Halogen-containing compounds used for 5'-end capping of nucleic acids and uses thereof

Abstract

The present invention provides a halogen-containing compound represented by the following formula (I) for use in 5'-end capping of nucleic acids, and uses thereof. The present invention also provides uses and effects of the compound in nucleic acid transcription, expression, etc.

Description

[Technical Field]

[0001] The present invention relates to the fields of biotechnology and synthetic chemistry, particularly to nucleoside compounds, and more particularly to halogen-containing compounds used for 5'-end capping of nucleic acids (RNA) and their uses. [Background technology]

[0002] In recent years, as the global COVID-19 infection situation has changed, messenger RNA (mRNA) technology and its vaccine drugs have made great strides from laboratory to clinical application. With the unprecedented success of COVID-19 vaccines, market expectations for mRNA technology have risen significantly. The application scope of mRNA technology is not limited to infectious diseases such as COVID-19, but also holds great potential for development in a wide range of fields, including preventive vaccines, therapeutic drugs, and even cell programming and regenerative therapy.

[0003] The cap structure is a type of RNA modification that is widely present in cells and plays an important role in maintaining mRNA stability and regulating protein translation. Currently, many studies are being conducted on further chemical modifications of the cap structure to further improve mRNA translation efficiency and reduce its immunogenicity. Summary of the Invention

[0004] The present invention provides halogen-containing compounds, their pharmaceutically acceptable salts, solvates, and stereoisomers, and uses thereof, which are used for 5'-end capping of nucleic acids (RNA). These compounds have various modified or unmodified 7-methylguanine bases at one terminal nucleoside and a halogen substituent at the 2' position of the other terminal nucleoside. The use of these compounds results in a high capping rate for the 5'-end capping reaction of mRNA, good in vitro transcription efficiency, and high translation expression efficiency at the cellular level and in vivo (e.g., in mice).

[0005] The present invention provides A halogen-containing compound used for 5'-end capping of nucleic acids is provided, having the structure of formula (I) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof: [ka] (In the formula, R0 is any one selected from F, Cl, Br, and I. R1 is -H, -OH, C 1-4 Alkyl groups and C 1-4 The group is selected from alkoxy groups. R2 is -H, -OH, C 1-6 Alkyl groups and C 1-6 It is any one selected from alkoxy groups. R1 and R2 may be linked by a chemical bond to form a ring, and -R1-R2- represents -(CH2) q -O-, -O-(CH2) q - and -(CH2) m -O-(CH2) n -, wherein q, m, and n are each independently 1, 2, or 3. R3 is H, -OH, -SH, -N3, -NH2, halogen, -CN, C 1-6 Alkoxy group, -O(CH2) s CN, -SR 3a , -O(CH2) p R 3b ,OCOR 3c , O(CH2) p COR 3c , -O(CH2) t SH, -O(CH2) p OH, -O(CH2) p N3, -O(CH2) p NH2, where t, p, and s each independently represent an integer of 1 to 6; 3a is C 1-6 is an alkyl group, and R 3b is one or more R 3d may be replaced by C 6-12an aryl group, or one or more R 3d may be replaced by C 5-12 is a heteroaryl group, R 3c is one or more R 3d may be replaced by C 1-10 an alkyl group, one or more R 3d may be replaced by C 1-10 an alkenyl group, one or more R 3d may be replaced by C 5-12 a cycloalkyl group, or one or more R 3d may be replaced by C 5-12 a cycloalkenyl group, where R is one or more R 3e and R 3d and R 3e is selected from the group consisting of an alkyl group, an alkenyl group, an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH. R4, R5, R6, and R7 are each independently any one selected from -H, -OH, -OCH3, halogen, -CN, and -SH. N 01 , N 02 , N 03 , N 04 are each independently selected from 0 or 1. J1, J2, J3, J4, and J5 are each independently selected from natural or modified pyrimidine nucleotide bases and natural or modified purine nucleotide bases. R P1 is a C1-C6 alkyl group, preferably a C1-C3 alkyl group, and the alkyl group may be substituted by -SH, -N3, a C2-C6 alkenyl group, or a C2-C6 alkynyl group. R P2 and R P3 each independently represents H, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, a PEG group, or a COR P4 , SO2R P4wherein said group is optionally substituted by -CN, -N, -SH, alkynyl groups, and R P4 is selected from H and C1-C6 alkyl groups. P2 and R P3 may be linked to form a ring. However, N 01 , N 02 , N 03 , N 04 are both 0, J5 is a guanine base, and R2 is -OH, then R3 is not a methoxy group.

[0006] In a preferred embodiment, the compound of the present invention has the structure of formula (I'). [ka]

[0007] wherein each group in formula (I') has the same meaning as described above with respect to formula (I). In a preferred embodiment, at least one of J1, J2, J3, J4, and J5 is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl-modified purine nucleotide base, more preferably 6-N-methyladenine.

[0008] In a preferred embodiment, R3 is -H, -OH, -SH, -N3, -NH2, halogen, -CN, C 1-6 Alkoxy group, -O(CH2) p CN, -SR 3a , -O(CH2) p R 3b ,OCOR 3c , O(CH2) p COR 3c , -O(CH2) p SH, -O(CH2) p OH, -O(CH2) p N3, -O(CH2) p NH2, wherein t, p, and s each independently represent an integer of 1 to 6, preferably an integer of 1 to 4; R3a is C 1-4 is an alkyl group, and R 3b is one or more R 3d may be replaced by C 6-10 an aryl group, or one or more R 3d may be replaced by C 5-10 is a heteroaryl group, R 3c is one or more R 3d may be replaced by C 5-10 a cycloalkyl group, or one or more R 3d may be replaced by C 5-10 a cycloalkenyl group, where R is one or more R 3e and R 3d and R 3e is C 1-4 Alkyl group, C 2-4 Alkenyl group, C 1-4 It is selected from the group consisting of an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH.

[0009] In another preferred embodiment, R and R are each independently -H, -OH, -SH, -N, -NH, halogen, -CN, or C. 1-3 Alkoxy group, -O(CH2) p CN, -SR 3a , -O(CH2) p R 3b ,OCOR 3c , O(CH2) p COR 3c , -O(CH2) p SH, -O(CH2) p OH, -O(CH2) p N3, -O(CH2) p NH2, where p and s are each independently an integer of 1 to 3, t is an integer of 1 to 4, and R 3a is a methyl group or an ethyl group, and R 3b is one or two R 3d may be replaced by C 5-10 is a heteroaryl group, R 3cis one or two R 3d may be replaced by C 5-10 a cycloalkyl group or one or two R 3d may be replaced by C 5-10 a cycloalkenyl group, where R is one or more R 3e and R 3d and R 3e is C 1-4 Alkyl group, C 2-4 Alkenyl group, C 1-4 It is selected from the group consisting of an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH.

[0010] In another preferred embodiment, R 3b is C 1-4 C5 or C6 heteroaryl group optionally substituted by an alkyl group, such as C 1-4 R is a tetrazinyl group optionally substituted by an alkyl group; 3c is C 1-4 C which may be substituted by alkyl groups, halogens, cyano groups, amino groups, or nitro groups 5-10 Cycloalkenyl groups, such as C 1-4 It is a norbornenyl group or a cyclooctenyl group which may be substituted by an alkyl group, a halogen atom, a cyano group, an amino group or a nitro group, and is, for example, an unsubstituted norbornenyl group or a cyclooctenyl group.

[0011] In preferred embodiments, the compound has the structure of Formula (Ia), Formula (Ib), or Formula (Ic). [ka]

[0012] In a preferred embodiment, R0 is -F or -Cl, and / or R4 and R5 are each independently any one selected from H, OH, OCH3, F, Cl, -CN, and -SH, and preferably any one of H, OH, OCH3, and F. In another preferred embodiment, the compound has the structure of formula (Id): [ka]

[0013] where: R3' has the defined meaning given above for R3.

[0014] The other radicals have the meanings given above.

[0015] In preferred embodiments, the compound has the structure of Formula (Ie), Formula (If), or Formula (Ig). [ka]

[0016] In a preferred embodiment, R0 is -F or -Cl, and / or R4 and R5 are each independently any one selected from H, OH, OCH3, F, Cl, -CN, and -SH, and preferably any one of H, OH, OCH3, and F.

[0017] In the most preferred embodiments, the compound has any one of the structures shown in Table 1.

[0018] In a preferred embodiment, the compounds of the present invention are present in the form of a pharmaceutically acceptable salt, preferably a triethylamine salt, a sodium salt, a potassium salt, an ammonium salt or a tris(hydroxymethyl)aminomethane hydrochloride salt.

[0019] Another aspect of the present invention relates to the use of the above compounds as RNA capping reagents by in vitro co-transcription.

[0020] Another aspect of the invention relates to RNA molecules that contain the above-described compounds as cap structures or cap structure fragments.

[0021] Another aspect of the present invention relates to a pharmaceutical composition comprising the above-described RNA molecule and a pharmaceutically acceptable carrier.

[0022] The present invention relates to a method for synthesizing an RNA molecule, comprising the steps of incubating the above-described compound with a polynucleotide template and transcribing the template.

[0023] The present invention also relates to a capping RNA transcription reaction system comprising a polynucleotide template, the above-mentioned compound, NTPs, and an RNA polymerase.

[0024] According to some specific embodiments of the invention, the compound (or cap analog) has any one of the structures shown in Table 1 below.

[0025] [ka] JPEG2026506832000008.jpg250170 JPEG2026506832000009.jpg250170 JPEG2026506832000010.jpg246170 JPEG2026506832000011.jpg244170 JPEG2026506832000012.jpg254170 JPEG2026506832000013.jpg244159 JPEG2026506832000014.jpg244165 JPEG2026506832000015.jpg251170 JPEG2026506832000016.jpg250164 JPEG2026506832000017.jpg247170 JPEG2026506832000018.jpg241170 JPEG2026506832000019.jpg248170 JPEG2026506832000020.jpg244170 JPEG2026506832000021.jpg246170 JPEG2026506832000022.jpg252170 JPEG2026506832000023.jpg182170 JPEG2026506832000024.jpg243170 JPEG2026506832000025.jpg245170 JPEG2026506832000026.jpg243170 JPEG2026506832000027.jpg247170 JPEG2026506832000028.jpg252170 JPEG2026506832000029.jpg245170 JPEG2026506832000030.jpg245170 JPEG2026506832000031.jpg247170 JPEG2026506832000032.jpg252170 JPEG2026506832000033.jpg248170 JPEG2026506832000034.jpg245170 JPEG2026506832000035.jpg248170 JPEG2026506832000036.jpg252170 JPEG2026506832000037.jpg252170 JPEG2026506832000038.jpg243170 JPEG2026506832000039.jpg254170 JPEG2026506832000040.jpg252170 JPEG2026506832000041.jpg250170 JPEG2026506832000042.jpg248170 JPEG2026506832000043.jpg247170 JPEG2026506832000044.jpg254170 JPEG2026506832000045.jpg253170 JPEG2026506832000046.jpg246170 JPEG2026506832000047.jpg250170 JPEG2026506832000048.jpg249170 JPEG2026506832000049.jpg250170 JPEG2026506832000050.jpg246170 JPEG2026506832000051.jpg246170 JPEG2026506832000052.jpg250170 JPEG2026506832000053.jpg238170 JPEG2026506832000054.jpg253170 JPEG2026506832000055.jpg249170 JPEG2026506832000056.jpg108170 JPEG2026506832000057.jpg249170 JPEG2026506832000058.jpg239170 JPEG2026506832000059.jpg106170 JPEG2026506832000060.jpg244170 JPEG2026506832000061.jpg106170 JPEG2026506832000062.jpg240170 JPEG2026506832000063.jpg247170 JPEG2026506832000064.jpg245170 JPEG2026506832000065.jpg250170 JPEG2026506832000066.jpg246170 JPEG2026506832000067.jpg249170 JPEG2026506832000068.jpg241170 JPEG2026506832000069.jpg250170 JPEG2026506832000070.jpg248170 JPEG2026506832000071.jpg249170 JPEG2026506832000072.jpg181170 JPEG2026506832000073.jpg187170 JPEG2026506832000074.jpg195170 JPEG2026506832000075.jpg183170 JPEG2026506832000076.jpg180170 JPEG2026506832000077.jpg200170 JPEG2026506832000078.jpg230170 JPEG2026506832000079.jpg212170 JPEG2026506832000080.jpg225170 JPEG2026506832000081.jpg218170

[0026] The present invention will now be further described with reference to the drawings. [Brief explanation of the drawings]

[0027] [Figure 1] Fluorescence imaging of mRNAs containing different cap analogs in HEK293T cells. [Figure 2] Fluorescence intensity of mRNAs with different cap analogs in HEK293T cells is shown. [Figure 3] Fluorescence intensity of mRNAs with different cap analogs in HepG2 cells is shown. [Figure 4] Figure 1 shows the expression efficiency of mRNAs with different cap analogs in different organs. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides halogen-containing compounds ("cap analogs" or "capping analogs") used for 5'-end capping of RNA, their pharmaceutically acceptable salts, solvates, and stereoisomers, and uses thereof. The use of such compounds results in a high capping rate for the 5'-end capping reaction of mRNA, good in vitro transcription efficiency, and high translation expression efficiency at the cellular level.

[0029] Before further describing the present invention, some terms used in the specification, examples, and appended claims are collected below. The definitions set forth herein should be read and understood by those skilled in the art based on the rest of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0030] definition Unless otherwise indicated, when any type of range is disclosed or claimed, it is intended to individually disclose or claim all possible values ​​that can reasonably be encompassed by that range, including any subranges subsumed therein. For example, a radical number 1 to 6 indicates integers within that range, where 1 to 6 is to be understood to include 1, 2, 3, 4, 5, 6, as well as the subranges 1 to 5, 1 to 4, and 1 to 3.

[0031] The specifications of the present disclosure should be construed in accordance with the laws and principles of chemical bonding. In some cases, hydrogen atoms can be removed to accommodate a substituent at a given position.

[0032] As used in this disclosure, the terms "comprise," "contain," "include," "comprise," and similar terms mean that the elements listed before the term include the elements listed after the term and their equivalents, but do not exclude elements not listed. As used herein, the terms "contain" or "include" can be used in any of an open, semi-closed, or closed sense. In other words, the terms also include "consist essentially of" or "consist of."

[0033] The term "pharmaceutically acceptable" as used herein means that a compound or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with the human or mammal in which it is used to prevent or treat a disease or condition.

[0034] Natural or modified pyrimidine nucleotide bases include, but are not limited to, uracil, thymine, cytosine, 5-methylcytosine, 5-fluorouracil, 5-fluorocytosine, and the like.

[0035] Natural or modified purine nucleotide bases include, but are not limited to, adenine, guanine, 6-N-methyladenine, 6-N,N-dimethyladenine, 2-N-methylguanine, 2-N,N-dimethylguanine, 7-methylguanine, and the like, wherein the structure of 6-N-methyladenine is [ka] is.

[0036] The nucleotide bases herein may be modified or substituted to provide an oligonucleotide. For example, these bases or synthetic and natural nucleotide bases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanine, or tuberculin) may be used as desired. Alternatively, substituted or modified analogs of any natural or synthetic base may be used. Examples include 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6,n6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine Cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N-4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl )-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinoalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkyla (aminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxoacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(Dithio)purine pyrimidines, 5-(alkyl)pseudouracils, 5-(methyl)pseudouracils, 5-(alkyl)-2-(thio)pseudouracils, 5-(methyl)-2-(thio)pseudouracils, 5-(alkyl)-4-(thio)pseudouracils, 5-(methyl)-4-(thio)pseudouracils, 5-(alkyl)-2,4-(dithio)pseudouracils, 5-(methyl)-2,4-(dithio)pseudouracils, 1-substituted pseudouracils, 1-substituted 2(thio)-pseudouracils, 1-substituted 4(thio)-pseudouracils. -(Thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2-(thio)pseudouracil, 1-(amino Alkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine- 1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinoalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinoalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl Nidinoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)naphthalene, inosine, xanthine, hypoxanthine, nubuline, tuberculin, isoguanosine, inosine group, 2-aza-inosine group, 7-deaza-inosine group, nitroimidazolyl group, nitropyrazolyl group, nitrobenzimidazolyl group, nitroindazolyl group, aminoindolyl group, pyrrolopyrimidinyl group, 3-(methyl)isocarbostyryl group, 5-(methyl (methyl)isocarbostyril group, 3-(methyl)-7-(propynyl)isocarbostyril group, 7-(aza)indolyl group, 6-(methyl)-7-(aza)indolyl group, iminopyridyl group, 9-(methyl)-iminopyridyl group, pyrrolopyrazinyl group, isocarbostyril group, 7-(propynyl)isocarbostyril group, propynyl-7-(aza)indolyl group, 2,4,5-(trimethyl)phenyl group, 4-(methyl)indolyl group, 4,6-(dimethyl)indolyl group, phenyl group, naphthyl group, anthryl group, phenanthryl group, pyrenyl group, stilbenyl group, tetraphenyl group, pentaphenyl group, difluorotolyl group, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, 2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole or any O- or N-alkylated derivative thereof.

[0037] "Stereoisomers" refer to compounds that have identical chemical constitution, but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0038] The phrase "linked by a chemical bond to form a ring" refers to two groups being linked by a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond, or the like to form a cyclic structure, and one or two hydrogen atoms may be removed from the corresponding group as needed.

[0039] The phrase "optionally substituted by" means that one, two, or more hydrogen atoms in the group may be substituted independently by the respective substituents, which may be selected from alkyl groups, alkenyl groups, alkoxy groups, halogens, cyano groups, amino groups, nitro groups, and -OH.

[0040] The term "alkyl group" refers to a saturated, straight or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. The alkyl group is, for example, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, pentyl, heptyl, or octyl. The alkyl group may be substituted.

[0041] The term "alkoxy group" includes -O-alkyl groups and alkyl groups in which the O atom is located within the alkyl chain (e.g., -CH2-O-CH3), having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. The alkoxy group may be optionally substituted.

[0042] The term "alkenyl group" includes both straight and branched chain alkyl groups containing at least two carbon atoms and at least one carbon-carbon double bond, and containing from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, and most preferably from 2 to 3 carbon atoms. The alkenyl group may be optionally substituted.

[0043] The term "alkynyl group" includes both straight and branched chain alkyl groups containing at least two carbon atoms and at least one carbon-carbon triple bond, and containing from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, and most preferably from 2 to 3 carbon atoms. The alkynyl group may be optionally substituted.

[0044] The terms "cycloalkyl group," "cycloalkenyl group," and "cycloalkynyl group," by themselves or in combination with other terms, represent cyclic forms of "alkyl group," "alkenyl group," and "alkynyl group," respectively, where preferably 3, 4, 5, 6, 7, 8, 9, or 10 atoms form the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclopropynyl, cyclobutynyl, cyclohexynyl, and cyclopentynyl groups, and the like, and the cycloalkenyl or cycloalkynyl group may be attached to other groups at any suitable position. The terms "cycloalkyl group," "cycloalkenyl group," and "cycloalkynyl group" are also intended to include bicyclic, tricyclic, and polycyclic forms thereof, which may be spirocyclic or bridged. "Cycloalkyl group," "cycloalkenyl group," and "cycloalkynyl group" may be substituted. Examples of cycloalkyl and cycloalkenyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and norbornenyl. The term "aryl group" refers to an aromatic monocyclic ring preferably containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms, or an aromatic tricyclic ring system containing 14 carbon atoms. Examples include phenyl, naphthyl, or anthryl groups. Aryl groups may be substituted.

[0045] The term "heteroaryl group" preferably refers to a 5- or 6-membered aromatic monocyclic ring in which at least one carbon atom is replaced by 1, 2, 3, or 4 (in the case of a 5-membered ring) or 1, 2, 3, 4, or 5 (in the case of a 6-membered ring) heteroatoms, which may be the same or different, and the heteroatoms are preferably selected from O, N, and S; an aromatic bicyclic ring system in which 8, 9, 10, 11, or 12 carbon atoms are replaced by 1, 2, 3, 4, 5, or 6 carbon atoms, which may be the same or different, and the heteroatoms are preferably selected from O, N, and S; or an aromatic tricyclic ring system in which 13, 14, 15, or 16 carbon atoms are replaced by 1, 2, 3, 4, 5, or 6 carbon atoms, which may be the same or different, and the heteroatoms are preferably selected from O, N, and S. Examples include an oxazolyl group, an isoxazolyl group, a 1,2,5-oxadiazolyl group, a 1,2,3-oxadiazolyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a 1,2,3-triazolyl group, a thiazolyl group, an isothiazolyl group, a 1,2,3-thiadiazolyl group, a 1,2,5-thiadiazolyl group, a pyridyl group, a pyrimidinyl group, a pyrazinyl group, a 1,2,3-triazinyl group, a 1,2,4-triazinyl group, a 1,3,5-triazinyl group, a 1,2,4,5-tetrazinyl group, a 1-benzofuranyl group, a 2-benzofuranyl group, an indole group, an aryl ... Examples of the alkyl group include a hydroxyl group, an isoindolyl group, a benzothienyl group, a 2-benzothienyl group, a 1H-indazolyl group, a benzimidazolyl group, a benzoxazolyl group, an indoloxazinyl group, a 2,1-benzoxazolyl group, a benzothiazolyl group, a 1,2-benzisothiazolyl group, a 2,1-benzisothiazolyl group, a benzotriazolyl group, a quinolyl group, an isoquinolyl group, a quinoxalinyl group, a quinazolinyl group, a quinolyl group, a 1,2,3-benzotriazinyl group, and a 1,2,4-benzotriazinyl group.

[0046] The term "PEG group" refers to a group comprising one or more -CH2-CH2-O- or CH3-CH2-O- units linked together, e.g., CH3-CH2-O-(CH2-CH2-O) x -, where x is selected from integers of 0 to 6, preferably integers of 0 to 4. The PEG group may be substituted.

[0047] The term "pharmaceutically acceptable salts" refers to relatively non-toxic addition salts of compounds of the present disclosure. See, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.

[0048] Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be, for example, acid addition salts of compounds of the present disclosure that have sufficient basicity and contain a nitrogen atom in the chain or ring, such as inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, or acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, The acid addition salts may be with organic acids such as pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

[0049] Other suitable pharmaceutically acceptable salts of the compounds of the present invention that are sufficiently acidic include alkali metal salts such as sodium salts or potassium salts, alkaline earth metal salts such as calcium salts or magnesium salts, ammonium salts, triethylamine salts, or salts with organic bases that provide physiologically acceptable cations (e.g., N-methylglucosamine, dimethylglucosamine, ethylglucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris(hydroxymethyl)aminomethane, aminopropane glycol, 1-amino-2,3,4-butanetriol). Alternatively, basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; long chain halides, e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aralkyl halides, e.g., benzyl and phenethyl bromides.

[0050] Those skilled in the art will also understand that acid addition salts of the claimed compounds can be prepared by reacting the compounds with an appropriate inorganic or organic acid by any of several known methods, and alkali metal and alkaline earth metal salts of acidic compounds of the disclosure are prepared by reacting with an appropriate base by a variety of known methods.

[0051] The present invention includes all possible salts of the compounds of the present disclosure, whether they be single salts or any mixture of said salts in any proportion.

[0052] The term "solvate" refers to a substance formed by combining, physically associating, and / or solvating a compound of the present invention with solvent molecules, including, for example, a disolvate, a monosolvate, or a hemisolvate (where the ratio of solvent molecules to compound of the present invention is about 2:1, about 1:1, or about 1:2, respectively). Such physical association involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (e.g., when one or more solvent molecules are incorporated into the lattice of a crystalline solid), the solvate may be isolated. Thus, solvates include solution-phase and isolable solvates. The compounds of the present invention may exist in solvated forms with pharmaceutically acceptable solvents (e.g., water, methanol, and ethanol), and the present application is intended to encompass both solvated and unsolvated forms of the compounds of the present invention. One solvate is a hydrate.

[0053] As used herein, the term "pharmaceutical composition" refers to a substance and / or combination of substances for identifying, preventing, or treating a tissue condition or disease. A pharmaceutical composition is formulated so that it is suitable for administration to a patient to diagnose, prevent, and / or treat a disease. A pharmaceutical composition is also a combination of an active agent with an inert or active carrier, making the composition suitable for therapeutic use.

[0054] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water or aqueous salt solutions in oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Salt solutions are preferred carriers when pharmaceutical compositions are administered intravenously. Salt solutions, aqueous glucose solutions, and glycerin solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc powder, sodium chloride, nonfat dry milk, glycerin, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Examples of suitable drug carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0055] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0056] The term "may" means that the situation may or may not occur. [Example]

[0057] Reagents and models used The starting materials in the examples are commercially available and / or can be prepared by various methods well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis can appropriately select the reaction conditions (including solvents, reaction atmospheres, reaction temperatures, experiment durations and post-treatments) in the following synthetic methods. Those skilled in the art of organic synthesis will understand that the functional groups present on each part of the molecule should be compatible with the proposed reagents and reactions.

[0058] All synthesized reagents and compounds were commercially available in China through common commercial channels, including Sigma-Aldrich (USA), Shanghai Zhaowei Technology Development Co., Ltd. (Trinlink Cleancap), and jetMESSENGER (Polyplus-transfection). (R) ), Shanghai Taitan Technology Co., Ltd.

[0059] Cell model: HEK293T cells were purchased from the Shanghai Chinese Academy of Sciences Cell Bank, and HepG2 cells were purchased from Wuhan PuNuoSai Biotechnology Co., Ltd.

[0060] Main equipment used: multi-microplate reader (MolecularDevices), flow cytometer (CytoFLEX S series).

[0061] Compound preparation and identification: Nuclear magnetic resonance spectrometer (Bruker 300MHz), liquid chromatography-mass spectrometer (Agilent 6150 / 1290), high-performance liquid chromatography (Agilent 1260). Cell experiments: inverted fluorescence microscope (Guangzhou Mingmei Optoelectronic Technology Co., Ltd.), cell incubator (Thermo Fisher Scientific).

[0062] The implementation process of the present invention and the resulting beneficial effects will be described in detail below using specific examples. However, these examples are intended to help readers better understand the substance and characteristics of the present invention and are not intended to limit the scope of the present application.

[0063] Synthesis of the final product: [ka]

[0064] J1 and J2 are any of the following five bases: [ka]

[0065] Final product synthesis method 1 0.4 g of compound 1 was dissolved in 8 mL of anhydrous DMSO, and 2 eq of compound 2 and 20 eq of anhydrous zinc chloride were added under argon gas protection. The reaction mixture was stirred at room temperature (25 °C) for 24 hours under argon gas protection. After monitoring the completion of the reaction by TLC, the reaction was quenched with 150 mL of 0.25 M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M ammonium bicarbonate. The product-containing eluate was collected and lyophilized to obtain the product.

[0066] Final product synthesis method 2 0.2 g of compound 2 was dissolved in 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then 0.2 g of compound 1 was added to the solution. The reaction mixture was stirred at room temperature at 25 °C for 16 hours. After monitoring the completion of the reaction by TLC, the reaction was quenched with 150 mL of 0.25 M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M ammonium bicarbonate. The product-containing eluate was collected and lyophilized to obtain the product.

[0067] Synthesis of representative structural compounds Example 1 Synthesis of compound 3 [ka]

[0068] Step 1: Compound 3-2 (2.38 g) was added to a solution of tetrazole (1.76 g) in acetonitrile (63 mL) in a three-neck flask. The mixture was purged with argon gas three times. Compound 3-1 (5 g) dissolved in 10 mL of acetonitrile was added to the above solution at room temperature (25 °C). The resulting solution was stirred at room temperature (25 °C) for 1 hour. No significant heat generation was observed, and TLC confirmed the disappearance of compound 3-1. A solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was then added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 hours, and the completion of oxidation was monitored by TLC. The reaction mixture was quenched by adding saturated aqueous sodium sulfite (10 mL), diluted with an additional 50 mL of water, and extracted with dichloromethane (50 mL × 2). The combined organic phase was washed once with water (50 mL) and concentrated to give product 3-3 (8 g, crude) as a pale yellow oil.

[0069] Step 2: Compound 3-3 (8 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water and stirred at 25 °C for 16 hours. TLC monitoring revealed the disappearance of compound 3-3 and the appearance of a highly polar spot. The reaction mixture was directly concentrated under vacuum. After concentration, an appropriate amount of silica gel and DCM were added, mixed, and purified (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 mL / min), and concentrated to give the white solid product 3-4 (2.8 g, two-step yield 51%).

[0070] Step 3: A 28 mL solution of tetrazole in acetonitrile (0.4 mmol / mL) was prepared. Compound 3-4 (2.8 g) was added to the above solution, followed by compound A1 (3 g) at room temperature (25 °C). The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature (25 °C) for 1 hour. The reaction was monitored for completion by TLC. The reaction mixture was cooled to below 10 °C in an ice-water bath, and a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise until the reaction mixture no longer faded. The completion of the oxidation reaction was monitored by TLC. The reaction mixture was quenched with 10 mL of saturated aqueous sodium sulfite solution and then diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The mixture was purified by adding an appropriate amount of silica gel and DCM (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 ml / min). The mixture was concentrated to give compound 3-6 (2.6 g, 78.2% yield) as a white foamy solid.

[0071] Step 4: Compound 3-6 (2.6 g) was dissolved in methanol (30 mL), and concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature (25°C) for 60 hours, and the completion of the reaction of compound 1-6 was monitored by TLC. The reaction mixture was concentrated in vacuo, and methanol was added and concentrated again to give compound 3-7 (2.4 g, crude product) as a pale yellow oil. The crude product was directly used in the next step.

[0072] Step 5: Compound 3-7 (2.4 g, crude) was dissolved in DMSO (3 mL), triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50 °C for 1 h. TLC showed that compound 3-7 had reacted completely. The reaction mixture was diluted to 50 mL with water, adjusted to pH 5.5 with 1 N aqueous NaOH, and loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions by vacuum concentration, and the remaining liquid was lyophilized to give the amine salt 3-8 (0.8 g, 33.7% yield). The product was a white solid.

[0073] Step 6: Compound 9a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 3-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions obtained by vacuum concentration. The remaining liquid was lyophilized to obtain the product compound 5 (65 mg) as a white powder, which is the ammonium salt. [Table 1]

[0074] Example 2 Synthesis of compound 35 [ka]

[0075] Step 1: Compound 35-2 (2.07 g) was added to a solution of tetrazole (1.6 g) in acetonitrile (56 mL) in a three-neck flask. The atmosphere was purged with argon gas three times. Compound 35-1 (5 g) dissolved in 10 mL of acetonitrile was added to the above solution. The resulting solution was stirred at room temperature (25 °C) for 1 h. No significant heat generation was observed, and TLC confirmed the disappearance of compound 2-1. Iodine solution (5 g of iodine was dissolved in 40 mL of a 0.5 mmol / mL solution in a THF:HO:pyridine = 8:1:1 mixture) was then added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 h and monitored for completion of oxidation by TLC. The reaction mixture was quenched by adding aqueous NaSO (10 mL), diluted with an additional 50 mL of water, and extracted with dichloromethane (50 mL × 2). The combined organic phases were washed once with water (50 mL) and concentrated to give product 35-3 (7.5 g, crude) as a pale yellow oil.

[0076] Step 2: Compound 35-3 (7.2 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water and stirred at 25 °C for 16 h. TLC monitoring revealed the disappearance of compound 35-3 and the appearance of a highly polar spot. The reaction mixture was directly concentrated under vacuum. After concentration, an appropriate amount of silica gel and DCM were added, mixed, and purified (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 mL / min), and concentrated to give white solid product 35-4 (2.8 g, two-step yield 54.8%).

[0077] Step 3: A 28 mL solution of tetrazole in acetonitrile (0.4 mmol / mL) was prepared. Compound 35-4 (2.8 g) was added to the above solution, followed by compound A1 (2.26 g) at room temperature (25 °C). The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature (25 °C) for 1 hour. Completion of the reaction was monitored by TLC. The reaction mixture was cooled to below 10 °C in an ice-water bath, and a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise until the reaction mixture no longer faded. Completion of the oxidation reaction was monitored by TLC. The reaction mixture was quenched with 10 mL of saturated aqueous sodium sulfite solution and further diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The mixture was purified by adding an appropriate amount of silica gel and DCM (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 ml / min). The mixture was concentrated to give compound 35-6 (3.1 g, yield 93.5%) as a white foamy solid.

[0078] Step 4: Compound 35-6 (3.1 g) was dissolved in methanol (30 mL), and concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature (25 °C) for 60 hours, and the completion of the reaction of compound 35-6 was monitored by TLC. The reaction mixture was concentrated in vacuo, and methanol was added and concentrated again to give compound 35-7 (2.4 g, crude product) as a pale yellow oil.

[0079] Step 5: Compound 35-7 (2.4 g, crude) was dissolved in DMSO (3 mL), triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50 °C for 1 h. TLC showed that compound 35-7 had reacted completely. The reaction mixture was diluted to 50 mL with water, adjusted to pH 5.5 with 1 N aqueous NaOH, and loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M TEAB. Most of the water was removed from the resulting fractions by vacuum concentration, and the remaining liquid was lyophilized to give the triethylamine salt 35-8 (1.5 g, 51% yield). The product was a white solid.

[0080] Step 6: Compound 35-8 (500 mg), compound 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added at room temperature (25 °C) under argon gas protection. Then, anhydrous DMSO (8 mL) was added via syringe and the mixture was allowed to react for 24 hours. TLC showed that most of the compound had reacted. The reaction solution was added to a solution of EDTA disodium salt (1.6 g, 80 mL) in water precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 35 as a white powder (ammonium salt, 120 mg). [Table 2]

[0081] Example 3 Synthesis of compound 68 [ka]

[0082] Step 1: Compound 3-2 (2.07 g) was added to a solution of tetrazole (1.6 g) in acetonitrile (56 mL) in a three-neck flask. The mixture was purged with argon gas three times. Compound 68-1 (5 g) dissolved in 10 mL of acetonitrile was added to the above solution. The resulting solution was stirred at room temperature (25 °C) for 1 h. No significant heat generation was observed, and TLC confirmed the disappearance of compound 68-1. Iodine solution (5 g of iodine dissolved in 40 mL of a THF:HO:pyridine = 8:1:1 mixture, 0.5 mmol / mL) was then added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 h and monitored for completion of oxidation by TLC. The reaction mixture was quenched by adding aqueous NaSO (10 mL), diluted with an additional 50 mL of water, and extracted with dichloromethane (50 mL × 2). The combined organic phase was washed once with water (50 mL) and concentrated to give product 68-3 (7.5 g, crude) as a pale yellow oil.

[0083] Step 2: Compound 68-3 (7.2 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water and stirred at 25 °C for 16 h. TLC monitoring revealed the disappearance of compound 68-3 and the appearance of a highly polar spot. The reaction mixture was directly concentrated under vacuum. After concentration, an appropriate amount of silica gel and DCM were added, mixed, and purified (40 g normal phase column, EA, 10 min, DCM:MeOH, 10–20%, 20 min, flow rate 30 mL / min), and concentrated to give white solid product 68-4 (2.8 g, two-step yield 54.8%).

[0084] Step 3: A 28 mL solution of tetrazole in acetonitrile (0.4 mmol / mL) was prepared. Compound 68-4 (2.8 g) was added to the above solution, followed by compound A1 (2.26 g) at room temperature (25 °C). The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature (25 °C) for 1 hour. The reaction was monitored for completion by TLC. The reaction mixture was cooled to below 10 °C in an ice-water bath, and a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise until the reaction mixture no longer faded. The completion of the oxidation reaction was monitored by TLC. The reaction mixture was quenched with 10 mL of saturated aqueous sodium sulfite solution and further diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The mixture was purified by adding an appropriate amount of silica gel and DCM (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 ml / min). The mixture was concentrated to give compound 68-6 (3.1 g, 93.5% yield) as a white foamy solid.

[0085] Step 4: Compound 68-6 (3.1 g) was dissolved in methanol (30 mL), and concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature (25 °C) for 60 hours, and the completion of the reaction of compound 68-6 was monitored by TLC. The reaction mixture was concentrated in vacuo, and methanol was added and concentrated again to give compound 68-7 (2.4 g, crude product) as a pale yellow oil.

[0086] Step 5: Compound 68-7 (2.4 g, crude) was dissolved in DMSO (3 mL), triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50 °C for 1 h. TLC showed that compound 68-7 was completely reacted. The reaction mixture was diluted to 50 mL with water, adjusted to pH 5.5 with 1 N aqueous NaOH, and loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M TEAB. Most of the water was removed from the resulting fractions by vacuum concentration. The remaining liquid was lyophilized to give the triethylamine salt 68-8 (1.5 g, 51% yield). The product was a white solid.

[0087] Step 6: Compound 68-8 (500 mg), 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added to the mixture at room temperature (25 °C) under argon gas protection. Then, anhydrous DMSO (8 mL) was added via syringe and the mixture was allowed to react for 24 h. TLC showed that most of the compound had reacted. The reaction mixture was added to a solution of EDTA disodium salt (1.6 g, 80 mL) in water precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to give the product compound 68 as a white powder (ammonium salt, 120 mg). [Table 3]

[0088] Example 4 Synthesis of compound 83 [ka]

[0089] Step 1: Compound 3-2 (2.1 g) was added to a solution of tetrazole (1.6 g) in acetonitrile (56 mL) in a three-neck flask. The mixture was purged with argon gas three times. Compound 83-1 (5 g) dissolved in 10 mL of acetonitrile was added to the above solution. The resulting solution was stirred at room temperature (25 °C) for 1 h. No significant heat generation was observed, and TLC confirmed the disappearance of compound 83-1. An iodine solution (5 g of iodine was dissolved in 40 mL of a THF:HO:pyridine = 8:1:1 mixture, 0.5 mmol / mL) was then added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 h and monitored for completion of the oxidation by TLC. The reaction mixture was quenched by adding aqueous NaSO (10 mL), diluted with an additional 50 mL of water, and extracted with dichloromethane (50 mL × 2). The combined organic phase was washed once with water (50 mL) and concentrated to give product 83-3 (7.5 g, crude) as a pale yellow oil.

[0090] Step 2: Compound 83-3 (7.4 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water and stirred at 25 °C for 16 h. TLC monitoring revealed the disappearance of compound 83-3 and the appearance of a highly polar spot. The reaction mixture was directly concentrated under vacuum. After concentration, an appropriate amount of silica gel and DCM were added, mixed, and purified (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 mL / min), and concentrated to give white solid product 83-4 (2.8 g, two-step yield 54.8%).

[0091] Step 3: A 28 mL solution of tetrazole in acetonitrile (0.4 mmol / mL) was prepared. Compound 83-4 (2.8 g) was added to the above solution, followed by the addition of A1 (2.26 g) at room temperature (25 °C). The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature (25 °C) for 1 hour. The completion of the reaction was monitored by TLC. The reaction mixture was cooled to below 10 °C in an ice-water bath, and a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise until the reaction mixture no longer faded. The completion of the oxidation reaction was monitored by TLC. The reaction mixture was quenched with 10 mL of saturated aqueous sodium sulfite solution and further diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The mixture was purified by adding an appropriate amount of silica gel and DCM (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 ml / min). The mixture was concentrated to give compound 83-6 (3.1 g, yield 93.5%) as a white foamy solid.

[0092] Step 4: Compound 83-6 (3.1 g) was dissolved in methanol (30 mL), and concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature (25° C.) for 60 hours, and the completion of the reaction of compound 83-6 was monitored by TLC. The reaction mixture was concentrated in vacuo, and methanol was added and concentrated again to give compound 83-7 (2.4 g, crude product) as a pale yellow oil.

[0093] Step 5: Compound 83-7 (2.4 g, crude) was dissolved in DMSO (3 mL), triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50 °C for 1 h. TLC showed that compound 83-7 had reacted completely. The reaction mixture was diluted to 50 mL with water, adjusted to pH 5.5 with 1 N aqueous NaOH, and loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M TEAB. Most of the water was removed from the resulting fractions by vacuum concentration. The remaining liquid was lyophilized to give the triethylamine salt 83-8 (1.5 g, 51% yield). The product was a white solid.

[0094] Step 6: Compound 83-8 (500 mg), compound 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added at room temperature (25 °C) under argon gas protection. Then, anhydrous DMSO (8 mL) was added via syringe and the mixture was allowed to react for 24 hours. TLC showed that most of the compound had reacted. The reaction solution was added to a solution of EDTA disodium salt (1.6 g, 80 mL) in water precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 83 as a white powder (ammonium salt, 120 mg). [Table 4]

[0095] Example 5 Synthesis of Compound 90 [ka]

[0096] Compound 9a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 90-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 90 (50 mg) as a white powder, which is the ammonium salt. [Table 5]

[0097] Example 6 Synthesis of Compound 103 [ka]

[0098] Compound 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 3-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product, compound 103, as a white powder (60 mg). [Table 6]

[0099] Example 7 Synthesis of Compound 119 [ka]

[0100] Compound 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 119-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 119 (65 mg) as a white powder, which is the ammonium salt. [Table 7]

[0101] Example 8 Synthesis of compound 127 [ka]

[0102] Compound 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 127-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 127 (82 mg) as a white powder, which is the ammonium salt. [Table 8]

[0103] Example 9 Synthesis of compound 167 [ka]

[0104] Compound 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 24-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product, compound 167, as a white powder (62 mg). [Table 9]

[0105] Example 10 Synthesis of Compound 180 [ka]

[0106] Compound 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 180-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 180 (95 mg) as a white powder, which is the ammonium salt. [Table 10]

[0107] Example 11 Synthesis of compound 203 [ka]

[0108] Compound 9c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 203-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 203 (ammonium salt) as a white powder (95 mg). [Table 11]

[0109] Example 12 Synthesis of compound 239 [ka]

[0110] Compound 9c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 239-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 239 (ammonium salt) as a white powder (95 mg). [Table 12]

[0111] Example 13 Synthesis of Compound 260 [ka]

[0112] Compound 9c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 260-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 260 (75 mg) as a white powder, which is the ammonium salt. [Table 13]

[0113] Example 14 Synthesis of compound 287 [ka]

[0114] 9c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 287-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 287 (75 mg) as a white powder, which is the ammonium salt. [Table 14]

[0115] Example 15 Synthesis of Compound 126 [ka]

[0116] 9d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 3-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 308 (60 mg) as a white powder, which is the ammonium salt. [Table 15]

[0117] Example 16 Synthesis of Compound 326 [ka]

[0118] Compound 9d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 326-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 326 (60 mg) as a white powder, which is the ammonium salt. [Table 16]

[0119] Example 17 Synthesis of Compound 352 [ka]

[0120] Compound 9d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 352-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 352 (74 mg) as a white powder, which is the ammonium salt. [Table 17]

[0121] Example 18 Synthesis of Compound 386 [ka]

[0122] Compound 9d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 386-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 386 (60 mg) as a white powder, which is the ammonium salt. [Table 18]

[0123] Example 19 Synthesis of Compound 403 [ka]

[0124] Compound 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 403-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 403 (55 mg) as a white powder, which is the ammonium salt. [Table 19]

[0125] Example 20 Synthesis of compound 443 [ka]

[0126] Compound 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 443-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 443 (70 mg) as a white powder, which is the ammonium salt. [Table 20]

[0127] Example 21 Synthesis of compound 467 [ka]

[0128] Compound 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 467-7 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 467 (75 mg) as a white powder, which is the ammonium salt. [Table 21]

[0129] Example 22 Synthesis of compound 480 [ka]

[0130] 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 480-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 480 (85 mg) as a white powder, which is the ammonium salt. [Table 22]

[0131] Example 23 Synthesis of Compound 501 [ka]

[0132] Compound 10b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 501-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 501 (92 mg) as a white powder, which is the ammonium salt. [Table 23]

[0133] Example 24 Synthesis of Compound 541 [ka]

[0134] Compound 10b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 541-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 541 (85 mg) as a white powder, which is the ammonium salt. [Table 24]

[0135] Example 25 Synthesis of Compound 574 [ka]

[0136] Compound 10b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 574-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 574 (ammonium salt) as a white powder (80 mg). [Table 25]

[0137] Example 26 Synthesis of Compound 589 [ka]

[0138] Compound 10b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 589-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 589 (58 mg) as a white powder, which is the ammonium salt. [Table 26]

[0139] Example 27 Synthesis of Compound 589 [ka]

[0140] Compound 10c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 589-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC showed the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 589 (88 mg) as a white powder, which is the ammonium salt. [Table 27]

[0141] Example 28 Synthesis of compound 664 [ka]

[0142] Compound 10c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 664-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 664 (60 mg) as a white powder, which is the ammonium salt. [Table 28]

[0143] Example 29 Synthesis of Compound 706 [ka]

[0144] Compound 10d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 706-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 706 (55 mg) as a white powder, which is the ammonium salt. [Table 29]

[0145] Example 30 Synthesis of Compound 760 [ka]

[0146] Compound 10d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 760-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 760 (70 mg) as a white powder, which is the ammonium salt. [Table 30]

[0147] Example 31 Synthesis of Compound 787 [ka]

[0148] Compound 10d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 787-7 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 787 (75 mg) as a white powder, which is the ammonium salt. [Table 31]

[0149] Example 32 Compound 828 synthesis [ka]

[0150] Compound 10e (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 828-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 828 (85 mg) as a white powder, which is the ammonium salt. [Table 32]

[0151] Example 33 Synthesis of Compound 880 [ka]

[0152] Compound 10e (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 880-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 880 (92 mg) as a white powder, which is the ammonium salt. [Table 33]

[0153] Example 34 Synthesis of Compound 903 [ka]

[0154] Compound 10f (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 903-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 903 (85 mg) as a white powder, which is the ammonium salt. [Table 34]

[0155] Example 35 Synthesis of Compound 974 [ka]

[0156] Compound 10f (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 974-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 974 (ammonium salt) as a white powder (80 mg). [Table 35]

[0157] Example 36 Synthesis of Compound 1040 [ka]

[0158] Compound 10g (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then compound 1040-1 (200 mg) was added to the solution. The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC showed the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the obtained fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1040 (ammonium salt) as a white powder (58 mg). [Table 36]

[0159] Example 37 Synthesis of Compound 1087 [ka]

[0160] Compound 10g (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then compound 1087-1 (200 mg) was added to the resulting solution. The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1087 (ammonium salt) as a white powder (88 mg). [Table 37]

[0161] Example 38 Synthesis of Compound 1108 [ka]

[0162] Compound 10h (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1108-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1108 (ammonium salt) as a white powder (88 mg). [Table 38]

[0163] Example 39 Synthesis of Compound 1130 [ka]

[0164] Compound 10h (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1130-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1130 (ammonium salt) as a white powder (88 mg). [Table 39]

[0165] Example 40 Synthesis of Compound 1130 [ka]

[0166] Compound 10h (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1130-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1130 (ammonium salt) as a white powder (88 mg). [Table 40]

[0167] Example 41 Synthesis of Compound 1140 [ka]

[0168] Compound 10i (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1140-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1140 (ammonium salt) as a white powder (88 mg). [Table 41]

[0169] Example 42 Synthesis of compound 1198 [ka]

[0170] Compound 10j (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1198-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1198 (ammonium salt) as a white powder (88 mg). [Table 42]

[0171] Example 43 Synthesis of Compound 1220 [ka]

[0172] Compound 10j (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1220-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1220 (ammonium salt) as a white powder (88 mg). [Table 43]

[0173] Example 44 Synthesis of Compound 1280 [ka]

[0174] Compound 10h (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1280-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1280 (ammonium salt) as a white powder (88 mg). [Table 44]

[0175] Example 45 Synthesis of compound 1307 [ka]

[0176] Compound 10i (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1307-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1307 (88 mg) as a white powder, which is the ammonium salt. [Table 45]

[0177] Example 46 Synthesis of compound 1373 [ka]

[0178] Compound 10k (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1373-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1373 (ammonium salt) as a white powder (88 mg). [Table 46]

[0179] Example 47 Synthesis of Compound 1400 [ka]

[0180] Compound 10k (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1400-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1400 (ammonium salt) as a white powder (88 mg). [Table 47]

[0181] Example 48 Synthesis of compound 1421 [ka]

[0182] Compound 10h (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1421-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1421 (ammonium salt) as a white powder (88 mg). [Table 48]

[0183] Example 49 Synthesis of compound 1449 [ka]

[0184] Compound 10i (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1449-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1449 (88 mg) as a white powder, which is the ammonium salt. [Table 49]

[0185] Example 50 Synthesis of compound 1449 [ka]

[0186] Compound 10i (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1449-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1449 (88 mg) as a white powder, which is the ammonium salt. [Table 50]

[0187] Example 51 Synthesis of compound 1495 [ka]

[0188] Compound 10l (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1495-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1495 (ammonium salt) as a white powder (88 mg). [Table 51]

[0189] Example 52 Synthesis of compound 1580 [ka]

[0190] Compound 10k (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1580-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1580 (ammonium salt) as a white powder (88 mg). [Table 52]

[0191] Example 53 Synthesis of Compound 1581 [ka]

[0192] Compound 11a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1581-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1581 (88 mg) as a white powder, which is the ammonium salt. [Table 53]

[0193] Example 54 Synthesis of compound 1617 [ka]

[0194] Compound 11b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1617-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1617 (ammonium salt) as a white powder (88 mg). [Table 54]

[0195] Example 55 Synthesis of Compound 1660 [ka]

[0196] Compound 11c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1600-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1600 (ammonium salt) as a white powder (88 mg). [Table 55]

[0197] Example 56 Synthesis of compound 1698 [ka]

[0198] Compound 11d (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1698-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1698 (ammonium salt) as a white powder (88 mg). [Table 56]

[0199] Example 57 Synthesis of compound 1728 [ka]

[0200] Compound 11e (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1728-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1728 (ammonium salt) as a white powder (88 mg). [Table 57]

[0201] Example 58 Synthesis of compound 1897 [ka]

[0202] Compound 11a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1897-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1897 (ammonium salt) as a white powder (88 mg). [Table 58]

[0203] Example 59 Synthesis of compound 1907 [ka]

[0204] Compound 11b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1907-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1907 (88 mg) as a white powder, which is the ammonium salt. [Table 59]

[0205] Example 60 Synthesis of compound 1925 [ka]

[0206] Compound 11c (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1925-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1925 (ammonium salt) as a white powder (88 mg). [Table 60]

[0207] Example 61 Synthesis of compound 1978 [ka]

[0208] Compound 11e (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1978-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1978 (ammonium salt) as a white powder (88 mg). [Table 61]

[0209] Example 62 Synthesis of compound 1981 [ka]

[0210] Compound 9a (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1981-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1981 (88 mg) as a white powder, which is the ammonium salt. [Table 62]

[0211] Example 63 Synthesis of compound 1988 [ka]

[0212] Compound 9b (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1988-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1988 (ammonium salt) as a white powder (88 mg). [Table 63]

[0213] Example 64 Synthesis of compound 1992 [ka]

[0214] Compound 9c (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1992-1 (200 mg). The reaction mixture was stirred at room temperature (25°C) for 16 hours. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0°C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 1992 (ammonium salt) as a white powder (88 mg). [Table 64]

[0215] Example 65 Synthesis of compound 1996 [ka]

[0216] Compound 9d (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 1996-1 (200 mg). The reaction mixture was stirred at room temperature (25°C) for 16 hours. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0°C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 1996 (ammonium salt) as a white powder (88 mg). [Table 65]

[0217] Example 66 Synthesis of compound 2005 [ka]

[0218] Compound 9e (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2005-1 (200 mg). The reaction mixture was stirred at room temperature (25°C) for 16 hours. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0°C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2005 (ammonium salt) as a white powder (88 mg). [Table 66]

[0219] Example 67 Synthesis of compound 2010 [ka]

[0220] Compound 9f (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 2010-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions by vacuum concentration. The remaining liquid was lyophilized to obtain the product compound 2010 (ammonium salt) as a white powder (88 mg). [Table 67]

[0221] Example 68 Synthesis of compound 2020 [ka]

[0222] Compound 10k (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganeseous chloride, followed by the addition of compound 2020-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions by vacuum concentration. The remaining liquid was lyophilized to obtain the product compound 2020 (ammonium salt) as a white powder (88 mg). [Table 68]

[0223] Example 69 Synthesis of compound 2025 [ka]

[0224] Compound 10d (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2025-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2025 (ammonium salt) as a white powder (88 mg). [Table 69]

[0225] Example 70 Synthesis of compound 2028 [ka]

[0226] Compound 10b (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2028-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2028 (ammonium salt) as a white powder (88 mg). [Table 70]

[0227] Example 71 Synthesis of compound 2031 [ka]

[0228] Compound 10f (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2031-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2031 (88 mg) as a white powder, which is the ammonium salt. [Table 71]

[0229] Example 72 Synthesis of compound 2037 [ka]

[0230] Compound 10a (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2037-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2037 (ammonium salt) as a white powder (88 mg). [Table 72]

[0231] Example 73 Synthesis of compound 2038 [ka]

[0232] Compound 12a (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2038-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2038 (ammonium salt) as a white powder (88 mg). [Table 73]

[0233] Example 74 Synthesis of compound 2039 [ka]

[0234] Compound 12b (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2039-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2039 (ammonium salt) as a white powder (88 mg). [Table 74]

[0235] Example 75 Synthesis of compound 2040 [ka]

[0236] Compound 12c (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2040-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2040 (ammonium salt) as a white powder (88 mg). [Table 75]

[0237] Example 76 Synthesis of compound 2050 [ka]

[0238] Compound 11c (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2050-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2050 (ammonium salt) as a white powder (88 mg). [Table 76]

[0239] Example 77 Synthesis of compound 2051 [ka]

[0240] Compound 11e (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2051-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2051 (88 mg) as a white powder, which is the ammonium salt. [Table 77]

[0241] Example 78 Synthesis of compound 2054 [ka]

[0242] Compound 11b (250 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of compound 2054-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2054 (ammonium salt) as a white powder (88 mg). [Table 78]

[0243] Example 79 Synthesis of compound 2064 [ka]

[0244] A three-neck flask was charged with 2064-2 (2.38 g) and a solution of tetrazole (1.76 g) in acetonitrile (63 mL). The mixture was purged with argon gas three times. Then, 2064-1 (5 g) dissolved in 10 mL of acetonitrile was added to the above solution. The resulting solution was stirred at room temperature (25 °C) for 1 h. No significant heat generation was observed, and TLC confirmed the disappearance of the starting material 2064-1. A solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was then added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 h and monitored for completion of oxidation by TLC. The reaction mixture was quenched by adding saturated aqueous sodium sulfite (10 mL), diluted with an additional 50 mL of water, and extracted with dichloromethane (50 mL x 2). The combined organic phase was washed once with water (50 mL) and concentrated to give a pale yellow oily product 2064-3 (8 g, crude).

[0245] Compound 2064-3 (8 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water and stirred at 25 °C for 16 hours. TLC monitoring revealed the disappearance of 3-3 and the appearance of a highly polar spot. The reaction mixture was directly concentrated under vacuum. After concentration, an appropriate amount of silica gel and DCM were added, mixed, and purified (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 mL / min), and concentrated to give white solid product 2064-4 (2.8 g, two-step yield 51%).

[0246] A 28 mL solution of tetrazole in acetonitrile (0.4 mmol / mL) was prepared. 2.8 g of 2064-4 was added to the above solution, followed by 3 g of A1 at room temperature (25 °C). The atmosphere was purged with nitrogen three times, and the reaction mixture was stirred at room temperature (25 °C) for 1 hour. The reaction was monitored for completion by TLC. The reaction mixture was cooled to below 10 °C in an ice-water bath, and a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise until the reaction mixture no longer faded. The completion of the oxidation reaction was monitored by TLC. The reaction mixture was quenched with 10 mL of saturated aqueous sodium sulfite and further diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. The mixture was purified by adding an appropriate amount of silica gel and DCM (40 g normal phase column, EA, 10 min, DCM:MeOH, 10-20%, 20 min, flow rate 30 ml / min). The mixture was concentrated to give compound 2064-6 (2.6 g, 78.2% yield) as a white foamy solid.

[0247] Compound 2064-6 (2.6 g) was dissolved in methanol (30 mL), and concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature (25 °C) for 60 hours, and the reaction completion of raw material 2064-6 was monitored by TLC. The reaction solution was concentrated in vacuo, and methanol was added and concentrated again to give compound 2064-7 (2.4 g, crude product) as a pale yellow oil. The crude product was directly used in the next step.

[0248] Compound 2064-7 (2.4 g, crude) was dissolved in DMSO (3 mL), triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50 °C for 1 h. TLC showed that starting material 3-7 was completely reacted. The reaction mixture was diluted to 50 mL with water, adjusted to pH 5.5 with 1 N aqueous NaOH, and loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the resulting fractions by vacuum concentration, and the remaining liquid was lyophilized to give the amine salt of the target compound 2064-8 (0.8 g, 33.7% yield). The product was characterized as a white solid.

[0249] 9a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then 2064-8 (200 mg) was added to the solution. The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC showed the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the obtained fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2064 (65 mg) as a white powder, which is the ammonium salt. [Table 79]

[0250] Example 80 Synthesis of compound 2078 [ka]

[0251] 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 2078-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2078 (55 mg) as a white powder, which is the ammonium salt. [Table 80]

[0252] Example 81 Synthesis of compound 2092 [ka]

[0253] 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then 2092-1 (200 mg) was added to the solution. The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC showed the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the obtained fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2092 (65 mg) as a white powder, which is the ammonium salt. [Table 81]

[0254] Example 82 Synthesis of compound 2098 [ka]

[0255] 9a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 2098-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2098 (70 mg) as a white powder, which is the ammonium salt. [Table 82]

[0256] Example 83 Synthesis of compound 2110 [ka]

[0257] 9b (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, followed by the addition of 2110-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC indicated the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the fractions in vacuo, and the remaining liquid was lyophilized to obtain the product compound 2110 (70 mg) as a white powder, which is the ammonium salt. [Table 83]

[0258] Example 84 Synthesis of compound 2123 [ka]

[0259] 10a (200 mg) was added to 16 mL of an aqueous solution (pH 7.0) containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganous chloride, and then 2123-1 (200 mg) was added to the solution. The reaction mixture was stirred at room temperature (25 °C) for 16 h. TLC showed the formation of the product. The reaction mixture was added to a solution of EDTA disodium salt (1.4 g, 80 mL) precooled to 0 °C, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0 to 1.0 M aqueous ammonium bicarbonate. Most of the water was removed from the obtained fractions under vacuum, and the remaining liquid was lyophilized to obtain the product compound 2123 (ammonium salt) as a white powder (50 mg). [Table 84]

[0260] Example 85 Detection of capping mRNA synthesis efficiency a) The plasmid was linearized and the DNA template was purified.

[0261] b) mRNA was synthesized by in vitro transcription, using the capping analogue of the present invention, Trilink CleanCap of Comparative Example 1, and the cap analogues of Comparative Examples 2 and 3. Here, Comparative Example 1 is a commercially available product, and Comparative Examples 2 and 3 were obtained by the method disclosed in WO2022 / 036858. [ka]

[0262] The minimum reaction system for preparing mRNA used in the present invention is shown in Table 1. [Table 85]

[0263] c) During the experiment, the above reagents were thoroughly mixed and incubated at 37°C. After 4 hours, deoxyribonuclease (DNase) was added and incubated for 30 minutes to remove the DNA template. After digestion was complete, LiCl solution was added, pre-cooled, and centrifuged at 16,000 rpm for 15 minutes, and the supernatant was removed. 70% ethanol was added and centrifuged, after which the supernatant was removed again. A certain amount of enzyme-free water was then added and stored. The purified mRNA sample was then quantitatively detected using Nanodrop One, and the results are shown in Table 2 below. The experimental results demonstrate that the product yield in mRNA synthesis using the halogenated cap analogs of the present invention was increased compared to the comparative example. [Table 86]

[0264] d) The purified mRNA was treated with enzyme digestion, and then oligonucleotide fragments of different sizes were separated and identified by liquid chromatography-mass spectrometry (LC-MS). This provided accurate molecular weight information for the enzyme digested fragments, which, combined with the theoretical molecular weight of the enzyme digested fragments, were assigned to determine the capping efficiency of the sample. The capping rate of mRNA synthesized using the cap analogues of the present invention is 90% to 98%. When viewed from the capped mRNA after purification, the compounds of the present invention all show good capping efficiency.

[0265] Example 86 Evaluation of the expression efficiency of green fluorescent protein mRNA with different capping analogs in different cells In this study, we investigated the expression efficiency of different capped green fluorescent protein mRNAs in HEK293T and HepG2 cells. Using the coding sequence of green fluorescent protein (GFP) as a DNA template and the cap analogs described in this study as the starting material, we performed in vitro mRNA transcription. Then, we transfected the different mRNA products into cells, and finally detected the fluorescent proteins in the cells using a flow cytometer.

[0266] a) The different cells listed above were cultured at 2 × 10 5 Cells were seeded in a 96-well plate.

[0267] b) 300 μL of mRNA buffer and 6 μg of RNA were mixed uniformly, and then 6 μL of transfection reagent (JetMESENG-ER) was added and mixed uniformly. After leaving the mixture for 10 minutes, the mixture was added to the cells in each well, and transfection medium (Opti-MEM) was added to 2 mL per well. The mixture was then incubated at 37°C, 5% CO2 for 6 hours.

[0268] c) After replacing the medium with fresh complete medium and continuing to culture under the same conditions for 24 hours, the GFP fluorescence intensity was observed under a fluorescence microscope. The results are shown in Figure 1. As can be seen from the figure, the mRNA expression efficiency in the present invention is higher than that in the comparative example.

[0269] d) The transfected cells were cultured for 24 hours, then processed and detected using a flow cytometer (CytoFLEX S series). The detected fluorescence intensity was proportional to the translation efficiency of the target protein, and the analytical results are shown in Figures 2 and 3.

[0270] FIG. 2 shows the fluorescence intensity analysis of mRNAs containing different cap analogs encoding green fluorescent protein in HEK293T cells. The horizontal axis represents the compound number of the cap analog, and the horizontal axis represents the fluorescence intensity value detected by a flow cytometer. The cap analogs of the present invention exhibit significantly higher fluorescence intensity values ​​in HEK293T cells than Comparative Examples 1 and 2. For example, the average fluorescence intensity value of compound 468 is 1.5 times that of Comparative Example 2 and 1.6 times that of Comparative Example 1, while the average fluorescence intensity value of compound 158 is 1.3 times that of Comparative Example 2 and 1.4 times that of Comparative Example 1.

[0271] Figure 3 shows the fluorescence intensity of mRNAs containing different cap analogs in Hep G2 cells. The results show that the efficiency of protein translation in Hep G2 cells using mRNAs containing the cap analogs of the present invention is higher than that of the control. The use of fluoronucleoside dimer cap analogs, such as compound 1982, and fluoronucleoside tetramer cap analogs, such as compound 2057, significantly improved protein expression.

[0272] Example 87 Testing the expression efficiency of mRNA synthesized with different cap analogs in mice a) mRNA encoding luciferase was prepared using the cap analog of the present invention, and the resulting mRNA was diluted in citrate buffer at pH 4.0. The cationic lipid DLin-MC3-DMA:DSPC:cholesterol:PEG lipid (DMG-PEG2000) was dissolved in ethanol at a molar ratio of 50:10:38.5:1.5.

[0273] b) 3 mL of mRNA buffer solution and 1 mL of lipid solution were placed in two 5 mL syringes, each of which was then set into a microfluidic injection pump. The flow rate of the injection pump was set, and the collected product was placed in a dialysis bag and then concentrated to the desired concentration by ultrafiltration. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter and stored until ready for use.

[0274] c) Luciferase mRNA-lipid nanoparticles containing 5 μg of mRNA were injected via the tail vein into 6-8 week-old female Balb / c mice. Five mice were injected with each luciferase mRNA-lipid nanoparticle in a parallel experiment. 24 hours later, luciferase substrate was injected and detected using a PerkinElmer small animal imaging system. The luminescence intensity is proportional to the translation efficiency of the target protein. Figure 4 shows the relative fluorescence intensity of mRNA in different organs of the mice. In the present invention, the mRNA expression efficiency in different organs using different halogenated cap analogs was significantly higher than in the comparative example.

Claims

1. A compound used for 5'-end capping of nucleic acids, characterized by having a structure of formula (I) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. 【Chemistry 1】 (In the formula, R 0 is any one selected from F, Cl, Br, and I. R 1 -H, -OH, C 1-4 Alkyl groups and C 1-4 The group is selected from alkoxy groups. R 2 -H, -OH, C 1-6 Alkyl groups and C 1-6 It is any one selected from alkoxy groups. R 1 and R 2 may be linked by a chemical bond to form a ring, and -R 1 -R 2 - is -(CH 2 ) q -O-, -O-(CH 2 ) q - and -(CH 2 ) m -O-(CH 2 ) n -, wherein q, m, and n are each independently 1, 2, or 3. R 3 -H, -OH, -SH, -N 3 , -NH 2 , halogen, -CN, C 1-6 Alkoxy group, -O(CH 2 ) s CN, -SR 3a , -O(CH 2 ) p R 3b ,OCOR 3c , O(CH 2 ) p COR 3c , -O(CH 2 ) t SH, -O(CH 2 ) p OH, -O(CH 2 ) p N 3 , -O(CH 2 ) p NH 2 wherein t, p, and s each independently represent an integer from 1 to 6; 3a is C 1-6 is an alkyl group, and R 3b is one or more R 3d may be replaced by C 6-12 an aryl group, or one or more R 3d may be replaced by C 5-12 is a heteroaryl group, R 3c is one or more R 3d may be replaced by C 1-10 an alkyl group, one or more R 3d may be replaced by C 1-10 an alkenyl group, one or more R 3d may be replaced by C 5-12 a cycloalkyl group, or one or more R 3d may be replaced by C 5-12 is a cycloalkenyl group, where R 3 is one or more R 3e and R 3d and R 3e is selected from the group consisting of an alkyl group, an alkenyl group, an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH. R 4 , R 5 , R 6 , R 7 are independently -H, -OH, and -OCH 3 , halogen, -CN, and -SH. N 01 , N 02 , N 03 , N 04 are each independently selected from 0 or 1. J 1 , J 2 , J 3 , J 4 , J 5 are each independently selected from natural or modified pyrimidine nucleotide bases, natural or modified purine nucleotide bases. R P1 is C 1 -C 6 alkyl group, preferably C 1 -C 3 The alkyl group is an alkyl group, and the alkyl group is -SH, -N 3 , C 2 -C 6 Alkenyl group or C 2 -C 6 It may be substituted by an alkynyl group. R P2 and R P3 are independently H, C 1 -C 6 Alkyl group, C 2 -C 6 Alkenyl group, C 2 -C 6 Alkynyl group, cycloalkyl group, cycloalkenyl group, cycloalkynyl group, PEG group, COR P4 , SO 2 R P4 and the groups are selected from -CN, -N 3 , -SH, alkynyl group, R P4 is H and C 1 -C 6 R is selected from alkyl groups. P2 and R P3 may be linked to form a ring. However, N 01 , N 02 , N 03 , N 04 are both 0, and J 5 is a guanine base, and R 2 is -OH, R 3 is not a methoxy group.)

2. 2. The compound according to claim 1, a pharmaceutically acceptable salt or solvate thereof, or a stereoisomer thereof, characterized in that it has the structure of formula (I'): 【Chemistry 2】 (Each group in formula (I') has the meaning as defined in claim 1.)

3. J 1 , J 2 , J 3 , J 4 , J 5 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, characterized in that at least one of the following is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl-modified purine nucleotide base, more preferably 6-N-methyladenine.

4. R 3 -H, -OH, -SH, -N 3 , -NH 2 , halogen, -CN, C 1-6 Alkoxy group, -O(CH 2 ) p CN, -SR 3a , -O(CH 2 ) p R 3b ,OCOR 3c , O(CH 2 ) p COR 3c , -O(CH 2 ) p SH, -O(CH 2 ) p OH, -O(CH 2 ) p N 3 , -O(CH 2 ) p NH 2 wherein t, p, and s are each independently an integer of 1 to 6, preferably an integer of 1 to 4; R 3a is C 1-4 is an alkyl group, and R 3b is one or more R 3d may be replaced by C 6-10 an aryl group, or one or more R 3d may be replaced by C 5-10 is a heteroaryl group, R 3c is one or more R 3d may be replaced by C 5-10 a cycloalkyl group, or one or more R 3d may be replaced by C 5-10 is a cycloalkenyl group, where R 3 is one or more R 3e and R 3d and R 3e is C 1-4 Alkyl group, C 2-4 Alkenyl group, C 1-4 selected from the group consisting of an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH; Preferably, R 3 -H, -OH, -SH, -N 3 , -NH 2 , halogen, -CN, C 1-3 Alkoxy group, -O(CH 2 ) p CN, -SR 3a , -O(CH 2 ) p R 3b ,OCOR 3c , O(CH 2 ) p COR 3c , -O(CH 2 ) p SH, -O(CH 2 ) p OH, -O(CH 2 ) p N 3 , -O(CH 2 ) p NH 2 wherein p and s are each independently an integer of 1 to 3, t is an integer of 1 to 4, and R 3a is a methyl group or an ethyl group, and R 3b is one or two R 3d may be replaced by C 5-10 is a heteroaryl group, R 3c is one or two R 3d may be replaced by C 5-10 a cycloalkyl group or one or two R 3d may be replaced by C 5-10 is a cycloalkenyl group, where R 3 is one or more R 3e and R 3d and R 3e is C 1-4 Alkyl group, C 2-4 Alkenyl group, C 1-4 selected from the group consisting of an alkoxy group, a halogen, a cyano group, an amino group, a nitro group, -OH, and -SH; More preferably, R 3b is C 1-4 C optionally substituted with an alkyl group 5 or C 6 Heteroaryl groups, such as C 1-4 R is a tetrazinyl group optionally substituted by an alkyl group; 3c is C 1-4 C which may be substituted by alkyl groups, halogens, cyano groups, amino groups, or nitro groups 5-10 Cycloalkenyl groups, such as C 1-4 a norbornenyl group or a cyclooctenyl group which may be substituted by an alkyl group, a halogen, a cyano group, an amino group, or a nitro group, for example, an unsubstituted norbornenyl group or a cyclooctenyl group; The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

5. The compound according to any one of claims 1 to 4, characterized in that it has a structure of formula (Ia), formula (Ib), or formula (Ic), or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. 【Transformation 3】

6. R 0 is -F or -Cl, and / or R 4 and R 5 are independently H, OH, and OCH 3 , F, Cl, —CN, —SH, preferably H, OH, OCH 3 , or F 6. The compound according to claim 5, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

7. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, characterized in that it has a structure of formula (Id): 【Chemistry 4】 (In the formula, R 3 ' is R in claim 1 or 4 3 has a limited meaning with respect to Other radicals have the meanings given in claims 1 to 4.

8. 8. The compound according to claim 7, characterized in that it has a structure of formula (Ie), formula (If), or formula (Ig), or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. 【Transformation 5】

9. R 0 is -F or -Cl, and / or R 4 and R 5 are independently H, OH, and OCH 3 , F, Cl, —CN, —SH, preferably H, OH, OCH 3 , or F 9. The compound according to claim 8, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

10. 2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, characterized in that it has any one of the structures shown in Table 1 of the specification.

11. The compound, solvate or stereoisomer thereof according to any one of claims 1 to 10, characterized in that it exists in the form of a pharmaceutically acceptable salt, preferably in the form of a triethylamine salt, a sodium salt, a potassium salt, an ammonium salt or tris(hydroxymethyl)aminomethane hydrochloride.

12. Use of a compound according to any one of claims 1 to 11 as an RNA capping reagent by in vitro co-transcription.

13. An RNA molecule comprising the compound according to any one of claims 1 to 11 as a cap structure or a cap structure fragment.

14. A pharmaceutical composition comprising the RNA molecule of claim 13 and a pharmaceutically acceptable carrier.

15. 12. A method for synthesizing an RNA molecule, comprising the steps of incubating a compound according to any one of claims 1 to 11 with a polynucleotide template and transcribing the template.

16. A capping RNA transcription reaction system comprising a polynucleotide template, the compound according to any one of claims 1 to 11, NTPs, and an RNA polymerase.

Images