Liver-targeting nitric oxide donor compound and application thereof

Abstract

The invention discloses a liver-targeting nitric oxide donor compound and application thereof. The compound is a derivative of 5-isosorbide mononitrate, and can be enriched in the liver and release nitric oxide. Compared with 5-isosorbide mononitrate, the compound disclosed by the invention is easier to metabolize after primary hepatocyte incubation of mice, effectively releases effector molecules such as nitric oxide and the like, shows good liver targeting, is expected to play a better effect in treatment of liver-related diseases, and improves the clinical application value of drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical medicines, and in particular to a liver-targeted nitric oxide donor compound and application thereof. Background Art

[0002] Portal hypertension is a clinical syndrome caused by increased pressure in the portal venous system, usually due to a variety of reasons leading to blood flow obstruction or increased blood flow. Clinically, the hepatic venous pressure gradient (HVPG) is used as an important indicator to assess portal pressure, and a normal value of <5mmHg or >10mmHg indicates portal hypertension. Portal hypertension can lead to some serious complications, such as esophageal varicose bleeding, refractory ascites, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, and hypersplenism, which require timely treatment.

[0003] Currently, the treatments for portal hypertension include drug therapy, endoscopic therapy, interventional therapy, and surgical treatment. In terms of drug therapy, the commonly used somatostatin and vasopressin are effective, but their side effects are serious, limiting their use. Studies have shown that the effect of the combination of 5-isosorbide mononitrate and propranolol is better than that of either alone, but the systemic vasodilator effect of nitrates (such as 5-isosorbide mononitrate, ISMN) may lead to increased portal blood flow, which is not conducive to disease control.

[0004] Although there have been some progress in existing treatments, there are still obvious defects: adverse reactions caused by the systemic effects of drugs, individual differences in treatment effects, and insufficient targeting of liver diseases. Therefore, it is urgent to modify existing drugs to improve their targeting of the liver to reduce the impact on peripheral blood vessels, thereby enhancing the safety and effectiveness of treatment. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a liver-targeted nitric oxide donor compound and its application. The present invention provides a 5-isosorbide mononitrate derivative that can be enriched in the liver and release nitric oxide, and has multiple effects such as improving intrahepatic hemodynamic properties, repairing liver damage, resisting portal hypertension and improving liver fibrosis.

[0006] The present invention provides a compound represented by formula (I) or a stereoisomer, tautomer, nitrogen oxide, deuterated product, hydrate, solvate, metabolite, pharmaceutically acceptable salt or isotope-labeled compound thereof;

[0007] DP(Ⅰ);

[0008] Wherein, D is selected from any one of the following structures:

[0009]

[0010] P is selected from any one of the following structures:

[0011] Preferably

[0012] Wherein, Ar is phenyl, 5- or 6-membered heteroaryl, and the 5- or 6-membered heteroaryl contains 1 to 2 heteroatoms selected from O, S, and N;

[0013] The Ar may be substituted by 0 to 4 substituents, and the substituents are independently:

[0014] H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl;

[0015] R 1 C 1-6 Alkyl, C 3-7 Cycloalkyl, phenyl, benzyl, 5-membered or 6-membered heteroaryl, wherein the heteroaryl contains 1 to 2 heteroatoms selected from O, S, and N;

[0016] The R 1 is further substituted by 0 to 4 substituents, the substituents being independently:

[0017] H, F, Cl, phenyl, C 1-4 Alkyl, C 1-6 Alkoxy;

[0018] R 2 , R 4a , R 4b Each independently selected from:

[0019] H.C 1-6 Alkyl or C 3-7 Cycloalkyl;

[0020] The C 1-6 Alkyl or C 3-7 The cycloalkyl group is further substituted by 0 to 4 substituents, which are independently:

[0021] F, Cl, C 1-6 Alkyl, C 1-6 Alkoxy;

[0022] R 3a , R 3b , R 3c Each independently selected from:

[0023] C 1-6 Alkyl, benzyl, C 3-7 Cycloalkyl;

[0024] The C 1-6 Alkyl, benzyl or C 3-7 The cycloalkyl group is further substituted by 0 to 4 substituents, which are independently:

[0025] F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Alkoxy.

[0026] In some embodiments, Ar is phenyl or pyridyl;

[0027] The Ar may be further substituted by 0 to 4 substituents, and the substituents are independently:

[0028] H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy;

[0029] R 1 Selected from C 1-6 Alkyl, C 3-7 Cycloalkyl, phenyl, benzyl, 5-membered or 6-membered heteroaryl;

[0030] The R 1 Optionally further substituted by 0 to 4 substituents, the substituents are independently:

[0031] H, F, Cl, phenyl, C 1-4 Alkyl, C 1-6 Alkoxy;

[0032] R 2 Selected from H, C 1-4 Alkyl, the C 1-4 The alkyl group may be further substituted by 0 to 4 independently selected groups selected from F, Cl, C 1-6 Alkyl or C 1-6 substituted by an alkoxy substituent;

[0033] R 3a Selected from C 1-4 Alkyl, benzyl, the R 3a can be further substituted by 0 to 4 independently selected from F, Cl, C 1-4 Alkyl or C 1-4 The alkoxy group is substituted with an alkoxy substituent.

[0034] In some embodiments, Ar is a phenyl group, which may be further substituted by 0 to 4 independently selected groups selected from H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy substituted;

[0035] R 1is substituted or unsubstituted methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, phenyl, benzyl, methylenecyclopropyl, methylenecyclobutyl, methylenecyclopentyl, methylenecyclohexyl;

[0036] When R 1 When substituted, it is substituted by 1 to 4 substituents, and the substituents are independently:

[0037] F, Cl, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy;

[0038] R 2 is H, methyl, ethyl or isopropyl;

[0039] R 3a is methyl, ethyl, isopropyl, cyclopropyl or benzyl.

[0040] In some embodiments, one or more embodiments of the present invention provide compounds selected from but not limited to the following structures:

[0041]

[0042]

[0043] Preferably:

[0044]

[0045]

[0046] The present invention also provides a pharmaceutical composition, which contains the compound or its stereoisomers, tautomers, nitrogen oxides, deuterated substances, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds.

[0047] In some embodiments, the pharmaceutical composition further comprises one or more pharmacologically acceptable excipients, which include conventional diluents, excipients, fillers, wetting agents, absorption enhancers, surfactants, lubricants or stabilizers in the pharmaceutical field.

[0048] The present invention also provides the use of the compound or its stereoisomers, tautomers, nitrogen oxides, deuterated products, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds in the preparation of drugs for treating hepatitis, liver fibrosis, liver cancer or portal hypertension.

[0049] The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. Unless otherwise specified, all stereoisomeric forms of the compounds of the present invention as well as mixtures thereof, including racemic mixtures, are part of the present invention.

[0050] The present invention includes all pharmaceutically acceptable isotope-labeled compounds of formula (I), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Substitution by heavier isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and therefore may be preferred in certain circumstances.

[0051] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0052] The C (carbon), H (hydrogen), O (oxygen), S (sulfur), N (nitrogen), F (fluorine), Cl (chlorine), Br (bromine), and I (iodine) involved in the groups and compounds of the present invention include their isotopes, and the C, H, O, S, or N involved in the groups and compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of C include 12 C. 13 C and 14 The isotopes of C and H include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called super tritium). The isotopes of O include 16 O. 17 O and 18 Isotopes of O and S include 32 S. 33 S. 34 S and 36 Isotopes of S and N include 14 N and 15 Isotopes of N and F include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Isotopes of Cl and Br include 79 Br and 81 Br.

[0053] "Alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, preferably an alkyl group of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms, and further preferably an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and various branched isomers thereof; when the alkyl group is substituted, it may be optionally further substituted by one or more substituents.

[0054] In various parts of this specification, the substituents of the compounds disclosed in the present invention are disclosed according to group types or ranges. It is particularly pointed out that the present invention includes each independent subcombination of the individual members of these group types and ranges. For example, the term "C 1-4 "Alkyl" specifically refers to methyl, ethyl, C3 alkyl, C4 alkyl as disclosed independently.

[0055] As used herein, the term "alkyl" and its prefix "alkane" include both straight and branched saturated carbon chains.

[0056] "Aryl" may be used alone or as a large part of "aralkyl", "aralkyloxy" or "aryloxyalkyl" to refer to monocyclic, bicyclic, and tricyclic carbon ring systems containing 6-14 ring atoms, or 6-12 ring atoms, or 6-10 ring atoms, wherein at least one ring system is aromatic, wherein each ring system contains 3-7 ring atoms and has one or more points of attachment to the rest of the molecule. The term "aryl" can be used interchangeably with the term "aromatic ring" or "aromatic ring", such as aromatic rings can include phenyl, naphthyl and anthracenyl. The aryl groups can independently be unsubstituted or substituted with one or more substituents described herein.

[0057] "Heteroaryl" refers to a substituted or unsubstituted aromatic ring, which can be a 3-8 membered (e.g., 3, 4, 5, 6, 7, 8 membered) monocyclic ring, a 5-12 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring, or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O or S, preferably a 5-8 membered heteroaryl. The 1 to 4 (e.g., 1, 2, 3, 4) N, S optionally substituted in the heteroaryl ring can be oxidized to various oxidation states. Heteroaryl can be connected to a heteroatom or a carbon atom, and can be a bridged ring or a spiro ring, and non-limiting examples include cyclopyridyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinyl benzimidazolyl, benzopyridinyl, pyrrolopyridinyl. Heteroaryl is optionally further substituted by one or more substituents.

[0058] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group whose ring can be a 3-10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic ring, a 4-12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10-20-membered (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-membered) polycyclic ring system, and the ring carbon atoms are preferably 3 to 10 carbon atoms, and more preferably 3 to 8 carbon atoms. Non-limiting examples of "cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,5-cyclooctadienyl, 1,4-cyclohexadienyl, and cycloheptatrienyl. When the cycloalkyl is substituted, it may be optionally further substituted by one or more substituents.

[0059] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

[0060] "Tautomers" refer to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0061] "N-oxide" means that when a compound contains several amine functional groups, one or more nitrogen atoms may be oxidized to form an N-oxide. In particular, the N-oxide may be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514).

[0062] "Deuterated" refers to a compound in which one or more of the hydrogen atoms are replaced by 2 The compound generated after H substitution.

[0063] "Hydrate" refers to an association formed when the solvent molecule is water.

[0064] "Solvate" refers to an association formed between one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0065] "Metabolite" refers to a product obtained by the metabolism of a specific compound or salt thereof in vivo. The metabolites of a compound can be identified by techniques known in the art, and their activity can be characterized by experimental methods as described herein. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a period of time.

[0066] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention which retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, or the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0067] Any structural formula given herein is also intended to represent unlabeled forms of these compounds as well as isotopically labeled forms. Isotopically labeled compounds have structures depicted by the general formula given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Exemplary isotopes that can be introduced into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 36 S, 37 Cl or 125 I.

[0068] On the other hand, the compounds of the present invention include compounds defined in the present invention that are labeled with various isotopes, for example, those compounds in which radioactive isotopes such as 3H, 14C and 18F are present, or in which non-radioactive isotopes such as 2H and 13C are present. Such isotopically labeled compounds can be used for metabolic studies (using 14C), reaction kinetic studies (using, for example, 2H or 3H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution determination, or can be used in radiotherapy of patients. 18F labeled compounds are particularly ideal for PET or SPECT studies. Isotopically labeled compounds of formula (I) can be prepared by conventional techniques familiar to those skilled in the art or as described in the examples and preparations of the present invention using an appropriate isotopically labeled reagent in place of the unlabeled reagent originally used.

[0069] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0070] 1. The 5-isosorbide mononitrate derivatives of the present invention are relatively stable in serum and plasma. Compared with 5-isosorbide mononitrate (ISMN), the derivatives of the present invention are more easily metabolized after incubation in primary mouse hepatocytes, and effectively release effector molecules such as nitric oxide, showing good liver targeting, and are expected to play a better effect in the treatment of liver-related diseases and enhance the clinical application value of the drug.

[0071] 2. The 5-isosorbide mononitrate derivatives of the present invention are abundantly distributed in hepatocytes and can be converted into polar metabolites. Since these metabolites have high polarity, their ability to penetrate the hepatocyte membrane is reduced, so that they are passively retained in the hepatocytes, which provides a new design idea for targeted drugs for the treatment of liver-related diseases such as portal hypertension, and helps to enhance the local effect of drugs and reduce systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0073] Figure 1 The results of the nitric oxide content test in the primary hepatocytes incubated in Example A of the present invention are as follows;

[0074] Figure 2 This is the result of the serum incubation experiment in Example B of the present invention. DETAILED DESCRIPTION

[0075] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0076] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0077] Example 1 (3R, 3aS, 6S, 6aS)-6-((4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)oxy)hexahydrofuran[3,2-b]furan-3-yl nitrate

[0078]

[0079] Step 1: Methyl 3-(3-chlorophenyl)-3-oxopropanoate

[0080] At room temperature, take a 250mL dry reaction bottle, add 100mL toluene, sodium hydrogen (5.17g, 129.37mmol), nitrogen protection, stir at room temperature for 10 minutes, then add 3-chloroacetophenone (5.00g, 32.34mmol), dimethyl carbonate (8.74g, 97.03mmol). Raise the temperature to 110℃ and react for 3 hours. Cool down 0-10℃, add water (80mL) dropwise to quench the reaction, adjust pH to 4-5 with 4M HCl (aq), extract with ethyl acetate (80mL×3), combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column (PE:EA=10:1) to obtain the target product as a yellow oil (6.0g, yield 87%).

[0081] Step 2: 1-(3-chlorophenyl)propane-1,3-diol

[0082] Take a 250mL reaction bottle, add sodium borohydride (4.40g, 116.6mmol), 45mL methanol, reduce the temperature to 0-10℃, take 3-(3-chlorophenyl)-3-oxopropionic acid methyl ester (3.10g, 14.6mmol) diluted in 10mL methanol, drop into the reaction bottle, react at room temperature for 20min, heat to 60℃ and react overnight. After the reaction is completed, remove methanol under reduced pressure, add 60mL water, extract with ethyl acetate (50mL×3), combine the organic phases, add anhydrous sodium sulfate to dry, and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (PE:EA=1:1) to obtain the target product (2.61g, yield 96%) as a colorless oily liquid.

[0083] Step 3: 4-(3-chlorophenyl)-2,2,8,8-tetramethyl-3,7-dioxo-2,8-disilanonane

[0084] Take a 50mL reaction bottle, add 1-(3-chlorophenyl)propane-1,3-diol (2.00g, 10.72mmol), 10mL tetrahydrofuran, hexamethyldisilazane (4.30g, 26.80mmol), nitrogen protection, and drop 2-4 drops of trimethylsilyl trifluoromethanesulfonate. React at room temperature for 1 hour, add 50mL water, extract with ethyl acetate (50mL×3), dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain a colorless oily crude target compound (3.62g, yield 102%), which is used directly in the next step without further purification.

[0085] Step 4: (2S,6S,7S,10R)-2-(3-chlorophenyl)-7-isopropyl-10-methyl-1,5-dioxopyrrolo[5,5]undecane

[0086] Take a 100mL dry reaction bottle, add 4-(3-chlorophenyl)-2,2,8,8-tetramethyl-3,7-dioxo-2,8-disilanonane (3.55g, 10.72mmol), 20mL dichloromethane, nitrogen protection, cool to -40°C, add (2S, 5R)-2-isopropyl-5-methylcyclohexanone (1.98g, 12.86mmol), add trimethylsilyl trifluoromethanesulfonate (285mg, 1.29mmol) dropwise, keep -20~-40°C to react for 3 hours. Add 6mL pyridine to quench the reaction, then add 100mL petroleum ether and 50mL water, separate the liquids, dry the organic phase with anhydrous sodium sulfate, concentrate the organic phase, and chromatograph the resulting residue on a silica gel column (triethylamine alkalized silica gel) (PE:DCM=5:1) to obtain the target compound (1.68g, yield 48%) as a colorless oil.

[0087] Step 5: (S)-1-(3-chlorophenyl)propane-1,3-diol

[0088] At room temperature, take a 50mL reaction bottle, add (2S, 6S, 7S, 10R)-2-(3-chlorophenyl)-7-isopropyl-10-methyl-1,5-dioxopyrrolo[5,5]undecane (1.68g, 5.20mmol), 10mL methanol, 3mL concentrated hydrochloric acid, and react at room temperature overnight. Concentrate under reduced pressure to remove methanol, add 30mL water, extract with ethyl acetate (50mL×3), combine the organic phases, dry over anhydrous sodium sulfate, concentrate the filtrate, and purify by column (PE:EA=1:1) to obtain the target compound (820mg, yield 84.4%) as a colorless oily liquid.

[0089] Step 6: (2R,4S)-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane 2-oxide

[0090] Take a 50mL reaction bottle, add (S)-1-(3-chlorophenyl)propane-1,3-diol (205mg, 1.10mmol), 4mL tetrahydrofuran, triethylamine (444mg, 4.39mmol), nitrogen protection, ice water bath cooling 0~5℃; then add 4-nitrophenyl phosphorus dichloride (506mg, 1.98mmol) dropwise at low temperature, keep the reaction at low temperature for 2h. Take a sample and point the plate, the raw material disappears, and two configurations of products are generated. Add triethylamine (350mg, 3.46mmol) and p-nitrophenol (460mg, 3.31mmol), react at room temperature overnight, point the plate, and all are converted into the target configuration compound. The tetrahydrofuran was removed under reduced pressure, 50 mL of ethyl acetate and 50 mL of 0.4 M aqueous sodium hydroxide solution were added, the liquids were separated, and the mixture was extracted twice with 50 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA=1:1) to obtain the target compound (330 mg, yield 81%) as an oil.

[0091] Mass spectrum data: MS-ESI (m / z) = 370.0 [M+H] + .

[0092] Step 7: (3R, 3aS, 6S, 6aS)-6-((4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)oxy)hexahydrofuran[3,2-b]furan-3-yl nitrate

[0093] Dissolve 5-isosorbide mononitrate (142 mg, 0.74 mmol) in 4 mL of tetrahydrofuran, cool to -20°C, add tert-butylmagnesium chloride (0.80 mL, 1.35 mmol, 1.7 M in THF) dropwise under nitrogen protection, and continue to react at -20°C with stirring for 0.5 hour; take (2R, 4S)-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphirane 2-oxide (250 mg, 0.68 mmol) and dissolve it in 1 mL of tetrahydrofuran, add it dropwise into the reaction system at 0°C, and continue to react for 3 hours after the temperature rises naturally to room temperature. 10 mL of saturated aqueous ammonium chloride solution was added to quench the reaction, and then 20 mL of water was added, extracted with ethyl acetate (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was chromatographed on a silica gel column (PE:EA=1:1) to give the target compound (100 mg, yield 35%) as a colorless oily liquid.

[0094] The structural characterization data of the compound are as follows: 1 H NMR (500 MHz, CDCl 3)δ7.42–7.29(m,3H),7.25–7.20(m,1H),5.63(dt,J=11.0,2.7Hz,1H),5. 36(td,J=5.5,3.0Hz,1H),5.02(td,J=7.3,4.1Hz,2H),4.73–4.60(m,2H), 4.50–4.40(m,1H),4.17(d,J=11.0Hz,1H),4.04–3.95(m,2H),3.92(dd,J= 11.2, 5.6Hz, 1H), 2.32 (dtd, J=16.1, 11.5, 4.8Hz, 1H), 2.15–2.06 (m, 1H).

[0095] Mass spectrum data: MS-ESI (m / z) = 422.0 [M+H] + .

[0096] Example 2 ((((3S, 3aS, 6R, 6aS)-6-(nitrogenoxy)hexahydrofuran[3,2-b]furan-3-yl)oxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0097]

[0098] Step 1: Isopropyl ((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine (GZBJ-1021-2)

[0099] Take a 100mL dry three-necked reaction bottle, N 2 Protection, at room temperature, add L-alanine isopropyl hydrochloride (3.00g, 17.91mmol), add 20mL of dichloromethane, cool to -60℃, add dichlorooxyphenyl phosphate (3.78g, 17.91mmol), and stir at low temperature for 10min; take triethylamine (5.50g, 54.35mmol) and dilute it in 10mL of dichloromethane, drop it into the reaction system at -60℃, a large amount of white solid precipitates during the addition process, keep warm at -20~-40℃ and react for 1.5 hours; add pentafluorophenol (3.63g, 19.70mmol) and triethylamine (2.72g, 26.86mmol), keep warm at -20℃ and react for 1 hour, and naturally return to room temperature and react for 2 hours. After the reaction is completed, filter, wash the filter cake with 50 mL of dichloromethane, concentrate the filtrate under reduced pressure, add 150 mL of tert-methyl ether for slurrying, filter, wash the filter cake with 50 mL of tert-methyl ether, concentrate the filtrate under reduced pressure, add 40 mL of mixed solvent (PE:EA=4:1) to the crude product, stir at room temperature for 1 hour, filter, take out the filter cake, and dry to obtain the target product as a white solid (2.11 g, yield 26.0%).

[0100] Mass spectrum data: MS-ESI (m / z) = 454.1 [M+H]+ ;

[0101] Step 2: ((((3S,3aS,6R,6aS)-6-(nitrogenoxy)hexahydrofuran[3,2-b]furan-3-yl)oxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0102] Dissolve 5-isosorbide mononitrate (371 mg, 1.94 mmol) in 10 mL tetrahydrofuran, cool to -20°C, add tert-butylmagnesium chloride (2.0 mL, 3.53 mmol, 1.7 M in THF) dropwise under nitrogen protection, and continue stirring and reacting at -20°C for 0.5 hours; dilute isopropyl((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine (800 mg, 1.76 mmol) in 3 mL tetrahydrofuran, dropwise add to the reaction system at 0°C, and naturally warm to room temperature for 3 hours. Add 10 mL saturated aqueous ammonium chloride to quench the reaction, add 50 mL water, extract with ethyl acetate (70 mL×3), combine the organic phases, dry over anhydrous sodium sulfate, concentrate the organic phases, and chromatograph on a silica gel column (PE:EA=1:1) to obtain the target compound as a colorless oily liquid.

[0103] The structural characterization data of the compound are as follows: 1 H NMR (500 MHz, CDCl 3 )δ7.32(t,J=7.9Hz,2H),7.22–7.12(m,3H),5.33(td,J=5.6,3.1Hz,1H), 5.02(dt,J=12.5,6.3Hz,1H),4.99–4.94(m,2H),4.65(d,J=4.8Hz,1H),4 .12(dd,J=8.4,5.8Hz,1H), 3.93(dddd,J=16.8,13.1,9.2,4.4Hz,4H), 3.67(t,J=10.3Hz,1H), 1.35(d,J=7.0Hz,3H), 1.24(dd,J=6.8,4.8Hz,6H).

[0104] Mass spectrum data: MS-ESI (m / z) = 461.1 [M+H] + .

[0105] Example 3 (3R, 3aS, 6S, 6aS)-6-(((((S)-1-(1,3-dioxacyclo-2-yl)ethyl)amino)(phenoxy)phosphoryl)oxy)hexahydrofuran[3,2-b]furan-3-yl nitrate

[0106]

[0107] Step 1: Benzyl (S)-(1-oxopropan-2-yl)carbamate

[0108] Take a 50mL dry three-necked reaction bottle, add benzyl (S)-(1-hydroxypropane-2-yl) carbamate (1.00g, 4.78mmol), add 15mL dichloromethane, protect with nitrogen, cool to 0-5°C, add Dess-Martin reagent (3.04g, 7.17mmol), return to room temperature naturally, and react at room temperature for 4 hours. After the reaction is completed, add 10mL saturated sodium thiosulfate solution to quench the reaction, add 25mL water, separate the liquids, extract three times with 30mL ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, concentrate the organic phase, and chromatograph on a silica gel column (PE:EA=2:1) ​​to obtain the target compound (780mg, yield 78%) as a colorless oily liquid.

[0109] Mass spectrum data: MS-ESI (m / z) = 208.1 [M+H] + .

[0110] Step 2: Benzyl (S)-(1-(1,3-dioxolan-2-yl)ethyl)carbamate

[0111] Take a 100mL reaction bottle, add benzyl (S)-(1-oxopropane-2-yl)carbamate (700mg, 3.38mmol), 10mL toluene, 1mL ethylene glycol, p-toluenesulfonic acid (116mg, 0.68mmol), heat to 90℃ and react overnight. After the reaction is completed, concentrate under reduced pressure, and obtain the target compound (520mg, yield 61%) as a colorless oily liquid through silica gel column chromatography (PE:EA=3:1).

[0112] Mass spectrum data: MS-ESI (m / z) = 252.1 [M+H] + .

[0113] Step 3: (S)-1-(1,3-dioxolan-2-yl)ethane-1-amine

[0114] Take a 50mL reaction bottle, add benzyl (S)-(1-(1,3-dioxolan-2-yl)ethyl)carbamate (520mg, 2.07mmol), 5mL methanol, 100mg 5% Pd / C, hydrogen balloon, replace air with hydrogen three times, heat to 50℃ and react overnight. After the reaction is completed, filter with diatomaceous earth, wash the filter cake with a small amount of methanol, and concentrate under reduced pressure to obtain the target compound (226mg, crude yield 93%) as a colorless oily liquid (Note: the product is easily pulled away during reduced pressure concentration).

[0115] Mass spectrum data: MS-ESI (m / z) = 118.1 [M+H] + .

[0116] Step 4: Perfluorophenyl ((S)-1-(1,3-dioxacyclohexane-2-yl)ethyl)phosphoramide

[0117] Take a 100mL dry three-necked reaction bottle, N 2 After protection, (S)-1-(1,3-dioxapentane-2-yl)ethane-1-amine (300 mg, 2.56 mmol), 10 mL of dichloromethane, triethylamine (777 mg, 7.68 mmol) were added, the temperature was lowered to -60 °C, dichlorooxyphenyl phosphate (540 mg, 2.56 mmol) was added, and the mixture was stirred at low temperature for 0.5 hours; pentafluorophenol (518 mg, 2.82 mmol) was added, and the mixture was naturally returned to room temperature for 2 hours. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with dichloromethane (40 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phases were concentrated. After silica gel column chromatography (PE: EA = 3: 1), the target compound (160 mg, yield 14%) was obtained as a white solid.

[0118] Mass spectrum data: MS-ESI (m / z) = 440.0 [M+H] + .

[0119] Step 5: (3R,3aS,6S,6aS)-6-(((((S)-1-(1,3-dioxacyclo[2-yl)ethyl)amino)(phenoxy)phosphoryl)oxy)hexahydrofuran[3,2-b]furan-3-yl nitrate

[0120] Take a 100mL dry three-necked reaction bottle, N 2 After protection, perfluorophenyl ((S)-1-(1,3-dioxocyclohexane-2-yl)ethyl)phosphoramide (510mg, 1.16mmol), 5-isosorbide mononitrate (266mg, 1.39mmol), 10mL tetrahydrofuran were added, the temperature was lowered to -20°C, tert-butylmagnesium chloride (1.36mL, 2.32mmol, 1.7M in THF) was added dropwise, stirred at low temperature for 0.5 hours, and the temperature was naturally raised to room temperature for reaction for 3 hours. After the reaction was completed, 10mL saturated aqueous ammonium chloride solution was added to quench the reaction, the tetrahydrofuran was removed by concentration under reduced pressure, 20mL water was added, and 30mL*4 ethyl acetate was extracted (40mL×3), the organic phases were combined, concentrated under reduced pressure, and purified by preparative liquid phase to obtain the target compound (250mg, yield 48%) as a colorless oily liquid.

[0121] The structural characterization data of the compound are as follows: 1 H NMR (500 MHz, CDCl 3)δ7.33(td,J=7.9,4.2Hz,2H),7.25–7.09(m,3H),5.33(dddd,J=11.6,8.6,5.8,3.1Hz,1H),5.03–4.88(m,2H),4.82–4.74(m,1H),4.63(dd ,J=77.9,4.7Hz,1H),4.18(dd,J=36.7,10.7Hz,1H),4.00–3.84(m,6H),3.60–3.44(m,1H),3.13–2.95(m,1H),1.15(dd,J=10.4,6.9Hz,3H).

[0122] Mass spectrum data: MS-ESI (m / z) = 447.1 [M+H] + .

[0123] Example 4 (3S, 3aR, 6R, 6aS)-6-(Nitrooxy)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0124]

[0125] Step 1: (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0126] Dissolve 5-isosorbide mononitrate (2.00 g, 10.47 mmol) in 20 mL tetrahydrofuran, add diethyl p-toluenesulfonyloxymethylphosphonate (3.71 g, 11.51 mmol), and cool the mixture to 0-5 ° C under nitrogen protection, then add sodium hydride (0.63 g, 15.70 mmol, 60%), stir at 5 ° C for 0.5 hours, and continue to stir for 3 hours after returning to room temperature. Add 30 mL of saturated ammonium chloride solution to quench the reaction, concentrate under reduced pressure to remove tetrahydrofuran, add 50 mL of water, extract with ethyl acetate (50 mL × 3), combine the organic phases, dry over anhydrous sodium sulfate, and concentrate the organic phase under reduced pressure. The residue is chromatographed on a silica gel column (PE: EA = 1: 2) to obtain a colorless oily liquid (1.66 g, yield 46.5%).

[0127] Mass spectrum data: MS-ESI (m / z) = 341.9 [M+H] + .

[0128] Step 2: (3R,3aS,6S,6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0129] (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (550 mg, 1.61 mmol) was dissolved in 5 mL of acetonitrile, and triethylamine (490 mg, 4.84 mmol) and trimethylsilyl bromide (987 mg, 6.45 mmol) were added. The resulting mixture was heated to 60°C for 2 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to remove the solvent, 5 ml of dichloromethane was added, nitrogen was protected, the temperature was lowered by 0-5°C, oxalyl chloride (614 mg, 4.84 mmol) was added, and the reaction was stirred at 5°C for 0.5 hours. After the temperature was naturally raised to room temperature, the reaction was continued to stir for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was directly used for the next step reaction at a yield of 100%.

[0130] Step 3: (3S,3aR,6R,6aS)-6-(Nitrooxy)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0131] Dissolve (3R, 3aS, 6S, 6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (crude product, 1.61mmol) and triethylamine (490mg, 4.84mmol) in 10mL dichloromethane, cool to -20℃ under nitrogen protection, add L-alanine isopropyl ester hydrochloride (189mg, 1.13mmol), stir and react at -20℃ for 0.5 hours, then add phenol (106mg, 1.13mmol), stir and react at -20℃ for 0.5 hours, and continue to react for 18 hours after naturally warming to room temperature. Add 50mL of water to the reaction solution, extract with dichloromethane (50mL×3), combine the organic phases, dry over anhydrous sodium sulfate, and concentrate the organic phase under reduced pressure. The residue is chromatographed on a silica gel column (PE:EA=1:2) to obtain the title compound as a light yellow solid (65mg, 8.5%)

[0132] The synthetic route is as follows:

[0133]

[0134] The characterization data of the compounds are as follows: 1 H NMR (500 MHz, CDCl 3)δ7.25(t,J=7.9Hz,2H),7.17–7.04(m,3H),5.26(td,J=5.5,3.0Hz,1H),4.93(dt,J=12.5,6.3Hz,1H),4.88(t,J=5.2Hz, 1H), 4.42 (d, J = 4.9Hz, 1H), 4.03–3.73 (m, 7H), 3.50 (t, J = 10.4Hz, 1H), 1.23 (d, J = 7.1Hz, 3H), 1.16 (dd, J = 6.2, 4.2Hz, 6H).

[0135] Mass spectrum data: MS-ESI (m / z) = 475.1 [M+H] + .

[0136] Example 5: (3R, 3aS, 6S, 6aR)-6-(((4S)-4-(3-chlorophenyl)-2-oxido-1,3,2-dioxophosphin-2-yl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0137]

[0138] Step 1: (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0139] Dissolve 5-isosorbide mononitrate (2.00g, 10.47mmol) in 20mL tetrahydrofuran, then add diethyl p-toluenesulfonyloxymethylphosphonate (3.71g, 11.51mmol), cool the mixture to 0-5°C, add sodium hydride (0.63g, 15.70mmol, 60%) under nitrogen protection, react at 5°C for 0.5 hours, and continue to stir and react for 3 hours after returning to room temperature naturally. Add 30mL of saturated ammonium chloride solution to the reaction solution to quench the reaction, concentrate under reduced pressure to remove tetrahydrofuran, add 50mL of water, extract with ethyl acetate (40mLx3), combine the organic phases, dry over anhydrous sodium sulfate, concentrate the organic phase, and obtain a colorless oily liquid (1.66g, yield 46.5%) by silica gel column chromatography (PE:EA=1:2). MS-ESI (m / z)=341.9[M+H] + .

[0140] Step 2: (3R,3aS,6S,6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0141] (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (1000 mg, 2.93 mmol) was dissolved in 10 mL of acetonitrile, and triethylamine (890 mg, 8.79 mmol) and trimethylsilyl bromide (1795 mg, 11.72 mmol) were added. The resulting mixture was heated to 60°C and stirred for reaction for 2 hours, and the solvent was removed by concentration under reduced pressure. 10 ml of dichloromethane was added, and the temperature was lowered by 0-5°C under nitrogen protection, and then oxalyl chloride (1115 mg, 8.79 mmol) was added. The reaction was carried out at low temperature for 0.5 hours, and the temperature was naturally raised to room temperature for reaction for 2 hours. After the reaction was completed, the reaction solution was removed and concentrated under reduced pressure to obtain a crude product, which was directly used for the next step reaction at a yield of 100%.

[0142] Step 3: (3R,3aS,6S,6aR)-6-(((4S)-4-(3-chlorophenyl)-2-oxido-1,3,2-dioxophosphin-2-yl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0143] Dissolve (3R, 3aS, 6S, 6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (crude product, 2.93mmol) in 20mL dichloromethane, add triethylamine (890mg, 8.79mmol), cool down to 0-5℃ under nitrogen protection, then add (S)-1-(3-chlorophenyl)propane-1,3-diol (273mg, 1.46mmol), stir at 5℃ for 0.5 hours, then naturally warm to room temperature for 18 hours. Add 80mL of water to the reaction solution, extract with dichloromethane (50mL×3), combine the organic phases, concentrate under reduced pressure, and the residue is chromatographed on a silica gel column (PE:EA=1:1) to obtain the title compound as a light yellow solid (230mg, 18%).

[0144] The structural characterization data of the compound are as follows: 1 HNMR (500MHz, CDCl 3 )δ7.30(s,1H),7.29–7.23(m,2H),7.16(d,J=6.4Hz,1H),5.52(d,J=11.2Hz,1H),5.27(td,J=5.5,3.0Hz,1H),4.88(t,J=5.2Hz,1H),4.58(t t,J=11.6,3.0Hz,1H),4.45(d,J=4.9Hz,1H),4.38–4.29(m,1H),4.11–3.81(m,7H),2.24(ddd,J=16.0,11.7,4.7Hz,1H),2.05–1.99(m,1H).

[0145] Mass spectrum data: MS-ESI (m / z) = 436.0 [M+H] + .

[0146] Example 6 (3R, 3aS, 6S, 6aR)-6-(S)-1-(1,3-dioxolan-2-yl)ethyl)amino)(phenoxy)phosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0147]

[0148] Step 1: (3R, 3aS, 6S, 6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0149] (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (2.10 g, 6.15 mmol) was dissolved in 20 mL of acetonitrile, and triethylamine (1.87 g, 18.46 mmol) and trimethylsilyl bromide (3.77 g, 24.62 mmol) were added. The resulting mixture was heated to 60°C and stirred for 2 hours. The reaction solution was removed, concentrated under reduced pressure to remove the solvent, 20 ml of dichloromethane was added, nitrogen was protected, the temperature was lowered by 0-5°C, oxalyl chloride (2.34 g, 18.46 mmol) was added, the reaction was carried out at low temperature for 0.5 hours, and the temperature was naturally raised to room temperature for 2 hours. After the reaction was completed, the reaction solution was removed and concentrated under reduced pressure to obtain 4.8 g of a crude product, which was directly used for the next step at a yield of 100%.

[0150] Step 2: (3R,3aS,6S,6aR)-6-(S)-1-(1,3-dioxolan-2-yl)ethyl)amino)(phenoxy)phosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate

[0151] Dissolve (3R, 3aS, 6S, 6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (crude product 4.8 g, 6.15 mmol) in 30 mL of dichloromethane, add triethylamine (2.34 g, 18.46 mmol), cool to -50°C under nitrogen protection, then add phenol (289 mg, 3.08 mmol), keep stirring at -50°C for 0.5 hour, then add (S)-1-(1,3-dioxolan-2-yl)ethan-1-amine (360 mg, 3.08 mmol), react at -50°C for 0.5 hour, and naturally heat to room temperature for 1 hour. After the reaction was completed, 100 mL of water was added, extracted with dichloromethane (50 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and chromatographed on a silica gel column (PE:EA=1:2) to give the title compound as a light yellow solid (90 mg, 3.2%).

[0152] The structural characterization data of the compound are as follows: 1 HNMR (500MHz, CDCl 3 ): δ7.32(t,J=7.9Hz,2H),7.22(d,J=8.5Hz,2H),7.16(t,J=7.3Hz,1H),5.32(td,J=5. 5,2.9Hz,1H),4.90(t,J=5.2Hz,1H),4.71(d,J=2.4Hz,1H),4.41(d,J=4.9Hz,1H),4.12 –4.06(m,2H),3.98(dd,J=11.2,2.8Hz,1H),3.93(dd,J=9.2,3.4Hz,2H),3.91–3.82(m, 6H), 3.65 (tdd, J=11.0, 6.8, 2.5Hz, 1H), 2.97 (t, J=11.2Hz, 1H), 1.10 (d, J=6.9Hz, 3H).

[0153] Mass spectrum data: MS-ESI (m / z) = 461.0 [M+H] + .

[0154] Example 7 (((3S, 3aR, 6R, 6aS)-6-(Nitrooxy)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)phosphonic acid

[0155]

[0156] Step 1: (3R, 3aS, 6S, 6aR)-6-((diethoxyphosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (800 mg, 2.34 mmol) was dissolved in 10 mL of acetonitrile, and triethylamine (712 mg, 7.03 mmol) and trimethylsilyl bromide (1436 mg, 9.38 mmol) were added. The resulting mixture was heated to 60°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and 10 mL of dichloromethane was added. The temperature was lowered to 0-5°C under nitrogen protection, and then oxalyl chloride (893 mg, 7.03 mmol) was added. The resulting mixture was stirred at 0°C for 0.5 hours, and then naturally heated to room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was directly subjected to the next step reaction at a yield of 100%.

[0157] Step 2: (((3S,3aR,6R,6aS)-6-(Nitrooxy)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)phosphonic acid

[0158] In a 100 mL dry three-necked reaction bottle, (3R, 3aS, 6S, 6aR)-6-((dichlorophosphoryl)methoxy)hexahydrofuro[3,2-b]furan-3-yl nitrate (crude product, 2.34 mmol) and 10 mL of water were added in sequence, and the reaction was stirred at room temperature for 0.5 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by high pressure liquid phase preparation to obtain the target compound (60 mg, colorless oil).

[0159] The structural characterization data of the compound are as follows: 1 H NMR (500MHz, DMSO): δ5.48(td,J=5.4,2.1Hz,1H), 4.89(t,J=5.3Hz,1H), 4.44(d,J=5.0Hz,1H), 4.13(d,J=3 .1Hz,1H),4.02–3.90(m,2H),3.83(dd,J=11.4,5.2Hz,1H),3.64(dd,J=10.3,3.3Hz,1H),3.61–3.55(m,2H).

[0160] Mass spectrum data: MS-ESI (m / z) = 286.0 [M+H] + .

[0161] Example 8 (((3S, 3aR, 6R, 6aR)-6-Hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)phosphoric acid

[0162]

[0163] Dissolve (((3S, 3aR, 6R, 6aS)-6-(Nitrooxy)hexahydrofurano[3,2-b]furan-3-yl)oxy)methyl)phosphonic acid (120 mg, 0.42 mmol) in 3 mL of methanol, add palladium hydroxide (50 mg), replace with hydrogen three times, and stir at room temperature for 18 hours under hydrogen atmosphere. Filter, and concentrate the filtrate under reduced pressure to obtain the title compound as an oil (80 mg, 79%).

[0164] The structural characterization data of the compound are as follows: 1 HNMR (500MHz, DMSO): δ4.42(d,J=3.9Hz,1H),4.34(t,J=4.4Hz,1H),4.09(d,J=5.1Hz,1H),4.04(s,1H),3.87( d,J=10.1Hz,1H),3.79–3.67(m,2H),3.53(d,J=8.4Hz,2H),3.29(t,J=8.0Hz,1H),1.17(dt,J=8.7,4.4Hz,1H).

[0165] Mass spectrum data: MS-ESI (m / z) = 241.1 [M+H] + .

[0166] Example A: Primary hepatocyte incubation experiment

[0167] This example uses a primary hepatocyte incubation experiment to explore the ability of the 5-isosorbide mononitrate derivative prepared in the example to release nitric oxide, and the specific steps are as follows:

[0168] 1. Accurately weigh appropriate amounts of the compounds prepared in Examples 1 to 6 and isosorbide 5-mononitrate (ISMN), prepare a 50 mM stock solution with DMSO, and store at -20°C.

[0169] 2. Dilute the compound stock solution to 200 μM using DMEM complete medium without phenol red, with a DMSO content of 0.4%.

[0170] 3. Primary hepatocytes were isolated and purified by in situ perfusion of C57BL / 6J mice. The cell density was adjusted to 666,700 cells per milliliter. 75 μL of cell suspension was added to each well of a 96-well cell culture plate (cells were resuspended in phenol red-free DMEM complete medium), equivalent to 50,000 cells per well.

[0171] 4. Add 75 μL of the corresponding 200 μM compound working solution to each well of the 96-well plate containing the primary hepatocyte suspension, mix well and place in an incubator at 37°C and 5% CO. 2 Incubate in the incubator for 1 hour.

[0172] 5. Collect the cell culture medium, centrifuge at 2000 rpm at 4°C for 5 minutes, and aspirate the supernatant.

[0173] 6. Prepare sodium nitrite standard solution with concentrations of 0μM, 1μM, 2μM, 5μM, 10μM, 20μM, 50μM, and 100μM. Pipette 50μL of standard or cell supernatant sample into a completely opaque white 96-well plate, add 50μL of 0.31mM 2,3-diaminonaphthalene (0.62M HCl dissolved) solution, mix well, and react at room temperature in the dark for 15 minutes. Add 25μL of 2.8M NaOH solution to the reaction solution.

[0174] 7. Detect the fluorescence signal of 96-well plate within 30 minutes, with the maximum excitation wavelength of 365nm and the maximum emission wavelength of 450nm. 2 replicate wells for each compound, and calculate the average value.

[0175] The results are as follows Figure 1 As shown, after the compounds prepared in Examples 1 to 6 were incubated with mouse primary hepatocytes, the fluorescence intensity was significantly higher than that of the parent compound 5-isosorbide mononitrate (ISMN), among which the compound in Example 5 had the highest fluorescence intensity. This indicates that the 5-isosorbide mononitrate derivatives prepared in this application have a stronger ability to release nitric oxide.

[0176] Example B Plasma incubation experiment

[0177] This example explores the stability of the compound prepared in the present application in serum, and the specific steps are as follows:

[0178] 1. Accurately weigh an appropriate amount of compound, prepare into 50 mM stock solution with DMSO, and store at -20℃.

[0179] 2. Use DMEM complete medium without phenol red to dilute the compound stock solution to 400 μM working solution with DMSO content of 0.8%.

[0180] 3. After anesthesia, blood was collected from the abdominal aorta of C57BL / 6J mice to obtain serum (without anticoagulant), which was centrifuged at 3000 rpm at 4°C for 10 minutes and the supernatant was obtained.

[0181] 4. Take 37.5 μL of serum supernatant, add 37.5 μL of 400 μM compound working solution, and incubate for 10 minutes to obtain the serum-compound working solution.

[0182] 5. Primary hepatocytes were isolated and purified by in situ perfusion of C57BL / 6J mice. The cell density was adjusted to 666,700 cells per milliliter. 75 μL of cell suspension was added to each well of a 96-well cell culture plate (cells were resuspended in phenol red-free DMEM complete medium), equivalent to 50,000 cells per well.

[0183] 6. Add 75 μL of the serum-compound working solution in step 4 to each well of the 96-well plate containing the primary hepatocyte suspension, mix well, and place in an incubator at 37°C and 5% CO. 2 Incubate in the incubator for 1 hour.

[0184] 7. Collect the cell culture medium, centrifuge at 2000 rpm for 5 minutes at 4°C, and aspirate the supernatant.

[0185] 8. Preparation of NaNO 2 Standard solution, concentrations are 0, 1, 2, 5, 10, 20, 50, 100 μM. Pipette 50 μL of standard or cell supernatant sample into a completely opaque white 96-well plate, add 50 μL of 0.31 mM 2,3-diaminonaphthalene (0.62 M HCl dissolved) solution, mix well, and react at room temperature in the dark for 15 minutes. Add 25 μL of 2.8 M NaOH solution to the reaction solution.

[0186] 9. Detect the fluorescence signal of 96-well plate within 30 minutes, with the maximum excitation wavelength of 365nm and the maximum emission wavelength of 450nm. 2 replicate wells for each compound, and calculate the average value.

[0187] The results are as follows Figure 2 As shown, the compounds prepared in Examples 1 to 6 had no significant difference in fluorescence values ​​compared with the 0.2% DMF group and the ISMN group, indicating that the compounds prepared in the examples of the present application were relatively stable in serum.

[0188] Example C Human plasma stability test

[0189] This example explores the stability of the compound prepared in this application in human plasma, and the specific steps are as follows:

[0190] 1. Prepare a 10 mM stock solution of the test compound with DMSO, and then dilute it with an appropriate solvent to form a working solution of the test compound for later use.

[0191] 2. Take an appropriate amount of blank human plasma in an EP tube, then add the working solution of the substance to be tested to a final concentration of 1 μM, and vortex to mix;

[0192] 3. Place the sample in a 37°C water bath in duplicate;

[0193] 4. Take out an appropriate amount of sample from the incubation system and add precipitant 3 times the volume of the incubation system for protein precipitation at 30 min, 1 h, 2 h, and 4 h;

[0194] 5. T0 sample (absolute zero point): Take an appropriate amount of blank plasma in an EP tube, first add 3 times the volume of the incubation system precipitant to precipitate protein, then add the working solution of the test substance, vortex to mix, and repeat 2 times in parallel;

[0195] 6. All samples were centrifuged at 15700×g for 10 min at 6°C.

[0196] 7. Take 150 μL of the supernatant and add 150 μL of water, vortex to mix, and inject for LC-MS / MS analysis;

[0197] 8. Data analysis: The remaining amount of original drug % = At / A0×100% (At: the amount of the analyte at the incubation time point; A0: the amount of the analyte in the 0h sample).

[0198] The results are shown in Table 1:

[0199] Table 1 Results of stability study in human plasma

[0200]

[0201] The results in Table 1 show that after the compound of the present invention was incubated with human plasma for 4 hours, a large amount of the compound still remained, indicating that the compound prepared in the example of the present application has good stability in human plasma.

[0202] Example D Hepatocyte Uptake Experiment

[0203] Take the compound, prepare it into a 10 mM stock solution with DMSO, dilute it to 500 μM with DMSO, take an appropriate amount of 500 μM working solution and further dilute it to 10 μM working solution of the test substance with cell incubation solution for use.

[0204] The information and preparation method of hepatocyte recovery medium are shown in Table 2:

[0205] Table 2 Information and preparation method of hepatocyte recovery medium

[0206]

[0207]

[0208] The hepatocyte incubation medium includes: 49.5 mL William's E Medium + 500 μL GlutaMAXTM-1 (100×).

[0209] Experimental steps:

[0210] (1) Remove the cells from the liquid nitrogen biocontainer, thaw the cells, count the cells using trypan blue staining, and then dilute them to a viable cell count of 2.0 × 10 6 cell / mL.

[0211] (2) Rat hepatocytes: 100 μL of hepatocyte suspension was incubated at 37°C for 3 minutes, then an equal volume of 100 μL of the drug solution was added. Each group was repeated in duplicate. Human hepatocytes: 50 μL of hepatocyte suspension was incubated at 37°C for 3 minutes, then an equal volume of 50 μL of the drug solution was added. Each group was repeated in duplicate.

[0212] (3) Shake gently to mix and incubate at 37°C for 1 hour;

[0213] (4) Treatment with dosing solution: dilute 100 μL of dosing solution + 100 μL of incubation solution, then take 100 μL of diluted dosing solution into a 96-well plate containing 400 μL of internal standard acetonitrile;

[0214] (5) After the incubation, transfer to an EP tube and centrifuge immediately at 900 rpm for 3 min;

[0215] (6) Transfer the supernatant to another EP tube for processing, take 100 μL of the supernatant to 400 μL of acetonitrile containing internal standard in a 96-well plate, and centrifuge the 96-well plate at 5500 g for 5 min;

[0216] (7) Add pre-cooled HBSS buffer to the cell pellet and gently wash once to terminate the reaction; centrifuge at 900 rpm for 3 min and discard the washing solution;

[0217] (8) Add 100 μL of ultrapure water to the cell pellet and freeze and thaw three times in liquid nitrogen at -37°C to lyse the cells;

[0218] (9) Add 400 μL of acetonitrile containing internal standard to 100 μL of cell lysate to precipitate protein, centrifuge at 15700 g for 5 min; take 150 μL of supernatant and add 150 μL of ultrapure water, vortex mix, and inject.

[0219] The LC-MS / MS analysis method of the analytes is shown in Table 3:

[0220] Table 3 LC-MS / MS parameters

[0221]

[0222] The test results are shown in Table 4:

[0223] Table 4 Hepatocyte uptake test results

[0224]

[0225]

[0226] Wherein, “*” means below the detection limit.

[0227] The results in Table 4 show that the concentration of the compound in the cell is much higher than that outside the cell, indicating that the compound prepared in the present application has a large distribution in the liver cells, and can be metabolized into polar metabolites, and because of its large polarity, it is difficult to penetrate the liver cells, so it is passively retained in the liver cells, and has an excellent liver targeting effect.

[0228] Example E Rat tissue distribution experiment

[0229] The compound prepared in Example 5 was dissolved in a mixed solvent of 5% DMSO + 60% PEG400 + 35% Saline, and intragastrically administered to male SD rats at a dose of 10 mg / kg. Blood and tissue samples were collected at 1 h, 4 h, and 8 h after administration (3 rats / time point):

[0230] 1. Plasma: Collect 0.2 mL of blood and place it in a labeled EDTA-2K anticoagulant tube. Gently invert the tube upside down to fully mix the anticoagulant (EDTA-2K) and blood, then immediately place it in wet ice and centrifuge it within 1 hour after blood collection to separate the plasma. The centrifugation conditions are set at 4°C, 6800g, and 6 minutes. The separated plasma after centrifugation is placed in a labeled EP tube and stored in an ultra-low temperature refrigerator as soon as possible until the sample is analyzed.

[0231] 2. Tissue: The animals were anesthetized with isoflurane and blood was drawn from the abdominal aorta. The liver tissue was obtained after blood was drawn from the abdominal aorta. The tissue was washed, weighed, and stored below -20°C.

[0232] 3. In an ice-water bath with yellow light, after the plasma sample is melted, vortex mix; after the liver sample is homogenized, vortex mix; add 20 μL of sample to a 96-well plate (blank sample, zero concentration sample, residual sample are replaced by blank matrix, and reagent blank is replaced by ultrapure water); add 300 μL of internal standard working solution (blank sample, residual sample, reagent blank are replaced by acetonitrile), vortex for 5 minutes; put the 96-well plate into a centrifuge, centrifuge at 5500g for 10 minutes at 6°C; add 150 μL of supernatant from a new 96-well plate to a 96-well plate containing 150 μL of ultrapure water, vortex mix, and inject for analysis.

[0233] Table 5 Rat tissue distribution test results

[0234]

[0235] The results in Table 5 show that the prototype compound can be detected in plasma, but not in the liver, and only downstream metabolites can be detected, indicating that the compound of the present invention is easily metabolized in the liver and releases the effector molecule nitric oxide, and has good liver targeting.

[0236] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound represented by formula (I) or its stereoisomers, tautomers, nitrogen oxides, deuterated derivatives, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds; DP(Ⅰ); in, D is selected from any one of the following structures: P is selected from any one of the following structures: Wherein, Ar is phenyl, 5- or 6-membered heteroaryl, and the 5- or 6-membered heteroaryl contains 1 to 2 heteroatoms selected from O, S, and N; The Ar may be substituted by 0 to 4 substituents, and the substituents are independently: H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl; R1 is C 1-6 Alkyl, C 3-7 Cycloalkyl, phenyl, benzyl, 5-membered or 6-membered heteroaryl, wherein the heteroaryl contains 1 to 2 heteroatoms selected from O, S, and N; The R1 is further substituted by 0 to 4 substituents, and the substituents are independently: H, F, Cl, phenyl, C 1-4 Alkyl, C 1-6 Alkoxy; R2, R 4a , R 4b Each independently selected from: H.C 1-6 Alkyl or C 3-7 Cycloalkyl; The C 1-6 Alkyl or C 3-7 The cycloalkyl group is further substituted by 0 to 4 substituents, which are independently: F, Cl, C 1-6 Alkyl, C 1-6 Alkoxy; R 3a , R 3b , R 3c Each independently selected from: C 1-6 Alkyl, benzyl, C 3-7 Cycloalkyl; The C 1-6 Alkyl, benzyl or C 3-7 The cycloalkyl group is further substituted by 0 to 4 substituents, which are independently: F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Alkoxy.

2. The compound of formula (I) according to claim 1 or its stereoisomers, tautomers, nitrogen oxides, deuterated substances, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds, characterized in that: Ar is phenyl or pyridyl; The Ar may be further substituted by 0 to 4 substituents, and the substituents are independently: H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy; R1 is selected from C 1-6 Alkyl, C 3-7 Cycloalkyl, phenyl, benzyl, 5-membered or 6-membered heteroaryl; The R1 is optionally further substituted by 0 to 4 substituents, and the substituents are independently: H, F, Cl, phenyl, C 1-4 Alkyl, C 1-6 Alkoxy; R2 is selected from H, C 1-4 Alkyl, the C 1-4 The alkyl group may be further substituted by 0 to 4 independently selected groups selected from F, Cl, C 1-6 Alkyl or C 1-6 substituted by an alkoxy substituent; R 3a Selected from C 1-4 Alkyl, benzyl, the R 3a can be further substituted by 0 to 4 independently selected from F, Cl, C 1-4 Alkyl or C 1-4 The alkoxy group is substituted with an alkoxy substituent.

3. The compound of formula (I) according to claim 2 or its stereoisomer, tautomer, nitrogen oxide, deuterated product, hydrate, solvate, metabolite, pharmaceutically acceptable salt or isotope-labeled compound, characterized in that: Ar is a phenyl group, which may be further substituted by 0 to 4 independently selected groups selected from H, F, Br, I, C 1-4 Alkyl, C 1-6 Alkoxy substituted; R1 is substituted or unsubstituted methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, phenyl, benzyl, methylenecyclopropyl, methylenecyclobutyl, methylenecyclopentyl, methylenecyclohexyl; When R1 is substituted, it is substituted by 1 to 4 substituents, which are independently: F, Cl, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy; R2 is H, methyl, ethyl or isopropyl; R 3a is methyl, ethyl, isopropyl, cyclopropyl or benzyl.

4. A compound represented by any of the following structures or its stereoisomers, tautomers, nitrogen oxides, deuterated derivatives, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds; 5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the compound according to any one of claims 1 to 4 or its stereoisomers, tautomers, nitrogen oxides, deuterated substances, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds.

6. The pharmaceutical composition according to claim 5, characterized in that It also includes one or more pharmacologically acceptable excipients, including conventional diluents, excipients, fillers, wetting agents, absorption promoters, surfactants, lubricants or stabilizers in the pharmaceutical field.

7. Use of the compound according to any one of claims 1 to 4 or its stereoisomers, tautomers, nitrogen oxides, deuterated derivatives, hydrates, solvates, metabolites, pharmaceutically acceptable salts or isotope-labeled compounds in the preparation of a medicament for treating hepatitis, liver fibrosis, liver cancer or portal hypertension.

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