Application of 20-Deacetyltaxuspine X in preparation of medicine for preventing and / or treating amyotrophic lateral sclerosis

Abstract

The invention discloses application of 20-Deacetyltaxupine X in preparation of a medicine for preventing and / or treating amyotrophic lateral sclerosis, and belongs to the technical field of medicinal chemistry. The novel serine-threonine kinase CDC7 inhibitor and CDC7 form a new hydrogen bond, and guidance is provided for design of CDC7 inhibitor drugs. The novel serine-threonine kinase CDC7 inhibitor has a protection effect on an amyotrophic lateral sclerosis cell model, and can be used as an amyotrophic lateral sclerosis resisting medicine. The novel serine-threonine kinase CDC7 inhibitor has a protective effect by inhibiting apoptosis of an amyotrophic lateral sclerotic cell model.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, in particular to the application of 20-Deacetyltaxuspine X in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis. BACKGROUND

[0002] Amyotrophic lateral sclerosis is a chronic and progressive neurological disease that mainly damages upper motor neurons and lower motor neurons and the muscles of the trunk, limbs and head and face. The cause of this disease is not clear, it may be related to genetic factors, lifestyle, exposure to toxic substances, excessive physical labor, low body mass index, head trauma history, metabolic diseases, autoimmune dysfunction and other factors. Amyotrophic lateral sclerosis, as a fatal neurodegenerative disease, has no cure at present.

[0003] In both familial and sporadic amyotrophic lateral sclerosis cases, post-translational modification of nuclear protein TDP-43 causes disruption of TDP-43 homeostasis and thus promotes its neurotoxicity. Among the kinases involved in these changes, serine-threonine kinase CDC7 plays an important role by directly phosphorylating TDP-43. Therefore, small molecule drugs targeting CDC7 may have protective effects on amyotrophic lateral sclerosis cell models, and CDC7 inhibitors can be used as new drug candidates for treating amyotrophic lateral sclerosis. Finding new CDC7 inhibitors has important scientific and clinical significance for the development of anti-amyotrophic lateral sclerosis drugs. SUMMARY

[0004] The purpose of the present application is to provide the application of 20-Deacetyltaxuspine X in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis, so as to solve the problems existing in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions: One of the technical solutions of the present application is the application of 20-Deacetyltaxuspine X in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis.

[0006] The second technical solution of the present application is a drug for preventing and / or treating amyotrophic lateral sclerosis, which comprises 20-Deacetyltaxuspine X, and the structural formula of 20-Deacetyltaxuspine X is as follows: .

[0007] The third technical solution of the present application is the application of 20-Deacetyltaxuspine X in the preparation of a CDC7 inhibitor.

[0008] The fourth technical solution of the present application is a CDC7 inhibitor, which comprises 20-Deacetyltaxuspine X, and the structural formula of 20-Deacetyltaxuspine X is as follows: .

[0009] Based on the above technical solution, the present application has the following technical effects: The present application first discloses that 20-Deacetyltaxuspine X can effectively protect neurons from damage by specifically inhibiting the activity of serine-threonine kinase CDC7 and blocking pathological TDP-43 protein phosphorylation, thereby providing a new molecular target and treatment strategy for amyotrophic lateral sclerosis, a fatal neurodegenerative disease. In vitro enzymatic experiments have confirmed that the inhibitory activity IC50 value of 20-Deacetyltaxuspine X on CDC7 kinase is as low as 1.22 μM, which shows stronger target inhibition capacity than the known CDC7 inhibitor Cdc7-IN-17 (IC50 = 7.83 μM), and has higher development potential. In the amyotrophic lateral sclerosis cell model, 40 μM of 20-Deacetyltaxuspine X can significantly increase the neuron survival rate from 65.27% of the model group to 81.96%, while reducing the apoptosis rate from 37.53% to 15.72%, effectively reversing the disease phenotype, and showing clear neuroprotective effect. The present application not only expands the medical use of 20-Deacetyltaxuspine X, but also provides experimental basis and lead compound for the development of anti-amyotrophic lateral sclerosis innovative drugs targeting CDC7, which is expected to break through the bottleneck of the lack of existing treatment methods, and has important scientific value and clinical transformation significance. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 A new mode of compound 20-Deacetyltaxuspine X ligand-CDC7 binding (PDB ID: 4F9B). DETAILED DESCRIPTION

[0011] The technical solution described in the present application is a conventional solution in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are publicly known.

[0012] The present application provides the use of 20-Deacetyltaxuspine X in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis.

[0013] In some specific embodiments, the 20-Deacetyltaxuspine X has the following structural formula: .

[0014] The present application also provides a medicine for preventing and / or treating amyotrophic lateral sclerosis, comprising 20-Deacetyltaxuspine X, and the 20-Deacetyltaxuspine X has the following structural formula: .

[0015] The present application also provides an application of 20-Deacetyltaxuspine X in preparing a CDC7 inhibitor.

[0016] In some specific embodiments, the 20-Deacetyltaxuspine X has the following structural formula: .

[0017] The present application also provides a CDC7 inhibitor, comprising 20-Deacetyltaxuspine X, and the 20-Deacetyltaxuspine X has the following structural formula: .

[0018] The new serine-threonine kinase CDC7 inhibitor forms a new hydrogen bond with CDC7, which provides guidance for the design of CDC7 inhibitor drugs. The new serine-threonine kinase CDC7 inhibitor has a protective effect on amyotrophic lateral sclerosis cell models, and can be used as an anti-amyotrophic lateral sclerosis drug. The new serine-threonine kinase CDC7 inhibitor produces a protective effect by inhibiting the apoptosis of amyotrophic lateral sclerosis cell models.

[0019] The new serine-threonine kinase CDC7 inhibitor forms a new ligand-protein binding mode with CDC7, and has good anti-amyotrophic lateral sclerosis application.

[0020] The new ligand-CDC7 binding mode of compound 20-Deacetyltaxuspine X: The carbonyl and hydroxyl groups of 20-Deacetyltaxuspine X can form four hydrogen bonds with amino acid residues Leu137, Glu66, Asn182, and Lys90 of the CDC7 protein, with distances of 2.7 Å, 2.4 Å, 1.9 Å, and 1.8 Å, respectively. 20-Deacetyltaxuspine X can form hydrophobic interactions with amino acid residues such as Met118, Val195, Met134, Pro135, and Leu137; and hydrophilic interactions with amino acid residues such as Tyr136, Gly67, Gly65, and Tyr412. The docking score of compound 20-Deacetyltaxuspine X is -8.747 kcal / mol. A new mode of ligand-CDC7 binding of compound 20-Deacetyltaxuspine X is described, such as... Figure 1 .

[0021] Example 1 The chemical structure of a novel serine-threonine kinase CDC7 inhibitor is as follows: .

[0022] This compound has been reported in the MCE compound library, and its English name is 20-Deacetyltaxuspine X.

[0023] 1. The CDC7 enzyme inhibitory activity of this compound Compound 20-Deacetyltaxuspine X was prepared into test sample solutions of 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, and 0.009765625 μM using biological-grade dimethyl sulfoxide (DMSO). 20 mM HEPES and 0.01% Triton X-100 were then added. TM A substrate buffer (pH 7.5) was prepared using 2 mM dithiothreitol, 4 μM FITC-labeled MCM2 peptide, 40 mM magnesium chloride, and 5 μM ATP. A 7 nM enzyme solution was prepared using a human CDC7 / human ASK protein mixture. In each well of a 96-well plate, 5 μL of the sample solution, 5 μL of the substrate buffer, and 10 μL of the enzyme solution were added. The plates were incubated at 37°C for 3 hours, and then 50 μL of enzyme stop solution was added. The phosphorylation of the substrate was determined using a LabChip EZ Reader II system (Caliper Life Sciences).

[0024] The positive control used in this experiment was the known CDC7 inhibitor Cdc7-IN-17, with CAS number 2253686-94-9. The experiment was repeated three times, and the IC50 of the enzyme inhibitory activity of the above compound was calculated using GraphPad software. 50 The values ​​are shown in Table 1.

[0025] Table 1. CDC7 enzyme inhibitory activity of the compounds

[0026] The experimental results in Table 1 show that compound 20-Deacetyltaxuspine X has stronger enzyme inhibitory activity than the known CDC7 inhibitor Cdc7-IN-17. Therefore, compound 20-Deacetyltaxuspine X is a novel serine-threonine kinase CDC7 inhibitor.

[0027] Example 2 Protective effect of compound 20-Deacetyltaxuspine X on a cell model of amyotrophic lateral sclerosis (ALS) NSC34 mouse neurons were cultured in DMEM high-glucose medium (10% FBS + 5% penicillin-streptomycin) and incubated at 37 ℃ in a 5% CO2 incubator. After passage to at least three times, logarithmically grown cells were harvested, trypsinized, centrifuged, and 8 × 10⁸ cells were collected. 4 Cells were seeded into 6-well plates and incubated at 37°C with 5% CO2 for transfection on day 2. The medium was replaced with fresh complete culture medium, and hSOD1 was added to each well. WT and hSOD1 G93A 16 μL of lentivirus containing empty plasmid was added and infected for 16 h. The culture medium containing viral particles was aspirated and replaced with fresh culture medium. A 2 mg / L solution was then used. -1 Screening was performed using puromycin, and the solution was changed every 1 day to contain 2 mg / L. -1 Fresh puromycin culture medium (DMEM + 10% FBS, penicillin-streptomycin-free) was used for screening for 2-3 weeks to obtain an amyotrophic lateral sclerosis (ALS) cell model. Stable hSOD1G93A expression was verified by qPCR, confirming the successful establishment of the ALS cell model (reference). https: / / doi.org / 10.1016 / j.ecoenv.2025.119301 , the literature title Lead exposure induces ferroptosis in ALS cell models by activating the MAPK / ERK signaling pathway).

[0028] Compound 20-Deacetyltaxuspine X was prepared into test sample solutions at concentrations of 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, and 0.009765625 μM using bio-grade DMSO. Normal cells or amyotrophic lateral sclerosis (ALS) cells in logarithmic growth phase were seeded into 96-well plates and cultured for 24 hours. The culture medium was then discarded, and the test sample solution was added. After 48 h, 20 μL of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) was added to each well. After incubation for another 2 h, the liquid was aspirated, 50 μL of DMSO was added, and the mixture was shaken well. The absorbance was measured at 490 nm using a microplate reader, and the inhibition rate was calculated using the following formula: Survival rate (%) = (absorbance value of the drug-treated group / absorbance value of the blank group) × 100%.

[0029] The experiment was repeated three times, and the results are shown in Table 2.

[0030] Table 2. Survival rate of compounds in amyotrophic lateral sclerosis (ALS) cell models

[0031] The experimental results in Table 2 showed that the cell survival rate in the model group was significantly lower than that in the normal group. Compared with the model group, cell viability increased with different concentrations of 20-Deacetyltaxuspine X. Cell survival rate significantly increased at a drug concentration of 40 μM. This indicates that a 40 μM concentration of 20-Deacetyltaxuspine X can significantly increase the survival rate of the amyotrophic lateral sclerosis (ALS) cell model. Therefore, the novel serine-threonine kinase CDC7 inhibitor 20-Deacetyltaxuspine X has a protective effect on the ALS cell model and can be used as an anti-ALS drug.

[0032] Example 3 Compound 20-Deacetyltaxuspine X inhibits apoptosis in an amyotrophic lateral sclerosis (ALS) cell model. Normal cells or amyotrophic lateral sclerosis (ALS) cells in the logarithmic growth phase were collected at a density of 2 × 10⁻⁶. 5 Seeds were inoculated per well in a 6-well plate. After intervention with 40 μM 20-Deacetyltaxuspine X for 48 h, the sample was digested and centrifuged, and 1×10⁻⁶ cells were collected. 5Cells were resuspended in phosphate buffer and then incubated with 195 μL Annexin V-FITC binding solution, 5 μL Annexin V-FITC, and 10 μL PI in sequence. The cells were then incubated at room temperature in the dark for 20 minutes. Cell apoptosis was detected using an FC500 flow cytometer. The experimental results are shown in Table 3.

[0033] Table 3 Effects of compounds on apoptosis in amyotrophic lateral sclerosis (ALS) cells

[0034] The experimental results in Table 3 show that the apoptosis rate in the model group was significantly increased compared to the normal group. Compared to the model group, administration of 40 μM 20-Deacetyltaxuspine X reduced the apoptosis rate in the amyotrophic lateral sclerosis (ALS) cell model. Therefore, 20-Deacetyltaxuspine X exerts a protective effect by inhibiting apoptosis in the ALS cell model.

[0035] Example 4 Animal experiments using 20-Deacetyltaxuspine X for the prevention and treatment of amyotrophic lateral sclerosis (ALS). The animal experiments of this invention use the most widely used amyotrophic lateral sclerosis (ALS) model: B6SJL-Tg(SOD1) G93A)1Gur / J (Jackson Laboratory, JAX stock #002726, USA) transgenic mice (References) https: / / doi.org / 10.1016 / j.neuroscience.2007.10.017 The literature, titled "Time-course and characterization of orolingual motor deficits in B6SJL-Tg(SOD1-G93A)1Gur / Jmice," describes a study in which model mice were divided into three groups: a model control group, a drug group, and a riluzole positive control group, with six mice in each group. The model control group received no intervention, the drug group received 20-Deacetyltaxuspine X (75 mg / kg), and the riluzole positive control group received riluzole (10 mg / kg). Administered via gavage once daily for 30 days. The gavage volume was 200 μL per mouse per administration. The model mice were housed in an SPF-grade environment with a room temperature maintained at (23±3)℃ and humidity between 40% and 70%, maintaining a 12-hour diurnal rhythm. Mice had free access to food and water.

[0036] Grasping force test: Grasping force was tested using a gripping force meter to assess the limb muscle strength of a mouse model of amyotrophic lateral sclerosis (ALS). The model mouse was placed on the grid of the gripping force meter, and its tail was pulled backward. The maximum pulling force at the moment the model mouse left the grid was recorded. Each model mouse was tested three times, and the average value was taken, with each test 30 minutes apart.

[0037] Rotary bar test: The motor coordination ability of a mouse model of amyotrophic lateral sclerosis (ALS) was assessed using a rotarod apparatus. The model mice were placed on a rotarod. The rotation speed of the rotarod was uniformly increased from 4 rpm to 40 rpm, and the fall time of the model mice was recorded. Each model mouse was tested three times, and the average value was taken, with each test 1 hour apart. The experimental results are shown in Table 4.

[0038] Table 4 Comparison of motor behavior after drug administration in each group

[0039] In the grip strength test, the 20-Deacetyltaxuspine X group significantly increased the grip strength of mice's hind limbs, and the increase in grip strength was superior to that of the positive control drug riluzole. In the rotarod test, the 20-Deacetyltaxuspine X group significantly increased the fall time of mice on the rotarod, and the fall time was longer than that of the positive control drug riluzole group. Therefore, animal experiments on amyotrophic lateral sclerosis (ALS) demonstrate that 20-Deacetyltaxuspine can improve the motor behavioral symptoms of ALS mice and can be used as an anti-ALS drug.

[0040] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. 20-Deacetyltaxuspine X in the preparation of drugs for the prevention and / or treatment of amyotrophic lateral sclerosis (ALS).

2. The application according to claim 1, characterized in that, The structural formula of 20-Deacetyltaxuspine X is as follows: 。 3. A drug for the prevention and / or treatment of amyotrophic lateral sclerosis (ALS), characterized in that, Including 20-Deacetyltaxuspine X, the structural formula of 20-Deacetyltaxuspine X is as follows: 。 4.20-Deacetyltaxuspine X in the preparation of CDC7 inhibitors.

5. The application according to claim 4, characterized in that, The structural formula of 20-Deacetyltaxuspine X is as follows: 。 6. A CDC7 inhibitor, characterized in that, Including 20-Deacetyltaxuspine X, the structural formula of 20-Deacetyltaxuspine X is as follows: 。

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