Plasmin inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a plasmin inhibitor as well as a preparation method and application thereof, in particular to a compound which can inhibit plasmin activity and delay fibrinolysis, has blood coagulation and hemostasis activity and is shown in a formula (I), pharmaceutically acceptable salts, hydrates, isomers, prodrugs and mixtures thereof, and X, R1 to R5 are defined in the specification.

Description

[0001] This application is a divisional application of the international patent application No.PCT / CN2021 / 143140, filed on December 30, 2021, and titled "A plasmin inhibitor, its preparation method and application", which entered the Chinese national phase on June 29, 2023, and the application number is 202180088567.7. TECHNICAL FIELD

[0002] The present application relates to the field of medicinal chemistry, in particular to a plasmin inhibitor, its preparation method and application in the field of pharmacy. BACKGROUND

[0003] Plasmin is a proteolytic enzyme that degrades fibrin. When tissue damage causes blood vessels to rupture, a hemostatic mechanism is triggered: blood vessels constrict, platelet emboli form, the coagulation process is initiated, and ultimately a stable fibrin clot is formed. At the same time, due to the deposition of fibrin, the fibrinolytic system is activated, which maintains a balance between the formation and lysis of fibrin, and plays a role in maintaining the patency of blood vessels and remodeling damaged tissues in the process of repairing damaged blood vessel walls (Tengborn L, M, Berntorp E. Thromb Res. 2015 Feb; 135(2): 231-42).

[0004] The fibrinolytic system includes plasminogen, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Plasminogen binds to lysine residues on the surface of fibrin, and is converted to plasmin by activators (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 diseases (Ng W, Jerath A, M. Anaesthesiol Intensive Ther. 2015; 47(4): 339-50). In early 1950, it was found that lysine amino acid inhibited the activation of plasminogen, but the effect was too weak to be used for the treatment of fibrinolytic hemorrhagic disease. In 1953, Shosuke Okamoto et al. research showed that several mercapto and amino carbonic acids had anti-plasma protein effects, and found that the synthetic derivative of lysine, epsilon-amino hexanoic 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, it also requires a large dose. In 1962, 4-amino-methyl-cyclohexane-carbonic acid (AMCHA) was found, which contains two stereoisomers, and further studies have shown that the trans form (trans-4-aminomethylcyclohexanecarboxylic acid, i.e. tranexamic acid, TXA) has anti-fibrinolytic ability, with an activity of about 10 times that of EACA, and has been proven to have stronger tolerance (Tengborn L, M, Berntorp E. Thromb Res. 2015 Feb; 135(2): 231-42).

[0005] Tranexamic acid is a synthetic lysine derivative and an antifibrinolytic agent that can form a reversible complex with plasminogen. By binding to plasminogen, it blocks the interaction of plasminogen and plasmin heavy chain with fibrin lysine residues, thereby preventing the binding of plasminogen to the fibrin surface, and thus delaying fibrinolysis. Tranexamic acid has been approved for the treatment of severe menstrual bleeding and various surgical hemorrhagic diseases, and is currently the most commonly used hemostatic drug in clinical practice. However, a large number of literature reports show that tranexamic acid is prone to cause gastrointestinal adverse reactions such as nausea, vomiting, diarrhea and indigestion after oral administration, and its dosage is large, which may cause complications such as epilepsy in patients after administration.

[0006] Other similar hemostatic drugs, such as aminohexanoic acid, have the problems of rapid excretion in the human body, weak hemostatic effect, short duration of action, and more toxic reactions, and when the dosage is too large, it can form thrombosis, limiting its application in those with a tendency to thrombosis or a history of thrombotic vascular disease and those with renal dysfunction. Tranexamic acid has the same mechanism as aminohexanoic acid, and its effect is 4-5 times stronger than that of aminohexanoic acid. It has a significant effect on general chronic bleeding, but has no hemostatic effect on traumatic bleeding and cancer bleeding. In addition, excessive dosage can also promote thrombosis. Aprotinin, a commonly used hemostatic drug in heart bypass surgery, was withdrawn from the market by FDA in 2008 due to the induction of renal failure, myocardial infarction, heart failure, etc.

[0007] Other hemostatic drugs of other mechanisms, such as carbazochrome acting on blood vessels, can induce epilepsy after repeated use; hemostatic drug thrombin promoting the coagulation process can only be applied to gastrointestinal bleeding or local bleeding.

[0008] In view of the fact that the clinically available hemostatic drugs are limited, and more or less have certain defects in dosage, clinical indications, etc., and the existing drugs of the same type have problems such as large dosage, many adverse reactions, and easy to induce epilepsy and other complications, it is necessary to develop a new hemostatic drug to better meet the clinical needs. SUMMARY

[0009] One of the purposes of the present application is to provide a new compound which can inhibit the activity of plasmin, delay fibrinolysis, and has coagulation and hemostatic activity.

[0010] Specifically, the present application provides a compound shown in the following formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug and mixture thereof:

[0011]

[0012] wherein X is selected from N or CR, and R = H or halogen;

[0013] R1 is independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl, or two R1 together with the carbon atom to which they are attached form a carbocyclic ring comprising 3 to 8 carbon atoms;

[0014] 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 aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl;

[0015] R3 is selected from hydrogen, halogen, substituted or unsubstituted alkyl;

[0016] R4 is selected from hydrogen, substituted or unsubstituted amino, hydroxyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl, substituted or unsubstituted aromatic heterocyclyl;

[0017] R5 is selected from hydrogen, substituted or unsubstituted alkyl, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl, alkylcarbonyloxyalkyl, alkoxy carbonyloxyalkyl.

[0018] In one embodiment, the present application relates to compounds as illustrated in the following structure of Formula (I'), pharmaceutically acceptable salts, hydrates, isomers, prodrugs, and mixtures thereof:

[0019]

[0020] wherein X is selected from N or CR, R = H or halogen;

[0021] R1is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl;

[0022] R2is selected from hydrogen, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl;

[0023] R3is selected from hydrogen, halogen, substituted or unsubstituted alkyl;

[0024] R4is selected from hydrogen, substituted or unsubstituted amino, hydroxyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl, substituted or unsubstituted aromatic heterocyclyl;

[0025] R5is selected from hydrogen, substituted or unsubstituted alkyl, haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aliphatic heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclyl.

[0026] In certain specific embodiments, X is N.

[0027] In certain specific embodiments, R1of the present application are independently selected from hydrogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, C1-C4haloalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered aliphatic heterocyclyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclyl, or two R1together with the carbon atom to which they are attached form a carbocyclic ring comprising 3 to 8 carbon atoms.

[0028] In certain specific embodiments, R2of the present application is selected from hydrogen, halogen, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, C1-C4haloalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered aliphatic heterocyclyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclyl.

[0029] In certain embodiments, R3of the present application is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, substituted or unsubstituted C1-C4alkyl.

[0030] In certain embodiments, R4of the present application is selected from the group consisting of hydrogen, substituted or unsubstituted amino, hydroxyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted 6-10 membered heteroaryl.

[0031] In certain embodiments, R5of the present application is selected from the group consisting of hydrogen, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4haloalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered heterocycloalkyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered heteroaryl, C1-C4alkylcarbonyloxy-C1-C4alkyl, C1-C4alkoxycarbonyloxy-C1-C4alkyl.

[0032] In certain embodiments, R1groups of the present application are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy; wherein the substituted C1-C6alkyl or substituted C1-C6alkoxy is substituted with one or more groups selected from hydroxyl, alkyl, cycloalkyl, alkoxy, aryl, or substituted aryl; in certain embodiments, the substituted C1-C6alkyl or substituted C1-C6alkoxy is substituted with one or more groups selected from hydroxyl, phenyl, C1-C4alkoxy, C1-C4alkoxy substituted phenyl, cyclohexyl.

[0033] In certain embodiments, R1groups of the present application are independently selected from the group consisting of hydrogen, -CH2OH, isobutyl, t-butyl, -O(CH2)2OH, -O(CH2)3OH, -(CH2)4OH, -CH2-O(CH2)3OH, phenethyl, propyl, isopentyl, 3,3-dimethylbutyl, cyclohexylmethyl, cyclohexylethyl, phenylpropyl, 4-methoxyphenethyl.

[0034] In certain embodiments, one of the R1groups is hydrogen.

[0035] In certain embodiments, two R1groups together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl, or cyclohexyl ring.

[0036] In certain embodiments, R2groups of the present application are selected from the group consisting of hydrogen, halogen, hydroxyl, hydroxyl substituted C1-C6alkoxy, 6 membered heterocycloalkyl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein the S heteroatom is optionally oxidized.

[0037] In certain embodiments, the R2group of the present application is selected from the group consisting of hydrogen, hydroxyl, -OCH2CH2OH,

[0038]

[0039] In certain embodiments, the R2group of the present application is selected from the group consisting of hydrogen.

[0040] In certain embodiments, the R3group of the present application is selected from the group consisting of hydrogen or fluoro.

[0041] In certain embodiments, the R4group of the present application is selected from the group consisting of hydroxyl, phenyl, C1-C6alkyl or phenyl substituted C1-C6alkyl.

[0042] In certain embodiments, the R4group of the present application is selected from the group consisting of hydroxyl, phenyl, ethyl or phenylethyl.

[0043] In certain embodiments, the R4group of the present application is selected from the group consisting of hydroxyl, phenyl or phenylethyl.

[0044] In certain embodiments, the R5group of the present application is selected from the group consisting of hydrogen, substituted or unsubstituted C1-C4alkyl, C1-C4alkylcarbonyloxy-C1-C4alkyl or C1-C4alkoxycarbonyloxy-C1-C4alkyl.

[0045] In certain embodiments, the R5group of the present application is selected from the group consisting of hydrogen, ethyl, methylcarbonyloxymethyl, isopropylcarbonyloxymethyl or methoxycarbonyloxymethyl.

[0046] In certain embodiments, the compound of formula I of the present application has the following structure:

[0047]

[0048]

[0049] Another object of the present application is to provide a pharmaceutical composition comprising at least one of the aforementioned compounds, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug and mixture thereof, and at least one pharmaceutically acceptable excipient.

[0050] Another object of the present application is to provide a use of the aforementioned compound, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug and mixture thereof, or a pharmaceutical composition, for the manufacture of a medicament. The medicament can effectively inhibit plasmin activity, delay fibrinolysis, exert excellent therapeutic activity of coagulation and hemostasis, and can be used for abnormal bleeding caused by hyperfibrinolysis, surgical operation and postoperative bleeding, etc.

[0051] It is another object of the present application to provide a method of treating and / or ameliorating a bleeding disorder or condition comprising administering to a patient in need thereof one or more of the aforementioned pharmaceutical compositions or a compound of Formula I or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof.

[0052] Definitions of Terms

[0053] The following terms and phrases, as used herein, are intended to have the following meanings unless otherwise indicated. A particular term or phrase should not be construed as undefined or unclear unless specifically defined, but should be interpreted in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or active ingredient thereof.

[0054] The term "pharmaceutically acceptable" as used herein, means only that which is useful for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0055] The term "pharmaceutically acceptable salt" means a salt of a compound of the present application that is found to possess the specific substituents of the compounds of the present application and is prepared from relatively non-toxic, acid or base addition salts. Base addition salts can be obtained by contacting the compound of the present application having an acidic functional group with a sufficient amount of the base in either a neat or inert solvent to produce the neutral form of the compound. Acid addition salts can be obtained by contacting the compound of the present application having a basic functional group with a sufficient amount of the acid in either a neat or inert solvent to produce the neutral form of the compound.

[0056] The compounds of the present application can exist in particular geometric or stereoisomeric forms. The present application contemplates all such compounds. The term "isomers" as used herein is intended to encompass diastereomers, enantiomers, racemates, (D)-isomers, (L)-isomers, and mixtures thereof, as would be apparent to one skilled in the art.

[0057] "Alkyl" means a straight-chain or branched-chain saturated aliphatic hydrocarbon group, e.g., C1-C4 alkyl and C1-C6 alkyl mean saturated aliphatic hydrocarbon groups having from 1 to 4 carbon atoms and 1 to 6 carbon atoms, respectively. Examples of alkyl groups according to the present application include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isopentyl, 3,3-dimethylbutyl, and the like, and various isomers thereof.

[0058] "Alkoxy" means -O-alkyl; for example, C1-C6 alkoxy means a straight or branched chain alkoxy group containing from 1 to 6 carbons, and C1-C3 alkoxy means a straight or branched chain alkoxy group containing from 1 to 3 carbons. Examples of alkoxy groups as described herein include, but are not limited to, methoxy, ethoxy, n-propyloxy, isopropyloxy, and the like.

[0059] "Cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. For example, "C3-C6 cycloalkyl" means a cycloalkyl group containing from 3 to 6 carbon atoms. Examples of cycloalkyl groups as described herein include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and the like.

[0060] "Heteroalicyclic" means a saturated monocyclic hydrocarbon substituent in which one or more ring atoms are replaced with a heteroatom selected from N, O, S, and the remaining ring atoms are carbon, and wherein the S heteroatom is optionally oxidized. For example, "3-8 membered heteroalicyclic" means a saturated cyclic hydrocarbon substituent containing from 3 to 8 ring atoms, in which one or more ring atoms are replaced with a heteroatom selected from N, O, S, and the remaining ring atoms are carbon, and wherein the S heteroatom is optionally oxidized. Examples of heteroalicyclic groups as described herein include, but are not limited to, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, and the like.

[0061] "Heteroaromatic" means an aromatic cyclic substituent in which one or more ring atoms are replaced with a heteroatom selected from N, O, S, and the remaining ring atoms are carbon. For example, "5-6 membered heteroaromatic" means an aromatic heterocyclic group containing from 5 to 6 ring atoms. Examples of heteroaromatic groups as described herein include, but are not limited to, pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, 1,2,4-oxadiazolyl.

[0062] "Aryl" means an aromatic ring group, for example, "6-10 membered aryl" means an aromatic ring group containing from 6 to 10 carbon ring atoms. Examples of aryl moieties as described herein include, but are not limited to, phenyl, naphthyl, and the like.

[0063] "Optionally" means that the event or circumstance described subsequently can, but need not, occur.

[0064] The abbreviations used in the present invention are known to those skilled in the art, and unless otherwise indicated, represent the meanings as informed in the art. For example: DMF means N,N-dimethylformamide; THF means tetrahydrofuran; Me means methyl.

[0065] The activity of the compound of the present application is determined by plasma clot degradation experiment and thrombelastogram (TEG) experiment. In the experiment, rtPA activates plasminogen in human plasma or whole blood, respectively, and the formed plasmin can degrade fibrin, which is specifically manifested as rapid degradation of plasma fibrin clot and whole blood clot. In the above two experiments, the compound of the present application can effectively inhibit the fibrinolysis process, prolong the plasma clot lysis time (CLT, clot lysis time), and exhibit excellent coagulation and hemostasis activity. The pharmacological activity and safety of the compound of the present application are obviously superior to those of the most widely used hemostatic drug tranexamic acid in the clinic, and the compound is convenient to prepare and suitable for industrialized mass production, can effectively reduce the drug cost, and has great clinical application value. DETAILED DESCRIPTION

[0066] The synthesis method of the compound and the intermediate of the present application is illustrated below by examples, and the following examples are only used as an example of the present application, but should not be regarded as a limitation to the scope of the present application. Unless otherwise specified, the raw materials and reagents involved in the present application can be obtained through commercial channels, and the specific channel source does not affect the implementation of the technical scheme of the present application.

[0067] Example 1: Preparation of (5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl) phosphonic acid hydrochloride

[0068]

[0069] Step 1: Preparation of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)- carboxylate

[0070]

[0071] 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyridine hydrochloride (0.9 g) was suspended in dichloromethane (15 mL), N,N-diisopropyl ethylamine (1.4 g) was added, followed by the addition of di-tert-butyl dicarbonate (1.15 g), and the reaction was carried out at room temperature for 1 h. TLC showed that the raw material was consumed, the reaction solution was diluted with water, dichloromethane was extracted, the combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained crude product was purified by column chromatography to obtain the target compound (1.12 g).

[0072] MS (ESI) m / z (M+H) + = 269.0.

[0073] Step 2: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate

[0074]

[0075] Tert-butyl 2-chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (100 mg) was dissolved in toluene (20 mL) under argon atmosphere, diethyl phosphite (102 mg), tris(dibenzylideneacetone)dipalladium (34 mg), l,l'-bis(diphenylphosphino)ferrocene (41 mg) and triethylamine (75 mg) were added, the system was reacted at 120 °C overnight. TLC showed that the starting material was consumed, diluted with ethyl acetate, filtered through diatomite, the filtrate was collected and concentrated, the crude product was purified by preparative TLC to obtain the target compound (70 mg).

[0076] MS (ESI) m / z (M+H) + = 371.1.

[0077] Step 3: Preparation of (5,6,7,8-tetrahydro-l,6-naphthyridin-2-yl)phosphonic acid hydrochloride

[0078]

[0079] Tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (70 mg) was dissolved in concentrated hydrochloric acid (5 mL), and reacted at 100 °C overnight. LCMS showed that the starting material was consumed, the reaction solution was concentrated, and the crude product was purified by pre-HPLC to obtain the target compound (30 mg).

[0080] MS (ESI) m / z (M+H) + = 215.0.

[0081] 1 H NMR (400 MHz, Deuterium Oxide) δ 8.35 (dd, J = 8.0, 2.4 Hz, 1H), 8.06 (t, J = 7.7 Hz, 1H), 4.59 (s, 2H), 3.67 (t, J = 6.0 Hz, 2H), 3.49 (t, J = 6.4 Hz, 2H).

[0082] Example 2: Preparation of (3-fluoro-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0083]

[0084] Step 1: Preparation of 2-chloro-5-fluoro-6-((4-methoxybenzyl)oxy)nicotinonitrile

[0085]

[0086] Dissolve 4-methoxybenzyl alcohol (3.95 g) in tetrahydrofuran (50 mL) and cool to -78 °C with stirring. Add potassium tert-butoxide (3.5 g) under a nitrogen atmosphere and stir at 0 °C for 0.5 h. Continue cooling to -78 °C and add a solution of 2,6-dichloro-5-fluoroisonicotinitrile (5.0 g) in tetrahydrofuran (50 mL) dropwise. After the addition is complete, allow the reaction to warm to room temperature overnight. TLC shows the reaction to be complete. Concentrate under reduced pressure, add ethyl acetate and water, separate the layers, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate. Purify the crude product by column chromatography to give the title compound (6.2 g).

[0087] MS (ESI) m / z (M+H) + = 293.1

[0088] Step 2: Preparation of 5-fluoro-6-((4-methoxybenzyl)oxy)-2-vinylisonicotinic acid

[0089]

[0090] Dissolve 2-chloro-5-fluoro-6-((4-methoxybenzyl)oxy)isonicotinic acid (6.0 g), vinylpotassium trifluoroborate (5.5 g), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.29 g), and cesium fluoride (6.23 g) in 1,4-dioxane (60 mL) and water (6 mL) under a nitrogen atmosphere and heat the reaction at 90 °C overnight. TLC shows the reaction to be essentially complete. Concentrate the reaction under reduced pressure, add ethyl acetate and water, separate the layers, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate. Purify the crude product by column chromatography to give the title compound (2.68 g).

[0091] MS (ESI) m / z (M+H) + = 285.1.

[0092] Step 3: Preparation of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-7,8-dihydro-1,6- naphthyridin-5(6H)-one

[0093]

[0094] Dissolve 5-fluoro-6-((4-methoxybenzyl)oxy)-2-vinylisonicotinic acid (2.68 g) in methanol (20 mL) and water (4 mL) and add benzylamine (12.44 g). Heat the reaction at 100 °C overnight. TLC shows the reaction to be essentially complete. Concentrate under reduced pressure, add dichloromethane and water, separate the layers, wash the organic layers with 1 M dilute hydrochloric acid, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate. Purify the crude product by column chromatography to give the title compound (2.37 g).

[0095] MS (ESI) m / z (M+H)+ = 393.1.

[0096] Step 4: Preparation of 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)- 5,6,7,8-tetrahydro-l,6-naphthyridine

[0097]

[0098] Under ice water bath, 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-7,8- dihydro-l,6-naphthyridin-5(6H)-one (1.19 g) was dissolved in tetrahydrofuran (20 mL), lithium aluminum hydride (0.29 g) was added portionwise, the system was heated to 70 °C for 4 hours, TLC showed the reaction was substantially complete. 0.5 mL water, 0.5 mL 15% sodium hydroxide aqueous solution and 1.5 mL water were added dropwise in turn under ice water bath, after stirring at room temperature for 15 minutes, anhydrous magnesium sulfate was added and stirred for 15 minutes. Filtered through diatomite and anhydrous sodium sulfate, the filter residue was washed with ethyl acetate, the filtrate was concentrated to give the title compound (1.15 g).

[0099] MS (ESI) m / z (M+H) + = 379.1.

[0100] Step 5: Preparation of 6-benzyl-2-chloro-3-fluoro-5,6,7,8-tetrahydro-l,6- naphthyridine

[0101]

[0102] Under ice water bath, 6-benzyl-3-fluoro-2-((4-methoxybenzyl)oxy)-7,8- dihydro-l,6-naphthyridin-5(6H)-one (1.19 g) was dissolved in tetrahydrofuran (20 mL), lithium aluminum hydride (0.29 g) was added portionwise, the system was heated to 70 °C for 4 hours, TLC showed the reaction was substantially complete. 0.5 mL water, 0.5 mL 15% sodium hydroxide aqueous solution and 1.5 mL water were added dropwise in turn under ice water bath, after stirring at room temperature for 15 minutes, anhydrous magnesium sulfate was added and stirred for 15 minutes. Filtered through diatomite and anhydrous sodium sulfate, the filter residue was washed with ethyl acetate, the filtrate was concentrated to give the title compound (1.15 g).

[0103] MS (ESI) m / z (M+H) + = 277.1.

[0104] Step 6: Preparation of 2-chloro-3-fluoro-5,6,7,8-tetrahydro-l,6-naphthyridine

[0105]

[0106] Under ice water bath condition, 6-benzyl-2-chloro-3-fluoro-5,6,7,8-tetrahydro-l,6- naphthyridine (0.82 g) was dissolved in 1,2-dichloroethane (8 mL), N,N- diisopropylethylamine (1.93 g) and 1-chloroethyl chloroformate (2.57 g) were added successively, the reaction was carried out at 80 °C for 1.5 h, TLC showed the reaction was almost complete. The system was concentrated, dissolved in methanol, the reaction was carried out at 60 °C for 1.5 h, TLC showed the reaction was almost complete. The system was concentrated under reduced pressure, the crude product was used directly for the next step.

[0107] MS (ESI) m / z (M+H) + = 187.1.

[0108] Step 7: Preparation of tert-butyl 2-chloro-3-fluoro-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate

[0109]

[0110] Under ice water bath condition, 2-chloro-3-fluoro-5,6,7,8-tetrahydro-l,6-naphthyridine (crude above) was dissolved in dichloromethane (10 mL), triethylamine (0.91 g) and di-tert-butyl dicarbonate (0.98 g) were added, the reaction was carried out at room temperature for 2 h, TLC showed the reaction was almost complete. Dichloromethane and water were added, the organic phase was separated, combined, dried over anhydrous sodium sulfate, filtered, concentrated. The crude product was purified by column chromatography to give the title compound (0.28 g).

[0111] MS (ESI) m / z (M+H) + = 287.1.

[0112] Step 8: Preparation of tert-butyl 2-(diethoxyphosphoryl)-3-fluoro-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0113]

[0114] Under nitrogen atmosphere, tert-butyl 2-chloro-3-fluoro-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (100 mg) was dissolved in toluene (8 mL), diethyl phosphite (97 mg), palladium acetate (16 mg), 1,1'-bis(diphenylphosphino)ferrocene (78 mg) and triethylamine (71 mg) were added successively, the reaction was carried out at 110 °C overnight. TLC showed the reaction was almost complete. The system was concentrated under reduced pressure, the crude product was purified by column chromatography to give the title compound (130 mg).

[0115] MS (ESI) m / z (M+H) + = 389.1.

[0116] Step 9: Preparation of (3-fluoro-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphinic acid hydrochloride

[0117]

[0118] tert-Butyl 2-(diethoxyphosphoryl)-3-fluoro-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (60 mg) was dissolved in concentrated hydrochloric acid (3 mL) and heated to 100 °C for 2 h in a sealed tube. TLC showed that the reaction was essentially complete. The system was concentrated and the crude product was purified by pre-HPLC to give the title compound (30 mg).

[0119] MS (ESI) m / z (M+H) + = 233.0.

[0120] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.77 (d, J = 7.1 Hz, 1H), 4.48 (s, 2H), 3.58 (s, 2H), 3.24 (s, 2H).

[0121] Example 3: Preparation of ethyl (5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphinic acid hydrochloride

[0122]

[0123] Step 1: Preparation of (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-l,6- naphthyridin-2-yl)phosphinic acid

[0124] tert-Butyl 2-chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (2 g), aniline phosphorodithioate (4.8 g), were dissolved in acetonitrile (40 mL), and then added with tris(dibenzylideneacetone)dipalladium (680 mg), diphenylphosphinyl ferrocene (830 mg), triethylamine (5.2 mL). After nitrogen replacement, the system was heated at 85 °C overnight, and then at 95 °C for 4 h. LC-MS monitoring showed that no starting material was left. The system was cooled to room temperature, and then adjusted to pH 3 with 2 M dilute hydrochloric acid. The residue was purified by reverse phase column to give the title compound (1.6 g).

[0125]

[0126] MS (ESI) m / z (M+H) + = 299.1.

[0127] Step 2: Preparation of methyl (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-l,6- naphthyridin-2-yl)phosphinate

[0128] Methyl (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-l,6-naphthyridin-2-yl)phosphinate (400 mg) was taken in dichloromethane (10 mL) and methyl chloroformate (0.2 mL) was added at room temperature under nitrogen atmosphere. Pyridine (0.2 mL) was added drop wise after some time and the reaction was stirred at 45 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and quenched with water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic extracts were dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography to afford the title compound (300 mg).

[0129]

[0130] MS (ESI) m / z (M+H) + = 313.1.

[0131] Step 3: Preparation of tert-butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0132] Methyl (6-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-l,6-naphthyridin-2-yl)phosphinate (50 mg) was taken in dry tetrahydrofuran (2 mL) and lithium hexamethyldisilylamide (0.19 mL, 1 M in THF) was added at -78 °C under nitrogen atmosphere. The reaction was stirred at -78 °C for 20 min and iodoethane (17 uL) was added drop wise. The reaction was stirred at room temperature for 1 h. The reaction was monitored by LC-MS. The reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography to afford the title compound (20 mg).

[0133]

[0134] MS (ESI) m / z (M+H) + = 341.1.

[0135] Step 4: Preparation of ethyl (5,6,7,8-tetrahydro-l,6-naphthyridin-2-yl)phosphinate hydrochloride

[0136] Tert-butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (20 mg) was taken in 6 M hydrochloric acid and the reaction was stirred at 105 °C for overnight. The reaction was monitored by LC-MS. The reaction mixture was cooled to room temperature and evaporated under reduced pressure. The residue was purified by pre-HPLC to afford the title compound (10 mg).

[0137]

[0138] MS (ESI) m / z (M+H) + = 227.1.

[0139] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.63 - 7.62 (m, 2H), 4.36 (s, 2H), 3.57 - 3.53 (t, J = 6.5 Hz, 2H), 3.18 - 3.15 (t, J = 6.4 Hz, 2H), 1.74 - 1.65 (dq, J = 15.3, 7.7 Hz, 2H), 0.85 - 0.76 (dt, J = 18.6, 7.9 Hz, 3H).

[0140] Example 4: Preparation of phenethyl(5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphinic acid hydrochloride

[0141]

[0142] Step 1: Preparation of tert-butyl 2-(methoxy(phenethenyl)phosphoryl)-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0143] tert-Butyl 2-(ethyl(methoxy)phosphoryl)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (50 mg), (2-bromovinyl)benzene (31 uL) were dissolved in toluene (3 mL), then tris(dibenzylideneacetone)dipalladium (15 mg), diphenylphosphinyl ferrocene (18 mg), triethylamine (5.2 mL) were added, after nitrogen replacement, the mixture was heated at 120 °C for 6 h, LC-MS monitoring no starting material left. After cooling to room temperature, the solvent was evaporated under reduced pressure, then the title compound (35 mg) was obtained by column purification.

[0144]

[0145] MS (ESI) m / z (M+H) + = 415.1.

[0146] Step 2: Preparation of tert-butyl 2-(methoxy(phenethyl)phosphoryl)-7,8-dihydro- l,6-naphthyridine-6(5H)-carboxylate

[0147] tert-Butyl 2-(methoxy(phenethenyl)phosphoryl)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (35 mg) was dissolved in ethanol (3 mL), then 10% palladium on carbon (20 mg, 55% water) was added, the mixture was stirred under hydrogen atmosphere at 60 °C overnight, LCMS monitoring reaction was complete. After cooling to room temperature, the mixture was filtered, then the filtrate was concentrated to obtain the title compound (40 mg crude).

[0148]

[0149] MS (ESI) m / z (M+H)+ = 417.1.

[0150] Step 3: Preparation of phenethyl(5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphinic acid hydrochloride

[0151] tert-Butyl 2-(methoxy(phenethyl)phosphoryl)-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate (40 mg crude) was weighed into and 3 mL of concentrated hydrochloric acid was added, and the reaction was stirred at 100 °C for 3 h. The reaction was monitored by LC-MS until completion. The reaction was cooled to room temperature and the solvent was evaporated under reduced pressure. The residue was purified by pre-HPLC to give the title compound (11.7 mg).

[0152]

[0153] MS (ESI) m / z (M-H) - = 301.0.

[0154] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.57 - 7.50 (m, 2H), 7.05 - 7.01 (m, 3H), 6.95 - 6.93 (dd, J = 7.5, 2.1 Hz, 2H), 4.30 (s, 2H), 3.49 - 3.46 (t, J = 6.4 Hz, 2H), 2.98 - 2.95 (t, J = 6.4 Hz, 2H), 2.67 - 2.59 (dt, J = 15.1, 7.6 Hz, 2H), 2.14 - 2.07 (dt, J = 15.2, 7.6 Hz, 2H).

[0155] Example 5: (8,8-difluoro-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0156]

[0157] Step 1: Preparation of tert-butyl 2-chloro-8,8-difluoro-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0158] tert-Butyl 2-chloro-8-oxo-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (0.3 g) was dissolved in dichloromethane (4 mL) and diethylamine sulfide trifluoride (342 mg) was added dropwise under ice bath. The reaction was stirred for 1 h under ice bath. The reaction was monitored by LC-MS until completion. The system was added water and dichloromethane was washed for 3 times. The organic phase was dried and concentrated to dryness. The title compound (225 mg) was purified by column chromatography.

[0159]

[0160] MS (ESI) m / z (M+H)+ = 304.0.

[0161] Step 2: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-8,8-difluoro-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0162] tert-Butyl 2-chloro-8,8-difluoro-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (100 mg) was weighed into a dry reaction flask, dissolved in toluene (10 mL), 1,1'- bis(diphenylphosphino)ferrocene palladium dichloride dichloromethane complex (54 mg), triethylamine (0.09 mL), di-tert-butyl phosphonate (128 mg) were added, the system was replaced with nitrogen for 3 times and heated to 100 °C. The reaction was allowed to proceed overnight. LC-MS indicated the reaction was complete, the system was concentrated to dryness. Column chromatography purification afforded the title compound (100 mg).

[0163]

[0164] MS (ESI) m / z (M+H) + = 463.2.

[0165] Step 3: Preparation of (8,8-difluoro-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0166] tert-Butyl 2-(di-tert-butoxyphosphoryl)-8,8-difluoro-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate (100 mg) was dissolved in dichloromethane (3 mL), 4M hydrochloric acid in 1.4-dioxane (3 mL) was added dropwise, stirred at room temperature for 1 hour, LC-MS indicated the reaction was complete, the solvent was evaporated under reduced pressure. Pre-HPLC purification afforded the title compound (20 mg).

[0167]

[0168] MS (ESI) m / z (M+H) + = 250.9.

[0169] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.89 (ddd, J = 23.5, 8.2, 4.8 Hz, 2H), 4.57 (s, 2H), 4.08 (t, J = 11.6 Hz, 2H).

[0170] Example 6 (5,6,7,8-Tetrahydro-l,6-naphthyridin-2-yl)phosphonic acid monohydrate

[0171]

[0172] (5,6,7,8-Tetrahydro-l,6-naphthyridin-2-yl)phosphonic acid hydrochloride was dissolved in 5 volumes of water, then the pH was adjusted to about 4.2 with 10% sodium hydroxide, and the solid was precipitated, filtered and dried to give the title compound.

[0173] MS (ESI) m / z (M+H) + = 215.0.

[0174] 1 H NMR (400 MHz, Methanol-d4) δ 7.72 (dd, J = 8.0, 5.6 Hz, 1H), 7.37 (dd J = 8.0, 3.6 Hz, 1H), 3.98 (s, 2H), 3.19 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 6.0 Hz, 2H).

[0175] Example 7: Preparation of (8-morpholino-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0176]

[0177] Step 1: Preparation of tert-butyl 2-chloro-8-((methylsulfonyl)oxy)-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0178] tert-Butyl 2-chloro-8-hydroxy-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (0.14 g) was dissolved in dichloromethane (10 mL), and triethylamine (0.15 g), methylsulfonyl chloride (0.11 g) were added successively. The reaction was allowed to proceed at room temperature for 1 hour, TLC showed that the reaction was substantially complete. The reaction was quenched by the addition of water (10 mL), the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness. The title compound (0.16 g) was purified by column chromatography.

[0179]

[0180] MS (ESI) m / z (M+H) + = 363.1.

[0181] Step 2: Preparation of tert-butyl 2-chloro-8-morpholino-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate

[0182] Take 2-chloro-8-((methylsulfonyl)oxy)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylic acid tert-butyl ester (0.16 g) dissolved in acetonitrile (3 mL) and N,N- dimethylformamide (3 mL), add potassium carbonate (0.12 g), morpholine (0.12 g), replace with nitrogen three times, react at 60 °C overnight, monitor the reaction completion by LCMS. Concentrate to remove acetonitrile, add water (10 mL) and ethyl acetate (10 mL), extract and separate the organic phase, dry and concentrate, purify the crude product by column chromatography to obtain the title compound (0.11 g).

[0183]

[0184] MS (ESI) m / z (M+H) + = 354.1.

[0185] Step 3: Preparation of 2-(diethoxyphosphoryl)-8-morpholino-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylic acid tert-butyl ester

[0186] Take 2-chloro-8-morpholino-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylic acid tert- butyl ester (0.11 g) dissolved in toluene (3 mL), add diethyl phosphite (86 mg), tris(dibenzylideneacetone)dipalladium (57 mg), l,l'-bis(diphenylphosphino)ferrocene (69 mg), triethylamine (62 mg) in sequence. Replace with nitrogen three times, react at 120 °C overnight under nitrogen atmosphere, LCMS shows the reaction is substantially complete. Concentrate to dryness under reduced pressure, purify the crude product by column chromatography to obtain the title compound (0.12 g).

[0187]

[0188] MS (ESI) m / z (M+H) + = 456.2.

[0189] Step 4: Preparation of (8-morpholino-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0190] Take 2-(diethoxyphosphoryl)-8-morpholino-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylic acid tert-butyl ester (60 mg), add concentrated hydrochloric acid (4 mL), react at 100 °C for 2 hours under seal, TLC shows the reaction is substantially complete. Concentrate the system to dryness, separate by pre-HPLC to obtain the title compound (11.7 mg).

[0191]

[0192] MS (ESI) m / z (M+H) + = 300.1.

[0193] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.70 (m, 2H), 5.14 (dd, J = 10.5, 6.3 Hz, 1H), 4.56-4.43 (m, 2H), 4.19 (dd, J = 12.7, 6.2 Hz, 1H), 3.98 (s, 4H), 3.90-3.81 (m, 1H), 3.40 (s, 2H), 3.27 (s, 2H).

[0194] Example 8: Preparation of (8-hydroxy-5,6,7,8-tetrahydro-l,6-naphthyridin-2-yl) phosphonic acid hydrochloride

[0195]

[0196] Step 1: Preparation of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-chloro-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0197] tert-Butyl 8-((tert-butyldimethylsilyl)oxy)-2-chloro-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate (0.20 g) was taken in toluene (10 mL), diethyl phosphite (138 mg), tris(dibenzylideneacetone)dipalladium (92 mg), 1,1'-bis(diphenylphosphino) ferrocene (110 mg), triethylamine (101 mg) were added successively. Nitrogen was purged for three times and the reaction was carried out at 110 °C under nitrogen atmosphere overnight. LCMS showed the reaction was completed. The reaction mixture was concentrated to dryness under reduced pressure and purified by column chromatography to get the title compound (0.16 g).

[0198]

[0199] MS (ESI) m / z (M+H) + = 399.2.

[0200] Step 2: Preparation of tert-butyl 8-((tert-butyldimethylsilyl)oxy)-2-(diethoxyphosphoryl)- 7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0201] tert-Butyl 8-((tert-butyldimethylsilyl)oxy)-2-chloro-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate (0.20 g) was taken in toluene (10 mL), diethyl phosphite (138 mg), tris(dibenzylideneacetone)dipalladium (92 mg), 1,1'-bis(diphenylphosphino) ferrocene (110 mg), triethylamine (101 mg) were added successively. Nitrogen was purged for three times and the reaction was carried out at 110 °C under nitrogen atmosphere overnight. LCMS showed the reaction was completed. The reaction mixture was concentrated to dryness under reduced pressure and purified by column chromatography to get the title compound (0.16 g).

[0202]

[0203] MS (ESI) m / z (M+H) + = 501.3.

[0204] Step 3: Preparation of (8-hydroxy-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0205] tert-Butyl 8-((tert-butyldimethylsilyl)oxy)-2-(diethoxyphosphoryl)-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate (70 mg) was weighed into and concentrated hydrochloric acid (4 mL) was added. The reaction was heated to 100 °C for 2 h under a seal. TLC indicated that the reaction was complete. The reaction was concentrated to dryness and purified by pre-HPLC to give the title compound (2.4 mg).

[0206]

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

[0208] 1 H 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-l,6- naphthyridin-2-yl)phosphonic acid hydrochloride

[0209] Step 1: Preparation of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2- chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0210] tert-Butyl 2-chloro-8-hydroxy-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (0.60 g) was dissolved in tetrahydrofuran (10 mL) and NaH (1.0 g) was added under an ice bath. After stirring for 5 min, (2-bromoethoxy)(tert-butyl)dimethylsilane was added and the reaction was stirred at room temperature for 2 h. TLC indicated that the reaction was complete. The reaction was quenched with ice water and extracted with ethyl acetate and water. The organic phase was collected, dried and concentrated to dryness under reduced pressure. The title compound was purified by column chromatography (0.40 g).

[0211]

[0212] MS (ESI) m / z (M+H) + = 443.2.

[0213] Step 2: Preparation of tert-butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2- (diethoxyphosphoryl)-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0214] tert-Butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-chloro-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate (0.40 g) was weighed into toluene (10 mL), diethyl phosphite (0.25 g), tris(dibenzylideneacetone)dipalladium (0.17 g), 1,1'- bis(diphenylphosphino)ferrocene (0.20 g), triethylamine (0.18 g) were added sequentially. Nitrogen was bubbled through the mixture three times and the reaction was heated at 110 °C under a nitrogen atmosphere overnight. LCMS showed the reaction was essentially complete. The reaction was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (0.44 g).

[0215]

[0216] MS (ESI) m / z (M+H) + = 545.3.

[0217] Step 3: Preparation of (8-(2-hydroxyethoxy)-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0218] tert-Butyl 8-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-(diethoxyphosphoryl)-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate (70 mg) was dissolved in 1,4- dioxane (5 mL), 1 M trimethylsilyl bromide (0.2 mL) was added and the reaction was refluxed at 75 °C overnight. LCMS showed the reaction was essentially complete. The reaction was concentrated to dryness and dissolved in 4 M hydrochloric acid in 1,4- dioxane and stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction was concentrated to dryness and the crude product was purified by pre-HPLC to give the title compound (30.0 mg).

[0219]

[0220] MS (ESI) m / z (M+H) + = 275.0.

[0221] 1H NMR (400 MHz, Deuterium Oxide) δ 7.79 (d, J = 5.0 Hz, 2H), 4.77 (s, 1H), 4.43 (q, J = 16.5 Hz, 2H), 3.93 (d, J = 13.5 Hz, 1H), 3.84 - 3.78 (m, 1H), 3.77 - 3.70 (m, 1H), 3.63 (t, J = 4.4 Hz, 2H), 3.49 (d, J = 13.6 Hz, 1H).

[0222] Example 10: Preparation of (8-oxidothiomorpholine-5,6,7,8-tetrahydro-l,6- naphthyridin-2-yl)phosphonic acid hydrochloride

[0223] Step 1: Preparation of tert-butyl 2-chloro-8-((methylsulfonyl)oxy)-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0224] tert-Butyl 2-chloro-8-((methylsulfonyl)oxy)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (0.30 g) was weighed into acetonitrile (5 mL) and N,N- dimethylformamide (5 mL), potassium carbonate (0.12 g) and thiomorpholine (0.12 g) were added, the reaction was purged with nitrogen three times and stirred at 60 °C overnight, the reaction was monitored by LCMS. The acetonitrile was removed by concentration, water (10 mL) and ethyl acetate (10 mL) were added, the organic phase was separated and dried, the crude product was purified by column chromatography to give the title compound (0.12 g).

[0225]

[0226] MS (ESI) m / z (M+H) + = 370.1.

[0227] Step 2: Preparation of tert-butyl 2-(di-tert-butyiphosphoryl)-8-thiomorpholine-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0228] tert-Butyl 2-chloro-8-thiomorpholine-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (0.23 g) was weighed into toluene (10 mL), di-tert-butyl phosphite (242 mg), tris(dibenzylideneacetone)dipalladium (114 mg), 1,1'- bis(diphenylphosphino)ferrocene (138 mg) and triethylamine (130 mg) were added sequentially, the reaction was purged with nitrogen three times and stirred at 120 °C overnight under a nitrogen atmosphere, the reaction was monitored by LCMS. The reaction was concentrated to dryness under reduced pressure and the product was purified by column chromatography to give the title compound (0.24 g).

[0229]

[0230] MS (ESI) m / z (M+H) + = 528.2.

[0231] Step 3: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-8-(1- oxidothiomorpholino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0232] tert-butyl 2-(di-tert-butoxyphosphoryl)-8-(1-oxidothiomorpholino)-7,8- dihydro-1,6-naphthyridine-6(5H)-carboxylate (90 mg) was weighed into acetic acid (2 mL), and urea hydrogen peroxide (242 mg) was added. The reaction was stirred at room temperature for 2 hours. LCMS showed that the reaction was substantially complete. Water (50 mL) and ethyl acetate (10 mL x 3) were added, and the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (60 mg).

[0233]

[0234] MS (ESI) m / z (M+H) + = 544.2.

[0235] Step 4: Preparation of (8-(1-oxidothiomorpholino)-5,6,7,8-tetrahydro-1,6- naphthyridin-2-yl)phosphonic acid hydrochloride

[0236] tert-butyl 2-(di-tert-butoxyphosphoryl)-8-(1-oxidothiomorpholino)-7,8- dihydro-1,6-naphthyridine-6(5H)-carboxylate (60 mg) was dissolved in dichloromethane (4 mL), and concentrated hydrochloric acid (0.1 mL) was added under ice bath. The reaction was stirred at ice bath for 0.5 hours. TLC showed that the reaction was substantially complete. The system was concentrated to dryness, and the title compound (2.94 mg) was isolated by pre-HPLC.

[0237]

[0238] MS (ESI) m / z (M+H) + = 332.0.

[0239] 1 H NMR (400 MHz, Deuterium Oxide) d 7.96 (m, 1H), 7.88 (m, 1H), 5.09 (dd, J = 10.8, 6.0 Hz, 1H), 4.61 - 4.49 (m, 2H), 4.20 (dd, J = 12.7, 6.0 Hz, 1H), 3.88 (m, 2H), 3.68 - 3.58 (m, 2H), 3.37 - 3.23 (m, 3H), 3.18 - 3.02 (m, 2H).

[0240] Example 11: Preparation of (7-isobutyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0241]

[0242] Step 1: Preparation of tert-butyl 2-chloro-7-(2-methylprop-l-en-l-yl)-7,8-dihydro- l,6-naphthyridine-6(5H)-carboxylate

[0243] Isopropyl triphenylphosphonium iodide (2.918 g) was weighed into a dry reaction flask and dissolved in N,N-dimethylformamide (10 mL). Sodium hydride (0.27 g) was added and the system was purged with nitrogen three times. The reaction was stirred at 0 °C for 20 min and tert-butyl 2-chloro-7-formyl-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (1 g) was added. The reaction was stirred for 3 h. The reaction was monitored by LC-MS and when complete, the system was quenched with saturated ammonium chloride solution and extracted with ethyl acetate three times. The organic phase was dried and concentrated to dryness. The title compound (280 mg) was purified by column chromatography.

[0244]

[0245] MS (ESI) m / z (M+H) + = 323.1.

[0246] Step 2: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7-(2-methylprop-l-en-l-yl)- 7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0247] Tert-butyl 2-chloro-7-(2-methylprop-l-en-l-yl)-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate (280 mg) was weighed into a dry reaction flask and dissolved in toluene (10 mL). Tris(dibenzylideneacetone)dipalladium (124.5 mg), l,l'-bis(diphenylphosphino) ferrocene (192.7 mg), triethylamine (0.0.24 mL), and diethyl phosphonate (240 mg) were added and the system was purged with nitrogen three times. The reaction was heated to 110 °C. The reaction was stirred for 4 h. The reaction was monitored by LC-MS and when complete, the system was concentrated to dryness. The title compound (300 mg) was purified by column chromatography.

[0248]

[0249] MS (ESI) m / z (M+H) + = 425.2.

[0250] Step 3: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7-isobutyl-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0251] Take 2-(diethoxyphosphoryl)-7-(2-methylprop-1-en-1-yl)-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate tert-butyl salt (300 mg), dissolve in methanol (15 mL), pass in hydrogen gas in a high pressure kettle, warm up to 40 °C and stir overnight, check reaction completion by LC-MS, filter through celite, evaporate solvent under reduced pressure. Purify by column chromatography to get the title compound (90 mg).

[0252]

[0253] MS (ESI) m / z (M+H) + = 427.2.

[0254] Step 4: Preparation of (7-isobutyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0255] Take 2-(diethoxyphosphoryl)-7-isobutyl-7,8-dihydro-1,6-naphthyridine-6(5H)- carboxylate tert-butyl salt (90 mg), dissolve in 12 M hydrochloric acid solution (5 mL), warm up to 100 °C and stir for 3 hours, check reaction completion by LC-MS, evaporate solvent under reduced pressure. Purify by pre-HPLC to get the title compound (50 mg).

[0256]

[0257] MS (ESI) m / z (M+H) + = 271.1.

[0258] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.71 (dd, J = 7.9, 3.7 Hz, 1H), 7.64 (t, J = 7.0 Hz, 1H), 4.39 (s, 2H), 3.70 (q, J = 10.7, 7.9 Hz, 1H), 3.32 (dd, J = 18.4, 4.7 Hz, 1H), 2.94 (dd, J = 18.1, 10.7 Hz, 1H), 1.75 (dt, J = 13.4, 6.8 Hz, 1H), 1.59 (t, J = 7.2 Hz, 2H), 0.85 (dd, J = 12.3, 6.4 Hz, 6H).

[0259] Example 12: Preparation of (7-propyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0260]

[0261] Step 1: Preparation of 2-methyl-N-(4-ylidene)propane-2-sulfmamide

[0262] Dissolve butyraldehyde (10 g) in dichloromethane (100 mL), add 2-methylpropane-2- sulfinamide (20 g), anhydrous magnesium sulfate (83.3 g) and pyridine 4-methylbenzenesulfonic acid (1.74 g) in sequence. Heat to 40 °C and stir for 24 h. Monitor the reaction by LC-MS. Cool to room temperature, filter, wash the filter cake with dichloromethane, concentrate the filtrate to dryness and purify by column chromatography to give the title compound (22.4 g).

[0263]

[0264] MS (ESI) m / z (M+H) + = 176.1

[0265] Step 2: Preparation of N-(l-(3-bromo-6-methoxypyridin-2-yl)-5-butane-2-yl)-2- methylpropane-2-sulfinamide

[0266] Dissolve butyraldehyde (10 g) in dichloromethane (100 mL), add 2-methylpropane-2- sulfinamide (20 g), anhydrous magnesium sulfate (83.3 g) and pyridine 4-methylbenzenesulfonic acid (1.74 g) in sequence. Heat to 40 °C and stir for 24 h. Monitor the reaction by LC-MS. Cool to room temperature, filter, wash the filter cake with dichloromethane, concentrate the filtrate to dryness and purify by column chromatography to give the title compound (22.4 g).

[0267]

[0268] MS (ESI) m / z (M+H) + = 377.1.

[0269] Step 3: Preparation of 2-(2-((tert-butylsulfinyl)amino)-5-butyl)-6-methoxynicotinic acid ethyl ester

[0270] Dissolve butyraldehyde (10 g) in dichloromethane (100 mL), add 2-methylpropane-2- sulfinamide (20 g), anhydrous magnesium sulfate (83.3 g) and pyridine 4-methylbenzenesulfonic acid (1.74 g) in sequence. Heat to 40 °C and stir for 24 h. Monitor the reaction by LC-MS. Cool to room temperature, filter, wash the filter cake with dichloromethane, concentrate the filtrate to dryness and purify by column chromatography to give the title compound (22.4 g).

[0271] Dissolve butyraldehyde (10 g) in dichloromethane (100 mL), add 2-methylpropane-2- sulfinamide (20 g), anhydrous magnesium sulfate (83.3 g) and pyridine 4-methylbenzenesulfonic acid (1.74 g) in sequence. Heat to 40 °C and stir for 24 h. Monitor the reaction by LC-MS. Cool to room temperature, filter, wash the filter cake with dichloromethane, concentrate the filtrate to dryness and purify by column chromatography to give the title compound (22.4 g).

[0272] MS (ESI) m / z (M+H) + = 371.1.

[0273] Step 4: Preparation of 2-methoxy-7-(3-propyl)-7,8-dihydro-l,6-naphthyridin-5(6H)-one

[0274] Ethyl 2-(2-((tert-butylsulfinyl)amino)-5-butyl)-6-methoxynicotinate (4 g) was weighed into acetonitrile (100 mL) and cesium carbonate (17.6 g) was added. The reaction was stirred at 80 °C overnight and monitored by LC-MS. The reaction was cooled to room temperature, suction filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated to dryness and purified by column chromatography to give the title compound (2.55 g).

[0275]

[0276] MS (ESI) m / z (M+H) + = 221.1.

[0277] Step 5: Preparation of 2-methoxy-7-(3-propyl)-5,6,7,8-tetrahydro-l,6-naphthyridine

[0278] 2-methoxy-7-(3-propyl)-7,8-dihydro-l,6-naphthyridin-5(6H)-one (2.55 g) was weighed into tetrahydrofuran (100 mL) and lithium aluminum hydride (2.6 g) was added under ice bath. The reaction was stirred at 70 °C for 8 hours and monitored by LC-MS. Water (2.6 mL), sodium hydroxide solution (15%, 2.6 mL), and water (7.8 mL) were added dropwise under ice bath. The reaction was stirred at room temperature for 20 minutes, dried over anhydrous magnesium sulfate, suction filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography to give the title compound (1.9 g).

[0279]

[0280] MS (ESI) m / z (M+H) + = 207.1.

[0281] Step 6: Preparation of 7-(3-propyl)-5,6,7,8-tetrahydro-l,6-naphthyridin-2-ol

[0282] 2-methoxy-7-(3-propyl)-5,6,7,8-tetrahydro-l,6-naphthyridine (1.9 g) was weighed into acetic acid (5 mL) and hydrobromic acid was added. The reaction was stirred at 80 °C for 5 hours and monitored by LC-MS. The solvent was removed under reduced pressure and the residue was slurried with ethyl acetate, filtered, and dried to give the title compound as a crude product (1.5 g).

[0283]

[0284] MS (ESI) m / z (M+H) + = 193.1.

[0285] Step 7: Preparation of 2-chloro-7-(3-propyl)-5,6,7,8-tetrahydro-l,6-naphthyridine

[0286] To a dry reaction flask, 7-(3-propyl)-5,6,7,8-tetrahydro-l,6-naphthyridin-2-ol (0.5 g) was weighed and phosphorous oxychloride (10 mL) was added. The reaction mixture was heated to 100 °C for 4 h. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. Ice water and dichloromethane were added to the reaction mixture to obtain the crude title compound.

[0287]

[0288] MS (ESI) m / z (M+H) + = 211.1.

[0289] Step 8: Preparation of tert-butyl 2-chloro-7-(3-propyl)-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0290] To the work-up system of Step 7, di-tert-butyl dicarbonate (1.29 mL) was added with sodium carbonate solution to adjust the pH to 8-9.

[0291] The reaction was stirred at room temperature for 1 h. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The title compound (0.5 g) was purified by column chromatography.

[0292]

[0293] MS (ESI) m / z (M+H) + = 311.1.

[0294] Step 9: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-7-propyl-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0295] To a dry reaction flask, tert-butyl 2-chloro-7-(3-propyl)-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate (93 mg) was weighed and dissolved in toluene (10 mL). Tris(dibenzylideneacetone)dipalladium (55 mg), l,l'-bis(diphenylphosphino) ferrocene (67 mg), triethylamine (61 mg), di-tert-butyl phosphonate (120 mg) were added. The system was purged with nitrogen for 3 times and heated to 115 °C. The reaction was allowed to proceed overnight. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The title compound (105 mg) was purified by column chromatography.

[0296]

[0297] MS (ESI) m / z (M+H) + = 469.2.

[0298] Step 10: Preparation of (7-propyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0299] tert-Butyl 2-(di-tert-butyloxyphosphoryl)-7-propyl-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate (105 mg) was dissolved in dichloromethane (4 mL) and 4M hydrochloric acid in 1.4-dioxane (4 mL) was added dropwise. The reaction was stirred at room temperature for 1 hour, and was complete by LC-MS. The solvent was removed under reduced pressure. Pre-HPLC purification gave the title compound (8 mg).

[0300]

[0301] MS (ESI) m / z (M+H) + = 257.0.

[0302] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.75 (dd, J = 8.0, 3.8 Hz, 1H), 7.68 (dd, J = 7.9, 6.2 Hz, 1H), 4.43 (s, 2H), 3.67 (ddd, J = 10.9, 5.3, 2.1 Hz, 1H), 3.34 (dd, J = 18.2, 4.8 Hz, 1H), 3.01 (dd, J = 18.2, 10.8 Hz, 1H), 1.74 (dtd, J = 14.7, 8.5, 6.7 Hz, 2H), 1.52 - 1.35 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0303] Example 13 Preparation of (7-phenethyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0304]

[0305] Step 1: Preparation of 2-methyl-N-(3-phenylpropylidene)propane-2- sulfmamide

[0306] Weigh 3-phenylpropanal (10.73 g) into dichloromethane (120 mL), add magnesium sulfate (41.2 g), 4-methylbenzenesulfonic acid pyridine (1.0 g), 2-methylpropane-2-sulfinamide (10.7 g), replace with nitrogen 3 times, reflux overnight. Monitor the reaction by LC-MS. When the reaction is complete, filter the system, wash with ethyl acetate 3 times, dry the organic phase, concentrate to dryness, and purify by column chromatography to give the title compound (12.44 g).

[0307]

[0308] MS (ESI) m / z (M+H) + = 338.1.

[0309] Step 2: Preparation of N-(l-(3-bromo-6-methoxypyridin-2-yl)-4-phenylbutan-2-yl)-2- methylpropane-2-sulfinamide

[0310] Weigh tetrahydrofuran (25 mL) into a dry reaction flask, replace with nitrogen 3 times, add 2M diisopropylamino lithium (12.4 mL), and cool to -78°C. Add a solution of 3-bromo-6-methoxy-2-methylpyridine (5 g) and tetrahydrofuran (5 mL), and stir at -78°C for 1 hour. Add a solution of 2-methyl-N-(3-phenylpropylidene)propane-2-sulfinamide (6.45 g) and tetrahydrofuran (15 mL), and stir at -78°C to -30°C for 2 hours. Monitor the reaction by LC-MS. When the reaction is complete, quench with saturated ammonium chloride solution, extract with ethyl acetate 3 times, dry the organic phase, concentrate to dryness, and purify by column chromatography to give the title compound (6.4 g).

[0311]

[0312] MS (ESI) m / z (M+H) + = 439.1.

[0313] Step 3: Preparation of ethyl 2-(2-((tert-butylsulfinyl)amino)-4-phenylbutyl)-6- methoxynicotinate

[0314] Weigh N-(l-(3-bromo-6-methoxypyridin-2-yl)-4-phenylbutan-2-yl)-2-methylpropane-2- sulfinamide (2 g) into a dry reaction flask, dissolve in ethanol (10 mL), add l,l'-ferrocenediyl- bis(diphenylphosphine)palladium dichloride (0.67 g), triethylamine (1.2 mL), replace with nitrogen 3 times, and heat to 110°C. Reflux overnight. Monitor the reaction by LC-MS. When the reaction is complete, concentrate the system to dryness. Purify by column chromatography to give the title compound (1 g).

[0315]

[0316] MS (ESI) m / z (M+H) + = 433.2.

[0317] Step 4: Preparation of 6-(tert-butylsulfinyl)-2-methoxy-7-phenethyl-7,8- dihydro-l,6-naphthyridin-5(6H)-one

[0318] Ethyl 2-(2-((tert-butylsulfinyl)amino)-4-phenylbutyl)-6-methoxynicotinate (1 g) was weighed into a dry reaction flask and dissolved in acetonitrile (10 mL). Cesium carbonate (124.5 mg) was added and the reaction was heated to 80 °C. The reaction was complete after 6 h as determined by LC-MS. The reaction mixture was filtered through celite and the filtrate was concentrated to dryness. The title compound (0.6 g) was purified by column chromatography.

[0319]

[0320] MS (ESI) m / z (M+H) + = 387.1.

[0321] Step 5: Preparation of 2-hydroxy-7-phenethyl-7,8-dihydro-l,6-naphthyridin-5(6H)- one

[0322] 6-(tert-Butylsulfinyl)-2-methoxy-7-phenethyl-7,8-dihydro-l,6-naphthyridin-5(6H)- one (600 mg) was dissolved in 33% hydrogen bromide in acetic acid (10 mL) and the reaction was heated to 80 °C with stirring for 2 h. The reaction was complete as determined by LC-MS. The solvent was removed under reduced pressure to give the title compound (1.2 g) as a crude product.

[0323]

[0324] MS (ESI) m / z (M+H) + = 269.1.

[0325] Step 6: Preparation of 2-chloro-7-phenethyl-7,8-dihydro-l,6-naphthyridin-5(6H)- one

[0326] 2-hydroxy-7-phenethyl-7,8-dihydro-l,6-naphthyridin-5(6H)-one (1.2 g) was dissolved in phosphorous oxychloride (10 mL) and the reaction was heated to 95 °C with stirring for 2 h. The reaction was complete as determined by LC-MS. The solvent was removed under reduced pressure. The residue was diluted with ethyl acetate and the pH was adjusted to 7-8 with sodium carbonate. The reaction mixture was extracted with ethyl acetate three times. The organic phase was concentrated and purified by column chromatography on silica gel to give the title compound (200 mg).

[0327]

[0328] MS (ESI) m / z (M+H) + = 287.1.

[0329] Step 7: Preparation of tert-butyl 2-chloro-5-oxo-7-phenethyl-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0330] tert-Butyl 2-chloro-7-phenethyl-7,8-dihydro-l,6-naphthyridin-5(6H)-one (185 mg) was dissolved in dichloromethane (10 mL), di-tert-butyl dicarbonate (423 mg), 4-dimethylaminopyridine (31.5 mg), triethylamine (391 mg) were added, and the mixture was stirred at 40 °C overnight. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The title compound (230 mg) was obtained by silica gel column purification.

[0331]

[0332] MS (ESI) m / z (M+H) + = 387.1.

[0333] Step 8: Preparation of tert-butyl 2-chloro-7-phenethyl-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylate

[0334] tert-Butyl 2-chloro-5-oxo-7-phenethyl-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate (230 mg) was dissolved in tetrahydrofuran (6 mL), and 2.5 M borane dimethyl sulfide (6 mL) was added. The mixture was stirred at 50 °C for 4 h. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The title compound (120 mg) was obtained by silica gel column purification.

[0335]

[0336] MS (ESI) m / z (M+H) + = 373.1.

[0337] Step 9: Preparation of tert-butyl 2-(di-tert-butyloxycarbonyl)-7-phenethyl-7,8- dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0338] Take 2-chloro-7-phenethyl-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylic acid tert-butyl ester (60 mg) in a dry reaction flask, dissolve in toluene (10 mL), add tris(dibenzylideneacetone)dipalladium (30 mg), 1,1'-bis(diphenylphosphino)ferrocene (35.7 mg), triethylamine (32 mg), di-tert-butyl phosphonate (63 mg), and sparge the system with nitrogen 3 times. Heat to 120 °C. Allow the reaction to proceed overnight. Check the reaction by LC-MS. Concentrate the system to dryness. Purify by column chromatography to give the title compound (20 mg).

[0339]

[0340] MS (ESI) m / z (M+H) + = 531.2.

[0341] Step 10: Preparation of (7-phenethyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2- yl)phosphonic acid hydrochloride

[0342] Take 2-(di-tert-butoxyphosphoryl)-7-phenethyl-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylic acid tert-butyl ester (20 mg), dissolve in dichloromethane (3 mL), add 4M hydrochloric acid in 1.4-dioxane (3 mL) dropwise, stir at room temperature for 1 hour, check the reaction by LC-MS, evaporate the solvent under reduced pressure. Pre-HPLC purification gives the title compound (5 mg).

[0343]

[0344] MS (ESI) m / z (M+H) + = 319.1.

[0345] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.88 - 7.63 (m, 3H), 7.37 - 7.17 (m, 4H), 4.51 - 4.29 (m, 2H), 3.61 (q, J = 7.4, 5.0 Hz, 1H), 3.39 (dd, J = 18.1, 4.8 Hz, 1H), 3.07 (dd, J = 18.1, 10.9 Hz, 1H), 2.88 - 2.67 (m, 2H), 2.20 - 2.08 (m, 1H), 2.02 (dt, J = 14.1, 7.9 Hz, 1H).

[0346] Example 14: Preparation of (7,7-diethyl-5,6,7,8-tetrahydro-naphthyridin-2- yl)phosphonic acid hydrochloride

[0347]

[0348] Step 1: Preparation of 2-methyl-N-(pentan-3-ylidene)propane-2-sulfmamide

[0349] Pentan-3-one (10.73 g) was weighed into a dry reaction flask and dissolved in tetrahydrofuran (300 mL). Tetraethyl orthotitanate (46 g) and 2-methylpropane-2-sulfmamide (12 g) were added and the flask was flushed with nitrogen three times. The reaction was heated to 65 °C for 20 hours. The reaction was monitored by LC-MS and was complete. Water (30 mL) was added to the reaction mixture and a large amount of solid precipitated. The organic phase was filtered and the solid was washed with tetrahydrofuran (100 mL). The filtrate was concentrated to dryness and the residue was purified by column chromatography to give the title compound (10.8 g).

[0350]

[0351] MS (ESI) m / z (M+H) + = 190.1.

[0352] Step 2: Preparation of N-(3-((3-bromo-6-methoxypyridin-2-yl)methyl)pentan-3-yl)-2- methylpropane-2-sulfmamide

[0353] Tetrahydrofuran (50 mL) was weighed into a dry reaction flask and flushed with nitrogen three times. 2M Diisopropylamino lithium (25 mL) was added and the reaction was cooled to -78 °C. A solution of 3-bromo-6-methoxy-2-methylpyridine (9.4 g) in tetrahydrofuran (50 mL) was added and the reaction was stirred at -78 °C for 1 hour. A solution of 2-methyl-N-(pentan-3-ylidene)propane-2-sulfmamide (8 g) in tetrahydrofuran (50 mL) was added and the reaction was stirred at -78 °C to -30 °C for 2 hours. The reaction was monitored by LC-MS and was complete. The reaction was quenched by the addition of saturated ammonium chloride solution and the reaction was extracted with ethyl acetate (3 x 100 mL). The organic phase was dried and concentrated to dryness. The residue was purified by column chromatography to give the title compound (11.3 g).

[0354]

[0355] MS (ESI) m / z (M+H) + = 391.1.

[0356] Step 3: Preparation of ethyl 2-(2-((tert-butylsulfmyl)amino)-2-ethylbutyl)-6- methoxynicotinate

[0357] Take N-(3-((3-bromo-6-methoxypyridin-2-yl)methyl)pentan-3-yl)-2-methylpropane-2- sulfinamide (11.3 g) in a dry reaction flask, dissolve in ethanol (10 mL), add 1,1'- bis(diphenylphosphino)ferrocene palladium dichloride dichloromethane complex (4.73 g), N,N- diisopropylethylamine (9.6 mL), and the system is replaced with nitrogen for 3 times and heated to 100 °C. The reaction is carried out overnight. LC-MS detects that the reaction is complete, and the system is concentrated to dryness. Column chromatography purification gives the title compound (9.3 g).

[0358]

[0359] MS (ESI) m / z (M+H) + = 385.2.

[0360] Step 4: Preparation of 7,7-diethyl-2-methoxy-7,8-dihydro-l,6-naphthyridin-5(6H)-one

[0361] Take 2-(2-((tert-butylsulfinyl)amino)-2-ethylbutyl)-6-methoxynicotinic acid ethyl ester (9.3 g) in a dry reaction flask, dissolve in acetonitrile (10 mL), add sodium hydroxide (4.8 g), heat to 100 °C, and react for 6 hours. LC-MS detects that the reaction is complete, and the system is filtered with diatomite, and the filtrate is concentrated to dryness. Column chromatography purification gives the title compound (4.6 g).

[0362]

[0363] MS (ESI) m / z (M+H) + = 235.1.

[0364] Step 5: Preparation of 7,7-diethyl-2-methoxy-5,6,7,8-tetrahydro-l,6-naphthyridine

[0365] Take 7,7-diethyl-2-methoxy-7,8-dihydro-l,6-naphthyridin-5(6H)-one (3 g), dissolve in tetrahydrofuran (100 mL), and add lithium aluminum hydride (1.9 g) in batches under ice bath. Warm to reflux and stir overnight, and LC-MS detects that the reaction is complete. The solvent is distilled off under reduced pressure. Silica gel column purification gives the title compound (2.8 g).

[0366]

[0367] MS (ESI) m / z (M+H) + = 221.1.

[0368] Step 6: Preparation of 7,7-diethyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2-ol

[0369] To a solution of 7,7-diethyl-2-methoxy-5,6,7,8-tetrahydro-l,6-naphthyridine (2.8 g) in 33% hydrogen bromide in acetic acid (20 mL) was added and heated to 80 °C overnight. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The crude product was slurried in acetonitrile to give the title compound (4.3 g).

[0370]

[0371] MS (ESI) m / z (M+H) + = 207.1.

[0372] Step 7: Preparation of 2-chloro-7,7-diethyl-5,6,7,8-tetrahydro-l,6-naphthyridin-7-ol

[0373] To a solution of 7,7-diethyl-5,6,7,8-tetrahydro-l,6-naphthyridin-2-ol (2.8 g) in phosphorous oxychloride (40 mL) was added and heated to 120 °C overnight. The reaction was monitored by LC-MS. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane and the pH was adjusted to 9-10 with sodium carbonate. The crude product was used directly in the next step without purification.

[0374]

[0375] MS (ESI) m / z (M+H) + = 225.1.

[0376] Step 8: Preparation of tert-butyl 2-chloro-7,7-diethyl-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylate

[0377] To the reaction mixture from the previous step was added di-tert-butyl dicarbonate (4.67 mL) and stirred at room temperature overnight. The reaction was monitored by LC-MS. The organic phase was extracted with dichloromethane three times and dried. The title compound (1.5 g) was obtained by purification on a silica gel column.

[0378]

[0379] MS (ESI) m / z (M+H) + = 325.1.

[0380] Step 9: Preparation of tert-butyl 2-(di-tert-butoxyphosphoryl)-7,7-diethyl-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0381] Take 2-chloro-7,7-diethyl-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylic acid tert-butyl ester (200 mg) in a dry reaction flask, dissolve in toluene (10 mL), add 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride dichloromethane complex (100 mg), triethylamine (0.17 mL), di-tert-butyl phosphonate (360 mg), the system is replaced by nitrogen 3 times, heated to 120 °C. The reaction is overnight. LC-MS detection of complete reaction, the system is concentrated to dryness. Column chromatography purification to give the title compound (80 mg).

[0382]

[0383] MS (ESI) m / z (M+H) + = 483.2.

[0384] Step 10: Preparation of (7,7-diethyl-5,6,7,8-tetrahydro-naphthyridin-2-yl)phosphonic acid hydrochloride

[0385] Take 2-(di-tert-butoxyphosphoryl)-7,7-diethyl-7,8-dihydro-l,6-naphthyridine-6(5H)- carboxylic acid tert-butyl ester (80 mg), dissolve in dichloromethane (4 mL), drop 4M hydrochloric acid 1.4-dioxane solution (4 mL), stir at room temperature for 1 hour, LC-MS detection of complete reaction, remove the solvent under reduced pressure. pre-HPLC purification to give the title compound (13 mg).

[0386]

[0387] MS (ESI) m / z (M+H) + = 271.1.

[0388] 1 H NMR (400 MHz, Deuterium Oxide) δ 7.88 - 7.57 (m, 2H), 4.39 (s, 2H), 3.11 (s, 2H), 1.84 - 1.42 (m, 4H), 0.90 (t, J = 7.5 Hz, 6H).

[0389] Preparation Example 1: Preparation of 2-chloro-8-hydroxy-7,8-dihydro-l,6-naphthyridine- 6(5H)-carboxylic acid tert-butyl ester

[0390]

[0391] Step 1: Preparation of 2-chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylic acid tert-butyl ester

[0392]

[0393] Dissolve 2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine hydrochloride (5 g) in dichloromethane (50 mL), add triethylamine (10 mL), slowly drop di-tert-butyl dicarbonate (6.7 mL), and react at room temperature for 3 hours. LC-MS shows that the reaction is complete. Concentrate the system to obtain an oily crude product, and separate the title compound (6 g) by column chromatography.

[0394] MS (ESI) m / z (M+H) + = 269.1.

[0395] Step 2: Preparation of 6-(tert-butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-l,6- naphthyridine- 1 -oxide

[0396]

[0397] Dissolve 2-chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-tert-butyl ester (9 g, 33.58 mmol) in dichloromethane (100 mL), and add m-chloroperbenzoic acid (11.7 g) in batches under ice bath. React at room temperature overnight. LC-MS shows that the reaction is complete. Add dichloromethane and water, separate the liquid phases, and concentrate the organic phase to dryness. Purify the obtained crude product by column chromatography to obtain the title compound (7.0 g).

[0398] MS (ESI) m / z (M+H) + = 285.1.

[0399] Step 3: Preparation of 8-acetyloxy-2-chloro-7,8-dihydro-l,6-naphthyridine-6(5H)-tert-butyl ester

[0400]

[0401] Dissolve 6-(tert-butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine- 1 -oxide (7 g) in acetic anhydride (80 mL), replace with nitrogen for 3 times, heat to 70°C, and react overnight. LC-MS shows that the reaction is complete. Concentrate under reduced pressure to remove a large amount of acetic anhydride, add ethyl acetate and water, extract with ethyl acetate for 3 times, wash with saturated sodium bicarbonate solution for 2 times, dry the organic phase, concentrate, and purify by column chromatography to obtain the title compound (5 g).

[0402] MS (ESI) m / z (M+H) + = 327.1.

[0403] Step 4: Preparation of 2-chloro-8-hydroxy-7,8-dihydro-l,6-naphthyridine-6(5H)-tert-butyl ester

[0404]

[0405] Charge 8-acetyloxy-2-chloro-7,8-dihydro-l,6-naphthyridine-6-(5H)-carboxylic acid tert-butyl ester (3 g) into methanol (30 mL), add potassium carbonate (635 mg), and react at room temperature for 0.5 hours. LC-MS shows that the reaction is complete. Add ethyl acetate and water, extract with ethyl acetate three times, dry the organic phase, and concentrate to obtain an oily crude product. Purify by column chromatography to obtain the title compound (1.9 g).

[0406] MS (ESI) m / z (M+H) + = 285.1.

[0407] Preparation Example 2: Preparation of 6-(tert-butyl) 7-methyl 2-chloro-7,8-dihydro-l,6- naphthyridine-6,7(5H)-dicarboxylate

[0408] Step 1: Preparation of 2,3-bis(methoxycarbonyl)pyridine 1-oxide

[0409]

[0410] Charge dimethyl pyridine-2,3-dicarboxylate (4.90 g) into acetonitrile (60 mL), add urea hydrogen peroxide (4.71 g) under ice bath, slowly drop trifluoroacetic anhydride (10.5 g), after the drop is completed, the system becomes a clear solution, and warm to room temperature to react for 4 hours. TLC shows that the reaction is basically complete. Add aqueous sodium disulfite to quench. Add dichloromethane and water, separate, extract the aqueous phase with a mixed solvent (DCM / MeOH). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness. Obtain the title compound (5.15 g).

[0411] MS (ESI) m / z (M+H) + = 212.1.

[0412] Step 2: Preparation of dimethyl 6-chloropyridine-2,3-dicarboxylate

[0413]

[0414] Charge 2,3-bis(methoxycarbonyl)pyridine 1-oxide (5.15 g) into phosphorus oxychloride (30 mL) under ice bath, warm to 105°C to react for 4 hours. TLC shows that the reaction is complete. Concentrate under reduced pressure, dilute with ethyl acetate, drop into crushed ice, add aqueous sodium carbonate to adjust pH = 10, extract with ethyl acetate, and wash the organic phase with aqueous sodium chloride. Concentrate the organic phase to dryness, purify the obtained crude product by column chromatography to obtain the title compound (3.52 g).

[0415] MS (ESI) m / z (M+H) += 230.1.

[0416] Step 3: Preparation of (6-chloropyridine-2,3-diyl)dimethanol

[0417]

[0418] Dissolve dimethyl 6-chloropyridine-2,3-dicarboxylate (3.50 g) in tetrahydrofuran (72 mL) and methanol (1.5 mL), and add lithium borohydride (0.84 g) portionwise with ice bath cooling. Allow the reaction to warm to room temperature and stir for 3 h. TLC shows most of the starting material has reacted. Pour the reaction into aqueous sodium bicarbonate solution, and extract with ethyl acetate. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness. This gives the title compound (2.63 g).

[0419] MS (ESI) m / z (M+H) + = 174.1.

[0420] Step 4: Preparation of 6-chloro-2,3-bis(chloromethyl)pyridine

[0421]

[0422] Dissolve (6-chloropyridine-2,3-diyl)dimethanol (2.63 g) in dichlorosulfoxide (40 mL) with ice bath cooling. Allow the reaction to warm to room temperature and stir for 3 h. There is some mono-chloro intermediate, so allow the reaction to warm to 35 °C and stir for 3 h. TLC shows the reaction is complete. Concentrate under reduced pressure, dilute with ethyl acetate, and add dropwise to crushed ice. Adjust the pH to 10 with aqueous sodium carbonate solution, and extract with ethyl acetate. Wash the organic phase with aqueous sodium chloride solution. Concentrate the organic phase to dryness, and purify the crude product by column chromatography to give the title compound (2.1 g).

[0423] MS (ESI) m / z (M+H) + = 210.1.

[0424] Step 5: Preparation of dimethyl 6-acetyl-2-chloro-5,8-dihydro-l,6- naphthyridine-7,7(6H)-dicarboxylate

[0425]

[0426] Dissolve 6-chloro-2,3-bis(chloromethyl)pyridine (2.10 g) in N,N- dimethylformamide (15 mL), and add dimethyl acetamidomalonate (2.17 g) and sodium hydride (0.40 g) sequentially with ice bath cooling. Allow the reaction to warm to room temperature and stir for 1 h. Add sodium hydride (0.40 g) with ice bath cooling, and allow the reaction to warm to room temperature and stir overnight. TLC shows the reaction is complete. Add ethyl acetate and water, and extract the phases. Concentrate the organic phase to dryness. Purify the crude product by column chromatography to give the title compound (1.74 g).

[0427] MS (ESI) m / z (M+H) + = 327.1.

[0428] Step 6: Preparation of 2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine-7-carboxylic acid hydrochloride

[0429]

[0430] Dissolve 6-acetyl-2-chloro-5,8-dihydro-l,6-naphthyridine-7,7(6H)-dicarboxylic acid dimethyl ester (1.74 g) in 6 M hydrochloric acid (15 mL) and heat to 100 °C for 4 h. TLC shows the reaction is complete. Concentrate the reaction mixture to dryness under reduced pressure to give the title compound (1.16 g).

[0431] MS (ESI) m / z (M+H) + = 213.1.

[0432] Step 7: Preparation of methyl 2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine-7-carboxylate hydrochloride

[0433]

[0434] Dissolve 2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine-7-carboxylic acid hydrochloride (1.16 g) in methanol (20 mL) and slowly add dichlorosulfoxide (1.67 g) dropwise at 0 °C. Heat to 70 °C and reflux for 2 h. TLC shows the reaction is complete. Concentrate the reaction mixture to dryness under reduced pressure to give the title compound (1.23 g).

[0435] MS (ESI) m / z (M+H) + = 227.1.

[0436] Step 8: Preparation of 6-(tert-butyl) 7-methyl 2-chloro-7,8-dihydro-l,6-naphthyridine-6,7(5H)-dicarboxylate

[0437]

[0438] Dissolve methyl 2-chloro-5,6,7,8-tetrahydro-l,6-naphthyridine-7-carboxylate hydrochloride (1.23 g) in dichloromethane (25 mL) and add triethylamine (1.89 g) and di-tert-butyl dicarbonate (1.53 g) sequentially. Stir at room temperature for 2 h. TLC shows the reaction is complete. Add dichloromethane and water and separate the layers. Concentrate the organic layer to dryness. Purify the crude product by column chromatography to give the title compound (1.19 g).

[0439] MS (ESI) m / z (M+H)+ = 327.1.

[0440] 1 H NMR (400 MHz, Chloroform-d) δ 7.42 (dd, J = 12.5, 8.0 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 5.34 (d, J = 6.7 Hz, 0.5H), 5.07 (dd, J = 7.2, 2.9 Hz, 0.5H), 4.79 (dd, J = 22.4, 17.0 Hz, 1H), 4.52 (dd, J = 31.0, 17.1 Hz, 1H), 3.68 (d, J = 7.7 Hz, 3H), 3.55 - 3.38 (m, 1H), 3.38 - 3.20 (m, 1H), 1.52 (d, J = 17.9 Hz, 9H).

[0441] Preparation Example 3: Preparation of tert-butyl 2-chloro-7-formyl-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate

[0442]

[0443] Step 1: Preparation of 6-(tert-butoxycarbonyl)-2-chloro-5,6,7,8-tetrahydro-1,6- naphthyridine-7-carboxylic acid

[0444]

[0445] Step 2: Preparation of tert-butyl 2-chloro-7-(methoxy(methyl)carbamoyl)-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate

[0446] MS (ESI) m / z (M+H) + = 313.1.

[0447] Step 2: Preparation of tert-butyl 2-chloro-7-(methoxy(methyl)carbamoyl)-7,8-dihydro-1,6- naphthyridine-6(5H)-carboxylate

[0448]

[0449] Step 2: Preparation of tert-butyl 2-chloro-7-(methoxy(methyl)carbamoyl)- 7,8-dihydro-l,6-naphthyridine-6(5H)-carboxylate

[0450] MS (ESI) m / z (M+H) + = 356.1.

[0451] Step 3: Preparation of tert-butyl 2-chloro-7-formyl-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0452]

[0453] Step 3: Preparation of tert-butyl 2-chloro-7-formyl-7,8-dihydro-l,6- naphthyridine-6(5H)-carboxylate

[0454] MS (ESI) m / z (M+H) + = 297.0

[0455] Using conventional and routine techniques known in the art and starting from the materials and reagents commercially available, the following compounds were prepared according to the procedures described in the previous examples and by using conventional separation and purification techniques:

[0456]

[0457]

[0458]

[0459]

[0460] Biological test

[0461] Experimental Example 1: Plasma clot lysis test

[0462] 1. Purpose of experiment

[0463] To determine the inhibitory effect of the compound of the present application on the plasma clot lysis of human.

[0464] 2. Materials and apparatus

[0465]

[0466] 3. Experimental procedure

[0467] 3.1 Fresh healthy human blood was collected, and 0.109 M trisodium citrate was used as an anticoagulant. The blood was mixed with the anticoagulant at a ratio of 1:9, centrifuged at 2000 x g for 20 minutes at room temperature, and the supernatant (i.e., plasma) was collected and stored at -80°C after aliquoting.

[0468] 3.2 On the day of the experiment, the plasma was thawed in a 37°C water bath, and all reagents except tPA were preheated at 37°C.

[0469] 3.3 In a 96-well plate, 12.5 μL of 80 mM CaCl2 (HEPES buffer, pH 7.4) was added, followed by the addition of 25 μL of the test compound diluted in physiological saline at different concentrations, and an equal volume of physiological saline was added to the negative control wells.

[0470] 3.4 50 μL of preheated plasma was mixed with 12.5 μL of 4 nM tPA (HEPES buffer, pH 7.4), and immediately added to the 96-well plate. The absorbance was measured at 405 nm, and the reading was taken every 2 minutes for a total of 15 hours.

[0471] 3.5 The absorbance value increased first and then decreased with time. The median absorbance value of the descending segment corresponds to the time of the median absorbance value of the ascending segment, which is the plasma clot lysis time. The plasma clot lysis time of the negative control wells was used as a reference to calculate the plasma clot lysis time in the wells containing different concentrations of the compound, and the inhibition rate was obtained:

[0472] Inhibition rate % = (1 - negative control well Clot lysis time / compound well Clot lysis time) x 100%

[0473] 3.6 Fitting the dose-effect curve

[0474] The log value of the compound concentration is taken as the X axis, and the percentage inhibition is taken as the Y axis, and the log (inhibitor) vs. response-variable slope (Variable slope) fitting dose-effect curve of the analysis software GraphPad Prism 5 is used to obtain the IC 50 value of the inhibition of plasma clot degradation of each compound.

[0475] The calculation formula is: Y = min + (max-min) / (1 + 10^((LogIC 50 -X) x Hillslope)).

[0476] The inhibition of plasma clot degradation of the compound of the present application is determined by the above test, and the IC 50 value of the compound of the present application is calculated to be lower than the IC 50 of tranexamic acid. For example, the IC 50 value of the compound of Example 1 of the present application for the inhibition of plasma clot degradation is 0.9 μM, which is much lower than that of the existing representative hemostatic drug tranexamic acid (IC 50 4.75 μM under the same test conditions). The relative coagulation activity (IC 50 ratio = IC 50实施例 / IC 50氨甲环酸 ) of the present application in vitro relative to tranexamic acid is shown in the following table:

[0477]

[0478] The test data show that the compound of the present application can effectively inhibit the degradation of plasma clot, has excellent coagulation and hemostatic activity, and its effective dose is much lower than the most frequently used hemostatic drug in the clinic, which can effectively avoid the adverse reactions and complications caused by high-dose medication, and has excellent drug prospects.

[0479] Experimental Example 2: Rat PK test

[0480] 1. Purpose of the experiment

[0481] The pharmacokinetic properties of the compound of the present application in rats in vivo are studied by determining the plasma drug concentration after intravenous administration in rats.

[0482] 2. Experimental animals

[0483] SD rats, SPF level, male, N = 3, source: Shanghai Xipu-Bike Experimental Animal Co., Ltd.

[0484] 3. Drug preparation and administration

[0485] The compound was weighed and dissolved in normal saline to prepare a 0.2 mg / mL intravenous administration solution.

[0486] The day before the experiment, rats were fasted overnight and fed 4 hours after dosing.

[0487] On the day of the experiment, rats were dosed according to the schedule below. After dosing, rats were bled from the jugular vein at various time points, approximately 200 μL into heparin sodium anticoagulation tubes. Blood samples were placed on ice after collection and centrifuged to separate plasma (centrifugation conditions: 6800g, 6 minutes, 2-8°C) within 1 hour. The separated plasma was stored in a -80°C freezer for bioanalytical analysis.

[0488]

[0489] 4. Bioanalysis

[0490] The method for determining the concentration of the compound in rat plasma is as follows:

[0491] Instrument: LC-MS / MS-19 (TQ5500, AB SCIEX, USA).

[0492] Internal standard: Warfarin.

[0493] Chromatographic column: ACQUITY UPLC BEH C18, model 1.7um 2.1*50mm, purchased from Shenzhen Noah Chemical Technology Co., Ltd.

[0494] Flow rate: 0.60 milliliters / minute.

[0495] Column temperature: 40°C.

[0496] Mobile phase A: 0.1% formic acid in water.

[0497] Mobile phase B: 0.1% formic acid in acetonitrile.

[0498] The elution gradient is shown in Table 3.

[0499] Table 3 Elution gradient

[0500] 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

[0501] MS detection conditions: Electrospray ion source (ESI), positive ion mode, MRM scan.

[0502] Take 30 μL of the plasma sample prepared in item “3” of this example, and perform protein precipitation with 300 μL of MeOH, which contains 100 ng / mL of internal standard. Vortex the mixture for 1 minute and centrifuge at 18000g for 7 minutes. Transfer the supernatant to a 96-well plate. Take 4 μL of the supernatant and inject it into LC-MS / MS for analysis.

[0503] The concentration of the compound in rat plasma was determined by using the above LC-MS / MS analysis method, and the pharmacokinetic parameters were calculated by using Phoenix WinNonlin 7.0 based on the blood concentration data at different time points.

[0504] The rat pharmacokinetic parameters of some compounds of the present application were determined by the above experiment, and are shown in the following table.

[0505] Table: In vivo pharmacokinetic study data of SD rats administered with test compounds by intravenous injection

[0506] Experimental Example 3: Human whole blood thrombelastogram experiment

[0507] 1. Experimental purpose

[0508] The anti-fibrinolytic effect of the compound of the present application in the high fibrinolysis state of human whole blood induced by rtPA (recombinant tissue plasminogen activator) was determined by using the thromboelastogram (TEG) method.

[0509] 2. Main experimental materials and instruments

[0510] Experimental materials

[0511]

[0512] Instruments

[0513]

[0514] Human blood source: All the human blood used in the experiment was provided by healthy volunteers.

[0515] 3. Experimental steps

[0516] (1) Test solution preparation: accurately weigh the test substance, and use normal saline to prepare the test substance into the following administration concentration (100x test concentration):

[0517] Tranexamic acid (μM): 3000, 1000, 300, 100, 30, 10, 0;

[0518] Example 6 (μM): 1000, 300, 100, 30, 10, 3, 0.

[0519] (2) rtPA (Alteplase for injection) preparation: use the water for injection in the rtPA package to prepare the active dry powder of rtPA into 25 μg / mL.

[0520] (3) Reaction system:

[0521] 392 μL sodium citrate anticoagulant whole blood + 4 μL rtPA + 4 μL test, room temperature reaction, TEG curve detection for 2 h, and CLT (clot lysis time) time parameter is obtained.

[0522] (4) Result calculation

[0523] Take the log value of the compound concentration as the X axis, and the CLT value as the Y axis, and use the analysis software GraphPad Prism 8 to fit the dose-effect curve of log (inhibitor) vs. response-variable slope (Variable slope):

[0524] Formula: Y = min + (max-min) / (1 + 10^((LogIC 50 -X) x Hillslope)).

[0525] Calculate the compound concentration corresponding to a doubling of the CLT time.

[0526] 4. Experimental results

[0527] The inhibitory effect of the compound of the present application on human whole blood fibrinolysis is determined by the above test, and the results are as follows:

[0528] CLT prolongs 1-fold drug concentration

[0529]

[0530] The experiments prove that, compared with the positive drug tranexamic acid which is the best active and most widely used in the clinic at present, the compound of the present application has significantly higher exposure in animals, lower clearance rate and longer half-life; in the plasma clot degradation experiment and thromboelastogram (TEG) experiment, the compound of the present application can effectively inhibit the fibrinolysis process, prolong the plasma clot degradation time (CLT, clot lysis time), and play a coagulation and hemostasis effect, which is significantly better than the positive control. These experiments show that the compound of the present application has the advantages of good hemostatic activity, small effective dose, long drug effect time, etc., can avoid various adverse reactions that may be caused by high-dose administration in the clinic, and improve the safety and effectiveness of drug administration for patients. Moreover, the compound of the present application is easy to prepare and can be mass-produced industrially, which can effectively reduce the cost of drug administration. The compound of the present application has good distribution, metabolism and excretion characteristics, and the possibility of drug interaction is low, which can meet the requirements of pharmacokinetic parameters required to achieve efficacy in the human body. In addition, the compound of the present application has low toxicity, no effect on the respiratory system, central nervous system and cardiovascular system, good tolerance in single and repeated dose toxicity tests, sufficient safety window, and no genetic toxicity. It has a broad clinical application prospect.

Claims

1. Compounds, selected from: (5,6,7,8-Tetrahydro-1,6-naphthid-2-yl)phosphonate salt; (3-Fluoro-5,6,7,8-Tetrahydro-1,6-Naphthid-2-yl)phosphonate salt; Ethyl(5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt; Phenylethyl (5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphinate salt; (8,8-Difluoro-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate salt; (5,6,7,8-Tetrahydro-1,6-naphthid-2-yl)phosphonic acid monohydrate; (8-morpholino-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate salt; (8-hydroxy-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate; (8-(2-hydroxyethoxy)-5,6,7,8-tetrahydro-1,6-naphthidin-2-yl)phosphonate; (8-oxothiomorpholine-5,6,7,8-tetrahydro-1,6-naphthidin-2-yl)phosphonate; (7-isobutyl-5,6,7,8-tetrahydro-1,6-naphthidin-2-yl)phosphonate; (7-propyl-5,6,7,8-tetrahydro-1,6-naphthid-2-yl)phosphonate salt; (7-Phenylacetyl-5,6,7,8-Tetrahydro-1,6-Naphthoidin-2-yl)phosphonate salt; (7,7-Diethyl-5,6,7,8-tetrahydro-naphthid-2-yl)phosphonate; 2. A pharmaceutical composition comprising the compound of claim 1 and at least one pharmaceutically acceptable excipient.

3. Use of the compound of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament.

4. The use according to claim 3, characterized in that... The drug has therapeutic activities of coagulation and hemostasis.

5. The use according to claim 4, 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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