Use of a bromobenzamide compound in antitumor drugs

Abstract

The application provides application of a bromobenzamide compound in an antitumor drug, and the bromobenzamide compound is applied in preparation of a drug for inhibiting the activity of fatty acid synthetase FAS-I. The bromobenzamide compound is a small molecule inhibitor of the dehydrase domain DH in the fatty acid synthetase FAS-I. The bromobenzamide compound provided by the application can be applied in preparation of a drug for inhibiting the activity of the fatty acid synthetase FAS-I, and the difficulty and complexity of treatment of tumor diseases such as breast cancer and prostate cancer are reduced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a bromobenzamide compound in antitumor drugs. Background Technology

[0002] Fatty acids are essential substances for life activities and play a key role in physiological processes such as energy storage. Fatty acid synthesis depends on fatty acid synthase (FAS). In mammals, type I fatty acid synthase (FAS-I) is concentrated in the liver and adipose tissue, synthesizing fatty acids from acetyl-CoA and maintaining energy metabolism balance under the regulation of nutrition and hormones. However, in the tumor microenvironment, metabolic reprogramming of cancer cells leads to a sustained increase in FAS-I expression, providing a sufficient material basis for the proliferation, invasion, and metastasis of cancer cells, increasing the complexity and difficulty of cancer treatment. FAS-I is a multifunctional homodimer with a molecular weight of 270 kDa. Its catalytic domains are precisely arranged in space, and the fatty acid biosynthesis process is relatively conserved. The process is divided into two stages: initial synthesis and elongation cycle. In the initial synthesis stage, the bifunctional malonyl-acetyltransferase domain MAT sequentially loads the terminal acyl groups of acetyl-CoA and malonyl-CoA onto the terminal thiol group of the Ppant arm of ACP, causing them to undergo transacyl and decarboxylation condensation reactions with the N-terminal ketoacyl synthase domain KS to generate the initial β-ketoacyl-ACP. In the elongation cycle stage, the ketoacyl reductase domain KR, the dehydratase domain DH, and the olefin reductase domain ER sequentially complete the reduction, dehydration, and re-reduction reactions to produce a saturated acyl product that has been extended by two carbon units. The product serves as the starting substrate for the next round of elongation, continuing the four-step cycle of "condensation-reduction-dehydration-re-reduction" until the continuously growing fatty acid chain reaches a length of 16 to 18 carbon atoms. The product is then released from ACP as a free fatty acid by the C-terminal thioesterase domain TE. While this integrated design sacrifices product diversity, it significantly improves catalytic efficiency. In addition, there are two non-enzymatic domains on the periphery of FAS-I. The first non-enzymatic domain is a pseudomethyltransferase (ΨME) homologous to methyltransferase. The second non-enzymatic domain is a truncated KR fold that dimers with the KR catalytic domain and is called pseudoketone reductase (ΨKR), which together participate in maintaining the complete function of the enzyme.

[0003] Clinical studies have shown that dysregulation of FAS-I expression is closely related to the occurrence of various human diseases. In metabolic diseases, abnormal expression can lead to non-alcoholic fatty liver disease, obesity, and the onset and exacerbation of type II diabetes. In the field of oncology, overexpression of FAS-I provides the fatty acid material basis for the proliferation, invasion, and metastasis of various malignant tumors such as breast cancer and prostate cancer, and is a significant metabolic characteristic that distinguishes tumor cells from normal cells. Chinese invention patent CN113041243B discloses a drug for inhibiting fatty acid synthase, which inhibits the activity of fatty acid synthase, but does not involve the inhibition of the activity of fatty acid synthase FAS-I.

[0004] Therefore, developing an anti-tumor drug targeting overexpressed FAS-I is a major technical problem that needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application of a bromobenzamide compound in an antitumor drug, wherein the bromobenzamide compound has the structural formula shown in Formula 1:

[0006]

[0007] The CAS number of the bromobenzamide compound is 954683-79-5.

[0008] In one embodiment, the bromobenzamide compound is used in the preparation of a medicament that inhibits the activity of fatty acid synthase FAS-I.

[0009] In one embodiment, the bromobenzamide compound is a small molecule inhibitor targeting fatty acid synthase FAS-I.

[0010] In one embodiment, the bromobenzamide compound is a small molecule inhibitor that targets the dehydratase domain DH in fatty acid synthase FAS-I.

[0011] In comparative experiments on the various domains of fatty acid synthase FAS-I, this application revealed a significant structural difference between the dehydratase domain DH, located at the central core of the "X"-shaped fatty acid synthase FAS-I, and its corresponding dehydratase FabA / FabZ in FAS-II. The inventors discovered that DH may possess a unique substrate catalytic recognition mechanism, which could provide new directions for the development of novel antitumor drugs, particularly regarding the physiological functions and pathogenic mechanisms of fatty acid synthase FAS-I. The DH domain of fatty acid synthase FAS-I plays a crucial role in the extended fatty acid synthesis cycle. Its structure and catalytic mechanism differ from the dehydratases in the prokaryotic FAS-II system, and its study contributes to the development of novel antitumor drugs.

[0012] During the experiment, the inventors discovered that the dehydratase domain DH is the core dehydratase domain of fatty acid synthase FAS-I, playing a crucial pivotal role in the extended fatty acid synthesis cycle. It catalyzes the dehydration reaction of β-hydroxyacyl-ACP from the acyl reductase domain KR to generate β-enoyl-ACP with a trans-unsaturated double bond, providing a substrate for the downstream enoyl reductase domain ER. This unique molecular mechanism, distinguishing it from FAS-II isoenzymes, is significant for understanding FAS-I and developing highly selective antitumor drugs. Structurally... In mammalian FAS-I dimers, the monomers contain fatty acid synthesis functional modules arranged in the order KS-MAT-DH-ψME-ψKR-ER-KR-ACP-TE. The dehydratase domain DH is located at the center, dynamically linked by DH1 (amino acids 858-960) and DH2 (amino acids 987-1102) through a flexible linker region (amino acids 961-986), forming a pseudo-dimer containing an "L"-shaped catalytic active pocket. This pseudo-dimer can synergistically catalyze the synthesis of long-chain saturated fatty acids with adjacent KR, ER, and KS domains. In prokaryotic FAS-II systems, FabA and FabZ exist as independent homodimers and hexamers composed of three homodimers, respectively. The monomers have a β+α "hot dog" structure, i.e., six antiparallel β-sheets enclosing a central α-helix. The two monomers interact to form two 180° centrosymmetric "L"-shaped catalytic active pockets.

[0013] To facilitate understanding of the technical solutions provided in this application, a schematic diagram of the fatty acid synthase FAS-I is shown below. Figure 1 The amino acid sequence diagram of the human dehydratase domain DH is shown below. Figure 2 .

[0014] Figure 1 Medium-dark yellow represents KS, light yellow represents MAT, red represents DH, green represents ΨME, light blue represents KR and ΨKR, dark blue represents ER, gray represents ACP, and purple represents TE.

[0015] Figure 2 DH1 and DH2 are the two functional subdomains that make up DH, and the connection region between DH1 and DH2 is marked in cyan.

[0016] During the experiment, the inventors discovered that in the critical extended cycle stage of fatty acid synthesis, both the dehydratase domain DH of fatty acid synthase FAS-I and the dehydratase domains FabZ / FabA of FAS-II undertake the catalytic task of the third-step dehydration reaction. By recognizing the β-hydroxyacyl-ACP produced by the upstream enzyme, they dehydrate the β-hydroxy group to generate a trans double bond, yielding trans-2-acyl-ACP. Among them, the dehydratase domains DH and FabZ have only a single dehydration function, while FabA, in addition to dehydration, can also catalyze the isomerization reaction of trans-2-acyl-ACP to cis-3-acyl-ACP, introducing cis-3-acyl-ACP into the synthesis of unsaturated fatty acids. The monofunctional FabZ prefers substrates with carbon chain lengths greater than 12C. When the substrate ACP is bound, its "L"-shaped catalytic active pocket is compressed into a "Y" shape due to structural changes, which can accommodate substrates of different lengths and assist in dehydration with the help of His and Glu amino acids from different monomers. The bifunctional FabA prefers substrates with carbon chain lengths less than 12C. The binding of ACP does not change the "L"-shaped pocket. The key catalytic amino acids from different monomers in its pocket become His and Asp. The Asp can deprotonate the C4 of trans-2-acyl-ACP, converting the trans-C2-C3 double bond into a cis-C3-C4 double bond.

[0017] To improve the effectiveness of bromobenzamide compounds in the application of antitumor drugs, the inventors discovered during experiments that the dehydratase domain DH of fatty acid synthase FAS-I plays an important role in the extended fatty acid synthesis cycle. The structure and catalytic mechanism of DH are different from those of the dehydratase in the prokaryotic FAS-II system. Inhibiting the activity of the dehydratase domain DH can improve the effectiveness of antitumor drugs.

[0018] In one embodiment, the process of inhibiting the activity of the dehydratase domain DH in the targeted fatty acid synthase FAS-I includes the following steps:

[0019] S1. Protein purification: Purification of the protein containing the dehydratase domain DH in fatty acid synthase FAS-I;

[0020] S2. Establishment of enzyme activity system: Establish a reversible dehydration-hydration reaction system for the dehydratase domain DH.

[0021] S3. Drug application: Bromobenzamide compounds are added to the enzyme activation system to inhibit the activity of the dehydratase domain DH in the target fatty acid synthase FAS-I.

[0022] In one embodiment, in S1, the protein containing the dehydratase domain DH of fatty acid synthase FAS-I is purified by column affinity chromatography.

[0023] In one embodiment, the protein purification process in S1 includes purifying the protein containing the dehydrase domain DH by Ni column affinity chromatography and Q column ion exchange chromatography.

[0024] In one embodiment, the protein purification process in S1 further includes size exclusion chromatography to purify the protein containing the dehydratase domain DH.

[0025] In one embodiment, the protein purification process in S1 includes purifying the protein containing the dehydrase domain DH by Ni column affinity chromatography, Q column ion exchange chromatography, and size exclusion chromatography.

[0026] In one embodiment, in step S3, a test solution with a molar concentration of 1-150 μM prepared from bromobenzamide compound is added to the enzyme activity system.

[0027] In one embodiment, the molar concentration of the bromobenzamide compound in the test solution is 3-100 μM.

[0028] During the experiment, the inventors discovered that a bromobenzamide compound can inhibit the activity of the dehydratase domain DH in fatty acid synthase FAS-I. By purifying the dehydratase domain DH, establishing an enzyme activation system, and adding the bromobenzamide compound to the enzyme activation system, the activity and thermal stability of the targeted fatty acid synthase FAS-I dehydratase domain DH can be inhibited.

[0029] Beneficial effects

[0030] 1. The protein purification process provided in this application can better purify proteins containing the dehydratase domain DH, providing purification conditions for verifying the inhibitory effect of the inhibitor on the dehydratase domain DH.

[0031] 2. The bromobenzamide compound provided in this application can be used to inhibit the activity of the dehydratase domain DH in fatty acid synthase FAS-I.

[0032] 3. This application, through protein purification of the dehydratase domain DH, establishment of an enzyme activation system, and the addition of bromobenzamide compounds to the enzyme activation system, can inhibit the activity and thermal stability of the dehydratase domain DH in the targeted fatty acid synthase FAS-I.

[0033] 4. By verifying the inhibitory effect on the dehydratase domain DH in the targeted fatty acid synthase FAS-I, this application enables the application of bromobenzamide compounds in the field of antitumor drugs, and demonstrates a good inhibitory effect on the dehydratase domain DH.

[0034] 5. The bromobenzamide compound provided in this application can be used in the preparation of drugs that inhibit the activity of fatty acid synthase FAS-I, thereby reducing the difficulty and complexity of treating tumors such as breast cancer and prostate cancer. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of fatty acid synthase FAS-I.

[0036] Figure 2 This is a schematic diagram of the amino acid sequence of the DH domain of human dehydratase.

[0037] Figure 3 This is a schematic diagram of the process of establishing an enzyme activation system.

[0038] Figure 4 The purification process and detection results of the dehydratase domain DH in fatty acid synthase FAS-I were obtained by Ni column affinity chromatography and Q column ion exchange chromatography.

[0039] Figure 5 This paper describes the protein purification process and detection results of the dehydratase domain DH in fatty acid synthase FAS-I using size exclusion chromatography.

[0040] Figure 6 The results are from the inhibition test of the dehydratase domain DH.

[0041] Figure 7 The results show the thermal stability of the dehydratase domain DH.

[0042] Figure 1 Medium-dark yellow represents KS, light yellow represents MAT, red represents DH, green represents ΨME, light blue represents KR and ΨKR, dark blue represents ER, gray represents ACP, and purple represents TE.

[0043] Figure 2 DH1 and DH2 are the two functional subdomains that make up DH, and the connection region between DH1 and DH2 is marked in cyan.

[0044] Figure 3 In the diagram, 'a' represents the reversible dehydration-hydration reaction of the dehydratase domain DH.

[0045] Figure 3 In Figure b, the dehydrase domain DH shows the preference screening results for different lengths of enoyl substrates carried by CoA.

[0046] Figure 4 In the figure, 'a' represents the Ni column affinity chromatography process for the dehydratase domain DH.

[0047] Figure 4Figure b shows the purity determination result of the dehydratase domain DH after purification by Ni column affinity chromatography using 13% SDS-PAGE.

[0048] Figure 4 In the diagram, c represents the Q-column ion exchange chromatography process of the dehydrase domain DH.

[0049] Figure 4 In the figure, d represents the 13% SDS-PAGE result of the dehydratase domain DH after purification by Q-column ion exchange chromatography.

[0050] Figure 5 In the diagram, 'a' represents the size exclusion chromatography process of the dehydratase domain DH.

[0051] Figure 5 In Figure b, the purity of the dehydratase domain DH after purification by size exclusion chromatography is determined by 13% SDS-PAGE. Detailed Implementation

[0052] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments of this application. Moreover, in the following embodiments, the description of each embodiment has its own emphasis, and for parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0053] Example 1

[0054] This embodiment provides an application of a bromobenzamide compound in an antitumor drug, the structural formula of which is shown in Formula 1:

[0055]

[0056] The CAS number of the bromobenzamide compound is 954683-79-5.

[0057] The bromobenzamide compound is used in the preparation of drugs that inhibit the activity of fatty acid synthase FAS-I.

[0058] The bromobenzamide compound is a small molecule inhibitor that targets the dehydratase domain DH in fatty acid synthase FAS-I.

[0059] The inhibition process of the dehydratase domain DH in the targeted fatty acid synthase FAS-I includes the following steps:

[0060] S1. Protein purification: Purification of proteins containing the dehydratase domain DH by column affinity chromatography;

[0061] S2. Establishment of enzyme activity system: Establish a reversible dehydration-hydration reaction system for the dehydratase domain DH.

[0062] S3. Drug application: Bromobenzamide compounds are added to the enzyme activation system to inhibit the activity of the dehydratase domain DH in the target fatty acid synthase FAS-I.

[0063] The protein purification process in S1 includes the purification of proteins with the dehydrase domain DH by Ni column affinity chromatography, Q column ion exchange chromatography, and size exclusion chromatography.

[0064] This application provides a schematic diagram of fatty acid synthase FAS-I (see attached diagram). Figure 1 A schematic diagram of the amino acid sequence of the human dehydratase domain DH is provided. Figure 2 .

[0065] A schematic diagram of the enzyme activation system establishment process is shown below. Figure 3 .

[0066] Figure 3 In Figure 'a', we see a schematic diagram of the reversible dehydration-hydration reaction of the dehydratase domain DH; the gray shaded area is labeled with the Ppant arm, and the location where the acyl base reacts on the ACP module is marked in red.

[0067] Figure 3 Figure b shows the preference of the dehydratase domain DH for different lengths of enoyl substrates carried by CoA. It can be seen from b that different carbon chain lengths (4C, 6C, 8C, 10C, 12C) in enoyl-CoA affect the relative activity of the dehydratase domain DH. As the carbon chain length increases, the relative activity of the dehydratase domain DH generally shows a decreasing trend.

[0068] Performance testing

[0069] 1. Protein expression assay:

[0070] The dehydratase domain DH of fatty acid synthase FAS-I was purified by Ni-column affinity chromatography, Q-column ion exchange chromatography, and size exclusion chromatography. The protein expression test results are shown below. Figure 4 and Figure 5 .

[0071] The purification process and detection results of the Ni-column affinity chromatography and Q-column ion exchange chromatography for the dehydratase domain DH in fatty acid synthase FAS-I are shown in the figure below. Figure 4 .

[0072] The protein purification process and detection results of the dehydratase domain DH in fatty acid synthase FAS-I by size exclusion chromatography are shown below. Figure 5 .

[0073] The protein was purified by Ni column affinity chromatography using the dehydratase domain DH of fatty acid synthase FAS-I, and the absorbance of the protein was detected at a wavelength of 280 nm.

[0074] After purifying the dehydratase domain DH of fatty acid synthase FAS-I by size exclusion chromatography, the elution status of the protein was monitored by detecting the absorbance at a wavelength of 280 nm (UV280).

[0075] pass Figure 4 As can be seen in Figure a, the signal peak of the target protein with the dehydratase domain DH eluted by the Ni column gradient indicates that the target protein was eluted in this elution stage.

[0076] pass Figure 4 In diagram b, we can see that ① represents the portion of the dehydratase domain DH flowing through the Ni column after sample loading, i.e., the portion that flows out directly without binding to the Ni column; ② represents the fraction eluted from the Ni column gradient, which is rich in the target protein DH (in the form of SUMO-DH fusion protein); ③ represents the protein in the SUMO-DH fusion state before ULP1 digestion; ④ represents the SUMO tag separating from the DH protein after overnight ULP1 digestion, showing separate bands for DH and SUMO, thus obtaining the digestion effect and protein purity.

[0077] pass Figure 4 As can be seen from Figure c, peaks ⑥ and ⑦ represent signal peaks of the target protein with the dehydratase domain DH eluted through different gradients of the Q column, showing the elution status of the target protein under different elution conditions.

[0078] pass Figure 4 As shown in 'd', ⑤ represents the protein composition before loading onto the Q column, allowing for comparison of the differences in protein composition before and after loading. ⑥ and ⑦ represent the fractions eluted from different gradients on the Q column, demonstrating that the target protein DH and impurity proteins were separated, indicating good purification performance.

[0079] pass Figure 5 As can be seen from Figure a, the purity of the dehydratase domain DH protein is good, and high-purity dehydratase domain DH protein can be obtained. Through... Figure 5 As shown in Figure b, the dehydratase domain DH protein purified by size exclusion chromatography is singular, with a clear DH band and no obvious impurities, indicating that the dehydratase domain DH protein was further purified by size exclusion chromatography with fewer impurities.

[0080] 2. Inhibition test of the dehydratase domain DH:

[0081] The bromobenzamide compound provided in Example 1 was used to perform an inhibitory test on the dehydratase domain DH in the targeted fatty acid synthase FAS-I. The results of the half-inhibitory concentration (IC50) test are shown below. Figure 6 .

[0082] pass Figure 6 It can be seen that when the molar concentration of the bromobenzamide compound is 5.76 μM, the activity of the dehydratase domain DH in the targeted fatty acid synthase FAS-I can be inhibited by 50%; the bromobenzamide compound has a good inhibitory effect on the activity of the dehydratase domain DH in the targeted fatty acid synthase FAS-I.

[0083] 3. Thermal stability test of the dehydratase domain DH:

[0084] The bromobenzamide compound provided in Example 1 was added to dimethyl sulfoxide to prepare test solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively. Dimethyl sulfoxide was used as a control group. Both the test solutions and dimethyl sulfoxide were added to the dehydratase domain DH of fatty acid synthase FAS-I, and the temperature was increased from 40 °C to 65 °C. The thermostability of the dehydratase domain DH was tested, and the results are shown in [Figure 1]. Figure 7 .

[0085] pass Figure 7 It can be seen that as the temperature increases, the fluorescence intensity of the dehydratase domain DH first increases and then decreases with temperature change; the peak positions and shapes of the curves with different concentrations of test solution are different from those of dimethyl sulfoxide; indicating that bromobenzamide compounds can affect the thermal stability of the dehydratase domain DH in fatty acid synthase FAS-I, and different concentrations of bromobenzamide compounds all affect the thermal stability of the dehydratase domain DH; compared with dimethyl sulfoxide, bromobenzamide compounds can improve the thermal stability of the dehydratase domain DH.

[0086] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0087] 1. The protein purification process provided in this application can better purify proteins containing the dehydratase domain DH, providing purification conditions for verifying the inhibitory effect of the inhibitor on the dehydratase domain DH.

[0088] 2. The bromobenzamide compound provided in this application can be used to inhibit the activity of the dehydratase domain DH in fatty acid synthase FAS-I.

[0089] 3. This application, through protein purification of the dehydratase domain DH, establishment of an enzyme activation system, and the addition of bromobenzamide compounds to the enzyme activation system, can inhibit the activity and thermal stability of the dehydratase domain DH in the targeted fatty acid synthase FAS-I.

[0090] 4. By verifying the inhibitory effect on the dehydratase domain DH in the targeted fatty acid synthase FAS-I, this application enables the application of bromobenzamide compounds in the field of antitumor drugs, and demonstrates a good inhibitory effect on the dehydratase domain DH.

[0091] 5. The bromobenzamide compound provided in this application can be used in the preparation of drugs that inhibit the activity of fatty acid synthase FAS-I, thereby reducing the difficulty and complexity of treating tumors such as breast cancer and prostate cancer.

[0092] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims. Furthermore, specific examples have been used in the specification to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application, and the content of this specification should not be construed as a limitation of this application.

Claims

1. The use of a bromobenzamide compound in an antitumor drug, characterized in that, The structural formula of the bromobenzamide compound is shown in Formula 1:

2. The use of the bromobenzamide compound as described in claim 1 in antitumor drugs, characterized in that, The bromobenzamide compound is used in the preparation of drugs that inhibit the activity of fatty acid synthase FAS-I.

3. The use of the bromobenzamide compound as described in claim 2 in antitumor drugs, characterized in that, The bromobenzamide compound is a small molecule inhibitor of fatty acid synthase FAS-I.

4. The use of the bromobenzamide compound as described in claim 3 in antitumor drugs, characterized in that, The bromobenzamide compound is a small molecule inhibitor of the dehydratase domain DH in fatty acid synthase FAS-I.

5. The use of the bromobenzamide compound as described in claim 4 in an antitumor drug, characterized in that, The inhibition process of the dehydratase domain DH in the fatty acid synthase FAS-I includes the following steps: S1. Protein purification: Purification of the protein containing the dehydratase domain DH in fatty acid synthase FAS-I. S2. Establishment of enzyme activity system: Establish a reversible dehydration-hydration reaction system for the dehydratase domain DH. S3. Drug application: Bromobenzamide compounds are added to the enzyme activation system to inhibit the activity of the dehydratase domain DH in the target fatty acid synthase FAS-I.

6. The use of the bromobenzamide compound as described in claim 5 in an antitumor drug, characterized in that, In S1, the protein containing the dehydratase domain DH of fatty acid synthase FAS-I is purified by column affinity chromatography.

7. The use of the bromobenzamide compound as described in claim 6 in an antitumor drug, characterized in that, The protein purification process in S1 includes purifying the protein containing the dehydratase domain DH by Ni column affinity chromatography and Q column ion exchange chromatography.

8. The use of the bromobenzamide compound as described in claim 7 in an antitumor drug, characterized in that, The protein purification process in S1 also includes size exclusion chromatography to purify proteins with the dehydratase domain DH.

9. The use of the bromobenzamide compound as described in claim 5 in an antitumor drug, characterized in that, In step S3, a test solution with a molar concentration of 1-150 μM prepared from bromobenzamide compound is added to the enzyme activity system.

10. The use of the bromobenzamide compound as described in claim 9 in an antitumor drug, characterized in that, The molar concentration of bromobenzamide compounds in the test solution is 3-100 μM.

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