A sulfur-containing 5-aminosalicylic acid prodrug and its uses

Abstract

This invention discloses a sulfur-containing 5-aminosalicylic acid prodrug and its uses, belonging to the field of biomedical technology. The sulfur-containing 5-aminosalicylic acid prodrug of this invention has the structure shown in general formula I. This type of compound has good intervention effects on inflammation-related diseases, exhibiting more ideal anti-inflammatory activity than 5-ASA and sulfasalazine. It can also inhibit the production of hypochlorous acid and related inflammatory mediators by inhibiting myeloperoxidase (MPO), and can be used to prepare drugs for the prevention and / or treatment of inflammatory bowel disease, and drugs for the prevention and / or treatment of diseases related to abnormal oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a sulfur-containing 5-aminosalicylic acid prodrug and its uses. Background Technology

[0002] Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn's disease (CD), affecting approximately 6 to 8 million people worldwide. As a chronic, progressive, and relapsing intestinal disease, IBD severely impacts patients' quality of life and daily activities. Currently available IBD treatments include non-targeted therapies (such as 5-aminosalicylic acid, glucocorticoids, and immunomodulators) and targeted therapies (such as monoclonal antibodies targeting TNFα and IL-12 / IL-23). ​​In clinical treatment, 5-aminosalicylic acid (5-ASA, also known as "mesalazine") and its prodrug derivatives are first-line drugs for treating mild to moderate ulcerative colitis. The mechanism of action of 5-ASA is complex; it is currently believed to primarily act on the colonic mucosa, inhibiting the activity of cyclooxygenase and lipoxygenase in the arachidonic acid metabolic pathway, thus reducing the levels of inflammatory mediators such as prostaglandins and leukotrienes. Furthermore, it can inhibit the activation of various inflammatory cells, reduce intestinal wall inflammation, and protect the intestinal mucosa. After oral administration, 5-ASA is mostly absorbed from the proximal small intestine, with a low amount specifically delivered to the colon, leading to repeated and high-dose administration to relieve symptoms. Furthermore, long-term, high-dose, frequent use can easily induce drug resistance.

[0003] To address the aforementioned issues, azoreductase-reactive prodrugs such as sulfasalazine, olsalazine, and balsalazine have been developed. These prodrugs rely on azoreductases specifically secreted by the colonic microbiota to release 5-ASA. Sulfasalazine, under enzymatic action, releases approximately 80% of its components in the colon as both 5-ASA and sulfapyridine; its active ingredient is 5-ASA, while sulfapyridine is more likely to cause adverse reactions. Orsalazine releases two molecules of 5-ASA, requiring only half the dosage of sulfasalazine, but it can lead to increased intestinal secretion and even diarrhea. It is noteworthy that although colonic targeting is increased, the anti-inflammatory effects of these drugs still primarily depend on the release of 5-ASA.

[0004] Therefore, there is a need to further develop novel 5-aminosalicylic acid prodrugs that simultaneously target the colon, have increased anti-inflammatory activity, and possess ideal safety profiles. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a novel sulfur-containing 5-aminosalicylic acid prodrug. This type of derivative has a good intervention effect on inflammation-related diseases and can be used to prepare drugs for the prevention and / or treatment of inflammatory bowel disease.

[0006] This invention designs and synthesizes a series of novel sulfur-containing 5-aminosalicylic acid prodrugs. Unexpectedly, this invention reveals that the described prodrugs not only exhibit more desirable anti-inflammatory activity than 5-ASA and sulfasalazine, but also possess novel and significant anti-inflammatory effects by inhibiting myeloperoxidase (MPO) and the generation of hypochlorous acid and related inflammatory mediators.

[0007] Technical solution: The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a compound of general formula I or a pharmaceutically acceptable salt, tautomer, meso compound, racemic compound, stereoisomer, metabolite, metabolic precursor, prodrug, or solvate thereof:

[0009]

[0010] in,

[0011] R 1 Selected from hydrogen, straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms, cyclic saturated hydrocarbon groups having 3-6 carbon atoms; or cyclic saturated hydrocarbon groups having 3-6 carbon atoms connected to straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms.

[0012] R 2 Selected from hydrogen, straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms, cyclic saturated hydrocarbon groups having 3-6 carbon atoms, or cyclic saturated hydrocarbon groups having 3-6 carbon atoms connected to straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms;

[0013] R 3 Selected from straight-chain or branched saturated hydrocarbon groups or aryl groups having 1 to 6 carbon atoms; said aryl group is selected from phenyl or naphthyl, each of which is optionally substituted by 1, 2 or 3 substituents, each substituent being independently selected from hydrogen, alkyl, cyano, halogen, nitro, haloalkyl, hydroxyl, mercapto, alkoxy, alkylthio or alkoxyalkyl.

[0014] R 4 Selected from hydrogen, COOH or COOR 5 ;

[0015] R 5 Selected from straight-chain or branched saturated hydrocarbon groups or benzyl groups having 1-6 carbon atoms;

[0016] n = 1, 2 or 3.

[0017] In some preferred embodiments,

[0018] R 1 Selected from hydrogen or straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms;

[0019] R 2 Selected from hydrogen or straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms;

[0020] R 3 Selected from straight-chain or branched saturated hydrocarbon groups having 1 to 6 carbon atoms, or phenyl; said phenyl is optionally substituted by 1, 2 or 3 substituents, each substituent being independently selected from hydrogen, alkyl, cyano, halogen, nitro, haloalkyl, hydroxy, mercapto, alkoxy, alkylthio or alkoxyalkyl.

[0021] R 4 Selected from hydrogen, COOH or COOR 5 ;

[0022] R 5 Selected from straight-chain or branched saturated hydrocarbon groups or benzyl groups having 1-6 carbon atoms;

[0023] n = 1, 2 or 3.

[0024] In some more preferred embodiments,

[0025] R 1 Selected from hydrogen or methyl;

[0026] R 2 Selected from hydrogen or methyl;

[0027] R 3 Selected from methyl, ethyl, isopropyl, or phenyl;

[0028] R 4 Selected from hydrogen, COOH or COOR 5 ;

[0029] R 5 Selected from methyl, ethyl, or isopropyl;

[0030] n is selected from 2 or 3.

[0031] In some preferred embodiments, the pharmaceutically acceptable salt includes, but is not limited to, acid addition salts formed by compounds of general formula I with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid; it also includes acid salts formed by compounds of general formula I with inorganic bases.

[0032] In some preferred embodiments, the pharmaceutically acceptable salt includes, but is not limited to, basic metal cation salts, alkaline earth metal cation salts, and ammonium cation salts.

[0033] The compounds of general formula I of the present invention are preferably the following compounds, as shown in Table 1:

[0034] Table 1 Preferred Compounds

[0035]

[0036] The compounds of general formula I described above in this invention can also exist in the form of their salts or solvates, which are converted into compounds of general formula I in vivo. For example, within the scope of this invention, the compounds of this invention are converted into pharmaceutically acceptable salt forms according to processes known in the art, and used in salt form.

[0037] All tautomers of compounds of general formula I of this invention are included within the scope of this invention. The compounds of this invention may exist in specific geometric or stereoisomer forms. Additional asymmetric carbon atoms may be present in alkyl or other substituents; all such isomers and mixtures thereof are included within the scope of this invention.

[0038] The present invention also provides a pharmaceutical composition comprising a compound of general formula I or a pharmaceutically acceptable salt thereof, a tautomer, a meso compound, a racemic compound, a stereoisomer, a metabolite, a metabolic precursor, a prodrug or solvate, and a pharmaceutically acceptable carrier or excipient.

[0039] The pharmaceutical compositions of the present invention can be administered locally or systemically, for example, for enteral administration, such as rectal or oral administration, or for parenteral administration to mammals (especially humans). The pharmaceutical compositions of the present invention can also be administered parenterally, for example, by inhalation, injection, or infusion, such as via intravenous, intra-arterial, intra-bone, intramuscular, intracerebral, extraventricular, intrasynovial, intrasternal, intrathecal, intralesional, intralesional, intracranial, intratumoral, intradermal, and subcutaneous injection or infusion.

[0040] The effective amount of the compound, pharmaceutical composition, or drug described in this invention depends on the species, weight, age, individual condition, individual pharmacokinetic parameters, the disease to be treated, and the route of administration of the mammal.

[0041] The effective amount of the compounds, pharmaceutical compositions, or drugs described in this invention can be easily determined by routine experiments. The most effective and convenient route of administration and the most appropriate formulation can also be determined by routine experiments.

[0042] To form a pharmaceutical composition in tablet form, any excipient known and widely used in the art can be used. Examples include carriers such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silica; binders such as water, ethanol, propanol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants such as dry starch, sodium alginate, agar powder, and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyvinyl sorbitol, sodium lauryl sulfate, glyceryl monostearate, starch, and lactose; disintegration inhibitors such as white sugar, glyceryl tristearate, coconut oil, and hydrogenated oil; adsorption promoters such as quaternary ammonium base and sodium lauryl sulfate; wetting agents such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silica; and lubricants such as pure talc, stearates, boric acid powder, and polyethylene glycol. It can also be made into sugar-coated tablets, gelatin-coated tablets, sausage-coated tablets, coated tablets, double-layered tablets and multilayered tablets by selecting common coating materials as needed.

[0043] To form a pharmaceutical composition in pellet form, any excipient known and widely used in the art may be used, such as carriers, like lactose, starch, coconut oil, hardened vegetable oil, kaolin, and talc; binders, like gum arabic, tragacanth, gelatin, and ethanol; and disintegrants, like agar and kelp powder.

[0044] To form a pharmaceutical composition in suppository form, any excipient known and widely used in the art may be used, such as polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0045] To prepare a pharmaceutical composition in injectable form, the solution or suspension can be sterilized and made into an isotonic injection with blood pressure similar to that of blood. Any commonly used carrier in the art can be used in the preparation of the injection. Examples include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyvinyl sorbitol. In addition, common solvents, buffers, and analgesics can be added.

[0046] In the pharmaceutical composition, the diluent may be a conventional diluent in the art.

[0047] The pharmaceutical composition may be in oral form or in the form of a sterile aqueous solution for injection, and may be prepared in accordance with any method known in the art for preparing pharmaceutical compositions.

[0048] The present invention also provides the use of compounds of general formula I or pharmaceutically acceptable salts, tautomers, meso compounds, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates thereof in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease.

[0049] This invention also provides the use of compounds of general formula I or pharmaceutically acceptable salts, tautomers, mesosomes, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates thereof in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal oxidative stress.

[0050] The diseases associated with abnormal oxidative stress include one or more of the following: autoimmune diseases, inflammatory diseases, allergic diseases, metabolic diseases, infection-based diseases, fibrotic diseases, cardiovascular diseases, respiratory diseases, kidney diseases, dermatological diseases, liver diseases, gastrointestinal diseases, oral diseases, or hematopoietic diseases.

[0051] Beneficial effects:

[0052] Compared to the existing first-line drug sulfasalazine, the prodrug described in this invention has a more ideal therapeutic effect on inflammatory bowel disease. The prodrug described in this invention exhibits more ideal anti-inflammatory activity than sulfasalazine, and it can also inhibit myeloperoxidase (MPO) activity, inhibit hypochlorous acid production, and inhibit the secretion of inflammatory factors, possessing a multidimensional and novel pharmacodynamic mechanism and significant anti-inflammatory efficacy. Therefore, the compounds of this invention can be used to prepare drugs for the prevention and / or treatment of inflammatory bowel disease, and for the prevention and / or treatment of diseases related to abnormal oxidative stress. Attached Figure Description

[0053] Figure 1 This is the hydrogen spectrum of compound 1.

[0054] Figure 2 This is the hydrogen spectrum of compound 2.

[0055] Figure 3 This is the hydrogen spectrum of compound 3.

[0056] Figure 4 This refers to changes in body weight and DAI during in vivo efficacy evaluation. Detailed Implementation

[0057] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0060] Example 1: Synthesis of (E)-5-((4-(((2-acetamidoethyl)dithioalkyl)methyl)phenyl)diazeninyl)-2-hydroxybenzoic acid (compound 1)

[0061]

[0062] 1. Synthesis of Compound 7

[0063] The raw materials benzyl alcohol (2.46 g, 20 mmol) and NaNO2 (1.4 g, 20 mmol) were placed in a single-necked flask (100 mL), dissolved in water, and concentrated HCl (5.5 mL, 150 mmol) was added dropwise under ice bath conditions. The mixture was reacted at low temperature for 10 min until the raw materials were exhausted.

[0064] The starting materials 2-hydroxybenzoic acid (2.76 g, 20 mmol), Na₂CO₃ (4.18 g, 30 mmol), and NaOH (400 mg, 10 mmol) were placed in another single-necked flask (500 mL), dissolved in water, and stirred under ice bath conditions. After the reaction in the previous step was complete, the resulting reaction solution was added dropwise to this reaction solution. The reaction was continued until the starting materials were completely reacted. The pH of the reaction solution was adjusted to 3-4 with 1 N hydrochloric acid, and the mixture was extracted with EA (3 × 100 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure, the crude product was subjected to column chromatography (dichloromethane:methanol = 20:1, v / v) to obtain a yellow powder, which was compound 4 (2.43 g, yield 45%).

[0065] Compound 4 (272 mg, 1 mmol) was dissolved in tetrahydrofuran, and PBr3 (76 μL, 0.8 mmol) was added dropwise under ice bath conditions until the benzyl alcohol reaction was complete as monitored by TLC. The mixture was extracted with EA (3 × 100 mL) and saturated sodium bicarbonate solution, and the organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure, the crude product was subjected to column chromatography (dichloromethane:methanol = 30:1, v / v) to give a yellow solid, which was compound 5 (83.5 mg, yield 25%).

[0066] Compound 5 (334 mg, 1 mmol), thiourea (78.8 mg, 0.8 mmol), and THF (3 mL) were placed in a dry sealed tube (15 mL), and stirred at room temperature for 2 h to remove oxygen until the reaction was complete as monitored by TLC. The reaction solution was then evaporated to dryness and slurried with dichloromethane to obtain an orange-yellow solid, which was the crude product of compound 6 (200 mg, yield 60%).

[0067] The crude compound 6 (413 mg, 1 mmol) was placed in a sealed tube, and Na₂S₂O₅ (776 mg, 4 mmol), THF (4 mL), and H₂O (2 mL) were added. The mixture was heated to 80 °C until the reaction was complete as monitored by TLC. Extraction was performed with EA (3 × 6 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The resulting crude product (i.e., compound 7) was unstable and used directly in the next step.

[0068] 2. Synthesis of Compound 8

[0069] Compound dithiodipyridine (220 mg, 1.2 mmol) and compound N-(2-mercaptoethyl)acetamide (119 mg, 1 mmol) were placed in a single-necked flask (25 mL), and then dissolved in methanol (10 mL). The reaction was carried out at room temperature for 6 h until the starting material was completely consumed. The solvent was removed by direct vacuum distillation, and the crude product was subjected to column chromatography (dichloromethane:methanol = 100:1, v / v) to give a clear oily liquid, which was compound 8 (109 mg, yield 48%).

[0070] 1 H NMR (300MHz, DMSO) δ (ppm) 8.47 (d, J = 4.8 Hz, 1H), 8.06 (s, 1H), 7.79 (s, 2H), 7.25 (s, 1H), 3.17 (d, J = 5.2Hz, 2H), 2.90 (d, J = 6.8Hz, 2H), 1.80 (s, 3H).

[0071] 3. Synthesis of Compound 1

[0072] Compound 7 (288 mg, 1 mmol) and compound 8 (214 mg, 1 mmol) were placed in a single-necked flask (25 mL), and methanol (5 mL) was added. The mixture was stirred overnight at room temperature. The solvent was removed directly under reduced pressure, and the crude product was subjected to column chromatography (dichloromethane:methanol = 20:1, v / v) to give an orange solid, compound 1 (100 mg, yield 18.8%). See attached chromatogram. Figure 1 .

[0073] 1 H NMR (500MHz, DMSO) δ8.33(d,J=2.5Hz,1H),8.02(s,1H),7.95-7.72(m,3H),7.53(d,J=8.3H z, 2H), 6.91 (d, J = 8.8Hz, 1H), 4.07 (s, 2H), 3.30 (d, J = 6.1Hz, 2H), 2.66 (s, 1H), 1.82 (s, 3H).

[0074] Example 2 Synthesis of (E)-5-((4-(((3-acetamido-4-methoxy-2-methyl-4-oxobutyl)dithioalkyl)methyl)phenyl)diazepine-2-hydroxybenzoic acid (compound 2)

[0075]

[0076] 1. Synthesis of Compound 10

[0077] (S)-2-acetamido-3-mercapto-3-methylbutyric acid (191.25 mg, 1 mmol) and 1,2-bis(benzo[d]thiazo-2-yl)dithione (498.7 mg, 1.5 mmol) were placed in a dry single-necked flask (25 mL), then dissolved in chloroform (10 mL), and stirred at room temperature for 16 hours. The reaction was monitored by TLC until complete. After the solvent was evaporated to dryness, a white solid was obtained. Column chromatography (dichloromethane:methanol = 100:1, v / v) yielded a white solid, which was compound 9 (83.5 mg, yield 25%).

[0078] Compound 9 (365 mg, 1 mmol) was dissolved in anhydrous methanol (5 mL) and placed in a dry double-necked flask (25 mL). TMSCl (217 μl, 3 mmol) was added dropwise to the flask, and the reaction was carried out at room temperature for 16 hours. The reaction was monitored by TLC until complete. The solvent was evaporated to dryness, and the crude product was subjected to column chromatography (petroleum ether: ethyl acetate = 1:1, v / v) to give compound 10 (83.5 mg, yield 25%) as a white solid.

[0079] 2. Synthesis of Compound 2

[0080] Following the method of Example 1, compound 8 in Example 1 was replaced with compound 10 to prepare compound 2 as an orange solid, with a yield of 21.4%. See attached spectrum. Figure 2 .

[0081] 1 H NMR (500MHz, DMSO) δ8.32(d,J=8.7Hz,1H),8.26(d,J=2.7Hz,1H),7.85-7.71(m,2H),7.47(d,J=8.3Hz,2H),6.75 (d,J=8.8Hz,1H),4.64(d,J=8.7Hz,1H),4.04(d,J=7.6Hz,2H),3.67(s,3H),1.94(s,3H),1.36(d,J=8.9Hz,6H).

[0082] Example 3 Synthesis of (E)-5-((4-(((3-acetamido-4-methoxy-2-methyl-4-oxobutyl)trithyl)methyl)phenyl)diazenin)-2-hydroxybenzoic acid (compound 3)

[0083]

[0084] 1. Synthesis of Compound 12

[0085] Compound (S)-2-acetamido-3-mercapto-3-methylbutyric acid (191.248 mg, 1 mmol) was placed in a double-necked flask, and Me3SiCl (172 μl, 2 mmol) was added dropwise under argon protection. The reaction was carried out at room temperature for 12 h. After the reaction was complete, the reaction solution was evaporated to dryness. The crude product was subjected to column chromatography (dichloromethane:methanol = 50:1, v / v) to give a white solid, which was compound 11 (62.5 mg, yield 30.5%).

[0086] Compound N,N-thiobis(imide) (324 mg, 1 mmol) was placed in a double-necked flask. Compound 11 (205 mg, 1 mmol) was dissolved in anhydrous DCM (3 mL) and added dropwise to the double-necked flask. The reaction was carried out at room temperature for 3 h. The reaction of compound 11 was monitored by TLC until it was completely reacted. The reaction solution was evaporated to dryness, and the crude product was subjected to column chromatography (dichloromethane:methanol = 30:1, v / v) to give compound 12 as a clear oily liquid (205 mg, yield 47.3%).

[0087] 1 H NMR (300MHz, CDCl3) δ7.89(d,J=8.1Hz,1H),7.80(d,J=7.8Hz,1H),7.46(t,J=7.2Hz,1H),7.36(t,J=7.6H z, 1H), 6.64 (d, J = 9.2Hz, 1H), 4.85 (d, J = 9.2Hz, 1H), 3.77 (s, 3H), 2.06 (s, 3H), 1.55 (s, 3H), 1.49 (s, 3H).

[0088] 2. Synthesis of Compound 3

[0089] Following the method of Example 1, compound 8 in Example 1 was replaced with compound 12 to prepare compound 3 as an orange solid, with a yield of 10.5%. See attached spectrum. Figure 3 .

[0090] 1H NMR (300MHz, DMSO) δ8.33(s,1H),8.07(dd,J=8.9,2.6Hz,2H),7.85(d,J=8.0Hz,4H),7.54(d,J=8.2Hz,3H), 7.14(d,J=8.9Hz,2H),4.65(d,J=8.6Hz,1H),4.24(s,3H),3.64(s,4H),1.89(s,4H),1.37(d,J=8.4Hz,9H).

[0091] Example 4: In vitro release experiment of the prodrug

[0092] Experimental Principle: The experiment was conducted according to the literature (ANTIOXIDANTS & REDOX SIGNALING. 2021, 34, 1407-1419). One molecule of the prodrug compound can be reduced by azoreductase to one molecule of 5-ASA and one molecule of sulfide. The sulfide can be captured by the trapping agent β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM). This invention investigated the release of the above-mentioned drug from a suspension of colonic contents using LC-MS / MS.

[0093]

[0094] Experimental methods:

[0095] (1) Preparation of solution:

[0096] ① Dilute the DMSO solution (20mM) of the sample to be tested and the capture agent with PBS to prepare a 1mM stock solution.

[0097] ② Colonic contents suspension: Add 5.6g sodium dihydrogen phosphate, 7.6g disodium hydrogen phosphate, and 1.5g D-glucose to 1000mL of water to prepare a 0.1M phosphate buffer (pH=6.8) for later use. Sacrifice SD rats (Shanghai Bikai Keyi Biotechnology Co., Ltd., 250g, 8-week-old male rats), collect their colonic contents, and dilute with the above phosphate buffer to a 10% (w / v) homogenate for later use.

[0098] ③ Working solution: The sample stock solution (10 μL, 1 mM) and the capture agent stock solution (HPE-IAM, 10 μL, 1 mM) were added sequentially to 990 μL of colonic contents suspension and diluted with 990 μL of phosphate buffer to prepare a working solution with a final concentration of 5 μM. HPE-IAM was purchased from COMBI-BLOCKS, USA.

[0099] (2) Experimental steps:

[0100] The prepared working solution of the test sample was incubated in a water bath at 37℃. At different time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 h), 50 μL of the working solution (n=3) was collected into EP tubes, followed by the addition of 150 μL of methanol. The reaction was terminated after mixing. After centrifugation twice at 12000 rpm, 70 μL of the supernatant was injected as an injection sample. The degradation of compounds, the release of 5-ASA, and persulfides in the test sample were detected by LC-MS / MS analysis.

[0101] (3) Liquid phase conditions:

[0102] The instrument used in this experiment was a Shimadzu LC 20A HPLC system (Kyoto Japan), with the following parameters set as follows: column: Waters XSelect-T3 column (3.5μm, 4.6×150mm, Ireland); flow rate of aqueous and organic phases: 0.6m / min; gradient elution using mobile phase A (0.01% ammonia) and mobile phase B (methanol); HPLC autosampler temperature set to 4℃; injection volume: 5μL.

[0103] The elution procedure is as follows, see Table 2:

[0104] Table 2 Gradient elution program

[0105] Time (min) 0.01 2.00 2.20 3.50 7.00 9.50 10.00 13.00 Mobile phase B (%) 1 1 15 15 90 90 1 STOP

[0106] (4) Mass spectrometry conditions

[0107] Mass spectrometry parameters are shown in Table 3:

[0108] Table 3 Mass Spectrometry MRM Parameters

[0109]

[0110] (5) Experimental Results

[0111] The in vitro release results of the prodrugs are shown in Table 4. The results indicate that compounds 1, 2, and 3 were all degraded in colonic contents suspension, releasing polysulfides and 5-ASA. This suggests that these prodrugs can be reduced and released by enzymes contained in the colonic contents. Notably, the yields of 5-ASA and sulfides from compound 3 were higher than those from the positive control sulfasalazine (SASP) and compounds 1 and 2. This indicates that the prodrugs designed in this invention have better release efficiency and significant advantages in in vitro metabolic properties. Furthermore, the LogP of compound 3 is close to that of SASP, indicating that their physicochemical properties are similar and they have a similar colonic distribution to SASP.

[0112] Table 4. In vitro release of prodrugs

[0113]

[0114] Example 5: Evaluation of the in vitro anti-inflammatory activity of the compound

[0115] Experimental principle: An inflammation model of mouse mononuclear macrophage leukemia cells RAW264.7 was induced by LPS.

[0116] Experimental methods:

[0117] RAW 264.7 cells (ATCC, 5 × 10⁻⁶) in good growth condition were selected. 4 Cells (cells / mL) were seeded in 96-well plates, 6 wells per group, and cultured at 37°C for 24 h in a DMEM medium, Gibco, high-glucose form. A blank group (+DMEM medium), an LPS stimulation group (+1 μg / mL LPS), and a drug treatment group (1 μg / mL LPS + drug (5, 25, 125 μM, diluted with DMEM medium)) were established. The drugs included compound 2, compound 3, and the positive control drug SASP (sulfasalazine). After 24 h of culture, the cell supernatant was used for NO content determination (NO kit, Nanjing Jiancheng Biotechnology Institute), and inflammatory factors were measured by ELISA (IL-β and IL-6 kit, KeyGEN, Nanjing). The experimental procedures are referenced in Toxicology and Applied Pharmacology, 387, 114846.

[0118] Experimental results:

[0119] The inhibitory effects of the compounds on LPS-induced NO and inflammatory factors are shown in Table 5:

[0120] Table 5. Inhibitory effects of compounds on LPS-induced NO and inflammatory factors.

[0121]

[0122] The results showed that, compared with the positive control drug SASP, compounds 2 and 3 both exhibited superior inhibitory effects on inflammatory factors (NO, IL-1β, and IL-6). This indicates that the compounds of the present invention possess excellent anti-inflammatory efficacy.

[0123] Example 6: Inhibitory effect of compound on HClO

[0124] Experimental Principle: Hypochlorous acid (HClO) is an important reactive oxygen species in inflammatory responses and a major culprit in promoting the occurrence and development of inflammation. Using the HClO fluorescent probe PZ-py (Chem. Commun. 2015, 51, 1442-1445) reported in the literature, an experiment was conducted to investigate the scavenging effect of compounds on intracellular HOCl in RAW264.7 cells.

[0125] Experimental procedure: Select RAW 264.7 cells in good growth condition (5×10⁻⁶ cells). 4 Cells were seeded in 96-well plates with 6 wells per group, and 6 samples were prepared for each group. The control group (+DMEM culture medium), the LPS stimulation group (+1 μg / mL LPS), and the drug-treated group (1 μg / mL LPS + 125 μM drug) were established. The drugs in the drug-treated group included compound 2, compound 3, and the positive control SASP (sulfasalazine). After 24 h of culture, in situ fluorescence detection was performed: the cell culture medium was removed, and the cells were washed once with PBS. Then, PBS solution (PZ-py, 25 μM) containing PMA (1 μg / mL) and HOCI probe was added. After incubation at room temperature in the dark for 10 min, the probe was removed, and the cells were washed three times with PBS. Cell fluorescence was then directly observed on a live-cell imaging system (Thermo Fisher Scientific).

[0126] After live-cell imaging, the cells were used for fluorescence intensity detection experiments: cell lysis buffer (E8061, Adamas Life) was added to wells containing the cells, the supernatant was collected, and the fluorescence intensity was measured using a fluorescence microplate reader. One well from each group of cells was used for protein content determination, and the fluorescence intensity was finally corrected using the protein content.

[0127] The percentage of fluorescent cells in the total number of cells in the live-cell imaging experiment is shown in the table below:

[0128] Table 6 shows the percentage of fluorescent cells in the total number of cells during live-cell imaging experiments.

[0129]

[0130] The average fluorescence intensity of RAW.264.7 cell lysates is shown in Table 7:

[0131] Table 7. Average fluorescence intensity of RAW.264.7 cell lysates

[0132]

[0133] Experimental conclusion: Compared with the positive control drug SASP, compounds 2 and 3 were able to better remove intracellular HClO. This indicates that the compounds of this invention can effectively alleviate the damage of HClO to living cells, have excellent anti-inflammatory effects, and have potential protective effects on cells and inflamed tissues.

[0134] Example 7 Evaluation of the inhibitory effect of the compound on MPO

[0135] Experimental principle: Myeloperoxidase (MPO) released by neutrophils can catalyze the production of hypochlorous acid. This invention evaluates the inhibitory effect of compounds on MPO activity in RAW264.7 cells, based on the literature (Acta Pharmacologica Sinica 2018, 39: 1633-1644).

[0136] Experimental methods: RAW 264.7 cells in good growth condition (5×10⁻⁶ cells) were selected. 4 Cells were seeded at 1 μg / mL in 96-well plates, with 6 wells per group and 6 samples per group. The groups included a blank control (+DMEM medium), an LPS stimulation group (+1 μg / mL LPS), and a drug-treated group (1 μg / mL LPS + 125 μM drug). The drug-treated group included compounds 2 and 3, and the positive control SASP (sulfasalazine). After 24 h of culture, 100 μl of cell supernatant was aspirated from each well into an EP tube for later use. The MPO content was then determined using an MPO kit (Nanjing Jiancheng Biotechnology Institute).

[0137] The experimental results are shown in the table below:

[0138] Table 8. Inhibitory effects of compounds on MPO

[0139]

[0140] Experimental Conclusion: This invention unexpectedly revealed that the prodrug compounds possess very significant MPO inhibitory activity. Compared with the positive control drug SASP, compounds 2 and 3 can better inhibit MPO enzyme activity. This indicates that the compounds of this invention can effectively alleviate MPO-induced cell damage, possess excellent anti-inflammatory effects, and have potential protective effects on cells and inflamed tissues.

[0141] Example 8: In vivo efficacy test in mice

[0142] Experimental principle: This invention selects sulfasalazine (SASP) as a positive control to investigate whether compound 3 can alleviate acute ulcerative colitis induced by sodium dextran sulfate (DSS) in mice.

[0143] Experimental animals: C57BL / 6Slac mice (Shanghai Bikai Keyi Biotechnology Co., Ltd., weighing 20g), 6-8 weeks old, n=8.

[0144] Experimental protocol: ① Modeling and drug administration (drug dosage is 100mg / kg, solvent is 0.5% CMC-Na, once daily, administered by gavage; except for the blank group, animals in each group have free access to 2.5% DSS solution): Modeling was carried out from day 0, and drug administration was performed simultaneously with modeling for 7 days; ② DSS was stopped and drinking water was replaced on day 8, while drug administration continued; ③ Animals were sacrificed on day 11 and samples were collected.

[0145] Experimental groups: (A) Blank group: daily gavage with solvent, free access to purified water; (B) Model group: daily gavage with solvent, free access to 2.5% DSS solution; (C) SASP group: daily gavage with SASP, free access to 2.5% DSS solution; (D) Compound 3 group: daily gavage with Compound 3, free access to 2.5% DSS solution.

[0146] Efficacy indicators:

[0147] ①DAI (Disease Activity Index) score, see Table 9:

[0148] Table 9 Scoring Criteria

[0149]

[0150] The fecal occult blood test uses the o-toluidine-glacial acetic acid method. The principle of this method is that heme in hemoglobin has peroxidase-like activity, which can catalyze hydrogen peroxide to release nascent oxygen, oxidizing o-toluidine to o-toluidine, which turns blue.

[0151] The experimental results are shown in Table 10 and appendix. Figure 4 :

[0152] Table 10 DAI scores 10 days after modeling

[0153]

[0154] Experimental conclusion: Compound 3 is more effective than the positive control SASP, and reduces the degree of bloody stool and loose stool in mice caused by DSS.

[0155] ② Weight changes

[0156] Experimental method: During the 10 days of drug administration, the weight of mice was measured and recorded daily. Finally, the weight change of mice on the last day of drug administration and the first day of drug administration was calculated.

[0157] The experimental results are shown in Table 11 and appendix. Figure 4 :

[0158] Table 11 Weight changes 10 days after modeling

[0159]

[0160] Experimental conclusion: Compound 3 can significantly inhibit DSS-induced weight loss in mice, and its remission effect is significantly better than that of the positive control drug SASP.

[0161] ③ Colon length

[0162] Experimental method: On day 11, the mice were sacrificed and their abdominal cavities were dissected along the midline. The cecal pouch and the lower end of the colon were removed. The length of the colon was measured and recorded using a ruler.

[0163] The experimental results are shown in Table 12:

[0164] Table 12 Colon length 10 days after modeling

[0165]

[0166] Experimental conclusion: The colon length of mice in the compound 3 group was significantly longer than that in the model group, and significantly better than that of the positive drug SASP. This indicates that compound 3 can better alleviate the colon shortening and damage caused by DSS in mice.

[0167] ④ MPO (myeloperoxidase) content in colon tissue

[0168] Experimental Methods: Mouse colon tissue was collected, and a 30 mg segment was cut into an EP tube. 300 μl of physiological saline was added to homogenize the tissue, preparing a 10 w / v colon homogenate. The homogenate was then centrifuged at 5000 g for 5 min, and 100 μl of the supernatant was collected into an EP tube. The MPO enzyme content in the tissue was determined by colorimetry. (Myeloperoxide assay kit (colorimetric method) from Nanjing Jiancheng Bioengineering Institute)

[0169] The experimental results are shown in Table 13:

[0170] Table 13 MPO content 10 days after modeling

[0171]

[0172] Experimental conclusions: The DSS-induced mouse ulcerative colitis model induces increased MPO activity in the mouse colon; while compound 3 can reduce MPO activity in the mouse colon, thereby inhibiting MPO-induced colonic damage. Compound 3 exhibits superior activity compared to SASP.

[0173] ⑤ Content of inflammatory factors in colon tissue

[0174] Experimental methods: The supernatant of the 10 w / v colon homogenate prepared above was diluted 10 times, and the content of inflammatory factors in colon tissue was determined by colorimetric method using an ELISA reader. The mouse interleukin-6 (IL-6) ELISA kit, mouse interleukin-1β (IL-1β) ELISA kit, and mouse tumor necrosis factor (TNF-α) ELISA kit were obtained from Elascience.

[0175] The experimental results are shown in Table 14:

[0176] Table 14. Content of inflammatory factors in colon tissue 10 days after modeling.

[0177]

[0178] Experimental conclusions: The levels of three inflammatory factors, IL-1β, IL-6, and TNF-α, in the colon tissue of the model group were significantly higher than those in the control group, indicating successful model establishment. Simultaneously, administration of compound 3 to the drug-treated group reduced inflammatory factors in the colon tissue of mice, with a better effect than the positive control group (SASP), indicating that compound 3 can significantly inhibit the production of inflammatory factors in the model mice.

[0179] ⑥ Serum inflammatory factor content

[0180] Experimental methods: On day 11, blood was collected from the orbital cavity of the mice before they were sacrificed. 1-1.5 mL of plasma was collected from each mouse into an EP tube. After standing for 2 hours, the tube was centrifuged at 5000g for 5 minutes and 10 μL of the supernatant serum was collected. The inflammatory factors in the serum were measured by colorimetric method using an enzyme-linked immunosorbent assay (ELISA) reader.

[0181] The experimental results are shown in Table 15:

[0182] Table 15 Serum inflammatory factor levels 10 days after modeling

[0183]

[0184] Experimental conclusions: The levels of three inflammatory factors, IL-1β, IL-6, and TNF-α, in the serum of the model group were significantly higher than those in the control group. Meanwhile, the administration of compound 3 to the drug-treated group reduced the levels of inflammatory factors in the mouse serum, and the effect was better than that of the positive control drug SASP. This indicates that compound 3 can significantly inhibit the production of inflammatory factors in the model mice and can alleviate colonic damage caused by DSS in mice.

[0185] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A compound of general formula I or a pharmaceutically acceptable salt or tautomer thereof: General Formula I in, R 1 Selected from hydrogen, straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms, cyclic saturated hydrocarbon groups having 3-6 carbon atoms; or cyclic saturated hydrocarbon groups having 3-6 carbon atoms connected to straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms. R 2 Selected from hydrogen, straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms, cyclic saturated hydrocarbon groups having 3-6 carbon atoms, or cyclic saturated hydrocarbon groups having 3-6 carbon atoms connected to straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms; R 3 Selected from straight-chain or branched saturated hydrocarbon groups or aryl groups having 1 to 6 carbon atoms; said aryl group is selected from phenyl or naphthyl, each of which is optionally substituted by 1, 2 or 3 substituents, each substituent being independently selected from hydrogen, alkyl, cyano, halogen, nitro, haloalkyl, hydroxyl, mercapto, alkoxy, alkylthio or alkoxyalkyl. R 4 Selected from hydrogen, COOH or COOR 5 ; R 5 Selected from straight-chain or branched saturated hydrocarbon groups or benzyl groups having 1-6 carbon atoms; n = 1, 2, or 3.

2. The compound according to claim 1, characterized in that, R 1 Selected from hydrogen or straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms; R 2 Selected from hydrogen or straight-chain or branched saturated hydrocarbon groups having 1-6 carbon atoms; R 3 Selected from straight-chain or branched saturated hydrocarbon groups having 1 to 6 carbon atoms, or phenyl; said phenyl is optionally substituted by 1, 2 or 3 substituents, each substituent being independently selected from hydrogen, alkyl, cyano, halogen, nitro, haloalkyl, hydroxy, mercapto, alkoxy, alkylthio or alkoxyalkyl. R 4 Selected from hydrogen, COOH or COOR 5 ; R 5 Selected from straight-chain or branched saturated hydrocarbon groups or benzyl groups having 1-6 carbon atoms; n = 1, 2, or 3.

3. The compound according to claim 2, characterized in that, R 1 Selected from hydrogen or methyl; R 2 Selected from hydrogen or methyl; R 3 Selected from methyl, ethyl, isopropyl, or phenyl; R 4 Selected from hydrogen, COOH or COOR 5 ; R 5 Selected from methyl, ethyl, or isopropyl; n is selected from 2 or 3.

4. The compound according to claim 1, characterized in that... Selected from: 。 5. A pharmaceutical composition, characterized in that: This includes the compound of any one of claims 1-4 or its pharmaceutically acceptable salt, tautomer, and pharmaceutically acceptable carrier or excipient.

6. Use of the compound according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease.

7. Use of the compound according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal oxidative stress.

8. The use according to claim 7, characterized in that, The diseases associated with abnormal oxidative stress include one or more of the following: autoimmune diseases, inflammatory diseases, allergic diseases, metabolic diseases, infection-based diseases, fibrotic diseases, cardiovascular diseases, respiratory diseases, kidney diseases, dermatological diseases, liver diseases, gastrointestinal diseases, oral diseases, or hematopoietic diseases.

Images