Nothing Special   »   [go: up one dir, main page]

CN118373866A - Compound for capping 5' end of nucleic acid and application thereof - Google Patents

Compound for capping 5' end of nucleic acid and application thereof Download PDF

Info

Publication number
CN118373866A
CN118373866A CN202310091020.3A CN202310091020A CN118373866A CN 118373866 A CN118373866 A CN 118373866A CN 202310091020 A CN202310091020 A CN 202310091020A CN 118373866 A CN118373866 A CN 118373866A
Authority
CN
China
Prior art keywords
compound
solution
added
water
pharmaceutically acceptable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310091020.3A
Other languages
Chinese (zh)
Inventor
胡勇
胡昭宇
姚君
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan Ruiji Biotechnology Co ltd
Shenzhen Ruiji Biotechnology Co ltd
Original Assignee
Wuhan Ruiji Biotechnology Co ltd
Shenzhen Ruiji Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan Ruiji Biotechnology Co ltd, Shenzhen Ruiji Biotechnology Co ltd filed Critical Wuhan Ruiji Biotechnology Co ltd
Priority to CN202310091020.3A priority Critical patent/CN118373866A/en
Priority to PCT/CN2024/073220 priority patent/WO2024153222A1/en
Publication of CN118373866A publication Critical patent/CN118373866A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention provides a 5' -end capping compound for nucleic acid and application thereof. The compound is shown as the following formula (I). The invention also provides the application and effect of the compound nucleic acid transcription and expression, including in vitro transcription, capping rate, cell and in vivo expression translation, and the like.

Description

Compound for capping 5' end of nucleic acid and application thereof
Technical Field
The invention relates to the technical fields of synthetic chemistry and bioengineering, in particular to a compound for capping a 5' end of nucleic acid (RNA) and application thereof.
Background
In recent years, with the change of the global new epidemic situation, the messenger RNA (mRNA) technology and vaccine medicaments thereof realize breakthrough from laboratory to clinical application. In the study of mRNA drugs and their functions, specific effects will only be apparent when the synthesized mRNA stably expresses the protein of interest in vivo. In the process of stably expressing the target protein, the capping reaction of the 5' end of mRNA is an indispensable modification process. In eukaryotic cells, the 5' end cap structure is capable of protecting the mRNA from degradation by exonucleases while preventing it from cleavage by recruited protein factors; the structure can also regulate protein synthesis; in addition, the 5' end cap structure also reduces mRNA immunogenicity and enhances mRNA in vivo stability.
In the in vitro transcription process, compared with the traditional enzymatic capping method, the mRNA co-transcription capping method is simpler and more convenient in process, and is easy to improve the cost efficiency and expand the mRNA drug productivity. At the same time, capping analogs have evolved from the first mCap to the second generation of ARCA and the third generation Cap of Cap1 analogs, with which transcription yields and capping efficiencies have been significantly improved.
Much research is currently devoted to chemical modification of the cap structure in an effort to further enhance the translation efficiency of mRNA while reducing its immunogenicity.
Disclosure of Invention
The invention provides a compound for capping the 5' end of RNA and application thereof, wherein the compound comprises pharmaceutically acceptable salts, solvates and stereoisomers thereof. The compound has higher capping rate and better in-vitro transcription efficiency on products obtained by capping the 5' end of mRNA, and has higher translation expression efficiency at the cellular and living levels.
The present invention provides a compound for capping the 5' end of a nucleic acid, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, having the structure of formula (I):
wherein:
R 0 is-OR 8, wherein R 8 is C 1-7 alkyl, C 2-7 alkenyl OR C 2-7 alkynyl;
R 1 is-H;
r 2 is-H, -OH, -O (any of CH 2)mCH3, wherein m is any integer from 0to 6;
Optionally R 1 and R 2 are linked by a chemical bond to form a ring, and-R 1-R2 -is any one of- (CH 2)q -O-or-O- (CH 2)q -), wherein q is 1,2 or 3;
R 3 is any one of H, -OH, -SH, -N 3,-NH2, halogen ,-CN,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2, wherein t is any one integer from 0 to 6, p is any one integer from 1 to 6, wherein R 3 is optionally substituted;
R 4、R5、R6、R7 is independently selected from any one of H, OH, OCH 3, halogen, -CN and-SH;
Each N 01、N02、N03、N04 is independently selected from 0 or 1, and the four are not simultaneously 0;
J 1、J2、J3、J4、J5 is each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base.
Embodiments of the invention include compounds of formula (I), or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein at least one of J 1、J2、J3、J4、J5 is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl modified purine nucleotide base, more preferably 6-N-methyladenine.
Embodiments of the present invention include compounds of formula (I), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein R 3 is any one of H,-SH,-N3,-NH2,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2, wherein t and p are each independently any one of integers from 1 to6, preferably any one of integers from 1 to 4.
Embodiments of the invention include compounds of formula (Ia), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein the groups in formula (Ia) have the meanings as indicated above:
Preferred embodiments of the present invention include compounds of formula (Ia), or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein,
R 0 is-OR 8, wherein R 8 is C 1-5 alkyl, preferably-CH 3, and/OR
R 4 is selected from any one of H, OH and halogen, preferably H or F.
Embodiments of the invention include compounds of formula (Ib), or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof:
R 3' is any one of H,-OH,-SH,-N3,-NH2,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2; the remaining groups have the meanings as described above.
Embodiments of the invention include compounds of formula (Ic), or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein the groups of formula (Ic) have the meanings as indicated above:
Preferred embodiments of the present invention include compounds of formula (Ic), or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein,
R 0 is-OR 8, wherein R 8 is C 1-5 alkyl, preferably-CH 3, and/OR
R 4 is selected from any one of H, OH and halogen, preferably H or F.
According to some embodiments of the invention, wherein the cap analogue has one of the structures shown below:
the invention also discloses application of the compound as an in vitro co-transcribed RNA capping reagent.
The invention also discloses an RNA molecule which comprises the compound as a cap structure or a cap structure fragment.
It will be appreciated that the RNA molecules described above can be used as either mRNA vaccines or RNA drugs, or in cell therapy in precision medicine.
The invention also discloses a pharmaceutical composition comprising the RNA molecule and a pharmaceutically acceptable carrier.
The invention also discloses a method for synthesizing RNA molecules, which comprises the following steps: the above compound is incubated with a polynucleotide template to perform template transcription.
The invention also discloses a capping RNA transcription reaction system, which comprises: polynucleotide templates, the above compounds, NTPs, RNA polymerase.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIGS. 1 to 3 show the expression efficiency of mRNA synthesized using cap analogues of the present invention in HEK293T, rat synovial cells and HUVEC, respectively.
FIG. 4 shows a fluorescence imaging of mice using mRNA synthesized with cap analogues of the present invention.
FIGS. 5 to 7 show the relative fluorescence intensities of mRNA synthesized using cap analogues of the present invention in different organs of mice, respectively.
Detailed Description
The invention provides a compound for capping the 5' end of RNA and application thereof, wherein the compound comprises pharmaceutically acceptable salts, solvates and stereoisomers thereof. The compound has higher capping rate and better in-vitro transcription efficiency on products obtained by capping the 5' end of mRNA, and has higher translation expression efficiency at the cellular and living levels.
Before further describing the present invention, certain terms used in the specification, examples, and appended claims are collected in the following sections. The definitions set forth herein should be read and understood by those skilled in the art in light of the remainder of the present invention. Unless defined otherwise, 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 belongs.
Definition of the definition
When any type of range is disclosed or claimed, it is intended that each possible value that the range may reasonably cover be disclosed or claimed separately, including any subrange encompassed therein, unless otherwise indicated. For example, a number of groups of 1 to 6 indicates an integer within this range, where 1-6 is understood to include 1, 2,3, 4, 5, 6, and also to include sub-ranges of 1-5, 1-4, and 1-3.
The description of the present disclosure should be construed as consistent with the principles and principles of chemical bonding. In some cases, it may be possible to remove a hydrogen atom in order to accommodate a substituent at a given position.
The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of … …", or "consisting of … …".
The term "pharmaceutically acceptable" in the present application means: the compound or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.
Natural or modified pyrimidine nucleotide bases include, but are not limited to: uracil, thymine, cytosine, 5-methylcytosine, 5-fluorouracil, 5-fluorocytosine, and the like.
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., wherein the 6-N-methyladenine has the structure of
"Stereoisomers" refer to compounds having the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.
"Connected into a ring through a chemical bond" means that two groups are connected through a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond, etc., to form a cyclic structure, and the corresponding groups can reduce 1-2 hydrogen atoms if necessary.
The expression "optionally substituted" means that one, two, three or more than three hydrogen atoms in the group may be substituted independently of each other by individual substituents. The substituents may be selected from alkyl, alkenyl, alkynyl, alkoxy, halo, cyano, amino, nitro, -OH.
The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain comprises 1 to 10 carbon atoms, i.e. 1, 2, 3,4,5, 6, 7, 8, 9 or 10 carbon atoms, preferably 1 to 6 carbon atoms, 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 is optionally substituted.
The term "alkoxy" includes-O-alkyl groups and alkyl groups in which the O atom is in an alkyl chain, such as-CH 2-O-CH3, which contains from 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, and most preferably from 1 to 3 carbon atoms. The alkoxy group is optionally substituted.
The term "alkenyl" includes both straight chain alkyl groups and branched alkyl groups containing at least two carbon atoms and at least one carbon-carbon double bond, containing from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, most preferably from 2 to 3 carbon atoms. The alkenyl group is optionally substituted.
The term "alkynyl" denotes a radical in which there is at least one unsaturated site, i.e. one carbon-carbon sp triple bond, comprising from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, most preferably from 2 to 3 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH 2 C≡CH), 1-propynyl (-C≡C-CH 3), and the like. The alkynyl group is optionally substituted.
The term "pharmaceutically acceptable salt" refers to the relatively non-toxic addition salts of the compounds of the present disclosure. See, for example, S.M. Bere et al, "Pharmaceutical Salts", J.Pharm. Sci.1977,66,1-19.
Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be acid addition salts of the compounds of the present disclosure having sufficient basicity, e.g., with inorganic acids such as: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or acid addition salts with organic acids such as: for example formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, persulphuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid or thiocyanic acid.
In addition, another suitable pharmaceutically acceptable salt of the compounds of the invention which is sufficiently acidic 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 with an organic base providing a physiologically acceptable cation, such as a salt with: n-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexamethylenediamine, ethanolamine, glucamine, sarcosine, serinol, tris-hydroxymethyl aminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol. Alternatively, the basic nitrogen-containing groups may be quaternized with the following agents: 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, and the like.
Those skilled in the art will also recognize that the acid addition salts of the claimed compounds can be prepared by any of a variety of known methods by reacting the compounds with an appropriate mineral or organic acid. Or alkali metal salts and alkaline earth metal salts of the acidic compounds of the present disclosure are prepared by reacting them with an appropriate base by various known methods.
The present invention includes all possible salts of the compounds of the present disclosure, which may be single salts or any mixture of the salts in any ratio.
The term "solvate" is a substance, such as a di-, mono-, or hemi-solvate, formed by combining, physically binding, and/or solvating a compound of the application with a solvent molecule, wherein the ratio of solvent molecules to the compound of the application is about 2:1, about 1:1, or about 1:2, respectively. This physical bonding involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (e.g., when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate may be isolated. Thus, solvates include solution phases and separable solvates. The compounds of the application may be in solvated form with pharmaceutically acceptable solvents (e.g., water, methanol, and ethanol), and the application is intended to cover both solvated and unsolvated forms of the compounds of the application. One solvate is a hydrate.
The term "pharmaceutical composition" as used herein refers to a substance and/or combination of substances for identifying, preventing or treating a tissue state or disease. The pharmaceutical compositions are formulated for administration to a patient for diagnosis, prevention and/or treatment of a disease. In addition, pharmaceutical compositions refer to combinations of an active agent with an inert or active carrier, rendering the composition suitable for therapeutic use.
As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as aqueous saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions as well as aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable drug carriers are described in "Remington's Pharmaceutical Sciences" of e.w. martin.
The term "halogen" refers to fluorine, chlorine, bromine, iodine, and astatine.
The term "optional" means that this situation may or may not occur.
Examples
Reagents and models used
The starting materials for the examples are commercially available and/or may be prepared by a variety of 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 solvents, reaction atmospheres, reaction temperatures, duration of the experiment, and post-treatments) in the synthetic methods described below. Those skilled in the art of organic synthesis will understand that the functional groups present on each part of the molecule should be compatible with the reagents and reactions presented.
All reagents, compounds synthesized are commercially available in China (without port Australia) and suppliers include Sigma-Aldrich (U.S.), shanghai megadimension technologies development Co., ltd (TRINLINK CLEANCAP) and jetMESSENGER
Cell model: HEK293T, rat synovial cells and HUVEC cells were purchased from the marsupenario life technologies limited.
The instrument used is as follows: multifunctional enzyme labeling instrument (MolecularDevices).
Compound preparation and identification: nuclear magnetic resonance (Bruker 300 MHz), liquid chromatography-mass spectrometer (Agilent 6150/1290), high performance liquid chromatograph (Agilent 1260).
Cell experiment: microscope, cell incubator (Thermo FISHER SCIENTIFIC).
Intermediate synthesis:
compound 8 used in each of the following examples was prepared by the following steps:
(1) 5g of Compound 1 was weighed out and dissolved in 10mL of anhydrous acetonitrile to obtain solution A. 4.5eq of tetrazole is dissolved in 120mL of anhydrous acetonitrile, 1eq of compound 2 is weighed and dissolved in the solution to obtain solution B, and then solution A containing compound 1 is dropwise added into solution B; reacting for 0.5 hour at room temperature of 25 ℃; after the reaction was completed, by Thin Layer Chromatography (TLC), 1.1eq of iodopyridine solution was added dropwise to the reaction solution; after the reaction is monitored, water is added for dilution, ethyl acetate is used for extraction, and the crude product of the compound 3 is obtained after concentration.
(2) 6.5G of crude compound 3 was weighed into 40mL of acetic acid and 10mL of water was added; the reaction solution reacts for 16 hours at the room temperature of 25 ℃; after the completion of the TLC monitoring reaction, the reaction solution was concentrated to dryness and purified by a silica gel column to give Compound 4.
(3) 3G of compound 4 was weighed and dissolved in 4.5eq of tetrazole acetonitrile solution, then 3eq of phosphine reagent 5 was added, and the reaction solution was stirred at room temperature 25 ℃ for 1 hour; after the TLC monitoring reaction is finished, 3.2eq of iodopyridine solution is added; after the completion of the monitoring reaction, water was added for dilution, followed by extraction with ethyl acetate, concentration and purification by silica gel column to obtain compound 6.
(4) 2G of Compound 6 was weighed and dissolved in 20mL of methanol and 20mL of concentrated ammonia, and the reaction solution was stirred at room temperature of 25℃for 48 hours; after the TLC monitoring reaction is finished, the reaction liquid is concentrated to obtain a crude product of the compound 7.
(5) 1.5G of crude compound 7 was weighed and dissolved in 1mL of DMSO, 1.2mL of TEA.3HF was added, and the reaction solution was stirred at 50℃for 1 hour; after the TLC monitoring reaction is finished, the reaction solution is cooled to room temperature, diluted by adding water and then the pH value is adjusted to 5.5 by using a saturated sodium carbonate aqueous solution; the mixture was loaded onto a DEAE Sephadex column and the mobile phase eluted with a linear gradient of 0-1.0M TEAB eluent to give compound 8.
And (3) synthesizing a final product:
method for synthesizing end product 1
0.4G of Compound 8 was weighed out and dissolved in 8mL of anhydrous DMSO, and 2eq of Compound 9 and 20eq of anhydrous zinc chloride were added under argon. Stirring the reaction solution for 24 hours at room temperature of 25 ℃ under the protection of argon; after the completion of TLC monitoring, the reaction was terminated with 150mL of a 0.25M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column; and (3) linearly gradient eluting the product by using 0.0-1.0M ammonium bicarbonate eluent, and collecting the eluent containing the product and freeze-drying to obtain the product.
Method for synthesizing end product 2
0.2G of Compound 10 was weighed out and dissolved in 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and pH 7.0, and then 0.2g of Compound 8 was added to the solution; the reaction solution was stirred at room temperature 25 ℃ for 16 hours; after completion of the TLC monitoring, the reaction was terminated with 150mL of 0.25M EDTA solution, and the mixture was loaded onto a DEAE Sephadex column; and (3) linearly gradient eluting the product by using 0-1.0M ammonium bicarbonate eluent, and collecting the eluent containing the product and freeze-drying to obtain the product.
Example 1
Synthesis of Compound 5
Step 1:
In a three-necked flask, compound 1-2 (2.38 g) was added to an acetonitrile solution (63 mL) of tetrazole (1.76 g), and argon was replaced three times; then, compound 1-1 (5 g) was dissolved in 10mL of acetonitrile solution at room temperature of 25℃and then added to the above solution, and the resulting solution was stirred at room temperature of 25℃for 1 hour, and no significant heat release was found, and the spot plate monitoring showed disappearance of starting material 1-1. To the solution was added dropwise a solution of pyridine/tetrahydrofuran/water of iodine (0.5 mmol/mL, pyridine: tetrahydrofuran: water = 1:8:1) until the solution was no longer discolored; the reaction was then stirred for a further 0.5 hour and the oxidation was monitored by a spot plate. After quenching the reaction mixture with saturated aqueous sodium sulfite (10 mL), it was diluted with 50mL of water, extracted with dichloromethane (50 mL. Times.2), the organic phases were combined, washed once with water (50 mL), and concentrated to give the product 1-3 (8 g, crude) as a pale yellow oil.
Step 2:
Compounds 1 to 3 (8 g, crude) were dissolved in 40mL of acetic acid and 10mL of water and stirred at 25℃for 16 hours, and the dot plates monitored the disappearance of compounds 1 to 3 with the occurrence of the polar dot. The reaction solution was directly concentrated in vacuo. After concentration, a suitable amount of silica gel and DCM were added for sample stirring and purification (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min) and concentration gave the product 1-4 as a white solid (2.8 g,51% two-step yield).
Step 3:
28mL of tetrazole in acetonitrile (0.4 mmol/mL) was prepared for use. Compounds 1 to 4 (2.8 g) were added to the solution, then compounds 1 to 5 (3 g) were added to the solution at room temperature of 25℃with nitrogen replaced three times, and the reaction solution was stirred at room temperature of 25℃for 1 hour, and the reaction was shown to be complete by dot-plate monitoring. The reaction solution was cooled to below 10 ℃ in an ice water bath, and a solution of pyridine/tetrahydrofuran/water (0.5 mmol/mL, pyridine: tetrahydrofuran: water=1:8:1) of iodine was added dropwise until the reaction solution did not fade, and the spot-on-plate monitoring showed that the oxidation reaction was complete. Then, 10mL of a saturated aqueous sodium sulfite solution was added to the reaction mixture to quench it, followed by dilution with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. Adding appropriate amount of silica gel and DCM, stirring, and purifying (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min). Concentration gave 1-6 as a white foamy solid (2.6 g,78.2% yield).
Step 4:
Compounds 1-6 (2.6 g) were dissolved in methanol (30 mL) and concentrated ammonia (30 mL) was added, and the resulting solution was stirred at 25℃for 60 hours at room temperature, and spot-on-plate monitoring showed complete reaction of compounds 1-6. The reaction mixture was concentrated in vacuo, and concentrated again with methanol to give 1-7 (2.4 g, crude) as pale yellow oily compound, which was directly taken to the next step.
Step 5:
Compounds 1-7 (2.4 g, crude) were dissolved in DMSO (3 mL) and triethylamine trihydrofluoride (3.5 mL) was added and the reaction stirred at 50℃for 1 hour, as indicated by the dot-dash panels indicating complete reaction of compounds 1-7. The reaction mixture was diluted to 50mL with water, the pH was adjusted to 5.5 with 1mol/L NaOH aqueous solution, and the mixture was loaded onto a DEAE Sephadex column. The product is eluted with linear gradient of 0-1.0M ammonium bicarbonate aqueous solution, the obtained component is concentrated in vacuum to obtain most of water, and the residual liquid is freeze-dried to obtain amine salt 1-8 (0.8 g,33.7% yield) of the target compound, and the product is white solid.
Step 6:
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine at pH 7.0 containing 0.2mol/L and manganous chloride at 0.2mol/L, and then compounds 1-8 (200 mg) were added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give the ammonium salt product compound 5 (65 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ7.98(d,J＝87.6Hz,2H),7.47(s,1H),5.62(d,J＝65.9Hz,4H),4.10(td,J＝70.5,63.9,40.6Hz,16H),3.79(s,3H),3.32(s,3H),2.84(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ-1.10,-10.65,-22.84。
Example 2
Synthesis of Compound 8
Step 1:
In a three-necked flask, compound 2-2 (2.07 g) was added to an acetonitrile solution (56 mL) of tetrazole (1.6 g), argon was replaced three times, then Compound 2-1 (5 g) was dissolved in 10mL of acetonitrile at room temperature of 25℃and then added to the above solution, and the resulting solution was stirred at room temperature of 25℃for 1 hour, and no significant heat release was found, and the point plate monitoring showed disappearance of raw material 2-1. Then, a solution of iodine (5 g of iodine was dissolved in 40mL of THF: H 2 O: pyridine=8:1:1 mixed solution to prepare a solution of 0.5 mmol/mL) was added dropwise to the solution until the solution was no longer discolored, and then the reaction solution was stirred for 0.5 hours, followed by monitoring the completion of oxidation on a spot plate. After quenching the reaction mixture with aqueous Na 2SO3 (10 mL), it was diluted with 50mL of water, extracted with dichloromethane (50 mL. Times.2), the organic phases were combined and washed once with water (50 mL), and concentrated to give 2-3 (7.5 g, crude) as a pale yellow oil.
Step 2:
compound 2-3 (7.2 g, crude) was dissolved in 40mL of acetic acid and 10mL of water, and the reaction was stirred at 25℃for 16 hours, with the dots of the plate monitoring the disappearance of compound 1-3, with the dots of the more polar nature. The reaction solution was directly concentrated in vacuo. After concentration, a suitable amount of silica gel and DCM were added for sample stirring and purification (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min) and concentration gave the product as a white solid 2-4 (2.8 g,54.8% two-step yield).
Step 3:
28mL of tetrazole in acetonitrile (0.4 mmol/mL) was prepared for use. Compound 2-4 (2.8 g) was added to the above solution, and then compound 2-5 (2.26 g) was added to the solution at room temperature of 25℃with nitrogen being replaced three times, and the reaction solution was stirred at room temperature of 25℃for 1 hour. Dot panel monitoring showed complete reaction. The reaction solution was cooled to below 10 ℃ in an ice water bath, and a solution of pyridine/tetrahydrofuran/water (0.5 mmol/mL, pyridine: tetrahydrofuran: water=1:8:1) of iodine was added dropwise until the reaction solution did not fade, and the spot-on-plate monitoring showed that the oxidation reaction was complete. Then, 10mL of saturated aqueous sodium sulfite solution was added to the reaction mixture to quench it, followed by dilution with water. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. Adding appropriate amount of silica gel and DCM, stirring, and purifying (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min). Concentration gave 2-6 (3.1 g,93.5% yield) as a white foamy solid.
Step 4:
compound 2-6 (3.1 g) was dissolved in methanol (30 mL), then concentrated ammonia (30 mL) was added, and the resulting solution was stirred at room temperature 25℃for 60 hours, and the reaction of compound 2-6 was complete on a spot plate monitor. The reaction mixture was concentrated in vacuo and concentrated again with methanol to give 2-7 (2.4 g, crude) as pale yellow oily compound.
Step 5:
Compound 2-7 (2.4 g, crude product) was dissolved in DMSO (3 mL), and triethylamine trihydrofluoride (3.5 mL) was added, and the reaction mixture was stirred at 50℃for 1 hour, and the dot-matrix showed complete reaction of starting material 2-7. The reaction mixture was diluted to 50mL with water, the pH was adjusted to 5.5 with 1N aqueous NaOH, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M TEAB eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give triethylamine salt 2-8 (1.5 g,51% yield) of the target compound as a white solid.
1H NMR(400MHz,Deuterium Oxide)δ8.48(s,1H),8.05(s,1H),7.82(s,1H),5.97(d,J＝6.4Hz,1H),5.78(d,J＝6.1Hz,1H),4.77(ddd,J＝6.3,4.9,1.3Hz,1H),4.71–4.67(m,1H),4.49(dd,J＝5.2,3.5Hz,1H),4.42–4.36(m,1H),4.26(t,J＝5.7Hz,1H),4.22–4.15(m,1H),4.05(h,J＝7.2Hz,2H),3.87(d,J＝3.9Hz,2H),3.27(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ3.63，δ-0.62。
Step 6:
Compounds 2-8 (500 mg), 9a (500 mg) and anhydrous zinc chloride (1.2 g) were added separately under argon at room temperature (25 ℃ C.) and then reacted with anhydrous DMSO (8 mL) using a syringe for 24 hours, the dot plates showing the majority of the starting material reaction. The reaction solution was added to EDTA disodium salt solution (1.6 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give the ammonium salt product compound 8 (120 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ8.16(s,1H),7.91(s,1H),7.74(s,1H),5.70(dd,J＝9.0,4.5Hz,3H),4.59(d,J＝4.6Hz,1H),4.48(dt,J＝12.4,4.8Hz,2H),4.38–4.29(m,3H),4.26(t,J＝5.1Hz,1H),4.22–4.01(m,8H),3.86(s,3H),3.26(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ-0.78,-11.58,-23.01。
Example 3
Synthesis of Compound 22
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine at pH 7.0 containing 0.2mol/L and manganous chloride at 0.2mol/L, and then Compound 6-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M ammonium bicarbonate aqueous eluent, and the remaining liquid was lyophilized to give ammonium salt product compound 22 (82 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ9.23(s,1H),8.40(d,J＝0.7Hz,1H),8.20(s,1H),7.58(dd,J＝7.3,1.8Hz,1H),6.18–6.14(m,2H),6.05(d,J＝7.3Hz,1H),5.95(ddd,J＝4.3,1.8,1.0Hz,1H),4.79–4.75(m,1H),4.72–4.65(m,1H),4.42(dtdd,J＝5.8,4.6,3.7,1.1Hz,2H),4.28–4.07(m,10H),4.05–3.97(m,4H),3.49(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22.
Example 4
Synthesis of Compound 28
Step 1:
In a three-necked flask, compound 4-2 (2.05 g) was added to an acetonitrile solution (56 mL) of tetrazole (1.6 g), argon was replaced three times, then Compound 4-1 (4 g) was dissolved in 10mL of acetonitrile at room temperature of 25℃and then added to the above solution, and the resulting solution was stirred at room temperature of 25℃for 1 hour, and no significant heat release was found, and monitoring on a spot plate showed disappearance of Compound 4-1. Then, a pyridine/tetrahydrofuran/water solution of iodine (0.5 mmol/mL, pyridine: tetrahydrofuran: water=1:8:1) was added dropwise to the solution until the solution was no longer discolored, and then the reaction solution was stirred for 0.5 hours, followed by monitoring the completion of oxidation on a spot plate. After quenching the reaction mixture with an aqueous solution of Na 2SO3 (10 mL), it was diluted with 50mL of water, extracted with dichloromethane, and the organic phase was washed once with water and concentrated to give 4-3 (5.6 g, crude) as a pale yellow oil.
Step 2:
Compound 4-3 (5.6 g, crude) was dissolved in 40mL of acetic acid and 10mL of water, and the reaction was stirred at 25℃for 16 hours, with the dot plates monitoring the disappearance of compound 4-3 and the occurrence of the polar dot. The reaction solution was concentrated in vacuo. After concentration, a suitable amount of silica gel and DCM were added for sample stirring and purification (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min), followed by concentration to give the product 4-4 as a white solid (2.4 g,54.8% two step yield).
Step 3:
24mL of tetrazole in acetonitrile (0.4 mmol/mL) was prepared for use. Compound 4-4 (2.4 g) was added to the above solution, and then compound 4-5 (1.96 g) was added to the solution at room temperature of 25℃with nitrogen replaced three times, and the reaction solution was stirred at room temperature of 25℃for 1 hour, and the reaction was monitored on a spot plate to show completion of the reaction. The reaction solution was cooled to below 10 ℃ in an ice water bath, and a solution of pyridine/tetrahydrofuran/water with iodine (5 g iodine in 40mL of mixed solution THF: H 2 O: pyridine=8:1:1, formulated as a solution of 0.5 mmol/mL) was added dropwise until the reaction solution did not fade, and the spot-on-board monitoring showed that the oxidation reaction was complete. The reaction mixture was quenched by adding 10mL of saturated aqueous sodium sulfite solution, and diluted with water. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. A proper amount of silica gel and DCM are added for sample stirring and purification (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min), and the white foam solid 4-6 (2.4 g,75.4% yield) is obtained by concentration.
Step 4:
Compound 4-6 (2.4 g) was dissolved in methanol (30 mL), then concentrated ammonia (30 mL) was added, and the resulting solution was stirred at 25℃for 60 hours at room temperature, and the reaction of compound 4-6 was complete on a spot plate monitor. The reaction mixture was concentrated in vacuo, diluted to 50mL with water, adjusted to pH 5.5 with dilute hydrochloric acid, and the mixture loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M TEAB eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give triethylamine salt 4-7 (1.5 g,51% yield) of the target compound as a white solid.
1H NMR(400MHz,Deuterium Oxide)δ8.48(s,1H),8.05(s,1H),7.82(s,1H),5.97(d,J＝6.4Hz,1H),5.78(d,J＝6.1Hz,1H),4.77(ddd,J＝6.3,4.9,1.3Hz,1H),4.71–4.67(m,1H),4.49(dd,J＝5.2,3.5Hz,1H),4.42–4.36(m,1H),4.26(t,J＝5.7Hz,1H),4.22–4.15(m,1H),4.05(h,J＝7.2Hz,2H),3.87(d,J＝3.9Hz,2H),3.27(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ3.63，δ-0.62。
Step 5:
Compounds 4-7 (500 mg), 9a (500 mg) and anhydrous zinc chloride (1.2 g) were added separately under argon at room temperature (25 ℃ C.) and then reacted with anhydrous DMSO (8 mL) using a syringe for 24 hours, the dot plates showing the majority of the starting material reaction. The reaction solution was added to EDTA disodium salt solution (1.6 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 28 (120 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ8.16(s,1H),7.91(s,1H),7.74(s,1H),5.70(dd,J＝9.0,4.5Hz,3H),4.59(d,J＝4.6Hz,1H),4.48(dt,J＝12.4,4.8Hz,2H),4.38–4.29(m,3H),4.26(t,J＝5.1Hz,1H),4.22–4.01(m,8H),3.86(s,3H),3.26(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ-0.78,-11.58,-23.01。
Example 5
Synthesis of Compound 38
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 2-8 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 38 (50 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ7.93–7.81(m,1H),7.62(s,1H),7.50(s,1H),5.45(dd,J＝4.4,2.8Hz,2H),5.41(d,J＝3.7Hz,1H),4.35(q,J＝7.1,6.1Hz,1H),4.25(t,J＝4.3Hz,2H),4.19(t,J＝5.1Hz,1H),4.11(d,J＝4.7Hz,1H),4.00–3.91(m,5H),3.88–3.78(m,4H),3.68(d,J＝5.1Hz,1H),3.63(s,3H),3.08(s,6H).
31P NMR(162MHz,Deuterium Oxide)δ-1.17,-11.63,-22.81。
Example 6
Synthesis of Compound 44
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 6-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M ammonium bicarbonate aqueous eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 44 (53 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ7.90(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.16(dt,J＝2.2,0.7Hz,1H),6.09–6.05(m,1H),5.86(ddt,J＝3.4,1.8,0.8Hz,1H),5.76(d,J＝7.9Hz,1H),4.77–4.67(m,3H),4.29–4.04(m,11H),4.03–3.98(m,4H),3.48(s,3H),3.45(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.23。
Example 7
Synthesis of Compound 48
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 7-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M ammonium bicarbonate aqueous eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 48 (65 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ7.86(s,1H),7.69(dd,J＝7.9,1.8Hz,1H),6.23–6.19(m,1H),6.16(dt,J＝2.2,0.7Hz,1H),5.79(d,J＝2.6Hz,1H),5.76(d,J＝7.9Hz,1H),4.74–4.64(m,2H),4.42–4.34(m,2H),4.29–4.07(m,11H),4.05–3.97(m,4H),3.49(s,3H),3.45(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.62,-10.29,-21.22。
Example 8
Synthesis of Compound 71
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine at pH 7.0 containing 0.2mol/L and manganous chloride at 0.2mol/L, and then Compound 8-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo for the most part using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give ammonium salt product compound 71 (82 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.29(s,1H),8.23(d,J＝0.7Hz,1H),7.86(s,1H),6.40(ddd,J＝25.2,1.8,0.8Hz,1H),6.25–6.19(m,1H),6.17–6.07(m,1H),5.37–5.19(m,2H),4.64–4.57(m,1H),4.53–4.49(m,1H),4.39(ddt,J＝6.1,3.0,1.5Hz,1H),4.36–4.31(m,1H),4.30–4.09(m,9H),4.02(s,3H),3.49(s,3H),3.04(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22。
Example 9
Synthesis of Compound 73
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 9-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was then loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo for the most part using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 73 (62 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.29(s,1H),8.23(d,J＝0.7Hz,1H),7.80(dd,J＝7.3,1.8Hz,1H),6.40(ddd,J＝25.2,1.8,0.8Hz,1H),6.20–6.11(m,1H),6.05(d,J＝7.3Hz,1H),5.91(ddt,J＝2.7,1.7,0.8Hz,1H),5.37–5.19(m,2H),4.69–4.58(m,1H),4.54–4.47(m,2H),4.37–4.05(m,11H),4.02(s,3H),3.48(s,3H),3.04(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22。
Example 10
Synthesis of Compound 76
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 10-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 76 (95 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.20(s,1H),8.19(s,1H),7.86(s,1H),6.40(ddt,J＝24.4,1.7,0.8Hz,1H),6.23–6.18(m,1H),6.17–6.12(m,1H),5.37–5.19(m,2H),4.64–4.56(m,1H),4.51(td,J＝3.2,2.3Hz,1H),4.41–4.08(m,11H),4.02(s,3H),3.49(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22。
Example 11
Synthesis of Compound 77
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 11-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give the ammonium salt product compound 77 (95 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.20(s,1H),8.19(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.40(ddt,J＝24.5,1.8,0.8Hz,1H),6.20–6.11(m,1H),5.86(ddt,J＝3.3,1.8,0.8Hz,1H),5.37–5.18(m,2H),4.65–4.58(m,1H),4.51–4.49(m,1H),4.37–4.04(m,11H),4.02(s,3H),3.48(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22。
Example 12
Synthesis of Compound 117
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 12-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, and the remaining liquid was lyophilized to give ammonium salt product compound 117 (95 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ7.74(dd,J＝7.9,1.8Hz,1H),7.70(dd,J＝7.4,1.7Hz,1H),6.15(dd,J＝2.8,0.8Hz,1H),6.04(d,J＝7.3Hz,1H),5.86(ddt,J＝3.4,1.8,0.8Hz,1H),5.76(d,J＝7.9Hz,1H),5.55–5.44(m,1H),5.25(ddd,J＝3.8,2.9,0.7Hz,1H),5.14–5.03(m,1H),4.65–4.56(m,1H),4.50–4.46(m,1H),4.36–4.31(m,1H),4.28(tdd,J＝3.1,2.3,0.8Hz,1H),4.22–4.04(m,9H),4.02(s,3H),3.48(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-10.29,-21.22。
Example 13
Synthesis of Compound 121
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 13-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, the mixture was loaded on a DEAE Sephadex column, followed by linear gradient elution with 0-1.0M aqueous ammonium bicarbonate eluent, and the resulting fraction was concentrated in vacuo to a large portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 121 (75 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.31(d,J＝21.6Hz,2H),7.86(s,1H),6.42–6.27(m,1H),6.23–6.18(m,1H),6.16–6.12(m,1H),5.00(dddd,J＝8.6,5.5,2.3,1.6Hz,1H),4.65–4.57(m,1H),4.39(tdq,J＝3.8,2.4,1.2Hz,1H),4.35–4.31(m,1H),4.29–4.05(m,10H),4.02(s,3H),3.49(s,3H),3.04(s,3H),2.70–2.50(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-0.90,-10.29,-21.22。
Example 14
Synthesis of Compound 122
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 9-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 122 (75 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.33(s,1H),8.29(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.42–6.31(m,1H),6.17–6.08(m,1H),5.86(ddt,J＝3.4,1.7,0.8Hz,1H),5.76(d,J＝7.9Hz,1H),5.00(dddd,J＝8.6,5.5,2.3,1.6Hz,1H),4.63–4.55(m,1H),4.35–4.30(m,1H),4.30–4.25(m,1H),4.23–4.03(m,10H),4.02(s,3H),3.48(s,3H),3.04(s,3H),2.75–2.52(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-1.11,-10.31,-21.25。
Example 15
Synthesis of Compound 126
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 15-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give product compound 126 as a white powder as an ammonium salt (60 mg).
1H NMR(500MHz,Deuterium Oxide)δ8.28(s,1H),8.20(s,1H),7.86(s,1H),6.39(ddt,J＝4.7,3.1,0.8Hz,1H),6.29–6.19(m,1H),6.18–6.11(m,1H),5.03–4.95(m,1H),4.65–4.57(m,1H),4.39(ddq,J＝5.9,2.8,1.4Hz,1H),4.36–4.31(m,1H),4.29–4.05(m,10H),4.02(s,3H),3.49(s,3H),2.71–2.55(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-0.9,-10.29,-21.27。
Example 16
Synthesis of Compound 127
Compound 9a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 16-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The resulting fractions were then concentrated in vacuo with a major portion of water using a linear gradient of 0-1.0M ammonium bicarbonate aqueous eluent, and the remaining liquid was lyophilized to give ammonium salt product compound 127 (60 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.28(s,1H),8.20(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.39(ddt,J＝4.8,3.1,0.8Hz,1H),6.18–6.11(m,1H),5.86(ddt,J＝3.3,1.8,0.8Hz,1H),5.76(d,J＝7.9Hz,1H),5.04–4.97(m,1H),4.65–4.57(m,1H),4.38–4.04(m,12H),4.02(d,J＝0.7Hz,3H),3.49(d,J＝1.4Hz,3H),2.89–2.36(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-0.78,-10.29,-21.22。
Example 17
Synthesis of Compound 146
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine at pH 7.0 containing 0.2mol/L and manganous chloride at 0.2mol/L, and then compound 17-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give the ammonium salt product compound 146 (74 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.33(s,1H),8.29(s,1H),7.86(s,1H),6.49–6.37(m,1H),6.24–6.18(m,1H),6.16(dt,J＝2.2,0.7Hz,1H),5.25–5.10(m,1H),4.72–4.66(m,1H),4.39(dtt,J＝5.9,2.8,1.3Hz,1H),4.29–4.05(m,10H),4.04–3.99(m,4H),3.49(s,3H),3.45(s,3H),3.04(s,3H),2.73–2.42(m,2H).
31P NMR(202MHz,Deuterium Oxide)-0.55,-10.32,-21.28。
Example 18
Synthesis of Compound 148
Compound 9b (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 18-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 148 (60 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.33(s,1H),8.29(s,1H),7.81(dd,J＝7.3,1.8Hz,1H),6.43(ddq,J＝3.9,2.3,0.8Hz,1H),6.16(dt,J＝2.3,0.8Hz,1H),6.05(d,J＝7.5Hz,1H),5.91(ddt,J＝2.6,1.7,0.8Hz,1H),5.23–5.14(m,1H),4.73–4.66(m,1H),4.25–4.05(m,11H),4.04–4.00(m,4H),3.49(d,J＝1.4Hz,3H),3.45(d,J＝1.6Hz,3H),3.05(d,J＝5.3Hz,3H),2.75–2.47(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-0.85,-10.09,-21.23。
Example 19
Synthesis of Compound 176
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 2-8 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 176 (55 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ8.11(s,1H),7.83(s,1H),7.77–7.58(m,1H),5.66(t,J＝4.6Hz,2H),5.49(s,1H),4.46(s,1H),4.41(d,J＝4.6Hz,1H),4.35(d,J＝10.8Hz,2H),4.26–4.04(m,8H),3.98(d,J＝11.4Hz,1H),3.91(d,J＝8.4Hz,1H),3.84(s,1H),3.83–3.73(m,3H),3.24(s,3H).
31P NMR(162MHz,Deuterium Oxide)δ-0.90,-11.41,-22.93。
Example 20
Synthesis of Compound 191
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 20-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give the ammonium salt product compound 191 (70 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ7.86(s,1H),7.59(dd,J＝7.3,1.8Hz,1H),6.38–6.31(m,1H),6.19(dd,J＝3.0,0.7Hz,1H),6.05(d,J＝7.3Hz,1H),6.01–5.92(m,1H),4.70–4.64(m,2H),4.45–4.23(m,8H),4.21–4.05(m,4H),4.02(d,J＝0.7Hz,3H),3.99(s,2H),3.49(d,J＝1.4Hz,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.50,-9.18,-10.26,-21.22。
Example 21
Synthesis of Compound 201
Step 1:
in a three-necked flask, compound 21-2 (1.84 g) was added to an acetonitrile solution (50 mL) of tetrazole (1.42 g), argon was replaced three times, then Compound 21-1 (4 g) was dissolved in 10mL of acetonitrile at room temperature of 25℃and then added to the above solution, and the resulting solution was stirred at room temperature of 25℃for 1 hour, and no significant heat release was found, and monitoring on a spot plate showed disappearance of Compound 21-1. Then, a pyridine/tetrahydrofuran/water solution of iodine (0.5 mmol/mL, pyridine: tetrahydrofuran: water=1:8:1) was added dropwise to the solution until the solution was no longer discolored, and then the reaction solution was stirred for 0.5 hours, followed by monitoring the completion of oxidation on a spot plate. After quenching the reaction mixture with aqueous sodium sulfite (10 mL), it was diluted with 50mL of water, extracted with dichloromethane, and the organic phase was washed once with water and concentrated to give 21-3 (5.8 g, crude) as a pale yellow oil.
Step 2:
Compound 21-3 (5.8 g, crude) was dissolved in 40mL of acetic acid and 10mL of water, and the reaction was stirred at 25℃for 16 hours, with the dot plate monitoring that 21-3 disappeared and a dot of great polarity was generated. The reaction solution was concentrated in vacuo. After concentration, a suitable amount of silica gel and DCM were added for sample stirring and purification (40 g normal phase column, EA,10min,DCM:MeOH,10-20%20min, flow rate 30 ml/min), and the white solid product 21-4 (2.8 g,54.8% two-step yield) was obtained by concentration.
Step 3:
30mL of tetrazole in acetonitrile (0.4 mmol/mL) was prepared for use. Compound 21-4 (2.8 g) was added to the above solution, and then compound 21-5 (2.5 g) was added to the solution at room temperature of 25℃with nitrogen replaced three times, and the reaction solution was stirred at room temperature of 25℃for 1 hour, and the reaction was monitored on a spot plate to show completion of the reaction. The reaction solution was cooled to below 10 ℃ in an ice water bath, and a solution of pyridine/tetrahydrofuran/water (0.5 mmol/mL, pyridine: tetrahydrofuran: water=1:8:1) of iodine was added dropwise until the reaction solution did not fade, and the spot-on-plate monitoring showed that the oxidation reaction was complete. The reaction mixture was quenched with 10mL of saturated aqueous sodium sulfite solution and diluted with water. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and filtered. A suitable amount of silica gel and DCM was added to stir and purify (40 g normal phase column, EA,10min, DCM/MeOH,10-20%20min, flow rate 30 ml/min) followed by concentration to give 21-6 as a white foam (2.4 g,71.2% yield).
Step 4:
compound 21-6 (2.4 g) was dissolved in methanol (30 mL), then concentrated aqueous ammonia (30 mL) was added, and the resulting solution was stirred at 25℃for 60 hours at room temperature, and the reaction of compound 21-6 was complete on a spot plate monitor. The reaction solution was concentrated in vacuo, diluted to 50mL with water, ph=5.5 adjusted with dilute hydrochloric acid, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M TEAB eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give the triethylamine salt 21-7 (2.1 g,85% yield) of the target compound as a white solid.
1H NMR(400MHz,Deuterium Oxide)δ8.55(s,1H),8.09(s,1H),7.87(s,1H),6.06(d,J＝6.9Hz,1H),5.81(d,J＝6.4Hz,1H),4.87(ddd,J＝7.2,4.7,2.1Hz,1H),4.58(ddd,J＝6.6,4.7,1.6Hz,1H),4.51(dd,J＝5.2,3.2Hz,1H),4.43(t,J＝2.3Hz,1H),4.31(dd,J＝6.4,5.2Hz,1H),4.21(dd,J＝3.4,1.9Hz,1H),4.04(dd,J＝5.1,3.6Hz,2H),3.87(t,J＝3.5Hz,2H),3.30(s,6H).
Step 5:
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 21-7 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give the ammonium salt product compound 201 (75 mg) as a white powder.
1H NMR(400MHz,Deuterium Oxide)δ8.25(s,1H),7.93(s,1H),7.80(s,1H),5.86(d,J＝5.7Hz,1H),5.73(d,J＝5.5Hz,1H),5.60(s,1H),4.78(dt,J＝8.2,4.0Hz,1H),4.51–4.45(m,2H),4.39(s,1H),4.35–4.23(m,5H),4.21–4.04(m,5H),3.96(d,J＝8.5Hz,1H),3.87(d,J＝3.6Hz,4H),3.29(d,J＝1.9Hz,6H).
31P NMR(162MHz,Deuterium Oxide)δ-0.99,-11.40,-23.06。
Example 22
Synthesis of Compound 221
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 22-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give ammonium salt product compound 221 (85 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.29(s,1H),8.23(d,J＝0.7Hz,1H),7.86(s,1H),6.40(ddt,J＝24.4,1.8,0.7Hz,1H),6.36–6.32(m,1H),6.22–6.15(m,1H),5.37–5.14(m,2H),4.67(t,J＝2.7Hz,1H),4.53–4.47(m,1H),4.44–4.09(m,10H),4.02(s,3H),3.99(s,2H),3.49(s,3H),3.04(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-9.01,-10.23,-21.17。
Example 23
Synthesis of Compound 226
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 23-1 (200 mg) was added to the solution. The reaction solution was stirred at 25℃for 16 hours at room temperature, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 226 (92 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.20(s,1H),8.19(s,1H),7.86(s,1H),6.40(ddt,J＝24.4,1.8,0.7Hz,1H),6.36–6.32(m,1H),6.20–6.15(m,1H),5.40–5.13(m,2H),4.67(t,J＝2.7Hz,1H),4.53–4.48(m,1H),4.43–4.40(m,1H),4.39–4.08(m,9H),4.02(s,3H),3.99(s,2H),3.49(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-9.10,-10.31,-21.17。
Example 24
Synthesis of Compound 227
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 24-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give the ammonium salt product compound 227 (85 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.20(s,1H),8.19(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.40(ddt,J＝24.5,1.8,0.8Hz,1H),6.35(d,J＝2.8Hz,1H),5.84(ddd,J＝3.3,1.7,0.7Hz,1H),5.76(d,J＝7.9Hz,1H),5.39–5.09(m,2H),4.67(t,J＝2.7Hz,1H),4.51(td,J＝3.2,2.3Hz,1H),4.40–4.29(m,2H),4.26(t,J＝3.0Hz,1H),4.25–4.13(m,5H),4.10(d,J＝8.6Hz,1H),4.08–4.06(m,1H),4.02(s,3H),3.99(s,2H),3.48(s,3H).
31P NMR(202MHz,Deuterium Oxide)δ0.60,-9.10,-10.22,-21.17。
Example 25
Synthesis of Compound 246
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 25-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g,80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions concentrated in vacuo to a major portion of water, and the remaining liquid was lyophilized to give ammonium salt product compound 246 (80 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.33(s,1H),8.29(s,1H),7.86(s,1H),6.39(ddt,J＝4.8,3.1,0.8Hz,1H),6.36–6.34(m,1H),6.21–6.15(m,1H),5.00(dddd,J＝8.6,5.5,2.3,1.6Hz,1H),4.67(t,J＝2.7Hz,1H),4.44–4.05(m,11H),4.02(s,3H),3.99(s,2H),3.49(d,J＝1.4Hz,3H),3.04(s,3H),2.69–2.53(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ-0.22,-9.07,-10.25,-21.27。
Example 26
Synthesis of Compound 247
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 26-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give ammonium salt product compound 247 (58 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.33(s,1H),8.29(s,1H),7.74(dd,J＝7.9,1.8Hz,1H),6.39(ddd,J＝4.1,2.6,0.8Hz,1H),6.35(d,J＝2.7Hz,1H),5.84(ddd,J＝3.3,1.7,0.7Hz,1H),5.76(d,J＝7.9Hz,1H),5.00(dddd,J＝8.6,5.5,2.3,1.6Hz,1H),4.67(t,J＝2.7Hz,1H),4.40–4.28(m,2H),4.26(t,J＝3.0Hz,1H),4.24–4.12(m,5H),4.11–4.09(m,1H),4.08(d,J＝1.1Hz,1H),4.07(td,J＝2.8,1.8Hz,1H),4.02(s,3H),3.99(s,2H),3.48(s,3H),3.04(s,3H),2.75–2.46(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ0.66,-9.13,-10.26,-21.25。
Example 27
Synthesis of Compound 251
Compound 10a (200 mg) was added to 16mL of an aqueous solution of N-methylmorpholine containing 0.2mol/L and manganous chloride containing 0.2mol/L and ph=7.0, and then compound 27-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature of 25℃for 16 hours, and the dot plate showed the formation of a product. The reaction solution was added to EDTA disodium salt solution (1.4 g, 80mL of water) cooled to 0℃in advance, and the mixture was loaded on a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M aqueous ammonium bicarbonate eluent, the resulting fractions were concentrated in vacuo to mostly water, and the remaining liquid was lyophilized to give ammonium salt product compound 251 (88 mg) as a white powder.
1H NMR(500MHz,Deuterium Oxide)δ8.28(s,1H),8.20(s,1H),7.86(s,1H),6.41–6.37(m,1H),6.37–6.33(m,1H),6.23–6.16(m,1H),5.05–4.93(m,1H),4.67(t,J＝2.7Hz,1H),4.43–4.05(m,11H),4.02(s,3H),3.99(s,2H),3.49(s,3H),2.71–2.41(m,2H).
31P NMR(202MHz,Deuterium Oxide)δ0.85,-9.12,-10.24,-21.26。
Example 28
Detection of mRNA capping synthesis efficiency
A) The plasmid was linearized and the DNA template was purified.
B) mRNA was synthesized by in vitro transcription using 35 capping analogues of the invention and comparative example TRILINK CLEANCAP, respectively, with the reaction system shown in Table 1; during the experiment, the above reagents were thoroughly mixed and incubated at 37 ℃. After 2 hours, deoxyribonuclease (DNase) was added and incubation was continued for 30 minutes to remove the DNA template, followed by quantitative detection of the purified mRNA sample using Nanodrop One.
C) And (3) carrying out mRNA purification after exonuclease treatment, and detecting the mRNA capping rate of the capping compound by adopting a liquid chromatography mass spectrometry (LC-MS), wherein the mRNA capping rate in the experimental example is between 90 and 98 percent. The compounds of the present invention all showed good capping efficiency after purification.
TABLE 1 in vitro transcription System
Component (A) Dosage of
T7 RNA polymerase 4μL(200U)
RNase inhibitor 0.5μL(20U)
Inorganic pyrophosphatase 1μL(0.5U)
100mM ATP 1.5μL
100mM CTP 1.5μL
100mM GTP 1.5μL
100mMψTP 1.5μL
100MM cap analogue 1.5μL
10×buffer 2μL
DNA template 1μL
Water and its preparation method 4μL
Totalizing 20μL
TABLE 2 final product obtained per 20. Mu.L of reaction system
Example 29
Evaluation test of expression efficiency of different capping analog luciferase mRNAs in different cells
The invention respectively tests the expression efficiency of different capped luciferase mRNAs in HEK293T and rat synovial cells. Taking a Luciferase coding sequence as a DNA template, performing in vitro transcription of mRNA by taking the cap analogue as a raw material, then carrying out cell transfection on different mRNA products, and finally quantitatively detecting the protein expression quantity of each cell by an enzyme-labeled instrument:
a) The different cells were plated in 0.5X10 5 cells (96 well plate);
b) Mixing 30. Mu.L of mRNA buffer with 0.6. Mu.g of RNA, adding 0.6. Mu.L of transfection reagent (jetMESSENGER), mixing, standing for 10min, adding the mixture into each well of cells, supplementing the transfection medium (Opti-MEM) to 100. Mu.L/well, and culturing for 6h at 37 ℃ under the condition of 5% CO 2;
c) Changing the culture medium to a fresh complete culture medium, and continuously culturing for 12 hours under the same conditions;
d) 100 μl of 1 Xfluorescein working fluid was added to each well, incubated at 37deg.C for 3min, and then detected using a microplate reader, as shown in FIGS. 1-3.
Example 30
Expression efficiency test of cap analogue synthesized mRNA in mice
Preparing mRNA encoding luciferase using the cap analogue of the invention, and diluting the mRNA obtained into a citric acid buffer with pH of 4.0; cationic lipid DLin-MC3-DMA: DSPC: cholesterol: PEG lipid (DMG-PEG 2000)) in a molar ratio of 50:10:38.5:1.5 in ethanol.
3ML of mRNA buffer solution and 1mL of lipid solution are respectively filled into two 5mL syringes, the two syringes are arranged on a microfluidic syringe pump, the flow rate of the syringe pump is set, the collected product is placed into a dialysis bag, then ultrafiltration and concentration are carried out to an ideal concentration, lipid nanoparticles are filtered by a 0.22 mu m sterile filter, the Luciferase mRNA-lipid nanoparticles containing 5 mu g of mRNA are injected into the tail of a mouse, 8 mice are injected into each Luciferase mRNA-lipid nanoparticle for parallel experiments, and the luminous intensity is directly proportional to the translation efficiency of the effective target protein after 24 hours, as shown in figure 4. The relative fluorescence intensities of mRNA in different organs of mice are shown in FIGS. 5 to 7.

Claims (14)

1. A compound for capping the 5' end of a nucleic acid, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, the compound having the structure of formula (I):
The method is characterized in that:
R 0 is-OR 8, wherein R 8 is C 1-7 alkyl, C 2-7 alkenyl OR C 2-7 alkynyl;
R 1 is-H;
r 2 is-H, -OH, -O (any of CH 2)mCH3, wherein m is any integer from 0to 6;
Optionally R 1 and R 2 are linked by a chemical bond to form a ring, and-R 1-R2 -is any one of- (CH 2)q -O-or-O- (CH 2)q -), wherein q is 1,2 or 3;
R 3 is any one of H, -OH, -SH, -N 3,-NH2, halogen ,-CN,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2, wherein t is any one integer from 0 to 6, p is any one integer from 1 to 6, wherein R 3 is optionally substituted;
R 4、R5、R6、R7 is independently selected from any one of H, OH, OCH 3, halogen, -CN and-SH;
Each N 01、N02、N03、N04 is independently selected from 0 or 1, and the four are not simultaneously 0;
J 1、J2、J3、J4、J5 is each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base.
2. The compound of claim 1, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein at least one of J 1、J2、J3、J4、J5 is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl modified purine nucleotide base, more preferably 6-N-methyladenine.
3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein,
R 3 is any one of H,-SH,-N3,-NH2,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2, wherein t and p are each independently any one of integers from 1 to 6, preferably any one of integers from 1 to 4.
4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein the compound has the structure of formula (Ia):
5. the compound of claim 4, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein,
R 0 is-OR 8, wherein R 8 is C 1-5 alkyl, preferably-CH 3, and/OR
R 4 is selected from any one of H, OH and halogen, preferably H or F.
6. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein the compound has the structure of formula (Ib):
Wherein,
R 3' is any one of H,-OH,-SH,-N3,-NH2,-O(CH2)tCH3,-O(CH2)pSH,-O(CH2)pN3,-O(CH2)pNH2;
the remaining groups have the meaning as defined in claim 1.
7. The compound of claim 6, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein the compound has the structure of formula (Ic):
8. The compound of claim 7, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein,
R 0 is-OR 8, wherein R 8 is C 1-5 alkyl, preferably-CH 3, and/OR
R 4 is selected from any one of H, OH and halogen, preferably H or F.
9. The compound of claim 1, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein the compound has one of the following structures:
10. use of a compound according to any one of claims 1-9 as an in vitro co-transcribed RNA capping reagent.
11. An RNA molecule comprising as cap structure or cap structure fragment a compound according to any one of claims 1-9.
12. A pharmaceutical composition comprising the RNA molecule of claim 11 and a pharmaceutically acceptable carrier.
13. A method of synthesizing an RNA molecule comprising the steps of: a compound according to any one of claims 1 to 9, co-incubated with a polynucleotide template for template transcription.
14. A capped RNA transcription reaction system, comprising: a polynucleotide template, a compound of any one of claims 1-9, NTPs and RNA polymerase.
CN202310091020.3A 2023-01-20 2023-01-20 Compound for capping 5' end of nucleic acid and application thereof Pending CN118373866A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202310091020.3A CN118373866A (en) 2023-01-20 2023-01-20 Compound for capping 5' end of nucleic acid and application thereof
PCT/CN2024/073220 WO2024153222A1 (en) 2023-01-20 2024-01-19 Compound for 5'-end capping of nucleic acid and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310091020.3A CN118373866A (en) 2023-01-20 2023-01-20 Compound for capping 5' end of nucleic acid and application thereof

Publications (1)

Publication Number Publication Date
CN118373866A true CN118373866A (en) 2024-07-23

Family

ID=91906497

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310091020.3A Pending CN118373866A (en) 2023-01-20 2023-01-20 Compound for capping 5' end of nucleic acid and application thereof

Country Status (2)

Country Link
CN (1) CN118373866A (en)
WO (1) WO2024153222A1 (en)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3529255A1 (en) * 2016-10-19 2019-08-28 Arcturus Therapeutics, Inc. Trinucleotide mrna cap analogs
EP4175967A2 (en) * 2020-07-02 2023-05-10 Life Technologies Corporation Trinucleotide cap analogs, preparation and uses thereof
CN113416762A (en) * 2020-08-20 2021-09-21 深圳市瑞吉生物科技有限公司 Cap analog with Cap2 structure 5' structure, and preparation method and application thereof
IL314442A (en) * 2022-01-27 2024-09-01 Trilink Biotechnologies Llc Trinucleotide cap analogs and methods of use thereof
EP4508201A1 (en) * 2022-04-14 2025-02-19 ModernaTX, Inc. Rna polymerase variants
CN114540444B (en) * 2022-04-23 2022-08-09 江苏申基生物科技有限公司 Capping composition, preparation method thereof and in-vitro transcription reaction system

Also Published As

Publication number Publication date
WO2024153222A1 (en) 2024-07-25

Similar Documents

Publication Publication Date Title
EP3908568B1 (en) Lipids for lipid nanoparticle delivery of active agents
EP3296306B1 (en) Base-protected oligonucleotide
CA2421040C (en) Methods for synthesizing nucleosides, nucleoside derivatives and non-nucleoside derivatives
US20190367484A1 (en) Process for the manufacturing of medicaments
WO2004106356A1 (en) Functionalized nucleotide derivatives
EP3398955A1 (en) Oligonucleotide production method, and nucleoside, nucleotide, or oligonucleotide
CN116003496A (en) Modified mRNA5' -cap analogues
CN116987137B (en) Capping compound and application thereof in mRNA capping
US20020150936A1 (en) Methods for synthesizing nucleosides, nucleoside derivatives and non-nucleoside derivatives
EP2609107B1 (en) Block synthesis of oligoribonucleotides
CN115109110A (en) Initial capped oligonucleotide primer containing hexa-membered sugar ring structure and preparation method and application thereof
WO2010150789A1 (en) Method for synthesizing nucleic acid
CN114656511A (en) Preparation method of acetylated cytosine triphosphate and intermediate thereof
EP0977769A2 (en) A process for the synthesis of modified p-chiral nucleotide analogues
CN118373866A (en) Compound for capping 5' end of nucleic acid and application thereof
WO2024208281A1 (en) Halogen-containing compounds used for capping the 5' end of nucleic acid and use thereof
CN100478349C (en) Nucleoside fluoride compound, its production and use
JP2017514802A (en) COMPOUND INCLUDING HEAT-labile Site, COMPOSITION CONTAINING THE COMPOUND, AND METHOD FOR PRODUCING AND USING THE COMPOUND
WO2024139896A1 (en) Sugar ring modified nucleoside phosphoramidite monomer, preparation method therefor and use thereof
WO2024093947A1 (en) Prodrug for delivering sirna into cell
EP4506353A1 (en) Mrna cap analog and uses thereof
WO2023219114A1 (en) Mrna cap analog and method for producing same, method for producing mrna, and kit
WO2014022839A1 (en) Exocylic nitrogen atom protected nucloside and method for producing and using the same
CN114174509A (en) Primer, double-stranded DNA production apparatus using the same, and double-stranded DNA production method
Sittiwong Synthesis of pseudouridine derivatives

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination