acid (3.8 g, 8 mmole) gave the title compound (2.55 g, 81%).

¹ H NMR (DMSO-d6)

9.96 (br.s, 1H), 7.62 (s, 1H), 6.27 (s, 2H), 5.83 (q, 1H), 3.98-3.80 (m, 4H), 3.51 (m, 2H).

Preparation of Monomer

Example 11

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- [2- (2-Nitro-phenyl ) -ethanesulfonyl] -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)

1.22 g (3.5 mmole) of 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride, 0.64 g (3.5 mmole) of thymine-1-yl acetic acid and 2.00 g of PyBOP ( 3.85 mmole) was suspended in 11 mL of N, N-dimethylformimide and 0.91 mL of diisopropylamine were added at room temperature. The reaction mixture was stirred at room temperature for about 2 hours and then slowly dropped into a mixture of water and ethanol to give a solid. The resulting solid was filtered, washed with ethanol and ethyl ether and dried to give 1.40 g (83% yield) of the target compound.

¹ H NMR (DMSO-d6)

11.3 (s, 1H), 8.03 (m, 1H), 7.72 (m, 1H), 7.60 (m, 1H), 7.55 (m, 1H), 7.34 (s, 0.6H), 7.30 (s, 0.4H) , 4.63 (s, 1.2H), 4.60 (s, 0.8H), 4.39 (s, 0.8H), 4.23 (s, 1.2H), 3.99-3.95 (m, 2.4H), 3.89-3.81 (m, 2.4 H), 3.72 (m, 1.6H), 3.33 (m, 1.6H)

Example 12

4-{[4-N- (benzyloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one ( 4-{[4-N- (Benzyloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (benzyloxycarbonyl) -cytosine-1- in the same manner as in Example 11 General] -acetic acid is used to obtain the title compound .

¹ H NMR (DMSO-d6)

8.04 (dd, 1H), 7.90 (dd, 1H), 7.72 (m, 1H), 7.61 (t, 1H), 7.55 (q, 1H), 7.42-7.33 (m, 5H), 7.00 (d, 1H) , 5.18 (d, 2H), 4.79 (s, 1.2H), 4.75 (s, 0.8H), 4.43 (s, 0.8H), 4.23 (s, 1.2H), 3.97 (m, 2H), 3.91-3.85 (br.m, 2.4H), 3.75 (br.s, 1.6H), 3.31 (m, 2H)

Example 13

4-{[4-N- (benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2- On (4-{[4-N- (Benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (benzhydryloxycarbonyl) -cytosine- in the same manner as in Example 11 1-yl] -acetic acid is used to obtain the title compound .

¹ H NMR (DMSO-d6)

8.04 (dd, 1H), 7.88 (dd, 1H), 7.71 (m, 1H), 7.60 (t, 1H), 7.54 (q, 1H), 7.45-7.26 (m, 10H), 6.95 (d, 1H) , 6.79 (s, 1H), 4.78 (s, 1.2H), 4.75 (s, 0.8H), 4.43 (s, 0.8H), 4.23 (s, 1.2H), 3.97 (m, 2H), 3.91-3.85 (br.m, 2.4H), 3.73 (br.s, 1.6H), 3.61 (m, 0.8H), 3.34 (m, 2.4H), 3.13 (m, 0.8H).

Example 14

4-{[4-N- (2-Methylthio-ethoxycarbonyl) -cytocin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazine 2-one (4-{[4-N- (2-Mehylthioethoxycarbonyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one ).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (2-methylthio-ethoxycarbonyl) -Cytocin-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.04 (dd, 1H), 7.90 (dd, 1H), 7.72 (m, 1H), 7.61 (t, 1H), 7.54 (q, 1H), 7.01 (d, 1H), 4.79 (s, 1.2H), 4.76 (s, 0.8H), 4.43 (s, 0.8H), 4.28-4.24 (m, 3.2H), 3.97 (m, 2H), 3.87 (br.d 2.4H), 3.74 (br.s, 1.6H ), 3.31 (br.s, 2 H), 2.73 (m, 2 H), 2.11 (s, 2 H).

Example 15

4-{[4-N- (4-methoxy-benzoyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2- On (4-{[4-N- (4-Methoxy-benzoyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (4-methoxy-benzoyl) -cytosine- in the same manner as in Example 11 1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.05 to 7.74 (m, 4H), 7.73 (m, 1H), 7.62 (t, 1H), 7.55 (q, 1H), 7.31 (br.d, 1H), 7.05 (d, 2H), 4.83 (s, 1.2H), 4.81 (s, 0.8H), 4.45 (s, 0.8H), 4.25 (s, 1.2H), 3.98 (m, 2H), 3.89 (br.d, 2.4H), 3.83 (s, 3H ), 3.75 (br.s, 1.6 H), 3.33 (m, 2H).

Example 16

4-{[4-N- (4-t-butyl-benzoyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazine-2 -One (4-{[4-N- (4-t-Butyl-benzoyl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2 -one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (4-t-butyl-benzoyl) -cytosine -1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.04 (dd, 1H), 8.00-7.96 (m, 3H), 7.72 (m, 1H), 7.62 (m, 1H), 7.56-7.52 (m, 3H), 7.32 (d, 1H), 4.85 (s, 1.2H), 4.81 (s, 0.8H), 4.45 (s, 0.8H), 4.25 (s, 1.2H), 3.99 (m, 2H), 3.89 (br.d, 2.4H), 3.75 (br.s , 1.6H), 3.32 (m, 2H), 1.30 (s, 9H).

Example 17

4-{[4-N- (isobutyryl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one (4 -{[4-N- (Isobutyryl) -cytosin-1-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (isobutyryl) -cytocin-1-yl in the same manner as in Example 11 ] -Acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.81 (s, 1H), 8.03 (d, 1H), 7.91 (dd, 1H), 7.73 (m, 1H), 7.62 (t, 1H), 7.55 (q, 1H), 7.22 (d, 1H), 4.77 (s, 1.2H), 4.74 (s, 0.8H), 4.44 (s, 0.8H), 4.24 (s, 1.2H), 3,98 (m, 2H), 3.89 (br.d, 2.4H), 3.74 (br.s, 1.6H), 3.32 (m, 2H), 2.71 (m, 1H), 1.06 (d, 6H).

Example 18

4-{[6-N- (benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one ( 4-{[6-N- (Benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (benzyloxycarbonyl) -adenin-9- General] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.60 (s, 0.4H), 8.59 (s, 0.6H), 8.35 (s, 0.4H), 8.33 (s, 0.6H), 8.04 (q, 1H), 7.73 (q, 1H), 7.62 (t, 1H), 7.55 (q, 5H), 7.47 ~ 7.32 (m, 5H), 5.36 (s, 1.2H), 5,32 (s, 0.8H), 5.22 (s, 2H), 4.54 (s, 0.8H ), 4.24 (s, 1.2H), 4.02-3.96 (m, 4H), 3.75 (br.s, 1.6H), 3.34 (m, 2.4H).

Example 19

4-{[6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2- On (4-{[6-N- (Benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (benzhydryloxycarbonyl) -adenine- 9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.95 (s, 1H), 8.59 (s, 0.6H), 8.58 (s, 0.4H), 8.34 (s, 0.6H), 8.32 (s, 0.4H), 8.04 (q, 1H), 7.73 (q, 1H), 7.62 (t, 1H), 7.58-7.27 (m, 11H), 6.81 (s, 1H), 5.36 (s, 1.2H), 5.32 (s, 0.8H), 4.54 (s, 0.8H), 4.24 (s, 1.2H), 4.01-3.95 (m, 4H), 3.76 (br.s, 1.6H), 3.35-3.30 (m, 2.4H)

Example 20

4-{[6-N- (4-methoxy-benzoyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2- On (4-{[6-N- (4-Methoxy-benzoyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one)

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (4-methoxy-benzoyl) -adenin- 9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.72 (s, 1H), 8.47 (s, 1H), 8.05 (d, 2H), 8.01 (m, 1H), 7.72 (m, 1H), 7.63-7.53 (m, 2H), 7.08 (d, 2H) , 7.02 (d, 1H), 5.40 (s, 1.2H), 5.37 (s, 0.8H), 4.56 (s, 0.8H), 4.25 (s, 1.2H), 4.01-3.97 (m, 4H), 3.85 (s, 3H), 3.79 (s, 1.6H), 3.35-3.31 (m, 2.4H).

Example 21

4-{[6-N- (4-t-butyl-benzoyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazine-2 -One (4-{[6-N- (4-t-Butyl-benzoyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2 -one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (4-t-butyl-benzoyl) -adenine -9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.72 (s, 1H), 8.44 (s, 1H), 8.06-7.99 (m, 3H), 7.72 (q, 1H), 7.63 (t, 1H), 7.58-7.53 (m, 3H), 5.40 (s, 1.2H), 5.37 (s, 0.8H), 4.56 (s, 0.8H), 4.26 (s, 1.2H), 4.01-3.95 (m, 4H), 3.76 (s, 1.6H), 3.35-3.30 (m , 2.4H), 1.33 (s, 9H).

Example 22

4-{[6-N- (isobutyryl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one (4 -{[6-N- (Isobutyryl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (isobutyryl) -adenin-9-yl ] -Acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.62 (s, 1H), 8.60 (s, 1H), 8.31 (s, 0.6H), 8.30 (s, 0.4H), 8.04 (t, 1H), 7.74 (q, 1H), 7.63 (t, 1H) , 7.56 (q, 1H), 5.35 (s, 1.2H), 5.32 (s, 0.8H), 4.55 (s, 0.8H), 4.25 (s, 1.2H), 4.03-3.97 (m, 4H), 3.76 (br.d, 1.6H), 3.35-3.31 (m, 2.4H), 2.95 (m, 1H), 1.13 (d, 6H)

Example 23

4-{[6-N- (2-Methylthio-ethoxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazine 2-one (4-{[6-N- (Benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (2-methylthio-ethoxycarbonyl) -Adenine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

8.65 (s, 1H), 8.50 (s, 0.6H), 8.49 (s, 0.4H), 8.05 (t, 1H), 7.73 (q, 1H), 7.63 (t, 3H), 7.55 (q, 1H) , 5.40 (s, 1.2H), 5.36 (s, 0.8H), 4.55 (s, 0.8H), 4.32 (t, 2H), 4.25 (s, 1.2H), 4.03-3.97 (m, 4.4H), 3.77 (br.d, 1.6H), 3.34 (m, 2H), 2.13 (s, 3H).

Example 24

4-{[2-N- (benzyloxycarbonyl) -guanine-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one ( 4-{[2-N- (Benzyloxycarbonyl) -guanin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (benzyloxycarbonyl) -guanine-9- in the same manner as in Example 11 General] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.44 (s, 1H), 11.38 (s, 0.6H), 11.36 (s, 0.4H), 8.04 (t, 1H), 7.81 (d, 1H), 7.72 (q, 1H), 7.61 (t, 1H) , 7.54 (q, 1H), 7.43 ~ 7.33 (m, 6H), 5.24 (s, 2H), .509 (s, 1.2H), 5.07 (s, 0.8H), 4.49 (s, 0.8H), 4.24 (s, 1.2H), 3.98 (m, 1.6H), 3.91 (s, 2.4H), 3.78-3.71 (m, 1.4H), 3.32 (m, 2.4H).

Example 25

4-{[2-N- (benzhydryloxycarbonyl) -guanine-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2- On (4-{[2-N- (Benzyloxycarbonyl) -guanin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (benzhydryloxycarbonyl) -guanine- in the same manner as in Example 11 9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.6 (s, 1H), 11.25 (s, 0.4H), 11.23 (s, 0.6H), 8.05 (t, 1H), 7.81 (d, 1H), 7.73 (q, 1H), 7.62 (t, 1H) , 7.55 (q, 1H), 7.46-7.28 (m, 10H), 6.85 (s, 1H), 5.12 (s, 1.2H), 5.09 (s, 0.8H), 4.50 (s, 0.8H), 4.24 ( s, 1.2H), 4.00-3.93 (m, 4H), 3.79-3.71 (m, 1.6H), 3.38 (m, 2.4H).

Example 26

4-{[2-N- (2-Methylthio-ethoxycarbonyl) -guanine-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazine 2-one (4-{[2-N- (2-Methylthio-ethoxycarbonyl) -guanin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2 -one).

In the same manner as in Example 11, 1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (2-methylthio-ethoxycarbonyl) -Guanine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.42 (s, 0.4H), 11.41 (s, 0.6H), 11.36 (s, 0.4H), 11.35 (s, 0.6H), 8.05 (t, 1H), 7.82 (s, 0.6H), 7.81 (s , 0.4H), 7.73 (q, 1H), 7.62 (t, 1H), 7.55 (q, 1H), 5.10 (s, 1.2H), 4.50 (s, 0.8H), 4.31 (t, 2H), 4.24 (s, 1.2H), 3.99 (m, 2H), 3.92 (s, 2H), 3.78-3.72 (m, 1.6H), 3.32 (m, 2.4H), 2.75 (t, 2H), 2.12 (s, 3H).

Example 27

4-{[2-N- (isobutyryl) -guanin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one (4 -{[2-N- (Isobutyryl) -guanin-9-yl] -acetyl} -1- [2- (2-nitro-phenyl) -ethanesulfonyl] -piperazin-2-one).

1- [2- (2-Nitro-phenyl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (isobutyryl) -guanine-9-yl in the same manner as in Example 11 ] -Acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

12.70 (s, 1H), 11.60 (s, 1H), 8.05 (t, 1H), 7.83 (s, 0.6H), 7.82 (s, 0.4H), 7.73 (q, 1H), 7.62 (t, 1H) , 7.56 (q, 1H), 5.14 (s, 1.2H), 5.12 (s, 0.8H), 4.50 (s, 0.8H), 4.26 (s, 1.2H), 4.02-3.92 (m, 4H), 3.81 ~ 3.73 (m, 1.6H), 3.32 (m, 2.4H), 2.76 (m, 1H), 1.10 (d, 6H).

Example 28

1- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -4-[(thimin-1-yl) -acetyl] -piperazin-2-one ( 1- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -4-[(thymin-1-yl) -acetyl] -piperazin-2-one).

1- [1- (6-Nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride 788 mg (2.0 mmole), thymine-1-yl acetic acid 368 mg (2.0 mmole) and 1.14 g (2.2 mmole) of PyBOP were suspended in 10 mL of N, N-dimethylformimide and 0.52 mL of diisopropylamine were added at room temperature. The reaction mixture was stirred at room temperature for about 2 hours and then slowly dropped into a mixture of water and ethanol to give a solid. The resulting solid was filtered, washed with ethanol and ethyl ether and dried to give 890 mg (85% yield) of the target compound.

¹ H NMR (DMSO-d6)

11.31 (s, 1H), 7.62 (s, 1H), 7.33 (s, 1H), 7.32 (s, 0.6H), 7.29 (s, 0.4H), 6.28 (s, 2H), 5.74 (m, 1H) , 4.59 (m, 2H), 4.46 (d, 0.4H), 4.27 (d, 0.4H), 4.24 (d, 0.6H), 4.20 (d, 0.6H), 3.85-3.72 (m, 1.6H), 3.68-3.60 (m, 1.6H), 3.46 (m, 0.4H), 3.34 (m, 0.4H), 1.77 (d, 3H), 1.75 (s, 3H).

Example 29

4-{[4-N- (benzyloxycarbonyl) -cytosine-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethane Sulfonyl] -piperazin-2-one (4-{[4-N- (Benzyloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol -5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (Benzyloxycarbonyl) -cytosine-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.78 (s, 1H), 7.89 (dd, 1H), 7.63 (s, 0.4H), 7.62 (s, 0.6H), 7.42-7.33 (m, 6H), 7.03 (d, 1H), 6.29 (d, 1H), 6.28 (s, 1H), 5.74 (q, 1H), 5.19 (s, 2H), 4.74 (m, 2H), 4.50. (d, 0.4H), 4.30 (d, 0.4H), 4.26 (d, 0.6H), 4.21 (d, 0.6H), 3.84 (m, 1.2H), 3.76 (m, 0.4H), 3.69 to 3.61 (m, 1.6H), 3.48 (m, 0.4H), 3.39 (m, 0.4H), 1.77 (d, 3H).

Example 30

4-{[4-N- (benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -Ethanesulfonyl] -piperazin-2-one (4-{[4-N- (Benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3 ] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (Benzhydryloxycarbonyl) -cytosine-1-yl] -acetic acid is used to give the desired title compound.

¹ H NMR (DMSO-d6)

10.97 (s, 1H), 7.88 (dd, 1H), 7.63 (s, 0.4H), 7.62 (s, 0.6H), 7.46-7.28 (m, 11H), 6.95 (d, 1H), 6.79 (s, 1H), 6.29 (d, 1H), 6.28 (s, 1H), 5.74 (m, 1H), 4.74 (m, 2H), 4.50 (d, 0.4H), 4.30 (d, 0.4H), 4.25 (d , 0.6H), 4.20 (d, 0.6H), 3.87 ~ 3.73 (m, 1.6H), 3.68 ~ 3.60 (m, 1.6H), 3.48 (m, 0.4H), 3.39 (m, 0.4H), 1.77 (d, 3H).

Example 31

4-{[4-N- (2-methylthio-ethoxycarbonyl) -cytosine-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5 -Yl) -ethanesulfonyl] -piperazin-2-one (4-{[4-N- (2-Methylthio-ethoxycarbonyl) -cytosin-1-yl] -acetyl} -1- [1- (6- nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (2-Methylthio-ethoxycarbonyl) -cytosine-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.73 (s, 1H), 7.89 (dd, 1H), 7.63 (s, 0.4H), 7.62 (s, 0.6H), 7.33 (s, 1H), 7.02 (d, 1H), 6.29 (d, 1H) , 6.28 (s, 1H), 5.75 (m, 1H), 4.74 (m, 2H), 4.50 (d, 0.4H), 4.33-4.19 (m, 3.6H), 3.84 (m, 1.2H), 3.69- 3.60 (m, 1.6H), 3.48 (m, 0.4H), 3.39 (m, 0.4H), 2.74 (t, 2H), 2.12 (s, 3H), 1.77 (d, 3H).

Example 32

4-{[4-N- (4-methoxy-benzoyl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -Ethanesulfonyl] -piperazin-2-one (4-{[4-N- (4-Methoxy-benzoyl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (4-methoxy-benzoyl) -cytosine-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.05 (s, 1H), 8.03 (d, 2H), 7.95 (t, 1H), 7.64 (s, 0.4H), 7.63 (s, 0.6H), 7.34 (s, 1H), 7.33 (d, 1H) , 7.04 (d, 2H), 6.30 (d, 1H), 6.29 (s, 1H), 5.76 (m, 1H), 4.79 (m, 2H), 4.52 (d, 0.4H), 4.33 (d, 0.4H ), 4.28 (d, 0.6H), 4.22 (d, 0.6H), 3.90 to 3.76 (m, 4.6H), 3.66 (m, 1.6H), 3.49 (m, 0.4H), 3.40 (m, 0.4H ), 1.78 (d, 3 H).

Example 33

4-{[4-N- (4-t-butyl-benzoyl) -cytocin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl ) -Ethanesulfonyl] -piperazin-2-one (4-{[4-N- (4-t-Butyl-benzoyl) -cytosin-1-yl] -acetyl} -1- [1- (6- nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (4-t-butyl-benzoyl) -cytosine-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.11 (s, 1H), 7.97 (d, 3H), 7.64 (s, 0.4H), 7.63 (s, 0.6H), 7.53 (d, 2H), 7.34 (s, 1H), 7.33 (d, 1H) , 6.29 (d, 1H), 6.29 (s, 1H), 5.75 (m, 1H), 4.80 (m, 2H), 4.53 (d, 0.4H), 4.33 (d, 0.4H), 4.28 (d, 0.6 H), 4.22 (d, 0.6H), 3.90-3.76 (m, 1.6H), 3.66 (m, 1.6H), 3.49 (m, 0.4H), 3.40 (m, 0.4H), 1.78 (d, 3H ).

Example 34

4-{[4-N- (isobutyryl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesul Ponyl] -piperazin-2-one (4-{[4-N- (Isobutyryl) -cytosin-1-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol- 5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [4-N- (Isobutyryl) -cytocin-1-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.82 (s, 1H), 7.90 (t, 1H), 7.63 (s, 0.4H), 7.62 (s, 0.6H), 7.34 (s, 1H), 7.22 (d, 1H), 6.29 (d, 1H) , 6.28 (s, 1H), 5.75 (m, 1H), 4.75 (m, 2H), 4.51 (d, 0.4H), 4.31 (d, 0.4H), 4.26 (d, 0.6H), 4.20 (d, 0.6H), 3.87-3.73 (m, 1.6H), 3.69-3.60 (m, 1.6H), 3.49 (m, 0.4H), 3.39 (m, 0.4H), 2.71 (m, 1H), 1.77 (d , 3H), 1.07 (d, 6H).

Example 35

4-{[6-N- (benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethane Sulfonyl] -piperazin-2-one (4-{[6-N- (Benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol -5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (Benzyloxycarbonyl) -adenine-9-yl] -acetic acid is used to obtain the desired title compound.

¹ H NMR (DMSO-d6)

10.67 (s, 1H), 8.60 (s, 1H), 8.30 (s, 1H), 7.66 (s, 0.4H), 7.64 (s, 0.6H), 7.47 ~ 7.34 (m, 6H), 6.29 (s, 1H), 6.27 (d, 1H), 5.76 (m, 1H), 5.31 (m, 2H), 5.22 (s, 2H), 4.60 (d, 0.4H), 4.42 (d, 0.4H), 4.27 (d , 0.6H), 4.20 (d, 0.6H), 3.99-3.87 (m, 1.2H), 3.78-3.65 (m, 2.0H), 3.49 (m, 0.4H), 3.38 (m, 0.4H), 1.79 (d, 3H)

Example 36

4-{[6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) Ethanesulfonyl] -piperazin-2-one (4-{[6-N- (Benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3 ] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (Benzhydryloxycarbonyl) -adenine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.91 (s, 1H), 8.59 (s, 1H), 8.32 (s, 0.6H), 8.31 (s, 0.4H), 7.66 (s, 0.4H), 7.64 (s, 0.6H), 7.53-7.29 ( m, 11H), 6.82 (s, 1H), 6.29 (s, 1H), 6.27 (d, 1H), 5.76 (m, 1H), 5.30 (m, 2H), 4.60 (d, 0.4H), 4.42 ( d, 0.4H), 4.27 (d, 0.6H), 4.20 (d, 0.6H), 3.99-3.87 (m, 1.2H), 3.78-3.66 (m, 2H), 3.50 (m, 0.4H), 3.37 (m, 0.4H), 1.79 (d, 3H).

Example 37

4-{[6-N- (2-Methylthio-ethoxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5 -Yl) -ethanesulfonyl] -piperazin-2-one (4-{[6-N- (2-Methylthio-ethoxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6- nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (2-Methylthio-ethoxycarbonyl) -adenin-9-yl] -acetic acid is used to give the desired title compound.

¹ H NMR (DMSO-d6)

10.57 (s, 1H), 8.59 (s, 1H), 8.29 (s, 1H), 7.66 (s. 0.4H), 7.64 (s, 0.6H), 7.36 (s, 0.6H), 7.35 (s, 0.4 H), 6.30 (s, 1H), 6.27 (d, 1H), 5.76 (m, 1H), 5.30 (m, 2H), 4.60 (d, 0.4H), 4.42 (d, 0.4H), 4.28 (t , 2H), 4,24 (d, 0.6H), 4.19 (d, 0.6H), 3.99-3.87 (m, 1.2H), 3.89-3.65 (m, 2H), 3.49 (m, 0.4H), 3.38 (m, 0.4H), 2.77 (t, 2H), 2.13 (s, 3H), 1.79 (d, 3H).

Example 38

4-{[6-N- (4-methoxy-benzoyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -Ethanesulfonyl] -piperazin-2-one (4-{[6-N- (4-Methoxy-benzoyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (4-Methoxy-benzoyl) -adenin-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.01 (s, 1H), 8.69 (s, 1H), 8.31 (s, 1H), 8.05 (d, 2H), 7.67 (s, 0.4H), 7.64 (s, 0.6H), 7.36 (s, 0.6H ), 7.35 (s, 0.4H), 6.30 (s, 1H), 6.28 (d, 1H), 5.77 (m, 1H), 5.33 (m, 2H), 4.62 (d, 0.4H), 4.44 (s, 0.4H), 4.29 (d, 0.6H), 4.11 (d, 0.6H), 4.00-3.82 (m, 1.2H), 3.86 (s, 3H), 3.80-3.68 (m, 2H), 3.50 (m, 0.4H), 3.40 (m, 0.4H), 1.80 (d, 3H).

Example 39

4-{[6-N- (4-t-butyl-benzoyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl ) -Ethanesulfonyl] -piperazin-2-one (4-{[6-N- (4-t-Butyl-benzoyl) -adenin-9-yl] -acetyl} -1- [1- (6- nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (4-t-Butyl-benzoyl) -adenine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.07 (s, 1H), 8.70 (s, 1H), 8.32 (s, 1H), 8.00 (d, 2H), 7.67 (s, 0.4H), 7.64 (s, 0.4H), 7.57 (d, 2H) , 7.36 (s, 0.6H), 7.35 (s, 0.4H), 6.30 (s, 1H), 6.28 (d, 1H), 5.77 (m, 1H), 5.35 (m, 2H), 4.62 (d, 0.4 H), 4.44 (d, 0.4H), 4.01-3.88 (m, 1.2H), 3.81-3.67 (m, 2H), 3.50 (m, 0.4H), 3.41 (m, 0.4H), 1.80 (d, 1H), 1.33 (s, 9H).

Example 40

4-{[6-N- (isobutyryl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesul Ponyl] -piperazin-2-one (4-{[6-N- (Isobutyryl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol- 5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [6-N- (Isobutyryl) -adenine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

10.62 (s, 1H), 8.61 (s, 1H), 8.30 (s, 1H), 7.66 (s, 0.4H), 7.64 (s, 0.6H), 7.36 (s, 0.6H), 7.35 (s, 1H ), 6.30 (s, 1H), 6.26 (d, 1H), 5.76 (m, 1H), 5.30 (m, 2H), 4.60 (d, 0.4H), 4.42 (d, 0.4H), 4.27 (d, 0.6H), 4.20 (d, 0.6H), 3.99-3.88 (m, 1.2H), 3.79-3.66 (m, 2H), 3.49 (m, 0.4H), 3.39 (m, 0.4H), 2.94 (m , 1H), 1.79 (d, 3H), 1.13 (d, 6H).

Example 41

4-{[2-N- (benzyloxycarbonyl) -guanine-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethane Sulfonyl] -piperazin-2-one (4-{[2-N- (Benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol -5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (Benzyloxycarbonyl) -guanine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.44 (s, 1H), 11.38 (s, 1H), 7.81 (s, 1H), 7.65 (s, 0.4H), 7.63 (s, 0.6H), 7.44-7.44 (m, 6H), 6.28 (s, 1H), 6.27 (d, 1H), 5.24 (s, 1H), 5.07 (s, 2H), 4.54 (d, 0.4H), 4.38 (d, 0.4H), 4.27 (d, 0.6H), 4.20 ( d, 0.6H), 3.95-3.84 (m, 1.2H), 3.75-3.67 (m, 2H), 3.49 (m, 0.4H), 3.40 (m, 1H), 1.78 (dd, 3H).

Example 42

4-{[2-N- (benzhydryloxycarbonyl) -guanine-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -Ethanesulfonyl] -piperazin-2-one (4-{[2-N- (Benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3 ] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (Benzhydryloxycarbonyl) -guanine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

11.60 (s, 1H), 11.24 (s, 1H), 7.81 (s, 1H), 7.66 (s, 0.4H), 7.64 (s, 0.6H), 7.46-7.28 (m, 11H), 6.86 (s, 1H), 6.29 (s, 1H), 6.27 (d, 1H), 5.76 (m, 1H), 5.09 (s, 1H), 4.55 (d, 0.4H), 4.39 (d, 0.4H), 4.28 (d , 0.6H), 4.20 (d, 0.6H), 3.95-3.85 (m, 1.2H), 3.76-3.66 (m, 2H), 3.49 (m, 0.4H), 3.40 (m, 0.4H), 1.79 ( dd, 3 H).

Example 43

4-{[2-N- (2-Methylthio-ethoxycarbonyl) -guanine-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5 -Yl) -ethanesulfonyl] -piperazin-2-one (4-{[2-N- (2-Methylthio-ethoxycarbonyl) -adenin-9-yl] -acetyl} -1- [1- (6- nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (2-Methylthio-ethoxycarbonyl) -guanine-9-yl] -acetic acid is used to give the desired title compound.

¹ H NMR (DMSO-d6)

11.50 (s, 1H), 11.36 (s, 1H), 7.81 (s, 1H), 7.66 (s, 0.4H), 7.64 (s, 0.6H), 7.34 (s, 1H), 6.29 (s, 1H) , 6.27 (d, 1H), 5.76 (m, 1H), 5.07 (s, 2H), 4.54 (d, 0.4H), 4.37 (s, 0.4H), 4.32 (t, 2H), 4.27 (d, 0.6 H), 4.20 (d, 0.6H), 3.95-3.84 (m, 1.2H), 3.75-3.65 (m, 2H), 3.49 (m, 0.4H), 3.39 (m, 0.4H), 2.76 (t, 2H), 2.13 (s, 3H), 1.78 (dd, 3H).

Example 44

4-{[2-N- (isobutyryl) -guanin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesul Ponyl] -piperazin-2-one (4-{[2-N- (Isobutyryl) -adenin-9-yl] -acetyl} -1- [1- (6-nitro-benzo [1,3] dioxol- 5-yl) -ethanesulfonyl] -piperazin-2-one).

In the same manner as in Example 28, 1- [1- (6-nitro-benzo [1,3] dioxol-5-yl) -ethanesulfonyl] -piperazin-2-one hydrochloride and [2-N- (Isobutyryl) -guanine-9-yl] -acetic acid is used to obtain the title compound.

¹ H NMR (DMSO-d6)

12.07 (s, 1H), 11.60 (s, 1H), 7.82 (s, 1H), 7.65 (s, 0.4H), 7.63 (s, 0.6H), 6.92 (s, 1H), 6.28 (d, 1H) , 5.76 (m, 1H), 5.10 (s, 2H), 4.54 (d, 0.4H), 4.38 (d, 0.4H), 4.28 (d, 0.6H), 4.21 (d, 0.6H), 3.91-3.85 (m, 1.2H), 3.76-3.66 (m, 2H), 3.50 (m, 0.4H), 3.42 (m, 0.4H), 2.76 (m, 1H), 1.78 (dd, 3H), 1.10 (d, 6H).

Oligomer manufacturing

The monomers synthesized above were used for direct oligomer synthesis. When preparing oligomers from the above-mentioned monomers, oligomers may be produced by those having the same protecting group, but when deprotecting under similar conditions, compounds having different protecting groups may be used. For example, the protecting group of cytosine is 4-methoxy-benzoyl group, the protecting group of adenine is 4- (t-butyl) -benzoyl group, and the protecting group of guanine is used in combination with isobutannoyl group to prepare an oligomer and under the same conditions. It may be deprotected. In addition, even if the deprotection conditions are different, two different deprotection methods can be used. The desired sequence was determined by the order in which the monomers were attached.

The synthesis process is as follows.

1.Remove amine protecting group by photoreaction to activate amine group

2.A linker or amino acid that can come out at the end, reacts first with amino acids and attaches monomers or attaches monomers directly

3. Wipe out unreacted material

4.Addition of anhydrous acetic acid (unreacted amine group capping)

5.propylamine added

6. Wipe reaction by-products

7.Sulfonyl protecting group remove by photoreaction

8.Wipe off reaction byproducts

9.Add monomer solution

Return to 10.3 and repeat 4, 5, 6, 7, 8, 9 to synthesize PNA oligomer

The reaction of the amine group and the monomer generated in the process of increasing the PNA oligomers one by one is dimethylformamide, N-methyl pyrrolidone dimethoxyethane, dichloromethane, dichloroethane, dimethyl sulfoxide, hexamethylphosphoamide, sulfolane and the like. And a mixture of two or three solvents described above can also be used. As reaction temperature, 0 ^o C ~ 60 ^o C is suitable. At this time, tetraalkyl ammonium fluoride such as mercuria acetate or tetrabutyl ammonium fluoride or the like can be used as a catalyst for increasing the reaction rate. Tetrabutyl ammonium fluoride is suitable.

Deprotector reaction by photoreaction

The rate of photolysis reaction to remove the sulfonyl protecting group was measured using a PNA monomer with thymine and using a HANOVIA PC451050 lamp in pyrex test tubes as shown in Scheme 23 below. Acetonitrile / water = 1: 1 or ethanol / water = 1: 1 were used as the solvent. The results are shown in Table 1 below.

Scheme 23

Table 1.

R Completion time of reaction 70 minutes in ethyl alcohol / water = 1/1 21 minutes CH ₃ CN / water = 1/1

The present specification abbreviates the reagents and solvents used for convenience of description, and the definitions of abbreviations used are described in Table 2 below.

Table 2.

Abbreviation Justice Aeg N- (2-aminoethylglycyl) An 4-methoxy-benzoyl Boc tert-butyloxycarbonyl BOP Benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate t-BuBz 4-tert-butyl-benzoyl Bn benzyl Cbz Benzyloxycarbonyl DCC Dicyclohexylcarbodiimide DIEA Ethyl diisopropylamine EDC 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide HOObt 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine Ibu Isobutyryl Mmt 4-methoxyphenyldiphenylmethyl NMM N-methylmorpholine PyBOP Benzotriazolyl-1-oxy-trispyrrolidinophosphonium hexafluorophosphate PyProp Bromo tris (pyrrolidino) phosphonium hexafluorophosphate TBTU 2- (1H-benzotriazol-1-yl) -1,1,3,3-yltetramethyluronium tetrafluoroborate TFA Trifluoroacetic acid TFMSA Trifluoromethanesulfonic acid THF Tetrahydrofuran TNTU 2- (5-norbornene-2,3-dicarboxyimido) -1,1,3,3-tetramethyluronium tetrafluoroborate TOTU O-[(cyano (ethoxycarbonyl) methylene) amino] -1,1,3,3-tetramethyluronium tetrafluoroborate TSTU O- (N-succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate

The monomer compound of the formula (1) according to the present invention contains a substituent having a sulfonyl group as a photolysis protecting group, and these substituents serve as protecting groups of the PNA backbone, activation of amide bond formation in PNA oligomer synthesis, and three types of exposure techniques. Play a role. The monomers of the present invention having these characteristics can be used as PNA oligomers, PNA / DNA chimeras, PNA / peptide conjugates and various labelable base compounds using various combinatorial chemistry techniques, and in particular can replace DNA chips. PNA chip can be useful for manufacturing through exposure technology.

Claims (8)

Compound of Formula (1): Formula (1) In the above formula, R is Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally. The compound of claim 1, wherein R is represented by the formula: Compound of formula (5): In the above formula, R is Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; HX is an inorganic acid or an organic acid. The compound of claim 3, wherein R is represented by the formula: A process for preparing a compound of formula (1), characterized by reacting a compound of formula (5) with a compound of formula (6) to form a compound of formula (1): Formula (1) Formula (5) BCH ₂ COOH Formula (6) In the above formula, R is Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; HX is an inorganic or organic acid; B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally. (a) reacting a compound of formula (6) with a compound of formula (2) to form a compound of formula (3), (b) hydrolyzing the compound of formula (3) produced in step (a) Reacting with di-t-butyl dicarbonate to produce a compound of formula (4), (c) cyclizing and deprotecting the compound of formula (4) to produce a compound of formula (5). Characterized in that for preparing a compound of formula (5): Formula (5) Formula (6) RSO ₂ Cl Formula (2) Formula (3) Formula (4) In the above formula, R is Wherein R 1, R 2, R 3 and R 4 are the same or different and are each independently hydrogen, nitro group, nitrile group, halogen atom, (C 1 -C 4) alkyl group, (C 1 -C 4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; HX is an inorganic or organic acid; R 6 is a (C ₁ -C ₄ ) alkyl or cyanoethyl group. (a) amidating a compound of formula (1-1) to a solid support to bind it; (b) irradiating light of a suitable wavelength to the amide-bonded compound to a solid phase support to remove the protecting group; Accordingly amidated by adding a compound of formula (1-2) to the primary amine group attached to the deprotected support, (d) again irradiating light of the appropriate wavelength to remove the protecting group, and (e) Method for producing a PNA oligomer, characterized in that to produce a PNA oligomer by repeating the amidation reaction and deprotection reaction: In the above formula, R is Wherein R1, R2, R3 and R4 are the same or different and each independently hydrogen, nitro group, nitrile group, halogen atom, (C1-C4) alkyl group, (C1-C4) alkoxy group, acyl group or An aryl group, or R 2 and R 3 form a five or six membered ring having two oxygen atoms; R 5 is H, a (C 1 -C 4) alkyl group or an aryl group; n is 0 or 1; B ₁ and B ₂ are nucleic acid bases identical or different from one another, protected or unprotected, and either naturally or non-naturally present. 8. The method of claim 7, wherein monomers are used to make PNA chips using exposure techniques.