An alpha-lipoic acid phenothiazine derivative, and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to an alpha-lipoic acid phenothiazine derivative, a preparation method and application thereof. The structural formula of the alpha-lipoic acid phenothiazine derivative is shown as formula 1. The application provides an alpha-lipoic acid phenothiazine derivative. Research shows that the alpha-lipoic acid phenothiazine derivative exhibits good anti-inflammatory activity, which is slightly better than alpha-lipoic acid. Moreover, the effect is stronger than that of edaravone, a positive drug currently used for ischemic stroke, and the alpha-lipoic acid phenothiazine derivative can be further developed as a therapeutic drug for iron death related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an α-thioctic acid phenothiazine derivative, its preparation method, and its application. Background Technology

[0002] Ferroptosis is an iron-dependent, programmed cell death process characterized by the accumulation of lipid peroxides, significantly different in morphology and mechanism from other forms such as apoptosis and necrosis. Numerous studies have confirmed its close association with various diseases, including neurodegenerative diseases, ischemia-reperfusion injury, spinal cord injury, and acute liver injury, suggesting that its inhibitors hold significant therapeutic potential. However, these diseases are not caused by a single mechanism; while ferroptosis is activated, a severe inflammatory response constitutes another core mechanism, forming a tightly intertwined and mutually amplifying "vicious cycle" with ferroptosis. A "dual-effect" protective strategy that simultaneously targets and inhibits both ferroptosis and the inflammatory response is more advantageous than single-target action. While the natural product α-lipoic acid (ALA) possesses both anti-ferroptosis and anti-inflammatory effects, theoretically making it an ideal lead compound, its anti-ferroptosis efficacy is insufficient. Therefore, developing an α-lipoic acid derivative with both high anti-ferroptosis activity and anti-inflammatory activity is of great significance. Summary of the Invention

[0003] To address the problem that while the natural product α-lipoic acid in existing technologies possesses both anti-ferroptosis and anti-inflammatory effects, its anti-ferroptosis efficacy is insufficient, this invention provides an α-lipoic acid phenothiazine derivative, its preparation method, and its applications. To achieve the above objectives, this invention adopts the following technical solution.

[0004] This invention provides an α-lipoic acid phenothiazine derivative, referred to as α-lipoic acid phenothiazine derivative D14 or compound D14, the structural formula of which is shown in Formula 1:

[0005] .

[0006] In Formula 1, R is selected from any one of the groups shown in Formulas 5 to 24:

[0007] .

[0008] The α-lipoic acid phenothiazine derivative provided by this invention is a novel ferroptosis inhibitor. Due to the synergistic effect of its unique tricyclic phenothiazine skeleton and α-lipoic acid moiety, as well as the structural optimization of the R group (such as trans-3-(Boc-amino)cyclobutanecarboxylic acid), it exhibits both anti-ferroptosis and anti-inflammatory effects, with strong anti-ferroptosis efficacy and low toxicity. The α-lipoic acid phenothiazine derivative provided by this invention achieves highly efficient and low-toxicity anti-ferroptosis and anti-inflammatory effects through the triple structural advantages of "phenothiazine-lipoic acid-R group".

[0009] Among them, these R groups (such as Boc-aminocyclobutane / cyclopentane / cyclohexanecarboxylic acid, Boc-amino acids, etc.) enhance drug efficacy and reduce toxicity through the following mechanisms:

[0010] (1) Increase membrane permeability: Boc-protective groups increase lipid solubility, promote drug transmembrane transport, and make it easier for drugs to enter cells (especially mitochondria and lysosomes) to exert their effects.

[0011] (2) Targeting mitochondria: Cycloalkyl acids or amino acid side chains can serve as mitochondrial targeting groups (such as cyclopentanecarboxylic acid, which has a structure similar to carnitine and can be recognized by mitochondrial membrane transport proteins), enhancing the accumulation of drugs in mitochondria.

[0012] (3) Reduce non-specific toxicity: The Boc-protecting group can mask the strong polarity of the amino group, reduce the excessive binding of the drug to plasma proteins or cell membranes, thereby reducing systemic toxicity.

[0013] The present invention also provides a method for preparing the α-lipoic acid phenothiazine derivative, comprising the following steps:

[0014] A raw material providing the R group, YADX-1, and a condensing agent are dissolved together in DMF to undergo a condensation reaction, generating intermediate product 1 with the structure shown in Formula 2; wherein, the structure of YADX-1 is shown in Formula 3:

[0015] .

[0016] .

[0017] Trifluoroacetic acid and dichloromethane were added to intermediate 1, and Boc was removed under ice bath conditions to generate intermediate 2 with the structure shown in Formula 4.

[0018] .

[0019] The intermediate product 2 and α-lipoic acid were mixed and condensed under the action of condensing agent 2 to obtain the α-lipoic acid phenothiazine derivative.

[0020] Preferably, the condensing agent includes at least one of PyBOP and DIEA.

[0021] The condensing agent 2 includes at least one of EDCI, HOBT, and DIEA.

[0022] The present invention also provides a pharmaceutical composition having the α-lipoic acid phenothiazine derivative or a pharmaceutically acceptable salt thereof as the active ingredient.

[0023] The present invention also provides the use of the α-lipoic acid phenothiazine derivative or the pharmaceutical composition in the preparation of ferroptosis inhibitors.

[0024] The present invention also provides the use of the α-lipoic acid phenothiazine derivative or the pharmaceutical composition in the preparation of anti-inflammatory and / or antioxidant agents.

[0025] The present invention also provides the use of the α-thioctic acid phenothiazine derivative or the pharmaceutical composition in the preparation of a therapeutic remedy for ferroptosis-related diseases.

[0026] Preferably, the iron death-related diseases include at least one of spinal cord injury, Alzheimer's disease, Parkinson's disease, liver injury, and ischemic stroke.

[0027] Preferably, the therapeutic agent comprises an α-lipoic acid phenothiazine derivative or a pharmaceutical composition, and pharmaceutically acceptable excipients.

[0028] Preferably, the excipients include any one or more of the following: excipients, fillers, compatibilizers, binders, humectants, disintegrants, slow solvents, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, or fragrances.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. This invention provides an α-lipoic acid phenothiazine derivative. The unique tricyclic phenothiazine skeleton and the synergistic effect of the α-lipoic acid moiety, along with the structural optimization of the R group (such as trans-3-(Boc-amino)cyclobutanecarboxylic acid), result in both anti-ferroptosis and anti-inflammatory effects, exhibiting strong anti-ferroptosis efficacy and low toxicity. The α-lipoic acid phenothiazine derivative provided by this invention achieves highly efficient and low-toxicity anti-ferroptosis and anti-inflammatory effects through the triple structural advantages of "phenothiazine-lipoic acid-R group," thus solving the problem that while the natural product α-lipoic acid (ALA) possesses both anti-ferroptosis and anti-inflammatory effects, its anti-ferroptosis efficacy is insufficient. Compared to existing α-lipoic acid, the α-lipoic acid phenothiazine derivative provided by this invention demonstrates stronger anti-ferroptosis ability, representing a significant advancement over existing technologies.

[0031] 2. This invention is based on the ferroptosis inhibitor Y2 (EC) reported in a previously published patent (patent number CN 118239942 B). 50 =94.3 nM), whose activity is significantly better than α-lipoic acid, but there is still room for improvement. Therefore, this invention systematically optimizes the R region in the Y2 molecule structure. First, YADX1 is condensed with one of 20 compounds (R substituents) under PyBOP, DIEA, and DMF conditions to obtain intermediates D1a-D20a. Then, compounds D1a-D20a are deBoc-treated by reacting in trifluoroacetic acid:dichloromethane (1:2) for 15 min to obtain intermediates D1b-D20b (1 eq). Finally, α-lipoic acid (1 eq) is added, and condensation is carried out under the action of EDCI (3 eq), HOBT (3 eq), and DIEA (3 eq) to finally obtain 20 α-lipoic acid phenothiazine derivatives. This invention screened 20 compounds for their antiferroptosis activity and ultimately selected compound D14 as the optimal compound. Among the antiferroptosis activity results, this α-lipoic acid phenothiazine derivative D14 exhibited the best antiferroptosis activity (EC). 50 =2.5nM), which is about 15,000 times more active than α-lipoic acid, about 37.7 times more active than the parent compound Y2, and 22 times more active than the positive control drug Ferrostatin-1 (Fer-1), and has good anti-inflammatory activity. Attached Figure Description

[0032] Figure 1 This is the synthetic route for the α-lipoic acid phenothiazine derivative in this invention.

[0033] Figure 2 This invention relates to the ferroptosis half-inhibitory activity of α-lipoic acid phenothiazine derivative (compound D14), ferroptosis-positive drugs Fer-1 and α-LA, and ischemic stroke-positive drug EDV; wherein, Figure 2 Figure A in the figure shows the dose-response curve of α-lipoic acid phenothiazine derivative (compound D14) inhibiting Erastin (1.1 μM)-induced ferroptosis; Figure 2 Figure B in the figure shows the dose-response curve of Fer-1 inhibiting Erastin (1.1 μM)-induced ferroptosis; Figure 2 Figure C in the figure shows the dose-response curve of α-lipoic acid inhibiting Erastin (1.1 μM)-induced ferroptosis; Figure 2 Figure D in the figure shows the dose-response curve of EDV inhibiting Erastin (1.1 μM)-induced ferroptosis.

[0034] Figure 3This study evaluates the toxicity of the α-lipoic acid phenothiazine derivative (compound D14) in BV2, HCT116, HepG2, PC12, and RAW164.7 cells. Figure 3 Figure A in the figure shows the toxicity evaluation of the α-lipoic acid phenothiazine derivative (compound D14) in BV2 cells; Figure 3 Figure B in the figure shows the toxicity evaluation of the α-lipoic acid phenothiazine derivative (compound D14) in HCT116 cells; Figure 3 Figure C in the figure shows the toxicity evaluation of the α-lipoic acid phenothiazine derivative (compound D14) in HepG2 cells; Figure 3 Figure D in the figure represents the toxicity evaluation of the α-lipoic acid phenothiazine derivative (compound D14) in PC12 cells; Figure 3 Figure E in the figure represents the toxicity evaluation of the α-lipoic acid phenothiazine derivative (compound D14) in RAW164.7 cells; standard deviation: n=3, ns, no significant difference; * P <0.05;** P <0.01; *** P <0.001.

[0035] Figure 4 This invention demonstrates the free radical scavenging and iron chelating abilities of the α-lipoic acid phenothiazine derivative (compound D14); wherein, Figure 4 Figure A shows the monitoring of cell-free antioxidant potential using relative DPPH absorbance; standard deviation: n=6, *** P <0.001; Figure 4 Figure B shows the iron chelation detection of the α-lipoic acid phenothiazine derivative (compound D14); standard deviation: n=3, ns, no significant difference; *** P <0.001.

[0036] Figure 5 Treatment with the α-lipoic acid phenothiazine derivative (compound D14) for 24 h in this invention has a protective effect against erastin-induced ferroptosis in PC12 cells; wherein, Figure 5 Figure A in the middle~ Figure 5 Figure C shows the effects of α-lipoic acid phenothiazine derivative (compound D14) on GSH, MDA, and Fe in erastin (5 μM)-induced ferroptosis in PC12 cells. 2+ The effect of level (n=3); Standard deviation: n=3, ns, no significant difference;* P <0.05;** P <0.01; *** P <0.001.

[0037] Figure 6Treatment with the α-lipoic acid phenothiazine derivative (compound D14) for 24 h in this invention has a protective effect against erastin-induced ferroptosis in PC12 cells; wherein, Figure 6 Figure A shows that α-lipoic acid phenothiazine derivative (compound D14) can inhibit the production of reactive oxygen species in the cytoplasm of PC12 ferroptosis cells induced by erastin. Figure 6 Figure B in the figure shows the statistical results of reactive oxygen species (n=2), bar=50μM; ##, p <0.01; ns, no statistical significance, *, p <0.05, **, p <0.01 compared to cells treated with Erastin alone.

[0038] Figure 7 The α-lipoic acid phenothiazine derivative (compound D14) in this invention has a protective effect against LPS-induced BV2 cell inflammation; wherein, Figure 7 Figure A shows the relative viability of BV2 cells treated with different concentrations of LPS for 24 hours; Dates are shown as mean ± SD (n=6). ns, no significant difference was found. P <0.01,***, P <0.001, compared to group 0; Figure 7 Figure B in the figure shows the relative cell viability of BV2 cells in an inflammation model after 24 hours of treatment with an α-lipoic acid phenothiazine derivative (compound D14); Dates are shown as mean ± SD (n=3). P <0.001, compared to Control.*** P <0.001,****, P <0.0001. There was no significant difference in ns compared with the no-drug group; Figure 7 Figure C in the figure shows the relative cell viability of BV2 cells in an inflammation model after 24 h of treatment with EDV and α-lipoic acid phenothiazine derivative (compound D14); Dates are shown as mean ± SD (n=3). P <0.001, no significant difference compared to Control (ns), * P <0.05,**, P <0.01,***, P <0.001, no significant difference in ns compared with the no-drug group.

[0039] Figure 8This is a dose-response curve showing the inhibition of LPS (900 ng / mL)-induced cellular inflammation by α-lipoic acid phenothiazine derivative (compound D14), α-lipoic acid, and EDV in this invention; wherein, Figure 8 Figure A in the figure shows the dose-response curve of α-lipoic acid phenothiazine derivative (compound D14) inhibiting LPS (900 ng / mL)-induced cellular inflammation; Figure 8 Figure B in the figure shows the dose-response curve of α-lipoic acid inhibiting LPS (900 ng / mL)-induced cellular inflammation; Figure 8 Figure C in the figure shows the dose-response curve of EDV inhibiting LPS (900 ng / mL)-induced cellular inflammation.

[0040] Figure 9 This invention aims to evaluate the in vivo side effects of the α-lipoic acid phenothiazine derivative (compound D14); wherein, Figure 9 Figure A in the diagram shows the weight gain curve during treatment. Figure 9 Image B in the figure shows H&E staining of major organs, bar=200μM; Figure 9 Figure C in the middle~ Figure 9 Figure E in the figure represents the evaluation of liver and kidney function in Balb / C mice; data are expressed as mean ± SD (n = 6); ns, no statistical significance.

[0041] Figure 10 This invention demonstrates the protective effect of the α-lipoic acid phenothiazine derivative (compound D14) on MCAO. Figure 10 Figure A in the middle~ Figure 10 Figure B in the image shows TTC staining after treatment and the analysis of infarct volume using ImageJ based on TTC staining. Figure 10 In Figure A, each row contains 5 parallel samples; Dates are shown as mean ± SD (n=3). P <0.001, compared to Sham.* P <0.05,**, P <0.01. Compared with the Vehicle group. ***, p <0.001,**, P <0.01; Figure 10 Figure C in the table shows the neurological score at 24 hours after ischemia / reperfusion (higher scores indicate more severe injury); Dates are shown as mean ± SD (n=6). P <0.001, compared to Sham.* P <0.05,**, P <0.01,***, P <0.001. Compared with the Vehicle group; Figure 10 Image D in the middle~ Figure 10 Figure F shows the levels of GSH, MDA, and Fe in the damaged hemisphere 24 hours after ischemia-reperfusion. 2+ Level; Date are shown as mean±SD (n=3); ###, P <0.001, compared to Sham.* P <0.05,**, P <0.01,***, P <0.001. Compared with the Vehicle group*, p <0.05,**, P <0.01.

[0042] Figure 11 The α-lipoic acid phenothiazine derivative (compound D14) in this invention 1 H NMR spectrum.

[0043] Figure 12 The α-lipoic acid phenothiazine derivative (compound D14) in this invention 13 C10 NMR spectrum. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1

[0046] 1. Preparation of α-Lipoic acid phenothiazine derivatives

[0047] Synthetic route: such as Figure 1 As shown. The synthesis method is as follows:

[0048] Synthesis of compound D14: Purchased raw materials providing the R group, DIEA (N,N-diisopropylethylamine), PyBOP (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), and compound YADX-1 were sequentially dissolved in DMF and reacted at room temperature for 12 hours to induce condensation. The mixture was then extracted with distilled water and dichloromethane, followed by purification by column chromatography (eluting ethyl acetate:petroleum ether, volume ratio = 1:2) to obtain intermediate D14a. The molar ratio of the raw materials providing the R group, DIEA, PyBOP, and compound YADX-1 was 1:2:1.1:1. Compound YADX-1 is described in Chinese Patent No. CN118239942 B. DIEA and PyBOP were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0049] Then, trifluoroacetic acid was added to the intermediate D14a, and the reaction was carried out under ice bath conditions for 15 min to remove Boc, yielding compound D14b. The molar ratio of trifluoroacetic acid to dichloromethane was 1:2.

[0050] Finally, α-lipoic acid, compound D14b, EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (hydrochloride)), HOBT (1-hydroxybenzotriazole), and DIEA were added sequentially and dissolved in dichloromethane. The mixture was then reacted at room temperature for 12 hours to induce condensation. Extraction was performed with distilled water and dichloromethane, followed by purification using thin-layer chromatography on silica gel plates (eluent:methanol:dichloromethane, volume ratio = 1:10). The final product was the α-lipoic acid phenothiazine derivative D14, denoted as compound D14. The molar ratio of α-lipoic acid, compound D14b, EDCI, HOBT, and DIEA was 1:1:3:3:3.

[0051] The synthesis methods of compounds D1 to D13 and compounds D15 to D20 are the same as those of compound D14, except that the "starting material that can provide the R group" and the corresponding intermediate products are different.

[0052] The "starting materials that can provide the R group" used to synthesize compounds D1 to D13 are, in order, Boc-glycine, Boc-β-alanine, N-BOC-γ-aminobutyric acid, N-tert-butoxycarbonyl-L-alanine, Boc-D-alanine, (S)-3-(tert-butoxycarbonylamino)-2-methylpropionic acid, (R)-3-(BOC-amino)-2-methylpropionic acid, Boc-L-threonine, N-Boc-L-allethreonine, N-Boc-3-hydroxy-D-valine, N-Boc-3-hydroxy-L-valine, Boc-L-proline, and Boc-D-proline.

[0053] The "starting materials that can provide R groups" used to synthesize compounds D15 to D20 are, in order, 3-Boc-aminocyclobutanecarboxylic acid, (1R,3R)-N-Boc-1-aminocyclopentane-3-carboxylic acid, (1R,3S)-N-tert-butoxycarbonyl-1-aminocyclopentane-3-carboxylic acid, (-)-(1R,3S)-N-Boc-3-aminocyclopentanecarboxylic acid, N-Boc-aminocyclohexylaminecarboxylic acid, and (1S,4S)-4-tert-butoxycarbonylaminocyclohexanecarboxylic acid.

[0054] The raw material compounds corresponding to the above-mentioned "raw materials that can provide R groups" were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0055] This invention screened the antiferroptosis activity of 20 compounds (compounds D1 to D20). The structural formulas of compounds D1 to D20 and their antiferroptosis activity results are shown in Table 1.

[0056] Table 1. Structural formulas of compounds D1 to D20 and their antiferroptosis activity results.

[0057] .

[0058] Based on the results in Table 1, compound D14 was ultimately selected as our optimal compound, and its antiferroptosis activity results are as follows: Figure 2 The results showed that the α-lipoic acid phenothiazine derivative D14 exhibited the best antiferroptosis activity (EC). 50 =2.5nM), which is about 15,000 times more active than α-lipoic acid, about 37.7 times more active than the parent compound Y2, and 22 times more active than the positive control drug Ferrostatin-1 (Fer-1).

[0059] The structure of compound D14 was identified as follows:

[0060] Compound D1 is a white solid with a 39% yield. mp 191.3℃~191.5℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.66 (s, 1H), 8.30 (s, 1H), 8.11 (s, 1H), 6.93 (s, 1H), 6.84 (s, 1H), 6.79 (s, 1H), 6.68 (d, J =6.2Hz, 2H), 6.58 (d, J=11.4Hz, 2H), 4.07 (d, J=5.8Hz, 2H), 3.67 (d, J=5.7Hz, 2H), 3.55 (s, 1H), 3.07 (s, 2H), 2.35 (s, 1H), 2.10 (s, 2H), 1.82 (s, 1H), 1.60 (s, 1H), 1.48 (s, 3H), 1.30 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6 )δ 172.87, 169.51, 142.54, 139.46, 127.92, 126.63, 126.43, 122.14, 121.17, 116.84, 11 5.02, 114.93, 113.87, 56.60, 42.48, 42.11, 40.38, 38.55, 35.46, 34.60, 28.82, 25.34. HRMS(ESI)m / z calcd for C 23 H 27 N3O2S3[M+H] + 474.1338 found: 474.1335.

[0061] Compound D2 is a pale yellow solid with a 46% yield. mp 210.4℃~210.6℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.70 (s, 1H), 8.39 (s, 1H), 7.93 (s, 1H), 6.96 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.72 (s, 2H), 6.61 (s, 2H), 4.10 (s, 2H), 3.27 (s, 2H), 3.16 (s, 2H), 2.37 (s, 1H), 2.30 (s, 2H), 2.04 (s, 2H), 1.83 (s, 1H), 1.65 (s, 1H), 1.46 (s, 4H), 1.23 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6 ) δ172.52, 170.86, 142.56, 142.54, 139.74, 127.92, 126.63, 126.37, 122.13, 121.12, 116.83, 114.94, 114.90, 113.80, 56.59, 42.02, 40.36, 38.55, 35.88, 35.78, 35.64, 34.60, 28.78, 25.53, HRMS (ESI) m / z calcd for C 24 H29 N3O2S3[M+Na] + 510.1314 found: 510.1315.

[0062] Compound D3 is a pale yellow solid with a 42% yield. mp 245.5℃~245.7℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.69 (s, 1H), 8.35 (s, 1H), 7.93 (s, 1H), 6.96 (s, 1H), 6.88 (s, 1H), 6.82 (s, 1H), 6.72 (d, J=4.9Hz, 2H), 6.61 (d, J=20.8Hz, 2H), 4.10 (s, 2H) , 3.15 (t, J=6.1Hz, 1H), 3.10 (d, J=4.3Hz, 1H), 3.04 (s, 2H), 2.39 (s, 1H), 2.13 (s, 2H), 2.06 (s, 2H), 1.62 (s, 4H), 1.51 (s, 4H), 1.32 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6 )δ 172.53, 172.25, 142.54, 142.51, 139.79, 127.94, 126.66, 126.46, 122.20, 121.11, 116.89, 114.94, 114. 89, 113.67, 56.58, 42.03, 40.40, 38.61, 38.56, 35.74, 34.58, 33.33, 28.78, 25.91, 25.53, HRMS (ESI) m / z calcd forC 25 H 31 N3O2S3[M+H] + 502.1651 found: 502.1649.

[0063] Compound D4 is a grayish-white solid with a 36% yield. mp 195.2℃~195.4℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.59 (s, 1H), 8.29 (s, 1H), 7.97 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.83 (s, 1H), 6.77~6.61 (m, 3H), 6.56 (s, 1H), 4.30 (s, 1H), 4.12 (s, 2H), 3.58 (s, 1H), 3.13 (d,J =18.9Hz, 2H), 2.38 (s, 1H), 2.12 (s, 2H), 1.85 (s, 1H), 1.64 (s, 1H), 1.50 (s, 3H), 1.33 (s, 2H), 1.21 (d, J =6.8Hz, 3H). 13 C NMR (101MHz, DMSO-) d 6 )δ 172.82, 172.23, 142.49, 139.54, 127.96, 126.67, 126.49, 122.19, 121.09, 116.87, 115.05, 114 .90, 113.76, 56.60, 48.51, 42.11, 40.37, 38.55, 35.36, 34.56, 28.80, 25.41, 18.92, HRMS (ESI) m / z calcd for C 24 H 29 N3O2S3[M+H] + 488.1495 found: 488.1499.

[0064] Compound D5 is a yellow solid with a 38% yield. mp 192.6℃~192.9℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.59 (s, 1H), 8.29 (s, 1H), 7.97 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.83 (s, 1H), 6.77~6.61 (m, 3H), 6.56 (s, 1H), 4.30 (s, 1H), 4.11 (s, 2H), 3.58 (s, 1H), 3.16 (d, J=11.3Hz, 2H), 2.37 (s, 1H), 2.13 (d, J =6.8Hz, 2H), 1.87 (s, 1H), 1.64 (s, 1H), 1.49 (d, J =6.3Hz, 3H), 1.34 (d, J =7.0Hz, 2H), 1.21 (d, J=6.8Hz, 3H). 13 C NMR (101MHz, DMSO-) d 6)δ 172.82, 172.24, 142.49, 139.54, 127.96, 126.67, 126.50, 122.19, 121.09, 116.87, 115.05, 114. 90, 113.77, 56.61, 48.51, 42.11, 40.37, 38.55, 35.36, 34.61, 28.80, 25.41, 18.92, HRMS (ESI) m / z calcd forC 24 H 29 N3O2S3[M+H] + 488.1495 found: 488.1500.

[0065] Compound D6 is a pale yellow solid in 44% yield. mp 194.1℃~194.3℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.54 (s, 1H), 8.28 (s, 1H), 7.76 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.82 (s, 1H), 6.74 (s, 1H), 6.64 (s, 2H), 6.59 (s, 1H), 4.06 (s, 2H), 3.54 ( s, 1H), 3.12 (s, 2H), 2.52 (s, 1H), 2.47 (s, 1H), 2.36 (s, 1H), 2.04 (s, 2 H), 1.83 (s, 1H), 1.60 (s, 1H), 1.47 (s, 4H), 1.29 (s, 2H), 1.01 (s, 3H). 13 C NMR (101MHz, DMSO-) d 6 )δ 174.67, 172.63, 142.52, 142.49, 139.92, 127.95, 126.66, 126.39, 122.17, 121.13, 116.89, 115.01, 114. 85, 113.82, 56.61, 42.41, 42.01, 40.36, 40.33, 38.55, 35.65, 34.61, 28.80, 25.56, 16.16, HRMS (ESI) m / z calcd forC 25 H 31 N3O2S3[M+H] + 502.1651 found: 502.1644.

[0066] Compound D7 is a pale pink solid with a 44% yield. mp 199.3℃~199.5℃.1 H NMR (400MHz, DMSO-) d 6 )δ 8.75 (s, 1H), 8.36 (s, 1H), 7.91 (s, 1H), 6.97 (s, 1H), 6.87 (s, 1H), 6.82 (s, 1H), 6.73 (s, 2H), 6.64 (s, 2H), 4.10 (s, 2H), 3.53 (s, 1H), 3.13 (s, 2H), 2.68 (s, 1H), 2.55 (s, 1H), 2.35 (s, 1H), 2.05 (s, 2H), 1.82 (s, 1H), 1.66 (s, 1H), 1.45 (s, 4H), 1.30 (s, 2H), 1.01 (s, 3H). 13 CNMR (101MHz, DMSO-) d 6 )δ 174.70, 172.69, 142.56, 139.91, 127.89, 126.60, 126.38, 126.31, 122.09, 121.04, 120.98, 116.84, 114. 91, 113.84, 56.60, 42.86, 42.48, 41.99, 40.36, 38.55, 35.63, 34.61, 28.80, 25.57, 16.15, HRMS (ESI) m / z calcd for C 25 H 31 N3O2S3[M+H] + 502.1651 found: 502.1649.

[0067] Compound D8 is a pink solid in 43% yield. mp 165.3℃~165.5℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.65 (s, 1H), 8.19 (s, 1H), 7.77 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.81 (s, 1H), 6.72 (d, J =7.9Hz, 2H), 6.67 (s, 1H), 6.60 (s, 1H), 4.99 (s, 1H), 4.21 (s, 1H), 4.13 (s, 2H), 4.02 (s, 1H), 3.11 (s, 2H), 2.38 (s, 1H), 2.22 (s, 2H), 1.85 (s, 1H), 1.61 (d, J=67.2Hz, 5H), 1.35 (s, 2H), 1.03 (s, 3H). 13C NMR (101MHz, DMSO-) d 6 )δ 172.84, 170.90, 142.55, 142.45, 139.47, 127.92, 126.63, 126.40, 122.14, 121.18, 116.86, 114.97, 114. 94, 113.96, 66.97, 58.81, 56.61, 42.26, 40.40, 38.55, 35.55, 34.61, 28.77, 25.51, 20.70, HRMS (ESI) m / z calcd forC 25 H 31 N3O3S3[M+Na] + 540.1420 found: 540.1428.

[0068] Compound D9 is a pink solid with a 51% yield. mp 170.1℃~170.6℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.54 (s, 1H), 8.27 (s, 1H), 7.85 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.81 (s, 1H), 6.78~6.63 ( m, 3H), 6.58 (s, 1H), 4.84 (s, 1H), 4.23 (s, 1H), 4.11 (d, J=4.9Hz, 2H), 3.85 (s, 1H), 3.13 (d, J =18.4Hz, 2H), 2.39 (s, 1H), 2.14 (d, J=6.5Hz, 2H), 1.85 (s, 1H), 1.57 (d, J =55.3Hz, 5H), 1.34 (d, J=6.7Hz, 2H), 1.04 (d, J =5.6Hz, 3H). 13 C NMR (101MHz, DMSO-) d 6 )δ 172.49, 170.92, 142.51, 142.38, 139.49, 127.95, 126.67, 126.41, 122.18, 121.22, 116.89, 114.98, 114. 91, 113.99, 67.31, 58.85, 56.61, 42.24, 40.40, 38.55, 35.44, 34.56, 28.75, 25.45, 20.44, HRMS (ESI) m / z calcd for C 25 H31 N3O3S3[M+H] + 518.1600 found: 518.1604.

[0069] Compound D10 is a pale yellow solid with a 47% yield. mp 175.2℃~175.6℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.69 (s, 1H), 8.32 (s, 1H), 7.94 (s, 1H), 6.96 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.77~6.65 (m, 3H), 6.63 (s, 1H), 4.94 (s, 1H), 4 .29 (s, 1H), 4.13 (s, 2H), 3.16 (s, 2H), 2.39 (s, 1H), 2.20 (s, 2H), 1.84 (s, 1H), 1.66 (s, 1H), 1.52 (s, 2H), 1.35 (s, 2H), 0.96 (d, J =93.0Hz, 8H). 13 C NMR (101MHz, DMSO-) d 6 )δ 172.54, 170.84, 142.55, 142.47, 139.36, 127.92, 126.63, 126.42, 122.14, 121.23, 116.83, 114.99, 114.96, 114.09, 71.44, 60.48, 56.64, 42.29, 40.40, 38.54, 35.45, 34.55, 28.76, 28.02, 26.72, 25.54, HRMS (ESI) m / z calcd forC 26 H 33 N3O3S3[M+Na] + 554.1576 found: 554.1580.

[0070] Compound D11 is a pale yellow solid in 54% yield. mp 158.1℃~158.3℃. 1H NMR (400MHz, DMSO) δ 8.68 (s, 1H), 8.32 (s, 1H), 7.93 (s, 1H), 6.96 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.76~6.65 (m, 3H), 6.63 (s, 1H), 4.92 (s , 1H), 4.32 (s, 1H), 4.11 (s, 2H), 3.15 (s, 2H), 2.37 (s, 1H), 2.20 (s, 2H), 1.84 (s, 1H), 1.67 (s, 1H), 1.51 (s, 4H), 1.02 (d, J =73.9Hz, 8H). 13 C NMR (101MHz, DMSO) δ 172.55, 170.84, 142.55, 142.47, 139.36, 127.91, 126.62, 126.42, 122.13, 121.23, 116.84, 115.00, 114.96, 114.09, 71.44, 60.48, 56.64, 42.30, 40.40, 38.54, 35.45, 34.55, 28.82, 28.02, 26.71, 25.59, HRMS (ESI) m / z calcd for C 26 H 33 N3O3S3[M+H] + 532.1757 found: 532.1751.

[0071] Compound D12 is a pale yellow solid with a 49% yield. mp 164.4℃~164.7℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.64 (s, 1H), 8.22 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.83 (s, 1H), 6.73 (s, 2H), 6.63 (s, 1H), 6.58 (s, 1H), 4.30 ( s, 1H), 4.10 (s, 2H), 3.12 (s, 2H), 2.28 (s, 4H), 1.87 (s, 6H), 1.64 (s, 1H), 1.53 (s, 2H), 1.40 (s, 2H), 1.23 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6)δ 172.35, 171.35, 142.55, 142.46, 139.73, 127.90, 126.62, 126.42, 122.13, 120.96, 116.88, 114.93, 114.17, 113.74, 59.96, 56.67, 47.22, 42.05, 40.39, 38.56, 34.72, 33.91, 29.94~28.95, 24.76, 22.89, HRMS (ESI) m / z calcd for C 26 H 31 N3O2S3[M+H] + 514.1651 found: 514.1649.

[0072] Compound D13. Pale yellow solid, 42% yield. mp 168.3-168.5℃. 1 H NMR (400 MHz, DMSO-) d 6 ) δ 8.63 (s, 1H), 8.24 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.83 (s, 1H), 6.71 (s, 2H), 6.6 1 (s, 2H), 4.29 (s, 1H), 4.11 (s, 2H), 3.58 (s, 1H), 3.45 (s, 1H), 3.12 (s, 2H), 2.33 (d, J = 46.2Hz, 2H), 1.86 (s, 6H), 1.66 (s, 1H), 1.51 (s, 2H), 1.39 (s, 2H), 1.23 (s, 2H). 13 C NMR (101 MHz, DMSO-) d 6 )δ 172.35, 171.31, 142.54, 142.45, 139.74, 127.93, 126.64, 126.45, 122.15, 121.30, 120.97, 116.87, 114.92, 113.71, 59.95, 56.66, 47.20, 42.03, 40.41, 38.56, 34.73, 33.95, 29.97, 28.98, 24.76, 24.41, HRMS (ESI) m / z calcd for C 26 H 31 N3O2S3[M+H] + 514.1651 found: 514.1651.

[0073] Compound D14 is a grayish-white solid with a 67% yield. Its mp values ​​range from 260.3℃ to 260.5℃. It has been identified as an α-lipoic acid phenothiazine derivative, and its structural formula is shown below:

[0074] .

[0075] The NMR data are as follows:

[0076] 1 H NMR (400MHz, DMSO-) d 6 )δ 8.61 (s, 1H), 8.21 (s, 1H), 8.06 (s, 1H), 6.97 (s, 1H), 6.89 (s, 1H), 6.83 (s, 1H), 6.74 (s, 2H), 6.64 (s, 1H), 6.57 (s, 1H), 4.36 (s, 1H), 4.12 (d, J =4.7Hz, 2H), 3.60 (s, 1H), 3.13 (ddd, J =16.6, 11.1, 6.9Hz, 2H), 2.90 (s, 1H), 2.46~2.30 (m, 3H), 2.05 (dd, J =18.3, 10.3Hz, 4H), 1.86 (s, 1H), 1.65 (s, 1H), 1.60~1.44 (m, 3H), 1.34 (d, J=7.2Hz, 2H). 1 The H NMR spectrum is shown in [reference]. Figure 11 .

[0077] 13 C NMR (101MHz, DMSO-) d 6 )δ 174.86, 171.55, 142.57, 142.50, 139.87, 127.95, 126.66, 126.49, 122.18, 121.21, 116.87, 115.0 4, 114.89, 113.73, 56.61, 42.58, 42.14, 40.39, 38.57, 35.66, 34.58, 33.49, 33.36, 28.81, 25.43. HRMS(ESI)m / z [M+H] + calcd forC 26 H 31 N3O2S2514.1650 found: 514.1650. 13 The C NMR spectrum is shown below. Figure 12 .

[0078] Compound D15 is a pink solid in 35% yield. mp 274.5℃~274.7 °C. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.57 (s, 1H), 8.19 (s, 1H), 8.04 (s, 1H), 6.97 (s, 1H), 6.88 (s, 2H), 6.69 (s, 2H ), 6.63 (s, 1H), 6.56 (s, 1H), 4.13 (s, 1H), 4.10 (s, 2H), 3.60 (s, 1H), 3.14 (d, J =18.0Hz, 2H), 2.67 (s, 1H), 2.39 (s, 4H), 2.02 (s, 3H), 1.86 (s, 1H), 1.49 (s, 4H), 1.32 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6 )δ 173.47, 171.54, 142.53, 142.50, 139.75, 127.95, 126.66, 126.49, 122.19, 121.19, 116.89, 115.04, 114.90, 113.78, 56.60, 42.21, 41.13, 40.37, 38.57, 35.53, 34.56, 33.93, 32.76, 32.39, 28.78, 25.40, HRMS (ESI) m / z calcd for C 26 H 31 N3O2S3[M+H] + 514.1651 found: 514.1651.

[0079] Compound D16 is a pale yellow solid in 32% yield. mp 205.1℃~205.3℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.71 (s, 1H), 8.37 (s, 1H), 7.95 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.83 (s, 1H), 6.73 (d, J =4.3Hz, 2H), 6.60 (d, J =22.5Hz, 2H), 4.11 (s, 2H), 3.59 (s, 1H), 3.16 (s, 2H), 3.11 (s, 1H), 2.67 (s, 1H), 2.38 (s, 1H), 2.03 (t, J=7.3Hz, 3H), 1.85 (s, 1H), 1.64 (d, J =16.4Hz, 2H), 1.57~1.38 (m, 6H), 1.37~1.27 (m, 3H). 13 C NMR (101MHz, DMSO-) d 6 )δ 175.44, 171.79, 142.57, 142.56, 139.79, 130.12, 127.92, 126.63, 126.47, 122.15, 121.04, 116.84, 114. 92, 113.69, 56.62, 50.61, 42.92, 42.16, 40.40, 38.56, 36.68, 35.74, 34.56, 32.62, 28.75, 27.97, 25.50. HRMS (ESI) m / z calcd for C 27 H 33 N3O2S3[M+H] + 528.1808 found: 528.1807.

[0080] Compound D17 is a white-yellow solid in 40% yield. mp 217.4℃~217.6℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.74 (s, 1H), 8.38 (s, 1H), 7.96 (s, 1H), 6.97 (s, 1H), 6.90 (s, 1H), 6.83 (s, 1H), 6.73 (d, J =6.5Hz, 2H), 6.61 (d, J =8.8Hz, 2H), 4.11 (d, J =4.8Hz, 2H), 3.59 (s, 1H), 3.17 (s, 1H), 3.11 (s, 1H), 2.69 (s, 1H), 2.39 (s, 1H), 2.04 (t, J =6.7Hz, 4H), 1.86 (s, 1H), 1.78 (s, 3H), 1.67 (s, 1H), 1.51 (dd, J =15.5, 8.1Hz, 5H), 1.23 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6) δ175.44, 171.80, 142.57, 139.79, 130.11, 127.91, 126.62, 126.46, 122.13, 121.03, 116.83, 114.94, 114.91, 11 3.69, 56.62, 50.61, 42.92, 42.15, 40.40, 38.56, 36.68, 35.74, 34.56, 32.62, 28.75, 27.96, 25.50, HRMS (ESI) m / z calcd for C 27 H 33 N3O2S3[M+H] + 528.1808 found: 528.1805.

[0081] Compound D18 is a pale yellow solid with a 42% yield. mp 210.3℃~210.5℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.63 (s, 1H), 8.33 (s, 1H), 7.88 (s, 1H), 7.00~6.93 (m, 1H), 6.88 (d, J =7.1Hz, 1H), 6.83 (d, J =7.8Hz, 1H), 6.71 (dd, J =17.3, 7.7Hz, 2H), 6.62 (d, J =7.9Hz, 1H), 6.57 (s, 1H), 4.11 (s, 2H), 3.57 (s, 1H), 3.12 (ddt, J =21.1, 10.9, 6.7Hz, 2H), 2.67 (s, 1H), 2.39 (s, 1H), 2.02 (s, 3H), 1.84 (s, 1H), 1.77 (s, 3H), 1.63 (s, 1H), 1.49 (s, 6H), 1.33 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6)δ 175.48, 171.84, 142.55, 142.52, 139.77, 127.95, 126.66, 126.50, 122.19, 121.07, 116.86, 114.97, 114. 91, 113.69, 56.62, 51.67, 50.62, 42.93, 42.18, 38.55, 36.69, 35.75, 34.56, 32.60, 28.74, 27.98, 25.49. HRMS (ESI) m / z calcd forC 27 H 33 N3O2S3[M+H] + 528.1808 found: 528.1814.

[0082] Compound D19 is a pale yellow solid in 44% yield. mp 276.7℃~276.9℃. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.78 (s, 1H), 8.30 (s, 1H), 7.77 (s, 1H), 6.96 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.73 (d, J =7.9Hz, 2H), 6.60 (d, J =11.6Hz, 2H), 4.07 (s, 2H), 2.40 (dd, J=12.3, 6.3Hz, 1H), 2.11 (t, J =11.4Hz, 1H), 2.01 (d, J =6.7Hz, 4H), 1.90~1.83 (m, 1H), 1.72 (s, 5H), 1.64 (s, 2H), 1.58~1.38 (m, 6H), 1.33 (d, J=6.5Hz, 2H). 13 C NMR (101MHz, DMSO-) d 6 )δ 175.24, 171.46, 142.59, 142.56, 139.92, 127.89, 126.60, 126.41, 122.11, 121.01, 116.85, 114.94, 114. 84, 113.68, 56.65, 47.55, 43.69, 41.88, 40.38, 38.57, 35.75, 34.57, 32.21, 28.74, 28.72, 25.64, 25.56. HRMS (ESI) m / z calcd for C 28 H 35 N3O2S3[M+H]+ 542.1964 found: 542.1964.

[0083] Compound D20 is a pale pink solid with a 42% yield. mp 269.2℃~269.4℃. 1 H NMR (400MHz, DMSO-) d 6 )δ 8.72 (s, 1H), 8.23 ​​(s, 1H), 7.75 (s, 1H), 6.97 (s, 1H), 6.87 (s, 1H), 6.82 (s, 1H), 6.73 (s, 2H), 6.61 (s, 2H), 4.11 (s, 2H), 3.59 (s, 1H), 2.41 (dt, J =12.4, 6.1Hz, 1H), 2.23 (s, 1H), 2.11 (s, 2H), 1.83 (s, 3H), 1.66 (s, 5H), 1.50 (s, 8H), 1.23 (s, 2H). 13 C NMR (101MHz, DMSO-) d 6 )δ 174.76, 171.51, 142.26, 142.2, 139.74, 127.53, 126.26, 126.06, 121.75, 120.61, 116.53, 114.59, 114.42, 113.32, 56.30, 44.41, 41.85, 41.58, 40.03, 38.22, 35.22, 34.23, 29.19, 28.41, 25.35, 24.77, HRMS (ESI) m / z calcd for C 28 H 35 N3O2S3[M+Na] + 564.1784found:564.1793.

[0084] Example 2

[0085] I. Method:

[0086] 1. Cell Culture

[0087] PC12 cells, BV2 cells, HepG2 cells, HCT116 cells, and RAW.264.7 cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Gibco), 100 units / mL penicillin (Gibco), and 100 units / mL streptomycin (Gibco), and grown in an incubator at 37°C and 5% CO2.

[0088] Among them, PC12 rat adrenal medullary pheochromocytoma cells, BV2 microglia, HepG2 human liver cancer cells, HCT116 human colon cancer cells, and RAW.264.7 mouse mononuclear macrophage leukemia cells were all purchased from Beyotime Biotechnology Co., Ltd.

[0089] 2. Cell viability assay

[0090] Cell viability was measured using the CCK8 (Beyotime Biotechnology) assay to collect PC12 cells in the logarithmic growth phase. Cells were then seeded at 5000 cells / well in 96-well plates (100 μL per well). After 12 h of culture, cells were treated with Erastin, Fer-1, or compound D14, alone or in combination at given concentration gradients, for 24 h. The original culture medium in the 96-well plates was then aspirated, and 100 μL of fresh culture medium containing 10 μL of CCK-8 solution was added to each well. Cells were incubated for 1 h, and the absorbance of the samples was measured at 450 nm using a molecular device. All data were calculated using GraphPad Prism software, and relative cell viability is expressed as a percentage of the control group.

[0091] 3. DPPH Measurement

[0092] Compounds Fer-1, α-lipoic acid, and D14 were dissolved in DMSO at a concentration of 0.05 mM, with each substance forming a separate group. Each group was then added to a 96-well plate in triplicate. A prepared DPPH solution (dissolved in methanol, concentration of 0.05 nM) was added to each well, and the mixture was incubated at room temperature for 10 min. Finally, the absorbance of the samples was measured at 517 nm using a microplate reader. All data were calculated using GraphPad Prism software.

[0093] The compound Fer-1 was sourced from Shanghai Maclean Biotechnology Co., Ltd.

[0094] 4. Determination of iron chelating activity

[0095] Compounds DFS, EDTA, H₂O₂, Fer-1, α-lipoic acid, and D14 were dissolved in DMSO to a concentration of 10 mM. Fenoxazine and ferrous sulfate were dissolved in purified water to concentrations of 5 mM and 1 mM, respectively. Then, 1 μL of the compound solution, 5 μL of FeSO₄·7H₂O solution, and 20 μL of fenoxazine solution were added to 100 μL of purified water and mixed thoroughly. The mixture was then added to 96-well plates, with three replicates. Finally, the absorbance was measured at 562 nM using a microplate reader. All data were calculated using GraphPad Prism software.

[0096] 5. Erastin-induced PC12 ferroptosis model

[0097] PC12 cells in the logarithmic growth phase were seeded at 5000 cells / well in 96-well plates, with five replicates per well. The control group, 2 μM Erastin group (MedChemexpress), 4 μM Erastin group, 6 μM Erastin group, 8 μM Erastin group, and 10 μM Erastin group were selected. After 12 hours of culture, 100 μL of fresh culture medium was added to the control group, while the other treatment groups received the prescribed concentrations. Cells were cultured at 37°C for 24 hours. Cell viability was then measured using the CCK8 assay, and the IC50 was calculated using GraphPadPrism software. 50 Values, relative cell viability, are expressed as a percentage of the control group.

[0098] 6. LPS-induced BV2 inflammation model

[0099] BV2 cells in the logarithmic growth phase were seeded at 30,000 cells / well in 96-well plates, with five replicates per group. The control group, 200 ng / mL LPS (Sigma-Aldrich), 400 ng / mL LPS, 600 ng / mL LPS, 800 ng / mL LPS, and 1000 ng / mL LPS groups were included. After 12 hours of culture, 100 μL of fresh medium was added to the control group, while the other groups received the prescribed drug concentrations. Cells were cultured at 37°C for 24 hours. Cell viability was then measured using the CCK8 assay, and the IC50 was calculated using GraphPad Prism software. 50 Values, relative cell viability, are expressed as a percentage of the control group.

[0100] 7. Determination of GSH content

[0101] 7.1 Cell Experiments:

[0102] Cells from each group were collected, and protein removal reagent S solution (3 times the cell volume) was added according to the Beyotime Total Glutathione Assay Kit instructions. Cells were sonicated (200W, 3s, 10s interval, repeated 30 times), incubated at 4°C for 10 minutes, and then centrifuged at 10000g at 4°C for 10 minutes. The supernatant was collected. Then, the Total Glutathione Assay Working Solution was added according to the instructions, mixed well, and incubated at room temperature for 5 minutes. Then, 0.5 mg / mL NADPH was added, and the mixture was incubated for 25 minutes. The absorbance of each sample at 412 nM was measured. All data were calculated using GraphPad Prism software, and the relative concentration of GSH was the percentage of the control group.

[0103] 7.2 Animal experiments:

[0104] Approximately 0.01 g of ischemic hemisphere brain tissue was collected and 100 μL of protein removal reagent S solution was added according to the manufacturer's instructions. The mixture was then homogenized on ice. After incubation at 4°C for 10 minutes, the mixture was centrifuged at 10000 g at 4°C for 10 minutes. The supernatant was collected, and then the total glutathione detection working solution was added according to the manufacturer's instructions. After mixing, the mixture was incubated at room temperature for 5 minutes. Then, 0.5 mg / mL NADPH was added, and the mixture was incubated for 25 minutes. The absorbance of each sample was measured at 412 nM. All data were calculated using GraphPad Prism software, and the relative concentration of GSH was the percentage of the control group.

[0105] 8. Determination of MDA content

[0106] The content of malondialdehyde (MDA) was determined based on the reactivity with thiobarbituric acid (TBA) using a malondialdehyde (MDA) detection kit.

[0107] 8.1 Cell Experiments:

[0108] Five million cells were collected, and 1 mL of extraction buffer was added. The cells were disrupted by sonication (200 W, 3 s, 10 s interval, repeated 30 times). The cells were then centrifuged at 8000 g at 4 °C for 10 min, and the supernatant was collected. Working solution was added according to the instructions of the MDA content assay kit (Solarbio), and the absorbance of each sample was measured at 532 nM and 600 nM. All data were calculated using GraphPad Prism software, and the relative concentration of MDA was the percentage of the control group.

[0109] 8.2 Animal experiments:

[0110] Rat serum was collected, and working solution was added according to the instructions of the MDA content assay kit. The absorbance of each sample was then measured at 532 nM and 600 nM. All data were calculated using GraphPad Prism software, and the relative concentration of MDA was the percentage of the control group.

[0111] 9. Fe 2+ Determination of content

[0112] The ferrous content was determined using a ferrous content detection kit. Ferrous iron (Fe2+) reacts with ferrous ions to form a purple-red compound. This colored substance has a characteristic absorption peak at 562 nm, which was then used to calculate the ferrous content.

[0113] 9.1 Cell Experiments:

[0114] Five million cells were collected, and 1 mL of extraction buffer was added according to the instructions of the ferrous iron content assay kit (mlbio). The cells were sonicated (ice bath, 200W power, sonication for 3 seconds, 10-second intervals, repeated 30 times), followed by centrifugation at 12000 rpm at 4°C for 10 minutes. The supernatant was collected. Working solution was added according to the instructions, and the absorbance of each sample was measured at 562 nm. All data were calculated using GraphPad Prism software. 2+ The relative concentration is a percentage of the control group.

[0115] 9.2 Animal experiments:

[0116] Approximately 0.1 g of ischemic hemisphere brain tissue was collected, and 1 mL of extraction buffer was added. The mixture was homogenized on ice. Centrifuged at 4℃ × 12000 rpm for 5 min, the supernatant was collected, and working solution was added. Finally, the absorbance of the sample at 562 nm was measured. All data were calculated using GraphPad Prism software. 2+ The relative concentration is a percentage of the control group.

[0117] 10. Measurement of reactive oxygen species

[0118] DCFH-DA can cross the cell membrane and enter the cell, where it is hydrolyzed to generate DCFH, which is then loaded into the cell. In the presence of reactive oxygen species (ROS), DCFH is oxidized to fluorescent DCF, and the intracellular ROS level is analyzed by detecting the fluorescence of DCF. The following groups were set up: control group, Erastin (5 μM), Erastin (5 μM) + DMSO group, Erastin (5 μM) + α-lipoic acid (1 μM) group, Erastin (5 μM) + Fer-1 (1 μM) group, Erastin (5 μM) + compound D14 (0.01 μM) group, Erastin (5 μM) + compound D14 (0.1 μM) group, and Erastin (5 μM) + compound D14 (1 μM) group. PC12 cells were seeded at 50,000 cells / well in 24-well plates containing cell spreaders. After 12 h of culture, the corresponding drugs and Erastin were added at a 1:1 ratio and co-cultured for 24 h. The experiment was then conducted according to the instructions of the reactive oxygen species detection kit (Solarbio), with detection performed under a confocal microscope. The fluorescence intensity was then analyzed using ImageJ, and the final data were calculated using GraphPad Prism software.

[0119] 11. Construction of the MCAO model in SD rats

[0120] 11.1 Animal Source

[0121] SPF-grade male Sprague-Dawley (SD) rats (250 g - 300 g) were purchased from Spif Bio-Technology Co., Ltd. (Beijing), and the animal license number is SCXK (Jing) 2024-0001. SPF-grade male Balb / c mice (18 g) were purchased from Shaanxi Shanyao Medical Biotechnology Co., Ltd., and the animal license number is SCXK (Shaan) 2024-004. All experimental operations were approved by the Laboratory Animal Ethics Committee of Yan'an University.

[0122] 11.2 Construction and grouping of the model

[0123] After 1 week of environmental adaptation, 36 male SD rats weighing about 250 g were divided into 6 groups: sham operation group, vehicle group, α-lipoic acid group, Fer-1 group, edaravone group, and D14 group. All drugs were dissolved in 5% (v / v) DMSO and 95% (v / v) physiological saline containing 20% (v / v) cyclodextrin. Among them, the treatment methods for each group were as follows:

[0124] (1) Sham operation group: The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed, and blunt separation of muscles and glands was performed, but no monofilament nylon was inserted. After surgery, 300 μL of physiological saline was injected into the tail vein. (2) Vehicle group: After surgery, 300 μL of 5% (v / v) DMSO and 95% (v / v) physiological saline containing 20% (v / v) cyclodextrin was injected into the tail vein. (3) α-lipoic acid group: After surgery, 300 μL of α-lipoic acid with a concentration of 5 mg / kg was injected into the tail vein; Fer-1 group: After surgery, 300 μL of Fer-1 with a concentration of 5 mg / kg was injected into the tail vein; Edaravone group: After surgery, 300 μL of edaravone with a concentration of 5 mg / kg was injected into the tail vein; D14 group: After surgery, 300 μL of compound D14 with a concentration of 5 mg / kg was injected into the tail vein.

[0125] Anesthesia was administered using a mixture of 3% isoflurane, 70% nitrous oxide, and 30% oxygen, and maintained with a 1% isoflurane solution using an anesthesia system. Mice were then immobilized in a supine position, and an incision was made along the mid-neck to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Muscles and glands were bluntly dissected. The ECA and CCA were then ligated proximally with 6-0 sutures, and a monofilament nylon suture selected according to the mouse's weight was inserted into the ICA through a small opening in the CCA to block blood flow to the CCA. 1.5 hours after MCAO, α-lipoic acid, edaravone, Fer-1, and compound D14 (all at 5 mg / kg) were administered intravenously, and the suture was removed, allowing for immediate reperfusion. Postoperatively, the mice's body temperature was maintained at 37°C, and they were provided with ample food and water. Throughout the procedure, a heating pad was used to maintain the animals' body temperature at 37°C ± 0.5°C. Body temperature and respiratory rate were monitored during the procedure. The procedure for the sham surgery group was the same as described above, except that a monofilament nylon was inserted.

[0126] 11.3 Neurological deficit score

[0127] Neurological deficits in each rat were assessed using a five-point scale: 0, no significant deficit; 1, flexion of the left forelimb, mild neurological dysfunction; 2, turning to the left and flexion of the left forelimb while walking, moderate neurological dysfunction; 3, more severe than level 2, turning to the left, severe neurological dysfunction; 4, unable to leave the problem area and losing consciousness; 5, death. Neurological scores were given by two individuals unaware of the experimental design.

[0128] 11.4 TTC staining

[0129] After 1.5 hours of MCAO followed by reperfusion for 24 hours, rats were weighed and euthanized with an overdose of isoflurane. The brain was quickly removed and placed at 20°C for 20 minutes. Starting from both poles of the frontal lobe, five coronal sections were prepared at 2 mm intervals. The brain sections were then immersed in 1% TTC solution at 37°C for 20 minutes.

[0130] 11.5 Quantitative measurement of cerebral infarction volume

[0131] After photographing rat brain slices with a digital camera, the infarct volume was calculated using ImageJ software. An observer unaware of the experimental group calculated the area of ​​the infarcted region (white staining) and the non-infarcted region (red staining), and corrected for the infarcted area by subtracting the area of ​​the edematous region. In summary, the corrected infarct area was ¼ the area of ​​the undamaged lateral brain region minus (the area of ​​the damaged lateral brain region minus the damaged area). The infarct volume of each brain slice was equal to the infarct area multiplied by the slice thickness.

[0132] 12. Internal toxicity

[0133] 12.1 Cytotoxicity grouping and body weight determination

[0134] After 1 week of acclimatization, 21 Balb / c mice weighing approximately 18g were divided into three groups: a control group (saline), a 100mg / kg D14 group, and a 250mg / kg D14 group. Each group received the drug via tail vein, three times every two days. Body weight was measured every two days for a total of 16 days. Compound D14 was dissolved in 5% (v / v) DMSO and 95% (v / v) saline containing 20% ​​(v / v) cyclodextrin.

[0135] The processing methods for each group are as follows:

[0136] Control (saline) group: 100 μL of saline was injected via tail vein; 100 mg / kg D14 group: 100 μL of compound D14 at a concentration of 100 mg / kg was injected via tail vein; 250 mg / kg D14 group: 100 μL of compound D14 at a concentration of 250 mg / kg was injected via tail vein.

[0137] 12.2 HE staining

[0138] Animals were divided into three groups: a control group (physiological saline), a 100 mg / kg D14 group, and a 250 mg / kg D14 group. After 16 days of continuous administration, heart, liver, spleen, lung, and kidney were harvested and fixed in 4% paraformaldehyde for 24 hours. They were then dehydrated sequentially in 15% and 30% sucrose solutions, followed by OCT embedding. Sections were then prepared using a cryostat and stained according to the HE staining kit (Solarbio) instructions. Finally, photographs were taken under an inverted fluorescence microscope (Carl Zeiss). The treatment methods for each group were the same as in section 10.1.

[0139] 12.3 Evaluation of Liver and Kidney Function

[0140] Animals were divided into three groups: a control group (physiological saline), a 100 mg / kg D14 group, and a 250 mg / kg D14 group. After 16 consecutive days of administration, ocular blood was collected from mice in each group and centrifuged at low temperature. Liver and kidney function were then evaluated according to the instructions of the BUN reagent (Jining Industrial Co., Ltd.), the CRE content assay kit (Jining Industrial Co., Ltd.), the GOT activity assay kit (Jining Industrial Co., Ltd.), and the GPT activity assay kit (Jining Industrial Co., Ltd.). The treatment methods for each group were the same as in section 10.1.

[0141] 13. In vitro toxicity

[0142] The cytotoxic effects of compound D14 on PC12, BV2, HCT116, RAW264.7 and HepG2 cells were detected using the Cell Counting Kit-8 (CCK-8).

[0143] Cells were loaded at 5 × 10 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight in 100 μL / well DMEM medium. They were then treated with compound D14 at a given concentration gradient for 12 h, followed by replacement of the medium with 100 μL of fresh DMEM medium containing 10 μL of CCK-8 solution, and incubated at 37°C for 1 h. OD values ​​were measured at 450 nm using a molecular device. Relative cell viability is expressed as a percentage of the control group. HCT116 cells were purchased from Beyotime Biotechnology Co., Ltd. RAW264.7 cells were purchased from Beyotime Biotechnology Co., Ltd. HepG2 cells were purchased from Beyotime Biotechnology Co., Ltd.

[0144] II. Results:

[0145] 1. Cytotoxicity test:

[0146] The results are as follows Figure 3 The results showed that when compound D14 was administered at a concentration of 60 μM, the viability of all five cell lines (PC12 and BV2) remained unchanged. This result was confirmed in HCT116, RAW264.7, and HepG2 cells. These results indicate that compound D14 exhibits low toxicity in vitro and has the potential for further investigation.

[0147] 2. Compound D14 is a free radical scavenging antioxidant that does not chelate Fe. 2+ :

[0148] Experimental results are as follows Figure 4 As shown in Figure A, compounds Y1, Fer-1, α-LA, Y2, and D14 all reduced DPPH levels. This indicates that compound D14 is a free radical scavenging antioxidant, and its antioxidant activity is significantly superior to that of compound Y2. To determine whether compound D14 is an iron chelating agent, this invention uses a colorimetric method based on ferriazine to detect its iron chelating ability. Figure 4 As shown in B, DFS and EDTA can chelate Fe. 2+ H2O2, Fer-1, α-LA, and compound D14 cannot chelate Fe. 2+ This indicates that compound D14 is a free radical scavenging antioxidant, not an iron chelating agent.

[0149] 3. Compound D14 has a protective effect against Erastin-induced PC12 ferroptosis model:

[0150] To further determine the protective mechanism of compound D14 in the PC12 ferroptosis model, this invention examined the GSH levels in cells. The invention found that, compared with the Fer-1 group and the α-LA group, treatment with compound D14 significantly increased the GSH levels in Erastin-treated PC12 cells in a concentration-dependent manner. Figure 5 (See Figure A in the original text). The invention also subsequently determined the ferroptosis-related biomarker Fe. 2+ And MDA. The results showed that the MDA content and Fe content of the compound D14 group were higher than those of the Erastin group. 2+ The content decreased significantly, and the effect was better than that of the Fer-1 group and the α-LA group, such as Figure 5 Figure B in the middle and Figure 5 Figure C in the diagram. Furthermore, this invention also examined intracellular reactive oxygen species (ROS) levels, and the results showed that compound D14 significantly reduced ROS levels in Erastin-treated PC12 cells (…). Figure 6 Figure A in the middle and Figure 6 (See Figure B in the diagram). In summary, compared with Y2, compound D14 is more protective against Erastin-induced PC12 ferroptosis.

[0151] 4. Compound D14 has a protective effect against LPS-induced BV2 inflammation models:

[0152] To test the anti-inflammatory ability of compound D14, this invention constructed a BV2 inflammation model using LPS ( Figure 7 (Figure A in the image). Figure 7 As shown in Figure B, compound D14's ability to rescue LPS-induced BV2 inflammation in the model is comparable to that of α-LA, and it exhibits a concentration-dependent effect. Furthermore, compared to EDV, compound D14 demonstrates stronger anti-inflammatory activity. Figure 7 (See Figure C in the original text). Furthermore, the anti-inflammatory activities of α-LA, EDV, and compound D14 were tested in this invention, with results as follows: Figure 8 As shown in the figure. The results indicate that the anti-inflammatory activity of compound D14 is close to that of α-LA and significantly higher than that of EDV. This suggests that compound D14 retains the anti-inflammatory ability of α-LA and has a protective effect against LPS-induced PC12 inflammation.

[0153] 5. Acute toxicity test:

[0154] To investigate the in vivo safety of compound D14, Balb / c mice were intravenously injected with compound D14 at doses of 100 mg / kg and 250 mg / kg for 16 consecutive days. Body weight was measured every two days. Results showed no significant difference in body weight between the control group and the treated group during the 16-day treatment period. Figure 9 (See Figure A in the original text). In addition, this invention also observed HE staining of the heart, liver, spleen, lungs, and kidneys of mice in the normal group and two different dose groups, with results as shown in Figure A. Figure 9 As shown in Figure B, there were no significant differences in HE staining results of the major organs among the three groups of mice. Finally, this invention also evaluated liver and kidney function in the three groups of mice 16 days after drug administration, measuring GOT, GPT, BUN, and CRE levels. The results showed no significant differences in GOT, GPT, and BUN levels among the three groups of mice. Figure 9 Figure C in the middle~ Figure 9 (See Figure E in the diagram). This demonstrates that compound D14 exhibits good tolerability at doses up to 250 mg / kg. Compound D14 exhibits low toxicity both in vivo and in vitro, indicating potential for further research.

[0155] 6. The in vivo effects of compound D14 on an ischemic stroke model:

[0156] A classic middle cerebral artery occlusion (MCAO) model was established to observe the effect of compound D14 on infarct volume, thereby evaluating the neuroprotective effect of compound D14 in vivo. Subsequently, the brains of rats in each group were stained with 2,3,5-triphenyltetrachloride (TTC staining), and the results are as follows: Figure 10 Figure A in the middle~ Figure 10 As shown in Figure B, this invention observed a reduction in infarct volume in the α-LA group (1 mg / kg), Fer-1 group (1 mg / kg), and edaravone group (1 mg / kg), but the effect was worse compared to the D14 group. This invention also conducted a modified mNSS neurological deficit assessment to systematically study the formation process of the rat MCAO model. The results showed that compared with the MCAO group, the scores of the α-LA group, Fer-1 group, edaravone group, and D14 group were all improved, with the most significant reduction in the D14 group. Figure 10 (See Figure C in the original text). Finally, this invention measured GSH, MDA, and Fe in the cerebral hemisphere of the injured side. 2+ The results showed that treatment with compound D14 significantly increased GSH levels and significantly decreased MDA and Fe levels. 2+ level( Figure 10 Image D in the middle~ Figure 10 (Figure F in the figure), this result is consistent with the results of in vitro experiments ( Figure 5 Figure A in the middle~ Figure 5(Figure C in the diagram).

[0157] As can be seen from the above, administration of compound D14 can effectively restore MCAO, reduce the infarct volume of ischemic hemispheres, and restore the walking ability of rats by inhibiting ferroptosis.

[0158] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0159] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. An α-lipoic acid phenothiazine derivative, characterized in that, The structural formula of the α-lipoic acid phenothiazine derivative is shown in Formula 1: ； In Formula 1, R is selected from any one of the groups shown in Formulas 5 to 8, 10 to 15, 18 to 21, and 23 to 24: ； Or the structural formula of the α-lipoic acid phenothiazine derivative is shown in any of the following formulas: 。 2. The method for preparing the α-lipoic acid phenothiazine derivative according to claim 1, characterized in that, Includes the following steps: A raw material providing the R group, YADX-1, and a condensing agent are dissolved together in DMF to undergo a condensation reaction, generating intermediate product 1 with a structure as shown in Formula 2-1, Formula 16-2, or Formula 17-2; wherein, the structure of YADX-1 is shown in Formula 3. ； ； Trifluoroacetic acid and dichloromethane were added to intermediate 1, and Boc was removed under ice bath conditions to generate intermediate 2 with the structure shown in Formula 4-1, Formula 16-4 or Formula 17-4. ； The intermediate product 2 and α-lipoic acid were mixed and condensed under the action of condensing agent 2 to obtain the α-lipoic acid phenothiazine derivative.

3. The preparation method according to claim 2, characterized in that, The condensing agent includes at least one of PyBOP and DIEA; The condensing agent 2 includes at least one of EDCI, HOBT, and DIEA.

4. A pharmaceutical composition, characterized in that, The active ingredient is the α-lipoic acid phenothiazine derivative of claim 1 or a pharmaceutically acceptable salt thereof.

5. The use of the α-lipoic acid phenothiazine derivative of claim 1 or the pharmaceutical composition of claim 4 in the preparation of ferroptosis inhibitors.

6. The use of the α-lipoic acid phenothiazine derivative of claim 1 or the pharmaceutical composition of claim 4 in the preparation of anti-inflammatory and / or antioxidant agents.

7. The use of the α-lipoic acid phenothiazine derivative of claim 1 or the pharmaceutical composition of claim 4 in the preparation of a therapeutic remedy for ferroptosis-related diseases.

8. The application according to claim 7, characterized in that, The iron death-related diseases include at least one of spinal cord injury, Alzheimer's disease, Parkinson's disease, liver injury, and ischemic stroke.

9. The application according to claim 7, characterized in that, The therapeutic agent includes the α-lipoic acid phenothiazine derivative or the pharmaceutical composition, and pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The excipients include any one or more of the following: excipients and flavoring agents.

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