Triazolothiadiazole compound as well as preparation method and application thereof

Abstract

The invention relates to a triazolothiadiazole compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The structure of the triazolothiadiazole compound disclosed by the invention is # imgabs0 #, wherein R1 is heterocyclic amino; and R2 is selected from aryl or heteroaryl. The triazolothiadiazole compound has remarkable inhibitory activity on ferroptosis inhibitory protein 1, has an inhibitory effect on proliferation of HT1080 cells, and has a good anti-tumor effect; therefore, the triazolothiadiazole compound can be used for preparing related diseases mediated by ferroptosis inhibition protein 1, and has potential application space in the aspect of development of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a triazolothiadiazole compound and a preparation method and application thereof. Background Art

[0002] There are many ways of programmed cell death, including apoptosis, pyroptosis, and autophagy. Ferroptosis is a new type of non-apoptotic programmed cell death caused by excessive accumulation of iron-dependent lipid peroxides discovered in 2012. This cell death mode is characterized by the fact that when the free iron in the cell increases, on the one hand, more reactive oxygen species will be produced through the Fenton reaction, oxidizing polyunsaturated fatty acids on the cell membrane into lipid peroxides; on the other hand, lipid peroxides can be further catalyzed into toxic lipid free radicals, leading to cell death. Ferroptosis has been found to be highly correlated with human diseases, such as neurodegenerative diseases, tissue damage during cold exposure, ischemia-reperfusion injury, and cancer. Inducing ferroptosis is a very promising method for treating malignant tumors. It has shown synergistic effects with cancer immunotherapy and can even kill drug-resistant and metastatic tumor cells.

[0003] Ferroptosis has attracted great interest because it has become a very promising target for cancer treatment. So far, it has been found that ferroptosis is regulated by at least three major redox-related systems, including the cysteine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) system, the guanosine triphosphate cyclohydrolase-tetrahydrobiopterin (BH4) system, and the ferroptosis suppressor protein 1 (FSP1)-CoQ system. 10 system. Among them, FSP1-CoQ 10 FSP1 inhibits ferroptosis by synergizing with GSH-GPX4. FSP1 inhibits ferroptosis by reducing ubiquinone to ubiquinol or vitamin K to hydroquinone. Studies have shown that inhibiting FSP1 can affect FSP1's ability to reduce lipid peroxides, thereby inducing cell ferroptosis and effectively preventing tumor recurrence. Therefore, the development of FSP1 inhibitors is of great significance for cancer treatment.

[0004] Although FSP1 inhibitors show great promise in tumor treatment, there are still certain challenges in developing small molecule inhibitors targeting FSP1. Not only are there few FSP1 inhibitors, but there are also no reports of FSP1 inhibitors entering clinical research. The reported FSP1 inhibitors mainly include NPD4928, iFSP1, icFSP1, FSEN1, FSEN3, etc. These reported FSP1 inhibitors have shown the efficacy of inducing ferroptosis in several cancer cell lines, but their inhibitory effects on FSP1 are not strong enough, and their pharmacokinetic properties are poor, making them unsuitable for in vivo use; in addition, they exhibit off-target effects at high concentrations. Therefore, new FSP1 inhibitors with improved pharmacokinetic and pharmacodynamic properties are urgently needed to fully explore the potential of FSP1 as a tumor treatment target. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of the FSP1 inhibitors in the prior art, such as a small selection range, insufficient inhibitory effect, and poor drugability.

[0006] In order to solve the above technical problems, the present invention provides a triazolothiadiazole compound and a preparation method and application thereof.

[0007] The first object of the present invention is to provide a triazolothiadiazole compound, the structure of which is shown in Formula V:

[0008]

[0009] Wherein, R1 is a heterocyclic amino group;

[0010] R2 is selected from aryl or heteroaryl.

[0011] In one embodiment of the present invention, the heterocyclic amino group is selected from

[0012] The aryl group is selected from: The heteroaryl group is

[0013] In one embodiment of the present invention, the triazolothiadiazole compound is selected from the following compounds:

[0014]

[0015] The second object of the present invention is to provide a method for preparing the triazolothiadiazole compound, comprising the following steps:

[0016] S1, a compound of formula I, carbon disulfide and potassium hydroxide react in a first organic solvent to obtain a compound of formula II;

[0017] S2, the compound of formula II reacts under the action of hydrazine hydrate to obtain a compound of formula III;

[0018] S3, the compound of formula III and chloroacetic acid react under the action of phosphorus oxychloride to obtain a compound of formula IV;

[0019] S4, reacting the compound of formula IV with the secondary amine H-R1 in a second organic solvent under the action of N,N-diisopropylethylamine to obtain the triazolothiadiazole compound of formula V;

[0020] Wherein, the structures of Formula I to Formula V are shown below:

[0021]

[0022] Wherein, R1 is a heterocyclic amino group;

[0023] R2 is selected from aryl or heteroaryl.

[0024] In one embodiment of the present invention, in S1, the first organic solvent is selected from one or more of methanol, ethanol, dichloromethane and ethyl acetate.

[0025] In one embodiment of the present invention, in S2, the reaction temperature is 115°C-125°C, and the reaction time is 16h-24h.

[0026] In one embodiment of the present invention, in S3, the reaction temperature is 95°C-115°C, and the reaction time is 8h-12h.

[0027] In one embodiment of the present invention, in S4, the reaction temperature is 85°C-95°C, and the reaction time is 10h-14h.

[0028] In one embodiment of the present invention, in S4, the second organic solvent is selected from one or more of acetonitrile, diethyl ether, dichloromethane and ethyl acetate.

[0029] In one embodiment of the present invention, the reaction formula of the triazolothiadiazole compound is as follows:

[0030]

[0031] The third object of the present invention is to provide a stereoisomer and / or a pharmaceutically acceptable salt of the triazolothiadiazole compound.

[0032] The fourth object of the present invention is to provide a pharmaceutical composition, the active ingredient of which is the triazolothiadiazole compound, the stereoisomer of the triazolothiadiazole compound and / or a pharmaceutically acceptable salt.

[0033] The fifth object of the present invention is to provide a triazolothiadiazole compound, a stereoisomer and / or a pharmaceutically acceptable salt of the triazolothiadiazole compound, and a pharmaceutical composition for use in preparing drugs for treating FSP1-related diseases.

[0034] In one embodiment of the present invention, the FSP1-related diseases include one or more of gastric cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, melanoma, lymphoma, leukemia, copper tolerance, severe acute pancreatitis, diabetes, Parkinson's disease, retinal degeneration, age-related macular degeneration, acute kidney injury, early brain injury, liver fibrosis and non-alcoholic fatty liver disease.

[0035] The technical solution of the present invention has the following advantages over the prior art:

[0036] (1) The present invention is the first to construct a compound system with triazolothiadiazole as the core skeleton. Compared with the thiazotriazole FSP1 inhibitors, the isosteric substitution of the thiadiazole ring produces a unique spatial topological structure. The nitrogen atom at position 2 forms a specific hydrogen bond interaction with the Lys355 residue in the FSP1 catalytic domain. Compound P3 also establishes a T-type π-π stacking interaction with Tyr296 through the side chain of methyl 4-(piperazine-1-acyl)benzoate, which increases the binding ability of the compound to FSP1 and enhances the inhibitory activity against FSP1.

[0037] (2) The triazolothiadiazole compounds of the present invention can enhance RSL3-induced ferroptosis in cancer cell lines from different tissues, and target and inhibit FSP1, blocking CoQ 10 , VK-type redox active molecules, thereby promoting the occurrence of phospholipid peroxidation and further promoting RSL3-induced cell ferroptosis; this shows that the triazolothiadiazole compound can be used as a sensitizer in combination therapy with GPX4 inhibitors, providing a new chemical entity for solving the problem of tumor resistance.

[0038] (3) The triazolothiadiazole compounds described in the present invention have significant inhibitory activity against ferroptosis inhibitory protein 1, and have an inhibitory effect on the proliferation of HT1080 cells, and have good anti-tumor effects; this indicates that the triazolothiadiazole compounds can be used to prepare treatments for ferroptosis inhibitory protein 1-mediated related diseases, and have potential application space in the development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0040] Figure 1 The binding mode prediction and interaction analysis of compound P3 and FSEN3 in Test Example 1 of the present invention; wherein A is the potential binding mode of FSEN3 and FSP1 (PDB ID: 8WIK), B is a three-dimensional close-up image of the intermolecular interaction between compound P3 and FSP1 (the residues around the ligand are white, the carbon atoms of the ligand are painted orange, the hydrophobic interaction is represented by a gray dashed line, the hydrogen bond is represented by a green dashed line, and the π-π interaction is represented by a yellow dashed line), and C and D are two-dimensional interaction images of FSEN3 and compound P3 with FSP1;

[0041] Figure 2 The test results of the compounds in Test Example 2 of the present invention for enhancing RSL3-induced ferroptosis; wherein AC is the cell viability of the indicated cancer cell lines treated with a specified concentration of RSL3 in the presence of 1 μM of a specified FSP1 inhibitor, and D is a representative image of A549 cells treated with 2.5 μM RSL3 in the presence of 1 μM of a specified FSP1 inhibitor or DMSO;

[0042] Figure 3 The test results of the compounds in Test Example 3 of the present invention for enhancing lipid peroxidation induced by RSL3; wherein, AC is the lipid peroxidation evaluation of the indicated cancer cell lines treated with 2.5 μM RSL3 or DMSO in the presence of 1 μM of the specified FSP1 inhibitor or DMSO, **p<0.01, ****p<0.0001, ns, not significant;

[0043] Figure 4 In Test Example 4 of the present invention, HT1080 cells were treated with 2 μM RSL3 to observe the effect of VK on cell survival rate. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0045] In the present invention, unless otherwise explained, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0046] In the present invention, unless otherwise stated, the term "and / or" used in the present invention includes any and all combinations of one or more of the associated listed items.

[0047] In the present invention, unless otherwise stated, the experimental methods used in the embodiments of the present invention are conventional methods unless otherwise stated, and the materials, reagents, etc. used are all commercially available unless otherwise stated.

[0048] Example 1

[0049] The triazolothiadiazole compound of the present invention and the preparation method thereof specifically comprise the following steps:

[0050]

[0051] S1. Synthesis of 4-amino-5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (IIIa): At room temperature, compound Ia (1.66 g, 10 mmol), potassium hydroxide (1.12 g, 20 mmol) and carbon disulfide (1.2 mL, 20 mmol) were added to ethanol and reacted at room temperature for 12 h. After the reaction was completed by TLC monitoring, ether was added, the mixture was filtered with suction, washed with water and ether, and dried to obtain white solid IIa (2.52 g, 90%). Compound IIa (2.52 g, 9 mmol) was dissolved in water, 85% hydrazine hydrate solution (4.36 mL, 90 mmol) was added dropwise with stirring, and refluxed at 120°C for 20 h. After the reaction was completed by TLC monitoring, water was added for dilution, the mixture was acidified with concentrated hydrochloric acid, cooled for crystallization, filtered with suction, washed with water, dried, and recrystallized with ethanol to obtain white solid IIIa (1.0 g, 50%).

[0052] 1 H NMR (400MHz, DMSO) δ13.84(s,1H),7.97(d,J=8.9Hz,2H),7.08(d,J=9.0Hz,2H),5.77(s,2H),3.82(s,3H).

[0053] 13 C NMR (101MHz, DMSO-d6) δ166.6,160.9,149.4,129.6,118.1,114.0,55.4.

[0054] HRMS(ESI)m / z:[M+H] + calculated for C9H 10 N4OS:223.0648; found:223.0653.

[0055] S2. Synthesis of 6-(chloromethyl)-3-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (IVa): At room temperature, compound IIIa (4.44 g, 20 mmol) and chloroacetic acid (2.27 g, 24 mmol) were added to phosphorus oxychloride, heated to 100°C for 10 h; the reaction was completed by TLC monitoring, cold water was added to quench, the pH was adjusted to neutral with 40% NaOH solution, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was separated by silica gel column chromatography and eluted with dichloromethane and methanol (dichloromethane: methanol = 50:1) to give a yellow solid IVa (2.24 g, 40%).

[0056] 1 H NMR (400MHz, CDCl3) δ8.24(d,J=8.9Hz,2H),7.04(d,J=8.9Hz,2H),4.86(s,2H),3.88(s,3H).

[0057] 13 C NMR (101MHz, Chloroform-d) δ165.6,161.3,154.0,146.5,128.0,117.8,114.3,55.4,39.6.

[0058] HRMS(ESI)m / z:[M+H] + calculated for C 11 H9ClN4OS:281.0258; found:281.0262.

[0059] S3. Synthesis of 6-(dihydroindole-1-ylmethyl)-3-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (P1): At room temperature, compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL), and dihydroindole (0.4 mmol, 45.7 μL) and N,N-diisopropylethylamine (0.8 mmol, 139 μL) were added, and then the temperature was raised to 90°C and refluxed for 12 h. After the reaction, the reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography and eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid P1 (57.4 mg, 79%).

[0060] 1H NMR (400MHz, CDCl3) δ8.27(d,J=8.9Hz,2H),7.18(d,J=7.2Hz,1H),7.12(t,J=7.7Hz,1H),7.05(d,J=8.9Hz,2H),6. 83(t,J=7.4Hz,1H),6.58(d,J=7.8Hz,1H),4.58(s,2H),3.89(s,3H),3.55(t,J=8.2Hz,2H),3.10(t,J=8.2Hz,2H).

[0061] 13 C NMR (101MHz, CDCl3) δ171.0,161.2,154.1,150.6,146.2,130.0,127.9,127.5,125.1,120.1,118.3,114.3,107.7,55.4,54.8,50.9,28.7.

[0062] HRMS(ESI)m / z:[M+H] + calculated for C 19 H 17 N5OS:364.1227; found:364.1224.

[0063] Example 2

[0064] The method is basically the same as Example 1, except that S3 specifically includes the following steps:

[0065]

[0066] S3. Synthesis of 5-((3-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (P2): Compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL) at room temperature, 4,5,6,7-tetrahydrothieno[3.2-c]pyridine (0.4 mmol, 48.6 μL) and N,N-diisopropylethylamine (0.8 mmol, 139 μL) were added at room temperature, and then the temperature was raised to 90°C and refluxed for 12 h. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography and eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1) ​​to obtain a yellow solid P2 (71.0 mg, 90%).

[0067] 1H NMR (600MHz, CDCl3) δ8.26(d,J=8.9Hz,2H),7.13(d,J=5.1Hz,1H),7.04(d,J=8.8Hz,2H),6.74(d, J=5.1Hz,1H),4.07(s,2H),3.88(s,3H),3.82(s,2H),3.01(t,J=5.6Hz,2H),2.96(t,J=5.7Hz,2H).

[0068] 13 C NMR (151MHz, CDCl3) δ172.1,161.1,154.3,146.1,133.0,132.4,127.9,125.0,123.3,118.3,114.3,56.4,55.4,53.2,50.8,25.0.

[0069] HRMS(ESI)m / z:[M+H] + calculated for C 18 H 17 N5OS2:384.0948; found:384.0948.

[0070] Example 3

[0071] The method is basically the same as Example 1, except that S3 specifically includes the following steps:

[0072]

[0073] S3. Synthesis of methyl 4-(4-((3-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl)piperazin-1-yl)benzoate (P3): At room temperature, compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL), methyl 4-(piperazin-1-yl)benzoate (0.4 mmol, 88.0 mg) and N,N-diisopropylethylamine (0.8 mmol, 139 μL) were added, and then the temperature was raised to 90°C and refluxed for 12 h. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography and eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1.5) to obtain a white solid P3 (80.1 mg, 86%).

[0074] 1H NMR (400MHz, CDCl3) δ8.26(d,J=8.9Hz,2H),7.94(d,J=9.0Hz,2H),7.04(d,J=8.9Hz,2H),6.89 (d,J=9.0Hz,2H),3.95(s,2H),3.88(s,3H),3.87(s,3H),3.45-3.38(m,4H),2.84-2.82(m,4H).

[0075] 13 C NMR (151MHz, DMSO-d6) δ172.7,166.1,160.7,154.0,153.7,145.0,130.7,127.4,118.3,118.0,114.6,113.5,56.2,55.4,52.3,51.5,46.7.

[0076] HRMS(ESI)m / z:[M+H] + calculated for C 23 H 24 N6O3S:465.1704; found:465.1704.

[0077] Example 4

[0078] Basically the same as Example 1, except that in S1, compound Ia is replaced with benzoyl hydrazide Ib to obtain 6-(dihydroindole-1-ylmethyl)-3-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (P4);

[0079]

[0080] 1 H NMR (600MHz, CDCl3) δ8.34(d,J=7.0Hz,2H),7.54(t,J=7.4Hz,2H),7.50(t,J=7.3Hz,1H),7.19(d,J=7.3Hz,1H),7.12(t ,J=7.7Hz,1H),6.84(t,J=7.4Hz,1H),6.58(d,J=7.8Hz,1H),4.59(s,2H),3.56(t,J=8.2Hz,2H),3.10(t,J=8.2Hz,2H).

[0081] 13C NMR (151MHz, CDCl3) δ171.4,154.6,150.5,146.3,130.3,130.0,128.9,127.5,126.3,125.6,125.1,120.1,107.7,54.9,50.9,28.7.

[0082] HRMS(ESI)m / z:[M+H] + calculated for C 18 H 15 N5S:334.1121; found:334.1119.

[0083] Example 5

[0084] Basically the same as Example 1, except that in S1, compound Ia is replaced with 4-chlorobenzoyl hydrazide Ic to obtain 3-(4-chlorophenyl)-6-(dihydroindole-1-ylmethyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (P5);

[0085]

[0086] 1 H NMR (400MHz, CDCl3) δ8.29(d,J=8.6Hz,2H),7.52(d,J=8.6Hz,2H),7.19(d,J=7.1Hz,1H),7.12(t,J=7.5Hz, 1H), 6.84 (t, J = 7.5Hz, 1H), 6.57 (d, J = 7.7Hz, 1H), 4.59 (s, 2H), 3.56 (t, J = 8.2Hz, 2H), 3.11 (t, J = 8.1Hz, 2H).

[0087] 13 C NMR (151MHz, CDCl3) δ171.8,154.7,150.5,145.4,136.3,130.0,129.2,127.6,127.5,125.1,124.1,120.2,107.7,54.9,50.9,28.7.

[0088] HRMS(ESI)m / z:[M+H] + calculated for C 18 H 14 ClN5S:368.0731; found:368.0731.

[0089] Example 6

[0090] Basically the same as Example 1, except that in S1, compound Ia is replaced with 2-furancarboxylic acid hydrazide Id to obtain 3-(furan-2-yl)-6-(dihydroindole-1-ylmethyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (P6);

[0091]

[0092] 1 H NMR (400MHz, CDCl3) δ7.68(d,J=1.8Hz,1H),7.25(d,J=4.0Hz,1H),7.18(d,J=7.3Hz,1H),7.12(t,J=7.1Hz,1H),6.83(t,J =7.4Hz,1H),6.62(dd,J=3.4,1.8Hz,1H),6.57(d,J=7.8Hz,1H),4.58(s,2H),3.55(t,J=8.2Hz,2H),3.10(t,J=8.2Hz,2H).

[0093] 13 C NMR (101MHz, CDCl3) δ172.3,153.9,150.5,147.3,144.6,140.7,129.9,127.6,125.1,120.2,111.7,111.6,107.7,54.9,50.9,28.7.

[0094] HRMS(ESI)m / z:[M+H] + calculated for C 16 H 13 N5OS:324.0914; found:324.0911.

[0095] Inhibitory activity of the compound of test example 1 on FSP1

[0096] The enzyme reaction was prepared using TBS buffer (50 nM Tris-HCl and 150 nM NaCl), which included the test molecules P1-P6 and 50 nM non-myr-FSP1, 200 nM NADH, and FSEN3 as a positive control. Then, 100 μM of fluoroquinone sodium salt (Sigma, cat. no. R7017) was added to the reaction system, and the fluorescence intensity (F) was measured every minute at 37°C using a SpectraMax iD5 microplate reader and SoftMax Pro v.7 software, with an excitation wavelength / emission wavelength of 540 / 590 nm. The IC was calculated using an equal volume of DMSO and a control reaction without fluoroquinone. 50After the experiment, GraphPadPrism 9 software was used for curve fitting and IC 50 The calculation results are shown in Table 1:

[0097] Table 1 Inhibitory activity of compounds on FSP1 protein

[0098] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> P1 0.05±0.02 P2 0.18±0.05 P3 0.035±0.016 P4 0.09±0.07 P5 0.07±0.00 P6 0.19±0.06 FSEN3 0.15±0.09

[0099] As can be seen from Table 1, the triazolothiadiazole compounds of the present invention have good inhibitory activity against FSP1. The FSP1 inhibitory activity of compounds P2 and P6 is comparable to that of FSEN3, and the FSP1 inhibitory activity of compounds P1, P3, P4, and P5 is better than that of FSEN3.

[0100] Compounds P3 and FSEN3 with the best enzyme inhibitory activity were selected for molecular docking with FSP1. Compounds P3 and FSEN3 both adopted similar binding modes. The predicted binding modes are as follows: Figure 1 As shown. Figure 1 It can be seen that the 4-methoxyphenyl ring of compounds P3 and FSEN3 is sandwiched by NAD+ and Val50, forming a bond π-π stacking interaction. Both compounds P3 and FSEN3 form hydrogen bonds with amino acid residue Ala295 through the nitrogen atom of the triazole ring. In addition, their R1 side chains extend into the hydrophobic pocket occupied by UQ1. Compared with FSEN3, the thiadiazole ring of compound P3 can form an additional hydrogen bond with Lys355. In addition, the 4-(piperazine-1-acyl)benzoic acid methyl ester side chain of compound P3 exhibits obvious π-π stacking interactions with Tyr296, presenting a t-shaped structure, which are the main reasons for its enhanced inhibitory effect on FSP1.

[0101] Test Example 2 Effect of Compounds on Cell Viability by Inhibiting FSP1 Combined with RSL3

[0102] FSP1 inhibitors can enhance RSL3-induced ferroptosis in cancer cell lines from different tissues, such as human non-small cell lung cancer cell line A549, human fibrosarcoma cell line HT1080, and human osteosarcoma cell line U2OS. Therefore, these three tumor cell lines were selected to determine whether the compounds can enhance RSL3-induced ferroptosis. A549, HT1080, and U2OS cells were cultured to a cell density of about 80%, the cells were digested and centrifuged, and the cells were resuspended and counted in 1 mL of fresh culture medium, and then the cells were inoculated into a 96-well plate at a number of 5000 cells / well. The 24 wells on the upper and lower edges were not inoculated with cells, and 200 μL PBS was added; the 96-well plate was placed in a cell culture incubator for culture. After about 12 hours, the cells were completely attached to the wall and drug treatment was performed. 1 μM of the test compounds P1 and P3 were added to all wells, and FSEN3 was used as a positive control; after adding the test compounds, RSL3 was set to a concentration gradient of 0, 0.01 μM, 0.03 μM, 0.05 μM, 0.1 μM, 0.3 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, and 10 μM, and added to the well plate in sequence, and 2 replicate wells were set for each concentration; after adding RSL3 and the test compounds, cell death was observed. After about 5 hours, the 96-well plate was taken out and the CellTiter The cells in the well plate were lysed by the Glo kit, and after thorough mixing, the cell ATP level was quantified by measuring the fluorescence value on the microplate reader (TECAN), and the relative viability was evaluated by the ATP reading value; GraphPad was used for data processing to calculate the effect of the test compound on cell viability by inhibiting FSP1 combined with RSL3. The results are shown in Figure 2 As shown. Figure 2 It can be seen that in human non-small cell lung cancer cell line A549, human fibrosarcoma cell line HT1080 and human osteosarcoma cell line U2OS, compounds P1 and P3 can significantly promote RSL3-induced ferroptosis.

[0103] Test Example 3 Effects of compounds on cellular lipid peroxidation by inhibiting FSP1 combined with RSL3

[0104] Since FSP1 exerts anti-ferroptosis activity by reducing RTA molecules such as CoQ or VK, and CoQ and VK can inhibit the propagation stage of phospholipid peroxidation and thus inhibit phospholipid peroxidation, the inhibition of FSP1 will theoretically promote the occurrence of phospholipid peroxidation. Based on this, the intracellular lipidROS level was detected by flow cytometry. A549, HT1080, and U2OS cells were cultured to a cell density of about 80%, digested and centrifuged, and resuspended and counted in 1 mL of fresh culture medium, and then the cells were inoculated into a 24-well plate at a number of 20,000 cells / well; the 24-well plate was placed in a cell culture incubator for culture, and after about 12 hours, the cells were completely attached to the wall, and drug treatment was performed, and the corresponding concentration of DMSO was added to the control wells, and 2.5 μM RSL3 (inducing ferroptosis) and 1 μM of the test compounds P1 and P3 were added to the experimental wells; after adding RSL3 and the test compounds, cell death was observed, and after about 5 hours, a final concentration of 5 μM BODIPY 581 / 591C11 dye was added, and cultured at 37°C for 30 minutes; the 24-well plate was taken out, the cells were digested and centrifuged, washed once with HBSS, and then resuspended in HBSS and transferred to a flow tube; after sufficient mixing, the cells were analyzed by flow cytometry (BD FACS The signal from the oxidation probe was counted through the FITC channel to determine the level of lipid peroxidation in the cells. At least 5 × 10 3 cells; Flowjo was used for data processing to calculate the effect of the test compound on cellular lipid peroxidation by inhibiting FSP1 combined with RSL3. The results are as follows Figure 3 As shown. Figure 3 It can be seen that when the cells were co-incubated with compounds P1, P3 and RSL3, the content of lipid peroxides increased significantly compared with cells treated with RSL3 alone, indicating that compounds P1 and P3 can enhance RSL3-induced lipid peroxidation.

[0105] Test Example 4 Effect of Compounds on HT1080 Cell Viability by Inhibiting FSP1

[0106] Assuming that adding a lethal dose of RSL3 to cells can induce ferroptosis, if VK, a substrate of FSP1, is added exogenously at this time, FSP1 can provide a continuous supply of VKH2 by reducing VK, thereby enabling tumor cells to resist a lethal dose of RSL3. To prove this hypothesis, ferroptosis was induced by adding 2μM RSL3 to HT1080 cells, and at the same time, gradient concentrations of VK were added. The results are shown in Figure 2. Figure 4 As shown. Figure 4It can be seen that about 0.3μM VK can keep cells alive. VK inhibits ferroptosis depends on the ability of intracellular FSP1 to reduce VK. If 2μM RSL3 and 0.3μM VK are added to keep cells alive and then FSP1 inhibitors are added exogenously to inhibit VK reduction, the cells will die because VK cannot be reduced. Using this system, the IC value of FSP1 inhibitors on FSP1 at the cellular level can be determined by adding different concentration gradients of FSP1 inhibitors. 50 , and it can also be demonstrated that the inhibitor can specifically target FSP1.

[0107] HT1080 cells were cultured until the cell density reached about 80%, and the cells were digested and centrifuged, and the cells were resuspended and counted in 1 mL of fresh culture medium, and then the cells were inoculated into a 96-well plate at a number of 5000 cells / well. Because the culture medium in the side wells of the 96-well plate evaporated quickly, the 24 wells on the upper and lower edges were not inoculated with cells, and were filled with 200 μL PBS; the 96-well plate was placed in a cell culture incubator for culture, and after about 12 hours, the cells were completely attached to the wall, and drug treatment was performed, and 2 μM RSL3 (inducing iron) was added to all wells. Death) and 0.3μM vitamin K (inhibit ferroptosis, vitamin K is reduced and regenerated by endogenous FSP1 in the cell to provide a continuous supply of reduced vitamin K, i.e. VKH2); after adding RSL3 and vitamin K, the test compounds P1, P3-P5 and positive control drugs iFSP1 and FSEN3 were set to 12 different concentration gradients and added to the well plate in sequence, and 2 replicates were set for each concentration; after adding the compounds, the cell death was observed, and after about 7h, the 96-well plate was taken out, and the cells in the well plate were lysed using the CellTiter Glo kit. After sufficient mixing, the cell ATP level was quantified by measuring the fluorescence value on the microplate reader (TECAN), and the relative viability was evaluated by the ATP reading; GraphPad was used for data processing to calculate the IC 50 The results are shown in Table 2:

[0108] Table 2 The results of the inhibition of HT1080 cell proliferation by the compounds

[0109] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> P1 0.4215 P3 0.3404 P4 0.7517 P5 0.9776 FSEN3 0.7322 iFSP1 0.8493

[0110] It can be seen from Table 2 that compounds P1 and P3 of the present invention have significant inhibitory effects on the proliferation of HT1080 cells, and compounds P4 and P5 also have certain inhibitory effects on HT1080 cells, indicating that the triazolothiadiazole compounds of the present invention have good anti-tumor effects and have broad application space as FSP1 inhibitors.

[0111] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A triazolothiadiazole compound, characterized in that: The structure is shown in Formula V: Wherein, R1 is a heterocyclic amino group; R2 is selected from aryl or heteroaryl.

2. The triazolothiadiazole compound according to claim 1, characterized in that: The heterocyclic amino group is selected from The aryl group is selected from: The heteroaryl group is 3. The triazolothiadiazole compound according to claim 1, characterized in that: The triazolothiadiazole compound is selected from the following compounds:

4. The method for preparing a triazolothiadiazole compound according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, a compound of formula I, carbon disulfide and potassium hydroxide react in a first organic solvent to obtain a compound of formula II; S2, the compound of formula II reacts under the action of hydrazine hydrate to obtain a compound of formula III; S3, the compound of formula III and chloroacetic acid react under the action of phosphorus oxychloride to obtain a compound of formula IV; S4, reacting the compound of formula IV with the secondary amine H-R1 in a second organic solvent under the action of N,N-diisopropylethylamine to obtain the triazolothiadiazole compound of formula V; Wherein, the structures of Formula I to Formula V are shown below: Wherein, R1 is a heterocyclic amino group; R2 is selected from aryl or heteroaryl.

5. The method for preparing a triazolothiadiazole compound according to claim 4, characterized in that: In S1, the first organic solvent is selected from one or more of methanol, ethanol, dichloromethane and ethyl acetate.

6. The method for preparing a triazolothiadiazole compound according to claim 4, characterized in that: In S4, the second organic solvent is selected from one or more of acetonitrile, diethyl ether, dichloromethane and ethyl acetate.

7. A stereoisomer and / or a pharmaceutically acceptable salt of the triazolothiadiazole compound according to any one of claims 1 to 3.

8. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is the triazolothiadiazole compound according to any one of claims 1 to 3, the stereoisomer of the triazolothiadiazole compound according to claim 7 and / or a pharmaceutically acceptable salt.

9. Use of the triazolothiadiazole compound according to any one of claims 1 to 3, the stereoisomer and / or pharmaceutically acceptable salt of the triazolothiadiazole compound according to claim 7, and the pharmaceutical composition according to claim 8 in the preparation of a drug for treating FSP1-related diseases.

10. The use according to claim 9, characterized in that: The FSP1-related diseases include one or more of gastric cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, melanoma, lymphoma, leukemia, copper tolerance, severe acute pancreatitis, diabetes, Parkinson's disease, retinal degeneration, age-related macular degeneration, acute kidney injury, early brain injury, liver fibrosis and non-alcoholic fatty liver disease.

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