A salicylaldehyde derivative and use thereof

Abstract

The application discloses a salicylaldehyde derivative and application thereof. The compound and pharmaceutically acceptable salt and ester thereof can inhibit the combination of STAT3 and phosphorylated polypeptide, and achieve the anti-tumor purpose. The salicylaldehyde derivative can be combined with the SH2 domain of target protein STAT3, and inhibit the combination of the target protein and phosphorylated polypeptide. Compound 25 shows good competitive inhibition activity. In the anti-proliferation activity of pancreatic cancer cells in vitro, the compound 25 has sub-micromolar anti-proliferation activity of pancreatic cancer cells, and can induce cancer cell apoptosis. In addition, the compound 25 can significantly inhibit the phosphorylation of STAT3 705 sites and 727 sites, can inhibit the proliferation of various tumor cells in vitro, and has low toxicity to normal cells. Therefore, the compound can be used for preparing a STAT3 inhibitor, and used for preparing a medicine for preventing and / or treating diseases related to tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a salicylaldehyde derivative and use thereof. BACKGROUND

[0002] Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a key role in a variety of cancers. Under normal circumstances, the activation of STAT3 is strictly regulated, usually showing a transient activation state, which is of great significance to maintain the normal function of cells. In tumor cells, STAT3 is often over-activated, becoming a promoting factor for tumor growth and progression. It can promote the occurrence and development of tumor cells by regulating genes related to cell cycle progression, inhibiting the expression of pro-apoptotic proteins, promoting tumor angiogenesis and regulating genes related to cell invasion and metastasis. Therefore, targeting inhibition of STAT3 activation is considered to be a feasible strategy for treating cancer.

[0003] With the in-depth study of STAT3 signaling pathway, it has been found that the activation of STAT3 depends on both the classical activation signaling pathway and the non-classical activation pathway. The classical and non-classical activation of STAT3 is characterized by phosphorylation of two key amino acid residues Tyr705 and Ser727 located in the transcriptional activation domain (TAD). Phosphorylation of these residues mediates the nuclear transcription and mitochondrial function of STAT3, thereby promoting the occurrence and development of tumor cells. Studies have shown that targeting the STAT3 SH2 domain can effectively inhibit the phosphorylation of Tyr705 and Ser727, thereby inhibiting the STAT3 signaling cascade.

[0004] However, existing small molecule SH2 domain inhibitors are either insufficiently active or have no statistically significant difference compared with the positive control drug. Therefore, designing and synthesizing new small molecule SH2 domain inhibitors plays an important role in blocking the STAT3 signaling cascade. SUMMARY

[0005] The present application aims to provide a salicylaldehyde derivative of formula I or a pharmaceutically acceptable salt or ester thereof:

[0006]

[0007] wherein,

[0008] A ring is selected from phenyl, naphthyl or a six-membered heterocycle or a five-membered heterocycle containing 1-3 heteroatoms, and the heteroatoms are selected from N, O or S;

[0009] B ring is selected from a five-membered heterocycle containing 1-3 heteroatoms, and the heteroatoms are selected from N, O or S;

[0010] R1, R2are each independently selected from hydrogen, halogen, C1-C3alkyl, nitro, amino, cyano, oxoacetylamino, halogenated C1-C3alkyl, C1-C3alkoxy, or halogenated C1-C3alkoxy;

[0011] n = 1 or 2.

[0012] In certain preferred embodiments,

[0013] A ring is selected from phenyl, naphthyl, or a six-membered heterocycle containing one heteroatom selected from N, O, or S;

[0014] B ring is selected from a five-membered heterocycle containing one to three heteroatoms selected from N, O, or S;

[0015] R1, R2are each independently selected from hydrogen, halogen, C1-C3alkyl, nitro, amino, cyano, oxoacetylamino, halogenated C1-C3alkyl, C1-C3alkoxy, or halogenated C1-C3alkoxy;

[0016] n = 1 or 2.

[0017] In certain more preferred embodiments,

[0018] A ring is selected from phenyl, naphthyl, or a six-membered heterocycle containing one heteroatom selected from N, O, or S;

[0019] B ring is selected from a five-membered heterocycle containing three heteroatoms selected from N, O, or S;

[0020] R1, R2are each independently selected from hydrogen, halogen, methyl, nitro, amino, cyano, oxoacetylamino, trifluoromethyl, or methoxy;

[0021] n = 1 or 2.

[0022] In some preferred embodiments, the pharmaceutically acceptable salts include, but are not limited to, acid addition salts of the compounds of Formula I with hydrochloric acid, hydrobromic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid; and also acid salts of the compounds of Formula I with inorganic bases.

[0023] In some more preferred embodiments, the pharmaceutically acceptable salts include, but are not limited to, alkali metal cation salts, alkaline earth metal cation salts, and ammonium cation salts.

[0024] The compounds of Formula I of the present application are preferably the following compounds:

[0025]

[0026] The compounds of the above general formula I according to the present application can also exist in the form of their salts, esters which are converted in the body to the compounds of formula I. For example, within the scope of the present application, the compounds of the present application are converted to the form of pharmaceutically acceptable salts according to processes known in the art and used as salts.

[0027] It is another object of the present application to provide a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, ester thereof and a pharmaceutically acceptable carrier or excipient.

[0028] The pharmaceutical compositions of the present application can be administered in a variety of known ways depending upon the type of condition to be treated or prevented, the stage of the condition, and the subject involved. The pharmaceutical compositions of the present application can be administered alone or in combination with other drugs known to be useful in treating or preventing the condition of interest. Oral compositions can be in any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions, and suspensions, dispersions and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, and the like.

[0029] Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[0030] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of factors including the activity of the particular compound of the present application or salt thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compound employed, the age, sex, weight, general health status, and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0031] It is another object of the present application to provide the use of a compound of general formula I or a pharmaceutically acceptable salt, ester thereof for the preparation of a STAT3 inhibitor.

[0032] Current studies have shown that knocking down Tyr705 and Ser727 phosphorylation of STAT3 at animal level can effectively inhibit the growth of tumors. The SH2 domain of STAT3 plays a key role in promoting the phosphorylation of Tyr705 and Ser727 and driving the formation of STAT3 homodimers. Therefore, targeting STAT3 to inhibit the phosphorylation of two sites has become a new strategy for the treatment of cancer. One of the purposes of the present application is to provide an inhibitor for effectively inhibiting the phosphorylation of two sites of STAT3 for the SH2 domain.

[0033] The pharmacological experiments of the present application prove that compound 25 has strong growth inhibition effect on pancreatic cancer cells PANC-1 and BxPC-3. Further mechanism verification shows that compound 25 can effectively bind to the SH2 domain of STAT3 protein, inhibit the formation of STAT3 homodimer, inhibit the proliferation of pancreatic cancer cells PANC-1 and BxPC-3, and induce the apoptosis of pancreatic cancer cells PANC-1 and BxPC-3.

[0034] Another purpose of the present application is to provide the use of the compound of formula I or a pharmaceutically acceptable salt, ester thereof in the preparation of a drug for preventing and / or treating diseases related to tumors.

[0035] The diseases related to tumors are pancreatic cancer, breast cancer, gastric cancer or colorectal cancer.

[0036] Beneficial effects:

[0037] The compound of general formula I and its pharmaceutically acceptable salt, ester prepared by the present application can inhibit the binding of STAT3 and phosphorylated polypeptide to achieve the purpose of anti-tumor. The salicylaldehyde derivative in the present application can bind to the SH2 domain of the target protein STAT3, and inhibit the binding of the target protein and the phosphorylated polypeptide. Compound 25 shows good competitive inhibition activity. In the in vitro anti-pancreatic cancer cell proliferation activity, compound 25 has sub-micromolar anti-pancreatic cancer cell proliferation activity and can induce apoptosis of cancer cells. In addition, compound 25 can significantly inhibit the phosphorylation of STAT3 705 site and 727 site. Specifically, it selectively inhibits the formation of p-STAT3, but does not affect the phosphorylation of related kinases. Compound 25 can inhibit the proliferation of various tumor cells in vitro, and has low toxicity to normal cells. Therefore, the compound of general formula I of the present application can be used for preparing STAT3 inhibitors, and for preparing drugs for preventing and / or treating diseases related to tumors. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The inhibition constant of compound 4-27 to STAT3.

[0039] Figure 2To verify the binding of the target compound 25 to the STAT3 SH2 domain by fluorescence polarization and to determine the binding force of the target compound 25 to the STAT3 protein by ITC.

[0040] Figure 3 To verify the anti-proliferation activity of the target compound 25 against pancreatic cancer cells and the toxicity of the target compound 25 to normal pancreatic cells Hpde6.

[0041] Figure 4 To verify the test results of the target compound 25 in inhibiting the activation of the STAT3 pathway.

[0042] Figure 5 To verify the test results of the target compound 25 in inducing apoptosis of tumor cells in vitro. DETAILED DESCRIPTION

[0043] The preparation methods of the compounds of general formula I of the present application are described below in conjunction with specific examples, but these specific methods do not constitute any limitation to the present application. The compounds of the present application can also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, which can be easily performed by those skilled in the art to which the present application belongs.

[0044] The starting materials and reaction reagents used in the specific examples of the present application are commercially available. The salted forms of the present application can be prepared by using the commonly used salt formation methods in the art, for example: the compound is dissolved in hydrochloric acid ethanol at room temperature to react to form a hydrochloride salt; or benzene sulfonic acid is added thereto to react to form a benzene sulfonic acid salt.

[0045] The synthesis route of compound 4-23 is as follows:

[0046]

[0047] 1. Synthesis of intermediate 4-(4-(methoxycarbonyl)-2-nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (compound 2)

[0048] Weigh 10.0 g of methyl 4-fluoro-3-nitrobenzoate (compound 1), 14.0 g of piperazine-1-carboxylic acid tert-butyl ester and 29.0 g of potassium carbonate into 200 mL of N,N-dimethylformamide, and stir overnight at 65°C. After monitoring the end of the reaction by TLC, the organic solvent is removed under reduced pressure, and the residue is extracted (dissolved in EtOAc, washed with H2O and saturated NaCl in turn). The organic phase is dried with anhydrous Na2SO4, evaporated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate 4-(4-(methoxycarbonyl)-2-nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (compound 2), 11.3 g, yellow solid, yield 62%. 1H NMR (300 MHz, Chloroform-d) d (ppm) 8.49 (d, J = 2.1 Hz, 1H, Ar-H), 8.11 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.10 (d, J = 8.8 Hz, 1H, Ar-H), 3.94 (s, 3H, COOCH3), 3.66-3.59 (m, 4H, piperazine-CH2CH2), 3.18 (dd, J = 6.3, 3.9 Hz, 4H, piperazine-CH2’CH2’), 1.50 (s, 9H, OC(CH3)3). ESI-MS calcd for C 17 H 23 N3O6Na (M+Na) + m / z: 388.1, found 388.1.

[0049] 2. Synthesis of intermediate 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3- nitrobenzoic acid (compound 3)

[0050] Weigh 10.0 g of intermediate (compound 2) into 163 mL of mixed solution (tetrahydrofuran: methanol: 1M NaOH solution = 1:1:1, v / v / v) and stir at room temperature for 2 h. After TLC monitoring reaction is completed, remove the organic solvent under reduced pressure, and adjust the remaining material to be acidic with 1M HC1 solution, and extract (dissolved in EtOAc, washed with H2O and saturated NaCl in turn). Dry the organic phase with anhydrous Na2SO4, and evaporate under reduced pressure to obtain intermediate (3), 8.9 g, yellow solid, yield 93%. 1 H NMR (400 MHz, DMSO-d6) d (ppm) 8.30 (s, 1H, Ar-H), 8.03 (d, J = 8.6 Hz, 1H, Ar-H), 7.33 (d, J = 8.7 Hz, 1H, Ar-H), 3.54-3.39 (m, 4H piperazine-CH2CH2), 3.13 (t, J = 5.0 Hz, 4H piperazine-CH2’CH2’), 1.42 (s, 9H, OC(CH3)3).

[0051] ESI-MS calcd for C 16 H 21 N3O6Na (M+Na) + m / z: 374.1, found 374.2.

[0052] 3. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-phenyl-1,2,4-oxadiazol-5- yl)phenyl)piperazine-1-carboxylate (5a)

[0053] To a solution of 0.6 g of benzonitrile (compound 4a), 3.0 g of hydroxylamine hydrochloride, 3.6 g of potassium carbonate in 50 mL of methanol, heat to reflux for 2 h. After monitoring the reaction by TLC, dryness under reduced pressure, extraction (dissolved in EtOAc, washed with H2O and saturated NaCl in turn). The organic phase was dried with anhydrous Na2SO4, and dryness under reduced pressure to obtain the intermediate benzamide oxime. Take 0.7 g of intermediate benzamide oxime, 1.4 g of intermediate 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-nitrobenzoic acid (compound 3) and 1.9 g of HATU dissolved in 20 mL of N,N-dimethylformamide, add 14 mL of DIPEA, stir at room temperature for 4 h, add water to precipitate a large amount of solid, filter, dry the solid, then dissolve the solid in 20 mL of DMF, heat to 120°C, react for 2 h. After monitoring the reaction by TLC, dryness under reduced pressure, silica gel column chromatography to obtain the intermediate (5a), 2.1 g, yellow solid, yield 95%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.27 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.18 (dd, J = 7.4, 2.2 Hz, 2H, Ar-H), 7.59-7.50 (m, 3H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 3.66 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.23 (t, J = 5.0 Hz, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated value C 23 H 26 N5O5 (M+H) + m / z: 452.2, found 452.2.

[0054] 4. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(pyridin-4-yl)-1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5b)

[0055] Referring to the preparation method of intermediate compound (5a), replace benzonitrile with 4-cyanopyridine to obtain intermediate (5b), 989 mg, yellow solid, yield 45%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 8.87-8.81 (m, 2H, Ar-H), 8.69 (dd, J = 2.2, 0.9 Hz, 1H, Ar-H), 8.26 (ddd, J = 8.8, 2.2, 0.8 Hz, 1H, Ar-H), 8.08-8.03 (m, 2H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 3.66 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.25 (s, 4H, piperazine-CH2CH2), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 22 H 25 N6O5 (M+H) + m / z: 453.2, found 453.2.

[0056] 5. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(pyridin-3-yl)-1,2,4-oxadiazol-5- yl)phenyl)piperazine-1-carboxylate (5c)

[0057] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 3-cyanopyridine, intermediate (5c) was obtained, 876 mg, yellow solid, yield 40%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 9.44-9.38 (m, 1H, Ar-H), 8.80 (dd, J = 4.9, 1.7 Hz, 1H, Ar-H), 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.46 (dt, J = 8.0, 1.9 Hz, 1H, Ar-H), 8.27 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.50 (dd, J = 8.0, 4.8 Hz, 1H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 3.66 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.25 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 22 H 25 N6O5 (M+H) + m / z: 453.2, found 453.2.

[0058] 6. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(pyridin-2-yl)-1,2,4-oxadiazol-5- yl)phenyl)piperazine-1-carboxylate (5d)

[0059] Referring to the method of preparation of compound (5a) of example 3, benzonitrile was replaced by 2-cyanopyridine to obtain intermediate (5d), 505 mg, yellow solid, yield 23%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 8.87 (d, J = 4.9 Hz, 1H, Ar-H), 8.74 (d, J = 2.1 Hz, 1H, Ar-H), 8.34 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.24 (dt, J = 7.8, 1.1 Hz, 1H, Ar-H), 7.92 (td, J = 7.8, 1.7 Hz, 1H, Ar-H), 7.50 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 3.65 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.24 (s, 4H, piperazine-CH2’CH2’), 1.50 (s, 9H, OC(CH3)3).

[0060] ESI-MS calc. for C 22 H 25 N6O5 (M+H) + m / z: 453.2, found 453.2.

[0061] 7. Synthesis of intermediate tert-butyl 4-(4-(3-(naphthalen-1-yl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-1-carboxylate (5e)

[0062] Referring to the method of preparation of compound (5a), benzonitrile was replaced by 1- naphthonitrile to obtain intermediate (5e), 446 mg, yellow solid, yield 18%. 1H NMR (400 MHz, Chloroform-d) δ (ppm) 8.98 (dd, J = 8.5, 1.1 Hz, 1H, Ar-H), 8.74 (d, J = 2.0 Hz, 1H, Ar-H), 8.37 (dd, J = 7.2, 1.1 Hz, 1H, Ar-H), 8.32 (dd, J = 8.7, 2.0 Hz, 1H, Ar-H), 8.06 (d, J = 8.2 Hz, 1H, Ar-H), 7.97 (d, J = 8.0 Hz, 1H, Ar-H), 7.72 - 7.58 (m, 3H, Ar-H), 7.24 (dd, J = 8.8, 1.1 Hz, 1H, Ar-H), 3.67 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.24 (s, 4H, piperazine-CH2’CH2’), 1.52 (s, 9H, OC(CH3)3).

[0063] ESI-MS calculated for C 27 H 28 N5O5 (M+H) + m / z: 502.2, found 502.1.

[0064] 8. Synthesis of intermediate tert-butyl 4-(4-(3-(naphthalen-2-yl)-1,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-1-carboxylate (5f)

[0065] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 2-naphthonitrile, intermediate (5f) was obtained, 1.6 g, yellow solid, yield 65%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.72 (d, J = 2.0 Hz, 2H, Ar-H), 8.29 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 8.22 (dd, J = 8.6, 1.5 Hz, 1H, Ar-H), 8.05 - 7.96 (m, 2H, Ar-H), 7.95 - 7.90 (m, 1H, Ar-H), 7.64 - 7.56 (m, 2H, Ar-H), 7.23 (dd, J = 8.8, 2.5 Hz, 1H, Ar-H), 3.66 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.24 (s, 4H, piperazine-CH2’CH2’), 1.52 (s, 9H, OC(CH3)3). ESI-MS calculated for C 27 H 28 N5O5 (M+H) + m / z: 502.2, found 502.2.

[0066] 9. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(4-(trifluoromethyl)phenyl)- 1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5g)

[0067] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 4-(trifluoromethyl)benzonitrile, intermediate (5g) was obtained, 1.8 g, yellow solid, yield 71%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.31 (d, J = 8.2 Hz, 2H, Ar-H), 8.27 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.80 (d, J = 8.2 Hz, 2H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 3.66 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.24 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 24 H 24 F3N5O5 (M+H) + m / z: 520.2, found 520.2.

[0068] 10. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(3-(trifluoromethyl)phenyl)- 1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5h)

[0069] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 3-(trifluoromethyl)benzonitrile, intermediate (5h) was obtained, 1.6 g, yellow solid, yield 63%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.46 (s, 1H, Ar-H), 8.37 (d, J = 7.8 Hz, 1H, Ar-H), 8.28 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.82 (d, J = 7.8 Hz, 1H, Ar-H), 7.68 (t, J = 7.8 Hz, 1H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 3.72-3.62 (m, 4H, piperazine-CH2CH2), 3.29-3.21 (m, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C24 H 24 F3N5O5(M+H) + m / z: 520.2, found 520.2.

[0070] 11. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(2-(trifluoromethyl)phenyl)- 1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5i)

[0071] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 2-(trifluoromethyl)benzonitrile, intermediate (5i) was obtained, 1.2 g, yellow solid, yield 47%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.66 (d, J = 2.1 Hz, 1H, Ar-H), 8.26 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.92-7.85 (m, 2H, Ar-H), 7.76-7.67 (m, 2H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 3.71-3.62 (m, 4H, piperazine-CH2CH2), 3.24 (t, J = 5.1 Hz, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 24 H 24 F3N5O5(M+H) + m / z: 520.2, found 520.2.

[0072] 12. Synthesis of intermediate tert-butyl 4-(4-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-1-carboxylate (5j)

[0073] Following the procedure for the preparation of intermediate compound (5a), replacing benzonitrile with 4-fluorobenzonitrile, intermediate (5j) was obtained, 1.9 g, yellow solid, yield 83%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.67 (d, J = 2.1 Hz, 1H, Ar-H), 8.25 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.21-8.14 (m, 2H, Ar-H), 7.26-7.18 (m, 3H, Ar-H), 3.73-61 (m, 4H, piperazine-CH2CH2), 3.23 (t, J = 5.0 Hz, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C23 H 25 FN5O5(M+H) + m / z: 470.2, found 470.2.

[0074] 13. Synthesis of intermediate tert-butyl 4-(4-(3-(3-fluorophenyl)-1,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-1-carboxylate (5k)

[0075] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 3-fluorobenzonitrile to obtain intermediate (5k), 2.1 g, yellow solid, yield 91%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.24 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.95 (dt, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.88-7.83 (m, 1H, Ar-H), 7.49 (td, J = 8.0, 5.6 Hz, 1H, Ar-H), 7.26-7.18 (m, 2H, Ar-H), 3.64 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.22 (s, 4H, piperazine-CH2’CH2’), 1.49 (s, 9H, OC(CH3)3). ESI-MS calcd for C 23 H 25 FN5O5(M+H) + m / z: 470.2, found 470.2.

[0076] 14. Synthesis of intermediate tert-butyl 4-(4-(3-(2-fluorophenyl)-1,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-1-carboxylate (5l)

[0077] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 2-fluorobenzonitrile to obtain intermediate (5l), 1.8 g, yellow solid, yield 78%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 8.69 (dd, J = 2.1, 1.3 Hz, 1H, Ar-H), 8.30-8.25 (m, 1H, Ar-H), 8.21-8.15 (m, 1H, Ar-H), 7.60-7.51 (m, 1H, Ar-H), 7.39-7.30 (m, 2H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 3.73-3.60 (m, 4H, piperazine-CH2CH2), 3.24 (t, J = 4.9 Hz, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 23 H 25 FN5O5 (M+H) + m / z: 470.2, found 470.2.

[0078] 15. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(4-nitrophenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5m)

[0079] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 4-nitrobenzonitrile to obtain intermediate (5m), 1.5 g, yellow solid, yield 62%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 8.69 (dd, J = 2.1, 1.3 Hz, 1H, Ar-H), 8.30-8.25 (m, 1H, Ar-H), 8.21-8.15 (m, 1H, Ar-H), 7.60-7.51 (m, 1H, Ar-H), 7.39-7.30 (m, 2H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 3.73-3.60 (m, 4H, piperazine-CH2CH2), 3.24 (t, J = 4.9 Hz, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 23 H 25 N6O7 (M+H) + m / z: 497.2, found 497.2.

[0080] 16. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(3-nitrophenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5n)

[0081] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 3-nitrobenzonitrile to obtain intermediate (5n), 1.9 g, yellow solid, yield 78%.1 H NMR (400 MHz, Chloroform-d) d (ppm) 9.04 (t, J = 1.9 Hz, 1H, Ar-H), 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.51 (dd, J = 7.7, 1.4 Hz, 1H, Ar-H), 8.41 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H, Ar-H), 8.28 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.75 (t, J = 8.0 Hz, 1H, Ar-H), 7.24 (d, J = 8.8 Hz, 1H, Ar-H), 3.67 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.26 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 23 H 25 N6O7 (M+H) + m / z: 497.2, found 497.1.

[0082] 17. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(3-(2-nitrophenyl)-1,2,4- oxadiazol-5-yl)phenyl)piperazine-1-carboxylate (5o)

[0083] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 2-nitrobenzonitrile to obtain intermediate (5o), 1.2 g, yellow solid, yield 49%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 8.62 (d, J = 2.1 Hz, 1H, Ar-H), 8.21 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.03 (dd, J = 7.9, 1.4 Hz, 1H, Ar-H), 7.94 (dd, J = 7.2, 1.9 Hz, 1H, Ar-H), 7.82-7.70 (m, 2H, Ar-H), 7.21 (d, J = 8.9 Hz, 1H, Ar-H), 3.65 (dd, J = 6.5, 3.7 Hz, 4H, piperazine-CH2CH2), 3.24 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 23 H 25 N6O7 (M+H) + m / z: 497.2, found 497.2.

[0084] 18. Synthesis of intermediate tert-butyl 4-(4-(3-(4-methoxyphenyl)-l,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-l-carboxylate (5p)

[0085] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 4-methoxybenzonitrile to obtain intermediate (5p), 2.1 g, yellow solid, yield 89%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 8.67 (dd, J = 2.1, 1.3 Hz, 1H, Ar-H), 8.25 (ddd, J = 8.7, 2.1, 1.2 Hz, 1H, Ar-H), 8.15-8.08 (m, 2H, Ar-H), 7.21 (dd, J = 8.8, 1.0 Hz, 1H, Ar-H), 7.08-6.98 (m, 2H, Ar-H), 3.91 (s, 3H, OCH3), 3.65 (t, J = 5.0 Hz, 4H, piperazine-CH2CH2), 3.22 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calcd for C 24 H 28 N5O6 (M+H) + m / z: 482.2, found 482.2.

[0086] 19. Synthesis of intermediate tert-butyl 4-(4-(3-(3-methoxyphenyl)-l,2,4-oxadiazol-5- yl)-2-nitrophenyl)piperazine-l-carboxylate (5q)

[0087] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 3-methoxybenzonitrile to obtain intermediate (5q), 1.9 g, yellow solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 8.68 (d, J = 2.1 Hz, 1H, Ar-H), 8.27 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 8.11 (dd, J = 7.7, 1.8 Hz, 1H, Ar-H), 7.59 - 7.49 (m, 1H, Ar-H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.17 - 7.07 (m, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.65 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.22 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 24 H 28 N5O6 (M+H) + m / z: 482.2, found 482.2.

[0088] 20. Synthesis of intermediate 4-(4-(3-(2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (5r)

[0089] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 2-methoxybenzonitrile to obtain intermediate (5r), 1.1 g, yellow solid, yield 47%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 8.68 (d, J = 2.1 Hz, 1H, Ar-H), 8.27 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 8.11 (dd, J = 7.7, 1.8 Hz, 1H, Ar-H), 7.59 - 7.49 (m, 1H, Ar-H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.17 - 7.07 (m, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.65 (t, J = 5.1 Hz, 4H, piperazine-CH2CH2), 3.22 (s, 4H, piperazine-CH2’CH2’), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated for C 24 H 28 N5O6 (M+H) + m / z: 482.2, found 482.2.

[0090] 21. Synthesis of intermediate tert-butyl 4-(4-(3-(4-cyano)-l,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-l-carboxylate (5s)

[0091] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by p-xylyl cyanide to obtain intermediate (5s), 671 mg, yellow solid, yield 29%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.57 (d, J = 2.2 Hz, 1H, Ar-H), 8.31-8.24 (m, 3H, Ar-H), 8.14-8.06 (m, 2H, Ar-H), 7.50 (d, J = 9.0 Hz, 1H, Ar-H), 3.50 (s, 4H, piperazine-CH2CH2), 3.29-3.23 (m, 4H, piperazine-CH2'CH2'), 1.43 (s, 9H, OC(CH3)3). ESI-MS calculated C 24 H 24 N6O5Na (M+H) + m / z: 499.2, found 499.2.

[0092] 22. Synthesis of intermediate tert-butyl 4-(4-(3-(3-cyano)-l,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-l-carboxylate (5t)

[0093] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by m-xylyl cyanide to obtain intermediate (5t), 543 mg, yellow solid, yield 23%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 8.68 (d, J = 2.1 Hz, 1H, Ar-H), 8.49 (t, J = 1.6 Hz, 1H, Ar-H), 8.42 (dt, J = 7.9, 1.4 Hz, 1H, Ar-H), 8.26 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.84 (dt, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.67 (t, J = 7.8 Hz, 1H, Ar-H), 7.24 (d, J = 8.8 Hz, 1H, Ar-H), 3.66 (t, J = 4.9 Hz, 4H, piperazine-CH2CH2), 3.25 (s, 4H, piperazine-CH2'CH2'), 1.51 (s, 9H, OC(CH3)3). ESI-MS calculated C 24 H 24 N6O5Na (M+H) +m / z: 499.2, found 499.2.

[0094] Example 1 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-phenyl-1,2,4-oxadiazol-5- yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 4)

[0095] Weigh 260 mg of intermediate (5a) into 1.5 mL mixed solution (trifluoroacetic acid: dichloromethane = 1:1, v / v), stir at room temperature for 2 h. After TLC monitoring reaction is completed, evaporate under reduced pressure, adjust the remaining residue to alkaline with saturated sodium bicarbonate, precipitate a large amount of solid, suction filter, and dry the solid. Dissolve the remaining residue in 3.0 mL of N,N-dimethylformamide, add 320 μL of DIPEA, stir at 0°C for 5 min, then add 69 mg of 5-(chloromethyl)-2-hydroxybenzaldehyde in N,N-dimethylformamide 3.0 mL dropwise. Then, continue to stir at room temperature for 105 min. After TLC monitoring reaction is completed, evaporate under reduced pressure, and purify the target compound (4) by silica gel column chromatography, 169 mg, yellow solid, yield 60%. Purity 97%. 1 H NMR (400 MHz, Chloroform-d) δ (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.23 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.20-8.14 (m, 2H, Ar-H), 7.60-7.49 (m, 5H, Ar-H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.00 (d, J = 8.5 Hz, 1H, Ar-H), 3.59 (s, 2H, Ar-CH2), 3.28 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.74-2.60 (m, 4H, piperazine-CH2’CH2’).

[0096] 13 C NMR (100 MHz, Chloroform-d) δ (ppm) 196.53, 173.83, 169.01, 161.01, 148.18, 140.64, 137.97, 133.98, 132.58, 131.34 (two), 128.90 (two), 127.52 (two), 126.96, 126.69, 120.37, 120.30, 117.75, 115.61, 61.67, 52.46 (two), 50.67 (two).

[0097] HRMS (ESI) calcd for C26 H 24 N5O5(M+H) + m / z 486.1777, found 486.1778.

[0098] Example 2 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(pyridin-4-yl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 5)

[0099] The target compound (5) was obtained by referring to the preparation method of compound (4) in Reference Example 1, using intermediate (5b) as the raw material, 177 mg, yellow solid, yield 63%. Purity 99%. 1 H NMR (300 MHz, Chloroform-d) d (ppm) 11.00 (s, 1H, Ar-CHO), 9.92 (s, 1H, Ar-OH), 8.82 (d, J = 5.9 Hz, 2H, Ar-H), 8.63 (d, J = 2.1 Hz, 1H, Ar-H), 8.21 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 8.06 - 7.97 (m, 2H, Ar-H), 7.59 - 7.50 (m, 2H, Ar-H), 7.21 (d, J = 8.9 Hz, 1H, Ar-H), 7.03 - 6.95 (m, 1H, Ar-H), 3.58 (s, 2H, Ar-CH2), 3.29 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.65 (t, J = 4.8 Hz, 4H, piperazine-CH2’CH2’).

[0100] 13 C NMR (75 MHz, Chloroform-d) d (ppm) 196.50, 174.63, 167.48, 161.00, 150.73 (two), 148.38, 140.37, 137.94, 134.16, 133.93, 132.58, 129.25, 127.17, 121.31 (two), 120.36, 120.30, 117.75, 114.76, 61.65, 52.42, 50.63.

[0101] HRMS (ESI) calcd for C 25 H 23 N6O5(M+H) + m / z 487.1730, found 487.1726.

[0102] Example 3 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(pyridin-3-yl)-1,2,4-oxadiazol-5- yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 6)

[0103] Referring to the preparation method of Example 1 Compound (4), the target compound (6) was obtained using intermediate (5c) as the raw material, 178 mg, yellow solid, yield 63%. Purity 98%. 1 H NMR (300 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 9.39 (dd, J = 2.2, 0.9 Hz, 1H, Ar-H), 8.79 (dd, J = 4.9, 1.7 Hz, 1H, Ar-H), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.44 (dt, J = 7.9, 1.9 Hz, 1H, Ar-H), 8.24 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.60-7.52 (m, 2H, Ar-H), 7.48 (ddd, J = 8.0, 4.9, 0.9 Hz, 1H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.00 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.30 (s, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (75 MHz, Chloroform-d) d (ppm) 196.51, 174.30, 167.09, 161.03, 152.18, 148.73, 148.32, 140.48, 139.77, 137.97, 134.78, 133.97, 132.65, 129.29, 129.21, 127.11, 123.71, 123.04, 120.37, 117.78, 61.64, 52.42 (two), 50.62 (two). HRMS (ESI) calcd for C 25 H 23 N6O5 (M+H) + m / z 487.1730, found 487.1729.

[0104] Example 4 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(pyridin-2-yl)-1,2,4-oxadiazol-5- yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 7)

[0105] The title compound (7) was obtained as a yellow solid, 113 mg, yield 40%, purity 95% by following the procedure of Example 1 compound (4) using intermediate (5d) as the starting material. 1 H NMR (300 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.92-8.69 (m, 1H, Ar-H), 8.65-8.48 (m, 1H, Ar-H), 8.36-8.12 (m, 2H, Ar-H), 7.97-7.75 (m, 1H, Ar-H), 7.60-7.52 (m, 2H, Ar-H), 7.51-7.39 (m, 1H, Ar-H), 7.25-7.10 (m, 1H, Ar-H), 7.00 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.28 (s, 4H, piperazine-CH2CH2), 2.66 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (75 MHz, Chloroform-d) d (ppm) 196.55, 174.67, 168.85, 161.05, 154.46, 150.52, 148.50, 146.23, 138.02, 137.16, 136.92, 134.50, 132.82, 128.24, 127.20, 125.69, 123.33, 121.54, 120.38, 117.78, 61.65, 52.44 (two), 50.60 (two). HRMS (ESI) calcd for C 25 H 23 N6O5 (M+H) + m / z 487.1730, found 487.1724.

[0106] Example 5 Synthesis of 2-hydroxy-5-((4-(4-(3-(naphthalen-1-yl)-1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazin-1-yl)methyl)benzaldehyde (compound 8)

[0107] The title compound (8) was obtained as a yellow solid, 73 mg, yield 24%, purity 100% by following the procedure of Example 1 compound (4) using intermediate (5e) as the starting material. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.68 (s, 1 H, Ar-CHO), 10.27 (s, 1 H, Ar-OH), 8.85 (d, J = 8.5 Hz, 1 H, Ar-H), 8.55 (d, J = 2.2 Hz, 1 H, Ar-H), 8.32 (d, J = 7.2 Hz, 1 H, Ar-H), 8.26 (dd, J = 8.9, 2.2 Hz, 1 H, Ar-H), 8.21 (d, J = 8.2 Hz, 1 H, Ar-H), 8.10 (d, J = 8.0 Hz, 1 H, Ar-H), 7.77 - 7.57 (m, 4 H, Ar-H), 7.53 - 7.45 (m, 2 H, Ar-H), 6.99 (d, J = 8.4 Hz, 1 H, Ar-H), 3.50 (s, 2 H, Ar-CH2), 3.32 (s, 4 H, piperazine-CH2CH2), 3.23 (s, 4 H, piperazine-CH2CH2). 13 C NMR (100 MHz, DMSO-d6) δ (ppm) 191.97, 173.52, 169.08, 160.43, 148.25, 139.74, 137.67, 133.97, 133.02, 132.57, 130.29, 129.91, 129.73, 129.33, 129.15, 128.32, 127.07, 127.00, 126.07, 125.89, 123.43, 122.36, 121.63, 117.67, 113.93, 61.22, 52.39 (two), 50.60 (two). HRMS (ESI) calcd for C 30 H 26 N5O5 (M+H) + m / z 536.1928, found 536.1921.

[0108] Example 6 Synthesis of 2-hydroxy-5-((4-(4-(3-(naphthalen-2-yl)-1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 9)

[0109] The target compound (9) was obtained from intermediate (5f) according to the preparation method of compound (4) in Reference Example 1, 132 mg, yellow solid, yield 43%. Purity 99%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.72 (d, J = 1.7 Hz, 1H, Ar-H), 8.69 (d, J = 2.1 Hz, 1H, Ar-H), 8.26 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.21 (dd, J = 8.5, 1.7 Hz, 1H, Ar-H), 8.06 - 7.95 (m, 2H, Ar-H), 7.95 - 7.88 (m, 1H, Ar-H), 7.65 - 7.51 (m, 4H, Ar-H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.6 Hz, 1H, Ar-H), 3.59 (s, 2H, Ar-CH2), 3.29 (t, J = 4.7 Hz, 4H, piperazine-CH2CH2), 2.76 - 2.59 (m, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.52, 173.87, 169.09, 161.02, 148.20, 140.63, 137.96, 134.71, 133.96, 133.05, 132.60, 129.26, 128.90, 128.78, 128.12, 127.90, 127.58, 127.01, 126.80, 124.01, 123.85, 120.37, 120.30, 117.76, 115.58, 61.67, 52.46 (two), 50.68 (two). HRMS (ESI) calcd for C 30 H 26 N5O5 (M+H) + m / z 536.1928, found 536.1932.

[0110] Example 7 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 10)

[0111] Referring to the preparation method of compound (4) of Example 1, the target compound (10) was obtained using intermediate (5g) as a raw material, 201 mg, yellow solid, yield 63%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) δ (ppm) 11.03 (s, 1H, Ar-CHO), 9.95 (s, 1H, Ar-OH), 8.67 (d, J = 2.1 Hz, 1H, Ar-H), 8.35 - 8.29 (m, 2H, Ar-H), 8.25 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.80 (d, J = 8.2 Hz, 2H, Ar-H), 7.61 (s, 1H, Ar-H), 7.57 (dd, J = 8.5, 2.1 Hz, 1H, Ar-H), 7.24 (d, J = 8.8 Hz, 1H, Ar-H), 7.02 (d, J = 8.5 Hz, 1H, Ar-H), 3.66 (s, 2H, Ar-CH2), 3.33 (s, 4H, piperazine-CH2CH2), 2.73 (s, 4H, piperazine-CH2'CH2'). 13 CNMR (100 MHz, Chloroform-d) δ (ppm) 196.50, 174.32, 168.03, 161.05, 148.32, 140.53, 137.98, 133.95, 133.21, 132.88, 132.61, 130.14, 127.89 (two), 127.10, 125.93, 125.89, 123.77 (q, J = 272.3 Hz), 120.55, 120.38, 117.85, 117.78, 61.66, 52.42 (two), 50.64 (two). HRMS (ESI) calculated for C 27 H 23 F3N5O5 (M+H) + m / z 554.1651, found 554.1642.

[0112] Example 8 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 11)

[0113] The target compound (11) was obtained from intermediate (5h) according to the preparation method of compound (4) in Reference Example 1, 185 mg, yellow solid, yield 58%. Purity 98%. 1H NMR (300 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, CHO), 9.93 (s, 1H, Ar-OH), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.37 (dd, J = 7.8, 1.5 Hz, 1H, Ar-H), 8.24 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.81 (d, J = 7.8 Hz, 1H, Ar-H), 7.67 (t, J = 7.8 Hz, 1H, Ar-H), 7.56 (d, J = 7.3 Hz, 2H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.00 (d, J = 8.6 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.30 (t, J = 4.9 Hz, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (75 MHz, Chloroform-d) d (ppm) 196.53, 174.29, 167.98, 166.31, 161.02, 148.32, 140.47 (d, J = 3.3 Hz), 137.97, 133.95, 132.63, 131.71, 131.27, 130.63, 129.52, 129.27, 127.91 (q, J = 3.6 Hz), 127.62, 125.55, 124.52 (q, J = 3.6 Hz), 123.74 (q, J = 272.7 Hz), 120.37, 120.30, 117.77, 61.66, 52.43 (two), 50.63 (two). HRMS (ESI) calcd for C 27 H 23 F3N5O5 (M+H) + m / z 554.1651, found 554.1646.

[0114] Example 9 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(2-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 12)

[0115] Referring to the preparation method of compound (4) in Example 1, the target compound (12) was obtained from intermediate (5i) as a yellow solid, 129 mg, yield 40%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.63 (d, J = 2.1 Hz, 1H, Ar-H), 8.23 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.92-7.85 (m, 2H, Ar-H), 7.77-7.65 (m, 2H, Ar-H), 7.56 (d, J = 8.4 Hz, 2H, Ar-H), 7.21 (d, J = 8.9 Hz, 1H, Ar-H), 7.00 (d, J = 8.3 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.35-3.25 (m, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’). 13 CNMR (100 MHz, Chloroform-d) d (ppm) 196.51, 173.81, 168.17, 162.54, 161.04, 148.30, 140.58, 137.97, 133.99, 132.66, 131.91, 131.80, 130.85, 129.64, 129.32, 128.42, 127.04, 126.96 (q, J = 5.4 Hz), 125.53, 123.40 (d, J = 273.6 Hz), 120.38, 117.77, 61.64, 52.42 (two), 50.62 (two). HRMS (ESI) calcd for C 27 H 23 F3N5O5 (M+H) + m / z 554.1651, found 554.1652.

[0116] Example 10 Synthesis of 5-((4-(4-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazin-1-yl)methyl)-2-hydroxybenzaldehyde (Compound 13)

[0117] Referring to the preparation method of compound (4) in Example 1, the target compound (13) was obtained from intermediate (5j) as a raw material, 199 mg, yellow solid, yield 68%, purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.63 (d, J = 2.1 Hz, 1H, Ar-H), 8.22 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.17 (dd, J = 8.9, 5.3 Hz, 1H, Ar-H), 7.62-7.50 (m, 2H, Ar-H), 7.26-7.17 (m, 3H, Ar-H), 7.00 (d, J = 8.5 Hz, 1H, Ar-H), 3.59 (s, 2H, Ar-CH2), 3.28 (s, 4H, piperazine-CH2CH2), 2.66 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.51, 173.92, 168.18, 164.65 (d, J = 251.7 Hz), 161.01, 148.22, 140.57, 137.96, 133.95, 132.56, 129.69 (d, J = 8.7 Hz) (two), 129.29, 126.99, 122.94 (d, J = 3.2 Hz), 120.37, 120.29, 117.76, 116.10 (d, J = 22.1 Hz) (two), 115.36, 61.66, 52.45 (two), 50.66 (two). HRMS (ESI) calcd for C 26 H 23 FN5O5 (M+H) + m / z 504.1683, found 504.1681.

[0118] Synthesis of 5-((4-(4-(3-(3-fluorophenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazin-1-yl)methyl)-2-hydroxybenzaldehyde (Compound 14)

[0119] Referring to the preparation method of compound (4) in Example 1, the target compound (14) was obtained using intermediate (5k) as the raw material, 166 mg, yellow solid, yield 57%. Purity 97%. 1H NMR (400 MHz, DMSO-d6) d (ppm) 10.68 (s, 1H, Ar-CHO), 10.27 (s, 1H, Ar-OH), 8.48 (d, J = 2.2 Hz, 1H, Ar-H), 8.18 (dd, J = 8.9, 2.2 Hz, 1H, Ar-H), 7.92 (dt, J = 7.8, 1.3 Hz, 1H, Ar-H), 7.80 (ddd, J = 9.5, 2.7, 1.5 Hz, 1H, Ar-H), 7.65 (td, J = 8.1, 5.9 Hz, 1H, Ar-H), 7.60 (d, J = 2.2 Hz, 1H, Ar-H), 7.52 - 7.42 (m, 3H, Ar-H), 6.99 (d, J = 8.4 Hz, 1H, Ar-H), 3.49 (s, 2H, Ar-CH2), 3.22 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.52 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, DMSO-d6) d (ppm) 191.96, 174.61, 167.76 (d, J = 2.9 Hz), 162.74 (d, J = 244.9 Hz), 160.43, 148.27, 139.59, 137.66, 132.92, 132.08 (d, J = 8.3 Hz), 129.73, 129.15, 128.67 (d, J = 8.6 Hz), 126.98, 123.79 (d, J = 2.7 Hz), 122.36, 121.57, 119.11 (d, J = 20.7 Hz), 117.67, 114.23 (d, J = 23.8 Hz), 113.64, 61.21, 52.36, 50.56. HRMS (ESI) calcd for C 26 H 23 FN5O5 (M+H) + m / z 504.1683, found 504.1686.

[0120] Example 12 Synthesis of 5-((4-(4-(3-(2-fluorophenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazin-1-yl)methyl)-2-hydroxybenzaldehyde (Compound 15)

[0121] The target compound (15) was obtained from intermediate (5l) according to the preparation method of compound (4) in Reference Example 1, 151 mg, yellow solid, yield 52%. Purity 97%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.24 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.17 (td, J = 7.5, 1.8 Hz, 1H, Ar-H), 7.55 (tdd, J = 8.6, 4.2, 1.9 Hz, 3H, Ar-H), 7.37-7.30 (m, 1H, Ar-H), 7.27 (dd, J = 8.8, 1.5 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.00 (d, J = 8.5 Hz, 1H, Ar-H), 3.59 (s, 2H, Ar-CH2), 3.28 (s, 4H, piperazine-CH2CH2), 2.66 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.52, 173.45, 165.88 (d, J = 5.5 Hz), 161.01, 160.74 (d, J = 257.6 Hz), 148.24, 140.57, 137.96, 133.95, 132.90 (d, J = 8.5 Hz), 132.63, 130.84, 130.82, 127.04, 124.50 (d, J = 3.6 Hz), 120.37, 120.29, 117.75, 116.74 (d, J = 21.1 Hz), 115.06, 114.94, 61.66, 52.44 (two), 50.66 (two). HRMS (ESI) calcd for C 26 H 23 FN5O5 (M+H) + m / z 504.1683, found 504.1684.

[0122] Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(4-nitrophenyl)-1,2,4-oxadiazol-5- yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 16)

[0123] Referring to the preparation method of compound (4) in Example 1, the target compound (16) was obtained using intermediate (5m) as the raw material, 174 mg, yellow solid, yield 57%. Purity 98%. 1H NMR (400 MHz, DMSO-d6) d (ppm) 10.69 (s, 1H, Ar-CHO), 10.27 (s, 1H, Ar-OH), 8.52 (d, J = 2.1 Hz, 1H, Ar-H), 8.43 (d, J = 8.9 Hz, 2H, Ar-H), 8.34 (d, J = 8.9 Hz, 2H, Ar-H), 8.22 (dd, J = 8.9, 2.2 Hz, 1H, Ar-H), 7.60 (d, J = 2.3 Hz, 1H, Ar-H), 7.53 - 7.45 (m, 2H, Ar-H), 6.99 (d, J = 8.4 Hz, 1H, Ar-H), 3.50 (s, 2H, Ar-CH2), 3.28 - 3.19 (m, 4H, piperazine-CH2CH2), 2.51 (s, 4H, piperazine-CH2’CH2’). HRMS (ESI) calcd for C 26 H 23 N6O7 (M+H) + m / z 531.1628, found 531.1619. 13 C NMR (100 MHz, DMSO-d6) d (ppm) 191.94, 175.04, 167.38, 160.43, 149.70, 148.36, 139.53, 137.69, 133.00, 132.34, 129.70, 129.14, 129.01 (two), 127.16, 124.96 (two), 122.36, 121.61, 117.67, 113.40, 61.21, 52.37, 50.56.

[0124] Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(3-nitrophenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 17)

[0125] Referring to the preparation method of compound (4) in Example 1, the target compound (17) was obtained using intermediate (5n) as the raw material, 182 mg, yellow solid, yield 59%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 9.03 (t, J = 1.9 Hz, 1H, Ar-H), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.51 (d, J = 7.8 Hz, 1H, Ar-H), 8.41 (dd, J = 7.9, 1.8 Hz, 1H, Ar-H), 8.25 (dd, J = 8.8, 1.9 Hz, 1H, Ar-H), 7.74 (t, J = 8.0 Hz, 1H, Ar-H), 7.56 (d, J = 8.5 Hz, 2H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.31 (s, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.51, 174.57, 167.38, 161.04, 148.62, 148.39, 140.41, 137.97, 133.94, 133.10, 132.70, 132.65, 130.10, 129.36, 128.56, 127.16, 125.84, 122.68, 120.38, 120.32, 117.78, 61.65, 52.41 (two), 50.61 (two). HRMS (ESI) calcd for C 27 H 23 F3N5O6 (M+H) + m / z 531.1628, found 531.1620.

[0126] Example 15 Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(3-(2-nitrophenyl)-1,2,4-oxadiazol-5-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 18)

[0127] The target compound (18) was obtained by referring to the preparation method of compound (4) in Reference Example 1, using intermediate (5o) as a raw material, 125 mg, yellow solid, yield 41%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.59 (d, J = 2.1 Hz, 1H, Ar-H), 8.18 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.02 (dd, J = 7.8, 1.6 Hz, 1H), 7.94 (dd, J = 7.4, 1.7 Hz, 1H, Ar-H), 7.82-7.67 (m, 2H, Ar-H), 7.62-7.50 (m, 2H, Ar-H), 7.20 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.29 (s, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’).

[0128] 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.52, 174.15, 166.82, 161.07, 149.01, 148.34, 140.48, 138.00, 137.24, 133.99, 132.78, 132.75, 131.81, 131.75, 131.47, 127.09, 124.54, 121.59, 120.38, 120.33, 117.78, 61.61, 52.39 (two), 50.57 (two).

[0129] HRMS (ESI) calcd for C 27 H 23 F3N5O6 (M+H) + m / z 531.1628, found 531.1622.

[0130] Synthesis of 2-hydroxy-5-((4-(4-(3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazin-1-yl)methyl)benzaldehyde (compound 19)

[0131] Referring to the preparation method of compound (4) of Example 1, the target compound (19) was obtained using intermediate (5p) as the raw material, 203 mg, yellow solid, yield 68%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.63 (d, J = 2.1 Hz, 1H, Ar-H), 8.22 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.14 - 8.06 (m, 2H, Ar-H), 7.56 (d, J = 8.3 Hz, 2H, Ar-H), 7.20 (d, J = 8.8 Hz, 1H, Ar-H), 7.07 - 6.96 (m, 3H, Ar-H), 3.90 (s, 3H, OCH3), 3.60 (s, 2H, Ar-CH2), 3.28 (t, J = 4.7 Hz, 4H, piperazine-CH2CH2), 2.66 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.52, 173.53, 168.70, 162.03, 161.02, 148.11, 140.70, 137.98, 133.98, 132.57 (two), 129.14 (two), 126.89 (two), 120.37, 120.30, 119.13, 117.76, 114.29 (two), 61.66, 55.42, 52.46 (two), 50.66 (two). HRMS (ESI) calcd for C 25 H 23 N6O5 (M+H) + m / z 516.1883, found 516.1876.

[0132] Example 17 Synthesis of 2-hydroxy-5-((4-(4-(3-(3-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 20)

[0133] Referring to the preparation method of compound (4) in Example 1, the target compound (20) was obtained using intermediate (5q) as the raw material, 194 mg, yellow solid, yield 65%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.65 (dd, J = 2.1, 0.8 Hz, 1H, Ar-H), 8.23 (dt, J = 8.8, 1.4 Hz, 1H, Ar-H), 7.77 (dt, J = 7.7, 1.2 Hz, 1H, Ar-H), 7.69 (dd, J = 2.7, 1.4 Hz, 1H, Ar-H), 7.61-7.52 (m, 2H, Ar-H), 7.44 (t, J = 8.0 Hz, 1H, Ar-H), 7.21 (d, J = 8.8 Hz, 1H, Ar-H), 7.09 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H, Ar-H), 7.00 (d, J = 8.5 Hz, 1H, Ar-H), 3.93 (s, 3H, OCH3), 3.59 (s, 2H, Ar-CH2), 3.28 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.66 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.52, 173.80, 168.95, 161.02, 159.91, 148.18, 140.62, 137.96, 133.98, 133.94, 132.59, 132.54, 130.02, 127.87, 126.98, 120.37, 120.29, 119.97, 117.87, 117.76, 112.03, 61.66, 55.50, 52.45 (two), 50.67 (two). ESI-MS calcd for C 25 H 23 N6O5 (M+H) + m / z 516.1883, found 516.1885.

[0134] Example 18 Synthesis of 2-hydroxy-5-((4-(4-(3-(2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 21)

[0135] Referring to the preparation method of compound (4) of Example 1, the target compound (21) was obtained using intermediate (5r) as a raw material, 189 mg, yellow solid, yield 63%. Purity 97%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.69 (s, 1 H, Ar-CHO), 10.27 (s, 1 H, Ar-OH), 8.48 (d, J = 2.2 Hz, 1 H, Ar-H), 8.19 (dd, J = 8.9, 2.2 Hz, 1 H, Ar-H), 7.93 (dd, J = 7.7, 1.8 Hz, 1 H, Ar-H), 7.64 - 7.55 (m, 2 H, Ar-H), 7.52 - 7.43 (m, 2 H, Ar-H), 7.25 (dd, J = 8.6, 1.0 Hz, 1 H, Ar-H), 7.13 (td, J = 7.5, 1.0 Hz, 1 H, Ar-H), 6.99 (d, J = 8.4 Hz, 1 H, Ar-H), 3.89 (s, 3 H, OCH3), 3.49 (s, 2 H, Ar-CH2), 3.21 (t, J = 4.7 Hz, 4 H, piperazine-CH2CH2), 2.52 (s, 4 H, piperazine-CH2'CH2'). 13 C NMR (100 MHz, DMSO-d6) δ (ppm) 191.97, 173.05, 167.20, 160.44, 158.19, 148.14, 139.80, 137.68, 133.25, 132.90, 131.33, 129.73, 129.17, 126.77, 122.37, 121.65, 121.01, 117.67, 115.51, 114.13, 112.83, 61.22, 56.33, 52.38 (two), 50.60 (two). ESI-MS calcd for C 25 H 23 N6O5 (M+H) + m / z 516.1883, found 516.1889.

[0136] Example 19 Synthesis of 4-(5-(4-(4-(3-formyl-4-hydroxybenzyl)piperazin-1-yl)-3- nitrophenyl)-1,2,4-oxadiazol-3-yl)benzonitrile (Compound 22)

[0137] Reference to the preparation method of Example 29, compound (4), the target compound (22) was obtained using intermediate (5s) as raw material, 83 mg, yellow solid, yield 28%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.94 (s, 1H, Ar-OH), 8.65 (d, J = 2.1 Hz, 1H, Ar-H), 8.33-8.27 (m, 2H, Ar-H), 8.23 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.83 (d, J = 8.4 Hz, 2H, Ar-H), 7.66-7.51 (m, 2H, Ar-H), 7.23 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 3.62 (s, 2H, Ar-CH2), 3.31 (s, 4H, piperazine-CH2CH2), 2.69 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.49, 174.49, 167.69, 161.11, 148.35, 140.51, 138.01, 132.70 (two), 132.60, 130.92, 128.08 (two), 128.01, 127.15, 127.10, 120.39, 120.29, 118.20, 117.88, 117.82, 114.86, 61.60, 52.37 (two), 50.55 (two). HRMS (ESI) calcd for C 27 H 23 N6O5 (M+H) + m / z 511.1730, found 511.1727.

[0138] Example 20 Synthesis of 3-(5-(4-(4-(3-formyl-4-hydroxybenzyl)piperazin-1-yl)-3- nitrophenyl)-1,2,4-oxadiazol-3-yl)benzonitrile (23)

[0139] The target compound (23) was obtained by referring to the preparation method of compound (4) in Reference Example 1, using intermediate (5t) as a raw material, 72 mg, yellow solid, yield 24%. Purity 96%. 1H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.93 (s, 1H, Ar-OH), 8.64 (d, J = 2.1 Hz, 1H, Ar-H), 8.48 (d, J = 1.7 Hz, 1H, Ar-H), 8.43-8.38 (m, 1H, Ar-H), 8.23 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.83 (dt, J = 7.8, 1.5 Hz, 1H, Ar-H), 7.67 (t, J = 7.8 Hz, 1H, Ar-H), 7.56 (d, J = 8.0 Hz, 2H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.30 (d, J = 5.8 Hz, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.50, 174.49, 167.38, 161.04, 148.37, 140.44, 137.97, 134.49, 133.97, 132.61, 132.57, 131.48, 131.12, 129.90, 129.87, 128.19, 127.12, 120.38, 118.00, 117.78, 114.84, 113.41, 61.64 (two), 52.41 (two), 50.61. HRMS (ESI) calcd for C 27 H 23 N6O5 (M+H) + m / z 511.1730, found 511.1734.

[0140] The synthesis route of compounds 24-25 is as follows:

[0141]

[0142] 23. Synthesis of intermediate 4-(4-(3-(3-hydroxyphenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (compound 7a)

[0143] Referring to the preparation method of intermediate compound (5a), benzonitrile is replaced by 3-hydroxybenzonitrile to obtain intermediate (7a), 905 mg, yellow solid, yield 40%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 9.88 (s, 1 H, Ar-OH), 8.54 (d, J = 2.2 Hz, 1 H, Ar-H), 8.24 (dd, J = 8.9, 2.2 Hz, 1 H, Ar-H), 7.54 - 7.46 (m, 3 H, Ar-H), 7.39 (t, J = 7.8 Hz, 1 H, Ar-H), 7.03 - 6.97 (m, 1 H, Ar-H), 3.50 (dd, J = 6.7, 3.6 Hz, 4 H, piperazine-CH2CH2), 3.27 - 3.21 (m, 4 H, piperazine-CH2'CH2'), 1.43 (s, 9 H, OC(CH3)3). ESI-MS calculated for C 23 H 26 N5O6 (M+H) + m / z: 468.2, found 468.2.

[0144] 24. Synthesis of intermediate tert-butyl 4-(4-(3-hydroxy-4- (trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)piperazin-1 -carboxylate (compound 7b)

[0145] Referring to the preparation method of intermediate compound (5a), benzonitrile was replaced by 3-hydroxy-4-(trifluoromethyl)benzonitrile to obtain intermediate (7b), 1.1 g, yellow solid, yield 42%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 9.88 (s, 1 H, Ar-OH), 8.54 (d, J = 2.2 Hz, 1 H, Ar-H), 8.24 (dd, J = 8.9, 2.2 Hz, 1 H, Ar-H), 7.54 - 7.46 (m, 3 H, Ar-H), 7.39 (t, J = 7.8 Hz, 1 H, Ar-H), 7.03 - 6.97 (m, 1 H, Ar-H), 3.50 (dd, J = 6.7, 3.6 Hz, 4 H, piperazine-CH2CH2), 3.27 - 3.21 (m, 4 H, piperazine-CH2'CH2'), 1.43 (s, 9 H, OC(CH3)3). ESI-MS calculated for C 24 H 25 F3N5O6 (M+H) + m / z: 536.2, found 536.1.

[0146] 25. Synthesis of intermediate tert-butyl 4-(4-(3-(2-amino-2-oxoethoxy)phenyl)- 1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazine-1 -carboxylate (compound 8a)

[0147] Weigh 300 mg of tert-butyl 4-(4-(3-(3-hydroxyphenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-1 -carboxylate (compound 7a), 115 mg of 2-bromoacetamide and 133 mg of potassium carbonate, dissolve in 5.5 mL of N,N-dimethylformamide, heat to 65°C and stir for 3 h. After monitoring the end of the reaction by TLC, it is evaporated under reduced pressure, extracted (dissolved in EtOAc, washed with H2O and saturated NaCl, in that order). The organic phase is dried with anhydrous Na2SO4, evaporated under reduced pressure and the intermediate (8a) is obtained, 312 mg, yellow solid, 93% yield. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.54 (d, J = 2.2 Hz, 1 H, Ar-H), 8.24 (dd, J = 8.9, 2.2 Hz, 1 H, Ar-H), 7.74-7.61 (m, 3H, Ar-H), 7.56-7.47 (m, 2H, CONH2), 7.44 (s, 1 H, Ar-H), 7.21 (ddd, J = 8.3, 2.7, 0.9 Hz, 1 H, Ar-H), 4.55 (s, 2H, OCH2CO), 3.50 (dd, J = 6.7, 3.6 Hz, 4H, piperazine-CH2CH2), 3.27-3.21 (m, 4H, piperazine-CH2'CH2'), 1.43 (s, 9H, OC(CH3)3). ESI-MS calculated for C 25 H 29 N6O7 (M+H) + m / z: 525.2, found 525.2.

[0148] 26. Synthesis of intermediate tert-butyl 4-(4-(3-(2-amino-2-oxoethoxy)-4- (trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-nitrophenyl)piperazine-1 -carboxylate (compound 8b) The synthesis of this intermediate is the same as that of intermediate 8a. Weigh 300 mg of tert-butyl 4-(4-(3-(2-amino-2-oxoethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-2- nitrophenyl)piperazine-1 -carboxylate (7b) into the reaction and the intermediate (8b) is obtained, 302 mg, yellow solid, 91 % yield. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 8.55 (d, J = 2.2 Hz, 1H), 8.25 (dd, J = 8.9, 2.2 Hz, 1H, Ar-H), 7.92 - 7.80 (m, 2H, Ar-H), 7.73 (s, 1H, Ar-H), 7.55 - 7.46 (m, 2H, CONH2), 7.37 (s, 1H, Ar-H), 4.80 (s, 2H, OCH2CO), 3.50 (dd, J = 7.1, 3.6 Hz, 4H, piperazine-CH2CH2), 3.29 - 3.18 (m, 4H, piperazine-CH2'CH2'), 1.43 (s, 9H, OC(CH3)3). ESI-MS calcd for C 26 H 28 F3N6O7 (M+H) + m / z: 593.3, found 593.2.

[0149] Synthesis of 2-(3-(5-(4-(3-formyl-4-hydroxybenzyl)piperazin-1-yl)-3- nitrophenyl)-1,2,4-oxadiazol-3-yl)phenoxy)acetamide (Compound 24)

[0150] The target compound (24) was obtained by referring to the preparation method of compound (4) in Reference Example 1, using intermediate (8a) as a raw material, 163 mg, yellow solid, yield 51%. Purity 96.31%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 10.69 (s, 1H, Ar-CHO), 10.27 (s, 1H, Ar-OH), 8.51 (d, J = 2.1 Hz, 1H, Ar-H), 8.21 (dd, J = 8.9, 2.2 Hz, 1H, Ar-H), 7.70 (dt, J = 7.7, 1.2 Hz, 1H), 7.67 - 7.63 (m, 2H, Ar-H), 7.61 (d, J = 2.2 Hz, 1H, Ar-H), 7.56 - 7.46 (m, 3H, Ar-H, CONH2), 7.45 - 7.41 (m, 1H, Ar-H), 7.21 (dd, J = 8.3, 2.0 Hz, 2H, Ar-H), 6.99 (d, J = 8.4 Hz, 1H, Ar-H), 4.54 (s, 2H, OCH2CO), 3.51 (s, 2H, Ar-CH2), 3.23 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.52 (s, 4H, piperazine-CH2'CH2'). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 191.96, 174.42, 170.07, 168.49, 160.45, 158.64, 148.24, 139.68, 137.70, 132.94, 130.99, 129.76, 127.72, 127.04, 126.95, 122.37, 121.64, 120.38, 118.40, 117.68, 113.89, 113.79, 67.22, 61.17, 52.36 (two), 50.55 (two). ESI-MS calcd for C 28 H 27 N6O7 (M+H) + m / z 559.1941, found 559.1934.

[0151] Synthesis of 2-(5-(5-(4-(4-(3-formyl-4-hydroxybenzyl)piperazin-l-yl)-3- nitrophenyl)-l,2,4-oxadiazol-3-yl)-2-(trifluoromethyl)phenoxy)acetamide (Compound 25)

[0152] The target compound (25) was obtained by referring to the preparation method of compound (4) in Reference Example 1, using intermediate (8b) as a raw material, 177 mg, yellow solid, yield 49%. Purity 97.00%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 10.68 (s, 1H, Ar-CHO), 10.27 (s, 1H, Ar-OH), 8.50 (d, J = 2.2 Hz, 1H, Ar-H), 8.20 (dd, J = 8.9, 2.2 Hz, 1H, Ar-H), 7.84 (q, J = 8.2 Hz, 2H, Ar-H), 7.72 (s, 1H, Ar-H), 7.60 (d, J = 2.3 Hz, 1H, Ar-H), 7.53-7.44 (m, 3H, Ar-H, CONH2), 7.36 (s, 1H, Ar-H), 6.99 (d, J = 8.4 Hz, 1H, Ar-H), 4.80 (s, 2H, OCH2CO), 3.50 (s, 2H, Ar-CH2), 3.22 (t, J = 4.8 Hz, 4H, piperazine-CH2CH2), 2.52 (s, 4H, piperazine-CH2’CH2’). 13C NMR (100 MHz, DMSO-d6) d (ppm) 191.97, 174.84, 169.12, 167.66, 162.75, 160.42, 156.70, 148.33, 139.55, 137.66, 132.96, 131.77, 129.72, 129.16, 128.46, 127.07, 126.44 (d, J = 273.1 Hz), 122.36, 121.59, 120.05, 117.66, 113.51, 112.39, 67.63, 61.21, 52.37 (two), 50.56 (two). ESI-MS calcd for C 29 H 26 F3N6O7 (M+H) + m / z 627.1810, found 627.1803.

[0153] The synthesis route of compounds 26-27 is as follows:

[0154]

[0155] 27. Synthesis of intermediate tert-butyl 4-(2-nitro-4-(2-(4- (trifluoromethyl)benzoyl)hydrazine-1-carbonyl)phenyl)piperazine-1-carboxylate (compound 9b)

[0156] Dissolve 10 mL of 80% hydrazine hydrate in 25 mL of acetone, stir for 10 min at 0 °C, then add 2.5 mL of 4-(trifluoromethyl)benzoyl chloride (9a) dropwise, and then continue to stir at room temperature for 60 min. After the reaction is completed, it is evaporated under reduced pressure, and purified by flash silica gel column chromatography to obtain the intermediate 4-(trifluoromethyl)benzohydrazide. Then dissolve the intermediate 4-(trifluoromethyl)benzohydrazide in 5.0 mL of dichloromethane solution, and add 1.4 g of intermediate 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-nitrobenzoic acid (3c), 1.9 mg of HATU and 1.4 mL of DIPEA in sequence, and stir at room temperature for 4 h. After the reaction is completed, it is evaporated under reduced pressure, and purified by silica gel column chromatography to obtain the intermediate (9b), 2.2 g, yellow solid, yield 24%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.81 (s, 1 H, CONH), 10.73 (s, 1 H, CONH’), 8.44 (d, J = 2.2 Hz, 1 H, Ar-H), 8.15 - 8.09 (m, 3 H, Ar-H), 7.93 (d, J = 8.3 Hz, 2 H, Ar-H), 7.41 (d, J = 8.9 Hz, 1 H, Ar-H), 3.52 - 3.45 (m, 4 H, piperazine-CH2’CH2’), 3.20 - 3.12 (m, 4 H, piperazine-CH2’CH2’), 1.43 (d, J = 3.2 Hz, 9 H, OC(CH3)3). ESI-MS calculated for C 24 H 26 F3N5O6 (M+H) + m / z: 538.2, found 538.1.

[0157] 28. Synthesis of tert-butyl 4-(2-nitro-4-(5-(4-(trifluoromethyl)phenyl)-1,3,4- thiadiazol-2-yl)phenyl)piperazine-1-carboxylate (compound 9c)

[0158] To a solution of 1.0 g of intermediate tert-butyl 4-(2-nitro-4-(2-(4- (trifluoromethyl)benzoyl)hydrazine-1-carbonyl)phenyl)piperazine-1-carboxylate (9b), 240 mg of Vilsmeier reagent and 1.5 g of Lawesson’s reagent in 40 mL of tetrahydrofuran was stirred at room temperature for 15 h. After the reaction was completed by TLC monitoring, it was evaporated under reduced pressure and purified by silica gel column chromatography to obtain the intermediate (9c), 617 mg, yellow solid, yield 61%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.46 (d, J = 2.3 Hz, 1 H, Ar-H), 8.24 (d, J = 8.1 Hz, 2 H, Ar-H), 8.18 (dd, J = 8.8, 2.3 Hz, 1 H, Ar-H), 7.96 (d, J = 8.2 Hz, 2 H, Ar-H), 7.47 (d, J = 8.8 Hz, 1 H, Ar-H), 3.49 (t, J = 5.0 Hz, 4 H, piperazine-CH2CH2), 3.22 - 3.15 (m, 4 H, piperazine-CH2’CH2’), 1.43 (s, 9 H, OC(CH3)3). ESI-MS calculated for C 24 H 24 F3N5O4S (M+H) + m / z: 536.2, found 536.1.

[0159] Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(5-(4-(trifluoromethyl)phenyl)-1,3,4- oxadiazol-2-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 26)

[0160] The target compound (26) was obtained by referring to the preparation method of compound (4) in Example 1, using intermediate (9b) as the raw material, 192 mg, yellow solid, yield 60%. Purity 97.63%. 1 H NMR (400 MHz, Chloroform-d) d (ppm) 11.01 (s, 1H, Ar-CHO), 9.94 (s, 1H, Ar-OH), 8.53 (d, J = 2.1 Hz, 1H, Ar-H), 8.29 (d, J = 8.2 Hz, 2H, Ar-H), 8.24 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.84 (d, J = 8.2 Hz, 2H, Ar-H), 7.64-7.51 (m, 2H, Ar-H), 7.24 (d, J = 8.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.5 Hz, 1H, Ar-H), 3.60 (s, 2H, Ar-CH2), 3.28 (s, 4H, piperazine-CH2CH2), 2.67 (s, 4H, piperazine-CH2’CH2’).

[0161] 13 C NMR (100 MHz, Chloroform-d) d (ppm) 196.51, 163.46, 161.08, 147.81, 140.83, 137.97, 133.98, 133.67, 133.34, 131.71, 129.37, 129.29, 127.27 (two), 126.22, 126.19, 125.47, 123.54 (q, J = 272.4 Hz), 120.74, 120.40, 117.81, 117.77, 61.65, 52.48 (two), 50.75 (two). HRMS (ESI) calcd for C 27 H 23 F3N5O5 (M+H) + m / z 554.1651, found 554.1645.

[0162] Synthesis of 2-hydroxy-5-((4-(2-nitro-4-(5-(4-(trifluoromethyl)phenyl)-1,3,4- oxadiazol-2-yl)phenyl)piperazin-1-yl)methyl)benzaldehyde (Compound 26)

[0163] The title compound (27) was obtained in reference to the preparation method of compound (4) in Example 1, using intermediate (9c) as the raw material, 210 mg, yellow solid, yield 64%. Purity 95.71%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 10.68 (s, 1H, Ar-CHO), 10.27 (s, 1H, Ar-OH), 8.41 (d, J = 2.3 Hz, 1H, Ar-H), 8.22 (d, J = 8.1 Hz, 2H, Ar-H), 8.14 (dd, J = 8.8, 2.3 Hz, 1H, Ar-H), 7.95 (d, J = 8.3 Hz, 2H, Ar-H), 7.60 (d, J = 2.2 Hz, 1H, Ar-H), 7.49 (dd, J = 8.5, 2.3 Hz, 1H, Ar-H), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 6.99 (d, J = 8.4 Hz, 1H, Ar-H), 3.49 (s, 2H, Ar-CH2), 3.32 (s, 4H, piperazine-CH2CH2), 3.16 (t, J = 4.8 Hz, 4H, piperazine-CH2’CH2’). 13 C NMR (100 MHz, DMSO-d6) δ (ppm) 192.00, 170.79, 167.26, 166.46, 160.42, 147.28, 140.64, 137.69, 133.57, 132.96, 131.64, 131.31, 129.74, 129.21, 128.86 (two), 126.94, 126.90, 125.85, 124.28 (q, J = 272.4 Hz), 122.37, 121.90, 60.22, 52.45 (two), 50.76 (two). HRMS (ESI) calcd for C 27 H 23 F3N5O4S (M+H) + m / z 570.1423, found 570.1415.

[0164] Biological activity test

[0165] Example 25 Fluorescence polarization (FP) test to determine the inhibition constant of target compounds 4-27 against STAT3

[0166] The present application uses Synergy H1 microplate reader (BioTek) to determine the inhibition constant of STAT3 of the compound, which is similar to the FP competition method, and uses the clinical phase I drug STAT3 inhibitor TTI-101 as a positive control compound (purchased from Adamas) (014156748). The fluorescent dye-labeled phosphopeptide 5-FAM-SpYLPQTV is custom synthesized from Genscript, and the purity of the polypeptide is >95%. The human recombinant STAT3 protein is purchased from Abeam (ab43618).

[0167] First, the required drug is diluted according to the concentration range (0-40 μM) at a ratio of 1:2, and then incubated with 300 nM human recombinant STAT3 protein at 37°C for 60 min (the buffer solution used: 10 mM Hepes, 50 mM NaCl, 0.1% Nonidet P-40 and 10% dimethyl sulfoxide, pH 7.6). Subsequently, 10 nM of fluorescently labeled peptide (5-FAM-SpYLPQTV) is added to the system, and incubation is continued for 30 min. The fluorescence polarization value is determined using a multifunctional microplate detector with a 480 nm excitation filter and a 530 nm emission filter, and the IC 50 .

[0168] The inhibition constant (K i value) of each compound on STAT3 is calculated according to the Cheng-Prusoff equation. The Cheng-Prusoff equation is: K i = IC 50 / (1+([STAT3] / K d ), where K d represents the dissociation equilibrium constant between the fluorescently labeled peptide and the STAT3 molecule, and the K d value of the tested fluorescently labeled peptide and STAT3 is 300 nM.

[0169] Figure 1 The inhibition constant of target compounds 4-27 on STAT3 is measured by FP experiment. From the data in the figure, it can be seen that the target compounds have good inhibition effect on STAT3, and the inhibition activity is better than that of the clinical phase I drug TTI-101. Among them, the activity of the target compound modified with hydrophobic group is better than that of the target compound modified with hydrophilic group. Representative compounds such as 9, 10 and 25 have significant inhibition activity on STAT3, and the K i values are 0.52 μM, 0.65 μM and 0.44 μM, respectively.

[0170] Example 26 Isothermal titration calorimetry experiment (ITC experiment)

[0171] Isothermal titration microcalorimetry (ITC) is an experimental technique used to measure the heat change of interactions between biological molecules. In this study, the MicroCal PEAQ-ITC (Malvern Panalytical) was used to determine the affinity of the compound to STAT3.

[0172] Compound 25 of the present application was dissolved in 100% DMSO and diluted to 100 mM (containing 10% DMSO) with a buffer solution (10 mM Hepes, 50 mM NaCl, 0.1% Nonidet P-40 and 10% dimethyl sulfoxide, pH 7.6), while human recombinant STAT3 protein was diluted to 6.5 mM (containing 10% DMSO) with the above buffer solution. 300 μL of 6.5 mM human recombinant STAT3 protein solution was injected into the sample cell, and 2 μL of 100 mM compound 25 solution was titrated every 120 s to return the titration peak to the baseline. Using MicroCal PEAQ-ITC analysis software and GraphPad Prism 9.0 software, the K d determined by single-point model. Control experiments were performed by titrating the test compound into the buffer solution to subtract background noise.

[0173] Figure 2 To verify that the target compound 25 binds to the STAT3 SH2 domain and determine the binding force of the target compound 25 to the STAT3 protein by ITC, among them, Figure 2 a is the optimal chemical structure of compound 25; Figure 2 b is the dose-dependent curve fitted from the FP experiment. The results show that the optimal compound 25 can competitively inhibit the binding of fluorescent polypeptide to the SH2 domain of STAT3, and has a good inhibition constant. Figure 2 c is the isothermal titration calorimetry curve of compound 25 and STAT3 protein measured by ITC experiment, Figure 2 d is the titration fitting curve of compound 25 and STAT3 protein. ITC experiment shows that compound 25 has a high binding affinity to STAT3 protein, with a K d value of 140 nM. In addition, the N value of compound 25 and STAT3 protein is 1.02, which proves that the high affinity of compound 25 to STAT3 is formed by single molecule binding. In summary, the optimal compound 25 is an effective inhibitor targeting the SH2 structure of STAT3.

[0174] Example 27 Anti-cell proliferation assay experiment (CCK-8 assay)

[0175] CCK8 is a chromogenic agent, which has been widely used in the detection of cell proliferation and toxicology evaluation. The CCK8 method is used to determine the optimal compound 25 anti-proliferative activity of different cells.

[0176] The specific operation is as follows: pancreatic cancer cells PANC-1, BxPC-3, Aspc-1, colorectal cancer cells HT29, breast cancer cells MDA-MB-231, gastric cancer cells AGS, normal pancreatic cells Hpde6, normal breast epithelial cells MCF-10A (cells purchased from the cell bank of Wuhan Institute of Virology, Chinese Academy of Sciences) are inoculated in 96-well plates at 5000 cells per well, incubated at 37℃, 5% CO2 for 24h, then treated with different concentrations (10, 5, 2.5, 1.25, 0.63, 0.31 μM) of inhibitors (compound 25). The cells are incubated for 48 hours, and the cytotoxicity is detected by CCK8 method. The absorbance is read at 450 nm using the above multifunctional microplate reader. The results are processed and analyzed by Excel and Graphpad Prism 9.0. Figure 3 The anti-proliferative activity of compound 25 on pancreatic cancer cells and the dose-dependent toxicity curve of normal pancreatic duct cells are compared. The experimental results are shown in Table 1, Table 2 and Figure 3 .

[0177] Table 1 Anti-proliferative activity of compound 25 on pancreatic cancer cells and toxicity of normal pancreatic duct cells

[0178]

[0179] a IC 50 represents the drug concentration required for 50% inhibition of pancreatic cancer cell proliferation, and the results are the average of three parallel experiments, ± indicates standard deviation (SD); b CC 50 represents the drug concentration required for 50% inhibition of pancreatic cancer cell proliferation, and the results are the average of three parallel experiments, ± indicates standard deviation (SD); cSI represents the CC of the inhibitor on Hpde6 50 IC 50 ratio of BxPC-3.

[0180] Table 2 Anti-proliferative activity of compound 25 on colorectal cancer cells, breast cancer cells, gastric cancer cells and toxicity on normal breast epithelial cells

[0181]

[0182] a IC 50 represents the drug concentration required for 50% inhibition of pancreatic cancer cell proliferation, and the results are the average of three parallel experiments, ± indicates standard deviation (SD);b CC 50 The results are the mean of triplicate experiments, ± SD (standard deviation).

[0183] From Table 1 and Figure 3 It can be seen that the optimal compound 25 has good anti-proliferative activity against two pancreatic cancer cell lines PANC-1 and BxPC-3 with high expression of STAT3 (IC 50 0.60 μM and 0.28 μM, respectively), and is superior to the positive control drug BBI-608 (IC 50 0.97 μM and 0.34 μM, respectively).

[0184] In addition, we also tested the anti-proliferative activity of the optimal compound 25 against the pancreatic cancer cell Aspc-1 with low expression of STAT3. From Table 1 and Figure 3 It can be seen that the anti-proliferative activity of the optimal compound 25 against Aspc-1 has a significant decrease, further proving that the optimal compound depends on the expression of STAT3 to exert strong anti-tumor activity. Compared with the positive control drug BBI-608, the optimal compound 25 exhibits good selectivity, with a SI of 45.1, which is much higher than that of BBI-608 (5.9). From the data in Table 1 and Table 2, it can be seen that the optimal compound 25 has good anti-proliferative activity against breast cancer, gastric cancer and colorectal cancer, and has low toxicity against normal breast cells, which indicates that it may be a broad-spectrum anti-tumor inhibitor.

[0185] Example 28 Western Blot experiment to explore the effect of compound 25 on STAT3 signaling pathway

[0186] Western blotting, also known as immunoblotting, is an experimental technique for detecting proteins using antibodies.

[0187] The specific operation is as follows: pancreatic cancer cells PANC-1 and BxPC-3 were seeded at 50 x 10 4The cells were inoculated in six-well plates and incubated at 37°C, 5% CO2 for 24 hours, then treated with DMSO or different concentrations (0.3, 0.6, 1.2, 2.4 μM) of the inhibitor (compound 25) for 48 hours, and then washed with cold PBS and collected. RIPA lysis buffer (Keygen) was added to lyse the cells at 0°C for 30 minutes, centrifuged at 140,000 rpm for 30 minutes, and the supernatant was used to determine the total protein concentration by BCA kit (Xin Saimei). 80 μg of protein sample was used for protein separation by 10% SDS-PAGE concentrated gel, and then transferred to a PVDE membrane (Millipore). After blocking with 5% skim milk powder for 2 hours, the corresponding primary antibodies (1:1000) (STAT3 (79D7) Rabbit mAb, Phospho-STAT3 (Tyr705) (D3A7) Rabbit mAb, Phospho-STAT3 (Ser727) Antibody, β-Actin Mouse mAb, Cyclin D1 Antibody, Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Phospho-Src (Tyr527) Antibody, Phospho-JAK2 Antibody and Bcl-2 Antibody were purchased from Cell Signaling Technology) were incubated at 4°C overnight; washed with TBST five times for 5 minutes each time, and then added with the corresponding secondary antibodies (1:10000) (Anti-mouse IgG (H+L) (DyLight 680 Conjugate) and Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) secondary antibodies purchased from Cell Signaling Technology), and incubated at room temperature for 2 hours, then washed with TBST five times for 5 minutes each time, and then developed with ECL luminescent solution (Xin Saimei). The bands were visualized using a Tanon 5200 full-automatic chemiluminescence image analysis system.

[0188] Figure 4 a is the effect of different concentrations of compound 25 on the STAT3 and its phosphorylation state in PANC-1 cells. Figure 4 b is the effect of different concentrations of compound 25 on the STAT3 and its phosphorylation state in BxPC-3 cells. Figure 4 c is the effect of compound 25 at different times on the STAT3 and its phosphorylation state in PANC-1 cells. Figure 4 d is the effect of compound 25 at different times on the STAT3 and its phosphorylation state in BxPC-3 cells. Figure 4 a andFigure 4 b shows that optimal compound 25 can dose-dependently inhibit STAT3 p-Tyr705 and p-Ser727 activation in pancreatic cancer cells PANC-1 and BxPC-3. In addition, Figure 4 c and Figure 4 d shows that optimal compound 25 can time-dependently inhibit STAT3 p-Tyr705 and p-Ser727 activation in pancreatic cancer cells PANC-1 and BxPC-3. In summary, compound 25 can inhibit STAT3 dual-site phosphorylation process in pancreatic cancer cells by binding STAT3 SH2 domain.

[0189] To further determine whether compound 25 directly acts on STAT3 rather than upstream kinases, we tested the effect of compound 25 on STAT3 upstream JAK2, Src and Erk phosphorylation by Western Blot experiment. Figure 4 e is the effect of compound 25 on JAK2, Src and Erk phosphorylation and on STAT3 downstream proteins in PANC-1 cells. Figure 4 f is the effect of compound 25 on JAK2, Src and Erk phosphorylation and on STAT3 downstream proteins in BxPC-3 cells. Figure 4 e and Figure 5 f results show that compound 25 does not inhibit JAK2, Src and Erk phosphorylation, indicating that the inhibitor does not inhibit STAT3 phosphorylation process by affecting the phosphorylation of upstream kinases. In addition, compound 25 can enhance the content of apoptosis-related protein Cleaved Caspase 3 and inhibit the expression level of downstream Cyclin D1 and BCL-xL proteins.

[0190] In summary, the above results show that compound 25 can exert anti-tumor proliferation activity by blocking the conduction of STAT3 signaling pathway.

[0191] Example 29 Apoptosis assay to explore the effect of compound 25 on cancer cell apoptosis Flow cytometry is a technique for rapid quantitative analysis of single cell physical and chemical properties.

[0192] The specific operation is as follows: pancreatic cancer cells PANC-1 and BxPC-3 are seeded at 3.0 x 10 5The cells were inoculated in six-well plates and incubated at 37℃, 5% CO2 for 24 hours. Then, the cells were treated with 0.1% DMSO or different concentrations of inhibitors for 48 hours, and then the cells were digested with trypsin without EDTA, washed twice with cold PBS, and finally resuspended in 1x binding buffer. The apoptosis of the cells was detected using the apoptosis detection kit Annexin V-FITC / 7-AAD (Procell) according to the instructions, and the cells were analyzed by flow cytometry. All experiments were set up in triplicate. All experimental data were processed by FlowJo 7.6.

[0193] Figure 5 a is the effect of compound 25 at different concentrations on the apoptosis of PANC-1 cells. Figure 5 b is the effect of compound 25 at different concentrations on the apoptosis of BxPC-3 cells. Figure 5 c is the statistical analysis of the effect of compound 25 at different concentrations on the apoptosis of PANC-1 cells. Figure 5 d is the statistical analysis of the effect of compound 25 at different concentrations on the apoptosis of BxPC-3 cells. Figure 5 a and Figure 5 b show that at a concentration of 1 μM, compound 25 can induce the apoptosis of pancreatic cancer cells PANC-1 and BxPC-3, and promote the apoptosis of tumor cells in a dose-dependent manner. Figure 5 c and ​ d show that compound 25 can significantly induce the apoptosis of pancreatic cancer cells.

[0194] In summary, the compound prepared in the present application has the activity of inhibiting the binding of STAT3 and phosphopolypeptide. Among them, the representative compound 25 can bind to the SH2 domain of STAT3 target protein, thereby mediating the formation of STAT3 homodimer. Compound 25 can selectively inhibit the JAK-STAT3 cell pathway, thereby inhibiting the proliferation of tumor cells and inducing the apoptosis of tumor cells, achieving the effect of anti-tumor. The compound has broad-spectrum anti-tumor activity and low toxicity, and has wide application prospect in the biological and pharmaceutical industries.

[0195] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is to be understood that such is by way of illustration and not of limitation. Various changes and modifications can be made therein without departing from the spirit and scope of the present application as defined in the appended claims.

Claims

1. A salicylaldehyde derivative of general formula I or a pharmaceutically acceptable salt thereof: General Formula I; in, Ring A is selected from phenyl, naphthyl, or a six-membered heterocycle containing one heteroatom, wherein the heteroatom is selected from N; Ring B is selected from a five-membered heterocycle containing three heteroatoms, wherein the heteroatoms are selected from N, O or S; R1 is selected from hydrogen, halogen, C1-C3 alkyl, nitro, amino, cyano, carbamoyl methoxy, halogenated C1-C3 alkyl, C1-C3 alkoxy, or halogenated C1-C3 alkoxy. R2 is selected from nitro; n = 1 or 2.

2. The compound according to claim 1, characterized in that: Ring A is selected from phenyl, naphthyl, or a six-membered heterocycle containing one heteroatom, wherein the heteroatom is selected from N; Ring B is selected from a five-membered heterocycle containing three heteroatoms, wherein the heteroatoms are selected from N, O or S; R1 is selected from hydrogen, halogen, methyl, nitro, amino, cyano, carbamoylmethoxy, trifluoromethyl, or methoxy. R2 is selected from nitro; n = 1 or 2.

3. The compound according to claim 1, characterized in that: The pharmaceutically acceptable salt is selected from the acid addition salts formed by compounds of general formula I with the following acids: hydrochloric acid, hydrobromic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid; and also selected from the acid salts formed by compounds of general formula I with inorganic bases.

4. The compound according to claim 3, characterized in that: The pharmaceutically acceptable salts are selected from basic metal cation salts, alkaline earth metal cation salts, and ammonium cation salts.

5. The compound according to claim 1, characterized in that... Selected from: ； 。 6. A pharmaceutical composition, characterized in that: This includes a therapeutically effective amount of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use of the compound according to any one of claims 1-5 in the preparation of a STAT3 inhibitor.

8. Use of the compound according to any one of claims 1-5 in the preparation of a medicament for the prevention and / or treatment of STAT3-related tumors.

9. The use according to claim 8, characterized in that, The tumors associated with STAT3 are pancreatic cancer, breast cancer, gastric cancer, or colorectal cancer.

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