WO2023071896A1 - Hydroxyprolyl-serine compound, and preparation and application thereof - Google Patents

Abstract

The present invention relates to the technical field of medicines, relates to a hydroxyprolyl-serine compound and a preparation method therefor, and further relates to a pharmaceutical composition comprising the compound and an application thereof in treating xerophthalmia. Provided are a hydroxyprolyl-serine compound having different stereoconfigurations, a preparation method therefor, a pharmaceutical composition comprising the compound, and an application of the hydroxyprolyl-serine having different stereoconfigurations in preparation of medicines for treating DED. The hydroxyprolyl-serine compound or salt thereof has the following structure (M).

Description

Hydroxyprolyl-serine compound and its preparation and application technical field

The invention belongs to the technical field of medicine, relates to a hydroxyprolyl-serine compound and a preparation method thereof, and also relates to a pharmaceutical composition containing the compound and its application in treating dry eye.

Background technique

Dry eye disease (DED) is a common ocular surface disease. The main pathological mechanisms include tear film instability, increased tear osmolarity, ocular surface inflammation and injury, and neurosensory abnormalities. The main clinical manifestations are dry and astringent eyes, foreign body sensation, blurred vision and other symptoms. Severe dry eye can also lead to corneal ulcers and even blindness. At present, according to the main components or functional abnormalities of tears, dry eye can be divided into tear deficiency, lipid abnormality, mucin abnormality, tear dynamics abnormality and mixed dry eye.

At present, the means of domestic treatment of dry eye are mainly drug therapy and surgical treatment. Commonly used clinical drugs mainly include artificial tears and immunosuppressants, such as sodium hyaluronate eye drops, glucocorticoid eye drops, cyclosporine eye drops, etc. For mild dry eye, the effect of artificial tears is better; for moderate dry eye, surgical treatment (such as punctal embolization, palpebral suture, etc.) or topical application of immunosuppressant eye drops Treatment; For severe dry eye, systemic anti-inflammatory or immunosuppressant drugs are often required. However, there are some drawbacks in the various existing therapeutic drugs and methods: artificial tears can only temporarily relieve eye symptoms but cannot cure them; long-term use of immunosuppressant drugs will cause serious side effects, such as systemic immunity ; Surgical treatment is traumatic and the curative effect in the later period is uncertain, so it cannot be widely used in clinical practice. Therefore, it is very necessary to develop a drug that can safely and effectively treat dry eye.

Trans-L-Hydroxyprolyl-L-serine is an antihepatitis drug

The dipeptide isolated from the injection has 3 chiral carbon atoms and 8 stereoisomers. After reviewing the literature, it was found that trans-L-hydroxyprolyl-L-serine has the potential to treat autoimmune hepatitis, and its mechanism of action is as follows: (1) Enhance liver tissue free radical scavenging ability, inhibit liver cell lipid peroxidation damage ; (2) Reduce the accumulation of inflammatory cells in areas of inflammation and tissue damage; (3) Inhibit the release of inflammatory cytokines. Currently, there is no report on the use of trans-L-hydroxyprolyl-L-serine in the treatment of DED.

In recent years, the study of chiral drugs has become a hot spot in the development of new drugs. Trans-L-hydroxyprolyl-L-serine is composed of two amino acids, trans-L-hydroxyproline and L-serine, in which hydroxyproline has 2 chiral carbon atoms and serine has 1 chirality carbon atom. The three-dimensional configuration of chiral drugs is closely related to the efficacy of drugs, and it is particularly important to study the pharmacodynamic differences between the enantiomers of chiral drugs. The relationship between isomers and DED efficacy can lay the foundation for the development of efficient and safe DED therapeutic drugs.

Contents of the invention

Aiming at the defects of the prior art, the present invention provides hydroxyprolyl-serine compounds of different stereoconfigurations and their preparation methods and pharmaceutical compositions containing the compounds, as well as the hydroxyprolyl-serine compounds of different stereoconfigurations. Application of serine in preparation of medicine for treating DED.

The present invention is achieved through the following technical solutions:

The present invention firstly provides a hydroxyprolyl-serine compound or a salt thereof with the following structure:

The present invention further provides a hydroxyprolyl-serine compound or a salt thereof with the following structure:

The present invention also provides a preparation method of the hydroxyprolyl-serine compound:

The preparation method of the hydroxyprolyl-serine compound comprises the following steps:

Step 1: Dissolve compound I, compound II, and N,N'-dicyclohexylcarboimide (DCC) in the reaction solvent, fill with nitrogen, react at 25-30°C, and rotate to evaporate to obtain compound III and compound The molar ratio of I and compound II is 0.1-10:1.

Step 2: Dissolve compound III and compound IV in a reaction solvent containing N,N-diisopropylethylamine (DIPEA), and react at 25-30°C for 2-24 hours to obtain compound V; compound III and compound IV The molar ratio is 0.1-10:1.

Step 3: Compound V is dissolved in different acids to obtain the target compound M.

Wherein, the reaction solvent described in the first step is one or two or more of dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylacetamide combination;

The reaction solvent described in the second step is one or a combination of two or more of dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, and N,N-dimethylacetamide;

The acid described in the third step is one or a combination of two or more of hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, p-toluenesulfonic acid.

The synthetic route is as follows:

can be

can be

a: N,N'-dicyclohexylcarboimide (DCC), reaction solvent (dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylethane amides);

b: N,N-diisopropylethylamine (DIPEA), reaction solvent (dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylacetamide) ;

c: Acids: hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, p-toluenesulfonic acid.

According to the different configurations of the reactants, the following compounds are obtained respectively: trans-4-L-hydroxyprolyl-L-serine shown in (A), trans-4-L-hydroxyprolyl shown in (B) -D-serine, trans-4-D-hydroxyprolyl-L-serine represented by (C), trans-4-D-hydroxyprolyl-D-serine represented by (D), (E) cis-4-L-hydroxyprolyl-L-serine shown, cis-4-L-hydroxyprolyl-D-serine shown in (F), cis-4-D shown in (G) One of the cis-4-D-hydroxyprolyl-D-serine shown in -hydroxyprolyl-L-serine, (H):

The present invention also provides a pharmaceutical composition, comprising the above-mentioned hydroxyprolyl-serine compound or its salt and a pharmaceutically acceptable carrier or excipient.

The invention provides the application of the hydroxyprolyl-serine compound or its salt in the preparation of medicine for treating dry eye syndrome.

The invention also provides the application of the pharmaceutical composition in the preparation of medicine for treating dry eye syndrome.

The beneficial effects of the present invention are as follows: based on the fact that hydroxyproline has 2 chiral carbon atoms and serine has 1 chiral carbon atom, due to the difference in stereoselectivity in the interaction between the chiral drug and the body, the Stereo configuration is closely related to pharmacodynamics. In the present invention, 4 different configurations of compound III are reacted with 2 different configurations of compound IV to form 8 different configurations of hydroxyprolyl-serine, and the effects of different enantiomers in treating DED are investigated. In a benzalkonium chloride (BAC)-induced dry eye model in rats, eight different configurations of hydroxyprolyl-serine had different efficacy against DED. Among them, the compound of Example 1 and the compound of Example 8 have the best therapeutic effects, that is, they can significantly improve dry eye symptoms, increase rat tear secretion, reduce corneal fluorescein staining scores, and play a good therapeutic effect. offers possibilities.

Description of drawings

Figure 1 is a single crystal X-ray diffraction characterization diagram of the compound of Example 1.

Figure 2 is a diagram of the dosing regimen of the compounds of Examples 1-8.

Fig. 3 is a comparison chart of tear secretion on the 14th day of the experiment (after 7 days of treatment) in DED rats treated with the compounds of Examples 1-8.

Fig. 4 is a score chart of corneal fluorescein sodium staining on the 14th day of the experiment (7 days after treatment) after the compound of Examples 1-8 treated DED rats.

Fig. 5 is the experiment 14th day (after 7 days of treatment) corneal fluorescein sodium staining microscopic observation figure of embodiment 1-8 compound to DED rat after treatment: (A) normal control group; (B) model group; (C) ) diquafosol sodium group; (D-K) compound groups of Examples 1-8.

Detailed ways

Example 1: Preparation of trans-4-L-hydroxyprolyl-L-serine (compound A):

Step 1: Dissolve compound I-1, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotary evaporate to obtain compound III-1 ;

Step 2: Dissolve compound III-1 and compound IV-1 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-1;

Step 3: Dissolve compound V-1 in hydrochloric acid, suction filter, wash, and lyophilize to obtain compound A (trans-4-L-hydroxyprolyl-L-serine).

Example 2: Preparation of trans-4-L-hydroxyprolyl-D-serine (compound B):

Step 1: Dissolve compound I-1, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotary evaporate to obtain compound III-1 ;

Step 2: Dissolve compound III-1 and compound IV-2 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-2;

Step 3: Compound V-2 was dissolved in hydrochloric acid, suction filtered, washed, and freeze-dried to obtain Compound B (trans-4-L-hydroxyprolyl-D-serine).

Example 3: Preparation of trans-4-D-hydroxyprolyl-L-serine (compound C):

Step 1: Dissolve compound I-2, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to evaporate to obtain compound III-2 ;

Step 2: Dissolve compound III-2 and compound IV-1 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-3;

Step 3: Dissolve compound V-3 in hydrochloric acid, filter with suction, wash, and lyophilize to obtain compound C (trans-4-D-hydroxyprolyl-L-serine).

Example 4: Preparation of trans-4-D-hydroxyprolyl-D-serine (compound D):

Step 1: Dissolve compound I-2, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to evaporate to obtain compound III-2 ;

Step 2: Dissolve compound III-2 and compound IV-2 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-4;

Step 3: Dissolve compound V-4 in hydrochloric acid, filter with suction, wash, and lyophilize to obtain compound D (trans-4-D-hydroxyprolyl-D-serine).

Embodiment 5: the preparation of cis-4-L-hydroxyprolyl-L-serine (compound E):

Step 1: Dissolve compound I-3, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to obtain compound III-3 ;

Step 2: Dissolve compound III-3 and compound IV-1 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-5;

Step 3: Compound V-5 was dissolved in hydrochloric acid, filtered with suction, washed, and freeze-dried to obtain Compound E (cis-4-L-hydroxyprolyl-L-serine).

Embodiment 6: the preparation of cis-4-L-hydroxyprolyl-D-serine (compound F):

Step 1: Dissolve compound I-3, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to obtain compound III-3 ;

Step 2: Dissolve compound III-3 and compound IV-2 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-6;

Step 3: Dissolve compound V-6 in hydrochloric acid, filter with suction, wash, and lyophilize to obtain compound F (cis-4-L-hydroxyprolyl-D-serine).

Example 7: Preparation of cis-4-D-hydroxyprolyl-L-serine (compound G):

Step 1: Dissolve compound I-4, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to obtain compound III-4 ;

Step 2: Dissolve compound III-4 and compound IV-1 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-7;

Step 3: Dissolve compound V-7 in hydrochloric acid, filter with suction, wash, and lyophilize to obtain compound G (cis-4-D-hydroxyprolyl-L-serine).

Example 8: cis-4-D-hydroxyprolyl-D-serine (compound H):

Step 1: Dissolve compound I-4, compound II, and N,N'-dicyclohexylcarboimide (DCC) in tetrahydrofuran, fill with nitrogen, react at 25°C for 2 hours, and rotate to obtain compound III-4 ;

Step 2: Dissolve compound III-4 and compound IV-2 in dichloromethane containing N,N-diisopropylethylamine (DIPEA), react at 25°C for 2h, evaporate the solvent under reduced pressure, and the residue is Dilute with dichloromethane, wash with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline successively, filter with suction, mix the filtrate with silica gel, and perform gradient elution with dichloromethane and methanol (100:1-1:100) through silica gel column chromatography , collecting the column chromatography and concentrating to obtain compound V-8;

Step 3: Compound V-8 was dissolved in hydrochloric acid, suction filtered, washed, and freeze-dried to obtain Compound H (cis-4-D-hydroxyprolyl-D-serine).

The identification information of the compounds of Examples 1-8 is shown in Table 1:

Table 1. Relevant information of the compounds of Examples 1-8

Embodiment 9: Pharmacodynamic study in SD rats

Dosing regimen

Taking healthy SD rats as model animals, 55 male SD rats with no abnormalities on the ocular surface were randomly divided into 11 groups, with 5 mice in each group, i.e. 10 eyes, respectively: normal control group, model group, positive drug group Diquafosol sodium group, embodiment 1 compound group, embodiment 2 compound group, embodiment 3 compound group, embodiment 4 compound group, embodiment 5 compound group, embodiment 6 compound group, embodiment 7 compound group, implement Example 8 Compound Group.

The rats in the normal control group did not receive any treatment, and the rats in the other 10 groups were instilled with 0.3% benzalkonium chloride (BAC) solution prepared in PBS, 10 μl per eye, twice a day (9am, 9pm), for 14 days , to establish a rat dry eye model.

The dosing regimen is as follows: according to its grouping, the positive drug diquafosol sodium eye drops and the compound solution (1 mg/ml) of Examples 1-8 are used for eye drop administration, each eye 10 μl, 4 times a day (11a. m., 1p.m., 5p.m., 7p.m.), administration began 7 days after modeling and continued for 7 days. The normal control group was not given eye drops, and the model group was replaced with eye drops of the same amount of normal saline. The dosing regimen is shown in Figure 2.

Detection indicators and methods

Basic tear secretion test (Schirmer Ⅰ test)

Lacrimal secretion was detected on the 0th, 7th, and 14th day of the experiment. Gently pull open the lower eyelid of the rat, and place one end of the special phenol red cotton thread for tear secretion test with a bent angle of about 1 mm in the lower fornix about one-third of the lateral corner of the rat, and take it out after 1 min without touching it during placement cornea. The length of the reddened part of the cotton thread is recorded as the result of the basic tear secretion test (mm). After successful modeling of dry eye, the basal tear secretion decreased.

Corneal fluorescein sodium staining score

Following the basic lacrimal secretion test, 8 μl of 1% sodium fluorescein solution was dropped into the conjunctival sac of the rat, and the eyes were artificially blinked gently. After 10 minutes, the excess staining was washed away with sterile saline, and the head of the rat was Placed under a slit lamp microscope, the upper and lower eyelids were separated to maintain the open state of the palpebral fissure, observed and photographed under a 25X slit lamp cobalt blue light, and evaluated according to the following scoring system: the cornea was divided into four quadrants, each quadrant The degree of staining is 0 to 3 points, and the total score is 0 to 12 points. Dry eye can lead to corneal epithelial damage and even secondary keratitis and corneal ulcers. After staining with sodium fluorescein, the corneal epithelial defect will be yellow-green, and the corresponding corneal fluorescein sodium staining score will be higher.

Table 2. Scores of corneal fluorescein sodium staining

statistical processing

The data were sorted out, processed by GraphPad Prism 8.0, and analyzed by independent sample t test. The results are shown in Figure 3 and Figure 4, where ### means p<0.005 compared with the normal control group; * means p<0.05 compared with the model group; ** means p<0.01 compared with the model group; * ** indicates p<0.005 compared with the model group.

result

Table 3. Therapeutic Effects of Embodiment 1-8 Compounds on DED Rats

in conclusion

It can be seen from the results of the above chart that when the compounds of Examples 1-8 are administered, the therapeutic effects of each compound are different. The test results of basic lacrimal secretion (Fig. 3) show that each compound increases the lacrimal secretion to varying degrees after treatment, especially, the lacrimal secretion of the compound in Example 1 is the closest to the result of the normal group after the compound treatment. There was no difference in the amount of tear secretion of the compound from the results of the positive control group, and compared with the model group, the tear secretion was significantly increased, indicating that they all had the effect of increasing tear secretion; the corneal fluorescein sodium staining score chart (Fig. The figure (Fig. 5) shows that the staining of the cornea of the compound of Example 1 is basically invisible after the drug treatment, which is equivalent to the results of the positive control group and the normal group, and the staining of the small area of the cornea can be seen in the compound of Example 8 (positive staining in the figure shows the brightness of the cornea) White area), both can significantly reduce the corneal fluorescein staining score, and there is a significant difference compared with the results of the model group. The final results show that: compared with the model group, each compound has the effect of improving dry eye symptoms and increasing tear secretion. In particular, the compounds of Example 1 and Example 8 have the best therapeutic effects, both of which can significantly improve dry eye symptoms, increase tear secretion, and reduce corneal fluorescein staining scores, providing the possibility for clinical treatment of DED.

Claims (10)

1. A hydroxyprolyl-serine compound or a salt thereof having the following structure:

   
2. The hydroxyprolyl-serine compound or its salt of claim 1, its structural formula is as follows:

   
3. The preparation method of hydroxyprolyl-serine compound as claimed in claim 1, is characterized in that:

   Step 1: Dissolve compound I, compound II, and N,N'-dicyclohexylcarboimide in a reaction solvent, fill with nitrogen, react, and rotary evaporate to obtain compound III;

   Step 2: dissolving compound III and compound IV in a reaction solvent containing N,N-diisopropylethylamine and reacting to obtain compound V;

   Step 3: dissolving compound V in acid to obtain target compound M;

   

   in,

   

   for

   

   

   for

   
4. The preparation method according to claim 3, wherein the reaction solvent in the first step or the second step is dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N, N-Dimethylacetamide.
5. The preparation method according to claim 3, wherein the acid in the third step is hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, p-toluenesulfonic acid.
6. The preparation method according to claim 3, characterized in that in the first step, the molar ratio of compound I to compound II is 0.1-10:1.
7. The preparation method according to claim 3, characterized in that, in the second step, the molar ratio of compound III to compound IV is 0.1-10:1.
8. A pharmaceutical composition, comprising one or more of the hydroxyprolyl-serine compounds or salts thereof as claimed in claim 1 or 2 and pharmaceutically acceptable carriers or excipients thereof.
9. A pharmaceutical preparation comprising one or more of the hydroxyprolyl-serine compounds or salts thereof according to claim 1 or 2 or the pharmaceutical composition according to claim 8.
10. One or more of the hydroxyprolyl-serine compounds described in claim 1 or 2 or the pharmaceutical composition described in claim 8 or the pharmaceutical preparation described in claim 9 in the preparation of the drug for the treatment of dry eye syndrome application.

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