Hydroxyprolyl-serine compounds, methods of making and using the same
By preparing hydroxyprolyl-serine compounds with different stereoconfigurations, the problems of low efficacy and large side effects of existing IBD treatment drugs were solved. The compound significantly improved colitis symptoms in mouse models, and the compound in Example 8 showed excellent therapeutic effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SUZHOU YUTAI PHARM TECH CO LTD
- Filing Date
- 2021-10-28
- Publication Date
- 2026-08-04
AI Technical Summary
Existing IBD treatments suffer from long treatment durations, low overall response rates, and severe side effects. The relationship between the stereoconfiguration and pharmacodynamics of chiral drugs has not been fully studied, and there are no reports of stereoisomer compounds being used to treat IBD in the current technology.
Hydroxyprolyl-serine compounds with different stereoconfigurations were prepared, and eight different hydroxyprolyl-serine compounds were formed through a combination reaction of specific solvents and acids for the treatment of inflammatory bowel disease.
In mouse models, some compounds significantly improved colitis symptoms, including improved weight loss, increased survival rate, reduced disease activity index, and increased colon length. In particular, the compound in Example 8 showed the best pharmacodynamic effects.
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Figure CN116041418B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, and relates to hydroxyprolyl-serine compounds and their preparation methods, as well as pharmaceutical compositions containing such compounds and their application in the treatment of inflammatory bowel disease. Background Technology
[0002] Inflammatory bowel disease (IBD) is a chronic, idiopathic enteritis that can affect the entire gastrointestinal tract and increase the risk of colorectal cancer. IBD mainly includes two types: ulcerative colitis (UC) and Crohn's disease (CD). Clinical manifestations mainly include diarrhea, abdominal pain, mucus and bloody stools, and weight loss, and are accompanied by extraintestinal diseases such as arthritis, erythema nodosum, iritis, ankylosing spondylitis, and sclerosing cholangitis.
[0003] Currently, the focus of IBD treatment is on controlling mucosal inflammation and suppressing overactive immune responses. Commonly used medications include aminosalicylic acids (such as sulfasalazine), glucocorticoids (such as methylprednisolone), and immunosuppressants (such as cyclosporine and tacomolidus). All of these drugs have drawbacks such as long treatment duration, low overall response rate, and severe side effects. Therefore, it is essential to develop highly effective and low-toxicity IBD drugs.
[0004] trans-L-hydroxyprolyl-L-serine is derived from anti-hepatitis drugs. The dipeptide isolated from the injection has certain anti-inflammatory activity. Trans-L-hydroxyprolyl-L-serine has a hepatoprotective effect against cholestatic hepatitis induced by α-naphthyl isothiocyanate (ANIT), and its mechanism of action is as follows: (1) enhancing the liver's free radical scavenging capacity and inhibiting lipid peroxidation damage in hepatocytes; (2) reducing the accumulation of inflammatory cells in areas of inflammation and tissue damage; and (3) inhibiting the release of inflammatory cytokines. Currently, there are no reports on the use of trans-L-hydroxyprolyl-L-serine for the treatment of IBD.
[0005] In recent years, research on chiral drugs has become a hot topic in new drug development. The stereoconfiguration of chiral drugs is closely related to their efficacy, making the study of efficacy differences between enantiomers of chiral drugs particularly important. Trans-L-hydroxyprolyl-L-serine is composed of two amino acids, trans-L-hydroxyproline and L-serine, which together have three chiral carbon atoms, resulting in eight stereoisomers. Currently, there are no reports of using stereoisomers to treat IBD. Furthermore, due to differences in compound configuration, their therapeutic effects on IBD also differ. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides hydroxyprolyl-serine compounds with different stereoconfigurations, their preparation methods, pharmaceutical compositions containing the compounds, and the application of the hydroxyprolyl-serine compounds with different stereoconfigurations in the preparation of drugs for treating IBD.
[0007] This invention is achieved through the following technical solution:
[0008] This invention first provides a hydroxyprolyl-serine compound or a salt thereof having the following structure:
[0009]
[0010] The present invention further provides hydroxyprolyl-serine compounds or salts thereof with the following structures:
[0011]
[0012] This invention also provides a method for preparing the aforementioned hydroxyprolyl-serine compound:
[0013] The preparation method of the hydroxyprolyl-serine compound includes the following steps:
[0014] Step 1: Dissolve compound I, compound II, and N,N'-dicyclohexylcarboimide (DCC) in a reaction solvent, purge with nitrogen, react at 25-30℃, and rotary evaporate to obtain compound III; the molar ratio of compound I to compound II is 0.1-10:1.
[0015] Step 2: Dissolve compound III and compound IV in a reaction solvent containing N,N-diisopropylethylamine (DIPEA) and react at 25-30℃ for 2-24 h to obtain compound V; the molar ratio of compound III to compound IV is 0.1-10:1.
[0016] Step 3: Dissolve compound V in different acids to obtain the target compound M.
[0017] The reaction solvent in the first step is one or a combination of two or more of the following: dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, and N,N-dimethylacetamide.
[0018] The reaction solvent in the second step is one or a combination of two or more of the following: dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, and N,N-dimethylacetamide.
[0019] The acid mentioned in the third step is one or a combination of two or more of the following: hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, and p-toluenesulfonic acid.
[0020] The synthesis route is as follows:
[0021]
[0022] for for a: N,N'-dicyclohexylcarboimide (DCC), reaction solvent (dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylacetamide);
[0023] b: N,N-Diisopropylethylamine (DIPEA), reaction solvent (dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylacetamide);
[0024] c: Acids: hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, p-toluenesulfonic acid.
[0025] Depending on the configuration of its reactants, the following compounds were obtained: one of the following: (A) trans-4-L-hydroxyprolyl-L-serine, (B) trans-4-L-hydroxyprolyl-D-serine, (C) trans-4-D-hydroxyprolyl-L-serine, (D) trans-4-D-hydroxyprolyl-D-serine, (E) cis-4-L-hydroxyprolyl-L-serine, (F) cis-4-L-hydroxyprolyl-D-serine, (G) cis-4-D-hydroxyprolyl-L-serine, and (H) cis-4-D-hydroxyprolyl-D-serine.
[0026]
[0027] The present invention also provides a pharmaceutical composition comprising the above-described hydroxyprolyl-serine compound or a salt thereof and a pharmaceutically acceptable carrier or excipient.
[0028] This invention provides the use of the hydroxyprolyl-serine compound or its salt in the preparation of a medicament for treating inflammatory bowel disease.
[0029] The present invention also provides the use of the pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease.
[0030] The inflammatory bowel disease mentioned is colitis.
[0031] The beneficial effects of this invention are as follows: Hydroxyproline has two chiral carbon atoms, and serine has one chiral carbon atom. Due to the stereoselective differences in the interaction between chiral drugs and the body, the stereoconfiguration of chiral drugs is closely related to their pharmacodynamics. This invention reacts four different configurations of compound III with two different configurations of compound IV to form eight different configurations of hydroxyprolyl-serine, and examines the effects of different enantiomers on treating IBD. In a mouse model of inflammatory bowel disease induced by dextran sulfate sodium (DSS), each compound showed varying degrees of improvement in the disease state of colitis. Compounds from Examples 1, 2, 4, and 8 effectively improved the disease state of colitis, specifically by reducing disease-induced weight loss, increasing survival rate, decreasing the Disease Activity Index (DAI) score, and prolonging colon length. Compound Example 8 exhibited the best pharmacodynamic effect, providing a potential treatment for DSS-induced IBD. Attached Figure Description
[0032] Figure 1 This is a single-crystal X-ray diffraction characterization pattern of the compound in Example 1.
[0033] Figure 2 This is a pharmacodynamic dosing regimen diagram for the compounds in Examples 1-8.
[0034] Figure 3 The graph shows the changes in mouse body weight before and after treatment with the compounds in Examples 1-8 for IBD.
[0035] Figure 4 The image shows the DAI score of mice after IBD treatment with the compounds in Examples 1-8.
[0036] Figure 5 This is a comparison of colon length in mice after IBD treatment with compounds from Examples 1-8. Detailed Implementation
[0037] Example 1: Preparation of trans-4-L-hydroxyprolyl-L-serine (compound A):
[0038]
[0039] Step 1: Compound I-1, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-1.
[0040] Step 2: Compound III-1 and compound IV-1 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-1 was obtained.
[0041] Step 3: Dissolve compound V-1 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound A (trans-4-L-hydroxyprolyl-L-serine).
[0042] Example 2: Preparation of trans-4-L-hydroxyprolyl-D-serine (compound B):
[0043]
[0044] Step 1: Compound I-1, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-1.
[0045] Step 2: Compound III-1 and compound IV-2 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-2 was obtained.
[0046] Step 3: Dissolve compound V-2 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound B (trans-4-L-hydroxyprolyl-D-serine).
[0047] Example 3: Preparation of trans-4-D-hydroxyprolyl-L-serine (compound C):
[0048]
[0049] Step 1: Compound I-2, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-2.
[0050] Step 2: Compound III-2 and compound IV-1 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-3 was obtained.
[0051] Step 3: Dissolve compound V-3 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound C (trans-4-D-hydroxyprolyl-L-serine).
[0052] Example 4: Preparation of trans-4-D-hydroxyprolyl-D-serine (compound D):
[0053]
[0054] Step 1: Compound I-2, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-2.
[0055] Step 2: Compound III-2 and compound IV-2 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-4 was obtained.
[0056] Step 3: Dissolve compound V-4 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound D (trans-4-D-hydroxyprolyl-D-serine).
[0057] Example 5: Preparation of cis-4-L-hydroxyprolyl-L-serine (compound E):
[0058]
[0059] Step 1: Compound I-3, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-3.
[0060] Step 2: Compound III-3 and compound IV-1 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-5 was obtained.
[0061] Step 3: Dissolve compound V-5 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound E (cis-4-L-hydroxyprolyl-L-serine).
[0062] Example 6: Preparation of cis-4-L-hydroxyprolyl-D-serine (compound F):
[0063]
[0064] Step 1: Compound I-3, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-3.
[0065] Step 2: Compound III-3 and compound IV-2 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-6 was obtained.
[0066] Step 3: Dissolve compound V-6 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound F (cis-4-L-hydroxyprolyl-D-serine).
[0067] Example 7: Preparation of cis-4-D-hydroxyprolyl-L-serine (compound G):
[0068]
[0069] Step 1: Compound I-4, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-4.
[0070] Step 2: Compound III-4 and compound IV-1 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-7 was obtained.
[0071] Step 3: Dissolve compound V-7 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound G (cis-4-D-hydroxyprolyl-L-serine).
[0072] Example 8: cis-4-D-hydroxyprolyl-D-serine (compound H):
[0073]
[0074] Step 1: Compound I-4, compound II, and N,N'-dicyclohexylcarboimide (DCC) were dissolved in tetrahydrofuran, purged with nitrogen, and reacted at 25°C for 2 hours. The mixture was then rotary evaporated to obtain compound III-4.
[0075] Step 2: Compound III-4 and compound IV-2 were dissolved in dichloromethane containing N,N-diisopropylethylamine (DIPEA) and reacted at 25°C for 2 hours. The solvent was removed by vacuum distillation, and the residue was diluted with dichloromethane and washed successively with 5% phosphoric acid aqueous solution, aqueous solution, and saturated saline solution. The mixture was filtered, and the filtrate was mixed with silica gel and subjected to silica gel column chromatography with gradient elution of dichloromethane and methanol (100:1-1:100). The column chromatogram was collected, concentrated, and compound V-8 was obtained.
[0076] Step 3: Dissolve compound V-8 in hydrochloric acid, filter, wash, and freeze-dry to obtain compound H (cis-4-D-hydroxyprolyl-D-serine).
[0077] The identification information of compounds in Examples 1-8 is shown in Table 1:
[0078] Table 1. Relevant information of compounds in Examples 1-8
[0079]
[0080]
[0081] Example 9: In vivo pharmacodynamic study in C57BL / 6 mice
[0082] Dosing regimen
[0083] Healthy C57BL / 6 mice were used as model animals and randomly divided into groups of 8. A mouse model of experimental colitis was established using sodium dextran sulfate (DSS, 3%). Except for the control group, which had free access to water, the other groups had free access to 3% DSS solution for 5 days. The drug administration regimen was as follows: Figure 2 As shown in the figure. The experiment consisted of 11 groups: a blank group, a model group, a positive control group (sulfasalazine), a group containing compounds from Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, and Example 8. Sulfasalazine (250 mg / kg) was administered orally once daily, while compounds from Examples 1-8 (25 mg / kg) were administered orally twice daily. The normal control group and the model group were administered an equal volume of physiological saline instead of the compounds from the examples to C57BL / 6 mice.
[0084] Detection indicators and methods
[0085] Weight change rate
[0086] The mice were weighed daily during the experiment. The rate of change in mouse body weight was calculated.
[0087] Mouse body weight change rate (%) = (mouse body weight - mouse initial body weight) / mouse initial body weight × 100%
[0088] Disease Activity Index Score
[0089] During the experiment, mouse body weight, fecal characteristics, and bloody stool were observed and scored daily, as shown in Table 2. The DAI index was the sum of the weight loss score, fecal characteristics score, and bloody stool score. The bloody stool score was based on the color development of the fecal occult blood test kit, and the specific steps were as follows: First, a fecal sample the size of a mung bean was placed on a white plate, and 2 drops (approximately 0.1 mL) of O-tolidine Solution were added to different locations on the feces. Then, 2 drops (approximately 0.1 mL) of freshly prepared oxidant were added, and the time was immediately recorded and the color change was observed. The reading was interpreted based on the color change within 2 minutes.
[0090] Table 2. Disease Activity Index Scores
[0091]
[0092] Measurement of colon length
[0093] The main characteristic of the DSS-induced ulcerative colitis model is the shortening of the colon accompanied by edema. Colon length can, to some extent, serve as an indicator of the severity of inflammation. The specific steps are as follows: the abdominal cavity is quickly opened, the colon and distal ileum are freed, the entire intestinal segment from the anus to the terminal cecum is removed, and the length of the entire intestinal segment from the anus to the terminal cecum is measured in each group.
[0094] Statistical processing
[0095] Data was processed using GraphPad Prism 5.0, and independent samples t-tests were used for data analysis. The results are as follows: Figure 3 , Figure 4 and Figure 5 As shown, ###, p<0.005 compared to the blank group; *, p<0.05 compared to the model group; **, p<0.01 compared to the model group; ***, p<0.005 compared to the model group.
[0096] result
[0097] Table 3. Experimental results of compounds in Examples 1-8 for the treatment of ulcerative colitis.
[0098]
[0099]
[0100] in conclusion
[0101] The results are as follows Figure 3 , 4 As shown in Figures 5 and 6, the results indicate that all compounds administered in Examples 1-8 exhibited a certain therapeutic effect on IBD. Compared to the model group, compounds in Examples 1, 2, 4, and 8 effectively improved the disease state of colitis, specifically by reducing weight loss caused by the disease. Figure 3 ), improve survival rate, and reduce disease activity index (DAI) score ( Figure 4 ), lengthen the colon ( Figure 5 Of these, the compound in Example 8 exhibited the best pharmacodynamic effects, offering a potential treatment for DSS-induced IBD.
Claims
1. The use of the following hydroxyprolyl-serine compounds or their salts in the preparation of drugs for treating inflammatory bowel disease: 。 2. The application as described in claim 1, characterized in that: The hydroxyprolyl-serine compound or its salt is prepared by the following method: Step 1: Dissolve compound I, compound II, and N,N'-dicyclohexylcarboimide in a reaction solvent, purge with nitrogen, react, and rotary evaporate to obtain compound III; Step 2: Dissolve compound III and compound IV in a reaction solvent containing N,N-diisopropylethylamine and react to obtain compound V; Step 3: Dissolve compound V in acid to obtain compound M; ； in, for , , ; for , .
3. The application as described in claim 2, characterized in that, The reaction solvent described in the first or second step is dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, or N,N-dimethylacetamide.
4. The application as described in claim 2, characterized in that, The acids mentioned in the third step are hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, and p-toluenesulfonic acid.
5. The application as described in claim 2, characterized in that, In the first step, the molar ratio of compound I to compound II is 0.1-10:
1.
6. The application as described in claim 2, characterized in that, In the second step, the molar ratio of compound III to compound IV is 0.1-10:
1.
7. The application as described in claim 1, characterized in that, The hydroxyprolyl-serine compound or its salt and its pharmaceutically acceptable carrier or excipient are prepared into a pharmaceutical composition.