Active aldehyde substance inhibitor based on AI design and application thereof

Abstract

The invention discloses an active aldehyde substance inhibitor based on AI design and application thereof. The active aldehyde substance inhibitor comprises a compound shown in a general formula (I) or a derivative thereof, and in the general formula (I), R1 and R2 are independently selected from any one of hydrogen, C2-6 unsaturated alkyl, C1-6 alkoxy, C2-6 halogenated alkyl, halogen, amino, C3-20 cycloalkyl or heterocyclic radical with 4-20 ring atoms; x is carbon or nitrogen; the derivative comprises at least one of a fluorescent marker, a spin marker, a heavy metal marker, an isotope marker and pharmaceutically acceptable salt. The active aldehyde substance (RASP) inhibitor provided by the invention has better capability and speed of complexing aldehyde, and can be quickly and effectively complexed with the active aldehyde substance after being applied to a diseased region, so that the content of RASP in the diseased region is reduced, and further, eye inflammation is reduced or avoided; the eye drops have potential prevention or treatment effects on various eye inflammatory diseases such as xerophthalmia and the like.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis and relates to an aldehyde scavenger, specifically an AI-designed active aldehyde inhibitor and its application. Background Technology

[0002] Dry eye disease (DED) refers to a series of conditions caused by various factors that lead to abnormalities in the quality and quantity of tears or abnormal tear dynamics, resulting in decreased tear film stability and accompanied by eye discomfort or ocular surface lesions. The causes of dry eye disease are complex, including environmental factors, lifestyle, immune diseases, eye surgery, medications, and age-related factors. Epidemiological surveys show that the global prevalence of dry eye is 8%-34%, with Asia having one of the highest prevalence rates globally, and China reaching 21%-30%.

[0003] Reactive aldehyde species (RASPs), such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), can be produced in vivo through various physiological processes. They covalently bind to the amino and sulfhydryl groups of receptors and kinases, thereby enhancing the cascade of upstream pro-inflammatory signals involving NF-κB, inflammasomes, scavenger receptor A, and other neurotransmitters (Higdon, A. et al., Biochem. J., 2012, 442:453-464; kalariya, N. Met al., Exp. Eye Res., 2008, 86:70-80; Kauppinen, A. et al., Immunol. Lett., 2012, 147:29-33; Sapkota, M. et al., Respir. Res., 2017, 18:36). Elevated RASP levels have been found in a variety of inflammatory eye diseases, including Behçet's disease, Sjögren's syndrome, non-infectious uveitis, allergic conjunctivitis, and dry eye (Sandikci, R. et al., Acta. Derm. Venereol., 2003, 83:342-346; Cejkova, J. et al., Mol. Vis., 2011, 17:443-447; Turk, A. et al., Ocul. Immunol. Inflamm., 2014, 22:127-132; Bacsi, A. et al., Allergy Clin. Immunol., 2005, 116:836-843; Choi, W. et al., Curr. Eye Res., 2016, 41:1143-1149). Studies have found elevated MDA levels in the tears of patients with dry eye syndrome, and the degree of MDA elevation is positively correlated with the severity of dry eye syndrome (Augustin, AJ, et al., 1995, 233:694-698). Another study also showed that, compared with the control group, the levels of MDA and HNE were elevated in the tears and conjunctival tissue biopsies of patients with dry eye syndrome, and were correlated with the severity of symptoms (Choi, W. et al., Curr. Eye Res., 2016, 41:1143-1149). In addition to pro-inflammatory signals, RASP also binds to phosphatidylethanolamine. Phosphatidylethanolamine is a key component of the tear lipid community and is essential for maintaining moisture in ocular surface tissues (Butovich, IA, et al., Prog. Lipid Res., 2011, 50:278-301). Therefore, RASP may serve as an important potential therapeutic target for dry eye syndrome.

[0004] RASP is closely related to the pathogenesis of various diseases and has been proven to be a target for major diseases such as dry eye, allergic conjunctivitis, uveitis, and Sjögren's syndrome. Therefore, RASP inhibitors have broad application prospects as drugs. However, no suitable RASP inhibitors have been marketed to date, and there are very few RASP inhibitors currently in clinical research. Therefore, finding RASP inhibitors with higher RASP inhibitory activity and lower toxicity has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide a drug that can inhibit the production of active aldehydes in the body (especially in the eyes) to effectively treat or prevent dry eye syndrome.

[0006] To achieve the above objectives, the present invention provides an active aldehyde inhibitor comprising a compound having the general formula I or a derivative thereof.

[0007]

[0008] in,

[0009] R1 and R2 are independently selected from hydrogen and C. 2-6 Unsaturated hydrocarbon group, C 1-6 Alkoxy, C 2-6 Halogenated alkyl, halogen, amino, C 3-20 Any one of cycloalkyl or heterocyclic groups having 4 to 20 ring atoms;

[0010] X is carbon or nitrogen;

[0011] The derivative comprises at least one of a fluorescent label, a spin label, a heavy metal label, an isotope label, and a pharmaceutically acceptable salt.

[0012] Optionally, R1 and R2 can form C6-C together with the carbon atoms they are attached to. 12 Aromatic or non-aromatic rings.

[0013] Optionally, R1 and R2 can form a benzene ring or an aliphatic heterocycle with 7-12 ring atoms together with the carbon atoms they are attached to.

[0014] Optionally, R1 and R2 are independently selected from hydrogen and C. 2-5 Unsaturated hydrocarbon group, C 2-5 Either alkoxy or amino.

[0015] Optionally, R1 and R2 are independently selected from hydrogen and C. 6-12 Any one of cycloalkyl or heterocyclic groups having 6 to 12 ring atoms.

[0016] Optionally, when R1 is a halogen, R2 is a C 2-6 Unsaturated hydrocarbon group.

[0017] Optionally, the compound represented by general formula I is selected from:

[0018] Any one of them.

[0019] The present invention also provides a pharmaceutical composition comprising, as an active ingredient, a compound of general formula I or a derivative thereof.

[0020] Optionally, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

[0021] Optionally, the pharmaceutical composition is a detection reagent or kit comprising at least one of a fluorescent label, spin label, heavy metal label, isotope label, and pharmaceutically acceptable salt of a compound represented by general formula I, for recognizing RASP ligands.

[0022] The present invention also provides an application of the above-mentioned active aldehyde inhibitor for the preparation of a drug as an aldehyde scavenger.

[0023] Optionally, the drug is used to treat or prevent a variety of diseases caused by active aldehydes.

[0024] Optionally, the disease refers to an inflammatory disease of the eye.

[0025] Optionally, the inflammatory eye disease includes at least one of dry eye syndrome, allergic conjunctivitis, Behcet's disease, Sjögren's syndrome, and uveitis.

[0026] Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0027] The active aldehyde (RASP) inhibitor provided by this invention has a good ability and speed to complex aldehydes. When applied to the lesion site, it can quickly and effectively complex with active aldehydes, reduce the content of RASP in the lesion site, and thus reduce or avoid eye inflammation. It has potential preventive or therapeutic effects in various inflammatory eye diseases such as dry eye syndrome. Attached Figure Description

[0028] Figure 1 This is a schematic diagram showing the results of the drug efficacy test in the animal model of allergic conjunctivitis in Example 28. Detailed Implementation

[0029] As described in the background section, RASP levels are elevated in ocular or systemic inflammatory diseases, potentially causing eye inflammation, reduced tear production, and altered tear lipid composition. For example, many patients with dry eye have high RASP levels. Inhibiting RASP can reduce ocular inflammation. Therefore, there is a need to design a compound that can inhibit RASP.

[0030] In the latest developments in AI technology, artificial intelligence can be used for the discovery, screening, and optimization of small molecule drugs. By integrating AI computing tools, databases (such as the CAS compound library), and receptor-ligand complex interaction information into efficient molecular / atomic training sets and AI digital workflows, and combining them with BT technologies (bioinformatics, in vitro target cell bioactivity detection, etc.), new therapeutic drugs can be invented.

[0031] As is well known, in the process of drug development, traditional small molecule compounds (drugs) require a lot of time to go from seed compounds to lead compounds and then to candidate compounds (usually taking 5-6 years or even longer). However, drug development driven by artificial intelligence (AIDrug Discovery & Design, AIDD) can perform data cross-comparison, molecular docking, molecular dynamics simulation, etc. in a shorter time (usually only 3-5 months), thereby accelerating the screening or de novo design of new compounds. Its core value lies in pioneering innovation and efficiency improvement.

[0032] Based on relatively well-defined mechanisms (such as the Schrödinger equation and Gibbs free energy change), AI can search multiple, broader, and more comprehensive databases of different compounds and proteomics, quickly completing searches and cross-matches, saving significant resources previously used for wet experiments. Wet experimental data can then be fed back into the AI ​​for iterative optimization of compound structures.

[0033] This invention is based on a computational structural biology technology platform built independently using an AI (dry experiment) + BT (wet experiment) interactive fusion R&D model. It combines various cutting-edge algorithms in medicinal chemistry and quantum chemistry with deep learning, natural language processing, and pre-trained models of various neural network frameworks to generate novel molecular structures with expected functional properties. Then, based on the target-drug related binding interface, molecular modeling, molecular docking, and molecular dynamics simulation are performed to obtain the optimal receptor-ligand binding free energy function, binding constant, and dissociation constant. This optimizes the screening of candidate compounds with the best in vitro cell activity and the most suitable overall evaluation. Meanwhile, through in-depth analysis of disease / drug correlation networks, AI servers, and workstations, drug deep learning and multi-threaded collaborative simulation can be performed simultaneously and independently. Alternatively, an AI computing power cluster can be formed to process large data of libraries and candidate compounds, deeply mining libraries, structure-pharmacodynamic group relationships (QSAR), and novel drug targets, saving innovative drug development costs and shortening R&D time. This forms a new R&D path of "dry and wet combination, seamless connection" for new drug development, which is expected to become a scientific paradigm for future innovative drug development, bringing a new breakthrough in thinking and experimental innovation to the past drug development process that was extremely dependent on wet experiments, which was time-consuming, laborious, and cumbersome.

[0034] This invention, after multiple rounds of iterative dry and wet closed-loop experiments for screening and verification, ultimately yielded an active aldehyde inhibitor comprising a compound or its derivative shown in general formula (I).

[0035]

[0036] in,

[0037] R1 and R2 are independently selected from hydrogen and C. 2-6 Unsaturated hydrocarbon group, C 1-6 Alkoxy, C 2-6 Halogenated alkyl, halogen, amino, C 3-20 Any one of cycloalkyl or heterocyclic groups with 4 to 20 ring atoms;

[0038] X is carbon or nitrogen;

[0039] The derivative comprises at least one of a fluorescent label, a spin label, a heavy metal label, an isotope label, and a pharmaceutically acceptable salt.

[0040] The "C" described in this invention 2-6"Unsaturated hydrocarbon group" refers to a straight-chain or branched hydrocarbon group with 2 to 6 carbon atoms having at least one carbon-carbon double or triple bond. A hydrocarbon group is a straight-chain or branched group containing several carbon atoms. Unsaturated hydrocarbon groups can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from one or more substituents selected from deuterium, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0041] The "C" described in this invention 1-6 "Alkoxy" refers to a group formed by attaching an alkyl group to an oxygen atom, wherein the oxygen atom has the ability to form bonds freely, such as methoxy, ethoxy, propoxy, butoxy, pentoxy, isopropoxy, tert-butoxy, cyclopropoxy, cyclohexyloxy, etc. Alkoxy groups can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from one or more substituents selected from deuterium, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0042] The "C" described in this invention 1-6 "Halogenated alkyl" includes monohalogenated alkyl and polyhalogenated alkyl (where all halogen atoms may be the same or different). Examples of halogenated alkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, etc.

[0043] The "halogen" mentioned in this invention refers to fluorine, chlorine, bromine, and iodine.

[0044] In this invention, "amino" refers to -NH2. The amino group can be substituted or unsubstituted, and when substituted, its substituent is preferably one or more of the following groups, which are independently selected from one or more substituents selected from alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0045] The term "cycloalkyl" as used in this invention refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10), and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0046] The term "spirocyclic" as used in this invention refers to a polycyclic group consisting of 5 to 20 quintiles, where the monocyclic rings share a single carbon atom (called a spiro atom), and may contain one or more double bonds. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles (e.g., 7, 8, 9, or 10 quintiles). Spirocyclic alkyl groups are classified into monospirocyclic alkyl groups, bispirocyclic alkyl groups, or polyspirocyclic alkyl groups based on the number of shared spiro atoms between the rings, with monospirocyclic alkyl groups and bispirocyclic alkyl groups being preferred. More preferably, they are 3 / 5 quintile, 3 / 6 quintile, 4 / 4 quintile, 4 / 5 quintile, 4 / 6 quintile, 5 / 5 quintile, or 5 / 6 quintile monospirocyclic alkyl groups. Non-limiting examples of spirocyclic alkyl groups include:

[0047] At least one of them.

[0048] The "fused ring" described in this invention refers to a 5- to 20-membered ring, wherein each ring shares an adjacent pair of carbon atoms with other rings in the structure as a fully carbon polycyclic group, and one or more rings may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused alkyl groups include:

[0049]

[0050] At least one of them.

[0051] The "bridged ring" described in this invention refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly connected carbon atoms, and may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged alkyl groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged alkyl groups include:

[0052]

[0053] At least one of them.

[0054] The polycyclic cycloalkyl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0055] The "heterocyclic group" as used in this invention refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 4 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), but does not include the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbon. Preferably, it contains 7 to 12 ring atoms, wherein 1 to 4 (e.g., 1, 2, 3, and 4) are heteroatoms; polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0056] The "spirocyclic heterocyclic group" described in this invention refers to a 5- to 20-membered polycyclic heterocyclic group in which the monocyclic rings share a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. It may contain one or more double bonds. Preferably, it is 6- to 14-membered. Spirocyclic heterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups according to the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic heterocyclic groups include:

[0057] At least one of them.

[0058] The "fused-ring heterocyclic group" described in this invention refers to a 4- to 20-membered polycyclic heterocyclic group in which each ring shares an adjacent pair of atoms with other rings in the structure. One or more rings may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring heterocyclic group, preferably bicyclic or tricyclic, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, and 6-membered / 6-membered bicyclic fused-ring heterocyclic groups. Non-limiting examples of fused-ring heterocyclic groups include:

[0059]

[0060]

[0061] At least one of them.

[0062] The "bridged heterocyclic group" described in this invention refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly connected atoms. It may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), with the remaining ring atoms being carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0063]

[0064] At least one of them.

[0065] The polycyclic heterocyclic group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently and arbitrarily selected from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0066] The "aromatic ring" as described in this invention refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (fused polycyclic) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aromatic ring can be substituted or unsubstituted; when substituted, it can be substituted at any usable junction. The substituent is preferably independently and arbitrarily selected from one or more of saturated or unsaturated hydrocarbon groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclic oxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups.

[0067] The term "pharmaceutically acceptable salt" or "medicinal salt" as used in this invention refers to salts of pharmaceutically acceptable acids and bases. Such pharmaceutically acceptable salts include salts formed with acids, including hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfinic acid, formic acid, p-toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, chain carboxylic acids such as acetic acid, and HOOC-(CH2). n -COOH (n=0-4), etc.; also includes salts formed with bases, whose cations include sodium, potassium, calcium, ammonium ions, etc.

[0068] In this invention, "substituted" means that an organic group (containing one or more bonds bonded to a hydrogen atom) as defined herein is replaced by one or more bonds bonded to a non-hydrogen atom or group of atoms, wherein the non-hydrogen atom or group of atoms is a substituent.

[0069] In this invention, "derivative" refers to a compound formed by replacing an atom or group of atoms in a compound of general formula (I) with other atoms or groups of atoms, including at least one of fluorescent markers, spin markers, heavy metal markers, isotope markers, and pharmaceutically acceptable salts.

[0070] On the other hand, the present invention also relates to fluorescently labeled, spin-labeled, heavy metal- or isotope-labeled derivatives of the compounds described herein, which can be used not only for imaging but also for the in vivo and in vitro detection, localization, and quantification of RASP in tissue samples (including humans), by inhibiting the binding of the labeled compound and identifying RASP ligands (ligands are groups that can bind to the labeled compound). Therefore, the present invention further provides RASP detection reagents or kits containing such labeled compounds.

[0071] This invention further provides isotopically labeled compounds of the compounds of this invention. The term "isotopically labeled compound" or "isotopically labeled" in this invention refers to the compound described herein, wherein one or more atoms are replaced by isotopic atoms whose atomic mass or mass number differs from that of naturally occurring atoms. Suitable radionuclides may include, but are not limited to, 2H (deuterium, also written as D), 3H (tritium, also written as T), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I, and 131I. The type of radioisotope contained in the isotopically labeled compound will depend on the specific application of the isotopically labeled compound. For example, for labeling and competition assays of in vitro IDO enzymes, compounds containing 3H, 14C, 82Br, 125I, 131I, or 35S are generally most useful. For isotope imaging applications, 11C, 18F, 125I, 123I, 124I, 131I, 75Br, 76Br, or 77Br are generally most useful.

[0072] The methods known in the prior art for labeling organic compounds with radioactive isotopes are also applicable to the compounds of the present invention.

[0073] When administered as a drug, the compound can be delivered in the form of a pharmaceutical composition. Therefore, in another aspect, this application provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0074] The term "composition" as used herein means a product containing a compound disclosed in this application or a pharmaceutically acceptable salt thereof as a specific active ingredient, as well as any other product directly or indirectly combined with said active ingredient.

[0075] Typically, the pharmaceutical composition contains at least one pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with other components in the formulation and is harmless to the subject. The carrier referred to herein is a substance used to improve the selectivity, efficacy, and / or safety of the drug during delivery. The carrier is primarily used to control drug release and may also be used to improve the pharmacokinetic properties of the drug, particularly bioavailability. The excipient refers to any substance in the pharmaceutical formulation other than the active ingredient, primarily used for long-term stability, filling solid dosage forms (therefore, it is often used specifically to refer to "filler"), or enhancing product efficacy (e.g., promoting absorption, reducing viscosity, or increasing solubility).

[0076] The raw materials or reagents used in the embodiments of the present invention are commercially available or prepared by synthetic methods commonly known in the art.

[0077] The full names of the reagent abbreviations used in the examples are as follows:

[0078] Pd(dppf)Cl2: 1,1-bis(diphenylphosphine)diferropalladium dichloride

[0079] DMF: N,N-Dimethylformamide

[0080] TMS: Tetramethylsilane

[0081] TEA: Triethylamine

[0082] NBS: N-bromosuccinimide

[0083] EA: Ethyl acetate

[0084] PE: Petroleum ether

[0085] LCMS detection: Combined detection by liquid chromatography and mass spectrometry

[0086] TLC detection: Thin-layer chromatography.

[0087] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0088] Example 1: Preparation of Compound 1

[0089]

[0090] Synthetic route of compound 1:

[0091]

[0092] Step 1: Preparation of intermediate 1a

[0093] At room temperature, methyl 3-bromo-2-oxopropionate (1.89 g, 10.45 mmol) was added dropwise to a pyridine (1.5 mL) solution in methanol (25 mL). The reaction mixture was stirred at room temperature for 1 hour, and then stirred at 60 °C for another 2 hours. After cooling to room temperature, 2-amino-5-bromobenzaldehyde (1.9 g, 9.50 mmol) and pyridine (5.0 mL) were added sequentially. The reaction mixture was heated to 85 °C and stirred for 4 hours, followed by the addition of pyrrole (2.0 mL), and the temperature was maintained for another 4 hours of stirring. The reaction was confirmed by LCMS. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with saturated sodium chloride (50 mL x 3). The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 3:1) to give intermediate 1a (1.0 g, yield: 37.45%).

[0094] MS(ESI): m / z 283.0 [M+2+H] + .

[0095] Step 2: Preparation of intermediate 1b

[0096] Under nitrogen protection, acetylenoltrimethylsilane (1.05 g, 10.67 mmol) and triethylamine (2.5 mL, 17.8 mmol) were added sequentially to a solution of intermediate 1a (1.0 g, 3.56 mmol), cuprous iodide (67.8 mg, 0.356 mmol), and palladium dichloride bis(triphenylphosphine) (250 mg, 0.356 mmol) in 1,4-dioxane (30 mL). The reaction mixture was stirred at 95 °C for 2 hours.

[0097] The reaction was detected by LCMS, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 4:1) to obtain intermediate 1b (1.0 g, yield: 94.34%).

[0098] MS(ESI): m / z 299.2 [M+H] + .

[0099] Step 3: Preparation of intermediate 1c

[0100] Potassium carbonate (1.39 g, 10.05 mmol) was added to a methanol (20 mL) solution of intermediate 1b (1.0 g, 3.35 mmol). The reaction mixture was stirred at room temperature for 30 minutes.

[0101] The reaction was confirmed by LCMS. The reaction solution was extracted with ethyl acetate (80 mL), diluted, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 2:1) to give intermediate 1c (550 mg, yield: 72.5%).

[0102] MS(ESI): m / z 227.1 [M+H] + .

[0103] Step 4: Preparation of Compound 1

[0104] Intermediate 1c (550 mg, 2.43 mmol) was dissolved in tetrahydrofuran (25 mL), cooled to -70 °C, and 3.0 M methyl magnesium bromide in 2-methyltetrahydrofuran solution (4 mL, 12.15 mmol) was added dropwise. The reaction solution was stirred for 30 minutes while maintaining the temperature.

[0105] The reaction was confirmed by LCMS. Saturated ammonium chloride (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparation (acetonitrile: 0.1% formic acid aqueous solution = 0% to 100%) to give compound 1 (100 mg, yield: 18.2%). MS (ESI): m / z 227.0 [M+H] + .

[0106] 1 H NMR (400MHz, DMSO-d6) δ7.76(d,J=2.0Hz,1H),7.69(d,J=8.4Hz,1H),7.30(dd,J1=2.0Hz,J2=7.6Hz,1H),7.20(s,1H),6.02(br s,2H),5.77(br s, 1H), 1.60 (d, J = 2.8Hz, 6H).

[0107] Example 2: Preparation of Compound 2

[0108]

[0109] Synthetic route of compound 2:

[0110]

[0111] Step 1: Preparation of intermediate 2a

[0112] 5-Bromo-3-nitrobutyronitrile (25 g, 109.65 mmol) was dissolved in concentrated sulfuric acid (200 mL), and the mixture was heated to 120 °C and stirred for 2 hours. The reaction solution was then cooled to 0 °C, and sodium nitrite aqueous solution (22.7 g, 329 mmol, dissolved in 50 mL of water) was added dropwise. After the addition was complete, the mixture was heated to 80 °C and stirred for 16 hours.

[0113] The reaction was confirmed by LCMS. After cooling to room temperature, the reaction solution was poured into ice water (800 mL) to obtain a white suspension. The suspension was filtered and washed with distilled water (600 mL). The filter cake was then vacuum dried to obtain intermediate 2a (25 g, yield: 92.3%). MS (ESI): m / z 247.0 [M+H] + .

[0114] Step 2: Preparation of intermediate 2b

[0115] Concentrated sulfuric acid (15 mL) was added to a methanol (200 mL) solution of intermediate 2a (25 g, 101.2 mmol). The reaction mixture was stirred at 80 °C for 16 hours.

[0116] The reaction was detected by LCMS. After the reaction solution was cooled to room temperature, it was diluted with ethyl acetate (800 mL), washed with saturated sodium bicarbonate (120 mL x 3) and saturated sodium chloride (120 mL x 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE:EA = 20:1 to 10:1) to give intermediate 2b (21.8 g, yield: 82.9%).

[0117] MS(ESI): m / z 261.0 [M+H] + .

[0118] 1 H NMR (400MHz, DMSO-d6) δ9.15 (d, J = 2.0 Hz, 1H), 8.97 (d, J = 1.6 Hz, 1H), 3.93 (s, 3H).

[0119] Step 3: Preparation of intermediate 2c

[0120] Intermediate 2b (800 mg, 3.09 mmol), potassium vinyltrifluoroborate (828 mg, 6.18 mmol), 1,1-bis(diphenylphosphine)dimerferropalladium dichloride (226 mg, 0.309 mmol), and triethylamine (2.2 mL, 15.45 mmol) were dissolved in ethanol (20 mL), and nitrogen gas was purged three times. The reaction mixture was stirred at 90 °C for 2 hours.

[0121] The reaction was confirmed by LCMS and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 15:1 to 3:1) to give intermediate 2c (400 mg, yellow oil, yield: 62.2%).

[0122] MS(ESI): m / z 208.8 [M+H] + .

[0123] Step 4: Preparation of intermediate 2d

[0124] Intermediate 2c (400 mg, 1.92 mmol) was dissolved in ethanol (20 mL), and iron powder (538 mg, 9.6 mmol) and ammonium chloride (514 mg, 9.6 mmol) were added. The reaction mixture was stirred at 50 °C for 1 hour.

[0125] The reaction was detected by LCMS, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 8:1) to give intermediate 2d (150 mg, yellow solid, yield: 43.9%).

[0126] MS(ESI): m / z 178.8 [M+H] + .

[0127] Step 5: Preparation of Compound 2

[0128] Compound 2 was prepared from intermediate 2d (150 mg, 0.843 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (4.2 mL, 4.215 mmol) according to step 4 of Example 1, with a yield of 21.3%.

[0129] MS(ESI): m / z 179.1 [M+H] + .

[0130] 1H NMR(400MHz,CD3OD)δ7.78(d,J=1.6Hz,1H),7.17(d,J=1.6Hz,1H),6.69-6.62(m,1H), 5.81(dd, J1=1.2Hz, J2=18.0Hz, 1H), 5.81(dd, J1=0.8Hz, J2=8.4Hz, 1H), 1.61(s, 6H).

[0131] Example 3: Preparation of Compound 3

[0132]

[0133] Synthetic route of compound 3:

[0134]

[0135] Step 1: Preparation of intermediate 3a

[0136] Intermediate 3a was prepared from methyl 3-amino-6-bromopyridinecarboxylate (1.0 g, 4.33 mmol) and potassium vinyltrifluoroborate (1.16 g, 8.66 mmol) according to step 3 of Example 2, with a yield of 90.9%.

[0137] MS(ESI): m / z 179.2 [M+H] + .

[0138] Step 2: Preparation of Compound 3

[0139] Compound 3 was prepared by the method in step 4 of Example 1, using intermediate 3a (200 mg, 1.12 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (3.7 mL, 11.2 mmol), with a yield of 45%.

[0140] MS(ESI): m / z 179.1 [M+H] + .

[0141] 1 H NMR (400MHz, MeOD) δ7.07 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.65 (dd, J = 10.8 Hz, 17.6 Hz, 1H), 5.88 (d, J = 17.2 Hz, 1H), 1.60 (s, 6H).

[0142] Example 4: Preparation of Compound 4

[0143]

[0144] Synthetic route of compound 4:

[0145]

[0146] Step 1: Preparation of intermediate 4a

[0147] Intermediate 4a was prepared from methyl 3-amino-5-bromopyridinecarboxylate (1.2 g, 5.19 mmol) and ethynyltrimethylsilane (1.53 g, 15.6 mmol) according to step 2 of Example 1, with a yield of 82%.

[0148] MS(ESI): m / z 249.2 [M+H] + .

[0149] Step 2: Preparation of intermediate 4b

[0150] Intermediate 4b was prepared from intermediate 4a (1.5 g, 6.04 mmol) according to step 3 of Example 1, with a yield of 84.8%.

[0151] MS(ESI): m / z 177.1 [M+H] + .

[0152] Step 3: Preparation of Compound 4

[0153] Compound 4 was prepared from intermediate 4b (150 mg, 0.852 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (4.3 mL, 4.26 mmol) according to step 4 of Example 1, with a yield of 53.7%.

[0154] MS(ESI): m / z 177.1 [M+H] + .

[0155] 1 H NMR (400MHz, CD3OD) δ7.80 (d, J = 2.0 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 3.55 (s, 1H), 1.57 (s, 6H).

[0156] Example 5: Preparation of Compound 5

[0157]

[0158] Synthetic route of compound 5:

[0159]

[0160] Step 1: Preparation of intermediate 5a

[0161] Methyl 2-amino-5-fluorobenzoate (1.0 g, 5.92 mmol) was dissolved in acetonitrile (10 mL), and N-bromosuccinimide (1.26 g, 7.10 mmol) was added. The mixture was stirred at room temperature for 16 hours.

[0162] The reaction was confirmed by TLC. The reaction solution was diluted with water and extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 5a (1.0 g, yellow oil, crude product). The crude product was used directly in the next reaction step.

[0163] Step 2: Preparation of intermediate 5b

[0164] Intermediate 5b was prepared from intermediate 5a (500 mg, 2.01 mmol) and potassium vinyltrifluoroborate (402 mg, 3.02 mmol) according to step 3 of Example 2, with a yield of 66.0%.

[0165] MS(ESI): m / z 195.8 [M+H] + .

[0166] Step 3: Preparation of Compound 5

[0167] Compound 5 was prepared from intermediate 5b (260 mg, 1.33 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (4.3 mL, 4.26 mmol) according to step 4 of Example 1, with a yield of 70.4%.

[0168] MS(ESI): m / z 178.1 [M+H-H2O] + .

[0169] 1 H NMR (400MHz, CD3OD) δ6.95-6.83 (m, 3H), 5.59 (d, J = 17.2Hz, 1H), 5.30 (dd, J1 = 0.8Hz, J2 = 10.8Hz, 1H), 1.60 (s, 6H).

[0170] Example 6: Preparation of Compound 6

[0171]

[0172] Synthetic route of compound 6:

[0173]

[0174] Step 1: Preparation of intermediate 6a

[0175] Intermediate 6a was prepared from methyl 2-amino-4-bromo-5-fluorobenzoate (500 mg, 2.0 mmol) and potassium vinyltrifluoroborate (536 mg, 4.0 mmol) according to step 3 of Example 2, with a yield of 76.5%.

[0176] MS(ESI): m / z 195.8 [M+H] + .

[0177] Step 2: Preparation of Compound 6

[0178] Compound 6 was prepared from intermediate 6a (300 mg, 1.54 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (7.7 mL, 7.7 mmol) according to step 4 of Example 1, yield: 20%.

[0179] MS(ESI): m / z 178.1 [M+H-H2O] + .

[0180] 1 H NMR (400MHz, CD3OD) δ6.86 (s, 1H), 6.83 (d, J = 6.8Hz, 1H), 6.78-6.70 (m, 1H), 5.75 (dd, J1=1.2Hz, J2=17.6Hz, 1H), 5.26 (dd, J1=1.2Hz, J2=11.2Hz, 1H), 1.58 (s, 6H).

[0181] Example 7: Preparation of Compound 7

[0182]

[0183] Synthetic route of compound 7:

[0184]

[0185] Step 1: Preparation of intermediate 7a

[0186] Intermediate 7a was prepared from methyl 2-amino-4-bromobenzoate (1.0 g, 4.34 mmol) and potassium vinyltrifluoroborate (750 mg, 5.64 mmol) according to step 3 of Example 2, with a yield of 78%.

[0187] MS(ESI): m / z 178.2 [M+H] + .

[0188] 1H NMR (400MHz, CDCl3) δ7.81 (d, J = 8.4Hz, 1H), 6.74 (dd, J = 8.4Hz, 1.6Hz, 1H), 6.66 (d, J = 1.6Hz, 1H) ,6.65-6.58(m,1H),5.79(dd,J=17.2Hz,0.4Hz,1H),5.33(dd,J=10.8Hz,0.4Hz,1H),3.87(s,3H).

[0189] Step 2: Preparation of Compound 7

[0190] Compound 7 was prepared from intermediate 7a (350 mg, 1.97 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (3.95 mL, 11.85 mmol) according to step 4 of Example 1, with a yield of 74.3%.

[0191] MS(ESI): m / z 160.1 [M+H-H2O] + .

[0192] 1 H NMR (400MHz, MeOD) δ7.07(d,J=8.4Hz,1H),6.78(d,J=1.6Hz,1H),6.67(dd,J=1.6Hz,8.0Hz,1H),6.58( dd,J=2.8Hz,17.6Hz,1H),5.67(dd,J=1.2Hz,17.6Hz,1H),5.11(dd,J=0.8Hz,10.8Hz,1H),1.60(s,6H).

[0193] Example 8: Preparation of Compound 8

[0194]

[0195] Synthetic route of compound 8:

[0196]

[0197] Step 1: Preparation of intermediate 8a

[0198] Intermediate 8a was prepared from methyl 2-amino-5-bromobenzoate (1.5 g, 6.52 mmol) and potassium vinyltrifluoroborate (1.3 g, 9.78 mmol) according to step 3 of Example 2, with a yield of 78%.

[0199] MS(ESI): m / z 178.2 [M+H] + .

[0200] Step 2: Preparation of Compound 8

[0201] Compound 8 was prepared from intermediate 8a (230 mg, 1.30 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (3.46 mL, 10.38 mmol) according to step 4 of Example 1, with a yield of 65.2%.

[0202] MS(ESI): m / z 160.1 [M+H-H2O] + .

[0203] 1 H NMR (400MHz, MeOD) δ7.17(d,J=2.0Hz,1H),7.10(dd,J=2.0Hz,8.0Hz,1H),6.64(d,J=8.0Hz,1H),6.57(d d, J=11.2Hz, 17.6Hz, 1H), 5.48 (dd, J=0.8Hz, 17.6Hz, 1H), 4.93 (dd, J=1.2Hz, 10.8Hz, 1H), 1.61 (s, 6H).

[0204] Example 9: Preparation of Compound 9

[0205]

[0206] Synthetic route of compound 9:

[0207]

[0208] Step 1: Preparation of intermediate 9a

[0209] Intermediate 9a was prepared from methyl 2-amino-4-bromobenzoate (1.0 g, 4.35 mmol) and ethynyltrimethylsilane (1.28 g, 13.05 mmol) according to step 2 of Example 1, with a yield of 93.1%.

[0210] MS(ESI): m / z 247.8 [M+H] + .

[0211] Step 2: Preparation of intermediate 9b

[0212] Intermediate 9b was prepared from intermediate 9a (1.0 g, 4.05 mmol) according to step 3 of Example 1, with a yield of 99%.

[0213] MS(ESI): m / z 175.8 [M+H] + .

[0214] Step 3: Preparation of Compound 9

[0215] Compound 9 was prepared from intermediate 9b (300 mg, 1.71 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (8.5 mL, 8.55 mmol) according to step 4 of Example 1, with a yield of 71%.

[0216] MS(ESI): m / z 158.1 [M+H-H2O] + .

[0217] 1 H NMR (400MHz, CD3OD) δ7.07 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 1.6 Hz, 1H), 6.70 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 1.59 (s, 6H).

[0218] Example 10: Preparation of Compound 10

[0219]

[0220] Synthetic route of compound 10:

[0221]

[0222] Step 1: Preparation of intermediate 10a

[0223] Intermediate 10a was prepared from 1-(2-amino-5-iodophenyl)ethane-1-one (700 mg, 2.69 mmol) and 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborane (904 mg, 5.38 mmol) according to step 3 of Example 2, with a yield of 27.6%.

[0224] MS(ESI): m / z 176.2 [M+H] + .

[0225] Step 2: Preparation of Compound 10

[0226] Compound 10 was prepared from intermediate 10a (130 mg, 0.743 mmol) and a 1.0 M tetrahydrofuran solution of methyl magnesium bromide (3.7 mL, 3.715 mmol) according to step 4 of Example 1, with a yield of 12.5%.

[0227] MS(ESI): m / z 174.1 [M+H-H2O] + .

[0228] 1H NMR (400MHz, CD3OD) δ7.08(d,J=1.6Hz,1H),7.02(dd,J1=2.0Hz,J2=8.4Hz,1H),7.02(dd,J1 =1.2Hz, J2=15.6Hz, 1H), 6.03-5.94 (m, 1H), 1.81 (dd, J1=1.6Hz, J2=6.8Hz, 1H), 1.61 (s, 9H).

[0229] Example 11: Preparation of Compound 11

[0230]

[0231] Synthetic route of compound 11:

[0232]

[0233] Step 1: Preparation of intermediate 11a

[0234] Methyl 2-amino-4-bromobenzoate (8.0 g, 34.72 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborane (8.75 g, 52.08 mmol), 1,1-bis(diphenylphosphine)diborane-iron palladium dichloride (2.54 g, 3.47 mmol), and potassium carbonate (14.4 g, 104.16 mmol) were dissolved in 1,4-dioxane (80 mL) and water (8 mL), and nitrogen gas was purged three times. The reaction mixture was stirred at 90 °C for 2 hours.

[0235] The reaction was detected by LCMS and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 20:1 to 15:1) to obtain intermediate 11a (4.1 g, yellow oil, yield: 61.8%).

[0236] MS(ESI): m / z 192.2 [M+H] + .

[0237] Step 2: Preparation of Compound 11

[0238] Compound 11 was prepared from intermediate 11a (4.1 g, 21.47 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methyl magnesium bromide (28.63 mL, 85.88 mmol) according to step 4 of Example 1, with a yield of 39.4%.

[0239] MS(ESI): m / z 174.1 [M+H-H2O] + .

[0240] 1H NMR (400MHz, CD3OD) δ7.07(d,J=8.4Hz,1H),6.84(d,J=2.0Hz,1H),6.76(dd,J1=2.0Hz, J2=8.0Hz,1H),5.29(d,J=0.8Hz,1H),4.97(t,J=1.6Hz,1H),2.07(s,3H),1.60(s,6H).

[0241] Example 12: Preparation of Compound 12

[0242]

[0243] Synthetic route of compound 12:

[0244]

[0245] Step 1: Preparation of intermediate 12a

[0246] Methyl 4-hydroxy-2-nitrobenzene (600 mg, 3.043 mmol), 1-chloro-2-methoxyethane (863 mg, 9.13 mmol), and potassium carbonate (1.26 g, 9.13 mmol) were dissolved in N,N-dimethylformamide (15 mL). The reaction mixture was stirred at 80 °C for 5 hours.

[0247] The reaction was detected by LCMS. After the reaction solution was cooled to room temperature, it was diluted with ethyl acetate (150 mL), washed with saturated sodium chloride (20 mL x 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 4:1) to obtain intermediate 12a (520 mg, yellow oil, yield: 67.01%).

[0248] MS(ESI): m / z 273.1 [M+H2O] + .

[0249] Step 2: Preparation of intermediate 12b

[0250] Intermediate 12a (520 mg, 2.04 mmol) and palladium on carbon (50 mg) were dissolved in methanol (15 mL). The reaction mixture was then purged with hydrogen and stirred at 60 °C for 16 hours.

[0251] The reaction was confirmed by LCMS. After the reaction solution was cooled to room temperature, it was filtered through diatomaceous earth, and the residue was washed with ethyl acetate (30 mL). The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 4:1) to obtain intermediate 12b (350 mg, yellow oil, yield: 76.25%).

[0252] MS(ESI): m / z 226.2 [M+H] + .

[0253] Step 3: Preparation of Compound 12

[0254] Compound 12 was prepared from intermediate 12b (150 mg, 0.666 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (1.76 mL, 5.32 mmol) according to step 4 of Example 1, with a yield of 56.6%.

[0255] MS(ESI): m / z 208.1 [M+H-H2O] + .

[0256] 1 H NMR(400MHz, DMSO_d6)δ6.86(d,J=8.4Hz,1H),6.17(d,J=2.8Hz,1H),6.04(dd,J=2.8Hz,8.8Hz,1H),5 .39(brs,2H),5.07(brs,1H),3.95(t,J=4.4Hz,2H),3.60(t,J=4.8Hz,2H),3.29(s,3H),1.45(s,6H).

[0257] Example 13: Preparation of Compound 13

[0258]

[0259] Synthetic route of compound 13:

[0260]

[0261] Step 1: Preparation of intermediate 13a

[0262] 6-Chloro-2-methyl-3-nitropyridine (20.0 g, 116 mmol) was dissolved in concentrated sulfuric acid (100 mL), cooled to 0 °C, and then chromium trioxide (34.8 g, 348 mmol) was added in portions. The mixture was stirred at room temperature for 12 hours under nitrogen protection.

[0263] The reaction was detected by LCMS. The reaction solution was slowly poured into ice water (200 mL) and extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give intermediate 13a (19.0 g, green solid, yield: 81%).

[0264] MS(ESI): m / z 202.9 [M+H] + .

[0265] Step 2: Preparation of intermediate 13b

[0266] Intermediate 13a (19.0 g, 93.8 mmol) was dissolved in methanol (100 mL), and thionyl chloride (10 mL) was slowly added dropwise. The mixture was stirred at room temperature for 12 hours under nitrogen protection.

[0267] The reaction was confirmed by LCMS, concentrated under reduced pressure, and extracted with ethyl acetate (100 mL) and saturated sodium bicarbonate aqueous solution (50 mL) (3 times). The organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA = 100:1 to 20:1) to give intermediate 13b (8.60 g, yellow oil, yield: 42%).

[0268] MS(ESI): m / z 217.0 [M+H] + .

[0269] Step 3: Preparation of intermediate 13c

[0270] Intermediate 13c was prepared from intermediate 13b (300 mg, 1.39 mmol) and 2-methoxyethane-1-ol (5 mL) according to step 1 of Example 12, yield: 48%.

[0271] MS(ESI): m / z 300.80 [M+NH4] + .

[0272] Step 4: Preparation of intermediate 13d

[0273] Intermediate 13d was prepared from intermediate 13c (200 mg, 0.666 mmol) according to step 2 of Example 12, with a yield of 89%.

[0274] MS(ESI): m / z 270.80 [M+H] + .

[0275] Step 5: Preparation of Compound 13

[0276] Compound 13 was prepared from intermediate 13d (160 mg, 0.592 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methyl magnesium bromide (2 mL, 6 mmol) according to step 4 of Example 1, yield: 28%.

[0277] MS(ESI): m / z 227.2 [M+H] + .

[0278] 1H NMR (400MHz, DMSO-d6) δ6.99(d,J=8.4Hz,1H),6.45(d,J=8.8Hz,1H),5.31(brs,1H),5.04(br s, 2H), 4.21 (t, J = 4.8Hz, 2H), 3.60 (t, J = 4.8Hz, 2H), 3.27 (s, 3H), 1.46 (s, 6H).

[0279] Example 14: Preparation of Compound 14

[0280]

[0281] Synthetic route of compound 14:

[0282]

[0283] Step 1: Preparation of intermediate 14a

[0284] Intermediate 14a was prepared from methyl 5-hydroxy-3-nitropyridinecarboxylate (500 mg, 2.52 mmol) and 1-chloro-2-methoxyethane (1.19 g, 12.62 mmol) according to step 1 of Example 12, with a yield of 31%.

[0285] MS(ESI): m / z 257.1 [M+H] + .

[0286] Step 2: Preparation of intermediate 14b

[0287] Intermediate 14b was prepared from intermediate 14a (200 mg, 0.78 mmol) according to step 2 of Example 12, with a yield of 82.9%.

[0288] MS(ESI): m / z 227.2 [M+H] + .

[0289] Step 3: Preparation of Compound 14

[0290] Compound 14 was prepared by the method in step 4 of Example 1, using intermediate 14b (130 mg, 0.575 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (1.9 mL, 5.75 mmol), with a yield of 57.69%.

[0291] MS(ESI): m / z 227.2 [M+H] + .

[0292] 1H NMR (400MHz, MeOD) δ7.54 (d, J = 2.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 4.25-4.23 (m, 2H), 3.77-3.75 (m, 2H), 3.41 (s, 3H), 1.66 (s, 6H).

[0293] Example 15: Preparation of Compound 15

[0294]

[0295] Synthetic route of compound 15:

[0296]

[0297] Step 1: Preparation of intermediate 15a

[0298] Intermediate 15a was prepared from methyl 6-chloro-3-nitropyridinecarboxylate (300 mg, 1.39 mmol) and (E)-2-(3-methoxypropyl-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane (412 mg, 2.08 mmol) according to step 1 of Example 11, yield: 77%.

[0299] MS(ESI): m / z 252.8 [M+H] + .

[0300] Step 2: Preparation of intermediate 15b

[0301] Intermediate 15b was prepared from intermediate 15a (270 mg, 1.07 mmol) according to step 4 of Example 2, with a yield of 29%.

[0302] MS(ESI): m / z 222.8 [M+H] + .

[0303] Step 3: Preparation of Compound 15

[0304] Compound 15 was prepared from intermediate 15b (70 mg, 0.315 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (1 mL, 3 mmol) according to step 4 of Example 1, yield: 28%.

[0305] MS(ESI): m / z 223.1 [M+H] + .

[0306] 1H NMR (400MHz, DMSO-d6) δ7.04(d,J=8.0Hz,1H),6.89(d,J=8.0Hz,1H),6.46(d,J=16Hz,1H),6.39-6.32(m,1H),5.62(br s,2H),5.45(br s, 1H), 4.01 (d, J = 5.6Hz, 2H), 3.25 (s, 3H), 1.49 (s, 6H).

[0307] Example 16: Preparation of Compound 16

[0308]

[0309] Synthetic route of compound 16:

[0310]

[0311] Step 1: Preparation of intermediate 16a

[0312] Intermediate 16a was prepared from 1-(2-amino-5-bromophenyl)ethyl ketone (2.5 g, 11.7 mmol) and (E)-2-(3-methoxypropyl-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane (2.54 g, 12.8 mmol) according to step 1 of Example 11, yield: 75%.

[0313] MS(ESI): m / z 206.2 [M+H] + .

[0314] Step 2: Preparation of Compound 16

[0315] Compound 16 was prepared from intermediate 16a (1.80 g, 8.77 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methyl magnesium bromide (23.4 mL, 70.2 mmol) according to step 4 of Example 1, with a yield of 43%.

[0316] MS(ESI): m / z 204.1 [M-H2O+H] + .

[0317] 1 H NMR (400MHz, CD3OD) δ7.17(s,1H),7.11(d,J=8.0Hz,1H),6.65(d,J=8.4Hz,1H),6.50( d,J=15.6Hz,1H),6.08-6.00(m,1H),4.04(d,J=6.4Hz,2H),3.35(s,3H),1.62(s,6H).

[0318] Example 17: Preparation of Compound 17

[0319]

[0320] Synthetic route of compound 17:

[0321]

[0322] Step 1: Preparation of intermediate 17a

[0323] Methyl 5-fluoro-2-nitrobenzoate (5.0 g, 25.1 mmol), potassium carbonate (10.4 g, 75.3 mmol), and 7-azaspiro[3.5]nonane hydrochloride (4.87 g, 30.1 mmol) were dissolved in N,N-dimethylformamide (50 mL). The reaction mixture was stirred at 80 °C for 12 hours.

[0324] The reaction was confirmed by LCMS. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 3:1) to give intermediate 17a (5.0 g, yellow solid, yield: 65%).

[0325] MS(ESI): m / z 305.2 [M+H] + .

[0326] Step 2: Preparation of intermediate 17b

[0327] Intermediate 17b was prepared from intermediate 17a (5.0 g, 16.4 mmol) according to step 2 of Example 12, with a yield of 67%.

[0328] MS(ESI): m / z 275.2 [M+H] + .

[0329] Step 3: Preparation of Compound 17

[0330] Compound 17 was prepared by the method in step 4 of Example 1 from intermediate 17b (3.0 g, 10.9 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (36.4 mL, 109 mmol), with a yield of 54%.

[0331] MS(ESI): m / z 275.1 [M+H] + .

[0332] 1H NMR(400MHz,CD3OD)δ6.89(s,1H),6.84-6.74(m,1H),6.68-6.60(m,1H),2.88 (s,4H),1.95-1.89(m,2H),1.84-1.80(m,4H),1.79-1.74(m,4H),1.60(s,6H).

[0333] Example 18: Preparation of Compound 18

[0334]

[0335] Synthetic route of compound 18:

[0336]

[0337] Step 1: Preparation of intermediate 18a

[0338] Intermediate 18a was prepared from methyl 2-amino-4-bromobenzoate (2.4 g, 10.4 mmol) and (E)-2-(3-methoxypropyl-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxorane (2.47 g, 12.48 mmol) according to step 1 of Example 11, with a yield of 75.8%.

[0339] MS(ESI): m / z 222.1 [M+H] + .

[0340] Step 2: Preparation of Compound 18

[0341] Compound 18 was prepared from intermediate 18a (1.75 g, 7.9 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methyl magnesium bromide (21.1 mL, 63.2 mmol) according to step 4 of Example 1, with a yield of 82.6%.

[0342] MS(ESI): m / z 204.1 [M+H] + .

[0343] 1 H NMR (400MHz, MeOD) δ7.07(d,J=8.0Hz,1H),6.77(s,1H),6.69(d,J=8.0Hz,1H),6.48( d,J=16.0Hz,1H),6.24-6.17(m,1H),4.05(d,J=6.0Hz,2H),3.35(s,3H)1.59(s,6H).

[0344] Example 19: Preparation of Compound 19

[0345]

[0346] Synthetic route of compound 19:

[0347]

[0348] Step 1: Preparation of intermediate 19a

[0349] Intermediate 19a was prepared from 1-(2-amino-5-bromophenyl)ethyl ketone (4.0 g, 18.7 mmol) and cyclohexyl-1-en-1-ylboranediol (3.53 g, 28.05 mmol) according to step 1 of Example 11, with a yield of 87%.

[0350] MS(ESI): m / z 216.3 [M+H] + .

[0351] Step 2: Preparation of intermediate 19b

[0352] Pd / C (350 mg) was added to a solution of intermediate 19a (3.5 g, 16.28 mmol) in EtOAc (20 mL). The reaction mixture was stirred at 20 °C under H2 for 30 minutes.

[0353] The reaction was confirmed by LCMS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 20:1 to 10:1) to give intermediate 19b (2.5 g, yellow solid, yield: 70.8%).

[0354] MS(ESI): m / z 218.6 [M+H] + .

[0355] Step 3: Preparation of Compound 19

[0356] Compound 19 was prepared from intermediate 19b (2.5 g, 11.5 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (19.2 mL, 57.5 mmol) according to step 4 of Example 1, with a yield of 42.61%.

[0357] MS(ESI): m / z 234.1 [M+H] + .

[0358] 1H NMR (400MHz, MeOD) δ6.96 (s, 1H), 6.85 (d, J = 8.4Hz, 1H), 6.63 (d, J = 8.0Hz, 1H), 2.35 (s, 1H), 1.80-1.71 (m, 4H), 1.60 (s, 6H), 1.44-1.24 (m, 6H).

[0359] Example 20: Preparation of Compound 20

[0360]

[0361] Synthetic route of compound 20:

[0362]

[0363] Step 1: Preparation of intermediate 20a

[0364] Methyl 5-fluoro-2-nitrobenzene (700 mg, 3.515 mmol), potassium carbonate (1.457 g, 10.55 mmol), and dimethylamine hydrochloride (458.61 mg, 5.624 mmol) were dissolved in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 60 °C for 1 hour.

[0365] The reaction was detected by LCMS. The reaction solution was diluted with ethyl acetate (120 mL), washed with saturated sodium chloride (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate 20a (600 mg, yellow oil, yield: 76.1%).

[0366] MS(ESI): m / z 247.1 [M+Na] + .

[0367] Step 2: Preparation of intermediate 20b

[0368] Pd / C (284.78 mg, 0.2676 mmol) was added to a solution of intermediate 20a (600 mg, 2.676 mmol) in methanol (20 mL). The reaction mixture was stirred at 50 °C under H2 for 2 hours.

[0369] The reaction was confirmed by LCMS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 10:1 to 4:1) to give intermediate 20b (450 mg, yellow solid, yield: 85.6%).

[0370] MS(ESI): m / z 195.2 [M+H] + .

[0371] Step 3: Preparation of Compound 20

[0372] Compound 20 was prepared by the method in step 4 of Example 1, using intermediate 20b (400 mg, 2.06 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methyl magnesium bromide (5.5 mL, 16.5 mmol), with a yield of 40%.

[0373] MS(ESI): m / z 195.1 [M+H] + .

[0374] 1 H NMR (400MHz, MeOD) δ6.73 (d, J = 38.4Hz, 3H), 2.74 (s, 6H), 1.60 (s, 6H).

[0375] Example 21: Preparation of compound 21

[0376]

[0377] Synthetic route of compound 21:

[0378]

[0379] Step 1: Preparation of intermediate 21a

[0380] Intermediate 21a was prepared from methyl 5-fluoro-2-nitrobenzoate (5.0 g, 25.1 mmol) and piperidine (4.28 g, 50.2 mmol) according to step 1 of Example 17, yield: 75%.

[0381] MS(ESI): m / z 264.80 [M+H] + .

[0382] Step 2: Preparation of intermediate 21b

[0383] Intermediate 21b was prepared from intermediate 21a (5.0 g, 18.9 mmol) according to the method in step 2 of Example 12, with a yield of 90%.

[0384] MS(ESI): m / z 235.2 [M+H] + .

[0385] Step 3: Preparation of Compound 21

[0386] Compound 21 was prepared from intermediate 21b (3.0 g, 12.8 mmol) and a 2-methyltetrahydrofuran solution of 3.0 M methylmagnesium bromide (42.7 mL, 128 mmol) according to step 4 of Example 1, with a yield of 47%.

[0387] MS(ESI): m / z 235.1 [M+H] + .

[0388] 1 H NMR (400MHz, CD3OD) δ6.90(s,1H),6.78(s,1H),6.68(s,1H),2.93(s,4H),1.80-1.72(m,4H),1.61(s,6H),1.55-1.48(m,2H).

[0389] Example 22: Preparation of compound 22

[0390]

[0391] Synthetic route of compound 22:

[0392]

[0393] Step 1: Preparation of intermediate 22a

[0394] Intermediate 22a was prepared from methyl 4-fluoro-2-nitrobenzoate (500 mg, 2.51 mmol) and dimethylamine hydrochloride (169.79 mg, 3.766 mmol) according to step 1 of Example 17, with a yield of 84.38%.

[0395] MS(ESI): m / z 225.2 [M+H] + .

[0396] Step 2: Preparation of intermediate 22b

[0397] Intermediate 22b was prepared from intermediate 22a (500 mg, 2.23 mmol) according to step 2 of Example 12, with a yield of 62.34%.

[0398] MS(ESI): m / z 195.0 [M+H] + .

[0399] Step 3: Preparation of Compound 22

[0400] Compound 22 was prepared from intermediate 22b (150 mg, 0.7723 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (1.3 mL, 3.86 mmol) according to step 4 of Example 1, with a yield of 23.92%.

[0401] MS(ESI): m / z 195.1 [M+H] + .

[0402] 1H NMR (400MHz, MeOD) δ6.97 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 2.8 Hz, 1H), 6.14 (dd, J = 2.4, 8.8 Hz, 1H), 2.83 (s, 6H), 1.57 (s, 6H).

[0403] Example 23: Preparation of compound 23

[0404]

[0405] Synthetic route of compound 23:

[0406]

[0407] Step 1: Preparation of intermediate 23a

[0408] Intermediate 23a was prepared from methyl 4-fluoro-2-nitrobenzoate (500 mg, 2.51 mmol) and piperidine (427.58 mg, 5.02 mmol) according to step 1 of Example 17, with a yield of 85.91%.

[0409] MS(ESI): m / z 265.2 [M+H] + .

[0410] Step 2: Preparation of intermediate 23b

[0411] Intermediate 23b was prepared from intermediate 23a (600 mg, 2.27 mmol) according to step 2 of Example 12, with a yield of 94.13%.

[0412] MS(ESI): m / z 235.2 [M+H] + .

[0413] Step 3: Preparation of Compound 23

[0414] Compound 23 was prepared from intermediate 23b (150 mg, 0.6402 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (1.1 mL, 3.2 mmol) according to step 4 of Example 1, with a yield of 65%.

[0415] MS(ESI): m / z 235.1 [M+H] + .

[0416] 1H NMR (400MHz, MeOD) δ6.99 (d, J=8.8Hz, 1H), 6.37 (d, J=2.4Hz, 1H), 6.30 (d, J= 2.8, 8.8Hz, 1H), 3.06 (t, J = 5.4Hz, 1H), 1.71-1.66 (m, 4H), 1.59-1.54 (m, 8H).

[0417] Example 24: Preparation of compound 24

[0418]

[0419] Synthetic route of compound 24:

[0420]

[0421] Step 1: Preparation of intermediate 24a

[0422] Intermediate 24a was prepared from ethyl 3,4-dihydroxybenzoate (4 g, 22 mmol) and piperidine 2-[2-(2-{[(4-methylbenzene)sulfonyl]oxy}ethoxy)ethoxy]4-methylbenzenesulfonate (11.1 g, 24.2 mmol) according to step 1 of Example 17, with a yield of 16.9%.

[0423] MS(ESI): m / z 297.2 [M+H] + .

[0424] Step 2: Preparation of intermediate 24b

[0425] Nitric acid (0.6 mL) and sulfuric acid (0.6 mL) were added to a 15 mL solution of intermediate 24a (1.1 g, 3.7 mmol) in acetic acid. The reaction mixture was stirred at room temperature for 16 hours.

[0426] The reaction was confirmed by LCMS. The reaction solution was diluted with ethyl acetate (200 mL), washed with saturated sodium chloride (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 3:1 to 1:1) to give intermediate 24b (950 mg, yellow solid, yield: 75.0%).

[0427] MS(ESI): m / z 342.2 [M+H] + .

[0428] Step 3: Preparation of intermediate 24c

[0429] Intermediate 24c was prepared from intermediate 24b (950 mg, 2.78 mmol) according to step 2 of Example 12, with a yield of 83.1%.

[0430] MS(ESI): m / z 312.2 [M+H] + .

[0431] Step 4: Preparation of Compound 24

[0432] Compound 24 was prepared from intermediate 24c (300 mg, 0.9636 mmol) and a 3.0 M methyl magnesium bromide solution in 2-methyltetrahydrofuran (6.4 mL, 19.3 mmol) according to step 4 of Example 1, with a yield of 8.73%.

[0433] MS(ESI): m / z 280.1 [M+H-H2O] + .

[0434] 1 H NMR (400MHz, MeOD) δ6.84(s,1H),6.42(s,1H),4.09-4.05(m,4H),3.84-3.82(m,2H),3.74-3.72(m,6H),1.57(s,6H).

[0435] Example 25: Preparation of Compound 25

[0436]

[0437] Synthetic route of compound 25:

[0438]

[0439] Step 1: Preparation of intermediate 25a

[0440] Intermediate 25a was prepared from 2-bromo-3-methylbut-2-ene (900 mg, 6.04 mmol) and methyl 2-amino-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzoate (2.175 g, 7.85 mmol) according to step 1 of Example 11, with a yield of 84.8%.

[0441] MS(ESI): m / z 220.2 [M+H] + .

[0442] Step 2: Preparation of Compound 25

[0443] Compound 25 was prepared from intermediate 25a (400 mg, 1.82 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (18.2 mL, 18.2 mmol) according to step 4 of Example 1, with a yield of 70%.

[0444] MS(ESI): m / z 220.1 [M+H] + .

[0445] 1 H NMR (400MHz, MeOD) δ6.87 (s, 1H), 6.76 (d, J = 7.6 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 1.89 (s, 3H), 1.77 (s, 3H), 1.60 (s, 6H), 1.56 (s, 3H).

[0446] Example 26: Preparation of Compound 26

[0447]

[0448] Synthetic route of compound 26:

[0449]

[0450] Step 1: Preparation of intermediate 26a

[0451] Intermediate 26a was prepared from 1-(2-amino-5-bromophenyl)ethyl ketone (500 mg, 2.33 mmol) and 4,4,5,5-tetramethyl-2-(2-methylprop-1-en-1-yl)-1,3,2-dioxaborane (552.86 mg, 3.037 mmol) according to step 1 of Example 11, with a yield of 86.0%.

[0452] MS(ESI): m / z 190.2 [M+H] + .

[0453] Step 2: Preparation of Compound 26

[0454] Compound 26 was prepared from intermediate 26a (380 mg, 1.85 mmol) and a 1.0 M methyl magnesium bromide tetrahydrofuran solution (9.2 mL, 9.2 mmol) according to step 4 of Example 1, with a yield of 55.8%.

[0455] MS(ESI): m / z 188.1 [M+H-H2O] + .

[0456] 1 H NMR (400MHz, MeOD) δ6.99 (s, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 1.84 (s, 3H), 1.81 (s, 3H), 1.60 (s, 6H).

[0457] In vitro activity evaluation

[0458] Example 27: In vitro aldehyde capture capacity experiment

[0459] Main principle of the experiment:

[0460] Elevated levels of reactive aldehydes have been found in various inflammatory eye diseases. If these aldehydes are not eliminated in time, they can exacerbate the symptoms of inflammation and worsen the eye disease. This experiment simulates the in vivo environment and selects relatively superior compounds based on their complexation ability with nonenal.

[0461] Experimental materials and reagents:

[0462] Sulfobutyl-β-cyclodextrin (Bide Pharmaceuticals, BD243603-10g), linoleic acid (Adamas Reagent, 69208F), trioleic acid glyceride (Sigma-Aldrich, T7140-10G), nonenal (Bide Pharmaceuticals, BD19638-1g).

[0463] Experimental steps:

[0464] S1. Dissolve 3 g of sulfobutyl-β-cyclodextrin in 12 mL of phosphate buffer to prepare a solution. Use a pipette to transfer 2.88 mL of trioleic acid glyceride and 2.88 mL of linoleic acid separately, and mix them thoroughly in a 1:1 ratio.

[0465] S2, at room temperature, add 48 μL of the above trioleic acid glyceride / linoleic acid mixture to a 2 mL Eppendorf tube, add a 5 μL solution of dimethyl sulfoxide (DMSO) containing 10 μmol, 1.0 equivalence of the compound prepared in the embodiments of the present invention, mix well, add 100 μL of the above sulfobutyl-β-cyclodextrin solution, and finally add a 10 μL solution of dimethyl sulfoxide (DMSO) containing 10 μmol, 1.0 equivalence of nonenal, and stir the reaction at 1000 rpm.

[0466] S3, after stirring for 0 minutes (without nonenal solution added to the reaction system, but with 1510 μL of methanol added), 10 minutes, 20 minutes, 40 minutes and 60 minutes respectively, 1500 μL of methanol was added to an Eppendorf tube. After high-speed vortexing, the solution was quickly centrifuged and transferred to a sample vial. The residual amount of nonenal in the reaction mixture was quantitatively monitored and analyzed by high performance liquid chromatography.

[0467] Experimental results:

[0468] The liquid chromatography conditions are shown in Table 1 below:

[0469] Table 1: Liquid phase gradient conditions

[0470]

[0471] The injector temperature was 15℃; the column temperature was 40℃; and the UV-Vis detection wavelength for nonenal was 220nm.

[0472] The concentrations (%) of nonenal at different time points are shown in Table 2 below:

[0473] Table 2: Nonenal concentration (%) at different time points

[0474]

[0475]

[0476] *: XRPR-5 is a similar control, Reproxalap.

[0477] At the same time point, the lower the concentration of nonenal (i.e., the lower the residual amount of nonenal), the stronger the compound's ability to complex aldehydes; when the concentration of nonenal is consistent or comparable, the smaller the time point, the faster the compound complexes aldehydes. As shown in Table 2 above, the compounds prepared in the embodiments of this invention, except for Examples 5, 14, and 24, all exhibit very significant aldehyde complexing ability and speed, and can be used to treat or prevent diseases with RASP-mediated pathological features, including but not limited to dry eye syndrome, Behçet's disease, Sjögren's syndrome, non-infectious uveitis, and allergic conjunctivitis, and other ocular inflammatory diseases.

[0478] Animal efficacy evaluation

[0479] Example 28: Drug Efficacy Test in an Animal Model of Allergic Conjunctivitis

[0480] Experimental Principle and Objective: C48 / 80 is a mast cell degranulation agent that can directly bind to receptors on the surface of mast cells, triggering intracellular signal transduction pathways. After mast cell degranulation, various inflammatory mediators are released. C48 / 80 and the inflammatory response it induces may directly damage the conjunctival epithelial barrier. Inflammatory mediators and proteases released by inflammatory cells can degrade junctional proteins between conjunctival epithelial cells, leading to impaired conjunctival epithelial integrity and making the conjunctiva more susceptible to invasion by external pathogens, while also exacerbating the inflammatory response within the tissue. Furthermore, the inflammatory process stimulates nerve endings in the conjunctival tissue. On one hand, inflammatory mediators such as histamine can activate sensory nerve endings, causing discomfort symptoms such as pain and itching. On the other hand, stimulated nerve endings release neuropeptides, such as substance P. Substance P has vasodilatory and pro-inflammatory effects, which can further aggravate conjunctival congestion and inflammation. This experiment uses C48 / 80 eye drops to induce a rat conjunctivitis model and examines the ameliorative effect of the compound of this invention on the rat conjunctivitis model.

[0481] Sample preparation: The positive control drug used was commercially available olopatadine hydrochloride eye drops (0.1%, Alcon, VEY89A); 1% Compound 13 eye drops were prepared by hydrochloric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate and water in a certain proportion.

[0482] Grouping: Female SD rats were used as the research subjects and were randomly divided into four groups: a normal control group, a model control group, an olopatadine hydrochloride group, and a compound 13 group. No model was established in the normal control group, while model establishment was performed in all other groups. Specific grouping information is shown in Table 4.

[0483] Table 4: Summary of Dosage and Administration Design for Each Group

[0484]

[0485]

[0486] Modeling, drug administration, and evaluation: Rats were randomly divided into groups. The rats were anesthetized. The normal control group was given 10 μL / eye saline solution instilled into the conjunctival sac. The other groups were given 10 μL / eye C48 / 80 solution (source: MCE; specification and concentration: 50 mg / vial; batch number: 312920; 200 mg / mL, prepared with 0.9% saline) instilled into the conjunctival sac. The eyelids were gently closed for 10 seconds to prevent the solution from overflowing, thus completing the modeling.

[0487] Ten minutes after model establishment, the first drug administration was performed: 15 μL of sample per eye was instilled into the conjunctival sac, and the eyelids were gently closed for 10 seconds to prevent leakage. The normal control group and the model control group were given an equal volume of physiological saline. At two time points, 20 minutes after the third drug administration and 24 hours after model establishment, eye examinations were performed under a slit lamp, and each animal's eyes were individually clinically scored according to the scoring criteria in Table 5.

[0488] Table 5: Scoring Rules for Conjunctivitis in Rats

[0489]

[0490] According to the scoring criteria in Table 5, the score values ​​were positively correlated with the severity of ocular inflammation. The experimental results are as follows: Figure 1 As shown, compound 13 of the present invention has a good therapeutic effect on allergic conjunctivitis induced by C48 / 80 in SD rats, wherein 1 represents the normal control group, 2 represents the model control group, 3 represents the olopatadine hydrochloride group, and 4 represents the compound 13 group.

[0491] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An AI-designed inhibitor of active aldehydes, characterized in that, It comprises compounds having the general formula (I) or derivatives thereof. in, R1 and R2 are independently selected from hydrogen and C. 2-6 Unsaturated hydrocarbon group, C 1-6 Alkoxy, C 2-6 Halogenated alkyl, halogen, amino, C 3-20 Any one of cycloalkyl or heterocyclic groups having 4 to 20 ring atoms; X is carbon or nitrogen; The derivative comprises at least one of a fluorescent label, a spin label, a heavy metal label, an isotope label, and a pharmaceutically acceptable salt.

2. The AI-designed active aldehyde inhibitor as described in claim 1, characterized in that, R1 and R2 can form C6-C together with the carbon atoms they are attached to. 12 Aromatic or non-aromatic rings.

3. The AI-designed active aldehyde inhibitor as described in claim 2, characterized in that, R1 and R2 can form a benzene ring or an aliphatic heterocycle with 7-12 ring atoms together with the carbon atoms they are attached to.

4. The AI-designed active aldehyde inhibitor as described in claim 1, characterized in that, R1 and R2 are independently selected from hydrogen and C. 2-5 Unsaturated hydrocarbon group, C 2-5 Either alkoxy or amino.

5. The AI-designed active aldehyde inhibitor as described in claim 1, characterized in that, R1 and R2 are independently selected from hydrogen and C. 6-12 Any one of cycloalkyl or heterocyclic groups having 6 to 12 ring atoms.

6. The AI-designed active aldehyde inhibitor as described in claim 1, characterized in that, When R1 is a halogen, R2 is a C 2-6 Unsaturated hydrocarbon group.

7. The AI-designed active aldehyde inhibitor as described in claim 1, characterized in that, The compounds represented by general formula (I) are selected from: Any one of them.

8. A pharmaceutical composition, characterized in that, The active ingredient comprises a compound of general formula (I) as described in any one of claims 1-7 or a derivative thereof.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is a detection reagent or kit containing at least one of a fluorescent label, spin label, heavy metal label, isotope label, and pharmaceutically acceptable salt of a compound of general formula (I) for recognizing RASP ligands.

11. An application of an AI-designed active aldehyde inhibitor according to any one of claims 1-7, characterized in that, Used in the preparation of drugs as aldehyde scavengers.

12. The application as described in claim 11, characterized in that, The drug is used to treat or prevent a variety of diseases caused by reactive aldehydes.

13. The application as described in claim 12, characterized in that, The disease in question refers to an inflammatory disease of the eye.

14. The application as described in claim 13, characterized in that, The ocular inflammatory disease includes at least one of dry eye syndrome, allergic conjunctivitis, Behcet's disease, Sjögren's syndrome, and uveitis.

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