阅读说明：本技术 一种芬乐胺21位代谢物及其制备和应用 (Fenleramine 21-site metabolite and preparation and application thereof ) 是由 贾振华 吴相君 杨青瑞 刘蕊 王宏涛 于 2020-06-05 设计创作，主要内容包括：本发明提供了一种芬乐胺21位代谢物及其制备和应用。本发明的芬乐胺21位代谢物具有良好的抗氧化活性、抗神经炎症及神经保护活性。所述芬乐胺21位代谢物如下式(I)所示：(The invention provides a phentermine 21-site metabolite and preparation and application thereof. The 21-bit metabolite of the fenle amine has good antioxidant activity, neuritis resistance and neuroprotective activity. The 21-position metabolite of the fenle amine is shown as the following formula (I):)

1. A phencyclamine 21-position metabolite, wherein the phencyclamine 21-position metabolite is represented by the following formula (I):

2. the method for producing a phentermine 21-position metabolite of claim 1, wherein the method comprises producing a phentermine 21-position metabolite represented by formula (I) using a compound of formula (7):

3. the method of claim 2, further comprising preparing a compound of formula (7) starting from a compound of formula (6) and a compound of formula (3):

4. the process of claim 3, further comprising preparing a compound of formula (6) starting from a compound of formula (5) and a compound of formula (5A):

5. the process of claim 4, further comprising preparing a compound of formula (5) starting from a compound of formula (4) and a compound of formula (4A):

6. the process according to claim 5, wherein the process further comprises reacting a compound of formula (2) with CCl₃CN as a raw material to prepare a compound of a formula (3):

7. the method of claim 6, wherein the step of preparing the composition is carried out in the presence of a catalystThe process further comprises reacting a compound of formula (1) with (NH)₂NH₂)₂COCH₃Preparing a compound of formula (2):

8. the method of manufacturing according to claim 2, wherein the method comprises the steps of:

9. the production method according to claim 8, wherein:

the solvent in the step 1 is selected from one or more of DMF, dichloromethane, acetonitrile, 1, 4-dioxane, DMSO and tetrahydrofuran;

the solvent in the step 2 is selected from one or more of dichloromethane, DMF, acetonitrile, 1, 4-dioxane, DMSO and tetrahydrofuran;

the solvent in the step 3 is acetic anhydride;

the solvents in the step 4 and the step 5 are respectively and independently selected from one or more of dichloromethane, DMF, acetonitrile, 1, 4-dioxane, DMSO and tetrahydrofuran;

the solvent of the step 6 is selected from methanol or one of the following mixed solutions: methanol/water, ethanol/water, acetonitrile/water, 1, 4-dioxane/water, DMF/water, tetrahydrofuran/water, and DMSO/water.

10. The production method according to claim 8 or 9, wherein:

step 2 is a compound of formula (2) and CCl in the presence of an organic base₃CN is used as a raw material to prepare a compound shown in a formula (3); preferably, the organic base is selected from one of DBU, triethylamine, diisopropylethylamine and N-methylimidazole;

step 3, preparing a compound shown in a formula (5) by taking a compound shown in a formula (4) and a compound shown in a formula (4A) as raw materials in the presence of a catalyst; preferably the catalyst is selected from triethylamine or diisopropylethylamine;

step 4, preparing a compound shown in the formula (6) by taking a compound shown in the formula (5) and a compound shown in the formula (5A) as raw materials in the presence of an organic base; preferably the organic base is selected from a mixture of one or more of triethylamine, diisopropylethylamine, N-methylimidazole and N-methylmorpholine;

step 5, in the presence of a catalyst, taking the compound of the formula (6) and the compound of the formula (3) as raw materials to prepare a compound of the formula (7); preferably the catalyst is selected from BF₃.Et₂O, TMSOTf and BF₃One of THF;

step 6 when the solvent is selected from one of methanol/water, ethanol/water, acetonitrile/water, 1, 4-dioxane/water, DMF/water, tetrahydrofuran/water and DMSO/water, step 6 is to prepare the compound of formula (I) by using the compound of formula (7) as a raw material in the presence of an alkaline substance; or, when the solvent is selected from methanol, the step 6 is to prepare the compound shown in the formula (I) by taking the compound shown in the formula (7) as a raw material in a methanol/sodium methoxide system or a sodium tert-butoxide/methanol system; preferably the alkaline substance is selected from a mixture of one or more of sodium carbonate, potassium carbonate and sodium hydroxide.

11. A pharmaceutical composition comprising the phentermine 21-position metabolite of claim 1, and one or more pharmaceutically acceptable carriers and/or excipients.

12. Use of the pheniramine 21-position metabolite of claim 1 for the preparation of anti-oxidant drugs, anti-neuritis drugs and neuroprotective drugs; preferably, the application is the application of the 21-bit metabolite of the phentermine in preparing the medicines for preventing and treating Parkinson's disease, improving learning and memory disorder and treating hypomnesis and Alzheimer's disease.

Technical Field

The invention relates to the field of medicines, in particular to a phentermine 21-site metabolite and preparation and application thereof.

Background

Fenamine is a derivative of annonaceous acetogenins, the structure of the compound is disclosed in Chinese patent CN1445211 (publication number), and the patent describes ' a novel annonaceous acetogenins derivative invented by the institute of medicine of Chinese academy of medical sciences ' and a preparation method, a pharmaceutical composition and application thereof '.

The molecular structural formula of the fenloramine (chemical name: trans-2- (2, 5-dimethoxyphenyl) -3- (4-hydroxy-3-methoxyphenyl) -N- (4-hydroxyphenylethyl) acrylamide) is as follows:

the pharmacodynamic action of the phentermine for treating the Parkinson Disease (PD) is similar to that of the known positive medicine levodopa, and the activity is stronger than that of the levodopa. The inventive phentermine has novel action mechanism, can resist nerve cell apoptosis, and has neuroprotective effect. However, the neuroprotective effect of the phentermine is not satisfactory, so that the development of a new phentermine substitute is a problem to be solved at present.

Disclosure of Invention

An object of the present invention is to provide a 21-position metabolite of phencyclamine;

another object of the invention is to provide a preparation method of the 21-position metabolite of the phentermine;

it is yet another object of the present invention to provide a pharmaceutical composition;

still another object of the present invention is to provide the use of the 21-position metabolite of phentermine.

To achieve the above objects, in one aspect, the present invention provides a 21-position metabolite of phentermine, wherein the 21-position metabolite of phentermine is represented by the following formula (I):

in another aspect, the invention also provides a preparation method of the phentermine 21-position metabolite, wherein the method comprises the step of preparing the phentermine 21-position metabolite shown in the formula (I) by using the compound shown in the formula (7) as a raw material:

according to some embodiments of the invention, the method further comprises preparing a compound of formula (7) starting from a compound of formula (6) and a compound of formula (3):

according to some embodiments of the invention, the method further comprises preparing a compound of formula (6) starting from a compound of formula (5) and a compound of formula (5A):

according to some embodiments of the invention, the method further comprises preparing a compound of formula (5) starting from a compound of formula (4) and a compound of formula (4A):

according to some embodiments of the invention, wherein the method further comprises administering to the subject a compound of formula (2) and CCl₃CN as a raw material to prepare a compound of a formula (3):

according to some embodiments of the invention, wherein the method further comprises administering a compound of formula (1) and (NH)₂NH₂)₂COCH₃Preparing a compound of formula (2):

according to some embodiments of the invention, the method comprises the steps of:

according to some embodiments of the invention, the solvent of step 1 is selected from one or more of DMF, dichloromethane, acetonitrile, 1, 4-dioxane, DMSO, and tetrahydrofuran.

According to some embodiments of the invention, the solvent of step 2 is selected from one or more of dichloromethane, DMF, acetonitrile, 1, 4-dioxane, DMSO, and tetrahydrofuran.

According to some embodiments of the invention, the solvent of step 3 is acetic anhydride.

According to some embodiments of the invention, the solvent of step 4 and step 5 is a mixture of one or more selected from dichloromethane, DMF, acetonitrile, 1, 4-dioxane, DMSO, and tetrahydrofuran.

According to some embodiments of the present invention, the solvent of step 6 is selected from methanol, or one of the following mixed solutions: methanol/water, ethanol/water, acetonitrile/water, 1, 4-dioxane/water, DMF/water, tetrahydrofuran/water, and DMSO/water.

According to some embodiments of the invention, step 2 is performed in an organic phaseIn the presence of a base, a compound of formula (2) and CCl₃CN is used as a raw material to prepare the compound shown in the formula (3).

According to some embodiments of the present invention, step 3 is a step of preparing the compound of formula (5) by using the compound of formula (4) and the compound of formula (4A) as raw materials in the presence of a catalyst.

According to some embodiments of the invention, step 4 is a step of preparing the compound of formula (6) by using the compound of formula (5) and the compound of formula (5A) as raw materials in the presence of an organic base.

According to some embodiments of the invention, step 5 is a step of preparing the compound of formula (7) by using the compound of formula (6) and the compound of formula (3) as raw materials in the presence of a catalyst.

According to some embodiments of the present invention, in step 6, when the solvent is selected from one of methanol/water, ethanol/water, acetonitrile/water, 1, 4-dioxane/water, DMF/water, tetrahydrofuran/water and DMSO/water, step 6 is to prepare the compound of formula (I) by using the compound of formula (7) as a raw material in the presence of a basic substance.

Or, when the solvent is selected from methanol, the compound of formula (7) is used as a raw material to prepare the compound of formula (I) in a methanol/sodium methoxide system or a sodium tert-butoxide/methanol system in the step 6.

According to some embodiments of the invention, the organic base of step 2 is selected from one of DBU (1, 8-diazabicycloundecen-7-ene), triethylamine, diisopropylethylamine, and N-methylimidazole.

According to some embodiments of the invention, wherein the catalyst of step 3 is selected from triethylamine or diisopropylethylamine.

According to some embodiments of the invention, the organic base of step 4 is selected from one or more of triethylamine, diisopropylethylamine, N-methylimidazole and N-methylmorpholine.

According to some embodiments of the invention, wherein the catalyst of step 5 is selected from BF₃.Et₂O, TMSOTf (trimethylsilyl trifluoromethanesulfonate) and BF₃One of THF.

According to some embodiments of the invention, the alkaline substance of step 6 is selected from a mixture of one or more of sodium carbonate, potassium carbonate and sodium hydroxide.

According to some embodiments of the invention, step 1 is carried out at a temperature of 10 ℃ to 30 ℃.

According to some embodiments of the invention, step 1 is carried out at room temperature.

According to some embodiments of the invention, step 2 is carried out at a temperature of 10 ℃ to 30 ℃.

According to some embodiments of the invention, step 2 is carried out at room temperature.

According to some embodiments of the invention, step 3 is carried out under reflux.

According to some embodiments of the invention, step 4 is carried out at 5 ℃ to 15 ℃.

According to some embodiments of the invention, step 5 is carried out at-30 ℃ to-10 ℃.

According to some embodiments of the invention, step 5 is carried out at-20 ℃.

According to some embodiments of the invention, step 6 is carried out at a temperature of 10 ℃ to 30 ℃.

According to some embodiments of the invention, step 6 is carried out at room temperature.

In yet another aspect, the present invention also provides a pharmaceutical composition comprising the phencyclamine 21-position metabolite of the present invention, and one or more pharmaceutically acceptable carriers and/or excipients.

In another aspect, the invention also provides application of the 21-bit metabolite of the fenhydramine in preparing anti-oxidation drugs, anti-neuritis drugs and neuroprotection drugs.

The invention also provides application of the phentermine 21-bit metabolite in preparing medicaments for preventing and treating Parkinson's disease, improving learning and memory disorder and treating hypomnesis and Alzheimer's disease.

In conclusion, the invention provides a phencyclamine 21-site metabolite, and preparation and application thereof. The 21-bit metabolite of the fenle amine has the following advantages:

the 21-bit metabolite of the fenle amine has good antioxidant activity, neuritis resistance and neuroprotective activity.

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.

Example 1

Step 1:

adding tetraacetylglucuronic acid (15.0g, 39.9mmol) and DMF (150mL) into a three-neck flask, adding hydrazine acetate (4.4g, 47.9mmol) under the protection of ice bath nitrogen, naturally warming to room temperature, stirring for 4 hours, TLC (thin layer chromatography) shows that the reaction is complete, pouring the system into water (500mL), EA (ethyl acrylate) (300mL) extracting, washing the organic phase with water for three times, washing with saturated common salt water once, drying with anhydrous sodium sulfate, and spin-drying to obtain the compound 2(11g, yield 83%).

¹HNMR CDCl₃δ:5.61-5.55(m,2H),5.23-5.16(m,1H),4.93-4.90(m,1H),4.61-4.58(d,1H,J＝10.0Hz),3.74(s,3H),2.05-2.04(m,9H)。

Step 2:

compound 2(12.0g, 35.9mmol) and dichloromethane (150mL) were added to a three-necked flask, trichloroacetonitrile (25.8g, 180mmol) and DBU (2.18g, 14.4mmol) were added under nitrogen protection in an ice bath, the solution turned brown, allowed to warm to room temperature naturally and stirred for 6 hours, TLC showed complete reaction, and triethylamine was added to stop the reaction. Direct wet silica gel column chromatography (silica gel column first wetted with 0.5% triethylamine/petroleum ether eluent) purification (petroleum ether/ethyl acetate 5: 1-3: 1-1: 1) and flash column chromatography to give crude compound 3 (12g, 70% yield).

¹HNMR CDCl₃δ:8.75(s,1H),6.66-6.65(d,1H,J＝2.8Hz),5.67-5.62(m,1H),5.29-5.26(m,1H),5.18-5.15(m,1H),4.53-4.51(d,1H,J＝10.4Hz),3.77(s,3H),2.10-2.03(m,9H)。

And step 3:

adding compound 4(20.0g, 102mmol), compound 4A (15.5g, 102mmol), acetic anhydride (47.8g, 469mmol), triethylamine (6.9g, 68.4mmol), heating at 140 ℃ for reflux for 15 hours, stopping reaction, cooling to 90 ℃, adding water (26mL) for reflux for 1 hour, evaporating the solvent under reduced pressure, diluting the residue with dichloromethane (100mL), washing with 1N hydrochloric acid (50mL), extracting the water layer with dichloromethane (100mL), combining the organic phases, washing the organic phases with 1N hydrochloric acid (50mL), drying with anhydrous sodium sulfate, filtering, evaporating the solvent, adding toluene for reflux dissolution, refrigerating overnight, filtering, washing the filter cake with ether to obtain light yellow compound 5(30g, 79% yield).

¹HNMR d6-DMSOδ:12.50-12.46(m,1H),7.71(s,1H),7.10-6.92(m,3H),6.90-6.66(m,2H),6.62-6.61(d,1H,J＝2.8Hz),3.84-3.73(m,6H),3.53(s,3H,),3.77(s,3H),2..27(s,3H)。

And 4, step 4:

compound 5(10.0g, 26.9mmol), DCM (dichloromethane) (200mL), Compound 5A (3.70g, 26.9mmol), HOBT (1-hydroxybenzotriazole) (4.40g, 32.3mmol), EDCI (1-ethyl-3 (3-dimethylpropylamine) carbodiimide) (7.70g, 40.3mmol), triethylamine (6.80g, 67.2mmol) were added to a three-necked flask and stirred at 10 ℃ for 7 hours, TLC indicated complete reaction. The reaction system was washed with water (50mL × 3), saturated sodium chloride (50mL), dried over anhydrous sodium sulfate, filtered and evaporated to remove the solvent to give a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 5:1) to give compound 6(6.00g, yield 45%).

¹HNMR CDCl₃δ:7.81(s,1H),6.93-6.87(m,5H),6.79-6.72(m,3H),6.63-6.59(m,1H),5.80-5.66(m,2H),3.71(s,3H),3.62(s,3H),3.54-3.31(m,2H),3.45(s,3H),2.73-2.69(m,2H),2..28(s,3H)。

And 5:

a three-necked flask was charged with compound 6(5.00g, 10.2mmol), compound 3(9.70g, 20.4mmol), 4A molecular sieve (24.0g), and anhydrous dichloromethane (1500mL), and BF was added dropwise under nitrogen protection at-20 deg.C₃.Et₂O (2.89g, 20.4mmol), after stirring for 2 hours at-20 deg.C, TLC indicated complete reaction and 1 drop of triethylamine was added to stop the reaction. Filtration, twoThe filter cake was washed with methyl chloride, and the organic phase was washed with water (30mL), saturated sodium chloride (30mL), dried over anhydrous sodium sulfate, filtered, evaporated to remove the solvent, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate: 6:1) to give compound 7(6g, yield 73%).

¹HNMR CDCl₃δ:7.78(s,1H),6.99-6.97(m,2H),6.93-6.85(m,5H),6.76-6.71(m,1H),6.60-6.57(m,2H),5.60-5.57(m,1H),5.35-5.25(m,3H),5.10-5.08(d,1H,J＝7.6Hz),4.19-4.09(m,1H),3.78-3.69(m,6H),3.59(s,3H),3.54-3.46(m,2H),3.43(s,3H),2.74-2.67(m,2H),2..25(s,3H),2.07-2.05(m,9H)。

Step 6:

compound 7(6.00g, 7.44mmol) was dissolved in methanol/water (135mL, v/v ═ 2:1) and Na was added₂CO₃(12.3g, 119mmol) and stirred at room temperature overnight. LCMS monitor reaction completion. The reaction was spun dry, dissolved in water, the pH of the reaction was adjusted to 5-6 with 1N hydrochloric acid, filtered, the cake was crude, and the crude was purified by prep-HPLC to give compound I (Target 1, T1) (1.1g, 23% yield).

¹HNMR CD₃ODδ:7.56(s,1H),7.03-7.05(m,4H),6.96-6.98(m,2H),6.70-6.73(m,1H),6.62-6.67(m,2H),6.44-6.48(m,2H),4.93-4.95(m,1H),3.99-4.01(d,1H),3.73(s,3H),3.61-3.67(m,4H),3.46-3.53(m,4H),3.43(s,3H),2.72-2.75(t,2H).

LC-MS:m/z＝626(M+1)。

Test example 1

This test example evaluates and compares the antioxidant, anti-neuritic and neuroprotective activity of phentermine (FLZ) and its inventive phentermine 21-position metabolite in vitro.

1. Experimental methods

1.1 determination of the content of Malondialdehyde (MDA) which is a product of lipid peroxidation

Preparing liver microsomes: rat liver tissue was weighed and 10% homogenate was prepared in Tris-HC1 buffer and liver microsomes were isolated by differential centrifugation.

The experiment is provided with a blank group, a model group, an FLZ group and an FLZ 21-bit metabolite group, and each group is provided with 3 multiple wells. PBS buffer, liver microsomes, cysteine and test drug solution were added to each tube. Test drug groupFLZ or FLZ21 metabolites were added to each tube to a final concentration of 1X 10^-4、1×10^-5And 1X 10^-6M, blank group, model group each tube added with the same volume of DMSO. Shaking in 37 deg.C water bath for 15min, adding 1 × 10 for each tube for model group and dosing group^-2M FeSO₄The solution and blank group were added with PBS buffer of the same volume and further shaken in a water bath at 37 ℃ for 15 min. Then adding TCA solution and TBA solution into each tube, and carrying out water bath at 100 ℃ for 10 min. Centrifuge at 8000rpm for 10 min. The supernatant was taken and absorbance (OD) was measured at 532 nm.

1.2, anti-neuritis administration and detection of Nitric Oxide (NO) content

BV₂The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 deg.C with 5% CO₂Cultured in an incubator. Cells in the logarithmic growth phase are selected and divided into a blank group, a model group, an FLZ group and an FLZ 21-bit metabolite group, and each group is provided with 3 multiple wells. Adding FLZ or FLZ metabolite at 21 position with different concentrations (final concentration 1 × 10)^-5，1×10^-6，1×10^-7M), adding DMSO with the same volume into a blank group and a model group, adding LPS with 500ng/mL into the model group and a dosing group after 1h, and incubating for 24h, and adding PBS buffer with the same volume into the blank group. Sucking 100 μ L of culture medium per well, adding prepared Griess reagent, and standing at room temperature for 20 min. The absorbance (OD) of each group was measured at 540 nm. The absorbance was converted to NO concentration value using a standard curve and calculated by substituting the formula.

NO inhibition rate ═ 1- (C)_{Test compound}-C_{Blank space})/(C_{Model (model)}-C_{Blank space}]×100％

1.3 neuroprotective drug delivery and cell viability assay

SH-SY5Y nerve cells are cultured in DMEM medium containing 10% fetal calf serum and placed at 37 ℃ and containing 5% CO₂Cultured in an incubator. Cells in logarithmic growth phase were selected for the experiments. SH-SY5Y cells were divided into blank, model, FLZ and FLZ 21-site metabolite groups, each group being provided with 3 duplicate wells. Adding FLZ or FLZ metabolite at 21 position with different concentrations (final concentration 1 × 10)^-5，1×10^-6，1×10^-7M), 1h post-model group and dosing group 8mM MPP was added⁺Incubate for 24h and add the same volume of PBS buffer to the blank. The supernatant was aspirated off, 100. mu.L of MTT (0.5mg/mL) was added to each well, incubation was continued for 4h, and absorbance (OD value) was measured on a microplate reader at a wavelength of 570 nm.

Effective rate%_{Test compound}-OD_{Model (model)})/(OD_{Blank space}-OD_{Model (model)})×100％

2. Results of the experiment

2.1 Effect of FLZ 21-position metabolites on lipid peroxidation

In an in vitro liver microsome lipid antioxidant experiment, FLZ21 site metabolite T1 has a certain inhibition effect on MDA generation and has a certain dose-effect relationship (Table 1).

TABLE 1 results of antioxidant activity in vitro of FLZ 21-site metabolites

2.2 Effect of FLZ and its metabolites at position 21 of FLZ on NO Release

After BV2 microglia is stimulated by LPS, the level of NO in the culture medium is obviously increased, FLZ and FLZ21 site metabolite T1 have certain inhibition effect on NO release of BV2 cells, and T1 has stronger inhibition effect on NO release than FLZ (Table 2).

TABLE 2 results of in vitro anti-neuritic Activity of FLZ and FLZ21 metabolites

2.3 Effect of FLZ and its FLZ 21-site metabolites on cell survival

Addition of MPP to SH-SY5Y cells⁺The survival rate of the later cells is obviously reduced, and the FLZ has a certain protective effect on cell injury and has a dose-effect relationship. T1 also showed some protection and T1 was superior to FLZ (Table 3).

TABLE 3 results of in vitro neuroprotective Activity of FLZ and FLZ21 metabolites

3. Conclusion

The FLZ21 site metabolite has certain antioxidant, anti-neuritis and neuroprotective activity, wherein the high concentration effect is better, and the dose dependence is realized. The FLZ21 site metabolite has higher drug effect on resisting neuritis and neuroprotection activity than FLZ.