A plasmin inhibitor, its preparation method and application

Abstract

This invention relates to compounds of formula (I) that can inhibit plasmin activity, delay fibrinolysis, and have coagulation and hemostatic activities, including pharmaceutically acceptable salts, hydrates, isomers, prodrugs, and mixtures thereof, wherein X, R1 to R5 are as defined in the specification.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, specifically to a plasmin inhibitor, its preparation method, and its application in the pharmaceutical field. Background Technology

[0002] Plasmin is a proteolytic enzyme that degrades fibrin. When tissue damage causes blood vessel rupture, it triggers hemostasis mechanisms: vasoconstriction, platelet embolism, and the initiation of coagulation, ultimately leading to the formation of stable fibrin. Simultaneously, due to fibrin deposition, the fibrinolytic system is activated. This system maintains a balance between fibrin formation and breakdown, playing a role in maintaining vascular patency and remodeling damaged tissue during the repair of damaged blood vessel walls (Tengborn L, M, Berntorp E. Thromb Res. 2015 Feb; 135(2):231-42).

[0003] The fibrinolytic system includes plasminogen, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). Plasminogen binds to lysine residues on the fibrin surface and is converted into plasmin via an activator (i.e., tPA) released from endothelial cells. Fibrinolysis inhibition can be used to treat bleeding. The use of antifibrinolytic drugs can reduce blood loss in cardiac surgery, trauma, orthopedic surgery, solid organ transplantation, obstetrics and gynecology, neurosurgery, and non-surgical conditions (Ng W, Jerath A, ...). M. Anaesthesiol Intensive Ther. 2015; 47(4):339-50). In the early 1950s, it was discovered that lysine amino acids inhibited the activation of plasminogen, but the effect was too weak to be used to treat fibrinolytic hemorrhage. In 1953, Shosuke Okamoto et al. showed that several thiol groups and aminocarbonates had anti-plasma protein effects, and found that the synthetic derivative of lysine, ε-aminohexanoic acid (EACA), had a strong inhibitory effect on plasminogen. EACA has been widely used in clinical practice, but in addition to mild gastrointestinal side effects such as nausea, a larger dose is required. In 1962, 4-amino-methyl-cyclohexane-carbonic acid (AMCHA) was discovered. This compound contains two stereoisomers. Further studies showed that its trans form (trans-4-aminomethylcyclohexanecarboxylic acid, i.e., tranexamic acid, TXA) has anti-fibrinolytic ability, with an activity about 10 times that of EACA, and has been shown to have stronger tolerance (Tengborn L, M, Berntorp E. Thromb Res. 2015 Feb; 135(2):231-42).

[0004] Tranexamic acid is a synthetic lysine derivative and antifibrinolytic agent that forms a reversible complex with plasminogen. By binding to plasminogen, it blocks the interaction between plasminogen and the plasmin heavy chain and fibrin lysine residues, thereby preventing plasminogen from binding to the fibrin surface and thus delaying fibrinolysis. Tranexamic acid has been approved for the treatment of severe menstrual bleeding and various surgical bleeding disorders, and is currently the most commonly used hemostatic drug in clinical practice. However, numerous literature reports indicate that oral administration of tranexamic acid easily causes gastrointestinal adverse reactions such as nausea, vomiting, diarrhea, and indigestion, and its dosage is relatively high; patients may also experience complications such as epilepsy after taking the drug.

[0005] Other similar hemostatic drugs, such as aminocaproic acid, have problems such as rapid excretion from the body, weak hemostatic effect, short duration of action, and more toxic reactions. Excessive dosage can lead to thrombosis, limiting their use in patients with a tendency to thrombosis, a history of thrombotic vascular disease, or renal insufficiency. Tranexamic acid has a similar mechanism to aminocaproic acid but is 4-5 times stronger. It is effective for general chronic bleeding but has no hemostatic effect on traumatic bleeding or cancerous bleeding. Furthermore, excessive dosage can also promote thrombosis. Aprotinin, a commonly used hemostatic drug in coronary artery bypass surgery, was withdrawn from the market by the FDA in 2008 due to its potential to induce renal failure, myocardial infarction, and heart failure.

[0006] Other hemostatic drugs with different mechanisms of action, such as carbazoline which acts on blood vessels, can induce epilepsy with repeated use; thrombin, a hemostatic drug that promotes the coagulation process, can only be used for gastrointestinal bleeding or local bleeding.

[0007] Given the very limited selection of hemostatic drugs available in clinical practice, and the deficiencies in dosage and clinical indications, as well as the problems of high dosage, numerous adverse reactions, and the potential to cause complications such as epilepsy, it is necessary to develop a new hemostatic drug to better meet clinical needs. Summary of the Invention

[0008] One of the objectives of this invention is to provide a novel compound that can inhibit plasmin activity, delay fibrinolysis, and has coagulation and hemostatic activities.

[0009] Specifically, the present invention provides compounds of Formula I, including pharmaceutically acceptable salts, hydrates, isomers, prodrugs, and mixtures thereof:

[0010]

[0011] Where X is selected from N or CR, and R = H or halogen;

[0012] R1 is independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic, or two R1 together with the attached carbon atom to form a carbon ring containing 3 to 8 carbon atoms.

[0013] R2 is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic.

[0014] R3 is selected from hydrogen, halogen, substituted or unsubstituted alkyl groups;

[0015] R4 is selected from hydrogen, substituted or unsubstituted amino groups, hydroxyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted alkyl groups, and substituted or unsubstituted aromatic heterocyclic groups.

[0016] R5 is selected from hydrogen, substituted or unsubstituted alkyl, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic, alkylcarbonyloxyalkyl, and alkoxycarbonyloxyalkyl.

[0017] In one embodiment, the present invention relates to compounds with structures representing formula (I'), including pharmaceutically acceptable salts, hydrates, isomers, prodrugs, and mixtures thereof:

[0018]

[0019] Where X is selected from N or CR, and R = H or halogen;

[0020] R1 is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic;

[0021] R2 is selected from hydrogen, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic.

[0022] R3 is selected from hydrogen, halogen, substituted or unsubstituted alkyl groups;

[0023] R4 is selected from hydrogen, substituted or unsubstituted amino groups, hydroxyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted alkyl groups, and substituted or unsubstituted aromatic heterocyclic groups.

[0024] R5 is selected from hydrogen, substituted or unsubstituted alkyl, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alicyclic or heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic.

[0025] In some specific implementations, X is N.

[0026] In some specific embodiments, the R1s of the present invention are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, C1-C4 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered alicyclic group, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclic group, or two R1s together with the attached carbon atom form a carbon ring containing 3 to 8 carbon atoms.

[0027] In some specific embodiments, R2 of the present invention is selected from hydrogen, halogen, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, C1-C4 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered alicyclic group, substituted or unsubstituted 6-10 membered aryl, and substituted or unsubstituted 6-10 membered aromatic heterocyclic group.

[0028] In some specific embodiments, R3 of the present invention is selected from hydrogen, fluorine, chlorine, bromine, substituted or unsubstituted C1-C4 alkyl groups.

[0029] In some specific embodiments, R4 of the present invention is selected from hydrogen, substituted or unsubstituted amino groups, hydroxyl groups, substituted or unsubstituted 6-10 aryl groups, substituted or unsubstituted C1-C6 alkyl groups, and substituted or unsubstituted 6-10 aryl heterocyclic groups.

[0030] In some specific embodiments, R5 of the present invention is selected from hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered alicyclic group, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclic group, C1-C4 alkylcarbonyloxy-C1-C4 alkyl, and C1-C4 alkoxycarbonyloxy-C1-C4 alkyl.

[0031] In some specific embodiments, the R1 groups of the present invention are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl groups, and substituted or unsubstituted C1-C6 alkoxy groups; wherein the substituted C1-C6 alkyl or substituted C1-C6 alkoxy groups are substituted by one or more groups selected from hydroxyl, alkyl, cycloalkyl, alkoxy, aryl, or substituted aryl groups; in some specific embodiments, the substituted C1-C6 alkyl or substituted C1-C6 alkoxy groups are substituted by one or more groups selected from hydroxyl, phenyl, C1-C4 alkoxy, C1-C4 alkoxy-substituted phenyl, and cyclohexyl groups.

[0032] In some specific embodiments, the R1 group of the present invention is independently selected from hydrogen, -CH2OH, isobutyl, tert-butyl, -O(CH2)2OH, -O(CH2)3OH, -(CH2)4OH, -CH2-O(CH2)3OH, phenylethyl, propyl, isopentyl, 3,3-dimethylbutyl, cyclohexylmethyl, cyclohexylethyl, phenylpropyl, 4-methoxyphenylethyl.

[0033] In some specific implementations, one of the R1 groups is hydrogen.

[0034] In some specific implementations, the two R1 atoms together with the attached carbon atom form a cyclobutyl, cyclopentyl, or cyclohexyl ring.

[0035] In some specific embodiments, the R2 group of the present invention is selected from hydrogen, halogen, hydroxyl, hydroxy-substituted C1-C6 alkoxy, and a 6-membered alicyclic group containing 1 to 3 heteroatoms selected from N, O and S, wherein the S heteroatoms may be selectively oxidized.

[0036] In some specific embodiments, the R2 group of the present invention is selected from hydrogen, hydroxyl, -OCH2CH2OH,

[0037] In some specific embodiments, the R2 group of the present invention is selected from hydrogen.

[0038] In some specific embodiments, the R3 group of the present invention is selected from hydrogen or fluorine.

[0039] In some specific embodiments, the R4 group of the present invention is selected from hydroxyl, phenyl, C1-C6 alkyl, or phenyl-substituted C1-C6 alkyl.

[0040] In some specific embodiments, the R4 group of the present invention is selected from hydroxyl, phenyl, ethyl or phenylethyl.

[0041] In some specific embodiments, the R4 group of the present invention is selected from hydroxyl, phenyl, or phenylethyl.

[0042] In some specific embodiments, the R5 group of the present invention is selected from hydrogen, substituted or unsubstituted C1-C4 alkyl, C1-C4 alkylcarbonyloxy-C1-C4 alkyl, or C1-C4 alkoxycarbonyloxy-C1-C4 alkyl.

[0043] In some specific embodiments, the R5 group of the present invention is selected from hydrogen, ethyl, methyl carbonyloxymethyl, isopropyl carbonyloxymethyl, or methoxycarbonyloxymethyl.

[0044] In some specific embodiments, the compound of formula I of the present invention has the following structure:

[0045]

[0046]

[0047] Another object of the present invention is to provide a pharmaceutical composition comprising at least one of the aforementioned compounds, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, and at least one pharmaceutically acceptable excipient.

[0048] Another object of the present invention is to provide the aforementioned compound or its pharmaceutically acceptable salts, hydrates, isomers, prodrugs, mixtures, or pharmaceutical compositions for use in the preparation of a medicament. The medicament can effectively inhibit plasmin activity, delay fibrinolysis, and exert excellent coagulation and hemostatic therapeutic activities, and can be used for abnormal bleeding caused by hyperfibrinolysis, surgical and postoperative bleeding, etc.

[0049] Another object of the present invention is to provide a method for treating and / or alleviating bleeding disorders or conditions, comprising administering to a patient the foregoing one or more of the aforementioned pharmaceutical compositions or compounds of Formula I or thereof, or pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof.

[0050] Definition of terms

[0051] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0052] The term "pharmaceutically acceptable" as used here means that it is intended for use only in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0053] The term "pharmaceutically acceptable salt" refers to the salt of the compounds of this invention, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this invention, with a specific substituent. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.

[0054] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds. The term "isomer" as used herein includes cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures, all of which are within the scope of this invention.

[0055] "Alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group, for example: C1-C4 alkyl and C1-C6 alkyl refer to saturated aliphatic hydrocarbon groups containing 1 to 4 carbon atoms and 1 to 6 carbon atoms, respectively. Examples of alkyl groups described in this invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isopentyl, 3,3-dimethylbutyl, and their various isomers.

[0056] "Alkoxy" refers to -O-alkyl; for example, C1-C6 alkoxy refers to straight-chain or branched alkoxy containing 1 to 6 carbons, and C1-C3 alkoxy refers to straight-chain or branched alkoxy containing 1 to 3 carbons. Examples of alkoxy compounds described in this invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0057] "Cycloalkyl" refers to a monocyclic or polycyclic cyclic hydrocarbon substituent that is saturated or partially unsaturated. For example, "C3-C6 cycloalkyl" refers to a cycloalkyl group containing 3 to 6 carbon atoms. Examples of cycloalkyl groups described in this invention include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl.

[0058] "Alicyclic group" refers to a saturated monocyclic hydrocarbon substituent in which one or more ring atoms are replaced by heteroatoms selected from N, O, and S, and the remaining ring atoms are carbon, wherein the S heteroatoms may be selectively oxidized. For example, "3-8 membered alicyclic" refers to a saturated cyclic hydrocarbon substituent containing 3-8 ring atoms, wherein one or more ring atoms are replaced by heteroatoms selected from N, O, and S, and the remaining ring atoms are carbon, wherein the S heteroatoms may be selectively oxidized. Examples of alicyclic groups described in this invention include, but are not limited to: oxabutyl, pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, etc. wait.

[0059] "Aromatic heterocyclic group" refers to an aromatic cyclic substituent in which one or more ring atoms are replaced by a heteroatom selected from N, O, and S, and the remaining ring atoms are carbon. For example, "5-6 membered aromatic heterocyclic" refers to an aromatic heterocyclic group containing 5 to 6 ring atoms. Examples of aromatic heterocyclic groups described in this invention include, but are not limited to, pyridinyl, pyrimidinyl, imidazoleyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and 1,2,4-oxadiazolyl.

[0060] "Aryl" refers to an aromatic cyclic group, such as "6-10 aryl" which refers to an aromatic cyclic group containing 6 to 10 carbon ring atoms. Examples of the aryl moiety described in this invention include, but are not limited to, phenyl, naphthyl, etc.

[0061] "Optional" means that the event or situation described below may occur but is not required to occur.

[0062] All abbreviations used in this invention are known to those skilled in the art and, unless otherwise stated, represent the meanings known in the art. For example: DMF refers to N,N-dimethylformamide; THF refers to tetrahydrofuran; and Me refers to methyl.

[0063] The activity of the compounds of this invention was determined by plasma clot degradation assay and thromboelastography (TEG) assay. In the experiments, rtPA was added to human plasma or whole blood to activate plasminogen. The resulting plasmin can degrade fibrin, specifically manifested as rapid degradation of plasma fibrin clots and whole blood clots. In both experiments, the compounds of this invention effectively inhibited the fibrinolytic process and prolonged the clot degradation time (CLT), exhibiting excellent coagulation and hemostatic activity. The pharmacological activity and safety of the compounds of this invention are significantly superior to tranexamic acid, the most widely used hemostatic drug in clinical practice. Furthermore, the compounds are easy to prepare, facilitating large-scale industrial production and effectively reducing drug costs, thus possessing significant clinical application value. Detailed Implementation

[0064] The following examples illustrate the synthesis methods of the compounds and intermediates of this invention. These examples are merely illustrative and should not be construed as limiting the scope of the invention. Unless otherwise specified, the raw materials and reagents involved in this invention are commercially available, and the specific source does not affect the implementation of the technical solution of this invention.

[0065] Example 1: Preparation of (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0066]

[0067] Step 1: Preparation of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthidine-6-(5H)-carboxylate

[0068]

[0069] 0.9 g of 2-chloro-5,6,7,8-tetrahydro-1,6-naphthylidine hydrochloride was suspended in 15 mL of dichloromethane. 1.4 g of N,N-diisopropylethylamine was added, followed by 1.15 g of ditert-butyl dicarbonate. The reaction was carried out at room temperature for 1 h. TLC showed that the starting material was completely consumed. The reaction mixture was diluted with water, extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain the target compound (1.12 g).

[0070] MS(ESI)m / z(M+H) + =269.0.

[0071] Step 2: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthylpyridine-6(5H)-carboxylate

[0072]

[0073] In an argon atmosphere, tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate (100 mg) was dissolved in toluene (20 mL), followed by the addition of diethyl phosphite (102 mg), tris(dibenzylacetone)dipalladium (34 mg), 1,1'-bis(diphenylphosphine)ferrocene (41 mg), and triethylamine (75 mg). The mixture was reacted overnight at 120 °C. TLC showed that the starting material was completely consumed. The mixture was diluted with ethyl acetate, filtered through diatomaceous earth, and the filtrate was collected and concentrated. The crude product was purified by preparative TLC to obtain the target compound (70 mg).

[0074] MS(ESI)m / z(M+H) + =371.1.

[0075] Step 3: Preparation of (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0076]

[0077] 70 mg of tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthylpyridine-6(5H)-carboxylic acid was dissolved in 5 mL of concentrated hydrochloric acid and reacted overnight at 100 °C. LC-MS showed that the starting material was completely consumed. The reaction solution was concentrated, and the crude product was purified by pre-HPLC to obtain the target compound (30 mg).

[0078] MS(ESI)m / z(M+H) + =215.0.

[0079] 1 H NMR (400MHz, Deuterium Oxide) δ8.35(dd,J=8.0,2.4Hz,1H),8.06(t,J=7.7Hz,1H),4.59(s,2H),3.67(t,J=6.0Hz,2H),3.49(t,J=6.4Hz,2H).

[0080] Example 2: Preparation of (3-fluoro-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0081]

[0082] Step 1: Preparation of 2-chloro-5-fluoro-6-((4-methoxybenzyl)oxy)nicotinonitrile

[0083]

[0084] 4-Methoxybenzyl alcohol (3.95 g) was dissolved in tetrahydrofuran (50 mL), and the mixture was cooled to -78 °C and stirred. Potassium tert-butoxide (3.5 g) was added under nitrogen protection, and the mixture was moved to 0 °C and reacted for 0.5 hours. The temperature was further lowered to -78 °C, and a tetrahydrofuran solution (50 mL) containing 2,6-dichloro-5-fluoronicotinonitrile (5.0 g) was added dropwise. After the addition was complete, the mixture was moved to room temperature and reacted overnight. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, ethyl acetate and water were added, and the mixture was extracted separately. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give the title compound (6.2 g).

[0085] MS(ESI)m / z(M+H) + =293.1

[0086] Step 2: Preparation of 5-fluoro-6-((4-methoxybenzyl)oxy)-2-vinylnicotinonitrile

[0087]

[0088] Under a nitrogen atmosphere, 2-chloro-5-fluoro-6-((4-methoxybenzyl)oxy)nicotinonitrile (6.0 g), potassium vinyltrifluoroborate (5.5 g), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (0.29 g), and cesium fluoride (6.23 g) were dissolved in 1,4-dioxane (60 mL) and water (6 mL). The system was reacted overnight at 90 °C, and TLC showed that the reaction was essentially complete. The system was concentrated under reduced pressure, ethyl acetate and water were added, and the mixture was extracted separately. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give the title compound (2.68 g).

[0089] MS(ESI)m / z(M+H) + =285.1.

[0090] Step 3: Preparation of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-7,8-dihydro-1,6-naphthidium-5(6H)-one

[0091]

[0092] 5-Fluoro-6-((4-methoxybenzyl)oxy)-2-vinylnicotinonitrile (2.68 g) was dissolved in methanol (20 mL) and water (4 mL), and benzylamine (12.44 g) was added. The reaction was carried out overnight at 100 °C, and TLC showed that the reaction was essentially complete. The mixture was concentrated under reduced pressure, and dichloromethane and water were added. The mixture was extracted separately, and the organic phase was washed with 1 M dilute hydrochloric acid. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give the title compound (2.37 g).

[0093] MS(ESI)m / z(M+H) + =393.1.

[0094] Step 4: Preparation of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-5,6,7,8-tetrahydro-1,6-naphthidine

[0095]

[0096] Under ice-water bath conditions, 1.19 g of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-7,8-dihydro-1,6-naphthidium-5(6H)-one was dissolved in 20 mL of tetrahydrofuran. Lithium aluminum hydride (0.29 g) was added in portions, and the mixture was heated to 70 °C for 4 hours. TLC showed that the reaction was essentially complete. Under ice-water bath conditions, 0.5 mL of water, 0.5 mL of 15% sodium hydroxide aqueous solution, and 1.5 mL of water were added dropwise. After stirring at room temperature for 15 minutes, anhydrous magnesium sulfate was added and stirred for another 15 minutes. The mixture was filtered through diatomaceous earth and anhydrous sodium sulfate. The residue was washed with ethyl acetate, and the filtrate was concentrated to give the title compound (1.15 g).

[0097] MS(ESI)m / z(M+H) + =379.1.

[0098] Step 5: Preparation of 6-benzyl-2-chloro-3-fluoro-5,6,7,8-tetrahydro-1,6-naphthidine

[0099]

[0100] Under ice-water bath conditions, 1.14 g of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-5,6,7,8-tetrahydro-1,6-naphthidine was dissolved in 10 mL of phosphorus oxychloride and reacted overnight at 100 °C. TLC showed that the reaction was essentially complete. The solution was concentrated under reduced pressure, diluted with ethyl acetate, and added dropwise to crushed ice. The pH was adjusted to approximately 10 with saturated sodium carbonate solution. The mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 0.82 g of crude product.

[0101] MS(ESI)m / z(M+H) + =277.1.

[0102] Step 6: Preparation of 2-chloro-3-fluoro-5,6,7,8-tetrahydro-1,6-naphthidine

[0103]

[0104] Under ice-water bath conditions, 0.82 g of 6-benzyl-2-chloro-3-fluoro-5,6,7,8-tetrahydro-1,6-naphthidine was dissolved in 8 mL of 1,2-dichloroethane. N,N-diisopropylethylamine (1.93 g) and 2.57 g of 1-chloroethyl chloroformate were added sequentially, and the reaction was carried out at 80 °C for 1.5 h. TLC showed that the reaction was essentially complete. The system was concentrated, dissolved in methanol, and reacted at 60 °C for 1.5 h. TLC showed that the reaction was essentially complete. The system was concentrated under reduced pressure to obtain a crude product, which was used directly for the next step.

[0105] MS(ESI)m / z(M+H)+ =187.1.

[0106] Step 7: Preparation of tert-butyl 2-chloro-3-fluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0107]

[0108] Under ice-water bath conditions, 2-chloro-3-fluoro-5,6,7,8-tetrahydro-1,6-naphthidine (the crude product above) was dissolved in dichloromethane (10 mL), and triethylamine (0.91 g) and di-tert-butyl dicarbonate (0.98 g) were added. The reaction was carried out at room temperature for 2 hours, and TLC showed that the reaction was essentially complete. Dichloromethane and water were added, and the mixture was extracted separately. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give the title compound (0.28 g).

[0109] MS(ESI)m / z(M+H) + =287.1.

[0110] Step 8: Preparation of tert-butyl 2-(diethoxyphosphoryl)-3-fluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0111]

[0112] Under a nitrogen atmosphere, tert-butyl 2-chloro-3-fluoro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate (100 mg) was dissolved in toluene (8 mL), followed by the addition of diethyl phosphite (97 mg), palladium acetate (16 mg), 1,1'-bis(diphenylphosphine)ferrocene (78 mg), and triethylamine (71 mg). The reaction was carried out overnight at 110 °C. TLC showed that the reaction was essentially complete. The system was concentrated under reduced pressure, and the crude product was purified by column chromatography to give the title compound (130 mg).

[0113] MS(ESI)m / z(M+H) + =389.1.

[0114] Step 9: Preparation of (3-fluoro-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0115]

[0116] 60 mg of tert-butyl 2-(diethoxyphosphoryl)-3-fluoro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylic acid was dissolved in concentrated hydrochloric acid (3 mL), and the mixture was heated to 100 °C and reacted under sealed conditions for 2 hours. TLC showed that the reaction was essentially complete. The system was concentrated, and the crude product was purified by pre-HPLC to obtain the title compound (30 mg).

[0117] MS(ESI)m / z(M+H) + =233.0.

[0118] 1 H NMR (400MHz, Deuterium Oxide) δ7.77 (d, J = 7.1Hz, 1H), 4.48 (s, 2H), 3.58 (s, 2H), 3.24 (s, 2H).

[0119] Example 3: Preparation of ethyl (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt

[0120]

[0121] Step 1: Preparation of (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonic acid

[0122] Weigh 2 g of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate and 4.8 g of aniline phosphite, dissolve them in acetonitrile (40 mL), then add tris(2,3-dibenzylacetone)palladium (680 mg), diphenylphosphine ferrocene (830 mg), and triethylamine (5.2 mL). After purging with nitrogen, react at 85 °C overnight, then at 95 °C for 4 h. LC-MS monitoring showed no reactants remaining. Cool to room temperature, adjust pH to 3 with 2 M dilute hydrochloric acid, evaporate to dryness, and purify the residue by reverse-phase column chromatography to obtain the title compound (1.6 g).

[0123]

[0124] MS(ESI)m / z(M+H) + =299.1.

[0125] Step 2: Preparation of methyl 6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,6-naphthidium-2-yl)phosphinic acid

[0126] Weigh 400 mg of (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonic acid, dissolve it in 10 mL of dichloromethane, and add 0.2 mL of methyl chloroformate at room temperature under nitrogen protection. Then, add 0.2 mL of pyridine dropwise. React at 45 °C for 1 h, and monitor the reaction until complete by TLC. Cool to room temperature, quench with water, and extract three times with dichloromethane. Combine the organic phases, dry to anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and column purify the residue to give the title compound (300 mg).

[0127]

[0128] MS(ESI)m / z(M+H) + =313.1.

[0129] Step 3: Preparation of tert-butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate

[0130] 50 mg of methyl phosphonate (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate was weighed and dissolved in dry tetrahydrofuran (2 mL). Under nitrogen protection, 0.19 mL of hexamethyldisilamide lithium (1 M in THF) was added at -78 °C, and the reaction was carried out at -78 °C for 20 min. Iodoethane (17 μL) was added dropwise, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by LC-MS to ensure complete reaction. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give the title compound (20 mg).

[0131]

[0132] MS(ESI)m / z(M+H) + =341.1.

[0133] Step 4: Preparation of ethyl (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt

[0134] Weigh 20 mg of tert-butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylic acid, add 6 M hydrochloric acid, and react overnight at 105 °C. The reaction was monitored by LC-MS until complete. After cooling to room temperature and removing the solvent under reduced pressure, the residue was purified by pre-HPLC column to obtain the title compound (10 mg).

[0135]

[0136] MS(ESI)m / z(M+H) + =227.1.

[0137] 1 H NMR(400MHz, Deuterium Oxide)δ7.63-7.62(m,2H),4.36(s,2H),3.57-3.53(t,J=6.5Hz,2H),3.18-3.15(t,J =6.4Hz,2H),1.74-1.65(dq,J=15.3,7.7Hz,2H),0.85-0.76(dt,J=18.6,7.9Hz,3H).

[0138] Example 4: Preparation of phenethyl (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt

[0139]

[0140] Step 1: Preparation of tert-butyl 2-(methoxy(styryl)phosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0141] Weigh 50 mg of tert-butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate and 31 μL of (2-bromovinyl)benzene, dissolve them in toluene (3 mL), then add tris(dibenzylacetone)palladium (15 mg), diphenylphosphine ferrocene (18 mg), and triethylamine (5.2 mL). After purging with nitrogen, react at 120 °C for 6 h. LC-MS monitoring showed no residual starting material. Cool to room temperature, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain the title compound (35 mg).

[0142]

[0143] MS(ESI)m / z(M+H) + =415.1.

[0144] Step 2: Preparation of tert-butyl 2-(methoxy(phenethyl)phosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0145] Weigh 35 mg of tert-butyl 2-(methoxy(styryl)phosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in ethanol (3 mL), add 20 mg of 10% palladium on carbon (55% water), and react overnight at 60 °C under a hydrogen atmosphere. The reaction was monitored by LCMS until complete. After cooling to room temperature, filter, and concentrate the filtrate to obtain the title compound (40 mg crude product).

[0146]

[0147] MS(ESI)m / z(M+H) + =417.1.

[0148] Step 3: Preparation of phenethyl (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt

[0149] Weigh 40 mg of crude tert-butyl 2-(methoxy(phenylethyl)phosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylic acid, add 3 mL of concentrated hydrochloric acid, and react at 100 °C for 3 h. The reaction was monitored by LC-MS until complete. After cooling to room temperature and removing the solvent under reduced pressure, the residue was purified by pre-HPLC to give the title compound (11.7 mg).

[0150]

[0151] MS(ESI)m / z(MH) - =301.0.

[0152] 1 H NMR(400MHz, Deuterium Oxide)δ7.57-7.50(m,2H),7.05-7.01(m,3H),6.95-6.93(dd,J=7.5,2.1Hz,2H),4.30(s,2H),3.49-3.46(t,J= 6.4Hz,2H),2.98-2.95(t,J=6.4Hz,2H),2.67-2.59(dt,J=15.1,7.6Hz,2H),2.14-2.07(dt,J=15.2,7.6Hz,2H).

[0153] Example 5: (8,8-difluoro-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0154]

[0155] Step 1: Preparation of tert-butyl 2-chloro-8,8-difluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0156] 0.3 g of tert-butyl 2-chloro-8-oxo-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in 4 mL of dichloromethane, and 342 mg of diethylaminosulfur trifluoride was added dropwise under ice bath conditions. The reaction was carried out under ice bath conditions for 1 hour. After the reaction was completed, water was added to the system, and the mixture was washed three times with dichloromethane. The organic phase was dried, concentrated to dryness, and purified by column chromatography to give the title compound (225 mg).

[0157]

[0158] MS(ESI)m / z(M+H) + =304.0.

[0159] Step 2: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-8,8-difluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0160] 100 mg of tert-butyl 2-chloro-8,8-difluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate was weighed into a dry reaction flask and dissolved in toluene (10 mL). 54 mg of 1,1′-ferrocene diyl-bis(diphenylphosphine)palladium dichloromethane dichloride complex, 0.09 mL of triethylamine, and 128 mg of di-tert-butyl phosphonate were added. The system was purged with nitrogen three times and heated to 100 °C. The reaction was allowed to proceed overnight. LC-MS was used to confirm the complete reaction, and the system was concentrated to dryness. The title compound (100 mg) was purified by column chromatography.

[0161]

[0162] MS(ESI)m / z(M+H) + =463.2.

[0163] Step 3: Preparation of (8,8-difluoro-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0164] Weigh 100 mg of tert-butyl 2-(di-tert-butoxyphosphoryl)-8,8-difluoro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in 3 mL of dichloromethane, add 3 mL of 4 M hydrochloric acid-1,4-dioxane solution, stir at room temperature for 1 hour, and check the reaction is complete by LC-MS. Remove the solvent under reduced pressure. The title compound (20 mg) is obtained by pre-HPLC purification.

[0165]

[0166] MS(ESI)m / z(M+H) + =250.9.

[0167] 1 H NMR (400MHz, Deuterium Oxide) δ7.89 (ddd, J=23.5, 8.2, 4.8Hz, 2H), 4.57 (s, 2H), 4.08 (t, J=11.6Hz, 2H).

[0168] Example 6: Preparation of (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonic acid monohydrate

[0169]

[0170] (5,6,7,8-Tetrahydro-1,6-naphthid-2-yl)phosphonate salt was dissolved in 5 times its volume of water, and then the pH was adjusted to about 4.2 with 10% sodium hydroxide. The solid precipitated, and the mixture was filtered and dried to obtain the title compound.

[0171] MS(ESI)m / z(M+H) + =215.0.

[0172] 1 H NMR (400MHz, Methanol-d4) δ7.72(dd,J=8.0,5.6Hz,1H),7.37(dd J=8.0,3.6Hz,1H),3.98(s,2H),3.19(t,J=6.0Hz,2H),2.98(t,J=6.0Hz,2H).

[0173] Example 7: Preparation of (8-morpholino-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0174]

[0175] Step 1: Preparation of tert-butyl 2-chloro-8-((methanesulfonyl)oxy)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0176] 0.14 g of tert-butyl 2-chloro-8-hydroxy-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in 10 mL of dichloromethane, followed by the addition of 0.15 g of triethylamine and 0.11 g of methanesulfonyl chloride. The reaction was allowed to proceed at room temperature for 1 hour, and TLC showed that the reaction was essentially complete. The reaction was quenched with 10 mL of water, separated, and the organic phase was collected. The organic phase was concentrated to dryness under reduced pressure using anhydrous sodium sulfate and purified by column chromatography to give the title compound (0.16 g).

[0177]

[0178] MS(ESI)m / z(M+H) + =363.1.

[0179] Step 2: Preparation of tert-butyl 2-chloro-8-morpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0180] 0.16 g of tert-butyl 2-chloro-8-((methanesulfonyl)oxy)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in acetonitrile (3 mL) and N,N-dimethylformamide (3 mL). Potassium carbonate (0.12 g) and morpholine (0.12 g) were added, and the mixture was purged with nitrogen three times. The reaction was carried out overnight at 60 °C, and the reaction was monitored by LC-MS until complete. The acetonitrile was removed by concentration, and water (10 mL) and ethyl acetate (10 mL) were added. The mixture was extracted and separated, and the organic phase was dried and concentrated. The crude product was purified by column chromatography to give the title compound (0.11 g).

[0181]

[0182] MS(ESI)m / z(M+H) + =354.1.

[0183] Step 3: Preparation of tert-butyl 2-(diethoxyphosphoryl)-8-morpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0184] Weigh 0.11 g of tert-butyl 2-chloro-8-morpholine-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate and dissolve it in toluene (3 mL). Then, add diethyl phosphite (86 mg), tris(dibenzylacetone)dipalladium (57 mg), 1,1'-bis(diphenylphosphine)ferrocene (69 mg), and triethylamine (62 mg) sequentially. The mixture was purged three times with nitrogen and reacted overnight at 120 °C under a nitrogen atmosphere. LC-MS showed that the reaction was essentially complete.

[0185] The solution was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.12 g).

[0186]

[0187] MS(ESI)m / z(M+H) + =456.2.

[0188] Step 4: Preparation of (8-morpholino-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0189] Weigh 60 mg of tert-butyl 2-(diethoxyphosphoryl)-8-morpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylic acid, add 4 mL of concentrated hydrochloric acid, and react at 100 °C for 2 hours under sealed conditions. TLC showed that the reaction was essentially complete. The system was concentrated to dryness and separated by pre-HPLC to obtain the title compound (11.7 mg).

[0190]

[0191] MS(ESI)m / z(M+H) + =300.1.

[0192] 1 H NMR(400MHz, Deuterium Oxide)δ7.70(m,2H),5.14(dd,J=10.5,6.3Hz,1H),4.56-4.43(m,2H),4.19(dd ,J=12.7,6.2Hz,1H),3.98(s,4H),3.90-3.81(m,1H),3.40(s,2H),3.27(s,2H).

[0193] Example 8: Preparation of (8-hydroxy-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0194]

[0195] Step 1: Preparation of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-chloro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0196] 0.16 g of tert-butyl 2-chloro-8-hydroxy-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate was dissolved in 10 mL of dichloromethane, and then 92 mg of 1-methyl-1H-imidazolium and 0.12 g of tert-butyldimethylchlorosilane were added sequentially. The reaction was carried out at room temperature for 2 hours, and TLC showed that the reaction was essentially complete. The solution was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.20 g).

[0197]

[0198] MS(ESI)m / z(M+H) + =399.2.

[0199] Step 2: Preparation of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0200] 0.20 g of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-chloro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in toluene (10 mL), and diethyl phosphite (138 mg), tris(dibenzylacetone)dipalladium (92 mg), 1,1'-bis(diphenylphosphine)ferrocene (110 mg), and triethylamine (101 mg) were added sequentially. The mixture was purged three times with nitrogen and reacted overnight at 110 °C under a nitrogen atmosphere. LC-MS showed that the reaction was essentially complete. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.16 g).

[0201]

[0202] MS(ESI)m / z(M+H) + =501.3.

[0203] Step 3: Preparation of (8-hydroxy-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0204] Weigh 70 mg of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylic acid, add 4 mL of concentrated hydrochloric acid, and react at 100 °C for 2 hours under sealed conditions. TLC showed that the reaction was basically complete.

[0205] The system was concentrated to dryness and separated by pre-HPLC to obtain the title compound (2.4 mg).

[0206]

[0207] MS(ESI)m / z(M+H) + =231.0.

[0208] 1 ¹H NMR (400 MHz, Deuterium Oxide) δ 7.71 (s, 2H), 5.00 (s, 1H), 4.40 (s, 2H), 3.59 (s, 2H). Example 9: Preparation of (8-(2-hydroxyethoxy)-5,6,7,8-tetrahydro-1,6-naphthidium-2-yl)phosphonate

[0209]

[0210] Step 1: Preparation of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-chloro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0211] 0.60 g of tert-butyl 2-chloro-8-hydroxy-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate was dissolved in 10 mL of tetrahydrofuran. NaH (1.0 g) was added in an ice bath, and the mixture was stirred for 5 minutes. Then, (2-bromoethoxy)(tert-butyl)dimethylsilane was added, and the mixture was reacted at room temperature for 2 hours. TLC showed that the reaction was essentially complete. The reaction was quenched with ice water, and the mixture was extracted with ethyl acetate and water. The organic phase was collected, dried, concentrated to dryness under reduced pressure, and purified by column chromatography to give the title compound (0.40 g).

[0212]

[0213] MS(ESI)m / z(M+H) + =443.2.

[0214] Step 2: Preparation of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0215] 0.40 g of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-chloro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in toluene (10 mL). Diethyl phosphite (0.25 g), tris(dibenzylacetone)dipalladium (0.17 g), 1,1'-bis(diphenylphosphine)ferrocene (0.20 g), and triethylamine (0.18 g) were added sequentially. The mixture was purged three times with nitrogen and reacted overnight at 110 °C under a nitrogen atmosphere. LC-MS showed that the reaction was essentially complete. The solution was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.44 g).

[0216]

[0217] MS(ESI)m / z(M+H) + =545.3.

[0218] Step 3: Preparation of (8-(2-hydroxyethoxy)-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0219] Weigh 70 mg of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylic acid and dissolve it in 5 mL of 1,4-dioxane. Add 0.2 mL of 1 M trimethylbromosilane and reflux overnight at 75 °C. The reaction was essentially complete as monitored by LCMS. Concentrate the system to dryness and dissolve it in a 1,4-dioxane solution of 4 M hydrochloric acid. React at room temperature for 2 hours. The reaction was complete as monitored by LCMS. Concentrate the system to dryness and separate the crude product by pre-HPLC to obtain the title compound (30.0 mg).

[0220]

[0221] MS(ESI)m / z(M+H) + =275.0.

[0222] 1 H NMR(400MHz, Deuterium Oxide)δ7.79(d,J=5.0Hz,2H),4.77(s,1H),4.43(q,J=16.5Hz,2H),3.93(d,J=13.5Hz,1 H),3.84–3.78(m,1H),3.77–3.70(m,1H),3.63(t,J=4.4Hz,2H),3.49(d,J=13.6Hz,1H).

[0223] Example 10: Preparation of (8-thiomorpholine-5,6,7,8-tetrahydro-1,6-naphthidin-2-yl)phosphonate

[0224]

[0225] Step 1: Preparation of tert-butyl 2-chloro-8-thiomorpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0226] 0.30 g of tert-butyl 2-chloro-8-((methanesulfonyl)oxy)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in acetonitrile (5 mL) and N,N-dimethylformamide (5 mL). Potassium carbonate (0.12 g) and thiomorpholine (0.12 g) were added, and the mixture was purged with nitrogen three times. The reaction was carried out overnight at 60 °C, and the reaction was monitored by LC-MS until complete. The acetonitrile was removed by concentration, and water (10 mL) and ethyl acetate (10 mL) were added. The mixture was extracted and separated, and the organic phase was dried and concentrated. The crude product was purified by column chromatography to give the title compound (0.12 g).

[0227]

[0228] MS(ESI)m / z(M+H) + =370.1.

[0229] Step 2: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-8-thiomorpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0230] 0.23 g of tert-butyl 2-chloro-8-thiomorpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate was dissolved in toluene (10 mL), followed by the addition of 242 mg of ditert-butyl phosphite, 114 mg of tris(dibenzylacetone)dipalladium, 138 mg of 1,1'-bis(diphenylphosphine)ferrocene, and 130 mg of triethylamine. The mixture was purged three times with nitrogen and reacted overnight at 120 °C under a nitrogen atmosphere. LC-MS showed that the reaction was essentially complete. The solution was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.24 g).

[0231]

[0232] MS(ESI)m / z(M+H) + =528.2.

[0233] Step 3: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-8-(1-thiomorpholine oxide)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0234] 90 mg of 2-(di-tert-butoxyphosphoryl)-8-thiomorpholine-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylic acid tert-butyl ester was weighed and dissolved in acetic acid (2 mL). Urea peroxide (242 mg) was added, and the reaction was carried out at room temperature for 2 hours. LC-MS showed that the reaction was essentially complete. Water (50 mL) and ethyl acetate (10 mL x 3 times) were added, and the mixture was extracted and separated. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and purified by column chromatography to give the title compound (60 mg).

[0235]

[0236] MS(ESI)m / z(M+H) + =544.2.

[0237] Step 4: Preparation of (8-(1-thiomorpholine)-5,6,7,8-tetrahydro-1,6-naphthidin-2-yl)phosphonate

[0238] Weigh 60 mg of tert-butyl 2-(di-tert-butoxyphosphoryl)-8-(1-thiomorpholine oxide)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in 4 mL of dichloromethane, add 0.1 mL of concentrated hydrochloric acid under ice bath conditions, and react under ice bath conditions for 0.5 hours. TLC showed that the reaction was essentially complete. The system was concentrated to dryness and separated by pre-HPLC to obtain the title compound (2.94 mg).

[0239]

[0240] MS(ESI)m / z(M+H) + =332.0.

[0241] 1 H NMR(400MHz, Deuterium Oxide)δ7.96(m,1H),7.88(m,1H),5.09(dd,J=10.8,6.0Hz,1H),4.61–4.49(m,2H),4.20(dd ,J=12.7,6.0Hz,1H),3.88(m,2H),3.68–3.58(m,2H),3.37–3.23(m,3H),3.18–3.02(m,2H).

[0242] Example 11: Preparation of (7-isobutyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0243]

[0244] Step 1: Preparation of tert-butyl 2-chloro-7-(2-methylprop-1-en-1-yl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0245] Isopropyltriphenyliodide (2.918 g) was dissolved in N,N-dimethylformamide (10 mL), sodium hydride (0.27 g) was added, the mixture was purged with nitrogen three times, and reacted at 0 °C for 20 min. Then, tert-butyl 2-chloro-7-formyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate (1 g) was added. The reaction was allowed to proceed for 3 hours. The reaction was monitored by LC-MS until completion. The system was quenched with saturated ammonium chloride solution, extracted three times with ethyl acetate, dried over the organic phase, concentrated to dryness, and purified by column chromatography to obtain the title compound (280 mg).

[0246]

[0247] MS(ESI)m / z(M+H) + =323.1.

[0248] Step 2: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7-(2-methylprop-1-en-1-yl)-7,8-dihydro-1,6-naphthidine-6(5H)-formate

[0249] Weigh 280 mg of tert-butyl 2-chloro-7-(2-methylprop-1-en-1-yl)-7,8-dihydro-1,6-naphthidium-6(5H)-carboxylate into a dry reaction flask, dissolve it in toluene (10 mL), and add tris(dibenzylindeneacetone)dipalladium (124.5 mg), 1,1′-ferrocenediyl-bis(diphenylphosphine) (192.7 mg), triethylamine (0.024 mL), and diethyl phosphonate (240 mg). Purge the system three times with nitrogen and heat to 110 °C. React for 4 hours. LC-MS analysis confirmed the reaction was complete, and the system was concentrated to dryness. Column chromatography purified the title compound (300 mg).

[0250]

[0251] MS(ESI)m / z(M+H) + =425.2.

[0252] Step 3: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7-isobutyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0253] Weigh 300 mg of tert-butyl 2-(diethoxyphosphoryl)-7-(2-methylprop-1-en-1-yl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate and dissolve it in 15 mL of methanol. In a high-pressure autoclave, hydrogen gas is introduced, and the mixture is heated to 40 °C and stirred overnight. The reaction is confirmed to be complete by LC-MS. The mixture is filtered through diatomaceous earth, and the solvent is removed under reduced pressure. Column chromatography is used to purify the compound to give 90 mg of the title compound.

[0254]

[0255] MS(ESI)m / z(M+H) + =427.2.

[0256] Step 4: Preparation of (7-isobutyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0257] Weigh 90 mg of tert-butyl 2-(diethoxyphosphoryl)-7-isobutyl-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate and dissolve it in 5 mL of 12 M hydrochloric acid solution. Heat to 100 °C and stir for 3 hours. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation. The title compound (50 mg) was obtained by pre-HPLC purification.

[0258]

[0259] MS(ESI)m / z(M+H) + =271.1.

[0260] 1 H NMR(400MHz, Deuterium Oxide)δ7.71(dd,J=7.9,3.7Hz,1H),7.64(t,J=7.0Hz,1H),4.39(s,2H),3.70(q,J=10.7,7.9Hz,1H),3.32(dd,J=18.4,4. 7Hz, 1H), 2.94 (dd, J=18.1, 10.7Hz, 1H), 1.75 (dt, J=13.4, 6.8Hz, 1H), 1.59 (t, J=7.2Hz, 2H), 0.85 (dd, J=12.3, 6.4Hz, 6H).

[0261] Example 12: Preparation of (7-propyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0262]

[0263] Step 1: Preparation of 2-methyl-N-(4-butylene)propane-2-sulfinamide

[0264] Butyraldehyde (10 g) was dissolved in dichloromethane (100 mL), and 2-methylpropane-2-sulfinylamine (20 g), anhydrous magnesium sulfate (83.3 g), and pyridine 4-methylbenzenesulfonic acid (1.74 g) were added sequentially. The mixture was heated to 40 °C and reacted for 24 hours. The reaction was monitored by LC-MS until completion. After cooling to room temperature, the mixture was filtered, the filter cake was washed with dichloromethane, and the filtrate was concentrated to dryness. The filtrate was purified by column chromatography to give the title compound (22.4 g).

[0265]

[0266] MS(ESI)m / z(M+H) + =176.1

[0267] Step 2: Preparation of N-(1-(3-bromo-6-methoxypyridin-2-yl)-5-butane-2-yl)-2-methylpropane-2-sulfinamide

[0268] 10 g of 3-bromo-6-methoxy-2-methylpyridine was weighed into a dry reaction flask, and 80 mL of anhydrous tetrahydrofuran was injected under a nitrogen atmosphere. The mixture was then cooled to -78 °C. A tetrahydrofuran solution of lithium diisopropylamino in 27.2 mL (2.0 M) was added dropwise, and the mixture was reacted at -78 °C for 40 min. 20 mL of tetrahydrofuran containing 9.53 g of 2-methyl-N-(4-butylene)propane-2-sulfinamide was added dropwise, and the mixture was reacted at -30 °C for 30 min, then slowly brought to room temperature. The reaction was confirmed to be complete by LC-MS. The reaction was quenched with saturated ammonium chloride solution, and ethyl acetate and water were added. The mixture was separated, extracted, and the organic phase was concentrated to dryness. The title compound (6.4 g) was purified by column chromatography.

[0269]

[0270] MS(ESI)m / z(M+H) + =377.1.

[0271] Step 3: Preparation of ethyl 2-(2-((tert-butylsulfinyl)amino)-5-butyl)-6-methoxynicotinic acid

[0272] Weigh out 6.4 g of N-(1-(3-bromo-6-methoxypyridin-2-yl)-5-butane-2-yl)-2-methylpropane-2-sulfinamide and dissolve it in 80 mL of ethanol. Add 2.48 g of [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride and 5.6 mL of N,N-diisopropylethylamine. After the addition is complete, the system is purged with carbon monoxide and heated to 100 °C in a carbon monoxide atmosphere and stirred for 24 hours. The reaction is confirmed to be complete by LCMS. The solvent is removed under reduced pressure. Column chromatography is used to purify the compound to give 4 g of the title compound.

[0273]

[0274] MS(ESI)m / z(M+H) + =371.1.

[0275] Step 4: Preparation of 2-methoxy-7-(3-propyl)-7,8-dihydro-1,6-naphthidium-5(6H)-one

[0276] 4 g of ethyl 2-(2-((tert-butylsulfinyl)amino)-5-butyl)-6-methoxynicotinic acid was dissolved in acetonitrile (100 mL), and 17.6 g of cesium carbonate was added. The mixture was heated to 80 °C and stirred overnight. The reaction was monitored by LC-MS until completion. After cooling to room temperature, the mixture was filtered, the filter cake was washed with dichloromethane, and the filtrate was concentrated to dryness. The filtrate was purified by column chromatography to give the title compound (2.55 g).

[0277]

[0278] MS(ESI)m / z(M+H) + =221.1.

[0279] Step 5: Preparation of 2-methoxy-7-(3-propyl)-5,6,7,8-tetrahydro-1,6-naphthidine

[0280] Weigh 2.55 g of 2-methoxy-7-(3-propyl)-7,8-dihydro-1,6-naphthidium-5(6H)-one and dissolve it in 100 mL of tetrahydrofuran. Under ice bath conditions, add 2.6 g of lithium aluminum hydride and stir at 70 °C for 8 hours. Monitor the reaction completion by LC-MS. Under ice bath conditions, add 2.6 mL of water, 2.6 mL of 15% sodium hydroxide solution, and 7.8 mL of water sequentially. After the additions are complete, stir at room temperature for 20 minutes. Dry the mixture on anhydrous magnesium sulfate, filter, wash the filter cake with dichloromethane, and concentrate to dryness under reduced pressure. Purify by column chromatography to give the title compound (1.9 g).

[0281]

[0282] MS(ESI)m / z(M+H) + =207.1.

[0283] Step 6: Preparation of 7-(3-propyl)-5,6,7,8-tetrahydro-1,6-naphthyl-2-ol

[0284] Weigh 1.9 g of 2-methoxy-7-(3-propyl)-5,6,7,8-tetrahydro-1,6-naphthidine, add 5 mL of hydrobromic acid in acetic acid solution, heat to 80 °C and stir for 5 hours. Monitor the reaction completion by LC-MS. Remove the solvent under reduced pressure, add ethyl acetate and slurry, filter, and dry to obtain the crude product of the title compound (1.5 g).

[0285]

[0286] MS(ESI)m / z(M+H) + =193.1.

[0287] Step 7: Preparation of 2-chloro-7-(3-propyl)-5,6,7,8-tetrahydro-1,6-naphthidine

[0288] Weigh 0.5 g of 7-(3-propyl)-5,6,7,8-tetrahydro-1,6-naphthidine-2-ol, add 10 mL of phosphorus oxychloride, heat to 100 °C and stir for 4 hours. Monitor the reaction completion by LC-MS. Remove the solvent under reduced pressure, add ice water and dichloromethane to obtain the crude product of the title compound.

[0289]

[0290] MS(ESI)m / z(M+H) + =211.1.

[0291] Step 8: Preparation of tert-butyl 2-chloro-7-(3-propyl)-7,8-dihydro-1,6-naphthyl-6-(5H)-carboxylate

[0292] Add 1.29 mL of ditert-butyl dicarbonate to the post-treatment system of step 7, using sodium carbonate solution at pH 8-9.

[0293] The mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LC-MS until completion. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography to obtain the title compound (0.5 g).

[0294]

[0295] MS(ESI)m / z(M+H) + =311.1.

[0296] Step 9: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-7-propyl-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate

[0297] 93 mg of tert-butyl 2-chloro-7-(3-propyl)-7,8-dihydro-1,6-naphthyl-6-(5H)-carboxylate was weighed into a dry reaction flask and dissolved in toluene (10 mL). Tris(dibenzylacetone)dipalladium (55 mg), 1,1'-bis(diphenylphosphine)ferrocene (67 mg), triethylamine (61 mg), and di-tert-butyl phosphonate (120 mg) were added. The system was purged with nitrogen three times and heated to 115 °C. The reaction was allowed to proceed overnight. LC-MS was used to confirm the completeness of the reaction, and the system was concentrated to dryness. Column chromatography was used to purify the title compound (105 mg).

[0298]

[0299] MS(ESI)m / z(M+H) + =469.2.

[0300] Step 10: Preparation of (7-propyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0301] Weigh 105 mg of tert-butyl 2-(di-tert-butoxyphosphoryl)-7-propyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in 4 mL of dichloromethane, add dropwise 4 mL of 1,4-dioxane hydrochloric acid solution, stir at room temperature for 1 hour, and check the reaction is complete by LC-MS. Remove the solvent under reduced pressure. The title compound (8 mg) is obtained by pre-HPLC purification.

[0302]

[0303] MS(ESI)m / z(M+H) + =257.0.

[0304] 1 H NMR(400MHz, Deuterium Oxide)δ7.75(dd,J=8.0,3.8Hz,1H),7.68(dd,J=7.9,6.2Hz,1H),4.43(s,2H),3.67(ddd,J=10.9,5.3,2.1Hz,1H),3.34(dd,J= 18.2,4.8Hz,1H),3.01(dd,J=18.2,10.8Hz,1H),1.74(dtd,J=14.7,8.5,6.7Hz,2H),1.52–1.35(m,2H),0.88(t,J=7.3Hz,3H).

[0305] Example 13 Preparation of (7-phenylethyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl) phosphonate

[0306]

[0307] Step 1: Preparation of 2-methyl-N-(3-phenylpropylidene)propane-2-sulfinamide

[0308] 10.73 g of 3-phenylpropanal was dissolved in 120 mL of dichloromethane. Magnesium sulfate (41.2 g), pyridine 4-methylbenzenesulfonic acid (1.0 g), and 2-methylpropane-2-sulfinamide (10.7 g) were added. The mixture was purged with nitrogen three times, and the reaction was refluxed overnight. The reaction was monitored by LC-MS until complete. The system was filtered, washed three times with ethyl acetate, dried over the organic phase, concentrated to dryness, and purified by column chromatography to give the title compound (12.44 g).

[0309]

[0310] MS(ESI)m / z(M+H) + =338.1.

[0311] Step 2: Preparation of N-(1-(3-bromo-6-methoxypyridin-2-yl)-4-phenylbut-2-yl)-2-methylpropane-2-sulfinamide

[0312] Measure 25 mL of tetrahydrofuran into a dry reaction flask, purge with nitrogen three times, add 12.4 mL of 2M diisopropylaminolithium, and cool to -78 °C. Add a solution of 5 g of 3-bromo-6-methoxy-2-methylpyridine and 5 mL of tetrahydrofuran, and stir at -78 °C for 1 hour. Add a solution of 6.45 g of 2-methyl-N-(3-phenylpropylidene)propane-2-sulfinamide and 15 mL of tetrahydrofuran, and slowly raise the temperature from -78 °C to -30 °C and stir for 2 hours. Monitor the reaction for completion by LC-MS, quench with saturated ammonium chloride solution, extract three times with ethyl acetate, dry the organic phase, concentrate to dryness, and purify by column chromatography to obtain the title compound (6.4 g).

[0313]

[0314] MS(ESI)m / z(M+H) + =439.1.

[0315] Step 3: Preparation of ethyl 2-(2-((tert-butylsulfinyl)amino)-4-phenylbutyl)-6-methoxynicotinic acid

[0316] 2 g of N-(1-(3-bromo-6-methoxypyridin-2-yl)-4-phenylbut-2-yl)-2-methylpropane-2-sulfinamide was weighed into a dry reaction flask and dissolved in 10 mL of ethanol. 0.67 g of 1,1′-ferrocene diyl-bis(diphenylphosphine)palladium dichloride and 1.2 mL of triethylamine were added. The system was purged with nitrogen three times and heated to 110 °C. The reaction was allowed to proceed overnight. LC-MS analysis confirmed the reaction was complete, and the system was concentrated to dryness. The title compound (1 g) was purified by column chromatography.

[0317]

[0318] MS(ESI)m / z(M+H) + =433.2.

[0319] Step 4: Preparation of 6-(tert-butylsulfinyl)-2-methoxy-7-phenethyl-7,8-dihydro-1,6-naphthidine-5(6H)-one

[0320] 1 g of ethyl 2-(2-((tert-butylsulfinyl)amino)-4-phenylbutyl)-6-methoxynicotinate was weighed into a dry reaction flask, dissolved in acetonitrile (10 mL), and cesium carbonate (124.5 mg) was added. The mixture was heated to 80 °C and allowed to react for 6 hours. The reaction was confirmed to be complete by LC-MS. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated to dryness. The title compound (0.6 g) was purified by column chromatography.

[0321]

[0322] MS(ESI)m / z(M+H) + =387.1.

[0323] Step 5: Preparation of 2-hydroxy-7-phenylethyl-7,8-dihydro-1,6-naphthidium-5(6H)-one

[0324] Weigh 600 mg of 6-(tert-butylsulfinyl)-2-methoxy-7-phenylethyl-7,8-dihydro-1,6-naphthidium-5(6H)-one, dissolve it in 10 mL of 33% hydrogen bromide acetic acid, heat to 80 °C and stir for 2 hours. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation to obtain the crude title compound (1.2 g).

[0325]

[0326] MS(ESI)m / z(M+H) + =269.1.

[0327] Step 6: Preparation of 2-chloro-7-phenylethyl-7,8-dihydro-1,6-naphthidium-5(6H)-one

[0328] Weigh 1.2 g of 2-hydroxy-7-phenylethyl-7,8-dihydro-1,6-naphthidium-5(6H)-one, dissolve it in 10 mL of phosphorus oxychloride, heat to 95 °C and stir for 2 hours. The reaction was confirmed to be complete by LC-MS, and the solvent was removed under reduced pressure. The solution was diluted with ethyl acetate, and the pH was adjusted to 7-8 with sodium carbonate. The solution was extracted three times with ethyl acetate. The organic phase was concentrated and purified by silica gel column chromatography to obtain the title compound (200 mg).

[0329]

[0330] MS(ESI)m / z(M+H) + =287.1.

[0331] Step 7: Preparation of tert-butyl 2-chloro-5-oxo-7-phenethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0332] Weigh 185 mg of 2-chloro-7-phenylethyl-7,8-dihydro-1,6-naphthidium-5(6H)-one and dissolve it in 10 mL of dichloromethane. Add 423 mg of ditert-butyl dicarbonate, 31.5 mg of 4-dimethylaminopyridine, and 391 mg of triethylamine. Heat to 40 °C and stir overnight. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation. Purify and separate by silica gel column chromatography to obtain the title compound (230 mg).

[0333]

[0334] MS(ESI)m / z(M+H) + =387.1.

[0335] Step 8: Preparation of tert-butyl 2-chloro-7-phenethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0336] Weigh 230 mg of tert-butyl 2-chloro-5-oxo-7-phenethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate and dissolve it in tetrahydrofuran (6 mL). Add 6 mL of 2.5 M borane dimethyl sulfide. Heat to 50 °C and react for 4 hours. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation. Purify and separate by silica gel column chromatography to obtain the title compound (120 mg).

[0337]

[0338] MS(ESI)m / z(M+H) + =373.1.

[0339] Step 9: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-7-phenethyl-7,8-dihydro-1,6-naphthidine-6(5H)-formate

[0340] 60 mg of tert-butyl 2-chloro-7-phenylethyl-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was weighed into a dry reaction flask and dissolved in toluene (10 mL). Tris(dibenzylindeneacetone)dipalladium (30 mg), 1,1′-ferrocene diyl-bis(diphenylphosphine) (35.7 mg), triethylamine (32 mg), and di-tert-butyl phosphonate (63 mg) were added. The system was purged with nitrogen three times and heated to 120 °C. The reaction was allowed to proceed overnight. The reaction was confirmed to be complete by LC-MS, and the system was concentrated to dryness. The title compound (20 mg) was purified by column chromatography.

[0341]

[0342] MS(ESI)m / z(M+H) + =531.2.

[0343] Step 10: Preparation of (7-phenylethyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate

[0344] Weigh 20 mg of tert-butyl 2-(di-tert-butoxyphosphoryl)-7-phenethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in 3 mL of dichloromethane, add dropwise 3 mL of 4 M hydrochloric acid-1,4-dioxane solution, stir at room temperature for 1 hour, and check the reaction is complete by LC-MS. Remove the solvent under reduced pressure. The title compound (5 mg) is obtained by pre-HPLC purification.

[0345]

[0346] MS(ESI)m / z(M+H) + =319.1.

[0347] 1 H NMR(400MHz, Deuterium Oxide)δ7.88–7.63(m,3H),7.37–7.17(m,4H),4.51–4.29(m,2H),3.61(q,J=7.4,5.0Hz,1H),3.39(dd,J=18.1, 4.8Hz,1H),3.07(dd,J=18.1,10.9Hz,1H),2.88–2.67(m,2H),2.20–2.08(m,1H),2.02(dt,J=14.1,7.9Hz,1H).

[0348] Example 14: Preparation of (7,7-diethyl-5,6,7,8-tetrahydro-naphthid-2-yl)phosphonate

[0349]

[0350] Step 1: Preparation of 2-methyl-N-(pentane-3-yl)propane-2-sulfinamide

[0351] 10.73 g of pentane-3-one was dissolved in 300 mL of tetrahydrofuran, and 46 g of tetraethyl titanate and 12 g of 2-methylpropane-2-sulfinamide were added. The mixture was purged with nitrogen three times and heated to 65 °C for 20 hours. LC-MS was used to monitor the reaction until completion. A large amount of solid precipitated upon the addition of 30 mL of water. The solid was filtered, dried, concentrated to dryness, and purified by column chromatography to give the title compound (10.8 g).

[0352]

[0353] MS(ESI)m / z(M+H) + =190.1.

[0354] Step 2: Preparation of N-(3-((3-bromo-6-methoxypyridin-2-yl)methyl)pent-3-yl)-2-methylpropane-2-sulfinamide

[0355] 50 mL of tetrahydrofuran was measured and placed in a dry reaction flask. The flask was purged with nitrogen three times, and 25 mL of 2M diisopropylaminolithium was added, followed by cooling to -78 °C. A solution of 9.4 g of 3-bromo-6-methoxy-2-methylpyridine and 50 mL of tetrahydrofuran was added, and the mixture was stirred at -78 °C for 1 hour. A solution of 8 g of 2-methyl-N-(pentane-3-yl)propane-2-sulfinamide and 50 mL of tetrahydrofuran was added, and the mixture was slowly heated from -78 °C to -30 °C and stirred for 2 hours. The reaction was monitored by LC-MS until complete. The reaction was quenched with saturated ammonium chloride solution, extracted three times with ethyl acetate, dried the organic phase, concentrated to dryness, and purified by column chromatography to obtain the title compound (11.3 g).

[0356]

[0357] MS(ESI)m / z(M+H) + =391.1.

[0358] Step 3: Preparation of ethyl 2-(2-((tert-butylsulfinyl)amino)-2-ethylbutyl)-6-methoxynicotinic acid

[0359] 11.3 g of N-(3-((3-bromo-6-methoxypyridin-2-yl)methyl)pent-3-yl)-2-methylpropane-2-sulfinamide was weighed into a dry reaction flask and dissolved in 10 mL of ethanol. 4.73 g of 1,1′-ferrocene diyl-bis(diphenylphosphine)palladium dichloromethane dichloride complex and 9.6 mL of N,N-diisopropylethylamine were added. The system was purged with nitrogen three times and heated to 100 °C. The reaction was allowed to proceed overnight. LC-MS analysis confirmed the reaction was complete, and the system was concentrated to dryness. The title compound (9.3 g) was purified by column chromatography.

[0360]

[0361] MS(ESI)m / z(M+H) + =385.2.

[0362] Step 4: Preparation of 7,7-diethyl-2-methoxy-7,8-dihydro-1,6-naphthidine-5(6H)-one

[0363] 9.3 g of ethyl 2-(2-((tert-butylsulfinyl)amino)-2-ethylbutyl)-6-methoxynicotinic acid was weighed into a dry reaction flask, dissolved in acetonitrile (10 mL), and sodium hydroxide (4.8 g) was added. The mixture was heated to 100 °C and reacted for 6 hours. The reaction was confirmed to be complete by LC-MS. The system was filtered through diatomaceous earth, and the filtrate was concentrated to dryness. The title compound (4.6 g) was purified by column chromatography.

[0364]

[0365] MS(ESI)m / z(M+H) + =235.1.

[0366] Step 5: Preparation of 7,7-diethyl-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthidine

[0367] Weigh 3 g of 7,7-diethyl-2-methoxy-7,8-dihydro-1,6-naphthidium-5(6H)-one and dissolve it in tetrahydrofuran (100 mL). Add 1.9 g of lithium aluminum hydride in portions over an ice bath. Heat to reflux and stir overnight. LC-MS was used to confirm the reaction was complete. The solvent was removed under reduced pressure. Purify and separate the compound using silica gel column chromatography to obtain the title compound (2.8 g).

[0368]

[0369] MS(ESI)m / z(M+H) + =221.1.

[0370] Step 6: Preparation of 7,7-diethyl-5,6,7,8-tetrahydro-1,6-naphthidine-2-ol

[0371] Weigh 2.8 g of 7,7-diethyl-2-methoxy-5,6,7,8-tetrahydro-1,6-naphthidine and dissolve it in 20 mL of 33% hydrogen bromide acetic acid. Heat the solution to 80 °C and stir overnight. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation. Crude acetonitrile was then slurried to obtain the title compound (4.3 g).

[0372]

[0373] MS(ESI)m / z(M+H) + =207.1.

[0374] Step 7: Preparation of 2-chloro-7,7-diethyl-5,6,7,8-tetrahydro-1,6-naphthidine

[0375] Weigh 2.8 g of 7,7-diethyl-5,6,7,8-tetrahydro-1,6-naphthidine-2-ol and dissolve it in 40 mL of phosphorus oxychloride. Heat to 120 °C and stir overnight. The reaction was confirmed to be complete by LC-MS. The solvent was removed by vacuum distillation. Dilute with dichloromethane, add water and adjust the pH to 9-10 with sodium carbonate. Proceed directly to the next reaction without purification to obtain the crude product of the title compound.

[0376]

[0377] MS(ESI)m / z(M+H) + =225.1.

[0378] Step 8: Preparation of tert-butyl 2-chloro-7,7-diethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0379] Take the reaction mixture from the previous step, add 4.67 mL of di-tert-butyl dicarbonate, stir overnight at room temperature, and check the reaction is complete by LC-MS. Extract three times with dichloromethane, and concentrate the organic phase after drying. Purify and separate by silica gel column chromatography to obtain the title compound (1.5 g).

[0380]

[0381] MS(ESI)m / z(M+H) + =325.1.

[0382] Step 9: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-7,7-diethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0383] Weigh 200 mg of tert-butyl 2-chloro-7,7-diethyl-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate into a dry reaction flask, dissolve in toluene (10 mL), add 100 mg of 1,1′-ferrocene diyl-bis(diphenylphosphine)palladium dichloride dichloromethane complex, triethylamine (0.17 mL), and di-tert-butyl phosphonate (360 mg), purge the system three times with nitrogen, and heat to 120 °C. React overnight. LC-MS was used to confirm the complete reaction, and the system was concentrated to dryness. Column chromatography was used to purify the compound to the title compound (80 mg).

[0384]

[0385] MS(ESI)m / z(M+H) + =483.2.

[0386] Step 10: Preparation of (7,7-diethyl-5,6,7,8-tetrahydro-naphthid-2-yl)phosphonate

[0387] Weigh 80 mg of tert-butyl 2-(di-tert-butoxyphosphoryl)-7,7-diethyl-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate, dissolve it in dichloromethane (4 mL), add dropwise 4 M hydrochloric acid 1,4-dioxane solution (4 mL), stir at room temperature for 1 hour, and check the reaction is complete by LC-MS. Remove the solvent under reduced pressure. The title compound (13 mg) is obtained by pre-HPLC purification.

[0388]

[0389] MS(ESI)m / z(M+H) + =271.1.

[0390] 1 H NMR (400MHz, Deuterium Oxide) δ7.88–7.57(m,2H),4.39(s,2H),3.11(s,2H),1.84–1.42(m,4H),0.90(t,J=7.5Hz,6H).

[0391] Preparation Example 1: Preparation of tert-butyl 2-chloro-8-hydroxy-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0392]

[0393] Step 1: Preparation of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0394]

[0395] 5 g of 2-chloro-5,6,7,8-tetrahydro-1,6-naphthidine hydrochloride was dissolved in 50 ml of dichloromethane. Triethylamine (10 ml) was added, followed by the slow addition of 6.7 ml of di-tert-butyl dicarbonate. The reaction was allowed to proceed at room temperature for 3 hours, and LC-MS showed that the reaction was complete. The system was concentrated to obtain an oily crude product, which was separated by column chromatography to obtain the title compound (6 g).

[0396] MS(ESI)m / z(M+H) + =269.1.

[0397] Step 2: Preparation of 6-(tert-Butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-1,6-naphthidine-1-oxide

[0398]

[0399] 9 g (33.58 mmol) of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was weighed and dissolved in 100 mL of dichloromethane. 11.7 g of m-chloroperoxybenzoic acid was added in portions under ice bath conditions. The reaction was allowed to proceed overnight at room temperature, and LC-MS showed complete reaction. Dichloromethane and water were added, and the mixture was extracted separately. The organic phase was concentrated to dryness. The crude product was purified by column chromatography to give the title compound (7.0 g).

[0400] MS(ESI)m / z(M+H) + =285.1.

[0401] Step 3: Preparation of tert-butyl 8-acetoxy-2-chloro-7,8-dihydro-1,6-naphthylpyridine-6(5H)-formate

[0402]

[0403] 7 g of 6-(tert-butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-1,6-naphthidine-1-oxide was dissolved in acetic anhydride (80 mL), purged three times with nitrogen, and heated to 70 °C overnight. LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure to remove a large amount of acetic anhydride, and ethyl acetate and water were added. The mixture was extracted three times with ethyl acetate, washed twice with saturated sodium bicarbonate solution, dried and concentrated the organic phase, and purified by column chromatography to obtain the title compound (5 g).

[0404] MS(ESI)m / z(M+H) + =327.1.

[0405] Step 4: Preparation of tert-butyl 2-chloro-8-hydroxy-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0406]

[0407] Weigh 3 g of tert-butyl 8-acetoxy-2-chloro-7,8-dihydro-1,6-naphthyl-6-(5H)-carboxylate, dissolve it in methanol (30 ml), add potassium carbonate (635 mg), and react at room temperature for 0.5 hours. LC-MS showed that the reaction was complete. Add ethyl acetate and water, extract three times with ethyl acetate, dry and concentrate the organic phase, concentrate the system to obtain an oily crude product, and purify by column chromatography to obtain the title compound (1.9 g).

[0408] MS(ESI)m / z(M+H) + =285.1.

[0409] Preparation Example 2: Preparation of 6-(tert-butyl)-7-methyl-2-chloro-7,8-dihydro-1,6-naphthidine-6,7(5H)-dicarboxylate

[0410]

[0411] Step 1: Preparation of 1-oxide of 2,3-bis(methoxycarbonyl)pyridine

[0412]

[0413] Dimethyl pyridine-2,3-dicarboxylate (4.90 g) was dissolved in acetonitrile (60 mL). Urea peroxide (4.71 g) was added under ice bath conditions, followed by slow dropwise addition of trifluoroacetic anhydride (10.5 g). After the addition was complete, the system became a clear solution. The mixture was heated to room temperature and reacted for 4 hours. TLC showed that the reaction was essentially complete. The reaction was quenched with an aqueous sodium metabisulfite solution. Dichloromethane and water were added, and the mixture was separated. The aqueous phase was extracted with a mixed solvent (DCM / MeOH). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The title compound (5.15 g) was obtained.

[0414] MS(ESI)m / z(M+H) + =212.1.

[0415] Step 2: Preparation of dimethyl 6-chloropyridine-2,3-dicarboxylate

[0416]

[0417] Weigh 5.15 g of 2,3-bis(methoxycarbonyl)pyridine 1-oxide, add 30 mL of phosphorus oxychloride in an ice bath, and react at 105 °C for 4 hours. TLC showed that the reaction was complete. Concentrate under reduced pressure, dilute with ethyl acetate, add dropwise to crushed ice, adjust the pH to 10 with sodium carbonate aqueous solution, extract with ethyl acetate, and wash the organic phase with sodium chloride aqueous solution. Concentrate the organic phase to dryness, and purify the crude product by column chromatography to give the title compound (3.52 g).

[0418] MS(ESI)m / z(M+H) + =230.1.

[0419] Step 3: Preparation of (6-chloropyridine-2,3-diyl)diethanol

[0420]

[0421] 3.50 g of dimethyl 6-chloropyridine-2,3-dicarboxylate was dissolved in 72 mL of tetrahydrofuran and 1.5 mL of methanol. Lithium borohydride (0.84 g) was added in portions under ice bath conditions, and the mixture was heated to room temperature for 3 hours. TLC showed that most of the starting material had reacted. The reaction mixture was poured into an aqueous sodium bicarbonate solution, ethyl acetate was added, and the mixture was extracted separately. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The title compound (2.63 g) was obtained.

[0422] MS(ESI)m / z(M+H) + =174.1.

[0423] Step 4: Preparation of 6-chloro-2,3-bis(chloromethyl)pyridine

[0424]

[0425] Weigh 2.63 g of (6-chloropyridine-2,3-diyl)diethanol and add 40 mL of thionyl chloride under ice bath conditions. React at room temperature for 3 hours. A portion of the monochlorinated intermediate was present; the reaction was then carried out at 35 °C for 3 hours, and TLC showed complete reaction. The mixture was concentrated under reduced pressure, diluted with ethyl acetate, and added dropwise to crushed ice. The pH was adjusted to 10 with sodium carbonate aqueous solution. The mixture was extracted with ethyl acetate, and the organic phase was washed with sodium chloride aqueous solution. The organic phase was concentrated to dryness, and the crude product was purified by column chromatography to give the title compound (2.1 g).

[0426] MS(ESI)m / z(M+H) + =210.1.

[0427] Step 5: Preparation of dimethyl 6-acetyl-2-chloro-5,8-dihydro-1,6-naphthidine-7,7(6H)-dicarboxylate

[0428]

[0429] 2.10 g of 6-chloro-2,3-bis(chloromethyl)pyridine was dissolved in 15 mL of N,N-dimethylformamide. Dimethyl acetaminomalonate (2.17 g) and sodium hydride (0.40 g) were added sequentially under ice bath conditions. The reaction was allowed to proceed for 1 hour at room temperature, followed by the addition of sodium hydride (0.40 g) under ice bath conditions, and the reaction was allowed to continue overnight at room temperature. TLC showed that the reaction was complete. Ethyl acetate and water were added, and the mixture was extracted separately. The organic phase was concentrated to dryness. The crude product was purified by column chromatography to give the title compound (1.74 g).

[0430] MS(ESI)m / z(M+H) + =327.1.

[0431] Step 6: Preparation of 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylate

[0432]

[0433] Weigh 1.74 g of dimethyl 6-acetyl-2-chloro-5,8-dihydro-1,6-naphthidine-7,7(6H)-dicarboxylate, add 15 mL of 6M hydrochloric acid, and react in a sealed container at 100 °C for 4 hours. TLC showed that the reaction was complete. The system was concentrated to dryness under reduced pressure to give the title compound (1.16 g).

[0434] MS(ESI)m / z(M+H) + =213.1.

[0435] Step 7: Preparation of methyl 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylate hydrochloride

[0436]

[0437] 1.16 g of 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylate was dissolved in 20 mL of methanol, and 1.67 g of thionyl chloride was slowly added dropwise under ice bath conditions. The mixture was refluxed at 70 °C for 2 hours, and TLC showed that the reaction was complete. The system was concentrated to dryness under reduced pressure to give the title compound (1.23 g).

[0438] MS(ESI)m / z(M+H) + =227.1.

[0439] Step 8: Preparation of 6-(tert-butyl)-7-methyl-2-chloro-7,8-dihydro-1,6-naphthidine-6,7(5H)-dicarboxylate

[0440]

[0441] 1.23 g of methyl 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylate hydrochloride was dissolved in 25 mL of dichloromethane, followed by the addition of 1.89 g of triethylamine and 1.53 g of ditert-butyl dicarbonate. The reaction was carried out at room temperature for 2 hours, and TLC showed complete reaction. Dichloromethane and water were added, and the mixture was extracted separately. The organic phase was concentrated to dryness. The crude product was purified by column chromatography to give the title compound (1.19 g).

[0442] MS(ESI)m / z(M+H) + =327.1.

[0443] 1 H NMR(400MHz,Chloroform-d)δ7.42(dd,J=12.5,8.0Hz,1H),7.21(d,J=8.1Hz,1H),5.34(d,J=6.7Hz,0.5H),5.07(dd,J=7.2,2.9Hz,0.5H),4.7 9(dd,J=22.4,17.0Hz,1H),4.52(dd,J=31.0,17.1Hz,1H),3.68(d,J=7.7Hz,3H),3.55–3.38(m,1H),3.38–3.20(m,1H),1.52(d,J=17.9Hz,9H).

[0444] Preparation Example 3: Preparation of tert-butyl 2-chloro-7-formyl-7,8-dihydro-1,6-naphthidine-6(5H)-formate

[0445]

[0446] Step 1: Preparation of 6-(tert-Butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylic acid

[0447]

[0448] At room temperature, 0.4 g of 6-(tert-butyl)-7-methyl-2-chloro-7,8-dihydro-1,6-naphthidine-6,7(5H)-dicarboxylate was dissolved in a tetrahydrofuran (3 mL) / methanol (3 mL) / water (3 mL) system, and 0.1 g of lithium hydroxide hydrate was added. The mixture was stirred for 1 hour, and the reaction was confirmed to be complete by LC-MS. Under ice bath conditions, the pH was adjusted to 4-5 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate / water. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to give the title compound (0.369 g).

[0449] MS(ESI)m / z(M+H) + =313.1.

[0450] Step 2: Preparation of tert-butyl 2-chloro-7-(methoxy(methyl)carbamoyl)-7,8-dihydro-1,6-naphthidine-6(5H)-carboxylate

[0451]

[0452] At room temperature, 0.374 g of 6-(tert-butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-1,6-naphthyl-7-carboxylic acid was dissolved in 20 mL of dichloromethane. Then, 1.25 mL of N,N-diisopropylethylamine, 0.494 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 0.235 g of methoxymethylamine hydrochloride were added sequentially, and the mixture was stirred overnight. TLC showed that the reaction was complete. The mixture was then extracted with dichloromethane / water, washed with saturated sodium chloride solution, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give the title compound (0.379 g).

[0453] MS(ESI)m / z(M+H) + =356.1.

[0454] Step 3: Preparation of tert-butyl 2-chloro-7-formyl-7,8-dihydro-1,6-naphthidine-6(5H)-formate

[0455]

[0456] 0.378 g of tert-butyl 2-chloro-7-(methoxy(methyl)carbamoyl)-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylate was dissolved in 20 mL of anhydrous tetrahydrofuran. The mixture was placed under a nitrogen atmosphere and cooled to -72 °C. 3.21 mL of 1 M diisobutylaluminum hydride solution was added. After the addition was complete, the mixture was slowly heated to room temperature and stirred for 3 hours. The reaction was confirmed to be complete by LC-MS. The reaction mixture was then placed in an ice bath and quenched with water for 10 minutes. 20 mL of saturated sodium potassium tartrate solution was added and stirred for 20 minutes. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to give the title compound (0.206 g).

[0457] MS(ESI)m / z(M+H) + =297.0

[0458] Using raw materials and reagents purchased through conventional commercial channels, and referring to the preparation method of the foregoing embodiments combined with conventional separation and purification methods in the art, the following compounds were prepared:

[0459]

[0460]

[0461]

[0462]

[0463] Biological tests

[0464] Experimental Example 1: Plasma Clot Degradation Experiment

[0465] 1. Experimental Objective

[0466] The inhibitory effect of the compounds of this invention on the degradation of human plasma clots was determined.

[0467] 2. Experimental materials and instruments

[0468]

[0469] 3. Experimental Procedure

[0470] 3.1 Collect fresh blood from healthy individuals, use 0.109M trisodium citrate as an anticoagulant, mix 1 part anticoagulant with 9 parts blood, centrifuge at 2000x g for 20 minutes at room temperature, collect the supernatant (i.e., plasma), aliquot and store at -80℃ for later use.

[0471] 3.2 On the day of the experiment, the plasma was thawed in a water bath at 37°C, and all reagents except tPA were preheated at 37°C.

[0472] 3.3 Add 12.5 μL of 80 mM CaCl2 (HEPES buffer, pH 7.4) to a 96-well plate, then add 25 μL of the test compound diluted with physiological saline at different concentrations. Add an equal volume of physiological saline to the negative control wells.

[0473] 3.4 Mix 50 μL of preheated plasma with 12.5 μL of 4 nM tPA (HEPES buffer, pH 7.4) and immediately add to a 96-well plate. Detect the absorbance at 405 nm, reading every 2 minutes for 15 hours.

[0474] 3.5 The absorbance value changes over time, first increasing and then decreasing. The time corresponding to the median absorbance value during the decreasing phase minus the time corresponding to the median absorbance value during the increasing phase is the plasma clot lysis time. Using the plasma clot lysis time of the negative control well as a reference, the inhibition rate was calculated relative to the plasma clot lysis time in wells with different compound concentrations.

[0475] Inhibition rate % = (1 - negative control well) Clot lysis time / compound pores Clot lysis time )×100%

[0476] 3.6 Fitting dose-response curve

[0477] Using the logarithm of compound concentration as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using the log(inhibitor) vs. response-variable slope method in GraphPadPrism 5 to derive the IC50 of each compound on the inhibition of plasma clot degradation. 50 value.

[0478] Calculation formula: Y = min + (max - min) / (1 + 10^(LogIC)) 50 -X)×Hillslope).

[0479] The inhibitory effect of the compound of the present invention on plasma clot degradation was determined by the above experiments, and the IC50 of the compound of the present invention was calculated. 50 The values ​​are all lower than the IC50 of tranexamic acid. 50 For example, the compound of Example 1 of this invention inhibits plasma clot degradation at an IC50 level. 50 The value was 0.9 μM, which is far lower than that of tranexamic acid, a representative hemostatic drug (IC50 under the same experimental conditions). 50 (4.75 μM). The relative coagulation activity (IC50) of this invention relative to tranexamic acid in vitro is [not specified]. 50 ratio = IC 50实施例 / IC 50氨甲环酸 See the table below:

[0480]

[0481] Experimental data show that the compound of the present invention can effectively inhibit the degradation of plasma clots, has excellent coagulation and hemostatic activity, and its effective dose is far lower than that of the most frequently used hemostatic drugs in clinical practice. It can effectively avoid adverse reactions and complications caused by high-dose medication and has excellent prospects for drug development.

[0482] Experimental Example 2: Rat PK Test

[0483] 1. Experimental Objective

[0484] The pharmacokinetic characteristics of the compound of the present invention in rats were studied by measuring the plasma drug concentration after intravenous administration.

[0485] 2. Laboratory animals

[0486] SD rats, SPF grade, male, N=3, source: Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

[0487] 3. Drug preparation and administration

[0488] Weigh the compound and dissolve it in physiological saline to prepare an intravenous administration solution of 0.2 mg / mL.

[0489] The rats were fasted overnight the day before the experiment and fed 4 hours after administration of the drug.

[0490] On the day of the experiment, the drugs were administered according to the protocol shown in the table below. At each time point after drug administration, approximately 200 μL of blood was collected from the jugular vein of the rats and placed in heparin sodium anticoagulant tubes. After collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6800g, 6 minutes, 2-8℃). The separated plasma was stored at -80℃ for use in biological sample analysis.

[0491]

[0492] 4. Bioanalysis

[0493] The specific method for determining the concentration of compounds in rat plasma is as follows:

[0494] Instrumentation: LC-MS / MS-19 (TQ5500, AB SCIEX, USA).

[0495] Internal label: Warfarin.

[0496] Chromatographic column: ACQUITY UPLC BEH C18, model 1.7um 2.1*50mm, purchased from Shenzhen Noyadi Chemical Technology Co., Ltd.;

[0497] Flow rate: 0.60 ml / min.

[0498] Column temperature: 40℃.

[0499] Mobile phase A: 0.1% formic acid aqueous solution.

[0500] Mobile phase B: 0.1% formic acid in acetonitrile solution.

[0501] The elution gradients are shown in Table 3.

[0502] Table 3 Elution gradient

[0503] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 0.60 12 88 1.10 12 88 1.11 98 2 1.40 98 2

[0504] MS detection conditions: electrospray ionization (ESI), cation mode, MRM scan.

[0505] Take 30 μL of the plasma sample prepared under item "3" of this embodiment and precipitate proteins with 300 μL of MeOH containing 100 ng / mL internal standard. Vortex the mixture for 1 minute and centrifuge at 18000 g for 7 minutes. Transfer the supernatant to a 96-well plate. Inject 4 μL of the supernatant into LC-MS / MS for analysis.

[0506] The concentrations of compounds in rat plasma were determined using the above-mentioned LC-MS / MS analysis method. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0 based on the blood drug concentration data at different time points.

[0507] The pharmacokinetic parameters of some compounds in this invention were determined through the above experiments, and the obtained parameters in rats are shown in the table below.

[0508] Table: In vivo pharmacokinetic data of the test compound administered intravenously to SD rats

[0509]

[0510] Experiment Example 3: Thromboelastography Experiment in Human Whole Blood

[0511] 1. Experimental Objective

[0512] The antifibrinolytic effect of the compound of the present invention in human whole blood hyperfibrinolysis induced by rtPA (recombinant tissue plasminogen activator) was determined by thromboelastography (TEG).

[0513] 2. Main experimental materials and instruments

[0514] Experimental materials

[0515]

[0516] instrument

[0517]

[0518] Human blood source: All human blood used in the experiments was provided by healthy volunteers.

[0519] 3. Experimental Procedure

[0520] (1) Preparation of test solution: Accurately weigh the test substance and prepare it to the following concentration (100 × test concentration) using physiological saline:

[0521] Tranexamic acid (μM): 3000, 1000, 300, 100, 30, 10, 0;

[0522] Example 6 (μM): 1000, 300, 100, 30, 10, 3, 0.

[0523] (2) Preparation of rtPA (alteplase for injection): Use the water for injection included in the rtPA package to prepare the active dry powder of rtPA to 25 μg / mL.

[0524] (3) Reaction system:

[0525] 392 μL of sodium citrate anticoagulated whole blood + 4 μL of rtPA + 4 μL of test substance were reacted at room temperature, and the TEG curve was detected after 2 hours to obtain the CLT (clot lysis time) parameter.

[0526] (4) Calculation of results

[0527] Using the logarithmic value of compound concentration as the X-axis and the CLT value as the Y-axis, the dose-response curve was fitted using the log(inhibitor) vs. response-variable slope method in GraphPad Prism 8 analysis software.

[0528] Formula: Y=min+(max-min) / (1+10^((LogIC 50 -X)×Hillslope).

[0529] Calculate the compound concentration that would require doubling the CLT time.

[0530] 4. Experimental Results

[0531] The inhibitory effect of the compound of this invention on fibrinolysis in human whole blood was determined by the above experiments, and the results are as follows:

[0532] CLT increases drug concentration by 1 time

[0533]

[0534] Experiments have demonstrated that, compared to tranexamic acid, the most active and widely used positive control drug currently available in clinical practice, the compound of this invention exhibits significantly higher exposure levels, lower clearance rates, and a prolonged half-life in animals. In plasma clot degradation and thromboelastography (TEG) experiments, the compound effectively inhibits fibrinolysis and prolongs clot degradation time (CLT), demonstrating superior hemostatic and coagulation effects compared to the positive control. These experiments indicate that the compound of this invention possesses advantages such as good hemostatic activity, low effective dose, and long duration of action, avoiding various adverse reactions that may occur with high-dose clinical administration and improving patient safety and efficacy. Furthermore, the compound is easy to prepare, facilitating large-scale industrial production and effectively reducing drug costs. The compound exhibits good distribution, metabolism, and excretion characteristics, with a low probability of drug-drug interactions, meeting the pharmacokinetic parameters required for therapeutic efficacy in humans. In addition, the compound exhibits low toxicity, with no effects on the respiratory, central nervous, and cardiovascular systems. Single-dose and repeated-dose toxicity tests show good tolerability, a sufficient safety window, and no genotoxicity. It has broad prospects for clinical application.

Claims

1. A pharmaceutically acceptable salt or stereoisomer of the compound represented by Formula I: in, X is N; R1 is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy or -CH2-O(CH2)3OH, wherein the substituted C1-C6 alkyl or substituted C1-C6 alkoxy is substituted by one or more groups selected from hydroxyl, phenyl, C1-C4 alkoxy, C1-C4 alkoxy-substituted phenyl and cyclohexyl, or the two R1s together with the attached carbon atom form a carbon ring containing 3 to 8 carbon atoms; R2 is selected from hydrogen, halogen, hydroxyl, hydroxy-substituted C1-C6 alkoxy, or a 6-membered alicyclic group containing 1 to 3 heteroatoms selected from N, O, and S, wherein the S heteroatoms may be selectively oxidized; R3 is selected from hydrogen and F; R4 is selected from hydroxyl, phenyl, C1-C6 alkyl, or phenyl-substituted C1-C6 alkyl; R5 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylcarbonyloxy-C1-C4 alkyl, or C1-C4 alkoxycarbonyloxy-C1-C4 alkyl.

2. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has the structure of formula I': in, X is N; R1 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy or -CH2-O(CH2)3OH, wherein the substituted C1-C6 alkyl or substituted C1-C6 alkoxy is substituted by one or more groups selected from hydroxyl, phenyl, C1-C4 alkoxy, C1-C4 alkoxy-substituted phenyl and cyclohexyl. R2 is selected from hydrogen, halogen, hydroxyl, hydroxy-substituted C1-C6 alkoxy, or a 6-membered alicyclic group containing 1 to 3 heteroatoms selected from N, O, and S, wherein the S heteroatoms may be selectively oxidized; R3 is selected from hydrogen and F; R4 is selected from hydroxyl, phenyl, C1-C6 alkyl, or phenyl-substituted C1-C6 alkyl; R5 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylcarbonyloxy-C1-C4 alkyl, or C1-C4 alkoxycarbonyloxy-C1-C4 alkyl.

3. The compound of claim 1, its pharmaceutically acceptable salt or stereoisomer thereof, wherein the two R1s together with the attached carbon atom form a cyclobutyl, cyclopentyl or cyclohexyl ring.

4. The compound according to claim 2, wherein its pharmaceutically acceptable salt or stereoisomer thereof, wherein the R1 group is selected from hydrogen, -CH2OH, isobutyl, tert-butyl, -O(CH2)2OH, -O(CH2)3OH, -(CH2)4OH, -CH2-O(CH2)3OH, phenethyl, propyl, isopentyl, 3,3-dimethylbutyl, cyclohexylmethyl, cyclohexylethyl, phenylpropyl, 4-methoxyphenylethyl.

5. The compound according to any one of claims 1-4, its pharmaceutically acceptable salt or stereoisomer thereof, wherein the R2 group is selected from hydrogen, hydroxyl, -OCH2CH2OH, ...

6. The compound according to claim 5, its pharmaceutically acceptable salt or stereoisomer thereof, wherein the R2 group is hydrogen.

7. The compound according to any one of claims 1-4, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the R4 group is selected from hydroxyl, phenyl, ethyl or phenylethyl.

8. The compound according to any one of claims 1-4, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the R5 group is selected from hydrogen, ethyl, methylcarbonyloxymethyl, isopropylcarbonyloxymethyl or methoxycarbonyloxymethyl.

9. The compound of formula I according to claim 1, characterized in that... It has the following structure:

10. A pharmaceutical composition comprising at least one compound according to any one of claims 1-9, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and at least one pharmaceutically acceptable excipient.

11. The use of the compound of any one of claims 1-9, its pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of claim 10, in the preparation of a medicament, characterized in that... The drug has therapeutic activities of coagulation and hemostasis.

12. The use according to claim 11, characterized in that... The drug is used to treat abnormal bleeding caused by hyperfibrinolysis, as well as bleeding during and after surgery.

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