阅读说明：本技术 一种化合物及其制法和在制备治疗糖代谢紊乱疾病的药物中的应用 (Compound, preparation method thereof and application thereof in preparing medicament for treating glycometabolism disorder diseases ) 是由 陈河如 刘志军 唐银莹 于 2020-09-14 设计创作，主要内容包括：本发明公开了一种化合物及其制法和在制备治疗糖代谢紊乱疾病的药物中的应用,该化合物的结构如式I所示。本发明的化合物对醛糖还原酶具有显著的抑制活性,并具有一定的抗氧化性。本发明的化合物在制备治疗糖代谢紊乱疾病的药物、治疗糖尿病并发症药物以及治疗由氧化应激所引起疾病中的应用为首次报道。相比于其它现有技术,本发明的化合物结构新颖、制备工艺简单、疗效优于阳性对照药依帕司他。(The invention discloses a compound, a preparation method thereof and application thereof in preparing a medicament for treating glycometabolism disorder diseases. The compound of the invention has obvious inhibitory activity to aldose reductase and certain oxidation resistance. The application of the compound in preparing the medicine for treating the glucose metabolism disorder diseases, the medicine for treating diabetic complications and the diseases caused by oxidative stress is reported for the first time. Compared with other prior art, the compound has novel structure, simple preparation process and better curative effect than a positive control drug epalrestat.)

1. A compound characterized by having the structure shown in formula I:

in the structure of formula I, the X group has a structure as shown in formula II-1, formula II-2 or formula II-3:

in the structures shown in the formula II-1, the formula II-2 and the formula II-3, R is monohydroxy substituent, dihydroxy substituent or hydroxy and hydroxymethyl substituent;

in the structure shown in the formula II-3, n is 0 and m is 1; or n is 1 and m is 0;

in the structure of formula I, the Y group has a structure as shown in formula III-1, formula III-2, formula III-3, formula III-4 or formula III-5:

the Z group is methyl, isopropyl or cyclopropyl;

when the X group is of formula II-1 and R is 4-hydroxy or 3, 4-dihydroxy, p.noteq.3 and 4 if the Y group is of formula III-1.

2. The compound of claim 1, wherein: r is a dihydroxy substitution, or when hydroxy and hydroxymethyl are disubstituted, an ortho substitution is performed.

3. The compound of claim 1, wherein:

in the structure shown in the formula III-1, p is 1-6;

in the structure shown in the formula III-2, q is 0-4.

4. The compounds shown in table 1:

TABLE 1

5. A pharmaceutically acceptable salt of a compound of any one of claims 1 to 4.

6. A synthetic intermediate of the compound of any one of claims 1 to 4.

7. A process for the preparation of a compound according to claims 1-3, characterized in that it comprises the following steps:

(1) preparation of N-t-Butoxyacyl protected substituted acyl diamine: dissolving carboxylic acid in a solvent, adding a coupling reagent with the molar weight of 1.0-3.0 times of that of the carboxylic acid, stirring and reacting for 10-40 min at the temperature of-20-0 ℃, then adding head and tail diamine with one end protected by N-tert-butoxy acyl and the organic base with the molar weight of 1.0-3.0 times of that of the carboxylic acid, stirring and reacting for 30-60 min at the temperature of-20-0 ℃, heating to 30-60 ℃, and continuing stirring and reacting for 24-36 h; after the reaction is finished, removing the solvent by rotary evaporation, and purifying the concentrate by silica gel column chromatography to obtain acyl head-tail diamine protected by one end of N-tert-butoxy acyl;

the carboxylic acid has a structure shown in a formula II-1 ', a formula II-2 ' or a formula II-3 ':

the head and tail diamine protected by one end of N-tert-butoxy acyl has a structure shown as a formula III-1 ', a formula III-2 ', a formula III-3 ', a formula III-4 ' or a formula III-5 ':

(2) mono-acyl head-to-tail diamine at one end: dissolving acyl head and tail diamine protected by one end of N-tert-butoxy acyl in a solvent, slowly adding a 4M hydrochloric acid solution of which the molar weight is 5-20 times that of the acyl head and tail diamine protected by one end of N-tert-butoxy acyl, and stirring and reacting for 3-6 hours at 0-40 ℃; after the reaction is finished, removing the solvent by rotary evaporation, and freeze-drying to obtain end-to-end monoacyl diamine;

(3) preparation of N-acylated-O-benzyl-D-serine: adding an acylation reagent with the molar weight of 1.0-5.0 times that of O-benzyl-D-serine into O-benzyl-D-serine, stirring and reacting for 4-9 h at 50-100 ℃, removing the solvent by rotary evaporation, and separating and purifying the concentrate by using high performance liquid chromatography to obtain N-acylation-O-benzyl-D-serine;

(4) preparation of a compound of the structure of formula I: dissolving N-acylation-O-benzyl-D-serine in a solvent, adding a coupling reagent with the molar weight of 1.0-3.0 times that of the N-acylation-O-benzyl-D-serine, and stirring and reacting at-20-0 ℃ for 10-40 min to obtain a solution A; dissolving the monoacyl end-to-end diamine prepared in the step (2) in a solvent, adding organic alkali with the molar weight 3.0-5.0 times that of the monoacyl end-to-end diamine, cooling to below-30 ℃, dropwise adding the solution A under stirring, keeping the temperature after dropwise adding, and stirring for 1-3 hours; heating to room temperature, and continuing to react for 20-40 h; after the reaction is finished, removing the solvent by rotary evaporation; purifying the concentrate by silica gel column chromatography to obtain a compound with a structure shown in formula I;

the acylating agent used in the step (3) is acid anhydride or acyl chloride.

8. Use of a compound according to any one of claims 1 to 4, a salt according to claim 5 or a synthetic intermediate according to claim 6 for the manufacture of a medicament for the treatment of disorders of carbohydrate metabolism.

9. Use of a compound according to any one of claims 1 to 4, a salt according to claim 5 or a synthetic intermediate according to claim 6 for the manufacture of a medicament for the treatment of a disease caused by oxidative stress.

10. Use of a compound according to any one of claims 1 to 4, a salt according to claim 5 or a synthetic intermediate according to claim 6 for the manufacture of a medicament for the treatment of diabetic complications.

11. Use according to claim 9, characterized in that: the diabetic complications comprise diabetic retinopathy, diabetic nerve ending disorder and diabetic senile dementia.

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a compound, a preparation method thereof and application thereof in preparing a medicament for treating glycometabolism disorder diseases.

Background

Diabetes Mellitus (DM) is an endocrine metabolic syndrome characterized primarily by hyperglycemia and Diabetes, due to insufficient insulin secretion or insulin resistance in the body. Diabetic patients are prone to induce diabetic complications when being in a hyperglycemic state for a long time, and the diabetic complications are mainly classified into three types: retinopathy, such as: glaucoma, cataract, and the like; peripheral neuropathies, such as: peripheral neuritis, foot ulcer, etc.; kidney disorders, such as: renal microangiopathy, which in severe cases can lead to renal failure, uremia, etc.

In addition, the heart and cerebral vessels are possibly diseased, and stroke, cardiac hypertrophy, congestive heart failure and the like can be caused. These complications are all caused by long-term hyperglycemia of the body, which damages the vascular wall and peripheral nerves and greatly increases the risk of cardiovascular diseases.

Although diabetes has always plagued patients' health, it is really life threatening to be a series of complications due to the persistent hyperglycemia in the body. These complications occur in association with excessive activation of Aldose Reductase (AR) in high sugar environments.

Inhibition of AR has been shown to be an effective approach for the treatment of diabetic complications. According to literature research, although many AR inhibitors have been developed, few aldose reductase inhibitors are available for clinical use, which are effective and have fewer adverse reactions. Among the aldose reductase inhibitors that are commercially available and chemically synthesized are epalrestat (epalrestat), astatin (alrestatin), ponalrestat (ponalrestat), sorbinil (sorbinel) and tolrestat (tolrestat). These drugs are also not ideal. The development of aldose reductase inhibitors with definite therapeutic effect and safety is urgent.

The Chinese patent of invention ZL201410723116.8 and ZL201710372369.9 both disclose a compound having aldose reductase inhibitory activity, but the compound of patent ZL201410723116.8 has less AR inhibitory activity than that of the positive control drug Epasitah and less antioxidant activity than Trolox; although the patent ZL201710372369.9 found candidate compounds with good AR inhibitory activity and antioxidant activity, its stability and bioavailability were deficient and further improvement was needed.

Disclosure of Invention

In view of the above, the primary object of the present invention is to provide a novel compound having both aldose reductase inhibitory activity and antioxidant activity.

Another object of the present invention is to provide a process for producing the above compound.

The invention also aims to provide the application of the compound in preparing medicaments for treating glucose metabolism disorder diseases, in particular to the application in preparing medicaments for treating diabetic complications.

The purpose of the invention is realized by the following technical scheme:

a compound having the structure shown in formula I:

in the structure of formula I, the X group has a structure as shown in formula II-1, formula II-2 or formula II-3:

in the structures shown in the formula II-1, the formula II-2 and the formula II-3, R is monohydroxy substituent, dihydroxy substituent or hydroxy and hydroxymethyl substituent;

preferably, when R is monohydroxy, the substitution position in the formula II-1 can be 2-, 3-, 4-, preferably 4-; the substitution positions thereof in the formula II-2 may be 5-, 6-, 7-, 8-, preferably 6-; in the formula II-3, the substitution position may be 5-, 6-, 7-, 8-, preferably 7-.

Preferably, R is a dihydroxy substitution, or when hydroxy and hydroxymethyl are disubstituted, an ortho substitution is provided;

further preferably, when R is dihydroxy, the substitution position in formula II-1 may be 2, 3-or 3, 4-; in formula II-2, the substitution position may be 5,6-, 6, 7-or 7, 8-; the substitution position thereof in the formula II-3 may be 5,6-, 6, 7-or 7, 8-.

Further preferably, when R is a hydroxyl group and a hydroxymethyl group, the substitution position in the formula II-1 is preferably 3-hydroxymethyl-4-hydroxyl; the substitution position thereof in the formula II-2 is preferably 6-hydroxy-7-hydroxymethyl; the substitution position thereof in the formula II-3 is preferably 6-hydroxymethyl-7-hydroxy.

In the structure shown in the formula II-3, n is 0 and m is 1; or n is 1 and m is 0.

In the structure of formula I, the Y group has a structure as shown in formula III-1, formula III-2, formula III-3, formula III-4 or formula III-5:

in the structure shown in the formula III-1, p is 1-6;

in the structure shown in the formula III-2, q is 0-4.

When the X group is of formula II-1 and R is 4-hydroxy or 3, 4-dihydroxy, p.noteq.3 and 4 if the Y group is of formula III-1.

The Z group is methyl, isopropyl or cyclopropyl.

Preferably, the compounds of the present invention are specific compounds as shown in table 1:

TABLE 1

The invention also claims pharmaceutically acceptable salts of the compounds having the structure shown in the formula I and the specific compounds shown in the table 1.

The invention also claims synthetic intermediates (or prodrugs) of compounds of the structure shown in formula I and specific compounds shown in Table 1.

A method for preparing a compound having a structure represented by formula I, comprising the steps of:

(1) preparation of N-t-Butoxyacyl protected substituted acyl diamine: dissolving carboxylic acid in a solvent, adding a coupling reagent with the molar weight of 1.0-3.0 times of that of the carboxylic acid, stirring and reacting for 10-40 min at the temperature of-20-0 ℃, then adding head and tail diamine with one end protected by N-tert-butoxy acyl and the organic base with the molar weight of 1.0-3.0 times of that of the carboxylic acid, stirring and reacting for 30-60 min at the temperature of-20-0 ℃, heating to 30-60 ℃, and continuing stirring and reacting for 24-36 h; after the reaction is finished, removing the solvent by rotary evaporation, and purifying the concentrate by silica gel column chromatography to obtain acyl head-tail diamine protected by one end of N-tert-butoxy acyl;

the carboxylic acid has a structure shown in a formula II-1 ', a formula II-2 ' or a formula II-3 ':

the head and tail diamine protected by one end of N-tert-butoxy acyl has a structure shown as a formula III-1 ', a formula III-2 ', a formula III-3 ', a formula III-4 ' or a formula III-5 ':

(2) mono-acyl head-to-tail diamine at one end: dissolving acyl head and tail diamine protected by one end of N-tert-butoxy acyl in a solvent, slowly adding a 4M hydrochloric acid solution of which the molar weight is 5-20 times that of the acyl head and tail diamine protected by one end of N-tert-butoxy acyl, and stirring and reacting for 3-6 hours at 0-40 ℃; after the reaction is finished, removing the solvent by rotary evaporation, and freeze-drying to obtain end-to-end monoacyl diamine;

(3) preparation of N-acylated-O-benzyl-D-serine: adding an acylation reagent with the molar weight of 1.0-5.0 times that of O-benzyl-D-serine into O-benzyl-D-serine, stirring and reacting for 4-9 h at 50-100 ℃, removing the solvent by rotary evaporation, and separating and purifying the concentrate by using high performance liquid chromatography to obtain N-acylation-O-benzyl-D-serine;

(4) preparation of a compound of the structure of formula I: dissolving N-acylation-O-benzyl-D-serine in a solvent, adding a coupling reagent with the molar weight of 1.0-3.0 times that of the N-acylation-O-benzyl-D-serine, and stirring and reacting at-20-0 ℃ for 10-40 min to obtain a solution A; dissolving the monoacyl end-to-end diamine prepared in the step (2) in a solvent, adding organic alkali with the molar weight 3.0-5.0 times that of the monoacyl end-to-end diamine, cooling to below-30 ℃, dropwise adding the solution A under stirring, keeping the temperature after dropwise adding, and stirring for 1-3 hours; heating to room temperature, and continuing to react for 20-40 h; after the reaction is finished, removing the solvent by rotary evaporation; purifying the concentrate by silica gel column chromatography to obtain a compound with a structure shown in formula I;

the solvent used in the steps (1) and (4) is N, N-Dimethylformamide (DMF) or Dichloromethane (DCM)/DMF mixed solution with the volume ratio of 10: 1.

The coupling reagent used in the steps (1) and (4) can be one of ethyl chloroformate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-diisopropyl carbodiimide (DIC) and benzotriazole-1-yl-oxy-tripyrrolidinyl phosphorus hexafluorophosphate (PyBOP) and N-hydroxybenzotriazole (HOBt) mixed according to a molar ratio of 1: 1.

The organic base used in the steps (1) and (4) is triethylamine or Diisopropylethylamine (DIPEA).

The solvent used in the step (2) is one of methanol, ethanol and DMF.

The acylating agent used in the step (3) is acid anhydride or acyl chloride.

In the step (4), the molar ratio of the N-acylated-O-benzyl-D-serine to the one-end monoacyl head-tail diamine is 1 (1-3), and preferably 1.0: 1.1.

In still another aspect, the invention also provides the application of the compounds (the compounds with the structure shown in the formula I and the compounds listed in the table 1) in the preparation of medicines for treating glucose metabolism disorder diseases, in particular to the application in the preparation of medicines for treating diabetic complications. These diabetic complications include diabetic retinopathy, diabetic peripheral neuropathy, and diabetic senile dementia.

The present invention has demonstrated that the above-mentioned compounds (structural compounds of formula I and compounds listed in Table 1) have excellent inhibitory activity against aldose reductase using the following experiments: the inhibition rate of alpha-cyano-3, 4-dihydroxycinnamic acid derivatives (namely the compounds of the invention) on the enzymatic hydrolysis activity of aldose reductase is determined by taking D, L-glyceraldehyde as a substrate, and Epalrestat (Epalrestat), a clinical drug, is taken as a positive control. The results showed that (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (. alpha. -cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G) had the best inhibitory activity against human aldose reductase, IC₅₀The value was 12.26. + -. 0.85nmol/L, which is greater than the epalrestat level (IC)₅₀＝75.64±4.22nmol/L) Stronger than CHR-532R (IC) reported in patent CN 201410723116.8₅₀89.71 +/-3.51 nmol/L) which is stronger than CHR-5N-D (IC) reported in patent CN201710372369.9₅₀41.02 ± 2.83 nmol/L). Other compounds of the invention also exhibit certain inhibitory activity against aldose reductase in vitro.

The compound (the compound with the structure shown in the formula I and the compound listed in the table 1) is inspected to eliminate the free radical generated by 1, 1-diphenyl-2-trinitrophenyl hydrazine (DPPH), and CHR-5N-F is found to show excellent antioxidation, and the half clearing concentration EC is₅₀The concentration is 8.81 +/-0.51 mu M and is obviously stronger than that of a positive control drug Trolox (EC)₅₀14.39 ± 0.23 μ M). Other compounds of the invention also exhibit some free radical scavenging ability.

The effect of CHR-5N-G as a test agent and Epalrestat as a positive control agent and Edaravone as an antioxidant on chick embryo survival, mortality, neural tube teratogenesis, and chick embryo morphology and body weight under a high sugar environment (0.4 mmol/eg) was examined by reference to experimental methods reported in the literature (Zhang L, Li YF, Yuan S, et al, scientific Reports,2016,6: 24942). The results show that the survival rate, the death rate, the total aberration rate and the body weight of the chick embryos treated by the CHR-5N-G are 78.8 percent, 20.7 percent, 11.3 percent and 243.2mg respectively at the concentration of 500 nM; the survival rate, the death rate, the total distortion rate and the body weight of the chick embryos treated by the epalrestat are 69.6 percent, 29.8 percent, 27.9.0 percent and 231.4mg respectively; the survival rate, the death rate, the total distortion rate and the body weight of the chicken embryos treated by the edaravone are 71.2 percent, 30.4 percent, 22.3 percent and 228.5mg respectively. The result shows that the effect of the tested medicament CHR-5N-G is generally superior to that of epalrestat and edaravone.

Meanwhile, the compound has specific inhibitory activity on AR, shows obvious enzyme inhibitory activity on ALR2, is superior to a positive control drug, and has consistent in-vitro activity result with the result of the in-vitro activity; the ALR1 does not show obvious inhibitory activity to the isozyme, so that the target compound has high selectivity on inhibiting AR. The compound remarkably inhibits the polyalcohol pathway of glucose metabolism by inhibiting the activity of AR, and prevents the generation of a metabolite sorbitol of the pathway. The in vivo antioxidant experiment also shows that the compound can obviously inhibit the formation of oxidation product MDA in embryoid bodies, and the ORAC level shows that the compound has good free radical scavenging capacity. The experiments strongly prove that the compound CHR-5N-G can play a good role in protecting the chicken embryonic neural tube deformity induced by high sugar, and secondly, the in-vivo enzyme inhibition activity and the in-vivo antioxidant activity of the compound are outstanding, which are consistent with the in-vitro experiments, and meanwhile, the expression of the regulatory protein Pax 3 can be obviously improved.

A diabetic peripheral neuropathy rat model is established according to a reference document (Neural Regeneration Research 2016,11(2),345 and 351), and CHR-5N-G has an obvious therapeutic effect on high-sugar induced peripheral neuropathy.

Therefore, the compounds (the structural compound of the formula I and the compounds listed in the table 1, and the pharmaceutically acceptable salts and synthetic intermediates thereof) can be used for preparing medicaments for treating glucose metabolism disorder diseases, and particularly can be used for preparing medicaments for treating diabetic complications such as diabetic retinopathy, diabetic senile dementia, nerve ending disorder and the like; the compounds can be used for preparing medicines for treating diseases caused by oxidative stress, especially diabetic complications.

Compared with the prior art, the invention has the following advantages and effects:

the inhibitory activity of the compound on aldose reductase and the application of the compound in preparing medicaments for treating glycometabolism disorder diseases, medicaments for treating diabetic complications and diseases caused by oxidative stress are reported for the first time. Compared with other prior art, the compound has novel structure, simple preparation process and better curative effect than a positive control drug epalrestat.

Drawings

FIG. 1 is a diagram of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (alpha-cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G)¹H NMR spectrum.

FIG. 2 is a diagram of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (alpha-cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G)¹³C MRC map.

FIG. 3 is an HR-ESI-MS spectrum of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (. alpha. -cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G).

FIG. 4 is a bar graph of the in vitro ALR2 enzyme inhibition rates of a portion of the compounds.

FIG. 5 is a bar graph of free radical scavenging rates for different concentrations of Trolox, CHR-5N-G, CHR-5N-F.

FIG. 6 is a graph showing the effect of CHR-5N-G on morphology of high-sugar-damaged chicken embryos.

FIG. 7 is a graph showing the effect of CHR-5N-G on the development of neural tubes of high-sugar-damage chicken embryos.

FIG. 8 is a graph showing the effect of CHR-5N-G compound on the brain tissue sugar content, aldose reductase (ALR2) activity, aldehyde reductase (ALR1) activity and Sorbitol content in EDD5 high-sugar chick embryo model.

FIG. 9 shows the effect of CHR-5N-G on the ORAC level of MDA content in EDD5 high-sugar chick embryo model.

FIG. 10 shows the Pax 3 protein expression levels in chicken embryos of each experimental group.

FIG. 11 is a graph of the neuroprotective effect of CHR-5N-G on diabetic peripheral neuropathy rats.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

(E) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (4-hydroxyphenyl) acrylamide (CHR-5F-B) comprising the following five steps:

(1) synthesis of (E) - α -cyano-4-hydroxycinnamic acid: 122.1mg (1.0mmol) of 4-hydroxybenzaldehyde is weighed into a dry 50mL round bottom flask, 85mg (1.0mmol) of cyanoacetic acid and 15.4mg (0.2mmol) of ammonium acetate are added, 48mg (0.8mmol) of glacial acetic acid is weighed into the reaction system by a syringe, 10mL of toluene is added as a solvent, and a small-sized water separator, a condenser tube and a drying tube are connected onto a reaction bottle. The reaction was stirred and refluxed for 18h in an oil bath at 120 ℃. After the reaction, the temperature is reduced to room temperature, then the mixture is kept stand at low temperature, filtered, and a filter cake is washed with Dichloromethane (DCM) for three times to obtain yellow solid with the yield of 92.8 percent.¹H NMR(300MHz,CD₃OD)δ:9.66(s,1H,Ar-OH),8.21(s,1H,＝CH-),7.46(d,2H,J＝8.3Hz,Ar-H),6.88(d,J＝8.3Hz,2H,Ar-H)；ESI-MS(m/z):188.3[M-H]^-。

(2) Preparation of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-4-hydroxycinnamamide: 1.89g (10.0mmol) of alpha-cyano-4-hydroxycinnamic acid was charged into a 100mL round bottom flask, 30mL of DCM/DMF 10:1(V/V) was added and dissolved with stirring, 1.15g (11.0mmol) of weighed EDC & HCl was added to the flask, reaction was carried out with stirring at 0 ℃ for 10min, then 0.82g (11.0mmol) of weighed HOBt was added to the reaction flask, reaction was carried out with stirring at 0 ℃ for 10min, and the free carboxyl group was sufficiently activated. After the carboxyl group was sufficiently activated, 2.29g (11.0mmol) of 4-N-Boc-aminoaniline and 4.6mL (26.4mmol) of DIPEA were measured by syringe, and injected into the reaction flask in sequence, stirred at 0 ℃ for reaction for about 30min, slowly warmed to room temperature, and reacted for 24 h. After the reaction, the solvent was removed, and the residue was separated and purified by silica gel column chromatography (chloroform: methanol: 40:1) to obtain 3.29g of a yellow solid, which was obtained in 86.7% yield.¹H NMR(400MHz,DMSO-d₆)δ:10.03(s,Ar-NH),9.34(s,Ar-NH),8.75(s,Ar-OH),8.45(s,＝CH),7.75(m,4H,Ar),7.45(d,J＝8.3Hz,2H,Ar-H),6.46(d,J＝8.3Hz,2H,Ar-H),1.48(CH₃,9H)；¹³C NMR(101MHz,DMSO-d₆)δ:163.8,157.7,152.5,150.4,143.2,133.6,133.2,124.7,121.8,115.8,106.9,79.5,28.4。

(3) Preparation of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide: (E) -N- (N-Boc-4-aminophenyl) - α -cyano-4-hydroxycinnamamide 1.14g (3.0mmol) was dissolved in 30mL of dry methanol, transferred to a 50mL round-bottomed flask, and 7.5mL of a 4mol/L HCl solution (30mmol) was slowly added dropwise thereto, and the reaction was stirred at room temperature for 4 hours. The solvent was removed to give a pale yellow solid in 97.3% yield.¹H NMR(400MHz,DMSO-d₆)δ:10.03(s,1H,Ar-NH),8.75(s,1H,Ar-OH),8.45(s,1H,＝CH),7.45(d,J＝8.3Hz,2H,Ar-H),7.38(d,J＝8.1Hz,2H,Ar-H),6.58(d,J＝8.3Hz,2H,Ar-H),6.32(d,J＝8.1Hz,2H,Ar-H),4.53(br,2H,Ar-NH₂)；¹³C NMR(101MHz,DMSO-d₆)δ:163.8,157.7,152.5,150.4,143.2,133.6,133.2,124.7,121.8,115.8,106.9。

(4) Preparation of N-acetyl-O-benzyl-D-serine: 195.2mg (1.0mmol) of O-benzyl-D-serine is weighed into a 50mL round-bottom flask, dissolved in 10mL redistilled methanol, and 0.3mg of acetic anhydride is weighed into a syringemL (about 3mmol) and 0.5mL (about 3mmol) of DIEA were added to the reaction system, and the reaction was refluxed at 70 ℃ for 7 hours. After the reaction, methanol in the reaction solution was removed under reduced pressure, and unreacted acetic anhydride was removed by freeze-drying. HPLC preparation (mobile phase methanol: water 50:50, 0.05% TFA, uv 254nm) gave a colorless oil in 74.5% yield.¹H NMR(400MHz,CDCl₃)δ:11.63(s,1H,-COOH),7.26(m,5H,Ar-H),7.04(d,J＝7.8Hz,1H,-CO-NH-),4.79-4.67(m,1H,-N-CH-),4.47(s,2H,Ar-CH₂-O-),3.82-3.78(m,2H,-CH₂-O-),2.01(s,3H,-CO-CH₃)；¹³C NMR(75MHz,CDCl3)δ172.98,171.69,137.45,128.61,128.09,127.90,73.53,69.66,52.91,22.92.ESI-MS(m/z):236.7[M-H]^-。

(5) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (4-hydroxyphenyl) acrylamide: weighing 118.7mg (0.5mmol) of N-acetyl-O-benzyl-D-serine in a penicillin bottle, sequentially adding 101.5mg (0.75mmol) of HOBt and 144mg (0.75mmol) of EDC & HCl, and 3-10 mL of DCM/DMF (10:1, V/V) as solvents, and stirring for reaction for 10min to obtain solution A. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) -alpha-cyano-4-hydroxycinnamamide is further weighed and dissolved in 3-10 mL of DCM/DMF, 0.4mL (2.2mmol) of DIPEA is added, the mixture is cooled to-30 ℃, the solution A is dropwise added under stirring, and after the dropwise addition is finished, the temperature is kept and the stirring is carried out for 1 hour. Then, the reaction was allowed to warm to room temperature and continued for 23 h. After the reaction is finished, the solvent is removed by rotary evaporation. The resulting extract was subjected to silica gel column chromatography (chloroform: methanol: 40:1, V: V) and gradient elution to obtain 181.1mg of a pale yellow solid with a yield of 66.1%.¹H NMR(300MHz,DMSO-d₆)δ:9.67(s,1H),8.17(d,J＝8.0Hz,3H),7.37–7.23(m,11H,＝CH,Ar-H),6.52(d,J＝8.7Hz,2H),4.65(d,J＝7.9Hz,1H),4.50(d,J＝3.8Hz,2H),3.61(s,1H),3.12(s,1H),1.88(s,3H,CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:171.80,169.49,167.79,144.76,138.32,138.21,128.37,128.36,127.67,127.65,127.62,127.59,121.25,114.12,72.19,70.13,69.75,53.79,53.34,52.44,42.02,22.68,22.51,18.27,16.92,12.63.ESI-MS(m/z):499.2for C₂₈H₂₆N₄O₅([M+H]⁺)；(C＝1,CH₃OH)。

The above data confirm that the compound is (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) -alpha-cyano-3- (4-hydroxyphenyl) acrylamide (CHR-5F-B), and the structure is shown below.

Example 2

(E) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) - α -cyano-3- (4-hydroxyphenyl) acrylamide (CHR-5F-E) comprising the following five steps.

(1) Preparation of (E) -alpha-cyano-4-hydroxycinnamic acid. Same procedure as in (1) of example 1

(2) Preparation of (E) -N- (N-Boc-4-aminocyclohexyl) - α -cyano-4-hydroxycinnamamide: the procedure was as in (2) of example 1. 2.29g (11.0mmol) of 4-N-Boc-aminocyclohexylamine was changed to 2.36g (11.0mmol) of 4-N-Boc-aminocyclohexylamine, giving 2.29g of a pale yellow solid in 59.5% yield.¹H NMR(300MHz,DMSO-d₆)δ:10.50(s,1H,Ar-OH),8.31(s,1H),8.15(s,1H),7.85(s,1H),7.77(d,J＝8.2Hz,2H),6.94(d,J＝8.2Hz,2H),3.70(m,1H),3.43(m,1H),1.66(m,4H),1.51(m,4H),1.38(s,9H)；¹³C NMR(75MHz,DMSO-d₆)δ:161.64,160.97,154.91,150.00,132.72,122.95,117.31,116.18,101.68,77.46,46.97,28.29,28.08,26.84。

(3) Preparation of (E) -N- (4-aminocyclohexyl) - α -cyano-4-hydroxycinnamamide: the procedure was as in (3) of example 1. The (E) -N- (N-Boc-4-aminocyclohexyl) - α -cyano-4-hydroxycinnamamide obtained in the above step was added to give 1.67g of a light brown solid, yield 89.6%.¹H NMR(300MHz,DMSO-d₆)δ:10.50(s,1H,Ar-OH),8.33(s,1H),8.17(s,1H),7.52(d,J＝8.1Hz,2H),6.63(d,J＝8.1Hz,2H),3.65(m,1H),2.58(m,1H),1.73(m,4H),1.52(br,2H),1.47(m,4H)；¹³C NMR(75MHz,DMSO-d₆)δ:161.65,160.96,150.01,132.73,122.97,117.33,116.17,101.69,46.98,28.32,28.10。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) - α -cyano-3- (4-hydroxyphenyl) acrylamide: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide was replaced by 156.9mg (0.55mmol) of (E) -N- (4-aminocyclohexyl) - α -cyano-4-hydroxycinnamamide. The resulting mixture was subjected to silica gel column chromatography (chloroform: methanol: 40:1, V: V) and gradient elution to obtain 174.3mg of a pale yellow solid with a yield of 62.8%.¹H NMR(300MHz,CDCl₃)δ:9.72(s,1H,Ar-OH),8.32(br,2H),8.16(s,1H),8.13(br,1H),7.89(d,J＝8.7Hz,2H,Ar-H),7.40-7.28(m,5H,Ar-H),6.98(d,J＝8.7Hz,2H,Ar-H),4.75(m,1H),4.63(s,2H),4.03-3.84(m,3H),3.64-3.46(m,2H),2.05(s,3H,CH₃),1.74(m,4H),1.51(m,4H)；¹³C NMR(75MHz,CDCl₃)δ:161.4,160.7,154.9,150.7,149.4,143.2,137.5,132.8,128.6,122.4,117.7,116.8,101.8,77.3,69.7,57.3,50.6,46.9,28.7,26.9.ESI-MS(m/z):503.6for C₂₈H₃₂N₄O₅([M-H]⁺)；(C＝2,CH₃OH)。

The above data confirm that this compound is (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) - α -cyano-3- (4-hydroxyphenyl) acrylamide (CHR-5F-E), and the structure is shown below.

Example 3

(E) Preparation of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (. alpha. -cyano-3- (4-hydroxyphenyl) acryloyl) piperazine (CHR-5F-G) comprising the following five steps:

(1) preparation of (E) -alpha-cyano-4-hydroxycinnamic acid. Same procedure as in (1) of example 1

(2) Preparation of (E) -4-Boc-1- (α -cyano-4-hydroxycinnamoyl) piperazine: the procedure was as in (2) of example 1. 4-N-Boc-aminoaniline 229g (11.0mmol) was changed to 2.05g (11.0mmol) of N-Boc-piperazine. 1.89g of light yellow solid is obtained, and the yield is 52.9%.¹H NMR(400MHz,CDCl₃)δ:9.68(s,1H),8.12(s,1H),7.83(d,J＝8.7Hz,2H),6.97(d,J＝8.7Hz,2H),3.55-3.51(m,8H),1.48(s,9H)；¹³C NMR(101MHz,CDCl₃)δ:163.97,160.14,154.43,152.81,132.69,124.48,116.60,116.15,101.31,80.56,50.91,48.82,28.21。

(3) Preparation of (E) - α -cyano-4-hydroxycinnamoylpiperazine: the procedure was as in (3) of example 1. The (E) -4-Boc-1- (alpha-cyano-4-hydroxycinnamoyl) piperazine obtained in the above step was charged to give 1.23g of a light brown solid, and the yield was 90.5%.¹H NMR(400MHz,CDCl₃)δ:9.69(s,1H),8.13(s,1H),7.82(d,J＝8.7Hz,2H),6.96(d,J＝8.7Hz,2H),3.36(m,4H),2.87(m,4H),1.23(br,1H)；¹³C NMR(101MHz,CDCl₃)δ:164.12,154.45,152.83,133.21,124.53,116.62,116.17,101.35,80.56,51.62,47.13。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (α -cyano-3- (4-hydroxyphenyl) acryloyl) piperazine: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamoyl amide was replaced with 141.5mg (0.55mmol) of (E) - α -cyano-4-hydroxycinnamoyl piperazine. Purifying by silica gel column chromatography (chloroform: methanol: 40:1, V: V) with gradient elution to obtain light yellow solid 132.6mg with yield of 50.6%.¹H NMR(400MHz,DMSO-d₆)δ:9.72(s,1H,Ar-OH),8.56(s,1H),8.15(s,1H),7.52(d,J＝8.7Hz,2H),7.42(m,5H,Ar-H),6.69(d,J＝8.7Hz,2H),4.76(m,1H),4.63(s,2H),3.62(m,1H),3.53(m,4H),3.41(m,4H),3.32(m,2H),2.04(s,3H,CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:171.73,169.75,168.12,157.87,151.43,144.25,138.52,129.61,128.82,127.40,124.70,115.80,115.60,106.90,72.30,70.0,54.80,49.40,48.90,22.90；ESI-MS(m/z):499.5for C₂₆H₂₈N₄O₅([M+Na]⁺)；(C＝2,CH₃OH).

The above data demonstrate that the compound is (E) -4- (N-acetyl-O-benzyl-D-serinyl) -1- (α -cyano-3- (4-hydroxyphenyl) acryloyl) piperazine (CHR-5F-G), having the structure:

example 4

(E) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (3, 4-dihydroxyphenyl) acrylamide (CHR-5N-B) comprising the following five steps:

(1) synthesis of (E) - α -cyano-3, 4-dihydroxycinnamic acid: 138mg (1.0mmol) of 3, 4-dihydroxybenzaldehyde is weighed into a dry 50mL round bottom flask, 85mg (1.0mmol) of cyanoacetic acid and 15.4mg (0.2mmol) of ammonium acetate are added, 48mg (0.8mmol) of glacial acetic acid is weighed into the reaction system by a syringe, 10mL of toluene is added as a solvent, and a small-sized water separator, a condenser tube and a drying tube are connected onto a reaction bottle. The reaction was stirred and refluxed for 18h in an oil bath at 120 ℃. After the reaction was completed, the temperature was lowered to room temperature, followed by standing at low temperature, filtration and washing of the filter cake with Dichloromethane (DCM) for three times to obtain 196.3mg of a yellow solid with a yield of 95.7%.¹H NMR(300MHz,CD₃OD)δ:9.52(s,2H,Ar-OH),8.21(s,1H,＝CH-),7.64(d,J＝8.3Hz,1H),7.36(s,1H,Ar-H),6.88(d,J＝8.3Hz,1H,Ar-H)；ESI-MS(m/z):204.3for C₁₀H₆NO₄([M-H]^-)。

(2) Preparation of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-3, 4-dihydroxycinnamamide: the same procedure as in (2) of example 1 was repeated except for replacing 1.89g (10.0mmol) of α -cyano-4-hydroxycinnamic acid with 2.05g (10.0mmol) of α -cyano-3, 4-dihydroxycinnamic acid. 2.82g of a yellow solid was obtained with a yield of 71.3%.¹H NMR(400MHz,DMSO-d₆)δ:10.03(s,1H),9.34(s,1H,Ar-NH),8.75(s,2H,2×Ar-OH),8.45(s,1H,＝CH),7.75(m,4H,Ar),7.66(d,J＝8.3Hz,1H),7.38(s,1H,Ar-H),6.89(d,J＝8.3Hz,1H,Ar-H),1.49(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:163.8,157.7,152.5,150.4,143.2,133.6,133.2,124.7,121.8,115.8,106.9,79.5,28.4。

(3) Preparation of (E) -N- (4-aminophenyl) - α -cyano-3, 4-dihydroxycinnamamide: the procedure was the same as in example 1, step (3), except that 1.14g (3.0mmol) of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-4-hydroxycinnamide was replaced with 1.19g (3.0mmol) of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-3, 4-dihydroxycinnamamide. Pale yellow solid was obtained in 96.4% yield.¹H NMR(400MHz,DMSO-d₆)δ:10.05(s,1H,Ar-NH-),8.77(s,2H,2×Ar-OH),8.46(s,1H,＝CH),7.76(d,J＝7.6Hz,2H,Ar-H),7.65(d,J＝8.3Hz,1H),7.36(s,1H,Ar-H),6.87(d,J＝8.3Hz,1H,Ar-H),6.53(d,J＝7.6Hz,2H,Ar-H),4.56(br,2H)；¹³C NMR(101MHz,DMSO-d₆)δ:163.9,151.8,150.2,142.6,133.4,132.8,124.6,121.7,115.6,106.8。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (3, 4-dihydroxyphenyl) acrylamide: 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamide was replaced with 162.4mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-3, 4-dihydroxycinnamamide, the procedure was as in example 1, step (5). After silica gel column chromatography (chloroform: methanol: 40:1, V: V), the mixture was separated and purified by gradient elution to obtain 152.2mg of a pale yellow solid, which was obtained in 53.8% yield.¹H NMR(300MHz,DMSO-d₆)δ:10.18(s,1H,-CONH-Ar),9.62(s,1H,Ar-NHCO-),9.47(s,2H,2×Ar-OH),8.34(d,J＝18.2Hz,1H),7.96(s,1H,＝CH),7.65(d,J＝6.2Hz,2H),7.59(d,J＝6.2Hz,2H),7.51(d,J＝8.5Hz,1H),7.25(m,5H,Ar-H),7.11(s,1H),6.85(d,J＝8.5Hz,1H),5.05(d,J＝13.1Hz,1H,CO-CH-N),4.65(s,2H,Ar-CH₂-O),3.55(s,1H,O-CH₂),3.07(s,1H,O-CH₂),1.80(s,3H,CH₃)；¹³C NMR(75MHz,DMSO-d₆)δ:172.81,171.23,163.92,150.56,146.92,144.76,137.53,134.31,133.42,129.33,128.62(2),127.87,127.65,127.62,123.20,121.25(4),117.29,115.76,115.32,109.51,72.29,69.75,56.63,22.92.ESI-MS(m/z):545.7for C₂₈H₂₆N₄O₆([M+Na]⁺).(C＝2，CH₃OH)。

The above data demonstrate that the compound is (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) -alpha-cyano-3- (3, 4-dihydroxyphenyl) acrylamide (CHR-5N-B) and has the following structure:

example 5

(E) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acrylamide (CHR-5N-C) comprising the following five steps:

(1) synthesis of (E) - α -cyano-3-hydroxymethyl-4-hydroxycinnamic acid: the procedure was carried out in the same manner as in example 4, step (1) except for replacing 138mg (1.0mmol) of 3, 4-dihydroxybenzaldehyde with 152.1mg (1.0mmol) of 3-hydroxymethyl-4-hydroxybenzaldehyde. 204.9mg of a yellow solid are obtained with a yield of 93.5%.¹H NMR(300MHz,CD₃OD)δ:9.62(s,H,Ar-OH),8.26(s,1H,＝CH-),7.54(d,J＝8.3Hz,1H),7.32(d,J＝8.3Hz,1H,Ar-H),7.14(s,1H,Ar-H),7.02(br,1H),4.63(s,2H)；ESI-MS(m/z):218.4for C₁₁H₈NO₄([M-H]^-)。

(2) Preparation of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-3-hydroxymethyl-4-hydroxycinnamamide: the same procedure as in example 1, step (2), was repeated except for replacing 1.89g (10.0mmol) of α -cyano-4-hydroxycinnamic acid with 2.19g (10.0mmol) of α -cyano-3-hydroxymethyl-4-hydroxycinnamic acid. 3.01g of a yellow solid was obtained in a yield of 73.2%.¹H NMR(400MHz,DMSO-d₆)δ:10.13(s,1H,-CO-NH-Ar),9.84(s,1H,Ar-NH-Boc-),9.72(s,1H,Ar-OH),8.45(s,1H,＝CH),7.75(m,4H,Ar),7.66(d,J＝8.3Hz,1H),7.38(d,J＝8.3Hz,1H,Ar-H),7.14(s,1H,Ar-H),7.06(br,1H),4.63(s,2H,Ar-CH₂-O),1.51(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:163.8,156.13,153.06,150.51,133.62,133.25,129.60,128.83,128.18,126.20,121.76(4),115.84,107.11,79.53,61.12,29.05(3)。

(3) (E) -N- (4-aminophenyl) -alpha-cyano-3-hydroxymethyl-4-hydroxycinnamoylPreparation of amine: the same procedure as in example 1, step (2), was repeated except for replacing 1.14g (3.0mmol) of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-3-hydroxymethyl-4-hydroxycinnamide with 1.23g (3.0mmol) of (E) -N- (N-Boc-4-aminophenyl) - α -cyano-4-hydroxycinnamide. This gave 88.7mg of a pale yellow solid in 95.6% yield.¹H NMR(400MHz,DMSO-d₆)δ:10.15(s,1H,Ar-NH-),9.77(s,1H,Ar-OH),8.21(s,1H,＝CH),7.52(d,J＝7.6Hz,1H,Ar-H),7.43(d,J＝8.3Hz,2H),7.37(d,J＝7.6Hz,1H,Ar-H),7.21(s,1H,Ar-H),6.33(d,J＝8.3Hz,2H,Ar-H),4.56(br,2H)；¹³C NMR(101MHz,DMSO-d₆)δ:163.9,156.14,150.52,133.63,133.26,129.63,128.85,128.17,126.22,121.78(4),115.85,107.12,61.13。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acrylamide: 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamide was replaced with 170.1mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-3-hydroxymethyl-4-hydroxycinnamide, and the procedure was the same as in step (5) of example 1. After silica gel column chromatography (chloroform: methanol: 40:1, V: V), the mixture was separated and purified by gradient elution to obtain 157.8mg of a pale yellow solid, which was obtained in 54.3% yield.¹H NMR(300MHz,DMSO-d₆)δ:10.13(s,1H,-CONH-Ar),9.71(s,1H,Ar-NHCO-),9.57(s,1H,Ar-OH),8.33(d,J＝18.2Hz,1H),8.16(s,1H,＝CH),7.66(d,J＝6.2Hz,2H),7.57(d,J＝6.2Hz,2H),7.53(d,J＝8.5Hz,1H),7.25(m,5H,Ar-H),7.14(s,1H),7.05(br,1H,Bn-OH),6.86(d,J＝8.5Hz,1H),5.07(d,J＝13.1Hz,1H,CO-CH-N),4.68(s,2H,Ar-CH₂-O-),4.63(s,2H,Ar-CH₂-O),3.56(s,1H,O-CH₂),3.08(s,1H,O-CH₂),1.82(s,3H,CH₃)；¹³C NMR(75MHz,DMSO-d₆)δ:172.83,171.23,164.01,154.87,138.02,134.21,133.34,129.56,128.74,128.63(2),128.06,127.82,127.45(2),126.14,121.85(4),115.12,107.11,72.32,69.11,61.25,57.14,53.34,22.93.ESI-MS(m/z):551.8for C₂₉H₂₈N₄O₆Na([M+Na]⁺).(C＝2，CH₃OH)。

The above data demonstrate that this compound is (E) -N- (4- (N-acetyl-O-benzyl-D-seryl) aminophenyl) - α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acrylamide (CHR-5N-C), and has the following structure:

example 6

(E) Preparation of-4- (N-acetyl-O-benzyl-D-seryl) -1- (α -cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G) comprising the following five steps:

(1) the procedure was as in (1) of example 4.

(2) Preparation of (E) -4-Boc-1- (α -cyano-3, 4-dihydroxycinnamoyl) piperazine: the procedure was as in (2) of example 4. 2.29g (11.0mmol) of 4-N-Boc-aminoaniline was changed to 2.05g (11.0mmol) of N-Boc-piperazine. 2.34g of pale yellow solid is obtained with a yield of 62.7%.¹H NMR(400MHz,CDCl₃)δ:9.57(s,2H,2×OH),8.15(s,1H,＝CH),7.52(d,J＝8.3Hz,1H,Ar-H),7.03(s,1H,Ar-H),6.86(d,J＝8.3Hz,1H,Ar-H),3.55-3.52(m,4H,CH₂),3.39(m,4H,CH₂),1.40(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:163.59,153.98,150.95,146.56,145.84,128.85,124.72,123.81,117.22,116.00,106.17,79.47,51.21(2),49.03(2),28.22(3)。

(3) Preparation of (E) - α -cyano-3, 4-dihydroxycinnamoyl piperazine: the procedure was as in (3) of example 1. The (E) -4-Boc-1- (alpha-cyano-3, 4-dihydroxycinnamoyl) piperazine obtained in the above step was charged to give 1.60g of a light brown solid, and the yield was 93.4%.¹H NMR(400MHz,CDCl₃)δ:9.63(s,2H,2×OH),8.16(s,1H),7.61(d,J＝8.7Hz,1H),7.03(s,1H,Ar-H),6.93(d,J＝8.7Hz,1H),3.36(m,4H),2.87(m,4H),1.23(br,1H)；¹³C NMR(101MHz,CDCl₃)δ:166.52,150.93,146.55,145.83,128.84,124.73,123.82,117.23,116.00,106.17,51.21(2),47.03(2)。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (α -cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide was replaced with 150.3mg (0.55mmol) of (E) - α -cyano-3, 4-dihydroxycinnamoylpiperazine. The resulting product was subjected to silica gel column chromatography (chloroform: methanol: 40:1, V: V) and gradient elution to obtain 145.7mg of a pale yellow solid, which was obtained in 53.8% yield.¹H NMR(400MHz,DMSO-d₆)δ:9.52(s,2H,2×Ar-OH),8.36(s,1H,Ac-NH-),8.13(s,1H,Ar-CH＝),7.53(d,J＝8.1Hz,1H),7.33(m,5H,Ar-H),7.08(s,1H,Ar-H),6.95(d,J＝8.1Hz,1H),4.75(m,1H,-CO-CH-N-),4.67(s,2H,Ar-CH₂ O-),3.62(m,1H),3.53(m,4H),3.39(m,4H),3.33(m,1H),1.86(s,3H,CH₃-CO-) (see fig. 1);¹³C NMR(101MHz,DMSO-d₆)δ:172.61,170.13,168.04,151.22,146.63,145.92,138.05,129.31,128.64(2),127.81,127.47(2),123.18,116.97,115.92,115.17,107.15,72.33,69.98,54.91,49.53(2),48.95(2),22.94.ESI-MS(m/z):493.4C₂₆H₂₉N₄O₆([M+H]⁺) (see FIG. 2); HR-ESI-MS (m/z):493.2087 (see FIG. 3);(C＝2,CH₃OH).

the above data demonstrate that the compound is (E) -4- (N-acetyl-O-benzyl-D-serinyl) -1- (α -cyano-3- (3, 4-dihydroxyphenyl) acryloyl) piperazine (CHR-5N-G) and has the following structure:

example 7

(E) Preparation of-4- (N-acetyl-O-benzyl-D-seryl) -1- (α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acryloyl) piperazine (CHR-5N-H) comprising the following five steps:

(1) the procedure was as in (1) of example 5.

(2) (E) -4-Boc-1- (. alpha. -cyano-3-hydroxymethyl-4-hydroxycinnamoylYl) piperazine preparation: the procedure was as in (2) of example 5. 2.29g (11.0mmol) of 4-N-Boc-aminoaniline was changed to 2.05g (11.0mmol) of N-Boc-piperazine. 2.55g of light yellow solid is obtained with a yield of 65.8%.¹H NMR(400MHz,CDCl₃)δ:9.63(s,1H,Ar-OH),8.13(s,1H,＝CH),7.48(d,J＝8.2Hz,1H,Ar-H),7.31(d,J＝8.2Hz,1H,Ar-H),7.13(s,1H,Ar-H),7.05(s,1H,Ar-H),4.65(s,2H,O-CH₂-Ar),3.36(m,4H,CH₂),3.32(m,4H,CH₂),1.43(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:167.52,155.46,154.81,150.65,129.85,128.73,128.15,126.32,117.22,116.07,115.85,106.94,79.86,51.03(2),48.81(2),28.52(3)。

(3) Preparation of (E) - α -cyano-3-hydroxymethyl-4-hydroxycinnamoylpiperazine: the procedure was as in (3) of example 1. The (E) -4-Boc-1- (alpha-cyano-3-hydroxymethyl-4-hydroxycinnamoyl) piperazine obtained in the above step was charged to give 1.75g of a light brown solid in a yield of 92.5%.¹H NMR(400MHz,CDCl₃)δ:9.65(s,1H,Ar-OH),8.16(s,1H,＝CH),7.45(d,J＝8.2Hz,1H,Ar-H),7.29(d,J＝8.2Hz,1H,Ar-H),7.12(s,1H,Ar-H),7.04(s,1H,Ar-H),4.62(s,2H,O-CH₂-Ar),3.25(m,4H,CH₂),2.82(m,4H,CH₂),1.08(br,1H)；¹³C NMR(101MHz,DMSO-d₆)δ:167.71,155.47,150.58,129.93,128.81,128.09,126.26,116.05,115.83,106.96,51.62(2),47.33(2)。

(4) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of (E) -4- (N-acetyl-O-benzyl-D-seryl) -1- (α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acryloyl) piperazine: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamoyl amide was replaced with 158.1mg (0.55mmol) of (E) - α -cyano-3-hydroxymethyl-4-hydroxycinnamoyl piperazine. After silica gel column chromatography (chloroform: methanol: 40:1, V: V), the mixture was separated and purified by gradient elution to obtain 153.8mg of a pale yellow solid with a yield of 55.2%.¹H NMR(400MHz,DMSO-d₆)δ:9.63(s,1H,Ar-OH),8.33(s,1H,Ac-NH-),8.12(s,1H,Ar-CH＝),7.49(d,J＝8.2Hz,1H),7.33(m,5H,Ar-H),7.29(d,J＝8.2Hz,1H),7.05(s,1H,Bn-OH),4.76(m,1H,-CO-CH-N-),4.66(s,2H,Ph-CH₂O-),4.63(s,2H,Ar-CH₂O-),3.59(m,1H),3.48(m,4H),3.36(m,4H),3.32(m,1H),1.87(s,3H,CH₃-CO-)；¹³C NMR(101MHz,DMSO-d₆)δ:170.73,169.84,167.23,155.45,150.56,137.52,129.61,128.75,128.62(2),128.03,127.83,127.52(2),126.18,116.06,115.83,107.13,72.35,70.02,60.51,54.93,49.46(2),48.94(2),22.95.ESI-MS(m/z):507.6C₂₇H₃₁N₄O₆([M+H]⁺)；(C＝2,CH₃OH).

The above data demonstrate that this compound is (E) -4- (N-acetyl-O-benzyl-D-serinyl) -1- (α -cyano-3- (3-hydroxymethyl-4-hydroxyphenyl) acryloyl) piperazine (CHR-5N-H), having the structure:

example 8

The preparation of N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) -6-hydroxy-beta-naphthamide (CHR-5Y-E1) comprising the following four steps:

(1) preparation of N- (N-Boc-4-aminocyclohexyl) -6-hydroxy- β -naphthylamide: the procedure was as in (2) of example 2. 1.89g (10.0mmol) of 4-hydroxy-alpha-cyanocinnamic acid was changed to 1.88g (10.0mmol) of 6-hydroxy-beta-naphthoic acid to give 3.63g of a pale yellow solid with a yield of 94.5%.¹H NMR(300MHz,DMSO-d₆)δ:9.99(s,1H,OH),8.33(s,1H,＝CH-CO),8.07(m,1H,Ar-H),7.85(m,2H,Ar-H),7.72(m,2H),7.46(m,1H),7.26(m,1H),3.88(m,1H),3.46(m,1H),1.78(s,4H,CH₂),1.59(s,4H,CH₂),1.39(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:166.95,157.73,154.99,134.94,131.48(2),130.92,129.09,127.41,126.65,125.75,124.70,119.32,108.62,77.44,53.76,51.61,28.71(2),28.63(2),28.41(3)。

(2) Preparation of N- (4-aminocyclohexyl) -6-hydroxy- β -naphthylamide: the procedure was as in (3) of example 1. The N- (N-Boc-4-amino ring obtained in the above step is addedHexyl) -6-hydroxy-beta-naphthylamide, 2.45g of a light brown solid was obtained in a yield of 91.2%.¹H NMR(300MHz,DMSO-d₆)δ:9.28(s,1H,OH),8.31(s,1H),8.08(m,1H,Ar-H),7.83(m,2H,Ar-H),7.72(m,1H),7.46(s,1H),7.28(m,1H),3.88(m,1H),3.55(m,1H),2.58(m,1H),1.78(m,2H,CH₂),1.73(m,2H),1.54(m,2H),1.51(m,2H)；¹³C NMR(101MHz,DMSO-d₆)δ:167.23,158.13,133.54,131.81,131.32,130.93,128.29,126.67,124.73,118.35,109.14,51.65,50.32,31.41(2),28.73(2)。

(3) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(4) Preparation of N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) -6-hydroxy- β -naphthylamide: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide was replaced by 156.4mg (0.55mmol) of N- (4-aminocyclohexyl) -6-hydroxy- β -naphthamide. The resulting extract was subjected to silica gel column chromatography (chloroform: methanol: 40:1, V: V) and gradient elution to obtain 181.4mg of a pale yellow solid with a yield of 65.5%.¹H NMR(300MHz,CDCl₃)δ:10.06(s,1H,OH),8.34(s,1H,Ac-NH-),8.31(s,1H,Ar-H),8.14(d,J＝7.4Hz,1H,-NH-CO-),8.08(d,J＝8.7Hz,1H,Ar-H),7.81(m,2H,Ar-H),7.72(d,J＝8.7Hz,1H,Ar-H),7.45(s,1H,Ar-H),7.34(m,5H,Ar-H),7.26(m,1H,Ar-H),4.67(m,1H,CO-CH-NH),4.62(s,2H,Ar-O-CH₂-),3.58(m,1H),3.53(m,2H),3.34(m,1H),1.88(s,3H,CH₃-CO-),1.81(m,2H),1.76(m,2H),1.53(m,2H),1.49(m,2H)；¹³C NMR(101MHz,DMSO-d₆)δ:171.42,170.69,167.18,158.14,138.25,135.95,130.49,129.07,128.20,127.45,126.62,125.75,124.76,119.36,108.63,72.03,70.29,52.49,46.84,45.22,28.01,27.47,22.56.ESI-MS(m/z):526.7C₂₉H₃₃N₃O₅([M+Na]⁺)； (C＝2,CH₃OH)。

The above data confirm that this compound is N- (4- (N-acetyl-O-benzyl-D-seryl) aminocyclohexyl) -6-hydroxy- β -naphthamide (CHR-5Y-E1), the structure of which is shown below.

Example 9

The preparation of 4- (N-acetyl-O-benzyl-D-seryl) -1- (6-hydroxy- β -naphthoyl) piperazine (CHR-5Y-G) comprises the following four steps:

(1) preparation of 4-Boc-1- (6-hydroxy- β -naphthoyl) piperazine: the procedure was as in (2) of example 3. 1.89g (10.0mmol) of 4-hydroxy-alpha-cyanocinnamic acid was replaced with 1.88g (10.0mmol) of 6-hydroxy-beta-naphthoic acid to give 2.34g of a pale yellow solid in 65.8% yield.¹H NMR(400MHz,CD₃OD)δ:9.23(s,1H,OH),8.31(s,1H,Ar-H),8.08(d,J＝8.5Hz,1H,Ar-H),7.77(d,J＝9.7Hz,1H,Ar-H),7.69(d,J＝8.5Hz,1H,Ar-H),7.41(s,1H,Ar-H),7.26(d,J＝9.7Hz,1H，Ar-H),3.61(m,4H,CH₂),3.35(m,4H),1.47(s,9H,3×CH₃)；¹³C NMR(101MHz,CD₃OD)δ:172.87,157.82,155.95,136.89,131.81,131.32,130.89,128.47,126.59,125.12,120.26,118.33,109.70,81.39,50.92(2),49.81(2),28.39。

(2) Preparation of 6-hydroxy- β -naphthoylpiperazine: the procedure was as in (3) of example 1. The 4-Boc-1- (6-hydroxy-. beta. -naphthoyl) piperazine obtained in the above step was charged to give 1.55g of a pale brown solid, yield 91.8%.¹H NMR(400MHz,CD₃OD)δ:9.21(s,1H,OH),8.29(s,1H,Ar-H),8.10(d,J＝8.3Hz,1H,Ar-H),7.79(d,J＝9.5Hz,1H,Ar-H),7.68(d,J＝8.3Hz,1H,Ar-H),7.43(s,1H,Ar-H),7.28(d,J＝9.5Hz,1H，Ar-H),3.48(m,4H,CH₂),2.92(m,4H)；¹³C NMR(101MHz,CD₃OD)δ:169.02,158.23,134.04,131.78,131.29,130.89,128.36,126.65,124.80,118.33,109.20,52.67(2),47.11(2)。

(3) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(5) Preparation of 4- (N-acetyl-O-benzyl-D-seryl) -1- (6-hydroxy- β -naphthoyl) piperazine: the procedure was as in (5) of example 1. Will (a) toE) 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide was replaced with 141.0mg (0.55mmol) of 6-hydroxy- β -naphthoylpiperazine. The resulting product was subjected to silica gel column chromatography (chloroform: methanol: 40:1, V: V) and gradient elution to separate and purify the product, to obtain 136.8mg of a pale yellow solid, with a yield of 52.3%.¹H NMR(400MHz,DMSO-d₆)δ:9.98(s,1H,Ar-OH),8.35(d,J＝8.1Hz,1H,Ar-H),8.30(s,1H,Ar-H),8.25(d,J＝8.2Hz,1H,Ar-H),7.84(d,J＝8.6Hz,1H,Ar-H),7.68(d,J＝8.2Hz,1H),7.32(m,5H,Ar-H),7.28(d,J＝8.6Hz,1H,Ar-H),4.75(m,1H,-CO-CH-N-),4.62(s,2H,Ar-CH₂-O),3.58(m,1H),3.61(m,4H),3.52(m,4H),3.35(m,1H),1.85(s,3H,CH₃CO-)；¹³C NMR(101MHz,DMSO-d₆)δ:170.59,169.72,168.85,158.19,137.56,134.07,131.18,130.93,128.65(2),128.27,127.61,127.53,127.46,126.90,126.71,126.13,124.87,119.43,108.66,72.19,69.64,50.02(2),49.42(2),22.93.ESI-MS(m/z):474.5C₂₇H₂₉N₃O₅([M-H]⁺)；(C＝2,CH₃OH).

The above data demonstrate that the compound is 4- (N-acetyl-O-benzyl-D-serinyl) -1- (6-hydroxy- β -naphthoyl) piperazine (CHR-5Y-G) and has the following structure:

example 10

The preparation of 3-N- (N-acetyl-O-benzyl-D-seryl) -7-hydroxy-3-quinolinecarboxylatepropanediamine (CHR-5H) comprises the following four steps:

(1) preparation of N- (3-N-Boc-aminopropyl) -7-hydroxy-3-quinolinecarboxamide: the procedure was as in (2) of example 1. 1.89g (10.0mmol) of 4-hydroxy- α -cyanocinnamic acid was replaced by 1.89g (10.0mmol) of 7-hydroxy-3-quinolinecarboxylic acid; 2.29g (11.0mmol) of 4-N-Boc-aminoaniline was replaced with 1.92g (11.0mmol) of N-Boc-1, 3-propanediamine to give 2.51g of a pale yellow solid in a yield of 72.7%.¹H NMR(400MHz,DMSO-d₆)δ:9.42(s,1H,Ar-OH),8.83(s,1H,-C-CH＝C-),8.63(s,1H,-N＝CH-),8.35(m,1H，ArCO-NH-),7.90(d,J＝8.9Hz,1H,NH),7.47(m,2H,Ar-H),6.80(m,1H,-NH-Boc),3.21(m,4H),1.78(m,2H),1.37(s,9H,3×CH₃)；¹³C NMR(101MHz,DMSO-d₆)δ:166.1,160.5,156.1,149.7,146.5,136.3,131.6,125.9,124.6,119.1,110.5,79.7,39.6,37.3,28.8,28.5(3)。

(2) Preparation of N- (3-aminopropyl) -7-hydroxy-3-quinolinecarboxamide: the procedure was as in (3) of example 1. The N- (3-N-Boc-aminopropyl) -7-hydroxy-3-quinolinecarboxamide obtained in the above step was charged to give 1.54g of a light brown solid in a yield of 95.3%.¹H NMR(400MHz,DMSO-d₆)δ:9.39(s,1H,Ar-OH),8.81(s,1H,-C-CH＝C-),8.64(s,1H,-N＝CH-),8.34(m,1H,ArCO-NH-),7.87(d,J＝8.9Hz,1H,NH),7.45(m,2H,Ar-H),3.19(m,2H),2.69(m,2H),1.89(m,2H)；¹³C NMR(101MHz,DMSO-d₆)δ:165.8,160.3,149.6,146.4,136.1,131.7,125.8,124.5,119.2,110.6,39.2,35.8,28.6。

(3) Preparation of N-acetyl-O-benzyl-D-serine: the procedure was as in (4) of example 1.

(4) Preparation of 3-N- (N-acetyl-O-benzyl-D-seryl) -7-hydroxy-3-quinolinecarboxylatepropanediamine: the procedure was as in (5) of example 1. 153.6mg (0.55mmol) of (E) -N- (4-aminophenyl) - α -cyano-4-hydroxycinnamamide was replaced by 134.9mg (0.55mmol) of N- (3-aminopropyl) -7-hydroxy-3-quinolinecarboxamide. After silica gel column chromatography (chloroform: methanol: 40:1, V: V), the mixture was separated and purified by gradient elution to obtain 167.8mg of a pale yellow solid with a yield of 65.7%.¹H NMR(400MHz,DMSO-d₆)δ:10.49(s,1H,OH),9.13(s,1H,＝CH-CO),8.65(s,1H,-N＝CH-),8.43(s,1H,ArCO-NH-),8.33(m,1H,Ac-NH-),8.03(m,1H,-NH-CO),7.90(d,J＝8.8Hz,1H,Ar-H),7.47(m,2H,Ar-H),7.32(m,5H),4.74(m,1H,CO-CH-N),4.61(s,2H,-CH₂-OAr),3.61(m,1H),3.32(m,1H),3.17(m,4H),2.36(m,2H),1.85(s,3H,CH₃CO-)；¹³C NMR(101MHz,DMSO-d₆)δ:173.32,170.91,165.73,160.22,150.65,149.16,137.90,136.15,131.64,128.56(2),127.69,127.42(2),125.48,124.38,120.88,119.06,110.11,73.00,70.11,57.44,39.52,37.35,28.67,22.25.ESI-MS(m/z):465.6C₂₅H₂₈N₄O₅([M+H]⁺)；(C＝2,CH₃OH).

The above data demonstrate that the compound is 3-N- (N-acetyl-O-benzyl-D-seryl) -7-hydroxy-3-quinolinecarboxylpropanediamine (CHR-5H), having the following structure:

according to the synthetic routes and methods of the compounds of formula I disclosed in the present application, and the synthetic methods of the specific compounds of examples 1-10, those skilled in the art can adjust the raw materials involved in the synthetic methods according to the structures of the target products, so as to synthesize other compounds listed in table 1, which are not listed here.

Example 11

Inhibitory Activity of Compounds in Table 1 against aldose reductase

In the present invention, Aldose Reductase (AR), reduced coenzyme II (NADPH), DL-glyceraldehyde were purchased from Sigma; epalrestat (Epalrestat) was purchased from tokyo chemical industry co.

The experimental steps are as follows: the enzyme reaction system is carried out on a 96-well plate, and comprises: PBS buffer (pH 6.2)100 μ L, 1.5mmol/L NADPH 20 μ L, 100mmol/L DL-glyceraldehyde 20 μ L, different concentration (Epalrestat or test sample) solutions 20 μ L, AR dilutions 20 μ L, distilled water 20 μ L. The experimental setup was blank control, standard control, enzyme metabolism group as in table 2-1.

TABLE 2-1 grouping and dosing

Note: all volume units above are μ L.

After the liquid is incubated at 37 ℃ for 5min, 20 mu L of AR is added into each group except a blank solvent group to start reaction, a 96-well plate is quickly placed into a fluorescence microplate reader, the temperature is 37 ℃, the detection wavelength is 340nm, the change condition of NADPH absorbance is continuously detected, the detection is carried out once every 30s, and the total detection time is 30 min.

According to the results measured by the experiment, the descending values A of blank group NADPH absorbance are respectively calculated₀Decrease in enzyme Activity group (No sample) NADPH Absorbance A₁NADPH absorbance decrease values A of the Epalrestat group and the test sample group_EPS、A₂. The inhibition rate was determined by using the change in NADPH absorbance of each group:

sample inhibition rate for AR/% - [1- (a)₂-A₀)/(A₁-A₀)]×100％。

According to the enzyme reaction experiment result, the final concentration of the sample is used as the abscissa, the inhibition rate is used as the ordinate, an inhibition curve is drawn, and the IC of different samples is calculated according to the kinetic curve₅₀. All data are usedData statistics are shown to be processed by the SPSS10.0 software, with the inter-group comparisons tested with t. IC of positive control Epalrestat was determined₅₀75.64 + -4.22. Obtaining IC of target end product by the above method₅₀The results are shown in Table 2-2:

TABLE 2-2 IC of the target Compounds₅₀Value of

As can be seen from Table 2-2 and FIG. 4, the compound CHR-5N-G has the best inhibitory activity on aldose reductase in vitro, which is significantly stronger than the positive control drug epalrestat, and is also significantly stronger than the compound CHR-532R reported in patent CN 201410723116.8 and the compound CHR-5N-D reported in patent CN 201710372369.9;

except that the compounds CHR-5N-E, CHR-5Y-1 and CHR-5H-E, CHR-5H-G, CHR-5H-H showed weaker enzyme inhibitory activity than that of the positive drug epalrestat, the in vitro inhibitory activity of other compounds on aldose reductase was stronger than that of the positive control drug.

Example 12

In vitro antioxidant Activity of the Compounds in Table 1

In this embodiment, Trolox (6-Hydroxy-2,5,7, 8-tetramethylhydroxychroman-2-carboxylic acid); 1, 1-diphenylyl-2-piperidinylhydrazyl (DPPH) was purchased from Sigma.

The experimental steps are as follows: the reaction system was performed in a 96-well plate, and 40. mu.L of each sample solution was added to the 96-well plate, and then 160. mu.L of DPPH solution was added in parallel to each well. A control group (40. mu.L methanol + 160. mu.L DPPH) and a blank group (40. mu.L test sample + 160. mu.L methanol) were also set. Shaking for 1min to mix well, standing 96-well plate in dark condition for 0.5 hr, quickly placing the plate in microplate reader, and detecting wavelength at 517 nm. The final absorbance (A) was measured and each sample was run in triplicate and the average taken. The clearance rate is calculated as follows:

radical scavenging rate (%) ═ a_Control－(A_{Sample (I)}－A_{Blank space}))/A_Control×100％

A_ControlAbsorbance of control group without sample;

A_{sample (I)}Is the absorbance of the reaction solution after the test sample is added;

A_{blank space}Absorbance of blank.

All data are usedData statistics are shown to be processed by the SPSS10.0 software, with the inter-group comparisons tested with t. The results are shown in Table 3-1.

TABLE 3-1 inhibition of DPPH by the target Compounds at a concentration of 100. mu.M

The inhibition of DPPH by the compounds examined at 100. mu.M is shown in Table 3-1. As can be seen from Table 3-1, the compounds examined all showed a certain degree of radical scavenging, wherein the in vitro antioxidant activity of CHR-5Y-2, CHR-5Y-3 and CHR-5Y-E2 is stronger than that of the positive control Trolox.

For compounds with outstanding inhibitory activity and positive controls, to facilitate comparison of their inhibitory activity, a histogram of concentration-inhibition at three concentrations of 25 μ M, 12.5 μ M, 6.25 μ M is generated as shown in FIG. 5:

the half clearing concentration of Trolox, CHR-5Y-2 and CHR-5Y-3, namely EC is obtained by linear regression analysis of the logarithm of the concentration and the inhibition rate of the compound₅₀The results are shown in Table 3-2.

TABLE 3-2 Compounds correspond to EC₅₀Value of

As can be seen from the results of Table 3-2, the compounds CHR-5Y-2 and CHR-5Y-3 are superior in oxidation resistance and stronger than the positive drug Trolox.

Example 13

The influence of the compound CHR-5N-G on the survival rate, the death rate, the total teratogenicity and the body weight of chick embryos incubated in a high-sugar environment, the inhibitory activity on aldose reductase (ALR2), the inhibitory activity on aldehyde reductase (ALR1), the radical scavenging capacity (ORAC level), the influence on the content of Malondialdehyde (MDA) in vivo, the blood sugar regulation capacity, the sorbitol production inhibition capacity, the chicken embryo development regulation protein Pax-3 expression and the like.

Experimental materials: fresh fertilized eggs were purchased from southern China university of agriculture.

The experimental steps are as follows: the incubation temperature of the fresh fertilized eggs is kept at 36-38 ℃ and the relative humidity is kept at 65-75%. The groups were randomized into normal (CON), Glucose (GLU), CHR-5N-G low and medium three dose groups (DL, DM, DH) and Epalrestat (EPS), Edaravone (EDA) two positive control groups (EPS, EDA), 20 per group. In the hatch of the 0 th weather room, DL, DM, DH (20nM, 100nM, 500nM) and EPS, EDA (500nM each) were administered separately. Day 1D-glucose (0.4mM) was administered to all groups except CON group. On day 5 of incubation, embryos were removed, body weight, survival rate, teratogenesis and body weight were recorded, and embryo morphology was photographed using a stereomicroscope.

The results are shown in Table 4-1 and FIG. 6.

TABLE 4-1 Effect of CHR-5N-G on chick embryo survival, mortality, overall teratogenicity and body weight in high sugar environments

(**P<0.01vs.CON；^##P<0.01,^#P<0.05vs.GLU)

Chick embryo death was judged by the absence of heartbeats. As a result, the survival rate of the chick embryos of the high-sugar group (glucose group) is obviously increased compared with that of the normal group (p < 0.01); and mortality, overall teratogenicity and neural tube teratogenicity (NTD) were significantly increased compared to the normal group (p < 0.01).

The survival rate of the CHR-5N-G group is improved along with the increase of the concentration, the death rate, the total distortion rate and the weight average of the body are reduced in a dose-dependent manner, and the sugar group with higher effect of the high-concentration group has statistical difference significance (p is less than 0.01).

Compared with the glucose group, the EPS, EDA and other groups have obvious recovery of the total deformity rate and the body weight (p is less than 0.01).

After chicken embryos are fixed by 4% paraformaldehyde, the chicken embryos are photographed under a somatic microscope, as shown in fig. 6 and 7, the chicken embryos and neural tube malformations can be caused under a high sugar environment (fig. 6B and 7B1), and CHR-5N-G, EPS and EDA have obvious recovery effects on the chicken embryos and neural tube developmental malformations. Under the concentration of 500nM, CHR-5N-G is superior to positive control drugs EPS and EDA in all indexes of chick embryo development in a high-sugar environment.

In order to further research the protective effect of the compound CHR-5N-G on the high-sugar-content damaged chicken embryos and neural tube deformity, the shape of the whole embryo is observed by a body type microscope after the embryo of EDD5 is extracted, the shape is photographed and recorded, and then the development conditions of the embryo and the neural tube are observed by methods of paraffin embedding, HE staining and slicing. As shown in fig. 6 and 7, fig. 6A shows chicken embryos of CON group, which were full, normal in development of eyes, brain vacuoles, body and tail. The cross section of the embryo is shown in FIG. 7A1, with normal neural tube closure and balanced development of spinal cord, ganglion, and neural tube fibrous tissue. FIG. 6B shows GLU group embryos with malformations throughout the embryo shape, delayed blastocyst development, significantly smaller eyes than normal chick embryos, and abnormal tail curvature. A cross-section of the embryo is shown in FIG. 7B1, where the dorsal aspect of the neural tube fails to close, a severe deformity is developed, and the neural tube epithelial cells are found to have a disordered array distribution relative to normal chick embryos. In addition, peripheral accessory structures of the neural canal such as spinal cord, spinal ganglia, etc. also exhibited significant abnormalities compared to the GLU group. Fig. 6C and 6D are EPS and EDA groups, respectively, which showed significant protection of the chicken embryos, with clear neural tube closure and good but not complete recovery of the surrounding accessory structures (fig. 7C1 and 7D 1). FIGS. 6F, 6G, and 6H show the administration of high dose group DH, medium dose group DM, and low dose group DL, respectively, of CHR-5N-D, showing that the development of chicken embryos is gradually improved with increasing administration dose; as can be seen from fig. 7F1, 7G1, 7H1, neural tube closure was gradually complete and protection was dose-dependent. The DH group has completely closed nerve tubes and obviously better effect than a positive control group, which shows that the compound CHR-5N-G can well protect chicken embryos from being damaged by a high-sugar environment and inhibit the development deformity of the nerve tubes when the compound CHR-5N-G is at a high concentration (500 nM).

Taking chick embryos of the fifth embryo age day, and determining aldose reductase (ALR2) inhibitory activity, aldehyde reductase (ALR1) inhibitory activity, free radical scavenging capacity (ORAC level) of a compound CHR-5N-G, influence on the content of Malondialdehyde (MDA) in vivo, blood sugar regulation capacity, sorbitol production inhibitory capacity, influence on chick embryo development regulation protein Pax 3 expression and the like by adopting a corresponding experimental method and a corresponding kit.

The results of the experiment are shown in fig. 8, and all the groups of drugs had no effect of lowering blood glucose levels (fig. 8A). As can be seen from FIG. 8C, the inhibitory activity of the compound CHR-5N-G on ALR2 is prominent, and the inhibitory activity of the high concentration group is superior to that of the EPS group; the inhibition rate of CHR-5N-G on ALR2 is in direct proportion to the administration concentration, and the activity is outstanding, so that the inhibitor is a valid ALR2 inhibitor; and CHR-5N-G has no obvious inhibition effect on ALR1 of the homologous enzyme ALR2, and the inhibition effect is not different from that of positive drugs and high-sugar groups (p is more than 0.05) (figure 8B), which shows that the compound CHR-5N-G has selective inhibition effect on ALR2 and has high specificity.

As shown in FIG. 8, compared with CON group, the high sugar environment significantly increased the sorbitol content in embryonic tissue, and the target compound significantly inhibited the production of sorbitol, because CHR-5N-G has stronger ALR2 inhibitory activity, thereby inhibiting the polyol pathway and reducing the production of sorbitol (p <0.01) (FIG. 7D). The experimental result is consistent with the ALR2 inhibitory activity, is dose-dependent and is stronger than EPS.

As shown in FIG. 9, CHR-5N-G can significantly inhibit the production of Malondialdehyde (MDA) (p <0.01), and the activity is dose-dependent, and the inhibitory activity of DH group is equivalent to that of Edaravone (EDA) (FIG. 9E). Meanwhile, ORAC level experiments also show that CHR-5N-G has good free radical scavenging capacity, and the scavenging capacity is increased in a dose-dependent mode better than that of EDA (free radical scavenger) (figure 9F).

Pax 3 is responsible for regulating the differentiation of tissues and organs during embryonic development, and mutation or expression disorder thereof can cause certain nerve-related birth defects and genetic syndromes, such as neural tube malformations and the like. The experimental result is shown in figure 10, the compound CHR-5N-G can significantly enhance the expression of the Pax 3 protein, as shown in the figure, compared with the CON group, exogenously added high-concentration glucose reduces the expression level of the Pax 3 protein (p is less than 0.01), the positive drugs EPS and EDA can obviously restore the expression of the Pax 3 protein (p is less than 0.01), the compound CHR-5N-G can better restore the expression of the Pax 3 protein (p is less than 0.01), and the compound CHR-5N-G is dose-dependent, and the activity of the compound is obviously stronger than that of the positive drugs EPS and EDA group (p is less than 0.01).

In chick embryos, CHR-5N-G showed specificity for the inhibition of ALR2, but did not show significant inhibitory activity against its isoenzyme ALR 1. Therefore, CHR-5N-G is highly selective for the inhibition of ALR 2. CHR-5N-G remarkably inhibits the polyalcohol pathway of glucose metabolism by inhibiting the activity of ALR2, and prevents the production of a metabolite sorbitol in the pathway. The in vivo antioxidant experiment also shows that the compound can obviously inhibit the formation of oxidation product MDA in embryoid bodies, and the ORAC level shows that the compound has good free radical scavenging capacity. The experiments strongly prove that the compound CHR-5N-G can play a good role in protecting the chicken embryonic neural tube deformity induced by high sugar, the in-vivo enzyme inhibition activity and the in-vivo antioxidant activity of the compound are outstanding, the in-vivo enzyme inhibition activity and the in-vivo antioxidant activity are consistent with those of in-vitro experiments, the expression of a regulatory protein Pax 3 can be obviously improved, and the compound CHR-5N-G has great potential for developing into an aldose reductase inhibitor.

Example 14

Protective effect of compound CHR-5N-G on Diabetic Peripheral Neuropathy (DPN)

Experimental materials: male Sprague-Dawley rats 8 weeks old and weighing 170-. Rats were acclimatized for 1 week.

In the first week, rats were randomly divided into a control group (30) and a diabetic group (46). Rats in the diabetic group were continuously fed high-sugar and high-fat for 4 weeks, followed by streptomycin injections at weeks 4 and 8. After 12 weeks, the two groups of rats were divided into 4 groups: non-diabetic group [ (nDM) (ARI)^-)]15 without CHR-5N-G treatment; (nDM) (ARI)⁺) Group, non-diabetic rats but treated with CHR-5N-G, 15; DPN (ARI)^-) The group was diabetic peripheral neuropathy rats, not treated with CHR-5N-G, 20; DPN (ARI)⁺) The group was diabetic peripheral neuropathy rats, treated with CHR-5N-G, 26.

Establishment of DPN animal model and drug treatment: 46 rats were fed a high fat, high sugar diet for 4 consecutive weeks. The composition of the food is as follows: 32% carbohydrate, 28% fat, 17% protein and 23% other food products capable of inducing insulin resistance. Streptomycin was dissolved in citric acid/sodium citrate buffer solution. The first time after feeding for 4 weeks is to inject streptomycin intraperitoneally at a dose of 25 mg/Kg; streptomycin was injected intraperitoneally at 40mg/Kg a second time at week 8. Blood glucose concentration was measured by pricking blood through the tip of the tail of a rat, and blood glucose concentration was measured using a glucometer. Rats with blood glucose concentrations greater than 16mM were identified as diabetic rats, and rats less than 16mM were identified as non-diabetic rats.

All rats were electrically charged 12 weeks after streptomycin injectionThe physiological test method identifies whether DPN is present. (nDM) (ARI)⁺) Group sum DPN (ARI)⁺) Rats in group (after identification of DPN) were injected intraperitoneally with CHR-5N-G continuously for six weeks at a dose of 100 mg/kg/d. Subsequently, all rats were sacrificed and the sciatic nerve was rapidly removed from the right leg and stored in a frozen tube.

The electrophysiological detection method comprises the following steps: all rats were anesthetized with 10% chloroacetaldehyde hydrate (300mg/Kg body weight) 12 weeks after the second streptomycin injection and examined for neuropathy with an electrical stimulator and muscle force multiplier. An input electrode was placed near the distal end of the right leg and the ischial notch and a reference electrode was placed at the tail, thereby stimulating the right sciatic nerve. Stimulating the rat with a pulse width of 0.1ms and a threshold value of 1.5 times; the body temperature of rats is maintained at a rectal temperature of 37.5-37.9 ℃; the computer records the intensity of the action from the stimulated nerve to the peripheral muscles. The electric stimulation was performed 3 times with 1min interval, and the average was taken.

And (3) observing by a transmission electron microscope: after six weeks of treatment with CHR-5N-G, the rats were sacrificed. The sciatic nerve of the rat was quickly removed and cut into specimens that could be placed under a projection electron microscope for observation. The sample preparation method comprises the following steps: the sciatic nerve is solidified for 2 hours by 1% osmium tetroxide at room temperature for three times by using a arsenate buffer solution, and then is rinsed for three times by using the arsenate buffer solution and is placed in the buffer solution overnight; the samples were dehydrated with 30%, 50%, 70%, 90% ethanol and 70% acetone, respectively, embedded in resin, cut into small pieces, stained with 2% uranium acetate for 30min, rinsed with distilled water, treated with lead citrate for 30min, and finally rinsed with distilled water. The samples were tested at an accelerating voltage of 80 kV.

The experimental results are as follows: as can be seen from FIG. 11, nDM (ARI)^-) And nDM (ARI)⁺) The myelin nerve fiber layer of the group rats (normal rats) is clear, and the myelin structure is smooth, full and complete without distortion variants; the Schwann cell membrane is complete, the cell nucleus is clear and visible, and the nuclear membrane is coherent and has no fault; for DPN (ARI)^-) In rats (diabetic peripheral neuropathy model group), the myelin cells in the nerve fibers of the sciatic nerve were disrupted, and neurite shrinkage, malformation, and heterogeneity were observed,schwann cells are indistinct in structure, vacuoles degenerate, but the integrity of the cell membrane is not lost; non-myelin cell swelling with irregular cell morphology and enlarged cell space, Schwann cell contraction; DPN (ARI)⁺) Group (drug-treated group) rats: after CHR-5N-G treatment, the pathological structures of neurites and myelin are obviously improved, disordered myelin cells are recovered to be normal, and the swelling state of Schwann cells is improved. The CHR-5N-G has obvious treatment effect on the diabetic peripheral neuropathy.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.