Protein tyrosine phosphatase inhibitor as well as pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of medical chemistry, and particularly discloses a protein tyrosine phosphatase inhibitor, such as type 2 non-receptor protein tyrosine phosphatase (PTPN2) and / or type 1 non-receptor protein tyrosine phosphatase (PTPN1) inhibitor, a pharmaceutical composition and application thereof. The invention discloses a method for treating related diseases such as cancers and metabolic diseases by using the protein tyrosine phosphatase inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a protein tyrosine phosphatase inhibitor or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, and uses thereof. Background Art

[0002] Protein tyrosine phosphatase non-receptor type 1 (PTPN1), also known as protein tyrosine phosphatase 1B (PTP1B), has been shown to play a key role in insulin and leptin signal transduction and is the main mechanism for downregulating the insulin and leptin receptor signal transduction pathways. Studies have found that animals lacking PTP1B have improved glucose regulation and lipid profiles and are resistant to weight gain when treated with a high-fat diet. Therefore, PTP1B inhibitors are expected to be used for the treatment of type 2 diabetes, obesity, and metabolic syndrome. Protein tyrosine phosphatase non-receptor type 2 (PTPN2), also known as T cell protein tyrosine phosphatase (TCPTP), is an intracellular member of a subclass of phospho-tyrosine specific phosphatases. The PTPN2 controls multiple cellular regulatory processes by removing phosphate groups from tyrosine substrates in the cytoplasm or nucleus. PTPN2 is ubiquitously expressed in cells, with the highest expression in hematopoietic stem cells and placental cells. In humans, PTPN2 expression is controlled post-transcriptionally by the presence of two splice variants: a 45 kDa form containing a nuclear localization signal at the C-terminus upstream of the splice junction, and a 48 kDa canonical form with a C-terminal ER retention motif. The two isoforms share an N-terminal phospho-tyrosine phosphatase catalytic domain, and the 45 kDa isoform can be passively imported into the cytosol under certain cellular stress conditions.

[0003] The catalytic domain of PTPN2 shares 74% sequence homology with PTPN1 and has similar enzyme kinetics. Protein tyrosine phosphatase PTPN2, as well as PTPN1, are both negative regulators of multiple cytokine signaling pathways and the T cell receptor (TCR) signaling pathway, and are key checkpoints for the inflammatory response. PTPN2 and PTPN1 (PTPN2 / N1) inhibit inflammation by removing the phosphorylation of JAK and STAT family members. PTPN2 is also a phosphatase of LCK and FYN downstream of TCR signaling, thus reducing the sensitivity of T cells to antigen recognition. Loss-of-function (LOF) single nucleotide polymorphisms at the PTPN2 locus are associated with several autoimmune diseases, which may be due to the central role of PTPN2 in inflammation regulation. Genetic ablation of PTPN2 or PTPN1 in tumor cells enhances the signal transduction of type I and type II IFNγ3, increasing downstream effects, including cell growth arrest, production of immunocyte chemotactic agents, and increased antigen presentation. In addition, the deletion of PTPN2 or PTPN1 enhances the expansion of T cells, the response to IL-2, and the ability to control tumors. Therefore, different from current treatment methods, PTPN2 / PTPN1 targeted therapy can achieve a dual anti-cancer mechanism by directly acting on tumor cells and increasing the anti-tumor activity of immune cells, showing its potential as an anti-cancer drug target.

[0004] Data from in vivo loss-of-function gene screening using CRISPR / Cas9 genome editing in a murine B16F10 transplantable tumor model showed that deletion of the PTPN2 gene in tumor cells improved the response to an immunotherapy regimen of secreted GM-CSF vaccine (GVAX) plus PD-1 checkpoint blockade. Deletion of the PTPN2 gene sensitizes tumors to immunotherapy by enhancing the IFNγ-mediated effects on antigen presentation and growth inhibition. The same screening data also revealed that genes known to be involved in immune evasion (including PD-L1 and CD47 blocking antibodies) were also depleted under the selective action of immunotherapy, while genes involved in the IFNγ signaling pathway (including IFNGR, JAK1, and STAT1) were enriched. These observations suggest that therapeutic strategies to enhance IFNy sensing and signaling have a significant role in enhancing cancer immunotherapy.

[0005] However, in drug discovery, phosphatases are considered undruggable due to their highly polar active sites. The challenges of identifying candidate drugs for phosphatase targets are well-known. On the one hand, the catalytic subunits of phosphatases are highly similar, making it difficult to develop inhibitors targeting a single phosphatase and prone to causing serious adverse reactions. On the other hand, the active sites of phosphatases have a special polar and charged environment, resulting in the need for highly polar active site inhibitors. However, such inhibitors have low potency and poor pharmacokinetic properties. Due to the importance of PTPN2 / PTPN1 in tumors and immune cells and the high homology of their active sites, researchers have attempted to discover a small molecule inhibitor targeting these two phosphatases.

[0006] On October 4, 2023, AbbVie published a preclinical study of the PTPN2 / PTPN1 inhibitor ABBV-CLS-484 in the journal Nature. AC484 is the first known active site phosphatase inhibitor to enter cancer immunotherapy clinical trials and is currently being evaluated in patients with advanced solid tumors. AC484 is a dual PTPN2 / N1 inhibitor that can increase the sensitivity of cancer cells to INF-γ, enhance the activation and effector functions of T cells and NK cells, and thus inhibit cell proliferation.

[0007] However, so far, there have been few reports on PTPN2 inhibitors. Developing PTPN2 inhibitors with high activity, high selectivity, and better druggability has good value prospects and is worthy of development. Summary of the Invention General Overview of the Invention

[0009] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a protein tyrosine phosphatase inhibitor, a pharmaceutical composition, and their uses with high activity, high selectivity, and better druggability, so as to improve the activity of anti-tumor immune cells. The compounds provided by the present invention have good inhibitory effects on the protein tyrosine phosphatase inhibitors PTPN2 / 1.

[0010] The present invention solves the above technical problems through the following technical solutions.

[0011] On the one hand, the present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0012]

[0013]

[0014] The present invention also provides a pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt thereof described in any one of the above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0015] The present invention also provides the use of the compound or its pharmaceutically acceptable salt as described in any one of the above, or the pharmaceutical composition, in the preparation of a drug for treating a disease or disorder by inhibiting PTPN1 / PTPN2.

[0016] As a preferred technical solution, wherein the disease or disorder is cancer, rheumatic disease, inflammatory disease, immune disease, metabolic disease, infectious disease, neurodegenerative disease, genetic disease, heart disease; the cancer is preferably selected from T-cell acute lymphoblastic leukemia, ovarian cancer, primary mediastinal B-cell lymphoma, bladder cancer, bone cancer, brain cancer, cardia cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spinal and cervical cancer, leukemia, liver cancer, lymphoma, melanoma, penile cancer, testicular germ cell cancer, thymoma cancer, thymic carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, multiple myeloma, melanoma, secretory cell carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, renal cell carcinoma; the metabolic disease is preferably selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, obesity, type 2 diabetes, heart disease, atherosclerosis, arthritis, cystinosis, phenylketonuria, proliferative retinopathy, metabolic syndrome or Kearns-Sayre disease; the infectious disease is preferably selected from bubonic plague; the genetic disease is preferably selected from Noonan syndrome, retinopathy.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention modifies (ABBV-CLS-484 analogues) through computer-aided design. Through computer simulation, molecular docking analysis, and drug structure-activity relationship research, a variety of novel-structured compounds are screened out. This series of compounds can specifically bind to protein tyrosine phosphatase and inhibit the function of PTPN2, and have extremely strong PTPN2 inhibitory activity. In vitro and in vivo experiments further show that this series of compounds can prevent and / or treat diseases such as pancreatic cancer, breast cancer, multiple myeloma, melanoma, secretory cell carcinoma, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, obesity, type 2 diabetes, heart disease, atherosclerosis, arthritis, cystinosis, phenylketonuria, proliferative retinopathy, metabolic syndrome or Kearns-Sayre disease. Detailed Description of the Invention

[0020] The following further describes each aspect and feature of the present invention.

[0021] As used in the specification and the appended claims, unless indicated to the contrary, the following terms have the meanings indicated below.

[0022] The abbreviations used herein have their conventional meanings within the fields of chemistry and biology. The chemical structures and chemical formulas shown herein are constructed according to the standard rules of chemical valence known in the field of chemistry.

[0023] As used herein, the term "pharmaceutically acceptable salt" means a salt that is not only physiologically acceptable to a subject but also refers to a synthetic substance that has utility in pharmacy, such as a salt formed as an intermediate during chiral resolution. Although such an intermediate salt cannot be directly administered to a subject, it can play a role in obtaining the end product of the present invention.

[0024] The term "excipient" refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable excipients that can be used in the manufacture of the pharmaceutical compositions of the present invention are any of those well known in the art of pharmaceutical formulation and include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrants, binders, preservatives, buffering agents, lubricants, and / or oils. Pharmaceutically acceptable excipients that can be used in the manufacture of the pharmaceutical compositions of the present invention include, but are not limited to, ion exchange agents, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, polyethylene glycol, and lanolin.

[0025] The compositions of the present invention can be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by an implantable depot. In some embodiments, the provided compounds or compositions are administered intravenously or orally.

[0026] As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. Preferably, the composition is administered orally, subcutaneously, intraperitoneally, or intravenously. The sterile injectable form of the compositions of the invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, in the form of a 1,3-butanediol solution. Acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly employed as a solvent or suspending medium.

[0027] The pharmaceutically acceptable compositions of the invention may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are typically also added. For oral administration in the form of capsules, diluents useful include lactose and dry corn starch. When an aqueous suspension for oral use is required, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring, or coloring agents may also be added as desired. In some embodiments, the oral formulations provided are formulated for immediate release or sustained / delayed release. In some embodiments, the composition is adapted for buccal or sublingual administration, including tablets, lozenges, and troches. The compounds disclosed herein may also be in microencapsulated form.

[0028] The compositions of the invention may be delivered by percutaneous, by topical routes, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral formulations include tablets, pills, powders, lozenges, capsules, liquids, troches, cachets, gels, syrups, slurries, suspensions, etc. suitable for ingestion by a patient. Solid form formulations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form formulations include solutions, suspensions, and emulsions such as water or water / propylene glycol solutions

[0029] The cancers of the present invention include those responsive to standard therapies such as surgery, radiotherapy, chemotherapy, and hormone therapy, etc.

[0030] "Cancer" includes T-cell acute lymphoblastic leukemia, ovarian cancer, primary mediastinal B-cell lymphoma, bladder cancer, bone cancer, brain cancer, gastric cardia cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine and neck cancer, leukemia, liver cancer, lymphoma, melanoma, penile cancer, testicular germ cell cancer, thymoma cancer, thymic carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, multiple myeloma, melanoma, secretory cell carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, renal cell carcinoma, etc.

[0031] The "metabolic diseases" include non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, obesity, type 2 diabetes, heart disease, atherosclerosis, arthritis, cystinosis, phenylketonuria, proliferative retinopathy, metabolic syndrome or Kearns-Sayre disease, etc.

[0032] The term "PTPN2" refers to non-receptor protein tyrosine phosphatase type 2. The term "PTPN1" refers to non-receptor protein tyrosine phosphatase type 1 (PTPN1), also known as protein tyrosine phosphatase-1B (PTP1B).

[0033] For the purpose of drug use and enhancing the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any well-known administration methods.

[0034] The compounds or compositions of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When there is a synergistic effect between the compounds of the present invention and other therapeutic drugs, the dosage should be adjusted according to the actual situation.

[0035] Beneficial technical effects

[0036] The inventors of the present invention found that the compounds in the present invention have good protein tyrosine phosphatase inhibitory activity, and the IC 50 or EC 50 is less than that of the positive control drug AC484. The present invention provides a class of protein tyrosine phosphatase inhibitor compounds with novel structures and strong activities, and such compounds have good application prospects in the prevention and / or treatment of indications related to the inhibition of protein tyrosine phosphatase PTPN2 / 1, such as cancer, metabolic diseases, etc. Detailed implementation manners

[0037] The following listed examples are helpful for those skilled in the art to better understand the technical solutions of the present invention, but do not limit the present invention in any way.

[0038] For all the following examples, standard operations and methods known to those skilled in the art can be used. Unless otherwise specified, all temperatures are expressed in °C (degrees Celsius). The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectrometry (MS).

[0039] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or / and liquid chromatography - mass spectrometry (LC - MS). The NMR chemical shift (δ) is in parts per million (ppm). Nuclear magnetic resonance was measured using a Bruker avance - 400 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO - d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3) as solvents, and tetramethylsilane (TMS) as the internal standard.

[0040] For liquid chromatography - mass spectrometry LC - MS measurement, the liquid phase part uses an ACQUITY UPLC ultra - high - pressure liquid chromatography, and the mass spectrometry part uses an Xevo G2 - S Qtof mass spectrometer.

[0041] The starting materials in the examples of the present invention are known and can be purchased on the market, or can also be synthesized using or according to methods known in the art.

[0042] Example 1: Synthesis of 3 - {[7 - (1,1 - dioxido - 4 - oxo - 1,2,5 - thiadiazolin - 2 - yl) - 8 - fluoro - 6 - hydroxy - 1,2,3,4 - tetrahydronaphthalen - 2 - yl]amino}-2,2 - dimethylpropanenitrile (Compound PTP - 01)

[0043] Intermediate: Synthesis of 5 - [3 - (benzyloxy) - 1 - fluoro - 7 - oxo - 5,6,7,8 - tetrahydronaphthalen - 2 - yl] - 1,2,5 - thiadiazoline - 3 - one - 1,1 - dioxide

[0044]

[0045] Step 1: Synthesis of Compound 6 - bromo - 8 - fluoro - 3,4 - dihydro - 1H - spiro[naphthalene - 2,2' - [1,3]dioxolane]

[0046] 6-Bromo-8-fluoro-3,4-dihydronaphthalen-2(1H)-one (30 g, 124.0 mmol, 1.0 eq.) and ethylene glycol (10.4 g, 743.9 mmol, 6.0 eq.) were added to toluene (300 mL). At room temperature, 4-toluenesulfonic acid hydrate (4.7 g, 24.8 mmol, 0.2 eq.) was added to the reaction system, and the reaction was carried out at 100 °C for 2 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the toluene was directly removed by concentration. The reaction solution was extracted with ethyl acetate, and the organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by silica gel column chromatography (75% petroleum ether, 25% ethyl acetate) to obtain the white solid product 6-bromo-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane] (19.2 g, yield 54.14%). LCMS (ESI) [M+H]+: 287.0. 1H NMR (400 MHz, CDCl3) δ 7.08 (t, J = 1.6 Hz, 1H), 7.02 (dd, J = 8.9, 1.9 Hz, 1H), 4.05–4.00 (m, 4H), 2.97 (t, J = 6.7 Hz, 2H), 2.85 (s, 2H), 1.92 (t, J = 6.7 Hz, 2H). 19F NMR (376 MHz, CDCl3) δ -116.12 (d, J = 8.8 Hz).

[0047] Step 2: Synthesis of compound 6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]

[0048] 6-Bromo-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane] (19.2 g, 67.1 mmol, 1.0 eq.), benzyl alcohol (10.2 g, 93.99 mmol, 1.4 eq.), copper(I) iodide (1.27 g, 6.71 mmol, 0.1 eq.), sodium tert-butoxide (7.73 g, 80.52 mmol, 1.2 eq.), N,N'-bis(2-phenylethyl)oxamide (1.99 g, 6.71 mmol, 0.1 eq.) were added to a 1,4-dioxane solution (200 mL). After purging with nitrogen, the reaction was carried out at 100 °C for 16 h. After completion of the reaction, silica gel was directly added for flash column chromatography purification (75% petroleum ether, 25% ethyl acetate) to obtain the white solid product 6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane] (12.1 g, yield 57.25%). LCMS (ESI) [M+H]+: 356.0. 1H NMR (400 MHz, DMSO-d6) δ 6.63–6.45 (m, 5H), 5.91–5.81 (m, 2H), 4.24 (s, 2H), 3.14–3.05 (m, 4H), 2.02 (t, J = 6.7 Hz, 2H), 1.88 (s, 2H), 1.00 (t, J = 6.7 Hz, 2H).

[0049] Step 3: Synthesis of 6-(benzyloxy)-7-bromo-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]

[0050] Dissolve 2,2,6,6 - tetramethylpiperidine (8.16 g, 57.78 mmol, 1.5 eq.) in tetrahydrofuran (500 mL) solution, and displace the system with nitrogen three times. At 0 °C, slowly add n - butyllithium (21.6 mL, 2.5 M, 53.93 mmol, 1.4 eq.) dropwise to the system, controlling the dropping time to be 40 minutes. After the addition, stir at 0 °C for 30 minutes, dilute the reaction mixture with tetrahydrofuran (500 mL), and cool to - 78 °C. Subsequently, dissolve 6 - (benzyloxy) - 8 - fluoro - 3,4 - dihydro - 1H - spiro[naphthalene - 2,2'-[1,3]dioxolane] (12.1 g, 38.52 mmol, 1.0 eq.) in tetrahydrofuran (500 mL), and slowly add it dropwise to the reaction system, controlling the dropping time within 30 minutes while keeping the internal temperature of the reaction system below - 70 °C. Stir for 2 hours, and finally slowly add 1,2 - dibromo - 1,1,2,2 - tetrafluoroethane (12.0 g, 46.22 mmol, 1.2 eq.) while keeping the internal temperature below - 60 °C. After complete addition, warm the reaction mixture to - 10 °C, then quench it with saturated ammonium chloride aqueous solution and dilute with water and ethyl acetate. Separate the layers, wash the organic layer with 1 M hydrochloric acid, saturated sodium bicarbonate aqueous solution and brine respectively, then dry over anhydrous sodium sulfate, filter and concentrate in vacuo. Dilute the obtained residue with isopropanol, then heat to 50 °C and slowly cool to ambient temperature, filter to collect the solid, and obtain yellow solid 6 - (benzyloxy) - 7 - bromo - 8 - fluoro - 3,4 - dihydro - 1H - spiro[naphthalene - 2,2'-[1,3]dioxolane] (7.95 g, yield 52.51%). LC - MS [M + H]+: 393.0. 1H NMR (400 MHz, DMSO - d6) δ 7.47–7.25 (m, 5H), 6.38 (d, J = 0.9 Hz, 1H), 5.11 (t, J = 0.8 Hz, 2H), 3.97–3.86 (m, 2H), 3.86–3.76 (m, 2H), 3.13 (s, 2H), 2.81–2.73 (m, 2H), 2.11–2.03 (m, 2H).

[0051] Step 4: Synthesis of methyl [6 - (benzyloxy) - 8 - fluoro - 3,4 - dihydro - 1H - spiro[naphthalene - 2,2'-[1,3]dioxolane] - 7 - yl]glycinate

[0052] 6-(Benzyloxy)-7-bromo-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane] (7.95 g, 20.2 mmol, 1.0 eq.), glycine methyl ester hydrochloride (3.29 g, 26.3 mmol, 1.3 eq.), (2-dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (1.83 g, 2.02 mmol, 0.1 eq.), 2-(dicyclohexylphosphino)-3,6-dimethoxy-2'-4'-6'-tri-I-propyl-11'-biphenyl (2.17 g, 4.04 mmol, 0.2 eq.) and cesium carbonate (13.16 g, 40.4 mmol, 2.0 eq.) were added to a solution of 1,4-dioxane (500 mL). After purging with nitrogen, the reaction was carried out at 100 °C for 16 h. After completion of the reaction, silica gel was directly added for sample mixing, and normal-phase purification (50% petroleum ether, 50% ethyl acetate) was performed to obtain the yellow solid product methyl [6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]glycinate (6.55 g, yield 62.14%). LCMS (ESI) [M+H]+: 402.2. 1H NMR (400 MHz, DMSO-d6) δ 7.88 (m, J = 5.9, 4.8 Hz, 1H), 7.51–7.20 (m, 5H), 6.34 (t, J = 0.8 Hz, 1H), 5.14 (t, J = 0.8 Hz, 2H), 4.07 (d, J = 5.9 Hz, 2H), 3.97–3.89 (m, 2H), 3.85–3.77 (m, 2H), 3.08 (d, J = 5.0 Hz, 2H), 2.80–2.65 (m, 2H), 2.11–2.02 (m, 2H).

[0053] Step 5: Synthesis of methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-[(N-tert-butoxycarbonyl)sulfamoyl]glycinate

[0054] Under an ice bath, sulfonyl chloride isocyanate (4.26 mL, 48.95 mmol, 3.0 eq.) was mixed with dichloromethane (100 mL), and tert-butanol (4.68 mL, 48.95 mmol, 3.0 eq.) was added dropwise. After reacting for 30 minutes, a mixed solution of methyl [6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]glycinate (6.55 g, 16.32 mmol, 1.0 eq.), triethylamine (9.07 mL, 65.27 mmol, 4.0 eq.) and dichloromethane (100 mL) was added dropwise to the reaction solution. After the addition was completed, the temperature was allowed to rise to room temperature naturally, and the reaction was continued for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate and water, washed three times with saturated brine, separated and dried, and the crude product of methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-[(tert-butoxycarbonyl)sulfamoyl]glycinate (7.27 g, crude) was obtained after concentration. LCMS (ESI) [M+H]+: 581.2.

[0055] Step 6: Synthesis of methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-sulfamoylglycinate

[0056] Methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-[(tert-butoxycarbonyl)aminosulfonyl]glycinate (7.27 g) was added to a solution of dichloromethane (70.0 mL). Under the condition of 0 °C, trifluoroacetic acid (35 mL) was slowly added. After the addition, the reaction was carried out at room temperature for 2 hours. After the reaction was completed, saturated sodium bicarbonate was added to neutralize to weak alkalinity, and the mixture was extracted with dichloromethane three times, dried over anhydrous sodium sulfate. After the organic phase was concentrated under reduced pressure, it was washed with a small amount of acetonitrile, filtered, and the filtrate was mixed with silica gel for normal-phase purification (40% petroleum ether, 60% ethyl acetate) to obtain the brown solid product methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-aminosulfonylglycinate (1.56 g, 25.91%). LCMS (ESI) [M+H]+: 481.2. 1H NMR (400 MHz, Chloroform-d) δ 7.45–7.26 (m, 5H), 6.39 (t, J = 0.9 Hz, 1H), 5.14 (t, J = 0.8 Hz, 2H), 4.70 (s, 2H), 3.97–3.86 (m, 2H), 3.88–3.78 (m, 2H), 3.72 (s, 3H), 3.08 (d, J = 5.0 Hz, 2H), 2.92 (m, J = 14.3, 6.6, 0.9 Hz, 1H), 2.80 (m, J = 14.5, 6.7, 0.9 Hz, 1H), 2.07 (d, J = 13.4 Hz, 2H).

[0057] Step 7: Synthesis of 5-{6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl}-1,2,5-thiadiazolidine-3-one 1,1-dioxide

[0058] To a solution of methyl N-[6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl]-N-sulfamoyl glycinate (1.56 g, 3.24 mmol, 1.0 eq.) in tetrahydrofuran (20 mL) was added a solution of sodium methoxide (1.2 mL, 5.4 M in MeOH, 6.48 mmol, 2.0 eq.). The reaction was carried out at room temperature for 1 hour. After completion of the reaction, the solid was removed by filtration and the product was purified by reverse phase (0.1% formic acid in water and acetonitrile system). When the acetonitrile was 55%, 5-{6-(benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl}-1,2,5-thiadiazolidine-3-one 1,1-dioxide (0.42 g, yield 28.80%) was obtained. LCMS (ESI) [M+H]+: 449.1. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.46–7.23 (m, 5H), 6.37 (t, J = 0.9 Hz, 1H), 5.15 (t, J = 0.9 Hz, 2H), 4.83 (s, 2H), 3.99–3.87 (m, 2H), 3.89–3.71 (m, 2H), 3.04 (s, 2H), 2.85–2.71 (m, 2H), 2.15–2.02 (m, 2H).

[0059] Step 8: Synthesis of 5-[3-(benzyloxy)-1-fluoro-7-oxo-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide

[0060] 5-{6-(Benzyloxy)-8-fluoro-3,4-dihydro-1H-spiro[naphthalene-2,2'-[1,3]dioxolane]-7-yl}-1,2,5-thiadiazolidin-3-one 1,1-dioxide (420 mg, 0.94 mmol, 1.0 eq.) was added to formic acid solution (2 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, brine was added to dilute the reaction system, and the solid was precipitated by standing. After filtration, the solid was washed with water and dried to obtain the product 5-[3-(benzyloxy)-1-fluoro-7-oxo-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidin-3-one-1,1-dioxide (0.36 g, yield 95.85%). LCMS (ESI) [M-H]+: 402.1. 1H NMR (400 MHz, DMSO-d6) δ 7.43 (d, J = 7.4 Hz, 2H), 7.41 - 7.25 (m, 3H), 7.00 (s, 1H), 5.16 (s, 2H), 4.38 (s, 2H), 3.44 (s, 2H), 3.08–3.01 (m, 2H), 2.51–2.48 (m, 2H).

[0061] Synthesis method of compound 3-{[7-(1,1-dioxo-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropionitrile (compound PTP-01)

[0062]

[0063] Step 1: Synthesis of compound 3-amino-2,2-dimethylpropionitrile

[0064] 3-Amino-2,2-dimethylpropanamide (1.0 g, 8.6 mmol, 1.0 eq.) was added to 1,2-dichloroethane (10 mL), and thionyl chloride (2.0 g, 17.2 mmol, 2.0 eq.) was added to the reaction system. The reaction system was purged with nitrogen and reacted at 90 °C for 5 hours. After the reaction was completed, the reactant was cooled to room temperature, and the reaction solution was diluted with 20 mL of dichloromethane. The organic phase was washed successively with water (20 mL) and brine (10 mL). The organic phases were combined, concentrated, and purified by normal phase chromatography (65% petroleum ether, 35% ethyl acetate) to obtain the white solid product 3-amino-2,2-dimethylpropionitrile (0.5 g, yield 59.3%). LCMS (ESI) [M+H] + : 99.1. 1 1H NMR (500 MHz, Chloroform-d) δ 2.92 (s, 2H), 1.34 (s, 6H), 1.32 (s, 2H).

[0065] Step 2: Synthesis of compound 3-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropanenitrile

[0066] Mix compound 5-[3-(benzyloxy)-1-fluoro-7-oxo-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide (360 mg, 0.89 mmol, 1.0 eq.), 3-amino-2,2-dimethylpropanenitrile (221 mg, 2.25 mmol, 2.5 eq.), triethylamine (270 mg, 2.68 mmol, 3.0 eq.) with ethanol (5 mL). After stirring for 15 minutes, add sodium cyanoborohydride (67 mg, 1.07 mmol, 1.2 eq.). Stir the mixture for 16 hours, then quench with a small amount of dilute hydrochloric acid, add N,N-dimethylformamide to assist dissolution and filter. After most of the solvent is removed by rotary evaporation, purify by reverse phase (0.1% formic acid in water and acetonitrile system) to obtain the product 3-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropanenitrile (220 mg, yield 51.3%). LCMS (ESI) [[M+H]] + : 487.16. 1 H NMR (500 MHz, Chloroform-d) δ 7.39–7.25 (m, 5H), 6.52 (t, J = 1.0 Hz, 1H), 5.16 (t, J = 1.0 Hz, 2H), 4.74 (s, 2H), 3.28 (s, 1H), 3.15 (s, 1H), 2.94–2.84 (m, 3H), 2.81–2.63 (m, 3H), 2.02 (s, 1H), 1.77 (s, 1H), 1.36 (s, 3H), 1.33 (s, 3H)

[0067] Step 3: Synthesis of compound 3-{[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropanenitrile

[0068] Dissolve 3-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropanenitrile (220 mg, 0.46 mmol, 1.0 eq.) in tetrahydrofuran (3.0 mL), add 5% palladium on carbon (55% water) (200 mg), purge with hydrogen three times, and react at room temperature for 2 hours under a hydrogen atmosphere. After completion of the reaction, filter off the solid, and purify by HPLC preparation to obtain the white solid 3-{[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-2,2-dimethylpropanenitrile (16.7 mg, yield 4.2%). LCMS (ESI) [[M+H]] + : 397.14. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 6.32 (t, J = 0.9 Hz, 1H), 4.83 (s, 2H), 3.64 (dt, J = 6.8, 3.4 Hz, 1H), 3.12 (m, J = 6.7, 5.9, 4.3 Hz, 1H), 3.02–2.64 (m, 7H), 1.96 (m, J = 13.6, 8.6, 5.9, 4.4 Hz, 1H), 1.70 (m, J = 13.5, 8.5, 5.9, 4.4 Hz, 1H), 1.36 (d, J = 12.0 Hz, 6H).

[0069] Example 2: Synthesis method of compound 2-(3,3-difluorocyclobutyl)-N-(7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (Compound PTP-02)

[0070]

[0071] Step 1: Synthesis of compound 5-[7-amino-3-(benzyloxy)-1-fluoro-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide

[0072] The compound 5-[3-(benzyloxy)-1-fluoro-7-oxo-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide (1.0 g, 2.47 mmol, 1.0 eq.), ammonium acetate (114 mg, 24.73 mmol, 10.0 eq.), acetic acid (103.9 mg, 1.73 mmol, 0.7 eq.) were mixed with 1,4-dioxane (10 mL). Stirred at room temperature for 30 minutes, then sodium triacetoxyborohydride (2.1 g, 9.89 mmol, 4.0 eq.) was added and the mixture was stirred for 16 hours. After the reaction was completed, the product 5-[7-amino-3-(benzyloxy)-1-fluoro-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide (350.0 mg, yield 35.0%) was obtained by reverse-phase purification (0.1 mol ammonium bicarbonate solution and acetonitrile system). LCMS (ESI) [M+H]+: 406.1. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.43–7.25 (m, 5H), 6.36 (t, J = 0.8 Hz, 1H), 5.15 (t, J = 0.9 Hz, 2H), 4.83 (s, 2H), 3.37–3.25 (m, 1H), 3.04–2.84 (m, 2H), 2.84–2.68 (m, 2H), 2.37 (d, J = 5.6 Hz, 2H), 2.04 (m, J = 14.3, 8.5, 5.9, 4.7 Hz, 1H), 1.79 (m, J = 14.2, 8.3, 5.7, 4.6 Hz, 1H).

[0073] Step 2: Synthesis of compound N-(6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-2-(3,3-difluorocyclobutyl)acetamide

[0074] 5-[7-Amino-3-(benzyloxy)-1-fluoro-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide (350.0 mg, 0.863 mmol, 1.0 eq.), 2-(3,3-difluorocyclobutyl)acetic acid (155.5 mg, 1.04 mmol, 1.2 eq.), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (426.7 mg, 1.12 mmol, 1.3 eq.), and triethylamine (262.0 mg, 2.59 mmol, 3.0 eq.) were dissolved in N,N-dimethylformamide (3.0 mL), and the reaction was carried out at room temperature for 2 hours. After completion of the reaction, the solid was removed by filtration and purified by HPLC preparation to obtain the white solid N-[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-2-(3,3-difluorocyclobutyl)acetamide (50.0 mg, yield 14.3%). LCMS (ESI) [[M+H]] - : 538.2. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.43–7.26 (m, 5H), 7.23 (d, J = 8.2 Hz, 1H), 6.36 (d, J = 1.0 Hz, 1H), 5.15 (t, J = 0.9 Hz, 2H), 4.83 (s, 2H), 3.85 (m, J = 8.2, 6.9, 5.3 Hz, 1H), 3.10 (m, J = 15.9, 6.9, 5.0 Hz, 1H), 2.97–2.73 (m, 3H), 2.41–2.26 (m, 2H), 2.26–2.16 (m, 2H), 2.20–1.95 (m, 4H), 1.77 (m, J = 13.9, 8.6, 6.0, 5.4 Hz, 1H).

[0075] Step 3: Synthesis of 2-(3,3-difluorocyclobutyl)-N-[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide

[0076] Dissolve N-[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-2-(3,3-difluorocyclobutyl)acetamide (50.0 mg, 0.093 mmol, 1.0 eq.) in tetrahydrofuran (1.0 mL), add 5% palladium on carbon (55% water) (50 mg), displace the gas with hydrogen three times, and react at room temperature for 2 hours under a hydrogen atmosphere. After completion of the reaction, filter off the solid, and purify by HPLC preparation to obtain the white solid 2-(3,3-difluorocyclobutyl)-N-[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide (5.3 mg, yield 10.6%). LCMS (ESI) [[M+H]] - : 448.1. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 7.23 (d, J = 8.2 Hz, 1H), 6.32 (t, J = 0.8 Hz, 1H), 4.83 (s, 2H), 3.85 (m, J = 8.2, 6.9, 5.3 Hz, 1H), 3.10 (m, J = 16.0, 7.0, 5.0 Hz, 1H), 2.95–2.72 (m, 3H), 2.41–2.26 (m, 2H), 2.26–2.16 (m, 2H), 2.20–1.95 (m, 4H), 1.77 (m, J = 13.8, 8.6, 6.0, 5.4 Hz, 1H).

[0077] Example 3: Synthesis method of compound 2-{[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide (compound PTP-03)

[0078]

[0079] Step 1: Synthesis of compound 2-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide

[0080] The compound 5-[3-(benzyloxy)-1-fluoro-7-oxo-5,6,7,8-tetrahydronaphthalen-2-yl]-1,2,5-thiadiazolidine-3-one 1,1-dioxide (300 mg, 0.74 mmol, 1.0 eq.), 2-amino-N,N-dimethylacetamide (114 mg, 1.11 mmol, 1.5 eq.), triethylamine (225 mg, 2.22 mmol, 3.0 eq.) were mixed with ethanol (3 mL). After stirring for 15 minutes, sodium cyanoborohydride (56 mg, 0.89 mmol, 1.2 eq.) was added. The mixture was stirred for 16 h, then quenched with a small amount of dilute hydrochloric acid, filtered after adding N,N-dimethylformamide to assist dissolution, and purified by reverse phase (0.1% formic acid in water and acetonitrile system) after most of the solvent was removed by rotary evaporation to obtain the product 2-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide (163 mg, yield 44.78%). LCMS (ESI) [M-H]+: 491.2. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.43 (m, J = 7.3, 1.6, 0.8 Hz, 2H), 7.36 (m, J = 7.2, 6.1, 0.8 Hz, 2H), 7.32–7.27 (m, 1H), 6.36 (t, J = 0.8 Hz, 1H), 5.15 (t, J = 0.9 Hz, 2H), 4.83 (s, 2H), 3.53 (dd, J = 15.6, 5.1 Hz, 1H), 3.44–3.36 (m, 1H), 3.34 (t, J = 5.1 Hz, 1H), 3.14 (m, J = 7.3, 6.4, 4.8 Hz, 1H), 2.99 (m, J = 15.7, 6.5, 4.9 Hz, 1H), 2.92–2.84 (m, 7H), 2.86–2.69 (m, 2H), 1.99 (m, J = 13.4, 8.5, 5.9, 4.8 Hz, 1H), 1.74 (m, J = 13.4, 8.5, 5.9, 4.9 Hz, 1H).

[0081] Step 2: Synthesis of compound 2-{[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide

[0082] Dissolve 2-{[6-(benzyloxy)-7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide (163 mg, 0.332 mmol, 1.0 eq.) in tetrahydrofuran (3.0 mL), add 5% palladium on carbon (55% water) (160 mg), displace the gas with hydrogen three times, and react at room temperature for 2 hours under a hydrogen atmosphere. After the reaction is completed, filter off the solid, and purify by HPLC preparation to obtain the white solid 2-{[7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-8-fluoro-6-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N,N-dimethylacetamide (11.2 mg, yield 8.40%). LCMS (ESI) [[M+H]] - : 401.1. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 6.32 (t, J = 0.9 Hz, 1H), 4.83 (s, 2H), 3.53 (dd, J = 15.6, 5.1 Hz, 1H), 3.46–3.31 (m, 2H), 3.14 (m, J = 7.3, 6.4, 4.8 Hz, 1H), 2.99 (m, J = 15.7, 6.5, 4.9 Hz, 1H), 2.92–2.81 (m, 7H), 2.80–2.70 (m, 2H), 1.99 (m, J = 13.4, 8.5, 5.9, 4.8 Hz, 1H), 1.81–1.71 (m, 1H).

[0083] The compounds listed in Table 1 below were prepared by a method similar to that described in Examples 1-3, with appropriate variations in the amounts of reactants, reagents, protection and deprotection, solvents, and reaction conditions. The characterization data of the compounds are summarized in Table 1 below.

[0084] Table 1: Structures and Characterizations of Some Compounds

[0085]

[0086]

[0087]

[0088] Experimental Example 1: Test on the Activity of the Compounds of the Present Invention against PTPN2 / 1 Protease

[0089] Experimental Purpose: The purpose of this test example is to test the effect of the compounds on the function of PTPN2 / 1 enzyme.

[0090] Background principle: Through in vivo CRISPR screening, it was found that PTPN2 and its closely related PTPN1 can serve as new targets for cancer immunotherapy. Knocking out PTPN2 can improve the effect of tumor immunotherapy by enhancing interferon-γ-mediated antigen presentation and growth inhibition. The phosphatases PTPN2 (also known as TC-PTP) and PTPN1 (also known as PTP-1B) are negative regulators of multiple cytokine signaling pathways and T cell receptor (TCR) signaling pathways, and are key checkpoints for inflammatory responses. p-Nitrophenyl phosphate (pNPP) is one of the most commonly used substrates for phosphatases. After adding the substrate to the microplate, under the action of phosphatase, PNPP generates a yellow water-soluble reaction product, and the solution turns yellow with p-nitrophenol. This reaction product has a large light absorption at 405 nm.

[0091] Experimental procedure:

[0092] Expression of PTPN2 / 1 protein: The PTPN2 sequence (residues 1-314) was cloned into the pET28A vector with a (His)6x tag; the PTPN1 sequence (residues 1-321) was cloned into the pET15B vector with a (His)6x tag. The constructed vectors were transformed into Escherichia coli BL21(DE3). When the culture OD reached 0.6, 1 mM IPTG was added and cultured at 12 °C for 16 h. The cells were collected, sonicated, centrifuged, and the supernatant was taken. The protein was purified by Ni-NTA, and the protein concentration and purity were measured after dialysis. Aliquots were stored at -80 °C.

[0093] Enzyme activity assay of PTPN2 / 1: In an experimental buffer containing 25 mM Tris HCl (pH 7.4), 50 mM NaCl and 1 mM DTT, the phosphatase activity of PTPN2 (or 1) was measured by monitoring the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol. In the reaction system, the enzyme concentration was fixed at 50 nM, the pNPP concentration was 50 μM, and the total reaction volume was 100 μL. 1. Mix the enzyme and the compound and incubate at 30 °C for 10 minutes; 2. Add pNPP to the enzyme-compound mixture and incubate at 30 °C for 30 minutes; 3. Add 100 μL of 1 M NaOH to stop the reaction, and then measure the absorbance at 405 nm using a microplate reader. The IC50 was calculated by processing with GraphPad software, and compounds were screened by comparing with the positive drug ABBV-CLS-484 (abbreviated as AC484).

[0094] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the antagonist being measured. It can indicate the half amount of a certain drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances included in this process, such as enzymes, cell receptors, or microorganisms). Using AC484 as the positive reference compound, its synthesis method refers to patent CN114025844B.

[0095] The structure of ABBV-CLS-484 is as follows:

[0096]

[0097] Determine the IC 50 value obtained for the protease functions of PTPN2 and PTPN1 for each compound.

[0098] The results show that the compounds designed in the present invention have good inhibitory effects on PTPN2 and PTPN1 enzymes.

[0099] Experimental Example 2: Determination of the effect of the compounds of the present invention on the proliferation of B16F10 cells

[0100] Experimental purpose: The purpose of this test example is to test the effect of the compounds on the proliferation of B16F10 cells

[0101] Background principle: IFNγ is a cytokine produced by immune system cells such as T cells or NK cells, which can inhibit the growth of certain cancer types, including melanoma. Inhibiting IFNγ signaling can promote tumor growth. On the contrary, enhancing IFNγ signaling can enhance tumor growth inhibition. Since PTPN2 and PTPN1 are negative regulators of cytokine signaling, including IFNγ signaling, through the dephosphorylation of JAK and STAT proteins, in the presence of IFNγ, an effective compound can inhibit the growth of tumor cells.

[0102] Experimental procedure:

[0103] B16F10 cells were cultured in DMEM + 10% FBS + 1% P / S growth medium. B16F10 cells in the logarithmic growth phase were seeded in a 96-well plate, 100 μl per well, 500 cells / well, and incubated overnight in a 37°C incubator. On the second day, 50 μl of mouse IFN-γ (final concentration 0.5 ng / ml) was added; 50 μl of compounds at different concentrations (final concentration of DMSO 1%) was added, and incubated in a 37°C incubator for 5 days. On the sixth day, the old medium was aspirated, 110 ul of medium (the ratio of medium to CCK8 is 100:10) was added, and incubated at 37°C for 1 - 4 h. The absorbance was measured at 450 nM, and the IC was calculated by processing with GraphPad software.50 , screening compounds by comparing with the positive drug AC484.

[0104] The results showed that the compounds designed in the present invention had good inhibitory effects on the proliferation of B16F10 cells.

[0105] Experimental Example 3: Determination of the effect of the compounds of the present invention on STAT1 phosphorylation in cells

[0106] Experimental purpose: The purpose of this test example is to test the determination of the effect of the compound on STAT1 phosphorylation in cells

[0107] Background principle: PTPN2 / N1 inhibits inflammation by removing the phosphorylation of JAK and STAT family members. PTPN2 also targets proximal TCR signaling molecules, thereby inhibiting the antigen sensitivity of T cells. Therefore, after incubation with the compound, the phosphorylation level of STAT in B16F10 cells can be detected by WB for the screening of inhibitors.

[0108] Experimental procedure:

[0109] B16F10 cells were cultured in DMEM + 10% FBS + 1% P / S growth medium. B16F10 cells in the logarithmic growth phase were seeded in a 24-well plate, 450 μl per well, 200,000 cells per well, and incubated overnight in a 37 °C incubator. On the second day, 50 μl of compounds at different concentrations (the final concentration of DMSO was 1%) were added and incubated at 37 °C for 3 h; then stimulated with 100 ng / ml mouse IFN-γ for 10 min, and the cells were collected for WB. Protein electrophoresis: The voltage of the stacking gel was set at 60 v, and the voltage of the separating gel was set at 120 v; after electrophoresis, electrotransfer was started. The electrotransfer conditions were set at 250 mA for 2 h; blocked with 5% BSA for 1 h; added with specific primary antibody and incubated overnight on a shaker at 4 °C; washed 4 times with TBST, 2.5 min each time; incubated with secondary antibody on a shaker at room temperature for 1 h; washed 4 times with TBST, 2.5 min each time; developed using ECL. The western blot bands were processed by image J and GraphPad software to calculate the IC 50 , screening compounds by comparing with the positive drug AC484.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

3. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claim 1 or the pharmaceutical composition according to claim 2 in the manufacture of a medicament for treating a disease or disorder by inhibiting PTPN1 / PTPN2.

4. The pharmaceutical use according to claim 3, wherein the disease or disorder is cancer, a rheumatic disease, an inflammatory disease, an immune disease, a metabolic disease, an infectious disease, a neurodegenerative disease, a genetic disease, a heart disease.

5. The pharmaceutical use according to claim 4, wherein the cancer is selected from T-cell acute lymphoblastic leukemia, ovarian cancer, primary mediastinal B-cell lymphoma, bladder cancer, bone cancer, brain cancer, cardia cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spinal and cervical cancer, leukemia, liver cancer, lymphoma, melanoma, penile cancer, testicular germ cell cancer, thymoma cancer, thymic carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, multiple myeloma, melanoma, secretory cell carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, renal cell carcinoma.

6. The pharmaceutical use according to claim 4, wherein the metabolic disease is selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, obesity, type 2 diabetes, heart disease, atherosclerosis, arthritis, cystinosis, phenylketonuria, proliferative retinopathy, metabolic syndrome or Kearns-Sayre disease.

7. The pharmaceutical use according to claim 4, wherein the infectious disease is selected from bubonic plague.

8. The pharmaceutical use according to claim 4, wherein the genetic disease is selected from Noonan syndrome, retinopathy.

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