Compound as well as preparation method and medical application thereof

Abstract

The invention relates to the technical field of medicinal chemistry and medicine, in particular to a novel compound obtained by alkaline degradation of a thyroid hormone receptor beta (THR-beta) agonist Resmetirom, a preparation method of the novel compound and a medicine containing the compound. The structural formula of the compound is shown in the specification. The compound is prepared by carrying out specific degradation on Resmetirom under a mild alkaline condition. The compound disclosed by the invention shows activity which is obviously superior to that of a parent compound Resmetirom in the aspects of inhibiting activation of hepatic stellate cells and inhibiting release of inflammatory factors TNF-alpha or IL-1beta.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry and medicine, in particular to a novel compound obtained by alkaline degradation of thyroid hormone receptor beta (THR-beta) agonist Resmetirom, a preparation method thereof, and a medicine comprising the compound. BACKGROUND

[0002] Liver fibrosis is a common pathological change in the progression of various chronic liver diseases, including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, etc., characterized by excessive deposition of extracellular matrix, leading to destruction of liver structure and loss of function. If not intervened, liver fibrosis can further develop into cirrhosis, liver failure and even hepatocellular carcinoma, seriously threatening human health. According to statistics, about one billion people worldwide are affected by chronic liver disease, and a considerable proportion of patients are accompanied by varying degrees of liver fibrosis. Due to the high prevalence of hepatitis B and the rapid increase in the incidence of non-alcoholic fatty liver, the disease burden of liver fibrosis continues to increase, becoming one of the major challenges in the field of public health.

[0003] Alpha-smooth muscle actin (α-SMA) is a specific marker of hepatic stellate cell activation, and its expression level directly reflects the progression of liver fibrosis. Inflammatory response plays a key driving role in the occurrence and development of liver fibrosis, among which pro-inflammatory cytokines TNF-alpha and IL-1 beta are particularly important. TNF-alpha can directly initiate an inflammatory cascade and induce cell damage, while IL-1 beta is directly involved in tissue damage and fibrosis formation. Therefore, it is of great significance to develop a compound that can inhibit the expression of α-SMA, TNF-alpha and IL-1 beta. SUMMARY

[0004] Based on the above, the purpose of the present application is to provide a compound that can inhibit the expression of α-SMA, TNF-alpha and IL-1 beta, a preparation method thereof, and a medicine comprising the compound.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions: One of the technical solutions of the present application is a compound with the following structural formula: .

[0006] The molecular formula of the compound of the present application is: C 18 H 15 Cl2N5O6; molecular weight: 468.25; property: light yellow powder; chemical name: 2-(1-cyano-2-methoxy-2-oxoethylidene)-1-[3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazine-3-yl)oxy)phenyl]hydrazine-1-carboxylic acid.

[0007] The second aspect of the present application is a preparation method of the compound, comprising the following steps: Step 1, Resmetirom is dissolved in an organic solvent, a base solution is added for reaction, then the pH of the reaction system is adjusted to neutral, after removing the organic solvent, freeze-drying is performed to obtain a salt-containing crude product; the salt-containing crude product is dissolved in water, centrifuged, the precipitate is collected and freeze-dried to obtain a crude product of a new compound; Step 2, the crude product of the new compound is dissolved in a solvent, and a preparation liquid chromatography system is used for purification to obtain the compound.

[0008] In a preferred embodiment of the present application, in step 1, the organic solvent is methanol; the base solution is a 1 M sodium hydroxide solution (solvent is water); the use amount ratio of Resmetirom, the organic solvent and the base solution is (1.6-2.4) mg: 2 mL: (0.8-1.2) mL.

[0009] In a preferred embodiment of the present application, in step 1, the reaction temperature is room temperature, and the reaction time is 45-75 min.

[0010] In a preferred embodiment of the present application, in step 1, the pH of the reaction system is adjusted to neutral by adding a 0.8-1.2 M hydrochloric acid solution (solvent is water) dropwise.

[0011] The present application does not make special limitations on the condition settings (pressure, temperature, time) of freeze-drying, and the freeze-drying condition setting parameters commonly used by those skilled in the art are adopted.

[0012] In a preferred embodiment of the present application, in step 2, the solvent is a methanol aqueous solution with a volume fraction of 65%-85%; the concentration of the crude product of the new compound in the solvent is 12-18 mg / mL.

[0013] In a preferred embodiment of the present application, in step 2, the purification condition settings are as follows: the chromatographic column is Agilent Eclipse XDB-C18; the mobile phase is a 60% (volume fraction) acetonitrile aqueous solution, the flow rate is 3 mL / min, the detection wavelength is 210 nm, the running time is 25 min; the fraction in the interval of 9.5 min to 10.7 min is collected.

[0014] The third aspect of the present application is an anti-inflammatory drug, characterized in that the effective component comprises the compound.

[0015] In a preferred embodiment of the present application, the anti-inflammatory drug is a drug for inhibiting the expression of inflammatory factors TNF-α and / or IL-1β.

[0016] The fourth technical solution of the present invention is a drug for inhibiting the expression of α-smooth muscle actin, the active ingredient of which includes the above-mentioned compounds.

[0017] The fifth technical solution of the present invention is a drug for treating liver fibrosis, the effective components of which include the above-mentioned compounds.

[0018] In a preferred embodiment of the present invention, the anti-inflammatory drug, the drug that inhibits the expression of α-smooth muscle actin, or the drug for treating liver fibrosis further includes pharmaceutically acceptable excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The compounds of the present invention exhibit significantly superior activity compared to their parent compound Resmetirom in inhibiting hepatic stellate cell activation and the release of inflammatory factors TNF-α or IL-1β. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a liquid chromatogram of the crude compound of the present invention.

[0022] Figure 2 This is a liquid chromatogram for the preparation of the compounds of this invention.

[0023] Figure 3 This is the ultraviolet absorption spectrum of the compound of the present invention.

[0024] Figure 4 This is a high-performance liquid chromatogram (purity detection) of the pure compound of the present invention.

[0025] Figure 5 This is a high-resolution mass spectrum of the compound of the present invention.

[0026] Figure 6 The proton nuclear magnetic resonance spectrum of the compound of this invention (NMR) 1 (H NMR) image.

[0027] Figure 7 The carbon NMR spectrum of the compound of this invention ( 13 (C NMR) plot.

[0028] Figure 8 This is a two-dimensional nuclear magnetic resonance spectrum of the compound of this invention.

[0029] Figure 9 The effect of the compound of this invention and Resmetirom on LPS-induced hepatic stellate cell (HSC-T6) viability (compared with the model group): <0.05, <0.01, <0.001; compared with the control group: ### P <0.001).

[0030] Figure 10 The effects of the compound of this invention and Resmetirom on the expression of α-SMA protein in HSC-T6 cells.

[0031] Figure 11 The effect of the compound of this invention and Resmetirom on the levels of TNF-α and IL-1β in the supernatant of HSC-T6 cells (compared with the control group): ### P <0.001; compared with the model group: 0.001). Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified, "room temperature" in this invention refers to 10-30°C.

[0038] Liver fibrosis is a core component of the progression of chronic liver disease, and currently, there are no highly effective specific treatments. Resmetirom, a known THR-β agonist, has been developed for the treatment of non-alcoholic steatohepatitis (NASH). However, the bioactivity of its degradation products has not been fully explored during drug development.

[0039] This invention unexpectedly revealed that Resmetirom undergoes specific degradation under mild alkaline conditions, yielding a novel compound. In vitro pharmacodynamic evaluation showed that this degradation product exhibited significantly superior activity compared to its parent compound, Resmetirom, in inhibiting hepatic stellate cell activation and suppressing the release of key inflammatory factors. This discovery not only provides a candidate compound with superior activity but also offers a new solution for treating liver inflammation and fibrosis.

[0040] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0041] The Resmetirom used in the embodiments of this invention was purchased from Shanghai Kalulan Technology Co., Ltd., and its molecular formula is C. 17 H 12 Cl2N6O4, with a molecular weight of 435.22, has the following structural formula: .

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1 (1) Preparation of crude product Accurately weigh 100 mg of Resmetirom into a 250 mL round-bottom flask, add 100 mL of methanol, and stir until completely dissolved. Then add 50 mL of 1 M sodium hydroxide solution and react at room temperature for 60 min. After the reaction is complete, slowly add 1 M hydrochloric acid solution dropwise while continuously stirring to adjust the pH to 7.0. Remove residual methanol by rotary evaporation, and freeze-dry the remaining aqueous solution to obtain a crude product containing salt. To remove salt, weigh an appropriate amount of the above crude product, dissolve it in water, prepare a solution with a concentration of 300 mg / mL, centrifuge at 3000 r / min for 20 min, discard the supernatant, collect the precipitate, and freeze-dry to obtain a crude new compound.

[0044] A suitable amount of the crude new compound was prepared into a 0.5 mg / mL sample solution using 75% (v / v) methanol aqueous solution. The sample was detected by high-performance liquid chromatography (HPLC) under the following conditions: Agilent 5 HC-C18 column (250 mm × 4.6 mm, 5 μm), flow rate 1.0 mL / min, and detection wavelength 210 nm. Mobile phase A was 0.045% trifluoroacetic acid aqueous solution–acetonitrile (90:10), and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–2 min, 10% B; 2–20 min, 10% B → 50% B; 20–30 min, 50% B → 54% B; 30–35 min, 54% B; 35–36 min, 54% B → 10% B; 36–45 min, 10% B. The HPLC chromatogram of the crude product is shown below. Figure 1 The results showed that the crude product mainly contained two components: new compounds and Resmetirom, with the new compounds accounting for 81.52%.

[0045] (2) Purification of crude product A suitable amount of the crude new compound was weighed and prepared into a solution with a concentration of 15 mg / mL using a 75% (v / v) methanol-water solution as the solvent and ultrasonically treated. Purification was performed using an Agilent 1260 preparative liquid chromatography system equipped with an automated fraction collector. The chromatographic column was an Agilent Eclipse XDB-C18 (250 × 9.4 mm, 5 μm), the mobile phase was a 60% (v / v) acetonitrile-water solution, the flow rate was set to 3 mL / min, the detection wavelength was 210 nm, and the run time was 25 min. The fractions collected between 9.5 min and 10.7 min were collected. The resulting fractions were combined, concentrated by rotary evaporation to remove acetonitrile, and finally, the sample solution was freeze-dried to obtain the pure new compound. The preparative liquid chromatogram is shown below. Figure 2 As shown.

[0046] Test Example 1: Structural Characterization of the New Compound Prepared in Example 1 Ultraviolet (UV) spectroscopy detection: Accurately weigh 10 mg of the new compound, dissolve and dilute it in 75% methanol aqueous solution to approximately 10 μg / mL, and scan in the wavelength range of 200–400 nm. The obtained UV spectrum is as follows. Figure 3 As shown, the maximum absorption wavelengths are 207 nm and 296 nm, respectively.

[0047] Liquid chromatography detection: Accurately weigh 10 mg of the new compound, dissolve and dilute it in 75% methanol aqueous solution to approximately 0.5 mg / mL. Detect according to the chromatographic conditions in the crude preparation of Example 1 (1). Detection results are as follows: Figure 4 As shown, the chromatographic purity of this compound is 99.1%.

[0048] High-resolution mass spectrometry (HMS) detection: 10 mg of the new compound was dissolved and diluted with 50% methanol aqueous solution to a concentration of 0.2 μg / mL. The molecular weight was determined using a Thermo Scientific Q Exactive high-resolution mass spectrometer (USA). The high-resolution mass spectrum is shown below. Figure 5 As shown.

[0049] Nuclear magnetic resonance (NMR) spectroscopy detection: 10 mg and 20 mg of the new compound were weighed out, dissolved in deuterated dimethyl sulfoxide (DMSO-d6), and then detected using a Bruker Avance III HD 500 NMR spectrometer (Germany). One-dimensional proton NMR spectra are shown below. Figure 6 As shown, the one-dimensional carbon spectrum is as follows Figure 7 As shown. The two-dimensional nuclear magnetic resonance spectrum is as follows. Figure 8 As shown.

[0050] Figures 5 to 8 The structural formula of the new compound prepared in Example 1 is as follows: .

[0051] Test Example 2: In vitro study of the anti-liver fibrosis activity of the new compound prepared in Example 1 To assess whether the new compound has the potential to combat liver fibrosis, this invention uses lipopolysaccharide (LPS) to stimulate rat hepatic stellate cells (HSC-T6) to establish an in vitro fibrosis model. By detecting changes in fibrosis-related indicators, the effect of the compound on LPS-induced cell activation is analyzed.

[0052] (1) Cell viability detection HSC-T6 cells were cultured at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum. Cells were grown at a density of 5 × 10⁶ cells per well. 3The cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h. Afterward, the cells were divided into four groups: a blank control group (no treatment), a model group (containing 1 μg / mL LPS), a new compound group (containing 1 μg / mL LPS + different concentrations of the new compound), and a Resmetirom group (containing 1 μg / mL LPS + different concentrations of Resmetirom). After another 24 h of culture, 100 μL of MTT solution (1 mg / mL) was added to each well, and the cells were incubated for 4 h. The supernatant was then discarded, and 150 μL of DMSO was added to each well. The cells were incubated at 37 °C in the dark for 30 min to allow the crystals to dissolve completely. The optical density (OD) at 490 nm was measured using a microplate reader.

[0053] The results are as follows Figure 9 As shown, compared with the blank control group, the OD value of the model group was significantly increased ( P The concentration of LPS (<0.001) indicates that LPS successfully induced the proliferation and activation of HSC-T6 cells. Compared with the model group, both the new compound and Resmetirom inhibited cell proliferation, with the inhibitory effects being particularly significant at concentrations of 20, 40, and 80 μg / mL. Considering the significant cytotoxicity of 40 and 80 μg / mL, 20 μg / mL was selected as the effective concentration for subsequent experiments. Notably, the inhibitory effect of the new compound on HSC-T6 cell proliferation was significantly stronger than that of Resmetirom.

[0054] (2) Detection of α-smooth muscle actin (α-SMA) expression α-Smooth muscle actin (α-SMA) is a specific marker of hepatic stellate cell activation, and its expression level directly reflects the progression of liver fibrosis. Therefore, detecting α-SMA protein expression is a key molecular basis for assessing whether drugs can reverse HSC activation.

[0055] Based on the cell viability detection results in (1) above, this invention selected 20 μg / mL as the intervention concentration of the new compound to further study its effect on HSC-T6 cell activation. Cell culture and grouping settings were the same as described in "(1) Cell Viability Detection" (including blank control group, model group, new compound group, and Resmetirom group). After 24 hours of treatment, cell pellets were collected, total protein was extracted, and quantified by BCA method. An equal amount of protein (30 μg) was taken for SDS-PAGE electrophoresis, transferred to a membrane, blocked, and incubated overnight at 4°C with α-SMA and GAPDH primary antibodies. After washing, the membrane was incubated at room temperature with HRP-labeled secondary antibody for 2 hours, and finally analyzed by ECL chemiluminescence method.

[0056] Western Blot results are as follows Figure 10As shown, α-SMA protein expression in the model group was significantly higher than that in the blank control group, indicating that LPS successfully induced HSC activation. Treatment with both the new compound and Resmetirom significantly inhibited α-SMA expression, with the new compound exhibiting a significantly stronger inhibitory effect than Resmetirom, further demonstrating that this compound can effectively reverse the HSC activation process at the protein level.

[0057] (3) Detection of inflammatory factors Inflammatory responses play a crucial driving role in the occurrence and development of liver fibrosis, with pro-inflammatory cytokines TNF-α and IL-1β being particularly important. TNF-α can directly initiate the inflammatory cascade and induce cell damage, while IL-1β directly participates in tissue damage and fibrosis formation. Therefore, if a new compound can reduce the expression of these two inflammatory factors, it not only demonstrates its anti-inflammatory activity but also helps to explain its inhibitory mechanism on hepatic stellate cell activation from an inflammatory perspective. Cell supernatants collected in “(2) α-smooth muscle actin (α-SMA) expression detection” were used to detect the concentrations of TNF-α and IL-1β according to the ELISA kit instructions.

[0058] The results are as follows Figure 11 As shown, after LPS stimulation, the levels of TNF-α and IL-1β in the cell supernatant of the model group were significantly increased, showing a highly significant difference compared with the blank control group. P <0.001). Both the new compound and Resmetirom intervention significantly inhibited the release of these two key inflammatory factors. More importantly, the new compound was more effective than Resmetirom in inhibiting the expression of inflammatory factors, further suggesting that it has strong anti-inflammatory potential, which may indirectly inhibit the activation process of hepatic stellate cells.

[0059] In LPS (lipopolysaccharide)-induced cell membrane-mediated inflammatory responses, the novel compound exhibits superior activity compared to its parent compound, Resmetirom. This is primarily due to the carboxylic acid group in the novel compound's structure, which imparts stronger polarity and hydrophilicity. This property allows it to more effectively accumulate and act on the "initiation site" of the inflammatory response—the cell membrane surface. The novel compound can more directly target and interfere with key membrane receptors (such as TLR4), thereby blocking the LPS-triggered inflammatory signaling pathway at its source and effectively inhibiting the production of downstream inflammatory factors such as TNF-α and IL-1β.

[0060] Based on the above research results, the novel compound obtained by liquid-phase purification in this invention exhibits significant anti-liver fibrosis potential in in vitro models. In in vitro models, it effectively inhibits the activation and proliferation of hepatic stellate cells and significantly downregulates the expression of α-SMA and key inflammatory factors TNF-α and IL-1β, with effects superior to the raw material drug Resmetirom. These results indicate that this compound not only has a clear mechanism of action but also possesses good development value and is expected to become a candidate compound for the treatment of liver fibrosis.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound, characterized in that, The structural formula is shown as follows: 。 2. A process for the preparation of a compound according to claim 1, characterized in that, The method comprises the following steps: Step 1, Resmetirom is dissolved in an organic solvent, a base solution is added for reaction, then the pH of the reaction system is adjusted to neutral, after removing the organic solvent, freeze-drying is performed to obtain a salt-containing crude product; the salt-containing crude product is dissolved in water, centrifuged, the precipitate is collected, and freeze-drying is performed to obtain a new compound crude product; Step 2, the new compound crude product is dissolved in a solvent, and a preparative liquid chromatography system is used for purification to obtain the compound.

3. The process for the preparation of a compound according to claim 2, characterized in that, In step 1, the organic solvent is methanol; the base solution is a 1 M sodium hydroxide solution; the use amount ratio of Resmetirom, the organic solvent and the base solution is (1.6-2.4) mg: 2 mL: (0.8-1.2) mL.

4. The method of claim 2, wherein the compound is prepared by the process comprising: In step 1, the reaction temperature is room temperature, and the reaction time is 45-75 min.

5. The method of claim 2, wherein the compound is prepared by the process comprising: In step 1, the pH of the reaction system is adjusted to neutral by dropwise adding a 0.8-1.2 M hydrochloric acid solution.

6. The method of claim 2, wherein the compound is prepared by the process comprising: In step 2, the solvent is a 65%-85% methanol aqueous solution by volume fraction; the concentration of the new compound crude product in the solvent is 12-18 mg / mL.

7. The method for preparing the compound according to claim 2, characterized in that, In step 2, the purification conditions are set as follows: the chromatographic column is an Agilent Eclipse XDB-C18; the mobile phase is a 60% acetonitrile aqueous solution by volume fraction, the flow rate is 3 mL / min, the detection wavelength is 210 nm, and the running time is 25 min; the fraction in the interval of 9.5 min to 10.7 min is collected.

8. An anti-inflammatory medicament, characterized by, The active ingredient comprises the compound of claim 1.

9. The anti-inflammatory medicament according to claim 8, characterized in that, The anti-inflammatory drug is a drug for inhibiting the expression of inflammatory factors TNF-α and / or IL-1β.

10. A medicament for inhibiting the expression of α-smooth muscle actin, characterized by, The active ingredient comprises the compound of claim 1. The active ingredient comprises the compound of claim 1.

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