Substituted aminoacetamide compounds containing ferrocene, preparation method and application thereof

Abstract

The embodiments of the present application relate to a substituted aminoacetamide compound containing ferrocene and a preparation method and application thereof, and belong to the field of pharmaceutical chemistry technology. The substituted aminoacetamide compound containing ferrocene is a compound shown in formula I, a pharmaceutically acceptable salt of the compound shown in formula I, a hydrate of the compound shown in formula I, a solvate of the compound shown in formula I, an optical isomer of the compound shown in formula I, or a derivative of the compound shown in formula I. The compound provided in the embodiments of the present application has a significant inhibitory effect on the activity of glutathione peroxidase 4 (GPX4).

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of pharmaceutical chemistry, and in particular to a ferrocene-containing substituted aminoacetamide compound, a preparation method and application thereof. Background Art

[0002] Malignant tumors are one of the world's major diseases and seriously threaten human health and life. Although the quality of current medical care has improved significantly, there is still a lack of effective countermeasures to treat cancer.

[0003] GPX4 is a member of the glutathione peroxidase family and is an enzyme containing a selenocysteine ​​core. Unlike other members of the family, GPX4 is the only peroxidase that can catalyze the reduction of lipid peroxides. Inhibition of GPX4 activity will directly lead to the obstruction of intracellular LPO clearance, thereby inducing ferroptosis. Therefore, inhibiting GPX4 activity is the most effective way to induce ferroptosis. Under the condition of glutathione (GSH) as a coenzyme, GPX4 can bioreduced LPO (PE-AA-OOH and PE-ADA-OOH, etc.) produced by various pathways in the cell to normal lipids. GPX4 is highly expressed in many tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cancer, and is associated with tumor MDR. Studies have shown that inhibiting GPX4 activity will block intracellular lipid peroxide metabolism and induce ferroptosis to overcome tumor MDR.

[0004] Ferrocene is a very important organometallic functional group with high stability, lipophilicity, low toxicity, unique sandwich structure and the ability to participate in redox reactions. It is widely used in pharmaceutical chemistry. The application of ferrocene in the field of anti-tumor drug chemistry mainly involves two aspects: first, using the unique three-dimensional spatial conformation of ferrocene to introduce it as a structural unit into the design of small molecule drugs, expanding the design of the chemical spatial structure of anti-tumor related molecules; second, based on the property that ferrocene can participate in redox reactions, studies have confirmed that ferrocene derivatives with specific structural substitutions can produce cytotoxic reactive oxygen free radicals through Fenton / Fenton-like reactions, thereby killing tumor cells. Therefore, screening and synthesizing compounds containing ferrocene structures and using them to promote the occurrence of ferroptosis are of great significance for the treatment of tumors.

[0005] Currently, the research on ferroptosis inducers is still in its early stages. Although a number of compounds have been found to have GPX4 inhibitory activity and ferroptosis inducing activity, such as RSL-3, Erastin and ML-210, their structures are single and they have problems such as insufficient ferroptosis inducing activity, lack of selectivity and poor drugability. No ferroptosis inducer has entered the clinic yet. Summary of the invention

[0006] In view of this, the embodiments of the present application provide a substituted aminoacetamide compound containing ferrocene, a preparation method and an application thereof. Experiments have shown that the compounds have a significant inhibitory effect on the activity of glutathione peroxidase 4 (GPX4), and can block the metabolism of intracellular lipid peroxides by inhibiting GPX4, so that toxic lipid peroxides such as LPO (PE-AA-OOH and PE-ADA-OOH) accumulate, and induce cell ferroptosis; in addition, GPX4 is highly expressed in a variety of tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer and renal cancer. The ferroptosis induced by the compounds effectively kills tumor cells, providing patients with more optional drugs; it can effectively overcome the defects of the above-mentioned prior art.

[0007] In a first aspect, an embodiment of the present application provides a substituted aminoacetamide compound containing ferrocene, wherein the compound is a compound of formula I, a pharmaceutically acceptable salt of the compound of formula I, a hydrate of the compound of formula I, a solvate of the compound of formula I, an optical isomer of the compound of formula I, or a derivative of the compound of formula I, and the structure of formula I is shown below:

[0008]

[0009] In the formula, R1 is selected from allyl, C 1-6 Alkyl, cycloalkyl, substituted or unsubstituted benzyl, phenethyl, phenethyl containing 1-2 substituents, substituted or unsubstituted aromatic ring, halogenated alkyl;

[0010] R2 is selected from the group consisting of nitrogen-containing aromatic heterocycles, biphenyl, saturated nitrogen-containing heterocycles, benzo five-membered heterocycles, benzo six-membered heterocycles, pyrido six-membered heterocycles, pyrido five-membered heterocycles, aliphatic heterocycles, naphthalene, anthracene, benzo five-membered heterocycles having 1-3 identical or different R3 substituents, and benzo six-membered heterocycles having 1-3 identical or different R3 substituents;

[0011] R3 is selected from hydrogen, hydroxy, alkoxy, halogen, C 1-6 Alkylamino, cyano, C 1-6 Alkyl, haloalkyl;

[0012] X is selected from S, O, NH;

[0013] Y is selected from CH, N.

[0014] In some embodiments that may include the above embodiments, R1 is selected from allyl, tert-butyl, cycloalkyl, benzyl, substituted or unsubstituted aromatic ring, phenethyl, phenethyl containing 1-2 substituents, substituted or unsubstituted aromatic ring, haloalkyl;

[0015] R2 is selected from the group consisting of a nitrogen-containing five-membered aromatic heterocycle, a saturated nitrogen-containing heterocycle, biphenyl, a benzo five-membered heterocycle, a benzo five-membered heterocycle having 1 to 3 identical or different R3 substituents, and a benzo six-membered heterocycle having 1 to 3 identical or different R3 substituents;

[0016] R3 is selected from hydrogen, alkoxy, halogen, C 1-6 Alkylamino, cyano, C 1-6 Alkyl, haloalkyl;

[0017] X is selected from S or O;

[0018] Y is selected from CH or N.

[0019] In some embodiments that may include the above embodiments, the compound is selected from N-(1-(ferrocen-3-yl)-2-oxo-2-(phenylethylamino)ethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(benzylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(4-(1H-imidazol-1-yl)phenyl)-N- (1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(4-(alkynyl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(4-(1H-pyrazol-1-yl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-fluorophenyl)thiazole-4-carboxamide, N-(1-(ferrocen- 3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethylphenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethoxyphenyl)thiazole-4-carboxamide, N-(4-(cyano)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-benzothienylphenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-benzothienylphenyl)thiazole-4-carboxamide, 4-(trifluoromethoxyphenyl)thiazole-4-carboxamide, N-(4-(cyano)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-benzothienylphenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethoxyphenyl)thiazole-4-carboxamide.

[0020] The second aspect of the embodiment of the present application further provides a method for preparing the above-mentioned ferrocene-containing substituted aminoacetamide compound, comprising the step of obtaining the compound shown in formula I according to the following reaction formula:

[0021]

[0022] In some embodiments that may include the above embodiments, compound 1 and compound 4 are dissolved in 0.6 mL of methanol, stirred for 15 mins, and then compound 2 and compound 3 are added to react for 8-10 h, and post-treated to obtain a substituted aminoacetamide compound containing ferrocene. Preferably, the post-treatment includes the following steps: after the reaction is completed, the mixture is concentrated under reduced pressure, and separated by preparing a thin layer plate to obtain a substituted aminoacetamide compound containing ferrocene.

[0023] The third aspect of the embodiments of the present application also provides a pharmaceutical composition comprising the ferrocene-containing substituted aminoacetamide compound as described above and one or more pharmaceutically acceptable additives or excipients.

[0024] The fourth aspect of the embodiments of the present application also provides the use of the above-mentioned ferrocene-containing substituted aminoacetamide compounds in the preparation of ferroptosis inducers.

[0025] The fifth aspect of the embodiments of the present application also provides the use of the above-mentioned ferrocene-containing substituted aminoacetamide compounds in the preparation of anti-tumor drugs.

[0026] In some embodiments that may include the above embodiments, the tumor is a ferroptosis-sensitive tumor, specifically including fibrosarcoma, lymphoma, breast cancer, lung cancer, and kidney cancer.

[0027] In some embodiments that may include the above embodiments, the compound inhibits GPX4 and increases the cellular reduced iron content to upregulate the oxidation level, thereby inducing ferroptosis, thereby inhibiting the survival of tumor cells and achieving an anti-tumor effect.

[0028] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0029] The ferrocene-containing substituted aminoacetamide compounds of the embodiments of the present application have a significant inhibitory effect on the activity of glutathione peroxidase 4 (GPX4), and can block the intracellular lipid peroxide metabolism by inhibiting GPX4, causing the accumulation of toxic lipid peroxides such as LPO (PE-AA-OOH and PE-ADA-OOH), thereby inducing cell ferroptosis; in addition, GPX4 is highly expressed in a variety of tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cancer. The ferroptosis induced by this type of compound effectively kills tumor cells, providing patients with more optional drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is a CETSA experiment diagram of Example 17 of the present application;

[0032] Figure 2 This is a flow cytometry experiment diagram of Example 17 of the present application;

[0033] Figure 3 This is a laser confocal microscopy cell experiment picture of Example 17 of the present application. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0035] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0036] The term "substituted" in the examples of the present application means that one or more hydrogen atoms in the group are independently replaced by a corresponding number of substituent groups.

[0037] In the examples of the present application, the additives or excipients are substances other than the active ingredients, which have been reasonably evaluated in terms of safety and are included in the pharmaceutical preparations, and may be gum arabic, syrup, lanolin, starch, magnesium chloride, cyclodextrin, sebacic acid, dextrin, medicinal calcium sulfate, glycerol, mannitol, sorbitol, inositol, thiol, tromethamine, phenol, m-cresol, benzyl alcohol, parabens, methyl paraben, tert-butyl alcohol, benzalkonium chloride, chlorobutanol, thimerosal, etc.

[0038] In the embodiments of the present application, optical isomers are a type of stereoisomers, also known as optical isomers, chiral isomers, optical isomers, mirror image isomers, enantiomers or chiral isomers, which cannot be completely superimposed on each other's stereoisomer mirror images.

[0039] The derivatives of the compounds of general formula I in the examples of the present application are used as prodrugs, which means that they themselves may have weak activity or even no activity, but after administration, they are converted into corresponding biologically active forms under physiological conditions (for example, by metabolism, solvent decomposition or other means).

[0040] The chemical structures of the examples of the present application were confirmed by 1H-NMR and 13C-NMR. Flash column chromatography was performed on silica gel H (10-40 μM).

[0041] Example 1

[0042] N-(1-(Ferrocen-3-yl)-2-oxo-2-(phenylethylamino)ethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide The preparation process is as follows:

[0043] Preparation of the target compound: Ferrocenecarboxaldehyde and 4-(oxazol-5-yl)aniline were dissolved in 0.6 mL of methanol, stirred for 15 mins, and then 2-(alkynyl)thiazole-4-carboxylic acid and the compound phenethyl isocyanide were added to react for 8-10 h. After post-treatment, the compound of Example 1 was obtained with a yield of 95%.

[0044] 1H NMR (400MHz, DMSO-d6) δ8.57(s,1H),8.38(s,1H),7.66(s,1H),7.60(s,1H),7.37(d,J=8.3Hz,2H),7.31(d,J=3.3Hz,4H),7.21(s,1H),7.0 0(s,2H),6.05(s,1H),4.83(s,1H),4.21(s,1H),4.15(s,5H),4.08(s ,1H),4.05(s,1H),3.97(s,1H),3.52–3.44(m,2H),2.93–2.85(m,2H).

[0045] 13C NMR (101MHz, DMSO) δ168.57,162.77,152.34,150.61,150.35,146.02,140.02,139.85,131.65,129.15,128.87,126.61,126 .34,125.99,125.02,123.58,122.84,121.40,86.75,81.69,76.29,70.60,70.09,69.24,68.45,68.09,60.40,41.10,35.67.

[0046] Example 2

[0047] N-(1-(ferrocen-3-yl)-2-(benzylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0048] 1H NMR (400MHz, DMSO-d6) δ9.03(t,J=5.7Hz,1H),8.37(s,1H),7.66(s,1H),7.59(s,1H),7.46(d,J=7.4Hz,2H),7.38–7.33(m,4H),7.25(t,J =7.3Hz,1H),7.07(s,2H),6.08(s,1H),4.83(s,1H),4.54–4.42(m,2H),4.21(s,1H),4.08(s,5H),4.06(s,1H),4.03(s,1H),3.94(s,1H).

[0049] 13C NMR (101MHz, DMSO) δ168.33,162.35,151.83,150.13,149.85,145.54,139.48, 139.24,131.30,128.24,127.76, 126.83,125.82,125.36,123.03,122.34,86.27,81.18,75.79,69.67,69.48,68.70,67.92,67.51,60.10,42.62.

[0050] Example 3

[0051] N-(1-(ferrocen-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0052] 1H NMR (400MHz, DMSO-d6) δ8.37(d,J=3.1Hz,2H),7.60(d,J=11.0Hz,2H),7.36(dJ=8.1Hz,2H),7.03(s,2H),6.03(s,1H),4.82(s,1H) ,4.19(s,6H),4.07(s,2H),3.96(s,1H),1.89(d,J=16.1Hz,3H),1.78–1.72(m,2H),1.60(d,J=12.0Hz,1H),1.36(d,J=20.2Hz,5H).

[0053] 13C NMR (101MHz, DMSO) δ167.02,162.21,151.81,150.18,149.86,145.48,139.50,131.30,125.80,125.24,122.9 9,122.31,86.22,81.32,75.77,69.55,69.51,68.76,67.88,67.53,59.81,48.11,32.45,32.32,25.25,24.75.

[0054] Example 4

[0055] N-(4-(1H-imidazol-1-yl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0056] 1H NMR (400MHz, DMSO-d6) δ8.20(s,1H),8.03(s,1H),7.66(d,J=10.4Hz,2H),7.36(d,J=7.9Hz,2H ),7.05(s,3H),6.06(s,1H),4.82(s,1H),4.22(s,6H),4.09(s,2H),4.00(s,1H),1.41(s,9H).

[0057] 13C NMR (101MHz, DMSO) δ167.50,162.20,150.18,145.50,137.63,135.24,135.16,132.00,129.98,125 .37,118.21,117.42,86.24,81.81,75.78,69.58,69.34,68.75,67.80,67.49,60.04,50.48,28.44.

[0058] Example 5

[0059] N-(4-(Alkynyl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0060] 1H NMR (400MHz, DMSO-d6) δ8.00(s,1H),7.62(s,1H),7.10(d,J=8.0Hz,2H),7.05–6.85(m,2H),6.02( s,1H),4.84(s,1H),4.21(s,5H),4.15(s,1H),4.12(s,1H),4.06(s,2H),3.99(s,1H),1.40(s,9H).

[0061] 13C NMR (101MHz, DMSO) δ167.46,162.05,150.14,145.48,139.96,130.77,125.39,120.13,86 .23,83.02,81.68,81.27,75.76,69.55,69.29,68.75,67.76,67.43,60.23,50.47,28.43.

[0062] Example 6

[0063] N-(4-(1H-pyrazol-1-yl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide The synthetic route of the application example is the same as that of Example 1.

[0064] 1H NMR (400MHz, DMSO-d6) δ8.39(d,J=2.2Hz,1H),8.02(s,1H),7.68(s,1H),7.60(s,1H),7.50(d,J=8.1Hz,2H),7.03(s,2H ), 6.49–6.47(m,1H),6.04(s,1H),4.82(s,1H),4.21(d,J=4.5Hz,6H),4.08(d,J=6.5Hz,2H),3.99(s,1H),1.41(s,9H).

[0065] 13C NMR (101MHz, DMSO) δ167.56,162.25,150.24,145.48,140.97,138.01,137.08,131.66,127.52,125 .08,116.70,107.94,86.23,81.79,75.79,69.60,69.32,68.74,67.74,67.47,60.12,50.46,28.45.

[0066] Example 7

[0067] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-fluorophenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0068] 1H NMR(400MHz,DMSO-d6)δ8.01(s,1H),7.62(s,1H),6.83(s,4H),5.99(s,1H),4.84(s,1H),4.21( s,5H),4.18–4.16(m,1H),4.08–4.06(m,1H),4.04–4.03(m,1H),3.99–3.98(m,1H),1.40(s,9H).

[0069] 13C NMR(101MHz,DMSO)δ167.59,162.21,150.24,145.40,135.57,132.70,125.20,114.13, 113.90,86.15,81.77,75.77,69.56,69.22,68.72,67.69,67.43,60.01,50.45,28.43.

[0070] Example 8

[0071] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethylphenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0072] 1H NMR (400MHz, DMSO-d6) δ8.06(s,1H),7.78(s,1H),7.38(d,J=8.3Hz,2H),7.15(s,2H),6.07(s ,1H),4.81(s,1H),4.22(s,5H),4.18(s,1H),4.07(d,J=4.7Hz,2H),4.00(s,1H),1.40(s,9H).

[0073] 13C NMR (101MHz, DMSO) δ167.42,161.90,149.96,145.51,143.34,131.27,127.34,127.03,126 .25,124.29,86.14,81.67,75.61,69.57,69.26,68.79,67.86,67.44,60.25,50.52,28.42.

[0074] Example 9

[0075] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethoxyphenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0076] 1H NMR(400MHz,DMSO-d6)δ8.03(s,1H),7.77(s,1H),6.99(d,J=6.7Hz,4H),6.01(s,1H),4 .80(s,1H),4.21(s,5H),4.17(s,1H),4.06(d,J=9.3Hz,2H),3.98(s,1H),1.40(s,9H).

[0077] 13C NMR (101MHz, DMSO) δ167.52,161.98,150.14,146.85,145.39,138.55,132.44,125.93,119 .72,118.58,86.00,81.75,75.59,69.56,69.22,68.75,67.73,67.42,60.10,50.47,28.42.

[0078] Example 10

[0079] N-(4-(Cyano)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0080] 1H NMR (400MHz, DMSO-d6) δ8.09(s,1H),7.81(s,1H),7.49(d,J=8.5Hz,2H),7.17(s,2H),6.06(s ,1H),4.84(s,1H),4.22(s,5H),4.15(s,1H),4.04(d,J=8.8Hz,2H),3.99(s,1H),1.40(s,9H).

[0081] 13C NMR (101MHz, DMSO) δ167.52,161.67,149.85,144.21,133.43,131.55,131.36,126.64,113 .44,109.29,86.31,81.61,75.67,69.59,69.20,68.84,67.94,67.46,60.45,50.57,28.44.

[0082] Embodiment 11

[0083] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-benzothienylphenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0084] 1H NMR (400MHz, DMSO-d6) δ8.11–8.05(m,2H),8.00(d,J=8.1Hz,1H),7.76–7.69(m,3H),7.54–7.49(m,1H),7.43(t,J=7.6Hz, 1H),7.14(s,2H),6.08(s,1H),4.81(s,1H),4.22(s,5H),4.20(s,1H),4.13(s,1H),4.07(s,1H),4.00(s,1H),1.42(s,9H).

[0085] 13C NMR (101MHz, DMSO) δ167.50,166.46,162.02,153.45,150.13,145.59,142.37,134.43,131.39,131.10,126.66,126 .18,125.78,125.55,122.80,122.30,86.29,81.75,75.76,69.63,69.39,68.80,67.86,67.50,60.40,50.52,28.46.

[0086] Example 12

[0087] N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-biphenylphenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0088] 1H NMR (400MHz, DMSO-d6) δ8.00(s,1H),7.70(d,J=8.5Hz,3H),7.66(d,J=8.8Hz,3H),7.59(s,1H),7.48–7.41(m,4H),7.36(t,J=7.3Hz,1H),7. 02(s,2H),6.07(s,1H),4.84(s,1H),4.24–4.22(m,1H),4.22(s,5H), 4.17–4.15(m,1H),4.11–4.09(m,1H),4.02–4.00(m,1H),1.41(s,9H).

[0089] 13C NMR (101MHz, DMSO) δ167.44,162.33,150.33,145.47,139.44,139.14,138.90,137.81,137.61,131.01,128.97,127.55, 127.08,126.85,126.49,125.23,125.08,86.23,81.90,75.83,69.61,69.49,68.76,67.76,67.49,60.20,50.48,28.46.

[0090] Embodiment 13

[0091] N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-(6-methylbenzothienyl)phenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0092] 1H NMR(400MHz, DMSO-d6)δ8.06(s,1H),7.88–7.86(m,2H),7.70(d,J=10.5Hz,3H),7.33(d,J=9.8Hz,1H),7.13(s,2H),6.07(s,1H), 4.82(s,1H),4.22(s,5H),4.20–4.19(m,1H),4.13–4.11(m,1H),4.08–4.06(m,1H),4.00–3.99(m,1H),2.43(s,3H),1.41(s,9H).

[0093] 13C NMR (101MHz, DMSO) δ167.51,165.32,162.03,151.62,150.13,145.59,142.16,135.38,134.59,131.37,131.23,128.16 ,126.02,125.73,122.38,121.81,86.31,81.75,75.78,69.64,69.38,68.80,67.86,67.50,60.40,50.53,28.47,21.06.

[0094] Embodiment 14

[0095] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiophene-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0096] 1H NMR (400MHz, DMSO-d6) δ8.43(s,1H),8.03(s,1H),7.70(s,1H),7.60–7.50(m,2H),7.05(d,J=4.1Hz,1H),6.4 5(d,J=4.0Hz,1H),5.98(s,1H),4.63(s,1H),4.20(s,6H),4.06–4.02(m,2H),3.96–3.94(m,1H),1.40(s,9H).

[0097] 13C NMR(101MHz,DMSO)δ167.74,159.64,152.08,149.70,139.00,138.79,132.73,132.31,131.97,127.19, 126.04,123.74,122.87,87.22,81.58,75.92,69.82,69.25,68.75,67.70,67.49,60.82,50.48,28.46.

[0098] Embodiment 15

[0099] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)furan-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0100] 1H NMR (400MHz, DMSO-d6) δ8.42(s,1H),8.05(s,1H),7.68(s,1H),7.52(s,2H),6.62(d,J=3.7Hz,1H),5.96(s,1H),5.42(d,J=3. 7Hz,1H),4.75(s,1H),4.21(s,5H),4.18–4.17(m,1H),4.06–4.04(m,1H),4.03–4.01(m,1H),3.97–3.95(m,1H),1.40(s,9H).

[0101] 13C NMR(101MHz,DMSO)δ167.69,156.70,152.01,149.79,146.73,138.98,136.36,131.72,126.80,123.55, 122.66,117.09,116.58,86.95,81.55,72.90,69.75,69.25,68.76,67.70,67.47,60.32,50.47,28.46.

[0102] Example 16

[0103] N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(3-chloro-4-methoxy)phenyl)thiazole-4-carboxamide The synthetic route of the present embodiment is the same as that of Example 1.

[0104] 1H NMR(400MHz,DMSO-d6)δ8.03(s,1H),7.62(s,1H),6.73(s,1H),5.97(s,1H),4.86(s,1H),4.2 1(s,5H),4.19(s,1H),4.09(s,1H),4.05(s,1H),4.02–4.00(m,1H),3.70(s,3H),1.40(s,9H).

[0105] 13C NMR (101MHz, DMSO) δ167.60,162.33,153.27,150.20,145.50,132.23,131.99,130.63,125.05,119 .09,110.79,86.27,81.77,75.82,69.56,69.19,68.75,67.75,67.48,59.97,55.99,50.47,28.44.

[0106] Embodiment 17

[0107] N-(1-(Ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide The synthetic route of the application example is the same as that of Example 1.

[0108] 1H NMR (400MHz, DMSO-d6) δ8.37(s,1H),8.01(s,1H),7.60(d,J=8.3Hz,2H),7.37(d,J=8.3Hz,2H),7.02(s,2 H), 6.04 (s, 1H), 4.82 (s, 1H), 4.21 (s, 5H), 4.19 (s, 1H), 4.08 (d, J = 8.5Hz, 2H), 3.99 (s, 1H), 1.41 (s, 9H).

[0109] 13C NMR (101MHz, DMSO) δ167.49,162.17,151.80,150.21,149.87,145.48,139.53,131.23,125.79,125 .25,123.01,122.29,86.24,81.76,75.78,69.59,69.34,68.74,67.76,67.44,60.22,50.47,28.44.

[0110] Application Examples

[0111] active:

[0112] Cytotoxicity: Most compounds showed significant inhibitory effects on human fibrosarcoma cells HT1080, with IC50 values ​​less than 100 nM.

[0113] As shown in Table 1, some compounds have extracellular inhibitory effects on GPX4: most of the compounds (1 μM) showed significant inhibitory effects on GPX4.

[0114] Table 1 Inhibitory activity of compounds on the growth of tumor cells HT1080

[0115]

[0116]

[0117] In the table: "A" indicates IC50≤100 nM, and "B" indicates 100 nM<IC50≤500 nM.

[0118] Studies on the inhibitory activity of compounds against the growth of tumor cells HT1080 showed that lipophilic antioxidants, such as Ferrostatin-1 (fer-1), can rescue cells from ferroptosis induced by GPX4 inhibition. Mesenchymal state GPX4-gene knockout cells can survive in the presence of fer-1, but in the absence of fer-1, these cells undergo ferroptosis. In addition, ferroptosis induced by GPX4 inhibitors can also be blocked by other small molecules, such as lipid ROS scavenger Liproxstatin, iron chelator DFO, etc. Therefore, the ability of GPX4 inhibitors to induce ferroptotic cell death can be indicated by the reversal of the addition of fer-1.

[0119] As shown in Table 2, the inhibitory activity of some compounds of the present application is significantly weakened in the presence of fer-1, and thus they are effective GPX4 inhibitors.

[0120] Table 2 Inhibitory activity of compounds on the growth of tumor cells HT1080

[0121] Compound <![CDATA[IC 50 (-do-)]]> <![CDATA[IC 50 (+do-)]]> Compound <![CDATA[IC 50 (-do-)]]> <![CDATA[IC 50 (+do-)]]> Example 1 62 4800 Example 10 <100 >1000 Example 2 >100 - Embodiment 11 <100 >1000 Example 3 34 >1000 Example 12 <100 >1000 Example 4 >100 - Embodiment 13 <100 >1000 Example 5 >100 - Embodiment 14 - - Example 6 <100 >1000 Embodiment 15 - - Example 7 >100 - Example 16 - - Example 8 <100 >1000 Embodiment 17 46 >1000 Example 9 <100 >1000

[0122] CETSA (Cellular Thermal Shift Assay) is an experiment to detect the efficiency of intracellular drug-target protein binding. Its principle is that target proteins usually become stable when bound to drug molecules. That is, as the temperature rises, the protein will degrade; when the protein binds to the drug, the amount of undegraded protein will increase at the same temperature.

[0123] In the CETSA experiment, HT1080 cells were washed three times with CETSA washing solution, and the washing solution was fully aspirated; 400 μL of NP40 lysis solution containing protease inhibitors was added to the dish, the cells were scraped off, and the cells were fully lysed on ice for 15 minutes, and then divided into a blank group and Example 17 group. Example 17 group was added with Example 17 to make the final concentration of 10 μM, and incubated for 1 hour; then, the blank group and Example 17 group were equally divided into 10 PCR tubes, each tube 100 μL. The 10 aliquot tubes of the blank group and Example 17 group were incubated at 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C for 3 minutes, respectively. Centrifuge at 100,000 rpm, 15 min, 4°C, pipette 80 μL of supernatant into a new clean PCR tube, add 20 μL of 5x loading buffer to each tube, and boil for denaturation for 5 min; the 10 samples of the blank group and Example 17 group were electrophoresed in 12% polyacrylamide gel according to a temperature gradient, and then silver stained; observe, and cut out the bands whose descent was significantly slowed down after the treatment of Example 17 for detection.

[0124] like Figure 1As shown, Example 17 of the present application ( Figure 1 In the CETSA experiment (repeated three times), the degradation of GPX4 was effectively reduced, thus proving that Example 17 effectively binds to the GPX4 protein and has a significant glutathione peroxidase 4 (GPX4) inhibitory effect.

[0125] The upregulation of intracellular ROS and LPO is an important sign of GPX4 inhibition and ferroptosis. In the presence of lipophilic antioxidants such as Ferrostatin-1 (fer-1), intracellular ROS and LPO are not regulated. Therefore, this type of compound can specifically inhibit GPX4-induced ferroptosis in tumor cells.

[0126] In flow cytometry experiments, HT1080 cells were plated at 5 × 10 4 The cells were seeded in a 12-well plate at a density of 100 μM and allowed to attach for 12 hours. Then, the cell culture medium was replaced with fresh culture medium containing Example 17 (0.5 μM) and 17 (0.5 μM) + Fer-1 (0.5 μM) respectively. After incubation for 6 hours, RSL3 (0.5 μM), 26a (0.5 μM), and 26a (0.5 μM) + Fer-1 (0.5 μM) were used as positive controls, and the non-administered group was used as a blank control (Control). The culture medium was removed, and the cells were incubated with a culture medium containing the DCFH-DA (10 μM) probe for 20 minutes. The culture medium was removed and the cells were washed three times with PBS; the cells were collected and analyzed by flow cytometry (FCM). HT1080 cells were plated in a 12-well plate (5×10 4 / well) for 24 hours. Then, the cell culture medium was replaced with fresh culture medium containing Example 17 (0.5 μM) and 17 (0.5 μM) + Fer-1 (0.5 μM) respectively, and incubated for 6 hours. RSL3 (0.5 μM), 26a (0.5 μM), 26a (0.5 μM) + Fer-1 (0.5 μM) were used as positive controls, and the non-administered group was used as a blank control (Control). After incubation for 6 hours, the cells were stained with C11-BODIPY probe for 20 minutes. Finally, the cells were collected and analyzed by flow cytometry (FCM).

[0127] like Figure 2 As shown, Example 17 of the present application ( Figure 2In the flow cytometry experiment, 01H is represented in Example 17) and Yangshen compound 26a (C.Xu, et al. J Med Chem, 64 (2021)) both effectively upregulated ROS and LPO in cells, while the ferroptosis inhibitor Ferrostatin-1 (fer-1) can effectively inhibit the upregulation of ROS and LPO in cells induced by Example 17 and Yangshen compound 26a. The experiment confirmed that Example 17 can significantly cause cell peroxide accumulation and specifically induce ferroptosis.

[0128] In the laser confocal microscopy (CLSM) cell experiment, HT1080 cells were inoculated on coverslips for 24 hours, and incubated with Example 17 (0.5 μM) or 17 (0.5 μM) + Fer-1 (0.5 μM) for 6 hours. The group without drug administration was used as the blank control (Control). Then, the cells were fixed with 4% paraformaldehyde, stained with DAPI, and finally observed with CLSM. HT1080 cells were plated at 1×10 5 / well were seeded on a glass cover in a 12-well plate and incubated for 24 hours. Example 17 (0.5 μM) or a mixture of Example 17 (0.5 μM) and fer-1 (0.5 μM) was added to the cells and incubated for 6 hours. The non-administered group was used as a blank control (Control). Then, the culture medium was removed and replaced with fresh culture medium containing DCFH or C11-BODIPY dye (1.0 μM). After incubation for 6 hours, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde. After staining with DAPI, the cells were collected and observed with CLSM.

[0129] like Figure 3 As shown, in the laser confocal microscopy cell experiment, Example 17 of the present application ( Figure 3 01H represents Example 17) The fluorescence intensity of the group was significantly enhanced compared with the blank control group, and in the presence of Ferrostatin-1 (fer-1), the fluorescence intensity was consistent with that of the model group, indicating that Example 17 can significantly induce the accumulation of cellular ROS and LPO, thereby selectively causing ferroptosis of tumor cells.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A ferrocene-containing substituted aminoacetamide compound, characterized in that: The compound is a compound of formula I or a pharmaceutically acceptable salt of the compound of formula I. The structure of formula I is shown below: Ⅰ In the formula, R1 is selected from benzyl, cyclohexyl, phenethyl, and tert-butyl; R2 is selected from the group consisting of nitrogen five-membered aromatic heterocycle, saturated nitrogen-containing heterocycle, ethynyl, cyano, benzothiophene, biphenyl, halogenated alkyl, F, trifluoromethoxy; X is S; Y is N.

2. The ferrocene-containing substituted aminoacetamide compound according to claim 1, characterized in that: The compound is selected from N-(1-(ferrocen-3-yl)-2-oxo-2-(phenylethylamino)ethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(benzylamino)-2-oxoethyl)-2-ethynyl-N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-ethynyl- N-(4-(oxazol-5-yl)phenyl)thiazole-4-carboxamide, N-(4-(1H-imidazol-1-yl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(4-(alkynyl)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(4-(1H-pyrazol-1-yl)phenyl)- N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-fluorophenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethylphenyl)thiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethylphenyl)thiazole-4-carboxamide, -3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-trifluoromethoxyphenyl)thiazole-4-carboxamide, N-(4-(cyano)phenyl)-N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynylthiazole-4-carboxamide, N-(1-(ferrocen-3-yl)-2-(tert-butylamino)-2-oxoethyl)-2-ethynyl-N-(4-benzothienylphenyl)thiazole-4-carboxamide.

3. A method for preparing a ferrocene-containing substituted aminoacetamide compound according to claim 1 or 2, characterized in that: The method comprises the steps of obtaining the compound shown in formula I according to the following reaction formula: 。 4. The preparation method according to claim 3, characterized in that: The specific steps include: Compound 1 and compound 4 were dissolved in 0.6 mL of methanol, stirred for 15 mins, and then compound 2 and compound 3 were added to react for 8-10 h. After post-treatment, the target compound was obtained.

5. A pharmaceutical composition for fibrosarcoma, comprising the ferrocene-containing substituted aminoacetamide compound according to claim 1 or 2 and one or more pharmaceutically acceptable additives or excipients.

6. Use of the ferrocene-containing substituted aminoacetamide compound according to claim 1 or 2 in the preparation of anti-fibrosarcoma drugs.

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