HyT compound, synthetic method thereof and application of HyT compound as TAU protein targeting degradation agent

Abstract

The invention discloses a HyT compound and a synthesis method thereof, and application of the HyT compound as a TAU protein targeting degradation agent, the HyT compound has a structure as shown in a formula I, and can be used for preparing a pharmaceutical preparation for preventing or treating TAU-mediated diseases, such as Alzheimer's disease (AD). # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of small molecule drugs, and in particular, to a HyT compound and a synthesis method thereof and an application thereof as a degradation agent targeting TAU protein. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that continuously affects memory and cognitive function as people age. It accounts for 60-80% of all dementia cases and is the fifth leading cause of death. Studies have shown that the two pathological characteristics of AD are the deposition of Aβ protein and neurofibrillary tangles formed by Tau protein. Currently, a variety of therapies targeting Aβ have been developed, such as reducing the production of Aβ through β- / γ-secretase inhibitors, removing Aβ by promoting its degradation or antibodies. However, the clinical effects of these strategies targeting Aβ are not ideal. At present, more and more studies have shown that Tau protein aggregation is common in AD patients, and Tau protein has become a new target for AD treatment.

[0003] Tau protein is a protein that regulates microtubule formation and stability, and plays an important role in the process of microtubule assembly. Under normal physiological conditions, the phosphorylation and dephosphorylation of Tau protein form a dynamic balance. However, once this balance is broken, excessive phosphorylation of Tau protein and separation of Tau protein from microtubules will occur, and the separated Tau will gradually evolve from a monomeric state into a neurotoxic aggregate. Therefore, if the pathogenic Tau protein is eliminated, it may effectively improve the health of AD patients.

[0004] Recently, the use of targeted protein degradation (TPD) strategies has been proposed to regulate the degradation of pathogenic Tau protein and prevent its further aggregation. Chu et al. designed and synthesized a hydrophobic tag-conjugated peptide (HyT-Tau-CPP) to selectively promote the degradation of Tau protein. HyT-Tau-CPP not only effectively reduces the level of Tau protein in vitro in a concentration- and time-dependent manner, but also reduces the level of Tau protein in the brain of AD mouse models. Jing et al. developed a peptide-PROTAC-C004019 by recruiting Keap1-Cul3 ubiquitin E3 ligase and applied it to the degradation of intracellular Tau protein

[25] The results showed that it accelerated the clearance of Tau through the ubiquitin-proteasome system (UPS) and improved the cognitive function of AD mice. At the same time, the identification of ubiquitination sites on soluble and insoluble highly phosphorylated Tau proteins demonstrated the important role of UPS in Tau clearance.

[27] Chang Hoon Ji et al. developed an AUTOTAC degrader that targets the aggregation-prone P301LTau mutant and targets the autophagic membrane for lysosomal degradation. The above studies show that Tau protein can be degraded through the proteasome pathway or the lysosomal pathway.

[0005] However, the activity of the small molecule inhibitors or degraders currently under development against TAU ​​is still not strong enough, and small molecule inhibitors can easily induce secondary drug resistance because they need to occupy the target protein for a long time; degraders also have many problems. Therefore, new drugs targeting TAU are still an urgent need for people's health and medical research. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art and to provide a HyT compound and a synthesis method thereof and an application thereof as a degradation agent targeting TAU protein.

[0007] The object of the present invention is achieved through the following technical solution: A HyT compound, i.e., a degradation agent targeting TAU, has the structure of the following formula I:

[0008]

[0009] in,

[0010] X is C, N, O or S;

[0011] L is a connecting chain, including a nonlinear chain, an aliphatic chain, an aromatic chain, and a heteroaromatic ring structure chain, and the two parts are connected by a covalent bond. The structure of some connecting chains is shown in the following formula:

[0012]

[0013] Wherein, m and n are independently selected from integers of 0-10;

[0014] R is the following structure:

[0015]

[0016] Furthermore, R is the following structure:

[0017] Furthermore, the present invention provides a degradation agent targeting TAU, which is a compound with the following structure:

[0018]

[0019] in,

[0020] X is a C or N atom;

[0021] n is independently selected from integers of 0-10.

[0022] Furthermore, the present invention provides a degradation agent targeting TAU, which is a compound with the following structure:

[0023] Compound II-1n=4,X=C;

[0024] Compound II-2n=3, X=N;

[0025] Compound II-3n=4,X=N.

[0026] The present invention also provides a method for preparing a degradation agent targeting TAU, and the reaction equation is as follows:

[0027]

[0028] The definitions of X and n are the same as above.

[0029] The present invention also provides a pharmaceutical application of a TAU-targeted degradation agent, which is specifically used for preparing a pharmaceutical preparation for preventing or treating a disease mediated by TAU protein, wherein the disease mediated by TAU is Alzheimer's disease (AD).

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The targeted protein degradation technology based on hydrophobic tagging (HyT) is to simulate the state of partial misfolding of the protein by introducing a larger hydrophobic group on the surface of the protein, so that it can be recognized by the molecular chaperone and achieve the degradation of the target protein. One end of the HyT bifunctional molecule is the ligand of the target protein, and the other end is a hydrophobic group, connected by a linker in the middle. Commonly used hydrophobic groups include phenyl series, adamantane, Boc3-Arg, norbornene, etc. Among them, adamantane has the advantages of small molecular weight and strong hydrophobicity compared to other molecules. The present invention solves the problem of drug resistance and insufficient activity of small molecule TAU inhibitors in the form of protein degradation. A TAU-targeted degrader provided by the present invention does not need to occupy the active site for a long time like an inhibitor, but only needs to be briefly combined with the TAU protein to induce the protein degradation effect. Since the protein needs to be resynthesized to restore its function after being degraded, this greatly delays the development of drug resistance; it is still effective for those proteins that have already developed drug resistance. Therefore, the TAU-targeted degradation agent provided by the present invention may be used in the preparation of pharmaceutical preparations for preventing or treating diseases mediated by TAU protein, such as Alzheimer's disease (AD). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1. Immunoblotting was used to detect the degradation effect of HyT on Tau in CHO-K1 cells;

[0033] Figure 2 . Clearance effect of different concentrations of Ⅱ-1 on total Tau and p-Tau in CHO-K1 cells

[0034] Figure 3 .Statistical histogram of the accuracy rate of spontaneous alternation of each group in the Y-maze experiment

[0035] Figure 4 . Immunoblotting was used to detect the clearance effect of Ⅱ-1 on Tau and p-Tau in the brain of 3×Tg-AD mice

[0036] Figure 5 .ELISA detection of changes in ILs and TNF-α in the brain of 3×Tg-AD mice DETAILED DESCRIPTION

[0037] The structure, preparation method and application of the present invention in preparing pharmaceutical preparations for preventing or treating diseases caused by excessive tubulin expression are further described below in conjunction with the examples, but the present invention is not limited thereto.

[0038] The analytical data of the samples were measured by the following instruments:

[0039] Thermometer uncalibrated; Bruker DRX400 NMR; Agilent 5975 mass spectrometer; Bruker Vector 22 infrared spectrometer.

[0040] Example 1: Synthesis of intermediates

[0041]

[0042] Step 1: Synthesis of intermediate a

[0043]

[0044] Weigh 2,6-dihydroxyquinoline (5.0 g, 31.05 mmol, 1 eq) into a 50 mL three-necked flask, dissolve the solid with 30 mL DMF, then pass N2 into the reaction flask to remove O2, slowly add POCl3 (11.23 mL, 155.23 mmol, 5 eq) at 0°C, and transfer to 70°C for overnight reaction. The next day, TLC detected that the reaction was complete, the reaction solution was placed in an open beaker, and ice cubes were added while stirring until no smoke was generated. A saturated aqueous solution of NaOH was added to adjust the base to pH = 7-8, and then extracted with saturated brine (150 mL) and ethyl acetate (100 mL×3), the organic phases were combined and washed with saturated brine (250 mL×3), anhydrous sodium sulfate was added to the organic phase for drying, filtering, concentrating, recrystallizing with ethyl acetate and petroleum ether, and filtering the filter cake to obtain 3.96 g of a yellow-brown solid a with a yield of 79.20%. 1 H NMR (400MHz, DMSO-D6) δ10.20 (s, 1H), 8.21 (d, J = 8.1Hz, 1H), 7.80 (d, J = 9.0Hz, 1H),7.42(d,J=8.6Hz,1H),7.36(dd,J=9.0,2.7Hz,1H),7.21(d,J=2.7Hz,1H). 13 CNMR(101MHz,DMSO-D6)δ156.12,146.30,142.26,138.19,129.36,128.24,123.06,122.22,108.74.

[0045] Step 2: Synthesis of intermediate b

[0046]

[0047] The intermediate a (3.90 g, 21.78 mmol, 1 eq) was placed in a 50 mL single-mouth bottle, dissolved with 25 mL THF, and vinyl ethyl ether (8.34 mL, 87.10 mmol, 4 eq) and 4-methylbenzenesulfonic acid pyridine (1.09 g, 4.36 mmol, 0.2 eq) were added in an ice bath, and the reaction was transferred to room temperature. After 4 hours, TLC detected that the reaction was complete, 100 mL of water was added to quench, ethyl acetate (50 mL × 3) was extracted, the organic phases were combined and washed with 150 mL of saturated brine, anhydrous sodium sulfate was added to the organic phase for drying, filtration, concentration, sand making, and column chromatography to obtain 5.04 g of yellow oily liquid b, with a yield of 90.10%.

[0048] Step 3: Synthesis of intermediate cd

[0049]

[0050] The intermediate b (2.50 g, 9.96 mmol, 1 eq) was placed in a 50 mL pressure tube, and the raw materials 4-(N, N-dimethylamino)phenylboronic acid pinacol ester (2.71 g, 10.96 mmol, 1.1 eq), K2CO3 (4.13 g, 29.88 mmol, 3 eq) and Pd(pph3)4 (1.16 g, 1 mmol, 0.1 eq) were added respectively, and dissolved with 25 mL of solvent (ethylene glycol dimethyl ether: water = 4:1), N2 was introduced to remove O2, and the mixture was placed in an 80 ° C oil bath for 9 hours. TLC detected that the reaction was complete, 100 mL of water was added to quench, ethyl acetate (50 mL × 3) was extracted, the organic phases were combined and washed with 150 mL of saturated brine, anhydrous sodium sulfate was added to the organic phase for drying, filtration, concentration, sand making, and column chromatography to obtain 3.08 g of light yellow solid c, with a yield of 92%.

[0051] The operation was the same as above, except that 4-(N,N-dimethylamino)phenylboronic acid pinacol ester was replaced with N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine to obtain 2.84 g of yellow solid d with a yield of 85%. 1 H NMR (400MHz, CHLOROFORM-D) δ8.87(d,J=2.5Hz,1H),8.33(dd,J=9.0,2.5Hz,1H),8.09–7.91(m,2H),7.72(d,J=8.8Hz,1H),7.39(dd,J=9.1,2.7Hz,1H ),6.62(d,J=9.0Hz,1H),5.52(d,J=5.3Hz,1H),3.82(dd,J=9.4,7.1Hz,1H) ,3.65–3.49(m,1H),3.14(s,6H),1.56(d,J=5.4Hz,3H),1.25–1.16(m,5H). 13 C NMR(101MHz,CHLOROFORM-D)δ159.47,154.48,153.85,147.13,144.75,136.21,135.76,13 0.77,127.62,123.35,118.14,110.50,105.87,99.69,61.41,38.28,24.92,20.24,15.32.

[0052] Step 4: Synthesis of intermediate ef

[0053]

[0054] The intermediate c (3.00 g, 9.98 mmol, 1 eq) was placed in a 50 mL single-mouth bottle, dissolved with 20 mL CHCl3, trifluoroacetic acid (3.71 mL, 49.9 mmol, 5 eq) was added under an ice bath, and reacted at 45°C for 2 hours. After 2 hours, the reaction was complete by TLC detection, and the pH was adjusted to about 8 with a saturated aqueous Na2CO3 solution, and 100 mL of water and dichloromethane (50 mL×3) were added for extraction, and the organic phases were combined and washed with 150 mL of saturated brine, and anhydrous sodium sulfate was added to the organic phase for drying, filtration, concentration, sand making, and column chromatography to obtain 1.66 g of red solid powder e, with a yield of 63%. 1 H NMR (400MHz, DMSO) δ10.20(s,1H),8.25(d,J=8.8Hz,1H),8.09(d,J=9.0Hz,2H),7.97(dd,J=8.9,5 .2Hz,2H),7.35(dd,J=9.1,2.7Hz,1H),7.19(d,J=2.6Hz,1H),6.83(d,J=9.1Hz,2H),3.00(s,6H). 13 C NMR (101MHz, DMSO) δ155.85,153.06,151.82,128.73,128.14,123.17,118.92,112.45,109.13.

[0055] The same operation was performed on intermediate d to obtain 1.77 g of yellow solid f with a yield of 67%. 1 H NMR (400MHz, DMSO) δ9.95(s,1H),8.93(d,J=2.4Hz,1H),8.34(dd,J=9.0,2.5Hz,1H),8.14(d,J=8.7Hz,1H),7.93(d,J=8 .7Hz,1H),7.84(d,J=9.0Hz,1H),7.28(dd,J=9.0,2.7Hz,1H),7.12(d,J=2.7Hz,1H),6.75(d,J=9.0Hz,1H),3.10(s,6H). 13 C NMR (101MHz, DMSO) δ159.48,155.44,152.11,147.03,143.23,135.89,135.53,130.64,128.25,122.80,122.46,118.15,108.84,106.01,38.14.

[0056] Step 5: Synthesis of intermediate gi

[0057]

[0058] The intermediate e (1.60 g, 6.06 mmol, 1 eq) and 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl 4-methylbenzenesulfonate (4.07 g, 9.09 mmol, 1.5 eq) were placed in a 50 mL single-mouth bottle, K2CO3 (1.67 g, 12.12 mmol, 2 eq) was added, dissolved with 20 mL DMF, and reacted at 60 ° C overnight. TLC detected that the reaction was complete, 100 mL of water was added to quench, ethyl acetate (50 mL × 3) was extracted, the organic phases were combined and washed with saturated brine (150 mL × 3), anhydrous sodium sulfate was added to the organic phase for drying, filtration, concentration, sand making, and column chromatography to obtain a light yellow solid g 1.93 g, with a yield of 59%.

[0059] The intermediate f was used to replace e and reacted with 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl 4-methylbenzenesulfonate. The post-treatment operation was the same to obtain 0.87 g of yellow solid h with a yield of 50%.

[0060] The intermediate f was used to replace e and reacted with 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridec-13-yl 4-methylbenzenesulfonate. The post-treatment operation was the same to obtain 0.97 g of a yellow solid i with a yield of 61%.

[0061] Step 6: Synthesis of intermediate jl

[0062]

[0063] The intermediate g (1.90 g, 3.53 mmol, 1 eq) was dissolved in an appropriate amount of DCM, 2N HCl-EA2.5 mL was gradually added dropwise thereto, the temperature was transferred to 50°C, and the reaction was completed after 2 hours of reaction by TLC. The base was adjusted to about pH=8 with saturated Na2CO3 aqueous solution, 100 mL of water and dichloromethane (50 mL×3) were added for extraction, the combined organic phases were washed with 150 mL of saturated brine, anhydrous sodium sulfate was added to the organic phase, dried, filtered, concentrated, sanded, and column chromatography was performed to obtain 1.08 g of yellow solid j with a yield of 70%. 1H NMR (400MHz, CHLOROFORM-D) δ8.13–7.89(m,4H),7.74(dd,J=8.5,3.8Hz,1H),7.34(dd,J=9.2,2.8Hz,1H),7.06(d,J=2.7H z,1H),6.81(d,J=9.0Hz,2H),4.23(t,J=4.7Hz,2H),3.71–3.51(m,10H),3.02(d,J=1.5Hz,6H),2.96(q,J=6.0,5.5Hz,2H). 13 C NMR(101MHz,CHLOROFORM-D)δ156.15,155.43,151.20,144.49,135.40,130.79,128.93,128.22,127.58,127. 52,125.99,122.12,118.71,112.39,106.41,77.35,70.78,70.55,70.17,69.73,67.78,40.86,40.49,29.79.

[0064] The same operation was performed on intermediate h to obtain 0.41 g of yellow solid k with a yield of 62%. 1 H NMR(400MHz,CHLOROFORM-D)δ8.85(d,J=2.5Hz,1H),8.31(dd,J=9.0,2.4Hz,1H),7.9 7(dd,J=16.5,8.9Hz,2H),7.71(dd,J=13.7,8.4Hz,2H),7.33(dd,J=9.4,2.7Hz,1H),7 .11(d,J=8.5Hz,1H),7.06(d,J=2.6Hz,1H),6.62(d,J=9.0Hz,1H),3.96–3.80(m,2H) ,3.74–3.68(m,3H),3.68–3.54(m,11H),3.15(d,J=1.5Hz,6H),3.01(t,J=5.0Hz,2H). 13 C NMR(101MHz,CHLOROFORM-D)δ159.46,156.31,153.50,147.09,144.49,142.21,140.20,136.13,135.66,130.74,128. 93,127.68,125.98,123.30,122.34,118.13,106.41,105.85,77.35,70.71,70.48,70.09,69.68,67.79,40.64,38.29.

[0065] The same operation was performed on intermediate i to obtain 0.43 g of yellow solid l with a yield of 59%. 1 H NMR (400MHz, DMSO-D6) δ8.92(d,J=2.5Hz,1H),8.32(dd,J=9.0,2.5Hz,1H),8. 19(d,J=8.7Hz,1H),7.97(d,J=8.7Hz,1H),7.86(d,J=9.0Hz,1H),7.32(t,J=3 .5Hz,2H),6.73(d,J=9.0Hz,1H),4.19(t,J=4.6Hz,4H),3.79(t,J=4.6Hz,2H) ,3.67–3.33(m,6H),3.07(s,6H),2.75(t,J=5.6Hz,2H),0.89(t,J=7.1Hz,1H). 13 C NMR(101MHz,DMSO-D6)δ159.13,155.90,152.52,146.76,135.51,130.19,127.48,1 25.53,122.16,117.89,106.53,105.56,70.32,69.90,69.67,68.89,67.49,37.67.

[0066] Example 2: Synthesis of Final Product II-1

[0067]

[0068] 1-adamantaneacetic acid (72.85 mg, 0.38 mmol, 1.5 eq) was placed in a single-mouth bottle, dissolved with an appropriate amount of DMF, HATU (142.59 mg, 0.38 mmol, 1.5 eq) and DIPEA (64.63 mg, 0.50 mmol, 2 eq) were added in sequence, stirred for 30 minutes, and intermediate j (100 mg, 0.25 mmol, 1 eq) was added, and the mixture was reacted at room temperature for 12 hours. TLC detected that the reaction was complete, 50 mL of water and ethyl acetate (30 mL × 3) were added for extraction, the organic phases were combined and washed with saturated brine (100 mL × 3), anhydrous sodium sulfate was added to the organic phase for drying, filtration, concentration, sand making, and column chromatography to obtain 78.41 mg of yellow oil II-1, with a yield of 51%. 1H NMR(400MHz,CHLOROFORM-D)δ8.10–7.97(m,4H),7.77(d,J=8.6Hz,1H),7.36(dd,J=9.2,2.8 Hz,1H),7.05(d,J=2.7Hz,1H),6.82(d,J=9.1Hz,2H),4.27–4.22(m,2H),3.96–3.89(m,2H), 3.80–3.73(m,2H),3.69(dd,J=6.2,3.4Hz,2H),3.67–3.63(m,2H),3.62–3.58(m,2H),3.53( t,J=5.2Hz,2H),3.46–3.39(m,2H),3.03(s,6H),1.92(d,J=13.8Hz,5H),1.72–1.54(m,12H). 13 C NMR(101MHz,CHLOROFORM-D)δ171.12,156.23,155.30,151.23,144.25,135.44,130.63,128.25,127.45,127.25,122.20,118.69,112.34,106.27,7 7.36,70.95,70.65,70.62,70.25,70.12,69.78,67.74,51.72,42.63,40. 44,39.07,36.83,32.75,31.99,31.57,30.19,29.76,29.42,28.69,22.76.

[0069] Example 3: Synthesis of Final Product II-2

[0070]

[0071] The same operation was performed on intermediate 1 to obtain 55.77 mg of yellow oil II-2 with a yield of 39%. 1H NMR (400MHz, CHLOROFORM-D) δ8.89 (s, 1H), 8.36 (d, J = 9.0Hz, 1H), 8.01 (t, J = 10.1Hz, 2H), 7. 75(d,J=8.6Hz,1H),7.37(d,J=9.3Hz,1H),7.06(s,1H),6.64(d,J=12.1Hz,1H),5.93(s,1H) ,4.25(s,2H),3.92(s,2H),3.74(d,J=2.9Hz,2H),3.67(d,J=2.9Hz,2H),3.57(t,J=5.1Hz,2 H),3.46(t,J=4.5Hz,2H),3.17(d,J=1.8Hz,6H),1.91(d,J=15.0Hz,5H),1.69–1.54(m,12H). 13 C NMR(101MHz,CHLOROFORM-D)δ171.11,159.37,156.40,153.47,146.95,144. 51,136.22,135.54,130.83,127.65,123.26,122.37,118.15,106.22,105.9 2,77.38,70.87,70.34,70.18,69.75,67.77,51.79,42.64,39.10,38.31,36.82,32.75,32.01,31.59,31.52,30.21,29.78,29.45,28.70,22.78,14.23.

[0072] Example 4: Synthesis of Final Product II-3

[0073]

[0074] The same operation was performed on intermediate k to obtain 67.76 mg of yellow oil II-3 with a yield of 44%. 1H NMR(400MHz,CHLOROFORM-D)δ8.87(d,J=1.6Hz,1H),8.34(dd,J=8.9,2.5Hz,1H),7.99(dd,J=13.0,9.4Hz,2H), 7.73(d,J=8.7Hz,1H),7.35(dd,J=9.1,2.7Hz,1H),7.05(d,J=2.7Hz,1H),6.63(d,J=9.0Hz,1H),4.27–4.20(m, 2H),3.95–3.88(m,2H),3.75(dd,J=5.8,2.9Hz,2H),3.68(dd,J=5.8,3.0Hz,2H),3.66–3.62(m,2H),3.62–3.57 (m,2H),3.53(t,J=5.2Hz,2H),3.42(q,J=5.2Hz,2H),3.15(s,6H),1.91(d,J=13.5Hz,5H),1.70–1.52(m,12H). 13 C NMR(101MHz,CHLOROFORM-D)δ171.10,159.38,156.42,153.46,146.96,144.51,136.21,135.55,130.80,127.65,123.28,12 2.40,118.12,106.23,105.90,70.99,70.66,70.28,70.14,69.80,67.77,51.77,42.66,39.08,38.31,36.85,32.77,28.71.

[0075] Example 5: Western Blot detection of protein degradation effect

[0076] For the new structure of Tau-HyT small molecules, CHO-K1 (P301L) Tau protein overexpression cell line was selected, the concentration was set to 5μM, the administration time was 24h, and Western Blot was used to evaluate the targeted protein degradation ability of these three types of compounds.

[0077] Degradation effect see attached Figure 1 .

[0078] It can be seen that the degradation efficiency of compound II-1 reached 50%, with no concentration dependence, and the degradation effect was verified in time-effect and dose-effect experiments; compound II-2 had an obvious degradation effect at 1 μM, and compound II-3 had no obvious degradation effect.

[0079] Example 6: Immunofluorescence detection of the clearing effect of II-1 on total Tau and p-Tau

[0080] After CHO-K1 cells were incubated with different concentrations of Ⅱ-1 for 24 hours, immunofluorescence analysis revealed that the levels of total Tau (Tau-5) and p-Tau decreased in a concentration-dependent manner (results shown in Figure 2 This indicates that II-1 can effectively reduce the levels of Tau-5 and p-Tau.

[0081] Example 7: HyT molecule II-1 improves short-term working memory in 3×Tg-AD mice

[0082] The Y-maze test mainly includes the spontaneous alternation test and the spatial recognition test, which can be used to evaluate the short-term cognitive function of mice.

[44] The Y-maze spontaneous alternation test can be used to detect short-term working memory in mice.

[45] In this study, the Y-maze spontaneous alternation test was used to test whether HyT molecule II-1 could improve the short-term working memory of 3×Tg-AD mice. The mice were injected into the tail vein for 7 consecutive days at a dose of 50 mg / kg, followed by behavioral tests.

[0083] Figure 3 Figure 3 is a schematic diagram of the Y-maze experiment. The spontaneous alternation accuracy rates of the four groups of mice, namely, WT, Model 1, Model 2, and drug administration (Ⅱ-1) group, were calculated to be 64.72±14.31%, 26.93±9.18%, 25.94±6.96%, and 53.19±10.67%, respectively. The data showed that the spontaneous alternation accuracy rates of mice in the Model 1 and Model 2 groups were significantly lower than those in the WT group, which was consistent with the actual situation of 3×Tg-AD mice and wild-type C57 mice, while the spontaneous alternation accuracy rate of mice in the drug administration (Ⅱ-1) group was significantly higher than that in the Model group, and was close to that in the WT group.

[0084] (Note: WT group is wild-type C57 mouse group, Model 1, Model 2, and Ⅱ-1 groups are all 3×Tg-AD mouse groups, M1 group is solvent-treated group, M2 group is oral borneol group, and Ⅱ-1 group is oral borneol and drug group. Borneol is used to enhance the ability of drugs to penetrate the blood-brain barrier. Compared with the WT group, ★P<0.05, ★★P<0.01, ★★★P<0.001; compared with the Model group, ▲▲P<0.01, n=5)

[0085] Example 8: HyT molecule II-1 clears pathogenic Tau protein in the brain of 3×Tg-AD mice

[0086] In order to explore whether HyT molecule II-1 can promote the clearance of Tau protein and p-Tau in vivo, Western Blot was used to detect the changes in the content of Tau protein and p-Tau in brain tissue.

[0087] The results are as follows Figure 4As shown, the content of Tau protein and p-Tau in brain tissue of the drug-treated group (Ⅱ-1) was significantly lower than that of the Model group. (Note: Compared with the WT group, ★P<0.05, ★★P<0.01; compared with the Model group, ▲P<0.05, n=3)

[0088] Example 9: HyT molecule II-1 alleviates brain neuroinflammation in 3×Tg-AD mice

[0089] ELISA (enzyme-linked immunosorbent assay) is an immunoassay method that is usually used to quantify the level of a specific target molecule in a sample. There is evidence that cytokines such as interleukins (ILs) and TNF-α are involved in the pathogenesis of AD and may become diagnostic or therapeutic targets for AD neurodegenerative lesions.

[46] Here, we used ELISA to detect the changes in the levels of ILs and TNF-α to reflect the effect of HyT molecule Ⅱ-1 in alleviating brain neuroinflammation in 3×Tg-AD mice.

[0090] like Figure 5 As shown, the content of neuroinflammatory factors in the Model group was significantly higher than that in the WT group, and the content of neuroinflammatory factors in the drug-treated (Ⅱ-1) group was significantly lower than that in the Model group. (Note: Compared with the WT group, ★★★P<0.001; compared with the Model group, ▲▲P<0.01, ▲▲▲P<0.001, n=3)

[0091] As can be seen from the above examples, on the basis of obtaining the target product, the target product was further studied for in vivo pharmacodynamics. Compound II-1 was administered by tail vein injection for 7 consecutive days at a dose of 50 mg / kg. The Y-maze spontaneous alternation experiment was used to detect whether II-1 could improve the short-term working memory ability of 3×Tg-AD mice. After the behavioral experiment, the mice were anesthetized, and the cerebral cortex and hippocampus of the mice were taken after intracardiac perfusion for subsequent HE staining, Thioflavin T staining, ELISA and tissue Western blot experiments to further investigate the ability of HyT molecule II-1 to clear Tau protein and alleviate p-Tau aggregation in vivo.

[0092] Behavioral experiment - Y-maze spontaneous alternation experiment statistical results show that the spontaneous alternation accuracy of mice in the drug administration (Ⅱ-1) group is significantly higher than that in the Model group. The spontaneous alternation accuracy of mice in the drug administration (Ⅱ-1) group is 105.05% higher than that in the Model group, which is almost the same as that in the WT group, indicating that Ⅱ-1 can significantly improve the short-term working memory ability of 3×Tg-AD mice; in HE staining, compared with the WT group and the Model group, the cell morphology of the Ⅱ-1 (drug administration) group has been significantly improved, and the cell outline and nucleolus have become clear; ELISA determined that there were significant differences in the content of neuroinflammatory factors ILs and TNF-α in 3×Tg-AD mice before and after administration; from the results of Thioflavin T staining, it can be seen that Ⅱ-1 can alleviate the aggregation of p-Tau protein in the brain of 3×Tg-AD mice; at the same time, Ⅱ-1 can promote the clearance of Tau protein in vivo. The above experimental results show the great application potential of the HyT technology of the present invention in the targeted degradation of Tau protein.

Claims

1. A HyT compound, characterized in that Has the following structure: in, L is a connecting chain, and its structure is shown below: Wherein, m and n are independently selected from integers of 0-10; X is C, N, O or S; R is the following structure:

2. The HyT compound according to claim 1, characterized in that L is selected from the following structures:

3. The HyT compound according to claim 1, characterized in that Select from the following structures: in, X is a C or N atom; n is independently selected from integers of 0-10.

4. The HyT compound according to claim 3, characterized in that For the following compounds: Compound II-1: n=4, X=C; Compound II-2: n=3, X=N; Compound II-3: n=4, X=N.

5. The method for synthesizing the HyT compound according to claim 1, characterized in that: The following steps are involved: The HyT compound is obtained by sequentially undergoing reactions v, vi, and vii. The reaction formula is as follows: Wherein, X and n in the reaction formula have the same meanings as above.

6. Use of the HyT compound according to any one of claims 1 to 4 as a degradation agent targeting TAU protein in the preparation of a pharmaceutical preparation for preventing or treating diseases mediated by TAU protein.

7. The use according to claim 6, characterized in that: The disease mediated by TAU protein is Alzheimer's disease.

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