Preparation method of FSP1 inhibitor FSEN3

Abstract

The invention discloses a preparation method of an FSP1 inhibitor FSEN3, which comprises the following steps: by taking 4-methoxybenzoyl hydrazine and allyl isothiocyanate as raw materials, forming a thiourea intermediate through nucleophilic addition reaction, carrying out cyclization reaction under the action of alkali, then reacting with liquid bromine to construct a thiazolotriazole skeleton, and finally preparing the FSP1 inhibitor FSEN3. And then carrying out substitution reaction with indoline to obtain a target product FSEN3 as shown in a formula (I). The total synthesis method provided by the invention is mild in condition, simple and convenient to operate and high in total yield. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a method for preparing an FSP1 inhibitor FSEN3. Background Art

[0002] Ferroptosis is a metabolic form of non-apoptotic cell death characterized by iron-dependent lipid peroxidation. It is highly relevant to human diseases such as neurodegenerative diseases, tissue damage during cold exposure, ischemia-reperfusion injury, and cancer. Studies have found that inducing ferroptosis is a very promising approach to treat malignant tumors, showing synergistic effects with cancer immunotherapy and can even kill drug-resistant and metastatic cancers.

[0003] Ferroptosis has attracted great interest because it has become a very promising target for cancer treatment. So far, ferroptosis has been found to be regulated by at least three major redox-related systems, including the cysteine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) system, the guanosine triphosphate cyclohydrolase-tetrahydrobiopterin (BH4) system, and the ferroptosis suppressor protein 1 (FSP1)-CoQ10 system. Among them, FSP1-CoQ10 synergistically inhibits ferroptosis with GSH-GPX4. FSP1 inhibits ferroptosis by reducing ubiquinone to ubiquinol or vitamin K to hydroquinone. Studies have shown that inhibiting FSP1 can affect the ability of FSP1 to reduce lipid peroxides, induce cell ferroptosis and prevent tumor recurrence, so the development of FSP1 inhibitors is of great significance for cancer treatment.

[0004] FSP1 inhibitors show great promise in tumor treatment. The reported FSP1 inhibitors mainly include NPD4928, iFSP1, and icFSP1. These FSP1 inhibitors have shown the ability to induce ferroptosis in several cancer cell lines, but their inhibitory activity on FSP1 is not ideal, and their pharmacokinetic parameters are poor, which makes them unsuitable for in vivo use, and they exhibit off-target effects at high concentrations. Therefore, there is an urgent need for new FSP1 inhibitors with improved pharmacokinetic parameters and pharmacodynamic properties to fully explore the potential of FSP1 as a target for tumor treatment. In 2023, the James A. Olzmann team at the University of California, Berkeley, identified several structurally different FSP1 inhibitors through a series of chemical screening. Among these compounds, the IC of 6-(indoline-1-methylene)-3-(4-methoxyphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazole (FSEN3 for short) is 1.54kDa. 50The optimal value is 90.1 nM. It is a non-competitive inhibitor that can selectively sensitize cancer cells to ferroptosis by targeting and inhibiting FSP1, and can also synergize with other ferroptosis inducers to further trigger ferroptosis.

[0005]

[0006] The discovery of this compound provides a new tool for promoting cancer treatment strategies that induce ferroptosis with FSP1 as the target. It can be used as a combination therapy for FSP1 and other ferroptosis defense pathways, and has great development value for use in anti-tumor drugs. However, so far, no synthetic route for FSEN3 has been reported, so it is necessary to develop an efficient method for preparing FSEN3 to provide support for the development of new FSP1 inhibitors. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing FSP1 inhibitor FSEN3.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0009] A method for preparing FSEN3, an FSP1 inhibitor, comprises the following steps:

[0010] S1, using 4-methoxybenzoylhydrazide represented by formula (II) and allyl isocyanate sulfate as raw materials, through nucleophilic addition reaction, to form a thiourea intermediate represented by formula (IIIa);

[0011] S2, the obtained thiourea intermediate undergoes a cyclization reaction under the action of a base to obtain a compound represented by formula (III);

[0012] S3, the compound represented by formula (III) is reacted with liquid bromine to obtain the compound represented by formula (IV);

[0013] S4. The compound represented by formula (IV) undergoes elimination reaction in an alkaline environment to obtain a compound represented by formula (V);

[0014] S5, the compound represented by formula (V) undergoes bromination reaction with liquid bromine to obtain a compound represented by formula (VI);

[0015] S6. The compound represented by formula (VI) undergoes a substitution reaction with dihydroindole to obtain the target compound represented by formula (I), which is a FSP1 inhibitor FSEN3;

[0016]

[0017] In one embodiment of the present invention, S1 comprises the following process: in an organic solvent, the compound represented by formula II reacts with allyl isothiocyanate to form the intermediate compound (IIIa) represented by formula (IIIa);

[0018]

[0019] In one embodiment of the present invention, in S1, the molar ratio of the compound represented by formula (II) to allyl isothiocyanate is 1:(1-1.5), and more preferably 1:1.2.

[0020] In one embodiment of the present invention, in S1, the reaction temperature is 80-100°C. More preferably, it is 90°C.

[0021] In one embodiment of the present invention, in S1, the reaction time is 2 to 4 hours, more preferably 3 hours.

[0022] In one embodiment of the present invention, in S1, the organic solvent is any one or more of the following: tetrahydrofuran, dichloromethane, dioxane, ethanol, and more preferably ethanol.

[0023] In one embodiment of the present invention, S2 comprises the following process: the compound represented by formula (IIIa) is cyclized under the action of an alkaline substance to obtain a compound represented by formula (III);

[0024]

[0025] In one embodiment of the present invention, in S2, the molar ratio of the compound represented by formula (IIIa) to the alkaline substance is 1:(10-30), and more preferably 1:20.

[0026] In one embodiment of the present invention, in S2, the alkaline substance is any one or more of the following: potassium carbonate, sodium hydroxide, potassium tert-butoxide, and more preferably sodium hydroxide.

[0027] In one embodiment of the present invention, in S2, specifically, an ethanol solution of an alkaline substance is mixed with the compound represented by formula (IIIa) for reaction.

[0028] In one embodiment of the present invention, in S2, the reaction temperature is 110-130°C, more preferably 120°C.

[0029] In one embodiment of the present invention, in S2, the reaction time is 3 to 5 hours, more preferably 4 hours.

[0030] In one embodiment of the present invention, S3 comprises the following process: in an organic solvent, the compound represented by formula (III) undergoes a bromination reaction with liquid bromine to obtain a compound represented by formula (IV);

[0031]

[0032] In one embodiment of the present invention, in S3, the molar ratio of the compound represented by formula (III) to the bromination reagent is 1:(1-1.5), and more preferably 1:1.2.

[0033] In one embodiment of the present invention, in S3, the reaction is first carried out at room temperature for 2 hours, and then heated to 40° C. for 2 hours.

[0034] In one embodiment of the present invention, in S3, the bromination reagent is any one or more of the following: N-bromosuccinimide, liquid bromine, tetrabromocycloketone, and more preferably liquid bromine.

[0035] In one embodiment of the present invention, in S3, the organic solvent is any one or more of the following: acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and more preferably acetonitrile.

[0036] In one embodiment of the present invention, S4 comprises the following process: the compound represented by formula (IV) undergoes an elimination reaction under the action of an alkaline substance to obtain a compound represented by formula (V);

[0037]

[0038] In one embodiment of the present invention, in S4, the molar ratio of the compound represented by formula (IV) to the alkaline substance is 1:(0.5-2), and more preferably 1:1.

[0039] In one embodiment of the present invention, in S4, the alkaline substance is any one or more of the following: sodium hydroxide, potassium hydroxide, potassium carbonate, and more preferably potassium hydroxide.

[0040] In one embodiment of the present invention, in S4, a basic substance, methanol, and a compound represented by formula (IV) are mixed and reacted.

[0041] In one embodiment of the present invention, in S4, the mass / volume ratio of the alkaline substance to methanol is 1:(15-30) in g / mL, and more preferably 1:24.

[0042] In one embodiment of the present invention, in S4, the reaction temperature is 50-60°C, more preferably 55°C.

[0043] In one embodiment of the present invention, in S4, the reaction time is 1 to 3 hours, more preferably 2 hours.

[0044] In one embodiment of the present invention, S5 comprises the following process: in an organic solvent, the compound represented by formula (V) undergoes a bromination reaction with liquid bromine to obtain a compound represented by formula (VI);

[0045]

[0046] In one embodiment of the present invention, in S5, the molar ratio of the compound represented by formula (V) to the bromination reagent is 1:(1-1.5), and more preferably 1:1.2.

[0047] In one embodiment of the present invention, in S5, the reaction is first carried out at room temperature for 2 hours, and then heated to 50° C. for 2 hours.

[0048] In one embodiment of the present invention, in S5, the bromination reagent is any one or more of the following: N-bromosuccinimide, liquid bromine, tetrabromocycloketone, and more preferably liquid bromine.

[0049] In one embodiment of the present invention, in S5, the organic solvent may specifically be carbon tetrachloride.

[0050] In one embodiment of the present invention, S6 comprises the following process: a compound represented by formula (VI) undergoes a substitution reaction with dihydroindole to obtain a target compound represented by formula I, namely, FSP1 inhibitor FSEN3.

[0051]

[0052] In one embodiment of the present invention, in S6, the molar ratio of the compound represented by formula (VI) to the indoline is 1:(1-1.5), and more preferably 1:1.2.

[0053] In one embodiment of the present invention, in S6, the reaction temperature is 40-60°C, more preferably 50°C.

[0054] In one embodiment of the present invention, in S6, the reaction time is 10 to 15 hours, more preferably 12 hours.

[0055] In one embodiment of the present invention, in S6, the reaction is carried out in a solvent, and the solvent is any one or more of tetrahydrofuran, acetonitrile, and diethyl ether, and acetonitrile is more preferably used.

[0056] In one embodiment of the present invention, the method specifically comprises the following synthetic reaction route:

[0057]

[0058] Beneficial effects:

[0059] The preparation method of the present invention has simple steps, does not require inert gas protection, has high safety, and has little pollution. The products do not need to be purified by column chromatography and can be obtained by recrystallization and beating. The product yield is high and is very suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the H NMR spectrum of the target product FSEN3.

[0061] Figure 2 This is the NMR carbon spectrum of the target product FSEN3. DETAILED DESCRIPTION

[0062] The present invention provides a method for synthesizing a FSP1 inhibitor FSEN3 compound, which is further explained below in conjunction with examples.

[0063] It should be noted that the embodiments of the present invention involve many compounds with complex structures. For the convenience of demonstration, the form of "compound + Roman numerals" is used to represent a certain compound in the explanation, for example: 3-(4-methoxyphenyl)-4-(prop-2-enyl)-5-thiodiphenyl-1H-1,2,4-triazole, which is compound III.

[0064] Example 1

[0065] The reaction equation for the synthesis of 6-(indoline-1-methylene)-3-(4-methoxyphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazole (FSEN3) is as follows:

[0066]

[0067] The specific steps for synthesizing FSEN3 are as follows:

[0068] (1) Synthesis of 3-(4-methoxyphenyl)-4-(prop-2-enyl)-5-thiolidene-1H-1,2,4-triazole (Compound III)

[0069] The reaction formula for this step is as follows:

[0070]

[0071] At room temperature, compound II (20.0 g, 120.5 mmol) was added to the reaction bottle, dissolved in anhydrous ethanol (120 mL), and allyl isothiocyanate (15.7 mL, 144.6 mmol) was added dropwise under stirring, and the reaction was refluxed for 3 h. After the reaction was completed, the solvent was dried, and the obtained solid was washed with ethyl acetate, filtered, dried, and used directly in the next step as a crude product.

[0072] The obtained crude product (14.0 g, 53 mmol) was placed in a flask, and a 2N NaOH ethanol solution (100 mL) was added. The mixture was refluxed at 120°C. After the reaction was completed, the reaction solution was cooled to room temperature, and concentrated hydrochloric acid was added dropwise until pH = 2. A white solid precipitated, which was filtered, dried, and recrystallized from anhydrous ethanol to obtain compound III (11.7 g, yield 90%), which was a white solid.

[0073] Characterization data: 1 H NMR (400MHz, CDCl3) δ11.63(s,1H),7.56(d,J=8.8Hz,2H),7.00(d,J=8.8Hz,2H),5.98(dd,J=17.2 ,10.5Hz,1H),5.31(d,J=11.2Hz,1H),5.09(d,J=17.3Hz,1H),4.70(d,J=4.9Hz,2H),3.87(s,3H). 13 C NMR(151MHz,DMSO-d6)δ167.3,161.0,151.3,131.9,130.0,118.2,117.1,114.4,55.4,45.9.HRMS(ESI)calculated for C 12 H 13 N3OS[M+H] + :248.0853,found 248.0853.

[0074] (2) Synthesis of 6-(bromomethyl)-3-(4-methoxyphenyl)-5,6-dihydro[1,3]thiazolo[2,3-c][1,2,4]triazole (Compound IV)

[0075] The reaction formula for this step is as follows:

[0076]

[0077] At room temperature, compound III (14.0 g, 57 mmol) was added to a flask and dissolved in acetonitrile (75 mL). A solution of bromine (3.5 mL, 68 mmol) in acetonitrile (10 mL) was added dropwise under stirring. The reaction solution was stirred at room temperature for 2 h, and then heated to 40 ° C for 2 h. After the reaction was completed, the solvent was concentrated to dryness, redissolved in water, and ammonia water was added dropwise to adjust the pH to 9. The mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude material was recrystallized with methanol to obtain a yellow solid, which was compound IV (13.7 g, yield 74%).

[0078] Characterization data: 1 H NMR(400MHz, CDCl3) δ7.69(d,J=8.8Hz,2H),7.01(d,J=8.9Hz,2H),4.73–4.64 (m,1H),4.52–4.38(m,2H),3.86(s,3H),3.80–3.71(m,1H),3.65–3.59(m,1H). 13 CNMR(101MHz,DMSO-d6)δ160.6,156.8,151.1,128.1,119.2,114.6,56.3,55.5,48.4,36.1.HRMS(ESI)calculated for C 12 H 12 BrN3OS[M+H] + :325.9958,found 325.9958.

[0079] (3) Synthesis of 3-(4-methoxyphenyl)-6-methylene-5,6-dihydro[1,3]thiazolo[2,3-c][1,2,4]triazole (Compound V) The reaction formula of this step is as follows:

[0080]

[0081] At room temperature, potassium hydroxide (1.03 g, 18.4 mmol) was placed in a reaction flask, dissolved in methanol (24.7 mL), and compound IV (6.0 g, 18.4 mmol) was added under stirring, and heated to 55°C for 2 h. After the reaction, the solvent was dried under vacuum, extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude material was slurried with methanol to obtain a yellow solid, which was compound V (3.33 g, yield 74%).

[0082] Characterization data: 1H NMR (400MHz, CDCl3) δ7.67(d,J=8.8Hz,2H),6.94(d,J=8.8Hz,2H),5.46(d,J=2.6Hz,1H),5.35(d,J=2.7Hz,1H),4.99(s,2H),3.82(s,3H). 13 C NMR(101MHz, CDCl3)δ161.0,156.6,151.6,142.3,127.7,118.8,114.4,109.8,55.3,50.4.HRMS(ESI)calculated for C 12 H 11 N3OS[M+H] + :246.0696,found 246.0693.

[0083] (4) Synthesis of 6-(bromomethyl)-3-(4-methoxyphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazole (Compound VI)

[0084] The reaction formula for this step is as follows:

[0085]

[0086] At room temperature, compound V (4.5 g, 18.3 mmol) was added to a flask and dissolved in carbon tetrachloride (15 mL). A solution of bromine (1.13 mL, 21.9 mmol) in carbon tetrachloride (3 mL) was added dropwise under stirring. The reaction solution was stirred at room temperature for 2 h, and then heated to 50 ° C for 2 h. After the reaction was completed, the solvent was concentrated, redissolved with water, and ammonia water was added dropwise to adjust the pH to 9. The mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude material was slurried with methanol to obtain a yellow solid, which was compound VI (5.38 g, yield 88%).

[0087] Characterization data: 1 H NMR (400MHz, DMSO-d6) δ8.51(s,1H),7.87(d,J=7.5Hz,2H),7.14(d,J=7.5Hz,2H),4.98(s,2H),3.85(s,3H). 13 C NMR(101MHz,DMSO-d6)δ160.7,156.4,146.7,133.0,128.1,118.7,117.5,114.7,55.5,26.5.HRMS(ESI)calculated for C 12 H 10 BrN3OS[M+H] +:323.9801,found 323.9801.

[0088] (5) Synthesis of 6-(indoline-1-methylene)-3-(4-methoxyphenyl)[1,3]thiazolo[2,3-c][1,2,4]triazole (FSEN3) (Compound Ⅰ)

[0089] The reaction formula for this step is as follows:

[0090]

[0091] At room temperature, compound VI (3.24 g, 10 mmol) was added to a flask, dissolved in acetonitrile (30 mL), and dihydroindole (2.29 mL, 20 mmol) was added dropwise under stirring. The reaction solution was stirred at 50°C for 12 h. After the reaction, the solvent was concentrated and recrystallized from methanol to obtain a yellow solid, which was the target product I (3.11 g, yield 96%).

[0092] Characterization data: 1 H NMR (600MHz, CDCl3) δ7.78(d,J=8.7Hz,2H),7.58(s,1H),7.15(d,J=7.3Hz,1H),7.11(t,J=7.6Hz,1H),7.05(d,J=8.8Hz, 2H), 6.78 (t, J=6.9Hz, 1H), 6.59 (d, J=7.8Hz, 1H), 4.35 (s, 2H), 3.88 (s, 3H), 3.41 (t, J=8.2Hz, 2H), 3.03 (t, J=8.2Hz, 2H). 13 C NMR (151MHz, CDCl3) δ161.0,157.2,151.0,147.0,135.6,130.1,128.1,127.4,124.9, 119.5,119.3,114.7,112.8,107.7,55.4,54.0,48.2,28.6.HRMS(ESI)calculatedfor C 20 H 18 N4OS[M+H] + :363.1275, found 363.1273.

[0093] The above is an exemplary description of the implementation methods of the technical solution of the present disclosure. It should be understood that the protection scope of the present disclosure is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present disclosure shall be included in the protection scope of the claims of this application.

Claims

1. A method for preparing a FSP1 inhibitor FSEN3, characterized in that: The steps include: S1, using 4-methoxybenzoic acid hydrazide represented by formula (II) and allyl isocyanate sulfate as raw materials, through nucleophilic addition reaction, to form a thiourea intermediate represented by formula (IIIa); S2, the obtained thiourea intermediate undergoes a cyclization reaction under the action of a base to obtain a compound represented by formula (III); S3, the compound represented by formula (III) is reacted with liquid bromine to obtain the compound represented by formula (IV); S4. The compound represented by formula (IV) undergoes elimination reaction in an alkaline environment to obtain a compound represented by formula (V); S5, the compound represented by formula (V) undergoes bromination reaction with liquid bromine to obtain a compound represented by formula (VI); S6. The compound represented by formula (VI) undergoes a substitution reaction with dihydroindole to obtain the target compound represented by formula (I), which is a FSP1 inhibitor FSEN3; 2. The method according to claim 1, characterized in that S1 comprises the following process: in an organic solvent, the compound represented by formula II reacts with allyl isothiocyanate to form the intermediate compound (IIIa) represented by formula II; The molar ratio of the compound represented by formula (II) to allyl isothiocyanate is 1:(1-1.5), and the organic solvent is any one or more of the following: tetrahydrofuran, dichloromethane, dioxane, ethanol; the reaction temperature is 80-100°C, and the reaction time is 2-4h.

3. The method according to claim 1, characterized in that S2 comprises the following process: the compound represented by formula (IIIa) is cyclized under the action of an alkaline substance to obtain a compound represented by formula (III); The molar ratio of the compound represented by formula (IIIa) to the alkaline substance is 1:(10-30), and the alkaline substance is any one or more of the following: potassium carbonate, sodium hydroxide, potassium tert-butoxide; the reaction temperature is 110-130° C., and the reaction time is 3-5 hours.

4. The method according to claim 1, characterized in that S3 comprises the following process: in an organic solvent, the compound represented by formula (III) undergoes a bromination reaction with liquid bromine to obtain a compound represented by formula (IV); The molar ratio of the compound represented by the formula (III) to the bromination reagent is 1:(1-1.5); the bromination reagent is any one or more of the following: N-bromosuccinimide, liquid bromine, tetrabromocycloketone; the organic solvent is any one or more of the following: acetonitrile, tetrahydrofuran, N,N-dimethylformamide.

5. The method according to claim 1, characterized in that In S3, the reaction was first carried out at room temperature for 2 h and then heated to 40° C. for 2 h.

6. The method according to claim 1, characterized in that S4 comprises the following process: the compound represented by formula (IV) undergoes elimination reaction under the action of an alkaline substance to obtain a compound represented by formula (V); The molar ratio of the compound represented by the formula (IV) to the alkaline substance is 1:(0.5-2); the alkaline substance is any one or more of the following: sodium hydroxide, potassium hydroxide, potassium carbonate; the reaction temperature is 50-60° C., and the reaction time is 1-3 hours.

7. The method according to claim 1, characterized in that In S4, specifically, an alkaline substance, methanol, and a compound represented by formula (IV) are mixed for reaction; in terms of g / mL, the mass / volume ratio of the alkaline substance to methanol is 1:(15-30).

8. The method according to claim 1, characterized in that S5 comprises the following process: in an organic solvent, the compound represented by formula (V) undergoes a bromination reaction with liquid bromine to obtain a compound represented by formula (VI); The molar ratio of the compound represented by formula (V) to the bromination reagent is 1:(1-1.5); the reaction is first carried out at room temperature for 2 hours, and then heated to 50° C. for 2 hours; the bromination reagent is any one or more of the following: N-bromosuccinimide, liquid bromine, tetrabromocycloketone; the organic solvent can be carbon tetrachloride.

9. The method according to claim 1, characterized in that: S6 comprises the following process: a compound represented by formula (VI) undergoes a substitution reaction with dihydroindole to obtain a target compound FSEN3 represented by formula I; The molar ratio of the compound represented by the formula (VI) to the indoline is 1:(1-1.5); the reaction is carried out in a solvent, which is any one or more of tetrahydrofuran, acetonitrile and diethyl ether.

10. The method according to any one of claims 1 to 9, characterized in that: In S6, the reaction temperature is 40-60° C., and the reaction time is 10-15 h.

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