TRIFLUOROACETYL HYDRAZIDE COMPOUNDS AND MEDICAL USES THEREOF

Abstract

The present invention relates to trifluoroacetyl hydrazide compounds and medical uses thereof. The compounds have a structure of the following formula:

##STR00001##

The compounds showed multifunctional mechanisms, including inhibition of glutamate excitotoxicity, activation of MEF2 transcriptional activity, clearance of free radicals, and promotion of nerve differentiation, and has a better protective effect on cells especially nerve cells. The compound can be used to prepare prophylactic or therapeutic medicaments with cytoprotective effects, for the prevention or treatment of diseases related to glutamate receptor activation, MEF2 disorders or excessive free radicals generation. The diseases include, for example, neurodegenerative diseases such as Alzheimer's disease, Parkinson and stroke, and the free radicals related diseases such as heart disease, myocardial ischemia, diabetes and other cardiovascular and cerebrovascular diseases.

Claims

1. A method of prevention or treatment of diseases, comprising administration of a therapeutically effectively amount of the compound of a trifluoroacetyl hydrazide compound of formula I: ##STR00004## wherein: R.sub.1, R.sub.2 and R.sub.3 are each independently hydrogen or alkyl group, but cannot be hydrogen at the same time; and X and Y are both N; wherein the diseases are related to glutamate excitotoxicity, MEF2 disorder, oxidative stress injury or excessive free radicals generation, or neurodegeneration.

2. The method of claim 1, wherein the alkyl group is a lower alkyl group.

3. The method of claim 1, wherein R.sub.1, R.sub.2 and R.sub.3 are methyl, and thus the compound has a structure of formula II: ##STR00005##

4. The method of claim 1, wherein the disease related to glutamate excitotoxicity is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular atrophic lateral sclerosis, myasthenia gravis, glaucoma, senile dementia, hyperthyroidism, hypertension, bronchial asthma, type IV hyperlipoproteinemia, and renal failure.

5. The method of claim 1, wherein the disease related to MEF2 disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, muscular atrophic lateral sclerosis, and myasthenia gravis.

6. The method of claim 1, wherein the disease related to oxidative stress injury or excessive free radicals generation is selected from the group consisting of stroke, brain trauma, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, hypoxic-ischemic brain injury, stroke, dementia, ischemic heart disease, vascular thrombosis, atherosclerosis, hyperlipidemia, emphysema, cataract, diabetes, acute pancreatitis, alcohol induced liver disease, kidney damage and cancer.

7. The method of claim 1, wherein the disease related to neurodegeneration is selected from the group consisting of cerebral ischemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, primary lateral sclerosis, and spinal muscular atrophy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1. Structure of compounds of TMP and T-006;

[0038] FIG. 2. Synthetic scheme of compound T-006;

[0039] FIG. 3. Protective effects of T-006 on IAA induced PC12 cell injury;

[0040] FIG. 4. Protective effects of T-006 on t-BHP induced PC12 cell injury;

[0041] FIG. 5. Protective effects of T-006 on IAA induced cortical neuronal injury;

[0042] FIG. 6. Protective effects of T-006 on t-BHP induced cortical neuronal injury;

[0043] FIG. 7. Protective effects of T-006 on glutamate induced cerebellar granule injury;

[0044] FIG. 8. Protective effects of T-006 on MPP.sup.+ induced cerebellar granule injury;

[0045] FIG. 9. T-006 can enhance MEF2 activity in PC12 cells.

[0046] FIGS. 10A and 10B. T-006 can significantly improve scopolamine-induced memory impairment in mice;

[0047] FIG. 11. T-006 can promote PC12 cells being differentiated into neurons with synapse growing and elongating;

[0048] FIGS. 12A to 12D. Scavenging effect of T-006 on free radicals;

[0049] FIGS. 13A to 13C. T-006 reduced the t-BHP-induced apoptosis of PC12 cells;

[0050] FIGS. 14A to 14D. T-006 reduced the t-BHP-induced increase of ROS and RNS in PC12 cells.

DETAILED DESCRIPTION OF EMBODIMENTS

[0051] Some specific embodiments or examples of the present invention will be described below. It will be understood that these specific embodiments or examples are only used for further explanation of the invention, rather than to limit the scope of the inventive subject matters as defined by the claims.

Example 1. Synthesis of Compound T-006 (FIG. 2)

[0052] In 10 mL of methanol was dissolved 2,4-dimethylphenylhydrazide hydrochloride (1.72 g, 10 mmol). To the solution, 3,5,6-trimethylpyrazine-2-carbaldehyde (1.50 g, 12 mmol) was added with an ice-bath under the protection of N.sub.2. After the mixture was stirred for 10 min, a lot of brick red precipitate showed and the reaction was then complete. The mixture was filtered through a suction funnel, and the brick red solid was transferred to a vacuum oven to be dried for using directly in the next reaction step of the reaction. The red brick intermediate (3.04 g, 10 mmol) was dissolved in 25 mL of methanol, and 1.68 ml of anhydrous triethylamine and 1.7 ml of trifluoroacetic anhydride were added respectively with an ice-bath under the protection of N.sub.2, and the reaction was run for 30 minutes. The reaction was monitored by TLC till completion. The resulting material was extracted with Ethyl acetate, dried over anhydrous Na2SO4, separated by a silica gel column (ethyl acetate:petroleum ether=1: 3) to get T-006 as a light yellow solid (3.25 g, 89.3%). ESI-MS: [M+H].sup.+ m/z 365.1. .sup.1H-NMR (CDCl.sub.3, 300 MHz) : 2.1 (s, 3H), 2.42 (s, 3H), 2.46 (s, 3H), 2.54 (s, 3H), 2.85 (s, 3H), 7.08 (d, j=7.05 Hz, 1H), 7.24 (d, J=7.21 Hz, 1H), 7.25 (s, 1H), 7.55 (s, 1H); Anal. (C.sub.18H.sub.19F.sub.3N.sub.4O) C, H, C; found C, 59.44%, H, 5.319%, N, 15.23%; requires: C, 59.33%, H, 5.26%, N, 15.38%.

Example 2. Protective Effects of T-006 on IAA-Induced PC12 Cell Injury. (FIG. 3)

[0053] PC12 cells were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 24 hours. Then 100 l of IAA (50 M) was added for induction for 2 hours. Two hours later, the medium was aspirated, and each of the compounds (T-006, TMP, Edaravone) was added and the cells were cultured for 24 hours. After 24 hours of incubation, MTT was added to each well. Four hours later, the absorbance was measured at 570 nm using a microplate reader. The results were shown in FIG. 3: T-006 had a significant protective effect on IAA-induced PC12 cell injury, and was much stronger than that of the parent compound TMP and Edaravone. Compared with the IAA group, **** P<0.0001.

Example 3. Protective Effect of T-006 on t-BHP-Induced PC12 Cell Injury (FIG. 4)

[0054] PC12 cells were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C., for 24 hours. Then each compound (T-006, TMP, Edaravone) was added in different concentration gradients to pre-incubated for 2 hours. Two hours later, the medium was aspirated, and 100 l t-BHP (100 M) was added to each well except those of the control group, then the cells were cultured in an incubator for 24 hours. Four hours later, MTT was added into each of the wells, and the absorbance was measured at 570 nm using a microplate reader. The results were shown in FIG. 4: T-006 had a significant protective effect on t-BHP-induced PC12 cell injury, and the effect is much stronger than that of the parent compound TMP or Edaravone. Compared with t-BHP group, **** P<0.0001.

Example 4. Protective Effects of T-006 on IAA-Induced Cortical Neuronal Injury (FIG. 5)

[0055] Cortical neurons of E16-18 day SD fetal rat were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 8 days. On day 8, each of the compounds (T-006, TMP, Edaravone) was added with different concentration gradients, and 50 l IAA (40 M) was added to each well except those of the control group. Then the neurons were set to incubator to cultivate for 24 hours, and then MTT was added to each well. Four hours later, the absorbance was measured at 570 nm using a microplate reader. The results were shown in FIG. 5: T-006 had a significant protective effect on IAA-induced cortical neuronal injury, and was much stronger than that of the parent compound TMP and Edaravone. Compared with the IAA group, **** P<0.0001.

Example 5. Protective Effects of T-006 on t-BHP-Induced Cortical Neuronal Injury (FIG. 6)

[0056] Cortical neurons of E16-18 days SD fetal rat were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 8 days. On day 8, each of the compounds (T-006, TMP, Edaravone) was added with different concentration gradients, and 50 l IAA(40 M) was added to each well except those of the control group. The neurons were set to incubator to cultivate for 24 hours, and then MTT was added to each well. Four hours later, the absorbance value was measured at 570 nm using a microplate reader. The results were shown in FIG. 6: T-006 had a significant protective effect on t-BHP-induced cortical neuronal injury, and was much stronger than that of the parent compound TMP and Edaravone. Compared with the t-BHP group, **** P<0.0001.

Example 6. Protective Effects of T-006 on t-BHP-Induced Cerebellar Granule Injury (FIG. 7)

[0057] Primary cerebellar granule cells, extracted from 7-8 day, 15-20 g SD fetal rat, were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 8 days. On day 8, 90 L of the compound (T-006) was added with different concentration gradients, then the cells were set to an incubator to cultivate for 2 hours. Then 10 L of glutamate (final concentration of 75 M) was added to each well except those of the control group. The cells were set to incubator to cultivate for 24 hours, and then MTT was added to each well. Four hours later, the absorbance value was measured at 570 nm using a microplate reader. The results were shown in FIG. 7: T-006 had a significant protective effect on glutamate-induced primary cerebellar granule injury, and showed a better concentration-dependent. Compared with the glutamate group, ** P<0.01.

Example 7. Protective Effects of T-006 on MPP.SUP.+.-Induced Cerebellar Granule Injury (FIG. 8)

[0058] Primary cerebellar granule cells, extracted from 7-8 days, 15-20 g SD fetal rat, were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 8 days. On day 8, 90 L of the compound (T-006) was added with different concentration gradients, then the cells were set to an incubator to cultivate for 2 hours. Then 10 L of MPP.sup.+ (final concentration of 35 M) was added to each well except those of the control group. The cells were set to incubator to cultivate for 24 hours, and then MTT was added to each well. Four hours later, the absorbance value was measured at 570 nm using a microplate reader. The results were shown in FIG. 8: T-006 had a significant protective effect on MPP.sup.+-induced Primary cerebellar granule injury, and showed a better concentration-dependent. Compared with the MPP.sup.+ group, ** P<0.01.

Example 8. T-006 Activates MEF2 Transcription Activity in PC12 Cells (FIG. 9)

[0059] The PC12 was transfected by cloning the MEF2 reporter gene pGreenFire 1TM-MEF2-EF1 lentivector. Stable transfected PC12 cells, as cultured in a conventional manner, were seeded on a 96-well plate with T-006 added in different concentrations, were incubated for 24 h. The activity was measured by the Luciferase reporter assay kit (Promega, USA). The results were shown in FIG. 9: T-006 significantly activates ME12 transcriptional activity in PC12 cells. Compared with the IAA group, ** P<0.01.

Example 9. T-006 can Significantly Improve Scopolamine-Induced Memory Impairment in Mice (FIGS. 10A and 10B)

[0060] In this example, Kunming mice were selected and divided into normal control group (Ctrl), Scopolamine group (Sco), Donepezil treatment group (Sco+3 mg/kg donepezil), 1 and 10 mg T-006 treatment group (Sco+1 mg/kg T-006 and Sco+10 mg/kg T-006). Intragastric administration was performed for two weeks, and after the final administration water maze test was conducted for 4 days. Thirty minutes before the test, abdominal injection of scopolamine (1 mg/kg) and model were performed. On day 5, space search test was conducted, and data were expressed as MeanSD (n=9 per group). The results were shown in FIG. 10A and FIG. 10B: Scopolamine-induced memory of mice was significantly impaired, the first incubation period of the platform was significantly prolonged, the number of times through the target platform decreased (compared with the normal control group, ## p<0.01); 1 and 10 mg/kg T-006 treatment group and 3 mg/kg donepezil treatment group significantly improved the memory impairment induced by scopolamine (compared with the scopolamine model group, ** p<0.01); and 10 mg/kg T-006 group was significantly better than 3 mg/kg donepezil group (.sup.$p<0.05).

Example 10. T-006 can Promote PC12 Cells being Differentiated into Neurons with Synapse Growing and Elongating (FIG. 11)

[0061] PC12 cells were seeded on 6-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 24 hours. Control group was unchanged; NGF control group: serum-free F-12K+50 ng/mL NGF; drug treatment group: serum-free F-12K with different concentrations of T-006. After 72 hours of incubation, picture was taken under an inverted microscope with 5 shoots at random for each hole. If the cell synapse length is more than 2 times the cell, it can be regarded as cell differentiation and synapse formation. The results were shown in FIG. 11: T-006 and positive control NGF could significantly promote differentiation of PC12 cells into neurons, synapse formation and growth, and showed a better concentration-dependent. Compared with the control group, ** P<0.01.

Example 11. Scavenging Effects of the Compound on OH.SUP.., O.SUB.2..SUP.., ONOO, and DPPH Free Radicals (FIGS. 12A-12D)

[0062] Hydroxyl radical (OH.sup.): o-Phenanthroline-metal ion-H.sub.2O.sub.2 were used to produce hydroxyl radicals via Fenton reaction (H.sub.2O.sub.2+Fe.sup.2+.fwdarw..OH+H.sub.2O+Fe.sup.3+), to promote the oxidation of o-phenanthroline-Fe.sup.2+ to o-phenanthroline-Fe.sup.3+, resulting in an aqueous solution disappearance at the maximum at the wavelength of 440 nm so that the clearance rate was calculated. The specific steps are: In a 48-well plate was added 300 L of double distilled water (blank control) or T-006, TMP, Edaravone in different concentrations (DMSO was used to prepare a 10 mM stock solution which was then diluted with double distilled water to 4 M, 20 M, 80 M, 320 M), and 50 L of 1.0 mM o-phenanthroline (1.0 mM dissolved in 50 mM NaCl solution) was added and then mixed with 125 L of 1.0 mM H.sub.2O.sub.2 and 125 L 2.0 mM Fe.sup.2+, respectively, and BioTek Synergy HT was used to measure the percentage of absorbance decreasing at 440 nm in 100 seconds. The hydroxyl radical scavenging rate was calculated as: clearance rate (%)=[1(A.sub.0A.sub.100)/A.sub.0]100%, A.sub.0 and A.sub.100 being the absorbance values at 0 seconds and 100 seconds, respectively.

[0063] Superoxide anionic radicals (O.sub.2.sup.): Pyrogallol autoxidation method was used, and the specific steps are: to a 48 well plate was added 250 L of 50 mM Tris-HCl buffer (pH 8.2), 300 L of double distilled water (control group) or T-006, TMP, Edaravone in different concentrations (DMSO was used to prepare a 10 mM stock solution which was then diluted with double distilled water to 4 M, 20M, 80 M, 320 M). Then 50 L of 2.0 mM of pyrogallol was added being mixed with a vortex mixer. At a wavelength of 320 nm, the absorbance of the blank control group was recorded every 30 seconds using a BioTek Synergy HT microplate reader for 30 minutes. The absorbance of the samples was measured under the same conditions, and the oxidation rate was the increment of absorbance per minute. By using linear regression method with time (seconds) as the abscissa and absorbance value as the ordinate, the linear relationship between absorbance value and time was obtained to calculate the pyrogallol autoxidation rate. The results are expressed in increments of dA/dt of the absorbance value per second, ie the a value in R.sup.2 in the linear regression equation of y=ax+b. Clearance rate (%)=(dA/dtdAs/dt)/(dA/dt), wherein dA/dt is the autoxidation rate of pyrogallol in the absence of sample, dAs/dt is the rate of pyrogallol in the presence of the sample.

[0064] Peroxynitrite free radicals (ONOO.sup.): The peroxynitrite free radical (ONOO) can be measured by SIN-1 simulation of the process of ONOO generation in the body. SIN-1 decomposes under weak alkaline conditions (PBS pH 7.4), which can then simultaneously produce superoxide anion radical (O.sub.2.sup.) and nitric oxide free radical (NO.sup.), and both of which instantaneously produce peroxynitrite Anion (ONOO.sup.). ONOO reacts with luminal by oxidization and excitation with emission at 425 nm. The luminous intensity can be measured to determine the generation of ONOO. Antioxidant (AH) competes with Luminol (L) in the reaction with ONOO, and thus reduces the reaction of ONOO with LH, and then reduces the luminous intensity. The luminous intensity is inversely correlated with the antioxidating capacity of the antioxidant, so that quantitative analysis can be made based on the change of the luminescence intensity before and after the reaction. The specific steps of the test are: In a 3 mL round bottom photometer tube were added 300 L 0.1 M PBS buffer solution (pH 7.4), 50 L 1 mM Luminol solution, 100 L PBS or T-006, TMP, Edaravone in different concentrations DMSO was used to prepare a 10 mM stock solution, which was then diluted into 4 M, 20 M, 80 M, 320 M). Then 50 L of 3 mg/mL SIN-1 hydrochloride solution was added and mixed with a vortex mixer. With the blank group as control, the photometric tube was placed with the temperature being controlled at 37 C., and the luminous value was recorded every 100 s with a luminometer continuously for 2000 s, and each measurement of the concentration is repeated at least three times and averaged. The clearance rate is calculated by the following formula: Clear rate rate (%)=(A.sub.ctrlA.sub.sample)/A.sub.ctrl100.

[0065] 1.1-Diphenyl-2-phenylhydrazide radicals (DPPH): The test of 1.1-Diphenyl-2-phenylhydrazide radicals (DPPH) spectrophotometry is based on that DPPH has a strong absorption at 517 nm, and has a deep violet color in methanol solution. When free radical scavenger is present, the absorption gradually disappears due to single electron pairing, and the extent of color fading is correlated quantitatively to the number of electrons received by the DPPH. Therefore, by detecting the change of optical absorbance of DPPH in with the samples in different concentrations, the clearance rate can be used as an indication of the free radical scavenging ability. The specific steps of the test are: Onto a 96 well plate was added 100 L of T-006, TMP, Edaravone (dissolved in DMSO to make a 10 mM stock solution and then diluted with double distilled water to 4 M, 20 M, 80 M, 320 M) in different concentrations or 100 L methanol (as blank control group), then quickly added with 100 L of 100 M DPPH solution in methanol (50 M of final concentration), each sample Concentration was repeated in 3-5 holes, vibrated evenly and then placed at room temperature under dark condition for one hour. The absorbance values were then measured at 517 nm using a microplate reader. Clearance rate (%)=(A.sub.ctrlA.sub.sample)/A.sub.ctrl100.

Example 12. T-006 Reduced t-BHP-Induced Apoptosis in PC12 Cells (FIGS. 13A-13C)

[0066] PC12 cells were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 24 hours, and then T-006 and TMP (100 M) in different concentration gradients were added to be pre-incubated for 2 hours. Then the medium was aspirated, and 100 l t-BHP (100 M) was added to each well except the wells of the control group, and the cells were cultured in an incubator for 24 hours. Hoechst working fluid was added and the number of PC12 cells was observed and counted under fluorescence microscope. (A) T-006 reduced t-BHP-induced PC12 cell apoptosis. (B) PC12 cell apoptosis statistics. (C) T-006 improved t-BHP-induced mitochondrial membrane potential disorder in PC12 cells. PC12 cells were seeded on a 96-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 24 hours, and then 7-006 and TMP (100 M) in different concentration gradients were added to be pre-incubated for 2 hours. 2 hours later, 100 l t-BHP (100 M) was added to each well except the control group for induction. After completion of the induction, JC-1 working solution was added to each well and mitochondrial membrane potential was monitored and analyzed using flow cytometry. Compared with the control group, ### p<0.001; compared with t-BHP, * p<0.05, ** p<0.01 and *** p<0.001.

Example 13. T-006 Reduced the Elevation of ROS and RNS in t-BHP-Induced PC12 Cells (FIGS. 14A-14D)

[0067] PC12 cells were seeded on 6-well plate and inoculated in a 5% CO.sub.2 incubator at 37 C. for 24 hours, and then T-006 and TMP (100 M) in different concentration gradients were added to be pre-incubated for 2 hours. Then the medium was aspirated, and 100 l t-BHP (100 M) was added to each well except the wells of the control group, and the cells were cultured in an incubator for 6 hours. Consequently, DCF-DA probes, HFP probes, DAF-FM probes and DHR123 probes were added respectively to determine the total ROS, hydroxyl radicals, nitric oxide radicals and peroxynitrite. (A) DCF-DA probe for determining intracellular total ROS. (B) HFP probe for determining intracellular hydroxyl radicals. (C) DAF-FM probe for determining nitric oxide free radicals. (D) DHR123 probe for determining intracellular peroxynitrite. Compared with the control group, ### p<0.001; compared with t-BHP, * p<0.05, ** p<0.01 and *** p<0.001.

[0068] While specific embodiments have been described in detail herein, this is only illustrative of the purpose of the invention and is not intended to limit the scope of the following claims. It should be understood that various substitutions, changes and modifications to the specific embodiments described herein are intended to be within the scope of the invention as defined by the appended claims without departing from the spirit and scope of the invention as defined by the appended claims.