CN112979675A

CN112979675A - Small-molecule sulfur-containing heterocyclic compound

Info

Publication number: CN112979675A
Application number: CN201911274951.7A
Authority: CN
Inventors: 沈旺; 党奎峰; 张超; 糜佳晨; 徐华萍; 陈春明; 王鑫; 孙亚波; 单立弘; 丁文; 沈家磊; 胡进忠; 盛夏鑫; 白海
Original assignee: Vivavision Biopharma Shanghai Co ltd
Current assignee: Weimu Biotechnology (Zhejiang) Co.,Ltd.
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2021-06-18
Anticipated expiration: 2039-12-12
Also published as: WO2021115495A1; CN112979675B

Abstract

The invention relates to a sulfur-containing heterocyclic compound, which is characterized in that: is a compound shown by the following structural formula or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotope derivative, a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt or a prodrug thereof;

Description

Small-molecule sulfur-containing heterocyclic compound

Technical Field

The invention relates to the field of small molecule compounds, in particular to a small molecule sulfur-containing heterocyclic compound and application thereof in treating glaucoma.

Background

Rho-associated coiled coil protein kinase (ROCK), belongs to the AGC (PKA/PKG/PKC) family of serine-threonine kinases. Two human isoforms of ROCK have been described, ROCK-I (also known as p160ROCK or ROK β) and ROCK-II (ROK α) are approximately 160kDa proteins containing an N-terminal Ser/Thr kinase domain followed by a coiled-coil structure, a pleckstrin homology domain and a cysteine-rich region at the C-terminus (Multifunctional kinases in cell biology. Nat. Rev. mol. cell biol.2003(4): 446-.

Rho belongs to a small molecule single-polymer GTPase superfamily, is a mammalian gene homolog of a Ras superfamily, and regulates the recombination of a cell actin framework through a main downstream effector Rho kinase (ROCK), so that Rho can be widely involved in a series of biological processes such as mitosis, cytoskeletal regulation, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration, apoptosis regulation and the like. Rho/ROCK can be activated to act on a variety of substrates, thereby generating a biological process. The two most prominent substrates are Myosin Light Chain (MLC), the level of phosphorylation of which is an important factor in determining the degree of smooth muscle contraction, and Myosin Light Chain Phosphatase (MLCP). Myosin Light Chain Kinase (MLCK) phosphorylates the Ser-19 site of MLC, leading to smooth muscle contraction; inhibition of MLCP can further enhance phosphorylation of MLC and contraction of smooth muscle. After the ROCK is activated, MLC can be phosphorylated to generate myofilament contraction; meanwhile, MLCP can be phosphorylated to inactivate the MLCP, so that the phosphorylation degree of MLC in cytoplasm of cells is increased, and myofilament contraction is indirectly promoted.

Inhibition of Rho kinase activity in animal models has shown various benefits in the treatment of human diseases including cardiovascular diseases such as pulmonary hypertension, atherosclerosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, and the like, and central nervous system disorders such as neuronal degeneration and the like, as well as tumors. ROCK expression and activity were shown to be elevated in spontaneously hypertensive rats, suggesting that it is associated with the development of hypertension in these animals (invasion of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. faseb j.,2001,15(6): 1062-4). The ROCK inhibitor Y-27632 can significantly lower blood pressure in three rat hypertension models (spontaneous hypertension, renal hypertension, deoxycorticosterone acetate type hypertension), while having less effect on blood pressure in control rats (Calcium sensitivity of small cardiac media hypertension by a Rho-associated protein kinase in hypertension [ J ] Nature,1997,389(6654): 990-4). It has also been shown that ROCK inhibitors have a better effect on pulmonary hypertension (Acute vasodialator effects of a Rho-kinase inhibitor, facial in tissues with segment pulmonary hypertension. Heart,2005:91(3): 391-2).

ROCK activity is an important signaling mechanism in leukocyte-platelet-endothelial interactions, leukocyte extravasation and edema. Over-activation of Rho kinase in endothelial cells can cause leakage due to disruption of cell-cell junctions that contribute to the recruitment of inflammatory cells. Taken together, these lines of evidence point to a role for ROCK in pathological conditions associated with acute and chronic inflammation, as well as in autoimmune diseases (Isoform-specific targeting of ROCK proteins in immune cells. Small GTPases.2016; 7(3): 173-. Validation of the beneficial effects of ROCK inhibition in experimental models of rheumatoid arthritis and lupus by animal experiments (Rho kinase inhibitors and the application to inflammatory disorders. Current. Top. Med. chem.2009 (9): 704-723; antibiotic effects of AS1892802, a novel Rho kinase inhibitor, in rat models of inflammatory and nasal disorders. J. Pharmacol. Exp. The.2010, 334: 955:. 963; Administration of drug, a ROCK inhibitor, activities disorders in cement NZB/W F1 enzymes, Lupus. 21(6): 656-61). The inhibition of T-cell migration by Fasudil may expand its clinical application as a novel therapy for multiple sclerosis (Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor. J Neurosci Res.2010; 88(8): 1664-72). Accumulating evidence also confirms that ROCK plays a key role in regulating 3 disease factors in the pathogenesis of Inflammatory Bowel Disease (IBD): disruption of the intestinal barrier, exposure of intestinal contents to mucosal immune cells, and abnormal immune responses (roll of rhokinase signal pathway in inflammatory bow disease. int J ClinExp Med. 2015; 8(3):3089 + 3097).

Currently marketed ROCK inhibitor drugs are Eril (suitable for the treatment of cerebral vasospasm) by Asahi Kasei; glanatec from Kowa (suitable for the treatment of ocular hypertension and glaucoma, approved only in japan), a new glaucoma drug rhopressa (netarsudil) from american aeire, was approved in 2017 in the united states for the treatment of patients suffering from open angle glaucoma or high ocular pressure, lowering their intraocular pressure. Rhopressa is a new once daily eye drop.

Therefore, research on developing targeted small molecule drugs acting on ROCK is carried out, and the ROCK inhibitor with better activity, higher selectivity, lower toxicity and side effect and more economy is obtained, thereby having very important social and economic significance.

Disclosure of Invention

The invention aims to provide a novel inhibitor with high activity on ROCK kinase, a preparation method and application thereof.

The invention provides a sulfur-containing heterocyclic compound, which is characterized in that: is a compound shown by the following structural formula or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotope derivative, a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt or a prodrug thereof;

wherein ring a is aryl or heteroaryl;

b is a fused heteroaryl group containing one N atom and one stream atom;

R¹hydrogen, hydroxyl, halogen, amino, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl of G2 substituted on any or several of the rings;

R²hydrogen, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy substituted or not in ring G1;

R³、R⁴each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl;

or, R³And R⁴Linked to form a substituted or unsubstituted cycloheteroalkyl group;

R⁵is any or several substituted hydrogen, hydroxyl, halogen, amino, cyano on A ringSubstituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl.

The preferable structure may be:

r is as defined above⁶Selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, NR^AR^B；

Wherein R is^AAnd R^BIndependently selected from substituted or unsubstituted alkyl, or R^AAnd R^BJoined to form a substituted or unsubstituted cycloheteroalkyl group.

Substituted alkyl in the present invention means that one or more of the hydrogen atoms of the alkyl carbon chain are substituted by other groups, which may be hydroxy, cycloalkyl (in analogy to the above-mentioned groups)

Spiro, fused ring, etc., any hydrogen atom on the cycloalkyl ring may be substituted with a group such as halogen, cyano, alkyl, hydroxyl, carboxyl, etc. (one or more carbons on the carbocyclic ring may be substituted with C (═ O)), heterocycloalkyl (i.e., at least one carbon atom on the alkyl ring is substituted with oxygen, sulfur, or nitrogen in addition to the above-mentioned cycloalkyl), halogen (F, Cl, Br, I), carboxyl, cyano (-CN), sulfonyl (-SO), etc₂R_a，R_aAlkyl, aryl, etc.), alkynyl (-C.ident.CH, -C.ident.CR)_b，R_bIs alkyl, aryl, etc.), an amide group (-C (O) NR)_xR_y，R_xR_yAlkyl, aryl, etc.), ester group (-C (O) O-R)_z，R_zIs alkyl, aryl, etc.), aryl, heteroaryl, etc. groupsOr one or more carbons of the carbon chain is substituted with C (═ O);

substituted or unsubstituted heteroalkyl in the present invention refers to: refers to the substituted or unsubstituted alkyl, one or more carbon atoms of the carbon chain of which are replaced by oxygen, sulfur, nitrogen.

Substituted cycloalkyl in the present invention refers to a cyclic ring wherein one or more of the hydrogen atoms are replaced by another group, which may be alkyl, substituted alkyl (as above), halo (F, Cl, Br, I), carboxy, cyano (-CN), sulfonyl (-SO)₂R_a，R_aAlkyl, aryl, etc.), alkynyl (-C.ident.CH, -C.ident.CR)_b，R_bIs alkyl, aryl, etc.), an amide group (-C (O) NR)_xR_y，R_xR_yHydrogen, alkyl, aryl, etc.), ester group (-C (O) O-R_z，R_zAlkyl, aryl, etc.), aryl, heteroaryl, etc., or one or more carbons of the carbon chain is substituted with C (═ O).

Substituted or unsubstituted heterocycloalkyl in the present invention means: refers to the substituted or unsubstituted cycloalkyl, one or more carbon atoms of the ring of which are replaced by oxygen, sulfur, nitrogen.

Substituted alkoxy in the context of the present invention means alkoxy(s) ((R))

n is selected from 0 or any natural number, m is a natural number including 0) one or more of the hydrogen atoms on the carbon chain are substituted with other groups, which may be cycloalkyl (in a manner similar to that of the cycloalkyl group)

Etc., any hydrogen atom on the cycloalkyl ring may also be substituted with a halogen, cyano, alkyl, hydroxy, carboxy, etc., heterocycloalkyl (i.e., in addition to the cycloalkyl groups described above, at least one carbon atom on the alkyl ring is replaced with oxygen, sulfur, nitrogen), halogen (F, Cl, Br, I), carboxy, cyano (-CN), sulfonyl (-SO), etc₂R_a，R_aAlkyl, aryl, etc.), alkynyl (-C.ident.CH, -C.ident.CR)_b，R_bIs alkyl, aryl, etc.), an amide group (-C (O) NR)_xR_y，R_xR_yHydrogen, alkyl, aryl, etc.), ester group (-C (O) O-R_z，R_zAlkyl, aryl, etc.), aryl, heteroaryl, etc., or one or more carbons of the carbon chain is substituted with C (═ O).

The sulfur-containing heterocyclic compound, or a stereoisomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug, or a metabolite thereof is used for preparing ROCK inhibitor medicines, and is used for preparing medicines for treating cardiovascular diseases, ocular hypertension, pulmonary hypertension and glaucoma.

The specific method comprises the following steps: the preparation is prepared by taking the compound, or the stereoisomer, or the pharmaceutically acceptable salt, or the solvate, or the prodrug, or the metabolite thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a preparation method of the sulfur-containing heterocyclic derivative, which is characterized in that the specific reaction equation is as follows:

wherein Re is selected from alkyl, and Prot is an amino protecting group.

The specific process of the reaction can be as follows: the compounds as shown in formulas IV-1 and IV-2 can be obtained from direct commercial sources or by known common synthetic methods. Formula IV-1 and formula IV-2 may be basic (e.g. K)₂CO₃Or DBU, etc.) at 30-150 deg.C to obtain the compound shown in formula IV-3. Hydrolysis of formula IV-3 (e.g., with NaOH in methanol and water solvent) converts to the acid shown in formula IV-4. The acid shown in formula IV-4 undergoes a Curtius rearrangement to give the amine shown in formula IV-5. Coupling of an amine of formula IV-5 and an intermediate Interm A (see, for synthesis, U.S. Pat. No. 4, 186917, Bioorg Med Chem Let,2016,26 (10): 2475-2480, U.S. Pat. No. 2010/22585) results in the formation of a compound of formula IV-6. Deprotection to obtainTo compounds shown by formula IIa 1. Formula IIa1 is mono-or di-alkylated to form a compound shown by formula IIa 2.

Prot is an amine protecting group.

The specific process of the reaction can be as follows: the compound represented by the formula Interm B can be synthesized by known methods (e.g., methods disclosed in US2007/112019, Journal of Medicinal Chemistry,2003, vol.46, p.3354-3370, US2002/86891, etc.). The compound shown in the formula Interm B reacts with trifluoromethanesulfonic anhydride under the condition of alkalinity (such as triethylamine and pyridine) to obtain the compound shown in the formula V-1. Amination of the compound shown by V-1 with palladium catalysis gives the compound shown by formula V-2. Hydrolysis of V-2 affords the compound of formula V-3. The reaction of a compound represented by formula V-3 with a compound represented by formula Interm A affords a compound represented by formula V-4. Deprotection provides the compound shown in formula IIIb1, and mono-or di-alkylation of formula IIIb1 provides the compound shown in formula IIIb 2.

Detailed Description

Some embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the claims in any way.

Example 1

3-amino-2- (4- (hydroxymethyl) phenyl) -N- (1H-indazol-6-yl) propanamide

The specific reaction equation is as follows:

step A: methyl 2- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) acetate (Compound 1.1)

Methyl 2- (4- (hydroxymethyl) phenyl) acetate (5.0g,27.8mmol) and imidazole (2.8g,41.7mmol) were dissolved in 50mL of N, N-dimethylformamide, followed by addition of t-butyldimethylchlorosilane (5.0g,33.4mmol) and reaction at room temperature overnight. TLC monitored the reaction complete. Water was added to quench the reaction, extracted with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give the product 1.1(8.1g, yield: 98%).

And B: methyl 2- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionate (Compound 1.2)

Dissolving compound 1.1(3.0g,10.2mmol) in 10mL of anhydrous tetrahydrofuran, replacing with nitrogen, cooling dry ice acetone to-78 ℃, slowly adding hexamethyldisilazane-based amino lithium (1.0M, 12.2mL,12.2mmol) dropwise, stirring the system at-78 ℃ for 1 hour after the dropwise addition is finished, dissolving N-bromomethylphthalimide (2.9g,12.2mmol) in 10mL of tetrahydrofuran, slowly adding dropwise into the system, controlling the dropwise addition temperature to be lower than-70 ℃, keeping the reaction at-78 ℃ for stirring for 3 hours after the dropwise addition is finished, and monitoring the reaction completion by TLC. The reaction was quenched by addition of saturated ammonium chloride solution, extracted with ethyl acetate (20mLX3), and the organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give the product 1.2(3.6g, yield: 78%).

And C: 2- ((2- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2-carboxyethyl) carbamoyl) benzoic acid (Compound 1.3)

Compound 1.2(3.6g,8.0mmol) was dissolved in 100mL tetrahydrofuran and 30mL water, lithium hydroxide monohydrate (1.0g,24.0mmol) was added at 0 deg.C, stirred at 0 deg.C for 3 hours, and the reaction was monitored for completion by LCMS. The organic solvent was distilled off under reduced pressure, the pH was adjusted to 3-4 with 1M aqueous hydrochloric acid, followed by extraction with ethyl acetate (20mLX3), washing with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to give crude product 1.3(3.4g, yield: 93%). LCMS ESI (+) m/z: 458.2(M +1).

Step D: 2- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionic acid (Compound 1.4)

Compound 1.3(3.4g,7.4mmol), 1-hydroxybenzotriazole (0.9g,7.4mmol) and triethylamine (2.2g,22.2mmol) were dissolved in 10mL of dichloromethane, and after cooling to 0 ℃ under nitrogen protection, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (1.4g,7.4mmol) was added to the reaction mixture, followed by stirring for a while to clarify the reaction mixture, slowly warming to room temperature, and reacting overnight. TLC monitored the reaction complete. The reaction mixture was diluted with water, extracted with dichloromethane (20mLX3), the organic phases were combined, washed with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give the product 1.4(1.5g, yield: 46%).

Step E: 2- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) -N- (1H-indazol-6-yl) propionamide (Compound 1.5)

Compound 1.4(100mg,0.23mmol) was dissolved in 2mL of N, N-dimethylformamide, and then 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (61mg,0.32mmol), 4-dimethylaminopyridine (39mg,0.32mmol) and 6-aminoindazole (36mg,0.27mmol) were added in this order and reacted at room temperature for 2 hours. TLC monitored the reaction complete. Adding water to quench the reaction, extracting with ethyl acetate (20mLX3), combining the organic phases, washing with saturated brine (50mL), drying the organic phase over anhydrous sodium sulfate, filtering, spin-drying, and purifying by column chromatography to obtain 1.5(50mg, yield: 39%).

Step F: 3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (hydroxymethyl) phenyl) -N- (1H-indazol-6-yl) propanamide (Compound 1.6)

Compound 1.5(50mg,0.09mmol) was dissolved in 5mL of tetrahydrofuran, and then 5mL of an aqueous hydrochloric acid solution (1.0M) was added to react at room temperature for 30 minutes. LCMS monitored reaction completion. After completion of the reaction, saturated sodium bicarbonate solution was added to neutralize the reaction solution, followed by extraction with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered and dried by spin-drying to obtain crude product 1.6(15mg, yield: 33%). LCMS ESI (+) M/z:441.1(M +1).

Step G: 3-amino-2- (4- (hydroxymethyl) phenyl) -N- (1H-indazol-6-yl) propanamide (Compound 1)

Compound 1.6(15mg,0.03mmol) was dissolved in 2mL of methylamine ethanol solution and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 1(9.2mg, yield: 77%) after lyophilization. LCMS ESI (+) M/z 311.1(M +1).

Example 2

3-amino-N- (benzo [ d ] isothiazol-6-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: 6-Methoxybenzo [ d ] isothiazole (Compound 2.1)

2-fluoro-4-methoxybenzaldehyde (20.0g,130.0mmol) was dissolved in 50mL of 2-methoxyethanol, and then sulfur (4.1g,130.0mmol) and 50mL of aqueous ammonia were added to react at 160 ℃ for 2 hours. LCMS monitored reaction completion. The reaction solution was diluted with water, extracted with dichloromethane (50mLX3), the organic phases were combined, washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 16.1(12.7g, yield: 59%). LCMS ESI (+) M/z 166.0(M +1).

And B: benzo [ d ] isothiazol-6-ol (Compound 2.2)

Compound 2.1(12.7g,77.0mmol) was dissolved in 100mL of dichloromethane, and then boron tribromide (14.5mL,154.0mmol) was slowly added dropwise at 0 ℃ to react at room temperature for 5 hours. TLC monitored the reaction complete. After completion of the reaction, the reaction mixture was slowly poured into ice water, extracted with dichloromethane (50mLX3), the organic phases were combined, washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered and spin-dried to obtain crude product 2.2(11.5g, yield: 98%).

And C: benzo [ d ] isothiazol-6-yl trifluoromethanesulfonate (Compound 2.3)

Compound 2.2(11.5g,76.0mmol) was dissolved in dichloromethane and diisopropylethylamine (48mL,129.0mmol) and trifluoromethanesulfonic anhydride (20mL,114.0mmol) were added dropwise at-78 deg.C and the reaction was allowed to react at-78 deg.C for 1 h. LCMS monitored reaction completion. The reaction was quenched by addition of saturated ammonium chloride, extracted with dichloromethane (50mLX3), the organic phases were combined, washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 2.3(8.1g, yield: 38%). LCMS ESI (+) M/z 284.0(M +1).

Step D: n- (diphenylmethylene) benzo [ d ] isothiazol-6-amine (Compound 2.4)

Compound 2.3(8.1g,28.6mmol), potassium phosphate (9.1g,42.9mmol), benzophenone imine (6.4g,35.8mmol) and 2-dicyclohexylphosphorus-2, 4, 6-triisopropylbiphenyl (2.7g,5.7mmol) were dissolved in ethylene glycol dimethyl ether and tris (dibenzylideneacetone) dipalladium (2.6g,2.8mmol) was added under nitrogen protection. The reaction was carried out overnight at 90 ℃ under nitrogen. LCMS monitored reaction completion. The reaction was quenched with water, extracted with ethyl acetate (50mLX3), the organic phases were combined, washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 2.4(8.4g, yield: 93%). LCMS ESI (+) M/z 315.1(M +1).

Step E: benzo [ d ] isothiazol-6-amine (Compound 2.5)

Compound 2.4(8.4g,26.7mmol) was dissolved in 50mL of tetrahydrofuran, and 50mL of aqueous hydrochloric acid (2.0M) was added at room temperature. The reaction was allowed to proceed overnight at room temperature. LCMS monitored reaction completion. After completion of the reaction, the reaction mixture was extracted with ethyl acetate to remove impurities, the aqueous phase was neutralized with saturated sodium hydrogencarbonate, extracted with ethyl acetate (50mLX3), and the organic layers were combined, washed with saturated brine (100mL) and dried over anhydrous sodium sulfate. Filtration and spin-drying gave 2.5(1.5g, yield: 37%) as a crude product. LCMS ESI (+) M/z 151.0(M +1).

Step F: n- (benzo [ d ] isothiazol-6-yl) -3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propanamide (Compound 2.6)

3- (1, 3-Dioxoisoindolin-2-yl) -2- (p-tolyl) propionic acid (62mg,0.20mmol) was dissolved in 5mL of N, N-dimethylformamide, and then 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (114mg,0.30mmol) and N, N-diisopropylethylamine (52mg,0.40mmol) were added in this order, stirred at room temperature for 5 minutes, followed by addition of compound 18.5(36mg,0.24mmol), and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, water was added to quench the reaction, and extraction was performed with ethyl acetate (10mLX3), and the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give product 2.6(82mg, yield: 93%). LCMS ESI (+) M/z:442.1(M +1).

Step G: 3-amino-N- (benzo [ d ] isothiazol-6-yl) -2- (p-tolyl) propanamide (Compound 2)

Compound 2.6(82mg,0.18mmol) was dissolved in 5mL of methylamine ethanol solution and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 2(1.3mg, yield: 2%) after lyophilization. LCMS ESI (+) M/z 312.1(M +1).

Example 3

3-amino-N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: (Z) -N- ((5-bromothien-2-yl) methylene) -2, 2-dimethoxyethylamine (Compound 3.1)

5-bromothiophene-2-carbaldehyde (1.0g,5.2mmol) and aminoacetaldehyde dimethyl acetal (546mg,5.2mmol) were dissolved in 10mL of toluene, heated under reflux for 3 hours, and water produced by the reaction was removed with a water separator, completion of the reaction was monitored by TLC, and the reaction solution was concentrated under reduced pressure to give crude product 3.1(1.4g, yield: 100%).

And B: ((5-bromothien-2-yl) (dimethoxyphosphoryl) methyl) (2, 2-dimethoxyethyl) carbamic acid ethyl ester (Compound 3.2)

Compound 3.1(1.4g,5.2mmol) was dissolved in 10mL of tetrahydrofuran, and methyl chloroformate (561mg,5.2mmol) was added dropwise under an ice salt bath, and after stirring for 5 minutes, the ice salt bath was removed, and trimethyl phosphite (645mg,5.2mmol) was added at room temperature, followed by reaction at room temperature overnight. TLC monitored the reaction completion, and concentrated under reduced pressure to give crude product 3.2(2.4g, yield: 100%).

And C: 2-Bromothieno [2,3-c ] pyridine (Compound 17.3)

Compound 3.2(2.4g, 5.2mmol) was dissolved in 10mL of methylene chloride, and titanium tetrachloride (3.5mL, 31.2mmol) was slowly dropped under ice bath to react at 40 ℃ for 24 hours. The reaction was monitored by LCMS for completion, and after completion of the reaction, the reaction mixture was slowly poured into a mixture of 20mL of ice water and 10mL of aqueous ammonia, titanium dioxide formed by the reaction was removed by filtration through Celite, the filter cake was washed with dichloromethane, the filtrate was extracted with dichloromethane (50mLX3), the organic phases were combined, washed with saturated brine (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to obtain the product 3.3(583mg, yield: 52%). LCMS ESI (+) M/z:213.9(M +1, 215.9(M +1)).

Step D: n- (diphenylmethylene) thieno [2,3-c ] pyridin-2-amine (Compound 3.4)

Compound 3.3(583mg, 2.7mmol), benzophenone imine (579mg, 3.2mmol),1,1 '-binaphthyl-2, 2' -bis-diphenylphosphine (336mg, 20 mol%) and potassium tert-butoxide (365mg, 3.8mmol) were dissolved in 10mL of toluene and tris (dibenzylideneacetone) dipalladium (247mg,10 mol%) was added under nitrogen. The reaction was carried out overnight at 80 ℃ under nitrogen. LCMS monitored reaction completion. The reaction mixture was concentrated under reduced pressure and purified by column chromatography to give the product 3.4(471mg, yield: 54%). LCMS ESI (+) M/z 315.1(M +1).

Step E: thieno [2,3-c ] pyridin-2-amine (Compound 3.5)

Compound 3.4(471mg,1.5mmol) was dissolved in 5mL of tetrahydrofuran, and 5mL of aqueous hydrochloric acid (2M) was added at room temperature. The reaction was allowed to proceed overnight at room temperature. LCMS monitored reaction completion. After completion of the reaction, the reaction mixture was extracted with ethyl acetate to remove impurities, the aqueous phase was neutralized with saturated sodium hydrogencarbonate, extracted with ethyl acetate again, and the organic layers were combined, washed with saturated brine (30mL) and dried over anhydrous sodium sulfate. Filtration and spin-drying gave crude product 3.5(153mg, yield: 68%). LCMS ESI (+) M/z 151.0(M +1).

Step F: 3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 3.6)

3- (1, 3-Dioxoisoindolin-2-yl) -2- (p-tolyl) propionic acid (100mg,0.30mmol) was dissolved in 5mL of N, N-dimethylformamide, and then 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (171mg,0.45mmol) and N, N-diisopropylethylamine (77mg,0.60mmol) were added in this order, stirred at room temperature for 5 minutes, followed by addition of Compound 3.5(54mg,0.36mmol), and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, water was added to quench the reaction, and extraction was performed with ethyl acetate (10mLX3), and the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give the product 3.6(92mg, yield: 70%). LCMS ESI (+) M/z:442.1(M +1).

Step G: 3-amino-N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 3)

Compound 3.6(92mg,0.21mmol) was dissolved in 5mL of methylamine ethanol solution and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 3(38mg, yield: 45%) after lyophilization. LCMS ESI (+) M/z 312.1(M +1).

Example 4

3-amino-N- (isoquinolin-6-yl) -2- (4-methyl-5, 6-dihydropyridin-1 (2H) -yl) propanamide

The specific reaction equation is as follows:

step A: 1- (2- (benzyloxy) -2-oxoethyl) -4-methylpyridin-1-ium (Compound 4.1)

Weighed 4-methylpyridine (4g, 42.95mmol) was dissolved in 120mL acetonitrile, benzyl 2-bromoacetate (10.8g, 47.25mmol) was added, and the reaction was stirred at 85 ℃ under nitrogen for 24 h. The reaction was concentrated under reduced pressure to give compound 4.1(10.4g, crude, brown oil). LCMS ESI (+) m/z: 242.1(M).

And B: benzyl 2- (4-methyl-5, 6-dihydropyridin-1 (2H) -yl) acetate (Compound 4.2)

Compound 4.1(10.4g, 42.92mmol) was dissolved in 100mL of ethanol, then sodium borohydride (1.62g, 42.92mmol) was added portionwise at 0 deg.C, and the reaction was stirred at room temperature for 3 hours. To the reaction mixture was added 40mL of a saturated ammonium chloride solution, followed by extraction with ethyl acetate (100mL) 3 times, and the organic phases were combined, washed with saturated brine (50mL) 3 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give compound 4.2(5g, yield: 48%, yellow oily substance). LCMS ESI (+) m/z: 246.1(M +1).

And C: benzyl 3- (1, 3-dioxoisoindolin-2-yl) -2- (4-methyl-5, 6-dihydropyridin-1 (2H) -yl) propionate (Compound 4.3)

Compound 4.2(3g, 12.23mmol) was dissolved in 50mL of anhydrous tetrahydrofuran, cooled to-78 deg.C, and under nitrogen protection, bis-trimethylsilyl aminolithium (13.4mL, 1M) was slowly added dropwise, stirred at-78 deg.C for 0.5 h, then 30mL of a tetrahydrofuran solution of N-bromomethylphthalimide (3.23g, 13.45mmol) was added dropwise to the reaction mixture, and stirred at-78 deg.C for 2.5 h. The reaction was quenched with a saturated solution of ammonium chloride (30mL), extracted 2 times with ethyl acetate (100mL), the organic phases were combined, washed 2 times with a saturated saline solution (50mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give compound 4.3(700mg, yield: 14%, yellow oily substance). LCMS ESI (+) m/z: 405.2(M +1).

Step D: 2- ((2-carboxy-2- (4-methyl-5, 6-dihydropyridin-1 (2H) -yl) ethyl) carbamoyl) benzoic acid (Compound 4.4)

Compound 4.3(580mg, 1.43mmol) was dissolved in 15mL of methanol, 15mL of tetrahydrofuran and 15mL of water, lithium hydroxide monohydrate (240mg,5.72mmol) was added, and the reaction mixture was stirred at 50 ℃ for 2 hours. To the reaction was added 1M hydrochloric acid solution to pH 3, and then the reaction was concentrated under reduced pressure to give the product 4.4(500mg, crude, yellow oil). LCMS ESI (+) m/z: 333.1(M +1).

Step E: 3- (1, 3-Dioxoisoindolin-2-yl) -2- (4-methyl-5, 6-dihydropyridin-1 (2H) -yl) propionic acid (Compound 4.5)

Compound 4.4(500mg, 1.50mmol) was dissolved in 30mL of N, N-dimethylformamide, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (570mg, 3.0mmol), 1-hydroxybenzotriazole (405mg, 3.0mmol) and triethylamine (608mg, 6.0mmol) were added. The reaction was stirred at room temperature for 2 hours. The reaction solution was adjusted to pH 4 with 4M hydrochloric acid/methanol solution, concentrated under reduced pressure, and the residue was purified by column chromatography and reverse preparation to give 4.5(130mg, yield: 28%, white solid). LCMS ESI (+) m/z:315.1(M +1).

Step F: 3- (1, 3-dioxoisoindolin-2-yl) -N- (isoquinolin-6-yl) -2- ((4-methyl-5, 6-dihydropyridin-1 (2H) -yl) methyl) propanamide (Compound 4.6)

Compound 4.5(120mg, 0.38mmol) was dissolved in 20mL of N, N-dimethylformamide, and diisopropylethylamine (148mg, 1.14mmol), 6-aminoisoquinoline (66mg, 0.45mmol) and 50% 1-propylphosphoric anhydride/N, N-dimethylformamide (488mg, 0.76mmol) were added. The reaction was stirred at room temperature for 2 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 4.6(50mg, yield: 29%, yellow oil). LCMS ESI (+) m/z: 455.2(M +1).

Step G: 3-amino-N- (isoquinolin-6-yl) -2- ((4-methyl-5, 6-dihydropyridin-1 (2H) -yl) methyl) propanamide (Compound 4)

Compound 4.6(50mg,0.11mmol) was dissolved in 4mL methylamine/ethanol and the reaction was stirred at 45 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 4(30mg, yield: 83%, white solid). LCMS ESI (+) m/z: 325.2(M +1).

Example 5

3-amino-N- (isoquinolin-6-yl) -2- (4-methyl-2-oxopyridin-1 (2H) -yl) propanamide

The specific reaction equation is as follows:

step A: 2- (4-methyl-2-oxopyridin-1 (2H) -yl) acetic acid methyl ester (Compound 5.1)

The weighed compound 4-methylpyridin-2 (1H) -one (600mg, 5.5mmol) was dissolved in N, N-dimethylformamide (10mL), and then methyl bromoacetate (925mg, 6.05mmol), lithium bromide (957mg, 11mmol) and cesium carbonate (2.15g, 6.6mmol) were added to a reaction flask, and the reaction solution was stirred at 75 ℃ for 3 hours. Completion of the reaction was confirmed by LCMS and TLC (PE: EA ═ 5: 1). Water was added to the reaction solution, followed by extraction with ethyl acetate (80mL), and the organic phases were combined for liquid separation, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give compound 5.1(230mg, yield: 23.1%, pale yellow oil). LCMS ESI (+) M/z:182.1(M +1).

And B: 3- (1, 3-Dioxoisoindolin-2-yl) -2- (4-methyl-2-oxopyridin-1 (2H) -yl) propionic acid methyl ester (Compound 5.2)

Compound 5.1(230mg, 1.27mmol) was dissolved in anhydrous tetrahydrofuran (15mL) at-78 deg.C, then bis-trimethylsilyl aminolithium (1.53mL, 1.53mmol) was slowly added dropwise to the reaction solution, and stirred at this temperature for 0.5 hour, followed by dissolving weighed amount of N-bromomethylphthalimide (335.3mg, 1.397mmol) in anhydrous tetrahydrofuran (5mL), then slowly adding dropwise to the reaction flask, followed by stirring at this temperature for 1 hour and stirring in ice bath for 1 hour. Completion of the reaction was confirmed by TLC (PE: EA ═ 1:1) and LCMS. The reaction was quenched with a saturated solution of ammonium chloride, extracted with ethyl acetate (60mL), the organic phases were combined for liquid separation, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give compound 5.2(130mg, yield: 30.1%, colorless oil). LCMS ESI (+) M/z 341.1(M +1).

And C: 2- ((2-carboxy-2- (4-methyl-2-oxopyridin-1 (2H) -yl) ethyl) carbamoyl) benzoic acid (Compound 5.3)

Compound 5.2(130mg, 0.382mmol) was dissolved in methanol (5mL), and then lithium hydroxide monohydrate (40mg, 0.955mmol) was dissolved in water (1mL) and added dropwise to the reaction mixture, followed by stirring at room temperature for 3 hours. The reaction was confirmed to be complete by TLC and LCMS. The reaction solution was concentrated in vacuo, the residue was dissolved in water and then acidified to pH 2-3 with 1M dilute hydrochloric acid, followed by extraction with ethyl acetate (50mL), liquid separation and combination of organic phases, drying over anhydrous sodium sulfate, filtration, and spin-drying to give compound 5.3(100mg, yield: 76%, white solid). LCMS ESI (+) M/z 345.1(M +1).

Step D: 3- (1, 3-Dioxoisoindolin-2-yl) -2- (4-methyl-2-oxopyridin-1 (2H) -yl) propionic acid (Compound 5.4)

Compound 5.3(100mg, 0.29mmol) was dissolved in 30mL of N, N-dimethylformamide, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (105mg, 0.55mmol), 1-hydroxybenzotriazole (75mg, 0.55mmol) and triethylamine (84mg, 0.83mmol) were added. The reaction was stirred at room temperature for 2 hours and then at 45 ℃ for 2 hours. The reaction solution was adjusted to pH 4 with 4M hydrochloric acid/methanol solution, concentrated under reduced pressure, and the residue was purified by column chromatography and reverse preparation to give a product 5.4(50mg, yield: 52%, white solid). LCMS ESI (+) m/z: 327.1(M +1).

Step E: 3- (1, 3-dioxoisoindolin-2-yl) -N- (isoquinolin-6-yl) -2- (4-methyl-2-oxopyridin-1 (2H) -yl) propanamide (Compound 5.5)

Compound 5.4(50mg, 0.15mmol) was dissolved in 6mL of N, N-dimethylformamide, and diisopropylethylamine (200mg, 1.5mmol), 6-aminoisoquinoline (33mg, 0.22mmol) and 50% 1-propylphosphoric anhydride/N, N-dimethylformamide (580mg, 0.9mmol) were added. The reaction solution was stirred at 60 ℃ for 1 hour. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 5.5(25mg, yield: 36%, yellow solid). LCMS ESI (+) m/z: 453.2(M +1).

Step F: 3-amino-N- (isoquinolin-6-yl) -2- (4-methyl-2-oxopyridin-1 (2H) -yl) propanamide (Compound 5)

Compound 5.5(25mg,0.06mmol) was dissolved in 10mL methylamine/ethanol and the reaction was stirred at 45 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 20(11mg, yield: 62%, white solid). LCMS ESI (+) m/z: 323.1(M +1).

Example 6

3- (1-aminocyclopropyl) -N- (isoquinolin-6-yl) -2-phenylpropionamide

The specific reaction equation is shown below

Step A: 2- (4-chlorophenyl) -3-cyanopropionic acid methyl ester (Compound 6.1)

Methyl p-chlorophenylacetate (3g, 16.2mmol) was dissolved in 30mL tetrahydrofuran and cooled to-78 ℃ under nitrogen. LiHMDS (29.2mL, 29.2mmol) was added dropwise thereto, and the mixture was stirred for 1 hour, bromoacetonitrile (1.7mL, 24.32mmol) was dissolved in 10mL of a tetrahydrofuran solution, and the solution was added dropwise thereto, and stirred for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with 50mL of ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to give the product 6.1(3.15g, yield: 87%). LCMS ESI (+) m/z: 224.0(M +1).

And B: 6- (4-chlorophenyl) -4-azaspiro [2.4] heptan-5-one (Compound 6.2)

Compound 6.1(3.15g, 14.06mmol) was dissolved in 30mL of tetrahydrofuran solution, tetraisopropyl titanate (5mL, 16.88mmol) was added, ethylmagnesium bromide (14mL, 28.12mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with 50mL of ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to give the product 6.2(1.6g, yield: 51%). LCMS ESI (+) m/z: 222.1(M +1).

And C: 6- (4-chlorophenyl) -5-oxo-4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (Compound 6.3)

Compound 6.2(1.6g, 7.2mmol) was dissolved in 20mL of tetrahydrofuran solution, LiHMDS (8.6mL, 8.6mmol) was added dropwise at-78 ℃ under nitrogen protection, and stirred for 30 minutes, di-tert-butyl dicarbonate (1.86mL, 7.9mmol) was dissolved in 5mL of tetrahydrofuran solution, added dropwise to the reaction mixture, and stirred for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with 50mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Spin-drying and purification by column chromatography gave the product 6.3(1.15g, yield: 50%). LCMS ESI (+) m/z: 322.1(M +1)

Step D: 3- (1- ((tert-butoxycarbonyl) amino) cyclopropyl) -2- (4-chlorophenyl) propanoic acid (Compound 6.4)

Compound 6.4 was synthesized using step C of example 5. LCMS ESI (+) m/z: 340.1(M +1).

Step E: (1- (2- (4-chlorophenyl) -3- (isoquinolin-6-ylamino) -3-oxopropyl) cyclopropyl) carbamic acid tert-butyl ester (Compound 6.5)

Compound 6.5 was synthesized using step E of example 5. LCMS ESI (+) m/z: 466.2(M +1).

Step F: tert-butyl (1- (3- (isoquinolin-6-ylamino) -3-oxo-2-phenylpropyl) cyclopropyl) carbamate (Compound 6.6)

Compound 6.5(140mg, 0.3mmol) was dissolved in 5mL of methanol solution, and palladium on carbon (20mg) and triethylamine (0.5mL) were added thereto, followed by stirring under a hydrogen atmosphere for 3 hours. The reaction solution was filtered through celite, spin-dried, and purified by column chromatography to give the product 6.6(100mg, yield: 77%). LCMS ESI (+) m/z:432.2(M +1).

Step G: 3- (1-Aminocyclopropyl) -N- (isoquinolin-6-yl) -2-phenylpropionamide (Compound 6)

Compound 6.6(100mg, 0.23mmol) was dissolved in 5mL of a dichloromethane solution, and trifluoroacetic acid (1mL) was added thereto and stirred at room temperature for 1 hour. Purification by rotary drying and reverse preparation gave example 6(60mg, yield: 99%). LCMS ESI (+) m/z: 332.2(M +1).

Example 7

3-amino-N- (4-chlorothieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: 4-Chlorothiopheno [2,3-C ] pyridine-2-carboxylic acid methyl ester (Compound 7.1)

3, 5-dichloroisonicotinal (2g, 11.36mmol) was dissolved in N, N-dimethylformamide (25mL), and cesium carbonate (3.89g, 11.93mmol) and methyl thioglycolate (1.81g, 17.04mmol) were added to the reaction solution, followed by stirring at room temperature for 3 hours. Completion of the reaction was confirmed by TLC and LCMS. After water was added to the reaction solution, a yellow solid precipitated, and the filtrate was filtered, and the filter cake was washed twice with water to spin-dry the water in the filter cake to obtain 7.1(2.2g, yield: 85%) as a pale yellow solid. LCMS ESI (+) M/z 228.0/230.0(M +1).

And B: (4-Chlorothiopheno [2,3-C ] pyridine-2-carboxylic acid (Compound 7.2)

Compound 7.1(2.2g, 9.69mmol) was dissolved in tetrahydrofuran (10mL) and methanol (10mL), and then lithium hydroxide monohydrate (1.02g, 24.23mmol) was dissolved in water (2mL) and added dropwise to the reaction mixture, followed by stirring at room temperature for 3 hours. The reaction was confirmed to be complete by TLC and LCMS. The reaction mixture was concentrated by a vacuum pump, water was added, the pH of the aqueous phase was adjusted to 3 to 4 with 2mol/L dilute hydrochloric acid, a solid precipitated, and the solid was collected by filtration to obtain 7.2(1.89g, yield: 92%) as an off-white solid. LCMS ESI (+) M/z 214.0/216.0(M +1).

And C: 4-chlorothieno [2,3-c ] pyridin-2-amine (Compound 7.3)

Compound 37.2(150mg, 0.7mmol) was dissolved in dry toluene (5mL) under ice bath, triethylamine (176mg, 1.75mmol) and diphenylphosphorylazide (250mg, 0.91mmol) were added under ice bath, stirring was continued at 0 ℃ for 0.5 hour, then stirring was continued at 85 ℃ for 2 hours under inert gas atmosphere, and then water (25mg, 1.4mmol) was added to the reaction flask, and stirring was continued at 85 ℃ overnight. The reaction was confirmed to be complete by LCMS. Water was added to the reaction solution, followed by extraction with ethyl acetate (15mL), and then the organic phases were combined, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and dried by spinning to obtain crude compound 7.3(70mg, yield: 54%). LCMS ESI (+) M/z 185.0/187.1(M +1).

Step D: n- (4-Chlorothiopheno [2,3-c ] pyridin-2-yl) -3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propanamide (Compound 7.4)

Compound 7.3(31mg, 0.16mmol) and 3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propionic acid (40mg, 0.129mmol) were dissolved in N, N-dimethylformamide (5mL), and then a solution of N, N-diisopropylethylamine (33mg, 0.25mmol) in N, N-dimethylformamide (123mg, 0.19mmol) containing 50% propylphosphoric anhydride was added to the reaction mixture, and the reaction was stirred at room temperature for 2 hours. The reaction was confirmed to be complete by LCMS. Water was added to the reaction solution, followed by extraction with ethyl acetate (12mL), and then the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give 7.4(15mg, yield: 24%) as a pale yellow oily product. LCMS ESI (+) M/z 476.1/478.1(M +1).

Step E: 3-amino-N- (4-chlorothieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 7)

Compound 7.4(15mg, 0.032mmol) was dissolved in ethanol (4mL), and hydrazine hydrate (16mg, 0.32mmol) was added to the reaction solutionThe reaction was stirred at 60 ℃ for 2 hours under an inert gas atmosphere. The reaction was confirmed to be complete by LCMS. The reaction mixture was directly concentrated and spin-dried by a vacuum pump, and the residue was dissolved in methanol (1.5mL) to prepare and purify by reverse rotation example 7(10mg, yield: 69%, purity: 98%) as a white solid. LCMS ESI (+) M/z 346.1/348.1(M +1).¹H NMR(400MHz,DMSO)δ12.26(s,1H),9.07(s,1H),8.46(s,1H),7.93(s,3H),7.25(dd,J＝18.6,8.2Hz,4H),7.02(s,1H),4.16–4.08(m,1H),3.58-3.56(m,1H),3.26–2.96(m,1H),2.29(s,3H).

Example 8

(S) -3-amino-2- (4- (hydroxymethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide

The specific reaction equation is shown below

Step A: 2- (4- (bromomethyl) phenyl) acetic acid (Compound 8.1)

Dissolving 4-methylphenylacetic acid (100g, 670mmol) in acetonitrile (1L), dissolving N-bromosuccinimide (125.2g, 703.5mmol) and azobisisobutyronitrile (2.2g, 13.4mmol) in a reaction solution, reacting at 80 ℃ for 3 hours under the protection of nitrogen, cooling the reaction solution to room temperature after the reaction is finished, separating out a large amount of solid, filtering, washing a filter cake with petroleum ether/ethyl acetate (1:1) (200mL), saturated sodium bisulfite (200mL), water (200mL) and petroleum ether (200mL), and drying at 50 ℃ to obtain a compound 8.1(82g, yield: 54%, white solid). LCMS ESI (+) M/z 229.0/231.0(M +1).

And B: 2- (4- (hydroxymethyl) phenyl) acetic acid (Compound 8.2)

Adding the compound 41.1(82g, 358.1mmol) into water (300mL), heating to 100 ℃ for reaction for 2 hours, making the reaction clear, then cooling to room temperature, further cooling to 0 ℃ in an ice bath, separating out a white solid, filtering, washing a filter cake for 2 times by using water, and drying at 50 ℃ to obtain the compound 8.2(40g, yield: 67%, white solid) which is directly used for the next reaction.

And C: 2- (4- (hydroxymethyl) phenyl) acetic acid methyl ester (Compound 8.3)

Compound 8.2(40g, 241.0mmol) was dissolved in methanol (400mL), sulfuric acid (2.4g, 24.1mmol) was added, the reaction was performed in ice bath for 2 hours, a saturated sodium bicarbonate solution (200mL) was added after completion of the reaction, the aqueous layer was extracted with ethyl acetate (200mL X3), the organic layers were combined, washed with a saturated brine (300mL), dried over anhydrous sodium sulfate, filtered, and dried by spinning to give compound 8.3(41g, yield: 95%, pale yellow oil). LCMS ESI (+) M/z 181.1(M +1).

Step D: methyl 2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) acetate (Compound 8.4)

Compound 8.3(41g, 226.5mmol) and imidazole (23.1g, 339.8mmol) were dissolved in N, N-dimethylformamide (400mL), heated to 0 ℃ in ice bath, triisopropylchlorosilane (48.1g, 249.2mmol) was added in portions, the temperature was naturally raised to room temperature and stirred overnight, after completion of the reaction, water (500mL) was added, the aqueous layer was extracted with ethyl acetate (200mL X3), the organic layers were combined, washed with saturated brine (300mL), dried over anhydrous sodium sulfate, filtered, and dried by spinning to give compound 8.4(72g, yield: 94%, colorless oil) which was used directly in the next reaction.

Step E: 2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) acetic acid (Compound 8.5)

Compound 8.4(72g, 214.0mmol) was dissolved in tetrahydrofuran (500mL), lithium hydroxide (10.8g, 256.8mmol) was added, the mixture was stirred at room temperature overnight, 1M hydrochloric acid solution (300mL) was added after completion of the reaction, the aqueous layer was extracted with ethyl acetate (200mL X3), the organic layers were combined, washed with saturated brine (300mL), dried over anhydrous sodium sulfate, filtered, and dried by spinning to give compound 8.5(67g, yield: 97%, colorless oil) which was used directly in the next reaction.

Step F: (R) -4-benzyl-3- (2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) acetyl) oxazolidine-2-one (Compound 8.6)

Dissolving a compound 8.5(67g, 207.8mmol) and anhydrous N, N-dimethylformamide (0.76g, 10.4mmol) in anhydrous dichloromethane, slowly adding oxalyl chloride (31.7g, 249.4mmol) dropwise under ice bath conditions, keeping ice bath to prepare acyl chloride, simultaneously adding (R) -4-benzyl-oxazolidinone (25.7g, 145.2mmol) into anhydrous tetrahydrofuran (300mL) in another three-necked bottle, replacing with nitrogen, cooling to-78 ℃ with dry ice acetone, slowly adding N-butyl lithium (2.5M, 64mL) dropwise, keeping the temperature at-78 ℃, continuing to stir at-78 ℃ for 2 hours after dropwise addition is finished, then slowly adding the anhydrous tetrahydrofuran solution of the acyl chloride prepared before dropwise through a constant pressure dropping funnel, keeping the dropwise adding temperature at-78 ℃, continuing to keep at-78 ℃ for reaction for 2 hours after dropwise addition is finished, then, the temperature was naturally raised to room temperature, after completion of the reaction, the reaction was extracted with a saturated aqueous ammonium chloride solution (400mL), the aqueous layer was extracted with ethyl acetate (300mL X3), the organic layers were combined, washed with a saturated aqueous sodium bicarbonate solution (300mL) and a saturated brine (300mL) in this order, dried over anhydrous sodium sulfate, filtered, dried by spinning, and purified by column chromatography to obtain compound 8.6(44g, yield: 44%, colorless oily substance).

Step G2- ((S) -3- ((R) -4-benzyl-2-oxooxazolidin-3-yl) -3-oxo-2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) propyl) isoindoline-1, 3-dione (Compound 8.7)

Dissolving a compound 8.6(44g, 91.3mmol) in anhydrous tetrahydrofuran (500mL), replacing with nitrogen, cooling dry ice acetone to-78 ℃, slowly dropwise adding LiHMDS (1M, 109mL), continuing to stir at-78 ℃ for 1 hour after the dropwise addition is finished, dissolving N-bromomethylphthalimide (26.3g, 109.6mmol) in anhydrous tetrahydrofuran (200mL), slowly dropwise adding the above system through a constant pressure dropping funnel, controlling the dropwise adding temperature to be lower than-70 ℃, keeping the reaction at-78 ℃ after the dropwise addition is finished, stirring for 3 hours, naturally heating to room temperature and stirring overnight, after the reaction is finished, extracting the reaction by using a saturated ammonium chloride aqueous solution (400mL), extracting the aqueous layer by using ethyl acetate (300mL X3), combining organic layers, sequentially washing by using a saturated saline solution (300mL), drying anhydrous sodium sulfate, filtering, spin-drying to obtain a yellow solid, it was recrystallized from ethyl acetate/petroleum ether system, a solid was precipitated, filtered, and the filter cake was washed with petroleum ether (100mL) and spin-dried to give compound 8.7(32g, yield: 55%, white solid).

Step H: (S) -2- ((2-carboxy-2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) ethyl) carbamoyl) benzoic acid (Compound 8.8)

Compound 8.7(32g, 49.9mmol) was dissolved in tetrahydrofuran (300mL), water (100mL) was added, the reaction was cooled in an ice bath to 0 ℃ and then lithium hydroxide (6.3g, 149.7mmol) was added, stirring was carried out at 0 ℃ for 1 hour, after completion of the reaction, the pH of the reaction solution was adjusted to 2-3 with 2M hydrochloric acid solution, the aqueous layer was extracted with ethyl acetate (200mL X4), the organic layers were combined, washed with saturated brine (300mL) in this order, dried over anhydrous sodium sulfate, filtered, and spin-dried to give compound 8.8(20g, yield: 80%, pale yellow solid). LCMS ESI (+) M/z:500.2(M +1).

Step I: (S) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) propanoic acid (Compound 8.9)

Compound 8.8(20g, 40mmol), 1-hydroxybenzotriazole (5.9g, 44mmol) and triethylamine (12.1g, 120mmol) were dissolved in N, N-dimethylformamide, the reaction was cooled to 0 ℃ in an ice bath, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (8.4g, 44mmol) was added to the reaction solution. Stirring for a while to clarify the reaction solution, slowly raising the temperature to room temperature overnight, after completion of the reaction, reducing the pressure to dryness, adding saturated aqueous ammonium chloride solution (200mL), extracting the aqueous layer with ethyl acetate (200mL X3), combining the organic layers, washing with saturated aqueous sodium bicarbonate solution (300mL), saturated brine (300mL), drying over anhydrous sodium sulfate, filtering, and spin-drying to obtain compound 8.9(17g, yield: 88%, yellow solid)

Step J: (S) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) propanamide (Compound 8.10)

(S) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) propanoic acid (65mg, 0.123mmol) was dissolved in 5mL of N, N-dimethylformamide, diisopropylethylamine (25. mu.l, 0.16mmol), 2- (7-oxybenzotriazole) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (77mg, 0.20mmol) were added, and stirring was carried out at room temperature for 10 minutes, thieno [2,3-c ] pyridin-2-amine (20mg, 0.135mmol) was added, and stirring was carried out for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with 30mL of ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to give the product 8.10(48mg, yield: 58%). LCMS ESI (+) m/z: 614.2(M +1).

Step K: (S) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (hydroxymethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 8.11)

Compound 8.10(48mg, 0.078mmol) was dissolved in 5mL of tetrahydrofuran solution, and 1mL of hydrochloric acid (2M) was added thereto, followed by stirring at room temperature for 0.5 hour. The solvent was directly spin-dried to give the product 8.11(30mg, yield: 100%). LCMS ESI (+) m/z: 458.1(M +1).

Step L: (S) -3-amino-2- (4- (hydroxymethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 8)

Compound 8.11(30mg, 0.066mmol) was dissolved in 5mL of methylaminoethanol solution, stirred at 60 ℃ for 3 hours, the solvent was dried by spinning, and preparative purification was performed in the reverse direction to give example 8(18mg, yield: 84%). LCMS ESI (+) m/z:328.1(M +1).

Example 9

(S) -3-amino-N- (benzo [ d ] isothiazol-6-yl) -2- (4- (hydroxymethyl) phenyl) propanamide

The specific reaction equation is shown below

Step A: compound 9.1 was synthesized by substituting benzo [ d ] isothiazol-6-amine for thieno [2,3-c ] pyridin-2-amine in step A of example 8. LCMS ESI (+) m/z: 614.2(M +1).

And B: compound 9.2 was synthesized using step B of example 8. LCMS ESI (+) m/z: 458.1(M +1).

And C: using step C of example 8, the synthesis gave example 9. LCMS ESI (+) m/z:328.1(M +1).

Example 10

3-amino-2- (4-hydroxyphenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide

The specific reaction equation is as follows:

step A: 2- (4- ((tert-butyldimethylsilyl) oxy) phenyl) acetic acid (Compound 10.1)

P-hydroxyphenylacetic acid (5.0g, 32.86mmol) was dissolved in 400mL of tetrahydrofuran, and imidazole (11g,164.3mmol) and tert-butyldimethylsilyl chloride (13.8g,92.00mmol) were added at 0 ℃ to stir the reaction solution at room temperature for 2 hours. 130mL of a saturated sodium carbonate solution was added to the reaction solution, and stirring was continued at room temperature for 1 hour. To the reaction solution was added 2M hydrochloric acid solution to pH 4, and extracted 2 times with ethyl acetate (500mL), the organic phases were combined, washed 2 times with brine (200mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give product 10.1(5.78g, yield: 66%, colorless oil).

And B: benzyl 2- (4- ((tert-butyldimethylsilyl) oxy) phenyl) acetate (Compound 10.2)

Compound 10.1(5.78g, 21.70mmol) was dissolved in 100mL of N, N-dimethylformamide, the solution was cooled to 0 deg.C, potassium carbonate (3.6g, 26.04mmol) and benzyl bromide (4.45g, 26.04mmol) were added, and the reaction solution was stirred at room temperature for 2 hours. 80mL of water was added to the reaction solution, and extracted 3 times with ethyl acetate (100mL), the organic phases were combined, washed 4 times with saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 10.2(3.66g, yield: 47%, colorless oil).

And C: benzyl 2- (4- ((tert-butyldimethylsilyl) oxy) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionate (Compound 10.3)

Compound 10.2(3.66g, 10.27mmol) was dissolved in 30mL of anhydrous tetrahydrofuran, the reaction solution was cooled to-78 deg.C, 1M lithium hexamethyldisilazide (12.3mL, 12.3mmol) was added dropwise under nitrogen protection, and the reaction solution was stirred at-78 deg.C under nitrogen protection for 1 hour. Then, 25mL of a tetrahydrofuran solution of 2- (bromomethyl) isoindole-1, 3-dione (2.96g, 12.3mmol) was added dropwise thereto, and the reaction mixture was stirred at-78 ℃ for 3 hours. To the reaction solution was added 30mL of water, and extracted 3 times with ethyl acetate (60mL), the organic phases were combined, washed 2 times with saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 10.3(3.9g, yield: 74%, colorless oil). LCMS ESI (+) m/z: 516.2(M +1).

Step D: 2- (4- ((tert-Butyldimethylsilyl) oxy) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionic acid (Compound 10.4)

Compound 10.3(3.9g, 7.56mmol) was dissolved in 30mL of tetrahydrofuran and 60mL of methanol, palladium on carbon (600mg, 10%) was added, and the reaction was stirred at room temperature under a hydrogen blanket for 3 hours. The reaction solution was filtered with celite, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 10.4(2.9g, yield: 90%, yellow solid). LCMS ESI (+) m/z: 426.2(M +1).

Step E: 2- (4- ((tert-Butyldimethylsilyl) oxy) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 10.5)

Compound 10.4(600mg, 1.41mmol) was dissolved in 20mL of N, N-dimethylformamide and diisopropylethylamine (276mg, 2.11mmol), 2- (7-oxide benzotriazol) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (648mg, 1.69mmol), and thieno [2,3-c ] pyridin-2-amine (233mg, 1.55mmol) were added. The reaction solution was stirred at 25 ℃ under nitrogen for 2 hours. Water (40mL) was added to the reaction, followed by extraction with ethyl acetate (60mL) 2 times, and the organic phases were combined, washed with saturated brine (30mL) 2 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give the product 10.5(780mg, yield: 99%, yellow solid). LCMS ESI (+) m/z: 558.2(M +1).

Step F: 3- (1, 3-dioxoisoindolin-2-yl) -2- (4-hydroxyphenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 10.6)

Compound 10.5(780mg, 1.40mmol) was dissolved in 50mL of tetrahydrofuran, 15mL of 2M aqueous hydrochloric acid was added, and the reaction mixture was stirred at 50 ℃ for 3 hours. The reaction solution was adjusted to pH 7 with saturated sodium bicarbonate solution, extracted 3 times with ethyl acetate (100mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 10.6(350mg, yield: 56%, white solid). LCMSISI (+) m/z: 444.1(M +1).

Step G: 3-amino-2- (4-hydroxyphenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 10)

Compound 10.6(15mg, 0.03mmol) was dissolved in 7mL methylamine/ethanol and the reaction was stirred at 50 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 10(8mg, yield: 80%, white solid). LCMS ESI (+) m/z: 314.1(M +1).

Example 11

3-amino-N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: 3, 5-Difluoropyridine-4-carbaldehyde (Compound 11.1)

Dissolving 3, 5-difluoropyridine (1g, 58.5mmol) in anhydrous tetrahydrofuran (15mL), cooling to-78 ℃, slowly dropping lithium diisopropylamide (10mL, 10.4mmol) into the reaction flask under the protection of nitrogen, continuing to stir at the temperature for 0.5 hour, dissolving ethyl formate (1.61g, 21.73mmol) in anhydrous tetrahydrofuran (5mL), slowly dropping into the reaction flask, and then stirring under ice bath for 2 hours. Completion of the reaction was confirmed by LCMS and TLC. Water was added to the reaction solution, followed by extraction with ethyl acetate (20mL), and then the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give 11.1(280mg, yield: 23%) as a yellow solid product. LCMS ESI (+) M/z 176.0(M +33).

And B: 4-Fluorothieno [2,3-C ] pyridine-2-carboxylic acid methyl ester (Compound 11.2)

Compound 11.1(280mg, 1.96mmol) was dissolved in N, N-dimethylformamide (6mL), and then cesium carbonate (703mg, 2.15mmol) and methyl thioglycolate (166mg, 1.568mmol) were added to the reaction solution under ice bath, followed by stirring at normal temperature for 3 hours. Completion of the reaction was confirmed by TLC and LCMS. Water was added to the reaction solution, followed by extraction with ethyl acetate (15mL), and then the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give 11.2(170mg, yield: 41%) as a white solid. LCMS ESI (+) M/z 212.0(M +1).

And C: (4-Fluorothieno [2,3-C ] pyridine-2-carboxylic acid (Compound 11.3)

Compound 11.2(170mg, 0.81mmol) was dissolved in tetrahydrofuran (6mL) and methanol (1mL), then lithium hydroxide monohydrate (85mg, 2.02mmol) was dissolved in water (2mL) and added dropwise to the reaction mixture, which was stirred at 40 ℃ for 0.5 h. The reaction was confirmed to be complete by TLC and LCMS. The reaction mixture was concentrated by a vacuum pump, water was added, the pH of the aqueous phase was adjusted to 3 to 4 with 2mol/L dilute hydrochloric acid, a solid precipitated, and the solid was collected by filtration and dried by spinning to give 11.3(130mg, yield: 82%) as an off-white solid. LCMS ESI (+) M/z:198.0(M +1).

Step D: 4-Fluorothieno [2,3-c ] pyridin-2-amine (Compound 11.4)

Compound 11.3(150mg, 0.7mmol) was dissolved in dry toluene (8mL) under ice bath, triethylamine (166mg, 1.65mmol) and diphenylphosphorylazide (236mg, 0.86mmol) were added under ice bath, stirring was continued at 0 ℃ for 0.5 hour, then stirring was continued at 85 ℃ for 2 hours under inert gas atmosphere, and then water (35mg, 1.98mmol) was added to the reaction flask, and stirring was continued at 85 ℃ overnight. The reaction was confirmed to be complete by LCMS. Water was added to the reaction solution, followed by extraction with ethyl acetate (15mL), and then the organic phases were combined, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain crude compound 11.4(100mg, yield: 90%). LCMSISI (+) M/z 169.0(M +1).

Step E: n- (4-Fluorothieno [2,3-c ] pyridin-2-yl) -3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propanamide (Compound 11.5)

Compound 11.4(26mg, 0.155mmol) and 3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propionic acid (40mg, 0.129mmol) were dissolved in N, N-dimethylformamide (5mL), and then triethylamine (26mg, 0.258mmol) and a solution of N, N-dimethylformamide (123mg, 0.1935mmol) containing 50% propylphosphoric anhydride were added to the reaction solution, and the reaction was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. Water was added to the reaction mixture, and after extraction with ethyl acetate (12mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spun-dried, and purified with silica gel column to give 11.5(20mg, yield: 34%) as a pale yellow oily product. LCMS ESI (+) M/z 460.1(M +1).

Step F: 3-amino-N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 11)

Compound 11.5(20mg, 0.043mmol) was dissolved in ethanol (5mL), and then hydrazine hydrate (22mg, 0.434mmol) was added to the reaction solution, and the reaction was stirred at 60 ℃ for 2 hours under an inert gas atmosphere. The reaction was confirmed to be complete by LCMS. The reaction mixture was directly concentrated and spin-dried by a vacuum pump, and the residue was dissolved in methanol (1.5mL) to prepare and purify by the reverse phase, thereby obtaining example 11(3mg (trifluoroacetate salt, yield: 21%, purity: 96%) as a white solid. LCMS ESI (+) M/z:330.1(M +1).

Example 12

3-amino-2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: 2- (4-Vinylphenyl) acetic acid methyl ester (Compound 12.1)

Methyl 2- (4-bromophenyl) acetate (11.0g,48.0mmol), potassium trifluoro (vinyl) borate (7.7g,57.6mmol) and cesium carbonate (31.3g,96.0mmol) were dissolved in 200mL of tetrahydrofuran and 20mL of water, followed by addition of bis (triphenylphosphine) palladium dichloride (674mg,0.96mmol) and reaction at 85 ℃ under nitrogen overnight. LCMS monitored reaction completion. Filtration, extraction of the filtrate with ethyl acetate (50mLX3) three times, combination of the organic phases, washing with saturated brine (100mL), drying of the organic phase over anhydrous sodium sulfate, filtration, spin-drying, and purification by column chromatography gave 12.1(7.0g, yield: 83%). LCMS ESI (+) M/z 177.1(M +1).

And B: methyl 2- (4- (2-hydroxyethyl) phenyl) acetate (Compound 12.2)

Compound 12.1(3.5g,19.9mmol) was dissolved in 20mL of tetrahydrofuran, borane (10.0M,4mL,40.0mmol) was added dropwise under ice bath, and the mixture was reacted at 0 ℃ for 1 hour and then at room temperature for 1 hour. Then, 60mL of an aqueous sodium hydroxide solution (1M) was added dropwise in an ice bath, 5mL of hydrogen peroxide (30%) was added dropwise, the reaction was carried out for 30 minutes in an ice bath, and the reaction was carried out for 30 minutes at room temperature. The starting material reaction was monitored by TLC to be complete. The reaction was quenched with water, extracted with ethyl acetate (50mLX3), the organic phases were combined, washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 12.2(1.5g, yield: 39%).

And C: methyl 2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) acetate (Compound 12.3)

Compound 12.2(1.5g,7.7mmol) was dissolved in 10mL of dichloromethane, and then 2, 6-lutidine (1.4mL,11.6mmol) and triisopropylsilyl trifluoromethanesulfonate (2.5mL,9.2mmol) were added and reacted at room temperature overnight. The starting material reaction was monitored by TLC to be complete. The reaction was quenched with water, extracted three times with dichloromethane (20mLX3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 12.3(2.1g, yield: 78%).

Step D: 3- (1, 3-Dioxoisoindolin-2-yl) -2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) propanoic acid methyl ester (Compound 12.4)

Dissolving compound 12.3(2.1g,6.0mmol) in 20mL of anhydrous tetrahydrofuran, replacing with nitrogen, cooling dry ice acetone to-78 ℃, slowly adding hexamethyldisilazane-based amino lithium (1.0M, 7.2mL, 7.2mmol) dropwise, stirring the system at-78 ℃ for 1 hour after dropwise addition is completed, dissolving N-bromomethylphthalimide (1.7g,7.2mmol) in 10mL of tetrahydrofuran, slowly adding dropwise into the system, controlling the dropwise addition temperature to be lower than-70 ℃, keeping the reaction at-78 ℃ after dropwise addition, stirring for 3 hours, and monitoring by TLC that the raw material is completely reacted. The reaction was quenched by the addition of saturated ammonium chloride solution (10mL), extracted with ethyl acetate (20mLX3), and washed with saturated brine (50 mL). Dried over anhydrous sodium sulfate, filtered, dried by spinning, and purified by column chromatography to obtain 12.4(1.0g, yield: 33%).

Step E: 2- ((2-carboxy-2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) ethyl) carbamoyl) benzoic acid (Compound 12.5)

Compound 12.4(1.0g,2.0mmol) was dissolved in 10mL tetrahydrofuran and 3mL water, lithium hydroxide monohydrate (252mg,6.0mmol) was added at 0 deg.C, stirred at 0 deg.C for 3 hours, and the reaction was monitored by LCMS for completion. The organic solvent was distilled off under reduced pressure, the pH was adjusted to 3-4 with 1M aqueous hydrochloric acid, extraction was performed with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to give the product 12.5(960mg, yield: 95%). LCMS ESI (+) m/z:514.2(M +1).

Step F: 3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) propanoic acid (Compound 12.6)

The compound 12.5(960mg,1.9mmol), 1-hydroxybenzotriazole (257mg,1.9mmol) and triethylamine (0.8mL,5.7mmol) were dissolved in 10mL dichloromethane, cooled to 0 ℃ under nitrogen protection, then 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (365mg,1.9mmol) was added to the reaction mixture, stirred for a while, the reaction mixture became clear, slowly warmed to room temperature, and reacted overnight. LCMS monitored reaction completion. The reaction mixture was diluted with water, extracted with methylene chloride (20mLX3), the organic phases were combined, washed with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give the product 12.6(510mg, yield: 54%). LCMS ESI (+) m/z: 496.2(M +1).

Step G: 3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) propanamide (Compound 12.7)

After 12.6(70mg,0.14mmol) was dissolved in 5mL of N, N-dimethylformamide, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (80mg,0.21mmol) and N, N-diisopropylethylamine (36mg,0.28mmol) were sequentially added thereto, and the mixture was stirred at room temperature for 5 minutes, followed by addition of 19.5(25mg,0.17mmol) and reaction at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, water was added to quench the reaction, and extraction was performed with ethyl acetate (10mLX3), and the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give the product 12.7(62mg, yield: 71%). LCMS ESI (+) M/z 628.3(M +1).

Step H: 3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 12.8)

Compound 12.7(62mg,0.10mmol) was dissolved in 5mL of tetrahydrofuran, and then added to 5mL of aqueous hydrochloric acid (1M) and reacted at 40 ℃ for 2 hours. LCMS monitored reaction completion. After completion of the reaction, saturated sodium bicarbonate solution was added to neutralize, followed by extraction with ethyl acetate (10mLX3), the organic phases were combined, washed with saturated brine (20mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and dried by spinning to give a crude product 12.8(47mg, yield: 100%). LCMS ESI (+) M/z 472.1(M +1).

Step I: 3-amino-2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 12)

Compound 12.8(47mg,0.10mmol) was dissolved in 5mL of methylamine ethanol solution and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 12(19mg, yield: 44%) after lyophilization. LCMS ESI (+) M/z 342.1(M +1).

Example 13

3-amino-2- (4- (3-hydroxypropoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: (2- (4-hydroxyphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 13.1)

3-amino-2- (4-hydroxyphenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (850mg, 2.71mmol) was dissolved in 40mL of N, N-dimethylformamide, triethylamine (550mg,5.42mmol) and BOC anhydride (770mg,3.52mmol) were added, and the reaction mixture was stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 13.1(1.1g, yield: 98%, yellow solid). LCMS ESI (+) m/z:414.1(M +1).

And B: (2- (4- (3- ((tert-butyldimethylsilyl) oxy) propoxy) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 13.2)

Compound 13.1(50mg, 0.12mmol) was dissolved in 10mL of N, N-dimethylformamide, potassium carbonate (34mg, 0.24mmol) and (3-bromopropoxy) (tert-butyl) dimethylsilane (46mg, 0.18mmol) were added, and the reaction solution was stirred at 100 ℃ under nitrogen for 10 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (40mL), the organic phases were combined, washed 3 times with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product 13.2(10mg, yield: 14%, yellow solid). LCMS ESI (+) m/z: 586.2(M +1).

And C: 3-amino-2- (4- (3-hydroxypropoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 13)

Compound 13.2(10mg, 0.02mmol) was dissolved in 2mL of dichloromethane, 1mL of trifluoroacetic acid was added, the reaction mixture was stirred at room temperature for 1 hour, the reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 13(5.8mg, yield: 91%, white solid). LCMS ESI (+) m/z: 372.1(M +1).

Example 14

3-amino-2- (4- (2, 3-dihydroxypropoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: (2- (4- (2, 3-dihydroxypropoxy) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 14.1)

Tert-butyl (2- (4-hydroxyphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamate (200mg, 0.48mmol) was dissolved in 18mL ethanol, triethylamine (244mg,2.42mmol) and glycidol (180mg,2.42mmol) were added, and the reaction was stirred at 80 ℃ under nitrogen for 60 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product 14.1(5mg, yield: 2%, yellow solid). LCMS ESI (+) m/z: 488.2(M +1).

And B: 3-amino-2- (4- (2, 3-dihydroxypropoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 14)

Compound 14.1(5mg, 0.01mmol) was dissolved in 2mL of dichloromethane, 1mL of trifluoroacetic acid was added, the reaction mixture was stirred at room temperature for 1 hour, the reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 49(0.8mg, yield: 20%, white solid). LCMS ESI (+) m/z: 388.1(M +1).

Example 15

3-amino-2- (4- ((1, 3-dihydroxypropan-2-yl) oxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide

The specific reaction equation is as follows:

step A: dimethyl 2- (4- (3- ((tert-butoxycarbonyl) amino) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) phenoxy) malonate (compound 15.1)

Tert-butyl (2- (4-hydroxyphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamate (100mg, 0.24mmol) was dissolved in 8mL of N, N-dimethylformamide, dimethyl 2-bromomalonate (76mg,0.36mmol) and potassium carbonate (67mg,0.48mmol) were added, and the reaction solution was stirred at 50 ℃ for 3.5 hours. To the reaction solution was added 25mL of water, and extracted 2 times with ethyl acetate (40mL), the organic phases were combined, washed 2 times with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 15.1(35mg, yield: 27%, yellow oil). LCMS ESI (+) m/z: 544.2(M +1).

And B: (2- (4- ((1, 3-dihydroxypropan-2-yl) oxy) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 15.2)

Compound 15.1(35mg, 0.06mmol) was dissolved in 8mL of tetrahydrofuran, the reaction solution was cooled to 0 deg.C, sodium borohydride (15mg, 0.36mol) was added, the reaction solution was stirred at 0 deg.C for 20 minutes, 4mL of methanol was added dropwise, and the reaction solution was stirred at room temperature for 2 hours. Lithium borohydride (28mg, 1.29mmol) was added to the reaction solution, and the reaction solution was stirred at 50 ℃ for 4 hours. To the reaction solution was added 1M hydrochloric acid solution to pH 7, the reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give 15.2(10mg, yield: 32%, white solid). LCMS ESI (+) m/z: 488.2(M +1).

And C: 3-amino-2- (4- ((1, 3-dihydroxypropan-2-yl) oxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 15)

Compound 15.2(10mg, 0.02mmol) was dissolved in 2mL of dichloromethane, 1mL of trifluoroacetic acid was added, the reaction was stirred at room temperature for 1 hour, the reaction was concentrated under reduced pressure, and the residue was purified by reverse preparation to give product 15(3.6mg, yield: 45%, white solid). LCMS ESI (+) m/z: 388.1(M +1).

Example 16

3-amino-N- (5-chlorobenzo [ d ] isothiazol-6-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: 5-chloro-2-fluoro-4-methoxybenzaldehyde (Compound 16.1)

2-fluoro-4-methoxybenzaldehyde (6g, 38.96mmol) was dissolved in glacial acetic acid (13mL), and sulfone chloride (10.52g, 77.92mmol) was slowly added to the reaction solution under ice bath, followed by stirring at room temperature overnight. The reaction was confirmed to be complete by LCMS. The reaction solution was added to ice water, after precipitation of a solid, the solid was filtered, the solid was dissolved in ethyl acetate, then the organic phase was washed with a saturated sodium bicarbonate solution and a saturated brine in this order, dried over anhydrous sodium sulfate, filtered, concentrated and spin-dried, and the crude solid was slurried with petroleum ether to give 16.1(4g, yield: 55%) as a white solid product after filtration. LCMS ESI (+) M/z:189.0/191.0(M +1).

And B: 5-chloro-6-methoxybenzo [ d ] isothiazole (Compound 16.2)

Compound 16.1(3.8g, 20.2mmol) was dissolved in ethylene glycol monomethyl ether (20mL), and then sulfur powder (646.4mg, 20.2mmol) and aqueous ammonia (20mL) were added to the reaction solution, followed by stirring in a sealed tube at 140 ℃ for 2 hours. Completion of the reaction (reaction with partially chlorinated molecules) was confirmed by TLC and LCMS. The reaction solution was added to ice water, extracted with ethyl acetate (20mL), and then the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give 16.2(1.88g, yield: 47%) as a pale yellow solid product. LCMS ESI (+) M/z 200.0/202.0(M +1).

And C: 5-chlorobenzo [ d ] isothiazol-6-ol (Compound 16.3)

Compound 51.2(1.88g, 9.5mmol) and pyridine hydrochloride (10g) were added to the reaction flask and stirred at 170 deg.C for 8 hours. Completion of the reaction was confirmed by TLC (PE: EA ═ 10:1) and LCMS. Water was added to the reaction flask and a solid precipitated out, which was filtered and dried by spinning to give 16.3 as a pale pink solid (1.57g, yield: 90%). LCMS ESI (+) M/z 186.0/188.0(M +1).

Step D: 5-chlorobenzo [ d ] isothiazol-6-yl trifluoromethanesulfonate (Compound 16.4)

Weighed compound 16.3(1.57g, 8.48mmol) was dissolved in dichloromethane (20mL), N-diisopropylethylamine (3.28g, 25.46mmol) and trifluoromethanesulfonic anhydride (3.59g, 12.73mmol) were added at-78 deg.C and stirring was continued for 1 hour maintaining this temperature. Completion of the reaction was confirmed by TLC (PE: EA ═ 10:1) and LCMS. The reaction solution was diluted with dichloromethane, washed with 1mol/L dilute hydrochloric acid, a saturated sodium bicarbonate solution and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give 16.4(1.37g, yield: 51%) as a pale yellow solid. LCMS ESI (+) M/z 318.1/320.1(M +1).

Step E: 5-chloro-N- (diphenylmethylene) benzo [ d ] isothiazol-6-amine (Compound 16.5)

Compound 16.4(640mg, 2.02mmol) was dissolved in 1, 4-dioxane (12mL), and benzophenone imine (402mg, 2.22mmol), tris (dibenzylideneacetone) dipalladium (185mg, 0.20mmol), 4, 5-bis diphenylphosphine-9, 9-dimethylxanthene (233mg, 0.404mmol), and cesium carbonate (1.32g, 4.04mmol) were added to the reaction solution, and the reaction was stirred at 90 ℃ overnight under an inert gas atmosphere. The reaction was confirmed to be complete by LCMS. Water was added to the reaction solution, followed by extraction with ethyl acetate (15mL), and then the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give 16.5(660mg, yield: 94%) as a yellow solid product. LCMS ESI (+) M/z:349.1/351.1(M +1).

Step F: 5-chlorobenzo [ d ] isothiazol-6-amine (Compound 16.6)

Compound 16.5(660mg, 0.043mmol) was dissolved in tetrahydrofuran (10mL), and then 3mol/L diluted hydrochloric acid (10mL) was added to the reaction solution, and the reaction was stirred at room temperature for 2 hours. Completion of the reaction was confirmed by TLC (PE: EA ═ 8: 1) and LCMS. Water was added to the reaction solution, and after extraction with ethyl acetate (15mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried to give a crude product, which was then slurried with petroleum ether for 10 minutes and filtered to give 16.6(295mg, yield: 85%, purity: 95%) as a yellow solid product. LCMS ESI (+) M/z 185.0/187.0(M +1).

Step G: n- (5-chlorobenzo [ d ] isothiazol-6-yl) -3- (1, 3-dioxoisoindolin-2-yl) -2- (p-tolyl) propanamide (Compound 16.7)

3- (1, 3-Dioxoisoindolin-2-yl) -2- (p-tolyl) propionic acid (20mg, 0.064mmol) was dissolved in dichloromethane (5mL), and N, N-dimethylformamide (1 drop) and oxalyl chloride (16mg, 0.13mmol) were added to the reaction solution under ice bath, and the reaction was stirred at room temperature for 2 hours. Completion of the reaction was confirmed by TLC. The reaction was directly spin dried in vacuo and transferred to a reaction flask with anhydrous pyridine (4mL), followed by addition of compound 51.6(13mg, 0.071mmol) and stirring at 80 deg.C for 8 hours under nitrogen. The reaction mixture was then directly dried in vacuo, the residue was dissolved in ethyl acetate (20mL), and the organic phase was washed with 1mol/L dilute hydrochloric acid and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, dried by spinning, and purified by column chromatography to give 16.7(20mg, yield: 65%, purity: 95%). LCMS ESI (+) M/z 476.1/478.1(M +1).

Step H: 3-amino-N- (5-chlorobenzo [ d ] isothiazol-6-yl) -2- (p-tolyl) propanamide (Compound 16)

Compound 16.7(20mg, 0.042mmol) was dissolved in ethanol (6mL), and then hydrazine hydrate (21mg, 0.42mmol) was added to the reaction solution, and the reaction was stirred at 60 ℃ for 2 hours under an inert gas atmosphere. The reaction was confirmed to be complete by LCMS. The reaction mixture was directly concentrated and spin-dried by a vacuum pump, and the residue was dissolved in methanol (1.5mL) to prepare and purify by reverse rotation example 16(6mg, yield: 31%, purity: 96%) as a white solid. LCMS ESI (+) M/z 346.1/348.1(M +1).

Example 17

3-amino-2- (4- (1, 2-dihydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: methyl 2- (4- (oxiran-2-yl) phenyl) acetate (Compound 17.1)

Weighed amount of methyl 2- (4-vinylphenyl) acetate (2.2g, 12.48mmol) was dissolved in 80mL of dichloromethane, m-chloroperoxybenzoic acid (7.6g, 37.44mmol) was added in portions under ice bath, and the reaction solution was stirred at 0 ℃ for 1 hour and then at room temperature for 16 hours. To the reaction solution was added 300mL of a saturated sodium sulfite solution, and the mixture was stirred for 1 hour, extracted 2 times with dichloromethane (300mL), and the organic phases were combined, washed 2 times with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give compound 17.1(1.84g, yield: 77%, colorless oily substance).

And B: methyl 2- (4- (2, 2-dimethyl-1, 3-dioxolan-4-yl) phenyl) acetate (Compound 17.2)

Compound 17.1(1.84g, 9.57mmol) was dissolved in 30mL of acetone, Amberlyst 15(2.5g, 12.44mmol) was added, and the reaction was stirred at room temperature for 20 h. The reaction solution was filtered, and 150mL of a saturated sodium bicarbonate solution was added to the filtrate, followed by extraction with ethyl acetate (300mL) 2 times, liquid separation and combination of organic phases, washing of the organic phase with a saturated saline solution (100mL) 1 time, drying over anhydrous sodium sulfate, filtration, spin-drying, and purification by column chromatography gave compound 17.2(1.5g, yield: 63%, colorless oily substance).

And C: methyl 2- (4- (2, 2-dimethyl-1, 3-dioxolan-4-yl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionate (Compound 17.3)

Compound 17.2(1.5g, 5.99mmol) was dissolved in anhydrous tetrahydrofuran (20mL) at-78 deg.C, then bis-trimethylsilyl aminolithium (7.2mL, 7.19mmol) was slowly added dropwise to the reaction solution, stirred at-78 deg.C under nitrogen for 0.5 h, then weighed amount of N-bromomethylphthalimide (1.73g, 7.19mmol) was dissolved in anhydrous tetrahydrofuran (10mL) and slowly added dropwise to the reaction flask, after which stirring at this temperature was continued for 1.5 h. The reaction was quenched with 20mL of saturated ammonium chloride solution, extracted 2 times with ethyl acetate (60mL), the organic phases were combined for liquid separation, washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 17.3(2g, yield: 82%, white solid). LCMS ESI (+) m/z: 410.1(M +1).

Step D: 2- ((2-carboxy-2- (4- (2, 2-dimethyl-1, 3-dioxolan-4-yl) phenyl) ethyl) carbamoyl) benzoic acid (Compound 17.4)

Compound 17.3(2g, 4.88mmol) was dissolved in 35mL of tetrahydrofuran and 30mL of water, lithium hydroxide monohydrate (615mg,14.64mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. To the reaction solution was added a saturated citric acid solution to pH 6, extracted 3 times with ethyl acetate (40mL), and the organic phases were combined, washed 2 times with a saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product 17.4(1.1g, yield: 55%, white solid). LCMS ESI (+) m/z:414.1(M +1).

Step E: 2- (4- (2, 2-dimethyl-1, 3-dioxolan-4-yl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) propionic acid (Compound 17.5)

Compound 17.4(1.1g, 2.66mmol) was dissolved in 30mL of N, N-dimethylformamide and 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (760mg, 3.99mmol), 1-hydroxybenzotriazole (540mg, 3.99mmol) and diisopropylethylamine (688mg, 5.32mmol) were added. The reaction solution was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure, 20mL of water was added to the residue, a saturated citric acid solution was added to pH 6, extraction was performed 3 times with ethyl acetate (40mL), the organic phases were combined, washed 2 times with a saturated saline solution (40mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 17.5(760mg, yield: 72%, yellow solid). LCMS ESI (+) m/z: 396.1(M +1).

Step F: 2- (4- (2, 2-dimethyl-1, 3-dioxolan-4-yl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] ] pyridin-2-yl) propanamide (Compound 17.6)

Compound 17.5(260mg, 0.66mmol) was dissolved in 12mL of N, N-dimethylformamide and diisopropylethylamine (127mg, 0.99mmol), 6-aminoisoquinoline (104mg, 0.69mmol) and 2- (7-oxybenzotriazole) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (300mg, 0.79mmol) were added. The reaction solution was stirred at room temperature for 1.5 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 17.6(260mg, yield: 75%, yellow solid). LCMS ESI (+) m/z: 528.1(M +1).

Step G:2- (4- (1, 2-dihydroxyethyl) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 52.7)

Compound 17.6(260mg,0.49mmol) was dissolved in 30mL of methanol and 30mL of acetonitrile, 3.3mL of 1.5M aqueous hydrochloric acid was added, the reaction mixture was stirred at 30 ℃ for 2 hours, and the reaction mixture was concentrated under reduced pressure to give product 17.7(240mg, yield: 100%, yellow solid). LCMS ESI (+) m/z: 488.1(M +1).

Step H: 3-amino-2- (4- (1, 2-dihydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 17)

Compound 17.7(25mg, 0.05mmol) was dissolved in 5mL methylamine/ethanol and the reaction was stirred at 50 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 17(11mg, yield: 61%, white solid). LCMS ESI (+) m/z: 358.1(M +1).

Example 18

3-amino-2- (4- (2-hydroxyethoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: (2- (4- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) phenyl) -3-oxo-3- (thieno [2, 3-c)]Pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 18.1)

Tert-butyl (2- (4-hydroxyphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamate (100mg, 0.24mmol) was dissolved in 8mL of N, N-dimethylformamide, and (2-bromoethoxy) (tert-butyl) dimethylsilane (115mg,0.48mmol) and potassium carbonate (100mg,0.72mmol) were added to stir the reaction solution at 100 ℃ for 12 hours. To the reaction solution was added 40mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 18.1(21mg, yield: 15%, yellow solid). LCMS ESI (+) m/z: 572.2(M +1).

And B: 3-amino-2- (4- (2-hydroxyethoxy) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 18)

Compound 18.1(21mg, 0.04mmol) was dissolved in 4mL of dichloromethane, 2mL of trifluoroacetic acid was added, and the reaction was stirred at 50 ℃ under nitrogen for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give example 18(3mg, yield: 23%, yellow solid). LCMS ESI (+) m/z: 358.1(M +1).

Example 19

3-amino-2- (4- (3-hydroxypropyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: 3-amino-2- (4- ((tert-butyldimethyl) oxy) phenyl) -N- (thieno [2, 3-c)]Pyridin-2-yl) propionamide (Compound 19.1)

2- (4- ((tert-butyldimethylsilyl) oxy) phenyl) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (1.5g,3.5mmol) was dissolved in 10mL of methylamine ethanol solution and reacted at 50 ℃ for 1.5 hours. LCMS monitored reaction completion. After completion of the reaction, water was added to quench the reaction, followed by extraction with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the product 19.1(1.0g, yield: 66%). LCMS ESI (+) M/z 428.2(M +1).

And B: (2- (4- ((tert-Butyldimethylsilyl) oxy) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 19.2)

Compound 19.1(1.0g,2.3mmol) and N, N-diisopropylethylamine (0.7mL,3.5mmol) were dissolved in 5mL of N, N-dimethylformamide, followed by the addition of di-tert-butyl dicarbonate (0.8mL,3.5mmol), reacted at room temperature for 1 hour and LCMS monitored for reaction completion. After completion of the reaction, water was added to quench the reaction, followed by extraction with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the product 19.2(1.2g, yield: 100%). LCMS ESI (+) M/z 528.2(M +1).

And C: (2- (4-hydroxyphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 19.3)

Compound 19.2(1.2g,2.3mmol) was dissolved in 10mL of methanol, followed by the addition of potassium carbonate (476mg,3.5mmol) and reaction at 50 ℃ for 2 hours and LCMS to monitor completion of the reaction. After the completion of the reaction, the reaction mixture was concentrated and purified by column chromatography to give the product 19.3(940mg, yield: 63%). LCMS ESI (+) M/z 414.1(M +1).

Step D: 4- (3- ((tert-Butoxycarbonyl) amino) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) phenyltriflate (Compound 19.4)

Compound 19.3(940mg,1.5mmol) and pyridine (474mg,6.0mmol) were dissolved in 5mL of dichloromethane, and trifluoromethanesulfonic anhydride (846mg,3.0mmol) was added dropwise at 0 deg.C, followed by reaction at room temperature for 30 minutes and LCMS to monitor completion of the reaction. After completion of the reaction, water was added to quench the reaction, followed by extraction with methylene chloride (20mLX3), and the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the product 19.4(220mg, yield: 27%). LCMS ESI (+) M/z:546.1(M +1).

Step E: (2- (4-allylphenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 19.5)

Compound 19.4(100mg,0.18mmol), 2-allyl-4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (37mg,0.22mmol) and cesium carbonate (117mg,0.36mmol) were dissolved in 9mL tetrahydrofuran and 1mL water, followed by addition of [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (6mg,5 mol%), nitrogen substitution, reaction overnight at 85 deg.C, and reaction completion monitored by LCMS. After completion of the reaction, water was added to quench the reaction, followed by extraction with ethyl acetate (10mLX3), and the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give 19.5(78mg, yield: 99%). LCMS ESI (+) M/z 438.2(M +1).

Step F: (2- (4- (3-hydroxypropyl) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 19.6)

Compound 19.5(78mg,0.17mmol) was dissolved in 2mL of tetrahydrofuran, borane (10.0M,0.02mL,0.34mmol) was added dropwise thereto while cooling on ice, and the mixture was reacted at 0 ℃ for 1 hour and then at room temperature for 1 hour. Then, 5mL of an aqueous sodium hydroxide solution (1M) was added dropwise in an ice bath, 1mL of hydrogen peroxide (30%) was added dropwise, the reaction was carried out for 30 minutes in an ice bath, and the reaction was carried out for 30 minutes at room temperature. LCMS monitored reaction completion. The reaction was quenched with water, extracted with ethyl acetate (10mLX3), the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 19.6(20mg, yield: 26%). LCMS ESI (+) M/z 456.2(M +1).

Step G: 3-amino-2- (4- (3-hydroxypropyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 19)

Compound 19.6(12mg,0.03mmol) was dissolved in 2mL of dichloromethane, and then 2mL of hydrochloric acid/1, 4-dioxane solution was added and reacted at room temperature for 1 hour. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 19(2mg, yield: 15%) after lyophilization. LCMS ESI (+) M/z:356.1(M +1).

Example 20

3-amino-2- (4- (4-hydroxybutyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide

The specific reaction equation is as follows:

step A: methyl 2- (4- (4-hydroxybut-1-yn-1-yl) phenyl) acetate (substance 20.1)

Methyl 2- (4-bromophenyl) acetate (4.3g,18.7mmol), 3-butyn-1-ol (2.6g,37.4mmol) were dissolved in 100mL of triethylamine, followed by addition of tetrakis (triphenylphosphine) palladium (864mg,4 mol%) and cuprous iodide (426mg,12 mol%), nitrogen substitution, and reaction at 100 ℃ for 2 hours. LCMS monitored reaction completion. Filtration, concentration of the filtrate under reduced pressure, spin-drying, and purification by column chromatography gave 20.1(2.8g, yield: 68%). LCMS ESI (+) M/z:219.1(M +1).

And B: methyl 2- (4- (4-hydroxybutyl) phenyl) acetate (Compound 20.2)

Compound 20.1(2.8g,12.8mmol) was dissolved in 20mL of methanol, followed by addition of palladium on carbon (280mg, 10%), replacement with hydrogen three times, and reaction at 40 ℃ overnight. LCMS monitored reaction completion. Filtering, concentrating the filtrate under reduced pressure, spin-drying, and purifying by column chromatography to obtain product 20.2(2.0g, yield: 70%). LCMS ESI (+) M/z:223.1(M +1).

And C: methyl 2- (4- (4- ((triisopropylsilyl) oxy) butyl) phenyl) acetate (Compound 20.3)

Compound 20.2(2.0g,9.0mmol) was dissolved in 10mL of dichloromethane, and then 2, 6-lutidine (1.6mL,13.5mmol) and triisopropylsilyl trifluoromethanesulfonate (3.0mL,10.8mmol) were added and reacted at room temperature overnight. The starting material reaction was monitored by TLC to be complete. Adding water to quench the reaction, extracting with dichloromethane (50mLX3), combining the organic phases, washing with saturated brine (100mLX3), drying the organic phase over anhydrous sodium sulfate, filtering, spin-drying, and purifying by column chromatography to obtain 20.3(3.4g, yield: 100%).

Step D: methyl 3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (4- ((triisopropylsilyl) oxy) butyl) phenyl) propanoate (Compound 20.4)

Dissolving compound 20.3(3.4g,9.0mmol) in 20mL of anhydrous tetrahydrofuran, replacing with nitrogen, cooling dry ice acetone to-78 ℃, slowly adding hexamethyldisilazane-based amino lithium (1.0M, 10.8mL, 10.8mmol) dropwise, stirring the system at-78 ℃ for 1 hour after the dropwise addition is completed, dissolving N-bromomethylphthalimide (2.6g,10.8mmol) in 10mL of tetrahydrofuran, slowly adding dropwise into the system, controlling the dropwise addition temperature to be lower than-70 ℃, keeping the reaction at-78 ℃ after the dropwise addition is completed, stirring for 3 hours, and monitoring that the raw materials are completely reacted by LCMS. The reaction was quenched by the addition of saturated ammonium chloride solution (10mL), extracted with ethyl acetate (50mLX3), and washed with saturated brine (100 mL). Dried over anhydrous sodium sulfate, filtered, dried by spinning, and purified by column chromatography to obtain 20.4(2.9g, yield: 60%). LCMS ESI (+) m/z: 538.3(M +1).

Step E: 2- ((2-carboxy-2- (4- (4- ((triisopropylsilyl) oxy) butyl) phenyl) ethyl) carbamoyl) benzoic acid (Compound 20.5)

Compound 20.4(2.9g,5.4mmol) was dissolved in 10mL tetrahydrofuran and 3mL water, lithium hydroxide monohydrate (680mg,16.2mmol) was added at 0 deg.C, stirred at 0 deg.C for 3 hours, and the reaction was monitored for completion by LCMS. The organic solvent was distilled off under reduced pressure, the pH was adjusted to 3-4 with 1M aqueous hydrochloric acid, extraction was performed with ethyl acetate (50mLX3), the organic phases were combined, washed with saturated brine (100mLX3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to give 20.5(2.8g, yield: 94%). LCMS ESI (+) m/z: 542.3(M +1).

Step F: 3- (1, 3-Dioxoisoindolin-2-yl) -2- (4- (4- ((triisopropylsilyl) oxy) butyl) phenyl) propanoic acid (Compound 20.6)

After compound 20.5(2.8g,5.1mmol), 1-hydroxybenzotriazole (688mg,5.1mmol) and triethylamine (2.1mL,15.3mmol) were dissolved in 10mL of dichloromethane, the temperature was reduced to 0 ℃ under nitrogen protection, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (979mg,5.1mmol) was added to the reaction mixture, and the mixture was stirred for a while to clarify the reaction mixture, slowly warmed to room temperature, and reacted overnight. LCMS monitored reaction completion. The reaction mixture was diluted with water, extracted with dichloromethane (50mLX3), the organic phases were combined, washed with saturated brine (100mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 20.6(2.3g, yield: 86%). LCMS ESI (+) m/z: 524.3(M +1).

Step G: 3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (4- (4- ((triisopropylsilyl) oxy) butyl) phenyl) propanamide (Compound 20.7)

After dissolving compound 20.6(300mg,0.57mmol) in 5mL of N, N-dimethylformamide, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (327mg,0.86mmol) and N, N-diisopropylethylamine (147mg,1.1mmol) were sequentially added thereto, and the mixture was stirred at room temperature for 5 minutes, followed by addition of compound 17.5(102mg,0.68mmol) and reaction at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, water was added to quench the reaction, followed by extraction with ethyl acetate (20mLX3), and the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give 20.7(373mg, yield: 100%). LCMS ESI (+) M/z:656.3(M +1).

Step H: 3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (4-hydroxybutyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 20.8)

Compound 20.7(373mg,0.69mmol) was dissolved in 2mL of tetrahydrofuran, and then added to 6mL of aqueous hydrochloric acid (1.0M) and reacted at 40 ℃ overnight. LCMS monitored reaction completion. After completion of the reaction, saturated sodium bicarbonate solution was added to neutralize, followed by extraction with ethyl acetate (20mLX3), the organic phases were combined, washed with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and dried by spinning to give a crude product 20.8(270mg, yield: 95%). LCMS ESI (+) M/z:500.2(M +1).

Step I: 3-amino-2- (4- (4-hydroxybutyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 20)

Compound 20.8(30mg,0.06mmol) was dissolved in 5mL of methylamine ethanol solution and reacted at room temperature for 2 hours. LCMS monitored reaction completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2mL of methanol, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 20(18mg, yield: 67%) after lyophilization. LCMS ESI (+) M/z 370.2(M +1).

Example 21

(S) -3-amino-2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide

The specific reaction equation is as follows:

step A: 2- (4-Vinylphenyl) acetic acid methyl ester (Compound 21.1)

Methyl 2- (4-bromophenyl) acetate (10g, 43.65mmol) was dissolved in 200mL of tetrahydrofuran and 20mL of water, potassium vinyltrifluoroborate (7g, 52.39mmol), cesium carbonate (28.6g, 87.31mmol) and bis (triphenylphosphine) palladium chloride (600mg, 0.87mmol) were added, and the reaction was stirred at 78 degrees under nitrogen for 16 hours. The reaction solution was cooled to room temperature, 100mL of water was added to the reaction solution, extraction was performed 2 times with ethyl acetate (400mL), the organic phases were combined, the organic phase was washed 2 times with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 21.1(5.6g, yield: 73%, colorless oil).

And B: 2- (4- (2-hydroxyethyl) phenyl) acetic acid (Compound 21.2)

Compound 21.1(5.6g, 31.78mmol) was dissolved in 50mL tetrahydrofuran, cooled to 0 deg.C, and 6.4mL of a 10M solution of borane dimethylsulfide was added dropwise. The reaction solution was stirred at 0 ℃ for 1 hour under nitrogen protection, and then stirred at 20 ℃ for 1 hour. The reaction solution was cooled to 0 ℃, and 56mL of 1M sodium hydroxide solution and 30% hydrogen peroxide were added dropwise. The reaction solution was stirred at 0 ℃ for 0.5 hour and then at 20 ℃ for 1 hour. To the reaction solution was added 400mL of a saturated sodium sulfite solution, followed by adjustment of pH to 2 with 1M hydrochloric acid solution, extraction with ethyl acetate (500mL) 5 times, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give compound 21.2(3.85g, yield: 67%, white solid).

And C: methyl 2- (4- (2-hydroxyethyl) phenyl) acetate (Compound 21.3)

Compound 21.2(3.85g, 21.37mmol) was dissolved in 45mL of dichloromethane and 10mL of methanol, cooled to 0 deg.C, 2M trimethylsilyl diazomethane (11.2 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 0.5 hour. The reaction mixture was concentrated under reduced pressure and purified by column chromatography to give compound 21.3(2.4g, yield: 58%, colorless oil).

Step D: methyl 2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) acetate (Compound 21.4)

Compound 21.3(2.4g, 12.36mmol) was dissolved in 40mL of dichloromethane, cooled to 0 ℃,2, 6-lutidine (1.99g, 18.54mmol) and triisopropylsilyl trifluoromethanesulfonate (4.56g, 14.83mmol) were added, and the reaction mixture was stirred at room temperature for 16 hours. To the reaction mixture was added 50mL of water, followed by extraction with dichloromethane (80mL) 2 times, followed by separation and combination of organic phases, and the organic phases were washed with saturated brine (30mL) 1 time, dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 21.4(3.7g, yield: 85%, colorless oily substance).

Step E: 2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) acetic acid (Compound 21.5)

Compound 21.4(3.7g, 10.55mmol) was dissolved in 15mL of water, 15mL of methanol and 40mL of tetrahydrofuran, lithium hydroxide monohydrate (1.33g,31.65mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. To the reaction solution was added a 1M hydrochloric acid solution to pH 3, extracted 2 times with ethyl acetate (100mL), and the organic phases were combined, washed 2 times with saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product 21.5(3.43g, yield: 97%, yellow solid).

Step F: 2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) acetyl chloride (Compound 21.6)

Compound 21.5(3.43g, 10.19mmol) was dissolved in 40mL of dichloromethane, the reaction was cooled to 0 deg.C, oxalyl chloride (1.55g, 12.23mmol) was added dropwise under nitrogen, and the reaction was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure to give the product 21.6(3.62g, yield: 100%, yellow oil).

Step G: (R) -4-benzyl-3- (2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) acetyl) oxazolidine-2-one (Compound 21.7)

(R) -4-Benzyloxazolidine-2-one (1.72g, 9.69mmol) was dissolved in 30mL of tetrahydrofuran, the reaction was cooled to-78 deg.C, 2.5M n-butyllithium (4.28mL, 10.71mmol) was added dropwise under nitrogen, and the reaction was stirred at-78 deg.C for 1 hour. Then, 15mL of a tetrahydrofuran solution of compound 21.6(3.62g, 10.20mmol) was added dropwise to the reaction mixture. The reaction solution was stirred at-78 ℃ for 2.5 hours. To the reaction solution was added 30mL of ammonium chloride solution, extracted 2 times with ethyl acetate (150mL), the organic phases were combined, washed 2 times with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 21.7(3.15g, yield: 62%, colorless oil). LCMS ESI (+) m/z: 496.3(M +1).

Step H: 2- ((R) -3- ((R) -4-benzyl-2-oxooxazolidin-3-yl) -3-oxo-2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) propyl) isoindole-1, 3-dione (Compound 21.8)

Compound 21.7(3.15g, 6.35mmol) was dissolved in 30mL of anhydrous tetrahydrofuran at-78 deg.C, then bis-trimethylsilyl aminolithium (7.63mL, 7.63mmol) was slowly added dropwise to the reaction solution, stirred at this temperature for 1 hour, and then weighed N-bromomethylphthalimide (1.83g, 7.63mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and slowly added dropwise to the reaction flask, followed by stirring at this temperature for 2 hours. The reaction was quenched with saturated ammonium chloride solution, extracted 2 times with ethyl acetate (200mL), the organic phases were combined for liquid separation, washed 2 times with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 21.8(2g, yield: 48%, white solid). LCMS ESI (+) M/z 655.3(M +1).

Step I: (R) -2- ((2-carboxy-2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) ethyl) carbamoyl) benzoic acid (Compound 21.9)

Compound 21.8(2g, 3.05mmol) was dissolved in 25mL of methanol and 25mL of tetrahydrofuran, and then lithium hydroxide monohydrate (385mg, 9.16mmol) was dissolved in water (20mL) and added dropwise to the reaction solution, followed by stirring at room temperature for 3 hours. To the reaction solution, 60mL of water was added, the aqueous phase was washed 2 times with ethyl acetate (40mL), the pH was adjusted to 4 with 1M hydrochloric acid solution, extracted 2 times with ethyl acetate (100mL), the organic phases were separated, combined, dried over anhydrous sodium sulfate, filtered, and dried by spinning to give compound 21.9(980mg, yield: 62%, white solid). LCMS ESI (+) M/z:514.2(M +1).

Step J: (R) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (2- ((triisopropylsilyl) oxy) ethyl) phenyl) propanoic acid (Compound 21.10)

Compound 21.9(980mg, 1.91mmol) was dissolved in 30mL of N, N-dimethylformamide, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (548mg, 2.86mmol), 1-hydroxybenzotriazole (387mg, 2.86mmol) and triethylamine (577mg, 5.72mmol) were added to stir the reaction solution at room temperature for 12 hours. The reaction solution was adjusted to pH 4 with 1M hydrochloric acid solution, extracted 2 times with ethyl acetate (60mL), the organic phases were combined, washed 3 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 21.10(840mg, yield: 89%, yellow oil). LCMS ESI (+) m/z: 496.2(M +1).

Step K: (S) -3- (1, 3-dioxoisoindolin-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (4- (2- ((triisopropylsilyl) oxy)) ethyl) phenyl) propanamide (Compound 21.11)

Compound 21.10(840mg, 1.69mmol) was dissolved in 30mL of N, N-dimethylformamide and diisopropylethylamine (438mg, 3.39mmol), 2- (7-oxide benzotriazol) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (967mg, 2.54mmol) and thieno [2,3-c ] pyridin-2-amine (305mg, 2.03mmol) were added. The reaction was stirred at room temperature for 2 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 21.11(580mg, yield: 55%, yellow solid). LCMS ESI (+) m/z:628.3(M +1).

Step L: (S) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 21.12)

Compound 21.11(580mg, 0.92mmol) was dissolved in 50mL of tetrahydrofuran, 20mL of 1.5M aqueous hydrochloric acid was added, and the reaction mixture was stirred at 40 ℃ for 2 hours. The reaction solution was cooled to room temperature, the pH was adjusted to 8 with a saturated sodium bicarbonate solution, extracted 2 times with ethyl acetate (100mL), the organic phases were combined, washed 3 times with a saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 21.12(350mg, yield: 80%, yellow solid). LCMS ESI (+) m/z:472.1(M +1).

Step M: (S) -3-amino-2- (4- (2-hydroxyethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (Compound 21)

Compound 21.12(100mg, 0.21mmol) was dissolved in 6mL of a 33% methylamine/ethanol solution, and the reaction was stirred at 50 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product, example 21(50mg, yield: 69%, white solid). LCMS ESI (+) m/z:342.1(M +1).

Example 22

(S) -3-amino-N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (4- (hydroxymethyl) phenyl) propanamide

The specific reaction equation is as follows:

step A: (S) -3- (1, 3-dioxoisoindolin-2-yl) -N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (4- (((triisopropylsilyl)) oxy) methyl) phenyl) propanamide (Compound 22.1)

(S) -3- (1, 3-dioxoisoindolin-2-yl) -2- (4- (((triisopropylsilyl) oxy) methyl) phenyl) propanoic acid (400mg, 0.832mmol) was dissolved in N, N-dimethylformamide (6mL), and then a solution of 50% 1-propylphosphoric anhydride in N, N-dimethylformamide (793mg, 1.24mmol), N-diisopropylethylamine (214mg, 1.66mmol) and 4-fluorothieno [2,3-c ] pyridin-2-amine (154mg, 0.92mmol) were added, and the reaction was stirred at room temperature for 2 hours. Completion of the reaction was confirmed by TLC (DCM: MeOH ═ 10:1) and LCMS. Water was added to the reaction flask, followed by extraction with ethyl acetate (12mL), washing with saturated brine, drying over anhydrous sodium sulfate, filtration, and purification by column chromatography to give 22.1(300mg, yield: 57%) as a white solid. LCMS ESI (+) M/z 632.2(M +1).

And B: (S) -3- (1, 3-dioxoisoindolin-2-yl) -N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (4- (hydroxymethyl) phenyl) propanamide (Compound 22.2)

Compound 22.1(300mg, 0.475mmol) was dissolved in tetrahydrofuran (12mL) and methanol (2mL), then 1.5mol/L dilute hydrochloric acid (5mL) was added to the reaction flask and the reaction was stirred in a 55 deg.C oil bath for 4 hours. The reaction was confirmed to be complete by LCMS. Saturated sodium bicarbonate solution was added to the reaction flask, followed by extraction with ethyl acetate (12mL), washing with saturated brine, drying over anhydrous sodium sulfate, filtration, and concentration in vacuo to give 22.2 as an off-white solid (185mg, yield: 82%). LCMS ESI (+) M/z 476.1(M +1).

And C: (S) -3-amino-N- (4-fluorothieno [2,3-c ] pyridin-2-yl) -2- (4- (hydroxymethyl) phenyl) propanamide (Compound 22)

Compound 22.2(125mg, 0.263mmol) was dissolved in 33% methylamine in ethanol (6mL) and the reaction stirred at 50 ℃ for 1.5 hours under an inert atmosphere. The reaction was confirmed to be complete by LCMS. The reaction mixture was directly concentrated and spin-dried by a vacuum pump, and the residue was dissolved in methanol (1.5mL) to prepare and purify by reverse rotation example 22 as a white solid (61mg, yield: 50%, purity: 98%). LCMS ESI (+) M/z 346.1(M +1).¹H NMR(400MHz,DMSO)δ12.28(s,1H),9.00(d,J＝1.4Hz,1H),8.41(d,J＝2.3Hz,1H),7.93(s,3H),7.38–7.31(m,4H),7.03(d,J＝0.6Hz,1H),4.48(s,2H),4.14(q,J＝8.8,5.6Hz,2H),3.62–3.54(m,1H),3.19–3.08(m,1H)。

Example 23

(S) -3-amino-N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: 2- (p-tolyl) acetyl chloride (Compound 23.1)

P-methylphenylacetic acid (5.0g, 33.29mmol) was dissolved in 100mL of dichloromethane, the reaction solution was cooled to 0 ℃, oxalyl chloride (5.07g, 39.95mmol) was added dropwise under nitrogen protection, and the reaction solution was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure to give 23.1(5.61g, yield: 100%, yellow oil).

And B: (R) -4-benzyl-3- (2- (p-tolyl) acetyl) oxazolidinebutan-2-one (Compound 23.2)

(R) -4-Benzyloxazolidine-2-one (5.6g, 31.61mmol) was dissolved in 60mL of tetrahydrofuran, the reaction was cooled to-78 deg.C, 2.5M n-butyllithium (13.97mL, 34.93mmol) was added dropwise under nitrogen, and the reaction was stirred at-78 deg.C for 1 hour. Then, 30mL of a tetrahydrofuran solution of Compound 1.1(5.61g, 33.27mmol) was added dropwise to the reaction mixture. The reaction was stirred at-78 ℃ for 2.5 hours, then gradually warmed to room temperature and stirred for 12 hours. To the reaction solution was added 50mL of ammonium chloride solution, extracted 2 times with ethyl acetate (300mL), the organic phases were combined, washed 2 times with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give 23.2(5.7g, yield: 55%, colorless oil). LCMS ESI (+) m/z: 310.1(M +1).

And C: 2- ((R) -3- ((R) -4-benzyl-2-oxooxazolidinebutan-3-yl) -3-oxo-2- (p-tolyl) propyl) isoindoline-1, 3-dione (Compound 23.3)

Compound 23.2(5.7g, 18.43mmol) was dissolved in 70mL of anhydrous tetrahydrofuran at-78 deg.C, then bis-trimethylsilyl aminolithium (22.1mL, 22.1mmol) was slowly added dropwise to the reaction solution, and stirred at this temperature for 1 hour, followed by dissolving weighed amount of N-bromomethylphthalimide (5.31g, 22.1mmol) in anhydrous tetrahydrofuran (35mL), slowly adding dropwise to the reaction flask, and then stirred at this temperature for 3 hours. The reaction was quenched with saturated ammonium chloride solution, extracted 2 times with ethyl acetate (200mL), the organic phases were combined for liquid separation, washed 2 times with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 23.3(7.8g, yield: 90%, white solid). LCMS ESI (+) M/z 469.2(M +1).

Step D: (R) -2- ((2-carboxy-2- (p-tolyl) ethyl) carbamoyl) benzoic acid (Compound 23.4)

Compound 23.3(7.8g, 16.65mmol) was dissolved in 25mL of methanol and 100mL of tetrahydrofuran, and then lithium hydroxide monohydrate (2.1g, 49.95mmol) was dissolved in water (40mL) and added dropwise to the reaction solution, followed by stirring at room temperature for 2 hours. 120mL of water was added to the reaction solution, the aqueous phase was washed 3 times with ethyl acetate (40mL), the pH was adjusted to 3 with 1M hydrochloric acid solution, extracted 2 times with ethyl acetate (100mL), the organic phases were separated, combined, dried over anhydrous sodium sulfate, filtered, and dried to give compound 23.4(4.6g, yield: 84%, white solid). LCMS ESI (+) M/z:328.1(M +1).

Step E: (R) -3- (1, 3-Dioxoisoindol-2-yl) -2- (p-tolyl) propionic acid (Compound 23.5)

Compound 23.4(4.6g, 14.05mmol) was dissolved in 80mL of N, N-dimethylformamide, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (4.0g, 21.07mmol), 1-hydroxybenzotriazole (2.84g, 21.07mmol) and triethylamine (5.26g, 42.15mmol) were added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was adjusted to pH 3 with 1M hydrochloric acid solution, extracted 2 times with ethyl acetate (100mL), the organic phases were combined, washed 3 times with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 23.5(2.8g, yield: 64%, white solid). LCMS ESI (+) m/z: 310.1(M +1).

Step F: (S) -3- (1, 3-Dioxoisoindol-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propionamide (Compound 23.6)

Compound 23.5(600mg, 1.94mmol) was dissolved in 30mL of N, N-dimethylformamide and diisopropylethylamine (664mg, 5.82mmol), 2- (7-oxide benzotriazol) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (1.1g, 2.91mmol) and thieno [2,3-c ] pyridin-2-amine (361mg, 1.94mmol) were added. The reaction was stirred at room temperature for 16 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 23.6(500mg, yield: 58%, yellow solid). LCMS ESI (+) m/z:442.1(M +1).

Step G: (S) -3-amino-N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 23)

Compound 23.6(500mg, 1.13mmol) was dissolved in 30mL of ethanol and 5mL of N, N-dimethylformamide, hydrazine hydrate (567mg, 11.30mmol) was added, and the reaction mixture was stirred at 50 ℃ for 4 hours. Cooling the reaction solution toAt room temperature, a solid precipitated, and was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give a crude product of 360mg, and 100mg of the sample was purified by reverse phase preparation to give the product of example 23(53mg, yield: 53%, white solid). LCMS ESI (+) m/z:312.1(M +1).¹H NMR(400MHz,DMSO-d6)δ13.51(s,1H),9.49(s,1H),8.52(d,J＝6.4Hz,1H),8.27(s,3H),8.16(d,J＝6.4Hz,1H),7.48-7.32(m,3H),7.20(d,J＝8.0Hz,2H),4.44(dd,J＝5.6,8.8Hz,1H),3.62(s,1H),3.17-3.04(m,1H),2.28(s,3H).

Example 24

(S) -3- (dimethylamino) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide

The specific reaction equation is as follows:

step A: (S) -3- (dimethylamino) -N- (thieno [2,3-c ] pyridin-2-yl) -2- (p-tolyl) propanamide (Compound 24)

Reacting (S) -3-amino-N- (thieno [2, 3-c)]Pyridin-2-yl) -2- (p-tolyl) propionamide (260mg, 0.83mmol) was dissolved in 30mL of methanol, 37% -40% aqueous formaldehyde (339mg, 4.17mmol), sodium cyanoborohydride (157mg, 2.50mmol) and acetic acid (150mg, 2.50mmol) were added, and the reaction was stirred at room temperature under nitrogen for 2 hours. To the reaction solution was added aqueous sodium hydrogencarbonate (20mL), extracted 2 times with ethyl acetate (70mL), and the organic phases were combined, washed 1 time with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product example 24(130mg, yield: 46%, pale yellow solid). LCMS ESI (+) m/z: 340.1(M +1).¹H NMR(400MHz,DMSO-d6)δ13.88(s,1H),10.56(s,1H),9.50(s,1H),8.53(d,J＝6.4Hz,1H),8.19(d,J＝6.4Hz,1H),7.49-7.37(m,3H),7.22(d,J＝8.0Hz,2H),4.82(dd,J＝4.4,8.8Hz,1H),4.03(dd,J＝8.8,12.8Hz,1H),3.47(d,J＝12.0Hz,1H),2.80(s,6H),2.28(s,3H).

Example 25

4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoic acid methyl ester

The specific reaction equation is as follows:

step A: methyl 2- (4-bromophenyl) -3- (1, 3-dioxoisoindol-2-yl) propionate (Compound 25.1)

Methyl p-bromophenylacetate (2.0g, 8.73mmol) was dissolved in 40mL of anhydrous tetrahydrofuran at-78 deg.C, then lithium bistrimethylsilylamide (1.75g, 10.48mmol) was slowly added dropwise to the reaction solution, stirred at this temperature for 1 hour, and then weighed N-bromomethylphthalimide (2.52g, 10.48mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and slowly added dropwise to the reaction flask, followed by stirring at this temperature for 2 hours. The reaction was quenched with saturated ammonium chloride solution, extracted 2 times with ethyl acetate (100mL), the organic phases were combined for liquid separation, washed 2 times with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give compound 25.1(2.64g, yield: 78%). LCMS ESI (+) M/z 388.0,390.0(M +1).

And B: 2- ((2- (4-bromophenyl) -2-carboxyethyl) carbamoyl) benzoic acid (Compound 25.2)

Compound 25.1(2.6g, 6.7mmol) was dissolved in 25mL of methanol and 25mL of tetrahydrofuran, and then lithium hydroxide monohydrate (482mg, 20.1mmol) was dissolved in water (10mL) and added dropwise to the reaction solution, followed by stirring at room temperature for 3 hours. To the reaction mixture was added 50mL of water, the pH was adjusted to 3 with 1M hydrochloric acid solution, and the mixture was extracted 2 times with ethyl acetate (60mL), and the organic phases were separated, combined, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain compound 25.2(2.63g, yield: 100%). LCMS ESI (+) M/z 392.0,394.0(M +1).

And C: 2- (4-bromophenyl) -3- (1, 3-dioxoisoindol-2-yl) propionic acid (Compound 25.3)

Compound 25.2(2.63g, 6.89mmol) was dissolved in 50mL of N, N-dimethylformamide, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (1.98g, 10.33mmol), 1-hydroxybenzotriazole (1.4g, 10.33mmol) and triethylamine (2.09g, 20.67mmol) were added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was adjusted to pH 3 with 1M hydrochloric acid solution, extracted 2 times with ethyl acetate (60mL), the organic phases were combined, washed 3 times with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 25.3(1.6g, yield: 62%). LCMS ESI (+) m/z: 274.0,276.0(M +1).

Step D: 2- (4-bromophenyl) -3- (1, 3-dioxoisoindol-2-yl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 25.4)

Compound 25.3(1.0g, 2.67mmol) was dissolved in 20mL of N, N-dimethylformamide and diisopropylethylamine (690mg, 8.02mmol), 2- (7-oxide benzotriazole) -N, N, N ", N' -tetramethyluronium hexafluorophosphate (1.53g, 4.01mmol) and thieno [2,3-c ] pyridin-2-amine (440mg, 2.94mmol) were added. The reaction was stirred at room temperature for 2 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 25.4(560mg, yield: 41%). LCMS ESI (+) m/z: 506.0,508.0(M +1).

Step E: 3-amino-2- (4-bromophenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propanamide (Compound 25.5)

Compound 25.4(560mg, 1.10mmol) was dissolved in 30mL of ethanol, hydrazine hydrate (551mg, 11.00mmol) was added, and the reaction mixture was stirred at 50 ℃ for 4 hours. The reaction mixture was cooled to room temperature, a solid precipitated, and the filtrate was filtered and concentrated under reduced pressure to give 25.5(415mg, yield: 100%). LCMS ESI (+) m/z: 376.0,378.0(M +1).

Step F: (2- (4-bromophenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamic acid tert-butyl ester (Compound 25.6)

Compound 25.5(415mg, 1.10mmol) was dissolved in 15mL of N, N-dimethylformamide, triethylamine (333mg, 3.30mmol) and BOC anhydride (480mg, 2.20mmol) were added, and the reaction mixture was stirred at room temperature for 3 hours. To the reaction solution was added 30mL of water, followed by extraction with ethyl acetate (60mL) 2 times, and the organic phases were combined, washed 3 times with saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 25.6(525mg, yield: 100%). LCMS ESI (+) m/z: 476.0,478.0(M +1).

Step G: 4- (3- ((tert-Butoxycarbonyl) amino) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoic acid methyl ester (Compound 25.7)

Compound 25.6(100mg, 0.21mmol) was dissolved in 2mL of N, N-dimethylformamide, 2mL of triethylamine and 6mL of methanol, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (44mg, 0.06mmol) was added, and the reaction solution was stirred at 80 ℃ under a carbon monoxide atmosphere for 12 hours. To the reaction solution was added 30mL of water, extracted 2 times with ethyl acetate (50mL), the organic phases were combined, washed 2 times with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 25.7(37mg, yield: 39%). LCMS ESI (+) m/z:456.2(M +1).

Step H: 4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoic acid methyl ester (Compound 25)

Compound 25.7(37mg, 0.08mmol) was dissolved in 5mL of methanol, 2mL of a 4M hydrochloric acid/methanol solution was added, and the reaction mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product, example 25(20mg, yield: 69%). LCMS ESI (+) m/z:356.1(M +1).

Example 26

4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoic acid

The specific reaction equation is as follows:

step A: 4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoic acid (Compound 26)

Methyl 4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoate (15mg, 0.04mmol) was dissolved in 5mL of tetrahydrofuran, 2mL of an aqueous solution of lithium hydroxide monohydrate (5.3mg, 0.12mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. A2M hydrochloric acid solution was added to the reaction solution to pH 5, the reaction solution was concentrated under reduced pressure, and the residue was purified by reverse preparation to give the product, example 26(7mg, yield: 50%). LCMS ESI (+) m/z:342.1(M +1).

Example 27

(S) -4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzyl benzoate

The specific reaction equation is as follows:

step A: (S) -benzyl 4- (3- (1, 3-Dioxoisoindol-2-yl) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoate (Compound 27.1)

(S) -3- (1, 3-Dioxoisoindol-2-yl) -2- (4- (hydroxymethyl) phenyl) -N- (thieno [2,3-c ] pyridin-2-yl) propionamide (80mg,0.175mmol) was dissolved in 2mL of pyridine, and benzoyl chloride (80uL,0.694mmol) was added under an ice-water bath, followed by warming to room temperature and stirring for 2.5 hours. LCMS monitored reaction completion. Water was added to quench the reaction, extracted with ethyl acetate (20mLX2), the organic phases were combined, washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified by column chromatography to give 27.1(90mg, yield: 92%).

And B: (S) -benzyl 4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzoate (Compound 27)

Compound 27.1(90mg,0.16mmol) was dissolved in 5mL of tetrahydrofuran, followed by addition of hydrazine hydrate (77uL,1.6mmol) and reaction at 55 ℃ for 3 hours. TLC and LCMS monitored the reaction was complete. After completion of the reaction, water (20mL) was added for dilution, extraction was performed with ethyl acetate (20mLX2), the organic phases were combined, washed with saturated brine (10mLX2), dried over anhydrous sodium sulfate, filtered, and spun-dried, and the obtained residue was dissolved in 5mL of methanol and an aqueous solution, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 27(36mg, yield: 52%, purity: 97%) after lyophilization. LCMS ESI (+) M/z:432.2(M +1).¹H NMR(400MHz,DMSO-d₆)δ13.56(s,1H),9.48(s,1H),8.52(d,J＝6.4Hz,1H),8.26(s,3H),8.15(d,J＝6.4Hz,1H),8.07–7.86(m,2H),7.66(t,J＝7.4Hz,1H),7.53(dt,J＝11.9,6.0Hz,6H),7.41(s,1H),5.34(s,2H),4.51(dd,J＝8.5,5.5Hz,1H),3.65(s,1H),3.18(d,J＝5.5Hz,1H).

Example 28

(S) -4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) 2, 4-dimethylbenzoic acid ester

Step A: (S) -dimethyl 4- (3- ((tert-butoxycarbonyl) amino) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzyl 2, 4-benzoate (Compound 28.1)

Tert-butyl (S) - (2- (4- (hydroxymethyl) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamate (100mg,0.234mmol) and 2, 4-dimethylbenzoic acid (38mg,0.257mmol) were dissolved in 3mL of N, N-dimethylformamide, and then 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (67mg,0.351mmol) and 4-dimethylaminopyridine (28mg,0.234mmol) were added and reacted at room temperature overnight. TLC monitored the reaction complete. Adding water to quench the reaction, extracting with ethyl acetate (20mLX2), combining the organic phases, washing with saturated brine (10mLX2), drying the organic phase over anhydrous sodium sulfate, filtering, spin-drying, and purifying by column chromatography to obtain 28.1(91mg, yield: 69%).

And B: (S) -4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) 2, 4-dimethylbenzoic acid ester (Compound 28)

Compound 28.1(89mg,0.16mmol) was dissolved in 5mL of methanol, 3mL of methanolic hydrochloric acid (4M) was added, the mixture was stirred at room temperature for 1 hour, then the system was warmed to 50 ℃ for 2 hours, and the reaction was monitored by TLC for completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 5mL of methanol and an aqueous solution, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 28(50mg, yield: 68%, purity: 98%) after lyophilization. LCMS ESI (+) M/z 460.2(M +1).¹H NMR(400MHz,DMSO-d₆)δ13.52(s,1H),9.47(s,1H),8.51(d,J＝6.4Hz,1H),8.26(s,3H),8.14(d,J＝6.4Hz,1H),7.77(d,J＝8.0Hz,1H),7.51(dd,J＝17.6,8.0Hz,4H),7.41(s,1H),7.27–6.92(m,2H),5.28(s,2H),4.51(dd,J＝8.4,5.6Hz,1H),3.65(s,1H),3.19(s,1H),2.49(s,3H),2.30(s,3H).

Example 29

(S) -4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) 3, 5-dimethylbenzoic acid ester

The specific reaction equation is as follows:

step A: (S) -dimethyl 4- (3- ((tert-butoxycarbonyl) amino) -1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) benzyl 3, 5-benzoate (Compound 29.1)

Tert-butyl (S) - (2- (4- (hydroxymethyl) phenyl) -3-oxo-3- (thieno [2,3-c ] pyridin-2-ylamino) propyl) carbamate (100mg,0.234mmol) and 3, 5-dimethylbenzoic acid (38mg,0.257mmol) were dissolved in 3mL of N, N-dimethylformamide, and then 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (67mg,0.351mmol) and 4-dimethylaminopyridine (28mg,0.234mmol) were added and reacted at room temperature overnight. TLC monitored the reaction complete. Adding water to quench the reaction, extracting with ethyl acetate (20mLX2), combining the organic phases, washing with saturated brine (10mLX2), drying the organic phase over anhydrous sodium sulfate, filtering, spin-drying, and purifying by column chromatography to obtain 29.1(96mg, yield: 73%).

And B: (S) -4- (3-amino-1-oxo-1- (thieno [2,3-c ] pyridin-2-ylamino) propan-2-yl) 3, 5-dimethylbenzoic acid ester (Compound 29)

Compound 29.1(96mg,0.17mmol) was dissolved in 5mL of methanol, 2mL of methanolic hydrochloric acid (4M) was added, the mixture was stirred at room temperature for 1 hour, then the system was warmed to 50 ℃ for 2 hours, and the reaction was monitored by TLC for completion. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 5mL of methanol and an aqueous solution, filtered, and the filtrate was purified by reverse phase preparation to obtain the objective product, example 29(46mg, yield: 60%, purity: 98%) after lyophilization. LCMS ESI (+) M/z 460.2(M +1).¹H NMR(400MHz,DMSO-d₆)δ13.34(s,1H),9.46(s,1H),8.51(d,J＝6.6Hz,1H),8.18(s,3H),8.13(d,J＝6.6Hz,1H),7.59(s,2H),7.51(s,4H),7.37(s,1H),7.29(s,1H),5.32(s,2H),4.45(dd,J＝8.8,5.6Hz,1H),3.64(s,1H),3.20(s,1H),2.31(s,6H).

Example 30: ROCK2 kinase inhibitor activity assay

< one > experimental procedure.

1) According to the experimental requirements, a 4X ROCK2 reaction solution, a 4X ATP reaction solution, a 4X substrate solution and a 4X compound solution are prepared.

2) To 384 multi-well plates were added 2.5. mu.L, 4 Xkinase solution, and 2.5. mu.L, diluted 4X test compound solution of different concentration gradients, each concentration being set at 2 duplicate wells, enzyme solution blank and negative control (DMSO group) being set at the same time, 1000 rotations, centrifugation for 1 minute, mixing enzyme and compound, to 384 multi-well plates was added 2.5. mu.L, 4X substrate solution, centrifugation for 1 minute at 1000 rotations, to 384 multi-well plates was added 2.5. mu.L, 4XATP solution, centrifugation for 1 minute at 1000 rotations to initiate enzyme reaction, and incubation at room temperature for 1.5 hours.

The final concentrations of the components of the ROCK2 reaction were: ROCK 2: 1nM, substrate: 500nM, ATP: 6uM, final concentration range of test compound: 100nM to 5pM

3) Preparing a 4X Streptavidin-XL-665 detection solution according to experimental requirements.

4) After the completion of the enzyme reaction, 5ul of 4X Streptavidin-XL-665 assay solution was added to each well of the 384 multi-well plate, and the mixture was centrifuged at 1000 rpm for 1 minute, and 5. mu.L of 4X STK Antibody-cryptate assay Antibody solution was added to each well of the 384 multi-well plate, and the mixture was centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 1 hour.

5) After the antibody incubation was complete, the signal values for each well were determined on an Envision plate reader using HTRF program.

< second > data analysis

1) The percent inhibition rate corresponding to each concentration of the test compound was calculated with the enzyme solution blank control group as 100% inhibition rate and the negative control group (DMSO group) as 0% inhibition rate.

2) The log concentration values and corresponding percent inhibition of the test compound were subjected to nonlinear regression analysis in GraphPad Prism software to obtain the median inhibitory concentration (IC50) of the test compound.

The activity (IC) of ROCK2 kinase inhibitor is detected by the method₅₀) As in the following table:

example 31: cell viability assay

< first > Experimental procedure

1) A7R5 cells in log phase were digested, 2E5 cells per well were seeded in 6-well plates, cultured overnight at 37 ℃ in 5% CO2, and after 24 hours the cell culture medium was replaced with starvation medium containing 0.05% FBS and the cells were starved for 20 hours.

2) Starvation medium was removed, cells were treated with the formulated compound concentration gradient for 60 min, and VIP-5021(10uM) was set as positive control and DMSO group as negative control.

3) After the compound treatment was completed, the compound solution was aspirated, washed with PBS 2 times, 100ul of cell lysate was added to each well, the cells were gently scraped with a cell scraper, and transferred to a centrifuge tube and lysed on ice for 60 minutes.

4, centrifuging at 14000rpm for 15 minutes at 4 ℃, collecting lysate supernatant, quantifying protein by adopting a BCA method, adjusting the protein concentration, adding a sample buffer according to the proportion, and treating for 10 minutes at 100 ℃ to denature the protein.

5) The electrophoresis apparatus was assembled, each sample was loaded with 20ug of total protein, fixed voltage 125V, electrophoresis was performed for 90 minutes, after the electrophoresis was completed, the transfer membrane system was assembled, fixed current 300mA was applied, the transfer membrane was performed for 120 minutes, after the transfer membrane was completed, the PVDF membrane was removed, PBS was washed 2 times, blocking solution was added, blocking solution was blocked at room temperature for 60 minutes, blocking solution was removed, and a solution containing primary antibody (Phospho-myostatin Light Chain2(Ser19)) was added (antibody dilution factor 1: 1000) GAPDH was selected as an internal control and incubated overnight at 4 ℃.

6) PBST was washed 3 times and HRP-antibody (secondary antibody dilution 1: 2000) the HRP chemiluminescent substrate was prepared by incubation for 60 minutes at room temperature and washed 3 times with PBST, dropped at 0.1 ml per square centimeter onto PVDF membrane for 30 seconds and the chemiluminescent signal collected using a gel imager (ChemiDoc XRS + (BIO-RAD)).

7) Each band was quantified using image J software, and the level of phosphorylated protein in Phospho-Myosin Light Chain2(Ser19) in each sample was corrected for GAPDH expression.

< second > data analysis

1) Percentage inhibition rates corresponding to the concentrations of the test compounds were calculated by taking the MLC2(Ser19) protein phosphorylation level of the highest concentration compound sample as 100% inhibition rate and the MLC2(Ser19) protein phosphorylation level of the negative control group (DMSO group) as 0% inhibition rate

2) The log concentration values and corresponding percent inhibition of the test compound were subjected to nonlinear regression analysis in GraphPad Prism software to obtain the half maximal inhibitory concentration (IC50) at the cellular level of the test compound.

The method can be used for detecting cell activity (IC) of the obtained compound₅₀) As in the following table:

Ex#	IC₅₀(nM)		Ex#	IC₅₀(nM)
					8	35		9	21

Claims

1. a sulfur-containing heterocyclic compound characterized by: is a compound shown by the following structural formula or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotope derivative, a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt or a prodrug thereof;

wherein ring a is aryl or heteroaryl;

b is a fused heteroaryl group containing one N atom and one sulfur atom;

R¹hydrogen, hydroxyl, halogen, amino, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy and substituted or unsubstituted heteroalkyl substituted on the B ring;

R²hydrogen, halogen, cyano, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy substituted or not substituted on the B ring;

R⁵is hydrogen, hydroxyl, halogen, amino, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl of any or several of A rings.

2. The sulfur-containing heterocyclic compound according to claim 1, which is a compound represented by the following structure or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotopic derivative, a hydrate, a solvate, a metabolite, and a pharmaceutically acceptable salt or prodrug thereof:

3. the sulfur-containing heterocyclic compound according to claim 1, which is a compound represented by the following structure or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotopic derivative, a hydrate, a solvate, a metabolite, and a pharmaceutically acceptable salt or prodrug thereof:

4. a sulfur-containing heterocyclic compound according to claim 3, wherein the compound of formula a is prepared by a specific method as follows:

s1, heating and reacting a compound IV-1 and a compound IV-2 under an alkaline condition to obtain a compound IV-3;

s2, hydrolyzing the compound IV-3 into a compound IV-4;

s3, carrying out Curtius rearrangement on the compound IV-4 to obtain a compound IV-5;

s4, carrying out coupling reaction on the compound IV-5 and an intermediate Interm A to form a compound IV-6;

s5, deprotecting the compound IV-6 to obtain a compound IIa 1;

s6, carrying out single or double alkylation reaction on the compound IIa1 to form a compound IIa 2;

wherein, the compounds Interm A, IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IIa1 and IIa2 are compounds shown in the following structures:

re is selected from alkyl, and Prot is an amine protecting group.

5. A sulfur-containing heterocyclic compound according to claim 3, wherein the compound of formula B is prepared by a specific method as follows:

s1, reacting a compound Interm B with trifluoromethanesulfonic anhydride under an alkaline condition to obtain a compound V-1;

s2, carrying out amination reaction on the compound V-1 by using palladium catalysis to obtain a compound V-2 in a formula;

s3, hydrolyzing the compound V-2 to obtain a compound V-3;

s4, reacting the compound V-3 with a compound Interm A to obtain a compound V-4;

s5, deprotecting the compound V-4 to obtain a compound IIIb 1;

s6, carrying out single or double alkylation reaction on the compound IIIb1 to form a compound IIIb 2;

wherein the compounds Interm B, Interm A, V-1, V-2, V-3, V-4, IIIb1 and IIIb2 are compounds shown in the following structures:

prot is an amine protecting group.

6. The sulfur-containing heterocyclic compound according to claim 1, which is a compound represented by the following structure or a stereoisomer, a geometric isomer, a tautomer, a racemate, a deuterated isotopic derivative, a hydrate, a solvate, a metabolite, and a pharmaceutically acceptable salt or prodrug thereof:

R⁶selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, NR^AR^B；

7. A sulfur-containing heterocyclic compound of any one of claims 1-6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, for use in the preparation of a ROCK inhibitor drug.

8. A sulfur-containing heterocyclic compound as described in any one of claims 1 to 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, for use in the manufacture of a medicament for the treatment of cardiovascular disease, ocular hypertension, pulmonary hypertension, glaucoma.

9. A preparation comprising the sulfur-containing heterocyclic compound according to any one of claims 1 to 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof as an active ingredient, together with a pharmaceutically acceptable excipient.