Novel ferrostatin-1 analogs, methods of making and uses

Abstract

The application belongs to the field of chemical medicine, and discloses a novel ferrostatin-1 analogue, a preparation method and application. The novel ferrostatin-1 analogue has a certain ferroptosis rescue effect on HK-2 cells in in-vitro anti-ferroptosis activity evaluation, and compared with ferrostatin-1, the above analogue has lower toxicity and higher activity, can be used as a potential anti-ferroptosis compound, and provides a novel and promising method for designing a novel ferroptosis inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine and relates to a novel class of carbon chain-extended ferrostatin-1 analogs, their preparation methods, and applications. Background Technology

[0002] Ferroptosis, discovered by Stockwell in 2012, is a unique form of cell death driven by iron-dependent lipid peroxidation. Unlike non-programmed cell death such as necrosis, ferroptosis, as a programmed cell death, is highly sensitive to molecular-level perturbations and can be regulated by controlling redox homeostasis. In cells, iron ions and lipid free radicals induce lipid peroxidation, leading to ferroptosis. Inhibiting ferroptosis, as a novel cell rescue mechanism, is playing an increasingly important role in the development and progression of major diseases such as neurodegenerative diseases, traumatic brain injury, and organ ischemia-reperfusion injury.

[0003] Ferrostatin-1, a typical lipophilic inhibitor, can exert its antioxidant effect by scavenging lipid hydroxyl radicals in cells and complexing iron ions in unstable iron pools, thereby inhibiting ferroptosis. However, ferrostatin-1 still has problems such as high toxicity and poor solubility, which affect its drug-like properties.

[0004] To discover new drugs with inhibitory activity against ferroptosis, ferrostatin-1 was used as a lead compound. Based on a summary of existing structure-activity relationship studies, analog modifications were performed targeting the primary and secondary amine sites in its structure. To simplify synthesis and facilitate subsequent derivatization, bromoacetyl bromide was creatively proposed as a carbon chain elongation group. The positions of the carbonyl and methylene groups were flexibly adjusted to increase lipophilicity. The effects of carbon chain elongation and electron-withdrawing groups on ferroptosis inhibitory activity were investigated, and a series of novel carbon chain-elongated ferrostatin-1 analogs were designed and synthesized. Compared with previous ferrostatin-1 derivatives, this method not only breaks with traditional design thinking but also provides a new perspective for further understanding the mechanism of action of ferroptosis inhibitors and offers a novel and promising method for designing new ferroptosis inhibitors. Summary of the Invention

[0005] The purpose of this invention is to design and synthesize a series of novel carbon chain-elongated ferrostatin-1 analogs and to provide a novel and promising method for designing novel ferroptosis inhibitors.

[0006] The technical solution of the present invention:

[0007] A novel class of ferrostatin-1 analogues exhibits inhibitory activity against ferroptosis in HK-2 cells (human renal tubular epithelial cells), with structural formulas shown as a1, a2, a3, and a4:

[0008]

[0009] A method for preparing a novel class of ferrostatin-1 analogs includes the following steps:

[0010] ① Dissolve 3-nitro-4-aminobenzoic acid in excess ethanol, add concentrated sulfuric acid, wherein the volume ratio of ethanol to concentrated sulfuric acid is 1:0.1, and react at 60℃ for 10 h to obtain compound 1;

[0011] ② Dissolve cyclohexylamine in dichloromethane, add DMAP and pyridine sequentially, and add bromoacetyl bromide dropwise in an ice-water bath. The molar ratio of cyclohexylamine:bromoacetyl bromide:pyridine:DMAP is 1:1.2:0.1~0.5:0.5~1. React at room temperature for 1 hour to obtain compound 2.

[0012] Compound 2 was dissolved in DMF, and then compound 1 and anhydrous K2CO3 were added sequentially, with the molar ratio of compound 1:compound 2:anhydrous K2CO3 being 1.2:1:3. The mixture was reacted at room temperature for 10 h to obtain a yellow solid compound 3.

[0013] Compound 3 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 3 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a1;

[0014]

[0015] ③ Dissolve compound 2 in dichloromethane, add DMAP and pyridine sequentially, and add bromoacetyl bromide dropwise in an ice-water bath. The molar ratio of compound 2:bromoacetyl bromide:pyridine:DMAP is 1:1.2:0.1~0.5:0.5~1. React at room temperature for 1 hour to obtain compound 4.

[0016] Compound 4 was dissolved in DMF, and cyclohexylamine and anhydrous K2CO3 were added sequentially, wherein the molar ratio of compound 4:cyclohexylamine:anhydrous K2CO3 was 1.2:1:3. The reaction was carried out at room temperature for 10 h to obtain a yellow solid compound 5.

[0017] Compound 5 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 5 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a2;

[0018]

[0019] ④ Dissolve compound 1 in THF, place in an ice bath, add cyclohexanediol chloride, and add two drops of triethylamine. The molar ratio of compound 1 to cyclohexanediol chloride is 1.2:1. React at room temperature for 2 hours to obtain a yellow solid compound 6.

[0020] Compound 6 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 6 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a3;

[0021]

[0022] ⑤ Ethyl 4-chloro-3-nitrobenzoate was dissolved in DMSO, followed by the addition of anhydrous K2CO3 and cyclohexylamine; wherein the molar ratio of ethyl 4-chloro-3-nitrobenzoate:cyclohexylamine:anhydrous K2CO3 was 1:1.2:2; the reaction was carried out at room temperature to 60℃ for 10 h to obtain compound 7;

[0023] Compound 7 was dissolved in methanol solution, protected by nitrogen gas, and palladium on carbon was added, wherein the mass ratio of compound 7 to palladium on carbon was 1:0.5. After reacting for 10 h, ferrostatin-1 was obtained.

[0024] Ferrostatin-1 was dissolved in DMF, and compound 2 and anhydrous K2CO3 were added sequentially. The molar ratio of compound 2:ferrostatin-1:anhydrous K2CO3 was 1:1 to 1.2:3. The reaction was carried out at room temperature under nitrogen protection for 12 h to obtain compound a4.

[0025]

[0026] The synthesized ferrostatin-1 analogues described above can be used as anti-ferroptosis drugs or as active ingredients in the preparation of related combination drugs that inhibit ferroptosis.

[0027] The beneficial effects of this invention are as follows: Compared with the original ferrostatin-1, the novel carbon chain-extended ferrostatin-1 analog described in this invention exhibits lower toxicity and better ferroptosis inhibition ability in HK-2 cells (human renal tubular epithelial cells) in in vitro antiferroptosis activity evaluation. It can be used as a potential antiferroptosis compound, which can help in the drug development of major diseases such as neurodegenerative diseases, traumatic brain injury, and organ ischemia-reperfusion injury. Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below in conjunction with the technical solution.

[0029] Example 1

[0030] Synthesis of compound a1

[0031] (1) Dissolve 152 mg of commercially available 3-nitro-4-aminobenzoic acid in 5 mL of ethanol. Then add 0.5 mL of concentrated sulfuric acid, and under nitrogen protection, raise the reaction temperature from room temperature to 60 °C. Monitor the reaction by TLC, and the reaction is complete in 10 h. Adjust the pH to weakly alkaline by adding saturated sodium bicarbonate solution dropwise, transfer to a separatory funnel, extract with ethyl acetate (3 × 10 mL), dry, and concentrate. Column chromatography with petroleum ether:ethyl acetate = 4:1 yields a yellow solid 1, with a yield of 85%.

[0032] ESI-MS m / z for C9H9N2O4[MH] - calcd 209.18.;found,208.85.

[0033] (2) Dissolve 226.6 mg of cyclohexylamine in 5 mL of dichloromethane solution, add 70 mg of DMAP, add 0.5 mL of pyridine dropwise, under nitrogen protection, in an ice-water bath for 10 minutes, add 239 μL of bromoacetyl bromide dropwise, remove the ice-water bath after half an hour, and react at room temperature for 1 hour to complete the reaction. Column chromatography with petroleum ether:ethyl acetate = 15:1 yielded a pale yellow solid 2, with a yield of 69%.

[0034] ESI-MS m / z for C8H 15 BrNO[M+H] + calcd 220.02; found, 219.94.

[0035] (3) Weigh 100 mg of compound 1 and dissolve it in 3 mL of anhydrous DMF. Under nitrogen protection, add 120 mg of compound 2 and 190 mg of anhydrous potassium carbonate sequentially. Monitor the reaction by TLC. The reaction is complete at room temperature for 10 hours. Transfer the reaction solution to a separatory funnel, extract with ethyl acetate (3 × 10 mL), dry, and concentrate. Column chromatography with petroleum ether:ethyl acetate = 4:1 yields a yellow solid 3, with a yield of 77%.

[0036] ESI-MS m / z for C 17 H 22 N3O5[MH] - calcd 348.16; found, 347.96.

[0037] (4) Weigh 50 mg of compound 3 and dissolve it in 5 mL of methanol solution, purging three times with nitrogen. Under nitrogen protection, add 25 mg of 10% Pd / C. React under a hydrogen atmosphere for 10 hours, and monitor the reaction by TLC until it ends. Filter palladium on carbon with diatomaceous earth, wash with ethyl acetate (3 × 10 mL), evaporate the filtrate to dryness, and perform column chromatography with petroleum ether:ethyl acetate = 8:1 to obtain a white solid a1, with a yield of 92%.

[0038] ESI-MS m / z for C 17 H 26 N3O3[M+H] + calcd 320.18; found, 320.07.

[0039] 1 H NMR (400MHz, CDCl3) δ7.54(d,J=8.2Hz,1H),7.43(d,J=2.0Hz,1H),6.46(d,J=8.2H z,1H),6.35–6.24(m,1H),4.77(d,J=6.2Hz,1H),4.30(q,J=7.1Hz,2H),3.82(d,J=4 .2Hz,2H),3.33–3.03(m,1H),1.91–1.77(m,2H),1.70–1.60(m,2H),1.56(d,J=9.0H z,1H),1.35(t,J=7.1Hz,3H),1.26(d,J=10.8Hz,2H),1.07(td,J=11.9,3.0Hz,3H).

[0040] Example 2

[0041] Synthesis of compound a2

[0042] (1) Weigh 120 mg of compound 1 into a 25 ml reaction flask, stir to dissolve in 3 ml of dichloromethane, under nitrogen protection, add 0.5 mL of pyridine, add 17.5 mg of DMAP, in an ice-water bath for 10 minutes, then add 200 μL of bromoacetyl bromide. After half an hour, remove the ice-water bath and react at room temperature for 1 hour to complete the reaction. Column chromatography with petroleum ether:ethyl acetate = 10:1 yielded a pale yellow solid 4, with a yield of 74%.

[0043] ESI-MS m / z for C 11 H 10 BrN2O5[MH] - calcd 329.98; found 329.91.

[0044] (2) Weigh 100 mg of 4 into a 25 mL reaction flask, stir to dissolve in 3 mL of anhydrous DMF, under nitrogen protection, add 36.14 mg of cyclohexylamine and 124.12 mg of anhydrous potassium carbonate sequentially, monitor the reaction by TLC, and allow the reaction to proceed to completion at room temperature for 10 hours. Extract with ethyl acetate (3 × 10 mL), dry, and concentrate. Column chromatography with petroleum ether:ethyl acetate = 4:1 yields a yellow solid 5, with a yield of 81%.

[0045] (3) Weigh 20 mg of compound 5 into a 25 ml reaction flask and dissolve it in 2 ml of methanol solution. Under nitrogen protection, add 10 mg of 10% Pd / C. React under hydrogen atmosphere for 10 hours, and monitor the reaction by TLC until it ends. Concentrate the reaction solution to obtain a pale yellow solution. Column chromatography with petroleum ether:ethyl acetate (8:1) yields a white solid a2, with a yield of 85%.

[0046] ESI-MS m / z for C 17 H 26 N3O3[M+H] + calcd 320.18; found, 320.03.

[0047] 1 H NMR (400MHz, CDCl3) δ9.57(s,1H),7.55(d,J=8.4Hz,1H),7.50(s,1H),7.49(s,1H),4.34(q,J=7.1Hz,2H),3.95–3.85(m,1H),3.43(s,2H),2.46( tt,J=7.8,3.7Hz,1H),1.92-2.03(m,2H),1.74(d,J=11.8Hz,2H),1.63( d,J=11.2Hz,1H),1.37(t,J=7.1Hz,3H),1.25(s,2H),1.11-1.21(m,3H).

[0048] Example 3

[0049] Synthesis of compound a3

[0050] (1) Weigh 50 mg of compound 1 into a 25 mL reaction flask and dissolve it in 3 mL of anhydrous THF with stirring. Under ice bath and nitrogen protection, after 10 minutes, add 28.976 mg of cyclohexanecarboxyl chloride, followed by dropwise addition of 0.033 mL of triethylamine. Gradually raise the reaction temperature to room temperature. Monitor the reaction by TLC; the reaction solution changes from yellow to orange-red, and the reaction is complete after 2 hours. Transfer the reaction solution to a separatory funnel, extract with ethyl acetate (3 × 10 mL), dry, and concentrate. Column chromatography with petroleum ether:ethyl acetate = 25:1 yields a yellow solid 6, with a yield of 27%.

[0051] ESI-MS m / z for C 16 H 19 N2O5[MH] - calcd 319.13; found, 318.93.

[0052] (2) Weigh 50 mg of compound 6 into a 25 ml reaction flask and dissolve it in 5 ml of methanol solution. Under nitrogen atmosphere, add 25 mg of 10% Pd / C. Replace with hydrogen three times and react for 10 hours. Monitor the reaction end by TLC. Concentrate the reaction solution to obtain a pale yellow solution. Column chromatography with petroleum ether:ethyl acetate = 8:1 yields a white solid a3, with a yield of 83%.

[0053] ESI-MS m / z for C 16 H 21 N2O3[MH] - calcd 289.16; found, 288.91.

[0054] 1 H NMR (600MHz, CDCl3) δ7.65(s,1H),7.54-7.52(m,2H),7.44(d,J=7.1Hz,1H),4.34(q,J=7.2Hz,2H),2.34(tt,J=10.9,2.8Hz,1H),1 .98(d,J=11.3Hz,2H),1.87–1.79(m,2H),1.71(d,J=12.2Hz,1H),1.54(d,J=11.8Hz,2H),1.37(t,J=7.2Hz,3H),1.33–1.24(m,3H).

[0055] Example 4

[0056] Synthesis of compound a4

[0057] (1) Weigh 200 mg of ethyl 4-chloro-3-nitrobenzoate and dissolve it in 2 mL of DMSO with stirring. Then add 240.8 mg of anhydrous potassium carbonate, under nitrogen protection, and slowly add 119.5 μL of cyclohexylamine. Raise the reaction temperature from room temperature to 60 °C, monitor the reaction by TLC, and the reaction is complete in 10 h. Transfer the reaction solution, extract with ethyl acetate (3 × 10 mL), dry, and concentrate. Column chromatography with petroleum ether:ethyl acetate = 20:1 separates the pale yellow solid to give compound 7, with a yield of 74%.

[0058] Weigh 130 mg of compound 7 into a 25 mL round-bottom flask, dissolve it in 5 mL of methanol solution, and purge with nitrogen for protection. Add 65 mg of Pd / C. React under a hydrogen atmosphere for 10 hours, and monitor the reaction for completion by TLC. Remove palladium on carbon by filtration with diatomaceous earth, wash with ethyl acetate (3 × 10 mL), concentrate the reaction solution to obtain a yellow solution, and separate by column chromatography with petroleum ether:ethyl acetate = 8:1 to obtain a white solid ferrostatin-1, in 91% yield.

[0059] ESI-MS m / z for C 15H 23 N₂O₂[M+H] + calcd 263.17; found 263.16.

[0060] 100 mg of ferrostatin-1 was weighed into a 25 mL reaction flask and dissolved in 3 mL of anhydrous DMF under stirring. Under nitrogen protection, 142 mg of compound 2 and 186 mg of anhydrous potassium carbonate were added. The reaction was monitored by TLC and allowed to proceed to completion at room temperature for 12 hours. Column chromatography with a petroleum ether:ethyl acetate ratio of 4:1 yielded a4, with a yield of 82%.

[0061] ESI-MS m / z for C 23 H 36 N3O3[M+H] + calcd 402.2678; found,402.2747.

[0062] 1 H NMR (500MHz, CDCl3) δ7.58(dd,J=8.3,1.9Hz,1H),7.24(d,J=1.9Hz,1H),6.64(d,J=8.4Hz,1H),6.22(d, J=8.3Hz,1H),4.30(q,J=7.1Hz,2H),3.81(dq,J=6.4,3.1Hz,1H),3.77(s,2H),3.73(s,1H),3.33(td,J= 8.4,5.0Hz,1H),2.12–1.97(m,2H),1.95–1.83(m,2H),1.81–1.72(m,2H),1.65(dt,J=8.8,4.1Hz,2H),1 .61–1.54(m,1H),1.44–1.28(m,7H),1.27–1.18(m,3H),1.12(td,J=11.3,3.6Hz,2H),0.98–0.85(m,1H).

[0063] Application Example 1

[0064] (1) In vitro toxicity evaluation of the target compound of this invention:

[0065] Dissolve the ferrostatin-1 analogue in dimethyl sulfoxide (cell culture grade) and store at -20°C. Before use, dilute with cell culture medium to a final concentration of less than 1‰ of dimethyl sulfoxide.

[0066] HK-2 cells (human renal tubular epithelial cells) in the logarithmic growth phase were collected and seeded into 96-well plates (5 × 10⁶ cells per well). 3Cells were incubated in an incubator (37℃, 5% CO2) for 24 hours, then the test compound was added. A blank control group and a control group were also set up. After co-incubating the cells and drug for 72 hours, 20 μM MTT solution was added to each well. After 4 hours of further incubation, the culture medium was discarded, and 200 μM dimethyl sulfoxide (chromatographic grade) was added. The absorbance at 490 nm was measured using a microplate reader, and the cell proliferation inhibition rate of the compound was calculated.

[0067]

[0068] Table 1. Cytotoxicity of Ferrostatin-1 and its analogues on HK-2 cells

[0069]

[0070] The results show that, compared with ferrostatin-1, the target compounds a1-a4 are less toxic to HK-2 cells and can be used as a basis for further evaluation of antiferroptosis activity.

[0071] (2) Evaluation of the in vitro antiferroptosis activity of the target compound of the present invention:

[0072] HK-2 cells (human renal tubular epithelial cells) in the logarithmic growth phase were collected and seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured in an incubator (37℃, 5% CO2) for 24 h, and then the ferroptosis inhibitory activity of the compound was detected. A blank group, a control group, a group with RSL3 alone, and a group incubated with both RSL3 and the test compound were set up. After co-incubating the cells and the drug for 72 h, 20 μM MTT solution was added to each well, and the cells were cultured for another 4 h. The culture medium was then discarded, and 200 μM dimethyl sulfoxide (chromatographic grade) was added. The absorbance at 490 nm was measured using a microplate reader, and the cell proliferation inhibition rate of the compound was calculated.

[0073] Table 2. Inhibitory activity of Ferrostatin-1 and its analogues against ferroptosis in HK-2 cells

[0074]

[0075] The ferroptosis inducer RSL3 successfully induced ferroptosis in HK-2 cells at 4 μM, with a cell viability of 22.7%. When ferrostatin-1 and its analogues were added, the cell viability increased and ferroptosis in HK-2 cells was inhibited.

[0076] In summary, this invention designed and synthesized four novel carbon-chain-extended ferrostatin-1 analogs, which exhibit certain ferroptosis inhibitory activity against HK-2 cells (human renal tubular epithelial cells). Compared with the original ferrostatin-1, these analogs have lower toxicity and higher activity, and can serve as potential anti-ferroptosis compounds, providing a novel and promising method for designing novel ferroptosis inhibitors.

Claims

1. A class of ferrostatin-1 analogues, characterized in that, The structural formulas of the ferrostatin-1 analogues are shown as a1, a2, a3, and a4: ； ； ； 。 2. A method for preparing a ferrostatin-1 analogue as described in claim 1, characterized in that, The steps include the following: ① Dissolve 3-nitro-4-aminobenzoic acid in excess ethanol, add concentrated sulfuric acid, wherein the volume ratio of ethanol to concentrated sulfuric acid is 1:0.1, and react at 60℃ for 10 h to obtain compound 1; ② Dissolve cyclohexylamine in dichloromethane, add DMAP and pyridine sequentially, and add bromoacetyl bromide dropwise in an ice-water bath. The molar ratio of cyclohexylamine:bromoacetyl bromide:pyridine:DMAP is 1:1.2:0.1~0.5:0.5~1. React at room temperature for 1 hour to obtain compound 2. Compound 2 was dissolved in DMF, and then compound 1 and anhydrous K2CO3 were added sequentially, with the molar ratio of compound 1:compound 2:anhydrous K2CO3 being 1.2:1:

3. The mixture was reacted at room temperature for 10 h to obtain a yellow solid compound 3. Compound 3 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 3 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a1; ； ③ Dissolve compound 1 in dichloromethane, add DMAP and pyridine sequentially, and add bromoacetyl bromide dropwise in an ice-water bath. The molar ratio of compound 2:bromoacetyl bromide:pyridine:DMAP is 1:1.2:0.1~0.5:0.5~1. React at room temperature for 1 hour to obtain compound 4. Compound 4 was dissolved in DMF, and cyclohexylamine and anhydrous K2CO3 were added sequentially, wherein the molar ratio of compound 4:cyclohexylamine:anhydrous K2CO3 was 1.2:1:

3. The reaction was carried out at room temperature for 10 h to obtain a yellow solid compound 5. Compound 5 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 5 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a2. ④ Dissolve compound 1 in THF, place in an ice bath, add cyclohexanediol chloride, and add two drops of triethylamine. The molar ratio of compound 1 to cyclohexanediol chloride is 1.2:

1. React at room temperature for 2 h to obtain a yellow solid compound 6. Compound 6 was dissolved in anhydrous methanol, palladium on carbon was added, and the reaction was carried out under hydrogen atmosphere, wherein the mass ratio of compound 6 to palladium on carbon was 1:0.5, and the reaction was carried out for 10 h to obtain compound a3; ⑤ Ethyl 4-chloro-3-nitrobenzoate was dissolved in DMSO, followed by the addition of anhydrous K2CO3 and cyclohexylamine; wherein the molar ratio of ethyl 4-chloro-3-nitrobenzoate:cyclohexylamine:anhydrous K2CO3 was 1:1.2:2; the reaction was carried out at room temperature to 60 °C for 10 h to obtain compound 7; Compound 7 was dissolved in methanol solution, protected by nitrogen gas, and palladium on carbon was added, wherein the mass ratio of compound 7 to palladium on carbon was 1:0.

5. The reaction was carried out under hydrogen atmosphere for 10 h to obtain ferrostatin-1. Ferrostatin-1 was dissolved in DMF, and compound 2 and anhydrous K2CO3 were added sequentially. The molar ratio of compound 2:ferrostatin-1:anhydrous K2CO3 was 1:1 to 1.2:

3. The reaction was carried out at room temperature under nitrogen protection for 12 h to obtain compound a4. 。 3. The use of the ferrostatin-1 analog obtained by the preparation method of claim 2 for the preparation of anti-ferroptosis drugs or related compositions for the preparation of drugs that inhibit ferroptosis.

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