Application of pyrroloquinoline-polyamine analogue combined with adriamycin in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of pyrroloquinoline-polyamine analogue and adriamycin in preparation of antitumor drugs. The pyrroloquinoline-polyamine analogue and adriamycin are combined for use, and are used for developing products such as medicines, preparations, kits and the like for treating colorectal cancer. Experimental results prove that when the pyrroloquinoline-polyamine analogue and adriamycin are independently used, a certain anti-tumor effect is achieved. After combined medication, the dosages of the two can be remarkably reduced, and the anti-tumor effect is remarkably improved. Animal experiments show that the in-vivo anti-tumor effect of the pyrroloquinoline-polyamine analogue and the adriamycin can be improved by combined medication compared with single medication. Combined medication of the pyrroloquinoline-polyamine analogue and adriamycin may become a novel method for treating patients with colorectal cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to an application of a pyrroloquinoline-polyamine analogue combined with doxorubicin in the preparation of an anti-tumor drug. Background Art

[0002] Cancer is one of the leading causes of death in humans. Colorectal cancer (CRC) is one of the most common malignant tumors in the world, and its mortality rate ranks second only to lung cancer, which has seriously endangered human survival and health. Although radiotherapy, chemotherapy, targeted therapy and immunotherapy have achieved certain results in colorectal cancer, the incidence and mortality of colorectal cancer continue to rise.

[0003] Doxorubicin (DOX) is a class of synthetic anthraquinone anti-tumor drugs that are effective against a variety of tumors, and has good therapeutic effects in cancers such as acute leukemia, breast cancer, sarcoma, lung cancer, bladder cancer, and malignant lymphoma. However, doxorubicin (DOX) has certain toxic side effects, such as affecting bone marrow hematopoietic function, manifested as thrombocytopenia and leukocytopenia; cardiotoxicity, heart failure may occur in severe cases, and nausea, vomiting, stomatitis, hair loss, high fever, phlebitis, and skin pigmentation may be seen; a few patients have fever, hemorrhagic erythema, and liver damage. After entering the cell, doxorubicin (DOX) binds to the chromosome, and its planar ring can be inserted into the DNA base pair, interfering with the synthesis of DNA, RNA, and protein, so that doxorubicin (DOX) can induce apoptosis of tumor cells. Existing reports show that doxorubicin (DOX) can have a synergistic effect with other anti-tumor drugs.

[0004] Quinoline, also known as benzopyridine, is a class of alkaline compounds containing nitrogen. Its unique and novel structure and diverse biological activities make it widely used in the field of medical research. Nearly 100 quinoline compounds currently on the market and in clinical trials have anti-tumor activity. Due to its important position in drug development, quinoline compounds are favored by researchers. Quinoline compounds can show excellent results through different mechanisms of action, such as inhibiting tumor growth by inducing cell cycle arrest, inducing cell apoptosis, inhibiting angiogenesis, destroying cell migration or regulating growth inhibitors.

[0005] Polyamines are a class of highly active small molecule aliphatic compounds containing two or more amino groups, which are widely distributed in prokaryotes and eukaryotes. Putrescine, spermidine and spermine are the most common and widely studied polyamines in organisms. Polyamines are involved in various cellular processes, including chromatin organization, gene regulation, cell proliferation and differentiation, cell death and immune system function. The dynamic balance of polyamines is a strictly regulated process, which is jointly regulated by the biosynthesis, catabolism and transport of polyamines. Cancer cells require a continuously elevated intracellular polyamine pool to maintain proliferation, and reducing polyamine levels can enhance anti-tumor immune responses. More importantly, polyamine modification can enhance the anti-tumor effect of drugs.

[0006] Although quinoline compounds have good anti-tumor effects, there is currently no polyamine-modified quinoline compound used in clinical practice, and there is no report on the combined use of polyamine-modified quinoline drugs and doxorubicin (DOX) to treat tumors. Clinically, many diseases are prone to drug resistance to quinoline drugs, causing the disease to worsen. Therefore, it is necessary to find a more effective and safe solution. Summary of the invention

[0007] In view of the problems existing in the prior art, the inventors have synthesized a novel pyrroloquinoline-polyamine analogue after long-term practical exploration, and proposed a pyrroloquinoline-polyamine analogue combined with doxorubicin (DOX) in the preparation of a product for treating colorectal cancer. The present invention uses a pyrroloquinoline-polyamine analogue in combination with doxorubicin, and is used to develop drugs, preparations, kits and other products for treating colorectal cancer. Experimental results show that after the pyrroloquinoline-polyamine analogue is used in combination with doxorubicin (DOX), it shows a strong activity in inhibiting colorectal cancer and a good synergistic tumor inhibition effect, and thus has a good practical application value.

[0008] Another object of the present invention is to provide an anti-tumor pharmaceutical composition containing pyrroloquinoline-polyamine analogs and doxorubicin.

[0009] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0010] Disclosed is an application of a pyrroloquinoline-polyamine analogue combined with doxorubicin in the preparation of an anti-tumor drug.

[0011] Specifically, the tumor includes but is not limited to human colorectal cancer, human lung adenocarcinoma, human liver cancer, human esophageal cancer, human pancreatic cancer, human prostate cancer, human breast cancer, human gastric cancer, human endometrial cancer, human bladder cancer, human skin cancer or human blood cancer.

[0012] Specifically, when the pyrroloquinoline-polyamine analogue and doxorubicin are used in combination, the pyrroloquinoline-polyamine analogue and doxorubicin can be used in a mixed manner or separately.

[0013] Specifically, when the pyrroloquinoline-polyamine analogue and doxorubicin are used in combination, the pyrroloquinoline-polyamine analogue and doxorubicin are added at once or added continuously for multiple times.

[0014] Specifically, when the pyrroloquinoline-polyamine analogue and doxorubicin are added at one time, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is (1-10):1, and the treatment time is 6-72 hours (preferably 6-24 hours); when the pyrroloquinoline-polyamine analogue and doxorubicin are added continuously for multiple times, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin each time is (2-200):1, and the addition is continuous for 7-14 times in total.

[0015] Preferably, when the pyrroloquinoline-polyamine analogue and doxorubicin are added at one time, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is 10:1, and the treatment time is 6-24 hours (preferably 24 hours); when the pyrroloquinoline-polyamine analogue and doxorubicin are added continuously for multiple times, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin each time is 10:1, and the addition is continuous for a total of 14 times.

[0016] Furthermore, the present invention also provides the use of pyrroloquinoline-polyamine analogs combined with doxorubicin in the preparation of drugs for inhibiting the proliferation of colon cancer cells or promoting their apoptosis, wherein the colon cancer cells are HT29, HCT116, CT26 or MC38 cells.

[0017] Specifically, when used alone, the pyrroloquinoline-polyamine analogs can inhibit the proliferation and / or number or size of colony formation of colorectal cancer cells at a concentration of 2.5-20 μM and a treatment time of 24-72 hours; or, can induce apoptosis of colorectal cancer cells.

[0018] Preferably, when used alone, the pyrroloquinoline-polyamine analogues can inhibit the proliferation and / or number or size of colony formation of colorectal cancer cells at a concentration of 2.5 or 20 μM and a treatment time of 24 hours; or, can induce apoptosis of colorectal cancer cells.

[0019] Specifically, when used in combination, the pyrroloquinoline-polyamine analogue at a concentration of 2.5-20 μM and a treatment time of 24-72 hours, and doxorubicin at a concentration of 0.1-2 μM and a treatment time of 24-72 hours can inhibit the proliferation and / or number or size of clones formed by colorectal cancer cells; or, can induce apoptosis of colorectal cancer cells.

[0020] Preferably, when used in combination, the pyrroloquinoline-polyamine analog is at a concentration of 2.5 μM, 10 μM, or 20 μM, and the treatment time is 24 h, 48 h, or 72 h; the concentration of doxorubicin is at 0.5 μM, 1 μM, or 2 μM, and the treatment time is 24 h, 48 h, or 72 h, which can inhibit the proliferation and / or the number or size of clones formed by colorectal cancer cells; or, can induce apoptosis of colorectal cancer cells.

[0021] Further preferably, when used in combination, when the concentration of pyrroloquinoline-polyamine analogue is 10 μM and the treatment time is 24 hours, and when the concentration of doxorubicin is 1 μM and the treatment time is 24 hours, they can inhibit the proliferation and / or the number or size of clones formed by colorectal cancer cells; or, they can induce apoptosis of colorectal cancer cells.

[0022] Furthermore, the present invention also provides the use of pyrroloquinoline-polyamine analogs combined with doxorubicin in the preparation of a drug for inhibiting tumor growth in a human or mouse transplanted tumor model of colon cancer.

[0023] Specifically, when the dosage of pyrroloquinoline-polyamine analogue is 2-20 mg / kg / day, the dosage of doxorubicin is 1-5 mg / kg / day, and the treatment time is 2-14 days, the growth of colorectal cancer mouse tumors can be inhibited.

[0024] Preferably, when the dosage of pyrroloquinoline-polyamine analogue is 20 mg / kg / day and the dosage of doxorubicin is 2 mg / kg / day, and the treatment time is 14 days, the growth of colorectal cancer mouse tumors can be inhibited.

[0025] Specifically, the mouse source is male Balb / c mice.

[0026] Furthermore, the preparation method of the pyrroloquinoline-polyamine analogue comprises the following steps:

[0027] (1) Using methyl-nitroaniline as the starting material, a substitution reaction is carried out with chloral hydrate and hydroxylamine hydrochloride to form a ring to obtain an intermediate product I;

[0028] (2) Intermediate product I undergoes a nucleophilic addition reaction with (benzoylmethylene)triphenylphosphine at room temperature (25±5°C) to obtain intermediate product II;

[0029] (3) Intermediate product II undergoes a nucleophilic addition reaction in the presence of a reducing agent and an organic solvent I to obtain intermediate product III;

[0030] (4) Intermediate product III undergoes nucleophilic substitution reaction with dibromobutane in the presence of an organic solvent II to obtain intermediate product IV;

[0031] (5) The intermediate product IV undergoes a nucleophilic substitution reaction with a polyamine compound in the presence of an organic solvent II to obtain a pyrroloquinoline-polyamine analog.

[0032] Specifically, the organic solvent I is ethanol, methanol, dichloromethane or acetonitrile.

[0033] Specifically, the organic solvent II is ethanol, methanol, dichloromethane or acetonitrile.

[0034] Specifically, the reducing agent is any one of stannous chloride, oxalic acid, potassium borohydride and sodium borohydride.

[0035] Specifically, the base is an organic base or an inorganic base. The inorganic base is, for example, any one of potassium carbonate, soda ash (sodium carbonate), sodium bicarbonate and potassium bicarbonate; the organic base is, for example, any one of dimethylamine, aniline, pyridine and triethylamine.

[0036] Specifically, the polyamine compound is any one of 1,3-propylenediamine, putrescine, spermidine and spermine.

[0037] Preferably, the structural formula of the pyrroloquinoline-polyamine analogue is as follows:

[0038]

[0039] Preferably, the synthetic route of the pyrroloquinoline-polyamine analogs is as follows:

[0040]

[0041] The specific preparation steps are:

[0042] 1) Preparation of compound 3: Methyl-nitroaniline (compound 1) is used as a starting material, and a substitution reaction is carried out with chloral hydrate (compound 2) and hydroxylamine hydrochloride to form a ring to obtain compound 3;

[0043] 2) Preparation of compound 4: Compound 3 undergoes a nucleophilic addition reaction with (benzoylmethylene)triphenylphosphine at room temperature (25±5°C) to obtain compound 4;

[0044] 3) Preparation of compound 5: Compound 4 undergoes a nucleophilic addition reaction in the presence of a reducing agent and an organic solvent I to obtain compound 5;

[0045] 4) Preparation of compound 6: Compound 5 is subjected to nucleophilic substitution reaction with 1,4-dibromobutane in the presence of an organic solvent II to obtain compound 6;

[0046] 5) Preparation of the target compound: Compound 6 undergoes nucleophilic substitution reaction with a polyamine compound in the presence of an organic solvent II to obtain the target compound.

[0047] Specifically, the organic solvent I is ethanol, methanol, dichloromethane or acetonitrile.

[0048] Specifically, the organic solvent II is ethanol, methanol, dichloromethane or acetonitrile.

[0049] Specifically, the reducing agent is any one of stannous chloride, oxalic acid, potassium borohydride and sodium borohydride.

[0050] Specifically, the base is an organic base or an inorganic base. The inorganic base is, for example, any one of potassium carbonate, soda ash (sodium carbonate), sodium bicarbonate and potassium bicarbonate; the organic base is, for example, any one of dimethylamine, aniline, pyridine and triethylamine.

[0051] Specifically, the polyamine compound is 1,3-propylenediamine; during the reaction, 1,3-propylenediamine can be protected by Boc.

[0052] Specifically, in step 1), the molar ratio of chloral hydrate to methyl-nitroaniline is (0.8-1.4):1; in step 1), the molar ratio of hydroxylamine hydrochloride to methyl-nitroaniline is (2.8-3.2):1.

[0053] Specifically, in step 5), the molar ratio of the polyamine compound to the compound 6 is (1-2):1, preferably 2:1.

[0054] Furthermore, the present invention also provides an anti-tumor pharmaceutical composition containing doxorubicin, comprising a pyrroloquinoline-polyamine analog or a salt thereof, and doxorubicin or a derivative thereof.

[0055] Specifically, in the anti-tumor pharmaceutical composition containing doxorubicin, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is (1-10):1.

[0056] Preferably, in the anti-tumor pharmaceutical composition containing doxorubicin, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is 10:1.

[0057] Further preferably, in the anti-tumor pharmaceutical composition containing doxorubicin, the pyrroloquinoline-polyamine analogue can promote the accumulation of doxorubicin in cells at a concentration of 10 μM.

[0058] Specifically, the structural formula of the pyrroloquinoline-polyamine analogue is:

[0059]

[0060] Specifically, when the anti-tumor pharmaceutical composition containing doxorubicin is used, the compound dissolved in physiological saline and doxorubicin dissolved in dimethyl sulfoxide (DMSO) are mixed according to the ratio at room temperature (25±5° C.).

[0061] Specifically, the present invention further provides a preparation of the above-mentioned pharmaceutical composition, which is composed of the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0062] Compared with the prior art, the beneficial effects of the present invention are:

[0063] The experimental results show that the pyrroloquinoline-polyamine analogs and doxorubicin described in the present invention have certain antitumor effects when used alone, but the in vitro antitumor effects of low concentrations or low doses of pyrroloquinoline-polyamine analogs and low concentrations or low doses of doxorubicin are not good. After combined use, the dosage of both can be significantly reduced, and the antitumor effect is significantly improved. Animal experiments show that compared with single use, combined use can improve the in vivo antitumor effects of pyrroloquinoline-polyamine analogs and doxorubicin. In summary, the combined use of pyrroloquinoline-polyamine analogs and doxorubicin of the present invention may become a new method for the treatment of colorectal cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Pyrroloquinoline-polyamine analog PQPA 13 C NMR spectrum;

[0065] Figure 2 Pyrroloquinoline-polyamine analog PQPA 1 H NMR spectrum;

[0066] Figure 3 is the HR-MS spectrum of pyrroloquinoline-polyamine analog PQPA;

[0067] Figure 4 The results of cell proliferation inhibition detected by MTT after 24, 48 and 72 hours of single administration of pyrroloquinoline-polyamine analog PQPA on human colorectal cancer cells HCT116, HT29 and mouse colorectal cancer cells CT26, MC38.

[0068] Figure 5 The results are as follows: the clone formation results after the pyrroloquinoline-polyamine analog PQPA was used alone on human colorectal cancer cells HCT116, HT29 and mouse colorectal cancer cells CT26, MC38;

[0069] Figure 6The results are shown in Figure 2. Pyrroloquinoline-polyamine analog PQPA was used alone to act on human colorectal cancer cells HCT116 and mouse colorectal cancer cells CT26. Cell apoptosis was detected by Annexin V-FITC / PI staining 24 hours later.

[0070] Figure 7 The results of the in vivo drug test in mice show that the pyrroloquinoline-polyamine analog PQPA was used alone (20 mg / kg / day) to measure the weight of solid tumors grown in mice 14 days after injection of CT26 cells.

[0071] Figure 8 This is the result of the body weight test of mice in an in vivo drug test in which the pyrroloquinoline-polyamine analog PQPA was used alone (20 mg / kg / day) to act on the mice after the injection of CT26 cells for 14 days;

[0072] Fig. 9 In the in vivo drug test in mice, the pyrroloquinoline-polyamine analog PQPA was used alone (20 mg / kg / day) to affect the organ index of mice grown for 14 days after injection of CT26 cells;

[0073] Fig.10 This is the result of hematoxylin-eosin staining of the heart, liver, spleen, lung, kidney and other tissues of mice injected with CT26 cells for 14 days in a drug test in mice.

[0074] Fig.11 The results of cell proliferation inhibition were detected by MTT after 24 hours when doxorubicin was used alone to act on human colorectal cancer cells HCT116 and HT29.

[0075] Fig.12 The combined drug index after 24 hours of different concentrations of pyrroloquinoline-polyamine analog PQPA and different concentrations of doxorubicin on human colorectal cancer cells HCT116 and HT29;

[0076] Fig.13 The results of the combined use of 10 μM pyrroloquinoline-polyamine analog PQPA and 1 μM doxorubicin on human colorectal cancer cells HCT116 were detected by MTT after 24 hours to inhibit cell proliferation.

[0077] Fig.14 The results of the combined use of 10 μM pyrroloquinoline-polyamine analog PQPA and 1 μM doxorubicin on human colorectal cancer cells HT29 were detected by MTT after 24 hours to inhibit cell proliferation.

[0078] Fig.15 The accumulation of PQPA and doxorubicin in human colorectal cancer cells HCT116 after 24 hours of combined use of 10 μM pyrroloquinoline-polyamine analog PQPA and 1 μM doxorubicin.

[0079] Fig.16 The in vivo growth inhibition of colorectal cancer by the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin. DETAILED DESCRIPTION

[0080] The present invention is described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise defined, all professional and scientific terms used herein are identical in meaning to those skilled in the art. In addition, any method and material similar to or equivalent to the recorded content can all be applied to the inventive method.

[0081] Doxorubicin (DOX) used in the present invention was purchased from MedChemExpress (MCE).

[0082] The invention mainly provides an application method of the combined use of pyrroloquinoline-polyamine analogs and doxorubicin. Before use, a pyrroloquinoline-polyamine analog stock solution is prepared by using physiological saline for standby use; and a doxorubicin (DOX) stock solution is prepared by using dimethyl sulfoxide (DMSO) for standby use.

[0083] The cancer cell lines selected for the following experiments of the present invention include: human colorectal cancer cells HCT116 and HT29, and mouse colorectal cancer cells CT26 and MC38.

[0084] Example 1: Preparation of pyrroloquinoline-polyamine analogs

[0085]

[0086] (I) At room temperature (25±5°C), chloral hydrate (compound 2, 29.8 g, 180.92 mmol) was added to 250 mL of an aqueous sodium sulfate solution (wherein the sodium sulfate content was 77 g (542.76 mmol)); after the solution was clarified, 125 mL of an aqueous solution of 2-methyl-5-nitroaniline (compound 1) (wherein the 2-methyl-5-nitroaniline content was 25 g (164 mmol)) and 12.5 mL of concentrated hydrochloric acid were added thereto; subsequently, 75 mL of an aqueous solution of hydroxylamine hydrochloride (wherein the hydroxylamine hydrochloride content was 34.2 g (493.42 mmol)) was added; a reaction mixture I was obtained, and the reaction mixture I was heated to 80°C for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature (25±5°C), diluted with 250 mL of water, filtered, washed with 100 mL of water, washed twice, and dried at 45°C for 24 hours to obtain 30 g of off-white solid.

[0087] Then, the off-white solid was added to hot sulfuric acid (120 mL) at 60°C in batches within 30 minutes to obtain a reaction mixture 2; the reaction mixture 2 was heated at 80°C for 30 minutes, cooled to room temperature (25±5°C), and quenched in ice water (1.4 L) within 30 minutes to obtain a reaction mixture 3. The reaction mixture 3 was placed at room temperature (25±5°C) for 24 hours to precipitate; the precipitated solid was filtered, washed with 150 mL of water, washed twice and then dried to obtain a crude product 1. A chloroform solution containing 0.8-2% methanol was used as an eluent, and the crude product 1 was purified by flash column chromatography (solid filler was silica gel, specification 100-200 mesh) to obtain 14.5 g of red solid compound 3 (yield 43%).

[0088] (II) Compound 3 was prepared into 105 mL of dichloromethane solution (wherein the content of compound 3 was 2.1 g (10 mmol)), and (benzoylmethylene)triphenylphosphine (3.87 g, 10 mmol) was added dropwise to the dichloromethane solution containing compound 3 at 0°C to obtain a reaction mixture 4. The reaction mixture 4 was stirred at room temperature (25±5°C) for 5 hours; the solvent was removed to obtain a crude product 2, the crude product 2 was ground with methanol (40 mL), filtered to obtain a solid, and then the solid was washed with methanol (10 mL), and air-dried to obtain 2.69 g of yellow solid compound 4 (yield 86%).

[0089] Compound 4 was prepared into 135 mL of ethanol solution (wherein the content of compound 4 was 2.7 g (9 mmol)). At room temperature (25 ± 5 °C), SnCl was added to the ethanol solution containing compound 4. 2 ·2H 2O (9.88 g, 44 mmol) to obtain reaction mixture 5. The reaction mixture 5 was heated to 80°C and kept warm for 5 hours. After the reaction was completed, the solvent was removed and water was added. The mixture was extracted with a dichloromethane solution containing 10% methanol, the organic layer was separated, and the mixture was washed with anhydrous Na 2 SO 4 The crude product 3 was dried, filtered and concentrated to obtain a crude product 3. The crude product 3 was purified by flash column chromatography (solid filler: silica gel, specification 100-200 mesh) using a dichloromethane solution containing 2-3% methanol as an eluent to obtain 1.96 g of yellow solid compound 5 (yield 86%).

[0090] 1.9 g of compound 5 (7.30 mmol) was dissolved in 133 mL of acetonitrile and K 2 CO 3 (1.2g, 8.76mmol) and 1,4-dibromobutane (1.04mL, 8.76mol) were added to obtain a reaction mixture of six. The reaction mixture of six was stirred at 85°C for 6 hours. Then it was cooled to room temperature (25±5°C), filtered and the filtrate was concentrated to obtain a crude product of four. The crude product of four was purified by ash column chromatography (solid filler is silica gel, specification 100-200 mesh), and the crude product of four was eluted with a dichloromethane solution containing 0-15% ethyl acetate to obtain 1.2g of yellow solid compound 6 (yield 42%).

[0091] (III) To 100 mL of anhydrous acetonitrile solution containing compound 7 (1 g, 5.74 mmol) and compound 6 (1.1 g, 2.87 mmol) was added 0.79 g of anhydrous K 2 CO 3 (5.74 mmol) to obtain reaction mixture VII. The reaction mixture VII was stirred at 45°C for 20 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, the residue was diluted with water, and extracted with ethyl acetate. The organic layer was washed with Na 2 SO 2 The mixture was dried, filtered and concentrated under reduced pressure to obtain a semisolid; the obtained semisolid was dissolved in 50 mL of methanol, and triethylamine (1.1 mL, 8.04 mmol) and di-tert-butyl dicarbonate (1.62 g, 7.45 mmol) were added to obtain a reaction mixture VIII. The reaction mixture VIII was stirred at room temperature (25±5°C) for 16 hours. After the reaction was completed, the reaction mixture VIII was concentrated under vacuum to obtain a crude product V, which was then precipitated with CH 2 Cl 2 The crude product was diluted and washed with brine solution. The organic layer was washed with Na 2 SO 2Dry, filter and concentrate under reduced pressure. Purify the residue by flash column chromatography using 20-45% ethyl acetate in petroleum ether as eluent to elute the crude product 5 to afford the Boc protected intermediate.

[0092] The compound 7 described in the present invention is obtained according to the method in the paper Li Jinghua, Tian Runguo, Ge Chaochao, Chen Ying, Liu Xiaodan, Wang Yuxia, Yang Yacheng, Luo Wen, Dai Fujun, Wang Senzhen, Chen Shuai, Xie Songqiang, and Wang Chaojie. Discovery of the Polyamine Conjugate with Benzo [cd] indol-2 (1H) -one as a Lysosome-Targeted Antimetastatic Agent. [J]. Journal of Medicinal Chemistry, 2018, 61, 6814-6829.

[0093] The obtained intermediate (1.75 g, 8.04 mmol) was dissolved in 8 mL of ethanol, and 24 mL of ethanol solution containing 4 M HCl was added dropwise at 0° C. to obtain a reaction mixture 9. The reaction mixture 9 was then stirred at room temperature (25±5° C.) for 16-24 hours, filtered, the precipitated solid was collected, washed with acetone, and vacuum dried to obtain the target compound PQPA (0.59 g, 26% yield) in the form of brown hydrochloride. The preparation result spectrum of the target compound PQPA is as follows: Figure 1 , 2 , as shown in Figure 3. 1 H NMR (300 MHz, D 2 O)δ7.44(s,1H),7.40-7.21(m,5H),7.16(d,J=9.0Hz,1H),7.02(d,J=9.0Hz,1H),3.54(s,2H ),3.12-2.88(m,6H),2.16(s,3H),2.05-1.93(m,2H),1.68-1.55(m,2H),1.52-1.39(m,2H); 13 C NMR (75MHz, D 2O) δ166.3,157.0,141.1,138.2,135.6,134.6,133.3,131.8,129.3,127.7,121.6 ,117.4,115.5,47.1,44.5,40.9,36.6,26.0,23.8,22.8,16.7; HRMS(ESI):calcd for C 24 H 28 N 4 O[M+H] + :389.2336,found:389.2335.

[0094] The flash column chromatography, ash column chromatography and related process parameters used in Example 1 can all adopt conventional methods and conventional settings in the prior art. The equipment and experimental materials used are also conventional commercially available products, and are not the inventive point of the present invention, so they will not be described in detail.

[0095] The following examples test the anti-tumor properties of the prepared pyrroloquinoline-polyamine analog PQPA and its anti-tumor properties in combination with doxorubicin.

[0096] Example 2: In vitro biological activity evaluation of pyrroloquinoline-polyamine analog PQPA alone

[0097] Test 1: Inhibition test on cancer cell proliferation:

[0098] Human colorectal cancer cells HCT116, HT29 and mouse colorectal cancer cells CT26, MC38 in the logarithmic growth phase were taken, the cancer cells were digested with trypsin, the cells were collected, centrifuged at 1000r / min for 5 minutes, the supernatant was discarded, and the cells were suspended with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibody), the number of cells was counted with a blood cell counting plate, and the cells were inoculated in a 96-well plate (5000 cells per well) and allowed to adhere to the wall for 24 hours. Then, the pyrroloquinoline-polyamine analog PQPA was added to a final concentration of 0μM, 2.5μM, 5μM, 10μM, and 20μM, and then incubated for 24 hours, 48 ​​hours, and 72 hours, respectively. After the designated incubation time, 50 μL of MTT solution (prepared by dissolving MTT in PBS buffer, wherein the MTT concentration is 2.5 mg / mL) was added to each well. After incubation for 4 hours, the liquid in the 96-well plate was removed, and 100 μL of dimethyl sulfoxide solution was added. The plate was gently shaken on a shaker for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell proliferation inhibition rate was calculated; wherein inhibition rate (%) = (OD 570 Control group-OD 570 Experimental group / OD 570Control group)×100%.

[0099] The test results are as follows Figure 4 As shown, Figure 4 The results showed that the pyrroloquinoline-polyamine analog PQPA was able to inhibit the proliferation of colorectal cancer cells with increasing incubation time.

[0100] Test 2: Inhibition test on the clone formation ability of cancer cells:

[0101] Human colorectal cancer cells HCT116 and HT29 and mouse colorectal cancer cells CT26 and MC38 in logarithmic growth phase were respectively obtained and digested with trypsin. The cells were collected and plated at 3×10 5 The cells were seeded into 6-well plates at a density of 1.5 × 10 μl and placed in a 5% CO 2 , 37°C incubator; then, the cells were treated with 0μM, 2.5μM, 5μM, 10μM, and 20μM pyrroloquinoline-polyamine analog PQPA for 24 hours, and then the cancer cells were digested with trypsin, the cells were collected, centrifuged, resuspended, counted, and plated; then, they were cultured in a 37°C incubator for 14 days, during which time the culture medium was replaced every 3 days. After 14 days, the original culture medium in the culture plate was discarded, the cells were washed 2-3 times with PBS, fixed with 4% (mass volume ratio) paraformaldehyde solution for 30 minutes, stained with 1% crystal violet for 5 minutes, and then washed with distilled water, placed in an oven for drying, photographed with a gel imager, and the number of cell clones was counted.

[0102] The test results are as follows Figure 5 As shown, the results showed that the pyrroloquinoline-polyamine analog PQPA significantly inhibited the colony-forming ability of colorectal cancer cells at concentrations of 10 μM and 20 μM compared with the control group.

[0103] Experiment 3: Inducing cancer cell apoptosis:

[0104] Human colorectal cancer cells HCT116 and mouse colorectal cancer cells CT26 in the logarithmic growth phase were digested, centrifuged, and resuspended using the above method. The cells were counted and plated at 3×10 5The cells were inoculated into a 6-well plate at a density of 10 μM and placed in a 37°C incubator. After the cells were completely attached, 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM of pyrroloquinoline-polyamine analog PQPA were added and cultured at 37°C for 24 hours. The supernatant was discarded, the plate was washed with PBS, and then trypsin was added for digestion. The cells were collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, the cells were resuspended with PBS, and centrifuged at 1500 rpm for another 10 minutes. The supernatant was discarded and the Annexin V-FITC / PI apoptosis detection kit (BD Company, USA) was used for testing according to the method in the paper Ge Chaochao, Wang Yuxia, Feng Yongli, WangSenzhen, Zhang Kemeng, Xu Xiaojuan, Zhang Zhiyang, Zhao Yuan, Wang Yanming, GaoLei, Dai Fujun, Xie Songqiang, Wang Chaojie. Suppression of oxidativephosphorylation and IDH2 sensitizes colorectal cancer to a naphthalimidederivative and mitoxantrone. [J]. Cancer Letters. 2021, 519: 30-45. Specifically, 300 μL binding buffer, 5 μL AnnexinⅤ-FITC solution and 5 μL PI solution were added to each sample, mixed evenly, incubated at 37°C in the dark for 20 minutes, and then centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, 700 μL of pre-cooled PBS was added, the cell pellet was gently blown, the cell pellet was sieved, and then the proportion of apoptotic cells was detected by flow cytometry.

[0105] The test results are as follows Figure 6 As shown, the results showed that compared with the control group, the pyrroloquinoline-polyamine analog PQPA could induce apoptosis in HCT116 colorectal cancer cells and CT26 mouse colorectal cancer cells.

[0106] Example 3: In vivo biological activity evaluation of pyrroloquinoline-polyamine analog PQPA alone

[0107] Test 4:

[0108] Take CT26 cells in the logarithmic growth phase, digest the cancer cells with trypsin, and then centrifuge at 1000 rpm for 5 minutes. Wash the cells twice with PBS, resuspend the cells with sterile pre-cooled saline, and adjust the cell density to 5×106 CT26 cells were injected into the armpits of mice (male Balb / c mice) (5×10 5 cells). Three days later, the mice were randomly divided into two groups, namely the control group and the treatment group, and normal saline (control group) and pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) were injected into the mice through the tail vein. After 14 consecutive days of administration, the experiment was terminated, the tumors were weighed, and statistical analysis was performed.

[0109] The test results are as follows Figure 7 As shown, the results showed that the pyrroloquinoline-polyamine analog PQPA was able to inhibit the in vivo growth of colorectal cancer compared with the control group, with an inhibition rate of 45.74%.

[0110] During the experiment, the weight changes of mice were counted. The results are as follows: Figure 8 As shown, the pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) had little effect on the body weight of mice compared with the control group.

[0111] After 14 days of continuous administration, the experiment was terminated, and the heart, liver, spleen, lung, kidney and other tissues were removed, weighed, and the organ index was calculated and analyzed. Fig. 9 As shown, compared with the control group, the pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) had no significant effect on the organ indexes of mice.

[0112] According to the method in the paper Ge Chaochao, Wang Yuxia, Feng Yongli, Wang Senzhen, Zhang Kemeng, Xu Xiaojuan, Zhang Zhiyang, Zhao Yuan, Wang Yanming, Gao Lei, Dai Fujun, Xie Songqiang, Wang Chaojie. Suppression of oxidative phosphorylation and IDH2sensitizes colorectal cancer to a naphthalimide derivative and mitoxantrone. [J]. Cancer Letters. 2021, 519: 30-45., the heart, liver, spleen, lung and kidney tissues of the above control group and the pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) group of mice were stained with hematoxylin-eosin to detect the effects of the drug on the tissues. The results are as follows Fig.10As shown, the results showed that the pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) alone had no significant effect on tissue morphology.

[0113] Example 4: In vitro biological activity evaluation of doxorubicin alone

[0114] Test 5:

[0115] Human colorectal cancer cells HCT116 and HT29 in the logarithmic growth phase were taken respectively, the cancer cells were digested with trypsin, centrifuged at a speed of 1000r / min for 5 minutes, the supernatant was discarded, and the cells were suspended with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibody), the number of cells was counted by a blood cell counting plate, and the cells were inoculated in a 96-well plate and allowed to adhere to the wall for 24 hours. Doxorubicin (DOX) was added to a final concentration of 0μM, 0.1μM, 0.5μM, 1μM, and 2μM, and then incubated for 24 hours, 48 ​​hours, and 72 hours, respectively. After the specified time of culture, 50μL of MTT solution (prepared by dissolving MTT in PBS buffer, wherein the MTT concentration was 2.5mg / mL) was added to each well, and the liquid in the 96-well plate was removed after incubation for 4 hours, and 100μL of dimethyl sulfoxide solution was added, and it was placed on a shaker and gently shaken for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell proliferation inhibition rate was calculated; where inhibition rate (%) = (OD 570 Control group-OD 570 Experimental group / OD 570 Control group)×100%.

[0116] The test results are as follows Fig.11 As shown, Fig.11 The results showed that doxorubicin (DOX) could inhibit the proliferation of colorectal cancer cells.

[0117] Example 5: In vitro mechanism of action of the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin

[0118] Experiment 6: Effect of combined drug therapy on cancer cell viability:

[0119] Human colorectal cancer cells HCT116 and HT29 in logarithmic growth phase were taken, and the cancer cells were digested with trypsin, the cells were collected, centrifuged at 1000r / min for 5 minutes, the supernatant was discarded, and the cells were suspended with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibody), the number of cells was counted with a blood cell counting plate, and the cells were inoculated in a 96-well plate (5000 cells per well) and allowed to adhere to the wall for 24 hours. Then, pyrroloquinoline-polyamine analogs PQPA were added at concentrations of 0μM, 5μM, 10μM, and 20μM, and doxorubicin (DOX) was added at concentrations of 0μM, 0.1μM, 0.5μM, 1μM, and 2μM, and the culture was continued for 24h. After the designated incubation time, 50 μL of MTT solution (prepared by dissolving MTT in PBS buffer, wherein the MTT concentration is 2.5 mg / mL) was added to each well. After incubation for 4 hours, the liquid in the 96-well plate was removed, and 100 μL of dimethyl sulfoxide solution was added. The plate was gently shaken on a shaker for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell proliferation inhibition rate was calculated; wherein inhibition rate (%) = (OD 570 Control group-OD 570 Experimental group / OD 570 Control group)×100%.

[0120] The combination index (CI) was used to determine the synergistic effect of the two drugs. The calculation formula is: CI = DA / ICX, A + DB / ICX, B, where A and B represent two different drugs, ICX, A, ICX, B are the drug concentrations when the growth inhibition rate reaches X when the two drugs are used alone, and DA and DB are the concentrations of the two drugs when the growth inhibition rate reaches X. CI is expressed as the mean ± standard deviation (SD) of three independent experiments. The CI value represents the interaction between the two drugs. CI = 1 is an additive effect, CI < 1 is a synergistic effect, and CI > 1 is an antagonistic effect.

[0121] The test results are as follows Fig.12 As shown, Fig.12 The results showed that the combined use of pyrroloquinoline-polyamine analogs PQPA and doxorubicin significantly enhanced the effect on cell viability, and the final concentration of pyrroloquinoline-polyamine analogs PQPA was determined to be 10 μM and the final concentration of doxorubicin (DOX) was determined to be 1 μM.

[0122] Experiment 7: Effect of combined drug therapy on cancer cell proliferation:

[0123] Take human colorectal cancer cells HCT116 in logarithmic growth phase, digest cancer cells with trypsin, collect cells, centrifuge at 1000r / min for 5 minutes, discard supernatant, suspend with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibody), count the number of cells with a blood cell counting plate, inoculate in 96-well plates (5000 cells per well), and let them adhere to the wall and grow for 24 hours. Then add pyrroloquinoline-polyamine analog PQPA to a final concentration of 10μM, and add doxorubicin (DOX) to a final concentration of 1μM, and continue incubation for 24h. After culturing for a specified time, add 50μL of MTT solution (prepared by dissolving MTT in PBS buffer, where the MTT concentration is 2.5mg / mL) to each well, remove the liquid in the 96-well plate after incubation for 4 hours, add 100μL of dimethyl sulfoxide solution, and place it on a shaker and shake it gently for 10 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell proliferation inhibition rate was calculated; where inhibition rate (%) = (OD 570 Control group-OD 570 Experimental group / OD 570 Control group)×100%.

[0124] The test results are as follows Fig.13 As shown, Fig.13 The results showed that the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin (DOX) significantly inhibited the proliferation of human colorectal cancer cells HCT116 compared with the single drug group.

[0125] Experiment 8: Effect of combined drug therapy on cancer cell proliferation:

[0126] The same experimental method as Experiment 7 was used, except that the targeted cancer cells were human colorectal cancer cells HT29.

[0127] The test results are as follows Fig.14 As shown, Fig.14 The results showed that the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin (DOX) significantly inhibited the proliferation of human colorectal cancer cells HT29 compared with the single drug group.

[0128] Test Nine:

[0129] Human colorectal cancer cells HCT116 in the logarithmic growth phase were taken, and the cancer cells were digested with trypsin, the cells were collected, centrifuged at a speed of 1000r / min for 5 minutes, the supernatant was discarded, and the cells were suspended with complete medium (RPMI 1640 medium + 10% fetal bovine serum + double antibody), the number of cells was counted with a blood cell counting plate, and the cells were inoculated in a confocal dish and allowed to adhere to the wall for 24 hours. Then, pyrroloquinoline-polyamine analog PQPA was added to a final concentration of 10μM, and doxorubicin (DOX) was added to a final concentration of 1μM, and the culture was continued for 6 hours. The supernatant was removed, PBS was added, and the confocal microscope was used to take pictures.

[0130] The test results are as follows Fig.15 As shown, Fig.15 The results showed that the combined use of pyrroloquinoline-polyamine analog PQPA and doxorubicin (DOX) can significantly enhance the fluorescence intensity of doxorubicin (DOX), indicating that it can effectively promote the cellular uptake of doxorubicin (DOX).

[0131] Example 6: In vivo biological activity evaluation of the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin

[0132] Test 10:

[0133] Mouse colorectal cancer cells CT26 in logarithmic growth phase were obtained, and the cancer cells were digested with trypsin. The cells were collected and centrifuged at 1000 r / min for 5 minutes, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS. CT26 cells were inoculated in the armpit of each mouse, and the number of cells was 5×10 5 The tumor-bearing mice were divided into four groups: control group, pyrroloquinoline-polyamine analog PQPA (20 mg / kg / day) group, doxorubicin (DOX, 2 mg / kg / day) group and combined drug group. The drugs were administered for 14 consecutive days. After the experiment, the tumors were removed, photographed, and the tumor weight was counted and analyzed.

[0134] The test results are as follows Fig.16 As shown, Fig.16 The results showed that the combination of pyrroloquinoline-polyamine analog PQPA and doxorubicin (DOX) can significantly inhibit the growth of colorectal cancer in mice compared with the control group. Statistical results showed that compared with the control group, the tumor inhibition rate of the pyrroloquinoline-polyamine analog group was 50.3%, the tumor inhibition rate of the doxorubicin (DOX) group was 61.2%, and the tumor inhibition rate of the combined drug group was 85.1%. These results show that compared with single drug use. Combination therapy can enhance the anti-tumor activity of single drug use.

[0135] The above experimental data show that compared with the single-drug group, the combination drug can significantly inhibit the in vivo growth of colorectal cancer cells and significantly improve the anti-tumor activity of the single-drug group.

Claims

1. The use of pyrroloquinoline-polyamine analogs combined with doxorubicin in the preparation of anti-tumor drugs, characterized in that: When the pyrroloquinoline-polyamine analogues are used alone, or when the pyrroloquinoline-polyamine analogues and doxorubicin are used in combination, they can inhibit the proliferation, migration, or number or size of clones formed by colorectal cancer cells, or can induce apoptosis of colorectal cancer cells.

2. The use according to claim 1, characterized in that: The colon cancer cells include HT29, HCT116, CT26 or MC38 cells.

3. The use according to claim 1, characterized in that: When the pyrroloquinoline-polyamine analogue and doxorubicin are used in combination, the pyrroloquinoline-polyamine analogue and doxorubicin are added at once or added continuously for multiple times.

4. The use according to any one of claims 1 to 3, characterized in that: When the pyrroloquinoline-polyamine analogue and doxorubicin are added at one time, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is (1-10):1, and the treatment time is 6-24 hours; when the pyrroloquinoline-polyamine analogue and doxorubicin are added continuously for multiple times, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin each time is (2-200):1, and the continuous addition is 7-14 times in total.

5. The use according to any one of claims 1 to 3, characterized in that: When used in combination, the pyrroloquinoline-polyamine analog at a concentration of 2.5-20 μM and a treatment time of 24-72 hours, and doxorubicin at a concentration of 0.1-2 μM and a treatment time of 24-72 hours can inhibit the proliferation and / or number or size of colony formation of colorectal cancer cells; or, can induce apoptosis of colorectal cancer cells.

6. The use according to any one of claims 1 to 3, characterized in that: Application of pyrroloquinoline-polyamine analogs combined with doxorubicin in the preparation of drugs for inhibiting tumor growth in human or mouse transplanted tumor models of colon cancer.

7. The use according to claim 6, characterized in that: When the dose of pyrroloquinoline-polyamine analogues is 2-20 mg / kg / day and the dose of doxorubicin is 1-5 mg / kg / day, and the treatment time is 2-14 days, the growth of colorectal cancer mouse tumors can be inhibited.

8. The use according to any one of claims 1 to 3, characterized in that: The pyrroloquinoline-polyamine analogues are prepared by the following steps: (1) Using methyl-nitroaniline as the starting material, a substitution reaction is carried out with chloral hydrate and hydroxylamine hydrochloride to form a ring to obtain an intermediate product I; (2) Intermediate product I undergoes a nucleophilic addition reaction with (benzoylmethylene)triphenylphosphine at room temperature to obtain intermediate product II; (3) Intermediate product II undergoes a nucleophilic addition reaction in the presence of a reducing agent and an organic solvent I to obtain intermediate product III; (4) Intermediate product III undergoes nucleophilic substitution reaction with dibromobutane in the presence of an organic solvent II to obtain intermediate product IV; (5) The intermediate product IV undergoes a nucleophilic substitution reaction with a polyamine compound using an organic solvent II as a solvent and under the condition of adding a base to obtain a pyrroloquinoline-polyamine analogue; The organic solvent I is ethanol, methanol, dichloromethane or acetonitrile; the organic solvent II is ethanol, methanol, dichloromethane or acetonitrile; the reducing agent is any one of stannous chloride, oxalic acid, potassium borohydride and sodium borohydride; The base is an organic base or an inorganic base, the inorganic base is any one of potassium carbonate, sodium carbonate, sodium bicarbonate and potassium bicarbonate; the organic base is any one of dimethylamine, aniline, pyridine and triethylamine; The polyamine compound is any one of 1,3-propylenediamine, putrescine, spermidine and spermine.

9. The use according to any one of claims 1 to 3, characterized in that: The structural formula of the pyrroloquinoline-polyamine analogue is as follows:

10. An anti-tumor pharmaceutical composition containing doxorubicin, characterized in that: It includes pyrroloquinoline-polyamine analogs or salts thereof, and doxorubicin or derivatives thereof, wherein the pyrroloquinoline-polyamine analogs have the structural formula: In the anti-tumor pharmaceutical composition containing doxorubicin, the molar ratio of the pyrroloquinoline-polyamine analogue to doxorubicin is (10-200):1.

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