Hydrazino-modified natural polyamine-naphthalimide conjugate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a hydrazino-modified natural polyamine-naphthalimide conjugate as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, introducing hydrazino to the tail end of a polyamine chain, further prolonging the length of the polyamine chain, and finally, conjugating parent nucleus naphthalimide to synthesize a series of hydrazino-modified natural polyamine-naphthalimide conjugates. The compound synthesized by the invention comprises a substituent-free type of parent nucleus naphthalimide, a 3-site amino substituted type of parent nucleus naphthalimide and a 4-site chlorine substituted type of parent nucleus naphthalimide. The conjugate disclosed by the invention solves the problems of poor solubility, relatively complicated clinical compatibility, poor patient immunity in clinical application of chemotherapeutic drugs and the like of the naphthalimide analogue represented by the dinafide in the prior art, and also has relatively good targeting property. According to the conjugate disclosed by the invention, the targeting property and bioavailability of a drug on tumor cells can be improved by utilizing the polyamine transporter highly expressed on the surfaces of the tumor cells, and the toxic and side effects on normal cells are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry, and specifically relates to a hydrazine-modified natural polyamine-naphthalimide conjugate, a preparation method and application thereof. Background Art

[0002] The main reasons for the failure of chemotherapy drugs in the treatment of cancer, especially metastatic cancer, are lack of targeting, drug resistance, weak anti-tumor metastasis activity and immunosuppression. Naphthalimide is a class of anticancer agents that have been proven to have broad application prospects. However, due to the intercalation of naphthalimide derivatives with antitumor activity into DNA, many clinical trials of naphthalimide derivatives have been terminated in Phase II, such as Amonafide, Mitonafide, DMP-840, etc.

[0003] As early as 1973, the Brana research group synthesized a series of naphthylimide-polyamine derivatives with anti-tumor activity. The study found that modifying the naphthylimide structure to enhance its targeting and solve its immunosuppression problems has become the focus of current chemotherapy drug research and development.

[0004] Polyamine analogs are compounds with similar structures to polyamines. They can be recognized and taken up by polyamine transporters (PTS). They cannot replace some physiological functions of polyamines, but they can inhibit the utilization of polyamines by tumor cells in various ways, thereby inducing cell apoptosis. At present, the focus of structural modification of naphthaleneimide is mainly on targeted modification of naphthaleneimide.

[0005] In order to synthesize clinically applicable naphthalimide anti-tumor drugs, the inventors have modified the structure of the parent core naphthalimide in previous experiments and have synthesized some naphthalimide-polyamine conjugates with good anti-tumor activity. Polyamines are selective drug carriers that can deliver drugs in a targeted manner.

[0006] In addition, during the research process, the inventors found that the representative hydrazine-containing drug procarbazine hydrochloride also exerts anti-tumor activity by acting on DNA; therefore, based on the characteristics and research status of naphthylimide, hydrazine, and polyamine, the inventors designed a series of naphthylimide-polyamine hydrazine conjugates. By modifying the active skeleton structure of natural polyamines with hydrazine groups and connecting the mother core naphthylimide with polyamines as carriers, it is expected to obtain anti-tumor lead compounds with stronger activity than polyamine structures without hydrazine group modification.

[0007] Based on this, the present invention introduces a hydrazine group at the end of the polyamine chain and then conjugates it with the polyamine chain to form a parent core naphthaleneimide, in order to obtain an anti-tumor lead compound with targeted action, strong anti-tumor activity and high research value. Summary of the invention

[0008] Based on the deficiencies in the prior art, the purpose of the present invention is to propose a hydrazine-modified natural polyamine-naphthalimide conjugate with high stability and high targeting. The hydrazine-modified natural polyamine-naphthalimide conjugate not only solves the problems of poor solubility, cumbersome clinical compatibility, and poor immunity of patients in clinical application of chemotherapy drugs of naphthalimide analogues represented by aminonaphthide, but also has good targeting. In addition, the present invention also studies whether the hydrazine-modified natural polyamine-naphthalimide conjugate exerts a dual inhibitory effect on subcellular organelles and cell nuclei.

[0009] The present invention also provides a method for preparing the hydrazine-modified natural polyamine-naphthalimide conjugate.

[0010] The present invention further provides the use of the hydrazine-modified natural polyamine-naphthalimide conjugate in the preparation of tumor therapeutic drugs.

[0011] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0012] A hydrazine-modified natural polyamine-naphthalimide conjugate, the general structural formula of which is shown in Formula I:

[0013]

[0014] Specifically, wherein R1 is H, NH2, Cl or Br; wherein R2 is H, NH2, Cl or Br.

[0015] Specifically, in the formula, R3 is

[0016] Specifically, the structural formula of the hydrazine-modified natural polyamine-naphthalimide conjugate is as follows:

[0017] or

[0018]

[0019] Specifically, the preparation method of the hydrazine-modified natural polyamine-naphthalimide conjugate comprises the following steps:

[0020] 1) dissolving a hydrazinolysis compound and a 1,8-naphthalimide derivative containing or not containing a substituent in a solvent I, reacting at 40 to 50° C. (preferably 45° C.) for 10 to 15 hours (preferably 12 hours), removing the solvent I after the reaction, extracting, washing, and drying the residue to obtain a product I;

[0021] 2) Dissolve the obtained product I in solvent II, and add a solution of hydrochloric acid in solvent II at -5 to 5°C (preferably 0°C), warm to room temperature and react for 10 to 15 hours (preferably 12 hours) to obtain a solid, which is then filtered, washed, and dried to obtain the final product.

[0022] Specifically, the hydrazinolysis compound in step 1) is or

[0023]

[0024] Specifically, the 1,8-naphthalimide derivative containing or not containing a substituent in step 1) is compound 4a, 4b, 4c, 4d, 4e, 4f, and the specific structural formula is as follows:

[0025]

[0026] Specifically, in step 1), the molar ratio of the hydrazinolysis compound to the 1,8-naphthalimide derivative containing or not containing a substituent is 1:(0.5-3), preferably 1:1.

[0027] Specifically, in step 1), solvent I is acetonitrile, ethanol or methanol.

[0028] Specifically, in step 2), solvent II is acetonitrile, ethanol or methanol.

[0029] Specifically, the room temperature refers to 25±5°C.

[0030] Furthermore, the specific synthesis route of the hydrazine-modified natural polyamine-naphthalimide conjugate is as follows:

[0031]

[0032]

[0033] Furthermore, the preparation method of the compound 3a or 3b comprises the following steps:

[0034] a. Dissolve di-tert-butyl dicarbonate (Boc)2O in solvent I, add hydrazine hydrate solution in solvent I, react at room temperature for 10 to 15 hours (preferably 12 hours), then remove solvent I, and purify by silica gel column chromatography to obtain 1,2-di-tert-butyloxycarbonylhydrazine;

[0035] Dissolve 1,2-di-tert-butyloxycarbonylhydrazine in solvent III, add cesium carbonate first, then add compound 1a or 1b, react at room temperature for 4 to 8 hours (preferably 6 hours), extract and dry after the reaction, remove solvent III, and purify by silica gel column chromatography to obtain compound 2a or 2b;

[0036] b. Dissolve the compound 2a or 2b obtained in step a in solvent I, add hydrazine hydrate, and react at room temperature for 6 to 10 hours (preferably 8 hours). After the reaction, remove solvent I, extract with an extractant, wash, and remove the extractant to obtain compound 3a or 3b.

[0037] Specifically, the structural formula of compounds 2a and 2b is

[0038] Specifically, the room temperature refers to 25±5°C.

[0039] Specifically, solvent I is acetonitrile, ethanol or methanol.

[0040] Specifically, solvent III is DMF.

[0041] Specifically, in step a, the molar ratio of di-tert-butyl dicarbonate (Boc)2O to hydrazine hydrate is 1:(0.2-0.5), preferably 1:0.425.

[0042] Specifically, in step a, the molar ratio of 1,2-di-tert-butyloxycarbonylhydrazine to compound 1a or 1b is 1:(1-1.5), preferably 1:1.1.

[0043] Specifically, in step a, the molar ratio of 1,2-di-tert-butyloxycarbonylhydrazine to cesium carbonate is 1:(1-1.5), preferably 1:1.1.

[0044] Specifically, in step b, the molar ratio of compound 2a or 2b to hydrazine hydrate is 1:(2-3), preferably 1:3.

[0045] Specifically, the extracting agent in step b is dichloromethane.

[0046] Further, the specific synthesis route of compound 3a or 3b is as follows:

[0047]

[0048] Furthermore, the preparation method of the compounds 8a-8d comprises the following steps:

[0049] a. Dissolve compound 3a or 3b and anhydrous K2CO3 in solvent I, then add compound 1a or 1b, and react at 40-45°C (preferably 45°C) for 10-15h (preferably 12h). After the reaction, remove solvent I, extract with an extractant, wash, and then remove excess water and extractant to obtain product II;

[0050] The product II is dissolved in solvent I, and then (Boc)2O is added to react at room temperature for 10 to 15 hours (preferably 12 hours). After the reaction is completed, solvent I is removed, and the residue is extracted with an extractant, washed, and then excess water and the extractant are removed, and purified by silica gel column chromatography to obtain compounds 7a-7d;

[0051] b. Dissolve the compound 7a-7d obtained in step a in solvent I, add hydrazine hydrate, and react at room temperature for 6 to 10 hours (preferably 8 hours). After the reaction, remove solvent I, extract with an extractant, wash, and remove the extractant to obtain compounds 8a-8d.

[0052] Specifically, the structural formula of compounds 7a-7d is

[0053] Specifically, the room temperature refers to 25±5°C.

[0054] Specifically, solvent I is acetonitrile, ethanol or methanol.

[0055] Specifically, the extractant is dichloromethane.

[0056] Specifically, in step a, the molar ratio of compound 3a or 3b to compound 1a or 1b is (4-5):1, preferably 4:1.

[0057] Specifically, in step a, the molar ratio of compound 3a or 3b to anhydrous K2CO3 is (2-2.5):1, preferably 2.5:1.

[0058] Specifically, in step a, the molar ratio of compound 3a or 3b to (Boc)2O is (2-2.5):1, preferably 2:1.

[0059] Specifically, in step b, the molar ratio of compound 7a-7d to hydrazine hydrate is 1:(2-3), preferably 1:3.

[0060] Furthermore, the specific synthetic routes of compounds 8a-8d are as follows:

[0061]

[0062] Furthermore, the preparation method of the compounds 4a, 4b, 4c, 4d, 4e, and 4f comprises the following steps:

[0063] 1,8-naphthalene diimide or 1,8-naphthalene diimide containing a substituent is mixed with anhydrous K2CO3, KI, 1,3-dibromopropane (or 1,4-dibromobutane) and dissolved in solvent IV, and reacted at room temperature for 10 to 15 hours (preferably 12 hours). After the reaction is completed, the solvent IV is removed by suction filtration, and purified by silica gel column chromatography to obtain compounds 4a-4d, compound 4e or compound 4f, respectively.

[0064] Specifically, the structural formula of the substituted 1,8-naphthalimide is

[0065] Specifically, the room temperature refers to 25±5°C.

[0066] Specifically, solvent IV is acetone.

[0067] Specifically, the molar ratio of 1,8-naphthalene dicarboximide or substituted 1,8-naphthalene dicarboximide to 1,3-dibromopropane (or 1,4-dibromobutane) is 1:(1-4), preferably 1:3.

[0068] Specifically, the molar ratio of 1,8-naphthalene diimide or substituted 1,8-naphthalene diimide to anhydrous K2CO3 is 1:(1-4), preferably 1:3.

[0069] Specifically, the molar ratio of 1,8-naphthalene dicarboximide or substituted 1,8-naphthalene dicarboximide to KI is 1:(1-2), preferably 1:1.5.

[0070] Further, the specific synthetic routes of compounds 4a, 4b, 4c, and 4d are as follows:

[0071]

[0072] Furthermore, the specific synthetic routes of compounds 4e and 4f are as follows:

[0073]

[0074] Furthermore, the present invention also provides the use of the hydrazine-modified natural polyamine-naphthalimide conjugate, or the hydrazine-modified natural polyamine-naphthalimide conjugate combined with cisplatin or oxaliplatin or 5-fluorouracil in the preparation of anti-tumor drugs.

[0075] Specifically, the tumor includes but is not limited to human colon cancer, human liver cancer, human breast cancer, human lung cancer or cisplatin-resistant human lung adenocarcinoma.

[0076] Furthermore, the present invention also provides the use of the hydrazine-modified natural polyamine-naphthalimide conjugate alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis, wherein the tumor cells are human colon cancer cells HCT-116, HT-29, human liver cancer cells Huh-7, HepG-2, SNU-739, human breast cancer cells MDA-MB-231, MCF-7, and human lung cancer cells A549.

[0077] Specifically, when used, when the final concentration of the hydrazine-modified natural polyamine-naphthalimide conjugate is (0.5-5) μM and the treatment time is 24-48 hours, it can inhibit the proliferation of tumor cells, induce their pyroptosis or promote their apoptosis.

[0078] Preferably, when used, when the final concentration of the hydrazine-modified natural polyamine-naphthalimide conjugate is 0.5 μM, 1 μM or 5 μM and the treatment time is 24 h, it can inhibit the proliferation of tumor cells, induce pyroptosis or promote apoptosis.

[0079] Furthermore, the present invention also provides the use of the hydrazine-modified natural polyamine-naphthalimide conjugate in the preparation of a drug for inhibiting tumor growth in a mouse orthotopic model of breast cancer. Specifically, the mouse used in the mouse source is a BALB / c mouse.

[0080] Specifically, the hydrazine-modified natural polyamine-naphthalimide conjugate can inhibit the metastasis of a breast adenocarcinoma mouse metastasis model tumor when the dosage is 2.5-5 mg / kg / day and the dosage is administered five times in total.

[0081] Preferably, the hydrazine-modified natural polyamine-naphthalimide conjugate can inhibit the metastasis of a breast adenocarcinoma mouse metastasis model tumor when the dosage is 10 mg / kg or 5 mg / kg every two days for a total of five administrations.

[0082] Compared with the prior art, the beneficial effects of the present invention are:

[0083] 1. The present invention first introduces a hydrazine group at the end of the polyamine chain, then further extends the length of the polyamine chain, and finally conjugates the parent core naphthalene imide to synthesize a series of hydrazine-modified natural polyamine-naphthalene imide conjugates. The compounds synthesized by the present invention include naphthalene imide-polyamine hydrazine conjugates in which the parent core naphthalene imide does not contain a substituent, the parent core naphthalene imide is substituted with an amino group at the 3rd position, and the naphthalene imide is substituted with a chlorine group at the 4th position. 1 H NMR, 13 C NMR and MS were used to confirm the structures of the compounds and the antitumor activities of the series of compounds were evaluated.

[0084] 2. The hydrazine-modified natural polyamine-naphthalimide conjugate of the present invention can utilize the polyamine transporter highly expressed on the surface of tumor cells to improve the targeting of drugs to tumor cells, improve bioavailability, and reduce toxic side effects on normal cells.

[0085] 3. The hydrazine-modified natural polyamine-naphthalimide conjugate of the present invention achieves dual targeting of subcellular organelles and cell nuclei through modification of the naphthalimide structure.

[0086] 4. The present invention improves the water solubility and fat solubility of naphthylimide drugs and increases the drug uptake by tumor cells.

[0087] 5. The hydrazine-modified natural polyamine-naphthalimide conjugate of the present invention is an anti-tumor compound. Its design, synthesis and anti-cancer activity test are aimed at preparing anti-cancer active molecules with broad spectrum, high efficiency and low toxicity, and providing new candidate drugs for clinical treatment of cancer. From the test results of the present invention, it can be seen that the hydrazine-modified natural polyamine-naphthalimide conjugate has good activity against tumor growth and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The inhibition of cell migration in breast cancer cells after 24 hours of incubation with different concentrations of hydrazine-modified natural polyamine-naphthalimide conjugate, cisplatin and combined administration in the wound healing experiment; *P<0.05; **P<0.01; ***P<0.001;

[0089] Figure 2 The inhibition of cell migration after 24 hours of incubation of breast cancer cells with different concentrations of hydrazine-modified natural polyamine-naphthalimide conjugate, cisplatin and combined administration in the Transwell chamber experiment; *P<0.05; **P<0.01; ***P<0.001;

[0090] Figure 3 This is a quantitative analysis of breast cancer cell apoptosis induced by different concentrations of hydrazine-modified natural polyamine-naphthalimide conjugates and 10 μM AAF after incubation for 24 hours;

[0091] Figure 4 This is an experiment to evaluate the anti-tumor activity of drugs in vivo; Figure 4 A indicates that 7 days after the Balb / c mice were inoculated with 4T1 cells, they were given aminefit and J11 treatment, once every 2 days, for a total of 4 times; Figure 4 B represents the quantitative analysis of the inhibitory effect of aminefide and J11 on tumor growth after treatment; Figure 4 C represents the changes in mouse body weight during treatment; Figure 4 D shows the changes in tumor volume during treatment; 4E shows the quantitative analysis of organ indexes of heart, liver, spleen, lung and kidney of mice after treatment. *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0092] The technical solution of the present invention is further described in detail below through specific examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, all reagents mentioned herein are commercially available high-purity reagents that meet the experimental requirements.

[0093] The abbreviations involved in the embodiments of the present invention include: Ac is acetyl, Me is methyl, DMSO is dimethyl sulfoxide, DMF is dimethylformamide, DCM is dichloromethane, Boc is tert-butyloxycarbonyl, and Et is ethyl. Cis. is cisplatin, Oxp. is oxaliplatin, and AF is aminonaphthalene. The compounds 1a, 1b, and 1,8-naphthalene diimide containing substituents 1,8-Naphthalimide, cisplatin, oxaliplatin, and aminonaphthenate are all common commercial products. You can also use the literature (Dai, Fujun, et al. "Design, synthesis, and biological evaluation of mitochondria-targeted flavone-naphthalimide-polyamine conjugates with antimetastatic activity." Journal of medical chemistry 60.5 (2017): 2071-2083; Wang, Yuxia, et al. "Nonhematotoxicnaphthalene diimide modified by polyamine: synthesis and Preparation according to the method in biologicalevaluation."Journal of medicinal chemistry 55.7(2012):3502-3512."

[0094] In the following examples, room temperature refers to 25±5°C.

[0095] Example 1

[0096] 1. Synthesis of compounds 2a and 2b

[0097]

[0098] Take 10mmol (2.18g) of di-tert-butyl dicarbonate (Boc)2O and dissolve it in 25mL of methanol. Stir it in an ice bath in a water bath for 10min. Slowly add 10mL of hydrazine hydrate (4.25mmol) methanol solution while stirring. Stir the reaction at room temperature for 12h after the addition is completed. Evaporate the reaction solvent under reduced pressure. Stir the remaining solvent into 100-200 mesh silica gel and mix the sample evenly. The amount of silica gel should be 1.5 times the amount of the sample. The column is packed using a dry method. A mixed solvent of petroleum ether (PE) and ethyl acetate (EA) is used as a developing agent to elute the sample. The ratio (volume ratio) of petroleum ether (PE) to ethyl acetate (EA) is 15:1. When synthesizing in large quantities, the sample can be recrystallized with a mixed solvent of PE:EA=15:1 to obtain 1,2-di-tert-butyloxycarbonylhydrazine.

[0099] Dissolve 10 mmol of 1,2-di-tert-butyloxycarbonylhydrazine in 10 mL DMF was placed in a round-bottom flask, 11 mmol of cesium carbonate was added thereto, magnetic stirring was performed for 5 min, and then 11 mmol of N-(3-bromoalkyl)phthalimide or N-(4-bromobutyl)phthalimide (i.e., compounds 1a and 1b) was added thereto. The mixture was reacted at room temperature for 6 h. The reaction was detected by TCL. The TCL detection results before and after the reaction were compared. After the reaction was completed, the reaction solution was diluted with 50 mL of water, and then extracted three times with 30 mL of ethyl acetate. The organic phase was dried over anhydrous Na2SO4, and then the reaction solvent DMF was removed by evaporation under reduced pressure. The remaining solvent 100-200 mesh silica gel was evenly mixed with the sample. The amount of silica gel was preferably 1.5 times the amount of the sample. The column was packed using a dry method, and a mixed solvent of petroleum ether (PE) and ethyl acetate (EA) was used as a developing solvent. Compounds 2a and 2b were obtained by purification by silica gel column chromatography (petroleum ether / ethyl acetate, V / V=10:1) with a yield of 83.57%-87.16%.

[0100] 2. Synthesis of compounds 3a and 3b

[0101]

[0102] 10 mmol of the above chromatogram (i.e., compounds 2a and 2b) was dissolved in 20 mL of anhydrous ethanol, 30 mmol of hydrazine hydrate was added, and the mixture was reacted at room temperature for 8 h. The reaction was then detected by TCL. The reaction solvent anhydrous ethanol was removed by evaporation under reduced pressure, and the mixture was extracted with 40 mL of dichloromethane. The extract was washed three times with 20 mL of dilute ammonia water, and the organic phase was removed from excess water with anhydrous Na2SO4. The organic layer of dichloromethane was evaporated to obtain a colorless oily liquid, i.e., compounds 3a and 3b, with a yield of 79.34%-84.28%.

[0103] 3. Synthesis of compounds 4a-4d, 4e, and 4f

[0104]

[0105] In a 100 mL round-bottom flask, 1.97 g (10 mmol) of 1,8-naphthalene diimide containing a substituent, 30 mmol of anhydrous K2CO3, KI (1.5 mmol), and 50 ml of acetone were added, and then 30 mmol of 1,3-dibromopropane (or 1,4-dibromobutane) was added dropwise; the reaction was stirred at room temperature for 12 h, and the reaction was monitored by TLC; the reaction solvent acetone was removed by suction filtration and evaporated under reduced pressure, and the residue was eluted by rapid column chromatography with petroleum ether / ethyl acetate to obtain N-(3-bromopropyl)-1,8-naphthalene diimide containing a substituent and N-(4-bromobutyl)-1,8-naphthalene diimide containing a substituent (i.e., compounds 4a-4d) in yields of 80.52%-89.26%, respectively.

[0106]

[0107] At the same time, the substituted 1,8-naphthalene diimide was replaced with 1,8-naphthalene diimide, and the non-substituted N-(3-bromopropyl)-1,8-naphthalene diimide (compound 4e) and the non-substituted N-(4-bromobutyl)-1,8-naphthalene diimide (compound 4f) were prepared in the same manner.

[0108] 4. Synthesis of Compounds 5a-5d

[0109]

[0110] Take 10mmol of compound 3b obtained by hydrazine hydrolysis and 30mmol of anhydrous K2CO3 and dissolve them in acetonitrile. React at room temperature for 15min. Control the temperature of the water bath at 45℃. Add 10mmol of compounds 4a-4d respectively while stirring. React at 45℃ for 12h. After the reaction, evaporate the reaction solvent acetonitrile under reduced pressure. Extract the residue with 30mL of dichloromethane. The product was washed three times with an aqueous solution of Na2CO3 (mass fraction of 10%), and the dichloromethane organic layer was collected. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to remove dichloromethane to obtain a light yellow oily substance. The oily substance was dissolved in 50 mL of methanol solvent, and 5 mmol (Boc)2O was added and stirred at room temperature for 12 h. TCL was used to detect whether the reaction was completed. After the reaction was completed, the methanol solvent was evaporated under reduced pressure, and the residue was extracted with 40 mL of dichloromethane. The extract was washed and extracted three times with 40 mL of water, and the dichloromethane organic layer was collected. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to remove dichloromethane. The product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, V / V=5:1) to obtain compounds 5a-5d, respectively, with yields of 57.78%-63.46%.

[0111]

[0112] At the same time, compounds 4e and 4f without substituents are reacted with compounds 3a and 3b to prepare compounds 5e, 5f, 5g and 5h. The synthesis method is the same as that of compounds 5a-5d.

[0113] 5. Synthesis of compounds J1-J4, J8-J9, J11-J12

[0114]

[0115] Take 5 mmol of compound 5a-5d obtained by the above column chromatography, dissolve them in appropriate amount of ethanol respectively, cool to 0°C, add 4 M hydrochloric acid ethanol solution (10 mL) respectively, warm to room temperature and stir to react for 12 h until a large amount of solid is generated, filter with filter paper, wash three times with redistilled anhydrous ethanol, and dry to obtain solid compounds J8-J9 and J11-J12, respectively, with yields of 37.89%-43.25%.

[0116]

[0117] Compounds 5e, 5f, 5g and 5h were simultaneously prepared to prepare compounds J1-J4, and the synthesis method was the same as that of compounds J8-J9 and J11-J12.

[0118] Spectral data of compounds J1-J4, J8-J9, J11-J12:

[0119] J1: 2-[3-(3-Propanhydrazine)-propyl]1H-benzissoquinoline-1,3-dione

[0120] yield: 42.97%; 1H NMR (300MHz, Deuterium Oxide)δ:7.39(d,J=7.3Hz,2H),7.29(d,J=8.2Hz,2H),6.87(t,J=7.7Hz,2H),3.52(t,J=7.2Hz,2H),3.21(m,6H),2.08(m,2H),1.94(m,2H); 13C NMR (75MHz, D2O) δ164.66,135.13,131.23,130.30,126.84,126.05,119.66,57.43,48.88,47.33,37.27,24.21,16.81; ESI-MS (positive ion mode):m / z[(M+H)+:calcd 327.26; obsd326.17.

[0121] J2: 2-[3-(4-Butylhydrazine)-propyl]1H-benzissoquinoline-1,3-dione

[0122] yield: 43.25%; 1H NMR (300MHz, Deuterium Oxide)δ7.91(d,J=8.2Hz,2H),7.40(d,J=7.8Hz,2H),7.33(t,J=7.0Hz,2H),4.6 1(t,J=8.0Hz,2H),3.11~3.85(m,6H),2.04~1.18(m,4H),1.11~1.16(m,2H); 13C NMR (75MHz, D2O) δ164.60,134.96,131.11,130.23,126.77,125.33,119.73,47.28,44.84,39.57,36.15,24.16,23.16,22.15; ESI-MS (positive ion mode):m / z[(M+H)+:calcd 341.29; obsd 340.19.

[0123] J3: 2-[4-(3-Propanhydrazine)-butyl]1H-benzissoquinoline-1,3-dione

[0124] yield: 39.17%; 1H NMR (300MHz, Deuterium Oxide)δ7.32(d,J=8.1Hz,2H),7.11(d,J=7.1Hz,2H),6.73(t,J=8.1Hz,2H),3.4 8(t,J=7.1Hz,2H),3.14~3.09(m,6H),2.06~1.48(m,4H),1.01~1.23(m,2H); 13C NMR (75MHz, D2O) δ164.43,134.98,131.04,130.06,126.68,125.78,119.39,50.04,46.97,45.14,37.18,24.09,22.74,21.52; ESI-MS (positive ion mode):m / z[(M+H)+:calcd 341.27; obsd 340.19.

[0125] J4: 2-[4-(4-Butylhydrazinyl)-butyl]1H-benzissoquinoline-1,3-dione

[0126] yield: 39.46%; 1H NMR (300MHz, Deuterium Oxide)δ7.93(d,J=7.9Hz,2H),7.53(d,J=7.5Hz,2H),7.41(d,J=7.4Hz,2H),3.7 3(t,J=3.7Hz,2H),3.53~3.00(m,6H),3.05~2.89(m,6H),1.60~0.93(m,2H); 13C NMR (75MHz, D2O) δ164.72,134.93,131.23,130.31,126.83,126.09,119.63,50.17,47.14,39.59,37.28,24.20,23.19,22.83,21.61; ESI-MS (positive ion mode):m / z[(M+H)+:calcd 355.30; obsd 354.21.

[0127] J8: 6-Chloro-2-[3-(4-butylhydrazinyl)-propyl]1H-benzissoquinoline-1,3-dione

[0128] yield: 40.74%; 1H NMR (300MHz, Deuterium Oxide)δ8.17(d,J=8.8Hz,1H),8.08(d,J=7.5Hz,1H),7.89(d,J=6.2Hz,1H),7.61(t ,J=5.6Hz,1H),7.40(d,J=5.1Hz,1H),3.85(t,J=8.2Hz,2H),2,71~3.29(m,6H),1.8 3~2.04(m,2H),1.34~1.77(m,4H); 13CNMR(75MHz,D2O)δ164.67,134.96,131.12,13 0.26,126.76,119.78,47.23,44.79,39.53,24.13,23.12,22.11; ESI-MS(positive ion mode):m / z[(M+H)+:calcd 374.70; obsd374.15.

[0129] J9: 6-Chloro-2-[4-(4-butylhydrazinyl)-butyl]1H-benzissoquinoline-1,3-dione

[0130] yield: 41.23%; 1H NMR (300MHz, Deuterium Oxide)δ8.48(d,J=8.5Hz,1H),8.17(d,J=8.1Hz,1H),7.77(d,J=7.8Hz,1H),7.45(t,J=7.5Hz,1H),7.41(d,J= 6.4Hz, 1H), 4.71 (t, J = 9.2Hz, 2H), 3.53~3.85 (m, 6H), 3.07~3.27 (m, 6H), 1.13~1.24 (m, 2H); ESI-MS (positive ion mode):m / z[(M+H)+:calcd 389.27; obsd 388.17.

[0131] J11: 5-amino-2-[4-(4-butylhydrazinyl)-butyl]1H-benzissoquinoline-1,3-dione

[0132] yield: 35.62%; 1H NMR (300MHz, Deuterium Oxide)δ8.32(d,J=7.4Hz,1H),8.22(d,J=7.2Hz,1H),8.07(t,J=8.0Hz,1H),8.04(d,J=6.8Hz,1H),7.95(t,J=8.0 Hz,1H),7.71(t,J=7.2Hz,1H),4.63(t,J=5.9Hz,2H),3.56~3.09(m,6H),3.04~2.63(m,4H),1.69~1.09(m,2H),13C NMR (75MHz, CDCl3) δ166.78,136.98,134.31,132.36,127.34,125.34,123.00,59.85,52.60,49.63,42.21,36.93,26.61,25.62,24.03; ESI-MS (positiveion mode):m / z[(M+H)+:calcd370.30; obsd 369.22.

[0133] J12: 5-amino-2-[3-(4-butylhydrazinyl)-propyl]1H-benzissoquinoline-1,3-dione

[0134] yield:42.29%; 1H NMR (300MHz, DMSO-d6) δ9.29(d,J=8.0Hz,1H),8.97(d,J=7.8Hz,1H),8.45(d,J=7,6Hz,1H),7.78(t,J=6.4Hz, 1H),7.60(d,J=6.2Hz,1H),4.25(t,J=5.8Hz,2H),1.40~1.59(m,2H),1.20~1.36(m,6H),1.01~1.16(m,2H); 13C NMR(75MHz,D2O)δ164.67,134.96,131.12,130.26,126.76,119.78,47.23,44.79,39.53,24.13,23.12,22.11; ESI-MS(positive ion mode):m / z[(M+H)+:calcd 355.29;obsd355.20.

[0135] Example 2

[0136] 1. Synthesis of compounds 7a-7d

[0137]

[0138] 10 mmol of compounds 3a and 3b obtained by hydrazine hydrolysis were dissolved in acetonitrile and added to a 500 mL round-bottom flask, and 0.55 g (4 mmol) of anhydrous K2CO3 was added. The mixture was stirred with a magnetic stirrer for 15 min. The water bath temperature was set to 45 °C, and 2.5 mmol of compounds 1a and 1b were added while stirring. The mixture was reacted in a water bath for 12 h. After the reaction was completed, acetonitrile was removed by rotary evaporation under reduced pressure. The remaining material in the round-bottom flask was extracted with 30 mL of dichloromethane, and the extract was washed three times with 30 mL of Na2CO3 (mass fraction 10%) aqueous solution. The dichloromethane organic layer was collected, and excess water was removed with anhydrous Na2SO4. The organic layer was rotary evaporated to remove dichloromethane, and finally a light yellow oil was obtained. The oil was dissolved in 50 mL of methanol solvent, and 5 mmol (Boc)2O was added and stirred at room temperature for 12 h. TCL was used to detect whether the reaction was completed. After the reaction was completed, the solvent was evaporated under reduced pressure, the residue was extracted with 40 mL of dichloromethane, and 40 mL of water was added to wash three times and extract. The dichloromethane organic layer was collected, excess water was removed with anhydrous Na2SO4, dichloromethane was removed by evaporation under reduced pressure, and silica gel column chromatography was used to separate compounds 7a-7d, respectively, with yields of 76.24%-78.61%.

[0139] 2. Synthesis of Compounds 8a-8d

[0140]

[0141] The synthesis method is the same as that of compounds 3a and 3b, with a yield of 77.39%-81.22%.

[0142] 3. Synthesis of compounds 3-9a, 3-9e, 3-9f, and 3-9g

[0143]

[0144] Take 10mmol of compound 8a-8d obtained by hydrazine hydrolysis and 30mmol of anhydrous K2CO3 and dissolve them in acetonitrile. Stir and react at room temperature for 15min. Keep the temperature in a water bath at 45℃, add 10mmol of compound 4a, 4e or 4f while stirring, and keep the temperature at 45℃ for 12h. After the reaction, evaporate the reaction solvent under reduced pressure, extract the residue with 30mL of dichloromethane, and wash the extract with 30mL of The mixture was extracted three times with an aqueous solution of Na2CO3 (mass fraction of 10%), and the dichloromethane organic layer was collected. The organic layer was dried over Na2SO4, and dichloromethane was removed by evaporation under reduced pressure to obtain a light yellow oily substance. The oily substance was dissolved in 30 mL of methanol, and 10 mmol (Boc)2O was added. The reaction was stirred at room temperature for 12 h. The reaction was monitored by TCL. After the reaction was completed, methanol was evaporated under reduced pressure. The residue was extracted with 40 mL of dichloromethane, and then extracted three times with 40 mL of water. The dichloromethane organic layer was collected, and the organic layer was dried over anhydrous Na2SO4. Dichloromethane was evaporated under reduced pressure, and the light yellow 3-9a or colorless oily compounds 3-9e, 3-9f, and 3-9g were separated and purified by silica gel column. The yields were 49.3%, 52.1%, 52.9%, and 54.8%, respectively.

[0145] 4. Synthesis of Compounds J5-J7 and J10

[0146]

[0147] Take 5 mmol of compound 3-9a or compound 3-9e, 3-9f, 3-9g obtained by column chromatography in the previous step, dissolve them in appropriate amount of ethanol, cool to 0°C, add 4M hydrochloric acid ethanol solution (10 mL), warm to room temperature and stir to react for 24 hours until a large amount of solid is generated, filter with filter paper, wash three times with redistilled anhydrous ethanol, and dry to obtain solid compounds J5-J7 and J10, respectively, with yields of 31.64%-36.89%.

[0148] Spectral data of compounds J5-J7 and J10:

[0149] J5: 2-{3-[3-(3-propylhydrazinyl)propylamino]propyl}1H-benzissoquinoline-1,3-dione

[0150] yield: 33.15%; 1H NMR (300MHz, Deuterium Oxide)δ7.94(d,J=7.9Hz,2H),7.78(d,J=7.8Hz,2H),7.45(d,J=7.5Hz,2H),3.8 6(t,J=7.0Hz,2H),3.14~2.97(m,10H),2.09~1.98(m,4H),1.98~1.87(m,2H); 13C NMR(75MHz,D2O)δ165.15,135.28,131.42,130.63,126.93,126.23,120.11,47 .09,45.41,45.01,44.50,37.19,36.73,24.20,22.62,22.13; ESI-MS(positive ion mode):m / z[(M+H)+:calcd 384.36; obsd 383.23.

[0151] J6: 2-{3-[4-(3-propylhydrazinyl)butylamino]propyl}1H-benzissoquinoline-1,3-dione

[0152] yield:36.89%; 1H NMR (300MHz, Chloroform-d) δ7.97 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 7. 8Hz,2H),3.85(t,J=7.3Hz,2H),2.98~3.09(m,10H),1.93(m,2H),1.53~1.82(m,6H); 13C NMR(75MHz,D2O)δ165.25,135.32,131.47,130.70,126.97,125.98,120.21,47. 21,46.90,45.26,44.86,37.24,36.21,24.23,22.75,22.12,;ESI-MS(positive ion mode):m / z[(M+H)+:calcd 398.38; obsd 397.25.

[0153] J7: 2-{4-[4-(4-butylhydrazinyl)butylamino]butyl}1H-benzissoquinoline-1,3-dione

[0154] yield: 31.64%; 1H NMR (300MHz, Deuterium Oxide) δ7.89 (d, J = 7.9Hz 2H), 7.81 (d, J = 7.8Hz 2H), 7.14 (t, J = 7.1Hz 2H), 3.74 (t, J = 7.1Hz 2H),3.09~3.02(m,10H),1.73~1.18(m,12H); 13C NMR(75MHz,D2O)δ164.73,134.98,131.14,130.31,126.78,126.10,119.84,50.02,47.11 ,46.82,46.57,38.62,37.87,24.14,23.86,23.47,22.75,22.63,21.54; ESI-MS (positive ion mode):m / z[(M+H)+:calcd 426.43; obsd 425.28.

[0155] J10: 6-Chloro-2-{3-[4-(3-propylhydrazinyl)butylamino]propyl}1H-benzissoquinoline-1,3-dione

[0156] yield: 40.37%; 1H NMR (300MHz, Deuterium Oxide)δ8.63(d,J=8.6Hz,1H),8.12(d,J=8.1Hz,1H),7.75(d,J=7.8Hz,1H),7.24(t,J=7.8Hz,1H),7.09(d,J=7.8Hz,1H ), 4.66 (t, J = 7.3Hz, 2H), 3.11 ~ 3.19 (m, 10H), 2.07 ~ 2.13 (m, 2H), 1.73 ~ 2.04 (m, 2H), 1.59 ~ 1.79 (m, 4H); ESI-MS (positive ion mode):m / z[(M+H)+:calcd 432.27; obsd 431.21.

[0157] Test Example 1: Evaluation of biological activity of target molecules

[0158] (1) In vitro antitumor activity test

[0159] The MTT method was used to test the in vitro antitumor activity of the hydrazine-modified natural polyamine-naphthalimide conjugates synthesized in the above examples against human breast cancer cells (MDA-MB-231, MCF-7), human liver cancer cells (HepG2, SNU-739) and human colon cancer cells (HCT116, HT-29).

[0160] Test method: Add 100 μL of cell suspension to a 96-well plate, control the cell density to 3000-5000 cells / well, leave the last column as a zero well, place in a 37°C cell culture incubator for 24 hours, then add 100 μL of the hydrazine-modified natural polyamine-naphthalimide conjugate culture medium solution with gradient concentrations (0.2, 0.6, 1.3, 3.2, 7.5, 17.8, 42.2, 100, unit μM) to the 96-well plate, and continue to place in a 37°C cell culture incubator for 48 hours. Then add 20 μL of 5 mg / mL MTT solution to each well of the 96-well plate, culture in a 37°C cell culture incubator for 4 hours, take out, remove the culture medium, add 150 μL of DMSO, and shake in a 37°C shaker in the dark for 20 minutes. Measure the absorbance of each well with an enzyme reader at a wavelength of 470 nm, and calculate its IC 50 Each experiment was repeated at least three times, and the measurement results are shown in Table 1. The positive control was aminonafide (AF).

[0161] Table 1 In vitro antitumor activity (IC 50 Unit: μM)

[0162]

[0163] It can be seen from the experimental results in Table 1 that in the hydrazine-modified natural polyamine-naphthalimide conjugates of the present invention, the activity of the 3-amino-substituted compound is better than the 4-chlorine-substituted compound; the length of the polyamine chain also has a certain influence on the activity of the compound, and the activity of the triamine chain hydrazine-substituted compound is better than the diamine chain hydrazine-substituted compound. The activity effects of the compounds in the triamine chain length are not much different, and the activity of the 4-4 chain length is the best among the naphthalimide-containing substituent compounds; comprehensively evaluating compound J11, it shows better cytotoxicity to all cell lines. Therefore, the present invention selects compound J11 for subsequent pharmacological activity evaluation. Experimental Example 2: Effect of the hydrazine-modified natural polyamine-naphthalimide conjugate J11 on the migration ability of breast cancer cells

[0164] (1) Wound healing experiment

[0165] In order to evaluate the inhibitory effect of compound J11 on breast cancer cell migration, the present invention used a wound healing assay to detect the effect of compound J11 on breast cancer cell migration. Figure 1 As shown, compared with the control group, the inhibitory effect of compound J11 on breast cancer cell migration was significantly higher than that of the AF (aminofate) control group, and the inhibitory effect was concentration-dependent.

[0166] (2) Transwell chamber experiment

[0167] In order to further verify the ability of compound J11 to inhibit breast cancer cell migration, the present invention used Transwell assay to detect the effect of compound J11 on the migration ability of breast cancer cells. The results are as follows: Figure 2 As shown. Compound J11 has an inhibitory effect on breast cancer cells, and the effect is enhanced in a dose-dependent manner. The inhibitory effect of compound J11 on breast cancer cell migration is significantly stronger than that of the AF control group, and the effect is enhanced in a dose-dependent manner.

[0168] Experimental Example 3: The hydrazine-modified natural polyamine-naphthalimide conjugate J11 induces apoptosis of breast cancer cells

[0169] In order to study the relationship between the anti-tumor activity of compound J11 and cell apoptosis, the present invention uses the Annexin V / PI double staining method to test the apoptosis of breast cancer cells induced by compound J11 and positive drug AF. The present invention incubates breast cancer cells with compound J11 at concentrations of 0.5μM, 1μM and 5μM and 5μM AF for 24 hours, collects the cells, and then uses flow cytometry to analyze the apoptosis induced by the compounds.

[0170] The results are as follows Figure 3 As shown, the apoptosis ratio of breast cancer cells induced by compound J11 was positively correlated with its concentration, that is, the ratio of early apoptotic cells was 11.0% and the ratio of late apoptotic cells was 0.18% at a concentration of 5 μM of compound J11, and the ratio of early apoptotic cells was 16.4% and the ratio of late apoptotic cells was 0.26% at a concentration of 10 μM of compound J11.

[0171] Experimental Example 4: In vivo antitumor activity of the hydrazine-modified natural polyamine-naphthalimide conjugate J11

[0172] (1) Compound J11 inhibits the growth of orthotopic tumors in breast cancer cells in mice

[0173] In vitro experiments have shown that the hydrazine-modified natural polyamine-naphthalimide conjugate J11 has a good killing effect on breast cancer cells, in order to verify the in vivo anti-tumor activity of compound J11. The present invention implants 4T1 cells (mouse breast cancer cells, 250,000 / mouse) into the armpits of six-week-old BALB / c mice to establish a breast cancer tumor animal model. Before the tumor cells are inoculated, the mice are placed in the same temperature, humidity, food, drinking water and other feeding environments for feeding. The mice inoculated with 4T1 cells were observed every other day. The tumor formation of all mice was checked on the third day after inoculation, and the mice that did not successfully form tumors were re-inoculated. On the seventh day after inoculation, the mice were evenly divided into 3 groups (10 in each group) and marked as the 0th day of administration, including PBS group, AF (10 mg / kg), and J11 (10 mg / kg). The drug was administered intraperitoneally once every two days for a total of 5 times, during which the mouse state was observed and the mouse weight was measured. After two days of observation after the end of the administration, the mice were killed by cervical dislocation and the brain tissue and internal organs were removed to evaluate the organ index of the mice (the ratio of the weight of each organ to the body weight of the corresponding mouse). The tumor inhibition rate was calculated as follows: inhibition rate (%) = [(average fluorescence value of tumor in PBS control group - average fluorescence value of tumor in drug treatment group or positive group) / average fluorescence value of tumor in control group] × 100%).

[0174] The results showed that during the entire animal experiment, the body weight and organ index of each group of mice ( Figure 4 ) showed no obvious abnormal changes. During the treatment period, the compound J11-administered group did not show obvious dehydration, anorexia, movement disorders or other toxicity-related symptoms. This suggests that compound J11 can inhibit the progression of breast cancer and has no strong toxic reaction to mice.

[0175] The hydrazine-modified natural polyamine-naphthalimide conjugate of the present invention is an anti-tumor compound. The purpose of its design, synthesis and anti-cancer activity test is to prepare a broad-spectrum, high-efficiency, low-toxic anti-cancer active molecule, and provide a new candidate drug for clinical cancer treatment. It can be seen from the test results of the present invention that the hydrazine-modified natural polyamine-naphthalimide conjugate has good activity against tumor growth and metastasis.

[0176] In summary, the present invention verifies the anti-tumor activity of the hydrazine-modified natural polyamine-naphthalimide conjugate through in vivo and in vitro experiments, and studies the mechanism of the hydrazine-modified natural polyamine-naphthalimide conjugate to clarify its anti-tumor mechanism and its effect on tumor internal immune metabolism. The present invention mainly obtains the following conclusions:

[0177] 1. The MTT method was used to screen the in vitro activity of the hydrazine-modified natural polyamine-naphthalimide conjugate. The results showed that the activity of the 3-amino-substituted compound was better than that of the 4-chlorine-substituted compound; the length of the polyamine chain also had a certain effect on the activity of the compound. The activity of the triamine chain hydrazine-substituted compound was better than that of the diamine chain hydrazine-substituted compound. The activity of each compound in the triamine chain length was not much different. Among the compounds containing naphthalimide substituents, the activity of the 4-4 chain length was the best. Through screening, the present invention found the best compound J11 as the anti-tumor lead compound.

[0178] 2. Wound healing experiments and Transwell experiments proved that compound J11 can effectively inhibit the migration of breast cancer cells.

[0179] 3. By establishing an in vivo breast cancer tumor animal model, it was found that compound J11 had a high inhibitory effect on the growth of breast cancer and had no effect on the body weight of mice, indicating that compound J11 has a relatively safe anti-tumor activity.

Claims

1. A hydrazine-modified natural polyamine-naphthalimide conjugate, characterized in that: Its general structural formula is shown in Formula I: wherein R1 is H, NH2, Cl or Br; wherein R2 is H, NH2, Cl or Br; Where R3 is 2. The hydrazine-modified natural polyamine-naphthalimide conjugate according to claim 1, characterized in that: The structural formula is as follows:

3. The method for preparing the hydrazine-modified natural polyamine-naphthalimide conjugate according to claim 1 or 2, characterized in that: The steps include: 1) dissolving a hydrazinolysis compound and a 1,8-naphthalimide derivative containing or not containing a substituent in a solvent I, reacting at 40-50° C. for 10-15 hours, removing the solvent I after the reaction, extracting, washing, and drying the residue to obtain a product I; 2) dissolving the obtained product I in solvent II, adding a solution of hydrochloric acid in solvent II at -5 to 5°C, warming to room temperature and reacting for 10 to 15 hours to obtain a solid, filtering, washing, and drying to obtain the final product; The hydrazinolysis compound in step 1) is or The 1,8-naphthalimide derivative containing or not containing a substituent in step 1) is compound 4a, 4b, 4c, 4d, 4e, 4f, and the specific structural formula is as follows:

4. The preparation method according to claim 3, characterized in that: In step 1), the molar ratio of the hydrazinolysis compound to the 1,8-naphthalimide derivative containing or not containing a substituent is 1:(0.5-3).

5. The preparation method according to claim 3, characterized in that: In step 1), solvent I is acetonitrile, ethanol or methanol.

6. The preparation method according to claim 3, characterized in that: In step 2), solvent II is acetonitrile, ethanol or methanol.

7. Use of the hydrazine-modified natural polyamine-naphthalimide conjugate according to claim 1 or 2, or the hydrazine-modified natural polyamine-naphthalimide conjugate combined with cisplatin, oxaliplatin or 5-fluorouracil in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor includes, but is not limited to, human colon cancer, human liver cancer, human breast cancer, human lung cancer or cisplatin-resistant human lung adenocarcinoma.

9. The use according to claim 7, characterized in that: The invention discloses an application of the hydrazine-modified natural polyamine-naphthalimide conjugate alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis. The tumor cells are human colon cancer cells HCT-116 and HT-29, human liver cancer cells Huh-7, HepG-2 and SNU-739, human breast cancer cells MDA-MB-231 and MCF-7, and human lung cancer cells A549.

10. Use of the hydrazine-modified natural polyamine-naphthalimide conjugate according to claim 1 or 2 in the preparation of a drug for inhibiting tumor growth in a mouse orthotopic breast cancer model.

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