Use of pyrrolocarbazole oxime derivatives for preventing or treating brain trauma

Abstract

The application belongs to the technical field of medicine, and discloses application of pyrano-carbazole oxime derivatives in prevention or treatment of brain trauma. Specifically, the application discloses a pyrano-carbazole compound as shown in general formula I. The compound is prepared by an artificial synthesis method, contains a pharmaceutical composition thereof, and is applied in anti-brain trauma.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to the use of pyranocarbazole oxime derivatives in the prevention or treatment of traumatic brain injury. Background Technology

[0002] Traumatic brain injury (TBI) specifically refers to brain tissue damage caused by external trauma, also known as craniocerebral injury or head trauma. TBI not only occurs at the moment of injury but also includes secondary injuries that occur minutes to days after the injury due to changes in cerebral blood flow or intracranial pressure. Statistics show that the incidence of TBI ranges from 20 to 536 per 100,000 people. Reducing the incidence, mortality, and disease burden of TBI presents an increasingly significant challenge and is a major medical problem that urgently needs to be solved.

[0003] Pyranocarbazole alkaloids possess a wide range of biological activities. This invention introduces oximes and nitrones into the pyranocarbazole alkaloid core to obtain pyranocarbazole oxime derivatives. Pharmacological experiments have confirmed that these derivatives can significantly improve the neurobehavioral function of experimental animals, suggesting that the compounds have significant anti-traumatic brain injury activity. Summary of the Invention

[0004] This invention discloses compounds of general formulas I and II. Pharmacological experiments have shown that the compounds and their formulations of this invention have a significant ameliorative effect on neurobehavioral function in a rat model of traumatic brain injury (WDI) induced by falling heavy objects. Therefore, the compounds of this invention can be used for the prevention and / or treatment of traumatic brain injury.

[0005] The technical problem solved by this invention is to provide the use of pyranocarbazole oxime compounds represented by general formulas I and II and their pharmaceutically acceptable salts in the prevention or treatment of traumatic brain injury.

[0006] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0007] The first aspect of this invention is the use of a class of pyranocarbazole oxime derivatives, as shown in general formula I, or pharmaceutically acceptable salts thereof, in the preparation of drugs for the prevention or treatment of traumatic brain injury:

[0008]

[0009] R1 and R2 are independent and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, respectively.

[0010] OCH2CH2CH3, CF3, OCF3;

[0011] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3.

[0012] The first aspect of this invention also provides the use of a class of pyranocarbazole nitroketone derivatives of general formula II or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment of traumatic brain injury:

[0013]

[0014] R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively.

[0015] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3;

[0016] R4 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn.

[0017] The compounds are selected from the following group:

[0018]

[0019] The pharmaceutically acceptable salt is selected from organic or inorganic acid salts, including hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, carbonate, oxalate, lactate, succinate, or gluconate.

[0020] The pyranocarbazooxime compounds mentioned above are artificially synthesized.

[0021] The second aspect of the present invention is to provide the use of a pharmaceutical composition in the preparation of a drug for the prevention or treatment of traumatic brain injury, characterized in that the pharmaceutical composition comprises the pyranocarbazole oxime derivatives described in the first aspect of the present invention, their pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.

[0022] The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

[0023] The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nano-suspensions.

[0024] The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nano-suspensions.

[0025] The pyranocarbazooxime compounds mentioned above are artificially synthesized.

[0026] This pharmaceutical composition is prepared according to methods known in the art. It can be formulated into any dosage form suitable for human or animal use by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The content of the compounds of the present invention in its pharmaceutical composition is typically 0.1-95% by weight.

[0027] The compounds of this invention or pharmaceutical compositions containing them can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0028] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0029] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0030] To formulate the compounds of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannose, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0031] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0032] To formulate the drug delivery unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. Various diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0033] To prepare the compounds of this invention into injectable formulations, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc. pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing lyophilized powder injections, mannitol, glucose, etc., can also be added as supporting agents.

[0034] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0035] To achieve the purpose of medication and enhance the therapeutic effect, the drug or drug composition of the present invention can be administered using any known method of administration.

[0036] The dosage of the pharmaceutical compositions of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally, the suitable daily dose range of the compounds of the present invention is 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above doses can be administered as a single dose unit or divided into several dose units, depending on the physician's clinical experience and the administration regimen, including the use of other treatment methods.

[0037] The compounds or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compounds of the present invention have a synergistic effect with other therapeutic drugs, their dosage should be adjusted according to the actual situation.

[0038] Beneficial technical effects

[0039] The pyranocarbazole oxime compound CXT-24 developed by this invention has shown significant therapeutic effects in both in vivo and in vitro experiments. In in vitro experiments, CXT-24 at a concentration of 10 micromoles (μM) effectively reduced the damage caused by hydrogen peroxide to SK-N-SH cells and significantly improved cell survival rate, indicating that it has a strong neuroprotective effect.

[0040] In in vivo experiments, compound CXT-24 at a dose of 10 mg / kg showed significant therapeutic effects on a SD rat model of traumatic brain injury induced by falling weights, specifically by increasing the drop time in the rotarod test and improving neurological function scores.

[0041] These results demonstrate that CXT-24 is effective in multiple models, has broad applicability and significant therapeutic potential, and provides a new and effective drug option for the treatment of traumatic brain injury. Attached Figure Description

[0042] Figure 1 Effects of compound CXT-24 on motor function in rats after TBI modeling

[0043] Figure 2 Effects of compound CXT-24 on behavioral deficits in rats after TBI modeling (mNSS score)

[0044] Figure 3 Effect of compound CXT-24 on cell survival in hydrogen peroxide-induced neuronal damage (n=9) Detailed Implementation

[0045] Pharmacological Experiment 1

[0046] Effects of 10 mg / kg dose of CXT-24 on neurobehavioral function in TBI rats

[0047] Experimental Materials and Methods

[0048] CXT-24 was provided by our synthesis laboratory and has a chemical purity >99.5%. During preparation, 0.5% CMC-Na was added, and the mixture was then ground evenly in a mortar.

[0049] Preparation of a rat model of traumatic brain injury (TBI):

[0050] After isoflurane anesthesia, rats underwent an incision in the scalp 1.5 mm posterior to the coronal suture and 2.5 mm lateral to the midline on the right side, and a 5 mm diameter bone hole was drilled. Using a modified Feeney free-fall injury device, a 40 g hammer was dropped from 25 cm to impact the striking rod to a depth of 6 mm, and the scalp was sutured. Control group rats underwent scalp incision, drilling, and scalp closure without hammer impact. Subsequently, drugs were administered at predetermined times according to the groups; the control and model groups received equal volumes of 0.5% CMC-Na solution.

[0051] Grouping and Dosing

[0052] The animals were divided into four groups of 10 each: a control group, a model group, a CXT-24 10 mg / kg double-dose group, and a butylphthalide 200 mg / kg group. All animals were administered the medication orally via gavage. The administration times for the control group, model group, and butylphthalide 200 mg / kg group were 0, 24, and 48 hours post-surgery, while the administration times for the CXT-24 10 mg / kg double-dose group were 0, 6, 24, 30, 48, and 54 hours post-surgery.

[0053] Neurobehavioral score

[0054] Behavioral observations were conducted at 24, 48, and 72 hours after modeling, and the evaluation method was mNSS scoring.

[0055] Spinning rod test:

[0056] The rats' motor abilities were assessed using a rotarod test at three time points: 0 h before modeling, 24 h after modeling, 48 h after modeling, and 72 h after modeling. The rats were placed on the rotarod and the initial speed was 5 rpm, which was uniformly accelerated to 25 rpm within 180 s. The longest latency of the fall was recorded. Each experiment was repeated 3 times and the average value was taken.

[0057] Inclusion and exclusion criteria for laboratory animals

[0058] This experiment used SD rats to model traumatic brain injury (TBI). The entire surgical (anesthesia) procedure took approximately 5 minutes per rat. All SD rats were allowed free access to water and food after recovery from anesthesia. The primary cause of death was intestinal distension during anesthesia. Exclusion criteria included:

[0059] (1) SD rats died within 24 hours after surgery due to bloating caused by anesthesia;

[0060] (2) The weight of the SD rats did not meet the experimental standard.

[0061] Statistical methods

[0062] All data were statistically analyzed using Graph Prism 8.0.1, and all results are expressed as mean ± SD. Statistical analysis was performed using t-tests to compare differences between groups; a p-value < 0.05 was considered statistically significant.

[0063] Experimental results

[0064] Effects of compound CXT-24 on motor function in SD rats after traumatic brain injury

[0065] In the model group, the rotarod drop time was significantly reduced (62.46±11.58s) 24 hours after brain injury, showing a significant difference compared to the control group (P<0.001), indicating that the TBI model in this experiment significantly impaired the motor function of rats. After administration of CXT-24 10mg / kg, the motor function of rats was significantly improved, with a drop time of 85.70±21.30s, which was significantly increased compared to the model group (P<0.01). The drop time in the butylphthalide 200mg / kg group was 69.26±27.43s, with no significant difference compared to the model group.

[0066] At 72 hours post-brain injury, the rotarod drop time in the model group was 114.95 ± 15.89 s. Compared with the control group, the rotarod drop time in the model group was significantly reduced at 72 hours post-brain injury (P < 0.001), indicating that the TBI model in this experiment caused motor function impairment in rats. After administration of CXT-24 (10 mg / kg), the rotarod drop time in rats increased slightly compared with the model group, but the difference was not statistically significant, with a drop time of 133.21 ± 24.72 s (P = 0.06). The drop time in the butylphthalide 200 mg / kg group was 118.47 ± 37.93 s, which was not significantly different from the model group. These results suggest that the CXT-24 10 mg / kg group can protect against TBI-induced motor function impairment 24 hours post-surgery.

[0067] Table 1. Effects of compound CXT-24 on motor function in rats after TBI modeling.

[0068]

[0069] **P<0.01 vs model.

[0070] Effects of compound CXT-24 on behavioral deficits after brain injury in SD rats

[0071] The mNSS score was used to determine the behavioral scores of rats after traumatic brain injury (TBI). The results showed that the model group rats had a significantly higher neurological function impairment score (7.30±1.06) 24 hours after brain injury, which was significantly different from the control group (P<0.001), indicating that the TBI model in this experiment caused significant neurological function impairment in rats. After administration of CXT-24 10mg / kg, the score was 7.70±0.95; the score of the butylphthalide 200mg / kg group was 7.80±1.03; no significant difference was found between these scores and those of the model group.

[0072] At 72 hours post-brain injury, the neurological function impairment score of the model group rats was 7.50±1.27. Compared with the control group, the neurological function impairment score of the model group rats at 72 hours post-brain injury was still significantly higher, showing a statistically significant difference (P<0.001), indicating that the TBI model in this experiment caused neurological function impairment in rats. After administration of CXT-24 10mg / kg, the neurological function impairment score of rats was significantly reduced, with a score of 6.40±0.70, which was significantly different from that of the model group (P<0.05). The butylphthalide 200mg / kg group showed a reduction compared with the model group, but the difference did not reach statistical significance. This suggests that the CXT-24 10mg / kg group can protect against TBI-induced neurological function impairment at 72 hours post-surgery.

[0073] Table 2. Effects of compound CXT-24 on behavioral deficits in rats after TBI modeling (mNSS score)

[0074]

[0075]

[0076] *P<0.05 vs model.

[0077] Pharmacological Experiment 2

[0078] Experimental Groups

[0079] The study included a blank control group, a hydrogen peroxide damage model group, and a hydrogen peroxide damage + test drug group (neuroprotective agent screening group).

[0080] Experimental process

[0081] (1) Based on a density of 6.5 × 10 4 SK-N-SH cells were seeded into 96-well culture plates at 100 μl / well.

[0082] (2) After cell adhesion, the medium was changed. The control group received 100 μl of complete culture medium per well, the model group received 90 μl of complete culture medium per well, and the neuroprotective agent screening group received 80 μl of complete culture medium per well. The neuroprotective agent screening group was then supplemented with 10 μl of the 100 μM compound to be screened. Subsequently, 10 μl of 15 mM hydrogen peroxide was added to both the model group and the neuroprotective agent screening group. Each group had 6 replicates, and the experiment was repeated 9 times.

[0083] (3) Incubate the cells at 37°C for 4 hours;

[0084] (4) Add MTT (5 mg / ml), 10 μl / well, and continue the reaction at 37°C for 4 h;

[0085] (5) Discard the culture medium from the 96-well plate. Add 150 μl of DMSO dissolving solution to each well and gently pipette to dissolve the crystals. Measure the optical density (OD) value of each well at a wavelength of 570 nm using a microplate reader. The measured OD value reflects the cell viability of each drug to be screened. Compounds that demonstrate a certain protective effect against this injury model are then selected for rescreening.

[0086] Calculate the relative viability of each group of cells compared to the model cells.

[0087] (1) First, calculate the cell survival rate of the model group:

[0088] (OD value of model group / OD value of control group) × 100%

[0089] (2) Then calculate the cell survival rate of each drug relative to the model group:

[0090] (Screening group cell survival rate - Model group cell survival rate) / Model group cell survival rate × 100%

[0091] Statistical methods

[0092] Statistical analysis was performed using the T-test method. P < 0.05 was considered statistically significant.

Claims

1. The use of a class of pyranocarbazole oxime derivatives, as shown in Formula I, or pharmaceutically acceptable salts thereof, in the preparation of drugs for the prevention or treatment of traumatic brain injury: R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively. R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3.

2. The use of a class of pyranocarbazole nitroketone derivatives of general formula II or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of traumatic brain injury: R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively. R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3; R4 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn.

3. The application according to any one of claims 1 and 2, characterized in that, The compounds are selected from the following group:

4. The application according to any one of claims 1-3, characterized in that, The pharmaceutically acceptable salt is selected from organic or inorganic acid salts, including hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, carbonate, oxalate, lactate, succinate, or gluconate.

5. The use of a pharmaceutical composition in the preparation of a drug for the prevention or treatment of traumatic brain injury, characterized in that, The pharmaceutical composition comprises the pyranocarbazole oxime derivatives as described in any one of claims 1-3, their pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

7. The application according to claim 5, characterized in that, The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nanosuspensions.

8. The application according to any one of claims 1 and 2, characterized in that, The pyranocarbazooxime compounds mentioned above are artificially synthesized.

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