Acetylcysteine derivatives, compositions comprising acetylcysteine derivatives and uses thereof

Abstract

The application discloses an acetylcysteine derivative, a composition containing the acetylcysteine derivative and application thereof. The acetylcysteine derivative has a structure shown in formula (I), and forms a composition with N-acetyl-L-cysteine, can synergistically induce liquid-liquid phase separation of MITF protein in a cell nucleus, makes the MITF protein form a transcription inactivation type aggregate, and thus inhibits the function of the MITF protein. Based on this, the application provides use of the composition as an MITF inhibitor, and thus is applied to the fields of cosmetics and medicines related to MITF activation. In addition, it is verified through experiments that the composition inhibits expression of melanin synthesis genes such as TYR, TYRP1 and DCT from a transcription source, and the inhibition rate is as high as 60% or more, the effect is remarkable, and the composition has application prospects of whitening, lightening spots or improving skin yellowing.

Description

Technical Field

[0001] This invention relates to the fields of cosmetics and pharmaceuticals, specifically to a novel acetylcysteine ​​derivative compound and a composition comprising the novel compound, which is used to induce liquid-liquid phase separation of MITF protein, thereby inhibiting MITF protein-related functions. Background Technology

[0002] Microphthalmia-associated transcription factor (MITF) is a key regulatory protein belonging to the bHLH-Zip (basic helix-loop-helix-leucine zipper) transcription factor family. The human MITF gene is located on chromosome 3, at 3p13. MITF is involved in maintaining the pigment phenotype and function of the retinocyte pigment epithelium (RPE) and is related to eye development, hence its name "microphthalmia-associated transcription factor." Furthermore, current research indicates that MITF can regulate mast cell differentiation and granule formation; MITF inhibition can reduce allergic reactions; MITF synergistically promotes the differentiation of osteoclast precursors into mature osteoclasts, which is related to osteosclerosis; MITF can activate key enzyme genes in melanin synthesis, regulating the development, differentiation, and survival of melanocytes.

[0003] In the prior art, for example, Chinese patent application CN116042617A discloses an siRNA that inhibits MITF gene expression and its application. This patent provides chemically modified siRNA for inhibiting the MITF gene and its encoded protein, thereby facilitating its use in the treatment of melasma. Another example is Chinese patent application CN120678924A, which discloses a drug that inhibits the target MITF, thereby using this drug to treat melanin metabolism-related fibrotic diseases. It is evident that the prior art employs multiple techniques to inhibit MITF function, thereby producing a variety of beneficial effects.

[0004] Therefore, more inhibitors of the MITF gene or MITF protein need to be discovered. Summary of the Invention

[0005] To inhibit the activity of the microphthalmia-associated transcription factor MITF protein, this invention provides an acetylcysteine ​​derivative, a composition containing the acetylcysteine ​​derivative, and its application.

[0006] The specific technical solution of this invention is as follows:

[0007] This invention provides a compound having the structure shown in formula (I):

[0008] Equation (I) is:

[0009] ;

[0010] Wherein, R is selected from -OH, , .

[0011] This invention provides a composition comprising:

[0012] (a) The compound represented by formula (I) or a pharmaceutically acceptable salt thereof;

[0013] (b) N-acetyl-L-cysteine ​​(NAC) or a pharmaceutically acceptable salt or solvate thereof.

[0014] This invention constructs a novel compound represented by formula (I). In one embodiment, experimental verification showed that the novel compound exhibited superior MITF protein aggregation promotion at a dose only half that of NAC, increasing the positive rate of MITF liquid-liquid phase separation in living cells. The combination of the novel compound and NAC significantly promoted MITF protein aggregation, with a significantly higher positive rate of MITF liquid-liquid phase separation in living cells compared to either the novel compound or NAC alone. This demonstrates that the combination of the novel compound and NAC can synergistically induce MITF formation of intranuclear punctate aggregates.

[0015] In one embodiment, experimental verification showed that after treatment with the combination of the novel compound and NAC, the rate of intranuclear condensate-positive cells in live cells reached over 40%, and could be as high as over 70%, while the rate of intranuclear condensate-positive cells in the single-drug treatment group of the novel compound and NAC was less than 5%. This demonstrates that the synergistic effect of the combination of the novel compound and NAC is significant.

[0016] Further validation revealed that after treating melanocytes in vitro with concentrations of 10-100 μM for 12-24 hours, immunofluorescence assays showed the formation of dot-like aggregates of MITF protein in the cell nucleus. Furthermore, in a fluorescence recovery after photobleaching (FRAP) experiment, after photobleaching a circular region approximately 1 μm in diameter with a 488 nm laser at 100% power for 1 second, the fluorescence recovery half-life was less than 10 seconds. The FRAP experiment confirmed that the aggregates possessed liquid-like physical properties (recovery half-life of 8.5 seconds). In addition, the aforementioned composition could remodel the MITF interaction network, replacing transcriptional coactivators with transcriptional repressor complexes.

[0017] Furthermore, in the composition, the molar ratio of (a) to (b) is 0.5 to 10:1.

[0018] Furthermore, the composition also contains pharmaceutically acceptable excipients.

[0019] Based on the above, the present invention also provides the use of the above-described compound or composition as an MITF inhibitor.

[0020] For example, the above-described compounds or compositions can be used to treat diseases caused by MITF overexpression by inhibiting MITF activity. These diseases include, but are not limited to: malignant melanoma, clear cell sarcoma, MiT family translocation tumors, gastrointestinal stromal tumors, breast cancer, allergies, and melasma.

[0021] For example, the above-mentioned compounds or compositions can be used to prepare cosmetics for whitening, fading dark spots, or improving dull skin by inhibiting the activity of MITF, or to prepare products for preventing or improving skin pigmentation. The above-mentioned compounds or compositions exert their whitening or therapeutic effects by inhibiting the MITF protein.

[0022] Furthermore, the pigmentation state is selected from post-inflammatory hyperpigmentation or solar lentigines.

[0023] Furthermore, in vivo experiments in guinea pigs showed that the above composition can significantly inhibit UVB-induced skin pigmentation, improve skin brightness, reduce epidermal melanin granule deposition, and downregulate the expression of key genes such as TYR and MITF.

[0024] In one embodiment, after treating melanocytes in vitro with the above composition at a concentration of 10-100 μM for 12-24 hours, chromatin immunoprecipitation (ChIP) showed that the level of histone H3 27 lysine acetylation (H3K27ac) in the promoter region of MITF target genes was reduced to less than 30% of that in the control group, and qPCR showed that the mRNA expression levels of MITF target genes TYR, TYRP1, and DCT were reduced to less than 40% of those in the control group.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] This invention is the first to construct the compound shown in formula (I) and the first to discover that the combination of this compound and N-acetyl-L-cysteine ​​induces phase separation in MITF, which directly leads to the transformation of MITF from a transcriptionally activated state to a transcriptionally repressed state. This invention is the first to discover that a small molecule composition induces phase separation in MITF.

[0027] For whitening, fading dark spots, or improving dull skin, this invention inhibits MITF, thereby inducing the expression of its downstream target genes TYR, TYRP1, and DCT, and exerts an excellent melanin-inhibiting effect. The composition provided by this invention has an inhibition rate of up to 60%-72% on genes TYR, TYRP1, and DCT, which is significantly higher than the inhibition rate of the MITF small molecule inhibitor ML329 (approximately 40%-50%). Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0029] Example 1: Preparation of S-(5-(1,2-dithiocyclopentan-3-yl)pentanoyl)-N-acetylcysteine

[0030] This embodiment describes the preparation of S-(5-(1,2-dithiocyclopentan-3-yl)pentanoyl)-N-acetylcysteine, and the reaction equation is as follows:

[0031]

[0032] The preparation steps are as follows:

[0033] Compound 1-a 1,2-dithiacyclopentane-3-pentanoyl chloride (247.5 mg, 1.1 mmol) was dissolved in anhydrous dichloromethane (10.0 mL), stirred for 10 min in an ice bath, and then triethylamine (4.15 mL, 3.0 mmol) was added. A dichloromethane solution containing acetylcysteine ​​1-b (165 mg, 1.0 mmol) was slowly added, and the mixture was stirred at room temperature for 1 h. After the reaction was complete, the dichloromethane was recovered under reduced pressure, and water was added to the remaining reaction mixture. The pH was adjusted to approximately 5 with dilute hydrochloric acid, and the mixture was filtered to obtain 260 mg of a white solid. The yield was calculated to be 75%.

[0034] The obtained white solid was characterized by NMR, and the characterization results are as follows: 1H NMR (400 MHz, DMSO) δ 12.39 (s, 1H, COOH), 8.32 (s, 1H, NH), 4.66- 4.32 (m, 1H, CH), 3.48 - 3.24 (m, 2H, CH2), 2.59 - 2.49 (m, 2H, CH2), 2.38 -2.34 (m, 3H, CH2+ CH), 1.96-1.87 (m, 2H, CH2), 1.84 (s, 3H, CH3), 1.60 - 1.51 (m, 4H, CH2), 1.25 (m, 2H, CH2); 13 C NMR (100 MHz, DMSO) δ 199, 178.8, 178.6, 56.3, 44.0, 40.2, 38.5,34.6, 30.7, 28.1, 25.8, 23.3; ESI (M+H + =352.0717.

[0035] Example 2: Preparation of S-(2-acetamido-3-((4-fluorophenyl)amino)-3-oxopropyl)5-(1,2-dithiocyclopentane-3-yl)pentylthioester

[0036] This embodiment describes the preparation of S-(2-acetamido-3-((4-fluorophenyl)amino)-3-oxopropyl)5-(1,2-dithiocyclopentan-3-yl)pentylthioester, and the reaction equation is as follows:

[0037]

[0038] The preparation steps are as follows:

[0039] Compound 1 (227.0 mg, 1.1 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (4.15 mL, 3.0 mmol) and HATU (418 mg, 1.1 mmol) were added. The mixture was stirred in an ice bath for 30 min, and then a dichloromethane solution containing p-fluoroaniline (200.0 mg, 1.0 mmol) was slowly added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the dichloromethane was recovered under reduced pressure, and the solution was separated into a white solid by silica gel column chromatography (dichloromethane:methanol = 50:1).

[0040] The obtained white solid was characterized by NMR, and the characterization results are as follows: 1H NMR (400 MHz, DMSO) δ 9.70 (s, 1H, NH), 8.32 (s, 1H, NH), 7.59 –7.14 (m, 4H, Ar-H), 4.89 (m, 1H, CH), 3.60 – 3.34 (m, 2H, CH2), 2.59 – 2.49(m, 2H, CH2), 2.38 (t, J = 7.1 Hz, 2H, CH2), 2.34 (m, 1H, CH), 1.96 – 1.70 (m,2H, CH2), 1.84 (s, 3H, CH3), 1.60 (m, 2H, CH2), 1.51 (m, 2H, CH2), 1.25 (m,2H, CH2); 13 C NMR (100 MHz, DMSO) δ 199.3, 172.7, 170.7, 162.9, 134.1, 120.6,120.6, 115.7, 115.7, 56.3, 53.2, 44.0, 40.2, 38.5, 34.6, 31.6, 28.1, 25.8,22.9; ESI (M+H + = 445.1086.

[0041] Example 3: Preparation of S-[2-acetamido-3-[(2-chlorobenzyl)amino]-3-oxopropyl]5-(1,2-dithiocyclopentane-3-yl)pentylthioester

[0042] This embodiment describes the preparation of S-[2-acetamido-3-[(2-chlorobenzyl)amino]-3-oxopropyl]5-(1,2-dithiocyclopentane-3-yl)pentylthioester, and the reaction equation is as follows:

[0043]

[0044] The preparation steps are as follows:

[0045] Compound 1 (227.0 mg, 1.1 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (4.15 mL, 3.0 mmol) and HATU (418 mg, 1.1 mmol) were added. The mixture was stirred in an ice bath for 30 min, and then a dichloromethane solution containing o-chlorobenzylamine (200.0 mg, 1.0 mmol) was slowly added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the dichloromethane was recovered under reduced pressure, and the mixture was separated by silica gel column chromatography (dichloromethane:methanol = 50:1) to obtain a white solid. The obtained white solid was characterized by NMR, and the characterization results were as follows: 1 H NMR (400 MHz, DMSO) δ 8.87 (s, 1H, NH), 8.32 (s, 1H, NH), 7.68 –7.21 (m, 4H, Ar-H), 4.73 (m, 1H, CH), 4.40 (m, 2H, CH2), 3.50 – 3.24 (m, 2H,CH2), 2.59 – 2.49 (m, 2H, CH2), 2.38 (t, J = 7.1 Hz, 2H, CH2), 2.34 (m, 1H,CH), 1.96 – 1.70 (m, 2H, CH2), 1.84 (s, 3H, CH3), 1.60 (m, 2H, CH2), 1.51 (m,2H, CH2), 1.25 (m, 2H, CH2); 13 C NMR (100 MHz, DMSO) δ 199.3, 172.0, 170.7, 142.4, 132.2, 128.6,128.3, 128.1, 126.6, 58.7, 56.3, 44.0, 40.2, 38.5, 38.5, 34.6, 31.6, 28.1,25.8, 22.9; ESI (M+H + = 475.0951.

[0046] Example 4: Formulation of the composition

[0047] This embodiment prepares multiple different compositions by mixing component A and component B in different proportions. Component A is obtained by the chemical synthesis method of Examples 1-3; component B is N-acetyl-L-cysteine ​​(NAC), which is commercially available. The different compositions are shown in Table 1.

[0048] Table 1. Composition information for different groups

[0049] The preparation method of the above composition is as follows:

[0050] Component A and combination B were dissolved in deionized water to obtain compound mother liquor and NAC mother liquor, respectively. The compound mother liquor and NAC mother liquor were mixed according to the molar ratio in Table 1, vortexed for 10 seconds, and deionized water was added to make up to 1000 μL. The mixture was then vortexed to obtain the above-mentioned combinations.

[0051] Example 5: Composition induces liquid-liquid phase separation in the MITF core

[0052] The composition prepared in Example 4 was used to induce the formation of droplet-like aggregates of MITF within the nucleus of living cells. The specific experimental method is as follows:

[0053] The experimental groupings for this embodiment are shown in Table 2. In Table 2, the molar number of the drug in the composition is expressed as the sum of the molar numbers of component A and component B; 5% 1,6-hexanediol is a phase separation inducing agent reported in the prior art.

[0054] Table 2 Experimental Groups

[0055] The experimental steps in this embodiment are as follows:

[0056] (1) Cell Culture and Transfection: Human epidermal melanocytes (HEM, purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee) were cultured in Melanocyte Medium 254 medium (containing 1% HMGS-2 additive) at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were transfected at a rate of 2 × 10⁻⁶ cells / cells. 5 HEM cells were seeded at a density of cells / well in 6-well plates pre-placed with sterile coverslips and incubated for 24 hours. The pEGFP-MITF plasmid (purchased from Addgene, model #38183) was transfected into HEM cells, and drug treatment was performed 24 hours after transfection.

[0057] (2) Drug treatment and immunofluorescence: According to the groups shown in Table 2, the corresponding drugs were added and treated for 12 hours. The culture medium was aspirated, and the slides were washed three times with pre-cooled PBS. 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. After washing three times with PBS, the slides were mounted with anti-fluorescence quenching mounting medium containing DAPI (Solarbio).

[0058] (3) Confocal microscopy observation and statistics: Observation was performed using a Zeiss LSM 880 confocal microscope (63× oil immersion). Five fields of view were randomly selected from each coverslip, and at least 200 cells were counted in each field of view. Cells positive for intranuclear aggregates were defined as those with ≥3 punctate aggregates of GFP-MITF with a diameter ≥0.5 μm in the nucleus. The experiment was independently repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA followed by Tukey's multiple comparison test was used for comparisons between groups.

[0059] Table 3. Percentage of positive cells for intranuclear aggregates in different treatment groups

[0060] Based on the above-mentioned intranuclear condensates, the positive cell rates of different treatment groups are shown in Table 3. The results show that the treatment group with the combination of compound 1 and NAC exhibited dose-dependent induction of condensate formation, as shown in Table 4.

[0061] Table 4. Dose-dependent induction of aggregate formation by the composition (NAC + compound 1)

[0062] The results above show that the positive cell rate was significantly higher in each composition treatment group compared to the single-component treatment groups. Specifically, the positive cell rate reached 71.2% in composition group 2, 78.5% in composition group 3, 75.5% in composition group 8, 68-70% in compositions group 6 and 7, and 40-42% in compositions group 1, 4, and 5, which were highly significant compared to the single-component groups (P<0.001). Furthermore, the positive cell rate was higher in each compound treatment group compared to the NAC monotherapy group.

[0063] Example 6: Composition-induced MITF condensates exhibit droplet properties

[0064] Liquid-liquid phase separation (LLPS) condensates exhibit typical liquid-state physical characteristics, with the most critical quantitative indicator being the rapid fluorescence recovery capability in FRAP experiments. Studies have shown that the fluorescence recovery half-life of LLPS condensates is typically on the order of several seconds to tens of seconds (e.g., t1 / 2 for P particles is approximately 5-10 seconds, and for nucleoli is approximately 10-30 seconds), while solid aggregates formed by protein denaturation show almost no fluorescence recovery (t1 / 2 > 120 seconds or no recovery at all).

[0065] The composition prepared in Example 4 was used to verify the performance of the MITF condensates induced by the composition. It was observed that the MITF condensates exhibited typical physical characteristics of liquid-liquid phase separation, namely, rapid fluorescence recovery. The specific experimental methods are as follows:

[0066] HEM cells were prepared according to the method in Example 5 and treated for 12 hours. FRAP experiments were performed using a Zeiss LSM 880 confocal microscope in a 37°C constant temperature culture chamber. A circular aggregate region with a diameter of approximately 1 μm was selected as the photobleaching area (ROI). The parameters were set as follows: 488 nm laser light was irradiated at 100% power for 50 times (approximately 1 second) for photobleaching. Fluorescence images were then acquired every 0.5 seconds for 120 seconds. The percentage of fluorescence recovery in the ROI region was calculated using the fluorescence intensity of the unbleached area as a reference. A single exponential function was used to fit the fluorescence recovery curve, and the recovery half-life (t) was calculated. 1 / 2 The experiment was independently repeated three times, with 10 aggregates selected for measurement each time. The results are shown in Table 5.

[0067] Table 5 FRAP fluorescence recovery parameters

[0068] The above measurements show that the fluorescence intensity recovered to 82.3% of its initial value within 30 seconds after bleaching, with a recovery half-life of only 8.5 seconds and a shift fraction of 86.5%, exhibiting typical droplet-like rapid recovery characteristics. This is consistent with the FRAP characteristics of classic liquid-liquid phase separation condensates (such as P particles and nucleoli), and contrasts sharply with traditional solid aggregates (which cannot recover). These results confirm that the MITF condensates induced by the combination of compounds 1, 2, 3, and NAC possess liquid-state physical properties and are indeed liquid-liquid phase separation condensates.

[0069] Example 7: Composition reshapes MITF interaction network

[0070] The composition prepared in Example 4 was used to identify the protein components in the MITF condensates induced by the composition, verifying their transformation from a transcriptional activation complex to a transcriptional repression complex. The specific steps are as follows:

[0071] Following the method in Example 5, HEM cells from the blank control group, NAC single-drug group, compound 1 single-drug group, and composition 2 group were treated. HEM cells were cultured in 150 mm culture dishes, and when cell confluence reached 80%, they were treated for 12 hours according to their respective groups. Cells were collected, and nucleoprotein was extracted using the NE-PER nucleoprotein extraction kit (Thermo Fisher). 500 μg of nucleoprotein and 2 μg of anti-MITF antibody (Abcam, ab12039) were incubated overnight at 4°C by rotation. 30 μL of Protein A / G magnetic beads (Thermo Fisher) were added, and incubation continued for 2 hours. After washing the magnetic beads 5 times with washing buffer, the following two assays were performed:

[0072] (1) Western blot validation: The immune complexes on the magnetic beads were denatured by boiling and then subjected to SDS-PAGE electrophoresis. After transfer to a membrane, the bands were detected using antibodies against HDAC1 (Abcam, ab7028), HDAC2 (Abcam, ab32117), anti-p300 (Abcam, ab10485), anti-CBP (Abcam, ab2832), and anti-BRD4 (Abcam, ab128874), with the input as an internal control. The grayscale values ​​of the bands were quantitatively analyzed using ImageJ software. The results are shown in Table 6.

[0073] Table 6. Western blot quantification results of MITF interacting proteins

[0074] (2) Mass spectrometry identification: The immune complexes on the magnetic beads were digested with trypsin and analyzed by LC-MS / MS using a Q-Exactive mass spectrometer (Thermo Fisher). The data were then used to identify the proteins by searching the human protein database using Proteome Discoverer 2.4 software. Screening criteria: peptide FDR <1%, containing at least 2 unique peptides. The identification results of the first 10 MITF interacting proteins in the two groups of compositions are shown in Table 7.

[0075] Table 7. Mass spectrometry identification of MITF interacting proteins (Top 10, 2 groups of combinations)

[0076] Furthermore, the analysis revealed that the top five interacting proteins identified by mass spectrometry in the blank control group were p300, CBP, BRD4, MED1, and TAF4 (transcriptional coactivator).

[0077] The results above show that, according to Western blot quantitative analysis, after the second treatment with the composition, the binding strength of MITF to HDAC1 / 2 increased to 5.82 times and 4.95 times that of the control group, respectively, while the binding strength to p300, CBP, and BRD4 decreased to 0.21, 0.18, and 0.15 times that of the control group, respectively. Mass spectrometry further confirmed that after the second treatment with the composition, the MITF interacting proteins changed from transcriptional coactivators (p300, CBP, etc.) to components of the transcriptional repressor complex (HDAC1 / 2, SIN3A, CHD4, etc.). These results indicate that the composition formed by compounds 1, 2, and 3 with NAC not only induces MITF phase separation but also completely reshapes its interaction network, transforming it from a transcriptionally activated state to a transcriptionally repressive state.

[0078] Example 8: Composition directly induces in vitro phase separation of MITF-IDR

[0079] This embodiment verifies in a cell-free system whether the composition directly acts on the intrinsic disorder region (IDR) of MITF to induce phase separation. The method is as follows:

[0080] The experimental grouping in this embodiment is the same as in Example 5. In addition, a BSA negative control group (equal concentration of bovine serum albumin instead of MITF-IDR) is added.

[0081] This embodiment follows the steps below:

[0082] (1) Protein expression and purification: The coding sequence of amino acids 1-120 (IDR region) at the N-terminus of the human MITF gene was cloned into the pET-28a vector and transformed into E. coli BL21(DE3). Expression was induced by 0.5 mM IPTG at 16℃ for 16 hours. His-MITF-IDR protein was purified by Ni-NTA affinity chromatography and further purified by Superdex 200 gel filtration chromatography. The final protein purity was >95% (verified by SDS-PAGE silver staining). The protein was concentrated to 5 mg / mL, aliquoted, and stored at -80℃.

[0083] (2) Turbidity determination: The MITF-IDR protein was diluted to a final concentration of 50 μM with phase separation buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% PEG3350, 1 mM DTT). 100 μL of the protein solution was added to a 96-well clear plate, and the corresponding drugs were added according to the groupings. The plate was incubated at 37°C for 30 minutes. The absorbance at 600 nm (OD) was measured using a BioTek Synergy H1 microplate reader. 600 The experiment was zeroed with blank buffer solution. The experiment was repeated independently three times. The results are shown in Table 8.

[0084] (3) Microscopic observation: Take 10 μL of the above reaction liquid droplet and add it to the glass slide, cover it with a coverslip, observe the droplet formation using an Olympus IX83 inverted microscope (40× objective lens), and take pictures to record.

[0085] The experimental results are shown in Tables 8 and 9.

[0086] Table 8. Turbidity determination of in vitro phase separation (OD600 × 10⁻⁶) -3 )

[0087] Table 9. Dose-dependent induction of in vitro phase separation by composition (NAC + compound 1)

[0088] The results showed that in the negative control group using BSA as a substrate, none of the treatments caused an increase in turbidity (OD).600 <3×10 -3 This excludes non-specific aggregation. In the MITF-IDR system, a single NAC or a single compound causes only a slight increase in turbidity (OD). 600 4-6×10 -3 The turbidity of the composition increased significantly to 38-43×10⁻⁶ after treatment. -3 It is approximately 7-9 times that of a single component. Microscopic observation reveals that the composition forms numerous spherical droplets, with some droplets fusing (a typical characteristic of phase separation). Dose-dependent experiments show that the effect of the composition is concentration-dependent, EC 100%. 50 The concentrations were approximately 22 μM for NAC and 11 μM for compound 1. This indicates that the combination of compounds 1, 2, and 3 with NAC can directly act on the IDR region of the MITF, synergistically inducing liquid-liquid phase separation.

[0089] Example 9: Competitive blocking of phosphorylation of MITF by MAPK by the composition

[0090] This embodiment verifies whether the composition competitively blocks MAPK-mediated phosphorylation modification by binding to phosphorylation site clusters within the MITF-IDR. The method is as follows:

[0091] The grouping in this embodiment is the same as in Example 5. In addition, the MAPK inhibitor U0126 (10 μM) is added as a positive control.

[0092] The experimental steps in this embodiment are as follows:

[0093] Purified MITF-IDR protein (1 μg) was reacted with active MAPK (ERK2, 50 ng, SignalChem), ATP (100 μM), and different concentrations of test samples in kinase reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT) at 30°C for 30 min. After the reaction was terminated, Phos-tag SDS-PAGE (containing 50 μM Phos-tag acrylamide) was performed. Phosphorylated proteins showed reduced migration and could be separated from non-phosphorylated proteins. Western blot analysis (using anti-MITF antibody) was performed after electrophoresis to calculate the percentage of phosphorylated bands in the total bands. The experiment was independently repeated three times. The results are shown in Tables 10 and 11.

[0094] Table 10 Inhibitory effect of the composition on MITF-IDR phosphorylation

[0095] Table 11. Dose-dependent inhibition of phosphorylation by the composition (NAC+ compound 1)

[0096] The results show that a single NAC or compound inhibits MITF-IDR phosphorylation by only 15-20%, while the combined composition achieves an inhibition rate of 69-74%. Dose-dependent experiments indicate that the inhibitory effect of the composition is concentration-dependent. MAPK phosphorylation of MITF is a crucial step in the transition of MITF from its transcriptionally active state to its phase-separated inactive state. These results confirm that the combination of compounds 1, 2, and 3 with NAC can synergistically block MAPK phosphorylation of MITF.

[0097] Example 10: Inhibition of MITF target gene expression and regulation of chromatin state by the composition

[0098] This embodiment verifies the transcriptional repression effect of the composition on key genes in melanin synthesis and confirms that this effect is related to histone deacetylation. The method is as follows:

[0099] The grouping in this embodiment is the same as in Example 5. In addition, an HDAC inhibitor TSA (tributamin A, 100 nM) intervention group is added, with ML329 as the positive control group.

[0100] The experimental steps in this embodiment are as follows:

[0101] (1) qPCR detection of gene expression: HEM cells were used at 1×10 5 Cells were seeded per well in 6-well plates and cultured for 24 hours. After 24 hours, cells were treated according to their grouping. Total RNA was extracted by TRIzol method and reverse transcribed into cDNA. The mRNA levels of TYR, TYRP1, DCT, and MITF were detected by SYBR Green qPCR, with GAPDH as an internal control. Primer information is shown in Table 12. The relative expression levels were calculated by the 2-ΔΔCt method, with the blank control group as 100% normalization. The experiment was independently repeated 3 times. (2) ChIP-qPCR detection of histone acetylation: After 24 hours of HEM cell treatment, chromatin was immunoprecipitated using the EpiQuik ChIP kit. Acetylated chromatin fragments were enriched using anti-H3K27ac antibody (Abcam, ab4729), with rabbit IgG as a negative control. The H3K27ac enrichment level in the promoter regions (-500 to +100 bp upstream of the transcription start site) of TYR, TYRP1, and DCT genes was detected by qPCR. The results are expressed as a percentage of input. The experiment was independently repeated 3 times.

[0102] The test results are shown in Tables 13 and 14.

[0103] Table 12 qPCR primer information

[0104] Table 13 Effects of the composition on the expression of MITF target genes (relative mRNA levels, % control)

[0105] Table 14 Effect of the composition on H3K27ac enrichment in the promoter region of MITF target genes (% Input)

[0106] The above experimental results clearly show that qPCR results indicate that compositions 1-3 inhibited TYR, TYRP1, and DCT by 60-72%, and MITF itself by 28-32% (indicating negative feedback regulation). When the HDAC inhibitor TSA was added, the target gene inhibition effect of the compositions was significantly reversed, recovering to 78-82%, confirming that this inhibitory effect depends on HDAC activity. ChIP-qPCR results showed that after treatment with the compositions, the H3K27ac enrichment levels in the promoter regions of TYR, TYRP1, and DCT genes decreased to 28%, 28%, and 28% of the control group, respectively. These results indicate that the compositions formed by compounds 1, 2, and 3 with NAC reduce histone acetylation levels in the promoter regions of MITF target genes by recruiting HDAC1 / 2, thereby shifting chromatin to a transcriptional repression state.

[0107] Example 11: Inhibitory effect of the composition on UVB-induced pigmentation in guinea pig skin

[0108] This embodiment evaluates the inhibitory effect of the composition on in vivo skin pigmentation using a UVB-induced guinea pig skin pigmentation model. The experimental method is as follows:

[0109] Common-grade male guinea pigs (Hartley strain), weighing 300-350 g, were acclimatized for one week. After back hair removal, they were subjected to cumulative UVB lamp (311 nm) irradiation at 3500 mJ / cm². 2 (Three times a week for two weeks) Induction of pigmentation model. Guinea pigs with successfully induced pigmentation were randomly divided into groups of six according to Table 15. Administration was a topical application once daily for 28 consecutive days.

[0110] Table 15 Experimental Groups

[0111] After 28 days of medication, perform testing according to the following steps:

[0112] (1) Skin color measurement: On days 0, 14 and 28 after administration, the L* value (brightness value, the higher the value, the lighter the skin color) of the back skin was measured using a skin colorimeter.

[0113] The test results are shown in Table 16. In Table 16, compared with the normal control group, the model control group showed P < 0.001; compared with the model control group, each drug administration group showed P < 0.01; and compared with the positive control group, the composition showed P < 0.05.

[0114] Table 16 Changes in L* values ​​of guinea pig skin in each group

[0115] The experimental results showed that the skin L value in the model control group was significantly lower than that in the normal control group (P < 0.001). The L values ​​of the compositions formed by compounds 1, 2, and 3 with NAC significantly increased after dose-gradient treatment, and this increase was dose-dependent. In the second composition group (NAC 50 μM + Compound 1 25 μM), the L* value on day 28 had recovered to a level not significantly different from the normal control group (P > 0.05), showing significantly better results than the positive control arbutin.

[0116] (2) Histological analysis: Skin tissue was taken after the drug administration was completed, and Masson-Fontana staining was used to observe the distribution of melanin granules. The results are shown in Table 17.

[0117] Table 17 Percentage of epidermal melanin granule area in each group

[0118] Masson-Fontana staining results showed that the model control group had a large amount of melanin granules deposited in the epidermis, accounting for 28.5% of the area. After treatment with the composition dose gradient group, the area of ​​melanin granules decreased to 7.5%, which was significantly better than the 15.2% of the positive control group (P < 0.01).

[0119] (3) qPCR detection: The expression levels of TYR and MITF mRNA in skin tissue were detected. The normal control group was set as 1.0. The relative expression levels of TYR and MITF mRNA in the skin of each group are shown in Table 18.

[0120] Table 18 Relative expression levels of TYR and MITF mRNA in skin of each group

[0121] The results showed that after treatment with the dosage gradient of the composition, the expression of TYR and MITF mRNA was significantly inhibited, with the inhibition rate of TYR reaching 78-80% and the inhibition rate of MITF reaching 68-71%, which was significantly better than that of the positive control group.

[0122] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A compound, characterized in that: It has the structure shown in equation (I): Equation (I) is: ； Wherein, R is selected from -OH, , .

2. A composition, characterized in that: It includes component (a) and component (b), wherein: Component (a) is: the compound of claim 1 or a pharmaceutically acceptable salt thereof; Component (b) is: N-acetyl-L-cysteine ​​or a pharmaceutically acceptable salt or solvate thereof.

3. The composition according to claim 2, characterized in that: In the composition, the molar ratio of component (a) to component (b) is 0.5 to 10:

1.

4. The composition according to claim 2, characterized in that: The composition also contains pharmaceutically acceptable excipients.

5. Use of the compound of claim 1 or the composition of any one of claims 2 to 4 as an MITF inhibitor.

6. The use of the compound of claim 1 or the composition of any one of claims 2 to 4 in the preparation of a medicament for treating diseases caused by MITF overexpression.

7. The application according to claim 6, characterized in that: The diseases mentioned include: malignant melanoma, clear cell sarcoma, MiT family translocation tumors, gastrointestinal stromal tumors, breast cancer, allergies, and melasma.

8. The use of the compound of claim 1 or the composition of any one of claims 2 to 4 in the preparation of cosmetics for whitening, fading spots or improving dull skin.

9. The use of the compound of claim 1 or the composition of any one of claims 2 to 4 in the preparation of a product for preventing or improving skin pigmentation, characterized in that: The pigmentation state is selected from post-inflammatory hyperpigmentation or solar lentigines.

10. The application according to claim 9, characterized in that: The compound or composition exerts its whitening or therapeutic effects by inhibiting the MITF protein.

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