PNA monomer and PNA oligomer including same

Abstract

The present invention relates to a novel PNA monomer, a PNA oligomer comprising same, and a use thereof, and more specifically, to a PNA monomer having a novel structure that can be dissolved in neutral and weak basic buffers while maintaining the advantages of PNA, and a PNA oligomer comprising same.

Description

PNA monomer and PNA oligomer comprising the same

[0001] The present invention relates to a novel PNA monomer and a PNA oligomer comprising the same.

[0002] Peptide nucleic acid (PNA) is an artificial nucleic acid with a polyamide backbone structure in which the phosphate-ribose backbone of DNA is replaced with N-(2-aminoethyl)-glycine (Aeg), an amide backbone similar to peptides. It has the property of strongly binding to RNA and DNA with complementary base sequences, and has the characteristic of being stable to enzymes such as nucleases and proteases. PNA has the advantage of not being easily degraded in vivo, and is actively used in the field of genetic diagnosis, and many researchers are continuously researching it for commercialization as a gene therapy.

[0003] In particular, the peptide nucleic acid is resistant to nucleases and has high biological stability, and research on therapeutic agents based on various oligonucleic acids is being conducted.

[0004] Since the PNA backbone is uncharged, unlike DNA and RNA, there is no (-) charge repulsion by phosphate groups like in DNA when forming a double helix. As a result, PNA / DNA binding is reported to be about 100 to 5000 times faster than DNA / DNA binding. This strong binding force increases the thermal stability of the PNA / DNA duplex, resulting in a high melting point (T m) and can be designed as a diagnostic probe using a short sequence. Due to these characteristics, PNA can effectively distinguish single nucleotide polymorphisms (SNPs), and is used as an important raw material in various molecular diagnostic industries, including companion diagnostics.

[0005] Despite these advantages, PNAs exhibit significant limitations in biological applications due to their low solubility in neutral and slightly alkaline buffers and problems such as precipitation. In contrast, phosphate-based synthetic nucleic acids, which are highly soluble in buffer compositions suitable for biological applications such as siRNA, have achieved significant success in various FDA-approved drugs and vaccines.

[0006] Accordingly, the inventors of the present invention sought to overcome the limitations of biological applications of PNA and to develop a new PNA structure that can be dissolved in neutral and weakly alkaline buffers while maintaining the advantages of PNA.

[0007] An object of one embodiment is to provide a novel PNA monomer and a PNA oligomer comprising the same.

[0008] Another object of one embodiment is to provide a chimeric compound comprising the PNA oligomer.

[0009] Another object of one embodiment is to provide a composition for regulating target gene expression comprising the PNA oligomer.

[0010] Another object of one embodiment is to provide a composition for preventing or treating a disease comprising the PNA oligomer.

[0011] Another object of one embodiment is to provide a diagnostic composition comprising the PNA oligomer.

[0012] Another embodiment provides a theranostics composition comprising the PNA oligomer.

[0013] To achieve the above purpose,

[0014] One embodiment provides a water-soluble PNA (peptide nucleic acid) monomer represented by the following chemical formula 1.

[0015] [Chemical Formula 1]

[0016]

[0017] In the above chemical formula 1,

[0018] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0019] L is C1-C10 alkylene;

[0020] R is or and;

[0021] L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with;

[0022] R 1a and R 1b are each independently C1-C10 alkyl;

[0023] a is an integer from 1 to 4;

[0024] L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0025] R 1c and R 1d are each independently C1-C10 alkyl;

[0026] c is an integer from 1 to 4;

[0027] B is a moiety comprising a nucleic acid base;

[0028] L 2 is a single bond or C1-C30 alkylene, and the above L 2 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0029] R 2a and R 2b are each independently C1-C10 alkyl;

[0030] b is an integer from 1 to 3;

[0031] P 1 is a protecting group for hydrogen or amine groups;

[0032] P 2 is a protecting group for hydrogen or hydroxyl groups.

[0033] Another embodiment provides a PNA oligomer comprising a structural unit represented by the following chemical formula 8.

[0034] [Chemical Formula 8]

[0035]

[0036] In the above chemical formula 8,

[0037] L is C1-C10 alkylene;

[0038] R is or and;

[0039] L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with;

[0040] R 1a and R 1b are each independently hydrogen or an alkali metal cation;

[0041] a is an integer from 1 to 4;

[0042] L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0043] R 1c and R 1d are each independently hydrogen or an alkali metal cation;

[0044] c is an integer from 1 to 4;

[0045] B is a moiety comprising a nucleic acid base;

[0046] L 2 is a single bond or C1-C30 alkylene, and the above L 2 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0047] R 2a and R 2b are each independently hydrogen or an alkali metal cation;

[0048] b is an integer from 1 to 3.

[0049] Another embodiment provides a chimeric compound comprising the PNA oligomer.

[0050] Another embodiment provides a composition for regulating target gene expression comprising the PNA oligomer.

[0051] Another embodiment provides a composition for preventing or treating a disease comprising the PNA oligomer.

[0052] Another embodiment provides a diagnostic composition comprising the PNA oligomer.

[0053] Another embodiment provides a theranostics composition comprising the PNA oligomer.

[0054] The present invention relates to novel PNA monomers and PNA oligomers, characterized by a backbone structure in which at least one phosphonate group is linked to the nitrogen atom of a glycine in a (2-aminoethyl)glycine backbone via a linking group. Due to this backbone structure, the PNA monomers possess physicochemical properties similar to DNA while maintaining the advantages of PNA itself, and thus have the potential to effectively replace phosphate-based nucleic acids currently used in various therapeutic and diagnostic fields, including gene therapy.

[0055] In addition, the PNA monomers and PNA oligomers of the present invention have high solubility in neutral and weakly basic buffer compositions, and thus can overcome limitations that have appeared in the biological applications of existing PNAs. Furthermore, the high solubility in neutral and weakly basic buffers can provide a basis for wide application in various life science and pharmaceutical fields such as diagnosis, gene therapy, targeted drug delivery, molecular diagnosis, and in vivo imaging by allowing PNA to bind and interact with various biopolymers such as intracellular environments, antibodies, proteins, and DNA with high yield.

[0056] Furthermore, the PNA monomers and oligomers of the present invention can improve the ease and stability of manufacturing and mass production processes due to the introduction of water-soluble linkers and monomers, thereby increasing industrial production efficiency and enhancing commercialization potential. In particular, when producing PNA chimera structures, the PNA of the present invention can stably bind to other biopolymers in high yields under neutral and slightly basic conditions, thereby significantly increasing the potential for use in the development of combination therapeutics and personalized medicine.

[0057] Figure 1 - HPLC and M / S results of SEQ 1 in Experimental Example 1

[0058] Figure 2 - HPLC and M / S results of SEQ 2 in Experimental Example 1

[0059] Figure 3 - Results of click reaction of PNA without introduction of water-soluble linker in Experimental Example 2

[0060] Figure 4 - Results of click reaction of PNA with water-soluble linker introduced in Experimental Example 2

[0061] Figure 5 - Solubility test results of Experimental Example 3

[0062] Figure 6 - Melting curve graph of Experimental Example 4

[0063] Figure 7 - Confocal analysis results of Experimental Example 6

[0064] Figure 8 - Digital PCR test results of Experimental Example 7

[0065] Hereinafter, the present invention will be described in detail. However, unless otherwise defined, the technical and scientific terms used herein have meanings commonly understood by a person of ordinary skill in the technical field to which this invention pertains, and in the following description, descriptions of well-known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

[0066] Meanwhile, the embodiments of the present invention may be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, the embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the relevant technical field. Furthermore, throughout the specification, the term "including" a certain component does not exclude other components, but rather means that other components may be included, unless specifically stated otherwise.

[0067] As used herein, the singular forms may be intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0068] A numerical range as used herein includes a lower bound and an upper bound and all values ​​within that range, increments logically derived from the shape and width of the defined range, all doubly delimited values, and all possible combinations of upper and lower bounds of numerical ranges delimited in different shapes.

[0069] The term "C" in this specification A -C B " means "having a carbon number of A or more and B or less", and the term "A to B" means "having a carbon number of A or more and B or less".

[0070] The "protecting group" described in the present invention is a functional group for protecting a specific functional group in an organic reaction, for example, a reactive group such as an amine or a hydroxyl group, and any functional group within the scope recognizable by a person skilled in the art of organic synthesis may be used, for example, acetyl (Ac), tosyl (Ts), benzyl (Bn), benzoyl (Bz), benzyloxy (Bn), isobutyryl, 2,2,2-trichloroethoxycarbonyl (Troc), allyloxycarbonyl (Alloc), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tert-butoxycarbonyl (Boc), methyl (Me) or tert-butyl (tBu), cyclohexyl, benzyloxycarbonyl (Cbz), It can be 9-fluorenylmethyloxycarbonyl (Fmoc), benzylhydryloxycarbonyl (Bhoc), monomethoxytrityl (Mmt), dimethoxytrityl (Dmt), nitrobenzenesulfonyl (Nosyl; Ns), 2-nitrophenylsulfenyl (Nps), or benzothiazole-2-sulfonyl (Bts).

[0071] The "nucleotide base" described herein refers to a nucleotide base known to those skilled in the art, including natural and non-natural nucleotides, and is not limited to specific examples. Natural nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Non-natural nucleotides refer to nucleotides that have been chemically or structurally modified based on the natural nucleotides, such as by introducing a substituent, introducing a protecting group, isomerization, ring expansion or contraction, or changing the position of a double bond.

[0072] The above nucleic acid bases include, for example, adenine (A), purine, 2-aminoadenine, N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Ethano-2,6-diaminopurine, 7-deazaadenine, 8-azapurine, guanine (G), O 6 -Protected-guanine, 7-deazaguanine, thymine (T), O 4 -Protected-thymine, uracil (U), dihydrouracil, 5-(C1-C6 alkyl)uracil, 5-(C2-C6 alkenyl)uracil, 5-(C2-C6 alkynyl)uracil, 5-(1-propargylamino)uracil, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 2-thiouracil, 4-thiouracil, cytosine (C), 6-(C1-C6 alkyl)cytosine, N 4 -Protected-cytosine, N 4 ,N 4 - It may be, but is not limited to, ethanocytosine, 6-(C2-C6 alkenyl)cytosine, 6-(C2-C6 alkynyl)cytosine, 5-(1-propargylamino)cytosine, pseudoisocytosine, 6-fluorocytosine, 6-chlorocytosine, 6-bromocytosine or 6-hydroxycytosine.

[0073] One embodiment provides a water-soluble PNA (peptide nucleic acid) monomer represented by the following chemical formula 1.

[0074] [Chemical Formula 1]

[0075]

[0076] In the above chemical formula 1,

[0077] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0078] L is C1-C10 alkylene;

[0079] R is or and;

[0080] L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with;

[0081] R 1a and R 1b are each independently C1-C10 alkyl;

[0082] a is an integer from 1 to 4;

[0083] L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0084] R 1c and R 1d are each independently C1-C10 alkyl;

[0085] c is an integer from 1 to 4;

[0086] B is a moiety comprising a nucleic acid base;

[0087] L 2 is a single bond or C1-C30 alkylene, and the above L 2The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0088] R 2a and R 2b are each independently C1-C10 alkyl;

[0089] b is an integer from 1 to 3;

[0090] P 1 is a protecting group for hydrogen or amine groups;

[0091] P 2 is a protecting group for hydrogen or hydroxyl groups.

[0092] In one embodiment, the water-soluble PNA monomer may be represented by the following chemical formula 2.

[0093] [Chemical Formula 2]

[0094]

[0095] In the above chemical formula 2, L, R, P 1 and P 2 is the same as the definition in the above chemical formula 1.

[0096] In one embodiment, the water-soluble PNA monomer may be represented by the following chemical formula 3.

[0097] [Chemical Formula 3]

[0098]

[0099] In the above chemical formula 3, L, R and P 1 is the same as the definition in the above chemical formula 1.

[0100]

[0101] In one embodiment, the water-soluble PNA monomer may be represented by the following chemical formula 4.

[0102] [Chemical Formula 4]

[0103]

[0104] In the above chemical formula 4,

[0105] L1 is a single bond, C1-C20 alkylene or And here the wave( ) is a bonding site with carbon (C), and the asterisk ( ) represents a binding site with P, and the L 1 The -CH2- of alkylene can be replaced with -O-;

[0106] L 1b is C1-C20 alkylene, and the above L 1b The -CH2- of alkylene can be replaced with -O-, and the above L 1b The alkylene of can be further substituted with;

[0107] R 1a and R 1b are each independently C1-C5 alkyl;

[0108] L 1a is C1-C20 alkylene, and the above L 1a The -CH2- of alkylene can be replaced with -O-;

[0109] R 1c and R 1d are each independently C1-C5 alkyl;

[0110] c is an integer of 1 or 2;

[0111] P 1 is a protecting group for hydrogen or amine groups;

[0112] P 2 is a protecting group for hydrogen or hydroxyl groups.

[0113] In one embodiment, the water-soluble PNA monomer may be represented by the following chemical formula 5.

[0114] [Chemical Formula 5]

[0115]

[0116] In the above chemical formula 5,

[0117] B is a moiety comprising a nucleic acid base;

[0118] L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-;

[0119] R 2a and R 2b are each independently C1-C5 alkyl;

[0120] b is an integer of 1 or 2;

[0121] q is an integer of 0 or 1;

[0122] P 1 is a protecting group for hydrogen or amine groups;

[0123] P 2 is a protecting group for hydrogen or hydroxyl groups.

[0124] In one embodiment, the water-soluble PNA monomer may be represented by the following chemical formula 6.

[0125] [Chemical Formula 6]

[0126]

[0127] In the above chemical formula 6,

[0128] B is a moiety comprising a nucleic acid base;

[0129] L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-;

[0130] R 2a and R 2b are each independently C1-Calkyl;

[0131] b is an integer of 1 or 2;

[0132] q is an integer of 0 or 1;

[0133] P 1 is a protecting group for the amine group.

[0134] In the water-soluble PNA monomers of the above chemical formulae 5 and 6 according to one embodiment, L 2a and L 2b are each independently C1-C20 alkylene or -(CH2CH2O) g - and g can be an integer from 1 to 6.

[0135] In one embodiment, the protecting group of the amine group and the protecting group of the hydroxy group are each independently selected from the group consisting of acetyl (Ac), tosyl (Ts), benzyl (Bn), benzoyl (Bz), benzyloxy (Bn), isobutyryl, 2,2,2-trichloroethoxycarbonyl (Troc), allyloxycarbonyl (Alloc), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tert-butoxycarbonyl (Boc), methyl (Me) or tert-butyl (tBu), alkyl, cyclohexyl, benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzylhydryloxycarbonyl (Bhoc), monomethoxytrityl (Mmt), dimethoxytrityl (Dmt), It can be nitrobenzenesulfonyl (Nosyl; Ns), 2-nitrophenylsulfenyl (2-nitrophenylsulfenyl; Nps) or benzothiazole-2-sulfonyl (Bts). Specifically, the protecting group of the amine group can be tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzylhydryloxycarbonyl (Bhoc) or benzothiazole-2-sulfonyl (Bts).

[0136] In one specific example, the P 1 is a protecting group of the amine group, R' is hydrogen, and R'' can be -OH.

[0137] In one specific example, the P 1 is a protecting group of the amine group, and R' and R'' can be connected to each other to form a ring.

[0138] In one specific example, the P 1It can be 9-fluorenylmethyloxycarbonyl (Fmoc) or benzothiazole-2-sulfonyl (Bts).

[0139] In one embodiment, the nucleic acid base may be a natural nucleic acid base including adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U) and an unnatural nucleic acid base. Specifically, the nucleic acid base may be adenine (A), purine, 2-aminoadenine, N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Ethano-2,6-diaminopurine, 7-deazaadenine, 8-azapurine, guanine (G), O 6 -Protected-guanine, 7-deazaguanine, thymine (T), O 4 -Protected-thymine, uracil (U), dihydrouracil, 5-(C1-C6 alkyl)uracil, 5-(C2-C6 alkenyl)uracil, 5-(C2-C6 alkynyl)uracil, 5-(1-propargylamino)uracil, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 2-thiouracil, 4-thiouracil, cytosine (C), 6-(C1-C6 alkyl)cytosine, N 4 -Protected-cytosine, N 4 ,N 4 - It may be, but is not limited to, ethanocytosine, 6-(C2-C6 alkenyl)cytosine, 6-(C2-C6 alkynyl)cytosine, 5-(1-propargylamino)cytosine, pseudoisocytosine, 6-fluorocytosine, 6-chlorocytosine, 6-bromocytosine or 6-hydroxycytosine.

[0140] In one specific example, the nucleic acid base is adenine (A), 2-aminoadenine, N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Protected-2-aminoadenine, guanine (G), O 6 -Protected-guanine, thymine (T), O 4-Protected-thymine, uracil (U), cytosine (C), or N 4 -may be a protected cytosine. Here, the N protecting group and the O protecting group are independently selected from the group consisting of C1-C6 alkyl such as acetyl (Ac), tosyl (Ts), benzyl (Bn), benzoyl (Bz), benzyloxy (Bn), isobutyryl, 2,2,2-trichloroethoxycarbonyl (Troc), allyloxycarbonyl (Alloc), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tert-butoxycarbonyl (Boc), methyl (Me) or tert-butyl (tBu), cyclohexyl, benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzylhydryloxycarbonyl (Bhoc), monomethoxytrityl (Mmt), dimethoxytrityl (Dmt), nitrobenzenesulfonyl (Nosyl; Ns), 2-nitrophenylsulfenyl (Nps), or benzothiazole-2-sulfonyl (Bts), but is not limited thereto. Specifically, the N protecting group may be benzyloxycarbonyl (Cbz) or tert-butoxycarbonyl (Boc), and the O protecting group may be benzyl (Bn) or C1-C6 alkyl.

[0141] In one specific example, the water-soluble PNA monomer of the above chemical formula 4 can be represented by the following chemical formula 4-1.

[0142] [Chemical Formula 4-1]

[0143]

[0144] In the above chemical formula 4-1,

[0145] L 1c is a single bond, C1-C20 alkylene or -(CH2CH2O) d - and the above L 1c The alkylene of can be further substituted with;

[0146] R 1a , R 1b , R 1c and R 1dare each independently C1-C5 alkyl;

[0147] L 1d is C1-C20 alkylene or -(CH2CH2O) e -and;

[0148] m is an integer of 0 or 1;

[0149] d and e are each independently integers from 1 to 6.

[0150] In one specific example, the water-soluble PNA monomer of Chemical Formula 4-1 may be represented by Chemical Formula 4-2 or Chemical Formula 4-3 below.

[0151] [Chemical Formula 4-2]

[0152]

[0153] [Chemical Formula 4-3]

[0154]

[0155] In the above chemical formulas 4-2 and 4-3,

[0156] L 1c is a single bond, C1-C10 alkylene or -(CH2CH2O) d -and;

[0157] L 1e is C1-C10 alkylene or -(CH2CH2O) f -and;

[0158] R 1a , R 1b , R 1e and R 1f are each independently C1-C5 alkyl;

[0159] d and f are each independently an integer from 1 to 5;

[0160] p is an integer from 1 to 3.

[0161] In one specific example, the water-soluble PNA monomer of the above chemical formula 5 can be represented by the following chemical formula 5-1.

[0162] [Chemical Formula 5-1]

[0163]

[0164] In the above chemical formula 5-1,

[0165] B is a moiety comprising a nucleic acid base;

[0166] R 2a and R 2b are each independently C1-C5 alkyl;

[0167] b is an integer of 1 or 2;

[0168] q is an integer of 0 or 1;

[0169] x and y are each independently integers from 1 to 10.

[0170] In one specific example, the water-soluble PNA monomer of the above chemical formula 6 can be represented by the following chemical formula 6-1.

[0171] [Chemical Formula 6-1]

[0172]

[0173] In the above chemical formula 6-1,

[0174] B is a moiety comprising a nucleic acid base;

[0175] R 2a and R 2b are each independently C1-C5 alkyl;

[0176] b is an integer of 1 or 2;

[0177] q is an integer of 0 or 1;

[0178] x and y are each independently integers from 1 to 10.

[0179] In one specific example, the water-soluble PNA monomer may be represented by Chemical Formula 7-1 or Chemical Formula 7-2.

[0180] [Chemical Formula 7-1]

[0181]

[0182] [Chemical Formula 7-2]

[0183]

[0184] In the above chemical formulas 7-1 and 7-2,

[0185] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0186] P 1 is a protecting group for hydrogen or amine group; P 2 is a protecting group for hydrogen or hydroxyl group;

[0187] P 3 is a protecting group for hydrogen or an amine group;

[0188] R 11 is a protecting group for hydrogen, amine group or and;

[0189] R 12 is a protecting group for hydrogen, amine group or and;

[0190] R 2a and R 2b are each independently C1-C5 alkyl;

[0191] x and y are each independently integers from 1 to 5.

[0192] In one specific example, the water-soluble PNA monomer may be represented by Chemical Formula 7-3 or Chemical Formula 7-4.

[0193] [Chemical Formula 7-3]

[0194]

[0195] [Chemical Formula 7-4]

[0196]

[0197] In the above chemical formulas 7-3 and 7-4,

[0198] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0199] P1 is a protecting group for hydrogen or amine group; P 2 is a protecting group for hydrogen or hydroxyl group;

[0200] L G Is or and;

[0201] P 4 is C1-C6 alkyl or benzyl;

[0202] R 2a and R 2b are each independently C1-C5 alkyl;

[0203] x and y are each independently integers from 1 to 5.

[0204] In one specific example, the water-soluble PNA monomer may be represented by Chemical Formula 7-5 or Chemical Formula 7-6.

[0205] [Chemical Formula 7-5]

[0206]

[0207] [Chemical Formula 7-6]

[0208]

[0209] In the above chemical formulas 7-5 and 7-6,

[0210] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0211] P 1 is a protecting group for hydrogen or amine group; P 2 is a protecting group for hydrogen or hydroxyl group;

[0212] L T Is or and;

[0213] P 5 is C1-C6 alkyl or benzyl;

[0214] R 2a and R 2bare each independently C1-C5 alkyl;

[0215] x and y are each independently integers from 1 to 5.

[0216] In one specific example, the water-soluble PNA monomer may be represented by Chemical Formula 7-7 or Chemical Formula 7-8.

[0217] [Chemical Formula 7-7]

[0218]

[0219] [Chemical Formula 7-8]

[0220]

[0221] In the above chemical formulas 7-7 and 7-8,

[0222] R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring;

[0223] P 1 is a protecting group for hydrogen or amine group; P 2 is a protecting group for hydrogen or hydroxyl group;

[0224] P 6 is a protecting group for hydrogen or an amine group;

[0225] R 2a and R 2b are each independently C1-C5 alkyl;

[0226] x and y are each independently integers from 1 to 5.

[0227] Above Moiety is or It could be.

[0228] The above P 3 , R 11 and P 6 The protecting groups of the amine group may each independently be benzyloxycarbonyl (Cbz) or tert-butoxycarbonyl (Boc).

[0229] Another embodiment provides a PNA oligomer comprising a structural unit represented by the following chemical formula 8.

[0230] [Chemical Formula 8]

[0231]

[0232] In the above chemical formula 8,

[0233] L is C1-C10 alkylene;

[0234] R is or and;

[0235] L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with;

[0236] R 1a and R 1b are each independently hydrogen or an alkali metal cation;

[0237] a is an integer from 1 to 4;

[0238] L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0239] R 1c and R 1d are each independently hydrogen or an alkali metal cation;

[0240] c is an integer from 1 to 4;

[0241] B is a moiety comprising a nucleic acid base;

[0242] L 2 is a single bond or C1-C30 alkylene, and the above L 2The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof;

[0243] R 2a and R 2b are each independently hydrogen or an alkali metal cation;

[0244] b is an integer from 1 to 3.

[0245] According to one embodiment, the PNA oligomer can be prepared by applying various combinatorial synthetic methods previously reported. Specifically, the PNA oligomer can be prepared through solid phase synthesis or solution phase synthesis, and repeated deprotection / coupling / capping processes can be included to achieve a desired PNA oligomer configuration, but are not limited thereto.

[0246]

[0247] In one embodiment, the PNA oligomer may include at least one of a structural unit represented by Chemical Formula 9 and a structural unit represented by Chemical Formula 10.

[0248] [Chemical Formula 9]

[0249]

[0250] [Chemical Formula 10]

[0251]

[0252] In the above chemical formulas 9 and 10,

[0253] L 1 is a single bond, C1-C20 alkylene or And here the wave( ) is a bonding site with carbon (C), and the asterisk ( ) represents a binding site with P, and the L 1 The -CH2- of alkylene can be replaced with -O-;

[0254] L 1bis C1-C20 alkylene, and the above L 1b The -CH2- of alkylene can be replaced with -O-, and the above L 1b The alkylene of can be further substituted with;

[0255] R 1a and R 1b are each independently hydrogen or an alkali metal cation;

[0256] L 1a is C1-C20 alkylene, and the above L 1a The -CH2- of alkylene can be replaced with -O-;

[0257] R 1c and R 1d are each independently hydrogen or an alkali metal cation;

[0258] B is a moiety comprising a nucleic acid base;

[0259] L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-;

[0260] R 2a and R 2b are each independently hydrogen or an alkali metal cation;

[0261] b is an integer of 1 or 2;

[0262] q is an integer of 0 or 1;

[0263] c is an integer of 1 or 2.

[0264] In the structural unit of the chemical formula 10 according to one embodiment, the L 2a and L 2b are each independently C1-C20 alkylene or -(CH2CH2O) g - and g can be an integer from 1 to 6.

[0265]

[0266] In one embodiment, the PNA oligomer may include at least one of a structural unit represented by Chemical Formula 11 and a structural unit represented by Chemical Formula 12.

[0267] [Chemical Formula 11]

[0268]

[0269] [Chemical Formula 12]

[0270]

[0271] In the above chemical formulas 11 and 12,

[0272] L 1c is a single bond, C1-C20 alkylene or -(CH2CH2O) d - and the above L 1c The alkylene of can be further substituted with;

[0273] R 1a , R 1b , R 1c and R 1d are each independently hydrogen or an alkali metal cation;

[0274] L 1d is C1-C20 alkylene or -(CH2CH2O) e -and;

[0275] B is a moiety comprising a nucleic acid base;

[0276] R 2a and R 2b are each independently hydrogen or an alkali metal cation;

[0277] b is an integer of 1 or 2;

[0278] q is an integer of 0 or 1;

[0279] m is an integer of 0 or 1;

[0280] d and e are each independently an integer from 1 to 6;

[0281] x and y are each independently integers from 1 to 10.

[0282] The structural unit of the above chemical formula 11 may be a structural unit represented by the following chemical formula 11-1 or chemical formula 11-2.

[0283] [Chemical Formula 11-1]

[0284]

[0285] [Chemical Formula 11-2]

[0286]

[0287] In the above chemical formulas 11-1 and 11-2,

[0288] L 1c is a single bond, C1-C10 alkylene or -(CH2CH2O) d -and;

[0289] L 1e is C1-C10 alkylene or -(CH2CH2O) f -and;

[0290] R 1a , R 1b , R 1e and R 1f are each independently hydrogen or an alkali metal cation;

[0291] d and f are each independently an integer from 1 to 5;

[0292] p is an integer from 1 to 3.

[0293] In one specific example, the PNA oligomer may include at least one of the structural units represented by Chemical Formulas 12-1 to 12-8 below.

[0294] [Chemical Formula 12-1]

[0295]

[0296] [Chemical Formula 12-2]

[0297]

[0298] [Chemical Formula 12-3]

[0299]

[0300] [Chemical Formula 12-4]

[0301]

[0302] [Chemical Formula 12-5]

[0303]

[0304] [Chemical Formula 12-6]

[0305]

[0306] [Chemical Formula 12-7]

[0307]

[0308] [Chemical Formula 12-8]

[0309]

[0310] In the above chemical formulas 12-1 to 12-8,

[0311] R 11 is hydrogen or and;

[0312] R 12 is hydrogen or and;

[0313] R 2a and R 2b are each independently hydrogen or an alkali metal cation;

[0314] x and y are each independently integers from 1 to 5.

[0315] In one specific example, the alkali metal cation is Na + , Li + or K + It could be.

[0316] A PNA oligomer according to one embodiment of the present invention comprises a structural unit having at least one selected from a phosphonate anion and a phosphonic acid.

[0317] A PNA oligomer comprising a PNA monomer and a structural unit thereof according to one embodiment of the present invention has a backbone structure in which at least one phosphonate group is bonded to a nitrogen atom of glycine in a (2-aminoethyl)glycine skeleton via a linking group, thereby maintaining the advantages of PNA itself while possessing properties similar to DNA, and can effectively replace phosphate-based nucleic acids used in the fields of gene therapy and diagnosis.

[0318] In addition, the PNA monomer and PNA oligomer according to one embodiment of the present invention have high solubility in neutral and weakly basic buffer compositions, and thus can overcome limitations that have appeared in the biological applications of existing PNAs.

[0319] In addition, the PNA oligomer according to one embodiment of the present invention has an advantage in that its solubility in neutral and weakly basic buffer compositions is not dependent on the base sequence, so that it can be used for therapeutic purposes.

[0320] In addition, the PNA oligomer according to one embodiment of the present invention can efficiently bind to polymers such as DNA and antibodies due to increased solubility under neutral and weakly basic buffer compositions, thereby enabling the mass production of various chimeric compounds, and can greatly contribute to the development of new therapeutic agents.

[0321] In addition, the PNA oligomer according to one embodiment of the present invention can obtain more accurate and effective results in diagnostic and therapeutic applications due to increased specificity, thereby greatly helping in the early detection and treatment of diseases and improving treatment outcomes for patients.

[0322] In addition, the PNA oligomer according to one embodiment of the present invention is easy to manufacture as a chimeric compound, exhibits improved nucleic acid binding affinity and high intracellular dissociation, has a structure that is less degraded within the cell and easily penetrates into the nucleus, and thus can have excellent advantages in applications such as spatial genomics analysis, live cell genetic imaging, protein analysis using chimeric compounds, and as a therapeutic material. Accordingly, it can play an important role in the development of real-time observation of intracellular changes, treatment methods targeting specific genes, missile therapy, pretargeting technology, and theranostics technology that enables simultaneous diagnosis and treatment.

[0323] Another embodiment provides a chimeric compound comprising the PNA oligomer.

[0324] Another embodiment provides a composition for regulating target gene expression comprising the PNA oligomer.

[0325] Another embodiment provides a composition for preventing or treating a disease comprising the PNA oligomer.

[0326] Another embodiment provides a composition for diagnosing a disease comprising the PNA oligomer.

[0327] In one embodiment, the disease may be, but is not necessarily limited to, cancer, an inflammatory disease, age-related macular degeneration, a rare disease, a serious disease, a cardiovascular disease, a metabolic disease, or a skin disease.

[0328] Another embodiment provides a composition for omics analysis comprising the PNA oligomer.

[0329] Another embodiment provides a theranostics composition comprising the PNA oligomer.

[0330] According to one embodiment, the PNA oligomer may be useful as a theranostics system that can be used simultaneously for pre-targeting imaging and therapy.

[0331] Hereinafter, specific examples and experimental examples of the present invention will be described. However, the examples and experimental examples described below are merely illustrative of a portion of the present invention, and the present invention is not limited thereto.

[0332] List of abbreviations

[0333] DMF dimethylformamide; EA ethyl acetate; THF tetrahydrofuran; MC methylene chloride; Fmoc 9-fluorenylmethyloxycarbonyl; DIEA N,N-diisopropylethylamine; PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; TFA trifluoroacetic acid; Boc tert-butoxycarbonyl; HOBt N-hydroxybenzotriazole; DCC 1,3-dicyclohexylcarbodiimide; DCU dicyclohexylurea; DMAP 4-dimethylaminopyridine; Cbz benzyloxycarbonyl; EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Bts benzothiazole-2-sulfonyl; Bhoc benzhydryloxycarbonyl; DIAD diisopropyl azodicarboxylate; CDI 1,1'-carbonyldiimidazole

[0334] Example I: Preparation of a water-soluble linker

[0335] [Example 1] Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(di-tert-butoxyphosphoryl)acetyl)glycine (Compound 1)

[0336]

[0337] 1.1. Preparation of ethyl 2-(di-tert-butoxyphosphoryl)acetate (1c)

[0338] Di-tert-butyl phosphite (1a) (5.0 g, 0.02575 mol) was dissolved in DMF (50 mL) and cooled in an ice bath. KOtBu (3.467 g, 0.03089 mol) was added and stirred in an ice bath for 30 minutes, after which ethyl chloroacetate (1b) was added via syringe. The ice bath was removed and stirred at room temperature for 18 hours. After completion of the reaction, excess EA was added and the mixture was washed three times with a saturated aqueous solution of NaHCO3. The EA layer was dried with anhydrous Na2SO4, filtered, and concentrated (7.1 g, 0.025 mol, 98.39% yield). The next reaction was carried out without further purification.

[0339] 1 H NMR (400 MHz, CDCl3) δ 4.19(q, 2H), 2.97(d, 2H), 1.30(t, 3H), 1.20(s, 9H); 13 C NMR (CDCl3) δ 167.8, 86.6, 61.0, 36.4, 29.4, 14.1

[0340] 1.2. Preparation of 2-(di-tert-butoxyphosphoryl)acetic acid (1d)

[0341] Ethyl 2-(di-tert-butoxyphosphoryl)acetate (1c) (7.1 g, 0.025 mol) was placed in a round-bottomed flask and dissolved in THF (75 mL). LiOH (3.189 g, 0.07599 mol) was placed in a 100 mL Erlenmeyer flask and dissolved in distilled water (50 mL). The LiOH solution was placed in the round-bottomed flask and stirred. A small amount of MeOH was added until the turbidity became clear. After the reaction was completed, an ice bath was installed and 1 N HCl was added until the pH became 3. Excess water was added and extracted twice using MC. The MC layer was dried with anhydrous Na2SO4, filtered, and concentrated (5.87 g, 0.023 mol, 91.87% yield). The next reaction was carried out without further purification.

[0342] 1 H NMR (400 MHz, CDCl3) δ 12.22(s, 1H), 2.97(d, 2H), 1.20(s, 9H); 13 C NMR (CDCl3) δ 167.8, 86.6, 36.4, 29.4

[0343] 1.3. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(di-tert-butoxyphosphoryl)acetyl)glycinate (1f)

[0344] 2-(di-tert-butoxyphosphoryl)acetic acid (1d) (5.87 g, 0.0233 mol) and Fmoc-aeg-OEt·HCl (1e) (9.43 g, 0.0233 mol) were placed in a round-bottomed flask and dissolved in DMF (100 mL). DIEA (20.29 mL, 0.1165 mol) was injected using a syringe, and PyBOP (14.544 g, 0.0279 mol) was added. After completion of the reaction, excess EA was added and washed three times with saturated aqueous NaHCO3 solution. The EA layer was dried with anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (MeOH / MC) to obtain the title compound (1f) (9.2 g, 0.0153 mol, 65.54% yield).

[0345] 1 H NMR (400 MHz, CDCl3) δ 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 4.19(q, 2H), 3.46(t, 2H), 3.42(t, 2H), 2.66(d, 2H), 1.21(t, 3H), 1.20(s, 18H); 13 C NMR (CDCl3) δ 169.4, 168.4, 157.8, 143.6, 142.6, 126.7, 126.2, 152.2, 120.5, 86.2, 67.3, 61.0, 50.4, 49.7, 47.0, 43.0, 38.1, 29.4, 14.1

[0346] 1.4. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(di-tert-butoxyphosphoryl)acetyl)glycine (1)

[0347] Ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(di-tert-butoxyphosphoryl)acetyl)glycinate (1f) (9.2 g, 0.0153 mol) was dissolved in isopropanol (212.52 mL). CaCl2 (26.95 g, 0.243 mol) was dissolved in water (91 mL) and then added. 1 N NaOH (45.797 mL, 0.0458 mol) was added. After completion of the reaction, an ice bath was installed and 1 N HCl was added until the pH reached 3. Excess water was added and extracted twice using MC. The MC layer was dried with anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (MeOH / MC) to obtain the title compound (1) (6.5 g, 0.011 mol, 72.34% yield).

[0348] 1 H NMR (400 MHz, CDCl3) δ 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 3.46(t, 2H), 3.42(t, 2H), 2.66(d, 2H), 1.20(s, 18H); 13 C NMR (CDCl3) δ 169.4, 168.4, 157.8, 143.6, 142.6, 126.7, 126.2, 152.2, 120.5, 86.2, 67.3, 50.4, 49.7, 47.0, 43.0, 38.1, 29.4

[0349] [Example 2] Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(3-(di-tert-butoxyphosphoryl)propanoyl)glycinate (Compound 2)

[0350]

[0351] 2.1. Preparation of methyl 3-(di-tert-butoxyphosphoryl)propanoate (2b)

[0352] The title compound (2b) was synthesized in the same manner as 1.1 of Example 1 using methyl 3-bromopropionate (6.67 mL, 0.0566 mol) instead of ethyl chloroacetate (12.5 g, 0.045 mol, 86.61% yield).

[0353] 1 H NMR (400 MHz, CDCl3) δ 4.15(q, 2H), 2.54(q, 2H), 1.99(m, 2H), 1.50(s, 18H). 1.28(t, 3H); 13 C NMR (CDCl3) δ 173.1, 86.9, 51.9, 29.4, 27.6, 22.0

[0354] 2.2. Preparation of 3-(di-tert-butoxyphosphoryl)propanoic acid (2c)

[0355] The title compound (2c) was synthesized (8.4 g, 0.032 mol, 70.74% yield) in the same manner as in 1.2 of Example 1 using methyl 3-(di-tert-butoxyphosphoryl)propanoate (2b) (12.5 g, 0.045 mol).

[0356] 1 H NMR (400 MHz, CDCl3) δ 3.60(s, 3H), 2.54(q, 2H), 1.99(m, 2H), 1.50(s, 18H); 13 C NMR (CDCl3) δ 173.1, 86.9, 29.4, 27.6, 22.0

[0357] 2.3. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(3-(di-tert-butoxyphosphoryl)propanoyl)glycinate (2d)

[0358] The title compound (2d) was synthesized (13.2 g, 0.021 mol, 67.81% yield) in the same manner as in 1.3 of Example 1 using 3-(di-tert-butoxyphosphoryl)propanoic acid (2c) (8.40 g, 0.032 mol).

[0359] 1 H NMR (400 MHz, CDCl3) δ 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 4.19(q, 2H), 3.46(t, 2H), 3.42(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.21(t, 3H), 1.20(s, 18H); 13 C NMR (CDCl3) δ 172.0, 168.4, 157.8, 143.6, 142.6, 126.7, 126.2, 125.2, 120.5, 86.9, 67.3, 61.0, 50.7, 50.0, 47.0, 38.1, 29.4, 28.5, 16.5, 14.1

[0360] 2.4. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(3-(di-tert-butoxyphosphoryl)propanoyl)glycinate (2)

[0361] The title compound (2) was synthesized (10.1 g, 0.017 mol, 79.37% yield) in the same manner as in 1.4 of Example 1 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(3-(di-tert-butoxyphosphoryl)propanoyl)glycinate (2d) (13.2 g, 0.021 mol).

[0362] 1 H NMR (400 MHz, CDCl3) δ 13.03(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.61(s, 2H), 4.46(s, 1H), 3.46(t, 2H), 3.42(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.20(s, 18H); 13 C NMR (CDCl3) δ 173.1, 172.0, 157.8, 143.6, 142.6, 126.7, 126.2, 125.2, 120.5, 86.9, 67.3, 52.9, 50.0, 47.0, 38.1, 29.4, 28.5, 16.5

[0363] [Example 3] Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)glycyl)glycine (Compound 3)

[0364]

[0365] 3.1. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((tert-butoxycarbonyl)glycyl)glycinate (3b)

[0366] The title compound (3b) was synthesized (5.0 g, 0.0095 mol, 64.20% yield) in the same manner as in 1.3 of Example 1 using Boc-Gly-OH (3a) (2.596 g, 0.0148 mol).

[0367] 1 H NMR (400 MHz, CDCl3) δ 7.76(d, 2H), 7.60(d, 2H), 7.39(t,2H), 7.31(t, 2H), 5.92(t, 1H), 5.38(s, 1H), 4.39(d, 2H), 4.23(q, 2H), 4.19(t, 1H), 4.01(s, 2H), 3.88(s, 2H), 3.45(t, 4H), 1.41(d, 9H), 1.28(t, 3H); 13 C NMR (CDCl3) δ 169.98, 169.02, 156.51, 155.78, 143.88, 141.26, 127.67, 127.05, 125.10, 119.95, 79.81, 67.01, 62.19, 49.63, 48.46, 47.19, 42.20, 39.12, 28.29, 14.09

[0368] 3.2. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-glycylglycinate·TFA salt (3c)

[0369] Ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((tert-butoxycarbonyl)glycyl)glycinate (3b) (5.0 g, 0.0095 mol) was placed in a round-bottom flask, and 50% TFA / MC (60 mL) was added. After completion of the reaction, the mixture was concentrated by rotary stirring, dried in vacuo, and then the next reaction was carried out without further purification.

[0370] 3.3. Preparation of methyl 3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoate (3e)

[0371] The title compound (3e) was synthesized in the same manner as 1.1 of Example 1 using 3-Bromo-2-(bromomethyl)propionate (3d) (2.951 mL, 0.0207 mol) instead of ethyl chloroacetate (10 g, 0.02 mol, 96.51% yield).

[0372] 1 H NMR (400 MHz, CDCl3) δ 3.70(s, 3H), 2.30(m, 1H), 2.10(dd, 4H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 173.0, 86.9, 52.2, 34.2, 29.4, 11.6

[0373] 3.4. Preparation of 3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoic acid (3f)

[0374] Methyl 3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoate (3e) was dissolved in MeOH (54.8 mL). 2 M NaOH (4.386 g, 0.1097 mol) was added, and the mixture was heated in a 50°C water bath while stirring. The reaction solution was concentrated to remove MeOH, and the aqueous layer was washed twice with ether. The aqueous layer was placed in an ice bath and lowered to pH 3 with 1 N HCl. Excess water was added, and extraction was performed twice using MC. The MC layer was dried with anhydrous Na2SO4, filtered, and concentrated (8.65 g, 0.018 mol, 91.82% yield). The next reaction was carried out without further purification.

[0375] 1 H NMR (400 MHz, CDCl3) δ 10.63(s, 1H), 2.44(m, 1H), 1.98(dd, 4H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 178.3, 86.9, 33.9, 29.4, 14.1

[0376] 3.5. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)glycyl)glycinate (3g)

[0377] The title compound (3g) was synthesized in the same manner as in 1.3 of Example 1, except that a 0.51 M solution (18.69 mL, 0.0095 mole) of 3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoic acid (3f) in DMF was added to ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-glycylglycinate·TFA salt (3c) (0.0095 mol) (6.3 g, 0.007 mol, 75.26% yield).

[0378] 1H NMR (400 MHz, CDCl3) δ 9.04(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 4.19(q, 2H), 4.09(s, 2H), 3.46(t, 2H), 3.42(t, 2H), 2.42(m, 1H), 1.99(m, 4H), 1.21(t, 3H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 175.3, 168.4, 164.5, 157.8, 143.6, 142.6, 126.7, 126.2, 125.2, 120.5, 86.9, 67.3, 61.0, 50.4, 49.7, 47.0, 42.6, 38.1, 34.8, 29.4, 14.1,11.9

[0379] 3.6. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)glycyl)glycine (3)

[0380] The title compound (3) was synthesized (4.7 g, 0.006 mol, 77.06% yield) in the same manner as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-((3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)glycyl)glycinate (3 g).

[0381] 1H NMR (400 MHz, CDCl3) δ 13.03(s, 1H), 9.04(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 4.09(s, 2H), 3.42(t, 2H), 2.42(m, 1H), 1.99(m, 4H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 175.3, 168.4, 164.5, 157.8, 143.6, 142.6, 126.7, 126.2, 125.2, 120.5, 86.9, 67.3, 50.4, 49.7, 47.0, 42.6, 38.1, 34.8, 29.4, 11.9

[0382] Example II: Preparation of water-soluble monomers

[0383] [Example 4] Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycine (Compound 4)

[0384]

[0385] 4.1. Preparation of 5-iodopyrimidine-2,4(1H,3H)-dione (4b)

[0386] Uracil (4a) (10.0 g, 0.089 mol) was placed in a 2-neck round-bottomed flask and KI (39.99 g, 0.241 mol), KHCO3 (11.61 g, 0.116 mol), and I2 (29.44 g, 0.116 mol) were added. Water (250 mL) was added, and a heating mantle and a reflux device were installed and refluxed for 3 hours and 30 minutes. After lowering the temperature in an ice bath, the solid was filtered using a small amount of water. The filtered solid was washed with a solution of acetone: H2O = 2:1 (300 mL), then washed with ether and dried. After drying, the title compound (4b) was obtained (13.7 g, 64.5%).

[0387] 1 H NMR (400 MHz, DMSO) δ 8.22(s, 1H), 11.15(s, 1H), 11.32(s, 1H); 13 C NMR (400 MHz, DMSO) δ 68.2, 146.9, 150.7, 161.0

[0388] 4.2. Preparation of ethyl 2-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4c)

[0389] 5-iodopyrimidine-2,4(1H,3H)-dione (4b) (16.04 g, 0.067 mol) was placed in a round bottom flask, and DMF (80 mL) and MeCN (160 mL) were added. DIEA (58.7 mL, 0.337 mol) and ethylbromoacetate (7.47 mL, 0.0674 mol) were added under an ice bath. After the reaction was allowed to proceed at room temperature for 3 h, the reaction solution was back-extracted using EA (240 mL) and water (240 mL). The aqueous layer was concentrated to obtain the title compound (4c) as a white solid (21.4 g, 98.6%).

[0390] 1H NMR (400 MHz, DMSO) δ 1.21(t, 3H), 4.15(q, 2H), 4.48(s, 2H), 8.22(s, 1H), 11.32(s, 1H); 13 C NMR (400 MHz, DMSO) δ 14.1, 50.3, 61.0, 68.2, 147.9, 150.8, 161.0, 167.5

[0391] 4.3. Preparation of ethyl 2-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4d)

[0392] Ethyl 2-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4c) (9.3 g, 0.029 mol) was placed in a round flask, and Pd(PPh3)4 (1.66 g, 0.0014 mol), CuI (0.55 g, 0.0029 mol), and Boc-propargyl amine (4.45 g, 0.029 mol) were added. After degassing the reaction flask, DMF (80 mL) and TEA (8 mL, 0.057 mol) were added using a syringe. The reaction flask was wrapped with aluminum foil and the reaction was carried out for 23 h, after which the solvent was removed using a rotary evaporator. Afterwards, MeOH was added to precipitate the Pd catalyst, which was filtered, and the filtrate was concentrated again using a rotary evaporator. After that, MC was added and the resulting solid was filtered. The filtrate was concentrated again and purified by column to obtain the title compound (4d) (7.9 g, 78.3%).

[0393] 1 H NMR (400 MHz, DMSO) δ 1.21(t, 3H), 1.42(s, 9H), 3.97(s, 2H), 4.15(q, 2H), 4.48(s, 2H), 7.79(s, 1H), 8.18(s, 1H), 11.32(s, 1H);13 C NMR (400 MHz, DMSO) δ 14.1, 28.4, 30.6, 50.8, 61.0, 68.6, 79.5, 89.5, 100.9, 142.7, 150.8, 155.1, 161.6, 167.5

[0394] 4.4. Preparation of ethyl 2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetate (4e)

[0395] Ethyl 2-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4d) (4.00 g, 0.011 mol) was placed in a round flask, and anhydrous MeOH (100 mL) was added. Pd / C (0.300 g) was added while stirring the reaction solution. After degassing, hydrogen gas was added using a hydrogen balloon. After 19 h, the Pd catalyst was removed using celite. Afterwards, the solvent was removed using a rotary evaporator to obtain the title compound (4e) (3.55 g, 90.8%).

[0396] 1 H NMR (400 MHz, DMSO) δ 1.21(t, 3H), 1.42(s, 9H), 1.69(quint, 2H), 2.41(t, 2H), 3.18(t, 2H), 4.15(q, 2H), 4.48(s, 2H), 6.76(s, 1H), 7.19(s, 1H), 11.32(s, 1H); 13 C NMR (400 MHz, DMSO) δ 14.1, 24.7, 27.0, 28.4, 40.2, 51.5, 61.0, 79.5, 113.1, 136.1, 150.8, 155.9, 166.3, 167

[0397] 4.5. Preparation of ethyl 2-(5-(3-aminopropyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4f)

[0398] Ethyl 2-(5-(3-aminopropyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4e) (2.0 g, 0.0057 mol) was placed in a round flask and dissolved in a small amount of MC. 20 mL of 50% TFA / MC was added under an ice bath. The ice bath was then removed, and the reaction was allowed to proceed at room temperature for 30 minutes. The solvent was removed using a rotary evaporator, and the residue was dried under vacuum and then proceeded with the next reaction without further purification.

[0399] 4.6. Preparation of 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl 3-(di-tert-butoxyphosphoryl)propanoate (4g)

[0400] 3-(di-tert-butoxyphosphoryl)propanoic acid (3.26 g, 0.0123 mol) was placed in a round-bottom flask, MC (50 mL) was added, and HOOBt(3-hydroxy-1,2,3-benzotriazin-4(3H)-one) (2.399 g, 0.0147 mol) and DCC (3.035 g, 0.0147 mol) were sequentially added and stirred for 30 min. After completion of the reaction, DCU was filtered off, and the filtrate was distilled under reduced pressure using a rotary evaporator to obtain 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl 3-(di-tert-butoxyphosphoryl)propanoate (4 g) (4.5 g, yield 88.93%).

[0401] 4.7. Preparation of ethyl 2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4h)

[0402] In a round bottom flask containing ethyl 2-(5-(3-aminopropyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4f) (1.98 g, 0.0056 mol) under an ice bath, DIEA (4.88 mL, 0.028 mol), 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl 3-(di-tert-butoxyphosphoryl)propanoate (4g) (2.31 g, 0.0056 mol), and DMF (80 mL) were added. The ice bath was then removed, and the reaction was allowed to proceed at room temperature for 30 min. The reaction solution was diluted with an excess of EA and washed three times with a half-diluted solution of saturated NaHCO3 aqueous solution. Finally, the reaction solution was washed once with brine, the moisture was removed with anhydrous Na2SO4, and the solvent was removed using a rotary evaporator. The residue was then purified using a column to obtain the title compound (4h) (2.45 g, 86.7%).

[0403] 1 H NMR (400 MHz, DMSO) δ 1.20(s, 18H), 1.21(t, 3H), 1.69(quint, 2H), 2.05(t, 2H), 2.41(t, 2H), 2.42(t, 2H), 3.42(t, 4H), 4.15(q, 2H), 4.48(s, 2H), 7.19(s, 1H), 7.70(s, 1H), 11.32(s, 1H); 13C NMR (400 MHz, DMSO) δ 14.1, 19.0, 24.7, 27.1, 28.2, 29.4, 40.0, 51.5, 61.0, 86.9, 113.1, 136.1, 150.8, 166.3, 167.5, 173.3

[0404] 4.8. Preparation of 2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetic acid (4i)

[0405] Ethyl 2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4h) (2.45 g, 0.0049 mol) was placed in a round flask and completely dissolved with 1 N NaOH (24.5 mL, 0.0245 mol). After lowering the pH to 3 with 1 N HCl in an ice-bath, extraction was performed twice using MC. After removing the moisture with anhydrous Na2SO4 and the solvent using a rotary evaporator, the title compound (4i) was obtained (2.21 g, 94.8%). The next reaction was carried out without further purification.

[0406] 4.9. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (4j)

[0407] The title compound (4j) was obtained (0.68 g, 76.1%) by synthesizing it in the same manner as in Example 1.3 using 2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (4i) (0.50 g, 0.0011 mol).

[0408] 1 H NMR (400 MHz, DMSO) δ 1.20(s, 18H), 1.21(t, 3H), 1.69(quint, 2H), 2.05(t, 2H), 2.41(t, 2H), 2.40(t, 2H), 3.42(t, 4H), 3.46(t, 2H), 4.19(q, 2H), 4.28(s, 2H), 4.46(s, 1H), 4.63(s, 2H), 4.70(d, 2H), 6.76(s, 1H), 7.19(s, 1H), 7.28(t, 2H), 7.38(t, 2H), 7.55(d, 2H), 7.70(s, 1H), 7.90(d, 2H), 11.32(s, 1H); 13 C NMR (400 MHz, DMSO) δ 14.1, 19.0, 24.7, 27.1, 28.2, 29.4, 38.1, 40.0, 47.0, 49.7, 50.4, 53.6, 61.0, 67.3, 86.9, 113.1, 120.5, 125.2, 126.2, 126.7, 136.1, 142.6, 147.0, 150.8, 157.8, 164.5, 166.3, 168.4, 173.3

[0409] 4.10. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetyl)glycine (4)

[0410] The title compound (4) was obtained (0.60 g, 90.6%) by the same method as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (4j) (0.68 g, 0.00083 mol).

[0411] 1 H NMR (500 MHz, DMSO) δ 1.28(s, 18H), 1.71(p, 2H), 2.22-2.11(m, 4H), 2.35-2.25(m, 2H), 3.23(dt, 2H), 3.35-3.29(m, 2H), 3.40(pd, 2H), 3.62(s, 1H), 3.98(s, 1H), 4.25(s, 1H), 4.46(t, 1H), 4.70(d, 2H), 4.94(s, 1H), 6.07(s, 1H), 7.19(s, 1H), 7.36(dtd, 4H), 7.54(dd, 2H), 7.75(dd, 2H), 8.05(s, 1H), 9.35(s, 1H)

[0412] [Example 5] Preparation of di-tert-butyl (3-((3-(1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)propyl)amino)-3-oxopropyl)phosphonate (Compound 5)

[0413]

[0414] 5.1. Preparation of tert-butyl 4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazine-1-carboxylate (5a)

[0415] tert-butyl 4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazine-1-carboxylate (5a) was synthesized (15.8 g, 91.2%) using the same method as in registered patent KR10-0464261.

[0416] 1 H NMR (400 MHz, DMSO) δ 1.42(s, 9H), 3.35(t, 2H), 3.46(t, 2H), 3.74(s, 2H), 7.53(m, 2H), 8.02(m, 1H), 8.18(m, 1H); 13 C NMR (400 MHz, DMSO) δ 28.4, 42.5, 49.8, 52.4, 79.8, 121.6, 121.8, 124.5, 125.3, 135.1, 153.5, 154.3, 156, 166.2

[0417] 5.2. Preparation of 1-(benzo[d]thiazol-2-ylsulfonyl)piperazin-2-one (5b)

[0418] tert-butyl 4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazine-1-carboxylate (5a) was treated with 50% TFA / MC in an ice bath, and the reaction was performed at room temperature for 30 minutes. The solvent was removed using a rotary evaporator, and the residue was dried in vacuo to obtain the title compound (5b) (15.56 g, 95.0%). The next reaction was carried out without further purification.

[0419] 5.3. Preparation of di-tert-butyl (3-((3-(1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropymidin-5-yl)propyl)amino)-3-oxopropyl)phosphonate (5)

[0420] 1-(benzo[d]thiazol-2-ylsulfonyl)piperazin-2-one(5b) (1.26 g, 0.0032 mol), 2-(5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (4i) (1.50 g, 0.0032 mol), and PyBOP (2.50 g, 0.0048 mol) were placed in a round flask and dissolved in DMF (15 mL). DIEA (2.07 mL, 0.016 mol) was then added and reacted for 2 hours. Excess EA was added to the reaction solution, and the mixture was washed three times with saturated NaHCO3 aqueous solution. Finally, the reaction solution was washed once with brine, the moisture was removed with anhydrous Na2SO4, and the solvent was removed using a rotary evaporator. Afterwards, the title compound (5) was obtained (1.50 g, 62.1%) through column purification.

[0421] 1 H NMR (400 MHz, DMSO) δ 1.20(s, 18H), 1.69(quint, 2H), 2.05(t, 2H), 2.41(t, 2H), 2.42(t, 2H), 3.42(t, 2H), 3.46(t, 2H), 3.59(t, 2H), 3.98(s, 2H), 4.28(s, 2H), 7.19(s, 1H), 7.53(m, 2H), 7.70(s, 1H), 8.02(m, 1H), 8.18(m, 1H) 11.32(s, 1H); 13 C NMR (400 MHz, DMSO) δ 19.0, 24.7, 27.1, 28.2, 29.4, 40.0, 42.6, 48.0, 50.6, 53.2, 86.9, 113.1, 121.6, 121.8, 124.5, 125.3, 135.1, 166.3, 150.8, 153.5, 156, 164.5, 166.2, 166.3, 173.3

[0422] [Example 6] Preparation of tert-butyl (1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (Compound 6)

[0423]

[0424] 6.1. Preparation of 4-amino-5-iodopyrimidin-2(1H)-one (6b)

[0425] KI (39.99 g, 0.241 mol), KHCO3 (11.61 g, 0.116 mol), and I2 (29.44 g, 0.116 mol) were dissolved in 250 mL of distilled water. A heating mantle was installed, and when the temperature was 35°C, Cytosine (6a) (10.0 g, 0.089 mol) was added. A reflux device was installed and refluxed for 2 hours. After completely cooling to room temperature, the solid was filtered. The filtered solid was slurried in a mixture of water and acetone (water:acetone = 100 mL:200 mL) for 1 hour. After filtering the solid, it was washed with acetone to obtain the title compound (6b) as an ivory solid (17.9 g, 84%).

[0426] 1 H NMR (DMSO-d6, δ ppm): 6.49(s, 1H), 7.77(s, 2H), 10.80(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 53.4, 143.5, 156.3, 158.9

[0427] 6.2. Preparation of tert-butyl (5-iodo-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (6c)

[0428] 4-amino-5-iodopyrimidin-2(1H)-one (6b) (3.0 g, 0.013 mol) was placed in a round flask and dissolved in DMSO (180 mL). Boc2O (4.14 g, 0.019 mol) and DMAP (0.16 g, 0.001 mol) were added to the round flask. After stirring for 30 minutes, EA (600 mL) and saturated aqueous NaHCO3 solution (300 mL) were added and washed three times. After adding brine (300 mL) and washing, anhydrous Na2SO4 (3 g) was added to the organic layer to remove moisture, filtered, and the filtrate was concentrated. The residue was purified by column chromatography to obtain the title compound (6c) as a white solid (2.1 g, 49.2%).

[0429] 1 H NMR (DMSO-d6, δ ppm): 6.67(s, 2H), 9.13(s, 1H), 10.99(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 53.4, 143.5, 156.3, 158.9

[0430] 6.3. Preparation of tert-butyl isopropyl (3-oxo-3-(prop-2-yn-1-ylamino)propyl)phosphonate (6d)

[0431] 3-(di-tert-butoxyphosphoryl)propanoic acid (2c) (5.0 g, 0.019 mol) was dissolved in MC (200 mL). Propagyl amine (1.43 mL, 0.023 mol) and DCC (4.65 g, 0.022 mol) were added and stirred for 1 hour. DCU was filtered off, and the filtrate was concentrated. The residue was purified by column chromatography to obtain the title compound (6d) as a white solid (5.0 g, 87.8%).

[0432] 1H NMR (DMSO-d6, δ ppm): 1.20(s, 18H), 2.05(m, 2H), 2.42(t, 2H), 3.08(t, 1H), 4.21(d, 2H), 8.18(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 18.1, 28.2, 29.4, 29.9, 73.2, 80.1, 86.9, 173.6

[0433] 6.4. Preparation of tert-butyl (5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)prop-1-yn-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (6e)

[0434] tert-Butyl (5-iodo-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (6c) (3.5 g, 0.010 mol), tert-butyl isopropyl (3-oxo-3-(prop-2-yn-1-ylamino)propyl)phosphonate (6d) (4.72 g, 0.016 mol), Pd(Pph3)4 (1.2 g, 0.001 mol), and CuI (0.40 g, 0.002 mol) were placed in a round flask and degassed. After adding anhydrous DMF (60 mL), TEA (4.34 mL, 0.03 mol) was added, and the mixture was stirred for 16 hours. The solution was distilled under reduced pressure, and MeOH (20 mL) was added, stirred for 20 minutes, and the Pd catalyst that had become solid was filtered. After concentration, column purification was performed to obtain the title compound (6e) as a yellow solid (1.3 g, 24.4%).

[0435] 1 H NMR (DMSO-d6, δ ppm): 1.20(s, 18H), 1.42(s, 9H), 2.05(m, 2H), 2.42(t, 2H), 4.21(s, 2H), 8.18(s, 1H), 8.70(s, 1H), 9.21(s, 1H), 10.99(s, 1H); 13C NMR (DMSO-d6, δ ppm): 16.1, 27.8, 28.2, 28.4, 29.4, 68.6, 77.8, 84.6, 86.9, 98.7, 133.0, 153.7, 156.3, 158.9, 173.6

[0436] 6.5. Preparation of ethyl 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)prop-1-yn-1-yl)-2-oxopyrimidin-1(2H)-yl)acetate (6f)

[0437] tert-Butyl (5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)prop-1-yn-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (6e) (3.0 g, 0.006 mol) was placed in a round flask, and DMF (40 mL) was added. KOtBu (0.77 g, 0.07 mol) was added and stirred for 30 minutes under an ice bath. Ethyl bromoacetate (0.91 mL, 0.082 mol) was added and stirred at room temperature for 15 hours. MC (250 mL) was placed in a separatory funnel, and saturated aqueous NaHCO3 solution (250 mL) was added, followed by extraction three times. Anhydrous Na2SO4 (2 g) was added to the organic layer to remove moisture, and the filtrate was concentrated after filtering. The residue was purified by column chromatography to obtain the title compound (6f) as a white solid (2.3 g, 65.9%).

[0438] 1 H NMR (DMSO-d6, δ ppm): 1.21(t, 3H), 1.30(s, 18H), 1.42(s, 9H), 2.05(m, 2H), 2.42(t, 2H), 4.15(q, 2H), 4.21(s, 2H), 4.28(s, 2H), 8.18(s, 1H), 8.70(s, 1H), 9.21(s, 1H); 13C NMR (DMSO-d6, δ ppm): 14.1, 18.1, 27.8, 28.2, 28.4, 29.4, 50.9, 61.0, 68.6, 77.8, 84.6, 86.9, 98.7, 142.4, 153.7, 159.0, 165.7, 173.6

[0439] 6.6. Preparation of ethyl 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (6g)

[0440] Ethyl 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)prop-1-yn-1-yl)-2-oxopyrimidin-1(2H)-yl)acetate (6f) (2.0 g, 0.003 mol) was placed in a round flask and dissolved in MeOH (100 mL). The mixture was stirred, Pd / C (0.10 g) was added, and degassed. After blowing H2 gas, degassed, and blown H2 gas again, the mixture was stirred for 16 hours. Celite 545 was placed on the filter and Pd / C was filtered out. The filtrate was concentrated to obtain the title compound (6g) as a white solid (2.0 g, 99.3%).

[0441] 1 H NMR (DMSO-d6, δ ppm): 1.21(t, 3H), 1.30(s, 18H), 1.42(s, 9H), 1.69(quintet, 2H), 2.05(m, 2H), 2.42(m, 4H), 3.42(t, 2H), 4.15(q, 2H), 4.28(s, 2H), 7.70(s, 1H), 8.10(s, 1H), 9.21(s, 1H); 13C NMR (DMSO-d6, δ ppm): 14.1, 19.0, 20.3, 27.9, 28.2, 28.4, 29.4, 40.4, 51.8, 61.0, 84.6, 86.9, 110.4, 135.3, 153.7, 159.0, 167.5, 169.2, 173.3

[0442] 6.7. Preparation of 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (6h)

[0443] Ethyl 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (6g) (2.0 g, 0.003 mol) was placed in a round flask and dissolved in THF (10 mL). 1.0 M NaOH aqueous solution (10 mL, 0.01 mol) was added and stirred for 30 minutes. After adjusting the pH to 3 with 1.0 M HCl, the mixture was extracted with MC. Na2SO4 (2 g) was added to the organic layer, filtered, and the filtrate was concentrated to obtain the title compound (6h) as a white solid (1.9 g, 99.6%).

[0444] 1 H NMR (DMSO-d6, δ ppm): 1.30(s, 18H), 1.42(s, 9H), 1.69(quintet, 2H), 2.05(m, 2H), 2.42(m, 4H), 3.42(t, 2H), 4.28(s, 2H), 7.70(s, 1H), 8.10(s, 1H), 9.21(s, 1H); 13C NMR (DMSO-d6, δ ppm): 19.0, 20.3, 27.9, 28.2, 28.4, 29.4, 40.4, 56.7, 84.6, 86.7, 110.3, 135.3, 153.7, 159.0, 169.2, 173.3, 175.9

[0445] 6.8. Preparation of tert-butyl (1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxo-1,2-dihydropymidin-4-yl)carbamate (6)

[0446] The title compound (6) as a white solid was obtained (1.8 g, 60.6%) by the same method as in Example 5.3 using 2-(4-((tert-butoxycarbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (6h) (2.0 g, 0.003 mol).

[0447] 1 H NMR (DMSO-d6, δ ppm): 1.30(s, 18H), 1.42(s, 9H), 1.69(quintet, 2H), 2.05(m, 2H), 2.42(m, 4H), 3.42(m, 4H), 3.60(q, 2H), 3.98(s, 1H), 4.08(s, 1H), 7.52(m, 2H), 7.70(s, 1H), 8.02(d, 1H), 8.10(s, 1H), 8.18(d, 1H), 9.21(s, 1H); 13C NMR (DMSO-d6, δ ppm): 19.0, 20.3, 27.9, 28.2, 28.4, 29.4, 40.4, 42.6, 48.0, 50.6, 53.5, 84.6, 86.9, 110.3, 121.6, 121.8, 124.5, 125.3, 135.1, 135.3, 153.5, 153.7, 156, 159.0, 164.5, 166.2, 169.2, 173.3

[0448] [Example 7] N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(4-((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopymidin-1(2H)-yl)acetyl)glycine (compound 7) Manufacturing

[0449]

[0450] 7.1. Preparation of tert-butyl (3-(4-amino-2-oxo-1,2-dihydropyrimidin-5-yl)prop-2-yn-1-yl)carbamate (7a)

[0451] The title compound (7a) was synthesized in the same manner as in 4.3 of Example 4 using 4-amino-5-iodopyrimidin-2(1H)-one (6b) (17.74 g, 0.075 mol) (white solid; 17.6 g, 88.8%).

[0452] 1 H NMR (DMSO-d6, δ ppm): 1.41(s, 9H), 3.95(d, 2H), 6.69(s, 1H), 7.35(t, 1H), 7.65(s, 2H), 10.85(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 28.65, 31.08, 74.91, 78.86, 88.50, 92.57, 146.21, 155.59, 155.80, 165.91

[0453] 7.2. Preparation of tert-butyl (3-(4-amino-2-oxo-1,2-dihydropyrimidin-5-yl)propyl)carbamate (7b)

[0454] The title compound (7b) was synthesized in the same manner as in Example 4.2 using tert-butyl (3-(4-amino-2-oxo-1,2-dihydropyrimidin-5-yl)prop-2-yn-1-yl)carbamate (7a) (14.57 g, 0.06 mol) (white solid; 14.8 g, 99%).

[0455] 1 H NMR (DMSO-d6, δ ppm): 1.39(s, 9H), 1.50(quintet, 2H), 2.20(t, 2H), 2.91(q, 2H), 6.85(t, 1H), 6.94(s, 1H), 7.22(s, 2H), 10.53(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 24.18, 28.75, 39.56, 77.91, 103.82, 140.69, 156.04, 157.14, 166.22

[0456] 7.3. Preparation of ethyl 2-(4-amino-5-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7c)

[0457] The title compound (7c) was synthesized in the same manner as in 4.4 of Example 4 using tert-butyl (3-(4-amino-2-oxo-1,2-dihydropyrimidin-5-yl)propyl)carbamate (7b) (14.80 g, 0.06 mol) (white solid; 14 g, 71.6%).

[0458] 1H NMR (DMSO-d6, δ ppm): 1.21(t, 3H), 1.39(s, 9H), 1.53(quintet, 2H), 2.21(t, 2H), 2.94(q, 2H), 4.12(q, 2H), 4.42(s, 2H), 6.86(s, 2H), 7.29(s, 1H), 7.42(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 14.52, 24.39, 28.75, 39.77, 50.20, 61.22, 77.93, 104.86, 144.36, 156.03, 156.07, 165.88, 169.27

[0459] 7.4. Preparation of ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7d)

[0460] DMAP (8.62 g, 0.07 mol) was placed in a round flask and dissolved in MC (200 mL). The temperature was lowered with ice / salt, and benzyl chloroformate (10.04 mL, 0.07 mol) was added. After 15 min at -15°C, ethyl 2-(4-amino-5-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7c) (10.0 g, 0.028 mol) was added. After 15 min at -15°C, the mixture was stirred at room temperature for 15 h. The reaction solution was concentrated and purified by column chromatography to obtain the title compound (7d) (white solid; 10.4 g, 75.45%).

[0461] 1H NMR (DMSO-d6, δ ppm): 1.23(t, 3H), 1.39(s, 9H), 1.58(quintet, 2H), 2.26(t, 2H), 2.92(t, 2H), 4.17(q, 2H), 4.58(s, 2H), 5.14(s, 2H), 6.87(t, 1H), 7.40(m, 5H), 7.68(s, 1H), 11.93(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 14.04, 23.49, 28.36, 28.55, 39.02, 49.23, 60.30, 62.11, 67.55, 78.73, 113.75, 128.10, 128.21, 128.42, 136.11, 142.46, 148.16, 156.13, 161.21, 163.71, 167.20, 171.07

[0462] 7.5. Preparation of ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7e)

[0463] The title compound (7e) was synthesized in the same manner as in 4.5 and 4.7 of Example 4 using ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7d) (14.0 g, 0.029 mol) (ivory solid; 16.1 g, 80.98%).

[0464] 1H NMR (DMSO-d6, δ ppm): 1.26(t, 3H), 1.45(s, 18H), 1.72(quintet, 2H), 1.96(m, 4H), 3.18(t, 2H), 4.22(q, 2H), 4.51(s, 2H), 5.19(t, 2H), 7.33(m, 5H), 7.55(s, 1H), 12.31(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 14.1, 19.0, 20.3, 29.4, 27.9, 28.2, 40.4, 51.8, 61.0, 66.5, 86.9, 110.3, 127.1, 127.6, 128.9, 135.3, 136.1, 159.0, 153.7, 167.5, 169.2, 173.3

[0465] 7.6. Preparation of 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (7f)

[0466] The title compound (7f) was synthesized in the same manner as in Example 4.8 using ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (7e) (10.0 g, 0.016 mol) (ivory solid; 8.7 g, 90.67%).

[0467] 1 H NMR (DMSO-d6, δ ppm): 1.45(s, 18H), 1.72(quintet, 2H), 1.96(m, 4H), 3.18(t, 2H), 4.51(s, 2H), 5.19(t, 2H), 7.33(m, 5H), 7.55(s, 1H), 12.31(s, 1H); 13C NMR (DMSO-d6, δ ppm): 19.0, 20.3, 27.9, 28.2, 29.4, 40.4, 56.7, 66.5, 86.9, 110.3, 127.1, 127.6, 128.9, 135.3, 136.1, 153.7, 159.0, 169.2, 173.3, 175.9

[0468] 7.7. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(4-((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetyl)glycinate (7g)

[0469] The title compound (7g) was synthesized in the same manner as in 1.3 of Example 1 using 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (7f) (10.0 g, 0.016 mol) (ivory solid; 11.6 g, 73.6%).

[0470] 1 H NMR (CDCl3, δ ppm): 1.29(t, 3H), 1.49(s, 18H), 1.69(quintet, 2H), 2.05(m, 2H), 2.41(m,4H), 3.17(t, 2H), 3.42(m, 4H), 4.08(s, 2H), 4.19(t, 2H), 4.46(t, 1H), 4.64(s, 2H), 4.70(d, 2H), 5.02(s, 2H), 6.76(s, 1H), 7.36(m, 9H), 7.55(t, 2H), 7.70(s, 1H), 7.90(d, 2H), 8.10(s, 1H), 9.21(s, 1H); 13C NMR (CDCl3, δ ppm): 14.1, 19.0, 20.3, 27.9, 28.2, 29.4, 38.1, 40.4, 47.0, 49.7, 50.4, 53.9, 61.0, 66.5, 67.3, 86.9, 110.3, 120.5, 125.2, 126.2, 126.7, 127.1, 127.6, 128.9, 135.3, 136.1, 142.6, 143.6, 153.7, 157.8, 159.0, 164.5, 168.4, 169.2, 173.3

[0471] 7.8. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetyl)glycine (7)

[0472] The title compound (7) was synthesized in the same manner as in 1.4 of Example 1 using ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetyl)glycinate (7g) (11.0 g, 0.011 mol) (white solid; 4.3 g, 40.3%).

[0473] 1H NMR (CDCl3, δ ppm): 1.49(s, 18H), 1.69(quintet, 2H), 2.05(m, 2H), 2.41(m,4H), 3.42(m, 4H), 4.08(s, 2H), 4.19(t, 2H), 4.46(t, 1H), 4.64(s, 2H), 4.70(d, 2H), 5.02(s, 2H), 6.76(s, 1H), 7.36(m, 9H), 7.55(t, 2H), 7.70(s, 1H), 7.90(d, 2H), 8.10(s, 1H), 9.21(s, 1H), 13.03(s, 1H); 13 C NMR (CDCl3, δ ppm): 19.0, 20.3, 27.9, 28.2, 29.4, 38.1, 40.4, 47.0, 49.7, 52.6, 53.9, 66.5, 67.4, 86.9, 110.3, 120.5, 125.2, 126.2, 126.7, 127.1, 127.6, 128.9, 135.3, 136.1, 142.6, 143.6, 153.7, 157.8, 159.0, 164.5, 169.2, 173.1, 173.3

[0474] [실시예 8] benzyl (1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate(8)의 제조

[0475]

[0476] The title compound (8) was synthesized in the same manner as in Example 5.3 using 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (7f) (8.0 g, 0.013 mol) (white solid; 6.9 g, 59.12%).

[0477] 1 H NMR (DMSO-d6, δ ppm): 1.47(s, 18H), 1.72(quintet, 2H), 1.96(m, 2H), 2.43(m, 4H), 3.99(q, 2H), 4.31(m, 4H), 4.53(d, 2H), 5.19(s, 2H), 6.96(t, 1H), 7.34(m, 6H), 7.54(quintet, 2H), 8.00(d, 1H), 8.18(d, 1H), 12.32(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 23.51, 25.15, 26.62, 28.03, 30.34, 37.88, 40.42, 42.96, 46.00, 47.47, 48.44, 48.69, 49.24, 67.69, 82.01, 82.10, 113.70, 122.29, 125.54, 127.82, 128.21, 128.38, 128.43, 128.52, 136.19, 137.11, 142.77, 148.36, 151.76, 161.21; 163.72, 164.80, 165.21, 171.90, 172.09

[0478] [Example 9] Preparation of N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (Compound 9)

[0479]

[0480] 9.1. Preparation of tert-butyl (3-((6-amino-9H-purin-2-yl)amino)propyl)carbamate (9d)

[0481] 2-Chloro-9H-purin-6-amine (9a) (20.0 g, 0.1179 mol) was placed in a round-bottom flask, and 1,3-diaminopropane (198 mL, 2.358 mol) was added. A heating mantle and a reflux device were installed, and refluxing was performed for 18 h. After monitoring the reaction, the heating mantle and reflux device were removed, and the unreacted 1,3-diaminopropane was concentrated and recovered. After recovery, toluene was added and azeotropic distillation was performed to remove the remaining 1,3-diaminopropane. THF (140 mL) and water (140 mL) were added to the mixture without purification. Boc2O (77 g, 0.3537 mol) was added, and the mixture was stirred for 5 h. After monitoring the reaction, THF was concentrated at low temperature, and an amount of NaHCO3 (12.6 g) sufficient to saturate the aqueous solution was added, followed by MeOH (140 mL), a heating mantle was installed, and the mixture was heated to 60°C. After 1 h, MeOH was completely removed, and the mixture was extracted with EA. The EA layer was dried with Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to obtain the title compound (9d) as a white solid (24.5 g, 0.0797 mol, 67.58%; 3 step overall yield).

[0482] 1 H NMR (400 MHz, DMSO-d6) δ 12.21(s, 1H), 7.67(s, 1H), 6.81(t, 1H), 6.66(s, 2H), 6.14(t, 1H), 3.24(q, 2H), 3.00(q, 2H), 1.62(m, 2H), 1.38(m, 9H); 13C NMR (DMSO-d6) δ 170.81, 159.97, 155.17, 152.80, 113.43, 77.91, 49.07, 38.90, 38.15, 28.71, 28.62

[0483] 9.2. Preparation of ethyl 2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetate (9e)

[0484] tert-Butyl (3-((6-amino-9H-purin-2-yl)amino)propyl)carbamate (9d) (24.5 g, 0.0797 mol) was placed in a round-bottom flask and dissolved in DMF (245 mL). An ice bath was set up, and 60% oil-coated NaH (4.783 g, 0.199 mol) was added. The ice bath was removed, and after 1 h, the ice bath was set up again, and ethyl bromoacetate (9.70 mL, 0.0877 mol) was added using a syringe. After 3 h of reaction at room temperature, a saturated NH4Cl aqueous solution (30 mL) was added. Excess EA was added, and the mixture was washed twice with a saturated NaHCO3 aqueous solution and once with brine. The EA layer was dried with anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to obtain the title compound (9e) as a white solid (20.3 g, 0.0516 mol, 64.73% yield).

[0485] 1 H NMR (400 MHz, DMSO-d6) δ 7.69(s, 1H), 6.80(t, 1H), 6.69(s, 2H), 6.26(t, 1H), 4.86(s, 2H), 4.17(q, 2H), 3.22(q, 2H), 2.86(q, 2H), 1.59(m, 2H), 1.38(m, 9H), 1.22(t, 3H); 13C NMR (DMSO-d6) δ 167.0, 160.2, 155.6, 152.0, 149.8, 142.0, 119.5, 79.2, 60.8, 53.0, 39.0, 37.2, 28.4, 28.0, 14.0

[0486] 9.3. Preparation of 2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (9f)

[0487] The title compound (9f) was synthesized (17.5 g, 0.0479 mol, 96.53% yield) in the same manner as in 1.2 of Example 1 using ethyl 2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetate (9e) (20.3 g, 0.05083 mol).

[0488] 1 H NMR (400 MHz, DMSO-d6) δ 11.7(s, 1H), 7.69(s, 1H), 6.80(t, 1H), 6.69(s, 2H), 6.26(t, 1H), 4.86(s, 2H), 4.17(q, 2H), 3.22(q, 2H), 2.86(q, 2H), 1.59(m, 2H), 1.38(s, 9H); 13 C NMR (DMSO-d6) δ 174.9, 160.0, 155.9, 152.0, 149.8, 142.0, 119.4, 79.5, 50.1, 39.4, 37.4, 28.7, 28.4

[0489] 9.4. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (9g)

[0490] The title compound (9g) was synthesized (2.78 g, 0.0039 mol, 64.49%) in the same manner as in 1.3 of Example 1 using 2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (9f) (2.39 g, 0.0060 mol).

[0491] 1 H NMR (400 MHz, DMSO-d6) δ 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H) 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(d, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H) 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.66(t, 2H) 1.39(s, 9H), 1.25(t, 3H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 157.8, 155.9, 152.0, 149.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 79.5, 67.3, 61.0, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 37.4, 28.7, 28.4, 14.1

[0492] 9.5. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (9i)

[0493] Ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (9g) (2.78 g, 0.0039 mol) was placed in a round bottom flask, and 50% TFA / MC (50 mL) was added under an ice bath. After 1 h, the reaction solution was concentrated and dried in vacuo to obtain ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-aminopropyl)amino)-9H-purin-9-yl)acetyl)glycinate (9h), which was then carried out to the next reaction without further purification. To ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-aminopropyl)amino)-9H-purin-9-yl)acetyl)glycinate (9h) (quantitative yield, 0.0039 mol) were added DMF (30 mL) and DIEA (3.38 mL, 0.0194 mol). Then, a solution of 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl 3-(di-tert-butoxyphosphoryl)propanoate (4 g) (1.6 g, 0.0039 mol) in DMF (5 mL) was added, and the mixture was reacted at room temperature for 2 h. After adding excess EA, the mixture was washed twice with saturated aqueous NaHCO3 solution and once with brine. The EA layer was dried with anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to obtain the title compound (9i) (2.0 g, 0.0023 mol, 59.69% yield).

[0494] 1H NMR (400 MHz, DMSO-d6) δ 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H) 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(d, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H) 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.81(m, 2H), 1.66(t, 2H) 1.39(s, 18H), 1.25(t, 3H); 13 C NMR (DMSO-d6) δ 173.3, 169.4, 168.4, 160.0, 157.8, 152.0, 159.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.3, 61.0, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 29.4, 28.8, 28.2, 19.0, 14.1

[0495] 9.6. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (9)

[0496] The title compound (9) was synthesized (1.4 g, 0.0017 mol, 60.9% yield) in the same manner as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (9i) (2.0 g, 0.0023 mol).

[0497] 1 H NMR (400 MHz, DMSO-d6) δ 11.2(s, 1H), 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H) 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(d, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H) 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.66(t, 2H) 1.39(s, 18H); 13 C NMR (DMSO-d6) δ 173.3, 169.4, 168.4, 160.0, 157.8, 152.0, 159.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.3, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 29.4, 28.8, 28.2, 19.0

[0498] [실시예 10] di-tert-butyl (3-((3-((6-amino-9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purin-2-yl)amino)propyl)amino)-3-oxopropyl)phosphonate (화합물 10)의 제조

[0499]

[0500] 10.1. ethyl N-(2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (10b)의 제조

[0501] The title compound (10b) was synthesized (19 g, 0.0209 mol, 66.99% yield) in the same manner as in 1.3 of Example 1 using 2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (9f) (15.0 g, 0.0411 mol) and ethyl (2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (10a) (14.1 g, 0.0411 mol).

[0502] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 4.20(q, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 1.85(m, 2H), 1.42(s, 9H), 1.21(t, 3H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 156, 155.9, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 79.5, 61.1, 50.4, 48.9, 39.6, 39.4, 37.7, 37.4, 28.7, 28.4, 14.1

[0503] 10.2. Preparation of ethyl N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (10d)

[0504] The title compound (10d) was synthesized (17.5 g, 0.0209 mol, 75.8% yield) in the same manner as in 9.5 of Example 9 using ethyl N-(2-(6-amino-2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (10b) (19.00 g, 0.0275 mol).

[0505] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 4.20(q, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.85(m, 2H), 1.42(s, 18H), 1.21(t, 3H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 156, 155.9, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 61.1, 50.4, 48.9, 39.6, 39.4, 37.7, 37.4, 28.7, 29.4, 28.2, 19.0, 14.1

[0506] 10.3. Preparation of N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycine (10e)

[0507] Ethyl N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (10d) (17.5 g, 0.0209 mol) was dissolved in THF (62.6 mL). LiOH (2.626 g, 0.0626 mol) was dissolved in water (62.6 mL) and then slowly added. After completion of the reaction, an ice bath was installed and 1 N HCl was added until the pH reached 3. Extraction was performed twice using MC. The MC layer was dried with anhydrous Na2SO4, filtered, and concentrated (9.2 g, 0.0113 mol, 54.4%). The next reaction was carried out without further purification.

[0508] 1 H NMR (400 MHz, DMSO-d6) δ 13.03(s. 1H), 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.85(m, 2H), 1.42(s, 18H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 156, 155.9, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 50.4, 48.9, 39.6, 39.4, 37.7, 37.4, 28.7, 29.4, 28.2, 19.0

[0509] 10.4. Preparation of di-tert-butyl (3-((3-((6-amino-9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purin-2-yl)amino)propyl)amino)-3-oxopropyl)phosphonate (10)

[0510] N-(2-(6-amino-2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycine (10e) (9.2 g, 0.0113 mol) was dissolved in DMF (60 mL), and EDC·HCl (3.261 g, 0.017 mol) was added. After reaction at room temperature for 2 h, excess EA was added to the separatory funnel and washed three times with saturated aqueous NaHCO3 solution and once with brine. The EA layer was dried with anhydrous Na2SO4, filtered, and concentrated (5.0 g, 0.0063 mol, 55.6%).

[0511] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 4.62(s, 2H), 3.98(s, 2H), 3.59(t, 2H), 3.49(t, 2H), 3.42(t, 2H), 3.35(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.85(m, 2H), 1.20(s, 18H); 13C NMR (DMSO-d6) δ 173.3, 169.4, 166.2, 160.0, 156, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 50.6, 48.0, 42.6, 39.4, 37.7, 37.3, 29.4, 28.8, 28.2, 19.0

[0512] [Example 11] Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)glycine (Compound 11)

[0513]

[0514] 11.1. Preparation of tert-butyl (2-((6-amino-9H-purin-2-yl)amino)ethyl)carbamate (11c)

[0515] The title compound (11c) was synthesized (11.0 g, 0.0375 mol; 3-step overall yield 63.59%) in the same manner as in 9.1 of Example 9 using 2-chloro-9H-purin-6-amine (9a) (10.0 g, 0.0590 mol) and ethylene diamine (79.2 mL, 1.1795 mol).

[0516] 1 H NMR (400 MHz, DMSO-d6) δ 12.09(s, 1H), 7.70(s, 1H), 6.85(t, 1H), 6.63(s, 2H), 6.12(t, 1H), 3.29(q, 2H), 3.12(dt, 2H), 1.39(m, 9H); 13 C NMR (DMSO-d6) δ 159.97, 156.23, 155.72, 152.72, 135.81, 113.60

[0517] 11.2. Preparation of ethyl 2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetate (11d)

[0518] The title compound (11d) was synthesized (11.01 g, 0.0290 mol, 77.31%) in the same manner as in 9.2 of Example 9 using tert-butyl (2-((6-amino-9H-purin-2-yl)amino)ethyl)carbamate (11c) (11 g, 0.0375 mol).

[0519] 1 H NMR (400 MHz, DMSO-d6) δ 7.73(s, 1H), 6.84(s, 1H), 6.76(s, 2H), 6.29(s, 1H), 4.90(s, 2H), 4.19(q, 2H), 3.30(d, 2H), 3.13(d, 2H), 1.40(s, 9H), 1.32(t, 3H); 13 C NMR (DMSO-d6) δ 168.68, 159.98. 156.47, 156.16, 152.27, 138.27, 113.20, 78.01, 61.64, 43.89, 41.66, 40.65, 28.71

[0520] 11.3. Preparation of 2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetic acid (11e)

[0521] The title compound (11e) was synthesized (8.8 g, 0.0250 mol, 86.39% yield) in the same manner as in 1.2 of Example 1 using ethyl 2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetate (11d) (11.01 g, 0.0290 mol).

[0522] 1 H NMR (400 MHz, DMSO-d6) δ 12.02(s, 1H), 7.73(s, 1H), 6.84(s, 1H), 6.76(s, 2H), 6.29(s, 1H), 4.90(s, 2H), 4.19(q, 2H), 3.30(d, 2H), 3.13(d, 2H), 1.40(s, 9H); 13 C NMR (DMSO-d6) δ 168.68, 159.98. 156.47, 156.16, 152.27, 138.27, 113.20, 78.01, 61.64, 43.89, 41.66, 40.65, 28.71, 14.49

[0523] 11.4. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetyl)glycinate (11f)

[0524] The title compound (11f) was synthesized (1.99 g, 0.0028 mol, 91.91 %) in the same manner as in 1.3 of Example 1 using 2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetic acid (11e) (1.08 g, 0.0031 mol).

[0525] 1H NMR (400 MHz, DMSO-d6) δ 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H), 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(dd, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H) 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.38(s, 9H), 1.20(dt, 3H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 157.8, 155.9, 152.0, 149.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 79.5, 67.3, 61.0, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 37.4, 28.4, 14.1

[0526] 11.5. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)glycinate (11h)

[0527] The title compound (11h) was synthesized (1.76 g, 0.00207 mol, 73.03% yield) in the same manner as in 9.5 of Example 9 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetyl)glycinate (11f) (1.99 g, 0.0028 mol).

[0528] 1H NMR (400 MHz, DMSO-d6) δ 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H) 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(dd, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H) 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.81(m, 2H), 1.39(s, 18H), 1.25(dt, 3H); 13 C NMR (DMSO-d6) δ 173.3, 169.4, 168.4, 160.0, 157.8, 152.0, 159.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.3, 61.0, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 29.4, 28.2, 19.0, 14.1

[0529] 11.6. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)glycine (11)

[0530] The title compound (11) was synthesized (1.4 g, 0.0017 mol, 82.2%) in the same manner as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)glycinate (11h) (1.76 g, 0.00207 mol).

[0531] 1 H NMR (400 MHz, DMSO-d6) δ 8.06(s, 1H), 7.89(d, 2H), 7.62(s, 1H), 7.51(s, 1H) 7.42(t, 2H), 7.34(t, 2H), 6.77(s, 2H), 6.67(s, 1H), 5.05-4.82(dd, 2H), 4.40(m, 2H), 4.23(m, 2H), 4.12(t, 1H), 4.09(q, 2H), 3.61-3.36(t, 4H), 3.20(t, 2H), 3.03(t, 2H), 1.81(m, 2H), 1.39(s, 18H); 13 C NMR (DMSO-d6) δ 173.3, 169.4, 168.4, 160.0, 157.8, 152.0, 159.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.3, 50.4, 49.7, 47.0, 39.4, 38.1, 37.7, 29.4, 28.2, 19.0

[0532] [실시예 12] di-tert-butyl (3-((2-((6-amino-9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purin-2-yl)amino)ethyl)amino)-3-oxopropyl)phosphonate (화합물 12)의 제조

[0533]

[0534] 12.1. ethyl N-(2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (12a)의 제조

[0535] The title compound (12a) was synthesized (4.3 g, 0.00635 mol, 72.73% yield) in the same manner as in 10.1 of Example 10 using 2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetic acid (11e) (3.068 g, 0.00874 mol).

[0536] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 4.20(q, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 1.42(s, 9H), 1.21(t, 3H); 13 C NMR (DMSO-d6) δ 169.4, 168.4, 160.0, 156, 155.9, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 79.5, 61.1, 50.4, 48.9, 39.6, 39.4, 37.7, 37.4, 28.4, 14.1

[0537] 12.2. Preparation of ethyl N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (12c)

[0538] The title compound (12c) was synthesized (4.1 g, 0.00487 mol, 78.2% yield) in the same manner as in 9.5 of Example 9 using ethyl N-(2-(6-amino-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (12a) (4.3 g, 0.00635 mol).

[0539] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.85(m, 2H), 1.42(s, 18H); 13 C NMR (DMSO-d6) δ 173.2, 169.4, 168.4, 160.0, 156, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 61.0, 50.4, 48.9, 39.6, 39.2, 37.7, 29.4, 28.2, 19.0, 14.1

[0540] 12.3. Preparation of N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycine (12d)

[0541] The title compound (12d) was synthesized (3.5 g, 0.00439 mol, 88.4%) in the same manner as in 10.3 of Example 10 using ethyl N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycinate (12c) (4.1 g, 0.00487 mol).

[0542] 1 H NMR (400 MHz, DMSO-d6) δ 13.03(s. 1H), 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 6.95(d, 2H), 4.62(d, 2H), 4.60(d, 2H), 3.46(t, 2H), 3.35(t, 2H), 3.18(t, 2H), 2.86(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.85(m, 2H), 1.42(s, 18H); 13 C NMR (DMSO-d6) δ 173.2, 169.4, 168.4, 160.0, 156, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 50.4, 48.9, 39.6, 39.2, 37.7, 29.4, 28.2, 19.0

[0543] 12.4. Preparation of di-tert-butyl (3-((2-((6-amino-9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purin-2-yl)amino)ethyl)amino)-3-oxopropyl)phosphonate (12)

[0544] The title compound (12) was synthesized (3.0 g, 0.00385 mol, 87.7% yield) in the same manner as in 10.4 of Example 10 using N-(2-(6-amino-2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycine (12d) (3.5 g, 0.00439 mol).

[0545] 1 H NMR (400 MHz, DMSO-d6) δ 8.18(d, 1H), 8.05(s, 1H), 7.72(s, 1H), 7.52(d, 2H), 7.12(s, 2H), 7.01(s, 1H), 4.62(s, 2H), 3.98(s, 2H), 3.59(t, 2H), 3.49(t, 2H), 3.42(t, 2H), 3.35(t, 2H), 2.42(t, 2H), 2.05(m, 2H), 1.20(s, 18H); 13 C NMR (DMSO-d6) δ 173.3, 169.4, 166.2, 160.0, 156, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 50.6, 48.0, 42.6, 39.4, 37.7, 37.3, 29.4, 28.2, 19.0

[0546] [Example 13] Preparation of N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (Compound 13)

[0547]

[0548] 13.1. Preparation of di-tert-butyl (((9H-purine-2,6-diyl)bis(azanediyl))bis(propane-3,1-diyl))dicarbamate (13d)

[0549] The title compound (13d) was synthesized (3.5 g, 0.0075 mol, 28.48% yield) in the same manner as in 9.1 of Example 9 using 2,6-dichloro-9H-purine (13a) (10.0 g, 0.0590 mol) and ethylene diamine (79.2 mL, 1.1795 mol).

[0550] 1 H NMR (400 MHz, CDCl3) δ 12.69(s, 1H), 7.64(s, 1H), 6.22(s, 1H), 5.54(s, 1H), 5.39(s, 1H), 5.28(s, 1H), 3.65(t, 2H), 3.46(t, 2H). 3.23(m, 4H), 1.76(m, 4H), 1.45(s, 18H); 13 C NMR (CDCl3) δ 155.9, 154.7, 152.7, 152.0, 138.7, 119.4, 79.5, 42.1, 39.4, 37.4, 28.7, 28.4

[0551] 13.2. Preparation of ethyl 2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetate (13e)

[0552] The title compound (13e) was synthesized (2.5 g, 0.00454 mol, 84.34% yield) in the same manner as in 9.2 of Example 9 using di-tert-butyl (((9H-purine-2,6-diyl)bis(azanediyl))bis(propane-3,1-diyl))dicarbamate (13d) (3.5 g, 0.0075 mol).

[0553] 1H NMR (400 MHz, CDCl3) δ 7.53(s, 1H), 6.56(s, 1H), 6.07(s, 1H), 5.84(s, 1H), 5.21(s, 1H), 4.83(s, 2H), 4.22(q, 2H), 3.59(t, 2H), 3.49(t, 2H), 3.17(t, 4H), 1.71(m, 4H), 1.46(s, 18H), 1.28(t, 3H); 13 C NMR (CDCl3) δ 167.71, 160.08, 156.10, 155.29, 137.06, 113.28, 79.21, 61.89, 60.29, 53.47, 43.74, 38.64, 38.24, 28.61, 14.11

[0554] 13.3. Preparation of 2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (13f)

[0555] The title compound (13f) was synthesized (1.72 g, 0.0036 mol, 61.16% yield) in the same manner as in 1.2 of Example 1 using ethyl 2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetate (13e) (2.50 g, 0.0454 mol).

[0556] 1 H NMR (400 MHz, CDCl3) δ 12.22(s, 1H), 7.53(s, 1H), 6.56(s, 1H), 6.07(s, 1H), 5.84(s, 1H), 5.21(s, 1H), 4.83(s, 2H), 3.59(t, 2H), 3.49(t, 2H), 3.17(t, 4H), 1.71(m, 4H), 1.46(s, 18H); 13C NMR (CDCl3) δ 167.71, 160.08, 156.10, 155.29, 137.06, 113.28, 79.21, 61.89, 53.47, 43.74, 38.64, 38.24, 28.61

[0557] 13.4. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (13g)

[0558] The title compound (13g) was synthesized (2.2 g, 0.0025 mol, 76.57%) in the same manner as in Example 1.3 using 2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (13f) (1.74 g, 0.0033 mol).

[0559] 1 H NMR (400 MHz, CDCl3) δ 8.05(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 7.01(s, 1H), 6.76(s, 3H), 6.05(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.62(s, 2H), 4.46(t, 1H), 4.19(q, 2H), 3.46(t, 2H), 3.42(t, 2H), 3.35(t, 4H), 3.18(t, 4H), 1.85(m, 4H), 1.42(s, 18H), 1.21(t, 3H); 13C NMR (CDCl3) δ 169.4, 168.4, 157.6, 155.9, 154.7, 152.0, 149.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 79.5, 67.3, 61.0, 50.4, 49.7, 47.4, 42.1, 39.4, 38.1, 37.7, 37.4, 28.7, 28.4, 14.1

[0560] 13.5. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (13i)

[0561] The title compound (13i) was synthesized (1.8 g, 0.0015 mol, 61.09 %) in the same manner as in 9.5 of Example 9 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (13g) (2.2 g, 0.0025 mol).

[0562] 1H NMR (400 MHz, CDCl3) δ 8.05(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 7.01(s, 1H), 6.76(s, 3H), 6.05(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.62(s, 2H), 4.46(t, 1H), 4.19(q, 2H), 3.46(t, 2H), 3.42(t, 2H), 3.35(t, 4H), 3.18(t, 4H), 2.42(m, 4H), 2.05(m, 4H), 1.85(m, 4H), 1.21(t, 3H) 1.20(s, 36H); 13 C NMR (CDCl3) δ 173.3, 169.4, 168.4, 157.8, 154.7, 152.0, 149.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.6, 61.0, 50.4, 49.7, 47.4, 42.1, 39.4, 38.1, 37.7, 37.4, 29.4, 28.8, 28.2, 19.1, 14.1

[0563] 13.6. N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (13)의 제조

[0564] The title compound (13) was synthesized (1.5 g, 0.0013 mol, 85.38% yield) in the same manner as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (13i) (1.8 g, 0.0015 mol).

[0565] 1 H NMR (400 MHz, CDCl3) δ 12.03(s, 1H), 8.05(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 7.01(s, 1H), 6.76(s, 3H), 6.05(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.62(s, 2H), 4.46(t, 1H), 3.46(t, 2H), 3.42(t, 2H), 3.35(t, 4H), 3.18(t, 4H), 2.42(m, 4H), 2.05(m, 4H), 1.85(m, 4H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 173.3, 169.4, 168.4, 157.8, 154.7, 152.0, 149.8, 143.6, 142.6, 142.0, 126.7, 126.2, 125.2, 120.5, 119.4, 86.9, 67.6, 50.4, 49.7, 47.4, 42.1, 39.4, 38.1, 37.7, 37.4, 29.4, 28.8, 28.2, 19.1

[0566] [Example 14] tetra-tert-butyl ((((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purine-2,6-diyl )bis(azanediyl))bis(propane-3,1-diyl))bis(azanediyl))bis(3-oxopropane-3,1-diyl))bis(phosphonate) Preparation of (Compound 14)

[0567]

[0568] 14.1. Preparation of ethyl N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (14a)

[0569] The title compound (14a) was synthesized (2.5 g, 0.0029 mol, 81.09 %) in the same manner as in 1.3 of Example 1 using 2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetic acid (13f) (1.9 g, 0.0036 mol).

[0570] 1 H NMR (400 MHz, CDCl3) δ 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.70(s, 1H), 7.51(q, 2H), 7.01(s, 1H), 6.76(s, 1H), 6.56(s, 1H), 6.05(s, 1H), 4.62(s, 2H), 4.55(s, 2H), 4.19(q, 2H), 3.46(t, 2H), 3.35(m, 4H), 3.18(m, 4H), 2.86(t, 2H), 1.85(q, 4H), 1.42(s, 18H), 1.21(t, 3H); 13C NMR (CDCl3) δ 169.4, 168.4, 155.9, 156, 154.7, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4. 79.5, 61.0, 50.4, 48.9, 42.1, 39.4, 37.7, 37.4, 28.7, 28.4, 14.1

[0571] 14.2. ethyl N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (14c) 의 제조

[0572] ethyl N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-((tert-butoxycarbonyl)amino)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (14a) (2.5 g, 0.0029 mol)을 9.5% pore size (2.65 g, 0.00232 mol, 14c). 78.11 % yield).

[0573] 1 H NMR (400 MHz, CDCl3) δ 8.18(d,1H), 8.05(s,1H), 8.02(d,1H), 7.70(s,1H), 7.51(q,2H), 7.01(s,1H), 6.76(s,1H), 6.56(s,1H), 6.05(s, 1H), 4.62(s, 2H), 4.55(s, 2H), 4.19(q, 2H), 3.46(t, 2H), 3.42(m, 4H), 3.35(m, 4H), 2.86(t, 2H), 2.42(m, 4H), 2.02(m, 4H), 1.85(q, 4H), 1.20(s, 36H), 1.21(t, 3H); 13C NMR (CDCl3) δ 173.3, 169.4, 168.4, 155.9, 156, 154.7, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 61.0, 50.4, 48.9, 42.1, 39.4, 37.7, 37.4, 29.4, 28.7, 28.2, 19.0, 14.1

[0574] 14.3. Preparation of N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (14d)

[0575] The title compound (14d) was synthesized (2.2 g, 0.002 mol, 85.1%) in the same manner as in 10.3 of Example 10 using ethyl N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycinate (14c) (2.65 g, 0.00232 mol).

[0576] 1 H NMR (400 MHz, CDCl3) δ 13.03(s, 1H), 8.18(d, 1H), 8.05(s, 1H), 8.02(d, 1H), 7.70(s, 1H), 7.51(q, 2H), 7.01(s, 1H), 6.76(s, 1H), 6.56(s, 1H), 6.05(s, 1H), 4.62(s, 2H), 4.55(s, 2H), 3.46(t, 2H), 3.42(m, 4H), 3.35(m, 4H), 2.86(t, 2H), 2.42(m, 4H), 2.02(m, 4H), 1.85(q, 4H), 1.20(s, 36H);13 C NMR (CDCl3) δ 173.3, 169.4, 168.4, 155.9, 156, 154.7, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4, 86.9, 50.4, 48.9, 42.1, 39.4, 37.7, 37.4, 29.4, 28.7, 28.2, 19.0

[0577] 14.4. tetra-tert-butyl ((((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-9H-purine-2,6-diyl )bis(azanediyl))bis(propane-3,1-diyl))bis(azanediyl))bis(3-oxopropane-3,1-diyl))bis(phosphonate) (14) Preparation

[0578] The title compound (14) was synthesized (1.8 g, 0.0016 mol, 83.2% yield) in the same manner as in 10.4 of Example 10 using N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (14d) (2.2 g, 0.002 mol).

[0579] 1H NMR (400 MHz, CDCl3) δ 8.18(d,1H), 8.05(s,1H), 8.02(d,1H), 7.70(s,1H), 7.51(q,2H), 7.01(s,1H), 6.76(s,1H), 6.56(s,1H), 6.05(s, 1H), 4.62(s, 2H), 3.98(s, 2H), 3.59(m, 2H), 3.46(t, 2H), 3.42(m, 4H), 3.35(m, 4H), 2.86(t, 2H), 2.42(m, 4H), 2.02(m, 4H), 1.85(q, 4H), 1.20(s, 36H); 13 C NMR (CDCl3) δ 173.3, 169.4, 166.2, 156, 154.7, 153.5, 152.0, 149.8, 142.0, 135.1, 125.3, 124.5, 121.8, 121.6, 119.4. 86.9, 50.9, 48.0, 42.6, 42.1, 39.4, 37.7, 37.4, 29.4, 28.7, 28.2, 19.0

[0580] [15 years] N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycine (화합물 15)Liver 제조

[0581]

[0582] 15.1. 2-chloro-1,9-dihydro-6H-purin-6-one (15a) was used

[0583] 2,6-dichloro-9H-purine (13a) (5.0 g, 0.0265 mol) was added 2.5 M NaOH(aq) (35 mL), and the mixture was refluxed for 5 hours in a heating mantle and reflux device. The reaction solution was cooled to 50–55°C, MeCN (15 mL) was added, and the mixture was cooled to room temperature. 6 M HCl(aq) was added until the pH reached 4. After stirring for 3 hours, the formed solid was filtered and washed sequentially with water and isopropanol (3.9 g, 86.4%). The next reaction was carried out without further purification.

[0584] 1 H NMR (DMSO-d6) δ 8.16(1H), 12.77(1H); 13 C NMR (DMSO-d6) δ 117.53, 141.55, 143.45, 154.25, 156,55

[0585] 15.2. Preparation of 2-((3-aminopropyl)amino)-1,9-dihydro-6H-purin-6-one (15b)

[0586] 2-chloro-1,9-dihydro-6H-purin-6-one (15a) (3.9 g, 0.0229 mol) was placed in a round-bottom flask, 1,3-diaminopropane (38 mL, 0.457 mol) was added, and a heating mantle and reflux device were installed to reflux for 18 h. The reaction solution was distilled under reduced pressure, and toluene was added to perform co-distillation. The next reaction was carried out without further purification (4.1 g, yield 84.01%).

[0587] 15.3. Preparation of tert-butyl (3-((6-oxo-6,9-dihydro-1H-purin-2-yl)amino)propyl)carbamate (15c)

[0588] 2-((3-aminopropyl)amino)-1,9-dihydro-6H-purin-6-one (15b) (4.1 g, 0.0197 mol) was dissolved in THF (40 mL) and water (40 mL), Boc2O (8.59 g, 0.0394 mol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was distilled under reduced pressure, and the resulting solid was filtered, dissolved in MeOH, refluxed for 1 hour, cooled to 30–40°C, filtered, dissolved in MeOH again, and refluxed for 1 hour. The reaction solution was distilled under reduced pressure, EA was added, refluxed for 1 hour, and the resulting solid was filtered (5.3 g, yield 87.3%).

[0589] 1 H NMR (DMSO-d6) δ 1.59(9H), 1.65(2H), 2.98(2H), 3.24(2H), 6.31(1H), 6.89(1H), 7.63(1H), 10.48(1H), 12.47(1H); 13 C NMR (DMSO-d6) δ 28.72, 29.79, 37.86, 38.45, 78.00, 116.93, 135.75, 151.97, 152.91, 156.19, 157.35

[0590] 15.4. Preparation of ethyl 2-(2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (15d)

[0591] tert-Butyl (3-((6-oxo-6,9-dihydro-1H-purin-2-yl)amino)propyl)carbamate (15c) (5.3 g, 0.0172 mol) was dissolved in DMF (50 mL). 60% oil-coated NaH(s) (1.71 g, 0.0716 mol) was added under an ice bath, and the mixture was stirred at room temperature for 1 h. Ethyl bromoacetate (2.09 mL, 0.019 mol) was then added under an ice bath. After completion of the reaction, the reaction solution was quenched with NH4Cl(s) (2.39 g, 0.045 mol) / 500 mL aqueous solution, and the resulting solid was filtered and washed sequentially with water and diethylether (5.8 g, yield 85.5%).

[0592] 1 H NMR (DMSO-d6) δ 1.22(3H), 1.39(9H), 1.61(2H), 2.96(2H), 3.24(2H), 4.17(2H), 4.88(2H), 6.44(1H), 6.86(1H), 7.68(1H), 10.59(1H); 13 C NMR (DMSO-d6) δ 14.50, 28.70, 29.72, 37.80, 38.38, 44.24, 61.72, 77.99, 116.48, 138.26, 151.57, 153.13, 156.15, 157.27, 168.43

[0593] 15.5. Preparation of ethyl 2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (15f)

[0594] The title compound (15f) was synthesized (4.3 g, yield 53.9%) in the same manner as in Example 4.6 using ethyl 2-(2-((3-((tert-butoxycarbonyl)amino)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (15d) (5.8 g, 0.0147 mol).

[0595] 1 H NMR (DMSO-d6) δ 1.22(3H), 1.43(18H), 1.64(2H), 1.79(2H), 2.26(2H), 3.10(2H), 3.25(2H), 4.18(3H), 4.88(1H), 6.63(1H), 7.69(1H), 7.95(1H), 10.69(1H); 13 C NMR (DMSO-d6) δ 14.48, 25.22, 29.26, 29.68, 30.41, 36.77, 38.60, 44.26, 61.69, 81.24, 116.43, 138.24, 151.58, 153.19, 157.28, 168.41, 171.19, 171.36

[0596] 15.6. Preparation of 2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (15g)

[0597] The title compound (15g) was synthesized in the same manner as in 1.2 of Example 1 using ethyl 2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (15f) (4.3 g, 0.00793 mol), and the following reaction was carried out without further purification (4.0 g, yield 98.1%).

[0598] 15.7. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycinate (15h)

[0599] The title compound (15h) was synthesized (3.2 g, 47.59%) in the same manner as in 1.3 of Example 1 using 2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (15g) (4.0 g, 0.0078 mol).

[0600] 1 H NMR (DMSO-d6) δ 1.17(3H), 1.41(18H), 1.62(2H), 1.80(2H), 2.26(2H), 3.42(2H), 3.46(2H), 3.64(2H), 3.82(2H), 4.08(2H), 4.19(2H), 4.35(2H), 4.84,5.03(2H), 6.40(1H), 7.28(2H), 7.40(2H), 7.56(1H), 7.67(2H), 7.90(2H), 8.00(1H), 10.57(1H); 13 C NMR (DMSO-d6) δ 14.1, 19.0, 28.2, 29.4, 30.3, 35.1, 37.4, 38.1, 47.0, 49.7, 50.4, 61.0, 67.3, 86.9, 117.1, 120.5, 125.2, 126.2, 126.7, 140.2, 142.6, 143.6, 149.6, 154.1, 157.1, 157.8, 168.4, 169.4, 173.3

[0601] 15.8. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycine (15)

[0602] The title compound (15) was synthesized (2.8 g, 90.4%) in the same manner as in 1.4 of Example 1 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycinate (15h) (3.2 g, 0.0037 mol).

[0603] 1 H NMR (DMSO-d6) δ 1.41(18H), 1.62(2H), 1.80(2H), 2.26(2H), 3.42(2H), 3.46(2H), 3.64(2H), 3.82(2H), 4.19(2H), 4.35(2H), 4.84,5.03(2H), 6.40(1H), 7.28(2H), 7.40(2H), 7.56(1H), 7.67(2H), 7.90(2H), 8.00(1H), 10.57(1H), 13.03(1H); 13 C NMR (DMSO-d6) δ 19.0, 28.2, 29.4, 30.3, 35.1, 37.4, 38.1, 47.0, 49.7, 52.6, 67.3, 86.9, 117.1, 120.5, 125.2, 126.2, 126.7, 140.2, 142.6, 143.6, 149.6, 154.1, 157.1, 157.8, 168.4, 169.4, 171.3,173.3

[0604] [Example 16] Preparation of di-tert-butyl (3-((3-((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)amino)propyl)amino)-3-oxopropyl)phosphonate (Compound 16)

[0605]

[0606] The title compound (16) was synthesized (1.4 g, 43.21%) in the same manner as in Example 5.3 using 2-(2-((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (15 g) (2.1 g, 0.00408 mol).

[0607] 1 H NMR (DMSO-d6) δ 1.41(18H), 1.64(2H), 1.85(2H), 2.26(2H), 3.25(2H), 3.27(2H), 3.87,3.90(2H), 4.27,4.28(2H), 4.32,4.57(2H), 4.96,5.06(2H), 6.57(1H), 7.55,7.56(2H), 7.72(2H), 8.03(1H), 8.25(1H), 8.35(1H), 10.69(1H); 13 C NMR (DMSO-d6) δ 26.29, 29.54, 29.78, 30.41, 38.44, 39.32, 40.37, 42.57, 43.85, 46.48, 47.65, 81.42, 81.50, 116.45, 123.86, 125.42, 128.49, 128.92, 137.06, 138.76, 151.64, 153.17, 157.44, 166.32, 166.87

[0608] [Example 17] Preparation of N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycine (Compound 17)

[0609]

[0610] 17.1. Preparation of 2-((2-aminoethyl)amino)-1,9-dihydro-6H-purin-6-one (17a)

[0611] The title compound (17a) was synthesized in the same manner as in 15.2 of Example 15 using ethylene diamine (39 mL, 0.5863 mol) and 2-chloro-1,9-dihydro-6H-purin-6-one (15a) (5.00 g, 0.0293 mol), and the following reaction was carried out without further purification (5.2 g, yield 91.3%).

[0612] 17.2. Preparation of tert-butyl (2-((6-oxo-6,9-dihydro-1H-purin-2-yl)amino)ethyl)carbamate (17b)

[0613] The title compound (17b) was synthesized (6.3 g, yield 80.1%) in the same manner as in Example 15.3 using 2-((2-aminoethyl)amino)-1,9-dihydro-6H-purin-6-one (17a) (5.2 g, 0.0267 mol).

[0614] 1 H NMR (DMSO-d6) δ 1.40(9H), 3.13(2H), 3.33(2H), 6.43(1H), 6.91(1H), 7.78(1H), 10.63(1H); 13C NMR (DMSO-d6) δ 28.72, 39.2, 39.7, 78.19, 114.49, 137.24, 152.88, 153.77, 156.23, 156.55

[0615] 17.3. Preparation of ethyl 2-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (17c)

[0616] The title compound (17c) was synthesized (6.7 g, yield 82.28%) in the same manner as in 15.4 of Example 15 using tert-butyl (2-((6-oxo-6,9-dihydro-1H-purin-2-yl)amino)ethyl)carbamate (17b) (6.3 g, 0.0214 mol).

[0617] 1 H NMR (DMSO-d6) δ 1.27(3H), 1.42(9H), 3.16(2H), 3.36(2H), 4.23(2H), 4.94(2H), 6.52(1H), 6.93(1H), 7.74(1H), 10.69(1H); 13 C NMR (DMSO-d6) δ 14.48, 28.68, 39.65, 40.78, 44.21, 61.71, 78.15, 116.58, 138.33, 151.52, 153.17, 156.19, 157.33, 168.41

[0618] 17.4. Preparation of ethyl 2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (17e)

[0619] The title compound (17e) was synthesized (5.9 g, yield 63.38%) in the same manner as in Example 4.7 using ethyl 2-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (17c) (6.7 g, 0.0176 mol).

[0620] 1 H NMR (DMSO-d6) δ 1.22(3H), 1.43(18H), 1.79(2H), 2.26(2H), 3.10(2H), 3.25(2H), 4.18(3H), 4.88(1H), 6.63(1H), 7.69(1H), 7.95(1H), 10.69(1H); 13 C NMR (DMSO-d6) δ 14.48, 25.22, 29.68, 30.41, 36.77, 38.60, 44.26, 61.69, 81.24, 116.43, 138.24, 151.58, 153.19, 157.28, 168.41, 171.19, 171.36

[0621] 17.5. Preparation of 2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (17f)

[0622] The title compound (17f) was synthesized in the same manner as in 1.2 of Example 1 using ethyl 2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetate (17e) (5.9 g, 0.0112 mol), and the following reaction was performed without further purification.

[0623] 17.6. Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycinate (17g)

[0624] 2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (17f) (5.0 g, 0.0100 mol) was synthesized using the same method as in Example 1.3 to obtain the title compound (17g) (4.2 g, yield 49.41%).

[0625] 1 H NMR (DMSO-d6) δ 1.17(3H), 1.41(18H), 1.80(2H), 2.26(2H), 3.42(2H), 3.46(2H), 3.64(2H), 3.82(2H), 4.08(2H), 6.40(1H), 7.28(2H), 7.40(2H), 7.56(1H), 7.67(2H), 7.90(2H), 8.00(1H), 10.57(1H); 13 C NMR (DMSO-d6) δ 14.1, 19.0, 28.2, 29.4, 30.3, 36.8, 38.1, 38.3, 47.0, 49.7, 50.4, 61.0, 67.3, 86.9, 117.1, 120.5, 125.2, 126.2, 126.7, 140.2, 142.6, 143.6, 149.6, 154.1, 157.1, 157.8, 168.4, 169.4, 173.3

[0626] 17.7. Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycine (17)

[0627] The title compound (17) was synthesized using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycinate (17g) (4.2 g, 0.00494 mol) in the same manner as in Example 15.8 (3.1 g, yield 76.33%).

[0628] 1 H NMR (DMSO-d6) δ 1.20(18H), 2.05(2H), 2.42(2H), 3.42(2H), 3.46(2H), 3.64(2H), 3.82(2H), 4.46(1H), 4.61(1H), 4.62(2H), 4.70(2H), 6.71(1H), 7.28(2H), 7.38(2H), 7.55(2H), 7.90(2H), 7.97(1H), 8.01(1H), 12.09(1H), 13.03(1H); 13 C NMR (DMSO-d6) δ 19.0, 28.2, 29.4, 30.3, 36.8, 38.1, 38.3, 47.0, 49.7, 52.6, 67.3, 86.9, 117.1 120.5, 125.2, 126.2, 126.7, 140.2, 142.6 143.6, 149.6, 154.1, 157.1, 157.8, 169.4, 173.1, 173.3

[0629] [Example 18] Preparation of di-tert-butyl (3-((2-((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)amino)ethyl)amino)-3-oxopropyl)phosphonate (Compound 18)

[0630]

[0631] 2-(2-((2-(3-(di-tert-butoxyphosphoryl)propanamido)ethyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetic acid (17f) (3.3 g, 0.00659 mol) was synthesized using the same method as in Example 5.3 to obtain the title compound (18) (2.4 g, yield 46.68%).

[0632] 1 H NMR (DMSO-d6) δ 1.41(18H), 1.86(2H), 2.28(2H), 3.26(2H), 3.29(2H), 3.87,3.90(2H), 4.27,4.28(2H), 4.32,4.57(2H), 4.96,5.06(2H), 6.57(1H), 7.55,7.56(2H), 7.72(2H), 8.03(1H), 8.25(1H), 8.35(1H), 10.69(1H); 13 C NMR (DMSO-d6) δ 26.29, 29.77, 30.41, 38.44, 39.32, 40.37, 42.57, 43.85, 46.48, 47.65, 81.42, 81.50, 116.45, 123.86, 125.42, 128.49, 128.92, 137.06, 138.76, 151.64, 153.17, 157.44, 166.32, 166.87

[0633] [Example 19] Preparation of tert-butyl (9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-6-yl)carbamate (Compound 19)

[0634]

[0635] 19.1. Preparation of benzyl 2-(6-chloro-2-(2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (19b)

[0636] Benzyl 2-(2-amino-6-chloro-9H-purin-9-yl)acetate (19a) (5.00 g, 0.0157 mol) was dissolved in CHCl3 (50 mL) and cooled in an ice bath. Pyridine (6.34 mL, 0.0787 mol) was added, and trifluoroacetic anhydride (4.37 mL, 0.0315 mol) was slowly added, followed by stirring at room temperature for 1 h. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, diluted with an excess of EA, and washed once with a saturated aqueous solution of NaHCO3 and once with brine. Anhydrous Na2SO4 was added to the organic layer to remove moisture, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (MeOH / MC) to obtain the title compound (19b) (5.5 g, 0.0133 mol, 84.47% yield).

[0637] 1 H NMR (400 MHz, CDCl3) δ 10.30(s, 1H), 8.05(s, 1H), 7.33(s, 4H), 7.32(s, 1H), 5.20(s, 2H), 4.69(s, 2H); 13C NMR (CDCl3) δ 166.1, 155.0, 152.3, 151.3, 142.0, 136.1, 128.9, 128.2, 127.6, 127.1, 125.0, 115.8, 66.1, 53.0

[0638] 19.2. Preparation of methyl 3-(di-tert-butoxyphosphoryl)propanoate (19c)

[0639] Di-tert-butyl phosphite (1a) (20.00 g, 0.1030 mol) was dissolved in DMF (100 mL), and K-OtBu (12.711 g, 0.1133 mol) was added under an ice bath. The flask inlet was closed with a septum. A balloon was connected to the septum and the mixture was stirred for 60 minutes while monitoring the gas produced. Methyl-3-bromoacetate (18.918 mL, 0.1133 mol) was then added using a syringe. The ice bath was removed and the mixture was stirred for 20 hours. The reaction solution was diluted with an excess of EA and washed three times with a solution diluted to half the volume of a saturated NaHCO3 aqueous solution. After washing the reaction solution once more with brine, anhydrous Na2SO4 was added to remove moisture, and the solution was concentrated under reduced pressure to obtain the title compound (19c) (25.23 g, 87%).

[0640] 1 H NMR (CDCl3, δppm): 1.50(s, 18H), 2.16(m, 2H), 2.49(t, 2H), 3.60(s, 3H); 13 C NMR (CDCl3, δppm): 22.01, 27.65, 29.49, 51.90, 86.94, 173.17

[0641] 19.3. Preparation of di-tert-butyl (3-hydroxypropyl)phosphonate (19d)

[0642] Methyl 3-(di-tert-butoxyphosphoryl)propanoate (19c) (25.23 g, 0.0900 mol) was dissolved in THF (270 mL). 1 M NaOH (270.0 mL, 0.2700 mol) was added, and a small amount of MeOH was added. After reacting for 30 minutes, the reaction solution was washed once with ether. The pH was then raised to 3 with 1 M HCl. Water was added in excess to the reaction solution, and the solution was extracted twice with MC. Anhydrous Na2SO4 was added to remove moisture, and the solvent was removed by distillation under reduced pressure. After THF (anhydrous, 400 mL) was added to dissolve the product, and CDI (38.182 g, 0.2355 mol) was added. The flask inlet was blocked with a septum, and a balloon was connected to monitor the gas produced. When no more gas was generated, an aqueous solution of NaBH4 (4.454 g, 0.1177 mol) dissolved in water (83.6 mL) was slowly added under an ice bath. After stirring for 10 minutes, the reaction solution was concentrated by distillation under reduced pressure. After adding 500 mL of 0.5 N HCl, the mixture was extracted twice with MC. After removing moisture by adding anhydrous Na2SO4, the solvent was removed by concentration under reduced pressure, and the mixture was purified by column chromatography. After purification, the moisture was removed by adding anhydrous Na2SO4, and the mixture was concentrated to obtain the title compound (19d) (16.8 g, 85%).

[0643] 1 H NMR (CDCl3, δppm): 1.49 (s, 18H), 1.77 (m, 4H), 3.65 (t, 2H), 4.13 (s, 1H); 13 C NMR (CDCl3, δppm): 26.29, 26.58, 26.54, 27.75, 30.31, 30.35, 62.18, 62.34, 81.56, 81.65

[0644] 19.4. Preparation of benzyl 2-(6-chloro-2-(N-(3-(di-tert-butoxyphosphoryl)propyl)-2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (19e)

[0645] Benzyl 2-(6-chloro-2-(2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (19b) (5.5 g, 0.0133 mol), PPh3 (4.53 g, 0.01728 mol), and di-tert-butyl (3-hydroxypropyl)phosphonate (19d) (4.025 g, 0.0160 mol) were placed in a round flask, degassed, and anhydrous THF (220 mL) was added. DIAD (3.5 g, 0.0174 mol) was slowly added under nitrogen. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, diluted with an excess of EA, and washed once with a saturated aqueous solution of NaHCO3 and once with brine. Anhydrous Na2SO4 was added to the organic layer to remove moisture, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (MeOH / MC) to obtain the title compound (19e) (6.7 g, 0.0103 mol, 77.78% yield).

[0646] 1 H NMR (400 MHz, CDCl3) δ 10.47(s, 1H), 8.05(s, 1H), 7.33(s, 4H), 7.32(s, 1H), 7.01(s, 1H), 5.20(s, 2H), 4.69(s, 2H), 3.35(t, 2H), 1.76(m, 2H), 1.75(m, 2H), 1.20(d, 18H); 13 C NMR (CDCl3) δ 166.1, 152.7, 152.0, 151.8, 149.8, 142.0, 136.1, 128.9, 127.6, 127.1, 123.5, 86.9, 79.5, 66.1, 53.0, 46.3, 30.5, 29.4, 28.4, 22.7

[0647] 19.5. Preparation of benzyl 2-(6-((tert-butoxycarbonyl)amino)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetate (19f)

[0648] Benzyl 2-(6-chloro-2-(N-(3-(di-tert-butoxyphosphoryl)propyl)-2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (19e) (6.7 g, 0.0103 mol), t-Butyl carbamate (1.82 g, 0.0155 mol), Xantphos (0.479 g, 0.0008 mol), Pd2(dba)3 (0.379 g, 0.0004 mol), and CsCO3 (6.737 g, 0.0207 mol) were placed in a round bottom flask, degassed, and anhydrous THF (135 mL) was added. The mixture was stirred under nitrogen until dissolved and refluxed at 65°C for 20 h. After completion of the reaction, the mixture was cooled to room temperature. After quenching with 10% NH4Cl aqueous solution, the mixture was extracted with EA. The organic layer was washed with water and brine, concentrated under reduced pressure, and purified by column chromatography (MeOH / MC) to obtain the title compound (19f) (2.5 g, 0.0040 mol, 38.22%).

[0649] 1 H NMR (400 MHz, CDCl3) δ 7.64(s, 1H), 7.33(s, 4H), 7.32(s, 1H), 5.22(s, 2H), 4.87(s, 2H), 3.45(t, 2H), 1.97(m, 2H), 1.75(m, 2H), 1.50(d, 18H); 13C NMR (CDCl3) δ 167.20, 159.73, 153.18, 150.11, 149.95, 139.00, 134.78, 128.72, 128.46, 114.83, 81.68, 81.54, 67.74, 43.74, 28.52, 28.17, 23.71

[0650] 19.6. Preparation of 2-(6-((tert-butoxycarbonyl)amino)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetic acid (19g)

[0651] Benzyl 2-(6-((tert-butoxycarbonyl)amino)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetate (19f) (2.5 g, 0.0040 mol) was dissolved in THF (25 mL). LiOH (0.497 g, 0.0119 mol) dissolved in water (12 mL) was slowly added. After completion of the reaction, THF was removed under reduced pressure. After lowering the pH to 3 by adding KHSO4 aqueous solution, extraction was performed twice using MC. After removing moisture from the MC layer with anhydrous Na2SO4 and filtering, the filtrate was concentrated using a rotary evaporator to obtain the title compound (19g) (2.1 g, 0.0039 mol, 97.95%).

[0652] 1 H NMR (400 MHz, CDCl3) δ 12.22(s, 1H), 10.47(s, 1H), 8.05(s, 1H), 7.01(s, 1H), 4.67(s, 2H), 3.35(t, 2H), 1.76(m, 2H), 1.75(m, 2H), 1.49(s, 9H), 1.20(d, 18H); 13C NMR (CDCl3) δ 174.9, 152.7, 152.0, 151.8, 149.8, 142.0, 123.5, 86.9, 79.5, 50.1, 46.3, 30.5, 29.4, 28.4, 22.7

[0653] 19.7. Preparation of tert-butyl (9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-6-yl)carbamate (19)

[0654] 2-(6-((tert-butoxycarbonyl)amino)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetic acid (19 g) (2.1 g, 0.00387 mol) was dissolved in DMF (21 mL). Bts-NH·TFA (0.758 g, 0.00387 mol) was added, followed by EDC·HCl (0.890 g, 0.0046 mol). After stirring for 1 hour, an excess of EA was added to the reaction solution, and the mixture was washed twice with saturated NaHCO3 aqueous solution, once with brine, and once with brine. Anhydrous Na2SO4 was added to the organic layer to remove moisture, filtered, and the filtrate was concentrated. The residue was purified by column chromatography to obtain the title compound (19) as a white solid (2.2 g, 0.0027 mol, 69.16%).

[0655] 1H NMR (400MHz, DMSO-d6) δ 10.47(s, 1H), 8.37(t, 1H), 8.28(t, 1H), 7.81(s, 1H), 7.74(m, 2H), 6.89(t, 1H), 5.12-5.01(d, 2H), 4.57(s, 1H), 4.31-4.28(t, 2H), 4.11(t, 2H), 3.89(t, 1H), 3.32(t, 2H), 1.72(m, 2H), 1.55(t, 2H), 1.48(s, 9H), 1.40(s, 18H); 13 C NMR (CDCl3) δ 166.83, 166.31, 166.01, 164.26, 159.45, 151.70, 150.40, 141.32, 137.10, 128.94, 128.62, 128.51, 125.44, 123.92, 80.90, 80.81, 80.07, 65.40, 49.49, 47.63, 46.75, 44.06, 43.90, 42.41, 42.14, 30.50, 30.46, 28.64, 28.40, 27.20, 23.81, 15.65

[0656] [실시예 20] di-tert-butyl (3-((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-6-(benzyloxy)-9H-purin-2-yl)amino)propyl)phosphonate (화합물 20)의 제조

[0657]

[0658] 20.1. 2-(2-amino-6-(benzyloxy)-9H-purin-9-yl)acetic acid (20a)의 제조

[0659] Finely chopped sodium(s) (2.026 g, 0.088 mol) was added to benzyl alcohol (200 mL) and stirred at room temperature for 1 hour. After 1 hour, benzyl 2-(2-amino-6-chloro-9H-purin-9-yl)acetate (19a) (10.0 g, 0.031 mol) was added and stirred at room temperature for 16 hours. After completion of the reaction, water (100 mL) was added, stirred for 1 hour, and washed twice with diethylether. After separating the aqueous layer, KHSO4 aqueous solution was added to lower the pH to 3, and the resulting white solid was filtered and dried to obtain the title compound (20a) (6.4 g, yield 67.94%).

[0660] 1 H NMR (400MHz, DMSO-d6) δ 4.67(2H), 5.16(2H), 6.33(2H), 7.32(1H), 7.40(2H), 7.48(2H), 8.05(1H), 12.22(1H); 13 C NMR (DMSO-d6) δ 50.1, 51.7, 115.3, 127.1, 127.6, 128.9, 136.7, 142.0, 149.8, 159.2, 160.6, 174.9

[0661] 20.2. Preparation of methyl 2-(2-amino-6-(benzyloxy)-9H-purin-9-yl)acetate (20b)

[0662] 2-(2-amino-6-(benzyloxy)-9H-purin-9-yl)acetic acid (20a) (6.4 g, 0.0214 mol) was slurried with MC (60 mL). MeOH (2.59 mL, 0.154 mol), DMAP (0.1 g, 0.00214 mol), and DCC (5.295 g, 0.0257 mol) were sequentially added and stirred for 1 h. After completion of the reaction, the produced solid was filtered, and the reaction solution was concentrated under reduced pressure and purified by column chromatography (EA / Hexane) to obtain the title compound (20b) (4.7 g, yield 70.1%).

[0663] 1 H NMR (400MHz, DMSO-d6) δ 3.66(3H), 4.69(2H), 5.16(2H), 6.33(2H), 7.32(1H), 7.40(2H), 7.48(2H), 8.05(1H); 13 C NMR (DMSO-d6) δ 51.6, 51.7, 52.7, 115.3, 127.1, 127.6, 128.9, 136.7, 142.0, 149.8, 159.2, 160.6, 166.1

[0664] 20.3. Preparation of methyl 2-(6-(benzyloxy)-2-(2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (20c)

[0665] Methyl 2-(2-amino-6-(benzyloxy)-9H-purin-9-yl)acetate (20b) (4.7 g, 0.0214 mol) was dissolved in CHCl3 (141.0 mL), and an ice bath was installed to lower the temperature. Pyridine (8.457 mL, 0.1069 mol) was injected, followed by slow addition of trifluoroacetic anhydride (8.983 mL, 0.0428 mol), and the mixture was stirred at room temperature for 1 h. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, dissolved in EA, and washed three times with 0.5 N HCl, NaHCO3(aq), and NaCl(aq) respectively. Anhydrous Na2SO4 was added to the organic layer to remove moisture, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (EA / Hexane) to obtain the title compound (20c) (3.2 g, yield 36.56%).

[0666] 1 H NMR (400MHz, DMSO-d6) δ 3.66(3H), 4.69(2H), 5.16(2H), 7.32(1H), 7.40(2H), 7.48(2H), 8.05(1H), 10.30(1H); 13 C NMR (DMSO-d6) δ 51.6, 52.7, 115.3, 127.1 127.6, 128.9, 136.7, 142.0, 149.8, 152.3, 155.0, 160.6. 166.1

[0667] 20.4. Preparation of methyl 2-(6-(benzyloxy)-2-(N-(3-(di-tert-butoxyphosphoryl)propyl)-2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (20d)

[0668] Methyl 2-(6-(benzyloxy)-2-(2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (20c) (3.2 g, 0.0078 mol) was added to a round bottom flask, PPh3 (2.66 g, 0.01016 mol), and di-tert-butyl (3-hydroxypropyl)phosphonate (19d) (2.37 g, 0.0094 mol), followed by degassing. After injecting anhydrous THF (80 mL) under nitrogen, DIAD (2.00 mL, 0.0102 mol) was slowly added, and the mixture was stirred for 40 min. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, diluted with excess EA, and washed twice with saturated aqueous NaHCO3 solution and once with brine. Anhydrous Na2SO4 was added to the organic layer to remove moisture, filtered, and concentrated. The residue was purified by column chromatography (EA / Hexane) to obtain the title compound (20d) (4.4 g, yield 87.0%).

[0669] 1 H NMR (400MHz, DMSO-d6) δ 1.44(18H), 1.64(2H), 1.86(2H), 3.77(3H), 4.05(2H), 4.97(2H), 5.63(2H), 7.34,7.49(5H), 8.08(1H); 13 C NMR (DMSO-d6) δ 22.00, 26.73, 30.30, 44.13, 49.35, 52.86, 69.00, 81,73, 111.98, 114.83, 117.97, 119.41, 120.55, 128.41, 135.50, 142.11, 152.81, 152.89, 158.97, 160.87, 167.04

[0670] 20.5. Preparation of 2-(6-(benzyloxy)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetic acid (20e)

[0671] Methyl 2-(6-(benzyloxy)-2-(N-(3-(di-tert-butoxyphosphoryl)propyl)-2,2,2-trifluoroacetamido)-9H-purin-9-yl)acetate (20d) (4.4 g, 0.00684 mol) was dissolved in THF (17.25 mL). LiOH·H2O(s) (0.8606 g, 0.0205 mol) dissolved in water (17.25 mL) was added and stirred for 20 minutes. After completion of the reaction, THF was concentrated under reduced pressure, and an ice bath was installed to lower the pH to 3. Then, it was extracted twice using MC. After that, the moisture was removed with anhydrous Na2SO4 and the solvent was removed using a rotary evaporator to obtain the title compound (20e) (3.26 g, yield 89.37%).

[0672] 1 H NMR (400MHz, DMSO-d6) δ 1.21(18H), 1.76(2H), 3.35(2H), 4.67(2H), 5.16(2H), 7.01(1H), 7.32(1H), 7.40(2H), 7.48(2H), 8.05(1H), 12.22(1H); 13 C NMR (DMSO-d6) δ 22.7, 29.4, 30.5, 46.3, 50.1, 51.7, 86.9, 115.3, 127.1, 127.6, 128.9, 136.7, 142.0, 149.8, 152.0, 160.6, 174.9

[0673] 20.6. Preparation of di-tert-butyl (3-((9-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-6-(benzyloxy)-9H-purin-2-yl)amino)propyl)phosphonate (20)

[0674] 2-(6-(benzyloxy)-2-((3-(di-tert-butoxyphosphoryl)propyl)amino)-9H-purin-9-yl)acetic acid (20e) (3.26 g, 0.00611 mol) was used to synthesize the title compound (20) as a white solid (3.28 g, yield 66.04%) in the same manner as in Example 5.3.

[0675] 1 H NMR (400MHz, DMSO-d6) δ 1.41(18H), 1.64(2H), 1.76(2H), 3.37(2H), 3.90,4,12,4.30(4H), 4.32,4.58(2H), 5.03,5.14(2H), 5.56(2H), 7.05(1H), 7.42,7.51(5H), 7.73(2H), 7.77(1H), 8.27(1H), 8.31(1H); 13 C NMR (DMSO-d6) δ 23.82 27.19, 28.63, 30.46, 40.01, 42.09, 42.41, 44.09, 46.47, 47.63, 49.49, 67.39, 113.59, 123.89, 125.43, 128.49, 128.78, 137.10, 137.29, 141.08, 151.71, 155.30, 159.24, 160.30, 166.31

[0676] [Example 21] Preparation of di-tert-butyl (3-(1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-4-methoxy-2-oxo-1,2-dihydropyrimidin-5-yl)propyl)phosphonate (Compound 21)

[0677]

[0678] 21.1. Preparation of ethyl 2-(5-iodo-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21a)

[0679] Ethyl 2-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (4c) (2.00 g, 0.00617 mol) was dissolved in DMF, KOtBU (0.831 g, 0.00741 mol) was added, and the mixture was stirred for 30 minutes under an ice bath. Dimethylsulfate (0.644 mL, 0.00679 mol) was added, and the mixture was reacted under an ice bath for 40 minutes. The reaction solution was diluted with an excess of EA, and washed twice with a solution diluted to half the volume of saturated NaHCO3 aqueous solution. Finally, the reaction solution was washed once with brine, dried with anhydrous Na2SO4, and the solvent was removed using a rotary evaporator. The residue was then purified by column chromatography to obtain the title compound (21a) (1.82 g, 87%).

[0680] 1 H NMR (CDCl3, δ ppm): 1.31(t, 3H), 3.42(s, 3H), 4.25(q, 2H), 4.51(s, 2H) 7.68(s, 1H); 13 C NMR (CDCl3, δ ppm): 14.10, 29.52, 49.96, 62.34, 67.92, 146.87, 151.16, 160.24, 167.26.

[0681] 21.2. Preparation of ethyl 2-(5-(3-hydroxyprop-1-yn-1-yl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21b)

[0682] Ethyl 2-(5-iodo-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21a) (5.22 g, 0.0154 mol), CuI (0.8821 g, 0.0046 mol), and Pd(PPh3)4 (1.784 g, 0.0015 mol) were mixed and degassed. DMF (70 mL), TEA (6.456 mL, 0.0463 mol), and propargyl alcohol (2.696 mL, 0.0463 mol) were added, and the mixture was reacted at room temperature for 30 minutes. The solvent was removed by distillation under reduced pressure. MeOH was added to precipitate the Pd catalyst, filtered, and the filtrate was concentrated by distillation under reduced pressure again. The concentrate was purified by column chromatography to obtain the title compound (21b) (2.66 g, 65%).

[0683] 1 H NMR (CDCl3, δ ppm): 1.31(t, 3H), 2.80(t, 1H), 3.37(s, 3H), 4.25(q, 2H), 4.45(d, 2H), 4.50(s, 2H) 7.45(s, 1H); 13 C NMR (CDCl3, δ ppm): 14.09, 28.57, 50.14, 51.41, 62.42, 78.41, 92.20, 99.08, 145.31, 150.58, 161.82, 167.14.

[0684] 21.3. Preparation of ethyl 2-(5-(3-hydroxypropyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21c)

[0685] Ethyl 2-(5-(3-hydroxyprop-1-yn-1-yl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21b) (2.66 g, 0.0100 mol) was dissolved in anhydrous MeOH (100 mL), and Pd / C (0.160 g) was slowly added while stirring the reaction solution. After degassing, hydrogen gas was added using a hydrogen balloon. After 17 hours of reaction, Pd / C was removed using celite. The title compound (21c) was obtained (2.4 g, 89%) by distillation under reduced pressure.

[0686] 1 H NMR (CDCl3, δ ppm): 1.31(t, 3H), 1.77(quint, 2H), 2.48(t, 2H), 3.36(s, 3H), 3.62(t, 2H), 4.25(q, 2H), 4.47(s, 2H), 7.01(s, 1H); 13 C NMR (CDCl3, δ ppm): 14.10, 18.38, 33.64, 51.88, 54.73, 61.06, 62.87, 109.07, 135.35, 155.39, 167.52, 169.83.

[0687] 21.4. Preparation of ethyl 2-(5-(3-bromopropyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21d)

[0688] Distilled MeCN (30 mL) and PBr3 (0.918 mL, 0.00977 mol) were added to ethyl 2-(5-(3-hydroxypropyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21c) (2.4 g, 0.0089 mol). The reaction solution was refluxed for 4 h. After cooling in an ice-bath, the pH was adjusted to neutral by adding saturated aqueous NaHCO3 solution. After adding excess water, extraction was performed twice with MC. Anhydrous Na2SO4 was added to remove moisture, and the residue was concentrated by distillation under reduced pressure. The residue was purified by column chromatography to obtain the title compound (21d) (1.26 g, 43%).

[0689] 1 H NMR (CDCl3, δ ppm): 1.18(t, 3H), 1.98(quint, 2H), 2.40(t, 2H), 3.22(s, 3H), 4.13(q, 2H), 4.39(s, 2H), 7.01(s, 1H); 13 C NMR (CDCl3, δ ppm): 14.09, 20.78, 30.33, 33.82, 51.89, 54.71, 61.04, 109.08, 135.36, 155.41, 167.51, 169.82.

[0690] 21.5. Preparation of ethyl 2-(5-(3-(di-tert-butoxyphosphoryl)propyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21e)

[0691] Di-tert-butyl phosphite (1a) (0.995 mL, 0.00492 mol) was dissolved in DMF (10 mL), KOtBU (0.552 g, 0.00492 mol) was added, and the mixture was stirred for 30 minutes in an ice-bath. Ethyl 2-(5-(3-bromopropyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21d) (1.26 g, 0.0038 mol) was dissolved in DMF (12 mL) and added to the reaction solution. After 3 hours of reaction, the reaction solution was diluted with an excess of EA, and washed three times with a solution diluted to half the volume of saturated NaHCO3 aqueous solution. The reaction solution was washed once with brine, and anhydrous Na2SO4 was added to remove water. Afterwards, the solvent was removed by distillation under reduced pressure and purified by column chromatography to obtain the title compound (21e) (1.15 g, 52%).

[0692] 1 H NMR (CDCl3, δ ppm): 1.30(t, 3H), 1.49(s, 18H), 1.65(m, 2H), 1.82(m, 2H), 2.46(t, 2H), 3.35(s, 3H), 4.24(q, 2H), 4.45(s, 2H), 7.04(s, 1H); 13 C NMR (CDCl3, δ ppm): 14.12, 22.00, 22.05, 27.53, 27.69, 28.02, 28.49, 29.94, 30.43, 30.47, 49.82, 62.06, 81.50, 81.58, 113.22, 138.93, 151.55, 163.40, 167.64.

[0693] 21.6. Preparation of 2-(5-(3-(di-tert-butoxyphosphoryl)propyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetic acid (21f)

[0694] Ethyl 2-(5-(3-(di-tert-butoxyphosphoryl)propyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetate (21e) (0.172 g, 0.00039 mol) was dissolved in THF (2 mL). 1 M NaOH (1.93 mL, 0.00193 mol) was added, and a small amount of MeOH was added. After stirring for 10 minutes, the pH was adjusted to 3 with 1 M HCl. Water was added in excess, and the mixture was extracted twice with MC. Anhydrous Na2SO4 was added to remove the water, and the mixture was distilled under reduced pressure to obtain the title compound (21f). The next reaction was carried out without further purification.

[0695] 21.7. Preparation of di-tert-butyl (3-(1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-4-methoxy-2-oxo-1,2-dihydropymidin-5-yl)propyl)phosphonate (21)

[0696] The title compound (21) was obtained (0.186 g, 69%) by synthesizing it in the same manner as in Example 5.3 using 2-(5-(3-(di-tert-butoxyphosphoryl)propyl)-4-methoxy-2-oxopyrimidin-1(2H)-yl)acetic acid (21f) (0.162 g, 0.000386 mol).

[0697] 1 H NMR (CDCl3, δ ppm): 1.47(s, 18H), 1.63(m, 2H), 1.81(m, 2H), 2.44(t, 2H), 3.30(d, 3H), 4.02(s, 2H), 4.23(s, 1H), 4.33(s, 2H), 4.37(s, 1H), 4.48(s, 1H), 4.58(s, 1H), 7.05(d, 1H), 7.63(quint, 2H), 8.01(d, 1H), 8.19(d, 1H); 13C NMR (CDCl3, δ ppm): 21.93, 21,98, 27.55, 27.71, 28.02, 28.52, 29.96, 30.43, 30.47, 40.32, 43.00, 46.08, 47.43, 48.90, 49.18, 49.33, 81.47, 81.56, 113.16, 122.30, 125.55, 127.80, 128.42, 137.13, 139.27, 151.70, 151.77, 163.31, 165.25.

[0698] [Example 22] Preparation of benzyl (1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (Compound 22)

[0699]

[0700] 22.1. Preparation of ethyl 2-(4-amino-5-(3-hydroxyprop-1-yn-1-yl)-2-oxopyrimidin-1(2H)-yl)acetate (22b)

[0701] Ethyl 2-(4-amino-5-iodo-2-oxopyrimidin-1(2H)-yl)acetate (22a) (3.74 g, 0.01159 mol), CuI (0.221 g, 0.00116 mol), and Pd(PPh3)4 (0.669 g, 0.00058 mol) were mixed and degassed. Then, DMF (50 mL), TEA (3.23 mL, 0.0232 mol), and propargyl alcohol (2.696 mL, 0.0463 mol) were added. After stirring at room temperature for 20 h, the solvent was removed by concentration under reduced pressure. Afterwards, MeOH was added to solidify, and the resulting solid was washed several times with MC to obtain the title compound (22b) (2.14 g, 74%).

[0702] 1 H NMR (DMSO-d6, δ ppm): 1.22(t, 3H), 4.14(q, 2H), 4.29(d, 2H), 4.49(s, 2H), 5.23(t, 1H), 6.89(s, 1H), 7.78(s, 1H), 7.98(s, 1H); 13 C NMR (DMSO-d6, δ ppm): 14.51, 50.19, 50.48, 61.46, 75.88, 89.66, 95.57, 149.92, 154.54, 165.48, 168.83.

[0703] 22.2. Preparation of ethyl 2-(4-amino-5-(3-hydroxypropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22c)

[0704] Ethyl 2-(4-amino-5-(3-hydroxyprop-1-yn-1-yl)-2-oxopyrimidin-1(2H)-yl)acetate (22b) (1.33 g, 0.00529 mol) was dissolved in MeOH (150 mL). Pd / C (0.100 g) was slowly added while stirring the reaction solution. After degassing, hydrogen gas was added using a hydrogen balloon. After stirring for 19 hours, Pd / C was removed using celite. The title compound (22c) was obtained (1.04 g, 91%) by distillation under reduced pressure.

[0705] 1 H NMR (DMSO-d6, δ ppm): 1.21(t, 3H), 1.60(quint, 2H), 2.28(t, 2H), 3.44(t, 2H), 4.14(q, 2H), 4.45(s, 2H), 4.55(m, 1H), 6.90(s, 1H), 7.39(s, 1H), 7.43(s, 1H) 13C NMR (DMSO-d6, δ ppm): 14.50, 23.26, 31.59, 50.27, 60.27, 61.26, 105.34, 144.15, 156.08, 166.01, 169.27.

[0706] 22.3. Preparation of ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-hydroxypropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22d)

[0707] DMAP (1.737 g, 0.01422 mol) was dissolved in DMF, cooled to below -15℃ using Ice / NaCl, and Cbz-Cl (benzyl chloroformate) (2.024 mL, 0.01422 mol) was added. After stirring at -15℃ for 15 minutes, ethyl 2-(4-amino-5-(3-hydroxypropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22c) (0.726 g, 0.002844 mol) was added to the reaction solution. After stirring at room temperature for 16 hours, the solution was diluted with an excess of EA, and washed three times with a solution diluted by half with a saturated NaHCO3 aqueous solution. After washing the reaction solution once with brine, the water was removed with anhydrous Na2SO4, and the solvent was removed using a rotary evaporator. The residue was then purified by column chromatography to obtain the title compound (22d) (0.550 g, 69%).

[0708] 1 H NMR (CDCl3, δ ppm): 1.30(t, 3H), 1.73(quint, 2H), 2.54(t, 2H), 3.56(t, 2H), 3.68(s, 1H), 4.24(q, 2H), 4.46(s, 2H), 5.20(s, 2H) 7.08(s, 1H), 7.35(m, 5H), 12.37(s, 1H); 13C NMR (CDCl3, δ ppm): 14.10, 22.51, 32.40, 49.22, 59.91, 62.39, 67.68, 114.49, 128.14, 128.16, 128.51, 136.04, 142.43, 148.06, 161.73, 163.46, 167.11.

[0709] 22.4. Preparation of ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-bromopropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22e)

[0710] Ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-hydroxypropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22d) (0.550 g, 0.0020 mol) was dissolved in distilled MeCN (10 mL) and PBr3 (0.249 mL, 0.0026 mol). The reaction solution was refluxed for 5 h. After cooling in an ice-bath, the pH was adjusted to neutral by adding saturated aqueous NaHCO3 solution. Water was added in excess to the reaction solution, and the mixture was extracted twice with MC. Anhydrous Na2SO4 was added to remove moisture, and the residue was purified by evaporation under reduced pressure and column chromatography to obtain the title compound (22e) (0.418 g, 45%).

[0711] 1 H NMR (CDCl3, δ ppm): 1.30(t, 3H), 1.83(quint, 2H), 2.41(t, 2H), 3.51(t, 2H), 4.15(q, 2H), 4.28(s, 2H), 5.02(s, 2H) 7.07(s, 1H), 7.34(m, 5H); 13C NMR (CDCl3, δ ppm): 14.10, 21.59, 30.03, 33.75, 51.82, 61.08, 66.47, 110.31, 128.13, 128.17, 128.50, 136.03, 142.43, 148.05, 161.74, 163.45, 167.10.

[0712] 22.5. Preparation of ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (22f)

[0713] Di-tert-butyl phosphite (1a) (0.243 mL, 0.0012 mol) was dissolved in DMF (3 mL), and KOtBU (0.135 g, 0.0012 mol) was added and stirred for 30 minutes in an ice-bath. Ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-bromopropyl)-2-oxopyrimidin-1(2H)-yl)acetate (22e) (0.418 g, 0.0009 mol) was dissolved in DMF (5 mL) and added to the reaction solution. After stirring for 5 hours, the reaction solution was diluted with an excess of EA and washed three times with a half-diluted solution of saturated NaHCO3 aqueous solution. The reaction solution was washed once with brine, and anhydrous Na2SO4 was added to remove water. After distillation under reduced pressure, the title compound (22f) was obtained by purification by column chromatography (0.291 g, 43%).

[0714] 1 H NMR (CDCl3, δ ppm): 1.21(t, 3H), 1.51(s, 18H), 1.64(m, 2H), 1.83(m, 2H), 2.41(t, 2H), 4.13(q, 2H), 4.28(s, 2H), 5.03(s, 2H) 7.06(s, 1H), 7.33(m, 5H); 13C NMR (CDCl3, δ ppm): 13.31, 14.12, 24.48, 29.43, 33.19, 51.76, 61.04, 66.50, 86.88, 110.34, 127.10, 127.64, 128.87, 159.03, 136.09, 153.71, 159.02, 167.46, 169.17

[0715] 22.6. Preparation of 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (22g)

[0716] Ethyl 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxopyrimidin-1(2H)-yl)acetate (22f) (0.582 g, 0.001 mol) was dissolved in THF (10 mL), 1 M NaOH (5.14 mL, 0.00514 mol) was added, and a small amount of MeOH was added. After stirring for 10 minutes, the pH was adjusted to 3 with 1 M HCl. After adding anhydrous Na2SO4, the reaction solution was extracted twice with MC. The title compound (22 g) was obtained by removing the water and concentrating under reduced pressure. The next reaction was carried out without further purification.

[0717] 22.7. Preparation of benzyl (1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (22)

[0718] The title compound (22) was obtained (0.463 g, 61%) by synthesizing it in the same manner as in Example 5.3 using 2-(4-(((benzyloxy)carbonyl)amino)-5-(3-(di-tert-butoxyphosphoryl)propyl)-2-oxopyrimidin-1(2H)-yl)acetic acid (22g) (0.5 g, 0.00093 mol).

[0719] 1 H NMR (CDCl3, δ ppm): 1.50(s, 18H), 1.57(m, 2H), 1.75(m, 2H), 2.41(t, 2H), 4.13(s, 2H), 4.28(s, 1H), 4.35(s, 2H), 4.39(s, 1H), 4.48(s, 1H), 4.58(s, 1H), 5.03(s, 2H) 7.06(d, 1H), 7.33(m, 5H) 7.62(quint, 2H), 8.02(d, 1H), 8.21(d, 1H); 13 C NMR (CDCl3, δ ppm): 13.31, 24.48, 29.46, 33.19, 42.62, 48.04, 50.61, 53.47, 66.55, 86.91, 86.94, 110.33, 121.58, 124.52, 125.27, 127.10, 127.62, 128.88, 135.11, 135.26, 153.44, 153.71, 159.05, 164.56, 166.21, 169.16

[0720] <Manufacturing Example 1> Preparation of Fmoc monomer

[0721] The following Fmoc-A-OH (compound 100), Fmoc-T-OH (compound 101), Fmoc-G-OH (compound 102), and Fmoc-C-OH (compound 103) monomers were prepared using the same method as in U.S. Patent No. 6,133,444.

[0722]

[0723] <Manufacturing Example 2> Preparation of Bts-cycle monomer

[0724] The following Bts-A-cycle (compound 104), Bts-T-cycle (compound 105), Bts-G-cycle (compound 106), and Bts-C-cycle (compound 107) monomers were prepared using the same method as in Korean Patent No. 10-0464261.

[0725]

[0726] <Manufacturing Example 3> Preparation of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)lysine (Compound 108; bP)

[0727] The following N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(3-(di-tert-butoxyphosphoryl)-2-((di-tert-butoxyphosphoryl)methyl)propanoyl)lysine (compound 108; bP) was prepared by the method of Nucleic Acids Research, 2008, 36(13), 4424-4432.

[0728]

[0729] [Example 23] N-(2-(2-(N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetamido)a cetamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (23) Preparation

[0730]

[0731] Preparation of N-(2-aminoethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (13-1)

[0732] N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (13) (10.0 g, 0.00867 mol) was dissolved in 20% Piperidine / MC (50 mL), stirred sufficiently at 0°C to lower the temperature, and then DBU (1,8-diazabicyclo[5.4.0]-7-undecane) (2.6 mL, 0.01752 mol) was added. After stirring for 6 minutes, the mixture was precipitated using an excess of ether, and the precipitate was filtered. The obtained solid was dissolved in AcOH (50 mL) and then subjected to the excess ether precipitation process three times to obtain N-(2-aminoethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (13-1). The next reaction was carried out without further purification.

[0733] N-(2-(2-(N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetamido)a cetamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (23) Preparation

[0734] N-(2-aminoethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (13-1) was placed in a round-bottom flask together with 1-(2-(4-(benzo[d]thiazol-2-ylsulfonyl)-3-oxopiperazin-1-yl)-2-oxoethyl)-5-methylpyrimidine-2,4(1H,3H)-dione (105) (4.5 g, 0.00979 mol) and dissolved in DMF (90 mL). DIEA (3.4 mL, 0.0196 mol) was added at 0°C and stirred for 18 hours. After completion of the reaction, 0.5 M HCl was used for precipitation and the precipitate was filtered. The obtained solid was dissolved in DMF, precipitated using an excess of EA, the precipitate was filtered, and dried using ether to obtain N-(2-(2-(N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetamido)ethyl)-N-(2-(2,6-bis((3-(3-(di-tert-butoxyphosphoryl)propanamido)propyl)amino)-9H-purin-9-yl)acetyl)glycine (23) (9.0 g, 0.00650 mol, 75% yield).

[0735] <Manufacturing Example 4> Preparation of N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycine (Compound 109)

[0736]

[0737] Preparation of ethyl N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetyl)glycinate (109b)

[0738] 2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (109a) was synthesized in the same manner as in Example 4.8 using ethyl 2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (4e) (2.26 g, 0.0064 mol). Then, the title compound (109b) was synthesized in the same manner as in Example 1.3 using Fmoc-aeg-Oet (1e) (2.58 g, 0.0064 mol) (2.8 g, 65.0 %).

[0739] 1 H NMR (DMSO-d6) δ 11.32(s, 1H), 7.90(d, 2H), 7.55(d, 2H), 7.38(t, 2H), 7.28(t, 2H), 7.19(s, 1H), 6.76(s, 1H), 4.70(d, 2H), 4.63(s, 2H), 4.46(t, 1H), 4.28(s, 2H), 4.19(q, 2H), 3.46(t, 2H), 3.42(t, 2H), 3.18(t, 2H), 2.41(t, 2H), 1.69(quint, 2H), 1.42(t, 9H), 1.21(s, 3H); 13C NMR (DMSO-d6) δ 168.4, 166.3, 164.5, 157.8, 155.9, 150.8, 143.6, 142.6, 136.1, 126.7, 1266.2, 125.2, 120.5, 113.1, 79.5, 67.3, 61.0, 53.6, 50.4, 49.7, 47.0, 40.2, 38.1, 28.4, 27.0, 24.7, 14.1

[0740] Preparation of N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetyl)glycine (109)

[0741] The title compound (109) was synthesized (1.8 g, yield 75.11%) in the same manner as in Example 1.4 using ethyl N-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-(5-(3-((tert-butoxycarbonyl)amino)propyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (109b) (2.5 g, 0.00369 mol).

[0742] 1H NMR (DMSO-d6) δ 9.42(s, 1H), 8.11(s, 1H), 7.91-7.77(m, 2H), 7.53-7.42(m, 2H), 7.42-7.37(m, 2H), 7.34-7.30(m, 2H), 7.19(s, 1H), 5.98(s, 1H), 4.73-4.67(m, 2H), 4.46(s, 1H), 4.36(s, 1H), 4.23(s, 1H), 4.13(s, 1H), 4.01(s, 1H), 3.49(s, 1H), 3.29(s, 1H), 3.20(s, 1H), 3.10(s, 1H), 3.04(s, 1H), 2.97(s, 1H), 2.22-2.17(m, 2H), 1.74-1.65(m, 2H), 1.49-1.45(m, 9H); 13 C NMR (DMSO-d6) δ 171.7, 169.3, 164.2, 158.3, 157.4, 152.0, 145.01-144.61, 140.9, 139.6-139.2, 128.8-128.4, 127.5-127.3, 124.4-124.0, 122.9-122.7, 106.2, 80.6, 66.5, 49.2, 48.0, 47.24, 46.7, 41.2, 38.6, 28.5-28.3, 26.9, 25.7

[0743] <제조예 5> N-(2-(2-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine) (화합물 110)의 제조

[0744]

[0745] Preparation of NH2-A(Bhoc)-OH (N-(2-aminoethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine)

[0746] N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)glycine (10.0 g, 0.0143 mol) was dissolved in DMF (49.2 mL) and stirred at 0°C. 4-methoxybenzenethiol (8.6 mL, 0.0701 mol) was added, followed by dropwise addition of N,N-diisopropylethylamine (12.2 mL, 0.3592 mol). After stirring for 15 min, the mixture was precipitated using excess ether and dried to obtain NH2-A(Bhoc)-OH as a solid (quantitative yield).

[0747] N-(2-(2-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine) Preparation of (Compound 110)

[0748] Bts-A(Bhoc)-cycle (104) (49.2 g, 0.07209 mol) and NH2-A(Bhoc)-OH (36.3 g, 0.07209 mol) were placed in a round-bottomed flask and dissolved in DMF (492 mL). After stirring at 0°C, DIEA (25.12 mL, 0.1442 mol) was added and stirred for 18 hours. After completion of the reaction, the residue was precipitated using an excess of EA and filtered. The filtered solid was dissolved in MC and THF and washed with 0.5 M HCl. The organic layer was completely concentrated, dissolved in MC, precipitated using an excess of ether, and filtered to obtain the title compound (110).

[0749] <Manufacturing Examples 6 to 10> Dimer block synthesis

[0750] 제조예 5와 동일한 방법으로 N-(2-(2-(N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetamido)ethyl)-N-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycine (111), N-(2-(2-(2-(4-(((benzhydryloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(4-(((benzhydryloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)acetyl)glycine (112), N-(2-(2-(2-(2-(((benzhydryloxy)carbonyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(2-(((benzhydryloxy)carbonyl)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)acetyl)glycine (113), N-(2-(2-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycine (114),N-(2-(2-(2-(4-(((benzhydryloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycine (115)을 합성하였다.,

[0751]

[0752] <제조예 11> 17-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-3,9-bis(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)-15-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-7,13,16-trioxo-3,6,9,12,15-pentaazaheptadecanoic acid (화합물 116)의 제조

[0753]

[0754] N-(2-(2-(N-(2-aminoethyl)-2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetamido)acetamido)ethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine (116a)의 제조

[0755] N-(2-(2-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-N-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)acetamido)acetamido)ethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine) (110) (50.0 g, 0.04215 mol) was placed in a round-bottom flask and dissolved in DMF (246 mL). After stirring at 0°C, 4-methoxybenzenethiol (43.05 mL) and DIEA (60.90 mL) were added and stirred for 1 hour. After completion of the reaction, the residue was precipitated using an excess of ether and filtered to obtain the title compound (116a).

[0756] 17-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)-3,9-bis(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-puri n-9-yl)acetyl)-15-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-7,13,16-trioxo-3,6,9,12,15-pentaazaheptadecanoic Preparation of acid (116)

[0757] Bts-A(Bhoc)-cycle (104) (31.1 g, 0.04550 mol) and N-(2-(2-(N-(2-aminoethyl)-2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetamido)acetamido)ethyl)-N-(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)glycine (116a) (45.0 g, 0.04550 mol) were placed in a round-bottom flask and dissolved in DMF (450 mL). After stirring at 0°C, DIEA (15.85 mL, 0.0910 mol) was added and stirred for 18 h. After completion of the reaction, precipitation was performed using 0.5 M HCl and filtered. The filtered solid was dissolved in DMF, precipitated using an excess of EA, filtered, washed with ether, and dried to obtain the title compound (116).

[0758] <Manufacturing Examples 12 to 13> Trimeric block synthesis

[0759] 3,9-bis(2-(6-(((benzhydryloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)-15-(2-(benzo[d]thiazole-2-sulfonamido)ethyl)-17-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7,13,16-trioxo-3,6,9,12,15-pentaazaheptadecanoic acid (117) using the same method as in Manufacturing Example 11. 17-(4-(((benzhydryloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-3-(2-(4-(((benzhydryloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)acetyl)-15-(2-(be nzo[d]thiazole-2-sulfonamido)ethyl)-9-(2-(5-methyl-2,4-dioxo-3,4-dihydropymidin-1(2H)-yl)acetyl)-7,13,16-trioxo-3,6,9,12,15-pentaazaheptadecanoic acid (118) was synthesized.

[0760]

[0761] [Example 20] Solid-phase oligomer synthesis

[0762] Using the monomers, dimer blocks, and trimer blocks synthesized in Examples 1 to 23 and Manufacturing Examples 1 to 13, PNA oligomers of SEQ 1, SEQ 2, and SEQ 5 to SEQ 19 in Table 1 below were synthesized in a solid phase by the method described in registered patents US6133444 and KR10-0464261.

[0763] Specifically, PNA oligomers are synthesized through the following steps.

[0764] ① Swelling: Add 100 mg of a suitable solid support such as Rink amide MBHA or rink amide resin to a 5 mL reactor (Libra tube) and swell for 30 minutes using a suitable solvent such as MC.

[0765] ② Washing: Filter the solution in the reactor and wash the resin with DMF.

[0766] ③ Coupling

[0767] - Bts monomer: Add 0.5-1 mL (0.3 M) of DMF to 10-20 eq. of Bts-cycle-monomer and completely dissolve. Place in a reactor, add 10-20 eq. of DIEA, and shake at 45°C for 30-60 minutes.

[0768] - Fmoc monomer: Add 0.5-1 mL (0.3 M) of DMF to 10-20 eq. of Fmoc monomer and 10-20 eq. of HBTU to completely dissolve and pre-activate. Place in a reactor, add 10-20 eq. of DIEA, and shake at room temperature for 30-60 minutes.

[0769] - Bts block: Add 0.5-1 mL (0.3 M) of DMF to 10-20 eq. of Bts block and 10-20 eq. of HBTU to completely dissolve and pre-activate. Place in a reactor, add 10-20 eq. of DIEA, and shake at room temperature for 30-60 minutes.

[0770] - Fmoc block: Add 0.5-1 mL (0.3 M) of DMF to 10-20 eq. of Fmoc block and 10-20 eq. of HBTU to completely dissolve and pre-activate. Place in a reactor, add 10-20 eq. of DIEA, and shake at room temperature for 30-60 minutes.

[0771] ④ Capping: DMF: acetic anhydride: lutidine=89:5:6 Add 1~2mL of capping solution and shake for 5 minutes.

[0772] ⑤ Washing: Filter the solution in the reactor and wash the resin with DMF.

[0773] ⑥ Deacetylation: When using Bts monomer or Bts block, add 1~2 mL of 10% piperidine / DMF and shake for 5 minutes to remove the acetyl group of the over-reacted sulfonamide.

[0774] ⑦ Washing: Filter the solution in the reactor and wash the resin with DMF.

[0775] ⑧ Deprotection

[0776] - Bts: Add 0.5~1 mL of 2M 4-methoxybenzenethiol / DMF and 0.5~1 mL of 2M DIEA / DMF and shake 3 times for 5 minutes each.

[0777] - Fmoc: Add 1~2mL of 20% piperidine / DMF and shake for 5 minutes.

[0778] ⑨ Return to ② and repeat steps 3-8 using appropriate monomers and blocks. SEQ1 is synthesized in the order of (5), (5), (5), T, A, A, A, C, T.

[0779] ⑩ Quencher conjugation: Dissolve DMF completely in an appropriate quencher (with HBTU, if necessary) and place in a reactor. Add DIEA and shake at room temperature for 1 hour.

[0780] ⑪ Talesin: A cocktail solution containing 87.5% Trifluoroacetic acid (TFA), 2.5% 2,2'-(Ethylenedioxy)diethanethiol (DODT), 2.5% water, 2.5% Thioanisole, 2.5% Triisopropylsilane (TIPS), and 2.5% m-cresol was treated for 0.5 h to simultaneously remove the protecting group of the amine of the nucleic acid base and perform the talesin reaction. The resin was filtered and washed with TFA. After dispersing in diethyl ether and centrifuging, the supernatant was removed by decanting. The separated solid was dispersed in diethyl ether, washed by centrifugation and supernatant removal, and dried to obtain a solid PNA oligomer.

[0781] ⑫ Fluorophore conjugation: Add DMF: DMSO=1:2 solution to completely dissolve and add the appropriate fluorescent substance. Add DIEA and shake at room temperature for 1 hour.

[0782] ⑬ Purification: Purify at 60℃ using a C18 column using an appropriate gradient from 0.1% TFA water and 0.1% TFA ACN buffer.

[0783] Additionally, PNA oligomers of SEQ 3 and SEQ 4 were synthesized as follows.

[0784] SEQ 3: Synthesis of Propargyl-PEG1-T(dab)GGTATGTTGTTCTO

[0785]

[0786] Fmoc-(109)T(Boc)PNA1(S1) synthesized according to steps ① to ⑨ described above was deprotected in step ⑧, and then 1 mL (0.3 M) of a DMF solution containing a mixture of Propargyl-PEG1-OH (20 eq) and PyBOP was added. DIEA (20 eq) was added and the mixture was shaken at room temperature for 60 min. The solution in the reactor was filtered, and the resin was washed with DMF. The resin was treated with a mixed solution of 87.5% Trifluoroacetic acid (TFA), 2.5% 2-2'-(Ethylenedioxy)diethanethiol (DODT), 2.5% water, 2.5% Thioanisole, 2.5% Triisopropylsilane (TIPS), and 2.5% m-cresol for 0.5 h to simultaneously remove the protecting group of the amine of the nucleobase and the deresinization reaction. The resin was filtered and washed with TFA. After dispersing in diethyl ether and centrifuging, the supernatant was removed by decanting. The separated solid was dispersed in diethyl ether, washed through the process of centrifuging and removing the supernatant, and dried to obtain S3 in a solid state.

[0787] After dissolving S3 in DMF without purification, 4-((4-(dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester (Dabcyl-SE) (1 umole) and diisopropylethylamine (DIPEA) (1 umole) were slowly added at room temperature. The mixed reaction product was stirred at room temperature for 1 hour, precipitated in an excess of diethyl ether, centrifuged, and the supernatant was removed by decantation. The separated solid was dispersed in diethyl ether, centrifuged, the supernatant was removed, and dried to obtain Propargyl-T(Dab)GGTATGTTGTTCTO, which was purified by HPLC. The HPLC purification was performed using Water / ACN (0.1% TFA) as the mobile phase and flowing ACN (0.1% TFA) from 10% to 40% for 30 minutes. A C18 (5um, 300Å, 4.6x250mm) column was used, and analysis was performed at a column temperature of 60℃. The newly generated peak was confirmed to be the product through maldi-tof mass analysis (mass 4628.556, crude purity 45.9%). After confirming the completion of the reaction, the product peak was fractionated under the same conditions and lyophilized to obtain 300 nmole of [Propargyl-T(Dab)GGTATGTTGTTCTO] (purity: 98.2%, mass 4628.556).

[0788] SEQ 4: Synthesis of Propargyl-PEG1-T(dab)GGTATGTTGTTCT(3)O

[0789]

[0790] SEQ 4 was synthesized using the same method as SEQ 3 (purity: 97.2%, Mass 5016.640).

[0791] [Table 1]

[0792]

[0793] [Experimental Example 1] Confirmation of the stability of PNA oligomers containing phosphonates.

[0794] The PNA oligomers of SEQ 1 and SEQ 2 in Table 1 were dissolved in 0.1% TFA / water and then subjected to HPLC analysis. The HPLC analysis was performed using an Agilent 1100 series instrument (C18 [5 μm, 300 Å, 4.6 × 250 mm; ODS-BIO] column) heated to 80°C under the analysis conditions described in Table 2 below. The results are shown in Figures 1 and 2.

[0795] [Table 2] HPLC analysis conditions (80 ℃)

[0796]

[0797] As a result of HPLC analysis, two major peaks were observed at retention times (RT) of 19.1 min and 19.91 min for SEQ 1, and a single major peak was observed at 21.4 min for SEQ 2. As a result of preparing each peak and analyzing it by Maldi-tof MS, a mass value of 11609.9 (P-80 adduct), which is expected to be the result of decomposition of phosphonate 1, was observed for SEQ 1 at 19.1 min, and a product mass value (11689.9) was observed at 19.91 min. On the other hand, only a product mass value (11732.0) was observed for SEQ 2, confirming its excellent stability.

[0798] [Experimental Example 2] Chimera Synthesis of Water-Soluble PNA

[0799] The click reaction tendency for chimeric synthesis according to the introduction of a water-soluble linker was tested using SEQ 3 and SEQ 4 of Table 1 above. The DNA-N3 moiety (FAM-TTCCTA-N3) for the click reaction was purchased from Bioneer Co., Ltd.

[0800]

[0801] (1) Synthesis of FAM-TTCCTA-[triazole]-T(Dab)GGTATGTTGTTCTO

[0802] After mixing water (30 uL) and 100 mM Phosphate buffer (30 uL), FAM-TTCCTA-[azide] (100 nmol), a DNA purchased from Bioneer, was added at room temperature. Then, the synthesized SEQ 3 [Propargyl-T(Dab)GGTATGTTGTTCTO] (100 nmol) was added, and 10 mM CuSO4 (100 nmol) and L-ascorbic acid (400 nmol) were added. After 2 hours of reaction at room temperature, the purity before and after purification was analyzed using HPLC. Water / ACN (0.1% TFA) was used as the mobile phase, and ACN (0.1% TFA) was flowed from 5% to 70% for 15 minutes. A C18 (5 μm, 300 Å, 4.6 × 250 mm) column was used, and the analysis was performed at a column temperature of 60℃. The newly generated peak was confirmed to be the product through maldi-tof mass analysis (10.03 min, M+1; 6466.288, crude purity 45.9%). After confirming the completion of the reaction, the product peak was collected under the same conditions and lyophilized to obtain 5 nmole of FAM-TTCCTA-[triazole]-T(Dab)GGTATGTTGTTCTO (purity: 50.1%). The results are shown in Fig. 3.

[0803] (2) Synthesis of FAM-TTCCTA-[triazole]-T(Dab)GGTATGTTGTTCT(3)O

[0804] Using the synthesized SEQ 4 above, the reaction was performed in the same manner as in (2), and the purity before and after purification was analyzed using HPLC. Using 0.05 M TEAA / ACN as the mobile phase, ACN was flowed from 0 to 50% for 20 min. A C18 (5 μm, 300 Å, 4.6 × 250 mm) column was used, and the analysis was performed at a column temperature of 60 °C. The newly generated peak was confirmed to be the product through maldi-tof mass analysis (13.21 min, M+1; 6703.468, crude purity: 77.0 %). After confirming the completion of the reaction, the product peak was collected under the same conditions and lyophilized to obtain 30 nmol of FAM-TTCCTA-[triazole]-T(Dab)GGTATGTTGTTCT(3)O (purity: 98.8%). The results are shown in Fig. 4.

[0805] From the results of (1) and (2) above, it was confirmed that SEQ 3, an acetylene derivative used in the click reaction with FAM-TTCCTA-N3, did not dissolve in a pH 7.5, 100 mM phosphate buffer because a water-soluble linker was not introduced, and thus the reaction did not proceed cleanly. On the other hand, in the case of SEQ 4, into which a water-soluble linker according to the present invention was introduced, it was confirmed that it dissolved well in a pH 7.5, 100 mM phosphate buffer, and that a chimeric compound was synthesized with high purity through the click reaction with FAM-TTCCTA-N3.

[0806] Therefore, when using PNA applied with a water-soluble linker and water-soluble monomer according to the present invention, chimeric compounds can be synthesized with high purity through various click reactions.

[0807] [Experimental Example 3] Solubility Test

[0808] Solubility tests were performed using SEQ 5, SEQ7, SEQ 9, and SEQ 19 of Table 1 above.

[0809] Each sample was eluted with ACN (0.1% TFA) from 10% to 40% for 30 minutes using the mobile phase Water / ACN (0.1% TFA). A C18 (5 μm, 300 Å, 4.6 × 250 mm) column was used, and the product peak was collected at a column temperature of 60 °C. The absorbance of the aliquoted solution of each sample was then measured at 260 nm using a nanodrop, and the mole content was calculated based on the absorbance at 260 nm.

[0810] After lyophilization of the aliquots of each sample, each lyophilized sample was added to 1X PBS buffer at a concentration of 100 uM. Thereafter, the concentration was calculated and the dissolution rate was calculated by measuring the absorbance at 260 nm using NanoDrop under the conditions of (a) to (e) [(a) during dissolution; (b) after vortexing; (c) after spin-down; (d) after sonication and heating; and (e) after centrifugation at 12,000 rpm for 3 min]. The same sample was stored frozen, and the absorbance measurement was repeated under the same conditions the next day to calculate the dissolution rate. The results are shown in Table 3 and Fig. 5 below.

[0811] [Table 3]

[0812]

[0813] While SEQ 5, a typical PNA, was hardly soluble in 1X PBS buffer, SEQ 7, SEQ 9, and SEQ 19, which had a water-soluble linker (3) or a water-soluble monomer (23, 20, or 21) introduced, all exhibited excellent solubility in 1X PBS buffer. In particular, SEQ 9, which had both a water-soluble linker and a water-soluble monomer introduced, showed a complete solubility close to 100%, and SEQ 19, which had water-soluble monomers (20 and 21) introduced, maintained a complete solubility of 100% under all treatment conditions.

[0814] Typically, experiments in the biomedical field are conducted in neutral or basic buffers. However, conventional PNAs are insoluble in neutral or basic buffers, which limits their biological applicability. However, the present invention effectively overcomes these problems, particularly solubility, by introducing water-soluble linkers and monomers, thereby providing PNA monomers and oligomers with excellent water solubility and stability even under neutral and slightly basic conditions.

[0815] That is, it was confirmed that the PNA into which the water-soluble linker and monomer according to the present invention are introduced has excellent solubility and storage stability in neutral and weakly basic environments, unlike the existing PNA, and has excellent solubility stability with minimal change in solubility rate even after various physical treatments.

[0816] [Experimental Example 4] Specificity Test

[0817] To confirm the specificity of SEQ 7, SEQ 8, SEQ 9, and SEQ 10 in Table 1 above, the melting temperature (Tm) was measured. Complementary DNA [TCTAAGGCAACCATA, TCTAAGGGAACCATA] for melting point measurement was purchased from Bioneer Co., Ltd.

[0818] <Tm 측정을 위한 샘플의 제조>

[0819] Each PNA and DNA was dissolved at 10 uM using 10 mM tris buffer.

[0820] It was prepared by mixing 10 uM PNA (2 uL), 10 uM DNA (2 uL), and 4x PCR premix (5 uL), and then adding triple-distilled water (11 uL).

[0821] <Tm 측정>

[0822] Using a CFX 96 real-time PCR device, the temperature was heated at 95°C for 3 minutes, then lowered to 20°C and maintained for 5 minutes. Afterwards, the fluorescence signal was measured at 1°C / 10 sec from 20°C to 90°C, and the Tm results in Table 4 below were obtained, and the melting curve graph is shown in Fig. 6.

[0823] [Table 4]

[0824]

[0825] From the melting curve results in Fig. 6, it was confirmed that SEQ 7 and SEQ 8, in which only a water-soluble linker was introduced and no water-soluble monomer was introduced, bound to both a full-matched sequence and a 1-mismatch sequence. On the other hand, SEQ 9 and SEQ 10, in which a water-soluble monomer was introduced, bound to a full-matched sequence but did not bind to a 1-mismatch sequence.

[0826] From the above, it can be seen that the PNA containing the water-soluble monomer according to the present invention can secure extremely high specificity. Therefore, the PNA containing the water-soluble monomer according to the present invention can be applied to signal amplification technologies requiring extremely high specificity in the field of diagnosis [e.g., HCR (hybridization chain reaction) technology, etc.], and can be applied as a therapeutic substance without off-target effects in the field of gene therapy.

[0827] [Experimental Example 5] Fluorescence Stability Test

[0828] To confirm the fluorescence stability according to the change in purification method for SEQ 11, SEQ 12, and SEQ 13 in Table 1 above, they were purified under acidic and basic conditions, respectively, through HPLC.

[0829] Specifically, acidic conditions were performed using mobile phase Water / ACN (0.1% TFA) and flowing ACN (0.1% TFA) from 5% to 70% for 15 minutes. A C18 (5 μm, 300 Å, 4.6 × 250 mm) column was used, and the purification was performed at a column temperature of 60 °C. Basic conditions were performed using mobile phase 0.05 M TEAA / ACN and flowing ACN from 0% to 50% for 20 minutes. A C18 (5 μm, 300 Å, 4.6 × 250 mm) column was used, and the purification was performed at a column temperature of 60 °C. PNA was purified under each condition and lyophilized. Stability tests were performed through HPLC purity analysis after storage at room temperature in a solid state (a) or a 100 μM solution state (100% DMSO (b); 1x PBS Buffer (c)). HPLC purity analysis was performed using a mobile phase of Water / ACN (0.1% TFA) with ACN (0.1% TFA) flowing from 5% to 70% for 15 minutes, using a C18 (5 um, 300 Å, 4.6 × 250 mm) column, and at a column temperature of 60°C.

[0830] As a result, it was confirmed that SEQ 11, SEQ 12, and SEQ 13 all maintained stability when purified under basic conditions. When purified under acidic conditions, it was confirmed that SEQ 11 and SEQ 13 maintained stability on the first day, but SEQ 13 decomposed.

[0831] [Table 5]

[0832]

[0833] Generally, PNA is stable in acid, but in the case of PNA conjugated with a fluorescent substance, it is known that the fluorescent substance, rather than the PNA portion, is easily decomposed in the presence of acid, showing an unstable behavior. In other words, the fluorescent substance is unstable under acidic conditions and stable under neutral or slightly basic conditions.

[0834] Conventional PNAs are insoluble in neutral and weakly alkaline conditions, making purification impossible under weakly alkaline conditions. However, the water-soluble PNA according to the present invention dissolves very well even in weakly alkaline conditions, enabling purification in basic buffer compositions, thereby enabling more stable production of fluorescent PNA.

[0835] [Experimental Example 6] Cell Permeability Test

[0836] Using SEQ 14, SEQ 15, SEQ 16, SEQ 17, and SEQ 18 of Table 1 above, nuclear penetration into A549 cells was confirmed using the method described in the reference [Nucleic Acids Research, 2008, 36, 4425]. PNA was labeled with Cy3 (Red), and nuclei were stained with DAPI (Blue), followed by confocal analysis. The results are shown in Fig. 7.

[0837] As shown in Figure 7, it was confirmed that PNA reached the inside of the nucleus in the case of SEQ 18 containing a water-soluble monomer.

[0838] [Experimental Example 7] Digital PCR (digital polymerase chain reaction; dPCR) test

[0839] Digital PCR was performed using SEQ 5 and SEQ 7 of Table 1 above.

[0840] Specifically, the experiment used a 10 μM forward primer (1.05 μL), a 10 μM reverse primer (2.1 μL), a 7.5 μM probe (0.7 μL), and 4X PCR buffer (10.5 μL) in one reaction with a total volume of 42 μL. The target was gDNA (10 ng, 0.1 ng) and the experiment was performed under NTC (Non-Template Control) conditions. Using QiAcuity dPCR equipment from QIAGEN, the initial denaturation was performed at 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds, 58°C for 45 seconds, and 74°C for 45 seconds.

[0841] The results are shown in Figure 8. In the reagent reaction including SEQ 5, no results were obtained for all samples, whereas in the reagent reaction including SEQ 7, results were detected only in samples containing the specific target. This shows that SEQ 5 without the soluble linker did not function as a dPCR-based probe, whereas SEQ 7 with the soluble linker did.

[0842] Although the present invention has been described with reference to specific details, limited embodiments, and drawings, these have been provided merely to aid in a more general understanding of the present invention, and the present invention is not limited to the above embodiments, and those skilled in the art to which the present invention pertains can make various modifications and variations based on this description. Therefore, the spirit of the present invention should not be limited to the described embodiments, and all things that are equivalent or equivalent to the following claims, as well as the claims, are considered to fall within the scope of the spirit of the present invention.

Claims

1. A water-soluble PNA (peptide nucleic acid) monomer represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, R' is hydrogen, and R'' is -OP 2 Alternatively, the above R' and R'' can be connected to each other to form a ring; L is C1-C10 alkylene; R is or and; L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with; R 1a and R 1b are each independently C1-C10 alkyl; a is an integer from 1 to 4; L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof; R 1c and R 1d are each independently C1-C10 alkyl; c is an integer from 1 to 4; B is a moiety comprising a nucleic acid base; L 2 is a single bond or C1-C30 alkylene, and the above L 2 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof; R 2a and R 2b are each independently C1-C10 alkyl; b is an integer from 1 to 3; P 1 is a protecting group for hydrogen or amine groups; P 2 is a protecting group for hydrogen or hydroxyl groups.

2. In paragraph 1, A water-soluble PNA monomer represented by the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 2, L, R, P 1 and P 2 is the same as the definition in claim 1.

3. In paragraph 1, A water-soluble PNA monomer represented by the following chemical formula 3: [Chemical Formula 3] In the above chemical formula 3, L, R and P 1 is the same as the definition in claim 1.

4. In paragraph 1, A water-soluble PNA monomer represented by the following chemical formula 4: [Chemical Formula 4] In the above chemical formula 4, L 1 is a single bond, C1-C20 alkylene or And here the wave( ) is a bonding site with carbon (C), and the asterisk ( ) represents a binding site with P, and the L 1 The -CH2- of alkylene can be replaced with -O-; L 1b is C1-C20 alkylene, and the above L 1b The -CH2- of alkylene can be replaced with -O-, and the above L 1b The alkylene of can be further substituted with; R 1a and R 1b are each independently C1-C5 alkyl; L 1a is C1-C20 alkylene, and the above L 1a The -CH2- of alkylene can be replaced with -O-; R 1c and R 1d are each independently C1-C5 alkyl; c is an integer of 1 or 2; P 1 is a protecting group for hydrogen or amine groups; P 2 is a protecting group for hydrogen or hydroxyl groups.

5. In paragraph 1, A water-soluble PNA monomer represented by the following chemical formula 5: [Chemical Formula 5] In the above chemical formula 5, B is a moiety comprising a nucleic acid base; L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-; R 2a and R 2b are each independently C1-C5 alkyl; b is an integer of 1 or 2; q is an integer of 0 or 1; P 1 is a protecting group for hydrogen or amine groups; P 2 is a protecting group for hydrogen or hydroxyl groups.

6. In paragraph 5, Above L 2a and L 2b are each independently C1-C20 alkylene or -(CH2CH2O) g - and g is an integer from 1 to 6, a water-soluble PNA monomer.

7. In paragraph 1, A water-soluble PNA monomer represented by the following chemical formula 6: [Chemical Formula 6] In the above chemical formula 6, B is a moiety comprising a nucleic acid base; L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-; R 2a and R 2b are each independently C1-Calkyl; b is an integer of 1 or 2; q is an integer of 0 or 1; P 1 is a protecting group for the amine group.

8. In paragraph 7, Above L 2a and L 2b are each independently C1-C20 alkylene or -(CH2CH2O) g - and g is an integer from 1 to 6, a water-soluble PNA monomer.

9. In paragraph 1, The protecting group of the above amine group and the protecting group of the hydroxy group are each independently selected from the group consisting of acetyl (Ac), tosyl (Ts), benzyl (Bn), benzoyl (Bz), benzyloxy (Bn), isobutyryl, 2,2,2-trichloroethoxycarbonyl (Troc), allyloxycarbonyl (Alloc), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tert-butoxycarbonyl (Boc), C1-C6 alkyl, cyclohexyl, benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzylhydryloxycarbonyl (Bhoc), monomethoxytrityl (Mmt), dimethoxytrityl (Dmt), nitrobenzenesulfonyl (Nosyl; Ns), A water-soluble PNA monomer, 2-nitrophenylsulfenyl (Nps) or benzothiazole-2-sulfonyl (Bts).

10. In paragraph 1, The above nucleic acid bases are adenine (A), purine, 2-aminoadenine, N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Ethano-2,6-diaminopurine, 7-deazaadenine, 8-azapurine, guanine (G), O 6 -Protected-guanine, 7-deazaguanine, thymine (T), O 4 -Protected-thymine, uracil (U), dihydrouracil, 5-(C1-C6 alkyl)uracil, 5-(C2-C6 alkenyl)uracil, 5-(C2-C6 alkynyl)uracil, 5-(1-propargylamino)uracil, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 2-thiouracil, 4-thiouracil, cytosine (C), 6-(C1-C6 alkyl)cytosine, N 4 -Protected-cytosine, N 4 ,N 4 - A water-soluble PNA monomer that is ethanocytosine, 6-(C2-C6 alkenyl)cytosine, 6-(C2-C6 alkynyl)cytosine, 5-(1-propargylamino)cytosine, pseudoisocytosine, 6-fluorocytosine, 6-chlorocytosine, 6-bromocytosine or 6-hydroxycytosine.

11. In paragraph 4, A water-soluble PNA monomer represented by the following chemical formula 4-1: [Chemical Formula 4-1] In the above chemical formula 4-1, L 1c is a single bond, C1-C20 alkylene or -(CH2CH2O) d - and the above L 1c The alkylene of can be further substituted with; R 1a , R 1b , R 1c and R 1d are each independently C1-C5 alkyl; L 1d is C1-C20 alkylene or -(CH2CH2O) e -and; m is an integer of 0 or 1; d and e are each independently integers from 1 to 6.

12. In paragraph 11, A water-soluble PNA monomer represented by the following chemical formula 4-2 or chemical formula 4-3: [Chemical Formula 4-2] [Chemical Formula 4-3] In the above chemical formulas 4-2 and 4-3, L 1c is a single bond, C1-C10 alkylene or -(CH2CH2O) d -and; L 1e is C1-C10 alkylene or -(CH2CH2O) f -and; R 1a , R 1b , R 1e and R 1f are each independently C1-C5 alkyl; d and f are each independently an integer from 1 to 5; p is an integer from 1 to 3.

13. In paragraph 5, A water-soluble PNA monomer represented by the following chemical formula 5-1: [Chemical Formula 5-1] In the above chemical formula 5-1, B is a moiety comprising a nucleic acid base; R 2a and R 2b are each independently C1-C5 alkyl; b is an integer of 1 or 2; q is an integer of 0 or 1; x and y are each independently integers from 1 to 10.

14. In paragraph 7, A water-soluble PNA monomer represented by the following chemical formula 6-1: [Chemical Formula 6-1] In the above chemical formula 6-1, B is a moiety comprising a nucleic acid base; R 2a and R 2b are each independently C1-C5 alkyl; b is an integer of 1 or 2; q is an integer of 0 or 1; x and y are each independently integers from 1 to 10.

15. PNA oligomer comprising a structural unit represented by the following chemical formula 8: [Chemical Formula 8] In the above chemical formula 8, L is C1-C10 alkylene; R is or and; L 1 is a single bond or C1-C30 alkylene, and the above L 1 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof, and the L 1 The alkylene of can be further substituted with; R 1a and R 1b are each independently hydrogen or an alkali metal cation; a is an integer from 1 to 4; L 1a is C1-C30 alkylene, and the above L 1a The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof; R 1c and R 1d are each independently hydrogen or an alkali metal cation; c is an integer from 1 to 4; B is a moiety comprising a nucleic acid base; L 2 is a single bond or C1-C30 alkylene, and the above L 2 The -CH2- of alkylene may be replaced by -O-, -C(=O)-, -NH- or a combination thereof; R 2a and R 2b are each independently hydrogen or an alkali metal cation; b is an integer from 1 to 3.

16. In paragraph 15, A PNA oligomer comprising at least one of a structural unit represented by the following chemical formula 9 and a structural unit represented by the following chemical formula 10: [Chemical Formula 9] [Chemical Formula 10] In the above chemical formulas 9 and 10, L 1 is a single bond, C1-C20 alkylene or And here the wave( ) is a bonding site with carbon (C), and the asterisk ( ) represents a binding site with P, and the L 1 The -CH2- of alkylene can be replaced with -O-; L 1b is C1-C20 alkylene, and the above L 1b The -CH2- of alkylene can be replaced with -O-, and the above L 1b The alkylene of can be further substituted with; R 1a and R 1b are each independently hydrogen or an alkali metal cation; L 1a is C1-C20 alkylene, and the above L 1a The -CH2- of alkylene can be replaced with -O-; R 1c and R 1d are each independently hydrogen or an alkali metal cation; B is a moiety comprising a nucleic acid base; L 2a and L 2b are each independently C1-C20 alkylene, and the above L 2a and L 2b The -CH2- of alkylene can be replaced with -O-; R 2a and R 2b are each independently hydrogen or an alkali metal cation; b is an integer of 1 or 2; q is an integer of 0 or 1; c is an integer of 1 or 2.

17. In paragraph 16, Above L 2a and L 2b are each independently C1-C20 alkylene or -(CH2CH2O) g - and g is an integer from 1 to 6, a PNA oligomer.

18. In paragraph 15, A PNA oligomer comprising at least one of a structural unit represented by the following chemical formula 11 and a structural unit represented by the following chemical formula 12: [Chemical Formula 11] [Chemical Formula 12] In the above chemical formulas 11 and 12, L 1c is a single bond, C1-C20 alkylene or -(CH2CH2O) d - and the above L 1c The alkylene of can be further substituted with; R 1a , R 1b , R 1c and R 1d are each independently hydrogen or an alkali metal cation; L 1d is C1-C20 alkylene or -(CH2CH2O) e -and; B is a moiety comprising a nucleic acid base; R 2a and R 2b are each independently hydrogen or an alkali metal cation; b is an integer of 1 or 2; q is an integer of 0 or 1; m is an integer of 0 or 1; d and e are each independently an integer from 1 to 6; x and y are each independently integers from 1 to 10.

19. In paragraph 15, The above nucleic acid bases are adenine (A), purine, 2-aminoadenine, N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Protected-2-aminoadenine, N 6 ,N 6 -Ethano-2,6-diaminopurine, 7-deazaadenine, 8-azapurine, guanine (G), O 6 -Protected-guanine, 7-deazaguanine, thymine (T), O 4 -Protected-thymine, uracil (U), dihydrouracil, 5-(C1-C6 alkyl)uracil, 5-(C2-C6 alkenyl)uracil, 5-(C2-C6 alkynyl)uracil, 5-(1-propargylamino)uracil, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 2-thiouracil, 4-thiouracil, cytosine (C), 6-(C1-C6 alkyl)cytosine, N 4 -Protected-cytosine, N 4 ,N 4 -PNA oligomers that are ethanocytosine, 6-(C2-C6 alkenyl)cytosine, 6-(C2-C6 alkynyl)cytosine, 5-(1-propargylamino)cytosine, pseudoisocytosine, 6-fluorocytosine, 6-chlorocytosine, 6-bromocytosine or 6-hydroxycytosine.

20. A composition for regulating target gene expression comprising the PNA oligomer of claim 15.

21. A composition for preventing or treating a disease comprising the PNA oligomer of Article 15.

22. A composition for omics analysis comprising the PNA oligomer of claim 15.

23. A pre-targeting imaging and therapeutic theranostics composition comprising the PNA oligomer of claim 15.

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