A halogenated compound for 5' end capping of nucleic acids and its applications

Abstract

This invention provides a halogenated compound for 5' end capping of nucleic acids and its applications, the compound being shown in formula (I). This invention also provides the uses and effects of the above-described compound on nucleic acid transcription and expression.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and synthetic chemistry, specifically to a class of nucleoside compounds, and more specifically to a halogenated compound used for 5' capping of nucleic acids (RNA) and its applications. Background Technology

[0002] In recent years, with the changing global COVID-19 pandemic, messenger RNA (mRNA) technology and its vaccines and drugs have achieved breakthroughs from laboratory to clinical applications. Following the unprecedented success of COVID-19 vaccines, market expectations for mRNA technology have surged. The applications of mRNA technology extend beyond infectious diseases like COVID-19; it holds immense potential in preventative vaccines, therapeutic drugs, and even cell programming and regenerative therapies.

[0003] Cap structures are a widespread RNA modification in cells, playing a crucial role in maintaining mRNA stability and regulating protein translation. Current research focuses on further chemically modifying cap structures to enhance mRNA translation efficiency while reducing its immunogenicity. Summary of the Invention

[0004] This invention provides a halogenated compound for 5' capping of nucleic acids (RNA), its pharmaceutically acceptable salt, its solvate, and its stereoisomers, as well as their applications. These compounds have various modified or unmodified 7-methylguanine bases at one end of the nucleoside and a halogen substituent at the 2' position of the nucleoside at the other end. Using these compounds results in a high capping rate for 5' capping of mRNA, good in vitro transcription efficiency, and high translational expression efficiency at both the cellular and in vivo levels, such as in mice.

[0005] This invention provides a halogenated compound for 5' capping of nucleic acids, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, having the structure of formula (I):

[0006]

[0007] in:

[0008] R0 is selected from any one of F, Cl, Br, and I;

[0009] R1 is selected from -H, -OH, C. 1-4 Alkyl and C 1-4 An alkoxy group;

[0010] R2 is selected from -H, -OH, C. 1-6 Alkyl and C 1-6 Any one of the alkoxy groups;

[0011] Optionally, R1 and R2 are linked together by a chemical bond to form a ring, and -R1-R2- is -(CH2). q -O-, -O-(CH2) q - and -(CH2) m -O-(CH2) n - any one of them, where q, m, and n are each independently 1, 2, or 3;

[0012] R3 can be H, -OH, -SH, -N3, -NH2, halogen, -CN, or C. 1-6 Alkyl 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 Any of NH2, where t, p, and s are each an independent integer from 1 to 6, R 3a C 1-6 Alkyl, R 3b For optional use by one or more R 3d Replacement C 6-12 aryl or optionally substituted with one or more R 3d Replacement C 5-12 heteroaryl, R 3c For optional use by one or more R 3d Replacement C 1-10 Alkyl group, optionally with one or more R 3d Replacement C 1-10 alkenyl, optionally with one or more R 3d Replacement C 5-12 cycloalkyl or optionally with one or more R 3d Replacement C 5-12 Cycloalkenyl, wherein R3 is optionally surrounded by one or more R 3e Replace, R 3d and R 3e Choose from the group consisting of alkyl, alkenyl, alkoxy, halogen, cyano, amino, nitro, -OH, and -SH;

[0013] R4, R5, R6, and R7 are each independently selected from any one of -H, -OH, -OCH3, halogen, -CN, and -SH;

[0014] N 01 N 02 N 03 N 04 Each can be independently selected from 0 or 1;

[0015] J1, J2, J3, J4, and J5 are each independently selected from natural or modified pyrimidine nucleotide bases and natural or modified purine nucleotide bases.

[0016] R P1 It is a C1-C6 alkyl group, preferably a C1-C3 alkyl group, which may optionally be substituted with -SH, -N3, C2-C6 alkenyl or C2-C6 alkynyl;

[0017] R P2 and R P3 Each is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, PEGyl, COR P4 SO2R P4 The group may optionally be substituted with -CN, -N3, -SH, or ynyl group, R P4 Selected from H and C1-C6 alkyl groups; R P2 and R P3 Optional connections to form a loop;

[0018] The premise is that when N 01 N 02 N 03 N 04 Both are 0. J5 is a guanine base. When R2 is -OH, R3 is not a methoxy group.

[0019] In a preferred embodiment, the compound of the present invention has the structure of formula (I'):

[0020]

[0021] In formula (I'), each group has the same meaning as described above regarding formula (I).

[0022] 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, and even more preferably 6-N-methyladenine.

[0023] In a preferred embodiment, R3 is -H, -OH, -SH, -N3, -NH2, halogen, -CN, or C. 1-6 Alkyl group, -O(CH2) p CN,-SR 3a , -O(CH2) p R 3b ,OCOR3c O(CH2) p COR 3c ,-O(CH2) p SH,-O(CH2) p OH,-O(CH2) p N3,-O(CH2) p Any of NH2, where t, p, and s are each independently 1-6, preferably any integer from 1-4, R 3a C 1-4 Alkyl, R 3b For optional use by one or more R 3d Replacement C 6-10 aryl or optionally substituted with one or more R 3d Replacement C 5-10 heteroaryl, R 3c For optional use by one or more R 3d Replacement C 5-10 cycloalkyl or optionally with one or more R 3d Replacement C 5-10 Cycloalkenyl, wherein R3 is optionally surrounded by one or more R 3e Replace, R 3d and R 3e Choose C freely 1-4 Alkyl, C 2-4 alkenyl, C 1-4 The group consisting of alkoxy, halogen, cyano, amino, nitro, -OH, and -SH.

[0024] In another preferred embodiment, R, R3 are -H, -OH, -SH, -N3, -NH2, halogen, -CN, C 1-3 Alkyl 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 Any of the following NH2 types, where p and s are each an independent integer from 1 to 3, t is an arbitrary integer from 1 to 4, and R 3a It is methyl or ethyl, R 3b For optional use by one or two R 3d Replacement C 5-10 heteroaryl, R 3cFor optional use by one or two R 3d Replacement C 5-10 Cycloalkyl or optionally with one or two R 3d Replacement C 5-10 Cycloalkenyl, wherein R3 is optionally surrounded by one or more R 3e Replace, R 3d and R 3e Choose C freely 1-4 Alkyl, C 2-4 alkenyl, C 1-4 The group consisting of alkoxy, halogen, cyano, amino, nitro, -OH, and -SH.

[0025] In another preferred embodiment, R 3b For optional C 1-4 Alkyl-substituted C5 or C6 heteroaryl groups, for example, optionally C 1-4 Alkyl-substituted tetrazinyl, R 3c For optional C 1-4 C-substituted with alkyl, halogen, cyano, amino, or nitro groups 5-10 Cycloalkenyl groups, for example, optionally C 1-4 Norbornyl or cyclooctenyl substituted with alkyl, halogen, cyano, amino, or nitro groups, such as unsubstituted norbornyl or cyclooctenyl.

[0026] In a preferred embodiment, the compound has the structure of formula (Ia), formula (Ib), or formula (Ic):

[0027]

[0028]

[0029] In a preferred embodiment, R0 is -F or -Cl, and / or

[0030] R4 and R5 are each independently selected from any one of H, OH, OCH3, F, Cl, -CN, and -SH, with H, OH, OCH3, and F being preferred.

[0031] In another preferred embodiment, the compound has the structure of formula (Id):

[0032]

[0033] in,

[0034] R3' has the meaning of the above-mentioned limitation of R3;

[0035] The remaining groups have the meanings described above.

[0036] In a preferred embodiment, the compound has the structure of formula (Ie), formula (If), or formula (Ig):

[0037]

[0038]

[0039] In a preferred embodiment, R0 is -F or -Cl, and / or

[0040] R4 and R5 are each independently selected from any one of H, OH, OCH3, F, Cl, -CN, and -SH, with H, OH, OCH3, and F being preferred.

[0041] In the most preferred embodiment, the compound has one of the structures shown in Table 1.

[0042] In a preferred embodiment, the compounds of the present invention are present in the form of pharmaceutically acceptable salts, preferably in the form of triethylamine salts, sodium salts, potassium salts, ammonium salts, or tris(hydroxymethyl)aminomethane hydrochloride.

[0043] Another aspect of the present invention relates to the use of the compounds described above as capping agents for in vitro co-transcription of RNA.

[0044] Another aspect of the present invention relates to an RNA molecule comprising the compounds described above as a cap structure or cap structure fragment.

[0045] Another aspect of the invention relates to a pharmaceutical composition comprising an RNA molecule as described above, and a pharmaceutically acceptable carrier.

[0046] The present invention also relates to a method for synthesizing RNA molecules, comprising the following steps: co-incubating the compound as described above with a polynucleotide template to perform template transcription.

[0047] The present invention also relates to a capped RNA transcription reaction system comprising: a polynucleotide template, the compounds described above, NTPs, and RNA polymerase.

[0048] According to some specific embodiments of the present invention, the compound (or cap analogue) has one of the structures shown in Table 1 below:

[0049] Table 1:

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Attached Figure Description

[0125] The invention will now be further described with reference to the accompanying drawings.

[0126] Figure 1 Fluorescence imaging of mRNAs with different cap analogs in HEK293T cells is shown.

[0127] Figure 2 The fluorescence intensity of mRNAs with different cap analogs in HEK293T cells is shown.

[0128] Figure 3 The fluorescence intensity of mRNAs with different cap analogs is shown in HepG2 cells.

[0129] Figure 4 The expression efficiency of mRNAs with different cap analogs in different organs is shown. Detailed Implementation

[0130] This invention provides a halogenated compound (“cap analog” or “capped analog”) for 5' capping of RNA, its pharmaceutically acceptable salt, its solvate, its stereoisomer, and its applications. Using this class of compounds results in a high capping rate for 5' capping of mRNA, good in vitro transcription efficiency, and high translational expression efficiency at the cellular level.

[0131] Before further describing the invention, certain terms used in the specification, embodiments, and appended claims are collected in the following sections. The definitions set forth herein are intended to be read and understood by those skilled in the art in accordance with the remainder of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0132] definition

[0133] Unless otherwise stated, when disclosing or claiming protection for any type of scope, the intent to separately disclose or claim protection for each possible value that scope may reasonably cover, including any sub-scopes included therein. For example, the number of groups 1 to 6 indicates integers within the scope, where 1-6 should be understood to include 1, 2, 3, 4, 5, 6, as well as sub-scopes of 1-5, 1-4, and 1-3.

[0134] This disclosure should be interpreted as consistent with the laws and principles of chemical bonding. In some cases, it may be necessary to remove a hydrogen atom to accommodate a substituent at a given position.

[0135] As used herein, the words “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described herein. The terms “containing” or “comprising (including)” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently composed of” or “composed of”.

[0136] The term "pharmaceutically acceptable" in this application means that the compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0137] Natural or modified pyrimidine nucleotide bases include, but are not limited to: uracil, thymine, cytosine, 5-methylcytosine, 5-fluorouracil, 5-fluorocytosine, etc.

[0138] 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, etc., among which the structure of 6-N-methyladenine is...

[0139] The nucleotide bases in this application may be modified or replaced to provide oligonucleotides. For example, they may be used with these bases or with synthetic and natural nucleotide bases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanine, or tuberculin) and optionally modified. Alternatively, analogs of substitutions or modifications of any natural or synthetic bases 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-(deazonyl)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-(isopentenyl)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-(deazinon)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halogenated)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deazinon)-5-(aza)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-(guanidinylalkyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halogenated)uracil, 5-(methoxy)uracilUracil-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 pyrimidine, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil 5-(alkyl)-2,4-(dithio)-pseudouracil, 5-(methyl)-2,4-(dithio)-pseudouracil, 1-substituted pseudouracil, 1-substituted 2-(thio)-pseudouracil, 1-substituted 4-(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)-2-(thio)- Pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-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)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-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)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl,7-(guanidinoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazine-1-yl, 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazine-1-yl, 1,3,5-(triaza)-2,6-(dioxa)naphthalene, inosine, xanthine Hypoxanthine, bubralin, tuberculin, isoguanosine, inosine, 2-aza-inosine, 7-deaza-inosine, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindole, pyrrolopyrimidinyl, 3-(methyl)isocarbazyl, 5-(methyl)isocarbazyl, 3-(methyl)-7-(propynyl)isocarbazyl, 7-(aza)indole, 6-(methyl)- 7-(aza)indolyl, iminopyridyl, 9-(methyl)-iminopyridyl, pyrrolopyrazinyl, isocarbostyryl, 7-(propynyl)isocarbostyryl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthraceneyl, phenanthryl, pyrene, stilbene, tetraphenyl, pentaphenyl, difluorotolyl, 4-( Fluorinated 6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymidine, 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-alkylated or N-alkylated derivative thereof.

[0140] "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.

[0141] "Connecting into a ring through chemical bonds" refers to connecting two groups through carbon-carbon bonds, carbon-oxygen bonds, carbon-nitrogen bonds, carbon-sulfur bonds, etc., to form a ring structure. If necessary, the corresponding group can reduce 1-2 hydrogen atoms.

[0142] The expression "optionally substituted" means that one, two, three, or more than three hydrogen atoms in a group can be substituted by various substituents independently of each other. The substituents can be selected from alkyl, alkenyl, alkoxy, halogen, cyano, amino, nitro, and -OH groups.

[0143] The term "alkyl" refers to a saturated straight-chain 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, more 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, or octyl. The alkyl group may optionally be substituted.

[0144] The term "alkoxy group" includes -O-alkyl groups and alkyl groups in which the O atom is within an alkyl chain, such as -CH2-O-CH3, which contain 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. The alkoxy group may optionally be substituted.

[0145] The term "alkenyl" includes both straight-chain alkyl groups and branched-chain alkyl groups containing at least two carbon atoms and at least one carbon-carbon double bond, comprising 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and most preferably 2 to 3 carbon atoms. The alkenyl group may optionally be substituted.

[0146] The term "alkynyl" includes both straight-chain alkyl groups and branched-chain alkyl groups containing at least two carbon atoms and at least one carbon-carbon triple bond, comprising 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and most preferably 2 to 3 carbon atoms. The alkynyl group may optionally be substituted.

[0147] The terms "cycloalkyl," "cycloalkenyl," and "cycloynyl," either alone or in combination with other terms, denote the cyclic form of "alkyl," "alkenyl," and "ynyl," respectively, preferably with 3, 4, 5, 6, 7, 8, 9, or 10 atoms forming a ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclopropynyl, cyclobutynyl, cyclohexynyl, cyclopentynyl, etc., wherein the cycloalkenyl or cycloynyl group can be attached to other groups at any suitable position. The terms "cycloalkyl," "cycloalkenyl," and "cycloynyl" are also intended to include their bicyclic, tricyclic, and polycyclic forms, which can be spirocyclic or bridged rings. "Cycloalkyl," "cycloalkenyl," and "cycloynyl" may optionally 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, norbornyl, etc.

[0148] The term "aryl" preferably refers to an aromatic monocyclic system containing 6 carbon atoms, an aromatic bicyclic system containing 10 carbon atoms, or an aromatic tricyclic system containing 14 carbon atoms. Examples are phenyl, naphthyl, or anthracene. The aryl group may optionally be substituted.

[0149] The term "heteroaryl" preferably refers to a five- or six-membered aromatic monocyclic ring in which at least one carbon atom is substituted by one, two, three, or four (for five-membered rings) or one, two, three, four, or five (for six-membered rings) identical or different heteroatoms, the heteroatoms preferably selected from O, N, and S; or an aromatic bicyclic system in which one, two, three, four, five, or six of eight, nine, ten, eleven, or twelve carbon atoms are substituted by identical or different heteroatoms, the heteroatoms preferably selected from O, N, and S; or an aromatic tricyclic system in which one, two, three, four, five, or six of thirteen, fourteen, fifteen, or sixteen carbon atoms are substituted by identical or different heteroatoms, the heteroatoms preferably selected from O, N, and S. Examples include oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrroleyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, and 1-benzofuranyl. 2-Benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indolazinyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzoisothiazolyl, 2,1-benzoisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0150] The term "PEG group" refers to a group consisting of one or more -CH2-CH2-O- or CH3-CH2-O- units linked together, such as CH3-CH2-O-(CH2-CH2-O). x - where x is selected from integers from 0 to 6, preferably integers from 0 to 4. The PEG group may optionally be substituted.

[0151] The term "pharmaceuticalally acceptable salt" refers to a relatively non-toxic addition salt of the compounds disclosed herein. See, for example, SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.

[0152] Suitable pharmaceutically acceptable salts of the compounds disclosed herein may be, for example, acid addition salts of the compounds of this disclosure that carry a nitrogen atom in the chain or ring and are sufficiently basic, such as acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, or acid addition salts formed with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, etc. 3-Hydroxy-2-naphtholic acid, nicotinic acid, pyruvic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, pentanoic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric 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, glucohepanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanate.

[0153] Alternatively, another suitable pharmaceutically acceptable salt of the compounds of the present invention having sufficient acidity is an alkali metal salt such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, an ammonium salt, a triethylamine salt, or a salt formed with an organic base that provides a physiologically acceptable cation, such as a salt formed with N-methylglucosamine, dimethylglucosamine, ethylglucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serine, trihydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternized using the following reagents: lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and dipentyl sulfate; long-chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, etc.

[0154] Those skilled in the art will also recognize that the acid addition salts of the claimed compounds can be prepared by reacting the compounds with a suitable inorganic or organic acid using any of a variety of known methods. Alternatively, the alkali metal and alkaline earth metal salts of the acidic compounds of this disclosure can be prepared by reacting them with a suitable base using a variety of known methods.

[0155] This invention includes all possible salts of the compounds disclosed herein, which may be a single salt or any mixture of said salts in any proportion.

[0156] The term "solvent" refers to a substance formed by combining, physically binding, and / or solvating a compound of the present invention with solvent molecules, such as a disolvent, monosolvent, or hemisolvent, wherein the ratio of solvent molecules to the compound of the present invention is about 2:1, about 1:1, or about 1:2, respectively. This physical binding involves, to varying degrees, ionization and covalent bonding (including hydrogen bonding). In some cases (e.g., when one or more solvent molecules are bound to the lattice of a crystalline solid), the solvate can be separated. Therefore, a solvate includes a solution phase and a separable solvate. The compounds of the present invention can be in a solvated form with pharmaceutically acceptable solvents (e.g., water, methanol, and ethanol), and this application is intended to cover both solvated and non-solvated forms of the compounds of the present invention. One solvate is a hydrate.

[0157] As used in this application, the term "pharmaceutical composition" refers to a substance and / or combination of substances used to identify, prevent, or treat a tissue condition or disease. A pharmaceutical composition is formulated to be administered to a patient for the diagnosis, prevention, and / or treatment of a disease. Additionally, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier, making the composition suitable for therapeutic use.

[0158] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or transporter that is administered with a therapeutic agent. Such a drug carrier can be a sterile liquid, such as a saline solution in water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions are preferred carriers when the drug composition is administered intravenously. Saline solutions, as well as aqueous solutions of glucose and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. Examples of suitable drug carriers are described in EWMartin's "Remington's Pharmaceutical Sciences".

[0159] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0160] The term "optional" means that the situation may or may not occur.

[0161] Example

[0162] Reagents and models used

[0163] The starting materials used in the examples are commercially available and / or can be prepared using various methods well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select the reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of the experiment, and post-treatment) from the synthetic methods described below. Those skilled in the art of organic synthesis will understand that the functional groups present on the various parts of the molecule should be compatible with the proposed reagents and reactions.

[0164] All reagents and compounds synthesized were available in China through general commercial channels. Suppliers included Sigma-Aldrich (USA), Trinlink Cleancap (Shanghai), and jetMESSENGER. Shanghai Titan Technology Co., Ltd., etc.

[0165] Cell models: HEK293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and HepG2 cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0166] Main instruments used: Multifunctional microplate reader (Molecular Devices), flow cytometer (CytoFLEX S series).

[0167] Compound preparation and identification: Nuclear magnetic resonance (NMR) instrument (Bruker 300MHz), liquid chromatography-mass spectrometry (Agilent 6150 / 1290), high performance liquid chromatography (Agilent 1260).

[0168] Cell experiments: Inverted fluorescence microscope (Guangzhou Mingmei Optoelectronic Technology Co., Ltd.), cell culture incubator (ThermoFisher Scientific).

[0169] The following detailed embodiments illustrate the implementation process and beneficial effects of the present invention, aiming to help readers better understand the essence and characteristics of the present invention, and are not intended to limit the scope of implementation of this case.

[0170] Synthesis of final product:

[0171]

[0172] Method 1 for synthesizing the final product

[0173] 0.4 g of compound 1 was dissolved in 8 mL of anhydrous DMSO. Under argon protection, 2 eq of compound 2 and 20 eq of anhydrous zinc chloride were added. The reaction mixture was stirred at room temperature (25 °C) for 24 hours under argon protection. After the reaction was completed by TLC monitoring, the reaction was terminated with 150 mL of 0.25 M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate. The eluent containing the product was collected and lyophilized to obtain the final product.

[0174] Method 2 for synthesizing the final product

[0175] 0.2 g of compound 2 was dissolved in 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, and then 0.2 g of compound 1 was added to the same solution. The reaction mixture was stirred at room temperature (25°C) for 16 hours. After the reaction was completed by TLC monitoring, the reaction was terminated with 150 mL of 0.25 M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate. The eluent containing the product was collected and lyophilized to obtain the product.

[0176] Synthesis of representative structural compounds

[0177] Example 1

[0178] Synthesis of Compound 3

[0179]

[0180] Step 1:

[0181] Compound 3-2 (2.38 g) was added to a 63 mL solution of tetrazolium (1.76 g) in acetonitrile in a three-necked flask, and the mixture was purged with argon three times. Then, at room temperature (25°C), compound 3-1 (5 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature (25°C) for 1 hour, and no significant exothermic reaction was observed. TLC showed that compound 3-1 disappeared. Then, a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise to the solution until the solution no longer faded. The reaction mixture was then stirred for 0.5 hours, and TLC showed that oxidation was complete. The reaction mixture was quenched with saturated sodium sulfite aqueous solution (10 mL), diluted with 50 mL of water, extracted with dichloromethane (50 mL x 2), and the combined organic phases were washed once with water (50 mL). The mixture was concentrated to obtain a pale yellow oily product 3-3 (8 g, crude product).

[0182] Step 2:

[0183] Compound 3-3 (8 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water. The reaction was stirred at 25 °C for 16 hours. TLC monitoring showed that compound 3-3 disappeared, and highly polar spots were formed. The reaction solution was directly concentrated under vacuum. After concentration, appropriate amounts of silica gel and DCM were added, and the mixture was purified (40 g normal phase column, EA, 10 min; DCM: MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white solid product 3-4 (2.8 g, 51% two-step yield).

[0184] Step 3:

[0185] Prepare 28 mL of tetrazolium in acetonitrile (0.4 mmol / mL). Add compound 3-4 (2.8 g) to the above solution, then add compound A1 (3 g) to the solution at room temperature (25°C). Purge with nitrogen three times, and stir the reaction solution at room temperature (25°C) for 1 hour. TLC monitoring showed the reaction was complete. Cool the reaction solution to below 10°C in an ice-water bath, and add dropwise a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) until the reaction solution no longer fades. TLC monitoring showed the oxidation reaction was complete. Quench the reaction solution with 10 mL of saturated sodium sulfite aqueous solution, then dilute with water. Extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amounts of silica gel and DCM for mixing and purification (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white, foamy solid compound 3-6 (2.6 g, 78.2% yield).

[0186] Step 4:

[0187] Compound 3-6 (2.6 g) was dissolved in methanol (30 mL), and then concentrated ammonia (30 mL) was added. The resulting solution was stirred at 25°C for 60 hours at room temperature. TLC showed that compounds 1-6 reacted completely. The reaction solution was concentrated under vacuum and then concentrated again with methanol to obtain a pale yellow oily liquid compound 3-7 (2.4 g, crude product). The crude product was directly added to the next step.

[0188] Step 5:

[0189] Compound 3-7 (2.4 g, crude) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction mixture was stirred at 50 °C for 1 hour, and TLC showed that compound 3-7 reacted completely. The reaction mixture was diluted with water to 50 mL, and the pH was adjusted to 5.5 with 1 N NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give the amine salt 3-8 of the target compound (0.8 g, 33.7% yield) as a white solid.

[0190] Step 6:

[0191] Compound 9a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by the addition of compounds 3-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 5, an ammonium salt (65 mg).

[0192] 1 H NMR(400MHz,D2O)δ8.08(s,1H),7.89(s,1H),7.65(s,1H),5.93(d,J=17.9Hz,1H),5.70( d,J=6.4Hz,2H),5.28–5.02(m,1H),4.58–3.99(m,14H),3.84(s,3H),3.00–2.70(m,3H).

[0193] 31 P NMR(162MHz,D2O)δ-0.80,-11.55,-23.03.

[0194] Example 2

[0195] Synthesis of Compound 35

[0196]

[0197] Step 1:

[0198] Compound 35-2 (2.07 g) was added to a 56 mL solution of tetrazolium (1.6 g) in acetonitrile in a three-necked flask. Argon gas was purged three times. Then, at room temperature (25°C), compound 35-1 (5 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature (25°C) for 1 hour. No significant exothermic reaction was observed, and TLC showed the disappearance of compound 2-1. Then, iodine solution (5 g iodine dissolved in 40 mL of a mixed solution of THF:H₂O:pyridine = 8:1:1, prepared to a 0.5 mmol / mL solution) was added dropwise until the solution no longer decolorized. The reaction mixture was then stirred for 0.5 hours, and TLC showed the oxidation was complete. After quenching the reaction solution with 10 mL of Na2SO3 aqueous solution, dilute with 50 mL of water, extract with dichloromethane (50 mL × 2), combine the organic phases, wash once with water (50 mL), and concentrate to obtain a pale yellow oily product 35-3 (7.5 g, crude product).

[0199] Step 2:

[0200] Compound 35-3 (7.2 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water. The reaction was stirred at 25 °C for 16 hours. TLC monitoring showed that compound 35-3 disappeared, and highly polar spots were formed. The reaction solution was directly concentrated under vacuum. After concentration, appropriate amounts of silica gel and DCM were added, and the mixture was purified (40 g normal phase column, EA, 10 min; DCM: MeOH, 10-20% for 20 min, flow rate 30 mL / min). Concentration yielded a white solid product 35-4 (2.8 g, 54.8% two-step yield).

[0201] Step 3:

[0202] Prepare 28 mL of tetrazolium in acetonitrile (0.4 mmol / mL). Add compound 35-4 (2.8 g) to the above solution, then add compound A1 (2.26 g) to the solution at room temperature (25°C). Purge with nitrogen three times, and stir the reaction mixture at room temperature (25°C) for 1 hour. TLC monitoring showed the reaction was complete. Cool the reaction mixture to below 10°C in an ice-water bath, and add dropwise a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) until the reaction mixture no longer fades. TLC monitoring showed the oxidation reaction was complete. Quench the reaction mixture with 10 mL of saturated sodium sulfite aqueous solution, then dilute with water. Extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amounts of silica gel and DCM for mixing and purification (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white, foamy solid 35-6 (3.1 g, 93.5% yield).

[0203] Step 4:

[0204] Compound 35-6 (3.1 g) was dissolved in methanol (30 mL), and then concentrated ammonia (30 mL) was added. The resulting solution was stirred at 25°C for 60 hours at room temperature. TLC showed that compound 35-6 reacted completely. The reaction solution was concentrated under vacuum and then concentrated again with methanol to give compound 35-7 (2.4 g, crude product), a pale yellow oily liquid.

[0205] Step 5:

[0206] Compound 35-7 (2.4 g, crude) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction mixture was stirred at 50 °C for 1 hour, and TLC showed that compound 35-7 reacted completely. The reaction mixture was diluted with water to 50 mL, and the pH was adjusted to 5.5 with 1 N NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0-1.0 M TEAB eluent. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give the triethylamine salt 35-8 of the target compound (1.5 g, 51% yield) as a white solid.

[0207] Step 5:

[0208] Under argon protection at room temperature (25°C), compounds 35-8 (500 mg), 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added separately. Anhydrous DMSO (8 mL) was then added via syringe, and the reaction proceeded for 24 hours. TLC showed that most of the compounds had reacted. The reaction mixture was then added to a pre-cooled 0°C solution of disodium EDTA (1.6 g, with 80 mL of water), and the mixture was loaded onto a DEAE-Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to obtain a white powder product, compound 35, an ammonium salt (120 mg).

[0209] 1 H NMR(400MHz,D2O)δ8.25(s,1H),7.98(s,1H),7.59(d,J=7.3Hz,1H),5.99–5. 53(m,4H),4.96–4.80(m,1H),4.46–3.99(m,14H),3.90(s,3H),3.41(s,3H).

[0210] 31 P NMR(162MHz,D2O)δ-1.25,-11.58,-22.98.

[0211] Example 3

[0212] Synthesis of Compound 68

[0213]

[0214] Step 1:

[0215] Compound 3-2 (2.07 g) was added to a 56 mL acetonitrile solution of tetrazolium (1.6 g) in a three-necked flask. Argon gas was purged three times. Then, at room temperature (25°C), compound 68-1 (5 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature (25°C) for 1 hour. No significant exothermic reaction was observed, and TLC monitoring showed that compound 68-1 disappeared. Then, iodine solution (5 g iodine dissolved in 40 mL of a mixed solution of THF:H₂O:pyridine = 8:1:1, prepared to a 0.5 mmol / mL solution) was added dropwise until the solution no longer decolorized. The reaction mixture was then stirred for 0.5 hours, and TLC monitoring showed that oxidation was complete. After quenching the reaction solution with 10 mL of Na2SO3 aqueous solution, 50 mL of water was added for dilution. The solution was then extracted with 50 mL of dichloromethane (50 mL × 2). The combined organic phases were washed once with 50 mL of water and concentrated to obtain a pale yellow oily product 68-3 (7.5 g, crude product).

[0216] Step 2:

[0217] Compound 68-3 (7.2 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water. The reaction was stirred at 25 °C for 16 hours. TLC monitoring showed that compound 68-3 disappeared, and highly polar spots were formed. The reaction solution was directly concentrated under vacuum. After concentration, appropriate amounts of silica gel and DCM were added, and the mixture was purified (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% for 20 min, flow rate 30 mL / min). Concentration yielded a white solid product 68-4 (2.8 g, 54.8% two-step yield).

[0218] Step 3:

[0219] Prepare 28 mL of tetrazolium in acetonitrile (0.4 mmol / mL). Add compound 68-4 (2.8 g) to the above solution, then add compound A1 (2.26 g) to the solution at room temperature (25°C). Purge with nitrogen three times, and stir the reaction mixture at room temperature (25°C) for 1 hour. TLC monitoring showed the reaction was complete. Cool the reaction mixture to below 10°C in an ice-water bath, and add dropwise a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) until the reaction mixture no longer fades. TLC monitoring showed the oxidation reaction was complete. Quench the reaction mixture with 10 mL of saturated sodium sulfite aqueous solution, then dilute with water. Extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amounts of silica gel and DCM for mixing and purification (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white, foamy solid, 68-6 (3.1 g, 93.5% yield).

[0220] Step 4:

[0221] Compound 68-6 (3.1 g) was dissolved in methanol (30 mL), and then concentrated ammonia (30 mL) was added. The resulting solution was stirred at 25°C for 60 hours at room temperature. TLC showed that compound 68-6 reacted completely. The reaction solution was concentrated under vacuum and then concentrated again with methanol to give compound 68-7 (2.4 g, crude product), a pale yellow oily liquid.

[0222] Step 5:

[0223] Compound 68-7 (2.4 g, crude) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction mixture was stirred at 50 °C for 1 hour, and TLC showed that compound 68-7 reacted completely. The reaction mixture was diluted with water to 50 mL, and the pH was adjusted to 5.5 with 1 N NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give the triethylamine salt 68-8 of the target compound (1.5 g, 51% yield) as a white solid.

[0224] Step 6:

[0225] Under argon protection at room temperature (25°C), compounds 68-8 (500 mg), 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added separately. Anhydrous DMSO (8 mL) was then added via syringe, and the reaction proceeded for 24 hours. TLC showed that most of the starting materials had reacted. The reaction mixture was then added to a pre-cooled 0°C solution of disodium EDTA (1.6 g, with 80 mL of water), and the mixture was loaded onto a DEAE-Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder product, compound 68, as an ammonium salt (120 mg).

[0226] 1 H NMR(400MHz,D2O)δ7.75(s,1H),7.61(d,J=8.1Hz,1H),5.97(dd,J=17.8,2.3Hz,1H),5.85–5.65(m,3H),5.28(ddd,J=52.2,4.7,2.3Hz,1H),5.01 (dt,J=51.5,3.4Hz,1H),4.57–4.51(m,2H),4.48(t,J=4.2Hz,1H),4.34 (t,J=5.1Hz,1H),4.24–4.07(m,7H),4.02(t,J=4.3Hz,2H),3.93(s,3H).

[0227] 31 P NMR (162MHz, D2O) δ -1.30, -11.57 (dd, J = 25.8, 18.4Hz), -22.98 (t, J = 18.3Hz).

[0228] Example 4

[0229] Synthesis of Compound 83

[0230]

[0231] Step 1:

[0232] Compound 3-2 (2.1 g) was added to a 56 mL acetonitrile solution of tetrazolium (1.6 g) in a three-necked flask. Argon gas was purged three times. Then, at room temperature (25°C), compound 83-1 (5 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature (25°C) for 1 hour. No significant exothermic reaction was observed, and TLC monitoring showed that compound 83-1 disappeared. Then, an iodine solution (5 g iodine dissolved in 40 mL of a mixed solution of THF:H₂O:pyridine = 8:1:1, prepared to a 0.5 mmol / mL solution) was added dropwise until the solution no longer decolorized. The reaction mixture was then stirred for another 0.5 hours, and TLC monitoring confirmed the oxidation was complete. After quenching the reaction solution with 10 mL of Na2SO3 aqueous solution, 50 mL of water was added for dilution. The solution was then extracted with 50 mL of dichloromethane (50 mL x 2). The combined organic phases were washed once with 50 mL of water and concentrated to obtain a pale yellow oily product 83-3 (7.4 g, crude product).

[0233] Step 2:

[0234] Compound 83-3 (7.4 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water. The reaction was stirred at 25 °C for 16 hours. TLC monitoring showed that compound 83-3 disappeared, and highly polar spots were formed. The reaction solution was directly concentrated under vacuum. After concentration, appropriate amounts of silica gel and DCM were added, and the mixture was purified (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% for 20 min, flow rate 30 mL / min). Concentration yielded a white solid product 83-4 (2.8 g, 54.8% two-step yield).

[0235] Step 3:

[0236] Prepare 28 mL of tetrazolium in acetonitrile (0.4 mmol / mL). Add compound 83-4 (2.8 g) to the above solution, then add Al (2.26 g) to the solution at room temperature (25°C). Purge with nitrogen three times, and stir the reaction solution at room temperature (25°C) for 1 hour. TLC monitoring showed the reaction was complete. Cool the reaction solution to below 10°C in an ice-water bath, and add dropwise a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) until the reaction solution no longer fades. TLC monitoring showed the oxidation reaction was complete. Quench the reaction solution with 10 mL of saturated sodium sulfite aqueous solution, then dilute with water. Extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amounts of silica gel and DCM for mixing and purification (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20%, 20 min, flow rate 30 mL / min). Concentration yielded a white, foamy solid 83-6 (3.1 g, 93.5% yield).

[0237] Step 4:

[0238] Compound 83-6 (3.1 g) was dissolved in methanol (30 mL), and then concentrated ammonia (30 mL) was added. The resulting solution was stirred at 25°C for 60 hours at room temperature. TLC showed that compound 83-6 reacted completely. The reaction solution was concentrated under vacuum and then concentrated again with methanol to give compound 83-7 (2.4 g, crude product), a pale yellow oily liquid.

[0239] Step 5:

[0240] Compound 83-7 (2.4 g, crude) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction mixture was stirred at 50 °C for 1 hour, and TLC showed that compound 83-7 reacted completely. The reaction mixture was diluted with water to 50 mL, and the pH was adjusted to 5.5 with 1 N NaOH aqueous solution. The mixture was then loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give the triethylamine salt 83-8 of the target compound (1.5 g, 51% yield) as a white solid.

[0241] Step 6:

[0242] Under argon protection at room temperature (25°C), compounds 83-8 (500 mg), 9a (500 mg), and anhydrous zinc chloride (1.2 g) were added separately. Anhydrous DMSO (8 mL) was then added via syringe, and the reaction proceeded for 24 hours. TLC showed that most of the starting materials had reacted. The reaction mixture was then added to a pre-cooled 0°C solution of disodium EDTA (1.6 g, with 80 mL of water), and the mixture was loaded onto a DEAE-Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder product, compound 83, an ammonium salt (120 mg).

[0243] 1 H NMR(400MHz,D2O)δ8.30(d,J=0.6Hz,1H),8.20(s,1H),8.02(d,J=0.7Hz,1H),6.59–6.30(m,2H),6.23–6.08(m,1H),5.2 2(dddd,J=46.4,7.0,1.7,0.7Hz,1H),5.08–4.90(m,1H),4.68–4.03(m,12H),3.98(d,J=0.7Hz,3H),2.76–2.48(m,2H).

[0244] 31P NMR (162MHz, D2O) δ -1.30, -11.57 (dd, J = 25.8, 18.4Hz), -22.98 (t, J = 18.3Hz).

[0245] Example 5

[0246] Synthesis of Compound 90

[0247]

[0248] Compound 9a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 90-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum, and the remaining liquid was lyophilized to give a white powder, compound 90, as an ammonium salt (50 mg).

[0249] 1 H NMR (400MHz, D2O) δ7.83 (d, J=0.6Hz, 1H), 7.70 (dd, J=7.4, 1.8Hz, 1H), 6.28 (ddq, J=3. 1,1.5,0.7Hz,1H),6.20–6.13(m,1H),6.04(d,J=7.3Hz,1H),5.40(dddd,J=25.2,3.5,1 .8,0.9Hz,1H),5.09–4.93(m,1H),4.81(dddd,J=46.4,7.1,3.7,0.7Hz,1H),4.61(ddd ,J=5.2,2.8,0.7Hz,1H),4.41–4.05(m,11H),3.98(d,J=0.7Hz,3H),2.81–2.38(m,2H).

[0250] 31 P NMR(162MHz,D2O)δ-1.17,-11.50,-22.81.

[0251] Example 6

[0252] Synthesis of Compound 103

[0253]

[0254] Compound 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by the addition of compounds 3-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 103, an ammonium salt (60 mg).

[0255] 1 H NMR(400MHz,D2O)δ8.33(d,J=0.5Hz,1H),8.29(s,1H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.2 ,1.4,0.7Hz,1H),6.17(dq,J=2.3,0.8Hz,1H),6.11(dq,J=3.1,0.8Hz,1H),5.22(dddd,J=46.4,7 .0,1.7,0.7Hz,1H),4.88–4.62(m,3H),4.45(dddd,J=25.2,6.8,5.0,0.7Hz,1H),4.33–4.09(m,9 H), 4.05 (ttd, J = 2.9, 1.5, 0.7Hz, 1H), 3.98 (d, J = 0.7Hz, 3H), 3.44 (d, J = 1.4Hz, 3H), 3.08 (s, 3H).

[0256] 31 P NMR (162MHz, D2O) δ0.60, -10.29, -21.23.

[0257] Example 7

[0258] Synthesis of Compound 119

[0259]

[0260] Compound 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 119-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum, and the remaining liquid was lyophilized to give a white powder, compound 119, as an ammonium salt (65 mg).

[0261] 1 H NMR(400MHz,D2O)δ7.81(dd,J=7.8,1.8Hz,1H),7.76(dd,J=7.8,1.8Hz,1H),6.17(dt,J=2.5,0.7Hz,1H),5.90(d,J=7.8Hz,1H),5.85–5. 73(m,2H),5.53(dddd,J=25.2,3.9,1.7,0.9Hz,1H),4.89–4.60(m,3H),4.46–4.02(m,12H),3.98(d,J=0.7Hz,3H),3.44(d,J=1.4Hz,3H).

[0262] 31 P NMR (162MHz, D2O) δ0.62, -10.29, -21.22.

[0263] Example 8

[0264] Synthesis of Compound 127

[0265]

[0266] Compound 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 127-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 127, an ammonium salt (82 mg).

[0267] 1 H NMR(400MHz,D2O)δ8.29(s,1H),8.24(d,J=0.7Hz,1H),8.21–8.18(m,2H),6.54–6.00(m,3H),5.27(dddd,J=4 6.5,7.0,4.0,0.6Hz,1H),4.97(dddd,J=8.0,4.4,2.9,0.7Hz,1H),4.77(tqd,J=4.4,1.5,0.7Hz,1H),4.71(d dd,J=3.9,2.6,0.7Hz,1H),4.45(dddd,J=25.2,6.8,5.1,0.7Hz,1H),4.35(qt,J=3.0,0.7Hz,1H),4.29–4.08 (m, 8H), 4.05 (ttd, J = 2.9, 1.5, 0.7Hz, 1H), 3.98 (d, J = 0.7Hz, 3H), 3.42 (dd, J = 18.0, 1.5Hz, 6H), 3.08 (s, 3H).

[0268] 31 P NMR (162MHz, D2O) δ0.60, -10.29, -21.22.

[0269] Example 9

[0270] Synthesis of Compound 167

[0271]

[0272] Compound 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 24-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 167, an ammonium salt (62 mg).

[0273] 1H NMR(400MHz,D2O)δ8.24(d,J=0.7Hz,1H),8.20(s,1H),7.81(dd,J=7.9,1.8Hz,1H),6.39(dd t,J=25.2,4.0,0.7Hz,1H),6.17(dq,J=2.3,0.7Hz,1H),5.90(d,J=7.8Hz,1H),5.56(dddt,J =25.3,3.4,1.8,0.9Hz,1H),5.43–4.99(m,3H),4.79–4.65(m,1H),4.56–4.38(m,2H),4.35– 4.06 (m, 8H), 4.05 (ddq, J = 4.6, 2.2, 1.1Hz, 1H), 3.98 (d, J = 0.7Hz, 3H), 3.44 (d, J = 1.4Hz, 3H).

[0274] 31 P NMR (162MHz, D2O) δ0.60, -10.29, -21.22.

[0275] Example 10

[0276] Synthesis of Compound 180

[0277]

[0278] Compound 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 180-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 180, an ammonium salt (95 mg).

[0279] 1H NMR(400MHz,D2O)δ8.35(d,J=0.6Hz,1H),8.29(s,1H),7.70(dd,J=7.4,1.8H z,1H),6.47–6.32(m,1H),6.17(dq,J=2.3,0.8Hz,1H),6.04(d,J=7.3Hz,1H), 5.54–5.33(m,1H),5.10–4.94(m,1H),4.91–4.64(m,2H),4.48–4.03(m,11H), 3.98(d,J=0.7Hz,3H), 3.44(d,J=1.4Hz,3H), 3.08(s,3H), 2.83–2.35(m,2H).

[0280] 31 P NMR (162MHz, D2O) δ0.60, -10.29, -21.22.

[0281] Example 11

[0282] Synthesis of Compound 203

[0283]

[0284] Compound 9c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 203-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 203, an ammonium salt (95 mg).

[0285] 1H NMR(400MHz,D2O)δ8.33(d,J=0.6Hz,1H),8.29(s,1H),8.02(d,J=0.7Hz,1H) ,6.45(ddq,J=25.2,1.4,0.7Hz,1H),6.23(dt,J=2.3,0.8Hz,1H),6.11(dq,J =3.1,0.8Hz,1H),5.22(dddd,J=46.4,7.0,1.7,0.7Hz,1H),5.00–4.68(m,4H ), 4.65–4.39 (m, 2H), 4.38–4.05 (m, 8H), 3.98 (d, J = 0.7Hz, 3H), 3.08 (s, 3H).

[0286] 31 P NMR (162MHz, D2O) δ0.60, -10.29, -21.22.

[0287] Example 12

[0288] Synthesis of Compound 239

[0289]

[0290] Compound 9c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 239-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 239, as an ammonium salt (95 mg).

[0291] 1 H NMR(400MHz,D2O)δ7.88(d,J=0.7Hz,1H),7.81(dd,J=7.8,1.8Hz,1H),6.33–6.06(m,2H),5.90(d,J=7.8Hz,1H),5.53( dddd,J=25.2,3.9,1.7,0.9Hz,1H),5.03–4.50(m,6H),4.43–4.07(m,9H),3.98(d,J=0.7Hz,3H),3.40(d,J=1.6Hz,3H).

[0292] 31P NMR (162MHz, D2O) δ0.60, -10.29, -21.22.

[0293] Example 13

[0294] Synthesis of Compound 260

[0295]

[0296] Compound 9c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 260-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 260, as an ammonium salt (75 mg).

[0297] 1 H NMR(400MHz,D2O)δ8.24(d,J=0.7Hz,1H),8.20(s,1H),7.70(dd,J=7.4,1.8Hz,1H),6.43(ddq,J=25.2,1.6,0.8Hz,1H),6 .27–6.20(m,1H),6.04(d,J=7.3Hz,1H),5.51–5.12(m,3H),5.05–4.70(m,3H),4.66–4.10(m,10H),3.98(d,J=0.7Hz,3H).

[0298] 31 P NMR(162MHz,D2O)δ-0.90,-10.29,-21.22.

[0299] Example 14

[0300] Synthesis of Compound 287

[0301]

[0302] 9c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, and then compound 287-1 (200 mg) was added to this solution. The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, added to 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder product, compound 287, as an ammonium salt (75 mg).

[0303] 1 H NMR(400MHz,D2O)δ8.24(d,J=0.7Hz,1H),8.20(s,1H),7.83(d,J=0.6Hz,1H),6.39(ddt,J=25.2,4.0,0.7Hz,1H),6.28(ddt,J=3.1,1.5,0.7Hz,1 H),6.26–6.15(m,1H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),5.11–4. 79(m,3H),4.65–4.03(m,10H),3.98(d,J=0.7Hz,3H),2.88–2.51(m,2H).

[0304] 31 P NMR(162MHz,D2O)δ-1.11,-10.31,-21.25.

[0305] Example 15

[0306] Synthesis of Compound 126

[0307]

[0308] 9d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 3-8 (200 mg) to the same solution. The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powdery product, compound 308, which was an ammonium salt (60 mg).

[0309] 1 H NMR(400MHz,D2O)δ8.28(d,J=0.6Hz,1H),8.20(s,1H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.3,1.6,0.9Hz,1H),6.21–6.07(m, 2H), 5.22(dddd,J=46.4,7.0,1.7,0.7Hz,1H),4.98–4.68(m,2H),4.57–4.03(m,11H),3.98(d,J=0.7Hz,3H),2.45–1.75(m,2H).

[0310] 31 P NMR(162MHz,D2O)δ-0.9,-10.29,-21.27.

[0311] Example 16

[0312] Synthesis of Compound 326

[0313]

[0314] Compound 9d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 326-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 326, as an ammonium salt (60 mg).

[0315] 1H NMR(400MHz,D2O)δ8.29(d,J=1.7Hz,3H),8.20(d,J=0.7Hz,1H),6.39(ddt,J=25.1, 3.9,0.7Hz,1H),6.29–6.04(m,2H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),4.97( dddd,J=8.0,4.4,2.9,0.7Hz,1H),4.77(tqd,J=4.4,1.5,0.7Hz,1H),4.61–4.06(m, 11H), 3.98 (d, J = 0.7Hz, 3H), 3.40 (d, J = 1.6Hz, 3H), 3.08 (s, 6H), 2.56–1.44 (m, 2H).

[0316] 31 P NMR(162MHz,D2O)δ-0.78,-10.29,-21.22.

[0317] Example 17

[0318] Synthesis of Compound 352

[0319]

[0320] Compound 9d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 352-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 352, an ammonium salt (74 mg).

[0321] 1 H NMR (400MHz, D2O) δ8.33–8.27(m,2H),8.24(d,J=0.6Hz,1H),8.20(s,1H),6.63–6.28(m,2H),6.17(dt,J=1. 3,0.7Hz,1H),5.57–5.04(m,3H),4.63–4.03(m,11H),3.98(d,J=0.7Hz,3H),3.08(s,3H),2.63–1.60(m,2H).

[0322] 31 P NMR(162MHz,D2O)-0.55,-10.32,-21.28.

[0323] Example 18

[0324] Synthesis of Compound 386

[0325]

[0326] Compound 9d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 386-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 386, as an ammonium salt (60 mg).

[0327] 1 H NMR (400MHz, D2O) δ8.38–8.22(m,2H),8.18(d,J=0.6Hz,1H),6.58–6.30(m,2H),6.17(t,J=1.0Hz,1H),5.27(dddd,J=46.5,7.0,4 .0,0.6Hz,1H),5.10–4.83(m,1H),4.60–4.07(m,11H),3.98(d,J=0.7Hz,3H),3.08(s,3H),2.69–2.46(m,2H),2.34–1.69(m,2H).

[0328] 31 P NMR(162MHz,D2O)δ-0.85,-10.09,-21.23.

[0329] Example 19

[0330] Synthesis of Compound 403

[0331]

[0332] Compound 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 403-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 403, an ammonium salt (55 mg).

[0333] 1 H NMR(400MHz,D2O)δ8.40–8.19(m,2H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.2,1.4,0.7Hz ,1H),6.33(dd,J=2.7,0.6Hz,1H),6.11(dq,J=3.0,0.7Hz,1H),5.22(dddd,J=46.4,7.0,1. 7,0.7Hz,1H),4.80(dddd,J=3.9,3.1,0.7Hz,1H),4.74(dddd,J=7.9,3.9,2.9,0.7Hz,1H),4 .64(t,J=2.8Hz,1H),4.51–4.08(m,10H),4.02(d,J=0.7Hz,2H),3.98(s,3H),3.08(s,3H).

[0334] 31 P NMR(162MHz,D2O)δ-0.90,-11.41,-22.93.

[0335] Example 20

[0336] Synthesis of Compound 443

[0337]

[0338] Compound 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 443-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 443, an ammonium salt (70 mg).

[0339] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),7.75(dd,J=7.8,1.8Hz,1H),6.45(ddq, J=25.2,1.6,0.9Hz,1H),6.33(dd,J=2.7,0.6Hz,1H),5.93–5.65(m,2H),5.22(ddd d,J=46.4,7.0,1.7,0.7Hz,1H),4.89(dddd,J=8.0,5.4,4.6,0.6Hz,1H),4.64(t,J =2.8Hz,1H),4.51–4.07(m,11H),4.02(d,J=0.7Hz,2H),3.98(s,3H),3.47(s,3H).

[0340] 31 P NMR (162MHz, D2O) δ0.50, -9.18, -10.26, -21.22.

[0341] Example 21

[0342] Synthesis of Compound 467

[0343]

[0344] Compound 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 467-7 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 467, as an ammonium salt (75 mg).

[0345] 1 H NMR(400MHz,D2O)δ8.32–8.10(m,2H),7.81(dd,J=7.9,1.8Hz,1H),6.54–6.21(m,2H),5.90( d,J=7.8Hz,1H),5.71–4.98(m,4H),4.73–4.09(m,11H),4.02(d,J=0.7Hz,2H),3.98(s,3H).

[0346] 31 P NMR(162MHz,D2O)δ-0.99,-11.40,-23.06.

[0347] Example 22

[0348] Synthesis of Compound 480

[0349]

[0350] Compound 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 480-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 480, as an ammonium salt (85 mg).

[0351] 1H NMR(400MHz,D2O)δ8.41–8.23(m,2H),7.70(dd,J=7.4,1.8Hz,1H),6.43–6.2 8(m,2H),6.04(d,J=7.3Hz,1H),5.56–5.21(m,1H),5.09–4.93(m,1H),4.81(d ddd,J=46.5,7.1,3.7,0.7Hz,1H),4.64(t,J=2.8Hz,1H),4.48–4.03(m,10H), 4.02(d,J=0.7Hz,2H), 3.98(d,J=0.7Hz,3H), 3.08(s,3H), 2.79–2.46(m,2H).

[0352] 31 P NMR (162MHz, D2O) δ0.60, -9.01, -10.23, -21.17.

[0353] Example 23

[0354] Synthesis of Compound 501

[0355]

[0356] Compound 10b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 501-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 501, an ammonium salt (92 mg).

[0357] 1H NMR(400MHz,D2O)δ8.41–8.14(m,4H),6.39(ddt,J=25.3,4.1,0.8Hz,1H),6.2 5–5.98(m,2H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),4.83–4.61(m,3H),4. 45(dddd,J=25.2,6.8,5.0,0.7Hz,1H),4.36–4.07(m,10H),3.98(d,J=0.7Hz, 3H), 3.69–3.48 (m, 2H), 3.08 (s, 6H), 2.90–2.61 (m, 2H), 1.84 (p, J = 6.5Hz, 2H).

[0358] 31 P NMR (162MHz, D2O) δ0.60, -9.10, -10.31, -21.17.

[0359] Example 24

[0360] Synthesis of Compound 541

[0361]

[0362] Compound 10b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 541-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 541, as an ammonium salt (85 mg).

[0363] 1H NMR(400MHz,D2O)δ8.38–8.21(m,2H),7.75(dd,J=7.8,1.8Hz,1H),6.50–6.30(m,1H),6.27–6. 10(m,1H),5.95–5.71(m,2H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),4.89(dddd,J=8.0,5.4, 4.7,0.6Hz,1H),4.71(ddd,J=3.3,2.5,0.7Hz,1H),4.52–4.04(m,12H),3.98(d,J=0.7Hz,3H),3 .75–3.54(m,2H),3.47(d,J=1.6Hz,3H),3.08(s,3H),2.90–2.51(m,2H),1.84(p,J=6.5Hz,2H).

[0364] 31 P NMR (162MHz, D2O) δ0.60, -9.10, -10.22, -21.17.

[0365] Example 25

[0366] Synthesis of Compound 574

[0367]

[0368] Compound 10b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 574-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 574, an ammonium salt (80 mg).

[0369] 1H NMR(400MHz,D2O)δ7.81(dd,J=7.8,1.8Hz,1H),7.70(dd,J=7.4,1.8Hz,1H) ,6.29–6.11(m,1H),6.04(d,J=7.3Hz,1H),5.90(d,J=7.8Hz,1H),5.63–5.4 2(m,2H),5.33–5.00(m,2H),4.89–4.64(m,2H),4.55–4.05(m,11H),3.98(d ,J=0.7Hz,3H),3.75–3.50(m,2H),2.89–2.50(m,2H),1.84(p,J=6.5Hz,2H).

[0370] 31 P NMR(162MHz,D2O)δ-0.22,-9.07,-10.25,-21.27.

[0371] Example 26

[0372] Synthesis of Compound 589

[0373]

[0374] Compound 10b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 589-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 589, an ammonium salt (58 mg).

[0375] 1 H NMR(400MHz,D2O)δ7.93–7.46(m,2H),6.31–6.11(m,2H),5.90(d,J=7.8Hz,1H),5.53(dddd,J=25.2,3.9,1.7,0.9Hz,1H),5.13–4.93(m,1H), 4.88–4.61(m,2H),4.43–4.04(m,11H),3.98(d,J=0.7Hz,3H),3.70–3. 28(m,2H),2.79–2.71(m,2H),2.70–2.46(m,2H),1.84(p,J=6.5Hz,2H).

[0376] 31 P NMR (162MHz, D2O) δ0.66, -9.13, -10.26, -21.25.

[0377] Example 27

[0378] Synthesis of Compound 589

[0379]

[0380] Compound 10c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 589-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 589, an ammonium salt (88 mg).

[0381] 1 H NMR(400MHz,D2O)δ7.97(dd,J=37.6,0.6Hz,2H),6.71–6.22(m,2H),6.10(dq, J=1.7,0.7Hz,1H),5.22(dddd,J=46.4,7.0,1.7,0.7Hz,1H),4.87(t,J=3.1Hz, 1H),4.74(qt,J=4.1,2.2Hz,2H),4.55–4.10(m,10H),4.02(s,2H),3.98(s,3H ),3.66(td,J=5.3,2.9Hz,2H),3.00–2.60(m,2H),1.85(tt,J=6.4,5.3Hz,2H).

[0382] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0383] Example 28

[0384] Synthesis of Compound 664

[0385]

[0386] Compound 10c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 664-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 664, an ammonium salt (60 mg).

[0387] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),7.81(dd,J=7.8,1.8Hz,1H),6.57–6.23(m,2H),5.90(d,J=7.8Hz,1H),5.71–5.42(m,1H),5.39–5.16(m, 2H),4.94–4.67(m,2H),4.56–4.08(m,10H),4.02(s,2H),3.98(s,3H),3. 66(td,J=5.3,2.9Hz,2H),2.98–2.65(m,2H),1.85(tt,J=6.4,5.3Hz,2H).

[0388] 31 P NMR(162MHz,D2O)δ-0.85,-10.09,-21.23.

[0389] Example 29

[0390] Synthesis of Compound 706

[0391]

[0392] Compound 10d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 706-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 706, an ammonium salt (55 mg).

[0393] 1 H NMR(400MHz,D2O)δ8.40–7.81(m,4H),6.84–5.90(m,3H),5.31(dddd,J=46. 5,7.0,4.0,0.6Hz,1H),4.76(dddd,J=46.0,3.3,2.8,0.7Hz,2H),4.54–4.1 8(m,9H),4.09–3.95(m,3H),3.92(d,J=0.6Hz,3H),3.56(td,J=6.4,1.5Hz, 2H), 3.23 (td, J=6.3, 0.8Hz, 2H), 3.10 (s, 3H), 1.94 (pd, J=6.3, 1.0Hz, 2H).

[0394] 31 P NMR(162MHz,D2O)δ-0.90,-11.41,-22.93.

[0395] Example 30

[0396] Synthesis of Compound 760

[0397]

[0398] Compound 10d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 760-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 760, as an ammonium salt (70 mg).

[0399] 1 H NMR(400MHz,D2O)δ8.28–8.06(m,2H),7.69(dd,J=7.4,1.8Hz,1H),6.47(ddt,J=25.2,1.5,0.8Hz, 1H),6.28(dq,J=2.3,0.8Hz,1H),6.15(d,J=7.3Hz,1H),5.60(dddt,J=25.3,3.6,1.8,0.8Hz,1H),5 .42(dddd,J=46.5,2.8,1.9,0.7Hz,1H),4.70(dddd,J=3.3,2.5,0.7Hz,1H),4.50–3.96(m,13H),3.9 2(d,J=0.6Hz,3H), 3.56(td,J=6.5,1.5Hz,2H), 3.36(t,J=6.2Hz,2H), 1.94(pd,J=6.3,1.0Hz,2H).

[0400] 31 P NMR (162MHz, D2O) δ0.50, -9.18, -10.26, -21.22.

[0401] Example 31

[0402] Synthesis of Compound 787

[0403]

[0404] Compound 10d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 787-7 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 787, as an ammonium salt (75 mg).

[0405] 1 H NMR(400MHz,D2O)δ8.49–7.98(m,2H),7.75(d,J=0.5Hz,1H),6.50–6.32(m,1H),6.28(dt d,J=3.2,1.6,0.8Hz,2H),5.31(dddd,J=46.5,7.0,4.0,0.6Hz,1H),4.70(dddd,J=3.3,2. 5,0.7Hz,1H),4.52–4.19(m,9H),4.14–3.95(m,2H),3.94–3.84(m,4H),3.56(td,J=6.4, 1.5Hz, 2H), 3.23 (td, J=6.3, 0.8Hz, 2H), 2.83–2.48 (m, 2H), 1.94 (pd, J=6.3, 1.0Hz, 2H).

[0406] 31 P NMR(162MHz,D2O)δ-0.99,-11.40,-23.06.

[0407] Example 32

[0408] Synthesis of Compound 828

[0409]

[0410] Compound 10e (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 828-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 828, an ammonium salt (85 mg).

[0411] 1 H NMR(400MHz,D2O)δ8.27(s,1H),8.19(d,J=0.6Hz,1H),7.95(d,J=0.7Hz,1H),6.63–6.29(m, 3H),5.28(dddd,J=46.4,6.9,1.6,0.7Hz,1H),4.62(ddqd,J=4.5,3.7,1.5,0.6Hz,1H),4.57 –4.40(m,2H),4.36–4.20(m,5H),4.16–3.95(m,5H),3.93–3.81(m,4H),3.73–3.51(m,3H),3 .37(d,J=1.6Hz,3H),3.23(td,J=6.3,0.8Hz,2H),3.10(s,3H),1.95(tt,J=6.3,5.3Hz,2H).

[0412] 31 P NMR (162MHz, D2O) δ0.60, -9.01, -10.23, -21.17.

[0413] Example 33

[0414] Synthesis of Compound 880

[0415]

[0416] Compound 10e (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 880-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 880, an ammonium salt (92 mg).

[0417] 1 H NMR(400MHz,D2O)δ8.35(d,J=0.6Hz,1H),8.27(s,1H),7.69(dd,J=7.4,1.8 Hz,1H),6.60–6.29(m,2H),6.15(d,J=7.3Hz,1H),5.89–5.26(m,1H),4.52(d d,J=11.6,8.5Hz,1H),4.44–3.84(m,16H),3.75–3.48(m,3H),3.23(td,J=6 .3,0.8Hz,2H),3.10(s,3H),2.87–2.56(m,2H),1.95(tt,J=6.3,5.3Hz,2H).

[0418] 31 P NMR (162MHz, D2O) δ0.60, -9.10, -10.31, -21.17.

[0419] Example 34

[0420] Synthesis of Compound 903

[0421]

[0422] Compound 10f (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 903-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 903, an ammonium salt (85 mg).

[0423] 1 H NMR(400MHz,D2O)δ8.48–8.18(m,2H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.2,1.6,0.8Hz,1H),6.25–6.00(m,2H),5.22(dddd, J=46.4,7.0,1.7,0.7Hz,1H),4.89–4.62(m,3H),4.59–4.09(m,13H),3.98(d,J=0.7Hz,3H),3.08(s,3H),2.42(t,J=3.0Hz,1H).

[0424] 31 P NMR (162MHz, D2O) δ0.60, -9.10, -10.22, -21.17.

[0425] Example 35

[0426] Synthesis of Compound 974

[0427]

[0428] Compound 10f (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 974-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 974, an ammonium salt (80 mg).

[0429] 1 H NMR(400MHz,D2O)δ7.76(ddd,J=44.0,7.5,1.8Hz,2H),6.20(dq,J=2.3,0.8Hz,1H),5.97(dd,J=56.9,7.5Hz,2H),5.64 –5.41(m,2H),5.34–4.98(m,2H),4.89–4.63(m,2H),4.55–4.08(m,13H),3.98(d,J=0.7Hz,3H),2.42(t,J=3.0Hz,1H).

[0430] 31 P NMR(162MHz,D2O)δ-0.22,-9.07,-10.25,-21.27.

[0431] Example 36

[0432] Synthesis of Compound 1040

[0433]

[0434] 10 g (200 mg) of compound was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 200 mg of compound 1040-1. The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1040, as an ammonium salt (58 mg).

[0435] 1 H NMR(400MHz,D2O)δ7.88(d,J=0.7Hz,1H),7.70(dd,J=7.4,1.8Hz,1H),6.47–6.14(m,2H),6.04(d,J=7.3Hz,1H),5.40(dddd,J=2 5.2, 3.5, 1.8, 0.9Hz, 1H), 5.10–4.64 (m, 4H), 4.43–4.08 (m, 12H), 4.02 (s, 2H), 3.98 (s, 3H), 3.40 (s, 3H), 2.42 (t, J = 3.0Hz, 1H).

[0436] 31P NMR (162MHz, D2O) δ0.66, -9.13, -10.26, -21.25.

[0437] Example 37

[0438] Synthesis of Compound 1087

[0439]

[0440] 10 g (200 mg) of compound was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 200 mg of compound 1087-1. The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1087, as an ammonium salt (88 mg).

[0441] 1 H NMR(400MHz,D2O)δ8.31–8.11(m,2H),7.83(d,J=0.6Hz,1H),6.88–6.07(m,3H),5.27(dddd,J=46.5,7.0,4.0,0.6H z,1H),5.05–4.88(m,2H),4.54–4.04(m,12H),4.02(s,2H),3.98(s,3H),2.77–2.52(m,2H),2.42(t,J=3.0Hz,1H).

[0442] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0443] Example 38

[0444] Synthesis of Compound 1108

[0445]

[0446] Compound 10h (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1108-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1108, an ammonium salt (88 mg).

[0447] 1 H NMR(400MHz,D2O)δ8.33–8.15(m,2H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.2,1.5,0.8Hz,1H),6.32–5.94(m,2H),5.22(dddd,J=46.4,7.0, 1.7,0.7Hz,1H),4.83–4.69(m,2H),4.56(dt,J=3.8,0.9Hz,1H),4.51–4.06(m,10H),3.98(d,J=0.7Hz,3H),3.65(ddd,J=3.6,2.6,0.6Hz,1H).

[0448] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0449] Example 39

[0450] Synthesis of Compound 1130

[0451]

[0452] Compound 10h (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1130-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1130, as an ammonium salt (88 mg).

[0453] 1 H NMR(400MHz,D2O)δ8.51–8.01(m,2H),7.70(dd,J=7.4,1.8Hz,1H),6.31–6.15 (m,2H),6.04(d,J=7.3Hz,1H),5.56–5.22(m,1H),4.97(dddd,J=8.0,4.4,2.9 ,0.7Hz,1H),4.90–4.69(m,2H),4.56(dt,J=3.8,0.9Hz,1H),4.48–4.10(m,10 H), 3.98 (s, 3H), 3.65 (ddd, J = 3.6, 2.6, 0.6Hz, 1H), 3.40 (s, 3H), 3.08 (s, 3H).

[0454] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0455] Example 40

[0456] Synthesis of Compound 1130

[0457]

[0458] Compound 10h (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1130-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1130, as an ammonium salt (88 mg).

[0459] 1 H NMR(400MHz,D2O)δ8.51–8.01(m,2H),7.70(dd,J=7.4,1.8Hz,1H),6.31–6.15 (m,2H),6.04(d,J=7.3Hz,1H),5.56–5.22(m,1H),4.97(dddd,J=8.0,4.4,2.9 ,0.7Hz,1H),4.90–4.69(m,2H),4.56(dt,J=3.8,0.9Hz,1H),4.48–4.10(m,10 H), 3.98 (s, 3H), 3.65 (ddd, J = 3.6, 2.6, 0.6Hz, 1H), 3.40 (s, 3H), 3.08 (s, 3H).

[0460] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0461] Example 41

[0462] Synthesis of Compound 1140

[0463]

[0464] Compound 10i (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1140-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1140, an ammonium salt (88 mg).

[0465] 1 H NMR(400MHz,D2O)δ8.18(d,J=1.3Hz,2H),7.69(dd,J=7.4,1.8Hz,1H),6.36–6.26(m,1H),6.20–6.09(m,2H),5.80– 5.38(m,1H),4.82(ddd,J=3.9,3.1,0.7Hz,1H),4.62(ddd,J=5.5,2.8,0.7Hz,1H),4.42–3.95(m,13H),3.92(s,3H).

[0466] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0467] Example 42

[0468] Synthesis of Compound 1198

[0469]

[0470] Compound 10j (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1198-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1198, an ammonium salt (88 mg).

[0471] 1H NMR(400MHz,D2O)δ8.28(d,J=0.5Hz,1H),8.20(s,1H),8.02(d,J=0.7Hz,1H),6 .45(ddq,J=25.2,1.5,0.8Hz,1H),6.22(dd,J=3.1,0.7Hz,1H),6.11(dd,J=3.1, 0.7Hz,1H),5.22(dddd,J=46.4,7.0,1.7,0.7Hz,1H),4.86–4.66(m,2H),4.59– 4.39(m,2H),4.36–4.06(m,9H),3.98(s,3H),2.80(ddd,J=5.1,4.0,0.7Hz,1H).

[0472] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0473] Example 43

[0474] Synthesis of Compound 1220

[0475]

[0476] Compound 10j (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1220-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1220, an ammonium salt (88 mg).

[0477] 1 H NMR(400MHz,D2O)δ8.29(s,1H),8.20(d,J=0.7Hz,1H),7.70(dd,J=7.4,1.8Hz,1H),6.32–6.16(m,2H),6.04(d,J=7.3Hz,1H),5.6 1–5.20(m,1H),5.07–4.65(m,3H),4.50–4.06(m,11H),3.98(s,3H),3.40(s,3H),3.08(s,3H),2.86(ddd,J=3.9,3.3,0.7Hz,1H).

[0478] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0479] Example 44

[0480] Synthesis of Compound 1280

[0481]

[0482] Compound 10h (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1280-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1280, as an ammonium salt (88 mg).

[0483] 1 H NMR(400MHz,D2O)δ8.24(d,J=0.7Hz,1H),8.20(s,1H),7.70(dd,J=7.4,1.8Hz,1H),6.43 (ddq,J=25.2,1.5,0.8Hz,1H),6.24(p,J=0.8Hz,1H),6.04(d,J=7.3Hz,1H),5.50–5.10(m ,3H),4.81(dddd,J=46.5,7.1,3.7,0.7Hz,1H),4.56(dt,J=3.8,0.9Hz,1H),4.48(tdd,J =3.5, 2.4, 0.8Hz, 1H), 4.44–4.09 (m, 9H), 3.98 (s, 3H), 3.65 (ddd, J = 3.6, 2.6, 0.6Hz, 1H).

[0484] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0485] Example 45

[0486] Synthesis of Compound 1307

[0487]

[0488] Compound 10i (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1307-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1307, an ammonium salt (88 mg).

[0489] 1 H NMR(400MHz,D2O)δ8.22–8.03(m,4H),6.60–6.35(m,2H),6.31–6.22(m,1H),5.54 –5.15(m,2H),4.62(ddd,J=5.5,2.8,0.7Hz,1H),4.51–4.01(m,12H),3.92(s,3H).

[0490] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0491] Example 46

[0492] Synthesis of Compound 1373

[0493]

[0494] Compound 10k (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1373-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1373, as an ammonium salt (88 mg).

[0495] 1H NMR(400MHz,D2O)δ8.40(d,J=0.5Hz,1H),8.20(s,1H),8.02(d,J=0.7Hz,1H),6.45(ddq,J=25.2,1.6,0.8H z,1H),6.35–5.85(m,2H),5.22(dddd,J=46.4,7.0,1.7,0.7Hz,1H),4.97(dddd,J=8.0,4.4,2.9,0.7Hz,1H) ,4.84–4.69(m,1H),4.58(ddd,J=3.8,1.6,0.7Hz,1H),4.45(dddd,J=25.2,6.8,5.0,0.7Hz,1H),4.36–4.30 (m,2H),4.30–4.05(m,7H),3.98(s,3H),3.61(dddd,J=3.8,2.6,1.8,1.1Hz,1H),3.40(s,3H),2.09(s,3H).

[0496] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0497] Example 47

[0498] Synthesis of Compound 1400

[0499]

[0500] Compound 10k (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1400-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1400, as an ammonium salt (88 mg).

[0501] 1H NMR(400MHz,D2O)δ8.43–8.11(m,2H),7.70(dd,J=7.4,1.8Hz,1H),6.43(ddq,J=25.2,1.6,0 .8Hz,1H),6.20(dt,J=1.6,0.8Hz,1H),6.04(d,J=7.3Hz,1H),5.49–5.13(m,3H),4.81(dddd, J=46.5,7.1,3.7,0.7Hz,1H),4.58(ddd,J=3.8,1.6,0.7Hz,1H),4.48(tdd,J=3.5,2.4,0.8H z,1H),4.42–4.03(m,9H),3.98(s,3H),3.61(dddd,J=3.8,2.5,1.8,1.1Hz,1H),2.09(s,3H).

[0502] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0503] Example 48

[0504] Synthesis of Compound 1421

[0505]

[0506] Compound 10h (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1421-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1421, as an ammonium salt (88 mg).

[0507] 1H NMR(400MHz,D2O)δ8.51–7.89(m,4H),6.46–6.31(m,2H),6.24(p,J=0.8Hz,1H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),5.08–4.88(m,1H),4. 56(dt,J=3.8,0.9Hz,1H),4.52–4.38(m,2H),4.36–4.04(m,8H),3.98(s ,3H),3.65(ddd,J=3.6,2.6,0.6Hz,1H),3.08(s,3H),2.80–2.52(m,2H).

[0508] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0509] Example 49

[0510] Synthesis of Compound 1449

[0511]

[0512] Compound 10i (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1449-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1449, as an ammonium salt (88 mg).

[0513] 1 H NMR(400MHz,D2O)δ8.40–8.08(m,2H),7.59–7.18(m,2H),6.48–6.34(m,1H),6.24(dq,J=4.7,0.8Hz,1H),5.98(dddt,J=25.2,4 .0,1.7,0.8Hz,1H),4.72(ddd,J=7.4,4.7,0.7Hz,1H),4.49–3.97(m,12H),3.95–3.80(m,4H),3.10(s,3H),2.93–2.53(m,2H).

[0514] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0515] Example 50

[0516] Synthesis of Compound 1449

[0517]

[0518] Compound 10i (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1449-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1449, as an ammonium salt (88 mg).

[0519] 1 H NMR(400MHz,D2O)δ8.40–8.08(m,2H),7.59–7.18(m,2H),6.48–6.34(m,1H),6.24(dq,J=4.7,0.8Hz,1H),5.98(dddt,J=25.2,4 .0,1.7,0.8Hz,1H),4.72(ddd,J=7.4,4.7,0.7Hz,1H),4.49–3.97(m,12H),3.95–3.80(m,4H),3.10(s,3H),2.93–2.53(m,2H).

[0520] 31 P NMR (162MHz, D2O) δ0.85, -9.12, -10.24, -21.26.

[0521] Example 51

[0522] Synthesis of Compound 1495

[0523]

[0524] Compound 10L (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1495-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1495, an ammonium salt (88 mg).

[0525] 1 H NMR(400MHz,D2O)δ8.30(d,J=0.6Hz,1H),8.20(s,1H),7.70(dd,J=7.4,1.8Hz,1H),6. 55–6.34(m,1H),6.16(dq,J=2.2,0.8Hz,1H),6.04(d,J=7.3Hz,1H),5.40(dddd,J=25. 2,3.4,1.8,0.9Hz,1H),5.12–4.93(m,1H),4.81(dddd,J=46.5,7.1,3.7,0.7Hz,1H),4 .41–4.04(m,11H),3.98(s,3H),3.63(ddd,J=4.6,3.1,0.7Hz,1H),2.81–2.48(m,2H).

[0526] 31 P NMR (162MHz, D2O) δ0.85, -10.30, -21.26.

[0527] Example 52

[0528] Synthesis of Compound 1580

[0529]

[0530] Compound 10k (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1580-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1580, as an ammonium salt (88 mg).

[0531] 1 H NMR(400MHz,D2O)δ7.73(ddd,J=24.9,7.2,1.8Hz,2H),6.35–6.15(m,2H),6.03(dd,J=12 .2,7.2Hz,2H),5.40(dddd,J=25.2,3.5,1.8,0.9Hz,1H),5.10(dddd,J=8.3,6.4,4.5,3 .7,0.7Hz,1H),4.81(dddd,J=46.5,7.1,3.7,0.7Hz,1H),4.58(dddd,J=3.8,1.6,0.7Hz,1 H),4.39–4.00(m,10H),3.98(s,3H),3.74–3.47(m,1H),2.61–2.28(m,2H),2.09(s,3H).

[0532] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0533] Example 53

[0534] Synthesis of Compound 1581

[0535]

[0536] Compound 11a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1581-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1581, an ammonium salt (88 mg).

[0537] 1 H NMR(400MHz,D2O)δ8.33–7.87(m,2H),7.60(dd,J=7.4,1.8Hz,1H),6.49–6.23(m,2H),6.15–5.73(m,2H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H), 5.00(dd,J=3.2,2.5Hz,1H),4.80–4.62(m,1H),4.53–4.32(m,4H),4.31–4 .10(m,6H),4.09(s,2H),3.98(s,3H),3.96(d,J=3.2Hz,1H),3.08(s,3H).

[0538] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0539] Example 54

[0540] Synthesis of Compound 1617

[0541]

[0542] Compound 11b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1617-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1617, an ammonium salt (88 mg).

[0543] 1 H NMR(400MHz,D2O)δ8.27(s,1H),8.22–8.17(m,2H),8.14(d,J=0.6Hz,1H),6.60–6.31 (m,3H),5.31(dddd,J=46.5,7.0,4.0,0.6Hz,1H),4.81(dd,J=4.8,2.0Hz,1H),4.66–4 .54(m,2H),4.46(dddd,J=25.2,6.8,5.0,0.7Hz,1H),4.36–4.21(m,6H),4.19–3.99( m, 4H), 3.92 (d, J = 0.6Hz, 3H), 3.83 (dd, J = 11.3, 8.5Hz, 1H), 3.37 (s, 3H), 3.10 (s, 3H).

[0544] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0545] Example 55

[0546] Synthesis of Compound 1660

[0547]

[0548] Compound 11c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1600-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1600, an ammonium salt (88 mg).

[0549] 1 H NMR(400MHz,D2O)δ7.88(d,J=0.7Hz,1H),7.70(dd,J=7.4,1.8Hz,1H),6.51–6.18(m,2H),6.04(d,J=7.3Hz,1H), 5.40(dddd,J=25.2,3.5,1.8,0.9Hz,1H),5.05–4.59(m,4H),4.48–4.09(m,9H),4.04–3.82(m,6H),3.40(s,3H).

[0550] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0551] Example 56

[0552] Synthesis of Compound 1698

[0553]

[0554] Compound 11d (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1698-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1698, an ammonium salt (88 mg).

[0555] 1H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),7.81(dd,J=7.3,1.8Hz,1H),6.63–6.39(m,2H),6.18–5.82(m,2H),5.22(dddd,J=46.4,7.0,1.7, 0.7Hz,1H),4.89(dddd,J=8.0,5.4,4.6,0.6Hz,1H),4.59–4.28(m,6H),4.25–4.03(m,7H),3.98(s,3H),3.46(s,3H),3.11(d,J=3.1Hz,1H).

[0556] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0557] Example 57

[0558] Synthesis of Compound 1728

[0559]

[0560] Compound 11e (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1728-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1728, an ammonium salt (88 mg).

[0561] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),7.81(dd,J=7.3,1.8Hz,1H),6.73–6.18(m,2H),6.18–5.82(m,2H),5.22(dddd,J=46.4,7.0,1.7,0.7 Hz,1H),5.02–4.79(m,2H),4.58–4.28(m,5H),4.27–4.01(m,7H),3.98( s, 3H), 3.93 (dq, J = 2.9, 1.5Hz, 1H), 3.46 (s, 3H), 2.10 (d, J = 1.4Hz, 3H).

[0562] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0563] Example 58

[0564] Synthesis of Compound 1897

[0565]

[0566] Compound 11a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1897-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1897, an ammonium salt (88 mg).

[0567] 1 H NMR(400MHz,D2O)δ8.32–8.11(m,2H),7.76(dd,J=7.0,1.8Hz,1H),6.46–6.31(m,2H),6.25(dddd ,J=5.2,3.3,1.7,0.8Hz,1H),6.01(d,J=7.2Hz,1H),5.27(dddd,J=46.5,7.0,4.0,0.6Hz,1H),5. 10(dddd,J=8.3,6.4,4.5,3.7,0.7Hz,1H),5.00(dd,J=3.2,2.5Hz,1H),4.54–4.30(m,3H),4.26 –4.17(m,4H),4.13–4.01(m,4H),3.98(d,J=0.7Hz,3H),3.96(d,J=3.2Hz,1H),2.60–2.26(m,2H).

[0568] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0569] Example 59

[0570] Synthesis of Compound 1907

[0571]

[0572] Compound 11b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1907-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1907, an ammonium salt (88 mg).

[0573] 1 H NMR(400MHz,D2O)δ8.18(s,1H),8.14(d,J=0.6Hz,1H),7.75(d,J=0.5Hz,1H),6.49(dd,J =2.0,0.7Hz,1H),6.47–6.35(m,1H),6.28(ddq,J=3.0,1.4,0.7Hz,1H),5.31(dddd,J=46. 5,7.0,4.0,0.6Hz,1H),4.81(dd,J=4.8,2.0Hz,1H),4.58(dd,J=11.3,8.5Hz,1H),4.50– 4.39(m,1H),4.37–4.19(m,6H),4.14–3.98(m,3H),3.96–3.76(m,5H),2.92–2.35(m,2H).

[0574] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0575] Example 60

[0576] Synthesis of Compound 1925

[0577]

[0578] Compound 11c (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1925-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1925, as an ammonium salt (88 mg).

[0579] 1 H NMR(400MHz,D2O)δ8.30(d,J=0.5Hz,1H),8.20(s,1H),7.70(dd,J=7.4,1.8Hz ,1H),6.42–6.36(m,1H),6.31(dd,J=2.0,0.6Hz,1H),6.04(d,J=7.3Hz,1H),5. 48–5.29(m,1H),5.18–4.94(m,1H),4.81(dddd,J=46.5,7.1,3.7,0.7Hz,1H),4 .66(t,J=2.2Hz,1H),4.46–4.05(m,9H),4.02–3.90(m,6H),2.92–2.35(m,2H).

[0580] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0581] Example 61

[0582] Compound 1978 Synthesis

[0583]

[0584] Compound 11e (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1978-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1978, an ammonium salt (88 mg).

[0585] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),7.76(dd,J=7.0,1.8Hz,1H),6.54–6.3 5(m,2H),6.25(dddd,J=5.2,3.3,1.7,0.8Hz,1H),6.01(d,J=7.2Hz,1H),5.37–5. 03(m,2H),4.88(dd,J=3.1,2.0Hz,1H),4.57–4.29(m,3H),4.26–4.01(m,8H),3. 98(d,J=0.7Hz,3H),3.93(dq,J=3.0,1.5Hz,1H),2.71–2.26(m,2H),2.10(s,3H).

[0586] 31 P NMR (162MHz, D2O) δ0.85, -10.24, -21.26.

[0587] Example 62

[0588] Synthesis of Compound 1981

[0589]

[0590] Compound 9a (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1981-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1981, an ammonium salt (88 mg).

[0591] 1 H NMR(400MHz,D2O)δ8.30–8.00(m,2H),6.39(ddd,J=25.2,4.0,0.7Hz,1H),6.25–5.90(m,1H),5.28(dddd,J=46.5,4 .9,4.1,0.6Hz,1H),4.69–4.49(m,2H),4.35(ddd,J=5.3,3.7,0.7Hz,1H),4.30–4.07(m,6H),3.98(d,J=0.7Hz,3H).

[0592] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0593] Example 63

[0594] Synthesis of Compound 1988

[0595]

[0596] Compound 9b (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1988-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1988, an ammonium salt (88 mg).

[0597] 1H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.43(ddd,J=25.2,1.5,0.8Hz,1H),6.17(dt,J=2.5,0.7Hz,1H),5.61–5.11(m,1 H),4.96–4.44(m,2H),4.30–4.22(m,4H),4.21–4.10(m,2H),4.06(dtt,J=4.2,2.1,1.1Hz,1H),3.98(s,3H),3.44(s,3H).

[0598] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0599] Example 64

[0600] Synthesis of Compound 1992

[0601]

[0602] Compound 9c (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1992-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1992, an ammonium salt (88 mg).

[0603] 1 H NMR(400MHz,D2O)δ7.81(dd,J=7.8,1.8Hz,1H),6.44–6.09(m,1H),5.90(d,J =7.8Hz,1H),5.53(dddd,J=24.4,3.9,1.8,0.9Hz,1H),5.00–4.94(m,1H),4.8 8–4.80(m,1H),4.78–4.69(m,1H),4.60(dtd,J=3.8,2.7,0.8Hz,1H),4.57–4. 51(m,1H),4.33(ddd,J=8.5,3.7,1.6Hz,2H),4.25–4.12(m,3H),3.98(s,3H).

[0604] 31P NMR(162MHz,D2O)δ-10.24,-21.26.

[0605] Example 65

[0606] Compound 1996 Synthesis

[0607]

[0608] Compound 9d (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 1996-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 1996, as an ammonium salt (88 mg).

[0609] 1 H NMR(400MHz,D2O)δ8.18(s,1H),8.14(d,J=0.6Hz,1H),6.41(ddq,J=25.2,4.0,0.8Hz,1H) ,6.31–6.22(m,1H),5.45–4.99(m,1H),4.79–4.50(m,2H),4.46–4.05(m,7H),3.92(s,3H).

[0610] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0611] Example 66

[0612] Synthesis of Compound 2005

[0613]

[0614] Compound 9e (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2005-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2005, an ammonium salt (88 mg).

[0615] 1 H NMR(400MHz,D2O)δ7.81(dd,J=7.8,1.8Hz,1H),6.24(p,J=0.8Hz,1H),5.90(d,J=7.8Hz,1H),5.74–5.30(m,1H),4.89– 4.64(m,1H),4.60–4.43(m,2H),4.43–4.37(m,1H),4.36–4.11(m,5H),3.98(s,3H),3.65(ddd,J=3.6,2.6,0.6Hz,1H).

[0616] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0617] Example 67

[0618] Synthesis of Compound 2010

[0619]

[0620] Compound 9f (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 200 mg of 2010-1. The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2010, an ammonium salt (88 mg).

[0621] 1H NMR (400MHz, D2O) δ7.70 (dd, J=7.3, 1.8Hz, 1H), 6.18 (dq, J=2.5, 0.8Hz, 1H), 6.0 4(d,J=7.3Hz,1H),5.40(dddt,J=25.1,3.4,1.6,0.8Hz,1H),4.83(dddd,J=46.3, 5.2,3.7,0.6Hz,1H),4.68(ddd,J=3.2,2.4,0.7Hz,1H),4.59–4.42(m,1H),4.30– 4.07 (m, 7H), 3.98 (d, J = 0.7Hz, 3H), 3.59 (p, J = 5.9Hz, 2H), 1.24 (t, J = 6.0Hz, 3H).

[0622] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0623] Example 68

[0624] Synthesis of Compound 2020

[0625]

[0626] Compound 10k (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2020-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2020, as an ammonium salt (88 mg).

[0627] 1 H NMR(400MHz,D2O)δ7.81(dd,J=7.8,1.8Hz,1H),6.21(dq,J=1.5,0.8Hz,1H),5.90(d,J=7.8Hz,1H),5.62–5.35(m,1H),5 .00–4.69(m,1H),4.62–4.41(m,2H),4.36–4.11(m,6H),3.98(d,J=0.7Hz,3H),3.69–3.47(m,1H),2.09(d,J=1.4Hz,3H).

[0628] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0629] Example 69

[0630] Synthesis of Compound 2025

[0631]

[0632] Compound 10d (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2025-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2025, as an ammonium salt (88 mg).

[0633] 1 H NMR(400MHz,D2O)δ7.95(d,J=0.7Hz,1H),6.49(ddt,J=25.2,1.5,0.8Hz,1H),6.28(dq,J=2.3,0.8Hz,1H ),5.31(dddd,J=46.3,5.0,1.6,0.7Hz,1H),4.70(dddd,J=3.3,2.5,0.7Hz,1H),4.58(dddd,J=25.2,4.9, 3.1,0.6Hz,1H),4.44–4.21(m,5H),4.13(qt,J=3.1,0.8Hz,1H),3.98(ddd,J=3.1,2.3,0.6Hz,1H),3.92 (d, J=0.6Hz, 3H), 3.56 (td, J=6.4, 1.5Hz, 2H), 3.23 (td, J=6.3, 0.8Hz, 2H), 1.94 (pd, J=6.3, 1.0Hz, 2H).

[0634] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0635] Example 70

[0636] Synthesis of Compound 2028

[0637]

[0638] Compound 10b (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2028-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2028, as an ammonium salt (88 mg).

[0639] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.43(ddd,J=25.2,1.5,0.8Hz,1H),6.27–6.03(m,1H),5.54–5.05(m,1H),4.71(ddd,J=3.3,2. 5,0.7Hz,1H),4.59–4.45(m,1H),4.37–4.06(m,7H),3.98(d,J=0.7Hz,3H),3.76–3.46(m,2H),3.06–2.68(m,2H),1.84(p,J=6.5Hz,2H).

[0640] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0641] Example 71

[0642] Synthesis of Compound 2031

[0643]

[0644] Compound 10f (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2031-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2031, as an ammonium salt (88 mg).

[0645] 1 H NMR (400MHz, D2O) δ7.70 (dd, J=7.3, 1.8Hz, 1H), 6.20 (dt, J=2.3, 0.7Hz, 1H), 6.04 (d, J=7.3Hz, 1H), 5.40 (dddd, J=25.2, 3. 5,1.7,0.8Hz,1H),4.97–4.69(m,2H),4.59–4.41(m,1H),4.35–4.09(m,9H),3.98(d,J=0.7Hz,3H),2.42(t,J=3.0Hz,1H).

[0646] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0647] Example 72

[0648] Synthesis of Compound 2037

[0649]

[0650] Compound 10a (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2037-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2037, an ammonium salt (88 mg).

[0651] 1H NMR(400MHz,D2O)δ8.42–8.05(m,2H),6.55–6.15(m,2H),5.28(dddd,J=46.4,7.0,3. 0,0.7Hz,1H),4.64(t,J=2.8Hz,1H),4.55–4.19(m,7H),4.00(dd,J=12.9,0.7Hz,5H).

[0652] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0653] Example 73

[0654] Synthesis of Compound 2038

[0655]

[0656] Compound 12a (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2038-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2038, an ammonium salt (88 mg).

[0657] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.43(ddt,J=25.2,1.6,0.8Hz,1H),6.27–5.99(m,1H),5.24(dddd,J=46.5,5.1,1.7,0.6Hz,1 H),4.86–4.45(m,2H),4.36–4.07(m,7H),3.98(d,J=0.7Hz,3H),3.83–3.48(m,2H),3.09–2.90(m,2H),2.79(s,3H),2.55–2.18(m,2H).

[0658] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0659] Example 74

[0660] Synthesis of Compound 2039

[0661]

[0662] Compound 12b (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2039-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2039, as an ammonium salt (88 mg).

[0663] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.43(ddd,J=25.2,1.6,0.8Hz, 1H),6.27–5.70(m,3H),5.42–5.07(m,1H),5.00–4.81(m,2H),4.65–4.43( m,2H),4.37–4.10(m,5H),3.98(d,J=0.7Hz,3H),3.38(ttdd,J=5.3,4.2, 1.8,1.0Hz,1H),3.11–2.53(m,2H),2.14–1.79(m,2H),1.62–1.17(m,2H).

[0664] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0665] Example 75

[0666] Synthesis of Compound 2040

[0667]

[0668] Compound 12c (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2040-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2040, as an ammonium salt (88 mg).

[0669] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.43(ddd,J=25.2,1.5,0.8Hz,1H),6.26–6.03(m,1H),5.69–5.39(m,2H),5.31–5.07(m,1H),4.84– 4.67(m,2H),4.63–4.46(m,1H),4.28–4.06(m,7H),3.98(d,J=0.7Hz,3 H),3.89–3.69(m,2H),2.58(td,J=7.2,2.9Hz,2H),2.26–1.11(m,10H).

[0670] 31 P NMR(162MHz,D2O)δ-10.25,-21.23.

[0671] Example 76

[0672] Synthesis of Compound 2050

[0673]

[0674] Compound 11c (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2050-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2050, an ammonium salt (88 mg).

[0675] 1 H NMR(400MHz,D2O)δ8.02(d,J=0.7Hz,1H),6.65–6.28(m,2H),5.39–5.11(m,1H),4.87(t,J=3.1Hz,1H),4.65–4.47(m,1H),4.39–4.21(m,5 H), 4.15 (d, J = 3.1Hz, 1H), 4.02 (s, 2H), 3.98 (d, J = 0.7Hz, 3H), 3.66 (td, J = 5.3, 2.9Hz, 2H), 2.96–2.52 (m, 2H), 1.85 (tt, J = 6.4, 5.3Hz, 2H).

[0676] 31 P NMR(162MHz,D2O)δ-10.21,-21.25.

[0677] Example 77

[0678] Synthesis of Compound 2051

[0679]

[0680] Compound 11e (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2051-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2051, an ammonium salt (88 mg).

[0681] 1 H NMR(400MHz,D2O)δ7.95(d,J=0.7Hz,1H),6.71–6.22(m,2H),5.31(dddd,J=46.3,5.0,1.6,0.7Hz,1H),4.67–4.45(m,2H),4.42–4 .21(m,2H),4.13(qt,J=3.1,0.8Hz,1H),4.06–3.85(m,7H),3.76–3.53(m,3H),3.36(t,J=6.2Hz,2H),1.95(tt,J=6.3,5.3Hz,2H).

[0682] 31 P NMR(162MHz,D2O)δ-10.34,-21.36.

[0683] Example 78

[0684] Synthesis of Compound 2054

[0685]

[0686] Compound 11b (250 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by compound 2054-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2054, an ammonium salt (88 mg).

[0687] 1 H NMR(400MHz,D2O)δ7.95(d,J=0.7Hz,1H),6.49(dd,J=2.0,0.7Hz,1H),6.44–6.1 9(m,1H),5.31(dddd,J=46.4,6.9,3.5,0.8Hz,1H),4.81(dd,J=4.8,2.0Hz,1H),4 .58(dd,J=11.3,8.5Hz,1H),4.42(dddd,J=25.2,7.1,3.2,0.7Hz,1H),4.36–4.2 3(m,3H),4.14–3.99(m,3H),3.92(d,J=0.6Hz,3H),3.83(dd,J=11.3,8.5Hz,1H).

[0688] 31 P NMR(162MHz,D2O)δ-10.24,-21.16.

[0689] Example 79

[0690] Synthesis of Compound 2064

[0691]

[0692] 2.38 g of 2064-2 was added to a 63 mL solution of acetonitrile (1.76 g) of tetrazolium in a three-necked flask. Argon gas was purged three times. Then, at room temperature (25°C), 5 g of 2064-1 was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature (25°C) for 1 hour. No significant exothermic reaction was observed, and TLC monitoring showed the disappearance of the starting material 2064-1. Then, a 0.5 mmol / mL solution of iodine in pyridine / tetrahydrofuran / water (pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise to the solution until the solution no longer decolorized. The reaction mixture was then stirred for another 0.5 hours, and TLC monitoring confirmed the oxidation was complete. After quenching the reaction solution with saturated sodium sulfite aqueous solution (10 mL), it was diluted with 50 mL of water, extracted with dichloromethane (50 mL x 2), and the combined organic phases were washed once with water (50 mL). The mixture was then concentrated to obtain a pale yellow oily product 2064-3 (8 g, crude product).

[0693] Compound 2064-3 (8 g, crude) was dissolved in 40 mL of acetic acid and 10 mL of water. The reaction was stirred at 25 °C for 16 hours. TLC monitoring showed the disappearance of the 3-3 spot and the formation of highly polar spots. The reaction solution was directly concentrated under vacuum. After concentration, appropriate amounts of silica gel and DCM were added, and the mixture was purified (40 g normal phase column, EA, 10 min; DCM: MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white solid product 2064-4 (2.8 g, 51% two-step yield).

[0694] Prepare 28 mL of tetrazolium in acetonitrile (0.4 mmol / mL). Add 2.8 g of 2064-4 to the above solution, then add 3 g of Al at room temperature (25°C). Replace with nitrogen three times, and stir the reaction solution at room temperature (25°C) for 1 hour. TLC monitoring showed the reaction was complete. Cool the reaction solution to below 10°C in an ice-water bath, and add dropwise a solution of iodine in pyridine / tetrahydrofuran / water (0.5 mmol / mL, pyridine:tetrahydrofuran:water = 1:8:1) until the reaction solution no longer fades. TLC monitoring showed the oxidation reaction was complete. Quench the reaction solution with 10 mL of saturated sodium sulfite aqueous solution, and then dilute with water. Extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amounts of silica gel and DCM for mixing and purification (40 g normal phase column, EA, 10 min; DCM:MeOH, 10-20% 20 min, flow rate 30 mL / min). Concentration yielded a white, foamy solid 2064-6 (2.6 g, 78.2% yield).

[0695] Compound 2064-6 (2.6 g) was dissolved in methanol (30 mL), and then concentrated ammonia (30 mL) was added. The resulting solution was stirred at 25°C for 60 hours at room temperature. TLC showed that the starting material 2064-6 reacted completely. The reaction solution was concentrated under vacuum and then concentrated again with methanol to obtain a pale yellow oily liquid compound 2064-7 (2.4 g, crude product). The crude product was directly added to the next step.

[0696] Compound 2064-7 (2.4 g, crude) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction mixture was stirred at 50 °C for 1 hour, and TLC showed that starting material 3-7 had reacted completely. The reaction mixture was diluted with water to 50 mL, and the pH was adjusted to 5.5 with 1 N NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution. The fraction obtained was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give the amine salt of the target compound, 2064-8 (0.8 g, 33.7% yield), as a white solid.

[0697] 9a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2064-8 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powdery product, compound 2064, as an ammonium salt (65 mg).

[0698] 1 H NMR(400MHz,D2O)δ8.33(s,1H),8.29(s,1H),8.04(s,1H),6.25(t,J=0.7Hz,1H),6.1 6(dt,J=3.0,0.7Hz,1H),6.11(dq,J=3.0,0.7Hz,1H),4.80(ddd,J=3.8,2.9,0.7Hz,1H ),4.74(dddd,J=7.9,3.9,2.9,0.7Hz,1H),4.61(dddd,J=5.2,2.8,0.7Hz,1H),4.55–4 .43(m,1H),4.41–4.32(m,2H),4.30–4.09(m,9H),3.98(d,J=0.7Hz,3H),3.08(s,3H).

[0699] 31 P NMR(162MHz,D2O)δ-0.80,-11.55,-23.03.

[0700] Example 80

[0701] Synthesis of Compound 2078

[0702]

[0703] 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2078-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2078, an ammonium salt (55 mg).

[0704] 1 H NMR(400MHz,D2O)δ8.39(s,1H),8.28(s,1H),8.19(s,1H),6.49(dq,J=1.5,0 .7Hz,1H),6.32–6.09(m,2H),5.30(ddd,J=2.6,1.8,0.7Hz,1H),5.25(ddd,J =3.6,2.9,0.7Hz,1H),4.72–4.60(m,2H),4.56–4.46(m,2H),4.44–4.36(m,2 H), 4.33 (d, J = 2.8Hz, 1H), 4.27–4.07 (m, 6H), 4.05–3.96 (m, 4H), 3.11 (s, 3H).

[0705] 31 P NMR(162MHz,D2O)δ-0.90,-11.50,-22.95.

[0706] Example 81

[0707] Synthesis of Compound 2092

[0708]

[0709] 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2092-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2092, an ammonium salt (65 mg).

[0710] 1 H NMR(400MHz,D2O)δ8.19(s,1H),8.08(s,1H),6.37–6.19(m,2H),6.09(dd,J=2.8,0.8Hz,1H),4.97(ddd,J=4.4,2.9,0.7Hz,1H),4.76–4.70(m ,1H),4.64(t,J=2.8Hz,1H),4.55–4.45(m,3H),4.44–4.35(m,2H),4.3 3(d,J=2.8Hz,1H),4.26–4.06(m,6H),4.05–3.97(m,4H),3.39(s,3H).

[0711] 31 P NMR(162MHz,D2O)δ-0.95,-11.70,-22.75.

[0712] Example 82

[0713] Synthesis of Compound 2098

[0714]

[0715] 9a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2098-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powder, compound 2098, an ammonium salt (70 mg).

[0716] 1 H NMR(400MHz,D2O)δ8.42(s,1H),8.18(s,1H),7.93(d,J=7.9Hz,1H),6.15(dq,J=4.1,0.7Hz,1H), 6.10(dd,J=2.9,0.7Hz,1H),6.01(ddd,J=2.7,1.7,0.8Hz,1H),5.91(d,J=7.8Hz,1H),5.02–4.88 (m,1H),4.77(dddd,J=5.9,4.3,1.5,0.7Hz,1H),4.55(dddd,J=5.3,2.9,0.6Hz,1H),4.48(qt,J=3 .3,0.8Hz,1H),4.36(ddd,J=7.3,2.8,0.6Hz,1H),4.30–4.04(m,10H),4.02(s,3H),3.39(s,3H).

[0717] 31 P NMR(162MHz,D2O)δ-0.95,-11.70,-22.75.

[0718] Example 83

[0719] Synthesis of compound 2110

[0720]

[0721] 9b (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2110-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum to remove most of the water, and the remaining liquid was lyophilized to give a white powdery product, compound 2110, as an ammonium salt (70 mg).

[0722] 1 H NMR(400MHz,D2O)δ8.41(s,1H),8.18(s,1H),6.31(dd,J=1.4,0.8Hz,1H),6.13(dd, J=2.5,0.8Hz,1H),4.72–4.53(m,2H),4.35–4.06(m,8H),4.02(s,3H),3.38(s,3H).

[0723] 31 P NMR(162MHz,D2O)δ-10.24,-21.26.

[0724] Example 84

[0725] Synthesis of compound 2123

[0726]

[0727] 10a (200 mg) was added to 16 mL of a pH 7.0 aqueous solution containing 0.2 mol / L N-methylmorpholine and 0.2 mol / L manganese chloride, followed by 2123-1 (200 mg). The reaction mixture was stirred at room temperature (25 °C) for 16 hours. TLC showed product formation. The reaction mixture was then added to a pre-cooled 0 °C solution of disodium EDTA (1.4 g, in 80 mL of water), and the mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate aqueous solution. The fraction was concentrated under vacuum, and the remaining liquid was lyophilized to give a white powder, compound 2123, an ammonium salt (50 mg).

[0728] 1 H NMR(400MHz,D2O)δ7.62(dd,J=7.4,1.8Hz,1H),6.24(dd,J=2.7,0.6Hz,1H),6.12 (ddt,J=2.6,1.6,0.7Hz,1H),5.96(d,J=7.3Hz,1H),4.64(t,J=2.8Hz,1H),4.49(d ,J=11.6Hz,1H),4.42(ddd,J=5.3,2.4,0.7Hz,1H),4.39(d,J=11.6Hz,1H),4.33(d ,J=2.8Hz,1H),4.27(td,J=5.1,0.7Hz,1H),4.21–4.05(m,4H),4.04–3.96(m,4H).

[0729] 31 P NMR(162MHz,D2O)δ-10.70,-20.75.

[0730] Example 85

[0731] Capped mRNA synthesis efficiency detection

[0732] a) Linearize the plasmid and purify the DNA template.

[0733] b) mRNA was synthesized by in vitro transcription using the capped analogues of this invention and the capped analogues of Comparative Example 1 (TrilinkCleanCap), Comparative Example 2, and Comparative Example 3. Comparative Example 1 was commercially available, while Comparative Examples 2 and 3 were obtained according to the methods disclosed in WO2022 / 036858.

[0734]

[0735] The minimum reaction system for mRNA preparation used in this invention is shown in Table 1.

[0736] Table 1. In vitro transcription system

[0737]

[0738]

[0739] c) During the experiment, after thoroughly mixing the above reagents, incubate at 37°C. After 4 hours, add deoxyribonuclease (DNase) and continue incubation for 30 minutes to remove the DNA template. After digestion, add LiCl solution, pre-cool, centrifuge at 16000 rpm for 15 minutes, and discard the supernatant. Add 70% ethanol, centrifuge again, discard the supernatant, and then add a certain amount of enzyme-free water for storage. The purified mRNA sample was then quantitatively detected using Nanodrop One, as shown in Table 2 below. The experimental results show that the product yield of the halogenated cap analog in this invention is improved compared to the comparative example in mRNA synthesis.

[0740] Table 2. Mass (mg) of final product obtained per 1 mL mRNA synthesis reaction system

[0741]

[0742] 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) to provide accurate molecular weight information of the digested fragments. Combined with the theoretical molecular weight of the digested fragments, the capping efficiency of the sample can be assigned and obtained.

[0743] The capping rates of the mRNA synthesized from the capping analogs of this invention are all between 90% and 98%. Based on the purified capped mRNA, the compounds of this invention all exhibit excellent capping efficiency.

[0744] Example 86

[0745] The expression efficiency of different capped analogues of green fluorescent protein mRNA was evaluated in different cells.

[0746] This invention investigated the expression efficiency of different capped green fluorescent protein (GFP) mRNAs in HEK293T and HepG2 cells. Using the GFP coding sequence as a DNA template, the capped analogue described in this invention was used as a raw material for in vitro mRNA transcription. Subsequently, different mRNA products were transfected into cells, and finally, the fluorescent protein in the cells was detected by flow cytometry.

[0747] a) The different cells mentioned above were divided into 2×10 5 Cells were plated in a 96-well plate.

[0748] b) Mix 300 μL mRNA buffer with 6 μg RNA, then add 6 μL transfection reagent (JetMESENG-ER) and mix well. After standing for 10 min, add the mixture to each well of cells, and add transfection medium (Opti-MEM) to 2 mL / well. Incubate at 37°C and 5% CO2 for 6 h.

[0749] c) After replacing the medium with fresh complete culture medium and culturing under the same conditions for another 24 hours, the GFP fluorescence intensity was observed using a fluorescence microscope. The results are as follows: Figure 1 As shown in the figure, it is clear that the mRNA expression efficiency in this invention is higher than that in the comparative example.

[0750] d) After culturing the transfected cells for 24 hours, they were processed and then detected using a flow cytometer (CytoFLEX S series). The detected fluorescence intensity was directly proportional to the translation efficiency of the target protein. The results are as follows: Figure 2 and 3 As shown.

[0751] in Figure 2 This is a fluorescence intensity analysis graph of the mRNA encoding different cap analogs of 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 flow cytometry. Compared with Comparative Examples 1 and 2, the fluorescence intensity values ​​of the cap analogs in this invention are significantly higher in HEK293T cells. 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.

[0752] Figure 3This is a statistical graph showing the fluorescence intensity of mRNAs with different cap analogs in Hep G2 cells. Comparatively, the efficiency of mRNA translation into protein expression in Hep G2 cells containing the cap analogs of this invention is higher than that of the control group. Fluorinated nucleoside dimer cap analogs, such as compound 1982, and fluorinated nucleoside tetramer cap analogs, such as 2057, also showed significant increases in protein expression.

[0753] Example 87

[0754] Expression efficiency test of mRNA synthesized from different cap analogs in mice

[0755] a) Prepare mRNA encoding luciferase using the cap analog of the present invention, and dilute the obtained mRNA in citrate buffer at pH 4.0; dissolve cationic lipid DLin-MC3-DMA:DSPC:cholesterol:PEG lipid (DMG-PEG2000) in ethanol at a molar ratio of 50:10:38.5:1.5.

[0756] b) Load 3 mL of mRNA buffer and 1 mL of lipid solution into two 5 mL syringes, attach them to a microfluidic injection pump, set the pump flow rate, put the collected product into a dialysis bag, then concentrate it to the ideal concentration by ultrafiltration, and then filter the lipid nanoparticles through a 0.22 μm sterile filter and store them for later use.

[0757] c) Luciferase mRNA-lipid nanoparticles containing 5 μg mRNA were administered to 6-8 week old females.

[0758] Balb / c mice were administered each type of Luciferase mRNA-lipid nanoparticle via tail vein injection in parallel experiments (5 mice per injection). Luciferase substrate was injected 24 hours later, and the results were detected using a PerkinElmer small animal imaging system. The fluorescence intensity was directly proportional to the effective translation efficiency of the target protein. The relative fluorescence intensity of mRNA in different mouse organs is shown in the figure. Figure 4 As shown in the figure, the expression efficiency of mRNAs with different halogenated caps in different organs in this invention is significantly higher than that in the comparative example.

Claims

1. A compound for 5' capping of nucleic acids, or a pharmaceutically acceptable salt thereof, said compound having the structure of formula (I): Its features are: R0 is selected from any one of F, Cl, Br, and I; R1 is selected from -H, -OH, C. 1-4 Alkyl and C 1-4 An alkoxy group; R2 is selected from -H, -OH, C. 1-6 Alkyl and C 1-6 Any one of the alkoxy groups; Optionally, R1 and R2 are linked together by a chemical bond to form a ring, and -R1-R2- is -(CH2). q -O-, -O-(CH2) q - and -(CH2) m -O-(CH2) n - any one of them, where q, m, and n are each independently 1, 2, or 3; R3 is H, -OH, -N3, a halogen, or C. 1-6 Alkoxy, -SR 3a , -O(CH2) p Any of N3, where p is any integer from 1 to 6, R 3a C 1-6 Alkyl group, wherein R3 is optionally surrounded by one or more R 3e Replace, R 3e Choose C freely 1-4 Alkyl, C 1-4 The group consisting of alkoxy groups; R4, R5, R6, and R7 are each independently selected from any one of -H, -OH, -OCH3, or halogens; N 01 N 02 Each can be independently selected from 0 or 1; N 03 N 04 Each is independently equal to 0; J1, J2, J3, J4, and J5 are each independently selected from natural pyrimidine nucleotide bases and natural or modified purine nucleotide bases, wherein the modified purine nucleotide bases are methyl-modified purine nucleotide bases. R P1 It is a C1-C6 alkyl group; R P2 and R P3 Each is independently selected from H and C1-C6 alkyl groups; The premise is that when N 01 N 02 N 03 N 04 Both are 0. J5 is a guanine base. When R2 is -OH, R3 is not a methoxy group.

2. The compound of claim 1, wherein its pharmaceutically acceptable salt is characterized in that, R P1 It is a C1-C3 alkyl group.

3. The compound of claim 1, wherein its pharmaceutically acceptable salt is characterized in that, The compound has the structure of formula (I'): Wherein, each group in formula (I') has the definition as described in claim 1.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, At least one of J1, J2, J3, J4, and J5 is a modified purine nucleotide base.

5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein the modified purine nucleotide base is 6-N-methyladenine.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein, R3 can be -H, -OH, -N3, a halogen, or C. 1-6 Alkoxy, -SR 3a , -O(CH2) p Any of N3, where p is any integer from 1 to 4, R 3a C 1-4 Alkyl group, wherein R3 is optionally surrounded by one or more R 3e Replace, R 3e Choose C freely 1-4 Alkyl, C 1-4 The group composed of alkoxy groups.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein, R3 can be -H, -OH, -N3, a halogen, or C. 1-3 Alkoxy, -SR 3a , -O(CH2) p Any of N3, where p is any integer from 1 to 3, R 3a It is methyl or ethyl, wherein R3 is optionally mixed with one or more R 3e Replace, R 3e Choose C freely 1-4 Alkyl, C 1-4 The group composed of alkoxy groups.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (Ia), formula (Ib), or formula (Ic):

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein, R0 is -F or -Cl, and / or R4 and R5 are each independently selected from any one of H, OH, OCH3, F, and Cl.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein, R4 and R5 are each independently selected from any one of H, OH, OCH3, and F.

11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (Id): in, R3' has the definition of R3 as defined in claim 1; The remaining groups have the definitions as described in claim 1.

12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (Ie), formula (If), or formula (Ig):

13. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein, R0 is -F or -Cl, and / or R4 and R5 are each independently selected from any one of H, OH, OCH3, F, and Cl.

14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein, R4 and R5 are each independently selected from any one of H, OH, OCH3, and F.

15. A compound for 5' capping of nucleic acids, or a pharmaceutically acceptable salt thereof, wherein said compound has one of the following structures:

16. The compound of any one of claims 1 to 15, wherein the compound is present in the form of a pharmaceutically acceptable salt.

17. The compound of claim 16, wherein the compound is present in the form of a triethylamine salt, sodium salt, potassium salt, ammonium salt, or tris(hydroxymethyl)aminomethane hydrochloride.

18. The use of the compound according to any one of claims 1 to 17 as an in vitro co-transcription RNA capping reagent.

19. An RNA molecule, characterized in that, The compound comprising any one of claims 1 to 17 is used as a cap structure or a cap structure fragment.

20. A pharmaceutical composition, characterized in that, It comprises the RNA molecule of claim 19, and a pharmaceutically acceptable carrier.

21. A method for synthesizing RNA molecules, characterized in that, The method includes the following steps: co-incubating the compound according to any one of claims 1 to 17 with a polynucleotide template to perform template transcription.

22. A capped RNA transcription reaction system, characterized in that, include: A polynucleotide template, a compound according to any one of claims 1 to 17, NTPs, and RNA polymerase.

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