Spirocyclic compounds and their pharmaceutical compositions and applications

Abstract

This invention discloses a spirocyclic compound having the structure of Formula 1, its pharmaceutical composition, and its applications, which also includes a pharmaceutically acceptable salt. This type of compound and its pharmaceutical composition exhibit excellent inhibitory / antagonistic effects against malignant proliferating cells and androgen receptors. It has wide applications and can be formulated as a drug for treating prostate cancer, benign prostatic hyperplasia, male pattern baldness, hypomuscular atrophy, hirsutism, and other diseases. It also possesses suitable pharmacokinetic properties, facilitating drug development.

Description

Technical Field

[0001] This invention relates to a spirocyclic compound, pharmaceutical compositions thereof, and applications, and more particularly to a spirocyclic compound that can be prepared as an androgen receptor antagonist drug, pharmaceutical compositions thereof, and applications. Background Technology

[0002] The androgen receptor (AR) is a member of the nuclear receptor family. The AR comprises four main regions: the N-terminal active transcriptional control domain (NTD), the DNA-binding domain (DBD), the hinge region, and the C-terminal ligand-binding domain (LBD). Since Huggins and Hodges discovered in the early 1940s that androgens promote prostate cancer growth, the AR has been an important target for prostate cancer treatment.

[0003] Androgen receptors play an important role in many androgen-related diseases, such as prostate cancer, benign prostatic hyperplasia, male hair loss, hypomuscular dystrophy, and hypertrichosis. For this reason, selective androgen receptor antagonists can be used for conditions and diseases including, but not limited to: male contraception; treatment of various androgen-related conditions such as hypersexuality and sexual deviation; treatment of conditions including benign prostatic hyperplasia, acne vugaris, androgenetic alopecia, and hirsutism; prevention of symptoms associated with decreased testosterone, such as hot flashes after castration; targeted prevention or counteracting of masculinization in transgender women undergoing sex reassignment therapy; as palliative, adjuvant, or neoadjuvant hormonal therapy in prostate cancer; and to reduce the incidence of prostate cancer, stop prostate cancer, or induce prostate cancer regression.

[0004] Prostate cancer (PCa) is one of the most common cancers worldwide. Endocrine therapy is the primary treatment for advanced prostate cancer. In the initial stages of endocrine therapy, various androgen deprivation therapies (ADTs) are effective. However, after a median time of 14-30 months, almost all patients will progress from androgen-dependent prostate cancer (HDPC) to androgen-independent prostate cancer (HIPC), also known as castration-resistant prostate cancer (CRPC). Argon expression is high in 80% of advanced CRPC cases. Currently approved oral medications for treating castration-resistant prostate cancer mainly include abiraterone and enzalutamide. Abiraterone is a novel androgen biosynthesis inhibitor that blocks androgen synthesis in the testes, adrenal glands, or tumor cells. Enzalutamide is an androgen receptor inhibitor that competitively inhibits the binding of androgens to their receptors. When enzalutamide binds to AR, it can further inhibit the nuclear transport of AR, thereby blocking the interaction between AR and DNA. Although enzalutamide and abiraterone are effective in the early stages, almost all patients develop resistance after 1-2 years of treatment, which seriously affects the efficacy of these drugs. There is currently no effective treatment for drug-resistant CRPC patients.

[0005] Common mechanisms of resistance to AR-targeted therapies include AR point mutations, AR amplification, and AR alternative splicing. For example, the F876L point mutation can convert high-dose enzalutamide from an antagonist to an agonist. Therefore, the development of novel AR antagonists with novel structures and mechanisms of action is of great significance in the treatment of drug-resistant prostate cancer. Summary of the Invention

[0006] Purpose of the invention: In view of the problems existing in existing drugs, the present invention aims to provide a spirocyclic compound, its pharmaceutical composition and application.

[0007] Technical solution: As a first aspect of the present invention, the spirocyclic compound of the present invention has the structure of Formula 1 and further comprises a pharmaceutically acceptable salt thereof.

[0008]

[0009] in:

[0010] X and U are selected from CH or N;

[0011] R 1 Selected from halogen, -CF3 or -OC 1-6 alkyl;

[0012] R 2 Selected from -CN, -NO2, -COR a-CONHR a -S(O)2R a or -S(O)2N(R) a )2;

[0013] R a Selected from H, -OH, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0014] R 3 Selected from H or -OC 1-6 alkyl;

[0015] Y is -CH2- or carbonyl;

[0016] m is selected from 0, 1, 2, or 3;

[0017] n is selected from 1, 2, or 3;

[0018] o can be selected from 1, 2, or 3;

[0019] p is selected from 1, 2, or 3;

[0020] Z is selected from -CH2-, -CONH-, -NHCO-, and -CO(CH2). q -、-(CH2) q CO-, -NH-, -N(CH3)-, -S(O)2-, -O- or carbonyl group;

[0021] q is selected from 1, 2, or 3;

[0022] When Z is -CH2-, r is selected from 0, 1, 2 or 3; when Z is any of the other groups described, r is selected from 0 or 1.

[0023] W is selected from chemical bonds, -CH2-, -NH-, -N(CH3)-, -O-, or carbonyl groups;

[0024] Ring D is selected from 5-10 heteroaryl groups containing 1 or 2 N or O atoms, or C atoms. 6-10 Aryl;

[0025] R 4 R 5 Selected from at least one H, halogen, -OH, -SH, -NH2, -NO2, -CN, -CONH2-, -CONHR 4A -COOR 4A -OCOR 4A -NHCOR 4A -OCONHR 4A -NHCONHR 4A -NHSO2R 4A -SO2NHR 4A-S(O)2-OR 4A -OSO2R 4A -OC 1-6 Alkyl, -CH2OH, -CH2SH, -OCH2CH2OH, -SC 1-6 Alkyl group, -(O=)S(=NH)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, C 1-6 Alkyl groups, 4-10 member heterocyclic groups containing 1 or 2 N or O atoms, 5-10 member heteroaryl groups containing 1 to 3 N, O, or S atoms, or C atoms. 6-10 Aryl, the C 1-6 Alkyl, 4-10 membered heterocyclic, 5-10 membered heteroaryl or C 6-10 Aryl groups are selectively coated with 1-3 R groups. 4A replace;

[0026] R 4A Selected from H, halogens, -OH, -NO2, -CN, -CD3, -CH2CD3, -CH2F, -CHF2, -CF3, C 1-6 Alkyl, -OC 1-6 Alkyl, -OC 3-6 cycloalkyl, -S(O)2-C 1-6 Alkyl or -S(O)2-C 3-6 Cycloalkyl.

[0027] Furthermore, the above-mentioned spirocyclic compounds have the structure of Formula 2 or Formula 3.

[0028]

[0029] in:

[0030] m is selected from 0 or 1;

[0031] n is selected from 1 or 2;

[0032] o is selected from 1;

[0033] p is selected from 1, 2, or 3;

[0034] Z is selected from -CH2-, -CONH-, -NHCO-, -NH-, -N(CH3)-, -S(O)2-, -O- or carbonyl;

[0035] When Z is -CH2-, r is selected from 0, 1, 2 or 3; when Z is any of the other groups described, r is selected from 0 or 1.

[0036] W is selected from chemical bonds, -CH2-, -NH-, -N(CH3)-, -O-, or carbonyl groups;

[0037] The ring D is selected from 5-6 heteroaryl or phenyl groups containing 1 or 2 N or O atoms.

[0038] Preferably, in the above structure:

[0039] R 1 Selected from halogens or -CF3;

[0040] R 2 Selected from -CN, -NO2, -COR a -CONHR a -S(O)2R a or -S(O)2N(R) a )2;

[0041] R a Selected from H, -OH, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0042] R 3 Selected from H or -OC 1-4 alkyl;

[0043] R 4 R 5 Selected from 1 or 2 H atoms, halogens, -OH, -SH, -NH2, -NO2, -CN, -CONH2-, -CONHR 4A -COOR 4A -OCOR 4A -NHCOR 4A -OCONHR 4A -NHCONHR 4A -NHSO2R 4A -S(O)2NHR 4A -S(O)2-OR 4A -OS(O)2R 4A -OC 1-4 Alkyl, -CH2OH, -CH2SH, -OCH2CH2OH, -SC 1-6 Alkyl group, -(O=)S(=NH)-C 1-6 Alkyl, -S(O)2-C 1-4 Alkyl, C 1-4 Alkyl or phenyl, wherein C 1-4 Alkyl or phenyl groups with 1-3 R groups 4A replace;

[0044] R 4A Selected from H, halogens, -OH, -NO2, -CN, -CD3, -CH2CD3, -CH2F, -CHF2, -CF3, C 1-4 Alkyl, -OC 1-4Alkyl, -OC 3-6 cycloalkyl, -S(O)2-C 1-4 Alkyl or -S(O)2-C 3-6 Cycloalkyl.

[0045] Selected from

[0046] Selected from

[0047] Selected from chemical bonds, carbonyl groups, -CONH-, -CH2C(O)NH-, -CH2-, or -NHCO-;

[0048] The ring D is selected from phenyl, pyridyl, pyrazolyl or isoxazolyl.

[0049] Further optimization is achieved in the above structure:

[0050] R 1 Selected from -CF3, R 2 Selected from -CN,R 3 Selected from H;

[0051] R 4 R 5 Phenyl group selected from one or two F, -OH, -NH2, -NO2, -CN, -CH3, -CH2OH, -OCH3, -OCH2CH2OH, -CH(OH)CH3, -C(O)NHCH3 or halogen, -OH, -NO2, -CN, -CF3 substituted phenyl groups.

[0052] Furthermore, the aforementioned spirocyclic compounds are preferably selected from any of the following compounds:

[0053]

[0054]

[0055]

[0056] Among them, the pharmaceutically acceptable salt of the above-mentioned spirocyclic compounds is the salt formed by its reaction with any of the following acids:

[0057] Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and acidic amino acids.

[0058] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0059] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0060] Preferably, the stereoisomer is an isomer introduced by chiral C and N.

[0061] Preferably, the tautomer is an isomer formed by double bond conjugation tautomerism in an unsaturated heterocycle, including carbon-carbon double bond tautomerism, carbon-heteroatom tautomerism, and heteroatom-heteroatom tautomerism, such as tautomers formed by double bond tautomerism in imidazole ring systems and pyrazole ring systems.

[0062] Preferably, the prodrug is an ester or amide prodrug introduced by a carboxyl, hydroxyl, or amino group, and more preferably a C1-C4 alkyl ester, C1-C4 carboxylic ester, or C1-C4 alkyl amide.

[0063] Preferably, the solvate is a small molecule bonded state formed by the compound and solvent molecules, more preferably a hydrate or alcohol; the solvate can further form a salt with the corresponding acid to obtain a salt of the solvate.

[0064] Preferably, the isotopic compound is a compound in which hydrogen is replaced by deuterium.

[0065] Preferably, the crystallization is a specific crystal structure formed by the compound during the crystallization process, including different crystal forms of the compound itself, as well as different crystal forms of its salts, solvates, and salts of solvates.

[0066] As a second aspect of the present invention, the above-mentioned spirocyclic compounds and pharmaceutically acceptable carriers constitute the pharmaceutical compositions of the present invention.

[0067] Preferably, the formulation is selected from tablets, capsules, powders, syrups, liquids, suspensions, lyophilized powder for injection, and injections.

[0068] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0069] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0070] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0071] As a third aspect of the present invention, the above-mentioned spirocyclic compounds and their pharmaceutical compositions are used in the preparation of androgen receptor antagonist drugs, specifically prepared as drugs for treating prostate cancer, benign prostatic hyperplasia, male pattern baldness, hypotonia, hirsutism, etc.

[0072] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0073] 1. These compounds and their drug compositions exhibit excellent inhibitory / antagonistic effects on malignant proliferating cells and androgen receptors, with high IC50 values ​​for cell inhibitory activity. 50 The optimal value is below 50 nM, and the optimal inhibition rate of receptor antagonism reaches 100%, inhibiting IC50. 50 The optimal value is below 20 nM;

[0074] 2. It has a wide range of applications and can be prepared into drugs for the treatment of prostate cancer, benign prostatic hyperplasia, male hair loss, hypomuscular dystrophy, hirsutism, etc. It also has suitable pharmacokinetic properties, which is conducive to drug development. Detailed Implementation

[0075] The technical solution of the present invention will be further described below with reference to the embodiments.

[0076] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are measured in units of 10⁻⁶. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., with tetramethylsilane (TMS) as the internal standard; "IC 50"Half-inhibitory concentration" refers to the concentration at which half of the maximum inhibitory effect is achieved.

[0077] Abbreviations:

[0078] DMF: N,N-dimethylformamide; Et3N and TEA: triethylamine; THF: tetrahydrofuran; DMSO: dimethyl sulfoxide; THF: tetrahydrofuran; DCM: dichloromethane; EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; NaOBu-t: sodium tert-butoxide; Pd(OAc)2: palladium acetate; EA: ethyl acetate; dioxane: dioxane; Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene; Ethanol: ethanol; Chloroform: chloroform; TFA: trifluoroacetic acid.

[0079] Example 1: Synthesis of I-1

[0080] Synthesis of intermediate S2:

[0081]

[0082] Step 1: Synthesis of S1

[0083] Under nitrogen protection, a mixture of 4-bromo-2-(trifluoromethyl)benzonitrile (663 mg, 2.65 mmol), 7-oxo-2,6-diazaspiro[3,4]octane-2-carboxylic acid tert-butyl ester (400 mg, 1.77 mmol), cesium carbonate (682 mg, 2.54 mmol), palladium acetate (16 mg, 0.071 mmol), Xantphos (123 mg, 0.21 mmol), and 1,4-dioxane (10 mL) was heated at 90 °C for 5 h. After cooling to room temperature, the mixture was extracted three times with ethyl acetate / water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (petroleum ether:ethyl acetate = 1:1) to give a white solid 1 (620 mg), yield 89%. ESI-MS m / z: 328.3 [M+H] + .1H NMR(300MHz,Chloroform-d)δ8.07(d,J=2.2Hz,1H),7.98(dd,J=8.6,2.3Hz,1H),7 .83(d,J=8.6Hz,1H),4.08(s,2H),4.02(d,J=1.6Hz,4H),2.93(s,2H),1.45(s,9H).

[0084] Step 2: Synthesis of S2

[0085] S1 (200 mg, 0.506 mmol) was dissolved in DCM (2 mL), and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was neutralized with 1 M NaOH aqueous solution, extracted with DCM (5 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give 120 mg of white solid, yield 80%. The solution was then directly added to the next step.

[0086]

[0087] A mixture of S2 (60 mg, 0.203 mmol), DMF (6 mL), triethylamine (56.4 μL, 0.406 mmol), and 4-(bromomethyl)benzonitrile (52 mg, 0.264 mmol) was stirred for 4 h at room temperature. After the reaction was complete, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a white solid I-1 (54 mg), yield 65%; ESI-MS m / z: 411.3 [M+H] + .1H NMR (300MHz, Chloroform-d) δ8.09(d,J=2.2Hz,1H),8.03(dd,J=8.7,2.3Hz,1H),7.83(d,J=8.6Hz,1H),7.63(d,J=7. 9Hz, 2H), 7.43 (d, J = 7.9Hz, 2H), 4.13 (s, 2H), 3.72 (s, 2H), 3.40 (d, J = 7.0Hz, 2H), 3.31 (d, J = 7.1Hz, 2H), 2.90 (s, 2H).

[0088] Example 2: Synthesis of I-2

[0089]

[0090] A mixture of S2 (160 mg, 0.542 mmol), dichloroethane (6 mL), 4-nitrobenzaldehyde (119.34 mg, 1.08 mmol), acetic acid (47 μL, 0.813 mmol), and anhydrous sodium sulfate (344.5 mg, 3.79 mmol) was heated and stirred at 55 °C for 5 h. After cooling to room temperature, sodium triacetoxyborohydride (538 mg, 1.63 mmol) was added, and the reaction was continued at room temperature for 8 h. After the reaction was complete, saturated sodium bicarbonate (aq) was added to adjust the pH to weakly alkaline. The mixture was extracted with dichloromethane / water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a yellow solid I-2 (145 mg), yield 71%; ESI-MS m / z: 430.9 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ8.40(d,J=2.1Hz,1H),8.19(dd,J=8.7,3.3Hz,3H),8.04(dd,J=8.7,2.2 Hz,1H),7.58(d,J=8.6Hz,2H),4.17(s,2H),3.75(s,2H),3.32(s,2H),3.28(s,2H),2.89(s,2H).

[0091] Example 3: Synthesis of I-3

[0092]

[0093] Following the synthetic method of Example 1, except that 4-(bromomethyl)benzonitrile was replaced with 1-(bromomethyl)-4-fluorobenzene, compound I-3 was prepared by the same method, yielding a white solid I-3 (41 mg), with a yield of 47%; ESI-MS m / z: 404.1 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.1Hz,2H),8.17(d,J=8.6Hz,2H),8.02(dd,J=8.7,2.2Hz,2H) ,7.40-7.26(m,4H),7.22-7.07(m,4H),4.13(s,4H),3.55(s,4H),3.28-3.13(m,8H),2.86(s,4H).

[0094] Example 4: Synthesis of I-4

[0095]

[0096] Under N2 protection, a mixture of S2 (80 mg, 0.271 mmol), p-fluorobenzonitrile (44 μL, 0.406 mmol), cesium carbonate (105 mg, 0.542 mmol), and DMF (10 mL) was heated and stirred at 55 °C for 7 h. After the reaction was complete, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a white solid I-4 (49 mg), yield 46%; ESI-MS m / z: 397.1 [M+H] + . 1 H NMR(300MHz,DMS O-d6)δ8.39(d,J=2.1Hz,1H),8.17(d,J=9.0Hz,1H),8.02(dd,J=8.6,2.2Hz,1H),7.41-7 .24(m,2H),7.20-7.07(m,2H),4.13(s,2H),3.55(s,2H),3.28-3.10(m,4H),2.86(s,2H).

[0097] Example 5: Synthesis of I-5

[0098]

[0099] p-Cyanobenzic acid (76 mg, 0.518 mmol), HATU (208 mg, 0.622 mmol), and DIPEA (61.5 μL, 0.78 mmol) were dissolved in dichloromethane (12 mL). The mixture was stirred at room temperature for 2 h, then S2 (100 mg, 0.471 mmol) was added, and stirring continued overnight. After the reaction was complete, the reaction mixture was extracted three times with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (evolving solvent: dichloromethane:methanol = 100:1) to give a white solid I-5 (134 mg), yield 83%; ESI-MS m / z: 425.2 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ8.33(d,J=2.1Hz,1H),8.20(d,J=8.6Hz,1H),8.01(dd,J=8.7,2.2Hz,1H),7 .98-7.91(m,2H),7.87-7.75(m,2H),4.40(s,2H),4.24(s,2H),4.17(s,2H),3.01(d,J=2.1Hz,2H).

[0100] Example 6: Synthesis of I-6

[0101]

[0102] Step 1: Synthesis of I-6-1

[0103] p-Aminobenzonitrile (5 g, 43.32 mmol), chloroacetyl chloride (3.71 mL, 47.65 mmol), and triethylamine (6.47 mL, 47.65 mmol) were dissolved in DCM and stirred at room temperature for 24 h. After the reaction was complete, water was added to precipitate the solid, which was filtered and washed with DCM to give a white solid 33 (7.3 g), yield 89%. 1 H NMR (300MHz, DMSO-d6) δ10.80(s,1H),7.88-7.71(m,4H),4.33(s,2H).

[0104] Step 2: Synthesis of I-6

[0105] S2 (80 mg, 0.27 mmol) and intermediate I-6-1 (58 mg, 0.30 mmol) were dissolved in DMF, and potassium carbonate (75 mg, 0.54 mmol) was added. The mixture was stirred at room temperature for 8 h. After the reaction was complete, water was added, and a solid precipitated. The solid was filtered, and the filter cake was recrystallized from methanol to give a white solid I-6 (64 mg), with a yield of 54%. ESI-MS m / z: 425.2 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ10.13(s,1H),8.39(d,J=2.2Hz,1H),8.19(d,J=8.6Hz,1H),8.03(dd,J=8.7,2.1Hz,1H ),7.81(q,J=8.9Hz,4H),4.17(s,2H),3.43(d,J=7.0Hz,2H),3.38(d,J=7.3Hz,2H),3.31(s,2H),2.91(s,2H).

[0106] Example 7: Synthesis of I-7

[0107]

[0108] Step 1: Synthesis of I-7-1

[0109] AIBN (533 mg, 3.244 mmol) and NBS (6.35 g, 35.684 mmol) were added to a carbon tetrachloride solution of 2-fluoro-4-methylbenzoic acid (5 g, 32.44 mmol), and the reaction was carried out at 90 °C for 3 h. After the reaction was completed, the mixture was cooled, filtered, and the filter cake was washed three times with dichloromethane. The solvent was then evaporated under reduced pressure to give a white solid I-7-1 (5.5 g), with a yield of 73%.1 H NMR (300MHz, DMSO-d6) δ11.38 (s, 1H), 7.86 (dd, J = 8.3, 7.1Hz, 1H), 7.47-7.30 (m, 2H), 4.72 (s, 2H).

[0110] Step 2: Synthesis of I-7-2

[0111] N-methylmorpholine (472 μL, 4.29 mmol) was slowly added dropwise to a DMF solution of I-7-1 (200 mg, 0.858 mmol), EDCI (247 mg, 1.287 mmol), and HOBt (174 mg, 1.287 mmol), and stirred at room temperature for 2 h. After the reaction was complete, water was added for dilution, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (petroleum ether:ethyl acetate = 1:1) to give I-7-2 (169 mg), yield 80%. 1 H NMR (300MHz, DMSO-d6) δ8.29 (s, 1H), 7.62 (t, J = 7.7Hz, 1H), 7.44-7.29 (m, 2H), 4.80 (s, 2H), 2.77 (d, J = 4.6Hz, 3H).

[0112] Step 3: Synthesis of I-7

[0113] Using intermediate 1 (80 mg, 0.271 mmol), intermediate 32 (57.6 mg, 0.542 mmol), DMF (6 mL), and potassium carbonate (75 mg, 0.542 mmol) as raw materials, the preparation method was the same as for I-1, yielding a white solid I-7 (52 mg), with a yield of 39%; ESI-MS m / z: 461.1 [M+H] + . 1 H NMR(300MHz,Chloroform-d)δ8.15-7.98(m,3H),7.84(d,J=8.6Hz,1H),7.18(dd,J=8.0,1.5Hz,1H),7.11(d,J=12.9Hz,1H),6.74( d,J=8.2Hz,1H),4.13(s,2H),3.69(s,2H),3.41(d,J=7.1Hz,2H),3.30(d,J=7.2Hz,2H),3.05(dd,J=4.8,1.1Hz,3H),2.89(s,2H).

[0114] Example 8: Synthesis of I-8

[0115]

[0116] Step 1: Synthesis of I-8-1

[0117] 5-Acetyl-1H-pyrazole-3-carboxylic acid (76 mg, 0.518 mmol), HATU (208 mg, 0.622 mmol), and DIPEA (61.5 μL, 0.78 mmol) were dissolved in dichloromethane (12 mL) and stirred at room temperature for 2 h. Then, intermediate 2 (100 mg, 0.471 mmol) was added, and stirring continued overnight. After the reaction was complete, the reaction mixture was extracted three times with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent:dichloromethane:methanol = 100:1) to give a white solid I-8-1 (134 mg), yield 83%; ESI-MS m / z: 432.1 [M+H] + .

[0118] Step 2: Synthesis of I-8

[0119] I-8-1 (130 mg, 0.301 mmol) was dissolved in methanol, and sodium borohydride (46 mg, 1.21 mmol) was added under ice bath conditions, followed by stirring for 1.5 h. After the reaction was complete, a small amount of saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted three times with ethyl acetate / water, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a white solid I-8 (119 mg), with a yield of 91%. 1 H NMR (300MHz, DMSO-d6) δ13.14(s,1H),8.38(d,J=2.2Hz,1H),8.20(d,J=8.6Hz,1H),8.01(dd,J=8.7,2.2Hz,1H),6.4 6(s,1H),5.46(s,1H),4.80(s,1H),4.64-4.45(m,2H),4.24(s,2H),4.10(s,2H),3.02(s,2H),1.38(d,J=6.5Hz,3H).

[0120] Example 9: Synthesis of I-9

[0121]

[0122] A mixture of S2 (90 mg, 0.345 mmol), dichloroethane (6 mL), 5-methyl-1H-pyrazole-3-carboxaldehyde (67.1 mg, 0.61 mmol), acetic acid (26 μL, 0.518 mmol), and anhydrous sodium sulfate (303 mg, 2.415 mmol) was stirred at room temperature for 5 h. Sodium triacetoxyborohydride (194 mg, 1.035 mmol) was added, and the reaction was continued for 8 h. After the reaction was complete, saturated sodium bicarbonate (aq) was added to adjust the pH to weakly alkaline. The mixture was extracted with dichloromethane / water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent:dichloromethane:methanol = 100:1) to give a yellow solid I-9 (63 mg), yield 53%; ESI-MS m / z: 390.1 [M+H] + . 1 H NMR(300MHz,Chloroform-d)δ8.09(d,J=2.2Hz,1H),8.01(dd,J=8.6,2.3Hz,1H),7.82(d,J=8. 6Hz,1H),5.98(s,1H),4.10(s,2H),3.66(s,2H),3.50-3.27(m,4H),2.87(s,2H),2.30(s,3H).

[0123] Example 10: Synthesis of I-10

[0124]

[0125] A mixture of S2 (90 mg, 0.345 mmol), dichloroethane (6 mL), 5-methylisoxazole-3-carboxaldehyde (67.1 mg, 0.61 mmol), acetic acid (26 μL, 0.518 mmol), and anhydrous sodium sulfate (303 mg, 2.415 mmol) was stirred at room temperature for 5 h. Sodium triacetoxyborohydride (194 mg, 1.035 mmol) was added, and the reaction was continued for 8 h. After the reaction was complete, saturated sodium bicarbonate (aq) was added to adjust the pH to weakly alkaline. The mixture was extracted with dichloromethane / water, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a white solid I-10 (71 mg), yield 60%; ESI-MS m / z: 391.1 [M+H] + . 1H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.1Hz,1H),8.18(d,J=8.7Hz,1H),8.02(dd,J=8.7,2.2Hz,1H),6.16( d,J=1.1Hz,1H),4.12(s,2H),3.56(s,2H),3.28(s,2H),3.26(s,2H),2.86(s,2H),2.37(d,J=0.9Hz,3H).

[0126] Example 11: Synthesis of II-1

[0127]

[0128] Using S2 (60 mg, 0.203 mmol), 3-(bromomethyl)benzonitrile (52 mg, 0.264 mmol), DMF (6 mL), and triethylamine (56.7 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid I-3 (49 mg) with a yield of 59%. 1 H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.1Hz,1H),8.18(d,J=9.0Hz,1H),8.03(dd,J=8.7,2.2Hz,1H),7.73(dp,J=5.0,1.6Hz,2H),7.63 (dt,J=7.8,1.5Hz,1H),7.54(t,J=7.9Hz,1H),4.16(s,2H),3.65(s,2H),3.30(d,J=7.0Hz,2H),3.23(d,J=7.0Hz,2H),2.87(s,2H).

[0129] Example 12: Synthesis of II-2

[0130]

[0131] Using S2 (60 mg, 0.203 mmol), 2-(bromomethyl)benzonitrile (52 mg, 0.264 mmol), DMF (6 mL), and triethylamine (56.7 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-2 (54 mg) with a yield of 65%. 1H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.1Hz,1H),8.17(d,J=8.6Hz,1H),8.03(dd,J=8.7,2.2Hz,1H),7.82(dd,J=7.7,1.3Hz,1H),7.69(t d,J=7.6,1.4Hz,1H),7.62-7.54(m,1H),7.46(td,J=7.6,1.3Hz,1H),4.16(s,2H),3.79(s,2H),3.33(s,2H),3.31(s,2H),2.88(s,2H).

[0132] Example 13: Synthesis of II-3

[0133]

[0134] Using S2 (60 mg, 0.203 mmol), 5-(bromomethyl)pyridinium (52 ​​mg, 0.264 mmol), DMF (6 mL), and triethylamine (56.7 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-3 (69 mg), with a yield of 83% and a purity of 96.77%. 1 H NMR (300MHz, DMSO-d6) δ8.68(d,J=2.0Hz,1H),8.38(d,J=2.2Hz,1H),8.17(d,J=8.6Hz,1H),8.08-7.98 (m,2H),7.95(dd,J=8.0,2.1Hz,1H),4.16(s,2H),3.74(s,2H),3.35-3.25(m,4H),2.88(s,2H).ESI-MS m / z:412.1[M+H] + .

[0135] Example 14: Synthesis of II-4

[0136]

[0137] Step 1: Synthesis of II-4-1

[0138] Under nitrogen protection, a mixture of 4'-methyl-[1,1'-biphenyl]-4-onitrile (50 mg, 0.259 mmol), BPO (62.7 mg, 0.259 mmol), NBS (51 mg, 0.285 mmol), and carbon tetrachloride (6 mL) was heated and stirred overnight at 55 °C. After the reaction was complete, the mixture was extracted with dichloromethane / water, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (electrolyte:PE:EA = 30:1) to give a white solid II-4-1 (58 mg), yield 82%.

[0139] Step 2: Synthesis of II-4

[0140] Using S2 (80 mg, 0.271 mmol), II-4-1 (57.6 mg, 0.542 mmol), DMF (6 mL), and potassium carbonate (75 mg, 0.542 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-4 (52 mg) with a yield of 39%. 1 H NMR(300MHz,Chloroform-d)δ8.16-7.96(m,5H),7.82(d,J=8.5Hz,3H),7.73(d,J=8.2Hz,5H),7.67(d,J=8.1Hz,5H),7.56(d,J=7.8Hz,5 H),7.40(d,J=7.9Hz,5H),7.27(s,2H),4.13(s,5H),3.71(s,5H),3.42(d,J=7.2Hz,5H),3.31(d,J=7.2Hz,5H),2.89(s,5H),1.25(s,1H).

[0141] Example 15: Synthesis of II-5

[0142]

[0143] Using S2 (90 mg, 0.305 mmol), 4-(bromomethyl)-3-fluorobenzonitrile (52 mg, 0.396 mmol), DMF (6 mL), and triethylamine (85 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid I-5 (51 mg) with a yield of 39%. 1H NMR (300MHz, DMSO-d6) δ8.38(d,J=2.1Hz,1H),8.18(d,J=8.6Hz,1H),8.03(dd,J=8.7,2.2Hz,1H),7.82( dd,J=9.9,1.5Hz,1H),7.74-7.56(m,2H),4.15(s,2H),3.70(s,2H),3.31(d,J=8.6Hz,4H),2.88(s,2H).

[0144] Example 16: Synthesis of II-6

[0145]

[0146] Using S2 (90 mg, 0.305 mmol), 4-(bromomethyl)-2-fluorobenzonitrile (52 mg, 0.396 mmol), DMF (6 mL), and triethylamine (85 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-6 (61 mg), with a yield of 47% and a purity of 99.89%. 1 H NMR (300MHz, DMSO-d6) δ8.41(d,J=2.2Hz,1H),8.20(d,J=8.7Hz,1H),8.05(dd,J=8.6,2.2Hz,1H),7.95-7.84(m,1H),7.51- 7.42(m,1H),7.36(dd,J=8.0,1.4Hz,1H),4.19(s,2H),3.73(s,2H),3.34(s,2H),3.28(d,J=7.0Hz,2H),2.90(s,2H).ESI-MS m / z:429.1[M+H] + .

[0147] Example 17: Synthesis of II-7

[0148]

[0149] Using S2 (90 mg, 0.305 mmol), 3-(bromomethyl)-4-fluorobenzonitrile (52 mg, 0.396 mmol), DMF (6 mL), and triethylamine (85 μL, 0.406 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-7 (58 mg) with a yield of 45%. 1H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.2Hz,1H),8.18(d,J=8.6Hz,1H),8.04(dd,J=8.8,2.2Hz,1H),7.87(ddd,J =10.6,7.0,2.1Hz,2H),7.44(dd,J=9.8,8.4Hz,1H),4.16(s,2H),3.66(s,2H),3.34-3.24(m,4H),2.88(s,2H).

[0150] Example 18: Synthesis of II-8

[0151]

[0152] Using S2 (190 mg, 0.643 mmol), 5-(bromomethyl)-2-fluorobenzonitrile (207 mg, 0.965 mmol), DMF (15 mL), and triethylamine (179 μL, 1.29 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid II-8 (157 mg) with a yield of 57%. 1 HNMR (300MHz, DMSO-d6) δ8.38(d,J=2.1Hz,1H),8.17(d,J=8.6Hz,1H),8.03(dd,J=8.7,2.2Hz,1H),7.81(dd,J=6. 4,2.2Hz,1H),7.74-7.65(m,1H),7.48(t,J=9.0Hz,1H),4.15(s,2H),3.62(s,2H),3.33-3.20(m,4H),2.87(s,2H).

[0153] Example 19: Synthesis of II-9

[0154]

[0155] Using S2 (200 mg, 0.677 mmol), 3-(bromomethyl)-5-fluorobenzonitrile (149 mg, 1.35 mmol), DCE (15 mL), acetic acid (58 μL, 1.02 mmol), anhydrous sodium sulfate (673 mg, 4.74 mmol) and sodium triacetoxyborohydride (431 mg, 2.03 mmol) as raw materials, the preparation method was the same as that for I-10, yielding a white solid II-9 (221 mg) with a yield of 87%. 1H NMR (300MHz, DMSO-d6) δ8.39(d,J=2.2Hz,1H),8.18(d,J=8.6Hz,1H),8.03(dd,J=8.7,2.2Hz,1H),7.73(d,J =8.5Hz,1H),7.62(s,1H),7.52(d,J=9.3Hz,1H),4.17(s,2H),3.66(s,2H),3.33-3.17(m,4H),2.88(s,2H).

[0156] Example 20: Synthesis of III-1

[0157]

[0158] Step 1: Synthesis of III-1-1

[0159] 400 mg (1.77 mmol) of tert-butyl 7-oxo-2,6-diazaspiro[3,4]octane-2-carboxylic acid was dissolved in THF (20 mL). Na₂H₂O (141 mg, 3.54 mmol) was added in an ice bath, and the mixture was stirred for 0.5 h under nitrogen protection. Then, 4-(bromomethyl)benzonitrile (485 mg, 2.47 mmol) was added, and stirring continued for 4 h at room temperature. After the reaction was complete, the mixture was quenched with water, extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 50:1) to give a white solid III-1-1 (586 mg), yield 97%.

[0160] Step 2: Synthesis of III-1-2

[0161] III-1-1 (350 mg) was dissolved in 7 mL of dichloromethane, and then 3 mL of dioxane hydrochloride (4 mol / L) was added. The mixture was stirred at room temperature for 4 h. At the end of the reaction, a large amount of white solid precipitated out. The solid was filtered, and the filter cake was dissolved in dichloromethane. The pH was adjusted to alkaline with sodium hydroxide, and the mixture was concentrated under vacuum. The residue was separated by silica gel chromatography (dichloromethane:methanol = 20:1 as the developing solvent) to give white solid III-1-2 (171 mg), with a yield of 69%.

[0162] Step 3: Synthesis of III-1

[0163] Using III-1-2 (150 mg, 0.622 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (176 mg, 0.932 mmol), DMF (6 mL), and potassium carbonate (172 mg, 1.24 mmol) as raw materials, the same method as I-4 was used to prepare a white solid III-1 (176 mg) with a yield of 69%. 1H NMR(300MHz,Chloroform-d)δ7.87(d,J=8.4Hz,1H),7.78(d,J=2.1Hz,1H),7.72-7.62(m,3H),7.39(d,J=8.2Hz,2H),4.67-4.45(m,2H),4 .44-4.17(m,2H),3.88(d,J=11.2Hz,1H),3.70(dd,J=11.0,1.7Hz,1H),3.48(d,J=10.4Hz,1H),3.31(d,J=10.4Hz,1H),2.69-2.45(m,2H).

[0164] Example 21: Synthesis of III-2

[0165]

[0166] Step 1: Synthesis of III-2-1

[0167] Using tert-butyl ester of 3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylic acid (500 mg, 1.97 mmol), 4-bromo-2-(trifluoromethyl)benzonitrile (737 mg, 2.95 mmol), cesium carbonate (961 mg, 2.95 mmol), palladium acetate (22 mg, 0.1 mmol), Xantphos (171 mg, 0.3 mmol), and 1,4-dioxane (40 mL) as raw materials, the preparation method was the same as in S1, yielding a white solid III-2-1 (812 mg) with a yield of 98%.

[0168] Step 2: Synthesis of III-2-2

[0169] Using III-2-1 (300 mg, 0.708 mmol) as the raw material, the same method as S2 was used to prepare a white solid III-2-2 (165 mg), with a yield of 72%.

[0170] Step 3: Synthesis of III-2

[0171] Using III-2-2 (150 mg, 0.622 mmol), 4-(bromomethyl)benzonitrile (176 mg, 0.932 mmol), DMF (6 mL), and potassium carbonate (172 mg, 1.24 mmol) as raw materials, the same method as I-1 was used to prepare a yellow solid III-2 (176 mg) with a yield of 69%. 1HNMR(300MHz,Chloroform-d)δ8.15(d,J=2.3Hz,1H),8.05(dd,J=8.7,2.3Hz,1H),7.86(d,J=8.6Hz,1H),7.67 (d,J=8.2Hz,2H),7.51(d,J=7.9Hz,2H),3.74(s,2H),3.64(s,2H),2.55(d,J=51.6Hz,6H),1.90-1.74(m,4H).

[0172] Example 22: Synthesis of III-3

[0173]

[0174] Step 1: Synthesis of III-3-1

[0175] Using tert-butyl ester of 3-oxo-2,7-diazaspiro[4.5]decane-7-carboxylic acid (500 mg, 1.97 mmol), 4-bromo-2-(trifluoromethyl)benzonitrile (737 mg, 2.95 mmol), cesium carbonate (961 mg, 2.95 mmol), palladium acetate (22 mg, 0.1 mmol), Xantphos (171 mg, 0.3 mmol), and 1,4-dioxane (40 mL) as raw materials, the preparation method was the same as in 1, yielding a white solid III-3-1 (771 mg) with a yield of 93%.

[0176] Step 2: Synthesis of III-3-2

[0177] Using III-3-1 (300 mg, 0.708 mmol) as the raw material, the same method as S2 was used to prepare a white solid III-3-2 (193 mg), with a yield of 84%.

[0178] Step 3: Synthesis of III-3

[0179] Using III-3-2 (66 mg, 0.204 mmol), 4-(bromomethyl)benzonitrile (60 mg, 0.306 mmol), DMF (6 mL), and potassium carbonate (56 mg, 0.408 mmol) as raw materials, the preparation method was the same as that of I-1, yielding a white solid III-3 (53 mg) with a yield of 59%. 1HNMR(300MHz,Chloro form-d)δ8.22(d,J=2.3Hz,1H),8.05-7.92(m,1H),7.82(d,J=8.7Hz,1H),7.63(d,J=7 .9Hz,2H),7.47(d,J=7.9Hz,2H),3.83(td,J=9.1,5.2Hz,1H),3.75(t,J=7.4Hz,3H),3. 66(d,J=14.0Hz,1H),3.49(d,J=13.8Hz,1H),2.89(d,J=11.2Hz,1H),2.56(d,J=11.0Hz ,1H),2.51-2.37(m,1H),2.28(d,J=11.0Hz,1H),2.23-2.08(m,2H),1.81-1.73(m,2H).

[0180] Example 23: Synthesis of III-4

[0181]

[0182] Step 1: Synthesis of III-4-1

[0183] p-Cyanobenzic acid (76 mg, 0.518 mmol), HATU (208 mg, 0.622 mmol), and DIPEA (61.5 μL, 0.78 mmol) were dissolved in dichloromethane (12 mL) and stirred at room temperature for 2 h. Then, tert-butyl 6-amino-2-azaspiro[3.3]heptane-2-carboxylic acid (100 mg, 0.471 mmol) was added, and stirring continued overnight. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (developing solvent: dichloromethane:methanol = 100:1) to give a white solid III-4-1 (134 mg), yield 83%.

[0184] Step 2: Synthesis of III-4-2

[0185] Using III-4-1 (300 mg, 0.708 mmol) as the raw material, the preparation method was the same as in 2, yielding a white solid III-4-2 (63 mg) with a yield of 74%.

[0186] Step 3: Synthesis of III-4

[0187] A mixture of III-4-2 (520 mg, 2.16 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (611 mg, 3.23 mmol), potassium carbonate (1.04 g, 7.54 mmol), and DMF (30 mL) was heated and stirred at 70 °C for 2.5 h. After the reaction was complete, 30 mL of water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 120:1) to give a white solid III-4 (386 mg), yield 44%. 1 H NMR (300MHz, DMSO-d6) δ8.92(d,J=7.2Hz,5H),8.06-7.92(m,20H),7.82-7.70(m,5H),6.73(d,J=2.3Hz,5H),6.65(dd,J=8.6,2.3Hz,5H),4 .35(h,J=8.1Hz,5H),4.08(d,J=32.5Hz,20H),2.61(ddd,J=10.2,7.7,2.9Hz,10H),2.44-2.29(m,10H),1.46(s,2H),1.23(d,J=6.9Hz,1H).

[0188] Example 24: Synthesis of III-5

[0189]

[0190] Step 1: Synthesis of III-5-1

[0191] Using tert-butyl ester of 2,6-diazaspiro[3,4]octane-2-carboxylic acid (500 mg, 2.36 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (668 mg, 3.53 mmol), potassium carbonate (651 mg, 4.71 mmol), and DMF (20 mL) as raw materials, the same method as for III-4 was used to prepare a white solid III-5-1 (670 mg), with a yield of 74%.

[0192] Step 2: Synthesis of III-5-2

[0193] Using III-5-1 (300 mg, 0.787 mmol) as the raw material, the preparation method was the same as in 2, yielding a white solid III-5-2 (142 mg) with a yield of 64%.

[0194] Step 3: Synthesis of III-5

[0195] Using III-5-2 (150 mg, 0.533 mmol), 5-(bromomethyl)-2-fluorobenzonitrile (157 mg, 0.8 mmol), DMF (15 mL), and triethylamine (149 μL, 1.07 mmol) as raw materials, the same method as I-1 was used to prepare a white solid III-5 (80 mg), with a yield of 38%. 1 H NMR (300MHz, DMSO-d6) δ7.78(dd,J=8.4,2.1Hz,3H),7.49(d,J=8.2Hz,2H),6.87(d,J=2.4Hz,1H),6.81(d d,J=8.9,2.5Hz,1H),3.69(s,2H),3.54(s,2H),3.41(d,J=6.8Hz,2H),3.20(s,4H),2.17(t,J=6.8Hz,2H).

[0196] Example 25: Synthesis of III-6

[0197]

[0198] Step 1: Synthesis of III-6-1

[0199] p-Cyanobenyl chloride (165 mg, 0.994 mmol) was dissolved in anhydrous DCM (15 mL), and tert-butyl 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid (136 μL, 0.663 mmol) and triethylamine (185 μL, 1.33 mmol) were added. The mixture was stirred at room temperature for 5 h. After the reaction was completed, the mixture was extracted three times with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (developing solvent: dichloromethane:methanol = 120:1) to give III-6-1 (180 mg). 1 ¹H NMR (300MHz, DMSO-d⁶) δ 8.88 (dd, J = 10.8, 7.1 Hz, 1H), 8.07–7.90 (m, 4H), 4.43 (h, J = 7.9 Hz, 1H), 3.32–3.16 (m, 4H), 2.34–2.19 (m, 2H), 2.16–2.03 (m, 2H), 1.95–1.77 (m, 2H), 1.39 (s, 9H). Yield 76%; Step 2: Synthesis of III-6-2

[0200] Using intermediate 13 (235 mg, 0.661 mmol) as the starting material, the same method as S2 was used to prepare a white solid III-6-2 (109 mg), with a yield of 65%.

[0201] Step 3: Synthesis of III-6

[0202] Using III-6-2 (168 mg, 0.658 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (187 mg, 0.987 mmol), potassium carbonate (182 mg, 1.32 mmol), and DMF (10 mL) as raw materials, the same method as for III-4 was used to prepare a white solid III-6 (197 mg), with a yield of 70.5%. 1 H NMR (400MHz, DMSO-d6) δ8.92(dd,J=7.3,3.5Hz,1H),8.09-7.93(m,4H),7.83-7.73(m,1H),6.98-6.75(m,2H),4.50(dh,J=16.1,8 .1Hz,1H),3.58-3.41(m,2H),3.39(d,J=2.8Hz,2H),2.40-2.30(m,2H),2.19(qd,J=8.8,2.6Hz,2H),2.07(dt,J=30.2,6.9Hz,2H).

[0203] Example 26: Synthesis of IV-1

[0204]

[0205] Step 1: Synthesis of IV-1-1

[0206] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (120 mg, 0.53 mmol), 5-acetyl-1H-pyrazole-3-carboxylic acid (90 mg, 0.583 mmol), HATU (60.5 mg, 0.636 mmol), and DIPEA (139 μL, 0.78 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a white solid IV-1-1 (130 mg) with a yield of 68%.

[0207] Step 2: Synthesis of IV-1-2

[0208] Using IV-1-1 (260 mg, 0.717 mmol) as raw material, the same preparation method as S2 was used to obtain white solid IV-1-2 (147 mg), with a yield of 78%.

[0209] Step 3: Synthesis of IV-1-3

[0210] Using IV-1-2 (270 mg, 1.03 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (293 mg, 1.55 mmol), potassium carbonate (500 mg, 3.62 mmol), and DMF (15 mL) as raw materials, the same method as III-4 was used to prepare a white solid IV-1-3 (263 mg), with a yield of 59%.

[0211] Step 4: Synthesis of IV-1

[0212] Intermediate 18 (130 mg, 0.301 mmol) was dissolved in methanol, and sodium borohydride (46 mg, 1.21 mmol) was added under ice bath conditions, followed by stirring for 1.5 h. After the reaction was complete, a small amount of saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted three times with ethyl acetate / water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was separated by silica gel chromatography (eluent: dichloromethane:methanol = 100:1) to give a white solid IV-1 (119 mg), yield 91%. 1 HNMR(300MHz,DMSO-d6)δ13.07(s,1H),8.32(t,J=7.3Hz,1H),7.78(d,J=8.7Hz,1H ),6.88-6.74(m,3H),6.45(s,1H),5.45(d,J=5.0Hz,1H),4.79(q,J=6.2Hz,1H),4. 47(q,J=9.0,8.2Hz,1H),3.51(s,1H),3.43(d,J=6.4Hz,2H),2.25(d,J=8.4Hz,4H) ,2.08(t,J=6.6Hz,2H),1.99(s,1H),1.39(d,J=6.5Hz,4H),1.18(t,J=7.1Hz,1H).

[0213] Example 27: Synthesis of IV-2

[0214]

[0215] Step 1: Synthesis of IV-2-1

[0216] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (120 mg, 0.53 mmol) and p-methylbenzoic acid (66 mg, 0.486 mmol) as raw materials, the preparation method was the same as that of III-4-1, and a white solid IV-2-1 (140 mg) was obtained with a yield of 92%.

[0217] Step 2: Synthesis of IV-2-2

[0218] Using IV-2-1 (140 mg, 0.406 mmol) as the raw material, the preparation method was the same as that of S2, and a white solid IV-2-2 (52 mg) was obtained with a yield of 52%.

[0219] Step 3: Synthesis of IV-2

[0220] Using IV-2-2 (70 mg, 0.286 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (81 mg, 0.430 mmol), potassium carbonate (198 mg, 1.43 mmol), and DMF (10 mL) as raw materials, the same method as III-4 was used to prepare a white solid IV-2 (70 mg) with a yield of 59%. 1 H NMR (300MHz, DMSO-d6) δ8.61(d,J=7.3Hz,1H),7.77(dd,J=8.6,2.6Hz,3H),7.27(d,J=7.8Hz,2H),6.98-6.68(m,2H),4.4 9(p,J=8.1,6.5Hz,1H),3.43(t,J=6.9Hz,2H),2.35(s,3H),2.30(d,J=9.2Hz,2H),2.24-2.14(m,2H),2.14-2.01(m,2H).

[0221] Example 28: Synthesis of IV-3

[0222]

[0223] Step 1: Synthesis of IV-3-1

[0224] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (100 mg, 0.442 mmol) and p-fluorobenzoic acid (68 mg, 0.486 mmol) as raw materials, the preparation method was the same as that in III-4-1, yielding white solid 21 (137 mg) with a yield of 89%.

[0225] Step 2: Synthesis of IV-3-2

[0226] Using IV-3-1 (137 mg, 0.393 mmol) as the raw material, the preparation method was the same as S2, and a white solid IV-3-1 (80 mg) was obtained with a yield of 82%.

[0227] Step 3: Synthesis of IV-3

[0228] Using IV-3-2 (80 mg, 0.322 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (91 mg, 0.483 mmol), potassium carbonate (223 mg, 1.61 mmol), and DMF (10 mL) as raw materials, the same method as III-4 was used to prepare IV-3 (73 mg) as a white solid, with a yield of 54%. 1H NMR(300MHz, DMSO-d6)δ8.78-8.60(m,1H),7.93(ddd,J=8.8,4.5,1.6Hz,2H),7.79(d,J=8.7Hz,1H),7.38-7.24(m,2H),6.95-6.76(m,2H),4.48 (hept,J=8.0Hz,1H),3.44(t,J=6.8Hz,2H),2.33(td,J=8.5,2.5Hz,2H),2.18(td,J=8.7,2.5Hz,2H),2.12(d,J=6.7Hz,1H),2.10-2.00(m,1H).

[0229] Example 29: Synthesis of IV-4

[0230]

[0231] Step 1: Synthesis of IV-4-1

[0232] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (140 mg, 0.618 mmol) and p-methoxybenzoic acid (103 mg, 0.68 mmol) as raw materials, the preparation method was the same as in 10, and a white solid IV-4-1 (194 mg) was obtained with a yield of 87%.

[0233] Step 2: Synthesis of IV-4-2

[0234] Using IV-4-1 (150 mg, 0.416 mmol) as the raw material, the preparation method was the same as that of S2, and a white solid IV-4-2 (84 mg) was obtained with a yield of 78%.

[0235] Step 3: Synthesis of IV-4

[0236] Using IV-4-2 (80 mg, 0.322 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (91 mg, 0.483 mmol), potassium carbonate (223 mg, 1.61 mmol), and DMF (10 mL) as raw materials, the same method as III-4 was used to prepare IV-4, yielding a white solid IV-4 (70 mg) with a yield of 52%. 1H NMR (300MHz, DMSO-d6) δ8.51(d,J=7.3Hz,1H),7.89-7.81(m,2H),7.78(d,J=8.6Hz,1H),7.04-6.95(m,2H),6.93-6.76(m ,2H),4.58-4.36(m,1H),3.81(s,3H),3.56-3.39(m,2H),3.37(s,2H),2.25(dt,J=42.1,10.0Hz,4H),2.13-2.02(m,2H).

[0237] Example 30: Synthesis of IV-5

[0238]

[0239] Step 1: Synthesis of IV-5-1

[0240] Using tert-butyl 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid (150 mg, 0.664 mmol) and 4'-cyano-[1,1'-biphenyl]-4-carboxylic acid (170 mg, 0.762 mmol) as raw materials, the same method as for III-4-1 was used to prepare IV-5-1 (262 mg) as a white solid, with a yield of 92%.

[0241] Step 2: Synthesis of IV-5-2

[0242] Using IV-5-1 (141 mg, 0.372 mmol) as the raw material, the preparation method was the same as that of S2, and a white solid IV-5-2 (96 mg) was obtained with a yield of 81%.

[0243] Step 3: Synthesis of IV-5

[0244] Using IV-5-2 (350 mg, 1.06 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (319 mg, 1.69 mmol), potassium carbonate (730 mg, 5.28 mmol), and DMF (25 mL) as raw materials, the same method as III-4 was used to prepare a white solid IV-5 (287 mg), with a yield of 54.3%. 1H NMR (300MHz, DMSO-d6) δ8.81(dd,J=7.4,3.1Hz,1H),8.04-7.97(m,2H),7.96(s,4H),7.92-7.83(m,2H),7.77(d,J=8.7Hz,1H),6.94- 6.73(m,2H),4.60-4.40(m,1H),3.43(t,J=6.7Hz,2H),2.43-2.28(m,2H),2.21(td,J=8.8,2.4Hz,2H),2.07(dt,J=21.4,6.7Hz,2H).

[0245] Example 31: Synthesis of IV-6

[0246]

[0247] Step 1: Synthesis of IV-6-1

[0248] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (120 mg, 0.53 mmol) and p-fluorobenzoic acid (97 mg, 0.583 mmol) as raw materials, the preparation method was the same as that of III-4-1, and a white solid IV-6-1 (180 mg) was obtained with a yield of 90%.

[0249] Step 2: Synthesis of IV-6-2

[0250] Using IV-6-1 (180 mg, 0.479 mmol) as the raw material, the same method as S2 was used to prepare white solid IV-6-2 (98 mg), with a yield of 75%.

[0251] Step 3: Synthesis of IV-6

[0252] Using IV-6-2 (80 mg, 0.322 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (91 mg, 0.483 mmol), potassium carbonate (223 mg, 1.61 mmol), and DMF (10 mL) as raw materials, the preparation method was the same as that for III-4, yielding a white solid IV-6 (73 mg) with a yield of 54%. 1H NMR(300MHz,DMSO-d6)δ9.00(d,J=7.1Hz,1H),8.40-8.22(m,2H),8.15-8.02(m,2H),7.76(d,J=8.7Hz,1H),6.97-6.70(m,2H),4.4 7(q,J=7.7Hz,1H),3.42(t,J=6.7Hz,2H),3.36(s,2H),2.33(t,J=9.5Hz,2H),2.18(t,J=9.7Hz,2H),2.05(dt,J=23.5,6.9Hz,2H).

[0253] Example 32: Synthesis of IV-7

[0254]

[0255] Step 1: Synthesis of IV-7-1

[0256] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (110 mg, 0.486 mmol) and benzoic acid (65 mg, 0.535 mmol) as raw materials, the preparation method was the same as that of III-4-1, and a white solid IV-7-1 (148 mg) was obtained with a yield of 98%.

[0257] Step 2: Synthesis of IV-7-2

[0258] Using IV-7-1 (148 mg, 0.449 mmol) as the raw material, the preparation method was the same as that of S2, and a white solid IV-7-2 (84 mg) was obtained with a yield of 81%.

[0259] Step 3: Synthesis of IV-7

[0260] Using IV-7-2 (80 mg, 0.322 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (91 mg, 0.483 mmol), potassium carbonate (223 mg, 1.61 mmol), and DMF (10 mL) as raw materials, the same method as III-4 was used to prepare a white solid IV-7 (81 mg) with a yield of 58%. 1H NMR (300MHz, DMSO-d6) δ8.76-8.59(m,1H),7.86(d,J=7.4Hz,2H),7.79(d,J=8.7Hz,1H),7.49(dt,J=14.7,7.5Hz,3H),6.99-6.73(m,2H) ,4.48(q,J=8.1Hz,1H),3.56-3.40(m,2H),2.54-2.46(m,2H),2.33(t,J=9.3Hz,2H),2.20(t,J=9.7Hz,2H),2.07(dt,J=22.8,7.2Hz,2H).

[0261] Example 33: Synthesis of IV-8

[0262]

[0263] Step 1: Synthesis of IV-8-1

[0264] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (120 mg, 0.476 mmol) and 4-trifluoromethylbenzoic acid (65 mg, 0.535 mmol) as raw materials, the preparation method was the same as that of III-4-1, and a white solid IV-8-1 (136 mg) was obtained with a yield of 95%.

[0265] Step 2: Synthesis of IV-8-2

[0266] Using intermediate IV-8-1 (120 mg, 0.431 mmol) as the raw material, the preparation method was the same as that of S2, yielding white solid IV-8-2 (81 mg), with a yield of 76%.

[0267] Step 3: Synthesis of IV-8

[0268] Using intermediate IV-8-2 (80 mg, 0.365 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (103 mg, 0.547 mmol), potassium carbonate (101 mg, 0.729 mmol), and DMF (10 mL) as raw materials, the preparation method was the same as that for III-4, yielding a white solid IV-8 (63 mg) with a yield of 58%. 1H NMR (300MHz, DMSO-d6) δ8.93(dd,J=7.5,2.5Hz,1H),8.06(d,J=8.1Hz,2H),7.86(d,J=8.2Hz,2H),7.78(d,J=8.4Hz,1H),6.96-6.74(m,2H),4.60-4.3 9(m,1H),3.50(d,J=22.1Hz,1H),3.43(d,J=7.0Hz,1H),2.36(dddd,J=10.4 ,8.3,6.2,2.8Hz,2H),2.21(t,J=9.2Hz,2H),2.07(dt,J=23.0,6.8Hz,2H).

[0269] Example 34: Synthesis of IV-9

[0270]

[0271] Step 1: Synthesis of IV-9-1

[0272] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (120 mg, 0.476 mmol) and p-hydroxybenzoic acid (70 mg, 0.495 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a white solid IV-9-1 (125 mg) with a yield of 91%.

[0273] Step 2: Synthesis of IV-9-2

[0274] Using intermediate IV-9-1 (125 mg, 0.431 mmol) as a raw material, the preparation method was the same as S2, yielding white solid IV-9-2 (81 mg), with a yield of 76%.

[0275] Step 3: Synthesis of IV-9

[0276] Using intermediate IV-9-2 (81 mg, 0.365 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (103 mg, 0.547 mmol), potassium carbonate (101 mg, 0.729 mmol), and DMF (10 mL) as raw materials, the preparation method was the same as that for III-4, yielding a white solid IV-9 (48 mg) with a yield of 40%. 1H NMR (300MHz, DMSO-d6) δ8.50-8.40(m,1H),7.87(d,J=8.7Hz,1H),7.80(dd,J=8.7,1.6Hz,2H),7.03-6.89(m,2H),6.86(dd,J=9. 1,2.4Hz,2H),4.54(q,J=8.1Hz,1H),3.66-3.48(m,4H),2.48-2.33(m,2H),2.26(t,J=9.8Hz,2H),2.15(dt,J=21.8,6.8Hz,2H).

[0277] Example 35: Synthesis of IV-10

[0278]

[0279] Step 1: Synthesis of IV-10-1

[0280] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (250 mg, 1.10 mmol) and monomethyl terephthalate (219 mg, 1.22 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a white solid IV-10-1 (370 mg) in a yield of 86%.

[0281] Step 2: Synthesis of IV-10-2

[0282] Using intermediate IV-10-1 (400 mg, 1.03 mmol) as a raw material, the preparation method was the same as S2, yielding white solid IV-10-2 (280 mg) with a yield of 94%.

[0283] Step 3: Synthesis of IV-10

[0284] Using intermediate IV-10-2 (280 mg, 0.971 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (202 mg, 1.07 mmol), and potassium carbonate (267 mg, 1.94 mmol) as raw materials, and DMF (3 mL) as solvent, the preparation method was the same as that of III-4, yielding a white solid IV-10 (304 mg) with a yield of 68%. 1H NMR(300MHz,DMSO-d6)δ8.88(d,J=7.2Hz,1H),8.05(dd,J=8.5,2.1Hz,2H),7. 97(d,J=8.5,2.1Hz,2H),7.78(d,J=8.7Hz,1H),6.86(d,J=2.4Hz,1H),6.81(dd ,J=8.7,2.4Hz,1H),4.58-4.40(m,1H),3.89(s,3H),3.44(t,J=6.7Hz,2H),3. 38(s,2H),2.34(t,J=8.6Hz,2H),2.20(t,J=8.6Hz,2H),2.11(t,J=6.7Hz,2H).

[0285] Example 36: Synthesis of IV-11

[0286]

[0287] IV-10 (150 mg, 0.328 mmol) was dissolved in THF, and 2 M LiOH aqueous solution was added. The reaction was carried out overnight at room temperature. After the reaction was completed by TLC monitoring, the solvent was evaporated under reduced pressure, acidified with 1 M HCl, filtered, and the filter cake was washed with water and recrystallized from methanol to give a white solid IV-11 (110 mg), with a yield of 76%. 1 H NMR (300MHz, DMSO-d6) δ13.22 (s, 1H), 8.86 (d, J = 7.4, 1H), 8.02 (dd, J = 8.5, 2. 1Hz,2H),7.95(dd,J=8.5,2.1Hz,2H),7.78(d,J=8.8Hz,1H),6.86(d,J=2.4Hz ,1H),6.80(dd,J=8.8,2.4Hz,1H),4.61-4.41(m,1H),3.44(t,J=6.7Hz,2H),3 .38(s,2H),2.43-2.28(m,2H),2.20(td,J=8.8,2.5Hz,2H),2.15-1.99(m,2H).

[0288] Example 37: Synthesis of IV-12

[0289]

[0290] Step 1: Synthesis of IV-12-1

[0291] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (200 mg, 0.884 mmol) and 4-carboxybenzoate ethyl ester (189 mg, 0.972 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a white solid IV-12-1 (320 mg) with a yield of 90%. 1 H NMR(300MHz,DMS O-d6)δ8.87-8.74(m,1H),8.03(d,J=8.3Hz,2H),7.97(d,J=8.3Hz,2H),4.53-4.40(m,1H),4.35(q,J=7.1Hz,2H),3.32-3.22(m,2H ),3.20(s,2H),2.27(dt,J=10.5,8.1Hz,2H),2.11(dt,J=10.5,5.2Hz,2H),1.96-1.76(m,2H),1.41(s,9H),1.34(t,J=7.1Hz,3H).

[0292] Step 2: Synthesis of IV-12-2

[0293] IV-12-1 (300 mg, 0.745 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed as monitored by TLC, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0294] Step 3: Synthesis of IV-12

[0295] The crude IV-12-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (155 mg, 0.819 mmol) and potassium carbonate (206 mg, 1.49 mmol) were added. The preparation method was the same as that for III-4, yielding a white solid IV-12 (304 mg) with a yield of 87%. 1 H NMR(300MHz,DM SO-d6)δ8.89(d,J=7.2Hz,1H),8.04(d,J=8.5Hz,2H),7.98(d,J=8.5Hz,2H),7.78( d,J=8.6Hz,1H),6.86(d,J=2.4Hz,1H),6.80(dd,J=8.6,2.4Hz,1H),4.61-4.41(m, 1H), 4.35 (q, J = 7.1Hz, 2H), 3.44 (t, J = 6.7Hz, 2H), 3.38 (s, 2H), 2.34 (td, J = 8.7, 2. 5Hz,2H),2.21(td,J=8.7,2.5Hz,2H),2.11(t,J=6.6Hz,2H),1.34(t,J=7.1Hz,3H).

[0296] Example 38: Synthesis of IV-13

[0297]

[0298] Step 1: Synthesis of IV-13-1

[0299] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (100 mg, 0.442 mmol) and 4-(4-methylpiperazine)benzoic acid (108 mg, 0.486 mmol) as raw materials, the same method as III-4-1 was used to prepare IV-13-1 (110 mg) as a yellow solid, with a yield of 95%. 1 H NMR (300MHz, DMSO-d6) δ8.43-8.21(m,1H),7.73(d,J=8.4Hz,2H),6.94(d,J=8.6Hz,2H),4.39(p,J=8.2Hz,1H) ,3.28-3.14(m,8H),2.44(t,J=5.0Hz,4H),2.22(s,5H),2.14-2.00(m,2H),1.85(p,J=7.0Hz,2H),1.39(s,9H).

[0300] Step 2: Synthesis of IV-13-2

[0301] Using intermediate IV-13-1 (110 mg, 0.257 mmol) as a raw material, the same method as S2 was used to prepare yellow solid IV-12-2 (80 mg), with a yield of 95%.

[0302] Step 3: Synthesis of IV-13

[0303] Using intermediate IV-13-2 (80 mg, 0.244 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (51 mg, 0.268 mmol), and potassium carbonate (68 mg, 0.487 mmol) as raw materials, and DMF (3 mL) as solvent, the preparation method was the same as that of III-4, yielding white solid IV-13 (58 mg) with a yield of 48%. 1H NMR(300MHz,DMSO-d6)δ8.37(d,J=7.4Hz,1H),7.78(d,J=8.7Hz,1H),7.76-7.70(m ,2H),7.00-6.91(m,2H),6.86(d,J=2.4Hz,1H),6.80(dd,J=8.7,2.4Hz,1H),4.55- 4.37(m,1H),3.43(t,J=6.7Hz,2H),3.37(s,2H),3.24(t,J=5.0Hz,4H),2.43(t,J= 5.1Hz,4H),2.37-2.25(m,2H),2.17(td,J=8.9,2.4Hz,2H),2.09(t,J=6.7Hz,2H).

[0304] Example 39: Synthesis of IV-14

[0305]

[0306] Step 1: Synthesis of IV-14-1

[0307] In a single-necked flask, methylamino hydrochloride (409 mg, 6.06 mmol), EDCI (1.45 g, 7.56 mmol), HOBt (7.62 mmol), and N-methylmorpholine (28 μL, 0.255 mmol) were added sequentially to a DMF solution of 2-fluoro-4-(methoxycarbonyl)benzoic acid (1 g, 5.05 mmol). The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC, 30 mL of water was added, and the mixture was extracted with EA (10 mL x 3). The organic phases were combined, washed with saturated brine, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 5:1) to give 1.02 g of white solid IV-14-1, with a yield of 96%. 1 H NMR (300MHz, DMSO-d6) δ8.46 (d, J = 5.3Hz, 1H), 7.83 (dt, J = 7.9, 1.7Hz, 1H), 7.79-7.70 (m, 2H), 3.88 (s, 3H), 2.80 (d, J = 4.6Hz, 3H).

[0308] Step 2: Synthesis of IV-14-2

[0309] IV-14-1 (430 mg, 2.04 mmol) was dissolved in a mixture of acetonitrile and water (acetonitrile:water = 10:1, 5.5 mL), and lithium bromide (354 mg, 4.07 mmol) and DIPEA (532 μL, 3.05 mmol) were added. The mixture was reacted overnight at room temperature. After the reaction was completed as monitored by TLC, the reaction mixture was filtered under reduced pressure. The filter cake was washed with acetonitrile, suspended in water (8 mL), acidified with concentrated hydrochloric acid (3 mL), and filtered under reduced pressure to give 270 mg of white solid IV-14-2, yield 67%. 1 H NMR (300MHz, DMSO-d6) δ13.31(s,1H),8.45(q,J=4.6Hz,1H),7.82(dd,J=8.0,1.5Hz,1H),7.76-7.67(m,2H),2.79(d,J=4.6Hz,3H).

[0310] Step 3: Synthesis of IV-14-3

[0311] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (150 mg, 0.663 mmol) and IV-14-2 (144 mg, 0.729 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding yellow solid IV-14-3 (220 mg) with a yield of 82%. 1 H NMR (400MHz, DMSO-d6) δ8.88-8.71(m,1H),8.45-8.31(m,1H),7.79-7.66(m,3H),4.50-4.34(m,1H),3.33- 3.16(m,4H),2.79(d,J=4.6Hz,3H),2.34-2.20(m,2H),2.17-2.03(m,2H),1.95-1.77(m,2H),1.40(s,9H).

[0312] Step 4: Synthesis of IV-14-4

[0313] Using intermediate IV-14-3 (200 mg, 0.493 mmol) as the raw material, the same method as S2 was used to prepare a pale yellow solid IV-14-4 (120 mg) with a yield of 80%.

[0314] Step 5: Synthesis of IV-14

[0315] Using intermediate IV-14-4 (120 mg, 0.393 mmol), 4-fluoro-2-(trifluoromethyl)benzonitrile (82 mg, 0.432 mmol), and potassium carbonate (109 mg, 0.786 mmol) as raw materials, and DMF (3 mL) as solvent, the same method as for III-4 was used to prepare a white solid IV-13 (160 mg), with a yield of 86%. 1 H NMR (300MHz, DMSO-d6) δ8.84(d,J=6.9Hz,1H),8.38(d,J=4.2Hz,1H),7.87-7.62(m,4H),6.96-6.72(m,2H),4.59-4. 36(m,1H),3.59-3.36(m,4H),2.79(d,J=4.2Hz,3H),2.34(t,J=9.0Hz,2H),2.20(t,J=9.0Hz,2H),2.14-1.99(m,2H).

[0316] Example 40: Synthesis of IV-15

[0317]

[0318] Step 1: Synthesis of IV-15-1

[0319] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (250 mg, 1.10 mmol) and 4-hydroxymethylbenzoic acid (185 mg, 1.22 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a colorless oil IV-15-1 (290 mg) with a yield of 73%.

[0320] Step 2: Synthesis of IV-15-2

[0321] IV-15-1 (278 mg, 0.771 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0322] Step 3: Synthesis of IV-15

[0323] The crude IV-15-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (160 mg, 0.845 mmol) and potassium carbonate (213 mg, 1.54 mmol) were added. The preparation method was the same as that for III-4, yielding a white solid IV-15 (270 mg). The yield of the two steps was 82%. 1H NMR(300MHz,DMSO-d6)δ8.61(dd,J=7.4,3.2Hz,1H),7.80(d,J=8.0Hz,2H),7 .76(d,J=8.5Hz,1H),7.37(d,J=8.0Hz,2H),6.94-6.72(m,2H),5.30(t,J=5.7 Hz,1H),4.53(d,J=5.7Hz,2H),4.50-4.38(m,1H),3.41(t,J=6.8Hz,2H),3.3 5(s,2H),2.32(t,J=9.2Hz,2H),2.17(t,J=9.2Hz,2H),2.04(t,J=6.8Hz,2H).

[0324] Example 41: Synthesis of IV-16

[0325]

[0326] Step 1: Synthesis of IV-16-1

[0327] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (200 mg, 0.884 mmol) and 6-methylnicotinic acid (134 mg, 0.972 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding a white solid IV-16-1 (250 mg) with a yield of 82%. 1 H NMR (300MHz, DMSO-d6) δ8.88(d,J=2.4Hz,1H),8.79-8.67(m,1H),8.08(dd,J=8.4,2.4Hz,1H),7.36(d,J=8.4Hz,1H),4.51-4 .34(m,1H),3.32-3.12(m,4H),2.52(s,3H),2.33-2.19(m,2H),2.10(td,J=8.8,2.6Hz,2H),1.94-1.78(m,2H),1.39(s,9H).

[0328] Step 2: Synthesis of IV-16-2

[0329] IV-16-1 (220 mg, 0.637 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed as monitored by TLC, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0330] Step 3: Synthesis of IV-16

[0331] The crude IV-16-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (133 mg, 0.700 mmol) and potassium carbonate (176 mg, 1.27 mmol) were added. The preparation method was the same as that for III-4, yielding a white solid IV-16 (230 mg). The yield of the two steps was 87%. 1 H NMR(300MHz,DMSO-d6)δ8.90(d,J=2.4Hz,1H),8.79(d,J=7.1Hz,1H),8.09(dd,J=8.1 ,2.4Hz,1H),7.78(d,J=8.7Hz,1H),7.36(d,J=8.1Hz,1H),6.86(d,J=2.4Hz,1H),6.80 (dd,J=8.7,2.4Hz,1H),4.58-4.38(m,1H),3.44(t,J=6.7Hz,2H),3.38(s,2H),2.52(s ,3H),2.34(td,J=8.9,2.5Hz,2H),2.19(td,J=8.9,2.5Hz,2H),2.11(t,J=6.7Hz,2H).

[0332] Example 42: Synthesis of IV-17

[0333]

[0334] Step 1: Synthesis of IV-17-1

[0335] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (200 mg, 0.884 mmol) and 4-(2-hydroxyethoxy)-benzoic acid (194 mg, 1.06 mmol) as raw materials, the same method as III-4-1 was used to prepare IV-17-1 (260 mg) as a pale yellow solid, with a yield of 75%. 1 H NMR (300MHz, DMSO-d6) δ8.53-8.35(m,1H),7.80(d,J=8.5Hz,2H),6.98(d,J=8.5Hz,2H),4.88(t,J=5.3Hz,1H),4.50-4.28(m,1H),4. 03(t,J=5.0Hz,2H),3.73(t,J=5.0Hz,2H),3.19-3.15(m,4H),2.31-2.15(m,2H),2.14-1.98(m,2H),1.94-1.75(m,2H),1.39(s,9H).

[0336] Step 2: Synthesis of IV-17-2

[0337] IV-17-1 (220 mg, 0.563 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0338] Step 3: Synthesis of IV-17

[0339] The crude IV-17-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (117 mg, 0.618 mmol) and potassium carbonate (156 mg, 1.13 mmol) were added. The preparation method was the same as that for III-4, yielding a white solid IV-17 (175 mg). The two-step yield was 68%. 1 H NMR (300MHz, Chloroform-d) δ7.75(dd,J=8.8,2.3Hz,2H),7.57(t,J=8.0Hz,1H),6.94(d,J=8.7Hz,2H),6.76(dd,J=11.5,2.4Hz,1H),6.59(td,J=11.5,8. 7,2.4Hz,1H),6.49-6.29(m,1H),4.75-4.53(m,1H),4.13(t,J=4.4Hz,2H),4 .00(t,J=4.4Hz,2H),3.56-3.27(m,5H),2.66-2.46(m,2H),2.26-2.0(m,4H).

[0340] Example 43: Synthesis of IV-18

[0341]

[0342] In a single-necked flask, deuterated methylamine hydrochloride (32 mg, 0.440 mmol) was added sequentially to a DMF solution of IV-11 (130 mg, 0.293 mmol) in the same manner as IV-14-1, to obtain a white solid IV-14-1 (90 mg), with a yield of 67%. 1 HNMR (300MHz, DMSO-d6) δ8.77(d,J=7.2Hz,1H),8.53(s,1H),7.90(s,4H),7.77(d,J=8.6Hz,1H),6.99-6.71( m,2H),4.60-4.36(m,1H),3.58-3.38(m,4H),2.34(t,J=9.5Hz,2H),2.21(t,J=9.7Hz,2H),2.14-1.91(m,2H).

[0343] Example 44: Synthesis of IV-19

[0344]

[0345] Step 1: Synthesis of IV-19-1

[0346] Using tert-butyl 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid (350 mg, 1.55 mmol) and 4-bromobenzoic acid (342 mg, 1.70 mmol) as raw materials, the same method as III-4-1 was used to prepare IV-19-1 (580 mg) as a pale yellow solid, with a yield of 92%. 1 H NMR(300MHz,Chloroform-d)δ7.77-7.62(m,2H),7.58-7.47(m,2H),7.13-6.86(m,1H),4.64-4 .43(m,1H),3.40-3.23(m,4H),2.36(s,2H),2.21-2.01(m,2H),1.96-1.78(m,2H),1.43(s,9H).

[0347] Step 2: Synthesis of IV-19-2

[0348] IV-19-1 (200 mg, 0.489 mmol) and pinarate 2-cyanopyridine-5-borate (135 mg, 0.586 mmol) were dissolved in dioxane (6 mL), followed by the addition of [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (18 mg, 0.024 mmol) and cesium carbonate (319 mg, 0.978 mmol). Then, 1 mL of water was added, and the reaction was carried out at 100 °C for 12 h under nitrogen protection. After the reaction was complete as monitored by TLC, the reaction solution was cooled to room temperature, quenched with water (10 mL), extracted with EA, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (PE:EA = 10:1) to give 170 mg of a pale yellow solid, with a yield of 80%. 1 HNMR(300MHz,DMSO-d6)δ9.17(d,J=2.3Hz,1H),8.82-8.68(m,1H),8.43(dd,J=8.2,2.3Hz,1H),8.16(d,J=8.2Hz,1H),8.01(d,J=8.3Hz,2H) ,7.96(d,J=8.3Hz,2H),4.53-4.36(m,1H),3.31-3.16(m,4H),2.27(t,J=9.3Hz,2H),2.13(t,J=9.7Hz,2H),1.96-1.77(m,2H),1.39(s,9H).

[0349] Step 3: Synthesis of IV-19-3

[0350] IV-19-2 (120 mg, 0.277 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0351] Step 3: Synthesis of IV-19

[0352] The crude IV-19-3 from step 3 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (58 mg, 0.305 mmol) and potassium carbonate (77 mg, 0.554 mmol) were added. The preparation method was the same as that for III-4, yielding a white solid IV-19 (118 mg). The two-step yield was 82%. 1 H NMR (300MHz, DMSO-d6) δ9.01(d,J=2.2Hz,1H),8.80(dd,J=7.3,3.0Hz,1H),8.34(dd,J=8. 2,2.2Hz,1H),8.18(s,1H),8.14(d,J=8.2Hz,1H),8.02(d,J=8.2Hz,2H),7.93(d,J=8.7Hz ,2H),7.78(d,J=8.7Hz,1H),7.74(s,1H),6.96-6.75(m,2H),4.63-4.42(m,1H),3.45(t,J =6.7Hz,2H),3.39(s,2H),2.35(t,J=9.7Hz,2H),2.24(d,J=9.7Hz,2H),2.16-1.98(m,2H).

[0353] Example 45: Synthesis of IV-20

[0354]

[0355] Step 1: Synthesis of IV-20-1

[0356] Using IV-19-1 (200 mg, 0.489 mmol) and 4-cyano-3-fluorophenylboronic acid pinacol ester (181 mg, 0.733 mmol) as raw materials, the same method as IV-19-2 was used to prepare IV-20-1 (145 mg) as a yellow solid, with a yield of 66%. 1H NMR(300MHz,Chloroform-d)δ7.91(d,J=8.0Hz,2H),7.73(dd,J=8.1,6.6Hz,1H),7.66(d,J=8.0Hz,2H),7.52(dd,J=8.1,1.5Hz,1H),7.47(dd,J=10 .0,1.5Hz,1H),6.46(s,1H),4.72-4.54(m,1H),3.47-3.26(m,4H),2.49( t,J=10.0Hz,2H),2.05(t,J=10.0Hz,2H),2.01-1.85(m,2H),1.47(s,9H).

[0357] Step 2: Synthesis of IV-20-2

[0358] IV-20-1 (130 mg, 0.289 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0359] Step 3: Synthesis of IV-20

[0360] The crude IV-20-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (66 mg, 0.347 mmol) and potassium carbonate (80 mg, 0.578 mmol) were added. The preparation method was the same as that of III-4, yielding a grayish-white solid IV-20 (105 mg). The yield of the two steps was 70%. 1 H NMR(300MHz,DMSO-d6)δ8.81(d,J=7.3Hz,1H),8.10-7.97(m,4H),7.94(d,J =8.2Hz,2H),7.84(dd,J=8.2,1.6Hz,1H),7.78(d,J=8.8Hz,1H),6.86(d,J=2 .4Hz,1H),6.81(dd,J=8.8,2.4Hz,1H),4.61-4.41(m,1H),3.44(t,J=6.7Hz ,2H),3.39(s,2H),2.42-2.29(m,2H),2.28-2.16(m,2H),2.15-2.00(m,2H).

[0361] Example 46: Synthesis of IV-21

[0362]

[0363] Step 1: Synthesis of IV-21-1

[0364] Using IV-19-1 (250 mg, 0.611 mmol) and 4-hydroxyethoxyphenylboronic acid pinacol ester (242 mg, 0.916 mmol) as raw materials, the same method as IV-19-2 was used to prepare a yellow solid IV-21-1 (252 mg), with a yield of 88%. 1 H NMR (300MHz, Methanol-d4) δ7.89(d,J=8.1Hz,2H),7.68(d,J=8.1Hz,2H),7.62(d,J=8.6Hz,2H),7.06(d,J=8.6Hz,2H),4.63-4.46(m,1H),4.11(t ,J=4.7Hz,2H),3.91(t,J=4.7Hz,2H),3.46-3.35(m,2H),3.32-3.27(m,2 H),2.49-2.34(m,2H),2.26-2.11(m,2H),2.06-1.87(m,2H),1.48(s,9H).

[0365] Step 2: Synthesis of IV-21-2

[0366] IV-21-1 (200 mg, 0.429 mmol) was dissolved in DCM, and 1 mL of HCl (4 M in 1,4-dioxane) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed as monitored by TLC, the solution was evaporated to dryness under reduced pressure and directly added to the next step.

[0367] Step 3: Synthesis of IV-21

[0368] The crude product IV-20-2 from step 2 was dissolved in DMF, and 4-fluoro-2-(trifluoromethyl)benzonitrile (96 mg, 0.505 mmol) and potassium carbonate (117 mg, 0.841 mmol) were added. The preparation method was the same as that for III-4, yielding a grayish-white solid IV-21 (65 mg). The yield of the two steps was 29%. 1H NMR(300MH z,DMSO-d6)δ8.70(d,J=7.2Hz,1H),7.94(d,J=8.0Hz,2H),7.80(d,J=8.8Hz,1H),7.74(d ,J=8.0Hz,2H),7.70(d,J=8.3Hz,2H),7.07(d,J=8.3Hz,2H),6.88(s,1H),6.82(d,J=8.8H z,1H),4.93(t,J=5.5Hz,1H),4.67-4.37(m,1H),4.06(q,J=5.0Hz,2H),3.76(q,J=5.0Hz, 2H),3.58-3.40(m,4H),2.38(t,J=10.1Hz,2H),2.24(t,J=9.5Hz,2H),2.17-1.99(m,2H).

[0369] Example 47: Synthesis of V-1

[0370]

[0371] Using intermediate III-6-2 (71 mg, 0.278 mmol), 5-bromo-3-(trifluoromethyl)-2-cyanopyridine (84 mg, 0.334 mmol), and potassium carbonate (77 mg, 0.557 mmol) as starting materials, and DMF (2 mL) as solvent, the reaction was carried out at 80 °C for 12 h. After the reaction was completed by TLC monitoring, the mixture was cooled to room temperature, and water (4 mL) and EA (3 mL × 3) were added. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 100:1) to give a white solid V-1 (30 mg), with a yield of 25%. 1 H NMR(300MHz,Chloroform-d)δ8.10(d,J=2.7Hz,1H),7.99-7.85(m,2H),7.83-7.72(m,2H),6.97(dd,J=8.0,2.7Hz,1H ),6.68(s,1H),4.85-4.61(m,1H),3.62-3.44(m,4H),2.59(dd,J=11.8,9.0Hz,2H),2.25(dq,J=15.9,8.1,6.7Hz,4H).

[0372] Example 48: Synthesis of V-2

[0373]

[0374] Under ice bath conditions, 60% NaH (29 mg, 0.706 mmol) was added to a 3 mL solution of anhydrous DMF (150 mg, 0.353 mmol) in volume V-1. After 30 min, under nitrogen protection, CH3I (33 μL, 0.529 mmol) was added, and the reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, 5 mL of water was added to quench the reaction, and the mixture was extracted with EA (5 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (PE:EA = 1:1) to give 106 mg of white solid, with a yield of 68%. 1 H NMR(300MHz,DMSO-d6)δ8.20(s,1H),7.95(d,J=7.9Hz,2H),7.58(s,2H),7.16(s,1H),4.09( s,1H),3.47(d,J=19.4Hz,4H),2.94(d,J=49.0Hz,3H),2.46-2.31(m,2H),2.31-1.79(m,4H).

[0375] Example 49: Synthesis of V-3

[0376]

[0377] Step 1: Synthesis of V-3-1

[0378] Using 6-(tert-butoxycarbonyl)-6-azaspiro[3.4]octane-2-carboxylic acid (150 mg, 0.588 mmol) and p-aminobenzonitrile (77 mg, 0.647 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding white solid V-3-1 (200 mg) with a yield of 96%. 1 H NMR (300MHz, DMSO-d6) δ10.32(d,J=5.6Hz,1H),7.82(d,J=8.8Hz,2H),7.76(d,J=8.7Hz,2H),3.7 0-3.52(m,1H),3.29-3.02(m,4H),2.28-2.04(m,4H),1.86(dq,J=28.8,6.4Hz,2H),1.40(s,9H).

[0379] Step 2: Synthesis of V-3-2

[0380] Using intermediate V-3-1 (200 mg, 0.563 mmol) as a starting material, the preparation method was the same as that for S2, and the white solid V-3-2 was directly used for the next reaction.

[0381] Step 3: Synthesis of V-3

[0382] Using intermediate V-3-2 (143 mg, 0.560 mmol) and 5-bromo-3-(trifluoromethyl)-2-cyanopyridine (155 mg, 0.617 mmol) as raw materials, potassium carbonate (155 mg, 1.12 mmol) was added, and DMF (3 mL) was used as solvent. The preparation method was the same as that for V-1, to obtain a pale yellow solid V-3 (60 mg). The yield of the two steps was 25%. 1 H NMR (300MHz, DMSO-d6) δ10.31(s,1H),8.23(dd,J=8.2,2.6Hz,1H),7.78(q,J=8.7Hz,4H),7.18(dd,J=6. 4,2.7Hz,1H),3.65-3.40(m,4H),3.27(p,J=8.3Hz,1H),2.42-2.15(m,4H),2.08(dt,J=29.7,6.5Hz,2H).

[0383] Example 50: Synthesis of V-4

[0384]

[0385] Step 1: Synthesis of V-4-1

[0386] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (150 mg, 0.663 mmol) and monomethyl terephthalate (132 mg, 0.730 mmol) as raw materials, the preparation method was the same as that of III-4-1, yielding white solid V-4-1 (210 mg) with a yield of 82%. 1 H NMR(300MHz,Chlorof orm-d)δ8.06(d,J=8.1Hz,2H),7.93-7.76(m,2H),6.31(s,1H),4.58(dq,J=15.7,8.1Hz,1H),3.92(s,3H),3.41 -3.23(m,4H),2.40(q,J=8.7Hz,2H),2.09(dd,J=19.5,10.6Hz,2H),1.90(dt,J=20.7,6.8Hz,2H),1.40(s,9H).

[0387] Step 2: Synthesis of V-4-2

[0388] Using intermediate V-4-1 (200 mg, 0.515 mmol) as a raw material, the same method as S2 was used to prepare white solid V-4-2 (60 mg), with a yield of 41%.

[0389] Step 3: Synthesis of V-4

[0390] Using intermediate V-4-2 (60 mg, 0.208 mmol) and 5-bromo-3-(trifluoromethyl)-2-cyanopyridine (58 mg, 0.229 mmol) as raw materials, potassium carbonate (58 mg, 0.417 mmol) was added, and DMF (2 mL) was used as solvent. The preparation method was the same as that for V-1, to obtain white solid V-4 (57 mg), with a yield of 60%. 1 H NMR(300MHz,Chloroform-d)δ8.12(d,J=8.0Hz,2H),8.07(s,1H),7.88(d,J=8.0Hz,2H) ,6.94(s,1H),6.76(d,J=7.7Hz,1H),4.75(p,J=8.3Hz,1H),3.96(s,3H),3.50(d,J=9.3

[0391] Hz,4H),2.58(t,J=9.9Hz,2H),2.36-2.14(m,4H). 13 C NMR(100MHz,DMSO-d6)δ166.16,16

[0392] 5.12,144.60,138.90,138.26,132.17,129.95(q, 2 J C-F =32.0Hz),129.54,128.11,122.85(q, 1 J C-F =273.0Hz), 116.80, 114.20(q, 3 J C-F =5.0Hz), 112.57(q, 3 J C-F =2.0Hz),59.14,52.81,47.06,39.18,38.71,36.13.

[0393] Example 51: Synthesis of V-5

[0394]

[0395] Step 1: Synthesis of V-5-1

[0396] Using 2-amino-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (150 mg, 0.663 mmol) and 5-carboxyphthalide (142 mg, 0.796 mmol) as raw materials, the preparation method was the same as that of III-4-1, and a white solid V-5-1 (243 mg) was obtained with a yield of 95%. 1HNMR (400MHz, DMSO-d6) δ8.92(dt,J=14.2,7.4Hz,1H),8.09(s,1H),8.04-7.98(m,1H),7.93(d,J=8.0Hz,1H),5.47(s,2H),4.45(qt,J=9 .2,4.8Hz,1H),3.33-3.22(m,2H),3.19(d,J=5.1Hz,2H),2.35-2.21(m,2H),2.17-2.03(m,2H),1.95-1.78(m,2H),1.40(d,J=8.5Hz,9H).

[0397] Step 2: Synthesis of V-5-2

[0398] Using intermediate V-5-1 (200 mg, 0.517 mmol) as a starting material, the same preparation method as S2 was used to obtain white solid V-5-2, which was directly used in the next reaction.

[0399] Step 3: Synthesis of V-5

[0400] Using intermediate V-5-2 (150 mg, 0.524 mmol) and 5-bromo-3-(trifluoromethyl)-2-cyanopyridine (145 mg, 0.576 mmol) as raw materials, potassium carbonate (145 mg, 1.05 mmol) was added, and DMF (3 mL) was used as solvent. The preparation method was the same as that for V-1, and a white solid V-5 (140 mg) was obtained. The two-step yield was 59%. 1 H NMR (300MHz, DMSO-d6) δ8.98(d,J=7.1Hz,1H),8.24(s,1H),8.10(s,1H),8.03(d,J=8.0Hz,1H),7.95(d,J=8.2Hz,1H),7.18(s,1H) ,5.48(s,2H),4.51(q,J=8.4,7.9Hz,1H),3.51(d,J=7.1Hz,2H),3.46(s,2H),2.37(t,J=9.5Hz,2H),2.18(dt,J=23.5,8.1Hz,4H).

[0401] Example 52: Synthesis of V-6

[0402]

[0403] Using III-6 (175 mg, 0.412 mmol) as a raw material, the same method as V-6 was used to prepare 130 mg of white solid, with a yield of 72%. 1H NMR(400MHz, DMSO-d6)δ7.93(d,J=7.8Hz,2H),7.78(d,J=8.7Hz,1H),7.66-7.46(m,2H),6.90-6.67(m,2H),4.96(s,0.4H,minor), 4.15(s,0.6H,major),3.41(s,3H),3.33(s,1H),3.01(s,2H),2.84(s,1H),2.38(t,J=9.1Hz,2H),2.28-1.95(m,3H),1.86(s,1H).

[0404] Example 53: Synthesis of V-7

[0405]

[0406] Using intermediate V-3-2 (55 mg, 0.167 mmol) and 4-fluoro-2-(trifluoromethyl)benzonitrile (35 mg, 0.185 mmol) as raw materials, potassium carbonate (47 mg, 0.335 mmol) was added, and DMF (3 mL) was used as solvent. The preparation method was the same as that for V-1, to obtain white solid V-7 (17 mg), with a yield of 24%. 1 H NMR (300MHz, DMSO-d6) δ10.29 (s, 1H), 7.80 (q, J = 8.3Hz, 5H), 7.01-6.71 (m, 2H), 3.51 (s,2H),3.46-3.37(m,2H),3.34-3.24(m,1H),2.41-2.17(m,4H),2.13-1.90(m,2H).

[0407] Example 54: In vitro activity evaluation

[0408] 1. Inhibitory effect of the compound on the proliferation of 22Rv1 cells

[0409] Experimental methods: The effect of the compounds in this application on the proliferation of 22Rv1 cells was evaluated using the CellTiter-Glo luminescence assay kit (CTG).

[0410] 22Rv1 cells (ATCC, catalog number: CRL-2505) were seeded into 384-well cell culture plates (Corning, CLS3764-100EA) and incubated overnight at 37°C in a 5% CO2 incubator (Note: 1% carbon-adsorbed serum was used). The next day, the diluted compound of this application was added to the culture plate using a nanoliter pipetting system (LABCYTE, P-0200). Finally, after co-incubating the compound of this application with the cells for 7 days, [the following was added]... The 2D reagent (Promega, G7573) was used, and the luminescence value was read using an Envision multi-functional microplate reader (the light signal is directly proportional to the amount of ATP in the system, and the ATP content directly represents the number of viable cells in the system). Finally, the IC50 of the compound was obtained using a nonlinear fitting formula with XLFIT software. 50 (Half-maximal inhibitory concentration). The specific activity test results for the test compounds are shown in Table 1.

[0411] 2. Inhibitory effect of the compound on LNCap cell proliferation

[0412] Experimental methods: The effect of the compounds in this application on the proliferation of LNCaP cells was evaluated using the CellTiter-Glo luminescence assay kit (CTG).

[0413] LNCap cells (ATCC, catalog number: CRL-1740) were seeded into 384-well cell culture plates (Corning, CLS3764-100EA) and incubated overnight at 37°C in a 5% CO2 incubator (Note: 1% carbon-adsorbed serum was used). The next day, the diluted test compound was added to the culture plate using a nano-pipette system (LABCYTE, P-0200) and incubated for 1 hour before adding R1881 (Xi'an Kaixin Biotechnology Co., Ltd., catalog number: K-ZJ-25741). Finally, after co-incubating the test compound with the cells for 7 days, [the following was added]... The 2D reagent (Promega, G7573) was used, and the luminescence value was read using an Envision multi-functional microplate reader (the light signal is directly proportional to the amount of ATP in the system, and the ATP content directly represents the number of viable cells in the system). Finally, the IC50 of the compound was obtained using a nonlinear fitting formula with XLFIT software. 50 (Half-maximal inhibitory concentration). The specific activity test results for the test compounds are shown in Table 1.

[0414] 3. Effects of the compound on AR antagonistic activity

[0415] (1) Materials and reagents

[0416]

[0417] (2) Instruments

[0418]

[0419] (3) Experimental methods

[0420] Day 1: Plate seeding, HEK-293 cells are digested and seeded into 10cm plates;

[0421] Day 2: Transfection. pBIND-AR / pGL4.35 / AR ORF plasmid was transfected into HEK293 cells;

[0422] Day 3: Collect transfected cells, resuspend them in DMEM medium without phenol red containing 5% carbon-adsorbed serum, count them, seed them into 96-well plates, add test compounds or positive compounds, incubate at 37°C for 30 min, add agonists, and then incubate at 37°C under 5% CO2 conditions.

[0423] Day 4: 22 hours after adding the drug, according to the Bright-Lite Luciferase Assay System kit instructions, add Bright-Glo Re agent to the experimental wells, lyse by shaking, and read the luminescence signal value using an HTS high-throughput drug screening multi-mode microplate reader.

[0424] 4. Experimental Results

[0425] The specific results of the corresponding activity tests for the tested compounds are shown in Table 1.

[0426] Table 1. Results of corresponding activity tests for compounds

[0427]

[0428]

[0429]

[0430] As shown in Table 1, the compounds of the present invention exhibit excellent inhibitory / antagonistic effects against malignant proliferating cells and androgen receptors, with high IC50 values ​​for cell inhibitory activity. 50 The optimal value is below 50 nM, and the optimal inhibition rate of receptor antagonism reaches 100%, inhibiting IC50. 50 The optimal value is below 20nM.

[0431] Example 55: Evaluation of the metabolic stability of the compound

[0432] 1. Experimental materials

[0433] Liver microsomes source: human liver microsomes (BiOIVT X008064), mouse liver microsomes (RILD LM-XS-02M).

[0434] 2. Experimental Procedure

[0435] (1) Solution preparation

[0436]

[0437] (2) Add 2.0 μL of 150 μM control or test compound solution to the culture plate. Diclofenac was used as a positive control in this study, and the final concentration of the test compound and diclofenac was 1 μM. Preheat the mixture at 37 °C for 10 minutes.

[0438] (3) Add 30 μL of 10 mM NADPH to start the reaction. The final NADPH concentration was 1 mM, with the NADPH-free composite sample NCF60 as a negative control. The incubation solution was incubated in water batches at 37°C.

[0439] (4) Take 30 μL of the reaction solution at 0, 5, 15, 30, and 60 minutes respectively. The reaction can be terminated by adding 200 μL of cold acetonitrile to IS (100 ng / mL Labetalol, 100 ng / mL tolbutamide). Centrifuge the sample at 1000 rpm for 10 minutes. Mix 50 μL of the supernatant with 150 μL of ultrapure water and perform LC-MS / MS analysis.

[0440] All data were calculated using Microsoft Excel software. Peak areas were detected by extracting ion spectra. The in vitro half-life (T0) of the parent drug was determined by linearly fitting the natural logarithm of the elimination percentage of the parent drug to time. 1 / 2 ).

[0441] in vitro half-life (T1) 1 / 2 ) Calculated using slope k:

[0442] in vitro T 1 / 2 =0.693 / k

[0443] In vitro clearance rate (CL) int (Unit: μL / min / mg protein) is calculated using the following formula:

[0444] in vitro CL int = k × incubation liquid volume / enzyme protein content;

[0445] 3. Experimental Results

[0446] T calculated using the above formula 1 / 2 and CL int The values ​​are shown in Table 2.

[0447] Table 2. Half-life and intrinsic clearance of compounds in liver microsomes

[0448]

[0449] As can be seen from Table 2, the compounds of the present invention have suitable pharmacokinetic properties, which is beneficial for drug development.

Claims

1. A spirocyclic compound, characterized in that, Having the structure of Formula 2 or Formula 3, it also contains its pharmaceutically acceptable salt. ， In Equation 2: Selected from or ; -(Z) r - Selected from -CH2- or carbonyl; In Equation 3: Selected from or ; -W-(Z) r -Selected from -CONH- or -NHCO-; In the structure of Equation 2 or Equation 3: X is selected from CH or N; Ring D is selected from phenyl or pyridinyl; R 1 Selected from -CF3, R 2 Selected from -CN,R 3 Selected from H; R 4 R 5 Phenyl group selected from one or two F, -OH, -NH2, -NO2, -CN, -CH3, -CH2OH, -OCH3, -OCH2CH2OH, -CH(OH)CH3, -C(O)NHCH3 or halogen, -OH, -NO2, -CN, -CF3 substituted phenyl groups.

2. A spirocyclic compound, characterized in that, Selected from any of the following compounds: 。 3. The spirocyclic compound according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound with any of the following acids: Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and acidic amino acids.

4. A pharmaceutical composition, characterized in that, It comprises the spirocyclic compound of claim 1 and a pharmaceutically acceptable carrier.

5. The use of a spirocyclic compound of claim 1 or a pharmaceutical composition of claim 4 in the preparation of a medicament for treating prostate cancer.

6. The use of a spirocyclic compound of claim 1 or a pharmaceutical composition of claim 4 in the preparation of an androgen receptor antagonist drug, wherein the drug is a drug for treating prostate cancer.

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