Btsa1 derivatives, methods for preparing the same, and uses thereof in the preparation of medicaments for treating a broad spectrum of solid tumors

Abstract

The application discloses a kind of BTSA1 derivatives and preparation method and application in preparation treatment broad-spectrum solid tumor drug thereof.The BTSA1 derivative is prepared by method one and method two, method one includes the following steps: amino thiourea is reacted with reagent I, to obtain intermediate I;Reagent III is reacted with intermediate II, to obtain intermediate III;Intermediate I is reacted with intermediate III, to obtain the BTSA1 derivative;Method two includes the following steps: amino thiourea is reacted with reagent I, to obtain intermediate I, intermediate I is reacted with reagent III, to obtain intermediate XI, intermediate XI is reacted with intermediate II, to obtain the BTSA1 derivative.The BTSA1 derivative shows strong anti-proliferation activity to a variety of solid tumors, with broad-spectrum anti-tumor characteristics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of BTSA1 derivatives, their preparation methods, and their application in the preparation of drugs for treating broad-spectrum solid tumors. Background Technology

[0002] Apoptosis plays a crucial role in the evolution of organisms, the stability of the internal environment, and the development of multiple systems. Bax proteins are among the key executors of apoptosis. In normal cells, Bax exists primarily as inactive monomers in the cytoplasm, in a dormant state. When cells are stimulated by DNA damage, hypoxia, or other factors, BH3 domain proteins (such as BIM and BID), acting as Bax activators, bind to the N-terminal trigger site of Bax, initiating conformational activation and causing Bax to transform from a dormant monomer into a toxic oligomer. This oligomer increases mitochondrial outer membrane permeability (MOMP), releasing mitochondrial proteins (such as cytochrome c, SMAC, and OMI), activating the caspase cascade, and thus initiating apoptosis.

[0003] However, in cancer cells, Bax is usually silenced, conformationally inactivated, retained in the cytoplasm and unable to translocate, or loses its function due to gene mutation, leading to cancer cells resisting apoptosis, proliferating indefinitely, and developing drug resistance.

[0004] Currently, clinical anticancer strategies primarily involve indirectly activating Bax by inhibiting Bcl-2 anti-apoptotic proteins. Venetoclax, the first novel apoptosis inducer and a Bcl-2 inhibitor, acts as a BH3 mimic, activating Bax by replacing proteins containing only the BH3 domain, thereby inducing apoptosis. However, this inducer has limitations, including limited therapeutic activity and resistance mechanisms associated with aberrant expression of other Bcl-2 anti-apoptotic proteins. Studies have shown that the vast majority of cancer cells (such as lung cancer, colon cancer, and breast cancer) contain inactive forms of Bax or Bax whose activity is inhibited by anti-apoptotic proteins. Therefore, directly activating Bax holds promise as a novel cancer treatment strategy and may overcome the limitations of existing BH3 mimics, possessing the potential to develop broad-spectrum anticancer drugs.

[0005] Researchers have identified several binding sites controlling Bax activation, including the trigger site S184 and the classical site, and have developed small-molecule Bax direct activators for cancer therapy. Gavathiotis et al. (Nature. 2008 October 23; 455(7216): 1076–1081. doi:10.1038 / nature07396) discovered the first gain-of-function direct Bax activator, BAM7 (IC50 = 3.31 μM), using a computer screening strategy. BAM7 selectively targets the trigger site and induces Bax conformational activation. Building on this, researchers further identified Bax activators containing a pyrazolone core structure, such as BTC-8 (Ki = 0.8 μM) and BTSA1 (a Bax trigger site activator with an IC50 of 3.31 μM). 50 = 247±30 nM). In addition, researchers have discovered that SMBA1 and its analogues, which specifically target the S184 site, can directly activate Bax and induce apoptosis in lung cancer cells. They have also identified a classic Bax activator through virtual screening, which can induce Bax-dependent apoptosis in lung cancer cells by promoting Bax insertion into the mitochondrial membrane.

[0006] However, no Bax activators have yet entered clinical trials. Existing research indicates that trigger site activators can mimic the basic characteristics of physiological BH3 domain proteins and induce all steps in the Bax activation pathway, suggesting that trigger sites may be the most promising drug binding sites for developing Bax activators. Among existing preclinical lead compounds, BTSA1 exhibits strong Bax functional activation ability with minimal toxicity, making it one of the most effective chemical tools in pharmacological research; its structural formula is shown below. However, BTSA1 has weak anti-proliferative activity against solid tumors, and its metabolite, aminothiazole, may lead to increased toxicity, limiting its further clinical application.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a class of BTSA1 derivatives, their preparation method and their application in the preparation of drugs for treating broad-spectrum solid tumors. The derivatives exhibit strong anti-proliferative activity against a variety of solid tumor cells and have broad-spectrum anti-tumor properties.

[0009] To address the above technical problems, this invention discloses a class of BTSA1 derivatives, the general structural formula of which is as follows:

[0010]

[0011] R1 is selected from one of hydrogen, hydroxyl, nitro, amino, acetoxy, halogen, unsubstituted or heteroatom-substituted hydrocarbon groups; the hydrocarbon group is a C1 to C25 saturated or unsaturated hydrocarbon group with or without a cyclic structure, the substituent in the substituted hydrocarbon group is a heteroatom or halogen, the heteroatom is O, N, S or P; the substitution position of R1 is any carbon atom on the benzene ring except for the carbon atom connected to the thiazole ring.

[0012] R2 is selected from hydrogen, heteroatoms, halogens, and alkyl groups; the heteroatoms are O, N, S, or P; the alkyl groups are C1 to C10 alkyl groups with or without cyclic structures; the substitution position of R2 is any carbon atom on the benzene ring except for the carbon atom attached to the pyrazolone.

[0013] R3 is selected from hydrogen, unsubstituted or substituted hydrocarbon groups; the hydrocarbon group is a C4 to C7 saturated or unsaturated hydrocarbon group with or without a cyclic structure, and the substituent in the substituted hydrocarbon group is a heteroatom, halogen or nitro group, and the heteroatom is O, N, S or P; when R3 is selected from hydrogen, no additional substituent is introduced on the thiazole ring in which it is located, and the parent structure of the thiazole ring is maintained; when R3 is selected from unsubstituted or substituted hydrocarbon groups, it is connected to the two carbon atoms indicated by the dashed line on the thiazole ring to form a cyclic structure.

[0014] R1, R2, and R3 are not all hydrogen.

[0015] The present invention provides a series of BTSA1 derivatives obtained based on BTSA1 through structure-activity relationships and prepared using appropriate methods.

[0016] This invention also discloses a method for preparing the aforementioned BTSA1 derivative.

[0017] Method 1, as shown in equation (I), includes:

[0018] The aminothiourea was reacted with reagent I to obtain intermediate I; reagent III was reacted with intermediate II to obtain intermediate III; intermediate I was reacted with intermediate III to obtain the BTSA1 derivative.

[0019]

[0020] (I)

[0021] Or, as shown in method (II), it includes:

[0022] The aminothiourea was reacted with reagent I to obtain intermediate I. Intermediate I was reacted with reagent III to obtain intermediate XI. Intermediate XI was reacted with intermediate II to obtain the BTSA1 derivative.

[0023]

[0024] (II)

[0025] When R1 in the BTSA1 derivative obtained by method one or method two is a hydroxyl group, the BTSA1 derivative is subjected to alkylation and esterification reactions with R4-X or R4-COOH, respectively, to obtain further substituted BTSA1 derivatives, as shown in formulas (III) and (IV); wherein R4 is selected from C1 to C20 alkyl groups and their substituted derivatives; wherein the substituents of the alkyl substituted derivatives are heteroatoms, and the heteroatoms are O, N, S or P; wherein X is a halogen;

[0026] .

[0027] Furthermore, Method 1 specifically includes the following steps:

[0028] (1) At room temperature, aminothiourea, reagent I, acetonitrile and sodium acetate are mixed and stirred to react; after the reaction is completed, the mixture is filtered, the solid is collected, washed successively with cold acetonitrile, deionized water and cold ethanol, and dried at 50-60℃ under vacuum for 4-6 hours to obtain intermediate I;

[0029] (2) At room temperature, reagent III was added to chemically pure ethanol and stirred until the solution was clear. Sodium acetate was then added, and the pH of the system was adjusted to 7-8 with sodium carbonate solution. The system was then cooled to 0-10°C, and intermediate II diazonium salt solution was added (under alkaline conditions, intermediate II diazonium salt is converted to intermediate II in situ, and then intermediate II reacts with reagent III) to suppress side reactions. After the addition was completed, the system was heated to 30-35°C and stirred at this temperature for 8-10 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the pH of the system was adjusted to 6.5-7.5 with saturated sodium bicarbonate. Extracted again with ethyl acetate, the organic layers were combined and washed with deionized water and saturated brine (generally washed twice with deionized water and once with saturated brine to remove residual salts and polar impurities). The mixture was dried with anhydrous sodium sulfate or anhydrous magnesium sulfate, filtered to remove the drying agent, and the filtrate was collected and concentrated under reduced pressure at 50–60°C to remove most of the ethanol and ethyl acetate, yielding the crude product. The crude product was purified by silica gel column chromatography, and the fractions containing the product were combined, dried under reduced pressure, to obtain intermediate III. The silica gel column chromatography used hexane containing 2–3% ethyl acetate as the mobile phase for eluting the crude product.

[0030] (3) At room temperature, intermediate I obtained in step (1), intermediate III obtained in step (2) and pure acetic acid are stirred evenly, heated to the reflux temperature of pure acetic acid, and stirred for 6 to 12 hours. After the reaction is completed, the mixture is cooled to room temperature, filtered under vacuum, the solid is collected, washed with deionized water and ethanol, and dried under vacuum at a temperature of 50 to 60°C (generally dried for 10 hours) to obtain the BTSA1 derivative.

[0031] Method 2 specifically includes the following steps:

[0032] (1) Dissolve potassium carbonate in a mixed solution of deionized water and ethanol to obtain potassium carbonate solution; mix reagent I, aminothiourea and potassium carbonate solution at room temperature and stir to react; after the reaction is completed, filter under vacuum, collect the solid, wash with cold ethanol and dry to obtain intermediate I;

[0033] (2) At room temperature, pure acetic acid and intermediate I obtained in step (1) are mixed to form a suspension. Then, reagent III is added and the mixture is heated to the reflux temperature of pure acetic acid and stirred to react. After the reaction is completed, the mixture is filtered under vacuum, the solid is collected, washed with deionized water and ethanol in sequence, and dried to obtain intermediate XI.

[0034] (3) At room temperature, intermediates XI and II obtained in step (2) and sodium nitrite are dissolved in chemically pure ethanol and reacted. After the reaction is completed, deionized water is added to the reaction mixture (the volume ratio of the reaction mixture to deionized water is 1:1), the pH value of the system is adjusted to 6.5-7 with hydrochloric acid, the mixture is filtered under vacuum, the solid is collected, and the solid is washed with deionized water and ethanol in sequence to obtain the BTSA1 derivative.

[0035] Both Method 1 and Method 2 involve monitoring the reaction progress using thin-layer chromatography.

[0036] Furthermore, when R1 in the BTSA1 derivative obtained by method one or method two is a hydroxyl group, the BTSA1 derivative is subjected to alkylation and esterification reactions with R4-X or R4-COOH, respectively, to obtain a further substituted BTSA1 derivative, specifically including the following steps:

[0037] When BTSA1 derivatives undergo alkylation with R4-X:

[0038] At room temperature, a BTSA1 derivative with R1 being a hydroxyl group is mixed with N,N-dimethylformamide and potassium carbonate, and heated to a certain temperature (generally 60°C) for reaction. After the reaction is completed, the mixture is cooled to room temperature, and R4-X is slowly added. After the addition is complete, the system is heated to reflux under a nitrogen atmosphere and stirred (generally for 10 hours). After the reaction is completed, the reaction mixture is cooled to room temperature, an ice-water mixture is added, and the mixture is extracted with ethyl acetate. The organic layers are combined, washed successively with deionized water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is collected and concentrated under reduced pressure to obtain the crude product. The crude product is purified by recrystallization or column chromatography to obtain the BTSA1 derivative with R1 being a heteroatom-substituted hydrocarbon group.

[0039] When BTSA1 derivatives undergo esterification with R4-COOH:

[0040] At room temperature, a BTSA1 derivative with R1 being a hydroxyl group is mixed with R4-COOH and anhydrous 1,4-dioxane and cooled to a certain temperature (generally 4-5°C). Triphenylphosphine is added to the mixture in batches, and diethyl azodicarbonate is added dropwise over 15 minutes. After the addition is complete, the system is heated to reflux under a nitrogen atmosphere and the reaction is stirred (generally for 9-10 hours). After the reaction is completed, the mixture is concentrated under reduced pressure to obtain a crude product. The crude product is separated with dichloromethane and recrystallized from ethanol to obtain a BTSA1 derivative with R1 being a heteroatom-substituted hydrocarbon group.

[0041] Further, in step (1) of method one, the mass-to-volume ratio of aminothiourea to acetonitrile is 1 g: 3 mL; the molar ratio of aminothiourea to sodium acetate and reagent I is 1: 2: 1; the volume ratio of cold acetonitrile, water, and cold ethanol is 1: 2: 1; and the temperature of the cold acetonitrile and cold ethanol is 0–5 °C. In step (2) of method one, the molar ratio of reagent III to chemically pure ethanol and sodium acetate is 1: 10: 3; the molar ratio of reagent III to diazonium salt of intermediate II is 1: 1.3; and the volume ratio of the reaction mixture to ethyl acetate is 1: 3. In step (3) of method one, the molar ratio of intermediate III to intermediate I and pure acetic acid is 1: 1.5: 5; and the reflux temperature of pure acetic acid is 117–120 °C.

[0042] Furthermore, in step (2) of method one, the intermediate II diazonium salt solution is an intermediate II diazonium hydrochloride solution or an intermediate II diazonium phosphate solution; when the intermediate II diazonium salt solution is an intermediate II diazonium hydrochloride solution, the preparation method of the intermediate II diazonium hydrochloride solution is as shown in formula (V), including:

[0043] Add reagent II to hydrochloric acid, stir until homogeneous, and then cool to a certain temperature (generally 4-5℃) to obtain a homogeneous solution. Keep the temperature constant and slowly add sodium nitrite aqueous solution to the obtained homogeneous solution dropwise under stirring conditions. The addition time is generally 10-15 minutes. After the addition is completed, continue stirring for a certain time (generally 40-50 minutes) to ensure that the diazonium salt is completely formed, and obtain intermediate II diazonium hydrochloride solution.

[0044]

[0045] (V)

[0046] When the intermediate II diazonium salt solution is an intermediate II diazonium phosphate solution, the preparation method of the intermediate II diazonium phosphate solution is as shown in formula (VI), including:

[0047] Add reagent II to the phosphoric acid aqueous solution, heat to 55-60℃, stir for a certain time, then cool the system to a certain temperature (generally 0-5℃), and slowly add sodium nitrite aqueous solution dropwise under stirring conditions. The dropwise addition time is generally 25-30 minutes. Keep the temperature constant and continue stirring to obtain intermediate II diazonium phosphate solution.

[0048] .

[0049] Furthermore, in the preparation method of intermediate II diazonium hydrochloride solution, the concentration of hydrochloric acid is 0.5-1.0M; the mass concentration of sodium nitrite aqueous solution is 30-50%; the molar ratio of reagent II to hydrogen chloride in hydrochloric acid is 1:1; and the molar ratio of reagent II to sodium nitrite is 1:1.1.

[0050] In the preparation method of intermediate II diazophosphate solution, the mass concentration of the phosphoric acid aqueous solution is 30-50%; the concentration of the sodium nitrite aqueous solution is 1.0-1.5M; the molar ratio of reagent II to phosphoric acid is 1:1.1-1.2; and the molar ratio of reagent II to sodium nitrite is 1:1.1-1.2.

[0051] Furthermore, in step (1) of method two, the molar ratio of reagent I to potassium carbonate and aminothiourea is 1:10:1.2; the volume ratio of deionized water to ethanol is 1:1; the mass concentration of the potassium carbonate solution is 30-50%; and the temperature of the cold ethanol is 0-5℃. In step (2) of method two, the molar ratio of intermediate I to acetic acid and reagent III is 1:6:1.2. In step (3) of method two, the molar ratio of intermediate XI to intermediate II, sodium nitrite, and ethanol is 1:1.2:3:60.

[0052] Furthermore, in the alkylation reaction of the BTSA1 derivative and R4-X, the molar ratio of the BTSA1 derivative with hydroxyl group R1 to N,N-dimethylformamide and potassium carbonate is 1:5:2.5; the molar ratio of the BTSA1 derivative with hydroxyl group R1 to R4-X is 1:1.2; the volume ratio of the ice-water mixture to the reaction mixture is 1:1; and the volume ratio of the ethyl acetate to the reaction mixture is 1:1.

[0053] In the esterification reaction of BTSA1 derivatives with R4-COOH, the molar ratio of BTSA1 derivatives with hydroxyl R1 to R4-COOH is 1:1; the molar ratio of BTSA1 derivatives with hydroxyl R1 to triphenylphosphine is 1:1.5; and the molar ratio of BTSA1 derivatives with hydroxyl R1 to diethyl azodicarbonate is 1.5:1.

[0054] The room temperature described in this invention is 20–25°C.

[0055] This invention further discloses a aforementioned BTSA1 derivative and its application in the preparation of drugs for treating broad-spectrum solid tumors using the aforementioned method. When R1 is a hydroxyl group, the derivative exhibits superior anti-solid tumor activity.

[0056] The beneficial effects of the present invention are as follows: The BTSA1 derivative prepared by the present invention exhibits strong anti-proliferative activity against a variety of solid tumors, has broad-spectrum anti-tumor properties, and has the potential to be further developed into a clinical treatment drug for solid tumors. Attached Figure Description

[0057] Figure 1 The hydrogen nuclear magnetic resonance spectrum of HZ-1 prepared in Example 1;

[0058] Figure 2 The carbon NMR spectrum of HZ-1 prepared in Example 1;

[0059] Figure 3 The 1H NMR spectrum of HZ-2 prepared in Example 1;

[0060] Figure 4 The carbon NMR spectrum of HZ-2 prepared in Example 1;

[0061] Figure 5 The 1H NMR spectrum of HZ-3 prepared in Example 1;

[0062] Figure 6 The carbon NMR spectrum of HZ-3 prepared in Example 1;

[0063] Figure 7 The hydrogen nuclear magnetic resonance spectrum of HZ-7 prepared in Example 2;

[0064] Figure 8 The hydrogen nuclear magnetic resonance spectrum of HZ-10 prepared in Example 5;

[0065] Figure 9 The carbon NMR spectrum of HZ-10 prepared in Example 5;

[0066] Figure 10 The 1H NMR spectrum of HZ-8 prepared in Example 7;

[0067] Figure 11 The carbon NMR spectrum of HZ-8 prepared in Example 7;

[0068] Figure 12 The 1H NMR spectrum of HZ-4 prepared in Example 8;

[0069] Figure 13 The image shows the carbon NMR spectrum of HZ-4 prepared in Example 8. Detailed Implementation

[0070] The present invention will be further explained below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] Example 1

[0072] (1) At 20°C, aminothiourea was added to a round-bottom reaction flask, along with acetonitrile (the mass-to-volume ratio of aminothiourea to acetonitrile was 1 g: 3 mL), 2.0 equivalents of sodium acetate, and 1.0 equivalents of 2-bromo-1-(4-hydroxyphenyl)ethyl ketone. The mixture was stirred for 3 hours, and the reaction was analyzed by thin-layer chromatography (TLC) (ethyl acetate / petroleum ether 1:1, R... f ≈0.4) Monitor the reaction process, the reaction formula is shown below.

[0073] After the reaction was complete, the mixture was filtered, and the precipitated solid was collected. Then, it was successively treated with approximately 5... o C's cold acetonitrile, deionized water and 5 o Washing with cold ethanol (cold acetonitrile, water, and cold ethanol in a volume ratio of 1:2:1) under vacuum at a temperature of approximately 55°C. o Drying at C for about 5 hours yields 2-(2-hydrazothiazol-4-yl)phenol, 3-(2-hydrazothiazol-4-yl)phenol, or 4-(2-hydrazothiazol-4-yl)phenol, as a pale yellow to off-white solid. The yield is 80%, and the melting point is 186–187°C. o C.

[0074]

[0075] 10 g of 2-aminothiazole was added to 100 mL of 1.0 M hydrochloric acid, stirred until homogeneous, and then cooled to approximately 5°C to form a homogeneous solution of 2-aminothiazole in hydrochloric acid. The reaction system temperature was maintained at approximately 5°C. o C. Slowly add 20.6 mL of a 30% sodium nitrite aqueous solution to the above homogeneous solution over 15 minutes to allow the 2-aminothiazole to undergo in-situ diazotization. After the addition is complete, heat the reaction mixture at approximately 5°C. o Continue stirring at C for 50 minutes to ensure complete formation of the diazonium salt, resulting in a 2-aminothiazole diazonium hydrochloride solution. This solution does not require separation and can be used directly in subsequent condensation steps.

[0076] (2) At room temperature, 25 g of ethyl 3-oxo-3-phenylpropionate was added to 75.8 mL of chemically pure ethanol. After stirring until the solution was clear, 30 g of sodium acetate was slowly added, and the pH of the system was adjusted to 7.5 with 1 M sodium carbonate aqueous solution to activate the methylene group. The system was then cooled to 5 °C. o C, and under stirring conditions, slowly add the obtained 2-aminothiazole diazonium hydrochloride solution to suppress side reactions. After the addition is complete, heat the system to approximately 35°C. o At temperature C, the mixture was stirred for 8 hours, and the reaction progress was monitored by thin-layer chromatography. The reaction formula is shown below. After the reaction was completed, the reaction mixture was cooled to room temperature, the pH of the system was adjusted to 7.0 with saturated sodium bicarbonate, and then extracted with ethyl acetate (the volume ratio of the reaction mixture to ethyl acetate was 1:3). The organic layers were combined, washed twice with deionized water, and once with saturated brine to remove residual salts and polar impurities. The mixture was then dried with anhydrous sodium sulfate, filtered to remove the drying agent, and the filtrate was collected. The solution was then heated to approximately 50 °C. o The solution was concentrated under reduced pressure at C to remove most of the ethanol and ethyl acetate, yielding a crude product. The crude product was purified by silica gel column chromatography, eluting with hexane containing 3% ethyl acetate as the mobile phase. The fractions containing the product were combined, the solvent was removed under reduced pressure, and the product was dried to give ethyl 3-oxo-3-phenyl-2-[2-(thiazol-2-yl)hydrazideyl]propionate in approximately 60.8% (based on ethyl 3-oxo-3-phenylpropionate).

[0077]

[0078] (3) At room temperature, add ethyl 3-oxo-3-phenyl-2-[2-(thiazol-2-yl)hydrazide]propionate and 5 equivalents of pure acetic acid to a dry reaction flask. Under stirring, slowly add 1.5 equivalents of 2-(2-hydrazidothiazol-4-yl)phenol obtained in step (1). After the addition is complete, heat the system to the reflux temperature of acetic acid (117-120 °C). oC), and stirred at this temperature for 6 hours, monitoring the reaction progress by thin-layer chromatography (TLC). The reaction formula is shown below. After the reaction is complete, the mixture is cooled to room temperature, filtered under vacuum, and the solid is collected. It is washed with deionized water and ethanol, and dried under vacuum at 60°C for 5 hours to obtain a substantially pure ortho-hydroxylated BTSA1 derivative, denoted as HZ-1, which requires no further column chromatography purification. The melting point of this derivative is 204–207 °C. o C, with a yield of 65%.

[0079] Using the same method as described above, but replacing 2-(2-hydrazothiazol-4-yl)phenol with 3-(2-hydrazothiazol-4-yl)phenol and 4-(2-hydrazothiazol-4-yl)phenol respectively, essentially pure para-hydroxylated BTSA1 derivatives and meta-hydroxylated BTSA1 derivatives were obtained, denoted as HZ-2 and HZ-3 respectively. The yield of the para-hydroxylated BTSA1 derivative was 58%, and the yield of the meta-hydroxylated BTSA1 derivative was 62%.

[0080] like Figures 1 to 6 As shown, the 1H NMR spectra of ortho, meta, and para-hydroxylated BTSA1 derivatives are... 1 H NMR) and carbon nuclear magnetic resonance (NMR) spectrum 13 The results of C NMR showed that all the above derivatives maintained the desired substitution pattern at the phenolic hydroxyl position.

[0081]

[0082] Example 2

[0083] Step (1) in this embodiment is the same as step (1) in embodiment 1.

[0084] 10 g of 2-aminobenzothiazole was added to 21 mL of a 30% (w / w) aqueous solution of phosphoric acid. The system was heated to 55 °C and stirred until the solution became clear, approximately 6 hours later. The system was then cooled to 5 °C, and 75 mL of a 1.0 M sodium nitrite aqueous solution was slowly added dropwise over 30 minutes. The mixture was stirred at 5 °C for another 1.5 hours to obtain a 2-aminobenzothiazole diazonium phosphate solution. This solution did not require separation and was used directly in the subsequent condensation step.

[0085] (2) At room temperature, 6.0 g of ethyl 3-oxo-3-phenylpropionate was added to 18.2 mL of chemically pure ethanol. After stirring until the solution was clear, 7.7 g of sodium acetate was added. The pH of the system was adjusted to approximately 7.5 with 1 M sodium carbonate aqueous solution. The system was then cooled to 5 °C, and the obtained 2-aminobenzothiazole diazonium phosphate solution was slowly added under stirring to suppress side reactions. After the addition was complete, the system was heated to approximately 35 °C. oAt temperature C, the mixture was stirred for approximately 9 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). The reaction formula is shown below. After the reaction was completed, the reaction mixture was cooled to room temperature, the pH of the system was adjusted to 7.0 with saturated sodium bicarbonate, and then extracted three times with approximately 30 mL of ethyl acetate (approximately 10 mL each time). The organic layers were combined, washed twice with deionized water, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure at approximately 50 °C to obtain the crude product. The obtained crude product was purified by silica gel column chromatography, using hexane containing 3% ethyl acetate as the mobile phase for elution. The fractions containing the product were combined, the solvent was removed under reduced pressure, and the product was dried to give ethyl 3-oxo-3-phenyl-2-[(benzo[d]thiazolyl)hydrazinoyl]propionate in a yield of 35% (based on ethyl 3-oxo-3-phenylpropionate).

[0086]

[0087] (3) At room temperature, ethyl 3-oxo-3-phenyl-2-[(benzo[d]thiazol-2-yl)hydrazino]propionate and 5 equivalents of pure acetic acid were added to a dry reaction flask. Under stirring, 1.5 equivalents of 2-(2-hydrazinothiazol-4-yl)phenol were slowly added. After the addition was complete, the system was heated to the reflux temperature of acetic acid (117-120 °C). o C), and stirred at this temperature for about 10 hours, monitoring the reaction progress by thin-layer chromatography (TLC). The reaction formula is shown below. After the reaction is complete, the mixture is cooled to room temperature, filtered under vacuum, the solid is collected, washed with deionized water and ethanol, and dried under vacuum at 60°C for 5 hours to obtain a substantially pure benzothiazole ortho-hydroxylated BTSA1 derivative, denoted as HZ-5, which does not require column chromatography purification.

[0088] Using the same method as above, but replacing 2-(2-hydrazothiazol-4-yl)phenol with 3-(2-hydrazothiazol-4-yl)phenol and 4-(2-hydrazothiazol-4-yl)phenol respectively, we can obtain benzothiazole para-hydroxylated BTSA1 derivatives and benzothiazole meta-hydroxylated BTSA1 derivatives, denoted as HZ-6 and HZ-7 respectively.

[0089] 1H NMR spectrum of benzothiazole ortho-hydroxylated BTSA1 derivative (HZ-5) 1 The H NMR data are as follows:

[0090] 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 8.21 (s, 2H), 7.77 (d, J =59.2 Hz,4H), 7.49 (d, J = 61.3 Hz, 4H), 6.94 (s, 2H) ppm.

[0091] 1 H D2O Exchange NMR (400 MHz, DMSO-d6) δ 8.14 (s, 2H), 7.97 – 7.60 (m,4H),7.59 – 7.05 (m, 6H), 6.92 (s, 2H) ppm.

[0092] 1H NMR spectrum of benzothiazole para-hydroxylated BTSA1 derivative (HZ-6) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from C NMR are as follows:

[0093] 1 H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.21 (s, 2H), 7.80 (t, J =47.4 Hz, 4H), 7.61 – 7.20 (m, 6H), 6.84 (s, 2H) ppm.

[0094] 1 H D2O Exchange NMR (400 MHz, DMSO-d6) δ 8.21 (s, 2H), 7.86 (t, J =39.4 Hz,4H), 7.64 – 7.17 (m, 6H), 6.84 (s, 2H) ppm.

[0095] 13 C NMR (101 MHz, DMSO-d6) δ 178.65, 157.37, 154.67, 152.72, 150.07,149.17,131.33, 130.28, 129.53, 128.47, 128.05, 127.40, 126.48, 125.68,124.33, 122.51,119.34, 115.44, 105.80 ppm.

[0096] like Figure 7 As shown, the 1H NMR spectrum of the benzothiazole meta-hydroxylated BTSA1 derivative (HZ-7) is... 1H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from C NMR are as follows:

[0097] 1 H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.26 (d, J = 7.2 Hz, 2H), 7.89 (d, J =7.7 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.63 – 7.46(m, 4H), 7.45 – 7.36(m, 2H), 7.34 (s, 1H), 7.29 – 7.24 (m, 2H), 6.82 – 6.71(m,1H).

[0098] 13 C NMR (101 MHz, DMSO-d6) δ 181.04, 157.74, 155.01, 152.83, 152.37,149.70,149.66, 135.74, 132.39, 132.30, 129.73, 129.06, 128.35, 128.14,125.83, 124.68,124.07, 122.04, 121.33, 116.73, 114.87, 112.98, 108.23 ppm.

[0099]

[0100] Example 3

[0101] Step (1) in this embodiment is the same as step (1) in embodiment 1.

[0102] 20 g of 6-nitrobenzothiazole-2-amine was added to 32 mL of a 30% (w / w) aqueous solution of phosphoric acid. The system was heated to 55 °C and stirred at this temperature for 8 hours to form a suspension. The system was then cooled to approximately 5 °C, and at this temperature, 8 g of sodium nitrite was slowly added dropwise over 30 minutes. The mixture was stirred at approximately 5 °C for another 100 minutes to obtain a 6-nitrobenzothiazole diazonium phosphate solution. This solution did not require separation and was used directly in the subsequent condensation step.

[0103] (2) At room temperature, 10.0 g of ethyl 3-oxo-3-phenylpropionate was added to 30.3 mL of chemically pure ethanol. After stirring until the solution was clear, 12.8 g of sodium acetate was added. The pH of the system was adjusted to approximately 7.5 with 1 M sodium carbonate aqueous solution. The system was then cooled to 5 °C, and the obtained 6-nitrobenzothiazole diazonium phosphate solution was slowly added under stirring to suppress side reactions. After the addition was complete, the system was heated to approximately 35 °C. o At temperature C, the mixture was stirred for approximately 9 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). The reaction mixture was shown below. After the reaction was completed, the reaction mixture was cooled to room temperature, the pH of the system was adjusted to 7.0 with saturated sodium bicarbonate, and then extracted twice with 40 mL of ethyl acetate (approximately 20 mL each time). The organic layers were combined, washed twice with deionized water, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at approximately 50 °C to obtain the crude product. The crude product was purified by silica gel column chromatography, using hexane containing 3% ethyl acetate as the mobile phase for elution. The fractions containing the product were combined, the solvent was removed under reduced pressure, and the product was dried to give ethyl 2-(2-(6-nitrobenzo[d]thiazo-2-yl)hydrazone)-3-oxo-3-phenylpropionate in 45% (based on ethyl 3-oxo-3-phenylpropionate).

[0104]

[0105] (3) At room temperature, add ethyl-2-(2-(6-nitrobenzothiazol-2-yl)hydrazino)-3-oxo-3-phenylpropionate and 5 equivalents of pure acetic acid to a dry reaction flask. Under stirring, slowly add 1.5 equivalents of 2-(2-hydrazinothiazol-4-yl)phenol obtained in step (1). After the addition is complete, heat the system to the reflux temperature of acetic acid (117-120 °C). o C), and stirred at this temperature for about 10 hours, monitoring the reaction progress by thin-layer chromatography (TLC). The reaction formula is shown below. After the reaction is complete, the mixture is cooled to room temperature, filtered under vacuum, and the solid is collected. It is washed with deionized water and ethanol, and dried under vacuum at 60°C for 5 hours to obtain the acetate of the ortho-hydroxylated BTSA1 derivative of 6-nitrobenzothiazole. The acetate is desalted by adjusting the pH with a saturated aqueous solution of sodium bicarbonate to obtain a free base, which is purified by column chromatography to obtain the ortho-hydroxylated BTSA1 derivative of 6-nitrobenzothiazole, designated as HZ-13, with a yield of 72.4%.

[0106] Using the same method as above, except that 2-(2-hydrazothiazol-4-yl)phenol was replaced with 4-(2-hydrazothiazol-4-yl)phenol, a 6-nitrobenzothiazole para-hydroxylated BTSA1 derivative, denoted as HZ-14, was obtained with a yield of 73.1%.

[0107] 1H NMR spectrum of 6-nitrobenzothiazole ortho-hydroxylated BTSA1 derivative (HZ-13) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from C NMR are as follows:

[0108] 1 H NMR (400 MHz, DMSO-d6) δ 8.87 (d, J = 2.2 Hz, 1H), 8.31 – 8.10 (m,3H), 7.96 (d, J = 7.8 Hz,1H), 7.89 – 7.74 (m, 2H), 7.57 (q, J = 8.9, 7.6 Hz,3H), 7.41 – 7.06 (m, 2H), 6.92 (dd, J = 18.2, 8.0 Hz, 2H) ppm.

[0109] 13 C NMR (101 MHz, DMSO-d6) 155.44, 129.35, 128.51, 128.05, 118.63, 116.98 ppm.

[0110] 1H NMR spectrum of 6-nitrobenzothiazole para-hydroxylated BTSA1 derivative (HZ-14) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from C NMR are as follows:

[0111] 1 H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.85 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 4.7, 2.0 Hz, 2H), 7.87 – 7.79 (m, 3H), 7.57 (t, J = 7.1 Hz,2H), 7.23 (s, 1H), 7.11 (s, 1H), 6.98 (s, 1H), 6.84 (d, J = 8.6 Hz, 2H) ppm.

[0112] 13C NMR (101 MHz, DMSO-d6) δ 185.69, 157.30, 154.68, 152.46, 149.93,149.41, 142.72, 132.83, 131.65, 129.32, 128.39, 128.11, 127.36, 125.74, 121.63, 120.47, 118.42, 115.40, 105.59 ppm.

[0113]

[0114] Example 4

[0115] Step (1) in this embodiment is the same as step (1) in embodiment 1.

[0116] 20 g of 6-fluorobenzothiazole-2-amine was added to 38 mL of a 30% (w / w) aqueous solution of phosphoric acid. The system was heated to approximately 60 °C and stirred at this temperature for 8 hours to form a suspension. The system was then cooled to approximately 5 °C, and 9.5 g of sodium nitrite was slowly added dropwise over 30 minutes. The mixture was stirred at approximately 5 °C for another 100 minutes to obtain a 6-nitrobenzothiazole diazonium phosphate solution. This solution did not require separation and was used directly in the subsequent condensation step.

[0117] (2) At room temperature, 10.0 g of ethyl 3-oxo-3-phenylpropionate was added to 30.3 mL of chemically pure ethanol. After stirring until the solution was clear, 12.8 g of sodium acetate was added. The pH of the system was adjusted to approximately 7.5 with 1 M sodium carbonate aqueous solution. The system was then cooled to 10 °C, and the obtained 6-nitrobenzothiazole diazonium phosphate solution was slowly added under stirring to suppress side reactions. After the addition was complete, the system was heated to approximately 35 °C. o At temperature C, the mixture was stirred for approximately 9 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). The reaction formula is shown below. After the reaction was completed, the reaction mixture was cooled to room temperature, the pH of the system was adjusted to 7.0 with saturated sodium bicarbonate, and then extracted twice with 200 mL of ethyl acetate (approximately 100 mL each time). The organic layers were combined, washed twice with deionized water, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure at approximately 50 °C to obtain the crude product. The obtained crude product was purified by silica gel column chromatography, using hexane containing 3% ethyl acetate as the mobile phase for elution. The fractions containing the product were combined, the solvent was removed under reduced pressure, and the product was dried to give 13 g of ethyl 2-(2-(6-fluoro-benzothiazol-2-yl)hydrazone)-3-oxo-3-phenylpropionate, with a yield of 40% (based on ethyl 3-oxo-3-phenylpropionate).

[0118]

[0119] (3) At room temperature, add ethyl 2-(2-(6-fluorobenzo[d]thiazol-2-yl)hydrazino)-3-oxo-3-phenylpropionate and 5 equivalents of pure acetic acid to a dry reaction flask. Under stirring, slowly add 1.5 equivalents of 2-(2-hydrazinothiazol-4-yl)phenol obtained in step (1). After the addition is complete, heat the system to the reflux temperature of acetic acid (117-120 °C). o C), and stirred at this temperature for approximately 10 hours. The reaction progress was monitored by thin-layer chromatography (TLC), as shown in the reaction formula below. After the reaction was completed, the mixture was cooled to room temperature, filtered under vacuum, and the solid was collected. It was washed with deionized water and ethanol, and dried under vacuum at 60°C for 5 hours to obtain the acetate of the 6-fluorofused ortho-hydroxylated BTSA1 derivative. This acetate was desalted by adjusting the pH with a saturated sodium bicarbonate solution to obtain a free base, which was purified by column chromatography to obtain the 6-fluorofused ortho-hydroxylated BTSA1 derivative, designated HZ-11. The melting point of this derivative is 255–256 °C. o C, with a yield of 53.0%.

[0120] Using the same method as described above, but replacing 2-(2-hydrazothiazol-4-yl)phenol with 4-(2-hydrazothiazol-4-yl)phenol, a 6-fluorofused para-hydroxylated BTSA1 derivative, denoted as HZ-12, was obtained. This derivative has a melting point of 168.2°C. o C, with a yield of 67.22%.

[0121] 1H NMR spectrum of 6-fluorofused ortho-hydroxylated BTSA1 derivative (HZ-11) 1 The H NMR data are as follows:

[0122] 1 H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 7.2 Hz, 2H), 7.97 (d, J =7.7 Hz, 1H), 7.88 (d, J = 2.5Hz, 2H), 7.72 (dd, J = 8.6, 4.9 Hz, 1H), 7.56(q, J = 11.1, 8.9 Hz, 3H), 7.29 (t, J = 9.0 Hz, 1H), 7.24 –7.17 (m, 2H), 7.08(s, 1H), 6.98 – 6.84 (m, 3H) ppm.

[0123] 1H NMR (400 MHz, DMSO-d6 D2O exchange) δ 8.18 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 6.1 Hz, 2H), 7.72 (dd, J = 8.9, 4.7 Hz, 1H), 7.56 (p, J = 7.0 Hz, 3H), 7.29 (td, J = 9.1, 2.4 Hz, 1H), 7.24 – 7.17(m, 1H), 6.99 – 6.87 (m, 2H) ppm.

[0124] 1H NMR spectrum of 6-fluorofused para-hydroxylated BTSA1 derivative (HZ-12) 1 The H NMR data are as follows:

[0125] 1 H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.18 (d, J = 6.8 Hz, 2H), 7.90 (dd, J = 8.6, 2.6 Hz, 1H), 7.81 (d, J = 8.6 Hz, 2H), 7.69 (dd, J = 8.8,4.7 Hz, 1H), 7.57 (d, J = 8.9 Hz, 4H), 7.31 (td, J = 9.0, 2.6 Hz, 1H), 7.23 –6.92 (m, 1H), 6.84 (d, J = 8.6 Hz, 2H) ppm.

[0126] 1 H NMR (400 MHz, DMSO-d6 D2O exchange) δ 8.17 (d, J = 6.8 Hz, 2H), 7.92 – 7.65 (m, 4H), 7.62 –7.47 (m, 4H), 7.30 (t, J = 9.0 Hz, 1H), 6.84 (d, J= 8.7 Hz, 2H) ppm.

[0127]

[0128] Example 5

[0129] At room temperature, 1.0 equivalent of the ortho-hydroxylated BTSA1 derivative prepared in Example 1, 5 equivalents of anhydrous N,N-dimethylformamide, and 2.5 equivalents of potassium carbonate were added to a dry round-bottom flask, and the system was heated to approximately 60°C. o The reaction proceeds at C. After the reaction is complete, the reaction mixture is cooled to room temperature, and 1.2 equivalents of 2-chloro-N-(2,6-dimethylphenyl)acetamide are slowly added to the reaction mixture. After the addition is complete, the mixture is heated to reflux (approximately 100 °C) under a nitrogen atmosphere. o C), and stirred for 10 hours. The reaction progress was monitored by thin-layer chromatography (TLC) (dichloromethane:methanol = 9.5:0.5), as shown in the reaction formula below. After the reaction was completed, the mixture was cooled to room temperature, and 50-60 mL of an ice-water mixture was added to quench the reaction. The mixture was then extracted three times with 90 mL of ethyl acetate (approximately 30 mL each time). The organic layers were combined and washed successively with 20 mL of deionized water and 20 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization from approximately 7 mL of acetone to obtain a pure ortho-alkylated BTSA1 derivative, designated HZ-9. The melting point of this derivative is 237°C. o C, with a yield of 47.05%.

[0130] 1H NMR spectrum of ortho-alkylated BTSA1 derivative (HZ-9) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from CNMR are as follows:

[0131] 1 HNMR (400MHz, DMSO-d6) δ9.65(s, 1H), 8.29–.11(m, 3H), 8.07(s, 1H),7.68(d, J=3.5Hz, 1H), 7.60–7.42(m, 3H), 7.41–7.34(m, 1H), 7.29(d, J=3.5Hz,1H), 7.21–7.12(m, 2H), 7.06(d, J=5.1Hz, 3H), 4.95(s, 2H), 2.14(s,6H) ppm.

[0132] 13CNMR(101MHz, DMSO) δ182.02, 166.44, 155.14, 154.02, 153.12, 149.55,145.71, 140.51, 135.27, 134.35, 132.49, 129.81, 128.82, 128.75, 128.10,127.72, 126.64, 123.54, 122.91, 121.37, 115.48, 112.77, 112.44, 67.49, 18.12ppm.

[0133] Using the same method as described above, but replacing the ortho-hydroxylated BTSA1 derivative with the para-hydroxylated BTSA1 derivative, a pure para-alkylated BTSA1 derivative was obtained, denoted as HZ-10. This derivative has a melting point of 69°C. o C, with a yield of 52.94%.

[0134] The 1H NMR spectrum of the para-alkylated BTSA1 derivative (HZ-10) 1 H NMR) and carbon nuclear magnetic resonance (NMR) spectrum 13 (C NMR) such as Figure 8 and Figure 9 As shown.

[0135]

[0136] Example 6

[0137] At room temperature, 1.0 equivalent of the ortho-hydroxylated BTSA1 derivative prepared in Example 1, 5 equivalents of anhydrous N,N-dimethylformamide, and 2.5 equivalents of potassium carbonate were added to a dry round-bottom flask, and the system was heated to approximately 60°C. o The reaction proceeds at C. After the reaction is complete, the reaction mixture is cooled to room temperature, and 1.2 equivalents of 2-oxo-2H-chromene-4-ylcarbamoyl chloride are slowly added to the reaction mixture. After the addition is complete, the mixture is heated to reflux (approximately 100 °C) under a nitrogen atmosphere. o C), and stirred for 10 hours. The reaction progress was monitored by thin-layer chromatography (TLC) (dichloromethane:methanol = 9.5:0.5), as shown in the following reaction formula. After the reaction was completed, the mixture was cooled to room temperature, and 50 mL of an ice-water mixture was added to quench the reaction. The mixture was then extracted three times with 90 mL of ethyl acetate (approximately 30 mL each time). The organic layers were combined, washed successively with 20 mL of deionized water and 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a pure ortho-BTSA1 coumarin ester derivative, designated HZ-15, with a yield of 45%.

[0138] Using the same method as above, except that the ortho-hydroxylated BTSA1 derivative was replaced with the para-hydroxylated BTSA1 derivative, a pure para-BTSA1 coumarin ester derivative was obtained, denoted as HZ-16, with a yield of 45%.

[0139] 1H NMR spectrum of ortho-BTSA1 coumarin ester derivative (HZ-15) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from CNMR are as follows:

[0140] 1 HNMR(400MHz, DMSO-d6) δ9.99(s, 1H), 8.63(s, 1H), 8.34–7.88(m, 4H),7.67(s, 2H), 7.55–7.20(m, 8H), 7.12(s, 2H), 6.87(d, J=41.2Hz, 1H), 5.05(s,2H).

[0141] 13 CNMR(101MHz, DMSO) δ201.87, 182.18, 167.92, 165.19, 157.46, 154.68,154.05, 153.06, 149.85, 145.56, 140.35, 132.38, 129.79, 128.86, 128.62,127.99, 124.92, 124.52, 123.84, 123.32, 121.40, 119.36, 115.83, 115.33,112.76, 112.22, 95.93, 79.43, 67.30, 30.73 ppm.

[0142] 1H NMR spectrum of para-BTSA1 coumarin ester derivative (HZ-16) 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from CNMR are as follows:

[0143] 1HNMR(400MHz, DMSO-d6) δ9.78(s, 1H), 8.67(s, 1H), 8.22(d, J=7.2Hz,2H), 7.96(d, J=8.7Hz, 2H), 7.79–7.71(m, 1H), 7.66(d, J=3.3Hz, 1H),7.57(s,1H), 7.55–7.47(m, 3H), 7.43(t, J= 7.8Hz, 2H), 7.35(t, J=7.5Hz, 1H), 7.27(d, J=3.5Hz, 1H), 7.10(d, J=8.8Hz, 2H), 4.94(s,2H).

[0144] 13 CNMR(101MHz, DMSO)δ182.48, 167.88, 157.64, 157.13, 155.27, 153.05,149.80, 149.58, 149.06, 141.15, 132.78, 129.94, 128.64, 128.31, 128.08,127.34, 125.10, 123.90, 123.80, 122.25, 119.43, 115.97, 115.49, 114.83,106.50, 66.92 ppm.

[0145]

[0146] Example 7

[0147] At room temperature, 1.0 mmol of the para-hydroxylated BTSA1 derivative prepared in Example 1 and 1 mmol of cholic acid were added to a dry round-bottom flask equipped with a magnetic stirrer, followed by 40–60 mL of anhydrous 1,4-dioxane. The flask was cooled to approximately 5 °C. 1.5 mmol of triphenylphosphine was added in approximately 5 equal portions, followed by 1.5 mmol of diethyl azodicarbonate dropwise over 15 minutes. After the addition was complete, the system was heated to reflux and stirred for approximately 9 hours under a nitrogen atmosphere. The reaction progress was monitored by thin-layer chromatography (TLC) (methanol:dichloromethane = 9.5:0.5), as shown in the reaction equation below. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. The crude product was separated with dichloromethane and recrystallized from ethanol to obtain pure type IV para-isomer cholic acid ester, designated HZ-8, with a melting point of 194–196 °C. o C, with a yield of 70%.

[0148] like Figure 10 and Figure 11As shown, the 1H NMR spectrum of the type IV para-BTSA1 derivative ( 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The data from C NMR are as follows:

[0149] 1 H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.18 – 8.12 (m, 2H), 7.80 (d, J = 8.6Hz, 2H), 7.54 (d, J = 7.9 Hz, 4H), 6.84 (d, J = 8.7 Hz, 2H), 3.77(s, 1H), 3.62 – 3.57(m, 1H), 3.44 (q, J = 7.0 Hz, 2H), 2.29 – 2.03 (m, 4H), 1.97 (td, J = 12.0, 7.3 Hz, 1H), 1.84 – 1.56 (m, 6H), 1.47 – 1.12 (m, 11H), 1.05 (t, J = 7.0 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.78 (s, 3H), 0.56 (s, 3H).

[0150] 13 C NMR (101 MHz, DMSO-d6) δ 178.66, 175.02, 157.46, 154.43, 150.29,148.80,132.11, 130.47, 130.04, 129.98, 128.53, 127.98, 127.41, 125.50,115.45, 114.02,106.10, 71.03, 70.48, 66.28, 56.07, 46.10, 45.77, 41.54,41.38, 35.33, 35.09, 34.89,34.40, 30.85, 30.44, 28.55, 27.32, 26.22, 22.83,22.62, 18.59, 16.96, 12.34 ppm.

[0151]

[0152] Example 8

[0153] (1) Potassium carbonate was dissolved in a mixed solution of deionized water and ethanol to prepare a 30% potassium carbonate solution. At room temperature, 20 g of 4-(2-bromoacetyl)phenylacetate, 8.5 g of aminothiourea, and the potassium carbonate solution were mixed and stirred for about 3 hours. The reaction progress was monitored by thin-layer chromatography (TLC), and the reaction formula is shown below. After the reaction was complete, the mixture was filtered under vacuum, the solid was collected, washed with cold ethanol, and dried to obtain 36 g of pure 4-(2-hydrazonethiazol-4-yl)phenylacetate, with a yield of 72.5%.

[0154] (2) At room temperature, pure acetic acid and 26g of 4-(2-hydrazonethiazol-4-yl)phenylacetic acid ester obtained in step (1) were mixed to form a suspension. Then, 25g of ethyl benzoate was added, and the mixture was heated to the reflux temperature of acetic acid. The mixture was stirred at this temperature for about 5 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the mixture was filtered under vacuum, and the solid was collected and washed successively with deionized water and ethanol to obtain 23g of 4-(2-(5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)phenylacetic acid ester, with a yield of 55%.

[0155] (3) At room temperature, 10 g of 4-(2-(5-oxo-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazolyl-4-yl)phenyl acetate obtained in step (2), 3.18 g of 2-aminothiazole, and 5.48 g of sodium nitrite were dissolved in 92.7 mL of chemically pure ethanol and reacted. The reaction was carried out overnight, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, 15 mL of deionized water was added to the reaction mixture, and the pH of the system was adjusted to 6.5–7.0 with 0.1 M hydrochloric acid. The mixture was filtered under vacuum, and the solid was collected and washed successively with deionized water and ethanol to obtain 6.2 g of (E)-4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)hydrazone)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)phenyl acetate, denoted as HZ-4, with a melting point of 204–207 °C. o C, with a yield of 69%.

[0156] (E)-4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)hydrazone)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)phenylacetate 1H NMR spectrum ( 1 H NMR) and carbon nuclear magnetic resonance (NMR) spectrum 13 (C NMR) such as Figure 12 and Figure 13 As shown.

[0157]

[0158] The compounds prepared in Examples 1 to 8 were subjected to in vitro cell activity tests, using five human solid tumor cell lines as representatives: A549 human non-small cell lung cancer cell line, MCF-7 human breast cancer cell line, NCI-H446 human small cell lung cancer cell line, MDA-MB-231 human triple-negative breast cancer cell line, and MDA-MB-468 human triple-negative breast cancer cell line. A549, NCI-H446, MCF-7, and MDA-MB-231 cells were purchased from Shanghai Beyotime Biotechnology Co., Ltd., while MDA-MB-468 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0159] The cell culture conditions were as follows: A549 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin / streptomycin, and 2 mML glutamine. NCI-H446 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin / streptomycin, and 2 mML glutamine. MCF-7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 10 μg / mL recombinant human insulin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U / mL penicillin / streptomycin, and 2 mML glutamine. MDA-MB-231 and MDA-MB-468 cells were both cultured in Leibovitz L-15 medium containing 10% fetal bovine serum, 100 U / mL penicillin / streptomycin, and 2 mML glutamine.

[0160] Each compound was dissolved in 100% DMSO to prepare an 8 mM stock solution, and then diluted in cell culture medium as needed for the experiment. The above-mentioned cancer cells (1–2 × 10⁻⁶) were then... 4 Cells (per well) were seeded into 96-well white transparent culture plates and treated with serially diluted compound or solvent control (1% DMSO) in fetal bovine serum-free medium for 2 hours. Fetal bovine serum was then added to a final concentration of 10%, with a final volume of 100 μL. After 72 hours of culture, the CCK-8 solution for testing was prepared according to the manufacturer's (Shanghai Beyotime Biotechnology Co., Ltd.) instructions at a ratio of 10:1 (complete culture:CCK-8). The original drug-containing medium in the 96-well cell culture plates was replaced, and the plates were incubated in a constant temperature CO2 cell culture incubator for 1–2 hours. The luminescence values ​​were then read using a Feyond-MF200 microplate reader (Hangzhou Ausen Instruments Co., Ltd.). Viability assays were performed at least three times, and data were normalized to the control wells treated with 1% solvent. The half-maximal inhibitory concentration (IC50) of each compound was calculated using Prism software (GraphPad) through nonlinear regression analysis. 50The values ​​are shown in Tables 1 to 4.

[0161] After testing the activity of all the synthesized compounds on five solid tumor cells, it was found that when R1 is a hydroxyl group, the different substitution positions of the hydroxyl group on the benzene ring have the most significant effect on antitumor activity. Therefore, the antiproliferative activity of derivatives with ortho, meta and para hydroxyl substitutions on the five cell types was further investigated.

[0162] Table 1 shows that the IC50 of HZ-1 in A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468 cells is [data missing]. 50 The values ​​were 4.65 μM, 8.34 μM, 6.99 μM, 1.06 μM, and 5.20 μM, respectively, all significantly lower than the corresponding values ​​of the parent compound BTSA1 (11.50 μM, 38.55 μM, 14.44 μM, 4.61 μM, and 12.36 μM). Especially in MDA-MB-231 cells, the IC50 of HZ-1 was significantly lower. 50 The concentration was as low as 1.06 μM, with an activity increase of approximately 4.3 times. These results indicate that HZ-1 exhibits strong anti-proliferative activity against five types of solid tumor cells, demonstrating good potential for broad-spectrum anti-solid tumor activity and warranting further development.

[0163] As shown in Table 1, compared with the parent compound BTSA1, HZ-2 exhibits significant cell dependence and weakened activity in A549 cells (IC50). 50 The value increased from 11.50 μM to 22.53 μM in MDA-MB-468 cells (IC50). 50 The value increased from 12.36 μM to 24.30 μM) and NCI-H446 cells (IC 50 The activity was slightly weakened in cells where the concentration increased from 14.44 μM to 17.82 μM; however, the activity was significantly enhanced in MCF-7 cells (IC50). 50 The value decreased from 38.55 μM to 15.06 μM), and its activity was also slightly enhanced in MDA-MB-231 cells (IC50). 50 The value decreased from 4.61 μM to 3.22 μM. The above results indicate that HZ-2 exhibits selective antiproliferative activity against all five solid tumor cell types, especially sensitive to MCF-7 cells and MDA-MB-231 cells, but its broad-spectrum activity is poor, which may be related to its intramolecular conformation.

[0164] As shown in Table 1, HZ-3 has an IC50 of 100% in A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468 cells. 50The values ​​were 47.98 μM, 211.3 μM, 38.54 μM, 33.28 μM, and 51.85 μM, respectively, all significantly higher than the corresponding values ​​of the parent compound BTSA1. These results indicate that HZ-3 exhibited weak anti-proliferative activity against all five solid tumor cell types, particularly showing extremely weak activity in MCF-7 cells, thus lacking further research value.

[0165] Table 1 shows that the IC50 of HZ-5 in A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468 cells is [data missing]. 50 The concentrations were 4.30 μM, 12.03 μM, 4.31 μM, 2.38 μM, and 4.88 μM, respectively. Compared with the corresponding values ​​of HZ-1, the activity was slightly improved in A549, NCI-H446, and MDA-MB-468 cells, and slightly decreased in MDA-MB-231 cells, but all were significantly better than the parent compound BTSA1. These results indicate that the ortho-hydroxyl configuration is well compatible with the benzene ring extension structure on the thiazole ring, maintaining or further enhancing the activity advantage, and has further development value.

[0166] Table 1 shows that the IC50 of HZ-6 in A549 and MDA-MB-231 cells is... 50 The values ​​were 10.08 μM and 7.42 μM, respectively. Compared with the corresponding values ​​of HZ-2 (22.53 μM and 3.22 μM), the activity was increased by about 2.2 times in A549 cells, but the activity was weakened in MDA-MB-231 cells.

[0167] The data in Tables 1 and 2 show that the IC50 values ​​of HZ-6 and HZ-12 in A549 and MCF-7 cells are... 50 The values ​​were all greater than 10 μM, showing weak anti-proliferative activity against A549 and MCF-7 cells; while the IC50 values ​​were higher in NCI-H446 and MDA-MB-231 cells. 50 The values ​​were all less than 10 μM, and both HZ-12 and HZ-6 exhibited strong anti-proliferative activity against NCI-H446 and MDA-MB-231 cells. These results indicate that HZ-12 and HZ-6 have similar activity ranges and specificity. The IC50 value of HZ-12 in MDA-MB-468 cells was... 50 The value was 8.48 μM, which, compared with the corresponding value of HZ-6 (11.71 μM), showed a slight increase in activity in MDA-MB-468 cells. This result indicates that introducing a fluorine atom onto the thiazole ring containing R3 is beneficial to enhancing the antiproliferative activity of the derivative against MDA-MB-468 cells.

[0168] Table 1 shows that the IC50 of HZ-7 in A549, NCI-H446, and MDA-MB-231 cells is... 50 The values ​​were 18.24 μM, 7.17 μM, and 6.02 μM, respectively. Compared with the corresponding values ​​of HZ-5 (4.30 μM, 4.31 μM, and 2.38 μM), the activities were all weakened, but compared with the corresponding values ​​of HZ-3 (A549 47.98 μM, NCI-H446 38.54 μM), the activities in A549 and NCI-H446 cells were increased by approximately 2.6-fold and 5.4-fold, respectively, and moderate activity was also observed in MDA-MB-231 cells. These results indicate that the fused ring of benzothiazole effectively offsets the activity loss caused by meta-substitution, and the phenyl extension on the thiazole ring may compensate for the adverse effects of meta-hydroxyl substitution by changing the molecular conformation.

[0169] Table 2 shows that the IC50 of HZ-11 in five cell types: A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468. 50 The values ​​were 5.65 μM, 5.35 μM, 6.12 μM, 1.91 μM, and 4.25 μM, respectively. Compared with the corresponding values ​​of the parent compound BTSA1, this indicates that HZ-11 exhibits good anti-proliferative activity against all five solid tumor cell types, demonstrating good broad-spectrum anti-solid tumor potential. Compared with the corresponding values ​​of HZ-5, the activity was enhanced in MCF-7, MDA-MB-231, and MDA-MB-468 cells, indicating that the introduction of a fluorine atom into the thiazole ring containing R3 is beneficial to enhancing the anti-proliferative activity of the derivative against MCF-7, MDA-MB-231, and MDA-MB-468 cells.

[0170] Table 2 shows that HZ-13 and HZ-14 are products obtained by nitro substitution of fluorine atoms in HZ-11 and HZ-12, respectively. Specifically, HZ-13 has an IC50 of [missing information - likely related to cell types A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468]. 50 The values ​​were 8.41 μM, 14.69 μM, 4.09 μM, 1.69 μM, and 5.59 μM, respectively; the IC50 values ​​of HZ-14 in five cell lines: A549, MCF-7, NCI-H446, MDA-MB-231, and MDA-MB-468. 50 The values ​​were 18.76 μM, 41.18 μM, 19.73 μM, 12.11 μM, and 15.55 μM, respectively. These results indicate that HZ-13 exhibited superior anti-proliferative activity against all five solid tumors compared to HZ-14, demonstrating good potential for broad-spectrum anti-solid tumor activity.

[0171] In summary, when R1 in a BTSA1 derivative is a hydroxyl group, the substitution position of this hydroxyl group on the benzene ring has a decisive influence on its anti-solid tumor activity, with ortho-substitution being the preferred configuration for enhanced activity. Further comparison of the activities of derivatives with different hydroxyl substitution positions (ortho, para, meta) after benzothiazole fused ring substitution of thiazole showed that ortho-hydroxyl substitution resulted in the most significant activity enhancement, followed by para-substitution, while meta-substitution contributed almost nothing and even led to decreased activity. The effect of benzothiazole fused ring on different substitution positions varied significantly, with only ortho-substitution maintaining high activity. From a structure-activity relationship (SAR) perspective, introducing a hydroxyl group onto the benzene ring containing R1 (enhancing hydrophilicity) and introducing a benzothiazole fused ring onto the thiazole ring containing R3 (enhancing hydrophobicity) both positively contributed to anti-solid tumor activity. These results provide effective strategies for further optimization of the BTSA1 skeleton and the preparation of derivatives with high anti-solid tumor activity.

[0172] Table 1. IC50 of the compounds synthesized in Examples 1 and 2 with BTSA1 50 Value comparison

[0173]

[0174] Table 2 shows the IC50 of the compounds synthesized in Examples 3 and 4 with BTSA1. 50 Value comparison

[0175]

[0176] Table 3 shows the IC50 of the compounds synthesized in Examples 5 and 6 with BTSA1. 50 Value comparison

[0177]

[0178] Table 4 shows the IC50 of the compounds synthesized in Examples 7 and 8 with BTSA1. 50 Value comparison

[0179]

[0180] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. A class of BTSA1 derivatives, characterized in that, The general structural formula of this derivative is as follows: R1 is selected from hydrogen, hydroxyl, nitro, amino, acetoxy, halogen, unsubstituted or substituted hydrocarbon group; the hydrocarbon group is a C1 to C25 saturated or unsaturated hydrocarbon group containing or not containing a cyclic structure, and the substituent in the substituted hydrocarbon group is a heteroatom or halogen, and the heteroatom is O, N, S or P. R2 is selected from hydrogen, heteroatoms, halogens, and alkyl groups; the heteroatoms are O, N, S, or P; the alkyl groups are C1 to C10 alkyl groups with or without cyclic structures. R3 is selected from hydrogen, unsubstituted or substituted hydrocarbon groups; the hydrocarbon group is a C4 to C7 saturated or unsaturated hydrocarbon group with or without a cyclic structure, and the substituent in the substituted hydrocarbon group is a heteroatom, halogen or nitro group, and the heteroatom is O, N, S or P. R1, R2, and R3 are not all hydrogen.

2. A method for preparing the BTSA1 derivative as described in claim 1, characterized in that, Method 1, as shown in equation (I), includes: The aminothiourea was reacted with reagent I to obtain intermediate I; reagent III was reacted with intermediate II to obtain intermediate III; intermediate I was reacted with intermediate III to obtain the BTSA1 derivative. （I） Or, as shown in method (II), it includes: The aminothiourea was reacted with reagent I to obtain intermediate I. Intermediate I was reacted with reagent III to obtain intermediate XI. Intermediate XI was reacted with intermediate II to obtain the BTSA1 derivative. (II) When R1 in the BTSA1 derivative obtained by method one or method two is a hydroxyl group, the BTSA1 derivative is subjected to alkylation and esterification reactions with R4-X or R4-COOH, respectively, to obtain further substituted BTSA1 derivatives, as shown in formulas (III) and (IV); wherein R4 is selected from C1 to C20 alkyl groups and their substituted derivatives; wherein the substituents of the alkyl substituted derivatives are heteroatoms, and the heteroatoms are O, N, S or P; wherein X is a halogen; 。 3. The preparation method according to claim 2, characterized in that, Method 1 specifically includes the following steps: (1) At room temperature, aminothiourea, reagent I, acetonitrile and sodium acetate are mixed and stirred to react; after the reaction is completed, the mixture is filtered, the solid is collected, washed successively with cold acetonitrile, deionized water and cold ethanol, and dried at 50-60℃ under vacuum for 4-6 hours to obtain intermediate I; (2) At room temperature, reagent III was added to chemically pure ethanol and stirred until the solution was clear. Sodium acetate was added, and the pH of the system was adjusted to 7-8 with sodium carbonate solution. The system was then cooled to 0-10°C, and intermediate II diazonium salt solution was added. After the addition was completed, the system was heated to 30-35°C and stirred at this temperature for 8-10 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the pH of the system was adjusted to 6.5-7.5 with saturated sodium bicarbonate. The mixture was then extracted with ethyl acetate, the organic layers were combined, washed with deionized water and saturated brine, dried with anhydrous sodium sulfate or anhydrous magnesium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure at 50-60°C to obtain the crude product. The crude product was purified by silica gel column chromatography, the fractions containing the product were combined, and the mixture was dried under reduced pressure to obtain intermediate III. (3) At room temperature, intermediate I obtained in step (1), intermediate III obtained in step (2) and pure acetic acid are stirred evenly, heated to the reflux temperature of pure acetic acid, and stirred for 6 to 12 hours. After the reaction is completed, the mixture is cooled to room temperature, filtered under vacuum, the solid is collected, washed with deionized water and ethanol, and dried under vacuum at a temperature of 50 to 60°C to obtain the BTSA1 derivative. Method 2 specifically includes the following steps: (1) Dissolve potassium carbonate in a mixed solution of deionized water and ethanol to obtain potassium carbonate solution; mix reagent I, aminothiourea and potassium carbonate solution at room temperature and stir to react; after the reaction is completed, filter under vacuum, collect the solid, wash with cold ethanol and dry to obtain intermediate I; (2) At room temperature, pure acetic acid and intermediate I obtained in step (1) are mixed to form a suspension. Then, reagent III is added and the mixture is heated to the reflux temperature of pure acetic acid and stirred to react. After the reaction is completed, the mixture is filtered under vacuum, the solid is collected, washed with deionized water and ethanol in sequence, and dried to obtain intermediate XI. (3) At room temperature, intermediates XI and II obtained in step (2) and sodium nitrite are dissolved in chemically pure ethanol and reacted. After the reaction is completed, deionized water is added to the reaction mixture, the pH of the system is adjusted to 6.5-7 with hydrochloric acid, the mixture is filtered under vacuum, the solid is collected, and the solid is washed with deionized water and ethanol in sequence to obtain the BTSA1 derivative.

4. The preparation method according to claim 2, characterized in that, When R1 in the BTSA1 derivative obtained by Method 1 or Method 2 is a hydroxyl group, the BTSA1 derivative is subjected to alkylation and esterification reactions with R4-X or R4-COOH, respectively, to obtain a further substituted BTSA1 derivative, specifically including the following steps: When BTSA1 derivatives undergo alkylation with R4-X: At room temperature, the BTSA1 derivative with R1 being a hydroxyl group was mixed with N,N-dimethylformamide and potassium carbonate, and the mixture was heated to a certain temperature to carry out the reaction. After the reaction was completed, the mixture was cooled to room temperature, and R4-X was slowly added. After the addition was completed, the system was heated to reflux under a nitrogen atmosphere and the reaction was stirred. After the reaction was completed, the reaction mixture was cooled to room temperature, an ice-water mixture was added, and the combined organic layers were extracted with ethyl acetate. The mixture was washed successively with deionized water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by recrystallization or column chromatography to obtain the BTSA1 derivative. When BTSA1 derivatives undergo esterification with R4-COOH: At room temperature, the BTSA1 derivative with R1 being a hydroxyl group was mixed with R4-COOH and anhydrous 1,4-dioxane. The mixture was cooled to a certain temperature, and triphenylphosphine was added to the mixture in batches. After the addition of diethyl azodicarbonate was completed, the system was heated to reflux under a nitrogen atmosphere and the reaction was stirred. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated with dichloromethane and recrystallized with ethanol to obtain the BTSA1 derivative.

5. The preparation method according to claim 3, characterized in that, In step (1) of Method 1, the mass-to-volume ratio of aminothiourea to acetonitrile is 1 g: 3 mL; the molar ratio of aminothiourea to sodium acetate and reagent I is 1: 2: 1; the temperature of the cold acetonitrile and cold ethanol is 0-5 °C; in step (2) of Method 1, the molar ratio of reagent III to chemically pure ethanol and sodium acetate is 1: 10: 3; the molar ratio of reagent III to diazonium salt of intermediate II is 1: 1.3; in step (3) of Method 1, the molar ratio of intermediate III to intermediate I and pure acetic acid is 1: 1.5: 5; the reflux temperature of pure acetic acid is 117-120 °C.

6. The preparation method according to claim 3, characterized in that, In step (2) of Method 1, the intermediate II diazonium salt solution is an intermediate II diazonium hydrochloride solution or an intermediate II diazonium phosphate solution; When the intermediate II diazonium salt solution is an intermediate II diazonium hydrochloride solution, the preparation method of the intermediate II diazonium hydrochloride solution is as shown in formula (V), including: Add reagent II to hydrochloric acid, stir until homogeneous, and then cool to a certain temperature to obtain a homogeneous solution. Keep the temperature constant and slowly add sodium nitrite aqueous solution to the homogeneous solution obtained above under stirring. After the addition is complete, continue stirring for a certain time to obtain intermediate II diazonium hydrochloride solution. （V） When the intermediate II diazonium salt solution is an intermediate II diazonium phosphate solution, the preparation method of the intermediate II diazonium phosphate solution is as shown in formula (VI), including: Add reagent II to an aqueous solution of phosphoric acid, heat to 55-60°C, stir for a certain time, then cool the system to a certain temperature, and slowly add an aqueous solution or solid sodium nitrite while stirring, keeping the temperature constant and continuing to stir to obtain intermediate II diazonium phosphate solution. 。 7. The preparation method according to claim 6, characterized in that, In the preparation method of intermediate II diazo hydrochloride solution, the concentration of hydrochloric acid is 0.5-1.0M; the mass concentration of sodium nitrite aqueous solution is 30-50%; the molar ratio of reagent II to hydrogen chloride in hydrochloric acid is 1:1; and the molar ratio of reagent II to sodium nitrite is 1:1.

1. In the preparation method of intermediate II diazophosphate solution, the mass concentration of the phosphoric acid aqueous solution is 30-50%; the concentration of the sodium nitrite aqueous solution is 1.0-1.5M; the molar ratio of reagent II to phosphoric acid is 1:1.1-1.2; and the molar ratio of reagent II to sodium nitrite is 1:1.1-1.

2.

8. The preparation method according to claim 3, characterized in that, In step (1) of method two, the molar ratio of reagent I to potassium carbonate and aminothiourea is 1:1:1.2; the volume ratio of deionized water to ethanol is 1:1; the mass concentration of the potassium carbonate solution is 30-50%; and the temperature of the cold ethanol is 0-5℃. In step (2) of method two, the molar ratio of intermediate I to acetic acid and reagent III is 1:6:1.

2. In step (3) of method two, the molar ratio of intermediate XI to intermediate II, sodium nitrite, and ethanol is 1:1.2:3:

60.

9. The preparation method according to claim 4, characterized in that, In the alkylation reaction of BTSA1 derivatives with R4-X, the molar ratio of BTSA1 derivatives with hydroxyl R1 to N,N-dimethylformamide and potassium carbonate is 1:5:2.5; the molar ratio of BTSA1 derivatives with hydroxyl R1 to R4-X is 1:1.

2. In the esterification reaction of BTSA1 derivatives with R4-COOH, the molar ratio of BTSA1 derivatives with hydroxyl R1 to R4-COOH is 1:1; the molar ratio of BTSA1 derivatives with hydroxyl R1 to triphenylphosphine is 1:1.5; and the molar ratio of BTSA1 derivatives with hydroxyl R1 to diethyl azodicarbonate is 1.5:

1.

10. The use of a BTSA1 derivative as described in claim 1 and a BTSA1 derivative prepared according to the method of any one of claims 2-9 in the preparation of a drug for treating broad-spectrum solid tumors.

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