A protein degradation agent with borneol as a hydrophobic group, a preparation method, a pharmaceutical composition and an application thereof

Abstract

The application belongs to the field of chemical drugs, and discloses a protein degrading agent with borneolamine as a hydrophobic group, a preparation method, a pharmaceutical composition and application thereof. The application provides a degradable AR protein degrading agent based on a hydrophobic label function of noradamantane as a hydrophobic group and an enzalutamide parent nucleus as a target protein ligand. The protein degrading agent prepared by the application can effectively induce degradation of AR and AR-V7 in a cancer cell line. Compared with a previously reported AR protein degrading agent based on adamantane as a hydrophobic label, the protein degrading agent has considerable improvement in plasma metabolic stability, can exhibit excellent in-vivo anti-prostate cancer effect in low-frequency drug administration, can be applied to preparation of an AR degrading agent, and can form a pharmaceutical composition, and is suitable for development of a cancer drug such as a prostate cancer drug.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceuticals, specifically relating to a protein degrading agent with norborneol as a hydrophobic group, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Hydrophobic tag (Hyt) bifunctional molecules consist of a target protein ligand, a linker, and a hydrophobic group. By attaching a large hydrophobic group to a small molecule that can bind to the target, this biheaded molecule, upon binding to the target, is mistakenly identified by intracellular protein repair mechanisms as a misfolded portion of the target protein. It is then folded by chaperone proteins and subsequently degraded by the proteasome. The hydrophobic group in Hyt molecules often has a small molecular weight, thus potentially exhibiting higher solubility and drug-like properties. Currently, the development of hydrophobic tag-based degraders is still in the exploratory stage. On the one hand, there are relatively few reported hydrophobic tag fragments, leaving considerable room for optimization in terms of degradation activity and physicochemical properties. On the other hand, the exact degradation mechanism remains unclear. Therefore, exploring more hydrophobic fragments with high activity and excellent physicochemical properties, and clarifying their corresponding degradation mechanisms, is crucial for the clinical application of hydrophobic tag-based fragments.

[0003] Androgen receptor (AR) is a clinically validated target for treating human prostate cancer. Androgen receptor antagonists are effective in treating metastatic castration-resistant prostate cancer (mCRPC), significantly improving patient survival and demonstrating good tolerability. However, current clinical androgen receptor antagonists (such as enzalutamide) often induce resistance within 18 months of treatment. In most patients who develop resistance to enzalutamide, the androgen receptor signaling pathway is still functioning; therefore, targeting androgen receptor protein degradation may be a very promising therapeutic strategy, potentially more effective than androgen receptor antagonists. In existing technologies, a research team has developed protein degraders SARD279 and SARD033 based on hydrophobic tagging technology, which can degrade AR. Figure 1 As shown, these two degrading agents are obtained by linking AR ligand RU59063 with polyethylene glycol of different lengths using adamantane as a hydrophobic tag. SARD279 was able to degrade 50% of AR protein (DC50) in LNCaP cells at a concentration of 1 μM. However, neither conventional PROTAC degrading agents nor these hydrophobic tag degrading agents had a significant effect on AR-V7 protein levels. Furthermore, conventional AR degrading agents have relatively low degradation efficiency, poor metabolic stability in liver microsomes and plasma, and poor solubility, making them difficult to administer orally. This hinders their use in further clinical treatment and development, necessitating the development of novel AR protein degrading agents with better metabolic stability to meet clinical needs. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a protein degrading agent with high plasma metabolic stability using norborneol as a hydrophobic group, its preparation method, pharmaceutical composition, and application. Specifically, the following technical solution is adopted:

[0005] According to a first aspect of the present invention, a protein degrading agent with norbornene as a hydrophobic group is provided, the structural formula of which is shown in formula (I):

[0006]

[0007] Linker can be any chemically feasible linker structure.

[0008] This invention provides a protein degrader with norborneol as a hydrophobic group. In the bifunctional molecule, norborneol is the hydrophobic group and enzalutamine is the target protein ligand. This invention screens protein degraders that can effectively degrade AR by replacing different linkers.

[0009] The protein degrader of AR prepared in this invention can effectively induce the degradation of AR and AR-V7 in human prostate cancer cell line (22Rv1 cell line) in a dose-dependent manner, has considerable plasma metabolic stability, and exhibits excellent in vivo anti-prostate cancer effect.

[0010] Preferably, the linker is a saturated fatty acid chain or an unsaturated fatty acid chain. By selecting ideal linkers of different types and lengths, the goal is to maintain the binding of the two proteins spatially without affecting their binding.

[0011] Preferably, the molecular structure of the AR protein degrader is any one of formula (II), formula (III), or formula (IV):

[0012]

[0013] In equation (II), m is any positive integer from 0 to 10; in equation (III), n is any positive integer from 0 to 10.

[0014] More preferably, the molecular structure of the protein degrading agent with norborneol as the hydrophobic group is shown in formula (IV):

[0015]

[0016] The compound shown in formula (IV) can effectively induce the degradation of AR and AR-V7 in human prostate cancer cell line 22Rv1 in a dose-dependent manner, exhibits considerable plasma metabolic stability, and demonstrates the best in vivo anti-prostate cancer effect with a tumor inhibition rate of 82.5%.

[0017] According to a second aspect of the present invention, a method for preparing the above-mentioned protein degrading agent with norbornene as a hydrophobic group is also provided. The method is simple, uses inexpensive and readily available raw materials, and has a high overall reaction yield. The preparation route is either Route 1 or Route 2.

[0018] Route 1: Norbornene A is reacted with dibromoalkane of different lengths (B1-B6) under TEA to obtain intermediates (C1-C6). Enzalutamine core D and compound E are substituted under K2CO3 and DMF. Subsequently, Boc is removed under TFA to obtain intermediate F. F reacts with compounds C1-C6 under Cs2CO3 and KI to obtain the final products E1-E6.

[0019]

[0020] Route 2: Norbornene (D) is amide condensed with alkynyl acids of different lengths (H1-H6) under the action of HATU to obtain intermediates I1-I6. Subsequently, the enzalutamine core D is substituted with 2-azidoethyl-4-methylbenzenesulfonate (compound J) under the action of K2CO3 and DMF to obtain intermediate K, which then undergoes a Click reaction with I1-I6 to obtain the final products L1-L6.

[0021]

[0022] According to a third aspect of the invention, the use of the above-described protein degrading agent with norbornene as a hydrophobic group or a pharmaceutically acceptable salt thereof in the preparation of AR degrading agents is also provided.

[0023] Preferably, the AR degrading agent can be used to prepare drugs for treating cancers associated with AR abnormalities. Associated cancers include: prostate cancer, breast cancer, ovarian cancer, and endometrial cancer. More preferably, the associated cancer is prostate cancer.

[0024] According to a fourth aspect of the present invention, a pharmaceutical composition is also provided, wherein the above-mentioned protein degrading agent with norborneol as a hydrophobic group or a pharmaceutically acceptable salt thereof is the main active ingredient.

[0025] Preferably, the pharmaceutical composition comprises an excipient, a solvent, and a pharmaceutical carrier. The excipient includes at least one selected from gum arabic, syrup, lanolin, and starch. This excipient is stable, has no incompatibilities with the active pharmaceutical ingredient, does not produce side effects, does not affect efficacy, is not easily deformed, cracked, moldy, or infested by insects at room temperature, is harmless to the human body, has no physiological effects, does not react chemically or physically with the active pharmaceutical ingredient, and does not affect the determination of the active pharmaceutical ingredient's content. The solvent includes water, glycerol, or ethanol.

[0026] The beneficial effects of this invention are as follows: This invention provides a protein degrading agent with norborneol as a hydrophobic group. The preparation process is simple and easy, and various protein degrading agents can be obtained through different pathways. These agents can be applied to the preparation of AR degrading agents. Compared with the positive control SARD279, the obtained protein degrading agents have higher plasma metabolic stability and lower metabolic risk. In in vivo experiments, the frequency of administration can be reduced, effectively improving drug efficacy. Furthermore, these agents can be formulated into pharmaceutical compositions, exhibiting certain inhibitory effects on the proliferation of various tumor cells, making them suitable for the development of drugs for treating prostate cancer and other cancers.

[0027] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments of the invention. Attached Figure Description

[0028] Figure 1 Structural diagrams of SARD279 and SARD033, protein degraders for degrading AR, developed based on hydrophobic tagging technology;

[0029] Figure 2 This is a screening diagram showing the concentration-dependent degradation of AR and AR-V7 proteins by compounds L1, L3, and L5 in the 22Rv1 cell line.

[0030] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0031] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] Example 1

[0034] The structures of a protein degrading agent with norborneol as a hydrophobic group (denoted as compound G1), compound A, compound B1, compound C1, compound D, compound E, and compound F are shown below:

[0035] Compound G1, Compound A, Compound B1, Compound C1, Compound D, Compound E, Compound F.

[0036] The specific preparation method is as follows:

[0037] 1) Preparation of compound C1: Compound A (307 mg, 2.00 mmol) and compound B1 (1,2-dibromoethane, 376 mg, 2.00 mmol) were dissolved in tetrahydrofuran (8 mL), and triethylamine (417 μL, 3.00 mmol) was added. The mixture was reacted at room temperature for 3 h. After the reaction was completed, the tetrahydrofuran was evaporated to dryness, diluted with water (10 mL), and extracted with ethyl acetate (3 × 10 mL). The organic phases were then combined and dried over anhydrous sodium sulfate. The mixture was concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain compound C1.

[0038] The detection results for compound C1 are as follows: HRMS (ESI) calculated for C 12 H 23 BrN + [M+H] + :260.1008,found.260.1010.

[0039] 2) Preparation of compound F: Compound D (405 mg, 1.00 mmol) and compound E (4-(2-bromoacetyl)piperazine-1-carboxylic acid tert-butyl ester, 307 mg, 1.00 mmol) were dissolved in N,N-dimethylformamide (3 mL), and potassium carbonate (277 mg, 2.00 mmol) was added. The mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was diluted with water (12 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phases were then combined and washed with saturated NaCl solution (12 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain compound C1.S. Subsequently, the compound was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at room temperature for 0.5 hours, and then the solvent was evaporated to obtain crude compound F.

[0040] The detection results for compound F are as follows: HRMS (ESI) calculated for C 25 H 25 F3N5O3S + [M+H] + :532.1625,found.532.1628.

[0041] 3) Preparation of compound G1: Compound C1 (130 mg, 0.500 mmol) and compound F (266 mg, 0.500 mmol) were dissolved in dioxane (7 mL), cesium carbonate (326 mg, 1.00 mmol) and potassium iodide (83.0 mg, 0.500 mmol) were added, and the mixture was refluxed overnight. After the reaction was completed, the dioxane was evaporated to dryness, diluted with water (8 mL), and extracted with ethyl acetate (3 × 8 mL). The organic phases were then combined and dried over anhydrous sodium sulfate. The mixture was concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 16:1) to obtain compound G1.

[0042] The results of the detection of compound G1 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.4Hz,1H),7.69(dd,J=7.5,2.1H z,1H),7.21–7.14(m,2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.77(s,1H), 2.71(d,J=12.4Hz,1H),2.64–2.51(m,3H),2.50(d,J=5.6Hz,5H),2.37(s,1H),1.79(d,J=13.0Hz, 1H),1.64–1.47(m,9H),1.32(d,J=13.0Hz,1H),1.25(d,J=13.0Hz,1H),0.91(s,3H),0.86(s,6H). 13 CNMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.32,136.87,135.57,133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70 ,106.85,67.50,66.95,61.42,58.89,53.54,50.91,49.37,48.13,45.99,44.73,34.91,34.36,27.15,23.78,20.91,16.32.HRMS(ESI)calculated for C 37 H 46 F3N6O3S + [M+H] + :711.3299,found.711.3303.

[0043] Example 2

[0044] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound G2) has the following structure:

[0045] The structure of compound G2 is as follows:

[0046]

[0047] The specific preparation method is as follows: In this embodiment, compound G2 is obtained by basically following the synthesis steps of compound G1 described in Example 1. The difference from Example 1 is that only 1,2-dibromoethane is replaced with 1,3-dibromopropane.

[0048] The results of the detection of compound G2 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.4Hz,1H),7.69(dd,J=7.5,2.1Hz,1H),7.21–7.14(m, 2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.87(s,1H),2.85–2.78(m,1H),2.64(d,J=12.4Hz,1H), 2.51–2.42(m,5H),2.37(d,J=12.5Hz,1H),2.16(d,J=0.7Hz,1H),1.79(d,J=12.9Hz,1H),1.74–1.66(m,4H),1.64–1.54 (m,3H),1.56(s,4H),1.51(d,J=13.0Hz,1H),1.32(d,J=13.0Hz,1H),1.25(d,J=13.0Hz,1H),0.91(s,3H),0.86(s,6H). 13 C NMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.32,136.87,135.57,133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70 ,106.85,67.50,66.95,63.17,55.28,53.72,50.91,49.37,48.16,47.34 ,44.73,34.91,34.36,27.85,27.15,23.78,20.91,16.32.HRMS(ESI):m / z calcd for C 38 H48 F3N6O3S + [M+H] + :725.3455; found725.3456.

[0049] Example 3

[0050] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound G3) has the following structure:

[0051] The structure of compound G3 is as follows:

[0052]

[0053] The specific preparation method is as follows: In this embodiment, compound G3 was obtained by basically following the synthesis steps of compound G1 described in Example 1. The difference from Example 1 is that only 1,2-dibromoethane was replaced with 1,4-dibromobutane.

[0054] The results of the detection of compound G3 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.5Hz,1H),7.69(dd,J=7.5,2.1Hz,1H), 7.21–7.14(m,2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.87(s,1H),2.77–2.69(m, 1H),2.63–2.55(m,1H),2.52–2.39(m,8H),1.79(d,J=13.0Hz,1H),1.68(s,1H),1.64–1.50(m,8H),1.51 (s,3H),1.49(d,J=2.8Hz,1H),1.32(d,J=13.0Hz,1H),1.25(d,J=13.0Hz,1H),0.91(s,3H),0.86(s,6H). 13C NMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.32,136.87,135.57,133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70 ,106.85,67.50,66.95,63.17,57.13,53.72,50.91,49.37,48.50,48.16 ,44.73,34.91,34.36,29.00,27.15,23.78,20.91,16.32.HRMS(ESI):m / z calcd for C 39 H 50 F3N6O3S + [M+H] + :739.3612; found 739.3611.

[0055] Example 4

[0056] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound G4) has the following structure:

[0057] The structure of compound G4 is as follows:

[0058]

[0059] The specific preparation method is as follows: In this embodiment, compound G4 was obtained by basically following the synthesis steps of compound G1 described in Example 1. The difference from Example 1 is that only 1,2-dibromoethane was replaced with 1,5-dibromopentane.

[0060] The detection results for compound G4 are as follows: 1H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.4Hz,1H),7.69(dd,J=7.5,2.1Hz,1H),7.21–7.1 4(m,2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.89(d,J=0.6Hz,1H),2.79(dd,J=12.5,0.6Hz ,1H),2.68–2.60(m,1H),2.49(d,J=4.0Hz,4H),2.44(d,J=1.2Hz,2H),2.40(s,1H),1.79(d,J=13.0Hz,1H),1.68( s,1H),1.64–1.41(m,13H),1.35(s,2H),1.32(d,J=13.1Hz,1H),1.25(d,J=13.0Hz,1H),0.91(s,3H),0.86(s,6H). 13 C NMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.32,136.87,135.57,133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70,10 6.85,67.50,66.95,63.17,57.29,53.72,50.91,49.37,48.68,48.16,44.7 3,34.91,34.36,29.66,27.79,27.15,23.78,20.91,16.32.HRMS(ESI):m / z calcd for C 40 H 52 F3N6O3S + [M+H] + :753.3768; found 753.3766.

[0061] Example 5

[0062] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound G5) has the following structure:

[0063] The structure of compound G5 is as follows:

[0064]

[0065] The specific preparation method is as follows: In this embodiment, compound G5 is obtained by basically following the synthesis steps of compound G1 described in Example 1. The difference from Example 1 is that only 1,2-dibromoethane is replaced with 1,6-dibromohexane.

[0066] The results of the detection of compound G5 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.5Hz,1H),7.69(dd,J=7.5,2.1Hz,1H),7.21– 7.14(m,2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.87(s,1H),2.79(dd,J=12.5,0.6Hz,1 H),2.68–2.60(m,1H),2.49(d,J=3.9Hz,4H),2.44(d,J=0.6Hz,2H),2.40(s,1H),1.79(d,J=13.0Hz,1H),1.68 (s,1H),1.64–1.42(m,12H),1.36–1.31(m,5H),1.30(s,1H),1.25(d,J=13.0Hz,1H),0.91(s,3H),0.86(s,6H). 13 C NMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.32,136.87,135.57, 133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70,117.70,106.8 5,67.50,66.95,63.17,57.29,53.72,50.91,49.37,48.70,48.16,44.73,34.91 ,34.36,30.38,29.36,28.54,28.21,27.15,23.78,20.91,16.32.HRMS(ESI):m / z calcd for C 41 H 54 F3N6O3S + [M+H] + :767.3925; found 767.3928.

[0067] Example 6

[0068] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound G6) has the following structure:

[0069] The structure of compound G6 is as follows:

[0070]

[0071] The specific preparation method is as follows: In this embodiment, compound G6 was obtained by basically following the synthesis steps of compound G1 described in Example 1. The difference from Example 1 is that only 1,2-dibromoethane was replaced with 1,7-dibromoheptane.

[0072] The results of the detection of compound G6 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.13(d,J=2.0Hz,1H),8.03(d,J=7.5Hz,1H),7.69(dd,J=7.5,2.1Hz,1H) ,7.21–7.14(m,2H),6.94–6.86(m,2H),4.77(s,2H),3.58(s,2H),3.51(s,2H),2.89(d,J=0.6Hz,1H),2. 79(dd,J=12.5,0.6Hz,1H),2.68–2.60(m,1H),2.49(d,J=3.9Hz,4H),2.44(d,J=0.6Hz,2H),2.40(s,1H) ,1.79(d,J=13.0Hz,1H),1.68(s,1H),1.64–1.42(m,14H),1.35–1.21(m,8H),0.91(s,3H),0.86(s,5H). 13 C NMR(100MHz,Chloroform-d)δ172.88,171.30,170.16,158.33,158.32,136.87,13 5.57,133.83,133.64,125.66,125.33,124.79,124.52,119.43,117.70,117.70,1 06.85,67.50,66.95,63.17,57.29,53.72,50.91,49.37,48.70,48.16,44.73,34. 91,34.36,30.38,30.08,28.82,28.22,27.15,23.78,20.91,16.32.HRMS(ESI):m / z calcd for C 42 H 56 F3N6O3S + [M+H] + :781.4081; found 781.4084.

[0073] Example 7

[0074] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L1), compound A, compound H1, compound I1, compound D, compound J, and compound K have the following structures:

[0075] Compound L1, Compound A, Compound H1, Compound I1, Compound D, Compound J, Compound K.

[0076] The specific preparation method is as follows:

[0077] 1) Preparation of compound I1: Compound A (307 mg, 2.00 mmol) was dissolved in N,N-dimethylformamide (6 mL) under ice bath conditions. N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (912 mg, 2.40 mmol), diisopropylethylamine (388 mg, 3.00 mmol), and compound H1 (propynic acid, 140 mg, 2.00 mmol) were added separately. After 5 minutes, the ice bath was removed, and the mixture was stirred overnight at room temperature. The liquid in the flask was diluted with ethyl acetate (20 mL), and then washed with 1N HCl solution (20 mL), saturated sodium bicarbonate solution (20 mL), and saturated saline solution (20 mL), respectively. The combined organic phases were dried with anhydrous sodium sulfate, concentrated, and subjected to silica gel rapid column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound I1.

[0078] The detection results for compound I1 are as follows: HRMS (ESI) calculated for C 13 H 20 NO + [M+H] + :206.3085,found.206.3083.

[0079] 2) Preparation of compound K: Compound D (811 mg, 2.00 mmol) and compound J (483 mg, 2.00 mmol) were dissolved in N,N-dimethylformamide (6 mL), potassium carbonate (553 mg, 4.00 mmol) was added, and the mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 × 20 mL). The organic phases were then combined and washed with saturated NaCl solution (30 mL). The organic phases were dried with anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 25:1) to obtain compound K.

[0080] The detection results for compound K are as follows: HRMS (ESI) calculated for C 21 H 18 F3N6O2S + [M+H] + :475.1159,found.475.1161.

[0081] 3) Preparation of compound L1: K (237 mg, 0.500 mmol) and compound I1 (103 mg, 0.500 mmol) were dissolved in a t-BuOH / H2O (67 mL, 1:1) mixed solvent. CuSO4·5H2O (62.5 mg, 0.250 mmol) and sodium L-ascorbate (89.1 mg, 0.450 mmol) were added. The mixture was reacted overnight at room temperature. After the reaction was completed, CuSO4·5H2O was removed by filtration. The filtrate was concentrated under vacuum and then separated by silica gel rapid column chromatography (dichloromethane:methanol = 18:1) to obtain compound L1.

[0082] The results of the detection of compound L1 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.24(s,1H),7.99–7.94(m,2H),7.83(dd,J=8.2,2.1Hz,1H),7.23 –7.20(m,2H),7.01(d,J=8.9Hz,2H),4.84(dd,J=6.2,4.1Hz,2H),4.42(t,J=4.9Hz,3H),2.40(d dt,J=13.8,11.2,4.1Hz,1H),1.81(d,J=4.1Hz,1H),1.75–1.59(m,3H),1.56(s,6H),1.46–1.39 (m,1H),1.27(dd,J=8.3,4.7Hz,1H),0.99(s,3H),0.97–0.92(m,1H),0.90(s,3H),0.86(s,3H). 13 C NMR(100MHz,Chloroform-d)δ180.2,175.0,159.9,158.4,143.9,137.2,135.2,133.8(q,J (CF) =29.8Hz),132.2,130.9,128.6,127.2(q,J (CF) =4.4Hz), 126.3(q,J (CF)=271.7Hz),123.2,120.5,115.6,114.8,66.3,53.6,49.9,49.8,48.3,44.9,37.5,28.4,28.0,23.6,19.8,18.7,13.7. 19 F NMR(376MHz,Chloroform-d)δ-61.9.HRMS(ESI):m / z calcd forC 34 H 37 F3N7O3S + [M+H] + :680.2625; found 680.2629.

[0083] Example 8

[0084] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L2) has the following structure:

[0085] The structure of compound L2 is as follows:

[0086]

[0087] The specific preparation method is as follows: In this embodiment, compound L2 is obtained by basically following the synthesis steps of compound L1 described in Example 7. The difference from Example 7 is that propynic acid is replaced with 1-butynic acid.

[0088] The results of the detection of compound L2 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.63(d,J=1.5Hz,1H),7.99–7.93(m,1H),7.68–7.62(m,2H),7.43 (s,1H),7.25(d,J=4.4Hz,2H),7.14–7.09(m,1H),6.55(s,1H),4.43(t,J=6.2Hz,2H),4.22(t,J= 6.7Hz,2H),3.83(t,J=6.9Hz,2H),2.23(tt,J=13.6,4.1Hz,1H),1.76–1.66(m,2H),1.58(s,6H), 1.43–1.25(m,2H),1.25–1.23(m,2H),1.13–1.06(m,1H),0.99(s,3H),0.82(s,3H),0.69(s,3H). 13 C NMR(100MHz,Chloroform-d)δ180.2,175.0,171.8,158.6,147.8,137.1,135.2,133.8(q,J(CF) =36.1Hz),132.2,130.9,128.3,127.1(q,J (CF) =4.6Hz), 123.2(q,J (CF) =266.1Hz),122.4,121.3,115.6,114.8,66.7,66.3,53.6,49.5,44.8,37.6,36.6,28.2,27.6,23.7,21.5,19.8,18.6,13.5. 19 F NMR(376MHz,Chloroform-d)δ-61.9.HRMS(ESI):m / z calcd forC 35 H 39 F3N7O3S + [M+H] + :694.2782; found 694.2783.

[0089] Example 9

[0090] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L3) has the following structure:

[0091] The structure of compound L3 is as follows:

[0092]

[0093] The specific preparation method is as follows: In this embodiment, compound L3 is obtained by basically following the synthesis steps of compound L1 described in Example 7. The difference from Example 7 is that only propynic acid is replaced with 1-pentynic acid.

[0094] The results of the detection of compound L3 are as follows: 1H NMR(400MHz,Chloroform-d)δ7.99–7.94(m,2H),7.83(dd,J=8.2,2.1Hz,1H),7.56(s,1H),7.21(d,J= 8.9Hz,2H),6.99(d,J=8.9Hz,2H),5.77(d,J=9.0Hz,1H),4.74(t,J=5.0Hz,2H),4.38(t,J=5.0Hz,2H), 3.05(t,J=6.9Hz,2H),2.64(t,J=7.0Hz,2H),2.25(tt,J=13.6,4.1Hz,1H),1.79–1.66(m,2H),1.56(s ,6H),1.40–1.28(m,2H),1.28–1.23(m,2H),1.13–1.06(m,1H),0.89(s,3H),0.82(s,3H),0.69(s,3H). 13 C NMR(100MHz,Chloroform-d)δ180.2,175.0,171.9,158.6,146.8,137.1,135.2,133.8(q,J (CF) =34.5Hz),132.2,130.9,128.3,127.2(q,J (CF) =5.3Hz), 123.2(q,J (CF) =273.0Hz),122.8,120.5,115.6,114.8,66.7,66.3,53.7,49.5,49.3,44.8,37.6,36.1,28.3,27.9,23.7,21.7,19.8,18.6,13.6. 19 F NMR(376MHz,Chloroform-d)δ-61.9.HRMS(ESI):m / z calcd for C 36 H 41 F3N7O3S + [M+H] + :708.2938; found 708.2939.

[0095] Example 10

[0096] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L4) has the following structure:

[0097] The structure of compound L4 is as follows:

[0098]

[0099] The specific preparation method is as follows: In this embodiment, compound L4 is obtained by basically following the synthesis steps of compound L1 described in Example 7. The difference from Example 7 is that propynic acid is replaced with 1-hexynic acid.

[0100] The detection results for compound L4 are as follows: 1 H NMR(400MHz,Chloroform-d)δ7.99–7.93(m,2H),7.83(dd,J=8.3,2.1Hz,1H),7.54(s,1H),7.24–7.19( m,2H),7.03–6.98(m,2H),6.09(d,J=9.0Hz,1H),4.76(t,J=5.0Hz,2H),4.40(t,J=5.0Hz,2H),2.78(t, J=7.1Hz,2H),2.39–2.30(m,1H),2.27(t,J=7.2Hz,2H),2.00(t,J=7.2Hz,2H),1.79(s,2H),1.64(t,J= 4.5Hz,1H),1.56(s,6H),1.41–1.34(m,1H),1.27–1.14(m,3H),0.93(s,3H),0.86(s,3H),0.80(s,3H). 13 C NMR(100MHz,Chloroform-d)δ180.2,175.0,172.7,158.6,147.6,137.2,135.2,133.4(q,J (CF) =33.9Hz),132.2,130.9,128.4,127.2(q,J (CF) =283.9Hz), 127.1(q,J (CF) =4.5Hz),122.3,115.6,114.8,110.2,66.7,66.3,53.7,49.4,48.2,44.9,37.7,35.8,28.4,28.1,25.8,24.4,23.6,19.8,18.6,13.8. 19 F NMR(376MHz,Chloroform-d)δ-61.9.HRMS(ESI):m / z calcd for C 37 H 43 F3N7O3S + [M+H] + :722.3095; found722.3097.

[0101] Example 11

[0102] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L5) has the following structure:

[0103] The structure of compound L5 is as follows:

[0104]

[0105] The specific preparation method is as follows: In this embodiment, compound L5 is obtained by basically following the synthesis steps of compound L1 described in Example 7. The difference from Example 7 is that only propynic acid is replaced with 1-heptynic acid.

[0106] The results of the detection of compound L5 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.00–7.94(m,2H),7.83(dd,J=8.2,2.1Hz,1H),7.50(s,1H),7.21(d,J=8 .9Hz,2H),7.01(d,J=8.9Hz,2H),5.51(d,J=9.0Hz,1H),4.75(d,J=5.2Hz,2H),4.40(t,J=5.0Hz,2H),2 .79–2.71(m,2H),2.37–2.29(m,1H),2.28–2.18(m,2H),1.72(ddd,J=19.2,10.9,3.6Hz,6H),1.56(s,6 H),1.47–1.36(m,2H),1.26(d,J=7.4Hz,2H),1.19–1.13(m,1H),0.93(s,3H),0.86(s,3H),0.79(s,3H). 13 C NMR(100MHz,Chloroform-d))δ180.2,175.0,172.8,158.6,137.2,135.2,133.7(q,J (CF) =31.5Hz),,133.2,132.2,130.9,128.4(q,J (CF) =264.5Hz), 127.7, 127.1(q,J) (CF) =5.4Hz),121.9,117.9,115.7,114.8,66.8,66.3,60.6,53.6,49.5,48.2,44.9,37.7,36.6,28.7,28.4,28.0,25.2,23.6,19.8,18.6,13.7. 19 F NMR(376MHz,Chloroform-d))δ-61.9.HRMS(ESI):m / z calcd for C 38 H45 F3N7O3S + [M+H] + :736.3251; found736.3253.

[0107] Example 12

[0108] A protein degrading agent with norborneol as a hydrophobic group (denoted as compound L6) has the following structure:

[0109] The structure of compound L6 is as follows:

[0110]

[0111] The specific preparation method is as follows: In this embodiment, compound L5 is obtained by basically following the synthesis steps of compound L1 described in Example 7. The difference from Example 7 is that propynic acid is replaced with 1-octynic acid.

[0112] The results of the detection of compound L6 are as follows: 1 H NMR(400MHz,Chloroform-d)δ7.99–7.92(m,2H),7.83(dd,J=8.3,2.1Hz,1H),7.48(s,1H),7.21(d,J =8.9Hz,2H),7.01(d,J=8.9Hz,2H),5.45(d,J=9.1Hz,1H),4.75(t,J=5.0Hz,2H),4.40(t,J=5.0Hz,2 H),2.72(t,J=7.6Hz,2H),2.37–2.29(m,1H),2.18(t,J=7.5Hz,2H),1.74(d,J=7.8Hz,6H),1.56(s,6 H),1.45–1.35(m,4H),1.29–1.21(m,2H),1.17–1.11(m,1H),0.93(s,3H),0.86(s,3H),0.78(s,3H). 13 C NMR(100MHz,Chloroform-d)δ180.2,175.0,172.9,158.6,148.4,137.2,135.2,133.7(q,J (CF) =31.7Hz),132.2,130.9,128.3(q,J (CF) =265.6Hz), 127.2(q,J (CF)=4.5Hz),123.2,121.9,115.7,114.8,110.2,66.8,66.3,53.5,49.4,48.1,4 4.9,37.7,36.9,29.1,28.7,28.4,28.0,25.6,25.4,23.6,19.8,18.6,13.7. 19 F NMR(376MHz,Chloroform-d)δ-61.9.HRMS(ESI):m / z calcd for C 39 H 47 F3N7O3S + [M+H] + :750.3408; found 750.3412.

[0113] Example 13

[0114] In this embodiment, the protein degrading agents with norborneol as the hydrophobic group (protein degrading agents prepared in Examples 1-12, G1-G6, L1-M6) were evaluated by Western blotting in the human prostate cancer cell line 22Rv1 to assess their ability to degrade AR and AR-V7. The AR protein degrading agent SARD279, as reported in the literature, was used as a positive control. All compounds used were at a concentration of 10 μM. The degradation efficiency results are shown in Table 1.

[0115] Table 1. Degradation efficiency of compounds on AR and AR-V7 in human prostate cancer cell line 22Rv1.

[0116]

[0117] The table shows that each compound has a certain ability to degrade AR and AR-V7. Among them, compounds L1, L3, and L5 showed degradation efficiencies comparable to or relatively higher than those of the positive control drug SARD279. Further degradation effects were evaluated using compounds L1, L3, and L5 at concentration gradients of 0, 1, and 10 μM to determine whether these preferred compounds could degrade AR and AR-V7 in a concentration-dependent manner. Results are shown below. Figure 1 The results showed that compounds L1, L3, and L5 could all degrade AR and AR-V7 in a concentration-dependent manner, with compound L3 exhibiting the highest degradation efficiency. Therefore, compound L3 was selected as the preferred compound for further evaluation.

[0118] Example 14

[0119] This embodiment evaluates the antitumor cell proliferation experiment of the protein degrading agents with norborneol as the hydrophobic group (the compounds synthesized in Examples 1-12). The experiment was carried out in the human prostate cancer cell line 22Rv1, with the AR protein degrading agent SARD279 reported in the literature as a positive control. After incubation with 1 μM of the drug for 6 days, the cells were counted. The results are shown in Table 2. It was found that the cell number decreased to varying degrees, and the cell number of compound L3 decreased the most significantly, which is consistent with the degradation efficiency results in Example 12.

[0120] To more intuitively compare the differences in compound activity, the remaining cell percentage was divided into four categories: 80% < percentage < 100% (*), 50% < percentage < 80% (**), and 50% > percentage (***). Specific test results are shown in Table 2 below.

[0121] Table 2. Degradation efficiency of compounds on AR and AR-V7 in human prostate cancer cell line 22Rv1.

[0122]

[0123] Example 15

[0124] This embodiment evaluates the plasma metabolic stability of compound L3. Previous reports have shown that the plasma metabolic instability of hydrophobic tag protein degraders typically limits their in vivo efficacy. This invention optimizes aspects such as the hydrophobic tag type and linker type, and the selected compound L3 is expected to exhibit higher metabolic stability. SARD279 was used as a positive control for evaluating plasma metabolic stability.

[0125] The specific method for evaluating plasma metabolic stability is as follows: Weigh a sample (2.00 mg), dissolve it in acetonitrile (1.00 ml), and prepare a standard solution with a concentration of 2.00 mg / mL. After sonication, filter through a 0.22 μL filter membrane. The solution is then visually clear and free of residue before use. The 2 mg / mL standard stock solution is successively diluted to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. The optimal peak time and peak shape (MeCN / H2O = 95:5) are determined using mobile phase conditions. The peak areas of samples at different concentrations are measured, and the linear regression equation of the standard curve is calculated.

[0126] A plasma sample of 0.800 mL was prepared by mixing SD rat plasma (0.800 mL) with 40 μL of a 1 mg / mL standard solution to obtain a plasma concentration of 0.095 mg / mL. Timing was started after adding the standard solution. Subsequently, 50 μL of plasma samples were collected at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h. 250 μL of acetonitrile precipitant was added, and the samples were vortexed and centrifuged at 12000 rpm (4℃, 12000 rpm, 12 min). The supernatant was filtered through a membrane and injected for analysis. The sample concentration at different time points was calculated using a linear regression equation based on a standard curve. The results are shown in Table 3. The results indicate that, compared to the positive control drug SARD279, compound L3 has higher plasma stability and is expected to exert better efficacy in vivo.

[0127] Table 3. Plasma stability results

[0128]

[0129] Example 16

[0130] In this embodiment, the in vivo efficacy of compound L3 was evaluated. 22Rv1 cells were used to establish a xenograft model, and SARD279 cells, as reported in the literature, were used as a positive control.

[0131] The specific method was as follows: 22Rv1 was injected into nu / nu immunodeficient mice. When the average tumor diameter reached 3 mm, the mice were randomly divided into four groups: control group (n=5), compound L3 intravenous administration group 1 (10 mpk, n=5, twice daily), compound L3 intravenous administration group 2 (10 mpk, n=5, once daily), compound L3 intravenous administration group 3 (10 mpk, n=5, every two days), SARD279 intravenous administration group 1 (10 mpk, n=5, twice daily), SARD279 intravenous administration group 2 (10 mpk, n=5, once daily), and SARD279 intravenous administration group 3 (10 mpk, n=5, every two days). After 18 days, there were no significant fluctuations in the body weight of the mice in the administration groups and the control group, and the mice showed no significant abnormalities, indicating that compound L3 had good safety. The tumor weight and tumor volume of the mice in the administration groups were significantly lower than those in the control group. The tumor inhibition rate ((1 - tumor weight of administration group / tumor weight of control group) * 100%) is shown in Table 4. The results showed that, compared with SARD279, compound L3 had stronger in vivo metabolic stability, longer in vivo retention time, and could reduce the frequency of administration. In the lower frequency administration group, it had an effect comparable to the daily administration group of the positive drug.

[0132] Table 4. 22Rv1 cell xenograft model TGI

[0133]

[0134] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0135] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A protein degrading agent using norborneol as a hydrophobic group, characterized in that, The molecular structure of the protein degrading agent is any one of formula (IV) or formula (V): Formula (IV) Formula (V).

2. A method for preparing a protein degrading agent with norbornene as a hydrophobic group, used to prepare the protein degrading agent with norbornene as a hydrophobic group as described in claim 1, characterized in that, The preparation method is Route 2: Route 2: 。 3. The application of the protein degrading agent according to claim 1 or the preparation method according to claim 2 in the preparation of AR degrading agents.

4. The application according to claim 3, characterized in that, The AR degrading agent is used to prepare drugs for treating cancers with AR abnormalities and resistance to AR inhibitors.

5. The application according to claim 4, characterized in that, The cancer in question is prostate cancer.

6. A pharmaceutical composition, characterized in that, The protein degrading agent with norborneol as a hydrophobic group as described in claim 1.

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