A triazole heterocyclic compound

Abstract

This invention discloses a Cbl-b inhibitor compound as shown in formula (I), its uses, and pharmaceutical compositions containing it. This type of inhibitor is a potential tumor immunotherapy agent and autoimmune disease treatment agent. This invention also discloses the use of this type of compound in the treatment of tumors, type I diabetes, Graves' disease, systemic lupus erythematosus, and multiple sclerosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a Cbl-b inhibitor compound, its use and a pharmaceutical composition containing the same. BACKGROUND

[0002] Cbl-b (Casitas B-lymphoma) is an E3 ligase containing Ring finger domain, mainly expressed in peripheral immune cells, and has the effect of regulating the function of various immune cells.

[0003] In T cells, Cbl-b exists in the cytoplasm and is recruited to the cell membrane after TCR signal stimulation, and plays a variety of negative regulatory roles on the TCR signal pathway, such as degradation of CD3ζ, reduction of LAT and Vav1 phosphorylation, inhibition of PLC-γ1 activation, etc. It can also act on CD28 downstream, ubiquitinate the p85β subunit of PI3K, thereby inhibiting the CD28-dependent PI3K pathway, so that the T cell activation threshold is lowered and the T cell can be activated without co-stimulatory signal. Cbl-b knockout T cells show significantly increased proliferation activity, increased expression of cytokines IL-2, etc., increased expression of activity markers, and decreased expression of exhaustion markers. Cbl-b knockout CAR-T cells show stronger tumor killing effect, and when Cbl-b knockout T cells are combined with immune checkpoint inhibitor CTLA-4 antibody, the tumor growth is better inhibited.

[0004] In B cells, downstream molecules of BCR signaling pathway such as Syk, PLC-γ2, p85 / PI3K, Vav and Grb2 are all Cbl-b substrates, and their knockout makes B cells in a state of persistent activation, such as increased Ca2+ flow and increased expression of activation marker CD69, etc. In addition, in natural immune cells such as macrophages, dendritic cells, NK cells and monocytes, Cbl-b also plays a regulatory role.

[0005] Systemic knockout of Cbl-b in mice shows no significant effect on their growth and reproduction, only presenting spontaneous autoimmune disease symptoms such as increased anti-double-stranded DNA autoantibodies in serum and inflammatory cell infiltration in multiple organs. And CBL-B knockout mice are also more sensitive to various autoimmune models. Due to the regulatory effect of Cbl-b on various immune cells, the spontaneous tumor incidence of Cbl-b knockout mice is significantly decreased. The growth of various transplanted tumor models including solid tumors and hematological tumors is also significantly inhibited.

[0006] In addition, single nucleotide polymorphisms (SNPs) and mutations of Cbl-b are associated with various human autoimmune diseases, such as type I diabetes, Graves' disease, systemic lupus erythematosus and multiple sclerosis.

[0007] In summary, Cbl-b is an important immune cell activity regulator and a potential therapeutic target for tumor immunotherapy and autoimmune diseases. SUMMARY

[0008] The present application provides a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] wherein R1 is independently C 1-3 alkyl, -CN, halogen or hydroxyl, n is 1 or 2

[0011] Further, when R1 is C 1-3 alkyl, R1 can be optionally substituted by halogen or -CN.

[0012] The compound as shown in formula (I) or a pharmaceutically acceptable salt thereof as described above can be:

[0013]

[0014] The present application also provides a use of the compound as shown above or a pharmaceutically acceptable salt thereof in the preparation of a Cbl-b inhibitor, such as tumor, type I diabetes, Graves' disease, systemic lupus erythematosus and multiple sclerosis, etc.

[0015] The present application also provides a pharmaceutical composition comprising the compound as shown above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0016] As described herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the present application. DETAILED DESCRIPTION

[0017] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0018] The structure of the compounds described in the following examples is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0019] The nuclear magnetic resonance (NMR) measurements were performed using a Bruker 400MHz NMR spectrometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), and hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS).

[0020] The abbreviations used in the nuclear magnetic resonance (NMR) data in the following examples have the following meanings:

[0021] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, m: multiplet, br: broad peak, J: coupling constant, Hz: Hertz, δ: chemical shift.

[0022] All chemical shift (δ) values ​​are given in parts per million (ppm).

[0023] The mass spectrometry (MS) measurements were performed using an Agilent 6120B mass spectrometer with an electrospray ionization (ESI) source.

[0024] Intermediate preparation examples

[0025] Synthesis of Int-A:

[0026]

[0027] Step 1:

[0028] Nitric acid (8.91 g, 141.32 mmol, 6.36 mL) was added to a solution of methyl 4-bromo-3-(trifluoromethyl)benzoate (20.0 g, 70.66 mmol) in 300.0 mL of sulfuric acid. The resulting mixture was stirred at 80 °C for 2 hours. TLC monitoring showed the formation of a major new spot (PE:EA = 5:1, R...). f =0.1). The mixture was slowly poured into ice water, filtered, and the filter cake was concentrated under vacuum to give crude product Int-A1 (20.0 g, 63.69 mmol, yield 90%) as a white solid, which was used directly in the next reaction.

[0029] Step 2:

[0030] Sulfuric acid (62.5 g, 636.90 mmol, 33.9 mL) was slowly added dropwise to a methanol (250.0 mL) solution of Int-A1 (20 g, 63.69 mmol). The mixture was stirred at 80 °C for 2 hours. TLC showed complete consumption of the reactants and detection of a new spot (PE:EA = 10:1, R0). f=0.8). The reaction mixture was poured into 200 mL of ice water, filtered, and the filter cake was concentrated under reduced pressure to give crude product Int-A2 (18.75 g, 57.32 mmol, yield 90%) as a white solid. 1 H NMR (DMSO-d6, 400MHz) δ = 8.77 (s, 1H), 8.39 (s, 1H), 3.94 (s, 3H).

[0031] Step 3:

[0032] Int-A2 (15.0 g, 45.73 mmol) was dissolved in ethanol (200.0 mL), followed by the addition of iron powder (7.66 g, 137.18 mmol), ammonium chloride (7.34 g, 137.18 mmol), and water (20.0 mL). The reaction mixture was stirred at 25 °C for 3 hours. LCMS showed that the reactants had been completely consumed. The reaction mixture was filtered, diluted with 200 mL of water, and extracted with 300 mL of ethyl acetate (150 mL * 2). The combined organic layers were washed with 200 mL of saturated brine (100 mL * 2), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude Int-A3 (10.0 g, 33.55 mmol, yield 73%) as a gray solid, which was used directly in the next step. LCMS (ESI) m / z = 298.0, 300.0 [M + H] + .

[0033] Step 4:

[0034] Potassium ethyl sulfonate (10.76 g, 67.10 mmol) was added to a pyridine (150 mL) solution of Int-A3 (10 g, 33.55 mmol). The reaction mixture was stirred at 110 °C for 12 hours. LCMS showed that the reactants were consumed. The reaction was quenched with 200 mL of ice water, then diluted with 100 mL of ethyl acetate and extracted with 400 mL (200 mL x 2) of ethyl acetate. The combined organic layers were washed with 200 mL (100 mL x 2) of saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to give a brown solid Int-A (4.0 g, 13.64 mmol, yield 41%), LCMS (ESI) m / z = 294.2 [M+H]. + .

[0035] Synthesis of Int-B:

[0036]

[0037] Step 1:

[0038] Iodomethane (14.53 g, 102.29 mmol) was added to a solution of Int-A (10 g, 34.097 mmol) and potassium carbonate (14.12 g, 102.29 mmol) in 100 mL of ethyl acetate. The reaction mixture was stirred at room temperature for 16 hours, then filtered to remove the solid. The solid was washed twice with ethyl acetate, the solvent was evaporated, and the solid was purified by column chromatography to give a yellow solid Int-B1 (2.0 g, yield 19%). LCMS (ESI) m / z = 307.9 [M+H] + .

[0039] Step 2:

[0040] Lithium aluminum hydride (0.74 g, 19.52 mmol) was added to a 20 mL solution of Int-B1 (3.0 g, 9.76 mmol) in tetrahydrofuran at 0 °C, and the reaction was carried out at 0 °C for 1 hour. The reaction was quenched with ice water, and then filtered through diatomaceous earth. The filter cake was washed several times with ethyl acetate, and the filtrate was separated into layers. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and dried to dryness to give a yellow solid Int-B2 (2.3 g, yield 84%). LCMS (ESI) m / z = 280.0 [M+H] + .

[0041] Step 3:

[0042] Manganese dioxide (7.16 g, 82.35 mmol) was added to a solution of Int-B2 (2.3 g, 8.23 ​​mmol) in dichloromethane (30 mL). The reaction was carried out at room temperature for 16 hours. The solvent was evaporated and the solution was purified by column chromatography to give a yellow solid Int-B3 (1.5 g, yield 66%). LCMS (ESI) m / z = 278.0 [M+H] + .

[0043] Step 4:

[0044] Triethylamine (1.39 g, 13.74 mmol) was added to a solution of Int-B3 (635 mg, 2.29 mmol) and (S)-3-methylpiperidine hydrochloride (465.94 mg, 3.44 mmol) in dichloroethane (20 mL). The reaction mixture was stirred at room temperature for 1 hour. Then, sodium triacetoxyborohydride (0.97 g, 4.58 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for another 3 hours. The solvent was then evaporated to dryness and purified by column chromatography to give a yellow oil, Int-B (680 mg, 82% yield), LCMS (ESI) m / z = 361.1 [M+H]. + .

[0045] Synthesis of Int-C:

[0046]

[0047] Step 1:

[0048] Int-A (10.0 g, 34.1 mmol) was dissolved in acetic acid (100.0 mL), and then iron powder (19.04 g, 341.0 mmol) was added. The reaction mixture was stirred at 110 °C for 16 hours. The mixture was then filtered through diatomaceous earth, and the filtrate was washed with 100 mL of ethyl acetate. The solution was then neutralized with 200 mL of saturated sodium bicarbonate solution and extracted with 200 mL (100 mL * 2) of ethyl acetate. The combined organic layers were washed with 200 mL (100 mL * 2) of saturated brine. The mixture was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 3 / 1) to give a pale yellow solid Int-C1 (6.5 g, 24.88 mmol, yield 73%), LCMS (ESI) m / z = 262.2 [M + H]. + .

[0049] Step 2:

[0050] Lithium aluminum tetrahydrofuran (581.2 mg, 15.31 mmol) was added to a tetrahydrofuran (200.0 mL) solution of Int-C1 (2.0 g, 7.66 mmol) at 0 °C. After reacting at 0 °C for 1 hour, the reaction was quenched by adding water (15 mL), 15% sodium hydroxide solution (15 mL), and water (45 mL) at 0 °C. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to give a yellow solid Int-C2 (1.2 g, 5.15 mmol, yield 67%), LCMS (ESI) m / z = 233.9 [M+H]. + Step 3:

[0051] Int-C2 (1.19 g, 5.10 mmol) was dissolved in dichloromethane (20.0 mL), and then thionyl chloride (7.3 g, 61.2 mmol, 4.4 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 1 hour, and LCMS monitoring showed that the reactants had been consumed. The reaction mixture was concentrated under reduced pressure and dried under vacuum to obtain a brown solid crude Int-C (1.2 g, 4.77 mmol, yield 93%), which was used directly in the next step. LCMS (ESI) m / z = 252.2 [M+H] + .

[0052] Synthesis of Int-D and Int-E:

[0053]

[0054] Step 1:

[0055] To a DMF (300.0 mL) solution of methyl 2-(3-bromophenyl)acetate (20.0 g, 87.31 mmol), NaH (13.9 g, 349.24 mmol, 60% purity) was added, and the mixture was stirred at 0 °C for 1 hour. Then, 1,3-dibromo-2-methylpropane (22.6 g, 104.77 mmol) was added to the reaction solution. After stirring at 20 °C for another 2 hours, the reaction was quenched at 0 °C with 400 mL of ammonium chloride aqueous solution, diluted with 200 mL of water, and extracted three times with ethyl acetate (300 mL x 3). The combined organic phases were washed three times with saturated saline solution (300 mL x 3) and dried over sodium sulfate. The residue was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1 to 3 / 1) to give a colorless oil, Int-D1 (20.5 g, 72.40 mmol, yield 83%), LCMS (ESI) m / z = 294.2 [M+H]. + . 1 H NMR (400MHz, CDCl3) δppm 7.51(dt,J=4.98,1.79Hz,1H)7.39(tdd,J=7.70,7.70,1.83,1.10Hz,1H)7.27(s,1H) )7.22-7.16(m,1H)3.71-3.60(m,3H)2.91-2.36(m,3H)1.81-1.62(m,2H)1.48(s,3H).

[0056] Step 2:

[0057] Int-D1 (20.0 g, 70.63 mmol) was dissolved in ethanol (100 mL), and hydrazine hydrate (41.6 g, 706.31 mmol, 40.39 mL, 85% purity) was added. The reaction mixture was stirred at 80 °C for 16 hours, then cooled to room temperature. The pH of the solution was adjusted to 3–4 with hydrochloric acid solution (1 M), and the solution was concentrated under reduced pressure. The residue was purified by preparative HPLC (alkaline conditions) to give a pale yellow oil, Int-D2 (14.5 g, 51.21 mmol, yield 73%), LCMS (ESI) m / z = 281.1, 283.1 [M+H]. + .

[0058] Step 3:

[0059] At room temperature, N,N-dimethylformamide dimethyl acetal (3.0 g, 25.43 mmol, 3.4 mL) was added dropwise to a solution of Int-D2 (6.0 g, 21.19 mmol) in acetonitrile (60.0 mL). The reaction mixture was stirred at 100 °C for 1 hour, then acetic acid (1.27 g, 21.19 mmol, 1.2 mL) was added, followed by dropwise addition of a methylamine solution in ethanol (5.26 g, 50.85 mmol, 30% purity). The reaction mixture was stirred at 100 °C for 1 hour. The reaction mixture was concentrated by vacuum distillation, and the residue was purified by preparative HPLC (FA) to give a white solid Int-D (3.0 g, 9.80 mmol, yield 46%), LCMS (ESI) m / z = 306.0, 308.0 [M+H]. + .

[0060] Step 4:

[0061] Int-D (500.0 mg, 1.63 mmol) was dissolved in dioxane (20.0 mL), and bis(pinacol) diboron (497.6 mg, 1.96 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (119.48 mg, 0.16 mmol), and potassium acetate (240.4 mg, 2.45 mmol) were added. The mixture was degassed and purged with nitrogen three times, then reacted at 100 °C for 1 hour under a nitrogen atmosphere. The reaction solution was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (FA conditions) to obtain a brown oily substance, Int-E (500.0 mg, 1.42 mmol, yield 87%), LCMS (ESI) m / z = 354.5 [M+H]. + .

[0062] Synthesis of Int-F:

[0063]

[0064] Step 1:

[0065] In a 1 L round-bottom flask, sodium hydride (7.57 g, 315.45 mmol) was dissolved in tetrahydrofuran (500 mL). A solution of trimethyl phosphoroacetate (31.60 g, 173.50 mmol) in tetrahydrofuran (50 mL) was added dropwise at 0 °C. The reaction was carried out under nitrogen protection at 0 °C for 1 hour. Then, a solution of 3-oxocyclobutyronitrile (15 g, 157.73 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred overnight at room temperature, quenched with ammonium chloride aqueous solution, and extracted with ethyl acetate. The organic phase was evaporated to dryness and purified by column chromatography (PE / EA = 5:1) to give a white solid Int-F1 (6.2 g, yield 26%), LCMS (ESI) m / z = 152.0 [M+H]. + .1 H NMR (400MHz, DMSO-d6) δ5.76 (p, J = 2.3Hz, 1H), 3.58–3.45 (m, 2H), 3.40–3.28 (m, 1H), 3.28–3.16 (m, 2H).

[0066] Step 2:

[0067] In a 250 mL round-bottom flask, Int-F1 (6.2 g, 41.015 mmol) was dissolved in 1,4-dioxane (60 mL), followed by a solution of potassium hydroxide (2.99 g, 53.32 mmol) in water (30 mL), (3-((tert-butoxycarbonyl)amino)phenyl)boronic acid (14.58 g, 61.52 mmol), and (1,5-cyclooctadiene)rhodium(I) dimer (1.01 g, 2.05 mmol). The mixture was stirred overnight under nitrogen protection at room temperature. The reaction mixture was quenched with ammonium chloride solution and extracted with ethyl acetate. The organic phase was evaporated to dryness and purified by column chromatography (PE / EA (5:1)) to give a white solid Int-F2 (4.9 g, 35% yield), LCMS (ESI) m / z = 345.0 [M+H]. + .

[0068] Step 3:

[0069] In a 250 mL round-bottom flask, Int-F2 (4.9 g, 14.23 mmol) was dissolved in tetrahydrofuran (50 mL), followed by the addition of lithium hydroxide (0.75 g, 17.78 mmol), water (25 mL), and methanol (12.5 mL). The mixture was stirred overnight under nitrogen atmosphere at room temperature. The pH of the reaction mixture was adjusted to 3 with hydrochloric acid solution, and then extracted with ethyl acetate. The organic phase was evaporated to dryness and then dried under vacuum to obtain crude Int-F3 (4.89 g) as a white solid. LCMS (ESI) m / z = 353.0 [M + Na] + .

[0070] Step 4:

[0071] In a 250 mL round-bottom flask, Int-F3 (4.89 g, 14.80 mmol) and 4-methylaminothiourea (2.33 g, 22.20 mmol) were dissolved in DMF (50 mL). DIEA (5.74 g, 44.40 mmol) and HATU (7.32 g, 19.24 mmol) were added. After reacting under nitrogen at room temperature for four hours, NaOH (1 M) (30 mL) was added, and the reaction was carried out overnight at 50 °C. The reaction mixture was quenched with ammonium chloride solution and extracted with ethyl acetate. The organic phase was evaporated to dryness to give a yellow oily substance, Int-F4 (5.3 g, 90% yield), LCMS (ESI) m / z = 400.1 [M+H]. + .

[0072] Step 5:

[0073] In a 250 mL round-bottom flask, Int-F4 (5.3 g, 13.27 mmol) was dissolved in dichloromethane (50 mL), and acetic acid (11.15 g, 185.72 mmol) was added. Hydrogen peroxide (30%) (1.13 g, 33.17 mmol) was added at 0 °C, and the reaction was carried out under nitrogen atmosphere at room temperature for two hours. The reaction mixture was quenched with sodium bicarbonate solution, then extracted with dichloromethane, and the organic phase was evaporated to dryness. The solution was purified by column chromatography (DCM / MeOH (10:1)) to obtain a yellow oily substance, Int-F5 (5.0 g, 100%), LCMS (ESI) m / z = 368.1 [M+H]. + .

[0074] Step 6:

[0075] In a 250 mL round-bottom flask, Int-F5 (5 g, 13.61 mmol) was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (50 mL) was added. The mixture was reacted under nitrogen at room temperature for half an hour. The solvent was evaporated and purified by reverse-phase column chromatography (column: C18 silica gel column; mobile phase: acetonitrile aqueous solution (0.1% FA), elution at a gradient of 10% to 50% for 10 minutes; UV monitoring at 254 nm) to obtain a yellow solid Int-F6 (2.5 g, yield 69%), LCMS (ESI) m / z = 268.2 [M+H]. + .

[0076] Step 7:

[0077] In a 100 mL round-bottom flask, Int-F6 (2.5 g, 9.35 mmol) and copper bromide (4.18 g, 18.70 mmol) were dissolved in acetonitrile (30 mL). Tert-butyl nitrite (1.45 g, 14.03 mmol) was slowly added at 0 °C. The reaction was carried out under nitrogen at 0 °C for one hour. The reaction solution was quenched with ammonia and extracted with dichloromethane. The organic phase was evaporated to dryness and purified by reverse-phase column chromatography (column: C18 silica gel column; mobile phase: acetonitrile aqueous solution (0.1% FA), elution at a gradient of 10% to 50% for 10 minutes; UV monitoring at 254 nm) to obtain a yellow solid Int-F (900 mg, yield 29%). LCMS (ESI) m / z = 331.0, 333.0 [M+H] + .

[0078] Synthesis of Int-G:

[0079] Following the synthesis method of Int-E, it was synthesized from Int-F. LCMS(ESI) m / z = 379.2 [M+H] + .

[0080] Synthesis of Int-H and Int-I:

[0081]

[0082] Following the synthetic methods for Int-D and Int-E respectively, brown oily compounds Int-H (LCMS(ESI) m / z = 318.2, 320.2 [M+H]) were synthesized using 1,1-bis(bromomethyl)cyclopropane as the corresponding starting material. + ). and Int-I(LCMS(ESI)m / z=366.1[M+H] + ).

[0083] Synthesis of Int-J and Int-K:

[0084]

[0085] Following the synthetic methods for Int-D and Int-E respectively, colorless oily substances Int-J (LCMS(ESI) m / z = 334.2, 336.2 [M+H)) were synthesized using 3-bis(bromomethyl)epoxybutane as the corresponding starting material. + ) and Int-K(LCMS(ESI)m / z=379.2[M+H] + ).

[0086] Synthesis of Int-L:

[0087]

[0088] Step 1:

[0089] At 0°C, isopropyl magnesium bromide (1M, 202.0 mL) was slowly added dropwise to a THF (300 mL) solution of m-bromophenylacetonitrile (18.0 g, 91.82 mmol), followed by the slow addition of epichlorohydrin (15.3 g, 165.27 mmol, 13.0 mL). The reaction mixture was stirred at 25°C for 45 minutes, and then isopropyl magnesium bromide (1M, 183.6 mL) was slowly added dropwise over 30 minutes. The reaction mixture was then slowly heated to 60°C and stirred for 14 hours. TLC showed complete consumption of the reactants and detection of a new spot (ethyl acetate / petroleum ether = 2:1, R0). f =0.4). The reaction solution was slowly poured into ice water, the pH was adjusted to 4 with 1M HCl, water (400 mL) was added, ethyl acetate (600 mL) was added for extraction, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain a pale yellow oily substance Int-L1 (12.2 g, yield 53%).

[0090] Step 2:

[0091] Compound Int-L1 (12.0 g, 47.60 mmol) was dissolved in ethanol (EtOH) (120 mL), and then an aqueous solution of sodium hydroxide (7.6 g, 190.40 mmol) (36 mL) was added. The reaction solution was slowly heated to 90 °C and stirred for 16 hours. The reaction was confirmed by LCMS. The pH of the reaction solution was adjusted to 1–2 with 2 M HCl, water (300 mL) was added, and extraction was performed with ethyl acetate (500 mL). The solution was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was further concentrated to remove the solvent, yielding a brown oily crude product Int-L2 (11.0 g, 85% yield), which was used directly in the next step without further purification. LCMS (ESI) m / z = 267.9 ({ 79 Br}MH) - ,236.9({ 81 Br}MH) - .

[0092] Step 3:

[0093] To a DMF solution (100 mL) of compound Int-L2 (11.0 g, 40.57 mmol), HATU (18.5 g, 48.69 mmol) and N,N-diisopropylethylamine (10.5 g, 81.15 mmol) were added, followed by N-methylhydrazine thioformamide (5.1 g, 48.69 mmol). The reaction mixture was stirred at 20°C for 30 minutes. The reaction was confirmed by LCMS. Water (400 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (600 mL). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was further purified by concentration. The residue was then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% ammonia), elution 20% to 70% for 15 minutes; detector: UV 254 nm) to obtain a pale yellow solid Int-L3 (4.0 g, yield 28%). LCMS(ESI)m / z=355.9({ 79 Br}MH) － 357.9({ 81 Br}MH) － .

[0094] Step 4:

[0095] Compound Int-L3 (3.8 g, 10.61 mmol) was added to an aqueous solution (20 mL) of sodium hydroxide (4.2 g, 106.07 mmol). The reaction mixture was stirred at 25°C for 16 hours. The reaction was confirmed by LCMS. The pH of the reaction mixture was adjusted to 1–2 with 1 M HCl, 150 mL of water was added, and 300 mL of ethyl acetate was added for extraction. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was concentrated to remove the solvent. The residue was then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% formic acid), elution 35%–65% for 12 minutes; detector: UV 254 nm) to give a pale yellow solid Int-L4 (3.0 g, yield 83%). LCMS (ESI) m / z = 340.0 ({ 79 Br}M+H) + ,342.0({ 81 Br}M+H) + .

[0096] Step 5:

[0097] Acetic acid (10.0 mL) and hydrogen peroxide (6.8 g, 59.96 mmol, 30% purity) were added to a dichloromethane solution (50.0 mL) of compound Int-L4 (3.0 g, 8.81 mmol). The reaction mixture was stirred at 25°C for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain a pale yellow oily crude product, Int-L5 (2.5 g, 81% yield), which was used directly in the next step without further purification. LCMS (ESI) m / z = 307.9 ({ 79 Br}M+H) + 309.9({ 81 Br}M+H) + .

[0098] Step 6:

[0099] To a 2 mL solution of compound Int-L5 (100.0 mg, 324.49 μmol) in dioxane, 1,1-bis(diphenylphosphine)ferrocene palladium chloride (23.7 mg, 32.45 μmol), potassium acetate (79.6 mg, 811.23 μmol), and bis(diphenylphosphine)boronic acid ester (90.6 mg, 356.94 μmol) were added. The mixture was purged three times with nitrogen, and the reaction solution was stirred at 100 °C for 1 hour. The reaction was confirmed by LC-MS. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain a dark brown solid crude Int-L (105.0 mg, 91% yield), which was used directly in the next step without further purification. LC-MS (ESI) m / z = 356.1 [M+H] + .

[0100] Synthetic route of Int-M

[0101]

[0102] Step 1:

[0103] NaH (9.18 g, 229.54 mmol, 60% purity) was added fractionally to a DMF (200 mL) solution of 15 g (76.51 mmol) of 3-bromophenylacetonitrile (0 °C). After addition, the mixture was stirred at 0 °C for 1 h, followed by the addition of 24.05 g (91.82 mmol) of 1,3-dibromo-2,2-dimethoxypropane at 0 °C. The resulting mixture was stirred at 60 °C for 11 h. TLC showed complete consumption of the reactants and detection of a new spot (ethyl acetate / petroleum ether = 20:1, R0). f =0.45). The reaction mixture was quenched at 0°C by adding it to a saturated ammonium chloride solution (500 mL), extracted with ethyl acetate (500 mL), washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by normal-phase column chromatography (mobile phase: petroleum ether / ethyl acetate, elution 20% to 50% for 10 min; detector: UV 254 nm) to give a yellow oily substance Int-M1 (5 g, yield 22%). 1 H NMR (400MHz, CD3OD) δppm 7.65(t,J=1.77Hz,1H),7.50(m,2H),7.32-7.39(m,1H),3.28(s,3H),3.18(s,3H),3.05-3.11(m,2H),2.71-2.78(m,2H)

[0104] Step 2:

[0105] At room temperature, a mixture of compound Int-M1 (5 g, 16.88 mmol), NaOH (1.35 g, 33.77 mmol), EtOH (50 mL), and water (15 mL) was stirred at 90°C for 15 hours. The reaction was confirmed by LCMS. The reaction mixture was then poured into water (200 mL) and extracted with ethyl acetate (200 mL). The pH of the aqueous phase was adjusted to approximately 5 with 1 M dilute hydrochloric acid. Further extraction with ethyl acetate (200 mL) was performed. The organic phase was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude yellow solid, Int-M2 (5.2 g, 97% yield). This was used directly in the next step without further purification. LCMS (ESI) m / z = 312.9 ({ 79 Br}MH) - ,314.9({ 81 Br}MH)- .

[0106] Step 3:

[0107] At room temperature, HATU (7.53 g, 19.80 mmol) and DIPEA (4.26 g, 33.00 mmol) were added to a DMF (50 mL) solution of Int-M2 (5.2 g, 16.50 mmol), followed by 4-methylaminothiourea (2.08 g, 19.80 mmol). The mixture was stirred at 20°C for 16 hours. The reaction was confirmed by LCMS. The reaction solution was added to an aqueous solution (300 mL), extracted with ethyl acetate (300 mL), and the organic phase was washed three times with saturated brine (300 mL). The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude yellow oily product, Int-M3 (6.6 g, 99% yield). This product was used directly in the next step without further purification. LCMS (ESI) m / z = 370.2 ({ 79 Br}M-MeO) + ,372.2({ 81 Br}M-MeO) + .

[0108] Step 4:

[0109] NaOH (4.9 g, 122.23 mmol) was added to 100 mL of an aqueous solution of Int-M3 (6.6 g, 16.41 mmol), and the mixture was stirred at 20 °C for 16 hours. The reaction was confirmed by LCMS. The reaction solution was then added to 300 mL of water, extracted with 300 mL of ethyl acetate, washed with 300 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude yellow oil Int-M4 (6.2 g, 98% yield). This was used directly in the next step without further purification. LCMS (ESI) m / z = 384.2 ({ 79 Br}M+H) + 386.2({ 81 Br}M+H) + .

[0110] Step 5:

[0111] HOAc (12 mL) and H2O2 (19.07 g, 168.19 mmol, 16.16 mL, 30% purity) were added to a DCM (60 mL) solution of Int-M4 (6.2 g, 17.17 mmol) and stirred at 20 °C for 1 hour. The reaction was detected by LCMS to indicate completion. The reaction solution was slowly added to a saturated sodium sulfite solution (300 mL) for quenching, and extracted with ethyl acetate (300 mL). The organic phase was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% FA), elution 30% to 70% for 10 minutes; detector: UV 254 nm). A colorless oily substance, Int-M5 (3.1 g, yield 51%), was obtained. LCMS (ESI) m / z = 352.3 ({ 79 Br}M+H) + ,354.3({ 81 Br}M+H) + .

[0112] Step 6:

[0113] At room temperature, hydrochloric acid (4M, 10mL) was added to a MeOH (40mL) solution of Int-M5 (2.8g, 7.95mmol), and the mixture was stirred at 80°C for 12 hours. The reaction was detected by LCMS. The reaction solution was then added to 1300mL of water, extracted with ethyl acetate (100mL), washed with saturated brine (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% FA), elution 30% to 70% for ten minutes; detector: UV 254nm). A yellow solid, Int-M (2.3g, yield 97%), was obtained. LCMS (ESI) m / z = 306.2. 79 Br}M+H) + ,308.2({ 81 Br}M+H) + .

[0114] Synthetic route of Int-N:

[0115]

[0116] Step 1:

[0117] t-BuOK (109.9 mg, 0.98 mmol) was added to a THF (10 mL) solution of Int-M (150.0 mg, 0.49 mmol) and diethyl cyanomethyl phosphate (104.2 mg, 0.59 mmol) at 0°C, and stirred at 20°C for 2 hours. The reaction was complete as determined by LC-MS. The reaction mixture was added to water (50 mL), extracted with ethyl acetate (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude yellow oil, Int-N1 (160 mg, 99% yield). This was used directly in the next step without further purification. LC-MS (ESI) m / z = 329.2 [M+H] + .

[0118] Step 2:

[0119] To a methanol (10 mL) solution of compound Int-N1 (140 mg, 425.28 μmol), NaBH4 (1.8 g, 47.31 mmol) was added in portions at 0°C. The mixture was stirred at 20°C for 12 hours. The reaction was confirmed by LCMS. The reaction mixture was then added to water (50 mL), extracted with ethyl acetate (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% FA), elution 30% to 70% for 10 min; detector: UV 254 nm). The result was a colorless oily substance, Int-N2 (45 mg, yield 31%). LCMS (ESI) m / z = 331.2 [M+H]. + .

[0120] Step 3:

[0121] To a 3 mL solution of compound Int-N2 (35 mg, 105.7 μmol) in 1,4-dioxane, B2Pin2 (32.20 mg, 126.81 μmol), KOAc (20.74 mg, 211.35 μmol), and Pd(dppf)Cl2 (7.73 mg, 10.57 μmol) were added. The reaction mixture was purged three times with nitrogen and stirred at 100°C for 1 h under a nitrogen atmosphere. The reaction was confirmed to be complete by LCMS. The reaction mixture was filtered, concentrated under reduced pressure, and a brown oily crude product, Int-N (39 mg, 97% yield), was obtained. This crude product was used directly in the next step without further purification. LCMS (ESI) m / z = 379.3 [M+H] + .

[0122] Synthetic route of Int-O

[0123]

[0124] Step 1:

[0125] Triethylamine (328.35 mg, 3.24 mmol) and methanesulfonic anhydride (423.94 mg, 2.43 mmol) were added to a dichloromethane solution (15 mL) of compound Int-N (500.0 mg, 1.62 mmol). The reaction mixture was stirred at 20 °C for 2 hours. The reaction was detected by LCMS. Water (150 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (300 mL). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was concentrated to remove the solvent. The residue was then purified by chromatographic separation using a C18 reversed-phase column (mobile phase: acetonitrile / H₂O, elution 35% to 70% for 13 minutes; detector: UV 254 nm) to give a pale yellow solid Int-O1 (500.0 mg, yield 80%). LCMS (ESI) + )m / z=385.9({ 79 Br}M+H) + 387.9({ 81 Br}M+H) + .

[0126] Step 2:

[0127] Potassium cyanide (252.9 mg, 3.88 mmol) and potassium carbonate (357.8 mg, 2.59 mmol) were added to a 10 mL dimethyl sulfoxide solution of compound Int-O1 (500.0 mg, 1.29 mmol). The solution was purged with nitrogen three times and stirred at 120 °C for 16 hours. The reaction was detected by LCMS. The reaction solution was quenched with 150 mL of sodium hypochlorite aqueous solution, then extracted with 100 mL of water and 300 mL of ethyl acetate. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was concentrated to remove the solvent. The residue was then purified by chromatographic separation using a C18 reversed-phase column (mobile phase: acetonitrile / H₂O (0.1% formic acid), elution 40% to 65% for 18 minutes; detector: UV 254 nm). This yielded pale yellow solids Int-O₂₁ (15.0 mg, yield 4%) and Int-O₂₂ (25.0 mg, yield 6%). LCMS (ESI) + )m / z=317.0({ 79 Br}M+H) + 319.0({ 81 Br}M+H) + .

[0128] Step 3:

[0129] To a 1 mL solution (15.0 mg, 47.29 μmol) of compound Int-O21 in dioxane, 1,1-bis(diphenylphosphine)ferrocene palladium chloride (3.5 mg, 4.73 μmol), potassium acetate (13.9 mg, 141.87 μmol), and bis(pinnatrol)boronic acid ester (14.4 mg, 56.75 μmol) were added. The mixture was purged three times with nitrogen, and the reaction solution was stirred at 100 °C for 1 hour. The reaction was confirmed by LC-MS. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain a dark brown solid crude Int-O (15.0 mg, 87% yield), which was used directly in the next step without further purification. LC-MS (ESI) + m / z = 365.1 [M+H] + .

[0130] Synthesis of Int-P

[0131]

[0132] Step 1:

[0133] At 0°C, a 1.6M, 10.21mL solution of magnesium methyl bromide in THF was added dropwise to a 6mL THF solution of compound Int-M (1.0g, 3.27mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction was confirmed by LCMS. The reaction mixture was then added to a 50mL saturated NH4Cl aqueous solution at 0°C, followed by extraction with 50mL ethyl acetate. The extract was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was further purified by solvent removal via concentration. The residue was then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H2O (0.1% FA), elution 17% to 37% for 10 minutes; detector: UV 254nm) to give a white solid Int-P1 (65.0mg, yield 8%). LCMS (ESI) + )m / z=322.0({ 79 Br}M+H) + ,324.0({ 81 Br}M+H) + .

[0134] Step 2:

[0135] Pd(dppf)Cl2 (13.6 mg, 18.62 μmol), KOAc (45.7 mg, 465.55 μmol), and B2Pin2 (52.0 mg, 204.84 μmol) were added to a 1 mL solution of dioxane (1 mL) of compound Int-P1 (60.0 mg, 186.22 μmol). The reaction mixture was stirred at 100 °C for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated under reduced pressure to obtain a black solid, Int-P (60.0 mg, 87% yield), which was used directly in the next step without further purification. LCMS (ESI) m / z = 370.0 [M+H] + .

[0136] Int-Q Synthesis Route

[0137]

[0138] Step 1:

[0139] At room temperature, H₂SO₄ (9.2 g, 91.93 mmol) was added to a solution of compound Int-M₂ (4.0 g, 12.69 mmol) in MeOH (50 mL), and the mixture was stirred at 80 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction solution was filtered, concentrated under reduced pressure, and then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H₂O (0.1% FA), elution 30% to 70% for 10 minutes; detector: UV 254 nm) to give a pale yellow solid Int-Q₁ (1.65 g, yield 45%). LC-MS (ESI) m / z = 283.0 [M+H] + .

[0140] Step 2:

[0141] BAST (1.41 g, 6.36 mmol) was added dropwise to an Int-Q1 (1.5 g, 5.30 mmol) DCM (20 mL) solution at 0°C. The mixture was stirred at this temperature for 1 hour. The resulting mixture was then stirred at 20°C for 16 hours. The reaction was confirmed by LCMS. The reaction mixture was filtered, the filter cake was washed with ethyl acetate and water, the filtrate was extracted, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was concentrated to remove the solvent, yielding a crude yellow oily product, Int-Q2 (1.6 g, 98% yield). This product was used directly in the next step without further purification. ¹H NMR (400 MHz, DMSO-d6) δppm 7.50–7.55 (m, 2H), 7.33–7.39 (m, 2H), 3.62 (s, 3H), 3.37–3.47 (m, 2H), 3.15–3.25 (m, 2H)

[0142] Step 3:

[0143] NaOH (419.48 mg, 10.49 mmol) was added to 15 mL of an aqueous solution of Int-Q2 (1.6 g, 5.24 mmol), and the mixture was stirred at 20 °C for 12 hours. The reaction was confirmed by LCMS. The reaction mixture was filtered, the filter cake was washed with ethyl acetate and water, the filtrate was extracted, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was further concentrated to remove the solvent, yielding a crude yellow oily product, Int-Q3 (1.43 g, 93% yield). This was used directly in the next step without further purification. LCMS (ESI) m / z = 288.9 [M+H] + .

[0144] Step 4:

[0145] HATU (2.24 g, 5.90 mmol, 1.2 eq) and DIPEA (1.27 g, 9.83 mmol) were added to a DMF (15 mL) solution of Int-Q3 (1.43 g, 4.91 mmol) and 4-methylaminothiourea (619.94 mg, 5.90 mmol) and stirred at 20 °C for 16 hours. The reaction was complete as determined by LC-MS. The reaction mixture was filtered, the filter cake was washed with ethyl acetate and water, the filtrate was extracted, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was further concentrated to remove the solvent, yielding a crude brown solid, Int-Q4 (1.85 g, 99% yield). This was used directly in the next step without further purification. LC-MS (ESI) m / z = 376.0 [M+H] + .

[0146] Step 5:

[0147] NaOH (1.9 g, 47.59 mmol) was added to 20 mL of an aqueous solution of Int-Q4 (1.8 g, 4.76 mmol), and the mixture was stirred at 20 °C for 16 hours. The reaction was confirmed by LCMS. The reaction mixture was washed with ethyl acetate and water to remove the filter cake. The filtrate was extracted, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was further concentrated to remove the solvent, yielding a crude yellow oily product, Int-Q5 (1.71 g, 100% yield). This product was used directly in the next step without further purification. LCMS (ESI) m / z = 360.2 [M+H] + .

[0148] Step 6:

[0149] Add 4 mL of HOAc to a 20 mL solution of Int-Q5 (1.7 g, 4.72 mmol) in DCM.

[0150] 2 mL of H₂O₂ was added and stirred at 20 °C for 1 hour. The reaction was confirmed by LCMS. Saturated sodium sulfite solution was added to quench the reaction mixture. The filter cake was washed with ethyl acetate and water. The filtrate was extracted, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was concentrated to remove the solvent, and then purified by C18 reversed-phase chromatography (mobile phase: acetonitrile / H₂O (0.1% FA), elution 30% to 70% for 10 minutes; detector: UV 254 nm) to obtain a colorless oily substance, Int-Q (585 mg, yield 37%). LCMS (ESI) m / z = 328.2

[0151] [M+H] + .

[0152] Example 1: Synthesis of compound Cpd 13

[0153]

[0154] Step 1:

[0155] At room temperature, 1-methylcyclobutyl-1-amine (724.8 mg, 5.96 mmol), K₂CO₃ (5.5 g, 39.74 mmol), and NaI (893.4 mg, 5.96 mmol) were added to a 20 mL solution of compound Int-C (1 g, 3.98 mmol). The reaction mixture was stirred at 20 °C for 12 hours. The reaction was confirmed by LC-MS. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate, 80% to 20% elution for 20 minutes) to give a yellow oily compound Cpd 13-1 (754 mg, 63% yield). LC-MS (ESI) m / z = 301.3 [M+H] + .

[0156] Step 2:

[0157] At room temperature, CbzOsu (938.6 mg, 3.76 mmol) and K2CO3 (1.04 g, 7.54 mmol) were added to a DMF (20 mL) solution of compound Cpd 13-1 (754 mg, 2.52 mmol). The reaction mixture was stirred at 20 °C for 2 hours. The reaction was confirmed by LCMS. Water (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate, 80% to 40% elution for 20 minutes) to give a yellow solid Cpd 13-2 (1 g, 91% yield). LCMS (ESI) m / z = 435.3 [M+H] + .

[0158] Step 3:

[0159] At room temperature, CCl4 (20 mL) was added to a DMF (20 mL) solution of compound Cpd 13-2 (1 g, 2.3 mmol), and then at 0 degrees Celsius, the following was added to the above system: t BuONa (442.3 mg, 4.60 mmol) was added, and the reaction mixture was stirred at 20°C for 2 hours. The reaction was confirmed by LCMS. Water (200 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (200 mL). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate, 80% to 40% eluent for 20 minutes) to give a yellow oily substance, Cpd 13-3 (571 mg, 52% yield). LCMS (ESI)

[0160] m / z = 469.2[M+H] + .

[0161] Step 4:

[0162] To a solution of compound Cpd 13-3 (360 mg, 0.76 mmol) in 1,4-dioxane (9 mL) and water (3 mL), Int-E-P1 (298 mg, 0.84 mmol), K2CO3 (318 mg, 2.30 mmol), and Pd(dppf)Cl2 (56.1 mg, 0.076 mmol) were added. The reaction mixture was purged three times with N2 and stirred at 100°C for 1 hour under N2 atmosphere. The reaction was detected by LCMS to indicate completion. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure and purified by chromatographic separation on a C18 reversed-phase column (mobile phase: acetonitrile / H2O (0.1% FA), elution 5% to 80% for 20 minutes; detector: UV 254 nm) to obtain a yellow oily substance Cpd 13-4 (243 mg, yield 47%). LCMS(ESI)m / z = 660.4[M+H] + .

[0163] Step 5:

[0164] Pd / C (200 mg) was added to 5 mL of a MeOH solution containing 243 mg (0.37 mmol) of compound Cpd 13-4. The reaction mixture was stirred at 20°C for 16 hours under a H2 atmosphere. The reaction was confirmed by LCMS. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure and then purified by chromatographic separation using a C18 reversed-phase column (mobile phase: acetonitrile / H2O (0.05% ammonia), elution 30% to 90% for 15 minutes; detector: UV 254 nm). A white solid Cpd 13 (138.6 mg, 70% yield) was obtained. LCMS (ESI) m / z = 526.4 [M+H]. + . 1 H NMR (400MHz, CD3OD) δppm 8.33(s,1H),8.29(s,1H),8.19(s,1H),8.05(br d,J=6.72Hz,1H),7.88(s,1H),7.57-7.65(m,2H),3.93(s,2H),3.32(br s,3H),2.94-3.08(m,2H),2.57-2.81(m,3H),2.07-2.26(m,2H),1.74-1.98(m,4H),1.42(s,3H),1.18(d,J=5.99Hz,3H).

[0165] The compounds corresponding to Examples 2-8 below were obtained by using a similar synthesis method as in Example 1, with the corresponding raw materials replaced.

[0166]

[0167]

[0168]

[0169] Effects Examples Biological Experimental Methods

[0170] The test compound was dissolved in 100% DMSO, with a stock solution concentration of 10 mM. The initial test concentration was 10 μM, with three-fold serial dilutions, and ten data points, each repeated twice.

[0171]

[0172] The binding constant of the compound to Cbl-b was determined by detecting the competition between the compound and the fluorescent probe (BODIPY labeled). The experiment was conducted using a TR-FRET platform in 384-well plates. Each well contained 0.5 nM biotin-labeled Cbl-b or 2.5 nM Cbl, 50 nM fluorescent probe, 1X Streptavidin-Tb (Cisbio), and 0.5% DMSO. The reaction buffer consisted of 20 mM Epps, pH 7.5, 150 mM NaCl, 0.01% TritionX-100, 0.01% BSA, and 0.5 M TCEP. The compound was incubated with Cbl-b or Cbl at 25 °C for 10 min, followed by the addition of the probe and Streptavidin-Tb. After incubation at 25 °C for 1 hour, the TR-FRET signal was read at 490 / 520 nm. Based on this reading, the percentage inhibition of probe binding by the compound was calculated, and the IC50 of the compound was calculated using a four-parameter fitting method. 50 See Table 1 for details:

[0173] Table 1

[0174]

[0175]

[0176] Where A represents IC 50 ≤100nM; B indicates 100nM < IC 50 ≤1000nM; C indicates 1000nM < IC 50 ≤3000nM, "-" indicates that it has not been tested.

Claims

1. A compound of the following formula: ###0001### or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition of claim 2, for the manufacture of a Cbl-b inhibitor.

4. Use of claim 3 for the treatment of tumors, Type I diabetes, Graves' disease, systemic lupus erythematosus, and multiple sclerosis.

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