2-acylamino-5-piperazine benzoic acid compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine synthesis, in particular to a 2-acylamino-5-piperazine benzoic acid compound as well as a preparation method and application thereof. The 2-acylamino-5-piperazine benzoic acid compound is selected from a compound represented by the following structural formula or pharmaceutically acceptable salts thereof: # imgabs0 #, in the formula, R2 is mono-substituted phenyl or mono-substituted benzyl, and R1 is hydrogen, C1-C5 unsubstituted alkyl or substituted acylamino. The compound can be effectively combined with a Skp2-Cks1 compound, so that the compound has a good treatment effect on breast cancer, gastric cancer and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to 2-amido-5-piperazinebenzoic acid compounds, a preparation method thereof and applications thereof. Background Art

[0002] Skp2 (S-phase kinase-associated protein 2) is an F-box protein that is a key component of the SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex and mainly mediates the ubiquitination and degradation of various cell cycle regulatory proteins. Abnormally high expression of Skp2 is closely related to a variety of human cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), etc. Studies have shown that Skp2 targets p27 Kip1 、p21 Cip1 Skp2 promotes cell cycle progression by degrading tumor suppressor proteins such as skp2 and skp2, and affects tumor cell proliferation, invasion, and drug resistance by regulating signaling pathways such as Akt and c-Myc. Therefore, Skp2 is considered a highly promising anti-tumor target.

[0003] Cks1 (Cyclin-dependent kinases regulatory subunit 1) is a small molecule regulatory protein that plays a crucial role in Skp2-mediated p27 Kip1 Cks1 plays a key role in the ubiquitination and degradation process. Cks1 can enhance the binding of Skp2 to p27^Kip1^, thereby accelerating its degradation and promoting cell cycle progression. Studies have found that abnormal expression of Cks1 not only affects p27 Kip1 The homeostatic regulation of Skp2 can also enhance the oncogenic activity of Skp2. Therefore, targeting the Skp2-Cks1 interaction and blocking the synergistic effect of the two has become a potential anti-tumor strategy.

[0004] Currently, research on Skp2 mainly focuses on the inhibition of its ubiquitination function, but reports have shown that inhibiting the ubiquitin ligase activity of Skp2 alone may not be able to completely block its carcinogenic effects. In addition, Skp2 inhibitors still face challenges such as selectivity, activity and toxic side effects in clinical applications. In contrast, interfering with the Skp2-Cks1 interaction can more directly inhibit Skp2-mediated protein degradation, thereby achieving more efficient and specific anti-tumor effects. Although research on Skp2-Cks1 inhibitors is still in its early stages, the development of small molecule Skp2-Cks1 inhibitors with strong selectivity, high activity and low toxicity is expected to provide new solutions for cancer treatment.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide 2-amido-5-piperazinebenzoic acid compounds, their preparation methods and applications. The present invention provides a novel compound that can effectively bind to the Skp2-Cks1 complex, thereby having a good therapeutic effect on breast cancer, gastric cancer, etc.

[0007] The present invention is achieved in that: In a first aspect, the present invention provides a 2-amido-5-piperazinebenzoic acid compound selected from the compounds represented by the following structural formula or pharmaceutically acceptable salts thereof: , Wherein, R2 is a monosubstituted phenyl group or a monosubstituted benzyl group, and R1 is hydrogen, a C1-C5 unsubstituted alkyl group or a substituted amide group.

[0008] In a second aspect, the present invention provides a method for preparing the 2-amido-5-piperazinebenzoic acid compound described in the aforementioned embodiment, which is synthesized according to the following synthetic route:

[0009] , X is halogen, R2 is monosubstituted phenyl or monosubstituted benzyl, and R1 is hydrogen, C1-C5 unsubstituted alkyl or substituted amide.

[0010] In a third aspect, the present invention provides a Skp2-Cks1 inhibitor, which includes the 2-amido-5-piperazinebenzoic acid compound described in the aforementioned embodiment.

[0011] In a fourth aspect, the present invention provides a use of the 2-amido-5-piperazinebenzoic acid compound described in the aforementioned embodiment in the preparation of a Skp2-Cks1 inhibitor or an anti-tumor drug.

[0012] In an optional embodiment, the tumor includes breast cancer, lung cancer, colon cancer, glioma and gastric cancer.

[0013] The present invention has the following beneficial effects: The embodiment of the present invention provides a new 2-amido-5-piperazinebenzoic acid compound that has a good inhibitory effect on the Skp2-Cks1 complex, and also has a good therapeutic effect on tumors such as breast cancer, gastric cancer and glioma, and can be used in the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is the titration curve of the 2-amido-5-piperazinebenzoic acid compound 91 provided in an example of the present invention and the recombinant Skp2-Cks1 protein complex. DETAILED DESCRIPTION

[0016] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0017] In a first aspect, an embodiment of the present invention provides a 2-amido-5-piperazinebenzoic acid compound selected from the compounds represented by the following structural formula or pharmaceutically acceptable salts thereof: , The salt may be a sodium salt or a potassium salt.

[0018] Specifically, R1 is hydrogen, C1-C5 unsubstituted alkyl or substituted amide, and the substituent of the substituted amide is selected from any one of C1-C5 unsubstituted alkyl, C2-C6 ether, halogen-substituted phenyl, cyano, C3-C5 cycloalkyl substituted C1-C5 alkyl, halogen-substituted C1-C5 alkyl and 3-membered to 6-membered heterocycloalkyl substituted C1-C5 alkyl.

[0019] For example, R1 is any one of hydrogen, methyl, ethyl, propyl, n-butyl substituted amide, isopropyl substituted amide, tert-butyl substituted amide, methyl ethyl ether substituted amide, methyl butyl ether substituted amide, para-halogen substituted phenyl substituted amide, cyclopropyl methylene substituted amide and chloroethyl substituted amide.

[0020] Preferably, R1 is a C1-C5 unsubstituted alkyl group, such as a methyl group, an ethyl group, etc. R1 can also be a para-halogen-substituted phenyl substituted amide group, a C3-C5 cycloalkyl substituted C1-C5 alkyl substituted amide group, a cyano substituted amide group, and a C1-C5 unsubstituted alkyl amide group.

[0021] R2 is a monosubstituted phenyl group or a monosubstituted benzyl group. Specifically, the substitution position of the monosubstituted phenyl group is located at the para position, meta position, and para position of the phenyl ring. The substituent of the monosubstituted phenyl group can be any one of halogen, C1-C3 unsubstituted alkyl, and C1-C3 alkoxy. For example, any one of fluorine, chlorine, methoxy, methyl, and ethyl.

[0022] The substituent of the monosubstituted benzyl group is selected from C1-C3 alkoxy groups, preferably methoxy groups.

[0023] It should be noted that the halogen mentioned above may be any one of fluorine, chlorine, bromine and iodine.

[0024] The C1-C3 unsubstituted alkyl group or the C1-C5 unsubstituted alkyl group may be a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group or the like.

[0025] The C1-C3 alkoxy group may be methoxy, ethoxy, propoxy or the like.

[0026] The C3-C5 cycloalkyl group may be a cycloalkyl group such as cyclopropyl, cyclobutyl and cyclopentyl.

[0027] The heteroatom of the 3-membered to 6-membered heterocycloalkyl group may be an oxygen, nitrogen, sulfur or other heteroatom.

[0028] Specifically, the 2-amido-5-piperazinebenzoic acid compound is selected from any one of the compounds represented by the following structural formulas: .

[0029] It should be noted that the numbered sections below the structural formulas correspond to the numbers of the embodiments.

[0030] In a second aspect, an embodiment of the present invention provides a method for preparing the above-mentioned 2-amido-5-piperazinebenzoic acid compound, comprising: synthesizing according to the following synthesis path: , That is to say, the amidation reaction can be carried out with different acyl halides first, and then the hydrolysis reaction can be carried out.

[0031] Specifically, the compounds represented by numbers 9a to 9o were synthesized according to the following synthetic routes.

[0032]

[0033] The experimental reagents, reaction conditions and reaction yields of the above synthesis route are as follows: (a) EDCI, HOBt, DCM, rt, 12-24h, yield: 40-60%; (b) TFA, DCM, 0℃-rt, 6-12h, yield: 50-70%; (c) Toluene, 60℃, 4-5h, yield: 87%; (d) DIPEA, NMP, 135℃, 24-48h, yield: 60-90%; (e) Zn, NH4Cl, MeOH, 60℃, 2h, yield: 75-90%; (f) Et3N, DCM, rt, 2-4h, yield:70-80%; (g) TFA, DCM, rt, 12-24h, yield: 60-75%.

[0034] Specifically, the compounds represented by numbers 9p-9q were synthesized according to the following synthetic route:

[0035] The experimental reagents, reaction conditions, and reaction yields of the above synthesis pathways are as follows: (d) DIPEA, NMP, 135℃, 24-48h, yield: 60-90%; (e) Zn, NH4Cl, MeOH, 60℃, 2h, yield: 75-90%; (f) Et3N, DCM, rt, 2-4h, yield: 70-80%; (g) TFA, DCM, rt, 12-24h, yield: 60-75%.

[0036] Specifically, the compound represented by No. 9r was synthesized according to the following synthetic route.

[0037]

[0038] Reagents and reaction conditions: (f) Et3N, DCM, rt, 2-4h, yield: 70-80%; (g) TFA, DCM, rt, 12-24h, yield: 60-75%.

[0039] In a third aspect, the present invention provides a Skp2-Cks1 inhibitor, which includes the 2-amido-5-piperazinebenzoic acid compound described in the aforementioned embodiment.

[0040] In a fourth aspect, the present invention provides a use of the 2-amido-5-piperazinebenzoic acid compound described in the aforementioned embodiment in the preparation of a Skp2-Cks1 inhibitor or an anti-tumor drug. For example, the tumor includes breast cancer, lung cancer, colon cancer, glioma, and gastric cancer.

[0041] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0042] Example 1-Example 15 Examples 1-15 provide a 2-amido-5-piperazinebenzoic acid compound, respectively, denoted as 9a-90, and the preparation methods thereof are as follows:

[0043] The specific steps are as follows: 1. Reaction a Under nitrogen protection, dichloromethane (30 mL) was added to a reaction flask containing 4-Boc-1-piperazineacetic acid (1) (1.00 g, 4.10 mmol, 1.0 equiv). Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.18 g, 6.15 mmol, 1.5 equiv) and 1-hydroxybenzotriazole (HOBt) (0.836 g, 6.15 mmol, 1.5 equiv) were added and activated by stirring at room temperature. The corresponding primary amine (12.30 mmol, 3.0 equiv) was added to the reaction system and continued to stir at room temperature for 12-24 h. After the reaction was completed, water (20 mL) was added to quench the reaction. After separation, the organic phase was washed with saturated brine (20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The intermediates 2a-1 were purified by silica gel column chromatography (BUCHI Pure C-810; eluent: petroleum ether / ethyl acetate = 3:1, v / v) in 40-60% yields.

[0044] 2. Reaction b Intermediate 2a-1 (1.0 mmol) was dissolved in dichloromethane (2 mL) and cooled to 0°C in an ice bath. Trifluoroacetic acid (10 mL) was slowly added dropwise, and the reaction mixture was then warmed to room temperature and stirred for 6-12 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure. Water (20 mL) was added to the residue, and the mixture was extracted and washed with ethyl acetate (30 mL x 3). After separation, the aqueous phase was concentrated under reduced pressure, and the residue was washed with methanol (50 mL x 3). Filtration and concentration of the filtrate under reduced pressure afforded the key intermediate 3a-1 in a 50-70% yield.

[0045] 3. Reaction c 5-Fluoro-2-nitrobenzoic acid (4) (1.0 g, 5.40 mmol, 1.0 equiv) and O-tert-butyl-N,N'-diisopropylisourea (4.85 mL, 21.60 mmol, 4.0 equiv) were dissolved in toluene (20 mL) and heated to 60°C for 4-5 h. A large amount of white solid precipitated during the reaction. After the reaction, the reaction solution was filtered and the filter cake was thoroughly washed with dichloromethane (50 mL). The filtrate was combined and concentrated under reduced pressure to obtain tert-butyl 5-fluoro-2-nitrobenzoate (5) as a yellow-green oil. This intermediate was used directly in the next reaction without further purification with a yield of 87%.

[0046] 4. Reaction d Intermediate 5 (482.44 mg, 2.0 mmol, 1.0 equiv) and intermediate 3a-1 (6.0 mmol, 3.0 equiv) were dissolved in N-methylpyrrolidone (15 mL). The reaction mixture was heated to 135°C and stirred for 24-48 hours. After the reaction, the mixture was diluted with water (100 mL) and stirred for another 3 hours. The precipitated solid was filtered, washed thoroughly with water, and then dried under vacuum at 60°C to yield intermediate 6a-1. This crude product was used directly in the next reaction without further purification, with a yield of 60-90%.

[0047] 5. Reaction e Intermediate 6a-1 (2.0 mmol, 1.0 equiv), zinc powder (1.96 g, 30.0 mmol, 15.0 equiv), and ammonium chloride (1.60 g, 30.0 mmol, 15.0 equiv) were dissolved in methanol (20 mL) and heated to 60°C for 2 h. After completion of the reaction, the reaction mixture was filtered through celite, and the filter cake was washed thoroughly with ethanol. The combined filtrates were concentrated under reduced pressure to afford intermediate 7a-1. This crude product was used directly in the next reaction without further purification, with yields of 75-90%.

[0048] 6. Reaction f To a solution of intermediate 7a-l (2.0 mmol, 1.0 equiv) in dichloromethane (10 mL) were added triethylamine (83.4 μL, 0.60 mmol, 0.3 equiv) and the corresponding acid chloride (3.0 mmol, 1.5 equiv) in sequence. The mixture was stirred at room temperature for 2-4 h. After completion of the reaction, the mixture was quenched by addition of saturated NaHCO₃ solution. After separation, the organic phase was separated and concentrated under reduced pressure. Purification by silica gel column chromatography (BUCHI Pure C-810; eluent: petroleum ether / ethyl acetate = 1:1, v / v) afforded intermediate 8a-o in 70-80% yield.

[0049] 7. Reaction g Trifluoroacetic acid (10 mL) was added to a solution of intermediate 8a-o (1 mmol) in dichloromethane (2 mL) at 0°C. The mixture was slowly warmed to room temperature and stirred for 12-24 hours. After completion of the reaction, the reaction solution was concentrated. Ethyl acetate (3 mL) was added to the residue and stirred for 1 hour. The solid was collected by filtration and washed thoroughly with ethyl acetate. The resulting solid was dried under vacuum at 60°C to afford the desired product 9a-o in approximately 60-75% yield.

[0050] Examples 16-17 Examples 16-17 provide a 2-amido-5-piperazinebenzoic acid compound, denoted as 9p-9q, respectively, and their preparation methods are as follows:

[0051] The specific steps are as follows: 1. Reaction d Intermediate 5 (482.44 mg, 2.0 mmol, 1.0 equiv) and intermediate 3m-o (6.0 mmol, 3.0 equiv) were dissolved in N-methylpyrrolidone (15 mL). The reaction mixture was heated to 135°C and stirred for 24-48 hours. After the reaction, the mixture was diluted with water (100 mL) and stirred for another 3 hours. The precipitated solid was filtered, washed thoroughly with water, and then dried under vacuum at 60°C to yield intermediate 6m-o. The crude product was used directly in the next reaction without further purification, with a yield of 60-90%.

[0052] 2. Reaction e Intermediate 6m-o (2.0 mmol, 1.0 equiv), zinc powder (1.96 g, 30.0 mmol, 15.0 equiv), and ammonium chloride (1.60 g, 30.0 mmol, 15.0 equiv) were dissolved in methanol (20 mL) and heated to 60°C for 2 h. After completion of the reaction, the reaction mixture was filtered through celite, and the filter cake was washed thoroughly with ethanol. The combined filtrates were concentrated under reduced pressure to afford intermediate 7m-o. This crude product was used directly in the next reaction without further purification, with a yield of 75-90%.

[0053] 3. Reaction f To a solution of intermediate 7m-o (2.0 mmol, 1.0 equiv) in dichloromethane (10 mL) were added triethylamine (83.4 μL, 0.60 mmol, 0.3 equiv) and the corresponding acid chloride (3.0 mmol, 1.5 equiv) in sequence. The mixture was stirred at room temperature for 2-4 hours. After completion of the reaction, the mixture was quenched by addition of saturated NaHCO₃ solution. After separation, the organic phase was separated and concentrated under reduced pressure. Purification by silica gel column chromatography (BUCHI Pure C-810; eluent: petroleum ether / ethyl acetate = 1:1, v / v) afforded intermediate 8p-q in 70-80% yield.

[0054] 4. Reaction g Trifluoroacetic acid (10 mL) was added to a solution of intermediate 8p-q (1 mmol) in dichloromethane (2 mL) at 0°C. The mixture was slowly warmed to room temperature and stirred for 12-24 hours. After completion of the reaction, the reaction solution was concentrated. Ethyl acetate (3 mL) was added to the residue and stirred for 1 hour. The solid was collected by filtration and washed thoroughly with ethyl acetate. The resulting solid was dried under vacuum at 60°C to afford the desired product 9p-q in a yield of approximately 60-75%.

[0055] Example 18 Example 18 provides a 2-amido-5-piperazinebenzoic acid compound, denoted as 9r, and its preparation method is as follows: . The specific process is as follows: 1. Reaction f To a solution of intermediate 7m (2.0 mmol, 1.0 equiv) in dichloromethane (10 mL) were added triethylamine (83.4 μL, 0.60 mmol, 0.3 equiv) and the corresponding acid chloride (3.0 mmol, 1.5 equiv) in sequence. The mixture was stirred at room temperature for 2-4 hours. After completion of the reaction, the mixture was quenched by addition of saturated NaHCO₃ solution. After separation, the organic phase was separated and concentrated under reduced pressure. Purification by silica gel column chromatography (BUCHI Pure C-810; eluent: petroleum ether / ethyl acetate = 1:1, v / v) afforded intermediate 8r in 70-80% yield.

[0056] 2. Reaction g Trifluoroacetic acid (10 mL) was added to a solution of intermediate 8r (1 mmol) in dichloromethane (2 mL) at 0°C. The mixture was slowly warmed to room temperature and stirred for 12-24 hours. After completion of the reaction, the reaction solution was concentrated. Ethyl acetate (3 mL) was added to the residue and stirred for 1 hour. The solid was collected by filtration and washed thoroughly with ethyl acetate. The resulting solid was dried under vacuum at 60°C to afford the desired product 9r in an approximately 60-75% yield.

[0057] Comparative Examples 1-5 Comparative Examples 1-5 provide a 2-amido-5-piperazine benzene compound (denoted as 13a-e), respectively, and their structural formulas are shown below: .

[0058] The synthesis method of the compounds of formula 13a-e is as follows, and the synthesis is carried out according to the following synthesis route:

[0059] Reagents and reaction conditions: (d) DIPEA, NMP, 135℃, 24-48h, yield: 60-90%; (e) Zn, NH4Cl, MeOH, 60℃, 2h, yield: 75-90%; (f) Et3N, DCM, rt, 2-4h, yield: 70-80%.

[0060] The specific steps are as follows: 1. Reaction d 4-Ethylpiperidine (compound 3m, 6 mmol, 3 equiv) and commercially available compounds 10a-e (2 mmol, 1 equiv) were dissolved in N-methylpyrrolidone (15 mL). The reaction mixture was heated to 135°C and stirred for 24-48 hours. Water (100 mL) was added to the reaction mixture for dilution, and stirring was continued for 3 hours. The precipitated solid was filtered, washed thoroughly with water, and then dried under vacuum at 60°C to obtain intermediates 11a-e. The crude product was used directly in the next reaction without further purification, with yields of 60-90%.

[0061] 2. Reaction e Intermediates 11a-e (2.0 mmol, 1.0 equiv), zinc powder (1.96 g, 30.0 mmol, 15.0 equiv), and ammonium chloride (1.60 g, 30.0 mmol, 15.0 equiv) were dissolved in methanol (20 mL) and heated to 60°C for 2 h. After completion of the reaction, the reaction mixture was filtered through celite, and the filter cake was washed thoroughly with ethanol. The combined filtrates were concentrated under reduced pressure to afford intermediates 7a-e. This crude product was used directly in the next reaction without further purification, with yields of 75-90%.

[0062] 3. Reaction f To a solution of intermediate 12a-e (2.0 mmol, 1.0 equiv) in dichloromethane (10 mL) were added triethylamine (83.4 μL, 0.60 mmol, 0.3 equiv) and the corresponding acid chloride (3.0 mmol, 1.5 equiv) in sequence, and the mixture was stirred at room temperature for 2-4 h. After completion of the reaction, the mixture was quenched by addition of saturated NaHCO₃ solution. After separation, the organic phase was separated and concentrated under reduced pressure. Purification by silica gel column chromatography (BUCHI Pure C-810; eluent: petroleum ether / ethyl acetate = 1:1, v / v) afforded intermediate 13a-e in 70-80% yields.

[0063] Comparative Example 6 This comparative example provides a method for preparing the compound represented by the following structural formula. The preparation method is the preparation method of the compound represented by the preparation method number 9p-9q, the only difference being that X represents C.

[0064] , which is recorded as 13f.

[0065] Comparative Examples 7-8 This comparative example provides a preparation method of a 2-amido-5-piperazinebenzoic acid compound. The preparation method is the same as the preparation method of the compound represented by 9r, and the only difference is that the corresponding raw materials are changed.

[0066] , which is recorded as 13g.

[0067] , which is recorded as 13h.

[0068] Characterization The characterization data of the compounds obtained in Examples 1-18 and Comparative Examples 1-6 are as follows: Example 1: 2-[(3-Chlorobenzoyl)amino]-5-[4-[2-[(2-methoxyethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9a).

[0069]

[0070] White powder, yield: 62%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.93 (s, 1H), 8.45(d, J = 9.1 Hz, 1H), 7.94 (t, J = 1.9 Hz, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.70 (dd, J = 7.9, 2.1 Hz, 1H), 7.56 (d, J = 2.9 Hz, 1H), 7.33 (dd, J = 9.1, 2.9 Hz, 1H), 3.73 – 3.69 (m, 2H), 3.43 – 3.36 (m, 8H), 3.29 (d, J = 21.3 Hz, 4H), 3.18 (s, 3H).

[0071] 13 C NMR (101 MHz, DMSO- d 6, ppm) δ.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94.

[0072] HRMS (ESI): calcd. C 23 H 27 ClN4O5, [M+H] + m / z : 475.1743; found: 475.1747. Example 2: Sodium 2-[[2-(4-methoxyphenyl)acetyl]amino]-5-[4-[2-[(1,1-dimethylethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoate (Compound 9b).

[0073]

[0074] Pale purple powder, yield: 41%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.62 (d, J = 9.2Hz, 1H), 7.75 (dt, J = 7.8, 1.1 Hz, 1H), 7.68 (d, J = 3.0 Hz, 1H), 7.64 (dt, J =9.7, 2.2 Hz, 1H), 7.53 (td, J = 8.0, 5.6 Hz, 1H), 7.32 (td, J = 8.4, 2.6 Hz, 1H),7.24 (dd, J = 9.2, 3.0 Hz, 1H), 4.03 (s, 2H), 3.55 – 3.43 (m, 13H), 3.36 (s, 3H), 2.01 (s, 1H). 13 C NMR (101 MHz, Methanol- d4, ppm) δ .81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94.

[0075] HRMS (ESI): calcd. C 26 H 33 N4NaO5, [M+H] + m / z : 505.2422; found: 505.2425.

[0076] Example 3: 2-[(4-Methoxybenzoyl)amino]-5-[4-[2-[(1-methylethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9c).

[0077]

[0078] White powder, yield: 57%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.93 (s, 1H), 8.59 (d, J = 9.1 Hz, 1H), 8.50 (d, J = 7.5 Hz, 1H), 7.93 – 7.87 (m, 2H), 7.58 (d, J =3.0 Hz, 1H), 7.33 (dd, J = 9.3, 3.0 Hz, 1H), 7.13 – 7.07 (m, 2H), 3.92 (q, 1H), 3.91 (s, 2H), 3.84 (s, 3H), 3.45 – 3.41 (m, 4H), 3.41 – 3.27 (m, 4H), 1.10(d, J = 6.6 Hz, 6H).

[0079] 13 C NMR (101 MHz, DMSO-d 6, ppm) δ , 170.47, 164.21, 163.75, 162.58,158.84, 158.53, 145.26, 134.81, 129.27, 127.23, 122.55, 121.61, 118.25,117.93, 114.60, 56.93, 55.91, 51.99, 46.25, 41.39, 22.57.

[0080] HRMS (ESI): calcd. C 24 H 30 N4O5, [M+H] + m / z : 455.2289; found: 455.2288.

[0081] Example 4: 2-[(3-Methylbenzoyl)amino]-5-[4-[2-(butylamino)-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9d).

[0082]

[0083] Pale purple powder, yield: 60%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.65 (d, J = 9.2Hz, 1H), 7.78 – 7.71 (m, 2H), 7.68 (d, J = 3.0 Hz, 1H), 7.39 (d, J = 4.7 Hz, 2H),7.24 (dd, J = 9.2, 3.0 Hz, 1H), 4.00 (s, 2H), 3.48 (dd, J = 14.8, 5.2 Hz, 8H),3.26 (t, J = 7.1 Hz, 2H), 2.42 (s, 3H), 1.58 – 1.47 (m, 2H), 1.45 – 1.31 (m,2H), 0.95 (t, J = 7.3 Hz, 3H).

[0084] 13 C NMR (101 MHz, Methanol- d 4, ppm)δ , 170.10, 165.74, 163.78, 144.99,138.61, 135.15, 134.59, 132.49, 128.45, 127.50, 123.97, 122.67, 121.16,118.54, 117.19, 56.61, 52.37, 46.35, 38.90, 30.97, 20.14, 19.66, 12.69.

[0085] HRMS (ESI): calcd. C 25 H 32 N4O4, [M+H] + m / z : 453.2497; found: 453.2500.

[0086] Example 5: 2-[(4-Fluorobenzoyl)amino]-5-[4-[2-[(2-methylpropyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9e).

[0087]

[0088] Yellow-green powder, yield: 63%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.92 (s, 1H), 8.62 (t, J = 5.9 Hz, 1H), 8.53 (d, J = 9.1 Hz, 1H), 8.04 – 7.96 (m, 2H), 7.58 (d, J = 3.0Hz, 1H), 7.40 (t, J = 8.8 Hz, 2H), 7.34 (dd, J = 9.2, 3.0 Hz, 1H), 4.02 (s, 2H), 3.68 – 3.26 (m, 8H), 2.99 (t, J = 6.3 Hz, 2H), 0.87 (d, J = 6.7 Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ8, 170.32, 165.93, 164.64, 163.62, 163.45,159.00, 158.68, 145.53, 134.25, 131.66, 131.63, 130.13, 130.04, 122.36,121.94, 119.04, 118.79, 117.92, 116.46, 116.24, 116.07, 56.74, 51.91, 46.53,46.08, 28.45, 20.44.

[0089] HRMS (ESI): calcd. C 24 H 29 FN4O4, [M+H] + m / z : 457.2246; found: 457.2252.

[0090] Example 6: 2-[(4-methoxybenzoyl)amino]-5-[4-[2-[(3-methoxypropyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9f).

[0091]

[0092] White powder, yield: 53%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.64 (d, J = 9.2Hz, 1H), 7.94 – 7.87 (m, 2H), 7.68 (d, J = 3.0 Hz, 1H), 7.24 (dd, J = 9.2, 3.1Hz, 1H), 7.04 – 6.97 (m, 2H), 4.03 (s, 2H), 3.85 (s, 3H), 3.53 (s, 4H), 3.49– 3.41 (m, 8H), 3.39 – 3.31 (m, 6H), 1.80 (p, J = 6.5 Hz, 2H). 13 C NMR (101 MHz, Methanol- d 4, ppm) δ, 170.08, 165.25, 163.74, 162.90,144.79, 135.48, 128.82, 126.56, 122.86, 121.10, 118.57, 116.80, 113.72,69.76, 60.16, 57.54, 56.51, 54.65, 52.39, 46.36, 36.53, 28.85.

[0093] HRMS (ESI): calcd. C 25 H 32 N4O6, [M+H] + m / z : 485.2395; found: 485.2398.

[0094] Example 7: 2-[(2-Fluorobenzoyl)amino]-5-[4-[2-[(4-fluorophenyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9g).

[0095]

[0096] Brown powder, yield: 36%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.88 (d, J = 5.4 Hz,1H), 10.46 (s, 1H), 8.58 (d, J = 9.2 Hz, 1H), 7.90 (td, J = 7.8, 1.8 Hz, 1H),7.66 (dd, J = 9.0, 5.0 Hz, 2H), 7.62 (dt, J = 8.4, 2.2 Hz, 1H), 7.59 (dd, J = 5.3,2.4 Hz, 1H), 7.45 – 7.34 (m, 2H), 7.31 (dd, J = 9.2, 3.0 Hz, 1H), 7.18 (t, J =8.8 Hz, 2H), 3.80 (s, 2H), 3.38 (t, J = 4.7 Hz, 4H), 3.15 (t, J = 5.0 Hz, 4H). 13C NMR (101 MHz, DMSO- d 6, ppm) δ , 170.02, 165.66, 161.56, 161.01,159.98, 158.93, 158.53, 157.59, 146.25, 135.09, 135.07, 134.18, 134.09,133.35, 131.23, 131.21, 125.49, 125.46, 123.51, 123.38, 122.21, 121.89,121.81, 121.76, 119.33, 117.63, 117.09, 116.86, 115.98, 115.76, 59.20, 52.44,47.14.

[0097] HRMS (ESI): calcd. C 26 H 24 F2N4O4, [M+H] + m / z : 495.1839; found: 495.1844.

[0098] Example 8: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(oxetan-3-ylmethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9h).

[0099]

[0100] Milky white powder, yield: 23%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.67 (d, J = 9.0Hz, 1H), 7.78 (dd, J = 7.6, 1.5 Hz, 1H), 7.73 (d, J = 2.9 Hz, 1H), 7.67 (dt, J =9.6, 2.2 Hz, 1H), 7.55 (td, J = 8.0, 5.5 Hz, 1H), 7.33 (ddt, J = 12.3, 7.0, 2.8Hz, 2H), 3.72 – 3.44 (m, 12H), 3.39 (d, J= 6.5 Hz, 2H), 3.32 (p, J = 1.6 Hz,1H).

[0101] 13 C NMR (101 MHz, Methanol- d 4, ppm) δ .42, 115.25, 113.21, 112.97,112.42, 59.58, 55.72, 51.60, 45.50, 42.59, 37.21.

[0102] HRMS (ESI): calcd. C 24 H 27 FN4O5, [M+H] + m / z : 471.2038; found: 471.2041.

[0103] Example 9: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(2-chloroethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9i).

[0104]

[0105] Dark brown powder, yield: 27%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 8.55 (d, J = 9.1 Hz,1H), 7.75 (d, J = 7.7 Hz, 1H), 7.65 (dt, J = 9.8, 2.2 Hz, 1H), 7.30 (dd, J = 9.2,3.1 Hz, 1H), 3.89 (s, 2H), 3.48 (t, J = 5.8 Hz, 2H), 3.43 (d, J = 5.6 Hz, 4H),3.35 (t, J= 5.0 Hz, 4H), 3.25 (t, J = 5.7 Hz, 2H).

[0106] 13 C NMR (101 MHz, DMSO- d 6, ppm) δ .97, 114.30, 114.07, 114.80, 114.70, 114.75, 114.80, 114.75, 114.81, 114.70, 114.10, 114.70, 114.11, 114.70, 114.80, 114.90, 114.70, 114.81, 114.70, 114.80, 114.90, 114.70, 114.80, 114.80, 114.90, 114.70, 114.80

[0107] HRMS (ESI): calcd. C 22 H 24 ClFN4O4, [M+H] + m / z : 463.1543; found: 463.1548.

[0108] Example 10: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(prop-2-ynyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9j).

[0109]

[0110] Light green powder, yield: 45%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.68 (d, J = 9.1Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 2.7 Hz, 1H), 7.68 (dt, J = 9.7, 2.2Hz, 1H), 7.57 (td, J = 8.0, 5.5 Hz, 1H), 7.40 – 7.27 (m, 2H), 4.08 (d, J = 2.6Hz, 1H), 4.04 (d, J= 9.6 Hz, 2H), 3.91 (dd, J = 5.6, 1.7 Hz, 1H), 3.54 – 3.50(m, 8H).

[0111] 13 C NMR (101 MHz, Methanol- d 4, ppm) δ 170.18, 164.16, 164.05, 163.96,163.88, 161.72, 145.32, 137.10, 137.03, 134.86, 133.42, 130.59,122.67, 122.54, 122.51, 121.27, 118.68, 118.59, 118.46, 117.41, 115.46,114.02, 113.79, 78.51, 71.46, 56.62, 56.54, 52.47, 52.43, 46.42, 41.40,28.17.

[0112] HRMS (ESI): calcd. C 23 H 23 FN4O4, [M+H] + m / z : 439.1776; found: 439.1776.

[0113] Example 11: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(cyanomethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9k).

[0114]

[0115] Pale yellow powder, yield: 31%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.87 (s, 1H), 8.49 (d, J = 9.1 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.73 – 7.60 (m, 2H), 7.58 (d, J =3.0 Hz, 1H), 7.47 (dd, J = 8.5, 2.6 Hz, 1H), 7.35 (dd,J = 9.2, 3.0 Hz, 1H), 7.15(s, 1H), 4.01 (s, 2H), 3.77 (t, J = 5.2 Hz, 2H), 3.64 – 3.13 (m, 7H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ .91, 52.03, 50.67, 46.08, 28.93, 170.58, 170.22, 167.53, 165.29,163.90, 163.35, 161.46, 158.81, 158.50, 145.79, 137.59, 133.80, 131.62,123.38, 122.23, 119.44, 117.84, 114.29, 56.91, 52.03, 50.67, 46.08, 28.93.

[0116] HRMS (ESI): calcd. C 22 H 22 FN5O4, [M+H] + m / z : 440.1729; found: 440.1729.

[0117] Example 12: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(cyclopropylmethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 91).

[0118]

[0119] White powder, yield: 43%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.63 (d, J = 9.1Hz, 1H), 7.79 – 7.73 (m, 1H), 7.68 (d, J = 3.0 Hz, 1H), 7.65 (dt, J = 9.6, 2.2Hz, 1H), 7.53 (td, J = 8.0, 5.6 Hz, 1H), 7.32 (td, J = 8.4, 2.7 Hz, 1H), 7.24(dd, J= 9.2, 3.1 Hz, 1H), 4.02 (s, 2H), 3.55 – 3.47 (m, 8H), 3.14 (d, J = 7.1Hz, 2H), 1.00 (ddd, J = 12.6, 6.1, 3.8 Hz, 1H), 0.53 (dt, 2H), 0.25 (dt, J = 6.2,4.5 Hz, 2H). 13 C NMR (101 MHz, Methanol- d 4, ppm) δ .36, 122.28, 122.18, 119.44, 119.23,119.10, 117.84, 114.51, 114.29, 56.91, 52.03, 50.67, 46.08, 28.93.

[0120] HRMS (ESI): calcd. C 24 H 27 FN4O4, [M+H] + m / z : 455.2089; found: 455.2093.

[0121] Example 13. 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(2-methoxyethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9m).

[0122]

[0123] White powder, yield: 59%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.62 (d, J = 9.2Hz, 1H), 7.75 (dt, J = 7.8, 1.1 Hz, 1H), 7.68 (d, J= 3.0 Hz, 1H), 7.64 (dt, J =9.7, 2.2 Hz, 1H), 7.53 (td, J = 8.0, 5.6 Hz, 1H), 7.32 (td, J = 8.4, 2.6 Hz, 1H),7.24 (dd, J = 9.2, 3.0 Hz, 1H), 4.03 (s, 2H), 3.55 – 3.43 (m, 12H), 3.36 (s, 3H). 13 C NMR (101 MHz, Methanol- d 4, ppm) δ .81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94, 118.65, 118.52, 118.43, 117.31, 114.05, 113.81,70.31, 57.55, 56.60, 52.39, 46.32, 38.94.

[0124] HRMS (ESI): calcd. C 23 H 27 FN4O5, [M+H] + m / z : 459.2038; found: 459.2040.

[0125] Example 14: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(1,1-dimethylethyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9n).

[0126]

[0127] White powder, yield: 34%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.62 (d, J = 9.2Hz, 1H), 7.79 – 7.72 (m, 1H), 7.67 (d, J= 3.0 Hz, 1H), 7.64 (dt, J = 9.4, 2.2Hz, 1H), 7.53 (td, J = 8.0, 5.6 Hz, 1H), 7.32 (td, J = 8.5, 2.6 Hz, 1H), 7.23(dd, J = 9.2, 3.0 Hz, 1H), 3.94 (s, 2H), 3.65 – 3.39 (m, 8H), 1.38 (s, 9H). 13 C NMR (101 MHz, Methanol- d 4, ppm) δ .83, 114.06,113.83, 60.16, 57.00, 52.35, 51.36, 46.30, 27.42, 122.59, 122.55, 122.52, 121.13, 118.65, 118.54, 118.44, 117.33, 114.06,113.83, 60.16, 57.00, 52.35, 51.36, 46.30, 27.42.

[0128] HRMS (ESI): calcd. C 24 H 29 FN4O4, [M+H] + m / z : 457.2246; found: 457.2248.

[0129] Example 15: 2-[(3-Fluorobenzoyl)amino]-5-[4-[2-[(4-fluorophenyl)amino]-2-oxoethyl]-1-piperazinyl]benzoic acid (Compound 9o).

[0130]

[0131] White powder, yield: 55%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.87 (s, 1H), 10.80 (s, 1H), 8.50 (d, J= 9.1 Hz, 1H), 7.81 – 7.75 (m, 1H), 7.74 – 7.57 (m, 5H), 7.48 (td, J = 8.5, 2.7 Hz, 1H), 7.37 (dd, J = 9.2, 3.0 Hz, 1H), 4.23 (s, 2H), 3.49 (s, 8H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ .81, 119.44, 119.23, 119.10, 117.88, 116.20, 115.98, 123.40, 123.37, 122.33, 122.24,121.89, 121.81, 119.44, 119.23, 119.10, 117.88, 116.20, 115.98, 114.53,114.30, 57.27, 52.08, 46.06.

[0132] HRMS (ESI): calcd. C 26 H 24 F2N4O4, [M+H] + m / z : 495.1839; found: 495.1841.

[0133] Example 16: 2-[(4-chlorobenzoyl)amino]-5-(4-ethyl-1-piperazinyl)benzoic acid (Compound 9p).

[0134]

[0135] Light green powder, yield: 68%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.87 (s, 1H), 8.51 (d, J = 9.2 Hz, 1H), 7.97 – 7.89 (m, 2H), 7.69 – 7.61 (m, 2H), 7.57 (d, J= 3.0Hz, 1H), 7.37 (dd, J = 9.2, 3.0 Hz, 1H), 3.92 – 3.12 (m, 6H), 1.30 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ 170.23, 163.65, 145.68, 137.24,134.10, 133.89, 129.47, 129.31, 122.60, 122.10, 118.81, 117.93, 50.94, 50.38,46.10, 9.25.

[0136] HRMS (ESI): calcd. C 20 H 22 ClN3O3, [M+H] + m / z : 388.1423; found: 388.1424.

[0137] Example 17: 2-[(3-Methylbenzoyl)amino]-5-(4-methyl-1-piperazinyl)benzoic acid (Compound 9q).

[0138]

[0139] White powder, yield: 62%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.96 (s, 1H), 8.60 (d, J = 9.1 Hz, 1H), 7.75 (s, 1H), 7.72 (dd, J = 5.7, 3.6 Hz, 1H), 7.60 (d, J = 3.0Hz, 1H), 7.49 – 7.40 (m, 2H), 7.36 (dd, J = 9.2, 3.1 Hz, 1H), 3.95 – 2.93 (m,8H), 2.88 (s, 3H), 2.39 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ8, 115.95, 52.66, 46.33, 42.50, 21.45, 170.44, 164.74, 159.46, 159.14,145.32, 138.71, 135.14, 134.65, 132.98, 129.25, 127.97, 124.47, 122.67,121.71, 118.91, 118.36, 118.08, 115.95, 52.66, 46.33, 42.50, 21.45.

[0140] HRMS (ESI): calcd. C 20 H 23 ClN3O3, [M+H] + m / z : 354.1812; found: 354.1814.

[0141] Example 18: 2-[(3-Fluorobenzoyl)amino]-5-(4-ethyl-1-piperazinyl)benzoic acid (Compound 9r).

[0142]

[0143] Pale yellow powder, yield: 63%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.90 (s, 1H),10.37 (s, 1H), 8.51 (d, J = 9.1 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.68 (dt, J =9.7, 2.2 Hz, 1H), 7.66 – 7.61 (m, 1H), 7.59 (d, J = 3.0 Hz, 1H), 7.47 (td, J =8.5, 2.6 Hz, 1H), 7.37 (dd, J = 9.2, 3.0 Hz, 1H), 3.73 (d, J = 101.6 Hz, 4H),3.22 (q, J = 7.3 Hz, 2H), 3.10 (d, J = 40.4 Hz, 4H), 1.27 (t, J = 7.3 Hz, 3H).

[0144] 13C NMR (101 MHz, DMSO- d 6, ppm) δ .33, 118.71,117.96, 115.76, 114.51, 114.29, 112.81, 51.08, 50.56, 46.24, 9.33.

[0145] HRMS (ESI): calcd. C 20 H 22 FN3O3, [M+H] + m / z : 372.1718; found: 372.1720.

[0146] Comparative Example 1: 9. Methyl 2-(4-chlorobenzamido)-5-(4-ethylpiperazin-1-yl)benzoate (Compound 13a).

[0147]

[0148] Yellow-green oily liquid, yield: 42%. 1 H NMR (400 MHz, Methanol- d 4, ppm) δ 8.66 (s,1H), 8.66 (d, J = 9.2 Hz, 1H), 7.66 (d, J = 3.0 Hz, 1H), 7.64 – 7.56 (m, 2H), 7.47 – 7.41 (m, 2H), 7.35 (dd, J = 9.2, 3.0 Hz, 1H), 3.99 (s, 3H), 3.43 – 3.37(m, 4H), 3.09 (t, J = 5.1 Hz, 4H), 2.93 (q, J = 7.3 Hz, 2H), 1.31 (t, J = 7.3 Hz,3H).

[0149] 13 C NMR (101 MHz, Methanol- d 4, ppm) δ .59, 122.59, 121.59, 117.33,116.81, 51.98, 51.75, 51.64, 51.52, 9.52, 9.53, 9.70, 9.81, 169.55, 168.71, 164.01, 146.31,137.84, 137.26, 133.93, 133.34, 132.97, 130.87, 130.81, 130.77, 128.86,128.75, 128.71, 128.63, 128.59,

[0150] HRMS (ESI): calcd. C 21 H 24 ClN3O3, [M+H] + m / z : 402.1579; found: 402.1581.

[0151] Comparative Example 2: 2-(4-chlorobenzamido)-5-(4-ethylpiperazin-1-yl)benzoyl cyanide (Compound 13b).

[0152]

[0153] White powder, yield: 42%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.10 (s, 1H), 8.16 (d, J = 9.1 Hz, 1H), 7.99 – 7.92 (d, 2H), 7.69 – 7.63 (d, 2H), 7.44 (d, J = 3.0Hz, 1H), 7.30 (dd, J = 9.1, 3.0 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 3.34 (s, 4H), 3.16 (d, J = 5.6 Hz, 4H), 1.04 (t, J = 7.2 Hz, 3H).

[0154] 13 C NMR (101 MHz, DMSO- d6, ppm) δ 170.23, 163.65, 145.68, 137.24,134.10, 133.89, 129.47, 129.31, 122.60, 122.10, 118.81, 117.93, 50.94, 50.38,46.10, 9.25.

[0155] HRMS (ESI): calcd. C 21 H 21 ClN4O2, [M+H] + m / z : 397.1426; found: 397.1429.

[0156] Comparative Example 3: 2-(4-chlorobenzamido)-N-cyano-5-(4-ethylpiperazin-1-yl)benzamide (Compound 13c).

[0157]

[0158] Pale yellow powder, yield: 18%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.87 (s, 1H), 8.51 (d, J = 9.2 Hz, 1H), 7.97 – 7.89 (m, 2H), 7.69 – 7.61 (m, 2H), 7.57 (d, J = 3.0Hz, 1H), 7.37 (dd, J = 9.2, 3.0 Hz, 1H), 3.92 – 3.12 (m, 6H), 1.30 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ 170.23, 163.65, 145.68, 137.24,134.10, 133.89, 129.47, 129.31, 122.60, 122.10, 118.81, 117.93, 50.94, 50.38,46.10, 9.25.

[0159] HRMS (ESI): calcd. C 21 H 22ClN5O2, [M+H] + m / z : 412.1535; found: 412.1537.

[0160] Comparative Example 4: 4-chloro-N-[4-(4-ethylpiperazin-1-yl)phenyl]benzamide (Compound 13d).

[0161]

[0162] White powder, yield: 20%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 10.19 (s, 1H), 7.98 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 6.98 (d, J =9.0 Hz, 2H), 3.33 (q, 2H), 3.22 – 2.76 (m, 8H), 1.19 (t, J = 6.9 Hz, 3H).

[0163] 13 C NMR (101 MHz, DMSO- d 6, ppm) δ 164.34, 136.63, 134.20, 131.62,129.98, 129.21, 128.88, 122.03, 116.39, 51.35.

[0164] HRMS (ESI): calcd. C 19 H 22 ClN3O, [M+H] + m / z : 344.1524; found: 344.1528.

[0165] Comparative Example 5: 4-Chloro-N-[6-(4-ethylpiperazin-1-yl)pyridin-3-yl]benzamide (Compound 13e).

[0166]

[0167] Light green powder, yield: 32%. 1H NMR (400 MHz, Methanol- d 4, ppm) δ 7.94 – 7.83 (m,4H), 7.51 (d, J = 8.1 Hz, 2H), 6.32 (d, J = 8.6 Hz, 1H), 3.57 (t, J = 4.9 Hz, 4H),2.61 (t, J = 5.1 Hz, 4H), 2.51 (q, J = 7.3 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H).

[0168] 13 C NMR (101 MHz, Methanol- d 4, ppm) δ 155.58, 155.28, 137.53, 135.13,133.05, 128.84, 128.42, 110.47, 97.61, 52.31, 52.14, 52.01, 44.64, 10.40.

[0169] HRMS (ESI): calcd. C 18 H 21 ClN4O, [M+H] + m / z : 345.1477; found: 345.1481.

[0170] Comparative Example 6: 2-(4-chlorobenzamide)-5-(4-ethylpiperidin-1-yl)benzoic acid (Compound 13f).

[0171]

[0172] White powder, yield: 37%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 11.87 (s, 1H), 8.51 (d, J = 9.2 Hz, 1H), 7.97 – 7.89 (m, 2H), 7.69 – 7.61 (m, 2H), 7.57 (d, J = 3.0Hz, 1H), 7.37 (dd, J= 9.2, 3.0 Hz, 1H), 3.92 – 3.12 (m, 6H), 1.30 (t, J = 7.3Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6, ppm) δ 170.23, 163.65, 145.68, 137.24,134.10, 133.89, 129.47, 129.31, 122.60, 122.10, 118.81, 117.93, 50.94, 50.38,46.10, 9.25. HRMS (ESI): calcd. C 21 H 23 ClN2O3, [M+H] + m / z : 387.1470; found: 387.1473.

[0173] Comparative Example 7: 5-(4-Ethylpiperazin-1-yl)-2-(propionylamino)benzoic acid (Compound 13g).

[0174]

[0175] Dark brown sticky solid, yield: 16%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 8.33 (s, 1H),7.34 (d, J = 7.0 Hz, 1H), 7.31 – 7.14 (m, 2H), 3.64 – 3.60 (m, 4H), 2.47 – 2.42(m, 4H), 2.34 (q, J = 7.2 Hz, 4H), 1.01 (t, J = 7.0 Hz, 6H).

[0176] 13 C NMR (101 MHz, DMSO- d 6, ppm) δ152.83, 148.50, 140.11, 139.60,139.48, 129.90, 129.61, 128.80, 128.67, 128.43, 127.61, 52.41, 52.13, 44.45,12.36.

[0177] HRMS (ESI): calcd. C 16 H 23 N3O3, [M+H] + m / z : 306.1812; found: 306.1814.

[0178] Comparative Example 8: 2-[(cyclohexylcarbonyl)amino]-5-(4-ethylpiperazin-1-yl)benzoic acid (Compound 13h).

[0179]

[0180] White powder, yield: 25%. 1 H NMR (400 MHz, DMSO- d 6, ppm) δ 10.86 (s, 1H), 10.00 (s, 1H), 8.38 (d, J = 9.1 Hz, 1H), 7.51 (d, J = 3.1 Hz, 1H), 7.30 (dd, J = 9.2, 3.1Hz, 1H), 3.81 (d, J = 12.9 Hz, 2H), 3.59 (d, J = 12.0 Hz, 2H), 3.21 (q, J = 7.2 Hz,2H), 3.11 (d, 2H), 2.98 (d, J = 12.7 Hz, 2H), 2.27 (ddt, J = 11.3, 7.1, 3.5 Hz,1H), 1.92 – 1.85 (m, 2H), 1.74 (dt, J = 12.4, 3.4 Hz, 2H), 1.64 (dt, J = 12.3,3.5 Hz, 1H), 1.50 – 1.28 (m, 3H), 1.25 (t, J = 7.3 Hz, 3H).

[0181] 13 C NMR (101 MHz, DMSO- d 6, ppm) δ 8, 51.08, 50.60,46.42, 46.16, 29.58, 25.87, 25.59, 21.19, 14.51, 9.37.

[0182] HRMS (ESI): calcd. C 20 H 29 N3O3, [M+H] + m / z : 360.2282; found: 360.2285.

[0183] Experimental Example 1 The in vitro antitumor activities of the compounds prepared in Examples 1-18 of the present invention and Comparative Examples 1-8 were detected using the Cell Counting Kit 8 (CCK8) method.

[0184] Among them, the cells used are as follows: six types of adherent cells, including A549 (human non-small cell lung cancer cells), HCT-116 (human colon cancer cells), U87 (human glioma cells), HGC-27 (human gastric cancer cells), MDB-MA-231 (human triple-negative breast cancer cells), MCF-7 (human breast cancer cells) and HUVEC (human umbilical vein endothelial cells).

[0185] The details are as follows: According to 1.5×10 5 cells·mL -1 A 96-well plate was inoculated with 100 μL of liquid per well. The edge of the plate was left uninoculated, and 100 μL of PBS buffer was added to the wells to prevent edge effects. After inoculation, the 96-well plate was transferred to an incubator and incubated for 24 hours before dosing.

[0186] The compounds synthesized in the Examples and Comparative Examples were set to five concentrations: 10 μM, 5.0 μM, 2.50 μM, 1.25 μM, and 0.625 μM. 10 μL of the corresponding compound was then added to each well of the cell plate (three wells per compound). In the blank control group, 10 μL of the corresponding complete culture medium was added to three wells, with three replicates. A blank control group was also set up. The administered compounds were then placed in an incubator and cultured for 48 hours.

[0187] Under a dark background, add 10 μL of CCK8 solution to each experimental well of the cell plate after 48 hours of culture (avoid bubbles during the addition process to prevent them from affecting the experimental results). Then, place the cell plate in an incubator and culture for 2 hours. Then, place the cell plate in a microplate reader to detect the OD value.

[0188] The calculation formula for cell viability is:

[0189] The results are shown in Table 1.

[0190] Table 1 In vitro anti-tumor cell proliferation activity

[0191] a All cells were treated with 10 μM of the compound for 72 hours, and the average inhibition rate was determined using the CCK-8 assay. All data are presented as the mean of three independent experiments.

[0192] As shown in Table 1, the 2-amido-5-piperazinebenzoic acid compounds provided in the embodiments of the present invention have a good known effect on tumor cells. However, if the structural formula of the compound is changed, for example, if the core structure of 13a-13e is changed to a non-benzoic acid, the tumor inhibition effect is extremely poor. Alternatively, if the substituent is changed, for example, R2 is a cycloalkane or alkane, the resulting compound has poor tumor inhibition effect.

[0193] Experimental Example 2 Study on the inhibitory activity of compound 9l against Skp2-Cks1 The specific process is as follows: 1. Instrument and Sample Preparation Before the experiment, the isothermal titration calorimeter was preheated to 37°C and a blank titration was performed using PBS buffer containing 10% DMSO to correct the baseline. The recombinant Skp2-Cks1 protein complex solution and compound 9l sample solution required for the experiment were also pre-equilibrated to 37°C to eliminate the thermal effect caused by the temperature difference between the ligand molecule and the receptor molecule.

[0194] 2. Experimental parameter setting 300 μL of 0.006 mM recombinant Skp2-Cks1 protein complex PBS solution was added to the sample cell and mixed thoroughly to ensure that there were no bubbles. 30 μL of 0.2 mM compound 9l PBS solution was aspirated into the syringe. The titrant concentration should be slightly higher than the protein solution concentration in the sample cell to ensure sufficient titration.

[0195] 3. Titration Procedure The titration was performed using a continuous injection method at a constant temperature of 37°C. For the experimental group, compound 9l solution was added to a sample cell containing the recombinant Skp2-Cks1 protein complex in 20 drops (2.5 μL per drop). For the control group, compound 9l solution was added to a PBS buffer containing 10% DMSO in 20 drops (2.5 μL per drop). After each addition, the solution was allowed to equilibrate for 2 minutes until the thermal signal peak returned to baseline, ensuring a stable thermodynamic response curve.

[0196] 4. Data Processing and Analysis The original titration data were integrated using Origin software, and the titration curve was obtained using the Independent model. Thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), binding constant (Ka), and dissociation constant (Kd) were obtained through further analysis. All experiments were repeated three times independently to ensure data reliability.

[0197] Results see Figure 1 .

[0198] Based on the independent model fitting, the titration curve of compound 9l and recombinant Skp2-Cks1 protein complex was obtained, and the thermodynamic parameters of the binding were obtained, including: 1. Kd (dissociation constant): The Kd value of compound 9l for the recombinant Skp2-Cks1 protein complex was 51.6 μM, indicating that compound 9l had a certain degree of binding affinity for the recombinant Skp2-Cks1 protein complex.

[0199] 2. n (stoichiometric number): The stoichiometric ratio of compound 9l binding to the recombinant Skp2-Cks1 protein complex was 1.649, close to a 2:1 ratio, suggesting that compound 9l may form a 1:2 complex with the recombinant Skp2-Cks1 protein complex.

[0200] 3. ΔH (enthalpy change) and ΔS (entropy change): The binding ΔH of compound 9l to the recombinant Skp2-Cks1 protein complex was -72.89 kJ / mol, and the ΔS was -163.5 J / mol·K, indicating that the binding process was mainly driven by enthalpy and may involve strong polar forces such as hydrogen bonds and electrostatic interactions.

[0201] From the above data, it can be seen that 2-amido-5-piperazinebenzoic acid compound 9l has a good inhibitory effect on the Skp2-Cks1 complex.

[0202] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A 2-amido-5-piperazinebenzoic acid compound, characterized in that: It is selected from the compounds represented by the following structural formula or pharmaceutically acceptable salts thereof: ， Wherein, R2 is a monosubstituted phenyl group or a monosubstituted benzyl group, and R1 is hydrogen, a C1-C5 unsubstituted alkyl group or a substituted amide group.

2. The 2-amido-5-piperazinebenzoic acid compound according to claim 1, wherein The substituent of the monosubstituted phenyl group is selected from any one of a C1-C3 unsubstituted alkyl group, a halogen group and a C1-C3 alkoxy group.

3. The 2-amido-5-piperazinebenzoic acid compound according to claim 1 or 2, characterized in that The substituent of the monosubstituted phenyl group is selected from any one of fluorine, chlorine, methoxy, methyl and ethyl.

4. The 2-amido-5-piperazinebenzoic acid compound according to claim 1, wherein The substituent of the monosubstituted benzyl group is selected from C1-C3 alkoxy groups, preferably methoxy groups.

5. The 2-amido-5-piperazinebenzoic acid compound according to claim 1, wherein The substituent of the substituted amide group is selected from any one of C1-C5 unsubstituted alkyl, C2-C6 ether, halogen-substituted phenyl, cyano, C3-C5 cycloalkyl-substituted C1-C5 alkyl, halogen-substituted C1-C5 alkyl and 3-membered to 6-membered heterocycloalkyl-substituted C1-C5 alkyl.

6. The 2-amido-5-piperazinebenzoic acid compound according to claim 1, characterized in that The 2-amido-5-piperazinebenzoic acid compound is selected from any one of the compounds represented by the following structural formulas: 。 7. A method for preparing the 2-amido-5-piperazinebenzoic acid compound according to claim 1, characterized in that: include: The synthesis was carried out according to the following synthesis route: ， X is halogen, R2 is monosubstituted phenyl or monosubstituted benzyl, and R1 is hydrogen, C1-C5 unsubstituted alkyl or substituted amide.

8. A Skp2-Cks1 inhibitor, characterized in that The invention comprises the 2-amido-5-piperazinebenzoic acid compound according to claim 1.

9. Use of the 2-amido-5-piperazinebenzoic acid compound according to claim 1 in the preparation of a Skp2-Cks1 inhibitor or an anti-tumor drug.

10. The use according to claim 9, characterized in that The tumors include breast cancer, lung cancer, colon cancer, glioma and gastric cancer.

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