Application of tetrahydroindazole derivative in preparation of drug for resisting micro-nano plastic toxicity

Abstract

The invention discloses an application of a tetrahydroindazole derivative in preparation of a drug for resisting micro-nano plastic toxicity, the tetrahydroindazole derivative is specifically N-((methylphenyl-3-) phenoxy-2-)-N '-(4, 5, 6, 7-tetrahydro-1H-indazole-5-) oxamide, and the structural formula of the tetrahydroindazole derivative is shown as a formula I. The invention further discloses a preparation method of the tetrahydroindazole derivative. By investigating the influence of N-((tolyl-3-) phenoxy-2-)-N '-(4, 5, 6, 7-tetrahydro-1H-indazole-5-) oxamide on the life of caenorhabditis elegans poisoned by the micro-nano plastic, the obtained experimental result shows that N-((tolyl-3-) phenoxy-2-)-N'-(4, 5, 6, 7-tetrahydro-1H-indazole-5-) oxamide has a relatively strong detoxifying effect on the micro-nano plastic. Therefore, the compound is expected to be applied to preparation and development of various micro-nano plastic toxicity resisting drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of a tetrahydroindazole derivative in the preparation of drugs resistant to micro- and nano-plastic toxicity. Background Technology

[0002] Microplastics refer to plastic particles with a diameter of less than 5 millimeters. Even smaller microplastics, such as those in the micrometer and nanometer sizes, are collectively known as micro- and nano-plastics. Micro- and nano-plastics are widely distributed in food, water, the atmosphere, and everyday consumer goods, and have even infiltrated human tissues and organs. Since the first report of microplastics in the human gut in 2018, their presence has been detected in lung, liver, spleen, and kidney tissues. Recent studies have also found that microplastics have reached the placenta. Once inside the human body, they may cause various health hazards, including cardiovascular damage, intestinal barrier dysfunction, promotion of cancer metastasis, and promotion of Alzheimer's disease.

[0003] In cell and animal models, low-dose exposure to polystyrene (PS) nanoplastics has been shown to act as catalytic surfaces, promoting the nucleation and oligomerization of Aβ peptides (especially low concentrations of Aβ40 and Aβ42), thereby enhancing neurotoxicity; it also induces (environmental) oxidative stress, mitochondrial dysfunction, and protein homeostasis imbalance. For individuals already experiencing protein homeostasis stress (such as those genetically susceptible to AD), microplastic exposure may produce additive or synergistic damaging effects, further exacerbating AD-like phenotypes such as motor behavioral deficits and cognitive impairment.

[0004] Therefore, developing protective drugs against microplastic toxicity is of great significance. In the early stages of drug screening, the *Caenorhabditis elegans* model has shown significant advantages. In particular, the transgenic strain CL2006, which specifically expresses human Aβ1-42 in body wall muscle cells, can reproduce typical toxic phenotypes such as Aβ-induced progressive paralysis and shortened lifespan. This model is sensitive to environmental stress, and when combined with wild-type N2, the efficacy of candidate drugs in addressing the combined toxicity (Aβ toxicity + environmental stress) mediated by MNPs can be systematically evaluated from both the overall level (motor capacity, lifespan) and the molecular level (oxidative stress response gene gst-4, cholinergic neurotransmitter-related gene unc-17, etc.).

[0005] To date, there are no literature reports on the antimicroplastic complex toxicity (Aβ toxicity + environmental stress) of tetrahydroindazole derivative specifically N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide. Summary of the Invention

[0006] In view of the technical problems existing in the background art, the purpose of this invention is to provide an application of tetrahydroindazole derivatives in the preparation of antimicro-nanoplastics toxic drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of a tetrahydroindazole derivative in the preparation of antimicro / nanoplastics toxicity drugs, wherein the tetrahydroindazole derivative is specifically N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide, and its structural formula is shown in formula (I): .

[0008] Preferably, the drug is a pharmaceutical composition consisting of the above-mentioned tetrahydroindazole derivative as the active ingredient and pharmaceutically acceptable excipients.

[0009] The present invention has the following beneficial effects: This invention investigates the effect of a tetrahydroindazole derivative, specifically N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide, on the lifespan of *Caenorhabditis elegans* poisoned by micro- and nano-plastics. Experimental results show that N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide has a strong detoxification effect on micro- and nano-plastics and holds promise for application in the preparation of various anti-micro- and nano-plastic toxicity drugs. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 The structural formula is N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide; Figure 2 Figure 1 shows the experimental results of the effect of N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide on the lifespan of Caenorhabditis elegans poisoned by micro- and nano-plastics. Figure 3 The figure shows the experimental results of the effect of N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide on the activity of Caenorhabditis elegans poisoned by micro- and nano-plastics. Detailed Implementation

[0012] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0013] Example 1 This embodiment uses the Caenorhabditis elegans model as a model to evaluate the anti-toxicity of N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide (commercially available) to micro / nanoplastics. The detection steps are as follows: (1) Solution preparation N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide was dissolved in DMSO to prepare a 10 mM mother liquor of compound 1 (Z27).

[0014] (2) Culture of Caenorhabditis elegans This study used wild-type N2 nematodes and the transgenic nematode CL2006 (dvIs2 [pCL12(unc-54 / Aβ1-42 peptide; pRF4)]). CL2006 nematodes express human Aβ1-42 protein in muscle cells and exhibit significant protein accumulation and paralysis phenotype with age. To ensure the consistency of experimental results, the nematodes underwent synchronization treatment before the experiment. All nematodes were first cultured on solid nematode growth medium (NGM) coated with E. coli OP50 in a constant temperature incubator at 20°C. Adult nematodes containing a large number of eggs were selected and treated with alkaline bleaching lysis buffer (1 M NaOH, 10% NaClO, M9 Buffer) for 8-10 minutes to obtain pure eggs. The eggs were then incubated overnight at 20°C in sterile M9 buffer to develop to the L1 stage, thereby achieving population synchronization. The formal experiment adopted a liquid culture mode, using K medium (32 mmol / L KCl, 51 mmol / L NaCl) as the exposure medium.

[0015] (3) Detoxification test and survival time detection of CL2006 Caenorhabditis elegans The lifespan experiment employed a combination of liquid exposure and solid plate culture, using the transgenic nematode CL2006 (dvIs2 [pCL12(unc-54 / Aβ1-42 peptide; pRF4)]). Early drug treatment phases used K-medium as the exposure medium, while later lifespan assays were performed on solid nematode growth medium (NGM) coated with E. coli OP50. Microplastic suspension preparation: 3.0 μm diameter polystyrene microspheres (PS, Sangon Biotech) were used. PS was added to K-medium to prepare a 1.0 mg / L working solution, which was ultrasonically treated for 1 hour before use to ensure uniform dispersion. Drug administration: Synchronized L4 stage CL2006 nematodes were transferred to K-medium containing PS. Group Z27 was supplemented with Z27 stock solution (dissolved in 10 mM DMSO) to achieve a final working concentration of 200 μM. All groups included 0.1% DMSO as a solvent control. To prevent interference from progeny, fluorouracil deoxyribonucleoside (FUdR) was added to the culture medium. During the 24-hour liquid exposure period, nematodes were fed 25 μL of E. coli OP50 bacterial suspension daily, and the mixture was shaken at 100 rpm on a 20°C constant-temperature shaker.

[0016] After exposure, i.e., on the first day of adulthood, the nematodes were transferred to fresh NGM solid medium coated with OP50 and incubated at 25°C. Fluorouracil deoxyribonucleoside (FUdR) was added to the medium to prevent interference with counts during offspring hatching. From the first day of adulthood, the number of surviving and dead nematodes was observed and recorded every 2 days until all nematodes died. To maintain environmental freshness, the nematodes were transferred to fresh NGM plates every 4 days. Survival curves were plotted using GraphPad Prism software, and log-rank test statistical analysis was performed to calculate the effect of the Z27 group of samples on prolonging the lifespan of *C. elegans* induced by plastic poisoning. The results are shown in [Figure number missing]. Figure 2 .

[0017] Depend on Figure 2 The results showed that the median survival time of the negative control group was 13.84 days, while the lifespan of nematodes in the PS-treated group was shortened to 10.81 days, exhibiting significant toxicity. However, the lifespan of nematodes in the Z27 (detoxification treatment) group recovered to 13.43 days, a result not only significantly longer than the PS group but also approaching that of the negative control group. This indicates that N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide has excellent microplastic detoxification activity. Therefore, it holds promise for use in the preparation and development of various micro / nanoplastics detoxification drugs.

[0018] Example 2 The resistance of N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide to micro / nanoplastics toxicity was evaluated using the locomotion ability of Caenorhabditis elegans.

[0019] (1) Solution preparation N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide was dissolved in DMSO to prepare a 10 mM sample stock solution. (2) Culture of CL2006 Caenorhabditis elegans This study used wild-type N2 nematodes and the transgenic nematode CL2006 (dvIs2 [pCL12(unc-54 / Aβ1-42 peptide; pRF4)]). CL2006 nematodes expressed human Aβ1-42 protein in muscle cells and exhibited significant protein accumulation and paralysis phenotype with age. The culture method for CL2006 *C. elegans* was the same as in Example 1.

[0020] (3) Detoxification test and locomotor ability test of CL2006 Caenorhabditis elegans L4-stage nematodes, after synchronization treatment, were placed in K medium containing 1.0 mg / L PS for 24 hours of liquid exposure. The experimental group received 200 μM Z27 solution simultaneously, while the control group received the same proportion of DMSO treatment. During exposure, the nematodes were shaken at 20°C and 100 rpm. Subsequently, the nematodes were transferred to fresh NGM plates, and phenotypic observations were performed every two days under constant temperature conditions of 20°C.

[0021] Motility was quantitatively assessed on days 2, 4, 6, and 8 of adulthood. Body bends per 30 seconds were manually counted and recorded under a stereomicroscope. A body bend was defined as a complete reciprocating oscillation of the midsection of the nematode relative to the axis of motion due to muscle contraction. Four biological replicates were set up, with at least 30 nematodes per group. By analyzing the rate of decrease in body bend counts at each time point, the study systematically evaluated whether Z27 (N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide) could inhibit PS-induced motility degradation. Culture plates were allowed to stand for 30 seconds before each count to eliminate mechanical stress interference. Results are shown below. Figure 3 .

[0022] Depend on Figure 3The results showed that the motility of nematodes was significantly reduced after PS treatment. However, after treatment with Z27, the motility of the nematodes was significantly restored, approaching that of the negative control group. This result indicates that Z27 (N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide) can alleviate the toxicity of PS and restore the motility of nematodes. Therefore, Z27 (N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide) shows promise for use in the preparation of antidotes for various micro / nanoplastics.

[0023] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. The application of a tetrahydroindazole derivative in the preparation of antimicro / nanoplastics toxicity drugs, characterized in that: The tetrahydroindazole derivative is specifically N-((tolyl-3-)phenoxy-2-)-N'-(4,5,6,7-tetrahydro-1H-indazole-5-)oxalamide, and its structural formula is shown in formula (I): 。 2. The application of the tetrahydroindazole derivative as described in claim 1 in the preparation of antimicro / nanoplastics toxicity drugs, characterized in that: The drug is a pharmaceutical composition consisting of the tetrahydroindazole derivative of claim 1 as the active ingredient and pharmaceutically acceptable excipients.

Images