7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative, preparation method therefor, and use thereof

Abstract

A 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative, a preparation method therefor, and the use thereof in the preparation of a protein phosphatase 5 (PP5) inhibitor. The 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative has good physicochemical properties, and is an active small molecule with good stability, safety, and druggability.

Description

7-Ozabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and specifically relates to a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, their preparation methods, and their application in the preparation of protein phosphatase 5 (PP5) inhibitors. Background Technology

[0002] Protein phosphatase 5 (PP5) is a serine / threonine protein phosphatase that primarily regulates protein dephosphorylation. Under normal physiological conditions, PP5 is usually in a state of autoinhibition. To overcome this autoinhibition, endogenous PP5 activation relies on two different pathways: TPR-dependent activation mediated by endogenous activators (such as Hsp90) and TPR-independent activation mediated by casein kinase 1δ (CK1δ), which puts PP5 in an excited state. Currently, most reported PP5 inhibitors are pan-inhibitors of serine / threonine phosphatases, such as macrolides like Nodularin R and Microcystin-LR, and cytotoxins like Okadaic acid, Fostriecin, and Cantharidin. However, these pan-inhibitors have not been further investigated due to their severe toxicity.

[0003] In addition, see the published literature D'Arcy BM, Swingle MR, Papke CM, et al. The Antitumor Drug LB-100 is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating with the Catalytic Metals in the Active Site of PP5C[J]. Molecular Cancer Therapeutics, 2019. The published LB-100 belongs to a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives. LB-100 has entered phase II clinical trials as a dual-target inhibitor of PP2A / 5 for the purpose of enhancing chemotherapy and immunotherapy drugs. However, due to the potential cytotoxicity caused by its inhibition of PP2A and the poor drug-like properties resulting from its poor stability, it is worth further research and solutions. The structural formula of LB-100 is as follows:

[0004] PP5 is expressed in all mammalian tissues, exhibiting extremely high levels in the brain, and overexpression has been found to varying degrees in many different cancer cells. For example, PP5 is closely related to the development of breast cancer; its overexpression may induce endoplasmic reticulum-independent proliferation and promote breast cancer cell development through endoplasmic reticulum dephosphorylation. PP5 is highly expressed in gliomas, so inhibiting PP5 to treat gliomas may be a potential research direction. Therefore, designing rational and effective PP5 inhibitors can reduce PP5 expression levels in tumor cells, thereby inhibiting the growth and proliferation of PP5-overexpressing tumor cells and achieving the goal of cancer treatment. Furthermore, combining PP5 inhibitors with marketed drugs or other investigational molecules to improve efficacy or overcome drug resistance is also a very promising design approach.

[0005] Summary of the Invention

[0006] To further improve the chemical structural stability of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives in the prior art, in a first aspect, the present invention provides a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives that are different from the existing disclosed structures. The structural formulas of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives of the present invention are shown in the following structural formulas I, II, or III.

[0007] Structural formula I is:

[0008] In structural formula I, R1 is selected from aryl or aldehyde groups;

[0009] Structural formula II is:

[0010] Structure III is:

[0011] In structural formula III, R2, R3, and R4 are independently selected from any one of hydrogen atom, halogen, alkyl, alkoxy, benzyloxy, piperazine, 2,2-difluoroethoxy, ethylenedioxy, nitro, amino, hydroxyl, cyano, aldehyde, and aryl.

[0012] Furthermore, structural formula III is preferably any one of the following structural formulas:

[0013] Current research on serine threonine phosphatase inhibitors mainly focuses on other subtypes, such as PP1 / 2A, with very little research on PP5 inhibitors. Besides the aforementioned stability issues, existing PP5 inhibitors also have the following problems: (1) Macrolide and toxin inhibitors, as pan-inhibitors of serine threonine phosphatase, are prone to off-target effects, leading to a series of toxic side effects. (2) The TPR domain inhibitor Ro90-7501 of PP5 has very weak inhibitory activity, making it difficult to efficiently inhibit PP5. Therefore, highly selective, potent, and druggable PP5 inhibitors can target PP5 without causing toxicity problems due to inhibition of other subtypes. They can also improve druggability as clinical candidates to treat cancers caused by PP5 overexpression. Furthermore, the biological mechanism of PP5 can act as an synergist to overcome or reverse resistance to marketed drugs, thereby further treating related cancers, showing great promise for clinical research.

[0014] Therefore, among the above-mentioned 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, further preferred options are... Designated as compound 4, compound 4 exhibits target selectivity for PP5 and is non-biotoxic. Through structural biology, structure-activity relationship studies, drugability evaluation, and in vivo pharmacodynamics, compound 4 has been verified to have the advantages of high selectivity, strong activity, and good drugability, and can be used as a clinical candidate drug for PP5 inhibitors.

[0015] Secondly, the present invention also provides a method for preparing 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, wherein,

[0016] The preparation method of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives of structural formula I and structural formula II includes the following steps: as shown in the chemical pathway below, raw material I-1 is dissolved in I-2 and undergoes a Diels-Alder reaction to generate intermediate I-3; I-3 is dissolved in ethyl acetate and undergoes a hydrogenation reduction reaction with 10% palladium on carbon as a catalyst to generate intermediate I-4; I-4 and different amines are reacted at room temperature for 6 h in a tetrahydrofuran environment to undergo an amide condensation reaction to generate compounds of structural formula I or structural formula II;

[0017] The preparation method of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative of structure III includes the following steps: after dissolving the above intermediate I-4 in dichloromethane, different amines, 4-dimethylaminopyridine and triethylamine are added and reacted at 45°C for 6 h to generate the compound of structure III by amide condensation reaction.

[0018] In another aspect, the present invention also provides the application of the above-mentioned 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives in the preparation of PP5 inhibitors.

[0019] In another aspect, the present invention provides the use of the above-described 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives and their pharmaceutically acceptable salts in the preparation of medicaments for the treatment / prevention of PP5-related diseases. Furthermore, PP5-related diseases are cancers caused by phosphatase overexpression or cancers with reduced efficacy due to resistance to clinically investigated / marketed drugs.

[0020] 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, excipients, diluents, adjuvants, mediators, or combinations thereof.

[0021] Definition of terminology: As described in this invention, compounds of this invention may optionally be substituted with one or more substituents, such as compounds of the general formula of this invention, or specific examples, subclasses, and a class of compounds included in this invention, as described in the embodiments. 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 specific group, the substituents may be substituted at the same or different positions.

[0022] As used in this invention, the term "alkyl" includes a monovalent hydrocarbon group consisting of a saturated straight-chain or branched chain of 1 to 6 carbon atoms. Some examples include methyl and ethyl.

[0023] As used in this invention, the term "alkoxy" refers to an -OR substituent, wherein R includes C. 1～6 Alkyl groups. Some examples are methoxy or ethoxy groups.

[0024] As used in this invention, the term "aryl" refers to an unsaturated aromatic group having 6 carbon atoms. The aryl group may be unsubstituted or substituted by one or two substituents independently selected from the group consisting of: (1) C1-C6 alkyl; (2) halogen; (3) C1-C6 alkoxy; (4) amino; (5) nitro; (6) cyano; and (7) trifluoromethyl.

[0025] As used in this article, the term "halogen" refers to a group selected from fluorine (-F), chlorine (-Cl), and bromine (-Br).

[0026] The term "amino" as used in this article refers to the -NH2 group.

[0027] The term "nitro" as used in this article refers to the -NO2 group.

[0028] The term "cyano" as used in this article refers to the -CN group.

[0029] The term "aldehyde" as used in this article refers to the -CHO group.

[0030] The term "hydroxyl group" as used in this article refers to the -OH group.

[0031] The term "benzyloxy" as used in this article refers to Group.

[0032] The term "piperazinyl" as used in this article refers to Group.

[0033] The term "2,2-difluoroethoxy" as used in this article refers to Group.

[0034] The term "ethylenedioxy" as used in this article refers to Group.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Beneficial effects: This invention provides a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives. Experiments show that they directly inhibit PP5 and have good physicochemical properties. They are a class of small active molecules with good stability, safety and drug-likeness. Some of the compounds also have significant PP5 selectivity and have shown good activity in various experiments. They can be used to treat cancer caused by phosphatase overexpression or as a synergist in drug combination to exert their efficacy. Attached Figure Description

[0039] Figure 1 shows the activity data of positive compounds LB-100, 4 and TMZ administered alone and in combination in U87 MG cells;

[0040] Figure 2 shows the contents of p53, p21 and p27 in U87 MG cells after administration of LB-100 and compound 4 at different concentrations (concentrations of 0, 6.25, 12.5, 25, 50 and 100 μM, respectively).

[0041] Figure 3 shows the levels of p53, Cyclin D1, and MGMT in U87 MG cells after administration of LB-100, compound 4, and TMZ alone and in combination (LB-100 and compound 4 concentrations were 25 μM; TMZ concentration was 100 μM).

[0042] Figure 4 shows the changes in tumor volume after administration of LB-100, compound 4, and TMZ alone and in combination. Figure A shows a comparison of tumor size for all samples in each group, and Figure B shows the curves of tumor volume changes in each group after accurate measurement. Figure 5 shows the changes in body weight of nude mice after administration of LB-100, compound 4, and TMZ alone and in combination. Detailed Implementation

[0043] 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 (300MHz) NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (Chloroform-d) as the solvent, and TMS as an 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.

[0044] Example 1: Preparation of compound (1S,2R,3S,4R)-3-(4-phenylpiperazine-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (1)

[0045] Preparation of (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (I-3): Maleic anhydride (I-1) (200 mg, 2.04 mmol) was dissolved in furan (I-2) (555 mg, 8.16 mmol), and stirred at room temperature 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 the white solid compound (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione, with a yield of 88.54%.

[0046] 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 (300MHz, Chloroform-d) δ5.04(t,J=2.8Hz,2H),3.18(s,2H),1.90(dh,J=6.2,3.2Hz,2H),1.65(t,J=6.8Hz,2H).

[0047] Preparation of compound (1S,2R,3S,4R)-3-(4-phenylpiperazine-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (1): Demethylcantharidin (280 mg, 1.67 mmol) was dissolved in tetrahydrofuran solution, and 1-phenylpiperazine (270 mg, 1.67 mmol) in tetrahydrofuran solution was added dropwise. The mixture was stirred at room temperature for 6 h. After the reaction was completed, 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 420 mg of white solid compound, with a yield of 76.31%. 1 H NMR (300MHz, DMSO-d6) δ7.22(dd,J=8.7,7.2Hz,2H),6.95(d,J=8.1Hz,2H),6.80(t,J=7.2Hz,1H),4.67(d,J=3.6Hz,2H), 3.68(t,J=14.4Hz,2H),3.49-3.36(m,2H),3.26-3.12(m,3H),3.10-2.93(m,3H),1.54(d,J=6.3Hz,4H).HRMS(ESI):calcd for C 18 H 22 N₂O₄[M+H] + ,331.1658;found,331.1651.

[0048] Example 2: Preparation of (1S,2R,3S,4R)-3-(4-formylpiperazine-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (2)

[0049] The preparation method of Example 1 is the same as that of Example 1, except that in this Example 2, the compound 1-phenylpiperazine is replaced with the compound piperazine-1-formaldehyde.

[0050] Compound 2 is a white solid with a yield of 81.35%. 1 H NMR(300MHz,DMSO-d6)δ8.04(d,J=3.6Hz,1H),4.69-4.64(m,2H),3.64-3.36(m,6H),3.29-3.18(m,4H),1.57-1.47(m,4H).HRMS(ESI):calcd for C 13 H 18 N₂O₅[M+H] + ,283.1294;found,238.1302.

[0051] Example 3: Preparation of (1S,2R,3S,4R)-3-(4-oxo-1,4-dihydropyridine-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (3)

[0052] The preparation method of Example 1 is the same as that of Example 1, except that the compound 1-phenylpiperazine is replaced by the compound pyridine-4(1H)-one in Example 3.

[0053] Compound 3 is a white solid with a yield of 73.86%. 1 H NMR (300MHz, DMSO-d6) δ11.89(s,1H),7.70(d,J=6.7Hz,2H),6.17(d,J=6.8Hz,2H),4.81-4.58(m,2H),2.95(d,J=28.6Hz,2H),1.65-1.39(m,4H).

[0054] Example 4: Preparation of (1S,2R,3S,4R)-3-(1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (4)

[0055] The preparation method of norcantharidin (I-4) is as described in Example 1. Norcantharidin (400 mg, 2.38 mmol) was dissolved in dichloromethane, and 1H-indole (279 mg, 2.38 mmol), 4-dimethylaminopyridine (29 mg, 0.24 mmol), and triethylamine (722 mg, 7.14 mmol) were added. The mixture was stirred at 45 °C for 6 h. After the reaction was completed, the mixture was concentrated to dryness under reduced pressure, and the solution was prepared by column chromatography to obtain 231 mg of off-white solid, with a yield of 34.04%. 1H NMR (300MHz, DMSO-d6) δ11.07(s,1H),7.53(d,J=8.0Hz,1H),7.42-7.36(m,1H),7.32(t,J=2.8Hz,1H),7.07(dd,J=8.2, 6.9Hz,1H),6.97(td,J=7.4,7.0,1.2Hz,1H),6.45-6.37(m,1H),4.89-4.84(m,2H),3.40(s,2H),1.65(d,J=2.0Hz,4H). 13 C NMR(75MHz,DMSO-d6)δ173.03,125.28,120.96,120.09,118.85,111.48,101.05,79.73,50.81,27.56.HRMS(ESI):calcd for C 16 H 15 NO4[M+H] + ,286.1079; found,286.1073.

[0056] Example 5: Preparation of (1S,2R,3S,4R)-3-(5-bromo-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (5)

[0057] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 5, compound 1H-indole is replaced by compound 5-bromo-1H-indole.

[0058] Compound 5 is an off-white solid with a yield of 23.11%. 1 H NMR (300MHz, DMSO-d6) δ11.32(s,1H),7.72(d,J=2.0Hz,1H),7.41(t,J=2.8Hz,1H),7.36(d,J=8.6Hz,1H),7.18(dd,J=8.6 ,2.0Hz,1H),6.42(d,J=2.7Hz,1H),4.86(t,J=2.1Hz,2H),3.51(s,1H),3.38(s,1H),1.69-1.62(m,4H).HRMS(ESI):calcd for C 16 H 14 BrNO4[M+H] + ,364.0184; found,364.0190.

[0059] Example 6: Preparation of (1S,2R,3S,4R)-3-(5-methoxy-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (6)

[0060] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 6, compound 1H-indole is replaced by compound 5-methoxy-1H-indole.

[0061] Compound 6 is an off-white solid with a yield of 25.37%. 1 H NMR(300MHz,DMSO-d6)δ10.93(s,1H),7.30-7.24(m,2H),7.03(d,J=2.5Hz,1H),6.71(dd,J=8.7,2.5Hz,1H), 6.32(t,J=2.7Hz,1H),4.86(t,J=2.1Hz,2H),3.74(s,3H),3.38(s,2H),1.69-1.61(m,4H).HRMS(ESI):calcd for C 17 H 17 NO5[M+H] + ,316.1185; found,316.1188.

[0062] Example 7: Preparation of (1S,2R,3S,4R)-3-(5-fluoro-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (7)

[0063] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 7, compound 5-fluoro-1H-indole is used instead of compound 1H-indole.

[0064] Compound 7 is an off-white solid with a yield of 35.16%. 1 H NMR (300MHz, DMSO-d6) δ11.17(s,1H),7.43-7.34(m,2H),7.28(dd,J=10.1,2.6Hz,1H),6.91(td,J=9.2,2.5H z,1H),6.41(dd,J=3.0,2.0Hz,1H),4.86(t,J=2.0Hz,2H),3.40(s,2H),1.70-1.61(m,4H).HRMS(ESI):calcd for C 16 H 14 FNO4[M+H] + ,304.0985; found,304.0978.

[0065] Example 8: Preparation of (1S,2R,3S,4R)-3-(5,6-dimethoxy-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (8)

[0066] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 8, compound 5,6-dimethoxy-1H-indole is used instead of compound 1H-indole.

[0067] Compound 8 is an off-white solid with a yield of 23.87%. 1 H NMR(300MHz,DMSO-d6)δ10.73(s,1H),7.12(t,J=2.7Hz,1H),7.03(s,1H),6.91(s,1 H), 6.26 (t, J = 2.6Hz, 1H), 4.86 (t, 2H), 3.74 (s, 6H), 3.40 (s, 2H), 1.69-1.61 (m, 4H).

[0068] Example 9: Preparation of (1S,2R,3S,4R)-3-(6-methoxy-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (9)

[0069] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 9, compound 1H-indole is replaced by compound 6-methoxy-1H-indole.

[0070] Compound 9 is an off-white solid with a yield of 24.17%. 1 H NMR (300MHz, DMSO-d6) δ10.86(s,1H),7.39(d,J=8.6Hz,1H),7.17(t,J=2.7Hz,1H),6.88(d,J=2.3Hz,1H),6.64(dd,J=8.6 ,2.3Hz,1H),6.32(t,J=2.7Hz,1H),4.91-4.80(m,2H),3.75(s,3H),3.40(s,2H),1.65(d,J=2.0Hz,4H).HRMS(ESI):calcd for C 17 H 17 NO5[M+H] + ,316.1185; found,316.1188.

[0071] Example 10: Preparation of (1S,2R,3S,4R)-3-(5-(benzyloxy)-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (10)

[0072] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 10, compound 5-(benzyloxy)-1H-indole is used instead of compound 1H-indole.

[0073] Compound 10 is an off-white solid with a yield of 34.67%. 1 H NMR(300MHz,DMSO-d6)δ10.92(s,1H),7.40(dd,J=38.5,19.1Hz,7H),7.15-7.06(m,1H),6.87-6 .72(m,1H),6.37-6.25(m,1H),5.08(s,2H),4.92-4.80(m,2H),3.40(s,2H),1.69-1.61(m,4H).

[0074] Example 11: Preparation of (1S,2R,3S,4R)-3-(3-methyl-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (11)

[0075] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 11, compound 1H-indole is replaced by compound 3-methyl-1H-indole.

[0076] Compound 11 is an off-white solid with a yield of 14.27%. 1 H NMR (300MHz, DMSO-d6) δ8.37(d,J=7.5Hz,1H),8.07(s,1H),7.62-7.54(m,1H),7.34(t,J=6.0Hz,2H),5.08(t,J=5.2 Hz,1H),4.89(d,J=5.0Hz,1H),4.12(s,1H),3.31(d,J=4.6Hz,1H),2.27(s,3H),1.77-1.41(m,4H).HRMS(ESI):calcd for C 17 H 17 NO4[M+Na] + ,322.1055;found,322.1064.

[0077] Example 12: Preparation of (1S,2R,3S,4R)-3-(5-methyl-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (12)

[0078] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 12, compound 5-methyl-1H-indole is used instead of compound 1H-indole.

[0079] Compound 12 is an off-white solid with a yield of 18.49%. 1 H NMR (300MHz, DMSO-d6) δ12.71(s,1H),8.29-8.20(m,2H),7.42(s,1H),7.17(dd,J=8.5,1.8Hz,1H),6.74(d,J=3.7Hz,1H),5.05(t,J =5.1Hz,1H),4.90(d,J=5.0Hz,1H),4.16(t,J=5.0Hz,1H),3.32(d,J=4.6Hz,1H),2.40(s,3H),1.76-1.28(m,4H).HRMS(ESI):calcd for C 17 H 17 NO4[M+Na] + ,322.1055;found,322.1064.

[0080] Example 13: Preparation of (1S,2R,3S,4R)-3-(5-(piperazin-1-yl)-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (13)

[0081] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 13, compound 5-(piperazin-1-yl)-1H-indole is used instead of compound 1H-indole.

[0082] Compound 13 is an off-white solid with a yield of 14.25%. 1H NMR (300MHz, DMSO-d6) δ8.90(s,1H),8.21(d,J=9.0Hz,1H),7.86(d,J=3.8Hz,1H),7.12(d,J=2.4Hz,1H),7.05(dd,J=9.1,2.4Hz,1H),6.59(d, J=3.6Hz,1H),4.93(s,1H),4.74(s,1H),3.80(d,J=9.4Hz,1H),3.40(d, J=9.4Hz,1H),3.37-3.23(m,8H),1.77-1.59(m,4H),1.54-1.50(m,1H).

[0083] Example 14: Preparation of (1S,2R,3S,4R)-3-(5,6-dimethoxy-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (14)

[0084] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 14, compound 5,6-diethoxy-1H-indole is used instead of compound 1H-indole.

[0085] Compound 14 is an off-white solid with a yield of 28.73%. 1 H NMR (300MHz, DMSO-d6) δ10.72(s,1H),7.12(t,J=2.7Hz,1H),7.02(s,1H),6.90(s,1H),6.24(t,J=2.5H z,1H),4.86(t,J=2.0Hz,2H),3.99(q,J=6.8Hz,4H),3.40(s,2H),1.69-1.62(m,4H),1.37-1.30(m,6H).

[0086] Example 15: Preparation of (1S,2R,3S,4R)-3-(5-isopropoxy-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (15)

[0087] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 15, compound 1H-indole is replaced by compound 5-isopropoxy-1H-indole.

[0088] Compound 15 is an off-white solid with a yield of 35.20%. 1H NMR (300MHz, DMSO-d6) δ10.90(s,1H),7.31-7.24(m,2H),7.05(d,J=2.4Hz,1H),6.72(dd,J=8.7,2.4Hz,1H),6.35-6.29(m, 1H),4.93-4.85(m,2H),4.51(p,J=6.0Hz,1H),3.42(s,2H),1.67(d,J=2.1Hz,4H),1.27(d,J=6.0Hz,6H).HRMS(ESI):calcd for C 19 H 21 NO5[M+Na] + ,366.1317;found,316.1339.

[0089] Example 16: Preparation of (1S,2R,3S,4R)-3-(2,3-dihydro-6H-[1,4]dioxy[2,3-f]indole-6-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (16)

[0090] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 16, compound 1H-indole is replaced by compound 2,3-dihydro-6H-[1,4]dioxy[2,3-f]indole.

[0091] Compound 16 is an off-white solid with a yield of 15.59%. 1 H NMR (300MHz, DMSO-d6) δ10.65(s,1H),7.15(t,J=2.8Hz,1H),6.93(s,1H),6.81(s,1H),6.21( d,J=2.7Hz,1H),4.86(t,J=2.0Hz,2H),4.19(d,J=2.2Hz,4H),3.40(s,2H),1.66-1.62(m,4H).

[0092] Example 17: Preparation of (1S,2R,3S,4R)-3-(5-(2,2-difluoroethoxy)-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (17)

[0093] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 17, compound 5-(2,2-difluoroethoxy)-1H-indole is used instead of compound 1H-indole.

[0094] Compound 17 is an off-white solid with a yield of 26.68%.1 H NMR (300MHz, DMSO-d6) δ10.99(s,1H),7.30(dd,J=6.6,3.7Hz,2H),7.12(d,J=2.4Hz,1H),6.78(dd,J=8.7,2.5Hz,1H),6 .34(t,J=2.5Hz,1H),4.86(t,J=2.1Hz,2H),4.26(td,J=14.7,3.6Hz,2H),3.51(s,1H),3.40(s,2H),1.69-1.62(m,4H).

[0095] Example 18: Preparation of (1S,2R,3S,4R)-3-(5-(cyclopentoxy)-1H-indole-1-carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (18)

[0096] The preparation method of Example 4 is the same as that of Example 4, except that in this Example 18, compound 5-(cyclopentoxy)-1H-indole is used instead of compound 1H-indole.

[0097] Compound 18 is an off-white solid with a yield of 25.23%. 1 H NMR (300MHz, DMSO-d6) δ10.89(s,1H),7.28-7.22(m,2H),6.99(d,J=2.4Hz,1H),6.67(dd,J=8.7,2.4Hz,1H),6.30(t, J=2.5Hz,1H),4.86(t,J=2.1Hz,2H),4.76(tt,J=5.5,2.5Hz,1H),3.40(s,2H),1.91-1.81(m,2H),1.74-1.54(m,10H).

[0098] Test Example 1: Phosphatase Activity Assay (pNPP Method and Serine / Threonine Phosphatase Kit)

[0099] Table 1. Inhibition activity of compounds on the PP5 catalytic domain of PP5

[0100] As shown in Table 1, the compounds in the examples all exhibited inhibitory activity against the PP5 catalytic domain. The IC50 of the positive compound LB-100 was tested. 50 The concentration of 6.3 μM is essentially the same as that reported in previous literature, indicating that the PP5 enzyme activity assay method of this patent is applicable and that the compounds in the examples have strong PP5 enzyme activity.

[0101] Table 2. Inhibitory activities of LB-100 and compound 4 on the PP1 / 2A / 5 isoform catalytic domain.

[0102] In Table 2, a PP1 / 5 enzyme activity was determined using the pNPP method; b PP2A enzyme activity was determined using a serine / threonine phosphatase assay kit. c The cLogP value was determined using ChemDraw Professional 16.0 software.

[0103] Among them, the IC50 of compound PP1 / 5 was tested using the pNPP method. 50 The procedure is as follows: First, prepare 50 mL of buffer solution containing 100 mM MOPS, 0.5 mM MnCl2 (pH = 7.0), and dissolve the test compound (DMSO content less than 10%), PP1 catalytic domain truncated somatic protein (1-330) or PP5 catalytic domain truncated somatic protein (169-499) and pNPP substrate in this buffer solution. Using 96-well clear plates, three replicates were set up. In each well, 50 μL of PP1 protein solution (100 nM) or PP5 protein solution (30 nM), 50 μL of compound solution (maximum concentration 500 μM, three-fold dilution for a total of seven concentrations) were added, followed by 50 μL of substrate solution (5 mg / mL). In the positive control wells, 50 μL of PP1 or PP5 protein solution, 50 μL of the inhibitor LB-100 solution, and 50 μL of substrate solution were added. In the blank wells, 50 μL of PP1 or PP5 protein solution, 50 μL of buffer solution, and 50 μL of substrate solution were added. The 96-well plates were incubated at room temperature (25 °C) for 30 min. Data were collected using an Elx800 absorbance microplate reader (BioTek, Vermont, USA) at a wavelength of 405 nm. Results were analyzed using GravaPad software.

[0104] The IC50 of compound PP2A was tested using a serine / threonine phosphatase kit. 50The procedure was as follows: Cells were collected from 6-well plates (eight concentrations total, three-fold diluted with the compound) and lysed for 20 min in RIPA lysis buffer containing a protease inhibitor complex, vortexing every 10 min. Cell lysates were filtered through a Sephadex G25 column to remove free phosphate, and protein concentration was then determined using the BCA method. 5 μg of cell protein was evenly distributed into 96-well clear plates in PP2A-specific reaction buffer (250 mM imidazole (pH 7.2), 1 mM EGTA, 0.1% β-mercaptoethanol, 0.5 mg / ml BSA). The 96-well plates were incubated at room temperature (37°C) for 30 min, and 50 μl of a molybdate dye / additive mixture was added. After development for 20 min, data were collected using an Elx800 absorbance microplate reader (BioTek, Vermont, USA) at a wavelength of 600 nm. Results were analyzed using GravaPad software.

[0105] As shown in Table 2, compound 4 has an IC50 value for the PP1 / 2A catalytic domain of other serine threonine phosphatases. 50 The concentrations were 19.3 μM and 33.8 μM, respectively, compared to the inhibitory activity of LB-100 on the PP1 / 2A catalytic domain (PP1 / 2AIC). 50 =5.0 / 1.0μM), achieving certain selectivity for the PP5 catalytic domain and more reasonable lipophilicity (cLogP=2.4), making it easier to formulate into a drug.

[0106] Test Example 2: Stability Test by High Performance Liquid Chromatography (HPLC)

[0107] Table 3. Stability of compounds in water

[0108] In Table 3, a This represents data obtained by HPLC analysis after the compound was incubated with water for 4 hours.

[0109] As shown in Table 3, the structure of LB-100 is unstable in water, with only 45.6% remaining after 4 hours. Compounds 1-18 disclosed in Examples 1-18 all exhibited better water stability than LB-100, with compound 4 showing virtually no hydrolysis in water (99.8%). This indicates that the compounds in the embodiments of this patent remain stable in water.

[0110] The procedure for testing the stability of the compound using HPLC is as follows: The compound is dissolved in methanol to prepare a 10 mM stock solution. 20 μL of the stock solution and 180 μL of the solution are incubated at 37 °C. Samples are taken before incubation (0 h) and after incubation (4 h) for analysis using HPLC (Shimadzu LC-20AT). The formula for calculating the amount of the compound remaining in water is as follows: Remaining (%) = Peak area (compound incubated for 4 h) / Peak area (compound incubated for 0 h). A larger remaining amount indicates a more stable compound.

[0111] Test Example 3: Cell Anti-proliferation Assay (CCK-8 assay)

[0112] The antiproliferative activity of compounds LB-100, 4, and TMZ, alone or in combination, against the U87 MG tumor cell line was determined using the CCK-8 assay. U87 MG cells were seeded in 96-well plates at 3000 cells per well and incubated for 24 h. Cells were then treated with serial dilutions of the compounds or DMSO for 48 h. The supernatant was then removed, and 100 μl of 10% CCK8 solution was added, followed by incubation at 37 °C for 4 h. Absorbance (OD) values ​​were measured at 450 nm using an Elx800 microplate reader (BioTek, Vermont, USA). The inhibitory rates of cell viability at each concentration were calculated and analyzed using Graphpad Prism software.

[0113] As shown in Figure 1A, compound 4 exhibited weak antiproliferative activity (IC50) in U87 MG cells. 50 =70.1μM), lower than the activity of LB-100 (IC50). 50 =25.6 μM); as shown in Figure B of Figure 1, but the activity of compound 4 after co-administration with temozolomide (TMZ) was significantly lower than that of TMZ alone (IC50 = 25.6 μM); 50 The significant increase in concentration (>200 μM) indicates that compound 4 has certain inhibitory activity in glioblastoma and can reverse resistance to first-line drug TMZ, thereby further inhibiting the proliferation of U87 MG tumors.

[0114] Test Example 4: Western blots to test the effect of PP5 inhibitors on intracellular PP5 substrate proteins

[0115] The effects of compound 4 in Example 4 of this invention on p53, p21, and p27 are as follows:

[0116] In contrast, as shown in Figure 2A, the test results indicate that LB-100 downregulates p21, p27, and p53 in a concentration-dependent manner, while as shown in Figure 2B, the test results indicate that compound 4 upregulates p21, p27, and p53 in a concentration-dependent manner, suggesting that compound 4 inhibits tumor growth through a biological mechanism different from that of LB-100.

[0117] The combined effects of the compounds in Example 4 of this invention on p53, Cyclin D1, and MGMT are as follows:

[0118] As shown in Figure 3, the test results indicate that compound 4, in combination with TMZ, upregulated p53 and downregulated Cyclin D1, thereby affecting the cell cycle and inhibiting the proliferation of U87 MG tumor cells. Furthermore, the combination administration also downregulated the increase in MGMT expression induced by TMZ administration, reversing TMZ resistance.

[0119] The operation method for Western blot testing is as follows:

[0120] 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 levels of p21, p27, p53, Cyclin D1, and MGMT were measured in the experiment.

[0121] Test Example 5: Pharmacokinetic Study of Compound 4

[0122] The pharmacokinetic study of compound 4 was conducted in SD rats. Rats were randomly divided into two groups (n=3 per group). Compound 4 was administered intravenously (5 mg / kg) and orally (25 mg / kg), respectively. 150 μL of blood samples were collected at predetermined time points (0, 0.017, 0.133, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, and 24 h) and immediately centrifuged at 4°C and 3000 rpm for 10 min. Plasma samples were analyzed using LC-MS / MS (SHIMADZU LCMS-8050), and the results were analyzed using Phoenix software.

[0123] Table 4. Pharmacokinetic parameters of compound 4

[0124] As shown in Table 4, after intravenous administration of compound 4 at 5 mg / kg, the plasma C levels were... max The concentration was 8622.26 ng / mL, T max The AUC is 0.02h. (0-∞)The concentration was 13279.89 h*ng / mL, and T 1 / 2 For 2.41 hours, MRT (0-∞) The duration of action was 5.52 h, Vz was 1318.25 mL / kg, and CLz was 378.08 mL / h / kg; after oral administration of 25 mg / kg, the plasma C... max The concentration was 11266.51 ng / mL, T max The AUC is 1.17h. (0-∞) The concentration was 54426.56 h*ng / mL, T 1 / 2 For 3.94 hours, MRT (0-∞) The activity was 5.00 h, Vz was 2628.04 mL / kg, CLz was 461.70 mL / h / kg, and the bioavailability F could reach 82.0%.

[0125] Test Example 6: In vivo pharmacodynamic evaluation of compound 4

[0126] In vivo pharmacodynamic studies of compound 4 were conducted in Balb / c-nu nude mice. Mice were fed standard laboratory rodent diet and water for 3 days under standard conditions (12 / 12h light / dark cycle, 22±3℃, 40% relative humidity). Then, U87 MG cells (1*10⁻⁶ cells) were subcutaneously injected into the right side of the mice. 7 (Number of tumors). When the average tumor volume reaches approximately 150 mm... 3 Mice were randomly divided into 7 groups (including a control group, a single-drug group, and a combined-drug group, n=6 in each group). The mice were administered the drug via gavage every other day (the control group received saline) for 18 consecutive days. Tumor volume and the weight of the nude mice were measured every other day. The mice were considered dead at the end of the 18-day period or when the tumor volume reached 1800 mmHg. 3 Afterwards, the nude rats were euthanized.

[0127] As shown in Figures 4 and 5, the tumor inhibition effects of LB-100, compound 4, and TMZ alone were generally moderate. However, the tumor inhibition rate (TGI) of the combined administration of LB-100 and TMZ (LB-100, 3 mg / kg and TMZ, 40 mg / kg) reached 46.2%. The TGI of the low-dose combined administration of compound 4 and TMZ (4, 1.5 mg / kg and TMZ, 40 mg / kg) was superior to that of the combined administration of LB-100 (70.9%). The high-dose combined administration of compound 4 and TMZ (4, 3 mg / kg and TMZ, 40 mg / kg) showed the best tumor inhibition rate, reaching 87.7%. In nude mouse weight measurements, no significant weight loss was observed in either the single-administered or combined-administered groups compared to the control group, indicating that compound 4 was not toxic after gavage administration, either alone or in combination.

[0128] In the experiments of Test Examples 1 to 5 above, the data are all shown as the average of three replicates, where the Western blot results are representative. In the experiment of Test Example 6 above, the data are shown as six replicates. In the paired samples two-tailed t test, P < 0.05 is considered statistically significant (ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Claims

1. A class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives, characterized in that, The structural formulas of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives are shown in the following structural formulas I, II, or III; Structural formula I is: In structural formula I, R1 is selected from aryl or aldehyde groups; Structural formula II is: Structure III is: In structural formula III, R2, R3, and R4 are independently selected from any one of hydrogen atom, halogen, alkyl, alkoxy, benzyloxy, piperazine, 2,2-difluoroethoxy, ethylenedioxy, nitro, amino, hydroxyl, cyano, aldehyde, and aryl.

2. The 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative as described in claim 1, characterized in that, The structural formula III is any one of the following structural formulas:

3. 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 of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives of structural formulas I and II includes the following steps: as shown in the following chemical pathway, raw material I-1 is dissolved in I-2, and a Diels-Alder reaction occurs to generate intermediate I-3; I-3 is dissolved in ethyl acetate, and a hydrogenation reduction reaction occurs using 10% palladium on carbon as a catalyst to generate intermediate I-4; I-4 and different amines react at room temperature for 6 hours in a tetrahydrofuran environment to undergo amide condensation reaction to generate compounds of structural formula I or structural formula II. The preparation method of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative of structure III includes the following steps: after dissolving the above intermediate I-4 in dichloromethane, different amines, 4-dimethylaminopyridine and triethylamine are added and reacted at 45°C for 6 h to generate the compound of structure III by amide condensation reaction.

4. The application of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives of claim 1 in the preparation of PP5 inhibitors.

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

6. A pharmaceutical composition, characterized in that, It includes a class of 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivatives as described in claim 1, or pharmaceutically acceptable salts thereof, or optionally pharmaceutically acceptable carriers, excipients, diluents, adjuvants, mediators, or combinations thereof.

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