A protein degrading agent with adamantane 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 taking noradamantane as a hydrophobic group, a preparation method, a pharmaceutical composition and application thereof. The application provides a protein degrading agent capable of degrading AR, which is based on noradamantane with a hydrophobic tag function as a hydrophobic group and an enzalutamide parent core 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 the AR protein degrading agent based on adamantane as a hydrophobic tag reported by the previous person, the protein degrading agent has considerable improvement in liver microsomal 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, 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 adamantane as a hydrophobic group, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Hydrophobic tags (Hyt) are bifunctional molecules composed 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 overcome the shortcomings of the prior art and provide a protein degrading agent with high metabolic stability in liver microsomes, using adamantane 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 adamantane as a hydrophobic group is provided, the structural formula of which is shown in formula (I):

[0006] Formula (I);

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

[0008] This invention provides a protein degrader with adamantane as the hydrophobic group. In the bifunctional molecule, adamantane is used as the hydrophobic group and enzalutamine is used as the target protein ligand. This invention screens for 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 hepatic microsomal 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 protein degrading agent is any one of formula (II), formula (III), or formula (IV):

[0012] Formula (II);

[0013] Formula (III);

[0014] Formula (IV);

[0015] In equation (II), n is any positive integer from 1 to 9; in equation (III), m is any integer from 2 to 10; and in equation (IV), o is any positive integer from 1 to 9.

[0016] More preferably, the molecular structure of the protein degrading agent with adamantane as the hydrophobic group is shown in formula (V):

[0017] Formula (V).

[0018] The compound shown in formula (V) can effectively induce the degradation of AR and AR-V7 in the human prostate cancer cell line 22Rv1 in a dose-dependent manner, exhibits considerable hepatic microsomal metabolic stability, and demonstrates the best in vivo anti-prostate cancer effect with a tumor inhibition rate of 76.3%.

[0019] According to a second aspect of the present invention, a method for preparing the above-mentioned protein degrading agent with adamantane 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 Route 1, Route 2, or Route 3.

[0020] Route 1: Arganic acid (A) is amide condensed with alkynes of different lengths (B1-B6) under HATU to obtain intermediates C1-C6. Enzalutamine core D is substituted with 2-azidoethyl-4-methylbenzenesulfonate (compound D1) under K2CO3 and DMF to obtain intermediate E, which is then reacted with C1-C6 to obtain the final products F1-F6.

[0021] ;

[0022] Route 2: Arganic acid (A) is amide condensed with aminoalkyl bromides (G1-G3) of different lengths under the action of HATU to obtain intermediates H1-H3, which are then substituted with enzalutamine core D under the action of K2CO3 and DMF to obtain intermediate final products I1-I3.

[0023] ;

[0024] Route 3: Using carboxyl alkyl bromides of different lengths (J1-J3) and N-Boc piperazine (K) in the action of HATU, intermediates L1-L3 are obtained. Then, they are substituted with the enzalutamine core D in the action of K2CO3 and DMF. Then, the Boc is removed in the action of TFA to obtain intermediates M1-M3. Finally, they are amide condensed with A in the action of HATU to obtain the final product N1-N3.

[0025] .

[0026] According to a third aspect of the invention, the use of the above-described protein degrading agent with a hydrophobic group of adamantane or a pharmaceutically acceptable salt thereof in the preparation of AR degrading agents is also provided.

[0027] 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.

[0028] According to a fourth aspect of the present invention, a pharmaceutical composition is also provided, wherein the above-mentioned AR protein degrader with a hydrophobic group of adamantane or a pharmaceutically acceptable salt thereof is the main active ingredient.

[0029] 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.

[0030] The beneficial effects of this invention are as follows: This invention provides a protein degrading agent with adamantane 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 hepatic microsomal metabolic stability, which can reduce the frequency of drug administration and effectively improve drug efficacy in in vivo experiments. 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.

[0031] 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

[0032] Figure 1 Structural diagrams of SARD279 and SARD033, protein degraders for degrading AR, developed based on hydrophobic tagging technology;

[0033] Figure 2 The diagram shows the concentration-dependent degradation of AR and AR-V7 proteins by high concentrations of compounds F1-F5 in the 22Rv1 cell line.

[0034] Figure 3 The diagram shows the concentration-dependent degradation of AR and AR-V7 proteins by low concentrations of compounds F1, F4, and F5 in the 22Rv1 cell line.

[0035] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] Example 1

[0039] A protein degrading agent with adamantane as a hydrophobic group (denoted as compound F1), compound A, compound B1, compound C1, compound D, compound D1, and compound E have the following structures:

[0040] Compound F1, Compound A, Compound B1, Compound C1, Compound D, Compound D1, Compound E.

[0041] The specific preparation method is as follows:

[0042] 1) Preparation of compound C1: Compound A (332 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 B1 (propargylamine, 110 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 1 N 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 = 2:1) to obtain compound C1.

[0043] The detection results for compound C1 are as follows: HRMS (ESI) calculated for C13 H 18 NO + [M+H] + 204.1383 was found. 204.1382.

[0044] 2) Preparation of compound E: Compound D (811 mg, 2.00 mmol) and compound D1 (483 mg, 2.00 mmol) were dissolved in N,N-dimethylformamide (6 mL), and potassium carbonate (553 mg, 4.00 mmol) was added. 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 E.

[0045] The detection results for compound E are as follows: HRMS (ESI) calculated for C 21 H 18 F3N6O2S + [M+H] + 475.1159, found. 475.1161.

[0046] 3) Preparation of compound F1: E (237 mg, 0.500 mmol) and compound C1 (102 mg, 0.500 mmol) were dissolved in... t In a 1:1 mixture of BuOH / H2O (7 mL), 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 complete, CuSO4•5H2O was removed by filtration. The filtrate was concentrated under vacuum and separated by silica gel rapid column chromatography (dichloromethane:methanol = 15:1) to obtain compound F1 (white solid, 190 mg, 56%).

[0047] The results of the detection of compound F1 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.70 (s, 1H), 7.73 – 7.66 (m,1H), 7.26 (d,J = 0.7 Hz, 1H), 7.14 – 7.06 (m, 2H), 6.89 – 6.81 (m, 2H), 4.44 –4.34 (m, 6H), 2.01 (s, 2H), 1.99 – 1.90 (m, 4H), 1.83 (d, J = 13.0 Hz, 2H), 1.69 (d, J = 12.9 Hz, 3H), 1.59 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100MHz, Chloroform- d ) δ 177.19, 172.88, 171.30, 158.13, 141.05, 136.87, 135.57,133.83, 133.64, 127.50, 125.66, 125.33, 124.79, 124.48, HRMS (ESI): m / z calcd for C 34 H 35 F3N7O3S + [M+H] + : 678.2469; found 678.2465.

[0048] Example 2

[0049] A protein degrading agent with adamantane as a hydrophobic group (denoted as compound F2) has the following structure:

[0050] The structure of compound F2 is as follows:

[0051] Compound F2.

[0052] The specific preparation method is as follows: In this embodiment, compound F2 was obtained by basically following the synthesis steps of compound F1 described in Example 1. The difference from Example 1 is that only the propargylamine was replaced with but-3-yn-1-amine.

[0053] The results of the detection of compound F2 are as follows: 1 H NMR (400 MHz, Chloroform- d) δ7.96 (d, J = 10.3 Hz, 2H), 7.83 (d, J = 8.2 Hz, 1H), 7.57 (s, 1H), 7.21 (d, J = 8.7Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 4.77 (t, J = 4.7 Hz, 2H), 4.39 (t, J = 4.7 Hz,2H), 3.73 (t, J = 6.2 Hz, 1H), 3.61 (q, J = 5.9 Hz, 2H), 2.93 (t, J = 6.3 Hz, 2H),2.59 (t, J = 6.7 Hz, 1H), 2.26 (s, 2H), 1.93 (d, J = 10.0 Hz, 2H), 1.88 – 1.80(m, 2H), 1.79 – 1.69 (m, 4H), 1.59 (s, 2H), 1.56 (s, 6H). 13 C NMR (100 MHz,Chloroform- d ) δ 180.19, 177.72, 174.99, 158.54, 137.16, 135.23, 133.73 (q, J F-C = 302.5 Hz), 133.57 (q, J F-C = 33.7 Hz), 132.22, 130.94, 128.41, 127.13 (q, J F-C =4.6 Hz), 123.25, 122.59, 115.61, 114.82, 110.18, 66.67, 66.32, 55.01, 49.58,47.45, 43.83, 43.41, 38.62, 37.58, 34.70, 25.49, 23.64. HRMS (ESI): m / z calcdfor C 35 H 37 F3N7O3S + [M+H] +: 692.2625; found 692.2628.

[0054] Example 3

[0055] A protein degrading agent with adamantane as a hydrophobic group (denoted as compound F3) has the following structure:

[0056] The structure of compound F3 is as follows:

[0057] Compound F3.

[0058] The specific preparation method is as follows: In this embodiment, compound F3 was obtained by basically following the synthesis steps of compound F1 described in Example 1. The difference from Example 1 is that only propargylamine was replaced with 4-pentyne-1-amine.

[0059] The results of the detection of compound F3 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ7.97 (d, J = 9.3 Hz, 2H), 7.84 (dd, J = 8.2, 1.7 Hz, 1H), 7.61 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 4.77 (t, J = 4.9 Hz, 2H), 4.41 (t, J = 4.9Hz, 2H), 3.34 (q, J = 6.5 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H), 2.63 (t, J = 6.7 Hz,1H), 2.30 (s, 2H), 1.94 (dt, J = 14.2, 8.2 Hz, 4H), 1.86 – 1.80 (m, 2H), 1.79 –1.74 (m, 2H), 1.65 (s, 1H), 1.63 (d, J = 2.8 Hz, 1H), 1.62 – 1.59 (m, 2H), 1.58(d, J = 6.3 Hz, 6H), 1.27 (d, J = 10.8 Hz, 1H). 13C NMR (100 MHz, Chloroform- d ) δ180.19, 177.76, 175.01, 158.62, 137.16, 135.24, 133.57 (q, J F-C = 33.6 Hz),132.23, 130.89, 129.61 (q, J F-C = 259.2 Hz), 128.32, 127.14 (q, J F-C = 5.2 Hz),127.11, 123.25, 115.66, 114.83, 110.19, 66.72, 66.34, 55.05, 49.52, 47.57,43.86, 43.40, 38.73, 37.62, 34.70, 29.70, 28.99, 23.64, 22.88. HRMS (ESI): m / z calcd for C 36 H 39 F3N7O3S + [M+H] + : 706.2782; found 706.2783.

[0060] Example 4

[0061] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound F4) has the following structure:

[0062] The structure of compound F4 is as follows:

[0063] Compound F4.

[0064] The specific preparation method is as follows: In this embodiment, compound F4 was obtained by basically following the synthesis steps of compound F1 described in Example 1. The difference from Example 1 is that only propargylamine was replaced with 5-hexyn-1-amine.

[0065] The results of the detection of compound F4 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ7.99 – 7.91 (m, 2H), 7.83 (dd, J = 8.2, 2.1 Hz, 1H), 7.50 (d, J = 4.0 Hz, 1H), 7.22 (d, J= 2.4 Hz, 1H), 7.19 (d, J = 2.7 Hz, 1H), 7.00 (dd, J = 9.3, 7.1 Hz, 2H),5.69 (t, J = 5.8 Hz, 1H), 4.76 (t, J = 4.9 Hz, 2H), 4.40 (q, J = 4.5, 4.0 Hz, 2H),3.29 (q, J = 6.6 Hz, 2H), 2.75 (t, J = 7.4 Hz, 2H), 2.29 (t, J = 3.7 Hz, 2H), 1.95(dq, J = 11.7, 3.1, 2.6 Hz, 2H), 1.76 (dtd, J = 21.4, 10.8, 8.7, 5.0 Hz, 8H),1.64 – 1.60 (m, 3H), 1.59 (d, J = 3.1 Hz, 2H), 1.56 (s, 6H). 13 C NMR (100 MHz,Chloroform- d ) δ 180.19, 177.59, 175.01, 158.64, 137.16, 135.35, 133.73 (q, J F-C = 303.0 Hz),133.57 (q, J F-C = 35.4 Hz), 130.91, 130.67, 128.33, 127.14 (q, J F-C =4.6 Hz), 123.24, 122.02, 115.65, 114.82, 110.19, 66.83, 66.78, 66.34, 49.49,47.59, 43.86, 43.38, 39.20, 37.63, 34.70, 29.70, 26.66, 25.12, 23.65. HRMS(ESI): m / z calcd for C 37 H 41 F3N7O3S + [M+H] +: 720.2938; found 720.2935.

[0066] Example 5

[0067] A protein degrading agent with adamantane as a hydrophobic group (denoted as compound F5) has the following structure:

[0068] The structure of compound F5 is as follows:

[0069] Compound F5.

[0070] The specific preparation method is as follows: In this embodiment, compound F5 is obtained by basically following the synthesis steps of compound F1 described in Example 1. The only difference from Example 1 is that propargylamine is replaced with 6-heptyne-1-amine.

[0071] The results of the detection of compound F5 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ7.96 (d, J = 8.5 Hz, 2H), 7.83 (dd, J = 8.2, 1.5 Hz, 1H), 7.49 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 4.75 (t, J = 4.7 Hz, 2H), 4.40 (t, J = 4.8Hz, 2H), 3.25 (q, J = 6.7 Hz, 2H), 2.72 (t, J = 7.4 Hz, 2H), 2.62 (t, J = 6.7 Hz,1H), 2.29 (s, 2H), 1.94 (d, J = 9.9 Hz, 2H), 1.90 – 1.67 (m, 8H), 1.67 – 1.49(m, 10H), 1.39 (p, J = 7.5, 7.1 Hz, 2H), 1.25 (d, J = 5.7 Hz, 1H). 13 C NMR (100MHz, Chloroform- d) δ 180.17, 177.48, 175.02, 158.64, 137.17, 135.22, 133.73(q, J F-C = 300.3 Hz), 133.57 (q, J F-C = 33.6 Hz), 132.24, 130.91, 128.35, 127.15(q, J F-C = 4.5 Hz), 121.92, 120.53, 115.67, 114.82, 110.18, 66.79, 66.35, 47.59,43.86, 43.37, 39.32, 37.63, 34.70, 29.70, 29.46, 28.95, 26.33, 25.61, 25.45,23.64. HRMS (ESI): m / z calcd for C 38 H 43 F3N7O3S + [M+H] + : 734.3095; found 734.3092.

[0072] Example 6

[0073] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound F6) has the following structure:

[0074] The structure of compound F6 is as follows:

[0075] Compound F6.

[0076] The specific preparation method is as follows: In this embodiment, compound F6 was obtained by basically following the synthesis steps of compound F1 described in Example 1. The difference from Example 1 is that only propargylamine was replaced with 7-octyne-1-amine.

[0077] The results of the detection of compound F6 are as follows:

[0078] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J =7.4 Hz, 1H), 7.69 (dd, J= 7.5, 2.1 Hz, 1H), 7.57 (s, 1H), 7.14 – 7.06 (m, 2H), 6.89 – 6.81 (m, 2H), 6.46 (d, J = 0.7 Hz, 1H), 4.40 (d, J = 15.4 Hz, 4H), 3.12(d, J = 0.6 Hz, 2H), 2.76 (s, 2H), 2.01 (s, 2H), 1.99 – 1.90 (m, 3H), 1.81 (d, J = 12.9 Hz, 2H), 1.73 – 1.65 (m, 6H), 1.59 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H), 1.49 (s, 2H), 1.35 (d, J = 6.6 Hz, 4H). 13 C NMR (100 MHz, Chloroform- d ) δ 177.66,172.88, 171.30, 158.13, 143.15, 136.87, 135.57, 133.83, 133.64, 128.43,125.66, 125.33, 124.79, 124.48, 119.43, 117.70, 106.85, 71.91, 66.95, 57.41,47.72, 43.23, 42.79, 42.06, 38.78, 37.29, 32.05, 30.83, 29.99, 29.44, 28.47,28.14, 23.78. HRMS (ESI): m / z calcd for C 39 H 45 F3N7O3S + [M+H] + : 748.3251; found748.3255.

[0079] Example 7

[0080] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound I1), compound A, compound G1, compound H1, and compound D have the following structures:

[0081] Compound I1, Compound A, Compound G1, Compound H1, Compound D,

[0082] The specific preparation method is as follows:

[0083] 1) Preparation of compound H1: Compound A (332 mg, 2.00 mmol) was dissolved in N,N-dimethylformamide (3 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 G1 (2-bromoethylamine, 248 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 1 N 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 = 2:1) to obtain compound H1.

[0084] The detection results for compound H1 are as follows: HRMS (ESI) calculated for C 12 H 19 BrNO + [M+H] + 272.0645 was found. 272.0647 was also found.

[0085] 2) Preparation of compound I1: Compound D (405 mg, 1.00 mmol) and compound H1 (272 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 completed, the mixture was diluted with water (15 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phases were then combined and washed with saturated NaCl solution (15 mL). The organic phases were dried with anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain compound I1.

[0086] The results of the detection of compound I1 are as follows:

[0087] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J= 2.0 Hz, 1H), 8.03 (d, J =7.5 Hz, 1H), 7.69 (dd, J = 7.5, 2.1 Hz, 1H), 7.13 – 7.07 (m, 2H), 6.87 – 6.80(m, 3H), 4.05 (s, 2H), 3.44 (s, 2H), 2.01 (s, 2H), 1.99 – 1.90 (m, 3H), 1.81(d, J = 13.0 Hz, 2H), 1.69 (d, J = 12.9 Hz, 4H), 1.59 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100 MHz, Chloroform- d ) δ 178.00, 172.88, 171.30, 157.27,136.87, 135.57, 133.83, 133.64, 125.66, 125.33, 124.79, 124.42, 119.43,117.70, 106.85, 68.72, 66.95, 47.91, 43.27, 42.79, 40.45, 38.78, 37.29,29.44, 23.78. HRMS (ESI): m / z calcd for C 31 H 32 F3N4O3S + [M+H] + : 597.2142; found597.2141.

[0088] Example 8

[0089] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound I2) has the following structure:

[0090] The structure of compound I2 is as follows:

[0091] Compound I2.

[0092] The specific preparation method is as follows: In this embodiment, compound I2 is obtained by basically following the synthesis steps of compound I1 described in Example 7. The difference from Example 7 is that only 2-bromoethylamine is replaced with 3-bromopropylamine.

[0093] The results of the detection of compound I2 are as follows:

[0094] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J =7.4 Hz, 1H), 7.69 (dd, J = 7.5, 2.1 Hz, 1H), 7.12 – 7.06 (m, 2H), 6.86 – 6.78(m, 2H), 6.54 (d, J = 0.7 Hz, 1H), 4.07 (s, 2H), 3.13 (s, 2H), 1.98 (dd, J =17.4, 6.2 Hz, 6H), 1.92 (s, 1H), 1.81 (d, J = 12.9 Hz, 2H), 1.69 (d, J = 12.9 Hz, 3H), 1.59 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100 MHz, Chloroform- d ) δ177.50, 172.88, 171.30, 159.06, 136.87, 135.57, 133.83, 133.64, 125.66,125.33, 124.79, 124.42, 119.43, 117.54, 106.85, 69.72, 66.95, 47.72, 43.23,42.79, 40.61, 38.78, 37.29, 30.53, 29.44, 23.78. HRMS (ESI): m / z calcd forC 32 H 34 F3N4O3S + [M+H] + : 611.2298; found 611.2295.

[0095] Example 9

[0096] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound I3) has the following structure:

[0097] The structure of compound I3 is as follows:

[0098] Compound I3.

[0099] The specific preparation method is as follows: In this embodiment, compound I3 is obtained by basically following the synthesis steps of compound I1 described in Example 7. The difference from Example 7 is that only 2-bromoethylamine is replaced with 4-bromobutylamine.

[0100] The results of the detection of compound I3 are as follows:

[0101] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J =7.4 Hz, 1H), 7.69 (dd, J = 7.5, 2.1 Hz, 1H), 7.13 – 7.06 (m, 2H), 6.86 – 6.78(m, 2H), 6.41 (d, J = 0.7 Hz, 1H), 4.01 (s, 2H), 3.16 (d, J = 0.6 Hz, 2H), 2.01(s, 2H), 1.99 – 1.90 (m, 3H), 1.85 – 1.77 (m, 4H), 1.73 – 1.63 (m, 5H), 1.59(d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100 MHz, Chloroform- d ) δ 177.66,172.88, 171.30, 159.04, 136.87, 135.57, 133.83, 133.64, 125.66, 125.33,124.79, 124.42, 119.43, 117.54, 106.85, 78.01, 66.95, 47.72, 43.23, 42.79,42.12, 38.78, 37.29, 30.56, 29.44, 27.76, 23.78. HRMS (ESI): m / z calcd forC 33 H 36 F3N4O3S + [M+H] + : 625.2455; found 625.2456.

[0102] Example 10

[0103] A protein degrading agent with noradaline as a hydrophobic group (denoted as compound N1), compound J1, compound K, compound L1, compound D, compound M1, and compound A have the following structures:

[0104] Compound N1, Compound J1 Compound K, Compound L1, Compound D Compound M1, Compound A,

[0105] The specific preparation method is as follows:

[0106] 1) Preparation of compound L1: Compound J1 (2-bromoacetic acid, 278 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 K (N-Boc piperazine, 371 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 1 N 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 = 3:2) to obtain compound L1.

[0107] The detection results for compound L1 are as follows: HRMS (ESI) calculated for C 11 H 20 BrN2O3 + [M+H] + 307.0652 was found. 307.0655.

[0108] 2) Preparation of compound M1: Compound D (405 mg, 1.00 mmol) and compound L1 (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 completed, the mixture was diluted with water (15 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phases were then combined and washed with saturated NaCl solution (15 mL). The organic phases were dried with anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain an intermediate, which was then dissolved in dichloromethane (5 mL). Trifluoroacetic acid (3 mL) was added, and the mixture was stirred at room temperature for 0.5 hours. The solvent was then evaporated to obtain crude compound M1.

[0109] The detection results for compound M1 are as follows: HRMS (ESI) calculated for C 25 H 25 F3N5O3S + [M+H] + 532.1625 was found. 532.1626.

[0110] 3) Preparation of compound N1: Compound M1 (266 mg, 0.500 mmol) was dissolved in N,N-dimethylformamide (2 mL) under ice bath conditions. N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (228 mg, 0.600 mmol), diisopropylethylamine (97.2 mg, 0.750 mmol), and compound A (83.0 mg, 0.500 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 (10 mL), and then washed with 1 N HCl solution (10 mL), saturated sodium bicarbonate solution (10 mL), and saturated saline solution (10 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 N1.

[0111] The results of the detection of compound N1 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.69 (dd, J= 7.5, 2.1 Hz, 1H),7.21 – 7.14 (m, 2H), 6.94 – 6.86 (m, 2H), 4.77 (s, 2H), 3.62 (d, J = 1.8 Hz, 4H), 3.53 (d, J = 13.5 Hz, 4H), 2.04 (d, J = 16.4 Hz, 3H), 1.96 (d, J = 13.0 Hz, 2H), 1.85 (d, J = 12.9 Hz, 2H), 1.73 (s, 1H), 1.61 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100 MHz, Chloroform- d ) δ 176.84, 172.88, 171.30, 170.74, 158.32,136.87, 135.57, 133.83, 133.64, 125.66, 125.33, 124.79, 124.52, HRMS (ESI): m / z calcd for C 35 H 37 F3N5O4S + [M+H] + : 680.2513;found 680.2515.

[0112] Example 11

[0113] A protein degrading agent (denoted as compound N2) with adamantane as a hydrophobic group has the following structure:

[0114] The structure of compound N2 is as follows:

[0115] Compound N2.

[0116] The specific preparation method is as follows: In this embodiment, compound N2 is obtained by basically following the synthesis steps of compound N1 described in Example 10. The difference from Example 10 is that only 2-bromoacetic acid is replaced with 3-bromopropionic acid.

[0117] The results of the detection of compound N2 are as follows:

[0118] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J =7.4 Hz, 1H), 7.69 (dd, J = 7.5, 2.1 Hz, 1H), 7.13 – 7.05 (m, 2H), 6.84 – 6.76(m, 2H), 4.20 (s, 2H), 3.59 (d, J = 15.2 Hz, 6H), 3.50 (s, 2H), 2.68 (s, 2H), 2.04 (d, J = 16.4 Hz, 3H), 1.96 (d, J = 13.0 Hz, 2H), 1.85 (d, J = 12.9 Hz, 2H), 1.72 (d, J = 12.9 Hz, 4H), 1.61 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100MHz, Chloroform- d ) δ 176.84, 172.96, 172.88, 171.30, 161.01, 136.87, 135.57,133.83, 133.64, 125.66, 125.33, 124.79, 124.42, 119.43, HRMS (ESI): m / z calcd for C 36 H 39 F3N5O4S + [M+H] + : 694.2669; found 694.2667.

[0119] Example 12

[0120] A protein degrading agent with adamantane as a hydrophobic group (denoted as compound N3) has the following structure:

[0121] The structure of compound N3 is as follows:

[0122] Compound N3.

[0123] The specific preparation method is as follows: In this embodiment, compound N3 is obtained by basically following the synthesis steps of compound N1 described in Example 10. The difference from Example 10 is that only 2-bromoacetic acid is replaced with 4-bromobutyric acid.

[0124] The results of the detection of compound N3 are as follows:

[0125] 1 H NMR (400 MHz, Chloroform- d ) δ 8.13 (d, J = 2.0 Hz, 1H), 8.03 (d, J =7.4 Hz, 1H), 7.69 (dd, J = 7.5, 2.1 Hz, 1H), 7.12 – 7.06 (m, 2H), 6.85 – 6.79(m, 2H), 4.05 (s, 2H), 3.61 (s, 2H), 3.56 (s, 4H), 3.50 (s, 2H), 2.52 (s,2H), 2.06 (d, J = 1.3 Hz, 4H), 2.02 (s, 1H), 1.96 (d, J = 13.0 Hz, 2H), 1.85 (d, J = 12.9 Hz, 2H), 1.72 (d, J = 12.9 Hz, 4H), 1.61 (d, J = 13.0 Hz, 2H), 1.53 (s, 6H). 13 C NMR (100 MHz, Chloroform- d) δ 176.84, 174.12, 172.88, 171.30, 159.24,136.87, 135.57, 133.83, 133.64, 125.66, 125.33, 124.79, 124.42, HRMS (ESI): m / z calcd for C 37 H 41 F3N5O4S + [M+H] + : 708.2826; found 708.2829.

[0126] Example 13

[0127] In this embodiment, the protein degrading agents with adamantane as the hydrophobic group (the protein degrading agents prepared in Examples 1-12, F1-F6, I1-I3, N1-N3) were evaluated using Western blotting to assess their ability to degrade AR and AR-V7 in the human prostate cancer cell line 22Rv1. The AR protein degrading agent SARD279, as reported in the literature, was used as the positive control. All compounds were used at a concentration of 10 μM. The degradation efficiency results are shown in Table 1.

[0128] Table 1. Degradation efficiency of compounds on AR and AR-V7 in human prostate cancer cell line 22Rv1

[0129]

[0130] The table shows that each compound has a certain ability to degrade AR and AR-V7. Among them, compounds F1-F5 showed comparable or relatively higher degradation efficiencies of AR and AR-V7 compared to the positive control drug SARD279. Further degradation efficacy evaluation was conducted using compounds F1-F5 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 2 The results showed that compounds F1-F5 could degrade AR and AR-V7 in a concentration-dependent manner, with compounds F1, F4, and F5 exhibiting the highest degradation efficiency. Therefore, compounds F1, F4, and F5 were selected as preferred compounds for further evaluation.

[0131] The degradation efficiency of the above compounds was evaluated at lower concentrations (0, 100, and 300 nM, respectively), and the results are shown in [Figure number missing]. Figure 3The results showed that compound F4 had the highest degradation efficiency. Therefore, compound F4 was selected as the preferred compound for further evaluation.

[0132] Example 14

[0133] This embodiment evaluates the anti-tumor cell proliferation experiment of the protein degrading agents with adamantane as the hydrophobic group (the compounds synthesized in Examples 1-12). The experiment was carried out in the human prostate cancer cell line 22Rv1. The AR protein degrading agent SARD279 reported in the literature was used as the positive drug. 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 F4 decreased the most significantly, which is consistent with the degradation efficiency results in Example 13.

[0134] 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.

[0135] Table 2

[0136]

[0137] Example 15

[0138] This embodiment evaluates the hepatic microsomal metabolic stability of compound F4. Previous reports have shown that the hepatic microsomal 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 F4 is expected to exhibit higher metabolic stability and in vivo bioavailability. SARD279 was used as a positive control for evaluating the hepatic microsomal metabolic stability.

[0139] The specific method for evaluating the metabolic stability of liver microsomes was as follows: Liver microsomes were removed from a -80 ℃ freezer and preheated for 3 min in a 37 ℃ water bath with a constant temperature shaker to thaw them before use. Then, a certain amount of NADPH was weighed and dissolved in an appropriate amount of magnesium chloride solution to prepare a 2 mM solution. A mixed solution of the incubation system (excluding NADPH) was prepared according to the composition ratio of the experimental incubation system (3 Mm MgCl2-PB solution, 1 μM compound F4, 0.5 mg / mL liver microsomes), and dispensed in 40 μL tubes. For the 0 min sample, 240 μL of internal standard working precipitant was added, followed by 40 μL of NADPH solution (40 μL of magnesium chloride solution was added to the negative control group). For other samples, 40 μL of NADPH solution was added to initiate the reaction (40 μL of magnesium chloride solution was added to the negative control group). After incubation at 37 °C for 5 min, 15 min, 30 min, and 60 min, 240 μL of internal standard precipitant was added. For the positive control group, 40 μL of NADPH solution was added to initiate the reaction, and after incubation at 37 °C for 5 min and 15 min, 240 μL of internal standard precipitant was added. All samples were vortexed and centrifuged, and 150 μL of the supernatant was collected. 150 μL of water was added, and the mixture was vortexed and analyzed by LC-MS / MS. Analysis showed that compound F4 exhibited moderate clearance efficiency in humans, mice, and rats, and had a lower in vivo metabolic clearance risk compared to SARD279. The results are shown in Table 3.

[0140] Table 3. Hepatic microsomal metabolic stability

[0141]

[0142] Example 16

[0143] In this embodiment, the in vivo efficacy of compound F4 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.

[0144] 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 F4 intravenous administration group 1 (10 mpk, n=5, daily administration), compound F4 intravenous administration group 2 (10 mpk, n=5, every two days), compound F4 intravenous administration group 3 (10 mpk, n=5, every three days), SARD279 intravenous administration group 1 (10 mpk, n=5, daily administration), SARD279 intravenous administration group 2 (10 mpk, n=5, every two days), and SARD279 intravenous administration group 3 (10 mpk, n=5, every three days). After 18 days, there were no significant fluctuations in the body weight of the mice in the administration groups and the body weight of the mice in the control group, and the mice showed no significant abnormalities, indicating that compound F4 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 F4 had stronger in vivo metabolic stability and a longer in vivo retention time, which could reduce the frequency of administration. In the lower dosing frequency group, it had an effect comparable to that of the positive drug daily dosing group.

[0145] Table 4. 22Rv1 cell xenograft model TGI

[0146]

[0147] 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.

[0148] 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 noradaline as a hydrophobic group, characterized in that, The molecular structure of the degrading agent is any one of formula (II), formula (III), or formula (IV): Formula (II); Formula (III); Formula (IV); In equation (II), n is any positive integer from 1 to 6; in equation (III), m is any integer from 2 to 3; and in equation (IV), o is any positive integer from 2 to 3.

2. The protein degrading agent with adamantane as a hydrophobic group according to claim 1, characterized in that, The molecular structure of the degrading agent is shown in formula (V): Formula (V).

3. A method for preparing a protein degrading agent with noradaline as a hydrophobic group, used to prepare a protein degrading agent with noradaline as a hydrophobic group as described in any one of claims 1-2, characterized in that, The preparation method is Route 1, Route 2, or Route 3, wherein: Route 1: Route 2: Route 3: 。 4. The application of the protein degrading agent with adamantane as a hydrophobic group according to any one of claims 1-2 in the preparation of AR degrading agents.

5. The application according to claim 4, characterized in that, The AR degrading agent can be used to prepare drugs for treating cancers with AR abnormalities.

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

7. A pharmaceutical composition, characterized in that, Includes the protein degrading agent with a hydrophobic group of adamantane as described in any one of claims 1-2.

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