Preparation method and application of a PTPRO inhibitor

Abstract

The present invention provides a method for preparing a PTPRO inhibitor and its application, belonging to the field of pharmaceutical technology. After pretreating mice with a PTPRO inhibitor and then establishing a sepsis model, it is found that the PTPRO inhibitor can significantly reduce the acute mortality rate of mice within 24 hours, reduce the total number of cells in the bronchoalveolar lavage fluid, and weaken the degree of damage to the blood-brain barrier permeability of mice. This indicates that the PTPRO inhibitor has good therapeutic effects in lung and cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a preparation method of a PTPRO inhibitor and application thereof. Background Art

[0002] Acute lung injury (ALI) and its more serious acute respiratory distress syndrome (ARDS) are inflammatory diseases characterized by acute lung tissue damage and pulmonary dysfunction caused by infectious or aseptic injury. They are characterized by diffuse alveolar damage, accumulation of inflammatory cells (macrophages, neutrophils), destruction of alveolar capillary membrane barrier function, and production of inflammatory factors. The pathogenesis is the necrosis and dysfunction of a large number of alveolar capillary endothelial and epithelial cells, the formation of a transparent membrane, and ultimately the formation of intra-alveolar thrombosis. A variety of diseases can lead to ALI / ARDS, including sepsis, infectious pneumonia, trauma, acute pancreatitis, and aspiration of gastric contents. Among the pathogenic factors, sepsis-related ALI has the highest incidence and mortality. Sepsis is a common fatal inflammatory disease defined as an uncontrolled inflammatory response of the host associated with multiple organ dysfunction. Pulmonary involvement is common in critically ill patients infected with sepsis. The inflammatory consequences of sepsis are particularly evident in the pulmonary circulation, and more than 40% of sepsis patients develop ALI.

[0003] At present, the treatment of ALI / ARDS is mainly symptomatic treatment such as mechanical ventilation and fluid management. Due to the lack of effective treatment strategies, most ALI / ARDS patients have a poor prognosis. The alveolar-capillary barrier is a tight barrier maintained between pulmonary endothelial cells and epithelial cells, which maintains efficient gas exchange while preventing fluid accumulation or inflammatory cell infiltration into the alveoli. It has been reported in the literature that clinically significant lesions (venous bacteria, lipopolysaccharide, and microemboli) can lead to increased pulmonary endothelial permeability; in experimental models of influenza pneumonia, persistent increases in pulmonary vascular permeability are associated with lung damage and slow recovery. In severe ALI, strong proinflammatory responses and infiltration of inflammatory cells into lung tissue can destroy the integrity of the alveolar capillary barrier.

[0004] Receptor-type protein tyrosine phosphatase O (PTPRO) is a receptor-type PTP that is widely expressed in multiple organs such as the breast, liver, lung, brain, and kidney. PTPRO with an intracellular PTP domain can catalyze the dephosphorylation of tyrosine peptides of target genes, thereby participating in biological processes such as apoptosis, differentiation, and proliferation. Aberrant expression of protein tyrosine phosphatases (PTPs) has been reported to be an important cause of cancer. The PTPRO gene is associated with various diseases and disorders, including cancer and neurological diseases, in which dysregulation of PTPRO activity can lead to significant pathological consequences. The PTPRO protein includes an extracellular domain with 8 type III fibronectin-like repeats, a transmembrane domain, and an intracellular catalytic PTP domain. PTPRO is widely expressed in multiple organs such as the breast, liver, lung, brain, and kidney. PTPRO with an intracellular PTP domain can catalyze the dephosphorylation of tyrosine peptides of target genes, thereby participating in biological processes such as apoptosis, differentiation, and proliferation. It has been reported that PTPRO is a tumor suppressor and an effective biomarker for the diagnosis and prognosis of various cancers. There is evidence that PTPRO plays a role in tumors such as breast cancer, hepatocellular carcinoma, lung cancer, and esophageal cancer through downregulation by methylation. PTPRO has also been found to be associated with immune infiltration in different cancers and acts as a tumor suppressor by polarizing macrophages into M1-like tumor-associated macrophages.

[0005] Currently, there are no commercially available PTPRO inhibitors on the market, and the therapeutic effects of PTPRO and PTPRO inhibitors on lung and cardiovascular and cerebrovascular diseases are still unclear. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a preparation method of a PTPRO inhibitor and its application in the treatment of lung and cardiovascular and cerebrovascular diseases.

[0007] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] A preparation method of a PTPRO inhibitor, using compound 1 as a raw material, and the synthesis route is as follows:

[0009]

[0010] The structural formula of compound 3a is

[0011] The structural formula of compound 4a is

[0012] Preferably, compound 3a is prepared as follows:

[0013]

[0014] Another object of the present invention is to provide the use of the PTPRO inhibitor obtained by the said preparation method in the preparation of a medicament for treating pulmonary inflammatory diseases.

[0015] Preferably, the pulmonary inflammatory disease is characterized by diffuse alveolar damage, aggregation of inflammatory cells, destruction of the alveolar-capillary membrane barrier function, and production of inflammatory factors.

[0016] Preferably, the PTPRO inhibitor elevates the expression level of neutrophil apoptosis signals.

[0017] Preferably, the PTPRO inhibitor inhibits the secretion of TNF-α by alveolar macrophages.

[0018] Another object of the present invention is to provide the use of the PTPRO inhibitor obtained by the said preparation method in the preparation of a medicament for improving the permeability of the blood-brain barrier.

[0019] Another object of the present invention is to provide the use of the PTPRO inhibitor obtained by the said preparation method in the preparation of a medicament for treating sepsis.

[0020] Another object of the present invention is to provide a medicament, the active ingredient of which comprises an effective dose of the PTPRO inhibitor obtained by the above preparation method or a pharmaceutically acceptable salt thereof.

[0021] Preferably, the medicament is used for treating pulmonary inflammatory diseases, improving the permeability of the blood-brain barrier, and treating sepsis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a preparation method of a PTPRO inhibitor. By using the prepared PTPRO inhibitor, it is first proposed that the PTPRO inhibitor has the effect of treating pulmonary and cardiovascular and cerebrovascular diseases. It is found that after treating mice with the PTPRO inhibitor and then establishing a sepsis model, it can reduce the acute mortality rate of mice within 24 hours, reduce the total number of cells in the bronchoalveolar lavage fluid, and weaken the degree of damage to the blood-brain barrier permeability of mice, indicating that the PTPRO inhibitor has a good therapeutic effect in pulmonary and cardiovascular and cerebrovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : 1H NMR spectrum of the PTPRO inhibitor;

[0025] Figure 2 : Mass spectrum of the PTPRO inhibitor;

[0026] Figure 3: Effects of PTPRO inhibitor on the mortality of mice with sepsis: Control is the control group, Pi is the PTPRO inhibitor group, LPS is the model group, and LPS+Pi is the model group after treatment with the PTPRO inhibitor;

[0027] Figure 4 : 2. Effects of PTPRO inhibitor on the changes in blood-brain barrier permeability in septic mice: Control is the control group, Pi is the PTPRO inhibitor group, LPS is the model group, and LPS+Pi is the model group after treatment with the PTPRO inhibitor;

[0028] Figure 5 : Effects of PTPRO inhibitor on the total number of cells in the bronchoalveolar lavage fluid of mice: Control is the control group, Pi is the PTPRO inhibitor group, LPS is the model group, and LPS+Pi is the model group after treatment with the PTPRO inhibitor;

[0029] Figure 6 : Effects of PTPRO inhibitor on neutrophils: Control is the control group, Pi is the PTPRO inhibitor group, LPS is the model group, and LPS+Pi is the model group after treatment with the PTPRO inhibitor;

[0030] Figure 7 : Effects of PTPRO inhibitor on the secretion of TNF-α by alveolar macrophages in mice: Control is the control group, Pi is the PTPRO inhibitor group, LPS is the model group, and LPS+Pi is the model group after treatment with the PTPRO inhibitor. Specific implementation methods

[0031] The present invention provides a preparation method of a PTPRO inhibitor, using compound 1 as a raw material, and the synthesis route is as follows:

[0032]

[0033] The structural formula of compound 3a described in the present invention is

[0034] The structural formula of compound 4a described in the present invention is

[0035] Compound 3a described in the present invention is prepared by the following method:

[0036]

[0037] The present invention also provides the application of the PTPRO inhibitor obtained by the above preparation method in the preparation of drugs for treating pulmonary inflammatory diseases.

[0038] The pulmonary inflammatory disease described in the present invention is characterized by diffuse alveolar damage, aggregation of inflammatory cells, disruption of the alveolar-capillary membrane barrier function, and production of inflammatory factors.

[0039] The PTPRO inhibitor described in the present invention elevates the expression level of neutrophil apoptosis signals.

[0040] The PTPRO inhibitor described in the present invention inhibits the secretion of TNF-α by alveolar macrophages.

[0041] In acute lung injury, pulmonary edema, endothelial cell and epithelial cell damage will further cause neutrophils to infiltrate into the interstitium and bronchoalveolar space. Neutrophils infiltrating into the lung contain a variety of antibacterial substances. Although neutrophil activation is crucial for host defense, over-activation can also lead to cytotoxicity and damage host tissue cells. The number and activity of neutrophils are strictly regulated by the apoptosis program. The apoptosis of neutrophils is extremely sensitive to stimuli in the inflammatory microenvironment (including lipopolysaccharide LPS, inflammatory cytokines, etc.). In particular, the apoptosis of neutrophils isolated from the peripheral circulation or lung of critically ill sepsis patients is significantly inhibited. Since the apoptosis process of these originally short-lived neutrophils is inhibited and they are forced to be in a persistent activation state for a long time, excessive NETs, etc. are produced, which will directly kill alveolar epithelial cells and endothelial cells while clearing foreign pathogens, ultimately aggravating the severe non-specific tissue damage in sepsis patients. The research of the present invention finds that the PTPRO inhibitor can elevate the expression level of neutrophil apoptosis signals, thereby regulating the apoptosis of neutrophils and reducing the severity of lung injury.

[0042] The present invention also provides the use of the PTPRO inhibitor obtained by the described preparation method in the preparation of a drug for improving blood-brain barrier permeability.

[0043] The present invention also provides the use of the PTPRO inhibitor obtained by the described preparation method in the preparation of a drug for treating sepsis.

[0044] The present invention also provides a drug, the active ingredient of which comprises an effective dose of the PTPRO inhibitor obtained by the above-described preparation method or a pharmaceutically acceptable salt thereof.

[0045] The drug described in the present invention is used for treating pulmonary inflammatory diseases, improving blood-brain barrier permeability, and treating sepsis.

[0046] The drug described in the present invention further comprises a pharmaceutically acceptable carrier. The present invention has no special limitation on other excipient components and drug dosage forms contained in the drug. Commonly used excipients and drug dosage forms in the art can be adopted. The content of the PTPRO inhibitor or its pharmaceutically acceptable salt in the drug described in the present invention is 0.1 - 99.9 wt%.

[0047] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

[0049] A preparation method of a PTPRO inhibitor

[0050] The synthetic route in this example is as follows:

[0051]

[0052] Synthesis of compound 3a:

[0053] Charge degassed THF (110 mL) into a 250 mL three-necked flask (R1); under N2 atmosphere, add compound 3a-1 (11.0 g, 59.5 mmol, 1.00 eq) to R1; under N2 atmosphere, add 3a-2 (20.7 g, 130 mmol, 2.20 eq), PPh3 (624 mg, 2.38 mmol, 0.04 eq), CuI (226 mg, 1.19 mmol, 0.02 eq), and TEA (12.0 g, 119 mmol, 16.6 mL, 2.00 eq) to R1; stir R1 at 25 °C for 15 minutes; under N2 atmosphere, add Pd(OAc)2 (534 mg, 2.38 mmol, 0.04 eq) to R1, and stir R1 at 25 °C for 12 hours; LCMS: product: RT = 1.933 min, MS(ESI) m / z = 263.0 [M+H] + It shows that compound 3a-1 is retained (RT = 1.326 minutes), compound 3a-2 is retained (RT = 1.792 minutes), and the expected mass is observed (RT = 1.933 minutes). TLC (petroleum ether / ethyl acetate = 100 / 1) shows that compound 3a-1 is retained (R f = 0.20), compound 3a-2 is retained (R f = 0.50) and a new main spot is observed (R f(= 0.10); The concentrated mixture was obtained to give a crude product; The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 50 / 1) to give compound 3a (14.0 g, 51.3 mmol, 86.3% yield, 96.1% purity) (1H NMR: (400 MHz, CDCl3). δ 10.02 (s, 1H), 7.87 - 7.85 (m, 2H), 7.68 - 7.66 (m, 2H), 7.48 - 7.46 (m, 2H), 7.20 - 7.18 (m, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.62 (m, 2H), 1.38 - 1.36 (m, 2H), 0.945 (t, J = 7.2 Hz, 3H).), which was a yellow solid.

[0054] Synthesis of PTPRO inhibitor:

[0055] 10 mL of acetone was added to a 250 mL three-necked flask (R1) filled with N2, and compound 1 (10.0 g, 54.6 mmol, 1.00 eq), TFA (92.1 g, 807 mmol, 60.0 mL, 14.8 eq), and TFAA (30.2 g, 144 mmol, 20.0 mL, 2.63 eq) were added. The mixture in R1 was stirred at 80 °C for 1 hour. Acetone (10 mL) was added to R1 at 80 °C, and the mixture in R1 was stirred at 80 °C for 12 hours. 1H NMR: (400 MHz, CDCl3). δ 8.58 (m, 1H), 8.52 (dd, J = 9.2, 2.4 Hz, 1H), 7.39 (m, 1H), 1.76 (s, 6H) indicated the formation of the desired product; R1 was cooled to 25 °C; Sat. NaHCO3 (400 mL) was added to a 2.00 L conical flask (R2); The reaction mixture was concentrated in R1; DCM (200 mL) was collected from R1; The mixture in R1 was added to R2; The mixture in R2 was stirred at 25 °C for 10 minutes; The mixture in R2 was transferred to a separate 1.00 L separatory funnel (R3); DCM (200 mL) was added to R3, and the organic layer was separated. The aqueous phase was extracted with DCM (100 mL × 2); The combined organic layers were washed with water (100 mL × 3) and brine (100 mL), dried over Na2SO4, filtered, and concentrated to give compound 2 (14.5 g, crude) as a yellow solid, 1H NMR: (400 MHz, CDCl3). δ 8.88 (m, 1H), 8.44 (dd, J = 8.8, 2.8 Hz, 1H), 7.13 (m, 1H), 1.80 (s, 6H).

[0056] Compound 2 (14.5 g, 65.0 mmol, 1.00 eq) was charged into a 500 mL autoclave (R1); ethanol (150 mL) and palladium / carbon (1.45 g, 1.36 mmol, 10% purity, 2.10 e -2 eq) were added to R1; R1 was degassed under vacuum, purged several times with argon, and then purged with H2; R1 was stirred at H2 (1 MPa) and 25 °C for 24 hours; LCMS: product: RT = 0.780 min, MS (ESI) m / z = 194.1 [M+H] + It was shown that Compound 2 was consumed and the desired mass (RT = 0.780 minutes) was observed; the reaction solution was filtered through diatomaceous earth; the filtrate was concentrated to obtain Compound 3 (13.7 g, 55.9 mmol, 86.0% yield, 78.8% purity)( 1 1H NMR: (400 MHz, CDCl3) δ 7.23 (m, 1H), 6.89 (m, 1H), 6.80 - 6.77 (m, 1H), 3.71 (br, 2H), 1.70 (s, 6H).), as a yellow solid.

[0057] In a 1.00 L three-necked flask (R1) filled with N2 gas, toluene (150 mL), Compound 3 (10.0 g, 51.76 mmol, 1.00 eq), and Compound 3a (13.6 g, 51.8 mmol, 1.00 eq) were added, and R1 was stirred at 100 °C for 3.5 hours; R1 was cooled to 0 °C; THF (150 mL) and methanol (150 mL) were added to R1, and NaBH4 (5.93 g, 157 mmol, 3.03 eq) was added to R1 at 0 °C; R1 was stirred at 0 °C for 1 hour; TLC (petroleum ether / ethyl acetate = 3 / 1) indicated that Reactant 1 was consumed and a new major spot (R f = 0.30) was observed. The reaction mixture was poured into brine (200 mL) and extracted with MTBE (2 × 100 mL); the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the crude product; the crude product (petroleum ether / ethyl acetate = 3 / 1, P1, R f = 0.30) was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 10 / 1) to obtain Compound 4 (10.5 g, crude), as a yellow solid.

[0058] In a 250 mL round-bottom flask (R1), add DCE (150 mL), compound 4 (10.5 g, 23.9 mmol, 1.00 eq), hexanal (3.83 g, 38.2 mmol, 4.59 mL, 1.60 eq), and sodium triacetoxyborohydride (16.2 g, 76.4 mmol, 3.20 eq); stir R1 at 80 °C for 12 h; LCMS: product: RT = 2.234 min, MS(ESI) m / z = 524.4 [M+H] + It was shown that compound 4 was consumed and the desired mass (RT = 2.234 min) was observed; cool R1 to 25 °C and wash the reaction mixture with H2O (100 mL); then transfer the mixture to a separate 1.00 L separatory funnel (R2); add DCM (100 mL) to R2, separate the organic layer, and extract the aqueous phase with DCM (100 mL × 2); wash the combined organic layers with water (50 mL × 3) and brine (100 mL), dry over Na2SO4, filter, and concentrate to obtain the crude product; the crude product (petroleum ether / ethyl acetate = 3 / 1, P1, R f = 0.50) was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 10 / 1) to give compound 5 (10.0 g, 18.4 mmol, crude) as a yellow oil.

[0059] In a 2.00 L round-bottom flask (R1), add compound 5 (10.0 g, 19.1 mmol, 1.00 eq), methanol (1.00 L), H2O (100 mL), NaOH (5.00 M, 16.0 mL, 4.20 eq), and stir R1 at 25 °C for 2 h; LCMS: product: RT = 0.844 min, MS(ESI) m / z = 498.2 [M+H] + It was shown that compound 5 was consumed and the desired mass (RT = 0.844 min) was observed; filter the mixture, concentrate the filter cake to obtain compound 6 (9.00 g, 17.1 mmol, crude) as a yellow solid.

[0060] In a 2.00 L round-bottom flask (R1), add compound 6 (9.00 g, 18.1 mmol, 1.00 eq), MeOH (900 mL), H2O (90 mL), aq. NaOH (5.00 M, 14.5 mL, 4.00 eq), and stir R1 at 60 °C for 12 h; LCMS: product: RT = 0.756 min, MS(ESI) m / z = 484.0 [M+H] +Compound 6 was shown to be consumed, and the desired mass (RT = 0.756 min) was observed; R1 was cooled to 25 °C, the reaction mixture was concentrated, and then HCl (5.00 M, 15 mL) was added to the mixture; the mixture was transferred to a 500 mL separatory funnel (R2); MTBE (200 mL) was added to R2, the organic layer was separated, and the aqueous phase was extracted with MTBE (50 mL × 2); the combined organic layers were washed with water (100 mL × 3) and brine (100 mL), dried over Na2SO4, filtered, and concentrated to give the crude product; the crude product (petroleum ether / ethyl acetate = 3:1, P1:R f = 0.30) was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 10 / 1) to give a yellow solid; the yellow solid was triturated with (DCM / MeOH = 1 / 3) at 25 °C for 2 h, then the mixture was filtered, and the cake was concentrated to give a white solid of compound 1 (PTPRO inhibitor) (1.00 g, 2.06 mmol, 11.4% yield, 99.8% purity), 1 1H NMR: (400 MHz, DMSO-d6) δ 7.42 - 7.47 (m, 4H), 7.26 - 7.22 (m, 4H), 7.03 - 6.96 (m, 2H), 6.78 - 6.80 (m, 1H), 4.47 (s, 2H), 2.59 (t, J = 7.6 Hz, 2H), 1.55 - 1.53 (m, 4H), 1.28 - 1.25 (m, 8H), 0.91 - 0.84 (m, 6H). LCMS: product: RT = 1.154 min, MS(ESI) m / z = 484.3 [M+H] + , HPLC: 99.7% purity (area%).

[0061] The 1H NMR spectrum of the PTPRO inhibitor prepared in this example is as Figure 1 shown, and the mass spectrum is as Figure 2 shown.

[0062] Example 2

[0063] Effect of PTPRO inhibitor on the mortality of murine sepsis

[0064] The PTPRO inhibitor in this example was obtained by the preparation in Example 1.

[0065] 50 mg of the PTPRO inhibitor was dissolved in DMSO to a final concentration of 16.67 mg / mL for standby.

[0066] Wild-type mice (C57BL / 6 strain) were randomly divided into four groups: control group (Control), PTPRO inhibitor treatment group (Pi), LPS treatment group (LPS), and PTPRO inhibitor + LPS treatment group (LPS+Pi). Mice in the control group were given intraperitoneal injection of PBS, mice in the PTPRO inhibitor treatment group were given intraperitoneal injection of 10 mg / kg PTPRO inhibitor, mice in the LPS treatment group were given intraperitoneal injection of 10 mg / kg LPS, and mice in the PTPRO inhibitor + LPS treatment group were first given intraperitoneal injection of PTPRO inhibitor (dose: 10 mg / kg), and then intraperitoneal injection of LPS (dose: 10 mg / kg) 0.5 hours later. 24 hours after intraperitoneal injection of LPS, the 24-hour mortality of the mice was recorded. This experiment was conducted three times, with 5 mice in each group per batch. The results are as Figure 3 shown.

[0067] According to Figure 3 it can be seen that in the three batches of experiments, there were no deaths in the control group and the PTPRO inhibitor group. In the first batch, 2 out of 5 mice died in the LPS treatment group, and 1 died in the PTPRO inhibitor + LPS treatment group; in the second batch, 2 out of 5 mice died in the LPS treatment group, and none died in the PPTPRO inhibitor + LPS treatment group; in the third batch, 3 out of 5 mice died in the LPS treatment group, and 2 died in the PTPRO inhibitor + LPS treatment group. It can be seen that the PTPRO inhibitor significantly reduced the acute mortality within 24 hours caused by LPS-induced sepsis.

[0068] Example 3

[0069] Effect of PTPRO inhibitor on the change of blood-brain barrier permeability in septic mice

[0070] The PTPRO inhibitor in this example was prepared as obtained in Example 1.

[0071] 50 mg of PTPRO inhibitor was dissolved in DMSO to a final concentration of 16.67 mg / mL for standby.

[0072] Wild-type mice (C57BL / 6 strain) were randomly divided into four groups: control group (Control), PTPRO inhibitor treatment group (Pi), LPS treatment group (LPS), and PTPRO inhibitor + LPS treatment group (LPS+Pi). Mice in the control group were given intraperitoneal injection of PBS, mice in the PTPRO inhibitor treatment group were given intraperitoneal injection of 10 mg / kg PTPRO inhibitor, mice in the LPS treatment group were given intraperitoneal injection of 10 mg / kg LPS, and mice in the PTPRO inhibitor + LPS treatment group were first given intraperitoneal injection of PTPRO inhibitor (dose: 10 mg / kg), and then intraperitoneal injection of LPS (dose: 10 mg / kg) 0.5 hours later.

[0073] After 24 hours of LPS injection in four groups of mice, the mice were perfused with cold PBS through the ventricle to remove the influence of proteins in the peripheral blood of the mice. Then, the brain tissues were taken, 1 mL of PBS was added, and after homogenization, the supernatant of the brain tissue homogenate was taken by centrifugation at 10,000 rpm. The expression level of albumin in the brain tissue homogenate of the mice was detected by Western blot to evaluate the influence of the PTPRO inhibitor on the degree of blood-brain barrier permeability damage (the expression level of albumin is positively correlated with the degree of blood-brain barrier permeability damage). This experiment was conducted three times, with 3 mice in each group per batch, and the results were the average values of the three batches of experiments. The results are as Figure 4 shown.

[0074] According to Figure 4 it can be seen that after the four groups of mice were treated, 24 hours after LPS injection, the expression levels of brain tissue albumin in the LPS treatment group and the PTPRO inhibitor + LPS treatment group were both higher than those in the control group, and the expression level of brain tissue albumin in the PTPRO inhibitor + LPS treatment group of mice was significantly lower than that in the LPS treatment group. It can be seen that the PTPRO inhibitor significantly weakens the degree of blood-brain barrier permeability damage in LPS-induced septic mice.

[0075] Example 4

[0076] Effect of PTPRO inhibitor on the total number of cells in the bronchoalveolar lavage fluid of mice

[0077] The PTPRO inhibitor in this example was prepared as obtained in Example 1.

[0078] 50 mg of the PTPRO inhibitor was dissolved in DMSO to a final concentration of 16.67 mg / mL for standby.

[0079] Wild-type mice (C57BL / 6 strain) were randomly divided into four groups: control group (Control), PTPRO inhibitor treatment group (Pi), LPS treatment group (LPS), and PTPRO inhibitor + LPS treatment group (LPS + Pi). The mice in the control group were given intraperitoneal injection of PBS, the mice in the PTPRO inhibitor treatment group were given intraperitoneal injection of 10 mg / kg of the PTPRO inhibitor, the mice in the LPS treatment group were given intraperitoneal injection of 10 mg / kg of LPS, and the mice in the PTPRO inhibitor + LPS treatment group were first given intraperitoneal injection of the PTPRO inhibitor (dose: 10 mg / kg), and 0.5 hours later, LPS (dose: 10 mg / kg) was given by intraperitoneal injection.

[0080] After 24 hours of LPS injection in four groups of mice, the fur around the neck bronchi of the mice was cut open. The fascia covering the bronchi was gently separated with forceps. A small incision was made on the bronchi with surgical scissors, and the indwelling needle was inserted into the bronchi along the incision. 1 mL of sterile PBS was injected into the lungs. When injecting the sterile PBS, it should be slowly pushed in. The 1 mL of sterile PBS was pushed out in about 30 s, and then stagnated for 10 s. Then, the previously injected sterile PBS was slowly withdrawn in about 30 s and collected into a centrifuge tube. Centrifugation was carried out at 4°C and 500 rpm for 20 min. The precipitate was resuspended with 200 μL of sterile PBS, and a counting plate was used to count the total number of cells in the bronchoalveolar lavage fluid (BAL). This experiment was carried out three times. Among them, there were 7 mice in each group of the LPS treatment group (LPS) and the PTPRO inhibitor + LPS treatment group (LPS+Pi) in each batch. The results were the mean values of the three batches of experiments. The results are as Figure 5 shown.

[0081] According to Figure 5 it can be seen that after the four groups of mice were treated and 24 hours after LPS injection, the total number of cells in the bronchoalveolar lavage fluid of the mice in the LPS treatment group and the PTPRO inhibitor + LPS treatment group was higher than that of the control group, and the total number of cells in the bronchoalveolar lavage fluid of the mice in the PTPRO inhibitor + LPS treatment group was significantly lower than that of the LPS treatment group. The PTPRO inhibitor of the present invention significantly weakened the infiltration of cells in the bronchoalveolar lavage fluid of LPS-induced septic mice, suggesting that the PTPRO inhibitor may weaken the degree of LPS-induced lung injury.

[0082] Example 5

[0083] Effect of PTPRO inhibitor on neutrophil apoptosis signal

[0084] The PTPRO inhibitor in this example was prepared as obtained in Example 1.

[0085] 50 mg of the PTPRO inhibitor was dissolved in DMSO to a final concentration of 20 nM for standby.

[0086] Normal human peripheral blood neutrophils and mouse bone marrow neutrophils were extracted and randomly divided into four groups: control group (Control), PTPRO inhibitor treatment group (Pi), LPS treatment group (LPS), and PTPRO inhibitor + LPS treatment group (LPS+Pi). Neutrophils in the control group were added with PBS, the PTPRO inhibitor treatment group was given 20 nM PTPRO inhibitor for stimulation, the LPS treatment group of cells was given 1 μg / ml LPS stimulation, and the cells in the PTPRO inhibitor + LPS treatment group were first given 20 nM PTPRO inhibitor for 1 h and then given LPS stimulation for 20 h. After stimulation, cell proteins were collected, and the expression levels of caspase-3 / cleaved caspase-3, / cleaved RARP, and Bcl-2 signals were detected by Western blot. The human neutrophil experiment was conducted three times, and neutrophils were extracted from the peripheral blood of 1 normal person each time for the experiment. The results were the average of three batches of experiments; the mouse bone marrow neutrophil experiment was conducted 6 times, and bone marrow neutrophils from 6 mice were taken each time for the experiment. The results are as Figure 6 shown.

[0087] As Figure 6 shown, after human neutrophils were treated with PTPRO inhibitor alone in vitro, the apoptosis signals (cleaved caspase 3 / PARP, bcl-2) were significantly enhanced. In addition, compared with the LPS treatment group, the apoptosis signals in the PTPRO inhibitor + LPS treatment group were also significantly increased, indicating that the PTPRO inhibitor can promote neutrophil apoptosis in vitro regardless of the presence of inflammation.

[0088] Example 6

[0089] Effect of PTPRO inhibitor on the inflammatory response of mouse alveolar macrophages

[0090] The mouse alveolar macrophage MSH cell line was randomly divided into four groups: control group (Control), PTPRO inhibitor treatment group (Pi), LPS treatment group (LPS), and PTPRO inhibitor + LPS treatment group (LPS+Pi). Neutrophils in the control group were added with PBS, the PTPRO inhibitor treatment group was given 20 nM PTPRO inhibitor for stimulation, the LPS treatment group of cells was given 10 ng / ml LPS stimulation, and the cells in the PTPRO inhibitor + LPS treatment group were first given 20 nM PTPRO inhibitor for 1 h and then given LPS stimulation for 24 h. After stimulation, cell supernatants were collected, and the expression level of the cytokine TNF-α secreted by the cells in the supernatant was detected by ELISA. There were 5 mice in each group of this experiment, and the results were the average of each group of experiments. The results are as Figure 7 shown.

[0091] AsFigure 7 As shown in the results, after LPS stimulation, the level of TNF-α secreted by mouse alveolar macrophages was significantly higher than that in the control group. PTPRO inhibitor stimulation alone did not cause mouse alveolar macrophages to secrete TNF-α. In addition, compared with the LPS treatment group, the level of TNF-α secreted by cells in the PTPRO inhibitor + LPS treatment group was significantly reduced, indicating that PTPRO inhibitor can inhibit the level of TNF-α secreted by alveolar macrophages in the inflammatory microenvironment, which may further protect against acute lung injury caused by sepsis.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a PTPRO inhibitor in the preparation of a drug for treating sepsis, characterized in that, The structural formula of the PTPRO inhibitor is as follows: The sepsis is induced by LPS.

2. Use of a PTPRO inhibitor in the preparation of a medicament for treating pulmonary inflammatory diseases, characterized in that, The structural formula of the PTPRO inhibitor is as shown in claim 1, and the pulmonary inflammatory disease is caused by LPS.

3. The application according to claim 2, characterized in that, The characteristics of the pulmonary inflammatory disease are diffuse alveolar damage, aggregation of inflammatory cells, destruction of the alveolar-capillary membrane barrier function, and production of inflammatory factors.

4. The application according to claim 2, wherein The PTPRO inhibitor increases the expression level of neutrophil apoptosis signals.

5. The application according to claim 2, wherein The PTPRO inhibitor inhibits the secretion of TNF-α by alveolar macrophages.

6. Use of a PTPRO inhibitor in the preparation of a drug for improving blood-brain barrier permeability, characterized in that, The structural formula of the PTPRO inhibitor is as shown in claim 1, and the blood-brain barrier permeability is caused by LPS-induced sepsis.

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