CN121648112B - Application of a benzimidazole compound in the preparation of drugs for treating intervertebral disc degeneration - Google Patents

Abstract

This invention discloses the application of a benzimidazolone compound in the preparation of a drug for treating intervertebral disc degeneration. This _{benzimidazolone} compound is a small molecule with the molecular formula _{C11H14N4O2 and} CAS number 881591-33-9. Starting from the fundamental molecular mechanism of intervertebral disc degeneration, this invention provides _an innovative treatment strategy that can intervene in the early stages of the disease and influence disease progression at the etiological level by targeting and activating SOD1 activity, a newly discovered pathogenic link.

Description

Application of benzimidazolone compound in preparation of medicines for treating intervertebral disc degeneration

Technical Field

The invention belongs to the technical field of chemical medicaments, and provides application of a benzimidazolone compound in preparation of a medicament for treating intervertebral disc degeneration.

Background

Disc degeneration (Intervertebral Disc Degeneration, IVDD) is the main pathological basis leading to lower back pain and spine related diseases, severely affecting the quality of life of patients and bringing about a huge socioeconomic burden. The occurrence of IVDD is a complex pathological process, mainly manifested by a decrease in nucleus pulposus cells and degradation of extracellular matrix. Recent studies have shown that oxidative stress plays a central driving role in the development of IVDD. Under the stimulation of microenvironments such as mechanical load, inflammatory factors or nutrition deprivation, active oxygen (ROS) in nucleus pulposus cells are overproduced and exceed the clearance capacity of the nucleus pulposus cells. Excessive ROS accumulation not only directly impairs mitochondrial function, but also activates downstream apoptosis signaling pathways and expression of matrix metalloproteinases, accelerating senescence and apoptosis of nucleus pulposus cells, ultimately leading to loss of disc structure and function. Therefore, scavenging excess ROS, restoring redox balance through antioxidant strategies, is considered an effective therapeutic approach to delay IVDD progression.

Superoxide dismutase 1 (Superoxide Dismutase, SOD 1) acts as the primary first-line antioxidant enzyme in cells, playing a vital role in protecting against ROS injury and maintaining the homeostasis of the intervertebral disc microenvironment. It was found that in degenerated disc tissues, SOD1 expression and activity tended to be significantly reduced, leading to breakdown of the cellular antioxidant defense system. Although direct supplementation of exogenous SOD1 proteins is theoretically possible, its clinical application is greatly limited due to the limitations of protein drugs, such as short half-life, immunogenicity risk, difficulty in penetrating dense disc matrix, etc. In contrast, the search for small molecule compounds that specifically target and enhance endogenous SOD1 activity is expected to be achieved, but the application in IVDD therapy has not been deeply reported.

Based on the background, the invention utilizes the Computer Aided Drug Design (CADD) technology, screens out a novel small molecular compound with potential SOD1 activation effect based on an active center area of SOD1 through high-throughput virtual screening, evaluates the effect of the novel small molecular compound on inhibiting oxidative stress and IVDD in vitro and in vivo, evaluates the biological safety of the novel small molecular compound, provides a novel lead compound for the treatment of the IVDD, and also provides a novel theoretical basis for the development of antioxidant drugs based on structural biology.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of benzimidazolone compounds in preparing medicines for treating disc degeneration, which aims to overcome the limitation of the existing disc degeneration treatment strategies, namely, the current clinical intervention is mostly focused on symptom relief and late structural repair, and the etiology treatment means aiming at the early key pathogenic mechanism of the disease is lacking, so that the disease process cannot be effectively blocked or reversed.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The invention provides an application of benzimidazolone compound or pharmaceutically acceptable salt thereof in preparing medicines for treating disc degeneration, wherein the Chinese name of the benzimidazolone compound is N- [ (1, 3-dimethyl-2-oxo-2, 3-dihydro-1H-benzimidazol-5-yl) methyl ] Urea, the English name is Urea, N- [ (2, 3-dihydro-1, 3-dimethy-2-oxo-1H-benzimidazol-5-y) methyl ] - (AC), the molecular formula is C ₁₁H₁₄N₄O₂, and the chemical structural formula is as follows:

。

As one of the preferred technical schemes, the benzimidazolone compound or the pharmaceutically acceptable salt thereof is used as an agonist of SOD1 for treating intervertebral disc degeneration.

As one of further preferable technical schemes, the benzimidazolone compound or pharmaceutically acceptable salt thereof is combined with a Lys123 site of an SOD1 active center, and the Lys123 site is the 123 rd lysine of a wild SOD1 protein with an amino acid sequence shown as SEQ ID NO. 1.

As one of the preferred technical schemes, the benzimidazolone compound or the pharmaceutically acceptable salt thereof has the following effects:

(A) Activating SOD1 enzyme activity of nucleus pulposus cells;

(B) Inhibiting accumulation of superoxide anions in nucleus pulposus cells;

(C) Inhibiting DNA damage of nucleus pulposus cells and accumulation of gamma-H2 AX;

(D) Inhibit accumulation of ROS in nucleus pulposus cells.

The invention also provides application of the pharmaceutical composition containing the benzimidazolone compound or the pharmaceutically acceptable salt thereof in preparing medicines for treating disc degeneration.

The invention also provides an intervertebral disc puncture injection, the active ingredient of which is benzimidazolone compound or pharmaceutically acceptable salt thereof.

The beneficial effects of the invention are as follows:

the invention provides application of a benzimidazolone compound in preparation of a medicament for treating disc degeneration, wherein the benzimidazolone compound is a small molecular compound with a molecular formula of C ₁₁H₁₄N₄O₂ and a CAS number of 881591-33-9. The invention starts from the fundamental molecular mechanism of intervertebral disc degeneration, provides an innovative treatment strategy which can intervene in early stage of diseases and influence the disease progress from the aspect of etiology through targeting the newly discovered pathogenic link of activating SOD1 activity.

Specifically, the invention solves the following technical problems:

1. Screening of compounds targeted to modulate SOD1 activity compounds that can target binding to the active center of SOD1 and modulate SOD1 activity were screened by computer-aided virtual screening using ChemDIV compound library.

2. The compounds are disclosed as inhibiting effects on oxidative stress of nucleus pulposus cells and disc degeneration, and their therapeutic effects are evaluated in animal models of nucleus pulposus cells cultured in vitro and disc degeneration in vivo.

3. The biological safety of the compound for treating the intervertebral disc degeneration is evaluated, and the biological safety of the compound in the treatment process is evaluated through indexes such as blood routine and the like.

The invention has the following advantages:

1. current disc degeneration treatments are mostly directed to symptoms (pain) or late structural destruction, lacking targeted therapies for early molecular events, and commonly used drugs (such as NSAIDs) only alleviate inflammation without altering disease progression. The invention takes SOD1 enzyme activity as a treatment target for the first time and intervenes aiming at the core driving factor (oxidative stress) of intervertebral disc degeneration.

The invention reveals the effectiveness of the small molecular compound C ₁₁H₁₄N₄O₂ as an SOD1 specific agonist for the first time, provides a brand-new targeting solution for treating the intervertebral disc degeneration, and has no report on the patent medicine of C ₁₁H₁₄N₄O₂ before.

2. Commonly used antioxidants (such as vitamins C, E) lack specificity and have limited effects, partial small molecule drugs have off-target effects, and adverse reactions are caused by systemic administration. C ₁₁H₁₄N₄O₂ intra-discal injection administration has good biological safety and provides a basis for future further transformation. In addition, C ₁₁H₁₄N₄O₂ specifically activates SOD1, has definite structure, small molecular weight and stable structure, and has local action of intra-discal administration, less systemic exposure and low systemic toxicity.

3. The technical means adopted by the invention has high specificity, high sensitivity and high repeatability, and the screened SOD1 specific activator C ₁₁H₁₄N₄O₂ can be used as a potential treatment strategy of the IVDD, thereby providing a new technical direction and experimental basis for the precise diagnosis and treatment of the IVDD.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.

Drawings

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic of the experimental procedure of high throughput virtual screening (A) and top 10 compounds with highest scores (B).

Fig. 2 shows a graph of SOD1 enzyme activity detection results, wherein the ordinate of the first 10 compounds directly intervenes in the histogram and is SOD1 enzyme activity, error bars represent standard deviations, P <0.001, and results show that the SOD1 enzyme activity of nucleus pulposus cells is obviously reduced after lactic acid treatment, and the SOD1 enzyme activity of a lactic acid+compound 3 (chemical formula C ₁₁H₁₄N₄O₂) treatment group is obviously recovered, so that C ₁₁H₁₄N₄O₂ can be proved to be effective in activating the SOD1 enzyme activity of nucleus pulposus cells.

FIG. 3 shows the molecular formula of compound C ₁₁H₁₄N₄O₂ and the binding scheme of SOD 1 molecular docking.

Fig. 4 shows the results of measurement of the content of superoxide anions in nucleus pulposus cells after lactic acid and C ₁₁H₁₄N₄O₂ treatment (DHE probe method), wherein the results comprise a cell imaging diagram (A) and a corresponding quantitative analysis histogram (B) under a fluorescence microscope, the red fluorescence intensity in the imaging diagram represents the content of the superoxide anions, the ordinate of the histogram is the fluorescence intensity of the probe, the error line represents the standard deviation, the error line represents P <0.001, the result shows that the red fluorescence intensity of nucleus pulposus cells after lactic acid treatment is obviously enhanced, the content of the superoxide anions is increased, and the DHE fluorescence intensity of a lactic acid+C ₁₁H₁₄N₄O₂ treatment group is obviously reduced, so that C ₁₁H₁₄N₄O₂ can be proved to effectively inhibit the accumulation of the superoxide anions in nucleus pulposus cells.

Fig. 5 shows the results of gamma-H2 AX content detection after the treatment of the nucleus pulposus cells with lactic acid and C ₁₁H₁₄N₄O₂, wherein the results comprise a nucleus imaging diagram (A) and a corresponding quantitative analysis histogram (B) under a fluorescence microscope, the red fluorescence intensity in the imaging diagram represents the gamma-H2 AX content, the ordinate of the histogram is gamma-H2 AX fluorescence intensity, the error line represents the standard deviation, the error line represents P <0.001, the results show that the red fluorescence intensity of the nucleus pulposus cells after the treatment of the lactic acid is obviously enhanced, the gamma-H2 AX content is increased, and the gamma-H2 AX fluorescence intensity of the lactic acid+C ₁₁H₁₄N₄O₂ treatment group is obviously reduced, so that C ₁₁H₁₄N₄O₂ can be proved to effectively inhibit the DNA damage and gamma-H2 AX accumulation of the nucleus pulposus cells.

FIG. 6 shows results of measurement of ROS content of nucleus pulposus cells after lactic acid and C ₁₁H₁₄N₄O₂ are treated (flow cytometry), wherein the results comprise a ROS flow detection chart (A) and a corresponding quantitative analysis histogram (B), the ordinate in the chart A represents the fluorescence intensity of ROS, the abscissa represents the number of cells containing ROS fluorescence, the ordinate in the chart B represents the average fluorescence intensity of ROS, error bars represent standard deviations and represent P <0.001, and the results show that the fluorescence intensity of nucleus pulposus cells after lactic acid treatment is remarkably enhanced, and the fluorescence intensity of ROS in a lactic acid+C ₁₁H₁₄N₄O₂ treatment group is remarkably reduced, so that C ₁₁H₁₄N₄O₂ can be proved to be capable of effectively inhibiting the accumulation of ROS in nucleus pulposus cells.

Fig. 7 is a graph of SO & FG staining of a sham rat, PIDD and C ₁₁H₁₄N₄O₂ -treated PIDD, containing a graph of SO & FG staining of disc tissue (a) and a corresponding histogram of histological scores (B), with the histogram ordinate representing the histological scores, error bars representing standard deviations, P <0.001, showing a significant decrease in nucleus tissue, a significant decrease in intervertebral height, a significant increase in histological scores, a significant disc degeneration, and a inversely proportional degree of disc degeneration of C ₁₁H₁₄N₄O₂ -treated group to the concentration of C ₁₁H₁₄N₄O₂, demonstrating that C ₁₁H₁₄N₄O₂ treatment is effective in inhibiting disc degeneration in a needled degeneration model of rats. Scale bar = 500 μm.

Fig. 8:C ₁₁H₁₄N₄O₂ shows a biosafety test indicator for disc degeneration treatment, error bars indicate standard deviations, ns represent no significant changes, (a-E) organ index values (n=6 rats) in groups receiving intra-discal injections at different concentrations C ₁₁H₁₄N₄O₂, (F-K) differences in blood normals (n=6 individual rats) in major indicators (RBC: red blood cells, WBC: white blood cells, PLT: platelets, HGB: hemoglobin, MCV: mean red blood cell volume, MCH: mean red blood cell hemoglobin), (L-M) liver function index (ALT: alanine aminotransferase; AST: aspartate aminotransferase), (n=6 rats).

Detailed Description

The invention is further described below in connection with the following detailed description. In which the drawings are for illustrative purposes only and are not intended to be construed as limiting the present patent, and in which certain elements of the drawings may be omitted, enlarged or reduced in order to better illustrate embodiments of the present invention, and not to represent actual product dimensions, it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.

1. Preparation of experimental materials

1. The rat nucleus pulposus cells are obtained by extracting the rat nucleus pulposus cells from the tail vertebral disc of an SD rat according to the method of 2016,35 (1-6) in the isolation, culture and identification of rat tail vertebral disc nucleus pulposus cells of applicant reference, culturing the rat nucleus pulposus cells in a DMEM/F12 culture medium containing 10% fetal bovine serum (FBS, gibco brand) and 1% green streptomycin mixed solution (solarbio brand) by volume percentage, placing the rat nucleus pulposus cells in a 37 ℃ constant temperature incubator with 5% CO ₂ for routine culture, and selecting logarithmic phase cells for subsequent experiments.

2. Tissue sample sources were 30 healthy SPF-grade SD rats (male, 8 weeks old, weight 200-220 g) purchased from Chengdu laboratory animal technologies Inc. Rats were randomly divided into 5 groups of sham surgery, PBS, small dose C ₁₁H₁₄N₄O₂, medium dose C ₁₁H₁₄N₄O₂, large dose C ₁₁H₁₄N₄O₂, 6 each. After the rats in the model group were effectively anesthetized, the prone position was fixed, 6-7 segments of the coccyx were positioned, the center of the coccyx was vertically penetrated by using a sterile 21G needle, the penetration depth was 5mm, the needle was slowly withdrawn after 30s stay, a model of degeneration of the rat tail disc was constructed, and then PBS (concentration 0.01M, ph=7.4), C ₁₁H₁₄N₄O₂ (5 μm, 25 μm, 50 μm) and the discs between the coccyx 6 and 7 were respectively injected by 26-gauge needle and microinjector injection. All rats are routinely bred after operation for 4 weeks, and the breeding environment temperature is 22-25 ℃, the humidity is 50-60%, and the rats are free to drink water. After 4 weeks of rearing, the rats were again anesthetized and the coccygeal 6-7 segments of the intervertebral discs were dissected apart. The experimental scheme is approved by the ethical committee of the unit experiment animal (ethical trial number: AMUWEC 20235167), and the experimental process strictly follows animal experiment welfare and ethical specifications.

3. Main reagents and instruments are C ₁₁H₁₄N₄O₂ compound (MedChemExpress), pancreatin (Promega brand), SOD enzyme activity detection kit, total antioxidant capacity (T-AOC) detection kit, superoxide anion detection kit (DHE probe method, all purchased from Biyun), modified SO & FG staining kit (Solarbio, cat# G1371). Inverted microscope (Olympus CKX 53), gallios flow cytometer (Beckman), molecular dynamics simulation: software: gromacs 2018.4, force field: amber14SB, water model: TIP3P, virtual screening platform: software: schr dinger Maestro 11.4, module: glide (HTVS/SP/XP).

2. Specific implementation steps

1. Cell model establishment

1.1 Primary nucleus pulposus cell separation, namely taking rat coccygeal nucleus pulposus tissue, digesting the tissue for 2 hours at 37 ℃ with 0.2% type II collagenase in mass concentration, centrifuging the tissue for 5 minutes at 400 Xg, and culturing the tissue by DMEM/F12+10% FBS+1% double antibody in mass concentration.

1.2 Lactic acid treatment model when cells passed to passage 2, medium containing lactic acid (2 mM, 6mM, 10 mM) was changed, and the medium was changed every 3 days, and treated for 7 days, and control group was normal medium.

2. High throughput virtual screening

Protein Structure preparation the crystal structure of the human SOD1 protein (PDB ID: 1PU 0) was obtained from RCSB Protein Data Bank. The electrostatic loop region (residues 122-144, which are conserved between human and rat species) where lysine (Lys 123) at position 123 of the active center is located was selected to be involved in directing the transfer of superoxide anions (O ₂ ^-) to SOD1 active center (copper ion binding site). The Protein Preparation Wizard module of Schr dinger Maestro software was used to perform hydrogen atom addition, charge distribution, water molecule removal and energy minimization treatments on the protein structure to obtain an optimized structure suitable for molecular docking.

2.2 Screening of compound library ChemDIV compound library (160 ten thousand small molecules), structural optimization of the compound using the LigPrep module of Schr dinger, including generating possible three-dimensional conformation, optimizing hydrogen bond network, ionization state prediction (pH 7.0.+ -. 2.0), distributing proper charge (OPLS 4 force field), removing repetitive structure, and retaining compound with good drug-like property and structural diversity for subsequent virtual screening.

2.3 Three-stage molecular Butt screening (A in FIG. 1)

2.3.1 Preliminary screening, namely, preliminary docking is carried out on all the pretreated compounds by using a Glide module, and 10% of the compounds with the strongest binding capacity with the Lys123 site of the SOD1 active center are screened out.

2.3.2 Standard Precision screening the compounds obtained from the preliminary screening were submitted to GLIDE STANDARD Precision docking, further assessing binding affinity and conformational stability, and selecting the first 10% of the compounds to go to the next round.

2.3.3 High-precision screening, namely adopting Glide Extra Precision mode to carry out refined butt joint on the residual compounds, and evaluating key parameters such as binding free energy, hydrogen bond network, hydrophobic interaction and the like.

2.3.4 Results ranking and candidate compound selection compounds were ranked according to GlideScore (binding energy score), the top 10 compounds with the highest scores were selected as potential activator candidates (B in fig. 1).

3.3 And (3) recovering and verifying the activity of the SOD1 enzyme, namely detecting whether the catalytic activity of the SOD1 in NPCs is recovered or not by adopting an SOD1 activity detection kit. Cell lysis, BCA assay, SOD activity assay kit operation, adding WST-8 working solution, adding enzyme working solution, incubating at 37deg.C for 30min, measuring absorbance at 450nm, and recovering SOD1 activity to normal level of 80% with compound No. 3, chemical formula C ₁₁H₁₄N₄O₂ (FIG. 2).

3.4. Analysis of binding patterns Thr40, his44, lys123, asn140 were the key binding residues (FIG. 3).

3. Cell experiments prove that the C ₁₁H₁₄N₄O₂ has the effect

3.1 Cell model construction SOD1 oxidative stress injury was induced by lactic acid treatment using rat Nucleus Pulposus Cells (NPCs) as an in vitro model.

3.4 Oxidative stress index detection, namely, detecting the ROS, namely, a total ROS detection kit and analyzing by a flow cytometer, and O ₂ ^- detection, namely, adopting dihydro ethidium (DHE) fluorescent staining (10 mu M DHE is incubated for 30 minutes at 37 ℃ in a dark place), and quantifying by confocal microscope observation and a fluorescence spectrophotometer, wherein the result is that the ROS is reduced by 50-60% by C ₁₁H₁₄N₄O₂.

3.5 Assessment of cell damage DNA damage by gamma-H2 AX immunofluorescence staining, positive cell count, cell senescence by beta-galactosidase staining, calculation of positive rate, and as a result, C ₁₁H₁₄N₄O₂ decreased DNA damage and cell senescence marker expression.

4. In vivo experiments demonstrated C ₁₁H₁₄N₄O₂ effect (FIG. 7)

7.1 PIDD model Male SD rats (2 months old, 200-220G), isoflurane inhalation anesthesia, coccyx 5-6 intervertebral space positioning (palpation or X-ray), 21G needle (with depth stop) penetrating 5mm depth vertically, rotating 360 DEG, stay for 30 seconds, postoperative disinfection, analgesic antibiotic treatment for 3 days.

7.2 Grouping treatment, control group, sham surgery (needle only), model group, PIDD+PBS injection, treatment group 1, PIDD+small dose C ₁₁H₁₄N₄O₂ injection (5. Mu.M), treatment group 2, PIDD+medium dose C ₁₁H₁₄N₄O₂ injection (25. Mu.M), treatment group 3, PIDD+large dose C ₁₁H₁₄N₄O₂ injection (50. Mu.M).

7.3 Dosing regimen direct injection with a 26G needle at the puncture site after modeling, hamilton microinjector C ₁₁H₁₄N₄O₂, dosing frequency 1 time per week for 4 weeks.

7.4 Evaluation of treatment effect:

Histological evaluation animals were sacrificed, disc tissue was removed, 4% paraformaldehyde fixed for 48 hours, EDTA decalcified for 2 weeks, paraffin embedded, 5 μm sections, modified SO & FG staining, three investigators blinded histological scores.

Results treatment group 3 scores improved by 30-50%.

8. Biological safety assessment (FIG. 8)

8.1 In vivo safety:

Continuous dosing test the therapeutic dose (50. Mu.M) was injected weekly for 4 weeks and the body weight and feeding were monitored. As a result, the body weight was steadily increased without abnormality, hematology was examined by taking blood 24 hours after the last administration, blood was routinely examined by a full-automatic blood cell analyzer, and liver function was examined by a biochemical analyzer.

As a result, each index was in the normal range. Histopathology, coring, liver, spleen, lung, kidney, 4% paraformaldehyde fixation, paraffin section, HE staining, pathologist blindness assessment. As a result, there is no pathological change in the main viscera, and local inflammation and necrosis are not caused by injection.

Summary of experimental results

According to the embodiment, through a series of experiments, the small molecular compound C ₁₁H₁₄N₄O₂ is successfully identified, through specific activation of SOD1 activity, the total antioxidant capacity of nucleus pulposus cells is improved, the accumulation of superoxide anions is inhibited, further oxidative stress damage of the nucleus pulposus cells is relieved, and the pathological process of the IVDD is effectively relieved. The experimental steps of the embodiment are clear and have strong repeatability, and the figure result intuitively verifies the core conclusion of the invention, thereby providing reliable experimental basis for the development of the follow-up IVDD treatment strategy.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

Claims (3)

1. The application of benzimidazolone compound or pharmaceutically acceptable salt thereof in preparing medicines for treating disc degeneration is characterized in that the Chinese name of the benzimidazolone compound is N- [ (1, 3-dimethyl-2-oxo-2, 3-dihydro-1H-benzimidazol-5-yl) methyl ] urea, the molecular formula is C ₁₁H₁₄N₄O₂, and the chemical structural formula is as follows:

。

2. Use of a pharmaceutical composition comprising a benzimidazolone compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating disc degeneration, wherein the benzimidazolone compound has the chinese name N- [ (1, 3-dimethyl-2-oxo-2, 3-dihydro-1H-benzimidazol-5-yl) methyl ] urea, and has the formula C ₁₁H₁₄N₄O₂ and the chemical structural formula:

。

3. an intervertebral disc puncture injection, characterized in that the active ingredient of the injection is the benzimidazolone compound or the pharmaceutically acceptable salt thereof according to claim 1, the Chinese name of the benzimidazolone compound is N- [ (1, 3-dimethyl-2-oxo-2, 3-dihydro-1H-benzimidazol-5-yl) methyl ] urea, the molecular formula is C ₁₁H₁₄N₄O₂, and the chemical structural formula is as follows:

。