Crystals of nitrogen-containing spiro compounds, methods for producing the same, and uses

Abstract

This invention discloses crystals of nitrogen-containing spiro compounds, methods for producing the same, and uses thereof. This invention provides a type I crystal of a nitrogen-containing spiro compound represented by formula A, in which the powder X-ray diffraction pattern displayed at a 2θ angle using Cu-Kα rays has diffraction peaks at positions 5.67±0.20°, 11.37±0.20°, 16.69±0.20°, 17.32±0.20°, and 19.73±0.20°. This invention also provides a type II crystal of a nitrogen-containing spiro compound represented by formula A, in which the powder X-ray diffraction pattern displayed at a 2θ angle using Cu-Kα rays has diffraction peaks at positions 11.32±0.20°, 11.70±0.20°, 11.93±0.20°, 18.03±0.20°, 18.81±0.20°, and 19.17±0.20°. The nitrogen-containing spiro compound crystals of the present invention exhibit good stability and complement factor D inhibitory activity, good inhibitory effect against rabbit erythrocyte hemolysis, high in vivo exposure levels, and high oral bioavailability. JPEG2026513298000037.jpg5469

Description

[Technical Field]

[0001] This application claims priority to Chinese patent application 2023103175276, filed on March 28, 2023, and Chinese patent application 2024102275411, filed on February 29, 2024. This application incorporates the full text of the aforementioned Chinese patent applications.

[0002] [Technical field] This invention relates to the crystals, manufacturing methods, and uses of nitrogen-containing heterocyclic compounds. [Background technology]

[0003] Complement is a protein widely present in the serum, tissue fluid, and cell membrane surfaces of humans and vertebrates. It mediates immune and inflammatory responses, is mostly glycoprotein, and is produced in a variety of cells, including hepatocytes, macrophages, and intestinal mucosal epithelial cells. Although it is named "complement" because it is a necessary condition for antibodies to exert their cytolytic effect, it actually mediates both specific and nonspecific immunity. The three activation pathways of the complement system include the classical pathway, the mannose-binding lectin (MBL) pathway, and the alternative (detour) pathway. Complement factor D plays an initial and core role in the activation of the complement alternative pathway cascade. Activation of the complement alternative pathway is triggered by the spontaneous hydrolysis of the thioester bond in C3 to produce C3(H2O), which then binds to factor B to form the C3(H2O)B complex. The action of complement factor D is to degrade factor B in the C3(H2O)B complex to form Ba and Bb. In addition to binding with C3b to form C3 convertase, Bb is involved in the proliferation of pre-activated B lymphocytes, while Ba inhibits this proliferation. Factor D is expressed at high levels in fat cells and can stimulate glucose transport, promote triglyceride accumulation in adipocytes, and inhibit lipolysis.

[0004] Complement system dysregulation plays a crucial role in the pathogenesis of IgA nephropathy (IgAN), lupus nephritis (LN), and paroxysmal nocturnal hemoglobinuria (PNH). Renal pathological examinations of IgAN and LN frequently reveal deposition of complement components and immune complexes. Complement is a direct cause of PNH hemolysis, C5aR is involved in amplifying complement system damage, and CFB and CFD are important components of the complement alternative pathway, directly involved in complement activation regulation. Therefore, C5aR, CFB, and CFD are closely related to the pathogenesis of IgAN, LN, and PNH.

[0005] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening blood disorder characterized by complement-driven hemolysis, thrombosis, and bone marrow dysfunction, causing anemia, fatigue, and other debilitating symptoms that can severely impact the patient's quality of life. Currently, the main treatments marketed for PNH are the monoclonal antibody drugs Soliris and Ultomiris. Of these, Soliris was first approved for market in 2007 and has been approved for a variety of ultra-rare diseases, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and neuromyelitis optica spectrum disorder (NMOSD). Ultomiris is an upgraded version of Soliris, a second-generation long-acting C5 complement inhibitor, and was first approved for market at the end of 2018, with approved indications including PNH and aHUS. Despite current standard anti-C5 therapy, many PNH patients experience anemia and are dependent on blood transfusions.

[0006] Currently, there are no small molecule inhibitors on the market that inhibit complement factor D. The target is the complement alternative pathway, a major cause of complement-driven renal disease (CDRD). Therefore, the development of small molecule inhibitors with superior biological activity is of great significance for the treatment of this disease. At the same time, developing pharmaceutical forms suitable for drug development of these compounds is a technical problem that must be addressed urgently. [Overview of the Initiative]

[0007] The technical problem that this invention aims to solve is to provide nitrogen-containing spirocrystalline compounds, a method for producing the same, and a method for using the same, in order to overcome the drawbacks of the limited variety and scarcity of crystals of existing complement factor D inhibitors. The nitrogen-containing spirocrystalline compounds of this invention have good stability and complement factor D inhibitory activity, good inhibitory effect against rabbit erythrocyte hemolysis, high in vivo exposure levels, and high oral bioavailability.

[0008] The present invention provides a type I crystal of a nitrogen-containing spiro compound represented by formula A, in which the powder X-ray diffraction (XRPD) pattern displayed at a 2θ angle using Cu-Kα rays has diffraction peaks at positions of 5.67±0.20°, 11.37±0.20°, 16.69±0.20°, 17.32±0.20°, and 19.73±0.20°.

[0009] [ka]

[0010] Using the Cu-Kα rays of the aforementioned type I crystal, the powder X-ray diffraction (XRPD) pattern, represented by a 2θ angle, also has diffraction peaks at one or more of the following positions: 12.01±0.20°, 13.89±0.20°, 17.05±0.20°, 19.42±0.20°, 23.41±0.20°, and 27.86±0.20°.

[0011] Using the Cu-Kα rays of the aforementioned type I crystal, the powder X-ray diffraction (XRPD) pattern, represented by a 2θ angle, also has diffraction peaks at one or more of the following positions: 18.04±0.20°, 18.43±0.20°, 22.94±0.20°, and 26.01±0.20°.

[0012] Using the Cu-Kα line of the type I crystal, the powder X-ray diffraction (XRPD) pattern represented by the 2θ angle has diffraction peaks at positions of 5.67±0.20°, 11.37±0.20°, 12.01±0.20°, 13.89±0.20°, 16.69±0.20°, 17.05±0.20°, 17.32±0.20°, 18.04±0.20°, 18.43±0.20°, 19.42±0.20°, 19.73±0.20°, 22.94±0.20°, 23.41±0.20°, 26.01±0.20° and 27.86±0.20°.

[0013] Using the Cu-Kα line of the type I crystal, the diffraction peak positions of the powder X-ray diffraction (XRPD) pattern represented by the 2θ angle are as shown in the following table.

[0014]

Table 1-1

[0015] Using the Cu-Kα line of the type I crystal, the diffraction peak positions and relative intensities of the powder X-ray diffraction (XRPD) pattern represented by the 2θ angle are as shown in the following table.

[0016]

Table 1-2

[0017] Using the Cu-Kα line of the type I crystal, the powder X-ray diffraction (XRPD) pattern represented by the 2θ angle is basically as shown in Figure 2.

[0018] The differential scanning calorimetry (DSC) pattern of the type I crystal has an endothermic peak at 231.1±2°C.

[0019] The differential scanning calorimetry (DSC) pattern of the type I crystal is basically as shown in Figure 3.

[0020] The thermogravimetric analysis (TGA) pattern of the type I crystal has no weight loss before 200±2°C.

[0021] The thermogravimetric analysis (TGA) pattern of the aforementioned type I crystal is basically as shown in Figure 3.

[0022] The present invention provides a type II crystal of a nitrogen-containing spicion compound represented by formula A, in which the powder X-ray diffraction (XRPD) pattern displayed at a 2θ angle using Cu-Kα rays has diffraction peaks at positions 11.32±0.20°, 11.70±0.20°, 11.93±0.20°, 18.03±0.20°, 18.81±0.20°, and 19.17±0.20°.

[0023] [ka]

[0024] Using the Cu-Kα rays of the aforementioned Type II crystal, the powder X-ray diffraction (XRPD) pattern, represented by a 2θ angle, also has diffraction peaks at one or more of the following positions: 5.54±0.20°, 12.37±0.20°, 13.78±0.20°, 14.11±0.20°, 15.78±0.20°, and 20.80±0.20°.

[0025] Using the Cu-Kα rays of the aforementioned Type II crystal, the powder X-ray diffraction (XRPD) pattern, represented by a 2θ angle, also has diffraction peaks at one or more of the following positions: 13.55±0.20°, 16.40±0.20°, 17.74±0.20°, 20.49±0.20°, and 22.06±0.20°.

[0026] Using the Cu-Kα rays of the aforementioned Type II crystal, the powder X-ray diffraction (XRPD) pattern, represented by a 2θ angle, also has diffraction peaks at the following positions: 5.54±0.20°, 11.32±0.20°, 11.70±0.20°, 11.93±0.20°, 12.37±0.20°, 13.55±0.20°, 13.78±0.20°, 14.11±0.20°, 15.78±0.20°, 16.40±0.20°, 17.74±0.20°, 18.03±0.20°, 18.81±0.20°, 19.17±0.20°, 20.49±0.20°, 20.80±0.20°, and 22.06±0.20°.

[0027] The diffraction peak positions of the powder X-ray diffraction (XRPD) pattern, displayed at a 2θ angle using the Cu-Kα line of the aforementioned Type II crystal, are as shown in the table below.

[0028] [Table 1-3]

[0029] The diffraction peak positions and relative intensities of the powder X-ray diffraction (XRPD) patterns, displayed at a 2θ angle using the Cu-Kα line of the aforementioned Type II crystal, are shown in the table below.

[0030] [Table 1-4]

[0031] The powder X-ray diffraction (XRPD) pattern, displayed at a 2θ angle using the Cu-Kα line of the aforementioned Type II crystal, is basically as shown in Figure 6.

[0032] The differential scanning calorimetry (DSC) pattern of the aforementioned Type II crystal has an endothermic peak at 230.7 ± 2°C.

[0033] The differential scanning calorimetry (DSC) pattern of the aforementioned Type II crystal has an endothermic peak at 159.1±2°C.

[0034] The differential scanning calorimetry (DSC) pattern of the aforementioned Type II crystal is basically as shown in Figure 7.

[0035] The thermogravimetric analysis (TGA) pattern of the aforementioned Type II crystal shows a 1.71% decrease in weight before 210±2℃.

[0036] The thermogravimetric analysis (TGA) pattern of the aforementioned Type II crystal is basically as shown in Figure 7.

[0037] The present invention provides a method for producing type I crystals of a nitrogen-containing spiro compound represented by formula A, comprising the steps of dissolving the nitrogen-containing spiro compound represented by formula A in N-methylpyrrolidone and adding a poor solvent to crystallize it, wherein the poor solvent is selected from one or more of nitrile solvents, ester solvents, benzene solvents, ether solvents, and water.

[0038] The nitrile solvent may be acetonitrile.

[0039] The aforementioned ester solvent is one or more of ethyl acetate, n-propyl acetate, and isopropyl acetate, for example, ethyl acetate and / or isopropyl acetate.

[0040] The benzene-based solvent is one or more of toluene, ethylbenzene, xylene, cumene, and chlorobenzene, for example, toluene.

[0041] The ether-based solvent is one or more of tert-butyl methyl ether, diethyl ether, cyclopentyl methyl ether, isopropyl ether, methyl tetrahydrofuran, and dioxane, for example, tert-butyl methyl ether.

[0042] In the method for producing the type I crystal, the dissolution method may be heating and stirring. The heating temperature may be 30 to 70°C, preferably 40 to 60°C, for example 50°C.

[0043] In the method for producing the type I crystal, the volume mass ratio of N-methylpyrrolidone to the nitrogen-containing spiro compound represented by formula A may be 15 to 50 mL / g, preferably 20 to 35 mL / g, for example, 26.7 mL / g.

[0044] In the method for producing the type I crystal, the volume mass ratio of the poor solvent to the nitrogen-containing spiro compound represented by formula A may be 50 to 350 mL / g, preferably 80 to 250 mL / g, for example 80 mL / g, 160 mL / g, or 213.3 mL / g.

[0045] In the method for producing the type I crystal, the volume ratio of the poor solvent to N-methylpyrrolidone may be (1 to 10):1, preferably (3 to 8):1, for example 3:1, 6:1, or 8:1.

[0046] The method for producing the type I crystal may include a further cooling step before adding the poor solvent. The cooling may be to 10-20°C, for example, 17°C. The cooling rate may be 0.2°C / min.

[0047] The present invention provides a method for producing type II crystals of a nitrogen-containing spiro compound represented by formula A, comprising the step of stirring the nitrogen-containing spiro compound represented by formula A in acetonitrile for 12 to 16 days.

[0048] In the method for producing the type II crystal, the volume mass ratio of acetonitrile to the nitrogen-containing spiro compound represented by formula A may be 15 to 50 mL / g, preferably 25 to 40 mL / g, for example, 33.3 mL / g.

[0049] In the method for producing the type II crystal, the stirring temperature may be a normal temperature used for such operations in the art, preferably 15 to 30°C, for example, 17°C.

[0050] In the method for producing the type II crystal, the stirring time may be 14 days.

[0051] The present invention relates to (1) a type I or type II crystal of a nitrogen-containing spiro compound represented by formula A, (2) To provide a pharmaceutical composition containing a pharmaceutically acceptable carrier.

[0052] The present invention provides the use of type I or type II crystals of a nitrogen-containing spiro compound represented by formula A in the manufacture of a medicament for treating and / or preventing a disease mediated by complement factor D, wherein the disease mediated by complement factor D is a blood disorder, kidney disease, cardiovascular disease, immune disorder, central nervous system disorder, respiratory disease, genitourinary tract disorder, or eye disease.

[0053] In the use of type I or type II crystals in the manufacture of a pharmaceutical product for treating and / or preventing a complement factor D-mediated disease, the complement factor D-mediated disease is cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoimmune hemolytic anemia due to warm antibodies, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus erythematosus or lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, organ transplant rejection, and severe Myasthenia gravis, Alzheimer's disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, emphysema, coronavirus infection (e.g., SARS-CoV, MERS-CoV, or SARS-CoV-2 infection), macular degeneration, age-related macular degeneration (AMD), macular edema, diabetic macular edema, choroidal neovascularization (CNV), uveitis, Behçet's disease uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, corneal transplant rejection, corneal dystrophy, autoimmune dry eye, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fuchs corneal endothelial dystrophy, retinal vein occlusion, or postoperative inflammation.

[0054] The present invention provides the use of type I or type II crystals of a nitrogen-containing spiro compound represented by formula A in the manufacture of a pharmaceutical product for treating and / or preventing a disease, the disease being a blood disorder, kidney disease, cardiovascular disease, immune disorder, central nervous system disorder, respiratory disease, genitourinary tract disorder, or eye disease.

[0055] In the use of type I or type II crystals in the manufacture of a pharmaceutical product for the treatment and / or prevention of a disease, the disease is cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoimmune hemolytic anemia due to warm antibodies, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus erythematosus or lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, organ transplant rejection, myasthenia gravis, or Alzheimer's disease. This may include respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, emphysema, coronavirus infection (e.g., SARS-CoV, MERS-CoV, or SARS-CoV-2 infection), macular degeneration, age-related macular degeneration (AMD), macular edema, diabetic macular edema, choroidal neovascularization (CNV), uveitis, Behçet's disease uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, corneal transplant rejection, corneal dystrophy, autoimmune dry eye, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fuchs corneal endothelial dystrophy, retinal vein occlusion, or postoperative inflammation.

[0056] The present invention provides the use of type I or type II crystals of a nitrogen-containing spiro compound represented by formula A in the production of a complement factor D inhibitor.

[0057] The complement factor D inhibitor can be used in vivo in mammals and can also be used in vitro, mainly for experimental purposes, for example, as a standard or control sample for comparison, or can be manufactured into a kit by conventional methods in the art to provide rapid detection of the inhibitory effect of complement factor D.

[0058] The crystals of the present invention can be identified through one or more solid-state analytical methods, such as powder X-ray diffraction, single-crystal X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. Those skilled in the art will understand that the peak intensity and / or peak configuration of powder X-ray diffraction may vary depending on experimental conditions. At the same time, there is an error of approximately ±0.20° in the measured 2θ values ​​due to differences in the accuracy of the measuring instruments. The relative intensity values ​​of the peaks depend more on specific characteristics of the measured sample, such as crystal size and purity, than on the peak position, and therefore the measured peak intensities may have a deviation of approximately ±20%. Despite the existence of experimental errors, instrument errors, and orientation preferences, those skilled in the art can obtain sufficient information from the powder X-ray diffraction data provided in this patent to identify each crystal. In DSC measurements, the onset temperature, maximum temperature, and heat of dissolution data of the endothermic peaks actually measured may vary to some extent depending on the heating rate, crystal morphology and purity, and other measurement parameters.

[0059] Explanation of terms: The term "basically" means that the peak position on the graph may change slightly due to minute variations in measuring equipment, measurement conditions, and the lot of the product being measured, and therefore should not be considered an absolute value.

[0060] The term "room temperature" refers to a range of 15-30°C, such as 17°C or 25°C.

[0061] The term "treatment" refers to therapeutic therapy. With respect to a particular disease, treatment means (1) alleviating one or more biological expressions of the disease or symptoms; (2) (a) interfering with one or more points in the biological cascade that leads to or causes the disease or (b) interfering with one or more biological expressions of the disease; (3) improving one or more symptoms, effects or side effects associated with the disease, or one or more symptoms, effects or side effects associated with the disease or its treatment; or (4) reducing the disease or one or more biological expressions of the disease.

[0062] The term "prevention" refers to reducing the risk of acquiring or developing a disease or disability.

[0063] The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, domestic rabbits, guinea pigs, monkeys, and humans, with humans being preferred.

[0064] The aforementioned preferred conditions can be combined in any way, without violating the ordinary knowledge of the art, to obtain each preferred embodiment of the present invention.

[0065] The reagents and raw materials used in this invention are commercially available.

[0066] Positive progressive effects of the present invention: The nitrogen-containing spiro compound crystals of the present invention have better stability and complement factor D inhibitory activity, exhibit good inhibitory effects against rabbit erythrocyte hemolysis, and have higher in vivo exposure and higher oral bioavailability. [Brief explanation of the drawing]

[0067] [Figure 1] This is the amorphous XRPD pattern of the compound represented by formula A. [Figure 2] This is the XRPD pattern of a type I crystal of the compound represented by formula A. [Figure 3] These are the TGA and DSC patterns of a type I crystal of the compound represented by formula A. [Figure 4] This is the 1H-NMR pattern (DMSO-d6) of a type I crystal of the compound represented by formula A. [Figure 5] This is the PLM pattern of a type I crystal of the compound represented by formula A. [Figure 6] This is the XRPD pattern of a type II crystal of the compound represented by formula A. [Figure 7] These are the TGA and DSC patterns of the type II crystal of the compound represented by formula A. [Figure 8] This is the 1H-NMR pattern (DMSO-d6) of a type II crystal of the compound represented by formula A. [Figure 9] This is a comparative XRPD pattern of type II and type I crystals of the compound represented by formula A. [Figure 10] This is a comparative DSC pattern of type II and type I crystals of the compound represented by formula A. [Figure 11] These are comparative XRPD patterns showing the crystallization changes of Type II crystals after heating to 140°C and 200°C. [Figure 12] These are the DSC and TGA patterns obtained by measuring the solid obtained after heating a type II crystal to 140°C. [Figure 13] These are comparative DSC patterns obtained by measuring the solids after heating Type II crystals to 140°C and 200°C. [Figure 14] This is the XRPD pattern of a wet polishing experiment of type I crystals in ethanol. [Figure 15] This is the XRPD pattern obtained from a wet polishing experiment of type I crystals in water. [Figure 16] This is the XRPD pattern of a tablet made from type I crystals. [Figure 17] This is the HPLC pattern of a type I crystal left open for 3 days. [Figure 18] This is the XRPD pattern of a type I crystal left open for 3 days. [Figure 19] This is the HPLC pattern of a type I crystal left open for 7 days. [Figure 20]This is the XRPD pattern of a type I crystal left open for 7 days. [Figure 21] This is the HPLC pattern of a type I crystal left open for 14 days. [Figure 22] This is the XRPD pattern of a type I crystal left open for 14 days. [Figure 23] This is the XRPD pattern of a solid formed by suspending a type I crystal in the biological solvent FaSSIF. [Figure 24] This is the XRPD pattern of a solid formed by suspending a type I crystal in the biological solvent FeSSIF. [Figure 25] This is the XRPD pattern of a solid formed by suspending a type I crystal in water. [Figure 26] This is the stability HPLC pattern of type I crystals in the biosolvent SGF. [Figure 27] This is the stability HPLC pattern of type I crystals in the biological solvent FaSSIF. [Figure 28] This is the stability HPLC pattern of type I crystals in the biological solvent FeSSIF. [Figure 29] This is the HPLC pattern of the stability of type I crystals in water. [Figure 30] This is the DVS pattern of a type I crystal. [Figure 31] This shows the XRPD patterns of a type I crystal before and after DVS measurement. [Modes for carrying out the invention]

[0068] The present invention will be further described below with reference to embodiments, but this does not limit the present invention to the scope of the above embodiments. In the following embodiments, experimental methods for which specific conditions are not described are selected according to conventional methods and conditions or according to the product description.

[0069] Abbreviation: ACN: Acetonitrile, DMSO: Dimethyl sulfoxide, IPA: Isopropyl alcohol, HPLC: High-performance liquid chromatography, NMP: N-methylpyrrolidone, RH: Relative humidity, aw: Water activity, PLM: Polarizing microscope, TGA: Thermogravimetric analysis, Tonset: Start temperature / Initial temperature. XRPD: Powder X-ray diffraction. DSC: Differential scanning calorimetry, DVS: Dynamic water adsorption.

[0070] Analysis method: Powder X-ray diffraction (XRPD) Powder X-ray diffraction patterns were obtained using a Bruker D2 Phaser, and the instrument parameters are as shown in the table below.

[0071] [Table 1-5]

[0072] Differential Scanning Calorimetry (DSC) The DSC curve is obtained by collecting samples using the DSC250 model of the TA instrument. The sample is weighed and placed in the sample pan, and after accurately measuring the weight, the weight is recorded. The sample is heated from 25°C to 250°C at a heating rate of 10°C / min.

[0073] Thermogravimetric analyzer (TGA) The TGA curve is obtained by collecting samples using the TGA550 TA instrument. The sample is weighed into a sample pan (Al2O3) and heated to 300°C at a heating rate of 10°C / min.

[0074] 1 1H NMR Nuclear magnetic field results were obtained using a Varian 400 MHz model, with deuterated DMSO as the solvent.

[0075] Polarizing microscope (PLM) PLM analysis is performed using an Otter optical microscope, BK-Pol. A small sample is taken and placed on a glass slide, silicone oil is added dropwise to disperse it, a coverslip is placed over it, and it is observed under the microscope.

[0076] LCMS The LCMS curve was obtained using a Shimadzu instrument. The instrumentation and parameters are shown in the table below.

[0077] [Table 1-6]

[0078] HPLC Shimadzu instruments were used for sample analysis and detection, and both stability and solubility experiments were measured using the methods described below. The HPLC instrument methods and parameters are shown in the table below.

[0079] [Table 1-7]

[0080] Dynamic moisture adsorbent (DVS) The sample is analyzed using an Intrinsic DVS (System Measurement System UK). A 20-30 mg test sample is taken, placed in a sample tray, and suspended in the measurement chamber. The chamber temperature is controlled between 25 ± 1°C, and the relative humidity is increased from 0% to 90% at a rate of 10% / h, then cooled to 0%. Quality data is recorded every 20 seconds. Each gradient is maintained for 60 minutes.

[0081] Example 1: Preparation of 2-(2-(3'-(aminomethyl)-5-(6-azaspiro[2.5]octan-6-yl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic acid (compound represented by formula A)

[0082] [ka]

[0083] 1.1: Synthesis of Compound 1-1 Compound 1-a (11.16 g, 60 mmol), triethylamine (11.00 g, 108 mmol), and dichloromethane (120 mL) were placed in a 500 mL reaction flask. Di-tert-butyl dicarbonate (15.71 g, 72 mmol) was added to the reaction system under an ice bath, and the reaction solution was allowed to react at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure, and ethyl acetate (150 mL) and water (200 mL) were added to the residue to separate it. The organic phase was separated by extraction. The aqueous phase was extracted with ethyl acetate (50 mL x 2), and the combined organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 (v / v)) to obtain compound 1-1.

[0084] 1.2: Synthesis of Compounds 1-2 Compound 1-1 (18.02 g, 60 mmol), bis(pinacolate)diborone (18.28 g, 72 mmol), potassium acetate (11.78 g, 120 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (4.40 g, 6 mmol), and 1,4-dioxane (120 mL) were added to a 500 mL reaction flask, purged three times with nitrogen gas, and the reaction solution was reacted at 80°C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Ethyl acetate (150 mL) and water (200 mL) were added, and the organic phase was separated by extraction. The aqueous phase was extracted with ethyl acetate (50 mL x 2), the combined organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1 (v / v)) to obtain compounds 1-2. LC / MS(ESI+)m / z:[M+Ht-Bu] + = 278.15.

[0085] 1.3: Synthesis of Compounds 1-3 Compounds 1-b (5.00 g, 18.8 mmol), 1-c (3.12 g, 18.8 mmol), and triphenylphosphine (9.87 g, 37.6 mmol) were sequentially dissolved in dichloromethane (70 mL). Under an ice bath, a solution of di-(4-chlorobenzyl)azodicarboxylic acid (13.7 g, 37.6 mmol) in dichloromethane (70 mL) was added dropwise to the reaction system. After the addition was complete, the reaction was continued at 0°C for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 10 / 1 (v / v)) to obtain compounds 1-3. LC / MS (ESI+) m / z: [M+H] + = 414.90.

[0086] 1.4: Synthesis of Compounds 1-4 Compounds 1-3 (2.00 g, 4.8 mmol), 1-2 (1.30 g, 3.9 mmol), potassium carbonate (1.30 g, 9.6 mmol), and dichloro[1,1'-bis(diphenylfospino)ferrocene]palladium(II) (0.35 g, 0.48 mmol) were added to 1,4-dioxane / water (30 mL / 3 mL), the mixture was purged three times with nitrogen gas, and the reaction solution was reacted at 80°C for 2 hours. After the reaction solution was cooled to room temperature, it was filtered, the filtrate was diluted with water (30 mL), and then extracted with ethyl acetate (30 mL x 2). The combined organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 5 / 1 (v / v)) to obtain compound 1-4. LC / MS(ESI+) m / z:[M+H-Boc] + = 439.95.

[0087] 1.5: Synthesis of Compounds 1-5 Compounds 1-4 (4.3g, 7.96 mmol), 6-aza-spiro[2.5]octane hydrochloride (compound 1-d, 1.4g, 9.55 mmol), potassium carbonate (3.3g, 23.87 mmol), tris(dibenzylideneacetone)dipalladium(0) (400 mg, 10% by weight), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (400 mg, 10% by weight) were sequentially added to 1,4-dioxane (50%). The mixture was purged three times with nitrogen gas, and the reaction solution was reacted at 90°C for 4 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compounds 1-5. LC / MS(ESI+) m / z:[M+H] + = 571.15.

[0088] 1.6: Synthesis of Compounds 1-6 Compound 1-5 (3.5 g, 6.1 mmol) was dissolved in dichloromethane (27 mL), trifluoroacetic acid (9 mL) was added, and the mixture was reacted at room temperature for 1 hour. The reaction solution was adjusted to pH 8 by adding saturated sodium bicarbonate aqueous solution, then extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1-6 (3.2 g, crude product), which was used directly in the next step. LC / MS (ESI+) m / z:[M+H] + = 471.10.

[0089] 1.7: Synthesis of Compound A Compound 1-6 (3.1 g, 6.5 mmol) and sodium hydroxide (1.05 g, 26.3 mmol) were added to tetrahydrofuran (10 mL), methanol (5 mL), and water (5 mL), and the reaction solution was allowed to react at 60°C for 4 hours. The reaction solution was purified by preparative HPLC, lyophilized, and then the amorphous form of the compound represented by formula A was obtained. The XRPD pattern is shown in Figure 1.

[0090] 1H NMR (400 MHz, DMSO-d6):δ 8.27 (s, 0.59H), 8.10 (s, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.37-7.40 (m, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.08-7.14 (m, 3H), 7.00 (s, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.81 (t, J = 7.6 Hz, 1H), 5.13 (s, 2H), 3.97 (s, 2H), 3.43 (s, 2H), 3.30 (t, J = 5.2 Hz, 4H), 1.48 (t, J = 5.2 Hz, 4H), 0.35 (s, 4H);LC / MS(ESI+) m / z:[M+H] + = 457.10.

[0091] Example 2: Preparation of type I crystals of the compound represented by formula A 2.1 Manufacturing of Type I Crystals Approximately 15 mg of the amorphous compound represented by formula A obtained in Example 1 was weighed into a sample bottle, 0.5 mL of n-heptane was added, and the mixture was stirred for 7 days and 14 days at room temperature (approximately 17°C) and 50°C, respectively. The resulting solid was used for XRPD analysis. The results showed that all the obtained solids were type I crystals. The specific experimental conditions and results are shown in Tables 1, 3, and 4 below.

[0092] [Table 1-8]

[0093] 2.2 Manufacturing of Type I Crystals Approximately 15 mg of the amorphous compound represented by formula A obtained in Example 1 was weighed and placed in a sample bottle. 0.4 mL of N-methylpyrrolidone was added at 50°C to completely dissolve the amorphous compound represented by formula A. After cooling to room temperature (approximately 17°C), a predetermined volume of poor solvent was added, and the mixture was stirred until a solid precipitated. The results showed that all the obtained solids were type I crystals. The specific experimental conditions and results are shown in Tables 2, 3, and 4 below.

[0094] [Table 2]

[0095] 2.3 The characterization data for the type I crystal is shown in Table 3 below, and the XRPD pattern analysis data for the type I crystal is shown in Table 4.

[0096] [Table 3]

[0097] [Table 4]

[0098] Example 3: Preparation of Type II crystals of the compound represented by formula A 3.1 Manufacturing of Type II Crystals Approximately 15 mg of the amorphous compound represented by formula A obtained in Example 1 was weighed and placed in a sample bottle. 0.5 mL of acetonitrile was added, and the mixture was stirred at room temperature (approximately 17°C) for 14 days. XRPD detection was then performed on the resulting solid product. The results indicated that the obtained solid was a type II crystal.

[0099] 3.2 The characterization data for the type II crystal is shown in Table 5 below, and the XRPD pattern analysis data for the type II crystal is shown in Table 6.

[0100] [Table 5]

[0101] [Table 6]

[0102] Example 4: Comparison of Type I and Type II crystals and study of their conversion relationships 4.1 Comparison of pattern data between Type I and Type II crystals When comparing type I crystals and type II crystals, there are significant differences in the range of the 2θ angle between the two patterns between 16 - 18° and 22 - 24°, as shown at the 2θ angle within the XRPD comparison pattern frame of type II crystals and type I crystals in Figure 9. The endothermic peak temperatures of both are similar, but type II crystals had one exothermic peak at 159℃ as shown in the DSC comparison pattern of Figure 10.

[0103] 4.2 Heating experiment of type II crystals A heating experiment was carried out on type II crystals. The type II crystal sample was heated to 140℃ using the TGA measurement method and to 200℃ using the DSC measurement method. XRPD, TGA, DSC, 1 and HNMR tests were performed on the obtained solid. The test results showed that when heated to 140℃, the XRPD pattern indicated that the crystal did not change, no weight loss was seen in TGA, 1 no acetonitrile solvent residue peak was seen in the HNMR pattern, and the exothermic peak at 159℃ could still be observed in the DSC pattern. When heated to 200℃, the XRPD pattern indicated that the obtained solid was type I crystal, 1 no residual solvent peak of acetonitrile was found in the H NMR pattern, and the endothermic peak at 159℃ disappeared in the DSC pattern. Therefore, it can be seen that the exothermic peak at 159℃ is the crystal conversion peak, and the endothermic peak at 230℃ is the melting peak of type I crystals. The XRPD comparison pattern of the heating experiment is as shown in Figure 11, the DSC and TGA patterns obtained by measuring the solid obtained after heating to 140℃ are as shown in Figure 12, and the DSC comparison pattern of the heating experiment is as shown in Figure 13.

[0104] Effect example 1: Evaluation of crystals 1. Moisture activity test Organic solvents and water were mixed in molar ratios of 0.001:0.999, 0.2:0.8, 0.5:0.5, 0.8:0.2, and 0.999:0.001. The type I crystals obtained in Example 2 were added to the solution to form a suspension, and the mixtures were stirred at 25°C and 50°C for 30 minutes, respectively. The mixtures were then filtered to obtain the filtrate. Approximately 10 mg of the type I crystals obtained in Example 2 was weighed and added to the filtrate. The mixtures were stirred for 5 days and 11 days, respectively, and the results were detected. The results showed that all solids obtained under the following conditions were type I crystals. The specific experimental conditions and results are shown in Tables 7 and 8 below.

[0105] [Table 7]

[0106] [Table 8]

[0107] 2. Polishing and crystal transition experiments 2.1 Wet polishing Approximately 20 mg of the type I crystals obtained in Example 2 were weighed and placed in a mortar. 0.04 mL of water or ethanol was added, and the crystals were manually polished for 2 minutes, 5 minutes, and 10 minutes. The XRPD was then measured for each step. Whenever the solvent completely evaporated during the polishing process, 0.04 mL of water or ethanol was added to the mortar. The results showed that the type I crystals remained unchanged even after 10 minutes of polishing, and the crystallinity did not significantly decrease. The XRPD patterns of the wet polishing experiment of type I crystals in ethanol are shown in Figure 14, and the XRPD patterns of the wet polishing experiment of type I crystals in water are shown in Figure 15.

[0108] 2.2 Tablet Compression Experiment Approximately 30 mg of type I crystals obtained in Example 2 were weighed, compressed into tablets at 40 MPa, and then subjected to XRPD analysis. The results showed that the type I crystals remained unchanged after tableting, and the crystallinity did not significantly decrease. The XRPD results are shown in Figure 16.

[0109] 3. Stability experiment An appropriate amount of the type I crystals obtained in Example 2 was weighed and placed in a sample bottle. The bottles were left open for 3 days, 7 days, and 14 days under conditions of 60°C, 25°C / 60%RH, 40°C / 75%RH, and 25°C / 90(±5)RH%, respectively, and then the purity was measured by HPLC. The results showed that after 14 days under the above conditions, the purity of the main peak did not change significantly, and the crystals themselves did not change. The stability conditions and results are shown in Table 9 below. The HPLC pattern of type I crystals left open for 3 days is shown in Figure 17. The XRPD pattern of type I crystals left open for 3 days is shown in Figure 18. The HPLC pattern of type I crystals left open for 7 days is shown in Figure 19. The XRPD pattern of type I crystals left open for 7 days is shown in Figure 20. The HPLC pattern of type I crystals left open for 14 days is shown in Figure 21. The XRPD pattern of type I crystals left open for 14 days is shown in Figure 22.

[0110] [Table 9]

[0111] Experimental conclusion: After leaving the samples under the above conditions for 14 days, the purity of the main peak of the type I crystals did not change significantly, and the crystals themselves remained unchanged, indicating good stability.

[0112] 4. Solubility and stability experiments in biological solvents and water 4.1 Solubility experiments in biological solvents and water Approximately 5 mg of the type I crystals obtained in Example 2 were weighed and placed in sample bottles. Suspensions with a concentration of 5 mg / mL were prepared by adding SGF, FaSSIF, FeSSIF, and water, respectively. After thoroughly stirring the SGF, FaSSIF, FeSSIF, and water for 0.5 hours, 2 hours, and 24 hours, the samples were removed, their solubility was measured by HPLC, and solid crystals in the suspension were detected by XRPD. The type I crystals dissolved completely in SGF and remained suspended in FaSSIF, FeSSIF, and water. The solubility of type I crystals was higher in SGF (>5 mg / mL), while their solubility in FaSSIF, FeSSIF, and water was less than 0.73 mg / mL. After stirring the FaSSIF, FeSSIF, and water for 24 hours, none of the crystals changed. The solubility results are shown in Table 10 below. The XRPD pattern of the suspension solid of type I crystals in the biosolvent FaSSIF is shown in Figure 23. The XRPD pattern of the suspension solid of type I crystals in the biosolvent FeSSIF is shown in Figure 24. The XRPD pattern of the suspension solid of type I crystals in water is shown in Figure 25.

[0113] [Table 10]

[0114] 4.2 Measurement of biosolvent stability Approximately 2.5 mg of the type I crystals obtained in Example 2 were weighed and added to 0.5 mL each of SGF, FaSSIF, FeSSIF, and water. After stirring thoroughly for 0.5 hours, 2 hours, and 24 hours, the mixed solutions were diluted to 10 mL, and their purity was measured by HPLC. The results showed that the major peak purity of the type I crystals did not decrease significantly with SGF, FaSSIF, and water, but with FeSSIF, the major peak purity gradually decreased from 99.56% to 98.04% as the stirring time increased. The stability conditions and results are shown in Table 11 below. The stability HPLC pattern of type I crystals in the biological solvent SGF is shown in Figure 26, the stability HPLC pattern of type I crystals in the biological solvent FaSSIF is shown in Figure 27, the stability HPLC pattern of type I crystals in the biological solvent FeSSIF is shown in Figure 28, and the stability HPLC pattern of type I crystals in water is shown in Figure 29. The main impurities are shown in the frames of Figures 26-29.

[0115] [Table 11]

[0116] 5. Hygroscopicity experiment The hygroscopicity of the type I crystals obtained in Example 2 was measured by DVS, and XRPD analysis was performed on the solids before and after the DVS measurement. The results showed that the type I crystals had slight hygroscopicity, with a weight increase of 0.55% under conditions of 0-80%RH, and the crystals did not change before and after the DVS measurement. The DVS results are shown in Figure 30, and the XRPD comparison patterns of the type I crystals before and after the DVS measurement are shown in Figure 31.

[0117] 6. Stability experiments of type I crystals in DMSO under high-temperature conditions Approximately 100 mg of the type I crystals obtained in Example 2 were weighed and placed in a sample bottle. DMSO (2 mL) was added to create a suspension, and the mixture was stirred at 50°C for 24 hours and 48 hours, respectively. LC-MS detection was then performed on the solid and filtrate. The results showed that after stirring the type I crystals with DMSO for 48 hours, the purity of the solid did not change significantly, but a small amount of impurities appeared in the filtrate. The measured purities are shown in Table 12 below.

[0118] [Table 12]

[0119] Experimental conclusion: The purity of the type I crystal of the present invention showed very little change under high-temperature conditions and demonstrated good stability.

[0120] Example of effect 2: Biological activity test 1. In vitro screening experiment of complement factor D inhibitory activity (C3b analysis) 1.1 Experimental materials and equipment V-bottom plate (AXYGEN), DMSO (Sigma), MicroVue Bb Plus ELISA kit (Quidel), complement factor C3b (abbreviated as factor C3b, Complement tech), complement factor B (abbreviated as factor B, Complement tech), complement factor D (abbreviated as factor D, Complement tech), EDTA (McLean), GVBo (Complement tech), EGTA (Aladdin), MgCl2 (Aladdin), NaOH (Sinopharm), microplate reader (Molecular Devices, SpectraMax i3x), microplate thermostatic shaker (Thermo, MB100-2A), pipette (Gilson).

[0121] 1.2 Preparation before the experiment 1.2.1 Manufacturing of Mg-EGTA 0.1M Mg-EGTA: 3.8g of EGTA, 0.9521g of MgCl2, and 0.7g of NaOH were weighed out, the pH was adjusted to 7.5 with NaOH, distilled water was added to 100mL, the mixture was filtered through a 0.2μm sterile filter, and the solution was divided into smaller portions and stored at 4°C.

[0122] 1.2.2 Preparation of the working solution Buffer: A 0.1M Mg-EGTA solution was diluted 10-fold with GVBo buffer to make a 10mM solution.

[0123] Factor B working solution: A 1 mg / mL factor B solution (10.75 μM) was diluted 6.7 times with buffer to a 1.6 μM solution.

[0124] Factor C3b working solution: A 1 mg / mL factor C3b solution (5.68 μM) was diluted five-fold with buffer to a concentration of 1.12 μM.

[0125] Factor D working solution: A 0.1 mg / mL factor D solution (4.17 μM) was diluted 1303 times with buffer to a concentration of 3.2 nM.

[0126] Note: a. The dilution ratios above are for reference only and must be adjusted according to the actual concentration indicated on the reagent.

[0127] b. Factor B: 93KDa, factor D: 24KDa, factor C3b: 176KDa.

[0128] 1.2.3 Preparation of stop fluid Stop solution: An appropriate amount of EDTA powder was weighed and dissolved in a predetermined amount of GVBo buffer. The pH was then adjusted to 7.5 with NaOH, and the mixture was stirred until clear to prepare a 10 mM solution.

[0129] 1.2.4 Manufacturing of Compounds Compound stock solution: A 40 mM compound solution was diluted with DMSO to prepare a 1 mM stock solution. The 1 mM stock solution was then diluted three times with DMSO to prepare eight different concentrations of compound stock solution.

[0130] Compound working solution: Compound solutions of various concentrations were obtained by diluting the compound 250-fold with buffer solution.

[0131] 1.3 Experimental Steps In a V-bottom plate, 10 μL of factor D solution and 10 μL of compound solution were added to the experimental group, 10 μL of factor D solution and a buffer containing 0.4% DMSO were added to the positive control group, and 20 μL of a buffer containing 0.2% DMSO was added to the blank control group. The samples were incubated at 37°C for 15 minutes. Factor B working solution and factor C3b working solution were mixed in a 1:1 ratio, and 20 μL of the mixture was added to each well. The samples were incubated at 37°C for 30 minutes, and the reaction was stopped by adding 40 μL of stop solution. The amount of Bb produced was detected using the MicroVue Bb Plus ELISA kit.

[0132] 1.4 ELISA detection 1.4.1 After removing the required ELISA plate wells and allowing them to equilibrate at room temperature, the remaining wells were repackaged and stored at 4°C.

[0133] 1.4.2 The samples were washed twice with 300 μL of 1× Wash Buffer, and incubated at 25°C for 1 minute during the first wash.

[0134] 1.4.3 The samples were diluted eightfold using Complement Specimen Diluent, and 100 μL of the sample was added to each well, after which they were incubated at 25°C for 30 minutes.

[0135] 1.4.4 The liquid in the wells was discarded, and the cells were washed five times with 300 μL of 1× Wash Buffer, with the cells incubated at room temperature for 1 minute during the first wash.

[0136] 1.4.5 50 μL of Bb Plus Conjugate was added to each well and incubated at 25°C for 30 minutes.

[0137] 1.4.6 The liquid in the wells was discarded, and the cells were washed five times with 300 μL of 1× Wash Buffer, with the cells incubated at room temperature for 1 minute during the first wash.

[0138] 1.4.7 100 μL of compound working solution (Substrate Solution) was added to each well and incubated at 25°C for 15 minutes.

[0139] 1.4.8 100 μL of stop solution was added to each well, and the absorbance at 450 nm was measured within 30 minutes.

[0140] 1.5 Data Analysis 1.5.1 Inhibition rates for each drug concentration:

[0141]

number

[0142] The PC group represents a 0% inhibition rate, and the NC group represents a 100% inhibition rate.

[0143] 1.5.2 Calculation of signal-to-background ratio (S / B): The average OD value of the PC group / the average OD value of the NC group indicates the size of the signal window.

[0144] 1.5.3 Z' factor: Calculation formula:

[0145]

number

[0146] The Z' factor must be greater than 0.4.

[0147] 1.5.4 IC of Compounds 50 : I C 50 : This represents the half-inhibitory concentration, indicating the concentration of a compound that inhibits the enzyme activity of complement factor D by 50%.

[0148] We collect data, calculate the log values ​​of inhibition rate and compound concentration, and use GraphPad Prism software for IC 50 The values ​​were calculated. The inhibitory activity of the compound represented by formula A obtained in Example 1 of the present invention against complement factor D is as shown in Table 13 below.

[0149] [Table 13]

[0150] Experimental conclusion: The compound represented by formula A of the present invention had a good inhibitory effect on complement factor D.

[0151] 2. Rabbit erythrocyte hemolysis experiment to evaluate the inhibitory activity of compounds against alternative pathways. 2.1 Experimental materials and equipment Normal human serum, NHS (collected from healthy individuals), normal human plasma, NHP (Shanghai Yuduo Biotechnology Co., Ltd.), 96-well ELISA plate (Jet Biofil), Japanese hare (Wuhan Wanqianjiaxing Biotechnology Co., Ltd.), Alceber solution (Procell), centrifuge (Thermo, PICO17), insulated shaker (Shanghai Fuma Laboratory Instrument Co., Ltd.), decolorizing shaker (Beijing Liuyi Biotechnology Co.,ltd.), cell counter (Invitrogen, Counter Countess II), (GVBo, EGTA, MgCl2, NaOH, microplate reader, microplate insulated shaker, pipettes, etc. are the same as in Experiment 1).

[0152] 2.2 Manufacturing of the working solution 48% NHP: 100% NHP was diluted to 48% with a buffer.

[0153] 26.4% NPS: Diluted to 26.4% with 100% NHS buffer.

[0154] Rabbit red blood cell suspension: Blood is collected from the ear veins of rabbits, treated with Alceber's solution in a 1:1 anticoagulation ratio, divided into smaller portions, and stored at 4°C for up to 4 weeks. Before use, 500g is centrifuged for 5 minutes to discard the Alceber's solution, then washed three times by centrifugation with the same volume of buffer at 500g for 5 minutes each, and finally the density is reduced to 6 × 10⁻⁶ with buffer. 8 The concentration was adjusted to 1 / mL.

[0155] Compound stock solution: A 40 mM compound solution was diluted with DMSO to prepare a 2 mM stock solution. This 2 mM stock solution was then diluted three times with DMSO to create 10 points, and compound stock solutions of various concentrations were prepared.

[0156] Compound working solution: Compound solutions of various concentrations were obtained by diluting the compound 500-fold with buffer solution.

[0157] 2.3 Experimental Steps In a 96-well ELISA plate, 50 μL of 48% NHP or 26.4% NHS and 50 μL of compound solution were added to the experimental group; 50 μL of 48% NHP or 26.4% NHS and 50 μL of buffer containing 0.2% DMSO were added to the positive control group; 50 μL of 48% inactive NHP or 26.4% inactive NHS and 0.2% DMSO buffer were added to the blank control group; and 100 μL of double-distilled water was added to the H2O group. The mixtures were incubated at 37°C for 15 minutes, 20 μL of rabbit erythrocyte suspension was added to each well, and the mixtures were incubated in a shaker at 37°C for 30 minutes. The mixtures were then centrifuged at 2000 g (3380 rpm) for 5 minutes, and 100 μL of the supernatant was transferred to a new 96-well ELISA plate. The absorbance was detected at 415 nm.

[0158] 2.4 Data Analysis 2.4.1 Hemolysis rate at each drug concentration:

[0159]

number

[0160] The PC group represents 100% hemolysis, while the NC group represents 0% hemolysis.

[0161] 2.4.2 Calculation of signal-to-background ratio (S / B): The average OD value of the PC group / the average OD value of the NC group indicates the size of the signal window.

[0162] 2.4.3 Z' factor: Calculation formula:

[0163]

number

[0164] 2.4.4 IC of Compounds 50 I C 50 : Half-inhibitory concentration. Data was collected, the log values ​​of hemolysis rate and compound concentration were calculated, and IC was performed using GraphPad Prism software. 50 The values ​​were calculated. The inhibitory activity of the compound represented by formula A obtained in Example 1 of the present invention against rabbit erythrocyte hemolysis is as shown in Table 14 below.

[0165] [Table 14]

[0166] Experimental conclusion: The compound represented by formula A of the present invention had a good inhibitory effect on rabbit erythrocyte hemolysis.

[0167] 3. Pharmacokinetic studies of compounds represented by formula A After acclimatizing SPF-grade SD rats, the compound represented by formula A obtained in Example 1 of the present invention was administered as a single oral dose or by tail vein injection at a dose of 1 mg / kg. Plasma was collected at specific time points after administration, and the compound concentration in the plasma was detected by LC-MS / MS (AB SCIEX Qtrap4500). The PK parameters of the compound were calculated using software to reflect its pharmacokinetic characteristics. The PK parameters of the compound represented by formula A of the present invention are shown in Table 15 below:

[0168] [Table 15]

[0169] Experimental conclusion: The compound represented by formula A of the present invention can achieve higher in vivo exposure and higher oral bioavailability at lower doses, and exhibits better overall pharmacokinetic properties.

Claims

1. A type I crystal of a nitrogen-containing spiro compound represented by formula A, characterized in that, using Cu-Kα rays, the powder X-ray diffraction pattern, expressed at a 2θ angle, has diffraction peaks at positions of 5.67±0.20°, 11.37±0.20°, 16.69±0.20°, 17.32±0.20°, and 19.73±0.20°. 【Chemistry 1】

2. (1) The type I crystal is subjected to Cu-Kα radiation, and the powder X-ray diffraction pattern, expressed at a 2θ angle, has diffraction peaks at one or more of the following positions: 12.01±0.20°, 13.89±0.20°, 17.05±0.20°, 19.42±0.20°, 23.41±0.20°, and 27.86±0.20°. The aforementioned Type I crystal, when analyzed using Cu-Kα rays, has a powder X-ray diffraction pattern expressed at a 2θ angle that exhibits diffraction peaks at one or more of the following positions: 18.04±0.20°, 18.43±0.20°, 22.94±0.20°, and 26.01±0.20°. Preferably, the type I crystal is subjected to Cu-Kα radiation, and the powder X-ray diffraction pattern, expressed at a 2θ angle, has diffraction peaks at the following positions: 5.67±0.20°, 11.37±0.20°, 12.01±0.20°, 13.89±0.20°, 16.69±0.20°, 17.05±0.20°, 17.32±0.20°, 18.04±0.20°, 18.43±0.20°, 19.42±0.20°, 19.73±0.20°, 22.94±0.20°, 23.41±0.20°, 26.01±0.20°, and 27.86±0.20°. More preferably, the type I crystal is analyzed using Cu-Kα radiation, and the positions of the diffraction peaks in the powder X-ray diffraction pattern represented by a 2θ angle are as shown in the table below. Table 1-1 Most preferably, the type I crystal is subjected to the conditions that Cu-Kα radiation is used, and the position and relative intensity of the diffraction peaks in the powder X-ray diffraction pattern represented by a 2θ angle are as shown in the table below. Table 1-2 (2) The condition under which the differential scanning calorimetry curve of the type I crystal has an endothermic peak at 231.1 ± 2°C, (3) The thermogravimetric analysis curve of the type I crystal shows no weight loss before 200 ± 2°C. The type I crystal according to claim 1, characterized in that it satisfies one or more of the following conditions.

3. (1) The type I crystal is subjected to the condition that the powder X-ray diffraction pattern, expressed as a 2θ angle using Cu-Kα rays, is basically as shown in Figure 2. (2) The condition that the differential scanning calorimetry curve of the type I crystal is basically as shown in Figure 3, and (3) The condition that the thermogravimetric analysis curve of the Type I crystal is basically as shown in Figure 3. The type I crystal according to claim 2, characterized in that it satisfies one or more of the following conditions.

4. A type II crystal of a nitrogen-containing spiro compound represented by formula A, characterized in that, using Cu-Kα rays, the powder X-ray diffraction pattern, expressed at a 2θ angle, has diffraction peaks at positions of 11.32±0.20°, 11.70±0.20°, 11.93±0.20°, 18.03±0.20°, 18.81±0.20°, and 19.17±0.20°. 【Chemistry 2】

5. (1) The type II crystal is subjected to Cu-Kα radiation, and the powder X-ray diffraction pattern, expressed at a 2θ angle, has diffraction peaks at one or more of the following positions: 5.54±0.20°, 12.37±0.20°, 13.78±0.20°, 14.11±0.20°, 15.78±0.20°, and 20.80±0.20°. The aforementioned Type II crystal, when subjected to Cu-Kα radiation, has a powder X-ray diffraction pattern expressed at a 2θ angle that exhibits diffraction peaks at one or more of the following positions: 13.55±0.20°, 16.40±0.20°, 17.74±0.20°, 20.49±0.20°, and 22.06±0.20°. Preferably, the type II crystal is subjected to Cu-Kα radiation, and the powder X-ray diffraction pattern, expressed as a 2θ angle, has diffraction peaks at the following positions: 5.54±0.20°, 11.32±0.20°, 11.70±0.20°, 11.93±0.20°, 12.37±0.20°, 13.55±0.20°, 13.78±0.20°, 14.11±0.20°, 15.78±0.20°, 16.40±0.20°, 17.74±0.20°, 18.03±0.20°, 18.81±0.20°, 19.17±0.20°, 20.49±0.20°, 20.80±0.20°, and 22.06±0.20°. More preferably, the type II crystal is analyzed using Cu-Kα radiation, and the positions of the diffraction peaks in the powder X-ray diffraction pattern represented by a 2θ angle are as shown in the table below. Table 1-3 Most preferably, the type II crystal is prepared using Cu-Kα radiation, and the position and relative intensity of the diffraction peaks in the powder X-ray diffraction pattern represented by a 2θ angle are as shown in the table below. Table 1-4 (2) The differential scanning calorimetry curve of the type II crystal has an endothermic peak at 230.7 ± 2°C. (3) The condition under which the differential scanning calorimetry curve of the type II crystal has an endothermic peak at 159.1 ± 2°C, (4) Conditions under which the thermogravimetric analysis curve of the type II crystal shows a 1.71% decrease in weight before 210 ± 2°C A type II crystal according to claim 4, characterized in that it satisfies one or more of the following conditions.

6. (1) The type II crystal is subjected to the condition that the powder X-ray diffraction pattern, expressed as a 2θ angle using Cu-Kα rays, is basically as shown in Figure 6. (2) The condition that the differential scanning calorimetry curve of the type II crystal is basically as shown in Figure 7, (3) Conditions under which the thermogravimetric analysis curve of the type II crystal is basically as shown in Figure 7 A type II crystal according to claim 5, characterized in that it satisfies one or more of the following conditions.

7. A method for producing a type I crystal according to any one of claims 1 to 3, comprising the steps of dissolving a nitrogen-containing spiro compound represented by formula A in N-methylpyrrolidone and adding a poor solvent to crystallize it, wherein the poor solvent is selected from one or more of nitrile solvents, ester solvents, benzene solvents, ether solvents, and water, Preferably, the method for producing the type I crystal is: (1) The dissolution method is heating and stirring, and the heating temperature may be 30 to 70°C, preferably 40 to 60°C, for example, 50°C. (2) The volume mass ratio of N-methylpyrrolidone to the nitrogen-containing spiro compound represented by formula A is 15 to 50 mL / g, preferably 20 to 35 mL / g, for example, 26.7 mL / g. (3) The volume mass ratio of the poor solvent to the nitrogen-containing spiro compound represented by formula A is 50 to 350 mL / g, preferably 80 to 250 mL / g, for example, 80 mL / g, 160 mL / g, or 213.3 mL / g. (4) The volume ratio of the poor solvent to N-methylpyrrolidone is (1 to 10):1, preferably (3 to 8):1, for example 3:1, 6:1 or 8:

1. (5) The process includes a further cooling step before adding the poor solvent, wherein the cooling may be to 10-20°C, for example, 17°C, and the cooling rate may be 0.2°C / min. (6) The nitrile solvent is acetonitrile, (7) The ester solvent is one or more of ethyl acetate, n-propyl acetate, and isopropyl acetate, for example, under the condition that it is ethyl acetate and / or isopropyl acetate. (8) The benzene-based solvent is one or more of toluene, ethylbenzene, xylene, cumene, and chlorobenzene, for example, under the condition that it is toluene, (9) The ether solvent is one or more of tert-butyl methyl ether, diethyl ether, cyclopentyl methyl ether, isopropyl ether, methyltetrahydrofuran, and dioxane, for example, under the condition that tert-butyl methyl ether is used. A method that satisfies one or more of the following conditions.

8. A method for producing type II crystals according to any one of claims 4 to 6, comprising the step of stirring a nitrogen-containing spiro compound represented by formula A in acetonitrile for 12 to 16 days, Preferably, the method for producing the type II crystal is: (1) The volume mass ratio of the acetonitrile to the nitrogen-containing spiro compound represented by formula A is 15 to 50 mL / g, preferably 25 to 40 mL / g, for example, 33.3 mL / g. (2) The stirring temperature is 15 to 30°C, for example, 17°C, and (3) Under the condition that the stirring time is 14 days, A method characterized by satisfying one or more of the following conditions.

9. (1) A type I crystal according to any one of claims 1 to 3 or a type II crystal according to any one of claims 4 to 6, (2) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

10. The use of a type I crystal according to any one of claims 1 to 3 or a type II crystal according to any one of claims 4 to 6 in the manufacture of a pharmaceutical product for treating and / or preventing a disease mediated by complement factor D, The diseases mediated by complement factor D include blood disorders, kidney diseases, cardiovascular diseases, immune diseases, central nervous system diseases, respiratory diseases, genitourinary diseases, or eye diseases. Preferably, the diseases mediated by complement factor D include cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoimmune hemolytic anemia due to warm antibodies, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus erythematosus or lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, organ transplant rejection, myasthenia gravis, Alzheimer's disease, respiratory distress syndrome, asthma, and chronic obstructive pulmonary disease. The following are conditions of use: pulmonary emphysema, coronavirus infection, macular degeneration, age-related macular degeneration (AMD), macular edema, diabetic macular edema, choroidal neovascularization (CNV), uveitis, Behçet's disease uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, corneal transplant rejection, corneal dystrophy, autoimmune dry eye, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fuchs corneal endothelial dystrophy, retinal vein occlusion, or postoperative inflammation, and the coronavirus infection is, for example, SARS-CoV, MERS-CoV, or SARS-CoV-2 infection.

11. The use of a type I crystal according to any one of claims 1 to 3 or a type II crystal according to any one of claims 4 to 6 in the manufacture of a pharmaceutical product for treating and / or preventing a disease, wherein the disease is a blood disorder, kidney disease, cardiovascular disease, immune disorder, central nervous system disorder, respiratory disease, genitourinary tract disorder or eye disease, Preferably, the diseases include cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), autoimmune hemolytic anemia due to warm antibodies, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus erythematosus or lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, organ transplant rejection, myasthenia gravis, Alzheimer's disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, emphysema, and cholera. Coronavirus infection, macular degeneration, age-related macular degeneration (AMD), macular edema, diabetic macular edema, choroidal neovascularization (CNV), uveitis, Behçet's disease uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, corneal transplant rejection, corneal dystrophy, autoimmune dry eye, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fuchs corneal endothelial dystrophy, retinal vein occlusion, or postoperative inflammation, and the coronavirus infection is, for example, SARS-CoV, MERS-CoV, or SARS-CoV-2 infection.

12. The use of type I crystals according to any one of claims 1 to 3 or type II crystals according to any one of claims 4 to 6 in the manufacture of a complement factor D inhibitor, The aforementioned complement factor D inhibitor can be used in mammals in vivo or in vitro.

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