Compound for specific targeted degradation of PIN1 protein and application

Abstract

The invention discloses a compound for specific targeted degradation of PIN1 protein and application, and belongs to the technical field of biological medicine, the structural general formula of the compound is as shown in formula I # imgabs0 #, X is any one of-CH2-,-NH-and-O-; linker is any structure of C3-12 alkyl, C3-12 alkoxy and a formula II, n represents any number between 1 and 4, A represents-N-or-CH-, and Cy represents an aromatic ring, an aromatic heterocyclic ring or a saturated carbocyclic ring with the carbon number of 3-6; the # imgabs1 # E3L is any one structure in a formula III; according to the present invention, the PIN1 protein can be subjected to targeted degradation in a variety of tumor cells so as to reduce the PIN1-mediated cancer promoting gene expression while up-regulating the PIN1-inhibited cancer suppressor gene.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a compound for specifically targeting and degrading PIN1 protein and its application. Background Art

[0002] Breast cancer is the most common cancer in women, with 2.26 million new cases worldwide in 2020. It is also the leading cause of cancer death in women worldwide, causing 684,996 deaths in 2020, with an age-adjusted mortality rate of 13.6 per 100,000 people. Although breast cancer incidence is highest in developed regions, the vast majority of breast cancer patients in high-income countries survive; in contrast, mortality rates are higher in middle-income and low-income countries, including China.

[0003] Current treatments for breast cancer include surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy. Although significant progress has been made, it still faces many challenges such as early diagnosis, drug resistance, individual differences, side effects, risk of recurrence, and high prices.

[0004] Targeted drugs are one of the important means of treating many cancers including breast cancer. However, there are many signal pathways in tumor cells that influence each other and have redundant functions. They are usually activated at the same time, so using targeted drugs to block a single pathway is sometimes difficult to work.

[0005] In many cancers, the peptidylprolyl cis-trans isomerase PIN1 is frequently overexpressed or abnormally activated, and is also significantly associated with poor prognosis. Through a feedback regulatory mechanism, PIN1 can both upregulate about 50 oncogenes and inhibit about 20 tumor suppressor genes, thereby simultaneously amplifying multiple pathways that drive cancer formation, playing a central role in these signaling pathways. PIN1-deficient mice do not form tumors under a variety of cancer-inducing conditions, including p53 mutations, HER2 / RAS overactivation, or sustained expression of c-Myc. At the same time, these mice can develop basically normally to adulthood. Therefore, chemical interventions targeting PIN1 can simultaneously block multiple cancer-driving pathways and are expected to become new targeted drugs.

[0006] Currently reported PIN1 inhibitors include juglone, KPT-6566, ATRA, ATO, sulfopin, pTide, cis-olefin peptidomimetics, etc., each of which has some shortcomings. Some of these inhibitors produce irreversible inhibition in a covalent manner, showing multiple off-target effects and potential toxicity; some are designed from simulated substrates, and the acidic groups in their structures lead to poor membrane permeability, which also affects their cell activity. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a compound and application for specifically targeting and degrading PIN1 protein to solve the above problems.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a compound for specifically targeting and degrading PIN1 protein, characterized in that the general structural formula of the compound is as shown in Formula I:

[0010]

[0011] X is any of -CH2-, -NH-, and -O-;

[0012] Linker for C 3-12 Alkyl, C 3-12 Alkoxy, any structure of Formula II, wherein n represents any number between 1 and 4, A represents -N- or -CH-, and Cy represents an aromatic ring, an aromatic heterocyclic ring, or a saturated carbocyclic ring having 3 to 6 carbon atoms;

[0013]

[0014] E3L is any structure in Formula III;

[0015]

[0016] Furthermore, the X is preferably -NH- or -O-.

[0017] Furthermore, the structural formula of the compound is any one of structures A1-A35:

[0018]

[0019]

[0020] Furthermore, the optical isomers and pharmaceutically acceptable salts of the compound.

[0021] Furthermore, the compound is used in combination with a specific drug to prepare a drug for treating tumor diseases.

[0022] Furthermore, the tumor disease is one or more of breast cancer, pancreatic cancer, colon cancer, prostate cancer, lung cancer, liver cancer, acute myeloid leukemia, and multiple myeloma.

[0023] Furthermore, the specific drug is at least one of RO3306, tamoxifen, toremifene, fulvestrant, abemaciclib, apellis, palbociclib, ribociclib, olaparib, beztifan, everolimus, thalidomide, lenalidomide, and pomadoamine.

[0024] Beneficial effects of the present invention:

[0025] 1. In the present invention, through TMT quantitative proteomics research, it was found that after treatment with the compounds of the present invention, the protein content of the cells that was most downregulated was PIN1 protein, and the difference was highly significant. This result shows that the compounds prepared by the present invention have high specificity for the degradation of PIN1. It is inferred that the compounds of the present invention can specifically target and degrade PIN1 protein in a variety of tumor cells, thereby reducing the expression of tumor-promoting genes mediated by PIN1 and upregulating tumor suppressor genes inhibited by PIN1.

[0026] 2. The compounds of the present invention exhibit killing effects on a variety of tumor cells, indicating that they have a wide range of applicability in the treatment of different cancers.

[0027] 3. The compound of the present invention has shown the effect of treating breast cancer in animal models and has good pharmacokinetic parameters, becoming the first PIN1 degrader molecule to show anti-tumor activity in vivo and has good drugability;

[0028] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0030] Figure 1 It is the degradation effect of some PROTAC molecules of the present invention on PIN1 protein in different tumor cells;

[0031] Figure 2 It is a further characterization of the PIN1 protein degradation properties of compound A1;

[0032] Figure 3 It is the inhibitory effect of compound A1 combined with other drugs on different tumor cells;

[0033] Figure 4 This is the experimental result that compound A1 specifically degrades PIN1 at the cellular level;

[0034] Figure 5 It is the therapeutic effect of the combination of Compound A1 on the xenograft animal model. DETAILED DESCRIPTION

[0035] The starting materials of the present invention can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein.

[0036] The structure of the compound was determined by nuclear magnetic resonance (NMR) 1 H-NMR / 13 C-NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR determination was performed using a Bruker AVANCE IV Dr (600 MHz) nuclear magnetic resonance spectrometer, and the determination solvent was deuterated chloroform (CDCl 3-d ), deuterated methanol (MeOD-d4), deuterated dimethyl sulfoxide (DMSO-d6), and TMS as the internal standard.

[0037] LC-MS determination was performed using an Agilent / 1260-6160 single quadrupole liquid chromatography-mass spectrometer, the preparative liquid phase model was Agilent 1260 Infinity II, and the silica gel column chromatography used 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.

[0038] Preparation of intermediates I-1 and I-2:

[0039]

[0040] Intermediate I-1 is: 3-((3-hydroxy-2,2-dimethylpropyl)amino)tetrahydrothiophene-1,1-dioxide, and its synthesis method is as follows:

[0041] 3-Aminotetrahydrothiophene-1,1-dioxide hydrochloride (3 g, 17.4 mmol, 1.0 eq) and triethylamine were dissolved in ultra-dry DMF (15 mL). After reacting at room temperature for 1 hour, 3-hydroxy-2,2-dimethylpropanal (1.95 g, 19.14 mmol, 1.1 equiv) and glacial acetic acid (200 μL, 17.4 mmol, 0.2 equiv) were added and stirring continued for 1 hour. Finally, sodium triacetoxyborohydride (7.74 g, 36.54 mmol, 2.1 equiv) was added in one go and stirring continued overnight. The pH was then adjusted to 7 using saturated sodium bicarbonate solution, extracted three times with dichloromethane, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography (100% ethyl acetate) to obtain a white solid, which was intermediate I-1 (4.38 g, yield: 78%).

[0042] 1H NMR (600MHz, DMSO-d6) δ4.00 (s, 1H), 3.64 (dd, J = 13.4, 8.1Hz, 1H), 3.47 (dd d,J=16.6,8.6,3.9Hz,2H),3.39–3.33(m,2H),3.20–3.17(m,1H),3.17–3.1 4(m,1H),2.88(d,J=7.0Hz,1H),2.79(d,J=13.1Hz,1H),2.68–2.60(m,1H), 2.37(dq,J=13.4,9.1Hz,1H), 1.28(d,J=66.6Hz,1H), 0.94(d,J=3.2Hz,6H).

[0043]

[0044] Intermediate I-2 is: 3-((3-amino-2,2-dimethylpropyl)amino)tetrahydrothiophene-1,1-dioxide, and its synthesis method is as follows:

[0045] 2,5-Dihydrothiophene-1,1-dioxide (5 g, 42.3 mmol, 1.0 equiv), 2,2-dimethylpropane-1,3-diamine (8.6 g, 84.6 mmol, 2.0 equiv), and cesium carbonate (10 mol%) were placed in a flask and stirred at 100° C. for 6 hours; the excess amine was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol = 95:5, v / v) to obtain a colorless oil, which is intermediate I-2 (5.47 g, yield: 59%).

[0046] 1 H NMR(600MHz, CDCl3-d)δ3.55(ddd,J=11.9,6.5,5.3Hz,1H),3.35–3.29(m,1H),3.29–3.24(m,1H),3.10–3.01(m,1H),2.96(ddt,J=13.3,5.7 ,1.0Hz,1H),2.62–2.53(m,2H),2.45(d,J=1.9Hz,2H),2.43–2.37(m,1H),2.12(dddd,J=14.7,13.7,7.2,1.0Hz,1H),0.89(d,J=4.9Hz,6H).

[0047] Example 1

[0048] Preparation of compound A1:

[0049]

[0050] Intermediate I-3 is: tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyrate, and its synthesis method is as follows:

[0051] 2-(2,6-dioxo-piperidin-3-yl)-4-hydroxy-isoindole-1,3-dione (5.13 g, 18.67 mmol, 1.0 equiv) was added to a DMF (30 mL) solution, followed by tert-butyl 4-bromobutyrate (5.00 g, 22.41 mmol, 1.2 equiv) and DIPEA (9.75 mL, 56.01 mmol, 3.0 equiv), stirred at 70°C for 2 days, the solvent was removed by rotary evaporation, the mixture was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, the organic phase was extracted with DCM, dried over anhydrous sodium sulfate, the filtrate was collected by suction filtration, and concentrated by rotary evaporation. The crude product was subjected to silica gel column chromatography (PE / EA=1:2) to obtain yellow solid I-3 (6.35 g, yield: 81%).

[0052] 1 H NMR (600MHz, CDCl3-d) δ8.49(s,1H),7.65(dd,J=8.5,7.3Hz,1H),7.43(d,J=7.3Hz,1H),7.22(d,J=8.5Hz,1H),4.95(dd, J=12.2,5.4Hz,1H),4.21(t,J=6.3Hz,2H),2.89–2.67(m,3H),2.48(t,J=7.2Hz,2H),2.17–2.06(m,3H),1.42(s,9H).13C NMR (151MHz, CDCl3-d) δ172.49,171.36,168.40,167.16,165.76,156.55,136.63,133 .88,119.16,117.29,116.00,80.67,68.39,49.19,31.53,31.46,28.19,24.45,22.70.

[0053] Intermediate I-4 is: 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyric acid, and its synthesis method is as follows:

[0054] Intermediate I-3 (6.35 g, 15.26 mmol) was dissolved in DCM (40 mL), TFA (8 mL, 20% v / v) was slowly added dropwise at 0°C, and the mixture was stirred overnight at room temperature. The solvent was removed by rotary evaporation to obtain crude product I-4, which was used directly in the next step without further purification.

[0055] Synthetic intermediate I-5: 3-((1,1-dioxidotetrahydrothiophene-3-yl)amino)-2,2-dimethylpropyl-4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyrate, the synthesis method is as follows:

[0056] I-1 (3.08 g, 13.93 mmol, 1.0 equiv) was dissolved in ultra-dry DCM (30 mL), and intermediate I-4 (5.50 g, 15.32 mmol, 1.1 equiv), DCC (4.31 g, 20.90 mmol, 1.5 equiv), EDC (4.00 g, 20.90 mmol, 1.5 equiv), DMAP (0.17 g, 1.39 mmol, 0.1 equiv), TEA (5.81 mL, 41.79 mmol, 3.0 equiv) were added in sequence at 0 ° C, and the reaction was stirred at room temperature for 8 hours; then washed with saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, extracted with DCM, dried over anhydrous sodium sulfate, and the filtrate was collected by suction filtration, concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography (EA) to obtain a white solid I-5 (1.70 g, yield: 22%).

[0057] Compound A1 is: 3-(2-chloro-N-(1,1-dioxidotetrahydrothiophene-3-yl)acetylamino)-2,2-dimethylpropyl-4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyrate, and its synthesis method is as follows:

[0058] The intermediate I-5 (1.70 g, 3.106 mmol, 1.0 equiv) was dissolved in ultra-dry DCM (15 mL), and DIPEA (2.36 mL, 13.57 mmol, 3.0 equiv) was added. Chloroacetyl chloride (0.36 mL, 4.52 mmol, 1.5 equiv) was dissolved in a small amount of ultra-dry DCM (2 mL) and added dropwise to the reaction solution at 0°C. The reaction was stirred at room temperature for 2 h. The mixture was then washed with saturated sodium bicarbonate solution and saturated sodium chloride solution in turn, extracted with DCM, dried over anhydrous sodium sulfate, and the filtrate was collected by suction and concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (PE: EA = 1:5) to obtain a white solid A1 (1.05 g, yield: 54%).

[0059] 1H NMR (600MHz, CDCl3-d) δ8.74(d,J=12.9Hz,1H),7.66(dd,J=8.4,7.3Hz,1H),7.44(d,J=7.3Hz,1H),7.21(d,J=8.5Hz,1H),5.01–4 .92(m,1H),4.25–4.20(m,2H),4.11(s,1H),3.87(pd,J=11.2,5.4Hz,3H),3.71–3.61(m,2H),3.36(dd,J=15.8,4.7Hz,1H),3.25(d ,J=15.8Hz,1H),3.13(td,J=13.2,8.0Hz,1H),3.03–2.97(m,1H),2.84(dd,J=12.5,2.8Hz,1H),2.79–2.73(m,2H),2.67(td,J=7. 3,2.5Hz,2H),2.50–2.44(m,2H),2.19(p,J=6.5Hz,2H),2.10(ddd,J=7.9,6.3,3.7Hz,1H),1.88(s,1H),0.98(t,J=6.2Hz,6H).13C NMR (151MHz, CDCl3-d) δ172.71,171.52,171.49,168.71,168.69,168.19,168.16,167.08,165.79,156.28,136.72,133.85,119.09,117.3 4,116.18,69.82,69.79,67.81,57.96,57.93,57.56,50.41,49.20,4 9.11,42.08,36.64,31.45,30.17,26.56,26.52,25.69,25.02,23.28.

[0060] Embodiment 2:

[0061] Preparation of Compound A15

[0062]

[0063] Intermediate I-6 is: tert-butyl (3-((1,1-dioxytetrahydrothiophene-3-yl)amino)-2,2-dimethylpropyl) glutarate, and its synthesis method is as follows:

[0064] The intermediate I-1 (290 mg, 1.31 mmol, 1.0 equiv) was dissolved in ultra-dry DMF, and 5-(tert-butoxy)-5-oxopentanoic acid (296 mg, 1.57 mmol, 1.02 equiv), DMAP (16 mg, 0.131 mmol, 0.1 equiv), DCC (405 mg, 1.965 mmol, 1.5 equiv), and triethylamine (546 μL, 3.93 mmol, 3.0 equiv) were added in sequence, and reacted at room temperature for 12 hours; water and dichloromethane were added to extract three times, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3: 7, v / v) to obtain a white solid, which is the intermediate I-6 (140 mg, yield: 27%).

[0065] Intermediate I-7 is: tert-butyl (3-(2-chloro-N-(1,1-dioxidotetrahydrothiophene-3-yl)acetylamino)-2,2-dimethylpropyl) glutarate, and its synthesis method is as follows:

[0066] The intermediate I-6 (140 mg, 0.36 mmol, 1 equiv) was dissolved in DMF, triethylamine (451 μL, 0.32 mmol, 9 equiv) was added, chloroacetyl chloride (114 mg, 1.44 mmol, 4 equiv) was dissolved in a little DMF, and added dropwise to the above solution at 0°C and then warmed to room temperature for 1 hour, and extracted three times with water and dichloromethane, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50: 50, v / v) to obtain a colorless oil, which is the intermediate I-7 (130 mg, yield: 78%).

[0067] Intermediate I-8 is: 5-(3-(2-chloro-N-(1,1-dioxidotetrahydrothiophene-3-yl)acetylamino)-2,2-dimethylpropoxy)-5-oxopentanoic acid, and its synthesis method is as follows:

[0068] The intermediate I-7 (130 mg, 0.27 mmol, 1 equiv) was dissolved in dichloromethane, and trifluoroacetic acid (150 μL, 2.16 mmol, 8 equiv) was added to react for 3 hours. The dichloromethane and excess trifluoroacetic acid were removed by rotary evaporation to obtain the crude intermediate I-8, which was directly used in the next step reaction.

[0069] Compound A15: 3-(2-chloro-N-(1,1-dioxidotetrahydrothiophen-3-yl)acetamido)-2,2-dimethylpropyl 5-(((2S)-1-((4S)-4-hydroxy-2-(((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-5-oxopentanoate, the synthesis method of which is as follows:

[0070] The intermediate I-8 (114 mg, 0.278 mmol, 1 equiv) was dissolved in ultra-dry dichloromethane, and HATU (158 mg, 0.417 mmol, 1.5 equiv), (S, R, S)-AHPC-Me hydrochloride (107 mg, 0.22 mmol, 0.8 equiv), and DIPEA (290 μL, 1.67 mmol, 6 equiv) were added at 0°C. DIPEA was added to adjust the pH to 7, and then the temperature was raised to room temperature for overnight reaction. Water and dichloromethane were added for extraction three times, and the mixture was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 20: 1, v / v) to obtain compound A15 (70 mg, yield: 30%).

[0071] 1H NMR (600MHz, CDCl3-d) δ8.74(dd,J=4.4,1.4Hz,1H),8.68(d,J=1.1Hz,1H),8.38(ddd,J=8.4 ,5.4,1.4Hz,1H),7.48(d,J=8.1Hz,1H),7.46–7.42(m,1H),7.41–7.38(m,2H),7.37(d,J=8. 4Hz,2H),6.77–6.61(m,1H),5.37(dd,J=13.4,6.8Hz,1H),5.33–5.26(m,1H),5.09(t,J=7.2 Hz,1H),4.69(dd,J=8.1,3.2Hz,1H),4.60(dd,J=8.9,2.4Hz,1H),4.51–4.48(m,1H),4.16–4 .08(m,1H),4.06–4.02(m,1H),3.95–3.91(m,1H),3.91–3.83(m,2H),3.74–3.68(m,1H),3.6 5–3.61(m,1H),3.60–3.53(m,1H),3.44–3.36(m,1H),3.34–3.14(m,2H),3.08–2.96(m,1H), 2.41–2.36(m,3H),2.34–2.29(m,1H),2.24(dt,J=14.6,7.0Hz,1H),2.14–2.05(m,1H),1.96 –1.91(m,2H),1.48(dd,J=6.9,1.8Hz,3H),1.05(d,J=3.4Hz,6H),1.03(d,J=4.0Hz,9H).13C NMR (151MHz, CDCl3-d) δ172.71,172.66,171.73,170.06,151.67,150.41,148.38,143.29,139.32,135.07,129.50,126.44 ,121.15,69.92,58.72,57.60,56.88,50.47,50.31,48.78,41.92,36.01,35.35,34.86,33.16,26.52,22.20,20.68,16.06

[0072] Example 3

[0073] Preparation of Compound A32

[0074]

[0075] Intermediate I-9 is: tert-butyl 4-(4-((3-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2,2-dimethylpropyl)carbamoyl)phenyl)piperazine-1-carboxylate, and its synthesis method is as follows:

[0076] The intermediate I-2 (0.40 g, 1.82 mmol, 1.0 equiv) was dissolved in anhydrous DCM (5 mL) and anhydrous DMF (1 mL), and HATU (1.04 g, 2.72 mmol, 1.5 equiv), tert-butyl 4-(4-carboxyphenyl)piperazine-1-carboxylate (0.50 g, 1.63 mmol, 0.9 equiv), and DIPEA (0.95 mL, 5.44 mmol, 3.0 equiv) were added, and the mixture was stirred at room temperature for 3 h, and washed with water, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, extracted with DCM, dried over anhydrous sodium sulfate, and the filtrate was collected by suction and concentrated. The crude product was purified by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain a white solid I-9 (0.59 g, yield: 64%).

[0077] 1 H NMR(600MHz, CDCl3-d)δ7.69(d,J=8.9Hz,2H),7.06(t,J=6.2Hz,1H),6.88–6.84(m,2H),3.56–3.52(m,4H),3.52–3.47(m,1H),3.34–3.20(m ,8H),3.02(dt,J=12.9,7.5Hz,1H),2.91(dd,J=13.3,5.9Hz,1H),2.43–2.36(m,3H),2.12–2.06(m,1H),1.46(s,9H),0.94(d,J=2.1Hz,6H).

[0078] Intermediate I-10 is: tert-butyl 4-(4-((3-(2-chloro-N-(1,1-dioxidotetrahydrothiophen-3-yl)acetylamino)-2,2-dimethylpropyl)carbamoyl)phenyl)piperazine-1-carboxylate, and its synthesis method is as follows:

[0079] The intermediate I-9 (0.59 g, 1.56 mmol, 1.0 equiv) was dissolved in ultra-dry DCM (6 mL), DIPEA (0.60 mL, 3.480 mmol, 3.0 equiv) was added, chloroacetyl chloride (0.18 mL, 2.32 mmol, 2.0 equiv) was dissolved in a small amount of ultra-dry DCM (1 mL), and added dropwise to the reaction solution at 0°C, and stirred at room temperature for 2 h. The mixture was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, extracted with DCM, dried over anhydrous sodium sulfate, and the filtrate was collected by suction filtration and concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (DCM: MeOH = 25: 1) to obtain a colorless oil I-10 (1.05 g, yield: 68%).

[0080] 1 H NMR (600MHz, CDCl3-d) δ7.74 (dd, J=54.3, 8.3Hz, 2H), 6.89 (d, J=8.5Hz, 2H), 6.37 (t, J= 6.6Hz,1H),4.06(d,J=2.9Hz,2H),3.98–3.89(m,1H),3.75–3.62(m,2H),3.60–3.55(m, 4H),3.40(d,J=15.8Hz,1H),3.34(d,J=6.7Hz,2H),3.26(t,J=6.1Hz,5H),3.14–3.09(m ,1H),3.01(dt,J=11.5,5.4Hz,1H),2.57–2.32(m,2H),1.47(s,9H),1.08–0.95(m,6H).

[0081] Intermediate I-11 is: N-(3-(2-chloro-N-(1,1-dioxidotetrahydrothiophene-3-yl)acetamido)-2,2-dimethylpropyl)-4-(piperazine-1-yl)benzamide, and the synthesis method is as follows:

[0082] Intermediate I-10 (0.44 g, 0.75 mmol) was dissolved in ultra-dry DCM (4 mL), TFA (0.8 mL, 20% v / v) was slowly added dropwise at 0°C, and stirred at room temperature for 4 h. The solvent was removed by rotary evaporation to obtain crude I-11, which was used directly in the next step without further purification.

[0083] Compound A32 is 4-(4-((3-(2-chloro-N-(1,1-dioxidotetrahydrothiophen-3-yl)acetylamino)-2,2-dimethylpropyl)carbamoyl)phenyl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)piperazine-1-carboxamide, and the synthesis method is as follows:

[0084] The intermediate I-11 (0.12 g, 0.27 mmol, 1.0 equiv) was dissolved in ultra-dry DCM (4 mL), and DIPEA (0.37 mL, 2.14 mmol, 8.0 equiv) and BTC (24 mg, 0.08 mmol, 0.3 euqiv) were added. The reaction was stirred at room temperature for 3 h. After the intermediate I-10 was completely consumed, (S, R, S)-AHPC-Me hydrochloride (0.10 g, 0.21 mmol, 0.8 euqiv) was added. The reaction was stirred at room temperature overnight, and the mixture was washed with water, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, and the filtrate was collected by suction and concentrated. The crude product was purified by silica gel column chromatography (DCM: MeOH = 25: 1) to give a white solid A32 (50 mg, yield: 24%).

[0085] 1 H NMR(600MHz, CDCl3-d)δ8.66(s,1H),7.67(d,J=8.4Hz,2H),7.57(d,J=7.9Hz,1H),7.39–7.34(m,5H),6.79(d,J=8.6Hz,2H),6.64–6.60(m ,1H),5.16(d,J=8.8Hz,1H),5.09–5.06(m,1H),4.70(t,J=8.1Hz,1H),4.45(d,J=4.1Hz,1H),4.40(d,J=8.9Hz,1H),4.11(d,J=11.5Hz,1H ),4.08–4.03(m,2H),3.94(p,J=8.4Hz,1H),3.71–3.61(m,2H),3.57(dd,J=11.4,3.6Hz,1H),3.53–3.44(m,5H),3.39(dd,J=16.1,2.6Hz, 1H),3.33(q,J=7.1Hz,2H),3.30–3.23(m,6H),2.48(s,3H),2.40(dt,J=10.3,4.9Hz,4H),1.47(d,J=6.9Hz,3H),1.04(d,J=10.0Hz,15H).13 C NMR (151MHz, CDCl3-d) δ173.23,169.96,167.95,167.88,157.86,152.95, 150.49,148.54,143.38,131.65,130.94,129.62,128.67,126.57,114.35, 70.07,59.12,58.94,58.58,57.70,56.65,50.51,49.20,48.91,48.59,47. 29,43.33,42.30,38.45,35.97,35.09,26.73,26.65,24.13,22.43,16.17.

[0086] Western-Blot experiments were performed to determine the degradation effects of compounds A1, A12, A14, A15, and A32 on PIN1 protein:

[0087] S1. Cells were seeded in 6-well plates and cultured to about 70% confluence, and compounds / DMSO were added to treat the cells. The final DMSO concentration was 1%;

[0088] S2. After the treatment, the cells were collected and transferred to RIPA buffer containing proteasome inhibitors and phosphatase inhibitors for lysis and centrifugation, and the expression level of PIN1 protein in the supernatant was detected by Western-Blot experiment.

[0089] like Figure 1 As shown, it can be observed from the Western-Blot experiment that in the MCF-7 cell line, compared with the DMSO blank control group, compounds A1, A12, A14, A15, and A32 have a significant degradation effect on PIN1 protein. The optimal compound A1 has a degradation efficiency of 90% at a low concentration of 2.2 μM. It should be pointed out that the degradation ability of A12 is reduced at high concentrations, which is consistent with the typical "Hook effect" of PROTAC molecules (see relevant literature: Moreau K, et al. Br J Pharmacol 2020; 177: 1709–18)

[0090] like Figure 1 As shown, in the PATU-8988t cell line, compounds A1, A12, A14, and A15 have a degradation effect on PIN1 protein. A1, A12, and A15 can show a high degradation ability at a low concentration of 2.2 μM, but the overall degradation effect is lower than that of the MCF-7 cell experimental group, and A1 and A12 both show Hook effect at high concentrations.

[0091] like Figure 2As shown, in the MCF-7 cell line, the degradation efficiency of compound A1 in a series of concentration gradients was measured to obtain its DC 50 =269nM. By measuring the PIN1 protein content at different time points after treatment, the degradation reached the maximum at 12 hours, after which the cells showed a PIN1 protein replenishment effect.

[0092] Determination of the inhibition of cell proliferation by the combination of Compound A1:

[0093] S1. Seed the cells in a 96-well white flat-bottom plate at a density of 1000 cells per well and incubate them in a cell culture incubator overnight.

[0094] S2. The next day, when the cells grew to about 80% confluence, they were treated with specific compounds, with the highest concentration generally being 20 μM, and three-fold gradient dilutions were performed, with a total of 6 concentration gradients, and the final volume per well was 200 μL, followed by incubation for 72 hours. Cell viability was assessed using the CellTiter-Glo luminescence assay;

[0095] S3. Return the 96-well plate to room temperature, remove 100 μL of culture medium from each well, then add 40 μL CellTiter-Glo reagent to each well, place the plate on a shaker for 30 minutes, ensure sufficient mixing, and measure the chemiluminescence intensity using the EnSight microplate reader;

[0096] S4. Drug concentration-effect curves were drawn using GraphPad Prism 9.0.

[0097] As attached Figure 3 As shown, compound A1 has inhibitory activity against MCF-7 and PATU8988t cells, among which the inhibitory activity against MCF-7 is stronger. When used in combination with the CDK1 inhibitor RO3306, the inhibitory activity against cell proliferation has an additive effect.

[0098] Determination of the specificity of compound A1 in regulating PIN1 degradation in cells:

[0099] S1. Compound A1 was added to the culture medium to treat the experimental group cells, and DMSO was added to the culture medium to treat the control group cells, and then the cell samples were collected and lysed to obtain the whole protein;

[0100] S2, centrifuge to remove cell debris, collect the protein in the supernatant, and use the BCA method to determine the protein concentration to ensure that the protein concentration of all samples is consistent;

[0101] S3. Reduce and alkylate the protein of each sample, use trypsin to enzymatically digest the protein, and use TMT (Tandem Mass Tag) to label the peptides in different samples, ensuring that each sample uses a different label;

[0102] S4. The combined samples are separated by high performance liquid chromatography and segmented according to the different characteristics of the peptides; the separated peptides are analyzed by mass spectrometry to detect peptides with different TMT labels and determine their relative abundance;

[0103] S5. Use Proteome Discoverer mass spectrometry analysis software for quantitative analysis, compare the protein expression levels between the treatment group and the control group, and perform statistical analysis;

[0104] like Figure 4 As shown in the figure, through TMT quantitative proteomics research, it was found that after treatment with A1, the protein content of PIN1 protein was the most downregulated in the cells, and the difference was highly significant. This result shows that A1 has a high specificity for the degradation of PIN1.

[0105] Determination of the anti-tumor effect of compound A1 combined with other drugs in mice:

[0106] S1. Establishment of MCF-7 cell xenograft model, which is used to evaluate the effectiveness of anticancer drugs and study tumor biological characteristics in breast cancer research;

[0107] S2. Five-week-old BALB / c-Nude female mice were selected and 10 μg of estradiol was injected subcutaneously in the neck. The next day, each nude mouse was inoculated with 5×10 6 MCF-7EGFP sorted cells were injected subcutaneously into the right shoulder and back during inoculation;

[0108] S3. After about two weeks, the appearance of a mass was observed and the drug was administered every other day by intraperitoneal injection (the dosage of the compound A1 used in the present invention was 50 mg / kg, and the dosage of the combined drug RO3306 was 20 mg / kg), and estradiol was administered once a week;

[0109] S4. Regularly check and measure the size of the tumor by caliper or imaging methods, and record the weight and health status of the mice to evaluate the toxicity and side effects of the drug;

[0110] S5. Evaluate the anti-tumor effect of the drug by comparing the changes in tumor volume between the drug-treated group and the control group; record the survival curve of mice to evaluate the effect of the drug on the overall survival of mice; at the end of the experiment, collect tumor tissues and other related tissues for histological examination.

[0111] like Figure 5As shown, compared with the blank control group, the tumor growth of mice treated with compound A1 and its combination group was significantly inhibited after 20 days of the experiment. The efficacy of compound A1 and RO3306 is basically equivalent, and the effect of the combination of the two is stronger than that of single drug. The mice in the drug-treated group survived, had stable body weight, and behaved normally.

[0112] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A compound that specifically targets and degrades PIN1 protein, characterized in that: The general structural formula of the compound is shown in Formula I: in: X is any of -CH2-, -NH-, and -O-; Linker for C 3-12 Alkyl, C 3-12 Alkoxy, any structure of Formula II, wherein n represents any number between 1 and 4, A represents -N- or -CH-, and Cy represents an aromatic ring, an aromatic heterocyclic ring, or a saturated carbocyclic ring having 3 to 6 carbon atoms; E3L is any structure in Formula III; 2. A compound for specifically targeting and degrading PIN1 protein according to claim 1, characterized in that: X is -NH- or -O-.

3. A compound for specifically targeting and degrading PIN1 protein according to claim 1, characterized in that: The structural formula of the compound is any one of structures A1-A35:

4. A compound for specifically targeting and degrading PIN1 protein according to claim 3, characterized in that: The invention also comprises optical isomers of the compounds according to claims 1 to 3, and pharmaceutically acceptable salts thereof.

5. Use of a compound for specifically targeting and degrading PIN1 protein as claimed in any one of claims 1 to 4, characterized in that: The compound is used in combination with a specific drug to prepare a drug for treating tumor diseases.

6. The use of the compound for specifically targeting and degrading PIN1 protein according to claim 5, characterized in that: The tumor disease is one or more of breast cancer, pancreatic cancer, colon cancer, prostate cancer, lung cancer, liver cancer, acute myeloid leukemia, and multiple myeloma.

7. The use of the compound for specifically targeting and degrading PIN1 protein according to claim 5, characterized in that: The specific drug is at least one of RO3306, tamoxifen, toremifene, fulvestrant, abemaciclib, apellis, palbociclib, ribociclib, olaparib, beztifan, everolimus, thalidomide, lenalidomide, and pomadoamine.

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