Use of n-phenylpiperazine derivatives for the preparation of medicaments for the treatment of parkinson's disease

Abstract

The application relates to the technical field of new uses of medicines, and particularly discloses application of an N-phenylpiperazine derivative in preparation of a medicine for treating Parkinson's disease, wherein the N-phenylpiperazine derivative is HS. + The N-phenylpiperazine derivative HS provided by the application can significantly improve the survival rate of SH-SY5Y cells induced by MPP + , shows a dose-dependent neuroprotective effect, can effectively improve the motor dysfunction of a model animal, increase the open field movement distance, prolong the time of staying on a rotating rod, and provides a new selection for developing an anti-Parkinson's disease treatment medicine with a neuroprotective effect.

Description

Technical Field

[0001] This invention relates to the field of novel pharmaceutical applications, specifically to the use of N-phenylpiperazine derivatives in the preparation of drugs for treating Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a chronic, progressive neurodegenerative disease, the second leading cause of neurodegeneration after Alzheimer's disease, and its global burden is rapidly increasing. Despite extensive research, there is currently a lack of disease-modifying therapies that can halt or reverse neuronal loss; clinical treatment is primarily symptomatic and fails to address the underlying cause. Dopamine D2-like receptors are a key hub in the development of motor symptoms in PD and the main target of existing treatments. However, most current drugs directly or indirectly stimulate dopamine D2-like receptors, thereby replacing the missing dopamine signal, but cannot halt or reverse the continued death of dopamine neurons. Against this backdrop, a series of N-phenylpiperazine derivatives have been synthesized previously. Although they have a protective effect against PC12 cell damage, the efficacy of these compounds in preventing and treating PD in animal models remains unclear, making them difficult to use for further drug development and application. Summary of the Invention

[0003] This invention provides the application of N-phenylpiperazine derivatives in the preparation of drugs for treating Parkinson's disease.

[0004] This invention provides the application of N-phenylpiperazine derivatives in the preparation of drugs for treating Parkinson's disease, wherein the N-phenylpiperazine derivative is HS, and its structural formula is: .

[0005] In this invention, the N-phenylpiperazine derivative HS can significantly improve MPP. + The induced SH-SY5Y cell survival rate showed a dose-dependent neuroprotective effect; in the MPTP-induced Parkinson's disease mouse model, HS could effectively improve the motor dysfunction of the model animals, increase the open field movement distance and prolong the rotarod dwell time, providing a new option for the development of anti-Parkinson's disease drugs with neuroprotective effects.

[0006] Furthermore, the drug is used to protect dopamine neurons, resist neuronal apoptosis, and / or improve motor dysfunction.

[0007] Furthermore, HS is an active ingredient in the drug.

[0008] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0009] Furthermore, the excipients include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0010] Furthermore, the dosage form of the drug is a solid dosage form or a solution dosage form. The solid dosage form includes granules, tablets, capsules, pills, and drop pills, and the solution dosage form includes oral liquid preparations, oral enemas, and injectable dosage forms.

[0011] Furthermore, the injectable drug is a solution composed of sesame oil and the HS, wherein the mass-to-volume ratio of the HS and sesame oil is 4-6 mg:1 mL.

[0012] The present invention also provides a drug for treating Parkinson's disease, the drug being a mixture of the HS and excipients, wherein the content of HS in the drug is 0.1 wt% to 99 wt%.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The N-phenylpiperazine derivative HS provided by this invention has a clear neuroprotective effect on a Parkinson's disease cell model. (In MPP) + In the induced SH-SY5Y cell Parkinson's disease model, HS significantly increased cell viability in a dose-dependent manner. Compared with the model group, low concentration of HS (1 μM) increased cell density, and the cell number in the high concentration of HS (10 μM) treatment group was even close to that of the normal control group, indicating that HS has significant potential to resist apoptosis and protect dopamine neurons.

[0014] The N-phenylpiperazine derivative HS provided by this invention has a significant effect on improving motor dysfunction in animal models of Parkinson's disease. In MPTP-induced Parkinson's disease mouse models, prophylactic administration of HS significantly improved the motor abilities of the model mice. Open field experiments showed that HS increased the total distance traveled by mice; rotarod experiments showed that HS prolonged the time mice spent on the rotating rod, improving their motor coordination and balance. These results demonstrate that HS has neuroprotective and motor function recovery effects in vivo. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0016] Figure 1 It is an HS structure.

[0017] Figure 2 Microscopic images and cell count statistics of each group of cells are presented in the figure; A represents microscopic images of each group of cells; B represents the cell count statistics of the DMSO group; C represents the MPP group.+ Cell count statistics for the group; D represents MPP. + Cell count in the +1 μM HS group; E represents MPP. + Cell count in the +10 μM HS group.

[0018] Figure 3 The figures show the results of a mouse behavioral assessment experiment; in the figure, A represents the experimental design; B represents the movement path of each group of mice; C represents the movement distance of each group of mice; and D represents the results of the mouse rotarod test. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0020] Example 1: Application of N-phenylpiperazine derivatives in the preparation of drugs for treating Parkinson's disease.

[0021] I. Experimental Methods 1. Establishment of PD cell model 1.1 Resuscitation of SH-SY5Y cells SH-SY5Y cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were removed from liquid nitrogen and quickly placed in a water bath preheated to 37°C for 2 min to lyse. In a clean bench irradiated with ultraviolet light for 30 min, the lysed cell suspension was transferred to a 5 mL EP tube and centrifuged at 1000 r / min for 5 min. The cell pellet was resuspended in 2 mL of complete culture medium and gently resuspended by repeated pipetting to disperse the cells. The cells were then transferred to a sterile culture flask, and complete culture medium was added to a final volume of 5 mL. The mixture was then stirred in a figure-eight motion on a horizontal surface to ensure even cell distribution. The flasks were then incubated at 37°C with 5% CO2. Cell adhesion was observed periodically, and the culture medium was changed after 24 h to facilitate better cell growth and maintain optimal cell condition.

[0022] 1.2 Cell passage Observe the density of SH-SY5Y cells under a microscope. When the cells reach 80% to 90% confluence with the bottom of the culture flask, passage them. Perform the procedure in a clean bench irradiated with ultraviolet light for 30 min. Discard the old culture medium, add 2 mL of 1×PBS solution to wash the cells twice gently. After discarding the PBS, add 400 μL of trypsin for digestion. Place the culture flask horizontally and let it stand for 2 min until the cells begin to detach. Gently shake the culture flask to remove all cells, then immediately add 1 mL of 10% FBS complete culture medium to stop the digestion. Gently pipette the bottom of the culture flask to resuspend all cells and transfer them to a 5 mL EP tube. Centrifuge at 1000 r / min for 5 min, discard the supernatant, add 2 mL of complete culture medium to resuspend, centrifuge again at 1000 r / min for 5 min, discard the supernatant, and wash with PBS to remove as much culture medium and trypsin as possible. Add 2 mL of complete culture medium and gently pipette to prepare a cell suspension. Subculture the cells into new culture flasks at a ratio of 1:2 to 1:4, with 5 mL of culture medium in each flask. Mix well in a figure-eight pattern and observe the number and state of cells under a microscope. Mark the flasks and place them in a cell culture incubator for culture (culture conditions: temperature 37℃, humidity 10%, CO2 concentration 5%).

[0023] 1.3 Cell cryopreservation SH-SY5Y cells in the logarithmic growth phase and in good condition were cryopreserved. A cell cryopreservation solution was prepared using complete culture medium, fetal bovine serum, and DMSO in a 7:2:1 ratio. Cells were digested with trypsin and collected in a clean bench according to the cell passage method. After centrifugation, a cell pellet was obtained, and 1 mL of the cell cryopreservation solution was added. The cells were gently pipetted to prepare an SH-SY5Y cell suspension, which was then transferred to specific cryopreservation tubes. The tubes were sealed with sealing film, and the cell line name and cryopreservation date were labeled on the tube or cap. The cryopreservation tubes were placed in a programmed cooling box, capped, and placed in a -80°C freezer, kept horizontal and avoiding contact with other materials. After freezing at -80°C for 6 hours, they were quickly transferred to liquid nitrogen for storage for subsequent experiments.

[0024] 1.4, MPP + preparation MPP + (Purchased from MCE, product number: HY-W008719) Prepare a 10 mM stock solution with ddH2O according to the instructions. Reserve 1 mL for use in a -20℃ freezer, and store the rest in a -80℃ freezer.

[0025] MPP was prepared with complete culture medium. + The storage fluid is configured as MPP +Concentration gradients: 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, with 1 mM being the final concentration used to treat SH-SY5Y cells for 24 h.

[0026] 1.5 Cell resuscitation and culture Thaw cryovials containing 1 mL of cell suspension rapidly by shaking in a 37°C water bath, add 5 mL of culture medium and mix well. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of culture medium and rehydrate, then add all cell suspensions to culture flasks and incubate overnight. Change the medium the next day and check cell growth and density. Take SHSY-5Y cells in logarithmic growth phase and in good condition, seed them into 6-well plates at 10,000 cells per well, with 1 mL of 10% FBS complete culture medium per well. After labeling, place the 6-well plates in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0027] 2. Select N-phenylpiperazine derivatives based on the dopamine D2-like receptor core structure. The specific steps are as follows: Weigh (E)-3-(4-(4-cyanobutoxy)phenyl)acrylic acid and (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine, and follow the same procedure as in Example 2.18 of patent CN108329282A to obtain a white solid, which is the phenylpiperazine derivative HS (HS structure as shown in Figure 108329282A). Figure 1 (As shown).

[0028] 3. Protective effect of HS on SH-SY5Y cell PD model After the cells adhered, the old culture medium was discarded, and the six-well plates were divided into DMSO group and MPP group. + Group (using 1mM MPP) + Induced SH-SY5Y cell PD model), MPP + +1 μM HS group, MPP + The cells in the +10 μM HS group were divided into crosses in each six-well plate. After culturing for 24 h at 37℃, 10% humidity and 5% CO2 concentration, the cells in each group were photographed at the marked fixed positions on the six-well plate under a microscope, and the cell count was counted.

[0029] 3. Establishment of PD mouse model The mice were SPF-grade 8-week-old male wild-type C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.). After purchase, the animals were housed in a laboratory environment for one week with free access to water and food, a 12-hour light / dark cycle, a temperature of 22℃, and a humidity of 58%. After one week of housing, the C57BL / 6J mice were intraperitoneally injected four times within 24 hours with MPTP (purchased from MCE, catalog number HY-15608) at a dose of 14 mg / kg to establish a PD mouse model.

[0030] 4. Neuroprotective effect of HS on PD mouse model HS was dissolved in sesame oil at a concentration of 5 mg / mL to obtain an HS solution. Experimental group, blank group, and control group were set up, and the specific grouping is shown below:

[0031] Control group (DMSO+SO): Wild-type C57BL / 6J mice were intraperitoneally injected with DMSO and sesame oil.

[0032] Model group (MPTP): PD model was established by intraperitoneal injection of MPTP at a concentration of 14 mg / kg four times within 24 h in wild-type C57BL / 6J mice.

[0033] Experimental group (MPTP+HS): Mice were intraperitoneally injected with HS solution at a concentration of 5 mg / kg 6 days before MPTP injection, and behavioral assessment began 8 days after MPTP was completed.

[0034] Positive control group (MPTP+Pram): Mice were intraperitoneally injected with a 1 mg / kg pramipexole solution (Pram, purchased from Yuan Ye Company, catalog number: 104632-26-0) 6 days before MPTP injection. Behavioral assessment was started 8 days after MPTP injection.

[0035] The operational steps for behavioral assessment experiments are as follows: Before the behavioral evaluation of the mice, they were acclimatized to the testing environment for two days. During this period, the mice were allowed into the testing device and underwent several trials to familiarize themselves with the rotating rod, reducing errors caused by excessive maladaptation to the experimental environment. During the acclimatization period, the rotation speed of the rod was generally slower than during the test, and the training time was 10 minutes, to help the mice gradually adapt to the movement of the rod.

[0036] 4.1 Open Field Activity Test Spontaneous activity, or spontaneous movement, is a commonly used indicator for detecting reduced movement after MPTP injury. Mice were placed in an open field (50 cm × 50 cm × 50 cm), and after 1 minute of acclimatization, 5 minutes of video was recorded. Software was used to analyze parameters such as total distance traveled and average speed. To eliminate olfactory cues, the surface of the field was wiped with 75% ethyl acetate wipes during the experiment.

[0037] 4.2 Rotating bar test The motor coordination ability of mice was assessed using a mouse rotarod apparatus (purchased from Shanghai Jiliang, specification JLBehv-RRTG). Mice were placed on a rotating rod with a diameter of 3 cm, and the rotation speed was adjusted to 30 r / min. Five mice were tested simultaneously at a time, one in each compartment. The time elapsed from the start of the rotarod's rotation to the moment the mouse fell off was recorded. This was repeated three times, with a 5-minute rest interval between each test. The duration of the rotarod before the mouse first fell off was also recorded.

[0038] II. Test Results 1. HS to MPP + Protective effect of induced SH-SY5Y cell PD model Cell culture results showed MPP + The cell density in the DMSO group was significantly reduced, and a large number of cells died (DMSO group: 115.8±6.6, n=5; MPP group: 115.8±6.6, n=5). + Group: 65.8±2.9, n=5; P<0.001) Figure 2 (A~C); while in the low-concentration HS treatment group, the cell density compared to MPP + The number of groups has increased (MPP) + Group: 65.8±2.9, n=5; MPP + +1 μM HS group: 90.6±7.5, n=5; P=0.0172) Figure 2 (A, C, D), and the high-dose HS group showed more significant effects (MPP). + Group: 65.8±2.9, n=5; MPP + +10 μM HS group: 124±4.9, n=5; P<0.001 Figure 2 (A, C, E). The results show that HS can increase MPP in a dose-dependent manner. + The cell survival rate of the group showed significant neuroprotective potential.

[0039] 2. Neuroprotective effect of HS on PD mouse model Figure 3 A represents the experimental design. Results of the open field activity test ( Figure 3 (B) showed that HS and Pram improved the motor abilities of MPTP-treated mice in the mine and rotundus tests and tended to increase spontaneous activity in the open field test. Motor abilities were compared by measuring the distance traveled by each group of mice within a fixed time period. The results (B) Figure 3The results (C) showed that MPTP treatment significantly reduced the total movement distance in mice, indicating that the PD model was successfully established and that dyskinesia was successfully induced in mice. Compared with the MPTP model group, the movement distance of mice in the HS-treated experimental group was significantly increased, indicating that HS can partially improve MPTP-induced dyskinesia. The movement distance of mice in the HS-treated positive control group recovered, and the effect was better than that of the Pram group, indicating that HS has a certain neuroprotective or functional improvement effect, but its efficacy needs further optimization. To evaluate the effect of HS on the coordination and balance ability of PD model mice, a rotundus test was conducted to detect their motor coordination ability: compared with the blank group, MPTP treatment significantly shortened the time mice spent on the rotundus, indicating impaired motor function; HS prophylactic administration significantly improved MPTP-induced dyskinesia, and the positive control drug Pram also significantly reversed the toxic effects of MPTP, increasing the time mice spent on the rotundus (C). Figure 3 (D).

[0040] The above results indicate that MPTP successfully established a PD mouse model and induced motor dysfunction in the mice. Both HS and Pram effectively improved MPTP-induced motor dysfunction and restored motor function in the mice.

[0041] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0042] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of N-phenylpiperazine derivatives in the preparation of drugs for treating Parkinson's disease, characterized in that: The N-phenylpiperazine derivative is HS, and its structural formula is: 。 2. The use of the N-phenylpiperazine derivative according to claim 1 in the preparation of drugs for treating Parkinson's disease, characterized in that, The drug is used to protect dopamine neurons, resist neuronal apoptosis, and / or improve motor dysfunction.

3. The use of the N-phenylpiperazine derivative according to claim 1 in the preparation of drugs for treating Parkinson's disease, characterized in that, HS is the active ingredient in the drug.

4. The use of the N-phenylpiperazine derivative according to claim 1 in the preparation of drugs for treating Parkinson's disease, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The use of the N-phenylpiperazine derivative according to claim 4 in the preparation of drugs for treating Parkinson's disease, characterized in that, The excipients include any one or more of the following: non-toxic fillers, stabilizers, diluents, and adjuvants.

6. The use of the N-phenylpiperazine derivative according to claim 1 in the preparation of a drug for treating Parkinson's disease, characterized in that, The drug is in the form of a solid dosage form or a solution dosage form. The solid dosage form includes granules, tablets, capsules, pills, and drop pills. The solution dosage form includes oral liquid preparations, oral enemas, and injectable dosage forms.

7. The use of the N-phenylpiperazine derivative according to claim 6 in the preparation of a drug for treating Parkinson's disease, characterized in that, The injectable drug is a solution composed of sesame oil and the HS, wherein the mass-volume ratio of the HS to the sesame oil is 4~6 mg:1 mL.

8. A drug for treating Parkinson's disease, characterized in that, The drug is a mixture of the compound HS described in claim 1 and excipients, wherein the content of HS in the drug is 0.1 wt% to 99 wt%.

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