Application of piperine or its derivatives

Abstract

The present invention discloses the use of piperine or its derivatives, relating to the field of pharmaceutical technology. The present invention utilizes natural piperine as a precursor compound and chemically modifies it to obtain a series of novel piperine derivatives. Pharmacodynamic evaluations in leukemia, ovarian cancer, and pancreatic cancer cell models in vitro and in vivo revealed that several novel piperine derivatives inhibit tumor cell proliferation, induce tumor cell apoptosis, and promote leukemia cell differentiation. The present invention provides a group of compounds with novel structures. The synthetic route employed utilizes green chemistry reactions, making them more environmentally friendly and more economical in terms of raw materials. These compounds can be used to develop new drugs for the treatment of leukemia, ovarian cancer, and pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of piperine or its derivatives. Background Art

[0002] Tumors are neoplasms formed by the proliferation of localized tissue cells in response to various tumorigenic factors. They are a serious threat to human health and the second leading cause of death. Typical characteristics of tumor cells are blocked apoptosis and malignant proliferation, while typical characteristics of leukemia cells are blocked apoptosis, malignant proliferation, and blocked differentiation. Therefore, identifying drugs that can promote tumor cell apoptosis, inhibit malignant proliferation, or promote tumor cell differentiation is an important approach to tumor treatment. Despite the emergence of new anti-tumor technologies such as immunotherapy and cell therapy, small molecule anti-tumor drugs remain an important cornerstone of tumor treatment.

[0003] Piperine, molecular formula C 17 H 19 NO3, chemical name is (E,E)-1-[5-(1,3-benzodioxolan-5-yl)-1-oxo-2,4-pentadienyl]-piperidine, the structural formula is as follows:

[0004]

[0005] Piperine is a white crystalline powder with a melting point of 130-133°C. It is soluble in acetic acid, benzene, ethanol, and chloroform, and slightly soluble in ether. It is primarily extracted from the dried, nearly mature or mature fruit of the Piper nigrum plant, a member of the Piperaceae family. It combines medicinal and edible properties, boasting a rich source and a long history of clinical use and consumption both domestically and internationally. Piperine is a broad-spectrum anticonvulsant, demonstrating effective antagonism to experimental electroconvulsive seizures in mice. It also exhibits varying degrees of antagonism to seizures and audiogenic seizures induced by pentylenetetrazol, picrotoxin, strychnine, and intraventricular injection of tubocurarine and glutamate. It is also effective for certain types of epilepsy. Piperine is more toxic to flies than pyrethrum. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide a group of piperine derivatives.

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned piperine derivatives.

[0008] The third object of the present invention is to provide the use of piperine or its derivatives in the preparation of anti-tumor drugs.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A group of piperine derivatives, comprising compounds Pa-3, Pa-4, Pa-6, Pa-7, Pa-8, Pb-1, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pb-10, Pc-2, Pc-3, Pc-4, Pc-6, Pc-7, Pc-8, Pc-9, Pd-2, Pd-3, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8 and Pd-9, wherein the chemical structural formulas of the piperine derivatives are shown below:

[0011]

[0012] The above piperine derivatives are pharmaceutically acceptable salts.

[0013] Furthermore, the anion of the pharmaceutically acceptable salt is an inorganic anion or an organic anion; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, toluenesulfonate, citrate, lactate or gluconate.

[0014] The preparation method of the piperine derivative is any one of the following methods:

[0015] (1) Using anhydrous dichloromethane as solvent, crotonyl chloride and different substituted amines are reacted in the presence of triethylamine at room temperature; the reactants are repeatedly extracted with water and dichloromethane, the organic layer is dried, spin-dried, and purified to obtain the target compound;

[0016] (2) Using anhydrous dichloromethane as solvent, crotonyl chloride and different substituted amines are reacted in the presence of triethylamine at room temperature to obtain an intermediate, which is then reacted with different substituted aldehydes in DMSO solvent under alkaline conditions overnight; the reactants are repeatedly extracted with water and dichloromethane, the organic layer is dried, spin-dried, and purified to obtain the target compound;

[0017] Wherein, the substituted amine is any one of morpholine, 4-piperidinylpiperidine, 4-methylpiperazine, and 4-ethylpiperazine;

[0018] The substituted aldehyde is any one of benzaldehyde, 3-methylbenzaldehyde, 4-methylbenzaldehyde, 4-methoxybenzaldehyde, 4-tert-butylbenzaldehyde, 4-phenylbenzaldehyde, 2-morpholinobenzaldehyde, 3,4-dimethoxybenzaldehyde, and 3,4-(methylenedioxy)benzaldehyde.

[0019] Furthermore, the molar ratio of crotonyl chloride to substituted amine is 2:1.

[0020] Furthermore, the reaction time at room temperature is 7±1 hours.

[0021] Furthermore, the molar ratio of the intermediate to the substituted aldehyde is 1:2.

[0022] Furthermore, the alkaline condition is achieved by adding NaOH, and the added amount is calculated as DMSO solvent: NaOH = 4 mL: 1 mL.

[0023] Use of piperine or a piperine derivative in the preparation of an anti-tumor drug, wherein the piperine or piperine derivative includes a pharmaceutically acceptable salt thereof; the application is any one or more of the following:

[0024] I. Use of piperine derivatives in the preparation of drugs for treating ovarian cancer;

[0025] II. Use of piperine or piperine derivatives in the preparation of drugs for the treatment of leukemia;

[0026] III. Use of piperine or a piperine derivative in the preparation of a medicament for treating pancreatic cancer.

[0027] The piperine derivatives include compounds having the structure shown in Formula I:

[0028]

[0029] Wherein, R1 is selected from any one of methyl, styryl, 3-methylstyryl, 4-methylstyryl, 4-methoxystyryl, 4-tert-butylstyryl, 4-phenylstyryl, 3,4-methylenedioxystyryl, 2-morpholinostyryl, and 3,4-dimethoxystyryl;

[0030] R2 is selected from any one of morpholinyl, 4-piperidinyl, 4-methylpiperazinyl and 4-ethylpiperazinyl.

[0031] Furthermore, the piperine derivative includes any one of compounds Pa-1, Pa-2, Pa-3, Pa-4, Pa-5, Pa-6, Pa-7, Pa-8, Pb-1, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pb-10, Pc-1, Pc-2, Pc-3, Pc-4, Pc-5, Pc-6, Pc-7, Pc-8, Pc-9, Pd-1, Pd-2, Pd-3, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8, and Pd-9; more preferably Pa-6 or Pb-7; compounds Pa-1 to Pd-9 are compounds having the following structures:

[0032]

[0033] Furthermore, the anion of the pharmaceutically acceptable salt is an inorganic anion or an organic anion; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, toluenesulfonate, citrate, lactate or gluconate.

[0034] Furthermore, the drug for treating ovarian cancer is a drug for treating paclitaxel-resistant ovarian cancer.

[0035] Furthermore, the drug for treating leukemia has the dual functions of promoting differentiation and apoptosis of leukemia cells.

[0036] Furthermore, the anti-tumor drug inhibits tumor cell proliferation by antagonizing the expression of BcL-2, XIAP, JAK1, JAK2, STAT1 and / or Cl-PARP proteins.

[0037] Furthermore, Application I is: use of any one of Pb-7, Pc-6, Pc-7, Pc-8, Pd-6, and Pd-7 in the preparation of a drug for treating ovarian cancer.

[0038] Furthermore, Application II is: the use of any one of Pa-1, Pa-2, Pa-3, Pa-4, Pa-5, Pa-6, Pa-7, Pa-8, Pb-1, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pc-1, Pc-2, Pc-3, Pc-4, Pc-5, Pc-6, Pc-7, Pc-8, Pd-1, Pd-2, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8, and Pd-9 in the preparation of a drug for treating leukemia.

[0039] Furthermore, Application III is: use of Pb-7 in preparing medicine for treating pancreatic cancer.

[0040] The anti-tumor drugs can be prepared in a variety of dosage forms, including solid dosage forms, semi-solid dosage forms, liquid dosage forms, and aerosol dosage forms. Specific dosage forms within these categories include tablets, pills, granules, lozenges, ointments, solutions, suppositories, injections, inhalants, and sprays. These dosage forms can be used for topical or systemic administration, as well as for rapid-release or sustained-release administration. There are many ways to administer these drugs, including oral, buccal, rectal, peritoneal, epidermal, subcutaneous, and intratracheal administration.

[0041] When the anti-tumor drug is administered by injection, the compound can be formulated into a solution, suspension or emulsion using a water-soluble or fat-soluble solvent. Fat-soluble solvents specifically include vegetable oils and similar oils, synthetic fatty acid glycerides, higher fatty acid esters and glycol esters.

[0042] When the anti-tumor drug is administered orally, it can be formulated into a complex with a pharmaceutically acceptable excipient using conventional techniques. These excipients allow the compound to be formulated into a variety of dosage forms acceptable to patients, such as tablets, pills, suspensions, gels, and the like. There are various methods for preparing oral dosage forms, such as first mixing the compound with a solid excipient, thoroughly grinding the mixture, adding appropriate excipients, and processing it into granules. Excipients that can be used to prepare oral dosage forms include: sugars such as lactose, niacin, mannitol, or sorbitol; cellulosics such as corn starch, wheat starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like.

[0043] The anti-tumor drug can also be made into a spray, which is achieved by using a pressurizer and a nebulizer or a dry powder inhaler. Suitable propellants such as dichlorodifluoromethane, fluorotrichloromethane, dichlorotetrafluoroethane, carbon dioxide, and dimethyl ether can be used as sprays. The dosage of the aerosol can be adjusted by the valve of the sprayer.

[0044] The present invention has the following advantages and effects compared to the prior art:

[0045] One of the innovative models for conducting innovative drug research is to use natural products as lead compounds, conduct structural modification studies using organic chemistry and medicinal chemistry, and find new drug lead compounds from them.

[0046] This study uses natural piperine as a precursor compound and chemically modifies it to produce a series of novel piperine derivatives. Pharmacodynamic evaluations in leukemia, ovarian cancer, and pancreatic cancer cell models in vitro and in vivo revealed that several of these novel piperine derivatives inhibit tumor cell proliferation, induce tumor cell apoptosis, and promote leukemia cell differentiation.

[0047] The present invention provides a group of compounds with novel structures. The synthetic route adopted utilizes green chemical reactions, which is more environmentally friendly and more economical in terms of raw materials. These compounds can be used to develop new drugs for treating leukemia, ovarian cancer, and pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The synthetic route of piperine derivatives.

[0049] Figure 2 This is a diagram showing the effects of compounds Pa-6 and Pb-7 on inducing differentiation of NB4 cells.

[0050] Figure 3 This is a graph showing the detection of apoptosis of NB4 cells induced by compounds Pa-6 and Pb-7.

[0051] Figure 4 Cell apoptosis (A), DNA content (B), mitochondrial membrane potential (C), and periodogram (D) were detected after compound Pb-7 was applied to SKOV3 cells.

[0052] Figure 5 This is the cell protein detection diagram after compound Pb-7 acts on SKOV3 cells for 48 hours.

[0053] Figure 6 This is a graph showing the inhibition of cell proliferation of PANC-1 cells by compound Pb-7 for 72 hours.

[0054] Figure 7 This is a graph showing the in vivo antitumor activity of compound Pb-7 in a nude mouse transplanted human SKOV3 cell model.

[0055] Figure 8 This is the HE staining image of nude mouse viscera in the in vivo anti-tumor activity evaluation of compound Pb-7.

[0056] Figure 9 This is the Ki67 staining image of nude mouse tumor tissue in the in vivo anti-tumor activity evaluation of compound Pb-7. DETAILED DESCRIPTION

[0057] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0058] In this disclosure, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0059] In the present invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or plural.

[0060] It should be understood that in various embodiments of the present invention, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0061] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "an", "the" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0062] The weights of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention.

[0063] Example 1: Preparation of Pa-1

[0064]

[0065] Morpholine (3.535 mL, 27.83 mmol) was measured and placed in a 100 mL round-bottom flask. 7 mL of anhydrous dichloromethane was added and stirred in an ice bath. 730 μL of triethylamine was then added. 4 mL of crotonyl chloride was dissolved in 6 mL of anhydrous dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to proceed for 8 hours. Saturated sodium bicarbonate solution was added dropwise to neutralize any unreacted crotonyl chloride. Extraction was performed, and the organic layers were combined to obtain intermediate Pa-1 as a yellow oil with a yield of 98%. Pa-1: 1 H NMR (400MHz, CDCl3) δ7.65(t,J=7.8Hz,1H),7.22(dd,J=15.6,6.9Hz,1H),7.06(d,J=7.4Hz,1H) ,6.91(d,J=8.0Hz,1H),6.04(dq,J=15.5,1.8Hz,1H),2.52(s,3H),1.95(dd,J=6.9,1.8Hz,3H). 13 CNMR(101MHz, CDCl3)δ164.36,158.05,157.20,147.74,139.48,121.77,121.32,113.21,23.98,18.23.HR-ESIMS m / z 156.1031[M+H] + ,calcd for C8H 14 NO2+ 156.1019.

[0066] Example 2: Preparation of Pb-1, Pc-1, and Pd-1

[0067] Replace morpholine with 4-piperidinylpiperidine or 4-methylpiperazine or 4-ethylpiperazine and use the method of Example 1 to obtain Pb-1, Pc-1, and Pd-1:

[0068]

[0069] Pb-1: 1 H NMR (400MHz, CDCl3) δ6.10 (d, J = 15.0Hz, 1H), 5.70 (d, J = 15.4Hz, 1H), 4.56

[0070] (d,J=13.8Hz,1H),3.91(d,J=15.5Hz,1H),2.84-2.76(m,1H),2.58(s,4H),1.88-1.78( m,2H),1.68(ddd,J=19.3,6.8,1.9Hz,5H),1.57(p,J=5.6Hz,5H),1.34(d,J=5.1Hz,3H). 13 C NMR (101MHz, CDCl3) δ171.37,165.10,141.31,140.11,126.66,121.07,61.94,49.01,24.16,23.32,17.84,17.28.HR-ESIMS m / z 237.2011[M+H] + ,calcd forC 14 H 25 N2O + 237.1961.

[0071] Pc-1: 1 H NMR (400MHz, CDCl3) δ6.92-6.80 (m, 1H), 6.24 (dd, J = 15.0, 1.9Hz, 1H), 3.67

[0072] (s,2H),3.55(s,2H),2.41-2.37(m,4H),2.30(s,3H),1.87(dd,J=6.9,1.9Hz,3H). 13 C NMR (101MHz, CDCl3) δ165.58,141.61,121.43,46.00,43.24,29.68,18.22.HR-ESIMS m / z 169.1335[M+H] +,calcd for C 10 H 19 N2O + 169.1341.

[0073] Pd-1: 1 H NMR (400MHz, CDCl3) δ6.79 (dd, J=14.9, 6.9Hz, 1H), 6.18 (d, J=15.0Hz, 1H),

[0074] 3.56(d,J=44.8Hz,4H),2.39-2.33(m,6H),1.80(d,J=6.9Hz,3H),1.02(t,J=7.3Hz,3H). 13 C NMR (101MHz, CDCl3) δ165.51,141.56,121.42,52.18,45.58,41.78,18.21,11.84.HR-ESIMS m / z 183.1463[M+H] + ,calcd for C 10 H 19 N2O + 183.1492.

[0075] Example 3: Preparation of Pa-2

[0076]

[0077] Benzaldehyde (3.16 mmol) was added to a 50 mL round-bottom flask, and 2 mL of DMSO was added with stirring to dissolve. 1 mL of saturated NaOH was added dropwise and stirred for 30 minutes. Intermediate Pa-1 (1.58 mmol) was dissolved in 2 mL of DMSO and added dropwise to the reaction system. The mixture was allowed to react overnight. DMSO was removed by repeated extraction with water and dichloromethane. Anhydrous sodium sulfate was added to the organic layer, which was then dried by spin drying. Pa-2 was isolated by column chromatography to obtain a yellow solid in a 50% yield. 1 H NMR (400MHz, CDCl3) δ7.50-7.41(m,3H),7.35(d,J=7.5Hz,2H),7.29(d,J=7.0Hz,1H),6.88(d,J=7.6Hz,2H),6.40(d,J=14.6Hz,1H),3.82-3.58(m,8H). 13C NMR (101MHz, CDCl3) δ165.54,143.27,139.33,136.15,128.69,126.94,126.56,119.49,66.74,46.08,42.32.HR-ESIMS m / z 244.1327[M+H] + ,calcd for C8H 14 NO2 + 244.1332.

[0078] Example 4: Preparation of Pa-3 to Pa-8

[0079]

[0080] Benzaldehyde was replaced by 3-methylbenzaldehyde, or 4-methylbenzaldehyde, or 4-methoxybenzaldehyde, 4-tert-butylbenzaldehyde, 4-phenylbenzaldehyde, or 2-morpholinobenzaldehyde, and Adol condensation was carried out with the intermediate Pa-1. The preparation method was the same as that in Example 3 to obtain Pa-3, Pa-4, Pa-5, Pa-6, Pa-7, and Pa-8, respectively.

[0081] Pa-3: 1 H NMR (400MHz, CDCl3) δ7.45 (dd, J=14.7, 9.9Hz, 1H), 7.25-7.18 (m, 3H), 7.07

[0082] (d,J=6.9Hz,1H),6.91-6.78(m,2H),6.38(d,J=14.6Hz,1H),3.61(d,J=41.9Hz,8H),2.32(s,3H). 13 C NMR (101MHz, CDCl3) δ165.57,143.33,139.46,138.25,136.14,129.56,128.59,127.74,126.45,124.09,119.40,66.75,21.30.HR-ESIMS m / z258.1493[M+H] + ,calcd for C 16 H 20 NO2 + 258.1489.

[0083] Pa-4: 1 H NMR (400MHz, CDCl3) δ7.50-7.41(m,1H),7.33(d,J=8.0Hz,2H),7.13(d,J=

[0084] 7.9Hz,2H),6.84(d,J=6.5Hz,2H),6.37(d,J=14.6Hz,1H),3.62(d,J=42.4Hz,8H),2.33(s,3H). 13 C NMR(101MHz,CDCl3)δ165.56,143.44,139.31,138.80,133.36,129.37,126.85,125.58,118.85,66.69,46.00,42.21,21.22.HR-ESIMS m / z 258.1491[M+H] + ,calcd for C 16 H 20 NO2 + 258.1489.

[0085] Pa-5: 1 H NMR(400MHz,CDCl3)δ7.39(dd,J=14.6,9.4Hz,1H),7.31(d,J=8.9Hz,2H),

[0086] 6.79(d,J=8.9Hz,2H),6.71(d,J=12.3Hz,2H),6.29(d,J=14.5Hz,1H),3.72(s,3H),3.55(d,J=39.6Hz,8H). 13 C NMR(101MHz,CDCl3)δ165.56,160.01,143.50,138.87,128.87,128.29,124.45,118.27,114.07,66.62,55.12.HR-ESIMS m / z 274.1348[M+H] + ,calcd for C 16 H 20 NO3 + 274.1438.

[0087] Pa-6: 1 H NMR(400MHz,CDCl3)δ7.48(dt,J=15.6,5.4Hz,1H),7.36(d,J=9.5Hz,4H),

[0088] 6.86(d,J=5.1Hz,2H),6.39(d,J=14.6Hz,1H),3.69(s,8H),1.31(s,9H). 13C NMR(101MHz,CDCl3)δ165.79,152.23,143.68,139.41,133.60,126.91,125.99,125.81,119.11,66.92,46.20,42.47,34.82,31.28.HR-ESIMS m / z 300.1961[M+H] + ,calcd forC 16 H 20 NO3 + 300.1958.

[0089] Pa-7: 1 H NMR(400MHz,CDCl3)δ7.65-7.55(m,4H),7.52(d,J=8.3Hz,2H),7.44(t,J=

[0090] 7.6Hz,3H),7.35(t,J=7.3Hz,1H),7.01-6.83(m,2H),6.42(d,J=14.6Hz,1H),3.70(s,8H). 13 C NMR(101MHz,CDCl3)δ165.70,143.42,141.55,140.43,138.99,135.37,128.94,127.68,127.59,127.50,127.01,126.78,119.68,66.93,29.78.HR-ESIMS m / z320.1647[M+H] + ,calcd for C 21 H 22 NO2 + 320.1645.

[0091] Pa-8: 1 H NMR(400MHz,CDCl3)δ7.57-7.52(m,1H),7.36(d,J=13.6Hz,1H),7.33-

[0092] 7.26(m,2H),7.07(q,J=7.9Hz,2H),6.89(dd,J=15.7,11.1Hz,1H),6.42(dd,J=15.1,5.0Hz,1H),3.85(dt,J=21.1,4.6Hz,4H),3.73(d,J=4.0Hz,6H),3.63(d,J=7.0Hz,2H),2.99-2.91(m,4H). 13C NMR (101MHz, CDCl3) δ165.83,151.39,144.15,136.64,130.61,129.69,127.09,126.70,123.41,119.09,118.94,67.33,52.98,29.85.HR-ESIMS m / z329.1861[M+H] + ,calcd for C 16 H 18 NO4 + 329.1860.

[0093] Example 5: Preparation of Pb-2

[0094]

[0095] Benzaldehyde (3.16 mmol) was added to a 50 mL round-bottom flask, and 2 mL of DMSO was added and stirred to dissolve. 1 mL of saturated NaOH was added dropwise and stirred for 30 min. Intermediate Pb-1 (1.58 mmol) was dissolved in 2 mL of DMSO and added dropwise to the reaction system. The mixture was allowed to react overnight. DMSO was removed by repeated extraction with water and dichloromethane. Anhydrous sodium sulfate was added to the organic layer, which was then dried by spin drying. Pb-2 was isolated by column chromatography to obtain a yellow solid in a 49% yield. 1 H NMR (400MHz, CDCl3) δ7.61(d,J=15.5Hz,1H),7.48(dd,J=7.8,1.9Hz,2H),7.33(d,J=7.5Hz,3H),6.86(d,J=15.4Hz,1H),4.84-4.63(m,3 H),4.24-4.08(m,1H),2.80(ddt,J=11.7,8.2,3.7Hz,1H),2.63(d,J=14.7Hz,4H),2.04(s,2H),1.69(p,J=5.5Hz,4H),1.57-1.43(m,4H). 13 C NMR (101MHz, CDCl3) δ165.26,142.74,135.07,129.53,128.68,127.64,116.98,62.58,49.70,29.57,24.94,23.83,21.44.HR-ESIMS m / z 325.2276[M+H] + ,calcd for C 21 H 29 N2O + 325.2274.

[0096] Example 6: Preparation of Pb-3 to Pb-10

[0097]

[0098] Benzaldehyde was replaced by 3-methylbenzaldehyde, or 4-methylbenzaldehyde, or 4-methoxybenzaldehyde, or 4-tert-butylbenzaldehyde, or 4-phenylbenzaldehyde, or 2-morpholinobenzaldehyde, or 3,4-dimethoxybenzaldehyde, or 3,4-(methylenedioxy)benzaldehyde, and Adol condensation was carried out with the intermediate Pb-1. The preparation method was the same as that in Example 5 to obtain Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, and Pb-10, respectively.

[0099] Pb-3: 1 H NMR (400MHz, CDCl3) δ7.37 (dd, J=15.2, 10.9Hz, 1H), 7.23-7.17 (m, 2H),

[0100] 7.05(d,J=6.6Hz,1H),6.90-6.73(m,2H),6.43(d,J=14.8Hz,1H),4.69(d,J=10.1Hz,1H),4.05(d,J=13.8Hz,1H) ,2.56(d,J=1.4Hz,7H),2.49-2.44(m,4H),2.31(s,1H),1.88-1.80(m,2H),1.59-1.50(m,4H),1.47-1.35(m,4H). 13 C NMR (101MHz, CDCl3) δ165.37,142.77,138.95,138.35,136.32,129.47,128.64,127.7 2,126.73,124.10,120.37,62.59,50.13,40.93,29.68,26.21,24.64,21.45.HR-ESIMS m / z 339.2438[M+H] + ,calcdfor C 22 H 31 N2O + 339.2431.

[0101] Pb-4: 1 H NMR (400MHz, CDCl3) δ7.39 (dd, J=14.8, 9.1Hz, 1H), 7.32 (d, J=8.0Hz, 2H),

[0102] 7.26(s,1H),7.12(d,J=8.0Hz,2H),6.82(d,J=9.4Hz,1H),6.42(d,J=14.6Hz,1H),4.70(d,J=13.6Hz,1H),4.12-4.00(m,1H),3.07-2.91(m,2H),2.57(s,1H),2.47(s,4H),2.32(s,3H),1.85(d,J=13.1Hz,2H),1.60-1.51(m,5H),1.44(d,J=12.4Hz,5H). 13 CNMR(101MHz,CDCl3)δ165.33,142.84,138.75,138.65,133.55,129.38,126.82,125.86,119.83,62.50,50.03,40.81,29.60,26.14,24.56,21.24.HR-ESIMS m / z 339.2436[M+H] + ,calcd for C 22 H 31 N2O + 339.2431.

[0103] Pb-5: 1 H NMR(400MHz,CDCl3)δ7.35(d,J=7.4Hz,2H),7.29(d,J=9.0Hz,2H),6.84(t,

[0104] J=6.1Hz,3H),6.75(s,1H),6.38(d,J=15.0Hz,1H),5.21(d,J=14.1Hz,1H),4.68(d,J=10.3Hz,1H),4.05(d,J=15.8Hz,1H),3.78(s,3H),2.64(s,1H),2.47(s,4H),1.84(d,J=13.5Hz,2H),1.58-1.53(m,4H),1.45-1.38(m,4H). 13 C NMR(101 MHz,CDCl3)δ165.52,160.09,143.16,138.56,128.38,127.12,124.83,119.24,114.23,62.60,55.31,49.70,40.89,29.69,26.14,24.61.HR-ESIMS m / z 355.2331[M+H] + ,calcd for C 22 H 31 N2O2+ 355.2380.

[0105] Pb-6: 1 H NMR(400 MHz,CDCl3)δ7.35(d,J=8.1 Hz,2H),7.28(s,1H),6.77(dt,J=14.9,

[0106] 7.3 Hz,1H),6.28(d,J=15.1 Hz,1H),4.77(t,J=6.4 Hz,1H),4.66(d,J=8.8Hz,1H),4.09(s,1H),3.62(d,J=10.1 Hz,4H),2.84(s,1H),2.55(s,4H),1.65-1.59(m,5H),1.45(d,J=5.4 Hz,5H),1.30(s,9H). 13 C NMR(101 MHz,CDCl3)δ165.57,150.83,141.98,140.99,125.78,125.59,123.39,73.02,70.76,62.70,50.10,46.18,42.50,41.14,34.73,31.57,29.91,25.85,24.55,23.56.HR-ESIMS m / z 381.2908[M+H] + ,calcd forC 25 H 37 N2O + 381.2900.

[0107] Pb-7: 1 H NMR(400 MHz,CDCl3)δ7.55(t,J=7.3 Hz,4H),7.47(d,J=8.3 Hz,2H),7.40(t,

[0108] J=6.8 Hz,3H),7.30(t,J=7.2 Hz,1H),6.95-6.81(m,2H),6.45(d,J=14.8 Hz,1H),4.69(s,1H),4.06(s,1H),3.25(s,1H),3.01(s,1H),2.75(s,1H),2.47(s,4H),1.85(d,J=13.0 Hz,2H),1.55(s,4H),1.48~1.37(m,4H). 13C NMR(101 MHz,CDCl3)δ165.26,142.61,141.22,140.30,138.22,135.38,128.80,127.40,127.32,126.85,120.57,62.53,50.09,40.90,29.65,28.82,27.44,26.18,24.61.HR-ESIMS m / z 401.2592[M+H] + ,calcdfor C 22 H 31 N2O2 + 401.2587.

[0109] Pb-8: 1 H NMR(400 MHz,CDCl3)δ7.50(d,J=7.6 Hz,1H),7.46-7.39(m,1H),7.29(d,J=

[0110] 8.8 Hz,1H),7.24(d,J=12.8 Hz,2H),7.20(s,1H),7.07-6.99(m,2H),6.85(dd,J=16.6,11.0Hz,1H),6.45(d,J=14.8 Hz,1H),4.72(s,1H),4.07(s,1H),3.85-3.82(m,4H),2.91(t,J=5.1Hz,4H),2.71(d,J=2.8 Hz,1H),2.51(s,4H),1.88(s,2H),1.58(s,4H),1.49-1.40(m,4H). 13 C NMR(101 MHz,CDCl3)δ165.54,151.30,143.53,137.44,136.05,130.72,129.54,128.98,126.93,126.12,123.38,122.18,119.99,118.88,67.33,62.69,52.94,50.18,41.03,29.76,26.15,24.64.HR-ESIMS m / z 410.2802[M+H] + ,calcd forC 22 H 31 N2O2 + 410.2802.

[0111] Pb-9: 1H NMR(400 MHz,CDCl3)δ7.36(dt,J=14.6,5.1 Hz,1H),6.95(d,J=10.6Hz,2H),

[0112] 6.79(d,J=8.3 Hz,2H),6.74(d,J=5.1 Hz,2H),6.40(d,J=14.6 Hz,1H),3.86(s,3H),3.85-3.83(m,3H),3.81(s,3H),2.64(s,1H),2.48-2.44(m,4H),1.84(d,J=12.5Hz,2H),1.57-1.52(m,4H),1.42(d,J=13.3 Hz,4H). 13 C NMR(101 MHz,CDCl3)δ165.48,149.77,149.11,142.99,138.73,129.50,125.08,120.80,119.43,111.18,109.02,62.60,55.94,50.11,40.91,29.68,26.14,24.60.HR-ESIMS m / z 385.2482[M+H] + ,calcd forC 23 H 33 N2O3 + 385.2486.

[0113] Pb-10: 1 H NMR(400 MHz,CDCl3)δ7.34(ddd,J=14.6,8.4,1.8 Hz,1H),6.92(d,J=1.8 Hz,

[0114] 1H),6.83(dd,J=8.1,1.8 Hz,1H),6.70(t,J=9.6 Hz,2H),6.36(d,J=14.6 Hz,1H),5.91(s,2H),4.74(d,J=12.6 Hz,1H),4.08(d,J=10.6 Hz,1H),3.12-2.96(m,1H),2.92-2.84(m,1H),2.71(s,2H),2.62-2.52(m,1H),2.09(s,1H),1.96(s,1H),1.75(t,J=4.9Hz,4H),1.49(d,J=17.5Hz,4H),1.32-1.15(m,2H). 13C NMR (101MHz, CDCl3) δ165.27,148.06,147.99,143.18,138.74,130.55,124.82,122.54,118.93,108. 30,105.44,101.15,62.83,49.64,44.76,41.19,29.50,27.79,26.14,24.36,23.38,22.50.HR-ESIMS m / z369.2179[M+H] + ,calcd for C 22 H 29 N2O3 + 369.2173.

[0115] Example 7: Preparation of Pc-2

[0116]

[0117] Benzaldehyde (3.16 mmol) was added to a 50 mL round-bottom flask, and 2 mL of DMSO was added and stirred to dissolve. 1 mL of saturated NaOH was added dropwise and stirred for 30 min. Intermediate Pc-1 (1.58 mmol) was dissolved in 2 mL of DMSO and added dropwise to the reaction system. The mixture was allowed to react overnight. DMSO was removed by repeated extraction with water and dichloromethane. Anhydrous sodium sulfate was added to the organic layer, which was then dried by spin drying. Pc-2 was isolated by column chromatography to obtain a yellow solid in a 51% yield. 1 H NMR(400MHz, CDCl3)δ7.42(t,J=7.5Hz,2H),7.36-7.29(m,4H),6.86(s,1H),6.44(d,J=14.6Hz,1H) ,4.60-4.42(m,1H),3.69(d,J=16.8Hz,2H),3.62(d,J=11.4Hz,2H),2.60(s,3H),2.43-2.38(m,4H). 13 C NMR (101MHz, CDCl3) δ165.46,142.92,139.02,128.73,128.31,127.83,126.96,120.13,54.61,45.94,40.92.HR-ESIMS m / z 257.1651[M+H] + ,calcd for C 16 H 21 N2O + 257.1648.

[0118] Example 8: Preparation of Pc-3 to Pc-10

[0119]

[0120] Benzaldehyde was replaced by 3-methylbenzaldehyde, or 4-methylbenzaldehyde, or 4-methoxybenzaldehyde, or 4-tert-butylbenzaldehyde, or 4-phenylbenzaldehyde, or 2-morpholinobenzaldehyde, or 3,4-dimethoxybenzaldehyde, and Adol condensation was carried out with the intermediate Pc-1. The preparation method was the same as that in Example 7 to obtain Pc-3, Pc-4, Pc-5, Pc-6, Pc-7, Pc-8, and Pc-9, respectively.

[0121] Pc-3: 1 H NMR (400MHz, CDCl3) δ7.43 (dd, J=14.8, 10.0Hz, 1H), 7.26-7.16 (m, 4H),

[0122] 7.10(t,J=7.0Hz,1H),6.84(d,J=6.6Hz,1H),6.43(d,J=14.8Hz,1H),3.72(s,2H),3.60(s,2H),2.44-2.39(m,4H),2.34(s,3H),2.30(s,3H). 13 C NMR (101MHz, CDCl3) δ165.61,143.17,139.34,138.44,136.37,132.43,129.63, 128.75,127.86,126.71,124.21,120.02,55.33,46.09,41.06,21.47.HR-ESIMS m / z 271.1815[M+H] + ,calcd for C 17 H 23 N2O + 271.1805.

[0123] Pc-4: 1 H NMR (400MHz, CDCl3) δ7.34(d,J=8.1Hz,2H),7.29(d,J=8.0Hz,1H),7.16(t,

[0124] J=7.8Hz,3H),6.84(d,J=3.3Hz,1H),6.41(d,J=14.6Hz,1H),3.72-3.59(m,3H),2.41(t,J=5.2Hz,4H),2.35(s,3H),2.30(s,3H). 13C NMR(101MHz,CDCl3)δ165.72,143.36,139.27,133.74,129.62,129.22,127.08,125.98,119.63,46.08,41.08,29.82,21.47.HR-ESIMS m / z271.1791[M+H] + ,calcd for C 17 H 23 N2O + 271.1805.

[0125] Pc-5: 1 H NMR(400MHz,CDCl3)δ7.45-7.38(m,1H),7.36(d,J=8.8Hz,2H),7.34-7.28

[0126] (m,1H),6.85(d,J=8.8Hz,2H),6.77(d,J=3.4Hz,1H),6.36(d,J=14.6Hz,1H),3.79(s,3H),3.63(d,J=43.1Hz,4H),2.39(t,J=5.1Hz,4H),2.28(s,3H). 13 C NMR(101MHz,CDCl3)δ165.74,160.19,143.50,138.88,129.21,128.46,124.77,118.89,114.29,55.39,54.73,46.03,40.96.HR-ESIMS m / z 287.1734[M+H] + ,calcd for C 17 H 23 N2O2 + 287.1754.

[0127] Pc-6: 1 H NMR(400MHz,CDCl3)δ7.39-7.34(m,1H),7.28(s,4H),6.77(d,J=7.8Hz,2H),

[0128] 6.33(d,J=13.4Hz,1H),3.63(s,2H),3.52(s,2H),2.32(t,J=5.2Hz,4H),2.21(s,3H),1.23(s,9H). 13C NMR(101MHz,CDCl3)δ164.65,151.13,142.33,138.08,132.67,125.87,125.12,124.79,118.64,54.32,45.05,40.03,33.81,30.29.HR-ESIMS m / z313.2269[M+H] + ,calcd for C 20 H 29 N2O + 313.2274.

[0129] Pc-7: 1 H NMR(400MHz,CDCl3)δ7.60-7.55(m,4H),7.50(d,J=8.5Hz,2H),7.47-7.39

[0130] (m,3H),7.33(t,J=6.7Hz,1H),6.97-6.84(m,2H),6.44(d,J=14.6Hz,1H),3.75-3.67(m,2H),3.64-3.57(m,2H),2.41(t,J=5.1Hz,4H),2.29(s,3H). 13 C NMR(101MHz,CDCl3)δ165.52,143.02,141.40,140.39,138.62,135.39,128.89,127.61,127.51,127.44,126.96,126.86,120.18,46.02,45.67,40.96.HR-ESIMS m / z 333.1957[M+H] + ,calcd forC 22 H 25 N2O + 333.1961.

[0131] Pc-8: 1 H NMR(400MHz,CDCl3)δ7.60-7.48(m,2H),7.33(dd,J=13.8,8.4Hz,2H),7.13

[0132] -7.05(m,2H),6.91(dd,J=15.8,11.1Hz,1H),6.49(d,J=14.6Hz,1H),3.91-3.88(m,4H),3.77(s,2H),3.66(s,2H),3.00-2.94(m,4H),2.49-2.43(m,4H),2.35(s,3H).13 CNMR(101MHz,CDCl3)δ165.62,151.26,143.70,136.23,130.61,129.54,127.01 ,126.78,123.33,119.59,118.84,67.25,60.40,52.89,45.98,40.93.HR-ESIMS m / z 342.2170[M+H] + ,calcd for C 20 H 28 N3O2 + 342.2176.

[0133] Pc-9: 1 H NMR (400MHz, CDCl3) δ7.41 (ddd, J=14.6, 7.7, 2.5Hz, 1H), 7.01-6.93 (m, 2H),

[0134] 6.81(d,J=8.4Hz,1H),6.79-6.74(m,2H),6.39(d,J=14.5Hz,1H),3.88(s,3H),3.87(s ,3H),3.69(d,J=7.0Hz,2H),3.58(d,J=4.6Hz,2H),2.39(t,J=5.2Hz,4H),2.28(s,3H). 13 C NMR (101MHz, CDCl3) δ165.67,149.84,149.14,143.31,139.03,129.48,125.02,120.88,119.10,111.18,109.04,56.00,55.89,46.04,40.99.HR-ESIMS m / z 317.1849[M+H] + ,calcd for C 18 H 25 N2O3 + 317.1860.

[0135] Example 9: Preparation of Pd-2

[0136]

[0137] Benzaldehyde (3.16 mmol) was added to a 50 mL round-bottom flask, and 2 mL of DMSO was added and stirred to dissolve. 1 mL of saturated NaOH was added dropwise and stirred for 30 min. Intermediate Pd-1 (1.58 mmol) was dissolved in 2 mL of DMSO and added dropwise to the reaction system. The mixture was allowed to react overnight. DMSO was removed by repeated extraction with water and dichloromethane. Anhydrous sodium sulfate was added to the organic layer, which was then dried and separated by column chromatography to obtain Pd-2 as a yellow solid in a 51% yield. Pd-2: 1 H NMR (400MHz, CDCl3) δ6.90 (ddq, J=28.8, 13.8, 6.9Hz, 2H), 6.39-6.18 (m, 4H), 5.82 (d, J=15.4H z,1H),3.70(d,J=40.9Hz,4H),2.64-2.59(m,4H),1.86(d,J=6.8Hz,4H),1.13(t,J=7.3Hz,3H). 13 C NMR (101MHz, CDCl3) δ170.58,165.77,144.87,144.81,142.47,123.85,121.70,121.13,51.94,18.37,18.04,10.80.HR-ESIMS m / z 271.1800[M+H] + ,calcd for C 17 H 23 N2O + 271.1805.

[0138] Example 10: Preparation of Pd-3 to Pd-9

[0139]

[0140] Benzaldehyde was replaced by 3-methylbenzaldehyde, or 4-methylbenzaldehyde, or 4-methoxybenzaldehyde, or 4-tert-butylbenzaldehyde, or 4-phenylbenzaldehyde, or 2-morpholinobenzaldehyde, or 3,4-dimethoxybenzaldehyde, and Adol condensation was carried out with the intermediate Pd-1. The preparation method was the same as that in Example 7 to obtain Pd-3, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8, and Pd-9, respectively.

[0141] Pd-3: 1 H NMR (400MHz, CDCl3) δ7.43 (dd, J=14.7, 10.1Hz, 1H), 7.27-7.19 (m, 3H),

[0142] 7.09(d,J=6.3Hz,1H),6.93-6.76(m,2H),6.43(d,J=14.6Hz,1H),3.67(d,J=48.7Hz,4H),2.48-2.40(m,6H),2.34(s,3H),1.09(t,J=7.3Hz,3H). 13 C NMR(101MHz,CDCl3)δ165.55,143.13,139.31,138.41,136.35,129.61,128.72,127.84,126.70,124.20,120.01,52.28,21.45,11.93.

[0143] Pd-4: 1 H NMR(400MHz,CDCl3)δ7.45-7.34(m,3H),6.85(d,J=8.6Hz,2H),6.76(d,J=

[0144] 8.8Hz,2H),6.38(d,J=14.6Hz,1H),3.79(s,3H),3.64(d,J=44.2Hz,4H),2.42(t,J=7.5Hz,6H),1.08(t,J=7.2Hz,3H). 13 C NMR(101MHz,CDCl3)δ165.55,160.09,143.31,138.71,129.12,128.37,124.72,118.89,114.19,64.48,55.28,52.16,45.58,11.02.HR-ESIMS m / z285.1976[M+H] + ,calcd for C 18 H 25 N2O + 285.1961.

[0145] Pd-5: 1 H NMR(400MHz,CDCl3)δ7.45-7.34(m,3H),6.85(d,J=8.6Hz,2H),6.76(d,J=

[0146] 8.8Hz,2H),6.38(d,J=14.6Hz,1H),3.79(s,3H),3.64(d,J=44.2Hz,4H),2.42(t,J=7.5Hz,6H),1.08(t,J=7.2Hz,3H). 13C NMR(101MHz,CDCl3)δ165.55,160.09,143.31,138.71,129.12,128.37,124.72,118.89,114.19,64.48,55.28,52.16,45.58,11.02.HR-ESIMS m / z301.1865[M+H] + ,calcd for C 18 H 25 N2O + 301.1911.

[0147] Pd-6: 1 H NMR(400MHz,CDCl3)δ7.38(d,J=3.6Hz,3H),7.37-7.34(m,1H),6.89-6.86

[0148] (m,2H),6.43(d,J=14.6Hz,1H),4.92(s,1H),3.69(d,J=43.7Hz,4H),2.51-2.45(m,6H),1.32(s,9H),1.11(t,J=7.3Hz,3H). 13 C NMR(101MHz,CDCl3)δ165.52,152.03,143.32,139.06,133.55,126.79,126.01,125.69,119.46,52.84,52.06,34.70,31.36,31.20,11.57.HR-ESIMS m / z 327.2436[M+H] + ,calcd for C 21 H 31 N2O + 327.2431.

[0149] Pd-7: 1 H NMR(400MHz,CDCl3)δ7.57(td,J=7.1,1.8Hz,4H),7.51-7.36(m,5H),7.35-

[0150] 7.30(m,1H),6.92-6.88(m,1H),6.42(d,J=14.6Hz,1H),3.69(d,J=47.8Hz,4H),2.55-2.43(m,6H),2.00(s,1H),1.09(t,J=7.3Hz,3H). 13C NMR(101MHz,CDCl3)δ174.87,165.45,143.15,141.34,140.29,138.70,135.30,128.84,127.56,127.48,127.36,126.88,126.76,119.92,51.97,41.53,21.76,11.32.HR-ESIMS m / z 347.2130[M+H] + ,calcd forC 23 H 27 N2O + 347.2118.

[0151] Pd-8: 1 H NMR(400MHz,CDCl3)δ7.30(d,J=7.3Hz,1H),7.08-6.98(m,2H),6.87-6.77

[0152] (m,2H),6.65(dd,J=15.6,11.1Hz,1H),6.22(d,J=14.6Hz,1H),3.67-3.59(m,4H),3.50(d,J=43.4Hz,4H),2.72-2.65(m,4H),2.36-2.23(m,6H),1.79(s,1H),0.90(t,J=7.3Hz,3H). 13 C NMR(101MHz,CDCl3)δ165.49,151.16,143.78,136.24,130.43,129.47,126.91,126.62,123.23,119.29,118.73,67.11,52.78,51.89,45.07,41.43,11.22.HR-ESIMS m / z 356.2290[M+H] + ,calcd for C 21 H 30 N3O2 + 356.2333.

[0153] Pd-9: 1 H NMR(400MHz,CDCl3)δ7.28-7.18(m,2H),7.15-6.99(m,4H),6.64(d,J=14.5

[0154] Hz,3H),4.14-4.10(m,6H),3.95(d,J=35.5Hz,4H),2.81-2.75(m,4H),2.25(s,2H),1.36(t,J=7.3Hz,3H). 13 C NMR (101MHz, CDCl3) δ174.91,165.53,149.71,148.95,143.49,139.13,134.08,129.22,124.73,120.77,119.07,118.5 1,111.04,110.85,110.29,108.94,64.49,55.78,55.69,55.64,51.75,44.75,41.12,40.46,21.65,10.95.HR-ESIMSm / z 331.2002[M+H] + ,calcd for C 19 H 27 N2O3 + 331.2016.

[0155] Example 11: Verification of the antitumor activity of piperine and piperine derivatives

[0156] Human chronic myeloid leukemia cell line K562, human histiocytic lymphoma cell line U937, human acute promyelocytic leukemia cell line NB4, human promyelocytic leukemia cell line HL60, human cervical cancer cell line HeLa, human pancreatic cancer cell line PANC-1, and human ovarian cancer cell line SKOV3 were cultured in a 37°C, 5% CO2 incubator. The culture medium for K562, U937, NB4, and HeLa cells consisted of RPMI-1640 medium supplemented with 10% FBS and 1% double-antibody. The culture medium for HL60 cells consisted of IMDM medium supplemented with 20% FBS and 1% double-antibody. The culture medium for PANC-1 and SKOV3 cells consisted of high-glucose DMEM supplemented with 10% FBS and 1% double-antibody.

[0157] 1) MTT assay steps: PANC-1 and SKOV3 cells in the logarithmic growth phase were digested and centrifuged, the supernatant was discarded, the cells were resuspended and counted, and the cells were counted according to 1×10 5 / mL of cells were seeded in a 96-well plate and cultured overnight until the cells adhered. The next day, the old culture medium was removed and 100 μL of culture medium containing the drug solution was added. After culturing in an incubator for a specific time, such as 72 hours, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated in an incubator for 4 hours. The supernatant was removed and 120 μL of DMSO was added to each well. The cells were shaken for 5 minutes and the absorbance was measured at a wavelength of 490 nm using a microplate reader. The inhibition rate was calculated as follows [= (OD 空白 -OD 实验) / OD 空白 ×100%】and IC 50 (Calculated using GraphPad software).

[0158] MTT results showed that piperine and the above-mentioned piperine derivatives can inhibit the proliferation of various tumor cells, and some piperine derivatives have better anti-ovarian cancer activity than piperine. Among them, the most active compound Pb-7 acts on ovarian cancer cells SKOV3 for 72 hours with an IC of 50 The IC values ​​of Pb-7 on pancreatic cancer cells PANC-1 for 72 h were 1.91-2.02 μM, which is comparable to that of the positive drug camptothecin (Table 1). 50 7.29 μM ( Figure 6 ).

[0159] 2) CCK8 experimental steps: K562, U937, HL60, and NB4 cells in the logarithmic growth phase were centrifuged, the supernatant was discarded, the cells were resuspended and counted, and the cells were counted according to 1×10 5 The cells were seeded at a density of 100 μL / mL in a 96-well plate, 50 μL of cell suspension was added to each well, and 50 μL of medium containing drug solution was added. After culturing in an incubator for a specific time of 72 hours, 10 μL of CCK8 solution was added to each well, and the cells were incubated in an incubator for 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The inhibition rate was calculated as [(OD 空白 -OD 实验 ) / (OD 空白 -OD 培养基 )×100%】and IC 50 (Calculated using GraphPad software).

[0160] The CCK8 results showed that piperine and its derivatives exhibited excellent anti-tumor activity, among which the most active compound Pb-7 had an IC of 1.57 for leukemia cells. 50 The range was 2.55-6.09 μM, which was comparable to the positive control camptothecin (Table 1).

[0161] Table 1 IC values ​​of piperine derivatives for inhibiting cell proliferation in tumor cells for 72 hours 50 (μM) Results

[0162]

[0163]

[0164] Note: CPT is camptothecin, Pip is piperine, a Not determined

[0165] Example 12: Novel piperine derivatives induce NB4 cell differentiation

[0166] The cell culture method is as described in Example 11. 5 Cells / well were seeded in a 6-well plate, and compounds (1, 2.5, 5 μM) were added for 72 hours. The cells were centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, the cells were collected, 200 μL of LCD11b-PE antibody dilution was added, mixed, and incubated at 37°C in the dark for 30 minutes. After incubation, the cells were collected by centrifugation, washed three times with PBS, the supernatant was discarded, the cells were collected, and finally 200 μL of PBS was added to resuspend them. Appropriate flow cytometer detection parameters were set and the flow cytometer was tested. The results are as follows Figure 2 and Figure 3 The results showed that compound Pb-7 increased CD11b expression in NB4 cells and significantly induced granulocytic differentiation of NB4 cells. Compound Pb-7 also induced apoptosis in NB4 cells. In summary, Pb-7 simultaneously induced differentiation and apoptosis in NB4 cells, thereby exerting an anti-leukemia effect.

[0167] Example 13: Novel piperine derivatives induce apoptosis in SKOV3 cells

[0168] The SKOV3 cell culture method is as described in Example 11. Cells in the logarithmic phase were seeded in 6-well plates, 1×10 5 Cells / well were added and treated with the compound for 48 hours. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were collected, and 100 μL Annexin VBinding Buffer was added to resuspend the cells. Then, 2.5 μL Annexin V-FITC Reagent and 2.5 μL PIReagent were added. After gentle vortexing, the cells were incubated at room temperature in the dark for 20 minutes, and then 400 μL Annexin V Binding Buffer was added to mix the samples. The appropriate flow cytometer detection parameters were set and the cells were tested. The results were as follows. Figure 4 As shown in (A), the results showed that Pb-7 (1, 2.5, 5 μM) could significantly induce apoptosis in SKOV3 cells.

[0169] Example 14: Effects of Novel Piperine Derivatives on DNA Content and Cell Cycle in SKOV3 Cells

[0170] The SKOV3 cell culture method is as described in Example 11. Cells in the logarithmic phase were seeded in 6-well plates, 1×10 5Cells / well were added and treated with the compound for 48 hours. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were collected, 500 μL of 70% pre-cooled ethanol was added and fixed at 4°C overnight, the cells were collected by centrifugation, 100 μL of RNase A solution was added, the cells were resuspended, and the cells were incubated at 37°C in a water bath for 30 minutes. 400 μL of PI staining solution was added and mixed, and the cells were incubated at 4°C in the dark for 30 minutes. Appropriate flow cytometer detection parameters were set and the cells were tested on the machine. The results are shown in Figure 2. Figure 4 As shown in (B) and (D), the results showed that after treatment with Pb-7, SKOV3 cells showed a distinct Sub-G1 peak, indicating apoptosis. Compound Pb-7 also caused G2 arrest in SKOV3 cells, indicating that compound Pb-7 inhibited the tumor cell cycle and, therefore, tumor cell proliferation.

[0171] Example 15: Effects of novel piperine derivatives on mitochondrial membrane potential in SKOV3 cells

[0172] The SKOV3 cell culture method is as described in Example 11. Cells in the logarithmic phase were seeded in 6-well plates, 1×10 5 Cells / well were added and treated with the compound for 48 hours. The cells were centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, the cells were collected, 0.5 mL of JC-1 staining working solution was added, and the mixture was mixed by inverting several times. After incubation at 37 ° C in a cell culture incubator for 20 minutes, the cells were centrifuged (600 × g, 4 ° C, 4 minutes) to collect the cells. The cells were washed twice with JC-1 staining buffer and finally resuspended with 200 μL of JC-1 staining buffer. The appropriate flow cytometer detection parameters were set and the cells were tested on the machine. The results are shown in the figure below. Figure 4 As shown in (C), the results showed that the mitochondrial membrane potential of SKOV3 cells was significantly reversed after Pb-7 treatment, indicating that Pb-7 promoted the apoptosis of tumor cells mediated by the mitochondrial pathway.

[0173] Example 16: Effects of Novel Piperine Derivatives on Protein Expression in SKOV3 Cells

[0174] The SKOV3 cell culture method is as described in Example 11. Cells in the logarithmic phase were seeded in 6-well plates, 1×10 5 Cells / well were added and treated with the compound for 48 hours. Cells were collected, protein samples were extracted, protein concentration was measured by BCA method, protein samples were denatured, electrophoresed, transferred to the membrane, blocked with 5% skim milk powder, incubated with primary and secondary antibodies, developed, and chemiluminescence was detected by ultrasensitive chemiluminescence instrument to obtain bands. The bands were analyzed and processed using Carestream software. Figure 5The results showed that Pb-7 inhibited the expression of tumor-promoting proteins BcL-2, XIAP, JAK1, JAK2, STAT1, and Cl-PARP in tumor cells in a concentration-dependent manner. In summary, Pb-7 exerts its anti-tumor effect by inhibiting the JAK / STAT pathway.

[0175] Example 17: Evaluation of the anti-tumor activity of novel piperine derivatives in vivo

[0176] The SKOV3 cell culture method was as described in Example 11. 8.0×10 6 SKOV3 cells in the logarithmic growth phase were cultured and the tumor volume was grown to 100 mm. 3 The mice were then divided into three groups: the drug administration group (low-dose and high-dose groups) and the control group (vehicle control group), with 6 mice in each group. All mice were housed in a laminar flow rack under SPF conditions. After grouping, the drug was administered daily, and the tumor volume (TV = 0.5 × a × b) was measured daily. 2 , where a and b represent the long diameter and short diameter respectively), the mice were weighed every day, and the mental state, lifespan and death of the mice were observed. After the 2-week experiment, the mice were anesthetized, and the tumor tissues and internal organs were taken. The tumor tissues were sliced ​​for Ki67 staining (completed by Wuhan Sevier Biotechnology Co., Ltd., and the company provided a paraffin section immunohistochemistry experimental report), and the internal organs were sliced ​​for HE staining (completed by Wuhan Sevier Biotechnology Co., Ltd., and the company provided a paraffin section and HE staining experimental report) to observe whether pathological changes occurred. Finally, the curves of tumor volume, tumor weight, and mouse weight changes were drawn. The results showed that continuous administration of 15 mg / kg and 30 mg / kg Pb-7 for 14 days significantly inhibited the growth of nude mouse tumors. Compared with the solvent control group, the tumor volume and body weight of the drug-treated group were significantly reduced, showing an in vivo anti-tumor effect, and the anti-tumor activity of the high-concentration group was better than that of the low-concentration group ( Figure 7 ) visceral HE staining showed that there were no significant changes in the heart, liver, spleen, lungs, and kidneys of the mice in the drug-treated group ( Figure 8 The Ki67 staining of tumor tissue showed that the proliferation activity of tumor tissue in the drug-treated group was significantly reduced ( Figure 9 In conclusion, Pb-7 exhibited good anti-tumor activity in vivo without obvious toxic side effects.

[0177] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A group of piperine derivatives, characterized in that: The piperine derivatives include compounds Pa-3, Pa-4, Pa-6, Pa-7, Pa-8, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pc-2, Pc-3, Pc-4, Pc-6, Pc-7, Pc-8, Pd-2, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8 and Pd-9, and the chemical structural formulas of the piperine derivatives are shown below: 。 2. The pharmaceutically acceptable salt of the piperine derivative according to claim 1, characterized in that: The anion of the pharmaceutically acceptable salt is an inorganic anion or an organic anion; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, toluenesulfonate, citrate, lactate or gluconate.

3. The method for preparing the piperine derivative according to claim 1, wherein: Any of the following methods (1) and (2): (1) Using anhydrous dichloromethane as solvent, crotonyl chloride and different substituted amines were reacted in the presence of triethylamine at room temperature; the reactants were repeatedly extracted with water and dichloromethane, the organic layer was dried, spin-dried, and purified to obtain the target compound; (2) Using anhydrous dichloromethane as solvent, crotonyl chloride and different substituted amines are reacted in the presence of triethylamine at room temperature to obtain an intermediate, which is then reacted with different substituted aldehydes in DMSO solvent under alkaline conditions overnight; the reactants are repeatedly extracted with water and dichloromethane, the organic layer is dried, spin-dried, and purified to obtain the target compound; wherein, The substituted amine is any one of morpholine, 4-piperidinylpiperidine, 4-methylpiperazine, and 4-ethylpiperazine; The substituted aldehyde is any one of benzaldehyde, 3-methylbenzaldehyde, 4-methylbenzaldehyde, 4-methoxybenzaldehyde, 4-tert-butylbenzaldehyde, 4-phenylbenzaldehyde, 2-morpholinobenzaldehyde, or 3, 4-dimethoxybenzaldehyde.

4. The method for preparing a piperine derivative according to claim 3, wherein: The molar ratio of crotonyl chloride to substituted amine is 2:1; The reaction time at room temperature is 7±1 hours; The molar ratio of the intermediate to the substituted aldehyde is 1:2; The alkaline condition is achieved by adding NaOH, and the added amount is calculated as DMSO solvent: NaOH = 4 mL: 1 mL.

5. The use of piperine derivatives in the preparation of anti-tumor drugs, characterized in that: The piperine derivatives include pharmaceutically acceptable salts thereof; the applications include any one or more of the following: I. Use of piperine derivatives in the preparation of drugs for the treatment of ovarian cancer; II. Use of piperine derivatives in the preparation of drugs for the treatment of leukemia; III. Use of piperine derivatives in the preparation of medicaments for treating pancreatic cancer; The piperine derivatives include any one of compounds Pa-1, Pa-2, Pa-3, Pa-4, Pa-5, Pa-6, Pa-7, Pa-8, Pb-1, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pc-1, Pc-2, Pc-3, Pc-4, Pc-5, Pc-6, Pc-7, Pc-8, Pd-1, Pd-2, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8, and Pd-9, each having the following structure: 。 6. The use according to claim 5, characterized in that: The anion of the pharmaceutically acceptable salt is an inorganic anion or an organic anion; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, toluenesulfonate, citrate, lactate or gluconate.

7. The use according to claim 6, characterized in that: The drug for treating ovarian cancer is a drug for treating paclitaxel-resistant ovarian cancer; The drug for treating leukemia has the dual functions of promoting differentiation and apoptosis of leukemia cells; The anti-tumor drug inhibits tumor cell proliferation by antagonizing the expression of BcL-2, XIAP, JAK1, JAK2, STAT1 and / or Cl-PARP proteins.

8. The use according to claim 7, characterized in that: Application I is: use of any one of Pb-7, Pc-6, Pc-7, Pc-8, Pd-6, and Pd-7 in the preparation of a drug for treating ovarian cancer; Application II is: use of any one of Pa-1, Pa-2, Pa-3, Pa-4, Pa-5, Pa-6, Pa-7, Pa-8, Pb-1, Pb-2, Pb-3, Pb-4, Pb-5, Pb-6, Pb-7, Pb-8, Pb-9, Pc-1, Pc-2, Pc-3, Pc-4, Pc-5, Pc-6, Pc-7, Pc-8, Pd-1, Pd-2, Pd-4, Pd-5, Pd-6, Pd-7, Pd-8, and Pd-9 in the preparation of a medicament for treating leukemia; Application III is: application of Pb-7 in the preparation of medicines for treating pancreatic cancer.

9. The use according to any one of claims 5 to 8, characterized in that: The dosage form of the anti-tumor drug is tablets, pills, granules, lozenges, ointments, solutions, suppositories, injections, inhalants or sprays; The anti-tumor drug is administered orally, buccally, rectally, intraperitoneally, topically, subcutaneously or intratracheally.

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