Tricyclic pyridine compound as SARM1 enzyme activity inhibitor and application thereof

Abstract

The invention provides application of an SARM1 enzyme activity inhibitor in treating neurodegenerative diseases or neurological diseases or symptoms. The invention particularly provides a compound as an SARM1 enzyme activity inhibitor and a pharmaceutical composition of the compound.

Description

Tricyclic pyridine compounds as SARM1 enzyme activity inhibitors and their applications

[0001] This application claims priority to Chinese patent application No. 202311222898.2, filed with the State Intellectual Property Office of China on September 20, 2023, entitled “Tricyclic pyridine compounds as SARM1 enzyme activity inhibitors and their applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application relates to compounds that can be used to inhibit SARM1 enzyme activity and the use of these compounds in treating and / or preventing neurodegenerative or neurological diseases or conditions associated with SARM1 enzyme activity. Background Art

[0003] Neurodegenerative diseases are a class of diseases that can severely harm humans, causing devastating damage, such as progressive neuronal cell death. Among the primary neurodegenerative diseases, known are central nervous system diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease, as well as peripheral nervous system diseases such as diabetes. While most are associated with aging, the incidence of these diseases increases with age, although some also occur in middle-aged individuals or even younger.

[0004] As a result of research on brain structure and function, the effects of neurotransmitters and neurotrophic factors have been gradually elucidated, but many local causes of neurodegeneration are still unclear. Only for Parkinson's disease, the relationship between this disease and the special neurotransmitter, i.e., dopamine, has been elucidated, and the precursor of dopamine, L-dopa, has been used as a medicine to alleviate neurological symptoms and restore neurological function. However, L-dopa cannot inhibit the development of neurodegeneration, and gradually loses its effect as the disease progresses, i.e., dopamine-based neuronal cell degeneration and defect. Similarly, Alzheimer's disease is also caused by the degeneration and defect of multiple nerve cells such as acetylcholine-based neurons, monoamine-based neurons, etc., and as medicines for the treatment of this disease, cholinesterase inhibitors have been put on the market or are being developed. However, the L-dopa for the treatment of Parkinson's disease is still limited to symptomatic treatment, to temporarily improve neurological symptoms.

[0005] Therefore, there is a lack of effective therapeutic drugs for neurodegenerative diseases.

[0006] Studies have found that axonal damage occurs in a variety of neurodegenerative diseases, accidental injuries, and other neurological diseases. Axonal degeneration can cause structural necrosis and functional disorders in the peripheral nervous system, ultimately leading to acquired or hereditary degenerative diseases of the central nervous system.

[0007] Although there is currently no very effective pharmacological method that can accurately assess the weight of the morbidity caused by axonal degeneration, it has been found in histopathological studies that significant axonal damage degradation has been observed in the early stages of various neuropathies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, and peripheral neuropathy, indicating that axonal degeneration plays an important role in the development of neuropathy (Fischer et al., Neuro-degenerative Diseases, 2007, 4:431-442). Therefore, by weakening or even blocking axonal degeneration, maintaining the integrity of neuronal structure and function may be a therapeutic solution that benefits various nervous system diseases.

[0008] In the absence of effective therapeutic drugs for neurodegenerative diseases, the existing technology urgently needs to research and develop new compounds, especially small chemical molecules, including compounds that have effects on axonal degeneration.

[0009] Summary of the Invention

[0010] After long-term research, the present inventors unexpectedly discovered a class of compounds with significant SARM1 enzyme activity inhibitory effects, and found that the compounds can improve axonal degeneration and be used to treat or prevent neurodegenerative diseases and related disorders.

[0011] The SARM1 (Sterile alpha and TIR motif containing 1) protein is composed of three domains: the ARM (Armadillo / HEAT repeat) domain at the nitrogen terminus, two tandem SAM (Sterile alpha motif) domains, and the TIR (Toll / Interleukin Receptor) domain at the carbon terminus. In addition, there is a mitochondrial localization signal peptide at the nitrogen terminus.

[0012] It is known that in wild-type neurons, axonal injury induces NAD + Depletion and axonal degeneration; SARM1 knockout inhibits axonal degeneration, and NAD +Maintained at normal levels, indicating that SARM1 promotes NAD + consumption, exacerbating axonal degeneration.

[0013] The Milbrandt team at the University of Washington School of Medicine prepared the TIR domain of SARM1 (SARM1-TIR) and found that it has NAD + Hydrolase activity. Further rigorous E. coli expression and purification experiments and cell-free expression systems were used to obtain high-purity SARM1-TIR, ultimately proving that SARM1-TIR can catalyze NAD + Produces adenosine 5'-diphosphate ribose (ADPR) and cyclic adenosine 5'-diphosphate ribose (cADPR).

[0014] SARM1 is a multifunctional signaling enzyme that catalyzes the conversion of multiple substrates to NAD + , NADP + and NA to generate signaling molecules such as cADPR, ADPR and NAADP. In a variety of neurodegenerative diseases, SARM1 is activated, leading to NAD + Depletion of SARM1 can activate a new cell death mechanism; knocking out SARM1 can inhibit axonal degeneration and disease progression, and is therefore considered a potential drug target for related neurological diseases, including TBI, AD, CIPN, ASL, etc.

[0015] In the present disclosure, the inventors prepared full-length SARM1 for NAD enzyme activity experiments, and used it to screen and obtain compound molecules with enzyme activity inhibitory ability of the present invention.

[0016] Therefore, based on the above findings, in a first aspect, the present invention provides compounds of formula (I) that can be used as inhibitors of SARM1 enzyme activity:

[0017] or its racemate, enantiomer, diastereomer, deuterated form or pharmaceutically acceptable salt, wherein,

[0018] X1 is selected from -O-, -CH 2- , -OCH2-, -NR3CH2-, -CH2NR3-, -N=CH-, -CH=N-, preferably -O-;

[0019] wherein R3 is selected from H and C1-C3 alkyl;

[0020] X2, X3, X4 are each independently selected from CH or N;

[0021] R1 is independently selected from H, halogen, C1-C3 alkyl, cyano, trifluoromethyl, amino, hydroxy, methoxy-C(O)NH2, preferably H, F, Cl, methyl, cyano and -C(O)NH2;

[0022] R2 is independently selected from H, halogen, C1-C3 alkyl, cyano, trifluoromethyl, amino, hydroxy, methoxy, C1-C3 alkyl-OC(O)-, C1-C3 alkyl-NHC(O)-, preferably H, cyano, Cl, methyl and methoxy;

[0023] M represents O, S or NH, preferably O;

[0024] L1 and L3 are each independently selected from -CH(R')-, -O-, -S- and -N(R')-, provided that at least one of L1 and L3 is -CH(R')-, wherein R' is hydrogen or C1-C3 alkyl;

[0025] L2 is selected from H, -CH3, -CH2-CH3, -CH2-CH2-CH3, -OR8, -NHR9;

[0026] wherein R8 and R9 are independently selected from H and C1-C3 alkyl;

[0027] A is selected from H, 5- to 10-membered aryl, 5- to 10-membered heteroaryl, 5- to 10-membered cycloalkyl, 5- to 10-membered heterocycloalkyl, 5- to 10-membered aryl and 3- to 6-membered cycloalkyl, 5- to 10-membered aryl and 3- to 6-membered heterocycloalkyl, wherein the heteroaryl or heterocycloalkyl may contain 1 or 2 heteroatoms selected from N, O and S, and the 5- to 10-membered aryl, 5- to 10-membered heteroaryl, 5- to 10-membered aryl and 3- to 6-membered cycloalkyl, 5- to 10-membered aryl and 3- to 6-membered heterocycloalkyl may be substituted by 1, 2 or 3 substituents selected from the group consisting of halogen, C1-C3 alkyl, C1-C3 alkoxy, halo-C1-C3 alkyl, halo-C1-C3 alkoxy, cyano, nitro and (R a R a ')N-, where R a and R a ' are each independently selected from hydrogen and C1-C3 alkyl; preferably, the substituent is selected from methyl, methoxy, trifluoromethyl, trifluoromethoxy, F, Cl, Br;

[0028] L1 may be linked to L2 to form a 5-membered ring or a 6-membered ring, preferably a 5-membered ring.

[0029] In a preferred aspect, the compound of formula (I) of the present invention has the following structure:

[0030] in,

[0031] X2, X3, X4, R1, R2, L3, A are as defined above;

[0032] X5 is selected from CH2, O, NH, preferably CH2.

[0033] In a preferred aspect, the compound of formula (I) of the present invention has the following structure:

[0034] in,

[0035] R1, R2, M, L1, L2, L3, A are as defined above.

[0036] In a preferred aspect, the compound of formula (I) of the present invention has the following structure:

[0037] in,

[0038] X2 and X3 are selected from CH and N, and X2 and X3 are not CH at the same time;

[0039] R1, R2, M, L1, L2, L3, A are as defined above.

[0040] In a preferred aspect, the compound of formula (II) of the present invention has the following structure:

[0041] in,

[0042] R1, R2, X2, X3, and A are as defined above.

[0043] In a preferred aspect, the compound of formula (III) of the present invention has the following structure:

[0044] in,

[0045] R1, R2, and A are as defined above.

[0046] In a preferred aspect, the compound of formula (IV) of the present invention has the following structure:

[0047] in,

[0048] X2 and X3 are selected from CH and N, and X2 and X3 are not CH at the same time;

[0049] R1, R2, and A are as defined above.

[0050] In some preferred embodiments, the compound of the present invention is selected from the following compounds, or racemates, enantiomers, diastereomers, deuterated compounds or pharmaceutically acceptable salts thereof:

[0051] Herein, when referring to the compound of formula (I), it also includes the pharmaceutically acceptable salt of the compound of formula (I) or its stereoisomers (including enantiomers, diastereomers, racemates) or tautomers or deuterated substances.

[0052] In another aspect, the present invention provides use of the SARM1 enzyme activity inhibitor of the present invention in the preparation of a SARM1 enzyme activity inhibitor.

[0053] In another aspect, the present invention provides the use of the SARM1 enzyme activity inhibitor of the present invention in the preparation of a medicament for treating or preventing axonal degeneration-related diseases or disorders.

[0054] In another aspect, the present invention provides use of the SARM1 enzyme activity inhibitor of the present invention in the preparation of a medicament for treating or preventing a neurodegenerative disease or a neurological disease or disorder.

[0055] Accordingly, the present invention also relates to a method for treating or preventing a neurodegenerative disease or a neurological disease or condition associated with neurodegeneration, comprising administering to a subject in need thereof a compound of the present invention that is an inhibitor of SARM1 enzyme activity. In particular, the present invention relates to a method for treating or preventing a disease or condition associated with axonal degeneration, comprising administering to a subject in need thereof a compound of the present invention that is an inhibitor of SARM1 enzyme activity. More particularly, the present invention relates to a method for inhibiting SARM1 enzyme activity, comprising administering to a subject in need thereof a compound of the present invention; more particularly, the present invention relates to a method for inhibiting axonal degeneration, comprising administering to a subject in need thereof a compound of the present invention. The compounds or compositions of the present invention can be administered to a subject or patient in need thereof in an effective amount.

[0056] Accordingly, the present invention also relates to the use of the compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers in the preparation of a method for treating or preventing a neurodegenerative disease or a neurological disease or condition. The present invention also relates to the use of the compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers in the preparation of a SARM1 enzyme activity inhibitor. The present invention also relates to the use of the compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers in the preparation of a method for treating or preventing axonal degeneration-related diseases or conditions. Preferably, the neurodegenerative disease or neurological disease or condition or axonal degeneration-related disease or condition is selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, and peripheral neuropathy. DETAILED DESCRIPTION

[0057] the term

[0058]

[0046] Reference herein to a "compound" having a particular structural formula is generally also intended to encompass pharmaceutically acceptable salts, stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives thereof.

[0059] Pharmaceutically acceptable salts of the present invention may be formed using, for example, the following inorganic or organic acids: "Pharmaceutically acceptable salts" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with human and mammalian tissues without undue toxicity, irritation, allergic reaction, or the like, at a reasonable benefit / risk ratio. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base or free acid with a suitable reagent. For example, the free base may be reacted with a suitable acid. Examples of pharmaceutically acceptable acid addition salts are salts of an amino group (amine group) formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include sodium alginate, ascorbate, benzenesulfonate, adipate, camphorsulfonate, aspartate, benzoate, bisulfate, borate, butyrate, camphorate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, heptanoate, hexanoate, hydroiodide, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0060] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in a water-miscible organic solvent (e.g., methanol, ethanol, acetone and acetonitrile), adding an excess of an organic acid or an aqueous inorganic acid solution thereto to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0061] The "stereoisomerism" mentioned in the present invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism (i.e., optical isomerism). Conformational isomerism refers to a stereoisomerism phenomenon in which the atoms or atomic groups of an organic molecule with a certain configuration have different spatial arrangements due to the rotation or distortion of carbon-carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair conformation and boat conformation that appear in the structure of cyclohexane. "Stereoisomers" refer to when the compounds of the present invention contain one or more asymmetric centers, and can thus be racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereoisomers. The compounds of the present invention have asymmetric centers, and each asymmetric center will produce two optical isomers. The scope of the present invention includes all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds.

[0062] In some cases, the compounds of the present invention can exist as tautomers, which have different hydrogen attachment points through one or more double bond displacements. For example, a ketone and its enol form are keto-enol tautomers. Amides and imines can also form tautomeric forms. Each tautomer and mixture thereof are included in the compounds of the present invention. Enantiomers, diastereomers, racemates, mesomorphs, cis-trans isomers, tautomers and mixtures thereof of all compounds are included within the scope of the present invention.

[0063] When the compound of the present invention is used in combination with another SARM1 enzyme activity inhibitor for the treatment or prevention of neurodegenerative diseases or related neurological diseases or conditions, or can be used in combination with another active drug for the treatment or prevention of neurodegenerative diseases or related neurological diseases or conditions, it is used to treat or prevent neurodegenerative diseases or related diseases or conditions.

[0064] The compound of the present invention or its mesomer, racemate, enantiomer, diastereomer or its pharmaceutically acceptable salt can be used as an active ingredient by oral or parenteral administration, and the scope of its effective amount is 0.1 to 2000 mg / kg body weight / day, preferably 0.1 to 100 mg / kg body weight / day in the case of mammals including humans (weighing about 70 kg), and is administered every day in single or divided doses, or with or without a predetermined time. The dosage of the active ingredient can be adjusted according to multiple relevant factors (such as the situation of the subject to be treated, the type of disease and severity, the rate of administration and the doctor's opinion). In some cases, the amount less than the above dosage may be suitable.

[0065] The compound of the present invention or its mesoform, racemate, enantiomer, diastereomer or pharmaceutically acceptable salt can be formulated into a pharmaceutical composition. The pharmaceutical composition can comprise the compound of the present invention or its mesoform, racemate, enantiomer, diastereomer or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0066] The pharmaceutical composition of the present invention can be formulated into dosage forms for oral administration or parenteral administration (including intramuscular, intravenous and subcutaneous routes, intratumor injection) according to any of the conventional methods, such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions or suspensions.

[0067] The pharmaceutical composition of the present invention for oral administration can be prepared by mixing the active ingredient with a pharmaceutically acceptable carrier such as cellulose, calcium silicate, magnesium stearate, calcium stearate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, surfactants, suspending agents, gelatin, talc, emulsifiers and diluents. Examples of carriers employed in the injectable composition of the present invention are water, glycerides, saline solutions, alcohols, glycols, glucose solutions, ethers (e.g., polyethylene glycol 400), oils, fatty acids, fatty acid esters, surfactants, suspending agents and emulsifiers.

[0068] If not stated otherwise, conventional methods of mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are used. In this application, if not stated otherwise, "or" or "and" may be used to mean "and / or".

[0069] In the specification and claims, a given chemical formula or name shall encompass all stereo and optical isomers and racemates in which such isomers exist. Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the present invention. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc. may exist in the compounds, and all such stable isomers are encompassed by the present invention.

[0070] The compounds of the present invention can be isolated in optically active or racemic forms. All methods for preparing the compounds of the present invention and the intermediates prepared therein are considered part of the present invention. When enantiomeric or diastereomeric products are prepared, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). It should be understood that all isomeric forms that may exist are included in the present invention.

[0071] Unless otherwise defined, when a substituent is marked as "optionally substituted", the substituent is selected from, for example, substituents such as C1-C3 alkyl, C3-C6 cycloalkyl, C5-C6 alkyl, C6-C 10Aryl, 3-10 membered heterocyclic group, halogen (including F, Cl, Br), hydroxyl, C1-C3 alkoxy, nitro, cyano, oxo (=O), C1-C3 alkyl acyl, C5-C 10 Aryloxy, C1-C3 alkylacyloxy, amino, C1-C3 alkylamino, C5-C 10 Arylamino, C1-C3 alkylthio, etc.

[0072] The term "alkyl" or "alkylene" as used herein is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. 12 Alkyl, C1-C 10 C1-C8 alkyl, C1-C6 alkyl, more preferably C1-C4 alkyl, particularly preferably C1-C3 alkyl. For example, "C1-C6 alkyl" means an alkyl group having 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl) and pentyl (e.g., n-pentyl, isopentyl, neopentyl). For the C1-C6 alkyl group in the present invention, 12 In the case of an alkyl group, 1 to 4 -CH2- units are optionally replaced by O atoms, S atoms or -NH-.

[0073] The term "carbonyl" refers to an organic functional group composed of carbon and oxygen atoms connected by a double bond (C=O or C(O)).

[0074] The term "aromatic ring" or "aryl" refers to an aromatic 5- to 10-membered ring structure. An aromatic carbocycle is an aromatic carbocyclic ring structure in which all ring members are composed of carbon atoms. A non-aromatic carbocyclic ring is a non-aromatic carbocyclic ring structure in which all ring members are composed of carbon atoms.

[0075] The term "heteroaryl" as used herein refers to a 5-10 membered aromatic group containing 1, 2 or 3 heteroatoms selected from O, N, S

[0076] "Halo" or "halogen" includes fluorine, chlorine, bromine and iodine; fluorine and chlorine are preferred.

[0077] The term "heterocycle" refers to a monocyclic or bicyclic heterocycloalkyl system, and may also include spiro heterocycles or bridged heterocycloalkyls. A monocyclic heterocycloalkyl refers to a 3-8 membered, preferably 3-6 membered, saturated or unsaturated, but non-aromatic, cyclic alkyl system containing at least one heteroatom selected from O, N, S, or P (preferably containing 1, 2, or 3 heteroatoms selected from O, N, and S). A bicyclic heterocycloalkyl system refers to a heterocycloalkyl fused to a phenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocycloalkyl group, or a heteroaryl group. An aromatic heterocycle refers to an aromatic heterocycle system, and a non-aromatic heterocycle refers to a non-aromatic heterocycle system.

[0078] When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3 R groups, then said group may be optionally substituted with up to three R groups, and at each occurrence R is independently selected from the definition of R. Furthermore, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0079] As used herein, the term "effective amount" means an amount of a drug or pharmaceutical agent (i.e., a compound of the present invention) that will cause a biological or medical response in a tissue, system, animal, or human being, such as that sought by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the above amount. An effective amount can be given in one or more administrations, applications, or dosages and is not intended to be limited by a specific formulation or route of administration. The term also includes within its scope an effective amount that enhances normal physiological function.

[0080] As used herein, the term "treating" includes any effect that results in improvement of a condition, disease, disorder, etc., such as alleviation, reduction, modulation, improvement, or elimination, or amelioration of the symptoms thereof.

[0081] The term "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response and / or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0082] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutical substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating substance, which is involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0083] The term "pharmaceutical composition" means a composition comprising a compound of the present invention and at least one other pharmaceutical carrier. "Pharmaceutical carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals (particularly mammals), including (i.e.) adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants, depending on the mode of administration and the nature of the dosage form.

[0084] The compounds or pharmaceutical compositions of the present invention can improve a disease, symptom, or condition after administration, particularly improving its severity, delaying its onset, slowing its progression, or reducing its duration, regardless of whether the administration is fixed or temporary, continuous or intermittent, and can be attributed to or related to the administration.

[0085] Route of administration

[0086] Suitable routes of administration for the compounds of the present invention or pharmaceutical compositions include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, auricular, nasal, and topical administration. In addition, parenteral administration, by way of example only, includes intramuscular, subcutaneous, intravenous, intramedullary, intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0087] In some embodiments, the administration of the compound of the present invention is topical administration. In other specific embodiments, the compound of the present invention can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In addition, in other specific embodiments, the compound of the present invention is administered by a targeted drug delivery system.

[0088] In the pharmaceutical compositions of the present invention, pharmaceutical carriers can be formulated based on a variety of factors well within the purview of those skilled in the art. These factors include, but are not limited to: the type and nature of the active agent being formulated; the subject to whom the composition containing the active agent is to be administered; the intended route of administration of the composition; and the targeted therapeutic indication. Pharmaceutical carriers include aqueous and non-aqueous liquid media and various solid and semisolid dosage forms.

[0089] Such carriers may include a variety of ingredients and additives in addition to the active agent, which are included in the formulation for various reasons known to those skilled in the art, such as stabilizing the active agent, binding agents, etc. Descriptions of suitable pharmaceutical carriers and factors involved in carrier selection can be found in a number of readily available sources, such as Allen LV Jr. et al. Remington: The Science and Practice of Pharmacy (2 Volumes), 22 nd Edition(2012),Pharmaceutical Press.

[0090] The compounds are usually administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as pharmaceutical carriers) appropriately selected according to the intended administration form (e.g., oral tablets, capsules, elixirs and syrups) and in accordance with conventional pharmaceutical practice.

[0091] While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0092] Kit / Product Packaging

[0093] Kits / product packaging are also described herein for use in treating the aforementioned indications. These kits can consist of a carrier, a pouch, or a container box, each of which can be divided into compartments to accommodate one or more containers, such as vials, test tubes, and the like, each containing a single component of the method. Suitable containers include bottles, vials, syringes, and test tubes. The containers can be made of acceptable materials such as glass or plastic.

[0094] For example, a container may contain one or more compounds described herein, either as a pharmaceutical composition or in admixture with other ingredients described herein. The container may have a sterile delivery port (e.g., an IV bag or bottle with a stopper pierceable by a hypodermic needle). Such a kit may include a compound and instructions, labeling, or operating instructions for use as described herein.

[0095] A typical kit may include one or more containers, each containing one or more materials (e.g., reagents, concentrated stock solutions, and / or instruments) to suit the commercialization and user needs of the compound. These materials include, but are not limited to, buffers, diluents, filters, needles, syringes, delivery devices, bags, containers, bottles, and / or test tubes, accompanied by a list of contents and / or instructions for use, including instructions for use within the packaging. The complete set of instructions should be included.

[0096] The features described above, or in the embodiments, may be combined in any combination. All features disclosed in this specification may be used in any combination, and each feature disclosed in this specification may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0097] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0098] The units used in the present invention for weight-volume percentages are well known to those skilled in the art, for example, referring to the weight of solute in 100 ml of solution. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0099] In the terms used in the present invention, "neurodegenerative disease" and "neurodegenerative disease" have the same meaning; "axonal degeneration" and "axonal degeneration" have the same meaning. Those skilled in the art will understand that the terms have commonly understood meanings.

[0100] The descriptions provided herein, including those provided in the Examples, are not intended to limit the scope of any claims. The following non-limiting examples are provided to further illustrate the present invention. Based on this disclosure, those skilled in the art will appreciate that many changes may be made to the specific embodiments disclosed and still achieve the same or similar results without departing from the spirit and scope of the present teachings.

[0101] Unless otherwise noted, all materials and reagents were obtained from commercial suppliers and used without further purification. Reactions were monitored by LC-MS and / or thin-layer chromatography (TLC) on silica gel 60F254 (0.2 mm) pre-coated glass backing and observed using UV light. The structures of the following example compounds were characterized by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0102] 1 H NMR spectra (400 MHz) were recorded at room temperature on a Bruker spectrometer using TMS or residual solvent peaks as internal standards. Chemical shift values ​​or peak shape multiples are given in (δ), and coupling constants (J) are given in absolute values ​​in Hertz (Hz). 1The multiplicity in HNMR spectra is abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br or broad (broadened).

[0103] Preparative HPLC purification was performed on a Shimadzu LC-6AD. All purifications were performed using a Shim-pack PREP-DDS(H)KIT column. The mobile phase consisted of water (containing 0.1% HCO₂H₂) and acetonitrile; all reagents used were HPLC grade. The flow rate was 10 ml / min.

[0104] LC-MS was performed on an Agilent 1260 infinity II; mobile phase: A: water (0.1% trifluoroacetic acid), B: ACN; 3.5 min column run; column: YMC-Triart C18 50*3 mm, 3 um; flow rate: 1.8 ml / min; oven temperature: 40°C; gradient: 5-100 (ACN%).

[0105] Preparative TLC was performed on Whatman LK6F Silica Gel 60A plates of size 20 x 20 cm with a thickness of 500 μm.

[0106] The following examples are intended to illustrate embodiments of the present invention but are not intended to limit the same in any way.

[0107] Synthesis route of intermediate BB1

[0108] Step 1: Intermediate BB1-C (5-bromo-2-(3-fluoropyridin-4-yl)phenyl)methanol

[0109] To a solution of Intermediate BB1-A (5-bromo-2-iodophenyl)methanol (5.0 g, 15.98 mmol) and Intermediate BB1-B (3-fluoropyridin-4-yl)-pinacol boronic acid (3.56 g, 15.98 mmol) in 1,4-dioxane (100 mL) and water (10 mL) was added PdCl2(dppf)·CH2Cl2 (1.3 g, 1.598 mmol) and sodium carbonate (4.23 g, 39.94 mmol) at room temperature. The reaction system was purged with nitrogen three times and stirred at 80°C for 12 hours. TLC monitored the reaction for completion. The reaction solution was cooled to room temperature, filtered through Celite, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 50% ethyl acetate in petroleum ether) to afford Intermediate BB1-C (5-bromo-2-(3-fluoropyridin-4-yl)phenyl)methanol (1.71 g) as a brown oil.

[0110] LC_MS:(ES+ ):m / z 281.95[M+H] + .

[0111] Step 2: Intermediate BB1 ​​8-bromo-6H-isochromeno[3,4-c]pyridine

[0112] To a solution of the intermediate BB1-C (5-bromo-2-(3-fluoropyridin-4-yl)phenyl)methanol (1.71 g, 6.061 mmol) in tetrahydrofuran (30 mL) was added sodium hydride (484.9 mg, 12.123 mmol, 60% purity) at 0°C. The reaction was stirred at room temperature for 12 hours. TLC confirmed the reaction was complete. The reaction solution was slowly poured into saturated ammonium chloride (30 mL) and extracted with ethyl acetate (30 mL x 2). The organic layers were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 33% to 50% ethyl acetate in petroleum ether) to afford the intermediate BB1 ​​as a yellow solid, 8-bromo-6H-isochromeno[3,4-c]pyridine (1.3 g).

[0113] LC_MS:(ES + ):m / z 261.94[M+H] + .

[0114] 1 H NMR (400MHz, CDCl3) δ8.38 (s, 1H), 8.33 (d, J = 5.1Hz, 1H), 7.63 (d, J = 8.3Hz, 1H), 7.60-7.52 (m, 2H), 7.38 (d, J = 1.4Hz, 1H), 5.19 (s, 2H).

[0115] Synthesis route of intermediate BB2

[0116] Step 1: Intermediate BB2 1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one

[0117] To a solution of Intermediate BB1 ​​(8-bromo-6H-isochromeno[3,4-c]pyridine) (1.3 g, 4.922 mmol) and Intermediate BB2-A (pyrrolidin-2-one) (418.9 mg, 4.922 mmol) in 1,4-dioxane (20 mL) was added Pd2(dba)3 (225.4 mg, 0.246 mmol), Xant-Phos (142.3 mg, 0.246 mmol), and cesium carbonate (3.2 g, 9.844 mmol) at room temperature. The reaction system was purged with nitrogen three times and heated to 100°C with stirring for 2 hours. TLC monitored the reaction for completion. The reaction solution was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: 7.2% methanol in dichloromethane) to give the intermediate BB2 1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one (1.14 g) as a yellow solid.

[0118] LC_MS:(ES + ):m / z 267.10[M+H] + .

[0119] 1 H NMR (400MHz, CDCl3) δ8.35(s,1H),8.29(d,J=5.0Hz,1H),7.74(d,J=8.5Hz,1H),7.69(s,1H),7.58(d,J=8.4 Hz,1H),7.53(d,J=5.0Hz,1H),5.22(s,2H),3.92(t,J=7.0Hz,2H),2.67(t,J=8.1Hz,2H),2.27-2.16(m,2H).

[0120] Synthesis route of intermediate BB3

[0121] Step 1: Intermediate BB3-B 2-bromo-5-nitrobenzyl alcohol

[0122] To a solution of intermediate BB3-A 2-bromo-5-nitrobenzoic acid (5 g, 20.32 mmol) in tetrahydrofuran (50 mL) was added BH3-THF (40.64 mL, 40.64 mmol) at room temperature. The reaction mixture was incubated at 60°C for 4 hours. TLC confirmed the reaction was complete. The reaction mixture was quenched with methanol (50 mL) and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 20% ethyl acetate in petroleum ether) to afford intermediate BB3-B 2-bromo-5-nitrobenzyl alcohol (4.2 g) as a beige solid.

[0123] 1H NMR (400MHz, CDCl3) δ8.45 (d, J = 2.7Hz, 1H), 8.04 (dd, J = 8.7, 2.7Hz, 1H), 7.74 (d, J = 8.7Hz, 1H), 4.85 (s, 2H).

[0124] Step 2: Intermediate BB3-D (2-(3-fluoropyridin-4-yl)-5-nitrophenyl)methanol

[0125] To a solution of 1,4-dioxane (60 mL) and water (10 mL) containing intermediate BB3-B (2-bromo-5-nitrobenzyl alcohol, 4.2 g, 18.1 mmol), intermediate BB3-C (3-fluoropyridine-4-boronic acid pinacol ester, 6.05 g, 27.15 mmol), and sodium carbonate (5.75 g, 54.3 mmol) was added Pd(dppf)Cl₂-CH₂Cl₂ (1.48 g, 1.81 mmol) at room temperature under nitrogen. The reaction system was purged with nitrogen three times and heated to 90°C for 18 hours. TLC confirmed the reaction was complete. The reaction solution was extracted with water (60 mL) and dichloromethane (120 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: 40% ethyl acetate in petroleum ether) to give a brown solid intermediate BB3-D (2-(3-fluoropyridin-4-yl)-5-nitrophenyl)methanol (4.1 g).

[0126] LC_MS:(ES + ):m / z 249.05[M+H] + .

[0127] 1 H NMR (400MHz, CDCl3) δ8.56(dd,J=17.5,2.7Hz,3H),8.25(dd,J=8.4,2.1Hz,1H),7.45(d,J=8.4Hz,1H),7.31(s,1H),4.66(s,2H).

[0128] Step 3: Intermediate BB3-E 8-nitro-6H-isochromeno[3,4-c]pyridine

[0129] To a solution of the intermediate BB3-D (2-(3-fluoropyridin-4-yl)-5-nitrophenyl)methanol (4 g, 16.11 mmol) in tetrahydrofuran (50 mL) was added sodium hydride (1.29 g, 32.23 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 18 hours. TLC confirmed the reaction completion. The reaction mixture was quenched by adding saturated ammonium chloride solution (50 mL) and extracted with dichloromethane (20 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 4.76% methanol in dichloromethane) to afford the intermediate BB3-E as a yellow solid, 8-nitro-6H-isochromeno[3,4-c]pyridine (3.3 g).

[0130] LC_MS:(ES + ):m / z 229.05[M+H] + .

[0131] Step 4: Intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine

[0132] Zinc powder (4.59 g, 70.11 mmol) was added portionwise to a solution of intermediate BB3-E 8-nitro-6H-isochromeno[3,4-c]pyridine (3.2 g, 14.02 mmol) and ammonium chloride (3.75 g, 70.11 mmol) in ethanol (50 mL) / water (10 mL) at 80°C. The reaction was stirred at 80°C for 18 hours. TLC monitored the reaction for completion. The reaction mixture was filtered, the filtrate concentrated, and extracted with water (20 mL) and dichloromethane (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was isolated and purified by silica gel column chromatography (eluent: 4.76% methanol in dichloromethane) to afford intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine (1.9 g) as a brown solid.

[0133] LC_MS:(ES + ):m / z 199.0[M+H] + .

[0134] 1 H NMR (400MHz, DMSO) δ8.16-8.10(m,2H),7.59(t,J=6.8Hz,2H),6.61(dd,J=8.4,2.3Hz,1H),6.41(d,J=2.2Hz,1H),5.71(s,2H),5.06(s,2H).

[0135] Synthesis route of intermediate BB4

[0136] Step 1: Intermediate BB4-C tert-butyl 3-(4-chlorobenzyl)-2-oxopyrrolidine-1-carboxylate

[0137] To a solution of intermediate BB4-A tert-butyl 2-oxopyrrolidine-1-carboxylate (4 g, 21.6 mmol) in tetrahydrofuran (60 mL) was slowly added LiHMDS (21.6 ml, 21.6 mmol) at -60°C under nitrogen. After reacting at -60°C for 1 hour, a solution of intermediate BB4-B 4-chlorobenzyl bromide (4.44 g, 21.6 mmol) in tetrahydrofuran (10 mL) was slowly added dropwise. The reaction mixture was reacted at -60°C for 2 hours, then warmed to room temperature and stirred for 18 hours. The reaction was monitored for completion by TLC. The reaction solution was quenched by adding saturated ammonium chloride solution (50 mL), extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was separated and purified by silica gel column chromatography (eluent: 10-15% ethyl acetate in petroleum ether) to give the intermediate BB4-C tert-butyl 3-(4-chlorobenzyl)-2-oxopyrrolidine-1-carboxylate (1.7 g) as a colorless oil.

[0138] 1 H NMR (400MHz, CDCl3) δ7.30-7.27(m,2H),7.15(d,J=8.4Hz,2H),3.69(ddd,J=11.2,8.7,2.7Hz,1H),3.59-3.51(m,1H),3. 23(dd,J=13.6,3.9Hz,1H),2.83-2.74(m,1H),2.68(t,J=6.8Hz,1H),2.06-1.99(m,1H),1.71-1.65(m,1H),1.55(s,9H).

[0139] Step 2: Intermediate BB4 3-(4-chlorobenzyl)pyrrolidin-2-one

[0140] Intermediate BB4-C tert-butyl 3-(4-chlorobenzyl)-2-oxopyrrolidine-1-carboxylate (1.7 g, 5.488 mmol) was added to a 4 M hydrochloric acid-dioxane solution (20 mL) at room temperature and stirred for 1 hour. The reaction was monitored for completion by TLC. Concentration under reduced pressure afforded the intermediate BB4 3-(4-chlorobenzyl)pyrrolidin-2-one (1.15 g) as a white solid.

[0141] LC_MS:(ES + ):m / z 210.06[M+H] +

[0142] Synthesis route of intermediate BB5

[0143] Step 1: Intermediate BB5-C tert-butyl 3-(4-fluorobenzyl)-2-oxopyrrolidine-1-carboxylate

[0144] Intermediate BB5-A tert-butyl 2-oxopyrrolidine-1-carboxylate (7404 mg, 40 mmol) was dissolved in THF (120 mL) and cooled to -78°C under nitrogen. Lithium bis(trimethylsilyl)amide (42 mmol, 42 mL) was added and the reaction was controlled at temperature for 1 hour. Intermediate BB5-B 1-(bromomethyl)-4-fluorobenzene (7518 mg, 40 mmol) was dissolved in tetrahydrofuran (40 mL) and added to the reaction solution within 5 minutes. The reaction was controlled at temperature for 1 hour. Saturated ammonium chloride (30 mL) was added to quench the reaction. The organic solvent was removed by concentration under reduced pressure. The aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with water (50 mL x 2) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: 27% ethyl acetate in n-hexane) to obtain intermediate BB5-C. tert-Butyl 3-(4-fluorobenzyl)-2-oxopyrrolidine-1-carboxylate (5240 mg, 18.22 mg), yield: 42.2%.

[0145] 1 H NMR (400MHz, Chloroform-d) δ7.21(dd,J=7.4,1.4Hz,1H),7.11(s,1H),7.06(d,J=7.5Hz,1H),4.67(s,3H),3.19(s,4H).

[0146] Step 2: Intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one

[0147] The intermediate BB5-C tert-butyl 3-(4-fluorobenzyl)-2-oxopyrrolidine-1-carboxylate (5240 mg, 18.22 mmol) was dissolved in dichloromethane (20 mL), cooled to 0°C, and a solution of hydrochloric acid in 1.4-dioxane (45 mL, 182.2 mmol) was added. The mixture was stirred at room temperature for 1 hour and concentrated to dryness under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: 7.6% methanol in dichloromethane) to obtain the intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one (2950 mg) in a yield of 84%.

[0148] LC_MS:(ES + ):m / z 194.07[M+H] +

[0149] Synthesis route of intermediate BB6

[0150] Step 1: Intermediate BB6 2-(bromomethyl)-5-chlorothiophene

[0151] Phosphorus tribromide (136.6 mg, 0.505 mmol) was added to a solution of intermediate BB6-A 5-chloro-2-thiophenemethanol (50 mg, 0.336 mmol) in dichloromethane (5 mL) at 0°C. The reaction mixture was stirred from 0°C to room temperature for 4 hours. TLC confirmed the reaction was complete. Ice water was added to the reaction solution, which was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative thin-layer chromatography (eluent: 5% ethyl acetate in petroleum ether) to afford intermediate BB6 2-(bromomethyl)-5-chlorothiophene (90 mg) as a brown oil.

[0152] Synthesis route of intermediate BB7

[0153] Step 1: Intermediate BB7-B (5-chlorofuran-2-yl) methanol

[0154] Intermediate BB7-A 5-chlorofuran-2-carboxaldehyde (100 mg, 0.77 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and placed in an ice bath. NaBH4 (30 mg, 0.79 mmol) was then added and the reaction was allowed to react at room temperature for 2 h. After completion of the reaction, saturated aqueous ammonium chloride (5 mL) was added dropwise, and the mixture was extracted three times with ethyl acetate (5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The crude intermediate BB7-B (5-chlorofuran-2-yl)methanol (100 mg) was used directly in the next step.

[0155] Step 2: Intermediate BB7 2-(Bromomethyl)-5-chlorofuran

[0156] Dissolve the intermediate BB7-B (5-chlorofuran-2-yl)methanol (100 mg, 0.77 mmol) in dichloromethane (2 mL) and add PBr3 (200 μL, 1.54 mmol) dropwise in an ice bath. After the addition is complete, allow to react at room temperature for 2 h. After the reaction is complete, spin dry the mixture, dilute it with dichloromethane (5 mL), place it in an ice bath, and add saturated aqueous sodium bicarbonate solution (5 mL) dropwise. Extract once with dichloromethane (5 mL), dry over anhydrous sodium sulfate, and filter. The resulting crude intermediate BB7 2-(bromomethyl)-5-chlorofuran (110 mg) is used directly in the next step.

[0157] Synthesis route of intermediate BB8

[0158] Step 1: Intermediate BB8-B bicyclo[4.2.0]octane-1(6),2,4-trien-3-ylmethanol

[0159] The synthesis of this step uses the intermediate BB8-A 4-formylbenzocyclobutene as the raw material and refers to the synthesis process of the first step of intermediate BB7.

[0160] 1 H NMR (400MHz, Chloroform-d) δ7.21(dd,J=7.4,1.4Hz,1H),7.11(s,1H),7.06(d,J=7.5Hz,1H),4.67(s,3H),3.19(s,4H).

[0161] Step 2: Intermediate BB8 3-(Bromomethyl)bicyclo[4.2.0]octane-1(6),2,4-triene

[0162] The synthesis of this step uses the intermediate BB8-B 4 bicyclo[4.2.0]octane-1(6),2,4-trien-3-ylmethanol as the raw material and refers to the synthesis process of the second step of intermediate BB7.

[0163] Synthesis route of intermediate BB9

[0164] Step 1: Intermediate BB9 3-(5-chlorothiophen-2-yl)propionic acid

[0165] Triethylamine (834.2 mg, 8.186 mmol) was added to formic acid (941.8 mg, 20.465 mmol) at room temperature and stirred for 30 minutes. Intermediate BB9-A 5-chlorothiophene-2-carboxaldehyde (1.0 g, 6.822 mmol) and Intermediate BB9-B 2,2-dimethyl-1,3-dioxane-4,6-dione (983 mg, 6.822 mmol) were then added, and the reaction mixture was heated to 95°C and stirred for 20 hours. LCMS confirmed the reaction was complete. The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 5% methanol in dichloromethane) to afford Intermediate BB9 3-(5-chlorothiophen-2-yl)propanoic acid (940 mg) as a yellow oil.

[0166] LC_MS:(ES - ):m / z 188.95[MH] - .

[0167] Synthesis route of intermediate BB10

[0168] Step 1: Intermediate BB10 3-(4-cyano-3-fluorophenyl)propionic acid

[0169] The synthesis of this step uses the intermediate BB10-A 2-fluoro-4-formylbenzonitrile as the raw material and refers to the synthesis process of the first step of intermediate BB9.

[0170] LC_MS:(ES - ):m / z 385.10[2M-H] - .

[0171] Synthesis route of intermediate BB11

[0172] Step 1: Intermediate BB11-C(E)-tert-butyl 3-(4-cyano-3,5-difluorophenyl)acrylate

[0173] Under nitrogen, palladium acetate (156.3 mg, 0.7 mmol) was added to N,N-dimethylformamide (10 mL) containing intermediate BB11-A (4-bromo-2,6-difluorobenzonitrile) (1 g, 4.6 mmol), intermediate BB11-B (tert-butyl acrylate) (882 mg, 6.9 mmol), DABCO (17 mg, 0.152 mmol), and potassium carbonate (565.83 mg, 4.1 mmol). The reaction system was purged with nitrogen three times and heated to 120°C for 2 hours. The reaction was monitored for completion by TLC. The reaction solution was extracted with ethyl acetate (30 mL x 2) and water (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: 0%-20% ethyl acetate in petroleum ether) to give a white solid intermediate BB11-C(E)-tert-butyl 3-(4-cyano-3,5-difluorophenyl)acrylate (350 mg).

[0174] 1 H NMR (400MHz, CDCl3) δ7.47(d,J=15.9Hz,1H),7.18(d,J=8.2Hz,2H),6.47(d,J=15.9Hz,1H),1.56(s,9H).

[0175] Step 2: Intermediate BB11-D tert-Butyl 3-(4-cyano-3,5-difluorophenyl)propionate

[0176] To a solution of the intermediate BB11-C(E)-tert-butyl 3-(4-cyano-3,5-difluorophenyl)acrylate (250 mg, 0.942 mmol) and cobalt chloride (25 mg, 0.192 mmol) in methanol (5 mL) was slowly added sodium borohydride (178.18 mg, 4.1 mmol) at 0°C. The reaction was stirred at 0°C for 3 hours. TLC monitored the reaction for completion. The reaction was quenched with saturated ammonium chloride (10 mL) and extracted with ethyl acetate (15 mL x 2) and water (10 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: 0%-20% ethyl acetate in petroleum ether) to afford the intermediate BB11-D as a white solid, tert-butyl 3-(4-cyano-3,5-difluorophenyl)propanoate (60 mg).

[0177] 1 H NMR (400MHz, CDCl3) δ6.93 (d, J = 8.3 Hz, 2H), 2.98 (t, J = 7.3 Hz, 2H), 2.58 (td, J = 7.4, 2.2 Hz, 2H), 1.44 (s, 9H).

[0178] Step 3: Intermediate BB11 3-(4-cyano-3,5-difluorophenyl)propionic acid

[0179] To a solution of the intermediate BB11-D, tert-butyl 3-(4-cyano-3,5-difluorophenyl)propanoate (60 mg, 0.224 mmol) in dichloromethane (1 mL), trifluoroacetic acid (200 μL) was added. The reaction mixture was stirred at room temperature for 5 hours. TLC confirmed the reaction was complete. The reaction mixture was concentrated to afford the intermediate BB11, 3-(4-cyano-3,5-difluorophenyl)propanoic acid (55 mg), which was used directly in the next reaction.

[0180] LC_MS:(ES - ):m / z 210.1[MH] - .

[0181] Synthesis route of intermediate BB12

[0182] Step 1: Intermediate BB12-C(E)-tert-butyl 3-(4-chloro-3,5-difluorophenyl)acrylate

[0183] The synthesis of this step uses the intermediate BB12-A 5-bromo-2-chloro-1,3-difluorobenzene as the raw material and refers to the synthesis process of the first step of intermediate BB11.

[0184] 1H NMR (400MHz, CDCl3) δ7.45 (d, J = 15.9 Hz, 1H), 7.17-7.10 (m, 2H), 6.36 (d, J = 15.9 Hz, 1H), 1.55 (s, 9H).

[0185] Step 2: Intermediate BB12-D tert-Butyl 3-(4-cyano-3,5-difluorophenyl)propionate

[0186] To a solution of the intermediate BB12-C(E)-tert-butyl 3-(4-chloro-3,5-difluorophenyl)acrylate (400 mg, 1.46 mmol) and nickel chloride (188.72 mg, 1.46 mmol) in methanol (10 mL) was slowly added sodium borohydride (165.24 mg, 4.37 mmol) at 0°C. The reaction was stirred at 0°C for 3 hours. TLC monitored the reaction for completion. The reaction was quenched with saturated ammonium chloride (10 mL) and extracted with ethyl acetate (10 mL x 2) and water (10 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: 0%-20% ethyl acetate in petroleum ether) to afford the intermediate BB12-D tert-butyl 3-(4-cyano-3,5-difluorophenyl)propanoate (380 mg) as a white solid.

[0187] 1 H NMR (400MHz, CDCl3) δ6.86 (d, J = 7.6Hz, 2H), 2.89 (t, J = 7.5Hz, 2H), 2.54 (dd, J = 8.6, 6.5Hz, 2H), 1.44 (s, 9H).

[0188] Step 3: Intermediate BB12 3-(4-chloro-3,5-difluorophenyl)propionic acid

[0189] The synthesis of this step uses the intermediate BB12-D tert-butyl 3-(4-cyano-3,5-difluorophenyl)propionate as the raw material and refers to the synthesis process of the third step of intermediate BB11.

[0190] LC_MS:(ES - ):m / z 219.2[MH] - .

[0191] Synthesis route of intermediate BB13

[0192] Step 1: Intermediate BB13-B (2-bromo-5-nitropyridin-3-yl)methanol

[0193] The synthesis of this step uses the intermediate BB13-A 2-bromo-5-nitronicotinic acid as the raw material and refers to the synthesis process of the first step of intermediate BB3.

[0194] LC_MS:(ES + ):m / z 232.9[M+H] + .

[0195] Step 2: Intermediate BB13-D (3'-fluoro-5-nitro-[2,4'-bipyridyl]-3-yl)methanol

[0196] The synthesis of this step uses intermediate BB13-B (2-bromo-5-nitropyridin-3-yl)methanol and intermediate BB13-C3-fluoropyridine-4-boronic acid pinacol ester as raw materials, referring to the synthesis process of intermediate BB3 in the second step.

[0197] LC_MS:(ES + ):m / z 250.0[M+H] + .

[0198] Step 3: Intermediate BB13-E 3-nitro-5H-pyrano[4,5-b:2,3-c']bipyridine

[0199] The synthesis of this step uses the intermediate BB13-D (3'-fluoro-5-nitro-[2,4'-bipyridyl]-3-yl)methanol as the raw material and refers to the synthesis process of the third step of intermediate BB3.

[0200] LC_MS:(ES + ):m / z 230.0[M+H] + .

[0201] Step 4: Intermediate BB13 5H-pyrano[4,5-b:2,3-c']dipyridin-3-amine

[0202] The synthesis of this step uses the intermediate BB13-E 3-nitro-5H-pyrano[4,5-b:2,3-c']bipyridine as the raw material and refers to the synthesis process of the fourth step of intermediate BB3.

[0203] LC_MS:(ES + ):m / z 200.1[M+H] + .

[0204] Synthesis route of intermediate BB14

[0205] Step 1: Intermediate BB14-B (5-bromo-2-nitropyridin-4-yl)methanol

[0206] The synthesis of this step uses the intermediate BB14-A 5-bromo-2-nitroisonicotinic acid as the raw material and refers to the synthesis process of the first step of the intermediate BB3.

[0207] LC_MS:(ES + ):m / z 232.9[M+H] + .

[0208] Step 2: Intermediate BB14-D (3'-fluoro-6-nitro-[3,4'-bipyridyl]-4-yl)methanol

[0209] The synthesis of this step uses intermediate BB14-B (5-bromo-2-nitropyridin-4-yl)methanol and intermediate BB14-C3-fluoropyridine-4-boronic acid pinacol ester as raw materials, referring to the synthesis process of intermediate BB3 in the second step.

[0210] LC_MS:(ES + ):m / z 250.0[M+H] + .

[0211] Step 3: Intermediate BB14-E 3-nitro-5H-pyrano[2,3-c:4,5-c']bipyridine

[0212] The synthesis of this step uses the intermediate BB14-D (3'-fluoro-6-nitro-[3,4'-bipyridyl]-4-yl)methanol as the raw material and refers to the synthesis process of the third step of intermediate BB3.

[0213] LC_MS:(ES + ):m / z 230.0[M+H] + .

[0214] Step 4: Intermediate BB14 5H-pyrano[2,3-c:4,5-c']dipyridin-3-amine

[0215] The synthesis of this step uses the intermediate BB14-E 3-nitro-5H-pyrano[2,3-c:4,5-c']bipyridine as the raw material and refers to the synthesis process of the fourth step of intermediate BB3.

[0216] LC_MS:(ES + ):m / z 200.1[M+H] + .

[0217] Synthesis route of intermediate BB15

[0218] Step 1: Intermediate BB15-B (2,5-dibromopyridin-3-yl)methanol

[0219] The synthesis of this step uses the intermediate BB15-A 2,5-dibromonicotinic acid as the raw material and refers to the synthesis process of the first step of intermediate BB3.

[0220] LC_MS:(ES + ):m / z 265.8[M+H] + .

[0221] Step 2: Intermediate BB15-D (5-bromo-3'-fluoro-[2,4'-bipyridyl]-3-yl)methanol

[0222] The synthesis of this step uses intermediate BB15-B (2,5-dibromopyridin-3-yl)methanol and intermediate BB15-C 3-fluoropyridine-4-boronic acid pinacol ester as raw materials, referring to the synthesis process of intermediate BB1 ​​in the first step.

[0223] LC_MS:(ES + ):m / z 283.0[M+H] + .

[0224] Step 3: Intermediate BB15 3-bromo-5H-pyrano[4,5-b:2,3-c']bipyridine

[0225] The synthesis of this step uses the intermediate BB15-D (5-bromo-3'-fluoro-[2,4'-bipyridyl]-3-yl)methanol as the raw material and refers to the synthesis process of the second step of intermediate BB1.

[0226] LC_MS:(ES + ):m / z 263.0[M+H] + .

[0227] Synthesis route of intermediate BB16

[0228] Step 1: Intermediate BB16-B (5-bromo-2-chloropyridin-4-yl)methanol

[0229] The synthesis of this step uses the intermediate BB16-A 5-bromo-2-chloroisonicotinic acid as the raw material and refers to the synthesis process of the first step of intermediate BB3.

[0230] LC_MS:(ES + ):m / z 221.9[M+H] + .

[0231] Step 2: Intermediate BB16-D (6-chloro-3'-fluoro-[3,4'-bipyridyl]-4-yl)methanol

[0232] The synthesis of this step uses intermediate BB16-B (5-bromo-2-chloropyridin-4-yl)methanol and intermediate BB16-C 3-fluoropyridine-4-boronic acid pinacol ester as raw materials, referring to the synthesis process of intermediate BB1 ​​in the first step.

[0233] LC_MS:(ES + ):m / z 239.0[M+H] + .

[0234] Step 3: Intermediate BB16 3-chloro-5H-pyrano[2,3-c:4,5-c']bipyridine

[0235] The synthesis of this step uses the intermediate BB16-D (6-chloro-3'-fluoro-[3,4'-bipyridyl]-4-yl)methanol as the raw material and refers to the synthesis process of the second step of intermediate BB1.

[0236] LC_MS:(ES + ):m / z 219.0[M+H] + .

[0237] Synthesis route of intermediate BB17

[0238] Step 1: Intermediate BB17-B (2-(3-fluoro-5-methylpyridin-4-yl)-5-nitrophenyl)methanol

[0239] The synthesis of this step uses intermediate BB3-B (2-bromo-5-nitrophenyl) methanol and intermediate BB17-A (3-fluoro-5-methylpyridin-4-yl) boronic acid as raw materials, referring to the synthesis process of intermediate BB3 in the second step.

[0240] LC_MS:(ES + ):m / z 263.1[M+H] + .

[0241] Step 2: Intermediate BB17-C 1-methyl-8-nitro-6H-isochromeno[3,4-c]pyridine

[0242] The synthesis of this step uses the intermediate BB17-B (2-(3-fluoro-5-methylpyridin-4-yl)-5-nitrophenyl)methanol as the raw material and refers to the synthesis process of the third step of intermediate BB3.

[0243] LC_MS:(ES+ ):m / z 243.1[M+H] + .

[0244] Step 3: Intermediate BB17 1-methyl-6H-isochromeno[3,4-c]pyridin-8-amine

[0245] This step of the synthesis uses the intermediate BB17-C 1-methyl-8-nitro-6H-isochromeno[3,4-c]pyridine as the starting material and refers to the synthesis process of the fourth step of intermediate BB3.

[0246] LC_MS:(ES + ):m / z 213.1[M+H] + .

[0247] Synthesis route of intermediate BB18

[0248] Step 1: Intermediate BB18-B (6-bromo-2-methyl-3-nitrophenyl)methanol

[0249] The synthesis of this step uses the intermediate BB18-A 6-bromo-2-methyl-3-nitrobenzoic acid methyl ester as the raw material and refers to the synthesis process of the first step of intermediate BB3.

[0250] LC_MS:(ES + ):m / z 246.0[M+H] + .

[0251] Step 2: Intermediate BB18-D (6-(3-fluoropyridin-4-yl)-2-methyl-3-nitrophenyl)methanol

[0252] The synthesis of this step uses intermediate BB18-B (6-bromo-2-methyl-3-nitrophenyl)methanol and intermediate BB18-C 3-fluoropyridine-4-boronic acid pinacol ester as raw materials, referring to the synthesis process of intermediate BB3 in the second step.

[0253] LC_MS:(ES + ):m / z 263.1[M+H] + .

[0254] Step 3: Intermediate BB18-E 7-methyl-8-nitro-6H-isochromeno[3,4-c]pyridine

[0255] The synthesis of this step uses the intermediate BB18-D (6-(3-fluoropyridin-4-yl)-2-methyl-3-nitrophenyl)methanol as the raw material and refers to the synthesis process of the third step of intermediate BB3.

[0256] LC_MS:(ES + ):m / z 243.1[M+H] + .

[0257] Step 4: Intermediate BB18 7-methyl-6H-isochromeno[3,4-c]pyridin-8-amine

[0258] This step of the synthesis uses the intermediate BB18-E 7-methyl-8-nitro-6H-isochromeno[3,4-c]pyridine as the starting material and refers to the synthesis process of the fourth step of intermediate BB3.

[0259] LC_MS:(ES + ):m / z 213.1[M+H] + .

[0260] Synthesis route of intermediate BB19

[0261] Step 1: Compound BB19-C tert-butyl 3-(4-fluorobenzyl)-2-oxopiperidine-1-carboxylate

[0262] The synthesis of this step refers to the synthesis process of the first step of intermediate BB4.

[0263] LC_MS:(ES + ):m / z 308.16[M+H] + .

[0264] Step 2: Compound BB19 3-(4-fluorobenzyl)piperidin-2-one

[0265] The synthesis of this step refers to the synthesis process of the second step of intermediate BB4.

[0266] LC_MS:(ES + ):m / z 208.11[M+H] + .

[0267] Synthesis route of intermediate BB20

[0268] Step 1: Compound BB20 3-(4-fluorobenzyl)pyrrolidin-2-one-3-d

[0269] To a solution of compound BB5-C (1 g, 3.41 mmol) in tetrahydrofuran (15 mL) was added LiHMDS (4.09 ml, 4.09 mmol) at 0°C. The reaction mixture was stirred at 0°C for 0.5 hours. Deuterated methanol (5 mL) was added, and the mixture was allowed to warm to room temperature with stirring for 2 hours. LCMS monitored the reaction for completion. The reaction mixture was poured into saturated ammonium chloride solution (30 mL) and extracted with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 0%-5% methanol in dichloromethane) to afford compound BB20 3-(4-fluorobenzyl)pyrrolidin-2-one-3-d (500 mg) as a white solid.

[0270] LC_MS:(ES + ):m / z 195.1[M+H] + .

[0271] 1 H NMR (400MHz, CDCl3) δ7.22-7.15(m,2H),7.02-6.95(m,2H),6.36(s,1H),3.32-3.21(m,2 H),3.17(d,J=14.0Hz,1H),2.70(t,J=10.6Hz,1H),2.19-2.11(m,1H),1.88-1.80(m,1H).

[0272] Synthesis route of intermediate BB21

[0273] Step 1: Intermediate BB21-C methyl 2-cyano-3-(4-fluorophenyl)propionate

[0274] Intermediate BB21-A methyl cyanoacetate (3.93 g, 0.04 mol) was dissolved in DMSO (20 mL). Potassium carbonate (5.48 g, 0.04 mol) was added and stirred at room temperature for 30 min. Then, intermediate BB21-B 4-fluorobenzyl bromide (5.0 g, 0.026 mol) was added at 0°C and stirred at room temperature for 6 h. The reaction was monitored for completion by TLC. The reaction solution was poured into water (60 mL) and extracted twice with ethyl acetate (50 mL). The combined organic phases were washed three times with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and spin-dried. The crude product was isolated and purified by silica gel column chromatography (eluent: PE:EtOAc = 20:1 → 15:1 → 10:1) to obtain intermediate BB21-C methyl 2-cyano-3-(4-fluorophenyl)propanoate (1.13 g, 20% yield) as a colorless oil.

[0275] 1 H NMR (400MHz, CDCl3): δ7.26-7.23(m,2H),7.06-7.02(m,2H),3.80(s,3H),3.74-3.70(m,1H),3.28-3.16(m,1H).

[0276] Step 2: Intermediate BB21-D methyl 2-(aminomethyl)-3-(4-fluorophenyl)propionate

[0277] Intermediate BB21-C methyl 2-cyano-3-(4-fluorophenyl)propionate (1.13 g, 5.4 mmol) was dissolved in ethanol (20 mL) and Raney nickel (1.5 g) was added. The mixture was reacted overnight at room temperature under a hydrogen balloon. The reaction was monitored for completion by TLC. The reaction solution was filtered through celite, the filter cake was washed with ethanol (20 mL), and the filtrate was collected and dried to give the crude intermediate BB21-D methyl 2-(aminomethyl)-3-(4-fluorophenyl)propionate (1.1 g, crude product) as a colorless oil. This product was used directly in the next step without purification.

[0278] LC_MS:(ES + ):m / z 212.10[M+H] + .

[0279] Step 3: Intermediate BB21 3-[(4-fluorophenyl)methyl]azetidin-2-one

[0280] Intermediate BB21-D methyl 2-(aminomethyl)-3-(4-fluorophenyl)propanoate (1.1 g, 5.2 mmol) was dissolved in anhydrous ether (10 mL) and cooled to 0°C under N2. Then, CH3MgI (7 mL, 20.8 mmol, 3 mol / L) was slowly added dropwise to the reaction mixture. The ice bath was removed and the mixture was stirred at room temperature overnight. LCMS monitored the reaction for completion. The reaction mixture was quenched with saturated sodium bicarbonate (20 mL) under ice bath, filtered, and the filter cake was washed with EtOAc. The mixture was separated and the aqueous phase was extracted with EtOAc (20 mL x 2). The organic phases were combined and washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and spin-dried. The crude product was purified by column chromatography (mobile phase DCM: CH3OH = 200:1 → 150:1), then stirred with n-hexane (5 mL) for 30 min and filtered to give the intermediate BB21 3-[(4-fluorophenyl)methyl]azetidin-2-one (125 mg, yield 13%) as a white solid.

[0281] LC_MS:(ES +):m / z 180.00[M+H] + .

[0282] 1 H NMR (400MHz, CDCl3): δ7.21-7.17(m,2H),6.99(t,J=8.8Hz,2H),5.66(brs,1H),3.53-3.5 1(m,1H),3.39(t,J=5.6Hz,1H),3.13-3.08(m,1H),3.04-3.02(m,1H),2.97-2.92(m,1H).

[0283] Synthesis route of intermediate BB22

[0284] Step 1: Intermediate BB22 1-(4-fluorobenzyl)imidazolidin-2-one

[0285] To a DMF solution containing BB22-A imidazolidin-2-one (910.9 mg, 10.58 mmol) was added sodium hydride (211.6 mg, 5.29 mmol, 60% wt) under ice-cooling. The mixture was stirred for half an hour, followed by the dropwise addition of BB22-B 1-(bromomethyl)-4-fluorobenzene (1.0 g, 5.29 mmol). The mixture was stirred at room temperature for 12 hours. LCMS confirmed the reaction was complete. The reaction mixture was poured into saturated ammonium chloride solution (20 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 50-70% ethyl acetate in petroleum ether) to afford the intermediate BB22 1-(4-fluorobenzyl)imidazolidin-2-one (350 mg) as a yellow solid.

[0286] LC_MS:(ES + ):m / z 195.1[M+H] + .

[0287] Synthesis route of intermediate BB23

[0288] Step 1: Intermediate BB23-B (5-bromo-2-iodophenyl)methane-d-ol

[0289] To a solution of intermediate BB23-A (5-bromo-2-iodobenzaldehyde) (1.0 g, 3.216 mmol) in tetrahydrofuran (15 mL) was added NaBD4 (134.6 mg, 3.216 mmol) at 0°C. The reaction was stirred at 25°C for one hour. TLC confirmed the reaction was complete. The reaction was quenched with water (20 mL) and extracted with ethyl acetate (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: 20% ethyl acetate in petroleum ether) to afford intermediate BB23-B (5-bromo-2-iodophenyl)methane-d-ol) (940 mg) as a white solid.

[0290] 1 H NMR (400MHz, CDCl3) δ7.67(d,J=8.3Hz,1H),7.65(dd,J=2.4,0.5Hz,1H),7.18-7.14(m,1H),4.64(s,1H),2.03(s,1H).

[0291] Step 2: Intermediate BB23-D (5-bromo-2-(3-fluoropyridin-4-yl)phenyl)methane-d-ol

[0292] The synthesis of this step refers to the synthesis process of the first step of intermediate BB1.

[0293] LC_MS:(ES + ):m / z 282.9[M+H] + .

[0294] Step 3: Intermediate BB23 8-bromo-6H-isochromeno[3,4-c]pyridine-6-d

[0295] The synthesis of this step refers to the synthesis process of the second step of intermediate BB1.

[0296] LC_MS:(ES + ):m / z 262.9[M+H] + .

[0297] Synthesis route of intermediate BB24

[0298] Step 1: Intermediate BB24-B (5-bromo-2-iodophenyl)methane-d2-ol

[0299] To a solution of intermediate BB24-A (5-bromo-2-iodobenzoic acid) (3 g, 9.18 mmol) in tetrahydrofuran (30 mL) was slowly added LiAlD4 (423.9 mg, 10.08 mmol) at 0°C. The reaction was stirred at 0°C for 3 hours. TLC confirmed the reaction was complete. The reaction was quenched by the addition of water (0.424 mL) and 10% sodium hydroxide solution (0.424 mL) at 0°C, filtered, and the filtrate concentrated. The crude product was isolated and purified by silica gel column chromatography (eluent: 0%-20% ethyl acetate in petroleum ether) to afford intermediate BB24-B (5-bromo-2-iodophenyl)methane-d2-ol) as a white solid (1.3 g).

[0300] 1 H NMR (400MHz, CDCl3) δ7.68(d,J=8.3Hz,1H),7.65(d,J=2.4Hz,1H),7.16(dd,J=8.3,2.4Hz,1H),2.01(s,1H).

[0301] Step 2: Intermediate BB24-D (5-bromo-2-(3-fluoropyridin-4-yl)phenyl)methane-d2-ol

[0302] The synthesis of this step refers to the synthesis process of the first step of intermediate BB1.

[0303] LC_MS:(ES + ):m / z 284.0[M+H] + .

[0304] Step 3: Intermediate BB24 8-bromo-6H-isochromeno[3,4-c]pyridine-6,6-d2

[0305] The synthesis of this step refers to the synthesis process of the second step of intermediate BB1.

[0306] LC_MS:(ES + ):m / z 263.9[M+H] + .

[0307] Synthesis route of intermediate BB25

[0308] Step 1: Intermediate BB25-B 1-(5-bromo-2-iodophenyl)ethanol-1-ol

[0309] To a solution of intermediate BB25-A (5-bromo-2-iodobenzaldehyde) (1 g, 3.21 mmol) in tetrahydrofuran (10 mL) was slowly added methylmagnesium bromide (1.07 mL, 3.21 mmol) at 0°C. The reaction was stirred at 0°C for 1 hour. TLC confirmed the reaction was complete. The reaction was quenched by addition of saturated ammonium chloride (15 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: 0%-20% ethyl acetate in petroleum ether) to afford intermediate BB25-B (1-(5-bromo-2-iodophenyl)ethanol-1-ol) as a yellow gum (960 mg).

[0310] Step 2: Intermediate BB25-D 1-(5-bromo-2-(3-fluoropyridin-4-yl)phenyl)ethanol-1-ol

[0311] The synthesis of this step refers to the synthesis process of the first step of intermediate BB1.

[0312] LC_MS:(ES + ):m / z 296.0[M+H] + .

[0313] Step 3: Intermediate BB25 8-bromo-6-methyl-6H-isochromeno[3,4-c]pyridine

[0314] The synthesis of this step refers to the synthesis process of the second step of intermediate BB1.

[0315] LC_MS:(ES + ):m / z 275.9[M+H] + .

[0316] Synthesis route of intermediate BB26

[0317] Step 1: Intermediate BB26-B (3-(((tert-Butyloxycarbonyl)amino)methyl)pyridin-4-yl)boronic acid

[0318] 3 g (14.4 mmol, 1.0 eq) of intermediate BB26-A was placed in a 100 mL three-necked flask. 15 mL of dry THF was added, the mixture was replaced with a nitrogen balloon, and the temperature was lowered to -60°C. 23 mL (30.3 mmol, 2.1 eq, 1.3 M in pentane) of tert-butyl lithium was slowly added dropwise to the flask at -60°C. After the addition, the temperature was slowly raised to -20°C, and while maintaining this temperature, 6.0 g of trimethyl borate (57.6 mmol, 4.0 eq) was slowly added dropwise. After the addition, the mixture was allowed to warm to room temperature and stirred overnight. LCMS monitoring indicated approximately 25% product. 1N dilute hydrochloric acid was slowly added to the reaction mixture until the pH reached 6. The low-boiling solvent was removed by concentration, and the residue was diluted to 30 mL with water and extracted twice with a 3:1 dichloromethane / isopropanol solution (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 3.1 g of crude intermediate BB26-B as a yellow oil, which was used directly in the next reaction.

[0319] LC_MS:(ES + ):m / z 253.00[M+H] + .

[0320] Step 2: Intermediate BB26-D (tert-butyl 4-(4-bromo-2-formylphenyl)pyridin-3-yl)methyl)carbamate

[0321] The crude intermediate BB26-B obtained in the previous step was placed in a 250 mL single-necked flask, along with 30 mL of 1,4-dioxane and 15 mL of water. Intermediate BB26-C, 5-bromo-2-iodobenzaldehyde (1.9 g, 6.13 mmol, 0.5 eq), sodium carbonate (1.3 g, 12.25 mmol, 2.0 eq), and tetrakis(triphenylphosphine)palladium (700 mg, 0.61 mmol, 0.1 eq) were then weighed in sequence. The mixture was replaced with a nitrogen balloon three times and allowed to react overnight at 90°C. LCMS confirmed the complete consumption of starting material 2. The reaction mixture was poured into 100 mL of water and extracted twice with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to yield 2 g of crude white viscous intermediate BB26-D, which was used directly in the next step.

[0322] LC_MS:(ES + ):m / z 390.75[M+H] + .

[0323] Step 3: Intermediate BB26-E 9-bromo-5H-benzo[c]pyrido[4,3-e]azepane

[0324] Dissolve the crude intermediate BB26-D in dichloromethane (20 mL), add trifluoroacetic acid (10 mL), and stir at room temperature for 1 hour. TLC monitoring indicates complete consumption of the starting material. Pour the reaction mixture into water (50 mL), adjust the pH to >7 with solid sodium bicarbonate, and extract with dichloromethane (20 mL). The organic phase is collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product is purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 1 / 1-1 / 2) to obtain 170 mg of the intermediate BB26-E (9-bromo-5H-benzo[c]pyrido[4,3-e]azepane) as a light yellow solid.

[0325] LC_MS:(ES + ):m / z 272.75[M+H] + .

[0326] Step 4: Intermediate BB26-F 9-bromo-6,7-dihydro-5H-benzo[c]pyrido[4,3-e]azepane

[0327] Intermediate BB26-E 9-bromo-5H-benzo[c]pyrido[4,3-e]azepane (200 mg, 0.73 mmol, 1.0 eq, 85% pure crude product) was dissolved in methanol (2 mL) and cooled to 0°C. Sodium borohydride (28 mg, 0.73 mmol, 1.0 eq) was then added in three portions. After addition, the mixture was warmed to room temperature and stirred for 1 hour. TLC monitoring indicated that intermediate BB26-E was completely consumed. The reaction mixture was then concentrated and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30 / 1 to 10 / 1) to obtain 220 mg of intermediate BB26-F 9-bromo-6,7-dihydro-5H-benzo[c]pyrido[4,3-e]azepane as a light yellow viscous product.

[0328] LC_MS:(ES + ):m / z 274.70[M+H] + .

[0329] Step 5: Intermediate BB26 9-bromo-6-methyl-6,7-dihydro-5H-benzo[c]pyrido[4,3-e]azepane

[0330] Dissolve the intermediate BB26-F (9-bromo-6,7-dihydro-5H-benzo[c]pyrido[4,3-e]azepane) (228 mg, 0.83 mmol, 1.0 eq) in 5 mL of acetonitrile. Then add paraformaldehyde (199 mg, 6.63 mmol, 8.0 eq), triethylsilane (293 mg, 2.57 mmol, 3.1 eq), and trifluoroacetic acid (288 mg, 2.49 mmol, 3.0 eq) in this order. Stir overnight at room temperature. TLC monitoring indicates that the starting material BB26-F has been consumed. Pour the reaction mixture into saturated sodium bicarbonate solution (50 mL) and extract with ethyl acetate (50 mL). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1-20 / 1) to obtain 210 mg of colorless viscous intermediate BB26 9-bromo-6-methyl-6,7-dihydro-5H-benzo[c]pyrido[4,3-e]azepane.

[0331] LC_MS:(ES + ):m / z 288.85[M+H] + .

[0332] 1 H NMR (400MHz, CDCl3): δ8.70 (d, J = 5.2Hz, 1H), 8.60 (s, 1H), 7.65-7.62 (m, 1H), 7.56(d,J=1.6Hz,1H),7.41-7.38(m,2H),3.39(d,J=10.8Hz,4H),2.50(s,3H).

[0333] Synthesis route of intermediate BB27

[0334] Step 1: Intermediate BB27-B ((2-bromo-5-nitrobenzyl)oxy)(tert-butyl)dimethylsilane

[0335] Intermediate BB27-A (1.0 g, 4.31 mmol, 1.0 eq) was dissolved in 10 mL of tetrahydrofuran, and TBSCl (780 mg, 5.17 mmol, 1.2 eq) and imidazole (734 mg, 10.78 mmol, 2.5 eq) were added. The mixture was stirred at room temperature overnight. TLC indicated a small amount of BB27-A remaining. The reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The concentrated crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 50 / 1) to obtain 1.39 g of intermediate BB27-B ((2-bromo-5-nitrobenzyl)oxy)(tert-butyl)dimethylsilane) as a colorless oily liquid in a 93% yield.

[0336] 1 H NMR (400MHz, CDCl3): δ8.26(d,J=2.8Hz,1H),7.83-7.80(m,1H),7.50(d,J=8.8Hz,1H),4.59(s,2H),0.83(s,9H),0.00(s,6H).

[0337] Step 2: Intermediate BB27-C tert-Butyldimethyl((5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)silane

[0338] Intermediate BB27-B ((2-bromo-5-nitrobenzyl)oxy)(tert-butyl)dimethylsilane (790 mg, 2.28 mmol, 1.0 eq) was dissolved in 8 mL of 1,4-dioxane. Diboronic acid pinacol ester (693 mg, 2.74 mmol, 1.2 eq), potassium acetate (447 mg, 4.56 mmol, 2.0 eq), and Pd(dppf)Cl2 (83 mg, 0.11 mmol, 0.05 eq) were then added sequentially. The mixture was placed in a reflux line, flushed with nitrogen three times, and reacted at 100°C for 3 hours. TLC monitoring indicated that BB27-B was completely consumed. The reaction solution was directly filtered to remove insoluble matter, and the filtrate was concentrated to obtain 1.8 g of black oily crude intermediate BB27-C tert-butyldimethyl((5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)silane, which was directly used in the next reaction.

[0339] Step 3: Intermediate BB27-E 4-(2-(((tert-Butyldimethylsilyl)oxy)methyl)-4-nitrophenyl)-3,5-dichloropyridine

[0340] The crude intermediate BB27-C tert-butyldimethyl((5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)silane (897 mg, 2.28 mmol, 1.0 eq), intermediate BB27-D 3,5-dichloro-4-iodopyridine (624 mg, 2.28 mmol, 1.0 eq) and sodium carbonate (725 mg, 6.84 mmol, 3.0 eq) were added to a mixture of 1,4-dioxane (12 mL) and water (2 mL). Pd(dppf)Cl2·DCM (186 mg, 0.23 mmol, 0.1 eq) was then added. The mixture was replaced with a nitrogen balloon three times and stirred at 70°C overnight. The reaction was complete after LCMS monitoring. The reaction solution was poured into water (50 mL) and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated crude product was separated and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 50 / 1-40 / 1) to obtain 258 mg of white solid intermediate BB27-E 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-nitrophenyl)-3,5-dichloropyridine.

[0341] 1 H NMR (400MHz, CDCl3): δ8.63 (brs, 2H), 8.53 (d, J = 2.4Hz, 1H), 8.25-8.22 (m, 1H), 7.22 (s, 1H), 4.45 (s, 2H), 0.89 (s, 9H), 0.00 (s, 6H).

[0342] Step 4: Intermediate BB27-F (2-(3,5-dichloropyridin-4-yl)-5-nitrophenyl)methanol

[0343] The intermediate BB27-E, 4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-nitrophenyl)-3,5-dichloropyridine (1.6 g, 3.87 mmol, 1.0 eq), was dissolved in 15 mL of tetrahydrofuran. TBAF (11.6 ml, 11.61 mmol, 3.0 eq, 1 M in THF) was added and stirred at room temperature for 1 hour. TLC monitoring indicated that the starting material BB27-E had been consumed. The reaction mixture was poured into saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (30 mL x 2). The product was dried over anhydrous sodium sulfate, and the concentrated crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1 to 2 / 1) to obtain 591 mg of the intermediate BB27-F (2-(3,5-dichloropyridin-4-yl)-5-nitrophenyl)methanol as a white solid in a 51% yield.

[0344] LC_MS:(ES+ ):m / z 298.65[M+H] + .

[0345] Step 5: Intermediate BB27-G 1-chloro-8-nitro-6H-isochromeno[3,4-c]pyridine

[0346] Intermediate BB27-F (2-(3,5-dichloropyridin-4-yl)-5-nitrophenyl)methanol (240 mg, 0.80 mmol, 1.0 eq) was dissolved in THF (5 mL) and cooled in an ice-water bath for 10 minutes. Sodium hydride (48 mg, 1.2 mmol, 1.5 eq) was quickly added. After nitrogen substitution, the mixture was stirred at room temperature for 1 hour. TLC analysis indicated that approximately half of the starting material remained. The reaction mixture was carefully poured into water (50 mL) and extracted with ethyl acetate (50 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1 to 2 / 1) to obtain 60 mg of intermediate BB27-G (1-chloro-8-nitro-6H-isochromeno[3,4-c]pyridine) as a yellow viscous oil in a 22% yield.

[0347] LC_MS:(ES + ):m / z 262.65[M+H] + .

[0348] Step 6: Intermediate BB27 1-chloro-6H-isochromeno[3,4-c]pyridin-8-amine

[0349] To a solution of the intermediate BB27-G, 1-chloro-8-nitro-6H-isochromeno[3,4-c]pyridine (20 mg, 76.15 μmol) in ethanol (2 mL) / H₂O (0.4 mL), iron powder (21.26 mg, 380.74 μmol) and ammonium chloride (20.37 mg, 380.74 μmol) were added. The reaction mixture was stirred at 80°C for 2 hours. LCMS monitored the reaction for completion. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethanol (2 mL x 2). The filtrate was concentrated. Water (2 mL) and dichloromethane (2 mL x 2) were added for extraction. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated and purified by preparative thin-layer chromatography (developing solvent: dichloromethane: methanol = 20:1) to obtain the intermediate BB27 1-chloro-6H-isochromeno[3,4-c]pyridin-8-amine (5 mg).

[0350] LC_MS:(ES + ):m / z 233.04[M+H] + .

[0351] Synthesis route of intermediate BB28

[0352] Step 1: Intermediate BB28-B 8-nitro-1-(prop-1-en-2-yl)-6H-isochromeno[3,4-c]pyridine

[0353] Intermediate BB27-G (1-chloro-8-nitro-6H-isochromeno[3,4-c]pyridine) (35 mg, 0.133 mmol, 1.0 eq), isopropenylboronic acid pinacol ester (33.5 mg, 0.200 mmol, 1.5 eq), and potassium phosphate (56 mg, 0.266 mmol, 2.0 eq) were added to a mixture of 1,4-dioxane (1 mL) and water (0.2 mL). Palladium acetate (3 mg, 0.013 mmol, 0.1 eq) and X-phos (13 mg, 0.027 mmol, 0.2 eq) were then added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 100°C for 2 hours. LCMS monitoring indicated that intermediate BB27-G had been completely consumed. The reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL), washed with saturated brine, and dried over anhydrous sodium sulfate. The concentrated crude product was separated and purified by preparative thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate / methanol = 25 / 8 / 1) to obtain 8 mg of white solid intermediate BB28-B 8-nitro-1-(prop-1-en-2-yl)-6H-isochromeno[3,4-c]pyridine, in a yield of 22%.

[0354] LC_MS:(ES + ):m / z 269.10[M+H] + .

[0355] Step 2: Intermediate BB28 1-isopropyl-8-nitro-6H-isochromeno[3,4-c]pyridine

[0356] Intermediate BB28-B 8-nitro-1-(prop-1-en-2-yl)-6H-isochromeno[3,4-c]pyridine (8 mg) was dissolved in 3 mL of methanol. 2 mg of palladium / carbon was carefully added, and the mixture was replaced with a hydrogen balloon three times. The mixture was stirred at room temperature for 24 hours. LCMS indicated that 30% of the intermediate remained. The mixture was heated to 45°C and stirred for 8 hours. LCMS indicated that the reaction was complete. The palladium on carbon was directly removed by filtration, and the filtrate was concentrated and purified by preparative thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate = 3 / 2, dichloromethane / methanol = 30 / 1) to obtain 3 mg of the off-white solid intermediate BB28 1-isopropyl-6H-isochromeno[3,4-c]pyridine in a 30% yield.

[0357] LC_MS:(ES + ):m / z 241.15[M+H] + .

[0358] 1 H NMR (400MHz, CD3OD): δ8.18(s,1H),7.99(s,1H),7.48(d,J=8.4Hz,1H),6.76-6.73( m,1H),6.61(d,J=2.4Hz,1H),4.86(s,2H),3.71-3.64(m,1H),1.39(d,J=6.8Hz,6H).

[0359] Example 1: Synthesis route of compound 1

[0360] Step 1: Compound 42 3-(4-chlorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one

[0361] Under nitrogen, to a solution of 1,4-dioxane (3 mL) containing intermediate BB1 ​​(8-bromo-6H-isochromeno[3,4-c]pyridine) (40 mg, 0.153 mmol), intermediate BB4 (3-(4-chlorobenzyl)pyrrolidin-2-one) (35.2 mg, 0.168 mmol), Xant-Phos (9.72 mg, 0.0168 mmol), and cesium carbonate (149.5 mg, 0.459 mmol) was added Pd2(dba)3-CHCl3 (7.82 mg, 0.008 mmol). The reaction system was purged with nitrogen three times and heated to 100°C for 2 hours. TLC monitored the reaction for completion. The reaction solution was extracted with ethyl acetate (5 mL x 2) and water (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified by preparative thin-layer chromatography (eluent: 4.76% methanol in dichloromethane) to give Compound 1 3-(4-chlorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one (38 mg) as a white solid.

[0362] LC_MS:(ES + ):m / z 391.1[M+H] + .

[0363] 1H NMR (400MHz, CDCl3) δ8.35(s,1H),8.30(d,J=4.4Hz,1H),7.75(d,J=8.6Hz,1H),7.70(d,J=2 .0Hz,1H),7.61-7.55(m,2H),7.33-7.29(m,2H),7.20(d,J=8.4Hz,1H),5.23(s,2H),3.83-3 .75(m,1H),3.68(td,J=9.1,3.1Hz,1H),3.27(dd,J=13.7,4.2Hz,1H),2.96(ddd,J=17.8,8. 9,4.2Hz,1H),2.84(dd,J=13.7,8.9Hz,1H),2.32-2.20(m,1H),1.90(dt,J=8.8,6.9Hz,1H).

[0364] Example 2: Synthesis route of compound 2

[0365] Step 1: Compound 42 3-benzyl-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one

[0366] Intermediate BB2 1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one (50 mg, 0.188 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). The system was cooled to -78°C under nitrogen protection. LDA (2M in THF, 0.141 ml) was slowly added dropwise. The reaction was stirred for half an hour. Then, a solution of compound 2-A benzyl bromide (32.1 mg, 0.188 mmol) in THF (5 mL) was injected along the wall of the reaction vessel with a syringe. The reaction was continued at -78°C for 1 hour. The reaction was completed by TLC monitoring, and the product was quenched with saturated ammonium chloride solution (10 mL). The product was extracted with ethyl acetate (10 mL x 2). The organic layers were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated and purified by Pre-TLC (eluted with dichloromethane solution containing 5% methanol) to give white solid compound 2 3-benzyl-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one (18 mg).

[0367] LC_MS:(ES + ):m / z 357.50[M+H] +

[0368] 1H NMR (400MHz, CDCl3) δ8.33 (s, 1H), 8.28 (d, J = 5.1Hz, 1H), 7.78-7.64 (m, 2H), 7.59-7.4 6(m,2H),7.34-7.21(m,5H),5.2(s,2H),3.88-3.69(m,1H),3.64(td,J1=9.0Hz,J2=3.4 Hz,1H),3.31(dd,J1=13.7Hz,J2=4.2Hz,1H),2.96(qd,J1=8.9Hz,J2=4.1Hz,1H),2.82( dd, J1=13.7Hz, J2=9.2Hz, 1H), 2.26-2.18(m, 1H), 1.90 (dq, J1=12.8Hz, J2=8.6Hz, 1H).

[0369] Example 3: Synthesis route of compound 42

[0370] Step 1: Compound 42 3-(4-chlorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)propionamide

[0371] HATU (69.1 mg, 0.182 mmol) was added to a solution of intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine (30 mg, 0.151 mmol), compound 42-A (33.5 mg, 0.182 mmol), and N,N-diisopropylethylamine (58.7 mg, 0.454 mmol) in DMF (1 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction, the mixture was diluted with water (5 mL) and extracted with ethyl acetate (5 mL x 2). The combined organic layers were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was isolated and purified by Pre-TLC (eluting with 5% methanol in dichloromethane) to afford compound 42 3-(4-chlorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)propanamide (46 mg) as a white solid.

[0372] LC_MS:(ES + ):m / z 365.40[M+H] + .

[0373] 1H NMR (400MHz, DMSO) δ10.18(s,1H),8.27-8.20(m,2H),7.89(d,J=8.5Hz,1H),7.76(d,J=5.0Hz,1H),7.66-7.5 3(m,2H),7.37-7.33(m,2H),7.29(d,J=8.5Hz,2H),5.21(s,2H),2.92(t,J=7.5Hz,2H),2.66(t,J=7.6Hz,2H).

[0374] Example 4: Synthesis route of compound 10

[0375] Step 1: Compound 10 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one

[0376] The synthesis of this step uses the intermediate BB1 ​​8-bromo-6H-isochromeno[3,4-c]pyridine and the intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one as raw materials, and refers to the synthesis process of the first step in Example 1.

[0377] LC_MS:(ES + ):m / z 375.2[M+H] + .

[0378] 1 H NMR(400MHz,DMSO-d6)δ8.33-8.21(m,2H),7.97(d,J=8.6Hz,1H),7.86-7.77(m,2H),7 .67(d,J=2.2Hz,1H),7.37-7.26(m,2H),7.18-7.07(m,2H),5.25(s,2H),3.74(dtd,J=1 8.3,9.5,6.9Hz,2H),3.12(dd,J=13.6,4.4Hz,1H),2.97(qd,J=9.2,4.4Hz,1H),2.74(d d,J=13.7,9.3Hz,1H),2.08(ddt,J=9.8,7.3,5.2Hz,1H),1.78(dq,J=12.1,8.8Hz,1H).

[0379] Example 5: Synthesis route of compound 45

[0380] Step 1: Compound 45 1-(4-fluorobenzyl)-3-(6H-isochromeno[3,4-c]pyridin-8-yl)urea

[0381] To a solution of intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine (30 mg, 0.151 mmol) in DMF (0.5 mL) were added CDI (24.5 mg, 0.151 mmol) and DIPEA (58.5 mg, 0.453 mmol). The reaction was stirred at room temperature for 30 minutes, followed by the addition of compound 45-A (4-fluorophenyl)methanamine (18.9 mg, 0.151 mmol), and the reaction was stirred at room temperature for 16 hours. After completion of the reaction, ice water was added dropwise to dilute the solution. The precipitated solid was filtered and washed with water. The residue was dissolved in DCM / MeOH (10:1) (2 mL) and isolated and purified by preparative thin-layer chromatography (eluting with 5% methanol in dichloromethane) to afford compound 45 1-(4-fluorobenzyl)-3-(6H-isochromeno[3,4-c]pyridin-8-yl)urea (18 mg).

[0382] LC_MS:(ES + ):m / z 350.1[M+H] + .

[0383] 1 H NMR(400MHz,Chloroform-d)δ8.32(s,1H),8.27(d,J=5.1Hz,1H),7.66(d,J=8.4Hz,1H),7.50(s,1H),7.43(d,J=2.2Hz,1H),7.33(dd,J=8.5,5 .4Hz,2H),7.24(dd,J=8.5,2.3Hz,1H),7.06(t,J=8.6Hz,2H),6.57(s,1H),5.17(s,2H),5.08(s,1H),4.46(d,J=5.7Hz,2H).MS:[M+H]+:350.1.

[0384] Example 6: Synthesis route of compound 49

[0385] Step 1: Compound 49 3-(4-fluorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)-N-methylpropionamide

[0386] To a solution of compound 29 (3-(4-fluorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)propionamide (20 mg, 0.0574 mmol) in DMF (1 mL) was added NaH (2.76 mg, 0.0689 mmol, 60% purity) under ice. After stirring for half an hour, iodomethane (9.8 mg, 0.0689 mmol) was added, and the mixture was allowed to warm to room temperature and stirred for 12 hours. LCMS monitoring confirmed the reaction was complete. The reaction solution was poured into saturated ammonium chloride (5 mL) and extracted with ethyl acetate (5 mL x 2). The organic layers were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated and purified by Pre-TLC (eluted with a dichloromethane solution containing 5% methanol) to give Compound 49 3-(4-fluorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)-N-methylpropanamide (15 mg) as a white solid.

[0387] LC_MS:(ES + ):m / z 363.12[M+H] + .

[0388] Example 7: Synthesis route of compound 52

[0389] Step 1: Compound 52-B (tert-butyl 1-((6H-isochromeno[3,4-c]pyridin-8-yl)amino)-3-(4-fluorophenyl)-1-oxopropan-2-yl)carbamate

[0390] This step of the synthesis uses compound 52-A 2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)propanoic acid and intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine as raw materials, and refers to the synthesis process of the first step in Example 3.

[0391] LC_MS:(ES + ):m / z 464.2[M+H] + .

[0392] Step 2: Compound 52 2-amino-3-(4-fluorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)propanamide

[0393] A solution of Compound 52-B (tert-butyl 1-((6H-isochromeno[3,4-c]pyridin-8-yl)amino)-3-(4-fluorophenyl)-1-oxopropan-2-yl)carbamate (25 mg, 0.0539 mmol) in HCl / dioxane (1 mL, 4 M) was stirred at room temperature for 1 hour. LCMS monitored the reaction for completion. The solution was then concentrated under reduced pressure to afford the hydrochloride salt of Compound 52-B (2-amino-3-(4-fluorophenyl)-N-(6H-isochromeno[3,4-c]pyridin-8-yl)propanamide (18 mg) as a white solid.

[0394] LC_MS:(ES + ):m / z 364.2[M+H] + .

[0395] 1 H NMR (400MHz, MeOH-d4): δ8.52(d,J=0.8Hz,1H),8.49-8.37(m,2H),8.12(d,J=8.5Hz,1H),7.83-7.69(m,2H ),7.47-7.33(m,2H),7.25-7.05(m,2H),5.49(s,2H),4.32(t,J=7.3Hz,1H),3.48-3.34(m,1H),3.20(dd,J 1=14.0Hz, J2=7.9Hz, 1H).

[0396] Example 8: Synthesis route of compound 53

[0397] Step 1: Compound 53-B (4-bromopyridin-3-yl)methanol

[0398] To a solution of Compound 53-A (4-bromonicotinic acid) (4.8 g, 23.761 mmol) and N-methylmorpholine (2.90 g, 28.513 mmol) in THF (50 mL) was added isobutyl chloroformate (3.24 g, 23.761 mmol) under ice. After stirring for half an hour, the resulting solid was filtered off, the mother liquor was dispersed in methanol (20 mL), and NaBH4 (1.35 g, 35.642 mmol) was added. The reaction was stirred at room temperature for 1 hour. LCMS monitored the reaction for completion. The reaction solution was concentrated under reduced pressure, water (30 mL) was added, and extraction was performed with ethyl acetate (30 mL x 2). The organic layers were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluted with petroleum ether containing 25% ethyl acetate) to give compound 53-B (4-bromopyridin-3-yl)methanol (860 mg) as a yellow solid.

[0399] LC_MS:(ES + ):m / z 188.02[M+H] + .

[0400] Step 2: Compound 53-C 4-bromo-3-(((tert-butyldimethylsilyl)oxy)methyl)pyridine

[0401] To a solution of compound 53-B (4-bromopyridin-3-yl)methanol (860 mg, 4.574 mmol) and imidazole (934.2 mg, 13.722 mmol) in DCM (20 mL) was added TBSCl (1.378 g, 9.148 mmol). The reaction was stirred at room temperature for 12 hours. TLC confirmed the completion of the reaction. The reaction solution was added with water (20 mL) and extracted with DCM (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was isolated and purified by silica gel column chromatography (eluting with petroleum ether containing 2-4% ethyl acetate) to afford compound 53-C (4-bromo-3-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidone) as a colorless oil (1.39 g).

[0402] LC_MS:(ES + ):m / z 302.03[M+H] + .

[0403] Step 3: Compound 53-D 3-(((tert-butyldimethylsilyl)oxy)methyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

[0404] To a solution of compound 53-C (4-bromo-3-(((tert-butyldimethylsilyl)oxy)methyl)pyridine (1.29 g, 4.267 mmol) and pinacol diboron (2.17 g, 8.535 mmol) in 1,4-dioxane (20 mL) was added PdCl(dppf)CHCl (348.5 mg, 0.4267 mmol) and potassium acetate (1.26 g, 12.802 mmol). The reaction was stirred at 100°C under a nitrogen atmosphere for 2 hours. LCMS confirmed the reaction was complete. The reaction solution was filtered through celite and concentrated under reduced pressure to give compound 53-D 3-(((tert-butyldimethylsilyl)oxy)methyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.491 g) as a yellow oil. The crude product was used directly in the next reaction without further purification.

[0405] LC_MS:(ES+ ):m / z 268.12[M-82+H] + .

[0406] Step 4: Compound 53-F (5-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)phenyl)methanol

[0407] To a solution of compound 53-D (3-((tert-butyldimethylsilyl)oxy)methyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.491 g, 4.267 mmol) and compound 53-E (5-bromo-2-iodophenyl)methanol (1.335 g, 4.267 mmol) in 1,4-dioxane (20 mL) and water (2 mL) was added PdCl(dppf)CHCl (348.5 mg, 0.4267 mmol) and sodium carbonate (1.13 g, 10.668 mmol). The reaction was stirred at 80°C under a nitrogen atmosphere for 12 hours. LCMS monitored the reaction for completion. The reaction solution was filtered through celite and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluted with petroleum ether containing 50% ethyl acetate) to give a crude yellow oily compound 53-F (5-bromo-2-(3-((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)phenyl)methanol (500 mg).

[0408] LC_MS:(ES + ):m / z 408.10[M+H] + .

[0409] Step 5: Compound 53-G (5-bromo-2-(3-(hydroxymethyl)pyridin-4-yl)phenyl)methanol

[0410] A solution of compound 53-F (5-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)phenyl)methanol (450 mg, 1.1 mmol) in trifluoroacetic acid (10 mL) was stirred at room temperature for 2 hours. LCMS monitored the reaction completion. The reaction solution was concentrated under reduced pressure to remove trifluoroacetic acid, and saturated NHCO3 solution (20 mL) was added and extracted with ethyl acetate (15 mL x 2). The organic layers were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluted with a dichloromethane solution containing 5% methanol) and Prep-HPLC to obtain compound 53-G (5-bromo-2-(3-(hydroxymethyl)pyridin-4-yl)phenyl)methanol (60 mg) as a yellow oil.

[0411] LC_MS:(ES + ):m / z 294.00[M+H] + .

[0412] Step 6: Compound 53-H 9-bromo-5,7-dihydrobenzo[5,6]oxabino[3,4-c]pyridine

[0413] To a solution of compound 53-G (5-bromo-2-(3-(hydroxymethyl)pyridin-4-yl)phenyl)methanol (50 mg, 169.98 μmol) in acetonitrile (2.5 mL) was added 48% hydrobromic acid (0.1 mL). The reaction mixture was stirred at 80°C for 1 hour. LCMS confirmed the reaction was complete. The reaction mixture was adjusted to pH 9 with saturated sodium bicarbonate solution and extracted with ethyl acetate (8 mL x 2). The combined organic layers were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was isolated and purified by Pre-TLC (eluting with 5% methanol in dichloromethane) to afford compound 53-H (9-bromo-5,7-dihydrobenzo[5,6]oxabino[3,4-c]pyridine) (5 mg) as a yellow oil.

[0414] LC_MS:(ES + ):m / z 276.10[M+H] + .

[0415] Step 7: Compound 53 1-(5,7-dihydrobenzo[5,6]oxirane[3,4-c]pyridin-9-yl)-3-(4-fluorobenzyl)pyrrolidin-2-one

[0416] The synthesis of this step uses compound 53-H 9-bromo-5,7-dihydrobenzo[5,6]oxabinol[3,4-c]pyridine and intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one as raw materials, and refers to the synthesis process of the first step in Example 1.

[0417] LC_MS:(ES + ):m / z 389.18[M+H] + .

[0418] Example 9: Synthesis route of compound 91

[0419] Step 1: Compound 91 3-(4-fluorophenyl)-N-(5H-pyrano[4,5-b:2,3-c']bipyridin-3-yl)propionamide

[0420] The synthesis of this step uses the intermediate BB13 5H-pyrano[4,5-b:2,3-c′]dipyridin-3-amine and compound 91-A 3-(4-fluorophenyl)propionic acid as raw materials, and refers to the synthesis process of the first step in Example 3.

[0421] LC_MS:(ES + ):m / z 350.1[M+H] + .

[0422] Example 10: Synthesis route of compound 112

[0423] Step 1: Compound 112 3-(4-fluorophenyl)-N-(5H-pyrano[2,3-c:4,5-c']bipyridin-3-yl)propionamide

[0424] The synthesis of this step uses the intermediate BB14 5H-pyrano[2,3-c:4,5-c′]dipyridin-3-amine and compound 112-A 3-(4-fluorophenyl)propionic acid as raw materials, and refers to the synthesis process of the first step in Example 3.

[0425] LC_MS:(ES + ):m / z 350.1[M+H] + .

[0426] Example 11: Synthesis route of compound 77

[0427] Step 1: Compound 77 1-(4-fluorobenzyl)-3-(6H-isochromeno[3,4-c]pyridin-8-yl)thiourea

[0428] To a solution of intermediate BB3 6H-isochromeno[3,4-c]pyridin-8-amine (30 mg, 151.34 μmol) and N,N-diisopropylethylamine (58.7 mg, 454.03 μmol) in DMF (1 mL) was added compound 77-A 4-fluorobenzylthiocyanate (25.3 mg, 151.34 μmol). The reaction mixture was heated to 100°C and stirred for 12 hours. LCMS monitored the reaction for completion. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (8 mL x 2). The organic layers were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by Pre-TLC (eluted with dichloromethane containing 5% methanol) to give compound 771-(4-fluorobenzyl)-3-(6H-isochromeno[3,4-c]pyridin-8-yl)thiourea (19.3 mg) as a yellow solid.

[0429] LC_MS:(ES + ):m / z 366.9[M+H] + .

[0430] 1 H NMR (400MHz, DMSO) δ9.87(s,1H),8.40(s,1H),8.27(s,1H),8.24(d,J=5.0Hz,1H),7.91(d,J=8.4Hz,1H),7.79(d,J=5.1Hz,1H), 7.53(dd,J=8.4,1.7Hz,1H),7.48(s,1H),7.40(dd,J=8.4,5.7Hz,2H),7.18(t,J=8.8Hz,2H),5.22(s,2H),4.74(d,J=4.6Hz,2H).

[0431] Example 12: Synthesis route of compound 101

[0432] Step 1: Compound 101 1-(4-fluorobenzyl)-3-(5H-pyrano[4,5-b:2,3-c']bipyridin-3-yl)urea

[0433] The synthesis of this step uses the intermediate BB13 5H-pyrano[4,5-b:2,3-c']bipyridin-3-amine and compound 101-A (4-fluorophenyl)methylamine as raw materials, and refers to the synthesis process of the first step in Example 5.

[0434] LC_MS:(ES + ):m / z 351.1[M+H] + .

[0435] Example 13: Synthesis route of compound 122

[0436] Step 1: Compound 122 1-(4-fluorobenzyl)-3-(5H-pyrano[2,3-c:4,5-c']bipyridin-3-yl)urea

[0437] The synthesis of this step uses the intermediate BB14 5H-pyrano[2,3-c:4,5-c']bipyridin-3-amine and compound 122-A (4-fluorophenyl)methylamine as raw materials, and refers to the synthesis process of the first step in Example 5.

[0438] LC_MS:(ES + ):m / z 351.1[M+H] + .

[0439] Example 14: Synthesis route of compound 130

[0440] Step 1: Compound 130 3-(4-fluorophenyl)-N-(1-methyl-6H-isochromeno[3,4-c]pyridin-8-yl)propanamide

[0441] The synthesis of this step was carried out by using the intermediate BB17 1-methyl-6H-isochromeno[3,4-c]pyridin-8-amine and compound 130-A 3-(4-fluorophenyl)propionic acid as raw materials, and referring to the synthesis process of the first step in Example 3.

[0442] LC_MS:(ES + ):m / z 363.1[M+H] + .

[0443] Example 15: Synthesis route of compound 131

[0444] Step 1: Compound 131 3-(4-fluorophenyl)-N-(7-methyl-6H-isochromeno[3,4-c]pyridin-8-yl)propionamide

[0445] This step of the synthesis uses the intermediate BB18 7-methyl-6H-isochromeno[3,4-c]pyridin-8-amine and compound 131-A 3-(4-fluorophenyl)propionic acid as raw materials, and refers to the synthesis process of the first step in Example 3.

[0446] LC_MS:(ES + ):m / z 363.1[M+H] + .

[0447] Example 16: Synthesis route of compound 139

[0448] Step 1: Compound 139 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidin-2-one-3d

[0449] The synthesis of this step uses the intermediate BB1 ​​8-bromo-6H-isochromeno[3,4-c]pyridine and the intermediate BB20 3-(4-fluorobenzyl)pyrrolidin-2-one-3-d as raw materials, and refers to the synthesis process of the first step in Example 1.

[0450] LC_MS:(ES + ):m / z 376.2[M+H] + .

[0451] 1 H NMR (400MHz, CDCl3) δ8.35(s,1H),8.30(d,J=5.0Hz,1H),7.74(d,J=8.6Hz,1H),7.70(d,J=2.0Hz,1H),7.59-7.53 (m,2H),7.26-7.20(m,2H),7.06-6.99(m,2H),5.22(s,2H),3.77(dt,J=15.9,7.9Hz,1H),3.66(td,J=9.1,3.3Hz, 1H),3.27(dd,J=13.8,6.2Hz,1H),2.85(dd,J=13.8,5.5Hz,1H),2.28-2.19(m,1H),1.95-1.85(m,1H).

[0452] Example 17: Synthesis route of compound 142

[0453] Step 1: Compound 142 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl)azetidin-2-one

[0454] The synthesis of this step uses the intermediate BB1 ​​8-bromo-6H-isochromeno[3,4-c]pyridine and the intermediate BB21 3-[(4-fluorophenyl)methyl]azetidin-2-one as raw materials and refers to the synthesis process of the first step in Example 1.

[0455] LC_MS:(ES + ):m / z 361.1[M+H] + .

[0456] 1 H NMR (400MHz, CDCl3) δ8.34 (s, 1H), 8.29 (d, J = 5.0Hz, 1H), 7.71 (d, J = 8.3Hz, 1H),7.51(d,J=5.0Hz,1H),7.29-7.20(m,4H),7.03(t,J=8.6Hz,2H),5.18(s ,2H),3.77(t,J=5.7Hz,1H),3.67(dtd,J=8.4,5.6,2.8Hz,1H),3.42(dd,J= 5.8, 2.6Hz, 1H), 3.24 (dd, J=14.6, 5.6Hz, 1H), 3.05 (dd, J=14.6, 8.8Hz, 1H).

[0457] Example 18: Synthesis route of compound 145

[0458] Step 1: Compound 145 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl-6-d)pyrrolidin-2-one

[0459] The synthesis of this step uses the intermediate BB23 8-bromo-6H-isochromeno[3,4-c]pyridine-6-d and the intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one as raw materials and refers to the synthesis process of the first step in Example 1.

[0460] LC_MS:(ES + ):m / z 376.1[M+H] + .

[0461] 1 H NMR (400MHz, DMSO) δ8.28(s,1H),8.25(d,J=5.0Hz,1H),7.97(d,J=8.6Hz,1H),7 .80(dd,J=12.2,3.4Hz,2H),7.67(s,1H),7.32(dd,J=8.1,5.8Hz,2H),7.13(t,J= 8.8Hz,2H),5.22(s,1H),3.81-3.65(m,2H),3.12(dd,J=13.6,4.1Hz,1H),3.02- 2.91(m,1H),2.74(dd,J=13.5,9.4Hz,1H),2.15-2.02(m,1H),1.85-1.71(m,1H).

[0462] Example 19: Synthesis route of compound 146

[0463] Step 1: Compound 146 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl-6,6-d2)pyrrolidin-2-one

[0464] The synthesis of this step uses the intermediate BB24 8-bromo-6H-isochromeno[3,4-c]pyridine-6,6-d2 and the intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one as raw materials, and refers to the synthesis process of the first step in Example 1.

[0465] LC_MS:(ES + ):m / z 377.1[M+H] + .

[0466] 1H NMR (400MHz, CDCl3) δ8.35 (s, 1H), 8.29 (d, J = 5.0Hz, 1H), 7.74 (d, J = 8.5Hz, 1H), 7.69 (d, J = 2. 0Hz,1H),7.61-7.52(m,2H),7.22(dd,J=8.4,5.5Hz,2H),7.01(t,J=8.7Hz,2H),3.76(dd,J=17 .0,8.3Hz,1H),3.66(td,J=9.1,3.2Hz,1H),3.27(dd,J=13.7,4.1Hz,1H),3.00-2.91(m,1H),2 .85(dd,J=13.7,8.8Hz,1H),2.24(ddd,J=11.6,8.4,3.9Hz,1H),1.90(dq,J=12.8,8.6Hz,1H).

[0467] Example 20: Synthesis of Compound 147

[0468] Step 1: Compound 147 3-(4-fluorobenzyl)-1-(6-methyl-6H-isochromeno[3,4-c]pyridin-8-yl)pyrrolidine

[0469] -2-Keto

[0470] The synthesis of this step uses the intermediate BB25 8-bromo-6-methyl-6H-isochromeno[3,4-c]pyridine and the intermediate BB5 3-(4-fluorobenzyl)pyrrolidin-2-one as raw materials, and refers to the synthesis process of the first step in Example 1.

[0471] LC_MS:(ES + ):m / z 389.1[M+H] + .

[0472] 1H NMR (400MHz, DMSO) δ8.26(s,1H),8.24(d,J=5.0Hz,1H),7.99(d,J=8.7Hz,1H),7.83(d,J=5.1Hz,1H),7 .79(dd,J=8.6,1.8Hz,1H),7.66(dd,J=3.8,2.2Hz,1H),7.36-7.29(m,2H),7.13(t,J=8.7Hz,2H),5.49 (q,J=6.5Hz,1H),3.85-3.69(m,2H),3.14(ddd,J=13.7,10.2,5.2Hz,1H),3.04-2.91(m,1H),2.73(ddd ,J=13.6,9.4,4.0Hz,1H),2.08(dt,J=9.0,6.4Hz,1H),1.85-1.72(m,1H),1.51(dd,J=6.5,3.5Hz,3H).

[0473] Example 21: Synthesis route of compound 143

[0474] Step 1: Compound 143 3-(4-fluorobenzyl)-1-(6H-isochromeno[3,4-c]pyridin-8-yl-6,6-d2)pyrrolidin-2-one-3d

[0475] The synthesis of this step uses intermediate BB24 8-bromo-6H-isochromeno[3,4-c]pyridine-6,6-d2 and intermediate BB20 3-(4-fluorobenzyl)pyrrolidin-2-one-3-d as raw materials, and refers to the synthesis process of the first step in Example 1.

[0476] LC_MS:(ES + ):m / z 378.1[M+H] + .

[0477] 1H NMR (400MHz, CDCl3) δ8.35(s,1H),8.29(d,J=5.0Hz,1H),7.74(d,J=8.6Hz,1H),7.69( d,J=2.2Hz,1H),7.59-7.52(m,2H),7.22(dd,J=8.5,5.5Hz,2H),7.01(t,J=8.7Hz,2H), 3.76(dd,J=16.7,8.6Hz,1H),3.66(td,J=9.3,3.3Hz,1H),3.27(dd,J=13.8,6.2Hz,1H ),2.86(dd,J=13.8,5.5Hz,1H),2.24(ddd,J=10.8,8.6,5.1Hz,1H),1.95-1.83(m,1H).

[0478] Example 22: Preparation of Compound 154 and Compound 155

[0479] Compound 10 (50 mg) was dissolved in methanol and separated by SFC to afford compounds 154 (21.2 mg) and 155 (22.3 mg). The separation method was as follows: chiral AD-H column, pressure: 5 MPa, mobile phase: 35% isopropanol / n-hexane, detection wavelength: 280 nm, flow rate: 2 mL / min, column temperature: 25°C.

[0480] Compound 156 and Compound 157, Compound 158 and Compound 159, Compound 160 and Compound 161 can also be obtained by the above-mentioned SFC preparation and separation method.

[0481] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 2, using corresponding reagents for synthesis:

[0482] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 3, using corresponding reagents for synthesis:

[0483] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 5, using corresponding reagents for synthesis:

[0484] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 9, using corresponding reagents for synthesis:

[0485] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 12, using corresponding reagents for synthesis:

[0486] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 10, using corresponding reagents for synthesis:

[0487] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 13, using corresponding reagents for synthesis:

[0488] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 1, using corresponding reagents for synthesis:

[0489] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 1, using corresponding reagents for synthesis:

[0490] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 1, using corresponding reagents for synthesis:

[0491] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 1, using corresponding reagents for synthesis:

[0492] The synthetic routes of the following compounds are as follows, referring to the synthetic method of the first step of Example 14, using corresponding reagents for synthesis:

[0493] Biological activity test example:

[0494] Biological Activity Example 1: Preparation of SARM1 Enzyme

[0495] Preparation of test compounds:

[0496] The test compounds were prepared at a stock concentration of 200 uM or 10 mM (in DMSO) and further diluted to the desired compound concentration for in vitro SARM1 enzyme assays and inhibitor screening.

[0497] Expression and purification of SARM1 protein

[0498] (1) Plasmid construction

[0499] The gene sequence of SARM1 protein was amplified by PCR, and the PCR amplification product was constructed into the pcDNA3.1-HIS-C plasmid. The above construction was completed by Youbao Bio (Hunan, China).

[0500] (2) Plasmid transfection

[0501] HEK293 cells were cultured in a 10 cm dish using DMEM, and the next day the cells were transfected with 15 μg of pcDNA3.1-SARM1-HIS-C expression plasmid.

[0502] (3) Cell collection and protein extraction

[0503] 48 hours after transfection, cells were collected and the SARM1 protein expressed in the cells was obtained by diginotin cleavage for in vitro activity assays.

[0504] The cells were digested with trypsin-EDTA and centrifuged at 1000 rpm for 5 minutes. The cells were washed once with PBS and then resuspended in PBS containing 100 μM diginotin (0.6 mL PBS / 10 cm2 of cells) for 5 minutes. The cells were then treated with trypan blue and observed under a microscope, indicating that >90% of the cells had been lysed. The cells were then centrifuged at 5000 rpm for 10 minutes, and the supernatant containing SARM1 protein was collected.

[0505] Biological Activity Example 2: In vitro biochemical test for inhibition of SARM1 enzyme activity (IC 50 ):

[0506] First, 200 μM of the compound was added to a 50 mM Tris-HCl (pH 7.5) solution containing 0.05 μg / ml SARM1, and then half was added to an equal volume of 50 mM Tris-HCl (pH 7.5) solution containing 0.05 μg / ml SARM1 and mixed. The drug was diluted 6 times in this way, with final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 μM, or 200, 50, 12.5, 3.125, 0.78, 0.195, 0.049 μM. The control group was incubated at room temperature for 10 minutes without adding inhibitors.

[0507] Then, 50 μM NAD and 50 μM PC6 as substrates and 50 μM NMN as an activator were added to the SARM1 protein after incubation with the inhibitor and reacted at room temperature for 30 minutes. The concentrations of each component are the final concentrations in the reaction system.

[0508] During the reaction, PC6 fluorescence kinetics were monitored using a microplate reader, with excitation and emission wavelengths at 390 nm and 520 nm, respectively. The reaction rate was used to represent protein activity, and the 50% inhibitory concentration (50%) was calculated. Higher reaction rates indicate greater protein activity and lower compound inhibition efficiency.

[0509] The dosage curve of compounds inhibiting SARM1 NAD enzyme activity was prepared using the above method.

[0510] The IC values ​​of these compounds in the assay are provided in Table 1 below. 50 Range:

[0511] IC for inhibition of SARM1 enzyme activity 50 Range: A<0.1μM; B:0.1-1μM; C:1-10μM

[0512] Table 1

[0513] Biological Activity Example 3: Axon Degeneration Index

[0514] For experimental methods, please refer to Gerdts J et al.

[0515] Mouse DRG explant culture

[0516] Dorsal root ganglia were dissected from two-month-old C57BL / 6 male mice and placed in a 35 mm culture dish containing pre-chilled HBSS buffer. Excess tissue from each dorsal root ganglion was removed using a scalpel and dissected into 2-3 explants. These were seeded into wells of a 96-well plate coated with 100 ng / mL Matrigel (Corning). 100 μL of DRG culture medium (Neurobasal medium (Gibco) containing 2% B-27 (Gibco), 1% GlutaMax (Gibco), and 10 ng / mL NGF (MCE)) was added and the plates were cultured in a 37°C incubator for one week. The medium was changed on the fourth day, and experiments were performed on the eighth day.

[0517] Axonal Wallerian degeneration assay

[0518] Dissolve 0.1 μL of 10 mM test compound in 100 μL DRG culture medium to prepare a new medium containing 10 μM test compound. The medium in the 96-well plate was aspirated and added to the new medium containing the test compound. The culture medium was then incubated with the DRG explants in a 37°C incubator for 2 hours to protect them. After incubation, axons were severed in an area adjacent to the tissue explant using a glass electrode. Brightfield images of axons in the same area were taken at 0 and 24 hours using a 20x microscope (Olympus). Axonal integrity was assessed using an in-house developed script.

[0519] The compounds of the present invention exhibited neuroprotective effects in an explant axon Wallerian degeneration assay at a concentration of 10 μM, and were classified according to the degree of protection: extreme protection (A) > 50% and partial protection (B) 20%-50%. The results are listed in Table 2 below.

[0520] Table 2

Claims

1. Compound of formula (I): or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, in, X1 is selected from -O-, -CH 2- , -OCH2-, -NR3CH2-, -CH2NR3-, -N=CH-, -CH=N-, preferably -O-; Wherein R3 is selected from H and C1-C3 alkyl; X2, X3, X4 are each independently selected from CH or N; R1 is independently selected from H, halogen, C1-C3 alkyl, cyano, trifluoromethyl, amino, hydroxy, methoxy, -C(O)NH2, preferably H, F, Cl, methyl, cyano and -C(O)NH2; R2 is independently selected from H, halogen, C1-C3 alkyl, cyano, trifluoromethyl, amino, hydroxy, methoxy, C1-C3 alkyl-OC(O)-, C1-C3 alkyl-NHC(O)-, preferably H, cyano, Cl, methyl and methoxy; M represents O, S or NH, preferably O; L1 and L3 are each independently selected from -CH(R')-, -O-, -S- and -N(R')-, provided that at least one of L1 and L3 is -CH(R')-, wherein R' is hydrogen or C1-C3 alkyl; L2 is selected from H, -CH3, -CH2-CH3, -CH2-CH2-CH3, -OR8, -NHR9; Wherein, R8 and R9 are independently selected from H and C1-C3 alkyl; A is selected from H, 5- to 10-membered aryl, 5- to 10-membered heteroaryl, 5- to 10-membered cycloalkyl, 5- to 10-membered heterocycloalkyl, 5- to 10-membered aryl and 3- to 6-membered cycloalkyl, 5- to 10-membered aryl and 3- to 6-membered heterocycloalkyl, wherein the heteroaryl or heterocycloalkyl may contain 1 or 2 heteroatoms selected from N, O and S, wherein The 5- to 10-membered aryl, 5- to 10-membered heteroaryl, 5- to 10-membered aryl and 3- to 6-membered cycloalkyl, and 5- to 10-membered aryl and 3- to 6-membered heterocycloalkyl may be substituted by 1, 2 or 3 substituents selected from the group consisting of halogen, C1-C3 alkyl, C1-C3 alkoxy, halogenated C1-C3 alkyl, halogenated C1-C3 alkoxy, cyano, nitro and (R a R a ')N-, where R a and R a 'are each independently selected from hydrogen and C1-C3 alkyl; preferably, the substituent is selected from methyl, methoxy, trifluoromethyl, trifluoromethoxy, F, Cl, Br; L1 may be linked to L2 to form a 5-membered ring or a 6-membered ring, preferably a 5-membered ring.

2. The compound of formula (I) according to claim 1, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the following structure of formula (II): in, X2, X3, X4, R1, R2, L3, A as defined in claim 1; X5 is selected from CH2, O, NH, preferably CH2.

3. The compound of formula (I) according to claim 1, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the following structure of formula (III): in, R1, R2, M, L1, L2, L3, A are as defined in claim 1.

4. The compound of formula (I) according to claim 1, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the following structure of formula (IV): in, X2 and X3 are selected from CH and N, and X2 and X3 are not CH at the same time; R1, R2, M, L1, L2, L3, A are as defined in claim 1.

5. The compound of formula (II) according to claim 2, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (II) has the following structure (V): in, R1, R2, X2, X3, A are as defined in claim 1.

6. The compound of formula (III) according to claim 3, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (III) has the following structure of formula (VI): in, R1, R2, and A are as defined in claim 1.

7. The compound of formula (IV) according to claim 4, or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof, wherein the compound of formula (IV) has the following structure of formula (VII): in, X2 and X3 are selected from CH and N, and X2 and X3 are not CH at the same time; R1, R2, and A are as defined in claim 1.

8. A compound, or a racemate, enantiomer, diastereomer, deuterated form or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from the group consisting of:

9. Use of the compound according to any one of claims 1 to 8 or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof in the preparation of a SARM1 enzyme activity inhibitor.

10. Use of the compound according to any one of claims 1 to 8 or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing axonal degeneration-related diseases or disorders.

11. Use of the compound according to any one of claims 1 to 8 or its racemate, enantiomer, diastereomer, deuterated substance or pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing a neurodegenerative disease or a neurological disease or condition.

12. Use according to claim 9 or 10 or 11, wherein the neurodegenerative disease or neurological disease or condition or axonal degeneration-related disease or condition is selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis and peripheral neuropathy.

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