A Novel Monomer For Synthesis of PNA Oligomer And A Process For Producing The Same

专利摘要:
The present invention relates to a novel monomer of the general formula (1) and a method for preparing the same for effectively synthesizing a peptide nucleic acid (PNA) oligomer: Wherein R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group); R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally; 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 process for preparing PNA oligomers from the novel monomers. The monomer of the formula according to the present invention is a 2-nitro-benzenesulfonamide derivative used as a protecting group of the backbone is stable in both acid and base, so not only serves as protecting group but also amide with primary amine It has the property to play two roles at the same time in the reaction. The monomer of the present invention having these characteristics is not only easily applied to oligomer synthesis through automated synthesis but also useful for making various libraries using combinatorial chemistry.
公开号:KR20030063848A
申请号:KR1020020004194
申请日:2002-01-24
公开日:2003-07-31
发明作者:김성기；이성희；임종찬；윤여홍；전재훈；최훈；이현일
申请人:주식회사 파나진；
IPC主号:

专利说明:

A novel monomer for synthesizing a PANA oligomer and a method of preparing the same {A Novel Monomer For Synthesis of PNA Oligomer And A Process For Producing The Same}
[6] The present invention is a novel monomer that is effective and economical in synthesizing a peptide nucleic acid (PNA) oligomer, which is not present in nature but can selectively complementarily bind to nucleic acids such as DNA and RNA, and It is about the manufacturing method thereof. The present invention also relates to a process for preparing PNA oligomers from the novel monomers.
[7] Many modified compounds that can be selectively complexed with DNA and / or RNA can be used for therapeutic purposes (antisense, antigene) or gene diagnostics or for basic research or commercial purposes in various biotechnologies. Has been developed. In the last few years, many chemicals have been reported that modify oligonucleotides with new physical properties (biochemical or physical), which have optimized the properties by changing the skeleton of nucleotides, phosphates or ribose in parts. One of the biggest conversions is the PNA (N- (2-aminoethyl) glycine), which replaces the ribose ring and phosphate reported by Buchardt, Nielsen, Egholm, Berg, and others, with N- (2-aminoethyl) glycine. peptide nucleic acid) (PE Nielsen, et al., Science, 1991, 254, 1497).
[8] PNAs are complementary pairs of DNA or RNA with Watson-Crick bases, and because PNAs are electrically neutral, PNA / DNA and PNA / RNA hybridization are DNA / DNA or DNA / Greater than the affinity of RNA complementary binding (M. Egholm, et al., Nature, 1993, 365, 566). Furthermore, PNAs can be parallel or anti-parallel complementary to DNA or RNA (E. Uhlmann, et al., Angew. Chem. Int. Ed., 1996, 35, 2632). It has the property of complementary binding regardless of salt concentration (S. Tomac, et al., J. Am. Chem. Soc., 1996, 118, 5544). Homopyrimidine PNAs combine with complementary DNA or RNA to form the structure of a triplex in the form of PNA ₂ / DNA or PNA ₂ / RNA (L. Betts, et al., Science , 1995, 270, 1838; MA Bonham, et el., Nucleic Acids Res., 1995, 23, 1197; H. Knudsen, et al., Nucleic Acids Res., 1996, 24, 494; JC Harvey, et al. , Science, 1992, 258, 1481). On the other hand, as affinity with DNA or RNA is more sensitive to mismatch in PNA / DNA or PNA / RNA complementary bonds.
[9] PNA is stable against nucleases or peptidases (V. Demidov, et al., Biochem. Pharmacol., 1994, 48, 1310), and DNA and RNA desorb in the presence of strong acid bases. While reactions such as depurination and hydrolysis occur, PNA is very stable in acids and fairly stable under basic conditions.
[10] The PNA structure can be divided into two parts, one of which is the nucleic acid base-methylenecarbonyl group in the skeleton portion of 2-aminoethylglycine and its amine portion, replacing the phosphodiester and ribose ring portions of DNA. It can be divided into nucleic acid base moieties linked by (methylenecarbonyl). PNA oligomers can be obtained through the continuous linking of monomers as in the synthesis of DNA oligomers. At this time, the continuous amidation reaction is similar to the method used in peptide synthesis chemistry, and thus oligomers can be synthesized by successive amidation and deprotection reactions with amine groups protected with Fmoc or t-Boc. have. On the other hand, the PNA monomer does not have an asymmetric carbon moiety to be obtained through the reaction of synthesizing 2-aminoglycine having a protecting group and introducing a nucleobase moiety thereto.
[11] Until now, various monomers for synthesizing PNA oligomers have been published, which are mainly composed of linear chains, and in terms of protecting groups introduced during oligomer synthesis, they are largely related to the form of the protecting group of the amino group of the backbone and the form of the protecting group of the nucleic acid base. Classified by Therefore, when the amino protecting group is unstable with an acid, the protecting group of the nucleic acid base uses an acid-stable protecting group, and when the amino protecting group is unstable with the base, the nucleic acid base uses a stable protecting group for the base. The use of conflicting protectors will limit the scope of application.
[12] In addition, a cyclic monomer may be used through cyclization rather than a linear chain. Results have been disclosed that can use cyclic monomers to minimize oligomers or prepare oligomers in other forms of reactions.
[13] Looking at these preceding results, the monomer having an amino group protecting group that is unstable in an acid is the first case where the amino group is protected with a t-butyloxycarbonyl (t-Boc) group, as represented by the following formula (where B is a nucleic acid base). (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; O. Buchardt et al. WO 92/20702):
[14]
[15] In this case, the protecting group of the nucleic acid base uses trifluoroacetic acid or benzyloxycarbonyl and benzyl groups stable in hydrochloric acid, which are used to remove the protecting group of the amino group, and the oligomer is synthesized. Phosphorus trifluoromethanesulfonic acid (Trifluorosulfonic acid) or fluoric acid (HF) to remove.
[16] When t-Boc is used as a protecting group, a relatively strong acid must be used to remove the protecting group from the amino group, which is not suitable because depurination reaction of DNA portion occurs when making PAN / DNA chimera. To improve this point, a monomethoxytrityl (Mmt) group that can be removed even in relatively weak acids, as represented by the following formula (where B is a nucleic acid base), is used as a protecting group for an amino group, and as a protecting group for a base: Oligo chimera is made by attaching oligo chimera to Controlled Pore Glass (CPG) using anisoyl group, isobutanoyl group and t-butylbenzoyl group when using DNA oligomer. (DW Will et al., Tetrahedron, 1995, 51, 12082; PJ Finn et al., Nucleic Acid Research 1996, 24, 3357; DA Stetsenko et al., Tetrahedron Lett. 1996) , 3571):
[17]
[18] Combinations of such protecting groups have continued to search for derivatives suitable for synthesizing oligomers and suitable for removing protecting groups after the completion of oligomer synthesis. A method of using a protecting group of a double amino group as a 4,4'-dimethylbenzhydryloxycarbonyl group and a protecting group of a nucleic acid base as (2-cyano-1,1-dimethyl) ethyloxycarbonyl has been disclosed (BD Gildea US 6063569 and US 6265559). These patents use a 4,4-dimethylbenzhydryloxycarbonyl group as a protecting group of an amino group as represented by the following formula (where B is a nucleic acid base), and an alkyl or aryl thioethoxycarbonyl ( Alkyl or aryl thioethoxycarbonyl) and the use of the oxidized form of the compound is described when removing the protecting group:
[19]
[20] In this case, the nucleic acid base is deprotected under the conditions of the base and the protecting group of the amino group of the skeleton is deprotected under the acidic condition.
[21] Compounds of the following formula (where B is a nucleic acid base), which is a monomer having an Fmoc group as an amino group protecting group that is unstable to a base, are N- (2-aminoethyl) glycine depending on the type of protecting group of the nucleic acid base. When the derivative is prepared, the form of the ester changes:
[22]
[23] That is, when a protecting group is used as a benzyloxycarbonyl group in a nucleic acid base, methyl or ethyl ester may be hydrolyzed in an alkali metal salt such as LiOH, NaOH, KOH, etc. Hydrolysis in trifluoroacetic acid using a butyl group is common (SA Thomson et al., Tetrahedron, 1996, 37, 6179). However, when a monomethoxytrityl (Mmt) group is used as the nucleic acid base, the monomethoxytrityl (Mmt) group is unstable in the presence of an acid such as trifluoroacetic acid. Hydrolyzable methyl or ethyl esters are used but the protecting groups of amino groups are also less effective because they are unstable under basic conditions (G. Breipohl et al., Bioorg. Med. Chem. Lett. 1996, 6, 665).
[24] As a result of various studies to introduce more effective protecting groups into monomers, automated synthesis to obtain oligomers efficiently requires protecting groups that can be easily removed, and solubility and reactivity in solvents are very important. It is common to use Fmoc as a protecting group in the amino group. For example, many studies on protecting an amino group with Fmoc and changing a protecting group of a nucleic acid base have been studied. Among them, protecting a nucleic acid group with a benzhydryloxycarbonyl group is most widely used (US 6172226B1 and US 6133444 by JM Coull et al.). .
[25] The methods described above deactivate the amino group through a protecting group, activate the carboxylic acid moiety, and then continue with the amidation reaction where the protecting group has been removed with an amide bond to obtain an oligomer.
[26] In addition, methods have been reported that can be cyclized rather than linear chains to minimize the protecting groups or to prepare oligomers in other forms of reactions. As a first example of this example, a method of synthesizing PNA oligomers using monomers represented by the following formula (where B is a nucleic acid base) is described in US Pat. No. 55,39083 and US 5831014 to P. D. Cook et al.
[27]
[28] The monomer of the above formula reacts with the amino group and the carbonyl group of the cyclized ester to open the ring to form an amide bond and to form a hydroxy group, which is troublesome to change the hydroxy group back to an amine group.
[29] As a second example of using a cyclic protecting group is a monomer represented by the formula: wherein B is a nucleic acid base:
[30]
[31] This monomer opens an activated ring to form an amide bond as in the case of the monomer described in the PD Cook et al. Patent, but the resulting compound has a protecting group because the amine group is protected with t-butyloxycarbonyl. Deprotection under conditions can give an amine group as a compound of an acid salt. There is no need to change the hydroxy group back to an amine group like the monomer described in PD Cook et al., But it is necessary to neutralize the acid salt in the reaction with other monomers, and the reaction time is long and heat is applied due to the low reactivity of the ring opening reaction. Has disadvantages (WO 00/02899 to S. Hahn).
[32] Among the various synthesis methods of the PNA oligomers described above, a commercially available method is a monomer in which the skeleton / nucleate group has a protecting group of t-butyloxycarbonyl / benzyloxycarbonyl group. In this case, when synthesizing the PNA oligomer, there is a process of removing successive t-butyloxycarbonyl groups at each step, which must be removed with a strong acid such as trifluoroacetic acid. At the same time, some benzyloxycarbonyl groups are removed at the same time, thereby lowering the yield of the oligomer, as well as benzyloxycarbonyl groups used as protecting groups for the nucleic acid base after synthesis of the desired oligomer. Very strong acids such as trifluoromethanesulfonic acid are used. These are not only difficult to handle, but also damage some amide bonds, resulting in lower yields and purity.
[33] Another commercially available method of synthesizing PNA oligomers is a monomer in which the backbone / nucleate group is protected with a protecting group of 9-fluorenylmethyloxycarbonyl (Fmoc) / benzhydryloxycarbonyl (Bhoc). In this case, the PNA oligomer synthesis has the advantage that it is very easy to remove the benzhydryloxycarbonyl protecting group, but the organic base is used during the amidation reaction, in which case 9-fluorenylmethyloxy carbonyl Often has the disadvantage of causing damage.
[34] Both methods described above undergo a process of making amide bonds, and the reagents used in the amidation reaction generally use reagents such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, and PyBrOP. These have the disadvantage of being expensive or very slow for some reagents. Therefore, there is an urgent need for a new type of monomer to synthesize the oligomer in a new way that does not use the expensive reagents described above for the amidation reaction and increases the reaction rate and is also suitable as a protecting group.
[35] The present invention provides a monomer represented by the following formula (1) used to effectively synthesize PNA oligomers:
[36]
[37] In the above formula,
[38] R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
[39] R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
[40] B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[41] In addition, the present invention provides a method for producing the monomer represented by the formula (1).
[42] The present invention also provides a compound of formula 6, which is a novel intermediate compound used to prepare the monomer represented by formula 1:
[43]
[44] In the above formula,
[45] R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
[46] R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
[47] R6 is a (C1-C4) alkyl or cyanoethyl group.
[48] In addition, the present invention provides a method for preparing a compound represented by the formula (6).
[49] The present invention also provides a compound of formula 9 or a free base thereof, which is a novel intermediate compound used to prepare the monomer represented by Formula 1 above:
[50]
[51] In the above formula,
[52] R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
[53] R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
[54] HY is an inorganic acid or an organic acid.
[55] In another aspect, the present invention provides a method for preparing a compound represented by the formula (9).
[56] The present invention also provides a compound of formula 10, which is a novel intermediate compound used to prepare the monomer represented by formula 1:
[57]
[58] In the above formula,
[59] R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
[60] R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
[61] R6 is a (C1-C4) alkyl or cyanoethyl group;
[62] B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[63] In addition, the present invention provides a method for preparing a compound represented by Formula 10.
[64] In addition, the present invention provides a method for preparing a PNA oligomer using the monomer represented by the formula (1).
[1] 1 shows the results of HPLC analysis of NsTaegNH ₂ .
[2] 2 shows the results of HPLC analysis of Ns (Taeg) ₂ NH ₂ .
[3] 3A is a chart showing the chemical formula of naturally occurring or non-naturally occurring nucleic acid bases useful in the present invention.
[4] 3B is a chart showing the chemical formula of naturally occurring or non-naturally occurring nucleic acid bases useful in the present invention.
[5] 3C is a chart showing the chemical formula of naturally occurring or non-naturally occurring nucleic acid bases useful in the present invention.
[65] The monomer of Formula 1 according to the present invention is a 2-nitrobenzenesulfonamide derivative used as a protecting group of the PNA backbone is stable in both the acid and the base, and thus not only acts as a protecting group but also activates the reaction in the acylation reaction with the primary amine. It has the property to play two roles at the same time. The monomer of the present invention having these characteristics is not only easily applied to oligomer synthesis through automated synthesis but also useful for making various libraries using combinatorial chemistry.
[66] Preparation of PNA Basic Backbone
[67] The first step for synthesizing the PNA monomer of the present invention is to use an arylsulfonyl group as a protecting group on the amino group of 2-aminoethylglycine, which is a PNA basic skeleton, as shown in Scheme 1 below. Sulfonylated derivatives of formula 6a prepared according to this scheme are important intermediates for the synthesis of monomers of formula 1 according to the invention.
[68]
[69] In the above formula,
[70] R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
[71] X is a halogen atom;
[72] R6 is a (C1-C4) alkyl or cyanoethyl group.
[73] In Scheme 1, the first step (conversion from compound 2 to compound 4) is sulfonylation of ethylenediamine in the presence of a base. The base used may be a tertiary amine organic base such as triethylamine, diisopropylethylamine, N-methylmorpholine, DBU, and an inorganic base such as potassium carbonate or cesium carbonate, but a tertiary amine organic base is preferable. Do. The reaction temperature is 0 ° C to 100 ° C, preferably 0 ° C to 50 ° C. Solvents used herein include water, alcohols (e.g. methanol and ethanol), aromatic hydrocarbons (e.g. benzene and toluene), esters (e.g. ethyl acetate), haloalkanes (e.g. dichloromethane and chloroform), ethers (e.g. Tetrahydrofuran, ethyl ether and isopropyl ether), ketones (e.g. acetone and methyl ethyl ketone), nitrile (e.g. acetonitrile and propionitrile), amides (e.g. dimethylformamide, dimethylacetamide and N-methyl Pyrrolidone), sulfoxides (eg dimethyl sulfoxide) and mixed solvents between the solvents are included. Preferred solvents are haloalkanes, nitriles and amides. The produced compound of formula 4 can be easily purified through conventional purification methods such as crystallization by adding column chromatography, organic acid, if necessary.
[74] In Chemical Formula 1, the compound of Chemical Formula 3 may be purchased and commercially available or may be simply prepared by conventional synthetic methods in the field of organic chemistry (D. D. Chapman et al., US 4204870).
[75] In Scheme 1, the second step (conversion from compound 4 to compound 6a) is an alkylation reaction in the presence of a base. X in the compound of formula 5 is substituted by an amino group as a halogen atom such as chlorine, bromine or iodine. The reaction proceeds at 0 ° C. to 50 ° C. in a solvent such as haloalkyl (eg dichloromethane) or dimethylformamide in the presence of a tertiary organic base such as triethylamine, the tertiary amine, to give a compound of formula 6a. . The resulting compound of Chemical Formula 6a can be easily purified through conventional purification methods such as column chromatography and crystallization, if necessary. R6 represented in the compounds of formulas (5) and (6a) is an alkyl group, such as methyl, ethyl, which can be easily hydrolyzed on alkali metal hydroxides such as LiOH, NaOH, KOH, etc., or t-butyl, which can be converted to a carboxylic acid in the presence of an acid, or organic. The cyanoethyl group etc. which can be converted into carboxylic acid from the base compound can be used.
[76] The compound of formula 6, including 6a, which is an important intermediate for synthesizing the monomer of formula 1 according to the present invention, can be prepared via another intermediate, which can be prepared by the following scheme 2. When R5 is H, it is prepared by a known method (SA Thomson et al., Tetrahedron, 1995, 6179) and when R5 is a side chain of amino acids except H (A. Puschl et al., Tetrahedron Lett. 1998, 39, 4707) and used as starting material.
[77]
[78] Wherein R and R6 are as defined above; R 5 is the side chain of hydrogen or an amino acid present naturally or non-naturally.
[79] In Scheme 2, the compound of formula 6 is a compound of formula 7 in the presence of a tertiary organic base such as triethylamine, which is a tertiary amine, water, alcohol (e.g. methanol and ethanol), aromatic hydrocarbons (e.g. benzene and toluene ), Esters (e.g. ethyl acetate), haloalkanes (e.g. dichloromethane and chloroform), ethers (e.g. tetrahydrofuran, diethyl ether and isopropyl ether), ketones (e.g. acetone and methyl ethyl ketone), nitrile (Eg acetonitrile and propionitrile), amides (eg dimethylformamide, dimethylacetamide and N-methylpyrrolidone), sulfoxides (eg dimethyl sulfoxide) and mixed solvents between the above solvents . Preferred solvents are haloalkanes, nitriles and amides and the reaction temperature proceeds from 0 ° C. to 50 ° C. to give the compound of formula 6.
[80] Scheme 3 shows a process for preparing the compound of formula 9 or a salt thereof used to prepare the monomer of formula 1 according to the invention from the compound of formula 6 shown in Scheme 1.
[81]
[82] Wherein R, R5 and R6 are as defined above; HY is an inorganic acid or an organic acid.
[83] The compound of formula 8 may be synthesized by hydrolyzing the compound of formula 6 under basic conditions and then reacting with di-t-butyl dicarbonate (t-Boc) ₂ O. R6 is most preferably used an alkyl group such as methyl or ethyl group which can be hydrolyzed under basic conditions or a cyanoethyl group which can be converted into an carboxylic acid in an organic base compound. As a solvent used when hydrolyzing an alkali metal hydroxide such as LiOH, NaOH, KOH, etc., water and an organic solvent are used together. Tetrahydrofuran, dioxane, dimethoxyethane and the like are suitable. The reaction temperature range may be 30 ° C to 70 ° C, but room temperature is preferred.
[84] Cyclization reaction (conversion from compound 8 to compound 9) is used to carry out the amidation reaction to form amides with carboxylic acid and amine groups in peptide synthesis such as HATU, HBTU, DCC, EDC, PyBOP, TOTU, HOObt, PyBrOP Coupling reagents may be used or isobutyl chloroformate and N-methylmorpholine may be used to react. At this time, dichloromethane, dimethylformamide, tetrahydrofuran, etc. are suitable as a solvent. The reaction temperature is suitable at -30 ° C to 50 ° C when using coupling reagents such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, PyBrOP, and using isobutyl chloroformate and N-methylmorpholine. When making it react, -20 degreeC-0 degreeC is suitable.
[85] When removing the t-butyloxycarbonyl protecting group, the salt of the compound of formula 9 is obtained using HCl / ethanol or HCl / ethyl acetate. Other acids that can be used to obtain such salts include, but are not limited to, halogen acids (HF, HBr, HI), sulfuric acid, nitric acid, alkanesulfonic acid, trifluoromethanesulfonic acid. At this time, the reaction temperature is performed at room temperature. Room temperature is 25 ° C unless otherwise specified.
[86] Preparation of PNA Monomers
[87] The monomer of formula 1 according to the invention can be prepared in two ways. One is to introduce the nucleic acid base moiety first and to prepare the cyclized piperazinone derivative through the cyclization reaction, the other is to synthesize the cyclized piperazinone derivative first and finally the nucleic acid base to the secondary amino group of the compound. Is to introduce a part.
[88] Scheme 4 below shows a method for preparing the monomer of Formula 1 according to the present invention by first introducing a nucleic acid base moiety and preparing a cyclized piperazinone derivative via a cyclization reaction.
[89]
[90] Wherein R, R5, R6 and B are as defined above.
[91] As can be seen in Scheme 4, by introducing a nucleic acid base moiety into a compound of formula 6, which is a linear derivative, a compound of formula 10 is produced and the compound of formula 10 is converted into a compound of formula 1 through a cyclization reaction. The conversion from Formula 6 to Formula 10 is carried out by an amidation reaction that forms an amide using coupling reagents such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, PyBrOP, as is well known in peptide chemistry. Is achieved. The reaction temperature in this amidation process is suitable 20-20 degreeC, The reaction solvent is suitable dichloromethane, tetrahydrofuran, dimethylformamide, etc., The base used is triethylamine, diisopropylethylamine. And N-methylmorpholine are suitable.
[92] The conversion of the compound of formula 10 to the compound of formula 1 is determined by what form the protecting group of the nucleic acid base is. That is, when the nucleic acid base is T or the protecting group of the other nucleic acid base is benzyloxycarbonyl, the range of R 6 is widened, and all the above-mentioned methods are possible. However, when the protecting group is benzhydryloxycarbonyl, the acid is unstable. It is difficult to use when R6 is t-butyl. Conversely, if the protecting group of the nucleic acid base is unstable to the base, it is preferable to use a compound in which R 6 is t-butyl. As the solvent used for hydrolysis, water and an organic solvent are used together. Tetrahydrofuran, dioxane, dimethoxyethane, ethyl alcohol, methyl alcohol and the like are suitable. The reaction temperature is preferably -30 ° C to 70 ° C. Cyclization reactions use coupling reagents such as HATU, HBTU, DCC, EDC, PyBOP, TOTU, BOObt, and PyBrOP to carry out the amidation reaction to form an amide with carboxylic acid and amine groups in peptide synthesis, or isobutyl It can be reacted with chloroformate and N-methylmorpholine. At this time, dichloromethane, dimethylformamide, tetrahydrofuran, etc. are suitable as a solvent. Reaction temperature is suitable at -30 ℃ to 50 ℃ when using coupling reagents such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, BOObt, PyBrOP, and isobutyl chloroformate and N-methylmorpholine are used. When reacting by making the reaction, -20 ° C to 0 ° C is suitable.
[93] Scheme 5 below shows a method for preparing a monomer of Formula 1 according to the present invention by first synthesizing a cyclized piperazinone derivative and finally introducing a nucleic acid base moiety to a secondary amino group of the derivative compound.
[94]
[95] Wherein R, HY, R5 and B are as defined above.
[96] According to Scheme 5, an amide compound of Formula 1 is synthesized through an amidation reaction using a coupling compound such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, PyBrOP, as well known in peptide synthesis chemistry. . In this process, the reaction temperature is appropriately -20 ° C to 30 ° C, and the reaction solvent is preferably dichloromethane, tetrahydrofuran, dimethylformamide, etc., and the base used may be triethylamine, diisopropylethylamine, N -Methyl morpholine is suitable.
[97] Nucleic acid base
[98] 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 various protecting groups (TW Greene and PGM Wuts, Protective Group in Organic Synthesis, 3 ^rd Edition, pp 494-653) that can be used as protecting groups for stable amines in bases. Include.
[99] As a preferred embodiment, the nucleic acid base of the present invention is specifically illustrated by thymine, guanine, cytosine and adenine.
[100] (A) Thymine (T)
[101] (Thimin-1-yl) -acetic acid represented by the following formula is known method (KL Dueholm et. Al., J. Org. Chem., 1994, 59, 5767; and O. Buchardt et. Al., WO9201219). Are synthesized.
[102]
[103] Scheme 6 below shows a method for preparing a monomer of Formula 1T wherein the nucleic acid base is thymine from the compound of Formula 6. Here, the amidation reaction of the compound of formula (6) with (thimin-1-yl) -acetic acid is carried out in a conventional manner. In this reaction, coupling reagents such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, PyProP and diisopropylethylamine can be used together, or (thimin-1-yl) -acetic acid is combined with pivaloyl chloride. An amide compound can be obtained by adding and reacting the anhydride obtained by reaction and diisopropylethylamine together. At this time, dimethylformamide is particularly suitable.
[104] The conversion from the compound of Formula 10T to the compound of Formula 1T can be used first of all with an alkyl derivative which R 6 can hydrolyze to alkali metal hydroxide or t-butyl which can be removed with acid. The second reaction, the cyclization reaction, is carried out using a coupling reagent such as HATU, HBTU, DCC, EDC, PyBOP, BOP, TOTU, HOObt, PyProP, etc., using a solvent such as dimethylformamide. Or isobutyl chloroformate and N-methylmorpholine. At this time, tetrahydrofuran is suitable as a solvent, and the reaction temperature is suitably 20 ° C to 30 ° C.
[105]
[106] Wherein R, R5 and R6 are as defined above.
[107] Scheme 7 below shows another method of making a T-monomer. According to Scheme 7, an amidated reaction of piperazinone of formula (9) and (thimin-1-yl) -acetic acid, protected by a sulfonyl group substituted with R, produces an amide monomer of formula (1T). Characteristically, the monomer of the present invention can be obtained by one-step reaction with the secondary amine portion of the skeleton and (thimin-1-yl) -acetic acid, which has the advantage that the monomer can be produced economically and efficiently. The amidation reaction of the piperazinone of the formula (9) with (thimin-1-yl) -acetic acid is as described above.
[108]
[109] Wherein R, R5 and HY are as defined above.
[110] (B) adenine, guanine and cytosine (A, G, C)
[111] (b-1) when the nucleic acid bases A, G and C have a protecting group of benzhydryloxycarbonyl
[112] When the nucleic acid base has a protecting group of benzhydryloxycarbonyl, the substituent which can be used as R6 is prepared by introducing an alkyl group such as methyl or ethyl or cyanoethyl, which can be hydrolyzed under basic conditions. Is carried out in the same way as for thymine.
[113] [4-N- (benzhydryloxycarbonyl) cytosin-1-yl] -acetic acid, a nucleic acid base derivative used to prepare the PNA monomers of the present invention, was synthesized by a known method as shown in Scheme 8 below. (JM Colull et al., US 6,172,226; and BD Gildea et al., US 6,265,559).
[114]
[115] [6-N- (benzhydryloxycarbonyl) adenin-9-yl] -acetic acid is prepared by known methods (J. M. Colull et al., US 6,172,226.) As shown in Scheme 9.
[116]
[117] [2-N- (benzhydryloxycarbonyl) guanine-9-yl] -acetic acid can be prepared as shown in Scheme 10, which is a modification of the method described in US 6,172,226 to JM Coull et al. will be. Nucleic acid base derivative for synthesizing desired monomer by using iodine group with more reactivity instead of chloro and hydrolysis reaction with LiOH using ethyl group instead of benzyl group at position 6 with cyano ethanol Phosphorus [2-N- (benzhydryloxycarbonyl) guanine-9-yl] -acetic acid is synthesized.
[118]
[119] (b-2) Monomer having a protecting group of benzhydryloxycarbonyl
[120] The monomer having a protecting group of benzhydryloxycarbonyl is prepared using the same method as in Scheme 6 and Scheme 7, and the preparation thereof is shown in Schemes 11 and 12 below.
[121]
[122] Wherein R, R5 and R6 are as defined above.
[123]
[124] Wherein R, R5 and HY are as defined above.
[125] (c-1) when the nucleic acid bases A, G and C have a protecting group of benzyloxycarbonyl (Cbz)
[126] Schemes 13, 14 and 15 show methods for preparing nucleic acid bases C, A and G, each having a protecting group of benzyloxycarbonyl as the nucleic acid base moiety used to prepare the PNA monomers of the invention.
[127] [4-N- (benzyloxycarbonyl) cytosine-1-yl] -acetic acid is prepared as shown in Scheme 13 below and methods for its preparation are described in Dueholm et al., J. Org. Chem., 1994, 59, 5767 and O. Buchardt et al. In WO9221702.
[128]
[129] [6-N- (benzyloxycarbonyl) adenine-9-yl] -acetic acid is prepared as shown in Scheme 9 below and methods for its preparation are described in S. A. Thomson et al., Tetrahedron, 1995, 6179.
[130]
[131] [2-N- (benzyloxycarbonyl) guanine-9-yl] -acetic acid is prepared as shown in Scheme 15 below, the preparation method of which is described in J. M. Coull et al. US Pat. No. 6,172,226. In the 6th position, iodine group, which is more reactive than chloro, was hydrolyzed with LiOH using ethyl group instead of benzyl group with ethyl group, and iodine group was prepared using cyanoethanol. (Benzyloxycarbonyl) guanine-9-yl] -acetic acid was synthesized.
[132]
[133] (c-2) Monomer having a protecting group of benzyloxycarbonyl
[134] Monomers having a protecting group of benzyloxycarbonyl are prepared using the same method as in Scheme 6 or 7, and the preparation thereof is shown in Schemes 16 and 17, respectively.
[135]
[136] Wherein R, R5 and R6 are as defined above.
[137]
[138] Wherein R, R5 and HY are as defined above.
[139] PNA oligomer preparation
[140] The monomers synthesized above are used for direct oligomer synthesis. As the resin for synthesizing the oligomer, one having an amine group at the end is used. The desired sequence is determined by the order in which the monomers are attached.
[141] Scheme 18 shows the synthesis of PNA oligomers, which can be summarized as follows:
[142] 1. Remove the protecting group of the amine group to activate the amine group.
[143] 2. React first with a linker or amino acid that can end the amine group and then attach the monomer or attach the monomer directly.
[144] 3. Wipe up unreacted material.
[145] 4. Remove the sulfonyl protecting group.
[146] 5. Wipe the reactants
[147] 6. Add monomer solution.
[148] 7. Go back to step 3 and repeat steps 4, 5 and 6 to synthesize PNA oligomers.
[149]
[150] Wherein R and R5 are as defined above; B1 and B2, which may be the same or different, each independently represent a protected or unprotected nucleic acid base that exists naturally or unnaturally; Indicates a resin.
[151] In the process of increasing the monomers to the PNA oligomer one by one, the reaction of the amine group and the monomers is performed by dimethylformamide, N-methylpyrrolidone, dimethoxyethane, dichloromethane, dichloroethane, dimethyl sulfoxide, hexamethylphosphoramide, and sulfolane. The solvent may be used and a mixture of two or three solvents described above may be used. The reaction temperature is preferably 0 ° C to 60 ° C. In this case, tetraalkylammonium fluoride such as mercuria acetate or tetrabutylammonium fluoride may be used as a catalyst for increasing the reaction rate, but tetrabutylammonium fluoride is suitable. As another additive, it is also possible to use HATU, HBTU, DCC, EDC, PyBOP, TOTU, HOObt, PyBrOP and the like. As the reaction for removing the sulfonyl protecting group, thiophenol having thiophenol and benzene rings having various substituents can be used. Preferably, thiophenol or 4-methoxythiophenol is used. At this time, as the solvent, tetrahydrofuran, dimethylformamide, dimethylacetamide, N-methylpyrrolidone dimethoxyethane, dichloromethane, dichloroethane, dimethyl sulfoxide, hexamethylphosphoamide, sulfolane, C1-C5 alkyl alcohol And mixtures of two or three solvents described above can also be used. As reaction temperature, 10 to 60 degreeC is suitable. As a solvent to clean after the reaction, dimethylformamide, N-methylpyrrolidone dimethoxyethane, dichloromethane, dichloroethane, dimethyl sulfoxide and the like are suitable.
[152] Abbreviations for the reagents or solvents used herein are as defined in the table below.
[153] AbbreviationJustice AegN- (2-aminoethylglycyl) AnAnisoil Boctert-butyloxycarbonyl BOPBenzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate t-BuBz4-tert-butylbenzoyl Bnbenzyl BzBenzoyl CbzBenzyloxycarbonyl DCCDicyclohexylcarbodiimide DIEAEthyldiisopropylamine EDC1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide, hydrochloride Fmoc (ONsu)9- (1H-Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro phosphate HATU2- (1H-Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro phosphate HBTU2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate HOObt3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine Mmt4-methoxyphenyldiphenylmethyl NMMN-methylmorpholine PyBOPBenzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate PyBrOPBromotris (pyrrolidino) phosphonium hexafluorophosphate TFATrifluoroacetic acid TFMSATrifluoromethanesulfonic acid THFTetrahydrofuran TOTUO-[(cyano (ethoxycarbonyl) methylene) amino] -1,1,3,3-tetramethyl uronium tetrafluoroborate
[154] The invention will be more specifically illustrated by the following examples. However, these examples are merely embodiments of the present invention and do not limit the scope of the present invention.
[155] Preparation of the base skeleton
[156] Example 1
[157] Preparation of N- (2-aminoethyl) -2nitrobenzenesulfonamide, acetic acid salt (N- (2-Am inoethyl) -2-nitro-benznesulfonamide, acetic acid salt)
[158] 2-nitro-benzenesulfonyl chloride (22.16 g, 0.100 mol) was dissolved in dichloromethane (200 ml), and a solution of ethylenediamine (60.1 g, 1.00 mol) in dichloromethane (1 l) was stirred at room temperature for 3 hours. Stir while dropwise. Saturated brine (1 l X 4) was added to the reaction solution, the organic layer was dried and filtered through anhydrous forget-me-not, and acetic acid (6.0 g, 0.1 mol) was added dropwise to solidify the title compound. The resulting solid was filtered, washed with dichloromethane (200 ml) and dried to give 23.1 g (76%) of the title compound as a yellow solid.
[159] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[160] δ 8.01 to 7.95 (m, 2H), 7.88 to 7.83 (m, 2H), 2.90 (t, 2H), 2.60 (t, 2H), 1.87 (s, 3H).
[161] Example 2
[162] Preparation of N- (2-aminoethyl) -4-chloro-2nitrobenzenesulfonamide, acetic acid salt (N- (2-Aminoethyl) -4-chloro-2-nitro-benznesulfonamide acetic acid salt)
[163] 4-Chloro-2-nitro-benzenesulfonyl chloride (25.61 g, 0.1 mol) was obtained in the same reaction manner as in Example 1 to obtain 22.5 g (66%) of the title compound as a yellow solid.
[164] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[165] δ 8.26 (d, 1H), 8.01 to 7.97 (m, 2H), 2.97 (t, 2H), 2.69 (t, 2H), 1.91 (s, 3H).
[166] Example 3
[167] Preparation of N- (2-aminoethyl) -4-fluoro-2nitrobenzenesulfonamide, acetic acid salt (N- (2-Aminoethyl) -4-fluoro-2-nitro-benznesulfonamide, acetic acid salt)
[168] 4-fluoro-2-nitro-benzenesulfonyl chloride (23.96 g, 0.100 mol) was obtained in the same manner as in Example 1 to obtain 21.7 g (67%) of the title compound as a yellow solid.
[169] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[170] 8.11 to 8.06 (m, 2H), 7.78 (m, 1H), 2.94 (t, 2H), 2.67 (t, 2H), 1.89 (s, 3H).
[171] Example 4
[172] N- (2-aminoethyl) -2nitro-4-trifluoromethyl-benzenesulfonamide, acetic acid salt of (N- (2-Aminoethyl) -2-nitro-4-trifluoromethyl-benznesul fonamide, acetic acid salt) Produce
[173] 2-nitro-4-trifluoromethyl-benzenesulfonyl chloride (28.96 g, 0.100 mol) was obtained in the same manner as in Example 1 to obtain 23.62 g (63%) of the title compound as a yellow solid.
[174] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[175] δ 8.56 (s, 1H), 8.28 (d, 1H), 8.24 (d, 1H), 2.92 (t, 2H), 2.65 (t, 2H), 1.87 (s, 3H).
[176] Example 5
[177] Preparation of [2- (2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (2-nitro-benznesulfonylamino) -ethylamino] -acetic acid ethyl ester)
[178] N- (2-aminoethyl) -2-nitro-benzenesulfonamide, acetic acid salt (15.15 g, 49.6 mmol) was suspended in dichloromethane (100 ml) and triethylamine (15.16 g, 0.15 mol) was added. Ethyl bromoacetate (16.7 g 0.100 mol) was added dropwise at a time while the suspended reaction solution was stirred at room temperature, and reacted for 1 hour. Saturated brine (100 ml) was added to the reaction solution, the organic layer was dried and filtered through anhydrous forget-me-not and concentrated under reduced pressure. The concentrated residue was purified by tube chromatography, containing the title compound and concentrated under reduced pressure. The concentrated residue was dissolved in ethyl ether (100 ml) and crystallized to give 8.1 g (49%) of the title compound as a white solid.
[179] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[180] δ 8.02 to 7.96 (m, 2H), 7.87 to 7.84 (m, 2H), 4.05 (q, 2H), 3.23 (s, 2H), 2.95 (t, 2H), 2.57 (t, 2H) 1.16 (t , 3H).
[181] Example 6
[182] [2- (4-Chloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (4-Chloro-2-nitro-benznesulfonylamino) -ethylami no] -acetic acid ethyl ester) Manufacture
[183] In the same manner as in Example 5, reaction was carried out using N- (2-aminoethyl) -4-chloro-2-nitro-benzenesulfonamide, acetic acid salt (16.79 g, 49.4 mmol), and the concentrated residue was then dissolved in ethyl ether. Crystallization gave 8.3 g (46%) of the title compound.
[184] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[185] δ 8.26 (s, 1H), 8.01 (d, 1H), 7.96 (d, 1H), 4.06 (q, 2H), 3.23 (s, 2H), 2.95 (t, 2H), 2.57 (t, 2H) , 1.16 (t, 3 H).
[186] Example 7
[187] [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (4-fluoro-2-nitro-benznesulfonylamino) -ethyl amino] -acetic acid ethyl ester Manufacturing
[188] In the same manner as in Example 5, reaction was carried out using N- (2-aminoethyl) -4-fluoro-2-nitro-benzenesulfonamide, acetic acid salt (16.79 g, 51.9 mmol), and the concentrated residue was then replaced by ethyl ether. Crystallization at gave 7.7 g (42%) of the desired title compound [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester.
[189] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[190] δ 8.08 (m, 2H), 7.76 (m, 1H), 4.06 (q, 2H), 3.24 (s, 2H), 2.95 (t, 2H), 2.57 (t, 2H), 1.16 (t, 3H)
[191] Example 8
[192] [2- (2-Nitro-4-trifluoromethyl-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (2-Nitro-4- trifluoromethyl-benznesulfonylamino) -ethylam ino] -acetic acid ethyl ester)
[193] After the reaction in the same manner as in Example 5 using N- (2-aminoethyl) -2-nitro-4-trifluoromethyl-benzenesulfonamide and acetic acid salt (18.76 g, 50.3 mmol), the concentrated residue was Crystallization in ethyl ether gave 9.23 g (46%) of the title compound.
[194] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[195] δ8.55 (s, 1H), 8.27 (d, 1H), 8.22 (d, 1H), 4.05 (q, 2H), 3.22 (s, 2H), 2.99 (t, 2H), 2.57 (t, 2H) , 1.16 (t, 3H)
[196] Example 9
[197] [2- (4-Methyl-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (4-Methyl-2-nitro-benzenesulfonylamino) -ethylam ino] -acetic acd ethyl ester) Manufacture
[198] Dichloromethane (50 ml) was prepared in two equivalents hydrochloride (1.10 g, 5.0 mmol) of (2-amino-ethylamino) -acetic acid ethyl ester, prepared by a known method (DW Will et al., Tetrahedron , 1995, 51 , 12069). ) And triethylamine (2.02 g, 20 mmol) were added. Suspended reaction solution was stirred at room temperature while 4-methyl-2-nitro-benzenesulfonyl chloride (1.19 g 5.0 mmol) was added dropwise for 5 minutes and reacted for 2 hours. Upon completion of the reaction, 30 ml of water was added to the reaction, the organic layer was dehydrated with anhydrous forget-me-not and concentrated under reduced pressure to obtain 1.60 g (92%) of the desired labeling compound.
[199] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[200] δ 7.87 (d, 1H), 7.81 (s, 1H), 7.66 (d, 1H), 4.06 (q, 2H), 3.24 (s, 2H), 2.91 (t, 2H), 2.57 (t, 2H) , 2.44 (s, 3 H), 1.17 (t, 3 H).
[201] Example 10
[202] [2- (4-Chloro-6-methyl-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (4-Chloro-6-methyl-2-nitro-benzenesulf onyl amino) -ethylam ino] -acetic acd ethyl ester)
[203] In the same manner as in Example 9, 2-equivalent hydrochloride (1.10 g, 5.0 mmol) of (2-amino-ethylamino) -acetic acid ethyl ester and 4-chloro-6-methyl-2-nitro-benzenesulfonyl chloride (1.36 g , 5.0 mmol) was used to obtain 1.72 g (90%) of the desired labeling compound.
[204] Example 11
[205] [2- (4,6-Dichloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester ([2- (4,6-Dichloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester)
[206] In the same manner as in Example 9 (2-amino-ethylamino)-2 equivalents hydrochloric acid salt of acetic acid ethyl ester (1.10 g, 5 mmol) and 4,6-dichloro-2-nitro-benzenesulfonyl chloride (1.46 g, 5 mmol) was used to obtain 1.83 g (87%) of the desired labeling compound.
[207] Example 12
[208] Preparation of N- [2- (2-Nitro-benzenesulfonylamino) -ethyl] -alanine ethyl ester (N- [2- (2-Nitro-benzenesulfonylamino) -ethyl] -alanine ethyl ester)
[209] Dichlorohydrochloride (1.165 g, 5.0 mmol) of N- (2-amino-ethyl) -alanine ethyl ester prepared by a known method (A. Puschl et al., Tetrahedron Lett. , 1998, 39 , 4707) Suspended in methane (50 ml) and triethylamine (2.02 g, 20 mmol) was added. 2-nitro-benzenesulfonyl chloride (1.11 g 5.0 mmol) was added dropwise for 5 minutes while the suspended reaction solution was stirred at room temperature and reacted for 2 hours. After the reaction was completed, 30 ml of water was added to the reaction, the organic layer was dehydrated with anhydrous forget-me-not and concentrated under reduced pressure to obtain 1.64 g (95%) of the title compound.
[210] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[211] δ 8.02 to 7.96 (m, 2H), 7.86 (m, 2H), 4.05 (q, 2H), 3.16 (q, 1H), 2.94 (t, 2H), 2.58 (m, 1H), 2.43 (m, 1H), 1.17 (t, 3H), 1.09 (d, 3H).
[212] Example 13
[213] Preparation of N- [2- (2nitro-benzenesulfonylamino) -ethyl] -phenylglycine methyl ester (N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -phenylglycine, methylester)
[214] N- (2-amino-ethyl) -phenylglycine methyl ester (416 mg, 2 mmol) was used in the same manner as in Example 12 to obtain 716 mg (91%) of the desired labeling compound.
[215] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[216] δ 8.00 to 7.74 (m, 2H), 7.87 to 7.81 (m, 2H), 7.34 to 7.26 (m, 5H), 4.32 (s, 1H), 3.57 (s, 3H), 2.98 (t, 2H), 2.54 to 2.41 (m, 2H)
[217] Example 14
[218] {t-Butyloxycarbonyl- [2- (2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (2-nitro-benzenesulfonylamino) -ethyl] -amino } -acetic acid)
[219] Lithium hydride dissolved in water (30 ml) in a solution of [2- (2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (6.66 g, 20.1 mmol) in tetrahydrofuran (50 ml) After addition of the hydroxide hydrate (1.64g, 40mmol), the mixture was stirred at room temperature for 1 hour. After the completion of the hydrolysis, di-t-butyl dicarbonate (6.55 g, 30 mmol) was added to the reaction solution, stirred for 30 minutes, and then dissolved in lithium hydroxide hydrate (0.82 g, 0.02). mol) was added dropwise for 30 minutes. After the reaction was completed, the insoluble substance was filtered and the filtrate was washed with ethyl ether (100 ml) and the aqueous layer was separated. The separated aqueous layer was adjusted to pH = 3 by adding 2N hydrochloric acid aqueous solution, and then extracted by adding dichloromethane (100 ml). The separated organic layer was dried and filtered through anhydrous forget-me-not and concentrated under reduced pressure to obtain 7.9 g (98%) of the title compound as a viscous oily solid.
[220] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[221] δ8.00 (m, 2H), 7.86 (m, 2H), 3.80 (s, 1H), 3.76 (s, 1H), 3.24 (m, 2H), 3.05 (m, 2H), 1.35 (s, 4.5H ), 1.31 (s, 4.5 H).
[222] Example 15
[223] {t-Butyloxycarbonyl- [2- (4-chloro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (4-chloro-2-nitro -benzenesulfonyl
[224] amino) -ethyl] -amino} -acetic acid)
[225] [2- (4-Chloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (7.35 g, 20 mmol) was prepared in the same manner as in Example 14 to give 8.1 g (92%) of the title compound. Got.
[226] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[227] δ 8.28 (s, 1H), 7.97 (s, 2H), 3.78 (s, 1H), 3.76 (s, 1H), 3.24 (m, 2H), 3.06 (m, 2H), 1.35 (s, 4.5H ) 1.31 (s, 4.5 H).
[228] Example 16
[229] {t-Butyloxycarbonyl- [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (4-fluoro-2- nitro-benzenesulfonyl amino) -ethyl] -amino} -acetic acid)
[230] [2- (4-Fluoro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (7.35 g, 21 mmol) was prepared in the same manner as in Example 14 to obtain 7.9 g (89%) of the title compound. )
[231] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[232] δ8.14 to 8.02 (m, 2H), 7.76 (m, 1H), 3.82 (s, 1H), 3.77 (s, 1H), 3.24 (m, 2H), 3.05 (m, 2H), 1.36 (s, 4.5H), 1.32 (s, 4.5H).
[233] Example 17
[234] {t-Butyloxycarbonyl- [2- (2-nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (2-nitro- Preparation of 4-trifluoromethyl-benzenesulfonylamino) -ethyl] -amino} -acetic acid)
[235] [2- (2-Nitro-4-trifluoromethyl-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (8.00 g, 20 mmol) was prepared in the same manner as in Example 14 to give 8.62 g of the title compound ( 91%).
[236] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[237] δ 8.57 (d, 1H), 8.41 (t, 0.5H), 8.35 (t, 0.5H), 8.19 (dd, 1H), 3.82 (s, 1H), 3.75 (s, 1H), 3.24 (t, 2H), 3.09 (m, 1H), 1.34 (s, 4.5H), 1.30 (s, 4.5H).
[238] Example 18
[239] {t-Butyloxycarbonyl- [2- (4-methyl-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (4-methyl-2-nitro -benzenesulfonylamino) -ethyl] -amino} -acetic acid)
[240] [2- (4-Methyl-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (6.91 g, 0.02 mol) was prepared in the same manner as Example 14 to yield 7.80 g (93%) of the title compound. Got.
[241] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[242] δ 7.99 to 7.73 (m, 1H), 7.85 (dd, 1H), 7.82 (s, 1H), 7.67 (d, 1H), 3.81 (s, 1H), 3.76 (s, 1H), 3.23 (m, 2H), 3.05 (m, 2H), 2.44 (s, 3H), 1.36 (s, 4.5H), 1.31 (s, 4.5H).
[243] Example 19
[244] {t-Butyloxycarbonyl-[(4-chloro-6-methyl-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (4-chloro-6 -methyl-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid)
[245] [2- (4-Chloro-6-methyl-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (380 mg, 0.01 mol) was prepared in the same manner as in Example 14 to obtain 413 mg of the title compound. (91%) was obtained.
[246] Example 20
[247] {t-Butyloxycarbonyl- [2- (4,6-dichloro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid ({t-Butyloxycarbonyl- [2- (4,6-dichloro -2-nitro-benzenesulfonyl amino) -ethyl] -amino} -acetic acid)
[248] [2- (4,6-Dichloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (400 mg, 1.0 mmol) was prepared in the same manner as Example 14 to obtain 417 mg (88) of the title compound. %) Was obtained.
[249] Example 21
[250] N- (t-butyloxycarbonyl) -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -alanine (N- (t-Butyloxycarbonyl) -N- [2- (2-nitro- benzenesulfonylamino) -ethyl] -alanine)
[251] Lithium hydroxide was added to a solution of N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -alanine ethyl ester (1.04 g, 3 mmol) in tetrahydrofuran (10 ml) in water (10 hydrate (252 mg, 6 mmol) dissolved in ml) was added, followed by stirring at room temperature for 1 hour. After completion of the hydrolysis, di-t-butyl dicarbonate (6.55 g, 0.03 mol) was added to the reaction solution, stirred for 5 hours, and then dissolved in lithium hydroxide hydrate (126 mg, 3). mmol) was added dropwise for 30 minutes. After the reaction was completed, the insoluble substance was filtered and the filtrate was washed with diethyl ether (20 ml) and the aqueous layer was separated. The separated aqueous layer was adjusted to pH = 3 by adding 2N hydrochloric acid aqueous solution, and then extracted by adding dichloromethane (30 ml). The separated organic layer was dried and filtered through anhydrous forget-me-not and concentrated under reduced pressure to obtain 1.16 g (93%) of the title compound as a viscous oily solid.
[252] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[253] δ 7.99 (m, 2H), 7.87 (m, 2H), 4.31 (q, 0.4H), 4.03 (q, 0.6H), 3.37 to 3.19 (m, 2H), 3.10 to 2.95 (m, 2H), 1.35, 1.33 (s, s, 9H), 1.30, 1.29 (d, d, 3H).
[254] Example 22
[255] N- (t-butyloxycarbonyl) -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -phenylglycine (N- (t-Butyloxycarbonyl) -N- [2- (2-nitro -benznesulfonylamino) -ethyl] -phenyl glycine)
[256] Hydrolysis and t-butyloxycarbonylation in the same manner as in Example 21 using N- [2- (2nitro-benzenesulfonylamino) -ethyl] -phenylglycine methyl ester (590 mg, 1.5 mmol) Proceeded for 48 hours to obtain 583 mg (81%) of the desired labeled compound.
[257] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[258] δ 7.97 to 7.82 (m, 4H), 7.35 to 7.30 (m, 3H), 7.23 to 7.21 (m, 2H), 5.58 (s, 0.6H), 5.40 (s, 0.4H), 3.20 to 2.85 (m , 4H), 1.46 (s, 4.5H), 1.37 (s, 4.5H).
[259] Example 23
[260] 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (2-Nitrobenzene
[261] sulfonyl) -piperazin-2-one, HCl salt)
[262] {t-Butyloxycarbonyl- [2- (2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (7.7 g, 19 mmol) was dissolved in dichloromethane (100 ml) and then dished at room temperature. Chlohexyl carbodiimide (5.16 g, 0.025 mol) was added and stirred 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 (50 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-ethyl acetate solution (100 ml) was added thereto. The reaction solution was stirred at room temperature for 10 hours, and the resulting solid was filtered, washed with ethyl acetate (500 ml) and dried to give 4.8 g (79%) of the title compound as a white solid.
[263] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[264] δ 9.95 (br.s, 2H), 8.38 (d, 1H), 8.04-7.95 (m, 2H), 4.09 (dd, 2H), 3.96 (s, 2H), 3.56 (dd, 2H).
[265] Example 24
[266] Preparation of 1-(-4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (4-Chloro-2-nitrobenzenesulfonyl) -piperazin-2-one, HCl salt)
[267] {t-butyloxycarbonyl- [2- (4-chloro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (7.92 g, 18 mmol) was dissolved in dichloromethane (100 ml). After reaction at room temperature using a dicyclohexyl carbodiimide (5.16 g, 0.025 mol) in the same manner as in Example 23 to obtain 4.3 g (67%) of the title compound as a white solid.
[268] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[269] δ 10.01 (br.s, 2H), 8.42 (d, 1H), 8.35 (dd, 1H), 8.06 (dd, 1H), 4.07 (t, 2H), 3.95 (s, 2H), 3.55 (t, 2H).
[270] Example 25
[271] Of 1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (4-Fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one, HCl salt) Produce
[272] Dissolve {t-butyloxycarbonyl- [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (7.61 g, 18 mmol) in dichloromethane (100 ml) After reacting at room temperature with dicyclohexyl carbodiimide (5.16 g, 0.025 mol) in the same manner as in Example 23 to obtain 4.2 g (69%) of the title compound as a white solid.
[273] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[274] δ9.91 (br.s, 2H), 8.43 (m, 1H), 8.27 (m, 1H), 7.87 (m, 1H), 4.06 (dd, 2H), 3.96 (s, 2H), 3.55 (dd, 2H).
[275] Example 26
[276] 1- (2-Nitro-4-trifluoromethyl-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (2-Nitro-4-trifluoromethyl-benzenesulfonyl) -piperazin-2-one, HCl salt Manufacturing
[277] {t-Butyloxycarbonyl- [2- (2-nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -amino} -acetic acid (4.71 g, 10 mmol) was diluted with dichloromethane (50 ml). After dissolving in, the reaction was carried out in the same manner as in Example 23 using dicyclohexyl carbodiimide (2.68 g, 0.013 mol) at room temperature to obtain 2.55 g (65%) of the title compound as a white solid.
[278] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[279] δ 10.16 (br.s, 2H), 8.71 (s, 1H), 8.60 (d, 1H), 8.35 (d, 1H), 4.13 (dd, 2H), 3.96 (s, 2H), 3.57 (dd, 2H).
[280] Example 27
[281] Preparation of 1- (4-methyl-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (4-Methyl-2-nitro-benzenesulfonyl) -piperazin-2-one, HCl salt)
[282] {t-butyloxycarbonyl- [2- (4-methyl-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (4.19 g, 0.01 mol) was dissolved in dichloromethane (50 ml). Subsequently, using a dicyclo hexyl carbodiimide (2.68 g, 0.013 mol) at room temperature to give a 2.40 g (71%) of the title compound as a white solid in the same manner as in Example 23.
[283] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[284] δ9.87 (br.s, 1H), 8.23 (d, 1H), 7.98 (s, 1H), 7.77 (d, 1H), 4.06 (dd, 2H), 3.95 (s, 2H), 3.55 (dd, 2H), 2.49 (s, 3H).
[285] Example 28
[286] 1- (4-Chloro-6-methyl-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (4-Chloro-6-methyl-2-nitro-benzenesulfonyl) -piperazin-2 -one, HCl salt)
[287] {t-Butyloxycarbonyl- [2- (4-chloro-6-methyl-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (4.54 g, 0.01 mol) was diluted with dichloromethane (50 ml ), And then reacted in the same manner as in Example 23 using dicyclohexyl carbodiimide (2.68 g, 0.013 mol) at room temperature to obtain 2.53 g (68%) of the title compound as a white solid.
[288] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[289] δ 10.02 (br.s, 2H), 8.20 (s, 1H), 7.96 (s, 1H), 4.04 (dd, 2H), 3.96 (s, 2H), 3.53 (dd, 2H), 2.67 (s, 3H).
[290] Example 29
[291] 1- (4,6-Dichloro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1- (4,6-Dichloro-2-nitro-benzenesulfonyl) -piperazin-2-one, HCl salt)
[292] {t-Butyloxycarbonyl- [2- (4,6-dichloro-2-nitro-benzenesulfonylamino) -ethyl] -amino} -acetic acid (4.74 g, 0.01 mol) in dichloromethane (50 ml) After dissolution, the reaction was carried out in the same manner as in Example 23 using dicyclohexyl carbodiimide (2.68 g, 0.013 mol) at room temperature, to obtain 2.51 g (64%) of the title compound as a white solid.
[293] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[294] delta 10.06 (br.s, 2H), 8.43 (s, 1H), 8.32 (s, 1H), 4.16 (t, 2H), 3.99 (s, 2H), 3.50 (t, 2H).
[295] Example 30
[296] Preparation of 1- (2-Nitro-benzenesulfonyl) -3-methyl-piperazin-2-one hydrochloride (1- (2-Nitro-benzenesulfonyl) -3-methyl-piperazin-2-one, HCl salt)
[297] N- (t-butyloxycarbonyl) -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -alanine (835 mg, 2 mmol) was dissolved in dichloromethane (10 ml) and then room temperature Dicyclo hexyl carbodiimide (619 mg, 3 mmol) was added thereto, followed by stirring for 12 hours. Upon completion of the reaction, the insoluble dicyclohexyl urea was filtered off and the solution removed was concentrated under reduced pressure. Ethyl acetate (10 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 (10 ml) was added thereto. After the reaction solution was stirred for 10 hours at room temperature, the resulting solid was filtered, washed with ethyl acetate (50 ml) and dried to give 450 mg (67%) of the title compound as a white solid.
[298] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[299] δ 8.38 (d, 1H), 8.14 (d, 1H), 8.03 (dd, 1H), 7.98 (dd, 1H), 4.30 (m, 1H), 4.13 (m, 2H), 3.70 (m, 1H) , 3.51 (m, 1 H), 1.39 (d, 3 H).
[300] Example 31
[301] 1- (2-Nitro-benzenesulfonyl) -3-phenyl-piperazin-2-one, hydrochloride (1- (2-Nitro-benzenesulfony l) -3-phenyl-piperazin-2-one, HCl salt)
[302] In the same manner as in Example 30 using N- (t-butyloxycarbonyl) -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -phenylglycine (480 mg, 1 mmol) 247 mg (63%) of the desired labeled compound were obtained.
[303] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[304] δ 8.35 (d, 1H), 8.15 (d, 1H), 8.02 (t, 1H), 7.94 (t, 1H), 7.40 (m, 5H), 5.47 (br s, 1H), 4.24 (m, 2H ), 3.69 (m, 1 H), 3.60 (m, 1 H).
[305] Preparation of Acetic Acid Derivatives of Nucleic Acid Bases
[306] (Timin-1-yl) -acetic acid (KL Dueholm et al, J. Org. Chem ., 1994, 59 , 5767; KL Dueholm et al, J. Org. Chem ., 1994, 59 , 5767; O. Buchardt et al., WO9201219) were prepared according to known methods. 6-N- (benzhydryloxycarbonyl) adenin-9-yl) -acetic acid, 6-N- (benzyloxycarbonyl) adenin-9-yl) -acetic acid, 4-N- (benzhydryloxycarbon Carbonyl) cytosine-1-yl) -acetic acid and 4-N- (benzyloxycarbonyl) cytosine-1-yl) -acetic acid were also prepared according to known methods. (JM Collul et al., US 6,172,226 )
[307] Example 32
[308] Preparation of (2-Amino-6-iodinepurin-9-yl) -acetic acid ethyl ester ((2-Amino-6-iodopurin-9-yl) -acetic acid ethyl ester)
[309] 2-amino-6-iodinepurine (78.3 g, 0.3 mol) was dissolved in 1960 ml of dimethylformamide and then ethyl bromoacetate (55.1 g, 0.33 mol) and potassium carbonate (82.9 g, 0.6 mol) were added. The solution was reacted at room temperature for about 12 hours, distilled under reduced pressure, and diluted with water. The resulting solid was filtered, washed with water and ethyl ether, and dried to give 98.4 g (95%) of the title compound.
[310] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[311] δ 8.06 (s, 1H), 6.90 (bs, 2H), 4.94 (s, 2H), 4.17 (q, 2H), 1.22 (t, 3H)
[312] Example 33
[313] [2- (Benzhydryloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid ethyl ester ([2- (Benzhydryloxycarbonyl) amino-6-iodopurine-9-yl] -aceticacid ethyl ester)
[314] 2-amino-6-iodinepurin-9-yl-acetic acid ethyl ester (13.9 g, 40 mmol) was dissolved in 280 ml of tetrahydrofuran and then cooled to about 0 ° C. and triphosgen (5.34 g, 18 mmol) After the addition, the mixture was stirred for about 5 minutes. Diisopropylethylamine (24.4 ml) was slowly added to the mixture, stirred at below 0 ° C. for about 30 minutes, and then benzhydrol (14.74 g, 80 mmol) was added. The reaction mixture was allowed to react for about 13 hours while gradually rising to room temperature, and then water and 1N HCl aqueous solution were added and stirred at room temperature for about 30 minutes. The aqueous layer was saturated with salt and sodium thiosulfate, the aqueous layer was neutralized with sodium hydrogen carbonate, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate, the two organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and distilled under reduced pressure to obtain a residue, which was then purified by column chromatography to give 15.15 g (68% of Yield).
[315] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[316] δ 10.88 (bs, 1H), 8.45 (s, 1H), 7.60 to 7.20 (m, 10H), 6.79 (s, 1H), 5.08 (s, 2H), 4.15 (q, 2H), 1.17 (t, 3H).
[317] Example 34
[318] Preparation of [2-N- (benzhydryloxycarbonyl) guanine-9-yl] -acetic acid ([2-N (Benzhydryl oxycarbonyl) guanin-9-yl] -acetic acid)
[319] 60% sodium hydride (5.04 g, 126 mmol) was suspended in tetrahydrofuran (110 ml) and cooled to 2 ° C. 3-hydroxypropionitrile (8.60 ml, 126 mmol) was added below 10 ° C. for about 10 minutes and stirred for 12 minutes. To the above solution, [2- (benzhydryloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid (11.12 g, 21 mmol) was added keeping the temperature below 5 ° C. The reaction mixture was slowly increased to room temperature and stirred for about 3.5 hours. After the reaction was completed, water (60 ml) and 20% aqueous citric acid solution (120 ml) were added, and the organic layer was separated by saturating the aqueous solution with salt. The aqueous layer was extracted with tetrahydrofuran, and each organic layer was mixed, dried over anhydrous sodium sulfate, filtered and distilled under reduced pressure, and the residue obtained was crystallized in ethanol. The matured solid was filtered, washed with ethanol and dried to give 9.00 g of the title compound.
[320] Example 35
[321] Preparation of [2- (benzyloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid ethyl ester ([2- (Benzyl oxycarbonyl) amino-6-iodopurine-9-yl] -acetic acid ethyl ester)
[322] (2-Amino-6-iodinepurin-9-yl) -acetic acid ethyl ester (13.9 g, 40 mmol) was dissolved in 280 ml of tetrahydrofuran and then cooled to about 0 ° C., triphosgen (5.34 g, 18 mmol) Was added and then stirred for about 5 minutes. Diisopropylethylamine (24.4 ml) was slowly added to the mixture and stirred at below 11 ° C. for about 30 minutes and benzylalcohol (8.8 ml, 80 mmol) was added at about 4 ° C. After the reaction mixture was slowly raised to room temperature and reacted for about 13 hours, water and 1N HCl aqueous solution were added, and the organic solvent was distilled off under reduced pressure. The aqueous layer was extracted twice with dichloromethane, the organic layer was dried over sodium sulfate, filtered and distilled under reduced pressure to give a brown residue, which was then precipitated with a white solid in anhydrous ethanol. The resulting solid was filtered, washed with ethanol and dried to give 12.42 g (64.4% yield) of the title compound.
[323] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[324] δ 10.83 (bs, 1H), 8.48 (s, 1H), 7.50 to 7.30 (m, 5H), 5.18 (s, 2H), 5.11 (s, 2H), 4.19 (q, 2H), 1.21 (t, 3H).
[325] Example 36
[326] Preparation of [2- (benzyloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid ([2- (Benzyl oxycarbonyl) amino-6-iodopurin-9-yl] -acetic acid)
[327] Dissolve [2- (benzyloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid ethyl ester (10.02 g, 20.8 mmol) in tetrahydrofuran (50 ml) and add water (50 ml) to suspension Made. The suspension was cooled to about 10 DEG C or lower, and 2.83 g (20.8 mmol) of lithium hydroxide hydrate was added and stirred at about 15 DEG C or lower for 0.5 hour. Aqueous 1N hydrochloric acid was added to the reaction to adjust the pH to about 3 to precipitate a solid, which was filtered, washed with water and ethyl ether and dried to obtain 9.81 g of the title compound.
[328] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[329] delta 10.75 (s, 1H), 8.44 (s, 1H), 7.50 to 7.30 (m, 5H), 5.17 (s, 2H), 4.97 (s, 2H).
[330] Example 37
[331] Preparation of [2-N- (benzyloxycarbonyl) guanine-9-yl] -acetic acid ([2-N- (Benzyloxycarbonyl) -guanin-9-yl] -acetic acid)
[332] 60% sodium hydride (4.80 g, 120 mmol) was suspended in tetrahydrofuran (90 ml) and cooled below 5 ° C. 3-hydroxypropionitrile (8.19 ml, 120 mmol) was added below about 10 ° C. and stirred for 18 minutes. To the above solution, 9.06 g (20 mmol) of [2- (benzyloxycarbonyl) amino-6-iodinepurin-9-yl] -acetic acid was added while maintaining below 10 ° C. The reaction mixture was slowly increased to room temperature and stirred for about 3.5 hours. After the reaction was completed, water (50 ml) and 20% aqueous citric acid solution (100 ml) were added, and the organic layer was separated by saturating the aqueous solution with salt. The aqueous layer was extracted with tetrahydrofuran and each organic layer was mixed. The white solid was crystallized from the mixed organic layers, filtered, washed with ethanol and dried to yield 5.44 g (79.2% yield) of the title compound.
[333] ¹ H NMR (DMSO-d6)
[334] delta 11.54 (s, 1H), 11.37 (s, 1H), 7.94 (s, 1H), 7.46-7.33 (m, 5H), 5.28 (s, 2H), 4.87 (s, 2H).
[335] Example 38
[336] N-[(Thymin-1-yl) -acetyl] -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycineethyl ester (N-[(Thymin-1-yl) -acetyl] Preparation of -N- [2- (2-nitro-benznesulfonylamino) -ethyl] -glycine ethyl ester
[337] [(2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (1.67 g, 5 mmol) and (0.92 g, 5 mmol) of (thimin-1-yl) -acetic acid were converted to dimethylformamide (15 ml) and PyBOP (3.12 g, 6 mmol) and diisopropylethylamine (1.31 ml, 7.5 mmol) were added sequentially at room temperature. The reaction mixture was stirred at room temperature for about 7 hours, and the residue obtained by distillation of the reaction solvent under reduced pressure was diluted with ethyl acetate (50 ml) and washed with an aqueous 1N hydrochloric acid solution and an aqueous sodium hydrogen carbonate solution. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and distilled under reduced pressure to obtain a residue. The residue was treated with ethanol and the resulting solid was filtered and dried to give 2.35 g (93% yield) of the title compound.
[338] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[339] δ 11.29 (s, 0.6H), 11.26 (s, 0.4H), 8.25 to 7.87 (m, 5H), 7.30 (s, 0.6H), 7.23 (s, 0.4H), 4.64 (s, 1.2H) , 4.46 (s, 0.8H), 4.29 (s, 0.8H), 4.16 (q, 0.8H), 4.07 (q, 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.21 (q, 1.2H), 3.12-3.01 (m, 1.6H), 1.75 (s, 3H), 1.22 (t, 1.2H), 1.17 (t, 1.8H).
[340] Example 39
[341] N-[(Thymin-1-yl) -acetyl] -N- [2- (4-chloro-2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester (N-[(Thymin-1-yl ) -acetyl] -N- [2- (4-chloro-2-nitro-benznesulfonylamino) -ethyl] -glycine ethyl ester)
[342] [2- (4-Chloro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (1.84 g, 5 mmol) and (thimin-1-yl) -acetic acid ( 0.92 g, 5 mmol) was suspended in dimethylformamide (15 ml) and obtained 2.46 g (92%) using PyBOP (3.12 g, 6 mmol) and diisopropylethylamine (1.31 ml, 7.5 mmol).
[343] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[344] δ 11.31 (s, 0.6H), 11.28 (s, 0.4H), 8.31 to 7.95 (m, 4H), 7.31 (s, 0.6H), 7.23 (s, 0.4H), 4.64 (s, 1.2H) , 4.46 (s, 0.8H), 4.28 (s, 0.8H), 4.16 (q. 0.8H), 4.07 (q. 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.22 (q, 1.2H), 3.12-3.01 (m, 1.6H), 1.74 (s, 3H), 1.22 (t, 1.2H), 1.17 (t, 1.8H)
[345] Example 40
[346] N-[(Thimin-1-yl) -acetyl] -N- [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester (N-[(Thymin-1- Preparation of yl) -acetyl] -N- [2- (4-fluoro-2-nitro-benznesulfonylami no) -ethyl] -glycine ethyl ester
[347] [(4-Fluoro-2-nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (1.76 g, 5 mmol) and (thimin-1-yl) -acetic acid (0.92) in the same manner as in Example 38 g, 5 mmol) was suspended in dimethylformamide (15 ml) and obtained 2.35 g (91%) using PyBOP (3.12 g, 6 mmol) and diisopropylethylamine (1.31 ml, 7.5 mmol).
[348] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[349] δ 11.30 (s, 0.6H), 11.27 (s, 0.4H), 8.28 to 8.05 (m, 2.8H), 7.78 (m, 1.2H), 7.31 (s, 0.6H), 7.23 (s, 0.4H ), 4.65 (s, 1.2H), 4.47 (s, 0.8H), 4.30 (s, 0.8H), 4.16 (q. 0.8H), 4.07 (q. 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.22 (q, 1.2H), 3.12-3.01 (m, 1.6H), 1.75 (s, 3H), 1.23 (t, 1.2H), 1.18 (t, 1.8H)
[350] Example 41
[351] N-[(Thimin-1-yl) -acetyl] -N- [2- (2-nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -glycine ethyl ester (N-[(Thymin- Preparation of 1-yl) -acetyl] -N- [2- (2-nitro-4-trifluoromethyl-benznesulfonylamino) -ethyl] -glycine ethyl ester)
[352] [(2-Nitro-4-trifluoromethyl-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (2.00 g, 5 mmol) and (thimin-1-yl) -acetic acid in the same manner as in Example 38 (0.92 g, 5 mmol) was suspended in dimethylformamide (15 ml) and obtained 2.69 g (95%) using PyBOP (3.12 g, 6 mmol) and diisopropylethylamine (1.31 ml, 7.5 mmol). .
[353] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[354] δ 11.31 (s, 0.55H), 11.28 (s, 0.45H), 8.57 (s, 0.55H), 8.54 (s, 0.45H), 8.50 (t, 0.55H), 8.38 (t, 0.45H), 8.29-8.18 (m, 2H), 7.31 (s, 0.55H), 7.22 (s, 0.45H), 4.64 (s, 1.1H), 4.46 (s, 0.9H), 4.21 (s, 0.9H), 4.16 (q. 0.9H), 4.07 (q. 1.1H), 3.95 (s, 1.1H), 3.49 (t, 1.1H), 3.37 (q, 1.1H), 3,25 (t, 0.9H), 3.09 (t, 0.9H), 1.75 (s, 3H), 1.23 (t, 1.35H), 1.18 (t, 1.65H).
[355] Example 42
[356] N-{[4-N- (benzhydryloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester ( Preparation of N-{[4-N- (Benzhydryloxy carbonyl) -cytosin-1-yl] -acetyl} -N- [2- (2-nitro-benznesulfonylamino) -ethyl] -glycine ethyl ester)
[357] [2- (2-Nitro-benzenesulfonylamino) -ethylamino] -acetic acid ethyl ester (1.33 g, 4 mmol) and 2- [4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -Acetic acid (1.51 g, 4 mmol) was suspended in dimethylformamide (13 ml) and PyBOP (2.60 g, 5 mmol) and diisopropylethylamine (1.05 ml, 6 mmol) were reacted in the same manner as in Example 38 This gave 2.45 g (88% yield) of the title compound.
[358] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[359] δ 10.99 (s, 0.6H), 10.98 (s, 0.4H), 8.30 to 7.80 (m, 5H), 7.50 to 7.25 (m, 10H), 6.94 (t, 1H), 6.79 (s, 1H), 4.79 (s, 1.2H), 4.61 (s, 0.8H), 4.33 (s, 0.8H), 4.15 (q, 0.8H), 4.05 (q, 1.2H), 4.01 (s, 1.2H), 3.52 (t, 1.2H), 3.36 (t, 0.8H), 3.24 (q, 1.2H), 3.02 (q, 0.8H), 1.23 (t, 1.2H), 1.15 (t, 1.8H).
[360] Example 43
[361] N-{[4-N- (benzyloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester (N- Preparation of {[4- (Benzyloxy carbonyl) -cytosin-1-yl] -acetyl} -N- [2- (2-nitro-benznesulfonylamino) -ethyl] -glycine ethyl ester)
[362] In the same manner as in Example 38, the title compound was prepared in a yield of 74%.
[363] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[364] δ8.15 to 8.06 (m, 1H), 7.84 to 7.57 (m, 5H), 7.38 to 7.72 (m, 6H), 7.14 (t, 0.7H), 6.44 (t, 0.3H), 5.20 (s, 2H ), 4.93 (s, 1.4H), 4.55 (s, 0.6H), 4.36 (s, 0.6H), 4.26 (q, 0.6H), 4.17 (q, 1.4H) 4.03 (s, 1.4H), 3.71 (t, 1.4H), 3.57 (t, 0.6H), 3.39 (q, 1.4H), 3.30 (q, 0.6H), 1.31 (t, 0.9H), 1.24 (t, 2.1H).
[365] Example 44
[366] N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (N- [2- (2-Nitro-benzenesulfonylamino) -ethyl] Preparation of -N-[(thymin-1-yl) -acetyl] -glycine)
[367] N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine ethyl ester (1.00 g, 2.0 mmol) was added to tetrahydrofuran (10 ml). ), The suspension was cooled to 10 ° C. or lower, and an aqueous solution of lithium hydroxide hydrate (210 mg, 5 mmol) dissolved in water (10 ml) was added to a clear solution, followed by stirring at the same temperature for about 1.5 hours. . 1N aqueous hydrochloric acid solution was added to the reaction to adjust the pH to 2-3, and the white solid produced was filtered to obtain 896 mg (95%) of the labeled compound.
[368] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[369] δ 11.30 (s, 0.6H), 11.27 (s, 0.4H), 8.22 to 7.85 (m, 5H), 7.31 (d, 0.6H), 7.23 (d, 0.4H), 4.63 (s, 1.2H) , 4.45 (s, 0.8H), 4.19 (s, 0.8H), 4.16 (q. 0.8H), 3.93 (s, 1.2H), 3.47 (t, 1.2H), 3.34 (t, 0.8H), 3.20 (q, 1.2H), 3.04 (q. 0.8H), 1.75 (s, 3H).
[370] Example 45
[371] N- [2- (4-Chloro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (N- [2- (4-Chloro-2 -nitro-benzenesulfonylamino) -ethyl] -N-[(thymin-1-yl) -acetyl] -glycine)
[372] In the same manner as in Example 44 N- [2- (4-chloro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine ethyl ester (1.60 g, 3.0 mmol) 1.40 g (93%) of the desired title compound were obtained.
[373] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[374] δ 11.31 (s, 0.55H), 11.28 (s, 0.45H), 8.33 to 7.95 (m, 4H), 7.31 (s, 0.55H), 7.23 (s, 0.45H), 4.63 (s, 1.1H) , 4.44 (s, 0.9H), 4.20 (s, 0.9H), 4.00 (s, 1.1H), 3.47 (t, 1.1H), 3.34 (t, 0.9H), 3.19 (q, 1.1H), 3.04 (q, 0.9H), 1.75 (s, 3H).
[375] Example 46
[376] N- [2- (4-Fluoro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (N- [2- (4-Fluoro- Preparation of 2-nitro-benzenesulfonylamino) -ethyl] -N-[(thymin-1-yl) -acetyl] -glycine)
[377] In the same manner as in Example 44 N- [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine ethyl ester (1.03 g, 2.0 mmol) was used Yielded 0.92 g (94%) of the title compound as desired.
[378] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[379] δ 11.30 (s, 0.6H), 11.28 (s, 0.4H), 8.29 to 8.05 (m, 2.8H), 7.78 (m, 1.2H), 7.31 (s, 0.6H), 7.23 (s, 0.4H ), 4.63 (s, 1.2H), 4.44 (s, 0.8H), 4.20 (s, 0.8H), 3.94 (s, 1.2H), 3.46 (t, 1.2H), 3.34 (t, 0.8H), 3.20 (q, 1.2 H), 3.04 (q. 0.8 H), 1.75 (s, 3 H).
[380] Example 47
[381] N- [2- (2-nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (N- [2- (2- Preparation of Nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -N-[(thymin-1-yl) -acetyl] -glycine)
[382] In the same manner as in Example 44 N- [2- (2-Nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine ethyl ester (1.14 g, 2.0 mmol) Was used to obtain 1.02 g (95%) of the title compound.
[383] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[384] δ 11.31 (s, 0.55H), 11.28 (s, 0.45H), 8.58 (s, 0.55H), 8.54 (s, 0.45H), 8.51 (t, 0.55H), 8.38 (t, 0.45H), 8.29 to 8.18 (m, 2H), 7.31 (s, 0.55H), 7.22 (s, 0.45H), 4.63 (s, 1.1H), 4.45 (s, 0.9H), 4.20 (s, 0.9H), 3.94 (s, 1.1H), 3.48 (t, 1.1H), 3.37 (t, 0.9H), 3,24 (q, 1.1H), 3.09 (q, 0.9H), 1.75 (s, 3H).
[385] Example 48
[386] N-{[4-N- (benzhydryloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine (N- Preparation of {[4-N- (Benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine)
[387] N-{[4-N- (benzhydryloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester ( 1.91 g, 2.75 mmol) was dissolved in tetrahydrofuran (10 ml) and diluted with water (9 ml) to form a suspension. The suspension was cooled to 10 ° C. or lower and lithium hydroxide hydrate (0.29 g, 6.9 mmol) was added to give a clear solution and stirred at the same temperature for about 1.5 hours. 1N hydrochloric acid aqueous solution was added to the reaction to adjust the pH to 3-4, and the aqueous layer was saturated with salt to separate the organic layer. The aqueous layer was washed with tetrahydrofuran and then the organic layers were mixed. The organic layer was dried over sodium sulfate, filtered and distilled under reduced pressure to obtain a residue. The resulting solid was filtered and dried to obtain 1.42 g (78%) of the title compound.
[388] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[389] δ 8.25 to 7.80 (m, 5H), 7.50 to 7.25 (m, 10H), 6.94 (d, 0.6H), 6.92 (d, 0.4H), 6,79 (1H, s), 4.78 (s, 1.2 H), 4.60 (s, 0.8H), 4.22 (s, 0.8H), 3.94 (s, 1.2H), 3.50 (t, 1.2H), 3.35 (t, 0.8H), 3.24 (q, 1.2H) , 3.02 (q, 0.8H).
[390] Example 49
[391] N-{[4-N- (benzyloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine (N-{[ Preparation of 4-N- (Benzyloxycarbonyl) -cytosin-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine)
[392] N-{[4-N- (benzyloxycarbonyl) -cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine ethyl ester In the same manner as in Example 48, the title compound was prepared in a yield of 70%.
[393] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[394] δ 8.26 to 7.83 (m, 5H), 7.44 to 7.33 (m, 5H), 7.02 (d, 0.6H), 7.00 (d, 0.4H), 5.19 (s, 2H) 4.80 (s, 1.2H), 4.61 (s, 0.8H), 4.23 (s, 0.8H), 3.94 (s, 1.2H), 3.50 (t, 1.2H), 3.35 (t, 0.8H), 3.24 (q, 1.2H), 3.02 ( q, 0.8H).
[395] Preparation of Monomer
[396] The method of preparing a monomer can be prepared by two methods, using a compound such as Examples 44, 45, 46, 47, 48, and 49 to form a ring by linking a sulfonamide and a carboxylic acid and a piperazinone already prepared. It can be divided into a method for producing acetic acid of a nucleic acid base using a derivative.
[397] 1.How to make a ring by linking sulfonamide and carboxylic acid
[398] Example 50
[399] 1- (2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (2-nitro-benzenesulfonyl) -4-[(thymin-1 -yl) -acetyl] -piperazin-2-one)
[400] N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (469 mg, 1.0 mmol) was dissolved in tetrahydrofuran (10 ml) N-methylmorpholine (330 mL, 3 mmol) is added at 20 ° C and stirred for 10 minutes. Isobutyl chloroformate (205 mg, 1.5 mmol) was added to the reaction solution. After slowly raising the temperature of the reaction mixture to room temperature, the mixture was stirred for about 1 hour to proceed with the reaction, and water (10 ml) was added to filter the resulting solid to prepare 442 mg (98%) of the title compound.
[401] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[402] δ 11.32 (s, 0.6H), 11.30 (s, 0.4H), 8.35 (m, 1H), 8.12 (dd, 1H), 8.04-7.96 (m, 2H), 7.35 (s, 0.6H), 7.28 (s, 0.4H), 4.67 (s, 1.2H), 4.59 (s, 0.8H), 4.41 (s, 0.8H), 4.27 (s, 1.2H), 4.04 (m, 1.2H), 3.95 (m , 1.2H), 3.90-3.85 (m, 1.6H), 1.75 (s, 3H).
[403] Example 51
[404] 1- (4-Chloro-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Chloro-2-nitro-benzenesulfonyl) Preparation of -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[405] In the same manner as in Example 50, N- [2- (4-chloro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (1.01 g, 2.0 mmol) was used to give 933 mg (96%) of the title compound.
[406] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[407] δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.40 (d, 1H), 8.33 (dd, 1H), 8.08 (dd, 1H), 7.34 (s, 0.6H), 7.28 (s , 0.4H), 4.66 (s, 1.2H), 4.59 (s, 0.8H), 4.41 (s, 0.8H), 4.26 (s, 1.2H), 4.02 (m, 1.2H), 3.94 (m, 1.2 H), 3.89 to 3.82 (m, 1.6H), 1.74 (s, 3H).
[408] Example 52
[409] 1- (4-Fluoro-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Fluoro-2-nitro-benzenesulfonyl ) -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[410] In the same manner as in Example 50, N- [2- (4-fluoro-2-nitro-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (487 mg, 1.0 mmol) was used to give 451 mg (96%) of the title compound.
[411] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[412] δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.41 (m, 1H), 8.25 (dd, 1H), 7.88 (m, 1H), 7.34 (s, 0.6H), 7.28 (s , 0.4H), 4.66 (s, 1.2H), 4.59 (s, 0.8H), 4.41 (s, 0.8H), 4.26 (s, 1.2H), 4.02 (m, 1.2H), 3.94 (m, 1.2 H), 3.88 to 3.82 (m, 1.6H), 1.74 (s, 3H).
[413] Example 53
[414] 1- (2-Nitro-4-trifluoromethyl-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (2-Nitro-4-trifluoromethyl Preparation of -benzenesulfonyl) -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[415] N- [2- (2-nitro-4-trifluoromethyl-benzenesulfonylamino) -ethyl] -N-[(thimin-1-yl) -acetyl] -glycine (537) in the same manner as in Example 50 mg, 1.0 mmol) was used to give 493 mg (95%) of the title compound.
[416] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[417] δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.69 (s, 1H), 8.56 (dd, 1H), 8.39 (dd, 1H), 7.34 (s, 0.6H), 7.28 (s , 0.4H), 4.67 (s, 1.2H), 4.59 (s, 0.8H), 4.42 (s, 0.8H), 4.27 (s, 1.2H), 4.05 (m, 1.2H), 3.95 (m, 1.2 H), 3.91-3.85 (m, 1.6H), 1.75 (s, 3H).
[418] Example 54
[419] 4-{[4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[4 Preparation of -N- (Benzhydryloxycarbonyl) -cytosin-1-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[420] N-{[4-N- (benzhydryloxycarbonyl) cytosin-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl] -glycine (1.20 g, 1.8 mmol) was dissolved in tetrahydrofuran (24 ml) and cooled to 5 ° C or lower. N-methylmorpholine (0.6 ml, 5.42 mmol) was added above solution and isobutyl chloroformate (0.30 ml, 2.31 mmol) at 4-5 캜 in turn. After slowly raising the temperature of the reaction mixture to room temperature, the mixture was stirred for about 1 hour, and then ethanol (3 ml) was added thereto. The reaction was distilled under reduced pressure to obtain a residue, which was dissolved in a mixed solvent of ethyl acetate and acetonitrile and washed with an acidic solution saturated with salt. The organic layer was dried over sodium sulfate, filtered, and the residue obtained by distillation under reduced pressure was treated with methanol to give a solid. The resulting solid was filtered, washed with methanol and dried to give 0.9 g (77%) of the title compound.
[421] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[422] δ 8.35 (t, 1H), 8.12 to 7.95 (m, 3H), 7.90 (d, 0.6H), 7.84 (d, 0.4H), 7.48 to 7.28 (m, 10H), 6.95 (d, 0.6H) , 6.94 (d, 0.4H), 6.79 (s, 1H), 4.82 (s, 1.2H), 4.73 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.10- 3.95 (m, 2.4H), 3.95-3.80 (m, 1.6H).
[423] Example 55
[424] 4-{[4-N- (benzyloxycarbonyl) -cytosin-1-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[4- Preparation of N- (Benzyloxycarbonyl) -cytosin-1-yl] acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[425] N-{[4-N- (benzyloxycarbonyl) cytosine-1-yl] -acetyl} -N- [2- (2-nitro-benzenesulfonylamino) -ethyl in the same manner as in Example 54 ] -Glycine was used to prepare the title compound in a yield of 90%.
[426] 1 H-NMR (500 MHz; DMSO-d6)
[427] δ 10.76 (s, 1H), 8.36 (t, 1H), 8.12 (dd, 1H), 8.02 to 7.81 (m, 3H), 7.41 to 7.31 (m, 5H), 7.03 (s, 0.4H), 7.02 (s, 0.6H), 5.17 (s, 2H), 4.82 (s, 1.2H), 4.74 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.06 (m, 1.2H), 3.98 (m, 1.2H), 3.91 (m, 0.8H), 3.84 (m, 0.8H).
[428] 2. Method for preparing from acetic acid of piperazinone derivative and nucleic acid base
[429] Example 56
[430] 1- (2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (2-nitro-benzenesulfonyl) -4-[(thymin-1 -yl) acetyl] -piperazin-2-one)
[431] 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1.13 g, 3.5 mmol), (thimin-1-yl) -acetic acid (0.64 g, 3.5 mmol) and PyBOP (2.00 g, 3.85 mmol) was suspended in dimethylformamide (11 ml) and diisopropylamine (0.91 ml) was 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 1.50 g (95%) of the title compound.
[432] NMR data is the same as in Example 50
[433] Example 57
[434] 1- (4-Chloro-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Chloro-2-nitro-benzenesulfonyl) Preparation of -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[435] 1- (4-Chloro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride salt (1.25 g, 3.5 mmol), (thimin-1-yl) -acetic acid (0.64 g, 3.5 mmol). The reaction was carried out in the same manner as in Example 56 using PyBOP (2.00 g, 3.85 mmol) and 0.91 ml of diisopropylamine, to obtain 1.60 g (94% yield) of the title compound.
[436] NMR data is the same as in Example 51
[437] Example 58
[438] 1- (4-Fluoro-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Fluoro-2-nitro-benzenesulfonyl ) -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[439] 1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (680 mg, 2.0 mmol) and (thimin-1-yl) -acetic acid (369 mg, 2.0 mmol) In the same manner as in Example 56, 900 mg (96%) of the title compound were obtained.
[440] NMR data is the same as in Example 52
[441] Example 59
[442] 1- (2-Nitro-4-trifluoromethyl-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (2-Nitro-4-trifluoromethyl Preparation of -benzenesulfonyl) -4-[(thymin-1-yl) -acetyl] -piperazin-2-one)
[443] 1- (2-Nitro-4-trifluoromethyl-benzenesulfonyl) -piperazin-2-one hydrochloride (780 mg, 2 mmol) and (thimin-1-yl) -acetic acid (369 mg, 2 mmol In the same manner as in Example 56, the title compound was obtained 980 mg (94%).
[444] NMR data is the same as in Example 53
[445] Example 60
[446] 1- (4-Methyl-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Methyl-2-nitro-benzenesulfonyl) Preparation of -4-[(thymin-1-yl) acetyl] -piperazin-2-one)
[447] 672 mg (2.0 mmol) of 1- (4-methyl-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride and (thiamin-1-yl) -acetic acid (369 mg, 2.0 mmol) In the same manner as in Example 56, 888 mg (95%) of the title compound were obtained.
[448] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[449] δ 11.33 (s, 0.6H), 11.32 (s, 0.4H), 8.22 (m, 1H), 7.96 (s, 1H), 7.78 (m, 1H), 7.35 (s, 0.6H), 7.28 (s , 0.4H), 4.66 (s, 1.2H), 4.58 (s, 0.8H), 4.39 (s, 0.8H), 4.24 (s, 1.2H), 4.01 (m, 1.2H), 3.93 (m, 1.2 H), 3.88 to 3.81 (m, 1.6H), 2.49 (s, 3H), 1.74 (s, 3H).
[450] Example 61
[451] 1- (4-Chloro-6-methyl-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (4-Chloro-6- Preparation of methyl-2-nitro-benzenesulfonyl) -4-[(thymin-1-yl) acetyl] -piperazin-2-one)
[452] 1- (4-Chloro-6-methyl-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (740 mg, 2.0 mmol) and (thimin-1-yl) -acetic acid (368 mg, 2.0 mmol) to give 913 mg (91%) of the title compound.
[453] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[454] δ 11.34 (s, 0.6H), 11.32 (s, 0.4H), 8.19 (s, 1H), 7.95 (s, 1H), 7.34 (s, 0.6H), 7.28 (s, 0.4H), 4.67 ( s, 1.2H), 4.59 (s, 0.8H), 4.40 (s, 0.8H), 4.26 (s, 1.2H), 3.99 (m, 1.2H), 3.92 (m, 1.2H), 3.82 (s, 1.6H), 2.64 (s, 3H), 1.74 (s, 3H).
[455] Example 62
[456] 1- (4,6-Dichloro-2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one (4- (4,6-Dichloro-6- Preparation of methyl-2-nitro-benzenesulfonyl) -4-[(thymin-1-yl) acetyl] -piperazin-2-one)
[457] 1- (4,6-Dichloro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (781 mg, 2.0 mmol) and (thimin-1-yl) -acetic acid (368 mg, 2.0 mmol) In the same manner as in Example 56, the title compound was obtained 919 mg (88%).
[458] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[459] δ 11.34 (s, 0.6H), 11.32 (s, 0.4H), 8.42 (d, 0.4H), 8.41 (d, 0.6H), 8.32 (d, 0.4H), 8.30 (d, 0.6H), 7.35 (s, 0.6H), 7.27 (s, 0.4H), 4.69 (s, 1.2H), 4.58 (s, 0.8H), 4.42 (s, 0.8H), 4.27 (s, 1.2H), 4.11 ( t, 1.2H), 3.97 (t, 0.8H), 3.93 (t, 1.2H), 3.82 (t, 0.8H), 1.74 (s, 3H).
[460] Example 63
[461] 4-{[6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[ Preparation of 6-N- (Benzhydryloxycarbonyl) -adenin-1-yl] acetyl} -1- (2-nitro-benzenesulfonyl) piperazin-2-one)
[462] [6-N- (benzhydryloxycarbonyl) adenin-9-yl] -acetic acid (1.0 g, 2.47 mmol), 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride 0.76 g (2.36 mmol) and PyBOP (1.35 g) are dissolved in dimethylformamide (20 ml) and cooled to 5 ° C. under nitrogen. Diisopropylethylamine (0.95ml) was slowly added to the reaction solution, and the reaction was performed at room temperature for 2 hours. After completion of the reaction, ethyl acetate (200 ml) and water (150 ml) were added, the organic layer was separated, washed successively with water, 5% sodium bicarbonate, 10% citric acid and saturated brine, dried over anhydrous magnesium sulfate, Filtered. The solution was concentrated, and column chromatography on silica gel (eluent; dichloromethane: methanol = 10: 1) gave 830 mg (58%) of the title compound.
[463] ¹ H NMR (DMSO-d ₆ )
[464] δ 10.95 (s, 1H), 8.59 (s, 0.6H), 8.58 (s, 0.4H), 8.40 to 8.36 (m, 1H), 8.33 (s, 0.6H), 8.31 (s, 0.4H), 8.14 (d, 1H), 8.06-7.96 (m, 2H), 7.54-7.27 (m, 10H), 6.82 (s, 1H), 5.39 (s, 1.2H), 5.30 (s, 0.8H), 4.56 ( s, 0.8H), 4.27 (s, 1.2H), 4.12-4.08 (m, 2.4H), 3.92-3.86 (m, 1.6H).
[465] Example 64
[466] 4-{[6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one ( Preparation of 4-{[6-N- (Benzhydryloxycarbonyl) -adenin-1-yl] acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[467] [6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetic acid (779 mg, 2.0 mmol) and 1- (4-chloro-2-nitro-benzenesulfonyl) -piperazine-2 The reaction was carried out as in Example 63 using-warm hydrochloride (712 mg, 2.0 mmol) to give 810 mg (56%) of the title compound.
[468] ¹ H NMR (DMSO-d ₆ )
[469] δ 11.05 (s, 1H), 8.60 (s, 0.6H), 8.59 (s, 0.4H), 8.43 to 8.41 (m, 1H), 8.37 to 8.32 (m, 2H), 8.08 (m, 1H), 7.53-7.27 (m, 10H), 6.82 (s, 1H), 5.39 (s, 1.2H), 5.31 (s, 0.8H), 4.56 (s, 0.8H), 4.27 (s, 1.2H), 4.12- 4.08 (br m, 2.4 H), 3.90-3.85 (m, 1.6 H).
[470] Example 65
[471] 4-{[6-N- (benzhydryloxycarbonyl) -adenin-9-yl] -acetyl} -1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one Preparation of (4-{[6-N- (Benzhydryloxycarbonyl) -adenin-9-yl] acetyl} -1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[472] [6-N- (benzhydryloxycarbonyl) adenin-9-yl] -acetic acid (807 mg, 2.0 mmol) and 1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazine-2 The reaction was carried out as in Example 63 using-warm hydrochloride (679 mg, 2.0 mmol) to give 805 mg (58%) of the title compound.
[473] ¹ H NMR (DMSO-d ₆ )
[474] δ 10.95 (s, 1H), 8.59 (s, 0.6H), 8.58 (s, 0.4H), 8.47-8.40 (m, 1H), 8.33 (s, 0.6H), 8.30 (s, 0.4H), 8.26 (m, 1H), 7.88 (m, 1H), 7.53-7.27 (m, 10H), 6.82 (s, 1H), 5.38 (s, 1.2H), 5.30 (s, 0.8H), 4.56 (s, 0.8H), 4.27 (s, 1.2H), 4.12-4.06 (m, 2.4H), 3.91-3.85 (br m, 1.6H).
[475] Example 66
[476] 4 -{[2-N- (benzhydryloxycarbonyl) -guanine-9-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[ Preparation of 2-N- (Benzhydryloxycarbonyl) -guanin-9-yl] acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[477] [2-N- (benzhydryloxycarbonyl) guan-9-9-yl] -acetic acid (1.89 g, 4.5 mmol), 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride ( 1.45 g, 4.5 mmol) and PyBOP (2.81 g, 5.4 mmol) were suspended in dimethylformamide (13 ml) and diisopropylethylamine (1.96 ml, 113 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for about 1 hour and then a 20% aqueous citric acid solution was added to the reaction solution to form a pH of 3-4 to form a solid. The solid was separated by filtration, dissolved in acetonitrile solvent and washed with dilute sodium hydrogen carbonate solution and saturated brine. The organic layer was left at room temperature to give a solid, filtered, washed with ethanol and dried to give 1.15 g (37%) of the title compound.
[478] ¹ H NMR (DMSO-d ₆ )
[479] δ 11.63 (s, 1H), 11.24 (s, 1H), 8.37 (m, 1H), 8.13 (dd, 1H), 8.06 to 7.97 (m, 2H), 7.82 (s, 0.6H), 7.78 (s , 0.4H), 7.46-7.28 (m, 10H), 6.86 (s, 1H), 5.15 (s, 1.2H), 5.07 (s, 0.8H), 4.52 (s, 0.8H), 4.27 (s, 1.2 H), 4.12-4.02 (m, 2.4H), 3.94-3.83 (m, 1.6H).
[480] Example 67
[481] 4-{[2-N- (benzhydryloxycarbonyl) -guanine-9-yl] -acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one ( Preparation of 4-{[2-N- (Benzhydryloxycarbonyl) -guanin-1-yl] acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[482] [2-N- (benzhydryloxycarbonyl) -guan-9-9-yl] -acetic acid (1.26 g, 3.0 mmol), 1- (4-chloro-2-nitro-benzenesulfonyl) -piperazine-2 -Warm hydrochloride (1.07 g, 3.0 mmol) and PyBOP (1.87 g, 3.6 mmol) were suspended in dimethylformamide (10 ml), cooled to 15 ° C. or lower, and then diisopropylethylamine (1.05 ml, 6.0 mmol). Was added and the temperature was slowly raised to room temperature and the reaction mixture was stirred for about 1 hour. After the reaction was completed, the residue obtained by distillation of the reaction solvent under reduced pressure was dissolved in ethyl acetate solvent, washed with saturated aqueous sodium hydrogen carbonate solution, water, 20% aqueous citric acid solution and saturated brine and distilled under reduced pressure to obtain a residue. The residue was purified by column chromatography to give 1.25 g (58%) of the title compound.
[483] ¹ H NMR (DMSO-d ₆ )
[484] δ 11.63 (br. s, 1H), 11.25 (br. s, 1H), 8.43 to 8.32 (m, 2H), 8.09 (m, 1H), 7.81 (s, 0.6H), 7.76 (s, 0.4H ), 7.46-7.28 (m, 10H), 6.86 (s, 1H), 5.14 (s, 1.2H), 5.06 (s, 0.8H), 4.52 (s, 0.8H), 4.27 (s, 1.2H), 4.10 to 4.00 (m, 2.4H), 3.92 to 3.82 (m, 1.6H)
[485] Example 68
[486] 4-{[4-N- (benzhydryloxycarbonyl) -cytocin-1-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[ Preparation of 4-N- (benzhydryloxycarbonyl) -cytosin-1-yl] acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[487] [4-N- (benzhydryloxycarbonyl) -cytocin-1-yl] -acetic acid (1.90 g, 5.0 mmol), 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1.61 g, 5.0 mmol) and PyBOP (2.86 g, 5.5 mmol) were suspended in dimethylformamide (14 ml) and diisopropylethylamine (1.31 ml) was added at room temperature. The reaction mixture was stirred at room temperature for about 1 hour, and then the solvent was distilled off under reduced pressure to obtain a residue, which was dissolved in ethyl acetate. The solution was washed sequentially with 20% aqueous citric acid solution, saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and distilled under reduced pressure to obtain a residue. The residue was purified by column chromatography to give 2.53 g (78%) of the title compound.
[488] NMR data is the same as in Example 54
[489] Example 69
[490] 4-{[4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one (4 Preparation of-{[4-N- (benzhydryloxycarbonyl) -cytosin-1-yl] acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[491] [4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -acetic acid (1.14 g, 3.0 mmol), 1- (4-chloro-2-nitro-benzenesulfonyl) -piperazine-2- The warm hydrochloride salt (1.07 g, 3.0 mmol) and PyBOP (1.87 g, 3.6 mmol) were suspended in dimethylformamide (10 ml) and cooled to 15 ° C. or below. Diisopropylethylamine (1.05 ml, 6.0 mmol) was added and the temperature was slowly raised to room temperature and the reaction mixture was stirred for 30 minutes. The residue obtained by distillation of the reaction solvent under reduced pressure was dissolved in ethyl acetate, washed sequentially with aqueous sodium bicarbonate solution, saturated brine, 20% citric acid solution and saturated brine, dried over anhydrous sodium sulfate, filtered and distilled under reduced pressure to obtain a residue. The residue was purified by column chromatography to give 0.56 g (27%) of the title compound.
[492] ¹ H NMR (DMSO-d ₆ )
[493] δ 10.98 (s, 1H), 8.40 (s, 1H), 8.32 (m, 1H), 8.07 (m, 1H), 7.89 (d, 0.6H), 7.85 (d, 0.4H), 7.46-7.28 ( m, 1H), 6.96 (m, 1H), 6.79 (s, 1H), 4.81 (s, 1.2H), 4.73 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H) , 4.07 to 3.97 (m, 2.4H), 3.90 to 3.80 (m, 1.6H).
[494] Example 70
[495] 4-{[4-N- (benzhydryloxycarbonyl) -cytocin-1-yl] -acetyl} -1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one Preparation of (4-{[4-N- (Benzhydryloxycarbonyl) -cytosin-1-yl] acetyl} -1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[496] [4-N- (benzhydryloxycarbonyl) cytocin-1-yl] -acetic acid (1.14 g, 3.0 mmol), 1- (4-fluoro-2-nitro-benzenesulfonyl) -piperazine-2 The-warm hydrochloride salt (1.02 g, 3.0 mmol) and PyBOP (1.87 g, 3.6 mmol) were suspended in dimethylformamide (10 ml) and cooled to 15 ° C. or below. Diisopropylethylamine (1.05 ml, 6.0 mmol) was added and the temperature was slowly raised to room temperature and the reaction mixture was stirred for 45 minutes. The residue obtained by distillation of the reaction solvent under reduced pressure was dissolved in tetrahydrofuran (15 ml) and slowly added to 10% aqueous citric acid solution (70 ml). The resulting solid was dissolved in ethyl acetate and dried over anhydrous sodium sulfate, Filtration and distillation under reduced pressure gave a residue, which was then purified by column chromatography to obtain 1.19 g (60%) of the title compound.
[497] ¹ H NMR (DMSO-d ₆ )
[498] δ 10.98 (s, 1H), 8.41 (m, 1H), 8.24 (dd, 1H), 7.87 (m, 2H), 7.50 to 7.25 (m, 10H), 6.96 (m, 1H), 6.79 (s, 1H), 4.81 (s, 1.2H), 4.73 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.06-3.95 (m, 2.4H), 3.90-3.80 (m , 1.6H)
[499] Example 71
[500] 4-{[6-N- (benzyloxycarbonyl) -adenin-9-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[6- Preparation of N- (Benzyloxycarbonyl) -adenin-9-yl] acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[501] [6-N- (benzyloxycarbonyl) adenine-9-yl] -acetic acid (655 mg, 2.0 mmol), 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (644 mg , 2.0 mmol) and PyBOP (1.25 g, 2.4 mmol) were suspended in dimethylformamide (10 ml) and cooled to 15 ° C or below. Diisopropylethylamine (0.7 ml, 4.0 mmol) was added and the temperature was slowly raised to room temperature and the reaction mixture was stirred for 45 minutes. The residue obtained by distillation of the reaction solvent under reduced pressure was dissolved in tetrahydrofuran (10 ml) and then slowly added to 50 ml of 10% aqueous citric acid solution. The resulting solid was dissolved in ethyl acetate, dried over anhydrous sodium sulfate, filtered and depressurized. Distillation gave a residue, which was then purified by column chromatography to give 731 mg (61%) of the title compound.
[502] ¹ H NMR (DMSO-d ₆ )
[503] δ 10.69 (s, 1H), 9.00 (m, 1H), 8.71 (m, 1H), 8.63-8.58 (m, 2H), 8.31 (s, 0.6H), 8.29 (s, 0.4H), 7.47- 7.33 (m, 5H), 5.39 (s, 1.2H), 5.29 (s, 0.8H), 5.21 (s, 2H), 4.57 (s, 0.8H), 4.29 (s, 1.2H), 4.15 to 4.09 ( m, 2.4H), 3.93-3.86 (m, 1.6H).
[504] Example 72
[505] 4-{[2-N- (benzyloxycarbonyl) -guanin-9-yl] -acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one (4-{[2- Preparation of N- (Benzyloxycarbonyl) -guanin-9-yl] acetyl} -1- (2-nitro-benzenesulfonyl) -piperazin-2-one)
[506] [2-N- (benzyloxycarbonyl) guanine-9-yl] -acetic acid (1.48 g, 4.5 mmol), 1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (1.45 g , 4.5 mmol) and PyBOP (2.81 g, 5.4 mmol) were suspended in dimethylformamide (13 ml) and diisopropylethylamine (1.96 ml, 113 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for about 40 minutes and then the reaction solution was slowly added to water (80 ml) to give a solid. The resulting solid was filtered, washed with ethanol, suspended in acetonitrile and tetrahydrofuran mixed solution, stirred and filtered again. The filtered solid was washed with ethanol and dried to give 1.56 g (58%) of the title compound.
[507] 1 H NMR (DMSO-d ₆ )
[508] δ 11.47 (s, 1H), 11.38 (s, 1H), 8.40 to 7.96 (m, 4H), 7.82 (s, 0.6H), 7.78 (s, 0.4H), 7.45 to 7.35 (m, 5H), 5.25 (s, 2H), 5.13 (s, 1.2H), 5.04 (s, 0.8H), 4.51 (s, 0.8H), 4.26 (s, 1.2H), 4.10 ~ 4.02 (m, 2.4H), 3.93 ~ 3.83 (m, 1.6H).
[509] Example 73
[510] 4-{[2-N- (benzyloxycarbonyl) -guanine-9-yl] -acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one (4- Preparation of {[2-N- (Benzyloxycarbonyl) -guanin-9-yl] acetyl} -1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one)
[511] [2-N- (benzyloxycarbonyl) guanine-9-yl] -acetic acid (1.15 g, 3.5 mmol), 1- (4-chloro-2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloric acid Salt (1.25 g, 3.5 mmol) and PyBOP (2.00 g, 3.85 mmol) were suspended in dimethylformamide (11 ml) and cooled to below 10 ° C. Diisopropylethylamine (0.91 ml, 5.25 mmol) was added and the temperature was slowly raised to room temperature and the reaction mixture was stirred for about 4 hours. After the reaction was completed, the residue obtained by distillation of the reaction solvent under reduced pressure was dissolved in an ethyl acetate solvent, washed successively with diluted aqueous sodium bicarbonate solution, water, 20% citric acid solution and saturated brine, dried over anhydrous sodium sulfate and filtered. After distillation under reduced pressure, a residue was obtained. The residue was crystallized in acetonitrile to give 1.32 g (60%) of the title compound.
[512] ¹ H NMR (DMSO-d ₆ )
[513] δ 11.46 (s, 1H), 11.37 (s, 1H), 8.44 ~ 8.31 (m, 2H), 8.09 (m, 1H), 7.82 (s, 0.6H), 7.78 (s, 0.4H), 7.46 ~ 7.33 (m, 5H), 5.25 (s, 2H), 5.12 (s, 1.2H), 5.04 (s, 0.8H), 4.51 (s, 0.8H), 4.26 (s, 1.2H), 4.10-4.00 ( m, 2.4H), 3.92-3.82 (m, 1.6H).
[514] Example 74
[515] 3-methyl-1- (2-nitro-benzenesulfonyl) -4-{(thimin-1-yl) -acetyl} -piperazin-2-one (3-Methyl-1- (2-nitro-benzensulfonyl) Preparation of -4-[(thymin-1-yl) acetyl] -piperazin-2-one)
[516] (Thimin-1-yl) -acetic acid (55 mg, 0.30 mmol), 3-methyl-1- (2-nitro-benzenesulfonyl) -piperazin-2-one hydrochloride (100 mg, 0.30 mmol) and PyBOP (156 mg, 0.45 mmol) was dissolved in dimethylformamide (1 ml) and diisopropylethylamine (0.08 ml) was added at room temperature. The reaction mixture was stirred at 30 to 40 ° C. for 2 hours, and then the solvent was distilled off under reduced pressure to obtain a residue. The residue was dissolved in dichloromethane and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and the residue obtained by distillation under reduced pressure was crystallized in dichloromethane to obtain 100 mg (72%) of the title compound.
[517] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[518] δ 11.31 (s, 1H), 8.33 (d, 1H), 8.10 (d, 1H), 8.04-7.73 (m, 2H), 7.35 (s, 1H), 4.85-4.60 (m, 2.5H), 4.55 -4.40 (m, 0.5H), 4.20-4.10 (m, 0.5H), 4.05-3.90 (m, 1.5H), 3.78-3.60 (m, 1H), 3.30 (m, 1H), 1.74 (s, 3H ), 1.42 (d, 1H), 1.27 (d, 2H)
[519] Example 75
[520] 1- (2-Nitro-benzenesulfonyl) -3-phenyl-4-[(thimin-1-yl) -acetyl] -piperazin-2-one (1- (2-Nitro-benzensulfonyl) -3-phenyl Preparation of -4-[(thymin-1-yl) acetyl] -piperazin-2-one)
[521] (Thimin-1-yl) -acetic acid (13 mg, 0.071 mmol), 1- (2-nitro-benzenesulfonyl) -3-phenyl-piperazin-2-one hydrochloride (28 mg, 0.070 mmol) and PyBOP (40 mg, 0.077 mmol) was dissolved in dimethylformamide (0.3 ml) and diisopropylethylamine (0.018 ml) was added at room temperature. The reaction mixture was stirred at 30-40 ° C. for about 3 hours, then slowly cooled to room temperature and stirred for about 1 day. The solvent was distilled off under reduced pressure to obtain a residue, which was then purified by column chromatography to obtain 16 mg (43%) of the title compound.
[522] ¹ H-NMR (500 MHz; DMSO-d ₆ )
[523] δ 11.33 (s, 1H), 8.36 (d, 1H), 8.13 (d, 1H), 8.05 to 7.94 (m, 3H), 7.39 to 7.29 (m, 5H), 5.93 (s, 1H), 4.87 ( d, 1H), 4.67 (d, 1H), 4.22-4.16 (m, 2H), 3.96 (m, 1H), 3.78 (m, 1H), 1.74 (s, 3H).
[524] Example of oligomer production method
[525] 1) NsTaegNH ₂ ({[2- (2-nitro-benzenesulfonylamino) -ethyl]-[(thimin-1-yl) -acetyl] -amino} -acetamide)
[526] The PAL resin (53 mg, 0.5 mmol / g) was removed for 20 minutes with a dimethylformamide solution (1.5 ml) of 20% piperidine. The solution was filtered off and washed alternately with dimethylformamide (2 ml) and dichloromethane (2 ml). After washing twice more in the same way, it was dried under nitrogen atmosphere. T monomer (1- (2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one) (6 mg) and dimethylformamide (500 ml) were added Then dimethylformamide solution (12 ml) of 1.0 M tetrabutylammonium fluoride was added. After reacting for 20 minutes at 30 ° C., the solution was filtered off and washed three times using dimethylformamide and dichloromethane in the above-described method. Drying under a nitrogen atmosphere for 5 minutes, acetic acid reaction with dimethylformamide solution (1.5 ml) of 5% acetic anhydride and 6% diisopropylethylamine for 20 minutes, and then using dimethylformamide and dichloromethane Washed three times. The mixture was dried for 5 minutes under a nitrogen atmosphere, reacted with a dimethylformamide solution of 10% n-propylamine for 10 minutes, and then the solution was filtered off. After washing three times with dimethylformamide and dichloromethane by the above method, it was dried for 5 minutes under a nitrogen atmosphere. A resin (2 mg) was taken and the mixture of trifluoroacetic acid and m-cresol (4/1) was treated for 90 minutes. The solvent was removed by blowing with nitrogen, washed twice with ethyl ether (1 ml), dissolved in methanol (300 ml), filtered and analyzed by HPLC. NsTaegNH ₂ : purity 95.7% (area%), retention time 8.76 minutes: HPLC conditions; Column: C18, developing solvent: water (0.1% TFA), acetonitrile (0.1% TFA), developing conditions; Gradient (0 min-water (0.1% TFA) / acetonitrile (0.1% TFA) = 90/10, 20 min-water (0.1% TFA) / acetonitrile (0.1% TFA) = 0/100), = 270 nm
[527] 2) Ns (Taeg) ₂ NH ₂
[528] The resin of 1) was reacted with a dimethylformamide solution (1.5 ml) of 0.5 M 4-methoxythiophenol and 0.5 M diisopropylethylamine for 1 hour to remove 2-nitrobenzenesulfon group, and the method of 1) was repeated. Washed once. After drying for 5 minutes under nitrogen atmosphere, T monomer (1- (2-nitro-benzenesulfonyl) -4-[(thimin-1-yl) -acetyl] -piperazin-2-one) (30 mg) and dimethylform Amide (500 ml) was added. A dimethylformamide solution (60 ml) of 1.0 M tetrabutylammonium fluoride was added and reacted at 30 ° C. for 20 minutes, and the solution was filtered off. After washing three times by the method of 1) and drying for 5 minutes under a nitrogen atmosphere, the mixture was treated by HPLC with trifluoroacetic acid and m-cresol (4/1) as in 1).
[529] NsTaegNH ₂ : purity 92.9% (area%), retention time 8.56 minutes: HPLC conditions; Column: C18, developing solvent: water (0.1% TFA), acetonitrile (0.1% TFA), developing conditions; Gradient (0 min-water (0.1% TFA) / acetonitrile (0.1% TFA) = 90/10, 20 min-water (0.1% TFA) / acetonitrile (0.1% TFA) = 0/100)
[530] The compounds of formula (7) according to the invention can be used as novel monomers for PNA oligomers to produce PNA oligomers effectively and economically.

权利要求:
Claims (18)
[1" claim-type="Currently amended] Compound of Formula 1:

In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[2" claim-type="Currently amended] The compound of claim 1, wherein R 5 is the side chain of naturally occurring amino acids.
[3" claim-type="Currently amended] The compound of claim 1, wherein B is protected or unprotected thymine (T), cytosine (C), guanine (G), adenine (A), or uracil (U).
[4" claim-type="Currently amended] The compound of claim 1, wherein R 1, R 2, R 3 and R 4 are each independently hydrogen.
[5" claim-type="Currently amended] The compound of claim 1, wherein R 2 is fluorine, chlorine, trifluoromethyl or methyl, and R 1, R 3 and R 4 are each independently hydrogen.
[6" claim-type="Currently amended] The compound of claim 1, wherein R 2 is chlorine, R 4 is chlorine or methyl, and R 1 and R 3 are each independently hydrogen.
[7" claim-type="Currently amended] A process for preparing the compound of formula 1, characterized in that the compound of formula 10 is hydrolyzed and the resulting hydrolyzate is cyclized to give the compound of formula 1.


In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group;
B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[8" claim-type="Currently amended] A process for preparing a compound of formula 1, characterized in that the compound of formula 9 is reacted with a compound of formula 11 to yield a compound of formula



In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
B is a nucleic acid base which is protected or unprotected and which exists naturally or unnaturally;
HY is an inorganic acid or an organic acid.
[9" claim-type="Currently amended] The method of claim 8, wherein the compound of formula 9 is reacted with the compound of formula 11 in the presence of a base.
[10" claim-type="Currently amended] Compound of Formula 10:

In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group;
B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[11" claim-type="Currently amended] A process for preparing a compound of formula 10, characterized in that the compound of formula 6 is reacted with a compound of formula 11 to produce a compound of formula 10:



In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group;
B is a nucleic acid base which is protected or unprotected and which exists either naturally or unnaturally.
[12" claim-type="Currently amended] Compound of Formula 6:

In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group.
[13" claim-type="Currently amended] A process for preparing a compound of formula 6, characterized in that the compound of formula 7 is reacted with a compound of formula 3 to produce a compound of formula 6:



In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group.
[14" claim-type="Currently amended] A compound of formula 6a, characterized in that ethylenediamine is reacted with a compound of formula 3 to produce a compound of formula 4, and the resulting compound of formula 4 is reacted with a compound of formula 5 to produce a compound of formula 6a How to manufacture:




In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R6 is a (C1-C4) alkyl or cyanoethyl group;
X is a halogen atom.
[15" claim-type="Currently amended] Compound of formula 9 or free base thereof:

In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
HY is an inorganic acid or an organic acid.
[16" claim-type="Currently amended] 16. The compound of claim 15, wherein HY is selected from the group consisting of halogen acid, sulfuric acid, nitric acid, alkanesulfonic acid, trifluoroacetic acid and trifluoromethanesulfonic acid.
[17" claim-type="Currently amended] The compound of formula 6 is hydrolyzed and the resulting hydrolyzate is reacted with di-t-butyl dicarbonate (t-Boc) ₂ O to produce the compound of formula 8, and the resulting compound of formula 8 is sequentially A process for preparing a compound of formula 9, characterized in that to undergo a chemical reaction and a deprotection reaction, to produce a compound of formula 9



In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
R6 is a (C1-C4) alkyl or cyanoethyl group;
HY is an inorganic acid or an organic acid.
[18" claim-type="Currently amended] Reaction of the compound having the amine group with the compound of formula (1) to produce a conjugate of formula (12), deprotection of the resulting bond to produce a conjugate of formula (13), and reacting the resulting compound of formula (13) with a compound of formula (1) And deprotecting the resulting conjugate to produce a dimeric conjugate of Formula 14, reacting the resulting dimer conjugate of Formula 14 with a compound of Formula 1, and deprotecting the resulting conjugate. Method for preparing a PNA oligomer, characterized in that performed by:



In the above formula,
R is substituted phenyl Wherein R 1, R 2, R 3 and R 4 are the same or different substituents, independently hydrogen, halogen atom, (C 1 -C 4) alkyl group, nitro group, nitrile group, (C 1 -C 4) alkoxy group, halogenated (C 1- C 4) alkyl group and halogenated (C 1 -C 4) alkoxy group);
R 5 is the side chain of hydrogen or an amino acid present naturally or unnaturally;
B is a nucleic acid base which is protected or unprotected and which exists naturally or unnaturally;
B1 and B2, which may be the same or different, each independently represent a protected or unprotected nucleic acid base that exists naturally or unnaturally;
Indicates a resin.

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DE60328866D1|2009-10-01|

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JP2005538928A|2005-12-22|

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DK1470114T3|2009-11-02|

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WO2003062212A1|2003-07-31|

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US7022851B2|2006-04-04|

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CN1642923A|2005-07-20|

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EP1470114B1|2009-08-19|

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EP1470114A4|2005-05-11|

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JP4394952B2|2010-01-06|

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KR100464261B1|2005-01-03|

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EP1470114A1|2004-10-27|

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US20030195332A1|2003-10-16|

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AT440089T|2009-09-15|

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CN100351243C|2007-11-28|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2002-01-24|Application filed by 주식회사 파나진

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2002-01-24|Priority to KR10-2002-0004194A

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2003-07-31|Publication of KR20030063848A

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2005-01-03|Application granted

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2005-01-03|Publication of KR100464261B1

优先权:

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申请号 | 申请日 | 专利标题

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KR10-2002-0004194A|KR100464261B1|2002-01-24|2002-01-24|A Novel Monomer For Synthesis of PNA Oligomer And A Process For Producing The Same|