Compounds and methods for treating glioblastoma

Abstract

The present application relates to compounds and methods for treating glioblastoma. Specifically, the invention provides application of a compound capable of being combined with a heterodimer formed by a mu-opioid receptor and a delta-opioid receptor in preparation of glioma drugs and application of the compound in screening of glioma treatment drugs.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to the application of a compound in the preparation of glioma drugs. Background Technology

[0002] Gliomas are neuroepithelial tumors originating from glial cells or supporting cells in the central nervous system. They commonly occur in various lobes of the brain, with a smaller proportion occurring in the brainstem and thalamus. Their biological behavior is complex, and treatment presents significant challenges. Among these, glioblastomas account for more than half, characterized by high invasiveness and rapid progression, resulting in generally poor prognoses for patients. The 5-year survival rate remains below 10%, making it a clinical challenge in the field of neuro-oncology that urgently needs to be addressed.

[0003] The treatment of gliomas faces many unique challenges: the tumors are located in the brain, approximately 98% of small molecule drugs and almost all large molecule drugs cannot cross the blood-brain barrier to enter brain tissue, and malignant cells can migrate to adjacent brain tissues; gliomas inherently exhibit resistance to traditional therapies; the tumor's blood supply is often obstructed to varying degrees, affecting drug delivery efficiency; capillary leakage from the tumor can lead to peritumoral fluid accumulation (peritumoral edema) and increased intracranial pressure, and may also induce seizures; treatment for gliomas often triggers neurotoxic reactions. These factors collectively lead to bottlenecks in traditional and targeted therapies that directly kill tumor cells, resulting in poor treatment efficacy, unfavorable patient prognosis, and a high risk of recurrence and mortality, prompting an urgent need to explore non-tumor-targeted treatment methods.

[0004] The development and progression of gliomas are closely related to their unique microenvironment. The close interaction between tumor cells and surrounding cells makes regulating the tumor microenvironment by modulating the function or role of these surrounding cells a promising avenue for exploration. Therefore, how to indirectly inhibit tumor progression by intervening in the local microenvironment upon which tumors depend for survival, thereby disrupting the conditions that promote growth, invasion, and drug resistance, rather than directly attacking the tumor cells themselves, is a pressing issue that needs to be addressed. Summary of the Invention

[0005] This application aims to provide a non-tumor-targeted method for alleviating and / or treating gliomas by modulating the tumor microenvironment. Specifically, this application discovers that heterodimers formed by acting on μ-opioid receptors and δ-opioid receptors in the tumor microenvironment can restore reactive astrocytes in the glioma tumor microenvironment to normal function, restoring their normal neuroprotective function, thereby effectively inhibiting the proliferation and invasion of glioma cells, ultimately achieving alleviation and / or treatment of gliomas.

[0006] On one hand, this application provides the use of a compound in the preparation of a medicament for relieving and / or treating glioma, wherein the compound is capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

[0007] On the other hand, this application provides a method for alleviating and / or treating glioma, the method comprising administering an effective amount of a compound to a subject in need, the compound being capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

[0008] In some embodiments, the compound can promote heterodimerization of μ-opioid receptors and δ-opioid receptors, and / or enhance the stability, function, and / or activity of the heterodimers formed by μ-opioid receptors and δ-opioid receptors.

[0009] In some embodiments, the heterodimer is expressed in the central nervous system.

[0010] In some implementations, the heterodimer is expressed in the brain.

[0011] In some embodiments, the heterodimer is expressed on the surface of glial cells.

[0012] In some embodiments, the heterodimer is expressed on the surface of astrocytes, oligodendrocytes, microglia, and / or ependymal cells.

[0013] In some embodiments, the compound is capable of restoring reactive astrocytes to normal astrocytes.

[0014] In some embodiments, the compound is able to upregulate the expression of glutamate transporter 1 (GLT-1) and / or increase its activity in astrocytes.

[0015] In some embodiments, the compound has one or more of the following properties:

[0016] (1) Increase the concentration of cyclic adenosine monophosphate (cAMP) in astrocytes;

[0017] (2) Increase the concentration of cAMP-response element-binding protein (pCREB) in astrocytes;

[0018] (3) Upregulates the expression of brain-derived neurotrophic factor (BDNF) in astrocytes;

[0019] (4) Upregulates the expression of glial fibrillary acidic protein (GFAP) in astrocytes;

[0020] (5) Reduce the expression of monoamine oxidase B (MAOB) in astrocytes;

[0021] (6) Upregulates the expression of the potassium channel Kir4.1 in astrocytes; and

[0022] (7) Reduce the concentration of glutamate in the intercellular spaces of the brain.

[0023] In some embodiments, the compound is able to alter the tumor microenvironment of the glioma.

[0024] In some embodiments, the μ-opioid receptor and / or δ-opioid receptor are derived from mammals.

[0025] In some embodiments, the μ-opioid receptor includes a μ1-opioid receptor, a μ2-opioid receptor, and / or a μ3-opioid receptor.

[0026] In some embodiments, the μ-opioid receptor is encoded by a gene with the Ensembl database number ENSG00000112038.

[0027] In some embodiments, the δ-opioid receptor includes δ1-opioid receptors and / or δ2-opioid receptors.

[0028] In some embodiments, the δ-opioid receptor is encoded by a gene with the Ensembl database number ENSG00000116329.

[0029] In some embodiments, the compound is a small molecule, polypeptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0030] In some embodiments, the compound is a metabolite of ketamine.

[0031] In some embodiments, the compound has the structure shown in Formula I:

[0032]

[0033] Formula I

[0034] in,

[0035] R 1Independently selected from hydrogen, halogen, hydroxyl, amino, nitro, cyano, amide, C1-C4 alkyl, C1-C4 alkoxy, mono- and di-C1-C4 alkylamino, C1-C2 haloalkyl, C1-C2 haloalkoxy, C6-C 10 Aryl or monocyclic or polycyclic heteroaryl;

[0036] R 2 Independently selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 acyl, C6-C 10 Aryl, monocyclic or polycyclic heteroaryl, aryl acyl or heteroaryl acyl;

[0037] R 3 It is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or halogen;

[0038] R 4 It is independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl carbonyl, C1-C8 acyl, aryl acyl or heteroaryl acyl;

[0039] And R 1 R 2 R 3 and R 4 The alkyl, alkynyl, or alkenyl groups in the definition are straight-chain or branched and are either unsubstituted or substituted by one or two independent substituents selected from the following: -F, -Cl, -Br, -I, -CN, -NCO, -NCS, -OH, -NH2, -NO2, benzene ring, pyridine, pyrrole, thiophene, pyran, or furan.

[0040] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0041] In some embodiments, the compound has the structure shown in Formula Ia:

[0042]

[0043] Formula Ia

[0044] , wherein R 1 R 2 R 3 and R 4 As defined in Equation I

[0045] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0046] In some implementations, the R 1It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkoxy or C1-C3 haloalkoxy.

[0047] In some implementations, the R 1 It is hydrogen, trifluoromethyl, or trifluoromethoxy.

[0048] In some implementations, the R 2 It is hydrogen or benzoyl.

[0049] In some implementations, the R 3 It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0050] In some implementations, the R 3 It can be hydrogen or chlorine.

[0051] In some implementations, the R 4 It is hydrogen.

[0052] In some embodiments, the pharmaceutically acceptable salt of the compound is a hydrochloride salt.

[0053] In some embodiments, the compound has the structure shown in Formula II:

[0054]

[0055] Formula II.

[0056] In some embodiments, the compound has a structure selected from the group consisting of:

[0057] , , , , , , ,

[0058] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0059] In some embodiments, the glioma includes WHO grade I, II, III and / or IV gliomas.

[0060] In some embodiments, the glioma includes astrocytoma, oligodendroglioma, ependymoma, and / or oligodendroastrocytoma.

[0061] In some embodiments, the glioma is a glioblastoma (GBM).

[0062] In some embodiments, the glioblastoma (GBM) includes primary GBM (IDH wild-type GBM) and / or secondary GBM (IDH mutant GBM).

[0063] In some embodiments, the glioma includes heterogeneous glioma.

[0064] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0065] On the other hand, this application provides a drug combination for alleviating and / or treating glioma, comprising compounds used in the purposes described in this application, as well as one or more other antitumor drugs.

[0066] In some embodiments, the other antitumor drugs are selected from the group consisting of chemotherapy drugs, molecularly targeted drugs, immune checkpoint inhibitors, and cell drugs.

[0067] In some embodiments, the drug combination described in this application further includes administering one or more other anticancer therapies, such as chemotherapy, radiotherapy, or immunotherapy, to the subject.

[0068] On the other hand, this application provides a method for identifying an agent for alleviating and / or treating glioma, the method comprising: (1) contacting a candidate compound with μ-opioid receptors and δ-opioid receptors, and (2) detecting the effect of the candidate compound on the dimerization of μ-opioid receptors and δ-opioid receptors or their downstream signaling pathways;

[0069] Specifically, when the dimerization or downstream signaling pathway is enhanced relative to the control compared to the untreated control, the candidate compound is identified as an agent that can be used to alleviate and / or treat glioma.

[0070] In some embodiments, the candidate compound is a small molecule, peptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0071] In some embodiments, the method according to this application includes, prior to step (1): providing a test system comprising a μ-opioid receptor and a δ-opioid receptor; and introducing the candidate compound into the test system such that it contacts the μ-opioid receptor and the δ-opioid receptor.

[0072] In some embodiments, the test system is a vector, cell, tissue, organ, or animal model expressing the μ-opioid receptor and / or the δ-opioid receptor.

[0073] In some embodiments, the cells are selected from astrocytes, oligodendrocytes, microglia, and / or ependymal cells.

[0074] In some embodiments, the cells are astrocytes.

[0075] In some embodiments, the dimerization includes the degree of dimerization and / or the stability, function, and / or activity of the dimer.

[0076] In some embodiments, the downstream signaling pathway includes one or more indicators selected from the group consisting of: (1) expression or activity of intracellular glutamate transporter 1, (2) concentration of intracellular cyclic adenosine monophosphate, (3) expression or activity of intracellular brain-derived neurotrophic factor, (4) expression or activity of intracellular glial fibrillary acidic protein, (5) expression or activity of intracellular monoamine oxidase B, (6) expression or activity of intracellular potassium channel Kir4.1, and (7) intercellular glutamate concentration in the brain.

[0077] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0078] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0079] Figure 1 The diagram shows the synthetic route for (2R, 6R)-hydroxydemethylketamine (HNK).

[0080] Figures 2-3 The figure shows the percentage of cells exhibiting GFP fluorescence due to HNK binding to the μ-δ-heterodimeric opioid receptor, as well as the increase or enhancement of the intensity of GFP fluorescence in each cell.

[0081] Figure 4 The display shows the blocking effect of HNK-induced GFP enhancement / increase by the short peptide PEP.

[0082] Figure 5 This shows the increase in intracellular cAMP concentration caused by HNK.

[0083] Figure 6 The display shows the changes in intracellular pCREB concentration / percentage caused by HNK.

[0084] Figure 7 The results show the blocking effect of PEP pretreatment on the changes in intracellular pCREB concentration / percentage caused by HNK.

[0085] Figure 8 The image shows the changes in GLT-1 and BDNF protein levels after HNK treatment, as detected by Western blot assay.

[0086] Figure 9 This shows the effect of HNK upregulating GLT-1 expression in vivo.

[0087] Figure 10 The image shows the effect of in vivo administration of HNK on reducing intercellular glutamate concentration.

[0088] Figures 11-12 The effect of HNK treatment on the survival time of nude mouse U87-MG GBM model animals is shown.

[0089] Figure 13 The image shows the effect of HNK treatment on weight loss caused by tumors in nude mice in the U87-MG GBM model.

[0090] Figure 14 The effect of HNK treatment on tumor growth rate in nude mouse U87-MG GBM model animals is shown.

[0091] Figures 15-17 The effect of HNK treatment on the survival time of nude mouse MU41 GBM model animals is shown.

[0092] Figure 18 The image shows the effect of HNK treatment on weight loss caused by tumors in nude mouse MU41 GBM model.

[0093] Figure 19 The effect of HNK treatment on the growth rate of tumors in nude mouse MU41 GBM model animals is shown.

[0094] Figures 20-22 The effect of HNK treatment on the survival time of GL261 GBM model animals is shown.

[0095] Figure 23 The image shows the effect of HNK treatment on weight loss in the GL261 GBM model caused by tumors.

[0096] Figure 24 The effect of HNK treatment on the growth rate of tumors in GL261 GBM model animals is shown.

[0097] Figure 25The figures show the duration of time animals remained on the rotundus and the maximum rotundus speed they could withstand without falling off the rotundus 3 days after HNK administration in the U87 glioma model.

[0098] Figure 26 The figures show the duration of time animals remained on the rotundus and the maximum rotundus speed they could withstand without falling off the rotundus 7 days after HNK administration in the U87 glioma model.

[0099] Figure 27 The figures show the duration of time animals remained on the rotundus before HNK administration and the maximum rotundus speed they could tolerate without falling off the rotundus in the MU41 glioma model.

[0100] Figure 28 The figures show the duration of time animals remained on the rotundus and the maximum rotundus speed they could withstand without falling off the rotundus 3 days after HNK administration in the MU41 glioma model.

[0101] Figure 29 The figures show the duration of time animals remained on the rotundus and the maximum rotundus speed they could withstand without falling off the rotundus 7 days after HNK administration in the MU41 glioma model.

[0102] Figures 30-32 The figure shows the effect of HNK treatment on the survival time of GL261 model animals.

[0103] Figure 33 This shows the effect of HNK treatment on weight loss in GL261 model animals caused by tumors.

[0104] Figure 34 The effect of HNK treatment on the growth rate of tumors in GL261 model animals is shown.

[0105] Figures 35-36 The results shown are from a Western blot analysis of protein quantification in GBM tumor tissue after HNK administration.

[0106] Figure 37 The effect of direct HNK treatment on GBM cell growth and proliferation is shown.

[0107] Figure 38 The effect of HNK treatment on GBM cells co-cultured with the astrocyte cell line (CTX-TNA2) is shown.

[0108] Figures 39-40 The image shows the results of pCREB immunofluorescence staining after in vivo administration of HNK.

[0109] Figure 41The figure shows the ratio of pCREB and GFAP co-staining after in vivo administration of HNK.

[0110] Figures 42-43 The image shows the changes in GFAP fluorescence intensity and morphological changes in GFAP-positive astrocytes after HNK treatment of U87-MG model mice.

[0111] Figures 44-45 This shows the changes in MAOB, a marker of reactive astrocytes, in GBM after HNK treatment.

[0112] Figures 46-48 The image shows the expression of GLT-1 and Kir4.1 on astrocytes surrounding GBM after HNK treatment.

[0113] Figures 49-51 The figure shows the change in the density of μ-δ ORs+S100β positive cells in GBM after HNK treatment. Detailed Implementation

[0114] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0115] Terminology Definition

[0116] In this application, the term "relief" generally refers to a reduction in the severity or duration of disease symptoms. Relief includes, but does not require, a complete recovery from or complete prevention of the disease or its symptoms, and includes improvement or reduction of at least one indicator, sign, or symptom of the disease or its symptoms; for example, improvement in the delay or slowing of the progression or severity of one or more indicators of the disease or its symptoms. The progression or severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0117] In this application, the term "treatment" generally refers to the application or administration of a therapeutic agent to a patient, or to the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient who has a disease, symptoms of a disease, or a predisposition to a disease, with the aim of treating, curing, alleviating, relieving, altering, remedying, improving, enhancing, or influencing the disease, symptoms of a disease, or a predisposition to a disease. This may include improving the disease state, eliminating lesions, or improving prognosis.

[0118] In this application, the term "glioma" may also be referred to as "neuroglioma," "brain glioma," or "glial cell tumor," generally referring to a tumor disease of the central nervous system. Histologically, it includes astrocytomas, oligodendrogliomas, ependymomas, and oligoastrocytomas, and covers all malignancy levels from I to IV in the World Health Organization classification system.

[0119] In this application, the term "opioid receptors" generally refers to a class of protein molecules capable of binding to endogenous opioid peptides and / or exogenous opioid-like substances and mediating their biological effects. This includes all known types of opioid receptors, such as the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR), as well as any isotypes, subtypes, splice variants, and homo / heterodimers or multimer complexes formed through homo- or hetero-oligomerization of these receptors. The opioid receptors described in this application encompass opioid receptors from any species, including but not limited to receptors derived from mammals such as humans and mice, as well as chimeric receptors obtained through genetic engineering. This includes native, complete proteins that exist in their natural state, as well as functional fragments (such as any form of truncated form) and variants (such as variants with point mutations, insertions, deletions, or substitutions), and receptors in different functional states, such as unbound, ligand-bound, etc. It also includes proteins that have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the full-length sequence of any known naturally occurring opioid receptor, and that retain ligand-binding capacity and / or signal transduction function.

[0120] In this application, the term "mu-opioid receptor" (MOR) generally refers to a member of the G protein-coupled receptor (GPCR) superfamily, encoded by the OPRM1 gene, including all known isotypes, subtypes, and splice variants. It encompasses mu-opioid receptors from any species, including but not limited to receptors of mammalian origin such as humans and mice, as well as chimeric receptors obtained through genetic engineering. It includes naturally occurring, complete proteins, as well as functional fragments (such as any form of truncated version) and variants (such as variants with point mutations, insertions, deletions, or substitutions), and receptors in different functional states, such as unbound and ligand-bound states. It also includes proteins that possess at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the full-length sequence of any known naturally occurring mu-opioid receptor, and retain ligand-binding capacity and / or signal transduction function.

[0121] In this application, the term "delta-opioid receptor" (DOR) generally refers to a member of the G protein-coupled receptor (GPCR) superfamily, encoded by the OPRD1 gene, including all known isotypes, subtypes, and splice variants. It encompasses delta-opioid receptors from any species, including but not limited to receptors of mammalian origin such as humans and mice, as well as chimeric receptors obtained through genetic engineering. It includes naturally occurring, complete proteins, as well as functional fragments (such as any form of truncated variants) and variants (such as variants with point mutations, insertions, deletions, or substitutions), and receptors in different functional states, such as unbound and ligand-bound states. It also includes proteins that possess at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the full-length sequence of any known naturally occurring delta-opioid receptor, and retain ligand-binding capacity and / or signal transduction function.

[0122] In this application, the term "heterodimer" generally refers to a molecule (e.g., a protein molecule) composed of two distinct members. The two members of a heterodimer can differ in structure, function, activity, and / or composition. For example, the two distinct members can comprise polypeptides that differ in the order, number, or type of amino acid residues that form these polypeptides. Each of the two distinct members of a heterodimer can independently comprise one, two, or more units, polypeptide chains, or portions. For example, a heterodimer can be a μ-δ-heterodimeric opioid receptor formed by the non-covalent binding of a μ-opioid receptor protein and a δ-opioid receptor protein.

[0123] In this application, the term "heterodimerization" generally refers to the process by which two molecules composed of different members (e.g., protein molecules) form a heterodimer through interaction, for example, through hydrophobic interactions, van der Waals forces, hydrogen bonds, or ionic bonds. For example, the process by which a μ-opioid receptor protein and a δ-opioid receptor protein bind non-covalently to form a μ-δ-heterodimer opioid receptor.

[0124] In this application, the term "central nervous system" generally refers to the core components of the nervous system, primarily composed of the brain and spinal cord. Its function is to integrate, process, and transmit neural information, and to regulate physiological processes such as sensation, movement, cognition, and autonomic nervous activity. The central nervous system is composed of two main cell types: neurons and glial cells, and is protected by the blood-brain barrier.

[0125] In this application, the term "glial cells" generally refers to a class of cells in the central nervous system that maintain neuronal function. They do not directly participate in the transmission of electrical signals, and their functions include structural support, nutrition and metabolism, myelination, and immune defense. Glial cells mainly include astrocytes, oligodendrocytes, microglia, and ependymal cells.

[0126] In this application, the term "astrocyte" generally refers to a star-shaped glial cell found in the brain and spinal cord. The astrocytes described in this application encompass primary cells isolated and cultured from animal or human brain tissue, as well as astrocyte cell lines (such as the CTX TNA2 cell line). This includes astrocytes derived from commonly used laboratory animals such as humans, non-human primates, and rodents (e.g., rats, mice).

[0127] In this application, the term "reactive astrocytes" generally refers to the abnormal state of astrocytes after injury or disease. Compared with normal astrocytes, reactive astrocytes often exhibit downregulated expression or impaired function of glutamate transporter 1 (GLT-1), altered expression profiles of brain-derived neurotrophic factor (BDNF), and downregulated function of potassium channels (Kir4.1). Studies have shown that reactive astrocytes can be hijacked by glioma cells to support their proliferation and invasion, and even impair neuronal function.

[0128] In this application, the term "glutamate transporter 1" (GLT-1) generally refers to a protein that is specifically highly expressed in astrocytes, also known as excitatory amino acid transporter 2 (EAAT2), encoded by the SLC1A2 (Solute Carrier Family 1 Member 2) gene. Its main function is to transport glutamate from the intercellular matrix into astrocytes to reduce the concentration of glutamate.

[0129] In this application, the term "cyclic adenosine monophosphate" (cAMP) generally refers to an intracellular second messenger that amplifies, transmits, and integrates signals from extracellular first messengers (such as hormones and neurotransmitters) by activating downstream effector proteins (such as protein kinase A, PKA), thereby broadly regulating various physiological and biochemical processes within the cell, including metabolism, gene expression, cell proliferation, and differentiation.

[0130] In this application, the term "cAMP-response element binding protein" (pCREB) generally refers to a widely expressed nuclear transcription factor that can bind to a specific sequence on DNA—the cAMP response element (CRE)—thereby initiating or regulating the transcription of downstream target genes.

[0131] In this application, the term "brain-derived neurotrophic factor" (BDNF) generally refers to a member of the neurotrophic factor family that plays a crucial regulatory role in neuronal survival, differentiation, growth, synaptic plasticity, and learning and memory by binding to specific receptors (primarily its high-affinity receptor TrkB) and activating downstream signaling pathways.

[0132] In this application, the term "glial fibrillary acidic protein" (GFAP) generally refers to a specific molecular marker of astrocytes, belonging to the cytoskeletal protein of type III intermediate filament protein, which plays a key role in maintaining the morphology, mechanical strength, and motility of astrocytes.

[0133] In this application, the term "monoamine oxidase B" (MAOB) generally refers to a flavin adenine dinucleotide (FAD)-dependent enzyme located mainly on the outer membrane of mitochondria, highly expressed in astrocytes. Its main function is to catalyze the oxidative deamination of various biogenic amines in the brain and peripheral tissues, inactivating them and producing corresponding aldehydes, hydrogen peroxide, and ammonia. It is one of the key enzymes for maintaining neurotransmitter homeostasis and metabolizing exogenous amines.

[0134] In this application, the term "potassium channel Kir4.1" generally refers to an inwardly rectifying potassium channel (Kir) encoded by the KCNJ10 gene, which is highly expressed in astrocytes, maintains the resting membrane potential of cells, is responsible for the spatial buffering of extracellular potassium ions (K⁺) in the brain, and participates in the homeostatic regulation of neuronal excitability and potassium ion homeostasis.

[0135] In this application, the term "glutamate" generally refers to α-aminoglutamate, an excitatory neurotransmitter involved in protein metabolism within the body. Excessive glutamate concentration in the intercellular matrix can lead to increased neuronal activity, making it more susceptible to inducing epilepsy. Prolonged high glutamate concentrations can result in synapse loss and ultimately neuronal apoptosis.

[0136] In this application, the term "tumor microenvironment (TME)" generally refers to the dynamic and complex ecosystem in which tumor cells reside, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and the extracellular matrix. Tumors and their surrounding environment are closely related and constantly interact. Tumors can influence their microenvironment by releasing cellular signaling molecules, promoting tumor angiogenesis and inducing immune tolerance, while immune cells in the microenvironment can affect cancer cell growth and development.

[0137] In this application, the term "ketamine" generally refers to 2-o-chlorophenyl-2-methylaminocyclohexanone, an N-methyl-D-aspartic acid (NMDA) receptor antagonist that inhibits excitatory signal transmission in the central nervous system by blocking NMDA receptors. "Metabolite" generally refers to the intermediate or final product generated after a series of enzymatic chemical reactions in a living organism. Hydroxynorketamine (HNK) is one of the main metabolites of ketamine in the human body.

[0138] In this application, the term "pharmaceutically acceptable salt" generally refers to a salt suitable for contact with mammalian, particularly human, tissues without excessive toxicity, irritation, allergic reactions, etc., and in a manner commensurate with a reasonable benefit / risk ratio, within the limits of reasonable medical judgment. This includes the modification of the parent compound by preparing a non-toxic acid or its base addition salt, and also refers to pharmaceutical solvates of these compounds and these salts, including hydrates. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid addition salts of basic residues such as amines; base or organic addition salts of acidic residues such as carboxylic acids; and combinations including one or more of the above salts. Pharmaceutically acceptable salts include non-toxic salts and quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. For example, non-toxic acidic salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.; other acceptable inorganic salts include metal salts such as sodium salts, potassium salts, cesium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc., and combinations including one or more of the above salts. Organic salts of compounds include those such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid, 2-acetylated benzoic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, and HOOC-(CH2). n Salts prepared from organic acids such as -COOH (where n is 0 to 4); organic amine salts, such as triethylamine salts, pyridinium salts, methylpyridinium salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, etc.; and amino acid salts, such as arginine salts, aspartic acid salts, glutamate salts, etc., as well as combinations including one or more of the above salts.

[0139] In this application, the term "stereoisomer" generally refers to compounds having the same chemical composition but with different spatial arrangements of atoms or groups, including enantiomers and diastereomers. "Enantiomer" refers to two stereoisomers of a compound that are not mirror images of each other. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur during chemical reactions or processes where stereoselectivity or stereoorientation has ceased. "Diastereomer" is a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. In the presence of resolving agents or chromatography, mixtures of diastereomers can be separated using high-resolution analytical steps such as electrophoresis and crystallization using chiral HPLC columns.

[0140] In this application, the term "tautomer" generally refers to two functional isomers that readily interconvert through the migration of atoms (e.g., hydrogen atoms) and double bond positions, with a rapid, reversible chemical equilibrium between them. The most common tautomer is the keto-enol tautomer, and other tautomers such as lactate-pyruvate and amide-imine tautomers are also included.

[0141] In this application, the term "astrocytoma" generally refers to a type of neuroepithelial tumor originating from astrocytes, which is the most common type of glioma. According to the World Health Organization (WHO) classification of tumors of the central nervous system, its malignancy ranges from low grade (WHO I-II) to high grade (WHO III-IV).

[0142] In this application, the term "oligodendroglioma" generally refers to a diffuse glioma that originates from or differentiates from oligodendroglial cells, is IDH-mutated, and has a combined deletion of 1p / 19q.

[0143] In this application, the term "ependymoma" generally refers to a neuroepithelial tumor originating from ependymal cells, typically located near the ventricular system or the central canal of the spinal cord.

[0144] In this application, the term "oligodendroastrocytoma" generally refers to a mixed glioma that histologically exhibits both oligodendroglial and astrocytic components.

[0145] In this application, the term "glioblastoma" generally refers to glioblastoma multiforme (GBM), which falls under the category of astrocytomas and is defined as a WHO grade IV astrocytoma according to the World Health Organization (WHO) classification of tumors of the central nervous system.

[0146] In this application, the term "primary GBM (IDH wild-type GBM)" generally refers to a glioblastoma with a short clinical history (usually less than 3 months), no prior history of low-grade astrocytoma, and which is WHO grade IV from de novo onset.

[0147] In this application, the term "secondary GBM (IDH-mutant GBM)" generally refers to WHO grade IV glioblastoma that gradually evolves from a known lower-grade astrocytoma (usually WHO grade II diffuse astrocytoma or WHO grade III anaplastic astrocytoma).

[0148] In this application, the term "heterogeneity" generally refers to the presence of multiple different subpopulations of cancer cells within a single tumor. "Heterogeneous glioma" refers to a glioma containing multiple different subpopulations of glioma cells.

[0149] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers may include, for example, buffers, antioxidants, peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions; and / or nonionic surfactants.

[0150] In this application, the term "molecularly targeted drug" generally refers to a drug that specifically acts on key molecular targets (such as specific gene mutations, abnormally activated kinases, growth factor receptors, or signaling pathways) in the process of tumor occurrence and development.

[0151] In this application, the term "cell drug" generally refers to a pharmaceutical preparation that uses living, therapeutically functional cells as its active ingredient, such as CAR-T cells and TCR-T cells.

[0152] In this application, the term "downstream signaling pathway" generally refers to a series of ordered biochemical reactions following a specific molecule or event in a continuous biochemical signal transduction chain. For example, in this application, the dimerization downstream signaling pathway of the μ-opioid receptor and δ-opioid receptor includes the expression or activity of intracellular glutamate transporter 1, the concentration of intracellular cyclic adenosine monophosphate (cAMP), the expression or activity of intracellular brain-derived neurotrophic factor (BDNF), the expression or activity of intracellular glial fibrillary acidic protein (GFAP), the expression or activity of intracellular monoamine oxidase B, the expression or activity of intracellular potassium channel Kir4.1, and the intercellular glutamate concentration, etc.

[0153] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0154] In this application, the term "and / or" should generally be understood to mean any one of the options or both of the options. Invention Details

[0156] Mechanism of action

[0157] Astrocytes exist in the tumor microenvironment of gliomas. When interacting with tumor cells, they can transform into tumor-associated astrocytes (TAAs), which strongly support tumor growth while impairing neuronal function and jeopardizing brain homeostasis. In the glioma microenvironment, the expression of two key proteins closely related to neuronal function in TAAs is decreased: glutamate transporter-1 (GLT-1) and the potassium channel Kir4.1. GLT-1 is responsible for uptake of glutamate from the synaptic cleft and brain parenchyma, maintaining a low extracellular glutamate concentration. Its decreased function leads to abnormal synaptic transmission and impaired plasticity, ultimately resulting in extracellular glutamate accumulation, triggering excitotoxicity, synaptic loss, and neuronal apoptosis, severely damaging brain function. Neuronal loss also creates space for tumor cell invasion, further promoting tumor spread. The Kir4.1 channel is responsible for regulating extracellular potassium ion concentration, which is crucial for maintaining the resting membrane potential of neurons and regulating excitability. Both GLT-1 and Kir4.1 are significantly downregulated in gliomas, leading to neuronal overexcitation, which in turn promotes tumor growth and invasion.

[0158] Therefore, this application restores the key functions of astrocytes by restoring TAA to its normal state, thereby inhibiting the proliferation and invasion of glioma cells.

[0159] On the one hand, this application provides a compound capable of binding to a μ-δ-heterodimeric opioid receptor formed by μ-opioid receptor and δ-opioid receptor, the compound being capable of being used to prepare a drug for alleviating and / or treating glioma.

[0160] In this application, the compound is capable of physically interacting with μ-δ-heterodimeric opioid receptors, for example, through hydrogen bonds, van der Waals forces, hydrophobic interactions, or ionic bonds. This binding can be agonistic, antagonistic, partially agonistic, anti-agonistic, or allosteric. The specificity and affinity of the binding can be determined using conventional techniques in the art, such as radioligand binding assays, surface plasmon resonance (SPR), biofilm layer interference (BLI), or fluorescence resonance energy transfer (FRET) to verify its selectivity for heterodimers.

[0161] In some embodiments, the compound can promote the binding of μ-opioid receptors and δ-opioid receptors to form heterodimers.

[0162] In some embodiments, the compound can enhance the stability of the heterodimer formed by the binding of μ-opioid receptors and δ-opioid receptors. For example, the compound can prolong the half-life of the formed heterodimer and prevent its dissociation.

[0163] In some embodiments, the compound can enhance the function of the heterodimer formed by the binding of α-opioid receptors and δ-opioid receptors. For example, it can enhance downstream signal transduction.

[0164] In some embodiments, the compound can enhance the activity of the heterodimer formed by the binding of α-opioid receptor and δ-opioid receptor.

[0165] In some embodiments, the μ-δ-heterodimeric opioid receptor is expressed on the surface of the central nervous system, for example, in the brain.

[0166] In some embodiments, the μ-δ-heterodimeric opioid receptor is expressed on the surface of glial cells. For example, it is expressed on the surface of astrocytes. For example, it is expressed on the surface of oligodendrocytes. For example, it is expressed on the surface of microglia. For example, it is expressed on the surface of ependymal cells.

[0167] In some embodiments, the compound can act on μ-δ-heterodimeric opioid receptors on astrocytes, restoring reactive astrocytes to normal astrocytes.

[0168] For example, the expression of glutamate transporter 1 (GLT-1) is downregulated in reactive astrocytes, and the compound is able to upregulate the expression of GLT-1 in astrocytes. For example, compared with the control group (saline group), the expression of GLT-1 is upregulated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher.

[0169] For example, the expression of cyclic adenosine monophosphate (cAMP) is decreased in reactive astrocytes, and the compound can increase the concentration of cAMP in astrocytes. For example, compared with the control group (saline group), cAMP expression is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more.

[0170] For example, the expression of cAMP-response element-binding protein (pCREB) is decreased in reactive astrocytes, and the compound can increase the concentration of pCREB in astrocytes. For example, compared with the control group (saline group), pCREB expression is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more.

[0171] For example, the expression of brain-derived neurotrophic factor (BDNF) is downregulated in reactive astrocytes. The compound is capable of upregulating BDNF expression in astrocytes. For example, compared to the control group (saline group), BDNF expression is upregulated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher.

[0172] For example, the expression of glial fibrillary acidic protein (GFAP) is downregulated in reactive astrocytes. The compound is capable of upregulating GFAP expression in astrocytes. For example, compared to the control group (saline group), GFAP expression is upregulated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher.

[0173] For example, the expression of monoamine oxidase B (MAOB) is elevated in reactive astrocytes. The compound is able to reduce MAOB expression in astrocytes. For example, compared with the control group (saline group), MAOB expression is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher.

[0174] For example, the expression of potassium channel Kir4.1 is downregulated in reactive astrocytes. The compound is capable of upregulating the expression of potassium channel Kir4.1 in astrocytes. For example, compared with the control group (saline group), the expression of potassium channel Kir4.1 is upregulated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher.

[0175] For example, the intercellular glutamate concentration is elevated in reactive astrocytes. The compound is able to reduce the intercellular glutamate concentration. For example, compared with the control group (saline group), the intercellular glutamate concentration is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more.

[0176] In some implementations, changes in the above-mentioned indicators can be detected using standard molecular biology techniques. For example, they can be detected by methods such as Western blot, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), or RNA in situ hybridization (RNAscope).

[0177] In some embodiments, the compound works by acting on the tumor microenvironment of glioma. For example, the compound does not directly kill glioma tumor cells, but rather inhibits tumor cell growth and migration by acting on the tumor microenvironment.

[0178] In some embodiments, the compound, when co-cultured with glioma tumor cells under in vitro conditions, fails to effectively induce tumor cell death. For example, the co-culture is performed in an environment lacking other cell types (such as astrocytes, immune cells). For example, the compound does not cause, or only causes negligible loss of glioma tumor cell membrane integrity. For example, the compound does not cause, or only causes negligible activation of key effector proteins involved in glioma tumor cell apoptosis. For example, the compound does not cause, or only causes negligible large-scale DNA breaks in glioma tumor cells. For example, the compound does not cause, or only causes negligible necrotic morphological changes in glioma tumor cells (such as cell swelling, rupture, etc.).

[0179] In some embodiments, when the compound is co-cultured with glioma tumor cells under in vitro conditions, it fails to effectively prevent the tumor cells from entering the cell cycle and dividing. For example, the co-culture is conducted in an environment lacking other cell types (such as astrocytes or immune cells). For example, the compound has no significant effect on the metabolic activity of glioma tumor cells. For example, the compound does not reduce the number of glioma tumor cells. For example, the compound has no significant effect on the DNA synthesis capacity of glioma tumor cells. For example, the compound has no significant effect on the cell cycle distribution of glioma tumor cells.

[0180] In some embodiments, the tumor cells of the glioma include human or murine glioma cell lines. For example, the tumor cells of the glioma may be U87-MG cells, GL261 cells, MU41 cells, U251 cells, T98G cells, SHG-44 cells, CT2A cells, LN-229 cells, or C6 cells, etc.

[0181] In some embodiments, the tumor cells of the glioma include tumor cells of astrocytoma, oligodendroglioma, ependymoma, or oligodendroastrocytoma.

[0182] In some embodiments, the tumor cells of the glioma include tumor cells of glioblastoma (GBM).

[0183] In some embodiments, the μ-opioid receptor and delta-opioid receptor are derived from mammals. For example, the μ-opioid receptor and delta-opioid receptor are derived from mice, rats, or non-human primates. For example, the μ-opioid receptor and delta-opioid receptor are derived from humans.

[0184] In some embodiments, the μ-opioid receptor includes μ1-opioid receptor, μ2-opioid receptor, and μ3-opioid receptor.

[0185] In some embodiments, the δ-opioid receptor includes δ1-opioid receptors and δ2-opioid receptors.

[0186] For example, the μ-δ-heterodimeric opioid receptor is a μ1-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ1-δ2-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ2-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ2-δ2-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ3-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ3-δ2-heterodimeric opioid receptor.

[0187] In some embodiments, the μ-opioid receptor comprises the amino acid sequence encoded by Ensembl gene database number ENSG00000112038 (OPRM1 gene), including but not limited to protein isoforms corresponding to various transcripts generated by alternative splicing of this gene, or variants having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity with the amino acid sequence encoded by any of the aforementioned transcripts, and retaining the function of forming heterodimers with the δ-opioid receptor. The variants may include naturally occurring allelic variations, splice variants, or engineered sequences.

[0188] In some embodiments, the delta-opioid receptor comprises the amino acid sequence encoded by Ensembl gene database number ENSG00000116329 (OPRD1 gene), including but not limited to protein isoforms corresponding to various transcripts generated by alternative splicing of this gene, or variants having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher sequence identity with the amino acid sequence encoded by any of the aforementioned transcripts, and retaining heterodimerization function with the μ-opioid receptor. The variants may include naturally occurring allelic variations, splice variants, or engineered sequences.

[0189] In some embodiments, the compound is a small molecule, polypeptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0190] For example, the small molecule can be a known opioid ligand or its derivative, or it can be a completely new chemical entity.

[0191] For example, the polypeptide can be a linear chain composed of natural or non-natural amino acids, or a cyclic peptide formed by disulfide bonds or other chemical bonds. It can also be a polypeptide obtained by modifying the sequence of opioid peptides (such as enkephalins, endorphins, and dynorphins).

[0192] For example, the protein may be an antibody or its antigen-binding fragment, a fusion protein, or an engineered protein.

[0193] For example, the nucleic acid can be DNA, RNA, antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), or a mimic thereof.

[0194] For example, the sugars may be polysaccharides or their conjugates (such as glycoproteins, glycolipids, etc.).

[0195] compound

[0196] In some embodiments, the compound is a metabolite of ketamine. For example, it may be norketamine or dehydronorketamine.

[0197] For example, the compound is hydroxynorketamine (HNK).

[0198] In some embodiments, the compound has the structure described in Formula I.

[0199]

[0200] Formula I

[0201] In some implementations, R 1 It is hydrogen.

[0202] In some implementations, R 1 It is a halogen. For example, it can be -F, -Cl, -Br, or -I.

[0203] In some implementations, R 1 It is a hydroxyl group.

[0204] In some implementations, R 1 It is an amino group.

[0205] In some implementations, R 1 It is a nitro group.

[0206] In some implementations, R 1 It is a cyano group.

[0207] In some implementations, R 1 It is an amide group.

[0208] In some implementations, R 1 It is a C1-C4 alkyl group. "alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups and has a specified number of carbon atoms. "C1-C4 alkyl" refers to a straight-chain or branched alkyl group with 1-4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl groups.

[0209] In some implementations, R 1 It is a C1-C4 alkoxy group. "Alkoxy" refers to an alkyl group as defined above that has a specified number of carbon atoms connected by oxygen bridges. For example, it can be a methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, or tert-butoxy group.

[0210] In some implementations, R 1 These are mono- and di-C1-C4 alkylamino groups. A mono-C1-C4 alkylamino group is one in which one hydrogen atom is replaced by a C1-C4 alkyl group, while a di-C1-C4 alkylamino group is one in which two hydrogen atoms are replaced by two (which may be the same or different) C1-C4 alkyl groups. For example, these groups can be methylamino, ethylamino, isopropylamino, dimethylamino, methylethylamino, or diethylamino groups.

[0211] In some implementations, R 1It is a C1-C2 haloalkyl group. "Halo" means that it is substituted by one or more halogen atoms (F, Cl, Br, I). For example, it can be a trifluoromethyl, chloromethyl, 2,2,2-trifluoroethyl, 1,1-difluoroethyl or 2-chloroethyl group.

[0212] In some implementations, R 1 It is a C1-C2 haloalkoxy group. A "C1-C2 haloalkoxy group" refers to a group formed when one or more hydrogen atoms in a "C1-C2 alkoxy group" are replaced by a halogen atom. For example, it can be a trifluoromethoxy, difluorochloromethoxy, 2,2,2-trifluoroethoxy, or 1,1,2,2-tetrafluoroethoxy group.

[0213] In some implementations, R 1 For C6-C 10 Aryl or monocyclic or polycyclic heteroaryl. "Aryl or heteroaryl" means a stable 5- or 6-membered monocyclic or polycyclic ring containing 1 to 4, or preferably 1 to 3, heteroatoms selected from N, O, and S, with the remaining ring atom being carbon. When the total number of S and O atoms in the heteroaryl exceeds 1, these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heteroaryl is not greater than 2. Particularly preferred is that the total number of S and O atoms in the heteroaryl is not greater than 1. Optionally, the nitrogen atom in the heterocycle may be quaternized. When specified, these heteroaryl groups may also be substituted with carbon or non-carbon atoms or groups. Such substitution may include fusion with a 5- to 7-membered saturated cyclic group optionally containing 1 or 2 heteroatoms independently selected from N, O, and S to form, for example, [1,3]dioxazolo[4,5-c]pyridyl. Examples of heteroaryl groups include, but are not limited to: pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, phenylthio, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrroleyl, pyrazolyl, benzo[b]phenylthio, isoquinolinyl, quinazolinyl, quinoxalinyl, thiophene, isoindolyl, or 5,6,7,8-tetrahydroisoquinoline.

[0214] In some implementations, R 2 It is hydrogen.

[0215] In some implementations, R 2 It is a C1-C8 alkyl group. "C1-C8 alkyl" refers to a straight-chain or branched alkyl group having 1-8 carbon atoms. For example, it can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 1,3-dimethylbutyl, n-heptyl, isoheptyl, n-octyl, or isooctyl, etc.

[0216] In some implementations, R 2It is a C2-C8 alkenyl group. "Alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more carbon-carbon double bonds (C=C). The number of carbon atoms in "C2-C8 alkenyl" is between 2 and 8. For example, it can be vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, allylmethyl, hexenyl, heptenyl, or octenyl, etc.

[0217] In some implementations, R 2 It is a C2-C8 ynyl group. "Yynyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more carbon-carbon triple bonds (C≡C). The number of carbon atoms in "C2-C8 ynyl" is between 2 and 8. For example, it can be ethynyl, propynyl, 1-butynyl, 2-butynyl, pentynyl, hexynyl, heptynyl, or octyynyl, etc.

[0218] In some implementations, R 2 It is a C1-C8 acyl group. "C1-C8 acyl group" refers to the acyl group of a carboxylic acid derivative containing 1-8 carbon atoms, with the general formula R-CO-, where R is a C1-C8 hydrocarbon group, such as formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, hexanoyl, heptanyl, or octanoyl, etc.

[0219] In some implementations, R 2 For C6-C 10 Aryl or monocyclic or polycyclic heteroaryl. For example, it can be pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, phenylthio, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrroleyl, pyrazolyl, benzo[b]phenylthio, isoquinolinyl, quinazolinyl, quinoxalinyl, thiophene, isoyindolyl, or 5,6,7,8-tetrahydroisoquinoline, etc.

[0220] In some implementations, R 2 These are aryl acyl or heteroaryl acyl groups. An "aryl acyl" group is a group formed by the direct attachment of an aryl group (Ar-) to a carbonyl group (C=O). A "heteroaryl acyl" group is a group formed by the direct attachment of a heteroaryl group (HeteroAr-) to a carbonyl group (C=O). Examples include benzoyl, naphthoyl, p-methylbenzoyl, p-nitrobenzoyl, p-chlorobenzoyl, m-chlorobenzoyl, o-hydroxybenzoyl, furanyl, thiophenecarboxyl, pyridinecarboxyl, pyrrolecarboxyl, thiazolecarboxyl, pyrimidinecarboxyl, or quinolinecarboxyl, etc.

[0221] In some implementations, R 3 It is hydrogen.

[0222] In some implementations, R3 It is a C1-C6 alkyl group. "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1-6 carbon atoms. For example, it can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, or neohexyl, etc.

[0223] In some implementations, R 3 It refers to a C1-C6 haloalkyl group. "C1-C6 haloalkyl" means a group formed by replacing one or more hydrogen atoms on a "C1-C6 alkyl" group with halogen atoms (F, Cl, Br, I). Examples include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, trichloromethyl, bromomethyl, 2-fluoroethyl, 1,1-dichloroethyl, 2,2,2-trifluoroethyl, 3-chloropropyl, 2-fluoroisopropyl, 4-bromobutyl, or 2-chloropentyl, etc.

[0224] In some implementations, R 3 It is a C1-C6 alkoxy group. "C1-C6 alkoxy" refers to a group in which a "C1-C6 alkyl" group is attached to the parent molecule through an oxygen atom. For example, it can be methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexyloxy, or isohexyloxy, etc.

[0225] In some implementations, R 3 It refers to a C1-C6 haloalkoxy group. A "C1-C6 haloalkoxy group" is a group formed when one or more hydrogen atoms in a "C1-C6 alkoxy group" are replaced by a halogen atom. Examples include difluoromethoxy, trifluoromethoxy, trichloromethoxy, 2-chloroethoxy, 2,2,2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 3-bromopropoxy, or heptafluoroisopropoxy, etc.

[0226] In some implementations, R 3 It is a halogen. For example, it can be -F, -Cl, -Br, -I, etc.

[0227] In some implementations, R 4 It is hydrogen.

[0228] In some implementations, R 4 It is a C1-C6 alkyl group. For example, it can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, or neohexyl, etc.

[0229] In some implementations, R 4It refers to a C1-C6 alkyl carbonyl group. A "C1-C6 alkyl carbonyl group" is a straight-chain or branched group formed by connecting a "C1-C6 alkyl" group to a carbonyl group (-C(=O)-). Examples include formyl, acetyl, propionyl, isopropionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, neovaleryl, or n-hexanoyl, etc.

[0230] In some implementations, R 4 It is a C1-C8 acyl group. For example, it can be formyl, acetyl, propionyl, isopropionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, hexanoyl, heptanyl, or octanoyl, etc.

[0231] In some implementations, R 4 It is an aryl acyl group. For example, it can be benzoyl, naphthoyl, p-methylbenzoyl, p-nitrobenzoyl, p-chlorobenzoyl, m-chlorobenzoyl, or o-hydroxybenzoyl, etc.

[0232] In some implementations, R 4 It is a heteroaryl acyl group. For example, it can be furanoyl, thiophenecarboxyl, pyridinecarboxyl, pyrrolocarboxyl, thiazolecarboxyl, pyrimidinecarboxyl, or quinolinecarboxyl, etc.

[0233] In some implementations, R 1 R 2 R 3 and R 4 The alkyl, ynyl, or alkenyl groups in the definition are straight-chain.

[0234] In some implementations, R 1 R 2 R 3 and R 4 The alkyl, alkynyl, or alkenyl groups in the definition are branched.

[0235] In some implementations, R 1 R 2 R 3 and R 4 The group defined is not substituted by a substituent.

[0236] In some implementations, R 1 R 2 R 3 and R 4 The group defined is substituted by one or two independent substituents selected from the following: -F, -Cl, -Br, -I, -CN, -NCO, -NCS, -OH, -NH2, -NO2, benzene ring, pyridine, pyrrole, thiophene, pyran, and furan.

[0237] In some embodiments, the compound is a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof of the structure described in Formula I.

[0238] In some embodiments, the compound has the structure described in formula Ia.

[0239]

[0240] Formula Ia

[0241] In some implementations, R in formula Ia 1 It is hydrogen.

[0242] In some implementations, R in formula Ia 1 It is a C1-C3 haloalkyl group. "C1-C3 haloalkyl" refers to a group formed by replacing one or more hydrogen atoms on a "C1-C3 alkyl" group with halogen atoms (F, Cl, Br, I). For example, it can be trifluoromethyl, chloromethyl, difluoromethyl or trichloromethyl, etc.

[0243] In some implementations, R in formula Ia 1 It is a C1-C3 alkoxy group. "C1-C3 alkoxy" refers to a group in which a "C1-C3 alkyl" group is attached to the parent molecule through an oxygen atom. For example, it can be methoxy, ethoxy, n-propoxy, or isopropoxy.

[0244] In some implementations, R in formula Ia 1 It is a C1-C3 haloalkoxy group. A "C1-C3 haloalkoxy group" is a group formed by replacing one or more hydrogen atoms in a "C1-C3 alkoxy group" with a halogen atom. For example, it can be a trifluoromethoxy or difluoromethoxy group.

[0245] In some implementations, R in formula Ia 2 It is hydrogen.

[0246] In some implementations, R in formula Ia 2 It is benzoyl.

[0247] In some implementations, R in formula Ia 3 It is hydrogen.

[0248] In some implementations, R in formula Ia 3 It is a C1-C3 alkyl group, for example, it can be methyl, ethyl, n-propyl or isopropyl, etc.

[0249] In some implementations, R in formula Ia 3 It is a C1-C3 alkoxy group, for example, it can be methoxy, ethoxy, n-propoxy or isopropoxy, etc.

[0250] In some implementations, R in formula Ia 3 It is a C1-C3 haloalkyl group, for example, it can be trifluoromethyl, chloromethyl, difluoromethyl or trichloromethyl, etc.

[0251] In some implementations, R in formula Ia 3 It can be a halogen, for example, -F, -Cl, -Br, -I, etc.

[0252] In some implementations, R in formula Ia 4 It is hydrogen.

[0253] In some implementations, R 2 For hydrogen, R 4 For hydrogen, R 1 Selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy, and R 3 It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0254] In some implementations, R 2 It is benzoyl, R 4 For hydrogen, R 1 Selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy, and R 3 It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0255] For example, the compound has the structure of formula Ia, wherein R 1 =Hydrogen, R 2 =Hydrogen, R 3 =hydrogen, and R 4 =Hydrogen.

[0256] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethyl, R 2 =Hydrogen, R 3 =hydrogen, and R 4 =Hydrogen.

[0257] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethoxy, R 2 =Hydrogen, R 3 =hydrogen, and R 4 =Hydrogen.

[0258] For example, the compound has the structure of formula Ia, wherein R 1 =Hydrogen, R 2 =benzoyl group, R3 =hydrogen, and R 4 =Hydrogen.

[0259] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethyl, R 2 =benzoyl group, R 3 =hydrogen, and R 4 =Hydrogen.

[0260] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethoxy, R 2 =benzoyl group, R 3 =hydrogen, and R 4 =Hydrogen.

[0261] For example, the compound has the structure of formula Ia, wherein R 1 =Hydrogen, R 2 =Hydrogen, R 3 =Chlorine, and R 4 =Hydrogen.

[0262] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethyl, R 2 =Hydrogen, R 3 =Chlorine, and R 4 =Hydrogen.

[0263] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethoxy, R 2 =Hydrogen, R 3 =Chlorine, and R 4 =Hydrogen.

[0264] For example, the compound has the structure of formula Ia, wherein R 1 =Hydrogen, R 2 =benzoyl group, R 3 =Chlorine, and R 4 =Hydrogen.

[0265] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethyl, R 2 =benzoyl group, R 3 =Chlorine, and R 4 =Hydrogen.

[0266] For example, the compound has the structure of formula Ia, wherein R 1 =trifluoromethoxy, R 2 =benzoyl group, R 3 =Chlorine, and R 4=Hydrogen.

[0267] In some embodiments, the compound is a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof of the structure of formula Ia.

[0268] This application is not limited to any specific tautomer, but includes all tautomer forms, even if the compounds exist in different tautomeric forms.

[0269] All the compounds mentioned above include compounds having all possible isotopes of the atoms appearing in the compound. Isotopes include those atoms having the same atomic number but different mass numbers. By general example, without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include... 11 C 13 C and 14 C.

[0270] Methods and uses

[0271] On the other hand, this application provides the use of compounds capable of binding to heterodimers formed by μ-opioid receptors and δ-opioid receptors in the preparation of medicaments for treating and / or alleviating gliomas.

[0272] On the other hand, this application provides compounds that can bind to heterodimers formed by μ-opioid receptors and δ-opioid receptors, which are used to treat and / or alleviate gliomas.

[0273] On the other hand, this application provides a method for treating and / or alleviating glioma, the method comprising administering an effective amount of a compound to a subject in need, the compound being capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

[0274] In some embodiments, the glioma is selected from WHO grade I, II, III and IV gliomas.

[0275] In some embodiments, the glioma is selected from astrocytoma, oligodendroglioma, ependymoma, and oligodendroastrocytoma.

[0276] In some embodiments, the glioma is a glioblastoma (GBM).

[0277] In some embodiments, the glioma is primary GBM (IDH wild-type GBM).

[0278] In some embodiments, the glioma is secondary GBM (IDH-mutant GBM).

[0279] In some embodiments, the glioma is a heterogeneous glioma.

[0280] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0281] In some embodiments, the carrier includes excipients and / or diluents, and must have sufficiently high purity and very low toxicity to be suitable for administration to a patient to be treated. The carrier may be inert or may itself have pharmaceutical benefits.

[0282] Carriers include, but are not limited to, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, flow aids, lubricants, preservatives, stabilizers, surfactants, tableting agents, or wetting agents. Some carriers may be listed in more than one category; for example, vegetable oils may be used as lubricants in some formulations and as diluents in others. Exemplary pharmaceutical carriers include sugars, starches, cellulose, tragacanth gum powder, malt, gelatin, talc, or vegetable oils. Optional active agents may be included in the pharmaceutical composition, which substantially do not affect the biological function of the compounds of this application.

[0283] On the other hand, this application provides a pharmaceutical combination for alleviating and / or treating gliomas, the pharmaceutical combination comprising the compounds described in this application, as well as one or more other antitumor drugs.

[0284] In some embodiments, the drug combination comprises chemotherapy drugs. For example, it may include temozolomide, carmustine, lomustine, nimustine, irinotecan, etoposide, carboplatin, cisplatin, or procarbazine.

[0285] In some embodiments, the drug combination comprises a molecularly targeted drug. In some embodiments, the molecularly targeted drug comprises a VEGF / VEGFR inhibitor, a BRAF inhibitor, a MEK inhibitor, an mTOR / PI3K pathway inhibitor, or an EGFR inhibitor. For example, it may comprise bevacizumab, dabrafenib, trametinib, everolimus, or gefitinib.

[0286] In some embodiments, the drug combination comprises an immune checkpoint inhibitor. For example, it may comprise a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0287] In some embodiments, the drug combination comprises cellular drugs. For example, it may comprise CAR-T cells, TCR-T cells, CAR-NK cells, or TIL cells.

[0288] In some embodiments, the drug combination further includes administering chemotherapy to the subject.

[0289] In some embodiments, the drug combination further includes administering radiation therapy to the subject.

[0290] In some embodiments, the drug combination further includes administering immunotherapy to the subject.

[0291] The combined administration of the drugs described in this application can be performed via simultaneous or sequential administration. When sequential administration is used, the dosing interval between the compound and the chemotherapeutic drug, molecularly targeted drug, immune checkpoint inhibitor, and cellular drug can be approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 12 hours, approximately 24 hours, approximately 48 hours, or approximately 72 hours. The specific dosing order and interval can be optimized based on the pharmacokinetic properties, pharmacodynamic interactions, and desired synergistic effects of the selected drugs. For example, the compound of this application can be administered first to pre-treat the tumor microenvironment, followed by the administration of an immune checkpoint inhibitor or CAR-T cells. This combination therapy regimen aims to enhance efficacy, overcome drug resistance, and potentially reduce the single-drug dosage of each drug.

[0292] The compounds, drugs, and / or drug combinations disclosed herein can be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, orally, rectally, as ophthalmic solutions, or otherwise, in dosage unit formulations comprising conventional pharmaceutical carriers. Drug combinations can be formulated into any pharmaceutical form, such as aerosols, creams, gels, pills, capsules, tablets, syrups, transdermal patches, or ophthalmic solutions. Some dosage forms, such as tablets and capsules, can be further subdivided into appropriate dosage unit formulations containing an appropriate amount of the active ingredient, such as an effective amount to achieve the desired effect.

[0293] On the other hand, this application provides a method for identifying agents for treating and / or alleviating gliomas, the method comprising: (1) contacting a candidate compound with μ-opioid receptors and δ-opioid receptors, and (2) detecting the effect of the candidate compound on dimerization of μ-opioid receptors and δ-opioid receptors or their downstream signaling pathways. Wherein, when the dimerization or downstream signaling pathway is enhanced relative to the control compared to an untreated control, the candidate compound is identified as an agent that can be used to alleviate and / or treat gliomas.

[0294] In some embodiments, the expression system for the μ-opioid receptor and the delta-opioid receptor is a cell line (e.g., an astrocyte cell line) that naturally expresses the μ-opioid receptor and the delta-opioid receptor. In some embodiments, the expression system for the μ-opioid receptor and the delta-opioid receptor is a host cell that has been genetically engineered to stably or transiently express the μ-opioid receptor and the delta-opioid receptor.

[0295] In some embodiments, after obtaining quantitative data by the method, when the detection signal value of the candidate compound treatment group shows a statistically significant enhancement relative to the untreated control group, the candidate compound is identified as an agent that can be used to alleviate and / or treat gliomas. For example, compared to the untreated control, the dimerization or downstream signaling pathway is enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more.

[0296] In some embodiments, the glioma includes astrocytoma, oligodendroglioma, ependymoma, or oligodendroastrocytoma.

[0297] In some embodiments, the glioma is a glioblastoma (GBM).

[0298] In some embodiments, the candidate compound is a small molecule, peptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0299] For example, the small molecule can be a known opioid ligand or its derivative, or it can be a completely new chemical entity.

[0300] For example, the polypeptide can be a linear chain composed of natural or non-natural amino acids, or a cyclic peptide formed by disulfide bonds or other chemical bonds. It can also be a polypeptide obtained by modifying the sequence of opioid peptides (such as enkephalins, endorphins, and dynorphins).

[0301] For example, the protein may be an antibody or its antigen-binding fragment, a fusion protein, an engineered protein, etc.

[0302] For example, the nucleic acid can be DNA, RNA, antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), or a mimic thereof.

[0303] For example, the sugars may be polysaccharides or their conjugates (such as glycoproteins, glycolipids, etc.).

[0304] In some embodiments, step (1) includes: providing a test system containing μ-opioid receptors and δ-opioid receptors; introducing the candidate compound into the test system so that it contacts the μ-opioid receptors and the δ-opioid receptors.

[0305] In some embodiments, the test system is a vector, cell, tissue, organ, or animal model expressing the μ-opioid receptor and / or the δ-opioid receptor.

[0306] In some embodiments, the cells are astrocytes. In some embodiments, the cells are oligodendrocytes. In some embodiments, the cells are microglia. In some embodiments, the cells are ependymal cells.

[0307] In some embodiments, the dimerization includes the degree of dimerization. For example, the degree of dimerization can be quantitatively or semi-quantitatively detected by techniques such as fluorescence resonance energy transfer (FRET), bioluminescent resonance energy transfer (BRET), bimolecular complementary fluorescence (BiFC), co-immunoprecipitation (Co-IP), or proximity linkage (PLA).

[0308] In some embodiments, the dimerization includes the stability of the formed dimer. For example, the stability of the dimer can be assessed by detecting the dimer dissociation constant (Kd) or the duration of dimer presence after agonist stimulation.

[0309] In some implementations, the dimerization includes the functionality of the formed dimer. For example, the intensity of its downstream signal transduction.

[0310] In some embodiments, the dimerization includes the activity of the formed dimer.

[0311] In some embodiments, the μ-opioid receptor and delta-opioid receptor are derived from mammals. For example, the μ-opioid receptor and delta-opioid receptor are derived from mice, rats, or non-human primates. For example, the μ-opioid receptor and delta-opioid receptor are derived from humans.

[0312] In some embodiments, the μ-opioid receptor includes μ1-opioid receptor, μ2-opioid receptor, and μ3-opioid receptor.

[0313] In some embodiments, the δ-opioid receptor includes δ1-opioid receptors and δ2-opioid receptors.

[0314] For example, the μ-δ-heterodimeric opioid receptor is a μ1-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ1-δ2-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ2-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ2-δ2-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ3-δ1-heterodimeric opioid receptor. For example, the μ-δ-heterodimeric opioid receptor is a μ3-δ2-heterodimeric opioid receptor.

[0315] In some embodiments, the μ-opioid receptor comprises the amino acid sequence encoded by Ensembl gene database number ENSG00000112038 (OPRM1 gene), including but not limited to protein isoforms corresponding to various transcripts generated by alternative splicing of this gene, or variants having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity with the amino acid sequence encoded by any of the aforementioned transcripts, and retaining the function of forming heterodimers with the δ-opioid receptor. The variants may include naturally occurring allelic variations, splice variants, or engineered sequences.

[0316] In some embodiments, the delta-opioid receptor comprises the amino acid sequence encoded by Ensembl gene database number ENSG00000116329 (OPRD1 gene), including but not limited to protein isoforms corresponding to various transcripts generated by alternative splicing of this gene, or variants having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher sequence identity with the amino acid sequence encoded by any of the aforementioned transcripts, and retaining heterodimerization function with the μ-opioid receptor. The variants may include naturally occurring allelic variations, splice variants, or engineered sequences.

[0317] In some embodiments, the downstream signaling pathway includes one or more indicators selected from the group consisting of: (1) expression or activity of intracellular glutamate transporter 1, (2) concentration of intracellular cyclic adenosine monophosphate, (3) expression or activity of intracellular brain-derived neurotrophic factor, (4) expression or activity of intracellular glial fibrillary acidic protein, (5) expression or activity of intracellular monoamine oxidase B, (6) expression or activity of intracellular potassium channel Kir4.1, and (7) intercellular glutamate concentration in the brain.

[0318] This application provides the following implementation scheme:

[0319] 1. Use of a compound in the preparation of a medicament for the relief and / or treatment of glioma, wherein the compound is capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

[0320] 2. The use according to embodiment 1, wherein the compound is capable of promoting heterodimerization of μ-opioid receptors and δ-opioid receptors, and / or enhancing the stability, function and / or activity of the heterodimers formed by μ-opioid receptors and δ-opioid receptors.

[0321] 3. The use according to any one of embodiments 1-2, wherein the heterodimer is expressed in the central nervous system.

[0322] 4. The use according to any one of embodiments 1-3, wherein the heterodimer is expressed in the brain.

[0323] 5. The use according to any one of embodiments 1-4, wherein the heterodimer is expressed on the surface of glial cells.

[0324] 6. The use according to any one of embodiments 1-5, wherein the heterodimer is expressed on the surface of astrocytes, oligodendrocytes, microglia and / or ependymal cells.

[0325] 7. The use according to any one of embodiments 1-6, wherein the compound is capable of restoring reactive astrocytes to normal astrocytes.

[0326] 8. The use according to any one of embodiments 1-7, wherein the compound is capable of upregulating the expression of glutamate transporter 1 (GLT-1) and / or increasing its activity in astrocytes.

[0327] 9. The use according to any one of embodiments 1-8, wherein said compound has one or more of the following properties:

[0328] (1) Increase the concentration of cyclic adenosine monophosphate (cAMP) in astrocytes;

[0329] (2) Increase the concentration of cAMP-response element-binding protein (pCREB) in astrocytes;

[0330] (3) Upregulates the expression of brain-derived neurotrophic factor (BDNF) in astrocytes;

[0331] (4) Upregulates the expression of glial fibrillary acidic protein (GFAP) in astrocytes;

[0332] (5) Reduce the expression of monoamine oxidase B (MAOB) in astrocytes;

[0333] (6) Upregulates the expression of the potassium channel Kir4.1 in astrocytes; and

[0334] (7) Reduce the concentration of glutamate in the intercellular spaces of the brain.

[0335] 10. The use according to any one of embodiments 1-9, wherein the compound is capable of altering the tumor microenvironment of the glioma.

[0336] 11. The use according to any one of embodiments 1-10, wherein the μ-opioid receptor and / or δ-opioid receptor are derived from mammals.

[0337] 12. The use according to any one of embodiments 1-11, wherein the μ-opioid receptor comprises a μ1-opioid receptor, a μ2-opioid receptor, and / or a μ3-opioid receptor.

[0338] 13. The use according to any one of embodiments 1-12, wherein the μ-opioid receptor is encoded by a gene with Ensembl database number ENSG00000112038.

[0339] 14. The use according to any one of embodiments 1-13, wherein the δ-opioid receptor comprises a δ1-opioid receptor and / or a δ2-opioid receptor.

[0340] 15. The use according to any one of embodiments 1-14, wherein the δ-opioid receptor is encoded by a gene with Ensembl database number ENSG00000116329.

[0341] 16. The use according to any one of embodiments 1-15, wherein the compound is a small molecule, polypeptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0342] 17. The use according to any one of embodiments 1-16, wherein the compound is a metabolite of ketamine.

[0343] 18. The use according to any one of embodiments 1-17, wherein said compound has the structure shown in Formula I:

[0344]

[0345] Formula I

[0346] in,

[0347] R1 is independently selected from hydrogen, halogen, hydroxyl, amino, nitro, cyano, amide, C1-C4 alkyl, C1-C4 alkoxy, mono- and di-C1-C4 alkylamino, C1-C2 haloalkyl, C1-C2 haloalkoxy, C6-C10 aryl or monocyclic or polycyclic heteroaryl.

[0348] R2 is independently selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 acyl, C6-C10 aryl, monocyclic or polycyclic heteroaryl, aryl acyl or heteroaryl acyl;

[0349] R3 is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or halogen;

[0350] R4 is independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl carbonyl, C1-C8 acyl, aryl acyl or heteroaryl acyl;

[0351] Furthermore, the alkyl, alkynyl, or alkenyl groups defined in R1, R2, R3, and R4 are straight-chain or branched, and are either unsubstituted or substituted by one or two independent substituents selected from the following: -F, -Cl, -Br, -I, -CN, -NCO, -NCS, -OH, -NH2, -NO2, benzene ring, pyridine, pyrrole, thiophene, pyran, or furan;

[0352] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0353] 19. The use according to any one of embodiments 1-18, wherein the compound has the structure shown in formula Ia:

[0354]

[0355] Formula Ia

[0356] Where R1, R2, R3, and R4 are as defined in Equation I,

[0357] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0358] 20. The use according to embodiment 19, wherein R1 is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

[0359] 21. The use according to any one of embodiments 19-20, wherein R1 is hydrogen, trifluoromethyl, or trifluoromethoxy.

[0360] 22. The use according to any one of embodiments 19-21, wherein the R2 is hydrogen or benzoyl.

[0361] 23. The use according to any one of embodiments 19-22, wherein the R3 is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkyl, C1-C3 alkoxy or halogen.

[0362] 24. The use according to any one of embodiments 19-23, wherein the R3 is hydrogen or chlorine.

[0363] 25. The use according to any one of embodiments 19-24, wherein the R4 is hydrogen.

[0364] 26. The use according to any one of embodiments 18-25, wherein a pharmaceutically acceptable salt of said compound is a hydrochloride salt.

[0365] 27. The use according to any one of embodiments 1-26, wherein said compound has the structure shown in Formula II:

[0366]

[0367] Formula II.

[0368] 28. The use according to any one of embodiments 1-26, wherein said compound has a structure selected from the group consisting of:

[0369] , , , , , , ,

[0370] Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

[0371] 29. The use according to any one of embodiments 1-28, wherein the glioma includes WHO grade I, II, III and / or IV gliomas.

[0372] 30. The use according to any one of embodiments 1-29, wherein the glioma includes astrocytoma, oligodendroglioma, ependymoma and / or oligodendroastrocytoma.

[0373] 31. The use according to any one of embodiments 1-30, wherein the glioma is glioblastoma (GBM).

[0374] 32. According to the use described in embodiment 31, the glioblastoma (GBM) includes primary GBM (IDH wild-type GBM) and / or secondary GBM (IDH mutant GBM).

[0375] 33. The use according to any one of embodiments 1-32, wherein the glioma includes heterogeneous glioma.

[0376] 34. The use according to any one of embodiments 1-33, wherein the drug further comprises a pharmaceutically acceptable carrier.

[0377] 35. A drug combination for alleviating and / or treating glioma, comprising the compound used in any one of embodiments 1-34, and one or more other antitumor drugs.

[0378] 36. The drug combination according to embodiment 35, wherein the other antitumor drugs are selected from the group consisting of: chemotherapy drugs, molecularly targeted drugs, immune checkpoint inhibitors, and cell drugs.

[0379] 37. The drug combination according to any one of embodiments 35-36, further comprising administering one or more other anticancer therapies, such as chemotherapy, radiotherapy or immunotherapy, to the subject.

[0380] 38. A method for alleviating and / or treating glioma, the method comprising administering an effective amount of a compound to a subject in need, the compound being capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

[0381] 39. A method for identifying an agent for alleviating and / or treating glioma, the method comprising: (1) contacting a candidate compound with μ-opioid receptors and δ-opioid receptors, and (2) detecting the effect of the candidate compound on the dimerization of μ-opioid receptors and δ-opioid receptors or their downstream signaling pathways;

[0382] Specifically, when the dimerization or downstream signaling pathway is enhanced relative to the control compared to the untreated control, the candidate compound is identified as an agent that can be used to alleviate and / or treat glioma.

[0383] 40. The method according to embodiment 39, wherein the candidate compound is a small molecule, peptide, protein, nucleic acid, carbohydrate, lipid, polymer or metal complex, or a combination thereof.

[0384] 41. The method according to any one of embodiments 39-40, wherein prior to step (1) the method comprises: providing a test system comprising a μ-opioid receptor and a δ-opioid receptor; and introducing the candidate compound into the test system such that it contacts the μ-opioid receptor and the δ-opioid receptor.

[0385] 42. The method according to embodiment 41, wherein the test system is a vector, cell, tissue, organ or animal model expressing the μ-opioid receptor and / or the δ-opioid receptor.

[0386] 43. The method according to embodiment 42, wherein the cells are selected from astrocytes, oligodendrocytes, microglia and / or ependymal cells.

[0387] 44. The method according to any one of embodiments 42-43, wherein the cell is an astrocyte.

[0388] 45. The method according to any one of embodiments 39-44, wherein the dimerization includes the degree of dimerization and / or the stability, function and / or activity of the dimer.

[0389] 46. ​​The method according to any one of embodiments 39-45, wherein the downstream signaling pathway comprises one or more indicators selected from the group consisting of: (1) expression or activity of intracellular glutamate transporter 1, (2) concentration of intracellular cyclic adenosine monophosphate, (3) expression or activity of intracellular brain-derived neurotrophic factor, (4) expression or activity of intracellular glial fibrillary acidic protein, (5) expression or activity of intracellular monoamine oxidase B, (6) expression or activity of intracellular potassium channel Kir4.1, and (7) intercellular glutamate concentration in the brain.

[0390] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the compounds, drugs, drug combinations, etc. of this application and are not intended to limit the scope of the invention.

[0391] Example

[0392] Example 1: Synthesis of (2R, 6R)-hydroxydemethylketamine (HNK)

[0393] The synthetic route of HNK is as follows Figure 1 As shown, the specific steps are as follows:

[0394] Step 1: In a round-bottom flask, add o-chlorobenzonitrile (200 mmol, 28.7 g) and CuBr (0.5 mmol, 86.64 mg), under argon protection, add 200 ml of THF, and slowly add cyclopentylmagnesium bromide (200 mmol, 200 ml) dropwise under ice bath conditions, stirring at room temperature for 16 h. Add 100 ml of water and 200 ml of 4N hydrochloric acid, stirring at room temperature for 16 h. Reduce THF to dryness, extract with EA, dry, and perform column chromatography (PE) to give compound H-1 29 g, yield 70%.

[0395] Step 2: Add compound H-1 (105 mmol, 22 g), NBS (116 mmol, 20 g), and TsOH (211 mmol, 40 g) to a round-bottom flask, add 400 ml of EA, heat to 50 °C and reflux for 6 h, evaporate EA to dryness, and perform column chromatography (PE) to obtain compound H-2 21 g, yield 70%.

[0396] Step 3: Pass ammonia gas (obtained by heating ammonia water) through 400 ml of ammonia solution until saturation. Add 20 g of compound H-2 and stir at room temperature for 24 h. The product precipitates out, is filtered, and dried to obtain a brown solid compound H-3. This product can be directly proceeded to the next step without purification.

[0397] Step 4: Dissolve compound H-3 in anhydrous THF, purge with HCl gas until pH 1, add concentrated sulfuric acid dropwise to sodium chloride to obtain HCl gas, evaporate THF to dryness, and obtain hydrochloride of compound H-4. Place the above H-4 hydrochloride in a round-bottom flask, under argon protection, heat the system to 175 °C and reflux for 20 min, cool to room temperature, add saturated sodium bicarbonate solution, extract with DCM, evaporate to dryness, and perform column chromatography (DCM) to obtain 6.5 g of racemic mixture of compound H-5 desmethylketamine. The yield of steps 2-4 is 40%. 1 H NMR (400 MHz, CDCl3) δ 7.70 (dd, J = 7.8, 1.7 Hz, 1H), 7.40 – 7.31 (m,2H), 7.30 – 7.22 (m, 1H), 2.83 – 2.71 (m, 1H), 2.64 – 2.55 (m, 1H), 2.52 –2.44 (m, 1H), 2.09 – 2.01 (m, 1H), 1.86 – 1.74 (m, 3H), 1.73 – 1.65 (m, 1H).

[0398] Step 5: Dissolve the racemic mixture of compound H-5 (norketamine) (29 mmol, 6.5 g) in anhydrous ethanol, add L-(S)-pyroglutamic acid (14.5 mmol, 1.8 g), stir, and heat under reflux for 5 min. A white solid precipitates during heating. Cool to room temperature, stir for 16 h, and filter. Dissolve the resulting white solid in anhydrous ethanol, stir, heat under reflux for 5 min, cool to room temperature, stir for 2 h, and filter. Dissolve the resulting white solid in anhydrous ethanol, heat under reflux for 5 min, stir, cool to room temperature, filter, and collect the white solid. Neutralize with 1N sodium hydroxide, extract with EA, to obtain compound H-6 (R)-norketamine 970 mg, yield 15%. The optical purity was 98% ee as determined by chiral HPLC. 1 H NMR (400 MHz, CDCl3) δ 7.69(dd, J = 7.8, 1.7 Hz, 1H), 7.39 – 7.31 (m, 2H), 7.25 (td, J = 7.6, 1.7 Hz,1H), 2.85 – 2.69 (m, 1H), 2.65 – 2.53 (m, 1H), 2.51 – 2.41 (m, 1H), 2.09 –2.04 (m, 1H), 1.88 – 1.72 (m, 3H), 1.71 – 1.61 (m, 1H).

[0399] HPLC: Chiralcel-AD-H column; mobile phase A: hexane; mobile phase B: isopropanol, A:B = 70:30; flow rate: 1 mL / min; retention time of the R-configuration isomer: 5.6 min; retention time of the S-configuration isomer: 5.1 min.

[0400] Step 6: Dissolve compound H-6(R)-norketamine (4 mmol, 900 mg) in THF, add TEA (8 mmol, 816 mg), then add (Boc)2O (8 mmol, 1761 mg), and heat under reflux for 6 h. Cool, evaporate to dryness, and perform column chromatography (PE) to give compound H-7 1.22 g. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.0 Hz, 1H), 7.41 –7.33 (m, 2H), 7.31 – 7.24 (m, 1H), 6.63 (s, 1H), 3.86 (d, J = 14.3 Hz, 1H),2.47 – 2.40 (m, 1H), 2.39 – 2.29 (m, 1H), 2.12 – 2.00 (m, 1H), 1.90 – 1.73 (m, 4H), 1.32 (s, 9H).

[0401] Step 7: Dissolve compound H-7 (3.7 mmol, 1.2 g) in anhydrous THF under argon protection. At -78 °C, add TMEDA (2.04 mmol, 237.3 mg), then slowly add LDA (11.14 mmol, 1.2 g). Stir at -78 °C for 1 h, then add TMSCl (11.14 mmol, 1.21 g), and stir at -78 °C for 1 h. After the reaction is complete as monitored by TLC, quench the reaction with saturated ammonium chloride solution, extract with EA, evaporate to dryness, and dry. The resulting yellow oily substance is dissolved in anhydrous DCM. At -15 °C, add mCPBA (11.14 mmol, 1.28 g), stir for 1 h, and after the reaction is complete as monitored by TLC, add saturated sodium thiosulfate and saturated sodium bicarbonate solution (1:1), extract with DCM, evaporate to dryness, and dry. A white solid was obtained, dissolved in anhydrous THF. TBAF (4.7 mmol, 1.22 g) was added at -15 °C, and the mixture was stirred for 30 min. After the reaction was complete as monitored by TLC, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was extracted with EA, evaporated to dryness, and subjected to column chromatography (PE:EA = 4:1). Compound H-8 was given in 692 mg. Yield: 55%. 1 H NMR (400 MHz, CDCl3) δ 7.83(d, J = 7.8 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.33 – 7.27 (m, 1H), 6.62 (s, 1H), 4.21 – 4.13 (m, 1H), 4.02 – 3.80 (m, 1H), 3.37 (d, J = 6.5 Hz, 1H), 2.44 –2.36 (m, 1H), 1.85 – 1.73 (m, 2H), 1.68 – 1.59 (m, 1H), 1.59 – 1.43 (m, 1H),1.33 (s, 9H).

[0402] Step 8: Dissolve compound H-8 (2 mmol, 690 mg) in anhydrous THF, purge with HCl gas until saturated, stir at room temperature for 4 h, add anhydrous diethyl ether, a white solid precipitates, filter to obtain 450 mg of hydrochloride of compound 2R,6R-hydroxydemethylketamine (HNK), yield 80%. 1 H NMR (400 MHz, MeOD) δ 8.01 – 7.82 (m, 1H), 7.65 – 7.56(m, 3H), 4.32 (dd, J = 11.6, 6.7 Hz, 1H), 3.29 – 3.22 (m, 1H), 2.38 – 2.29(m, 1H), 2.04 – 1.87 (m, 2H), 1.86 – 1.58 (m, 2H).

[0403] Example 2: HNK acts on μ-δ-heterodimeric opioid receptors

[0404] To demonstrate that HNK can effectively enhance the function of μ-δ-heterodimeric opioid receptors, μ and δ opioid receptors were expressed in HeLa cells and linked to complementary GFP (split GFP). The results are as follows: Figures 2-3 As shown, GFP fluorescence only appears or its intensity increases when μ-δ-heterodimeric opioid receptors are formed. Figure 2 The results showed that HNK effectively increased green fluorescence (GFP) compared to the control (saline; Sal) group, indicating increased formation or enhanced stability of μ-δ-heterodimeric opioid receptors. Figure 3 The quantitative results showed that the percentage of cells with GFP fluorescence and the fluorescence intensity of each cell with GFP fluorescence were increased or enhanced, proving that HNK binding to the μ-δ-heterodimeric opioid receptor can significantly increase the formation of the μ-δ-heterodimeric opioid receptor or enhance its stability.

[0405] PEP is a short peptide that has been shown to effectively block the formation of μ-δ-heterodimeric opioid receptors. Incubation with PEP revealed that the HNK-induced enhancement / increase in GFP, including increased fluorescence intensity and an increased percentage of cells with GFP fluorescence, was blocked by PEP (e.g., Figure 4 As shown in the figure, this result also demonstrates that HNK functions through the μ-δ heterodimeric opioid receptor.

[0406] Example 3: HNK activates downstream pathways via μ-δ-heterodimeric opioid receptors

[0407] This embodiment uses an enzyme-linked immunosorbent assay (ELISA) kit to detect changes in cAMP concentration in cells. Prepare standard working solutions, washing buffer, biotinylated antibody working solution, and enzyme conjugate working solution in advance according to the kit instructions. Add the standards and samples sequentially to the ELISA plate, 50 μL per well. Immediately add 50 μL of the pre-prepared biotinylated antibody working solution to each well. Cover the plate with a membrane and incubate at 37 °C for 45 minutes. Discard the liquid from the wells, add 350 μL of washing buffer to each well, soak for 1 minute, then discard the liquid and tap the plate on absorbent paper. Repeat three times. Add 100 μL of enzyme conjugate working solution to each well, cover with a membrane, and incubate at 37 °C for 30 minutes. Wash the plate 5 times. Add 90 μL of substrate solution to each well and incubate at 37 °C in the dark for 15 minutes. Add 50 μL of stop solution to each well to stop the reaction. Immediately after stopping the reaction, measure OD450 using a microplate reader. A standard curve of a four-parameter logistic function was plotted using Origin software, and the cAMP concentration in the sample was calculated based on the standard curve. Western blotting was used to detect the expression levels of p-CREB and CREB proteins, with the p-CREB / CREB ratio representing the CREB phosphorylation level.

[0408] The results are as follows Figures 5-7 As shown, HNK treatment significantly increased intracellular cAMP concentration in astrocytes, further leading to upregulation of p-CREB levels / proportions. This change could be effectively blocked by PEP pretreatment, indicating that HNK mediates increased cAMP concentration and upregulation of p-CREB by binding to and activating the μ-δ heterodimeric opioid receptor, thereby triggering subsequent signaling pathways.

[0409] Example 4: HNK alters protein expression on astrocytes

[0410] This embodiment further investigates the major protein changes caused by the activation of μ-δ-heterodimeric opioid receptors on astrocytes (CTX TNA2). Frozen tissue samples were added to RIPA lysis buffer (with protease and phosphatase inhibitors pre-added) and homogenized using an ultrasonic lysis device. After centrifugation at 12000 × g for 20 minutes at 4 °C, the supernatant was used for the next experiment or stored at -80 °C. Total protein quantification was performed using a BCA kit. SDS-PAGE electrophoresis and gel transfer were performed. After transfer, the membrane was placed in blocking buffer and shaken on a low-speed shaker for 1 hour. The blocked membrane was cut to the target band size and placed in the corresponding primary antibody. It was shaken on a low-speed shaker and incubated overnight at 4 °C or 2 hours at room temperature. The primary antibody was aspirated, and the membrane was washed with TBST buffer. The membrane was shaken on a high-speed shaker for 10 minutes each time, washing at least 3 times. Secondary antibodies were prepared according to the source of the primary antibody, and the membrane was incubated in the secondary antibody buffer for 1 hour. After removing the secondary antibody, the membrane was washed. Finally, it was exposed and images were acquired using an e-blot developer.

[0411] The results are as follows Figure 8 As shown, in astrocytes, the levels of highly expressed and specifically expressed GLT-1 and BDNF proteins were significantly increased and significantly upregulated with increasing drug concentration, showing a clear concentration-dependent gradient.

[0412] Example 5: Validation of HNK binding to μ-δ-heterodimeric opioid receptors and activation of downstream pathways using a depression model.

[0413] In a mouse model of depression induced by the stress hormone ACTH, HNK was found to bind to μ-δ-heterodimeric opioid receptors in the brain and induce activation of downstream signaling pathways. The results are as follows: Figures 9-10 As shown. Studies related to gliomas have shown that elevated glutamate concentrations in the intercellular spaces of the brain are significantly associated with glioma growth, migration, and toxicity. GLT-1 is highly expressed on astrocytes, transporting glutamate from the intercellular spaces of the brain into the astrocytes. This transport effectively reduces intercellular glutamate concentrations, thereby protecting nerve cells. In vivo administration of HNK rapidly upregulates GLT-1 expression (e.g., Figure 9 As shown), and reduce the intercellular glutamate concentration (e.g. Figure 10 (As shown).

[0414] Example 6: Validation of HNK's Inhibition of Tumor Growth in Three Glioma Models

[0415] Different mouse glioma models were constructed. The xenograft model was established by implanting human glioma cell line (U87-MG) or patient-derived primary tumor cells (MU41) into nude mice; the syngeneic model was established by implanting mouse glioma cell line GL261 into C57BL / 6 mice. To facilitate monitoring tumor growth, all U87-MG, MU41, and GL261 cells carried the luciferase gene. Cell lines were cultured to appropriate growth stages, digested with trypsin for cell counting, and diluted to 50,000 cells / μL. Six- to eight-week-old mice were fixed in a stereotaxic apparatus, positioned to the target area (AP +1.5 mm, ML +1 mm, DV -3.5 mm), and a microdrill was used to drill a hole, avoiding damage to the dura mater. One μL of cells was injected, the puncture site was sealed with tissue glue, and the skin was sutured. In vivo imaging was performed after wound healing to serve as a baseline. Intraperitoneal drug administration began once the tumor reached a suitable size.

[0416] Tumor growth was monitored using bioluminescence imaging (BLI). D-Luciferin sodium was prepared as a 15 mg / mL stock solution (dissolved in PBS, sterilized with a 0.22 μm filter, and stored at -20°C protected from light) and dissolved before use. Each mouse was intraperitoneally injected with 150 mg / kg of D-Luciferin sodium. Ten to thirty minutes later, the mice were anesthetized and placed prone in a black-bottomed culture dish within the imaging chamber. The imaging system was activated, and bioluminescence and brightfield images were acquired. Image analysis was performed using IVIS Spectrum software to calculate the relative tumor size.

[0417] The results showed that in the nude mouse GBM model constructed using the U87-MG GBM cell line, HNK significantly enhanced the survival time of the model animals (e.g., Figures 11-12 As shown), it significantly reduced weight loss in animals caused by tumors (e.g. Figure 13 As shown), and slowing the growth rate of tumors (such as...). Figure 14 (As shown).

[0418] In the nude mouse GBM model constructed using the MU41 cell line, HNK significantly enhanced the survival time of the model animals (e.g., Figures 15-17 As shown), it significantly reduced weight loss in animals caused by tumors (e.g. Figure 15 , Figure 18 As shown), and slowing the growth rate of tumors (such as...). Figure 15 , Figure 19 (As shown).

[0419] The GBM model constructed using the GL261 cell line against a C57 mouse background is a mouse model with a healthy immune system. Results showed that in the C57 mouse GBM model constructed using the GL261 cell line, HNK significantly enhanced the survival time of the model animals (e.g., Figures 20-22 As shown), it significantly reduced weight loss in animals caused by tumors (e.g. Figure 20 , Figure 23 As shown), and slowing the growth rate of tumors (such as...). Figure 20 , Figure 24 (As shown).

[0420] Example 7: HNK alleviates the brain function decline caused by GBM.

[0421] The rotarod test is a widely used animal behavioral experiment to assess motor coordination, balance, and muscle endurance. In this application, it is used to detect the impact of gliomas on motor function. In pre-training, the rotarod was set to a constant low speed (5 rpm) for 1 minute. The animal was gently placed on the rotarod, and the instrument was started. This was repeated 3 times, with 10-minute intervals between each repetition. In the formal test, the parameters were set as follows: initial speed: 4 rpm, final speed: 40 rpm, acceleration time: 300 seconds (5 minutes). After the animal was placed, the acceleration program was started. The speed, time, and total distance traveled when the animal fell were recorded.

[0422] To test whether HNK could significantly slow down the decline in brain function caused by GBM, GBM cells were seeded in the motor cortex to construct three GBM tumor models as described in Example 5.

[0423] In the U87-MG glioma model, as the tumor grows, the animal's motility significantly decreases. Motility is manifested in the length of time the animal can remain on the rotundus and the maximum rotundus speed it can withstand without falling off the rotundus (e.g., ...). Figures 25-26 As shown). After 3 days of administration, there was no difference between the HNK group and the control group (as shown). Figure 25 (As shown), but after 7 days of administration, the rate of decline in exercise capacity in the HNK group was significantly lower than that in the control group (as shown). Figure 26 (As shown).

[0424] In the MU41 glioma model, there was no significant difference between the control group and the MU41 GBM group (e.g., Figures 27-28 (As shown). After 3 and 7 days of administration, the rate of decline in motor function in the HNK group was significantly lower than that in the control group (as shown). Figures 28-29 (As shown).

[0425] In the GL261 model, HNK was administered when the GBM size reached a fluorescence intensity of 10,000. Results showed that HNK significantly enhanced the survival time of the model animals (e.g., Figures 30-32As shown), it significantly reduced weight loss in animals caused by tumors (e.g. Figure 33 As shown), and slowing the growth rate of tumors (such as...). Figure 30 , Figure 34 (As shown).

[0426] The above results all indicate that HNK can slow down the decline in brain function caused by GBM.

[0427] Example 8: HNK exerts its anti-cancer effect through astrocytes rather than directly acting on tumor cells.

[0428] To evaluate the effect of HNK administration on GBM cells, Western blot protein quantification analysis was performed on GBM tumor tissue. The results are as follows: Figures 35-36 As shown, key marker proteins associated with GBM (including CD44, TGF-β, β-catenin, NeuN, and CD31) were all significantly reduced, indicating a significant weakening of tumor growth, migration, and toxicity.

[0429] To further investigate whether HNK directly affects GBM cells, cultured GBM cells were directly incubated with HNK, and the effect of HNK on GBM cell growth and proliferation was assessed by measuring cell density. The results showed no significant changes observed in any of the four GBM cell types (e.g., Figure 37 As shown in the figure, this indicates that HNK does not directly act on GBM cells.

[0430] Subsequently, GBM cells were co-cultured with the astrocyte cell line (CTX-TNA2) and then incubated with HNK. The results showed that HNK significantly reduced GBM cell density in a concentration-dependent manner (e.g., Figure 38 As shown in the figure, this indicates that HNK exerts its function through coexisting astrocytes.

[0431] Example 9: HNK targets μ-δ-heterodimeric opioid receptors on astrocytes.

[0432] p-CREB was detected using immunofluorescence staining. First, experimental mice were anesthetized and perfused: 1% sodium pentobarbital was injected intraperitoneally to confirm anesthesia. Brain tissue was then collected and fixed and dehydrated sequentially in 4% paraformaldehyde solution, 20% sucrose solution, and 30% sucrose solution, followed by OCT embedding. Brain sections with a thickness of 30 μm were cut using a cryostat and collected in six-well plates containing PBS for immunoassay or stored at 4°C (if the brain tissue was fluorescently labeled, it needed to be stored in the dark).

[0433] Brain slice permeabilization: Transfer brain slices to 24-well plates, add 1 mL of PBST, and wash three times on a shaker for 10 minutes each time. Then add immunofluorescence blocking buffer and shake on a shaker for 2 hours. After blocking, aspirate the blocking buffer, dilute the primary antibody with blocking buffer according to the manufacturer's instructions, and incubate overnight at 4 °C or for 2 hours at room temperature. After aspirating the primary antibody, wash three times with PBST solution for 10 minutes each time. Dilute the secondary antibody with blocking buffer according to the manufacturer's instructions and incubate at room temperature for 2 hours. Repeat the washing process.

[0434] Arrange the brain slices neatly on a glass slide, let them dry, add an antiquenching agent, cover with a coverslip, and seal the edges of the coverslip with colorless nail polish. Take pictures using a confocal microscope to acquire images.

[0435] Following in vivo administration of HNK, immunofluorescence staining results showed a significant increase in the intensity and density of p-CREB fluorescent staining (e.g., Figures 39-40 As shown). The proportion of P-CREB and GFAP co-stained also increased significantly (e.g. Figure 41 As shown in the figure, this increase in pCREB mainly occurs on astrocytes.

[0436] Further investigation was conducted into the density and morphology of GFAP-positive astrocytes. In U87-MG model mice, the fluorescence intensity of GFAP was significantly increased in the HNK group (e.g., ...). Figures 42-43 (As shown). The morphology of GFAP-positive astrocytes also showed significant changes, and the cell branch length in the HNK group was significantly shortened (e.g., Figure 43 As shown in the figure, this indicates a significant improvement in the reactivity of astrocytes.

[0437] MAOB, an important marker of reactive astrocytes, was significantly elevated in the GBM marginal region and significantly decreased in the HNK group (e.g., Figures 44-45 As shown in the figure), this indicates a significant decrease in the responsiveness of astrocytes. Furthermore, GLT-1 (e.g., [unclear text - possibly related to GLT-1 expression]) expressed on astrocytes surrounding GBM in the HNK group... Figure 46 , Figure 48 (as shown) and Kir4.1 (as shown) Figures 47-48 The levels shown (as indicated) were significantly higher than the control group, indicating that glutamate reuptake in astrocytes was related to K+. + Enhanced reuptake function.

[0438] Example 10: HNK acts directly on μ-δ-heterodimeric opioid receptors in GBM

[0439] Following in vivo administration of HNK, immunofluorescence staining of brain slices was performed to detect the expression of astrocyte markers S100β and μ-δ-heterodimer. The results showed a significant increase in the proportion of co-stained samples (e.g., ...). Figures 49-51(As shown). Previous results have shown that HNK can pharmacologically activate μ-δ-heterodimers on astrocytes, and this result indicates that the density of μ-δ-heterodimers in astrocytes increased in the HNK-treated group, providing strong support for the effective targeting of HNK in vivo.

Claims

1. Use of a compound in the preparation of a medicament for the relief and / or treatment of glioma, wherein the compound is capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

2. The use according to claim 1, wherein the compound is capable of promoting heterodimerization of μ-opioid receptors and δ-opioid receptors, and / or enhancing the stability, function and / or activity of the heterodimers formed by μ-opioid receptors and δ-opioid receptors.

3. The use according to any one of claims 1-2, wherein the heterodimer is expressed on the surface of astrocytes, oligodendrocytes, microglia and / or ependymal cells.

4. The use according to any one of claims 1-3, wherein the compound is capable of restoring reactive astrocytes to normal astrocytes.

5. The use according to any one of claims 1-4, wherein the compound is capable of upregulating the expression of glutamate transporter 1 (GLT-1) and / or increasing its activity in astrocytes.

6. The use according to any one of claims 1-5, wherein the compound is capable of altering the tumor microenvironment of the glioma.

7. The use according to any one of claims 1-6, wherein the μ-opioid receptor is encoded by a gene with Ensembl database number ENSG00000112038, and the δ-opioid receptor is encoded by a gene with Ensembl database number ENSG00000116329.

8. The use according to any one of claims 1-7, wherein the compound has the structure shown in Formula I: Formula I in, R 1 Independently selected from hydrogen, halogen, hydroxyl, amino, nitro, cyano, amide, C1-C4 alkyl, C1-C4 alkoxy, mono- and di-C1-C4 alkylamino, C1-C2 haloalkyl, C1-C2 haloalkoxy, C6-C 10 Aryl or monocyclic or polycyclic heteroaryl; R 2 Independently selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 acyl, C6-C 10 Aryl, monocyclic or polycyclic heteroaryl, aryl acyl or heteroaryl acyl; R 3 It is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or halogen; R 4 It is independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl carbonyl, C1-C8 acyl, aryl acyl or heteroaryl acyl; And R 1 R 2 R 3 and R 4 The alkyl, alkynyl, or alkenyl groups in the definition are straight-chain or branched and are either unsubstituted or substituted by one or two independent substituents selected from the following: -F, -Cl, -Br, -I, -CN, -NCO, -NCS, -OH, -NH2, -NO2, benzene ring, pyridine, pyrrole, thiophene, pyran, or furan. Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

9. The use according to any one of claims 1-8, wherein the compound has the structure shown in formula Ia: Formula Ia, Wherein R 1 R 2 R 3 and R 4 As defined in Equation I Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

10. The use according to claim 9, wherein the R 1 It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkoxy or C1-C3 haloalkoxy.

11. The use according to any one of claims 9-10, wherein the R 1 It is hydrogen, trifluoromethyl, or trifluoromethoxy.

12. The use according to any one of claims 9-11, wherein the R 2 It is hydrogen or benzoyl.

13. The use according to any one of claims 9-12, wherein the R 3 It is selected from hydrogen, C1-C3 haloalkyl, C1-C3 alkyl, C1-C3 alkoxy or halogen.

14. The use according to any one of claims 9-13, wherein the R 3 It can be hydrogen or chlorine.

15. The use according to any one of claims 9-14, wherein the R 4 It is hydrogen.

16. The use according to any one of claims 1-15, wherein the compound has the structure shown in Formula II: Formula II.

17. The use according to any one of claims 1-15, wherein the compound has a structure selected from the group consisting of: 、 、 、 、 、 、 ， Or a pharmaceutically acceptable salt, stereoisomer, tautomer, or mixture thereof.

18. The use according to any one of claims 1-17, wherein the glioma is glioblastoma (GBM).

19. The use according to any one of claims 1-18, wherein the medicament further comprises a pharmaceutically acceptable carrier.

20. A pharmaceutical combination for alleviating and / or treating glioma, comprising the compound used in any one of claims 1-19, and one or more other antitumor drugs.

21. A method for alleviating and / or treating glioma, the method comprising administering an effective amount of a compound to a subject in need, the compound being capable of binding to a heterodimer formed by μ-opioid receptors and δ-opioid receptors.

22. A method for identifying an agent for alleviating and / or treating glioma, the method comprising: (1) Contact the candidate compound with μ-opioid receptors and δ-opioid receptors, (2) detect the effect of the candidate compound on the dimerization of μ-opioid receptors and δ-opioid receptors or their downstream signaling pathways; Specifically, when the dimerization or downstream signaling pathway is enhanced relative to the control compared to the untreated control, the candidate compound is identified as an agent that can be used to alleviate and / or treat glioma.

23. The method of claim 22, wherein the dimerization includes the degree of dimerization and / or the stability, function and / or activity of the dimer.

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