CINNAMOYL AMINO ACID COMPOUND AND USE THEREOF

Abstract

Cinnamoyl amino acid compounds and a use thereof, which relate to the fields of medicinal chemistry and pharmacotherapeutics, and specifically relate to cinnamoyl amino acid compounds having inducible nitric oxide synthase (iNOS) inhibitory activity or a pharmaceutically acceptable salt thereof, a pharmaceutical composition including the compounds, and a medical use thereof, particularly an application thereof in the prevention and treatment of neurodegenerative diseases related to nerve cell damage, including ischemic stroke, Parkinson's disease, and so on.

Claims

1. A 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine, being a compound shown in structural formula I or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00003##

2. An application of the 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine according to claim 1 in preparation of a drug for prevention and treatment of neurodegenerative diseases related to nerve cell damage.

3. The application according to claim 2, wherein the drug is taken orally or injected.

4. The application according to claim 2, wherein the neurodegenerative diseases comprise ischemic stroke and Parkinson's disease.

5. A pharmaceutical composition, comprising a therapeutically effective amount of one or more of the 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanines according to claim 1 and/or a pharmaceutically acceptable salt thereof.

6. An application of the pharmaceutical composition according to claim 5 in preparation of a drug for prevention and treatment of neurodegenerative diseases related to nerve cell damage.

7. The application according to claim 6, wherein the neurodegenerative diseases comprise ischemic stroke and Parkinson's disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows an IC.sub.50 result of an iNOS inhibition rate of a compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application.

[0015] FIG. 2 shows anti-Parkinsonian effect results of the compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application, a compound 4-hydroxy-3-methoxycinnamoyl-L-tyrosine and a positive drug levodopa.

[0016] FIG. 3 shows effects of the compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application, the compound 4-hydroxy-3-methoxycinnamoyl-L-tyrosine and a positive drug butyphthalide on the size of cerebral ischemia-induced cerebral infarction.

DETAILED DESCRIPTION

[0017] This application will be further described below in conjunction with the specific examples.

[0018] For the preparation of 4-hydroxy-3-methoxycinnamoyl-L-tyrosine, reference may be made to: Hu, X. L., Lin, J., Lv, X. Y., Feng, J. H., Zhang, X. Q., Wang, H., Ye, W. C., 2018. Synthesis and biological evaluation of clovamide analogues as potent anti-neuroinflammatory agents in vitro and in vivo. European Journal of Medicinal Chemistry 151, 261-271.

EXAMPLE 1

Synthesis of Compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of the Present Disclosure

[0019] Experimental reagents: Thionyl chloride, methanol, ethanol, potassium carbonate and dipotassium phosphate purchased from Sinopharm Chemical Reagent Co., Ltd .; 4-(4,6-dimethoxy-triazin-2-yl)-4-methyl morpholinium chloride (DMTMM) purchased from Macklin Biological Company; and 4-fluoro-L-phenylalanine and 4-trifluoromethylcinnamic acid purchased from Adamas Reagent Company.

[0020] The synthetic route was as follows:

##STR00002##

[0021] Experimental process: The amino acid methyl ester hydrochloride was prepared from a thionyl chloride/methanol system first, and a condensing agent DMTMM, which is resistant to protic solvents, was selected, and amide condensation was completed in aqueous ethanol. Finally, the methyl ester of the amino acid part is removed by a methanol/water/potassium carbonate system to expose the carboxyl group to obtain the target compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine.

[0022] Physicochemical properties: White amorphous powder (yield 61%), easily soluble in methanol, acetone, ethyl acetate, soluble in dichloromethane, insoluble in petroleum ether, water, dark spots at UV 254 nm, melting point 236-238? C., [?].sup.20=11.2, ESI-MS m/z:[M-H].sup.? 380, [2M-H].sup.? 761, molecular formula: C.sub.19H.sub.15F.sub.4NO.sub.3

[0023] Spectral data: .sup.1H-NMR (300 MHz, DMSO-d.sub.6) ?.sub.H: 12.79 (1H, s), 8.51 (1H, d, J=8.1 Hz), 7.77 (4H, s), 7.47 (1H, d, J=15.9 Hz), 7.28 (2H, m), 6.84 (1H, d, J=15.9 Hz), 4.58 (1H, m), 3.14 (1H, dd, J=13.9 Hz, 4.9 Hz), 2.94 (1H, dd, J=13.9 Hz, 9.3 Hz) .sup.13C-NMR (75 MHz, DMSO-d.sub.6) ?.sub.C:175.78, 167.45, 165.72, 162.52, 141.92, 140.63, 136.69, 134.03, 133.92, 132.55, 131.25 (2C), 128.86 (2C), 127.50 (2C), 118.10, 117.82, 56.72, 39.05

EXAMPLE 2

In Vitro iNOS Inhibitory Effect of Compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of the Present Disclosure

[0024] Experimental materials: BV-2 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences), DMEM cell culture fluid (Sigma), fetal bovine serum (Gibico), 96-well cell culture plate (Corning), DMSO (Sigma), LPS (Sigma), iNOS activity detection kit (Beyotime), test drug 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine prepared in Example 1, and microplate reader.

[0025] Experimental method: The BV-2 cells were seeded in the 96-well plate at a density of 1?10.sup.4 cells/well, and a blank group, an LPS model group, and an LPS+compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine (0.01-10 ?M) group were set and cultured for 24 hours. Then according to the group, the drug or blank culture fluid was added to the corresponding group for culture for 4 hours. Finally, except the blank group, each group was stimulated with LPS with a final concentration of 500 ng/mL for 24 hours. The iNOS activity assay was performed according to the requirements of the iNOS detection kit.

[0026] Results and discussion: As shown in FIG. 1, the compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application (Compound 2 shown) has a very strong iNOS inhibitory effect, with an IC.sub.50 value of 110 nM, and is the compound with the strongest iNOS inhibitory activity among natural products or synthetic compounds by far.

EXAMPLE 3

In Vivo Anti-Parkinsonian Activity of Compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of the Present Disclosure

[0027] Experimental materials: 10-week-old C57BL/6 male mice (Nanjing Qinglongshan Farm), rota-rod treadmill (Beijing Zhongshi Technology), levodopa and MPTP (Sigma), test drug 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine prepared in Example 1 (Compound 2 shown), and test drug 4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown).

[0028] Experimental groups: Mice were randomly divided into 5 groups, 10 mice in each group: a blank group, a model group, a 4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown, 2 mg/kg) group, a 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine (Compound 2 shown, 2 mg/kg) group and a levodopa (15 mg/kg) group. Pre-dosing by gavage was carried out for 12 consecutive days. The blank group and the model group were given equal doses of PBS, and modeling was performed on the 13.sup.th day. The model group and the drug group were intraperitoneally injected with MPTP (20 mg/kg), once every 2 h, for 4 consecutive injections. After modeling, behavioral tests were carried out. The behavioral performance of mice was tested with a YLS-4C mouse rota-rod treadmill at 4 h, 24 h, 48 h, and 72 h after MPTP modeling. The time from the start of rotation of the rota-rod to the time leaving the rota-rod of the test mice was taken as the latency. The test time was 180 s, and each mouse was tested 3 times to obtain an average value. Training was carried out for 3 consecutive days before the test, twice a day.

[0029] Results and Discussion: FIG. 2 shows the Parkinsonian mouse rota-rod experiment. The compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application (Compound 2 shown) can effectively alleviate the dyskinesia of the Parkinsonian mice at a lower dose (2 mg/kg), and increase the running time of the Parkinsonian mice. The effect is obviously better than that of the compound 4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown) and the listed drug levodopa. The experimental result shows that the compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application (Compound 2 shown) can be used for preparing a drug for treatment and prevention of Parkinson's disease.

EXAMPLE 4

In Vivo Anti-Ischemic Brain Injury Activity of Compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of the Present Disclosure (Compound 2 Shown)

[0030] Experimental materials: Male SD rats, weighing 250-300 g, provided by the Model Animal Research Center of Nanjing University and raised in the Experimental Animal Center of China Pharmaceutical University; triphenyl tetrazolium chloride (TTC) and butyphthalide purchased from Sigma; test drug 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine prepared in Example 1 (Compound 2 shown), and test drug 4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown).

[0031] Experimental method: The rats were randomly divided into 5 groups, i.e., a blank group, a model group (MCAO group), an MCAO+4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown, 2 mg/kg) group, an MCAO+4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine (Compound 2 shown, 2 mg/kg) group and an MCAO+positive drug butyphthalide (5 mg/kg) group. After successful modeling, administration of different doses of compounds by gavage was carried out every day for 15 consecutive days. After the administration, brains were harvested, subjected to TTC staining, and photographed and recorded.

[0032] Experimental result: As shown in FIG. 3, the rat ischemia-reperfusion in-vivo model was used in combination with the TTC staining method to verify the effect of the test compounds on the size of cerebral infarction after 24 hours of ischemia reperfusion in rats. The experimental result shows that: 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine (Compound 2 shown) can effectively reduce the increase in the size of cerebral infarction caused by ischemia reperfusion at a very low dose (2 mg/kg). The effect is better than that of the positive drug and 4-hydroxy-3-methoxycinnamoyl-L-tyrosine (Compound 1 shown). On the one hand, this result confirms the in vivo effectiveness of the compound 4-trifluoromethyl-cinnamoyl-4-fluoro-L-phenylalanine of this application, and on the other hand, it can also indirectly verify the in vitro experimental result.