Compound with amino-terminated structure as well as preparation method, pharmaceutical composition and application of compound

Abstract

The invention discloses a compound with an amino-terminated structure as well as a preparation method, a pharmaceutical composition and application of the compound. The structure of the compound is as shown in formula (I), and the compound utilizes a ubiquitin-proteasome system, has good VHL targeting affinity on the molecular level, and can effectively degrade VHL protein; on the cellular level, the cell activity can be effectively protected, the mitochondrial function can be improved, then neurons are protected, and a new strategy is provided for treatment of neurodegenerative diseases and mitochondrial defect diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a compound with a terminal amino structure, a preparation method, a pharmaceutical composition and an application thereof, and in particular to a compound with a terminal amino structure as a VHL small molecule degrader, a preparation method, a pharmaceutical composition and an application thereof. Background Art

[0002] Neurodegenerative diseases are a type of progressive disease that occurs frequently in the elderly population, mainly including Alzheimer's disease (AD), Parkinson's disease (PD), etc. PD is a common neurodegenerative disease. Its main pathological features are the degeneration of dopaminergic neurons in the substantia nigra, the lack of dopamine in the striatum, and the formation of Lewy bodies. Its clinical manifestations mainly include motor symptoms represented by resting tremor and hypokinesia, and non-motor symptoms such as cognitive impairment. AD is the most common of all neurodegenerative diseases. Its main features are memory loss and cognitive dysfunction in patients. It is one of the main causes of dementia. This type of disease is characterized by progressive dysfunction of the nervous system as a whole. As time goes by, the patient's condition will continue to worsen. Since the occurrence and development of neurodegenerative diseases are affected by many factors and the pathological mechanism is complex, there is no effective method to completely reverse the patient's neurological damage at this stage.

[0003] In recent years, significant progress has been made in the study of PD, however, the current options for the treatment of PD are still limited. The pathogenesis of PD is complex and there are many influencing factors. Among them, mitochondrial dysfunction plays a key role in the occurrence and progression of PD, and maintaining mitochondrial homeostasis may become an effective strategy to improve PD pathology. The applicant's previous studies have shown that the loss of VHL-1 (human VHL homolog) in Caenorhabditis elegans can effectively reverse the degeneration of dopaminergic neurons caused by defects in the mitochondrial respiratory chain complex. In the PD mouse model, knocking down VHL by transgenic means can improve its movement and coordination function by maintaining mitochondrial homeostasis and alleviate a variety of PD-related pathological changes. The functional loss of VHL exerts anti-PD effects through a wide range of downstream pathways. Therefore, directly reducing the level of VHL protein may be more meaningful for the treatment of PD than simply antagonizing the binding of VHL to the substrate.

[0004] VHL can interact with Elongin B (EloB), Elongin C (EloC) and Cullin2 to form a VCB complex with E3 ubiquitin ligase function, which acts as a recruitment subunit of the target protein to induce ubiquitination and degradation of the target protein. A homologous bivalent degrader targeting VHL has been reported. This strategy induces VHL to degrade its “same species” by linking two VHL ligands together. This approach provides a new strategy for regulating VHL levels to treat neurodegenerative diseases and other related diseases. However, the high molecular weight of the bivalent molecule leads to challenges in membrane permeability and bioavailability, limiting the application of homologous degraders in the treatment of neurodegenerative diseases. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a compound with a terminal amino structure having VHL degradation activity, the second purpose is to provide a method for preparing the compound, the third purpose is to provide a pharmaceutical composition containing the compound, and the fourth purpose is to provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0006] Technical solution: The compound containing a terminal amino structure of the present invention has a structure of formula (I),

[0007]

[0008] in:

[0009] R 1 , R 2 are each independently selected from hydrogen, methyl, halogen, methoxy or propargyloxy;

[0010] R 3 is selected from hydrogen or methyl;

[0011] A is selected from -CH2- or -NH-;

[0012] L represents a connecting chain with a length of 3 to 13 atoms, and the main chain atoms of the connecting chain are selected from carbon, nitrogen, oxygen or sulfur.

[0013] Preferably, in the structure:

[0014] R 1 , R 2 are each independently selected from hydrogen, methyl, halogen, methoxy or propargyloxy;

[0015] R 3 is selected from hydrogen or methyl;

[0016] A is selected from -CH2- or -NH-;

[0017] L is selected from a linear or branched C 3-13Alkylene chain, -(CH2CH2O) n1 -(CH2) n2 -、-(CH2CH2S) n1 -(CH2) n2 -、-(CH2) n1 -Triazole-(CH2) n2 -、-(CH2) n1 -CONH-(CH2) n2 -、-(CH2) n1 -C≡C-(CH2) n2 -or-(CH2) n1 -piperazine-(CH2) n2 -, wherein n1 and n2 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0018] Preferably, in the structure:

[0019] L is selected from

[0020] Preferably, the compound of the present invention is selected from any one of the following compounds:

[0021]

[0022]

[0023]

[0024] Preferably, the pharmaceutically acceptable salt of the present invention is a salt formed by the compound of the present invention and any one of the following acids: hydrochloric acid, hydrobromic acid, carbonic 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 or ferulic acid.

[0025] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents discovered by the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds 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 amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds 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, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Citric acid, tartaric acid and methanesulfonic acid and similar acids; organic acid salts also include amino acids (such as arginine, etc.), glucuronic acid and other organic acid salts. Certain specific compounds of the present invention contain basic and acidic functional groups, and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and 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 solubility in polar solvents.

[0026] The "pharmaceutically acceptable salts" of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0027] The applicant's previous studies found that knocking out VHL-1 (human VHL homolog) in a nematode model with defects in the mitochondrial respiratory chain complex can reverse the degeneration of dopaminergic neurons. In addition, inhibiting VHL by various means in cell and mammalian neurodegenerative disease models can protect mitochondrial homeostasis, thereby alleviating various neural injuries and improving related symptoms.

[0028] E2 ubiquitin conjugating enzyme has the characteristic of low selectivity and is easy to ubiquitinate primary amine groups in the environment. The present invention utilizes this characteristic to connect VHL ligand to a specific primary amine group, which can be ubiquitinated by E2 enzyme recruited by VHL. This modification can convert VHL ligand into an inducer of VHL degradation, and utilize the ubiquitin-proteasome system to degrade VHL.

[0029] The compounds designed by the present invention have suitable molecular weight and good physical and chemical properties, can protect neurodegenerative disease cell models, and show good therapeutic effects on both PD and AD models, and this effect is related to improving mitochondrial function. There are no drugs targeting VHL in clinical practice. The present invention is expected to provide new ideas for the prevention and treatment of neurodegenerative diseases and mitochondrial-related diseases and drug design, and has broad application prospects.

[0030] The compound I of the present invention is prepared by condensation and Boc protection removal of compound II:

[0031]

[0032] Among them, R 1 , R 2 , R 3 ,A,L are defined as above;

[0033] The compound I prepared by the above method is salified with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

[0034] The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

[0035] The pharmaceutically acceptable carrier can be an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical adjuvant can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical adjuvant can include one or more of the following adjuvants: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0036] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0037] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations 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 lozenges.

[0038] The compound or pharmaceutically acceptable salt thereof or pharmaceutical composition thereof of the present invention is used in preparing medicine for treating neurodegenerative diseases or mitochondrial defect diseases.

[0039] Preferably, the drug is a drug for treating Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Leigh syndrome or mitochondrial enzyme deficiency.

[0040] Preferably, the drug is a drug having VHL degradation activity.

[0041] Specifically, the VHL small molecule degrader of the present invention can effectively reduce the level of VHL protein, thereby maintaining mitochondrial homeostasis, promoting energy production, and improving neurodegenerative diseases or mitochondrial defect diseases.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0043] The compounds designed by the present invention utilize the ubiquitin-proteasome system, have good VHL targeting at the molecular level, and have an affinity with the VHL protein at the nanomolar concentration level. They can effectively degrade the VHL protein, and the protein degradation rate can reach more than 50%. At the cellular level, they can effectively protect cell vitality and improve mitochondrial function, thereby protecting neurons. They have broad application prospects in the treatment of neurodegenerative diseases and mitochondrial defect diseases, and provide a new strategy for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The effect of VHL small molecule degraders at different concentrations on VHL protein levels in SH-SY5Y cells;

[0045] Figure 2 The improvement effect of VHL small molecule degrader on abnormal cell death of SH-SY5Y PD model. The Control group is the control group, and the MPP + Group is the model group, MPP + +I-5 group is the small molecule degrader group to be tested;

[0046] Figure 3 The improvement effect of VHL small molecule degrader on abnormal cell death of SH-SY5Y cells in AD model. The Control group is the control group, and Aβ 1-42 The model group, Aβ 1-42 +I-5 group is the small molecule degrader group to be tested;

[0047] Figure 4 The effect of VHL small molecule degrader on mitochondrial membrane potential of SH-SY5Y cells in the PD model. The Control group is the control group, and the MPP + Group is the model group, MPP + +I-5 group is the small molecule degradation agent group to be tested. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below in conjunction with embodiments.

[0049] Example 1: Preparation of Compound I-1

[0050] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-[4-(4-methylthiazol-5-yl)benzyl]pyrrolidine-2-carboxamide (50 mg, 0.1163 mmol) was dissolved in DMF (5 mL), and tert-butoxycarbonyl-6-aminohexanoic acid (27 mg, 0.1163 mmol), TEA (24 mg, 0.232 mmol), and HATU (53 mg, 0.1395 mmol) were added in sequence, and the reaction was stirred for 1 h. After the reaction was completed, water (5 mL) was added to quench the reaction, and ethyl acetate (5 mL × 3) was added for extraction. The organic extracts were combined, washed with water (5 mL × 3), and then washed with saturated brine (10 mL × 1). The extract was dried over anhydrous magnesium sulfate and concentrated to obtain a crude product, which was purified by silica gel (200-300 mesh) column chromatography and eluted with 9% methanol in dichloromethane to obtain a white powdery solid, which was dissolved in 2 mL of ethyl acetate, the temperature was controlled at 0-5°C in an ice-water bath, and 4M HCl ethyl acetate solution (5 mL) was slowly added dropwise to precipitate a white solid. Filtering gave 37 mg of a white powdery solid, and the total yield was 60%.

[0051] 1 H NMR(400MHz,DMSO-d6)δ7.66-7.60(m,1H),7.52(d,J=10.1Hz,1H),7.29-7.22(m,1H) ,4.36-4.30(m,1H),4.33-4.25(m,0H),3.98(d,J=5.1Hz,0H),3.79(d,J=5.1Hz,0H), 3.65(d,J=9.5Hz,0H),3.42(d,J=9.5Hz,0H),2.74-2.64(m,1H),2.50(s,1H),2.22-2 .09(m,1H),1.75(s,1H),1.56-1.45(m,1H),1.38(d,J=0.9Hz,1H),0.97(s,3H).EI-MS m / z:544[M] + .

[0052] Example 2: Preparation of Compound I-2

[0053] The preparation method of compound I-2 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with tert-butoxycarbonyl-8-aminooctanoic acid to obtain 36 mg of a white powdery solid with a total yield of 55%.

[0054] 1H NMR (400MHz, DMSO-d6) δ8.77 (s, 1H), 7.66-7.60 (m, 2H), 7.52 (d, J = 10.1Hz, 2H), 7.28-7.23 (m, 2H), 4.4 5(s,1H),4.36-4.25(m,3H),3.98(d,J=5.1Hz,1H),3.79(d,J=5.1Hz,1H),3.65(d,J=9.5Hz,1H),3.42(d ,J=9.5Hz,1H),2.69(d,J=5.5Hz,2H),2.50(s,2H),2.36(s,1H),2.22-2.15(m,2H),2.12(d,J=11.2Hz,1 H),1.54(d,J=1.1Hz,2H),1.50(d,J=0.9Hz,2H),1.32(d,J=4.6Hz,4H),1.29(s,2H),0.97(s,7H).EI-MS m / z:572[M] + .

[0055] Example 3: Preparation of Compound I-3

[0056] The preparation method of compound I-3 is the same as compound I-1, except that tert-butoxycarbonyl-6-aminohexanoic acid is replaced with tert-butoxycarbonyl-11-aminoundecanoic acid to obtain 35 mg of a white powdery solid with a total yield of 48%.

[0057] 1 H NMR (400MHz, DMSO-d6) δ8.77(s,1H),7.66-7.60(m,2H),7.52(d,J=10.1Hz,2H),7.28-7.23(m,2H),4.45( s,1H),4.36-4.25(m,3H),3.98(d,J=5.1Hz,1H),3.79(d,J=5.1Hz,1H),3.65(d,J=9.5Hz,1H),3.42(d,J= 9.5Hz,1H),2.69(d,J=5.5Hz,2H),2.50(s,2H),2.36(s,1H),2.22-2.15(m,2H),2.12(d,J=11.2Hz,1H),1 .54(d,J=1.1Hz,2H),1.50(d,J=0.9Hz,2H),1.34-1.27(m,8H),1.28(d,J=1.6Hz,4H),0.97(s,7H).EI-MS m / z:614[M] + .

[0058] Example 4: Preparation of Compound I-4

[0059] The preparation method of compound I-4 is the same as compound I-1, except that tert-butyloxycarbonyl-6-aminohexanoic acid is replaced with 12-(Boc-amino)dodecanoic acid to obtain 36 mg of a white powdery solid with a total yield of 50%.

[0060] 1 H NMR (400MHz, DMSO-d6) δ8.77 (s, 1H), 7.66-7.60 (m, 2H), 7.52 (d, J = 10.1Hz, 2H), 7.28-7.22 (m, 2H), 4.45 (s,1H),4.36-4.25(m,3H),3.98(d,J=5.1Hz,1H),3.79(d,J=5.1Hz,1H),3.65(d,J=9.5Hz,1H),3.42(d, J=9.5Hz,1H),2.69(d,J=5.5Hz,2H),2.50(s,2H),2.36(s,1H),2.22-2.15(m,2H),2.12(d,J=11.2Hz,1H ),1.54(d,J=1.1Hz,2H),1.50(d,J=0.9Hz,2H),1.34-1.27(m,8H),1.30-1.25(m,6H),0.97(s,7H).EI-MS m / z:628[M] + .

[0061] Example 5: Preparation of Compound I-5

[0062] The preparation method of compound I-5 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with 5-tert-butoxycarbonylamino-3-oxavalanic acid to obtain 25 mg of a white powdery solid with a total yield of 40%.

[0063] 1 H NMR(400MHz,DMSO-d6)δ8.77(s,1H),7.86(s,1H),7.66-7.60(m,2H),7.53(s,1H),7.29-7.22( m,2H),4.45(s,1H),4.36-4.30(m,2H),4.30-4.25(m,1H),4.10-4.00(m,2H),3.98(d,J=5.1Hz ,1H),3.79(d,J=5.1Hz,1H),3.69-3.63(m,2H),3.55(d,J=12.5Hz,1H),3.42(d,J=9.5Hz,1H), 2.98(d,J=2.7Hz,2H),2.64(s,1H),2.57(s,1H),2.50(s,2H),2.36(s,1H),0.97(s,7H).EI-MS m / z:532[M]+ .

[0064] Example 6: Preparation of Compound I-6

[0065] The preparation method of compound I-6 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid to obtain 35 mg of a white powdery solid with a total yield of 52%.

[0066] 1 H NMR(400MHz,DMSO-d6)δ8.77(s,1H),7.86(s,1H),7.66-7.60(m,2H),7.53(s,1H),7 .29-7.22(m,2H),4.45(s,1H),4.38-4.25(m,2H),4.13-4.03(m,2H),3.98(d,J=5.1 Hz,1H),3.79(d,J=5.1Hz,1H),3.68-3.51(m,6H),3.42(d,J=9.5Hz,1H),2.95(d,J= 0.7Hz,2H),2.64(s,1H),2.57(s,1H),2.50(s,2H),2.36(s,1H),0.97(s,6H).EI-MS m / z:576[M] + .

[0067] Example 7: Preparation of Compound I-7

[0068] The preparation method of compound I-7 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with N-tert-butoxycarbonyl-tetraethylene glycol-acetic acid to obtain 40 mg of a white powdery solid with a total yield of 51%.

[0069] 1H NMR(400MHz,DMSO-d6)δ8.77(s,1H),7.66-7.60(m,2H),7.53(s,1H),7.48(s,1H),7.29-7.22(m,2H) ,4.45(s,1H),4.36-4.31(m,2H),4.28(dt,J=12.4,1.3Hz,1H),3.98(d,J=5.1Hz,1H),3.79(d,J=5.1H z,1H),3.74(d,J=12.4Hz,1H),3.69-3.50(m,17H),3.42(d,J=9.5Hz,1H),2.95(d,J=0.7Hz,2H),2.64 (s,1H),2.57(s,1H),2.51(d,J=12.3Hz,3H),2.44(d,J=12.5Hz,1H),2.36(s,1H),0.97(s,7H).EI-MS m / z:678[M] + .

[0070] Example 8: Preparation of Compound I-8

[0071] The preparation method of compound I-8 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with 2-[(2-{[(tert-butoxy)carbonyl]amino}ethyl)sulfanyl]acetic acid to obtain 37 mg of a light yellow oily liquid with a total yield of 51%.

[0072] 1 H NMR(500MHz,Chloroform-d)δ8.76(s,1H),7.93(s,1H),7.69(s,1H),7.43(dd,J=7.5,2.0Hz,1H),7.18(dt,J=7.5,0.9Hz,1 H),6.96(d,J=2.0Hz,1H),4.82(s,2H),4.44(d,J=6.2Hz,2H),4.42-4.36(m,1H),4.28(dt,J=12.3,0.7Hz,1H),3.98(d,J=5 .1Hz,1H),3.79(d,J=5.1Hz,1H),3.65(d,J=9.5Hz,1H),3.42(d,J=9.5Hz,1H),3.27(d,J=12.5Hz,1H),3.22(d,J=12.3Hz,1 EI-MS m / z:602[M] + .

[0073] Example 9: Preparation of Compound I-9

[0074] The preparation method of compound I-9 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminohexanoic acid is replaced with 7-[(tert-butoxycarbonyl)amino]hept-5-ynoic acid to obtain 47 mg of a light yellow oily liquid with a total yield of 67%.

[0075] 1 HNMR (400MHz, DMSO-d6) δ8.83(s,1H),8.14(d,J=0.8Hz,1H),7.84(d,J=0.6Hz,1H),7.44(dd,J=7.5,2.0Hz,1H ),7.14(dt,J=7.5,1.1Hz,1H),7.09(d,J=1.9Hz,1H),4.53(d,J=5.0Hz,1H),4.44(s,1H),4.38-4.24(m,3H),3. 91(d,J=5.0Hz,1H),3.82(s,3H),3.72(t,J=2.0Hz,2H),3.58(d,J=9.5Hz,1H),3.36(d,J=9.5Hz,1H),2.36(dt ,J=12.5,2.1Hz,1H),2.34-2.26(m,2H),2.24-2.12(m,2H),2.09(s,1H),1.88-1.75(m,2H),0.97(s,8H).EI-MS m / z:618[M] + .

[0076] Example 10: Preparation of Compound I-10

[0077] The preparation method of compound I-10 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminohexanoic acid is replaced with 4-{4-[2-((tert-butoxycarbonyl)amino)ethyl]-4H-114,2,3-triazol-1-yl}butyric acid to obtain 34 mg of a light yellow oily liquid with a total yield of 47%.

[0078] 1H NMR (400MHz, DMSO-d6) δ8.81(s,1H),8.29(s,1H),7.84(d,J=0.6Hz,1H),7.55(d,J=7.5Hz,1H),7.23(dt,J=2.1 ,1.0Hz,1H),7.13(ddt,J=7.4,1.9,1.0Hz,1H),4.53(d,J=5.0Hz,1H),4.44(s,1H),4.37-4.24(m,3H),4.03(d, J=0.6Hz,2H),3.91(d,J=5.0Hz,1H),3.58(d,J=9.5Hz,1H),3.36(d,J=9.5Hz,1H),2.81(d,J=2.2Hz,2H),2.69( d,J=0.7Hz,1H),2.54(d,J=0.7Hz,1H),2.31(s,1H),2.22(s,2H),2.09(s,1H),1.94(s,2H),0.97(s,8H).EI-MS m / z:629[M] + .

[0079] Example 11: Preparation of Compound I-11

[0080] The preparation method of compound I-11 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminocaproic acid is replaced with 3-{3-[(tert-butoxycarbonyl)amino]propylamino}propionic acid to obtain 50 mg of a light yellow oily liquid with a total yield of 73%.

[0081] 1 HNMR(400MHz, DMSO-d6)δ8.83(s,1H),8.19(d,J=0.6Hz,1H),7.98(d,J=8.8Hz,2H),7.67-7.59(m,2H),7.25-7.18 (m,2H),5.07(d,J=0.8Hz,1H),4.96(q,J=0.8Hz,1H),4.82(d,J=0.7Hz,1H),4.53(d,J=5.0Hz,1H),4.40(s,1H),4 .33(d,J=0.6Hz,1H),3.91(d,J=5.0Hz,1H),3.58(d,J=9.5Hz,1H),3.34(d,J=9.5Hz,1H),3.22-3.13(m,2H),2.77 EI-MS m / z:587[M] + .

[0082] Example 12: Preparation of Compound I-12

[0083] The preparation method of compound I-12 is the same as that of compound I-1, except that tert-butoxycarbonyl-6-aminohexanoic acid is replaced with {2-[4-((((tert-butoxycarbonyl)amino)methyl)piperazin-1-yl]ethyl}carbamic acid to obtain 40 mg of a light yellow oily liquid with a total yield of 56%.

[0084] 1 H NMR (400MHz, DMSO-d6) δ8.83 (s, 1H), 8.24 (d, J = 0.8Hz, 1H), 7.67-7.59 (m, 2H), 7.29-7.21 (m, 2H), 6 .65(d,J=0.6Hz,1H),6.13(d,J=0.7Hz,1H),4.53(d,J=5.0Hz,1H),4.44(s,1H),4.35(d,J=0.6Hz,1H ),4.34-4.21(m,2H),3.91(d,J=5.0Hz,1H),3.63-3.51(m,3H),3.34(d,J=9.5Hz,1H),3.24(s,1H),3 .22(s,1H),3.14(d,J=9.2Hz,1H),2.56-2.45(m,12H),2.31(s,1H),2.09(s,1H),0.97(s,8H).EI-MS m / z:615[M] + .

[0085] Example 13: Preparation of Compound I-13

[0086] The preparation method of compound I-13 is the same as that of compound I-1, except that tert-butyloxycarbonyl-6-aminocaproic acid is replaced with 2-(2-aminoethoxy)acetic acid to obtain 43 mg of a light yellow oily liquid with a total yield of 64%.

[0087] 1HNMR(400MHz, DMSO-d6)δ8.83(s,1H),8.19(d,J=0.6Hz,1H),7.98(d,J=8.8Hz,2H),7.67-7.59(m,2H),7.25-7.18 (m,2H),5.07(d,J=0.8Hz,1H),4.96(q,J=0.8Hz,1H),4.82(d,J=0.7Hz,1H),4.53(d,J=5.0Hz,1H),4.40(s,1H),4 .33(d,J=0.6Hz,1H),3.91(d,J=5.0Hz,1H),3.58(d,J=9.5Hz,1H),3.34(d,J=9.5Hz,1H),3.22-3.13(m,2H),2.77 EI-MS m / z:586[M] + .

[0088] Example 14: Preparation of Compound I-14

[0089] The preparation method of compound I-14 is the same as compound I-1, except that tert-butyloxycarbonyl-6-aminocaproic acid is replaced with 5-aminopentanoic acid to obtain 30 mg of a light yellow oily liquid with a total yield of 45%.

[0090] 1 HNMR(400MHz, DMSO-d6)δ8.83(s,1H),8.19(d,J=0.6Hz,1H),7.98(d,J=8.8Hz,2H),7.67-7.59(m,2H),7.25-7.18 (m,2H),5.07(d,J=0.8Hz,1H),4.96(q,J=0.8Hz,1H),4.82(d,J=0.7Hz,1H),4.53(d,J=5.0Hz,1H),4.40(s,1H),4 .33(d,J=0.6Hz,1H),3.91(d,J=5.0Hz,1H),3.58(d,J=9.5Hz,1H),3.34(d,J=9.5Hz,1H),3.22-3.13(m,2H),2.77 EI-MS m / z:576[M] + .

[0091] Example 15: Evaluation of the affinity of the compound to the target protein VCB - using an affinity-based fluorescence polarization (FP) activity test method

[0092] The ability of the test compound to compete with the fluorescent group-labeled HIF-1α peptide (FAM-DEALAHyp-YIPD) to bind to VCB protein was tested. A 384-well black microplate was used in the test. The final test volume was 60 μL. The tested compound and FAM-DEALAHyp-YIPD were dissolved in DMSO and pure water respectively for use. The compound was diluted with assay buffer in 12 concentration gradients and 20 μL of the diluted 300 nM VCB protein was added to each well. Two replicate wells were set for each compound concentration. A blank control (20 μL FAM-DEALAHyp-YIPD + 40 μL assay buffer) and a negative control (20 μL FAM-DEALAHyp-YIPD + 20 μL FIH + 20 μL assay buffer) were set for each test. Incubate at room temperature for 1 hour, scan the plate with a Synergy plate reader, and set the excitation wavelength to 490 nm and the emission wavelength to 520 nm. Calculation formula: % inhibition rate = 100 × [1-(measured value-blank) / (negative value-blank)], the inhibition rate corresponding to the specific concentration was obtained. The obtained data was imported into Graphpadprism 8.0 for analysis and fitting to obtain IC 50 The affinity test results of the compounds are shown in Table 1.

[0093] Example 16: Evaluation of the ability of compounds to degrade VHL - Western blot detection

[0094] Compound group: SH-SY5Y neural cells were cultured to the logarithmic growth phase, and the cells were collected and inoculated in a 6-well plate and placed in a cell culture incubator (37°C, 5% CO2) for 24 hours. The cells were co-incubated with 1 μM of different compounds and cultured for another 24 hours. The cells were lysed and the protein was collected. The protein concentration was determined by the BCA method and the corresponding volume of PBS was added. Loading buffer was added to the protein solution and the sample was boiled in a boiling water bath for 10 minutes. After electrophoresis, the protein was transferred to the PVDF membrane by wet transfer. The PVDF membrane was placed in a blocking solution (5% skim milk powder) and blocked at room temperature for 2 hours; the primary antibody was incubated at 4°C overnight; the secondary antibody was incubated at room temperature for 2 hours. After the PVDF membrane was treated with ECL luminescent solution, the bands were exposed by the chemiluminescence imaging system. Image J software was used for quantification and calculation of the degradation rate of VHL protein. The results are shown in Table 1.

[0095] Table 1 Affinity of each compound in the present invention for VHL and degradation rate of VHL protein

[0096] serial number <![CDATA[K i (nM)]]> VHL protein degradation rate (%) I-1 ＜300 68 I-2 ＜300 80 I-3 ＜300 51 I-4 ＜300 67 I-5 ＜300 82 I-6 ＜300 39 I-7 ＜300 63 I-8 ＜300 56 I-9 ＜300 32 I-10 ＜300 40 I-11 ＜300 54 I-12 ＜300 32 I-13 ＜300 66 I-14 ＜300 28

[0097] As shown in Table 1, the affinity of the compounds designed by the present invention to the VHL protein is at the nanomolar level, showing good target affinity. In addition, at the cellular level, the compounds at a concentration of 1 μM can degrade the VHL protein well, among which 9 compounds (I-1 to I-5, I-7, I-8, I-11, I-13) have a degradation rate of more than 50% for VHL.

[0098] Example 17: Evaluation of the ability of compound I-5 to degrade VHL - Western blot detection

[0099] Test compound group: SH-SY5Y neural cells were cultured to the logarithmic growth phase, the cells were collected and inoculated into 6-well plates, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 hours. The cells were co-incubated with different concentrations of compound I-5 and cultured for another 24 hours. The VHL protein content was then characterized by the method in Example 16.

[0100] The results are as follows Figure 1 As shown, after SH-SY5Y neurons were treated with compound I-5 for 24 hours, the VHL protein level decreased in a dose-dependent manner, indicating that the degrading agent has significant degradation activity on VHL. The above results show that the compounds designed by the present invention can significantly reduce the intracellular VHL protein level, providing a new method for VHL targeted degradation.

[0101] Example 18: Evaluation of the protective effect of VHL small molecule degraders on abnormal cell death in PD models

[0102] Compound I-5 group: SH-SY5Y neural cells were cultured to the logarithmic growth phase, and the cells were collected and inoculated into 6-well plates, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 hours. + The solution was co-incubated with 1 μM I-5 and cultured for 24 hours. The original culture medium in the 6-well plate was discarded and pre-cooled PBS was added for rinsing. Repeat 3 times. Hoechst and PI were mixed and diluted with DMEM high-glucose medium to prepare the staining working solution. Then 1 mL of staining working solution was added to each well and incubated on ice for 25 minutes. After the incubation, the well plate was rinsed 3 times with pre-cooled PBS and transferred to an inverted fluorescence microscope for observation and photography.

[0103] Compared with the compound I-5 group, the control group did not add MPP + and compound I-5, while the model group did not add compound I-5.

[0104] The results are as follows Figure 2 As shown, compound I-5 can significantly alleviate the abnormal death of PD cell model.

[0105] Example 19: Evaluation of the protective effect of VHL small molecule degraders on abnormal cell death in AD models

[0106] Compound I-5 group: SH-SY5Y neural cells were cultured to the logarithmic growth phase, and the cells were collected and inoculated into 6-well plates, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 hours. 1-42 The solution was co-incubated with 1 μM I-5 and cultured for 24 hours. The original culture medium in the 6-well plate was discarded, and pre-cooled PBS was added to rinse, and repeated 3 times. Hoechst and PI were mixed and diluted with DMEM high-glucose medium to prepare the staining working solution, and then the staining working solution was added to each well at a volume of 1 mL per well, and incubated on ice for 25 minutes. After the incubation, the well plate was rinsed 3 times with pre-cooled PBS, and transferred to an inverted fluorescence microscope for observation and photography.

[0107] Compared with the compound I-5 group, the control group did not add Aβ 1-42 and compound I-5, while the model group did not add compound I-5.

[0108] The results are as follows Figure 3 As shown, compound I-5 can significantly alleviate the abnormal death of AD cell models.

[0109] Example 20: Evaluation of the protective effect of VHL small molecule degraders on mitochondrial membrane potential of PD model cells

[0110] Compound I-5 group: SH-SY5Y cells were cultured to the logarithmic growth phase, and the cells were collected and inoculated into 6-well plates, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h. The cells were incubated with 0.2 mM MPP + The solution was co-incubated with 1 μM I-5 and cultured for 24 hours. The original culture medium in the 6-well plate was discarded, and pre-cooled PBS was added for rinsing. TMRE was diluted with DMEM high-glucose medium to prepare the staining working solution, and then the staining working solution was added to each well according to the volume of 1 mL per well, and incubated in a cell culture incubator (37°C, 5% CO2) for 30 minutes. After the incubation, the cells were rinsed 3 times with pre-cooled DMEM high-glucose medium, and observed and photographed under an inverted fluorescence microscope.

[0111] Compared with the compound I-5 group, the control group did not add MPP + and compound I-5, while the model group did not add compound I-5.

[0112] The results are as follows Figure 4 As shown, compound I-5 can significantly improve MPP +The induced mitochondrial membrane potential damage in the PD cell model indicates that the VHL small molecule degrader designed and synthesized by the present invention can improve mitochondrial function and thus protect neurons.

Claims

1. A compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof, in: R 1 , R 2 are each independently selected from hydrogen, methyl, halogen, methoxy or propargyloxy; R 3 is selected from hydrogen or methyl; A is selected from -CH2- or -NH-; L represents a connecting chain with a length of 3 to 13 atoms, and the main chain atoms of the connecting chain are selected from carbon, nitrogen, oxygen or sulfur.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: In the structure: R 1 , R 2 are each independently selected from hydrogen, methyl, halogen, methoxy or propargyloxy; R 3 is selected from hydrogen or methyl; A is selected from -CH2- or -NH-; L is selected from a linear or branched C 3-13 Alkylene chain, -(CH2CH2O) n1 -(CH2) n2 -、-(CH2CH2S) n1 -(CH2) n2 -、-(CH2) n1 -Triazole-(CH2) n2 -、-(CH2) n1 -CONH-(CH2) n2 -、-(CH2) n1 -C≡C-(CH2) n2 -or-(CH2) n1 -piperazine-(CH2) n2 -, wherein n1 and n2 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: In the structure: L is selected from 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from any one of the following compounds:

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, carbonic 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 or ferulic acid.

6. A method for preparing the compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound I is prepared by condensation and Boc removal reaction of compound II: Among them, R 1 , R 2 , R 3 , A, L are defined as in claim 1; The compound I prepared by the above method is salified with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

7. A pharmaceutical composition, characterized in that Comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

8. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating neurodegenerative diseases or mitochondrial defect diseases.

9. The use according to claim 8, characterized in that: The drug is a drug for treating Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Leigh syndrome or mitochondrial enzyme deficiency.

10. The use according to claim 8, characterized in that: The drug is a drug with VHL degradation activity.

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