Hedgehog signaling pathway inhibitors

Abstract

The present application relates to a kind of novel Hedgehog signal pathway inhibitor and preparation method thereof, the Hedgehog signal pathway inhibitor has the structure shown in formula (I).The Hedgehog signal pathway inhibitor of the present application can effectively inhibit SMO receptor activity, and block SHH signal pathway. With good effect of blocking the seizure caused by excessive or super-synchronous neuron discharge in brain. As epilepsy treatment candidate drug has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a novel Hedgehog signaling pathway inhibitor, its preparation method, and its application. Background Technology

[0002] Epilepsy is one of the most common chronic central nervous system disorders. Although the causes of epilepsy are diverse, they are typically characterized by repetitive, spontaneous seizures caused by excessive or hypersynchronous neuronal discharge in the brain. Drug therapy is the first-line treatment for epilepsy patients, and 70-80% of patients experience sustained remission of seizure symptoms after treatment with existing antiepileptic drugs. However, these drugs do not prevent or reverse the pathological processes behind human epilepsy (or other clinical manifestations of epilepsy), meaning they cannot stop the development of epilepsy. Approximately half of patients report at least one adverse reaction during first-line antiepileptic drug treatment, such as ataxia, liver or kidney damage. Over the past thirty years, the emergence of many new antiepileptic drugs has provided more treatment options. However, a significant drawback of new antiepileptic drugs is that they do not offer better efficacy than first-generation drugs and have barely reduced the proportion of drug-resistant epilepsy patients; approximately 20-30% of epilepsy patients still experience seizures. Therefore, there is an urgent need for new drugs with fewer side effects and better efficacy than existing medications.

[0003] The Hedgehog (HH) gene family comprises a series of highly hydrophobic secretory proteins. After secretion, these proteins diffuse to form a concentration gradient, leading to a concentration-dependent effect in cells. By regulating the transcription of related genes, they are widely involved in embryonic development and organ differentiation. In mammals, the HH family includes three homologous genes: Sonic Hedgehog (SHH), Desert Hedgehog (DHH), and Indian Hedgehog (IHH). Although the distribution and function of these three homologous genes differ in vivo, their signaling pathways are similar. The SHH signaling pathway plays a crucial role in the development and homeostasis of the central nervous system. SHH mediates its function by binding to the 12-transmembrane receptor PTCH on the cell membrane, releasing its inhibitory effect on the 7-transmembrane G protein-coupled receptor Smoothened (Smo).

[0004] In the absence of Shh ligands, downstream Hedgehog signaling is maintained in an inhibited state by the Hedgehog receptor Patched (PTCH1). PTCH1 is a 12-transmembrane domain protein (displaying a transporter-like structure) located at the base of the cilia. Although the mechanism by which PTCH1 inhibits downstream signal transduction is not fully elucidated, studies have shown that free PTCH1 (not bound by Shh) inhibits the activity of the 7-transmembrane G protein-coupled receptor Smoothened (Smo) by preventing its translocation to the cilia. This leads to the formation of a complex of downstream GLI transcription factors with the suppressor of fused (SUFU) in the cytoplasm, which is then hydrolyzed by the proteasome, inhibiting the transcription of the target gene. Classical Shh signaling is triggered by Shh ligands: when Shh binds to PTCH1, it simultaneously relieves the inhibition of Smo and localizes Smo to the cilia. Activation of Smo and its translocation to the cilia involve the association of Smo with G protein-coupled receptor kinase 2 (GRK2) and β-arrestins. Studies have shown that GRK2 phosphorylation of Smo and recruitment of β-arrestin lead to Smo endocytosis. Serine / threonine kinases (CK1α) also play a crucial role in the phosphorylation of Smo in mammals. Shh signaling recruits CK1α to initiate Smo phosphorylation, which in turn increases the binding of CK1α and GRK2 to Smo, forming a positive feedback loop that further enhances Smo phosphorylation levels. Furthermore, β-arrestins promote the translocation of active (phosphorylated) Smo to cilia by mediating their interaction with Kif3A in mammalian cells. However, the mechanism by which the Shh pathway dissociates the downstream GLI-UFU complex remains unclear, but there is evidence that the SUFU-Gli2 and SUFU-Gli3 complexes accumulate in cilia. Upon contact with Shh ligands, they independently synthesize new proteins, leading to the immediate transport of Gli2 and Gli3 to the cilia, where they are subsequently phosphorylated and dissociated from SUFU. Through a series of complex signal transduction processes, the full-length activated form of Gli1 migrates to the cell nucleus, where it binds to the promoters of target genes, promoting transcription of these genes, including Ptch and Gli1 itself. Activation of Gli1 amplifies downstream responses of Shh and is itself a target gene of Shh, providing a convenient and intuitive method for studying this pathway. Gli2 primarily functions as a transcriptional activator and has been shown to have repressive functions in certain specific environments, such as the development of skeletal muscle and the central nervous system. Gli3 retains its dual potential activity, functioning as a transcriptional repressor in dorsal interneurons, but also playing an activating role during embryonic development.

[0005] Because activation of the Shh signaling pathway is widespread in neurological diseases such as brain injury and brain tumors, one of the current research focuses is designing and synthesizing Shh signaling pathway inhibitors or agonists for targeted therapy of brain injuries caused by medulloblastoma or cerebral ischemia. Studies have shown that the seven-transmembrane protein SMO, a member of the G-protein-coupled receptor (GPCR) family, is a major transducer of the Shh signaling pathway, regulating cerebellar development and serving as an important drug target. Many SMO inhibitors are currently in clinical trials for cancer treatment. Among them, GDC-0449 / vismodegib was approved by the FDA in 2012 for the treatment of basal cell carcinoma with abnormal activation of the Shh signaling pathway; however, treatment was unsuccessful due to drug resistance caused by the Smo-D473H mutation. Another Smo inhibitor, LDE-225 (sonidegib), was also approved by the FDA in 2015. However, in phase I clinical trials for medulloblastoma, while LDE-225 effectively inhibited the SHH signaling pathway and induced medulloblastoma regression, it also exhibited drug resistance and various adverse reactions such as muscle tremors and taste disturbances. Therefore, the development of novel, highly effective, and low-toxicity drugs targeting the SHH signaling pathway remains a pressing challenge.

[0006] In atypical type I signaling pathways, PTCH1 regulates cell proliferation and apoptosis in the absence of Smo and GLI. In the absence of Hh ligand, the PTCH1 receptor binds to phosphorylated Cyclin B1 and the pro-apoptotic complex; ectopic expression of PTCH1 in the absence of Hh ligand induces apoptosis in neuroepithelial cells. Evidence suggests that the interaction between PTCH1 and the pro-apoptotic complex depends on caspase-3 cleavage of the C-terminal domain of PTCH1, leading to activation of caspase-9. Activated caspase-9 further promotes the formation of the pro-apoptotic complex by activating caspase-3, thereby leading to apoptosis. This relationship is disrupted by the presence of Hh ligand; Hh binding to the PTCH1 receptor inhibits its interaction with the pro-apoptotic complex through conformational changes in the PTCH1 receptor, resulting in enhanced cell survival and proliferation.

[0007] In type II atypical signaling pathways, Smo regulates actin cytoskeleton activity via small GTPase families such as RhoA and Rac1. Reports indicate that Shh stimulates Rac1 and RhoA via a Gi protein and phosphatidylinositol 3-kinase (PI3K)-dependent mechanism, a known mechanism for inducing fibroblast cell migration. In axonal guidance, Shh activates Smo by binding to its receptors PTCH1 and Boc, subsequently activating Src family kinases (SFK) and Fyn in a hierarchical manner. Recent studies have found that stimulation of arachidonic acid metabolites, such as leukotrienes synthesized by Shh, can induce Smo-dependent actin cytoskeleton remodeling, and this effect is independent of GLI.

[0008] In a 2011 article published in *Neuroscience*, Fang M described the development of shh in epileptic foci. This study used human samples from the epileptogenic zone excised during temporal lobe resection in 30 patients with drug-resistant temporal lobe epilepsy. Immunohistochemical staining revealed significantly increased shh expression in the lesions of patients with refractory temporal lobe epilepsy. In animal experiments, they established a pirocarpine-induced chronic epilepsy rat model and detected elevated shh expression at the tissue, cellular, and protein levels. Shh protein levels increased from the third day of induction and continued to rise, peaking on the thirtieth day. These studies in patients with refractory temporal lobe epilepsy and experimental animals all demonstrate that increased shh protein expression in temporal lobe epileptic foci is likely a result of the development of epileptic latency rather than a triggering factor for chronic drug-resistant epilepsy.

[0009] Another article, published in *EMBO Reports* in 2016 by Feng SJ and Ma SR, confirmed the role of the Shh signaling pathway in epilepsy development. This study first showed that Shh protein expression levels were significantly increased under epileptic stimulation in in vivo mouse models, acute brain slices, and in vitro neuronal models. Pretreatment with the Shh signaling pathway antagonist Cyclopamine significantly reduced the frequency of epileptiform discharges and the proportion of epileptic neurons in acute brain slices and cultured neurons, indicating that inhibiting the Shh signaling pathway can effectively suppress epileptiform discharges. Finally, in a pirocarpine mouse model, administration of a Shh signaling pathway-targeting inhibitor or gene knockout of the key transmembrane protein Smo in the Shh pathway both delayed the onset of epilepsy and reduced the frequency of seizures. These results, at the cellular, brain slice, and whole-animal levels, all demonstrate that inhibiting the Shh pathway has a significant anti-epileptic effect, and Smo could be a potential drug target for treating epilepsy.

[0010] Therefore, screening for compounds that can both target the Sonic hedgehog signaling pathway and inhibit abnormal neuronal discharges may provide new directions for future epilepsy treatment. Summary of the Invention

[0011] The purpose of this invention is to provide a novel Hedgehog signaling pathway inhibitor and its preparation method, which can effectively inhibit Smo receptor activity, block the SHH signaling pathway, and has a good effect on blocking epileptic seizures caused by excessive or hypersynchronous neuronal discharge in the brain.

[0012] In one aspect of the invention, compounds of formula (I) or pharmaceutically acceptable salts thereof, isomers thereof, or solvates are provided:

[0013]

[0014] In the formula,

[0015] R 1 Selected from -C(O)R 3 -S(O)2R 3 or -P(O)(R 3 )2;

[0016] R 3 Selected from hydroxyl, amino, or -C 1-3 alkyl;

[0017] R 2 Selected from -C 1-6 alkyl;

[0018] X is selected from F, Cl, Br or I.

[0019] In one implementation scheme, R 1 It is selected from carboxyl, formyl, acetyl, propionyl, methanesulfonyl, ethanesulfonyl, propionyl, or phosphate groups.

[0020] Preferably, R 2 Selected from -C 1-3 alkyl.

[0021] Preferably, X is selected from Cl or Br.

[0022] In one embodiment, the compound has the following structure:

[0023]

[0024] In another aspect, the present invention provides a method for preparing the above-mentioned compound, comprising the following steps:

[0025]

[0026] The coupling reaction of compound (IA) with compound (IB) yields compound (I).

[0027] Among them, X and R 1 R 2 Define compounds of formula (I).

[0028] X' is a halogen; preferably, X' is Br.

[0029] In one embodiment, the compound of formula (IA) is prepared by a method comprising the following steps:

[0030] (1)

[0031] The compound of formula (IA-1) and the compound of formula (IA-2) undergo a condensation reaction to give the compound of formula (IA-3);

[0032] (2)

[0033] Compound (IA-3) undergoes a cyclization reaction to give compound (IA).

[0034] In one embodiment, the compound of formula (IB) is prepared by a method comprising the following steps:

[0035]

[0036] The catalytic hydrogenation reaction of formula (IB-1) yields compound (IB).

[0037] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof, an isomer thereof or a solvate thereof, and a pharmaceutically acceptable excipient.

[0038] In another aspect of the invention, the use of the above-described compound or a pharmaceutically acceptable salt thereof, its isomers or solvates, or the above-described pharmaceutical composition in the preparation of a medicament for inhibiting the Hedgehog signaling pathway is provided.

[0039] Preferably, its use in the preparation of a medicament for inhibiting the Sonic Hedgehog signaling pathway.

[0040] In another aspect, the present invention provides the use of the above-described compound or a pharmaceutically acceptable salt thereof, isomers thereof or solvates thereof, or the above-described pharmaceutical composition in the preparation of a medicament for treating chronic brain diseases.

[0041] Preferably, the chronic brain disease is a brain disease with the Smo D473H mutation.

[0042] Preferably, the chronic brain disease includes epilepsy.

[0043] In another aspect, the present invention provides the use of Sonici (LDE225) in the preparation of a medicament for treating chronic brain diseases or in the preparation of an antitumor medicament.

[0044] Preferably, the chronic brain disease includes epilepsy.

[0045] According to the research of this invention, Sinodeji, in addition to inhibiting βarr2-GFP aggregation, inhibiting Shh-CM-induced Gli1 expression, and effectively inhibiting abnormal epileptiform discharges of neurons, can better overcome the blood-brain barrier compared to other Smo inhibitors, achieving sufficient exposure in the central nervous system and exerting a good therapeutic effect on central nervous system-related diseases such as epilepsy. It is also well absorbed, exhibiting a better brain / plasma ratio, higher peak concentration, shorter time to peak concentration, good bioavailability, lower clearance rate, and good metabolic stability, demonstrating superior pharmacodynamic and pharmacokinetic advantages and therefore promising clinical application prospects.

[0046] In another aspect, the present invention provides a method for treating a patient in need of a condition mediated by the Hedgehog signaling pathway, comprising administering to the patient the aforementioned compound or a pharmaceutically acceptable salt thereof, an isomer thereof or a solvate thereof, sinedig or the aforementioned pharmaceutical composition.

[0047] In another aspect, the present invention provides the use of the above-described compound or a pharmaceutically acceptable salt thereof, its isomers or solvates, or the above-described pharmaceutical compositions in the preparation of antitumor drugs. Attached Figure Description

[0048] Figure 1 Confocal images of βarr2-GFP translocating to SMO in U2OS cells are shown. Arrows indicate βarr2-GFP aggregation within vesicles. Scale bar, 10 μm. ±SEM (t-test), ***P<0.001.

[0049] Figure 2 The inhibition of intracellular accumulation of βarr2-GFP by TT22 is shown in scale bar, 20 μm.

[0050] Figure 3 The novel Smo inhibitor TT22 was identified in U2OS cells. Arrows indicate βarr2-GFP accumulation within vesicles. Scale bar, 20 μm. ±SEM (t-test), ***P<0.001.

[0051] Figure 4The results show that TT22 can competitively displace the binding of Bodipy-cyclopamine to Smo. Bodipy-cyclopamine binding (green) was analyzed using flow cytometry. All data are presented as mean ± SEM (t-test). ***P < 0.001.

[0052] Figure 5 The results show that TT22 can block HH-induced Smo accumulation on primary cilia. Scale bar, 5 μm. ± SEM (t-test), ***P<0.001.

[0053] Figure 6 The results showed that TT22 could inhibit Shh-CM-induced Gli1 expression. All data are presented as mean ± SEM (t-test). ***P < 0.001.

[0054] Figure 7 TT22 was shown to inhibit SAG-induced Gli1 expression. All data are presented as mean ± SEM (t-test). ***P < 0.001.

[0055] Figure 8 TT22 was shown to attenuate epileptiform discharges. All data are presented as mean ± SEM (t-test). *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0056] I. Definition

[0057] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0058] The range of numbers in this article refers to the integers within a given range. For example, "C 1-6 "" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0059] The term "SHH" refers to Sonic Hedgehog, and the term "HH" refers to Hedgehog.

[0060] The term "Gli" refers to Ci (Cubitus interruptus, Gli in vertebrates), a transcription factor in the Hedgehog signaling pathway; Gli1 is a downstream target gene of SHH signaling and is a marker of whether the SHH signaling pathway is activated.

[0061] The term "SMO" stands for Smoothened. Hedgehog (HH) signaling is controlled by two receptors on the target cell membrane: Patched (Ptc) and Smoothened (Smo). The Ptc receptor, encoded by the tumor suppressor gene Patched, is a single polypeptide chain consisting of 12 transmembrane domains. It binds directly to ligands and negatively regulates HH signaling. The Smo receptor, encoded by the proto-oncogene Smothened, is homologous to G protein-coupled receptors. It is a single polypeptide chain consisting of 7 transmembrane domains, with the N-terminus located extracellularly and the C-terminus intracellularly. The amino acid sequence of the transmembrane domains is highly conserved, and the serine and threonine residues at the C-terminus are phosphorylation sites, which bind phosphate groups during protein kinase catalysis.

[0062] Drugs or drug compositions

[0063] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications in proportion to a reasonable benefit / risk ratio.

[0064] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of a particular compound as a free acid or base without any adverse biological effects. Examples include acid (including organic and inorganic acids) addition salts or base addition salts (including organic and inorganic bases).

[0065] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0066] The medicaments or pharmaceutical compositions of the present invention can be administered orally, topically, parenterally, or mucosally (e.g., sublingually, by inhalation, or rectally) in dosage units comprising conventional, non-toxic, pharmaceutically acceptable carriers. Oral administration is generally preferred. The active agent can be administered orally in capsule, tablet, or other forms (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0067] The term "pharmaceutical composition" means a composition comprising the compound described in this invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and dosage form: carrier, diluent, adjuvant, excipient, preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, fragrance, antibacterial agent, antifungal agent, lubricant, dispersant, thermosensitive material, temperature regulator, adhesive, stabilizer, suspending agent, etc.

[0068] abbreviation:

[0069] X-phos: 2-Bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl;

[0070] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0071] DCM: Dichloromethane;

[0072] DIEA: N-ethyldiisopropylamine;

[0073] DMF: N,N-dimethylformamide dimethyl acetal;

[0074] AcOH: Acetic acid; Pd(aba)3: Tris(dibenzylacetone)palladium.

[0075] II. Specific Implementation Examples

[0076] 1. Materials and Reagents

[0077] 1.1 Compounds:

[0078] All compounds were stored as solid powders at -20°C. The compounds were dissolved in DMSO to a concentration of 10 mmol / L as a stock solution and stored at -20°C. Before use, the solutions were diluted with complete culture medium to the required concentration.

[0079] Positive control 1 is GDC-0449, whose structural formula is:

[0080] Positive control 2 is LDE-225, whose structural formula is:

[0081] 1.2 Main Instruments and Equipment

[0082]

[0083]

[0084] 2. Experimental Methods

[0085] 2.1 Cell transfection

[0086] (1) Preparation: VigoFect 0.4ml at 4℃, prepare 150mM NaCl (prepared with ultrapure water, sterilized by autoclaving or filtration) or physiological saline for injection as diluent for VigoFect and DNA, and DNA solution to be transfected (high purity, concentration 0.1-2μg / μl).

[0087] (2) Operation method:

[0088] Preparing to culture cells:

[0089] 1) 24 hours before transfection, collect cells in the logarithmic growth phase, digest and resuspend them in a single-cell suspension, and seed an appropriate amount of cells based on the estimated cell growth rate. The cell density at transfection should be 40-60% (80-90% is also acceptable).

[0090] 2) One hour before transfection, replace with fresh complete culture medium and incubate at 37°C in a 5% CO2 incubator.

[0091] Prepare transfection working solution: (6-well plate or 35mm petri dish, 2ml culture medium)

[0092] 3) Take 5-8 μg of DNA (starting amount 5 μg), add it to the dilution buffer to a total volume of 100 μl, mix gently, and let stand at room temperature.

[0093] 4) Take 1-4 μl of VigoFect (starting volume 2 μl), add it to the diluent until the total volume is 100 μl, mix gently, and let stand at room temperature for 5 minutes.

[0094] 5) Add the diluted VigoFect dropwise to the diluted DNA solution, mix gently, and let the resulting transfection working solution stand at room temperature for 15 minutes.

[0095] 6) Gently mix the transfection working solution, add it dropwise to 2 ml of culture medium, gently mix the culture medium, and incubate at 37°C in a 5% CO2 incubator.

[0096] Subsequent cell processing:

[0097] 7) Remove the culture medium containing the transfection solution 3-6 hours after transfection and replace it with fresh complete culture medium.

[0098] 8) Observe or collect the cells after 24-48 hours.

[0099] 9) When the transfection is stable, digest the cells 24-48 hours after transfection and divide them into 3-5 culture dishes, add appropriate concentrations of the corresponding antibiotic (such as G418) for selection.

[0100] Table 1: Initial Transfection Conditions

[0101]

[0102] 2.2 Total RNA extraction and real-time quantitative PCR

[0103] Trizol is a reagent for extracting total RNA directly from cells or tissues, and it maintains the integrity of RNA when cells are broken down and dissolved.

[0104] (1) After experimental treatment, adherent cells were washed twice with 1×PBS in 6-well plates, and 1 ml of Trizol was added to each well. The cells were repeatedly lysed by pipetting.

[0105] (2) Transfer the Trizol lysis buffer of the above cells to EP tubes and place at room temperature for 2-5 min (15-30℃). At the same time, pre-cool with 75% ethanol and pre-cool the centrifuge.

[0106] (3) Add chloroform at a rate of 200 μl per 1 ml TrizolL, vortex vigorously for 15 seconds, place at room temperature for 2-3 minutes, and centrifuge at 12000 rpm or higher (4℃) for 10 minutes (from bottom to top, the layers are chloroform (protein), intermediate layer (DNA), and aqueous phase (RNA)).

[0107] (4) Take the upper aqueous phase into a new EP tube, add 0.3-0.5 ml of isopropanol (equal volume), mix well, place at -20℃ for 10-30 minutes (lowering the temperature to increase the yield), and centrifuge at 12000 rpm (4℃) for 10 minutes.

[0108] (5) Discard the supernatant, being careful not to discard the precipitate. Add 400 μl of 75% ethanol (pre-cooled) for washing, centrifuge at 12000 rpm (4℃) for 2 min, and gently pour off the ethanol; repeat once; centrifuge again to remove residual ethanol from the wall, and then blot dry.

[0109] (6) Allow the RNA precipitate to dry at room temperature, then dry it in a clean bench.

[0110] (7) After air drying, add 20-50 μl of DEPC water (RNA-free water) to dissolve the RNA precipitate.

[0111] (8) The RNA concentration was detected using a Nano micro-volume nucleic acid quantification instrument. The A260 / A280 value was measured. A value between 1.8 and 2.0 indicates that the RNA is relatively pure and the protein content is low. The measured RNA value was X ng / ul.

[0112] (9) Total RNA was stored in a -80°C freezer for later use.

[0113] Real-time PCR

[0114] 1. Genomic DNA removal reaction

[0115] Reaction system (10 μl):

[0116] Tatol mRNA: 1μg

[0117] DNA Eraser: 1 μl

[0118] 5×gDNA Eraser Buffer: 2μl

[0119] Add DEPC water to a final volume of 10 μl and mix well.

[0120] The PCR instrument was used to react at 42℃ for 5 min;

[0121] 2. RT (Reverse Transcription, mRNA → cDNA)

[0122] Reaction system (20 μl):

[0123] 1. Reaction solution: 10 μl

[0124] 5×Prime Script Buffer 2: 4μl

[0125] Prime Script RT Enzyme MixⅠ:1μl

[0126] RT Primer Mix: 1μl

[0127] DEPC water: 4μl

[0128] The PCR reaction was performed at 37°C for 30 min, followed by a reaction at 85°C for 5 s. The synthesized cDNA was diluted 1:1 with DEPC water and stored at -20°C.

[0129] 3. RT-PCR

[0130] The PCR amplification conditions are as follows (total reaction volume: 20 μl):

[0131] cDNA (<100ng): 1μl

[0132] Forward Primer (10μM): 0.8μl

[0133] Reverse Primer (10μM): 0.8μl

[0134] SYBR Premix Ex TaqⅡ(2×): 10μl

[0135] Rox II: 0.4 μl

[0136] RNase-Free ddH2O: 7 μl

[0137] The procedure was performed according to the kit (QuantiTect™ SYBR Green PCR kits) instructions. Each sample was repeated three times, with negative and positive controls included.

[0138] Primers for amplifying specific regions are

[0139] Mouse Gli1:

[0140] F: 5′-CTCAACTGCCCAGCTTA ACCC-3′ (SEQ ID NO: 1)

[0141] R: 5′-TGCGGCTGACTGTGTAAGCAGA-3′ (SEQ ID NO: 2)

[0142] Mouse Actin:

[0143] F: 5′GCAAGTGCTTCTAGGCGGAC-3′ (SEQ ID NO: 3)

[0144] R: 5′-AAGAAAGGG TGTAAA ACGCAGC-3′ (SEQ ID NO: 4)

[0145] 2.3 Western-Blotting

[0146] (1) Sample preparation: Detection of Gli1 and α-tubulin in NIH3T3 cells

[0147] Extraction of intracellular proteins by lysis method:

[0148] Perform the procedure on ice. Mix RIPA lysis buffer (containing 100×cocktail) with 100×PMSF at the specified ratio. Remove the culture medium from the treated NIH3T3 cells. Wash each well of a 12-well plate with 1 ml of 1×PBS. Add 100 μl of RIPA to each well. Use a cell scraper to scrape off the cells in one direction and transfer them to a 1.5 ml EP tube. Incubate on ice for 15 min, shaking every 3-4 min for mechanical lysis. Centrifuge at maximum speed for 10 min (4℃). Aspirate the supernatant and transfer it to a new EP tube, labeling it accordingly.

[0149] BCA protein quantification principle: Under alkaline conditions, Cu 2+ Reduced to Cu by proteins +Monovalent copper ions can interact with two molecules of BCA to form a blue-violet complex. This complex is water-soluble and exhibits strong absorbance at a wavelength of 562 nm. Within a certain range, the absorbance shows a good linear relationship with protein concentration, allowing the creation of a standard curve. Therefore, protein concentration can be calculated by measuring the absorbance at 562 nm.

[0150] 1) Preparation of BCA working solution: Based on the quantity of standard and sample, prepare BCA working solution by mixing BCA reagent and Cu reagent at a ratio of 50:1 and then mix thoroughly for later use.

[0151] 2) Diluting the standard: Dilute the 5 mg / mL protein standard with PBS at a ratio of 1:9 to 5 mg / mL. Add the standard to a 96-well plate at concentrations of 0, 2, 4, 6, 8, 12, 16, and 20 μl, respectively. Then add 20, 18, 16, 14, 12, 8, 4, and 0 μl of PBS to a final concentration of 20 μl. Set up two replicates for each concentration.

[0152] 3) Add 200 μl of BCA working solution to each well and incubate at 37°C for 20 min. Measure the absorbance at 562 nm using a microplate reader. Create a standard curve based on the absorbance at 562 nm and the protein concentration.

[0153] 4) Protein sample concentration determination: Take a 96-well plate, add 19 μl of PBS diluent, 200 μl of BCA working solution and 1 μl of protein sample to each well, set two replicates for each sample, incubate in a 37℃ incubator for 20 min, and then measure the absorbance at 562 nm. Calculate the protein concentration based on the protein standard curve.

[0154] 5) Deflate in a 95℃ constant temperature heating block for 5 minutes, then store at -20℃ for later use.

[0155] (2) SDS-PAGE gel electrophoresis (polyacrylamide gel electrophoresis)

[0156] 1) Preparation of polyacrylamide gel:

[0157] Clean the glass plate: Wipe the clean glass plate with alcohol and let it air dry.

[0158] Leak test: Place the two glass plates into the slots and secure them. Fill the glass tank with pure water and let it stand for 5 minutes. Observe whether the water level in the tank drops. If the device does not leak, pour out the pure water, use clean filter paper to absorb any remaining water droplets in the glass tank, and set it aside. Otherwise, rebuild the device.

[0159] Preparation of polyacrylamide gel: Different concentrations of separating gel are prepared according to the molecular weight of the target protein to be measured. Generally, the larger the molecular weight of the protein, the lower the concentration of the separating gel required. To prepare 10 ml of 12% separating gel, rinse a 50 ml centrifuge tube with double-distilled water. Add 3.3 ml of pure water, 4 ml of 30% acrylamide solution, 2.5 ml of 1.5 M Tris-HCl (pH 8.8), 0.1 ml of 10% (m / v) SDS, 0.1 ml of 10% AP (m / v) AP, and 0.004 ml of TEMED. Mix thoroughly. Add approximately 7 ml of the separating gel to a 1.5 mm thick glass trough, then slowly add 1 ml of isopropanol to seal the gel, ensuring the surface is level. After approximately 15 minutes, the separating gel will solidify. Pour off the isopropanol from the gel surface and blot away any remaining solvent with clean filter paper. Prepare 8 ml of 5% stacking gel by adding 5.4 ml of pure water, 1.3 ml of 30% propylene glycol, 1 ml of 1.5 M Tris-HCl (pH 6.8), 0.08 ml of 10% (m / v) SDS, 0.08 ml of 10% (m / v) AP, and 0.008 ml of TEMED. Mix thoroughly and fill the glass tank. Quickly insert a 1.0 mm, 10-tooth comb into the stacking gel. After approximately 15 minutes, the stacking gel will solidify. Fill the electrophoresis tank with electrophoresis buffer and gently remove the comb, keeping the sample wells vertical to avoid interference between lanes during electrophoresis.

[0160] 2) Sample loading: Calculate the required sample volume based on the protein concentration measured using the Bradford standard curve. Inject the protein sample sequentially into the wells using the pipette tip, and inject a protein marker next to it to indicate the protein molecular weight. Generally, the wells at both ends are not used; to avoid edge effects, inject a volume approximately equal to the sample volume of 1×Loading Buffer.

[0161] 3) Gel electrophoresis: Inject an appropriate amount of electrophoresis buffer into the outer tank of the gel electrophoresis tank, and start gel electrophoresis at a constant voltage of 80V. After about 30 minutes of electrophoresis, the protein sample will be concentrated into a straight line and gradually enter the separating gel from the stacking gel. Due to the increase in resistance, the constant voltage will be increased to 120V. Continue electrophoresis for about 40 minutes until the proteins of each molecular weight are fully separated.

[0162] 4) Transfer: After gel electrophoresis, cut off a gel sample with a molecular weight range of 40-200 kDa and immerse it in distilled water. Thoroughly soak the upper filter paper, 0.45 μm NC membrane, and lower filter paper in Top Buffer, Balance Buffer, and Down Buffer, respectively. Then, in the order from top to bottom, lay the upper filter paper, gel, 0.45 μm NC membrane, and lower filter paper flat in the electrophoresis tank. During this process, ensure that there are no air bubbles between each layer. Cover the electrophoresis tank and insert it into the semi-dry transfer apparatus. Set the transfer time to approximately 10 minutes. Because the transfer process involves high current and high resistance, it generates a lot of heat. Prolonged high heat can damage proteins, so after transfer, the membrane must be quickly removed and transferred to distilled water or 1×TBST buffer.

[0163] 5) Blocking with Milk: After the transfer is complete, Ponceau S can quickly bind to the proteins on the NC membrane and develop color, thereby verifying the success of the transfer and determining the position of the protein bands. Dissolve skim milk powder in 1×TBST to prepare a milk blocking solution with a mass-volume ratio of 5%, then block the bands with milk and incubate at room temperature for 1 hour.

[0164] 6) Incubation with primary antibody: Dilute Gli1 (1:1000) and α-tubulin (1:2000) with antibody dilution buffer (1×TBST and BSA at a mass-to-volume ratio of 5%) and incubate the corresponding antibody bands in a shaker overnight at 4°C.

[0165] 7) Secondary antibody incubation: After primary antibody incubation, recover the primary antibody, wash the protein band three times with 1×TBST to remove unbound primary antibody, 10 minutes each time. Add horseradish peroxidase-labeled secondary antibody (1:5000) corresponding to the primary antibody species diluted with blocking buffer (5% milk TBST), and hybridize on a shaker at room temperature for 1 hour. Discard the secondary antibody, wash the membrane three times with 1×TBST, 10 minutes each time.

[0166] 8) Exposure: Prepare the ECL chemiluminescence solution (solution A:solution B = 1:1). Place the protein bands into the chemiluminescence imager, blot off any remaining TBST on the bands with filter paper, and then evenly coat the ECL chemiluminescence solution onto the band surface. Automatically sense the exposure time and save the exposed image in 600 dpi TIFF format. Use ImageJ software for grayscale analysis.

[0167] 2.4 Bodipy-cyclopamine competitive binding assay

[0168] HEK293 cells were transfected with human Flag Smo WT or the Smo mutant (D473H). After 24 h, the cells were trypsinized, washed with phenol red-free DMEM containing 0.5% fetal bovine serum, fixed with 4% paraformaldehyde at room temperature for 10 min, and incubated with 5 nM BODIPY-Cyclopamine and different concentrations of indicator compounds at 37 °C for 2 h. The treated cells were centrifuged, and the fluorescence signal was analyzed by flow cytometry.

[0169] Construction of the 2.5Smo-D473H mutant strain

[0170] The FLAG Smo-D473H point mutation was established using the Fast Mutagenesis System (TRANGEN BIOTECH). The primers are as follows: F: 5′-AGCTGCCACTTCTAC (SEQ ID NO:5), CACTTCTTCAA-3′ (SEQ ID NO:6), R: 5′-GGTAGAAGTGGCAGC (SEQ ID NO:7)TGAAGGTAATG-3′ (SEQ ID NO:8).

[0171] 2.6 Primary cortical neuron culture

[0172] (1) Preparations before collecting materials from the original source:

[0173] 1) Autoclaving: The day before the primary neuron culture is taken, the surgical instruments, culture dishes, ddH2O, pipette tips, etc. are autoclaved.

[0174] 2) Preparation of neuronal seeding medium: DMEM high glucose medium + fetal bovine serum (FBS, 10%) + horse serum (HS, 5%) + penicillin / streptomycin antibiotics (1%).

[0175] 3) Preparation of neuronal maintenance medium: neurobasal medium + B27 (2%) + penicillin / streptomycin antibiotics (1%).

[0176] 4) Coating culture plates: Dilute 10× poly-L-lysine stock solution with autoclaved ddH2O to obtain a working solution of 0.1 mg / ml and coat the culture plates. After incubation at room temperature overnight, discard the coating solution in a clean bench, air dry, and wash twice with ddH2O for 5 min each time. Then add the inoculation medium and place in a clean bench for later use.

[0177] 5) Before sampling, place the dissecting microscope, high-pressure surgical instruments, culture dishes, etc. into the sampling laminar flow hood and irradiate with ultraviolet light for 1 hour.

[0178] (2) Experimental steps for primary fetal rat cortical neuron culture:

[0179] 1) Anesthesia: SD rats gestation days 16-18 were anesthetized by intraperitoneal injection of 10% chloral hydrate (400 μl / 100g).

[0180] 2) Removal of the rat uterus: After disinfection with 75% alcohol, the rat's abdomen was dissected layer by layer into the abdominal cavity using autoclaved large scissors in a fume hood. Hemostatic forceps were used to pull the uterus out to expose the surgical field. The rat uterus was pulled out, and the mesentery and blood vessels were severed with surgical scissors, keeping the uterus as intact as possible, avoiding damage to the intestines, and avoiding contact with the rat's fur to prevent contamination. After removal, the uterus was quickly placed in a disposable sterile culture dish containing low-temperature DMEM medium, the lid was closed, and the dish was transferred to the laminar flow hood in an ice box.

[0181] 3) Fetal mouse separation: Prepare surgical instruments, ice packs, etc. in the biosafety cabinet. Pour the biosafety DMEM medium containing 1% penicillin-streptomycin antibiotics into four culture dishes. Use forceps to tear open the uterus and remove the fetal mice, placing them in culture dishes containing low-temperature biosafety DMEM medium. Separate the placenta and umbilical cord of all fetal mice. Place the fetal mice in a new culture dish containing low-temperature biosafety DMEM medium.

[0182] 4) Separation of the fetal mouse cortex: Hold the fetal mouse's head still with forceps in your left hand, and separate the skull and dura mater with forceps in your right hand. Peel off the cerebral hemispheres and place them in a new culture dish containing the sample culture medium. Under a microscope, remove the thalamus and brainstem, leaving only the bilateral cerebral hemispheres. Remove all the fetal mouse cortex and place it in a new culture dish.

[0183] 5) Removal of the meninges and blood vessels: Under a microscope, use micro-forceps to remove the meninges and blood vessels one by one, ensuring that all the meninges and blood vessels are completely removed. Otherwise, in order to prepare a single-cell suspension after digestion, the force and frequency of pipetting will be forced to increase, which may cause neuronal damage. After separating all cortical meninges and blood vessels, cover the culture dish and transfer it to a clean bench in the intercellular space. To reduce neuronal metabolism and ensure cell viability, the entire process is performed on ice.

[0184] 6) Digestion and Termination: In a laminar flow hood, the brain tissue was minced to approximately 1 mm using sterile micro-scissors. 4 ml of preheated 0.25% trypsin was added, and the mixture was placed in a 37°C incubator for 10 minutes, shaking every 5 minutes. Digestion was terminated with DMEM medium containing 10% FBS and 5% HS.

[0185] 7) Filtration: Gently blow the mixture several times with a pipette, avoiding the formation of air bubbles that could damage neurons. Aspirate the digested cell suspension from the supernatant and filter it through a 70-mesh cell sieve. Centrifuge the filtered liquid at 1000 rpm for 5 minutes.

[0186] 8) Inoculation: After centrifugation, remove the supernatant, add 1 ml of inoculation medium to resuspend the cells, and gently pipette to mix. Take 10 μl of the cell suspension, add it to a cell counting chamber for counting, and then inoculate it into a culture plate pre-coated with poly-L-lysine. The inoculation density should not be too high, otherwise contact inhibition may occur, nor should it be too low, as neurons are unlikely to survive or grow poorly if the density is too low.

[0187] 9) Shaking: After inoculation, shake the culture plate in a "cross" motion to distribute the cells evenly. Avoid shaking in circles, otherwise the cell distribution density will be uneven.

[0188] 10) Culture and Medium Change: Place the culture plate in a cell culture incubator at 37℃ and 5% CO2. After 4 hours, most of the cells will adhere to the plate. Replace the entire medium with serum-free maintenance medium and continue culturing. Be gentle during the medium change to avoid blowing the cells away. Replace half the medium every other day thereafter. After 7 days of culture, the neurons will show good growth, with plump cells, strong refractive properties, and neural network formation, ready for subsequent experiments.

[0189] Regarding the timing of medium replacement, some studies recommend replacing the medium 12 hours after inoculation. However, at this time, glial cells begin to divide, resulting in relatively low purity of the cultured neurons. Adding cytarabine to inhibit glial cell growth to improve purity can also negatively impact neuronal growth due to its toxicity. This experiment found that replacing the medium after 4 hours yielded better results. If the replacement time is less than 4 hours, the neurons do not fully adhere to the culture vessel, and the neurons may be blown up during the replacement, leading to uneven density.

[0190] 2.7 Electrophysiology of Hippocampal Neurons

[0191] Whole-cell recordings of primary neurons incubated with different doses of drugs were performed at room temperature (23-24℃) using an Olympus 40X immersion lens (Olympus, Japan, BX51WI) infrared differential interference contrast microscope. All recordings were acquired using a patch-clamp amplifier (MultiCLAMP 700B) under the control of pCLAMP Clampex 10.3 software. The data acquisition frequency was 10 kHz, and the low-pass filter frequency was 1 kHz. Recording electrodes (resistance 3-6 MΩ) were prepared using borosilicate glass capillaries (BF150-86-75, Sutter Instruments, USA) with a vertical pipette puller (PC100NARISHIGE, Japan). The electrode internal solution (mM) was prepared as follows: potassium gluconate 120, potassium chloride 20, HEPES 10, EGTA 10, MgCl2 2, Na2ATP 2 (pH adjusted to 7.3 with KOH, osmotic pressure 300 mOsm). The standard extracellular solution consists of (unit: mM): 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 4 MgCl2, 2 CaCl2 (pH adjusted to 7.35 with NaOH, 310 mOsm).

[0192] When I = 0, the resting membrane potential (Vm) is measured. Using 0Mg... 2+ / HighK+(8K + Epileptiform discharges were recorded using an epileptogenic solution instead of standard extracellular fluid. Recording was performed continuously for 30 minutes in current mode. Epileptiform discharges can be categorized into two forms: one is a large depolarization shift (≥10 mV, ≥300 ms) with at least 5 action potentials; the other is an action potential characterized by a high frequency with an amplitude significantly higher than baseline and / or signal. Cells were transferred to the epileptogenic extracellular fluid, and recording of these events began when the cells reached a stable epileptiform state within 10–20 minutes. The "solvent" group received pretreatment with an equal volume of solvent solution (DMSO), while the "drug" group received pretreatment with different concentrations of drugs. Electrophysiological recording was performed 20 minutes later.

[0193] 2.8 Statistical Analysis

[0194] All statistical analyses were performed from at least three independent experiments. Comparisons of two or more samples were performed using the two-tailed Student's t-test and one-way ANOVA. *P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.).

[0195] Example 1: Synthesis of compound TT22

[0196] (1)

[0197] HATU (16.3 g, 42.8 mmol, 2.0 eq), DIEA (5.5 g, 42.8 mmol, 2.0 eq), and compound 2 (2.6 g, 21.4 mmol, 1.0 eq) were added to a solution of compound 1 dissolved in DCM (50 mL) (5.0 g, 21.4 mmol, 1.0 eq). The reaction mixture was stirred at room temperature for 2 hours. After stirring, H₂O (30 mL) was added and the mixture was extracted with DCM (20 mL x 3), washed with brine (10 mL x 3), dried over Na₂SO₄, and filtered. The combined organic phases were separated and concentrated under vacuum to give compound 3 (8.0 g, crude), which was used directly for the next step without further purification. LCMS: 339.0, 341.0 ([M+H]+).

[0198] (2)

[0199] Compound 3 (8.0 g, 23.7 mmol, 1.0 eq) was refluxed in AcOH (30 mL) solution for 1 h. After completion, the reaction mixture was cooled to room temperature, H2O (60 mL) was added, and the mixture was extracted with DCM (20 mL x 3), washed with brine (10 mL x 3), dried over Na2SO4, and filtered. The combined organic phases were separated and concentrated under vacuum. The residue was dissolved in DMSO (5.0 mL) and purified by reversed-phase preparative-grade HPLC (Waters XBridge Prep C18 OBD; CH3CN:H2O = 50-65-100-10 min, 5 mmol NH4HCO3 as additive) to give compound 4 (1.0 g, 14.5%, two steps) as a white solid. 1 H NMR (300MHz, DMSO-d6): δppm 8.24(d,J=1.8Hz,1H),7.89(d,J=2.1Hz,1H),7.83(d,J=8.1Hz,1H),7.71- 7.63(m,2H),7.35-7.24(m,2H),3.90(s,3H).LCMS:321.1,323.2([M+H]+).

[0200] (3)

[0201] CuI (743 mg, 3.9 mmol, 0.1 equivalent), K3PO4 (10.6 g, 50.0 mmol, 1.3 equivalent), and L-proline (886 mg, 7.7 mmol, 0.2 equivalent) were added to a mixed solution of compound 5 (8.0 g, 38.5 mmol, 1.0 equivalent) and compound 6 (5.1 g, 50.0 mmol, 1.3 equivalent) dissolved in dimethyl sulfoxide (80 mL). The mixture was heated at 110 °C overnight. After heating, the reaction mixture was cooled to room temperature, H2O (200 mL) was added, and the mixture was extracted with DCM (50 mL x 3), washed with brine (30 mL x 3), dried over Na2SO4, and filtered. The combined organic phases were separated and concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 1:1) to give compound 7 (3.0 g, 37.5%) as a white solid. 1 H NMR (300MHz, CDCl3): δppm 9.41(s,1H),8.72(d,J=5.7Hz,1H),8.53(s,1H),8.20(d,J=8.7Hz,1H),8.07 (d,J=8.7Hz,1H),7.83(d,J=5.7Hz,1H),3.15(s,3H).LCMS:208.1([M+H]+).

[0202] (4)

[0203] PtO2 (0.6 g, 20% wt) was added to a solution of compound 7 (3.0 g, 14.5 mmol, 1.0 eq) dissolved in EtOH (30 mL) at room temperature to give a mixture. The mixture was stirred overnight at room temperature under a H2 atmosphere. After completion, the mixture was filtered and the filtrate was concentrated to give a white solid compound 8 (2.0 g, 65.4%). The crude product was used for the next step without further purification. 1 HNMR (300MHz, CDCl3): δppm 7.67-7.65(m,2H),7.19(d,J=8.7Hz,1H),4.07(s,2H),3.15(t,J=6.0Hz,2H),3.03(s,3H),2.87(t,J=6.0Hz,2H).LCMS:212.1([M+H]+).

[0204] (5)

[0205] Compound 4 (2.3 g, 7.3 mmol, 1.0 eq), Pd2(dba)3 (1.4 g, 1.5 mmol, 0.2 eq), X-phos (1.7 g, 2.9 mmol, 0.4 eq), and Cs2CO3 (4.7 g, 14.6 mmol, 2.0 eq) were added to a mixture of compound 8 (2.0 g, 9.5 mmol, 1.3 eq) dissolved in DMF (20 mL). The mixture was heated at 85 °C for 3 hours. After heating, the reaction mixture was cooled to room temperature and filtered. H2O (60 mL) was added to the filtrate, and the mixture was extracted with DCM (20 mL x 3), washed with brine (15 mL x 3), dried over Na2SO4, and filtered. The combined organic phases were separated and concentrated under vacuum. The crude product was purified by reversed-phase preparative HPLC (WatersXBridge Prep C18 OBD; CH3CN:H2O = 50-58-100-10 min, 5 mmol NH4HCO3 as additive) to obtain a white solid Bellen00064255-N (236.0 mg, 7.2%). 1H NMR (400MHz, CDCl3): δppm7.88(d,J=6.8Hz,1H),7.78(s,1H),7.74(d,J=8.0Hz,1H),7.68(s,1H),7.58(d,J=8.0Hz,1H),7. 45-7.32(m,5H),4.47(s,2H),3.91(s,3H),3.52(t,J=5.6Hz,2H),3.17(t,J=5.6Hz,2H),3.06(s,3H).LCMS:452.0([M+H]+).

[0206] Example 2: Inhibition of βarr2-GFP punctate aggregation in cells by compound TT22

[0207] 1.1 Construction of Smo protein inhibitor screening platform

[0208] When βarr2-GFP is expressed alone in U2OS cells, it is uniformly distributed in the cytoplasm. U2OS cells were selected for high-throughput cancer drug screening. U2OS cells were cultured at 37°C for 24 h in solvents DMSO (group B), 1 μM Cyclo (group C), 1 μM Cyclo, and 1 μM SAG (group D), respectively. The percentage of βarr2-GFP aggregates was observed and detected by confocal microscopy.

[0209] Figure 1 A represents untreated U2OS cells. Co-expression of βarr2-GFP with SMO-633 leads to a redistribution of βarr2-GFP expression into intracellular aggregates / vesicles, resulting in bright aggregates around the nucleus. Figure 1 B). Treatment with the SMO antagonist cyclopamine (Cyclo) resulted in the disappearance of green vesicles. Figure 1 C). In the presence of Cyclo, the addition of the Smo agonist SAG leads to the reappearance of green vesicle aggregates. Figure 1 D). Based on this, a preliminary high-throughput screening method was established to screen out suitable small molecule libraries and small molecules that can prevent βarr2-GFP from accumulating in vesicles.

[0210] 1.2TT22 inhibits intracellular accumulation of βarr2-GFP

[0211] Add gradient concentrations (10) to U2OS cells that stably express βarr2-GFP and SMO-633. -3 -10 4 TT22 (nM), positive control 1, and positive control 2 were cultured at 37℃ for 24 h. The percentage of βarr2-GFP aggregates was quantitatively detected, and the IC50 was calculated. 50 .

[0212] Compound TT22 can inhibit the punctate aggregation of βarr2-GFP in cells. Figure 2 A). IC50 of TT22, positive control 1, and positive control 2 inhibiting βarr2-GFP aggregation 50 The values ​​were 4.7 nM, 44.6 nM, and 26.8 nM, respectively. Figure 2 B).

[0213] 1.3 Identification of a novel Smo inhibitor TT22 in U2OS cells

[0214] According to the screening method constructed in 1.1, U2OS cells were cultured at 37℃ for 24 h using solvents DMSO (group A), 1 μM Cyclo (group B), 100 nM TT22 (group C), 100 nM TT22, and 1 μM SAG (group D), respectively. The percentage of βarr2-GFP aggregates was then detected. Figure 3 E).

[0215] from Figure 3 It is evident that TT22 treatment can lead to the disappearance of green vesicles. Figure 3 C), in the presence of TT22, the addition of the Smo agonist SAG leads to the reappearance of green vesicle aggregates. Figure 3 D). The Smo / βarr2-GFP internalization experiment further verified the activity of TT22 for Smo, with Cyclo and SAG as controls.

[0216] Example 3: Competitive binding of compound TT22 to Bodipy-cyclopamine

[0217] To further verify the binding of TT22 to Smo, a Bodipy-cyclopamine competitive binding assay was performed. Specifically, gradient concentrations of the Smo antagonist TT22 and positive control 1 were used to competitively bind with Smo in HEK293 cells transiently transfected with human Smo-WT. It was found that the known Smo antagonist (Cyclopamine, positive control 1) and TT22 could replace 5 nM Bodipy-cyclopamine in Smo with similar affinity. Figure 4 A).

[0218] Recent studies have reported that the Smo mutation in Smo-D473H leads to resistance to treatment in positive controls 1 and 2. To investigate whether TT22 can bind to Smo-D473H, Smo-D473H was overexpressed in HEK293 cells, and a Bodipy-cyclopamine competition assay was performed in HEK293 cells transiently transfected with human Smo-D473H. The results of Bodipy-cyclopamine binding (green) were analyzed by flow cytometry. All data are presented as mean ± SEM (t-test). ***P < 0.001.

[0219] The results showed that TT22 could effectively replace 5 nM bodipy-cyclopamine in Smo-D473H. However, positive control 1 could not bind to the mutant Smo-D473H at low concentrations, but showed partial activity at high concentrations. Figure 4 B). The above results indicate that TT22 can competitively bind to WT-Smo and Smo-D473H receptors with Cyclopamine.

[0220] Example 4: Blocking of Smo aggregation on cilia by compound TT22

[0221] Smo tested the inhibitory effect of TT22 on Sonic Hedgehog (Shh) signaling by mediating the Shh signaling pathway through localization on cilia.

[0222] PTCH- was treated with DMSO, 5 μM positive control 1, and 5 μM TT22. / -MEF cells were stained for 24 hours and immunohistochemically with antibodies against Smoothened and ARL13B to observe the regulatory effect of TT22 on SMO accumulation in protocilia. Specifically, serum-starved PTCH cells were used. - / -MEF cells were treated at 37°C for 24 h with DMSO (Vehicle group), 5 μM TT22, and 5 μM positive control 1. Cells were then stained with a smoothed (green) antibody against the primary ciliary marker ARL13B (red). The percentage of Smo in primary cilia was subsequently quantified (n = 100).

[0223] The results showed that, compared with the DMSO control, TT22 had a similar effect on inhibiting Smo accumulation in primary cilia as positive control 1, and positive control 1 could also inhibit Smo accumulation in primary cilia. Figure 5 It is evident that TT22 can block HH-induced Smo accumulation on primary cilia.

[0224] Example 5: Inhibition of Shh signal transduction by compound TT22

[0225] Gli1 is a downstream target gene of Shh signaling and serves as a measure of Shh signaling pathway activity. To verify the inhibitory effect of TT22 on Shh signaling, Gli1 transcription was enhanced using N-Shh conditioned medium (Shh-CM) or the known Smo agonist SAG. First, Shh-CM was prepared, and the optimal effective concentration of SAG (100 nM) was explored through gradient treatment. Then, serum-starved NIH3T3 cells were stimulated with 20% Shh-CM or 100 nM SAG, resulting in a significant increase in Gli1 mRNA levels. To investigate the inhibitory effect of TT22, serum-starved NIH3T3 cells were induced for 24 h with 20% Shh-CM, DMSO, TT22 (0.01, 0.1, 1, 10 μM), or positive control 2 (1 μM) and positive control 1 (1 μM).

[0226] Figure 6 A represents the mRNA level of Gli1 in NIH3T3 cells 24 hours after treatment with Shh-CM and starvation. Figure 6 B and Figure 6 C represents the results of detecting Gli1 mRNA (6B) and protein expression levels (6C) in NIH3T3 cells that have been treated with serum-starved cells for 24 hours with TT22, 1μM positive control 2, or 1μM positive control 1. Figure 6 D represents the dose-dependent inhibition test results of TT22 on Shh-stimulated HH pathway activity, measured by quantitative measurement of Gli1 protein levels. TT22 was observed to reduce Gli1 mRNA and protein levels in a dose-dependent manner. Positive controls 1 and 2 also inhibited Gli1 expression. Therefore, TT22 can inhibit Shh-CM-induced Gli1 expression.

[0227] Example 6: Inhibition of SAG-induced Gli1 expression by compound TT22

[0228] We demonstrated, using a method similar to Shh-CM, that Shh activity stimulated by the Smo agonist SAG could be inhibited by TT22.

[0229] Figure 7 A represents the detection results of Gli1 mRNA levels in NIH3T3 cells induced by serum starvation with DMSO or SAG for 24 hours. Figure 7 B and Figure 7 C is used for 10- 7 M(100nM)SAG and TT22, 1μM positive control 2 or 1μM positive control 1 induced serum starvation of NIH3T3 cells for 24h, intracellular Gli1 mRNA level ( Figure 7 B) and protein levels ( Figure 7 C) test results. Figure 7 D represents the results of a dose-dependent inhibitory effect of TT22 on SAG-stimulated HH pathway activity, measured at Gli1 protein levels. This indicates that TT22 can inhibit SAG-induced Gli1 expression. TT22 can act as an effective inhibitor, dose-dependently suppressing the expression of target genes in the Shh signaling pathway.

[0230] Example 7: Inhibition of abnormal epileptiform discharges of neurons by compound TT22

[0231] Through such Figure 8 The method shown in A evaluates the effect of Smo inhibitors on neuronal electrophysiological responses.

[0232] Hippocampal neurons (n=7-14) cultured in vitro were treated with DMSO, TT22, positive control 1, and positive control 2 at 37°C for 30 min, respectively. The neurons were then transferred to a patch-clamp platform and perfused with HighK. + / 0Mg 2+ Extracellular fluid was used to induce epileptiform discharges until complete cell ejaculation. Cells were then stabilized and recorded using patch clamps for 30 minutes. Data from 10-20 minutes of recording were analyzed. (See figure). Figure 8 B) Treatment with TT22, positive control 1, and positive control 2 did not affect the resting membrane potential of neurons.

[0233] The electrical activity of neurons treated with TT22, positive control 1, and positive control 2 to suppress epileptiform discharges was recorded using whole-cell patch-clamp techniques. After 30 min of incubation with the indicated drugs, neurons were transferred to standard extracellular fluid for whole-cell recording. The resting membrane potential (Vm) of neurons was measured at I=0. Representative recordings of neuronal electrical activity under the specified treatments are shown below. Figure 8 C. See the expanded view of a single burst (indicated by the arrow). Figure 8 D.

[0234] In addition, the firing frequency of neurons ( Figure 8 E) and average discharge interval ( Figure 8 Quantitative measurements were performed on F, and the number of epileptiform discharges was recorded over 10-20 minutes. Figure 8 G). TT22, along with positive controls 1 and 2, can reduce the frequency of epileptiform discharges (G). Figure 8 E), and increase the interval between seizures (E), and increase the interval between seizures (E). Figure 8 F). Furthermore, data recorded between 10 and 20 minutes showed that pre-incubation with 1 μM TT22, 10 μM TT22, and 1 μM positive control 1 and 1 μM positive control 2 inhibited epileptiform discharges (F). Figure 8 G). These results indicate that TT22, positive control 1, and positive control 2 can effectively inhibit abnormal epileptiform discharges in neurons.

Claims

1. The following compounds or their pharmaceutically acceptable salts: 。 2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

3. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for inhibiting the Hedgehog signaling pathway.

4. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for inhibiting the Sonic Hedgehog signaling pathway.

5. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for treating brain diseases caused by Smo D473H mutations.

6. The application according to claim 5, wherein, The brain disease mentioned is epilepsy.

7. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of an antitumor drug.

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