WO2024153222A1 - Compound for 5'-end capping of nucleic acid and use thereof - Google Patents

Abstract

Provided are a compound for 5'-end capping of a nucleic acid and use thereof. The compound is represented by formula (I) below. Further provided are use and effects of the above compound in nucleic acid transcription and expression, etc., including in-vitro transcription, capping rate, cell and living body expression and translation, etc.

Description

A compound for capping the 5' end of nucleic acid and its application Technical Field

The present invention relates to the field of synthetic chemistry and bioengineering technology, and in particular to a compound for capping the 5' end of nucleic acid (RNA) and application thereof.

Background technique

In recent years, as the global COVID-19 situation has changed, messenger RNA (mRNA) technology and its vaccine drugs have achieved breakthroughs from laboratory to clinical application. In the study of mRNA drugs and their functions, specific effects will only appear when the synthesized mRNA stably expresses the target protein in vivo. In the process of stable expression of the target protein, the 5' end capping reaction of mRNA is an indispensable modification process. In eukaryotic cells, the 5' end cap structure can protect mRNA from degradation by nucleases and prevent it from being sheared by recruited protein factors; this structure can also regulate protein synthesis; in addition, the 5' end cap structure can also reduce the immunogenicity of mRNA and enhance the stability of mRNA in vivo.

In the in vitro transcription process, compared with the traditional enzymatic capping method, the mRNA co-transcriptional capping method is simpler in terms of process, and is easy to improve cost-effectiveness and expand the production capacity of mRNA drugs. At the same time, capping analogs have gradually developed from the initial mCap to the ARCA second-generation capping analog and the Cap1 third-generation capping analog, and at the same time, their transcription yield and capping efficiency have also been significantly improved.

Currently, many studies are devoted to chemical modification of the cap structure in order to further enhance the translation efficiency of mRNA while reducing its immunogenicity.

Summary of the invention

The present invention provides a compound for capping the 5' end of RNA and its application, wherein the compound includes pharmaceutically acceptable salts, solvates and stereoisomers thereof. The product after capping the 5' end of mRNA using the compound has a high capping rate and good in vitro transcription efficiency, and at the same time, has a high translation expression efficiency at the cell and in vivo levels.

The present invention provides a compound for capping the 5' end of a nucleic acid, or a pharmaceutically acceptable salt thereof, or a solvent thereof. The compound has the structure of formula (I):

in:

R ₀ is -OR ₈ , wherein R ₈ is C _1-7 alkyl, C _2-7 alkenyl or C _2-7 alkynyl;

_R1 is -H;

R ₂ is any one of -H, -OH, -O(CH ₂ ) _m CH ₃ , wherein m is any integer from 0 to 6;

Optionally, R ₁ and R ₂ are linked to form a ring through a chemical bond, and -R ₁ -R ₂ - 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 , -NH ₂ , halogen, -CN, -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ , wherein t is any integer from 0 to 6, and p is any integer from 1 to 6, wherein R ₃ is optionally substituted;

R ₄ , R ₅ , R ₆ , and R ₇ are each independently selected from any one of H, OH, OCH ₃ , halogen, -CN, and -SH;

N ₀₁ , N ₀₂ , N ₀₃ , and N ₀₄ are each independently selected from 0 or 1, and the four are not 0 at the same time;

J ₁ , J ₂ , J ₃ , J ₄ , and J ₅ are each independently selected from natural or modified pyrimidine nucleotide bases, natural or modified of purine nucleotide bases.

Embodiments of the present invention include compounds of formula (I), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein at least one of J ₁ , J ₂ , J ₃ , J ₄ , and J ₅ is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl-modified purine nucleotide base, and more preferably 6-N-methyladenine.

An embodiment of the present invention includes a compound of formula (I), or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein R ₃ is any one of H, -SH, -N ₃ , -NH ₂ , -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ , wherein t and p are each independently any integer of 1-6, preferably any integer of 1-4.

Embodiments of the present invention include compounds of formula (Ia), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein the groups in formula (Ia) have the meanings shown above:

Preferred embodiments of the present invention include compounds of formula (Ia), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein:

R ₀ is -OR ₈ , wherein R ₈ is C _1-5 alkyl, preferably -CH ₃ , and/or

_R4 is selected from any one of H, OH, and halogen, preferably H or F.

Embodiments of the present invention include compounds of formula (Ib), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof:

R ₃ ' is any one of H, -OH, -SH, -N ₃ , -NH ₂ , -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ ; the remaining groups have the same meanings as described above.

Embodiments of the present invention include compounds of formula (Ic), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein the groups in formula (Ic) have the meanings shown above:

Preferred embodiments of the present invention include compounds of formula (Ic), or pharmaceutically acceptable salts, or solvates, or stereoisomers thereof, wherein:

R ₀ is -OR ₈ , wherein R ₈ is C _1-5 alkyl, preferably -CH ₃ , and/or

_R4 is selected from any one of H, OH, and halogen, preferably H or F.

According to some specific embodiments of the present invention, the cap analog has one of the structures shown below:

The invention also discloses the application of the compound as an in vitro co-transcribed RNA capping reagent.

The invention also discloses an RNA molecule, comprising the above compound as a cap structure or a cap structure fragment.

It can be understood that the above-mentioned RNA molecules can be used as mRNA vaccines, RNA drugs, or in cell therapy for precision medicine.

The present invention also discloses a pharmaceutical composition, comprising the above RNA molecule and a pharmaceutically acceptable carrier.

The invention also discloses a method for synthesizing RNA molecules, comprising the following steps: co-incubating the above compound with a polynucleotide template to perform template transcription.

The invention also discloses a capping RNA transcription reaction system, comprising: a polynucleotide template, the above compound, NTPs, and RNA polymerase.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings.

1 to 3 show the expression efficiency of mRNA synthesized using the cap analog of the present invention in HEK293T, rat synoviocytes and HUVEC, respectively.

FIG. 4 shows fluorescent imaging of mice using mRNA synthesized using the cap analogs of the present invention.

5 to 7 respectively show the relative fluorescence intensity of mRNA synthesized using the cap analog of the present invention in different organs of mice.

Detailed ways

The present invention provides a compound for capping the 5' end of RNA and its application, wherein the compound includes its pharmaceutically acceptable salt, solvate and stereoisomer. The product after capping the 5' end of mRNA using the compound has a high capping rate and good in vitro transcription efficiency, and at the same time, has a high translation expression efficiency at the cell and living body 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 listed herein should be read and understood by those skilled in the art in light of the remainder of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

definition

Unless otherwise stated, when disclosing or claiming any type of range, it is intended to disclose or claim individually each possible value that the range may reasonably cover, including any sub-ranges contained therein. For example, the number of groups is 1 to 6 indicating integers within the range, wherein 1-6 should be understood to include 1, 2, 3, 4, 5, 6, and also include sub-ranges of 1-5, 1-4, and 1-3.

The description of the present disclosure should be interpreted in accordance with the laws 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 words "include", "contain" or "comprises" and the like used in the present disclosure mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used herein may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or with humans or mammals for the prevention or treatment of a disease or condition.

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 structure of 6-N-methyladenine is

"Stereoisomers" refer to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans) isomers, atropisomers, and the like.

"Connected to form a ring through chemical bonds" means connecting two groups through a carbon-carbon bond, carbon-oxygen bond, carbon-nitrogen bond, carbon-sulfur bond, etc. to form a ring structure. If necessary, the corresponding group can reduce 1-2 hydrogen atoms.

The expression "optionally substituted" means that one, two, three or more than three hydrogen atoms in the group may be replaced independently of one another by respective substituents. The substituents may be selected from alkyl, alkenyl, alkynyl, alkoxy, halogen, cyano, amino, nitro, -OH.

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

The term "alkoxy" includes -O-alkyl groups and alkyl groups wherein the O atom is within the 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 is optionally substituted.

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

The term "alkynyl" means a group having at least one unsaturated site, i.e., one carbon-carbon sp triple bond, containing 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and most preferably 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 relatively non-toxic addition salts of the disclosed compounds. See, for example, S. M. Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19.

Suitable pharmaceutically acceptable salts of the disclosed compounds may be acid addition salts of the disclosed compounds having a sufficiently basic nature, for example, carrying a nitrogen atom in a chain or ring, 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 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, 3-Hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric 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, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of the compound of the present invention having sufficient acidity is an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt or a magnesium salt, an ammonium salt, a triethylamine salt, or a salt formed with an organic base providing a physiologically acceptable cation, such as a salt formed with the following substances: N-methylglucose Amine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternized with 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 diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, etc.

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 by any of a variety of known methods. Alternatively, alkali metal salts and alkaline earth metal salts of the acidic compounds of the present disclosure can be prepared by reacting them with a suitable base by various known methods.

The present invention includes all possible salts of the disclosed compounds, either as a single salt or as any mixture of said salts in any ratio.

The term "solvate" is a substance formed by combining, physically combining and/or solvating the compounds of the present invention with solvent molecules, such as a disolvate, a monosolvate or a hemisolvate, wherein the ratio of the solvent molecules to the compounds of the present invention is about 2:1, about 1:1 or about 1:2, respectively. This physical combination involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid), the solvate can be separated. Therefore, solvates include solution phases and separable solvates. The compounds of the present invention can be in solvated form with pharmaceutically acceptable solvents (such as water, methanol and ethanol), and the application is intended to cover solvated and non-solvated forms of the compounds of the present invention. A solvate is a hydrate.

The term "pharmaceutical composition" as used in this application 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 suitable for administration to a patient to diagnose, prevent and/or treat a disease. Additionally, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier that makes the composition suitable for therapeutic use.

As used herein, the term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, 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 pharmaceutical composition is administered intravenously. Saline solutions as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, Sugar, 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 necessary, the composition may also contain a small amount of wetting agent or emulsifier or pH buffer. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

The term "halogen" refers to fluorine, chlorine, bromine, iodine and astatine.

The term "optionally" means that the situation may or may not occur.

Example

Reagents and models used

The starting materials of the embodiments are commercially available and/or can be prepared by a variety of methods known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of experiment and post-treatment) in the following synthetic methods. Those skilled in the art of organic synthesis will appreciate that the functional groups present in each part of the molecule should be compatible with the proposed reagents and reactions.

All reagents and compounds synthesized can be purchased through general commercial channels in China (excluding Hong Kong, Macao and Taiwan), including Sigma-Aldrich (USA), Shanghai Zhaowei Technology Development Co., Ltd. (Trinlink Cleancap) and jetMESSENGER

Cell model: HEK293T cells, rat synovial cells and HUVEC cells were purchased from Wuhan Pronocell Life Science Co., Ltd.

Instrument used: Multifunctional microplate reader (Molecular Devices).

Preparation and identification of compounds: nuclear magnetic resonance (Bruker 300 MHz), liquid chromatography-mass spectrometry (Agilent 6150/1290), and high performance liquid chromatography (Agilent 1260).

Cell experiments: microscope, cell culture incubator (Thermo Fisher Scientific).

Intermediate synthesis:

The compounds 8 used in the following examples were prepared by the following steps:

(1) Weigh 5 g of compound 1 and dissolve it in 10 mL of anhydrous acetonitrile to obtain solution A. Dissolve 4.5 eq of tetrazole in 120 mL of anhydrous acetonitrile, and then weigh 1 eq of compound 2 and dissolve it in the solution to obtain solution B, then dropwise add solution A containing compound 1 into solution B; react at room temperature 25°C for 0.5 hour; after monitoring the reaction by thin layer chromatography (TLC), dropwise add 1.1 eq of iodine pyridine solution into the reaction solution; after monitoring the reaction, dilute with water, extract with ethyl acetate, and concentrate to obtain a crude product of compound 3.

(2) Weigh 6.5 g of the crude product of compound 3 and dissolve it in 40 mL of acetic acid, then add 10 mL of water; the reaction solution is reacted at room temperature at 25° C. for 16 hours; after the reaction is completed as monitored by TLC, the reaction solution is concentrated to dryness and purified using a silica gel column to obtain compound 4.

(3) Weigh 3 g of compound 4 and dissolve it in 4.5 eq of tetrazole in acetonitrile solution, then add 3 eq of phosphine reagent 5, and stir the reaction solution at room temperature 25°C for 1 hour; after monitoring the reaction by TLC, add 3.2 eq of iodine pyridine solution; after monitoring the reaction, dilute with water, then extract with ethyl acetate, concentrate and purify with silica gel column to obtain compound 6.

(4) Weigh 2 g of compound 6 and dissolve it in 20 mL of methanol and 20 mL of concentrated aqueous ammonia. The reaction solution is stirred at room temperature (25° C.) for 48 hours. After the reaction is completed as monitored by TLC, the reaction solution is concentrated to obtain a crude product of compound 7.

(5) Weigh 1.5 g of crude compound 7 and dissolve it in 1 mL of DMSO. Add 1.2 mL of TEA·3HF, the reaction solution was stirred at 50°C for 1 hour; after the reaction was completed under TLC monitoring, the reaction solution was cooled to room temperature, diluted with water, and the pH was adjusted to 5.5 with saturated sodium carbonate aqueous solution; the mixture was loaded onto a DEAE Sephadex column, and the mobile phase was linearly eluted with 0-1.0 M TEAB eluent to obtain compound 8.

Final product synthesis:

Final product synthesis method 1

Weigh 0.4g of compound 8 and dissolve it in 8mL of anhydrous DMSO. Add 2eq of compound 9 and 20eq of anhydrous zinc chloride under argon protection. Stir the reaction solution at room temperature 25℃ for 24 hours under argon protection. After the reaction is completed by TLC monitoring, terminate the reaction with 150mL of 0.25M EDTA solution, and then load the mixture onto a DEAE Sephadex column. Use 0.0-1.0M ammonium bicarbonate eluent to linearly gradient elute the product, collect the product-containing eluent and freeze-dry to obtain the product.

Final product synthesis method 2

0.2 g of compound 10 was weighed and 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 8 was added to the solution; the reaction solution 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 linearly eluted using a 0-1.0 M ammonium bicarbonate eluent, and the product-containing eluent was collected and lyophilized 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, at room temperature 25°C, compound 1-1 (5 g) was dissolved in 10 mL of acetonitrile solution and added to the aforementioned solution. The resulting solution was stirred at room temperature 25°C for 1 hour, and no obvious heat release was found. The spot plate monitoring showed that the raw material 1-1 disappeared. 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; then the reaction solution was stirred for 0.5 hours, and the spot plate monitoring showed that the oxidation was complete. Saturated aqueous sodium sulfite solution (10 mL) was added to the reaction solution to quench it, and then 50 mL of water was added to dilute it. It was extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed once with water (50 mL), and concentrated to give a light yellow oily product 1-3 (8 g, crude product).

Step 2:

Compound 1-3 (8 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water, stirred at 25 ° C for 16 hours, and the spot plate monitoring showed that compound 1-3 disappeared and a spot with high polarity was generated. The reaction solution was directly concentrated in vacuo. After concentration, appropriate amount of silica gel and DCM were added for sample mixing and purification (40 g normal phase column, EA, 10 min, DCM: MeOH, 10-20% 20 min, flow rate 30 ml/min) to obtain a white solid product 1-4 (2.8 g, 51% two-step yield).

Step 3:

Prepare 28mL of tetrazole in acetonitrile solution (0.4mmol/mL) for standby use. Add compound 1-4 (2.8g) to the solution, then add compound 1-5 (3g) to the solution at room temperature 25℃, replace nitrogen three times, stir the reaction solution at room temperature 25℃ for 1 hour, and the spot plate monitoring shows that the reaction is complete. The reaction solution is cooled to below 10℃ in an ice water bath, and a solution of iodine pyridine/tetrahydrofuran/water (0.5mmol/mL, pyridine:tetrahydrofuran:water=1:8:1) is added dropwise until the reaction solution does not fade and the spot plate monitoring shows that the oxidation reaction is complete. Then add 10mL of saturated sodium sulfite aqueous solution to the reaction solution to quench, and then dilute with water. Extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amount of silica gel and DCM for sample mixing and purification (40g normal phase column, EA, 10min, DCM:MeOH, 10-20% 20min, flow rate 30ml/min). Concentration gave 1-6 (2.6 g, 78.2% yield) as a white foamy solid.

Step 4:

Compound 1-6 (2.6 g) was dissolved in methanol (30 mL), and then concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature at 25°C for 60 hours. The spot plate monitoring showed that compound 1-6 was completely reacted. The reaction solution was concentrated in vacuo and concentrated again with methanol to obtain a light yellow oily liquid compound 1-7 (2.4 g, crude product), which was directly used in the next step.

Step 5:

Compound 1-7 (2.4 g, crude product) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction solution was stirred at 50 ° C for 1 hour. The dot plate showed that compound 1-7 was completely reacted. The reaction solution was diluted to 50 mL with water, and the pH was adjusted to 5.5 with 1 mol/L NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was linearly eluted with a 0-1.0 M ammonium bicarbonate aqueous solution eluent. Most of the water was concentrated in the obtained components under vacuum. After the remaining liquid was freeze-dried, the amine salt 1-8 of the target compound (0.8 g, 33.7% yield) was obtained. The product was a white solid.

Step 6:

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH 7.0, and then compound 1-8 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution, and the resulting components were concentrated in vacuo. Most of the water was removed and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product, Compound 5 (65 mg).

¹ H 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).

³¹ P 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), and argon was replaced three times. Then, at room temperature of 25°C, compound 2-1 (5 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The resulting solution was stirred at room temperature of 25°C for 1 hour. No obvious heat release was found, and the spot plate monitoring showed that the raw material 2-1 disappeared. Then, iodine solution (5 g iodine was dissolved in 40 mL of a mixed solution of THF:H ₂ O:pyridine = 8:1:1, to prepare a 0.5 mmol/mL solution) was added dropwise to the solution until the solution no longer faded, and then the reaction solution was stirred for 0.5 hours. The spot plate monitoring showed that the oxidation was complete. The reaction mixture was quenched by adding aqueous Na ₂ SO ₃ 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) and concentrated to give a light yellow oily product 2-3 (7.5 g, crude product).

Step 2:

Compound 2-3 (7.2 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water, and the reaction was stirred at 25 ° C for 16 hours. The spot plate monitoring showed that compound 1-3 disappeared and a spot with high polarity was generated. The reaction solution was directly concentrated in vacuo. After concentration, appropriate amount of silica gel and DCM were added for sample mixing and purification (40 g normal phase column, EA, 10 min, DCM: MeOH, 10-20% 20 min, flow rate 30 ml/min) and concentrated to obtain a white solid product 2-4 (2.8 g, 54.8% two-step yield).

Step 3:

Prepare 28mL of tetrazole in acetonitrile solution (0.4mmol/mL) for standby use. Add compound 2-4 (2.8g) to the above solution, then add compound 2-5 (2.26g) to the solution at room temperature 25℃, replace nitrogen three times, and stir the reaction solution at room temperature 25℃ for 1 hour. Spot plate monitoring shows that the reaction is complete. The reaction solution is cooled to below 10℃ in an ice water bath, and a solution of iodine pyridine/tetrahydrofuran/water (0.5mmol/mL, pyridine:tetrahydrofuran:water=1:8:1) is added dropwise until the reaction solution does not fade, and the spot plate monitoring shows that the oxidation reaction is complete. Subsequently, 10mL of saturated sodium sulfite aqueous solution is added to the reaction solution to quench, and then diluted with water. Extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amount of silica gel and DCM to mix the sample and purify (40g normal phase column, EA, 10min, DCM:MeOH, 10-20% 20min, flow rate 30ml/min). Concentrate to obtain white foam solid 2-6 (3.1g, 93.5% yield).

Step 4:

Compound 2-6 (3.1 g) was dissolved in methanol (30 mL), and then concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature 25°C for 60 hours, and the plate monitor showed that compound 2-6 was completely reacted. The reaction solution was concentrated in vacuo, and methanol was added to concentrate again to obtain a light yellow oily liquid compound 2-7 (2.4 g, crude product).

Step 5:

Compound 2-7 (2.4 g, crude product) was dissolved in DMSO (3 mL), and then triethylamine trihydrofluoride (3.5 mL) was added. The reaction solution was stirred at 50 ° C for 1 hour. The dot plate showed that the raw material 2-7 was completely reacted. The reaction solution was diluted to 50 mL with water, and the pH was adjusted to 5.5 with 1N NaOH aqueous solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0M TEAB eluent. Most of the water was concentrated in the obtained components under vacuum. After the remaining liquid was freeze-dried, the triethylamine salt 2-8 of the target compound (1.5 g, 51% yield) was obtained. The product was a white solid.

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ3.63, δ-0.62.

Step 6:

At room temperature (25°C) under argon protection, compound 2-8 (500 mg), compound 9a (500 mg) and anhydrous zinc chloride (1.2 g) were added respectively, and then anhydrous DMSO (8 mL) was added with a syringe to react for 24 hours. The dot plate showed that most of the raw materials reacted. The reaction solution was added to the EDTA disodium salt solution (1.6 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was linearly eluted with a 0-1.0M ammonium bicarbonate aqueous solution eluent. Most of the water was concentrated in the obtained components in vacuo, and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 8 (120 mg).

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ -0.78, -11.58, -23.01.

Example 3

Synthesis of compound 22

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH 7.0, and then compound 6-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. A linear gradient elution was performed using a 0-1.0 M aqueous ammonium bicarbonate solution eluent, and most of the water was concentrated in vacuo in the resulting component. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 22 (82 mg).

¹ H 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).

³¹ P 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 a tetrazole (1.6 g) acetonitrile solution (56 mL), and argon was replaced three times. Then, at room temperature 25°C, compound 4-1 (4 g) was dissolved in 10 mL acetonitrile and added to the above solution. The resulting solution was stirred at room temperature 25°C for 1 hour. No obvious heat release was found. The spot plate monitoring showed that compound 4-1 disappeared. Then, iodine pyridine/tetrahydrofuran/water solution (0.5 mmol/mL, pyridine:tetrahydrofuran:water = 1:8:1) was added dropwise to the solution until the solution no longer faded. Then the reaction solution was stirred for 0.5 hours. The spot plate monitoring showed that the oxidation was complete. After adding Na ₂ SO ₃ aqueous solution (10 mL) to the reaction solution to quench, 50 mL of water was added to dilute it, and it was extracted with dichloromethane. The organic phase was washed once with water and concentrated to obtain a light yellow oily product 4-3 (5.6 g, crude product).

Step 2:

Compound 4-3 (5.6 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water, and the reaction was stirred at 25 ° C for 16 hours. The spot plate monitoring showed that compound 4-3 disappeared and a spot with high polarity was generated. The reaction solution was directly concentrated in vacuo. After concentration, appropriate amount of silica gel and DCM were added for sample mixing and purification (40 g normal phase column, EA, 10 min, DCM: MeOH, 10-20% 20 min, flow rate 30 ml/min), and then concentrated to obtain a white solid product 4-4 (2.4 g, 54.8% two-step yield).

Step 3:

Prepare 24mL of tetrazole acetonitrile solution (0.4mmol/mL) for standby. Add compound 4-4 (2.4g) to the above solution, then add compound 4-5 (1.96g) to the solution at room temperature 25°C, replace nitrogen three times, stir the reaction solution at room temperature 25°C for 1 hour, and the spot plate monitoring shows that the reaction is complete. The reaction solution is cooled to below 10°C in an ice-water bath, and a solution of iodine pyridine/tetrahydrofuran/water (5g iodine is dissolved in 40mL of a mixed solution of THF: _H2O :pyridine＝8:1:1, and a 0.5mmol/mL solution is prepared) is added dropwise until the reaction solution does not fade, and the spot plate monitoring shows that the oxidation reaction is complete. Add 10mL of saturated sodium sulfite aqueous solution to the reaction solution to quench, and then dilute with water. Extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate, and filter. Add appropriate amount of silica gel and DCM for sample mixing and purification (40g normal phase column, EA, 10min, DCM:MeOH, 10-20% 20min, flow rate 30ml/min), and concentrate to obtain white foamy solid 4-6 (2.4g, 75.4% yield).

Step 4:

Compound 4-6 (2.4 g) was dissolved in methanol (30 mL), and then concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature at 25 ° C for 60 hours. The spot monitor showed that compound 4-6 was completely reacted. The reaction solution was concentrated in vacuo, diluted to 50 mL with water, and the pH was adjusted to 5.5 with dilute hydrochloric acid. The mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M TEAB eluent. Most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain the triethylamine salt of the target compound 4-7 (1.5 g, 51% yield). The product was a white solid.

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ3.63, δ-0.62.

Step 5:

At room temperature (25°C) and under argon protection, compound 4-7 (500 mg), compound 9a (500 mg) and anhydrous zinc chloride (1.2 g) were added respectively, and then anhydrous DMSO (8 mL) was added with a syringe to react for 24 hours. The dot plate showed that most of the raw materials reacted. The reaction solution was added to a disodium EDTA solution (1.6 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous solution of ammonium bicarbonate was used as an eluent. Most of the water was concentrated in vacuo in the obtained components, and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 28 (120 mg).

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ -0.78, -11.58, -23.01.

Example 5

Synthesis of compound 38

Compound 9b (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 2-8 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous ammonium bicarbonate solution was used as an eluent, and most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 38 (50 mg).

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ -1.17, -11.63, -22.81.

Example 6

Synthesis of compound 44

Compound 9b (200 mg) was added to 16 mL of pH 7.0 containing 0.2 mol/L N-methylmorpholine and 0.2mol/L manganese chloride aqueous solution, and then compound 6-1 (200mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to EDTA disodium salt solution (1.4g, 80mL of water) cooled to 0°C in advance, and the mixture was loaded on a DEAE Sephadex column. Subsequently, a linear gradient elution of 0-1.0M ammonium bicarbonate aqueous solution was used as an eluent, and most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 44 (53mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -10.29, -21.23.

Example 7

Synthesis of compound 48

Compound 9b (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 7-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous ammonium bicarbonate solution was used as an eluent, and most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 48 (65 mg).

¹ H 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, 0.7 Hz,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.62, -10.29, -21.22.

Example 8

Synthesis of compound 71

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, and then compound 8-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous ammonium bicarbonate solution was used as an eluent, and most of the water was concentrated in the resulting component under vacuum. After the remaining liquid was freeze-dried, a white powdery ammonium salt product compound 71 (82 mg) was obtained.

¹ H 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.

Embodiment 9

Synthesis of compound 73

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 9-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was then loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous ammonium bicarbonate solution was used as the eluent, and the resulting components were concentrated in vacuo Most of the water was removed and the remaining liquid was freeze-dried to obtain the ammonium salt product compound 73 (62 mg) as a white powder.

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -10.29, -21.22.

Example 10

Synthesis of compound 76

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 10-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution was performed using a 0-1.0 M aqueous ammonium bicarbonate solution eluent, and most of the water was concentrated in the obtained component under vacuum. After the remaining liquid was freeze-dried, a white powdery ammonium salt product compound 76 (95 mg) was obtained.

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -10.29, -21.22.

Embodiment 11

Synthesis of compound 77

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganous chloride at pH = 7.0, and then compound 11-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution was performed using a 0-1.0 M aqueous ammonium bicarbonate solution eluent, and most of the water was concentrated in vacuo in the resulting component. After the remaining liquid was freeze-dried, a white powdery ammonium salt product compound 77 (95 mg) was obtained.

¹ H NMR (500MHz, Deuterium Oxide) δ8.20 (s, 1H), 8.19 (s, 1H), 7.74 (dd, J = 7.9, 1.8 Hz, 1H), 6.40 (ddt, J = 24.5, 1.8, 0.8 Hz,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -10.29, -21.22.

Example 12

Synthesis of compound 117

Compound 9b (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 12-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25 ° C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0 ° C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution of 0-1.0 M ammonium bicarbonate aqueous solution was used as an eluent, and most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 117 (95 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -10.29, -21.22.

Embodiment 13

Synthesis of compound 121

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 13-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column, followed by linear gradient elution using a 0-1.0 M aqueous ammonium bicarbonate solution eluent. Most of the water was concentrated in vacuo from the resulting component, and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 121 (75 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ -0.90, -10.29, -21.22.

Embodiment 14

Synthesis of compound 122

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganous chloride at pH = 7.0, and then compound 9-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 122 (75 mg).

¹ H 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(dd dd,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ -1.11, -10.31, -21.25.

Embodiment 15

Synthesis of compound 126

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 15-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. After the remaining liquid was lyophilized, a white powder product compound 126 was obtained, which was an ammonium salt (60 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ -0.9, -10.29, -21.27.

Example 16

Synthesis of compound 127

Compound 9a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 16-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. Subsequently, a linear gradient elution with a 0-1.0 M aqueous ammonium bicarbonate solution was used as an eluent, and most of the water was concentrated in vacuo in the resulting component. After the remaining liquid was freeze-dried, a white powdery ammonium salt product compound 127 (60 mg) was obtained.

¹ H NMR (500MHz, Deuterium Oxide) δ8.28 (s, 1H), 8.20 (s, 1H), 7.74 (dd, J = 7.9, 1.8 Hz, 1H), 6.39 (ddt, J = 4.8, 3.1, 0.8 Hz,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 ).

³¹ P NMR (202MHz, Deuterium Oxide) δ -0.78, -10.29, -21.22.

Embodiment 17

Synthesis of compound 146

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, and then compound 17-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was linearly eluted with a 0-1.0 M aqueous ammonium bicarbonate solution eluent. Most of the water in the resulting component was concentrated in vacuo, and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 146 (74 mg).

¹ H 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.

Embodiment 18

Synthesis of compound 148

Compound 9b (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 18-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution, and the resulting components were concentrated in vacuo. Most of the water was removed and the remaining liquid was freeze-dried to obtain the ammonium salt product compound 148 (60 mg) as a white powder.

¹ H NMR (500MHz, Deuterium Oxide) δ8.33 (s, 1H), 8.29 (s, 1H), 7.81 (dd, J = 7.3, 1.8 Hz, 1H), 6.43 (ddq, J = 3.9, 2.3, 0.8 Hz,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ -0.85, -10.09, -21.23.

Embodiment 19

Synthesis of compound 176

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 2-8 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 176 (55 mg).

¹ H 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＝11.4 Hz, 1H), 3.91 (d, J = 8.4Hz, 1H), 3.84 (s, 1H), 3.83–3.73 (m, 3H), 3.24 (s, 3H).

³¹ P NMR (162MHz, Deuterium Oxide) δ -0.90, -11.41, -22.93.

Embodiment 20

Synthesis of compound 191

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 20-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 191 (70 mg).

¹ H NMR (500MHz, Deuterium Oxide) δ7.86 (s, 1H), 7.59 (dd, J = 7.3, 1.8 Hz, 1H), 6.38–6.31 (m, 1H), 6.19 (dd, J = 3.0, 0.7 Hz,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.50, -9.18, -10.26, -21.22.

Embodiment 21

Synthesis of compound 201

step 1:

In a three-necked flask, compound 21-2 (1.84 g) was added to a solution of tetrazole (1.42 g) in acetonitrile (50 mL), and the argon gas was replaced three times. Then, at room temperature (25°C), compound 21-1 (4 g) was dissolved in 10 mL of acetonitrile and added to the above solution. The solution was stirred at room temperature 25°C for 1 hour, and no obvious heat release was found. The spot plate monitoring showed that compound 21-1 disappeared. Then, a solution of iodine in pyridine/tetrahydrofuran/water (0.5mmol/mL, pyridine:tetrahydrofuran:water=1:8:1) was added dropwise to the solution until the solution no longer faded. The reaction solution was then stirred for 0.5 hours. The spot plate monitoring showed that the oxidation was complete. After adding sodium sulfite aqueous solution (10mL) to the reaction solution for quenching, 50mL of water was added for dilution, and the organic phase was extracted with dichloromethane. The organic phase was washed once with water and concentrated to obtain a light yellow oily product 21-3 (5.8g, crude product).

Step 2:

Compound 21-3 (5.8 g, crude product) was dissolved in 40 mL of acetic acid and 10 mL of water, and the reaction was stirred at 25 ° C for 16 hours. The spot plate monitoring 21-3 disappeared and a polar spot was generated. The reaction solution was directly concentrated in vacuo. After concentration, appropriate amounts of silica gel and DCM were added for sample mixing and purification (40 g normal phase column, EA, 10 min, DCM: MeOH, 10-20% 20 min, flow rate 30 ml/min), and concentrated to obtain a white solid product 21-4 (2.8 g, 54.8% two-step yield).

Step 3:

Prepare 30mL of tetrazole in acetonitrile solution (0.4mmol/mL) for standby. Add compound 21-4 (2.8g) to the above solution, then add compound 21-5 (2.5g) to the solution at room temperature 25℃, replace nitrogen three times, stir the reaction solution at room temperature 25℃ for 1 hour, and the spot plate monitoring shows that the reaction is complete. The reaction solution is cooled to below 10℃ in an ice water bath, and iodine pyridine/tetrahydrofuran/water solution (0.5mmol/mL, pyridine:tetrahydrofuran:water=1:8:1) is added dropwise until the reaction solution does not fade, and the spot plate monitoring shows that the oxidation reaction is complete. The reaction solution is quenched with 10mL of saturated sodium sulfite aqueous solution, and then diluted with water. Extract with ethyl acetate three times, combine the organic phases, dry them with anhydrous sodium sulfate, and filter. Appropriate amount of silica gel and DCM were added for sample mixing and purification (40 g normal phase column, EA, 10 min, DCM/MeOH, 10-20% 20 min, flow rate 30 ml/min), followed by concentration to obtain a white foamy solid 21-6 (2.4 g, 71.2% yield).

Step 4:

Compound 21-6 (2.4 g) was dissolved in methanol (30 mL), and then concentrated aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature at 25 ° C for 60 hours. The spot plate monitor showed that compound 21-6 was completely reacted. The reaction solution was concentrated in vacuo, diluted with water to 50 mL, and pH was adjusted to 5.5 with dilute hydrochloric acid. The mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M TEAB eluent. Most of the water was concentrated in vacuo. The remaining liquid was freeze-dried to obtain the triethylamine salt of the target compound 21-7 (2.1 g, 85% yield). The product was a white solid.

¹ H 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 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 21-7 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 201 (75 mg).

¹ H 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).

³¹ P NMR (162MHz, Deuterium Oxide) δ -0.99, -11.40, -23.06.

Embodiment 22

Synthesis of compound 221

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 22-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25 ° C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0 ° C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution, and the resulting components were concentrated in vacuo Most of the water was removed and the remaining liquid was freeze-dried to obtain a white powdery ammonium salt product, Compound 221 (85 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -9.01, -10.23, -21.17.

Embodiment 23

Synthesis of compound 226

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 23-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature at 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. After the remaining liquid was freeze-dried, a white powdery ammonium salt product compound 226 (92 mg) was obtained.

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -9.10, -10.31, -21.17.

Embodiment 24

Synthesis of compound 227

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 24-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 227 (85 mg).

¹ H NMR (500MHz, Deuterium Oxide) δ8.20 (s, 1H), 8.19 (s, 1H), 7.74 (dd, J = 7.9, 1.8 Hz, 1H), 6.40 (ddt, J = 24.5, 1.8, 0.8 Hz,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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.60, -9.10, -10.22, -21.17.

Embodiment 25

Synthesis of compound 246

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, and then compound 25-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M aqueous ammonium bicarbonate solution, and most of the water in the obtained fraction was concentrated in vacuo. The remaining liquid was lyophilized to obtain a white powdery ammonium salt product, Compound 246 (80 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ -0.22, -9.07, -10.25, -21.27.

Embodiment 26

Synthesis of compound 247

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganese chloride at pH = 7.0, and then compound 26-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 247 (58 mg).

¹ H NMR (500MHz, Deuterium Oxide) δ8.33 (s, 1H), 8.29 (s, 1H), 7.74 (dd, J = 7.9, 1.8 Hz, 1H), 6.39 (ddd, J = 4.1, 2.6, 0.8 Hz,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),4.6 7(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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.66, -9.13, -10.26, -21.25.

Embodiment 27

Synthesis of compound 251

Compound 10a (200 mg) was added to 16 mL of an aqueous solution containing 0.2 mol/L N-methylmorpholine and 0.2 mol/L manganous chloride at pH = 7.0, and then compound 27-1 (200 mg) was added to the solution. The reaction solution was stirred at room temperature 25°C for 16 hours, and the plate showed that the product was generated. The reaction solution was added to a disodium EDTA solution (1.4 g, plus 80 mL of water) cooled to 0°C in advance, and the mixture was loaded onto a DEAE Sephadex column. The product was eluted with a linear gradient of 0-1.0 M ammonium bicarbonate aqueous solution eluent, and most of the water was concentrated in the obtained component under vacuum. The remaining liquid was freeze-dried to obtain a white powdery ammonium salt product compound 251 (88 mg).

¹ H 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).

³¹ P NMR (202MHz, Deuterium Oxide) δ0.85, -9.12, -10.24, -21.26.

Embodiment 28

Detection of mRNA capping efficiency

a) Linearize the plasmid and purify the DNA template.

b) In vitro transcription synthesis of mRNA, using 35 capping analogs of the present invention and comparative example Trilink CleanCap, respectively, the reaction system is shown in Table 1; during the experiment, the above reagents were fully mixed and incubated at 37°C. After 2 hours, deoxyribonuclease (DNase) was added and incubated for 30 minutes to remove the DNA template, and then the purified mRNA samples were quantitatively detected using Nanodrop One.

c) After exonuclease treatment, mRNA was purified and the mRNA capping rate of the capping compound was detected by liquid chromatography mass spectrometry (LC-MS). The mRNA capping rates in the above experimental examples were all between 90% and 98%. After purification, the compounds of the present invention all showed good capping efficiency.

Table 1. In vitro transcription system

Table 2 Final products obtained per 20 μL reaction system

Embodiment 29

Evaluation of the expression efficiency of luciferase mRNA with different capping analogs in different cells

The present invention experiments the expression efficiency of different capped luciferase mRNA in HEK293T and rat synovial cells. The luciferase coding sequence is used as a DNA template, and the cap analogs of the present invention are used as raw materials for in vitro transcription of mRNA, and then different mRNA products are transfected into cells, and finally the protein expression of each cell is quantitatively detected by an ELISA instrument:

a) The above-mentioned different cells were plated at 0.5×10 ⁵ cells (96-well plate);

b) Mix 30 μL mRNA buffer with 0.6 μg RNA, then add 0.6 μL transfection reagent (jetMESSENGER) and mix well. After standing for 10 minutes, add the mixture to each well of cells. Opti-MEM was added to 100 μL/well and incubated at 37°C and 5% CO ₂ for 6 h;

c) Replace with fresh complete medium and continue culturing for 12 h under the same conditions;

d) Add 100 μl of 1× luciferin working solution to each well, incubate at 37°C for 3 min, and then detect using an ELISA reader. The results are shown in Figures 1 to 3.

Embodiment 30

Expression efficiency test of mRNA synthesized by cap analogs in mice

The cap analog of the present invention is used to prepare mRNA encoding luciferase, and the obtained mRNA is diluted into a citric acid buffer at pH 4.0; the cationic lipid DLin-MC3-DMA: DSPC: cholesterol: PEG lipid (DMG-PEG2000)) is dissolved in ethanol at a molar ratio of 50:10:38.5:1.5.

3 mL of mRNA buffer and 1 mL of lipid solution were loaded into two 5 mL syringes, respectively, and installed on a microfluidic syringe pump. The flow rate of the syringe pump was set, and the collected product was placed in a dialysis bag, and then ultrafiltration was concentrated to the ideal concentration. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter, and Luciferase mRNA-lipid nanoparticles containing 5 μg of mRNA were injected into the tail vein of mice. Each Luciferase mRNA-lipid nanoparticle was injected into 8 mice for parallel experiments. Luciferase substrate was injected 24 hours later, and the luminescence intensity was proportional to the effective target protein translation efficiency, as shown in Figure 4. The relative fluorescence intensity of mRNA in different organs of mice is shown in Figures 5 to 7.

Claims (14)

A compound for capping the 5' end of a nucleic acid, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the compound has a structure of formula (I):
Features: R ₀ is -OR ₈ , wherein R ₈ is C _1-7 alkyl, C _2-7 alkenyl or C _2-7 alkynyl; _R1 is -H; R ₂ is any one of -H, -OH, -O(CH ₂ ) _m CH ₃ , wherein m is any integer from 0 to 6; Optionally, R ₁ and R ₂ are linked to form a ring through a chemical bond, and -R ₁ -R ₂ - 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 , -NH ₂ , halogen, -CN, -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ , wherein t is any integer from 0 to 6, and p is any integer from 1 to 6, wherein R ₃ is optionally substituted; R ₄ , R ₅ , R ₆ , and R ₇ are each independently selected from any one of H, OH, OCH ₃ , halogen, -CN, and -SH; N ₀₁ , N ₀₂ , N ₀₃ , and N ₀₄ are each independently selected from 0 or 1, and the four are not 0 at the same time; J ₁ , J ₂ , J ₃ , J ₄ and J ₅ are each independently selected from a natural or modified pyrimidine nucleotide base, or a natural or modified purine nucleotide base. The compound according to claim 1, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, characterized in that at least one of J ₁ , J ₂ , J ₃ , J ₄ , and J ₅ is a modified nucleotide base, preferably a modified purine nucleotide base, more preferably a methyl-modified purine nucleotide base, and more preferably 6-N-methyladenine. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein: R ₃ is any one of H, -SH, -N ₃ , -NH ₂ , -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ , wherein t and p are each independently any integer of 1-6, preferably any integer of 1-4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the compound has a structure of formula (Ia):
The compound according to claim 4, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein: R ₀ is -OR ₈ , wherein R ₈ is C _1-5 alkyl, preferably -CH ₃ , and/or _R4 is selected from any one of H, OH, and halogen, preferably H or F. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the compound has a structure of formula (Ib):
in, R ₃ ' is any one of H, -OH, -SH, -N ₃ , -NH ₂ , -O(CH ₂ ) _t CH ₃ , -O(CH ₂ ) _p SH, -O(CH ₂ ) _p N ₃ , -O(CH ₂ ) _p NH ₂ ; The remaining radicals have the meanings as defined in claim 1. The compound according to claim 6, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the compound has a structure of formula (Ic):
The compound according to claim 7, or a pharmaceutically acceptable salt, or a solvate, or a isomers, among which R ₀ is -OR ₈ , wherein R ₈ is C _1-5 alkyl, preferably -CH ₃ , and/or _R4 is selected from any one of H, OH, and halogen, preferably H or F. The compound according to claim 1, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the compound has one of the following structures:











Use of the compound as described in any one of claims 1 to 9 as an in vitro co-transcribed RNA capping agent. An RNA molecule, characterized in that it comprises the compound according to any one of claims 1 to 9 as a cap structure or a cap structure fragment. A pharmaceutical composition, characterized in that it comprises the RNA molecule according to claim 11 and a pharmaceutically acceptable carrier. A method for synthesizing RNA molecules, characterized in that it comprises the following steps: co-incubating the compound described in any one of claims 1 to 9 with a polynucleotide template to perform template transcription. A capped RNA transcription reaction system, characterized in that it comprises: a polynucleotide template, the compound according to any one of claims 1 to 9, NTPs and RNA polymerase.