A hydrazine-modified natural polyamine-naphthalimide conjugate, its preparation method and application

Abstract

This invention belongs to the field of medicinal chemistry, specifically relating to a hydrazine-modified natural polyamine-naphthalimide conjugate, its preparation method, and its applications. This invention first introduces a hydrazine group at the end of the polyamine chain, then further extends the polyamine chain length, and finally conjugates it with a parent naphthimide core to synthesize a series of hydrazine-modified natural polyamine-naphthalimide conjugates. The compounds synthesized in this invention include conjugates with no substituents in the parent naphthimide core, conjugates with amino substitution at the 3-position, and conjugates with chlorine substitution at the 4-position. The conjugates of this invention solve problems such as poor solubility, cumbersome clinical compatibility, and poor patient immunity in the clinical application of chemotherapy drugs, as seen with previous naphthimide analogs represented by aminonaphthylfenitide, while also exhibiting good targeting properties. The conjugates of this invention can utilize the highly expressed polyamine transport proteins on the surface of tumor cells to improve the drug's targeting and bioavailability to tumor cells, and reduce toxic side effects on normal cells.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a hydrazine-modified natural polyamine-naphthalimide conjugate, its preparation method and application. Background Technology

[0002] The main reasons for the failure of chemotherapy drugs in treating 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 has been shown to have broad application prospects. However, due to the DNA intercalation of naphthalimide derivatives with antitumor activity, many clinical trials of naphthalimide derivatives have been terminated in Phase II, such as Amonafide, Mitonafide, and DMP-840.

[0003] As early as 1973, the Brana research group synthesized a series of naphthalimide-polyamine derivatives with anti-tumor activity. The study found that modifying the structure of naphthalimide 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 structures similar to polyamines that can be recognized and taken up by polyamine transporters (PTS). While they cannot replace the physiological functions of polyamines themselves, they can inhibit the utilization of polyamines by tumor cells through various mechanisms, thereby inducing apoptosis. Currently, the focus of naphthalimide structural modification is primarily on targeted modification of naphthalimide.

[0005] In order to synthesize naphthalimide antitumor drugs with clinical applicability, the inventors previously modified the structure of the parent naphthalimide nucleus in experiments and synthesized some naphthalimide-polyamine conjugates with good antitumor activity. The polyamines, as drug carriers, have selectivity and can target drug delivery.

[0006] In addition, during the research process, the inventors discovered that procarbazine hydrochloride, a representative drug containing a hydrazine group, also exerts antitumor activity by acting on DNA. Therefore, based on the characteristics and current research status of naphthalimide, hydrazine, and polyamines, the inventors designed a series of naphthalimide-polyamine hydrazine conjugates. By modifying the active backbone structure of natural polyamines with a hydrazine group, and using polyamines as carriers to link the parent naphthalimide core, they hope to obtain antitumor lead compounds with stronger activity than those without hydrazine-modified polyamine structures.

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

[0008] To address the shortcomings of existing technologies, this invention aims to propose a hydrazine-modified natural polyamine-naphthalimide conjugate with high stability and high targeting. This hydrazine-modified natural polyamine-naphthalimide conjugate solves problems such as poor solubility, cumbersome clinical compatibility, and poor patient immunity in the clinical application of chemotherapy drugs, as seen with previous naphthalimide analogs like aminonaphthylfenitide. Furthermore, this invention investigates whether the hydrazine-modified natural polyamine-naphthalimide conjugate exerts a dual inhibitory effect on subcellular organelles and the cell nucleus.

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

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

[0011] To achieve the above objectives, the technical solution created by this 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, R1 in the formula is H, NH2, Cl or Br; R2 in the formula is H, NH2, Cl or Br.

[0015] Specifically, R3 in the formula 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 includes the following steps:

[0020] 1) The hydrazine compound and a 1,8-naphthalenediamine derivative with or without a substituent are dissolved in solvent I and reacted at 40-50°C (preferably 45°C) for 10-15 h (preferably 12 h). After the reaction is completed, solvent I is removed, and the residue is extracted, washed, and dried to obtain product I.

[0021] 2) Dissolve the obtained product I in solvent II, and add the hydrochloric acid solution of solvent II at -5 to 5°C (preferably 0°C). Raise the temperature to room temperature and react for 10 to 15 hours (preferably 12 hours) to obtain a solid. Filter, wash, and dry to obtain the final product.

[0022] Specifically, the hydrazine hydrolysis compound mentioned in step 1) is or

[0023]

[0024] Specifically, the 1,8-naphthalenediamine derivatives with or without substituents mentioned in step 1) are compounds 4a, 4b, 4c, 4d, 4e, and 4f, with the following specific structural formulas:

[0025]

[0026] Specifically, the molar ratio of the hydrazine compound described in step 1) to the 1,8-naphthalenediamine derivative, whether substituted or unsubstituted, 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℃.

[0030] Furthermore, the specific synthetic route for the hydrazine-modified natural polyamine-naphthalimide conjugate is shown below:

[0031]

[0032]

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

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

[0035] 1,2-Di-tert-butoxycarbonylhydrazine was dissolved in solvent III, cesium carbonate was added first, followed by compound 1a or 1b, and the reaction was carried out at room temperature for 4-8 hours (preferably 6 hours). After the reaction was completed, the mixture was extracted, dried, and then solvent III was removed. The mixture was purified by silica gel column chromatography to obtain compound 2a or 2b.

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

[0037] Specifically, the structural formulas of compounds 2a and 2b are as follows:

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

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

[0040] Specifically, solvent III is DMF.

[0041] Specifically, in step a, the molar ratio of ditert-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-butoxycarbonylhydrazine to compound 1a or 1b is 1:(1 to 1.5), preferably 1:1.1.

[0043] Specifically, in step a, the molar ratio of 1,2-di-tert-butoxycarbonylhydrazine 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 extractant in step b is dichloromethane.

[0046] Furthermore, the specific synthetic routes for compounds 3a or 3b are shown below:

[0047]

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

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

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

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

[0052] Specifically, the structural formulas of compounds 7a-7d are as follows:

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

[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 to 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 compounds 7a-7d to hydrazine hydrate is 1:(2-3), preferably 1:3.

[0060] Furthermore, the specific synthetic routes for compounds 8a-8d are shown below:

[0061]

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

[0063] 1,8-naphthalenediamide, or 1,8-naphthalenediamide containing a substituent, is mixed with anhydrous K2CO3, KI, and 1,3-dibromopropane (or 1,4-dibromobutane) and dissolved in solvent IV. The mixture is reacted at room temperature for 10–15 h (preferably 12 h). After the reaction is complete, the mixture is filtered to remove solvent IV and purified by silica gel column chromatography to obtain compounds 4a-4d, 4e, or 4f, respectively.

[0064] Specifically, the structural formula of the substituted 1,8-naphthalenedimide is as follows:

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

[0066] Specifically, solvent IV is acetone.

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

[0068] Specifically, the molar ratio of 1,8-naphthalenediamide, or 1,8-naphthalenediamide containing a substituent, to anhydrous K2CO3 is 1:(1-4), preferably 1:3.

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

[0070] Furthermore, the specific synthetic routes for compounds 4a, 4b, 4c, and 4d are shown below:

[0071]

[0072] Furthermore, the specific synthetic routes for compounds 4e and 4f are shown below:

[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 antitumor drugs.

[0075] Specifically, the tumors include, but are 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 drugs that inhibit tumor cell proliferation, induce pyroptosis, or promote 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, in application, when the final concentration of the hydrazine-modified natural polyamine-naphthalimide conjugate is (0.5-5) μM and the treatment time is 24-48 h, it can inhibit the proliferation of tumor cells, induce their pyroptosis, or promote their apoptosis.

[0078] Preferably, 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 their pyroptosis or promote their apoptosis.

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

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

[0081] Preferably, the hydrazine-modified natural polyamine-naphthalimide conjugate, when administered at a dose of 10 mg / kg or 5 mg / kg every two days for a total of five times, can inhibit the metastasis of tumors in a mouse model of breast adenocarcinoma.

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

[0083] 1. This invention first introduces a hydrazine group at the end of a polyamine chain, then further extends the polyamine chain length, and finally conjugates the parent nucleus naphthalimide to synthesize a series of hydrazine-modified natural polyamine-naphthalimide conjugates. The compounds synthesized in this invention include naphthalimide-polyamine hydrazine conjugates with unsubstituent parent nucleus naphthalimide, parent nucleus naphthalimide with 3-amino substitution, and parent nucleus naphthalimide with 4-chlorine substitution. This invention employs... 1 H NMR, 13 The structures of the compounds were confirmed by C NMR and MS, and their antitumor activity was evaluated.

[0084] 2. The hydrazine-modified natural polyamine-naphthalimide conjugate of the present invention can utilize the polyamine transporter protein highly expressed on the surface of tumor cells to improve the drug's targeting of tumor cells, increase 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 the cell nucleus through modification of the naphthalimide structure.

[0086] 4. This invention improves the water solubility and lipid solubility of naphthalimide drugs, thereby increasing the uptake of drugs by tumor cells.

[0087] 5. The hydrazine-modified natural polyamine-naphthalimide conjugate of this invention is an antitumor compound. The design, synthesis, and anticancer activity testing of this compound aim to prepare a broad-spectrum, highly efficient, and low-toxicity anticancer molecule, providing novel candidate drugs for clinical cancer treatment. The experimental results of this invention show that the hydrazine-modified natural polyamine-naphthalimide conjugate exhibits good activity against tumor growth and metastasis. Attached Figure Description

[0088] Figure 1 To investigate 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 the combined drug administration group in a wound healing experiment; *P<0.05; **P<0.01; ***P<0.001;

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

[0090] Figure 3 This study aimed to quantitatively analyze the effects of different concentrations of hydrazine-modified natural polyamine-naphthalimide conjugates and 10 μMAF on the induction of apoptosis in breast cancer cells after 24 hours of incubation.

[0091] Figure 4 This is an in vivo antitumor activity evaluation experiment for the drug; among which Figure 4 A indicates that Balb / c mice were treated with amenophenate and J11 7 days after being inoculated with 4T1 cells, and were given the medication once every 2 days for a total of 4 times; Figure 4 B represents the quantitative analysis of the tumor growth inhibition effect after treatment with amenofiltrate and J11; Figure 4 C represents the change in mouse body weight during treatment; Figure 4 D represents the change in tumor volume during treatment; 4E represents the quantitative analysis of organ indices such as heart, liver, spleen, lung, and kidney in mice after treatment. *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation

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

[0093] The abbreviations used in the embodiments of this invention include: Ac for acetyl, Me for methyl, DMSO for dimethyl sulfoxide, DMF for dimethylformamide, DCM for dichloromethane, Boc for tert-butyloxycarbonyl, Et for ethyl, Cis. for cisplatin, Oxp. for oxaliplatin, and AF for aminonaphthylfine. Compounds 1a, 1b, and the substituted 1,8-naphthalenedimide are also mentioned. 1,8-Naphthalenediamine, cisplatin, oxaliplatin, and aminonaphthylfine are all commercially available 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℃.

[0095] Example 1

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

[0097]

[0098] Dissolve 10 mmol (2.18 g) of di-tert-butyl dicarbonate (Boc)₂O in 25 mL of methanol. Stir in an ice bath for 10 min in a water bath. While stirring, slowly add 10 mL of hydrazine hydrate (4.25 mmol) in methanol. After the addition is complete, stir the reaction at room temperature for 12 h. Evaporate the reaction solvent under reduced pressure. Mix the remaining solvent with 100-200 mesh silica gel and mix thoroughly. The amount of silica gel should be 1.5 times the sample volume. Use dry packing for column packing. Use a mixed solvent of petroleum ether (PE) and ethyl acetate (EA) as the developing solvent to elute the sample. The ratio (volume ratio) of petroleum ether (PE) to ethyl acetate (EA) is 15:1. For large-scale synthesis, the sample can be recrystallized using a mixed solvent with a PE:EA ratio of 15:1 to obtain 1,2-di-tert-butyloxycarbonylhydrazine.

[0099] Dissolve 10 mmol of 1,2-di-tert-butoxycarbonylhydrazine in 10 mL DMF was placed in a round-bottom flask, and 11 mmol of cesium carbonate was added. The mixture was stirred magnetically 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. The mixture was reacted at room temperature for 6 h, and the reaction was detected by TCL method. The TCL detection results before and after the reaction were compared to confirm that the reaction was complete. 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 with anhydrous Na2SO4, and the reaction solvent DMF was removed by vacuum evaporation. The remaining solvent was mixed evenly with 100-200 mesh silica gel. The amount of silica gel should be 1.5 times the amount of sample. Dry column packing was used, and a mixed solvent of petroleum ether (PE) and ethyl acetate (EA) was used as the developing solvent. The silica gel column was purified by purification (petroleum ether / ethyl acetate, V / V = 10:1) to obtain compounds 2a and 2b, with yields of 83.57%-87.16%.

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

[0101]

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

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

[0104]

[0105] 1.97 g (10 mmol) of substituted 1,8-naphthalenediamine, 30 mmol of anhydrous K₂CO₃, 1.5 mmol of KI, and 50 mL of acetone were added to a 100 mL round-bottom flask, followed by the addition of 30 mmol of 1,3-dibromopropane (or 1,4-dibromobutane). The reaction was stirred at room temperature for 12 h, and the reaction was monitored by TLC. The mixture was filtered, and the acetone solvent was removed by evaporation under reduced pressure. The residue was eluted by rapid column chromatography with a gradient of petroleum ether / ethyl acetate to obtain substituted N-(3-bromopropyl)-1,8-naphthalenediamine and substituted N-(4-bromobutyl)-1,8-naphthalenediamine (i.e., compounds 4a-4d), with yields of 80.52%-89.26%.

[0106]

[0107] Meanwhile, by replacing the substituted 1,8-naphthalenediamine with 1,8-naphthalenediamine, unsubstituted N-(3-bromopropyl)-1,8-naphthalenediamine (compound 4e) and unsubstituted N-(4-bromobutyl)-1,8-naphthalenediamine (compound 4f) were prepared in the same manner.

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

[0109]

[0110] 10 mmol of compound 3b obtained from hydrazine hydrolysis and 30 mmol of anhydrous K2CO3 were dissolved in acetonitrile. The reaction was carried out at room temperature for 15 min. Then, 10 mmol of compounds 4a-4d were added to the mixture while stirring in a water bath at 45 °C. The reaction was carried out at 45 °C for 12 h. After the reaction was completed, the acetonitrile solvent was evaporated under reduced pressure. The residue was extracted with 30 mL of dichloromethane. The extract was then purified with 30 mL of dichloromethane. The organic layer of dichloromethane was washed three times with an aqueous solution of Na₂CO₃ (10% by mass). The organic layer was dried over anhydrous Na₂SO₄ and the dichloromethane was removed by evaporation under reduced pressure, yielding a pale yellow oil. This oil was dissolved in 50 mL of methanol, and 5 mmol of (Boc)₂O was added. The mixture was stirred at room temperature for 12 h. The reaction was monitored by TCL to determine if it was complete. After the reaction was complete, the methanol solvent was evaporated under reduced pressure. The residue was extracted with 40 mL of dichloromethane. The extract was washed three times with 40 mL of water. The organic layer of dichloromethane was collected and dried over anhydrous Na₂SO₄. The dichloromethane was removed by evaporation under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate, V / V = 5:1) to give compounds 5a-5d, with yields of 57.78%-63.46%.

[0111]

[0112] Simultaneously, compounds 5e, 5f, 5g, and 5h were prepared by reacting unsubstituent compounds 4e and 4f with compounds 3a and 3b, using the same synthetic method as compounds 5a-5d.

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

[0114]

[0115] 5 mmol of compounds 5a-5d obtained by column chromatography were dissolved in an appropriate amount of ethanol, cooled to 0°C, and 10 mL of 4 M hydrochloric acid ethanol solution was added. The mixture was then heated to room temperature and stirred for 12 h until a large amount of solid was formed. The mixture was filtered through filter paper, washed three times with redistilled anhydrous ethanol, and dried to obtain solid compounds J8-J9 and J11-J12, respectively, with yields of 37.89%-43.25%.

[0116]

[0117] Simultaneously, compounds 5e, 5f, 5g, and 5h were used to prepare compounds J1-J4, using the same synthetic methods as compounds J8-J9 and J11-J12.

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

[0119] J1: 2-[3-(3-propanehydrazyl)propyl]1H-benzisoquinoline-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-Butylhydrazyl)propyl]1H-benzisoquinoline-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-propanehydrazine)-butyl]1H-benzisoquinoline-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-Butylhydrazyl)-Butyl]1H-Benzisoquinoline-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-Butylhydrazyl)propyl]1H-Benzisoquinoline-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-Butylhydrazyl)-Butyl]1H-Benzisoquinoline-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-Butylhydrazyl)-Butyl]1H-Benzisoquinoline-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-Butylhydrazyl)propyl]1H-Benzisoquinoline-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. Then, 0.55 g (4 mmol) of anhydrous K2CO3 was added, and 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 the water bath for 12 h. After the reaction was complete, acetonitrile was removed by rotary evaporation under reduced pressure. The remaining material in the round-bottom flask was extracted with 30 mL of dichloromethane. The extract was washed three times with 30 mL of 10% Na2CO3 aqueous solution. The dichloromethane organic layer was collected, and excess water was removed with anhydrous Na2SO4. The organic layer was then removed by rotary evaporation to remove dichloromethane, finally yielding a pale yellow oil. This oil was dissolved in 50 mL of methanol, and 5 mmol of (Boc)2O was added. The mixture was stirred at room temperature for 12 h. The reaction was monitored by TCL to determine if it was complete. After the reaction was complete, the solvent was evaporated under reduced pressure. The remaining material was extracted with 40 mL of dichloromethane. The mixture was washed three times with 40 mL of water and extracted again. The dichloromethane organic layer was collected, and excess water was removed with anhydrous Na2SO4. The dichloromethane was then removed by evaporation under reduced pressure. The mixture was separated by silica gel column chromatography to obtain compounds 7a-7d, with yields of 76.24%-78.61%.

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

[0140]

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

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

[0143]

[0144] 10 mmol of compounds 8a-8d obtained from hydrazine hydrolysis and 30 mmol of anhydrous K2CO3 were dissolved in acetonitrile. The mixture was stirred at room temperature for 15 min. The mixture was then placed in a water bath at 45 °C, and 10 mmol of compounds 4a, 4e, or 4f were added while stirring. The reaction was continued at 45 °C for 12 h. After the reaction was completed, the solvent was evaporated under reduced pressure. The residue was extracted with 30 mL of dichloromethane. The extract was then diluted with 30 mL of dichloromethane. The product was extracted three times with an aqueous solution of Na₂CO₃ (10% by mass), and the dichloromethane organic layer was collected. The organic layer was dried with Na₂SO₄, and the dichloromethane was removed by vacuum evaporation, yielding a pale yellow oily substance. This oily substance was dissolved in 30 mL of methanol, and 10 mmol of (Boc)₂O was added. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by TCL. After the reaction was completed, the methanol was removed by vacuum evaporation. 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 with anhydrous Na₂SO₄. The dichloromethane was removed by vacuum evaporation, and the product was purified by silica gel column chromatography to obtain pale yellow 3-9a or colorless oily compounds 3-9e, 3-9f, and 3-9g, with yields of 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 compounds 3-9e, 3-9f, and 3-9g obtained from the previous column chromatography and dissolve them in an appropriate amount of ethanol. Cool to 0°C and add 10 mL of 4 M hydrochloric acid in ethanol solution. Heat to room temperature and stir for 24 h until a large amount of solid is formed. 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-propanehydrazyl)propylamino]propyl}1H-benzisicoquinoline-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-propanehydrazyl)butanyl]propyl}1H-benzisicoquinoline-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-buthydrazyl)butanyl]butyl}1H-benzisicoquinoline-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-propanehydrazyl)butanoyl]propyl}1H-benzisicoquinoline-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] Experimental Example 1: Evaluation of the Bioactivity of the Target Molecule

[0158] (1) In vitro antitumor activity test

[0159] The in vitro antitumor activity of the hydrazine-modified natural polyamine-naphthalimide conjugates synthesized in the aforementioned 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) was tested using the MTT assay.

[0160] Test method: 100 μL of cell suspension was added to each well of a 96-well plate, controlling the cell density to 3000-5000 cells / well, with the last column reserved as a zeroing well. After incubation at 37℃ for 24 h, 100 μL of the hydrazine-modified natural polyamine-naphthalimide conjugate culture medium solution at gradient concentrations (0.2, 0.6, 1.3, 3.2, 7.5, 17.8, 42.2, 100, unit μM) was added to each well, and the plate was incubated at 37℃ for another 48 h. Then, 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was incubated at 37℃ for 4 h. After removal, the culture medium was aspirated, and 150 μL of DMSO was added. The plate was then shaken at 37℃ in the dark for 20 min. The absorbance of each well was measured at 470 nm using a microplate reader, and the IC50 was calculated. 50 The values ​​were measured at least three times for each group of experiments, and the results are shown in Table 1. The positive control was aminonaphthylamine (AF).

[0161] Table 1. In vitro antitumor activity (IC50) of hydrazine-modified natural polyamine-naphthalimide conjugates. 50 Unit: μM

[0162]

[0163] As shown in Table 1, among the hydrazine-modified natural polyamine-naphthalimide conjugates of this invention, the 3-amino-substituted compounds exhibit better activity than the 4-chlorine-substituted compounds. The length of the polyamine chain also has a certain influence on the activity of the compounds; the triamine chain hydrazine-substituted compounds show better activity than the diamine chain hydrazine-substituted compounds. Within the triamine chain lengths, the activity of each compound is not significantly different. Among the naphthalimide-substituent compounds, the 4-4 chain length shows the best activity. Overall, compound J11 exhibits better cytotoxicity against all cell lines. Therefore, compound J11 was selected for subsequent pharmacological activity evaluation in this invention. 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] To evaluate the inhibitory effect of compound J11 on breast cancer cell migration, this invention employed a wound healing assay to examine the influence of compound J11 on the migration ability of breast cancer cells. The results are as follows: Figure 1 As shown, compared with the control group, compound J11 had a significantly higher inhibitory effect on breast cancer cell migration than the AF (aminonaphthylfenoxate) control group, and the inhibitory effect was concentration-dependent.

[0166] (2) Transwell chamber experiment

[0167] To further verify the inhibitory effect of compound J11 on breast cancer cell migration, this invention employed a Transwell assay to examine 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 this effect increases 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 this effect also increases in a dose-dependent manner.

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

[0169] To investigate the relationship between the antitumor activity of compound J11 and apoptosis, this invention employed Annexin V / PI double staining to test the apoptosis-inducing effect of compound J11 and the positive control drug AF on breast cancer cells. Breast cancer cells were incubated with 0.5 μM, 1 μM, and 5 μM concentrations of compound J11 and 5 μM of AF for 24 hours, and the cells were collected. Flow cytometry was then used to analyze the apoptosis-inducing effect of the compounds.

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

[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 mouse orthotopic tumors of breast cancer cells.

[0173] In vitro experiments demonstrated that the hydrazine-modified natural polyamine-naphthalimide conjugate J11 exhibited good killing activity against breast cancer cells. To verify the in vivo antitumor activity of compound J11, this invention established a breast cancer tumor animal model by implanting 4T1 cells (250,000 murine breast cancer cells / mouse) into the axilla of six-week-old BALB / c mice. Before inoculation with tumor cells, the mice were housed in the same environment with identical temperature, humidity, food, and water. The mice inoculated with 4T1 cells were observed every other day. On the third day after inoculation, the tumor formation of all mice was checked, and mice that failed to form tumors were re-inoculated. On the seventh day after inoculation, the mice were divided into three groups (n=10 per group) and marked as day 0 of drug administration, including the PBS group, the AF group (10 mg / kg), and the J11 group (10 mg / kg). The drugs were administered intraperitoneally every two days for a total of five times, during which the mice's condition and weight were observed. After two days of observation following drug administration, the mice were euthanized by cervical dislocation, and brain tissue and internal organs were removed. The organ index (the ratio of the weight of each organ to the corresponding mouse body weight) was assessed. The tumor inhibition rate was calculated using the formula: Inhibition rate (%) = [(mean fluorescence value of tumors in the PBS control group - mean fluorescence value of tumors in the drug treatment group or positive group) / mean fluorescence value of tumors in the control group] × 100%.

[0174] The results showed that throughout the animal experiment, the body weight and organ index of mice in each group were relatively stable. Figure 4 No significant abnormal changes were observed in any of the mice. During treatment, no significant dehydration, anorexia, dyskinesia, or other toxicity-related symptoms were observed in the compound J11 group. This suggests that compound J11 can inhibit the progression of breast cancer and has no strong toxic effects in mice.

[0175] The hydrazine-modified natural polyamine-naphthalimide conjugate of this invention is an antitumor compound. The purpose of its design, synthesis, and anticancer activity testing is to prepare a molecule with broad-spectrum, high-efficiency, and low-toxicity anticancer activity, providing novel drug candidates for clinical cancer treatment. The experimental results of this invention show that the hydrazine-modified natural polyamine-naphthalimide conjugate exhibits good activity against tumor growth and metastasis.

[0176] In summary, this invention verified the antitumor activity of the hydrazine-modified natural polyamine-naphthalimide conjugate through in vivo and in vitro experiments. By investigating the mechanism of the hydrazine-modified natural polyamine-naphthalimide conjugate, its antitumor mechanism and its influence on tumor-mediated immune metabolism were elucidated. The main conclusions of this invention are as follows:

[0177] 1. The in vitro activity screening of the hydrazine-modified natural polyamine-naphthalimide conjugates was performed using the MTT assay. The results showed that compounds with an amino substitution at the 3-position exhibited better activity than those with a chlorine substitution at the 4-position. The length of the polyamine chain also had a certain influence on the compound activity; compounds with a triamine chain and hydrazine substitution showed better activity than those with a diamine chain and hydrazine substitution. Within the triamine chain length range, the activity of various compounds was not significantly different. Among the naphthalimide-containing substituents, compounds with a 4-4 chain length showed the best activity. Through screening, this invention identified compound J11 as the most effective antitumor lead compound.

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

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

Claims

1. The application of a hydrazine-modified natural polyamine-naphthalimide conjugate in the preparation of antitumor drugs, characterized in that, The tumors involved are human colon cancer cells HT-29, human liver cancer cells SNU-739, or human breast cancer cells MCF-7. The hydrazine-modified natural polyamine-naphthalimide conjugate has the following structural formula: .

2. The application according to claim 1, characterized in that, The application of the hydrazine-modified natural polyamine-naphthalimide conjugate in the preparation of drugs that inhibit tumor cell proliferation, induce pyroptosis, or promote apoptosis, wherein the tumor cells are human colon cancer cells HT-29, human liver cancer cells SNU-739, or human breast cancer cells MCF-7.

3. The application according to claim 2, characterized in that, When applied, if the final concentration of the hydrazine-modified natural polyamine-naphthalimide conjugate is (0.5-5) μM and the treatment time is 24-48 h, it can inhibit the proliferation of tumor cells, induce their pyroptosis, or promote their apoptosis.

4. The application according to claim 1 or 2, characterized in that, The hydrazine-modified natural polyamine-naphthalimide conjugate was prepared by the following method: 1) The hydrazine compound and a 1,8-naphthalenediamine derivative with or without a substituent were dissolved in solvent I and reacted at 40-50°C for 10-15 h. After the reaction was completed, solvent I was removed, and the residue was extracted, washed, and dried to obtain product I. 2) Dissolve the obtained product I in solvent II, and add hydrochloric acid solution of solvent II at -5~5℃. Raise the temperature to room temperature and react for 10~15h to obtain a solid. Filter, wash and dry to obtain the final product. The hydrazine compound mentioned in step 1) is ; The 1,8-naphthalenediamine derivatives with or without substituents mentioned in step 1) are compounds 4b, 4c, and 4d, with the following specific structural formulas: .

5. The application according to claim 4, characterized in that, The molar ratio of the hydrazine compound described in step 1) to the 1,8-naphthalenediamine derivative, whether substituted or unsubstituted, is 1:(0.5~3).

6. The application according to claim 4, characterized in that, In step 1), solvent I is acetonitrile, ethanol, or methanol.

7. The application according to claim 4, characterized in that, In step 2), solvent II is acetonitrile, ethanol, or methanol.

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