Piperidinyl spiro derivative as well as preparation method and application thereof

Abstract

The invention relates to a piperidyl spiro derivative and a preparation method and application thereof. The invention provides a piperidinyl spiro derivative # imgabs0 # with the following structural general formula, and also provides a preparation method of the piperidinyl spiro derivative # imgabs0 # and application of the piperidinyl spiro derivative # imgabs0 # in complement system mediated diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a piperidinyl spiro derivative and a preparation method and medical use thereof. Background Art

[0002] The complement system is mainly composed of a variety of proteins synthesized by the liver, circulates in the blood and tissues, and accounts for about 10% of the globulin in the serum. It is an important component of the innate immune system and is essential for defending against microbial infections and clearing immune complexes and damaged cells. Some complement proteins are in an activated state for a long time, so they can monitor the body's immune status for a long time. When the complement system is activated, the proteases in it will cut specific complement proteins to release cytokines and trigger further cascade reactions. In normal host cells, complement is strictly controlled by many cell surface proteins to avoid damage to its own tissues; while foreign pathogens and damaged host cells are vulnerable to such attacks. When the complement system is abnormally activated or overactivated, it can cause cell and tissue damage, causing autoimmune diseases and inflammatory diseases.

[0003] The complement system is mainly composed of complement activating factors, regulatory factors and complement receptors. Complement activating factors are also called intrinsic complement components, including C1-C9, factor B (FB), factor D (FD) and mannose binding lectin. Under physiological conditions, these components exist in the form of inactive enzyme precursors; when the complement system is activated, a cascade amplification reaction will occur, eventually generating a membrane attack complex, which acts on the cell membrane to form pores, and eventually leads to the death of target cells. Complement regulatory factors are a class of proteins that regulate complement activation in a soluble or membrane-bound form, mainly including complement 1 inhibitory factor, factor I (FI), factor H (FH), complement component 4 binding protein, properdin and decay accelerating factor. Complement regulatory factors can finely regulate each link of complement activation, such as: strictly controlling the activity of C3 convertase to avoid continuous activation of complement; converting anaphylatoxins C3a and C5a into inactive or low-activity substances. Complement receptors are mainly expressed on the surface of immune cell membranes and are a type of membrane protein, including anaphylatoxin receptors (C5aR and C3aR), C3b and its derivative-related complement receptors (CR1-4), and C1q receptors (cC1qR, qC1qR, and VLA-2), etc. Complement receptors can bind to activated complement components to recruit white blood cells to the site of inflammation, promote the phagocytosis of pathogenic microorganisms, and clear immune complexes produced in the blood system.

[0004] The complement system is activated by three pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). These three pathways have common terminal pathways C3 and C5-9.

[0005] The classical pathway and the lectin pathway are initiated by recognizing signals on the cell surface, including non-host cells and damaged cells. In both pathways, C4 and C2 molecules are cleaved and C4b2a is formed. This reaction of the classical pathway is achieved through the C1 complex, while the lectin pathway activates serine proteases (MASPs) through some pathogen-associated molecular patterns PAMPs (such as mannose-binding lectin MBL, fibrin gel protein Hcolin, etc.), MASP-1 and MASP-2 further form a complex, promote the cleavage of C4 and C2, and finally form C3 convertase (C4b2a).

[0006] The alternative pathway is different from the above two pathways. It is through the hydrolysis of C3, exposing the thioester domain, and under the action of complement factor B (CFB) and complement factor D (CFD), it combines with the activated Bb molecule to form an active C3 convertase (C3 (H 2 C3a and C3b are further cleaved by C3 convertase. C3b deposited on the surface can form a new C3 convertase (C3bBb) under the action of CFB, thus forming a C3 reaction loop.

[0007] In addition, the excess C3b molecules can combine with C3 convertase to form C5 convertase (including C4b2aC3b and C3bBbC3b), which splits C5 molecules into C5a and C5b. C5b combines with C6, C7, C8 and C9 to form a membrane attack complex (C5b9), which ultimately causes damage to target cells.

[0008] The components of the alternative complement pathway mainly include serine protease factor D, gamma globulin properdin also known as factor P (FP) and serine protease factor B (i.e. complement factor B, FB). FB is a trypsin-like serine protease with a molecular weight of 93kDa and consists of three domains: a three-module complement control protein (CCP), a vonWillebrand factor A domain (VWA) and a C-terminal serine protease domain (SP). The interaction between FB and pathogen-bound C3b is mediated by its A domain, which triggers the conformational change of FB and eventually forms the "C3 convertase" of the alternative complement pathway. As a key node in the alternative complement pathway, FB is indispensable for the formation of C3 and C5 convertases. It is mainly produced in the liver and macrophages and circulates in human blood in a latent form. FB can bind to C3b or C3 to generate the C3 convertase proenzyme C3bB complex, making the complex a substrate for FD. FD cleaves the Arg234-Lys235 peptide bond of FB in the complex, releasing the N-terminal fragment Ba, while the remaining FB subunit Bb containing the serine protease domain becomes a key component of the fully activated C3 convertase. C3 convertase can cleave additional C3 to produce more C3b, thereby further amplifying the local complement response. Therefore, complement factor B inhibitors can be used to treat a variety of complement-mediated diseases.

[0009] Current studies have found that many diseases such as hematological, autoimmune, inflammatory and neurodegenerative diseases are related to abnormal regulation of the complement system, such as paroxysmal nocturnal hemoglobinuria (PNH), immunoglobulin A nephropathy (IgAN), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), dense deposit disease (DDD), ANCA-associated vasculitis (AAV), immune thrombocytopenia (ITP), cold agglutinin disease (CAD), etc.

[0010] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell clonal disease, with clinical manifestations of varying degrees of paroxysmal intravascular hemolysis, paroxysmal hemoglobinuria, bone marrow hematopoietic failure, and venous thrombosis, ultimately leading to progressive end-organ damage and death. It is currently believed that the pathogenesis of PNH is that the glycosylated phosphatidylinositol-A (PIG-A) gene located on the X chromosome (Xp22.1) undergoes somatic mutation at the hematopoietic stem cell level and produces a PNH clone with glycosylated phosphatidylinositol (GPI) deficiency; at the same time, due to factors such as selective immune attack, hematopoietic damage or hematopoietic failure occurs, allowing the PNH clone to gain a proliferation advantage and exceed the normal clone. There are more than ten proteins on the surface of red blood cells that inhibit the activation of the complement pathway, all of which are anchored on their cell membranes through GPI. Among them, the loss of complement decay accelerating factor CD55 and membrane attack complex inhibitor CD59 is considered to be the main mechanism of intravascular hemolysis in PNH patients. CD55 and CD59 are physiologically expressed on the cell surface. CD55 inhibits complement activation by accelerating the decay of C3 and C5 convertases, while CD59 directly inhibits the formation of membrane attack complex (MAC) by preventing C9 from inserting into C5b-8 complex during the assembly of MAC, thereby inhibiting the terminal attack response of complement. Therefore, the lack of any complement inhibitor on the surface of red blood cells will cause abnormal complement activation, making cells more susceptible to complement-mediated damage and hemolysis.

[0011] Immunoglobulin A nephropathy (IgAN) refers to a primary glomerulopathy characterized by the deposition of IgA in the mesangial region, with or without the deposition of other immunoglobulins in the mesangial region. The types of lesions include focal segmental lesions, endocapillary proliferative lesions, mesangial proliferative lesions, crescentic lesions, and sclerotic lesions. The clinical manifestations of IgAN are recurrent macroscopic hematuria or microscopic hematuria, which may be accompanied by varying degrees of proteinuria. Some patients may develop severe hypertension or renal insufficiency. IgAN is one of the main causes of chronic kidney disease. 25-30% of patients progress to end-stage renal disease after 25-30 years, which seriously endangers the life and health of patients. Studies have found that IgAN is caused by a combination of factors. The most widely accepted international theory is the "quadruple-hit theory", which states that genetic or environmental factors lead to insufficient glycosylation of circulating IgA1, forming defective O-glycans, which results in an increase in galactose-deficient IgA1 (Gd-IgA1); the presence of Gd-IgA1 triggers an autoimmune response, producing specific anti-Gd-IgA1 antibodies; Gd-IgA1 binds to antibodies to form pathogenic immune complexes containing IgA; these immune complexes pass through the fenestrae of glomerular endothelial cells and deposit in the mesangial region, activating the complement pathway and inflammatory response, leading to glomerular damage. In this process, complement activation is one of the most common downstream events after the Gd-IgA1 immune complex is deposited in the mesangial region of the kidneys of IgAN patients. Studies have found that the levels of multiple factors involved in the activation of the complement alternative pathway in the plasma and urine of IgAN patients are significantly higher than those in normal healthy people. Multiple factors of the complement alternative pathway are deposited in the renal tissue of IgAN patients, suggesting that abnormal activation of the complement alternative pathway is related to the degree of clinical and pathological changes.

[0012] The U.S. FDA has approved Iptacopan, a drug for the treatment of paroxysmal nocturnal hemoglobinuria in adults, in the form of 200 mg capsules, taken twice a day. The U.S. FDA has approved the marketing application for Iptacopan for the treatment of IgA nephropathy in adults. Although Iptacopan has significant efficacy, the drug still has many defects, such as serious adverse reactions and short duration of efficacy. The most common adverse reactions of the drug include serious infections with encapsulated bacteria (such as Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, etc.), hyperlipidemia, etc. In particular, infections with encapsulated bacteria may be life-threatening, and patients should be fully vaccinated with relevant vaccines within 2 weeks before starting medication.

[0013] C3 glomerulopathy (C3G) is a type of disease in which complement C3 is abnormally deposited in the glomeruli due to inherited or acquired regulatory defects in the complement alternative pathway. As a highly heterogeneous disease, C3G has diverse clinical manifestations, which may present as asymptomatic hematuria and / or proteinuria, or acute nephritic syndrome, nephrotic syndrome, rapidly progressive nephritic syndrome, etc. Clinically, the prognosis of C3G is poor, with 70% of pediatric patients developing end-stage renal disease within 10 years after diagnosis, and 30% to 50% of adult patients developing end-stage renal disease within 10 years after diagnosis. Studies have found that dysregulation of the complement alternative pathway is the main driving factor in the pathogenesis of C3G, and its causes include acquired factors (autoantibodies) and genetic factors (genetic variations). Acquired factors mainly refer to the presence of autoantibodies in the patient's body that can prevent the activation of complement alternative pathway regulatory proteins or directly activate the complement alternative pathway, resulting in continuous activation of the alternative pathway. C3 nephritic factor and C5 nephritic factor are the most common autoantibodies, which can stabilize C3 and C5 convertases, prolong their half-lives, and lead to continuous activation of the complement alternative pathway. Genetic factors mainly include the inherent components of the complement alternative pathway and abnormal genes encoding complement regulatory proteins, including C3, CFB, CFH, CFI, CFHR gene mutations, genetic polymorphisms of CFH, C3, CFB and MCP genes, and genomic rearrangements of the CFH-CFHR locus, which cause protein mutations and make it unable to inhibit C3 lyase activity, resulting in continuous activation of the complement alternative pathway, and then inducing the deposition of activated components and degradation products in the kidneys, causing renal tissue damage. In terms of treatment, C3G lacks a clinically effective treatment plan, and optimized supportive treatment is usually recommended. The treatment principle is to reduce proteinuria and delay the progression of renal function. Drugs commonly used in treatment include hormones and immunosuppressants (cyclophosphamide, tacrolimus, cyclosporine A, etc.), blood pressure control drugs, diuretics, antiplatelet aggregation drugs, plasma exchange and kidney transplantation, etc. However, the above drugs lack specificity and effectiveness, and the long-term prognosis is poor. In clinical practice, it is urgent to develop more targeted and efficient drugs for precise treatment.

[0014] Atypical hemolytic uremic syndrome (aHUS) is a rare and severe clinical syndrome with clinical manifestations of microangiopathic hemolytic anemia, thrombocytopenia and organ damage, especially acute kidney injury. It is currently believed that congenital abnormalities of complement alternative pathway proteins or autoantibodies against complement factors or complement pathway regulatory proteins, which lead to persistent activation of the complement alternative pathway, are the main pathogenic mechanisms of aHUS. Activation of the complement alternative pathway can affect all cells that are in direct contact with plasma, with vascular endothelial cells being particularly susceptible to damage, leading to activation of the coagulation cascade, arteriolar microthrombosis and organ damage. Innate mutations in complement alternative pathway genes can be detected in about 50% of patients. The treatment strategy for aHUS is usually to correct the dysregulation of the complement system. Before the application of complement inhibitors, plasma therapy was the first-line option, but plasma therapy was not effective for patients with complement gene abnormalities and could not solve problems such as high recurrence rate and progression of renal damage. Eculizumab is a recombinant humanized monoclonal antibody that inhibits the cleavage of C5 into C5a and C5b and prevents the formation of the terminal complement complex C5b-9, retaining the function of upstream complement factors (such as C3a and C3b). The use of eculizumab has greatly improved the prognosis of patients with aHUS and has become a first-line treatment for aHUS. Observational studies have shown that for some patients with good efficacy, eculizumab does not need to be used lifelong. However, some patients face the risk of recurrence after discontinuation of the drug, so it is necessary to continue to closely monitor indicators such as blood routine, renal function, and urine routine. For patients with rare mutations in complement genes such as complement factor H, complement factor I, complement factor B, membrane cofactor, and C3, discontinuation of the drug is accompanied by a higher risk of recurrence. Regardless of whether there is a gene mutation, long-term maintenance medication should be considered once recurrence occurs.

[0015] Age-related macular degeneration (AMD) is a degenerative disease caused by multiple factors. It is the main cause of low vision and even blindness in the elderly. The number of AMD patients worldwide is expected to reach 288 million in 2040. The prevalence of AMD in people over 70 years old is 20.2%. With the aging of the population, the number of AMD patients is also continuing to rise. According to the fundus manifestations, AMD is generally divided into two types: exudative (wet) and atrophic (dry). Dry AMD (dAMD) is more common, accounting for about 80% of all AMD patients. In the late stage of dAMD, it can manifest as geographic atrophy (GA), which is characterized by irreversible vision loss due to the loss of retinal pigment epithelium (RPE), photoreceptors and capillaries in the macula. At present, large-scale studies have confirmed that complement activation plays an important role in the pathogenesis of AMD. The complement system is a system that destroys and attacks foreign invaders, interacts with various proteins, and leads to humoral immunity. Inhibition of the complement system, an approach that involves inhibiting complement proteins to downregulate the complementary pathway and the formation of the membrane attack complex, is a potential treatment for dAMD.

[0016] ANCA-associated vasculitis (AAV) is a group of systemic diseases characterized by small vessel immune necrotizing inflammation. AAV includes granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA). AAV is a global disease that can occur at any age, but is more common in people aged 30 to 50 years old, with an incidence of 10 to 20 per million people. AAV is characterized by the presence of antibodies against neutrophil proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA). These antibodies can activate neutrophils and the complement system, leading to inflammation and damage of the vascular wall. Activation of the complement alternative pathway is a key link in the pathogenesis of MPO-ANCA-associated small vessel vasculitis. Studies have found that the concentrations of C3a, C5a, and Bb in the circulation and urine of AAV patients are significantly higher in the acute phase than in the remission phase. Among them, the interaction between the complement activation product C5a and its related receptor C5aR1 (CD88) has become a recognized key link in inducing inflammatory response and a key target for therapeutic intervention.

[0017] Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease involving multiple organs, characterized by the production of multiple pathogenic autoantibodies and extensive deposition of immune complexes (ICs). 50% to 60% of SLE patients develop lupus nephritis (LN) within 10 years after onset, and LN is one of the main causes of poor prognosis in SLE patients. Clinically, glucocorticoids, cyclophosphamide, and new immunosuppressants such as mycophenolate mofetil and tacrolimus are currently commonly used drugs for the treatment of LN, but they have large adverse reactions. A considerable number of refractory LN patients have persistent disease activity, and more than 25% of LN patients progress to end-stage renal failure after 10 years. Studies have found that the formation and deposition of a large number of immune complexes and the excessive activation or imbalance of the complement system are closely related to the onset and progression of LN. Complement activation is an important sign of LN disease activity and recurrence.

[0018] Immune thrombocytopenia (ITP) is a disease characterized by decreased platelet counts due to increased platelet destruction and decreased platelet production. The annual incidence of ITP in adults is (2-10) / 100,000, with the elderly over 60 years old being the most susceptible group, and women of childbearing age being slightly higher than men of the same age group. The clinical manifestations of ITP are somewhat heterogeneous. Many patients are asymptomatic or only experience mild mucocutaneous bleeding, and 5-6% of patients experience severe bleeding, which greatly affects the quality of life of patients. A large amount of experimental and clinical evidence shows that the production of autoantibodies that accelerate platelet clearance is the core of the pathogenesis of ITP, and these IgM and some IgG antiplatelet antibodies activate complement in vivo, leading to complement cascade amplification reactions that aggravate disease progression. Early studies have shown that inhibiting C5 terminal complement activation can improve platelet counts and rapidly reduce the occurrence of thromboembolic complications.

[0019] Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia (AIHA) caused by cold agglutinins (CAs) that are affected by lower temperatures. According to statistics, CAD accounts for 15-30% of AIHA cases, with an overall prevalence of 5-20 cases per million people. In this disease, the complement system in the immune system mistakenly attacks the body's healthy red blood cells and causes them to rupture (hemolysis). CAD patients may experience chronic anemia, severe fatigue, acute hemolytic crisis, and other potential complications, including an increased risk of thromboembolic events and early death. Currently, treatment for CAD includes non-drug therapy and drug therapy. Mild patients can be improved through non-drug therapy such as warming, warming of infusions, and prevention and treatment of infections. Drug therapy can be selected when non-drug therapy is ineffective, symptomatic anemia, transfusion dependence, or circulatory system symptoms that seriously affect life occur. Drug therapy includes general treatment, treatment targeting B cells, and treatment with complement inhibitors. General treatment includes glucocorticoids, cytotoxic drugs, splenectomy, etc., with poor efficacy. Summary of the invention

[0020] Since the current drugs used to treat diseases caused by dysregulation of the complement system still have many defects, there is still an urgent need for the research and development of more therapeutic drugs in clinical practice.

[0021] The purpose of the present invention is to provide drugs that can be used to treat diseases caused by abnormal regulation of the complement system. To this end, the present invention provides a series of new compounds.

[0022] In order to achieve the above-mentioned purpose, the invention provides the following technical solutions.

[0023] A piperidinyl spiro derivative, the general chemical structure of which is as follows:

[0024]

[0025] In the formula,

[0026] X=CH 2 , O, S, NH, CO, or the CH 2 One hydrogen atom on the CH is replaced by methyl, ethyl, cyclopropyl, cyclobutyl, trifluoromethyl, trideuterated methyl, or halogen, or the CH 2 The two hydrogens on or the H on the NH is substituted by a methyl group;

[0027] Y=CH 2 , O, S, NH, CO, or the CH 2 One hydrogen atom on the CH is replaced by methyl, ethyl, cyclopropyl, cyclobutyl, trifluoromethyl, trideuterated methyl, or halogen, or the CH2 The two hydrogens on or the H on the NH is substituted by a methyl group;

[0028] And X and Y are not the same;

[0029] n = 0, 1, or 2;

[0030] R 1 =H, D, halogen, CH 3 , NH 2 , or OH;

[0031] R 2 =H, D, halogen, CH 3 , NH 2 , or OH;

[0032] R 3 =H, D, halogen, CH 3 , NH 2 , or OH;

[0033] R 4 =H, OH, NH 2 , CH 3 , CD 3 , OCH 3 , or OEt;

[0034] R 5 =H, OH, NH 2 , CH 3 , CD 3 , OCH 3 , or OEt;

[0035] R 6 =CH 3 , CH 2 CH 3 , OCF 3 , dimethyl, cyclopropyl, cyclobutyl, halocyclobutyl, cyclopropyloxy, cyclobutyloxy, or halocyclobutyloxy;

[0036] or,

[0037] R 6 for Among them, R 7 =H, D, CH 3 , or halogen; R 8 =H, D, CH 3 , or halogen;

[0038] or,

[0039] R 6 for Among them, R9 =H,CH 3 , CH 2 CH 3 , cyclopropyl, cyclobutyl, or halocyclobutyl;

[0040] or,

[0041] R 6 is a halogenated phenyl group or an aniline carbonyl group;

[0042] or,

[0043] R 6 For R 10 , R 11 A three-membered ring of two substituents, and the three-membered ring and the group to which it is connected share a carbon atom; wherein R 10 =H, D, CH 3 , or halogen; R 11 =H, D, CH 3 , or halogen;

[0044] or

[0045] R 6 For R 12 , R 13 A four-membered ring of two substituents, and the four-membered ring and the group to which it is connected share a carbon atom; wherein R 12 =H, D, CH 3 , or halogen; R 13 =H, D, CH 3 , or halogen;

[0046] or,

[0047] R 6 is an aza four-membered ring, and the four-membered ring shares one carbon atom with the group to which it is connected;

[0048] or,

[0049] R 6 It is an oxygen-containing four-membered ring, and the four-membered ring and the group to which it is connected share one carbon atom.

[0050] The above piperidinyl spiro derivative, when X is CH 2 When the CH 2 The hydrogen can be replaced by deuterium.

[0051] The above piperidinyl spiro derivative, when Y is CH 2 When the CH 2 The hydrogen can be replaced by deuterium.

[0052] In the above piperidinyl spirocyclic derivative, when X is NH, the hydrogen on the NH may be substituted by deuterium.

[0053] In the above piperidinyl spirocyclic derivative, when Y is NH, the hydrogen on the NH may be substituted by deuterium.

[0054] The above piperidinyl spiro derivative, when R 6 CH 2 CH 3 When the CH 2 CH 3 The hydrogen atoms on the residue may be deuterated, for example, compound A107 described in the present invention.

[0055] The above piperidinyl spiro derivative, when R 6 for R 9 CH 3 When R 6 The hydrogen in may be deuterated, for example, compound A129 described in the present invention.

[0056] The above piperidinyl spiro derivative, when R 6 for R 9 CH 3 When R6 is present, the hydrogen on the carbon atom connected to R6 may be deuterated, such as compound A127 of the present invention.

[0057] The above piperidinyl spiro derivative, R 6 The halogenated cyclobutyl group is 3,3-difluorocyclobutyl.

[0058] The above piperidinyl spiro derivative, R 6 The halogenated cyclobutyloxy group is 3,3-difluorocyclobutyloxy.

[0059] The above piperidinyl spiro derivative, R 9 The halogenated cyclobutyl group is 3,3-difluorocyclobutyl.

[0060] The above piperidinyl spiro derivative, R 6 The halogenated phenyl group is 2,3-difluorophenyl.

[0061] In the compounds provided by the present invention, D refers to deuterium.

[0062] In the compounds provided by the present invention, the substituent R6 connected to the piperidinyl parent ring is a three-membered ring or a four-membered ring with a substituent, and when the membered ring structure and the group connected thereto share a carbon atom, the membered ring and the piperidinyl parent ring form a spiro ring structure. Compounds A4, A32, etc. provided by the present invention are one of the cases of such a structure.

[0063] The present invention lists the structural formula of the above-mentioned general formula compound, including:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] The compounds provided by the present invention can be prepared into various pharmaceutically acceptable salts, solvates, deuterated substances, stereoisomers, tautomers, etc. as required.

[0072] The compounds provided by the present invention can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, granules, suspensions, sustained-release preparations, etc., by adding auxiliary materials as needed.

[0073] The compounds provided by the present invention can be used to treat diseases mediated by the complement system, especially diseases caused by abnormal regulation of the complement system or abnormal function of the complement system.

[0074] The compounds provided by the present invention are preferably used for the treatment of diseases associated with the complement pathway, especially diseases associated with the alternative pathway of the complement system.

[0075] The compounds provided by the present invention are further preferably used for the preparation of drugs for inhibiting complement factor B.

[0076] The diseases mediated by the complement system described in the present invention include: paroxysmal nocturnal hemoglobinuria, immunoglobulin A nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome, age-related macular degeneration, ANCA-associated vasculitis, systemic lupus erythematosus, immune thrombocytopenia, cold agglutinin disease, etc.

[0077] The C3 glomerulopathy described in the present invention includes dense deposit disease, primary C3 glomerulonephritis, familial type III membranoproliferative glomerulonephritis, and complement factor H-related protein 5 nephropathy.

[0078] Experimental studies have confirmed that the compounds provided by the present invention have significant pharmacological activity in regulating complement system abnormalities. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below with reference to specific embodiments.

[0080] The room temperature in the experiment refers to 20-25°C.

[0081] Example 1 Preparation of (4S, 4'-4'-ethoxy-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (Compound A2)

[0082]

[0083] Synthesis route:

[0084]

[0085] Step 1:

[0086]

[0087] 7-Bromo-4-dihydrochromanone (23.0 g, 101 mmol) and (R)-2-methylpropane-2-sulfinamide (14.7 g, 121 mmol) were dissolved in anhydrous tetrahydrofuran (460 mL), tetraethyl titanate (34.6 g, 152 mmol) was added dropwise at room temperature, the reaction solution was heated to 80°C, reacted for 12 hours, and the reaction process was monitored by LCMS. The reaction solution was cooled to room temperature, water (500 mL) was added, filtered, the aqueous phase was extracted with ethyl acetate (200 mL*3), the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Silica gel column chromatography was used to separate and purify to obtain (R, Z)-N-(7-bromochroman-4-ylidene)-2-methylpropane-2-sulfonamide (15.0 g, yield 44.8%).

[0088] Step 2:

[0089]

[0090] Methyl acetate (1.12 g, 15.1 mmol) was dissolved in anhydrous tetrahydrofuran (7.00 mL), cooled to -70 ° C, lithium diisopropylamide (2M, 4.54 mL) was added dropwise, and the mixture was reacted at -70 ° C for 0.5 hour. Then, the (R, Z)-N-(7-bromobenzodihydropyran-4-ylidene)-2-methylpropane-2-sulfonamide (1.00 g, 3.03 mmol) prepared in step 1 was added in anhydrous tetrahydrofuran (10.0 mL). The temperature was controlled at -70 to -65 ° C, and the reaction was carried out for 1 hour. The reaction process was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride solution (10.0 mL) at 0°C, and then extracted with ethyl acetate (10.0 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 4-((S)-7-bromo-4-((R)-tert-butylsulfinyl)amino)benzopyran-4-yl)-3-oxobutanoic acid methyl ester (0.26 g).

[0091] Step 3:

[0092]

[0093] The methyl 4-((S)-7-bromo-4-((R)-tert-butylsulfinyl)amino)benzopyran-4-yl)-3-oxobutanoate (0.60 g, 1.34 mmol) prepared in step 2 was dissolved in anhydrous tetrahydrofuran (6.00 mL). After replacing nitrogen three times, the system was cooled to -70 ° C, zinc borohydride was added dropwise, and then the temperature was raised to 25 ° C under nitrogen protection for 12 hours. TLC monitored the reaction process. The reaction solution was cooled to 0 ° C, quenched with methanol (10.0 mL), and concentrated under reduced pressure. 4-((S)-7-bromo-4-((R)-tert-butylsulfinyl)amino)benzopyran-4-yl)-3-hydroxybutyrate (0.20 g, yield 50.1%) was obtained by separation and purification by silica gel column chromatography.

[0094] Step 4:

[0095]

[0096] The methyl 4-((S)-7-bromo-4-((R)-tert-butylsulfinyl)amino)benzopyran-4-yl)-3-hydroxybutyrate (0.20 g, 446 mmol) prepared in step 3 was dissolved in dioxane (0.20 mL), and then hydrochloric acid / dioxane (2 M, 0.50 mL) was added to the system and reacted at 25° C. for 1 hour. LCMS showed that the starting material was completely consumed. The mixture was concentrated under reduced pressure to give methyl 4-((S)-4-amino-7-bromobenzopyran-4-yl)-3-hydroxybutyrate hydrochloride (0.14 g).

[0097] Step 5:

[0098]

[0099] The 4-((S)-4-amino-7-bromobenzopyran-4-yl)-3-hydroxybutyric acid methyl ester hydrochloride (0.14 g, 406 mmol) prepared in step 4 was dissolved in methanol (0.50 mL), and then the system was added to ammonia methanol (7 M, 0.50 mL) at room temperature, heated to 45°C, and reacted for 12 hours. LCMS showed that the raw material was completely consumed. Concentrated under reduced pressure to obtain (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-6'-one (0.94 g, yield 74.0%).

[0100] Step 6:

[0101]

[0102] The (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-6'-one (0.10 g, 224 mmol) prepared in step 5 was dissolved in anhydrous tetrahydrofuran (1 mL), and nitrogen was replaced three times. The system was cooled to -40°C, and borane tetrahydrofuran complex (2M, 0.15 μL) was added dropwise at -40°C for 1 hour. LCMS showed that the raw material was completely consumed. Water (1.00 mL) was added at -10°C to quench, and the mixture was extracted with ethyl acetate (10.0 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure to obtain (4S)-7-bromospiro[benzopyran-4,2'-piperidine]-4'-ol (0.10 g).

[0103] Step 7:

[0104]

[0105] The (4S)-7-bromospiro[benzopyran-4,2'-piperidine]-4'-ol (0.08 g, 268 μmol) prepared in step 6 was dissolved in anhydrous tetrahydrofuran (1 mL), di-tert-butyl dicarbonate (0.12 g, 123 μmol) was added, and the mixture was reacted at 15-25° C. for 1 hour. LCMS showed that the raw material was completely consumed. The mixture was concentrated under reduced pressure to obtain (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.10 g).

[0106] Step 8:

[0107]

[0108] The (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.12 g, 306 μmol) prepared in step 7 was dissolved in N-methylpyrrolidone (1.00 mL), replaced with nitrogen three times, cooled to 0°C, and sodium hydrogen hydride (30.6 mg, 768 μmol) was added, and the temperature was controlled at 0-5°C during the addition. After the addition was completed, the reaction was carried out at room temperature for 2 hours, and then iodoethane (162 mg, 1.04 mmol) was added and reacted at room temperature for 2 hours. LCMS showed that the raw material was completely consumed. The system was cooled to 0°C, quenched with saturated aqueous ammonium chloride solution (5.00 mL), diluted with water (5.00 mL), extracted with ethyl acetate (10.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (4S)-7-bromo-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.02 g, yield 46.7%).

[0109] Step 9:

[0110]

[0111] Bistriphenylphosphine palladium dichloride (19.1 mg, 23.4 μmol) was added to anhydrous methanol (5.00 mL), and (4S)-7-bromo-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.10 g, 234 μmol) and triethylamine (94.9 mg, 938 μmol) prepared in step 8 were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, the system was reacted in carbon monoxide (2 MPa) at 125°C for 12 hours, and TLC showed that the raw material was completely consumed. The reaction solution was filtered and concentrated under reduced pressure to obtain 1'-(tert-butyl) 7-methyl (4S)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-1', 7-dicarboxylic acid (0.03 g).

[0112] Step 10:

[0113]

[0114] The 1'-(tert-butyl)7-methyl(4S)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylic acid (0.03 g, 74.0 μmol) prepared in step 9 was dissolved in ethyl acetate (0.50 mL), and then hydrochloric acid / ethyl acetate (2M, 0.75 mL) was added to the system, and the reaction was carried out at 25°C for 12 hours. LCMS showed that the raw material was completely consumed. The mixture was concentrated under reduced pressure to obtain (4S)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (0.022 g).

[0115] Step 11:

[0116]

[0117] The (4S)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (0.02 g, 72.0 μmol) prepared in step 10 was dissolved in N,N-dimethylformamide (1.00 mL), and potassium iodide (11.9 mg, 72.0 μmol), potassium carbonate (19.9 mg, 144 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (33.4 mg, 108 μmol) were added in sequence. The reaction was carried out at room temperature for 30 minutes, and the reaction process was monitored by LCMS. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10.0 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give methyl (4S)-1'-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylate (0.04 g).

[0118] Step 12:

[0119]

[0120] The (4S)-1'-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylate (40.0 mg, 69.1 μmol) prepared in step 11 was dissolved in methanol (2.00 mL) and water (0.50 mL), and lithium hydroxide monohydrate (29.0 mg, 691 μmol) and potassium carbonate (19.9 mg, 144 μmol) (33.4 mg, 108 μmol) were added. The reaction was carried out at room temperature for 30 minutes. LCMS showed that the raw material was completely consumed. The mixture was concentrated under reduced pressure, diluted with water (5.00 mL), and then the pH of the system was adjusted to 2-3 with hydrochloric acid (0.50 M), extracted with ethyl acetate (10.0 mL*3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4'-4'-ethoxy-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (4.43 mg, yield 10.8%), i.e., compound A2.

[0121] 1H NMR: (400MHz, CDCl3), δppm: 8.14 (s, 1H), 7.71–7.67 (m, 1H), 7.56 (d, J = 8.00 Hz,1H),7.25(s,1H),6.82–6.81(m,1H),6.69(s,1H),4.48–4.35(m,1H).4.3 4–4.30(m,1H),3.84–3.78(m,1H),4.34–4.30(m,1H),3.46(td,J=7.60Hz,3H ),2.82–2.81(m,4H),2.48(s,1H),2.05–1.90(m,2H),3.46(t,J=7.20Hz,3H).

[0122] Example 2 Preparation of (R)-3,3-difluoro-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid (Compound A8)

[0123]

[0124] Synthesis route:

[0125]

[0126] Step 1:

[0127]

[0128] Dissolve (R)-7-methyl-4'-methylenespiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.4 g, 1.07 mmol) in hydrochloric acid / ethyl acetate (2M, 8 mL) and react at 35-40°C for 1 hour. Monitor the reaction process by LCMS. The reaction solution is cooled to room temperature and concentrated under reduced pressure to obtain (R)-7-methyl-4'-methylenespiro[benzopyran-4,2'-piperidine]-carboxylate hydrochloride (0.37 g).

[0129] Step 2:

[0130]

[0131] The (R)-7-methyl-4'-methylene spiro[benzopyran-4,2'-piperidine]-carboxylate hydrochloride (0.37 g, 1.19 mmol) prepared in step 1 was dissolved in tetrahydrofuran (4 mL) and water (0.4 mL), and then sodium bicarbonate (710 mg, 8.45 mmol) and benzyl chloroformate (710 mg, 8.45 mmol) were added in sequence, and the reaction was carried out at 35-40°C for 1 hour. LCMS was used to monitor the reaction process. The reaction solution was diluted with water (5 mL) at 20-25°C, and then extracted with ethyl acetate (5 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 1'-benzyl 7-methyl (R)-4'-methylene spiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylate (476 mg, yield 97.81%).

[0132] Step 3:

[0133]

[0134] The 1'-benzyl 7-methyl (R)-4'-methylene spiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylate (0.28 g, 573 μmol) prepared in step 2 was dissolved in methanol (3 mL) and acetonitrile (6 mL), and triethylamine (232 mg, 2.30 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (213 mg, 261 μmol) were added, and the carbon monoxide gas was replaced three times, and the temperature was raised to 130-140°C, and the reaction was carried out under a carbon monoxide atmosphere for 36 hours. The reaction process was monitored by LCMS until the raw material was completely consumed. The reaction solution was cooled to 20° C. and concentrated under reduced pressure to give 1-benzyl 7-methyl (4R)-2,2-dichloro-3-oxadispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (70.0 mg, yield 26.1%).

[0135] Step 4:

[0136]

[0137] The 1-benzyl 7-methyl (4R)-2,2-dichloro-3-oxodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (605 mg, 1.17 mmol) prepared in step 3 was dissolved in methanol (12 mL) and reacted at 40-45° C. for 1 hour. LCMS showed that the raw material was completely consumed and the product was generated. The mixture was concentrated under reduced pressure to obtain 1-benzyl 7-methyl (R)-3-oxodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (60.0 mg, yield 99.2%).

[0138] Step 5:

[0139]

[0140] The 1-benzyl 7-methyl (R)-3-oxodispiro [benzopyran-4,2-piperidine-4,1-cyclobutane] -1,7-dicarboxylic acid (0.13 g, 289 μmol) prepared in step 4 was dissolved in dichloromethane (4 mL), and diethylaminosulfur trifluoride (234 mg, 1.45 mmol, 191 μL) was added at 0 ° C. The reaction solution was reacted at 20-25 ° C for 12 hours. LCMS monitored the reaction process. The reaction solution was poured into a saturated potassium carbonate solution (10 mL) at 10 ° C, and then extracted with ethyl acetate (2 mL x 2) and concentrated to obtain 1-benzyl 7-methyl (R) -3,3-difluorobipyridine [benzopyran-4,2-piperidine-4,1-cyclobutane] -1,7-dicarboxylic acid (60 mg, yield 44.00%).

[0141] Step 6:

[0142]

[0143] The 1-benzyl 7-methyl (R)-3,3-difluorobipyridine [benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (60 mg, 127 μmol) prepared in step 5 was dissolved in ethanol (2 mL), and palladium carbon (40 mg, 37.6 μmol, 10% purity) was added under nitrogen protection. The reaction solution was replaced with hydrogen three times and reacted for 2 hours at 15-20° C. under a hydrogen atmosphere (15 Psi). LCMS showed that the raw material was completely consumed and the product was generated. The reaction solution was filtered and concentrated to obtain (R)-3,3-difluorobispiro [benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid methyl ester (42.9 mg, yield 99.93%).

[0144] Step 7:

[0145]

[0146] The (R)-3,3-difluorodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid methyl ester (55.0 mg, 163 μmol) prepared in step 6 was dissolved in N,N-dimethylformamide (3.00 mL), potassium iodide (27.5 mg, 188 μmol, 1.13 eq), potassium carbonate (55.0 mg, 398 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (82.5 mg, 266 μmol) were added in sequence, and the mixture was reacted at 15-25° C. for 30 minutes. LCMS showed that the raw material was completely consumed. Water (10.0 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10.0 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by thin layer chromatography to give methyl (R)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-3,3-difluorodipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylate (65 mg, yield 65.3%).

[0147] Step 8:

[0148]

[0149] The methyl (R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-3,3-difluorodipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylate (65.0 mg, 106 μmol, 1.00 eq) prepared in step 7 was dissolved in methanol (2.00 mL) and water (1.00 mL), and lithium hydroxide monohydrate (45.5 mg, 1.08 mol, 10.1 eq) was added. The reaction solution was reacted at 50-60° C. for 10 hours. LCMS showed that the raw material was completely consumed. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 mins) to obtain (R)-3,3-difluoro-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid (46.7 mg, yield 85.3%), i.e., compound A8.

[0150] 1H NMR: (400MHz, CDCl3), δppm: 7.72 (d, J = 8.00Hz, 1H), 7.43 (d, J = 8.00Hz, 1H), 7.24 (d, J = 2.80Hz, 1 H),7.21(d,J=1.20Hz,1H),6.57(s,2H),4.33-4.20(m,2H),3.68(s,3H),3.56(d,J=12.0Hz,1H),3 .37(d,J=11.6Hz,1H),2.59-2.52(m,2H),2.41(s,3H),2.38-2.31(m,2H),2.27-2.18(m,1H),2.11 (d,J=13.2Hz,1H),1.91(d,J=14.4Hz,1H),1.81(d,J=13.6Hz,1H),1.75(s,2H),1.61-1.52(m,2H)

[0151] Example 3 Preparation of (R)-3-fluoro-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid (Compound A10)

[0152]

[0153] Synthesis route:

[0154]

[0155] Step 1:

[0156]

[0157] Tert-butyl (R)-7-methyl-4'-methylene[benzopyran-4,2'-piperidine]-1'-carboxylate (830 mg, 2.22 mmol) was dissolved in hydrochloric acid / ethyl acetate (2M, 16.0 mL), and the reaction solution was heated to 30-35°C and reacted for 1 hour. The reaction process was monitored by LCMS. The reaction solution was directly concentrated under reduced pressure to obtain (R)-7-methyl-4'-methylenespiro[benzopyran-4,2'-piperidine]-carboxylate (701 mg).

[0158] Step 2:

[0159]

[0160] The (R)-7-methyl-4'-methylene spiro[benzopyran-4,2'-piperidine]-carboxylate (700 mg, 2.26 mmol) prepared in step 1 was dissolved in tetrahydrofuran (7 mL) and water (0.7 mL), and then sodium bicarbonate (1.34 g, 15.9 mmol) and benzyl chloroformate (1.17 g, 6.88 mmol) were added in sequence, and the mixture was reacted at 35-40°C for 2 hours, and the reaction process was monitored by LCMS. The reaction solution was cooled to room temperature, diluted with water (10 mL), and extracted with ethyl acetate (10 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was separated and purified by column chromatography to obtain 1-benzyl 7-methyl (R)-4-methylene spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylate (920 mg, yield 99.9%).

[0161] Step 3:

[0162]

[0163] The 1-benzyl 7-methyl (R)-4-methylene spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylate (0.92 g, 2.26 mmol) prepared in step 2 was dissolved in ethylene glycol dimethyl ether (12 mL), and copper-zinc alloy (1.30 g, 20.3 mmol) was added to the reaction solution. After heating to 40-45°C, trichloroacetyl chloride (2.00 g, 11.0 mmol) was added. After replacing nitrogen three times, the reaction was carried out at 40-45°C for 1.5 hours, and the reaction process was monitored by LCMS. The reaction solution was cooled to room temperature, washed with saturated sodium bicarbonate aqueous solution (40 mL) and extracted with ethyl acetate (10 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 1-Benzyl 7-methyl (4R)-2,2-dichloro-3-oxadispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (1.17 g) was obtained.

[0164] Step 4:

[0165]

[0166] The 1-benzyl 7-methyl (4R)-2,2-dichloro-3-oxodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (1.17 g, 2.26 mmol) prepared in step 3 was dissolved in methanol (20 mL), and ammonium chloride (1.57 g, 29.3 mmol) and zinc powder (1.48 g, 22.6 mmol) were added, and the reaction was carried out at 40-45°C for 1 hour. LCMS showed that the raw material was completely consumed and the product was generated. The reaction solution was filtered through a diatomaceous earth pad, and the filtrate was concentrated and diluted with water (20 mL), extracted with ethyl acetate (20 mL*2), and the organic phase was washed with a saturated sodium bicarbonate aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 1-Benzyl 7-methyl (R)-3-oxodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (813 mg, yield 80.1%) was obtained by separation and purification by column chromatography.

[0167] Step 5:

[0168]

[0169] The 1-benzyl 7-methyl (R)-3-oxodispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (813 mg, 1.81 mmol) prepared in step 4 was dissolved in methanol (10 mL), and sodium borohydride (380 mg, 10.0 mmol) was slowly added in batches at 15-20°C. After replacing nitrogen three times, the mixture was reacted at 15-20°C for 1 hour. LCMS showed that the raw material was completely consumed and the product was generated. At room temperature, the reaction solution was quenched with saturated aqueous ammonium chloride solution (10 mL), concentrated to remove methanol, extracted with ethyl acetate (5 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 1-benzyl 7-methyl (R)-3-hydroxydipyridine[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylate (767 mg, yield 93.9%) was obtained.

[0170] Step 6:

[0171]

[0172] The 1-benzyl 7-methyl (R)-3-hydroxybipyridine [benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylate (767 mg, 1.70 mmol) prepared in step 5 was dissolved in dichloromethane (5 mL), cooled to 0 ° C in an ice-water bath, and diethylaminosulfur trifluoride (602 mg, 3.73 mmol) was added. The reaction was reacted at 15-20 ° C for 2 hours. LCMS monitoring showed that the product was generated and the raw materials were completely consumed. The reaction was cooled to room temperature, the reaction solution was quenched with saturated potassium carbonate aqueous solution (10 mL) and extracted with ethyl acetate (10 mL*2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was separated and purified by a preparative plate to obtain 1-benzyl 7-methyl (R)-3-fluorobispiro [benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (184 mg, yield 23.9%).

[0173] Step 7:

[0174]

[0175] The 1-benzyl 7-methyl (R)-3-fluorobispiro[benzopyran-4,2-piperidine-4,1-cyclobutane]-1,7-dicarboxylic acid (80.0 mg, 176 μmol) prepared in step 6 was dissolved in dichloromethane (2 mL), and trimethylsilyl iodide (177 mg, 886 μmol) was added. After replacing nitrogen, the mixture was reacted at 15-20°C for 1 hour. LCMS showed that the raw material was completely consumed. Saturated potassium carbonate aqueous solution (5 mL) was added to the reaction solution to quench, and the mixture was diluted with water (5 mL) and extracted with dichloromethane (5 mL*2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. (R)-3-fluorobipyridine[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid methyl ester (60.0 mg) was obtained.

[0176] Step 8:

[0177]

[0178] The (R)-3-fluorobipyridine [benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid methyl ester (55.0 mg, 172 μmol) prepared in step 7 was dissolved in N,N-dimethylformamide (3 mL), potassium iodide (44.0 mg, 265 μmol), potassium carbonate (77.0 mg, 557 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (165 mg, 533 μmol) were added in sequence and reacted at 15-25 ° C for 30 minutes. LCMS showed that the raw material was completely consumed. Water (10 mL) was added to the reaction system, extracted with ethyl acetate (5 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was separated and purified by TLC to give methyl (R)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-3-fluorobipyridine[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylate (32.0 mg, yield 31.4%).

[0179] Step 9:

[0180]

[0181] The methyl (R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-3-fluorobipyridine[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylate (22.0 mg, 37.1 μmol) prepared in step 8 was dissolved in methanol (0.5 mL), tetrahydrofuran (0.5 mL) and water (0.5 mL), and lithium hydroxide monohydrate (10.0 mg, 418 μmol) was added. The reaction solution was reacted at 55-60° C. for 12 hours. LCMS showed that the raw material was completely consumed. After concentrating under reduced pressure to remove the organic solvent, the pH was adjusted to 7 with 1M HCl. Neutral reverse phase high performance liquid chromatography was used for separation and purification (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (R)-3-fluoro-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclobutane]-7-carboxylic acid (1.01 mg, yield 5.26%), i.e., compound A10.

[0182] 1H NMR: (400MHz, CDCl3) δppm: 8.04 (d, J = 8.00Hz, 1H), 7.93 (s, 1H), 7.65 (d, J = 7.20Hz, 1H), 7.52 (s, 1H), 6 .27(s,1H),6.79(s,1H),6.69(s,1H),5.06-5.04(m,1H),4.33-4.20(m,2H),3.68(s,3H),3.56(d,J=12. 0Hz,1H),3.37(d,J=11.6Hz,1H),2.59-2.52(m,2H),2.41(s,3H),2.38-2.31(m,2H),2.27-2.18(m,1H) ,2.11(d,J=13.2Hz,1H),1.91(d,J=14.4Hz,1H),1.81(d,J=13.6Hz,1H),1.75(s,2H),1.61-1.52(m,2H)

[0183] Example 4 Preparation of (4S, 4'S)-4-(2,2-difluorocyclopropyl)methoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[4,2-piperidine]-7-carboxylic acid (Compound A11)

[0184]

[0185] Synthesis route:

[0186]

[0187] Step 1:

[0188]

[0189] Dissolve (4S, 4'S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (300 mg, 753 μmol) in anhydrous tetrahydrofuran (3 mL), slowly add sodium hydride (100 mg, 2.50 mmol, 60% purity) and allyl bromide (274 mg, 2.26 mmol) in batches at 0°C, warm the reaction solution to 20-25°C, react for 20 hours, and monitor the reaction process with LCMS. The reaction solution was cooled to 10-15°C, quenched with saturated aqueous ammonium chloride solution (5 mL), diluted with water (5 mL), extracted with ethyl acetate (6 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was separated and purified using a preparative plate to give (4S,4'S)-4-(allyloxy)-7-bromospiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (262 mg, yield 79.3%).

[0190] Step 2:

[0191]

[0192] The (4S, 4'S)-4-(allyloxy)-7-bromospiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (260 mg, 593 μmol) and (bromodifluoromethyl)trimethylsilane (247 mg, 1.22 mmol) prepared in step 1 were dissolved in anhydrous toluene (4 mL), and tetrabutylammonium bromide (20.0 mg, 62.3 μmol) was added, and the reaction was carried out at 95-100 ° C for 4 hours, and the reaction process was monitored by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (10 mL) at 20-25 ° C, and then extracted with ethyl acetate (10 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. (4S, 4'S)-7-bromo-4-(2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (0.28 g) was obtained.

[0193] Step 3:

[0194]

[0195] The (4S, 4'S)-7-bromo-4-(2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (0.28g, 573μmol) prepared in step 2 was dissolved in methanol (3mL) and acetonitrile (6mL), and triethylamine (232mg, 2.30mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (213mg, 261μmol) were added to the reaction solution. After replacing carbon monoxide gas three times, the temperature was raised to 130-140°C and reacted for 36 hours under carbon monoxide gas atmosphere. LCMS monitoring found that the product was generated and the raw materials were completely consumed. The reaction solution was cooled to 20°C and concentrated under reduced pressure. The concentrate was separated and purified using a preparative plate to give 1-(tert-butyl)7-methyl(4S,4'S)-4((2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (70.0 mg, yield 26.1%).

[0196] Step 4:

[0197]

[0198] The 1-(tert-butyl)7-methyl(4S,4'S)-4((2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (70.00 mg, 149.73 μmol) prepared in step 3 was dissolved in hydrochloric acid / dioxane (2M, 3.50 mL) and reacted at 40-45° C. for 1 hour. LCMS showed that the raw material was completely consumed and the product was generated. The solution was concentrated under reduced pressure to obtain (4S,4'S)-4((2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (60.0 mg, yield 99.2%).

[0199] Step 5:

[0200]

[0201] The (4S, 4'S)-4((2,2-difluorocyclopropyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (60.0 mg, 148 μmol, HCl) prepared in step 4 was dissolved in N,N-dimethylformamide (1 mL), and potassium carbonate (123 mg, 891 μmol), potassium iodide (36.0 mg, 217 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (200 mg, 646 μmol) were added in sequence, and the mixture was reacted at 15-20°C for 1.5 hours. LCMS showed that the raw material was completely consumed and the product was generated. At room temperature, the reaction solution was diluted with water (5 mL), extracted with ethyl acetate (2 mL*2), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was separated and purified using a preparative plate to give methyl (4S,4'S)-1-((1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-((2,2-difluorocyclopropyl)methoxy)spiro[4,2-piperidine]-7-carboxylate (42.0 mg, yield 44.1%).

[0202] Step 6:

[0203]

[0204] The methyl (4S, 4'S)-1-((1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-((2,2-difluorocyclopropyl)methoxy)spiro[4,2-piperidine]-7-carboxylate (42.0 mg, 65.6 μmo) prepared in step 5 was dissolved in methanol (0.5 mL), tetrahydrofuran (0.5 mL) and water (0.5 mL), lithium hydroxide (17.0 mg, 709 μmol) was added, and the system was heated to 50-55°C for 12 hours. LCMS showed that the raw material was completely consumed and the product was generated. After the reaction solution was cooled to room temperature, the pH was adjusted to 7 with 1M hydrochloric acid aqueous solution and directly concentrated under reduced pressure. The concentrate was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4'S)-4-(2,2-difluorocyclopropyl)methoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[4,2-piperidine]-7-carboxylic acid (20.0 mg, yield 57.9%), i.e., compound A11.

[0205] 1H NMR: (400MHz, CDCl3) δppm: 8.12 (s, 1H), 8.10 (s, 1H), 8.09 (d, J = 3.60Hz, 1H), 7.67 (s, 1H), 7. 22(s,1H),6.79(s,1H),6.69(s,1H),4.46-4.45(m,1H),4.42-4.34(m,1H),3.68(s,3H),3.56 -3.50(m,4H),3.47-3.44(m,1H),2.78-2.72(m,4H),2.70(s,3H),2.69-2.47(m,1H),1.84-1.80(m,4H),1.78-1.77(m,1H),1.10-1.09(m,1H)

[0206] Example 5 Preparation of (4S, 4'S)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-propoxyspiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A31)

[0207]

[0208] Synthesis route:

[0209]

[0210] Step 1:

[0211]

[0212] Dissolve (4S, 4'S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (370 mg, 929 μmol) in anhydrous N-methylpyrrolidone (2 mL), add sodium hydride (185 mg, 4.63 mmol, 60% purity) at 0-10°C, and stir the reaction solution at 0-10°C for 30 minutes. Add 1-iodopropane (370 mg, 2.18 mmol, 213 μL) to the reaction solution and heat to 15-25°C for 4 hours. Monitor the reaction process with LCMS. Quench the reaction solution with saturated ammonium chloride solution (20 mL), then extract with ethyl acetate (10.0 mL*3), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The concentrate was separated and purified by thin layer chromatography to give (4S,4'S)-7-bromo-4-propoxyspiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (325 mg, 738 μmol, yield 79.4%).

[0213] Step 2:

[0214]

[0215] The (4S, 4'S)-7-bromo-4-propoxyspiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (170 mg, 386 μmol) prepared in step 1 was dissolved in a mixed solution of anhydrous methanol (5.00 mL) and acetonitrile (5.00 mL), and bis(diphenylphosphino)ferrocenepalladium dichloride (31.9 mg, 43.56 μmol) and triethylamine ((156 mg, 1.54 mmol, 21 5μL), the reaction system was replaced with argon three times, and then replaced with carbon monoxide three times. The system was reacted in carbon monoxide (2Mpa) at 120-130°C for 10 hours. The reaction solution was concentrated to obtain a crude product. 1-(tert-butyl)7-methyl(4S,4'S)-4-propoxyspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (10.0mg, 23.8μmol, yield 6.17%) was obtained by separation and purification using a thin layer chromatography plate.

[0216] Step 3:

[0217]

[0218] The 1-(tert-butyl)7-methyl(4S,4'S)-4-propoxyspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (40.0 mg, 95.4 μmol, 1.00 eq) prepared in step 2 was dissolved in hydrochloric acid / dioxane (2M, 1.00 mL) and reacted at 30-40°C for 2 hours. LCMS showed that the raw material was completely consumed. After concentration under reduced pressure, the solution was diluted with ethyl acetate and the pH was adjusted to 7-8 with a saturated aqueous sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Methyl (4S,4S)-4-propoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylate (40 mg) was obtained.

[0219] Step 4:

[0220]

[0221] The (4S, 4S)-4-propoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (40.0 mg, 125 μmol) prepared in step 3 was dissolved in N,N-dimethylformamide (3.00 mL), and potassium iodide (23.9 mg, 144 μmol), potassium carbonate (35.8 mg, 259 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (97.0 mg, 313 μmol) were added in sequence and reacted at 15-25°C for 30 minutes. LCMS showed that the raw material was completely consumed. Water (10.0 mL) was added to the reaction body, extracted with ethyl acetate (10.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The compound was separated and purified by thin layer chromatography to obtain methyl (4S,4'S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-propoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (50 mg, yield 67.4%).

[0222] Step 5:

[0223]

[0224] The methyl (4S, 4'S)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-propoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (50.0 mg, 84.4 μmol, 1.00 eq) prepared in step 4 was dissolved in methanol (2.00 mL) and water (0.50 mL), and lithium hydroxide monohydrate (35.4 mg, 844 μmol, 422 μL, 10.0 eq) was added, and the reaction solution was reacted at 50-60° C. for 5 hours. LCMS showed that the raw material was completely consumed. Concentrate under reduced pressure to obtain a crude product. Neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) was used to obtain (4S, 4'S)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-propoxyspiro[benzopyran-4,2-piperidine]-7-carboxylic acid (10.2 mg, yield 24.7%), i.e., compound 31.

[0225] LCMS(ESI):[M+H] + =479.25

[0226] 1H NMR: (400MHz, CDCl3), δppm: 9.56 (s, 1H), 7.98 (d, J = 8.40Hz, 1H), 7.77 (d, J = 8.80Hz 1H),7.51(s,1H),7.34(s,1H),6.77(s,1H),6.51(s,1H),4.51-4.45(m,2 H),4.32(t,J=9.60Hz,2H),4.19(s,1H),3.78(s,3H),3.76-3.73(m,2H),3 .54(t,J=9.60Hz,2H),3.40-3.34(m,1H),3.30-3.28(m,1H),2.50(s,3H), 1.60-1.52(m,4H),1.31-1.29(m,2H),1.10(s,2H),0.94(t,J=7.20Hz,3H)

[0227] Example 6 Preparation of (R)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylic acid (Compound A32)

[0228]

[0229] Synthesis route:

[0230]

[0231] Step 1:

[0232]

[0233] Add diethylzinc toluene solution (1M, 20.1mL) to dichloromethane (15.0mL), then cool to 0-10°C, add diiodomethane (10.8g, 40.2mmol) to the reaction system, and then stir at -5 to 5°C for 1 hour. Dissolve 1'-(tert-butyl)7-methyl(R)-4'-methylenespiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylate (1.50g, 4.02mmol) in dichloromethane (15.0mL) and then dropwise add to the reaction system. After the addition is complete, stir at 15-25°C for 24 hours. LCMS shows that the raw material is completely consumed. Add saturated ammonium chloride solution (100mL) to the reaction system, then collect the organic phase and concentrate under reduced pressure. The concentrated product was separated and purified by acidic reverse-phase high performance liquid chromatography (column specification: CD07-DaisogelSP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (R)-bipyridine[benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylic acid methyl ester (480 mg).

[0234] Step 2:

[0235]

[0236] The (R)-dipyridine [benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylic acid methyl ester (480 mg, 1.48 mmol) prepared in step 1 was dissolved in N, N-dimethylformamide (6.00 mL), potassium iodide (492 mg, 2.96 mmol), potassium carbonate (205 mg, 1.48 mmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (768 mg, 2.48 mmol) were added in sequence, and then reacted at 15-25° C. for 30 minutes. LCMS showed that the raw material was completely consumed. Ethyl acetate (3.00 mL) and saturated brine (3 mL) were added to the reaction system, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give methyl (R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylate (800 mg).

[0237] Step 3:

[0238]

[0239] The methyl (R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylate (250 mg, 1.43 mmol) prepared in step 2 was dissolved in a mixed solution of methanol (6.00 mL), tetrahydrofuran (4.00 mL) and water (1.00 mL), and then lithium hydroxide monohydrate (599 mg, 14.3 mmol) was added, and the temperature was raised to 60-70°C for 6 hours. LCMS showed that the raw material was completely consumed. The reaction solution was adjusted to pH 6-7 with 1M dilute hydrochloric acid and then concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (R)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)dipyrrole[benzopyran-4,2-piperidine-4,1-cyclopropane]-7-carboxylic acid (314.15 mg), i.e., compound A32.

[0240] 1 H NMR: (400MHz, CDCl3), δppm 0.18 (br t, J = 6.63Hz, 2H), 0.37 (br d, J = 5.13Hz, 1H), 0.50 (br s, 1H), 0.74 (br d, J = 13.13Hz, 1H), 1.33 (br d,J=14.01Hz,1H),2.07-2.17(m,1H),2.33(br d,J=13.63Hz,1H),2.47(s,3H),2.51-2.62(m,2H),2.65-2.72(m,1H),2.80(br d,J=10.88Hz,1H),3.60(br d,J=12.63Hz,1H),3.74(br d,J=13.01Hz,1H),3.78(s,3H),4.18(br t,J=10.26Hz,1H),4.40-4.48(m,1H),6.70(s,1H),6.91(br s,1H),7.23(br s,1H),7.52(s,1H),7.67(br d,J=8.38Hz,1H),7.93(br s,1H),8.10(br d,J=8.13Hz,1H)

[0241] Example 7 Preparation of (4R)-4-ethyl-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A33)

[0242]

[0243] Synthesis route:

[0244]

[0245] Step 1:

[0246]

[0247] Ethyl triphenylphosphonium iodide (670 mg, 1.60 mmol) was dissolved in tetrahydrofuran (6 mL), and n-butyl lithium (2.50 M, 660 μL) was added at -70 to -60 ° C, and the reaction solution was stirred at -70 to -60 ° C for 30 minutes. (S)-7-bromo-4'-oxopyridine [benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (400 mg, 1.01 mmol) was added to the reaction solution, and the reaction was carried out at -70 to -60 ° C for 0.5 hours. LCMS monitored the reaction process. The reaction solution was quenched with saturated ammonium chloride solution (10 mL), then extracted with ethyl acetate (50.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was separated and purified by TLC to obtain (R)-7-bromo-4-ethylidenepyridine[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (255 mg, 624 μmol, yield 61.9%).

[0248] Step 2:

[0249]

[0250] The (R)-7-bromo-4-ethylidenepyridine[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (200 mg, 490 μmol) prepared in step 1 was dissolved in a mixed solution of anhydrous methanol (3.00 mL) and acetonitrile (6.00 mL), and bis(diphenylphosphino)ferrocenepalladium dichloride (40 mg, 50.0 μmol) and triethylamine ((200 mg, 1.92 mmol, 275 μL) were added. The reaction system was replaced with argon three times and then replaced with carbon monoxide three times. The system was reacted at carbon monoxide (3 MPa) at 120-130° C. for 10 hours. TLC showed that the raw material was completely consumed, and the reaction solution was concentrated to obtain a crude product. The (R)-4-ethylidene-7-methylspiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (170 mg, 438 μmol, yield 89.6%) was obtained by separation and purification using a thin layer chromatography plate.

[0251] Step 3:

[0252]

[0253] The (R)-4-ethylidene-7-methylspiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (150 mg, 387 μmol) prepared in step 2 was dissolved in ethyl acetate (5.00 mL) solution, palladium carbon (150 mg, 10% purity) was added, and the reaction system was replaced with argon 3 times and then replaced with hydrogen 3 times. The system was reacted for 2 hours at 10-20°C in hydrogen (15 Psi). LCMS showed that the raw material was completely consumed. The reaction solution was filtered, the filtrate was collected, and the filtrate was concentrated to obtain (4R)-4'-ethyl-7-methylspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (157 mg).

[0254] Step 4:

[0255]

[0256] The (4R)-4'-ethyl-7-methylspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (157 mg, 403 μmol) prepared in step 3 was dissolved in hydrochloric acid / ethyl acetate (2M, 4.00 mL) and reacted at 30-40°C for 0.5 hours. LCMS showed that the starting material was completely consumed. The reaction solution was concentrated to obtain (4R)-4'-ethylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (130 mg, HCl).

[0257] Step 5:

[0258]

[0259] The (4R)-4'-ethylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (130 mg, 399 μmol) prepared in step 4 was dissolved in N,N-dimethylformamide (3.00 mL), potassium iodide (70.0 mg, 422 μmol), potassium carbonate (200 mg, 1.45 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (200 mg, 645 μmol) were added in sequence and reacted at 10-20°C for 30 minutes. LCMS showed that the raw material was completely consumed. Water (10.0 mL) was added to the reaction system, extracted with ethyl acetate (10.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give methyl (4R)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethylspiro[benzopyran-4,2-piperidine]-7-carboxylate (150 mg).

[0260] Step 6:

[0261]

[0262] The methyl (4R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethyl spiro[benzopyran-4,2-piperidine]-7-carboxylate (150.0 mg, 166 μmol) prepared in step 5 was dissolved in methanol (4.00 mL) and water (2.00 mL), and lithium hydroxide monohydrate (120 mg, 2.86 mmol) was added. The reaction solution was reacted at 50-60° C. for 5 hours. LCMS showed that the raw material was completely consumed. The crude product was obtained by concentration under reduced pressure. The product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10min), to obtain (4R)-4-ethyl-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (78.0 mg, yield 63.3%), i.e., compound A33.

[0263] LCMS(ESI):[M+H] + =449.24

[0264] 1 H NMR: (400MHz, CDCl3), δppm: 8.09 (d, J=8.00Hz, 1H), 7.95-7.90 (m, 1H), 7.68 (td, J=8.40Hz, 1.60Hz, 1H), 7.55 (d d,J=6.80Hz,1.60Hz,1H),7.24-7.20(m,1H),6.86-6.79(m,1H),6.70(d,J=8.80Hz,1H),4.42-4.24(m,2H),3.87 -3.82(m,0.5H),3.7(s,3H),3.72-3.67(m,0.5H),3.60(d,J=12.4Hz,1H),2.96-2.81(m,2H),2.66-2.57(m,1H), 2.46(s,3H),2.15-2.06(m,1H),2.16-1.96(m,1H),1.85-1.52(m,2H),1.45-1.16(m,4H),0.85(t,J=7.20Hz,3H).

[0265] Example 8

[0266] Preparation of (4R, 4'S)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (Compound A34-1)

[0267] Preparation of (4R, 4'R)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (Compound A34-2)

[0268]

[0269] Synthesis route:

[0270]

[0271] Step 1:

[0272]

[0273] At 15-25°C, (4S, 4'S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (6.00g, 15.1mmol) was dissolved in dichloromethane (60.0mL), and then the temperature was controlled at 0-5°C, and Dess-Martin oxidant (16.0g, 37.7mmol) was added in batches, and then stirred at 15-25°C for 2 hours. LCMS showed that the raw material was completely consumed. The system was cooled to 0°C, and saturated sodium bicarbonate solution (100mL) and sodium thiosulfate (15.0g) were added to the reaction system respectively, stirred until the system was non-oxidizing, and then separated, and the organic phase was collected and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography gave (S)-7-bromo-4'-oxopyridine[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (5.53g).

[0274] Step 2:

[0275]

[0276] Slowly add anhydrous tetrahydrofuran (35.0 mL) to a mixture of potassium tert-butoxide (1.98 g, 17.7 mmol) and triphenylmethylphosphine iodide (5.36 g, 13.3 mmol) at -5 to 0 ° C. After the addition is complete, stir at -5 to 0 ° C for 1 hour. Add (S)-7-bromo-4'-oxopyridine [benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (3.50 g, 8.83 mmol) prepared in step 1 to the reaction system, then return to the temperature of 15-20 ° C and stir for 2 hours. LCMS shows that the raw material is completely consumed. Control the temperature at 0-10 ° C, add saturated ammonium chloride solution (20 mL) to the reaction system, then stand and separate, and concentrate the organic phase under reduced pressure to obtain a crude product. The residue was separated and purified by column chromatography to obtain (R)-tert-butyl 7-bromo-4'-methylenespiro[benzopyran-4,2'-piperidine]-1'-carboxylate (3.00 g).

[0277] Step 3:

[0278]

[0279] The (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (2.20 g, 5.58 mmol) prepared in step 2 was added to anhydrous methanol (10.0 mL) and acetonitrile (20.0 mL), and [1,1-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane (1.37 g, 1.67 mmol)) and triethylamine (1.69 g, 16.74 mmol) were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, and the system was reacted at carbon monoxide (3 MPa) at 125-135 ° C for 6 hours. LCMS showed that the raw material was completely consumed. The reaction solution was filtered and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to obtain 1-(tert-butyl) 7-methyl (R)-4-methylene spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (1.80 g).

[0280] Step 4:

[0281]

[0282] 1-(tert-butyl)7-methyl(R)-4-methylenespiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (200 mg, 536 μmol) was dissolved in tetrahydrofuran (8.00 mL), and then wet palladium carbon (10%, 40 mg, 38 μmol) was added, argon was replaced three times, hydrogen was replaced three times, and the system was reacted for 6 hours under hydrogen (50 Psi) at 45-50 ° C. LCMS showed that the raw material was completely consumed. The reaction solution was filtered and the filtrate was concentrated under reduced pressure. The compound 1-(tert-butyl)7-methyl(4R)-4-methylspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (210 mg) was obtained.

[0283] Step 5:

[0284]

[0285] The 1-(tert-butyl)7-methyl (4R)-4-methylspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (200 mg, 533 μmol) prepared in step 4 was dissolved in hydrochloric acid / ethyl acetate (2M, 10.0 mL) and reacted at 15-25° C. for 12 hours. LCMS showed that the starting material was completely consumed. The product was concentrated under reduced pressure, and the concentrated product was extracted with ethyl acetate (10.0 mL) and saturated sodium bicarbonate solution (5.00 mL). The organic phase was collected and concentrated under reduced pressure to obtain (4R)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (150 mg).

[0286] Step 6:

[0287]

[0288] The (4R)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (150 mg, 545 μmol) prepared in step 5 was dissolved in N,N-dimethylformamide (5.00 mL), potassium iodide (181 mg, 1.09 mmol), potassium carbonate (75.3 mg, 545 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (282 mg, 911 μmol) were added in sequence, and then reacted at 15-25° C. for 30 minutes. LCMS showed that the starting material was completely consumed. Ethyl acetate (3.00 mL) and saturated brine (3.00 mL) were added to the reaction system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give methyl (4R)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-methylspiro[benzopyran-4,2-piperidine]-7-carboxylate (307 mg).

[0289] Step 7:

[0290]

[0291] The methyl (4R)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-methylspiro[benzopyran-4,2-piperidine]-7-carboxylate (300 mg, 547 μmol) prepared in step 6 was dissolved in methanol (3.00 mL), and then an aqueous lithium hydroxide solution (4.0 M, 1.37 mL) was added, and the temperature was raised to 60-70°C for 6 hours. LCMS showed that the raw material was completely consumed. The temperature was lowered to 15-20°C, and the pH of the reaction system was adjusted to 7-8 with 1 M dilute hydrochloric acid, and then concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specifications: CD07-Daisogel SP-100-8-ODS-PK150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10min; carbon monoxide), to obtain (4R, 4S)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid LB2138-1 (27.66 mg) (compound 34-1), and (4R, 4'R)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-methylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid LB2138-2 (34.42 mg) (compound 34-2).

[0292] 1 H NMR: (400MHz, CDCl3), δppm 0.94(d,J=6.63Hz,3H)1.05-1.17(m,1H)1.51(brdd,J=13.07,7.82Hz,1H)1.65-1. 75(m,1H)1.85-2.02(m,3H)2.42(s,3H)2.64-2.75(m,2H)2.76-2.84(m,1H)3.40(br d,J=12.63Hz,1H)3.63-3.72(m,4H)4.27-4.41(m,2H)6.58-6.63(m,1H)6.66(s,1 H)7.25-7.30(m,2H)7.49(dd,J=8.13,1.63Hz,1H)7.86(d,J=8.13Hz,1H)10.81(br s,1H)12.30-13.49(m,1H).

[0293] 1H NMR: (400MHz, CDCl3), δppm 0.83 (br d, J = 6.25Hz, 3H) 0.96-1.10 (m, 1H) 1.30 (br t, J = 12.76Hz, 1H) 1.49 (br d, J = 11.88Hz, 1H) 1.67-1.80 (m, 1H) 1.91 (br d,J=13.13Hz,1H)2.10(br d,J=14.13Hz,1H)2.35-2.47(m,5H)2.57(br d,J=11.63Hz,1H)3.34(br s,1H)3.54(br d,J=12.26Hz,1H)3.68(s,3H)4.14-4.38(m,2H)6.60(br s,1H)6.64(s,1H)7.27(s,2H)7.51(d,J=8.13Hz,1H)7.95(d,J=8.13Hz,1H)10.80(br s,1H)12.00-13.40(m,1H).

[0294] Example 9 Preparation of (4S, 4'S)-4-(cyclopropylmethoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A36)

[0295]

[0296] Synthesis route:

[0297]

[0298] Step 1:

[0299]

[0300] Sodium hydrogen (30.6 mg, 768 μmol) was dissolved in N-methylpyrrolidone (6.00 mL), and the temperature was controlled at 0-10 ° C. (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (0.33 g, 829 μmol) was dissolved in N-methylpyrrolidone (2.00 mL) and added dropwise to the reaction system, and then stirred at 15-25 ° C for 1 hour, followed by the addition of iodomethylcyclopropane (483 mg, 2.65 mmol), and stirred at 15-25 ° C for 7 hours. LCMS showed that the raw material was completely consumed. The system was cooled to 0 ° C, quenched with saturated aqueous ammonium chloride solution (6.00 mL), diluted with saturated brine (5.00 mL), extracted with ethyl acetate (5.00 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to give (4S)-7-bromo-4'-(cyclopropylmethoxy)spiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (313 mg).

[0301] Step 2:

[0302]

[0303] The (4S)-7-bromo-4'-(cyclopropylmethoxy)spiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (300 mg, 663 μmol) prepared in step 1 was added to anhydrous methanol (4.00 mL) and acetonitrile (6.00 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (108 mg, 133 μmol)) and triethylamine (201 mg, 1.99 mmol) were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, and the system was reacted at carbon monoxide (3 MPa) at 120-130°C for 30 hours. TLC showed that the raw materials were completely consumed. The reaction solution was filtered and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give 1-(tert-butyl)7-methyl(4S)-4-(cyclopropylmethoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (250 mg).

[0304] Step 3:

[0305]

[0306] The 1-(tert-butyl)7-methyl(4S)-4-(cyclopropylmethoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (250 mg, 579 μmol) prepared in step 2 was dissolved in hydrochloric acid / ethyl acetate (2M, 25.0 mL) and reacted at 15-25° C. for 2 hours and 10 minutes. LCMS showed that the raw material was completely consumed. The mixture was concentrated under reduced pressure, and the concentrate was extracted with ethyl acetate (10 mL) and saturated sodium bicarbonate solution (5 mL). The organic phase was collected and concentrated under reduced pressure to give (4S)-4-(cyclopropylmethoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (200 mg).

[0307] Step 4:

[0308]

[0309] The (4S)-4-(cyclopropylmethoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (200 mg, 603 μmol) prepared in step 3 was dissolved in N,N-dimethylformamide (4.00 mL), potassium iodide (200 mg, 1.21 mmol), potassium carbonate (83 mg, 603 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (280 mg, 905 μmol) were added in sequence, and then reacted at 15-25° C. for 30 minutes. LCMS showed that the starting material was completely consumed. Ethyl acetate (3.00 mL) and saturated brine (3 mL) were added to the reaction system, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to give methyl (4S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(cyclopropylmethoxy)spiro[4,2-piperidine]-7-carboxylate (260 mg).

[0310] Step 5:

[0311]

[0312] The methyl (4S)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(cyclopropylmethoxy)spiro[4,2-piperidine]-7-carboxylate (250 mg, 413 μmol) prepared in step 4 was dissolved in methanol (5.00 mL), and then lithium hydroxide aqueous solution (4.0 M, 1.03 mL, 4.13 mmol) was added, and the temperature was raised to 60-70° C. for reaction for 30 minutes. LCMS showed that the starting material was completely consumed. The reaction solution was filtered, the filtrate was concentrated, and the concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4'S)-4-(cyclopropylmethoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (142.84 mg), i.e., compound A36.

[0313] 1 H NMR: (400MHz, CDCl3), δppm 0.12-0.22(m,2H)0.48-0.55(m,2H)0.96-1.06(m,1H)1.76(br d,J=12.88Hz,1H)1.92(br d,J=1.13Hz,2H)2.34(br d,J=14.38Hz,1H)2.44(s,3H)2.61(br t,J=10.38Hz,1H)2.76-2.95(m,3H)3.16-3.28(m,2H)3.60-3.81(m,6H)4.25-4.33(m,1H)4.38-4.46(m,1H)6.65(s,1H)6.72(br s,1H)7.16(br s,1H)7.51(s,1H)7.58(br s,1H)8.11(br d,J=7.63Hz,1H)8.20(br s,1H).

[0314] Example 10 Preparation of (4S, 4'S)-4-(cyclobutylmethoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A37)

[0315]

[0316]

[0317] Step 1:

[0318]

[0319] At 15-25°C, sodium hydrogen (66.3 mg, 1.66 mmol) was dissolved in N-methylpyrrolidone (5.00 mL). The temperature was controlled at 0-10°C, (4S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (300 mg, 753 μmol) was dissolved in N-methylpyrrolidone (2.00 mL) and then added dropwise to the reaction system, and then stirred at 15-25°C for 1 hour, followed by the addition of 4-methylbenzenesulfonic acid cyclobutyl methyl ester (453 mg, 1.88 mmol), and stirred at 45-55°C for 6 hours. LCMS showed that the raw material was completely consumed. The system was cooled to 0°C, quenched with saturated aqueous ammonium chloride solution (5.00 mL), diluted with saturated brine (6.00 mL), extracted with ethyl acetate (10.0 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give (4S)-7-bromo-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (150 mg).

[0320] Step 2:

[0321]

[0322] The (4S)-7-bromo-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (150 mg, 322 μmol) prepared in step 1 was added to anhydrous methanol (4.00 mL) and acetonitrile (8.00 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (26.3 mg, 32.2 μmol)) and triethylamine (97.6 mg, 965 μmol) were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, and the system was reacted at carbon monoxide (3 MPa) at 120-130°C for 6 hours. TLC showed that the raw materials were completely consumed. The reaction solution was filtered and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give 1-(tert-butyl)7-methyl(4S)-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (140 mg).

[0323] Step 3:

[0324]

[0325] The 1-(tert-butyl)7-methyl (4S)-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (140 mg, 314 μmol) prepared in step 2 was dissolved in hydrochloric acid / ethyl acetate (2M, 14.0 mL) and reacted at 15-25° C. for 2 hours. LCMS showed that the starting material was completely consumed. The mixture was concentrated under reduced pressure, and the concentrated product was extracted with ethyl acetate (10 mL) and saturated sodium bicarbonate solution (5 mL). The organic phase was collected and concentrated under reduced pressure to obtain (4S)-4'-(cyclobutylmethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (100 mg).

[0326] Step 4:

[0327]

[0328] The (4S)-4'-(cyclobutylmethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (100 mg, 290 μmol) prepared in step 3 was dissolved in N,N-dimethylformamide (4.00 mL), potassium iodide (96.1 mg, 579 μmol), potassium carbonate (40.0 mg, 290 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (280 mg, 905 μmol) were added in sequence, and then reacted at 15-25° C. for 30 minutes. LCMS showed that the starting material was completely consumed. Ethyl acetate (10.00 mL) and saturated brine (6 mL) were added to the reaction mixture, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified using a thin layer chromatography plate to give methyl (4S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylate (150 mg).

[0329] Step 5:

[0330]

[0331] Methyl (4S)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(cyclobutylmethoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylate (150 mg, 242 μmol) was dissolved in methanol (2.00 mL), and then lithium hydroxide aqueous solution (4.0 M, 606 μL) was added, and the temperature was raised to 60-70° C. for 6 hours. LCMS showed that the starting material was completely consumed. The temperature was lowered to 15-20° C., the pH of the reaction system was adjusted to 7-8 with 1 M dilute hydrochloric acid, and then concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4'S)-4-(cyclobutylmethoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (73.66 mg), i.e., compound A37.

[0332] 1 H NMR: (400MHz, CDCl3), δppm 1.72-2.05(m,9H)2.34-2.59(m,6H)2.77-2.91(m,3H)3.26-3.38(m,2H)3.63(br s,2H)3.75(s,4H)4.28-4.37(m,1H)4.39-4.45(m,1H)6.66(s,1H)6.77(br s,1H)7.18(br s,1H)7.48-7.65(m,2H)8.07-8.26(m,2H).

[0333] Example 11 Preparation of (4S, 4'S)-4-(3,3-difluorocyclobutyl)methoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A38)

[0334]

[0335] Synthesis route:

[0336]

[0337] Step 1:

[0338]

[0339] The compound (3,3-difluorocyclobutyl)methanol (3.00 g, 24.6 mmol) was dissolved in dichloromethane (30.0 mL), and methanesulfonic anhydride (8.56 g, 49.1 mmol) and triethylamine (8.56 g, 49.1 mmol) were added at 15-25°C. The reaction solution was stirred at 15-25°C for 1 hour, and the product formation was monitored by LCMS. The reaction solution was washed twice with hydrochloric acid solution (0.2 M, 30 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. (3,3-difluorocyclobutyl)methanesulfonic acid methyl ester (4.20 g, 20.9 mmol, yield 85.4%) was obtained.

[0340] Step 2:

[0341]

[0342] Dissolve (4S, 4'S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (370 mg, 929 μmol) in anhydrous N-methylpyrrolidone (5 mL), add sodium hydride (185 mg, 4.63 mmol, 60% purity) at 0-10 ° C, and stir the reaction solution at 0-10 ° C for 30 minutes. Add (3,3-difluorocyclobutyl) methanesulfonic acid methyl ester (370 mg, 1.85 mmol) prepared in step 1 to the reaction solution and heat to 15-25 ° C and react for 2 hours. LCMS monitoring found that the product was generated. The reaction solution was quenched with saturated ammonium chloride solution (20.0 mL), then extracted with ethyl acetate (10.0 mL*3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give (4S,4'S)-7-bromo-4-(3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (140 mg, 278 μmol, yield 30.0%).

[0343] Step 3:

[0344]

[0345] The (4S, 4'S)-7-bromo-4-(3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (140 mg, 279 μmol) prepared in step 2 was dissolved in tetrahydrofuran (4.00 mL), nitrogen was replaced 3 times, n-butyl lithium (2.50 M, 167 μL) was added at -70 to -78 ° C, and then carbon dioxide was introduced. After the completion of the introduction, the reaction was carried out at -70 to -78 ° C for 0.5 hours. LCMS showed that the raw material was completely consumed. Saturated ammonium chloride (10.0 mL) was added to the reaction solution for quenching, ethyl acetate was added for extraction (10.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give (4S,4'S)-1-(tert-butyloxycarbonyl)-4-(3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (50 mg, 107 μmol, yield 38.4%).

[0346] Step 4:

[0347]

[0348] The (4S, 4'S)-1-(tert-butyloxycarbonyl)-4-(3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (50.0 mg, 107 μmol) prepared in step 3 was dissolved in dichloromethane (2 mL) and methanol (1 mL), and then (trimethylsilyl)diazomethane (24.4 mg, 214 μmol) was added to react at 15-25°C for 0.5 hours. LCMS showed that the raw material was completely consumed. After adding glacial acetic acid (0.10 mL) to the reaction solution for quenching, saturated sodium bicarbonate (10.0 mL) was added, and ethyl acetate was added to the reaction solution for extraction (10.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 1-(tert-Butyl)7-methyl(4S,4'S)-4((3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (21.0 mg, 43.6 μmol, yield 40.8%) was obtained.

[0349] Step 5:

[0350]

[0351] The 1-(tert-butyl)7-methyl(4S,4'S)-4((3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (20.0 mg, 41.5 μmol) prepared in step 4 was dissolved in hydrochloric acid / ethyl acetate (2M, 2.00 mL) and reacted at 15-25° C. for 6 hours. LCMS showed that the starting material was completely consumed. The reaction solution was directly concentrated to obtain (4S,4'S)-4((3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (20.0 mg).

[0352] Step 6:

[0353]

[0354] The (4S, 4'S)-4((3,3-difluorocyclobutyl)methoxy)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (20.0 mg, 47.9 μmol) prepared in step 5 was dissolved in N,N-dimethylformamide (2.00 mL), potassium iodide (8.00 mg, 48.2 μmol), potassium carbonate (20.0 mg, 144 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (30.0 mg, 96.8 μmol) were added in sequence and reacted at 15-25°C for 30 minutes. LCMS showed that the starting material was completely consumed. Water (10.0 mL) was added to the reaction mass, extracted with ethyl acetate (10.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give methyl (4S,4'S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(3,3-difluorocyclobutyl)methoxy)spiro[4,2-piperidine]-7-carboxylate (35.0 mg).

[0355] Step 7:

[0356]

[0357] The methyl (4S, 4'S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-(3,3-difluorocyclobutyl)methoxy)spiro[4,2-piperidine]-7-carboxylate (35.0 mg, 53.5 μmol, 1.00 eq) prepared in step 6 was dissolved in methanol (2.00 mL) and water (0.50 mL), and lithium hydroxide monohydrate (23.3 mg, 556 μmol) was added. The reaction solution was reacted at 50-60° C. for 5 hours. LCMS showed that the raw material was completely consumed. Concentration under reduced pressure gave a crude product. Neutral reverse phase high performance liquid chromatography was used for separation and purification (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 mins) to obtain (4S, 4'S)-4-(3,3-difluorocyclobutyl)methoxy)-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (15.2 mg, yield 50.2%), i.e., compound A38.

[0358] 1 H NMR: (400MHz, CDCl3), δppm: 10.82 (br s,1H),7.97(d,J=8.00Hz,1H),7.50(d,J=7.60Hz,1H),7.27(d,J=6.40Hz,2H),6 .64(d,J=12.4Hz,2H),4.42-4.34(m,1H),4.28(t,J=11.6Hz,1H),3.69(s,3H),3. 67-3.51(m,3H),3.46-3.41(m,2H),2.70-2.55(m,4H),2.47-2.44(m,1H),2.42(s ,3H),2.39-2.27(m,4H),1.69(d,J=14.0Hz,2H),1.62-1.54(m,1H),1.23(s,1H).

[0359] Example 12 Preparation of (4S, 4-cyclopropyloxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A39)

[0360]

[0361] Synthesis route:

[0362]

[0363] Step 1:

[0364]

[0365] Dissolve (4S, 4'S)-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (400 mg, 1.00 mmol) in vinyl n-butyl ether (4.00 mL), then add 1,10-phenanthroline (54.3 mg, 301 μmol), palladium acetate (67.6 mg, 301 μmol), triethylamine (203 mg, 2.01 mmol) to the above solution, and react at 85-95°C for 6 hours. LCMS monitors the reaction process. The mixture was diluted with water (20 mL) at room temperature and extracted with dichloromethane (50 mL*2). The organic phase was washed with a saturated aqueous sodium bicarbonate solution and dried over anhydrous sodium sulfate. The mixture was concentrated to give (4S, 4'S)-7-bromo-4-(vinyloxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (291 mg, yield 68.3%).

[0366] Step 2:

[0367]

[0368] Diiodomethane (720 mg, 2.69 mmol) was dissolved in dichloromethane (3.00 mL), and then diethylzinc / n-hexane solution (1.00 M, 2.69 mL) was dissolved in dichloromethane (3.00 mL), and stirred at 0-5° C. for 1 hour; then (4S, 4'S)-7-bromo-4-(vinyloxy)spiro[benzopyran-4,2-piperidine]-1-carboxylic acid tert-butyl ester (380 mg, 895 μmol) prepared in step 1 and dissolved in dichloromethane (3.00 mL) was added to the above reaction solution at 0-5° C., and then stirred at 20-25° C. for 1 hour. LCMS monitoring showed that the raw material was completely consumed and the product was generated. At room temperature, the reaction solution was quenched with saturated aqueous ammonium chloride solution (50 mL), extracted with ethyl acetate (50 mL*2), and the organic phase was dried over anhydrous sodium sulfate and concentrated to give (4S, 4'S)-7-bromo-4'-cyclopropyloxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (240 mg).

[0369] Step 3:

[0370]

[0371] The (4S, 4'S)-7-bromo-4'-cyclopropyloxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (200 mg, 456 mmol) prepared in step 2 was dissolved in acetonitrile (5.00 mL) and methanol (5.00 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (37.3 mg, 45.6 μmol) and triethylamine (2.28 mmol, 317 μL) were added to the reaction solution. After replacing nitrogen three times, the temperature was adjusted to 125 ° C under carbon monoxide (2.00 MPa) and reacted for 36 hours. LCMS monitoring found that the product was generated and the raw materials were completely consumed. The reaction solution was concentrated under reduced pressure. 1-(tert-butyl) 7-methyl (4S, 4'S)-4-cyclopropyloxyspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (60 mg) was obtained.

[0372] Step 4:

[0373]

[0374] The 1-(tert-butyl)7-methyl(4S,4'S)-4-cyclopropyloxyspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (60.0 mg, 143 μmol) prepared in step 3 was dissolved in ethyl acetate (2 mL), and dioxane hydrochloride (2.00 M, 3.00 mL) was added to the reaction solution, and the reaction was carried out at 20-25° C. for 1 hour. LCMS monitored the reaction process. The reaction solution was concentrated under reduced pressure to obtain compound (4S,4'S)-4'-cyclopropyloxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (40.0 mg).

[0375] Step 5:

[0376]

[0377] The (4S, 4'S)-4'-cyclopropyloxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (40.0 mg, 126 μmol) prepared in step 4 and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (52.6 mg, 190 μmol) were dissolved in N, N-dimethylformamide (4.00 mL), potassium iodide (20.9 mg, 126 μmol) and potassium carbonate (34.8 mg, 252 μmol) were added, and the reaction was reacted at 25° C. for 0.5 hours. LCMS monitoring showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to obtain methyl (4S,4'S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-cyclopropoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (50.0 mg).

[0378] Step 6:

[0379]

[0380] The methyl (4S, 4'S)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-cyclopropyloxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (50.0 mg, 84.6 μmol) prepared in step 5 was dissolved in methanol (2.00 mL) and water (2.00 mL), and lithium hydroxide (10.1 mg, 423 μmol) was added to the above reaction solution, and the reaction was carried out at 60° C. for 2 hours. LCMS showed that the raw material was completely consumed and the product was generated. Concentration under reduced pressure gave a crude product. Neutral reverse phase high performance liquid chromatography was used for separation and purification (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4-cyclopropyloxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (14.0 mg, yield 31.4%), i.e., compound A39.

[0381] 1 H NMR (400 MHz, METHANOL-d 4 )δppm 0.35-0.43(m,2H)0.46(br d,J=3.44Hz,2H)1.13-1.27(m,2H)1.93(s,3H)2.38(br s,3H)2.41-2.50(m,1H)2.59-2.71(m,1H)2.97(brd,J=12.76Hz,1H)3.25(s,2H)3.60-3.68(m,3H) 3.74-3.82(m,1H)3.82-3.90(m,1H)3.96-4.07(m,1H)4.10-4.21(m,1H)4.27-4.38(m,1H)6.34(br s,1H)6.58-6.67(m,1H)7.19(brd,J=3.03Hz,1H)7.36-7.44(m,1H)7.60(br s,2H).

[0382] Example 13 Preparation of (4R, 4'S)-4'-cyclopropyl-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (Compound A41)

[0383]

[0384] Synthesis route:

[0385]

[0386] Step 1:

[0387]

[0388] At 15-25°C, (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (1.40 g, 3.55 mmol) was added to a hydrochloric acid ethyl acetate solution (14 mL) and stirred for 1 hour. LCMS showed that the starting material was completely consumed. The system was concentrated under reduced pressure until there was no obvious fraction, and then extracted with saturated sodium bicarbonate solution (20 mL) and ethyl acetate (20 mL). The organic phase was collected and then concentrated under reduced pressure to obtain (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine] (1.00 g).

[0389] Step 2:

[0390]

[0391] The (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine] (1.00 g, 3.40 mmol) prepared in step 1 and sodium bicarbonate (1.14 g, 13.6 mmol) were added to tetrahydrofuran (10.0 mL) and water (1.00 mL), and then benzyl chloroformate (1.74 g, 10.2 mmol) was slowly added dropwise to the reaction system at 15-25°C, and stirred at 15-25°C for 2 hours after the addition was complete. LCMS showed that the raw material was completely consumed. The reaction solution was concentrated under reduced pressure, and the concentrated product was separated and purified by column chromatography to obtain benzyl (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine]-1'-carboxylate (1.07 g).

[0392] Step 3:

[0393]

[0394] Benzyl (R)-7-bromo-4'-methylene spiro[benzopyran-4,2'-piperidine]-1'-carboxylate (800 mg, 1.87 mmol) prepared in step 2 was dissolved in tetrahydrofuran (40.0 mL), and borane tetrahydrofuran complex (1 M, 2.80 mL) was added dropwise to the reaction system at 0-10°C, and then stirred at 0-10°C for 1 hour. Then, sodium hydroxide aqueous solution (2 M, 1.87 mL) and hydrogen peroxide aqueous solution (718 μL, 30%) were added to the reaction system at 0-10°C, and then stirred at 0-10°C for 1 hour. LCMS showed that the starting material was completely consumed. A saturated sodium sulfite solution (15.0 mL) was added to the reaction system, followed by stirring for 20 minutes, followed by standing for separation, collecting the organic phase, and concentrating the organic phase under reduced pressure. The concentrated product was separated and purified by column chromatography to obtain benzyl (4R)-7-bromo-4-(hydroxymethyl)spiro[benzopyran-4,2-piperidine]-1-carboxylate (670 mg).

[0395] Step 4:

[0396]

[0397] The benzyl (4R)-7-bromo-4-(hydroxymethyl)spiro[benzopyran-4,2-piperidine]-1-carboxylate (600 mg, 1.34 mmol) prepared in step 3 was dissolved in dichloromethane (10.0 mL), the temperature was controlled at 0-10°C, Dess-Martin oxidant (684 mg, 1.61 mmol), and then reacted at 0-10°C for 2 hours. LCMS showed that the raw material was completely consumed. Saturated aqueous sodium bicarbonate solution (10.0 mL) and saturated aqueous sulfurous acid solution (10.0 mL) were added to the reaction system, the organic phase was collected and concentrated under reduced pressure, and the concentrated product was separated and purified by thin layer chromatography to obtain benzyl (4R)-7-bromo-4'-formylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (507 mg).

[0398] Step 5:

[0399]

[0400] Anhydrous tetrahydrofuran (10.0 mL) was slowly added dropwise to a mixture of potassium tert-butoxide (253 mg, 2.25 mmol) and triphenylmethylphosphine iodide (682 mg, 1.69 mmol) at -0-5°C. After the addition was complete, the mixture was stirred at 0-5°C for 1 hour. Benzyl (4R)-7-bromo-4'-formylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (500 mg, 1.13 mmol) prepared in step 4 was added to the reaction system, and then the mixture was warmed to 15-20°C and stirred for 2 hours. LCMS showed that the starting material was completely consumed. The temperature was controlled at 0-10°C, saturated ammonium chloride solution (10 mL) was added to the reaction system, and then the mixture was allowed to stand for separation. The organic phase was concentrated under reduced pressure, and the concentrated product was separated and purified by column chromatography to obtain benzyl (4R)-7-bromo-4'-vinylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (417 mg).

[0401] Step 6:

[0402]

[0403] A toluene solution of diethylzinc (1M, 3.39 mL) was added to dichloromethane (3.00 mL), and then the temperature was lowered to 0-10°C. Diiodomethane (1.82 g, 6.78 mmol) was added to the reaction system, and then stirred at 0-10°C for 0.5 hours. Benzyl (4R)-7-bromo-4'-vinyl spiro[benzopyran-4,2'-piperidine]-1'-carboxylate (300 mg, 678 μmol) prepared in step 5 was dissolved in dichloromethane (3.00 mL), and then added dropwise to the reaction system. After the addition was complete, the mixture was stirred at 15-25°C for 12 hours. LCMS showed that the raw material was completely consumed. Saturated ammonium chloride solution (4.00 mL) was added to the reaction system, and the organic phase was collected and concentrated under reduced pressure. The concentrated product was separated and purified by acidic reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain benzyl (4R)-7-bromo-4'-cyclopropylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (120 mg).

[0404] Step 7:

[0405]

[0406] The benzyl (4R)-7-bromo-4'-cyclopropyl spiro[benzopyran-4,2'-piperidine]-1'-carboxylate (70 mg, 153 μmol) prepared in step 6 was added to anhydrous methanol (5.00 mL) and acetonitrile (8.00 mL), and [1,1-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane (12.5 mg, 15.3 μmol) and triethylamine (46.6 mg, 460 μmol) were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, and the system was reacted at carbon monoxide (3 MPa) at 120-130°C for 36 hours. TLC showed that the raw material was completely consumed. The reaction solution was filtered and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to obtain 1-benzyl 7-methyl (4R)-4-cyclopropyl spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylate (70 mg).

[0407] Step 8:

[0408]

[0409] The 1-benzyl 7-methyl (4R)-4-cyclopropyl spiro[benzopyran-4,2-piperidine]-1,7-dicarboxylate (70 mg, 160 μmol) prepared in step 8 was added to tetrahydrofuran (2.00 mL), wet palladium carbon (70 mg, 65.8 μmol) was added, argon was replaced three times, hydrogen was replaced three times, and the system was reacted under hydrogen (15 Psi) at 20-25 ° C for 2 hours. LCMS showed that the raw material was completely consumed. The reaction solution was filtered and concentrated under reduced pressure to obtain (4R)-4'-cyclopropyl spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (35 mg).

[0410] Step 9:

[0411]

[0412] The (4R)-4'-cyclopropylspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (35 mg, 116 μmol) prepared in step 8 was dissolved in N,N-dimethylformamide (3.00 mL), potassium iodide (38.6 mg, 232 μmol), potassium carbonate (16.0 mg, 116 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (60.2 mg, 194 μmol) were added in sequence, and then reacted at 15-25° C. for 30 minutes. LCMS showed that the starting material was completely consumed. Ethyl acetate (15.0 mL) and saturated brine (6.00 mL) were added to the reaction system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to give methyl (4R)-1'-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-cyclopropylspiro[benzopyran-4,2'-piperidine]-7-carboxylate (45 mg).

[0413] Step 10:

[0414]

[0415] The methyl (4R)-1'-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-cyclopropylspiro[benzopyran-4,2'-piperidine]-7-carboxylate (45 mg, 78.3 μmol) prepared in step 9 was dissolved in a mixed solution of methanol (1.00 mL), tetrahydrofuran (1.0 0 mL) and water (0.20 mL), and then an aqueous solution of lithium hydroxide (32.9 mg, 783 μmol) was added, and the temperature was raised to 55-65° C. for reaction for 3 hours. LCMS showed that the starting material was completely consumed. The temperature was lowered to 15-20°C, and the pH of the reaction system was adjusted to 6-7 with 1 M dilute hydrochloric acid, followed by concentration under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 mins) to obtain (4R, 4'S)-4'-cyclopropyl-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (24.79 mg), i.e., compound A41.

[0416] 1H NMR: (400MHz, CDCl3), δppm 0.11-0.21(m,2H)0.39-0.48(m,2H)0.50-0.61(m,1H)1.12-1.25(m,1H)1 .64-1.77(m,1H)1.89-2.07(m,2H)2.28-2.48(m,2H)2.51(s,3H)2.77-2. 89(m,1H)3.36-3.53(m,2H)3.77(s,3H)4.15-4.45(m,4H)6.39(d,J=3.00 Hz,1H)6.77(s,1H)7.31(d,J=3.25Hz,1H)7.54(s,1H)7.72-7.79(m,2H).

[0417] Embodiment 14

[0418] Preparation of (4R, 4'R)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid (Compound A54-1) Preparation of (4R, 4'S)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid (Compound A54-2) Preparation of (4S, 4'R)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid (Compound A54-3) Preparation of (4S, 4'S)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid compound (A54-4)

[0419]

[0420] Synthesis route:

[0421]

[0422] Step 1:

[0423]

[0424] 1-(tert-butyl)7-methyl4-ethoxyspiro[isochromane-4,2-piperidine]-1,7-dicarboxylic acid (10.0 mg, 24.6 μmol) was dissolved in ethyl acetate (1.00 mL), and then hydrochloric acid / ethyl acetate (2M, 123 μL) was added to the system at 25°C, and the reaction solution was heated to 50°C for 4 hours. TLC showed that the raw material was completely consumed. Concentrated under reduced pressure to obtain 4'-ethoxyspiro[isochromane-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (8.43 mg).

[0425] LCMS (ESI, m / z): [M+H] + =306.0

[0426] Step 2:

[0427]

[0428] The 4'-ethoxyspiro[isochromane-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (8.40 mg, 24.5 μmol) prepared in step 1 was dissolved in N,N-dimethylformamide (1.00 mL), and potassium iodide (4.08 mg, 24.5 μmol), potassium carbonate (10.1 mg, 73.7 μmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (15.2 mg, 49.1 μmol) were added in sequence and reacted at 25 °C for 12 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (20.0 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified using a thin layer chromatography plate to obtain 1'-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[isochromane-4,2-piperidine]-7-carboxylic acid methyl ester (14.0 mg).

[0429] LCMS (ESI, m / z): [M+H] + =579.3

[0430] Step 3:

[0431]

[0432] The 1'-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[isochromane-4,2-piperidine]-7-carboxylic acid methyl ester (14.0 mg, 14.5 μmol) prepared in step 2 was dissolved in methanol (2.50 mL) and water (0.50 mL), and lithium hydroxide monohydrate (6.09 mg, 145 μmol) was added at 25°C. The reaction solution was heated to 60°C and reacted for 12 hours. The mixture was concentrated under reduced pressure, diluted with water (5.00 mL), and then the pH of the system was adjusted to 7 with hydrochloric acid (1 M), extracted with ethyl acetate (10.0 mL*6), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse-phase high performance liquid chromatography (column specifications: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain four configuration compounds.

[0433] Configuration 1:

[0434] (4R, 4'R)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid, ie, compound A54-1.

[0435] LCMS (ESI, m / z): [M+H] + =465.2

[0436] 1 H NMR: (400MHz, CDCl 3 )δ8.64-8.35(m,1H),8.30–8.19(m,1H),7.94-7.75(m,1H),7.66(s,1H),7.07(s,1H),6.59(s, 1H),6.59(s,1H),6.37-6.35(m,1H),4.97-4.87(m,1H),4.85-4.75(m,1H),4.75-4.66(m,1H), 4.12-3.96(m,2H),3.90-3.79(m,1H),3.65(s,4H),3.49-3.38(m,2H),3.07-2.96(m,2H),2.41 (s,3H),2.32-2.20(m,1H),2.12-2.01(m,2H),1.77(d,J=14.0Hz,1H),1.22(t,J=7.00Hz,3H).

[0437] Configuration 2:

[0438] (4R, 4'S)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid, ie, compound 54-2.

[0439] LCMS (ESI, m / z): [M+H] + =465.2

[0440] 1 H NMR: (400MHz, CDCl 3 )δ8.18-8.09(m,1H),8.07–8.01(m,1H),8.00-7.94(m,1H),7.75(s,1H),6.72-6.63(m,2H),4.90-4.81(m, 1H),4.80-4.70(m,1H),4.54(d,J=12.2Hz,1H),3.98(d,J=10.2Hz,1H),3.87-3.79(m,1H),3.77-3.70(m,3 H),3.65(d,J=12.8Hz,1H),3.54-3.44(m,3H),2.91(d,J=12.4Hz,1H),2.63-2.56(m,1H),2.43(s,3H),2.3 1-2.26(m,1H),1.95(d,J=9.76Hz,1H),1.69(t,J=12.2Hz,1H),1.61-1.47(m,1H),1.16(t,J=6.94Hz,3H).

[0441] Configuration three:

[0442] (4S, 4'R)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid, ie, compound A54-3.

[0443] LCMS (ESI, m / z): [M+H] + =465.2

[0444] 1 H NMR: (400MHz, CDCl 3)δ8.39-8.12(m,2H),8.02-7.79(m,1H),7.68(s,1H),7.12(s,1H),6.63(s,1H),6.54-6.39(m,1 H),4.90-4.79(m,2H),4.77-4.70(m,1H),4.16-4.13(m,1H),4.07-3.95(m,1H),3.87-3.77(m,1H ),3.74-3.62(m,4H),3.56-3.48(m,1H),3.45-3.38(m,1H),3.04-2.98(m,1H),2.92-2.86(m,1H ), 2.42 (s, 3H), 2.23-2.10 (m, 2H), 2.07-1.90 (m, 1H), 1.85-1.71 (m, 1H), 1.23 (t, J = 6.94Hz, 3H).

[0445] Configuration 4:

[0446] (4S, 4'S)-4'-ethoxy-1'-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[isochromane-4,2'-piperidine]-7-carboxylic acid, ie, compound A54-4.

[0447] LCMS (ESI, m / z): [M+H] + =465.2

[0448] 1 H NMR: (400MHz, CDCl 3 )δ8.15(d,J=8.26Hz,1H),8.04(d,J=8.38Hz,1H),7.94(s,1H),7.76(s,1H),7.19(s,1H),6.74-6.65(m,2H),4.9 2-4.83(m,1H),4.81-4.72(m,1H),4.54(d,J=11.8Hz,1H),4.00(d,J=11.8Hz,1H),3.84(d,J=12.8Hz,1H),3.75( s,3H),3.66(d,J=12.6Hz,1H),3.50(q,J=7.06Hz,3H),2.92(d,J=11.8Hz,1H),2.64-2.57(m,1H),2.46(s,3H),2 .30(d,J=11.8Hz,1H),1.96(d,J=12.6Hz,1H),1.68(t,J=12.0Hz,1H),1.61-1.49(m,1H),1.16(t,J=7.00Hz,3H).

[0449] Example 15 Preparation of 4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A64)

[0450]

[0451]

[0452] Synthesis route:

[0453]

[0454] Step 1:

[0455]

[0456] Dissolve tert-butyl 7-bromo-4'-hydroxy-2-methylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (6.00 g, 14.5 mmol) in N-methylpyrrolidone (60.0 mL), replace nitrogen three times, cool to 0-5°C, add sodium hydrogen (5.82 g, 145 mmol, 60% purity), react at 0-5°C for 2 hours, add iodoethane (22.7 g, 145 mmol, 11.6 mL), and react at 25°C for 12 hours. LCMS shows that the raw material is completely consumed. The system is cooled to 0-5°C, quenched with saturated aqueous ammonium chloride solution (300 mL), extracted with ethyl acetate (100 mL*2), and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to obtain tert-butyl 7-bromo-4'-ethoxy-2-methylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (4.46 g, yield 69.6%).

[0457] Step 2:

[0458]

[0459] Pd(dppf)Cl 2(556mg, 681μmol) was added with anhydrous methanol (15.0mL) and acetonitrile (15.0mL), and tert-butyl 7-bromo-4'-ethoxy-2-methylspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (1.50g, 3.41mmol) and triethylamine (1.38g, 13.6mmol, 1.90mL) prepared in step 1 were added in sequence, argon was replaced three times, carbon monoxide was replaced three times, and the system was in carbon monoxide (3Mpa), 135°C, and reacted for 24 hours. LCMS showed that the raw materials were completely consumed and the product was generated. After cooling to room temperature, it was diluted with ethyl acetate (50mL), filtered, and the filtrate was concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography (petroleum ether / ethyl acetate = 5:1) to obtain 1-(tert-butyl) 7-methyl 4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (1.00g).

[0460] Step 3:

[0461]

[0462] The 1-(tert-butyl)7-methyl4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-1,7-dicarboxylic acid (216 mg, 514 μmol, 1.00 eq) prepared in step 2 was dissolved in ethyl acetate (6.00 mL), and then hydrochloric acid / ethyl acetate (2M, 5.15 mL, 20.0 eq) was added to the system and reacted at 25°C for 1 hour. The mixture was concentrated under reduced pressure to obtain 4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (160 mg).

[0463] Step 4:

[0464]

[0465] The 4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylic acid methyl ester (160 mg, 449 μmol) prepared in step 3 was dissolved in N,N-dimethylformamide (4.00 mL), potassium iodide (149 mg, 899 μmol), potassium carbonate (186 mg, 1.35 mmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (208 mg, 674 μmol) were added in sequence, and the reaction solution was reacted at 25°C for 2 hours, and the reaction process was monitored by LCMS. Water (12.0 mL) was added to the reaction system, extracted with ethyl acetate (5.00 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give methyl 1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylate (100 mg, yield 37.5%).

[0466] Step 5:

[0467]

[0468] The methyl 1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxy-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylate (100 mg, 168 μmol) prepared in step 4 was dissolved in MeOH (10.0 mL) and water (3.00 mL), and lithium hydroxide monohydrate (70.8 mg, 1.69 mmol) was added. The reaction solution was reacted at 60°C for 12 hours. LCMS showed that the raw material was completely consumed and the product was generated. The reaction solution was adjusted to pH 7-8 with hydrochloric acid (1.00 M) and then concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min), and the product was lyophilized to obtain 4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-2-methylspiro[benzopyran-4,2-piperidine]-7-carboxylic acid (9.21 mg, 19.2 μmol, yield 11.4%), i.e., compound A64.

[0469] H NMR: (400MHz, CDCl 3)δ8.12-8.07(m,2H),7.73(s,1H),7.62(s,1H),7.13(s,1H),6.65(s,1H),6.52(s,1H),4. 22-4.17(m,1H),3.84(d,J=11.2Hz,1H),3.74(s,3H),3.61(s,2H),3.51-3.38(m,2H),3.00 (d,J=15.2Hz,1H),2.87(t,J=12.2Hz,1H),2.75-2.63(m,1H),2.43(s,3H),2.04-1.93(m, 1H), 1.91-1.76 (m, 3H), 1.75-1.65 (m, 1H), 1.78 (d, J = 5.88Hz, 3H), 1.22 (t, J = 6.88Hz, 3H).

[0470] Example 16 Preparation of (4S, 4-ethoxy-1-(6-fluoro-5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A66)

[0471]

[0472] Synthesis route:

[0473]

[0474] Step 1:

[0475]

[0476] Dissolve (4S, 4'S)-4'-ethoxyspiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester (25 mg, 73.1 μmol) in N,N-dimethylformamide (2.00 mL), add potassium iodide (12.0 mg, 72.3 μmol), potassium carbonate (32.0 mg, 231 μmol) and tert-butyl 4-(chloromethyl)-6-fluoro-5-methoxy-7-methyl-1H-indole-1-carboxylate (22.0 mg, 67.1 μmol) in sequence, and react at 15-25°C for 4 hours. LCMS shows that the starting material is completely consumed. Add water (10.0 mL) to the reaction system, extract with ethyl acetate (10.0 mL*2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The concentrated product was separated and purified by TLC to give methyl (4S,4'S)-1-(1-(tert-butoxycarbonyl)-6-fluoro-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (10 mg, yield 22.9%).

[0477] Step 2:

[0478]

[0479] The methyl (4S, 4'S)-1-(1-(tert-butoxycarbonyl)-6-fluoro-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (10.0 mg, 16.7 μmol) prepared in step 1 was dissolved in methanol (2.00 mL) and water (0.50 mL), and lithium hydroxide monohydrate (7 mg, 167 μmol) was added. The reaction solution was reacted at 50-60° C. for 4 hours. LCMS showed that the raw material was completely consumed. The pH was adjusted to 7-8 with hydrochloric acid (0.1 M) and concentrated under reduced pressure to obtain a crude product. Neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) was used to obtain (4S, 4-4-ethoxy-1-(6-fluoro-5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (3.44 mg, yield 42.3%), i.e., compound A66.

[0480] H NMR: (400MHz, CDCl 3 )δppm:8.08(d,J=8.00Hz,1H),8.01(s,1H),7.68(d,J=8.40Hz,

[0481] 1H),7.53(d,J=1.20Hz,1H),7.20(s,1H),6.85(s,1H),4.49-4.42(m,1H) ,4.38-4.29(m,1H),3.76-3.72(m,4H),3.66(s,1H),3.57(d,J=12.4Hz,1H ),3.47-3.38(m,2H),2.84-2.76(m,2H),2.60-2.53(m,2H),2.34(bs,1H), 2.39 (s, 3H), 1.82 (s, 1H), 1.76 (d, J = 2.00Hz, 2H), 1.19 (t, J = 7.20Hz, 3H).

[0482] Example 17 Preparation of (4S)-4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (Compound A106)

[0483]

[0484] Synthesis route:

[0485]

[0486] Step 1:

[0487]

[0488] Dissolve (4S)-4'-ethoxyspiro[benzopyran-4,2'-piperazine]-7-carboxylic acid methyl ester (670 mg, 1.96 mmol) in N,N-dimethylformamide (7.00 mL), add potassium iodide (325 mg, 1.96 mmol), potassium carbonate (813 mg, 5.88 mmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (917 mg, 294 mmol) in sequence, and react at room temperature for 1 hour. TLC shows that new spots are generated. Add water (5.0 mL) to the reaction body, extract with ethyl acetate (10.0 mL*3), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The concentrated product was separated and purified by TLC to give methyl (4S)-1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (1.20 g).

[0489] LCMS (ESI, m / z): [M+H] + =581.3

[0490] Step 2:

[0491]

[0492] The methyl (4S)-1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxyspiro[benzopyran-4,2-piperidine]-7-carboxylate (900 mg, 1.55 mmol) prepared in step 1 was dissolved in methanol (10.0 mL) and water (1.00 mL), and lithium hydroxide monohydrate (650 mg, 15.5 mmol) was added, and the mixture was reacted at 60°C for 12 hours. The mixture was concentrated under reduced pressure, diluted with water (10.00 mL), and then the pH of the system was adjusted to 7 with hydrochloric acid (0.50 M), extracted with ethyl acetate (30.0 mL*3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 mins) to obtain (4S)-4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2-piperidine]-7-carboxylic acid (234 mg, yield 30.8%), i.e., compound A106.

[0493] LCMS (ESI, m / z): [M+H] + =467.2

[0494] 1 H NMR: (400MHz, DMSO) δ8.10-8.05(m,1H),7.96-7.95(m,1H),7.67-7.65(m,1H),7.52(s,1H) ,7.20-7.18(m,1H),6.80-6.78(m,1H),6.69(s,1H),4.47-4.40(m,1H),4.37-4.29(m,1H),3 .77(s,3H),3.65-3.60(m,1H),3.43-3.40(m,2H),2.82-2.76(m,2H),2.68-2.65(m,1H),2. 63-2.55(m,2H),2.46(s,3H),2.33-2.30(m,1H),1.81-1.80(m,1H),1.19(t,J=6.90Hz,3H).

[0495] Example 18 Preparation of (4S, 4'S)-4'-(deuterated ethoxy)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (Compound A107)

[0496]

[0497] Synthesis route:

[0498]

[0499] Step 1:

[0500]

[0501] Dissolve (4S)-4-tert-butyl-7-bromo-4'-hydroxyspiro[benzopyran-4,2'-piperidine]-1'-carboxylate (1.50 g, 3.77 mmol) in N-methylpyrrolidone (15.0 mL), replace nitrogen three times, cool to 0°C, add sodium hydrogen (377 mg, 9.42 mmol), control the temperature at 0-5°C, react at room temperature for 2 hours, add deuterated iodoethane (2.06 g, 12.8 mmol), and react at room temperature for 1 hour. Thin layer chromatography monitoring shows that the raw material is completely consumed. The system is cooled to 0°C, quenched with saturated aqueous ammonium chloride solution (20.0 mL), diluted with water (10.0 mL), extracted with ethyl acetate (50.0 mL*3), and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by column chromatography to obtain (4S)-tert-butyl 7-bromo-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-1'-carboxylate (1.00 g, yield 61.5%).

[0502] LCMS (ESI, m / z): [M+H] + =431.1

[0503] Step 2:

[0504]

[0505] Bistriphenylphosphine palladium dichloride (123 mg, 150 μmol) was added to anhydrous methanol (20.0 mL) and acetonitrile (20.0 mL), and (4S)-7-bromo-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (650 mg, 1.51 mmol)) prepared in step 1 and triethylamine (610 mg, 6.03 mmol) were added in sequence, and argon was replaced three times, and carbon monoxide was replaced three times. The system was reacted in carbon monoxide (2 MPa) at 125°C for 12 hours. TLC (petroleum ether / ethyl acetate = 2:1) showed that the raw material was completely consumed. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The concentrated product was separated and purified by column chromatography to obtain 1'-(tert-butyl)7-methyl(4S)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylic acid (600 mg, yield 97.0%).

[0506] LCMS (ESI, m / z): [M+H]+ =411.2

[0507] Step 3:

[0508]

[0509] The 1'-(tert-butyl)7-methyl(4S)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-1',7-dicarboxylic acid (550 mg, 1.34 mmol) prepared in step 2 was dissolved in ethyl acetate (1.00 mL), and then hydrochloric acid / ethyl acetate (2M, 6.70 mL) was added to the system and reacted at 25°C for 12 hours. TLC showed that the raw material was completely consumed. Concentrated under reduced pressure to obtain (4S)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (458 mg).

[0510] LCMS (ESI, m / z): [M+H] + =347.1

[0511] Step 4:

[0512]

[0513] (4S)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid methyl ester hydrochloride (458 mg, 1.32 mmol) was dissolved in N,N-dimethylformamide (5.00 mL), potassium iodide (219 mg, 1.32 mmol), potassium carbonate (730 mg, 5.28 mmol) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (613 mg, 1.98 mmol) were added in sequence and reacted at room temperature for 30 minutes. The product was generated by TLC monitoring. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10.0 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified using a thin layer chromatography plate to obtain methyl (4S)-1'-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylate (800 mg).

[0514] LCMS (ESI, m / z): [M+H] + =584.3

[0515] Step 5:

[0516]

[0517] Methyl (4S)-1'-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4'-(deuterated ethoxy)spiro[benzopyran-4,2'-piperidine]-7-carboxylate (500 mg, 856 μmol) was dissolved in methanol (4.00 mL) and water (1.00 mL), and lithium hydroxide monohydrate (359 mg, 8.57 mmol) was added, and the mixture was reacted at 60°C for 3 hours. The mixture was concentrated under reduced pressure, diluted with water (5.00 mL), and then the pH of the system was adjusted to 7 with hydrochloric acid (0.50 M), extracted with ethyl acetate (30.0 mL*3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain (4S, 4'S)-4'-(deuterated ethoxy)-1'-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)spiro[benzopyran-4,2'-piperidine]-7-carboxylic acid (320 mg, yield 76.6%), i.e., compound A107.

[0518] LCMS (ESI, m / z): [M+H] + =470.2

[0519] 1 H NMR: (400MHz, DMSO), δ10.8(s,1H),7.96(d,J=8.40Hz,1H),7.49(d,J=8.00Hz, 1H),7.27-7.25(m,2H),6.65(s,1H),6.62-6.61(m,1H).4.41-4.39(m,1H),4.38 -4.26(m,1H),3.68(s,3H),3.61-3.58(m,2H),3.32(s,1H),2.67-2.50(m,2H),2 .49-2.46(m,5H),2.49-2.46(m,5H),2.43(d,J=10.0Hz,1H),1.67-1.59(m,3H).

[0520] Embodiment 19

[0521] Preparation of 4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylic acid (Compound A65)

[0522]

[0523] Synthesis route:

[0524]

[0525] Step 1:

[0526]

[0527] 7-Bromo-1-methylspiro[isochromane-4,2'-piperidine]-4'-ol (380 mg, 1.22 mmol) was dissolved in tetrahydrofuran (5.00 mL), di-tert-butyl dicarbonate (531 mg, 2.43 mmol, 559 μL) and triethylamine (369 mg, 3.65 mmol, 508 μL) were added, and the reaction solution was reacted at 25°C for 8 hours. Water (10.0 mL) was added to the reaction solution at 25°C to dilute it, and it was extracted with ethyl acetate (20.0 mL*2), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by silica gel column chromatography to obtain tert-butyl 7-bromo-4'-hydroxy-1-methylspiro[isochromane-4,2'-piperidine]-1'-carboxylate (257 mg, yield 51.2%).

[0528] LCMS (ESI, m / z): [M+H] + =412.1

[0529] Step 2:

[0530]

[0531] The tert-butyl 7-bromo-4'-hydroxy-1-methylspiro[isochromane-4,2'-piperidin]-1'-carboxylate (257 mg, 623 μmol) prepared in step 1 was dissolved in N-methylpyrrolidone (3.00 mL), replaced with nitrogen three times, cooled to 0-5°C, added with sodium hydrogen (249 mg, 6.23 mmol, 60.0% purity), reacted at 0-5°C for 2 hours, and then added with iodoethane (972 mg, 6.23 mmol, 498 μL), and reacted at 25°C for 8 hours. The system was cooled to 0-5°C, quenched with saturated aqueous ammonium chloride solution (10.0 mL), extracted with ethyl acetate (10.0 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give tert-butyl 7-bromo-4'-ethoxy-1-methylspiro[isochromane-4,2'-piperidine]-1'-carboxylate (160 mg, yield 58.3%).

[0532] LCMS (ESI, m / z): [M+H] + =440.1

[0533] Step 3:

[0534]

[0535] Pd(dppf)Cl 2 (59.3mg, 72.7μmol) was added to anhydrous methanol (4.00mL), and the compound 7-bromo-4'-ethoxy-1-methylspiro[isochromane-4,2'-piperidine]-1'-carboxylic acid tert-butyl ester (160mg, 363μmol) and triethylamine (147mg, 1.45mmol, 202μL) were added in sequence, and argon was replaced three times, and carbon monoxide was replaced three times. The system was in carbon monoxide (3Mpa), 135°C, and reacted for 24 hours. Water (10mL) was added to the reaction solution at 25°C to dilute it, and it was extracted with ethyl acetate (10.0mL*2). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by thin layer chromatography to obtain 1-(tert-butyl) 7-methyl 4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-1,7-dicarboxylic acid (107mg).

[0536] Step 4:

[0537]

[0538] The 1-(tert-butyl)7-methyl-4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-1,7-dicarboxylic acid (34.0 mg, 81.1 μmol) prepared in step 3 was dissolved in ethyl acetate (1.00 mL), and then hydrochloric acid / ethyl acetate (2.00 M, 2.00 mL) was added to the system and reacted at 25° C. for 1 hour. The mixture was concentrated under reduced pressure to obtain methyl 4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylate (23.0 mg).

[0539] Step 5:

[0540]

[0541] The 4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylic acid methyl ester (28.0 mg, 87.7 μmol, 1.00 eq) prepared in step 4 was dissolved in N,N-dimethylformamide (1.00 mL), potassium iodide (43.7 mg, 263 μmol, 3.00 eq), potassium carbonate (36.4 mg, 263 μmol, 3.00 eq) and tert-butyl 4-(chloromethyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (27.2 mg, 87.7 μmol, 1.00 eq) were added in sequence, and the reaction solution was reacted at 25° C. for 8 hours. Water (5.00 mL) was added to the reaction system, extracted with ethyl acetate (5.00 mL*2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was separated and purified by TLC to give methyl 1-(1-(tert-butoxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylate (20.0 mg).

[0542] Step 6:

[0543]

[0544] The 1-(1-(tert-butyloxycarbonyl)-5-methoxy-7-methyl-1H-indol-4-yl)methyl)-4-ethoxy-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylic acid methyl ester (7.00 mg, 11.8 μmol) prepared in step 5 was dissolved in MeOH (2.00 mL) and water (0.50 mL), and lithium hydroxide monohydrate (29.0 mg, 691 μmol) was added, and the reaction solution was reacted at 60° C. for 8 hours. The reaction solution was adjusted to pH 7-8 with hydrochloric acid (2.00 M) and then concentrated under reduced pressure. The concentrated product was separated and purified by neutral reverse phase high performance liquid chromatography (column specification: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; time: 10 min) to obtain 4-ethoxy-1-(5-methoxy-7-methyl-1H-indol-4-yl)methyl)-1-methylspiro[isochromane-4,2-piperidine]-7-carboxylic acid (0.62 mg, yield 10.5%), i.e., compound A65.

[0545] LCMS (ESI, m / z): [M+H] + =479.2

[0546] H NMR: (400MHz, CDCl 3)δ8.19(d,J=7.2Hz,1H),7.95-7.99(m,2H),7.79-7.81(m,1H),

[0547] 7.20(s,1H),6.77(s,1H),6.78(s,1H),4.89(d,J=6.8Hz,1H),4.76(d,J=11.2Hz,1H) ,4.31(d,J=12.4Hz,1H),3.86(d,J=13.2Hz,1H),3.73-3.76(m,5H),3.63-3.64(m,1H ),3.41(t,J=8.2Hz,1H),2.92(t,J=11.2Hz,1H),2.70(d,J=9.6Hz,1H),2.46-2.51(m ,4H),1.98-1.99(m,1H),1.60(d,J=6.0Hz,1H),1.25-1.27(m,3H),1.23-1.25(m,2H).

[0548] Example 20 Preparation of Ipracopan

[0549]

[0550] Synthesis route:

[0551]

[0552] Step 1:

[0553]

[0554] Trans-4-ethoxy-2-(4-(propionyloxy)phenyl)piperidine-1-carboxylic acid benzyl ester (5.00 g, 24.8 mmol) was purified by supercritical fluid chromatography (SFC) (chromatographic column model: (s, s) WHELK-O1 (250 mm*30 mm, 10 um); mobile phase: [carbon dioxide-methanol (0.1% ammonia water); flow rate: 20 ml / min] to obtain (2S, 4S)-4-ethoxy-2-(4-(propionyloxy)phenyl)piperidine-1-carboxylic acid benzyl ester (8.60 g).

[0555] Step 2:

[0556]

[0557] The (2S, 4S)-4-ethoxy-2-(4-(propionyloxy)phenyl)piperidine-1-carboxylic acid benzyl ester (4.30 g, 10.4 mmol) prepared in step 1 was dissolved in tetrahydrofuran (50 mL), wet palladium / carbon (430 mg, 404 μmol, 10% purity) was added and hydrogen was introduced. Then the reaction was carried out at a hydrogen pressure of 15 Psi and 25-30° C. for 1 hour (LCMS monitoring showed that the raw material was completely consumed and the product was detected). The reaction solution was filtered and the filtrate was concentrated to obtain 4-((2S, 4S)-4-ethoxypiperidin-2-yl)phenylpropanoate (5.60 g).

[0558] Step 3:

[0559]

[0560] The 4-((2S, 4S)-4-ethoxypiperidin-2-yl)phenylpropanoate (5.00 g, 18.0 mmol) prepared in step 2 was dissolved in dichloroethane (50 mL), and tert-butyl 4-formyl-5-methoxy-7-methyl-1H-indole-1-carboxylate (10.4 g, 36.0 mmol) and magnesium sulfate (6.51 g, 54.1 mmol) were added. Then the reaction was carried out at 25-30°C for 12 hours. Then sodium triacetoxyborohydride (13.4 g, 63.1 mmol) was added and the reaction was carried out at 25-30°C for 1 hour (LCMS monitoring the reaction process). The reaction solution was filtered, and then ethyl acetate (400 mL) and saturated sodium bicarbonate aqueous solution (400 mL) were added and stirred at 10-20°C for 5 minutes. The organic phase was separated and concentrated to obtain a crude product (15.0 g, crude product). The crude product was purified by column chromatography (silica, petroleum ether:ethyl acetate = 1:0 to 3:1) to give tert-butyl 4-(((2S,4S)-4-ethoxy-2-(4-(propionyloxy)phenyl)piperidin-1-yl)methyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (4.50 g).

[0561] Step 4:

[0562]

[0563] The tert-butyl 4-(((2S, 4S)-4-ethoxy-2-(4-(propionyloxy)phenyl)piperidin-1-yl)methyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate (4.50 g, 8.17 mmol) prepared in step 3 was dissolved in tetrahydrofuran (22.5 mL) and methanol (45 mL), and lithium hydroxide (1M, 32.7 mL) was added. Then the reaction was carried out at 50-60°C for 12 hours. LCMS showed that the product was detected. The reaction solution was filtered, and the filtrate was adjusted to pH 7-8 using a saturated aqueous citric acid solution. The crude product was concentrated to obtain a crude product. The crude product was purified by reverse phase preparation (chromatographic column model: CD07-Daisogel SP-100-8-ODS-PK 150*25*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 22%-52%; flow rate: 20ml / min; time: 10mins). The compound 4-((2S,4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic acid hydrochloride (3.55 g, yield 94.2%) was obtained.

[0564] Step 5:

[0565]

[0566] The 4-((2S, 4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic acid hydrochloride (3.30 g, 7.19 mmol, hydrochloride) prepared in step 4 was dissolved in water (10 mL), and sodium bicarbonate (605 mg, 7.20 mmol, 280 μL) was added. Then the reaction was carried out at 20-25° C. for 10 minutes (LCMS monitored the reaction process). Concentration gave 4-((2S, 4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic acid (3.80 g, yield 87.5%).

[0567] 1 H NMR: (400MHz, D 2O)δppm 1.11(br t,J=6.88Hz,3H)1.43-1.66(m,2H)1.79(brs,2H)2.12(s,3H)2.61-2.89(m,2H)2.99(br d,J=12.38Hz,1H)3.30(s,3H)3.33(br d,J=7.13Hz,3H)3.52(br s,1H)3.83-4.11(m,1H)5.85(br s,1H)6.24(s,1H)7.05(br d,J=2.13Hz,1H)7.34(br d,J=7.38Hz,2H)7.94(br d,J=7.88Hz,2H)

[0568] Experimental example

[0569] The inventors investigated the pharmacological activity of the compounds prepared in the examples, and conducted affinity experiments, in vitro enzyme activity experiments (CVF-Bb experiments, AP-deposition experiments, PNH-Like hemolysis experiments), and rat pharmacokinetic experiments with reference to the preclinical experimental data of ipracopan, and conducted parallel comparisons with ipracopan.

[0570] The preclinical experimental data of ipracopan demonstrated through CVF-Bb experiment and AP-deposition experiment that the drug has the potential to treat IgA nephropathy and other complement pathway-related kidney diseases and blood diseases by targeting the complement pathway. The inventors used the same experimental items to investigate the compound provided by the present invention, and compared it with ipracopan to prove the pharmacological activity of the compound of the present invention.

[0571] 1. Affinity experiment

[0572]

Purpose

[0573] The affinity of the compound of the present invention to complement factor B is detected, and the binding strength of the compound of the present invention to factor B is evaluated.

[0574]

Experimental materials / instruments

[0575] Complement factor B (Sino Biological), NTA sensor (Sartorius)

[0576]

Experimental samples

[0577] Compounds A2, A8, A10, A11, A31, A32, A33, A34-1, A34-2, A36, A37, A38, A39, A41, A54-1, A54-2, A54-3, and A54-4 were prepared according to Examples 1-20.

[0578] Ipracolans prepared according to Example 20.

[0579]

Experimental methods

[0580] (1) Take n compounds to conduct experiments, divide 2*(n+1) NTA sensors into 2 groups, and pre-wet them in phosphate buffered saline (PBST) containing Tween 80 for more than 10 minutes. Among them, (n+1) sensors are solidified, and the other (n+1) sensors are used as reference sensors and do not need to be solidified (no more than 7 compounds per experiment). In a 96-well sample plate, add PBST to the first column and 50μg / mL complement system factor B to the second column. Set baseline 60s, loading 3000s, so that the solidification height reaches more than 4nm, and then balance with baseline 60s.

[0581] (2) Take a new 96-well plate and set up the Buffer and Sample wells. Add PBST+0.1% DMSO buffer to the Buffer wells and add the corresponding compounds to the Sample wells. The drug concentrations are 4.12, 12.35, 37.04, 111.11, 333.33, and 1000 nM from left to right.

[0582] (3) After solidification, first set a relatively long equilibration step (more than 10 minutes), and then enter the baseline / association / dissociation cycle. The Assay Definition is set as follows: baseline 60 seconds, association 120 seconds, dissociation 360 seconds.

[0583] (4) After setting the curing sensor cycle, add a set of reference sensor cycles. The cycle program is consistent with the curing sensor cycle program.

[0584] (5) After setting, set the position of the sensor in Sensor Assignment. The sensors are divided into two columns, one group of solidified sensors and one group of reference sensors, with (n+1) sensors in each column. After checking that everything is correct in Review Experiment, set the save path, file name, temperature, etc. in Run Experiment and click GO to run.

[0585] (6) Use Octet analysis software Data Analysis software to perform kinetic and steady-state analysis on the raw data.

[0586]

Experimental results

[0587] The affinity test results are shown in Table 1.

[0588] Table 1

[0589]

[0590]

[0591] From the results in Table 1, it can be seen that the affinity KD values ​​of compounds A2, A8, A10, A11, A32, A33, A34-1, A34-2, A36, and A37 for complement factor B are basically consistent with those of ipracopan, indicating that these 10 compounds have strong binding to complement factor B and have the potential to treat complement pathway-related kidney diseases (such as IgA nephropathy) and blood diseases by targeting the complement pathway.

[0592] 2. CVF-Bb Experiment

[0593] Cobra venom factor (CVF) is an anti-complement factor isolated from cobra venom. It is closely related to the complement system and has similar functions to the C3 degradation product C3b fragment. It can reversibly bind to factor B in serum at a ratio of 1:1 both in vivo and in vitro to form a stable C3 splice CVF-B complex. Under the hydrolytic activation of factor D, the CVF-B complex is cleaved into CVF-Bb with C3 convertase function. Subsequently, CVF-Bb can cleave C3 to generate two fragments, C3b and C3a, which further activate the alternative pathway of complement, thereby triggering a cascade amplification reaction, resulting in massive activation and depletion of complement.

[0594]

Purpose

[0595] The inhibitory effect of the compound of the present invention on the activity of complement factor B is evaluated by detecting the content of the downstream molecule C3a after the compound of the present invention binds to CVF-B.

[0596]

Experimental materials / instruments

[0597] Complement factor B recombinant protein (Purified human factor B, Factor B): ComplementTechnology

[0598] Complement factor D recombinant protein (Purified human factor D, Factor D): ComplementTechnology Cobra Venom Factor (Cobra Venom Factor, Factor B, CVF): quidel

[0599] C3 Protein: quidel

[0600] Anti-C3a / C3a des Arg antibody

[2991] : Abcam Goat Anti-Mouse IgG H&L(HRP) : Abcam QuantaBlu Fluorogenic Peroxidase Substrate Kits: thermofisher StartingBlock TM T20 (PBS) blocking buffer (ThermoScientific TM StartingBlock TM T20(PBS)1X):thermofisher

experimental sample

[0601] Compounds A2, A8, A11, A31, A32, A33, A34-1, A34-2, A36, A37, A38, A39, and A54-3 were prepared according to Examples 1-19.

[0602] Ipracolans prepared according to Example 20.

[0603]

Experimental methods

[0604] (1) Add Factor B, Factor D and CVF to the experimental wells respectively, mix well and incubate at 37°C for 3 hours.

[0605] (2) Add the compound of the present invention to the reaction well, mix well, and incubate at 37° C. for 1 hour.

[0606] (3) Add human complement factor C3 protein to the reaction well, mix well, and incubate at 37° C. for 2 hours to prepare a C3 reaction sample.

[0607] (4) Add 97 μL of coating buffer and 3 μL of reaction sample to a 96-well black adsorption plate, mix well, seal the plate, and incubate at 4°C overnight.

[0608] (5) Wash three times with 300 μL washing solution and add 300 μL startingBlock TM Blocking buffer and incubate at room temperature for 15 minutes.

[0609] (6) Wash three times with 300 μL washing buffer and add 100 μL Anti-C3a / C3a des Arg antibody and incubate at 37°C for 1 hour.

[0610] (7) Wash three times with 300 μL of washing buffer and add 100 μL of Goat Anti-Mouse IgG H&L (HRP) and incubate at 37°C for 30 min.

[0611] (8) Wash three times with 300 μL washing solution. Add 100 μL Quantablu Substrate Solution and incubate at room temperature for 20 minutes.

[0612] (9) Add 100 μL Quantablu Stop Solution.

[0613] (10) Use an ELISA reader to measure the absorbance at 320 nm and 420 nm.

[0614]

Experimental results

[0615] The results of CVF-Bb enzyme activity are shown in Table 2.

[0616] Table 2

[0617] Experimental samples <![CDATA[IC 50 Value]]> Ipracopan 33.62nM A2 28.62nM A8 35.41nM A11 40.14nM A31 206nM A32 9.42nM A33 31.22nM A34-1 38.2nM A34-2 26.06nM A36 40.84nM A37 65.77nM A38 44.7nM A39 205nM A54-3 711.6nM

[0618] It can be seen from the results in Table 2 that the compounds A2, A8, A11, A33, A34-1, A34-2, A36, A37, and A38 of the present invention have an inhibitory effect on the C3 spliceosome enzyme activity. 50 Basically consistent with ipracopan, the inhibitory activity of compound A32 is 3.6 times that of ipracopan, indicating that compound A32 has a significant inhibitory effect on the complement alternative pathway and has the potential to treat complement pathway-related kidney diseases (such as IgA nephropathy) and blood diseases by targeting the complement pathway.

[0619] 3. Complement alternative pathway deposition experiment (AP-deposition experiment)

[0620] In this experiment, lipopolysaccharide (LPS) was used as an activation initiator to activate the complement alternative pathway. LPS can directly bind to C3b and form C3 convertase with the participation of factors such as Factor B and Factor D. C3 convertase can cleave C3 into C3a and C3b fragments, and then bind to the cleaved C3b to form C5 convertase. C5 convertase cleaves C5 into C5a and C5b fragments. C5b reacts with C6, C7, C8 and C9 in the liquid phase in turn, and finally forms the terminal complement complex (TCC, SC5b-9), thereby exerting the effect of lysing cells.

[0621]

Purpose

[0622] The inhibitory effect of the compounds of the present invention on Factor B activity was evaluated by measuring the concentration of terminal complement complex (TCC) after LPS activated the alternative complement pathway.

[0623]

Experimental materials / instruments

[0624] Normal Human Serum: Complement Technology

[0625] WIESLAB Complement System Alternative Pathway Detection Kit: Svarlifescience

[0626]

Experimental samples

[0627] Compounds A2, A8, A10, A11, A32, A33, A34-1, A34-2, A36 and A37 were prepared according to Examples 1-19.

[0628] Ipracolans prepared according to Example 20.

[0629]

Experimental methods

[0630] (1) Add 95 μL human serum and 5 μL compound to each experimental well. Add 100 μL sample diluent to each control well as blank control, LPS-activated normal human serum as positive control, and LPS-untreated normal human serum as negative control. Mix well and incubate at 37°C for 1 hour.

[0631] (2) Wash three times with 300 μL of washing solution, pat the plate dry for the last time, add 100 μL of C5b-9 conjugated antibody and incubate at room temperature for 30 minutes.

[0632] (3) Wash three times with 300 μL of washing solution, pat the plate dry for the last time, add 100 μL of substrate solution and incubate at room temperature for 30 minutes.

[0633] (4) Use an ELISA reader to measure the absorbance at 405 nm.

[0634]

Experimental results

[0635] The AP-deposition enzyme activity results are shown in Table 3.

[0636] Table 3

[0637] Experimental samples <![CDATA[IC 50 Value]]> Ipracopan 15.08nM A2 13.37nM A8 30.76nM A10 68.97nM A11 18.28nM A32 6.165nM A33 14.13nM A34-1 18.84nM A34-2 22.7nM A36 18.23nM A37 27.59nM

[0638] As can be seen from Table 3, the compounds A2, A8, A11, A33, A34-1, A34-2, A36, and A37 of the present invention inhibit the AP-deposition enzyme activity IC 50 Basically consistent with ipracopan, the inhibitory activity of compound A32 is about 2.4 times that of ipracopan, indicating that compound A32 of the present invention has significant inhibitory activity on the complement alternative pathway and has the potential to treat complement pathway-related kidney diseases (such as IgA nephropathy) and blood diseases by targeting the complement pathway.

[0639] 4. Paroxysmal nocturnal hemoglobinuria-like hemolysis test (PNH-Like hemolysis test)

[0640] Under acidic conditions (pH 6.4-6.5), the complement system in serum is easily activated. Since the red blood cells of PNH patients lack CD55 and CD59, they cannot effectively prevent the attack of the terminal complement complex, resulting in hemolysis; while normal red blood cells can resist this attack, so hemolysis does not occur. In this experiment, CD55 and CD59 antibodies were used to neutralize CD55 and CD59 on the surface of normal red blood cells to simulate the characteristics of PNH red blood cells, and EGTA was used to chelate Ca in serum. 2+ It blocks the classical complement pathway and the lectin pathway, leaving only the alternative complement pathway active.

[0641]

Purpose

[0642] The inhibitory effect of the compound of the present invention on the activity of Factor B was evaluated by detecting the hemolysis of red blood cells.

[0643]

Experimental Materials

[0644] CD55 antibody, EDTA, PBS (Thermo), anti-CD59 antibody [MEM-43] (abcam), EGTA (Solerbo), whole blood and serum from healthy individuals.

[0645]

Experimental samples

[0646] Compounds A2, A8, A10, A11, A32, A34-2, A36, A37 and A38 were prepared according to Examples 1-19.

[0647] Ipracolans prepared according to Example 20.

[0648]

Experimental methods

[0649] (1) Collect 1 ml of fresh blood from healthy volunteers using a heparin anticoagulant tube and 15 ml of blood using a procoagulant tube.

[0650] (2) Centrifuge at 2000 rpm at room temperature for 5 min to remove the plasma and other upper solutions, then add 10 times the volume of sodium chloride solution and wash three times.

[0651] (3) Dilute RBCs to 2×10 with PBS solution. 9 cells / ml.

[0652] (4) Take 100ul 2×10 9 cells / ml red blood cells, add 1ul 1mg / ml mouse anti-human CD 55 and 3ul 1mg / ml CD 59 monoclonal antibodies respectively, and incubate with shaking at 37℃ for 30min.

[0653] (5) Wash RBCs three times with PBS to remove excess antibodies.

[0654] (6) Dilute RBCs to 1×10 with PBS solution. 8 cells / ml.

[0655] (7) Collect serum (NHS) from the same healthy individual as a complement source. Collect blood using a coagulant tube and centrifuge to obtain serum at 4°C, 3000 g, for 15 min.

[0656] (8) Add EGTA and MgCl to the serum 2 The classical complement pathway was blocked (final concentrations were 8 mM and 2.85 mM, respectively); and the pH was acidified to 6.4 with 0.25 M HCL to activate the AP system.

[0657] (9) Acidified serum was used to prepare a series of drug concentrations and pre-incubated on ice for 10 min. A baseline group (final concentration of 10 mM EDTA group) and a maximum hemolysis group (pure water group) were also set up.

[0658] (10) Drug incubation: Take 200ul of acidified serum containing drugs and 20ul of 1×10 8 The cells / ml red blood cells were mixed and incubated with shaking at 37°C for 6 h.

[0659] (11) Termination of reaction: Add 200 μl of E-PBS solution (final EDTA concentration: 15 mM) to each tube and centrifuge at 4000 rpm for 5 min at room temperature.

[0660] (12) Hemolysis assay: 100 μl of supernatant was added to a 96-well plate and tested at OD 405 nm.

[0661]

Experimental results

[0662] The results of serum hemolysis are shown in Table 4.

[0663] Table 4

[0664] Experimental samples <![CDATA[Hemolysis inhibition IC 50 value]]> Ipracopan 157.4nM A2 193nM A8 646nM A10 338.5nM A11 135.3nM A32 232nM A34-2 153.9nM A36 118.2nM A37 144.9nM A38 156.8nM

[0665] It can be seen from the results in Table 4 that the hemolytic inhibition IC values ​​of compounds A2, A11, A32, A34-2, A36, A37, and A38 of the present invention are 50 The results are basically consistent with those of ipracopan, suggesting that these compounds have potential pharmacological activity for paroxysmal nocturnal hemoglobinuria.

[0666] 5. Pharmacokinetics Experiment in Rats

[0667] Based on the results of affinity experiments, CVF-Bb experiments, AP-deposition experiments, and PNH-Like hemolysis experiments, compounds A2, A11, A32, A33, A34-1, A34-2, A36, and A37 were screened for rat pharmacokinetic experiments.

[0668]

Purpose

[0669] The pharmacokinetic characteristics of the compound of the present invention in rats after intragastric administration of 1 mg / kg were investigated.

[0670]

Experimental Materials

[0671] Acetonitrile, methanol, isopropanol (chromatographic grade Merck), formic acid (McLean), PEG400 (Solabol), DMSO (Aladdin).

[0672]

Experimental samples

[0673] Compounds A2, A11, A32, A33, A34-1, A34-2, A36 and A37 were prepared according to Examples 1-19.

[0674] Ipracolans prepared according to Example 20.

[0675] Storage conditions: 0-5℃, dry and sealed.

[0676] Purity of Ipracolos: 99.9%.

[0677] Purity of compound A2: 95.653%.

[0678] Purity of compound A11: 98.67%.

[0679] Purity of compound A32: 96.35%.

[0680] Purity of compound A33: 97.08%.

[0681] Purity of compound A34-1: 97.95%.

[0682] Purity of compound A34-2: 95.39%.

[0683] Purity of compound A36: 98.59%.

[0684] Purity of compound A37: 98.43%.

[0685] Experimental animals

[0686] Twenty-seven male SD rats were randomly divided into two groups, with 3 rats in each group.

[0687]

Dosage regimen

[0688] Each group of animals was given the drug by gavage.

[0689] Table 5

[0690]

[0691]

Experimental methods

[0692] (1) Drug administration and sampling in rats: All 9 groups of animals were gavaged with a dosage volume of 5 mL / kg. 0.1 mL of blood was collected from the test animals at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. The blood was placed in a disposable anticoagulant tube and centrifuged at 4000 rpm for 10 min. The supernatant was stored in a refrigerator at -20 °C for testing.

[0693] (2) The plasma sample processing method is as follows: take 20 μL of plasma sample into a 1.5 mL centrifuge tube, add 180 μL of internal standard working solution (1 ng / mL LB102 acetonitrile solution), vortex for 10 min at gear 5, centrifuge at 12000 rpm for 10 min, take the supernatant, add it to the injector vial for LC-MS / MS analysis, and record the chromatogram.

[0694]

Experimental results

[0695] The pharmacokinetic results are shown in Table 6.

[0696] In the table, AUClast is the area under the drug concentration-time curve from time zero to the last measured time point; AUCINF_obs is the area under the drug concentration-time curve from time zero to infinity; Vz_F_obs is the apparent distribution volume; Cl_F_obs is the clearance; MRTlast is the mean residence time calculated from time zero to the last measured time point; MRTINF_obs is the mean residence time calculated from time zero to infinity.

[0697] Table 6

[0698]

[0699]

Claims

1. A piperidinyl spiro derivative, the general chemical structure of which is as follows: In the formula, X=CH2, O, S, NH, CO, or one hydrogen on CH2 is replaced by methyl, ethyl, cyclopropyl, cyclobutyl, trifluoromethyl, trideuterated methyl, or halogen, or two hydrogens on CH2 are replaced by or the H on the NH is substituted by a methyl group; Y=CH2, O, S, NH, CO, or one hydrogen on the CH2 is replaced by methyl, ethyl, cyclopropyl, cyclobutyl, trifluoromethyl, trideuterated methyl, or halogen, or two hydrogens on the CH2 are replaced by or the H on the NH is substituted by a methyl group; And X and Y are not the same; n = 0, 1, or 2; R1=H, D, halogen, CH3, NH2, or OH; R2=H, D, halogen, CH3, NH2, or OH; R3=H, D, halogen, CH3, NH2, or OH; R4=H, OH, NH2, CH3, CD3, OCH3, or OCH2CH3; R5=H, OH, NH2, CH3, CD3, OCH3, or OCH2CH3; R6=CH3, CH2CH3, OCF3, dimethyl, cyclopropyl, cyclobutyl, halogenated cyclobutyl, cyclopropyloxy, cyclobutyloxy, or halogenated cyclobutyloxy; or, R6 is in, R7=H, D, CH3, or halogen; R8=H, D, CH3, or halogen; or, R6 is Wherein, R9=H, CH3, CH2CH3, cyclopropyl, cyclobutyl, or halogenated cyclobutyl; or, R6 is a halogenated phenyl group or an aniline carbonyl group; or, R6 is R 10 , R 11 A three-membered ring of two substituents, and the three-membered ring and the group to which it is connected share a carbon atom; wherein R 10 =H, D, CH3, or halogen; R 11 =H, D, CH3, or halogen; or R6 is R 12 , R 13 A four-membered ring of two substituents, and the four-membered ring and the group to which it is connected share a carbon atom; wherein R 12 =H, D, CH3, or halogen; R 13 =H, D, CH3, or halogen; or, R6 is an aza four-membered ring, and the four-membered ring and the group to which it is connected share a carbon atom; or, R6 is an oxygen four-membered ring, and the four-membered ring and the group to which it is connected share one carbon atom.

2. The piperidinyl spiro derivative according to claim 1, characterized in that When R6 is CH2CH3, the hydrogen on the CH2CH3 is deuterium.

3. The piperidinyl spiro derivative according to claim 1, characterized in that: When R6 is When R9 is CH3, the hydrogen in R6 is deuterium.

4. The piperidinyl spiro derivative according to claim 1, characterized in that: When R6 is When R9 is CH3, the hydrogen on the carbon atom connected to R6 is deuterium.

5. The piperidinyl spiro derivative according to claim 1, characterized in that: When X is CH2, the hydrogen on the CH2 is deuterium.

6. The piperidinyl spiro derivative according to claim 1, characterized in that: When Y is CH2, the hydrogen on the CH2 is deuterium.

7. The piperidinyl spiro derivative according to claim 1, characterized in that: When X is NH, the hydrogen on the NH is deuterium.

8. The piperidinyl spiro derivative according to claim 1, characterized in that: When Y is NH, the hydrogen on the NH is deuterium.

9. The piperidinyl spiro derivative according to claim 1, characterized in that: The halogenated cyclobutyl group in R6 is 3,3-difluorocyclobutyl group, the halogenated cyclobutyloxy group in R6 is 3,3-difluorocyclobutyloxy group, and the halogenated phenyl group in R6 is 2,3-difluorophenyl group.

10. The piperidinyl spiro derivative according to claim 1, characterized in that: The halogenated cyclobutyl group in R9 is 3,3-difluorocyclobutyl group.

11. The piperidinyl spiro derivative according to claim 1, characterized in that: The derivative is selected from:

12. A salt, solvate, stereoisomer, tautomer, or deuterated product of the piperidinyl spiro derivative according to any one of claims 1 to 11.

13. The pharmaceutical preparation of the piperidinyl spiro derivative according to any one of claims 1 to 11, comprising: Tablets, capsules, granules, sustained-release preparations, controlled-release preparations.

14. Use of the piperidinyl spiro derivative according to any one of claims 1 to 11 in the preparation of a medicament for treating diseases mediated by the complement system.

15. Use of the piperidinyl spiro derivative according to any one of claims 1 to 11 in the preparation of a medicament for treating a disease caused by dysregulation of the complement system or dysfunction of the complement system.

16. Use of the piperidinyl spiro derivative according to any one of claims 1 to 11 in the preparation of a drug for treating diseases associated with the complement pathway.

17. Use of the piperidinyl spiro derivative according to any one of claims 1 to 11 in the preparation of a drug for inhibiting complement factor B.

18. The use according to claim 14, characterized in that: The diseases mediated by the complement system include: paroxysmal nocturnal hemoglobinuria, immunoglobulin A nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome, age-related maculopathy, ANCA-associated vasculitis, systemic lupus erythematosus, immune thrombocytopenia, and cold agglutinin disease.

19. The use according to claim 18, characterized in that: The C3 glomerulopathy includes: dense deposit disease, primary C3 glomerulonephritis, familial type III membranoproliferative glomerulonephritis, and complement factor H-related protein 5 nephropathy.

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