Crystal form of nitrogen-containing spiro compound as well as preparation method and application thereof

Abstract

The invention discloses a nitrogen-containing spiro compound crystal form and a preparation method and application thereof. The invention provides a crystal form I of a nitrogen-containing spiro compound as shown in a formula A. The crystal form I of the nitrogen-containing spiro compound is radiated by Cu-K alpha, and an X-ray powder diffraction pattern expressed by a 2 theta angle has diffraction peaks at 5.67 + / -0.20 degrees, 11.37 + / -0.20 degrees, 16.69 + / -0.20 degrees, 17.32 + / -0.20 degrees and 19.73 + / -0.20 degrees. The invention provides a crystal form II of a nitrogen-containing spiro compound as shown in a formula A. The crystal form II is radiated by Cu-K alpha, and an X-ray powder diffraction pattern expressed by a 20 angle has diffraction peaks at the positions of 11.32 + / -0.20 degrees, 11.70 + / -0.20 degrees, 11.93 + / -0.20 degrees, 18.03 + / -0.20 degrees, 18.81 + / -0.20 degrees and 19.17 + / -0.20 degrees. The crystal form of the nitrogen-containing spiro compound has good stability and complement factor D inhibitory activity, has a good inhibitory effect on rabbit red blood cell hemolysis, and has high in-vivo exposure and high oral bioavailability.

Description

A crystal form of a nitrogen-containing spiro compound, its preparation method and application

[0001] This application claims priority to Chinese Patent Application No. 2023103175276, filed on March 28, 2023, and Chinese Patent Application No. 2024102275411, filed on February 29, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention relates to a crystal form of a nitrogen-containing spiro compound, a preparation method and application thereof. Background Art

[0003] Complement is a protein found widely in the serum, tissue fluid, and cell membranes of humans and vertebrates, mediating immune and inflammatory responses. Most of these proteins are glycoproteins and are produced by a variety of cells, including hepatocytes, macrophages, and intestinal epithelial cells. Complement is named complement because it is a necessary condition for antibodies to achieve their cytolytic effects, but it actually mediates both specific and nonspecific immunity. The three activation pathways of the complement system include the classical pathway, the mannan-binding lectin (MBL) pathway, and the alternative (alternative) pathway. Complement factor D plays an early and central role in the activation of the alternative complement pathway. Activation of the alternative complement pathway is initiated by the spontaneous hydrolysis of the thioester bond within C3 to produce C3(H2O), which then associates with factor B to form the C3(H2O)B complex. Complement factor D cleaves factor B within the C3(H2O)B complex to form Ba and Bb. In addition to binding to C3b to form the C3 convertase, Bb also participates in the proliferation of preactivated B lymphocytes, while Ba inhibits their proliferation. Factor D is expressed at high levels in fat, which can stimulate glucose transport, promote the accumulation of triglycerides in fat cells, and inhibit fat decomposition.

[0004] Complement system dysregulation plays a crucial role in the pathogenesis of IgA nephropathy (IgAN), lupus nephritis (LN), and paroxysmal nocturnal hemoglobinuria (PNH). Deposition of complement components and immune complexes is often seen in renal pathology of IgAN and LN. Complement is the direct cause of hemolysis in PNH, while C5aR participates in amplifying complement system damage. CFB and CFD are key components of the alternative complement pathway, directly involved in regulating complement activation. 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 impaired bone marrow function, leading to anemia, fatigue, and other debilitating symptoms that can severely impact patients' quality of life. Currently marketed treatments for PNH primarily include the monoclonal antibody drugs Soliris and Ultomiris. Soliris was first approved for marketing in 2007 and is currently 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, an upgraded version of Soliris, is a second-generation, long-acting C5 complement inhibitor. It was first approved for marketing in late 2018 for indications including PNH and aHUS. Despite treatment with current standard-of-care anti-C5 therapies, a significant proportion of PNH patients remain anemic and transfusion-dependent.

[0006] Currently, there are no marketed small-molecule inhibitors of complement factor D. This target is the alternative complement pathway, a key driver of complement-driven renal disease (CDRD). Therefore, the development of small-molecule inhibitors with good bioactivity is of great significance for the treatment of these diseases. At the same time, developing a drug form suitable for these compounds has become a pressing technical challenge.

[0007] Summary of the Invention

[0008] The present invention addresses the technical problem of the limited variety and crystal forms of existing complement factor D inhibitors. The invention provides a crystal form of a nitrogen-containing spiro compound, a preparation method, and its application. The crystal form of the nitrogen-containing spiro compound of the present invention exhibits good stability and complement factor D inhibitory activity, exhibits a strong inhibitory effect on rabbit erythrocyte hemolysis, and exhibits high in vivo exposure and oral bioavailability.

[0009] The present invention provides a crystalline form I of a nitrogen-containing spiro compound as shown in formula A, which has diffraction peaks at 5.67±0.20°, 11.37±0.20°, 16.69±0.20°, 17.32±0.20° and 19.73±0.20° in an X-ray powder diffraction (XRPD) pattern expressed in 2θ angles using Cu-Kα radiation;

[0010] The crystalline form I may also have an X-ray powder diffraction (XRPD) pattern using Cu-Kα radiation and expressed in 2θ angles, which may have diffraction peaks at one or more of 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] The X-ray powder diffraction (XRPD) pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles may also have diffraction peaks at one or more of 18.04±0.20°, 18.43±0.20°, 22.94±0.20° and 26.01±0.20°.

[0012] The crystalline form I may have an X-ray powder diffraction (XRPD) pattern using Cu-Kα radiation and expressed in 2θ angles, with diffraction peaks at 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] The X-ray powder diffraction (XRPD) pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles can be shown in the following table:

[0014] The X-ray powder diffraction (XRPD) pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles, and its diffraction peak positions and relative intensities can be shown in the following table:

[0015] The X-ray powder diffraction (XRPD) pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles can be basically as shown in Figure 2.

[0016] The differential scanning calorimetry (DSC) diagram of the crystalline form I may have an endothermic peak at 231.1±2°C.

[0017] The differential scanning calorimetry (DSC) diagram of the crystalline form I can be basically shown in Figure 3.

[0018] The thermogravimetric analysis (TGA) diagram of the crystalline form I can show no weight loss before 200±2°C.

[0019] The thermogravimetric analysis (TGA) diagram of the crystalline form I can be basically shown in Figure 3.

[0020] The present invention provides a crystalline form II of a nitrogen-containing spiro compound as represented by formula A, which has diffraction peaks at 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° in an X-ray powder diffraction (XRPD) pattern using Cu-Kα radiation and expressed in 2θ angles;

[0021] The X-ray powder diffraction (XRPD) pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles may also have diffraction peaks at one or more of 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°.

[0022] The X-ray powder diffraction (XRPD) pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles may also have diffraction peaks at one or more of 13.55±0.20°, 16.40±0.20°, 17.74±0.20°, 20.49±0.20° and 22.06±0.20°.

[0023] The crystalline form II may have an X-ray powder diffraction (XRPD) pattern using Cu-Kα radiation and expressed in 2θ angles, with diffraction peaks at 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°.

[0024] The X-ray powder diffraction (XRPD) pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles has diffraction peak positions as shown in the following table:

[0025] The X-ray powder diffraction (XRPD) pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles, and its diffraction peak positions and relative intensities can be shown in the following table:

[0026] The X-ray powder diffraction (XRPD) pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles can be basically as shown in FIG6 .

[0027] The differential scanning calorimetry (DSC) diagram of the crystalline form II may have an endothermic peak at 230.7±2°C.

[0028] The differential scanning calorimetry (DSC) diagram of the crystalline form II may have an exothermic peak at 159.1±2°C.

[0029] The differential scanning calorimetry (DSC) diagram of the crystalline form II can be basically shown in Figure 7.

[0030] The thermogravimetric analysis (TGA) diagram of the crystal form II shows a weight loss of 1.71% before 210±2°C.

[0031] The thermogravimetric analysis (TGA) diagram of the crystal form II can be basically shown in Figure 7.

[0032] The present invention provides a method for preparing the crystalline form I of the nitrogen-containing spiro compound represented by formula A, comprising the following steps: dissolving the nitrogen-containing spiro compound represented by formula A in N-methylpyrrolidone, adding an antisolvent, and crystallizing; the antisolvent is selected from one or more of a nitrile solvent, an ester solvent, a benzene solvent, an ether solvent, and water.

[0033] The nitrile solvent can be acetonitrile.

[0034] The ester solvent may be one or more of ethyl acetate, n-propyl acetate and isopropyl acetate, such as ethyl acetate and / or isopropyl acetate.

[0035] The benzene solvent may be one or more of toluene, ethylbenzene, xylene, isopropylbenzene and chlorobenzene, such as toluene.

[0036] The ether solvent may be one or more of methyl tert-butyl ether, diethyl ether, cyclopentyl methyl ether, isopropyl ether, methyltetrahydrofuran and dioxane, for example, methyl tert-butyl ether.

[0037] In the preparation method of the crystal form I, the dissolution method can be heating and stirring. The heating temperature can be 30-70°C, preferably 40-60°C, for example 50°C.

[0038] In the preparation method of the crystalline form I, the volume mass ratio of the N-methylpyrrolidone to the nitrogen-containing spiro compound represented by formula A can be 15 to 50 mL / g, preferably 20 to 35 mL / g, for example 26.7 mL / g.

[0039] In the preparation method of the crystalline form I, the volume mass ratio of the antisolvent to the nitrogen-containing spiro compound represented by formula A can 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.

[0040] In the preparation method of the crystalline form I, the volume ratio of the antisolvent to N-methylpyrrolidone can be (1-10):1, preferably (3-8):1, for example 3:1, 6:1 or 8:1.

[0041] In the method for preparing Form I, the temperature may be lowered before adding the anti-solvent. The temperature may be lowered to 10-20° C., for example, 17° C. The rate of the temperature reduction may be 0.2° C. / min.

[0042] The present invention provides a method for preparing the crystalline form II of the nitrogen-containing spiro compound as shown in formula A, which comprises the following steps: stirring the nitrogen-containing spiro compound as shown in formula A in acetonitrile for 12 to 16 days.

[0043] In the preparation method of the crystalline form II, the volume mass ratio of the acetonitrile to the nitrogen-containing spiro compound represented by formula A can be 15 to 50 mL / g, preferably 25 to 40 mL / g, for example 33.3 mL / g.

[0044] In the preparation method of the crystal form II, the stirring temperature can be a conventional temperature for such operations in the art, preferably 15 to 30°C, for example 17°C.

[0045] In the preparation method of the crystal form II, the stirring time can be 14 days.

[0046] The present invention provides a pharmaceutical composition comprising: (1) the crystalline form I or crystalline form II of the nitrogen-containing spiro compound represented by formula A; and

[0047] (2) Pharmaceutically acceptable carrier.

[0048] The present invention provides a use of a crystalline form I or a crystalline form II of a nitrogen-containing spiro compound as represented by formula A in the preparation 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 disease, a kidney disease, a cardiovascular disease, an immune disorder, a central nervous system disease, a respiratory system disease, a genitourinary system disease, or an eye disease.

[0049] The use of the crystal form I or crystal form II in the preparation of a drug for treating and / or preventing diseases mediated by complement factor D, wherein the diseases mediated by complement factor D may be cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), warm antibody-type autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranous proliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus 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, coronavirus infection (such as 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 uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, corneal transplant rejection, corneal dystrophies, autoimmune dry eye disease, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fisher's endothelial corneal dystrophy, retinal vein occlusion, or postoperative inflammation.

[0050] The present invention provides a use of a crystalline form I or a crystalline form II of a nitrogen-containing spiro compound as represented by formula A in the preparation of a medicament for treating and / or preventing a disease, wherein the disease is a blood disease, a kidney disease, a cardiovascular disease, an immune disorder, a central nervous system disease, a respiratory system disease, a genitourinary system disease, or an eye disease.

[0051] The use of the crystal form I or crystal form II in the preparation of a drug for treating and / or preventing a disease, wherein the disease may be cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), warm antibody autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranous proliferative glomerulonephritis (MPGN), dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus or lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, organ transplant rejection, myasthenia gravis, Alzheimer's disease, respiratory Respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, emphysema, coronavirus infection (such as 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 uveitis, proliferative diabetic retinopathy, non-proliferative diabetic retinopathy, glaucoma, hypertensive retinopathy, corneal neovascularization, post-keratoplasty rejection, corneal dystrophies, autoimmune dry eye disease, Stevens-Johnson syndrome, Sjögren's syndrome, environmental dry eye, Fisher's endothelial corneal dystrophy, retinal vein occlusion, or post-operative inflammation.

[0052] The present invention provides a use of the crystalline form I or crystalline form II of the nitrogen-containing spiro compound represented by formula A in the preparation of a complement factor D inhibitor.

[0053] The complement factor D inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the field to provide rapid detection of the inhibitory effect of complement factor D.

[0054] The crystal forms of the present invention can be identified using one or more solid-state analytical methods, such as X-ray powder diffraction, single crystal X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. Those skilled in the art will appreciate that the peak intensities and / or peak configurations of X-ray powder diffraction may vary depending on experimental conditions. Furthermore, due to varying instrumental precision, the measured 2θ values ​​may have an error of approximately ±0.20°. The relative peak intensity depends more on certain properties of the sample being measured, such as crystal size and purity, than the peak position. Therefore, the measured peak intensity may vary by approximately ±20%. Despite experimental and instrumental errors, as well as orientation bias, those skilled in the art can still obtain sufficient information to identify individual crystal forms from the X-ray powder diffraction data provided in this patent. In DSC measurements, the measured onset temperature, peak temperature, and heat of fusion of the endothermic peak are subject to a certain degree of variability, depending on the heating rate, crystal shape and purity, and other measurement parameters.

[0055] Explanation of terms:

[0056] The term “substantially” means that the positions of the peaks in the graph may vary slightly with slight variations in the measuring equipment, measuring conditions, and batches of the product to be measured, and are not to be regarded as absolute values.

[0057] The term "room temperature" refers to 15-30°C, for example 17°C, 25°C.

[0058] The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0059] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0060] The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., preferably humans.

[0061] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0062] The reagents and raw materials used in the present invention are commercially available.

[0063] The positive progress of the present invention is that the crystalline form of the nitrogen-containing spiro compound of the present invention has good stability and complement factor D inhibitory activity, has a good inhibitory effect on rabbit erythrocyte hemolysis, and has a high in vivo exposure and high oral bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG1 is an XRPD spectrum of the amorphous form of the compound represented by Formula A.

[0065] FIG2 is an XRPD spectrum of Form I of the compound represented by Formula A.

[0066] FIG3 is the TGA and DSC spectra of Form I of the compound represented by Formula A.

[0067] Figure 4 is a diagram of the crystalline form I of the compound represented by formula A 1 H-NMR spectrum (DMSO-d6).

[0068] Figure 5 is a PLM spectrum of Form I of the compound represented by Formula A.

[0069] FIG6 is an XRPD spectrum of Form II of the compound represented by Formula A.

[0070] FIG7 is a TGA and DSC spectrum of the crystalline form II of the compound represented by formula A.

[0071] Figure 8 is a diagram of the crystalline form II of the compound represented by formula A 1 H-NMR spectrum (DMSO-d6).

[0072] Figure 9 is a comparative XRPD spectrum of Form II and Form I of the compound represented by Formula A.

[0073] FIG10 is a comparative DSC spectrum of Form II and Form I of the compound represented by Formula A.

[0074] FIG11 is a comparative XRPD spectrum of the crystal form changes of Form II after heating to 140° C. and 200° C.

[0075] FIG12 is the DSC and TGA spectra of the solid obtained after Form II was heated to 140° C.

[0076] FIG13 is a comparative DSC spectrum of the solid obtained after Form II was heated to 140° C. and 200° C.

[0077] FIG14 is an XRPD spectrum of the wet grinding experiment of Form I in ethanol.

[0078] FIG15 is an XRPD spectrum of Form I obtained by wet grinding in water.

[0079] FIG16 is an XRPD spectrum of Form I tableting experiment.

[0080] FIG17 is an HPLC spectrum of Form I after being left open for 3 days.

[0081] FIG18 is an XRPD spectrum of Form I after being left open for 3 days.

[0082] FIG19 is an HPLC spectrum of Form I after being left open for 7 days.

[0083] Figure 20 is the XRPD spectrum of Form I after being exposed for 7 days.

[0084] Figure 21 is the HPLC spectrum of Form I after being left open for 14 days.

[0085] Figure 22 is the XRPD spectrum of Form I after being exposed for 14 days.

[0086] FIG23 is an XRPD spectrum of Form I suspended in the biosolvent FaSSIF.

[0087] FIG24 is an XRPD spectrum of Form I suspended in the biological solvent FeSSIF.

[0088] FIG25 is an XRPD spectrum of Form I suspended in water.

[0089] FIG26 is an HPLC spectrum of the stability of Form I in the biological solvent SGF.

[0090] FIG27 is an HPLC spectrum showing the stability of Form I in the biosolvent FaSSIF.

[0091] FIG28 is an HPLC spectrum showing the stability of Form I in the biological solvent FeSSIF.

[0092] FIG29 is an HPLC spectrum of the stability of Form I in water.

[0093] Figure 30 is a DVS spectrum of Form I.

[0094] Figure 31 is a comparison of XRPD spectra of Form I before and after DVS determination. DETAILED DESCRIPTION

[0095] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0096] Abbreviations:

[0097] ACN: acetonitrile; DMSO: dimethyl sulfoxide; IPA: isopropyl alcohol; HPLC: high-performance liquid chromatography; NMP: N-methylpyrrolidone; RH: relative humidity; aw: water activity; PLM: polarizing light microscopy; TGA: thermogravimetric analysis; Tonset: initial temperature / onset temperature. XRPD: X-ray powder diffraction. DSC: differential scanning calorimetry; DVS: dynamic water sorption.

[0098] Analytical methods:

[0099] X-ray powder diffraction (XRPD)

[0100] The X-ray powder diffraction pattern was acquired by a Bruker D2 Phaser instrument. The instrument parameters are shown in the following table:

[0101] Differential Scanning Calorimetry (DSC)

[0102] DSC curves were acquired using a TA Instruments DSC250. The sample was weighed accurately onto a sample pan and the weight recorded. The temperature was increased from 25°C to 250°C at a rate of 10°C / min.

[0103] Thermogravimetric Analyzer (TGA)

[0104] The TGA curve was acquired by TA Instruments TGA550. The sample was weighed into a sample pan (Al2O3) and heated to 300°C at a heating rate of 10°C / min.

[0105] 1 H NMR

[0106] The NMR spectra were acquired using a Varian 400 MHz spectrometer using deuterated DMSO as the solvent.

[0107] Polarized Light Microscopy (PLM)

[0108] PLM analysis was performed using an Otto optical microscope, a BK-Pol. A small amount of sample was placed on a glass slide, silicone oil was added, and the sample was dispersed. The sample was then covered with a coverslip and observed under a microscope.

[0109] LCMS

[0110] LCMS curves were acquired using a Shimadzu instrument. The instrument methods and parameters are shown in the following table:

[0111] LCMS method parameters

[0112] HPLC

[0113] Shimadzu instruments were used for sample analysis and testing. Stability and solubility experiments were performed using the following methods. The HPLC instrument methods and parameters are shown in the table below:

[0114] Dynamic Water Sorption Analyzer (DVS)

[0115] Samples were analyzed using an Intrinsic DVS (System Measurement System UK). Approximately 20-30 mg of test sample was placed in a sample basket hung in the measurement chamber. The chamber temperature was controlled at 25 ± 1°C, and the relative humidity was increased from 0% to 90% and then to 0% at a rate of 10% / h. Mass data were recorded every 20 seconds. Each ramp dwell time was 60 minutes.

[0116] 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 (Formula A compound)

[0117] 1.1 Synthesis of Compound 1-1

[0118] Compound 1-a (11.16 g, 60 mmol), triethylamine (11.00 g, 108 mmol), and dichloromethane (120 mL) were added to a 500 mL reaction flask. Di-tert-butyl dicarbonate (15.71 g, 72 mmol) was added to the above system under an ice bath. The reaction solution was reacted at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure. Ethyl acetate (150 mL) and water (200 mL) were added to the residue, and the organic phase was separated. The aqueous phase was further extracted with ethyl acetate (50 mL × 2). The combined organic phases were 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.

[0119] 1.2 Synthesis of compound 1-2

[0120] Compound 1-1 (18.02 g, 60 mmol), pinacol diboronate (18.28 g, 72 mmol), potassium acetate (11.78 g, 120 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (4.40 g, 6 mmol), and 1,4-dioxane (120 mL) were added to a 500 mL reaction flask. The atmosphere was purged with nitrogen three times and the reaction mixture was allowed to react at 80°C for 4 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and ethyl acetate (150 mL) and water (200 mL) were added for separation. The organic phase was separated. The aqueous phase was further extracted with ethyl acetate (50 mL x 2). The combined organic phases were 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 = 8 / 1 (v / v)) to obtain compound 1-2. LC / MS (ESI+) m / z: [M+Ht-Bu] + =278.15.

[0121] 1.3 Synthesis of Compounds 1-3

[0122] Compound 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 dissolved in dichloromethane (70 mL) in sequence. A solution of bis(4-chlorobenzyl)azodicarboxylate (13.7 g, 37.6 mmol) in dichloromethane (70 mL) was added dropwise to the above system under an ice bath. 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 compound 1-3. LC / MS (ESI+) m / z: [M+H] + =414.90.

[0123] 1.4 Synthesis of Compounds 1-4

[0124] Compound 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 [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.35 g, 0.48 mmol) were added sequentially to 1,4-dioxane / water (30 mL / 3 mL). The atmosphere was purged with nitrogen three times, and the reaction mixture was incubated at 80°C for 2 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic phases were 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 to 5 / 1 (v / v)) to obtain compound 1-4. LC / MS (ESI+) m / z: [M+H-Boc] + =439.95.

[0125] 1.5 Synthesis of Compounds 1-5

[0126] Compound 1-4 (4.3 g, 7.96 mmol), 6-aza-spiro[2.5]octane hydrochloride (compound 1-d, 1.4 g, 9.55 mmol), potassium carbonate (3.3 g, 23.87 mmol), tris(dibenzylideneacetone)dipalladium (400 mg, 10 wt%), and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (400 mg, 10 wt%) were added to 1,4-dioxane (50 mL) in sequence. The atmosphere was purged with nitrogen three times, and the reaction mixture was reacted at 90°C for 4 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1-5. LC / MS (ESI+) m / z: [M+H] + =571.15.

[0127] 1.6 Synthesis of Compounds 1-6

[0128] Compound 1-5 (3.5 g, 6.1 mmol) was dissolved in dichloromethane (27 mL), and trifluoroacetic acid (9 mL) was added. The reaction mixture was allowed to react at room temperature for 1 hour. Saturated aqueous sodium bicarbonate solution was added to the reaction solution to adjust the pH to 8, and then extracted with ethyl acetate (30 mL x 3). The combined organic phases were 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.

[0129] 1.7 Synthesis of Compound A

[0130] 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). The reaction mixture was reacted at 60°C for 4 hours. The reaction mixture was purified by preparative HPLC and lyophilized to obtain an amorphous compound of Formula A. The XRPD pattern is shown in Figure 1.

[0131] 1 H NMR (400MHz, DMSO-d6): δ8.27(s,0.59H),8.10(s,1H),7.67(d,J=7.6Hz,1H),7 .37-7.40(m,2H),7.28(d,J=7.2Hz,1H),7.08-7.14(m,3H),7.00(s,1H),6.92(d ,J=8.0Hz,1H),6.81(t,J=7.6Hz,1H),5.13(s,2H),3.97(s,2H),3.43(s,2H),3. 30(t,J=5.2Hz,4H),1.48(t,J=5.2Hz,4H),0.35(s,4H); LC / MS(ESI+)m / z: [M+H] + =457.10.

[0132] Example 2: Preparation of Form I of the Compound of Formula A

[0133] 2.1 Preparation of Form I

[0134] Approximately 15 mg of the amorphous compound of Formula A obtained in Example 1 was weighed into a sample vial, 0.5 mL of n-heptane was added, and the mixture was stirred at room temperature (approximately 17°C) and 50°C for 7 and 14 days, respectively. The resulting solid was then subjected to XRPD analysis. The results showed that the resulting solids were all Form I. Specific experimental conditions and results are shown in Tables 1, 3, and 4 below:

[0135] Table 1. Experimental conditions and results of crystallization in n-heptane using amorphous form of compound A as starting material

[0136] 2.2 Preparation of Form I

[0137] Approximately 15 mg of the amorphous compound of Formula A obtained in Example 1 was weighed into a sample vial. 0.4 mL of N-methylpyrrolidone was added at 50°C to completely dissolve the amorphous compound of Formula A. The mixture was brought to room temperature (approximately 17°C), and a certain volume of antisolvent was added. The mixture was stirred until solids precipitated. The results showed that the resulting solids were all Form I. Specific experimental conditions and results are shown in Tables 2, 3, and 4 below:

[0138] Table 2. Experimental conditions and results of crystallization of amorphous compound of formula A as the starting material and anti-solvent

[0139] 2.3 Characterization data of Form I are shown in Table 3 below, and XRPD pattern analysis data of Form I are shown in Table 4:

[0140] Table 3. Characterization data of Form I

[0141] Table 4. XRPD pattern analysis data of Form I

[0142] Example 3: Preparation of Form II of the Compound of Formula A

[0143] 3.1 Preparation of Form II

[0144] About 15 mg of the amorphous compound of Formula A obtained in Example 1 was weighed into a sample bottle, 0.5 mL of acetonitrile was added, and the mixture was stirred at room temperature (about 17° C.) for 14 days. The resulting solid product was then subjected to XRPD analysis. The results showed that the obtained solid was Form II.

[0145] 3.2 The characterization data of Form II are shown in Table 5 below, and the XRPD pattern analysis data of Form II are shown in Table 6:

[0146] Table 5. Characterization data of Form II

[0147] Table 6. XRPD pattern analysis data of Form II

[0148] Example 4: Comparison and transformation relationship study between crystal forms I and II

[0149] 4.1 Comparison of spectral data between Form I and Form II

[0150] Comparing Form II with Form I, the 2θ angle of the spectra of the two is 16-18 o and 22-24 o There are significant differences within this range, as shown in the 2θ angle box in the XRPD comparison spectra of Form II and Form I in Figure 9. The endothermic peak temperatures of the two are similar, but Form II has an exothermic peak at 159°C, as shown in the DSC comparison spectra in Figure 10.

[0151] 4.2 Heating experiment of Form II

[0152] A heating experiment was conducted on Form II. The Form II sample was heated to 140°C using the TGA method and to 200°C using the DSC method. The obtained solids were subjected to XRPD, TGA, DSC and 1 HNMR test. The test results show that when heated to 140℃, the XRPD spectrum shows that the crystal form has not changed, and TGA has not found any weight loss. 1 No residual peak of acetonitrile solvent was found in the H NMR spectrum, and an exothermic peak at 159°C was still observed in the DSC spectrum. When heated to 200°C, the XRPD spectrum showed that the obtained solid was Form I. 1 No residual acetonitrile solvent peaks were observed in the H NMR spectrum, and the endothermic peak at 159°C disappeared in the DSC spectrum. Therefore, it can be concluded that the exothermic peak at 159°C is the crystallization peak, and the endothermic peak at 230°C is the melting peak of Form I. A comparative XRPD spectrum from the heating experiment is shown in Figure 11, and the DSC and TGA spectra of the solid obtained after heating to 140°C are shown in Figure 12. A comparative DSC spectrum from the heating experiment is shown in Figure 13.

[0153] Effect Example 1: Evaluation of Crystal Form

[0154] 1. Water activity experiment

[0155] The organic solvent was mixed with water at a molar ratio of 0.001:0.999, 0.2:0.8, 0.5:0.5, 0.8:0.2, and 0.999:0.001, and the Form I obtained in Example 2 was added to the suspension. The solution was stirred at 25°C and 50°C for 30 minutes, respectively, and filtered to retain the filtrate. About 10 mg of the Form I obtained in Example 2 was weighed and added to the filtrate. The results were tested after stirring for 5 days and 11 days, respectively. The results showed that the solids obtained under the following conditions were all Form I. The specific experimental conditions and results are shown in Tables 7 and 8 below:

[0156] Table 7. Experimental conditions and results of water activity at 25°C

[0157] Table 8. Experimental conditions and results of water activity at 50°C

[0158] 2. Grinding and crystal transformation experiment

[0159] 2.1 Wet grinding

[0160] Approximately 20 mg of Form I obtained in Example 2 was weighed into a mortar, 0.04 mL of water or ethanol was added, and the mixture was manually ground for 2, 5, and 10 minutes, followed by XRPD measurements. If the solvent evaporated completely during the grinding process, 0.04 mL of water or ethanol was added to the mortar each time. The results showed that the crystal form of Form I remained unchanged after grinding for 10 minutes, and no significant decrease in crystallinity was observed. The XRPD spectrum of the wet grinding experiment of Form I in ethanol is shown in Figure 14, and the XRPD spectrum of the wet grinding experiment of Form I in water is shown in Figure 15.

[0161] 2.2 Tablet compression experiment

[0162] Approximately 30 mg of Form I obtained in Example 2 was weighed and tableted at 40 MPa. XRPD analysis was then performed. The results showed that Form I exhibited no change in crystalline form after tableting, and no significant decrease in crystallinity was observed. The XRPD results are shown in Figure 16.

[0163] 3. Stability test

[0164] An appropriate amount of the crystal form I obtained in Example 2 was weighed into a sample bottle and left exposed for 3 days, 7 days, and 14 days at 60°C, 25°C / 60%RH, 40°C / 75%RH, and 25°C / 90(±5)RH%, respectively. The purity was then determined by HPLC. The results showed that after 14 days under the above conditions, the main peak purity did not change significantly, and the crystal form did not change. The stability conditions and results are shown in Table 9 below. The HPLC spectrum of the crystal form I left exposed for 3 days is shown in Figure 17, the XRPD spectrum of the crystal form I left exposed for 3 days is shown in Figure 18, the HPLC spectrum of the crystal form I left exposed for 7 days is shown in Figure 19, the XRPD spectrum of the crystal form I left exposed for 7 days is shown in Figure 20, the HPLC spectrum of the crystal form I left exposed for 14 days is shown in Figure 21, and the XRPD spectrum of the crystal form I left exposed for 14 days is shown in Figure 22.

[0165] Table 9. Stability test results

[0166] Experimental conclusion: After 14 days of storage under the above conditions, the purity of the main peak of Form I did not change significantly, and the crystal form did not change, indicating good stability.

[0167] 4. Solubility and stability tests in biosolvents and water

[0168] 4.1 Solubility experiments in biosolvents and water

[0169] About 5 mg of the crystalline form I obtained in Example 2 was weighed into a sample bottle, and SGF, FaSSIF, FeSSIF and water were added respectively to prepare a suspension with a concentration of 5 mg / mL. After being fully stirred in SGF, FaSSIF, FeSSIF and water for 0.5 hour, 2 hours and 24 hours, the suspension was taken out, its solubility was determined by HPLC, and the crystal form of the solid in the suspension was detected by XRPD. Crystal form I was completely dissolved in SGF and was in a suspended state in FaSSIF, FeSSIF and water. The solubility of crystal form I in SGF was relatively large (>5 mg / mL), and the solubility in FaSSIF, FeSSIF and water was less than 0.73 mg / mL. After stirring for 24 hours in FaSSIF, FeSSIF and water, the crystal form did not change. The solubility results are shown in Table 10 below, the XRPD spectrum of Form I as a solid suspended in the biosolvent FaSSIF is shown in Figure 23, the XRPD spectrum of Form I as a solid suspended in the biosolvent FeSSIF is shown in Figure 24, and the XRPD spectrum of Form I as a solid suspended in water is shown in Figure 25.

[0170] Table 10. Solubility test results in biosolvents and water

[0171] Note: LOQ stands for limit of quantification, which refers to the lowest amount of the analyte in the sample that can be quantitatively determined.

[0172] 4.2 Biosolvent Stability Determination

[0173] Approximately 2.5 mg of Form I obtained in Example 2 was weighed and added to 0.5 mL of SGF, FaSSIF, FeSSIF, and water, respectively. After thorough stirring for 0.5, 2, and 24 hours, the mixed solution was diluted to 10 mL and its purity was determined by HPLC. The results showed that the main peak purity of Form I in SGF, FaSSIF, and water did not show a significant decrease. However, in FeSSIF, the main peak purity gradually decreased from 99.56% to 98.04% with increasing stirring time. The stability conditions and results are shown in Table 11 below. The HPLC spectrum of the stability of Form I in the biosolvent SGF is shown in Figure 26, the HPLC spectrum of the stability of Form I in the biosolvent FaSSIF is shown in Figure 27, the HPLC spectrum of the stability of Form I in the biosolvent FeSSIF is shown in Figure 28, and the HPLC spectrum of the stability of Form I in water is shown in Figure 29. The main impurities are shown in the boxes in Figures 26-29.

[0174] Table 11. Biosolvent stability test results

[0175] 5. Hygroscopicity test

[0176] Hygroscopicity of Form I obtained in Example 2 was determined using DVS, and XRPD analysis was performed on the solid before and after DVS. The results showed that Form I was slightly hygroscopic, with a weight gain of 0.55% under 0-80% RH conditions. The crystal form remained unchanged before and after DVS. The DVS results are shown in Figure 30, and comparative XRPD spectra of Form I before and after DVS are shown in Figure 31.

[0177] 6. Stability of Form I in DMSO at high temperature

[0178] Approximately 100 mg of Form I obtained in Example 2 was weighed into a sample vial and suspended in DMSO (2 mL). After stirring at 50°C for 24 and 48 hours, the solid and filtrate were analyzed by LCMS. The results showed that after stirring Form I in DMSO for 48 hours, the purity of the solid did not change significantly, while a small amount of impurities appeared in the filtrate. The measured purity is shown in Table 12 below.

[0179] Table 12. Stability test results of Form I in DMSO under high temperature conditions

[0180] Experimental conclusion: The purity of Form I of the present invention changes little under high temperature conditions and has good stability.

[0181] Effect Example 2: Biological Activity Test

[0182] 1. In vitro screening of complement factor D inhibitory activity (C3b method)

[0183] 1.1 Experimental materials and instruments

[0184] V-bottom plates (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), GVB° (Complement tech), EGTA (Aladdin), MgCl2 (Aladdin), NaOH (Sinopharm), microplate reader (Molecular Devices, SpectraMax i3x), microplate constant temperature shaker (Thermo, MB100-2A), pipette (Gilson)

[0185] 1.2 Preparation before the experiment

[0186] 1.2.1 Preparation of Mg-EGTA

[0187] 0.1M Mg-EGTA: Weigh 3.8 g EGTA, 0.9521 g MgCl2, and 0.7 g NaOH. Adjust the pH to 7.5 with NaOH. Make up to 100 mL with distilled water. Filter through a 0.2 μm sterile filter. Aliquot and store at 4°C.

[0188] 1.2.2 Preparation of working fluid

[0189] Buffer: 0.1 M Mg-EGTA solution was diluted 10-fold with GVB° buffer to 10 mM;

[0190] Factor B working solution: 1 mg / mL Factor B solution (10.75 μM) was diluted 6.7-fold with buffer to 1.6 μM;

[0191] Factor C3b working solution: 1 mg / mL factor C3b solution (5.68 μM) was diluted 5-fold with buffer to 1.12 μM;

[0192] Factor D working solution: 0.1 mg / mL Factor D solution (4.17 μM) was diluted 1303-fold with buffer to 3.2 nM.

[0193] Note: a. The above dilution ratios are for reference only and should be adjusted according to the actual concentration marked on the reagent;

[0194] b. Factor B: 93KDa, Factor D: 24KDa, Factor C3b: 176KDa.

[0195] 1.2.3 Preparation of stop solution

[0196] Stop solution: Weigh an appropriate amount of EDTA powder, dissolve it in a certain amount of GVB° buffer, adjust the pH to 7.5 with NaOH, stir until clear, and prepare a 10 mM solution.

[0197] 1.2.4 Compound preparation

[0198] Compound stock solution: 40 mM compound solution was diluted with DMSO to 1 mM stock solution, and the 1 mM stock solution was diluted 3-fold with DMSO to prepare 8 points of compound stock solution at each concentration;

[0199] Compound working solution: Dilute 250 times with buffer to obtain compound solutions of various concentrations.

[0200] 1.3 Experimental steps

[0201] In a V-bottomed 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 0.4% DMSO buffer were added to the positive control group; 20 μL of 0.2% DMSO buffer were added to the blank control group; the plates were incubated at 37°C for 15 min; Factor B working solution and Factor C3b working solution were mixed 1:1, 20 μL of the mixture was added to each well, and the plates were incubated at 37°C for 30 min; 40 μL of stop solution was added to terminate the reaction; and the amount of product Bb produced was detected using a MicroVue Bb Plus ELISA kit.

[0202] 1.4 ELISA

[0203] 1.4.1 Remove the required ELISA plate wells and return them to room temperature. Rewrap the remaining wells and store at 4°C.

[0204] 1.4.2 Wash twice with 300 μL 1× Wash Buffer, incubating the first wash at 25°C for 1 min.

[0205] 1.4.3 Dilute the sample 8-fold with Complement Specimen Diluent, add 100 μL of sample to each well, and incubate at 25°C for 30 min.

[0206] 1.4.4 Discard the liquid in the wells and wash five times with 300 μL 1× Wash Buffer. Incubate the first wash at room temperature for 1 min.

[0207] 1.4.5 Add 50 μL of Bb Plus Conjugate to each well and incubate at 25°C for 30 min.

[0208] 1.4.6 Discard the liquid in the wells and wash five times with 300 μL 1× Wash Buffer. Incubate the first wash at room temperature for 1 min.

[0209] 1.4.7 Add 100 μL of the compound working solution (Substrate Solution) to each well and incubate at 25°C for 15 min.

[0210] 1.4.8 Add 100 μL of Stop Solution to each well and measure the absorbance at 450 nm within 30 min.

[0211] 1.5 Data Analysis

[0212] 1.5.1 Inhibition rate of each drug concentration:

[0213] The PC group represents 0% inhibition rate, and the NC group represents 100% inhibition rate.

[0214] 1.5.2 Calculate the signal-to-background ratio (S / B):

[0215] The average OD value of the PC group / the average OD value of the NC group represents the size of the signal window.

[0216] 1.5.3 Z' factor:

[0217] Calculation formula:

[0218] The Z' factor should be greater than 0.4.

[0219] 1.5.4 Compound IC 50 :

[0220] IC 50 : half-inhibitory concentration, representing the concentration of the compound that inhibits the enzyme activity of complement factor D by 50%;

[0221] Collect data to calculate the inhibition rate and the log value of compound concentration, and use GraphPad Prism software to calculate IC 50 The inhibitory activity of the compound represented by formula A obtained in Example 1 of the present invention on complement factor D is shown in Table 13 below:

[0222] Table 13. Inhibitory activity of the compound of formula A against complement factor D

[0223] Experimental conclusion: The compound represented by formula A of the present invention has a good inhibitory effect on complement factor D.

[0224] 2. Rabbit erythrocyte hemolysis method to evaluate the inhibitory activity of compounds on the alternative pathway

[0225] 2.1 Experimental materials and instruments

[0226] Normal Human Serum, NHS (collected from healthy individuals), Normal Human Plasma, NHP (Shanghai Yuduo), 96-well ELISA plate (Jet Biofil), Japanese big-eared white rabbit (Wuhan Wanqian Jiaxing), Aldrich solution (Punosai), centrifuge (Thermo, PICO17), constant temperature shaker (Shanghai Fuma), decolorization shaker (Beijing Liuyi), cell counter (Invitrogen, Counter Countess II) (GVB°, EGTA, MgCl2, NaOH, microplate reader, microplate constant temperature shaker, pipette, etc. are the same as those in Experiment 1).

[0227] 2.2 Preparation of working solution

[0228] 48% NHP: 100% NHP was diluted to 48% with buffer;

[0229] 26.4% NPS: 100% NHS diluted to 26.4% with buffer;

[0230] Rabbit red blood cell suspension: Blood was collected from the rabbit ear vein, anticoagulated with Aldrich solution (1:1), aliquoted and stored at 4°C. It can be stored for 4 weeks. Before use, centrifuge at 500g for 5 minutes and discard the Aldrich solution. Wash three times with an equal volume of buffer (500g) and centrifuge at 500g for 5 minutes. Finally, adjust the density to 6×10 8 pieces / mL.

[0231] Compound stock solution: 40 mM compound solution was diluted with DMSO to 2 mM stock solution, and the 2 mM stock solution was diluted 3-fold with DMSO, 10 points, to prepare the compound stock solution of each concentration;

[0232] Compound working solution: Dilute 500 times with buffer to obtain compound solutions of various concentrations.

[0233] 2.3 Experimental steps

[0234] 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% inactivated NHP or 26.4% inactivated NHS and 0.2% DMSO buffer were added to the blank control group; 100 μL of double-distilled water was added to the H2O group; the plates were incubated at 37°C for 15 min; 20 μL of rabbit erythrocyte suspension was added to each well, and the plates were incubated on a shaker at 37°C for 30 min; the plates were centrifuged at 2000 g (3380 rpm) for 5 min, 100 μL of the supernatant was transferred to a new 96-well ELISA plate, and the absorbance was measured at 415 nm.

[0235] 2.4 Data Analysis

[0236] 2.4.1 Hemolysis percentage at each drug concentration:

[0237] The PC group represents 100% hemolysis, and the NC group represents 0% hemolysis.

[0238] 2.4.2 Calculate the signal-to-background ratio (S / B):

[0239] The average OD value of the PC group / the average OD value of the NC group represents the size of the signal window.

[0240] 2.4.3 Z' factor:

[0241] Calculation formula:

[0242] 2.4.4 Compound IC 50

[0243] IC 50 : Half inhibitory concentration. Data were collected to calculate the log value of hemolysis percentage and compound concentration, and IC was calculated using GraphPad Prism software. 50 The inhibitory activity of the compound represented by formula A obtained in Example 1 of the present invention on rabbit erythrocyte hemolysis is shown in Table 14 below:

[0244] Table 14. Inhibitory activity of the compound of formula A on rabbit erythrocyte hemolysis

[0245] Experimental conclusion: The compound represented by formula A of the present invention has a good inhibitory effect on rabbit erythrocyte hemolysis.

[0246] 3. In vivo pharmacokinetic study of the compound represented by formula A

[0247] After SPF-grade SD rats were acclimated, 1 mg / kg of the compound represented by Formula A obtained in Example 1 of the present invention was administered via single oral gavage or tail vein bolus injection. Plasma was collected at specific time points after administration, and the compound concentration in plasma was measured by LC-MS / MS (AB SCIEX Qtrap4500). The PK parameters of the compound were calculated using software to reflect the pharmacokinetic properties of the compound in animals. The PK parameters of the compound represented by Formula A of the present invention are shown in Table 15 below:

[0248] Table 15. PK parameters of compounds of formula A

[0249] Experimental conclusion: The compound represented by formula A of the present invention can achieve higher in vivo exposure and higher oral bioavailability at a lower dose, and has better overall pharmacokinetic properties.

Claims

1. A crystalline form I of a nitrogen-containing spiro compound as shown in formula A, characterized in that: Its X-ray powder diffraction pattern using Cu-Kα radiation and expressed in 2θ angles has diffraction peaks at 5.67±0.20°, 11.37±0.20°, 16.69±0.20°, 17.32±0.20° and 19.73±0.20°; 2. The crystalline form I according to claim 1, characterized in that The crystal form I satisfies one or more of the following conditions: (1) The X-ray powder diffraction pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles also has diffraction peaks at one or more of 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 X-ray powder diffraction pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles may also have diffraction peaks at one or more of 18.04±0.20°, 18.43±0.20°, 22.94±0.20° and 26.01±0.20°; Preferably, the crystalline form I has an X-ray powder diffraction pattern using Cu-Kα radiation and expressed in 2θ angles at 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 X-ray powder diffraction pattern of the crystalline form I using Cu-Kα radiation and expressed in 2θ angles has diffraction peak positions as shown in the following table: Optimally, the crystal form I uses Cu-Kα radiation and an X-ray powder diffraction pattern expressed in 2θ angles, and its diffraction peak positions and relative intensities are shown in the following table: (2) The differential scanning calorimetry of the crystalline form I has an endothermic peak at 231.1±2°C; and (3) The thermogravimetric analysis of the crystal form I shows no weight loss before 200±2°C.

3. The crystalline form I according to claim 2, characterized in that The crystal form I satisfies one or more of the following conditions: (1) The X-ray powder diffraction pattern of Form I using Cu-Kα radiation and expressed in 2θ angles is substantially as shown in FIG2 ; (2) The differential scanning calorimetry diagram of Form I is substantially as shown in FIG3 ; and (3) The thermogravimetric analysis diagram of Form I is basically as shown in Figure 3.

4. A crystalline form II of a nitrogen-containing spiro compound as shown in formula A, characterized in that: Its X-ray powder diffraction pattern using Cu-Kα radiation and expressed in 2θ angles has diffraction peaks at 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°; 5. The crystal form II according to claim 4, characterized in that The crystal form II satisfies one or more of the following conditions: (1) The X-ray powder diffraction pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles also has diffraction peaks at one or more of 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 X-ray powder diffraction pattern of the crystalline form II using Cu-Kα radiation and expressed in 2θ angles may also have diffraction peaks at one or more of 13.55±0.20°, 16.40±0.20°, 17.74±0.20°, 20.49±0.20° and 22.06±0.20°; Preferably, the crystalline form II has an X-ray powder diffraction pattern using Cu-Kα radiation and expressed in 2θ angles at 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 X-ray powder diffraction pattern of the crystal form II using Cu-Kα radiation and expressed in 2θ angles has diffraction peak positions as shown in the following table: Optimally, the crystal form II uses Cu-Kα radiation and an X-ray powder diffraction pattern expressed in 2θ angles, and its diffraction peak positions and relative intensities are shown in the following table: (2) The differential scanning calorimetry of the crystal form II has an endothermic peak at 230.7±2°C; (3) The differential scanning calorimetry of the crystal form II has an exothermic peak at 159.1±2°C; and (4) The thermogravimetric analysis of the crystal form II showed a weight loss of 1.71% before 210±2°C.

6. The crystal form II according to claim 5, characterized in that The crystal form II satisfies one or more of the following conditions: (1) The X-ray powder diffraction pattern of Form II using Cu-Kα radiation and expressed in 2θ angles is substantially as shown in FIG6 ; (2) The differential scanning calorimetry diagram of Form II is substantially as shown in FIG7 ; and (3) The thermogravimetric analysis diagram of Form II is basically as shown in FIG7 .

7. A method for preparing the crystalline form I according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: dissolving the nitrogen-containing spiro compound as shown in formula A in N-methylpyrrolidone, adding an anti-solvent, and crystallizing; the anti-solvent is selected from one or more of a nitrile solvent, an ester solvent, a benzene solvent, an ether solvent and water; Preferably, the preparation method of the crystalline form I satisfies one or more of the following conditions: (1) The dissolving method is heating and stirring; the heating temperature can be 30 to 70° C., preferably 40 to 60° C., for example 50° C.; (2) The volume mass ratio of the 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 anti-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 anti-solvent to N-methylpyrrolidone is (1-10):1, preferably (3-8):1, for example 3:1, 6:1 or 8:1; (5) The step of cooling the temperature before adding the anti-solvent may be to a temperature of 10 to 20° C., for example, 17° C. 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, such as ethyl acetate and / or isopropyl acetate; (7) the benzene solvent is one or more of toluene, ethylbenzene, xylene, isopropylbenzene and chlorobenzene, such as toluene; and (8) The ether solvent is methyl tert-butyl ether, diethyl ether, cyclopentyl methyl ether, isopropyl ether, methyltetrahydrofuran and dioxane One or more of, for example, methyl tert-butyl ether.

8. A method for preparing the crystal form II according to any one of claims 4 to 6, characterized in that: The method comprises the following steps: stirring the nitrogen-containing spiro compound as shown in formula A in acetonitrile for 12 to 16 days; Preferably, the preparation method of the crystal form II satisfies one or more of the following conditions: (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) The stirring time is 14 days.

9. A pharmaceutical composition, characterized in that It includes: (1) the crystalline form I according to any one of claims 1 to 3 or the crystalline form II according to any one of claims 4 to 6; as well as (2) A pharmaceutically acceptable carrier.

10. Use of the crystalline form I according to any one of claims 1 to 3 or the crystalline form II according to any one of claims 4 to 6 in the preparation 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 disease, a kidney disease, a cardiovascular disease, an immune disorder, a central nervous system disease, a respiratory system disease, a urogenital system disease or an eye disease; Preferably, the disease mediated by complement factor D is cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody-associated vasculitis, warm antibody-type autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranous proliferative glomerulonephritis, dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus 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, lung Emphysema, coronavirus infection, macular degeneration, age-related macular degeneration, macular edema, diabetic macular edema, choroidal neovascularization, uveitis, Behcet's 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, Fisher's corneal endothelial dystrophy, retinal vein occlusion or postoperative inflammation; the coronavirus infection may be SARS-CoV, MERS-CoV or SARS-CoV-2 infection.

11. Use of the crystalline form I according to any one of claims 1 to 3 or the crystalline form II according to any one of claims 4 to 6 in the preparation of a medicament for treating and / or preventing a disease, wherein the disease is a blood disease, a kidney disease, a cardiovascular disease, an immune disorder, a central nervous system disease, a respiratory system disease, a urogenital system disease or an eye disease; Preferably, the disease is cold agglutinin disease, catastrophic antiphospholipid syndrome, hemolytic anemia, antineutrophil cytoplasmic antibody-associated vasculitis, warm antibody-type autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, membranous proliferative glomerulonephritis, dense deposit disease, C3 glomerulonephritis, focal segmental glomerulosclerosis, diabetic nephropathy, systemic lupus 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, coronavirus infection, macular degeneration, age-related macular degeneration, macular edema, diabetic macular edema, choroidal neovascularization, uveitis, Behcet's 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, Fisher's corneal endothelial dystrophy, retinal vein occlusion or postoperative inflammation; the coronavirus infection may be SARS-CoV, MERS-CoV or SARS-CoV-2 infection.

12. Use of the crystalline form I according to any one of claims 1 to 3 or the crystalline form II according to any one of claims 4 to 6 in the preparation of a complement factor D inhibitor; The complement factor D inhibitor can be used in vivo or in vitro in mammals.

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