13-OD carbamate derivatives and their preparation and application

Abstract

The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to 13-OD carbamate derivatives and their preparation and application. The present invention prepares 13-OD carbamate derivatives with anti-lung cancer activity by chemical semi-synthesis modification of 13-OD, and provides a preparation method of the derivative; the preparation method provided by the present invention has the advantages of being simple and easy to operate, low raw material cost and suitable for large-scale production. In addition, the present invention also provides the application of the 13-OD carbamate derivative in the preparation of tumor treatment drugs; the 13-OD carbamate derivative provides a new idea for the preparation of drugs for treating tumors; it has great research value and clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to 13-OD carbamate derivatives and preparation and application thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide, with approximately 10 million deaths in 2020, accounting for nearly one-sixth of all deaths. The most common cancers are breast, lung, colon, rectal, and prostate cancers (Global Cancer Observatory: Cancer Today. Lyon: International Agency for Research on Cancer; 2020). Cancer originates from a multi-stage process in which normal cells transform into tumor cells, usually progressing from precancerous lesions to malignant tumors. These changes are the result of the interaction between an individual's genetic factors and three external factors: physical carcinogens, such as ultraviolet light and ionizing radiation; chemical carcinogens, such as tobacco smoke, alcohol, aflatoxins, and arsenic; and biological carcinogens, such as infections caused by certain viruses, bacteria, or parasites. In addition, approximately one-third of cancer deaths are related to smoking, obesity, alcohol abuse, low fruit and vegetable intake, and lack of exercise. Cancer has posed a huge threat to human health, and early detection and treatment of cancer are urgent.

[0003] Most cancers have a long incubation period and are difficult to diagnose in the early stages. They are often discovered in the middle and late stages. Clinically, surgical resection, radiotherapy, and chemotherapy are often used alone or in combination to treat patients with middle and late stage cancer. However, most patients often use anti-tumor drugs to improve their quality of life and prolong their overall survival due to tumor burden or inappropriate surgical treatment. Therefore, the discovery and development of new anti-cancer drugs is crucial. On the market, molecular targeted drugs sorafenib and gefitinib are first-line drugs for the treatment of primary liver cancer and non-small cell lung cancer, but they can only prolong the median survival of patients by about 1 year (Cancer Research and Treatment, 2019, 51 (2): 502-509). It is urgent to find anti-tumor drugs with stronger activity and less toxic side effects.

[0004] Gan Sui is the dried root of Euphorbia kansui, a plant of the Euphorbia genus of the Euphorbiaceae family. It is widely distributed in Gansu, Shanxi and other places. It tastes bitter, is cold in nature, and enters the lung and kidney meridians. It has the effects of purging water, removing fluid, reducing swelling and dispersing nodules. Modern pharmacological experiments have shown that Gan Sui has multiple pharmacological activities, such as anti-cancer, anti-viral, anti-inflammatory and anti-asthmatic. The inonoterpenoids present in Gan Sui are its active ingredients and have good anti-tumor effects. 13-Oxyingenoldodecanoate (13-OD) belongs to inonoterpenoids, has a 5 / 7 / 7 / 3 tetracyclic system and a highly oxidized structure, and has a long-chain fatty acyl group at the C-13 position. The molecular formula is as follows:

[0005]

[0006] The esterified derivatives of 13-OD are widely found in Euphorbiaceae plants and show good anti-tumor activity. According to the literature, the anti-tumor activity of the compound can be improved after esterification of its 3-OH. However, there are few studies on 13-OD, and there are no reports of 13-OD derivatives being marketed as tumor treatment drugs.

[0007] Therefore, with 13-OD as the core, based on the combination of active fragments and the principle of bioelectronic isostery, a series of derivatives with significant anti-tumor activity and an ABPP photoaffinity probe for target fishing were designed and synthesized. Summary of the invention

[0008] To solve the above technical problems, the present invention uses 13-OD as the mother nucleus and performs chemical semi-synthetic modification to obtain 13-OD carbamate derivatives, and provides a preparation method of the above derivatives. In addition, it also provides the use of the 13-OD carbamate derivatives in the preparation of tumor treatment drugs.

[0009] Specifically, the present invention is realized through the following technical solutions:

[0010] The present invention provides a ganicane-type diterpene derivative from kansui with anti-non-small cell lung cancer activity, which is a 13-OD carbamate derivative, wherein the 13-OD carbamate derivative has a chemical structure shown in formula (I), and the 13-OD photoaffinity probe has a chemical structure shown in formula (II).

[0011]

[0012] Wherein, in formula (I), R1 is selected from C1-C9 alkyl, unsaturated alkenyl, substituted or unsubstituted halogenated aromatic hydrocarbon, cycloalkane and nitrogen-containing heterocycle or its salt;

[0013] In formula (II), R2 is a saturated aliphatic hydrocarbon or unsaturated alkyne containing a diazirine group or an aromatic hydrocarbon or a salt thereof containing diazirine, and X is selected from NH, CH2 and O atoms;

[0014] Preferably, R1 in the above formula can be selected from: CH3,

[0015]

[0016] Preferably, R2 in the above formula can be selected from:

[0017] Further preferably, in formula (I), R1 is: In formula (II), R2 is:

[0018] Most preferably, the 13-OD carbamate derivative having anti-non-small cell lung cancer activity is selected from any one of the following compounds:

[0019]

[0020] In a second aspect, the present invention provides a method for preparing the 13-OD carbamate derivative, the method comprising the following steps:

[0021]

[0022] Using 13-Oxyingenol dodecanoate as the starting material, intermediate 1 is prepared through step a, and then intermediate 1 reacts with a formyl activator to obtain active intermediate 2, and reacts with different amines to obtain intermediates B1_1-B11_1, and finally the intermediates are ring-opened in 2M hydrochloric acid methanol solution to generate B1-B11;

[0023] Specifically, the preparation method comprises the following steps:

[0024] (1) Synthesis of intermediate 1: 13-OD was dissolved in a solvent, p-toluenesulfonic acid was added under stirring, and the reaction was carried out at 0°C. After the reaction was complete, the solvent was removed, extracted, washed, dried, filtered, and purified to obtain intermediate 1;

[0025] (2) Synthesis of intermediate 2: Dissolve intermediate 1 in a solvent, add a formyl activator and an acid binding agent under stirring, and react at room temperature. After the reaction is complete, perform post-treatment in the same manner as step (1) to obtain intermediate 2.

[0026] (3) Synthesis of intermediates B1_1-B11_1: Dissolve intermediate 2 in a solvent, add different organic amine reagents and organic bases under stirring, react at room temperature, and perform post-treatment after the reaction is complete in the same manner as step (1) to obtain intermediates B1_1-B11_1.

[0027] (4) Synthesis of intermediate 13-OD-P_1: Dissolve intermediate 2 in a solvent, add diazirine reagent and organic base under stirring, react at room temperature, and perform post-treatment after the reaction is complete in the same manner as step (1) to obtain intermediate 13-OD-P_1.

[0028] (5) Synthesis of B1-B11: Dissolve the intermediate B1_1-B11_1 in a solvent, add 2M hydrochloric acid methanol solution under stirring, react at room temperature, and post-treat after the reaction is complete in the same manner as step (1).

[0029] (6) Synthesis of 13-OD-P: The intermediate 13-OD-P_1 was dissolved in a solvent, and a 2M methanol solution of hydrochloric acid was added under stirring. The reaction was allowed to react at room temperature. After the reaction was complete, the post-treatment was performed in the same manner as in step (1).

[0030] The above preparation method, wherein:

[0031] The solvent is anhydrous acetonitrile, anhydrous acetone, N,N'-dimethylformamide, tetrahydrofuran or dichloromethane.

[0032] The organic base is triethylamine, N,N'-diisopropylethylamine, pyridine, 4-dimethylaminopyridine or N-methylmorpholine;

[0033] The formyl activator in step (2) is carbonyldiimidazole, carbonyltriazole, phosgene or triphosgene; the R group in the intermediate 2 is 1,2,4-triazole, imidazole or Cl atom.

[0034] The acid binding agent in step (2) is triethylamine, N,N'-diisopropylethylamine, pyridine or 4-dimethylaminopyridine.

[0035] The organic amine in step (3) is cycloalkaneamine, aliphatic amines of different lengths or benzylamine substituted with different groups. In a third aspect of the present invention, the use of the 13-OD carbamate derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors is provided.

[0036] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises a 13-OD carbamate derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0037] The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, wherein the oral dosage form is a capsule, a tablet, a granule, an oral liquid, a sustained-release preparation or a controlled-release preparation.

[0038] In a fifth aspect, the present invention provides use of the 13-OD carbamate derivative or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a drug for treating lung cancer.

[0039] Preferably, the lung cancer is non-small cell lung cancer.

[0040] As used in the present invention, the term "pharmaceutically acceptable carrier" refers to one or more of a diluent, a lubricant, a binder, a disintegrant, a stabilizer or a solvent.

[0041] The diluent is selected from starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, and glucose; the lubricant is selected from magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer; the binder is selected from water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone; the disintegrant is selected from starch effervescent mixture, i.e., sodium bicarbonate and citric acid, tartaric acid, and low-substituted hydroxypropyl cellulose; the stabilizer is selected from polysaccharides such as acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan; and the solvent is selected from water and a balanced salt solution.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention designs and prepares a series of 13-OD carbamate derivatives or pharmaceutically acceptable salts thereof by structurally modifying 13-OD. The preparation method of the 13-OD carbamate derivative provided by the present invention has the advantages of being simple and easy to operate, easy to purify, and having low raw material cost. The 13-OD carbamate derivative or pharmaceutically acceptable salt thereof provided by the present invention has good non-small cell lung cancer inhibitory activity, is expected to become a new drug for treating non-small cell lung cancer, and has great research value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The changes in mitochondrial membrane potential were observed by JC-1 double-staining fluorescent probe. (A) Flow cytometric graph of the changes in mitochondrial membrane potential studied by JC-1 double-staining fluorescent probe; (B) Fluorescence graph of mitochondrial membrane potential detected by JC-1 staining;

[0045] Figure 2 Figure 1 shows the pharmacological validation results of mitophagy and ferroptosis;

[0046] Figure 3 (A) Intracellular Ca after 8 h of 13-OD treatment 2+Confocal photography of TMBIM6 content; (B) RT-PCR study of the effect of TMBIM6 mRNA level; (C) Regulation of TMBIM6 protein expression by 13-OD and B6; (D) 13-OD and B6 regulate the interaction between TMBIM6 and VDAC1;

[0047] Figure 4 The CETSA method was used to detect the effects of 13-OD and B6 on the stability of TMBIM6 protein at 46–66°C; the stability of 13-OD and B6 at 54–60°C;

[0048] Figure 5 (A) RNA interference and transfection; (B) Fe 2+ Determination of content;

[0049] Figure 6 Photoaffinity probe 13-OD-P was used for target fishing through ABPP strategy; (A) Cytotoxic effects of 13-OD and 13-OD-P on A549 cells; (B) In situ competition between 13-OD and 13-OD-P in A549 cells; (C) Overall flow chart of ABPP mass spectrometry analysis to identify the target of 13-OD;

[0050] Figure 7 13-OD and its derivatives to measure the intracellular GSH, LPO and MDA contents: (A, B) the expression levels of several autophagy markers under the intervention of 13-OD and B6; (C, D) the expression levels of SLC7A11, GPX4, Keap1, Nrf2 and HO-1 under the intervention of 13-OD and B6;

[0051] Figure 813-OD preparation flow chart. DETAILED DESCRIPTION

[0052] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] Example 1: Preparation of 13-OD:

[0055] The method for preparing 13-OD is as follows Figure 8As shown. 50 kg of dried root tubers of Euphorbia kansui, a plant of the Euphorbia genus of the Euphorbiaceae family, were soaked in 85% ethanol for 24 hours, condensed and refluxed for 2 hours, extracted four times, and the four extraction solutions were combined and the solvent was evaporated to obtain 4.5 kg of crude extract. The crude extract was extracted with ethyl acetate and n-butanol respectively, and the ethyl acetate layer was concentrated to obtain 648.0 g of crude fraction. The crude fraction was dissolved in anhydrous methanol, and anhydrous barium hydroxide was added at room temperature for hydrolysis, maintaining pH = 9, and reacted overnight. After the reaction was completed, 2M hydrochloric acid was added to adjust the pH to neutral, the solvent was evaporated, and extracted with ethyl acetate, filtered, and evaporated to obtain Fr.1. The fractions were separated using macroporous adsorption resin HP-20 with different concentrations of ethanol (40%, 70%, 90%) to obtain three fractions, Fr.A, B, and C, of ​​which Fr.C was 170.8 g. Fr.C was separated using 200-mesh silica gel and eluted with petroleum ether:ethyl acetate = 80:1-1:1 to obtain Fr.A-Fr.E. The Fr.E fraction was concentrated, dissolved in acetonitrile, filtered through a 0.45 μm filter membrane, and eluted using high performance liquid chromatography (acetonitrile:water = 85:15) to obtain 13-OD with a purity of 95%, totaling 3.1 g.

[0056] 13-OD: 1 H NMR(600MHz, CDCl3) δ6.02(dd,J=4.8,1.2Hz,1H),5.89(q,J=1.2Hz,1H),4.41(s,1H),4.18(d,J=12.6Hz ,1H),4.12(d,J=12.6Hz,1H),4.06(dd,J=12.0,4.1Hz,1H),3.84(s,1H),2.73(dd,J=16.8,3.0Hz,1H),2. 46(m,1H),2.21(t,J=7.8Hz,2H),2.17(overlap,1H),1.86(d,J=1.2Hz,3H),1.55(overlap,2H),1.29–1 .23(m,16H),1.28(overlap,1H),1.23(s,3H),1.08(s,3H),0.97(d,J=7.2Hz,3H),0.88(t,J=7.0Hz,3H);

[0057] 13C NMR (150MHz, CDCl3) δ206.9,174.2,141.0,139.6,128.8,126.4,84.1,80.2,75.3,72.8,68.9,66.8, 43.3,38.5,35.3,34.5,30.3,29.7,29.6,29.5,29.4,29.3,28.3,24.9,22.6,18.6,16.8,15.5,14.2.

[0058] HR-ESI-MS:547.3629[M+H] + ,(calcd for C 32 H 50 O7,547.3629).

[0059] Example 2: Preparation of Compound B2

[0060]

[0061] 13-OD (10 mg, 0.018 mmol) was dissolved in 5 mL of anhydrous acetone, p-TsOH·H2O was added, and the mixture was reacted at 0°C for 30 min. After TLC monitoring, the reaction was complete, water was added to quench the mixture, the solvent was evaporated under reduced pressure, dichloromethane was added for extraction, the mixture was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 1. Intermediate 1 (10 mg, 0.017 mmol) was dissolved in anhydrous dichloromethane, N,N'-carbonylbis(1,2,4-triazole) (6 eq), 4-dimethylaminopyridine (1 eq), and 4-methylbenzylamine (6 eq) were added to react at room temperature for 16 h. After TLC detection, no raw material spots were found. The mixture was extracted with dichloromethane, washed with water, and dried over anhydrous sodium sulfate to obtain intermediate B2_1. The intermediate B2_1 (10 mg) was dissolved in 5 mL of anhydrous methanol and 100 ul of 2M HCl was added dropwise. The mixture was reacted at room temperature for 6 h. After TLC showed that there was no raw material point, saturated sodium bicarbonate solution was added to adjust the pH to 7. B2 was purified by HPLC. The structural identification data are as follows:

[0062] B2: yellow oil, yield: 34%

[0063] 1H NMR (600MHz, CDCl3) δ7.19–7.15(m,4H),6.03(d,J=4.0Hz,1H),5.98(d,J=1.5Hz,1H),5.29(s,1H),4.39–4.3 1(m,2H),4.13(s,2H),4.08(dd,J=12.1,4.1Hz,1H),4.06(s,1H),2.74(dd,J=12.0,3.0Hz,1H),2.61–2.59(m ,1H),2.34(s,3H),2.21(t,J=7.2Hz,2H),2.20(overlap,1H),1.81(d,J=1.2Hz,3H),1.55(overlap,2H),1.2 8(overlap,1H),1.26–1.22(m,16H),1.21(s,3H),1.07(s,3H),0.94(d,J=7.2Hz,3H),0.88(t,J=7.2Hz,3H).

[0064] 13 C NMR (150MHz, CDCl3) δ205.9,174.0,157.3,139.7,137.5,136.0,134.7,131.3,129.5,127.6,127.1,84.3,83.8,76.8,72.0, 69.0,67.1,45.0,42.6,38.3,35.3,34.4,30.2,29.6,29.4,29.3,29.3,29.2,28.3,24.8,22.5,21.1,18.4,16.7,15.4,14.1.

[0065] HR-ESI-MS:716.4196[M+Na] + (calcd for C 41 H 59 NNaO8,716.4196).

[0066] Example 3: Preparation of Compound B4

[0067]

[0068] 13-OD (10 mg, 0.018 mmol) was dissolved in 5 mL of anhydrous acetone, p-TsOH·H2O was added, and the mixture was reacted at 0°C for 30 min. After TLC monitoring, the reaction was complete, water was added to quench the mixture, the solvent was evaporated under reduced pressure, dichloromethane was added for extraction, the mixture was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 1. Intermediate 1 (10 mg, 0.017 mmol) was dissolved in anhydrous dichloromethane, N,N'-carbonylbis(1,2,4-triazole) (6 eq), 4-dimethylaminopyridine (1 eq), and aminocyclopentane (6 eq) were added to react at room temperature for 16 h. After TLC detection, no raw material point was found, the mixture was extracted with dichloromethane, washed with water, and dried over anhydrous sodium sulfate to obtain intermediate B4_1. The intermediate B4_1 (10 mg) was dissolved in 5 mL of anhydrous methanol and 100 ul of 2M HCl was added dropwise. The mixture was reacted at room temperature for 6 h. After TLC detection showed that there was no raw material point, saturated sodium bicarbonate solution was added to adjust the pH to 7. B4 was purified by HPLC. The structural identification data are as follows:

[0069] B4: yellow oil, yield: 38%

[0070] 1 H NMR (600MHz, CDCl3) δ6.02(d,J=4.0Hz,1H),5.97(s,1H),5.21(s,1H),4.13(s,2H),4.05(dd,J=12.6,4.2H z,1H),3.95(m,1H),3.80(s,1H),2.74(dd,J=16.8,3.0Hz,1H),2.60(m,1H),2.20(t,J=7.5Hz,2H),2.20(o verlap,1H),1.99(m,2H),1.80(d,J=1.2Hz,3H),1.72-1.59(m,4H),1.55(m,2H),1.43(overlap,2H),1.30 (overlap,1H),1.29-1.26(m,16H),1.20(s,3H),1.06(s,3H),0.94(d,J=7.2Hz,3H),0.88(t,J=7.2Hz,3H).

[0071] 13C NMR (150MHz, CDCl3) δ206.13,174.20,157.08,140.01,136.17,131.21,127.05,84.31,83.71,77.55,72.23,69.09,67.25,53.22,42 .69,37.37,35.56,34.52,33.34,30.30,29.73,29.60,29.47,29.40,29.34,28.53,24.95,23.71,22.86,18.58,16.81,15.49,14.27.

[0072] HR-ESI-MS:680.4105[M+Na] + (calcd for C 38 H 59 NNaO8,680.4132).

[0073] Example 4: Preparation of Compound B6

[0074]

[0075] 13-OD (10 mg, 0.018 mmol) was dissolved in 5 mL of anhydrous acetone, p-TsOH·H2O was added, and the mixture was reacted at 0°C for half an hour. The reaction was complete after TLC monitoring. Water was added to quench the mixture, and the solvent was evaporated under reduced pressure. Dichloromethane was added for extraction, and the mixture was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 1. Intermediate 1 (10 mg, 0.017 mmol) was dissolved in anhydrous dichloromethane, and N,N'-carbonylbis(1,2,4-triazole) (6 eq), 4-dimethylaminopyridine (1 eq), and n-propylamine (6 eq) were added to react at room temperature for 16 h. After TLC detection, no raw material point was found. The mixture was extracted with dichloromethane, washed with water, and dried over anhydrous sodium sulfate to obtain intermediate B6_1. The intermediate B6_1 (10 mg) was dissolved in 5 mL of anhydrous methanol and 100 ul of 2M HCl was added dropwise. The mixture was reacted at room temperature for 6 h. After TLC detection showed that there was no raw material point, saturated sodium bicarbonate solution was added to adjust the pH to 7. B6 was purified by HPLC. The structural identification data are as follows:

[0076] B6: yellow oil, yield: 35%

[0077] 1H NMR(600MHz, CDCl3)δ6.03(d,J=4.3Hz,1H),5.98(d,J=1.8Hz,1H),5.26(s,1H),4.15–4.07(m,2H),4.05 (dd,J=12.6,4.2Hz,1H),4.03(s,1H),3.21–3.13(m,2H),2.74(dd,J=16.2,1.8Hz,1H),2.61–2.58(m,1H ),2.22(t,J=7.1Hz,3H),2.21(overlap,1H),1.81(d,J=1.4Hz,3H),1.57–1.53(m,4H),1.31–1.28(m,16 H),1.25(overlap,1H),1.21(s,3H),1.06(s,3H),0.95–0.93(m,6H),0.93(s,3H),0.88(t,J=7.0Hz,3H).

[0078] 13 C NMR (150MHz, CDCl3) δ206.0,174.1,157.5,139.8,136.0,131.1,127.0,84.2,83.6,77.0,72.0,69.0,67.1,43.0,42 .6,37.3,35.4,34.4,30.2,29.6,29.4,29.3,29.3,29.2,28.4,24.8,23.0,22.7,22.6,18.4,16.7,15.3,14.1,11.2.

[0079] HR-ESI-MS:654.3970[M+Na] + (calcd for C 36 H 57 NNaO8,654.3976).

[0080] Example 5: Preparation of Compound 13-OD-P

[0081]

[0082] 13-OD (10 mg, 0.018 mmol) was dissolved in 5 mL of anhydrous acetone, p-TsOH·H2O was added, and the reaction was carried out at 0°C for 30 min. After TLC monitoring, the reaction of the raw material was complete, water was added for quenching, the solvent was evaporated under reduced pressure, extracted with dichloromethane, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 2. Intermediate 2 (10 mg, 0.017 mmol) was dissolved in anhydrous dichloromethane, N,N'-carbonylbis(1,2,4-triazole) (6 eq), 4-dimethylaminopyridine (1 eq), 2-(3-But-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine (6 eq) were added to react at room temperature for 16 hours, TLC detection showed no raw material spot, extracted with dichloromethane, washed with water, and dried over anhydrous sodium sulfate to obtain intermediate 13-OD-P_1. The intermediate 13-OD-P_1 (10 mg) was dissolved in 5 mL of anhydrous methanol and 100 uL of 2N HCl was added dropwise. The mixture was reacted at room temperature for 6 hours. After TLC analysis showed that there was no raw material point, saturated sodium bicarbonate solution was added to adjust the pH to 7. 13-OD-P was purified by high performance liquid chromatography. The structural identification data are as follows:

[0083] 1 H NMR (600MHz, CDCl3) δ6.02(1H,d,J=4.2Hz),5.99(1H,d,J=1.8Hz),5.27(1H,s),4.13(2H,s),4.08(1H,dd,J =11.8,3.7Hz),4.02(1H,s),3.11–3.05(2H,m),2.74(1H,dd,J=16.2,2.4Hz),2.61–2.59(1H,m),2.48–2.45 (1H,m),2.20(2H,t,J=7.5Hz),2.20(1H,overlap),2.04–2.02(2H,m),1.82(3H,s),1.68–1.64(4H,m),1.55 (2H,overlap),1.29–1.24(16H,m),1.19(3H,s),1.07(3H,s),0.96(3H,d,J=7.1Hz),0.87(3H,t,J=7.0Hz).

[0084] 13C NMR (150MHz, CDCl3) δ206.0,174.2,157.3,139.8,136.1,131.5,127.3,84.5,83.8,82.8,72.1,69.7,69.1,67.3,42.8,37.6,36 .1,35.4,34.5,32.9,32.4,32.0,30.4,29.7,29.6,29.5,29.4,29.3,28.5,26.8,24.9,22.8,22.7,18.5,16.9,15.5,14.3,13.4.

[0085] HR-ESI-MS:732.4194[M+Na] + ,(calcd for C 39 H 53 FNaO8,732.4198).

[0086] Example 6: Pharmaceutical Screening

[0087] In order to prove that the 13-OD carbamate derivatives of the present invention have the potential to treat non-small cell lung cancer, the prepared compounds were screened for lung cancer pharmacodynamics.

[0088] The specific anti-lung cancer compound screening method and results are as follows:

[0089] The CCK8 method was used to determine the cytotoxicity of the compounds on three cell lines: HepG2, A549, and H460. The cells were seeded in 96-well plates with 8,000 cells per well. Then 70-80% of the cells were treated with different concentrations of drugs for 24, 48, and 72 hours. Cell viability was determined by adding CCK8, and the absorbance of the solution at 450 nm was measured using a multi-template reader (PerkinElmer, USA). The calculation formula is as follows:

[0090]

[0091] The concentration at which the growth inhibition rate is 50% is calculated as IC 50 The results showed that most of the compounds had considerable activity in inhibiting the proliferation of lung cancer cells, as shown in Table 1.

[0092] Table 1 Anti-lung cancer cell proliferation activity of compounds a .

[0093]

[0094] a Determination of IC of several tumor cells at 72h 50 , b ND Not determined.

[0095] For the compound B6 with better activity, we conducted a cytotoxicity test on normal human lung cells and found that the cytotoxicity of this compound is about half of that of the positive drug oxaliplatin, and it shows almost no toxicity to normal human lung cells. The activity data are shown in the following table.

[0096] Table 2 Cytotoxicity test of compound B6 on normal human lung cells

[0097]

[0098] SI a =BEAS-2B / A549.

[0099] Example 7: 13-OD and B6 induce mitochondrial damage in A549 cells

[0100] (1) Analysis of mitochondrial membrane potential (Δψm)

[0101] The JC-1 kit (Beyotime) was used to detect the change of Δψm. When the mitochondrial membrane potential was high, JC-1 aggregated in the mitochondrial matrix and produced red fluorescence. When the mitochondrial membrane potential was low, JC-1 could not aggregate in the mitochondrial matrix. At this time, JC-1 was a monomer and produced green fluorescence. Therefore, the change of mitochondrial membrane potential can be measured by fluorescence color shift. A549 cells (1×10 5 Cells were cultured in 6-well plates with 13-OD and its carbamate derivatives for 6 h, then incubated with 10 μM JC-1 in the dark for 20 min, washed twice with PBS buffer, then digested with EDTA-free trypsin, washed twice with PBS, resuspended with 400 uL PBS buffer, and quantitatively analyzed by flow cytometry (Beckman, USA). According to the selection of probe excitation and emission wavelengths, FITC and PE dual channels were used for monitoring. Total proteins were extracted from A549 cells with RIPA lysis buffer (Beyotime, China) supplemented with 1× protease inhibitor cocktail and separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Then, they were electrotransferred to PVDF membranes. Then, the membranes were blocked with 5% bovine serum albumin (BSA) and incubated with the corresponding primary and secondary antibodies at room temperature for 1 h at 4 °C. Finally, protein bands were visualized using enzyme-linked chemiluminescence (ECL) (Thermo Fisher, USA). Protein amounts were semi-quantified using Image J and normalized to the corresponding controls.

[0102] The results showed that 13-OD and its derivatives could reduce mitochondrial membrane potential in a dose-dependent manner and cause mitochondrial damage. Combined with confocal fluorescence microscopy, the transformation of mitochondrial JC-1 polymers to monomers was captured. Figure 1 shown.

[0103] Example 8: 13-OD and B6 induce mitochondrial autophagy and ferroptosis in A549 cells

[0104] (1) Analysis of ROS and lipid ROS

[0105] DCFH-DA and C11-BODIPY probes were used to detect ROS and lipid ROS levels. A549 cells (1×10 5 Cells were cultured in 6-well plates (100 μg / mL). The cells were treated with 13-OD and its carbamate derivatives for 6 h, then incubated with 5 μM DCFH or C11-BODIPY in the dark for 20 min, washed twice with PBS buffer, digested with EDTA-free trypsin, washed twice with PBS, resuspended in 400 μL PBS buffer, and detected by flow cytometry (Beckman, USA). Quantitative analysis. According to the selection of probe excitation and emission wavelengths, the FITC channel was used for monitoring. The results are shown in Figure 2 shown.

[0106] (2) Cellular and mitochondrial Ca 2+ Determination of content

[0107] A549 cells (1×10 5 / mL) were cultured in a 6-well plate. After incubation with 1μM Rhod-2AM probe in the dark for 20 minutes, the cells were washed twice with PBS buffer, then digested with EDTA-free trypsin, washed twice with PBS, and then resuspended with 400uL PBS buffer, and quantitatively analyzed using a flow cytometer (Beckman, USA). The vertical axis was set to the PE channel and the horizontal axis to the Time channel according to the wavelength of the probe. Then, the baseline was collected for 2 minutes, the collection was paused, and 100μM 13-OD and its carbamate derivatives were immediately added to the cell suspension. After mixing, the content changes were continued to be monitored on the flow cytometer machine. The results are as follows Figure 3 shown.

[0108] (3) CETSA assay (in situ)

[0109] The cells were cultured to 80% confluence, treated with 13-OD, B6 (100 μM) and DMSO for 2 h, and then lysed with RIPA lysis buffer. The soluble protein lysate was collected into a PCR tube, heated at the indicated temperature (46-66°C and 54-60°C) for 3 min, then cooled in a thermal cycler (Applied Biosystems, Inc., USA) for 3 min, and centrifuged at 4°C for 20 min. The soluble supernatant was subjected to Western blot analysis, and the results were shown in Figure 2. Figure 4 shown.

[0110] (4) RNA interference and transfection

[0111] GenePharma was used to design and synthesize the complementary oligonucleotide sequence of si-TMBIM6: GGCCGAACAUGGAGAUCAATT, antisense: UUGAUCUCCAUGUUCGGCCTT. Lipofectamine 2000 was used to transfect si-RNA or negative control (si-NC) vector into A549 cells. The results are shown in Figure 5 As shown in A.

[0112] (5) Fe 2+ Determination of content

[0113] A549 cells (1×10 5 Cells were cultured in 6-well plates with 13-OD and its carbamate derivatives for 6 h, then incubated with 1 μM ferrous ion probe in the dark for 20 min, washed twice with PBS buffer, digested with trypsin without EDTA, washed twice with PBS, resuspended in 400 μL PBS buffer, and quantitatively analyzed by flow cytometry (Beckman, USA). The PE channel was used to monitor the results according to the selection of probe excitation and emission wavelengths. Figure 5 As shown in B.

[0114] Embodiment 9: Target fishing

[0115] (1) In situ fluorescence labeling analysis of cells

[0116] After the cells were inoculated in a 6-well plate and cultured to 70-80% confluence, different concentrations of competitive 13-OD were added and incubated for 2h, then 13-OD-P (4μM) was added and incubated for 2h, and finally transferred to UV 365nm for 20min, the culture medium was discarded, and the cells were washed 3 times with PBS solution. Cell lysis buffer (200μL PBS buffer containing 1% protease inhibitor (Thermo Fisher, USA) and 0.1% Triton X-100) was added, the cells were collected, and ultrasonic treatment was performed in an ice bath until clarified. Soluble protein was obtained by centrifugation at 4°C and 20,000r for 10min, and the protein concentration was determined using a BCA kit. Equal amounts of protein lysate (200mg) of each group were incubated with click reaction mixture (1mmolNaVc, 100μmol THPTA, 1mmol CuSO4 and 20μmol TAMRA-azide). The reaction was vigorously shaken for 2h at room temperature. The sample at the bottom of the tube was dissolved in 50 μL of 1× loading buffer, separated by SDS-PAGE gel electrophoresis, and scanned and analyzed by a laser scanner (Azure Sapphire RGBNIR, USA). Finally, the gel was stained with instantaneous blue Coomassie brilliant blue for diagnosis, as shown in the following procedure. Figure 6 shown.

[0117] (2) Pull-down and target identification by LC-MS / MS

[0118] The cells were incubated with 13-OD (24 μM) for 2 h. After incubation with 13-OD-P for 2 h, soluble protein lysates were extracted, and the cells were irradiated under 365 nm ultraviolet light for 20 min for click chemistry treatment. The dried precipitate at the bottom of the tube was precipitated with ice acetone and centrifuged, then dissolved in PBS solution with 1.5% SDS, and then SDS was diluted to 0.1% with PBS buffer and centrifuged at room temperature for 10 min. The sample was incubated with 50 μL of streptavidin-adsorbed beads at room temperature for 6 h, and then washed 3 times with 5 mL of 1% SDS, 0.1% SDS and 6 M urea. The sample was then reduced with dithiothreitol (100 mM DTT) and alkylated with iodoacetamide (400 mM IAA). Finally, the sample was incubated with trypsin overnight at 37 ° C to fully digest the peptides. The peptide solution was eluted on a C18 column and after determination of the peptide concentration, the samples were analyzed using LC-MS / MS (Orbitrap Fusion Lumos, Thermo, USA).For pull-down analysis, the concentration of bound protein was determined using Western immunoblotting according to the above procedure.

[0119] (3) Western blot method

[0120] Total protein of A549 cells was extracted with RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor cocktail, and the total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was then electrotransferred to a PVDF membrane. Then, the membrane was blocked in 5% bovine serum albumin (BSA) and incubated with the corresponding primary and secondary antibodies for 1 h at room temperature and overnight at 4°C. Finally, the protein bands were visualized by enzyme-linked chemiluminescence (ECL) (Thermo Fisher, USA). The protein amount was semi-quantified using Image J and normalized with the corresponding control.

[0121] Combined with the WB experimental results Figure 7 As shown in (A, B, C, D), we found that 13-OD and B6 can mediate oxidative stress and produce a large amount of reactive oxygen species by activating the Nrf2-Keap1-HO-1 axis signaling pathway. All these results indicate that 13-OD and B6 can turn on the "lipid ROS" ferroptosis switch. WB experiments verified that 13-OD and B6 can promote ferroptosis by downregulating the expression of SLC7A11 and GPX4 proteins. si-RNA interference experiments proved that reducing the expression of TMBIM6 protein can promote autophagy and ferroptosis.

[0122] In summary, by applying the above pharmacological experiments to screen antitumor drugs for lung cancer cell proliferation, compound B6 was obtained, and the inhibitory activity of the compound on normal human lung cells was tested. It was found that its inhibitory activity on lung cancer A549 cell line was about 10 times that of normal human lung cell BEAS-2B cell line, which has the value of further development. In addition, it was found that 13-OD and its carbamate derivatives can regulate the function of TMBIM6 protein and affect the endoplasmic reticulum Ca 2+ release, causing mitochondrial Ca 2+ Overload, membrane potential depolarization, triggering mitochondrial autophagy and triggering ferroptosis. Therefore, the 13-OD carbamate derivatives of the present invention can be further developed into drugs for treating lung cancer.

Claims

1. A 13-OD carbamate derivative, characterized in that The 13-OD carbamate derivative is selected from any one of the following compounds:

2. The method for preparing the 13-OD carbamate derivative according to claim 1, characterized in that: The following steps are involved: R is a 1,2,4-triazolyl group, an imidazole group or a chlorine atom; (1) Synthesis of intermediate 1: 13-OD was dissolved in a solvent, p-toluenesulfonic acid was added under stirring, and the reaction was carried out at 0°C. After the reaction was complete, the solvent was removed, extracted, washed, dried, filtered, and purified to obtain intermediate 1; (2) Synthesis of intermediate 2: Dissolve intermediate 1 in a solvent, add a formyl activator and an acid binding agent under stirring, react at room temperature, and perform post-treatment after the reaction is complete in the same manner as step (1) to obtain intermediate 2; (3) Synthesis of intermediates B1_1, B3_1-B11_1: Dissolve intermediate 2 in a solvent, add different organic amine reagents and organic bases under stirring, react at room temperature, and perform post-treatment after the reaction is complete in the same manner as step (1) to obtain intermediates B1_1, B3_1-B11_1; (4) Synthesis of intermediate 13-OD-P_1: Dissolve intermediate 2 in a solvent, add diazirine reagent and organic base under stirring, react at room temperature, and perform post-treatment after the reaction is complete. The post-treatment method is the same as step (1) to obtain intermediate 13-OD-P_1; (5) Synthesis of B1, B3-B11: Dissolve the intermediates B1_1, B3_1-B11_1 in a solvent, add 2M methanol solution of hydrochloric acid under stirring, react at room temperature, and perform post-treatment after the reaction is complete in the same manner as step (1); (6) Synthesis of 13-OD-P: The intermediate 13-OD-P_1 was dissolved in a solvent, and a 2M methanol solution of hydrochloric acid was added under stirring. The reaction was allowed to react at room temperature. After the reaction was complete, the post-treatment was performed in the same manner as in step (1).

3. The method for preparing the 13-OD carbamate derivative according to claim 2, characterized in that: The solvent is anhydrous acetonitrile, anhydrous acetone, N,N'-dimethylformamide, tetrahydrofuran or dichloromethane; the organic base is triethylamine, N,N'-diisopropylethylamine, pyridine, 4-dimethylaminopyridine or N'-methylmorpholine; the formyl activator in step (2) is carbonyldiimidazole, carbonyltriazole, phosgene or triphosgene; the acid binding agent in step (2) is triethylamine, N,N'-diisopropylethylamine, pyridine or 4-dimethylaminopyridine.

4. A pharmaceutical composition, characterized in that It comprises the 13-OD carbamate derivative or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form; the oral dosage form is a capsule, a tablet, a granule, an oral liquid, a sustained-release preparation or a controlled-release preparation.

6. Use of the 13-OD carbamate derivative or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to any one of claims 4 to 5 in the preparation of a drug for treating non-small cell lung cancer.

7. Use of the 13-OD carbamate derivatives B1, B4, B6, B8-B11 or pharmaceutically acceptable salts thereof according to claim 1 in the preparation of a drug for inhibiting H460 cell proliferation.

8. Use of the 13-OD carbamate derivatives B1-B9 or pharmaceutically acceptable salts thereof according to claim 1 in the preparation of a drug for inhibiting HepG2 cell proliferation.

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