A derivative of memantine, its preparation method and application

Abstract

The present application provides a derivative of memantine, wherein at least one hydrogen atom on the amino group of memantine is replaced by a peptide chain containing 5-10 amino acids, and the peptide chain at least contains the sequence of Arg-Glu-Arg-Met-Ser. The derivative of memantine modified by the RERMS pentapeptide has lower cytotoxicity, higher neurotrophic activity, and can promote the expression of Bcl-2 protein, compared with memantine.

Description

TECHNICAL FIELD

[0001] The present application relates to the medical field, in particular to a derivative of memantine, a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairment and memory impairment. It is estimated that by 2030, the economic burden of AD in China will reach 254 million US dollars, and by 2050, it will reach 912 million US dollars. AD is progressive within 5-10 years of onset, and is also fatal, which is a heavy burden on society and family, and also brings great pain to patients.

[0003] The treatment method for the disease is to intervene in the progress of the disease by drugs to alleviate the symptoms of patients. The existing single target drug has certain side effects, and it is difficult to effectively control or cure the disease by using a single drug or intervening in a single target. Multi-target drugs can use biological structure information and pharmacophore models to obtain the required multiple biological activities, while removing the unwanted biological activities, thereby reducing neuronal damage, significantly delaying the progression of neuronal degenerative diseases and repairing damaged neurons. Therefore, developing new cognitive dysfunction drugs with high efficiency, low toxicity and small toxic side effects is one of the important topics of new drug research.

[0004] The inventors found that memantine hydrochloride, as a non-competitive N-methyl-D-aspartate receptor antagonist, is used for treating AD. In clinical application, taking an overdose of memantine hydrochloride tablets can produce side effects such as hallucinations, confusion, dizziness, headache and fatigue, and at the same time, it is not recommended for patients with moderate to severe renal function impairment to use the drug, which greatly limits its application range. SUMMARY

[0005] The inventors found that APP17 peptide has a neurotrophic effect, but due to its complex structure, it is not easy to modify the structure, but the arginine-glutamic acid-arginine-methionine-serine (RERMS) in the sequence is the key sequence to maintain this activity. APP5 peptide and its analogs have a neurotrophic protective effect and an effect of improving learning and memory in animal models of various learning and memory disorders. The inventors found through experimental research that the derivative of memantine obtained by modifying memantine through RERMS pentapeptide has reduced cytotoxicity and enhanced cell neurotrophic activity, thereby proposing the present application.

[0006] According to an aspect of the present application, a derivative of memantine is provided, in which at least one hydrogen atom on the amino group is replaced by a peptide chain containing 5-10 amino acids, and the peptide chain at least contains the sequence of Arg-Glu-Arg-Met-Ser.

[0007] In some embodiments, the derivative has the structure shown in Formula I:

[0008]

[0009] In some embodiments, the derivative is prepared by sequentially dehydrating and condensing memantine with Ser, Arg-Met dipeptide, Glu and Arg, or the amino-protected products of these amino acids or dipeptides. Such condensation sequence is the optimal scheme obtained by the inventors after multiple attempts, effectively reducing the occurrence of side reactions, saving synthesis steps, and maximizing the yield of the final product, memantine derivative.

[0010] In some embodiments, the present application provides a method for preparing the memantine derivative as follows:

[0011] (1) condensing Boc-Arg(NO2) with Met-OBzl in anhydrous tetrahydrofuran in the presence of dicyclohexyl carbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Met-OBzl;

[0012] (2) saponifying Boc-Arg(NO2)-Met-OBzl to Boc-Arg(NO2)-Met in the presence of NaOH in methanol;

[0013] (3) condensing Boc-Ser with memantine amine (MEM) in anhydrous N,N-dimethylformamide (DMF) solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Ser-MEM;

[0014] (4) removing Boc from Boc-Ser-MEM in hydrogen chloride-ethyl acetate solution to form Ser-MEM;

[0015] (5) condensing Boc-Arg(NO2)Met with Ser-MEM in anhydrous DMF solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Met-Ser-MEM;

[0016] (6) removing Boc from Boc-Arg(NO2)-Met-Ser-MEM in hydrogen chloride-ethyl acetate solution to form Arg(NO2)-Met-Ser-MEM;

[0017] (7) Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM is condensed with Boc-Arg(NO2) in anhydrous DMF solution in the presence of 2-(7-oxadiazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM; and

[0018] (8) Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM is deprotected to form Glu(OBzl)-Arg(NO2)-Met-Ser-MEM in a hydrogen chloride-ethyl acetate solution;

[0019] (9) Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM is condensed with Boc-Arg(NO2) in anhydrous DMF solution in the presence of 2-(7-oxadiazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM; and

[0020] (10) Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM is deprotected to form Arg-Glu-Arg-Met-Ser-MEM in a trifluoromethanesulfonic acid and trifluoroacetic acid solution.

[0021] According to another aspect of the present application, there is also provided a composition comprising a memantine derivative and a pharmaceutically acceptable carrier, for use in the treatment of a neurodegenerative disease. In some embodiments, at least one hydrogen atom on the amino group of the memantine derivative is replaced with a peptide chain containing 5-10 amino acids, the peptide chain comprising at least the sequence Arg-Glu-Arg-Met-Ser. In other embodiments, the memantine derivative has the structure shown in Formula I above.

[0022] According to another aspect of the present application, there is also provided the use of the memantine derivative described above in the manufacture of a medicament for the treatment of a neurodegenerative disease, wherein the treatment of the neurodegenerative disease comprises one or more of reducing neuronal damage, slowing the progression of neurodegenerative pathology, and repairing damaged neurons.

[0023] The memantine derivative provided by the present application has lower cytotoxicity than memantine, and higher neurotrophic activity than memantine.

[0024] In some embodiments, the administration of the memantine derivative causes one or more of the following:

[0025] In some embodiments, the administration of the memantine derivative causes one or more of the following:

[0026] reducing neural cell injury;

[0027] enhancing neuronal cell survival;

[0028] preventing extracellular stress-induced apoptosis and cell death; or

[0029] inhibiting neurodegeneration. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 shows the synthetic route of Arg-Glu-Arg-Met-Ser-MEM, wherein i) N-hydroxybenzotriazole (HOBt), N,N-dicyclohexylcarbodiimide (DCC), N-methylmorpholine (NMM); ii) aqueous NaOH (2 M), dilute hydrochloric acid; iii) 2-(7-oxabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), HOBt, NMM; iv) hydrogen chloride in ethyl acetate (4 M); v) triflic acid, trifluoroacetic acid;

[0031] Figure 2 shows the results of MTT cytotoxicity evaluation;

[0032] Figure 3 shows the results of CCK8 cytotoxicity evaluation;

[0033] Figure 4 shows the effect of MEM and RERMS-MEM on cell viability at a concentration of 10 μΜ in a cell viability experiment;

[0034] Figure 5 shows the effect of MEM and RERMS-MEM on cell viability at a concentration of 50 μΜ in a cell viability experiment;

[0035] Figure 6 shows the effect of RERMS-MEM on cell viability at concentrations of 0.01, 0.1 and 1 μΜ in a cell viability experiment (48 hours and 96 hours);

[0036] Figure 7 shows the results of LDH release experiment;

[0037] Figure 8 shows the effect of compound RERMS-MEM on Bcl-2 protein expression. DETAILED DESCRIPTION

[0038] The application will be further described in conjunction with specific examples. It should be noted that these representative examples are only used to explain and illustrate the technical solutions of the application, and the application is not limited to these specific examples. Any combination, change, modification of the examples according to the essential content of the application all belong to the scope of the application.

[0039] In order to make the application more easily understood, the definitions of some terms used in the application will be set forth in the description of the application.

[0040] For the sake of brevity, the application uses the abbreviation "MEM" to represent "memantine" or its main structure, and uses the common amino acid abbreviation name to represent the corresponding amino acid or its residue, for example, the compound abbreviation name "Arg-Glu-Arg-Met-Ser-MEM" or "RERMS-MEM" both represent a memantine derivative in which one hydrogen atom on the amino group of memantine is replaced by a pentapeptide of arginine-glutamic acid-arginine-methionine-serine, wherein the serine residue (Ser or S) is directly connected to the amino N atom of memantine, and according to the distance from the memantine parent, the amino acid sequence on the pentapeptide chain is serine, methionine, arginine, glutamic acid, and arginine in turn from near to far.

[0041] According to the synthesis route of RERMS-MEM shown in Figure 1 The preparation of RERMS-MEM is carried out according to the steps described in Examples 1-10.

[0042] Example 1 Preparation of Boc-Arg(NO2)-Met-OBzl

[0043] To a solution of 5 g (15.7 mmol) of Boc-Arg(NO2) in 50 mL of dry tetrahydrofuran, 2.5 g (18.5 mmol) of N-hydroxybenzotriazole (HOBt) and 3.3 g (18.5 mmol) of N,N-dicyclohexylcarbodiimide (DCC) in 30 mL of dry tetrahydrofuran were added successively with stirring in an ice bath. The resulting solution was stirred for 0.5 h. Then, 5.5 g (15.7 mmol) of Tos-Met-OBzl in 30 mL of dry tetrahydrofuran was added to the solution. The resulting reaction mixture was adjusted to pH 9 with N-methylmorpholine (NMM) and stirred at room temperature for 6 h. TLC (dichloromethane / methanol, 20 / 1) showed complete disappearance of Tos-Met-OBzl. Dicyclohexylurea (DCU) was removed by filtration. The filtrate was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The resulting solution was washed successively three times with saturated aqueous NaHCO3, three times with saturated aqueous NaCl, three times with 5% aqueous KHSO4, three times with saturated aqueous NaCl, three times with 5% aqueous NaHCO3, and three times with saturated aqueous NaCl. The collected ethyl acetate layer was dried over anhydrous Na2SO4for 12 h, filtered, and the filtrate was concentrated to dryness under reduced pressure. The resulting crude product was purified by column chromatography to give 6.2 g (74%) of the title compound as a colorless powder. ESI-MS (m / e): 541 [M+H] + .

[0044] Example 2 Preparation of Boc-Arg(NO2)-Met

[0045] To a solution of 5 g (9.2 mmol) of Boc-Arg(NO2)-Met-OBzl in 50 mL of methanol, aqueous NaOH (2 M) was added slowly dropwise with stirring in an ice bath for 6 h. TLC (dichloromethane / methanol, 40 / 1) showed complete disappearance of Boc-Arg(NO2)-Met-OBzl. The pH was adjusted to 7 slowly dropwise with stirring in an ice bath by adding saturated aqueous KHSO4. The methanol was removed by concentration under reduced pressure. To the remaining aqueous solution, dilute hydrochloric acid was added slowly dropwise with stirring in an ice bath to adjust the pH to 2. The resulting solution was extracted three times with 200 mL of ethyl acetate. The combined ethyl acetate layer was washed three times with 200 mL of saturated aqueous NaCl. The ethyl acetate layer was collected and dried over anhydrous Na2SO4for 12 h. It was filtered and the filtrate was concentrated under reduced pressure to give 4.08 g (98%) of the title compound. ESI-MS (m / e): 449 [M-H] - .

[0046] Example 3 Preparation of Boc-Ser-Met

[0047] To a solution of 4.1 g (20.0 mmol) of Boc-Ser in 50 mL of dry N,N- dimethylformamide (DMF), 3 g (22.2 mmol) of HOBt and 8.44 g (22.2 mmol) of 2-(7-oxazolylbenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in 30 mL of dry DMF were added successively under stirring in an ice bath. The resulting solution was stirred for 0.5 h. Then, 5.25 g (24.4 mmol) of memantine hydrochloride (HCl MEM) in 30 mL of dry DMF was added to the solution. The resulting reaction mixture was adjusted to pH 9 with NMM and stirred at room temperature for 6 h. TLC (dichloromethane / methanol, 30 / 1) showed complete disappearance of HCl MEM. The filtrate was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The resulting solution was washed successively three times with saturated aqueous NaHCO3, three times with saturated aqueous NaCl, three times with 5% aqueous KHSO4, three times with saturated aqueous NaCl, three times with 5% aqueous NaHCO3, and three times with saturated aqueous NaCl. The collected ethyl acetate layer was dried over anhydrous Na2SO4for 12 h, filtered, and the filtrate was concentrated to dryness under reduced pressure. This resulted in 5.71 g (78%) of the title compound. ESI-MS (m / e): 367 [M+H] + .

[0048] Example 4: Preparation of Ser-MEM

[0049] Boc-Ser-MEM 5.644 g (15.4 mmol) was weighed into a reaction flask and dissolved in 15 mL of dichloromethane. 2.5 mL of trifluoroacetic acid was added dropwise and the reaction was allowed to proceed for 1 h. The dichloromethane was removed by concentration under reduced pressure. The concentrated product was dissolved in ethyl acetate and washed three times with saturated aqueous NaHCO3and three times with saturated aqueous NaCl. The collected ethyl acetate layer was dried over anhydrous Na2SO4for 12 h, filtered, and the filtrate was concentrated to dryness under reduced pressure. This resulted in 3.137 g (76.5%) of the product as an oil. ESI-MS (m / e): 265 [M-H] - .

[0050] Example 5: Preparation of Boc-Arg(NO2)-Met-Ser-MEM

[0051] 4.16 g (9.2 mmol) of Boc-Arg(NO2)-Met was dissolved in 50 mL of anhydrous DMF. Under ice bath and stirring, 1.5 g (11.1 mmol) of HOBt and 4.2 g (11.1 mmol) of HATU in 30 mL of anhydrous DMF were added sequentially, and the mixture was stirred thoroughly for 0.5 h. Then, 2.46 g of Ser-MEM (9.2 mmol) in 30 mL of anhydrous DMF was added to the solution. The resulting reaction mixture was adjusted to pH 9 with N-methylmorpholine (NMM) and stirred at room temperature for 6 h. The TLC (dichloromethane / methanol, 20 / 1) Tos·Met-OBzl completely disappeared. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was washed three times with saturated NaHCO3 aqueous solution, three times with saturated NaCl aqueous solution, three times with 5% KHSO4 aqueous solution, three times with saturated NaCl aqueous solution, three times with 5% NaHCO3 aqueous solution, and three times with saturated NaCl aqueous solution. The collected ethyl acetate layer was dried over anhydrous Na₂SO₄ for 12 h, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography to give 2.34 g (36.7%) of the title compound as a colorless powder. ESI-MS (m / e): 697 [MH] - .

[0052] Example 6: Preparation of Arg(NO2)-Met-Ser-MEM

[0053] 2.063 g (2.95 mmol) of Boc-Arg(NO2)-Met-Ser-MEM was weighed into a reaction flask and dissolved in 5 mL of anhydrous ethyl acetate. 30 mL of a 4 M solution of hydrogen chloride in ethyl acetate was added to the solution under ice bath conditions with stirring, and the mixture was stirred for 2 h. TLC (dichloromethane / methanol, 15 / 1) showed complete disappearance of Boc-Arg(NO2)-Met-Ser-MEM. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was diluted with 30 mL of anhydrous ethyl acetate and then concentrated to dryness under reduced pressure. This operation was repeated three times. The resulting residue was diluted with 30 mL of anhydrous diethyl ether and then concentrated to dryness under reduced pressure. This operation was repeated three times to give the title compound. ESI-MS (m / e): 599 [M+H] + .

[0054] Example 7 Preparation of Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM

[0055] Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM 0.516 g (0.56 mmol) was weighed into a reaction flask and dissolved in 3 mL of dry ethyl acetate. To this solution was added 20 mL of hydrogen chloride in ethyl acetate (4 M) with stirring in an ice bath. The reaction was stirred for 2 h. TLC (methylene chloride / methanol, 15 / 1) showed complete disappearance of Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM. The reaction mixture was concentrated to dryness under reduced pressure and the residue was diluted with 20 mL of dry ethyl acetate and concentrated to dryness under reduced pressure. This procedure was repeated three times. The resulting residue was diluted with 20 mL of dry diethyl ether and concentrated to dryness under reduced pressure. This procedure was repeated three times to give the title compound. ESI-MS (m / e): 818 [M+H] - .

[0056] Example 8 Preparation of Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM

[0057] Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM 0.516 g (0.56 mmol) was weighed into a reaction flask and dissolved in 3 mL of dry ethyl acetate. To this solution was added 20 mL of hydrogen chloride in ethyl acetate (4 M) with stirring in an ice bath. The reaction was stirred for 2 h. TLC (methylene chloride / methanol, 15 / 1) showed complete disappearance of Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM. The reaction mixture was concentrated to dryness under reduced pressure and the residue was diluted with 20 mL of dry ethyl acetate and concentrated to dryness under reduced pressure. This procedure was repeated three times. The resulting residue was diluted with 20 mL of dry diethyl ether and concentrated to dryness under reduced pressure. This procedure was repeated three times to give the title compound. ESI-MS (m / e): 818 [M+H] + .

[0058] Example 9 Preparation of Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM

[0059] To a solution of 0.193 g (0.605 mmol) of Boc-Arg(NO2) in 30 mL of dry DMF, 0.125 g (0.93 mmol) of HOBt and 0.36 g (0.93 mmol) of HATU in 30 mL of dry DMF were added successively under stirring in an ice bath. The resulting solution was stirred for 0.5 h. Then, 0.43 g (0.58 mmol) of Glu(OBzl)-Arg(NO2)-Met-Ser-MEM in 10 mL of dry DMF was added to the solution. The resulting reaction mixture was adjusted to pH 9 with NMM and stirred at room temperature for 6 h. TLC (dichloromethane / methanol, 15 / 1) showed complete disappearance of Arg(NO2)-Met-Ser-MEM. The filtrate was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The resulting solution was washed successively three times with saturated aqueous NaHCO3solution and three times with saturated aqueous NaCl solution. The collected ethyl acetate layer was dried over anhydrous Na2SO4for 12 h, filtered and the filtrate was concentrated to dryness under reduced pressure. The resulting crude product was purified by column chromatography to give 2.34 g (36.7%) of the title compound as a colorless powder. ESI-MS (m / e): 1117 [M-H] - . 1 H NMR (DMSO-d6, 300 MHz) δ / ppm = 8.596 (m, 2H), 8.287 (m, 1H), 8.159 (m, 1H), 7.970 (m, 2H), 7.821 (m, 1H), 7.558 (m, 1H), 7.366 (m, 5H), 5.090 (m, 2H), 4.415 (m, 2H), 4.209 (m, 3H), 3.896 (m, 1H), 3.792 (m, 1H), 3.164 (m, 4H), 2.812 (m, 2H), 2.646 (m, 2H), 2.170 (m, 2H), 2.049 (m, 2H), 1.990 (m, 2H), 1.896 (m, 3H), 1.722 (m, 4H), 1.546 (m, 9H), 1.377 (m, 9H), 1.283 (m, 2H), 1.235 (m, 4H), 1.080 (m, 2H), 0.794 (m, 6H).

[0060] Example 10 Preparation of Arg-Glu-Arg-Met-Ser-MEM

[0061] Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM 0.3 g (0.26 mmol) was weighed into a reaction flask and dissolved in 3 mL trifluoroacetic acid with stirring for 3 min in an ice bath. 1 mL trifluoromethanesulfonic acid was added dropwise. The mixture was stirred for 30 min. The reaction was diluted with a large volume of ether and concentrated to dryness under reduced pressure. This procedure was repeated six times. After purification by Pre-HPLC, 50 mg (22.2%) of the title compound was obtained. ESI-MS (m / e): 835 [M-H] - . 1 H NMR (DMSO-d6, 300 MHz) δ / ppm = 8.876 (m, 2H), 8.664 (m, 2H), 8.441 (m, 1H), 8.205 (m, 1H), 7.917 (m, 2H), 7.567 (m, 1H), 4.439 (m, 4H), 3.788 (m, 2H), 3.623 (m, 1H), 3.160 (m, 4H), 2.524 (m, 2H), 2.190 (m, 2H), 2.048 (m, 7H), 1.766 (m, 4H), 1.536 (m, 9H), 1.305 (m, 2H), 1.234 (m, 4H), 1.080 (m, 2H), 0.804 (m, 6H).

[0062] Example 1 Evaluation of the cytotoxic effect of the compound Arg-Glu-Arg-Met-Ser-MEM

[0063] 1) MTT method for cytotoxicity evaluation

[0064] SH-Sy5y (human neuroblastoma cell parent cell) cells in good growth condition and in logarithmic growth phase were inoculated in 96-well plates at a density of 3 x 10 4 4 h in a 37 °C, 5% CO2 incubator, and the test samples after sterilization were added at a preset concentration gradient, and the control group was added with the same volume of solvent for dissolving the samples. After 48 h of continuous culture, 25 μL of MTT solution with a concentration of 5 mg / mL was added to each well, and the incubator was incubated for four hours. After the supernatant was carefully removed, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the purple precipitate (formazan) was dissolved by shaking for about 15 min. The O.D. (optical density) value was detected on the microplate reader at a wavelength of 570 nm.

[0065] The results are shown in Table 1 and Figure 2As shown in Table 2 and Figure 2, with the increase of the concentration of the memantine group, the OD value gradually decreased, and the cell number decreased. When the dose was 1 μM, the cell number was not different from the blank group. With the increase of the concentration, the OD value was significantly reduced at the concentrations of 10 μM and 50 μM, indicating that it had a certain cytotoxic effect. The OD value of the compound RERMS-MEM at three different concentrations was equivalent to that of the blank group, indicating that it had no cytotoxic effect. It can be seen that the memantine derivative modified by RERMS pentapeptide has a reduced cytotoxic effect compared with the memantine parent compound (especially at a concentration of 50 μM).

[0066] Table 1 Effect of compounds at different concentrations on cell proliferation (OD value, )

[0067]

[0068] 2) CCK8 method for cytotoxicity evaluation

[0069] Respectively, the SH-Sy5y (human neuroblastoma cell parent cell) cells in good growth state and in logarithmic growth phase were inoculated in 96-well plates at a density of 3 x 10 4 individuals / mL, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 4 h. The preset concentration gradient was added to the tested and sterilized samples, and the same volume of solvent for dissolving the sample was added to the control group. After 48 h of continuous culture, 10 μL of CCK-8 detection reagent was added to each well, and the incubator was incubated for four hours. The O.D. (absorbance) value was detected on the microplate reader at a wavelength of 450 nm.

[0070] As shown in Table 2 and Figure 3 With the increase of the concentration of the memantine group, the OD value gradually decreased, and the cell number decreased. When the dose was 1 μM, the cell number was more than that of the blank group. With the increase of the concentration, the OD value at the dose of 10 μM was equivalent to that of the blank group. When the dose was 50 μM, the OD value was significantly less than that of the blank group, indicating that at this dose, memantine could inhibit cell growth and had a certain cytotoxic effect. The OD value of the compound RERMS-MEM at three different concentrations was greater than that of the blank group, indicating that it not only had no cytotoxic effect, but also had a certain cell nutrition effect and promoted cell proliferation.

[0071] Table 2 Effect of compounds at different concentrations on cell proliferation (OD value, )

[0072]

[0073] Experimental Example 2 Evaluation of the cell neurotrophic effect of the compound Arg-Glu-Arg-Met-Ser-MEM: cell viability experiment and LDH (lactate dehydrogenase) release experiment

[0074] 1) Cell viability experiment

[0075] The Muse Count & Viability Kit reagent of the MUSE all-in-one cell quality control instrument was used for cell viability detection. The reagent contains a cell nucleus dye and a cell viability dye. The cell nucleus dye has a membrane permeable dye that distinguishes between cells and cell debris. The cell viability dye is a non-permeable dye that enters dead cells with damaged cell membranes or cells in the apoptosis state to bind to nuclear DNA and develop color. Through the combination of the two dyes, live cells, apoptotic cells, dead cells, and cell debris can be distinguished. The obtained live cell data is more accurate.

[0076] The SH-Sy5y (human neuroblastoma cell mother cell) cells in good growth state and in logarithmic growth phase were respectively inoculated in a 24-well plate at a density of 3x10 4 μL in a 37℃, 5% CO2 incubator for 4h, and the test, sterilized sample was added at a preset concentration gradient, and the control group was added with the same volume of solvent for dissolving the sample. After 48h / 96h of continuous culture, the cells were prepared into a single cell suspension. 450μL of Muse Count & Viability Kit reagent was aspirated, 50μL of cell liquid was added, and after 5min of incubation at room temperature in the dark, the machine was detected. The results are shown in Table 3 and Figures 4-6 As shown in the table, after 48h of drug intervention, the number of live cells in each group increased, and the number of live cells of the compound RERMS-MEM increased with the increase of the concentration when the concentration was 0.01μM, 0.1μM and 1μM, and when the concentration was 10μM and 50μM, the number of live cells no longer increased and decreased, and remained stable, and the number of live cells in each group was greater than that in the blank control group; the number of live cells of the compound MEM was less than that in the blank group when the concentration was 10μM and 50μM, and the greater the concentration, the fewer the number of live cells. After 96h of drug intervention, the number of cells in each group increased, and the number of live cells of the compound RERMS-MEM increased with the increase of the concentration when the concentration was 0.01μM, 0.1μM and 1μM, and when the concentration was 10μM and 50μM, the number of live cells no longer increased and decreased, and remained stable, and the number of live cells in each group was greater than that in the blank control group; the number of live cells of the compound MEM was less than that in the blank group when the concentration was 10μM and 50μM, and the greater the concentration, the fewer the number of live cells.

[0077] The above results show that, compared with memantine, the RERMS pentapeptide modified memantine derivative has increased neurotrophic activity, and can significantly increase the cell metabolic rate at concentrations of 1, 10 and 50 μM; and can significantly increase the number of living cells at concentrations of 0.01, 0.1, 1, 10 and 50 μM. The increased neurotrophic activity plays an important role in improving cognitive dysfunction.

[0078] Table 3 Change in the number of living cells after drug intervention

[0079]

[0080] 2) LDH release experiment

[0081] Lactate dehydrogenase (LDH) is a glycolytic enzyme present in all tissues of the body. The destruction of the cell membrane structure caused by apoptosis or necrosis will lead to the release of enzymes in the cytoplasm into the culture medium, including LDH which has relatively stable enzyme activity. By detecting the activity of LDH released from the cells with ruptured plasma membranes into the culture medium, the damage to the cells can be reflected. The lactate dehydrogenase detection kit is used, and NAD+ and strong color formazan generated by catalysis are generated, and an absorption peak is generated at a wavelength of 490 nm, so as to determine the lactate dehydrogenase activity. The absorbance is linearly and positively correlated with the lactate dehydrogenase activity. The greater the absorbance, the stronger the lactate dehydrogenase activity, and the greater the degree of cell damage.

[0082] The SH-Sy5y (human neuroblastoma cell mother cell) cells in a good growth state and in a logarithmic growth phase were respectively inoculated in a 96-well plate at a density of 3×10 4 The cells were cultured in a 37℃, 5% CO2 incubator for 4 h, and the pre-set concentration gradient of the tested and sterilized sample was added, and the same volume of the solvent for dissolving the sample was added to the control group. After 24 h of continuous culture, the LDH activity was detected. First, the LDH detection working solution was prepared according to the reagent instructions, then 60 μL of the LDH detection working solution was added to each well, mixed, incubated at room temperature for 30 min, and the absorbance was measured at 490 nm. The results are shown in Table 4 and Figure 7 As shown in Table 4 and FIG. 2, the absorbance of the compound RERMS-MEM at different concentrations is less than that of the blank control group. When the concentration is 0.01 μM, 0.1 μM, 1 μM, the absorbance gradually decreases with the increase of the drug concentration, and the cell nutrition gradually increases. When the concentration is 10 μM and 50 μM, the absorbance increases, but is still less than that of the blank control group, indicating that it still has a certain cell nutrition effect, but compared with the concentration of 1 μM, the effect is no longer enhanced.

[0083] The above results show that the memantine derivative provided by the application can inhibit the activity of LDH and reduce cell damage.

[0084] Table 4 Effect of compound RERMS-MEM on cell LDH release

[0085]

[0086] Experimental Example 3 Evaluation of the effect of compound RERMS-MEM on Bcl-2

[0087] Bcl-2 is a mitochondrial membrane protein mainly distributed in the cytoplasm and plays an important role in cell signal transduction. Bcl-2 protein can enhance neuronal cell survival, prevent apoptosis and cell death caused by extracellular stress, and is involved in inhibiting neurodegeneration, thus being a protein with an inhibitory effect on apoptosis.

[0088] Western-blot is a method for obtaining information on the expression of a specific protein in the analyzed cells or tissues by coloring the cell or biological tissue sample subjected to gel electrophoresis with a specific antibody and analyzing the coloring position and coloring depth, and is an immunological assay method that can be used to analyze sample components, and is widely used in the fields of molecular biology, biochemistry and immunogenetics. The experimental method is as follows:

[0089] 1. Total protein extraction in cells

[0090] Respectively, the SH-Sy5y (human neuroblastoma cell mother cell) cells in good growth state and in logarithmic growth phase were inoculated in a 6-well plate at a density of 3x10 4 μL per well in a 37℃, 5% CO2 incubator for 4h, and the test and sterilized samples were added at a preset concentration gradient, and the control group was added with the same volume of solvent for dissolving the sample. After 24h of continuous culture, the cells were prepared into a single cell suspension. After centrifugation, the cell culture solution was discarded, PBS was added to wash the cells gently, and the washing was repeated for 3 times. After the PBS was discarded, 0.5ml RIPA tissue lysis solution (with protease inhibitor PMSF) was added, and the protein lysis was carried out on a shaker at 0℃ for 15min. Low-temperature centrifuge, 4℃, 12000r / min centrifugation for 10min. The supernatant was transferred to a 0.5ml centrifuge tube, and was stored at -80℃ for standby. Because repeated freezing and thawing can cause protein degradation, the extracted protein can be stored for easy use after being divided into portions.

[0091] 2. Protein concentration detection

[0092] According to the operation instruction of BCA protein kit, the protein concentration in the cells was determined.

[0093] (1) According to the sample quantity, 50 volumes of BCA reagent A were added with 1 volume of reagent B (50:1) to prepare an appropriate amount of BCA working solution (stable within 24h at room temperature, and it is recommended to prepare and use it now), and it was mixed thoroughly.

[0094] (2) Fully dissolved protein standard, take 10 μl diluted to 100 μl, the final concentration is 0.5 mg / m1.

[0095] (3) The standard is added to the standard well of the 96-well plate according to 0, 1, 2, 4, 8, 12, 16, 20 μL, and an appropriate amount of PBS buffer is added to make up to 20 μl.

[0096] (4) Add 1 μl sample to the sample well of the 96-well plate, and add 19 μl PBS buffer to make up to 20 μl.

[0097] (5) Add 200 μl BCA working solution to each well, and place at 37℃ for 30 min. Different concentrations of light blue to blue-violet color can be seen in each well.

[0098] (6) Detect the absorbance value at 562 nm with a microplate reader. According to the relationship between the absorbance value of the standard and the concentration, a standard curve is drawn, and the regression equation of the standard curve is calculated. The absorbance value of each sample is substituted into this equation to calculate the protein concentration in the cell.

[0099] According to the protein concentration of each sample, an appropriate amount (to ensure that the concentration of each sample after denaturation is consistent) is added to 5x SDS-PAGE protein loading buffer at a ratio of 1:4, and boiled in water at 100℃ for 10 min, and stored in a refrigerator at -20℃ for standby.

[0100] 3、Western-blot

[0101] (1) Electrophoresis

[0102] Check the assembly of the gel preparation device, two glass plates (one thin and one thick), sample comb and gel preparation frame. Wash the glass plates with double distilled water and dry them before fixing them on the gel preparation frame. Test the tightness of the seal and make sure there is no leakage. Prepare 10% separation gel according to the molecular weight of the protein to be separated. Slowly pour the separation gel solution between the two fixed glass plates. Add slowly from the edge of the gel plate to avoid air bubbles. When the gel surface rises to about 2 / 3 of the height of the glass plate, change to distilled water to maintain the flatness of the gel surface. Add water slowly to prevent deformation of the gel. Place at room temperature for 30-40 minutes (the higher the temperature, the shorter the gel time). When a refractive line appears between the water and the gel, it indicates that the separation gel has completely solidified. Pour off the distilled water on the gel surface and carefully absorb the remaining water with filter paper, taking care not to disturb the flatness of the gel surface. Prepare 5% concentrated gel. Fill the remaining space between the glass plates with concentrated gel solution along the edge of the gel plate. Quickly insert the sample comb vertically (used to form sample wells) with the lower edge of the comb teeth more than 1 cm above the separation gel, taking care to avoid air bubbles. Place at room temperature for 30-40 minutes (the higher the temperature, the shorter the gel time). After the concentrated gel has completely solidified, gently pull out the sample comb vertically by holding the two sides of the comb (if not used immediately, store the comb in the 4°C refrigerator and retrieve it before use). Carefully remove the glass plates from the gel preparation frame and fix them on the electrophoresis tank support (thin glass plate facing inwards and thick glass plate facing outwards). Place the glass plates in the electrophoresis tank and add Tris-glycine electrophoresis buffer to submerge the gel.

[0103] Take the denatured protein with consistent concentration out of the refrigerator and warm it to room temperature. Add it to the corresponding gel sample well. Add the protein Marker to the two sample wells. Do not suck in air bubbles when loading to avoid inaccurate loading. Cover the tank and connect the power supply to start electrophoresis. Set the concentrated gel to a constant voltage of 80V for electrophoresis. When the bromophenol blue of the Marker reaches the boundary between the concentrated gel and the separation gel, the Marker proteins will spread out and multiple colored bands will appear. At this time, increase the voltage to 120V and continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel. Turn off the power supply and the electrophoresis is complete.

[0104] (2) Transfer

[0105] After the glass plate is disassembled, the concentrated glue is discarded, and according to the marker position, the gel piece where the target protein and the internal reference protein are located is gently peeled off. The size of the gel piece is measured and placed in the transfer buffer for 15 min. Take a PVDF membrane with the same size as the gel piece, cut the mark, and soak it in methanol for 20-60 s for activation. Place the transfer clamp, PVDF membrane, sponge pad, and filter paper in the transfer buffer for soaking. Open the transfer clamp and place the sponge pad, three layers of filter paper, the above-mentioned balanced gel piece, PVDF membrane, three layers of filter paper, and sponge pad on it in order, ensuring that each layer is tightly attached and aligned. Pay attention to keep it moist with transfer buffer and use a glass rod to remove air bubbles between each layer, especially between the gel piece and the PVDF membrane. Close the transfer clamp. At this time, the white side (negative) to the black side (positive) of the transfer clamp is in the order of sponge pad, filter paper, PVDF membrane, gel piece, filter paper, and sponge pad.

[0106] Install the transfer clamp on the support of the electrophoresis tank, place it in the electrophoresis tank, and pour the pre-cooled transfer solution. Under ice-cold conditions, set the transfer voltage and time according to the molecular weight of the protein to be transferred in the gel piece (the larger the protein molecular weight, the slightly higher the voltage and the slightly longer the time). After the transfer is completed, remove and open the transfer clamp, place the gel piece in the Coomassie brilliant blue staining solution, and place the PVDF membrane in the TBST.

[0107] Coomassie brilliant blue staining solution is used to stain the gel piece, which is slowly shaken on a horizontal shaker for 1 h or more at room temperature. Then the gel piece is placed in the Coomassie brilliant blue decolorizing solution and decolorized at room temperature for 4 h or more. The decolorizing solution is replaced 3-4 times during this period until the blue background is basically removed, and the presence of blue bands is observed to evaluate the transfer effect. If there are blue bands, it means that the gel piece still contains protein components, which may be due to insufficient voltage or time set for the electrotransfer.

[0108] The PVDF membrane is washed in TBST for 3 times, each for 5 min, and then washed with methanol. To determine the transfer effect and consider whether to continue the next step of the experiment, check whether there is transferred protein on the membrane by staining the membrane with the Ponceau red staining solution and slowly shaking it on a horizontal shaker for 5 min. Then wash off the staining solution that has not been stained with water. If there are red bands on the membrane, it means that protein has been transferred to the membrane. Ponceau red staining of protein is reversible and can be removed by washing with water several times. After the Ponceau red is removed, the membrane can be subjected to subsequent operations.

[0109] (3) Blocking

[0110] After washing the membrane, immerse it in 5% skimmed milk for blocking, slowly shake it on a horizontal shaker, and incubate it at room temperature for 1 h. Then rinse it with TBST for 3 times, each for 5-10 min.

[0111] (4) Primary antibody incubation

[0112] The primary antibody was diluted with antibody diluent at a specific ratio, and the PVDF membrane was placed in the solution. After incubating on a shaker at room temperature for 1 hour, it was then incubated overnight at 4°C. The next day, the primary antibody was recovered, and the membrane was placed on a TBST and gently shaken on a horizontal shaker for 10 minutes each time. β-actin was selected as an internal reference.

[0113] (5) Secondary antibody incubation

[0114] Place the PVDF membrane in a secondary antibody solution diluted to a certain ratio corresponding to the primary antibody, place it on a horizontal shaker and shake slowly, incubate at room temperature for 1 hour, and then wash it 3 times with TBST for 15 minutes each time.

[0115] (6) Color Development / Exposure

[0116] Place the PVDF membrane in a pre-cooled gel imaging instrument dark chamber with the protein side facing up, and evenly drop ECL hypersensitive chemiluminescence solution (mix solutions A and B in the kit in equal volumes in a container and use immediately), avoiding the formation of air bubbles. Close the dark chamber and turn on the imaging system to expose and take pictures.

[0117] The results showed that after drug intervention in SHSY5Y cells, the content of Bcl-2 protein in the cells was measured, such as... Figure 8 As shown, when the concentrations of MEM and RERMS-MEM were both 10 μmol / L, statistical tests revealed that, compared with the blank control group, the expression of Bcl-2 was increased in both the MEM and RERMS-MEM groups. Furthermore, the expression of Bcl-2 protein in the RERMS-MEM group was higher than that in the MEM group, indicating that RERMS-MEM has superior activity compared to MEM in enhancing neuronal cell survival, preventing apoptosis and cell death caused by extracellular stress, and inhibiting neurodegeneration.

[0118] The present invention has been described in detail above with reference to preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are merely illustrative explanations of the invention and do not constitute any limitation on the scope of protection of the invention. Various improvements, equivalent substitutions, or modifications can be made to the technical content and embodiments of the present invention without departing from the spirit and scope of protection of the invention, and all such modifications fall within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A derivative of memantine, characterized in that, The derivative has a structure shown in formula I:

2. The derivative according to claim 1, characterized in that, The derivative is prepared by dehydrating and condensing memantine with Ser, Arg-Met dipeptide, Glu and Arg or the amino-terminally protected products of these amino acids or dipeptides in sequence.

3. The derivative according to claim 1 or 2, characterized in that, The derivative is prepared by the following method: (1) condensing Boc-Arg(NO2) with Met-OBzl in anhydrous tetrahydrofuran in the presence of dicyclohexyl carbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Met-OBzl; (2) saponifying Boc-Arg(NO2)-Met-OBzl to Boc-Arg(NO2)-Met in the presence of NaOH in methanol; (3) condensing Boc-Ser with memantine (MEM) in anhydrous N,N-dimethylformamide (DMF) solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Ser-MEM; (4) removing Boc from Boc-Ser-MEM in hydrogen chloride-ethyl acetate solution to form Ser-MEM; (5) condensing Boc-Arg(NO2)Met with Ser-MEM in anhydrous DMF solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Met-Ser-MEM; (6) removing Boc from Boc-Arg(NO2)-Met-Ser-MEM in hydrogen chloride-ethyl acetate solution to form Arg(NO2)-Met-Ser-MEM; (7) condensing Boc-Glu(OBzl) with Arg(NO2)-Met-Ser-MEM in anhydrous DMF solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM; (8) removing Boc from Boc-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM in hydrogen chloride-ethyl acetate solution to form Glu(OBzl)-Arg(NO2)-Met-Ser-MEM; (9) condensing Boc-Arg(NO2) with Glu(OBzl)-Arg(NO2)-Met-Ser-MEM in anhydrous DMF solution in the presence of 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-hydroxybenzotriazole (HOBt) to form Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM; and (10) Boc-Arg(NO2)-Glu(OBzl)-Arg(NO2)-Met-Ser-MEM is deprotected in trifluoromethanesulfonic acid and trifluoroacetic acid solution to give Arg-Glu-Arg-Met-Ser-MEM.

4. A composition comprising the memantine derivative according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier for use in the treatment of a neurodegenerative disease.

5. Use of the memantine derivative according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of a neurodegenerative disease, characterized in that, Treating a neurodegenerative disease comprises one or more of reducing neuronal damage, slowing the progression of neurodegenerative pathology, repairing damaged neurons.

6. Use according to claim 5, characterized in that, The memantine derivative has lower cytotoxicity than memantine.

7. Use according to claim 5, characterized in that, The memantine derivative has higher neurotrophic activity than memantine.

8. Use according to claim 5, characterized in that, The memantine derivative promotes expression of Bcl-2 protein.

9. Use according to claim 5, characterized in that, Administration of the memantine derivative results in one or more of: reducing neuronal damage; enhancing neuronal cell survival; preventing apoptosis and cell death caused by extracellular stress; or inhibiting neurodegeneration.

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