7-oxabicyclo [2.2. 1] heptane-2, 3-dicarboxylic acid derivatives and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and discloses a 7-oxabicyclo [2.2. 1] heptane-2, 3-dicarboxylic acid derivative and an application of the 7-oxabicyclo [2.2. 1] heptane-2, 3-dicarboxylic acid derivative in preparation of a protein phosphatase 5 degradation agent. The compound can directly target PP5 and induce the PP5 to degrade, has the advantages of novel structure and excellent physical and chemical properties, and shows high selectivity to PP5. In various biological evaluations, the molecules all show remarkable anti-proliferative activity and degradation effect.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives and their application in the preparation of PP5 degrading agents. Background Technology

[0002] Protein phosphatase 5 (PP5) is a serine / threonine protein phosphatase primarily responsible for regulating protein dephosphorylation. Under physiological conditions, PP5 typically exists in an autoinhibitory conformation, and its activation depends on endogenous mechanisms, including a TPR-dependent activation pathway mediated by the molecular chaperone Hsp90 and a TPR-independent activation pathway mediated by casein kinase 1δ (CK1δ). PP5 is widely expressed in various mammalian tissues, particularly in brain tissue. Studies have shown that PP5 is overexpressed in various tumor cells; for example, its expression level is closely related to breast cancer progression, possibly promoting tumor cell proliferation by dephosphorylating endoplasmic reticulum-related proteins. PP5 is also highly expressed in gliomas, suggesting its potential as a therapeutic target for tumors.

[0003] Currently, research on serine / threonine phosphatase inhibitors mainly focuses on subtypes such as PP1 and PP2A, while highly selective inhibitors targeting PP5 are still relatively scarce. Existing PP5 inhibitors suffer from several key drawbacks: First, macrolides and toxic compounds (such as Nodularin R and Okadaic acid) are mostly pantothenate phosphatase inhibitors, exhibiting significant off-target effects and leading to severe toxic side effects; second, inhibitors targeting the TPR domain (such as Ro 90-7401) have weak activity and are difficult to efficiently inhibit PP5 function; third, the dual-target inhibitor LB-100, which has entered phase II clinical trials, has a low PP5 IC50 threshold. 50 =6.5 μM, HCT116 IC 50 =10.1 μM, causing cytotoxicity due to simultaneous inhibition of PP2A, and exhibiting poor stability and drug-likeness; furthermore, recent studies have also found that even catalytic domain inhibitors such as P053 or highly selective inhibitors such as DDO-4222 have low PP5 IC50 values. 50 =0.9 μM, HCT116IC 50While 26.5 μM achieves inhibitory activity at the enzyme level, its antiproliferative activity in various tumor cell types is generally higher than 20 µM, resulting in limited efficacy when administered alone. Currently, it is only suitable as a potentiator in combination therapy. Overall, existing PP5 inhibitors generally suffer from insufficient selectivity, significant toxicity, weak cell viability, or poor drug-likeness. As can be seen above, although some PP5 inhibitor regulation strategies have been proposed, such as developing them as potentiators in combination therapy, existing methods still have significant limitations, mainly manifested in insufficient single-drug activity, toxicity problems due to poor selectivity, and poor stability. Traditional inhibitors are mostly pan-phosphatase inhibitors, which have significant toxic side effects; while dual-target inhibitors entering clinical trials also face challenges in drug-likeness due to insufficient selectivity and physicochemical stability. Therefore, there is an urgent need to develop novel, efficient, and highly selective PP5-targeting regulation strategies to provide new avenues for anti-tumor therapy. It should be noted that the structural formula of the aforementioned dual-target inhibitor LB-100 is:

[0004]

[0005] Loss of PP5 function can trigger multiple cellular regulatory abnormalities, including inducing G0 / G1 phase cell cycle arrest, checkpoint dysfunction, and weakened DNA damage repair capabilities, particularly exacerbating genomic instability by interfering with ATM / ATR pathway activation. Simultaneously, PP5 knockout enhances pro-apoptotic effects, disrupts autophagy homeostasis, and induces metabolic disorders, all contributing to disease progression. In oncology, PP5 knockout significantly inhibits cancer cell proliferation; for example, in breast and prostate cancer models, it can induce cell cycle arrest and increase apoptosis rates, highlighting its potential as a therapeutic target.

[0006] In recent years, protein-targeted degradation technology has provided a new approach to overcome the limitations of traditional inhibitors. Unlike enzyme activity inhibition, degradative agents achieve complete clearance of target proteins through the ubiquitin-proteasome system, effectively overcoming the selectivity and toxicity problems caused by the conservation of catalytic sites, representing a significant upgrade in mechanism of action. This strategy has been successfully validated in other phosphatases such as SHP2, where it induces target protein degradation through allosteric regulation, significantly improving subtype selectivity and representing a breakthrough in overcoming the insufficient activity of existing inhibitors.

[0007] Based on the key biological functions of PP5 in tumors and the significant anti-proliferative effect of its knockout, developing PP5-targeted degradative agents is highly feasible. This strategy holds promise for obtaining therapeutic molecules with lower toxicity and longer-lasting effects, not only opening up new directions for PP5-targeted drug development but also providing a promising new means of precision intervention in tumor treatment. Summary of the Invention

[0008] Using the clinical phase II compound LB-100 as a lead compound, the inventors aimed to improve its target selectivity for PP5 and its inhibitory activity against tumor cells. They provided a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives with high selectivity, strong activity and PP5 degradation mechanism, and also provided its application in the preparation of protein phosphatase 5 degrading agents.

[0009] In a first aspect, the present invention provides a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, the structural formula of which is shown below:

[0010]

[0011] In the above structural formula, R1, R2, R3, R4 and R5 are each independently selected from any one of hydrogen atom, halogen, alkyl, hydroxyl, trifluoromethyl and acetyl groups.

[0012] This invention also discloses a method for preparing 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, the preparation method comprising the following preparation route:

[0013]

[0014] The preparation method includes the following steps: dissolving raw material I-1 in I-2 and reacting it at room temperature (25°C~32°C) for 12 hours to generate intermediate I-3 via a Diels-Alder reaction; dissolving intermediate I-3 in ethyl acetate and reacting it at room temperature (25°C~32°C) for 6 hours using 10% palladium on carbon as a catalyst to generate intermediate I-4 via a hydrogenation reduction reaction; and dissolving intermediate I-4 and a tetrahydroisoquinoline derivative in tetrahydrofuran to generate a compound as shown in Formula I via an amide condensation reaction. The preferred tetrahydroisoquinoline derivatives are 1,2,3,4-tetrahydroisoquinoline, 1-methyl-1,2,3,4-tetrahydroisoquinoline, 6-methyl-1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroisoquinoline-6-ol, 6-fluoro-1,2,3,4-tetrahydroisoquinoline, 6-chloro-1,2,3,4-tetrahydroisoquinoline, 6-bromo-1,2,3,4-tetrahydroisoquinoline, 6-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline, and 1-(1,2,3,4-tetrahydroisoquinoline-6-yl). Any one of ethane-1-one, 7-methyl-1,2,3,4-tetrahydroisoquinoline, 7-fluoro-1,2,3,4-tetrahydroisoquinoline, 7-chloro-1,2,3,4-tetrahydroisoquinoline, 7-bromo-1,2,3,4-tetrahydroisoquinoline, 7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline, 8-chloro-1,2,3,4-tetrahydroisoquinoline, 8-bromo-1,2,3,4-tetrahydroisoquinoline, 5-chloro-1,2,3,4-tetrahydroisoquinoline, and 5-bromo-1,2,3,4-tetrahydroisoquinoline.

[0015] As a preferred embodiment of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives described in the first aspect of this invention, with consideration of target selectivity for PP5 and inhibitory activity against tumor cells, while prioritizing the reduction of its potential toxicity, clinical candidate compound 12 was validated through structural biology, structure-activity relationship studies, druggability evaluation, and in vivo pharmacodynamic evaluation. The structural formula of compound 12 is as follows:

[0016] ;

[0017] Secondly, another object of the present invention is to provide the use of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives and their pharmaceutically acceptable salts in the preparation of PP5 degrading agents.

[0018] Abnormally high expression of PP5 is closely related to the progression of various diseases, including various cancers such as breast cancer, colon cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, prostate cancer, and osteosarcoma, as well as non-cancerous diseases such as obesity, cardiomyopathy, Alzheimer's disease, and sleep phase syndrome. Therefore, based on the second aspect mentioned above, the present invention also provides the use of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives in the preparation of drugs for the treatment or prevention of PP5-related diseases.

[0019] In another aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention, or pharmaceutically acceptable salts thereof, or optionally pharmaceutically acceptable carriers, diluents, excipients, mediators, or combinations thereof.

[0020] Definition of terminology:

[0021] As described in this invention, the compounds of this invention may optionally be substituted with one or more substituents, such as the general formula compounds of this invention, or the specific examples, subclasses, and classes of compounds included in this invention, as described in the embodiments. It should be understood that the terms "substituted or unsubstituted" and "optionally substituted" are used interchangeably. Generally, unless otherwise indicated, an optional substituent group may have one substituent substituted at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a particular group, the substituents may be substituted at the same or different positions.

[0022] The term "alkyl" as used in this invention includes a monovalent hydrocarbon group consisting of a saturated straight-chain or branched chain of 1 to 6 carbon atoms, such as methyl or ethyl in some of the embodiments.

[0023] The term "halogen" as used in this article refers to any one of the following groups: fluorine-F, chlorine-Cl, and bromine-Br.

[0024] The term "amino" as used in this article refers to the -NH2 group.

[0025] The term "hydroxyl group" as used in this article refers to the -OH group.

[0026] The term “trifluoromethyl” as used in this article refers to the -CF3 group.

[0027] The term "acetyl" as used in this article refers to Group.

[0028] Unless otherwise indicated, the structural formulas described in this invention include all isomers such as enantiomers, diastereomers, and geometric isomers (or conformational isomers); for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compounds of this invention, or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers), are within the scope of this invention.

[0029] In this invention, "pharmaceutically acceptable" means any substance that does not interfere with the bioactivity of the active ingredient and is non-toxic to the host to which it is given.

[0030] Unless otherwise stated, all tautomeristic forms of the compounds of this invention are included within the scope of this invention. Furthermore, unless otherwise stated, the structural formulas of the compounds described in this invention comprise enriched isotopes of one or more different atoms.

[0031] Technical Effects: This invention provides a novel class of compounds that can serve as PP5 degrading agents. Experimental results show that these compounds can directly target PP5 and induce its degradation, exhibiting novel structures and excellent physicochemical properties. The obtained active small molecules demonstrate good stability, safety, and drug development potential, while also showing high selectivity for PP5. In various biological evaluations, these molecules have shown significant antiproliferative activity and degradation effects, and are expected to be used to treat various cancers caused by PP5 overexpression, or as synergists in combination therapies to enhance the efficacy of existing treatments. Attached Figure Description

[0032] Figure 1A Activity data of HCT 116 cells after administration of compound 12 and the PP5 inhibitor LB-100;

[0033] Figure 1B Activity data of PP5 knockout HCT 116 cells after administration of compound 12;

[0034] Figure 2A Analysis of PP5 protein levels and grayscale values ​​in HCT116 cells after administration of different concentrations of compound 12;

[0035] Figure 2B DC for the degradation of PP5 by compound 12 50 ;

[0036] Figure 2C PP5 protein levels were measured at different time points after HCT116 cells were treated with compound 12.

[0037] Figure 3AChanges in the protein levels of PP5, PPARγ, GR, and pGR in HCT116 cells after administration of the PP5 inhibitor LB-100;

[0038] Figure 3B Changes in the protein levels of PP5, PPARγ, GR, and pGR in HCT116 cells after administration of compound 12; Figure 4A Changes in CDK4 / 6 and CDC37 protein levels in HCT116 cells after administration of the PP5 inhibitor LB-100;

[0039] Figure 4B Changes in CDK4 / 6 and CDC37 protein levels in HCT116 cells after administration of compound 12;

[0040] Figure 5 The results show the selective inhibition of PP1, PP2A, PP4, and PP6 by compound 12. Detailed Implementation

[0041] Unless otherwise specified, the raw materials may be obtained commercially, or prepared by methods known in the art, or prepared according to the methods described herein. The structure of the compounds was determined by nuclear magnetic resonance (NMR). 1 Confirmation was performed using 1H-NMR and / or mass spectrometry (MS). NMR measurements were performed using a Varian INOVA (300 MHz) or Bruker Advance (400 MHz) NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (Chloroform-d) as the solvent, and TMS as the internal standard. MS measurements were performed using a Waters-Q-Tof miniature mass spectrometer. Silica gel column chromatography was performed using 200-300 mesh silica gel from Qingdao Marine Chemical Plant.

[0042] Example 1: Preparation of (1S,2R,3S,4R)-3-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (1)

[0043] Preparation of (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (I-3): Maleic anhydride (200 mg, 2.04 mmol) was dissolved in furan (555 mg, 8.16 mmol), and stirred at room temperature (25 °C) for 12 h. After the reaction was completed, a solid was observed to precipitate in the reaction mixture. The mixture was filtered, and the filter cake was washed three times with methyl tert-butyl ether to obtain 312 mg of a white solid compound (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione, with a yield of 88.54%.

[0044] Preparation of (3aR,4S,7R,7aS)-hexahydro-4,7-epoxyisobenzofuran-1,3-dione (norcantharidin, I-4): (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (300 mg, 1.81 mmol) was dissolved in ethyl acetate solution, 10% by mass of 10% palladium on carbon (10 w%) was added, H2 was bubbled in, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the solution was concentrated to dryness under reduced pressure to give 291 mg of white solid norcantharidin, with a yield of 95.84%. 1 H NMR (300 MHz, Chloroform-d) δ 5.04 (t, J = 2.8 Hz, 2H), 3.18 (s, 2H), 1.90 (dh, J = 6.2,3.2 Hz, 2H), 1.65 (t, J = 6.8 Hz, 2H).

[0045] Preparation of compound (1S,2R,3S,4R)-3-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxaxabicyclo[2.2.1]heptane-2-carboxylic acid (1): Demethylcantharidin (200 mg, 1.19 mmol) was dissolved in tetrahydrofuran solution, and a tetrahydrofuran solution of 1,2,3,4-tetrahydroisoquinoline (158 mg, 1.19 mmol) was added dropwise. The mixture was stirred at room temperature for 6 h. After the reaction was completed, a solid was observed to precipitate in the reaction mixture. The mixture was filtered, and the filter cake was rinsed with tetrahydrofuran 3 times to obtain 301 mg of white solid compound, with a yield of 84.0%. 1 H NMR (300 MHz, DMSO-d6) δ 7.24–7.09 (m, 4H), 4.72–4.53 (m, 3H), 3.84–3.57 (m, 1H), 3.45 (ddd, J = 17.9, 8.8, 4.9 Hz, 1H), 3.27 (dd, J = 9.6, 3.5Hz, 1H), 3.09 (dd, J = 9.6, 3.4 Hz, 1H), 2.83 (q, J = 7.0, 5.7 Hz, 1H), 2.72(t, J = 5.9 Hz, 1H), 1.55 (d, J = 6.0 Hz, 4H). 13HRMS (ESI): calcd for C 17 H 19 NO4 [M + H] + , 302.1392; found,302.1381. Purity: 95.090% by HPLC (MeOH / H2O = 70:30, t R = 6.397 min).

[0046] Example 2: Preparation of 3-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (2)

[0047]

[0048] Example 2 follows the same preparation method as Example 1, except that in Example 2, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 1-methyl-1,2,3,4-tetrahydroisoquinoline.

[0049] Compound 2 is a white solid with a yield of 41.6%. 1 H NMR (300 MHz, DMSO-d6) δ 7.19 (tdd, J =12.9, 7.4, 3.7 Hz, 4H), 4.69 (t, J = 2.4 Hz, 2H), 4.29 (q, J = 6.7 Hz, 1H), 3.63 – 3.59 (m, 1H), 3.29 (dt, J = 11.6, 5.6 Hz, 1H), 3.10 (ddd, J = 12.6,7.7, 5.4 Hz, 1H), 2.99 – 2.81 (m, 2H), 1.78–1.74 (m, 1H), 1.57–1.48 (m, 4H),1.47 (d, J = 6.6 Hz, 3H). MS (ESI): calcd for C 18 H 21 NO4 [M + Na] +, 338.1369; found, 338.1686. Purity: 99.404% by HPLC (MeOH / H2O = 70:30, t R = 4.551 min).

[0050] Example 3: Preparation of 3-(6-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (3)

[0051]

[0052] Example 3 is prepared using the same method as Example 1, except that in Example 3, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 6-methyl-1,2,3,4-tetrahydroisoquinoline.

[0053] Compound 3 is a white solid with a yield of 72.1%. 1 H NMR (300 MHz, DMSO-d6) δ 7.00 (d, J =1.8 Hz, 2H), 6.97 (s, 1H), 4.70 (t, J = 2.4 Hz, 2H), 4.04 (s, 2H), 3.18 (t, J= 6.1 Hz, 2H), 2.80 (d, J = 11.4 Hz, 4H), 2.25 (s, 3H), 1.56 – 1.47 (m, 4H).MS (ESI): calcd for C 18 H 21 NO4 [M + Na] + , 338.1369; found, 338.1555. Purity:97.316% by HPLC (MeOH / H2O = 70:30, t R = 4.537 min).

[0054] Example 4: Preparation of 3-(6-hydroxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxocyclic[2.2.1]heptane-2-carboxylic acid (4)

[0055]

[0056] Example 4 is prepared using the same method as Example 1, except that in Example 4, compound 1,2,3,4-tetrahydroisoquinoline-6-ol is used instead of compound 1,2,3,4-tetrahydroisoquinoline.

[0057] Compound 4 is a white solid with a yield of 54.2%. 1 H NMR (300 MHz, DMSO-d6) δ 6.92 (dd, J =25.8, 8.1 Hz, 1H), 6.65–6.49 (m, 2H), 4.68 (t, J = 6.9 Hz, 2H), 3.83–3.57 (m,1H), 3.43 (td, J = MS (ESI):calcd for C 17 H 19 NO5 [M + H] + , 318.1341; found, 318.1461. Purity: 91.936% byHPLC (MeOH / H2O = 70:30, t R = 4.491 min).

[0058] Example 5: Preparation of 3-(6-fluoro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (5)

[0059]

[0060] Example 5 is prepared using the same method as Example 1, except that in Example 5, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 6-fluoro-1,2,3,4-tetrahydroisoquinoline.

[0061] Compound 5 is a white solid in 83.7% yield. ¹H NMR (300 MHz, DMSO-d6) δ 7.15 (dd, J = 9.3, 5.7 Hz, 1H), 7.01 (ddd, J = 9.3, 6.1, 2.8 Hz, 2H), 4.77–4.63 (m, 2H), 4.01 (s, 2H), 3.12 (t, J = 6.1 Hz, 2H), 2.82 (d, J = 7.3 Hz, 4H), 1.62–1.42 (m, 4H). MS (ESI): calcd for C 17 H 18 FNO4 [M + H] +, 320.1298; found, 319.8189.Purity: 91.565% by HPLC (MeOH / H2O = 70:30, t R = 4.565 min).

[0062] Example 6: Preparation of 3-(6-chloro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (6)

[0063]

[0064] Example 6 is prepared using the same method as Example 1, except that in Example 6, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 6-chloro-1,2,3,4-tetrahydroisoquinoline.

[0065] Compound 6 is a white solid with a yield of 78.7%. 1 H NMR (300 MHz, DMSO-d6) δ 7.30–7.09 (m,3H), 4.76–4.47 (m, 3H), 4.07–3.69 (m, 1H), 3.69–3.37 (m, 1H), 3.28–3.04 (m,2H), 2.94–2.66 (m, 3H), 1.68–1.40 (m, 4H). MS (ESI): calcd for C 17 H 18 35 ClNO4 [M+ H] + , 336.1002; found, 335.9540. Purity: 92.289% by HPLC (MeOH / H2O = 70:30,t R = 4.481 min).

[0066] Example 7: Preparation of 3-(6-bromo-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxobicyclo[2.2.1]heptane-2-carboxylic acid (7)

[0067]

[0068] Example 7 is prepared using the same method as Example 1, except that in Example 7, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 6-bromo-1,2,3,4-tetrahydroisoquinoline.

[0069] Compound 7 is a white solid with a yield of 76.6%.1 H NMR (300 MHz, DMSO-d6) δ 7.34 (d, J =8.9 Hz, 2H), 7.06 (d, J = 8.0 Hz, 1H), 4.68 (t, J = 2.4 Hz, 2H), 3.97 (s,2H), 3.09 (t, J = 6.0 Hz, 2H), 2.82 (d, J = 3.5 Hz, 4H), 1.51 (dt, J = 10.1,4.9 Hz, 4H). MS (ESI): calcd for C 17 H 18 79 BrNO4 [M + H] + , 380.0497; found,380.9802. Purity: 95.188% by HPLC (MeOH / H2O = 70:30, t R = 4.087 min).

[0070] Example 8: Preparation of 3-(6-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (8)

[0071]

[0072] Example 8 is prepared using the same method as Example 1, except that in Example 8, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 6-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline.

[0073] Compound 8 is a white solid with a yield of 71.8%. 1 H NMR (300 MHz, DMSO-d6) δ 7.53 (d, J =7.5 Hz, 1H), 7.39 (dt, J = 15.2, 7.5 Hz, 2H), 4.71–4.64 (m, 2H), 4.06 (s,2H), 3.06 (t, J = 6.0 Hz, 2H), 2.87 (d, J = 6.1 Hz, 4H), 1.51 (dt, J = 9.9,4.9 Hz, 4H). MS (ESI): calcd for C 18 H 18 F3NO4 [M + H] +, 392.1086; found,392.3185. Purity: 90.760% by HPLC (MeOH / H2O = 70:30, t R = 7.865 min).

[0074] Example 9: Preparation of 3-(6-acetyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (9)

[0075]

[0076] Example 9 is prepared using the same method as Example 1, except that in Example 9, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 1-(1,2,3,4-tetrahydroisoquinoline-6-yl)ethane-1-one.

[0077] Compound 9 is a white solid with a yield of 14.27%. (73.1%) 1 H NMR (300 MHz, DMSO-d6) δ 7.77–7.69 (m, 2H), 7.24 (d, J = 8.4 Hz, 1H), 4.68 (t, J = 2.4 Hz, 2H), 4.07 (s,2H), 3.13 (t, J = 6.0 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.82 (s, 2H), 2.55(s, 3H), 1.58–1.46 (m, 4H). MS (ESI): calcd for C 19 H 21 NO5 [M + H] + , 344.1498; found, 344.2289. Purity: 93.653% by HPLC (MeOH / H2O = 70:30, t R = 4.680 min).

[0078] Example 10: Preparation of 3-(7-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxocyclic[2.2.1]heptane-2-carboxylic acid (15)

[0079]

[0080] Example 10 was prepared using the same method as Example 1, except that in Example 10, compound 1,2,3,4-tetrahydroisoquinoline was replaced with compound 7-methyl-1,2,3,4-tetrahydroisoquinoline.

[0081] Compound 10 is a white solid with a yield of 67.8%. 1 H NMR (300 MHz, DMSO-d6) δ 7.09 – 6.87(m, 3H), 4.65 (dt, J = 17.1, 6.0 Hz, 3H), 3.87–3.69 (m, 1H), 3.62–3.37 (m,2H), 3.25 (dd, J = 9.6, HRMS (ESI): calcd for C 18 H 21 NO4 [M + Na] + , 338.1369; found, 338.1390.Purity: 95.979% by HPLC (MeOH / H2O = 70:30, t R = 3.657 min).

[0082] Example 11: Preparation of 3-(7-fluoro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (16)

[0083]

[0084] Example 11 is prepared using the same method as Example 1, except that in Example 11, compound 1,2,3,4-tetrahydroisoquinoline is replaced with compound 7-fluoro-1,2,3,4-tetrahydroisoquinoline.

[0085] Compound 11 is a white solid with a yield of 64.9%. 1H NMR (300 MHz, DMSO-d6) δ 7.17 (dd, J =8.4, 5.8 Hz, 1H), 7.00 (ddt, J = 12.6, 6.2, 2.7 Hz, 2H), 4.68 (t, J = 2.4 Hz,2H), 4.02 (s, 2H), 3.12 (t, J = 6.0 Hz, 2H), 2.80 (d, J = 7.6 Hz, 4H), 1.60–1.41 (m, 4H). MS (ESI): calcd for C 17 H 18 FNO4 [M + Na] + , 342.1118; found,341.8507. Purity: 95.070% by HPLC (MeOH / H2O = 70:30, t R = 4.856 min).

[0086] Example 12: Preparation of (1S,2R,3S,4R)-3-(7-chloro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxobicyclo[2.2.1]heptane-2-carboxylic acid (12)

[0087]

[0088] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 12, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 7-chloro-1,2,3,4-tetrahydroisoquinoline.

[0089] Compound 12 is a white solid in 79.5% yield. [α] 20 D = –13.0 (c = 0.5, CHCl3). 1H NMR (300 MHz, DMSO-d6) δ 7.27 (d, J = 23.5 Hz, 1H), 7.19 (d, J = 7.9 Hz, 2H), 4.77 – 4.63 (m, 3H), 3.81 (dt, J = 11.6, 5.2 Hz, 1H), 3.58 (q, J = 6.5 Hz, 1H), 3.50 – 3.39 (m, 1H), 3.24 (t, J = 8.7 Hz, 1H), 3.10 (t, J = 4.9 Hz, 1H), 2.82 (q, J = 6.4, 5.2 Hz, 1H), 2.69 (t, J = 6.0 Hz, 1H), 1.68 – 1.45 (m, 4H). 13 C NMR (75 MHz, DMSO-d6) δ 172.73, 170.51, 136.34, 134.45, 134.09, 130.99,130.71, 126.65, 126.36, 78.49, 78.05, 53.09, 49.32, 44.30, 43.02, 29.38,28.68, 27.71. HRMS (ESI): calcd for C 17 H 18 35 ClNO4 [M + H] + , 336.1002; found,336.0986. Purity: 99.089% by HPLC (MeOH / H2O = 70:30, t R = 4.540 min).

[0090] Example 13: Preparation of 3-(7-bromo-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (13)

[0091]

[0092] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 13, the compound bromo-1,2,3,4-tetrahydroisoquinoline is replaced with the compound 7-bromo-1,2,3,4-tetrahydroisoquinoline.

[0093] Compound 13 is a white solid with a yield of 74.2%. 1H NMR (300 MHz, DMSO-d6) δ 7.48 – 7.32(m, 2H), 7.12 (t, J = 8.4 Hz, 1H), 4.80–4.57 (m, 4H), 3.81 (dt, J = 11.4, 5.3Hz, 1H), 3.58 (q, J = 6.4 Hz, 1H), 3.24 (t, J = 9.3 Hz, 1H), 3.10 (dd, J =9.6, 6.3 Hz, 1H), 2.79 (d, J = 5.4 Hz, 1H), 2.67 (t, J = 6.0 Hz, 1H), 1.59–1.50 (m, 4H). HRMS (ESI): calcd for C 17 H 18 79 BrNO4 [M + H] + , 380.0497; found,380.0490. Purity: 94.858% by HPLC (MeOH / H2O = 70:30, t R = 4.410 min).

[0094] Example 14: Preparation of 3-(7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (14)

[0095]

[0096] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 14, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline.

[0097] Compound 14 is a white solid with a yield of 67.2%. 1H NMR (300 MHz, DMSO-d6) δ 7.58 (d, J =23.7 Hz, 1H), 7.49 (s, 1H), 7.37 (q, J = 8.0 Hz, 1H), 4.87–4.60 (m, 3H),4.10–3.43 (m, 2H), 3.26 (t, J = 8.8 Hz, 1H), 3.16–3.05 (m, 1H), 2.85 (q, J =13.2 Hz, 3H), 1.52 (dt, J = 14.9, 7.4 Hz, 4H). HRMS (ESI): calcd forC 18 H 18 F3NO4 [M + H] + , 370.1266; found, 370.1262. Purity: 97.975% by HPLC (MeOH / H2O = 70:30, t R = 7.711 min).

[0098] Example 15: Preparation of 3-(8-chloro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (15)

[0099]

[0100] The preparation method of Example 1 is the same as that of Example 1, except that in Example 15, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 8-chloro-1,2,3,4-tetrahydroisoquinoline.

[0101] Compound 15 is a white solid with a yield of 65.8%. 1 H NMR (300 MHz, DMSO-d6) δ 7.31 (d, J =7.5 Hz, 1H), 7.20 (dt, J = 10.6, 8.1 Hz, 2H), 4.75 – 4.19 (m, 4H), 3.92 –3.67 (m, 1H), 3.46 (dq, J = MS (ESI): calcd for C 17 H18 35 ClNO4 [M + Na] + , 358.0822; found, 357.9175.Purity: 98.098% by HPLC (MeOH / H2O = 70:30, t R = 3.780 min).

[0102] Example 16: Preparation of 3-(8-bromo-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxocyclic[2.2.1]heptane-2-carboxylic acid (16)

[0103]

[0104] The preparation method of Example 1 is the same as that of Example 1, except that in Example 16, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 8-bromo-1,2,3,4-tetrahydroisoquinoline.

[0105] Compound 16 is a white solid with a yield of 75.3%. 1 H NMR (300 MHz, DMSO-d6) δ 7.47 (d, J =7.7 Hz, 1H), 7.17 (dt, J = 18.9, 7.7 Hz, 2H), 4.73–4.67 (m, 2H), 4.67–4.15(m, 2H), 3.92–3.78 (m, 1H), 3.44 (ddd, J = 13.3, 7.7, 4.9 Hz, 1H), 3.29 (d, J= 9.5 Hz, 1H), 3.14–3.03 (m, 1H), 2.94–2.72 (m, 2H), 1.63–1.46 (m, 4H). MS(ESI): calcd for C 17 H 18 79 BrNO4 [M + H] + , 380.0497; found, 380.9807. Purity:98.733% by HPLC (MeOH / H2O = 70:30, t R = 4.945 min).

[0106] Example 17: Preparation of 3-(5-chloro-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (17)

[0107]

[0108] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 17, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 5-chloro-1,2,3,4-tetrahydroisoquinoline.

[0109] Compound 17 is a white solid with a yield of 70.9%. 1 H NMR (300 MHz, DMSO-d6) δ 8.12 – 7.93(m, 2H), 7.37 (dd, J = 8.4, 5.5 Hz, 1H), 4.74–4.62 (m, 2H), 4.20 – 3.89 (m,2H), 3.11 (dt, J = 23.1, 5.8 Hz, 2H), 3.01–2.69 (m, 4H), 1.76 – 1.39 (m, 4H).MS (ESI): calcd for C 17 H 18 35 ClNO4 [M + Na] + , 358.0822; found, 357.9131. Purity:91.849% by HPLC (MeOH / H2O = 70:30, t R = 3.519 min).

[0110] Example 18: Preparation of 3-(5-bromo-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (18)

[0111]

[0112] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 18, compound 1,2,3,4-tetrahydroisoquinoline is replaced by compound 5-bromo-1,2,3,4-tetrahydroisoquinoline.

[0113] Compound 18 is a white solid with a yield of 53.7%. 1H NMR (300 MHz, DMSO-d6) δ 7.48 (dd, J =5.6, 3.5 Hz, 1H), 7.13 (q, J = 3.1, 2.1 Hz, 2H), 4.80–4.59 (m, 2H), 3.98 (s,2H), 3.12 (d, J = 7.1 Hz, 2H), 2.83 (s, 2H), 2.70 (d, J = 7.5 Hz, 2H), 1.51 (dt, J = 10.0, 5.0 Hz, 4H). MS (ESI): calcd for C 17 H 18 79 BrNO4 [M + H] + ,380.0497; found, 381.0195. Purity: 98.136% by HPLC (MeOH / H2O = 70:30, t R =4.854 min)

[0114] Test Example 1: PP5 Degradation Activity Experiment (Western blot method)

[0115] Western blots were used to determine the degradation rate of PP5 in HCT 116 cells by LB-100 and the products prepared in Examples 1-18 (i.e., compounds 1-18) at two different concentrations. LB-100 was purchased from AbMole, CAS number 1632032-53-1. HCT 116 cells are a human colon cancer cell line. The HCT 116 cells used in Example 1 were purchased from the Cell Bank of the Chinese Academy of Sciences, model CBP60028. The two different compound concentrations were 20 μM and 50 μM. PP5 degradation rate = 100 - PP5 expression level.

[0116] The Western blot assay was performed according to standard experimental procedures. The specific experimental process included cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The degradation rates of PP5 by the different compounds tested at two different concentrations are shown in Table 1.

[0117] Table 1. Compound activity against PP5 degradation

[0118] Entry Compound name or number Degradation rate of PP5 at a compound concentration of 20 μM (%) Degradation rate of PP5 at a compound concentration of 50 μM (%) Comparative Example 1 LB-100 0 0 Example 1 1 0 53 Example 2 2 28 54 Example 3 3 21 33 Example 4 4 23 51 Example 5 5 5 16 Example 6 6 24 16 Example 7 7 3 36 Example 8 8 12 26 Example 9 9 3 17 Example 10 10 4 38 Example 11 11 7 42 Example 12 12 36 66 Example 13 13 16 45 Example 14 14 6 17 Example 15 15 14 19 Example 16 16 17 22 Example 17 17 6 20 Example 18 18 10 15

[0119] Test Example 2: Cell Anti-proliferation Assay (CCK-8 assay)

[0120] The antiproliferative activity of the compounds against different tumor cell lines was determined using the CCK-8 assay. HCT116 cells and HCT116 PP5 KO cells (HCT116 cells purchased from the Cell Bank of the Chinese Academy of Sciences, model CBP60028; HCT116 PP5 KO cells are a knockout cell line of the HCT116 cell line with the PP5 gene removed, purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.) were seeded in two 96-well plates, with 3000 cells per well and incubated for 24 h. Then, HCT116 cells and HCT116 PP5 KO cells were treated with serially diluted compounds or DMSO for 48 h, respectively. The supernatant was then removed, and 100 µl of 10% CCK-8 solution was added, incubated at 37°C. o Incubate at C for 4 h. Absorbance (OD) values ​​were measured at 450 nm using an Elx800 microplate reader (BioTek, Vermont, USA). The inhibitory rate of the compound on cell viability at each concentration was calculated and analyzed using Graphpad Prism software.

[0121] like Figure 1A As shown, compound 12 exhibited strong anti-proliferative activity in HCT 116 cells, with an IC50 value of [missing value]. 50 =6.9 µM, stronger than the previously reported IC50 value of the PP5 inhibitor LB-100 for HCT 116 activity assay. 50 = 25.6 µM; indicating that compound 12 has certain inhibitory activity in colorectal cancer. Meanwhile, as Figure 1B As shown, compound 12 completely lost its antiproliferative activity in PP5 knockout HCT 116 cells (IC5 knockout). 50 (>200 µM), which also indicates that the antiproliferative effect of compound 12 on HCT 116 cells depends on the degradation of PP5.

[0122] Test Example 3: Western blot analysis of the time- and dose-dependent effects of PP5 degrading agents on intracellular PP5.

[0123] like Figure 2A The test results shown indicate that compound 12 can reduce the intracellular PP5 protein expression level in a significant concentration-dependent manner. Figure 2B As shown, quantitative calculations based on grayscale analysis revealed that compound 12 affects the DC degradation of PP5. 50 The value is 6.9 µM. Meanwhile, as... Figure 2CAs shown, the expression level of PP5 protein gradually decreased with prolonged treatment time, reaching a degradation efficiency of over 50% at 8 h. The experimental results fully demonstrate that compound 12 possesses significant PP5 degradation activity, exhibiting good concentration- and time-dependent characteristics.

[0124] The Western blot assay was performed according to standard experimental procedures. The specific procedures included cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gels, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The expression level of PP5 was measured in this experiment.

[0125] Test Example 4: Western blots to test the effect of PP5 degrading agents on intracellular PP5 substrate proteins.

[0126] The effects of compound 12 of this invention on PP5, GR, pGR, and PPARγ are as follows: Figure 3B As shown, the test results indicate that compound 12 downregulates PP5, PPARγ, GR, and pGR in a concentration-dependent manner, while... Figure 3A As shown, the PP5 inhibitor LB-100 upregulated pGR in a concentration-dependent manner, but had no significant effect on PP5, PPARγ, and GR, indicating that compound 12 inhibits tumor growth through a biological mechanism different from that of the PP5 inhibitor LB-100.

[0127] The effects of compound 12 of this invention on CDC37 and its substrate CDK4 / 6 are as follows: Figure 4B As shown, the test results indicate that compound 12 downregulates CDC37 and its substrate CDK4 / 6 in a concentration-dependent manner, while Figure 4A As shown, the PP5 inhibitor LB-100 has no significant effect on CDC37 and its substrates CDK4 / 6.

[0128] The Western blot assay was performed according to the standard experimental procedure. The specific experimental process included cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The expression levels of PP5, PPARγ, GR, pGR, CDC37, and CDK4 / 6 were measured in the experiment.

[0129] Test Example 5: Western blots to test the effect of PP5 degraders on intracellular protein phosphatase family members

[0130] The effects of compound 12 of this invention on other members of the protein phosphatase family are as follows: Figure 5As shown, the test results indicate that compound 12 has no significant effect on PP1, PP2A, PP4, and PP6, indicating that compound 12 has no degradation effect on other members of the protein phosphatase family to which PP5 belongs, and that compound 12 is selective for PP5.

[0131] The procedure for Western blot analysis is as follows: The procedure follows the standard experimental protocol. The specific steps include cell drug administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation, and result scanning. The expression levels of PP1, PP2A, PP4, and PP6 were measured in this experiment.

Claims

1. A class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, characterized in that, The structural formula of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative is shown below: ； In the above structural formula, R1, R2, R3, R4 and R5 are each independently selected from any one of hydrogen atom, halogen, alkyl, hydroxyl, trifluoromethyl and acetyl groups.

2. The method for preparing a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives as described in claim 1, characterized in that, The preparation method includes the following preparation path: ； The preparation method includes the following steps: dissolving raw material I-1 in I-2 and reacting it at room temperature (25°C~32°C) for 12 hours to prepare intermediate I-3 via a Diels-Alder reaction; dissolving intermediate I-3 in ethyl acetate and reacting it at room temperature (25°C~32°C) for 6 hours using 10% palladium on carbon as a catalyst to generate intermediate I-4 via a hydrogenation reduction reaction; and dissolving intermediate I-4 and a tetrahydroisoquinoline derivative in tetrahydrofuran to generate a compound as shown in Formula I via an amide condensation reaction.

3. The 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative as described in claim 1, characterized in that, The structural formula of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative is shown below: 。 4. The use of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives of claim 1 and their pharmaceutically acceptable salts in the preparation of PP5 degrading agents.

5. Use of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative of claim 1 and its pharmaceutically acceptable salts in the preparation of medicaments for the treatment or prevention of PP5-related diseases.

6. The use as described in claim 5, characterized in that, The PP5-related diseases are any one of the following: breast cancer, colon cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, prostate cancer, osteosarcoma, as well as obesity, cardiomyopathy, Alzheimer's disease, and sleep phase syndrome.

7. A pharmaceutical composition comprising the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative of claim 1, or a pharmaceutically acceptable salt thereof, or optionally a pharmaceutically acceptable carrier, diluent, excipient, medium, or combination thereof.

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