Application of Nogo-B protein in preparation of medicine serving as anti-osteosarcoma target

Abstract

The invention relates to application of Nogo-B protein in preparation of a medicine serving as an anti-osteosarcoma target spot. The invention discloses an alpha-hydroxyl / alkoxy / amino-beta-amino derivative, a synthesis method thereof and application of Nogo-B protein serving as a new osteosarcoma target, an osteosarcoma diagnostic reagent and a small-molecule inhibitor 4v serving as an osteosarcoma targeting drug. A diazo compound, water / alcohol / amide and imine are used as raw materials, rhodium acetate is used as a catalyst, a product is obtained in an organic solvent through a one-step reaction with high selectivity and high yield, then the small-molecule inhibitor 4v is obtained through osteosarcoma cell surface screening, a new osteosarcoma target Nogo-B is proposed based on a proteomics analysis technology, and the new target Nogo-B is used for inhibiting osteosarcoma. The effects of inhibiting cell proliferation and migration and tumor growth can be achieved through experiments such as knockout of Nogo-B protein. The invention discloses a novel application of Nogo-B protein. The Nogo-B protein is used for preventing, relieving and treating osteosarcoma and osteosarcoma-related complications of individuals in need. Also disclosed are methods of diagnosing, treating, and monitoring osteosarcoma or methods of inhibiting osteosarcoma, and methods of screening for osteosarcoma inhibitors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical biology technology, and relates to the use of Nogo-B protein in the preparation of a drug as an anti-osteosarcoma target. BACKGROUND

[0002] Osteosarcoma, also known as osteogenic sarcoma, has a relatively high incidence rate in children and adolescents, followed by the population over the age of fifty. Osteosarcoma is one of the common primary malignant tumors, accounting for about 60% of childhood malignant bone tumors. According to relevant research, osteosarcoma still accounts for a high proportion in the mortality rate of adolescent malignant tumors. Osteosarcoma is derived from osteogenic mesenchymal cells, and the pathogenesis is not yet clear.

[0003] There is evidence that osteosarcoma has a high risk of metastasis, especially lung metastasis, in which more than 90% of patients with lung metastasis osteosarcoma, the brain metastasis rate of patients is low, but according to clinical data statistics, the brain metastasis rate of patients with lung metastasis rises to 10-15%, and the prognosis is poor, with a 5-year survival rate of only about 20%. Early osteosarcoma patients may have symptoms such as stage bone pain (night pain aggravated), fever of unknown origin, skin swelling and hyperemia, etc.

[0004] Due to the high invasiveness and high metastasis of osteosarcoma, and the fact that it often occurs in adolescents under the age of 20, it is the second highest cause of death from childhood malignant tumors, seriously threatening the life and health of children and adolescents. There are ten subtypes in clinical pathology histology, and the prognosis of patients is very poor. The pathogenesis is very complex, the cause of the disease is not clear, and effective drug targets have not been found, and no targeted drugs are currently on the market. For a long time, people have tried to study the potential targets of osteosarcoma at the genomic level by using osteosarcoma driver genes, main signal pathways involved, immune expression microenvironment, multi-omics methods, etc., but the effect is very small. Compared with other adolescent malignant tumors (mutation load about 0.1 Mut / Mb), osteosarcoma has a higher mutation load (1.2 Mut / Mb), but these mutations are more scattered, showing high complexity of structural heterogeneity, extensive chromothripsis and kataegis phenomenon, and no single, common high-frequency driver gene has been found. Literature reports two potential targets RB1 and P53 by gene sequencing, but no corresponding targeted drug molecules have been found so far.

[0005] Small molecule targeted therapy may be a more effective treatment for osteosarcoma. The efficient synthesis of a large number of structurally diverse molecules through synthetic chemistry, combined with high-throughput screening technology, provides a highly efficient approach for phenotypic screening of osteosarcoma, leading to the discovery of novel targets. Specifically, this involves first obtaining highly active and selective small molecule compounds through high-throughput screening; then, using proteomics molecular techniques and mass spectrometry analysis, potential targets can be identified, followed by biological validation of these potential targets.

[0006] The RTN protein family is a newly discovered gene family in recent years. To date, the function of the RTN family remains unclear. However, the RTN1 / NSP isoform has been reported as an independent marker distinguishing small cell lung cancer (SCLC) from non-small cell lung cancer (non-SCLC). RTN4 / Nogo is another recently discovered member of the human RTN family. Similar to RTN1 and RTN2, it also has three isoforms with the same C-terminus (RTN4A, 4B, and 4C). The RTN4 isoform shares a common C-terminus with the RTN1 / NSP, RTN2, and RTN3 isoforms, and all four show high homology in their C-terminus. Research literature reports high expression of this protein in various tumor tissues, but there are no reports of it being used as a drug target in osteosarcoma. Summary of the Invention

[0007] Based on the current status and technology of osteosarcoma, this invention proposes a small molecule inhibitor (S,R)-4v obtained through osteosarcoma cell phenotypic screening, which can be used as an osteosarcoma inhibitor. Combined with Abbreviated Proteomics Profiling (ABPP) technology, a novel osteosarcoma target, Nogo-B, is proposed, along with new applications for Nogo-B protein as an anti-osteosarcoma target. These applications are used for the prevention, alleviation, and treatment of osteosarcoma and its related complications in individuals with specific needs. Specifically, this invention relates to the use of Nogo-B protein as a novel tumor target, tumor diagnostic and differential diagnostic reagents targeting Nogo-B, and its use in the preparation of new anti-tumor drugs.

[0008] This invention discloses an original Nogo-B inhibitor (S,R)-4v and uses this inhibitor as a tool to reveal Nogo-B as a therapeutic target for osteosarcoma or osteosarcoma-related tumors. The Nogo-B inhibitor is an α-hydroxy / alkoxy / amino-β-amino derivative. This invention also proposes Nogo-B inhibitors and other osteosarcoma treatment agents for combination therapy to prevent, alleviate, and treat osteosarcoma and osteosarcoma-related complications in individuals in need. This invention also proposes methods for diagnosing, treating, and monitoring osteosarcoma, as well as methods for screening osteosarcoma inhibitors or methods for inhibiting osteosarcoma.

[0009] This invention proposes a biomarker called Nogo-B.

[0010] The Nogo-B protein is a membrane-bound protein primarily found in the endoplasmic reticulum (ER). It participates in the formation of tubular ER, membrane transport, inhibition of axon growth, and apoptosis. There are three main isotypes: RTN4A (Nogo-A, operating at ~200 kDa), RTN4B (Nogo-B, 40-55 kDa), and RTN4C (Nogo-C, 22-25 kDa). The amino acid sequence of Nogo-B is as follows:

[0011] MEDLDQSPLVSSSDSPPRPQPAFKYQFVREPEDEEEEEEEEEEDEDEDLEELEVLERKPAAGLSAAPVPTAPAAGAPLMDFGNDFVPPAPRGPLPAAPPVAPERQPSWDPSPVSSTVPAPSPLSAAAVSPSKLPEDDEPPARPPPPPPASVSPQAEPVWTPPAPAPAAPPSTPAAPKRRGSSGSVVVD LLYWRDIKKTGVVFGASLFLLLSLTVFSIVSVTAYIALALLSVTISFRIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVDSLKFAVLMWVFTYVGALFNGLTLLILALISLFSVPVIYERHQAQIDHYLGLANKNVKDAMAKIQAKIPGLKRKAE(SEQ ID NO.1).

[0012] The present invention also proposes the application of the aforementioned biomarker Nogo-B as a drug target in the preparation of drugs for the prevention, relief and / or treatment of osteosarcoma and osteosarcoma-related complications.

[0013] Among them, the biomarker Nogo-B inhibits the proliferation, migration, metastasis and growth of osteosarcoma cells and osteosarcoma growth; affects the expression of related proteins and other physiological processes in the body; and significantly inhibits the proliferation rate of osteosarcoma cells and the tumor growth curve of tumor-bearing animals.

[0014] The osteosarcoma cells in question are type 143B osteosarcoma cells.

[0015] This invention also proposes an inhibitor of a biomarker, wherein the inhibitor is an α-hydroxy / alkoxy / amino-β-amino derivative, the structure of which is shown in formula (I).

[0016]

[0017] In the aforementioned formula (I),

[0018] R 1 The phenyl groups are H, halogen, alkyl, aryl, nitro, alkoxy, trifluoromethyl, cyano, ester, carboxyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, and ester-substituted phenyl.

[0019] R 2 The substituted phenyl groups are H, alkyl, aryl, benzyl, alkynyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, and aromatic heterocyclic-substituted phenyl.

[0020] R 3 R 4 Each of the following is independently alkyl, benzyl, phenyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, methyl 2-benzoate, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, olefin-substituted phenyl, alkyne-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, acetylamide-substituted phenyl, steroid, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, furan ring, thiophene ring, indole ring, pyrrole ring;

[0021] R 5 The substituted phenyl groups are H, alkyl, alkylacyl, phenyl, benzoyl, heterocyclic acyl, cyclopropionyl, benzyl, benzoyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, and acetyleneamide-substituted phenyl.

[0022] Preferably,

[0023] R 1 The phenyl groups are H, F, Cl, Br, C1-C10 alkyl, C1-C10 alkoxy, nitro, trifluoromethyl, cyano, ester, carboxyl, phenyl, halogen-substituted phenyl, C1-C10 alkyl-substituted phenyl, C1-C10 alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, and ester-substituted phenyl.

[0024] R 2The substituted phenyl groups are H, C1-C10 alkyl, benzyl, phenyl, halogen-substituted benzyl, C1-C10 alkoxy-substituted benzyl, C1-C10 alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, C1-C10 alkyl-substituted phenyl, C1-C10 alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, and aromatic heterocyclic-substituted phenyl.

[0025] R 3 R 4 The compounds are C1-C10 alkyl, benzyl, phenyl, halogen-substituted benzyl, C1-C10 alkoxy-substituted benzyl, C1-C10 alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, C1-C10 alkyl-substituted phenyl, C1-C10 alkoxy-substituted phenyl, methyl 2-benzoate, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, olefin-substituted phenyl, alkyne-substituted phenyl, steroid, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, furan ring, thiophene ring, indole ring, and pyrrole ring.

[0026] R 5 The substituted phenyl groups are H, C1-C10 alkyl, benzyl, phenyl, halogen-substituted benzyl, C1-C10 alkoxy-substituted benzyl, C1-C10 alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, C1-C10 alkyl-substituted phenyl, C1-C10 alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, and aromatic heterocyclic-substituted phenyl.

[0027] More preferably,

[0028] R 1 A halogen-substituted phenyl group;

[0029] R 2 Methyl, ethyl, and benzyl groups;

[0030] R 3 R 4 These are phenyl, halogen-substituted phenyl, and C1-C10 alkyl-substituted phenyl, respectively.

[0031] R 5 It can be H, methyl, ethyl, benzyl, benzoyl, halogen-substituted benzoyl, or C1-C10 alkyl-substituted benzoyl.

[0032] The inhibitor is a small molecule inhibitor α-hydroxy / alkoxy / amino-β-amino derivative compound 4v, with the structure shown below:

[0033]

[0034] The present invention also proposes a drug / drug composition comprising the biomarker as described above, or comprising a compound that can bind to or react with the biomarker as described above.

[0035] The present invention also proposes a detection reagent / kit that contains biomarkers that can be used to detect the biomarkers described above.

[0036] The present invention also proposes the use of the inhibitors of the biomarkers, the drugs / drug compositions, or the detection reagents / kits of the invention in the preparation, relief and / or treatment of osteosarcoma and osteosarcoma-related complications.

[0037] The inhibitors, drugs / drug compositions, and test reagents / kits can be used alone or in combination with other drugs.

[0038] This invention also proposes a method for synthesizing the α-hydroxy / alkoxy / amino-β-amino derivatives, using diazo compound 1, water / alcohol / amide 2, and imine 3 as raw materials, and rhodium acetate as a catalyst, in an organic solvent to react and obtain the α-hydroxy / alkoxy / amino-β-amino derivatives shown in formula (I); the reaction process is shown in formula (II):

[0039]

[0040] Among them, R 1 R 2 R 3 R 4 R 5 The definition is the same as that described in formula (I).

[0041] The reaction temperature is 0–40°C; preferably, it is 25°C.

[0042] The reaction time is 1–5 hours; preferably, it is 2 hours.

[0043] The molar ratio of the diazo compound represented by formula (1), water / alcohol / amide, and imine represented by formula (3) is 1.2:5:1-2.0:5:1; preferably, it is 1.2:5:1.

[0044] The amount of rhodium acetate catalyst used is based on the imine shown in formula (3), and is 0.5 mol% to 2.0 mol% of the imine shown in formula (3); preferably, it is 1.0 mol%.

[0045] The organic solvent is selected from one or more of DCM, DCE, EA, toluene, etc.; preferably, it is DCM.

[0046] The amount of organic solvent is based on the imine represented by the substrate formula (3), such that the concentration of the imine represented by formula (3) in the organic solvent is in the range of 0.05-0.15M; preferably, it is 0.1M.

[0047] The method specifically includes the following steps: first, dissolve the imine and Rh2(OAc)4 shown in formula (3) in the organic solvent, and add the diazo compound shown in formula (1) and the water / alcohol / amide organic solvent solution shown in formula (2) at 25°C to react and obtain the α-hydroxy / alkoxy / amino-β-amino derivative shown in formula (I).

[0048] Further, the α-hydroxy / alkoxy / amino-β-amino derivatives obtained from the reaction are separated and purified by column chromatography using a solution of ethyl acetate:petroleum ether at a volume ratio of 1:20 to 1:3.

[0049] This invention also proposes the application of the aforementioned α-hydroxy / alkoxy / amino-β-amino derivatives in the preparation of antitumor active drugs.

[0050] The tumors mentioned include osteosarcoma, lung cancer, and colorectal cancer.

[0051] The osteosarcoma cells are one or more of the following types: MG63 osteosarcoma cells, SJSA-1 osteosarcoma cells, U2OS osteosarcoma cells, SW1353 osteosarcoma cells, MNNG / HOS osteosarcoma cells, HOS osteosarcoma cells, and 143B osteosarcoma cells.

[0052] The present invention also proposes a method for screening candidate drugs and / or inhibitors for the prevention, mitigation and / or treatment of osteosarcoma and osteosarcoma-related complications, the method comprising detecting the effect of the candidate drugs and / or inhibitors on the biological effects of said biomarkers in a subject or a sample obtained from the subject.

[0053] The present invention also proposes a method for preventing, alleviating, and / or treating osteosarcoma and osteosarcoma-related complications in subjects in need, or a method for assessing the severity of patients with osteosarcoma and osteosarcoma-related complications, or a method for predicting the recurrence time of osteosarcoma and osteosarcoma-related complications in patients, or a method for predicting or assessing the suitability of Nogo-B treatment for said patients. The method includes: determining the Nogo-B level from blood or osteosarcoma tissue samples from said patient or subject, wherein the level is used to prevent, alleviate, and / or treat osteosarcoma in said subject, or to assess the severity of osteosarcoma in patients, or to predict whether a patient will develop osteosarcoma, or to predict or assess the suitability of Nogo-B-targeted treatment for said patients.

[0054] The beneficial effects of this invention include: the α-hydroxy / alkoxy / amino-β-amino derivative small molecule compound (S,R)-4v proposed in this invention has a high inhibitory effect on osteosarcoma; the diamino ester compound acts as an osteosarcoma inhibitor targeting Nogo-B; knockdown of Nogo-B can induce apoptosis of tumor cells; targeting Nogo-B causes changes in the expression of PI3K / AKT / NF-κB pathway proteins.

[0055] The beneficial effects of this invention also include: this invention provides a new target for tumor treatment, involving the role of Nogo-B in osteosarcoma and its application in the preparation of tumor-targeted drugs, and the technical solution of this invention has important clinical application value. Attached Figure Description

[0056] Figure 1-1 α-hydroxy / alkoxy / amino-β-amino derivatives 4v 1 H NMR and 13 Schematic diagram of C NMR;

[0057] Figure 1-2 α-hydroxy / alkoxy / amino-β-amino derivatives 4w 1 H NMR and 13 Schematic diagram of C NMR;

[0058] Figure 1-3 α-hydroxy / alkoxy / amino-β-amino derivatives 4a 1 H NMR and 13 Schematic diagram of C NMR;

[0059] Figure 1-4 α-hydroxy / alkoxy / amino-β-amino derivatives 4b 1 H NMR and 13 Schematic diagram of C NMR;

[0060] Figure 1-5 α-hydroxy / alkoxy / amino-β-amino derivatives 4c 1 H NMR and 13 Schematic diagram of C NMR;

[0061] Figure 1-6 α-hydroxy / alkoxy / amino-β-amino derivatives 4d 1 H NMR and 13 Schematic diagram of C NMR;

[0062] Figure 1-7 α-hydroxy / alkoxy / amino-β-amino derivatives 4e 1 H NMR and 13Schematic diagram of C NMR;

[0063] Figure 1-8 α-hydroxy / alkoxy / amino-β-amino derivatives 4f 1 H NMR and 13 Schematic diagram of C NMR;

[0064] Figure 1-9 4g of α-hydroxy / alkoxy / amino-β-amino derivatives 1 H NMR and 13 Schematic diagram of C NMR;

[0065] Figure 1-10 α-hydroxy / alkoxy / amino-β-amino derivatives 4h 1 H NMR and 13 Schematic diagram of C NMR;

[0066] Figure 1-11 α-hydroxy / alkoxy / amino-β-amino derivatives 4i 1 H NMR and 13 Schematic diagram of C NMR;

[0067] Figure 1-12 α-hydroxy / alkoxy / amino-β-amino derivatives 4j 1 H NMR and 13 Schematic diagram of C NMR;

[0068] Figure 1-13 α-hydroxy / alkoxy / amino-β-amino derivatives 4k 1 H NMR and 13 Schematic diagram of C NMR;

[0069] Figure 1-14 α-hydroxy / alkoxy / amino-β-amino derivatives 4l 1 H NMR and 13 Schematic diagram of C NMR;

[0070] Figure 1-15 α-hydroxy / alkoxy / amino-β-amino derivatives 4m 1 H NMR and 13 Schematic diagram of C NMR;

[0071] Figure 1-16 α-hydroxy / alkoxy / amino-β-amino derivatives 4n 1 H NMR and 13 Schematic diagram of C NMR;

[0072] Figure 1-17α-hydroxy / alkoxy / amino-β-amino derivatives 4o 1 H NMR and 13 Schematic diagram of C NMR;

[0073] Figure 1-18 α-hydroxy / alkoxy / amino-β-amino derivatives 4p 1 H NMR and 13 Schematic diagram of C NMR;

[0074] Figure 1-19 α-hydroxy / alkoxy / amino-β-amino derivatives 4q 1 H NMR and 13 Schematic diagram of C NMR;

[0075] Figure 1-20 α-hydroxy / alkoxy / amino-β-amino derivatives 4r 1 H NMR and 13 Schematic diagram of C NMR;

[0076] Figure 1-21 α-hydroxy / alkoxy / amino-β-amino derivatives 4S 1 H NMR and 13 Schematic diagram of C NMR;

[0077] Figure 1-22 α-hydroxy / alkoxy / amino-β-amino derivatives 4t 1 H NMR and 13 Schematic diagram of C NMR;

[0078] Figure 1-23 α-hydroxy / alkoxy / amino-β-amino derivatives 4u 1 H NMR and 13 Schematic diagram of C NMR;

[0079] Figure 1-24 α-hydroxy / alkoxy / amino-β-amino derivatives 5a 1 H NMR and 13 Schematic diagram of C NMR;

[0080] Figure 1-25 Inhibitory activity of 4v in osteosarcoma cell lines;

[0081] Figure 1-26 Separation of small molecule 4V and probe 4W and their inhibitory activity in the 143B cell line;

[0082] Figure 2-1 WB images obtained from probe 4w via ABPP experiment;

[0083] Figure 2-2 Scatter plot analysis of potential target sites detected by mass spectrometry and Venn diagram of protein data from three mass spectrometry detections;

[0084] Figure 2-3 The potential target Nogo-B and the active molecule (S,R)-4v showed a concentration- and time-dependent relationship.

[0085] Figure 2-4 (S,R)-4v inhibits 143B cell migration;

[0086] Figure 2-5 (S,R)-4v inhibits the proliferation of 143B cells;

[0087] Figure 2-6 The knockdown of the Nogo-B protein was validated.

[0088] Figure 2-7 Knocking down Nogo-B protein inhibited 143B cell migration;

[0089] Figure 2-8 Knocking down Nogo-B protein inhibited the proliferation of 143B cells;

[0090] Figure 3-1 (S,R)-4v inhibits the expression of PI3K / AKT / NF-κB pathway proteins in 143B cells;

[0091] Figure 3-2 Knockdown of Nogo-B protein reduced the expression of PI3K / AKT / NF-κB pathway proteins in 143B cells;

[0092] Figure 3-3 Nogo-B related pathway diagram. Detailed Implementation

[0093] The invention will be further described in detail below with reference to specific embodiments. Except for the content specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0094] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0095] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0096] Preparation of main solutions:

[0097] 1) 10×TBS solution: Weigh 60.55g Tris and 87.5g NaCl, add 800mL ddH2O, adjust the pH to 7.5, and then bring the volume to 1L.

[0098] 2) TBST solution: Take 100 mL of the above TBS solution, dilute it with 900 mL of ddH2O, add 1 mL of Tween 20, mix well and set aside.

[0099] 3) 5% blocking solution: Weigh 2g of skim milk and add TBST solution to make up to 40g.

[0100] 4) Cell lysis buffer: Measure 10 mL of RIPA Lysis Buffer, add 100 μL of 100 mM PMSF, and mix well.

[0101] 5) 10× Electrophoresis Buffer

[0102] Bring the volume to 2L, and dilute to 1× before use.

[0103]

[0104] 6) 10× wet transfer buffer

[0105] Bring the volume to 2L. When needed, dilute to 1× wet transfer buffer: 200mL of 10× wet transfer buffer, 100mL of methanol, and 700mL of ddH2O to bring the volume to 1L.

[0106] 7) Separating gel buffer (5 mL, freshly prepared)

[0107]

[0108] 8) Stacking gel buffer (2 mL, prepare fresh before use)

[0109]

[0110] Main instruments and equipment:

[0111] Table 3. Equipment Names and Sources:

[0112]

[0113] This invention synthesized a large number of structurally diverse small molecule compounds, which were then screened for cell phenotype. The α-hydroxy / alkoxy / amino-β-amino derivative compound (S,R)-4v was found to have high activity in the 143B cell line. Its activity was further confirmed by the determination of cell IC50. The results showed that the α-hydroxy / alkoxy / amino-β-amino derivative small molecule compound (S,R)-4v had good inhibitory activity in the osteosarcoma cell line 143B.

[0114] This invention further modifies inhibitors to obtain small molecule probe compounds for target studies. The aforementioned small molecule probe compounds are co-incubated with cells, and the target protein is enriched using ABPP technology. Proteomic profiling is performed, and Western blotting (WB) confirms the discovery of a new target, Nogo-B.

[0115] Furthermore, this invention, through the study of the migration ability of tumor cells by targeting Nogo-B, discovered using a small molecule inhibitor that (S,R)-4v reduces the migration ability of osteosarcoma cells by targeting Nogo-B; the shNogo-B transfection experiment observed the phenotype and apoptosis of osteosarcoma cells by knocking down Nogo-B, and found that knocking down Nogo-B can cause changes in cell phenotype and increase apoptosis.

[0116] Furthermore, Western blot analysis was performed on the expression levels of five proteins, Nogo-B, PI3K, AKT, NF-Kb, and Bcl-2, in the presence of different concentrations of (S,R)-4v. The results showed that the small molecule inhibitor (S,R)-4v reduced the expression of all five proteins in a dose-dependent manner.

[0117] Furthermore, by comparing the expression levels of four proteins—PI3K, AKT, NF-Kb, and Bcl-2—in normal 143B cells and 143B cells with Nogo-B protein knockdown, it was proposed that the expression of PI3K, AKT, NF-Kb, and Bcl-2 all decreased after Nogo-B protein knockdown.

[0118] Example 1: Synthesis of small molecule compounds of α-hydroxy / alkoxy / amino-β-amino derivatives and determination of their IC50 for cell activity.

[0119] (1) A 4V synthesis is shown in the following formula:

[0120]

[0121] The raw material imine is reacted with the catalyst dimer rhodium acetate and After weighing, the molecular sieve was transferred to a flask. The gas in the flask was displaced with nitrogen, and the flask was sealed with a rubber stopper. Anhydrous dichloromethane was then injected. The diazo reactants and acetylene amide were dissolved in the anhydrous dichloromethane and injected into the reaction system at a rate of 1 mL / h using a peristaltic pump. The reaction was allowed to proceed overnight. After the reaction was completed, the target product was obtained by column chromatography for separation and purification. In this example, the determination of compound 4v was performed using high-resolution mass spectrometry (molecular weight) and nuclear magnetic resonance (NMR) instruments (H1N and C1N spectrometry). The determination results are as follows: Figure 1-1 As shown.

[0122] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((2-chlorophenyl)oxy)-2-(2,4-dichlorophenyl)succinate (4v)

[0123] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((2-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)succinate(4v)

[0124]

[0125] 4.82(d,J=12.68Hz,1H),4.66(d,J=12.75Hz,1H),4.18–4.04(m,2H),3.72(s,3H),1.11(t,J=7.16Hz,3H). 13 C NMR (101MHz, CDCl3) δ170.5,169.0,163.4,143.4,135.1,133.8,132.9,132.1,131.3,131.1,130.7,129 .5,129.1,128.8,128.7,126.9,126.8,121.6,114.6,85.5,65.7,61.5,52.8,13.9.HRMS(ESI)Calcd.for C 29 H 28 Cl3N2O6(M+H) + 605.1007, found 605.1001.

[0126] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-phenyl succinate (4a)

[0127] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-phenylsuccinate(4a)

[0128]

[0129] J=9.58Hz,2H),4.86–4.73(m,2H),4.52(d,J=11.79Hz,1H),4.41(d,J=12.06Hz,1H),4.33(d,J=9.68Hz,1H) ,4.16–4.07(m,2H),4.02(m,1H),3.90(s,2H),3.86(s,3H),1.15(t,J=7.13Hz,3H),1.07(t,J=7.09Hz,2H). 13 C NMR (101MHz, CDCl3) δ171.4,170.9,170.6,170.3,163.3,163.2,143.9,143.5,138 .5,138.3,134.5,134.3,131.2,129.3,128.9,128.7,128.4,128.3,128.2,127.9, 127.5,127.3,127.2,127.0,126.9,126.7,121.7,121.5,114.5,114.4,86.7,86.2 ,67.9,67.8,65.2,64.4,61.7,61.2,52.6,52.4,13.9,13.8.HRMS(ESI)Calcd.for C 29 H 31 N₂O₆(M+H) + 503.2177, found 503.2179.

[0130] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(4-chlorophenyl)succinate (4b)

[0131] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(4-chlorophenyl)succinate(4b)

[0132]

[0133] 5.83Hz,2H),5.69–5.60(m,2H),4.94(d,J=7.38Hz,1H),4.90–4.70(m,3H),4.42(dd,J=32.07,11.86Hz,2H),4.2 4(d,J=9.61Hz,1H),4.18–3.98(m,3H),3.87(d,J=12.39Hz,5H),1.16(t,J=7.12Hz,3H),1.08(t,J=7.10Hz,2H). 13 CNMR (101MHz, CDCl3) δ171.0,170.5,170.3,170.1,163.4,143.6,143.3,138.1,13 7.9,134.9,134.8,133.2,132.9,131.3,129.7,129.6,129.1,128.6,128.4,128.3 ,128.1,127.5,127.3,127.0,126.8,126.6,121.7,121.6,114.7,114.5,86.3,85. 9,68.0,67.9,65.4,64.4,61.8,61.4,52.8,52.6,14.0,13.9.HRMS(ESI)Calcd.for C 29 H 30 ClN2O6(M+H) + 537.1787, found 537.1788.

[0134] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(4-bromophenyl)succinate (4c)

[0135] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(4-bromophenyl)succinate(4c)

[0136]

[0137] 6.64(d,J=8.38Hz,1H),6.51(d,J=8.38Hz,2H),6.34(d,J=16.57Hz,2H),6.20(m,2H),5.64(d,J=10.19Hz,2H),4.93(s,1H),4.76(d,J=28.5 7Hz, 2H), 4.42 (dd, J = 32.44, 11.70Hz, 2H), 4.10 (d, J = 7.48Hz, 3H), 3.86 (d, J = 13.42Hz, 5H), 1.15 (t, J = 7.29Hz, 3H), 1.09 (t, J = 6.93Hz, 2H). 13 CNMR (101MHz, CDCl3) δ170.9,170.5,170.3,170.2,163.6,163.5,143.6,143.4,138.1,137.9,133.7,133. 5,131.5,131.3,131.0,130.0,129.7,129.5,129.4,129.3,128.5,128.48,128.43,128.40,128.36,128.33 ,128.1,128.0,127.5,127.3,127.1,127.0,126.9,126.8,126.7,126.6,123.2,123.1,121.8,121.6,114.7 ,114.5,86.3,86.0,68.1,68.0,65.3,65.2,64.3,61.8,61.5,52.8,52.6,14.0,13.9.HRMS(ESI)Calcd.for C 29 H 30 BrN2O6(M+H) + 581.1282, found 581.1287.

[0138] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(4-(trifluoromethyl)phenyl)succinate (4d)

[0139] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(4-(trifluoromethyl)phenyl)succinate(4d)

[0140]

[0141] 6.66(d,J=8.47Hz,1H),6.53(d,J=8.41Hz,2H),6.38–6.27(m,2H),6.19(s,2H),5.65(d,J=10.13Hz,2H),4.98(s,1H),4.87–4.71(m ,3H),4.46(dd,J=29.68,11.74Hz,2H),4.17–3.98(m,4H),3.88(d,J=14.43Hz,5H),1.15(t,J=7.07Hz,3H),1.05(t,J=7.22Hz,2H). 13 C NMR (101MHz, CDCl3) δ170.7,170.2,170.1,170.0,163.4,143.5,143.3,138.8,13 8.6,137.9,137.7,131.2,129.9,129.7,128.7,128.4,128.3,128.1,127.6,127. 4,127.0,126.9,126.6,125.3,125.2,124.8,124.7,121.7,121.6,114.8,114.7, 86.3,86.1,68.2,65.6,64.3,61.8,52.8,52.6,13.9,13.7.HRMS(ESI)Calcd.for C 20 H 30 F3N2O6(M+H) + 571.2050, found 571.2043.

[0142] 4-Ethyl-1-methyl-2-([1,1'-biphenyl]-4-yl)-3-((4-acrylamidophenyl)amino)-2-(phenoxy)succinate (4e)

[0143] 4-ethyl 1-methyl 2-([1,1'-biphenyl]-4-yl)-3-((4-acrylamidophenyl)amino)-2-(benzyloxy)succinate(4e)

[0144]

[0145] 4.92–4.74(m,3H),4.52(dd,J=41.05,11.70Hz,2H),4.17–3.95(m,4H),3.89(d,J=15.96Hz,5H),1.15(t,J=7.12Hz,3H),1.06(t,J=7.13Hz,2H). 13C NMR (101MHz, CDCl3) δ171.4,170.9,170.6,170.3,163.3,143.8,143.5,141.7,141.5,141 .4,140.3,140.1,138.5,138.3,133.5,133.4,131.3,128.9,128.8,128.7,128.3,128.2, 128.0,127.5,127.4,127.2,127.1,126.9,126.7,126.6,121.7,121.6,114.6,114.5,86. 6,86.2,68.0,67.9,65.3,64.4,61.7,61.3,52.7,52.5,14.0,13.9.HRMS(ESI)Calcd.for C 35 H 35 N₂O₆(M+H) + 579.2490, found 579.2488.

[0146] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(3,4-difluorophenyl)succinate (4f)

[0147] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(3,4-difluorophenyl)succinate(4f)

[0148]

[0149] 5.65(dd,J=10.30,3.88Hz,2H),5.00(s,1H),4.89–4.75(m,3H),4.57(d,J=11.74Hz,1H),4.47(d,J=12.07Hz,1H) ,4.17–4.09(m,2H),4.07–3.97(m,1H),3.91(s,2H),3.87(s,3H),1.16(t,J=7.14Hz,3H),1.07(t,J=7.12Hz,2H). 13CNMR (101MHz, CDCl3) δ171.4,170.9,170.6,170.3,163.2,143.8,143.5,141.7,141.4,140.3 ,140.1,138.5,138.3,133.5,133.3,131.3,128.9,128.8,128.7,128.3,128.2,128.0,127.6, 127.5,127.4,127.2,127.14,127.11,127.0,126.9,126.7,126.6,121.7,121.5,114.6,114.5 ,86.6,86.2,68.0,67.9,65.3,64.4,61.7,61.3,52.7,52.5,14.0,13.9.HRMS(ESI)Calcd.for C 29 H 29 F2N2O6(M+H) + 539.1988, found 539.1986.

[0150] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(4-chloro-3-fluorophenyl)succinate (4g)

[0151] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(4-chloro-3-fluorophenyl)succinate(4g)

[0152]

[0153] 3.87(d,J=9.71Hz,5H), 1.17(t,J=7.20Hz,3H), 1.09(t,J=7.26Hz,2H). 13CNMR (101MHz, CDCl3) δ170.5,170.1,169.9,163.5,163.4,159.1,158.8,156.6,156.3,143.4,143.3,137. 8,137.6,135.6,135.5,131.3,130.4,129.9,129.7,128.5,128.4,128.3,127.6,127.5,127.0,126.9,126 .9,126.7,126.6,124.8,124.7,124.1,124.1,121.8,121.7,121.6,121.5,121.3,117.1,116.9,116.5,11 6.3,114.7,114.6,86.0,85.7,68.2,65.7,64.4,61.9,61.6,52.9,52.7,14.0,13.9.HRMS(ESI)Calcd.for C 29 H 29 ClFN2O6(M+H) + 555.1693, found 555.1699.

[0154] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(2,4-dichlorophenyl)succinate (4h)

[0155] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(2,4-dichlorophenyl)succinate(4h)

[0156]

[0157] 4.12(q,J=6.98Hz,2H),4.03–3.90(m,1H),3.83(s,1H),3.73(s,3H),1.13(t,J=7.12Hz,3H),1.04(t,J=7.11Hz,1H). 13C NMR (101MHz, CDCl3) δ170.6,170.5,170.1,169.0,163.4,163.3,143.4,142.8,137.2,135 .5,135.0,134.7,133.6,133.3,132.5,131.3,131.1,131.0,131.1,130.6,129.4,128.4, 128.3,127.8,127.6,127.4,127.2,127.0,126.9,126.8,121.9,121.6,114.5,114.0,85. 1,83.4,68.6,67.7,61.5,61.4,61.3,60.2,53.0,52.8,13.9,13.8.HRMS(ESI)Calcd.for C 29 H 29 Cl2N2O6(M+H) + 571.1397, found 571.1393.

[0158] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(naphth-1-yl)succinate (4i)

[0159] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(naphthalen-1-yl)succinate(4i)

[0160]

[0161] 16.86,10.17Hz,1H),5.67–5.62(m,1H),5.04(d,J=8.61Hz,1H),4.80(d,J=12.48Hz,1H),4.54(d,J=11.73 Hz,1H),4.41–4.30(m,1H),4.12–4.05(m,2H),3.90(s,3H),1.11(t,J=7.14Hz,3H),1.01(t,J=7.16Hz,2H). 13C NMR (101MHz, CDCl3) δ171.5,170.9,170.7,170.3,163.2,163.1,143.8,138.5,138.2,133.2,133 .1,132.8,132.6,132.1,131.2,129.8,129.2,128.5,128.3,128.2,127.8,127.6,127.5,127.48, 127.44,127.2,127.0,126.9,126.8,126.7,126.4,126.2,125.7,124.4,121.6,121.5,114.7,114 .5,86.9,86.4,68.1,68.0,65.2,64.7,61.7,61.3,52.7,52.5,13.9,13.8.HRMS(ESI)Calcd.forC 33 H 33 N₂O₆(M+H) + 553.2333, found 553.2332.

[0162] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(phenoxy)-2-(4-chlorophenyl)succinate (4j)

[0163] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(benzyloxy)-2-(4-chlorophenyl)succinate(4j)

[0164]

[0165] J=10.19Hz,2H),4.94(s,1H),4.82–4.72(m,2H),4.42(dd,J=32.20,11.78Hz,2H),4.10(d ,J=7.81Hz,4H),3.86(d,J=12.50Hz,5H),1.15(t,J=7.36Hz,3H),1.09(t,J=7.47Hz,2H). 13C NMR (101MHz, CDCl3) δ171.0,170.5,170.3,170.2,163.5,143.6,143.3,138.1,137. 9,134.9,134.8,133.2,133.0,131.3,129.8,129.7,129.1,128.6,128.4,128.3,128 .1,127.5,127.3,126.9,126.8,126.6,121.7,121.6,114.6,114.5,86.3,85.9,68. 0,68.0,65.4,64.4,61.8,61.4,52.7,52.5,14.1,14.0,13.9.HRMS(ESI)Calcd.forC 29 H 30 ClN2O6(M+H) + 537.1787, found 537.1779.

[0166] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((4-chlorophenyl)oxy)-2-(4-chlorophenyl)succinate (4k)

[0167] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((4-chlorobenzyl)oxy)-2-(4-chlorophenyl)succinate(4k)

[0168]

[0169] Hz,1H),6.19(dd,J=16.84,10.14Hz,1H),5.71(dd,J=10.42,4.69Hz,1H),5.15(d,J=8.38Hz,1H),4.82(d,J=12.68Hz,1H), 4.69–4.60(m,1H),4.35(d,J=10.04Hz,1H),4.16–4.05(m,2H),3.73(s,3H),1.12(t,J=7.14Hz,3H),1.03(t,J=7.13Hz,1H). 13C NMR (101MHz, CDCl3) δ170.4,168.9,163.1,143.5,135.2,133.8,132.9,132.1,131.2,131.1,130.7,129 .0,128.8,128.7,127.0,126.9,126.8,121.6,114.6,65.7,61.4,58.5,52.7,13.8.HRMS(ESI)Calcd.for C 29 H 29 Cl2N2O6(M+H) + 571.1397, found 571.1393.

[0170] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((4-bromophenyl)oxy)-2-(4-chlorophenyl)succinate (4l)

[0171] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((4-bromobenzyl)oxy)-2-(4-chlorophenyl)succinate(4l)

[0172]

[0173] 6.35(dd,J=16.87,5.42Hz,2H),6.20(m,2H),5.65(dd,J=10.36,4.02Hz,2H),4.89(d,J=8.49Hz,1H),4.79(s,1H),4.76–4.66(m,2H),4.39(d,J =12.03Hz,1H),4.32(d,J=11.97Hz,2H),4.11(dd,J=7.16,4.41Hz,4H),3.88(d,J=8.61Hz,5H),1.16(t,J=7.13Hz,3H),1.08(t,J=7.13Hz,2H). 13C NMR (101MHz, CDCl3) δ170.8,170.5,170.2,170.0,163.5,163.4,143.6,143.2,137. 2,137.1,135.1,134.9,133.0,132.8,131.5,131.4,131.3,129.8,129.7,129.6,129 .0,128.6,128.5,128.3,128.1,127.0,121.7,121.6,121.3,121.1,114.5,86.4,86 .0,67.4,67.3,65.3,64.6,61.9,61.5,52.8,52.6,14.0,13.9.HRMS(ESI)Calcd.for C 29 H 29 BrClN2O6(M+H) + 615.0892, found 615.0890.

[0174] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((2-bromobenzyl)oxy)-2-(4-chlorophenyl)succinate (4m)

[0175] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((2-bromobenzyl)oxy)-2-(4-chlorophenyl)succinate(4m)

[0176]

[0177] J=16.82,5.04Hz,1H),6.19(m,2H),5.70(dd,J=10.39,3.85Hz,1H),4.93(s,1H),4.89–4.73(m,2H),4.52(d,J=13.14Hz,1H),4.43 (d,J=13.51Hz,1H),4.18–4.09(m,2H),4.07–3.95(m,1H),3.89(d,J=7.30Hz,4H),1.18(t,J=7.12Hz,3H),1.09(t,J=7.14Hz,1H). 13C NMR (101MHz, CDCl3) δ170.7,170.4,170.0,163.2,143.7,143.4,137.5,137.4, 134.9,132.6,132.3,132.2,131.2,129.6,128.8,128.7,128.7,128.6,128.3, 128.2,127.5,127.4,127.1,121.8,121.7,121.5,114.6,114.6,86.4,86.2,67 .5,67.5,65.6,64.6,61.9,61.4,52.8,52.6,13.9,13.8.HRMS(ESI)Calcd.for C 29 H 29 BrClN2O6(M+H) + 615.0892, found 615.0890.

[0178] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((2-chlorophenyl)oxy)-2-(4-chlorophenyl)succinate (4n)

[0179] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((2-chlorobenzyl)oxy)-2-(4-chlorophenyl)succinate(4n)

[0180]

[0181] Hz,2H),6.35(dd,J=16.77,4.58Hz,2H),6.18(dd,J=17.16,9.17Hz,2H),5.66(d,J=10.15Hz,1H),4.99–4.75(m,3H),4.5 2(dd,J=37.19,13.33Hz,2H),4.20–3.94(m,3H),3.88(d,J=7.88Hz,5H),1.17(t,J=7.23Hz,3H),1.08(t,J=7.25Hz,1H). 13C NMR (101MHz, CDCl3) δ170.7,170.3,170.1,163.3,143.6,143.3,135.8,133.2,131.9,131.7,131.3,131.1,129.9,129.1,129.0,128.4,128.2, 128.0,127.0,126.9,126.8,123.2,121.7,121.6,114.6,86.5,86.2,65.5,65.2,64.6,61.9,61.5,52.8,52.6,13.9,13.8.HRMS(ESI)Calcd.for C 29 H 29 Cl2N2O6(M+H) + 571.1397, found 571.1393.

[0182] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((3-chlorophenyl)oxy)-2-(4-chlorophenyl)succinate (4o)

[0183] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((3-chlorobenzyl)oxy)-2-(4-chlorophenyl)succinate(4o)

[0184]

[0185] 22.30,11.87Hz,2H),4.40(d,J=12.10Hz,1H),4.25(d,J=9.65Hz,1H),4.06(dd,J=9.6 1,5.72Hz,2H),3.91(d,J=3.05Hz,4H),1.14(t,J=7.22Hz,3H),1.06(t,J=7.14Hz,2H). 13C NMR (101MHz, CDCl3) δ171.0,170.5,170.3,170.1,163.4,163.4,143.6,143.3,138. 1,137.9,134.9,134.8,133.2,132.9,131.3,129.7,129.6,129.1,128.6,128.3,128 .3,128.1,127.5,127.3,127.0,126.8,126.6,121.7,121.6,114.7,114.5,86.3,85. 9,68.0,67.9,65.4,64.4,61.8,61.4,52.8,52.6,14.0,13.9.HRMS(ESI)Calcd.forC 29 H 29 Cl2N2O6(M+H) + 571.1397, found 571.1393.

[0186] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-(furan-2-ylmethoxy)succinate (4p)

[0187] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-(furan-2-ylmethoxy)succinate(4p)

[0188]

[0189] J=12.11Hz,2H),4.19–4.10(m,1H),4.08–3.97(m,2H),3.90(d,J=2.78Hz,4H),1.14(t,J=7.18Hz,3H),1.05(t,J=7.16Hz,2H). 13C NMR (101MHz, CDCl3) δ171.0,170.5,170.1,170.0,163.3,163.2,151.3,151.2,143 .7,143.4,142.7,142.6,135.0,134.8,133.1,132.8,131.2,129.8,129.5,129.1, 128.6,128.1,127.0,121.6,121.5,114.6,110.4,110.3,109.2,108.9,86.2,85.8 ,65.3,64.4,61.7,61.3,61.1,60.9,52.8,52.6,13.9,13.8.HRMS(ESI)Calcd.for C 27 H 28 ClN2O7(M+H) + 527.1580, found 527.1587.

[0190] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-(thiophen-2-ylmethoxy)succinate (4q)

[0191] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-(thiophen-2-ylmethoxy)succinate(4q)

[0192]

[0193] 6.33(s,2H),6.18(m,2H),5.67(d,J=9.84Hz,2H),4.94–4.84(m,3H),4.64–4.51(m,2H),4.19 –4.08(m,2H),4.06–3.98(m,1H),3.91(s,4H),1.18(t,J=7.14Hz,3H),1.08(t,J=7.13Hz,2H). 13C NMR (101MHz, CDCl3) δ170.9,170.6,170.2,169.9,163.4,163.3,143.7,143.3,140.7, 140.6,135.0,134.9,133.1,132.8,131.2,129.8,129.6,129.5,129.0,128.6,128.1,1 27.0,126.5,126.4,125.7,125.68,125.63,125.5,121.7,121.5,114.6,114.4,86.5, 86.0,65.5,64.6,63.9,63.8,61.9,61.4,52.8,52.6,14.0,13.9.HRMS(ESI)Calcd.for C 27 H 28 ClN2O6S(M+H) + 543.1351, found 543.1354.

[0194] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-methoxysuccinate (4r)

[0195] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(4-chlorophenyl)-2-methoxysuccinate(4r)

[0196]

[0197] (dd,J=16.76,3.45Hz,2H),6.19(m,2H),5.71(d,J=10.18Hz,2H),4.83(dd,J=14.08,10.16Hz,2H),4.69(d,J=10.56Hz,1 H),4.32–4.00(m,5H),3.89(d,J=5.61Hz,5H),3.38(s,3H),3.34(s,2H),1.19(t,J=7.16Hz,3H),1.09(t,J=7.16Hz,2H). 13 C NMR (101MHz, CDCl3) δ171.7,163.3,142.7,136.0,134.2,131.2,130.3,129.7,129.1,1 28.9,128.6,127.0,121.9,113.7,60.2,58.4,53.0,18.4,14.1.HRMS(ESI)Calcd.forC 23 H26 ClN2O6(M+H) + 461.1474, found 461.1476.

[0198] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-((4-chlorophenyl)oxy)-2-(2,4-dichlorophenyl)succinate (4S)

[0199] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-((4-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)succinate(4s)

[0200]

[0201] 1.40Hz,1H),5.11(d,J=8.08Hz,1H),4.70(d,J=11.32Hz,1H),4.45(d,J=11.35Hz,1H), 4.34(q,J=7.96Hz,1H),4.11(m,2H),3.85(s,1H),3.74(s,3H),1.11(t,J=7.13Hz,3H). 13 C NMR (101MHz, CDCl3) δ170.4,169.0,163.33,143.4,135.8,135.1,133.7,133.5,133.1,131.3,131.0,130.7, 129.5,128.7,128.6,128.5,127.0,126.8,121.6,114.6,85.5,67.9,61.4,52.8,13.9.HRMS(ESI)Calcd.for C 29 H 28 Cl3N2O6(M+H) + 605.1007, found 605.1001.

[0202] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(2,4-dichlorophenyl)-2-((2-methoxy)oxy)succinate (4t)

[0203] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(2,4-dichlorophenyl)-2-((2-methoxybenzyl)oxy)succinate(4t)

[0204]

[0205] 6.38–6.30(m,1H),6.18(dd,J=16.88,9.79Hz,1H),5.66(dd,J=10.43,5.19Hz,1H),5.10(d,J=10.12Hz,1H),4.71(d,J=12.21Hz,1H),4.61 (d,J=11.97Hz,1H),4.40(d,J=10.29Hz,1H),4.18–4.05(m,2H),3.78(s,3H),3.73(s,3H),1.13(t,J=7.12Hz,3H),0.99(t,J=7.12Hz,1H). 13 C NMR (101MHz, CDCl3) δ170.73,169.04,163.27,156.53,143.50,134.92,133.74,133.52,131.35,131.28,130.47,129.32,128.73,128. 53,128.07,126.94,126.71,125.80,121.64,120.58,114.48,110.01,84.83,63.72,61.38,55.29,52.73,13.92.HRMS(ESI)Calcd.for C 30 H 31 Cl2N2O7(M+H) + 601.1503, found 601.1504.

[0206] 4-Ethyl-1-methyl-3-((4-acrylamidophenyl)amino)-2-(2,4-dichlorophenyl)-2-((4-methylbenzyl)oxy)succinate (4u)

[0207] 4-ethyl 1-methyl 3-((4-acrylamidophenyl)amino)-2-(2,4-dichlorophenyl)-2-((4-methylbenzyl)oxy)succinate(4u)

[0208]

[0209] J=29.25Hz,1H),4.69(d,J=10.76Hz,1H),4.46(d,J=10.79Hz,2H),4.12(t,J=7.18Hz,2H),4.02–3.90(m ,1H),3.82(s,1H),3.74(s,3H),2.34(d,J=5.27Hz,4H),1.14(t,J=7.12Hz,3H),1.04(t,J=7.11Hz,1H). 13 C NMR (101MHz, CDCl3) δ170.7,170.5,170.1,169.0,163.4,163.3,143.4,142.8,137.6,1 37.3,135.5,134.9,134.6,134.2,133.6,133.5,132.7,131.3,131.1,131.0,130.5,129 .4,129.1,129.0,127.6,127.3,127.0,126.9,126.7,121.9,121.6,114.5,114.0,84.9 ,83.4,68.6,67.7,61.5,61.4,61.2,60.2,52.9,52.8,13.9,13.8.HRMS(ESI)Calcd.for C 30 H 31 Cl2N2O6(M+H) + 585.1554, found 585.1555.

[0210] 4-Ethyl-1-(prop-2-yn-1-yl)3-((4-acrylamidophenyl)amino)-2-((2-chlorophenyl)oxy)-2-(2,4-dichlorophenyl)succinate (4w)

[0211] 4-ethyl 1-(prop-2-yn-1-yl)3-((4-acrylamidophenyl)amino)-2-((2-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)succinate(4w)

[0212]

[0213] 16.83,10.17Hz,1H),5.69(d,J=10.17Hz,1H),5.22(d,J=8.07Hz,1H),4.85(dd,J=22.71,14.12Hz,2H ), 4.68 (d, J = 13.29Hz, 2H), 4.36 (d, J = 9.74Hz, 1H), 4.10 (s, 2H), 2.48 (s, 1H), 1.09 (t, J = 7.13Hz, 3H). 13 C NMR (101MHz, CDCl3) δ170.2,167.9,163.2,143.5,135.3,135.2,133.9,132.4,132.1,131.2,131.1,130.7,129.4,12 9.0,128.8,128.7,127.1,126.9,121.6,114.8,85.9,76.3,76.0,65.8,61.7,61.6,53.3,13.8.HRMS(ESI)Calcd.for C 31 H 28 Cl3N2O6(M+H) + 629.1007, found 629.1012.

[0214] (Methacrylamide-2)-6-chloropyridin-3-yl-3-(4-methoxyphenyl)3-(phenylamino)propionate (5a)

[0215] Methyl2-acrylamido-2-(6-chloropyridin-3-yl)-3-(4-methoxyphenyl)-3-

[0216] (phenylamino)propanoate(5a)

[0217]

[0218] 5.79(d,J=10.0Hz,1H), 5.58(d,J=6.6Hz,1H), 3.77(d,J=1.7Hz,6H). 13 C NMR (101MHz, CDCl3) δ170.5,165.9,159.6,151.1,148.7,146.3,138.0,130.9,130.0,129.5,1 29.1,129.1,128.8,124.0,117.1,114.1,112.6,69.6,61.5,55.2,54.3.HRMS(ESI)Calcd.for C 25 H 25ClN3O4(M+H) + 466.1528, found 466.1524.

[0219] (2) The cell viability test procedure at 4V is as follows:

[0220] ① Prepare a 6cm culture dish of 143B cells. After trypsin digestion, count the cells and prepare a cell suspension of 3×10⁴ / mL. Add 100μL evenly to each well of a 96-well plate to maintain a cell count of 3×10⁴ / mL. 3 / hole, after the hole is completely attached to the wall, proceed with subsequent experiments;

[0221] ② On the second day, after the cells adhered, the culture medium was discarded: MEM culture medium containing different drug concentrations (4v) was added to each well, and the cells were placed in an incubator at 37°C and 5% CO2 for three days of culture before the CCK8 experiment was performed.

[0222] ③ Cell OD value determination: The CCK8 reagent was removed from the 4℃ refrigerator beforehand and allowed to equilibrate to room temperature. A CCK8 solution was prepared using MEM complete medium: 100 μL medium + 10 μL CCK8 solution, mixed well, and set aside. The 96-well plate was removed from the incubator, the previous medium was discarded, and 100 μL of the above mixed solution was added to each well. The 96-well plate was returned to the incubator and incubated for 1 hour. The OD value was measured at 450 nm using a microplate reader, and the data was recorded for subsequent analysis. The compound 4v prepared in Example 1 of this invention has an activity of 1360 nM. Figure 1-3 As shown.

[0223] Example 2: Confirmation of a New Target for Osteosarcoma

[0224] 4v inhibitor probe modification: The structure-activity relationship of compound 4v prepared in Example 1 of this invention was studied. The acetylene group was modified without affecting its approximate activity to obtain the modified probe molecule 4w. Then, compound 4v and probe molecule 4w were chirally separated to obtain (R,S)-4v, (S,R)-4v and (R,S)-4w, (S,R)-4w for osteosarcoma target research experiments.

[0225] Exploring new targets for osteosarcoma through a combination of protein proteomics analysis (ABPP):

[0226] ① Cell culture: 143B cells were cultured in MEM complete medium (90% MEM + 10% FBS, with 0.1% glutamine and 0.1% antibiotics, penicillin and streptomycin added if necessary) and placed in a 37℃, 5% CO2 incubator.

[0227] ② Cell drug delivery: Cultured 143B cells were co-incubated with probe-modified probe (S,R)-4w for 3 hours, and the probe concentration was set at IC50. 50 The amount of DMSO was 10 times that of the target compound, and a blank control and a competition group were set up. The blank control group used an equal amount of DMSO, and the competition control group used the active compound (R,S)-4w without probe modification.

[0228] ③ Cell collection: Remove the cell culture medium, wash the cells 2-3 times with pre-cooled PBS, add trypsin to digest the cells and collect them in a 1.5mL EP tube, centrifuge at 1200rpm for 5min, discard the supernatant and place on ice.

[0229] ④ Cell disruption: Add 300 μL of RIPA cell lysis buffer to the EP tube for cell collection, incubate on ice for 30 min, then disrupt the cells using an ultrasonic disruptor, collect the cell supernatant, and place it on ice.

[0230] ⑤ Bioorthogonal reaction: Add CuSO4, SA, Biotin-PEG2-N3 and TBTA at 10 times the probe concentration to the cell lysis buffer and react at room temperature for 2 hours.

[0231] ⑥ Ultrafiltration centrifugation: After the reaction is completed, the reaction solution is subjected to ultrafiltration centrifugation. The ultrafiltration centrifuge tube has a pore size of 10 kDa. Centrifuge at 4°C and 12,000 rpm for 10 min. Wash the protein solution after ultrafiltration centrifugation with ultrapure water twice. Repeat the above ultrafiltration centrifugation operation and transfer the remaining protein solution to a 1.5 mL EP tube.

[0232] ⑦ Magnetic bead enrichment: Purify the protein solution after ultrafiltration and centrifugation, add streptomycin affinity magnetic beads to enrich the target protein, and gently mix on a shaker for 2 hours at room temperature. After enrichment, place a 1.5 mL EP tube on a magnetic rack for 1-2 minutes to adsorb the protein, and discard the supernatant. Wash the remaining magnetic beads with 500 μL of ultrapure water, gently mix on a shaker for 10 minutes, and repeat the above operation 2-3 times after magnetic rack adsorption. Add 20-50 μL of 1×Loading Buffer to the magnetic beads after the last enrichment, boil for 5-10 minutes, and then cool to room temperature for later use.

[0233] ⑧ Electrophoresis: Take 5 mL of the lower gel buffer into a 1 mm gel casting plate, add ultrapure water and seal for 30 min, then discard the ultrapure water and add about 1 mL of the upper gel. Insert the casting comb and let it stand for 30 min. After the gel is prepared, take 5-10 μL of the above protein solution and load it onto the plate. Electrophore at 80 V for 30 min in constant voltage mode on the electrophoresis apparatus, and then electrophore at 120 V for 60 min to obtain well separated protein bands.

[0234] ⑨ Staining: Heat the gel strip for 2-3 minutes, then add Coomassie Brilliant Blue staining solution and stain for 30 minutes. After staining, decolorize overnight.

[0235] ⑩ Exposure: Take photos of the stained adhesive strip after exposure, such as... Figure 2-1 As shown, the results indicate that there is a clearly marked band at 50 kDa, and the effect is dose- and time-dependent.

[0236] Identification of the potential target protein Nogo-B using mass spectrometry: Mass spectrometry identification of proteins within a gel requires decolorization and liquid removal of the gel strip. Detailed experimental procedures are as follows:

[0237] ① Cutting the rubber: Cut the rubber strip into several small pieces.

[0238] ② Decolorization: Add sufficient amount of 50mM NH4CO3 (100-200ul) containing 50% ACN, vortex for 5 minutes, remove the liquid, and repeat 3 times. On the last time, let stand for 2-3 hours until the strip is colorless.

[0239] ③ Dehydration: Add sufficient ACN (100-200ul), vortex for 5 minutes, remove the liquid, and repeat 3 times.

[0240] ④ Drying: Remove ACN and dry by rotation; or let stand for 10 minutes.

[0241] ⑤ Reduction: Add sufficient 20mM DTT (100-200ul), react at 55℃ for 45min, cool and centrifuge, and remove the liquid.

[0242] ⑥ Alkylation: Add sufficient amount of 55mM IAA (100-200ul), and react at room temperature in the dark for 45min.

[0243] ⑦ Liquid removal: Aspirate the liquid, add sufficient ACN (100-200 μL), vortex for 5 minutes, aspirate the liquid, and repeat 3 times. Remove the ACN and rotate to dry; or let stand for 10 minutes.

[0244] ⑧ Enzymatic hydrolysis: Add 100 μL of 50 mM NH4CO3 and 2 μg of Trypsin, and react at 37°C for 12–16 h.

[0245] ⑨ Reconstitute: Reconstitute with 30 μl of 0.1% FA, centrifuge at 14000 g / min for 15 min, collect the supernatant and put it into a sample tube, take 1 μl of sample for mass spectrometry identification.

[0246] The mass spectrometry data files were searched using MaxQuant (Andromeda) and Proteome Discoverer software (Version PD 1.4, ThermoScientific, USA). The protein database was sourced from UniProt, with a maximum deviation of 10 ppm for the molecular weight of the precursor ion and 0.02 Da for the molecular weight of the daughter ion. The peptide false discovery rate (FDR) was set to <1%.

[0247] Venn diagram analysis of three mass spectrometry data ( Figure 2-2 Nogo-B was found to be recurring and highly abundant in mass spectrometry searches, such as... Figure 2-2 As shown.

[0248] Immunoblot assay of Nogo-B protein: After co-incubating live cells with different concentrations of the (S,R)-4v compound prepared in Example 1 of this invention, protein samples from cell lysis were used for SDS-polyacrylamide gel separation and transferred to a PVDF membrane. The membrane was then blocked with 5% skim milk in TBST at room temperature for 1 h. After blocking, the membrane was incubated with a monoclonal mouse antibody against Nogo-B for 2 h. After incubation, the PVDF membrane was washed with TBST (4 × 10 min) and then incubated with the corresponding secondary antibody for 1 h. The antibody dilution concentration was 1:2000. After incubation, the membrane was washed again with TBST, and then the protein on the PVDF membrane was exposed and photographed with chemiluminescent solution. The results showed that, compared with the control group, the Nogo-B protein in the treatment group showed a high-intensity band at 50 kDa.

[0249] Example 3: The role of Nogo-B protein in tumors

[0250] This study investigated the effect of Nogo-B on the migration ability of tumor cells: Migration and invasion are characteristics of tumor cells, and osteosarcoma cells have a strong ability to metastasize to the lungs. Scratch assays can explore the effect of drugs on the migration ability of tumor cells. This experiment was conducted on 143B cells. The specific protocol is as follows:

[0251] 143B cells were seeded at 350,000 cells per well in a six-well plate and cultured overnight. The culture medium was discarded, and a 10 μL pipette was used to make cross-shaped incisions perpendicular to the cell plane in each well. The cells were then washed three times with PBS, and serum-free medium diluted with the drug solution was added. The (S,R)-4v drug concentration prepared in Example 1 of this invention was set to 0, 100, 300, and 1000 nM. Photos were taken every 12 hours. Figure 2-3 As shown, try to ensure that the shooting location is the same each time, and record the data.

[0252] According to relevant literature, dysregulation of Nogo-B protein function has been found to induce the expression of PI3K / AKT / NF-κB-related proteins in cells, resulting in, for example... Figure 3-1 As shown. The specific experiment is shown below:

[0253] ① Add four 6cm dishes of 143B cells to four groups of culture medium containing different concentrations of (S,R)-4v drug prepared in Example 1 of this invention. The concentrations of each group are set to 0, 0.3, 1, and 3 μM, respectively. After adding the medium, place the cells in an incubator at 37°C and 5% CO2. After culturing for 24 hours, the cells are lysed and the cell protein albumen is collected.

[0254] ② After determining the protein concentration, take the same amount of supernatant, boil for 5-10 minutes and then perform electrophoresis. After electrophoresis, perform immunoblotting experiments on proteins such as PI3K / AKT / NF-κB, and follow the same procedure as the immunoblotting experiment on Nogo-B protein described above.

[0255] Based on the experimental data provided above by this invention, the following conclusions can be drawn:

[0256] (1) The α-hydroxy / alkoxy / amino-β-amino derivative small molecule compound (S,R)-4v prepared in this invention can effectively inhibit the growth and migration of osteosarcoma 143B cells.

[0257] (2) Nogo-B protein is a direct target of the small molecule compound (S,R)-4v, an α-hydroxy / alkoxy / amino-β-amino derivative, which inhibits the growth and migration of osteosarcoma 143B cells.

[0258] (3) Knocking down Nogo-B protein can inhibit the growth and migration of osteosarcoma 143B cells;

[0259] (4) The small molecule compound (S,R)-4v, an α-hydroxy / alkoxy / amino-β-amino derivative prepared in this invention, promotes osteosarcoma cell apoptosis by targeting Nogo-B and causing changes in the expression of PI3K / AKT / NF-κB pathway proteins.

[0260] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0261] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0262] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0263] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention are covered within the scope of protection of the present invention.

[0264] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. Application of the biomarker Nogo-B as a drug target in the preparation of drugs for the prevention, relief and / or treatment of osteosarcoma and osteosarcoma-related complications.

2. The application as described in claim 1, characterized in that, The biomarkers inhibit the proliferation, migration, and metastasis of osteosarcoma cells and the growth of osteosarcoma; they also affect the expression of related proteins in the body. And / or, the amino acid sequence of the Nogo-B is shown in SEQ ID NO.

1.

3. An inhibitor of a biomarker, characterized in that, The inhibitor is an α-hydroxy / alkoxy / amino-β-amino derivative, the structure of which is shown in formula (I). In the aforementioned formula (I), R 1 H, halogen, alkyl, aryl, nitro, alkoxy, trifluoromethyl, cyano, ester, carboxyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, ester-substituted phenyl; R 2 The substituted phenyl groups are H, alkyl, aryl, benzyl, alkynyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, and aromatic heterocyclic-substituted phenyl. R 3 R 4 Each of the following is independently alkyl, benzyl, phenyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, methyl 2-benzoate, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, olefin-substituted phenyl, alkyne-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, acetylamide-substituted phenyl, steroid, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, furan ring, thiophene ring, indole ring, pyrrole ring; R 5 The following are substituted for H, alkyl, alkylacyl, phenyl, benzoyl, heterocyclic acyl, cyclopropionyl, benzyl, benzoyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, and acetyleneamide-substituted phenyl. Preferably, the structure of the inhibitor is as follows:

4. A drug / drug composition, characterized in that, It contains the biomarker as described in claim 1, or contains a compound that can bind to or react with the biomarker as described in claim 1.

5. A detection reagent / kit, characterized in that, It contains biomarkers that can be used to detect the biomarkers as described in claim 1.

6. The use of the inhibitor of the biomarker as described in claim 3, the drug / drug composition as described in claim 4, or the detection reagent / kit as described in claim 5 in the preparation, relief and / or treatment of osteosarcoma and osteosarcoma-related complications.

7. The application as described in claim 6, characterized in that, The osteosarcoma is one or more of the following types of osteosarcoma cells: MG63 type, SJSA-1 type, U2OS type, SW1353 type, MNNG / HOS type, HOS type, and 143B type.

8. A method for synthesizing α-hydroxy / alkoxy / amino-β-amino derivatives, characterized in that, Using diazo compound 1, compound 2, and imine 3 as raw materials, and rhodium acetate as a catalyst, in an organic solvent, the α-hydroxy / alkoxy / amino-β-amino derivatives shown in formula (I) are reacted to obtain the derivatives shown in formula (I); the reaction process is shown in formula (II): in, R 1 H, halogen, alkyl, aryl, nitro, alkoxy, trifluoromethyl, cyano, ester, carboxyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, ester-substituted phenyl; R 2 The substituted phenyl groups are H, alkyl, aryl, benzyl, alkynyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, and aromatic heterocyclic-substituted phenyl. R 3 R 4 Each of the following is independently alkyl, benzyl, phenyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, methyl 2-benzoate, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, olefin-substituted phenyl, alkyne-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, acetylamide-substituted phenyl, steroid, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, furan ring, thiophene ring, indole ring, pyrrole ring; R 5 The following are substituted for H, alkyl, alkylacyl, phenyl, benzoyl, heterocyclic acyl, cyclopropionyl, benzyl, benzoyl, halogen-substituted benzyl, alkoxy-substituted benzyl, alkyl-substituted benzyl, trifluoromethyl-substituted benzyl, nitro-substituted benzyl, halogen-substituted phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, trifluoromethyl-substituted phenyl, nitro-substituted phenyl, heterocyclic-substituted phenyl, aromatic heterocyclic-substituted phenyl, acrylamide-substituted phenyl, chloroacetamide-substituted phenyl, and acetyleneamide-substituted phenyl. Preferably, the reaction temperature is 0–40 °C; and / or the reaction time is 1–5 h; and / or the molar ratio of the diazo compound represented by formula (1), the compound represented by formula (2), and the imine represented by formula (3) is 1.2:5:1 to 2.0:5:1; and / or the amount of the catalyst rhodium acetate is based on the imine represented by formula (3) and is 0.5 mol% to 2.0 mol% of the imine represented by formula (3); and / or the organic solvent is selected from one or more of DCM, DCE, EA, and toluene; and / or the amount of the organic solvent is based on the imine represented by the substrate formula (3) such that the concentration of the imine represented by formula (3) in the organic solvent is in the range of 0.05–0.15 M.

9. A method for screening candidate drugs and / or inhibitors for the prevention, mitigation, and / or treatment of osteosarcoma and osteosarcoma-related complications, characterized in that, The method includes detecting the effect of the candidate drug and / or inhibitor on the biological effects of the biomarkers as described in claim 1 in a subject or a sample obtained from the subject.

10. A method for preventing, alleviating, and / or treating osteosarcoma and osteosarcoma-related complications in subjects in need, or a method for assessing the severity of osteosarcoma and osteosarcoma-related complications in patients, or a method for predicting the recurrence time of osteosarcoma and osteosarcoma-related complications in patients, or a method for predicting or prognostically assessing whether said patients are suitable for Nogo-B treatment, characterized in that, The method includes: determining the Nogo-B level from a blood or osteosarcoma tissue sample of the patient or subject, wherein the level is used to prevent, alleviate and / or treat osteosarcoma in the subject, or to assess the severity of osteosarcoma in a patient, or to predict whether a patient will develop osteosarcoma, or to predict or assess whether the patient is suitable for Nogo-B-targeted therapy.

Images