Ferroptosis-inducing urea derivatives as well as preparation method and anti-tumor application thereof

Abstract

The invention belongs to the technical field of chemical medicines, and provides a ferroptosis-inducing urea derivative as well as a preparation method and anti-tumor application thereof. The ferroptosis-inducing urea derivatives are oxyurea, thiourea and squaramide sorafenib derivatives, the anti-proliferative activity on HT1080 human fibrosarcoma cells is remarkably enhanced by modifying the structure of a sorafenib mother nucleus, and in-vitro experiments show that the inhibition effect of the ferroptosis-inducing urea derivatives is more than one time higher than that of sorafenib. The series of derivatives play an anti-tumor role by activating a ferroptosis pathway, and can potentially overcome the drug resistance problem of sorafenib. The invention further provides a synthetic route, a purification method and an in-vitro pharmacodynamic evaluation system of the derivative, and a structure optimization strategy and candidate compounds are provided for developing novel antitumor drugs of a targeted ferroptosis pathway.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and relates to a class of urea derivatives that induce ferroptosis, and a preparation method and anti-tumor application thereof. Background Art

[0002] In today's society, cancer has become one of the leading causes of death worldwide. Although traditional tumor treatments such as surgical resection, chemotherapy and radiotherapy have certain therapeutic effects, chemotherapy drugs generally have drug resistance problems, which often lead to tumor metastasis and invasion. Therefore, the development of new anti-tumor drugs with high efficiency and low toxicity, especially to solve the problem of tumor resistance, has become a key scientific problem that needs to be broken through in the global medical field.

[0003] In 2012, Brent R. Stockwell's team first defined and named "ferroptosis", a new type of cell death, which is characterized by lipid peroxidation caused by iron-dependent reactive oxygen species (ROS) accumulation. Recent studies have shown that ferroptosis induction can effectively reverse tumor drug resistance, and its mechanism of action mainly involves three major metabolic pathways: iron metabolism, lipid metabolism, and amino acid metabolism. Normal cells maintain metabolic balance by precisely regulating iron homeostasis, while iron metabolism disorders in tumor cells are closely related to tumor occurrence and development.

[0004] The ferroptosis inducers discovered so far mainly work through the following mechanisms: (1) reducing intracellular glutathione levels; (2) promoting ROS accumulation; (3) regulating cellular iron content; and (4) targeting GPX4 protein. Typical system Xc-inhibitors include Erastin, Sorafenib, etc. Among them, Sorafenib is the first oxurea-structured multikinase inhibitor approved for the treatment of advanced liver cancer. It works by inhibiting tumor angiogenesis, and its aryl urea pharmacophore has become an important template for drug design.

[0005] Although sorafenib has definite clinical efficacy, it has defects such as strong drug resistance and weak ferroptosis induction activity. Existing literature has carried out structural optimization through the following strategies: (Chen J N, Li T, Cheng L, et al. Synthesis and in vitro anti-bladder cancer activity evaluation of quinazolinyl-arylurea derivatives[J]. Eur J Med Chem, 2020, 205: 112661.) The arylurea derivatives 7j-a1 with quinazoline structures introduced have good activity against a variety of cancer cells, but a relatively high concentration is required to induce ferroptosis in cells, and the activity against common cancer cells such as HepG2 (human liver cancer cells), MDA-MB-231 (human breast cancer cells) and HT-1080 (human fibrosarcoma cells) is not high.

[0006] Based on the above background, the present invention systematically investigated the core structure of sorafenib, introduced pyrrolo[1,2-a]triazine or quinazoline as the core structure, and oxyurea or thiourea or formamidine as the linking group, improving the activity against cancer cells. Experiments confirmed that the introduction of the new group significantly enhanced the ferroptosis induction activity and anti-proliferation effect, providing a new structural optimization strategy for overcoming tumor drug resistance. Summary of the Invention

[0007] Aiming at the technical defects of existing sorafenib drugs such as strong drug resistance and insufficient ferroptosis induction activity, the present invention aims to provide a class of novel structural derivatives to enhance anti-tumor activity and overcome the drug resistance problem.

[0008] The technical solution of the present invention:

[0009] A class of urea derivatives that induce ferroptosis, namely oxyurea derivatives, thiourea derivatives or formamidine derivatives represented by the following general formula;

[0010]

[0011]

[0012] Among them,

[0013] A is quinazoline or pyrrolo[1,2-a]triazine;

[0014] B is 3,4-dichlorophenyl or p-chlorophenyl;

[0015] The structural general formula (Ⅰ) is an oxyurea derivative, the structural general formula (Ⅱ) is a thiourea derivative, and the structural general formula (Ⅲ) is a formamidine derivative. The specific structures are as follows:

[0016] (a) Oxamide derivatives, with the structural formulas shown as N-2-a1 and N-2-a2;

[0017] (b) Thiourea derivatives, with the structural formulas shown as 7j-b1 and 7j-b2;

[0018] (c) Squaramide derivatives, with the structural formulas shown as N-2-c1, N-2-c2, 7j-c1 and 7j-c2;

[0019]

[0020]

[0021] A preparation method of a class of urea derivatives that induce ferroptosis, comprising the following steps:

[0022] (1) Synthesis of intermediate N-1:

[0023] Dissolve compound N and 3-nitrobenzyl bromide in a DMF solvent at a molar ratio of 1:1 - 1.5, react at room temperature for 22 - 26 hours, and then obtain the white flaky solid intermediate N-1 through recrystallization with DMF and methanol;

[0024]

[0025] (2) Synthesis of intermediate N-2:

[0026] Dissolve the intermediate N-1 obtained in step (1) in a mixed solvent of ethanol and water with a volume ratio of 2:1, then add iron powder and ammonium chloride, where the molar ratio of intermediate N-1, iron powder to ammonium chloride is 1:6 - 10:0.5 - 1, react at 78 - 82 °C for 1.5 - 2.5 hours, and then obtain the white powdery intermediate N-2 through recrystallization with ethyl acetate and petroleum ether;

[0027]

[0028] (3) Synthesis of intermediate 7j:

[0029] Dissolve compound j and 3-aminobenzylamine in dichloromethane at a molar ratio of 1:1.2 - 1.5, react at room temperature for 24 hours, extract with saturated sodium bicarbonate and dichloromethane, and perform pulping treatment with ethyl acetate and petroleum ether to obtain the white solid powder intermediate 7j;

[0030]

[0031] (4) Synthesis of intermediate a1 and intermediate a2:

[0032] Compound 1 and Compound 2 were separately reacted in the presence of triphosgene, with the molar ratio of Compound 1 to triphosgene being 1:0.37 - 0.4 and the molar ratio of Compound 2 to triphosgene being 1:0.37 - 0.4. They were reacted in a mixed solvent of dichloromethane and saturated sodium bicarbonate aqueous solution with a volume ratio of 1:1 under an ice bath condition of 0 - 5°C for 10 - 20 minutes. After liquid separation, drying, and concentration respectively, intermediate a1 and intermediate a2 were obtained;

[0033]

[0034] (5) Synthesis of intermediate b1 and intermediate b2:

[0035] Compound 1 and Compound 2 were separately reacted in the presence of thiophosgene, with the molar ratio of Compound 1 to thiophosgene being 1:1.2 - 1.5 and the molar ratio of Compound 2 to thiophosgene being 1:1.2 - 1.5. They were reacted in a mixed solvent of dichloromethane and saturated sodium bicarbonate aqueous solution with a volume ratio of 1:1 - 1.2 under an ice bath condition of 0 - 5°C for 10 - 20 minutes. After liquid separation, drying, and concentration respectively, intermediate b1 and intermediate b2 were obtained;

[0036]

[0037] (6) Synthesis of intermediate c1 and intermediate c2:

[0038] Compound 1 and Compound 2 were separately reacted in the presence of butyl squarate, with the molar ratio of Compound 1 to butyl squarate being 1:1.2 - 1.5 and the molar ratio of Compound 2 to butyl squarate being 1:1.2 - 1.5. They were reacted in anhydrous ethanol solvent under an ice bath condition of 0 - 5°C. After the reaction was completed, it was transferred to room temperature and reacted for 22 - 26 hours. Intermediate c1 and intermediate c2 were obtained by pulping with ethyl acetate and petroleum ether;

[0039]

[0040] (7) Synthesis of target compounds N - 2 - a1 and N - 2 - a2:

[0041] Intermediate N - 2 was respectively dissolved in anhydrous tetrahydrofuran with intermediate a1 or intermediate a2 at a molar ratio of 1:1.2 - 1.5. The reaction was started at 0 - 5°C and then raised to room temperature and reacted for 22 - 26 hours. After purification by pulping with ethyl acetate and petroleum ether, target compounds N - 2 - a1 and N - 2 - a2 were respectively prepared;

[0042]

[0043] (8) Synthesis of target compounds 7j - b1 and 7j - b2:

[0044] Intermediate 7j was dissolved in anhydrous tetrahydrofuran with intermediate b1 or intermediate b2 in a molar ratio of 1:1.2 - 1.5, and the reaction was initiated at 0 - 5 °C and then raised to room temperature for 22 - 26 hours. After purification by pulping with ethyl acetate and petroleum ether, the target compounds 7j - b1 and 7j - b2 were obtained respectively;

[0045]

[0046] (9) Synthesis of target compounds N - 2 - c1, N - 2 - c2, 7j - c1 and 7j - c2:

[0047] Intermediate N - 2 or intermediate 7j was dissolved in absolute ethanol with intermediate c1 or intermediate c2 in a molar ratio of 1:1.2 - 1.5, and the reaction was carried out at 118 - 122 °C for 22 - 26 hours. After purification by pulping with ethyl acetate and petroleum ether, the target compounds N - 2 - c1, N - 2 - c2, 7j - c1 and 7j - c2 were obtained respectively.

[0048]

[0049] Advantages of the present invention: For the compounds N - 2 - a1 and N - 2 - a2 of the present invention, the activity of the compounds is improved by the introduction of pyrrolo - triazine. For the compounds 7j - b1, 7j - b2, 7j - c1 and 7j - c2, the activity of the compounds is also improved by the introduction of thiourea or formamidine structure. The activity of the compounds N - 2 - c1 and N - 2 - c2 is significantly improved by the co - introduction of pyrrolo - triazine and formamidine, and it is more selective for inducing ferroptosis in HT1080 cells. In summary, the application of the compounds in anti - tumor drugs, especially suitable for the treatment of ferroptosis - sensitive human fibrosarcoma. Description of the Drawings

[0050] Figure 1 It is the anti - proliferation activity diagram of N - 2 - c1, 7j - c1 and the positive control sorafenib.

[0051] Figure 2 It is the ferroptosis rescue experiment of N - 2 - c1, 7j - c1 and the positive control sorafenib (*p < 0.05). Detailed Embodiments

[0052] The following further illustrates the detailed embodiments of the present invention in combination with the drawings and technical solutions.

[0053] Example 1

[0054] Synthesis of Intermediate N - 1

[0055] Dissolve compound N (3.7 mmol) in 3 mL of DMF, add 3-nitrobenzyl bromide (3.7 mmol), and react at room temperature for 24 h to ensure that compound N reacts as completely as possible. The reaction can be monitored using a TLC plate with petroleum ether:ethyl acetate = 1:1. After the reaction is completed, concentrate the reaction solution. Recrystallize using a DMF and methanol system to obtain 344 mg of a white flaky solid with a yield of 34%.

[0056] 1 H NMR (600 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.17–8.13 (m, 2H), 8.11 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.31 (dd, J = 2.7, 1.7 Hz, 1H), 6.97 (s, 1H), 6.38 (dd, J = 4.2, 2.7 Hz, 1H), 5.25 (s, 2H).

[0057] Example 2

[0058] Synthesis of Intermediate N-2

[0059] Dissolve compound N-1 (0.8 mmol) in a mixed system of 8 mL of ethanol and 4 mL of water, add iron powder (5.1 mmol) and ammonium chloride (0.6 mmol), and react at 80 °C for 2 h. The reaction can be monitored using a TLC plate with petroleum ether:ethyl acetate = 2:1, and the formation of the product can be judged by ninhydrin color development. After the reaction is completed, filter under atmospheric pressure, concentrate the reaction solution, adjust the pH to 8 - 9 with saturated sodium bicarbonate solution, extract with ethyl acetate, and dry the organic phase with anhydrous magnesium sulfate to obtain 150 mg of a white solid powder with a yield of 80%.

[0060] 1 H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.87 (s, 1H), 7.29 (s, 1H), 7.08–6.87 (m, 2H), 6.47 (m, 3H), 6.37 (t, J = 3.5 Hz, 1H), 5.08 (s, 2H), 5.00 (s, 2H).

[0061] Example 3

[0062] Synthesis of Intermediate 7j

[0063] Dissolve compound j (1.1 mmol) in 25 mL of dichloromethane, add 3-aminobenzylamine (1.5 mmol), and react at room temperature for 24 h to ensure that compound j reacts as completely as possible. The reaction can be monitored using a TLC plate with petroleum ether:ethyl acetate = 1:2. After the reaction is complete, concentrate the reaction solution and adjust the pH to about 8 - 9 with saturated NaHCO3 solution. Extract with dichloromethane. Pulp with an ethyl acetate and petroleum ether system to obtain 205 mg of white solid powder with a yield of 77%.

[0064] 1 H NMR (400 MHz, DMSO-d6) δ 8.76 (t, J = 5.8 Hz, 1H), 8.46 (s, 1H), 8.33 (d, J = 8.2 Hz, 1H), 7.84–7.73 (m, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 6.95 (t, J = 7.7 Hz, 1H), 6.55 (s, 1H), 6.51 (d, J = 7.5 Hz, 1H), 6.43 (d, J = 7.9 Hz, 1H), 5.01 (s, 2H), 4.68 (d, J = 5.8 Hz, 2H).

[0065] Example 4

[0066] Synthesis of intermediate a1

[0067] Dissolve compound 1 (1.0 mol) in 10 mL of dichloromethane, add 10 mL of saturated aqueous NaHCO3 solution to form a two-phase system. Stir and dissolve thoroughly in an ice bath. Stop stirring, dissolve BTC (triphosgene) (0.4 mmol) in 1 mL of dichloromethane solution and quickly add it to the dichloromethane layer of the reaction solution. Stir for 10 - 15 min and observe through a TLC plate with a petroleum ether:ethyl acetate = 1:1 system until the raw materials react completely. Collect the organic phase, dry and concentrate it with magnesium sulfate, and no further treatment is required. The product is 130 mg of a red-brown solid with a yield of 70%.

[0068] 1 H NMR (400 MHz, Chloroform-d) δ 7.41 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 2.5 Hz, 1H), 6.97 (dd, J = 8.6, 2.5 Hz, 1H).

[0069] Example 5

[0070] Synthesis of intermediate a2

[0071] Dissolve compound 2 (4.0 mmol) in 40 mL of dichloromethane, add 40 mL of saturated aqueous NaHCO3 solution to form a two-phase system. Stir and dissolve well under an ice bath. Stop stirring, dissolve BTC (triphosgene) (1.6 mmol) in 5 mL of dichloromethane solution and quickly add it to the dichloromethane layer of the reaction solution. Stir for 10 - 15 min and observe through a TLC plate with a petroleum ether:ethyl acetate = 1:1 system until the raw materials react completely. Collect the organic phase, dry and concentrate it with magnesium sulfate. Without the need for the next step, the product is a light green solid of 371 mg with a yield of 60%.

[0072] 1 H NMR (400 MHz, Chloroform-d) δ 7.28 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H).

[0073] Example 6

[0074] Synthesis of intermediate b1

[0075] Dissolve compound 1 (2.5 mmol) in 20 mL of dichloromethane, add 20 mL of saturated aqueous NaHCO3 solution to form a two-phase system. Stir and dissolve well under an ice bath. Stop stirring, dilute CSCl2 (3.4 mmol) with 5 mL of dichloromethane solution and quickly add it to the dichloromethane layer of the reaction solution. Stir for 10 - 15 min and observe through a TLC plate with a petroleum ether:ethyl acetate = 1:1 system until the raw materials react completely. Collect the organic phase, dry and concentrate it with magnesium sulfate. Without the need for the next step, the product is a reddish-brown solid of 456 mg with a yield of 90%.

[0076] 1 H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J = 8.6 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H).

[0077] Example 7

[0078] Synthesis of intermediate b2

[0079] Dissolve compound 2 (2.1 mmol) in 40 mL of dichloromethane, add 40 mL of saturated aqueous NaHCO3 solution to form a two-phase system. Stir and dissolve well under an ice bath. Stop stirring, dilute CSCl2 (3.0 mmol) with 5 mL of dichloromethane solution and quickly add it to the dichloromethane layer of the reaction solution. Stir for 10 - 15 min and observe through a TLC plate with a petroleum ether:ethyl acetate = 1:1 system until the raw materials react completely. Collect the organic phase, dry and concentrate it with magnesium sulfate. Without the need for the next step, the product is a light yellow solid of 689 mg with a yield of 98%.

[0080] 1 H NMR (400 MHz, Chloroform-d) δ 7.32 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H).

[0081] Example 8

[0082] Synthesis of Intermediate c1

[0083] Dissolve Compound 1 (1.00 eq) in 5 mL of ethanol and stir in an ice bath. Add Compound n-butyl squarate (1.50 eq), and stir in the ice bath for 1 h. Then transfer to room temperature and react for 24 h to ensure that Compound 1 reacts as completely as possible. It can be monitored using a TLC plate under the condition of petroleum ether:ethyl acetate = 4:1. At the same time, it can be slurried with a petroleum ether and ethyl acetate system to obtain 190 mg of a yellow precipitate with a yield of 30%.

[0084] 1 H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 7.67 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.41–7.24 (m, 1H), 4.75 (t, J = 6.5 Hz, 2H), 1.77 (p, J = 6.5 Hz, 2H), 1.44 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H).

[0085] Example 9

[0086] Synthesis of Intermediate c2

[0087] Dissolve Compound 2 (1.0 mmol) in 5 mL of ethanol and stir in an ice bath. Add Compound n-butyl squarate (1.5 mmol), and stir in the ice bath for 1 h. Then transfer to room temperature and react for 24 h to ensure that Compound 6 reacts as completely as possible. It can be monitored using a TLC plate under the condition of petroleum ether:ethyl acetate = 2:1. At the same time, it can be slurried with a petroleum ether and ethyl acetate system to obtain 219 mg of a yellow precipitate with a yield of 78%.

[0088] 1 H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 7.40 (s, 4H), 4.74 (t, J = 6.6 Hz, 2H), 1.77 (p, J = 6.6 Hz, 2H), 1.42 (h, J = 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H).

[0089] Example 10

[0090] Synthesis of Target Compound N-2-a1

[0091] Dissolve compound a1 (0.26 mmol) in 2 mL of anhydrous tetrahydrofuran and stir in an ice bath. Add compound N-2 (0.12 mmol) dissolved in 3 mL of anhydrous tetrahydrofuran, and stir in the ice bath for 1 h. Transfer to room temperature and react for 24 h to ensure that compound N-2 reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1. At the same time, ninhydrin color development can be used to judge whether compound N-2 has reacted completely. Pulping with an ethyl acetate and petroleum ether system gives 20 mg of white solid powder with a yield of 40%.

[0092] MS (ESI Positive) m / z: calcd for C 20 H 17 Cl2N6O [M+H] + 427.08; found: 426.94. 1 1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 9.32 (s, 1H), 8.06 (s, 1H), 7.97 (s, 1H), 7.87 (s, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.37–7.28 (m, 2H), 7.24 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 5.6 Hz, 2H), 6.38 (s, 1H), 5.11 (s, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 152.91, 148.72, 140.49, 140.47, 139.77, 134.76, 131.49, 131.08, 129.93, 124.87, 123.42, 120.86, 119.34, 118.51, 118.45, 117.02, 114.17, 112.01, 111.57, 51.61.

[0093] Example 11

[0094] Synthesis of target compound N-2-a2

[0095] Dissolve compound a2 (0.82 mmol) in 2 mL of anhydrous tetrahydrofuran and stir in an ice bath. Add compound N-2 (0.55 mmol) dissolved in 3 mL of anhydrous tetrahydrofuran, stir in the ice bath for 1 h, transfer to room temperature and react for 24 h to ensure that compound N-2 reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1. At the same time, ninhydrin color development can be used to judge whether compound N-2 has reacted completely. Pulping with an ethyl acetate and petroleum ether system gives 175 mg of a white solid powder with a yield of 81%.

[0096] MS (ESI Positive) m / z: calcd for C 20 H 18 ClN6O [M + H] + 393.12; found: 392.99. 1 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 9.32 (s, 1H), 8.22 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.51 - 7.46 (m, 3H), 7.35 - 7.26 (m, 4H), 7.03 (d, J = 7.2 Hz, 1H), 6.68 (d, J = 3.2 Hz, 1H), 6.59–6.40 (m, 1H), 5.17 (s, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 160.29, 152.94, 142.57, 141.24, 140.26, 139.28, 139.02, 137.67, 129.61, 129.07, 126.52, 125.93, 121.68, 121.43, 120.54, 120.41, 118.05, 117.77, 114.38, 111.04, 109.83, 49.55.

[0097] Example 12

[0098] Synthesis of target compound 7j-b1

[0099] Dissolve compound b1 (0.87 mmol) in 3 mL of anhydrous tetrahydrofuran and stir in an ice bath. Add compound 7j (0.55 mmol) dissolved in 2 mL of anhydrous tetrahydrofuran, stir in the ice bath for 1 h, transfer to room temperature and react for 24 h to ensure that compound 7j reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1. At the same time, ninhydrin color development can be used to judge whether compound 7j has reacted completely. Pulping with an ethyl acetate and petroleum ether system gives 160 mg of a light yellow solid powder with a yield of 65%.

[0100] MS (ESI Positive) m / z: calcd for C 22 H 18 Cl2N5S [M+H] + 454.06; found: 453.99. 1 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.93 (s, 1H), 8.92 (t, J = 5.9 Hz, 1H), 8.46 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 2.5 Hz, 1H), 7.79 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.75–7.68 (m, 1H), 7.54 (dd, J = 8.4, 6.5 Hz, 2H), 7.41 (dt, J = 7.2, 2.5 Hz, 3H), 7.30 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 4.81 (d, J = 5.9 Hz, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 179.94, 159.87, 155.41, 149.26, 140.51, 140.21, 139.59, 133.16, 130.84, 130.53, 128.92, 127.74, 126.29, 126.25, 125.07, 124.07, 123.85, 123.15, 122.70, 122.49, 115.31, 43.79, 40.25.

[0101] Example 13

[0102] Synthesis of target compound 7j-b2

[0103] Dissolve compound b2 (1.19 mmol) in 5 mL of anhydrous tetrahydrofuran and stir in an ice bath. Add compound 7j (0.68 mmol) dissolved in 5 mL of anhydrous tetrahydrofuran, stir in the ice bath for 1 h, transfer to room temperature and react for 24 h to ensure that compound 7j reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1, and ninhydrin color development can be used to judge whether compound 7j has reacted completely. Pulp with an ethyl acetate and petroleum ether system to obtain 150 mg of light yellow solid powder, with a yield of 52%.

[0104] MS (ESI Positive) m / z: calcd for C 22 H 19 ClN5S [M+H] + 420.10; found: 420.10.1 HNMR(400 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.80 (s, 1H), 8.87 (t, J = 5.0 Hz, 1H), 8.46 (s, 1H), 8.31 (d, J = 8.2 Hz, 1H), 7.78 (t, J = 7.4 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.43 (s, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 8.1 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 4.80 (d, J = 5.5 Hz, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 179.99, 159.85, 155.51, 149.54, 140.46, 139.81, 138.93, 133.09, 128.83, 128.68, 128.63, 127.93, 126.19, 125.61, 123.83, 123.14, 122.58, 122.39, 115.37, 43.79.

[0105] Example 14

[0106] Synthesis of the target compound N-2-c1

[0107] Dissolve compound N-2 (0.43 mmol) in 5 mL of ethanol and stir in an ice bath. Add compound c1 (0.52 mmol), heat under reflux to ensure that compound N-2 reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1, and at the same time, ninhydrin color development can be used for judgment. Pulp in the petroleum ether and ethyl acetate system to obtain 172 mg of yellow precipitate, with a yield of 85%.

[0108] MS(ESI Positive) m / z: calcd for C 23 H 17 Cl2N6O2 [M + H] + 479.07; found: 479.07. 1HNMR (400 MHz, DMSO-d6) δ 10.02 (s, 2H), 8.33 (s, 1H), 8.00 (s, 1H), 7.83 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.42–7.30 (m, 3H), 7.27 (s, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.98 (d, J = 3.0 Hz, 1H), 6.49–6.33 (m, 1H), 5.15 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 182.42, 182.20, 166.53, 165.36, 148.40, 141.38, 139.24, 138.97, 132.09, 131.54, 130.04, 125.28, 123.00, 120.53, 120.23, 119.11, 118.26, 118.08, 117.53, 109.86, 106.46, 56.49, 48.44, 19.03.

[0109] Example 15

[0110] Synthesis of the target compound N-2-c2

[0111] Dissolve compound N-2 (0.37 mmol) in 5 mL of ethanol and stir in an ice bath. Add compound c2 (0.44 mmol), heat under reflux to ensure that compound N-2 reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1, and at the same time, ninhydrin color development can be used to judge. Pulp in the petroleum ether and ethyl acetate system to obtain 159 mg of yellow precipitate, with a yield of 97%.

[0112] MS (ESI Positive) m / z: calcd for C 23 H 18 ClN6O2 [M + H] + 445.11; found: 445.11. 1 HNMR (400 MHz, ) δ 9.96 (s, 2H), 8.26 (s, 1H), 8.00 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.37–7.32 (m, 2H), 7.28 (s, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.99 (dd, J = 4.2, 1.7 Hz, 1H), 6.41 (dd, J = 4.2, 2.7 Hz, 1H), 5.15 (s, 2H). 1313C NMR (101 MHz, DMSO-d6) δ 182.11, 166.22, 165.77, 148.43, 141.31, 139.15, 138.04, 130.07, 129.66, 127.63, 122.80, 120.62, 120.47, 117.99, 117.35, 110.08, 106.70, 60.22, 48.60, 21.24, 14.56.

[0113] Example 16

[0114] Synthesis of target compound 7j-c1

[0115] Dissolve compound 7j (0.24 mmol) in 5 mL of ethanol and stir in an ice bath. Add compound c1 (0.28 mmol), heat under reflux to ensure that compound 7j reacts as completely as possible. Monitor with TLC plate under the condition of petroleum ether: ethyl acetate = 1:1, and at the same time, use ninhydrin color development to judge. Pulp with petroleum ether and ethyl acetate system to obtain 105 mg of yellow precipitate, with a yield of 89%.

[0116] MS (ESI Positive) m / z: calcd for C 25 H 18 Cl2N5O2 [M + H] + 490.08; found: 490.08. 1 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.98 (s, 1H), 9.04 (s, 1H), 8.50 (s, 1H), 8.34 (d, J = 8.1 Hz, 1H), 7.93–7.77 (m, 2H), 7.72 (d, J = 8.1 Hz, 1H), 7.64–7.51 (m, 2H), 7.46 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 11.3 Hz, 3H), 7.11 (d, J = 7.3 Hz, 1H), 4.83 (d, J = 5.1 Hz, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 182.31, 182.03, 166.56, 165.32, 160.14, 154.83, 147.45, 140.98, 139.25, 138.94, 133.75, 132.08, 131.53, 129.89, 126.73, 126.46, 125.22, 123.44, 122.81, 120.41, 119.00, 117.65, 117.35, 115.02, 44.05.

[0117] Example 17

[0118] Synthesis of Target Compound 7j-c2

[0119] Dissolve compound 7j (0.22 mmol) in 5 mL of ethanol and stir in an ice bath. Add compound c2 (0.26 mmol), heat under reflux to ensure that compound 7j reacts as completely as possible. It can be monitored by TLC plate under the condition of petroleum ether: ethyl acetate = 1:1, and can also be judged by ninhydrin color development. Pulp in a petroleum ether and ethyl acetate system to obtain 43 mg of yellow precipitate with a yield of 41%.

[0120] MS (ESI Positive) m / z: calcd for C 25 H 19 ClN5O2 [M+H] + 456.12; found: 456.12. 1 1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 10.13 (s, 1H), 9.33 (s, 1H), 8.58 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 8.3 Hz, 3H), 7.40 (d, J = 8.8 Hz, 3H), 7.35 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 4.86 (d, J = 5.4 Hz, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 181.96, 166.28, 165.76, 160.33, 154.40, 140.70, 139.15, 138.10, 134.19, 129.93, 129.66, 128.97, 127.57, 127.09, 125.49, 123.64, 122.65, 120.51, 117.57, 117.23, 114.80, 44.21.

[0121] Application Example 1

[0122] In vitro activity evaluation of the compounds of the present invention.

[0123] 1. Experimental principle: The MTT method is used for in vitro toxicity evaluation of compounds. MTT is a yellow dye with the trade name thiazolyl blue. After cells are incubated with drugs, the mitochondria of surviving cells can use succinate dehydrogenase and cytochrome c to metabolize and reduce MTT into insoluble blue-violet crystalline formazan, while dead cells cannot form it, thus distinguishing between living and dead cells. The formed crystals are soluble in DMSO, so the survival rate of cells can be calculated based on the absorbance value measured at 490 nm by an enzyme-linked immunosorbent assay (ELISA) reader.

[0124] 2. Experimental method: Weigh the compounds N-2-a1, N-2-a2, 7j-b1, 7j-b2, N-2-c1, N-2-c2, 7j-c1, 7j-c2 of the present invention and the positive control substances 7j-a1 and sorafenib respectively, add corresponding volumes of cell culture-grade DMSO, and prepare stock solutions with a final concentration of 10 mM for standby. The stock solutions are stored at 0 °C. When used, dilute them with cell culture medium to the required concentrations.

[0125] Collect HepG2 (human liver cancer cells), MDA-MB-231 (human breast cancer cells), and HT-1080 (human fibrosarcoma cells) in the logarithmic growth phase and inoculate them into 96-well plates (5×10 3 per well), place them in an incubator (37 °C, 5% CO2) and culture for 24 h, then add the test compounds. At the same time, set up a blank group and a control group. After co-incubating the cells with the drugs for 24 h, add 200 μM of the culture medium containing 20 μL of MTT solution to each well, continue to culture for 4 h, then aspirate the culture medium, add 200 μM of dimethyl sulfoxide (chromatographic grade), and use an ELISA reader to detect the absorbance value at 490 nm wavelength and calculate the proliferation inhibition rate of the compounds on the cells.

[0126] 3. Experimental results: As shown in Table 1, the compounds containing quinazoline skeletons generally have certain selectivity for the proliferation inhibition of MDA-MB-231 cells, while the compounds containing formamidine fragments have better selectivity for HT1080 cells sensitive to ferroptosis; the activity order of the compounds with different linking groups is that those containing formamidine are generally higher than those containing biuret and thiourea structures; the compound N-2-c1 containing pyrrolo[1,2-a]triazine structure and formamidine group has the strongest activity, and its activity is better than that of the control compounds sorafenib and 7j-a1. In summary, N-2-c1 and 7j-c1 show good cell activity.

[0127] Table 1 Cytotoxicity of sorafenib and its analogs against HepG2, MDA-MB-231 and HT-1080 cells

[0128]

[0129] Application Example 2

[0130] Evaluation of the in vitro anti-proliferative activities of N-2-c1, 7j-c1 with good activities and the positive control sorafenib in the compounds of the present invention.

[0131] 1. Experimental principle: The cell colony formation assay is a method for evaluating the ability of drugs to affect tumor growth in vitro, which can effectively reflect the dependence and proliferation ability of cell populations.

[0132] 2. Experimental method: HT1080 cells were seeded in 6-well plates at a concentration of 400 cells / mL. After culturing in an incubator for 12 h, they were cultured with culture medium containing different concentrations of drugs, and the fresh drug-containing medium was changed every three days. After obvious cell clusters could be observed under a microscope, the culture medium was aspirated, and the cells were rinsed once with PBS. 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 60 min, then rinsed once with PBS and stained with crystal violet for 20 min. After rinsing three times with PBS, the cells were air-dried and photographed. In this colony formation assay, HT1080 cells were treated with compound N-2-c1 at concentrations of 2 μM, 3 μM, 4 μM, 7j-c1 at 4 μM, and sorafenib.

[0133] 3. Experimental results: As Figure 1 , it can be seen from the cell colony formation experiment that the drugs N-2-c1 and 7j-c1 modified from sorafenib significantly weakened the anti-proliferative ability of HT1080 cells, which is very likely related to their induction of a relatively high proportion of ferroptosis in cells.

[0134] Application Example 3

[0135] Ferroptosis rescue experiments of N-2-c1, 7j-c1 with good activities and the positive control sorafenib in the compounds of the present invention.

[0136] 1. Experimental principle: To verify the activity of compound N-2-c1 in inducing ferroptosis in cells, we performed a ferroptosis rescue experiment using ferrostatin-1 (Fer-1). Fer-1 is the most commonly used ferroptosis inhibitor, which can rescue cells undergoing ferroptosis and has no obvious cytotoxicity at 10 μM.

[0137] 2.2. Experimental method: Concentrations near the 50% survival rate of each compound of sorafenib / 7j-c1 / N-2-c1 were selected for related cell proliferation activity evaluation experiments.

[0138] 3. Experimental results: Figure 2At the same Fer-1 (5 μM) concentration, the cell survival rate of Sorafenib ranged from 48.16% to 53.66%, that of compound 7j-c1 ranged from 52.67% to 59.70%, and that of compound N-2-c1 ranged from 67.72% to 91.73%. The maximum rescue amplitude was close to 74%, indicating that in the case of compound N-2-c1, it was easier to induce ferroptosis in cells. (N-2-c1: 74.4%, 7j-c1: 14.8%, Sorafenib: 10.6%) [(Ratio of cells rescued by ferroptosis inhibitor-1 / Ratio of cells that died without ferroptosis inhibitor) x 100%]. It can be seen from this that N-2-c1 can make the HT1080 cell line more prone to ferroptosis.

[0139] The present invention designed and synthesized 12 novel sorafenib analogues, which have certain inhibitory activities against HepG2 (human liver cancer cells), MDA-MB-231 (human breast cancer cells) and HT-1080 (human fibrosarcoma cells). Compared with the original sorafenib, N-2-c1 and 7j-c1 have higher activities among the above analogues and can be used as potential ferroptosis-inducing compounds, providing a new and promising method for designing novel ferroptosis inducers.

[0140] The above embodiments illustrate the specific implementation process of the present invention patent. The changes and improvements in the present invention patent are also covered by the protection scope of the present invention.

Claims

1. A class of urea derivatives that induce ferroptosis, characterized in that, The urea derivatives that induce ferroptosis are alloxan derivatives represented by the following general formula; wherein, B is 3,4-dichlorophenyl or p-chlorophenyl; The structural formula of the alloxan derivative is shown as N-2-a1 and N-2-a2; 2. A class of urea derivatives that induce ferroptosis, characterized in that, The urea derivatives that induce ferroptosis are thiourea derivatives represented by the following general formula; wherein, B is 3,4-dichlorophenyl or p-chlorophenyl; The thiourea derivative, and its structural formula is shown as 7j-b1 and 7j-b2; 3. A class of urea derivatives that induce ferroptosis, characterized in that, The urea derivatives that induce ferroptosis are formamidine derivatives represented by the following general formula; wherein, A is quinazoline or pyrrolo[1,2-a]triazine; B is 3,4-dichlorophenyl or p-chlorophenyl; The formamidine derivatives, and their structural formulas are shown as N-2-c1, N-2-c2, 7j-c1 and 7j-c2; 4. A method for preparing the urea derivatives for inducing ferroptosis according to claim 1, characterized in that, Comprising the following steps: (1) Synthesis of intermediate N-1: Dissolve compound N and 3-nitrobenzyl bromide in a molar ratio of 1:1 - 1.5 in DMF solvent, react at room temperature for 22 - 26 hours, and then perform recrystallization with DMF and methanol to obtain the white flaky solid intermediate N-1; (2) Synthesis of intermediate N-2: Dissolve the intermediate N-1 obtained in step (1) in a mixed solvent of ethanol and water with a volume ratio of 2:1, and then add iron powder and ammonium chloride, wherein the molar ratio of intermediate N-1, iron powder to ammonium chloride is 1:6 - 10:0.5 - 1, react at 78 - 82 °C for 1.5 - 2.5 hours, and then perform recrystallization with ethyl acetate and petroleum ether to obtain the white powdery intermediate N-2; (3) Synthesis of intermediate a1 and intermediate a2: In the presence of triphosgene, react compound 1 and compound 2 respectively, wherein the molar ratio of compound 1 to triphosgene is 1:0.37 - 0.4, the molar ratio of compound 2 to triphosgene is 1:0.37 - 0.4, in a mixed solvent of dichloromethane and saturated sodium bicarbonate aqueous solution with a volume ratio of 1:1, react under an ice bath condition of 0 - 5 °C for 10 - 20 minutes, and respectively obtain intermediate a1 and intermediate a2 after liquid separation, drying and concentration; (4) Synthesis of the target compounds N-2-a1 and N-2-a2: Dissolve intermediate N-2 and intermediate a1 or intermediate a2 in a molar ratio of 1:1.2 - 1.5 in anhydrous tetrahydrofuran, start the reaction at 0 - 5 °C and then raise the temperature to room temperature and react for 22 - 26 hours, and purify by pulping with ethyl acetate and petroleum ether to respectively prepare the target compounds N-2-a1 and N-2-a2; 5. A method for preparing the urea derivatives for inducing ferroptosis according to claim 2, characterized in that, Comprising the following steps: (1) Synthesis of intermediate 7j: Dissolve compound j and 3-aminobenzylamine in a molar ratio of 1:1.2 - 1.5 in dichloromethane, react at room temperature for 24 hours, extract with saturated sodium bicarbonate and dichloromethane, and perform pulping treatment with ethyl acetate and petroleum ether to obtain the white solid powder intermediate 7j; (2) Synthesis of intermediate b1 and intermediate b2: Compound 1 and Compound 2 are separately reacted in the presence of thiophosgene. The molar ratio of Compound 1 to thiophosgene is 1:1.2 - 1.5, and the molar ratio of Compound 2 to thiophosgene is 1:1.2 - 1.

5. They are reacted in a mixed solvent of dichloromethane and saturated sodium bicarbonate aqueous solution with a volume ratio of 1:1 - 1.2 under an ice bath condition at 0 - 5 °C for 10 - 20 minutes. After liquid separation, drying, and concentration respectively, intermediate b1 and intermediate b2 are obtained; (3) Synthesis of target compounds 7j - b1 and 7j - b2: Intermediate 7j and intermediate b1 or intermediate b2 are dissolved in anhydrous tetrahydrofuran at a molar ratio of 1:1.2 - 1.

5. After starting the reaction at 0 - 5 °C, the temperature is raised to room temperature and the reaction proceeds for 22 - 26 hours. After purification by pulping with ethyl acetate and petroleum ether, target compounds 7j - b1 and 7j - b2 are respectively prepared; 6. A method for preparing the urea derivatives for inducing ferroptosis according to claim 3, characterized in that, It includes the following steps: (1) Synthesis of intermediate N - 1: Compound N and 3 - nitrobenzyl bromide are dissolved in DMF solvent at a molar ratio of 1:1 - 1.

5. After reacting at room temperature for 22 - 26 hours, the white flaky solid intermediate N - 1 is obtained after recrystallization with DMF and methanol; (2) Synthesis of intermediate N - 2: The intermediate N - 1 obtained in step (1) is dissolved in a mixed solvent of ethanol and water with a volume ratio of 2:

1. Then, iron powder and ammonium chloride are added. The molar ratio of intermediate N - 1, iron powder, and ammonium chloride is 1:6 - 10:0.5 - 1. After reacting at 78 - 82 °C for 1.5 - 2.5 hours, the white powdery intermediate N - 2 is obtained after recrystallization with ethyl acetate and petroleum ether; (3) Synthesis of intermediate 7j: Compound j and 3 - aminobenzylamine are dissolved in dichloromethane at a molar ratio of 1:1.2 - 1.

5. After reacting at room temperature for 24 hours, it is extracted with saturated sodium bicarbonate and dichloromethane, and then treated by pulping with ethyl acetate and petroleum ether to obtain the white solid powder intermediate 7j; (4) Synthesis of intermediate c1 and intermediate c2: Compound 1 and Compound 2 are separately reacted in the presence of butyl squarate. The molar ratio of Compound 1 to butyl squarate is 1:1.2 - 1.5, and the molar ratio of Compound 2 to butyl squarate is 1:1.2 - 1.

5. They are reacted in anhydrous ethanol solvent. After the reaction is completed under an ice bath condition at 0 - 5 °C, it is transferred to room temperature and the reaction proceeds for 22 - 26 hours. After treatment by pulping with ethyl acetate and petroleum ether, intermediate c1 and intermediate c2 are obtained; (5) Synthesis of target compounds N - 2 - c1, N - 2 - c2, 7j - c1 and 7j - c2: Intermediate N - 2 or intermediate 7j and intermediate c1 or intermediate c2 are dissolved in anhydrous ethanol at a molar ratio of 1:1.2 - 1.

5. After reacting at 118 - 122 °C for 22 - 26 hours, after purification by pulping with ethyl acetate and petroleum ether, target compounds N - 2 - c1, N - 2 - c2, 7j - c1 and 7j - c2 are respectively prepared; 7. Use of the urea derivatives for inducing ferroptosis as described in claims 1 - 3 in the preparation of drugs for treating breast cancer tumors, liver cancer tumors or human fibrosarcoma tumors.

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